FIELD OF THE INVENTION

This invention relates to amino-substituted furostenes, and derivatives and analogs thereof, including compositions and methods of making and using these compounds.

This work was supported in part by the National Institute of Health, under Grants NIH/NIDDKD #30-1-8410. Through these grants, the U.S. Government has rights in this invention.

BACKGROUND OF THE INVENTION Cholesterol can prevent a reduction in the fluidity of cells and tissues by preventing the fatty acyl chains of the membrane lipids from binding with each other. See, e.g., Darnell, et al. , Molecular Cell Biology, Scientific American Books, New York: 576-577 (1986) . Cholesterol synthesis occurs according to an enzyme-mediated biosynthetic pathway, in which the rate limiting enzyme is believed to be hydroxymethylglutaryl-Co-A reductase

(hereinafter "HMG-CoA reductase") . See, for example, Javitt,

U.S. Pat. No. 4,427,668. This enzyme catalyzes the formation of mevalonic acid, a cholesterol precursor, from hydroxymethyl glutaric acid. Cholesterol production is therefore believed to be decreased by inhibiting HMG-CoA reductase. See Goodman & Gilman, The Pharmacological Basis of Therapeutics. 7th ed., Macmillan, New York: 841-843 (1985) ; and Kandustsch et al . Science 201: 498 (1978) . Cholesterol is transported through the bloodstream by a carrier, due to its insolubility. The most clinically signifi¬ cant carrier is low-density lipoprotein (LD ) . See Darnell, supra. , at 648-650. Most cells manufacture specific receptors that will bind LDL to the cell membrane. Thus, it is believed that cholesterol accumulation (e.g., cellular uptake) and synthesis can also be regulated by interfering with LDL binding.

The excessive accumulation of cholesterol has been implicat¬ ed as the primary causal factor in a number of diseases, including arteriosclerosis, a disease of the arteries, the most important type of which is atherosclerosis, which is character¬ ized by an abnormal hardening and thickening of the arterial

walls due to the accumulation and deposition of fatty materials, including cholesterol. Atherosclerosis, in turn, can lead to thrombosis and infarction, and is involved in several other related diseases, including hypertension, hypothyroidism, obstructive jaundice, nephrosis, diabetes mellitus, pancreatitis, multiple myeloma, porphyria and obstructive liver disease. See, e.g., The Merck Manual 15th edition, Chs. 85 and 25-30 (1987) .

Although it was initially thought that the amount of cholesterol in tissues regulated its own production, more recent evidence indicates that certain precursors and metabolites of cholesterol are much more potent inhibitors of the rate-limiting enzyme HMG-CoA reductase. Many of these potent inhibitors have been classified as "oxysterols" such as 25-hydroxycholesterol and 26-hydroxycholesterol. The latter compound was previously shown to be naturally occurring and also to be an inhibitor of HMG-CoA reductase. See, e.g., Javitt, U.S. Patent No. 4,427,668. The product 26-aminocholesterol was also found to be an even more potent inhibitor of HMG-CoA reductase. See, e.g., Javitt, U.S. Patent No. 4,939,134. It should also be noted that there is no reliable correla¬ tion between liver or plasma cholesterol levels and the amount of HMG-CoA reductase and LDL receptor binding. For example, the known drug mevinolin has been observed to inhibit hepatic cholesterol synthesis even though it increases the amount of HMG-CoA reductase. The enzyme is less active in the presence of the drug, which is believed to modify mevinolin\' s three-dimen¬ sional conformation. From cell culture studies it is known that this drug (and others) also upregulates LDL receptors in fibroblasts and other non-hepatic cells. In contrast, 26-hydroxycholesterol and 26-amino-cholesterol have been found to inhibit HMG-CoA reductase activity in fibroblasts and reduce LDL binding, thereby decreasing the rate at which tissue cells can produce and accumulate cholesterol . See also, Javitt et al J. Lipid Res. , 26:950-954 (1985) ; Lorenzo et al FEBS Letters 218:77-80 (1987) ; and U.S. Patent No. 4,939,134.

The drugs currently in use for the inhibition of cholesterol synthesis have an impact primarily on the liver, and are believed

to function by causing upregulation of the LDL receptors of the liver (to increase liver uptake of cholesterol and decrease serum levels) or by inhibiting HMG-CoA reductase, so that cholesterol production is reduced. For example, 26-hydroxycholesterol and 26-aminocholesterol have been found to inhibit HMG-CoA reductase activity in fibroblasts and reduce LDL binding, thereby decreasing the rate at which tissue cells can produce and accumulate cholesterol. See Javitt et al J. Lipid Res. , 26: 950-954 (1985) ; Lorenzo et al FEBS Letters 218:77-80 (1987) ; and U.S. patent No. 4,939,134.

Representative of known drugs that are currently in use, which cause the upregulation of LDL receptors in the liver are

^\' mevinolin and cholestyramine. Mevinolin, which is an allosteric inhibitor of HMG-CoA reductase, achieves this regulation effect by inhibiting cholesterol synthesis in the liver. This stimu¬ lates increased LDL receptor activity in the liver, causing hepatic uptake of cholesterol, which in turn reduces plasma cholesterol. Cholestyramine increases the need for bile acid synthesis from cholesterol in the liver, which in some individu- als will result in upregulation of LDL receptors.

Drugs like cholestyramine are not absorbed in the intestine and also bind numerous other drugs used for cardiovascular diseases (such as chlorothiazide, phenylbutazone, phenobarbital, anticoagulants, digitalis) as well as fat soluble vitamins (A, D, E and K) . Consequently, these drugs and vitamins must be taken before and after administration of cholestyramine, or the effect of these drugs or vitamins is lost. Accordingly, it would be preferable to have an alternative drug to cholestyramine that acts systemically and does not deplete the body of concomitantly administered drugs, as well as fat-soluble vitamins. See Goodman and Gilman, supra, at 834-847.

Diosgenin, a natural plant sterol, is the starting material for the production of many different synthesis sterols used as drugs. In addition to the basic four ring structure that is common to diosgenin, and to all genins, a 5th ring, referred to as a furane ring, and a 6th ring, that can be referred to as a spiroketal ring, also are present. Virtually all- steroids used as medications that are produced from diosgenin require elimina-

tion of the 5th and 6th ring and shortening of the hydrocarbon side chain. Opening of the 6th ring only has not been taught or suggested for use as a drug.

