Cyclodextrin dimers, their compositions, and their uses

Abstract

To provide methods of making a cyclodextrin dimers.SOLUTION: A method of making a CD dimer comprises: (a) reacting a dialkylating agent with β-cyclodextrin whose upper base is protected, thereby producing an upper base-protected βCD dimer linked through a lower base; (b) deprotecting the upper base-protected βCD dimer, thereby producing a deprotected βCD dimer; and (c) linking the deprotected βCD dimer to one or more hydroxypropyl, methyl, succinyl, sulfobutyl and / or quaternary ammonium or -CH2CH(OH)CH2 N(CH3)3+ groups, thereby producing the cyclodextrin dimer. The CD dimer has an average degree of substitution of 1-40, and includes 28 or less methyl groups.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 787,869, filed on Jan. 3, 2019 (Attorney Docket No. 48731.1600) and U.S. Provisional Application No. 62 / 850,334, filed on May 20, 2019 (Attorney Docket No. 48731.1601). These are hereby incorporated by reference in their entirety as if fully set forth herein.

Background Art

[0002] 7 - Ketocholesterol (7KC) is an oxysterol that results from the non - enzymatic reaction of oxygen radicals with cholesterol. 7KC can be formed in vivo or ingested from food, but is potentially toxic and thought to serve no useful purpose in humans and other eukaryotes. 7KC, like cholesterol, is found in atherosclerotic plaques. 7KC is the most abundant non - enzymatically - produced 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] Cyclodextrin (CD) is a cyclic oligosaccharide polymer consisting of six (αCD), seven (βCD), or eight (γCD) sugar rings (FIG. 1A). Alpha, beta, and gamma cyclodextrins are the most common forms and have many pharmaceutical, industrial, household, and food - related uses. Cyclodextrin has been used in a variety of applications, for example, as a dietary fiber as a food additive. Cyclodextrin has also been used in pharmaceutical compositions as an aerosolizing agent and excipient for low - molecular - weight hydrophobic drugs, typically in combination with an active pharmaceutical ingredient.

[0004] Hydroxypropyl-β-cyclodextrin (HPβCD) is a βCD in which a certain number of hydroxypropyl (HP) groups are added to O2, O3, or O6 oxygen (or an atom substituted for the oxygen) in some or all of the seven glucose monomers that make up β-cyclodextrin. Hydroxypropylation of cyclodextrin improves its water solubility and safety to such an extent that it is used in humans, especially as an excipient for active drugs, for various purposes. It has obtained the HPβCD GRAS (Generally Recognized as Safe) list designation by the FDA. The most commercially available HPβCDs have an average of 4 to 9 HP substituents, and all available products contain a mixture of substitution numbers and positions, which are usually reflected in the advertised average degree of substitution (DS).

[0005] Other CD substituents include, among others, methyl, succinyl, sulfobutyl, maltosyl, carboxymethyl, and quaternary ammonium, which can create CDs that are highly water-soluble and low in cytotoxicity, regardless of whether they are charged or neutral groups. Commercially available βCDs can have various degrees of substitution, which can vary from as low as about 1 to maximum substitution (21 substituents), depending on the specific substituent and the vendor. Summary of the Invention

[0006] The present disclosure describes the design and testing of various cyclodextrin (CD) dimers, including, inter alia, HPβCD dimers, methylβCD dimers, succinylβCD dimers, sulfobutylβCD dimers, and quaternary ammonium dimers as CD dimers. It has been demonstrated that certain dimers have a dramatically increased affinity for 7KC and cholesterol compared to monomeric CDs. 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. The molecular modeling experiments described below show the predicted interaction mechanism. Further, the examples confirm the predicted ability of the novel substituted cyclodextrin dimers to solubilize sterols, including the selective solubilization of 7KC compared to cholesterol.

[0007] In one aspect, the present disclosure provides a CD dimer having the structure CD-L-CD, wherein each CD is 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 functional group, such as methyl, hydroxypropyl (HP), sulfobutyl (SB), succinyl (SUCC), -CH2CH(OH)CH2N(CH3)3 + and the like, a quaternary ammonium (QA), or a combination thereof. Typically, each CD monomer is composed of D-type glucose monomers. The CD dimer is substituted with a functional group and typically has a degree of substitution (DS) of 1 to 28, which indicates the total number of such functional group substitutions present in both CD subunits. The substituents may be present in one or both of the CD subunits. The linker length can be 2 to 8 atomic lengths, for example 4 to 8 atomic lengths, along 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, and the linker is optionally substituted. Examples of CD dimers are those of Formulas I to IX (Figures 3B to 3J, respectively). Optionally, the CD dimer is further substituted.

[0008] In another aspect, the present disclosure provides a βCD dimer having the structure CD-L-CD, where each CD is 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 D-type glucose monomers. The βCD dimer is substituted with hydroxypropyl (HP), typically having a degree of substitution (DS) of 1 to 40, which indicates the total number of substituents present in both CD subunits. The substituents may be present in one or both of the CD subunits. The linker length can be 4 to 8 atomic lengths along 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, and the linker is optionally substituted. Examples of the βCD dimer are those of formula I, formula II, or formula III (FIGS. 3B - 3D, respectively). Optionally, the βCD dimer is further substituted.

[0009] 7KC is thought to be involved in heart disease, cystic fibrosis, liver injury and liver failure, as well as hypercholesterolemia complications. When suffering from hypercholesterolemia, 7KC may diffuse through the cell membrane, in which case 7KC affects receptor and enzyme functions. The high prevalence of dementia in hypercholesterolemia has been associated with 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 improves rapid reactive oxygen species production. The pathogenesis of other age-related diseases, such as age-related macular degeneration (AMD - atrophic type), Alzheimer's disease, and lysosomal storage diseases such as Niemann - Pick disease type C (NPC), has also been linked to increased levels of 7KC. Oxysterols, including 7KC, are also involved in the increase in free radical levels, which in turn affects lipid circulation in cystic fibrosis. The increase in free radicals caused by oxysterols such as 7KC is thought to be involved in apoptosis, cytotoxicity, endothelial dysfunction, and the regulation of enzymes involved in inflammation and fatty acid metabolism.

[0010] 7KC is formed from the non-enzymatic reaction of oxygen radicals and cholesterol, indicating that 7KC formation may not be beneficial. In fact, 7KC is thought to enhance the production of free radicals throughout the body, but this is of particular concern in the heart and vascular tissues. Free radicals affect cell and enzyme reactions that are important for cholesterol-mediated tissue damage, which is particularly critical in the heart and vascular tissues. Free radicals are thought to enhance inflammation in the vasculature. 7KC is thought to cause mitochondrial and lysosomal dysfunction by disrupting the function of cells and organelle membranes and is thought 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 resolve plaques, but if they are filled 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 diseases including, for example, atherosclerosis, AMD, arteriosclerosis, coronary atherosclerosis due to coronary artery calcified 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, neuronal ceroid lipofuscinosis in children, 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, Gaucher's disease, Stargardt's disease, 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 or hypercholesterolemia-related dementia. Suitable cyclodextrin (e.g., HPβCD, MeβCD, SUCCβCD, QAβCD, or SBβCD) dimers are selective for 7KC (compared to cholesterol). Preferably, the CD 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 invention provide for the use of cyclodextrin (e.g., HPβCD, MeβCD, SUCCβCD, QAβCD, or SBβCD) dimers for solubilizing and / or removing 7KC, which can be performed either in vitro or in vivo.

[0013] In an exemplary embodiment, the cyclodextrin dimer (e.g., HPβCD, MeβCD, SUCCβCD, QAβCD, or SBβCD) exhibits a higher binding affinity and / or solubilization for 7KC than for cholesterol. The higher specificity for 7KC than for cholesterol is most evident at sub-saturating concentrations, but at higher concentrations, solubilization of both sterols may approach 100%. This specificity enables the use of the cyclodextrin dimer for the purpose of preferentially solubilizing and removing 7KC.

