CROSS-REFERENCE TO RELATED APPLICATION

[001] This application claims priority to United States Provisional Patent Application No. 63/403,831, filed September 05, 2022, the entire contents of which is incorporated herein by reference in its entirety.

FIELD

[002] The present subject matter relates to treatment and prevention of endocrine, autoimmune and neurological disorders including type 1 diabetes mellitus.

BACKGROUND

[003] Most of the autoimmune and neurological disorders have been linked to a combination of genetic and environmental factors. Many neurological disorders are complex and can harbor a variety of characteristics such as neuro -inflammation and autoimmunity. Examples of neurological and autoimmune disorders include Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD), Stiff-person syndrome (SPS), multiple sclerosis (MS) and type 1 diabetes mellitus (T1DM).

[004] Neurological disorders are primarily caused by neurons and neurotransmission in the human brain and central nervous system (CNS). Neurons are excitable cells that communicate through a combination of electrical and chemical signals. 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 postsynaptic neuron's specialized proteins called neurotransmitter receptors. The neurotransmitter molecules affect the postsynaptic neuronal function through binding to the receptor proteins.

[005] Glutamate and gamma aminobutyric acid (GABA) are major neurotransmitters in the brain, CNS and the pancreas. Inhibitory GABA and excitatory glutamate work together to control many processes, including the overall level of excitation in the cells and apoptosis. Neuronal excitability is characterized by a balance between excitatory input of glutamate and counterbalancing inhibitory transmission of GABA. During neuronal signaling a neuron releases the neurotransmitter at its synapse and the message is conveyed to the adjacent neuron though activation of specific receptors on its surface. A balanced interaction is required to maintain physiological homeostasis, while prolonged imbalance can lead to a variety of diseases. [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] Both glutamatergic and GABAergic neurons are highly diversified in a variety of electrically excitable cells, thus maintaining appropriate and balanced neurotransmission is essential in controlling not just the level of excitation in the CNS, but also in the pancreas through extra-neuronal GABAergic signaling. Dysfunction in balance or transmission of GABA and glutamate in the CNS is known to be associated with a number of neurological and autoimmune conditions such as epilepsy, SPS and MS.

[007] Glutamate is the major excitatory neurotransmitter and, as such, it inevitably plays a role in optimizing and synchronizing cellular responses in neurons, endocrine and immune cells alike. In neurons, excess glutamate is known to induce 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. Several glutamate receptors and glutamate transporters in the CNS are known to mediate glutamate effects and regulate extracellular glutamate levels. The function of a-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMD A) receptors is enhanced in the hippocampus of kindled rodents and in the cerebral cortex of patients with focal epilepsy.

[008] Additionally, in electrically excitable cells of the pancreatic islets of Langerhans, AMPA and NMDA receptors are particularly important in islet cell survival due to their function regulating glucagon, insulin and somatostatin secretion. 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 Ca ²⁺ 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] .

[009] 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] 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 is produced from glutamate by the enzyme glutamic acid decarboxylase (GAD) in neurons, B lymphocytes and pancreatic P-cells in the islets of Langerhans, where it functions as a paracrine and autocrine signaling molecule controlling the function of islet endocrine cells. Antibodies to GAD have been linked to a variety of neurological diseases, such as SPS, cerebellar ataxia, and epilepsy. These antibodies were first discovered in 1988, although there are still various questions surrounding GAD autoimmunity. GABA plays a significant part in a range of bodily functions, and while its significance in many processes remains unclear, it is present at varied low amounts in all human tissues and organ systems. 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. [Oi l] Notably, 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, T1DM 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]. Maintaining appropriate and balanced glutamate/GABA transmission is essential in controlling the level of excitation and survival of the CNS cells, activation and proliferation of the immune cells and the function and viability of endocrine cells in pancreatic islets promoting glucose- and incretin- stimulated insulin secretion.

[012] 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] .

[013] 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 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].

[014] T1DM is a disease characterized by the destruction of pancreatic islet P-cells by autoimmunity. T1DM affects over 1.6 million individuals in the USA alone and some 18 million worldwide, according to the Centers for Disease Control and Prevention. It is anticipated that the prevalence of T1DM in the United States will grow threefold by 2050, based on projections of a linear increase in incidence over the next 30 years.

[015] Treatment options are extremely limited, and the majority of T1DM patients rely solely on insulin therapy for life. The high cost of new technologies based on enhanced insulin therapies makes them less universally accessible. Due to a variety of limiting factors, including their great complexity, donor availability, requirement for life-long immunosuppression, and high expense, procedures like islet or pancreas transplantation cannot be considered scalable and consistently reliable therapeutic choices. Future therapeutic options rely extensively on islet transplants produced from autologous stem cells.

[016] In most patients, some P-cell function is retained for a short time after T1DM onset, however this is insufficient to maintain normoglycemia without exogenous insulin administration. Even with slightly preserved P-cell function, diabetes complications can be avoided [Roep BO, Thomaidou S, van Tienhoven R, Zaldumbide A. Type 1 diabetes mellitus as a disease of the P-cell (do not blame the immune system?). Nat Rev Endocrinol. 2021 ; 17(3): 150-161. doi:10.1038/s41574-020-00443-4; Leighton E, Sainsbury CA, Jones GC. A Practical Review of C-Peptide Testing in Diabetes. Diabetes Ther. 2017;8(3):475-487. doi: 10.1007/s 13300-017-0265-4, the entire contents of which is incorporated herein by reference]. Therefore, any therapy that can protect and preserve residual P-cell function is of paramount importance.

[017] Maintenance treatment of T1DM involves lifelong injections of insulin. While insulin provides an option of a chronic therapy that is managing the patient’s survival, it is not a cure and is usually accompanied by severe complications. The risk of hypoglycemia, which contributes to the development of brain damage, dementia, cardiovascular complications, atherosclerosis and arterial hypertension, is particularly high. Nevertheless, without insulin the disease progresses rapidly and leads to severe complications such as diabetic retinopathy, polyneuropathy, diabetic foot ulcers, ketoacidosis, diabetic coma, diabetic cardiomyopathy, kidney failure, all of which can lead to disability or result in the patient’s death.

