Cyclodextrin dimers, compositions thereof, and uses thereof

Abstract

A novel class of synthetic cyclodextrin dimers is disclosed. Exemplary cyclodextrin dimers can treat atherosclerotic plaques by targeting various forms of cholesterol both intracellularly and extracellularly. Also provided are methods for removing atherosclerotic plaques from cholesterol, cholesterol esters, 7-ketocholesterol, and 7-ketocholesterol esters by treatment with the cyclodextrins. Also described is a dimer subclass with high specificity for 7-ketocholesterol.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 787,869 (Attorney Docket No. 48731.1600), filed January 3, 2019, and U.S. Provisional Application No. 62 / 850,334 (Attorney Docket No. 48731.1601), filed May 20, 2019, each of which is hereby incorporated by reference in its entirety. [Background technology]

[0002] 7-Ketocholesterol (7KC) is an oxysterol that is formed by the nonenzymatic reaction of oxygen radicals with cholesterol. 7KC can be formed in vivo or ingested from food, but is thought to be potentially toxic and of no use in humans and other eukaryotes. Like cholesterol, 7KC is found in atherosclerotic plaques. 7KC is the most abundant nonenzymatically generated oxysterol in atherosclerotic plaques and may contribute to the pathogenesis of atherosclerosis and other age-related diseases. 7KC is also thought to contribute to the pathogenesis of lysosomal storage diseases such as Niemann-Pick disease type C (NPC).

[0003] Cyclodextrins (CDs) are cyclic oligosaccharide polymers consisting of six (αCD), seven (βCD), or eight (γCD) sugar rings (Figure 1A). Alpha, beta, and gamma cyclodextrins are the most common forms and have many pharmaceutical, industrial, consumer, and food-related applications. Cyclodextrins have been used in a variety of applications, including as dietary fiber as a food additive. Cyclodextrins have also been used in pharmaceutical compositions as aerosolizing agents and excipients for small hydrophobic drugs, typically in combination with active pharmaceutical ingredients.

[0004] Hydroxypropyl-beta-cyclodextrin (HPβCD) is a βCD in which some number of hydroxypropyl (HP) groups are attached to the O2, O3, or O6 oxygens (or to atoms substituted for those oxygens) in some or all of the seven glucose monomers that make up beta-cyclodextrin. Hydroxypropylation of cyclodextrin improves its water solubility and safety to a degree that allows it to be used in humans for a variety of purposes, particularly as an excipient for active pharmaceutical agents. It has earned HPβCD GRAS (Generally Recognized as Safe) listing by the FDA. Most commercially available HPβCDs have an average of 4-9 HP substituents, with all available products containing a mixture of substitution numbers and positions, which is usually reflected in the advertised average degree of substitution (DS).

[0005] Other CD substituents include methyl, succinyl, sulfobutyl, maltosyl, carboxymethyl, and quaternary ammonium, among others, which can produce CDs with very high water solubility and low cytotoxicity, whether charged or neutral. Commercially available βCDs can have a variety of degrees of substitution, which can vary from as low as about 1 to maximum substitution (21 substituents), depending on the particular substituent and manufacturer. Summary of the Invention

[0006] This disclosure describes the design and testing of various cyclodextrin (CD) dimers, including, among others, HPβCD dimer, methylβCD dimer, succinylβCD dimer, sulfobutylβCD dimer, and quaternary ammonium dimer. Certain dimers demonstrate dramatically increased affinity for 7KC and cholesterol compared to monomeric CD. The exemplified dimers represent a novel class of linked substituted cyclodextrin dimers with improved properties, including the ability to selectively interact with and solubilize sterols. Molecular modeling experiments described below demonstrate the predicted interaction mechanism. Furthermore, the examples confirm the predicted ability of the novel substituted cyclodextrin dimers for sterol solubilization, including the selective solubilization of 7KC compared to cholesterol.

[0007] In one embodiment, the disclosure provides a CD dimer of the structure CD-L-CD, where each CD is a beta cyclodextrin, L is linked to the C2 or C3 carbon of each CD monomer, and one or both of the CD monomers contain at least one functional group, e.g., methyl, hydroxypropyl (HP), sulfobutyl (SB), succinyl (SUCC), -CH2CH(OH)CH2N(CH3)3. + or a combination thereof. Typically, each CD monomer is composed of a D-type glucose monomer. The CD dimer is substituted with a functional group, typically having a degree of substitution (DS) of 1-28, which indicates the total number of functional group substitutions present on both CD subunits. The substitutions may be present on one or both CD subunits. The linker length can be 2-8 atoms long, e.g., 4-8 atoms long, for the shortest path through the linker connecting the two CD subunits of the cyclodextrin dimer. The linker can include an alkyl (e.g., butyl) linker and / or a triazole linker, which is optionally substituted. Examples of CD dimers are those of Formulae I-IX (FIGS. 3B-3J, respectively). Optionally, the CD dimer is further substituted.

[0008] In another aspect, the disclosure provides a βCD dimer of structure CD-L-CD, where each CD is a beta cyclodextrin, L is linked to the C2 or C3 carbon of each CD monomer, and one or both of the CD monomers are substituted with at least one hydroxypropyl group. Typically, each CD monomer is composed of a D-form glucose monomer. The βCD dimer is substituted with hydroxypropyl (HP) and typically has a degree of substitution (DS) of 1-40, which indicates the total number of substituents present on both CD subunits. The substituents may be present on one or both CD subunits. The linker length can be 4-8 atoms long, the shortest path through the linker connecting the two CD subunits of the cyclodextrin dimer. The linker can include an alkyl (e.g., butyl) linker and / or a triazole linker, which is optionally substituted. Examples of βCD dimers are those of Formula I, Formula II, or Formula III (FIGS. 3B-3D, respectively). Optionally, the βCD dimers are further substituted.

[0009] 7KC is thought to be involved in heart disease, cystic fibrosis, liver injury and failure, and hypercholesterolemia complications. In hypercholesterolemia, 7KC can diffuse through cell membranes, where it affects receptor and enzyme function. High rates of dementia in hypercholesterolemia have been linked to 7KC accumulation. In the liver, 7KC affects tissue fenestration and porosity, which increases with age. 7KC also promotes the translocation of cytoplasmic NADPH oxidase components to the membrane of neutrophils (white blood cells) and enhances rapid reactive oxygen species production. The pathogenesis of other age-related diseases, such as age-related macular degeneration (AMD-dry type), Alzheimer's disease, and lysosomal storage diseases such as Niemann-Pick disease type C (NPC), has also been linked to elevated levels of 7KC. Oxysterols, including 7KC, have also been implicated in increasing free radical levels, which in turn affect circulating lipids in cystic fibrosis. The increase in free radicals caused by oxysterols such as 7KC is thought to be involved in apoptosis, cytotoxicity, impaired endothelial function, and regulation of enzymes involved in inflammation and fatty acid metabolism.

[0010] 7KC is formed from a non-enzymatic reaction of oxygen radicals with cholesterol, which indicates that 7KC formation may not be beneficial. Indeed, 7KC is believed to enhance the production of free radicals anywhere in the body, but in cardiac and vascular tissues, this is of particular concern. Free radicals affect cellular and enzymatic reactions that are important for cholesterol-mediated tissue damage, which is particularly critical in cardiac and vascular tissues. Free radicals are believed to enhance inflammation in the vasculature. 7KC is believed to cause mitochondrial and lysosomal dysfunction by disrupting the function of cell and organelle membranes, and is believed to be involved in increasing the frequency of foam cell formation from macrophages in atherosclerotic plaques. The scavenging function of these macrophages is expected to help ameliorate plaques, but when they are loaded with cholesterol and oxysterols, they may instead become part of the plaque.

[0011] Exemplary embodiments provide for the treatment of diseases associated with and / or exacerbated by 7KC accumulation, such as, for example, atherosclerosis, AMD, arteriosclerosis, coronary artery disease due to calcified coronary artery lesions, heart failure (all stages), Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, vascular dementia, multiple sclerosis, Smith-Lemli-Opitz syndrome, childhood neuronal ceroid lipofuscinosis, lysosomal acid lipase deficiency, and the like. In some embodiments, the cyclodextrin dimer is a cyclodextrin that is selective for 7KC (relative to cholesterol). Advantageously, the cyclodextrin dimer preferentially solubilizes 7KC while minimizing or avoiding potential harmful or toxic effects that may result from excessive removal of cholesterol.

[0012] Exemplary embodiments of the present invention provide the use of cyclodextrin (e.g., HPβCD, MeβCD, SUCCβCD, QAβCD, or SBβCD) dimers to solubilize and / or remove 7KC, which can be done in vitro or in vivo.

[0013] In exemplary embodiments, the cyclodextrin (e.g., HPβCD, MeβCD, SUCCβCD, QAβCD, or SBβCD) dimers exhibit higher binding affinity and / or solubilization for 7KC than for cholesterol. The higher specificity for 7KC than for cholesterol is most evident at subsaturating concentrations, but at higher concentrations, solubilization of both sterols can approach 100%. This specificity allows the use of the cyclodextrin dimers to preferentially solubilize and remove 7KC.

[0014] In an exemplary embodiment, the disclosure provides a cyclodextrin dimer having the following structure:

[0015] CD-L-CD

[0016] where L is the C2 carbon of each CD subunit (R 1 instead of) and / or through the C3 carbon (R 2 instead of ) connecting the large face (bottom) of each CD molecule;

[0017] wherein CD has the structure of formula X: [ka] (Formula X)

[0018] wherein L has a length of 8 or less atoms in the shortest path through the linker connecting the two CD subunits of the dimer, each of said 8 or less atoms being preferably C, N, O, or S;

[0019] and CD is substituted with 1 to 40 groups, for example with 1 to 28 groups, optionally with 2 to 15 groups, or 4 to 20 groups. The number of substitutions is determined by the number of R 1 , R 2 , and / or R 3 The total number of groups is indicated. The CD may have one or more additional substituents.

[0020] The R1 , R 2 , and R 3 are each independently H, methyl, hydroxypropyl, sulfobutyl, succinyl, -CH2CH(OH)CH2N(CH3)3 + Quaternary ammonium, such as alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamido, aminocarbonyloxyalkyl, aminosulfonyl, ammonium, ammonia, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyloxy, azido, bromo, chloro, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene , cycloalkylalkylene, deoxy, glucosyl, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroaralkyloxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocycloamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, iodo, ureido, carbamate, carboxy, sulfate, sulfuryl, sulfonamido, nitro, nitrito, cyano, phosphate, phosphoryl, phenoxy, acetyl, palmitoyl, or other fatty acid, monosaccharide, or disaccharide. In an exemplary embodiment, the substituent is preferably a maltosyl or carboxymethyl group.

[0021] In an exemplary embodiment, the R 1 , R 2 , and / or R 3 The groups are each independently H, methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium (-CH2CH(OH)CH2N(CH3)3 + etc.), glucosyl, palmitoyl, phosphate, phosphoryl, amino, azido, sulfate, sulfuryl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromo, and chloro, provided that R 1 , R 2 , and R 3 1 to 40, for example 1 to 28, or optionally 2 to 15 or 4 to 20, of the groups are not H.

[0022] In an exemplary embodiment, the R 1 , R 2 , and R 3 The groups are each independently H, methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, -CH2CH(OH)CH2N(CH3)3 + and the like, provided that R 1 , R 2 , and R 3 Of the groups, 1 to 40, for example 1 to 28, are not H and are optionally 1 , R 2 , and R 3 Of the groups, 2 to 15 or 4 to 20 are not H. 1 , R 2 , and R 3 The group may include one or more maltosyl or carboxymethyl groups.

[0023] In a further exemplary embodiment, the disclosure provides a CD dimer having the following structure:

[0024] CD-L-CD

[0025] where L is the C2 carbon of each CD subunit (R 1 instead of) and / or through the C3 carbon (R 2 instead of ) connecting the large face (bottom) of each CD molecule;

[0026] wherein CD has the structure of formula X: [ka] (Formula X)

[0027] wherein L has a length of 8 or less atoms in the shortest path through the linker connecting the two CD subunits of the dimer, each of said 8 or less atoms being preferably C, N, O, or S;

[0028] CD is HP-substituted with 1-28 hydroxypropyl (HP) groups, optionally with 2-15 HP groups or 4-20 HP groups, preferably with 2-5 HP groups, and optionally the CD has one or more additional substituents. The CD can have 2-4 HP groups, or it can have 2 HP groups, 3 HP groups, 4 HP groups, or 5 HP groups.

[0029] In a further exemplary embodiment, the disclosure provides a CD dimer having the following structure:

[0030] CD-L-CD

[0031] where L is the C2 carbon of each CD subunit (R 1 instead of) and / or through the C3 carbon (R 2 instead of ) connecting the large face (bottom) of each CD molecule;

[0032] wherein CD has the structure of formula X: [ka] (Formula X)

[0033] wherein L has a length of 8 or less atoms in the shortest path through the linker connecting the two CD subunits of the dimer, each of said 8 or less atoms being preferably C, N, O, or S;

[0034] CD is Me-substituted with 1-40 methyl (Me) groups, optionally 1-28 Me groups, optionally 2-15 Me groups or 4-20 Me groups, preferably 2-10 Me groups, and optionally the CD has one or more additional substituents. Without wishing to be bound by theory, it is believed that methyl groups are particularly suitable for substituting such CD dimers with a large number of substituents, because the size of the methyl group is particularly small and therefore does not interfere with the access of guests (such as 7KC or cholesterol) to the CD dimer binding cavity. It is further envisioned that one or more methyl substituents can be added to any of the cyclodextrin dimers of the present disclosure, including a number greater than those specified in the general formulas herein, for example up to a total of 40 non-hydrogen substituents when including both non-methyl and additional methyl substituents.

[0035] In a further exemplary embodiment, the disclosure provides a CD dimer having the following structure:

[0036] CD-L-CD

[0037] where L is the C2 carbon of each CD subunit (R 1 instead of) and / or through the C3 carbon (R 2 instead of ) connecting the large face (bottom) of each CD molecule;

[0038] wherein CD has the structure of formula X: [ka] (Formula X)

[0039] wherein L has a length of 8 or less atoms in the shortest path through the linker connecting the two CD subunits of the dimer, each of said 8 or less atoms being preferably C, N, O, or S;

[0040] CD is sulfobutyl-substituted with 1 to 28 sulfobutyl groups, e.g., 1 to 14 sulfobutyl groups, optionally with 2 to 10 sulfobutyl groups, preferably 2 to 5 sulfobutyl groups, and optionally the CD has one or more additional substituents. The CD can have 2 to 4 sulfobutyl groups, or it can have 2 sulfobutyl groups, 3 sulfobutyl groups, 4 sulfobutyl groups, or 5 sulfobutyl groups.

[0041] In a further exemplary embodiment, the disclosure provides a CD dimer having the following structure:

[0042] CD-L-CD

[0043] where L is the C2 carbon of each CD subunit (R 1 instead of) and / or through the C3 carbon (R 2 instead of ) connecting the large face (bottom) of each CD molecule;

[0044] wherein CD has the structure of formula X: [ka] (Formula X)

[0045] wherein L has a length of 8 or less atoms in the shortest path through the linker connecting the two CD subunits of the dimer, each of said 8 or less atoms being preferably C, N, O, or S;

[0046] CD is succinyl-substituted with 1-28 succinyl groups, optionally with 2-15 succinyl groups or 4-20 succinyl groups, preferably with 2-5 succinyl groups, and optionally with one or more additional substituents. CD can have 2-4 succinyl groups, or can have 2 succinyl groups, 3 succinyl groups, 4 succinyl groups, or 5 succinyl groups.

[0047] In a further exemplary embodiment, the disclosure provides a CD dimer having the following structure:

[0048] CD-L-CD

[0049] where L is the C2 carbon of each CD subunit (R 1 instead of) and / or through the C3 carbon (R 2 instead of ) connecting the large face (bottom) of each CD molecule;

[0050] wherein CD has the structure of formula X: [ka] (Formula X)

[0051] wherein L has a length of 8 or less atoms in the shortest path through the linker connecting the two CD subunits of the dimer, each of said 8 or less atoms being preferably C, N, O, or S;

[0052] CD is substituted with 1 to 28 quaternary ammonium groups, optionally with 2 to 15 quaternary ammonium groups or with 4 to 20 quaternary ammonium groups, preferably with 2 to 5 quaternary ammonium groups, the quaternary ammonium groups being -CH2CH(OH)CH2N(CH3)3 +For example, -CH2CH(OH)CH2N(CH3)3Cl, and optionally the CD has one or more additional substituents. The CD can have 2 to 4 quaternary ammonium groups, or can have 2 quaternary ammonium groups, 3 quaternary ammonium groups, 4 quaternary ammonium groups, or 5 quaternary ammonium groups. Of course, any pharma- ceutically acceptable salt of the quaternary ammonium is within the scope of the present disclosure.

[0053] L may have the structure: [ka]

[0054] wherein each R is independently selected from H, X, SH, NH, NH2, or OH, or may be absent;

[0055] the connection between each CD and the linker is independently through an O, S, or N bond connected to the C2 or C3 carbon of CD, or through an acetal bond through two adjacent oxygens of CD;

[0056] each X is a substituted or unsubstituted alkane, alkene, or alkyne;

[0057] each A is independently selected from a single, double, or triple covalent bond, S, N, NH, O, or a substituted or unsubstituted alkane, alkene, or alkyne; and

[0058] B is a substituted or unsubstituted five- or six-membered ring, S, N, NH, NR, O, or absent.

[0059] The linker may be 2-7, 3-6, 4-7, 4-6, 4-5, or 4, or 2-3 in length.

[0060] The linker can be an unsubstituted alkyl, for example, an unsubstituted butyl.

[0061] The linker may be a substituted or unsubstituted butyl linker.

[0062] The linker may comprise a triazole.

[0063] The linker has the following structure: [ka] (Formula XI) in which n1 and n2 are each 1-8 or 1-4, preferably in which n1 is 1 and n2 is 3.

[0064] In an exemplary embodiment, when the linker comprises a triazole, e.g., has the structure of formula XI, the linker L can be linked at the O2 position of each CD monomer, where n1 and n2 are each 0 to 8, e.g., 1 to 4, and preferably the total length of the linker can be 8 or less, e.g., 8, 7, 6, 5, 4, 3, or any range therebetween, and in a preferred embodiment, n1 is 1 and n2 is 3.

[0065] In exemplary embodiments, when the linker L comprises a substituted or unsubstituted alkyl and is preferably 8 atoms or less in length, e.g., 2-7, 2-6, or 4-7 or 4-6 or 4-5, or 8, 7, 6, 5, 4, 3, or 2 in length, or any range of values ​​therebetween, the linker L can be linked at the O2 position of each CD monomer, at the O2 position of one CD monomer and the O3 position of the other CD monomer, or at the O3 position of both CD monomers, although preferably the linker is a substituted or unsubstituted butyl, more preferably unsubstituted butyl.

[0066] The linker can have a single point of attachment to each CD monomer. The linker can have a single point of attachment to one CD monomer and multiple (two or more) points of attachment to the other CD monomer. The linker can have multiple (two or more) points of attachment to each CD monomer. The linker can include any of the linkers depicted in FIG. 8D. It should be understood that the depicted linkers have oxygen atoms at each end that form part of the cyclodextrin to which the linker is attached. The oxygen atoms are not considered to be part of the linker for purposes of determining the linker length. Similarly, for linkers that connect at multiple sites to one or both cyclodextrin monomers, the connections shown on the left connect to one monomer and the connections shown on the right connect to the other monomer.

[0067] In an exemplary embodiment, the disclosure provides a CD dimer having the following structure:

[0068] CD-L-CD

[0069] where L is the C2 carbon of each CD subunit (R 1 instead of) and / or through the C3 carbon (R 2 instead of ) connecting the large face (bottom) of each CD molecule;

[0070] wherein CD has the structure of formula X: [ka] (Formula X)

[0071] wherein L is a triazole having a length of 8 or less atoms, each of said 8 or less atoms being preferably C, N, O, or S;

[0072] CD is substituted with 0-28 groups, optionally the number of groups is 0, or optionally, CD has one or more substituents.

[0073] The linker has the following structure: [ka] (Formula XI) in which n1 and n2 are each 1-8 or 1-4, preferably in which n1 is 1 and n2 is 3.

[0074] The linker may be 3-7, 3-6, 4-7, 4-6, or 5-6 in length.

[0075] The linker may be 4-5 in length.

[0076] The cyclodextrin may further comprise (a) at least one methyl, hydroxypropyl, sulfobutyl, or succinyl group, and / or (b) at least one alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamido, aminocarbonyloxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyloxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, heteroalkyl, heteroaryl, hetero arylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroaralkyloxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocycloamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, ureido, carbamate, carboxy, sulfonamido, nitro, cyano, phenoxy, acetyl group, ammonium, ammonia, azido, bromo, chloro, deoxy, glucosyl, iodo, sulfate, sulfuryl, nitrito, phosphate, phosphoryl, palmitoyl group, fatty acid, monosaccharide, or disaccharide, and / or (c) at least one methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium (e.g., -CHCH(OH)CHN(CH) +), glucosyl, palmitoyl, phosphate, phosphoryl, amino, azido, sulfate, sulfuryl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromo, or chloro groups.

[0077] The cyclodextrin dimer can have a structure according to any one of Formulas I through IX (FIGS. 3B through 3J, respectively).

[0078] Each R 1 , each R 2 , and each R 3are independently: (a) methyl, H, hydroxypropyl, sulfobutylether, succinyl, succinyl-hydroxypropyl, quaternary ammonium, carboxymethyl, carboxymethyl-hydroxypropyl, hydroxyethyl, maltosyl, acetyl, carboxyethyl, sulfated, sulfopropyl, sodium phosphate, or glucosyl, and / or (b) hydrogen, alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamido, aminocarbonyloxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl , alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyloxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroaralkyloxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocycloamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkylalkyl, hydroxyalkylcarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, ureido, carbamate, carboxy, sulfonamide, nitro, cyano, phenoxy, or acetyl group.

[0079] L can be linked to the C2 carbon of each CD monomer, to the C3 carbon of each CD monomer, or to the C2 carbon of one CD monomer and the C3 of the other CD monomer. If the linker has multiple points of attachment to a single CD monomer, the linker can be linked to the C2 carbon, the C3 carbon, or a combination of the C2 and C3 carbons of that monomer. A particular arrangement may be preferred based on the reaction utilized to form the linkage, the purification process, and / or based on the structure of the linker.

[0080] The cyclodextrin dimer may exhibit a higher affinity for 7KC than for cholesterol, which may be determined using the turbidity assay disclosed herein.

[0081] The cyclodextrin dimer may exhibit at least 1.1, 1.5, 2, 3, 4, 5, or 10 times greater affinity for 7KC than for cholesterol. The cyclodextrin dimer may exhibit at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% or more reduction in relative turbidity of 7KC compared to cholesterol in a turbidity assay.

[0082] In an exemplary embodiment, the disclosure provides a composition comprising a mixture of cyclodextrin dimers as disclosed herein, optionally with an average degree of substitution of the mixture can be 2-10, e.g., 2-8, e.g., 3-7, or 2-5. The composition can comprise a mixture of CD dimers having a degree of substitution of 2-5 with hydroxypropyl, sulfobutyl, succinyl, or quaternary ammonium groups, e.g., about 2, about 3, about 4, or about 5 of such substituents. The composition can comprise a mixture of CD dimers having a degree of substitution of 2-10 with methyl groups. The degree of substitution can be measured by NMR. The degree of substitution can be measured by mass spectrometry, e.g., MALDI.

[0083] In an exemplary embodiment, the present disclosure provides a composition comprising a mixture of cyclodextrin dimers as disclosed herein, for example, according to Formulas I-III (FIGS. 3B-3D, respectively).

[0084] In an exemplary embodiment, the present disclosure provides a pharmaceutical composition comprising a cyclodextrin dimer or composition thereof as disclosed herein and a pharma- ceutically acceptable carrier. The cyclodextrin dimer may be the only active ingredient in the composition. The pharmaceutical composition may consist of, or consist essentially of, the cyclodextrin dimer and the pharma- ceutically acceptable carrier.

[0085] In an exemplary embodiment, the present disclosure provides a method of treatment comprising administering to a subject in need of treatment an effective amount of a cyclodextrin dimer or composition thereof as disclosed herein. The subject in need of treatment may be suffering from the harmful or toxic effects of 7KC.

[0086] In an exemplary embodiment, the present disclosure provides a method for reducing the amount of 7KC in a subject in need of such reduction, comprising administering to the subject an effective amount of a cyclodextrin dimer as disclosed herein.

[0087] The cyclodextrin dimer can be administered to the patient via parenteral (e.g., subcutaneous, intramuscular, or intravenous), topical, transdermal, oral, sublingual, or buccal administration, preferably intravenously.

[0088] The method may include administering to the patient about 1 mg to 10 g of the cyclodextrin dimer, e.g., 10 mg to 1 g, 50 mg to 200 mg, or 100 mg. In exemplary embodiments, 1 to 10 g of the cyclodextrin dimer may be administered, e.g., about 2 g, about 3 g, about 4 g, or about 5 g. In exemplary embodiments, 50 mg to 5 g of the cyclodextrin dimer may be administered, e.g., 100 mg to 2.5 g, 100 mg to 2 g, 250 mg to 2.5 g, e.g., about 1 g.

[0089] The method is for treating atherosclerosis, arteriosclerosis, coronary artery sclerosis due to coronary artery calcification, heart failure (all stages), Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, vascular dementia, multiple sclerosis, Smith-Lemli-Opitz syndrome, childhood neuronal ceroid lipofuscinosis, lysosomal acid lipase deficiency, cerebrotendinous xanthomatosis, X-linked adrenoleukodystrophy, sickle cell disease, Niemann-Pick disease type A, Niemann-Pick disease The present invention may be useful in preventing, treating, and / or ameliorating symptoms of one or more of type B, Niemann-Pick disease type C, Gaucher disease, Stargardt disease, age-related macular degeneration (dry type), idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, liver injury, liver failure, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, and / or hypercholesterolemia, preferably atherosclerosis.

[0090] The method may further comprise administering to the patient a second treatment, which may be administered simultaneously or sequentially in any order.

[0091] The second therapy may include one or more of an anti-cholesterol drug, such as a fibrate or a statin drug, an antiplatelet drug, an antihypertensive drug, or a nutritional supplement. Such statins may include ADVICOR® (niacin extended-release / lovastatin), ALTOPREV® (lovastatin extended-release), CADUET® (amlodipine-atorvastatin combination), CRESTOR® (rosuvastatin), JUVISYNC® (sitagliptin / simvastatin), LESCOL® (fluvastatin), LESCOLXL (fluvastatin extended-release), LIPITOR® (atorvastatin), LIVALO® (pitavastatin), MEVACOR® (lovastatin), PRAVACHOL® (pravastatin), SIMCOR® (niacin extended-release / simvastatin), VYTORIN® (ezetimibe / simvastatin), or ZOCOR® (simvastatin).

[0092] The second treatment may include anti-cholesterol and anti-hypertensive drugs.

[0093] In an exemplary embodiment, the disclosure provides a method for purifying an oxysterol, the method comprising: contacting a composition comprising an oxysterol with a cyclodextrin dimer as disclosed herein, thereby solubilizing the oxysterol in the cyclodextrin dimer; and recovering the cyclodextrin dimer and the solubilized oxysterol. The oxysterol comprises or consists of 7KC. The method may further comprise measuring the concentration of 7KC in the solubilized oxysterol, thereby determining the relative concentration of 7KC in the composition. The composition may comprise a patient sample. The method may be used to measure the concentration of 7KC in a patient sample, which may be used for diagnosis and / or treatment planning.

[0094] In an exemplary embodiment, the disclosure provides an in vitro method of removing oxysterols from a sample, the method comprising: contacting a sample containing an oxysterol with a cyclodextrin dimer as disclosed herein, thereby solubilizing the oxysterol in the cyclodextrin dimer; and separating the sample from the cyclodextrin dimer and the solubilized sterol.

[0095] In an exemplary embodiment, the disclosure provides a method for producing a cholesterol-reduced product, the method comprising: contacting a cholesterol-containing product with a cyclodextrin dimer as disclosed herein, thereby solubilizing the cholesterol in the cyclodextrin dimer; and removing the cyclodextrin dimer and solubilized cholesterol from the product. The product can be a food product, such as a meat product and / or a dairy product.

[0096] In another aspect, the disclosure provides a method for making a cyclodextrin dimer as described herein, for example, a cyclodextrin dimer having an unsubstituted or substituted alkyl linker, the method comprising: (a) reacting a protected β-cyclodextrin with a dialkylating agent, thereby producing a protected βCD dimer linked through a lower base, and optionally purifying the protected βCD dimer; (b) deprotecting the protected βCD dimer, thereby producing a deprotected βCD dimer, and optionally purifying the deprotected βCD dimer; and (c) hydroxypropylating the deprotected βCD, thereby producing a cyclodextrin dimer, and optionally purifying the cyclodextrin dimer. The protected β-cyclodextrin may comprise heptakis(6-O-tert-butyldimethylsilyl)-β-cyclodextrin. The dialkylating agent may comprise a dibromoalkane, optionally 1,4-dibromobutane. Step (a) may be carried out under anhydrous conditions and / or using sodium hydride as base. The purification in step (a) may comprise isotropic normal phase chromatography. Step (b) may be carried out using tetrabutylammonium fluoride in tetrahydrofuran (THF). The purification in step (b) may comprise isotropic normal phase chromatography. Step (c) may comprise reacting the deprotected βCD dimer with a hydroxypropylating agent such as propylene oxide, a methylating agent such as methyl iodide, a succinylating agent such as succinic anhydride, a sulfobutylating agent such as 1,4-butane sultone, and / or a quaternary ammonium linking agent such as glycidyltrimethylammonium chloride.

[0097] Step (c) may be carried out in aqueous conditions and may optionally include sodium hydroxide as a base. Step (c) may include one or more of ion exchange resin treatment, activated charcoal clarification, and dialysis.

[0098] In another aspect, the disclosure provides a method for making a cyclodextrin dimer, e.g., a cyclodextrin dimer having a triazole linker, as described herein, comprising: (a) reacting 2-O-(n-azidoalkyl)-βCD with 2-O-(n-alkyne)-βCD, thereby forming a βCD-triazole-βCD dimer having the structure βCD-alk1-triazole-alk2-βCD, and optionally (b) purifying the βCD-triazole-βCD dimer. Step (a) may be performed using a copper(I) catalyst, optionally about 15 mM copper(I). Step (a) may be performed in an aqueous solution. The aqueous solution may include dimethylformamide (DMF), optionally about 50% DMF (v / v). Step (b) may include chromatography. The method may further include, prior to step (a), producing the 2-O-(n-azidoalkyl)-βCD, the method comprising: (1) reacting an n-azido-1-bromo-alkane with β-cyclodextrin, optionally using a catalytic amount of lithium iodide, to thereby produce the 2-O-(n-azidoalkyl)-βCD; and (2) optionally purifying the 2-O-(n-azidoalkyl)-βCD. Step (2) may comprise chromatography. The method may further include forming 2-O-(n-alkyne)-βCD prior to step (a), the method including: (i) reacting n-bromo-1-alkyne with β-cyclodextrin, optionally using a catalytic amount of lithium iodide, to thereby form the 2-O-(n-alkyne)-βCD, and (ii) optionally purifying the 2-O-(n-alkyne)-βCD. Step (2) may include silica gel chromatography. Step (1) may be performed in dry DMSO. The reaction of step (1) may include lithium hydride. The βCD-triazole-βCD dimer has the following structure: [ka] (Formula XII), where n1 can be 1-8 and / or n2 can be 1-8, optionally n1 can be 1, 2, 3, or 4, and / or n2 can be 1, 2, 3, or 4, preferably n1 is 1 and n2 is 3. The length of the triazole linker can be 5-8. The method may further include hydroxypropylating the βCD-triazole-βCD dimer, thereby producing a cyclodextrin dimer, and optionally purifying the cyclodextrin dimer. Step (c) may include reacting the βCD-triazole-βCD dimer with a hydroxypropylating agent such as propylene oxide, a methylating reagent such as methyl iodide, a succinylating reagent such as succinic anhydride, a sulfobutylating reagent such as 1,4-butane sultone, and / or a quaternary ammonium linking reagent such as glycidyl trimethylammonium chloride.

[0099] Step (c) may be carried out in aqueous conditions and may optionally include sodium hydroxide as a base. The purification in step (c) may include one or more of ion exchange resin treatment, activated charcoal clarification, membrane filtration, and dialysis.

[0100] The present invention provides compositions and methods for treating or preventing atherosclerosis. 7KC is the most abundant non-enzymatically produced oxysterol in atherosclerotic plaque and is believed to contribute to the pathogenesis of atherosclerosis. Treatment with a CD dimer of the present invention (e.g., HPβCD or another CD of the present disclosure) is expected to be beneficial in preventing and / or reversing atherosclerotic plaque formation.

[0101] The present invention provides compositions and methods for treating or preventing diseases and conditions in which 7KC has been implicated, including, but not limited to, age-related diseases such as atherosclerosis, AMD, arteriosclerosis, coronary artery sclerosis due to coronary artery calcification, heart failure (all stages), Alzheimer's disease, Parkinson's disease, vascular dementia, chronic obstructive pulmonary disease, non-alcoholic fatty liver disease, and / or hypercholesterolemia or hypercholesterolemia-related dementia. Other sporadic and / or congenital disorders that are also associated with 7KC accumulation include Huntington's disease, multiple sclerosis, Smith-Lemli-Opitz syndrome, childhood neuronal ceroid lipofuscinosis, lysosomal acid lipase deficiency, amyotrophic lateral sclerosis, cerebrotendinous xanthomatosis, X-linked adrenoleukodystrophy, sickle cell anemia, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Gaucher disease, Stargardt disease, idiopathic pulmonary fibrosis, cystic fibrosis, liver injury, liver failure, nonalcoholic steatohepatitis, ulcerative colitis, Crohn's disease, and other irritable bowel syndromes.