Julian, U.S. patent No. 3,019,220, discloses isolation of sapogenins, including diosgenin, from plant material. Staba, U.S. patent No. 3,628,287, discloses the use of tissue culture of Dioscera seeds for the production and isolation of diosgenin, for use in the further synthesis of corticosteroid hormones and derivatives, but Staba does not provide any specific steroid syntheses from diosgenin. Eyssen, U.S. patent No. 3,640,848 discloses microbial reduction of delta steroids, including diosgenin, as presenting a Δ ⁵ -stigmastene structure, to yield 5, 6-dihydro-5-/3 products. Jeger et al, U.S. patent No. 3,723,418, discloses the production of 17/5-hydroxy-androstanes by treating an α_, -oxido-steroid ketone with triakylstannine in the presence of UV light and a suitable solvent using diosgenin as a starting material. While Pettit and Bowyer, J. Amer. Chem. Soc. 25:84-86 (1960) discloses the opening of ring 6 of a diosgenin to form 3jδ-acetoxy-5-furostene-26-ethylenethioketal, this and related compounds are not taught or suggested to be used as a drug, nor are amino substituted furostene compounds taught or suggested. One furostene compound, unsubstituted 26-amino- furost-5en-3j8-ol may be disclosed by Tschesche et al. Phytochem- istry, 14:435-438 (1975) ; but no teaching or suggestion is made for its use in inhibiting cholesterol synthesis or accumulation. Accordingly, there is a need to provide additional anti- cholesterol or anti-hyperlipoproteinemic drugs that function systemically and do not suffer from the negative side effects of known anti-cholesterol drugs or intestinal cholesterol clearing drugs, such as cholestyramine.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome the deficiencies of the related art.

It is also an object of the present invention to provide novel 26-aminofurostene compounds other than unsubstituted 26- amino-furost-5-en-33-ol, compositions and methods for inhibiting cholesterol synthesis, uptake and accumulation in cells and tissues, in vitro, in situ, and in vivo.

It is another object of the present invention to provide analogs and derivatives of 2β-aminofurostene compounds. 3jS-ol, of the present invention.

It is still another object of the present invention to reduce abnormally high levels of serum cholesterol.

Yet another object of the present invention is to provide compounds, compositions and methods for the prevention and treatment of atherosclerosis and other diseases associated with excessive cholesterol uptake, accumulation and/or synthesis, by administering an effective cholesterol-inhibiting amount of an aminofurostene, and a pharmaceutically acceptable carrier. A further object of the present invention is to provide compounds, compositions and methods for regulating cholesterol synthesis, uptake, and accumulation in a subject, including a mammal or a bird, such as a human.

A further object of the present invention is to provide pharmaceutical preparations containing 26-aminofurostenes according to formula (I) in an amount sufficient for use in one or more of the embodiments of this invention.

According to one aspect of the present invention, aminofuro¬ stene compounds are provided according to formula (I) :

wherein (a) R ¹ is hydroxy or keto, such that at least one of C3

and C4 and C4 and C5 form a double bond when R ¹ is keto and C5 and C6 form a double bond when R ¹ is hydroxy; (b) R ² is sulfur, oxygen or nitrogen; and (c) R ³ is a C1-C3 alcohol, aldehyde or acid, or a pharmaceutically acceptable ester, ether, sulfate, carbonate, glucuronide, cyclodextrin complex or salt thereof, which compounds provide inhibitory effects on the uptake, synthesis and/or accumulation of cholesterol in cells and/or tissues of an animal.

In a preferred embodiment, R ¹ is hydroxyl. In another preferred embodiment, R ² is oxygen. In still another preferred embodiment, R ⁵ is selected from a methyl, a methyl alcohol, a formaldehyde and a formic acid.

Compounds according to formula (I) can be selected from substituted 26-amino-furost-5-en-3 -ols, and substituted and unsubstituted 26 -amino- furost- 5 - en- 3 α - o 1 s , 26-amino-furost-4-en-3-ones, 26-amino-furost- 4, 6-dien-3-ones, wherein 26-amino-furost-5-en-3/3-ol is more preferred.

According to another aspect of the present invention, a pharmaceutical composition is provided, comprising a compound according to formula (I) , or a pharmaceutically acceptable ester, ether, sulfate, carbonate, glucuronide or salt thereof, and a pharmaceutically acceptable carrier.

According to still another aspect of the present invention, a pharmaceutical composition of the present invention further comprises a drug selected from a cardiac glycoside, an antidys- rythinic, a diuretic, an antihypertensive, an antianginal, an anticoagulant, and a lipid lowering agent.

According to a further aspect of the present invention, a method for inhibiting cholesterol by reducing the rate of cholesterol synthesis uptake or accumulation by cells or tissue in an animal is provided, comprising administering an effective cholesterol inhibiting amount of a pharmaceutical composition comprising a compound according to formula (I) . In a preferred embodiment, the composition is administered to provide the compound in an amount ranging from about 0.1 to 3 mg/kg, and also preferably in an amount ranging from about 0.15 to 1 mg/kg per 24 hour period. In another preferred embodiment, the animal is selected from a mammal and a bird, and, more preferably the

mammal is a human.

In still another aspect of the present invention, a method is provided for treating a patient suffering from a pathology involving elevated cholesterol synthesis uptake or accumulation in cells or a tissue of a subject, comprising administering to the subject an amount effective to inhibit the cholesterol synthesis uptake or accumulation of a pharmaceutical composition comprising a compound according to formula (I) . In a preferred embodiment, the pathology is selected from arteriosclerosis, atherosclerosis, hypertension, hypothyroidism, obstructive jaundice, nephrosis, diabetes mellitus, pancreatitis, multiple myeloma, porphyria and obstructive liver disease.

Other objects of the present invention will be apparent to skilled practitioners from the following detailed description and examples of the present invention provided below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to substituted and unsubsti- tuted 26-amino-substituted furostenes. Additionally, the present invention relates to compositions and methods of making and using these compounds. These compounds have been unexpectedly discovered to provide anti-cholesterol activity, by inhibiting cholesterol uptake, synthesis and/or accumulation in cells and tissues of an animal, such as a human.

