WO2022105220A1

WO2022105220A1 - New use of sphingosine 1-phosphate receptor modulator in preparation of medicament for treating diabetes

Info

Publication number: WO2022105220A1
Application number: PCT/CN2021/100638
Authority: WO
Inventors: 马忠民; 吴彦卓
Original assignee: 睿阜隆(杭州)生物医药有限公司
Priority date: 2020-11-20
Filing date: 2021-06-17
Publication date: 2022-05-27
Also published as: CN112402610A

Abstract

Provided is the use for preparing a medicament for treating diabetes. The use comprises administering to a subject a therapeutically effective amount of a sphingosine 1-phosphate (S1P) receptor modulator. The diabetes is type 1 diabetes or type 2 diabetes. The sphingosine 1-phosphate receptor is S1PR1 and/or S1PR5. The sphingosine 1-phosphate receptor modulator is selected from Siponimod, SEW2871, KRP-203, CS-0777, Ponesimod, Ozanimod, Ceralifimod, GSK2018682, and MT-1303 or a pharmaceutically acceptable salt thereof.

Description

New use of sphingosine 1-phosphate receptor modulator in the preparation of medicaments for the treatment of diabetes

This application requires a Chinese patent application with an application number of 202011308797.3 and an invention titled "New Use of Sphingosine-1-Phosphate Receptor Modulators in the Preparation of Medicines for the Treatment of Diabetes" filed with the China Patent Office on November 20, 2020 Priority, the entire contents of which are incorporated herein by reference.

technical field

The present invention generally relates to the field of medical technology. Specifically, it relates to the application of using Siponimod and other sphingosine 1-phosphate (S1P) receptor modulators to prepare medicines for treating diabetes.

Background technique

Diabetes is a class of metabolic diseases, and diabetes can be divided into three main types: type 1 diabetes (T1D), type 2 diabetes (T2D), and gestational diabetes. T1D was once called "insulin-dependent diabetes mellitus" or "juvenile diabetes mellitus", which is hyperglycemia caused by autoimmunity that specifically destroys pancreatic β-cells and cannot produce insulin. T2D, once known as "non-insulin-dependent diabetes," typically begins with insulin resistance. During the initial stage of the disease, the body's pancreatic beta-cells produce extra insulin to compensate for insulin resistance. However, over time, functional pancreatic beta-cells are lost and cannot make enough insulin to keep blood sugar at normal levels. Gestational diabetes is the third major form of intolerance to high blood sugar levels in pregnant women. In addition, people with prediabetes have higher-than-normal blood sugar levels, not high enough to be diagnosed with diabetes, but they are at extremely high risk for T2D and other health problems, such as heart disease and stroke.

Medications used to treat diabetes are limited. T1D can only be treated with insulin or synthetic insulin analogs. Several groups of drugs have been developed for T2D, such as alpha-glucosidase inhibitors (acarbose and miglitol), biguanides (eg metformin), dopamine agonists (eg bromocriptine), DPP-4 inhibitors (eg, alogliptin), glucose-like peptides, meglitinic acid, glucose transporter 2 inhibitors (eg, dapagliflozin), sulfonylureas (eg, glimepiride), and thiazolidinediones (eg, Luo glitazone). However, these drugs need to be taken consistently over a long period of time to control blood sugar levels, and some of these drugs can cause serious side effects. Therefore, there is a continuing need for effective drugs and therapeutic methods for the treatment of diabetes.

Sphingosine 1-phosphate (S1P) is a class of bioactive sphingolipid metabolites that act as growth factors, stimulating cell proliferation and survival by interacting with a group of G protein-coupled receptors (GPCRs). Using diabetic db/db mice as a model, the first-generation analog of S1P, FTY720, can induce islet β-cell regeneration and normalize blood glucose in db/db mice. The db/db mouse, one of the most widely used models of obese type 2 diabetes, is characterized by a progressive loss of insulin in the β-cells that synthesize insulin severely with age.

The English name of Siponimod is Siponimod, and the Chinese name is Siponimod. Code BAF-312, chemical name is 3-[[4-[(1E)-1-[[[4-cyclohexyl-3-(trifluoromethyl)phenyl]methoxy]imino]ethyl] -2-Ethylphenyl]-methyl]-3-azetidine carboxylic acid fumarate, a selective sphingosine 1-phosphate (S1P) receptor modulator for the treatment of relapse type of multiple sclerosis. Structurally, ciponimod is an analog of S1P and is a sphingosine-1-phosphate receptor agonist, which is highly selective for S1PR1 and S1PR5, and is more selective for the first two than for S1PR2, S1PR3 and S1PR4 (EC50 for inhibition of the above five sphingosine 1-phosphate receptors in a radioligand binding assay of 0.39, 0.98, >10000, >1000 and 750 nM, respectively). Ciponimmod has been shown to initiate and complete its biological functions by binding to the S1PR1 receptor and activating downstream signaling pathways. There is currently no relevant validation of the association of activating S1PR1 with diabetes. Fingolimod has been found to be an agonist of S1PR1, S1PR3, S1PR4 and S1PR5, but it has not been determined whether S1PR1 or S1PR5 is involved in the control of diabetes.