[0102] In another exemplary embodiment, the present disclosure provides a cycloalkyl group having a degree of substitution of 1 to 40, for example, 1 to 28 or 4 to 20, preferably 2 to 15, and the substituents are methyl, hydroxypropyl, sulfobutyl, succinyl, -CH2CH(OH)CH2N(CH3)3 +Quaternary ammonium, alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamido, aminocarbonyloxyalkyl, aminosulfonyl, ammonium, ammonia, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyloxy, azido, bromo, chloro, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, deoxy, glucosyl, heteroalkyl, hetero A cyclodextrin dimer composition is provided in which the aryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroaralkyloxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocycloamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, iodo, ureido, carbamate, carboxy, sulfate, sulfuryl, sulfonamido, nitro, nitrito, cyano, phosphate, phosphoryl, phenoxy, acetyl, palmitoyl, or other fatty acid, monosaccharide, or disaccharide, the composition comprising a cyclodextrin dimer of the structure CD-L-CD, where L is (R 1 instead of) and / or through the C3 carbon (R 2 (instead of R 1 , R 2 , and / or R3 wherein L is eight atoms or less in length, each of the eight or less atoms being preferably C, N, O, or S. The substituents can be carboxymethyl or maltosyl. The substituents are preferably methyl, hydroxypropyl, sulfobutyl, succinyl, quaternary ammonium (e.g., -CH2CH(OH)CH2N(CH3)3 + ) The degree of substitution can be measured by NMR. The degree of substitution can be measured by mass spectrometry, e.g., MALDI.

[0103] In another exemplary embodiment, the present disclosure provides a cycloaliphatic alkyl ester having a degree of substitution of 1 to 40, for example, 1 to 28 or 4 to 20, preferably 2 to 15, and the substituents are methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium (e.g., -CHCH(OH)CHN(CH) + ), glucosyl, palmitoyl, phosphate, phosphoryl, amino, azido, sulfate, sulfuryl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromo, or chloro, the composition comprising a cyclodextrin dimer of structure CD-L-CD, where L is connected through the C2 carbon of each CD subunit (R 1 instead of) and / or through the C3 carbon (R 2 (instead of R 1 , R 2 , and / or R 3 wherein L is 8 atoms in length or less, each of the 8 or less atoms being preferably C, N, O, or S. The degree of substitution can be measured by NMR. The degree of substitution can be measured by mass spectrometry, e.g., MALDI.

[0104] In another exemplary embodiment, the present disclosure provides a cycloalkyl group having a degree of substitution of 1 to 40, for example, 1 to 28 or 4 to 20, preferably 2 to 15, and the substituents are methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, or -CH2CH(OH)CH2N(CH3)3. + and the like, wherein the composition comprises a cyclodextrin dimer of the structure CD-L-CD, where L is bonded through the C2 carbon of each CD subunit (R 1 instead of) and / or through the C3 carbon (R 2 (instead of R 1 , R 2 , and / or R 3 wherein L is 8 atoms in length or less, each of the 8 or less atoms being preferably C, N, O, or S. The degree of substitution can be measured by NMR. The degree of substitution can be measured by mass spectrometry, e.g., MALDI.

[0105] In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a degree of substitution of 1-40, e.g., 1-28 or 4-20, preferably 2-15, more preferably 2-5, and even more preferably 2-4, and bearing hydroxypropyl substituents, the composition comprising a cyclodextrin dimer of the structure CD-L-CD, where L is a cyclodextrin dimer having a substituent (R 1 instead of) and / or through the C3 carbon (R 2 (instead of R 1 , R 2 , and / or R 3 wherein L is 8 atoms in length or less, each of the 8 or less atoms being preferably C, N, O, or S. The degree of substitution can be measured by NMR. The degree of substitution can be measured by mass spectrometry, e.g., MALDI.

[0106] In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a degree of substitution of 1-40, e.g., 1-28 or 4-20, preferably 2-15, more preferably 2-10, and bearing methyl substituents, the composition comprising a cyclodextrin dimer of the structure CD-L-CD, where L is a cyclodextrin dimer having a substituent (R 1 instead of) and / or through the C3 carbon (R 2 (instead of R 1 , R 2 , and / or R 3 wherein L is 8 atoms in length or less, each of the 8 or less atoms being preferably C, N, O, or S. The degree of substitution can be measured by NMR. The degree of substitution can be measured by mass spectrometry, e.g., MALDI.

[0107] In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a degree of substitution of 1-40, e.g., 1-28 or 4-20, preferably 2-15, more preferably 2-5, and even more preferably 2-4, and having sulfobutyl substituents, the composition comprising a cyclodextrin dimer of the structure CD-L-CD, where L is a cyclodextrin dimer having a sulfobutyl substituent through the C2 carbon of each CD subunit (R 1 instead of) and / or through the C3 carbon (R 2 (instead of R 1 , R 2 , and / or R 3 wherein L is 8 atoms in length or less, each of the 8 or less atoms being preferably C, N, O, or S. The degree of substitution can be measured by NMR. The degree of substitution can be measured by mass spectrometry, e.g., MALDI.

[0108] In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a degree of substitution of 1-40, e.g., 1-28 or 4-20, preferably 2-15, more preferably 2-5, and even more preferably 2-4, and having succinyl substituents, the composition comprising a cyclodextrin dimer of the structure CD-L-CD, where L is a bond through the C2 carbon of each CD subunit (R 1 instead of) and / or through the C3 carbon (R 2 (instead of R 1 , R 2 , and / or R 3 wherein L is 8 atoms in length or less, each of the 8 or less atoms being preferably C, N, O, or S. The degree of substitution can be measured by NMR. The degree of substitution can be measured by mass spectrometry, e.g., MALDI.

[0109] In another exemplary embodiment, the present disclosure provides a quaternary ammonium substituent, preferably -CHCH(OH)CHN(CH), having a degree of substitution of 1-40, e.g., 1-28 or 4-20, preferably 2-15, more preferably 2-5, even more preferably 2-4. + The present invention provides a cyclodextrin dimer composition having the structure CD-L-CD, where L is a cyclodextrin dimer having a structure represented by the formula: 1 instead of) and / or through the C3 carbon (R 2 (instead of R 1 , R 2 , and / or R 3 wherein L is 8 atoms in length or less, each of the 8 or less atoms being preferably C, N, O, or S. The degree of substitution can be measured by NMR. The degree of substitution can be measured by mass spectrometry, e.g., MALDI.

[0110] In another exemplary embodiment, the disclosure provides a cyclodextrin dimer composition having a degree of substitution of 0-40, the composition comprising a cyclodextrin dimer of the structure CD-L-CD, where L is a bond through the C2 carbon of each CD subunit (R 1 instead of) and / or through the C3 carbon (R 2 (instead of L) to the large face (bottom) of each CD molecule; where each CD has the structure of formula X, optionally substituted with one or more substituents, where L is 8 atoms or less in length, each of the 8 atoms or less being preferably C, N, O, or S. The cyclodextrin dimer composition can be used to synthesize cyclodextrin dimer compositions substituted with one or more substituents. The degree of substitution can be measured by NMR. The degree of substitution can be measured by mass spectrometry, e.g., MALDI.

[0111] The linker L may have the structure: [ka]

[0112] wherein each R is independently selected from H, X, SH, NH, NH2, or OH, or is absent;

[0113] the connection between each CD and the linker is independently through an O, S, or N bond connected to the C2 or C3 carbon of CD, or through an acetal bond through two adjacent oxygens of CD;

[0114] each X is a substituted or unsubstituted alkane, alkene, or alkyne;

[0115] each A is independently selected from a single, double, or triple covalent bond, S, N, NH, O, or a substituted or unsubstituted alkane, alkene, or alkyne; and

[0116] B is a substituted or unsubstituted five- or six-membered ring, S, N, NH, NR, O, or absent.

[0117] The length of the linker may be 2 to 7. The length of the linker may be 3 to 6. The length of the linker may be 2 or 3. The length of the linker may be 4 to 7. The length of the linker may be 4 to 6. The length of the linker may be 4 to 5. The length of the linker may be 4.

[0118] The linker may be a substituted or unsubstituted alkyl, for example, an unsubstituted alkyl, for example, an unsubstituted butyl. The linker may comprise a triazole.

[0119] The linker has the following structure: [ka] (Formula XI). n1 and n2 can each be 0 to 8, for example, 1 to 4. Preferably, the total length of the linker can be 8 or less, for example, 8, 7, 6, 5, 4, or any range therebetween. In a preferred embodiment, n1 is 1 and n2 is 3.

[0120] In an exemplary embodiment, when the linker comprises a triazole, e.g., has the structure of formula XI, the linker L can be linked at the O2 position of each CD monomer, where n1 and n2 are each 0 to 8, e.g., 1 to 4, and preferably the total length of the linker can be 8 or less, e.g., 8, 7, 6, 5, 4, or any range therebetween, and in a preferred embodiment, n1 is 1 and n2 is 3.

[0121] In exemplary embodiments, when the linker comprises a substituted or unsubstituted alkyl and is preferably 8 atoms or less in length, e.g., 2-7, 2-6, or 4-7 or 4-6 or 4-5, or 8, 7, 6, 5, 4, 3, or 2 in length, or any range of values ​​therebetween, the linker L can be linked at the O2 position of each CD monomer, the O2 position of one CD monomer and the O3 position of the other CD monomer, or the O3 position of both CD monomers, although preferably the linker is a substituted or unsubstituted butyl, more preferably unsubstituted butyl.

[0122] The linker can include any of the linkers depicted in Figure 8D, except that the oxygen atoms depicted at each end of each linker form part of the cyclodextrin monomer to which the linker is attached.

[0123] The cyclodextrin dimer composition may further comprise a cyclodextrin dimer comprising: (a) at least one of methyl, hydroxypropyl, sulfobutyl, succinyl, or —CHCH(OH)CHN(CH) +and / or (b) at least one alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamido, aminocarbonyloxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyloxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl ... and / or (c) at least one of a fatty acid, monosaccharide, or disaccharide, such as a methyl, hydroxypropyl, methyl, ethyl ... +), glucosyl, palmitoyl, phosphate, phosphoryl, amino, azido, sulfate, sulfuryl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromo, and chloro groups.

[0124] The cyclodextrin dimer composition can include a cyclodextrin dimer having a structure according to any one of Formulas I through IX (FIGS. 3B through 3J, respectively).

[0125] Each R 1 , each R 2 , and each R 3 are, unless otherwise specified, independently: (a) methyl, H, hydroxypropyl, sulfobutyl ether, succinyl, succinylhydroxypropyl, -CH2CH(OH)CH2N(CH3)3 +quaternary ammonium such as carboxymethyl, carboxymethylhydroxypropyl, hydroxyethyl, maltosyl, acetyl, carboxyethyl, sulfated, sulfopropyl, sodium phosphate, or glucosyl, and / or (b) hydrogen, alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamido, aminocarbonyloxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl ... The alkyl group may be selected from arylsulfonyl, aryloxy, aralkyloxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroaralkyloxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocycloamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, ureido, carbamate, carboxy, sulfonamido, nitro, cyano, phenoxy, or acetyl groups.

[0126] The linker L can be linked to the C2 carbon of each CD monomer. The linker L can be linked to the C3 carbon of each CD monomer. The linker L can be linked to the C2 carbon of one CD monomer and the C3 carbon of the other CD monomer.

[0127] The cyclodextrin dimer composition may exhibit a higher affinity for 7KC than for cholesterol, optionally as measured by a turbidity test.

[0128] The cyclodextrin dimer compositions may exhibit at least 1.1, 1.5, 2, 3, 4, 5, or 10 times greater affinity for 7KC than for cholesterol. The cyclodextrin dimers may exhibit at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% or greater reduction in relative turbidity of 7KC compared to cholesterol in a turbidity assay.

[0129] The degree of substitution may be 2. The degree of substitution may be 3. The degree of substitution may be 4. The degree of substitution may be 5. The degree of substitution may be 6. The degree of substitution may be 7. The degree of substitution may be 8. The degree of substitution may be 9. The degree of substitution may be 10.

[0130] The cyclodextrin dimer composition may comprise a mixture of cyclodextrin dimer molecules each having a different number of substituent groups and / or different linker attachment points, where the average degree of substitution of the composition is as specified.

[0131] In another aspect, the present disclosure provides a pharmaceutical composition comprising a cyclodextrin dimer composition as disclosed herein and a pharma- ceutically acceptable carrier. The pharmaceutical composition may be suitable for administration to a subject, for example, parenterally (e.g., subcutaneously, intramuscularly, or intravenously), topically, transdermally, orally, sublingually, or buccally, preferably intravenously or subcutaneously, more preferably intravenously. The cyclodextrin dimer composition may be the only active ingredient in the composition. The pharmaceutical composition may consist of, or consist essentially of, the cyclodextrin dimer and the pharma- ceutical acceptable carrier.

[0132] In another aspect, the disclosure provides a method of treatment comprising administering an effective amount of a cyclodextrin composition as disclosed herein to a subject in need of treatment, the subject may be suffering from adverse or toxic effects of 7KC or a condition associated with the adverse or toxic effects of 7KC.

[0133] In another aspect, the present disclosure provides a method for reducing the amount of 7KC in a subject in need of such a reduction, comprising administering to the subject an effective amount of a cyclodextrin dimer composition as disclosed herein or a pharmaceutical composition comprising a cyclodextrin dimer composition as disclosed herein.

[0134] The cyclodextrin dimer composition can be administered to the subject via parenteral (e.g., subcutaneous, intramuscular, or intravenous), topical, transdermal, oral, sublingual, or buccal administration, preferably intravenously.

[0135] The method can include (a) administering to the subject about 1 mg to 20 g, e.g., 10 mg to 1 g, 50 mg to 200 mg, or 100 mg, of the cyclodextrin dimer composition, or (b) 1 to 10 g, e.g., about 2 g, about 3 g, about 4 g, or about 5 g of the cyclodextrin dimer composition, or (c) 50 mg to 5 g, e.g., 100 mg to 2.5 g, 100 mg to 2 g, or 250 mg to 2.5 g of the cyclodextrin dimer composition.

[0136] The method is for treating atherosclerosis / coronary artery disease, arteriosclerosis, coronary artery sclerosis due to coronary artery calcification, heart failure (all stages), Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, vascular dementia, multiple sclerosis, Smith-Lemli-Opitz syndrome, childhood neuronal ceroid lipofuscinosis, lysosomal acid lipase deficiency, cerebrotendinous xanthomatosis, X-linked adrenoleukodystrophy, sickle cell disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Neumann-Pick disease type C, Neumann-Pick disease type D, Neumann-Pick disease type E, Neumann-Pick disease type F ... The method may be used to prevent, treat, and / or ameliorate symptoms of one or more of the following: Type C N-Pick disease, Gaucher disease, Stargardt disease, age-related macular degeneration (dry type), idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, liver injury, liver failure, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, and / or hypercholesterolemia, optionally where the treatment is administered in combination with another treatment. The method may include administering to the subject a second treatment, which may be administered simultaneously or sequentially in either order.

[0137] The method may be for preventing, treating, and / or ameliorating symptoms of atherosclerosis. The cyclodextrin dimer composition may be administered in combination with another therapy for treating or preventing atherosclerosis, such as, but not limited to, an anticholesterol drug, an antihypertensive drug, an antiplatelet drug, a nutritional supplement, or a surgical or behavioral intervention, including those described herein. The anticholesterol drug may include a fibrate or statin drug, an antiplatelet drug, an antihypertensive drug, or a nutritional supplement. Such statins may include ADVICOR® (niacin extended-release / lovastatin), ALTOPREV® (lovastatin extended-release), CADUET® (amlodipine-atorvastatin combination), CRESTOR® (rosuvastatin), JUVISYNC® (sitagliptin / simvastatin), LESCOL® (fluvastatin), LESCOL XL (fluvastatin extended-release), LIPITOR® (atorvastatin), LIVALO® (pitavastatin), MEVACOR® (lovastatin), PRAVACHOL® (pravastatin), SIMCOR® (niacin extended-release / simvastatin), VYTORIN® (ezetimibe / simvastatin), or ZOCOR® (simvastatin).

[0138] The method may be for preventing, treating, and / or ameliorating symptoms of dry age-related macular degeneration. The method may be for preventing, treating, and / or ameliorating symptoms of Stargardt's disease. The cyclodextrin dimer composition may be administered in combination with another treatment for treating or preventing dry AMD or Stargardt's disease, such as LBS-008 (Belite Bio) (a non-retinoid antagonist of retinol binding protein 4), AREDS dietary supplements including vitamin C and vitamin E, beta-carotene, zinc, and copper, AREDS2 dietary supplements including vitamin C and vitamin E, zinc, copper, lutein, zeaxanthin, and omega-3 fatty acids, or combinations thereof.

[0139] The method can be for preventing, treating, and / or ameliorating symptoms of Niemann-Pick disease. The cyclodextrin dimer composition can be administered in combination with another therapy for treating or preventing Niemann-Pick disease, such as one or more of miglustat (ZAVESCA®), HPβCD (TRAPPSOL CYCLO, VTS-270), and physical therapy.

[0140] The method can be for preventing, treating, and / or ameliorating symptoms of Alzheimer's disease. The cyclodextrin dimer composition can be administered in combination with another therapy for the treatment or prevention of Alzheimer's disease, such as a cholinesterase inhibitor (ARICEPT®, EXELON®, RAZADYNE®), and memantine (NAMENDA®), or a combination thereof.

[0141] The method may be for preventing, treating, and / or ameliorating symptoms of heart failure. The cyclodextrin dimer composition may be administered in combination with another therapy for treating or preventing heart failure, such as one or more aldosterone antagonists, ACE inhibitors, ARBs (angiotensin II receptor blockers), ARNIs (angiotensin receptor neprilysin inhibitors), beta blockers, vasodilators, calcium channel blockers, digoxin, diuretics, heart pump drugs, potassium, magnesium, selective sinus node inhibitors, or combinations thereof.

[0142] In another aspect, the disclosure provides a method for making a cyclodextrin dimer composition as described herein, for example a cyclodextrin dimer composition having an unsubstituted or substituted alkyl linker, the method comprising: (a) reacting a protected β-cyclodextrin with a dialkylating agent, thereby producing a protected βCD dimer linked through a lower base, and optionally purifying the protected βCD dimer; (b) deprotecting the protected βCD dimer, thereby producing a deprotected βCD dimer, and optionally purifying the deprotected βCD dimer; and (c) hydroxypropylating the deprotected βCD, thereby producing a cyclodextrin dimer composition, and optionally purifying the cyclodextrin dimer composition. The protected β-cyclodextrin may comprise heptakis(6-O-tert-butyldimethylsilyl)-β-cyclodextrin. The dialkylating agent may comprise a dibromoalkane, optionally 1,4-dibromobutane. Step (a) may be performed under anhydrous conditions and / or using sodium hydride as a base. The purification of step (a) may comprise isotropic normal phase chromatography. Step (b) may be performed using tetrabutylammonium fluoride in tetrahydrofuran (THF). The purification of step (b) may comprise isotropic normal phase chromatography. Step (c) may comprise reacting the deprotected βCD dimer with a hydroxypropylating agent such as propylene oxide, a methylating agent such as methyl iodide, a succinylating agent such as succinic anhydride, a sulfobutylating agent such as 1,4-butane sultone, and / or a quaternary ammonium linking agent such as glycidyl trimethylammonium chloride. The cyclodextrin dimer composition may be a cyclodextrin dimer composition as disclosed herein. The cyclodextrin dimer composition may have a degree of substitution of 1 to 40, for example, 1 to 28 or 4 to 20, preferably 2 to 15, and more preferably 2 to 5 or 2 to 10.

[0143] Step (c) may be carried out in aqueous conditions and may optionally include sodium hydroxide as a base. Step (c) may include one or more of ion exchange resin treatment, activated charcoal clarification, and dialysis.

[0144] In another aspect, the disclosure provides a method for making a cyclodextrin dimer composition as described herein, e.g., a cyclodextrin dimer composition having a triazole linker, comprising: (a) reacting 2-O-(n-azidoalkyl)-βCD with 2-O-(n-alkyne)-βCD, thereby forming a βCD-triazole-βCD dimer having the structure βCD-alk1-triazole-alk2-βCD, and optionally (b) purifying the βCD-triazole-βCD dimer. Step (a) may be performed using a copper(I) catalyst, optionally about 15 mM copper(I). Step (a) may be performed in an aqueous solution. The aqueous solution may include dimethylformamide (DMF), optionally about 50% DMF (v / v). Step (b) may include chromatography. The method may further include, prior to step (a), producing the 2-O-(n-azidoalkyl)-βCD, the method comprising: (1) reacting an n-azido-1-bromo-alkane with β-cyclodextrin, optionally using a catalytic amount of lithium iodide, to thereby produce the 2-O-(n-azidoalkyl)-βCD; and (2) optionally purifying the 2-O-(n-azidoalkyl)-βCD. Step (2) may comprise chromatography. The method may further include forming 2-O-(n-alkyne)-βCD prior to step (a), the method including: (i) reacting n-bromo-1-alkyne with β-cyclodextrin, optionally using a catalytic amount of lithium iodide, to thereby form the 2-O-(n-alkyne)-βCD, and (ii) optionally purifying the 2-O-(n-alkyne)-βCD. Step (2) may include silica gel chromatography. Step (1) may be performed in dry DMSO. The reaction of step (1) may include lithium hydride. The βCD-triazole-βCD dimer has the following structure: [ka] (Formula XII), where n1 can be 1-8 and / or n2 can be 1-8, optionally n1 can be 1, 2, 3, or 4, and / or n2 can be 1, 2, 3, or 4, preferably n1 is 1 and n2 is 3. The length of the triazole linker can be 5-8. The method may further include hydroxypropylating the βCD-triazole-βCD dimer composition, thereby producing a cyclodextrin dimer composition, and optionally purifying the cyclodextrin dimer composition. Step (c) may include reacting the βCD-triazole-βCD dimer with a hydroxypropylating agent such as propylene oxide, a methylating agent such as methyl iodide, a succinylating agent such as succinic anhydride, a sulfobutylating agent such as 1,4-butane sultone, and / or a quaternary ammonium linking agent such as glycidyl trimethylammonium chloride. The cyclodextrin dimer composition may be a cyclodextrin dimer composition as disclosed herein. The cyclodextrin dimer composition may have a degree of substitution of 1 to 40, for example, 1 to 28 or 4 to 20, preferably 2 to 15, more preferably 2 to 5 or 2 to 10.

[0145] Step (c) may be carried out in aqueous conditions and may optionally include sodium hydroxide as a base. The purification in step (c) may include one or more of ion exchange resin treatment, activated charcoal clarification, membrane filtration, and dialysis.

[0146] In another aspect, the disclosure provides a pharmaceutical composition comprising the CD (eg, an HPβCD or another CD of the disclosure) dimer.

[0147] In another aspect, the disclosure provides a pharmaceutical composition comprising a cyclodextrin dimer as disclosed herein and a hydrophobic drug. The hydrophobic drug may comprise a hormone or sterol, e.g., estrogen, an estrogen analog, etc. The cyclodextrin dimer can be present in an amount effective to solubilize the hydrophobic drug.

[0148] The phrase "pharmacologically acceptable" is used herein to indicate compounds, materials, compositions, and / or dosage forms that are suitable for administration to a living organism or living biological tissue, within the scope of reasonable medical judgment, and preferably without significant toxicity, irritation, or allergic reaction. The present invention encompasses methods comprising administering to a patient a cyclodextrin dimer, wherein the cyclodextrin dimer is contained in a pharmaceutical composition. The pharmaceutical compositions of the present invention are formulated with pharma- ceutically acceptable carriers, excipients, and other agents that provide suitable transport, delivery, tolerance, and the like. Numerous suitable formulations can be found in formularies known to medicinal chemists, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Such formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN™), DNA complexes, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and carbowax-containing semi-solid mixtures. (See also Powell et al., J. Pharm. Sci. Technol., 52:238-311, (1998)).

[0149] The phrase "pharmaceutical acceptable carrier" as used herein generally refers to a pharma-ceutically acceptable composition, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, useful for introducing an active agent into the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients in the formulation and not harmful to the patient. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0150] Other examples of materials which may be pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; and (10) glycols. , such as propylene glycol, (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffer solutions, (21) polyesters, polycarbonates, and / or polyanhydrides, and (22) other non-toxic, compatible substances used in pharmaceutical formulations.

[0151] Various auxiliary agents, such as wetting agents, emulsifiers, lubricants (e.g., sodium lauryl sulfate and magnesium stearate), coloring agents, release agents, coating agents, sweeteners, flavoring agents, preservatives, and antioxidants, can also be included in the pharmaceutical compositions. Some examples of pharma- ceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc. In some embodiments, the pharmaceutical formulations include excipients selected from, for example, cellulose, liposomes, micelle-forming agents (e.g., bile acids), and polymeric carriers, for example, polyesters and polyanhydrides. Suspensions may contain, in addition to the active compound, suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, and mixtures thereof. Prevention of microbial activity on the active compound may be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonicity agents, for example, sugars, sodium chloride, and the like, in the composition. Agents that delay absorption, for example, aluminum monostearate and gelatin, may also be included to provide an injectable form that is absorbed over a long period of time.

[0152] The pharmaceutical formulations of the present invention can be prepared by any method known in the pharmaceutical art. The amount of active ingredient (i.e., CD dimer, e.g., HPβCD dimer of the present disclosure or another CD dimer) that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of compound that produces a therapeutic effect. The amount of active compound may range from about 0.1 to 99.9%, more typically about 80 to 99.9%, more typically about 99%. The amount of active compound may range from about 0.1 to 99%, more typically about 5 to 70%, more typically about 10 to 30%. In an exemplary embodiment, a dosage form for intravenous administration is provided that is an aqueous solution at a concentration of 0.5% to 0.001%, e.g., 0.12% to 0.0105%, e.g., about 0.01% (W / V). In an exemplary embodiment, a dosage form is provided for intravenous administration that is an aqueous solution at a concentration of 2.5% to 0.25%, such as 2% to 0.5%, for example about 1% (W / V). In an exemplary embodiment, a dosage form is provided for intravenous administration of up to 500 mL of a 1% solution (W / V), resulting in a dosage of up to 5 grams.

[0153] In an exemplary embodiment, the cyclodextrin dimer can be administered to a patient in an amount of 1 mg to 10 g, for example, 10 mg to 1 g, 100 mg to 500 mg. In an exemplary embodiment, about 400 mg of the cyclodextrin dimer can be administered. In an exemplary embodiment, 1 to 10 g of the cyclodextrin dimer can be administered, for example, about 2 g, about 3 g, about 4 g, or about 5 g. In an exemplary embodiment, 50 mg to 5 g of the cyclodextrin dimer can be administered, for example, 100 mg to 2.5 g, 100 mg to 2 g, 250 mg to 2.5 g, for example, about 1 g.

[0154] Exemplary embodiments provide a single dosage form, which can include the above amounts of cyclodextrin dimer, which can be packaged for individual administration, and optionally further includes a pharma- ceutically acceptable carrier or excipient. The total amount of the cyclodextrin dimer in the single dosage form can be as set forth above, for example, 1 mg to 10 g of cyclodextrin dimer, for example, 10 mg to 1 g, 100 mg to 500 mg, 1 to 10 g, 50 mg to 5 g, 100 mg to 2.5 g, 100 mg to 2 g, 250 mg to 2.5 g, for example, about 1 g, 2 g, about 3 g, about 4 g, or about 5 g.

[0155] Formulations of the present invention suitable for oral administration can be in the form of capsules, cachets, pills, tablets, lozenges (with a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (with an inert base, e.g., gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, each containing a predetermined amount of a compound of the present invention as an active ingredient. The active compound can also be administered as a bolus, electuary, or paste.

[0156] The preparation of such formulations or compositions generally comprises the step of mixing the compound of the present invention with a carrier and, optionally, with one or more auxiliary agents. In the case of solid dosage forms (e.g., capsules, tablets, pills, powders, granules, lozenges, etc.), the active compound is mixed with a finely divided solid carrier and typically can be shaped, e.g., pelletized, tableted, granulated, powdered, or coated. In general, solid carriers can include, for example, sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia gum; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; and (5) solution retarding agents. (6) absorption enhancers, such as quaternary ammonium compounds and surfactants, such as poloxamers and sodium lauryl sulfate, (7) wetting agents, such as cetyl alcohol, glycerol monostearate, and nonionic surfactants, (8) absorbents, such as kaolin and bentonite clay, (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof, (10) coloring agents, and (11) release-controlling agents, such as crospovidone or ethylcellulose. In the case of capsules, tablets, and pills, the pharmaceutical compositions may also include buffering agents. Solid compositions similar to those used to fill soft and hard shell gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols, may also be used.

[0157] A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be formulated with binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface active agents, or dispersing agents.

[0158] Tablets and other solid dosage forms of active agents, such as capsules, pills, and granules, can be optionally scored or formulated with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical manufacturing field. Such dosage forms can also be formulated to provide slow or controlled release of the active ingredient in the dosage form, for example, by using hydroxypropylmethylcellulose in various proportions to provide the desired release profile, or by using other polymer matrices, liposomes, and / or microspheres. Alternatively, such dosage forms can be formulated for rapid release, for example, lyophilized.

[0159] Generally, the dosage form needs to be sterile. For this purpose, the dosage form can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other type of sterile injectable medium immediately before use. The pharmaceutical composition can also contain an opacifying agent, and can be a composition that releases the active ingredient(s) only or preferentially in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, optionally with one or more of the above-mentioned excipients.

[0160] Liquid dosage form is typically the pharma- ceutical acceptable emulsion, microemulsion, solution, suspension, syrup or elixir of active agent.In addition to active ingredient, liquid dosage form can contain inert diluent commonly used in the art, such as water or other solvent, solubilizer and emulsifier, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol and sorbitan fatty acid ester, and mixtures thereof.

[0161] Dosage forms specifically intended for external or transdermal administration can be in the form of, for example, powders, sprays, ointments, pastes, creams, lotions, gels, solutions, or patches. Ophthalmic formulations, such as eye ointments, powders, solutions, and the like, are also contemplated herein. The active compound can be mixed under sterile conditions with a pharma- ceutical acceptable carrier, and with any preservatives, buffers, or propellants, if necessary. External or transdermal dosage forms can contain, in addition to the active compound of the present invention, one or more excipients, such as those selected from animal and vegetable oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide, and mixtures thereof. Sprays can also contain conventional propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0162] For the purposes of the present invention, transdermal patches can offer the advantage of being able to deliver the compounds of the present invention to the body in a controlled manner. Such dosage forms can be made by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. Such flux rates can be controlled by either using rate-controlling membranes or dispersing the compound in a polymer matrix or gel.

[0163] Pharmaceutical compositions of the invention suitable for parenteral administration will generally contain one or more compounds of the invention in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, or with sterile powders which can be reconstituted into sterile injectable solutions or dispersions before use; such sterile powders may contain sugars, alcohols, antioxidants, buffers, bacteriostats, or solutes which render the formulation isotonic with the blood of the intended recipient.

[0164] In some cases, it is desirable to slow the absorption of drugs from subcutaneous or intramuscular injections in order to prolong the effect of the drug. This can be accomplished by using a liquid suspension of crystalline or amorphous material with poor water solubility. In this case, the rate of absorption of the drug depends on the dissolution rate and may therefore depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered dosage form can be accomplished by dissolving or suspending the drug in an oil vehicle.

[0165] Injectable depot forms can be made by forming microencapsulated matrices in which the active compound is contained in biodegradable polymers such as polylactide-polyglycolide. Depending on the drug to polymer ratio and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

[0166] The pharmaceutical composition may be in the form of a microemulsion, which may improve the bioavailability of the active agent. See, for example, (Dorunoo et al., Drug Development and Industrial Pharmacy, 17(12):1685-1713 (1991)) and (Sheen et al., J. Pharm. Sci., 80(7):712-714, (1991)), the contents of which are incorporated herein by reference in their entirety.

[0167] The pharmaceutical compositions may also contain micelles formed from the compounds of the present invention and at least one amphiphilic carrier, where the micelles have an average diameter of less than about 100 nm, in some embodiments, the micelles have an average diameter of less than about 50 nm, or an average diameter of less than about 30 nm, or an average diameter of less than about 20 nm.

[0168] Although any suitable amphiphilic carrier is contemplated herein, the amphiphilic carrier is generally one that has been granted Generally Recognized As Safe (GRAS) status and is capable of both dissolving the compound of the present invention and subsequently microemulsifying it when the solution comes into contact with a complex aqueous phase (e.g., one found in living biological tissue). Typically, amphiphilic components that meet these requirements have an HLB (hydrophilic to lipophilic balance) value of 2 to 20 and their structure contains straight chain aliphatic radicals ranging from C-6 to C-20. Some examples of amphiphilic agents include polyethylene-glycolized fatty glycerides and polyethylene glycols.

[0169] Particularly suitable amphiphilic carriers are saturated and monounsaturated polyethylene glycolated fatty acid glycerides, such as those obtained by full or partial hydrogenation of various vegetable oils. The oils may advantageously consist of tri-, di-, and mono fatty acid glycerides, as well as di- and monopolyethylene glycol esters of the corresponding fatty acids, with particularly suitable fatty acid compositions including 4-10% capric acid, 3-9% capric acid, 40-50% lauric acid, 14-24% myristic acid, 4-14% palmitic acid, and 5-15% stearic acid. Another useful class of amphiphilic carriers includes partially esterified sorbitan and / or sorbitol with saturated or monounsaturated fatty acids (SPAN series) or the corresponding ethoxylated analogues (TWEEN series). Commercially available amphiphilic carriers are specifically contemplated, such as the Gelucire® series, Labrafil®, Labrasol®, or Lauroglycol®, PEG-monooleate, PEG-dioleate, PEG-monolaurates and dilaurates, lecithin, polysorbate 80.

