CN112402610A - New use of sphingosine-1-phosphate receptor modulator in preparation of medicine for treating diabetes - Google Patents

Abstract

The invention belongs to the technical field of medicines, and provides application of a medicine for treating diabetes, which comprises the step of administering a therapeutically effective amount of sphingosine-1-phosphate (S1P) receptor modulator to an individual. The diabetes is type 1 diabetes or type 2 diabetes, wherein the sphingosine-1-phosphate receptor is S1PR1 and/or S1PR 5. Wherein the sphingosine-1-phosphate receptor modulator is selected from the group consisting of Siponimod (Siponimod), SEW2871, KRP-203, CS-0777, beneximod, ozanimod, selapilimomod, GSK2018682, and MT-1303 or a pharmaceutically acceptable salt thereof.

Description

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

Technical Field

The present invention relates generally to the field of medical technology. In particular to application of Siponimod (Siponimod) and other sphingosine-1-phosphate (S1P) receptor modulators in preparing a medicament for treating diabetes.

Background

Diabetes is a group of metabolic diseases and can be divided into three main types: type 1 diabetes (T1D), type 2 diabetes (T2D), and gestational diabetes. T1D was previously referred to as "insulin-dependent diabetes" or "juvenile onset diabetes" and is a hyperglycemic condition caused by autoimmune specific destruction of islet beta cells and the inability to produce insulin. T2D, once referred to as "non-insulin dependent diabetes mellitus," generally begins with insulin resistance. During the initial stages of the disease, the beta cells of the islets of langerhans of the body produce additional insulin to compensate for the insulin resistance. However, over time, functional islet beta cells are lost and insufficient insulin is produced to maintain blood glucose at normal levels. Gestational diabetes is the third major form of intolerance of pregnant women to high blood glucose levels. Furthermore, people with pre-diabetes have blood glucose levels above normal levels, which are not enough to be diagnosed as diabetic, but are at a high risk of type 2 diabetes and other health problems, such as heart disease and stroke.

There are limited drugs available for the treatment of diabetes. Only insulin or synthetic insulin analogues can be used for T1D. Several groups of drugs have been developed for T2D, such as alpha-glucosidase inhibitors (acarbose and miglitol), biguanides (e.g., metformin), dopamine agonists (e.g., bromocriptine), DPP-4 inhibitors (e.g., alogliptin), glucose-like peptides, meglitinide, glucose transport 2 inhibitors (e.g., dapagliflozin), sulfonylureas (e.g., glimepiride) and thiazolidinediones (e.g., rosiglitazone). However, these drugs require long-term continuous administration to control blood glucose levels, and some of them may cause serious side effects. Thus, there is a continuing need for effective drugs and treatments for diabetes.

Sphingosine-1-phosphate (S1P) is a class of bioactive sphingolipid metabolites that act as growth factors to stimulate cell proliferation and survival through interaction with a group of G protein-coupled receptors (GPCRs). Using diabetic db/db mouse as model, FTY720, the first generation analogue of S1P, was able to induce islet beta-cell regeneration and normalize blood glucose in db/db mouse. The db/db mouse is one of the most widely used models of obese type 2 diabetes, and is characterized by severe impairment of insulin-synthesizing beta-cells with age, and gradual loss of insulin.

Siponimod is known by the english name Siponimod and by the chinese name Siponimod. Code BAF-312, chemical name 3- [ [4- [ (1E) -1- [ [ [ 4-cyclohexyl-3- (trifluoromethyl) phenyl ] methoxy ] imino ] ethyl ] -2-ethylphenyl ] -methyl ] -3-azetidinecarboxylic acid fumarate, is a selective sphingosine-1-phosphate (S1P) receptor modulator for the treatment of relapsing multiple sclerosis. Structurally, siponimod is an analog of S1P, is a sphingosine-1-phosphate receptor agonist with a high degree of selectivity for S1PR1 and S1PR5 over S1PR2, S1PR3 and S1PR4 (EC 50 inhibiting the five sphingosine-1-phosphate receptors mentioned above is 0.39, 0.98, >10000, >1000 and 750nM, respectively, in an radioligand binding assay). Siponimod is shown to initiate and complete its biological functions by binding to the S1PR1 receptor, activating downstream information 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 possible to determine whether S1PR1 or S1PR5 is involved in controlling diabetes.

