CN1684971A - Novel structures and compositions comprising sterols and/or stanols and specific classes of anti-inflammatory agents and use thereof in treating or preventing cardiovascular disease, its underlying co - Google Patents

Abstract

The present invention provides, in one aspect, novel derivatives comprising sterols and/or stanols and an NSAID selected from salicylic acids and arylalkanoic acids, including salts of these derivatives, and having one or more of the following formulae: a) R2-(CH2)n-CO-OR b) R2-R c) R2-CO-CO-OR d) formula (I), wherein R is a sterol or stanol moiety, R2 is derived from a salicylic acid or an arylalkanoic acid and n=1-5. Also provided are pharmaceutical compositions comprising one or more of these novel derivatives and methods of treating or preventing cardiovascular disease and its underlying conditions including, without limitation, atherosclerosis, hypercholesterolemia, hyperlipidemia, hypertension, thrombosis, coronary artery disease, and for treating and reducing inflammation including coronary plaque inflammation, bacterial-induced inflammation, viral induced inflammation and inflammation associated with acute pain and surgical procedures which comprises administering to an animal, particularly a human, a non-toxic and therapeutically effective amount of one or more of these compounds or a biologically acceptable salt thereof.

Description

Novel structures and compositions including sterols and/or stanols, and special classes of anti-inflammatory drugs and their use in the treatment or prevention of cardiovascular disease and its underlying conditions, including the treatment of hyperlipidemia and other diseases in which inflammation is partly the cause disease or symptoms

technical field

The field of the invention is that of sterols and stanols and their novel derivatives and their use in the treatment and prevention of cardiovascular diseases and other disorders.

Background technique

Although recent advances in science and technology have helped improve the quality and length of life of humans, there has been no success in finding a way to prevent atherosclerosis, the underlying condition of cardiovascular disease ("CVD"). Atherosclerosis is a degenerative process caused by the interplay of genetic (gene) factors and environmental factors such as diet and lifestyle. Research so far suggests that cholesterol may play an important role in atherosclerosis, where it forms atherosclerotic plaques in blood vessels that eventually cut off blood supply to the heart muscle, or optionally to the brain or extremities (depending on where the plaque is in the arterial network ( 1, 2) position). The general perception is that a 1% reduction in serum cholesterol in humans reduces the risk of coronary artery disease by 2% (3). Statistics indicate that a 10% reduction in mean serum cholesterol (eg, from 6.0 mmol/L to 5.3 mmol/L) could prevent 100,000 deaths per year in the United States (4).

Sterols are naturally occurring compounds that are used for many extremely important cellular functions. Phytosterols such as campesterol, stigmasterol and β-sitosterol in plants, ergosterol in fungi and cholesterol in animals are major components in cellular and subcellular membranes in their respective cell types. Dietary sources of phytosterols in humans come from plants, namely vegetables and vegetable oils. It is estimated that the daily phytosterol content in a conventional Western diet is about 60-80mg, whereas a vegetarian diet can provide about 500mg phytosterol per day.

Phytosterols have attracted much attention because of their ability to lower serum cholesterol levels when fed to many species of mammals, including humans. Although the exact mechanism of action is unknown, the relationship between cholesterol and phytosterols is apparently due in part to similarities in their chemical structures (differences in the side chains of the molecules). It is estimated that phytosterols displace cholesterol in the micellar phase thereby reducing absorption, or may compete for receptor and/or carrier sites during cholesterol absorption.

According to one report (5), forty years ago the company Eli Lilly sold sterol preparations from tall oil and later from soybean oil called Cytellin ^™ and found that these preparations reduced serum cholesterol by about 9%. Various subsequent studies investigated the effect of sitosterol preparations on plasma lipid and lipoprotein concentrations (6), and the effect of sitosterol and campesterol from soybean and tall oil on serum cholesterol (7). A phytosterol composition which was found to be highly effective in reducing serum cholesterol is disclosed in U.S. Patent 5,770,749 to Kutney et al., the composition comprising no more than 70% by weight of β-sitosterol, at least 10% by weight of campesterol and Stigmasterol (beta-sitostanol). It is noted in this patent that some form of synergy between the phytosterol components provides better cholesterol-lowering results than previously possible.

Recently, the role of inflammation in cardiovascular disease has become more apparent. Ridker et al. (8) described the possible role of inflammation in CVD. J. Boyle (9) described a link between plaque rupture and atherosclerotic inflammation.

Prostaglandins play an important role in the inflammatory process, and prostaglandin inhibitor products, especially the products PGG2, PGH2 and PGE2, have become common targets in the development of anti-inflammatory drugs. Conventional non-steroidal anti-inflammatory drugs (NSAID's), however, are active in reducing prostaglandin-induced pain and inflammation-induced swelling, and can affect processes regulated by other prostaglandins. Thus, high doses of even the most common NSAIDs can produce severe side effects, including life-threatening ulcers, thus limiting their therapeutic potential. An alternative to NSAID’s are corticosteroids which can have serious side effects, especially if long-term treatment is required.

NSAIDs have been found to prevent prostaglandin production by inhibiting enzymes in the human arachidonic acid/prostaglandin pathway, including cyclooxygenase (COX).(10). It is now well known that COX has two isomers, cyclooxygenase-1 (COX-1) cyclooxygenase-2 (COX-2) or "prostaglandin G/H synthase II t. COX-1 is Constitutive isoforms, present in blood vessels, stomach, and kidney, whereas COX-2 is induced in the context of cytokines and inflammatory mediators during inflammation. Different fates of PGG2/PHG2 cyclooxygenase products among tissues Depending on the activity of specific PGG2/PHG2 metabolic enzymes, arachidonic acid can also be converted to 12-HPETE and 12-HETE by 12-lipoxygenase, or converted to various white three by 5-lipoxygenase Aspirin and other NSAIDs inhibit cyclooxygenase enzymes and prostaglandin production; they do not inhibit the lipoxygenase pathway and, therefore, do not inhibit leukotriene production.

It is an object of the present invention to alleviate compounds known in the art for the treatment of CVD and its underlying conditions, including disorders or conditions in which inflammation is part of the etiology or condition.

Summary of the invention

One aspect of the present invention provides a novel derivative comprising sterol and/or stanol and an NSAID (non-steroidal anti-inflammatory drug) selected from salicylic acid and aryl alkanoic acid, including salts of these derivatives , which have one or more of the following formulas:

a) R ₂ -(CH2) _n -CO-OR

b)R ₂ -R

c) R ₂ -CO-CO-OR

d)

wherein R is a sterol or stanol moiety, _R2 is derived from salicylic aryl alkanoic acid and n=1-5.

In another aspect the present invention also provides a composition comprising at least one sterol and/or stanol and at least one NSAID selected from salicylic acid and aryl alkanoic acid.

The present invention also includes processes for the production of novel derivatives of the above formula.

The present invention also includes a pharmaceutical composition for the treatment or prevention of CVD and its underlying conditions, including but not limited to atherosclerotic hypercholesterolemia, hyperlipidemia, hypertension, thrombosis, coronary artery disease, Including coronary plaque inflammation, bacterial inflammation, viral inflammation and inflammation associated with acute pain and surgery, which contains one or more sterols and/or stanols and selected from salicylic acid and aryl chain NASID derivatives of alkanoic acid, which have one or more of the above molecular formulas, and a pharmaceutically acceptable carrier.

The present invention also includes a pharmaceutical composition for the treatment or prevention of CVD and its underlying conditions, including but not limited to atherosclerotic hypercholesterolemia, hyperlipidemia, hypertension, thrombosis, coronary artery disease, For the treatment and reduction of coronary plaque inflammation (coronaryplaque inflammation), bacterial inflammation, viral inflammation and inflammation associated with acute pain (acute pain) and surgery, including one or more sterols and / or stanols and Derivatives of NASID selected from salicylic acid and aryl alkanoic acid having one or more of the above molecular formulas, and a pharmaceutically acceptable carrier.

The invention also includes foods, beverages and nutritional supplements comprising sterols and/or stanols and derivatives of NASID selected from salicylic acid and arylalkanoic acids, or compositions thereof having one or more of the above formulas .

The invention also includes foods, beverages and nutritional supplements comprising a composition comprising one or more sterols and/or stanols and a NASID selected from salicylic acid and arylalkanoic acids.

The present invention also provides a method for treating or preventing CVD and its underlying conditions, including but not limited to atherosclerosis, hypercholesterolemia, hyperlipidemia, hypertension, thrombosis, coronary artery disease, for the treatment of and reduction including coronary plaque inflammation, bacterial inflammation, viral inflammation and inflammation associated with severe pain and surgery, which comprises administering to animals a non-toxic and therapeutically effective amount of one or more sterols and/or stanols selected from NASID derivatives of salicylic acid and arylalkanoic acid having one or more of the above formulas.

