RU2334518C2 - Method for treatment of sugar diabetes including states associated with sugar diabetes, and sugar diabetes complications - Google Patents

Abstract

FIELD: medicine, endocrinology.

SUBSTANCE: therapeutically efficient quantities of phitostanol characterised by general formula I are introduced.

EFFECT: efficient treatment of sugar diabetes due to an increase in cell sensitivity to glucose and insulin and enhancing insulin secretion by beta-cells of pancreas.

4 ex, 10 dwg, 18 cl

Description

FIELD OF TECHNOLOGY

This invention relates to methods for treating diabetes in animals, in particular in humans.

BACKGROUND OF THE INVENTION

Diabetes mellitus is a heterogeneous primary violation of carbohydrate metabolism with numerous etiological factors, which usually include insulin deficiency or insulin resistance, or both. Type 1 diabetes, or juvenile diabetes (which develops mainly in young people), or insulin-dependent diabetes mellitus, is present in patients with little or no absolute endogenous insulin secretion. These patients develop extreme hyperglycemia (the accumulation of glucose in the bloodstream) and they are completely dependent on exogenous insulin therapy for life. Type 2 diabetes, which affects mainly people in adulthood and non-insulin-dependent diabetes mellitus ("NIDDM", "NIDDM"), occurs in patients who retain some ability to secrete endogenous insulin, but most of these patients suffer both from insulin deficiency (relative insulin deficiency) and insulin resistance. Insulin resistance may be due to insufficient expression of insulin receptors, a decrease in the affinity for insulin binding (a decrease in the number and affinity of insulin receptors), or any abnormality at any stage of the insulin signal transmission pathway (1).

In general, in the United States, the prevalence of diabetes in the population is probably 2–4 percent, with type 1 diabetes accounting for 7–10 percent of all patients with diabetes. Secondary complications of diabetes are characterized by serious clinical manifestations. About 25 percent of all new cases of end-stage renal failure occur in patients with diabetes mellitus. About 20,000 amputations (toes, feet, and lower extremities) are performed in patients with diabetes mellitus, which is approximately half of the non-traumatic amputations in the United States. In addition, diabetes is the main cause of new cases of blindness, with approximately 5,000 new cases occur each year.

Carbohydrates from food are broken down in the intestines to glucose and other simple sugars. These sugars are absorbed into the bloodstream and enter (along with other nutrients) into all cells of the body. However, in order to absorb glucose from the blood, cells need insulin, a hormone produced in the pancreas. The pancreas, a large gland located behind the stomach, performs many functions. It produces digestive enzymes - proteins that help break down food in the intestines. In addition, it contains specialized groups of cells called "islets of Langerhans." The cells of these islets are of several types, each type of cell producing a specific hormone. There are two main hormones that regulate blood glucose levels - insulin and glucagon. Both are produced in the islets of Langerhans - glucagon by alpha cells, and insulin by beta cells.

As glucose enters the bloodstream after ingestion, an increase in blood glucose signals beta cells to secrete insulin. Insulin (along with glucose) is carried by the bloodstream to cells throughout the body. Various body tissues, in particular muscle, liver and adipose tissue, have specialized molecules on their cell surfaces called insulin receptors. Insulin binds to these receptors, like a key in a lock - opening channels that allow glucose to enter cells.

After glucose enters the cells, it can be used as a source of energy (chemical energy released during catabolism), necessary for the biological work of cells and the growth of the body. Excess glucose is stored in the liver in the form of a complex carbohydrate called glycogen. In the meantime, as blood glucose levels drop, insulin secretion slows down. When the blood glucose level begins to drop significantly (below the physiological level), this serves as a signal to alpha cells to secrete glucagon. Glucagon, in turn, transmits a signal from the liver to turn glycogen back into glucose - a process called glycogenolysis. This prevents the blood glucose level from dropping to a too low value, thereby providing the cells of the body with a steady supply of glucose between meals. In addition, glucagon stimulates the production of new glucose by the liver from other (non-carbohydrate) nutrients, such as amino acids (the blocks that make up the protein), in a way called gluconeogenesis, thereby ensuring a backup (duplicate) source of glucose until the next meal.

The interaction of glucose, insulin and glucagon usually ensures that blood glucose levels stay within certain limits (physiological level). In the case of diabetics, however, this delicate balance is disturbed.

Today, insulin therapy is the main treatment for all patients with type 1 diabetes and for many patients with type 2 diabetes. Depending on the number of injections per day and the type (s) of insulin used, the treatment regimen may be more or less intense. The most intensive method of treatment involves the continuous supply of insulin to the subcutaneous site of the abdominal wall using an open-loop delivery device consisting of a small pump with insulin that the patient must wear for essentially 24 hours daily. Oral hypoglycemic agents, such as sulfoureas, are effective for patients with type 2 diabetes, however, approximately 10 to 20 patients (of them) do not respond or stop responding 12-24 months after the treatment with the indicated drugs.

Effective monitoring of glucose levels over long periods of time is difficult to achieve even in the case of the most careful regimen of insulin therapy in the most conscious patients. As a potential treatment, transplantation of the pancreas or islet cells, which normally produce insulin, continues to be intensively investigated. In addition, work continues on the development of the latest and most suitable external devices or implantable insulin delivery devices, integrated (with built-in) glucose sensor.

Type 2 diabetes is usually observed in people of mature and senile age (usually after the age of 40), but it can also occur in younger people. As a rule, in people suffering from this condition, the pancreas produces insulin, but the cells (tissues) of the body are not able to respond effectively to it. This condition is called insulin resistance. A person can have insulin resistance without diabetes, as long as there is enough insulin to overcome resistance. However, if insulin resistance continues to increase and / or insulin production falls below the level required to achieve compensation, diabetes can develop.