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

[0015] CD-L-CD

[0016] wherein L is linked to the large face (bottom base) of each CD molecule through the C2 carbon (instead of R 1 ) and / or through the C3 carbon (instead of R 2 );

[0017] wherein CD has the structure of formula X:

Chemical formula

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

[0019] and CD is substituted with 1 to 40 groups, for example, 1 to 28 groups, optionally 2 to 15 groups, or 4 to 20 groups. The number of substitutions indicates the total number of R 1 , R 2 , and / or R 3 groups that are not H. The CD can 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 + and other 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, alkylsulfonylamide, aminocarbonyloxyalkyl, aminosulfonyl, ammonium, ammonia, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyl oxy, azide, bromo, chloro, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, deoxy, glucosyl, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroaralkyl oxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocycloamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, iodo, ureido, carbamate, carboxy, sulfate, sulfinyl, sulfonamide, nitro, nitrito, cyano, phosphate, phosphoryl, phenoxy, fatty acids such as acetyl group, palmitoyl group, monosaccharide, or disaccharide. In an exemplary embodiment, the substituent is preferably a maltosyl group or a carboxymethyl group.

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

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

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

[0024] CD-L-CD

[0025] In the formula, L is connected to the major face (lower base) of each CD molecule through the C2 carbon of each CD subunit (instead of R 1 ), and / or through the C3 carbon (instead of R 2 );

[0026] In the formula, CD has the structure of formula X:

Chemical formula

[0027] In the formula, L has a length of 8 or fewer atoms along the shortest path through the linker connecting the two CD subunits of the dimer, and each of the 8 or fewer atoms is preferably C, N, O, or S;

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

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

[0030] CD-L-CD

[0031] In the formula, L is connected to the major face (lower base) of each CD molecule through the C2 carbon of each CD subunit (instead of R 1 ), and / or through the C3 carbon (instead of R 2 );

[0032] In the formula, CD has the structure of formula X:

Chemical formula

[0033] In the formula, L has a length of 8 or fewer atoms along the shortest path through the linker connecting the two CD subunits of the dimer, and each of the 8 or fewer atoms is preferably C, N, O, or S;

[0034] CD is substituted with 1 to 40 methyl (Me) groups, optionally 1 to 28 Me groups, optionally 2 to 15 Me groups or 4 to 20 Me groups, preferably 2 to 10 Me groups, and optionally the CD has one or more additional substituents. Without wishing to be bound by theory, methyl groups are thought to be particularly suitable for substituting such CD dimers with a large number of substituents. This is because the size of the methyl group is particularly small and thus does not interfere with the entry of a guest (such as 7KC or cholesterol) into the CD dimer binding cavity. Further, it is assumed that one or more methyl substituents can be added to any of the cyclodextrin dimers of the present disclosure, and this assumption includes a number up to a maximum of 40 substituents other than hydrogen when including both substituents other than methyl and the added methyl substituents as substituents that are not hydrogen in total.

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

[0036] CD-L-CD

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

[0038] In the formula, CD has the structure of formula X:

Chemical formula

[0039] In the formula, L has a length of 8 or fewer atoms along the shortest path through the linker connecting the two CD subunits of the dimer, and each of the 8 or fewer atoms is preferably C, N, O, or S;

[0040] CD is substituted with sulfobutyl groups having 1 to 28 carbon atoms, such as sulfobutyl groups having 1 to 14 carbon atoms, optionally sulfobutyl groups having 2 to 10 carbon atoms, preferably sulfobutyl groups having 2 to 5 carbon atoms, and optionally the CD has one or more additional substituents. The CD can have 2 to 4 sulfobutyl groups, or can have 2, 3, 4, or 5 sulfobutyl groups.

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

[0042] CD-L-CD

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

[0044] In the formula, CD has the structure of formula X:

Chemical formula

[0045] In the formula, L has a length of 8 or fewer atoms along the shortest path through the linker connecting the two CD subunits of the dimer, and each of the 8 or fewer atoms is preferably C, N, O, or S;

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

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

[0048] CD-L-CD

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

[0050] wherein CD has the structure of formula X:

Chemical formula

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

[0052] The CD is substituted with 1 to 28 quaternary ammonium groups, optionally 2 to 15 quaternary ammonium groups or 4 to 20 quaternary ammonium groups, preferably 2 to 5 quaternary ammonium groups, and the quaternary ammonium group is -CH2CH(OH)CH2N(CH3)3 +, for example, containing -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 pharmaceutically acceptable salt of the quaternary ammonium is included within the scope of the present disclosure.

[0053] L may have the following structure:

Chemical formula

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

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

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

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

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

[0059] The length of the linker can be 2 to 7, 3 to 6, 4 to 7, 4 to 6, 4 to 5, or 4, or 2 to 3.

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

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

[0062] The linker may contain a triazole.

[0063] The linker has the following structure:

Chemical formula

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

[0065] In an exemplary embodiment, when the linker L contains a substituted or unsubstituted alkyl, preferably having a length of 8 atoms or less, for example, 2 to 7, 2 to 6, or 4 to 7 or 4 to 6 or 4 to 5, or having a length of 8, 7, 6, 5, 4, 3, or 2, or any numerical range 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 positions of both CD monomers, provided that preferably the linker is a substituted or unsubstituted butyl, more preferably an unsubstituted butyl.

[0066] The linker can have a single binding point with each CD monomer. The linker can have a single binding point with one CD monomer and multiple (two or more) binding points with the other CD monomer. The linker can have multiple (two or more each) binding points with each CD monomer. The linker can include any of the linkers depicted in FIG. 8D. Of course, the depicted linker has oxygen atoms at each end, and these oxygen atoms form part of the cyclodextrin to which the linker is attached. The oxygen atom is not considered part of the linker with respect to the purpose of determining the linker length. Similarly, in the case of a linker that connects to one or both cyclodextrin monomers at multiple sites, the connection shown on the left is connected to one monomer, and the connection shown on the right is connected to the other monomer.

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

[0068] CD-L-CD

[0069] wherein L is connected to the major face (bottom) of each CD molecule through the C2 carbon (instead of R 1 and / or through the C3 carbon (instead of R 2 );

[0070] wherein CD has the structure of formula X:

Chemical formula

[0071] wherein L is a triazole and has a length of 8 or fewer atoms, and each of the 8 or fewer atoms is preferably C, N, O, or S;

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

[0073] When the linker has the following structure: [Chemical formula] It may have (Formula XI), where n1 and n2 are each 1 to 8 or 1 to 4, and preferably in the formula, n1 is 1 and n2 is 3.

[0074] The length of the linker may be 3 to 7, 3 to 6, 4 to 7, 4 to 6, or 5 to 6.

[0075] The length of the linker may be 4 to 5.

[0076] When the cyclodextrin further has (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, alkylsulfonylamide, aminocarbonyloxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyl oxy, 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, sulfonamide, nitro, cyano, phenoxy, acetyl group ammonium, ammonia, azide, bromo, chloro, deoxy, glucosyl, iodo, sulfate, sulfinyl, nitrito, phosphate, phosphoryl, fatty acid such as palmitoyl group, monosaccharide, or disaccharide, and / or (c) at least one methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium (e.g., -CH2CH(OH)CH2N(CH3)3 +) may be substituted with a glucosyl, palmitoyl, phosphate, phosphoryl, amino, azido, sulfate, sulfuryl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromo, or chloro group.