[0170] A CD (e.g., an HPβCD or another CD of the present disclosure) dimer can be administered by any suitable means. Suitable routes of administration include parenteral (e.g., subcutaneous, intramuscular, or intravenous), topical, transdermal, oral, sublingual, or buccal. The administration may also be ocular (e.g., in the form of eye drops), intravitreal, retroorbital, subretinal, or subscleral, which may be suitable for ocular diseases such as AMD.

[0171] A CD (e.g., an HPβCD or another CD of the present disclosure) dimer may be administered to a subject or used in vitro, e.g., on cells or tissue removed from an animal. The cells or tissue can then be introduced into a subject, whether from the subject or another individual from whom the cells or tissue was removed, preferably of the same species.

[0172] The subject (i.e., patient) to be treated is typically an animal, generally a mammal, preferably a human. The subject can also be a non-human animal, including all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. In some embodiments, the subject is a livestock animal, such as cows, pigs, sheep, poultry, and horses, or a companion animal, such as dogs and cats. The subject can be genetically male or female. The subject can be of any age, for example, elderly (generally at least 60, 70, or 80 years old or older), subjects in the transitional stage of adulthood to elderly age, adults, subjects in the transitional stage of pre-adulthood to adulthood age, and pre-adults, including young people (e.g., 13 to 16, 17, 18, or 19 years old), children (generally under 13 years old or prepubertal), and infants. The subject can also belong to any racial group or genotype. Some examples of human racial groups include Caucasian, Asian, Latin American, African, African American, Native American, Semitic, and Pacific Islander. The method of the present invention may be more suitable for some racial groups, for example, Caucasian, particularly Northern European, and Asian.

[0173] The present disclosure includes further substitutions of the dimeric CDs described herein (e.g., the HPβCD of the present disclosure or other CDs). Chemical modification can be performed before or after dimerization. Chemical modification of cyclodextrins can be performed directly on the native beta cyclodextrin ring by reacting a chemical reagent (nucleophile or electrophile) with an appropriately functionalized cyclodextrin (Adair-Kirk et al., Nat. Med., 14(10):1024-5, (2008)); (Khan, et al., Chem. Rev., 98(5):1977-1996, (1998)). To date, more than 1,500 cyclodextrin derivatives have been made by chemical modification of native cyclodextrins. Cyclodextrins can also be prepared by de novo synthesis, which starts with glucopyranose-linked oligopyranosides. Such synthesis can be accomplished using a variety of chemical reagents or biological enzymes, such as cyclodextrin transglycosylase. A review of chemically modified cyclodextrins as drug carriers in drug delivery systems is described, for example, in (Stella, [et al.], Toxicol. Pathol., 36(1):30-42, (2008)), the disclosures of which are incorporated herein by reference in their entirety. U.S. Patent Nos. 3,453,259 and 3,459,731 describe electrically neutral cyclodextrins, the disclosures of which are incorporated herein by reference in their entirety. Other derivatives include cyclodextrins of cationic nature, as disclosed in U.S. Patent No. 3,453,257; insoluble crosslinked cyclodextrins, as disclosed in U.S. Patent No. 3,420,788; and cyclodextrins of anionic nature, as disclosed in U.S. Patent No. 3,426,011, the disclosures of all of which are hereby incorporated by reference in their entirety. Among the cyclodextrin derivatives of anionic nature, carboxylic acids, phosphorous acids, phosphinic acids, phosphonic acids, phosphoric acids, thiophosphonic acids, thiosulfinic acids, and sulfonic acids have been added to the parent cyclodextrin, for example, as disclosed in U.S. Patent No. 3,426,011.Sulfoalkyl ether cyclodextrin derivatives are also described, for example, in U.S. Patent No. 5,134,127, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, cyclic oligosaccharides can have one or more monosaccharide units replaced with a triazole ring, which can be synthesized by an azide-alkyne Huisgen cycloaddition reaction ((Bodine, [et al.], J. Am. Chem. Soc., 126(6):1638-9, (2004)).

[0174] The dimeric cyclodextrins of the present disclosure are connected by a linker. Methods that can be used to connect the CD subunits and the linker are described in the Examples. Additional methods for connecting the CD subunits and the linker are known in the art. (Georgeta et al., J. Bioact. Compat. Pol., 16:39-48. (2001)), (Liu et al., Acc. Chem. Res., 39:681-691. (2006)), (Ozmen et al., J. Mol. Catal. B-Enzym., 57:109-114. (2009)), (Trotta et al., Compos. Interface, 16:39-48. (2009)), each of which is hereby incorporated by reference in its entirety. For example, a linker group having a moiety reactive to hydroxyl groups (e.g., a carboxyl group, which can be activated by a carbodiimide) can be reacted with a cyclodextrin to form a covalent bond with the cyclodextrin. In another example, one or more hydroxyl groups of the cyclodextrin can be activated by known methods (e.g., tosylation) and reacted with a reactive group (e.g., an amino group) of the linker.

[0175] Generally, the linker initially has two reactive moieties that react and bond with each CD monomer. In one embodiment, the linker is first attached to a cyclodextrin to form a linker-cyclodextrin compound, which is isolated, and then the remaining reactive moiety of the linker of the linker-cyclodextrin compound is subsequently reacted with a second cyclodextrin. The second reactive moiety of the linker can be protected during the reaction of the first reactive group, but protection may not be used if the first and second reactive moieties of the linker react differently with the two molecules. The linker can react with both molecules simultaneously to link them together. In other embodiments, the linker can have an additional reactive group for the purpose of linking with other molecules.

[0176] Several linkers are known in the art. Such linkers can be used to link together any of a variety of groups that carry or are functionalized to carry a group that can react and link with a reactive linker. Some groups that can react with a dual reactive linker include amino, thiol, hydroxyl, carboxyl, ester, and alkyl halide groups. For example, aminoamino coupling reagents can be used to link cyclic oligosaccharides and polysaccharides (or any of these groups with, for example, fluorophores, or to each other) where each group being linked carries at least one amino group. Some examples of aminoamino coupling reagents include diisocyanates, alkyl dihalides, dialdehydes, disuccinimidyl suberate (DSS), disuccinimidyl tartrate (DST), and disulfosuccinimidyl tartrate (sulfo-DST), all of which are commercially available. In other embodiments, aminothiol coupling agents can be used to link a thiol group of one molecule to an amino group of another molecule. Some examples of aminothiol coupling reagents include succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) and sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC). In yet other embodiments, thiol-thiol coupling agents can be used to link groups bearing at least one thiol group.

[0177] In some embodiments, the linker is as small as one atom in length (e.g., -O-, -CH2-, or -NH- linkage), or as small as two or three atoms in length (e.g., amide, ureido, carbamate, ester, carbonate, sulfone, ethylene, or trimethylene linkage). In other embodiments, the linker is at least 4, 5, 6, 7, or 8 atoms in length up to, e.g., 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 atoms in length, thereby providing greater freedom of movement. Suitable linker lengths are 2-12 atoms, or 4-8 atoms. In exemplary embodiments, the linker is a C4 alkyl, which may be unsubstituted. In exemplary embodiments, the linker comprises a triazole.

[0178] atherosclerosis

[0179] The exemplary cyclodextrin dimers described herein are useful for preventing or treating diseases such as atherosclerosis. The combination of cyclodextrin dimers with one or more active agents, such as those described herein (e.g., antihyperlipidemic drugs such as statins), is useful for treating any atherosclerosis, as well as the signs, symptoms, or complications of atherosclerosis. Atherosclerosis (also known as arteriosclerotic vascular disease or ASVD and coronary artery disease or CAD) is a condition in which the arterial wall thickens as a result of the accumulation of fatty substances such as cholesterol. Atherosclerosis is a chronic disease that may remain asymptomatic for decades. Atherosclerosis is a syndrome that affects arterial blood vessels, in which a chronic inflammatory response in the arterial wall is largely due to the accumulation of macrophage white blood cells, which is thought to be driven by low density lipoproteins (plasma proteins that carry cholesterol and triglycerides) if fats and cholesterol are not properly removed from the macrophages by functional high density lipoproteins (HDL). Atherosclerosis is commonly referred to as hardening or furring of the arteries. It is caused by the formation of multiple plaques within the arteries.

[0180] The pathophysiology of atherosclerotic lesions is complex, but generally, stable atherosclerotic plaques, which tend to be asymptomatic, are rich in extracellular matrix and smooth muscle cells, whereas unstable plaques are rich in macrophages and foam cells, and the extracellular matrix (also known as the fibrous capsule) that separates the lesion from the arterial lumen is usually fragile and prone to rupture. Rupture of the fibrous capsule exposes thrombogenic materials, such as collagen, to the blood circulation, ultimately inducing luminal thrombus formation. Upon formation, intraluminal thrombi may be completely occluding (e.g., coronary artery occlusion), but more frequently, they may detach and enter the blood circulation, eventually occluding smaller downstream tributaries and causing thromboembolism (e.g., thrombus formation in the carotid artery often causes stroke). Apart from thromboembolism, chronic expansion of atherosclerotic lesions may result in complete occlusion of the lumen. Chronic expansion of the lesion is often asymptomatic until luminal narrowing becomes severe enough that the downstream tissue(s) is inadequately supplied with blood, resulting in ischemia.

[0181] These complications of advanced atherosclerosis are chronic, progressive, and cumulative. In some cases, soft plaques suddenly rupture, causing the formation of a thrombus, which rapidly slows or stops blood flow, resulting in the death of tissues supplied by the artery (infarction). Coronary thrombosis of the coronary arteries is also a common complication that can lead to myocardial infarction. Blockage of the artery going to the brain can result in stroke. In advanced arteriosclerotic disease, claudication due to inadequate blood supply to the legs can occur, which can typically be caused by a combination of both stenosis and aneurysmal segments narrowed by blood clots.

[0182] Although atherosclerosis can affect the entire arterial tree, typically the larger, high pressure vessels, such as the coronary, renal, femoral, cerebral, and carotid arteries, are at higher risk.

[0183] Signs, symptoms, and complications of atherosclerosis include, but are not limited to, elevated plasma total cholesterol, VLDL-C, LDL-C, free cholesterol, cholesterol esters, triglycerides, phospholipids, and the presence of arterial lesions (e.g., plaque), as discussed above. In some cases, elevated cholesterol (e.g., total cholesterol, free cholesterol, and cholesterol esters) may be found in one or more of plasma, aortic tissue, and aortic plaque.

[0184] Some individuals may be predisposed to atherosclerosis. Thus, the present disclosure relates to a method of administering a cyclodextrin dimer alone or in combination with one or more therapeutic agents (e.g., antihyperlipidemic agents such as statins) to a subject to prevent atherosclerosis or its signs, symptoms, or complications. In some embodiments, a subject predisposed to atherosclerosis may exhibit one or more of the following characteristics: advanced age, family history of heart disease, biological conditions, high blood cholesterol. In some embodiments, the biological conditions include high levels of low-density lipoprotein cholesterol (LDL-C) in the blood, low levels of high-density lipoprotein cholesterol (HDL-C) in the blood, high blood pressure, insulin resistance, diabetes, excess weight, obesity, sleep apnea, contributing lifestyle choice(s), and / or contributing behavioral habit(s). In some embodiments, the behavioral habit includes smoking and / or alcohol consumption. In some embodiments, the lifestyle choices include a sedentary lifestyle and / or high stress levels.

[0185] Exemplary embodiments provide for administering the cyclodextrin dimer of the present disclosure, optionally in combination with one or more additional agents, to a patient suffering from atherosclerosis.The patient may exhibit one or more signs or symptoms of atherosclerosis.Atherosclerosis can be diagnosed based on one or more of Doppler ultrasound, ankle-brachial index, electrocardiogram, stress test, angiogram (optionally in combination with cardiac catheterization), computed tomography (CT), magnetic resonance angiography (MRA), or other arterial imaging or blood flow measurement methods.

[0186] Exemplary embodiments provide for the administration of combination therapy comprising the cyclodextrin dimer of the present disclosure and one or more additional therapies. These combination therapies for treating atherosclerosis can include the cyclodextrin dimer of the present disclosure and another therapy for treating or preventing atherosclerosis, such as anticholesterol drugs, antihypertensive drugs, antiplatelet drugs, dietary supplements, or surgery or behavioral intervention, including but not limited to those described below. Further combination therapy includes the CD dimer of the present disclosure and another therapy for treating heart failure, such as one or more aldosterone antagonists, ACE inhibitors, ARBs (angiotensin II receptor blockers), ARNIs (angiotensin receptor neprilysin inhibitors), beta blockers, vasodilators, calcium channel blockers, digoxin, diuretics, heart pump drugs, potassium, magnesium, selective sinus node inhibitors, or combinations thereof. Combination therapies for treating dry age-related macular degeneration (AMD) or Stargardt's disease include the CD dimers of the present disclosure and another therapy for treating AMD, such as LBS-008 (Belite Bio) (a non-retinoid antagonist of retinol binding protein 4), AREDS dietary supplements including vitamin C and vitamin E, beta-carotene, zinc, and copper, AREDS2 dietary supplements including vitamin C and vitamin E, zinc, copper, lutein, zeaxanthin, and dietary supplements having omega-3 fatty acids, or combinations thereof. Combination therapies for treating Alzheimer's disease include the CD dimers of the present disclosure and one or more cholinesterase inhibitors (ARICEPT®, EXELON®, RAZADYNE®) and memantine (NAMENDA®), or combinations thereof. Combination therapy for Niemann-Pick disease includes a CD dimer of the present disclosure and one or more of miglustat (ZAVESCA®), HPβCD (TRAPPSOL CYCLO, VTS-270), and physical therapy. The combination therapy may be administered simultaneously, essentially simultaneously, or sequentially in any order.The combination therapy may be administered simultaneously in a single formulation or separately, optionally in a dosage kit or pack containing each formulation of the combination therapy, for example in a conveniently metered manner to provide one or more single doses of each drug of the combination therapy. The combination therapy may exhibit synergistic effects, in which the effect of the combined therapy exceeds the effect of each treatment alone. The combination therapy generally involves administering an effective amount of the CD dimer and the combined therapy, but the combination therapy may provide effective treatment with reduced dosages of the CD and / or the combined therapy, which may advantageously reduce the side effects normally associated with (non-combined) dosages.

[0187] The combination therapy may include a therapy for the treatment or prevention of a disease or condition associated with atherosclerosis, such as coronary artery disease, angina, heart attack, cerebrovascular disease, transient ischemic attack, and / or peripheral arterial disease. The combination therapy may include a therapy for the treatment or prevention of a condition that may contribute to worsening atherosclerosis formation and / or prognosis, such as hypertension, hypercholesterolemia, hyperglycemia, and diabetes.

[0188] In an exemplary embodiment, the cyclodextrin dimers of the invention are used in combination with anticholesterol drugs, e.g., fibrates or statins, such as ADVICOR® (niacin extended release / lovastatin), ALTOPREV® (lovastatin extended release), CADUET® (amlodipine-atorvastatin combination), CRESTOR® (rosuvastatin), JUVISYNC® (sitagliptin / simvastatin), LESCOL® (flu ... XL (fluvastatin extended release), LIPITOR® (atorvastatin), LIVALO® (pitavastatin), MEVACOR® (lovastatin), PRAVACHOL® (pravastatin), SIMCOR® (niacin extended release / simvastatin), VYTORIN® (ezetimibe / simvastatin), and / or ZOCOR® (simvastatin). The anticholesterol agent may be administered in an amount effective to prevent or treat hypercholesterolemia.

[0189] In an exemplary embodiment, the cyclodextrin dimer of the invention is co-administered with an antiplatelet agent, such as aspirin.

[0190] In an exemplary embodiment, the cyclodextrin dimers of the invention are co-administered with an antihypertensive drug, such as a beta-blocker, angiotensin-converting enzyme (ACE) inhibitor, a calcium channel blocker, and / or a diuretic.

[0191] In an exemplary embodiment, the cyclodextrin dimer of the present invention is co-administered with one or more of a dietary supplement, such as alpha-linolenic acid (ALA), barley, beta-sitosterol, black tea, psyllium, calcium, cocoa, cod liver oil, coenzyme Q10, fish oil, folic acid, garlic, green tea, niacin, oat bran, omega-3 fatty acids (such as eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA)), sitostanol, and / or vitamin C.

[0192] Exemplary combination therapies also include patient behavioral and / or lifestyle interventions, such as counseling and / or support with smoking cessation, exercise, and a healthy diet, e.g., a diet low in low-density lipoprotein (LDL) and optionally high in high-density lipoprotein (HDL).

[0193] Exemplary combination therapies also include surgical intervention, such as angioplasty, stent placement, or both.

[0194] The method of the present invention is useful for treating or preventing atherosclerosis in human subjects. In some cases, the patient is healthy except for the presence of atherosclerosis. For example, the patient may not present any other risk factors for cardiovascular, thrombotic, or other diseases or disorders at the time of treatment. However, in other cases, the patient is selected based on being diagnosed with a disease or disorder caused by or correlated with atherosclerosis, or being at risk of developing such a disease or disorder. For example, at the time of administration of the pharmaceutical composition of the present invention or before administration, the patient may be diagnosed with or identified as being at risk of developing a cardiovascular disease or disorder, such as coronary artery disease, acute myocardial infarction, asymptomatic carotid atherosclerosis, stroke, peripheral arterial occlusive disease, etc. In some cases, the cardiovascular disease or disorder is hypercholesterolemia.

[0195] In other cases, at the time of administration of the pharmaceutical composition of the invention or prior to its administration, the patient may have been diagnosed with atherosclerosis or identified as being at risk for developing atherosclerosis.

[0196] In still other cases, patients to be treated with the methods of the invention are selected based on one or more factors selected from the group consisting of age (e.g., greater than 40, 45, 50, 55, 60, 65, 70, 75, or 80 years of age), race, sex (male or female), exercise habits (e.g., regular exercise or not), other pre-existing medical conditions (e.g., type II diabetes, high blood pressure, etc.), and current medical condition (e.g., current use of statins, e.g., cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, etc., beta blockers, niacin, etc.).

[0197] In the following figures, the following abbreviations are used: Me or ME or me or met: methyl; SB: sulfobutyl; QA = quaternary ammonium, e.g., -CH2CH(OH)CH2N(CH3)3 + , for example -CH2CH(OH)CH2N(CH3)3Cl; SUCC: succinyl; DMSO: dimethylsulfoxide. [Brief description of the drawings]