Most type 2 diabetics can be treated with oral medications that increase pancreatic insulin secretion and / or reduce insulin resistance. But after some time, pancreatic beta cells can become less and less sensitive, while secreting less insulin, while blood glucose levels are high. Ultimately, a person may need insulin injections in order to control blood glucose levels.

Because persistent hyperglycemia can interfere with tissue healing and immunity, in some people, the first sign of diabetes can be slow healing ulcers (wounds), frequent infections, or gum disease. Or the first manifesting symptoms may be symptoms of organ damage, such as, for example, heart disease or neuropathy. Common symptoms of diabetic neuropathy include tingling (numbness), loss of sensation, or burning pain in the feet and lower extremities. Although the exact causes have not been established, diabetes mellitus appears to be a consequence of the interaction of genes and lifestyle factors.

One of the most important risk factors for diabetes is overweight (2). The study showed that overweight and a diet with a high fat content are associated with the development of insulin resistance (3). This is especially true when excess adipose tissue is localized in the upper body, around the abdominal cavity - the "apple shape" (as opposed to the "pear shape" with excess fat below the waist). Although the reasons for this have not yet been elucidated, the researchers believe that insulin resistance is increased by hormones (such as leptin and resistin), which are secreted by fat cells.

Active research is ongoing within this area to develop safe and effective diabetes treatments. The purpose of the present invention is to eliminate or mitigate the disadvantages and deficiencies known in this field.

SUMMARY OF THE INVENTION

The present invention relates to a method for treating diabetes mellitus and conditions associated with diabetes in an animal, said method comprising administering a non-toxic and therapeutically effective amount of one or more of the following compounds:

where R is part of phytosterol or phytostanol, R _{2 is} derived from ascorbic acid, R ₃ is hydrogen or any metal, alkaline earth metal or alkali metal, and all their salts.

In addition, the present invention relates to the use of said compounds for regulating serum glucose levels, for increasing cellular sensitivity to insulin, and for increasing glucose sensitivity and insulin secretion by pancreatic beta cells.

The compounds of the present invention have been found to be surprisingly effective in enhancing glucose tolerance in animals. Until now, there has been no assessment of these types of compounds regarding their manifestation of the aforementioned effects.

One significant advantage of using the particular compounds of the present invention for this purpose is their solubility in water. In particular, it was found that the solubility in aqueous solutions, such as water, is excellent, thereby allowing for oral administration per se without any further improvements or modifications. Accordingly, the compounds of the present invention can be prepared and used as such, or they can easily be incorporated into foods, drinks, pharmaceuticals and nutraceuticals, regardless of whether these “excipients” are water based. This increased solubility usually translates into lower doses of the compounds for administration necessary to achieve the desired therapeutic or prophylactic effect.

These effects and other significant advantages are described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by the following non-limiting drawings, in which:

Figure 1 - schematic representation of a method for producing phytostanol phosphate ascorbate and its sodium salt;

Figure 2 is a schematic representation of a method for producing phytostanol carbonate ascorbate and its sodium salt;

Figure 3 - schematic representation of a method for producing phytostanoloxalate ascorbate and its sodium salt;

Figure 4 is a graph showing the effect of treatment with sitostanolphosphoryl scororbate ("FM-VP4") on body weight in (in an animal model) Zucer lean and obese rats. Data are presented as mean ± standard deviation; in each group treated, n = 6. * p <0.05 versus obese;

Fig. 5 is a graph showing the effect of treatment with sitostanol phosphoryl ascorbate ("FM-VP4") on morning blood glucose levels in lean and obese Zucker rats. Data are presented as mean ± standard deviation; in each group treated, n = 6. * p <0.05 versus obese;

6 is a graph showing the effect of FM-VP4 after 30 days of treatment on plasma leptin levels in lean and obese Zucker rats. Data are presented as mean ± standard deviation; in each group treated, n = 6. * p <0.05 against DO (treatment);

7 is a graph showing the effect of FM-VP4 after a 30-day treatment on glucose tolerance in lean Zucker rats. Data are presented as mean ± standard deviation; in each group treated, n = 6. * p <0.05 vs Skinny control;

Fig. 8 is a graph showing the effect of FM-VP4 after a 30-day treatment on glucose tolerance in Zucker mast rats (VDF). Data are presented as mean ± standard deviation; in each group treated, n = 6. * p <0.05 against VDF control;

Fig. 9 is a graph showing the effect of FM-VP4 after 30 days of treatment on OGTT insulin in lean Zucker rats. Data are presented as mean ± standard deviation; in each group treated, n = 6. * p <0.05 vs Skinny control; and

Figure 10 is a graph showing the effect of FM-VP4 after 30-day treatment on OGTT insulin in Zucker mast rats. Data are presented as mean ± standard deviation; in each group treated, n = 6. * p <0.05 versus obese.

PREFERRED EMBODIMENTS

The following detailed description is provided in order to help those skilled in the art to practice the present invention. However, this detailed description should not be construed as limiting the scope of the present invention. Various modifications and changes may be made to the embodiments of the invention set forth herein by those of ordinary skill in the art without departing from the spirit or scope of the present invention.

The present invention provides a method for treating diabetes mellitus and conditions associated with diabetes mellitus in an animal, the method comprising administering a non-toxic and therapeutically effective amount of one or more of the following compounds:

where R is part of phytosterol or phytostanol, R _{2 is} derived from ascorbic acid, R ₃ is hydrogen or any metal, alkaline earth metal or alkali metal, and all their salts.