[018] Efforts in this direction are aimed at finding new drugs that can save the patient from the daily administration of insulin by preserving the P-cells and even regenerating the P-cell pool. As such, T1DM can greatly benefit from enhancing GABA uptake and transmission, as it functions within the islets in the regulation of islet cell function and secretion. It protects and promotes P-cell regeneration against apoptosis induced by cytokines, drugs, and other stresses, and has anti-inflammatory and immunoregulatory activities. GABA was identified as a key compound to promote the misexpression of Pax4/ARX inhibition and initiate conversion of a- cells into P-like cells. It was concluded that GABA influences the a-cells through the GAB A(A) receptor in order to inhibit ARX activities, such inactivation leading to their transformation into P-like cells. [GABA signaling stimulates a-cell-mediated P-like cell neogenesis (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501192/), the entire contents of which is incorporated herein by reference]. Under prolonged and consistent exposure to GABA P-like cell mass will be increased potentially restoring insulin secreting function.

SUMMARY

[019] 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.

[020] According to one aspect of the present subject matter, there is provided a composition for use in the treatment and prevention of neurodegenerative, endocrine and autoimmune diseases that are caused by, or linked to, glutamate/GABA imbalance and excito toxicity, the composition comprising: a first composition comprising gamma-aminobutyric acid (GABA) or a GABA- receptor agonist; a second composition comprising at least one decapeptyl peptidase (DPP-4) inhibitor; a third composition comprising at least one proton pump inhibitor (PPI); and a fourth composition comprising at least one Glucagon like peptide 1 (GLP-1) receptor agonist.

[021] According to one embodiment, the disease is type 1 diabetes.

[022] According to one embodiment, the GABA, or the GABA-receptor agonist, is administered in a daily dose of substantially 375 to 3,000 mg.

[023] According to one embodiment, the at least one DPP-4 inhibitor is selected from the group consisting of: at least one CD45 inhibitor, at least one CD3 inhibitor, at least one antiinterleukin (IL)-21 antibody, at least one anti-interleukin (IL)- 18 antibody, and any combination thereof.

[024] According to one embodiment, the at least one DPP-4 inhibitor is selected from the group consisting of: alogliptin, linagliptin, saxagliptin, sitagliptin, vildagliptin, and any combination thereof.

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

[026] According to one embodiment, the at least one DPP-4 inhibitor is administered orally at a daily dose of substantially 2.5 to 400 mg. [027] According to one embodiment, the at least one PPI is selected from the group consisting of: omeprazole, pantoprazole, lansoprazole, rabeprazole, esomeprazole, and combination thereof.

[028] According to one embodiment, the at least one PPI is omeprazole.

[029] According to one embodiment, the PPI is administered orally at a daily dose of substantially 10 to 360 mg.

[030] According to one embodiment, the PPI is administered orally at a daily dose of substantially 10 to 120 mg.

[031] According to another aspect of the present subject matter, there is provided a kit for treating and preventing neurodegenerative, endocrine and autoimmune diseases that are caused by, or linked to, glutamate/GABA imbalance and excito toxicity, the kit comprising the composition for use described above.

[032] According to one embodiment, the disease is type 1 diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS

[033] Embodiments are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the embodiments. In this regard, no attempt is made to show structural details in more detail than is necessary for a fundamental understanding, the description taken with the drawings making apparent to those skilled in the art how several forms may be embodied in practice.

[034] In the drawings:

Fig. 1 presents a graph showing survival percentage of model mice of type 1 diabetes following treatment with combinations of drugs, during a study. Fig. 2 presents a graph showing blood glucose levels (median, 25% percentile) of model mice of type 1 diabetes following treatment with combinations of drugs, during a study.

Fig. 3 presents a graph showing a statistical analysis of blood glucose levels (median, 25% percentile) at the last day of the study of model mice of type 1 diabetes following treatment with combinations of drugs.

Fig. 4 presents a graph showing percentage of healthy animals among model mice of type 1 diabetes following treatment with combinations of drugs, during a study.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[035] 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.

[036] For clarity, non-essential elements were omitted from some of the drawings.

[037] The endocrine cells of pancreatic islets and the neurons of the central nervous system have many features in common. They express similar genes and cell surface receptors including GLP-1 receptors, glutamate receptors and GABA receptors. Endocrine cells, immune cells and neurons are also similar in that they are capable of releasing neurotransmitters such as GABA.

[038] Amino acids, which play pivotal roles in fast neuronal signaling, have also been proposed to act as signaling molecules in the islets of Langerhans. 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 not just the level of excitation in CNS, but also in the pancreas through extra-neuronal GABAergic signaling. Dysfunction in balance or transmission of GABA and glutamate in the CNS is known to be associated with a number of neurological and neurodegenerative conditions such as epilepsy and seizures, while dysfunction in extra-neuronal signaling of glutamate and GABA in pancreatic islets of Langerhans can result in a-cell and P-cell malfunction, excitotoxicity and apoptosis, triggering conditions such as T1DM.

[039] The present subject matter provides a composition comprising a combination of therapeutic agents for treating and preventing a variety of neurodegenerative, endocrine and autoimmune diseases that are caused by, or linked to, glutamate/GAB A imbalance, for example substantial glutamate elevation, and excito toxicity. For the sake of simplicity, the term "composition" will occasionally be used hereinafter instead of the phrase "composition comprising a combination 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, glutamate/GAB A imbalance, for example substantial glutamate elevation, excitotoxicity and transmission dysfunction, with the aforementioned composition.

[040] The present subject matter provides a composition for treating and preventing diseases that are caused by, or linked to, GABAergic transmission dysfunction, the composition comprising:

Glucagon like peptide 1 (GLP-1) receptor agonist; a proton pump inhibitor (PPI); gamma-aminobutyric acid (GABA) or a GABA-receptor agonist; and a decapeptyl peptidase (DPP-4) inhibitor.