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

[0078] Each R 1 、each R 2 、and each R 3is independently selected from (a) methyl, H, hydroxypropyl, sulfobutyl ether, 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, alkylsulfonylamide, aminocarbonyl-oxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyl-oxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroaralkyl-oxy, heterocyclic-alkoxy, halogen, haloalkyl, haloalkoxy, heterocyclicamino, heterocyclic, heterocyclicalkyl, heterocyclicoxy, heterocyclicalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, 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, the C3 carbon of each CD monomer, or the C2 carbon of one CD monomer and the C3 of the other CD monomer. When the linker has multiple binding points for a single CD monomer, the linker can be linked to the C2 carbon, C3 carbon, or a combination of the C2 and C3 carbons of that monomer. Depending on the reaction used to form the linkage, the purification process, and / or the structure of the linker, a particular configuration may be preferred.

[0080] The cyclodextrin dimer may exhibit a higher affinity for 7KC than for cholesterol. The higher affinity can be identified using the turbidity test disclosed herein.

[0081] The cyclodextrin dimer may exhibit an affinity for 7KC that is at least 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold stronger than for cholesterol. The cyclodextrin dimer may exhibit a decrease in the relative turbidity of 7KC compared to cholesterol in the turbidity test of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, or more.

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

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

[0084] In an exemplary embodiment, the present disclosure provides a pharmaceutical composition comprising a cyclodextrin dimer or a composition thereof as disclosed herein and a pharmaceutically 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 pharmaceutically acceptable carrier.

[0085] In an exemplary embodiment, the present disclosure provides a treatment method comprising administering to a subject in need of treatment an effective amount of a cyclodextrin dimer or a 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 reducing the amount of 7KC, 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 preferably intravenously via parenteral (e.g., subcutaneous, intramuscular, or intravenous), topical, transdermal, oral, sublingual, or buccal administration.

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

[0089] The method may be capable of preventing, treating, and / or alleviating the symptoms of one or more of atherosclerosis, arteriosclerosis, coronary atherosclerosis due to coronary artery calcification 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, neuronal ceroid lipofuscinosis in children, lysosomal acid lipase deficiency, cerebrotendinous xanthomatosis, X-linked adrenoleukodystrophy, sickle cell anemia, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Gaucher's disease, Stargardt disease, age-related macular degeneration (atrophic 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 for atherosclerosis.

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

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

[0092] The second treatment may include an anti-cholesterol drug and an antihypertensive drug.

[0093] In an exemplary embodiment, the present disclosure provides a method for purifying oxysterol, the method comprising: contacting a composition comprising 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 include measuring the concentration of 7KC in the solubilized oxysterol, thereby determining the relative concentration of 7KC in the composition. The composition may include a patient sample. The method can be used for measuring the concentration of 7KC in a patient sample, and this concentration can be used for diagnosis and / or treatment planning.

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

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

[0096] In another aspect, the present disclosure provides a method for preparing a cyclodextrin dimer as described herein, for example, a cyclodextrin dimer having an unsubstituted or substituted alkyl linker, the method comprising: (a) reacting a β-cyclodextrin with a protected upper base with a dialkylating agent to thereby produce an upper base-protected βCD dimer linked through the lower base, and optionally purifying the upper base-protected βCD dimer; (b) deprotecting the upper base-protected βCD dimer to thereby produce a deprotected βCD dimer, and optionally purifying the deprotected βCD dimer; and (c) hydroxypropylating the deprotected βCD to thereby produce a cyclodextrin dimer, and optionally purifying the cyclodextrin dimer. The β-cyclodextrin with a protected upper base may include heptakis(6-O-tert-butyldimethylsilyl)-β-cyclodextrin. The dialkylating agent may include dibromoalkane, and optionally 1,4-dibromobutane. Step (a) may be carried out under anhydrous conditions and / or using sodium hydride as a base. The purification in step (a) may include normal-phase chromatography with elution by a homogeneous solvent. Step (b) may be carried out using tetrabutylammonium fluoride in tetrahydrofuran (THF). The purification in step (b) may include normal-phase chromatography with elution by a homogeneous solvent. Step (c) may include reacting the deprotected βCD dimer with a hydroxypropylation agent such as propylene oxide, a methylation reagent such as methyl iodide, a succinylation reagent such as succinic anhydride, a sulfobutylation reagent such as 1,4-butanesultone, and / or a quaternary ammonium linking reagent such as glycidyltrimethylammonium chloride.

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

[0098] In another aspect, the present disclosure provides a method for preparing a cyclodextrin dimer as described herein, for example, a cyclodextrin dimer having a triazole linker, the method 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 carried out using a copper(I) catalyst, optionally one having about 15 mM of copper(I). Step (a) may be carried out in an aqueous solution. The aqueous solution may contain dimethylformamide (DMF), optionally about 50% DMF (v / v). Step (b) may include chromatography. The method may further include, prior to step (a), generating the 2-O-(n-azidoalkyl)-βCD, and the method for generating may include: (1) reacting an n-azido-1-bromo-alkane with β-cyclodextrin using an optionally catalytic amount of lithium iodide, thereby generating the 2-O-(n-azidoalkyl)-βCD; and (2) optionally purifying the 2-O-(n-azidoalkyl)-βCD. Step (2) may include chromatography. The method may further include, prior to step (a), generating 2-O-(n-alkyne)-βCD, and the method for generating may include: (i) reacting an n-bromo-1-alkyne with β-cyclodextrin using an optionally catalytic amount of lithium iodide, thereby generating 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 carried out in dry DMSO. The reaction of step (1) may include lithium hydride. The βCD-triazole-βCD dimer has the following structure:

Chemical formula

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

[0100] Embodiments of the present invention provide compositions and methods for treating or preventing atherosclerosis. 7KC is the most abundantly non-enzymatically produced oxysterol in atherosclerotic plaques and is thought to contribute to the pathogenesis of atherosclerosis. Treatment with the CD (e.g., HPβCD of the present disclosure or another CD) dimer of the present invention is expected to be beneficial for the prevention and / or reversal of atherosclerotic plaque formation.

[0101] Embodiments of the present invention provide compositions and methods for treating or preventing diseases and conditions that have been associated with 7KC. Such diseases and conditions include, but are not limited to, age-related diseases such as atherosclerosis, AMD, arteriosclerosis, coronary atherosclerosis due to coronary artery calcified lesions, 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. Also, other sporadic and / or congenital diseases associated with 7KC accumulation include Huntington's disease, multiple sclerosis, Smith-Lemli-Opitz syndrome, 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's disease, Stargardt's disease, idiopathic pulmonary fibrosis, cystic fibrosis, liver injury, liver failure, non-alcoholic steatohepatitis, ulcerative colitis, Crohn's disease, and other irritable bowel syndromes.

[0102] In another exemplary embodiment, the present disclosure has a degree of substitution of 1 to 40, such as 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, alkylsulfonylamide, aminocarbonyloxyalkyl, aminosulfonyl, ammonium, ammonia, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyl oxy, azide, bromo, chloro, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, deoxy, glucosyl, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroaralkyloxy, heterocyclilalkoxy, halogen, haloalkyl, haloalkoxy, heterocycloamino, heterocyclil, heterocyclilalkyl, heterocyclilo xy, heterocyclilalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, iodo, ureido, carbamate, carboxy, sulfate, sulfinyl, sulfonamide, nitro, nitrito, cyano, phosphate, phosphoryl, phenoxy, acetyl group, fatty acids such as palmitoyl group, monosaccharides, or disaccharides, and provides a cyclodextrin dimer composition selected from the group consisting of, the composition contains a cyclodextrin dimer of the structure CD-L-CD, wherein, L is, through the C2 carbon of each CD subunit (instead of R 1 ), and / or through the C3 carbon (instead of R 2 ), is linked to the major face (lower base) of each CD molecule; wherein each CD is, R 1 , R 2 , and / or R3 has the structure of formula X having the substituent on one or more of them, provided that L has a length of 8 or fewer atoms, and each of the 8 or fewer atoms is preferably C, N, O, or S. The substituent can be carboxymethyl or maltosyl. The substituent is preferably methyl, hydroxypropyl, sulfobutyl, succinyl, quaternary ammonium (e.g., -CH2CH(OH)CH2N(CH3)3 + ). The degree of substitution is measurable by NMR. The degree of substitution is measurable by mass spectrometry, e.g., MALDI.