The term “therapeutically effective” is intended to indicate the amount of compound (s) to be administered that is necessary in order to achieve one or more of the following objectives:

a) treating conditions associated with diabetes mellitus, such as hyperglycemia and insulin resistance, including acquired insulin resistance;

b) treating complications of diabetes mellitus, such as insulin resistance, including congenital insulin resistance, impaired glucose tolerance and hyperinsulinemia;

c) treatment of conditions associated with insulin resistance, including polycystic ovary syndrome, steroid-induced insulin resistance and diabetes in pregnant women;

d) treating complications associated with diabetes mellitus, including kidney disease, especially kidney disease associated with type 2 diabetes, neuropathy and retinopathy. Renal diseases associated with type 2 diabetes, including nephropathy, glomerulonephritis, glomerulosclerosis, hypertensive nephrosclerosis, and end-stage renal disease. Additional renal diseases associated with type 2 diabetes, including nephrotic syndrome;

e) improving glucose tolerance;

f) regulation (control) of serum glucose levels;

g) increased cellular sensitivity to insulin;

h) increased sensitivity (sensitivity) to glucose and the level of insulin secretion by pancreatic beta cells;

i) treatment of conditions prior to diabetes.

Diabetes mellitus is (in the present description) preferably type 2 diabetes mellitus.

Below will be described in more detail the components of the compounds. It should be noted that, throughout this description, the terms “compound”, “derivative”, “structure” and “analog” can be used interchangeably to describe the above structures that bind both phytosterol or phytostanol and ascorbic acid and which, like have been found to be effective in treating diabetes mellitus, conditions associated with diabetes mellitus, and complications associated with diabetes mellitus.

Phytosterols / Phytostanols

As used herein, the term “phytosterol” includes all phytosterols, without limitation, for example, sitosterol, campesterol, stigmasterol, brassicasterol, desmosterol, halinosterol, poriferirasterol, clionasterol, and all its natural or synthetic forms and its derivatives, including isomers. The term “phytostanol” includes all saturated or hydrogenated phytosterols and all its natural or synthetic forms and its derivatives, including isomers. It must be borne in mind that modifications in relation to phytosterols and phytostanols, i.e. the inclusion of side chains also does not go beyond the boundaries (scope) of the present invention. In addition, it must be borne in mind that, in case of doubt about the use of the above two terms throughout the present description, the term phytosterol includes both phytosterol and phytostanol, i.e. These terms may be used interchangeably unless otherwise specified.

Phytosterols and phytostanols used to produce derivatives in accordance with this invention can be extracted from several natural sources. For example, they can be obtained by processing vegetable oils (including aquatic plant oils) such as corn oil and other vegetable oils, wheat germ oil, soybean extract, rice extract, rice bran, rapeseed oil, sunflower oil, sesame oil and fish fats (and fats from other sources related to the marine world). The present invention should not be limited to any one source of phytosterols. US patent No. 4420427 discloses the preparation of sterols from vegetable oilseed fuse (tank sludge) using solvents such as methanol. Alternatively, phytosterols and phytostanols can be obtained from tall oil gum (a fatty mixture of saponifiables) or soap, by-products from forestry processing, as described in US Pat. No. 5,770,749, which is incorporated herein by reference.

In one preferred form, the derivative of the present invention is prepared with beta-sitosterol, campestanol, sitostanol and campesterol, naturally occurring or synthetically prepared, and each of these derivatives thus obtained can then be intervened in the composition before delivery in various ratios. In another preferred form, the derivative of the present invention is prepared with sitostanol, naturally occurring or synthetically prepared, or with campestanol, naturally occurring or synthetically prepared, or mixtures thereof.

In a preferred form, sitostanol is phytostanol. In a most preferred form, the compound is any phytostanol phosphoryl ascorbate or a salt thereof.

R ₂

R ₂ includes ascorbic acid or any derivative thereof. What is achieved within the scope of the present invention is the creation of a new structure or compound, where part of the phytosterol or phytostanol is chemically bound to ascorbic acid. Combining benefits and improves both (component) parts of this new structure. A portion of phytosterol, formerly poorly soluble, becomes, as part of a new derivative, substantially more readily soluble in an aqueous or non-aqueous medium, such as oils and fats. Accordingly, it becomes possible to introduce phytosterol without any further improvements associated with its modification to ensure its delivery to the body.

R ₃

R ₃ may represent hydrogen or may convert the starting compound into a salt. The consideration on which the choice of the appropriate salt depends is that these salts must be pharmaceutically, nutraceutically, or acceptable for use in foods, drinks, and the like. Such salts must have a suitable anion or cation. Within the scope of the present invention, suitable acid addition salts include salts derived from inorganic acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, sulfonic and sulfuric acids, and organic acids such as acetic, benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric, gluconic, glyconic, glycolic, isothionic, dairy, lactobionic, maleic, malic, methanesulfonic, succinic, toluenesulfonic and wine.

Suitable base salts include ammonium salts or any metal, alkaline earth metal or alkali metal salt. Preferably, R _{3 is} selected from: calcium, magnesium, manganese, copper, zinc, sodium, potassium, and lithium. Most preferably, R ₃ is sodium.

In the most preferred form of the present invention, the compound has the structure 1 noted above, phytostanol is sitostanol, R ₃ is sodium.