The present invention also provides a method of treating or preventing CVD and its underlying conditions, including but not limited to atherosclerosis, hypercholesterolemia, hyperlipidemia, hypertension, thrombosis, coronary artery disease, including coronary plaque Inflammation, bacterial inflammation, viral inflammation and inflammation associated with severe pain and surgery, comprising administering to animals a non-toxic and therapeutically effective amount of Compositions of NASID derivatives of arylalkanoic acids.

It has been found that the derivatives and compositions of the present invention exhibit excellent activity in the treatment or prevention of CVD and its underlying conditions, especially hyperlipidemia and the treatment of conditions in which inflammation is part of the etiology or disease. There may be additive or synergistic effects in both areas. Equally importantly, it is believed that when salicylic acid and/or aryl alkanoic acid, whether derivatized with a sterol/stanol component as described herein or only in combination with a sterol/stanol The expected curative effect can be obtained by lower doses of selected NSAIDs. This is important because side effects of long-term use of many anti-inflammatory drugs, such as NSAIDs (non-steroidal anti-inflammatory drugs) consisting of salicylic acid and aryl alkanoic acids, have been reported in the literature. These therapeutic effects and other significant advantages will be more apparent in the following description.

Description of drawings

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

Figure 1 is a diagram showing the formation of a derivative of the present invention, phytostanyl-acetylsalicylate, by reaction of hydrochloric acid with the hydroxyl group of the phytostanol component;

Figure 2 is a diagram showing the formation of another derivative of the present invention, acetoxy phytostanyl salicylate, by reaction of active phytostanyl chloride with the carboxyl group of the salicylic acid component;

Figure 3 is a bar graph showing inhibition of cholesterol absorption by a derivative of the present invention;

Fig. 4 is a bar graph showing the percent inhibition of COX-1 in the presence of "FDC-2-4" of the present invention. SC-560 is a COX-1 inhibitor with IC ₅₀ of 10nM. FDC-2-4 was expressed as (PL+) when treated with PL/C but not (PL-) when treated with PL/C. "PLonly" groups are treated with PL/C alone (not drug). The data are expressed as the percentage of mean absorption inhibition relative to 100% activity ± standard error, n=3, * means p<0.05vs.0.45mM ASA;

Fig. 5 is a bar graph showing the percent inhibition of COX-2 in the presence of "FDC-2-4" of the present invention. DuP-697 is a COX-2 inhibitor with _IC50 of 50nM. FDC-2-4 was expressed as (PL+) when treated with PL/C but not (PL-) when treated with PL/C. The "PLonly" group is only treated with PL/C (non-drug) alone, and the data is represented by the percentage of mean absorption inhibition relative to 100% activity ± standard error, n=3, * indicates p<0.05vs.0.45mM ASA;

Fig. 6 is a digital image of a 1 ml aqueous solution containing 4 mg of a compound FDC-24 of the present invention and 120 mg of Tween 80 against a black background.

Figure 7 is a transmission electron microscope image of an aqueous solution containing FDC-24 (4mg/ml) and Tween 80 (120mg/ml), which may be in the form of micelles;

The graph of Fig. 8 represents the particle size distribution of the Tween 80 aqueous solution of 4mg/ml FDC-24 120mg/ml;

The graph of Figure 9 represents the particle size distribution of 120mg/ml Tween 80 solution (control group); and

Figure 10 shows the structure of a preferred compound "FDC-2-4" of the present invention.

Preferred Embodiments of the Invention

The following detailed description is intended to assist those of ordinary skill in the art to practice the present invention. However, the detailed description should not be construed as unduly limiting the scope of the invention. Modifications and changes to the embodiments discussed herein may be made by persons of ordinary skill in the art without departing from the spirit or scope of the invention.

According to one aspect of the present invention there are provided novel derivatives of sterols and/or stanols and NASIDs selected from salicylic acid and arylalkanoic acids, which are themselves suitable for use in the treatment or prevention of CVD and its underlying conditions, including and Not limited to atherosclerosis, hypercholesterolemia, hyperlipidemia, hypertension, thrombosis, coronary artery disease, including coronary plaque inflammation, bacterial inflammation, viral inflammation, and inflammation associated with severe pain and surgery .

Derivatives of the present invention are represented by one or more of the following molecular formulas:

a) R2-(CH2)n-CO-OR

b) R2-R

c) R2-CO-CO-OR

d)

wherein R is a sterol or stanol moiety, _R2 is derived from an NSAID selected from salicylic acid or arylalkanoic acid and n=1-5.

It should be noted that in the disclosure the terms "derivative", "structure" and "analogue" and "compound" are used interchangeably to describe a novel single group of compounds.

According to another aspect of the present invention there is provided novel compositions comprising sterols and/or stanols and an NSAID selected from salicylic acid and arylalkanoic acids, suitable per se for the treatment or prevention of CVD and its underlying conditions , including but not limited to atherosclerosis, hypercholesterolemia, hyperlipidemia, hypertension, thrombosis, coronary artery disease, including coronary plaque inflammation, bacterial inflammation, viral inflammation and associated with severe pain and surgery concomitant inflammation.

Sterols/Stanols

The term "sterol" as used herein includes all sterols such as, but not limited to, sitosterol, campesterol, stigmasterol, brassicasterol (including dihydrobrassicasterol), streptosterol, chalinosterol, porous sterol , coprosterol, ergosterol, coprosterol, codisterol, isofucosterol, fucosterol, erythrosterol, neurosterol, 7-enecholesterol, stellasterol, spinasterol (spinasterol), chondrillasterol, peposterol, avenasterol, isoavenasterol, fecosterol, pollen sterol, and cholesterol and all natural or synthetic forms or derivatives thereof, including isomers. The term "stanols" refers to saturated or hydrogenated sterols, including all natural or synthetic forms and derivatives, and isomers thereof. It should be understood that modified sterols and stanols, including side chains, also fall within the scope of this invention. For example, 24β-ethylcholestanol, 24-α-ethyl-22-dihydrocholestanol are clearly included within the scope of the present invention. It is also clear that, in the case of doubt in the specification, unless otherwise stated, the term "sterol" includes sterols and stanols.

The sterols and stanols that form the derivatives of the present invention can be obtained from a variety of natural sources. For example, obtained from the production of vegetable oils (including aquatic plants), 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 oil (and other seafood) . They can be of fungal origin, such as ergosterol, or of animal origin, such as cholesterol.

Accordingly, the present invention is not limited to any one source of sterols. US Patent 4,420,427 teaches how to produce sterols from vegetable sludges using solvents such as methanol. Alternatively, phytosterols and phytostanols may be obtained from tall oil pitch or soap, a by-product of forestry production as described in US Patent No. 5,770,749, which is incorporated herein.

In a preferred embodiment of the present invention, the derivatives of the present invention are formed from naturally derived or synthetic β-sitosterol, campestanol, sitostanol, cholesterol or campesterol, and the formed derivatives Each can be incorporated into compositions to deliver at different ratios. In another preferred embodiment, the derivatives according to the invention are formed from naturally derived or synthetic sitostanol or naturally derived or synthetic campestanol or mixtures thereof. Most preferred forms of the derivatives of the present invention include sitostanyl esters and campesteryl or cholestanyl esters, as further described herein.

Salicylic acid/aryl alkanoic acid

Suitable anti-inflammatory agents within the scope of the present invention are selected from the group consisting of specific NSAIDs, ie drugs exhibiting anti-inflammatory activity in animals, especially humans, but without steroid-like structural elements. More specifically, "aryl alkanoic acids" herein are intended to include:

·Aryl acetic acid compounds such as acemetacin, amfenac sodium, bentacin, glumethacin, oxameacin;

Aryl propionate compounds such as alminoprofen, ibuprofen, ketoprofen, flurbiprofen, fenoprofen, oxaprozine,

• Arylbutyric compounds such as bumadizon, butibufen, fenbufen, and xenbucin; and arylvaleric compounds including all salts thereof.

The term "salicylic acid" as used herein is intended to include:

Salicylic acid compounds such as acetylsalicylic acid (ASA), ASA aluminum, ASA sodium, ASA glycolate, salicylic acid, salicylic acid glycolate, salicin, salicortin, special Lisin (tremulacin), acetylaminosalo, balsalazide, benoate, gentisic acid, imidazole salicylic acid, lysine acetylsalicylic acid, 5-amino salicylic acid, morpholine salicylic acid, Naphthyl salicylic acid, olsalazine, parsalimide, phenyl salicylate, salicyloyl sulfate, trisalicylate, and other salts, diflunisal, etelsalate, phossalicylic acid, salo, dihydrate Salicylate, salicylamine, salicylsalicylic acid, sulfasalazine, olsalazone, including all such compounds both synthetically and naturally derived;

with all its salt.

For example, naturally derived salicylates or salts may be extracted from the bark of Salix alba, S. prupurea L, S. fragilis L (also known as willow, a deciduous shrub) according to techniques well known in the art.