[0103] In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a degree of substitution of 1 to 40, e.g., 1 to 28 or 4 to 20, preferably 2 to 15, and the substituent is selected from methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium (e.g., -CH2CH(OH)CH2N(CH3)3 + ), glucosyl, palmitoyl, phosphate, phosphoryl, amino, azide, sulfate, sulfuryl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromo, or chloro, and the composition comprises a cyclodextrin dimer of the structure CD-L-CD, wherein L is connected through the C2 carbon of each CD subunit (instead of R 1 ) and / or through the C3 carbon of each CD subunit (instead of R 2 ) to the large face (bottom) of each CD molecule; wherein each CD has the structure of formula X having the substituent on one or more of R 1 , R 2 , and / or R 3 , provided that L has a length of 8 or fewer atoms, and each of the 8 or fewer atoms is preferably C, N, O, or S. The degree of substitution is measurable by NMR. The degree of substitution is measurable by mass spectrometry, e.g., MALDI.

[0104] In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a degree of substitution of 1 to 40, such as 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, selected from quaternary ammonium. The composition includes a cyclodextrin dimer having the structure CD-L-CD, wherein L is connected to the large face (lower base) of each CD molecule through the C2 carbon of each CD subunit (instead of R 1 ), and / or through the C3 carbon (instead of R 2 ); wherein each CD has the structure of formula X with the substituent on one or more of R 1 , R 2 , and / or R 3 , provided that L has a length of 8 or fewer atoms, and each of the 8 or fewer atoms is preferably C, N, O, or S. The degree of substitution is measurable by NMR. The degree of substitution is measurable by mass spectrometry, such as MALDI.

[0105] In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a degree of substitution of 1 to 40, such as 1 to 28 or 4 to 20, preferably 2 to 15, more preferably 2 to 5, even more preferably 2 to 4, and having hydroxypropyl substituents. The composition includes a cyclodextrin dimer having the structure CD-L-CD, wherein L is connected to the large face (lower base) of each CD molecule through the C2 carbon of each CD subunit (instead of R 1 ), and / or through the C3 carbon (instead of R 2 ); wherein each CD has the structure of formula X with the substituent on one or more of R 1 , R 2 , and / or R 3 , provided that L has a length of 8 or fewer atoms, and each of the 8 or fewer atoms is preferably C, N, O, or S. The degree of substitution is measurable by NMR. The degree of substitution is measurable by mass spectrometry, such as MALDI.

[0106] In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a degree of substitution of 1 to 40, such as 1 to 28 or 4 to 20, preferably 2 to 15, more preferably 2 to 10, and having methyl substituents. The composition includes a cyclodextrin dimer having the structure CD-L-CD, where L is linked to the major face (bottom) of each CD molecule through the C2 carbon of each CD subunit (instead of R 1 and / or) and / or through the C3 carbon of each CD subunit (instead of R 2 and / or); where each CD has the structure of Formula X having the substituent at one or more of R 1 R 2 and / or) and / or R 3 , provided that L has a length of 8 or fewer atoms, and each of the 8 or fewer atoms is preferably C, N, O, or S. The degree of substitution is measurable by NMR. The degree of substitution is measurable by mass spectrometry, such as MALDI.

[0107] In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a degree of substitution of 1 to 40, such as 1 to 28 or 4 to 20, preferably 2 to 15, more preferably 2 to 5, even more preferably 2 to 4, and having sulfobutyl substituents. The composition includes a cyclodextrin dimer having the structure CD-L-CD, where L is linked to the major face (bottom) of each CD molecule through the C2 carbon of each CD subunit (instead of R 1 and / or) and / or through the C3 carbon of each CD subunit (instead of R 2 and / or); where each CD has the structure of Formula X having the substituent at one or more of R 1 R 2 and / or) and / or R 3 , provided that L has a length of 8 or fewer atoms, and each of the 8 or fewer atoms is preferably C, N, O, or S. The degree of substitution is measurable by NMR. The degree of substitution is measurable by mass spectrometry, such as MALDI.

[0108] In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a degree of substitution of 1 to 40, such as 1 to 28 or 4 to 20, preferably 2 to 15, more preferably 2 to 5, even more preferably 2 to 4, and having a succinyl substituent. The composition comprises a cyclodextrin dimer of the structure CD-L-CD, wherein L is linked to the major face (bottom) of each CD molecule through the C2 carbon of each CD subunit (instead of R 1 (instead) and / or through the C3 carbon of each CD subunit (instead of R 2 ); wherein each CD has the structure of formula X having the substituent at one or more of R 1 , R 2 , and / or R 3 , provided that L has a length of 8 or fewer atoms, and each of the 8 or fewer atoms is preferably C, N, O, or S. The degree of substitution is measurable by NMR. The degree of substitution is measurable by mass spectrometry, such as MALDI.

[0109] In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a degree of substitution of 1 to 40, such as 1 to 28 or 4 to 20, preferably 2 to 15, more preferably 2 to 5, even more preferably 2 to 4, and having a quaternary ammonium substituent, preferably -CH2CH(OH)CH2N(CH3)3 + . The composition comprises a cyclodextrin dimer of the structure CD-L-CD, wherein L is linked to the major face (bottom) of each CD molecule through the C2 carbon of each CD subunit (instead of R 1 (instead) and / or through the C3 carbon of each CD subunit (instead of R 2 ); wherein each CD has the structure of formula X having the substituent at one or more of R 1 , R 2 , and / or R 3 , provided that L has a length of 8 or fewer atoms, and each of the 8 or fewer atoms is preferably C, N, O, or S. The degree of substitution is measurable by NMR. The degree of substitution is measurable by mass spectrometry, such as MALDI.

[0110] In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a degree of substitution of 0 to 40, the composition comprising a cyclodextrin dimer of the structure CD-L-CD, wherein L is through the C2 carbon of each CD subunit (instead of R 1 (instead of) and / or through the C3 carbon (instead of R 2 (instead of) is linked to the major face (bottom) of each CD molecule; wherein each CD has a structure of formula X optionally substituted with one or more substituents, provided that L has a length of 8 or fewer atoms, and each of the 8 or fewer atoms is preferably C, N, O, or S. The cyclodextrin dimer composition can be used for the synthesis of a cyclodextrin dimer composition substituted with one or more substituents. The degree of substitution is measurable by NMR. The degree of substitution is measurable by mass spectrometry, for example, MALDI.

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

Chemical formula

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

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

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

[0115] Each A is independently selected from a single bond, a double bond, or a triple bond 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 is 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 contain a triazole.

[0119] The linker has the following structure:

Chemical formula

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

[0121] In an exemplary embodiment, the linker comprises substituted or unsubstituted alkyl, preferably having a length of 8 atoms or less, such as 2 to 7, 2 to 6, or 4 to 7 or 4 to 6 or 4 to 5, or having a length of 8, 7, 6, 5, 4, 3, or 2, or any numerical range 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 positions of both CD monomers, provided that preferably the linker is substituted or unsubstituted butyl, more preferably unsubstituted butyl.