GLP-1 receptor (GLP-1R) agonist

[041] GLP-1 R 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 GAB A(A) 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. GLP-1 and GLP-1 R agonists (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] .

[042] 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/S0006 2952183032417via%3Dihub), the entire contents of which is incorporated herein by reference]. Administration of a DPP-4 inhibitor will suppress hyperthermia-induced neuronal excitability through activation of the GLP-1R pathway, and will also enhance GABAergic transmission.

[043] 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.

[044] According to one embodiment, the GLP-1 R agonist is in a form of a soluble pharmaceutical composition for parenteral administration. According to another embodiment, the GLP-1R agonist is in a form of an injectable suspension for parenteral administration. According to another embodiment, the GLP-1 R agonist is in a form of tablets configured to be orally administered to a patient.

[045] According to one embodiment, a formulation of the GLP-1R agonist comprises an insulinotropic GLP-1 compound, which is an analog of human GLP-1 and acts as a GLP-1 agonist, and pharmaceutically acceptable additives.

[046] According to one embodiment, the GLP-1R agonist is a short-acting Exenatide (Byetta), administered in an amount of substantially 5 pg subcutaneously twice daily within substantially 1 hour before meals. According to another embodiment, based on clinical response, the dose of Byetta can be increased to substantially 10 pg twice daily after substantially 1 month of therapy. [047] According to one embodiment, the GLP-1R agonist is Lixisenatide (Adlyxin), 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. On day 15, the amount of Lixisenatide is increased to substantially 20 pg once daily.

[048] According to one embodiment, the GLP-1R agonist is immediate-acting Liraglutide (Victoza/Saxenda) 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. After one week at substantially 0.6 mg per day, the dose is increases to substantially 1.2 mg daily. 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 yet another embodiment, if additional glycemic control is required, the dose is increased to substantially 2.4 mg daily after at least one week of treatment with the substantially 1.8 mg daily dose. According to yet another embodiment, if additional glycemic control is required, the dose is increased to substantially 3 mg daily after at least one week of treatment with the substantially 2.4 mg daily dose.

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

[050] According to one embodiment, the GLP-1R agonist is Albiglutide (Tanzeum) administered in an amount of substantially 30 mg subcutaneously once weekly. The amount of Albiglutide can be increased to substantially 50 mg subcutaneously once weekly.

[051] According to one embodiment, the GLP-1R agonist is Dulaglutide (Trulicity) administered in an amount of substantially 0.75 mg subcutaneously once weekly. The amount of Dulaglutide can be increased to substantially 4.5 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]. [052] According to one embodiment, the GLP- 1R agonist is Semaglutide Injection, known as Ozempic, or Wegovy (Novo Nordisk).

[053] According to one embodiment, Semaglutide is first 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 4 weeks on the substantially 0.25 mg dose, the dosage is increased to substantially 0.5 mg once weekly. If additional glycemic control is needed after at least 4 weeks on the substantially 0.5 mg dose, the dosage can be increased to substantially 1 mg once weekly. According to another embodiment, if additional glycemic control is needed after at least 4 weeks on the substantially 1 mg dose, the dosage can be increased to substantially 2 mg once weekly.

[054] According to one embodiment, Semaglutide is 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.

[055] Table 1: An exemplary dose escalation schedule of Semaglutide:

[056] According to one embodiment, if a patient does not tolerate a dose of Semaglutide 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 Semaglutide, the dose of Semaglutide 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 Semaglutide, it should be considered to discontinue the administration of Semaglutide to the patient. [057] According to one embodiment, the GLP-1R agonist is Semaglutide Tablets, also known as Rybelsus (Novo Nordisk). According to another embodiment, Semaglutide Tablets are firstly administered at a dose of substantially 3 mg once daily for 30 days. The substantially 3 mg dose of Semaglutide Tablets is for treatment initiation and is not effective for glycemic control. The dose of Semaglutide Tablets can be increased to substantially 7 mg once daily. If after 30 days on the substantially 7 mg daily dose there is a need for an additional glycemic control, the dose of Semaglutide Tablets can be increased to substantially 14 mg once daily. It should be noted that the presently commercially available Semaglutide Tablets (Rybelsus) are poorly absorbed, namely a very low bioavailability. Therefore, Semaglutide Tablets (Rybelsus) are currently administered in high dosages as compared to the parenteral route of administration. However, there is a possibility that the bioavailability of this drug will be improved as a result of research and development efforts and consequently the orally administered quantities of this drug would decrease accordingly. Therefore, the parameter that should be considered in this regard is the level of this drug in the plasma of the patient. Thus, any dosage of future developed Semaglutide Tablets that gives rise to levels of the drug in the plasma of the patient that are similar to the levels of the drug in the plasma that are currently obtained, is under the scope of the present subject matter.

Proton pump inhibitor (PPI)

[058] PPIs are described in United States patent No. US4786505 and European patent No. EP2201952, the entire contents of which is incorporated herein by reference. Some exemplary PPIs include: omeprazole, pantoprazole, lansoprazole, rabeprazole, and esomeprazole. The PPI is administered orally at a daily dose of 10 to 360 mg, depending on the indication and body weight.

Gamma-amino butyric add (GABA)

[059] The composition can comprise GABA, or GABA-receptor agonist. Some exemplary GABA-receptor agonists include: most anti-epileptic medications, Gamma-Pantene acid, medical (legal) forms of Marijuana and the like.

[060] GABA has been shown to provide the regeneration of P-cells in mouse and human cell cultures. [061] United States patent No. US9463174, the entire contents of which is incorporated herein by reference, discloses a method for treating type 1 diabetes, including administering a therapeutically effective amount of GABA and an effective amount of a DPP-4 inhibitor, for example, Sitagliptin, to a type 1 diabetes patient. GABA and the DPP-4 inhibitor are used in a single-dosage form or separate-dosage forms. The dosage includes GABA in 0.002 to 2 mg/kg of body weight. This combination does not lead to a significant recovery of beta cells of the pancreas.

