WO2021137808A1 - Utilisation de peptides de bêta-casomorphine dans la sclérose en plaques (sep) - Google Patents

Utilisation de peptides de bêta-casomorphine dans la sclérose en plaques (sep) Download PDF

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Publication number
WO2021137808A1
WO2021137808A1 PCT/TR2020/051302 TR2020051302W WO2021137808A1 WO 2021137808 A1 WO2021137808 A1 WO 2021137808A1 TR 2020051302 W TR2020051302 W TR 2020051302W WO 2021137808 A1 WO2021137808 A1 WO 2021137808A1
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WO
WIPO (PCT)
Prior art keywords
beta
disease
casomorphin
molecule
use according
Prior art date
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PCT/TR2020/051302
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English (en)
Inventor
Ebru SAATCI
Ahmet Eken
Yousif Ahmed Ataallah AL ANI
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T. C. Erciyes Universitesi
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication of WO2021137808A1 publication Critical patent/WO2021137808A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
    • A61K38/018—Hydrolysed proteins; Derivatives thereof from animals from milk
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids

Definitions

  • 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.
  • MS Multiple sclerosis
  • MS disease is a chronic inflammatory demyelinating disease of the central nervous system.
  • 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.
  • 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.
  • MPO myeloperoxidase
  • MS disease 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.
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • beta-casomorphin-7 biological molecule Two aspects of the beta-casomorphin-7 biological molecule are used in the treatment of MS disease.
  • the beta-casomorphin-7 molecule increases the activity of MPO in the body.
  • 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.
  • 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).
  • mice 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.
  • mice in groups (a) and (c) 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mice in group (d) are shown to be increased (p ⁇ 0.05).
  • FIG. 2 shows that 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.
  • 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).
  • mice The number and frequency of leukocytes, CD4+ T cells and subgroups infiltrating the central nervous system (CNS) of mice were determined.
  • IL-22, IL-17 and GM-CSF tests were performed with commercial ELISA kits.
  • 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.
  • 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).
  • 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.
  • 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

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne l'utilisation d'une molécule biologique thérapeutique pour prévenir et traiter la sclérose en plaques ou pour soulager les symptômes provoqués par cette maladie. Ainsi, la molécule biologique, qui est mentionnée dans l'invention sans effets secondaires thérapeutiques ni effets toxiques, agit par réduction significative et statistique du nombre de lymphocytes infiltrant le système nerveux central, un sous-ensemble de lymphocytes T et en particulier, des cellules produisant GM-CSF, IL-17 et IL-22 conjointement avec IFN-g, IFN-g et IL-17A.
PCT/TR2020/051302 2019-12-30 2020-12-15 Utilisation de peptides de bêta-casomorphine dans la sclérose en plaques (sep) WO2021137808A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
TR2019/22305 2019-12-30
TR2019/22305A TR201922305A2 (tr) 2019-12-30 2019-12-30 Multi̇pl skleroz (ms) hastaliğinda beta-kazomorfi̇n pepti̇tleri̇ni̇n kullanilmasi

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002016410A2 (fr) * 2000-08-21 2002-02-28 Apitope Technology (Bristol) Limited Procede de selection de peptide

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002016410A2 (fr) * 2000-08-21 2002-02-28 Apitope Technology (Bristol) Limited Procede de selection de peptide

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PANITCH HILLEL S, HIRSCH ROBERT L, SCHINDLER JOHN, JOHNSON KENNETH P: "Treatment of multiple sclerosis with gamma interferon: exacerbations associated with activation of the immune system", NEUROLOGY, vol. 37, no. 7, 1 July 1987 (1987-07-01), pages 1097 - 1102, XP055839017, DOI: 10.1212/wnl.37.7.1097 *
YOUSIF AHMED ATAALLAH AL-ANI: "Investigating the effect of beta-casomorphin-7 peptide on Th17 associated cytokines in model of multiple sclerosis", THESIS, November 2019 (2019-11-01), pages 1 - 82, XP055839013 *

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