[0122] The linker can comprise any of the linkers depicted in FIG. 8D, provided 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 further comprises that the cyclodextrin dimer is (a) at least one of methyl, hydroxypropyl, sulfobutyl, succinyl, or -CH2CH(OH)CH2N(CH3)3 +quaternary ammonium groups such as, 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, alkylsulfonylamide, aminocarbonyloxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyl oxy, 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, sulfonamide, nitro, cyano, phenoxy, acetyl group, ammonium, ammonia, azide, bromo, chloro, deoxy, glucosyl, iodo, sulfate, sulfinyl, nitrito, phosphate, phosphoryl, fatty acids such as palmitoyl group, monosaccharide, or disaccharide, and / or (c) at least one methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium (e.g., -CH2CH(OH)CH2N(CH3)3 +) It can be substituted with glucosyl, palmitoyl, phosphate, phosphoryl, amino, azido, sulfate, sulfuryl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromo, chloro groups.

[0124] The cyclodextrin dimer composition can contain a cyclodextrin dimer having a structure according to any one of Formulas I to IX (FIGS. 3B to 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, succinyl hydroxypropyl, -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, alkylsulfonylamide, 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, sulfonamide, nitro, cyano, phenoxy, or acetyl group.

[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, and optionally, the affinity is measured by a turbidity test.

[0128] The cyclodextrin dimer composition may exhibit an affinity for 7KC that is at least 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold stronger than for cholesterol. The cyclodextrin dimer may exhibit a decrease in the relative turbidity of 7KC compared to cholesterol in a turbidity test of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, or more.

[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 contain a mixture of cyclodextrin dimer molecules having different numbers of substituents and / or different linker attachment points, in which case 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 pharmaceutically acceptable carrier. The pharmaceutical composition may be suitable for administration to a subject, for example, by parenteral (e.g., subcutaneous, intramuscular, or intravenous), topical, transdermal, oral, sublingual, or buccal administration, preferably by intravenous or subcutaneous administration, more preferably by intravenous administration. 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 pharmaceutically acceptable carrier.

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

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

[0136] The method may be used to prevent, treat, and / or alleviate the symptoms of one or more of atherosclerosis / coronary artery disease, arteriosclerosis, coronary atherosclerosis due to coronary artery calcified 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, neuronal ceroid lipofuscinosis in children, 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, Gaucher disease, Stargardt disease, age-related macular degeneration (atrophic 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. Optionally, the treatment is administered in combination with another treatment. The method may include administering a second treatment to the subject, and the second treatment can be administered simultaneously or sequentially in any order.

[0137] The method may be for preventing, treating, and / or alleviating the symptoms of atherosclerosis. The cyclodextrin dimer composition may be co-administered with another treatment for the treatment or prevention of atherosclerosis, such as, for example, an anti-cholesterol drug, an antihypertensive drug, an antiplatelet drug, a dietary supplement, or surgery or behavioral intervention, and examples of such other treatments include, but are not limited to, those described herein. The anti-cholesterol drug may include a fibrate or statin drug, an antiplatelet drug, an antihypertensive drug, or a dietary supplement. The statin drugs may include ADVICOR® (niacin extended release / rosuvastatin), ALTOPREV® (rosuvastatin 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 alleviating the symptoms of atrophic age-related macular degeneration. The method may be for preventing, treating, and / or alleviating the symptoms of Stargardt's disease. The cyclodextrin dimer composition may be co-administered with another treatment for the treatment or prevention of atrophic AMD or Stargardt's disease, such as, for example, LBS-008 (Belite Bio) (a non-retinoid antagonist of retinol-binding protein 4), vitamin C and vitamin E, beta-carotene, zinc, and copper-containing AREDS dietary supplement formulation, vitamin C and vitamin E, zinc, copper, lutein, zeaxanthin, and omega-3 fatty acid-containing dietary supplement formulation including the AREDS2 dietary supplement formulation, or a combination thereof.

[0139] The method may be for preventing, treating, and / or alleviating the symptoms of Niemann-Pick disease. The cyclodextrin dimer composition may be administered in combination with one or more other treatments for the treatment or prevention of Niemann-Pick disease, such as miglustat (ZAVESCA(R)), HPβCD (TRAPPSOL CYCLO, VTS-270), and physical therapy.

[0140] The method may be for preventing, treating, and / or alleviating the symptoms of Alzheimer's disease. The cyclodextrin dimer composition may be administered in combination with one or more other treatments for the treatment or prevention of Alzheimer's disease, such as cholinesterase inhibitors (ARICEPT(R), EXELON(R), RAZADYNE(R)), and memantine (NAMENDA(R)) or combinations thereof.

[0141] The method may be for preventing, treating, and / or alleviating the symptoms of heart failure. The cyclodextrin dimer composition may be administered in combination with one or more other treatments for the treatment or prevention of 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 present disclosure provides a method for preparing 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 β-cyclodextrin with a protected upper base with a dialkylating agent to thereby form an upper base-protected βCD dimer linked through the lower base, and optionally purifying the upper base-protected βCD dimer; (b) deprotecting the upper base-protected βCD dimer to thereby form a deprotected βCD dimer, and optionally purifying the deprotected βCD dimer; and (c) hydroxypropylating the deprotected βCD to thereby form a cyclodextrin dimer composition, and optionally purifying the cyclodextrin dimer composition. The β-cyclodextrin with a protected upper base may include heptakis(6-O-tert-butyldimethylsilyl)-β-cyclodextrin. The dialkylating agent may include dibromoalkane, optionally 1,4-dibromobutane. Step (a) may be carried out under anhydrous conditions and / or using sodium hydride as a base. The purification in step (a) may include normal-phase chromatography with elution by a homogeneous solvent. Step (b) may be carried out using tetrabutylammonium fluoride in tetrahydrofuran (THF). The purification in step (b) may include normal-phase chromatography with elution by a homogeneous solvent. Step (c) may include reacting the deprotected βCD dimer with a hydroxypropylation agent such as propylene oxide, a methylation reagent such as methyl iodide, a succinylation reagent such as succinic anhydride, a sulfobutylation reagent such as 1,4-butanesultone, and / or a quaternary ammonium linking reagent such as glycidyltrimethylammonium chloride. The cyclodextrin dimer composition may be a cyclodextrin dimer composition as disclosed herein. The cyclodextrin dimer composition may have a degree of substitution by substituents 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.

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

[0144] In another aspect, the present disclosure provides a method for preparing a cyclodextrin dimer composition as described herein, for example, a cyclodextrin dimer composition having a triazole linker, the method 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 carried out using a copper(I) catalyst, optionally one having about 15 mM of copper(I). Step (a) may be carried out in an aqueous solution. The aqueous solution may contain dimethylformamide (DMF), optionally about 50% DMF (v / v). Step (b) may include chromatography. The method may further include, prior to step (a), generating the 2-O-(n-azidoalkyl)-βCD, and the method for generating may include: (1) reacting an n-azido-1-bromo-alkane with β-cyclodextrin using an optionally catalytic amount of lithium iodide, thereby generating the 2-O-(n-azidoalkyl)-βCD; and (2) optionally purifying the 2-O-(n-azidoalkyl)-βCD. Step (2) may include chromatography. The method may further include, prior to step (a), generating 2-O-(n-alkyne)-βCD, and the method for generating may include: (i) reacting an n-bromo-1-alkyne with β-cyclodextrin using an optionally catalytic amount of lithium iodide, thereby generating 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 carried out in dry DMSO. The reaction of step (1) may include lithium hydride. The βCD-triazole-βCD dimer has the following structure: [Chemical Formula] (Formula XII) may be included, where n1 can be from 1 to 8, and / or n2 can be from 1 to 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 from 5 to 8. The method may further include hydroxypropylation of 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 hydroxypropylation agent such as propylene oxide, a methylation reagent such as methyl iodide, a succinylation reagent such as succinic anhydride, a sulfobutylation reagent such as 1,4-butanesultone, and / or a quaternary ammonium linking reagent such as glycidyltrimethylammonium chloride. The cyclodextrin dimer composition may be a cyclodextrin dimer composition as disclosed herein. The cyclodextrin dimer composition may have a degree of substitution by substituents 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 under 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 carbon clarification, membrane filtration, and dialysis.