[0198] [Figure 1A] Structure of cyclodextrin (CD), a cyclic oligosaccharide polymer composed of six (αCD), seven (βCD), or eight (γCD) sugar rings (from left to right). All sugar rings in all CDs are D-glucose molecules. [Figure 1B] In the structure of a substituted CD, R1, R2, and R3 are substituents: [Figure 1C] The structure of a substituted CD is βCD(DS0), i.e., when each of R1, R2, and R3 is hydrogen, [Figure 1D] The structure of the substituted CD, which is hydroxypropyl BCD (DS4); [Figure 1E] The structure of the substituted CD, methyl βCD (DS6), [Figure 1F] The structure of the substituted CD, sulfobutyl BCD (DS4); [Figure 1G]The structure of substituted CD, in which the C2, C3, or C6 position of BCD is substituted with a quaternary ammonium (DS3) group; [Figure 1H] The structure of substituted CD, in which the C2, C3, or C6 position of BCD is substituted with a succinyl (DS1) group; [Figure 1I] The structure of substituted CD, in which the C2, C3, or C6 position of BCD is substituted with a carboxymethyl (DS4) group; and [Figure IJ] The structure of substituted CDs in which the C2, C3, or C6 positions of BCD are substituted with a maltosyl (DS1) group. [Figure 2A] Solubilization of various cholesterol derivatives by HPβCD (DS4.5) monomer was assessed by relative turbidity, tested in PBS, where the absorbance of an aqueous suspension containing 300 uM sterol is defined as 100. Shown in Figure 2A are the results for cholesterol (diamonds), 7KC (squares), vitamin D2 (triangles), vitamin D3 (X), and desmosterol (+). In this and subsequent figures, the data points have been connected by smooth curves to make the results easier to see. [Figure 2B] Solubilization of various sterols by hydroxypropyl-beta cyclodextrin (DS4.5) monomer was assessed by relative turbidity, tested in PBS, where the absorbance of an aqueous suspension containing 300 uM sterol is defined as 100. Figure 2B shows the results for 7-ketocholesterol (7KC (lined X)), 4-beta hydroxycholesterol (4-BOH (squares)), 25-hydroxycholesterol (25OH (triangles)), cholesterol epoxide (diamonds), and 27-hydroxycholesterol (27OH (circles)). [Figure 2C] Solubilization of 7KC by various forms of hydroxypropyl-beta cyclodextrin monomer was assessed by relative turbidity. DS = average number of hydroxypropyl substitutions per molecule. [Figure 2D]Solubilization of cholesterol by various forms of hydroxypropyl-beta cyclodextrin monomer was assessed by relative turbidity. DS = average number of hydroxypropyl substitutions per molecule. [Figure 2E] Predicted relative affinity of HPβCD molecule calculated by molecular docking. DS indicates the number of hydroxypropyl substitutions per molecule. [Figure 2F] In vitro cholesterol solubilization by MeβCD with various degrees of substitution was assessed by relative turbidity. [Figure 2G] In vitro 7KC solubilization by MeβCD with various degrees of substitution was assessed by relative turbidity. [Figure 2H] In vitro cholesterol solubilization by various monomeric βCDs assessed by relative turbidity. [Figure 2I] In vitro solubilization of 7KC by various monomeric forms of βCD assessed by relative turbidity. [Figure 3A] The HPβCD dimer structure of the present disclosure. The beta cyclodextrin monomers are linked through the major face (bottom), i.e., the linker is linked to the C2 or C3 carbon of each CD subunit. The HP substituents are linked (typically in combination) to the C2, C3, and / or C6 carbons. [Figure 3B] Formula I. C2-C2 cyclodextrin dimer with a triazole linker. [Figure 3C] Formula II. C2-C3 cyclodextrin dimer with triazole linker. [Figure 3D] Formula III. C3-C3 cyclodextrin dimer with triazole linker. [Figure 3E] Formula IV. Lower-bottom linked methyl substituted BCD with linker L. [Figure 3F] Formula V. Substrate-linked sulfobutyl-substituted BCD with linker L. Although the sodium salt is depicted, other salts are encompassed by the compounds of the present disclosure. [Figure 3G]Formula VI. Lower-bottom linked succinyl-substituted BCD with linker L. [Figure 3H] Formula VII. Lower base linked maltosyl substituted BCD with linker L. [Figure 3I] Formula VIII. Lower base linked quaternary ammonium substituted BCD with linker L. [Figure 3J] Formula IX. A carboxymethyl substituted BCD linked at the bottom with a linker L. Although the sodium salt is depicted, other salts are encompassed by the compounds of the present disclosure. [Figure 4A] Structural models of the association of HPβCD monomer with a sterol (top) or HPβCD butyl-linked dimer with a sterol (bottom), shown as an illustration of the monomer-sterol and dimer-sterol host-guest interactions. [Figure 4B] Predicted relative affinities of butyl- and triazole-linked dimers towards cholesterol and 7KC. Docking calculations were performed with linked HPβCD dimers with various degrees of hydroxypropylation. [Figure 4C] Illustration of the measurements used in the molecular dynamics simulations. The cyclodextrin and sterol descriptive systems are included to define the O4 atom of the CD (indicated by an arrow), the bottom and top bases of the CD, and the head and tail groups of the sterol. The angle between the O4 face and the ligand indicates how well the ligand fits inside the CD cavity. 30 degrees corresponds to a solubilized "up" configuration (the sterol head associates with the bottom base of the CD and the tail with the top base), and 150 degrees corresponds to a solubilized "down" configuration (the sterol tail associates with the bottom base of the CD and the head with the top base). [Figure 4D]MD simulation of βCD in DS0: Distance between the center of gravity of all O4 oxygens and the center of gravity of the ligand in the up and down directions of the ligand in the GROMOS force field for the native (i.e., unsubstituted) monomeric beta-CD (top); Angle between the vector perpendicular to the plane formed by the O4 atoms of CD and the main axis of the ligand (middle); Lennard-Jones and Coulomb energies of interaction between cyclodextrin and the ligand (bottom). In the graphs included in Figures 4D-4LL, the light line graphs are the results for cholesterol and the dark line for 7KC. [Figure 4E] Ligand solubilization by native monomeric βCD in DS0 in the GROMOS force field. [Figure 4F] Visualization of trajectories of complex formation of 7KC and cholesterol with native βCD in DS0 in both orientations (GROMOS force field). [Figure 4G] The distance between the center of mass of all O4 oxygens and the center of mass of the ligand in the up and down direction of the ligand in the AMBER force field for natural monomeric beta-cyclodextrin DS0; the angle between the vector perpendicular to the plane formed by the O4 atoms of CD and the principal axis of the ligand; the Lennard-Jones and Coulomb energies of interaction between cyclodextrin and the ligand. [Figure 4H] Ligand solubilization by native monomeric βCD in DS0 in the AMBER force field. [Figure 4I] Visualization of trajectories of complex formation of 7KC and cholesterol with native βCD in DS0 in both orientations (AMBER force field). Abbreviations used: "ms": microseconds. [Figure 4J] The distance between the center of mass of all O4 oxygens and the center of mass of the ligand in the up and down direction of the ligand in the GROMOS force field for the translated native monomeric beta-cyclodextrin (DS0); the angle between the vector perpendicular to the plane formed by the O4 atoms of CD and the principal axis of the ligand; the Lennard-Jones and Coulomb energies of interaction between cyclodextrin and the ligand. [Figure 4K]Ligand solubilization by translated monomeric βCD in the GROMOS force field. [Figure 4L] Visualization of trajectories of complex formation with 7KC and cholesterol in both directions and with native βCD (DS0) translated in the GROMOS force field. [Figure 4M] The distance between the center of mass of all O4 oxygens and the center of mass of the ligand in the up and down directions of the ligand in the AMBER force field for the translated native monomeric beta-cyclodextrin (DS0); the angle between the vector perpendicular to the plane formed by the O4 atoms of CD and the principal axis of the ligand; the Lennard-Jones and Coulomb energies of interaction between cyclodextrin and the ligand. [Figure 4N] Ligand solubilization by translated monomeric βCD in the AMBER force field. [Figure 4O] Visualization of trajectories of complex formation of 7KC and cholesterol with native βCD in DS0 in both orientations (AMBER force field). [Figure 4P] The distance between the center of mass of all O4 oxygens and the center of mass of the ligand in the up and down direction of the ligand in the GROMOS force field for natural monomeric beta-cyclodextrin DS0; the angle between the vector perpendicular to the plane formed by the O4 atoms of CD and the main axis of the ligand; the Lennard-Jones and Coulomb energies of interaction between cyclodextrin and the ligand. [Figure 4Q] Ligand solubilization by native monomeric βCD in the GROMOS force field. [Figure 4R] Visualization of trajectories of complex formation of 7KC and cholesterol with native monomeric βCD in both directions (GROMOS force field). [Figure 4S] The distance between the center of mass of all O4 oxygens and the center of mass of the ligand in the up and down direction of the ligand in the AMBER force field between HPβCD(DS5) and cholesterol or 7KC; the angle between the vector perpendicular to the plane formed by the O4 atoms of CD and the principal axis of the ligand; the Lennard-Jones and Coulomb energies of interaction. [Figure 4T]Ligand solubilization with HPβCD(DS5) in the AMBER force field. [Figure 4U] Visualization of trajectories of complex formation of 7KC and cholesterol with HPβCD (DS5) in both directions (AMBER force field). [Figure 4V] The distance between the center of mass of all O4 oxygens and the center of mass of the ligand in the up and down direction of the ligand in the GROMOS force field between HPβCD(DS5) and cholesterol or 7KC; the angle between the vector perpendicular to the plane formed by the O4 atoms of CD and the principal axis of the ligand; the Lennard-Jones and Coulomb energies of interaction. [Figure 4W] Ligand solubilization by monomeric HPβCD (translation) in the GROMOS force field. [Figure 4X] Visualization of trajectories of complex formation of 7KC and cholesterol with monomeric HPβCD (DS5, translation) in both directions (GROMOS force field). [Figure 4Y] Between HPβCD (DS5, translation) and cholesterol or 7KC in the AMBER force field, the distance between the center of mass of all O4 oxygens and the center of mass of the ligand in the up and down direction of the ligand; the angle between the vector perpendicular to the plane formed by the O4 atoms of CD and the principal axis of the ligand; the Lennard-Jones and Coulomb energies of interaction. [Figure 4Z] Ligand solubilization by monomeric HPβCD (DS5, translation) in the AMBER force field. [Figure 4AA] Visualization of trajectories of complex formation of 7KC and cholesterol with monomeric HPβCD (DS5, translation) in both directions (AMBER force field). [Figure 4BB] Between butyl-dimerized HPβCD(DS5) and cholesterol or 7KC, the distance between the center of mass of all O4 oxygens and the center of mass of the ligand in the up and down direction of the ligand in the GROMOS force field; the angle between the vector perpendicular to the plane formed by the O4 atoms of CD and the principal axis of the ligand; the Lennard-Jones and Coulomb energies of interaction. [Figure 4CC]Solubilization of 7KC and cholesterol by butyl-dimerized HPβCD (DS5) in the GROMOS force field. [Figure 4DD] Visualization of trajectories of complex formation of butyl-dimerized HPβCD (DS5) with 7KC and cholesterol in both orientations (GROMOS force field). [Figure 4EE] Distance between the center of mass of all O4 oxygens and the center of mass of the ligand in the up and down direction of the ligand in the AMBER force field for butyl-dimerized HPβCD (DS5); angle between the vector perpendicular to the plane formed by the O4 atoms of CD and the principal axis of the ligand; Lennard-Jones and Coulomb energies of interaction between cyclodextrin and the ligand. [Figure 4FF] Ligand solubilization with butyl-dimerized HPβCD (DS5) in the AMBER force field. [Figure 4GG] Visualization of trajectories of complex formation of 7KC and cholesterol with butyl-dimerized HPβCD (DS5) in both orientations (AMBER force field). [Figure 4HH] The distance between the center of mass of all O4 oxygens and the center of mass of the ligand in the up and down direction of the ligand in the GROMOS force field for dimerized hydroxypropyl beta-cyclodextrin (DS5, translation); the angle between the vector perpendicular to the plane formed by the O4 atoms of CD and the principal axis of the ligand; the Lennard-Jones and Coulomb energies of interaction between cyclodextrin and the ligand. [Figure 4II] Solubilization of 7KC and cholesterol by butyl-dimerized HPβCD (DS5, translation) in the GROMOS force field. [Figure 4JJ] Visualization of trajectories of complex formation of 7KC and cholesterol with butyl-dimerized HPβCD (DS5, translation) in both directions (GROMOS force field). [Figure 4KK]The distance between the center of mass of all O4 oxygens and the center of mass of the ligand in the up and down direction of the ligand in the AMBER force field for butyl-dimerized hydroxypropyl beta-cyclodextrin (DS5, translation); the angle between the vector perpendicular to the plane formed by the O4 atoms of CD and the principal axis of the ligand; the Lennard-Jones and Coulomb energies of interaction between cyclodextrin and the ligand. [Figure 4LL] Solubilization of 7KC and cholesterol by butyl-dimerized HPβCD (DS5, translation) in the AMBER force field. [Figure 4MM] Visualization of trajectories of complex formation of 7KC and cholesterol with butyl-dimerized HPβCD (DS5, translation) in both directions (AMBER force field). [Figure 4NN] For unsubstituted (DS0) butyl-dimerized beta-cyclodextrin, the distance between the center of mass of all O4 oxygens and the center of mass of the ligand in the up and down direction of the ligand in the GROMOS force field; the angle between the vector perpendicular to the plane formed by the O4 atoms of CD and the principal axis of the ligand; the Lennard-Jones and Coulomb energies of interaction between cyclodextrin and the ligand. [Figure 4OO] Visualization of trajectories of complex formation of 7KC and cholesterol with unsubstituted (DS0) butyl-dimerized βCD in both orientations (AMBER force field). [Figure 4PP] MD analysis of triazole-linked DS0 cyclodextrin. The angles between the vector perpendicular to the plane formed by the O4 atom of CD and the ligand principal axis in the ligand up- and down-direction in the GROMOS force field for unsubstituted (DS0) dimerized beta-cyclodextrin, as well as the Lennard-Jones and Coulomb energies of interaction between cyclodextrin and ligand. [Figure 4QQ] The trajectories of complex formation of 7KC and cholesterol with triazole dimerized βCD (DS0) in both orientations were visualized. [Figure 4RR]MD analysis of triazole-linked HPβCD(DS4). The angles between the vector perpendicular to the plane formed by the O4 atom of CD and the ligand's principal axis in the ligand's up- and down-directions in the GROMOS force field for the translated dimerized hydroxypropyl beta cyclodextrin (DS4), as well as the Lennard-Jones and Coulomb energies of interaction between the cyclodextrin and the ligand. [Figure 4SS] Trajectories were visualized for 100 ns in both directions for the interaction between triazole-linked hydroxypropyl βCD dimer (DS4) and 7KC / cholesterol. [Figure 5A] Relative affinities of a wide range of possible dimerized MeβCD molecules predicted by molecular docking. Affinity of butyl-linked (left) and triazole-linked (right) dimers for sterols. Docking calculations were performed with varying degrees of methylation of the linked MeβCD dimers. Comparison with cholesterol (dotted line) and 7KC (solid line). [Figure 5B] MD simulation describing the interaction between butyl-linked methyl βCD dimer (DS4) and 7KC / cholesterol for 100 ns in both up and down directions. Legend: 7KC (dark line) and cholesterol (light grey line), dashed lines are down direction, solid lines are up direction. [Figure 5C] The trajectories of butyl-linked methyl-βCD dimer (DS4) and 7KC / cholesterol in both the up and down directions were visualized. [Figure 5D] MD simulation describing the interaction between triazole-linked methyl-βCD dimer (DS4) and 7KC / cholesterol for 100 ns in both the up and down directions. Legend as in Figure 5B. [Figure 5E] The trajectories of triazole-linked methyl-βCD dimer (DS4) and 7KC / cholesterol in both the up and down directions were visualized. [Figure 6A]Relative affinities of a wide range of possible dimerized sulfobutylated βCD molecules predicted by molecular docking. Affinities of butyl-linked and triazole-linked dimers towards sterols. Docking calculations were performed with varying degrees of sulfobutylation of linked SBβCD dimers. Comparison with cholesterol (dotted line) and 7KC (solid line). [Figure 6B] MD simulation describing the interaction between butyl-linked sulfobutyl βCD dimer (DS4) and 7KC / cholesterol for 100 ns in both the up and down directions. Legend as in Figure 5B. [Figure 6C] The trajectories of butyl-linked sulfobutyl βCD dimer (DS4) and 7KC / cholesterol in both the up and down directions were visualized. [Figure 6D] MD simulation describing the interaction between triazole-linked sulfobutyl βCD dimer (DS4) and 7KC / cholesterol for 100 ns in both the up and down directions. Legend as in Figure 5B. [Figure 6E] The trajectories of triazole-linked sulfobutyl βCD dimer (DS4) and 7KC / cholesterol in both the up and down directions were visualized. [Figure 7A] MD simulation describing the interaction between butyl-linked quaternary ammonium βCD dimer (DS4) and 7KC / cholesterol for 100 ns in both up and down orientations. Legend as in Figure 5B. [Figure 7B] The trajectories of butyl-linked quaternary ammonium βCD dimer (DS4) and 7KC / cholesterol in both the up and down directions were visualized. [Figure 7C] MD simulation describing the interaction between triazole-linked quaternary ammonium βCD dimer (DS4) and 7KC / cholesterol for 100 ns in both up and down orientations. Legend as in Figure 5B. [Figure 7D] The trajectories of triazole-linked quaternary ammonium βCD dimer (DS4) and 7KC / cholesterol in both the up and down orientations were visualized. Legend is as in Fig. 5B. [Figure 8A] The hydroxypropylation sites of the triazole-linked and butyl-linked dimers of DS8 and DS4 were varied, including hydroxypropylation of only the minor or major face. Docking calculations were performed with various hydroxypropylation sites of the HPβCD dimer to determine the effect of changing the position of the hydroxypropyl group on sterol binding. The site of hydroxypropylation varies in reality due to the stochastic nature of the substitutions on a nearly symmetric molecule. Labels "C", "D", and "E" indicate mutant structures that have equal HP group distribution between the minor and major faces of the CD monomer but are distinct from each other. Legend: The top (light grey) bar represents values ​​for cholesterol, and the bottom (dark) bar represents values ​​for 7KC. [Figure 8B] Docking calculations were performed with alkyl-linked HPβCD dimers (DS5) of various lengths, various degrees of hydroxypropylation sites, and various lengths of carbon-only linkers. The bars in each cluster are, from top to bottom, DS20, DS16, DS12, DS8, DS4, and DS0. [Figure 8C] Varying the length of the triazole-linked HPβCD dimer (DS5). Docking calculations were performed with varying lengths of the triazole linker by changing the number of carbons on either side of the triazole ring. The length of each side of the linker is identified as n1 or n2, cholesterol is represented by a striped bar, while 7KC is represented by a solid bar. The bars within each cluster, from top to bottom, are N1=2 and 7KC; N1=2 and cholesterol; N1=3 and 7KC; N1=3 and cholesterol; N1=4 and 7KC; and N1=4 and cholesterol. [Figure 8D]Linkers tested in docking calculations (FIG. 8E) to identify linker-dependent variability in sterol binding. Linked HPβCD dimer compositions for DS4 and DS8 hydroxypropyl dimers were compared with a 4-carbon linker (Linker W, where n=3 carbons) and triazole-linked dimers (Linker U, where n=1 carbon, and Linker V, where n=1 carbon) based on various side chain, ring, double bond additions, and / or substitutions with sulfur, nitrogen, and / or oxygen atoms in the linker composition. [Figure 8E] Docking results for various HPβCD dimers with different linkers. 7KC preference of linked HPβCD dimers for DS4 and DS8 hydroxypropyl dimers compared with 4-carbon linker (Linker W, where n=3 carbons) and triazole linked dimers (Linker U, where n=1 carbon, and Linker V, where n=1 carbon) based on linkers A-W (FIG. 8D). Legend: Top (light grey) bars represent values ​​for cholesterol, bottom (dark) bars represent values ​​for 7KC. [Figure 8F] Molecular docking predictions of the effect of the CD binding sites of triazole- and butyl-linked dimers on the predicted affinity for cholesterol and 7KC. Docking calculations were performed for symmetric butyl- and triazole-linked dimers, thus allowing three possible linkages: C2-C2, C3-C3, and C2-C3, which is the same as the C3-C2 linked dimer due to the symmetry of the linkers. Legend: The top (light grey) bar represents the value for cholesterol, and the bottom (dark) bar represents the value for 7KC. [Figure 8G]Varying the attachment points of the asymmetric linkers. Docking calculations were performed on dimers linked with asymmetric 4-atom linkers C, D, K, N, and R (see FIG. 8D). For these asymmetric linkers, four possible linkages are possible: C2-C2, C3-C3, C2-C3, and C3-C2. In these cases, C3-C2 is not the same as C2-C3 due to the asymmetry of the linkers. Legend: Each group of sticks, from top to bottom, represents cholesterol with C3 / C2 linkage; cholesterol with C2 / C3 linkage; 7KC with C3 / C2 linkage, and 7KC with C2 / C3 linkage. [Figure 8H] MD simulation describing the interaction between nitrogen-linked hydroxypropyl βCD dimer (DS4) and 7KC / cholesterol for 100 ns in both orientations (linker O). Legend as in Figure 5B. [Figure 8I] The trajectories of nitrogen-linked hydroxypropyl βCD dimer (DS4) and 7KC / cholesterol in both orientations were visualized (Linker O). [Figure 9A] 7KC specificity predicted by molecular docking for a wide range of linked dimers. 7KC specificity is maintained across a wide range of linker and substituent types for βCD dimers. The order of the sticks within each cluster, from left to right, is: sulfobutyl (DS4); hydroxypropyl (DS4); methyl (DS4); quaternary ammonium (DS4); succinyl (DS4); carboxymethyl (DS4); maltosyl (DS4). [Figure 9B] Sterol affinity for alkyl linkers of various lengths with hydroxypropyl, methyl, and sulfobutyl substitutions (DS4) modeled by molecular docking. The order of the rods within each cluster, from top to bottom, is methyl, sulfobutyl, and hydroxypropyl. [Figure 9C] Sterol affinity for triazole linkers of various lengths with hydroxypropyl, methyl, and sulfobutyl substitutions (DS4) was modeled by molecular docking. The order of the sticks is as in Figure 9B. [Figure 9D]Predicted 7KC specificity of butyl- and triazole-linked βCD dimers at multiple substitution positions, modeled by molecular docking. The x-axis indicates the affinity for 7KC relative to cholesterol. In each population, the top bar represents triazole and the bottom bar represents butyl. [Figure 9E] Docking screening of other βCD variants. 7KC specificity is seen for butyl- and triazole-linked βCD dimers, even with the combination of substituents; as modeled by molecular docking. X-axis indicates affinity for 7KC over cholesterol. Bar order is as in Figure 9D. [Figure 10A] A synthetic strategy for hydroxypropylated dimers connected with one linker unit based on 1,4-dibromobutane (leading to butyl-linked HPβCD dimers). [Figure 10B] A synthetic strategy for hydroxypropylated dimers connected with one linker unit based on 3-azido-1-bromo-propane (leading to triazole-linked HPβCD dimers). [Figure 10C] TLC analysis was used to estimate the reaction progress and conversion. [Figure 10D] MALDI spectrum of TBDMS-βCD-pig-βCD-TBDMS. [Figure 10E] TLC analysis was used to estimate the reaction progress and conversion. [Figure 10F] MALDI spectrum of synthetic large surface butyl-linked beta-cyclodextrin (βCD-buta-βCD), DS=0. [Figure 10G] MALDI spectrum of synthetic large surface butyl-linked hydroxypropyl beta-cyclodextrin HP (βCD-buta-βCD), DS approximately 3. Some peaks are not labeled due to crowding, but show the expected molecular weight. [Figure 10H] MALDI spectrum of synthetic large surface butyl-linked hydroxypropyl beta-cyclodextrin HP (βCD-buta-βCD), DS approximately 6. Some peaks are not labeled due to crowding, but show the expected molecular weight. [Figure 10I] MALDI spectrum of synthetic large surface butyl-linked hydroxypropyl beta-cyclodextrin HP (βCD-buta-βCD), DS approximately 8. [Figure 10J] 1H-NMR spectrum of HP(βCD-pig-βCD) (D2O, 298K), signals are labeled. [Figure 10K] The structure of one of the predicted isomers of HP(βCD-pig-βCD) and DS8, as well as the nomenclature system of the linker, are given. [Figure 10L] DEPT-edited HSQC spectrum of HP(βCD-Pig-βCD) (D2O, 298K). [Figure 10M] DEPT-edited HSQC spectrum of HP(βCD-pig-βCD), linker frequencies assigned by thermal mapping (D2O, 298 K). [Figure 10N] DEPT-edited HSQC spectrum of HP(βCD-Pig-βCD), fully assigned (D2O, 298K). [Figure 10O] MALDI spectrum of synthetic large triazole-linked beta-cyclodextrin (βCD-(triazole)1-BCD, DS=0). [Figure 10P] MALDI spectrum of synthetic large triazole-linked beta-cyclodextrin HP (βCD-triazole-βCD), DS ~ 3. Some peaks are not labeled due to crowding, but show the expected molecular weight. [Figure 10Q] MALDI spectrum of synthetic large triazole-linked beta-cyclodextrin HP (βCD-triazole-βCD), DS ~ 7. Some peaks are not labeled due to crowding, but show the expected molecular weight. [Figure 10R] DEPT-edited HSQC spectrum of HP(βCD-triazole-βCD), DS ∼7, with the linker assigned (DO, 298 K) (left), and TLC with the linker fraction (right). [Figure 10S] TLC plate showing reaction monitoring, with spots assigned. [Figure 10T] MALDI spectrum of 2-O-propargyl-β-CD. [Figure 10U] 1H-NMR spectrum of 2-O-propargyl-β-CD, with some peaks highlighted (DMSO-d6, 298K). [Figure 10V] BCD-(triazole) 1H-NMR spectrum of the BCD dimer (D2O, 298K). [Figure 10W] H-NMR spectrum (DO, 298 K) of HP(βCD-triazole-βCD) with labeled signals, corresponding to the molecule labeled CD-triazole-CD, DS3 in Figure 16B and elsewhere. [Figure 10X] 1H-NMR spectrum (DO, 298 K) of HP(βCD-triazole-βCD) with labeled signals. This corresponds to the molecule labeled CD-triazole-CD, DS6 in Figure 16B. [Figure 10Y] 1H-NMR spectrum (DO, 298 K) of HP(βCD-triazole-βCD) with labeled signals, corresponding to the molecule labeled CD-triazole-CD, DS7 in Figure 16B. [Figure 11A] Synthesis scheme of methylated βCD dimer. [Figure 11B] TLC analysis was used to estimate the reaction progress and conversion. [Figure 11C] MALDI spectrum of the final compound obtained in reaction (A). [Figure 11D] MALDI spectrum of the final compound obtained in the reaction in (B). [Figure 11E] MALDI spectrum of the final compound obtained in reaction (C). [Figure 11F] (D) MALDI spectrum of the final compound obtained in the reaction. [Figure 11G] MALDI spectral overlay of reaction traces: Reaction A (DS0), Reaction B (DS1), Reaction C (DS2), and Reaction D (DS4, 5, 6). [Figure 11H]MALDI spectrum of Me(βCD-triazole-βCD) dimer. [Figure 11I] Zoomed-in MALDI spectrum of Me(βCD-triazole-βCD) dimer. [Figure 11J] Structure of one of the possible isomers of the Me(βCD-triazole-βCD) dimer, with atoms numbered. [Figure 11K] HNMR spectrum of Me(βCD-triazole-βCD) dimer, all frequencies assigned. [Figure 11L] HNMR spectrum and integration values ​​of Me(βCD-triazole-βCD) dimer. [Figure 11M] DEPT-edited HSQC spectrum of Me(βCD-triazole-βCD) dimer, fully assigned. [Figure 11N] COSY-NMR spectrum of Me(βCD-triazole-βCD) dimer, assigned. [Figure 12A] Synthesis scheme of sulfobutylated βCD dimer. [Figure 12B] TLC analysis was used to estimate the reaction progress and conversion rate of the SBβCD prototype. [Figure 12C] Fingerprint chromatogram overlay analysis used to estimate the DS of SB-βCD prototype reaction A. [Figure 12D] Fingerprint chromatogram overlay analysis used to estimate the DS of SB-βCD prototype reaction B. [Figure 12E] MALDI of SB-βCD dimer (low DS). [Figure 12F] One of the possible isomers of the SB-βCD dimer, with atoms numbered. [Figure 12G] HNMR spectrum of the sulfobutylated dimer (low DS), fully assigned (D2O, 298K). [Figure 12H] HNMR spectrum and integrals (D2O, 298K) of sulfobutylated dimer (low DS). Calculation of DS value based on NMR is explained. [Figure 12I] DEPT-edited HSQC spectrum of SB dimer (low DS), fully assigned (D2O, 298K). [Figure 12J] COSY spectrum of the SB dimer (low DS), fully assigned (D2O, 298K). [Figure 12K] MALDI spectrum of SB dimer (high DS). [Figure 12L] Structure of one of the possible isomers of the SB dimer (DS3), with atoms numbered. [Figure 12M] HNMR spectrum of SB dimer (high DS), fully assigned (D2O, 298K). [Figure 12N] HNMR spectrum and integral value (D2O, 298K) of SB dimer (high DS). Calculation of DS value based on NMR is explained. [Figure 12O] DEPT-edited HSQC spectrum of SB dimer (high DS), fully assigned (D2O, 298K). [Figure 12P] This is a COSY spectrum of the SB dimer (high DS), all assigned (D2O, 298K). [Figure 13A] Synthesis scheme of quaternary ammonium β-cyclodextrin dimer. [Figure 13B] MALDI spectrum of quaternary ammonium β-cyclodextrin dimer reaction A. [Figure 13C] MALDI spectrum of quaternary ammonium β-cyclodextrin dimer reaction B. [Figure 13D] MALDI spectrum of quaternary ammonium β-cyclodextrin dimer reaction C. [Figure 13E] MALDI spectrum of quaternary ammonium β-cyclodextrin dimer reaction D. [Figure 13F] MALDI spectrum of quaternary ammonium β-cyclodextrin dimer. [Figure 13G] Structure of one of the possible isomers of the QA dimer isomer (DS3), with atoms numbered. [Figure 13H]HNMR spectrum of the QA dimer, fully assigned (D2O, 298K). [Figure 13I] HNMR spectrum and integrals of QA dimer (D2O, 298K). Calculation of DS value based on NMR is explained. [Figure 13J] DEPT-edited HSQC spectrum of the QA dimer, fully assigned (D2O, 298K). [Figure 13K] Partially assigned COSY spectrum of the QA dimer (D2O, 298 K). [Figure 14A] Synthesis scheme of succinylated dimer. [Figure 14B] MALDI of succinylated dimer reaction A. [Figure 14C] MALDI of succinylated dimer reaction B. [Figure 14D] MALDI of succinylated dimer reaction C. [Figure 14E] MALDI of succinylated dimer reaction D. [Figure 14F] MALDI of succinylated dimers. [Figure 14G] Structure of one of the possible isomers of the SUCC dimer isomer (DS3), with atoms numbered. [Figure 14H] HNMR spectrum of the succinylated dimer, fully assigned (D2O, 298K). [Figure 14I] HNMR spectrum and integrals of the succinylated dimer (D2O, 298K). Calculation of DS value based on NMR is explained. [Figure 14J] DEPT-edited HSQC spectrum of the succinylated dimer, fully assigned (D2O, 298K). [Figure 14K] Partially assigned COSY spectrum of the succinylated dimer (D2O, 298 K). [Figure 15A] 7KC hemocyte efflux concentration after incubation with DS8 HPβCD dimer. [Figure 15B] 7KC hemocyte efflux concentration after incubation with HPβCD monomer. [Figure 15C] Plasma cholesterol is not perturbed by incubation with HPβCD dimers Plasma cholesterol was measured by mass spectrometry to determine cholesterol efflux from blood cells caused by incubation with HPβCD dimers. [Figure 15D] Hemolysis assay as a measure of potential cytotoxicity of various butyl- and triazole-linked HPβCD and methyl dimers. [Figure 15E] Hemolysis assay as a measure of potential cytotoxicity of various triazole-linked βCD dimers: unsubstituted βCD, SBβCD (low and high DS), QAβCD, and succinylated βCD dimer. [Figure 16A] Butyl-linked HPβCD dimers are much superior to monomeric HPβCD in solubilizing 7KC and cholesterol. Dimers with degrees of substitution of about 3, about 6, and about 8 were tested. [Figure 16B] The triazole-linked HPβCD dimer is much superior to monomeric HPβCD in solubilizing 7KC and cholesterol. Dimers with degrees of substitution of 0, about 3, about 5, and about 6 were tested. HPBCD denotes monomeric HPβCD and CD-triazole-CD denotes the triazole-linked dimer with the indicated degrees of substitution. [Figure 16C] Solubilization of various cholesterol derivatives and oxysterols by butyl-linked HPβCD dimer (DS ~ 8). Results are shown for cholesterol, 7-ketocholesterol (7KC), vitamin D2, vitamin D3, desmosterol, 27-hydroxycholesterol (27OH), 4-beta-hydroxycholesterol (4BOH), 25-hydroxycholesterol (25OH), and cholesterol epoxide. [Figure 16D] Compound solubilization with butyl-linked HPBCD dimer (DS approximately 8). The sterol hormones tested were estradiol, estriol, estrone, pregnenolone, and progesterone. [Figure 16E]Butyl-linked HPβCD dimer (DS approx. 3) ("DS3 butyl dimer") has affinity and specificity for 7KC. HPBCD denotes monomeric HPβCD. [Figure 16F] The triazole-linked HPβCD dimer (DS approximately 3) has affinity and specificity for 7KC. [Figure 16G] The triazole-linked MeβC dimer (DS approx. 3) ("Methyl dimer DS3") is as effective as the HPβCD dimer (DS approx. 3) ("HPBCD dimer DS3") at solubilizing 7KC and cholesterol. [Figure 16H] Triazole-linked unsubstituted βCD ("CD-triazole-CD, DS0"), triazole-linked SBβCD dimer (DS 3.4) ("SBCD-triazole-CD, DS3.4"), triazole-linked QaβCD dimer (DS 2) (QACD-triazole-CD, DS2"), and triazole-linked succinylated βCD dimer (DS 2) ("SUCC CD-triazole-CD, DS2") all have specificity for 7KC over cholesterol in vitro. Triazole-linked SBβCD dimer (DS 14.6) ("SB CD-triazole-CD, DS14.6) has low affinity for both cholesterol and 7KC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0199] definition

[0200] Unless otherwise stated, the following terms used in this Application, including the specification and claims, have the definitions given herein.

[0201] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0202] Linker length. As used herein, the length of a linker or equivalently "linker length" refers to the number of linker atoms in the shortest path through the linker connecting two CD subunits of a cyclodextrin dimer. For clarity, the length of the linker does not include the oxygen atoms (or other atoms that may replace the oxygen) of each CD subunit to which the linker is attached. For example, in FIG. 3B, the linker length is 3+n1+n2, reflecting the shortest path through the triazole ring. If the linker is attached at multiple points to one or both of the cyclodextrin monomers, the linker length is the shortest path connecting the two cyclodextrins among all possible paths that may start and end at different locations on each cyclodextrin.

[0203] Head-to-Head Cyclodextrin Dimer. As used herein, the term "head-to-head cyclodextrin dimer" refers to a CD dimer in which two CD monomers are typically linked through the C2 and / or C3 carbons of each CD monomer and linked through the large face (bottom) of the cyclodextrin.

[0204] Tail-to-Tail Cyclodextrin Dimer. As used herein, the term "tail-to-tail cyclodextrin dimer" refers to a CD dimer in which two CD monomers are typically linked through the C6 carbon of each CD monomer and bonded through the small face (top and bottom) of the cyclodextrin.

[0205] Head-to-Tail Cyclodextrin Dimer. As used herein, the term "head-to-tail cyclodextrin dimer" refers to a CD dimer in which two CD monomers are attached at opposite ends, i.e., one monomer is attached from the small face (top base), typically through the C6 carbon, and the other monomer is attached from the large face (bottom base), typically through the C2 and / or C3 carbons.

[0206] Degree of Substitution (DS). As used herein, "degree of substitution" or "DS" refers to the number of a given subgroup attached to a monomer or dimer. For example, MeβCD(DS3) refers to a βCD having an average of three methyl R groups attached to O2, O3, or O6 of the CD, while HPβCD(DS3) refers to a monomer or dimer having an average of three hydroxypropyl groups attached to O2, O3, or O6 of the CD. When referring to a CD dimer, unless otherwise stated, DS is used to refer to the total average substituents of both constituent monomers, and all substituents are included (e.g., in the case of mixed substituents, such as a mixture of hydroxypropyl and methyl substituents, all are included in the number). Terms such as "degree of substitution with a substituent X" refer to the average number of that substituent X per CD dimer, i.e., other substituents, although they may be present, are not included in the number. DS can be measured by mass spectrometry (e.g., matrix-assisted laser desorption ionization, "MALDI") or by NMR. MALDI is preferred for cyclodextrin derivatives with substituents that give a more typical Gaussian distribution of ions in the mass spectrum, as shown, for example, in Figures 10G-I, 10P-Q, 11C-G, 11I, 12E, and 12K for methyl, hydroxypropyl, and sulfobutyl substituents. The average DS, when determined by MALDI, is calculated by averaging the peak heights of the peaks corresponding to each DS type of the CD in question. In other instances, there may be less commonly seen ion peak patterns, for example due to the formation of various adducts, fragmentation, cleavage products, etc. Other mass spectrometry techniques can be utilized to potentially circumvent such problems. Alternatively, NMR can be used to determine DS values, which was preferred for succinyl and quaternary ammonium groups, given the more complex MS spectra observed with MALDI. Thus, to achieve the calculation of the average degree of substitution (DS), the peaks corresponding to the protons of the core dimer are first identified and the measured values ​​are scaled so that the peak areas correspond to the known number of such protons in the structure. The signals corresponding to the protons in the substituents are then examined and appropriately scaled to obtain the average degree of substitution.In simpler cases, the clearly resolved peaks corresponding to the substituent protons are identified and already scaled as above, then divided by the number of protons present in the peak to obtain the average number of substituents. For example, in the case of hydroxypropyl substituents, the peaks identified as corresponding to the 14 protons of the core structure (aromatic region of glucopyranose) are identified and normalized to 14 by signaling, then the peaks corresponding to the 3 protons of the methyl substituents are identified, and finally the area of ​​the peaks is divided by 3 to obtain the average number of hydroxypropyl groups present per molecule. In other cases, the substituent peaks and the cyclodextrin core peaks may be very close together or overlap. In this case, the number of contributing protons of the cyclodextrin core structure is identified and then subtracted from the peak area (the peak area is already scaled to an integrated area of ​​1 per proton), and then the remaining area is divided by the number of contributing protons to obtain the average degree of substitution. For example, in the case of the methyl substituent (shown in Figures 11K-11L), a collection of peaks was identified that corresponded to the three methyl hydrogens of the substituent and, additionally, to a cluster of 86 protons of the core cyclodextrin dimer structure. As with the hydroxypropyl substituent example, the peaks identified as corresponding to 14 protons of the core structure (the aromatic region of glucopyranose) were identified and normalized to 14 by signaling; the area of ​​the peak containing the methyl hydrogens and the core cyclodextrin hydrogens was determined to be 92.77, and subtracting the 86 protons of the core cyclodextrin structure from this signal left 6.77; after dividing by the three protons of each methyl group, the average degree of substitution was estimated to be 2.26. For HP and ME substituted CDs, the integrals were divided by 3, for QA, the integrals were divided by 9, for SB, the integrals were divided by 2, and for SUCC, the integrals were divided by 4. The above calculations are straightforward to apply to other types of substituents based on the identification of peaks corresponding to protons in the substituent structure. NMR-based DS calculations are illustrated in Figures 10X-10Y, 11L, 12H, 12N, 13I, and 14I.CD compositions, such as CD dimer compositions (defined below), may contain mixtures of molecules each substituted with a different number of substituents, in which case the DS value is expressed as the average (median) of the number of substitutions. Fractional DS values ​​reflect the case where the median value can be between integer substituents. Unless otherwise stated, integer DS values ​​refer to CD compositions with DS numbers rounded to the nearest integer. For example, DS4 indicates a DS value of at least 3.5 and less than 4.5.

[0207] Average degree of substitution with hydroxypropyl groups. As used herein, the term "average degree of substitution with hydroxypropyl groups" refers to the degree of substitution as defined above, disregarding any substituents other than the hydroxypropyl group. Similarly, references to the average degree of substitution with a specified substituent refer to the degree of substitution as defined above, but disregarding other types of substituents.

[0208] Hydroxypropyl (HP or Hp)-Substituted Cyclodextrins (CDs). As used herein, the term "hydroxypropyl-substituted cyclodextrins" or "HP-substituted CDs" refers to cyclodextrins linked with hydroxypropyl groups, i.e., -CH2-CH(OH)-CH3. Typically, the HP groups are linked to oxygen atoms linked to the C2, C3, and / or C6 carbons of the CDs (most commonly, these attachment sites are mixed).

[0209] Hydroxypropyl beta cyclodextrin, abbreviated as HPβCD, HPBCD, HPβCD, HPBCD, HP-BCD, HP-BCD, HP-βCD, HP-βCD, 2-HPβCD, and similar terms, refers to a beta cyclodextrin substituted with one or more hydroxypropyl groups, i.e., -CH2-CH(OH)-CH3, which are typically attached to oxygen atoms attached to the C2, C3, and / or C6 carbons of CD (most commonly, these attachment sites are mixed).

[0210] Hydroxypropyl beta cyclodextrin dimer, abbreviated as HP(CD-L-CD) or HP(CD-L-CD) or HP(βCD-L-βCD) or HP(βCD-L-βCD)HP and similar terms, refers to hydroxypropyl beta cyclodextrin dimers covalently linked with a linker L. Substituents can be present in a particular average number, for example, DS4 indicates that on average, four HP groups are present. Additional substituents can be present as further described herein.

[0211] A similar convention is used for other substituted cyclodextrins and cyclodextrin dimers, such as methyl (Me), quaternary ammonium (QA), succinyl (SUCC), sulfobutyl (SB), etc. For example, MeβCD refers to methyl beta cyclodextrin, and so on. Similarly, methyl beta cyclodextrin dimers are sometimes abbreviated as Me(CD-L-CD) or Me(CD-L-CD) or Me(βCD-L-βCD) or Me(βCD-L-βCD)Me and similar terms, which refer to methyl beta cyclodextrin dimers covalently linked with a linker L. Substituents can be present in a particular average number, for example, DS4 indicates that there are an average of four Me groups. Additional substituents can be present, as described further herein.

[0212] Cyclodextrin dimer compositions. As used herein, the term "cyclodextrin dimer composition" or "CD dimer composition" refers to a mixture of cyclodextrin dimers, e.g., a mixture of CD dimers substituted with the same substituents but in different numbers. Typically, CD dimer compositions are characterized by having a specified degree of substitution with a specified substituent. CD dimer compositions can result from a synthetic process in which substituents are added to CD dimers in a stochastic manner such that individual CD molecules vary in the number and position of the substituents, due in large part to the symmetry of the CD molecule. Furthermore, CD dimer compositions can include a mixture of individual molecules with different linker attachment sites (e.g., O2 and O2, O2 and O3, O3 and O2, or O3 and O3) or can be homogenous in the linker attachment sites (e.g., only O2 and O2, only O2 and O3, only O3 and O2, or only O3 and O3). The degree of substitution of the CD dimer composition can be determined by NMR and / or mass spectrometry, for example, as described above.

[0213] Terms such as "specifically bind" mean that a molecule, for example, a cyclodextrin dimer of the present disclosure, forms a relatively stable complex with a binding partner, for example, cholesterol (oxysterol, e.g., 7KC, etc.) under physiological conditions. Methods for identifying whether a molecule specifically binds to a binding partner are well known in the art, and such methods include, for example, equilibrium dialysis, surface plasmon resonance, and the like. In exemplary embodiments, the cyclodextrin dimer of the present disclosure binds to cholesterol, oxysterol, or 7KC at about 5 μM to about 100 μM, about 10 μM to about 90 μM, about 20 μM to about 80 μM, about 30 μM to about 70 μM, about 40 μM to about 60 μM, about 0.5 μM to about 50 μM, about 1 μM to about 40 μM, about 2 μM to about 30 μM, about 3 μM to about 20 μM, about 4 μM to about 10 μM, or about 5 μM to about 10 μM. K of less than about 1000 μM, less than about 500 μM, less than about 300 μM, less than about 200 μM, less than about 100 μM, less than about 90 μM, less than about 80 μM, less than about 70 μM, less than about 60 μM, less than about 50 μM, less than about 40 μM, less than about 30 μM, less than about 20 μM, less than about 10 μM, less than about 5 μM, less than about 4 μM, less than about 3 μM, less than about 2 μM, less than about 1 μM, or less than about 0.5 μM D Combine with.

[0214] Higher affinity for 7KC than for cholesterol. As used herein, the term "higher affinity for 7KC than for cholesterol" indicates that a compound (e.g., cyclodextrin) has a higher ability to solubilize 7KC than for cholesterol. Higher affinity can be predicted by molecular docking, by molecular dynamics simulation, or measured by calorimetry. In exemplary embodiments, the cyclodextrin dimer has a binding affinity for 7KC that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 8 times, at least 10 times, at least 15 times, at least 20 times, at least 30 times, or at least 50 times stronger than its binding affinity for cholesterol, which may be determined, for example, by comparing the concentration at which 50% of 7KC in suspension is solubilized using the procedure described in the Examples herein. In exemplary embodiments, the cyclodextrin dimer has a binding affinity for 7KC that is at least 1.1, 1.5, 2, 3, 4, 5, or 10 times stronger than its binding affinity for cholesterol, which is optionally measured by a computed or measured binding affinity for cholesterol (K D ) divided by the computed binding affinity to 7KC.

[0215] Higher affinity for one compound than another, for example, higher affinity for 7KC than for cholesterol, can be determined using a "turbidity test" performed in an aqueous suspension containing 3% ethanol in PBS, 300 uM sterol, and 1 mM cyclodextrin to be tested. This single concentration of cyclodextrin is used to normalize the test results. To perform the test, the sample is incubated at 37°C for 30 minutes, and then the absorbance at 350 nm is measured, for example, using a spectrophotometer plate reader. The relative turbidity is determined by dividing the turbidity measured in the presence of cyclodextrin by the baseline turbidity without cyclodextrin. If the relative turbidity of the 7KC suspension is lower than that of the cholesterol solution, the cyclodextrin has a higher affinity for 7KC than for cholesterol.

[0216] Hydrophobic drugs. As used herein, the term "hydrophobic drug" refers to a drug that does not dissolve in water unless some surfactant or other solvent is present. Hydrophobic drugs include, but are not limited to, hormones, such as estrogen, progesterone, and testosterone. The cyclodextrin dimers of the present disclosure can be used as excipients for hydrophobic drugs. Additional examples of hydrophobic drugs include dextromethorphan HBr (DXM), diphenhydramine HCl (DPH), lidocaine HCl (LDC), heparin, bendroflumethiazide, acyclovir, revaprazan, curcumin, and testosterone propionate (TP), to name a few. The cyclodextrin dimer can be present in an amount sufficient to increase the solubility of the molecule and / or aid in better drug delivery. The molecular ratio of drug to cyclodextrin can be a 1:1 ratio or can be greater than 1:1.

[0217] An amount effective to solubilize the hydrophobic drug. As used herein, the phrase "an amount effective to solubilize the hydrophobic drug" typically refers to the concentration of a substance (e.g., a cyclodextrin dimer) capable of solubilizing the hydrophobic drug in an aqueous composition, e.g., phosphate buffered saline (PBS) or water. Solubilization can be measured by spectrophotometric or other means known in the art. Solubilization can be measured at room temperature, physiological temperature (37°C), or another suitable temperature (e.g., 0-4°C).

[0218] "Alkyl" means the monovalent linear or branched saturated hydrocarbon moiety, consisting solely of carbon and hydrogen atoms, having from one to twelve carbon atoms.

[0219] "Lower alkyl" refers to an alkyl group having 1 to 6 carbon atoms, e.g., C3 alkyl. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, sec-butyl, tert-butyl, pentyl, n-hexyl, octyl, dodecyl, and the like.