Disclosure of Invention

The invention provides a use for preparing a medicament for treating diabetes, which comprises administering a therapeutically effective amount of a sphingosine-1-phosphate receptor modulator to a subject.

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

In some embodiments, the sphingosine-1-phosphate receptor of the invention is S1PR1 and/or S1PR 5.

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

The chemical name of the free state of siponimod in the 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 is 5- (4-phenyl-5-trifluoromethyl thiophene-2-yl) -3- (3-trifluoromethyl phenyl) -1,2, 4-oxadiazole.

The free state of CS-0777 is chemically 1- [5- [ (3R) -3-amino-4-hydroxy-3-methylbutyl ] -1-methyl-1H-pyrrol-2-yl ] -4- (4-methylphenyl) -1-one.

The chemical name of the free state of the ozanimod (RPC1063) is 5- (3- [ (1S) -1- [ (2-hydroxyethyl) amino ] -2, 3-dihydro-1H-inden-4-yl ] -1,2, 4-oxadiazol-5-yl) -2- [ (propane-2-yl) oxy ] benzonitrile.

The chemical name of the free state of the selaphanim (ONO-4641, also called selamemod) is 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-1, 2-dihydroxy quinoline-3-amide.

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

The free chemical name of MT-1303 is 2- (4- (heptyloxy) -3-trifluoromethylphenethyl) -2-amino-propane-1, 3-diol.

The Invitrogen name of the bordeauximod is Ponesimod (ACT-128800), the CAS number is 854107-55-4, and the chemical name of the free state of the beneximod is (Z) -2- ((Z) -3-chloro-4- ((R) -2, 3-dihydroxypropoxy) benzylidene) -5- (propylamino) -4- (oxy-tolyl) dihydroxythiophene-3 (2H) -ketone.

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

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

In certain embodiments, the sphingosine 1-phosphate receptor modulators of the present invention are administered once daily, once a week, twice a week or three times a week.

The invention also provides a composition comprising the sphingosine-1-phosphate receptor modulator of claim 1, wherein the receptor modulator is selected from siponimod, SEW2871, KRP-203, CS-0777, bostemmod, ozanimod, selapilimod, GSK2018682, MT-1303 or a pharmaceutically acceptable salt thereof.

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

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

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

The invention also discloses a pharmaceutical preparation or a pharmaceutical composition containing the medicine, wherein the composition contains the sphingosine-1-phosphate receptor modulator and pharmaceutically acceptable auxiliary materials, and is prepared into tablets, pills, capsules, liquid, gel, syrup, serous fluid, emulsifiable paste, aerosol, powder or suspension.

In another aspect, the present disclosure provides methods for maintaining or increasing the quality of functional beta cells in a subject having diabetes. The method comprises administering to the subject an effective amount of a sphingosine 1-phosphate receptor modulator, preferably the sphingosine 1-phosphate receptor modulator is siponimod, or a composition comprising a sphingosine 1-phosphate receptor modulator.

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

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

In the present invention, a new generation of S1PR1 and/or S1PR5 receptor modulators, such as siponimod (BAF312), demonstrated the ability to prevent or reverse elevated blood glucose in DB/DB mice, using DB/DB mice as a model. It has also been shown that modulators of the S1PR1 and/or S1PR5 receptors are able to prevent the development of diabetes in non-obese diabetic (NOD) mice. NOD resembles many of the genetic and immunological properties of human type 1 diabetes, and NOD mice are the mouse model of the most widely used type 1 diabetes (T1D) study today.

Detailed Description

Before the present invention is described in more detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the claims, since the scope of the present invention will be limited only by the appended claims. If a dosage range is provided, it is understood that each intervening value, to the extent that there is no intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within 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 that do not include one or both of the limits are also included in the disclosure.