In most preferred forms of the invention, the NSAID is a salicylic acid derivative, more preferably one of ASA and its derivatives.

In one embodiment of the invention, the derivative is formed between the salicylic acid compound and the sterol/stanol moiety, having one of the following structures:

i)

wherein R is selected from H and CH ₃ , and R ₁ , R ₂ , R ₃ , R ₄ , R ₅ are each independently selected from OH, acetyl, halogen (Cl, Br, I or F) and have 1-5 Alkyl moieties of carbon atoms;

ii)

wherein R is selected from H and CH ₃ , and R ₁ , R ₂ , R ₃ , R ₄ , R ₅ are each independently selected from OH, acetyl, halogen (Cl, Br, I or F) and have 1-5 Alkyl moieties of carbon atoms;

iii)

wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ are each independently selected from OH, acetyl, halogen (Cl, Br, I or F) and alkyl moieties having 1-5 carbon atoms; and

iv)

Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ are each independently selected from OH, acetyl, halogen (Cl, Br, I or F) and alkyl moieties with 1-5 carbon atoms.

In most preferred forms, the derivatives of the present invention are selected from the group consisting of phytostanyl acetylsalicylate, phytostanyl acetylsalicylate, acetoxy phytostanyl acetylsalicylate, acetyloxy Phytostanyl salicylate, acetoxy phytostanyl acetate, cholestanyl salicylate, acetoxycholestane salicylate, and acetoxy phytostanylamino salicylate Ester, represented by the following molecular formula:

Phytosteryl acetylsalicylate (where R=H or CH ₃ )

Phytostanyl salicylate (where R=H or CH ₃ )

Acetoxyphytostanyl acetylsalicylate (where R=H or CH ₃ )

Acetoxyphytostanyl salicylate (where R = H or CH ₃ )

Acetoxyphytostanyl acetate (where R = H or CH ₃ )

Cholesteryl Salicylate

Acetoxycholestane Salicylate

Acetoxyphytostanylaminosalicylate (where R=H or CH ₃ )

derivative formation process

a) Formation of esters

There are many ways to form new structures including sterols and/or stanols and selected anti-inflammatory drugs. In one approach, selected sterols or stanols (or halogenated phosphates; halogenated carbonates or their halogenated oxalate derivatives) and anti-inflammatory drugs are mixed together under reaction conditions such that the "acid" moiety Condensation with "alcohol" (phytosterol) moiety. The reaction conditions are the same as those used in other general ester condensation reactions, wherein acid chlorides form alcohol components from acid moieties, which can be reacted directly or in the presence of appropriate acid catalysts, such as mineral acids, sulfuric acid, phosphoric acid, p-toluenesulfonic acid. The organic solvents generally used in this type of ester condensation reaction are esters such as diethyl ester, tetrahydrofuran, or benzene, toluene or similar aromatic solvents, and the temperature varies from room temperature to high temperature, depending on the reactivity of the reactants to carry out the reaction .

In a preferred embodiment, the method of forming an ester derivative includes "protecting" the hydroxyl group of the anti-inflammatory drug or its derivative as an ester (such as acetate) or ether (such as methyl ether), and then Under reaction conditions, the protected anti-inflammatory drug is subjected to a condensation reaction (or halophosphate; halocarbonate or its halogenated oxalate derivatives) with the reactive sterol/stanol. Generally, such condensation reactions are carried out in organic solvents such as diethyl ether, tetrahydrofuran or benzene, toluene or similar aromatic solvents. Depending on the nature or reactivity of the reactants, the reaction temperature can vary from low temperature (-15°C) to high temperature.

By way of example, Figure 1 is a diagram showing the condensation reaction of a protected anti-inflammatory drug (acetylsalicylic acid chloride) and a phytostanol to a novel derivative of the present invention.

Alternatively, as shown in the diagram in Figure 2, in general, active chlorine is generated in the sterol or sterane moiety, which subsequently reacts with the carboxylic acid carboxyl unit of the or anti-inflammatory drug, in order to obtain the ester to be formed. In this way, a carboxyl "link" is established between the anti-inflammatory drug and the sterol/stanol. Linkages preferably comprise 1-5 carbon atoms. In Example 2 below, monochloroacetic acid is reacted with a mixture of stanols to form sterane monochloroacetate. Similarly, monochloropropionic, monochlorobutyric, and monochlorovaleric acids, or similar acids, can be used to grow carbon chains on demand. Although the mechanism of the reaction is unclear, in fact these derivatives containing carboxyl carbon chains were found to be extremely effective in lipid regulation (lowering cholesterol), as well as in reducing deleterious inflammatory effects.

As far as the formation of these derivatives is concerned, it is clear that the different derivatives disclosed and claimed can be prepared by many other methods when the synthetic method chosen is designed. Once a particular derivative has been selected, it can be synthesized using techniques routinely available in the art, within the knowledge of those of ordinary skill in the chemical arts. For this reason, the synthesis of all claimed derivatives is not described one by one.

Where possible, (ie, when the parent contains free hydroxyl groups), the invention encompasses biologically acceptable metal, alkaline earth metal, or alkali metal salts of the disclosed derivatives. These salts are more soluble in water than the corresponding parent compounds, thus improving their in vitro and in vivo potency and evaluation.

The derivatives of the present invention can be easily formed into salts, and the corresponding alkali metal salts can be prepared by treating any parent compound containing phenolic hydroxyl groups with a series of bases (such as sodium methoxide or other metal alkoxides). Other metal salts of calcium, magnesium, manganese, copper, zinc and the like can also be obtained by reacting the precursor with the appropriate metal alkoxide.

The term "FDC2-4" as used herein has a structure shown in Figure 10, which is a stanol derivative (mixture of sitostanol and campestanol linked to ASA). According to the invention it is a preferred compound.

Instructions

The present invention provides a method: 1) treating or preventing CVD and its latent conditions, including but not limited to atherosclerosis, hypercholesterolemia, hyperlipidemia, hypertension, thrombosis, coronary artery disease, and coronary plaque inflammation; 2) treatment and improvement of general inflammation, including bacterial inflammation, viral inflammation and inflammation associated with severe pain and surgery, including giving animals one or more non-toxic and therapeutically effective compositions , comprising one or more sterols or stanols and at least one NSAID selected from salicylic acid and aryl alkanoic acids or one or more sterols and/or stanols and selected from salicylic acid and aryl chains NSAID derivatives of alkanoic acids having one or more of the following structures:

a) R ₂ -(CH2) _n -CO-OR

b)R ₂ -R

c) R ₂ -CO-CO-OR

d)

wherein R is a sterol or stanol moiety, _R2 is derived from salicylic acid or arylalkanoic acid and n=1-5.

The present invention also includes the use of any of the disclosed compounds for the treatment of these indications.

The term "therapeutically effective" is intended to ensure that the amount of compound or composition administered is sufficient to achieve one or more of the following goals:

a) treatment of conditions commonly associated with CVD;

b) treatment of atherosclerosis;

c) treatment of hypercholesterolemia;

d) treatment of hyperlipidemia;

e) treatment of high blood pressure;

f) treatment of thrombosis;

g) treatment of coronary artery disease;

h) treatment of coronary plaque inflammation;

i) treatment of any inflammation, including bacterial inflammation, viral inflammation and inflammation associated with severe pain and surgery; and/or

j) inhibiting COX-1 and/or COX-2 activity,

In particular, the compounds of the present invention have been found to be very effective in combating at least two factors that contribute significantly to the multifactorial appearance of cardiovascular disease: elevated cholesterol levels and inflammation. It has been reported in the literature that endothelial inflammatory responses, together with plasma cholesterol levels, play an important role in atherosclerosis (11). Accordingly, it would be highly advantageous to administer a compound that simultaneously reduces cholesterol absorption, thereby lowering serum cholesterol, and simultaneously reduces the concomitant inflammation thought to be part of disease progression. No other compound was shown to have a beneficial dual effect.

The desired effects described here can be obtained in a number of ways. The compounds and compositions of the present invention may be administered by any conventional means, suitable for use in combination with drugs, nutritionals, foods, beverages and the like.

The amount of compound or composition needed to obtain the desired therapeutic effect will depend on various factors such as the particular compound or composition chosen, the mode of administration and the condition of the patient.

The compounds and compositions of the present invention can be administered by themselves alone or in pharmaceutical compositions in which they are mixed with suitable carriers and excipients.

In practicing the present invention, it is within the scope of the invention to formulate the compounds and compositions disclosed herein with pharmaceutically acceptable carriers so that they are suitable for systemic administration. With appropriate choice of carrier and manufacturing practice, the compounds and compositions of the invention, particularly those formulated as solutions, may be administered parenterally, eg, by intravenous injection. The compounds and compositions are readily formulated using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds and compositions of this invention to be formulated into tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient.