In making this discovery, it has been found that, contrary to what was taught or suggested in the related art, the side chain common to naturally occurring cholesterol-type steroids, as well as synthetic analogs, is not essential to providing the biological activity of inhibiting cholesterol synthesis, accumulation and uptake, as previously taught or suggested in the art. It has also been discovered that the furane or 5th ring of diosgenin need not be eliminated to produce a series of potent biologic steroids as 26-amino furostenes according to the present invention.

Thus, the novel set of 26-aminofurostene compounds in accordance with the present invention have been discovered that have useful therapeutic properties.

26-aminofurostene compounds according to the present

invention include compounds according to formula (I) :

wherein (a) R ¹ is hydroxy or keto, such that at least one of C3 and C4 and C4 and C5 form a double bond when R ¹ is keto and C5 and C6 form a double bond when R ¹ is hydroxy; (b) R ² is sulfur, oxygen or nitrogen; and (c) R ³ is a C1-C3 alcohol, aldehyde or acid, or a pharmaceutically acceptable ester, ether, sulfate, carbonate, glucuronide, cyclodextrin complex or salt thereof, which provide inhibitory effects for the synthesis and/or accumulation of cholesterol in cells and/or tissues of an animal. 26-aminofurostene compounds of the present invention according to formula (I) may also include mono and di-esters, sulfates, carbonates, glucuronides and ether or fluoro substitu¬ tions of such 26-aminofurostenes, which can be readily prepared according to known method steps of pharmaceutical technology, based on the teaching and guidance presented in the present specification. See, e.g., The U. S . Pharmacopeia, the National Formulary, 22nd Edition, U.S. Pharmacopeial Convention, Inc., Rockville, Maryland, and Suppl. 1-6, (1989); and Parrott, Pharmaceutical Technology, Burgess Publ. Co., Minneapolis, Minn. (1970) .

According to the present invention, the C3-C5 and C5-C6 containing cyclic rings can be cyclohexane or cyclohexene, wherein (a) R ¹ is hydroxy or keto, such that at least one of C3 and C4 and C4 and C5 form a double bond when R ¹ is keto and C5 and C6 form a double bond when R ¹ is hydroxy; (b) R ² is sulfur, oxygen or nitrogen; and (c) R ³ is a C1-C3 alcohol, aldehyde or acid, or a pharmaceutically acceptable ester, ether, sulfate, carbonate, glucuronide, cyclodextrin complex or salt thereof, to provide aminofurost-5-enes, aminofurost-4-enes, and aminofurost-

4-,6-dienes, according to formula (I) .

Such aminofurostenes of the present invention also include 3-keto and 3 -hydroxy alkyl-substituted aminofurostenes, wherein the 27-methyl group can be an alcohol, an aldehyde or an acid, such that R ³ is a C1-C3 alkyl, alcohol, aldehyde or acid. Additionally, aminofurostenes of the present invention can include 26-aminofurostene compounds having substituted 28-methyl or 28-ethyl groups, wherein R ³ is a C2-C3 alkyl, alcohol, aldehyde or acid, including an ethyl or propyl alcohol, aldehyde or acid. It is preferred that R ¹ is hydroxyl such that C5 and C6 form a double bond, as in aminofurost-5-ene-3-ols .

Additionally, 26-aminofurostenes of the present invention, as described herein, include sulfur and nitrogen substitutions for oxygen, in the heterocyclic ring, wherein R ² is preferably oxygen, but where R ² can also be sulfur or nitrogen.

Preferred compounds according to formula (I) can be selected from alkyl substituted 26-amino-furost-5-en- 3/3-ols, unsubsti- tuted or alkylsubstituted 26-amino-furost- 5-en-3α-ols, substi¬ tuted or unsubstituted amino-furost- 4-en-3-ones, and unsubsti- tuted or alkylsubstituted 26- amino-furost-4, 6-dien-3-ones, which compounds can include but are not limited to a compound selected from 26-amino-furost-5-en-3/3-ol, 26-amino-furost-5-en-3α_-ol, 26-amino-furost-5-en-27, 3/3-diol, 26-amino-furost-5-en- -27, 3/3-diol, 26-amino-furost-5-en-27-al-33-ol, 26-amino-furost-5-en-27-al-3α_-ol, 26-amino-furost-5-en-27-oic-33-ol, 26-amino-furost-5-en-27-oic-3α_-ol, 28-methyl, 26-amino-furost-5-en-3/3-ol , 28-methyl, 26-amino-furost-5-en-3α_-ol,

28-methyl, 26-amino-furost-5-en-28 , 3/3-diol, 28-methyl,26-amino-furost-5-en-28 , 3α_-diol, 28-methyl ,26-amino-furost-5-en-28-al-3/3-ol, 28-methyl , 26-amino-furost-5-en-28-al-3o;-ol, 28-methyl , 26-amino-furost-5-en-28-oic-3/3-ol, 28-methyl, 26-amino-furost-5-en-28-oic-3α_-ol , 28-ethyl,26-amino-furost-5-en-3/3-ol , 28-ethyl ,26-amino-furost-5-en-3α_-ol ,

28 - ethyl , 26 - amino- furost - 5 - en- 29 , 3/3 - diol ,

28-ethyl,26-amino-furost-5-en-29 , 3α_-diol,

28-ethyl, 26-amino-furost-5-en-29-al-3/3-ol,

28-ethyl,26-amino-furost-5-en-29-al-3α-ol, 28-ethyl,26-amino-furost-5-en-29-oic-3/3-ol,

28-ethyl,26-amino-furost-5-en-29-oic-3α-ol,

26-amino-furost-4-en-3-one, 26-amino-furost-4-en-27-ol-3-one,

26-amino-furost-4-en-27-al-3-one,

26-amino-furost-4-en-27-oic-3-one, 28-methyl,26-amino-furost-4-en-3-one,

28-methyl,26-amino-furos -4-en-28-ol-3-one,

28-methyl,26-amino-furost-4-en-28-al-3-one,

28-methyl,26-amino-furost-4-en-28-oic-3-one,

28-ethyl,26-amino-furost-4-en-3-one, 28-ethyl,26-amino-furost-4-en-29-ol-3-one,

28-ethyl,26-amino-furost-4-en-29-al-3-one,

28-ethyl,26-amino-furost-4-en-29-oic-3-one,

26-amino-furost-4, 6-dien-3-one,

26-amino-furost-4,6-dien-27-ol-3-one, 26-amino-furost-4, 6-dien-27-al-3-one,

26-amino-furost-4,6-dien-27-oic-3-one,

28-methyl,26-amino-furost-4,6-dien-3-one,

28-methyl,26-amino-furos -4, 6-die -28-ol-3-one,

28-methyl, 26-amino-furost-4, 6-dien-28-al-3-one, 28-methyl,26-amino-furost-4, 6-dien-28-oic-3-one,