SUMMARY OF THE INVENTION

The present invention provides an application for preparing a medicament for treating diabetes, comprising administering to an individual a therapeutically effective amount of a sphingosine-1-phosphate receptor modulator.

In some embodiments, the diabetes of the present invention is type 1 diabetes or type 2 diabetes.

In some embodiments, the sphingosine-1-phosphate receptors described herein are S1PR1 and/or S1PR5.

The present invention In some embodiments, the sphingosine-1-phosphate receptor modulator of the present invention is ciponimod (BAF-312), SEW2871, KRP-203, CS-0777, ciponimod , ozanimod, serafimod, GSK2018682, MT-1303 or a pharmaceutically acceptable salt thereof.

The chemical name of the free state of siponimod in the present invention is 3-[[4-[(1E)-1-[[[4-cyclohexyl-3-(trifluoromethyl)phenyl]methoxy ]imino]ethyl]-2-ethylphenyl]-methyl]-3-azetidinecarboxylic acid.

The chemical name of the free state of SEW2871 described in the present invention is 5-(4-phenyl-5-trifluoromethylthiophen-2-yl)-3-(3-trifluoromethylphenyl)-1,2, 4-oxadiazole.

The chemical name of the free state of CS-0777 described in the present invention is 1-[5-[(3R)-3-amino-4-hydroxy-3methylbutyl]-1-methyl-1H-pyrrole-2- yl]-4-(4-methylphenyl)-1-one.

The chemical name of the free state of ozanimod (RPC1063) described in the present invention is 5-(3-[(1S)-1-[(2-hydroxyethyl)amino]-2,3-dihydro-1H -Inden-4-yl]-1,2,4-oxadiazol-5-yl)-2-[(propan-2-yl)oxy]benzonitrile.

The chemical name of the free state of Serafimod (ONO-4641, also known as Seraphim) described in the present invention is 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl -1,2-Dihydroxyquinoline-3-amide.

The chemical name of the free state of GSK2018682 described in the present invention is 4-[5-chloro-6-(1-methylethoxy)-3-pyridyl]-1,2,4-oxadiazol-3-yl ]-1H-indole-1-butyric acid.

The chemical name of the free state of MT-1303 described in the present invention is 2-(4-(heptyloxy)-3-trifluoromethylphenethyl)-2-amino-propane-1,3-diol.

The English name of the Ponesimod of the present invention is Ponesimod (ACT-128800), the CAS number is 854107-55-4, and the chemical name of its free state (Z)-2-((Z)-3-chloro- 4-((R)-2,3-Dihydroxypropoxy)benzylidene)-5-(propylamino)-4-(oxy-tolyl)dihydroxythiophen-3(2H)-one.

In some embodiments, the sphingosine-1-phosphate receptor modulator of the present invention is ciponimod, its free form, or a pharmaceutically acceptable salt thereof with other acid bases.

In certain embodiments, the therapeutically effective amount of the sphingosine-1-phosphate receptor modulator of the present invention is about 0.01 mg to about 10 mg/kg body weight per dose. In certain embodiments, the therapeutically effective amount is about 0.001 mg/kg body weight, 0.01 mg/kg body weight, 0.1 mg/kg body weight, about 0.5 mg/kg body weight, about 1 mg/kg body weight, about 1.5 mg/kg body weight per dose. kg body weight, about 2 mg/kg body weight, about 2.5 mg/kg body weight, about 3 mg/kg body weight, about 3.5 mg/kg body weight, about 4 mg/kg body weight, about 4.5 mg/kg body weight, about 5 mg/kg body weight, about 5.5 mg/kg body weight, or about 6 mg/kg body weight.

In certain embodiments, the sphingosine 1-phosphate receptor modulators described herein are administered once a day, once a week, twice a week, or three times a week.

The present invention also provides a composition comprising the sphingosine-1-phosphate receptor modulator described in the above technical solution, wherein the receptor modulator is selected from ciponimod, SEW2871, KRP-203, CS-0777, Bonesimod, Ozanimod, Serafimod, GSK2018682, MT-1303 or a pharmaceutically acceptable salt thereof.

In some embodiments, the sphingosine-1-phosphate receptor modulator of the present invention is ciponimod (BAF-312), or a pharmaceutically acceptable salt thereof.

In certain embodiments, the unit dose of the composition comprising the sphingosine 1-phosphate receptor modulator is from about 0.001 mg to about 10 mg.

In certain embodiments, the sphingosine 1-phosphate receptor modulators of the present invention are administered by injection, orally or mucosally, preferably intravenously, subcutaneously, orally, intramuscularly, intraventricularly, or by inhalation medicine.