Pharmaceutical compositions, comprising one or more compounds of the invention, include compositions wherein the active ingredient is included in an effective amount to achieve a therapeutic purpose. Determination of effective amounts is well known to those of ordinary skill in the art, especially where details have been disclosed herein.

In addition to the active ingredients, these pharmaceutical compositions may also contain appropriate pharmaceutically acceptable carriers, including excipients and adjuvants, which facilitate the processing of the active ingredients into pharmaceutically usable preparations. The prepared oral preparations can be tablets, dragees, capsules or solutions.

The pharmaceutical compositions of the present invention can be prepared by known methods, for example by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or freeze-drying processes.

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

Suitable fatty solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty oleates, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to obtain highly concentrated solution preparations.

Formulations for oral use can be obtained by combining the active compounds in a solid excipient, optionally grinding the resulting mixture, and processing it, after adding suitable auxiliaries, to the form of granules, if desired, into tablets or dragee cores. Suitable excipients include lactose, sucrose, mannitol, sorbitol, corn starch, barley starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethyl-cellulose, carboxymethyl Sodium cellulose, and polyvinylpyrrolidone (PVP). If desired, a disintegrant may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee inner cores are provided with suitable coatings. Concentrated sugar solutions may be used for coating, optionally containing gum arabic, talc, polyvinylpyrrolidone, carbomer gum, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures . Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, and soft, sealed capsules made of gelatin and a plasticizer, such as glycerol and sorbitol. The push-fit capsules can contain the active ingredients in admixture with 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 acids, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may also be added.

Oral liquid preparations may be presented as emulsions, syrups, or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, such as sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gum, hydrogenated edible fats; Emulsifiers such as lecithin, sorbitan monooleate, or gum arabic; non-hydrophilic carriers (among them deuterated oils), such as almond oil, fractions of coconut oil, oily esters such as glycerides, propylene glycol esters, or ethyl alcohol esters; preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid; conventional flavoring and coloring agents may also be added if desired.

Because the sterol/stanol moiety is very poorly water soluble, the compounds of the invention can be formulated to be almost completely soluble in the carrier, making the compound more water soluble or fat soluble.

Preferably, a selected solubilizing agent is added to the compound of the present invention, mixed with a non-toxic organic solvent, and subjected to one or more steps of heating, ultrasonication and evaporation to dissolve the compound and remove the solvent.

Where the compounds of the present invention (predominantly lipophilic) are expected to be dissolved in aqueous solutions, preferred selected solubilizers have a Hydrophile/Lipophile Balance ("HLB") of 12 or greater. Where it is contemplated that the compounds of the invention will be dissolved in a lipid such as a fat or oil medium, it is preferred that the selected solubilizer has an HLB of 8 or less. The HLB value range indicates a molecule's lipophilic propensity relative to its solvency, ie the ratio of polar and non-polar groups in the molecular formula.

Preferred solubilizers for increasing the solubility of the aqueous and lipid phases include, but are not limited to, surfactants such as Tween-80 and Tween-60 (trademarks Tween-80 ^™ and Tween-60 ^™ respectively), poly(ethylene base oxide)-poly(propylene oxide) tri-block copolymer surfactant (Pluron: C ^TM such as: Pluronic P-85, Pluronic F-127, and Pluronic F-108, poly(vinyl oxide) ( PEO)-containing non-ionic surfactant) and macrogolycerol (C ₈ -C ₁₈ Glycerides; Fatty Acid C8-C18 Ethoxylated) are collectively referred to as Gelcire ^TM and specifically as Gelcire 44/14. For Gelucire 44/14, 44 refers to a melting point of 44 °C, while 14 refers to an HLB value of 14.

The organic solvent is selected from any commonly used non-toxic solvents, including but not limited to all halogenated aliphatic hydrocarbons and all linear and branched C ₃ -C ₅ fatty alcohols. More preferably, the organic solvent is selected from the group consisting of propanol, isopropanol, butanol, isobutanol, pentanol, isoamyl alcohol, chloroform, methylene chloride (methylene dichloride) and dimethylsulfoxide ("DMSO") ).

Depending on the particular solubilizing agent and the organic solvent chosen, heating of the solvent may be required to dissolve the compounds of the invention. In such cases, the solution may be heated to from about 25-75°C, more preferably from about 50-70°C, most preferably to about 65°C. As an alternative to heating, ultrasound can be used to dissolve compounds of the invention in organic solvents of choice without exposing the compounds to detrimentally elevated temperatures.

Removal of the organic solvent can be by any suitable type of evaporation including, but not limited to, nitrogen evaporation and rotary or roto-evaporation.

In operation, there are two preferred modes of dissolving the compounds of the present invention in water or lipid carriers, as follows:

Preferred Mode A:

1) One or more compounds of the present invention are mixed with a selected solubilizer, preferably Tween-80;

2) adding an organic solvent, preferably isopropanol, at about room temperature;

3) dissolving the compound and the solubilizing agent in the solvent by heating (preferably heating to about 65° C.), ultrasonication or other dissolution methods,

4) evaporation of the organic solvent; and

6) Add water and vortex or thoroughly mix the solution by any suitable means.

Preferred mode B:

1) One or more compounds of the present invention are mixed with a selected solubilizer, preferably Tween-80;

2) adding an organic solvent, preferably chloroform, at about room temperature, and mixing;

3) evaporation of the organic solvent, preferably by rotary evaporation; and

6) Add water and vortex or thoroughly mix the solution by any suitable means.

In another form of the present invention, the compounds and compositions can be taken in the form of food, drink and nutritional supplements, including but not limited to the following:

1) Dairy products - such as cheese, butter, milk and other dairy beverages, spread and dairy mixtures, ice cream and yoghurt;

2) Fatty products - such as margarine, spreads, coatings, shortenings, cooking and frying oils, and condiments;

3) cereal products - including grains (such as bread and batter) whether they are cooked, baked or otherwise prepared;

4) Confectionery - such as chocolate, candied fruit, chewing gum, desserts, non-dairy toppings (such as Cool Whip), ice water with fruit juice, ice and other fillings;

5) Beverages - whether alcoholic or non-alcoholic, including cola and other soft drinks, fruit juice drinks, dietary supplements, meal replacement drinks such as those sold under the trade names Boost ^TM and Ensure ^TM ; and

6) Miscellaneous products - including eggs and egg products, processed foods such as soups, pre-made batter sauces, pre-cooked meals and the like.

The compounds and compositions of the present invention can be directly added to foods, nutraceuticals or beverages without modification by various techniques such as mixing, soaking, injection, blending, dispersion, emulsification, dipping, spraying and kneading. Alternatively, the compounds and compositions may be ingested by consumers directly in foods and beverages. These are simple and economical delivery modes.

EXAMPLES - The invention is described by the following non-limiting examples.

Synthesis of embodiment 1-phytosteryl acetylsalicylate

R = Me, H

i Synthesis of acetylsalicylic acid chloride

Acetylsalicylic acid (1 g) was suspended in oxalyl chloride (5 ml) and the mixture was refluxed for 1 hour. The remaining oxalyl chloride was recovered by distillation, and the residue was dried in vacuo overnight, and then yellow wax (1.1g) was added.

ii Synthesis of phytosteryl acetylsalicylate

To the chloride prepared above were added a stanol mixture (2 g, campestanol: 36.4% w/w; sitostanol: 62.3% w/w) and pyridine (10 ml), and the mixture was stirred at room temperature overnight. The red solution was poured into water (100ml) and filtered to collect the solid (2.1g). The crude product was applied to a silica gel column (100ml) and eluted with petroleum (30-50°C):ethyl acetate (100:3) to give a white powder (1.5g, yield 55%).

Synthesis of embodiment 2-acetoxy phytostanyl acetylsalicylate

R = Me, H

(i) Synthesis of phytostanyl monochloroacetate.

A mixture of stanols (4 g, campestanol: 36.4% w/w; sitostanol: 62.3% w/w) in monochloroacetic acid (10 ml) was stirred and heated at 120°C for 3 hours. After being cooled to room temperature, water (50ml) was added to the reacted mixture, and the precipitate was collected and washed with water (10ml × 2), dried in vacuo to obtain phytostanyl monochloroacetate (4.5g, yield 95%).

¹ H NMR (CDCl ₃ ): 4.75 (1H, m), 4.00 (2H, s).

MS(E1): 492 (M ⁺ _{sitostanol esters} ), 478 (M ⁺ _{campestanol esters} ).

(ii) Synthesis of acetoxyphytostanol acetylsalicylate

Sodium O-acetylsalicylate (0.5 g) and dry DMF (10 ml) were added to the phytostanyl monochloroacetate (1.5 g) prepared above, and the mixture was stirred and heated at 140° C. for 1 hour. After cooling to room temperature, the mixture was poured into water (100ml), and the off-white solid was collected and weighed dry (1.8g, yield 93.4%). The crude product was purified by silica gel chromatography with ethyl acetate and hexane=100:5 as the eluent to obtain a white powder (1.2 g).