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

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

[0148] The phrase "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that are suitable for administration to a living organism or living tissue within the scope of reasonable medical judgment, preferably without significant toxicity, irritation, or allergic response. The present invention encompasses methods that include administering a cyclodextrin dimer to a patient, wherein the cyclodextrin dimer is included in a pharmaceutical composition. The pharmaceutical compositions of the present invention are formulated with pharmaceutically acceptable carriers, excipients, and other agents that provide for appropriate transport, delivery, tolerance, and the like. Suitable formulations can be found in abundance in formularies known to pharmaceutical chemists, such as 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] As used herein, the term "pharmaceutically acceptable carrier" generally refers to a pharmaceutically acceptable composition useful for introducing an active agent into the body, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium stearate, calcium stearate or zinc stearate, or stearic acid), or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients in the formulation and not injurious 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), as well as suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersants, and by using surfactants.

[0150] Other examples of materials that can serve as pharmaceutically acceptable carriers include the following: (1) saccharides such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, 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; (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) buffering agents 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 and compatible substances used in pharmaceutical formulations.

[0151] Various auxiliaries, such as wetting agents, emulsifiers, lubricants (e.g., sodium lauryl sulfate and magnesium stearate), colorants, release agents, coating agents, sweeteners, flavoring agents, preservatives, and antioxidants, etc., can also be included in the pharmaceutical composition. Some examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, 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 chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc. Depending on the embodiment, the pharmaceutical formulation may include excipients selected from, for example, cellulose, liposomes, micelle-forming agents (e.g., bile acids), and polymeric carriers, such as polyesters and polyanhydrides. Suspending agents may contain, in addition to the active compound, suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, crystalline cellulose, aluminum hydroxide, bentonite, agar and tragacanth, and mixtures thereof. The activity of microorganisms on the active compound can sometimes be reliably prevented by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenolsorbic acid, etc. It may also be desirable to include in the composition isotonic agents, such as sugars, sodium chloride, etc. Also, an agent that delays absorption, such as aluminum monostearate and gelatin, can be included to provide an injectable form that is absorbed over a long period.

[0152] The pharmaceutical formulations of the present invention can be prepared by any method known in the pharmaceutical art. The amount of the active ingredient (i.e., the CD dimer, such as the HPβCD dimer of the present disclosure or another CD dimer, etc.) that can be mixed with a carrier material to produce a single dosage form will vary depending on the host to be treated and the particular mode of administration. The amount of the active ingredient that can be mixed with a carrier material to produce a single dosage form will generally be the amount of the compound that produces a therapeutic effect. The amount of the active compound can range from about 0.1% to 99.9%, more typically from about 80% to 99.9%, and more typically about 99%. The amount of the active compound can range from about 0.1% to 99%, more typically from about 5% to 70%, and more typically from about 10% to 30%. In an exemplary embodiment, a dosage form for intravenous administration is provided that is an aqueous solution having a concentration of 0.5% to 0.001%, such as 0.12% to 0.0105%, such as about 0.01% (W / V). In an exemplary embodiment, a dosage form for intravenous administration is provided that is an aqueous solution having a concentration of 2.5% to 0.25%, such as 2% to 0.5%, such as about 1% (W / V). In an exemplary embodiment, a dosage form for intravenous administration of a maximum of 500 mL of a 1% solution (W / V) is provided, which results in a maximum dosage of 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, such as 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, such as about 2 g, about 3 g, about 4 g, or about 5 g, can be administered. In an exemplary embodiment, 50 mg to 5 g of the cyclodextrin dimer, such as 100 mg to 2.5 g, 100 mg to 2 g, 250 mg to 2.5 g, such as about 1 g, can be administered.

[0154] The exemplary embodiments provide a single dosage form which can contain the above amount of the cyclodextrin dimer, which can be packaged for individual administration and optionally further contains a pharmaceutically acceptable carrier or excipient. The total amount of the cyclodextrin dimer in the single dosage form can be as presented above, for example, 1 mg to 10 g of the cyclodextrin dimer, such as 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] The formulations of the present invention suitable for oral administration can be in the form of capsules, cachets, pills, tablets, lozenges (using a flavoring base, usually sucrose and gum acacia or tragacanth), powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as water-in-oil or oil-in-water liquid emulsions, or as elixirs or syrups, or as troches (using an inert base, for example, gelatin and glycerin, or sucrose and gum acacia), and / or in the form of mouthwashes, etc., each containing a predetermined amount of the compound of the present invention as an active ingredient. The active compound can also be administered as a bolus, a lickable preparation, or a paste.

[0156] The preparation method of such formulations or compositions generally includes the step of mixing the compound of the present invention with a carrier and optionally one or more auxiliaries. In the case of solid dosage forms (e.g., capsules, tablets, pills, powders, granules, lozenges, etc.), the active compound is mixed with a micronized solid carrier and can typically be formed, for example, pelletized, tabletted, granulated, powdered, or coated. Generally, examples of solid carriers can include 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, etc.; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia gum, etc.; (3) humectants, such as glycerol, etc.; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, etc.; (5) solution retarding agents, such as paraffin, etc.; (6) absorption promoters, such as quaternary ammonium compounds and surfactants, such as poloxamer and sodium lauryl sulfate, etc.; (7) wetting agents, such as cetyl alcohol, glycerol monostearate, and nonionic surfactants, etc.; (8) absorbents, such as kaolin and bentonite clay, etc.; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof, etc.; (10) coloring agents, and (11) release control agents, such as crospovidone or ethylcellulose, etc. In the case of capsules, tablets, and pills, the pharmaceutical composition can also include a buffering agent. Solid compositions of the same type as the fillers for soft and hard shell gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycol, etc. may also be used when applicable.

[0157] Tablets can be manufactured by compression or molding, optionally together with one or more auxiliary components. Compressed tablets can be formulated using a binder (e.g., gelatin or hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), a surfactant, or a dispersant.

[0158] Tablets, and other solid dosage forms of the active agent, such as capsules, pills, and granules, can be optionally surface processed (scored) or formulated using coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical manufacturing art. Such dosage forms can also be formulated to provide for the sustained or controlled release of the active ingredient in the dosage form, for example, by using hydroxypropylmethylcellulose in various ratios to provide a desired release profile, or by using other polymer matrices, liposomes, and / or microspheres. Such dosage forms can alternatively be formulated for rapid release, for example, by lyophilization.

[0159] Generally, it is necessary for the dosage form to be sterile. For this purpose, the dosage form can be sterilized, for example, by filtration through a bacteria-trapping filter or by incorporating a sterilizing agent in the form of a sterile solid composition that is soluble in sterile water or other suitable sterile injection medium immediately prior to 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 particular part of the gastrointestinal tract, optionally in a delayed manner. Examples of embeddable 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 excipients described above.

[0160] Liquid dosage forms are typically pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, or elixirs of active agents. Liquid dosage forms can contain, in addition to the active ingredient, inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 - butylene glycol, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters, and mixtures thereof.

[0161] Dosage forms specifically intended for topical or transdermal administration can be, for example, in the form of powders, sprays, ointments, pastes, creams, lotions, gels, solutions, or patches. Ophthalmic formulations, such as ophthalmic ointments, powders, solutions, etc. are also contemplated herein. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and, if necessary, any preservative, buffer, or propellant. Topical or transdermal dosage forms can contain, in addition to the active compounds of the present invention, one or more excipients selected from, for example, animal and vegetable fats and oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, and mixtures thereof. Sprays can also contain conventional propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0162] For the purposes of the present invention, transdermal patches can offer the advantage of providing controlled delivery of the compounds of the present invention to the body. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium. Absorption promoters can also be used to increase the flow of the compound across the skin. Such flow rates can be controlled either by using a rate - controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0163] The pharmaceutical compositions of the present invention suitable for parenteral administration generally comprise one or more compounds of the present invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions prior to use, such sterile powders being capable of containing saccharides, alcohols, antioxidants, buffers, bacteriostatic agents, or solutes that render the formulation isotonic with the blood of the intended recipient.