[0220] "Alkylene" means a divalent linear or branched saturated hydrocarbon radical having one to twelve carbon atoms, or a divalent branched saturated hydrocarbon radical having three to six carbon atoms, e.g., methylene, ethylene, 2,2-dimethylethylene, propylene, 2-methylpropylene, butylene, pentylene, and the like.

[0221] "Alkenyl" means a monovalent straight chain hydrocarbon radical of 2 to 12 carbon atoms or a monovalent branched chain hydrocarbon radical of 3 to 12 carbon atoms having at least one double bond. Examples of alkenyl groups include, but are not limited to, ethenyl (vinyl, -CH=CH2), 1-propenyl (-CH=CH-CH3), 2-propenyl (allyl, -CH-CH=CH2) moieties, methoxy, ethoxy, iso-propoxy, and the like.

[0222] "Alkoxyalkyl" means a moiety of the formula Ra-O-Rb-, where Ra is alkyl and Rb is alkylene, as defined herein. Examples of alkoxyalkyl groups include, for example, 2-methoxyethyl, 3-methoxypropyl, 1-methyl-2-methoxyethyl, 1-(2-methoxyethyl)-3-methoxypropyl, and 1-(2-methoxyethyl)-3-methoxypropyl.

[0223] "Alkoxyalkoxyalkyl" means a group of the formula -RO-R'-O-R'', where R and R' are each alkylene and R'' is alkyl, as defined herein.

[0224] "Alkylcarbonyloxyalkyl" means a group of the formula -ROC(O)-R', where R is alkylene and R' is alkyl, as defined herein.

[0225] "Alkylcarbonyl" means a moiety of the formula -R'-R'', where R' is -C(=O)- and R'' is alkyl, as defined herein.

[0226] "Alkylsulfonyl" means a moiety of the formula -R'-R'', where R' is -SO2- and R'' is alkyl, as defined herein.

[0227] "Alkylsulfonylalkyl" means a moiety of the formula -R'-R''-R''', where R' is alkyl, R'' is -SO2-, and R''' is alkyl, as defined herein.

[0228] "Alkylamino" means a moiety of the formula -NR-R', where R is hydrogen or alkyl and R' is alkyl, as defined herein.

[0229] "Alkoxyamino" means a moiety of the formula -NR-OR', where R is hydrogen or alkyl and R' is alkyl, as defined herein.

[0230] "Alkylsulfanyl" means a moiety of the formula -SR, where R is alkyl as defined herein.

[0231] "Alkali metal ion" means a monovalent ion of a Group I metal, such as lithium, sodium, potassium, rubidium, or cesium, preferably sodium or potassium.

[0232] "Alkaline earth metal ion" means a divalent ion of a Group II metal, such as beryllium, magnesium, calcium, strontium, or barium, preferably magnesium or calcium.

[0233] "Amino" refers to the group -NR'R'', where R' and R'' are each independently hydrogen or alkyl. Thus, "amino", as used herein, includes "alkylamino" and "dialkylamino".

[0234] "Alkylaminoalkyl" means the group -R-NHR' where R is alkylene and R' is alkyl. Alkylaminoalkyl include methylaminomethyl, methylaminoethyl, methylaminopropyl, ethylaminoethyl, and the like.

[0235] "Dialkylaminoalkyl" means the group -R-NR'R'', where R is alkylene and R' and R'' are alkyl, as defined herein. Dialkylaminoalkyl include dimethylaminomethyl, dimethylaminoethyl, dimethylaminopropyl, N-methyl-N-ethylaminoethyl, and the like.

[0236] "Aminoalkyl" means the group -R-R', where R' is amino and R is alkylene, as defined herein. "Aminoalkyl" includes aminomethyl, aminoethyl, 1-aminopropyl, 2-aminopropyl, and the like.

[0237] "Aminoalkoxy" means the group -OR-R1, where R' is amino and R is alkylene, as defined herein.

[0238] "Alkylsulfonylamido" means a moiety of the formula -NR'SO2-R, where R is alkyl and R' is hydrogen or alkyl.

[0239] "Aminocarbonyloxyalkyl" or "carbamylalkyl" means the group -ROC(=O)-R', where R' is amino and R is alkylene, as defined herein.

[0240] "Aminosulfonyl" means the group -SO2-NR'R'', where R' and R'' are each independently hydrogen or alkyl. Thus, "aminosulfonyl", as used herein, includes "alkylaminosulfonyl" and "dialkylaminosulfonyl".

[0241] "Alkynylalkoxy" means a group of the formula -OR-R', where R is alkylene and R' is alkynyl, as defined herein.

[0242] "Aryl" refers to a monovalent cyclic aromatic hydrocarbon moiety consisting of a monocyclic, bicyclic, or tricyclic aromatic ring. The aryl group can be optionally substituted as defined herein. Examples of aryl moieties include, but are not limited to, optionally substituted phenyl, naphthyl, phenanthryl, fluorenyl, indenyl, pentalenyl, azulenyl, oxydiphenyl, biphenyl, methylenediphenyl, aminodiphenyl, diphenylsulfidyl, diphenylsulfonyl, diphenylisopropylidenyl, benzodioxanyl, benzofuranyl, benzodioxylyl, benzopyranyl, benzoxazinyl, benzoxazinonyl, benzopiperazinyl, benzopiperazinyl, benzopyrrolidinyl, benzomorpholinyl, methylenedioxyphenyl, ethylenedioxyphenyl, and the like, as well as their partially hydrogenated derivatives.

[0243] "Arylalkyl" and "aralkyl" may be used interchangeably and refer to the radical -RaRb, where Ra is an alkylene group and Rb is an aryl group, as defined herein. For example, phenylalkyl, such as benzyl, phenylethyl, 3-(3-chlorophenyl)-2-methylpentyl, etc., are examples of arylalkyl.

[0244] "Arylsulfonyl" means a group of the formula -SO2-R, where R is aryl as defined herein.

[0245] "Aryloxy" means a group of the formula -OR, where R is aryl as defined herein.

[0246] "Aralkyloxy" or "arylalkyloxy" means a group of the formula -OR-R'', where R is alkylene and R' is aryl, as defined herein.

[0247] "Cyanoalkyl" means a moiety of the formula -R'-R'', where R' is alkylene as defined herein and R'' is cyano or nitrile.

[0248] "Cycloalkyl" means a monovalent saturated carbocyclic moiety consisting of a monocyclic or bicyclic ring. Cycloalkyl can be optionally substituted with one or more substituents, each of which, unless specifically indicated otherwise, is independently hydroxy, alkyl, alkoxy, halo, haloalkyl, amino, monoalkylamino, or dialkylamino. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like, as well as partially unsaturated derivatives thereof.

[0249] "Cycloalkenyl" means a monovalent unsaturated carbocyclic moiety consisting of a monocyclic or bicyclic ring and having at least one double bond. Cycloalkenyl can be optionally substituted with one or more substituents, each of which, unless specifically indicated otherwise, is independently hydroxy, alkyl, alkoxy, halo, haloalkyl, amino, monoalkylamino, or dialkylamino. Examples of cycloalkenyl moieties include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0250] "Cycloalkylalkyl" means a moiety of the formula -R'-R'', where R' is alkylene and R'' is cycloalkyl, as defined herein.

[0251] "Cycloalkylene" means a divalent saturated carbocyclic radical consisting of mono- or bicyclic rings. The cycloalkylene can be optionally substituted with one or more substituents, each of which, unless specifically stated otherwise, is independently hydroxy, alkyl, alkoxy, halo, haloalkyl, amino, monoalkylamino, or dialkylamino.

[0252] "Cycloalkylalkylene" means a moiety of the formula -R'-R''-, where R' is alkylene and R'' is cycloalkylene, as defined herein.

[0253] "Heteroalkyl" means an alkyl radical, as defined herein, in which one, two, or three hydrogen atoms are replaced by a substituent independently selected from the group consisting of -ORa, -NRbRc, and -S(O)nRd, where n is an integer from 0 to 2, provided that the point of attachment of the heteroalkyl radical is through a carbon atom, and where Ra is hydrogen, acyl, alkyl, cycloalkyl, or cycloalkylalkyl; Rb and Rc are, independently of each other, hydrogen, acyl, alkyl, cycloalkyl, or cycloalkylalkyl; when n is 0, Rd is hydrogen, alkyl, cycloalkyl, or cycloalkylalkyl, and when n is 1 or 2, Rd is alkyl, cycloalkyl, cycloalkylalkyl, amino, acylamino, monoalkylamino, or dialkylamino. Representative examples include, but are not limited to, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxy-1-hydroxymethylethyl, 2,3-dihydroxypropyl, 1-hydroxymethylethyl, 3-hydroxybutyl, 2,3-dihydroxybutyl, 2-hydroxy-1-methylpropyl, 2-aminoethyl, 3-aminopropyl, 2-methylsulfonylethyl, aminosulfonylmethyl, aminosulfonylethyl, aminosulfonylpropyl, methylaminosulfonylmethyl, methylaminosulfonylethyl, methylaminosulfonylpropyl, and the like.

[0254] "Heteroaryl" means a monocyclic or bicyclic radical having 5-12 ring atoms and at least one aromatic ring containing 1, 2, or 3 ring heteroatoms selected from N, O, or S, with the remaining ring atoms being C, and the point of attachment of the heteroaryl radical will be on the aromatic ring. The heteroaryl ring can be optionally substituted as defined herein. Examples of heteroaryl moieties include, but are not limited to, optionally substituted imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyrazinyl, thienyl, benzothienyl, thiophenyl, furanyl, pyranyl, pyridyl, pyrrolyl, pyrazolyl, pyrimidyl, quinolinyl, isoquinolinyl, benzofuryl, benzothiophenyl, benzothiopyranyl, benzimidazolyl, benzoxazolyl, benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl, benzopyranyl, indolyl, isoindolyl, triazolyl, triazinyl, quinoxalinyl, purinyl, quinazolinyl, quinolizinyl, naphthyridinyl, pteridinyl, carbazolyl, azepinyl, diazepinyl, acridinyl, and the like, as well as partially hydrogenated derivatives thereof.

[0255] "Heteroarylalkyl" or "heteroaralkyl" means a group of the formula -R-R', where R is alkylene and R' is heteroaryl, as defined herein.

[0256] "Heteroarylsulfonyl" means a group of the formula -SO2-R, where R is heteroaryl as defined herein.

[0257] "Heteroaryloxy" means a group of the formula -OR, where R is heteroaryl as defined herein.

[0258] "Heteroaralkyloxy" means a group of the formula -OR-R'', where R is alkylene and R' is heteroaryl, as defined herein.

[0259] "Heterocyclylalkoxy" means a group of the formula -OR-R', where R is alkylene and R' is heterocyclyl, as defined herein.

[0260] The terms "halo," "halogen," and "halide" may be used interchangeably and refer to a substituent fluoro, chloro, bromo, or iodo. In some embodiments, halo refers to a fluoro substituent.

[0261] "Haloalkyl" refers to an alkyl, as defined herein, in which one or more hydrogens are replaced with the same or different halogens. In some embodiments, the haloalkyl is a fluoroalkyl. In some embodiments, the haloalkyl is a perfluoroalkyl. Examples of haloalkyl include -CH2Cl, -CH2CF3, -CH2CCl3, perfluoroalkyl (e.g., -CF3), and the like.

[0262] "Haloalkoxy" means a moiety of the formula -OR, where R is a haloalkyl moiety as defined herein. In some embodiments, the haloalkoxy is a fluoroalkoxy. In some embodiments, the haloalkoxy is a perfluoroalkoxy. An example of a haloalkoxy is difluoromethoxy.

[0263] "Heterocycloamino" means a saturated ring in which at least one ring atom is N, NH, or N-alkyl, and the remaining ring atoms form an alkylene group.

[0264] "Heterocyclyl" means a monovalent saturated moiety, consisting of one to three rings and incorporating one, two, or three, or four heteroatoms (chosen from nitrogen, oxygen, or sulfur). The heterocyclyl ring can be optionally substituted as defined herein. Examples of heterocyclyl moieties include, but are not limited to, optionally substituted piperidinyl, piperazinyl, homopiperazinyl, azepinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, pyridinyl, pyridazinyl, pyrimidinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinuclidinyl, quinolinyl, isoquinolinyl, benzimidazolyl, thiadiazolidinyl, benzothiazolidinyl, benzoazolidinyl, dihydrofuryl, tetrahydrofuryl, dihydropyranyl, tetrahydropyranyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, dihydroquinolinyl, dihydroisoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0265] "Heterocyclylalkyl" means a moiety of the formula -R-R', where R is alkylene and R' is heterocyclyl, as defined herein.

[0266] "Heterocyclyloxy" means a moiety of the formula -OR, where R is heterocyclyl as defined herein.

[0267] "Heterocyclylalkoxy" means a moiety of the formula -OR-R', where R is alkylene and R' is heterocyclyl, as defined herein.

[0268] "Hydroxyalkoxy" means a moiety of the formula -OR, where R is hydroxyalkyl as defined herein.

[0269] "Hydroxyalkylamino" means a moiety of the formula -NR-R', where R is hydrogen or alkyl and R' is hydroxyalkyl, as defined herein.

[0270] "Hydroxyalkylaminoalkyl" means a moiety of the formula -R-NR'-R'', where R is alkylene, R' is hydrogen or alkyl, and R'' is hydroxyalkyl, as defined herein.

[0271] "Hydroxyalkyl" means an alkyl moiety, as defined herein, substituted with one or more, preferably one, two, or three, hydroxy groups, provided that the same carbon atom does not have more than one hydroxy group. Representative examples include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl, 2-hydroxy-1-hydroxymethylethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutyl, and 2-(hydroxymethyl)-3-hydroxypropyl.

[0272] "Hydroxycarbonylalkyl" or "carboxyalkyl" means a group of the formula -R-(CO)-OH, where R is alkylene as defined herein.

[0273] "Hydroxyalkyloxycarbonylalkyl" or "hydroxyalkoxycarbonylalkyl" means a group of the formula -RC(O)-OR-OH, where each R is alkylene and may be the same or different.

[0274] "Hydroxyalkyl" means an alkyl moiety, as defined herein, substituted with one or more, preferably one, two, or three, hydroxy groups, with the proviso that no same carbon atom has more than one hydroxy group. Representative examples include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-(hydroxy-5-methyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl, 2-hydroxy-1-hydroxymethylethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutyl, and 2-(hydroxymethyl)-3-hydroxypropyl.

[0275] "Hydroxycycloalkyl" means a cycloalkyl moiety, as defined herein, in which one, two, or three hydrogen atoms of the cycloalkyl radical are replaced by a hydroxy substituent. Representative examples include, but are not limited to, 2-, 3-, or 4-hydroxycyclohexyl, and the like.

[0276] "Urea" or "ureido" means a group of the formula -NR'-C(O)-NR''R''', where R, R'', and R''' are each independently hydrogen or alkyl.

[0277] "Carbamate" means a group of the formula -OC(O)-NR'R'', where R' and R'' are each independently hydrogen or alkyl.

[0278] "Carboxy" means a group of the formula -C(O)OH.

[0279] "Sulfonamide" means a group of the formula -SO2-NR'R'', where R', R'', and R'' are each independently hydrogen or alkyl.

[0280] "Nitro" means --NO2.

[0281] "Cyano" means --CN.

[0282] "Phenoxy" means a phenyl ring substituted with at least one --OH group.

[0283] "Acetyl" means -C(=O)-CH3.

[0284] "Cn-m-" is used as a prefix before a functional group, for example, C1-12-alkyl or C5-12-heteroaryl, where "n" and "m" are filled in with integer values ​​(i.e., 0, 1, 2, 12). This prefix indicates the number or range of carbon atoms present in the functional group. In the case of ring systems, this prefix indicates the number or range of ring atoms, whether the ring atoms are carbon atoms or heteroatoms. In the case of a functional group that consists of cyclic and non-cyclic portions (i.e., "arylalkyl" consists of aryl and alkyl portions), this prefix is ​​used to indicate how many carbon atoms and ring atoms are present in total. For example, in the case of arylalkyl, "C7-arylalkyl" can be used to indicate "phenyl-CH2-". In some example functional groups, there may be zero carbon atoms present, for example, C0-aminosulfonyl (i.e., -SO2-NH2, where both possible R groups are hydrogen), where "0" indicates that there are no carbon atoms.

[0285] "Peptide" refers to an amide derivative resulting from two or more amino acids by bonding between the amino and carboxyl groups of one amino acid. A "monopeptide" refers to one amino acid, a "dipeptide" refers to an amide compound containing two amino acids, a "tripeptide" refers to an amide compound containing three amino acids, etc. The C-terminus of a "peptide" can be linked to another moiety via an ester functionality.

[0286] "Optionally substituted," when used with respect to "aryl," "phenyl," "heteroaryl," "cyclohexyl," or "heterocyclyl," means an aryl, phenyl, heteroaryl, cyclohexyl, or heterocyclyl that is optionally substituted independently with one to four substituents, preferably one or two substituents, the substituents being alkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, hydroxyalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino, acylamino, monoalkylamino, dialkylamino, haloalkyl, haloalkoxy, heteroalkyl, -CO R, where R is hydrogen, alkyl, phenyl, or phenylalkyl; -(CR'R'')n-COOR, where n is an integer from 0 to 5, R' and R'' are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl, or phenylalkyl; or -(CR'R'')n-CONRaRb, where n is an integer from 0 to 5, R' and R'' are independently hydrogen or alkyl, and Ra and Rb are each independently hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl, or phenylalkyl.

[0287] "Leaving group" means the group with the meaning conventionally associated with the term in synthetic organic chemistry, i.e., an atom or group capable of being displaced under substitution reaction conditions. Examples of leaving groups include, but are not limited to, halogen, alkane or arylenesulfonyloxy, such as methanesulfonyloxy, ethanesulfonyloxy, thiomethyl, benzenesulfonyloxy, tosyloxy, and thienyloxy, dihalophosphinoyloxy, optionally substituted benzyloxy, isopropyloxy, acyloxy, and the like.

[0288] "Modulator" means a molecule that interacts with a target, including, but not limited to, agonist, antagonist, etc., as defined herein.

[0289] "Optional" or "optionally" means that the subsequently described event or circumstance may, but does not necessarily, occur, and that the description includes cases where the event or circumstance occurs and cases where the event or circumstance does not occur.

[0290] "Disease" and "Disease state" mean any disease, condition, symptom, disorder, or indication.

[0291] "Inert organic solvent" or "inert solvent" means that the solvent is inert under the conditions of the reaction described therewith, and examples of such solvents include benzene, toluene, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, chloroform, methylene chloride or dichloromethane, dichloroethane, diethyl ether, ethyl acetate, acetone, methyl ethyl ketone, methanol, ethanol, propanol, isopropanol, tert-butanol, dioxane, pyridine, etc. Unless otherwise stated, the solvents used in the reactions of the present disclosure are inert solvents.

[0292] "Pharmaceutically acceptable" generally means something that is safe, non-toxic, and not biologically or otherwise undesirable, and is useful in the preparation of pharmaceutical compositions, and includes veterinary acceptable as well as human pharmaceutical use.

[0293] "Pharmaceutically acceptable salts" of a compound means salts that are pharma-ceutically acceptable, as defined herein, and that retain the desired pharmacological activity of the parent compound. Such salts include acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids, such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalene-sulfonic acid, propionic acid, salicylic acid, succinic acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, and the like; or salts formed when an acidic proton present in the parent compound is replaced with a metal ion, such as an alkali metal ion, an alkaline earth ion, or an aluminum ion; or salts formed by the coordination of an organic or inorganic base. Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, trimethylamine, tromethamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide. Suitable pharmaceutically acceptable salts are salts formed with acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, maleic acid, phosphoric acid, tartaric acid, citric acid, sodium, potassium, calcium, zinc, and magnesium. All references to pharmaceutically acceptable salts include solvent addition forms (solvates) or crystalline polymorphs (polymorphs) of the acid addition salts as defined herein. In general, when a particular salt is included in a formula herein, it is understood that other pharmaceutically acceptable salts can be substituted within the scope of this disclosure, for example, in the case of the quaternary ammonium salt of formula VIII, chloride or another negative ion or combinations thereof can be included, and similarly, in the case of the carboxymethyl sodium salt of formula IX, another positive ion can be substituted for the indicated sodium.

[0294] "Protective group" or "protecting group" refers to a group that selectively blocks one reactive site in a multifunctional compound so that a chemical reaction can selectively occur at another unprotected reactive site, in the sense traditionally associated with the term in synthetic chemistry. Certain processes of the present disclosure rely on a protecting group to block reactive nitrogen and / or oxygen atoms present in the reactants. For example, the terms "amino protecting group" and "nitrogen protecting group" are used interchangeably herein and refer to an organic group intended to protect a nitrogen atom from undesired reactions during a synthetic procedure. Examples of nitrogen protecting groups include, but are not limited to, trifluoroacetyl, acetamide, benzyl (Bn), benzyloxycarbonyl (carbobenzyloxy, CBZ), p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, tert-butoxycarbonyl (BOC), and the like. Those skilled in the art will know how to select a group for ease of removal and for resistance to subsequent reactions.

[0295] "Subject" refers to mammals and non-mammals. Mammals refer to any member of the class Mammalia, including, but not limited to, humans; non-human primates, such as chimpanzees and other apes, as well as monkey species; pastoral animals, such as cows, horses, sheep, goats, and pigs; livestock animals, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds. The term "subject" does not denote a particular age or sex.

[0296] "Therapeutically effective amount" means the amount of a compound that, when administered to a subject for treating a disease state, is sufficient to effect that treatment for the disease state. A "therapeutically effective amount" will vary depending on the compound, the disease state being treated, the severity of the disease being treated, the age and relative health of the subject, the route and mode of administration, the judgment of the attending physician or veterinarian, and other factors.

[0297] The terms "as defined above" and "as defined herein," when referring to a variable, incorporate by reference the broad definition of that variable, as well as the preferred, more preferred, and particularly preferred definitions, if any.

[0298] "Treating" a disease state or "treatment" of a disease state includes: (i) preventing the disease state, i.e., preventing the development of clinical symptoms of the disease state in a subject who may have or be predisposed to the disease state, but who has not yet experienced or become symptomatic of the disease state; (ii) inhibiting the disease state, i.e., arresting the development of the disease state or its clinical symptoms; or (iii) alleviating the disease state, i.e., causing a temporary or permanent regression of the disease state or its clinical symptoms.

[0299] Any unshared pair of electrons appearing on a carbon, oxygen, sulfur, or nitrogen atom in the structures herein indicates the presence of a hydrogen atom. EXAMPLES

[0300] Example 1. Solubilization of Compounds with HPβCD

[0301] Example 1 demonstrates the ability of HPβCD(DS4.5) monomer to solubilize various sterols, vitamins, oxysterols, and steroid hormones (FIGS. 2A-B). Lower turbidity indicates a higher ability to solubilize a given sterol. FIGS. 2A-B show the solubilization of various sterols and sterol derivatives by HPβCD(DS4.5) monomer, as assessed by relative turbidity.

[0302] We also tested variants of HPβCD by testing different numbers of hydroxypropyl groups on HPβCD. We tested a range of 3.7 to 21 (maximum number of possible substituents). Although the data was noisy, the ability to solubilize 7KC and cholesterol decreased with increasing degree of substitution (Figure 2C-Figure 2D). This was strongly supported by molecular docking of monomeric HPβCD with a wide range of substituents (Figure 2E). The monomers and sterols were designed in PyMOL based on known chemical properties. The most likely arrangement of each hydroxypropyl group was taken, and the top 20 conformations were considered to identify the affinity score for each pair. If any of the atoms of the sterol passed through the plane formed by the O4 oxygen of the cyclodextrin, that conformation was included in the calculation. HPβCD with lower DS showed preferential solubilization of 7KC over cholesterol, suggesting that such HPβCD has specificity for 7KC. Without wishing to be bound by theory, one possible explanation is that a maximum number of hydroxyl groups are available to hydrogen bond with the keto group at position 7 of 7KC, however, this theory is not necessary to practice the present invention.

[0303] Example 2. Computer modeling of the interactions of cyclodextrin monomers and dimers with cholesterol and 7KC

[0304] overview

[0305] In this example, molecular modeling and computer simulations are described. These molecular modeling and computer simulations were performed to investigate the mechanism by which CDs bind sterols, to predict the relative binding ability of cyclodextrin dimers to cholesterol and 7KC, and to identify cyclodextrin dimers that are predicted to have a higher affinity for 7KC than for cholesterol. Presumably, the configuration in which sterols are completely encapsulated by CDs or CD dimers shields the hydrophobic sterols from hydrophilic solvents, thus allowing the sterols to go into solution.

[0306] For initial docking analysis (Figure 2E [monomer], Figure 4B [dimer]), HPβCD monomers and dimers with various substitution levels were constructed using the computer modeling program PyMOL (PyMOL Molecular Graphics System, version 2.0, Schrodinger, LLC.), and then the interactions of these hypothetical CD molecules with 7KC or cholesterol were modeled using the extended AutoDock Vina (Trott et al., J. Comput. Chem., 31(2):455-61. (2010)) developed at Scripps Research Institute (LaJolla, CA, USA). Autodock Vina is a molecular docking software that shows significant improvements in accuracy and speed over its predecessor Autodock4. This software uses a scoring function to approximate the standard chemical potential of the system and predicts non-covalent binding between molecules to predict energetically favorable conformations as well as binding affinities. It was generally found that hydroxypropyl dimers and monomers with a DS of about 2-6 showed the best specificity for 7KC.

[0307] Three beta-cyclodextrin derivatives, native monomeric (DS0) beta-cyclodextrin (βCD), monomeric hydroxypropyl-beta-cyclodextrin (DS5, HPβCD), and dimerized hydroxypropyl-beta-cyclodextrin of DS5 (two HPβCD monomers are linked via a butyl chain through the O2 oxygen of the DS2 monomer and the O3 oxygen of the DS3 monomer, resulting in a total DS of 5), were subjected to molecular dynamics simulations using GROMACS 2018 (University of Groningen, Groningen, Netherlands; Bekker et al., World Scientific (1993); and Berendsen et al., Comp. Phys. Comm., 91:43-56. (1995)) in addition to docking simulations using AutoDock Vina for binding with either 7KC or cholesterol. Both of these ligands are asymmetric, so simulations were performed with the ligands oriented both up and down. These simulations were then repeated in the AMBER force field and at translated positions to establish which positions / force fields provided the most informative data for these new molecules (initial MD analysis, Figures 4D-4MM). The initial positions in the GROMOS force field were identified as being the most effective at capturing the CD dimer-sterol interactions, so this force field and position were used in subsequent abbreviated MD simulations of other CD dimers (subsequent MD analysis, Figures 4NN-4SS, 5B-C, 6B-7B).

[0308] In general, it was found that the addition of hydroxypropyl groups resulted in less stable complexes, but at the same time, it provided some specificity for 7KC over cholesterol that was different from that seen with native, unsubstituted βCD. This was seen because 7KC was able to form and reform somewhat stable complexes in both the up and down orientations, while cholesterol was less able to form stable complexes, possibly because cholesterol did not appear to be as fully enclosed in βCD as 7KC, especially in the "down" orientation. Dimerization of βCD provided significantly higher affinity for sterol targets, e.g., 7KC and cholesterol. This was manifested by the formation of stable dimeric complexes with strong energetic interactions for all ligands and orientations, where the ligand fits inside the hydrophobic core of the CD dimer, allowing it to be solubilized in aqueous solutions.

[0309] To further analyze the effect of small modifications on the βCD dimer, additional docking and molecular dynamics simulations were performed for various linkers and degrees of substitution of HPβCD (Figure 8). We extended this analysis to include other types of selected substituents and other selected linkers (Figure 9), and found that, in general, DS of about 2-6 showed the best 7KC specificity for a wide range of substituents and linker types tested.

[0310] Based on this extensive computational analysis, we believe that dimerization of βCD is optimal for forming strong and stable complexes with sterols, regardless of the type or position of the substituent or linker used. A wide variety of dimerized βCD molecules have been tested, and they show much higher sterol affinity than monomeric βCD for many types of substituents and linkers, even though they are chemically very different from each other.

[0311] Computer Calculation Method

[0312] Initial docking simulation

[0313] The inventors have developed a faster and simpler method to predict the binding of cyclodextrins to various sterols by computation using AutoDock Vina without analyzing the entire trajectory, which is very time-consuming and computationally expensive. Applying this technique to the cyclodextrin system, it has been possible to perform hundreds of docking simulations with a wide variety of cyclodextrins designed by the inventors. This type of computational modeling presents the possible interactions between various cyclodextrins and various sterols, providing both spatial information and binding affinity data.

[0314] These structural predictions can be made as modeling of several different sterol and / or CD derivatives may reveal potential mechanistic features. We have several binding hypotheses and hope to test these using computational techniques. We have developed different models of HPβCD to test our binding theory:

[0315] Monomer-sterol association: To help determine whether sterols are more likely to be bound by monomeric or dimeric HPβCD and whether the monomer shows specificity for 7KC or cholesterol, we examined monomer versus sterol affinity compared to dimer association (Figure 4A).

[0316] Linked dimer-sterol: To test new potential molecules as well as eliminate the need for multiple steps, two monomers were covalently linked with multiple linkers and associated with sterols to study the affinity and properties of these prelinked dimers (Figure 4A).

[0317] In order to make the output of these files comparable to each other, a scoring system for complexation with sterols was developed. In this system, the most favorable affinity was adjusted based on whether the dimer was in head-to-head configuration (if possible) and whether the sterol was actually in the barrel-shaped HPβCD cavity. This number of "complexed conformations" (out of a maximum of 20 configurations) was then added to the absolute value of the most favorable affinity. Thus, an association that resulted in 15 / 20 configurations complexed with sterols (head-to-head and / or with the sterol in the CD cavity) and with the highest affinity of -10 kJ / mol would receive a score of 25 (|-10|+15=25). In this computation, a ligand was considered to be in complex if any atom of the ligand crossed the plane formed by the O4 atoms of the CD, regardless of the angle or degree of insertion into the cavity. The resulting value is called the "affinity score".

[0318] This docking analysis was then extended to include a wider variety of substituents (including charged groups) and linkers to determine whether 7KC specificity was influenced by these factors. Sulfobutyl and methyl substituents, along with triazole and butyl linkers, were tested over the full DS range of 0-20 and showed a similar pattern to hydroxypropyl, where the highest DS for 7KC specificity was around 4 (Figures 5A and 6A). Therefore, other cyclodextrins, such as quaternary ammonium and carboxymethylated, were only tested at low DS (around 4).

[0319] Initial molecular dynamics simulation (Figure 4D to Figure 4MM)

[0320] This initial set of simulations was performed using GROMACS 2018 (University of Groningen, Groningen, Netherlands) with both the GROMOS 54a7 and AMBER 99SB force fields, resulting in two reproductions of these simulations that served to determine the consistency of the observed interactions. These two reproductions for each of the three CD molecules and each orientation of the ligand were then repeated with different initial structures. In each iteration, the ligand was moved to determine the dependence of these calculations on the initial structure as well as the force field. The resulting 48 trajectories of the hydroxypropyl dimer were then analyzed using GROMACS tools.

[0321] Molecular dynamics, unlike docking, allows the simulated molecules to interact in a time-dependent manner, rather than simply a snapshot of an energetically favorable conformation as docking provides. For each of the first three CD-sterol complexes, the simulation was extended to 1 microsecond (a very long time for MD simulations) to allow enough time for the complexes to stabilize. The output was then analyzed to determine the distance between the center of mass of all O4 oxygens (the center of the CD cavity for both dimers and monomers) and the center of mass of the ligand, the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the main axis of the ligand (see Figure 4C), and both the Lennard-Jones and Coulomb energies of interaction between the cyclodextrin and the ligand.

[0322] Thus, the distance indicates the closeness of the ligand to the cyclodextrin, the angle indicates how well the ligand fits inside the CD cavity, and the interaction energy represents how strongly the two molecules interact (the more negative the interaction energy, the stronger the interaction). Figure 4C shows how useful the "angle" measurement is in identifying how well the ligand is shielded from the surrounding water molecules: zero or 180 degrees would indicate that the ligand is perfectly perpendicular to the plane of the cyclodextrin, while 90 degrees would indicate that the ligand is parallel to the CD plane and therefore not complexed within the cavity. In these simulations, we chose 30 degrees to correspond to starting complexed in the "up" configuration (sterol heads meet the bottom base of the CD, tails meet the top base, and the entire ligand is inserted into the CD cavity), and 150 degrees to correspond to initially complexed in the "down" configuration (sterol tails meet the bottom base of the CD, heads meet the top base, and the entire ligand is inserted into the CD cavity). Note that for dimers, only the face of one CD monomer is considered in the angle between the CD and the ligand, but if the dimer were perfectly formed, this face would be the mirror face of the other monomer face.

[0323] The number of water molecules within 3 Å of the ligand was determined over time to determine how well the CDs shield the ligand from the surrounding solvent. Presumably, more water molecules around the ligand indicates that the ligand is less well shielded from the surrounding water and therefore not in solution. All these simulations were extended to 1 microsecond (1000 ns), which should be long enough to accurately describe the interactions between the CDs and the sterols.

[0324] This extensive initial analysis provides evidence that the simulation adequately captures the interactions of the CD monomers and dimers with the sterol ligands and can therefore be extended to other CD monomers and dimers without the need for such laborious methods.