28-ethyl,26-amino-furost-4,6-dien-3-one,

28-ethyl,26-amino-furost-4,6-dien-29-ol-3-one,

28-ethyl,26-amino-furost-4,6-dien-29-al-3-one,

28-ethyl,26-amino-furost-4,6-dien-29-oic-3-one, and derivatives or analogs thereof, and wherein the heterocyclic oxygen between C16 and C22 of the above compounds is replaced with sulfur or nitrogen.

Such compounds according to formula (I) are structurally analogous to oxysterols that repress HMG-CoA reductase, but are sufficiently dissimilar as to be unpredictable in their biologi¬ cal properties, such as the inhibition of cholesterol biosynthe¬ sis. Such furostene compounds according to the present invention have been discovered to provide unpredictable results involving

cholesterol inhibition, including inhibition of cholesterol uptake, synthesis and/or accumulation in vivo and in si tu, in comparison to naturally occurring oxygenated or hydroxy steroids. Unlike other compounds that primarily affect cholesterol synthesis in the liver to lower blood plasma cholesterol, 26-aminofurostene compounds of the present invention have been discovered to protect cells from the accumulation of cholesterol irrespective of the plasma cholesterol level, as well as inhibiting uptake and synthesis of cholesterol. Aminofurostene compounds of the present invention can also be used therapeuti- cally, either alone or in combination with other drugs and/or pharmaceutical carriers, as a means for regulating cholesterol levels or for preventing and treating diseases related to abnormal cholesterol synthesis, uptake and accumulation. For example, 26-amino-furost-5-en-3/3-ol has been found to inhibit de novo cholesterol synthesis in human hepatocytes, as presented herein.

This discovery is supported by the analogous finding in substituted cholesterol that thio (-SH) substitution of a cholesterol side chain results in a reduction in cholesterol inhibition in comparison to both -OH and -NH ₂ , apparently because the thio group (-SH) is less polar than both the hydroxy and amino groups. Another explanation is that the receptor site for the cholesterol inhibitor compound has a higher affinity for proton acceptors, such as NH ₂ (a base) than for OH, or for SH (an acid) .

Compounds of formula (I) can be synthesized by one of ordinary skill in the art by routine experimentation using known methods, as taught herein. For example, substitution at a terminal carbon of the sterol can be achieved by lithium aluminum hydride (LAH) reduction according to the procedure set forth herein for the preparation of 26-amino-furost-5-en-3/3-ol . Starting materials and intermediates are readily available or can be readily made, and the variations in this method that would be suitable for each substituted compound are well within the skill of an ordinary synthetic chemist, based on the guidance and teachings presented herein.

The following scheme is representative of the manner in

which compounds according to the present invention can be obtained: Synthesis of Substituted or Unsubstituted 26-Aminofurostenes According to Formula (I) from Diosgenin 1. Formation of a 26-hydroxy-furostene-3-ol

A1C1 ₃ Diosgenin + LiAlH ₄ > 26-hydroxy-furostene-3-ol

2. Protection of a 3-ol as an acetate a. 26-hydroxy-furostene-3-ol + triphenylmethyl chloride (trityl) > 26-0-trityl-furostene-3-ol b. 26-0-trityl-furostene-3-ol + acetic anhydride > 26-0-trityl-furostene-3-acetate c. 26-0-trityl-furostene-3-acetate + HCl/ethanol > 26-hydroxy-furostene-3-acetate

3. Formation of 26-amino a. 26-hydroxy-furostene-3-acetate + prosylchloride > 26-tosyl-furostene-3-acetate

DMF b. 26-tosyl-furostene-3-acetate + NaH ₃ -acetate >

26-azido-furostene-3-acetate c. 26-azido-furostene-3-acetate + LiAlH ₄ —

26-amino-furostene-3-acetate

4. Hydrolysis of acetate to form 3-ol Methanol a. 26-amino-furostene-3-acetate >

K04

26-amino-furostene-3-ol 26-aminofurostene-3-ones, converted from the above-described synthesis of 26-amino-furostene-3-ols, can readily be formed by known methods, such as by the use of chromic acid, such as 26- amino-furost-4-ene-3-ones or 26-amino-furost-4, 6-diene-3-ones .

Additionally, the above scheme can also take into account the presence of additional hydroxy, aldehyde, acid (such as 27-, 28-, or 29- OH, -al, or -oic) or alkyl groups which are protected

(e.g., as an acetate) or unprotected (e.g., using methanol/KOH) as the 3-ol presented above, or which can be alternatively protected and/or unprotected by known methods readily determina-

ble by routine experimentation. For example, the 27-, 28-, or 29-acid substituents can be reacted with an alcohol to form the acetate protecting group.

Alternatively, other known steps can be used to synthesize 26-aminofurostene compounds according to formula (I) , which known steps, based on the teaching herein, could be combined with the knowledge of one of ordinary skill in the art to make and use compounds according to known methods.

Compounds according to formula (I) , such as 26-amino-furost-5-en-3/3-ol, can inhibit cholesterol uptake, accumulation and synthesis, e.g., by acting as a downregulator of LDL receptors and consequently as an inhibitor of the rate-limiting enzyme HMG-CoA reductase. In theory, the bio- isosteric substitution of an amino group at a terminal aliphatic carbon (as in formula (I) ) , and preferably the N-26 position of a sterol with a five carbon side chain, provides a marked increase in capacity to interact with specific configurations on a cytostolic oxysterol carrier protein that is believed to participate in the biosynthetic pathway for cholesterol produc- tion.