Decapeptyl peptidase (DPP-4) inhibitor

[062] Any agent that is configured to cause immunomodulation through inhibition of membrane and serum forms of DPP4 is under the scope of the present subject matter. Some exemplary agents that inhibit DPP4 include at least one Dipeptidyl peptidase-4 (DPP-4) inhibitor, or at least one CD45 inhibitor (CD45-binding molecules capable of binding to human and non-human CD45), at least one CD3 inhibitor (CD3-binding molecules capable of binding to human and non-human CD3), at least one anti-interleukin (IL)-21 antibody, at least one antiinterleukin (IL)- 18 antibody, or any combination thereof.

[063] 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.

[064] 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 and commercially available with sitagliptin (Januvia) as the first approved and most investigated DPP-4 inhibitory agent for the treatment of patients with type 2 diabetes.

[065] 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.gov/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].

[066] 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), the entire of which is incorporated herein by reference]. 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.

[067] 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 DPP-4 inhibitor is administered orally at a daily dose in a range of substantially 25 to 400 mg. [068] 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.

[069] According to one embodiment, the DPP-4 inhibitor is orally administered to the patient, for example as a tablet, capsule, powder, granules, oral suspension and the like, once daily. According to another embodiment, the DPP-4 inhibitor is orally administered to the patient, for example as a tablet, capsule, powder, granules, oral suspension and the like, twice daily.

[070] The drug sitagliptin (Januvia) has been registered in many countries in the world. Sitagliptin can serve as a classic representative of a family of DPP-4 inhibitors. Sitagliptin increases the concentration of two known hormones of the incretin family: GLP-1 and glucosedependent insulinotropic peptide (GIP). Hormones of the family of incretins are secreted in the intestine during the day, with levels rising in response to food intake. Incretins are part of the internal physiological system of regulation of glucose homeostasis. At normal or elevated blood glucose levels, the hormones of the incretin family promote an increase in insulin synthesis, as well as its secretion by beta cells of the pancreas, due to the intracellular signaling mechanisms associated with cyclic AMP. A double -blind, randomized, cross-over, 8-week, preliminary study in adult patients with type 1 diabetes showed that the drug had significantly reduced blood glucose levels despite a reduction of total and lunch dose of insulin [Diabet. Med. 2011 Oct; 28 (10): 1176-81 Effect of Sitagliptin on glucose control in an adult patient with Type 1 diabetes: a pilot, double -blind, randomized, crossover trial. Ellis SL, Moser EG, Snell-Bergeon JK, Rodionova AS, Hazenfield RM, Garg SK., the entire contents of which is incorporated herein by reference]. Sitagliptin is now being considered for introduction into the clinical practice of type 1 diabetes treatment.

[071] In addition, attempts have been made to create combined medications based on sitagliptin and other DPP-4 inhibitors for the treatment of diabetes.

[072] In particular, in an article by Griffin KJ, Thompson PA, Gottschalk M, Kyllo JH, Rabinovitch A., Combination therapy with Sitagliptin and lansoprazole in patients with recentonset type 1 diabetes (REPAIR-T1D): 12-month results of a multicentre, randomized, placebo- controlled, phase 2 trial. // Lancet Diabetes Endocrinol. 2014 Sep; 2 (9): 710-8, the entire contents of which is incorporated herein by reference, it is postulated that sitagliptin and lansoprazole will maintain the beta-cell function in patients with type 1 diabetes. However, in the analysis, it was noted that not all participants were able to increase their GLP-1 and gastrin concentrations.

[073] International patent application publication No. W02006000567, the entire contents of which is incorporated herein by reference, discloses the use of a GLP-1 receptor agonist and/or a DPP-4 inhibitor and a proton pump inhibitor such as omeprazole or esomeprazole, in the manufacture of a medicament for the treatment of type 1 diabetes. However, this combination does not provide a significant reduction of the insulin dose.

[074] CD3-binding molecules are described in United States patent No. US9587021B2, the entire contents of which is incorporated herein by reference.

[075] CD45-binding molecules are described in US20180237521A1, the entire contents of which is incorporated herein by reference.

[076] Anti-interleukin (IL)-21 antibody as described in US20150266954A1, the entire contents of which is incorporated herein by reference.

[077] Anti-interleukin (IL)- 18 antibody as described in US9255144B2, the entire contents of which is incorporated herein by reference.

Embodiments of the composition

[078] According to one embodiment, the DPP-4 inhibitor is administered orally at a daily dose of substantially 2.5 to 400 mg, depending on body weight, and renal (kidney) function. Examples of drugs of this group, which are suitable to be used in the present composition, are the DPP-4 inhibitors: alogliptin, linagliptin, saxagliptin, sitagliptin, and vildagliptin. The use of sitagliptin (Januvia) in particular is preferable in the practice of the present subject matter. Any type of DPP-4, and preferably sitagliptin, is under the scope of the present subject matter, including tablets of sitagliptin that are manufactured by various companies. According to another embodiment, the DPP-4 inhibitor may be administered as a pharmaceutically acceptable salt or ester or as free base. [079] According to one embodiment, the PPI is administered orally at a daily dose of substantially 10 to 120 mg, depending on body weight. According to another embodiment, the PPI is administered orally at a daily dose of substantially 10 to 360 mg, depending on body weight. Some exemplary PPIs include: omeprazole, pantoprazole, lansoprazole, rabeprazole, and esomeprazole. Omeprazole is preferred in the practice of the present subject matter. Any type of PPI, and preferably omeprazole, is under the scope of the present subject matter, for example in the form of delayed-release tablets, granules, or capsules, or mixed with an alkali substance, for example sodium bicarbonate, in order to protect the PPI from degradation by stomach acid. According to an additional embodiment, the PPI may be administered as a parenteral solution by intravenous infusion.