The present invention also discloses a pharmaceutical preparation or pharmaceutical composition containing the above-mentioned drugs, the composition comprising the sphingosine-1-phosphate receptor modulator and pharmaceutically acceptable auxiliary materials, and formulated into tablets and pills , capsules, liquids, gels, syrups, serums, creams, aerosols, powders or suspensions.

In another aspect, the present invention provides methods for maintaining or increasing the mass of functional pancreatic beta-cells in a subject with diabetes. The method comprises administering to the individual an effective amount of a sphingosine 1-phosphate receptor modulator or a composition comprising a sphingosine 1-phosphate receptor modulator, preferably a sphingosine 1-phosphate receptor modulator The agent is ciponimod.

In another aspect, the present invention provides a method for increasing insulin levels in a subject with diabetes, the method comprising administering to the individual an effective amount of a sphingosine 1-phosphate receptor modulator or comprising Compositions of sphingosine-1-phosphate receptor modulators, preferably, the sphingosine-1-phosphate receptor modulator is ciponimod.

In certain embodiments, the subject of the present invention is a human or other mammal, preferably a human.

In the present invention, using db/db mice as a model, it is demonstrated that a new generation of S1PR1 and/or S1PR5 receptor modulators, such as siponimod (BAF312), can prevent or reverse db/db mice Elevated blood sugar. It was also demonstrated that S1PR1 and/or S1PR5 receptor modulators can prevent the development of diabetes in non-obese diabetic (NOD) mice. NOD shares many genetic and immunological properties with human type 1 diabetes, and NOD mice are currently the most widely used mouse model for type 1 diabetes (T1D) research.

Detailed description of the invention

Before the present invention is described in greater detail, it should be understood that this invention is not limited to the particular embodiments described and as such may vary. It is also to be understood that since the scope of the invention is limited only by the appended claims, the terminology used herein is used to describe specific embodiments only and should not be construed as limiting the scope of the claims. If a dosage range is provided, it should be understood that each intervention value between the upper and lower limits of the range and any other specified value or intervention value within the specified range is encompassed by the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. If the stated range includes one or both of the limits, ranges excluding either or both of those limits are also included in the disclosure.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated herein by reference and are incorporated herein by reference to disclose and describe the methods described and/or materials. Contacts related to publications. The citation of any publication is for its disclosure prior to its filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. In addition, publication dates provided may differ from actual publication dates and require independent confirmation.

It will be apparent to those skilled in the art upon reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that may be readily separated or combined with the features of any other several. Embodiments without departing from the scope or spirit of the invention. Any recited method can be performed in the order of events recited or in any other order that is logically possible.

definition

The following definitions are provided to assist the reader. Unless otherwise defined, all artistic terms, symbols and other scientific or medical terms or terms used herein have the meanings commonly understood by those skilled in the chemical and medical arts. In some cases, terms with commonly understood meanings are defined herein for clarity and/or ease of reference, and these definitions contained herein should not necessarily be construed as being substantially different from commonly understood definitions of terms in the art.

As used herein, the singular forms "a" and "an", "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the word "about" refers to plus or minus 10% of the number in which it is used. Thus, about 5 mg/kg body weight means within the range of 4.9 mg/kg to 5.1 mg/kg body weight.

Definition of S1PR1 and/or S1PR5, on the S1P signaling pathway, some drugs can change the physiological function of S1P in different diseases by agonizing or inhibiting S1P receptors, S1P receptor data G protein-coupled receptors, there are 5 Subtypes, namely S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5, S1PR1, S1PR2, S1PR3 are widely expressed in various tissues, S1PR4 is mainly expressed in the lymphatic system and blood system, and S1PR5 is mainly expressed in the central nervous system. Regulate different subtype pathways and their corresponding enzymatic activities, thereby playing the role of treating different diseases. In the present invention, S1PR1 and/or S1PR5 refer to S1P regulators that are effective for either or both of S1PR1 or S1PR5.

It should be noted that in the present invention, terms such as "comprising", "containing" and the like have the meanings given in Chinese Patent Law; they are inclusive or open-ended, and do not exclude other unrecited elements or method steps. Terms such as "consisting essentially of" have the meanings in Chinese patent law; they allow the inclusion of other components or steps that do not materially affect the basic and novel characteristics of the claimed invention. The terms "consisting of" have the meanings ascribed to them in Chinese Patent Law; that is, these terms are closed-ended.

As used herein, "sphingosine 1-phosphate receptor modulators" include, but are not limited to, chemicals, compounds, and proteins, or compositions that have the ability to modulate sphingosine 1-phosphate receptor activity. S1P is a biologically active lysophospholipid metabolite that acts as an intercellular lipid mediator. There are five known sphingosine-1-phosphate receptors, namely S1PR1-5. The sphingosine-1-phosphate receptors show overlapping or different expression patterns in various cells and tissues, thus, various cells of S1P Function has been assigned to the sphingosine-1-phosphate receptor subtype. Various modulators of the sphingosine-1-phosphate receptor have been discovered, including agonists and antagonists. Some of these sphingosine-1-phosphate receptor modulators exhibit sphingosine-1-phosphate receptor subtype-selective activity.