Further support can be found in experiments which show that other oxygenated sterols tend to downregulate DNA synthesis and cell growth through a mechanism that appears to be independent of HMG-CoA reductase activity. Whatever the mechanism of action, it has been discovered that compounds according to formula (I) , such as 26-amino-furost-5-en-3/3-ol, inhibit cholesterol synthe¬ sis, and therefore have a direct or indirect effect of inhibiting cholesterol uptake, synthesis and/or accumulation in vivo and in si tu . Pharmaceutical Administration

For administration to a patient or subject, compounds according to formula (I) of the present invention can be provided as a pure compound, or as mono and di-esterified derivatives and other pharmaceutically acceptable derivatives thereof, such as mono and di-ethers, and especially complexed with cyclodextrin, wherein 2 - hydroxyp ropy1 - β - cyc 1 odext rin and 2-hydroxypropyl -y-cyclodextrin are preferred. Cyclodextrin-- aminofurostane complexes can be formed by adding the cyclodextrin

in a 20-60% aqueous solution to one or more 26-aminofurostenes of the present invention, and letting the mixture stand for 12 to 72 hours, e.g., 24 hours, such that the aminofurostene molecules are partially enclosed by the cyclodextrin molecules. Such complexes can have a maximum solubility of about 5-30 mg/ml of aqueous aminofurostene, such as 5, 7, 10, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 27, 28 or 30 mg/ml, depending on the cyclodextrin, aminofurostene and solubilization conditions used.

Cyclodextrins which can be used in 26-aminofurostene containing compositions of the present are preferably those that: provide sufficient solubility for administration to subjects to

^\' be treated or tested; maintain to a useful degree the cholesterol inhibitory activity; and which are readily preparable. Such preparation, e.g., can be provided by mixing a 20-60% aqueous solution of a cyclodextran with a 26-aminofurostene compound according to formula (I) , in a final concentration of about 5 to 30 mg/ml, and letting the solution stand for 12-72 hours until the cyclodextran molecules have sufficiently associated with the molecules of a compound according to formula (I) to become soluble to substantially remain in solution at a temperature selected from about room temperature (e.g., 18-28 °C) to about 40"C.

For example, 26-amino-furost-5-en-3/3-ol according to formula (I) has a maximum, stable solubility of 20 mg/ml as a 45% aqueous solution of 2-hydroxypropyl-/3-cyclodextrins at about room temperature to about 39°C. The cyclodextrin can be selected from a naturally occurring or chemically modified cyclodextrin. Naturally occuring cyclodextrins include, e.g., a- , β - , and y -cyclodextrins. Chemically modified cyclodextrins can be selected from a methyl cyclodextrin, a hydroxyethyl cyclodextrin, a 2-hydroxypropyl cyclodextrin, a glucosyl cyclodextrin and a maltosyl cyclodextrin. In compositions of the present invention, 2-hydroxypropyl- β - and 2-hydroxypropyl-7-cyclodextrans are preferred, and more preferably, 2-hydroxypropyl- β -cyclodextrans. Cyclodextans useful for compositions of the present invention are readily made by known methods and are also available from commerical sources, such as MOLECULSOL ^® , from

Pharmatec (Lathua, Florida) .

Alternatively, or in addition to, the use of cyclodextrins (as presented herein) , naturally occurring fatty acids, or other organic or inorganic esters can be formed using known pharmaceu- tically acceptable substituents, such as sulfates, carbonates and glucuronides . Esterification and/or etherification can be at the 3- and/or 26-, 27-, 28-, and 29- positions. Aryl and/or alkyl ethers, such as methyl, ethyl or cycloalleles (e.g., cyclopentyl ethers) can also be used in the present invention. Furthermore, acid salts and other pharmaceutically accept¬ able substitutions can be used. A particular aspect of the present invention comprises a compound of the present invention in an effective unit dose form. By "effective unit dose" is meant a predetermined amount sufficient to bring about the desired cholesterol inhibitory effect, including inhibition of uptake, synthesis and/or accumulation of cholesterol in vivo, in si tu and in vi tro, which unit dose form can be readily determined by one skilled in the art.

The dosage of the compounds of the present invention or their pharmaceutically acceptable salts or derivatives will depend, of course, on the degree of cholesterol inhibition desired. Dosages of pharmaceutically active compounds such as those of the present invention are conventionally given in amounts sufficient to bring about the desired inhibition of cholesterol uptake, synthesis or accumulation, relative to the condition being treated (e.g., arteriosclerosis, atherosclerosis, hypertension, hypothyroidism, obstructive jaundice, nephrosis, diabetes mellitus, pancreatitis, multiple myeloma, porphyria and obstructive liver disease) without causing undue burden on the treated subject.

Compounds of the present invention according to formula (I) , can be administered to a subject in amounts preferably ranging from about 0.1 to 3.0 mg/kg per day orally, parenterally, subcutaneously, intramuscularly, intraveneously or as a dermal patch. For example, oral administration is preferably 0.1 to 2 mg/kg, and more preferably, 0.2 to 0.6 mg/kg, 0.7 to 1.2 mg/kg or 1.3 to 1.8 mg/kg, per day. Oral administration can require a higher dosage than intravenous administra io: ^-1 , due to loss in

the digestive tract. Parenteral administration is preferably 0.1 to 3 mg/kg, and more preferably 0.2 to 0.3 mg/kg, 0.4 to 0.7 mg/kg, 0.8 to 1.2 mg/kg, 1.4 to 1.7 mg/kg, 2.0 to 2.5 mg/kg or 2.6 to 2.9 mg/kg, per day. Dermal patch administration is preferably 0.1 to 2.0 mg/kg, and more preferably 0.15 to 0.3 mg/kg, 0.5 to 0.8 mg/kg, or 1.0 to 1.5 mg/kg per day.

The precise dose in each instance will depend on which compound according to formula (I) is used and the degree cholesterol inhibition, the mass of the patient, the condition of the patient, the concurrent or additional use of other drugs and their accompanying side effects, and the like, which factors will be readily determinable and evaluatabls using routine experimentation by skilled practitioners and clinicians.

Animals and birds, as well as humans, are suitable subjects for use of pharmaceutical compositions of the present invention, which composition comprises a compound according to formula (I) . Animals, in the context of the present invention include mammals birds and fish, and mammalian use includes use, e.g., in rodents, livestock, primates, horses, dogs, cats and humans. Pharmaceutical preparations including a compound of the present invention according to formula (I) can be administered as a solid or liquid, or as an injectable preparation, in combination with suitable pharmaceutical carriers. Suitable pharmaceutical preparations include tablets, capsules, oral liquid, intravenous and parenteral injectables and other equivalent means of administration. In a preferred embodiment, the compound is administered as an oral liquii or intravenous glucose solution al using conventional diluents and excipients, wherein the 26-amino furostene has been solubilized in cyclo- dextrin, as presented herein.