[080] According to one embodiment, the GABA, or the GABA-receptor agonist is administered in a daily dose of substantially 375 to 3,000 mg which may be administered in a single daily dose or divided into twice or thrice daily dosing, the total dose depending on body weight. Some exemplary formulations of GABA that can be used include oral tablets, or capsules of GABA with gelatin or cellulose, as disclosed in United States patent No. US5698155, the entire contents of which is incorporated herein by reference, or tablets of GABA, for example Aminalon. According to another embodiment, GABA agonists, for example baclofen, aminophenylbutiric acid (Phenibut), hopantenic acid (Pantogam), and antiepileptic medications such as valproic acid (Depalept), topiramate (Topamax), carbamazepine, benzodiazepines, gabapentin, pregabalin, including their salts and esters and medical cannabis, can be used instead of GABA. According to another embodiment, the GABA or GABA agonists may be administered in a modified release dosage form in the form of extended release oral tablets, capsules, powders, granules or suspension or the like. According to yet another embodiment, the GABA or GABA agonist may be administered in an oral dosage form that enhances biovailability of the GABA or GABA agonist.

[081] It should be mentioned that the doses of the GABA agonists that should be administered to the patients are lower than the doses of the GABA mentioned above, because the GABA agonists are significantly more potent than GABA. Some exemplary dosages of GABA agonists include: alprazolam, which is used therapeutically in depression and is administered at a starting daily dose of 0.75 mg/day and reaching a maximum of 4 mg/day in 3 divided doses; and clonazepam, which is used in seizure disorders and is administered at a dosage starting at 1.5 mg/day in 3 divided doses, up to a maximum of 20 mg/day. Thus, according to one embodiment, any medically approved dosage range of each GABA agonist is under the scope of the present subject matter.

[082] 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].

[083] 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.

[084] 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].

[085] 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] .

[086] 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] .

[087] 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] . [088] According to one embodiment, GABA, the at least one GABA receptor agonist, or the at least one GABA analogue, or any combination thereof, is administered to a patient in a daily dose of up to substantially 3000 mg, depending on body weight. Any type of oral solid or liquid administration form can be used, for example capsules, or tablets, or oral powder, or oral granules of GABA, for example tablets of Aminalon. According to another embodiment, GABA is formulated in a manner that allows modification of absorption of the GABA by increasing the bioavailability of GABA, for example in order to reduce the dose of GABA that is 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.

[089] 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].

[090] Any type of GABA agonist is under the scope of the present subject matter. Some exemplary types of GABA agonist include baclofen aminophenylbutiric acid (Phenibut), hopantenic acid (Pantogam), clonazepam, alprazolam and zolpidem. In addition, anti-epileptic medications can be used instead of GABA, for example valproic acid (Depalept), valproate sodium, topiramate (Topamax), carbamazepine, gabapentin, pregabalin, their salts and esters and medical cannabis.

[091] According to one embodiment, oral forms of the therapeutic agents include any pharmaceutically acceptable dosage forms, powders, granules, capsules, tablets, microcapsules suspensions, and the like. The therapeutic agents can be administered as four separate single drugs; or any two drugs together in a single dosage unit, and the other two as separate single drugs; or any two drugs together in a single dosage unit, and the other two together as separate single dosage unit; or any three of the drugs in a single dosage unit and the other drug as a single drug; or all four drugs together in a single dosage form, for example in the form of tablets, capsules, powder, granules, suspension, or any other pharmaceutically appropriate dosage form. The GLP-1 agonist may be administered as a parenteral (e.g., subcutaneous) injection or as an oral solid dosage form. The PPI can be administered as a parenteral, for '? example intravenous, injection, or as an oral dosage form. According to another embodiment, the four therapeutic agents, as described herein, can be provided as a kit.

[092] The technical result of the present subject matter lies in the fact that the administration of the drugs in this combination allows for a significant reduction in the need for insulin in T1DM patients, and in some cases, it is even possible to stop insulin injections completely.

[093] The DPP-4 inhibitors, for example sitagliptin, vildagliptin, saxagliptin. alogliptin, linagliptin and the like, help in the regeneration of P-cells. This is due in particular to the fact that medications of the DPP-4 inhibitors family are capable of modulating the immune response by limiting autoimmune activity, reducing the inflammatory component (insulitis) and ultimately achieving the regeneration of the P-cells of the pancreas.

[094] DPP-4 inhibitors block the activity of the DPP-4 enzyme, which leads to an increase in both concentration and duration of action of GLP-1 and GIP. The DPP-4 inhibitors are taken orally and provide a healthy physiological level of incretins in the blood.

[095] A critically important feature of the effect of GLP-1 on the function of pancreatic P- and a-cells is its glucose-dependent nature. This means that the GLP-1 stimulates insulin secretion and, on the other hand, suppresses the production of glucagon only under conditions of hyperglycemia. Once plasma glucose drops to a regular level, the effects above of GLP-1 subside, making it a reliable physiological mechanism for preventing the development of a hypoglycemic state.

[096] GABA affects both P- and a-cells, their functions and the viability of the pancreas as a whole. GABA is widely used as a food supplement. In a-cells, GABA induces hyperpolarisation of the membrane and suppresses glucagon, whereas in P-cells GABA induces membrane depolarization and increases insulin secretion. Also, GABA has a multi-directional positive effect on P-cells, which includes the stimulation of cell proliferation and antiapoptosis.

[097] Any type of pharmaceutically acceptable formulation of the PPI is under the scope of the present subject matter. Some exemplary types of formulation include orally administered delayed-release tablets, capsules, or granules, or parenteral like intravenous (IV) injection, and the like.

[098] According to one embodiment, the PPI is 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. According to one embodiment, the PPI is omeprazole, and is administered in a range of substantially 10 to 360 mg daily. According to another embodiment, the PPI is lansoprazole, and is administered daily in a range of substantially 15-60 mg daily. According to yet another embodiment, the PPI is pantoprazole, and is administered in a range of substantially 20 to 80 mg daily. According to still another embodiment, the PPI is esomeprazole, and is administered in a range of substantially 20 to 240 mg daily. It should be noted that the PPIs can be administered to treat Pathological Hypersecretory Conditions as well. This is done by gradual increase of the administered dosage and adjusting the dosage according to the patient's needs. For example, administering omeprazole to a patient under Pathological Hypersecretory Conditions a starting dose of substantially 60 mg once daily, and adjusting the dosage according to patient needs up to 120 mg three times daily, wherein daily dosages greater than substantially 80 mg are administered in divided doses.