[0325] Additional molecular dynamics simulations (Figures 4NN-4SS, 5B-5C, 6B-6C, 7A-7B, 8H-8I)

[0326] Based on the initial HPβCD simulations, it was concluded that the non-translated positions in the GROMOS force field gave the best and most dynamic results for these complexes. This lengthy initial analysis was important to establish a precedent for modeling these novel molecules, which would allow for shorter and more targeted simulations of other dimers. Thus, the molecular dynamics analysis was extended with various promising linkers and substituents. First, docking calculations were performed on methyl (Figure 5A) and sulfobutyl (Figure 6A) βCD dimers at various DS. This demonstrated that low DS (~4) showed the most promising results in terms of dimers with the best 7KC specificity. Therefore, additional MD simulations were performed on βCD dimers with DS4, with triazole and butyl linkers (Figures 4RR-4SS, 5B-5C, 6B-6C, 7A-7B). We also performed simulations on βCD dimers with DS0 (Figures 4NN-4QQ). These simulations were performed for 100 ns and were analyzed only in terms of angles and interaction energies to assess the major differences or similarities between these molecular interactions and those in the butyl-linked hydroxypropyl dimer.

[0327] Additional docking simulations

[0328] After initial simulations demonstrated similar promise for a range of possible substituents and linkers, many more linkers, substituents, and even substitution positions were screened using the same docking techniques described above. This analysis helps to show that the effectiveness of these molecules is largely, if not entirely, driven by actual dimerization of βCD and not dependent on the type of linker or substituent.

[0329] Computer calculation results and conclusions

[0330] docking:

[0331] We first investigated whether HPβCD could bind cholesterol and 7KC as a monomer (Fig. 2E), and then investigated whether HPβCD could bind cholesterol and 7KC as a dimer (Fig. 4B).

[0332] We found that HPβCD monomers (Figure 2E) have high affinity for both cholesterol and 7KC at low degrees of substitution (DS), but as DS increases, the affinity for both sterols appears to decrease. This is likely due to crowding of hydroxypropyl groups, which prevents sterols from entering the core of the monomer. Furthermore, fewer hydroxyl groups are available to hydrogen bond with the carbonyl group of 7KC at the inner surface of the CD. The best specificity (but not the best affinity) is seen as a spike at DS4, while the preference for 7KC extends from DS2 to DS6, switching to cholesterol at DS7 and above. From DS10 onwards, little or no affinity is observed in these models.

[0333] The butyl-linked dimers showed higher affinity for sterols when compared to the monomeric CDs, with the best affinity / specificity for 7KC being in dimerized DS10 and DS4 (Figure 4B). However, this specificity appears to be present only in dimers of a particular DS for these calculations, and the variation between the different DSs shown in these calculations is significant. The triazole-linked dimers showed better specificity overall, with the exception of DS6, which has similar affinity to the butyl-linked dimers. This specificity is hypothesized to be due to the formation of an additional hydrogen bond with 7KC between the hydrogen bond donor nitrogen and the hydrogen bond withdrawing ketone of 7KC.

[0334] Initial molecular dynamics analysis:

[0335] Figures 4D-O support the hypothesis that native (unsubstituted, DS0) monomeric βCD can complex with both 7KC and cholesterol in the up and down orientations, but 7KC maintains a more stable complex than cholesterol in the down orientation and vice versa in the up orientation. Cholesterol shows less variability throughout the trajectory in the up orientation, and shows how cholesterol leaves and reassociates with CD multiple times in the up orientation (notice the large angle change at about 150 ns, when cholesterol rotates around and associates in the opposite orientation) (Figure 4D). This angle change indicates that the down orientation is significantly more stable, so much so that cholesterol leaves the cavity and rotates 180 degrees before reassociating, and the overall affinity of cholesterol is very high since it is able to complete this large move in the simulation.

[0336] On the other hand, 7KC does not reassociate once the complex is dissociated in either orientation, but the down orientation is significantly more stable for more than half of the trajectories, supporting the hypothesis that the down orientation is preferred for 7KC. This indicates that both 7KC and cholesterol prefer the down orientation, where the head group associates with the top base and the tail with the bottom base, but in this preferred conformation, only cholesterol can actually disassociate from and reassociate with the CD. This could explain why native CD, although very good at solubilizing cholesterol and its derivatives, does not show specificity for 7KC. This slight preference for cholesterol shown by native, monomeric βCD is expected and is consistent with reported experimental results (Zidovetzki et al., Biochim. Biophys. Acta., 1768(6):1311-1324. (2007)) and is further supported by the number of water molecules surrounding the ligand (Figure 4E). Cholesterol has much less water than 7KC, especially in the "up" direction.

[0337] The AMBER force field (Figure 4G-I) showed a significantly stronger interaction between native βCD and the sterol. Both ligands remained inside the cyclodextrin ring throughout the trajectory in both directions, and little preference for 7KC or cholesterol was observed. The AMBER force field showed a stronger and longer interaction between the two molecules than the GROMOS force field, and the solubilization of the sterol by native βCD in the AMBER force field appears to be nearly the same between the two ligands in both the up and down directions. Despite this strong and stable interaction, the AMBER force field may not fully capture the interaction between βCD and the sterol, since the complex does not separate at all. Although some movement is required to fully resolve the interactions that are occurring, this is good evidence that a strong complex is indeed formed between the two molecules.

[0338] Even when the ligand was translated deeper inside the CD cavity (Figure 4J-O), the native complex still formed effectively in both force fields, but again, less consistently in GROMOS than in AMBER. The GROMOS force field showed a significant preference for the "up" orientation for 7KC and the "down" orientation for cholesterol, whereas AMBER only showed strong interactions between both ligands and the CD. This indicates that 7KC and cholesterol interact similarly and strongly with native βCD, which is consistent with the experimental data, although the orientation of the ligand appears to make a difference in the observed complex formation. The subtle differences in these trajectories are explained in detail below.

[0339] The monomeric HPβCD of DS5 (Figure 4P-4AA) shows less consistent interactions between CD and sterols in the GROMOS force field than the native CD, but at the same time, it also appears to prefer the down orientation for 7KC, as seen in Figure 4P. The AMBER force field (Figure 4S, 4Y) again showed stronger and more consistent interactions, but the stable complexes formed were still the same in both force fields. Overall, it can be seen that the addition of hydroxypropyl groups to the cyclodextrin monomer reduces the likelihood of complexation for both ligands in both force fields, but 7KC is more consistent in forming and reforming stable complexes than cholesterol. Cholesterol does not appear to complex with HPβCD as readily as 7KC, generally due to cholesterol having more accessible water molecules than 7KC in both force fields. This is evident in Figure 4R, where 7KC forms and reforms complexes in the "down" orientation, while cholesterol does not complex in the same way. The trajectory visualization also shows how 7KC strongly prefers the "up" orientation, yet still forms complexes in the "down" orientation at about 500 ns.

[0340] When translating the ligand, the deeper the initial ligand was buried in the CD cavity, the more effectively HPβCD could complex with both sterols. For this translation trajectory in GROMOS (Figure 4V), the preference for 7KC over cholesterol was even more evident than in the previous simulations, with 7KC able to form stable complexes in both orientations, whereas cholesterol could only form stable complexes in the "down" orientation. Furthermore, 7KC in the up orientation was initially outside the cavity and was able to associate with it, forming a very stable complex within 300 ns. The AMBER force field again showed a significantly stronger interaction between HPβCD and the sterols, but still formed the same stable complexes for both ligands, and preferred the up orientation, with somewhat less water generally surrounding 7KC throughout the entire trajectory (see Figure 4T). This is probably because the "down" orientation shows the sterol head group protruding out of the cavity more than in the "up" orientation. This is consistent with our experimental data (Figure 2), as we showed that the HPβCD monomer has some specificity for 7KC while still forming stable, apparently solubilized complexes with both KC and cholesterol. All of these simulations are described in detail below.

[0341] Our novel butyl-linked hydroxypropyl β-cyclodextrin dimer of DS5 was then modeled with 7KC and cholesterol in the GROMOS and AMBER force fields, as seen in Figures 4B-4MM. Contrast between these trajectory plots and those for monomeric HPβCD and native βCD provides clear evidence that the dimerized version consistently binds sterols significantly more securely than its corresponding monomeric version, hydroxypropylated or not. This is consistent with our experimental data (Figure 16). The angles, distances, and energies, as well as the water molecules surrounding the ligand, are all in much more stable and apparently more solubilized configurations than in the monomeric simulations. The GROMOS force field showed that there was less than 5 Å between the ligand and the center of mass of the CD when the complex was fully formed in the down orientation (Figure 4B), while the GROMOS force field monomer consistently showed more than 5-10 Å between the molecules when the complex was formed. The AMBER force field also showed a very strong interaction between the sterol and the dimerized CD, with the interaction energy approaching −300 kJ / mol in the down orientation compared to approximately −150 kJ / mol for the monomer (Figure 4BB), indicating that the dimer forms very strong and stable complexes with both ligands, especially in the down orientation, especially when compared to the monomeric βCD.

[0342] The AMBER force field results (Figure 4EE, KK) support the findings from the GROMOS force field simulations that dimerization of HPβCD creates stronger and more stable interactions between the CDs and the sterols, with a much smaller distance between the two molecules and a much larger interaction energy. The dimerized CDs again consistently showed less than five water molecules surrounding the ligand, especially in the down direction, whereas the monomeric CDs showed more than 10 water molecules surrounding the ligand (Figure 4CC). Overall, the presence of water around the sterols was significantly reduced by dimerization, although this was sometimes reached for the monomers and for 7KC and cholesterol. The dimerization of HPβCD also confers some specificity to 7KC, as evidenced by the fact that 7KC always remained associated with at least one of the two linked CDs during the entire trajectory, regardless of the force field or translation, whereas cholesterol generally dissociated from both monomeric pairs during at least a portion of the trajectory, and even produced distorted head-to-tail dimer configurations in which cholesterol could not be fully enclosed by the dimer. These trajectories are described in detail in the following sections.

[0343] These simulations provide strong evidence that dimerization of HPβCD promotes complexation with sterols by forming an inclusion complex that shields the hydrophobic sterol from the surrounding water molecules. The data imply that dimerized HPβCD has a much stronger sterol affinity overall than the monomer, and that it has a preference for 7KC, as 7KC is associated with at least one of the CDs significantly longer than cholesterol. From this method, we can conclude that although strong complex formation with the AMBER force field is good evidence for the validity of our complex formation and stability, more useful information can be gleaned from the GROMOS force field. This is because, unlike AMBER, the GROMOS force field shows dynamic interactions between molecules, rather than just one very (and sometimes unrealistically) stable complex.

[0344] The details of 48 trajectories of the hydroxypropyl-beta cyclodextrin dimer, each spanning 1 microsecond, are detailed below.

[0345] Detailed description of the initial molecular dynamics trajectory (Figure 4):

[0346] Native monomeric βCD and 7KC, up orientation, GROMOS force field:

[0347] In Figure 4F, 7KC starts with its head group inserted into the CD cavity and its tail extending out of the bottom base. At 134 ns, the complex dissociates and 7KC moves towards the bottom base and rotates out of the cavity. 7KC then moves its head group in and out of the cavity while still associated with the bottom base, and finally at 150 ns the complex dissociates completely and 7KC moves around the box to reassociate with the top base. 7KC continues to associate with and dissociate from the top base but does not reenter the cavity for the remainder of the trajectory.

[0348] Native monomeric βCD and cholesterol, up orientation, GROMOS force field:

[0349] FIG. 4F shows that cholesterol (up) starts with its tail inserted into the CD cavity and its head group stretched out of the bottom base. At about 150 ns, the complex separates, as seen by the large change in the "angle" of cholesterol, which rotates outward out of the cavity and parallels the cyclodextrin, then reassociates in the opposite direction, with its tail stretched out of the bottom base. Cholesterol then reinserts its head group and cycles between inserting its head group and paralleling the CD for about 200 ns, as seen by the change in angle, energy, and distance of cholesterol (up) in FIG. 4D. At about 300 ns, the complex separates completely (corresponding to the cholesterol spike in FIG. 4D), and cholesterol moves randomly around the CD molecule. The two molecules briefly reassociate from 310 ns for about 1 nanosecond, with cholesterol now lying parallel to the top base of the CD. Cholesterol then resumes its random motion for another 2 ns, finally reassociating with the bottom at 330 ns, with the cholesterol tail gently inserting into the CD cavity. Then, at about 400 ns, cholesterol turns around and associates its head group with the CD cavity, and this configuration remains relatively stable, with the head group regularly associating and dissociating, until the complex dissociates again at about 560 ns. At this point, cholesterol briefly moves around the CD randomly, then associates its tail with the bottom of the CD. By 580 ns, the cholesterol tail is snugly inserted into the CD molecule, with the head group extending out from the bottom of the CD. Then, at 582 ns, the complex dissociates again, and finally, at 610 ns, the head group is inserted from the bottom to form the complex again. At about 680 ns, the complex separates again, reforms at 750 ns, then separates again at 880 ns, reforms at 920 ns, and continues to separate and reform approximately every 10 ns until the end of the trajectory (but always associates as seen at 920 ns). The fact that cholesterol completely exits the CD cavity within the simulation time and then reassociates indicates that the program was able to associate the two molecules by itself and not due to any external circumstances.This provides strong evidence that this interaction is rational, recurrent, and effectively captured by the simulation.

[0350] Native monomeric βCD and 7KC, down orientation, GROMOS force field:

[0351] In Figure 4F, 7KC starts with its tail inserted into the CD cavity and its head group extended out of the top base. The complex maintains this conformation, with 7KC moving back and forth in the cavity, tilting. The complex does not separate until 600 ns, at which point 7KC quickly exits the cavity and wraps around to the bottom base. 7KC begins to float around the simulation box, periodically and briefly associating with the CD in a conformation similar to that at 720 ns. Overall, the complex remains dissociated until the end of the simulation. Despite this dissociation, the complex is stable for 600 ns, indicating that once 7KC is inside the cavity of the CD, it is fixed there by interaction forces. This trajectory is quantifiable in Figure 4D, as the plot of 7KC (up) remains relatively flat until about 600 ns, at which point the complex separates and assumes a random motion.

[0352] Native monomeric βCD and cholesterol, down orientation, GROMOS force field:

[0353] In Figure 4F, cholesterol starts in the down position with its head group inside the CD cavity and its tail extending out of the bottom base. It remains stable until about 125 ns, at which point cholesterol rotates out of the cavity. However, cholesterol continues to periodically insert its head group from the bottom base into the CD cavity for the next 200 ns. At about 340 ns, the complex dissociates entirely and cholesterol floats around the simulation box, but finally, at about 560 ns, it reassociates with the bottom base in the same manner as before. Then, about 30 ns later, cholesterol dissociates and reassociates parallel to the top base. Cholesterol then alternates between associating with the top base in this manner and floating randomly for the remainder of the trajectory, like a pendulum.

[0354] Native monomeric βCD and 7KC, up orientation, AMBER force field:

[0355] The interactions seen in the AMBER force field in Figure 4I support the strong solubilization of sterols by native monomeric βCD. Both ligands remain inside the cyclodextrin ring in both orientations throughout the trajectory, with little preference for 7KC or cholesterol. 7KC (up) starts with the center of the molecule inside the CD cavity, the head group slightly outside the bottom base, while the tail group slightly outside the top base. 7KC remains closely fitted inside the CD cavity throughout the trajectory, but does rock back and forth a bit, visible as a slight fluctuation of the generally flat line in Figure 4G. This indicates that a stable conformation is formed and does not dissociate. This is consistent with the experimental data, although the AMBER force field shows a stronger and longer interaction between the two molecules than the GROMOS force field.

[0356] Native monomeric βCD and cholesterol, up orientation, AMBER force field:

[0357] In Figure 4I, cholesterol (up) starts with the center of the molecule inside the CD cavity, the head group a little outside the bottom base, while the tail group a little outside the top base. This complex remains stable throughout the entire trajectory. Cholesterol never leaves the cavity, nor does it change orientation, it just rocks back and forth inside the cavity. These small variations in position correspond to the small protrusions in Figure 4G, especially at the angles.

[0358] Native monomeric βCD and 7KC, down orientation, AMBER force field:

[0359] In Figure 4I, 7KC(down) starts with the center of the molecule inside the CD cavity, with the head groups slightly outside the top base, while the tail groups slightly outside the bottom base. This complex remains stable throughout the entire trajectory. 7KC never leaves the cavity, nor does it change orientation, it just rocks back and forth inside the cavity. These small variations in position correspond to the small protrusions in Figure 4G, especially at the angles.

[0360] Native monomeric βCD and cholesterol, down orientation, AMBER force field:

[0361] Figure 4I shows that cholesterol (down) starts with the center of the molecule inside the CD cavity, with the head group slightly outside the top base, while the tail group slightly outside the bottom base. This complex remains stable throughout the entire trajectory. Cholesterol never leaves the cavity, nor does it change orientation, it just rocks back and forth inside the cavity. These small variations in position correspond to the small protrusions in Figure 4G, especially at the angles.

[0362] Translated native monomeric βCD and 7KC, up direction, GROMOS force field:

[0363] In FIG. 4L, 7KC starts with the center of the molecule inside the CD cavity, with the head groups slightly outside the bottom base, while the tail groups slightly outside the top base. The complex is stable until about 710 ns, at which point 7KC moves out of the bottom base, rotates, and associates parallel to the bottom base. Then, at 715 ns, 7KC rotates fully and inserts the head groups, so that the head groups extend toward the top base and the tail extends outside the bottom base. 7KC then associates and dissociates the head groups from the CD cavity several times, until the complex completely dissociates at about 850 ns. The complex remains dissociated for the remainder of the trajectory.

[0364] Translated native monomeric βCD and cholesterol, up direction, GROMOS force field:

[0365] FIG. 4L shows that cholesterol starts out associated with the CD with its head group extending out of the bottom base while the tail group extends out of the top base. At about 120 ns the complex dissociates as cholesterol migrates to the top base of the CD and inserts its head group to rotate in and out of the cavity at the top base, only to finally dissociate completely again at about 160 ns. At about 163 ns cholesterol reassociates with the bottom base, only to finally rotate back to the top base 5 ns later. Cholesterol then alternates between association with the bottom or top base and random movement, finally somewhat reforming the complex at the very end of the trajectory for the last 3 nanoseconds. This continued formation and transformation in silico shows that in reality it has a strong tendency to form.

[0366] Translated native monomeric βCD and 7KC, down direction, GROMOS force field:

[0367] FIG. 4L shows that 7KC starts in the down position with its head group stretched out from the top base. At 40 ns, 7KC retracts and exits the cavity, assembles parallel to the bottom base, reinserts its head group after 2 ns, then retracts and exits again. At 45 ns, the complex completely separates, at which point 7KC floats around the simulation box, reassembles with the top base at 47 ns, briefly inserts its head group, then rotates back to be parallel to the top base, and finally the complex separates again at 51 ns. At 210 ns, the complex reforms, with its head group inserted from the bottom base and its tail stretched out, as in the initial configuration. The complex remains stable until 268 ns, at which point 7KC retracts and exits the CD again, assembles parallel to the bottom base. The complex completely separates again, but briefly reforms at 360 ns. After this, 7KC occasionally associates parallel to one of the two faces in a conformation similar to that at 710 ns, but does not re-enter the CD cavity. This trajectory is somewhat unclear because 7KC associates with the cavity only for 100 ns, but the complex still forms freely in the simulation. This indicates that the complex is likely to form in reality, even though the interaction forces seem not to be very consistent.

[0368] Native monomeric βCD and cholesterol translated in the down direction, GROMOS force field:

[0369] In Figure 4L, cholesterol starts with its head group associated with the top base and its tail extending out of the bottom base. Cholesterol wobbles laterally within the cavity, until the complex dissociates at about 15 ns. At 17 ns, cholesterol reinserts its head group and continues to rotate between being parallel to the bottom base of the CD and inserting (always with its head group) into the cavity from the bottom base until about 675 ns, when the complex dissociates completely. This indicates a strong interaction and tendency for cholesterol to form an apparently stable complex with native βCD, but the complex does not reassociate once it has completely dissociated from the bottom base of the CD at 675 ns.

[0370] Translated native monomeric βCD and 7KC, up direction, AMBER force field:

[0371] In Figure 4O, 7KC begins with its head base extended out the base below and its tail extended out the base above. The complex remains stable throughout the entire trajectory, but 7KC exhibits more extreme bending than seen in the down direction. 7KC remains bent around the CD ring for a significant portion of the trajectory.

[0372] Translated native monomeric βCD and cholesterol, up direction, AMBER force field:

[0373] Figure 4O shows how cholesterol starts out with its head group extending out the bottom base and its tail extending out the top base. This complex remains stable throughout the entire trajectory. Cholesterol moves significantly back and forth within the cavity, but the angle inside the cavity remains relatively constant.

[0374] Translated native monomeric βCD and 7KC, down direction, AMBER force field:

[0375] As seen in Figure 4O, 7KC starts with its head group extended out the upper base and its tail extended out the lower base. The complex remains stable throughout the entire trajectory, and 7KC does not move appreciably inside the CD cavity, as evidenced by the constant horizontal plot in Figure 4M.

[0376] Native monomeric βCD and cholesterol translated in the down direction, AMBER force field:

[0377] Figure 4O shows how cholesterol starts out with its head group extending out the top base and its tail extending out the bottom base. The complex remains stable throughout the entire trajectory, with cholesterol not moving appreciably inside the CD cavity, as evidenced by the constant horizontal graph in Figure 4M.

[0378] Monomeric hydroxypropyl βCD and 7KC, up orientation, GROMOS force field:

[0379] 7KC in the up position (Figure 4R) starts with its tail inside the cavity of HPβCD and its head extended out of the base. At about 13 ns, 7KC rotates out of the base and associates parallel to the base. At 28 ns, the head group of 7KC reassociates with the cavity, but then rotates back out several times, until 7KC remains associated parallel to the base, at which point the complex dissociates completely. 7KC then cycles between associating parallel to one of the faces and moving randomly around the simulation box for the remainder of the trajectory. No stable complex is formed.

[0380] Monomeric hydroxypropyl βCD and cholesterol, up orientation, GROMOS force field:

[0381] In Figure 4R, cholesterol starts with its tail inserted into the CD cavity and its head group extending out of the bottom base. The complex is stable until about 3 ns, at which point cholesterol finally rotates out of the bottom base and parallel to the CD, and then by 7 ns, turns around and goes to the top base. Cholesterol then moves randomly around the simulation box, occasionally associating parallel to either the top or bottom base, but never stabilizing inside the cavity, except for a brief period at about 300 ns. This lack of strong association is evident by the wild fluctuations in Figure 4P and is supported by experimental evidence.

[0382] Monomeric hydroxypropyl βCD and 7KC, down orientation, GROMOS force field:

[0383] Figure 4R shows that 7KC starts somewhat outside the monomer cavity, initially floating randomly around the simulation box. By 29 ns, 7KC associates with its head group inside the cavity of HPβCD with its tail extending out from the bottom. It remains stable until 35 ns, at which point the complex completely dissociates. The complex remains dissociated until 320 ns, at which point the complex finally reforms with its head group again inside the cavity and its tail extending out from the bottom. The complex remains associated until about 470 ns, at which point it finally dissociates again, remaining dissociated until the end of the trajectory.

[0384] Monomeric hydroxypropyl βCD and cholesterol, down orientation, GROMOS force field:

[0385] Figure 4R shows that cholesterol in the down position starts with its tail inserted into the cavity and its head extended out of the top base. This complex remains stable until about 300 ns, at which point cholesterol rotates out of the bottom base and associates parallel to the CD, and then the complex completely dissociates and dissociates from the CD. Cholesterol then moves around the CD, sometimes associating parallel to the bottom base, and finally associating with the top base at about 100 ns. Cholesterol then continues its random movement around the CD, sometimes associating with one face or rotating as if entering the cavity, similar to the conformation at 275 ns, but cholesterol never fully re-enters the cavity no matter how long it takes. These trajectories suggest a preference for the up orientation. In these trajectories, the only stable complex formed is 7KC-up, which formed independently after fully dissociating in the simulation, and cholesterol-down, which remained stable from its initial configuration. This suggests a strong preference for 7KC in the up-direction and some interaction with cholesterol in the down-direction.

[0386] Monomeric hydroxypropyl βCD and 7KC, up orientation, AMBER force field:

[0387] 7KC(up) starts with the center of the molecule inside the CD cavity, the head groups slightly outside the bottom base, while the tail groups slightly outside the top base. Figure 4U shows how 7KC remains in the cavity of HPβCD during the entire trajectory; although it does move up and down slightly, it never moves very far outside the cavity at either end. The complex does not dissociate.

[0388] Monomeric hydroxypropyl βCD and cholesterol, up orientation, AMBER force field:

[0389] The AMBER force field shows much more consistent interactions and much more stable complexes than the GROMOS force field for both native and HPβCD. In Figure 4U, cholesterol (up) starts with the center of the molecule inside the CD cavity, the head group a little outside the bottom base, while the tail group a little outside the top base. This complex remains stable during the entire trajectory. Cholesterol never leaves the cavity, nor does it change orientation, it just rocks back and forth inside the cavity. The most favorable conformation occurs between 500 and 700 ns, as visible in Figure 4S, but cholesterol and CD remain complexed during the entire trajectory. These small variations in position correspond to the small bumps in Figure 4S, especially at the angles.

[0390] Monomeric hydroxypropyl βCD and 7KC, down orientation, AMBER force field:

[0391] 7KC(down) starts with the center of the molecule inside the CD cavity, with the head groups protruding slightly outside the top base, while the tail groups protrude slightly outside the bottom base. Figure 4U shows how the heads of 7KC protrude farther out of the cavity than in the up orientation, but also shows that the complex remains intact for the entire trajectory. This preference for the up orientation is visible in Figure 4S, where the "up" plot is much less variable than the "down" plot, although both are still significantly less variable than HPβCD in GROMOS.

[0392] Monomeric hydroxypropyl βCD and cholesterol, down orientation, AMBER force field:

[0393] Figure 4U shows that cholesterol (down) starts inside the CD cavity with its head group extending out of the top base while its tail group extends out a little from the bottom base. Noticeably, the cholesterol head group occasionally moves out of the cavity significantly further than in the up orientation, yet the complex remains stable throughout the entire trajectory. Cholesterol never leaves the cavity completely, nor does it change direction. These small variations in position correspond to the small bumps in Figure 4S, especially at the angles. There is noticeably more lateral movement through the CD cavity and less radial rocking motion compared to the other complexes.

[0394] Parallel translation of monomeric hydroxypropyl βCD and 7KC, up orientation, GROMOS force field:

[0395] Figure 4X shows that the translated up-direction 7KC starts with its tail inserted into the CD cavity and its head group extended out of the bottom. At about 105 ns, 7KC rotates out of the cavity, and then 7KC swings between having its head group inserted into the CD and being parallel to the CD about every 5-10 s, seemingly spending more time in a conformation with its head group in the cavity. At about 415 ns, the conformation stabilizes and the head group is inserted, but finally at 700 ns, the conformation separates again and dissociates completely. The complex then remains dissociated for the remainder of the trajectory except for one brief reassociation at 726 ns, at which time the head group of 7KC is inserted into the large surface of the CD. The interaction energies here are comparable to those at 400 ns when the complex is briefly formed. Because the complex appears to be able to form and dissociate easily, this interaction is likely to be realistic, strong, and captured by simulations.

[0396] Translated monomeric hydroxypropyl βCD and cholesterol, up orientation, GROMOS force field:

[0397] Cholesterol starts with its head group inserted into the cavity and its tail extending out of the top base. This complex is stable for 60 ns before cholesterol rotates out of the bottom base and associates parallel to the CD. Cholesterol then moves randomly around the simulation box completely out of the CD, eventually reassociating its tail with the CD cavity and its head group again extending out of the bottom base at about 215 ns. This is stable for about 30 ns before cholesterol finally exits the CD again and then rapidly reassociates its head group into the CD cavity at 280 ns, this time with the head group in the cavity and the tail extending out of the bottom base. This complex remains stable for the remainder of the trajectory. This indicates that the complex formed at the end of the trajectory is very stable and likely to form, as seen in Figure 4X.

[0398] Parallel translation of monomeric hydroxypropyl βCD and 7KC, down direction, GROMOS force field:

[0399] The translated down 7KC starts with its head groups inserted into the CD cavity and its tail extended out of the bottom base. At about 105 ns, 7KC rotates out of the cavity, and then 7KC swings between its head groups inserted into the CD and parallel to the CD about every 5-10 ns, seemingly spending more time in a conformation with its head groups inside the cavity. At about 415 ns, the conformation stabilizes and the head groups are inserted, but finally, at 700 ns, the conformation separates again and completely dissociates. The complex then remains dissociated for the remainder of the trajectory, as seen in Figure 4X.

[0400] Translated monomeric hydroxypropyl βCD and cholesterol, down direction, GROMOS force field:

[0401] For cholesterol translated down with respect to HPβCD, cholesterol starts with its tail inserted into the CD cavity and its head group extended out of the top base. At 50 ns, the complex separates but cholesterol maintains association with the top base, with the tail cycling in and out of the cavity before dissociating completely at 88 ns. The cholesterol molecule then associates with the bottom base of the CD and then resumes random movement around the simulation box. The trajectory cycles between association with one of the two faces and random movement until at 215 ns, at which point the cholesterol tail re-enters the cavity from the top base for the next 25 ns. Cholesterol then resumes random movement around the CD. At 275 ns, the cholesterol head group enters the cavity from the top base and remains there until the complex dissociates completely at about 410 ns. From this point onwards, until about 490 ns, cholesterol moves randomly around the simulation box, at which point it rotates towards the bottom and inserts its head group into the cavity. The complex maintains this conformation until about 530 ns, at which point it moves out of the cavity, rotates, and reinserts its tail group back into the cavity from the bottom. By 540 ns, cholesterol has resumed random movement. It never reenters the cavity, but is frequently in close association with either face of the CD. As is evident in Figure 4X, cholesterol never forms a stable complex with HPβCD no matter how long it is taken, so the interaction between HPβCD and cholesterol, even in the translated position, appears to be transient and not as strong as that between HPβCD and 7KC.

[0402] Translated monomeric hydroxypropyl βCD and 7KC, up orientation, AMBER force field:

[0403] Figure 4AA shows that 7KC starts with its head group extended out the upper base and its tail facing outwards at the lower base, with the center of 7KC located within the CD cavity. The complex remains stable throughout the entire trajectory, with 7KC having no noticeable movement inside the CD cavity, as evidenced by the constant horizontal graph in Figure 4Y.

[0404] Translated monomeric hydroxypropyl βCD and cholesterol, up direction, AMBER force field:

[0405] Figure 4AA shows that cholesterol starts with its head group extended out the top base, its tail facing out the bottom base, and the center of cholesterol located in the cavity of the CD. The complex remains stable throughout the entire trajectory, and cholesterol does not move or shift appreciably inside the CD cavity, as evidenced by the constant horizontal graph in Figure 4Y.

[0406] Parallel translation of monomeric hydroxypropyl βCD and 7KC, down direction, AMBER force field:

[0407] Figure 4AA shows that 7KC starts with its head group significantly extended out of the top base and its tail pointing out of the bottom base, but with the tail completely in the cavity. The complex remains stable throughout the entire trajectory, and 7KC does not move appreciably inside the CD cavity, as evidenced by the constant horizontal graph in Figure 4Y. 7KC shows greater lateral movement in this direction than in the up direction.

[0408] Monomeric hydroxypropyl βCD and cholesterol translated in the down direction, AMBER force field:

[0409] Figure 4AA shows that cholesterol starts with its head group extended out the top base, its tail facing out the bottom base, and the center of cholesterol located in the cavity of the CD. This complex remains stable throughout the entire trajectory, but cholesterol moves significantly inside the cavity, often with only the tail associated and the head group outside the CD. This is seen in Figure 4Y by the larger fluctuations in the down direction than in the up direction, especially in distance.

[0410] Dimerized hydroxypropyl βCD and 7KC, up orientation, GROMOS force field:

[0411] In FIG. 4DD, 7KC starts out inside the dimer and well trapped. At about 100 ns, the dimer begins to stretch, but 7KC remains in a barrel structure inside the two CDs despite this stretching. At 111 ns, the head group dissociates from the associated monomer (in this discussion, the term "monomer" refers to a CD subunit regardless of whether it is part of a covalently linked dimer), but the tail remains associated with the cavity of the other monomer. After 5 ns, the head group of 7KC proceeds to interact with the large face (not the cavity) of one monomer, while the tail remains tethered to the other. At 120 ns, the tail is released from the associated monomer and the head group enters the cavity of the other monomer. This configuration remains stable, and the sterol-associated monomer swings around the empty monomer until the end of the trajectory.

[0412] Dimerized hydroxypropyl βCD and cholesterol, up orientation, GROMOS force field:

[0413] The trajectory of cholesterol (up) starts with cholesterol wrapped around the dimer. At about 22 ns, the dimer starts to move, but cholesterol moves with it, remaining inside the dimer cavity. At about 200 ns, the monomer associated with the cholesterol head group separates and dissociates from the dimer, but cholesterol remains associated with one of the monomers (head group aligned with the bottom base and tail aligned with the top base). This arrangement is maintained until, at 355 ns, cholesterol completely dissociates from the cavity and rotates toward the bottom base. Cholesterol then remains between the two monomers, with its head group occasionally loosely associated with one of the monomers, before finally breaking away completely and floating around the simulation box. As can be seen in Figure 4D-D, cholesterol continues to interact intermittently with one of the CD monomers, but the dimer-cholesterol complex never fully reforms.

[0414] Dimerized hydroxypropyl βCD and 7KC, down orientation, GROMOS force field:

[0415] The 7KC in the down position starts trapped inside the dimer as shown in Figure 4DD. The dimer does not begin to deform until about 600 ns, at which point one monomer extends away from the other while the 7KC remains between the two. At about 820 ns, the 7KC dissociates its tail from one of the monomers, but the head group remains in the cavity of the other monomer. This configuration remains stable, and the sterol-associated monomer swings around the empty monomer until the end of the trajectory.