[0164] In some cases, for the purpose of prolonging the effect of the drug, it is desirable to slow down the drug absorption from subcutaneous or intramuscular injections. This can be achieved by using a liquid suspension of a poorly water-soluble crystalline or amorphous material. In that case, the drug absorption rate depends on the dissolution rate and thus may depend on the crystal size and crystal form. Alternatively, the absorption delay of parenterally administered dosage forms is achieved by dissolving or suspending the drug in an oily vehicle.

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

[0166] The pharmaceutical composition can also be in the form of a microemulsion. In the form of a microemulsion, the bioavailability of the active agent may be improved. (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)) are cited for reference, and the entire contents of these are hereby incorporated by reference in their entirety.

[0167] The pharmaceutical composition can also contain micelles formed from the compound of the present invention and at least one amphiphilic carrier, in which case the micelles have an average diameter of less than about 100 nm. Depending on the embodiment, the micelles have an average diameter of less than about 50 nm, or less than about 30 nm, or less than about 20 nm.

[0168] Although any suitable amphiphilic carrier is contemplated herein, the amphiphilic carrier generally has an approved Generally Recognized as Safe (GRAS) status and is capable of both dissolving the compound of the present invention and microemulsifying it when the solution comes into contact with a complex aqueous phase (such as those found in living biological tissues) at a later stage. Usually, the amphiphilic components that meet these requirements have an HLB (hydrophilic-lipophilic balance) value of 2 to 20, and their structures contain linear aliphatic radicals in the range of C-6 to C-20. Some examples of amphiphilic agents include polyethylene-glycolated fatty glycerides and polyethylene glycol.

[0169] Particularly preferred amphiphilic carriers are saturated and monounsaturated polyethylene glycolated fatty acid glycerides, such as those obtained by fully or partially hydrogenating various vegetable oils. The oil may advantageously consist of tri, di, and mono fatty acid glycerides, as well as di and mono polyethylene glycol esters of the corresponding fatty acids. Particularly preferred fatty acid compositions contain 4 - 10% capric acid, 3 - 9% caprylic 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 using saturated or monounsaturated fatty acids (SPAN series) or the corresponding ethoxylated analogs (TWEEN series). Commercially available amphiphilic carriers are particularly contemplated, and such carriers include Gelucire® series, Labrafil®, Labrasol®, or Lauroglycol®, PEG - monooleate, PEG - dioleate, PEG - monolaurate and dilaurate, lecithin, polysorbate 80.

[0170] CD (e.g., HPβCD of the present disclosure or another CD) dimers 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 be for the eye (e.g., in the form of eye drops), intravitreal, retrobulbar, subretinal, sub - scleral, which may be suitable in cases of eye diseases such as AMD.

[0171] CD (e.g., HPβCD of the present disclosure or another CD) dimers may be administered to a subject or used in vitro, e.g., on cells or tissues removed from an animal. The cells or tissues can then be introduced into a subject, whether the subject from which the cells or tissues were removed or another individual, provided that the other individual is preferably of the same species.

[0172] The subject to be treated (i.e., the patient) is typically an animal, generally a mammal, preferably a human. The subject can also be a non-human animal, and examples of non-human animals include all vertebrates, such as mammals and non-mammals, for example, non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. In some embodiments, the subject is a domestic 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 at the age of transition from adult to elderly, adults, subjects at the age of transition from pre-adult to adult, and pre-adults including juveniles (e.g., from 13 years old to 16, 17, 18, or 19 years old), children (generally 13 years old or younger or pre-pubertal), and infants. The subject can also belong to any racial group or genotype. Examples of human racial groups include Caucasian, Asian, Latin American, African, African American, Native American, Semitic, and Pacific Islander ethnic groups. The methods of the present invention may be more suitable for some racial groups, such as the Caucasian race, particularly the Nordic racial group, and the Asian racial group.

[0173] The present disclosure includes further substitution of the dimeric CDs (e.g., the HPβCD of the present disclosure or another CD) described herein. The chemical modification may be performed either before or after dimerization. Chemical modification of cyclodextrin can be performed directly on the native beta-cyclodextrin ring by reacting a chemically appropriate functionalized cyclodextrin with a chemical reagent (nucleophilic or electrophilic reagent) (Adair-Kirk [et al.], Nat. Med., 14(10):1024-5, (2008)); (Khan, [et al.], Chem. Rev., 98(5):1977-1996, (1998)). To date, over 1,500 cyclodextrin derivatives have been created by chemical modification of native cyclodextrin. Cyclodextrin can also be prepared by novel synthesis, which starts from glucopyranose-linked oligopyranosides. Such synthesis can be achieved using various chemical reagents or biological enzymes, such as cyclodextrin transglycosylase. A review of chemically modified cyclodextrin as a drug carrier in drug delivery systems is described, for example, in (Stella, [et al.], Toxicol. Pathol., 36(1):30-42, (2008)), the disclosure of which is hereby incorporated by reference in its entirety. U.S. Patent Nos. 3,453,259 and 3,459,731 describe electrically neutral cyclodextrin, the disclosures of which are hereby incorporated by reference in their entirety. Other derivatives include cationic cyclodextrin, as disclosed in U.S. Patent No. 3,453,257; insoluble crosslinked cyclodextrin, as disclosed in U.S. Patent No. 3,420,788; and anionic cyclodextrin, 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 anionic cyclodextrin derivatives, carboxylic acid, phosphorous acid, phosphinic acid, phosphonic acid, phosphoric acid, thiophosphonic acid, thiosulfinic acid, and sulfonic acid 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, the cyclic oligosaccharide can have one or more monosaccharide units replaced with triazole rings, which can be synthesized by azide-alkyne Huisgen cycloaddition reactions ((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 subunit and the linker are described in the examples. Additional methods for connecting the CD subunit 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 that reacts with a hydroxyl group (e.g., a carboxyl group, which can be activated by carbodiimide) can be reacted with cyclodextrin to form a covalent bond with the cyclodextrin. In another example, one or more hydroxyl groups of the cyclodextrin can be activated by a known method (e.g., tosylation) and reacted with a reactive group of the linker (e.g., an amino group).

[0175] Generally, a linker initially has two reactive moieties that react and bind to each CD monomer. In one embodiment, first, the linker is attached to 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, although 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 additional reactive groups for the purpose of linking to other molecules.

[0176] Multiple linkers are known in the art. Such linkers can be used to link together any collection of various groups that carry or are functionalized to carry a group that can react and link with a reactive linker. Some of the groups that can react with a dual-reactive linker include amino, thiol, hydroxyl, carboxyl, ester, and alkyl halide groups. For example, when each of the groups to be linked carries at least one amino group, an amino-amino coupling reagent can be used to link a cyclic oligosaccharide and a polysaccharide (or any of these groups with a fluorophore, for example, or with each other). Some examples of amino-amino coupling reagents include diisocyanate compounds, alkyldihalides, dialdehydes, disuccinimidyl suberate (DSS), disuccinimidyl tartrate (DST), and disulfosuccinimidyl tartrate (sulfo-DST), all of which are commercially available. In other embodiments, an amino-thiol coupling agent can be used to link a thiol group of one molecule with an amino group of another molecule. Some examples of amino-thiol 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, a thiol-thiol coupling agent can be used to link groups having at least one thiol group.