[099] Any type of H2-receptor antagonist is under the scope of the present subject matter. According to one embodiment, the H2-receptor antagonist is a type of stomach acid reducer. Any type of stomach acid reducer is under the scope of the present subject matter, for example an antacid. Some exemplary H2-receptor antagonists include: cimetidine, ranitidine, nizatidine and famotidine.

[100] The PPIs inhibit Na ⁺ /K ⁺ -ATPase (a proton pump) on the apical membrane of parietal cells of the gastric mucosa and ensure the achievement of clinical, endoscopic remission in all acid-dependent diseases, including those requiring prolonged or continuous therapy. For an extended period during the day, the PPIs maintain pH values in the stomach within limits favorable for the healing of stomach or duodenal ulcers. The general effect of the pharmacological action of the PPI drugs group is the increase of Gastrin in the blood and the pancreatic tissue. Gastrin is a natural stimulant for the recovery (regeneration) of pancreatic cells.

[101] Any type of bombesin-like peptide is under the scope of the present subject matter. [102] According to one embodiment, the bombesin-like peptide is a gastrin-releasing peptide

(GRP), or gastrin-releasing peptide receptor agonist. 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].

[103] The present subject matter further provides a kit for treating and preventing diseases that are caused by, or linked to, GABAergic transmission dysfunction. According to one embodiment, the kit comprises the composition described herein.

[104] In the light of the above, the use of the composition of the present subject matter, that includes a DPP-4 inhibitor, a PPI, GABA or a GABA-receptor agonist, and a GLP-1R agonist, appears to be promising for maintaining pancreatic function and for controlling type 1 diabetes in human patients. [105] The present subject matter provides a medication for treating patients with endocrine, autoimmune and neurological disorders, including type 1 diabetes, comprising a single dose form comprising essentially therapeutically effective amounts of: dipeptidyl peptidase (DPP- 4) inhibitor, a GLP-1R agonist, a proton pump inhibitor (PPI) and gamma-aminobutyric acid preparation (GABA) or GABA receptor agonist in powder, granule, tablet, capsule, microcapsule or suspension form for oral administration. The GLP-1R agonist can be administered parenterally, for example by subcutaneous injection.

[106] According to one embodiment, the amount of DPP-4 inhibitor is in the range of substantially 2.5-400 mg per day, the amount of PPI is in the range of substantially 10-360 mg per day, the amount of GABA or GABA receptor agonist is in the range of substantially 375- 3,000 mg per day and the amount of GLP-1R agonist is in the ranges described above, depending on the specific peptide and dosage form.

[107] According to one embodiment, the DPP-4 inhibitor is sitagliptin.

[108] According to one embodiment, the PPI is omeprazole.

[109] According to one embodiment, the DPP-4 inhibitor is sitagliptin and the PPI is omeprazole.

[110] The present subject matter further provides a kit for daily oral administration, for treating patients with type 1 diabetes, the kit comprising therapeutically effective amounts of: a first composition comprising dipeptidyl peptidase (DPP-4) inhibitor; a second composition comprising a proton pump inhibitor (PPI); a third composition comprising gamma-aminobutyric acid preparation (GABA) or GABA receptor agonist; and a fourth composition comprising a GLP-1R agonist.

[111] According to one embodiment, the kit comprising: the first composition comprising a dose in a range of substantially 2.5-400 mg/day of DPP-4 inhibitor; the second composition comprising a dose in a range of substantially 10-360 mg/day of PPI; the third composition comprising a dose in a range of substantially 375-3,000 mg/day of GABA; and the fourth composition comprising a safe and effective dosage of GLP-1R agonist, as described above.

[112] According to one embodiment, the DPP-4 inhibitor is sitagliptin and the PPI is omeprazole.

EXAMPLES

Treatment of type 1 diabetes in a model of type 1 diabetes in mice with GABA, a DPP-4 inhibitor, a proton pump inhibitor and a GLP-1R agonist

Introduction

[113] Type 1 diabetes is an autoimmune disease of the pancreas. The basis of this disease is the lack of insulin production by endocrine cells (P-cells of the islets of Langerhans of the pancreas). Diabetes most often develops in childhood or adolescence. As a rule, with early diagnosis of the disease, some function of P-cells is preserved for a short period, although this is not enough to maintain normoglycemia without injections of exogenous insulin. It is well known that even minimally preserved function of beta cells prevents the development of complications of diabetes. Therefore, even a small amount of protection and maintenance of residual P-cell function is critical.

Abbreviations of drugs

[114] For the sake of simplicity only, the following abbreviations of the drugs will be occasionally used:

Drug A - GABA

Drug B - a DPP-4 inhibitor, in a form of Sitagliptin

Drug C - a proton pump inhibitor, in a form of Omeprazole

Drug D - a GLP-1R agonist, in a form of Liraglutide (comprising GLP-1)

Purpose of the study [115] To study the specific pharmacological activity of a quadruple combination of drugs A+B+C+D in comparison with A+B+C combination in a model of type 1 diabetes in C57BL/6 mice, both compared to a diabetes control group and an untreated healthy control group of mice.

[116] To investigate the development, severity and course of diabetes mellitus in C57BL/6 mice to confirm the efficacy of the four-component combination.

Objectives

[117] The objectives of the study were:

To investigate blood glucose levels and their dynamics; and To investigate survival rate of the treated animals.

Overview of the study

[118] It is hypothesized that a combination of Gamma-aminobutyric acid (GABA), Sitagliptin (a dipeptidyl peptidase-4 inhibitor), Omeprazole (a proton-pump inhibitor) and Liraglutide, [a glucagon-like peptide- 1 receptor agonist (GLP-1)] can induce regenerative effects on beta cells in a diabetic mouse model. It is assumed that joint administration of the aforementioned four drugs can cause a higher therapeutic effect, compared to joint administration of only GABA, sitagliptin and omeprazole. To test this hypothesis, a streptozotocin (STZ) induced multiple beta-cell injury model in mice was used.