As used herein, "individual" refers to mammals or humans such as guinea pigs, mice, rats, gerbils, cats, rabbits, dogs, monkeys, chimpanzees, stump macaques, humans and non-humans, primates.

As used herein, "salt" refers to pharmaceutically acceptable salts and salts suitable for use in industrial processes, such as the preparation of compounds.

As used herein, "treating" with respect to a disorder may refer to preventing the disorder, reducing or ending symptoms associated with the disorder, resulting in complete or partial recovery of the disorder. "Prevention" as used herein with respect to a disorder may refer to the prevention, in whole or in part, of the disorder or symptoms associated with the disorder. For example, "prevention" may refer to completely halting the development of a disease or its symptoms, or delaying the onset or development of a disease, delaying the onset or development of symptoms associated with a disease, or reducing the risk of developing a disease.

As used herein, the term "therapeutically effective amount" refers to a dose or concentration of a sphingosine 1-phosphate receptor modulator, or a pharmaceutically acceptable salt thereof, that is capable of preventing, delaying the onset, and ameliorating the symptoms of diabetes. A therapeutically effective amount of a sphingosine-1-phosphate receptor modulator, or a pharmaceutically acceptable salt thereof, provided herein will depend on various factors known in the art, such as body weight, age, previous medical history, existing medications, health situation. Subjects and cross-reactions, likelihood of allergies, sensitivities and adverse side effects, and route of administration and extent of diabetes development.

As used herein, the term "pharmaceutically acceptable" means suitable for use in mammals, indicating that the specified carrier, vehicle, diluent, excipient and/or salt is generally chemically and/or physically compatible with another compatible. The ingredients of the formulation are contained and are physiologically compatible with their recipients.

As used herein, the term "unit dose" refers to a single unit of the carrier in which the contents are administered as a single dose directly from the carrier to the subject for convenience, safety or monitoring. In certain embodiments, unit-dose parenteral formulations are packaged in needled ampoules, vials, or syringes. All formulations for parenteral administration should be sterile and apyrogenic, as is known and practiced in the art. A unit dose of a compound refers to the weight of the compound without the weight of the carrier, when a carrier is used.

As used herein, the term "weekly" refers to a unit dose, eg, a unit dose of a sphingosine-1-phosphate receptor modulator, administered once over a seven-day period, preferably on the same day of each week. In a weekly dosing regimen, unit doses are typically administered about every seven days. A non-limiting example of administration every seven days is administration of a unit dose of a sphingosine 1-phosphate receptor modulator every Sunday.

As used herein, the term "once-daily" refers to a unit dose, eg, a unit dose of a sphingosine-1-phosphate receptor modulator, administered once during the course of the day, preferably at the same time each day. In a once-daily dosing regimen, a unit dose is typically administered every 24 hours. A non-limiting example of a once-daily dosing regimen is to administer a unit dose of a sphingosine-1-phosphate receptor modulator at 9:00 am each day.

The present disclosure provides methods of treating diabetes using BAF312 and other sphingosine-1-phosphate receptor modulators. In one aspect, the method prevents or ameliorates autoimmune damage to islet beta-cells in type 1 diabetes, increasing the number of beta-cells. In another aspect, the method prevents or ameliorates inflammatory damage to pancreatic beta-cells in type 1 or type 2 diabetes. In another aspect, the method promotes islet β-cell regeneration in type 1 or type 2 diabetes and is able to promote the survival of insulin-secreting cells in vitro.

In one aspect, the present invention provides a method of treating a diabetic patient comprising administering to the patient a therapeutically effective amount of siponimod is administered to the patient.

In another aspect, the present invention provides a method of treating a diabetic patient comprising administering to the patient a compound comprising a therapeutically effective amount of a sphingosine 1-phosphate receptor modulator.

In certain embodiments, the sphingosine-1-phosphate receptor modulator is SEW2871, KRP-203, CS-0777, ACT-128800, RPC1063, ONO-4641, GSK2018682, MT-1303 or a pharmaceutically acceptable one thereof Salt.