Intravenous and parenterally administered compositions according to the present invention may be given in combination with conventional injectable liquid carriers such as sterile pyrogen-free water, cyclodextrins, sterile peroxide-free ethyl oleate, dehydrated alcohol, or propylene glycol. Conventional pharmaceutical adjuvants for injection solution such as stabiliz¬ ing agent, solubilizing agents and buffers, such as cyclodextrin, ethanol, complex forming agents such as ethylene diamine

tetraacetic acid, tartrate and citrate buffers, and high-mole¬ cular weight polymers such as polyethylene oxide for viscosity regulation may be added, with cyclodextrin, wherein 2-hydroxy¬ propyl - β- and 2-hydroxypropyl -y-cyclodextrins are preferred. In a preferred embodiment, the cyclodextrin can be provided as 20-60% cyclodextrin aqueous solution containing 0.1-30 mg/ml of stably solubilized aminofurostene compounds of the present invention, such that the solution can be diluted to the apporp- riate dosage concentration prior to administration. Such compositions may be administered intramuscularly, subcutaneously, a intraperitoneally, intravenously, or from a dermal patch.

Further non-limiting examples of carriers and diluents include albumin and/or other plasma protein components such as low density lipoproteins, high density lipoproteins and other lipids and lipoproteins with which these serum proteins are associated. These lipids can include phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine and neutral lipids such as triglycerides. Lipid carriers also include, without limitation, tocopherol and retinoic acid. Additional lipid and lipoprotein drug delivery systems that may be included herein are described more fully in "Biological Approaches to Controlled Delivery of Drugs," Annals of the New York Academy of Sciences , 507, 775-88, 98-103, and 252-271, which disclosure is hereby incorporated by reference. Compositions of the present invention may also be formulated into orally administrable compositions containing one or more physiologically compatible carriers or excipients, and may be in solid or liquid in form. These compositions may, if desired, contain conventional ingredients such as binding agents, for example, syrups, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, such as lactose, mannitol, starch, calcium phosphate, sorbitol, cyclodextran, or methylcellulose; lubricants such as magnesium stearate, high molecular weight polymers such as polyethylene glycols, high molecular weight fatty acids such as stearic acid or silica; disintegrants such as starch; and acceptable wetting agents as, for example, sodium lauryl sulfate.

The oral compositions may assume any convenient form, such

as tablets, capsules, lozenges, aqueous or oily suspensions, emulsions, or dry products suitable for reconstitution with water or other liquid medium prior to use. The liquid oral forms are preferred and may, of course, contain flavors, sweeteners, preservatives such as methyl or propyl p-hydroxybenzoates; suspending agents such as sorbitol, glucose or other sugar syrup, methyl, hydroxymethyl, or carboxymethyl celluloses or gelatin; emulsifying agents such as lecithin or sorbitan monooleate or thickening agents. Concentrated oral forms are also preferred, wherein 20-60% cyclodextrin aqueous solutions containing 0.2-30 mg/ml solubilized aminofurostene compounds of the present invention, such that the concentrate can be diluted to the appropriate dosage concentration prior to administration. Non-aqueous compositions may also be formulated which comprise edible oils as, for example, fish-liver or vegetable oils. These liquid compositions may conveniently be encapsulated in, for example, gelatin capsules in a unit dosage amount.

The pharmaceutical compositions according to the present invention may also be administered, if appropriate, either topically as an aerosol or, formulated with conventional bases as a cream or ointment.

Pharmaceutical compositions of the present invention can also be administered by incorporating the active ingredient into colloidal carriers, such as liposomes. Liposome technology is well known in the art, having been described by Allison et al. in Nature 252: 252-254 (1974) and Dancy et al. , J. Immunol. 120: 1109- 1113 (1978) .

Alternatively, active-targeting vesicles can be used as carriers for the active components of the present invention by placing a recognition sequence, i.e., from an antibody, onto the vesicles such that it is taken up more rapidly by certain cell types which are known to uptake, synthesize, store or accumulate cholesterol (cf. Papahadjopoulou et al. , Annals of the New York Academy of Sciences, 507: 67-74 (1987)). As further embodiments of the present invention, the active components can be adminis¬ tered in the form of sustained release products, by incorporating the active components in a suitable polymer. Polymer systems found to be useful in delivering compounds according to formula

(I) of the present invention in a time-dependent manner are known in the art and include subdermal reservoir implants composed of non-degradable polymers that release steroids for extended periods of time (Folkman et al . , J. Sur . Res. 4: 139 (1964) , entirely incorporated by reference) and subdermal implants or injectable microspheres comprising degradable materials, such as lactide-glycoside copolymers and polycaprolactones (Langer, Science 249: 1527-1532 (1990) entinrely incoporated by reference) . For use of such polmer systems, cyclodextrin is preferred, as presented herein, due to the suitable solubility and and lack of toxicity using cyclodextrin, such as β - and 7-cyclodextrin are preferred and wherein 2-hydroxy - β -cyclodextrin and 2-hydroxy-7-cyclodextrin are especially preferred.

It also will be understood by the skilled practitioner that the compositions of the present invention may be administered in conjunction with, as well as formulated with, at least one other therapeutic agent to produce a combination composition and/or therapy effective for ameliorating certain elevated cholesterol pathologies, e.g., arteriosclerosis, atherosclerosis, hyperten- sion, hypothyroidism, obstructive jaundice, nephrosis, diabetes mellitus, pancreatitis, multiple myeloma, porphyria and obstruc¬ tive liver disease.