[119] Male C57BL/6J mice were randomized into four groups:

• Treatment group A+B+C: GABA + Omeprazole + Sitagliptin + STZ

• Treatment group A+B+C+D): GABA + Omeprazole + Sitagliptin + Liraglutide + STZ

• Diabetes control: STZ

• Untreated healthy control

Oral drug administration started 1 week prior to STZ injection and continued for 6 weeks.

Materials and methods

Animals

[120] The study used 132 male C57BL/6 mice, weighing 18-20 grams, aged 10-11 weeks. Conditions for keeping the animals

[121] During the experiment, the animals were kept under controlled conditions: ambient air temperature 20-26 °C; relative humidity 55+15%; light-dark cycle (08:00-20:00 - day, 20:00-08:00 - night).

Bedding

[122] Sawdust of soft non-coniferous wood species (OOO Laboratorkorm, Russia) was used as bedding.

Food and water

[123] The diet included complete granulated feed for laboratory animals (produced by Provimi LLC) (produced in accordance with GOST 34566-2019 Complete feed for laboratory animals. Specifications - GOST of August 23, 2019). Animals received water from the central water supply (corresponding to SanPiN 2.1.3684-21) and had free access to food and water.

Animal cages

[124] The animals were kept in polycarbonate cages, 325x210x180 mm in size, with an area of 682.5 cm ² of the IVK- Vent-Bio- IM system with individual ventilation, on a bedding, equipped with steel lattice covers with a feed recess, steel feed dividers and steel label holders. The number of animals in one cage was 5 mice.

Animal identification and grouping

[125] During the study the specific pharmacological activity of the four drugs (A+B+C+D), compared to the effect of three of the drugs (A+B+C) was tested on a STZ model of type 1 diabetes in male C57BL/6 mice. After the animals had passed 6 days of acclimatization, each animal was assigned an individual number, according to which the animal was given a coloring label. On the label of a cage of a certain color, the group, animal number, label, study code and name of the research leader were indicated.

[126] The animals were divided into groups randomly, using body weight as a criterion, so that the individual weight of animals did not differ by more than 10% from the average weight of animals in one group. The animals were divided into groups, in accordance with the doses of the administered drugs, as detailed in Table 2.

[127] Table 2: Distribution of animals into groups in the study of the specific pharmacological activity of a 4-component therapy in an STZ model of type 1 diabetes in C57BL/6 mice.

Induction of type 1 diabetes with STZ

[128] Type 1 diabetes was induced in mice groups A+B+C, A+B+C+D and Diabetes by intraperitoneal administration of STZ at a dose of 80 mg/kg, in a 1% starch solution, every other day for 5 days. A solution of only 1% starch was administered to the Healthy mice group (Control) every other day for 5 days.

Administration of drugs

[129] The drugs were administered to animals for 7 weeks, on a daily basis, as follows:

• GABA (drug A) - 300 mg/kg;

• Sitagliptin (drug B) - 30 mg / kg;

• Omeprazole (Drug C) - 15 mg/kg;

• Liraglutide (Drug D) - 1 mg/kg.

Observation period

[130] The follow-up period for the animals was 52 days.

Clinical examination

[131] Throughout the study, a clinical examination of the following parameters in the animals was carried out: number and timing of death of animals (if any); respiratory indicators (difficulty breathing, cyanosis, rapid breathing, nasal discharge); motoric activity (increased / decreased, drowsiness, loss of balance, sensitivity, catalepsy, ataxia, unusual movements, prostration, tremor, fasciculation); convulsions (clonic, tonic, tonic-clonic, asphyxic); reflexes (corneal, balance, myotatic, light, startle reflex); eye signs (lacrimation, miosis, mydriasis, exophthalmos, ptosis, clouding, iritis, conjunctivitis, chromodactriorrhea, weakening of the nictitating membrane); increased salivation; condition of the coat (pilo-leiomyoma, alopecia); analgesia; muscle tone (hypotension, hypertension); gastrointestinal indicators (soft stools, diarrhea, vomiting, polyuria, rhinorrhea).

Body weight

[132] The animals were weighed at the time of group formation, as well as every week for 6 weeks from the start of the study.

Blood glucose level

[133] To determine the effectiveness of tested therapeutic regimes, blood glucose levels were measured in the animals twice a week for 7 weeks by using Accu-Chek test strips. The determination of blood glucose levels was not performed on an empty stomach.

Data analysis

[134] Statistical data processing was carried out using IBM SPSS Statistics 17.0 software and Microsoft Excel (version 14.0, 32-bit). For all data, descriptive statistics were applied: the mean value and standard deviation were calculated, which are presented in the final tables. The Kruskal- Wallis (KU) test was used as a nonparametric test. In case of revealing differences between the groups by the KU criterion, the Bonferroni criterion was applied. Differences were determined at a 0.05 significance level and marked with

Results

[135] Diabetes was induced in the mice by fractional administration of STZ at a dose of 80 mg/kg every other day for five days. Upon reaching a blood glucose level above 13 mmol/1 the animals were considered diabetic. Blood glucose levels are shown in Fig. 2, hereinafter. Survival score

[136] Based on considerations of humanity, on day 21 all animals who had blood glucose levels above the upper detection limit of the device (33.3 mmol/1) and were hypodynamic and thin, were euthanized. All mice in the Diabetes group, one in A+B+C and none of A+B+C+D group and the Healthy group (Control) met these criteria. The results of the survival assessment are presented in Fig. 1.

[137] Referring now to Fig. 1, presenting a graph showing survival percentage of model mice of type 1 diabetes following treatment with combinations of drugs, during a study. The X-axis of the graph is the time in days. The Y-axis of the graph is percentage of survival. Curve 12 shows the survival percentage of the A+B+C group. Curve 14 shows the survival percentage of the A+B+C+D group. Curve 16 shows the survival percentage of the Diabetes group. Curve 18 shows the survival percentage of the Healthy group (Control).