The official names and target selectivity of SEW2871, KRP-203, BAF312, CS-0777, ACT-128800, RPC1063, ONO-4641, GSK2018682, and MT-1303 are shown in Table 1 below. Table 1. Names and structural formulas of free states of 1-sphingosine phosphate receptor modulators

As noted above, the therapeutically effective amount of ciponimod or other sphingosine-1-phosphate receptor modulator provided herein will depend on various factors known in the art, such as subject, body weight, target disease, route of administration, etc. In some embodiments, the therapeutically effective amount of the sphingosine 1-phosphate receptor modulator is from about 0.001 mg/kg body weight to about 10 mg/kg body weight. In some embodiments, the therapeutically effective amount is about 0.001 mg/kg body weight, about 0.01 mg/kg body weight, about 0.1 mg/kg body weight, about 0.2 mg/kg body weight, about 0.3 mg/kg body weight, about 0.4 mg/kg body weight body weight, about 0.5 mg/kg body weight, about 0.6 mg/kg body weight, about 0.7 mg/kg body weight, about 0.8 mg/kg body weight, about 0.9 mg/kg body weight, about 1 mg/kg body weight, about 1.1 mg/kg body weight , about 1.2mg/kg body weight, about 1.3mg/kg body weight, about 1.4mg/kg body weight, about 1.5mg/kg body weight, about 2mg/kg body weight, about 2.5mg/kg body weight, about 3mg/kg body weight, about 3.5 mg/kg body weight, about 4 mg/kg body weight, about 4.5 mg/kg body weight, about 5 mg/kg body weight, about 5.5 mg/kg body weight, about 6 mg/kg body weight, about 6.5 mg/kg body weight, about 7 mg/kg body weight, About 7.5 mg/kg body weight, about 8 mg/kg body weight, about 8.5 mg/kg body weight, about 9 mg/kg body weight, about 9.5 mg/kg body weight, or about 10 mg/kg body weight.

In some embodiments, the unit dose of a ciponimod or other sphingosine-1-phosphate receptor modulator compound of the invention is from about 0.001 mg to about 10 mg. For example, a unit dose of a composition of the present invention is about 0.001 mg, about 0.01 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg mg, about 5 mg, about 5.5 mg, about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg, or about 10 mg. In a preferred embodiment, the unit dose of a compound of the present invention is about 5 mg.

In some embodiments, ciponimod or other compounds of the present invention may be administered to a subject in need thereof by an appropriate route, including but not limited to oral, injection (eg, intravenous, intramuscular, subcutaneous, intradermal, Intracardiac, intrathecal, intrapleural, intraperitoneal), etc., mucosal (such as nasal cavity, intraoral administration, etc.), sublingual, rectal, transdermal and pulmonary administration. In some embodiments, the compounds can be administered intravenously, subcutaneously, orally, intramuscularly, intracerebroventricularly, or by the respiratory route.

In some embodiments, the ciponimod or other sphingosine-1-phosphate receptor modulator compound is an injectable formulation. Injectable preparations include sterile aqueous solutions or dispersions, suspensions or emulsions. In all cases, preparations for injection should be sterile and should be fluid for easy injection. It should be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Injectable preparations should maintain proper fluidity. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by the use of surfactants, and the like. The infectious action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.

In some embodiments, the ciponimod or other sphingosine-1-phosphate receptor modulator compound is an oral formulation. Oral formulations include, but are not limited to, capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as solutions in aqueous or non-aqueous liquids or as a suspension, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a lozenge (with intercalating bases such as gelatin and glycerol, or sucrose and acacia) and/or as a mouthwash and analog.

In solid dosage forms for oral administration (eg, capsules, tablets, pills, dragees, powders, granules, etc.), ciponimod or other sphingosine-1-phosphate receptor modulators are combined with a or multiple pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol and/or or silicic acid; (2) binders such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants such as glycerin; (4) disintegrants , such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retarders, such as paraffin; (6) absorption enhancers, such as quaternary ammonium compounds; (7) Wetting agents, such as acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, Sodium lauryl sulfate and mixtures thereof; (10) colorants.

In liquid dosage forms for oral administration, ciponimod or other sphingosine-1-phosphate receptor modulators are mixed with any of the following: pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, Syrups and elixirs. In addition to the sphingosine-1-phosphate receptor modulator, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate , benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (especially cottonseed, peanut, corn, olive oil, castor oil and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol and dehydration Fatty acid esters of sorbitol, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

In some embodiments, the composition is an oral spray formulation or a nasal spray formulation. Spray formulations include, but are not limited to, aqueous aerosols, non-aqueous suspensions, liposomal formulations or solid particle formulations, and the like. Aqueous aerosols are prepared by mixing an aqueous solution or suspension of the agent with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary according to the requirements of the particular compound, but generally include nonionic surfactants (Tween or polyethylene glycol), oleic acid, lecithin, amino acids such as glycine, buffer solutions, salts, sugars or sugar alcohols. Aerosols are usually prepared from isotonic solutions and can be delivered by a nebulizer.

In some embodiments, the therapeutically effective dose of ciponimod or other compound is about 0.001 mg/kg body weight, about 0.01 mg/kg body weight, about 0.1 mg/kg body weight, about 0.2 mg/kg body weight, about 0.3 mg /kg body weight, about 0.4 mg/kg body weight, about 0.5 mg/kg body weight, about 0.6 mg/kg body weight, about 0.7 mg/kg body weight, about 0.8 mg/kg body weight, about 0.9 mg/kg body weight, or about 1 mg /kg body weight, wherein the composition is administered once a day.