Unless defined otherwise, 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 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 by reference and were incorporated by reference to disclose and describe the methods and/or materials. A connection to a publication. The citation of any publication is a disclosure prior to the filing date thereof 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, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

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

Definition of

The following definitions are provided to assist the reader. Unless defined otherwise, all art terms, notations and other scientific or medical terms or terms used herein have the meanings commonly understood by those of skill in the chemical and medical arts. In some instances, terms are defined herein with a commonly understood meaning for the sake of clarity and/or ease of reference, and such definitions contained herein do not necessarily have to be construed as substantially different from the definition of the term as commonly understood in the art.

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

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

The definition of S1PR1 and/or S1PR5, on the S1P signaling pathway, some drugs can alter the physiological function of S1P in different diseases by agonizing or inhibiting S1PR 1P receptor, S1P receptor data G protein coupled receptor, there are 5 subtypes, i.e., S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5, where S1PR1, S1PR2, S1PR3 are widely expressed in various tissues, S1PR4 is mainly expressed in lymphatic system and blood system, and S1PR5 is mainly expressed in central nervous system. The invention relates to a method for regulating different subtype pathways and corresponding enzyme activities thereof, and the method plays a role in treating different diseases, wherein S1PR1 and/or S1PR5 refers to an S1P regulator which is effective for one or both of S1PR1 and S1PR 5.

It should be noted that in the present invention, terms such as "comprising," "including," "containing," and the like have the meanings given in chinese patent law; they are inclusive or open-ended and do not exclude additional unrecited elements or method steps. Terms such as "consisting essentially of and" consisting essentially of have the meaning of 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 and" consisting of have the meaning assigned to them in chinese patent law; i.e. these terms are closed.

As used herein, a "sphingosine 1-phosphate receptor modulator" or "sphingosine 1-phosphate receptor modulator" includes, but is not limited to, chemicals, compounds and proteins, or compositions having the ability to modulate the activity of sphingosine 1-phosphate receptors. S1P is a bioactive lysophospholipid metabolite that acts as an intercellular lipid mediator. Five sphingosine-1-phosphate receptors are known, namely S1PR 1-5. Sphingosine-1-phosphate receptors exhibit overlapping or different expression patterns in various cells and tissues, and thus, various cellular functions of S1P have been assigned to sphingosine-1-phosphate receptor subtypes. Various sphingosine-1-phosphate receptor modulators, including agonists and antagonists, have been discovered. Some of these sphingosine-1-phosphate receptor modulators exhibit sphingosine-1-phosphate receptor subtype selective activity.

As used herein, "subject" refers to a mammal or a human, e.g., guinea pig, mouse, rat, gerbil, cat, rabbit, dog, monkey, chimpanzee, stub macaque, human and non-human, primate.

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 can refer to preventing the disorder, reducing or ending symptoms associated with the disorder, resulting in complete or partial recovery of the disorder. As used herein, "prevention" with respect to a disorder can refer to the prevention, in whole or in part, of the disorder or a symptom associated with the disorder. For example, "preventing" can refer to completely halting the development of a disease or a symptom thereof, or delaying the onset or development of a disease, delaying the onset or development of a symptom 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 onset, 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, past medical history, current drugs, health status. Subjects and the likelihood of cross-reactivity, allergies, sensitivity and adverse side effects, as well as the route and extent of administration for the development of diabetes.

As used herein, the term "pharmaceutically acceptable" means suitable for use in a mammal, indicating that the specified carrier, vehicle, diluent, excipient and/or salt is generally chemically and/or physically compatible with another. Comprises the ingredients of the formulation and is physiologically compatible with its recipient.