¹ H NMR (CDCl ₃ ): 8.12 (1H, d), 7.55 (1H, t), 7.30 (1H, t), 7.10 (1H, d), 4.80 (1H, m), 4.75 (2H, s).

¹³ C NMR (CDCl ₃ ): 169.58, 167.02, 163.71, 150.85, 134.19, 131.96, 126.02, 123.83, 122.56, 75.29, 61.39.

MS(E1): 636 (M ⁺ _{sitostanol esters} ), 622 (M ⁺ _{campestanol esters} ), 594 (M ⁺ _s -15), 580 (M ⁺ _c -15).

IR (cm ^-1 ): 2934.5 (CH), 1764.03 (C=O), 1731.08 (C=O).

Synthesis of Example 3-Acetoxycholestanol Salicylate

(iii) Synthesis of cholestanol monochloroacetate

Cholesteranol (4 g) in monochloroacetate (10 ml) was stirred and heated at 120° C. for 3 hours. Cool to room temperature, add water (50ml) to the mixture after the reaction, collect the precipitate, wash with water (10ml×2), and vacuum-dry to obtain cholestanol monochloroacetate (4.5g, yield 96%).

¹ H NMR (CDCl ₃ ): 4.75 (1H, m), 4.00 (2H, s).

(iv) Synthesis of Acetoxycholestanol Acetylsalicylate

Sodium O-acetylsalicylate (0.5 g) and dry DMF (10 ml) were added to the cholestanol monochloroacetate (1.5 g) prepared above, and the mixture was stirred and heated at 140° C. for 1 hour. After cooling to room temperature, the mixture was poured into water (100ml), extracted with hexane (150ml), dried over sodium sulfate (20g), concentrated to recover the solvent and collected as a waxy solid. After drying, the crude product (2.8 g) was recrystallized from ethyl acetate:methanol solution to give a white powder (2.2 g).

¹ H NMR (CDCl ₃ ): 10.4 (1H, s), 7.90 (1H, d), 7.50 (1H, t), 7.00 (1H, d), 6.90 (1H, t), 4.80 (1H, m), 4.80(2H,s).

¹³ C NMR (CDCl ₃ ): 169.3, 166.8, 161.71, 136.1, 130.2, 119.3, 126.02, 117.6, 111.8, 75.5, 61.4.

MS (E1): 566 (M ⁺ ). IR (cm ^-1 ): 2930.5 (CH), 1759 (C=O), 1687.4 (C=O). Example 4-Acetoxyphytostanyl Salicyl Synthesis of esters

R = H, Me

(v) Synthesis of Acetoxy Phytostanyl Salicylate

Sodium salicylate (0.5 g) in dry DMF (10 ml) was added to the phytostanol monochloroacetate (1.5 g) prepared above, and the mixture was stirred and heated at 140° C. for 1 hour. After cooling to room temperature, the mixture was poured into water (100ml), and the off-white solid was collected and weighed dry (1.8g, yield 93.4%). After recrystallization from methanol, a white powder (1.5 g) was obtained.

¹ H NMR (CDCl ₃ ): 10.4 (1H, s), 7.90 (1H, d), 7.46 (1H, t), 7.00 (1H, d), 6.90 (1H, t), 4.80 (1H, m), 4.80(2H,s).

¹³ C NMR (CDCl ₃ ): 169.3, 166.8, 161.7, 136.1, 130.3, 131.96, 119.3, 117.6, 111.7, 75.5, 61.4.

MS(E1): 594 (M ⁺ _{sitostanol esters} ), 580 (M ⁺ _{campestanol esters} ).

IR (cm ^-1 ): 2934.0 (CH), 1755.9 (C=O), 1680.6 (C=O).

Synthesis of embodiment 5-acetoxy phytostanyl acetate

R = Me, H

Except replacing sodium salicylate with sodium acetate, other preparation methods are exactly the same as those shown in Example 4.

¹ H NMR (CDCl ₃ ): 4.90 (1H, m), 4.52 (2H, s), 2.15 (3H, s).

¹³ C NMR (CDCl ₃ ): 170.3, 167.3, 75.1, 60.9, 56.4, 56.1, 54.1.

MS(E1): 516 (M ⁺ _{sitostanol esters} ), 502 (M ⁺ _{campestanol esters} ).

IR (cm ^-1 ): 2933.0 (CH), 1765.9 (C=O), 1741.8 (C=O).

Synthesis of embodiment 6-acetoxy phytostanyl amino salicylate

R = H, Me

Except that the sodium salicylate was replaced by 4-aminosalicylate sodium, the other preparation methods were exactly the same as those shown in Example 4.

¹ H NMR (CDCl ₃ ): 10.6 (1H, s), 7.68 (1H, d), 6.30 (2H, s), 4.78 (1H, m), 4.72 (2H, s), 4.10 (2H, br).

¹³ C NMR (CDCl ₃ ): 169.0, 167.3, 163.7, 153.4, 132.0, 107.2, 102.4, 100.9, 75.3, 60.9, 56.4, 56.1, 54.1.

MS(E1): 609 (M ⁺ _{sitostanol esters} ), 595 (M ⁺ _{campestanol esters} ).

IR (cm ^-1 ): 3377.2(NH), 2933.1(CH), 1742(C=O), 1660(C=O).

Example 7 - Cholesterol lowering assay in rats

Male adult Sprague Dawley rats (-350 g) were exposed to a 12-hour light (0700-1900)/dark cycle and given a standard diet and ad libitum water prior to the start of the experiment.

The overnight fasted rats (12-16 hours) were subsequently divided into two groups: the control group (n=3) and the new compound (acetoxyphytostanyl salicylate designated as "FDC-2-4") ) group (n=3). At 0700, both groups received single-dose oral feeding. Blood samples were collected by cardiac puncture 10 hours after gavage. After gavage, animals were fasted. Throughout the experiment, water was given ad libitum. Blood obtained by cardiac puncture was collected in EDTA-coated tubes and centrifuged. [3H]cholesterol in plasma samples was analyzed radiometrically.

Administration of an oral formulation of a derivative of the invention to male adult Sprague Dawley rats comprising: 20 mg/kg sterol/stanol analogue, 1 mg unlabeled cholesterol in a 10% emulsion containing Intralipid Animals were fasted 12-16 hours before and 10 hours after oral feeding was completed. After oral gavage, blood samples were collected by cardiac puncture for 10 hours. Analysis of [3H]cholesterol in plasma samples obtained by centrifugation showed an inhibition rate of intestinal absorption of cholesterol.

Development of oral formulations of phytostanol [3H] cholesterol.

To solubilize exogenous radiolabeled cholesterol [25 μCi (approximately 227 ng) an emulsion containing 10% Intralipid (Kabi Pharmacia) was used.

Intralipid is a sterilized non-pyrogenic fat emulsion used as a pyrogen and essential fatty acid for administration. It consists of 10% Soybean Oil (refined natural product containing neutral triglycerides and unsaturated fatty acids), 1.2% egg yolk phospholipids, 2.25% glycerin and water. In addition, sodium hydroxide is added to adjust the pH value, so that the pH value of the final product is 8.0; the pH value range is 6.0-8.9. The major constituent fatty acids are linoleic acid (50%), oleic acid (26%), palmitic acid (10%), linolenic acid (9%) and stearic acid (3.5%). Rats in the control group were given the same dose of vehicle.

Cardiac puncture

Blood samples were collected by cardiac puncture 10 hours after oral feeding and centrifuged at 40C and 4,000 rpm for 15 minutes to prepare plasma. [3H]cholesterol in plasma samples was analyzed by radiometric method, and the rate of inhibition of cholesterol absorption of neosterol/stanol compounds compared with control group.

Figure 3 shows the results of FDC-2-4 compared to a control group, as compared to another potent cholesterol absorption inhibitor called VP4. The results clearly showed that FDC-2-4 was significantly stronger than the control group and VP4. The extent to which FDC-2-4 reduces cholesterol absorption is surprising and unexpected.

Inhibition of cyclooxygenase by embodiment 8-FDC-2-4

The purpose of this study was to determine whether pancreatic lipase/co-lipase-activated acetoxyphytostanyl salicylate derivatives of the present invention (designated "FDC-2-4") were able to inhibit cyclooxygenase (COX ) activity.

In summary, FDC-2-4 was activated by a 1:1 ratio of porcine pancreatic lipase and co-lipase (PL/C). Inhibition of cyclooxygenase was determined using a cyclooxygenase inhibitor (sheep) colorimetric screening assay. The conversion of arachidonic acid to prostaglandin H2 is monitored by a colorimetric reaction. PL/C-activated FDC-2-4 concentrations were 0, 0.45, and 4.5-μM in 1 μM hemoglobin pH 8.0 in 1M Tris-HCl buffer. The reaction was initiated after the addition of 100-μM arachidonic acid and 100-μM tetramethyl-p-phenylenediamine (TMPD-colorimetric matrix). The optical density of the dyed product was measured at 620 nm. Acetylsalicylic acid was used as a positive control in all experiments.