"In conjunction" is defined herein to mean the compositions of the present invention may be administered first and other therapeutic agents later, or vice versa. Therapeutic agents include, by way of non-limiting examples, cardiac glycosides, antidysrythmics, diuretics, antihypertensives, antianginals, anticoagulants, and lipid lowering agents. Sucn agents are well known in the art and the use of such drugs in connection with a pharmaceutical composition of the present invention can be accomplished by one of ordinary skill in the art using routine experimentation, based on the teachings and guidance presented herein. See, MacLeod et al Principles of Medical Pharmacology Toronto:University of Toronto 303-320, 343-362, 373-381 (1980) ; Goodman and Gil an The Pharmacological Basis of Therapeutics New York:Macmillan 793-818, 834-847, 885-915, 1347-1366 (1980) ; Ewy and Bressler, eds . Cardiovascular Drugs and the Management of Heart Disease New York:Raven 635-645 (1982) , the entire contents

of which references are incorporated by reference.

Example 1: Preparation of an Aminofurostene

26-amino-furost-5-en-3/3-ol was prepared by lithium aluminum hydride (LAH) reduction of diosgenin to form 26-amino-furost-5-en-3/3-ol, followed by substitution of the

26-hydroxy group with an amine; by the following synthesis scheme using known steps:

Synthesis of 26-Amino-furost-5-ene-3/3-ol from Diosgenin

1. Formation of 26-hydroxy-furost-5-ene-3/3-ol A1C1,

Diosgenin LAH >

26-hydroxy-furost-5-ene-3/3-ol

2. Protection of 3/3-ol as an acetate a. 26-hydroxy- furost-5-ene-3/3-ol + triphenylmethyl chloride (trityl) >

26-0-trityl-furost-5-ene-3/3-ol b. 26-0-trityl-furost-5-ene-3/3-ol + acetic anhydride —> 26-0-trityl-furost-5-ene-3-acetate c. 26-0-trityl-furost-5-ene-3-acetate + HCl/ethanol —> 26-hydroxy-furost-5-ene-3-acetate

3. Formation of 26-amino a. 26-hydroxy-furost-5-ene-3-acetate + prosylcholoride — 26-1osyl-furost-5-ene-3-acetate

DMF b. 26-tosyl-furost-5-ene-3-acetate + NaH ₃ -acetate > 26-azido-furost-5-ene-3-acetate c. 26-azido-furost-5-ene-3-acetate + LiAlH ₄ > 26-amino-furost-5-ene-3-acetate

4. Hydrolysis of acetate to form 3/3-ol Methanol a. 26-amino- furost-5-ene-3 -acetate ->

K04 26-amino-furost-5-ene-3/3-ol .

Melting points were determined on a Thomas-Hoover capillary melting-point apparatus and are uncorrected. NMR spectra

[ ( ² H) chloroform] were run on a Nicolet QE-300 spectrometer. IR spectra were obtained on a Matteson Polaris FT-IR instrument.

26-hydroxyfurost-5-ene-3/3-ol was prepared by lithium aluminum hydride reduction of Diosgenin, followed by formation of an acetate of the 3/3-ol by reaction with trityl to form 26-0-trityl-furost-5-ene-3/3-ol, followed by reaction with the 26-0-trityl with acetic anhydride to form 26-0-trityl-furost-5-ene-3-acetate and reaction of the 3-acetate with HCl/ethanol to form 26-hydroxyl-furost-5-ene-3-acetate. 26 amino-furost-5-ene-3-jSol was then formed by reacting the 26 hydroxy-furost-5-ene-3-acetate with proylchloride to form 26 - tosyl - furost - 5 - ene- 3 acetate .

26-Tosylfurost-5-ene-3-acetate (0.66 mmol) was dissolved in 25 ml of DMF containing 1.5 equiv. of NaN ₃ and 1 ml of water. The solution was heated to between 90 and 100°C for 2 h, cooled to room temperature and then worked up as usual. Rotary evaporation yield (yield > 80%) of 26-azidocholesterol 3-acetate as a white powder, which was subsequently recrystallized (with ethanol) to yield white waxy crystals. Lithium aluminum hydride (20 mg, 527 mmol) reduction of 26-azidofurost-5-ene 3-acetate (100 mg) in diethyl ether, followed by usual workup, afforded >80% yield of 26-aminofurost-5-ene-3/3-ol crude powder that was purified by flash chromatography [chloroform/ethanol/29% (w/v NH ₄ 0H, 93:7:1 (by vol.) (m.p. 149-152°C) .

26-aminofurost-5-ene-3/3-ol is purified usings a capillary gas liquid chromatography using a Shimadzu 14-a capillary GLC using WCOT fused silica cp-sil-5-CB having a 50 meter lenght a 2.25 mm I.D., a 0.39 O.D. and a 0.12 micromfilm thickness, at a flow carrier rate of 1.5 mils per minute of helium with a temperature program of 265 initial with 1 °C/min raised 285 °C/min raised to 1 °C/min raised to 285 °C. Peak 2 was found to be th 26 amino furost-5-ene-3/3-ol with a 96.4% purity and a elution time of 23.742 minutes.

Example 2 ; Cholesterol Inhibition by an Aminofurostene

HepG2 Cells were obtained from Dr. Barbara Knowles of the Wistar Institute, Pennsylvania, and are readily available from

commercial sources, e.g., the ATCC in Rockville, Maryland. Stock HepG2 monolayers were maintained in T-75 disposable Nunc flasks, in a medium comprising 10 ml of DMEM (Dulbecco\'s Eagle\'s Minimum Essential Medium with Earl\'s salts, pH 7.4) supplemented with 10% (v/v) fetal bovine serum (FBS) and human serum plasma, 2% (v/v) non-essential and essential amino acids, 1% (v/v) DMEM vitamins, 1% L-glutamine (292 ug/ml) , 1% penicillin (100 U/ml) -streptomycin (100 ug/ml) . The HepG2 stock was maintained at 37°C. in a humidified atmosphere of 5% C02 and 95% air. The medium was routinely changed every 3-4 days and the cells were passaged

(1:3) every 7 days. The ingredients for these media were supplied primarily by Gibco Chemical Co, and are also readily available from other commercial sources, su h as SIGMA and

ALDRICH. For experiments using HepG2 cells, confluent stock mono¬ layers were washed once with Dulbecco\'s PBS, dissociated with 0.05-0.02% trypsin-EDTA followed by treatment with deoxyribonu- clease I (0.1%), and were seeded (3.0 x 10 <6> ) under identical conditions. The experimental DMEM contained 25% (v/v) D ₂ 0, 9% (v/v) delipidated FBS, and 1% (v/v) each of heat-inactivated FBS, penicillin-streptomycin, essential-and non-essential amino acids, L-glutamine and 20 mM HEPES.