Derivative Derivation

a) Obtaining ester

There are many methods by which structures including phytosterols and / or phytostanols and ascorbic acid can be obtained. In general, the selected phytosterol or stanol (or its halogen phosphate, halogen carbonate or halogen oxalate derivatives) and ascorbic acid are mixed together under appropriate reaction conditions, which allow the condensation of the "acid" part with "alcohol" (phytosterol). These conditions are the same as used in conventional esterification reactions, such as the Fischer esterification method, in which the acid component and the alcohol component are reacted directly or in the presence of a suitable acid catalyst, such as inorganic (mineral) acid, sulfuric acid, phosphoric acid , p-toluenesulfonic acid. Organic solvents commonly used in such esterification reactions are ethers, such as diethyl ether, tetrahydrofuran, or benzene, toluene or similar aromatic solvents, and temperatures can vary from room temperature to elevated temperatures, depending on the reactivity of the reactants subjected to interaction.

In a preferred embodiment, a method for producing an ester derivative comprises “protecting” the hydroxyl groups of ascorbic acid or its derivatives in the form of esters (eg, as acetate esters) or ethers (eg, methyl esters) and then condensation of the protected ascorbic acid with halogen phosphate , halocarbonate or halooxalate phytosterol / phytostanol under suitable reaction conditions. In general, the above condensation reactions are carried out in an organic solvent such as diethyl ether, tetrahydrofuran, or benzene, toluene or similar aromatic solvents. Depending on the nature and reactivity of the reacting substances, reaction temperatures can vary from low (-15 ° C) to elevated temperatures.

Figure 1 is a schematic representation of the preparation of "protected" ascorbic acid (step a), the preparation of an intermediate product of a chlorophosphate / stanol derivative (step b), and the condensation reaction (alternatively, step c or d) to produce one of the new derivatives of the present invention, corresponding to formula I: phytostanol phosphate ascorbate (indicated as structure 6).

In more detail, the method presented in FIG. 1 is as follows: first, ascorbic acid is protected from decomposition by the formation of 5,6-isopropylidene ascorbic acid (structure 2). This can be achieved by mixing acetone with ascorbic acid and an acid catalyst, such as sulfuric acid or hydrochloric acid, under suitable reaction conditions (reference to example 1 below). Chlorophosphate phytostanol (structure 4) is obtained by preparing a solution of phytostanol in toluene and pyridine (although other nitrogen-containing bases such as aliphatic and aromatic amines can alternatively be used) and treating the resulting solution with a phosphorus derivative such as phosphorus oxychloride. The residue obtained after filtration and concentration of the mother liquor is chlorophosphatephytostanol (structure 4). The latter is then mixed with 5,6-isopropylidene-ascorbic acid, and after the addition of a suitable alcohol, such as ethanol, and HCl (step d) is concentrated. Alternatively, a pyridine / THF (THF) mixture may be added (step c) and the product concentrated. After the final washing and drying (step e), the resulting new product of both steps c or d is phytostanol phosphate ascorbate (structure 6).

Alternatively, ascorbic acid is protected on hydroxyl groups, but not in the form of 5,6-isopropylidene ascorbic acid, but in the form of esters (for example, in the form of acetates, phosphates, etc.). Then the latter is condensed with phytosterols or phytostanols, derivatized, as described above, using known methods of esterification, ultimately, obtaining the structure of this invention. The formation of mono- and diphosphates of ascorbic acid is thoroughly described in the literature. For example, in U.S. Patent No. 4,939,128, the contents of which are incorporated by reference in their disclosure, Kato et al. Disclose the production of phosphoric acid esters of ascorbic acid esters. Similarly, US Pat. No. 4,999,437 to Dobler et al., The entire contents of which are incorporated herein by reference in their entirety, describes the preparation of ascorbic acid 2-phosphate. In Dobler et al., The main phosphorylation reaction of ascorbic acid or ascorbic acid derivatives with POCl ₃ in the presence of tertiary amines (described in German Patent Laid-Open Application DOS 2719303) was improved by adding a magnesium compound, preferably aqueous, to the reaction solution magnesium compound solution. Any of the aforementioned known ascorbic acid derivatives may be used within the scope of the present invention.

FIG. 2 is a schematic representation of the preparation of “protected” ascorbic acid (step a), the preparation of the intermediate chlorocarbonate / stanol derivative (step b), and the condensation reaction (optionally step c or d) to give structure 9 (10 is the same), one of derivatives of the present invention, corresponding to the formula II: phytostanol carbonate ascorbate. These chlorocarbonate derivatives can be obtained in the same manner as described in detail above according to Figure 1; however, phosphorus oxychloride is replaced (as shown in step b of FIG. 2) with phosgene.

FIG. 3 is a schematic representation of the preparation of “protected” ascorbic acid (step a), the preparation of the intermediate chloroxalate / stanol derivative (step b), and the condensation reaction (optionally step c or d) to give a new structure 13 (same as 14), one of the derivatives of the present invention, corresponding to formula III: phytostanoloxalate ascorbate (indicated as structure 14). These chloroxalate derivatives can be obtained in the same manner as described in detail above according to Figure 1; however, phosphorus oxychloride is replaced (as shown in step b of FIG. 3) with oxalyl chloride.

b) Salt production

The present invention encompasses not only the parent (parent) structures including phytosterols or phytostanols and ascorbic acid (for example, preferred structures shown as structures 5 and 6 in FIG. 1; structures 9 and 10 in FIG. 2 and structures 13 and 14 in FIG. .3), but also their salts. These salts are even more soluble in water than the corresponding starting compounds, and therefore their effectiveness and (objective) assessment of both in vitro and in vivo conditions is significantly improved.