As used herein, the term "unit dose" refers to a single unit vehicle from which the contents are administered directly to the individual as a single dose for convenience, safety, or monitoring. In certain embodiments, the unit dose of parenteral formulation is packaged in a needle-containing ampoule, vial, or syringe. All formulations for parenteral administration should be sterile and non-febrile, as 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 "once weekly" refers to a unit dose, e.g., of a sphingosine 1-phosphate receptor modulator, administered once during seven days, preferably on the same day of the week. In a once-weekly dosing regimen, the unit dose is typically administered about once every seven days. A non-limiting example of once every seven days is the administration of a unit dose of a sphingosine 1-phosphate receptor modulator every weekday.

As used herein, the term "once-a-day" refers to a unit dose, e.g., a unit dose of a sphingosine 1-phosphate receptor modulator, administered once during a day, preferably at the same time of day. In a once-a-day dosing regimen, unit doses are typically administered every 24 hours. A non-limiting example of a once-daily dosing regimen is the administration of a unit dose of a sphingosine-1-phosphate receptor modulator at 9:00 am per 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 beta cells in type 1 diabetes, increasing the number of beta cells. In another aspect, the method prevents or ameliorates inflammatory injury of beta cells in type 1 or type 2 diabetes. In another aspect, the method promotes beta cell regeneration in type 1 or type 2 diabetes and is capable of promoting the survival of insulin secreting cells cultured in vitro.

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

In another aspect, the 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 salt thereof.

Formal names and target selectivities for SEW2871, KRP-203, BAF312, CS-0777, ACT-128800, RPC1063, ONO-4641, GSK2018682, and MT-1303 are given in Table 1 below.

TABLE 1 name and structural formula of the free form of sphingosine-1-phosphate receptor modulators

As noted above, a therapeutically effective amount of siponimod or other sphingosine-1-phosphate receptor modulator provided herein will depend upon a variety of factors known in the art, e.g., subject, body weight, disease of interest, route of administration, and the like. In some embodiments, a therapeutically effective amount of a sphingosine-1-phosphate receptor modulator is from about 0.001mg/kg body weight to about 10mg/kg body weight. In some embodiments, the therapeutically effective amount is about 0.001mg/kg body weight, about 0.01mg/kg body weight, about 0.1mg/kg body weight, about 0.2mg/kg body weight, about 0.3mg/kg body weight, about 0.4mg/kg body weight, about 0.5mg/kg body weight, about 0.6mg/kg body weight, about 0.7mg/kg body weight, about 0.8mg/kg body weight, about 0.9mg/kg body weight, about 1mg/kg body weight, about 1.1mg/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 3.5mg/kg body weight, about 4mg/kg body weight, about 4.5mg/kg body weight, about 5mg/kg body weight, about 5.5mg/kg body weight, about 6mg/kg body weight, about 6.5mg/kg body weight, about 7mg/kg body weight, about 7.5mg/kg body weight, about 8mg/kg body weight, about 8.5mg/kg body weight, about 9mg/kg body weight, about 9.5mg/kg body weight, or about 10mg/kg body weight.

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

In some embodiments, siponimod-based other compounds of the invention may be administered to a subject in need thereof by an appropriate route, including, but not limited to, oral, injection (e.g., intravenous, intramuscular, subcutaneous, intradermal, intracardiac, intrathecal, intrapleural, intraperitoneal) and the like, mucosal (e.g., nasal, buccal, etc.), sublingual, rectal, transdermal and pulmonary. In some embodiments, the compound may be administered intravenously, subcutaneously, orally, intramuscularly, intracerebroventricularly or by the respiratory route.

In some embodiments, siponimod or other sphingosine-1-phosphate receptor modulator compounds are injectable formulations. Injectable preparations include sterile aqueous solutions or dispersions, suspensions or emulsions. In all cases, the injectable formulations should be sterile and should be fluid to facilitate injection. It should be stable under the conditions of manufacture and storage and must be protected from 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), and suitable mixtures thereof and/or vegetable oils. The injectable preparation should maintain proper fluidity. For example, proper fluidity can be maintained by the use of a coating 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, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

In some embodiments, siponimod or other sphingosine-1-phosphate receptor modulator compounds are oral formulations. Oral formulations include, but are not limited to, capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as lozenges (using an intervening matrix such as gelatin and glycerin, or sucrose and acacia) and/or as mouthwashes and the like.

In solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules, etc.), siponimod or other sphingosine-1-phosphate receptor modulator is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, for example starch, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binding agents, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retarders, such as paraffin; (6) absorption promoters, such as quaternary ammonium compounds; (7) wetting agents, such as acetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; (10) a colorant.

In a liquid dosage form for oral administration, siponimod or other sphingosine-1-phosphate receptor modulator is mixed with any of the following: pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the sphingosine 1-phosphate receptor modulator, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular cottonseed, groundnut, corn, olive oil, castor oil, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to 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, nonaqueous suspensions, liposomal formulations, solid particulate 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. The carrier and stabilizer vary according to the requirements of a 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 typically prepared from isotonic solutions and can be delivered by nebulizers.

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

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

In some embodiments, the subject of the present invention is primarily intended to include humans and 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 invention is a human. May be male or female, may be elderly, and may be adult, adolescent, child or infant. The human subject may be caucasian, african, asian, emmett or other ethnic background, or a mixture of such ethnic backgrounds.

Drawings

FIG. 1: effect of siponimod on blood glucose in db/db mice

FIG. 2: effect of siponimod on glucose tolerance in db/db mice

FIG. 3: effect of siponimod on islet beta cell mass in db/db mice

FIG. 4: effect of siponimod on the incidence of diabetes in NOD mice

FIG. 5: effect of different sphingosine-1-phosphate receptor modulators on INS-1 cell growth.

Detailed Description

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

All of the specific compositions, materials and methods described below are included, in whole or in part, within the scope of the 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 may develop equivalent compositions, materials and methods without departing from the scope of the invention, and without inventive faculty. It should be understood that many variations in the procedures described herein may be made while still remaining within the scope of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.

Example 1 assay to assess the effects of siponimod or other sphingosine-1-phosphate receptor modulators on blood glucose in diabetic mice

Five week old female db/db mice (BKS. Cg-m +/-Leprdb) were purchased from Jackson Laboratories (Bar Harbor, Maine). Mice were placed under controlled light (12 hours light/12 hours dark) and temperature conditions and were free to access food (normal rodent chow) and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and were performed according to the guidelines for experimental animal care and use (ILAR, National academyes Press, USA 2011). After 1 week, fasting blood glucose levels were measured in 6-week-old mice. Mice with fasting plasma glucose in the Glc >7mM range were randomized into control (n ═ 10) and siponimod test groups (n ═ 10). Mice in the treatment group were fed 10mg/kg of siponimod daily via the feeding tube 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, administration was stopped and each index was measured for 2 weeks (X, n ═ 10). t-test method was used to analyze statistical significance, P <0.05 was statistically different.

The results are shown in FIG. 1: it was shown that oral administration of siponimod prevented an increase in fasting blood glucose levels in db/db mice. The fasting blood glucose levels of db/db mice in the control group are constantly elevated.

EXAMPLE 2 intraperitoneal glucose tolerance test in mice

For the Glucose Tolerance Test (GTT), mice were fasted for 16 hours and injected intraperitoneally with 10% glucose (1mg/g body weight). Glucose levels were then measured by tail-end blood sampling via a Glucometer Elite (Bayer Corp., Elkhart, Ind.) after 0,15, 30,60,90, and 120 minutes. The results are shown in the attached figure 2:

glucose Tolerance Test (GTT) is shown for control and test mice. Oral administration of siponimod greatly improved glucose tolerance in db/db mice. After 10 weeks, the control group (O, n ═ 10) and the test group (. cndot., n ═ 10) were fasted for 12 hours, and blood glucose levels were measured by intraperitoneal injection of 10% glucose (1mg/g body weight) at 0,15, 30,60,90, 120min tail blood collection. P <0.05 was statistically different.