Pancrelipase/co-lipase activation: Enzyme reaction in a final volume of 50 mL of assay buffer (30 mM Tris-HCl, pH 8.0, 1.0 mM CaCl ₂ , 4 mM taurodeoxycholate), 0.312 mM friolein in the presence of Or in the absence of FDC-2-4 of the present invention. The solution was vortexed for 2 minutes and sonicated for 5 minutes before adding 2.5 mg porcine pancreatic lipase and 2.5 mg porcine co-lipase. The reaction was incubated at room temperature for 2 hours.

Cyclooxygenase Inhibition: Inhibition was determined using the (Ovine) Cyclooxygenase Inhibitor Colorimetric Screening Assay (Cayman Chemicals). The conversion of arachidonic acid to prostaglandin H2 is monitored by a colorimetric reaction. The cyclooxygenase was exposed to the inhibitor for 5 minutes. The reaction was initiated after the addition of 100-μM arachidonic acid and 100-μM tetramethyl-p-phenylenediamine (TMPD-colorimetric matrix). Five minutes after the initiation of the reaction, the optical density of the dyed product was measured at 620 nm. In 1 μM hemoglobin/1M Tris-HCl buffer at pH 8.0, the PL/C-activated FDC-2-4 concentrations were 0, 0.45, and 4.5-μM. The cyclooxygenase inhibitory effects of PL/C-activated FDC-2-4, non-PL/C-activated FDC-2-4 and the control (for adding FDC-2-4) were compared. Acetylsalicylic acid (ASA) was used as a positive control in all experiments. Statistical analysis was performed for all treatment groups using ANOVA and Tukey Posthoc test.

The cyclooxygenase inhibitory effects of PL/C-activated FDC-2-4, non-PL/C-activated FDC-2-4 and the control (for adding FDC-2-4) were compared. Compared with FDC-2-4 not activated with PL/C (20%) and control substance (5%), n=3, the inhibitory activity of 0.45uM PL/C-activated FDC-2-4 on COX-1 was 69%. The inhibitory activity of FDC-2-4 activated by 4.5μM PL/C on COX-2 was 88%.

Figures 4 and 5 show that FDC-2-4 can effectively inhibit two isoforms of cyclooxygenase (COX-1 and COX-2). PL/C-activated FDC-2-4 showed a stronger inhibitory effect than FDC-2-4 not activated with PL/C. These findings imply that FDC-2-4 is a potent anti-inflammatory agent activated by pancreatic lipase/co-lipase.

Without further elaboration, the foregoing is so sufficient to illustrate the invention that others may, by applying present or future knowledge, make the same modifications under the various conditions described or claimed herein.

Example 9—Using a solubilizer to dissolve water-insoluble FDC-2-4 (a phytostanyl analog with the chemical name of phytostanyl-O-acetylsalicylic acid oxyacetate, and a molecular weight of 631.66 g/ mol) formulated as 4mg/ml to 8mg/ml aqueous solution.

General Procedure: Dissolve the tested FDC-2-4 and solubilizer in a suitable organic solvent; heating if necessary. The organic solvent was recovered by nitrogen evaporation and water was added to the test tube. Visually inspect the mixture for turbidity or particle size. The resulting formulations were characterized using light microscopy, particle size measurements, zeta potential measurements and transmission electron microscopy.

Special method:

Solubility determination of FDC2-4 in various organic solvents:

1. Turn on the nitrogen evaporator and adjust the temperature to 65°C (the nitrogen evaporator without nitrogen is initially used as a hot water bath)

2. Put 4mg FDC2-4 into a 16×100mm test tube, and add 120mg Tween 80 with a glass pipette.

3. Prepare the reference substance, put 120mg Tween 80 into a 16×100mm test tube (there is no FDC2-4 in the reference substance)

4. Add 7 mL of isopropanol to each test tube using a 5 mL pipette. Tubes were covered with parafilm and vortexed thoroughly.

5. Place sample in nitrogen evaporator, cover closed - nitrogen evaporator initially used as 65°C hot water bath.

6. After approximately 45-60 minutes, FDC2-4 was dissolved in isopropanol - although the time varied (vortex the tube 1 or 2 times within this time frame to speed up the dissolution process).

7. Once the FDC2-4 has been dissolved by visual inspection, the parafilm on the test tube is removed, the temperature of the nitrogen evaporator is lowered to 60° C., and the nitrogen gas is turned on. The nitrogen pressure was initially about 7 psi for about 30 minutes to ensure that no solution was splashed out. After 30 minutes, the psi dropped to 15-20. The samples were placed in the nitrogen evaporator for a total of approximately 2.5 hours.

8. After 2.5 hours, close the nitrogen valve, but keep the nitrogen evaporator at 60°C. The samples were placed in a nitrogen evaporator. A sample was removed and 1 mL of water was quickly added. Samples were immediately vortexed until dissolved.

Visually inspect turbidity and particle size

Light microscopy, particle size and zeta potential energy measurements, and transmission electron microscopy were performed.

Table 1: Solubility profile of FDC2-4 in different solvents solvent Solvent amount Amount of FDC2-4 temperature dissolve? Butanol 5ml 4mg 65°C yes Chloroform - - room temperature yes DMSO 1ml 2mg 70°C yes DMSO 2ml 4mg 80°C yes Isopropanol 5ml 4mg 65°C yes

Table 2: Solubility profile of different solubilizers used to solubilize FDC2-4 into aqueous formulations. excipient Solubility of FDC2-4 proposal Pluronic P-85 Yes (FDC2-4:Pluronic ratio, 1:25-1:35) Organic solvents: chloroform and isopropanol Gelucire 44/14 Yes (FDC2-4:Gelucire ratio, 1:20-1:75) Organic solvent: chloroform Pluronic P85 and Pluronic F127 yes Organic solvent: chloroform Tween 80 yes Organic solvent: isopropanol

Results: After testing many surfactants and organic solvents, a more successful formulation was obtained using Tween 80. Solutions containing 4mg and 8mg of FDC-2-4 were prepared respectively, wherein Tween 80 was 120mg and 240mg (per milliliter of preparation).

Solution concentration: 1 mL preparation contains 4 mg FDC2-4 and 120 mg Tween 80 or 8 mg FDC2-4 and 240 mg Tween 80. Therefore, the mass ratio of FDC2-4 and Tween 80 in the preparation is 1:30.

Visual observation: the solution is clear and slightly yellow. No precipitate or particles appeared. Control and FDC2-4 looked identical.

Shown in Figure 6 is a digital image of 1 mL of an aqueous solution containing 4 mg of FDC2-4 and 120 mg of Tween 80, shown as a clear solution against a black background.

Light microscopy at 40x and 100x resolution: Microscopy at 40x and 100x magnification showed no difference in the amount or type of particulate matter between the selected FDC-2-4 formulation and the control.

Particle Size Measurement: The formulation particle size was measured in a Zetasizer 3000HS. The average of the peak heights analyzed by the amount in the 4 mg/mL formulation (particle size average) was 4.3 nm. This can be shown that the compound of the present invention and the selected solubilizer, Tween 80, form micelles, as shown in FIG. 7 . The average particle size of the 120mg/ml polysorbate 80 reference substance was 5.7nm. Figure 8 and Figure 9 show the similarity of the particle size distribution of the FDC-2-4 formulation and the reference product.

Conclusion: The compounds of the present invention are more soluble in aqueous solution.

Qualitative of the preparation of embodiment 10-embodiment 9 and cholesterol absorption experiment in rats

Rats were administered the formulation of the invention to measure cholesterol absorption in the presence of FDC-2-4.

Overview of the method for the rat fasting tracking model:

1. Prepare three formulations containing 4 mg FDC2-4, 120 mg polysorbate 80 and 1 mL water

2. Prepare three points of reference substance containing only 120mg polysorbate 80 ( ^Tween 80) and 1mL water

3. Samples were spiked with radiolabeled and unlabeled cholesterol and administered orally to Sprague-Dawley male fasted rats

4. After 10 hours, the rats were sacrificed by cardiac puncture and their blood was collected

5. Determination of [ ³ H]cholesterol (radiolabeled cholesterol) plasma concentration.

in conclusion:

Table 3: [ ³ H]cholesterol plasma concentrations 10 hours after a single oral ingestion of [ ³ H]cholesterol, unlabeled cholesterol, and FDC 2-4 combined with Tween 80 administered to Sprague-Dawley male fasted rats . compound Dose (mg/kg) Time after administration (hours) [ ³ H]cholesterol plasma concentration (pg/ml) percent change from control Tween 80 control (n=5) - 10 hours 4980+/-968 ----- FDC2-4/Tween 80 (n=6) 10 10 hours 2708+/-1379** -45.6%

^** p<0.05 vs. Tween 80 control (Tween control substance is a mixture of 120 and 240 mg). Rats weighed between 360-400 g.