The formation of salts of the derivatives of the present invention can be easily carried out by treating the starting compound with a number of bases (for example, sodium methoxide or alkoxides of other metals) to obtain the corresponding alkali metal salts. Salts of other metals, calcium, magnesium, manganese, copper, zinc, etc., can be generated by reacting the starting compound with alkoxides of the corresponding metals. As for formula I, R ₃ represents either hydrogen (starting compound) or any metal, alkaline earth or alkali metal (salt).

c) Catalytic (hydrogenation) and chemical reduction

Optionally, the phytosterol derivatives of the present invention or their components (either phytosterol or ascorbic acid) can be hydrogenated or saturated before or after the derivative is obtained. Hydrogenation of heterocyclic structures into partially and fully reduced analogs is a known method. For example, the catalytic and / or chemical reduction of the ascorbic acid cycle to the corresponding dihydro analog is readily carried out in a hydrogen atmosphere and in the presence of a metal catalyst such as platinum, palladium or Raney nickel. In general, the reaction is carried out in an organic solvent such as ethanol, ethyl acetate or similar media and either under atmospheric pressure or at low pressure (3-5 lbs / ⁱⁿ²⁾ at room temperature or slightly elevated temperatures.

Chemical reductions of these systems include the reduction of a family of hydride reagents such as sodium borohydride, lithium aluminum hydride and their analogs. These reduction reactions are usually carried out in an anhydrous inert medium, including ethyl ether, tetrahydrofuran, dioxane or benzene, toluene or similar aromatic solvents, at temperatures from room temperature to reflux temperature.

Similar catalytic or chemical methods (modifications) can be applied to all phytosterol analogues of the present invention. Accordingly, the compounds used in the methods of the present invention include all fully or partially reduced derivatives, wherein the ascorbic acid cycle is partially or fully reduced and / or where part of the phytosterol is fully or partially hydrogenated.

Derivatives

The present invention includes a method for treating diabetes mellitus and conditions associated with diabetes mellitus in an animal, a method for controlling (controlling) serum glucose levels and increasing cellular insulin sensitivity and a method for increasing glucose sensitivity and insulin secretion by pancreatic beta cells, and this method includes the introduction of one or more of the derivatives containing phytosterol and / or phytostanol and ascorbic acid, including their salts, represented by general formulas:

where R represents a part of phytosterol or phytostanol; R _{2 is} derived from ascorbic acid, R ₃ is hydrogen or any metal, alkaline earth metal or alkali metal. Most preferably, the compounds are derivatives of halophosphate, halocarbonate and halooxalate / phytostanol / ascorbate, illustrated in Figures 1-3 as structures 5, 6, 7, 9, 10, 11, 13, 14 and 15. It should be very obvious however, that these structures are only a sample of many new derivatives that do not go beyond the boundaries (volume) covered by formulas I, II and III, and which can be used in accordance with the methods of the present invention. In addition, it must be borne in mind that although structures 7, 11 and 15 (see Figs. 1-3) represent sodium salts of the claimed (starting) compounds, other salts are also included in the scope of the invention, as described above.

Mechanism of action

Without delving into any theory regarding the rationale for the various therapeutic efficiencies of the compounds described herein, it seems that much relies on the improved glucose tolerance provided by these compounds.

The data provided in the examples below show that there is a very significant improvement in glucose tolerance in a group of obese animals that exhibit a glucose tolerance curve inherent in diabetics. Although the insulin response to oral glucose does not show any significant change in non-diabetic lean animals, a change in the profile of insulin secretion after treatment with FM-VP4 is observed in the group of obese animals. Obese animals are extremely insulin resistant and hyperinsulinemic. It is believed that hyperinsulinemia is an attempt to compensate for the lack of effectiveness of insulin on its target tissues (insulin resistance). These animals are also characterized by a blunted response of insulin to glucose. This is proved by the almost flat response of insulin to oral glucose, as seen in Figure 5 (pre-study group). Obviously, the compounds of the present invention improve glucose tolerance in obese (genetically) diabetic rats.

Ways to use

The desired effects described herein can be achieved in various ways. These compounds can be administered by any conventional method suitable for use in combination with pharmaceuticals, nutraceuticals, food products, beverages, and the like.

The amount of compound that is necessary to achieve the desired effects will, of course, depend on a number of factors, such as the particular compound selected, route of administration and condition of the patient.

The compounds of the present invention can be administered to the patient either as such or in pharmaceutical compositions, where they are mixed with suitable carriers or excipients.

The use of pharmaceutically acceptable carriers for formulating the compounds of this invention to practice the invention in dosage forms suitable for systemic administration is within the scope of this invention. With respect to the appropriate choice of carrier and a suitable preparation method, the compounds of the present invention, in particular the compounds formulated as solutions, can be administered parenterally, such as by intravenous injection. The compounds can be easily formulated using pharmaceutically acceptable carriers well known in the art, in dosage forms suitable for oral administration. Such carriers make it possible to formulate the compounds of this invention in the form of tablets, pills, capsules, liquids, gels, syrups, suspensions, suspensions, and the like. for swallowing by patients (oral administration by oral route), which is to be treated.

Pharmaceutical compositions comprising one or more compounds of the present invention include compositions in which the active components are contained in an effective amount sufficient to achieve a predetermined purpose. The determination of effective amounts is the responsibility of qualified specialists in this field, especially in light of the detailed description of the invention presented here.

In addition to the active components, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers, including excipients and excipients, which facilitate the processing of the active compounds into preparations that can be used pharmaceutically. Drugs formulated for oral administration may be in the form of tablets, dragees, capsules or solutions.