[138] As can be seen in Fig. 1, curve 14 of the A+B+C+D group and curve 12 of the A+B+C group appear to have a similar profile. However, the percentage of deaths in the A+B+C group 12 was higher (46%), while the percentage of deaths in the A+B+C+D group 14 did not exceed 15%.

Blood glucose level

[139] Referring now to Fig. 2, presenting a graph showing blood glucose levels (median, 25% percentile) of model mice of type 1 diabetes following treatment with combinations of drugs, during a study. The X-axis of the graph is the time in days. The Y-axis of the graph is blood glucose level (mmol/1). Curve 22 shows the blood glucose level of the A+B+C group. Curve 24 shows the blood glucose level of the A+B+C+D group. Curve 26 shows the blood glucose level of the Diabetes group. Curve 28 shows the blood glucose level of the Healthy group (Control).

[140] As can be seen in Fig. 2, from the first glucose measurement after the start of therapy, the curves of blood glucose levels began to diverge. The dynamics of the discrepancy persisted until the end of the study. Though the blood glucose level in the A+B+C+D group 24 did not reach the level of normoglycemia, it was significantly lower than in the A+B+C group 22 and the Diabetes groups 26. [141] Referring now to Fig. 3, presenting a graph showing a statistical analysis of blood glucose levels (median, 25% percentile) at the last day of the study of model mice of type 1 diabetes following treatment with combinations of drugs. The X-axis of the graph shows the treatment group. The Y-axis of the graph is blood glucose level (mmol/1). Column 32 shows statistical analysis results of blood glucose levels of the A+B+C group. Column 34 shows statistical analysis results of blood glucose levels of the A+B+C +D group. Column 36 shows statistical analysis results of blood glucose levels of the Diabetes group at day 20. Curve 38 shows statistical analysis results of blood glucose levels of the Healthy group (Control).

[142] The level of blood glucose in the four-treatment group at day 52 of the study was statistically analyzed. Comparison of groups using the Kruskal-Wallace test revealed a statistically significant difference between groups, p=<0.001. Further pairwise comparison of groups using the Bonferroni test revealed the following:

In the A+B+C group 32, there was significantly higher blood glucose levels compared with the A+B+C +D group 34 and the Healthy (Control) group 38 (Bonferroni test, p=<0.001).

The blood glucose levels in the A+B+C+D group 34 were significantly lower compared with the A+B+C group 32 and the Diabetes group 36 (Bonferroni test, p=<0.001). In addition, the blood glucose levels in the A+B+C+D group 34 were significantly higher compared with the Healthy (Control) group 38 (Bonferroni test, p=<0.001).

Thus, the combination of drugs A+B+C+D showed a greater efficiency in terms of lowering blood glucose levels compared to the combination of drugs A+B+C. However, the combination of drugs A+B+C+D was not able to lower the blood glucose levels to the levels of the Healthy group (Control).

Percentage of healthy animals

[143] Referring now to Fig. 4, presenting a graph showing percentage of healthy animals among model mice of type 1 diabetes following treatment with combinations of drugs, during a study. The X-axis of the graph is the time in days. The Y-axis of the graph is percentage of healthy animals. Curve 42 shows the percentage of healthy animals in the A+B+C group. Curve 44 shows the percentage of healthy animals in the A+B+C+D group. Curve 46 shows the percentage of healthy animals in the Diabetes group. Curve 48 shows the percentage of healthy animals in the Healthy group (Control). A healthy animal was considered as having a blood glucose level lower than 13.9 mmol/1. [144] The results of the evaluation of the recovery of the animals are presented in Fig. 4 as percentage of healthy animals in each group. As can be seen in the Fig. 4, the curve constructed for the A+B+C+D group 44 indicates an increase in the percentage of recovered animals compared to the A+B+C group 42 and the Diabetes group 46 (A+B+C+D - 20%, A+B+C - 0%, Diabetes - 0%). The A+B+C+D group 44 was the only group at the end of the study to have healthy mice.

Conclusions

[145] To summarize: the percentage of death in A+B+C+D group was the lowest compared to the other test groups. (A+B+C+D -15% death, A+B+C - 46% death, Diabetes - 100% death).

From the first glucose measurement after the start of therapy, the curves of glucose levels began to diverge. The dynamics of the discrepancy persisted until the end of the study. Comparison of groups using the Kruskal-Wallace test revealed a statistically significant difference between groups, p=<0.001. Though the glucose level in the A+B+C+D group did not reach the level of normoglycemia, it was significantly lower than the glucose level in the A+B+C group (Bonferroni test, p=<0.001) and the Diabetes group (Bonferroni test, p=<0.001).

The A+B+C+D group was the only therapy group to have healthy mice (having a blood glucose lever lower than 13.9 mmol/1) at the end of the study (A+B+C+D - 20% healthy mice, A+B+C - 0% healthy mice, Diabetes - 0% healthy mice).

To conclude, the A+B+C+D group showed significantly lower Blood glucose levels, a better survival rate, and had more healthy mice than the A+B+C group and the Diabetes group.

[146] According to all the parameters that were tested in the present in vivo study, the combination of the four drugs - GABA, sitagliptin, omeprazole and liraglutide (group A+B+C+D), had a superior therapeutic effect in the treatment of type 1 diabetes, compared to the combination of only the three drugs - GABA, sitagliptin and omeprazole (A+B+C). These results support the main idea of the present subject matter - that a combination of GABA, a DPP-4 inhibitor, a proton pump inhibitor and a GLP-1R agonist, has a superior therapeutic effect on endocrine, autoimmune and neurological disorders. In the present in vivo feasibility study, the inventors used GABA, sitagliptin as an exemplary DPP-4 inhibitor, omeprazole as an exemplary proton pump inhibitor and liraglutide as an exemplary GLP-1R agonist, for treating type 1 diabetes as a model of an endocrine, autoimmune and neurological disorder. [147] 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.

[148] 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.