In some embodiments, the effective therapeutic dose is about 0.01 mg/kg body weight, about 0.1 mg/kg body weight, about 0.2 mg/kg body weight, about 0.3 mg/kg body weight, about 0.4 mg/kg body weight, about 0.5 mg/kg body weight kg body weight, about 0.6 mg/kg body weight, about 0.7 mg/kg body weight, about 0.8 mg/kg body weight, about 0.9 mg/kg body weight, about 1 mg/kg body weight, about 2 mg/kg body weight, about 3 mg/kg body weight, about 4 mg/kg body weight, about 5 mg/kg body weight, or about 10 mg/kg body weight, wherein the composition is administered once a week.

In some embodiments, individuals described herein are primarily meant to include humans or other mammals. The subject of the invention may also be domestic animals such as cattle, pigs, sheep, poultry and horses, or domestic animals such as dogs and cats. Preferably, the subject of the present invention is human. Can be male or female, can be elderly, and can be adults, teenagers, children or infants. The human subject can be of Caucasian, African, Asian, Semitic or other ethnic background, or a mixture of such ethnic backgrounds.

Instruction drawings

Figure 1: The effect of siponimod on blood glucose in db/db mice;

Figure 2: The effect of siponimod on glucose tolerance in db/db mice;

Figure 3: The effect of siponimod on the amount of islet β-cells in db/db mice;

Figure 4: The effect of siponimod on the incidence of diabetes in NOD mice;

Figure 5: Effects of different sphingosine-1-phosphate receptor modulators on INS-1 cell growth.

Detailed ways

The following examples are provided to better illustrate the claimed invention and should not be construed to limit the scope of the invention.

All specific compositions, materials and methods described below are included in whole or in part within the scope of the present invention. These specific compounds, materials and methods are not intended to limit the invention, but merely to illustrate specific embodiments included within the scope of the invention. Those skilled in the art can develop equivalent compositions, materials and methods without the ability to invent inventive step without departing from the scope of the present invention. It should be understood that many changes can be made in the procedures described herein while remaining within the scope of the invention. The inventors intend that such variations are included within the scope of the present invention.

Example 1 Assessing the effect of ciponimod or other sphingosine-1-phosphate receptor modulators on blood sugar in diabetic mice

Five-week-old female db/db mice (BKS.Cg-m+/-Leprdb) were purchased from Jackson Laboratories (BarHarbor, Maine). Mice were placed under controlled light (12 h light/12 h dark) and temperature conditions and had free access to food (normal rodent chow) and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and performed in accordance with the Guide for the Care and Use of Laboratory Animals (ILAR, National Academies Press, USA 2011). After 1 week, the fasting blood glucose levels of 6-week-old mice were measured. Mice with fasting blood glucose in the range of Glc>7mM were randomly divided into control group (n=10) and siponimod test group (n=10). Mice in the treatment groups were fed 10 mg/kg siponimod daily via feeding tubes, and food intake and body weight were measured twice weekly. Fasting blood glucose levels were measured at the end of each week. After 8 weeks of administration, the administration was stopped, and the measurement of each index was continued for 2 weeks (X, n=10). The t-test method was used to analyze statistical significance, and *P<0.05 was considered statistically significant.

Results are shown in Figure 1 : show that oral administration of ciponimod prevented the increase in fasting blood glucose levels in db/db mice. The fasting blood glucose level of the control group db/db mice increased continuously.

Example 2 Intraperitoneal glucose tolerance test in mice

For glucose tolerance test (GTT), mice were fasted for 16 hours and injected intraperitoneally with 10% glucose (1 mg/g body weight). Glucose levels were then measured by a Glucometer Elite (Bayer Corp., Elkhart, Ind.) caudal to blood after 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min. The result is shown in Figure 2:

Glucose tolerance test (GTT) of control and test mice is shown. Oral administration of siponimod greatly improved glucose tolerance in db/db mice. After 10 weeks, the control group (●, n=10) and the test group (○, n=10) were fasted for 12 hours and injected with 10% glucose (1 mg/g body weight) by intraperitoneal injection at 0 min, 15 min, 30min, 60min, 90min, 120min tail blood was collected to measure blood glucose level. *P<0.05 indicates statistical difference.

Example 3 Experiment on the effect of ciponimod on the area of pancreatic islets in mouse pancreas after 6 weeks of treatment

Quantitative assessment of islet region in mice

To assess islet area in the pancreas after 6 weeks of treatment with ciponimod or other sphingosine-1-phosphate receptor modulators, six serial paraffin sections (10 μm) from the pancreas were used (5 pancreas in the control group, The treatment group was 5 pancreases), and immunofluorescence staining for insulin was performed. All insulin-stained areas (i.e., islet areas) on the entire section were photographed at X10 magnification by an Axioplan2 microscope, and each islet area was measured by the Java-based image processing program ImageJ (National Institutes of Health, Bethesda, Md.), and all The islet region in the sum of islet regions section is considered to be the islet region of each pancreas.