The hepatoblastoma cells were cultured in media containing 25% D20 and 1% FBS in order to determine de novo cholesterol synthesis under conditions that permit receptor mediated cholesterol transport, according to the method of Easterman & Javitt et al J. Lipid Res. 26:950-954 (1985) . Dish cultures of each cell type were prepared, and 26-amino-f^rost-5-en-33-ol, dissolved in N,N-dimethylformamide (DMF, 9 μl per dish, 0.3% v/v) was added to each culture.

For binding studies, D ₂ 0 was used during all cholesterol synthesis, provided as 15% of the growth medium. After this incubation, the culture dishes were placed on ice, the medium was removed, and the cell monolayers were washed as described in Goldstein & Brown J. Biol. Chem. 249:5153-5162 (1974) . The

, monolayers were then scraped off the dishes with a teflon policeman and were solubilized with 1 ml of 0.1 N NaOH.

Cell-associated radioactivity was measured using the following

assays. Cholesterol assay

After 96 h incubation, the medium was removed and the cells were washed with Dulbecco\'s PBS and harvested with 0.05% trypsin/0.02% EDTA. The proteinase was de=activated with an equal volume of soybean trypsin inhibitor and the resulting cell suspension was centrifuged for 5 min at 500 g. An internal standard of 5 beta-cholestan-3/3-ol was added to the pellets before extraction with chloroform/methanol/water (8:4:3, by vol. ; Folch mixture) . A portion of the extract dried in a current of n i t r o g e n a n d s i l y a t e d [N-methyl-N(trimethylsily) tri-fluoroacetamide; (from Pierce, Inc. ) /toluene/pyridine (2:1:1, by vol.)

Free cholesterol was then quantified by g.l.c. on a Perkin-Elmer capillary gas-liquid chromatograph (model 8320) fitted wiht a 20 mDe-1 methylsilicone glass column (Supelco) . The remaining extract was dried and saponified with 2% ethanolic KOH at 80°C for 1 hr. The sterols were then re-extracted, and total cholesterol was measured by g.l.c. ar ^\' ter silylation. Cholesterol ester was calculated as the difference between total and free cholesterol determinations. Newly synthesized choles¬ terol was quantified by a g.l.c.-.s. isotpe mass-ratio method using a Hewlet-Packard g.c- m.s. spectrometer (model 5890) fitted with DB-1 methylsilicone 0.18 m (internal diameter) x 20 m long glass column (Supelco) according to a known method. See, e.g., Esterman et al, J. Lipid Res. 26:950-954 (1985) , the entire contents of which is herein incorporated by reference.

The results of cholesterol experiments are shown in the following tables, with Table I and Table II showing results for cholesterol synthesis inhibition in HepG2 cells.

TABLE I

EFFECTS OF A 26-AMINO-FUROSTENE ON CHOLESTEROL SYNTHESIS IN HEP G2 CELL CELL CULTURE ³

26-AMINO-FUROST-

5-ENE-3/3-OL (2) 74 2.73 27.1 34 25

26-AMINOCHOL-

ESTEROL (2) 76 2.84 26.7 34 26

( ) number of flasks

"Cells plated at high density; DMEM 450 mg/dl glucose enriched with 10% delipitated FBS and human plasma containing total cholesterol = 200 μg/ 5ml meidu. 7-day study; medium changed x 1 at 4 days; total = 10 ml.

•"Medium contained 15% D ₂ 0; therefor all the cholesterol synthe¬ sized during the 7-day period is deuterated. ^c all compounds dissolvled in 2-hyroxypropy1-/3-cyclodextrin; final concentration in medium = 1 μM

TABLE II

PER CENT INHBITION OF CHOLESTEROL SYNTHESIS

FUROST-5-ENE-3/3, 26-DI0L (34-31) x 100/34 = 8.8% 26-AMINO-FUROST-5-ENE-3j3-0L (34-25) X 100/34 = 26.5% 26-AMINOCHOLESTEROL (34-26) X 100/34 = 24%

These data reveal that 26-aminofurostene compounds according to formula (I) act as potent inhibitors of cholesterol synthesis, since they result in significant reductions in de novo cholester¬ ol synthesis in human in HepG2 cells. Therefore, 26-amino-furo- st-5-en-3/3-ol appear to be selective inhibitors of de novo cholesterol synthesis.

Example 3; Pharmaceutical Administration of an Aminofurostene

A pharmaceutical composition containing 26-amino-furo- st-5-en-3/3-ol was prepared as a stable aqueous solution having a maximum solubility of 20 mg/ml in a 45% aqueous solution of 2-hydroxypropyl- β-cyclodextrin. 26-amino-furost-5-en-3/3-ol, prepared, e.g., as presented in Example 1 above, was added to provide a final concentration of 20 mg/ml in a sterile 45% aqueous solution of 2-hydroxypropyl - β-cyclodextrin and left standing for 24 hours, which was then suitable for administration to a subject in by diluting to an appropriate concentration. Cyclodextrins are well known to be safe for administration to mammals and birds. Additionally, the present composition was also found to lack toxic effects when given in a range of concentrations of up to about 45 mg/kg of an aminofurostene of the present invention, as 26-amino-furost-5-en- iβ - ol (e.g., 5 mg dose to a llOg hamster) .

For pharmaceutical administration in human or animal subjects, the above 20 mg/ml composition is added in the amount of 1 ml to 1 liter of a 5% glucose IV solution and administered intravenously over a period of approximately 24 hours (e.g., 1 mg per hour or about 0.25-0.3 mg/kg per day) for 3-7 days following cardiac sugery, such as angioplasty. The administra¬ tion of 26-aminofurostene compounds according to the present invention is useful for inhibiting the uptake, synthesis and/or accumulation of cholesterol and thus is helpful for keeping cardiac arteries from additional or re-accumulation of cholester¬ ol related or unrelated to corrective cardiac surgery, such as angioplasty.

All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the contents of the references cited within the references cited herein are also entirely incorporated by reference.

Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an

admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fully reveal the general nature of the present invention that others can, by applying\' knowledge within the skill of the art

(including the contents of the references cited herein) , readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the generic concept of the present invention. Therefore, such adaptations and modifications are intended to be comprehend¬ ed within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein.