TECHNICAL FIELD

The invention relates to the use of casomorphin peptides which are biological molecules for the prevention and treatment of multiple sclerosis disease or for relieving the symptoms resulting from this disease.

PRIOR ART

Multiple sclerosis (which may be referred to as MS) is a chronic inflammatory demyelinating disease of the central nervous system. Particularly MS disease is a chronic common disease seen in young adults and characterized by findings affecting the spinal cord white substance in multiple localizations.

MS disease is also a disease that reduces the quality of life with physical and psychological damage in young people and places economic burdens on the health system. MS disease generally begins in the form of relapses and remissions and progresses to a progressive phase in some of the patients, while some are progressive from the onset of the disease.

Caseins, known as the main proteins of cow's milk, are the part that does not precipitate after the reaction of milk with acid. Said proteins are held together by non-covalent bonds and consist of 4 different (as ¹ -, as ² -, b- and kappa) caseins. Opioid active peptides released from beta-caseins are potent exogenous agonists of opioid receptors with the greatest affinity for m-opioid receptors.

The effects of beta-caseins on gastrointestinal motility and mucus secretion, lymphocyte proliferation and histamine release from peripheral leukocytes are known in the art. Beta- caseins have also been reported to make the intestinal wall susceptible to infection by affecting the lymphocyte proliferation in the lamina propria. It has also been reported that beta-caseins can trigger the formation of autoimmune diseases by increasing the amount of autoantibodies.

Beta-casomorphin peptides cross the blood-brain barrier and act on signal pathways in the central nervous system. Beta-casomorphins have been the subject of study in the technical field related to their effects on other immune cells. The effect of beta-casomorphins on myeloperoxidase (MPO) released from polymorphonuclear leukocytes was investigated. It has been reported that beta-casomorphins cause the transformation of ThO CD4+ T cells to Th2 by increasing IL-4 production in the medium.

Many anti-inflammatory, immunomodulatory and immunosuppressive drugs used in the treatment of MS disease reduce relapses but have limited effect in progressive disease. There is no agent that can stop or slow the progressive disease yet. In this sense, it is necessary to discover new agents that can show an axon-sparing approach to treat or prevent MS disease.

The drug treatments used in the treatment of MS have many side effects. Many anti inflammatory, immunomodulatory and immunosuppressive drugs used in the treatment of MS reduce relapses but have limited effect in progressive disease. There is no completely curative effect in the disease.

As a result, all the problems mentioned above require obtaining anti-inflammatory, immunomodulatory and immunosuppressive substances with reduced side effects and no toxic effects for the prevention and treatment of multiple sclerosis disease or for relieving the symptoms caused by this disease.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to the use of biological molecules for the prevention and treatment of multiple sclerosis disease or for relieving the symptoms resulting from this disease to eliminate the disadvantages mentioned above and to bring new advantages to the relevant technical field.

An object of the invention is to provide a natural non-toxic biological molecule suitable for use in MS disease.

An object of the invention is to provide a biological molecule which has anti-inflammatory, immunomodulatory and immunosuppressive properties.

The invention relates to the use of beta-casomorphin-7 peptides which are biological molecules for the prevention and treatment of multiple sclerosis disease or for relieving the symptoms resulting from this disease in order to realize all the objectives which are mentioned above and will emerge from the detailed description below Thus, the biological molecule that acts on T cell substrates known to be involved in the treatment of MS disease is revealed.

A possible embodiment of the invention is that the beta-casomorphin-7 molecule is in a form suitable for oral administration.

A possible embodiment of the invention is that the biological molecule is in the form of gel capsules, tablets, lozenges or pills.

A possible embodiment of the invention is that beta-casomorphin-7 molecule is used at a dose in the range of 1 x1 O ⁸ to 5x1 O ⁸ mol/day.

A possible embodiment of the invention is that beta-casomorphin-7 molecule is used at a dose of 3x1 O ^-8 mol/day.

A possible embodiment of the invention is that the biological molecule is administered daily, weekly, bi-weekly or monthly.

A possible embodiment of the invention is that the beta-casomorphin-7 molecule acts by significantly and statistically reducing the number of lymphocytes infiltrating the central nervous system, T cell subset and especially, IL-17, IL-22 and GM-CSF producing cells together with IFN-g, IFN-g and IL-17A.

A possible embodiment of the invention is that it is beneficial to reduce the annual recurrence rate in patients.

A possible embodiment of the invention is that the decrease in the rate of disease progression is based on a decrease in the progression of the motor impairment.

BRIEF DESCRIPTION OF THE DRAWINGS

In Figure 1 , the comparative graph of the disease scores and disease incidence of the mice in the EAE control group and the control group given BKM-7 molecules are presented. In Figure 2, the comparative graphs of ICC (which can also be referred as intracellular staining), the absolute number differences of the total leukocyte, CD4+ and IL-17A+ T cells accumulated in the central nervous system of the group (d) and the control groups (c) given the BKM-7 peptide given in the examples are presented.

In Figure 3, comparative graphs of the percentage and absolute number differences in total CD4+ cells of IFNg+ and IFNg+ IL-17A+ T cells accumulated in the central nervous system of control groups (d) and (c) to whom BKM-7 is given in the examples are presented.

In Figure 4, comparative graphs of the percentage and absolute number differences in ICC (with intracellular staining) total CD4+ cells of IL-22+ T cells accumulated in the central nervous system of control groups (d) and (c) to whom BKM-7 is given in the examples are presented.

In Figure 5, comparative graphs of IL-22 ELISA levels of control groups (a), (b), (c) and (d) are presented.

In Figure 6, comparative graphs of GM-CSF ELISA levels of control groups (a), (b), (c) and (d) are presented.

In Figure 7, comparative graphs of IL-17A ELISA levels of control groups (a), (b), (c) and (d) are presented.

DETAILED DESCRIPTION OF THE INVENTION

In this detailed description, the subject of the invention relates to the therapeutic use of beta- casomorphin molecules in the prevention and treatment of multiple sclerosis disease or in relieving symptoms resulting from this disease and it is only described with examples that do not have any limiting effect for a better understanding of the subject.

Caseins, known as the main proteins of cow's milk, are the part that does not precipitate after the reaction of milk with acid. These proteins are held together by non-covalent bonds. There are four different (as ¹ -, as ² -, b- and kappa) casein molecules in cow's milk.

Beta-casomorphin-7 (also shown as BKM-7) molecules are released as a result of gastrointestinal digestion of b-casein contained in cow milk of type A1 (containing the Pro- His mutation). BKM-7 is not released as a result of the breakdown of A2 type b-casein. b- casomorphins show their opioid agonist effects similar to morphin by binding to opioid receptors in the body.

In our present invention, beta-casomorphin-7 molecule is used for the prevention and treatment of multiple sclerosis disease or for relieving the symptoms caused by this disease. Said beta casomorphin-7 structure is represented by Formula 1 below. Beta casomorphin-7 amino acid sequence is NFI2-YPFPGPI-COOFI, the molecular weight of the peptide being 789.9 daltons.

Formula 1. Structural formulation of beta casomorphin-7 molecule

Two aspects of the beta-casomorphin-7 biological molecule are used in the treatment of MS disease. First, the beta-casomorphin-7 molecule increases the activity of MPO in the body. As it is known from the literature, increasing MPO activity inhibits the formation of adaptive immunity by acting on dendritic cells (DC), suppressing DC activation, Ag uptake/processing and transition to LNs and thereby reduces the formation of pathological tissue inflammation. Another effect of the mentioned beta-casomorphin-7 molecule is its protective effect on EAE. Accordingly, the protective effect on EAE is predicted to be a signal transduction-mediated effect through opioid receptors.

Beta-casomorphin-7 molecule is a biological molecule with anti-inflammatory, immunomodulatory and immunosuppressive properties. It is a great advantage that the peptide is a natural food. Beta casomorphin molecule does not have toxic effects.

The invention will now be described with reference to the following examples, which are for exemplary purposes only and should not be construed as limiting the scope of the present invention in any way. Example

C57BL/6 female mice were kept under pathogen-free conditions in Erciyes University, Kayseri, Turkey Experimental Research and Application Center of the animals in the facility, according to welfare conditions, were kept under pathogen-free conditions.

Mice were kept in plastic propylene cages and in a sterile condition at room temperature. The mice used in the study are 6 to 8 weeks old and weigh 20 to 25 g. All transactions were approved by the Institutional Animal Ethics Committee (HADYEK, Erciyes University).

Under experimental conditions, it includes: group (a), which included five mice and received water in which the peptide was dissolved; control group (b) consisting of five mice receiving BKM-7 peptide; reference group (c) consisting of twelve mice in the group for which EAE developed but not given BKM-7 peptide; the control group (d) of thirteen mice in which the effect of EAE-BKM-7 peptide was studied.

100 mI_ of water is given orally to mice in groups (a) and (c) to prevent them from being affected by treatment stress. BKM-7 peptide is not administered to mice in the aforementioned groups.

100 mI_ of BKM-7 peptide is fed orally to mice in groups (b) and (d).

First BKM-7 molecule was synthesized chemically. Then, the mice in groups (b) and (d) were give 3x10 ^-8 mol/day/animal once a day. Said amounts of molecules are administered to mice in groups (b) and (d) for ten days. The BKM-7 molecule obtained is 99% pure in HPLC test and does not contain any other substance.

In the next experimental step, mice in groups (a) and (b) are euthanized on the eleventh day. Organ parts are taken from mice for experimental studies. Organ parts are taken from the brain, spinal cord, small and large intestines.

On the eleventh day, EAE indication procedures are applied to mice in groups (c) and (d). EAE indication procedures continue for four days. BKM-7 molecule is administered to mice in group (d) for a total of fifteen days. Later, when disease scores are three, mice in groups (c) and (d) are euthanized. The daily symptoms of EAE are taken as clinical signs. Accordingly, it s determined as follows: 0, no signs of illness; tail weakness: 1 ; weakness in the hind feet and a loss of the ability to turn the mouse when inverted: 2; paralysis in one of the hind feet: 2.5; paralysis of the two hind feet: 3; paralysis of the forefoot: 3.5; paralysis of the two front feet: 4; moribund: 5.

EAE disease comparison between groups is performed by a) disease scoring, b) disease incidence, c) quantification of lymphocyte subtypes infiltrating the central nervous system (cerebrospinal cord) and cytokine production, and it is described in detail below.

Disease Scoring

As shown in Figure 1 , the group (d) containing the mice given the BKM-7 molecule and the diseased mice in the group (c) where EAE indication procedures were applied and the BKM- 7 molecule was not given were determined.

Nine of the mice in group (c) became ill and 3 remained healthy. Three of the mice administered with BKM-7 molecule in group (d) became ill and ten remained healthy. Test results are shown in Figure 1.

Flow Cytometry Technique

Flow cytometry technique is a technique used to detect and measure the size, shape, and proteins produced by a population of cells or particles.

PMA/lonomycin/Golgi Plug mixture is used to test the cytokine production of cells obtained from the brain/spinal cord or lymph nodes of EAE-developed mice (groups d and c). The mentioned PMA/lonomycin/Golgi Plug mixture is prepared in the ratio of 50ng/ml:1pm/ml:1pm. Cells obtained from mice were stimulated with the mixture for 4 hours and at 37°C. Then, surface staining is performed on the leukocyte cells with CD4 antibody for 30 minutes. In the next step, permabilization followed by staining of IFN-g, IL-17A, GM-CSF and IL-22 was performed. The protocol of the BD Fixation/Permabilization kit was followed.

As shown in Figure 2, the leukocyte values of mice in group (d) are shown to be increased (p<0.05).

As shown in Figure 2, CD4 and IL-17A cell numbers of mice in group (d) decreased significantly compared to mice in group (c) (p <0.05). Figure 3 shows the absolute number difference graphs of IFN-g and IFN-g+IL-17A. As shown in Figure 3, BKM-7 feeding with oral nutrition has been shown to significantly reduce the number of total CD4+IFN-g, CD4+IL-17A+IFN-g double positive cells infiltrating the central nervous system (p<0.05).

As seen in Figure 4, oral feeding with BKM-7 significantly reduces the total number of CD4 and IL-22 positive cells infiltrating the central nervous system (p<0.05).

The number and frequency of leukocytes, CD4+ T cells and subgroups infiltrating the central nervous system (CNS) of mice were determined. The results obtained revealed that BKM-7 feeding greatly reduced the absolute number of CD4+ T cells infiltrating the central nervous system. Moreover, it is seen that the absolute number of IL-17+, CD4+ cells, IL-17+IFN- g+CD4+ cells, IFN-g+CD4+ cells and finally IL-22+CD4+ cells was significantly reduced after being fed with BKM-7 compared to the control group (p <0.05). These results show that feeding with BKM-7 molecules results in a better EAE course, possibly by reducing the infiltration of CD4+ lymphocytes into the CNS.

ELISA ASSAYS

IL-22, IL-17 and GM-CSF tests were performed with commercial ELISA kits. In order to understand the effect of BKM-7 feeding on the inflammatory cytokine production associated with Th17 response, a 1cm piece taken from the intestines of all groups (a,b,c,d) was cultured in Complete Medium (10% FBS, RPMI 1640) at 37°C for 24 hours. The supernatant was collected.

In Figure 5, IL-22 ELISA levels are determined on the intestinal samples taken from groups (a), (b), (c) and (d). Accordingly, it is shown that the IL-22 ELISA values are the highest in the samples taken from group (c).

In Figure 6, GM-CSF ELISA levels are examined on intestinal samples taken from groups (a), (b), (c) and (d). Accordingly, the highest value is seen in the mice in the (a) group, the lowest in the mice in the (d) and (c) groups.

In Figure 7, IL-17A ELISA levels are examined on intestinal samples taken from groups (a), (b), (c) and (d). Accordingly, the highest levels are obtained from mice in groups (a) and (c). The lowest values are obtained from mice in groups (b) and (d). Beta-casomorphin-7 biological molecules have a protective effect on Autoimmune Encephalomyelitis (to be shown by EAE), as can be seen from the results of the experiments conducted in the invention. Said protective effect is realized by the signal transmission of BKM-7 biological molecules through opioid receptors.

Beta-casomorphin-7 biological molecule functioned by reducing the total number of CD4+ T cells affecting the central nervous system and the number of IFNg+IL-17A+ or IL-17A+ T cells previously shown to be pathogenic in the prevention and treatment of EAE, which is a mouse model of multiple sclerosis disease, or in relieving the symptoms caused by this disease

The scope of protection of the invention is specified in the appended claims and it cannot be limited to what is explained in this detailed description for the purpose of example. Because it is clear that a person skilled in the art can come up with similar embodiments in the light of the above, without departing from the main theme of the invention.