The mice of Example 1 were taken, and after the last measurement of fasting blood glucose, the pancreas was removed from the db/db mice, fixed in 4% formaldehyde solution overnight, and embedded in paraffin. Paraffin sections (10 μm thick) were rehydrated and subjected to antigen retrieval using microwaves in 10 mM _sodium citrate solution, followed by blocking of endogenous peroxidase in ₃ % H2O2 solution. The following primary antibodies were used: guinea pig anti-pig insulin (1:300; DAKO Corp., Carpinteria, CA), rabbit anti-glucagon (1:200; purchased from Thermo Fisher Scientific, Fremont, CA), mouse trans Cyclic peptide D3 (1:40; purchased from Vector Laboratories, Boehringham, CA), mouse anti-BrdU (1:10; purchased from BD), rabbit anti-Ki67 (1:100; purchased from Abcam, Cambridge) , Mass.) and rabbit anti-p57KIP2 (1:100; purchased from Abcam, Cambridge, Mass.). Goat anti-mouse/rabbit IgG and goat anti-guinea pig-mouse/rabbit IgG conjugated to ALEXA FLUOR dye (ALEXA FLUOR 488 and ALEXAFLUOR, Invitrogen) were used for secondary antibodies. All images were captured by a Zeiss Axioplan 2 microscope.

The results are shown in Figure 3, which shows the stereological quantitative analysis of insulin-positive areas of db/db mice in the control group and the siponimod test group. Oral ciponimod increased the amount of insulin-secreting cells. Five serial paraffin sections (10 μm) were immunofluorescently stained for insulin from each pancreas (5 control pancreases and 5 pancreases from siponimod-tested db/db mice). All insulin-stained areas (islet areas) on the entire section were photographed by an Axioplan 2 microscope at × 10x magnification, each islet area was measured by the Java-based image processing program ImageJ, and the sum of each islet area was considered to be the islet area of a pancreas. *p<0.05 is statistically significant.

Example 4 Experiment in which siponimod prevents the development of diabetes in non-obese diabetic (NOD) mice

NOD mice, a model of the autoimmune disease type 1 diabetes mellitus, were selected and placed under controlled light (12 h light/12 h dark) and temperature conditions with free access to food (normal rodent chow) ) and water. From the age of 13 weeks, NOD mice were randomly divided into two groups, NOD mice in the control group (n=12) were given normal saline, and the test group (n=12) was given 1.0 mg/kg ciponimo De (from CAYMAN CHEMICALS, Ann Arbor, Michigan) once a day. Dosing was discontinued at 37 weeks of age. Fasting blood glucose was measured every 2-3 weeks, and NOD mice were defined as diabetic with blood glucose exceeding 260 mg/dL.

The results are shown in Figure 4, showing the effect of oral administration of ciponimod on the incidence of diabetes in non-obese diabetic (NOD) mice. Oral administration of ciponimod effectively prevented the development of diabetes in NOD mice.

Example 5 The effect of sphingosine-1-phosphate receptor modulators such as ciponimod on INS-1 cells cultured in vitro

Islet β-cell line (INS-1 cells) cultured in medium (RPMI1640 medium containing 10% fetal bovine serum, 2mM L-glutamine, 1% sodium pyruvate, 50 μM β-mercaptoethanol, 100 units/ml) , containing penicillin and streptomycin each 100μg/ml). For treatment of INS-1 cells, cells were cultured overnight in RPMI medium and changed to Hanks' balanced salt solution containing 2 mM glucose and 0.5% FBS, ie, overnight without growth factors. Cells were then treated for 24 hours in the same medium containing 1 mg/ml of SEW2871, KRP-203, CS-0777, ACT-128800, RPC1063, ONO-4641, GSK2018682, MT-1303, respectively. Cell proliferation was then tested with SIGMA's Assay Cell Growth Kit (MTT). The assay is based on tetrazolium salt MTT cleavage in the presence of an electron coupling reagent. The resulting water-insoluble methoxy nitrogen salt. Cells grown in 96-well tissue culture plates were incubated with MTT solution for approximately 4 hours. After this incubation period, a water-insoluble dye was formed, which was quantified using a scanning porous spectrophotometer. The measured absorbance (O.D.) has a linear relationship with the number of viable cells, that is, the larger the O.D. value, the more cells for several months. The results are shown in Figure 5, in which 10% FBS is the positive control, and (-FBS) is the negative control.

Figure 5 shows the effect of ciponimod and other sphingosine-1-phosphate receptor modulators on the growth of INS-1 cells. Insulin-secreting cells INS-1 cells were cultured in RPMI-1640 medium, then treated with 10 μM sphingosine 1-phosphate receptor modulator, and 24 hours later, cells were analyzed for growth using the Cell Proliferation Assay Kit (XTT). Absorbance (A492nm-A690nm) was measured by a microplate reader. The sphingosine-1-phosphate receptor modulators tested were: SEW2871, KRP-203, BAF312, CS-0777, ACT-128800, RPC1063, ONO-4641, GSK2018682, MT-1303. Ciponimod and other sphingosine-1-phosphate receptor modulators promote INS-1 cell growth to varying degrees.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims

An application for the preparation of a medicament for the treatment of diabetes, the application comprising administering to an individual a therapeutically effective amount of a sphingosine 1-phosphate receptor modulator, the diabetes being type 1 diabetes or type 2 diabetes, wherein sphingosine 1-phosphate The receptors are S1PR1 and/or S1PR5.
The application according to claim 1, characterized in that, the sphingosine-1-phosphate receptor modulator is selected from the group consisting of ciponimod, SEW2871, KRP-203, CS-0777, Bonesimod, Ozani Maude, Serafimod, GSK2018682, MT-1303 or a pharmaceutically acceptable salt thereof.
The use according to claim 2, wherein the sphingosine-1-phosphate receptor modulator is ciponimod or a pharmaceutically acceptable salt thereof.
The application according to claim 1, wherein each dose of the therapeutically effective amount is 0.001 mg/kg body weight to 10 mg/kg body weight.
The application according to claim 1, wherein each dose of the therapeutically effective amount is 0.001 mg/kg body weight, 0.01 mg/kg body weight, 0.1 mg/kg body weight, 0.5 mg/kg body weight, 1 mg/kg body weight, 1.5mg/kg body weight, 2mg/kg body weight, 2.5mg/kg body weight, 3mg/kg body weight, 3.5mg/kg body weight, 4mg/kg body weight, 4.5mg/kg body weight, 5mg/kg body weight, 5.5mg/kg body weight, or 6mg/kg body weight.
The application according to claim 1, characterized in that the administration is administered once a day, once a week, twice a week or three times a week.
A composition comprising a sphingosine-1-phosphate receptor modulator in the application of claim 1, wherein the sphingosine-1-phosphate receptor modulator is selected from ciponimod, SEW2871, KRP -203, CS-0777, Bonesimod, Ozanimod, Serafimod, GSK2018682 or MT-1303 or a pharmaceutically acceptable salt thereof.
The pharmaceutical preparation comprising the medicine for treating diabetes in the application of claim 1, the pharmaceutical preparation comprising the sphingosine-1-phosphate receptor modulator and pharmaceutically acceptable adjuvant in the application of claim 1, and formulated into a tablet , pills, capsules, liquids, gels, syrups, serums, creams, aerosols, powders or suspensions in unit doses of 0.001 mg to 10 mg.
A use for maintaining or increasing functional pancreatic beta-cells in an individual suffering from diabetes, the application comprising administering to the individual an effective amount of a sphingosine-1-phosphate receptor modulator or comprising sphingosine-1-phosphate Compositions of alcohol receptor modulators.
The application according to claim 9, wherein the sphingosine-1-phosphate receptor modulator is siponimod.
A use for increasing insulin levels in an individual suffering from diabetes, the use comprising administering to the individual an effective amount of a sphingosine 1-phosphate receptor modulator or a sphingosine 1-phosphate receptor modulator comprising combination.
The use according to claim 11, wherein the sphingosine-1-phosphate receptor modulator is ciponimod.
A method of preventing and/or treating diabetes, the method comprising administering to an individual a therapeutically effective amount of a sphingosine 1-phosphate receptor modulator, the diabetes being type 1 diabetes or type 2 diabetes, wherein sphingomyelin 1-phosphate The amino alcohol receptor is S1PR1 and/or S1PR5; the sphingosine 1-phosphate receptor modulator is selected from the group consisting of ciponimod, SEW2871, KRP-203, CS-0777, Bonesimod, Ozani Maude, Serafimod, GSK2018682 or MT-1303 or a pharmaceutically acceptable salt thereof.
14. The method of claim 13, wherein the composition is administered by injection, orally, or via mucosa.
15. The method of claim 14, wherein the composition is administered intravenously, subcutaneously, orally, intramuscularly, intraventricularly, or by inhalation.
Use of a sphingosine 1-phosphate receptor modulator in the preparation of a medicament for the prevention and/or treatment of diabetes mellitus, which is type 1 diabetes or type 2 diabetes, wherein the sphingosine 1-phosphate receptor is S1PR1 and/or S1PR5; the sphingosine-1-phosphate receptor modulator is selected from ciponimod, SEW2871, KRP-203, CS-0777, ponesimod, ozanimod, serafimod, GSK2018682 or MT-1303 or a pharmaceutically acceptable salt thereof.