Compared with the control group, administration of the 4 mg/ml preparation to rats resulted in 45.6% inhibition of cholesterol absorption.

Concentration values of 4 mg/mL-8 mg/mL were chosen because a similar water-soluble phytostanol ("FM-VP4") known to the inventors exhibited the best cholesterol-suppressing effect at a dose of 10 mg/kg- 20mg/kg. The weight of the rats used was 400 g due to the dose of 4 mg-8 mg. In the rat fasting follower model, rats are administered 1 mL of drug; for this purpose, 4 mg/mL-8 mg/mL FDC 2-4 water-soluble formulations are prepared.

references:

1. Law M.R., Wald N.J., Wu., Hacksaw ZA., Bailey A.; Systemic underestimation of association between serum cholesterol concentration and ischemic heart disease in Observational studies: Data from BUPA Study: Br.Med.J.1994:338:3063

2. Law M.R., Wald N.J., Thompson S.G.; By how much and how quickly does reduction in serum cholesterol concentration lower risk of ischemic heart disease? Br.Med.J.1994;308:367-373

3. La Rosa J.C., Hunninghake D..Bush D.et al.; The cholesterol facts: A summary of the evidence relating dietary fats, serum cholesterol and coronary heart disease: A joint statement by the American Heart Association and the National Heart, Lung Blood Institute. Circulation 1990;81:1721-1733

4. Havel R.J., Rapaport E.. Drug Therapy: Management of Primary Hyperlipidemia. New England Journal of Medicine, 1995; 332: 1491-1498

5. Kuccodkar et al.; Effects of plant sterols oncholesterol metabolism. Atherosclerosis, 1976; 23:239-248

6. Lees R.S., Lees A.M. Effects of sitosteroltherapy on plasma lipid and lipoprotein concentrations. In: Greten H (Ed) Lipoprotein Metabolism. Springer-Verlag, Berlin, Heidelberg, New York, 1976: 119-124

7. Lees A.M., Mok H.Y.I., Lees R.S., McCluskey M.A., Grundy S.M. Plant sterols as cholesterol-lowering agents: clinical trials in patients with hypercholesterolemia and studies of sterol balance. New 1975. Atherosclerosis. , 336, 973-9 (1999)

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Claims (60)

1. the compound that comprises sterol or stanols, comprising its biologically acceptable salt, this compound has one or more following molecular formula:

a)R ₂-(CH2) _n-CO-OR

b)R ₂-R

c)R ₂-CO-CO-OR

d)

Wherein R is sterol or stanols part, R ₂Aryl-alkanoic derived from Whitfield's ointment or n=1-5.

2. the compound of claim 1, wherein sterol is selected from Sitosterol, campesterol, Stigmasterol, brassicasterol (comprising Neospongosterol,dihydro-), desmosterol, spongosterol, poriferasterol, clionasterol, ergosterol, stercorin, codisterol, isofucosterol, fucosterol, Clerosterol, neural sterol, 7-alkene cholesterol, stellasterol, spinasterol, spinasterol, peposterol, avenasterol, different avenasterol, stercorin, pollen sterol, and cholesterol.

3. the compound of claim 1, wherein stanols is selected from sitostanol, campestanol, stigmastanol, vegetable seeds stanols (comprising dihydro vegetable seeds stanols), chain stanols, the sponge stanols, the porous stanols is worn shellfish sponge stanols, ergostanol, coprostanol, codistanol, different rock algae stanols, rock algae stanols, red paulownia stanols, neural stanols, lathosterol, star fish stanols, the spinach stanols, spinach stanols (chondrillastanol), pepostanol, oat stanols, different oat stanols, coprostanol, pollen stanols, and Dihydrocholesterol.

4. the compound of claim 1, wherein sterol and stanols are natural or synthetic.

5. the compound of claim 1, wherein sterol and stanols are their any one isomer.

6. the compound of claim 1, wherein aryl-alkanoic is selected from aromatic acid, fragrant acetate, fragrant propionic acid, fragrant butyric acid and fragrant valeric acid.

7. the compound of claim 1, wherein aryl-alkanoic is selected from acemetacin, Fenazox, Bendazac, indomethacin glucosamide, oxametacin, alminoprofen, Ibuprofen BP/EP, Ketoprofen, flurbiprofen, fenoprofen, the Ao Shapu piperazine, bumadizone, Butibufen, fenbufen, and xenbucin.

8. the compound of claim 1, wherein Whitfield's ointment is selected from acetylsalicylic acid (ASA), ASA aluminium, ASA sodium, the ASA oxyacetate, Whitfield's ointment, Whitfield's ointment oxyacetate, saligenin, salicortin, Tremulacine, choline magnesium trisalicylate, diflunisal, etherylate, Fosfosal, salol, salsalate, salacetamide, the acid of salicyl salicylyl, sulfasalazine, and olsalazine (olsalazone).

9. the compound of claim 1 has following structural formula:

Wherein R is selected from H and CH ₃, R ₁, R ₂, R ₃, R ₄, R ₅Be selected from OH independently of one another, ethanoyl, halogen (Cl, Br, I or F) and have the moieties of 1-5 carbon atom.

10. the compound of claim 1 has following structural formula:

Wherein R is selected from H and CH ₃, R ₁, R ₂, R ₃, R ₄, R ₅Be selected from OH independently of one another, ethanoyl, halogen (Cl, Br, I or F) and have the moieties of 1-5 carbon atom.

11. the compound of claim 1 has following structural formula:

R wherein ₁, R ₂, R ₃, R ₄, R ₅Be selected from OH independently of one another, ethanoyl, halogen (Cl, Br, I or F) and have the moieties of 1-5 carbon atom.

12. the compound of claim 1 has following structural formula:

R wherein ₁, R ₂, R ₃, R ₄, R ₅Be selected from OH independently of one another, ethanoyl, halogen (Cl, Br, I or F) and have the moieties of 1-5 carbon atom.

13. the compound of claim 1, be selected from plant gonane base acetylsalicylate, plant gonane base salicylate, acetoxyl group plant gonane base acetylsalicylate, acetoxyl group plant gonane base salicylate, acetoxyl group plant gonane yl acetate, cholestane base salicylate, acetoxyl group cholestane base salicylate and acetoxyl group plant gonane base aminosallcylic acid ester.

14. the compound of claim 1 has structural formula:

Wherein R is selected from H and CH ₃

15. the compound of claim 1 has structural formula:

Wherein R is selected from H and CH ₃

16. the compound of claim 1 has structural formula:

Wherein R is selected from H and CH ₃

17. the compound of claim 1 has structural formula:

Wherein R is selected from H and CH ₃

18. the compound of claim 1 has structural formula:

Wherein R is selected from H and CH ₃

19. the compound of claim 1 has structural formula:

20. the compound of claim 1 has structural formula:

21. the compound of claim 1 has structural formula:

22. a pharmaceutical composition is used for the treatment of or prevents cardiovascular diseases and the method for the situation of hiding, and includes, without being limited to atherosclerosis, blood cholesterol is too high, hyperlipidaemia, hypertension, thrombosis, coronary artery disease is used for the treatment of and reduces inflammation, comprises the coronary pluques inflammation, bacterial inflammation, viral inflammation and the inflammation that accompanies with severe pain and surgical operation, described composition comprises at least a compound, this compound has one or more following molecular formula:

a)R ₂-(CH2) _n-CO-OR

b)R ₂-R

c)R ₂-CO-CO-OR

d)

Wherein R is sterol or stanols part, R ₂Derived from Whitfield's ointment or aryl-alkanoic and n=1-5, comprise its all biologically acceptable salt and pharmaceutically acceptable carriers.

23. the composition of claim 22, wherein aryl-alkanoic is selected from aromatic acid, fragrant acetate, fragrant propionic acid, fragrant butyric acid and fragrant valeric acid.

24. the composition of claim 22, wherein aryl-alkanoic is selected from acemetacin, Fenazox, Bendazac, indomethacin glucosamide, oxametacin, alminoprofen, Ibuprofen BP/EP, Ketoprofen, flurbiprofen, fenoprofen, the Ao Shapu piperazine, bumadizone, Butibufen, fenbufen, and xenbucin.

25. the composition of claim 22, wherein Whitfield's ointment is selected from acetylsalicylic acid (ASA), ASA aluminium, ASA sodium, the ASA oxyacetate, Whitfield's ointment, Whitfield's ointment oxyacetate, saligenin, salicortin, Tremulacine, choline magnesium trisalicylate, diflunisal, etherylate, Fosfosal, salol, salsalate, salacetamide, the acid of salicyl salicylyl, sulfasalazine, and olsalazine (olsalazone).

26. the compound of claim 22, wherein compound has following molecular formula:

Wherein R is selected from H and CH ₃, R ₁, R ₂, R ₃, R ₄, R ₅Be selected from OH independently of one another, ethanoyl, halogen (Cl, Br, I or F) and have the moieties of 1-5 carbon atom.

27. the compound of claim 22, wherein compound has following molecular formula:

Wherein R is selected from H and CH ₃, R ₁, R ₂, R ₃, R ₄, R ₅Be selected from OH independently of one another, ethanoyl, halogen (Cl, Br, I or F) and have the moieties of 1-5 carbon atom.

28. the composition of claim 22, wherein compound has following molecular formula:

R wherein ₁, R ₂, R ₃, R ₄, R ₅Be selected from OH independently of one another, ethanoyl, halogen (Cl, Br, I or F) and have the moieties of 1-5 carbon atom.

29. the composition of claim 22, compound wherein has following molecular formula:

R wherein ₁, R ₂, R ₃, R ₄, R ₅Be selected from OH independently of one another, ethanoyl, halogen (Cl, Br, I or F) and have the moieties of 1-5 carbon atom.

30. the composition of claim 22, wherein compound is selected from plant gonane base acetylsalicylate, plant gonane base salicylate, acetoxyl group plant gonane base acetylsalicylate, acetoxyl group plant gonane base salicylate, acetoxyl group plant gonane yl acetate, cholestane base salicylate, acetoxyl group cholestane base salicylate and acetoxyl group plant gonane base aminosallcylic acid ester.

31. a method for the treatment of or preventing the cardiovascular diseases and the situation of hiding thereof includes, without being limited to atherosclerosis, blood cholesterol is too high, hyperlipidaemia, hypertension, thrombosis, coronary artery disease, be used for the treatment of and reduce inflammation, comprise the coronary pluques inflammation, bacterial inflammation, viral inflammation and the inflammation that accompanies with severe pain and surgical operation, comprising giving animal one or more compounds nontoxic and the treatment significant quantity, it is as follows to have molecular formula:

a)R ₂-(CH2) _n-CO-OR

b)R ₂-R

c)R ₂-CO-CO-OR

d)

Wherein R is sterol or stanols part, R ₂Derived from Whitfield's ointment or aryl-alkanoic and n=1-5, or its any biologically acceptable salt.

32. the method for claim 31, wherein aryl-alkanoic is selected from aromatic acid, fragrant acetate, fragrant propionic acid, fragrant butyric acid and fragrant valeric acid.

33. the method for claim 31, wherein aryl-alkanoic is selected from acemetacin, Fenazox, Bendazac, indomethacin glucosamide, oxametacin, alminoprofen, Ibuprofen BP/EP, Ketoprofen, flurbiprofen, fenoprofen, the Ao Shapu piperazine, bumadizone, Butibufen, fenbufen, and xenbucin.

34. the method for claim 31, wherein Whitfield's ointment is selected from acetylsalicylic acid (ASA), ASA aluminium, ASA sodium, the ASA oxyacetate, Whitfield's ointment, Whitfield's ointment oxyacetate, saligenin, salicortin, Tremulacine, choline magnesium trisalicylate, diflunisal, etherylate, Fosfosal, salol, salsalate, salacetamide, the acid of salicyl salicylyl, sulfasalazine, and olsalazine (olsalazone).

35. the method for claim 31, wherein compound has molecular formula:

Wherein R is selected from HCH ₃, R ₁, R ₂, R ₃, R ₄, R ₅Be selected from OH independently of one another, ethanoyl, halogen (Cl, Br, I or F) and have the moieties of 1-5 carbon atom.

36. the method for claim 31, wherein compound has molecular formula:

Wherein R is selected from H and CH ₃, R ₁, R ₂, R ₃, R ₄, R ₅Be selected from OH independently of one another, ethanoyl, halogen (Cl, Br, I or F) and have the moieties of 1-5 carbon atom.

37. the method for claim 31, wherein compound has molecular formula:

R wherein ₁, R ₂, R ₃, R ₄, R ₅Be selected from OH independently of one another, ethanoyl, halogen (Cl, Br, I or F) and have the moieties of 1-5 carbon atom.

38. the method for claim 31, wherein compound has molecular formula:

R wherein ₁, R ₂, R ₃, R ₄, R ₅Be selected from OH independently of one another, ethanoyl, halogen (Cl, Br, I or F) and have the moieties of 1-5 carbon atom.

39. compound comprises sterol or stanols, and biologically acceptable salt, has one or more following molecular formula:

a)R ₂-(CH2) _n-CO-OR

b)R ₂-R

c)R ₂-CO-CO-OR

d)

Wherein R is sterol or stanols part, R ₂Derived from the aryl-alkanoic of Whitfield's ointment or n=1-5, almost completely be dissolved in fat and be dissolved in the aqueous phase solution mutually or by following manner,

I) compound and selected solubilizing agent are mixed;

Ii) add at least a organic solvent;

Iii) compound and solubilizing agent are dissolved in organic solvent; With

Iv) therefrom evaporate organic solvent.

40. the compound of claim 39, wherein to be selected from those hydrophilic coefficients (" HLB ") be 12 or greater than 12 compound to solubilizing agent.

41. the compound of claim 39, wherein to be selected from those hydrophilic coefficients (" HLB ") be 8 or less than 8 compound to solubilizing agent.

42. the compound of claim 39, wherein solubilizing agent is selected from tensio-active agent such as tween 80 and polysorbate60, poly-(ethylene oxide)-poly-(propylene oxide) three-block copolymer surfactant such as PluronicP-85, PluronicF-127, and PluronicF-108, and macrogolycerol (C ₈-C ₁₈Glyerides; Fatty Acids C8-C18Ethoxylated) as Gelcire ^TM

43. the compound of claim 39, wherein organic solvent is selected from halogenation aliphatic hydrocarbon and all side chain and straight chain C ₃-C ₅Fatty Alcohol(C12-C14 and C12-C18).

44. the compound of claim 39, organic solvent wherein are selected from propyl alcohol, Virahol, butanols, isopropylcarbinol, amylalcohol, primary isoamyl alcohol, chloroform, methylene dichloride (carrene) and methyl-sulphoxide (" DMSO ").

45. the compound of claim 39, step I ii) in compound use heating or ultrasonic method to be dissolved in the solvent.

46. the compound of claim 39, wherein step I v) in the evaporation organic solvent use vaporized nitrogen or rotary evaporation.

47. dissolve the method for one or more following compounds in the fat base or the aqueous solution, compound comprises sterol or stanols, and biologically acceptable salt, has one or more following molecular formula:

a)R ₂-(CH2) _n-CO-OR

b)R ₂-R

c)R ₂-CO-CO-OR

d)

Wherein R is sterol or stanols part, R ₂Derived from the aryl-alkanoic of Whitfield's ointment or n=1-5, comprising:

I) compound and selected solubilizing agent are mixed;

Ii) add at least a organic solvent in addition;

Iii) compound and solubilizing agent are dissolved in organic solvent; With

Iv) therefrom evaporate organic solvent.

48. the method for claim 47, wherein to be selected from those hydrophilic coefficients (" HLB ") be 12 or greater than 12 compound to solubilizing agent.

49. the method for claim 47, wherein to be selected from those hydrophilic coefficients (" HLB ") be 8 or less than 8 compound to solubilizing agent.

50. the method for claim 47, wherein solubilizing agent is selected from tensio-active agent such as tween 80 and polysorbate60, poly-(ethylene oxide)-poly-(propylene oxide) three-block copolymer surfactant such as PluronicP-85, PluronicF-127 and PluronicF-108, and macrogolycerol (C ₈-C ₁₈Glycerides; Fatty Acid C8-C18Ethoxylated) as Gelcire ^TM

51. the method for claim 47, wherein organic solvent is selected from halogenation aliphatic hydrocarbon and all side chain and straight chain C ₃-C ₅Fatty Alcohol(C12-C14 and C12-C18).

52. the method for claim 47, wherein organic solvent is selected from propyl alcohol, Virahol, butanols, isopropylcarbinol, amylalcohol, primary isoamyl alcohol, chloroform, methylene dichloride (carrene) and methyl-sulphoxide.

53. the method for claim 47, wherein step I ii) in compound use heating or ultrasonic method to be dissolved in the solvent.

54. the method for claim 47, wherein the v) middle evaporation organic solvent of step I uses vaporized nitrogen or rotary evaporation.

55. the method for claim 47, wherein solubilizing agent is tween 80, and organic solvent is a Virahol.

56. the method for claim 55, wherein step I ii) in, compound is in the high dissolving during to 65 ℃ of heating.

57. the method for claim 55 is wherein used vaporized nitrogen in step I in v).

58. the method for claim 47, wherein solubilizing agent is tween 80, and organic solvent is a chloroform.

59. the method for claim 58, wherein step I ii) in, compound is at room temperature or approximately room temperature condition dissolving down.

60. the method for claim 58 is wherein used rotary evaporation in step I in v).

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