Example 3 effects of siponimod on islet area in mouse pancreas 6 weeks after treatment

Quantitative assessment of the islet region of mice

To assess islet area in the pancreas after 6 weeks of treatment with siponimod or other sphingosine-1-phosphate receptor modulators, insulin immunofluorescent staining was performed using six serial paraffin sections (10 μm) from the pancreas (5 pancreases in the control group and 5 pancreases in the treatment group). All insulin-stained areas (i.e., islet areas) across the section were photographed by an Axioplan 2 microscope at X10 magnification, each islet area was measured by the Java-based image processing program ImageJ (National Institutes of Health, Bethesda, Md.), and the islet areas in one section were considered to be the islet areas of each pancreas.

The mice of example 1 were taken and, after the last fasting blood glucose measurement, the pancreas was removed from db/db mice, fixed overnight in 4% formaldehyde solution and embedded in paraffin. Paraffin sections (10 μm thick) were rehydrated and antigen retrieval was performed using microwaves in 10mM sodium citrate solution, followed by blocking of endogenous peroxidase in 3% H2O 2 solution. The following primary antibodies were used: guinea pig anti-porcine insulin (1: 300; DAKO Corp., Carpinteria, CA), rabbit anti-glucagon (1: 200; purchased from Thermo Fisher Scientific, Fremont, CA), mouse trans-cyclopeptide D3(1: 40; purchased from Vector laboratories, Boringer, CA), mouse anti-BrdU (1: 10; purchased from BD), rabbit anti-Ki 67 (1: 100; purchased from Abcam, Cambridge, Mass.), and rabbit anti-p 57KIP2 (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 ALEXA FLUOR, Invitrogen) were used for the secondary antibodies. All images were captured by a Zeiss Axioplan 2 microscope.

The results are shown in FIG. 3, which shows the stereological quantitative analysis of the insulin positive region in db/db mice in the control group and the siponimod test group. Oral administration of siponimod increases the amount of insulin secreting cells. 5 consecutive paraffin sections (10 μm) insulin immunofluorescent staining was performed from each pancreas (5 control pancreases and 5 pancreases from the siponimod test group db/db mice). All insulin-stained areas (islet areas) across the section, taken by an Axioplan 2 microscope at x10 magnification, were measured per islet area by the Java-based image processing program ImageJ, and the sum of each islet area was considered to be the islet area per pancreas. P <0.05 is statistically different.

Example 4 Studimod inhibition of non-obese diabetic (NOD) mice from developing diabetes

NOD mice, a model of the autoimmune disease type 1 diabetes, were selected and placed under controlled light (12 hours light/12 hours dark) and temperature conditions and were free to access food (normal rodent chow) and water. Starting at 13 weeks of age, NOD mice were randomly divided into two groups, with control NOD mice (n-12) given saline and test groups (n-12) given 1.0mg/kg siponimod (from CAYMAN CHEMICALS, ann, michigan) once a day. Dosing was stopped at 37 weeks of age. Fasting blood glucose was measured every 2-3 weeks, and blood glucose in NOD mice exceeding 260 mg/dl was defined as diabetes.

The results are shown in figure 4, which shows the effect of oral sibonimod on the incidence of diabetes in non-obese diabetic (NOD) mice. Oral administration of siponimod effectively prevented NOD mice from developing diabetes.

Example 5 Effect of sphingosine-1-phosphate receptor modulators such as siponimod on INS-1 cells cultured ex vivo

Islet cells (INS-1 cells) (containing 10% fetal bovine serum, 2mM L-glutamine, 1% sodium pyruvate, 50. mu.M. beta. -mercaptoethanol, 100 units/ml RPMI 1640 medium containing penicillin and streptomycin, each 100. mu.g/ml) were cultured in medium. For INS-1 cell treatment, cells were cultured overnight in RPMI medium and changed to Hanks' balanced salt solution containing 2mM glucose and 0.5% FBS, i.e., cultured overnight without growth factors. The cells were then treated for 24 hours in the same medium containing 1mg/ml of SEW2871, KRP-203, CS-0777, ACT-128800, RPC1063, ONO-4641, GSK2018682, MT-1303, respectively. Cell proliferation was then tested using the assay cell growth kit for SIGMA (MTT). The assay is based on MTT cleavage of the tetrazolium salt 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 about 4 hours. After this incubation period, a water-insoluble dye was formed and quantified using a scanning multi-well spectrophotometer. The measured absorbance (o.d.) is linear with the number of living cells, i.e. the larger the o.d. value, the more months the cells. The results are shown in FIG. 5, in which 10% FBS is a positive control and (-FBS) is a negative control.