[0416] Dimerized hydroxypropyl βCD and cholesterol, down orientation, GROMOS force field:

[0417] Cholesterol in the down position (Figure 4DD) starts inside the dimer cage. At about 50 ns, the complex starts to stretch and twist, but cholesterol remains tethered inside the dimer for the entire trajectory. This is evident in Figure 4BB, where the plot of cholesterol angle is very flat and constant for the entire trajectory. This is the only complex in the GROMOS force field that remained intact for the entire trajectory. Figure 4BB shows molecular dynamics analysis on our novel butyl-linked hydroxypropyl β-cyclodextrin dimer (DS5), which forms a very stable complex with 7KC and cholesterol. Contrasting these graphs with those of monomeric HPβCD provides clear evidence that the dimerized version consistently binds sterols significantly more robustly than its monomeric counterpart. In the down orientation, the energies, angles, and distances all remain very consistent with only minimal fluctuations, indicating that there is a stable, apparently solubilized complex for both 7KC and cholesterol that does not change significantly over time. The same can be seen in the up orientation, but with some more variability, especially for cholesterol. This suggests that 7KC binds most effectively in the down orientation, with a strong preference for the down orientation, as seen by the angle flip at about 350 ns in the up orientation. The angle flip at about 350 ns is where 7KC comes out of the dimer and reassociates in the down orientation. Cholesterol is similar, but the complex is not as stable as the one formed with 7KC, indicating that 7KC appears to be able to form a more stable down complex from a less stable up complex, whereas cholesterol does not have the same ability as 7KC.

[0418] Dimerized hydroxypropyl βCD and 7KC, up orientation, AMBER force field:

[0419] Figure 4GG shows in detail how 7KC remains located inside the cavity formed by the two monomers throughout the entire trajectory. Although the complex shows some movement and 7KC moves slightly inside the cavity, 7KC remains complexed with the CD dimer during the entire trajectory.

[0420] Dimerized hydroxypropyl βCD and cholesterol, up orientation, AMBER force field:

[0421] In Figure 4GG, cholesterol remains located between the two monomers throughout the entire trajectory: the monomers maintain association with each other and with the cholesterol, and the complex moves but never separates.

[0422] Dimerized hydroxypropyl βCD and 7KC, down orientation, AMBER force field:

[0423] Figure 4GG shows that 7KC remains located inside the cavity formed by the two monomers throughout the entire trajectory. Although the complex shows some movement and 7KC moves slightly inside the cavity, 7KC remains complexed with the CD dimer during the entire trajectory.

[0424] Dimerized hydroxypropyl βCD and cholesterol, down orientation, AMBER force field:

[0425] Figure 4GG shows that cholesterol remains located inside the cavity formed by the two monomers throughout the entire trajectory. Although the complex shows some movement and cholesterol shifts slightly inside the cavity, cholesterol remains complexed to the CD dimer during the entire trajectory.

[0426] Translated dimerized hydroxypropyl βCD and 7KC, up orientation, GROMOS force field:

[0427] Figure 4JJ shows that the dimerized complex starts with the translated up-oriented 7KC positioned snugly within the cavities of both CD monomers. At about 140 ns, the complex stretches, causing the first oscillation at this point in Figure 4GG, but quickly recovers. The complex continues to cycle through stretches and deformations as seen from the oscillations in Figure 4GG, but the 7KC remains inside both cavities until the tail is released from its monomer at about 700 ns. The 7KC does not re-enter both cavities simultaneously for the remainder of the trajectory.

[0428] Translated dimerized hydroxypropyl βCD and cholesterol, up orientation, GROMOS force field:

[0429] Figure 4JJ shows that cholesterol starts in a translated up orientation complexed to a dimer, and at about 100 ns the complex begins to deform (apparently more than the 7KC complex). The large change in angle at about 180 ns for cholesterol in Figure 4GG occurs when one monomer associated with the cholesterol head through its bottom base rotates a full 180 degrees to the opposite bottom of the second monomer, and the somewhat deformed bottom base (associated with cholesterol) associates with the somewhat deformed top base of the second monomer, thus generating a somewhat deformed head-tail dimer. This head-tail dimer never fully forms a complex with cholesterol, but the cholesterol head group remains associated with the monomer with which it was originally associated. This is the only trajectory that produces a head-tail dimer, and this configuration does not appear to effectively complex cholesterol.

[0430] Translated dimerized hydroxypropyl βCD and 7KC, down direction, GROMOS force field:

[0431] The translated down position of 7KC starts in the center of the CD dimer, associated with both monomers. Figure 4JJ shows that at 230 ns, one monomer extends away from the tail of 7KC, and then at 355 ns, 7KC leaves the dimer completely (note that the consistency in Figure 4GG is also broken here). At 400 ns, 7KC reassociates with one monomer at its head group. The head of 7KC remains associated with this monomer for the remainder of the trajectory, but the tail never reinserts into the second monomer.

[0432] Translated dimerized hydroxypropyl βCD and cholesterol, down direction, GROMOS force field:

[0433] Cholesterol in the translated down position complexes with the CD dimer for about 162 ns. At this point, the dimerization complex begins to stretch and deform, and then at 190 ns, the cholesterol head group separates from its monomer. Until 210 ns, cholesterol does not associate with the cavity of either monomer, but remains between the two separated monomers. Cholesterol remains closely associated with the dimer until 320 ns, at which time it completely dissociates. As can be seen in Figure 4JJ, cholesterol does not re-enter either cavity for the remainder of the trajectory, nor does the dimerization complex completely reform, although cholesterol occasionally associates with the bottom of one of the monomers with its head group, similar to the configuration at 640 ns.

[0434] Translated dimerized hydroxypropyl βCD and 7KC, up orientation, AMBER force field:

[0435] Figure 4MM shows in detail how 7KC remains located inside the cavity formed by the two monomers throughout the entire trajectory. Although the complex shows some movement around 7KC, 7KC maintains almost exactly the same location during the entire trajectory.

[0436] Translated dimerized hydroxypropyl βCD and cholesterol, up direction, AMBER force field:

[0437] Figure 4MM shows in detail how cholesterol remains located inside the cavity formed by the two monomers throughout the entire trajectory. The complex and cholesterol move some during the trajectory, especially the monomer associated with the cholesterol head, but cholesterol never completely dissociates from either monomer. Cholesterol remains complexed to the CD dimer throughout the entire trajectory.

[0438] Translated dimerized hydroxypropyl βCD and 7KC, down direction, AMBER force field:

[0439] Figure 4MM shows in detail how 7KC remains located inside the cavity formed by the two monomers throughout the entire trajectory. Although the complex shows some movement around 7KC, 7KC maintains almost exactly the same location during the entire trajectory. 7KC is complexed with the CD dimer throughout the entire trajectory.

[0440] Translated dimerized hydroxypropyl βCD and cholesterol, down direction, AMBER force field:

[0441] Figure 4MM shows in detail how cholesterol remains located inside the cavity formed by the two monomers throughout the entire trajectory. Although the complex shows some movement around the cholesterol, cholesterol maintains almost exactly the same location during the entire trajectory. Cholesterol is complexed with the CD dimer throughout the entire trajectory.

[0442] Additionally, abbreviated analyses were performed on both βCD dimers of DS0 with butyl and triazole linkers (Figures 4NN-4QQ) as well as the hydroxypropyl dimer with triazole linker (Figures 4RR-4SS). Simulations of DS0 indicate that the triazole linker allows for some additional specificity for 7KC, albeit somewhat destabilizing the complex. The slightly different but still strong and favorable interactions bode well for both types of linkers.

[0443] The triazole-linked HPβCD dimer (Figure 4RR) showed a slightly weaker interaction than the butyl-linked hydroxypropylated dimer, and a strong preference for 7KC in the down orientation. The cholesterol interaction was weaker than that with 7KC, indicating some specificity for 7KC. 7KC in the down orientation forms the most stable complex so far. Addition of the triazole group made 7KC stable in the down orientation, but all other complexes dissociated at some point.

[0444] Further MD analysis Further, abbreviated MD analysis was also performed on triazole-linked as well as butyl-linked methyl βCD, sulfobutyl βCD, and quaternary ammonium βCD, all of which were DS4 (Figures 5B-C, 6B-C, 7A-B). The methyl dimer formed the most stable complex with the butyl linker, and appeared to prefer the up orientation for both linkers, but the interactions were very similar for the two methyl dimers tested. Both linkers readily complexed with both ligands, although it was difficult to distinguish which was more specifically effective with respect to the methyl substituent. The trajectories revealed that the head group of 7KC was not fully contained within the cavity of the dimer, but was stably maintained between the two sister monomers. The complex with 7KC in the down orientation remained associated for approximately 50 ns, after which 7KC moved out of the cavity, with only the head group remaining associated with one of the monomers for the remainder of the trajectory.

[0445] The negatively charged sulfobutyl dimer shows a similar pattern to the methyl and hydroxypropyl dimers, where the triazole linker creates a slightly less stable complex but allows for 7KC specificity. The charged and bulky sulfobutyl group appears to interact very favorably with both 7KC and cholesterol, but for both linkers the only separate complex is with cholesterol. This indicates that the sulfobutyl dimer is likely to have very good specificity for 7KC compared to the methyl and hydroxypropyl.

[0446] To further evaluate the use of charged substituents, MD analysis of the positively charged quaternary ammonium βCD of DS4 was performed. These trajectories revealed strong binding between QAβCD and sterols, as no sterol was released at any time point for either linker. For both ligands and linkers, the presence of strong interaction energies and association with at least one sister monomer throughout the trajectories suggests that QAβCD of DS4 is well suited to bind and solubilize sterols, much like other types of substituents.

[0447] In the final MD analysis, HPβCD with a single O-linker (Figure 8H) was tested. The O-linked dimer (Figure 8H) showed good 7KC specificity as only the up-oriented 7KC remained complexed for the entire 100 ns. Although the interaction energy is slightly lower in magnitude for the O-linkage compared to the butyl linkage, the overall specificity appears to be better for the linker O since both cholesterol complexes separate by 100 ns. The interactions are similar to the butyl-linked dimers, but they appear to confer slightly better 7KC specificity. This is apparently due to the nitrogen of the linker interacting with the carbonyl of the 7KC.

[0448] Further docking screening

[0449] Docking simulations allow for rapid modeling of many different candidate molecules without the need for their synthesis. For this reason, these docking techniques were used to "screen" many different substituent types, linker types, numbers of substitutions, and substitution positions (Figures 8-9). This screening allows for the determination of whether a particular modification improves or worsens specificity for 7KC.

[0450] FIG. 8E illustrates the evaluation of how our HPβCD dimers are sensitive to linker composition and attachment point, varying hydroxypropylation site, varying linker length, and varying linker chemical composition. Linker attachment site was examined computationally since it is not easily controlled during chemical synthesis of cyclodextrin. Docking calculations were performed for various hydroxypropylation sites (FIG. 8A), carbon-only linkers of various lengths (chain lengths from 2 to 8 carbons, FIG. 8B) and triazole linkers (varying n1 and n2 valences around the triazole ring, FIG. 8C), as well as different attachment points to the O2 and / or O3 oxygen(s) of dimerized HPβCD (FIG. 8F-FIG. 8G), and all different linker types (FIG. 9A). The results show that varying the position of the hydroxypropyl group has little effect on 7KC preference and minimal effect on overall sterol binding. Linker lengths of 3 to 5 carbons showed the greatest affinity and specificity for 7KC (Figure 8B).

[0451] Various triazole linkers modeled in AutoDock are shown in Figure 8C. For these linked dimers, n1 indicates the number of carbons on the right side of the azide ring, while n2 indicates the number of carbons on the left side of the azide ring. Based on these results, triazole linkers with lengths less than 4 on either side of the ring are predicted to have the greatest affinity for 7KC.

[0452] In Figure 8E, docking calculations were performed between the HPβCD dimer and 7KC for 23 different possible alternative linkers (shown in Figure 8D). Based on these results, most of the linked dimers tested are predicted to maintain good affinity for 7KC.

[0453] We also considered the fact that the linker can be attached to the base of the cyclodextrin at either the C2 or C3 carbon. We tested by molecular docking whether this would affect the predicted affinity (Figure 8F). We also considered whether there would be more obvious differences in affinity for sterols by linking with asymmetric linkers at various binding sites. These calculations show the tendency to bind 7KC and cholesterol for all three possible linkage sites, all present in roughly equal amounts in a typical synthesis. These calculations show the tendency to bind 7KC and cholesterol for all four possible linkages, present in the synthesis of dimers linked with five different asymmetric linkers. In general, no significant differences were observed between the C2 and C3 binding sites.

[0454] Our molecular modeling revealed differences in the level of 7KC specificity for different numbers of substituents. Of particular interest were HPβCDs linked with three, four, or five hydroxypropyl groups, which showed the greatest 7KC specificity for any of the butyl dimers modeled (Figure 4B). We synthesized a variety of butyl- and triazole-linked HPβCD dimers, including a DS of about 3. Consistent with our predictions, HPβCD-butyl-DS3 and HPβCD-triazole-DS3 had higher 7KC specificity than for cholesterol (Figures 16A-C).

[0455] Upon completing the hydroxypropyl CD dimer docking analysis, a wide variety of CD dimers with different linkers and varying degrees of substitution were docked against 7KC and cholesterol to examine how these factors affect 7KC and cholesterol binding (Figures 5A, 6A, and 9). Methyl and sulfobutyl substituents were tested with butyl and triazole linkers as DS1-DS20 (Figures 5A, 6A), and the results were promising enough to prompt further molecular dynamics analysis and ultimately synthesis.

[0456] In Figures 5A and 6A, it is observed that 7KC specificity is best at low DS (2-6) for both sulfobutyl and methyl substituents. MeβCD and SBβCD with DS4 behave most similarly to HPβCD with DS5, where 7KC is well solubilized but cholesterol is not. 7KC specificity appears to become less and less pronounced at higher DS for both linkers and all substituents. Only DS4 was tested for the other linker types, as it appeared to give the greatest 7KC specificity for all of the substituent types tested.

[0457] Substituents other than hydroxypropyl, methyl, or sulfobutyl were tested only with low DS and only with butyl, triazole, O, and R linkers (Figure 9A). Although some linker or substituent types show higher or lower specificity than others, most still show at least some specificity for 7KC. This suggests that among the compounds tested, 7KC specificity does not depend on the type of linker or substituent, but on the number of substituents on the βCD ring. Although a few substituent types showed negative specificity with a few linker types, the average 7KC specificity was still well above 0 for these 23 linkers and 7 substituent types with low DS (4).

[0458] Using molecular docking, we were able to test how the length of the triazole or alkyl linker affects the 7KC specificity of cyclodextrin dimers with hydroxypropyl, methyl, and sulfobutyl substituents (Figures 9B-9C). As the linker length increases, the specificity is shown to decrease. Without wishing to be bound by theory, it is believed that the longer the linker length, the greater the distance the CD subunits can be separated and therefore spend less time in a conformation that can effectively encompass a molecule the size of 7KC or cholesterol. Based on these results, we conclude that dimers with linker lengths that allow the guest (7KC or cholesterol) to fit into two CD subunits, e.g., linker lengths of 7 atoms or less, will show better solubilization of the molecule.

[0459] We also tested whether the specificity of CD dimers for 7KC depends on the substitution position by creating a number of different substitution patterns using sulfobutyl, hydroxypropyl, and methyl substituents, as well as combinations of the three (Figures 9D-E). We found that 7KC specificity was largely maintained when a single or even multiple substituent types were present on a CD dimer, with a DS of about 4. The type and position of these substituents did not significantly affect 7KC specificity. The results of the docking simulations suggest that although the composition of both the linker and the substituents influences how well a given CD can solubilize guests, the degree of specificity for 7KC is most dependent on the number of substituents on the CD ring. As seen in Figures 4B and 5A-B, the butyl-linked dimer showed the highest 7KC specificity with a DS of about 2-5 for methyl, sulfobutyl, and hydroxypropyl substituents. This was true for the triazole linker as well, supporting the idea that multiple linker and substituent types may exhibit similar specificity for 7KC, with degrees of substitution ranging from 2 to 5. Additionally, a wide range of different substitution patterns / combinations consisting of 23 different linkers and 14 different substituents were docked to determine whether the linker or substituent pattern had an effect on 7KC specificity (Figure 9A). Both of these analyses showed varying degrees of 7KC specificity, but the average specificity was still well above 0.

[0460] The docking and molecular dynamics analysis screens performed served to identify whether certain linker species or the number, type, and location of substituents influenced 7KC specificity. The only modification that had a significant effect on binding (affinity) was the actual dimerization of the cyclodextrin (compared to the docked monomer, Figure 2E, the dimer showed much better binding to the sterol). In contrast, the number of substituents present in the dimer had the greatest effect on 7KC binding specificity. Docking simulations indicate that when βCD is dimerized and substituted with about four compatible functional groups, the specificity for 7KC is largely maintained for a wide variety of substituent types, patterns, and linkers.

[0461] Because the methyl, sulfobutyl, and hydroxypropyl groups are all very different from one another and the range of linkers tested included significant diversity, we believe it is not unreasonable to expect that other substituent types with linker lengths similar to the sterol guest will behave similarly to the butyl-linked CD dimer with a hydroxypropyl group. The specificity for 7KC is expected to be present in other molecules of this class as well, although the type of substituent and linker may have some effect on other properties such as solubility and toxicity.

[0462] Example 3. Synthesis of HPβCD-substituted cyclodextrin dimer

[0463] 3A-3D illustrate the molecules synthesized in FIG. 10 below.

[0464] This example describes the synthesis of a substituted cyclodextrin dimer, first linked with a butyl linker and then with a triazole-containing linker.

[0465] For DS measurements, 1H and 2D NMR spectra are recorded on a Varian VXR-600 at 600 MHz, using residual solvent signals as internal reference. To elucidate the structure, samples are dissolved in DMSO-d6 / D2O. The FID signals are recorded with at least 16 scans to obtain a spectral window that includes at least 0 ppm to +10 ppm. Calculation of the average degree of substitution (DS) can be achieved by setting the integral of the anomeric region to 14 (14 is the number of anomeric protons in the beta-cyclodextrin dimer) and dividing the integral of the alkyl region by 3 (see Figure 10J).

[0466] General Description of Synthesis and Characterization

[0467] HP(βCD-pig-βCD)

[0468] The preparation of hydroxypropylated β-cyclodextrin dimer was achieved through a three-step synthesis (see Figure 10A). The starting material is monomeric β-cyclodextrin protected at the top with a tert-butyldimethylsilyl group (TBDMS-βCD, CycloLab, Budapest, Hungary).

[0469] The bottom dimerization was achieved by using TBDMS-βCD, anhydrous conditions, and sodium hydride as the base. The dialkylating agent was added dropwise to the heterogeneous reaction mixture and allowed to react to completion at room temperature.

[0470] The capped βCD dimer (TBDMS-βCD-Buta-βCD-TBDMS) was purified by chromatography using isocratic elution (chloroform:methanol:water=50:8:0.8 (v / v / v) as eluent). MALDI analysis of the compound confirmed the identity of the product (Figure 10D).

[0471] Desilylation was carried out with tetrabutylammonium fluoride in THF at room temperature. The βCD dimer (βCD-buta-βCD) was purified by chromatography using isocratic elution (1,4-dioxane:NH3 = 10:7 (v / v) as eluent). MALDI and TLC analysis of the compound confirmed the identity of the product (Figure 10E-F).

[0472] Hydroxypropylation of the βCD dimer was achieved using sodium hydroxide as a base in aqueous conditions at room temperature. Purification of the hydroxypropylated βCD dimer (HP(βCD-pig-βCD)) utilized ion exchange resin treatment, activated charcoal clarification, and extensive dialysis. MALDI and TLC analysis of the compounds confirmed the identity and structure of the products (Figures S10G-S10N).

[0473] HP(βCD-triazole-βCD)

[0474] The preparation of hydroxypropylated β-cyclodextrin dimers connected through the lower base with one triazole moiety can be carried out in a four-step procedure (Figure 10B). The first step is the preparation of the azido linker (3-azido-1-bromo-propane), since this reagent is not commercially available. The second step is the preparation of two βCD monomers, 2-O-propargyl-β-CD and 2-O-(3-azidopropyl)-βCD, respectively. The third step of the synthesis is the construction of the dimeric core by copper-catalyzed azide-alkyne cycloaddition, and the final step is the preparation of a series of 2-hydroxypropylated triazole-linked dimers by a classical alkylation approach.

[0475] In particular, the preparation of the azido linker can be achieved by strictly limiting the amount of sodium azide and extending the addition time of the limiting reagent, and the azido linker is then characterized by NMR spectroscopy and TLC (Figure 10R).

[0476] The synthesis of the two monomers is achieved by using lithium hydride as a selective base to deprotect the lower base. Specifically, this approach activates only the hydroxyl group located at C2. As a result, the monomers prepared by this method are substituted only at O2 (they are single isomers). The two monomers are characterized by NMR spectroscopy, MALDI, and TLC (Figures 10S-10U).

[0477] The preparation of the dimeric core is then achieved by reacting the two monomers. The resulting compound, a single isomer, (BCD-(triazole)1-BCD, DS=0), is characterized by NMR spectroscopy (FIG. 10V) and MALDI (FIG. 10O).

[0478] Hydroxypropylation of βCD-triazole-βCD was achieved using propylene oxide and alkaline aqueous conditions. A series of hydroxypropylated compounds were characterized by MALDI (Figures 10P-Q).

[0479] Detailed Description of the Synthesis (HP(βCD-Buta-βCD))

[0480] Step 1: Lower base dimerization of TBDMS-βCD

[0481] Dry TBDMS-βCD (10 g, 5.17 mmol) was dissolved in THF (400 mL) under an inert atmosphere and sodium hydride (2.5 g, 50 mmol) was carefully added in portions (over 30 min). The addition of sodium hydride caused the formation of hydrogen and the suspension foamed vigorously. After 15 min of stirring, the reaction mixture gelled and became difficult to stir. To break up the gel, the reaction mixture was heated to a gentle reflux and maintained at reflux for 30 min. The yellowish heterogeneous suspension became easier to stir and the gel-like structure disappeared. The reaction mixture was cooled to room temperature in a water bath. The alkylating agent 1,4-dibromobutane (1.25 mL, 2.25 g, 10.5 mmol) was added dropwise (15 min) and the reaction mixture became dark orange in color.

[0482] The brownish suspension was stirred overnight under inert atmosphere. The conversion was estimated by TLC to be 10-15% (eluent: chloroform:methanol:water = 50:10:1, v / v / v, see Figure 10C), and therefore was deemed suitable for workup.

[0483] The reaction mixture was quenched with methanol (30 mL), concentrated under reduced pressure (approximately 20 mL), and precipitated with water (200 mL). The crude reaction was filtered through a sintered glass filter and washed thoroughly with water (3×300 mL). The crude material was dried in a dry box in the presence of KOH and P2O5 to constant weight (12.1 g).

[0484] The crude reaction was purified by chromatography and fractions containing the product based on TLC analysis (FIG. 10C) were collected and evaporated to dryness in vacuo to give a white material, which was dried to constant weight in a dry box in the presence of KOH and PO (TBDMS-βCD-pig-βCD-TBDMS, 3.5 g).

[0485] Step 2: Deprotection of the TBDMS-βCD butyl-linked dimer

[0486] Dry TBDMS-βCD-Buta-βCD-TBDMS (3.5 g, 0.89 mmol) was dissolved in THF (250 mL) under inert atmosphere, and tetrabutylammonium fluoride (8.75 g, 33.47 mmol) was added in one portion to the yellowish solution. After stirring at room temperature for 30 min, the reaction mixture became dark green in color. The reaction mixture was stirred at room temperature overnight. TLC analysis (1,4-dioxane:NH3=10:7 (v / v)) revealed that the reaction was not complete, so a second portion of tetrabutylammonium fluoride (4 g, 13.3 mmol) was added to the vessel. The reaction mixture was heated to a gentle reflux and refluxed for 2 h. At this stage, the conversion of the reaction was complete as there was no starting material detectable by TLC. The reaction mixture was cooled to room temperature, concentrated under reduced pressure (to about 10 mL), and methanol (200 mL) was added to obtain a white precipitate. The solid was filtered off, analyzed by TLC, and dried to constant weight in a dry box in the presence of KOH and P2O5 (1.2 g). By TLC analysis, this material contained negligible (≦3%) amounts of tetrabutylammonium fluoride. The mother liquor was concentrated under reduced pressure (to about 10 mL) and purified by chromatography (eluent: 1,4-dioxane:NH3=10:7 v / v) to collect the fractions containing the product and evaporated to dryness under reduced pressure to give a white material. This was dried to constant weight in a dry box in the presence of KOH and P2O5 (βCD-buta-βCD, 0.55 g).

[0487] Step 3: Hydroxypropylation of βCD-Buta-βCD

[0488] βCD-Buta-βCD (0.5 g, 0.21 mmol) was suspended in water (10 mL) and sodium hydroxide (0.1 g, 2.5 mmol) was added to the reaction vessel, the color of the mixture became a slightly yellow solution. The reaction mixture was cooled in a water bath (10° C.) and propylene oxide (0.5 mL, 0.415 g, 7.14 mmol) was added in one portion. The reaction vessel was flushed with argon, sealed, and stirred at room temperature for 2 days. The reaction mixture was concentrated under reduced pressure to a viscous syrup, which was precipitated by the addition of acetone (50 mL). The white solid was filtered through a sintered glass filter and washed thoroughly with acetone (3×15 mL). This material was dissolved in water (50 mL), treated with ion exchange resin (to remove salts), clarified with activated charcoal, membrane filtered, and dialyzed against purified water for 1 day. The residue was evaporated to dryness in vacuo to give a white solid (0.8g).

[0489] Detailed Description of the Synthesis (HP(βCD-triazole-βCD))

[0490] Step 1: Preparation of the azide-linker

[0491] Dissolve 1,3-dibromopropane (10 mL, 20.18 g, 0.1 mol) in 40 mL of DMSO under vigorous stirring. Prepare a solution of sodium azide (6.7 g, 0.1 mol) in DMSO (240 mL) and add it dropwise (addition over 2 h) to the dihalopropane solution. Stir the solution at room temperature overnight. The crude reaction is then extracted with n-hexane (3 x 100 mL), back-extract the collected n-hexane phase with water (3 x 50 mL) and carefully evaporate the organic phase under reduced pressure (exactly 40°C and 400 mbar, otherwise the target compound may be distilled off). The oily residue is purified by chromatography (n-hexane-EtAc = 98:2 as eluent, isocratic elution). Appropriate fractions are collected and concentrated under reduced pressure to give the target compound as a viscous oil, which can be stored under inert atmosphere in a light-proof refrigerated container. The compound is visualized by immersing the TLC plate in a solution of triphenylphosphine in dichloromethane (10%) for about 15 seconds, drying the TLC plate below 60° C., immersing the TLC in a solution of ninhydrin in ethanol (2%) for about 15 seconds, and finally drying the TLC plate below 60° C. The target compound appears as a purple spot on the TLC plate.

[0492] Step 2.1: Preparation of 2-O-propargyl-βCD

[0493] Lithium hydride (212 mg, 26.432 mmol) is added to a solution of β-cyclodextrin (20 g, 17.62 mmol) in dry DMSO (300 mL). This is stirred at room temperature under N2 until the resulting suspension becomes clear (12-24 h). Propargyl bromide (1.964 mL, 17.62 mmol) and a catalytic amount of lithium iodide (ca. 20 mg) are then added and the mixture is stirred at 55 °C for 5 h protected from light. The product is characterized using TLC (10:5:2 CH3CN:H2O:25% v / v in NH3 water) showing spots corresponding to monopropargylated and non-propargylated β-cyclodextrin, respectively. The solution is poured into acetone (3.2 L) and the precipitate is filtered and washed thoroughly with acetone. The resulting solid is transferred to a round-bottom flask and dissolved in a minimum volume of water. Silica gel (40 g) is added and the solvent is removed under reduced pressure until a powdery residue is obtained. The crude mixture is loaded onto the top of a silica column (25 x 6 cm) and chromatographed (10:5:2 CH3CN:H2O:25% v / v NH3 in water) to give, after lyophilization, 2-O-propargyl-β-CD as a solid, which was analyzed by MALDI and NMR (Figure 10T and Figure 10U).

[0494] Step 2.2: Synthesis of 2-O-(3-azidopropyl)-βCD

[0495] Lithium hydride (212 mg, 26.432 mmol) is added to a solution of β-cyclodextrin (20 g, 17.62 mmol) in dry DMSO (300 mL). This is stirred at room temperature under N2 until the resulting suspension becomes clear (12-24 h). 3-azido-1-bromo-propane (3 mL) and a catalytic amount of lithium iodide (ca. 20 mg) are then added and the mixture is stirred at 55 °C for 5 h protected from light. The product is characterized using TLC (10:5:2 CH3CN:H2O:25% v / v in NH3 water) showing spots corresponding to 2-O-(3-azidopropyl)-βCD and βCD. The solution is poured into acetone (3.2 L) and the precipitate is filtered and washed thoroughly with acetone. The resulting solid is transferred to a round-bottom flask and dissolved in a minimum volume of water. Silica gel (40 g) is added and the solvent is removed under reduced pressure until a powdery residue is obtained. The crude mixture is loaded onto the top of a silica column and chromatographed (10:5:2 CH3CN:H2O:25% v / v aqueous NH3) to give, after lyophilization, 2-O-(3-azidopropyl)-β-CD as a solid.

[0496] Step 3: Synthesis of βCD-triazole-βCD dimer

[0497] Dissolve 2-O-propargyl-β-CD and 2-O-(3-azidopropyl)-β-CD in water (300 mL) with vigorous stirring (each at a concentration of about 8-12 mM). Add dimethylformamide (DMF) (about 300 mL) to this suspension in order to completely dissolve the heterogeneous mixture (addition of DMF is a slightly exothermic process). Add copper bromide (2 g, 13.49 mmol) to the solution. Stir the suspension at room temperature for 1 h. The reaction is monitored by TLC and is expected to have reacted in about 1 h (eluent: CH3CN:H2O:NH3 = 10:5:2). Filter the crude reaction and concentrate the mother liquor under reduced pressure (60 °C). Dilute the gel-like material with water and add silica (15 g). Concentrate the heterogeneous mixture under reduced pressure to dryness. The crude mixture is loaded onto the top of a silica column and chromatographed (10:5:2 CHCN:H2O:25% v / v NH3 in water) to give, after drying, the BCD-(triazole)1-BCD dimer, whose preparation was characterized by NMR (Figure 10V).

[0498] Step 4: HP (βCD-triazole-βCD)

[0499] βCD-(triazole)1-βCD dimer (1 g, 0.418 mmol), obtained by steps 1-3 above or by other methods, was suspended in water (50 mL) and sodium hydroxide (DS3=0.32 g, 8 mmol; DS6=0.74 g, 18.5 mmol; DS7=0.87 g, 21.75 mmol) was added to the reaction vessel, resulting in a slightly yellow solution. The reaction mixture was cooled in a water bath (10° C.) and propylene oxide (DS3=0.49 mL, 0.42 g, 7.25 mmol; DS6=1.21 mL, 1.04 g, 17.9 mmol; DS7=1.46 mL, 1.7 g, 29.3 mmol) was added in one portion. The reaction vessel was flushed with argon, sealed, and stirred at room temperature for 2 days. The solution was concentrated under reduced pressure until a viscous syrup was obtained, which was precipitated with acetone (50 mL). The white solid was filtered through a sintered glass filter and washed thoroughly with acetone (3×15 mL). This material was dissolved in water (50 mL), treated with ion exchange resin (to remove salts), clarified with activated charcoal, membrane filtered, and dialyzed against purified water for 1 day. The residue was evaporated to dryness under reduced pressure to give a white solid (0.8 g). The HP(βCD-triazole-βCD) products were analyzed by NMR (FIG. 10W, FIG. 10X, and FIG. 10Y), and their degrees of substitution were calculated as shown in the figures, respectively.

[0500] Example 4. Synthesis of methyl-substituted cyclodextrin dimers

[0501] FIG. 3E illustrates the molecule to be synthesized.

[0502] This example describes the synthesis of a methyl-substituted cyclodextrin dimer with a triazole-containing linker.

[0503] Methyl (βCD-(triazole)1-βCD) dimer (synthesis example)

[0504] The preparation of the methylated β-cyclodextrin dimer was accomplished in a one-step reaction (see FIG. 11A). The βCD-(triazole)1-βCD dimer core is prepared by the synthetic strategy described in Example 3 above.

[0505] synthesis

[0506] The βCD-(triazole)1-βCD dimer core (1.1 g, 0.46 mmol) was suspended in deionized HO (100 mL) with vigorous stirring, and sodium hydroxide (0.35 g, 8.8 mmol) was added. The resulting slightly yellow suspension was stirred for 30 min until complete dissolution. Once the temperature of the yellowish clear solution stabilized at about 20 °C, methyl iodide (0.5 mL, 1.14 g, 8.03 mmol) was added in one portion with vigorous stirring (Note: methyl iodide is not miscible with the reaction mixture, therefore vigorous stirring was used for efficiency). The reaction mixture was stirred at room temperature for 24 h, and then it was treated with ion exchange resin (H+ resin (6 g) and OH- (6 g) resin were added to the solution, stirred for 15 min, and filtered off (resin was washed with 3 x 15 mL deionized water)). The resulting filtrate (final pH=7) was clarified with activated charcoal (activated charcoal (0.2 g) was added to the solution under vigorous stirring, stirred for 30 min, and filtered off (the activated charcoal pad was washed with 3×15 mL of deionized water). The colorless solution was evaporated under reduced pressure (40° C.) to give the title compound as a white powder (approximately 1 g).

[0507] Characterization

[0508] The reaction process was monitored by TLC (FIG. 11B) and the resulting materials were characterized by MALDI-TOF and NMR analysis as shown in FIGS. 11C-11N.

[0509] Example 5. Synthesis of sulfobutyl-substituted cyclodextrin dimer

[0510] Figure 12F illustrates the molecule to be synthesized.

[0511] This example describes the synthesis of a sulfobutyl-substituted cyclodextrin dimer with a triazole-containing linker.

[0512] The preparation of the SB dimer was achieved in a one-step reaction (Figure 12A).

[0513] Synthesis (SB low DS)

[0514] βCD-(triazole)1-βCD dimer core (1.2 g, 0.5 mmol) was suspended in deionized H2O (60 mL) under vigorous stirring. Sodium hydroxide (0.39 g, 9.75 mmol) was added to the mixture and the resulting solution was heated to 60°C. Butane sultone (0.88 mL, 1.17 g, 8.6 mmol) was added dropwise at 60°C and the solution was heated at the same temperature for 3 h. The reaction was then heated to 90°C for an additional 1 h to decompose the remaining butane sultone. The reaction mixture was cooled and treated with ion exchange resin. Cation exchange resin (H+ resin, 2 g) and anion exchange resin (OH- resin, 2 g) were added to the solution, stirred for 15 min and filtered off (resin was washed with 3×15 mL deionized water). The resulting filtrate (final pH=7) was clarified with activated charcoal (activated charcoal (0.3 g) was added to the solution under vigorous stirring, stirred for 30 min, and filtered off (the activated charcoal pad was washed with 3×15 mL of deionized water)).

[0515] The colorless solution was evaporated under reduced pressure (40° C.) to give a white powder (approximately 1.47 g).

[0516] Characterization

[0517] The reaction was monitored by TLC analysis (Figure 12B) and the resulting materials were characterized by MALDI-TOF and NMR analysis as shown in Figures 12C-12K.

[0518] Synthesis (high DS)

[0519] (βCD-(triazole)1-βCD) dimer core (1.2 g, 0.5 mmol) was suspended in deionized H2O (60 mL) under vigorous stirring. Sodium hydroxide (1.22 g, 30.5 mmol) was added to the mixture and the resulting solution was heated to 60°C. Butane sultone (2.8 mL, 3.72 g, 27.35 mmol) was added dropwise at 60°C and the solution was heated at the same temperature for 3 h. The reaction was then heated at 90°C for an additional hour to decompose the remaining butane sultone. The reaction mixture was cooled and treated with ion exchange resin. Cation exchange resin (H+ resin, 4 g) and anion exchange resin (OH- resin, 4 g) were added to the solution, stirred for 15 min and filtered off (resin was washed with 3×15 mL deionized water). The resulting filtrate (final pH=7) was clarified with activated charcoal (activated charcoal (0.5 g) was added to the solution under vigorous stirring, stirred for 30 min, and filtered (the activated charcoal pad was washed with 3×15 mL of deionized water). The colorless solution was evaporated under reduced pressure (40° C.) to give a white powder (1.51 g).

[0520] Characterization The resulting materials were characterized by MALDI-TOF and NMR analysis as shown in Figures 12M-P.

[0521] Example 6. Synthesis of quaternary ammonium substituted cyclodextrin dimer

[0522] Figure 3I and Figure 13G illustrate the molecules that are synthesized.

[0523] This example describes the synthesis of a quaternary ammonium substituted cyclodextrin dimer with a triazole-containing linker.

[0524] Quaternary ammonium (βCD-(triazole)1-βCD) dimer (synthesis example)

[0525] Preparation of the QA dimer was accomplished in a one-step reaction (see FIG. 13A). The βCD-(triazole)1-βCD dimer core is prepared by the synthetic strategy described in Example 2 above.

[0526] synthesis

[0527] The (BCD-(triazole)1-BCD) dimer core (1.2 g, 0.5 mmol) was suspended in deionized HO (100 mL) with vigorous stirring and sodium hydroxide (0.39 g, 9.8 mmol) was added. The resulting slightly yellow suspension was stirred for 30 min until complete dissolution. Once the temperature of the yellowish clear solution had stabilized at 5-10 °C, glycidyltrimethylammonium chloride (1.17 mL, 1.32 g, 8.7 mmol) was added in one portion with vigorous stirring. The reaction mixture was stirred at room temperature for 24 h, then once the temperature of the solution had stabilized at 5-10 °C, a second portion of glycidyltrimethylammonium chloride (0.4 mL, 0.45 g, 3 mmol) was added with vigorous stirring. The reaction mixture was heated at 50° C. for 3 h, then cooled and treated with ion exchange resins (H+ resin (6 g) and OH- (6 g) resin were added to the solution, stirred for 15 min, and filtered off (resin was washed with 3×15 mL deionized water)). The resulting filtrate (final pH=7) was clarified with activated charcoal (activated charcoal (0.2 g) was added to the solution with vigorous stirring, stirred for 30 min, and filtered off (charcoal pad was washed with 3×15 mL deionized water)). The colorless solution was evaporated under reduced pressure (40° C.) to give the title compound as a white powder (approximately 800 mg).

[0528] Characterization

[0529] The resulting material was characterized by MALDI-TOF and NMR analysis as shown in Figures 13B-13K.

[0530] In the case of QA-BCD derivatives, the typical Gaussian distribution with a regular pattern observed for randomly substituted derivatives is lost, while an irregular pattern of fragmentation is detected. Identification / assignment of these irregular peaks is challenging, since no simple pattern of fragmentation can be predicted. The irregular pattern observed in the MALDI spectra is most likely due to the instability of the trimethylammonium moiety under the experimental conditions. In detail, the desorption products (see Figure 2) are the result of trimethylammonium moiety cleavage, whereas the demethylation products (see Figure 2) are the result of progressive cleavage of the methyl group from the cationic side chain. Due to the generation of uninformative peaks during laser desorption, it is reasonable to conclude that MALDI conditions are not adequate to identify the DS of QA-BCD derivatives. However, the DS of QA-BCD derivatives could be identified by NMR (Figure 13I), which was estimated to be about 2.1.

[0531] Example 7. Synthesis of succinyl-substituted cyclodextrin dimers

[0532] The molecules synthesized are illustrated in Figure 3G and Figure 14G. Preparation of the succinyl-substituted dimer (Succ dimer) was accomplished in a one-step reaction (see Figure 14A).

[0533] synthesis

[0534] The (βCD-(triazole)1-βCD) dimer core (1.2 g, 0.5 mmol) was suspended in pyridine (23 mL) under inert atmosphere with vigorous stirring. In an attempt to increase the solubility of the (βCD-(triazole)1-βCD) dimer, the suspension was heated at 40° C. for 1 h, but this did not result in complete dissolution. A second portion of pyridine (23 mL) was added to the suspension, but dilution did not further improve the solubility of the (βCD-(triazole)1-βCD) dimer. Succinic anhydride (0.1 g, 1 mmol) was added at room temperature and the reaction mixture was stirred for 24 h. The crude reaction was concentrated under reduced pressure, dissolved in water (50 mL) (no clear solution was obtained), and treated with ion exchange resin (H+ resin (2 g) and OH- (2 g) resin were added to the solution, stirred for 15 min, and filtered (resin was washed with 3×15 mL deionized water)). The resulting filtrate (final pH=7) was clarified with activated charcoal (activated charcoal (0.5 g) was added to the solution under vigorous stirring, stirred for 30 min, and filtered (the activated charcoal pad was washed with 3×15 mL of deionized water)). The colorless solution was evaporated under reduced pressure (40° C.) to give the title compound as a white powder (approximately 900 mg).

[0535] Characterization

[0536] The resulting material was characterized by MALDI-TOF and NMR analysis as shown in Figures 14B-14K.

[0537] As with the QA dimer, MALDI analysis proved inadequate for determining the DS, which was determined by NMR (Figure 14I) and estimated to be approximately 2.1.

[0538] Example 8. Extraction of 7KC and cholesterol from blood cells using βCD dimer and monomer

[0539] method

[0540] Blood was collected from healthy donors by a qualified phlebotomist. Test substances or PBS only (negative control) were added to whole blood at various concentrations and incubated at 37°C for 3 hours. Blood was then spun down and serum was collected. Serum was frozen and then processed for mass spectrometry.

[0541] Plasma-free 7-ketocholesterol was measured by LC-MS / MS after protein precipitation, extraction with acetonitrile, and derivatization with a novel quaternary aminooxy (QAO) mass tag reagent, Amplifex Keto Reagent (AB Sciex, Framingham, MA, USA), which has been used previously for the analysis of testosterone (Star-Weinstock et al., Analytical Chemistry, 84(21):9310-9317. (2012)).

[0542] 50 μL of plasma samples were spiked with 0.5 ng of the internal standard d7-7-ketocholesterol (Toronto Research Chemicals, North York, Ontario, CA). d7-7-ketocholesterol was prepared as a 0.1 ng / μL solution in ethanol. Samples were treated with 250 μL of acetonitrile, vortex mixed, and centrifuged at 12,000×g for 10 min to remove protein. The supernatant was vacuum dried and then treated with 75 μL of QAO reagent. Working reagent was prepared by mixing 0.7 mL of Amplifex keto reagent with 0.7 mL of Amplifex keto diluent to prepare a 10 mg / mL stock solution. This stock solution was then diluted 1:4 with 5% acetic acid in methanol to a final working concentration of 2.5 mg / mL. The mixture was allowed to react at room temperature for 2 days before LC-MS / MS analysis.

[0543] 7-Ketocholesterol standards (Toronto Research Chemicals, North York, Ontario, CA) were prepared at 1–100 ng / ml in charcoal-stripped plasma, SP1070, (Golden West Biological, Temecula, CA, USA) and in phosphate-buffered saline. Standards prepared in PBS were used because there was detectable residual 7-ketocholesterol in charcoal-stripped plasma.

[0544] The QAO-7-ketocholesterol derivatives were analyzed using electrospray ionization (ESI) in positive mode on a 4000 Q-TRAP hybrid / triple quadrupole linear ion trap mass spectrometer (SCIEX, Framingham, MA, USA) interfaced with a Shimadzu (Columbia, MD) SIL-20AC XR autosampler followed by a 2LC-20AD XR LC pump.

[0545] The instrument was operated with the following settings: power supply voltage 4500 kV, GS1 50, GS2 50, CUR 20, TEM 550, and CAD gas medium. Compounds were quantified using multiple reaction monitoring (MRM) and transitions optimized by injection of pure derivatized compounds as shown in Table 1 below. Transitions in bold were used for quantification. [Table 1]

[0546] Separation was achieved using a Gemini 3μ C6-phenyl 110 Å, 100×2 mm column (Phenomenex, Torrance, CA, USA) maintained at 35° C. in a Shimadzu (Columbia, MD) CTO-20AC column oven. The gradient mobile phase was delivered at a flow rate of 0.5 ml / min. The gradient mobile phase consisted of two solvents, A: water with 0.1% formic acid, B: acetonitrile with 0.1% formic acid. The initial concentration of solvent B was 20%, followed by a linear increase to 60% B in 10 min, followed by 95% B in 0.1 min and held there for 3 min, then decreased over 0.1 min back to the original 20% B, and held there for 4 min. The retention time of 7-ketocholesterol was 8.46 min.

[0547] Data were acquired using Analyst 1.6.2 (SCIEX, Framingham, MA, USA) and analyzed with Multiquant 3.0.1 (SCIEX, Framingham, MA, USA) software. Sample values ​​were calculated from a standard curve constructed from the peak area ratios of analyte to internal standard versus analyte concentration fitted to a linear equation using 1 / x weighting. The lower limit of quantification was 1 ng / mL with an accuracy of 102% and a precision (relative standard deviation) of 8.5%. The signal-to-noise (S / N) was 19:1. At a concentration of 100 ng / mL, the accuracy was 98% and the precision was 0.5%, with a S / N of 24:1.

[0548] result

[0549] Figures 15A and 15B demonstrate that HPβCD dimers (DS of about 8 as determined by both MALDI and NMR, see Figures 10I and 10J) are able to remove 7KC from blood cells (whole blood) much more efficiently than HPβCD monomers. Although this is an ex vivo assay in human subjects, it allows to obtain results that can predict the effect in human patients much more accurately than experiments in non-human animals. Figure 15C demonstrates that HPβCD dimers do not appreciably affect plasma cholesterol levels, implying that they do not remove cholesterol to a large extent from blood cells. Removing too much cholesterol from cells could potentially lead to the destruction of cell and organelle membranes and cause cell death. We wanted to address this directly, and therefore performed a hemolysis assay.

[0550] Example 9. Hemolysis induced only by high concentrations of cyclodextrin dimer

[0551] method

[0552] For the test solutions, the amount of PBS was varied depending on the concentration of cyclodextrin being tested. Samples were tested in triplicate. To each sample, 50 μL of blood was added along with PBS and cyclodextrin solution (stock solution was also made in PBS) to the appropriate concentration in a final volume of 200 ul. 5% TritonX-100 was used as the positive control and PBS was the negative control. Once all samples were mixed, they were placed in a 37°C incubator and left for 3 hours with agitation. The positive control was 100% hemolyzed with TritonX-100 detergent. After removal from incubation, the samples were diluted by the same factor in a 96 Hydrograde plate and normalized to the positive control absorbance. The positive control absorbance is around 1.1. The absorbance is read at 540 nm. The sample average was then corrected by subtracting the negative samples. The experiment was performed in triplicate. Error bars are standard deviation of the mean (Melanga et al., Journal of Pharmaceutical Sciences, 105(9):2921-31. (2016)), (Kiss et al., European Journal of Pharmaceutical Sciences, 40(4):376-80. (2010)).

[0553] Figures 15D-E demonstrate that the toxicity of butyl-linked and triazole-linked dimers to blood cells remains very low, with no noticeable toxicity in the pharmacological range below 1 mM. Figure 15D shows hemolysis with butyl-linked HP dimers with three different DS (DS identified by MALDI in Figures 10G-I and NMR in Figure 10J), triazole-linked HP dimers with DS ≈3 (characterized in Figures 10P and 10W; labeled based on MALDI), and triazole-linked Me dimers with DS ≈3 (characterized in Figures 11I and 11L). At higher concentrations, only the three butyl-linked dimers showed measurable hemolysis. In Figure 15E, we tested hemolysis with various other substituents of the triazole-linked βCD dimer. Tested were unsubstituted quaternary ammonium (DS approx. 2, characterized in FIG. 13I), succinyl (DS approx. 2, characterized in FIG. 14I), and sulfobutyl (DS characterized by both NMR and MALDI in FIG. 12E, FIG. 12H, FIG. 12K, and FIG. 12N; MALDI DS is labeled). Only the unsubstituted dimer was tested up to 7.5 mM. At this concentration, about 5% hemolysis could be detected. The other dimers were tested only up to 5 mM, but no significant hemolysis was detected at any of the concentrations tested.

[0554] Although the triazole dimerized form of βCD appears to be less hemolytic than the HPβCD butyl dimer tested, even at high concentrations, both linkers and all substituent types showed very low solubility, suggesting low toxicity.

[0555] Example 10. Solubilization of sterols and sterol-like compounds by cyclodextrin dimers

[0556] The solubilization of lipophilic compounds by the dimers described in Examples 2 to 6 was examined. Test compounds included a cholesterol precursor (desmosterol), other oxysterols, steroid hormones, and sterol vitamins.

[0557] Method of in vitro solubility assay (turbidity assay)

[0558] Sterol stock solutions (containing oxysterols, hormones, and vitamins) were suspended in 100% ethanol. Final concentration of suspension: 3% ethanol, 300 uM sterol in PBS with various concentrations of cyclodextrin. Samples were incubated at 37° C. for 30 minutes and then absorbance was measured at 350 nm in a spectrophotometer plate reader. Samples were prepared in quadruplicate using a Beckman Biomek2000 liquid handler and hydrophilic coated plates were used to minimize sterol binding to the well surface. All experiments were performed at least three times. Error bars are standard deviation of the mean.

[0559] Turbidity values ​​were normalized based on the percentage of turbidity measured without cyclodextrin.

[0560] result

[0561] We tested our novel dimers against 7-ketocholesterol in an in vitro spectrophotometric assay. In FIG. 16A, DS3 is a butyl-linked dimer with an average of about 3 hydroxypropyl groups (quantified by MALDI in FIG. 10G), DS6 is a butyl-linked dimer with an average of about 6 substituents (MALDI, FIG. 10H), and DS8 is a butyl-linked dimer with an average of about 8 hydroxypropyl substituents (MALDI, FIG. 10I). Sterol concentrations were always constant at 300 μM and were tested against various concentrations of HPβCD dimer. HP(CD-triazole-CD) is a triazole-linked cyclodextrin dimer with the indicated average number of substitutions as determined by MALDI (FIG. 10P), while HP(CD-pig-CD) shows a butyl-linked dimer with the indicated DS.

[0562] Figures 16A-B show that all of our synthesized HPβCD dimers solubilize both 7KC and cholesterol much more efficiently than the HPβCD monomer. This is consistent with our computational models and predictions that explain how two linked monomers completely surround the sterol, protect it from water, maintain binding over time, and can restore binding if lost. At some low concentrations of dimer, it is possible to compare the solubilization achieved at that concentration with that achieved at high concentrations of monomer and estimate that the same solubilization is achieved at about one-tenth the molar concentration. This suggests that the affinity for cholesterol / 7KC may be about ten times higher than that of the monomer, but the exact identity of the affinity constant must await the results of other experiments. We then further attempted to determine whether these dimerized HPβCDs could bind 7KC with suitable affinity.

[0563] We found that several different HPβCD dimers could indeed preferentially bind 7KC (Figures 16A-B). Figure 16B shows that triazole dimers labeled with DS3 bind 7KC with higher specificity than DS6 or DS7 dimers. These DS values ​​were determined by MALDI. We further found that these HPβCDs could preferentially bind 7KC over cholesterol. It should be noted that some dimers appeared to preferentially solubilize 7KC over others, which is explored in Figures 16E-H.

[0564] As shown above in Figure 15C, we found that in human blood, DS8 HPβCD dimers removed significant amounts of 7KC from donor cells, while serum cholesterol levels appeared to be stable, implying that the affinity for cholesterol was insufficient to perturb plasma cholesterol levels outside the normal range, although it may have resulted in cholesterol removal from cells at the concentrations tested.

[0565] Figures 16C-D show how the dimer interacts with a variety of other sterols and steroid hormones with differing affinities as defined by relative turbidity.

[0566] Figure 16C shows that HP(βCD-(butyl)1-βCD) dimers can efficiently encapsulate vitamin D3 (cholecalciferol), but not vitamin D2. It has been previously observed that βCD monomers can encapsulate vitamin D3 (Szejtli et al., Drugs of the Future, 9:675-676. (1984)), but our dimers appear to solubilize vitamin D3 many times more efficiently than HPβCD monomers (Figure 2A vs. Figure 16C, although the concentration range is 10-fold lower for the dimer experiments).

[0567] We also wished to test the ability of our dimers to solubilize oxysterols other than 7KC.

[0568] Figure 16C shows that the HPβCD-butyl linked dimer (DS8) solubilizes various oxysterols to different degrees and appears to solubilize cholesterol epoxide particularly well.

[0569] Figure 16D demonstrates the ability of the butyl dimer to bind various hormones. As with monomeric HPβCD, our dimer binds the three estrogens variably well. Note that although progesterone solubilization appears dramatic this time, progesterone solubility is originally much higher than the other hormones tested, so this data normalization method is somewhat deceptive in this example.

[0570] We observed that the dimer with the lowest DS had the highest specificity for 7KC over cholesterol. We therefore performed a more detailed analysis of the least substituted molecules of each linked dimer. Figures 16E and 16F go into more detail on the two HP dimers that showed the best specificity for 7KC. In deeper detail, we confirmed that both head-to-head linked cyclodextrin dimers with approximately 3 HP substituents preferentially solubilized 7KC over cholesterol. These dimers show substantial affinity and specificity for 7KC at concentrations below 0.5 mM.

[0571] We further noticed that CD dimers substituted with alternative groups that confer solubility and low toxicity significantly increased the affinity of CD for 7KC (Figure 16G-H). The methylated triazole-linked dimers had a similar number of substituents (approximately 3) as the HPβCD dimer in Figure 16F. We retested the HPβCD dimer (DS3) together with the methyl dimer (DS3) and found that they had very similar abilities to solubilize both 7KC and cholesterol while maintaining similar specificity for 7KC.

[0572] Based on the prediction that dimerized βCD with other substituents and similar degrees of substitution may also bind 7KC and cholesterol with similar affinity and specificity, novel substituted triazole-linked dimers were synthesized (Examples 5-7 above). We used a set of charged functional groups typically used as substituents on cyclodextrins (quaternary ammonium (QA), sulfobutyl (SB), and succinyl (SUCC)). These low-substituted compounds yielded comparable or improved affinity and specificity for 7KC (Figure 16H) when compared to unsubstituted, hydroxypropyl, or methyl-substituted triazole-linked dimers (Figure 16B, Figure 16E-G). Conversely, highly substituted SB dimers bound neither cholesterol nor 7KC well. This was likely caused by the many bulky SB groups restricting access to the binding cavity of the CD dimer.

[0573] Combining the turbidity data for the monomers and dimers with the computational data, two generalized conclusions can be made. One is that low substitution (substitutions at the bottom are likely to be particularly important) promotes specificity for certain interactions, especially with 7KC. The modeling data indicate that hydrogen bonds between the bottom hydroxyl group and the 7-keto group may promote this specificity. Furthermore, in general, the modeling data indicate that if a bulky substituent is present at a high enough DS level, it may indiscriminately block access to the cavity of any potential guest molecule. That is, non-bulky groups such as methyl groups added at high substitution levels to the CD dimer are predicted to bind sterol molecules such as cholesterol and 7KC with high affinity, but not with particular selectivity for 7KC compared to cholesterol, whereas low-substituted methyl beta cyclodextrin dimers are predicted to bind 7KC with high specificity compared to cholesterol. Conversely, cyclodextrin dimers with bulky substituents such as SB are predicted to bind specifically to 7KC over cholesterol at low substitution levels, but at high substitution levels, they are predicted to bind neither cholesterol nor 7KC, and likely other sterols, due to blocked access to the binding cavity. Less bulky groups such as HP are predicted to behave similarly to SB, but in general, a greater number of HP groups than S...

Claims

1. The structure: CD-L-CD A cyclodextrin (CD) dimer having the formula: wherein a linker (L) is attached to the large face (bottom) of each CD molecule, and said attachment of each CD to said linker is, independently, through an O, S, or N attached to the C2 or C3 carbon of each CD subunit, or through an acetal bonded to two adjacent oxygens of said CD subunit; wherein each CD has the structure of Formula X: 【Chemistry 1】 (Formula X) wherein L has a length of 1 to 20 atoms, each of said 1 to 20 atoms being C, N, O, or S; In the formula, R １ , R ２ , and R ３ each independently represents H, methyl, hydroxypropyl, sulfobutyl, succinyl, quaternary ammonium, -CH ２ CH(OH)CH ２ N (CH ３ ) ３ ＋ , maltosyl, carboxymethyl, palmitoyl, ethyl, propyl, isopropyl, butyl, isobutyl, alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamido, aminocarbonyloxyalkyl, aminosulfonyl, ammonium, ammonia, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyloxy, azido, bromo, chloro, cyanoalkyl, cycloalkyl, cycloalkenyl, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkyl ... cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, deoxy, glucosyl, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroaralkyloxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocycloamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, iodo, ureido, carbamate, carboxy, sulfate, sulfuryl, sulfonamido, nitro, nitrito, cyano, phosphate, phosphoryl, phenoxy, acetyl, fatty acid, palmitoyl, monosaccharide, or disaccharide, with the proviso that R １ , R ２ , and R ３ 1 to 40 of the groups are not H and are optionally １ , R ２ , and R ３ 1 to 28 of the groups are not H and are optionally １ , R ２ , and R ３ 2-15 or 4-20 of the groups are not H; and, optionally, the CD monomer bears one or more additional substituents; wherein the CD dimer has an average degree of substitution of 1 to 40; and wherein the CD dimer contains up to 28 methyl groups. The cyclodextrin dimer.

2. 2. The cyclodextrin dimer of claim 1 , (Formula X), (a) R １ , R ２ , and R ３ each independently represents H, methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium, -CH ２ CH(OH)CH ２ N (CH ３ ) ３ ＋ , glucosyl, palmitoyl, phosphate, phosphoryl, amino, azido, sulfate, sulfuryl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromo, chloro; or (b) R １ , R ２ , and R ３ each independently represents H, methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium, -CH ２ CH(OH)CH ２ N (CH ３ ) ３ ＋ and (c) the CD dimer is HP-substituted with 1-28 hydroxypropyl (HP) groups, or 2-15 HP groups, or 4-20 HP groups, or 2-5 HP groups; (d) the CD dimer is Me-substituted with 1-20 methyl (Me) groups, 2-15 Me groups, or 2-10 Me groups; (e) the CD dimer is sulfobutyl-substituted with 1-28 sulfobutyl groups, 1-14 sulfobutyl groups, optionally 2-10 sulfobutyl groups, or 2-5 sulfobutyl groups; (f) the CD dimer is succinyl-substituted with 1-28 succinyl groups, optionally 2-15 succinyl groups or 4-20 succinyl groups, or 2-5 succinyl groups; or (g) the CD dimer is quaternary ammonium substituted with 1 to 28 quaternary ammonium groups, 2 to 15 quaternary ammonium groups, 4 to 20 quaternary ammonium groups, or 2 to 5 quaternary ammonium groups, said quaternary ammonium groups being -CH ２ CH(OH)CH ２ N (CH ３ ) ３ ＋ Including, The cyclodextrin dimer.

3. L has the following structure: 【Chemistry 2】 having each R is independently selected from H, X, SH, NH, NH2, or OH, or is absent; each X is a substituted or unsubstituted alkane, alkene, or alkyne; each A is independently selected from a single, double, or triple covalent bond, S, N, NH, O, or a substituted or unsubstituted alkane, alkene, or alkyne; B is a substituted or unsubstituted 5- or 6-membered ring, S, N, NH, NR, O, or absent; and / or the length of the linker is 2-7 atoms, 3-6 atoms, 4-7 atoms, 4-6 atoms, 4-5 atoms, 4, 3, or 2 atoms; The cyclodextrin dimer according to claim 1 or 2.

4. The cyclodextrin dimer of claim 1 , wherein the linker has a length of 4.

5. The cyclodextrin dimer of claim 1 , wherein the linker is an unsubstituted alkyl.

6. The cyclodextrin dimer of claim 1 , wherein the linker is a substituted or unsubstituted butyl linker.

7. (a) the linker comprises a triazole; (b) the linker has the formula: 【Chemistry 3】 (Formula XI) wherein n1 and n2 are each 1 to 8, or 1 to 4, or wherein n1 is 1 and n2 is 3; or (c) the linker has the formula A-W: 【change】 wherein n is 1, 2, 3, 4 or 5 carbons, and wherein the depicted oxygen atom at each end of each linker forms part of the cyclodextrin monomer to which the linker is attached. The cyclodextrin dimer according to any one of claims 1 to 6.

8. 2. The cyclodextrin dimer of claim 1 , (Formula X), (a) the linker has the formula: 【Chemistry 4】 (Formula XI) wherein n1 and n2 are each 1-8, or 1-4, or wherein n1 is 1 and n2 is 3; or wherein the linker is 4-7 atoms, 4-6 atoms, or 4-5 atoms in length; (b) R １ , R ２ , and R ３ are each independently H, methyl, hydroxypropyl, sulfobutyl, succinyl or quaternary ammonium, -CH ２ CH(OH)CH ２ N (CH ３ ) ３ ＋ wherein R １ , R ２ , and R ３ 1 to 20 of the groups are not H; and (c) the CD dimer has an average degree of substitution of 1 to 28, 1 to 20, 2 to 15, 2 to 10, 2 to 8, or 2 to 5; The cyclodextrin dimer.

9. moreover, (a) at least one methyl, hydroxypropyl, sulfobutyl, succinyl, quaternary ammonium group, or —CH ２ CH(OH)CH ２ N (CH ３ ) ３ ＋ and / or (b) at least one of alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamido, aminocarbonyloxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyloxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, heteroalkyl ... heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroaralkyloxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocycloamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, ureido, carbamate, carboxy, sulfonamido, nitro, cyano, phenoxy, acetyl, ammonium, ammonia, azido, bromo, chloro, deoxy, glucosyl, iodo, sulfate, sulfuryl, nitrito, phosphate, phosphoryl, fatty acid, palmitoyl, monosaccharide, or disaccharide, and / or (c) The cyclodextrin dimer of any one of claims 1 to 8, substituted with at least one maltosyl, carboxymethyl, amino, azido, ethyl, propyl, isopropyl, butyl, or isobutyl group.

10. 10. The cyclodextrin dimer of claim 1, wherein: (i) L is linked to the C2 carbon of each CD monomer; (ii) L is linked to the C3 carbon of each CD monomer; or (iii) L is linked to the C2 carbon of one CD monomer and the C3 carbon of the other CD monomer.

11. 11. The cyclodextrin dimer of claim 1, wherein the cyclodextrin dimer (i) exhibits a higher affinity for 7KC than for cholesterol, optionally wherein the higher affinity is determined by a turbidity test; or (ii) exhibits at least a 1.1-fold to 10-fold higher affinity for 7KC than for cholesterol.

12. 12. A composition comprising a mixture of cyclodextrin dimers according to any one of claims 1 to 11, having an average degree of substitution of 2 to 10; or an average degree of substitution with hydroxypropyl, sulfobutyl, succinyl, or quaternary ammonium groups of 2 to 5; or an average degree of substitution with methyl groups of 2 to 10, wherein the average degree of substitution is determined by NMR or by mass spectrometry.

13. A pharmaceutical composition comprising the cyclodextrin dimer according to any one of claims 1 to 11 or the composition according to claim 12, and a pharma- ceutically acceptable carrier, A pharmaceutical composition, wherein the cyclodextrin dimer is the only active ingredient in the composition, and the pharmaceutical composition consists or consists essentially of the cyclodextrin dimer and the pharma- ceutically acceptable carrier.

14. 14. The pharmaceutical composition of claim 13, (i) for administration to a subject in need of treatment; or (ii) for reducing the amount of 7KC in a subject suffering from harmful or toxic effects of 7KC and in need of reducing the amount of 7KC.

15. (a) administered to said subject via parenteral (subcutaneous, intramuscular, or intravenous), topical, transdermal, oral, sublingual, or buccal administration; or (b) the cyclodextrin dimer is administered intravenously; and (c) the pharmaceutical composition comprises about 1 mg to 20 g, 1 to 10 g, or 50 mg to 5 g of the cyclodextrin dimer; A pharmaceutical composition according to claim 13 or 14.

16. Atherosclerosis / coronary artery disease, arteriosclerosis, coronary artery sclerosis due to coronary artery calcification, heart failure (all stages), Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, vascular dementia, multiple sclerosis, Smith-Lemli-Opitz syndrome, childhood neuronal ceroid lipofuscinosis, lysosomal acid lipase deficiency, cerebrotendinous xanthomatosis, X-linked adrenoleukodystrophy, sickle cell disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Goat's disease, 16. The pharmaceutical composition of any one of claims 13 to 15, for preventing, treating or ameliorating symptoms of one or more of the following: Chronic Obstructive Pulmonary Disease, Chronic Obstructive Pulmonary Disease, Chronic Hepatitis, Chronic Kidney ...

17. 17. A pharmaceutical composition according to any one of claims 13 to 16 for preventing, treating or ameliorating symptoms of atherosclerosis.

18. 18. The pharmaceutical composition of claim 17, further comprising a second treatment, applied simultaneously or sequentially in any order, said second treatment comprising one or more of an anticholesterol drug selected from fibrates or statins, an antiplatelet drug, an antihypertensive drug, or a nutritional supplement.

19. 19. The pharmaceutical composition of claim 18, wherein the statin drug comprises a statin drug or combination of drugs including: (i) niacin sustained release and lovastatin, (ii) lovastatin sustained release, (iii) amlodipine-atorvastatin combination, (iv) rosuvastatin, (v) sitagliptin and simvastatin, (vi) fluvastatin, (vii) fluvastatin sustained release, (viii) atorvastatin, (ix) pitavastatin, (x) statins including lovastatin, pravastatin, (xi) niacin sustained release and simvastatin, (xii) ezetimibe and simvastatin, or (xiii) simvastatin.

20. 12. A method for purifying an oxysterol, comprising contacting a composition containing an oxysterol with a cyclodextrin dimer of any one of claims 1 to 11, thereby solubilizing the oxysterol in the cyclodextrin dimer; and recovering the cyclodextrin dimer and the solubilized oxysterol.

21. (a) the oxysterol comprises or consists of 7KC; (b) the method further comprises measuring the amount or concentration of 7KC in the solubilized oxysterol, thereby determining the relative concentration of 7KC in the composition; and / or (c) the composition comprises a patient sample.

21. The method of claim 20.

22. 12. An in vitro method for removing oxysterols from a sample comprising contacting a sample containing an oxysterol with a cyclodextrin dimer according to any one of claims 1 to 11, thereby solubilizing the oxysterol in the cyclodextrin dimer; and separating the sample from the cyclodextrin dimer and solubilized sterol, and optionally reintroducing the sample to the subject from whom the sample was obtained.

23. 12. A method for producing a cholesterol-reduced product, comprising contacting a product containing cholesterol with the cyclodextrin dimer of claim 1, thereby solubilizing the cholesterol in the cyclodextrin dimer; and removing the cyclodextrin dimer and solubilized cholesterol from the product.

24. 24. The method of claim 23, wherein the product is a food product or the food product is a meat product and / or a dairy product.

Images