The pharmaceutical compositions of the present invention can be obtained by a method that is itself known, for example by conventional mixing, dissolving, granulating, preparing dragees, grinding into powder, emulsification, encapsulation, capture (capture) or lyophilization.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.

Suitable lipophilic solvents or excipients include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspensions, such as carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may additionally contain suitable stabilizers or agents that increase the solubility of the compounds, allowing the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral administration can be obtained by combining the active compounds with a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable excipients, if necessary, to obtain tablets or dragee cores. Suitable excipients are, in particular, excipients such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and / or polyvinylpyrrolidone (PVP). If necessary, disintegrants, such as crosslinked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, varnish solutions and suitable organic solvents or solvent mixtures. Colorants (dyes) or pigments may be added to tablet or dragee coating compositions for identification or to characterize various combinations of doses of the active compound.

Pharmaceutical preparations that can be used orally include (hard) capsules with push-fit connectors made from gelatin, as well as soft, sealed capsules made from gelatin and a plasticizer such as glycerin or sorbitol. Hard capsules may contain the active ingredients in admixture with a filler, such as lactose, binders, such as starches, and / or lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty (non-volatile) oils, liquid paraffin or liquid polyethylene glycols. In addition, stabilizers can be added.

Oral liquid preparations may be in the form of, for example, emulsions, syrups or elixirs, or they may be presented as a dry product to be reconstituted with water or other suitable excipient before use. Said liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitol monooleate or acacia; non-aqueous fillers (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters, such as glycerol, propylene glycol esters, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired, conventional flavoring agents or coloring agents.

In another form of the present invention, the compounds of this invention can be introduced using food products, beverages and nutraceuticals, including, without limitation, the following:

1) dairy products - such as cheeses, butter, milk and other dairy drinks, pasty and milk mixtures, ice cream and yogurt;

2) fat-based products - such as margarines, pastes, mayonnaise, shortenings, cooking fats and oils for frying and dressing (seasoning);

3) cereal-based products - including cereals (for example, bread and pasta), regardless of whether these products (food) are boiled, baked or obtained as a result of other processing;

4) confectionery products - such as chocolate, sweets (candies), chewing gum, desserts (sweet dish), non-dairy cream (for example, Cool Whip ™), sorbets, glazes and other confectionery fillings;

5) drinks - regardless of whether they are alcoholic or not containing alcohol, including sausages (carbonated) and other non-alcoholic drinks, juices (nectars), food additives and liquid food substitutes, such as, for example, sold under the trademarks Boost ™ and Ensure ™; and

6) mixed (dissimilar) products - including eggs and egg-added products; processed foods, such as soups, pre-cooked pasta sauces, convenience foods (pre-cooked foods), etc.

The compounds of this invention can be incorporated directly and without further modification to food, using methods such as mixing, infusion, injection, dispersion, emulsification, immersion, spraying (spraying) and kneading (churning). Alternatively, the compounds may be applied directly to a food product or a beverage by a consumer prior to swallowing (ingestion). The above options are simple and economical methods of delivery of the claimed compounds.

EXAMPLES

The present invention is described by the following examples, which are only illustrative of it, but not limitation.

Example 1

Ascorbic Acid Protection

Oleum (24%, 8.3 g) was added dropwise to acetone (50 ml). Ascorbic acid (12 g) was added to the mixture at 0 ° C and the reaction mixture was stirred at 0 ° C for 6 hours. The resulting crystals are filtered off with suction and the filtered precipitate is pressed to dryness and then washed with acetone (30 ml). The product obtained is 5,6-isopropylidenene ascorbic acid (14 g).

Example 2

Joining Phytostanols

A solution of a mixture (25 g) of phytostanols (campestanol: 36.4%; sitostanol: 62.3%) in toluene (500 ml) and pyridine (25 ml) was added dropwise to a mixture of phosphorous oxychloride (9 ml) in toluene (200 ml ) at 0 ° C. The mixture was stirred at room temperature for 3 hours. Pyridine hydrochloride is filtered off and the mother liquor is concentrated to recover toluene. The residue was dissolved in dry THF (THF) (100 ml) and a solution of the above protected ascorbic acid (14 g) in dry THF (THF) (400 ml) was added dropwise at 0 ° C. Stirring at room temperature is maintained for 1 hour. The solution was concentrated to remove the solvent. Ethanol (400 ml) and 3N HCl (200 ml) were added, the mixture was heated to 50 ° C for 30 minutes and concentrated. Ethyl acetate (600 ml) was added, the resulting solution was washed with water (3 × 300 ml), dried over sodium sulfate, concentrated and 22 g of product (phytostanol phosphate ascorbate) were obtained as a white powder.

Example 3

Conversion to Sodium Salt

The above acid (17 g) was dissolved in ethanol (100 ml) and a solution of sodium methoxide (2.7 g) in ethanol (50 ml) was added with stirring and at room temperature. Stirring is maintained for 30 minutes after addition. The resulting white precipitate was filtered off, dried and weighed, yielding 20 g of a white powder (phytostanol phosphate ascorbate sodium).

Example 4

Zucker Fat Rat Study (fa / fa)

Adult obese (fa / fa) and skinny Zucker male rats (weighing 340-410 g) were used both in treatment for 4 weeks according to the protocol and in studies on absorption (absorption) in the gastrointestinal tract (FA) , GI) [ ³ H] -cholesterol. The rat was an appropriate animal model for studies of cholesterol absorption in GI (GI) after oral administration due to similarities in intestinal characteristics (i.e. anatomical, metabolic and biochemical characteristics) and in intestinal processing and absorption of dietary cholesterol between rats and humans. Similar processing and absorption of dietary (food-injected) cholesterol is observed in Zucker rats.

Determination of plasma glucose, insulin, leptin and lipid concentrations

Glucose is determined using a blood glucose monitoring system Surestep (Lifescan Canada). Insulin and leptin levels are measured by radioimmunoassay (RIA LINCO corp kits). Plasma cholesterol and triglycerides are determined using enzyme kits (Sigma, St. Louis, MO) as previously described.

Building a study. This study uses a group of labeled obese male rats (n = 6) and lean VDF rats (n = 6), 12 years old. Prior to the administration of one of the compounds of the present invention designated as “FM-VP4” (sitostanol phosphate ascorbate), the animals were weighed and fasted overnight, blood samples (on an empty stomach) were taken to determine: glucose, insulin, leptin, cholesterol and triglycerides. The animals are then treated with 250 mg / kg FM-VP4 twice daily by feeding through a gastric tube for 30 days (using a 2% solution of FM-VP4 dissolved in drinking water). Every two days, animals are weighed and their morning blood glucose levels determined. At the end of 30 days, all animals were fasted overnight and a second oral glucose tolerance test (PGTT) was performed. Fasting blood samples are taken from all animals to determine glucose, insulin and leptin. This is followed by an oral glucose tolerance test (PGTT, OGTT), performed on conscious, naturally behaving animals, as described previously. Animals received glucose (1 g / kg as a 40% solution) using feeding through a gastric tube, and blood samples were taken for 10, 20, 30, 60 and 120 minutes to determine glucose and insulin.

Plasma [ ³ H] -cholesterol concentration after administration of FM-VP4

Development of the composition of FM-VP4- [ ³ H] -cholesterol for feeding through an oral probe. The compositions consisted of 25 μCi [ ³ H] -cholesterol (corresponding to 227.3 ng of cholesterol, based on the specific activity of 110 mCi / mg; Amersham, Mississauga, ON, Canada), 1 mg of unlabeled (radioactive isotope) cholesterol (6.7) and increasing amounts of FM-VP4 (1-20 mg). The formulations are mixed with 1 ml Intralipid® (Clintec Nutritional Company; Deerfield, IL, USA) on the day of the study and gently shaken. Intralipid® is a sterile, pyrogen-free, fat emulsion obtained for administration as a source of calories and essential fatty acids and which is used as a filler for solubilization and co-administration with exogenous [ ³ H] -cholesterol and FM-VP4 in tangible ( palpable) oral composition. Analysis by a complex method, which is a combination of liquid chromatography and mass spectrometry, revealed a minimum total cholesterol content and a plant stanol content of 10% Intralipid® before the administration of exogenous cholesterol (labeled and unlabeled) and FM-VP4, as previously published.

Differences in body weight, blood glucose, plasma leptin, glucose and insulin levels after an oral glucose tolerance test and absorption in FA [ ³ H] -cholesterol between the treated groups and the control groups were determined using analysis of variance (PCANOVA; Human Dynamic Systems). Statistical differences were determined using the Newman Keuls post-hoc test. Differences were considered significant if p <0.05. All data are expressed as mean ± standard deviation.

results

Body mass. Body mass profiles during the 30-day period of treatment with the drug (Figure 4) do not show after treatment any significant change in body weight either in the group of lean rats or in the group of obese rats.

Morning blood glucose. The profiles of morning blood glucose during the 30-day period of treatment with the drug (Figure 5) do not show after treatment any significant change in the morning level of glucose in the blood (not determined when not fasting), neither in the group of lean rats nor in the group of obese rats. There is always variability (variability) in these indications, since animals can differ in nutritional status relative to the time of glucose measurement (8 am). However, in these studies, blood glucose levels remain relatively consistent throughout the study. In addition, FM-VP4 treatment does not change the daily diet and / or water intake of lean and obese Zucker rats (data not shown).

Plasma leptin levels. Levels of satiety of the hormone-leptin did not significantly change after treatment with the drug in any group (Figure 6). It should be noted that in obese Zucker rats, there was a primary violation, which involved a mutation in the central leptin receptors, leading to abnormal appetite regulation through the hypothalamus. As a consequence of fa / fa, obese animals have extremely elevated leptin levels. There is evidence of a decrease in leptin levels after treatment with FM-VP4 in obese animals, but these differences are not significant.

Glucose data according to the oral glucose tolerance test (PHTT, OGTT). Figures 7 and 8 show glucose data according to the PGTT (OGTT) for lean and obese animals, respectively, before and after treatment with FM-VP4. While for the group of non-diabetic, with normal glycemia, skinny rats there is no change in glucose tolerance, in the group of obese animals that show the glucose tolerance curve inherent in diabetics, there is a very significant improvement in glucose tolerance (Fig. 8).

Data on insulin according to the oral glucose tolerance test (PHTT, OGTT). Figures 9 and 10 show insulin data according to the TGTT for lean and obese animals, respectively, before and after treatment with FM-VP4. The response of insulin to oral glucose does not undergo any significant change in non-diabetic lean animals (Figure 9), while in the group of obese animals after treatment with FM-VP4 there is a change in the profile of insulin secretion (Figure 10).

Discussion

The purpose of these studies was to determine the effects of phytostanolphosphoryl scorbate (FM-VP4) on insulin resistance and hyperglycemia in obese (fa / fa) and lean Zucker rats. Treatment (FM-VP4) did not change body weight, morning glucose and leptin levels. The data obtained indicate that FM-VP4 does not make changes in the systemic and (hereditarily) genetically predisposed glucose metabolism and hormone-regulated appetite.

In addition, while for the group of non-diabetic, with normal glycemia, skinny rats there is no change in glucose tolerance, in the group of obese animals that exhibit the glucose tolerance curve inherent in diabetics, there is a very significant improvement in glucose tolerance. However, although the response of insulin to oral glucose does not undergo any significant change in non-diabetic lean animals, in the group of obese animals there is a change in the profile of insulin secretion after treatment with FM-VP4. Obese animals are extremely insulin resistant and hyperinsulinemic. It is believed that hyperinsulinemia is an attempt to compensate for the lack of effectiveness of insulin on its target tissues (insulin resistance). These animals are characterized by a blunted response of insulin to glucose. The latter is manifested in the almost flat response of insulin to oral glucose, as can be seen in Fig. 8 (group before the study).

Interestingly, an early peak in insulin secretion over a 10-minute period after administration of oral glucose is accompanied by an improvement in glucose tolerance in these animals after treatment with FM-VP4. This indicates an increase in glucose sensitivity of pancreatic beta cells secreting insulin in these animals as a result of such treatment. The loss of glucose-stimulated secretion of early phase insulin is a criterion (sign) of the occurrence of type 2 diabetes (the onset of diabetes in adulthood) in humans and animal models of type 2 diabetes, including the Zucker mast rat colony used in the study. Regardless of the mechanism for improving glucose tolerance in treatment with FM-VP4 (not due to anorexia or weight loss, see Figure 4), a decrease in hyperglycemia probably has an effect on reducing the severity of insulin resistance, and this, in turn, , reduces the need for insulin and leads to a more sensitive (reactive) pancreatic function.

In conclusion, the administration of FM-VP4 causes a significant improvement in glucose tolerance within Zucker obese rats without changing body weight and morning glucose, insulin and leptin levels in both lean and obese rats.

LINKS

1. Olefsky, 1988, in "Cecil Textbook of Medicine", 18th Ed., 2: 1360-81.

2. De Fronzo R.A. Ferrannini E. Insulin resistance: A multifaceted syndrome response for NIDDM, obesity, hypertension, dyslipidemia and atherolsclerotic cardiovascular disease. Diabetes Care: 14: 173; 1991.

3. Beck-Nielsen H., Pedersen O., Sorensen N.S. Effects of dietary changes on cellular insulin binding and in vivo insulin sensitivity. Metabolism 29: 482: 1994.

Claims (18)

1. A method of treating diabetes mellitus and conditions associated with diabetes mellitus in an animal, comprising administering a non-toxic and therapeutically effective amount of the following compound:

where R is part of phytostanol, R _{2 is} derived from ascorbic acid, and R ₃ is hydrogen or any metal, alkaline earth metal or alkali metal; and all its salts. 2. The method according to claim 1, where the phytostanol is selected from the group consisting of sitostanol, campestanol, stigmastanol, brassicastanol, desmostanol, chalinostanol, poriferastanol, clionastanol and all its natural or synthetic forms and its derivatives, including isomers. 3. The method according to claim 1, where the phytostanol is sitostanol. 4. The method according to claim 1, where R ₃ selected from the group consisting of calcium, magnesium, manganese, copper, zinc, sodium, potassium and lithium. 5. The method according to claim 1, where the animal is a human. 6. A method of controlling serum glucose levels in an animal, said method comprising administering a non-toxic and therapeutically effective amount of the following compound:

where R is part of phytostanol, R _{2 is} derived from ascorbic acid, and R ₃ is hydrogen or any metal, alkaline earth metal or alkali metal; and all its salts. 7. The method according to claim 6, where the phytostanol is selected from the group consisting of sitostanol, campestanol, stigmastanol, brassicastanol, desmostanol, chalinostanol, poriferastanol, clionastanol and all its natural or synthetic forms and its derivatives, including isomers. 8. The method according to claim 6, where the phytostanol is sitostanol. 9. The method according to claim 6, where R ₃ selected from the group consisting of calcium, magnesium, manganese, copper, zinc, sodium, potassium and lithium. 10. The method according to claim 6, where the animal is a human. 11. A method of increasing cellular sensitivity to insulin in an animal, said method comprising administering a non-toxic and therapeutically effective amount of the following compound:

where R is part of phytostanol, R _{2 is} derived from ascorbic acid and R ₃ is hydrogen or any metal, alkaline earth metal or alkali metal; and all its salts. 12. The method according to claim 11, where the phytostanol is selected from the group consisting of sitostanol, campestanol, stigmastanol, brassicastanol, desmostanol, chalinostanol, poriferastanol, clionastanol and all its natural or synthetic forms and its derivatives, including isomers. 13. The method according to claim 11, where the phytostanol is sitostanol. 14. The method according to claim 11, where R ₃ selected from the group consisting of calcium, magnesium, manganese, copper, zinc, sodium, potassium and lithium. 15. A method of increasing glucose sensitivity and the level of insulin secretion in an animal by pancreatic beta cells, said method comprising administering a non-toxic and therapeutically effective amount of the following compound:

where R is part of phytostanol, R _{2 is} derived from ascorbic acid and R ₃ is hydrogen or any metal, alkaline earth metal or alkali metal; and all its salts. 16. The method according to clause 15, where the phytostanol is selected from the group consisting of sitostanol, campestanol, stigmastanol, brassicastanol, desmostanol, chalinostanol, poriferastanol, clionastanol and all its natural or synthetic forms and its derivatives, including isomers. 17. The method according to clause 15, where the phytostanol is sitostanol. 18. The method according to clause 15, where R ₃ selected from the group consisting of calcium, magnesium, manganese, copper, zinc, sodium, potassium and lithium.

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