FIG. 5 shows the effect of siponimod and other sphingosine-1-phosphate receptor modulators on INS-1 cell growth. INS-1 cells, insulin-secreting cells, were cultured in RPMI-1640 medium and then analyzed for cell growth 24 hours later using a cell proliferation assay kit (XTT) with 10. mu.M sphingosine 1-phosphate receptor modulator. The absorbance (A492 nm-A690 nm) was measured by a microplate reader. Among the sphingosine-1-phosphate receptor modulators tested are: SEW2871, KRP-203, BAF312, CS-0777, ACT-128800, RPC1063, ONO-4641, GSK2018682, MT-1303. Siponimod and other sphingosine-1-phosphate receptor modulators promote INS-1 cell growth to varying degrees.

The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.

Claims (10)

1. Use of a therapeutically effective amount of a sphingosine 1-phosphate receptor modulator for the manufacture of a medicament for the treatment of diabetes mellitus, said diabetes mellitus being type 1 diabetes or type 2 diabetes mellitus, wherein the sphingosine 1-phosphate receptor is S1PR1 and/or S1PR5, to said subject.

2. Use according to claim 1, wherein the sphingosine-1-phosphate receptor modulator is selected from siponimod, SEW2871, KRP-203, CS-0777, bosiimod, ozanimod, selapilimod, GSK2018682, MT-1303 or a pharmaceutically acceptable salt thereof;

preferably, the sphingosine-1-phosphate receptor modulator is siponimod or a pharmaceutically acceptable salt thereof.

3. The use according to claim 1, wherein the therapeutically effective amount is from 0.001mg/kg body weight to 10mg/kg body weight per dose.

4. The use according to claim 1, wherein the therapeutically effective amount is 0.001mg/kg body weight, 0.01mg/kg body weight, 0.1mg/kg body weight, 0.5mg/kg body weight, 1mg/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 per dose.

5. The composition of claim 1, administered once daily, once a week, twice a week or three times a week.

6. A composition comprising a sphingosine-1-phosphate receptor modulator according to claim 1, wherein the sphingosine-1-phosphate receptor modulator is selected from siponimod, SEW2871, KRP-203, CS-0777, bostemmod, ozanimod, selapilimod, GSK2018682 or MT-1303 or a pharmaceutically acceptable salt thereof;

preferably, the sphingosine-1-phosphate receptor modulator is siponimod or a pharmaceutically acceptable salt thereof.

7. The composition of claim 6, administered by injection, orally, or through a mucosal membrane, preferably intravenously, subcutaneously, orally, intramuscularly, intraventricularly, or by inhalation.

8. A pharmaceutical preparation comprising the drug of claim 1, which comprises the sphingosine-1-phosphate receptor modulator of claim 1 and pharmaceutically acceptable excipients, formulated as a tablet, pill, capsule, liquid, gel, syrup, slurry, cream, aerosol, powder or suspension, in a unit dose of 0.001mg to 10 mg.

9. Use of maintaining or increasing functional beta cells in a subject having diabetes, the use comprising administering to the subject an effective amount of a sphingosine 1-phosphate receptor modulator or a composition comprising a sphingosine 1-phosphate receptor modulator, preferably the sphingosine 1-phosphate receptor modulator is siponimod.

10. Use of increasing insulin levels in a subject having diabetes, the use comprising administering to the subject an effective amount of a sphingosine 1-phosphate receptor modulator or a composition comprising a sphingosine 1-phosphate receptor modulator, preferably the sphingosine 1-phosphate receptor modulator is siponimod.

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