[0177] Depending on the embodiment, the linker is so small that it has a length of only one atom (e.g., -O-, -CH2-, or -NH- linkage), or two or three atoms (e.g., amide, ureido, carbamate, ester, carbonate, sulfone, ethylene, or trimethylene linkage). In other embodiments, the linker has a length of at least four, five, six, seven, or eight atoms and up to, for example, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 atoms, thereby providing a higher degree of freedom of movement. Suitable linker lengths are 2 to 12 atoms, or 4 to 8 atoms. In an exemplary embodiment, the linker is a C4 alkyl, which may be unsubstituted. In an exemplary embodiment, the linker contains a triazole.

[0178] Atherosclerosis

[0179] The exemplary cyclodextrin dimers described herein are useful for preventing or treating diseases such as atherosclerosis. Combinations of cyclodextrin dimers with one or more active agents, such as those described herein (e.g., antihyperlipidemic agents such as statin drugs), are 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 as 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 be asymptomatic for decades. Atherosclerosis is a syndrome that affects arterial blood vessels, and the chronic inflammatory response in the arterial wall is largely due to the accumulation of macrophage white blood cells, and this chronic inflammatory response is thought to be promoted by low-density lipoprotein (a plasma protein that transports cholesterol and triglycerides) if the fatty acids and cholesterol are not properly removed from the macrophages by functional high-density lipoprotein (HDL). Atherosclerosis is generally referred to as hardening of the arteries or furring of the arterial wall. This is caused by the formation of multiple plaques within the artery.

[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, while unstable plaques are rich in macrophages and foam cells, and the extracellular matrix (also known as the fibrous cap) separating the lesion from the arterial lumen is usually fragile and prone to rupture. The rupture of the fibrous cap exposes thrombogenic materials such as collagen to the blood circulation, ultimately inducing thrombosis in the lumen. During formation, luminal thrombi can potentially cause complete occlusion (e.g., coronary artery occlusion), but more frequently, they break off, enter the blood circulation, and can ultimately occlude smaller downstream branches, causing thromboembolism (e.g., stroke is often caused by thrombosis in the carotid artery). Apart from thromboembolism, the chronic expansion of atherosclerotic lesions can potentially cause complete occlusion of the lumen. The chronic expansion of the lesion is often asymptomatic until the stenosis of the lumen becomes severe enough to cause insufficient blood supply to downstream tissue(s), resulting in ischemia.

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

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

[0183] As atherosclerotic signs, symptoms, and complications, as discussed above, there are included, but not limited to, elevated total plasma cholesterol, VLDL-C, LDL-C, free cholesterol, cholesterol ester, triglyceride, phospholipid, and the presence of arterial lesions (e.g., plaques). In some cases, an increase in cholesterol (e.g., total cholesterol, free cholesterol, and cholesterol ester) may be seen in one or more of plasma, aortic tissue, and aortic plaque.

[0184] Some individuals may have a predisposition to atherosclerosis. Accordingly, the present disclosure relates to a method of administering a cyclodextrin dimer alone or in combination with one or more therapeutic agents (e.g., an antihyperlipidemic agent such as a statin agent) to a subject for preventing atherosclerosis or its signs, symptoms, or complications. In some embodiments, a subject having a predisposition to atherosclerosis may exhibit one or more of the following characteristics: advanced age, family history of heart disease, biological status, high blood cholesterol. In some embodiments, the biological status includes high levels of low-density lipoprotein cholesterol (LDL-C) in the blood, low levels of high-density lipoprotein cholesterol (HDL-C) in the blood, hypertension, insulin resistance, diabetes, excessive weight, obesity, sleep apnea, contributing lifestyle choices (s), and / or contributing behavioral habits (s). In some embodiments, the behavioral habits include smoking and / or alcohol consumption. In some embodiments, the lifestyle choices include an inactive lifestyle and / or a high stress level.

[0185] An exemplary embodiment provides administering the cyclodextrin dimer of the present disclosure, optionally in combination with one or more additional agents, to a patient having atherosclerosis. The patient may exhibit one or more signs or symptoms of atherosclerosis. Atherosclerosis is diagnosable based on one or more of Doppler ultrasound examination, ankle brachial blood pressure ratio, 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 therapies comprising a 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 an alternative therapy for treating or preventing atherosclerosis, such as an anti-cholesterol drug, an antihypertensive drug, an antiplatelet drug, a dietary supplement, or surgery or behavioral intervention, etc. Examples of alternative therapies include, but are not limited to, those described below. Further combination therapies include the CD dimer of the present disclosure and an alternative 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 atrophic age-related macular degeneration (AMD) or Stargardt disease include the CD dimer of the present disclosure and an alternative therapy for treating AMD, such as LBS-008 (Belite Bio) (a non-retinoid antagonist of retinol-binding protein 4), vitamin C and vitamin E, beta-carotene, zinc, and copper-containing AREDS dietary supplement formulation, an AREDS2 dietary supplement formulation having vitamin C and vitamin E, zinc, copper, lutein, zeaxanthin, and omega-3 fatty acids, or combinations thereof. Combination therapies for treating Alzheimer's disease include the CD dimer of the present disclosure and one or more cholinesterase inhibitors (ARICEPT(R), EXELON(R), RAZADYNE(R)) and memantine (NAMENDA(R)), or combinations thereof. Combination therapies for Niemann-Pick disease include the CD dimer of the present disclosure and one or more of miglustat (ZAVESCA(R)), HPβCD (TRAPPSOL CYCLO, VTS-270), and physical therapy. The combination therapies may be administered simultaneously, essentially simultaneously, or sequentially in any order.Combination therapy may be administered simultaneously in a single formulation or separately, and in the latter case, optionally, in the form of a dosing kit or pack containing each prescription of the combination therapy, and the kit or pack is, for example, in a conveniently quantified manner providing one or more single doses of each drug of the combination therapy. Combination therapy may exhibit a synergistic effect, in which case the effect of the combined therapy exceeds the effect of each individual treatment alone. Combination therapy generally involves administering a CD dimer and the combined therapy in effective amounts, but combination therapy may result in effective treatment while reducing the dosage of the CD and / or the combined therapy, which advantageously may reduce the side effects associated with the normal (non-combination) dosage.

[0187] Combination therapy may include therapies for the treatment or prevention of diseases or conditions associated with atherosclerosis, such as coronary artery disease, angina, heart attack, cerebrovascular disease, transient ischemic attack, and / or peripheral artery disease. Combination therapy may include therapies for the treatment or prevention of conditions that may contribute to the development and / or progression of atherosclerosis, such as hypertension, hypercholesterolemia, hyperglycemia, and diabetes.

[0188] In an exemplary embodiment, the cyclodextrin dimer of the present invention is co-administered with an anti-cholesterol drug, such as a fibrate drug or a statin drug, such as ADVICOR® (niacin extended release / rosuvastatin), ALTOPREV® (rosuvastatin 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), and / or ZOCOR® (simvastatin), etc. The anti-cholesterol drug can be administered in an amount effective to prevent or treat hypercholesterolemia.

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

[0190] In an exemplary embodiment, the cyclodextrin dimer of the present invention is co-administered with an antihypertensive drug. Examples of antihypertensive drugs include beta blockers, angiotensin-converting enzyme (ACE) inhibitors, calcium channel blockers, and / or diuretics.

[0191] In an exemplary embodiment, the cyclodextrin dimer of the present invention is co-administered with a dietary supplement, such as one or more of alpha-linolenic acid (ALA), barley, beta-sitosterol, black tea, tobacco, 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), etc.), sitostanol, and / or vitamin C.

[0192] The exemplary combination therapy also includes interventions to the patient's behavior and / or lifestyle, such as interventions including smoking cessation, exercise, and advice and / or support on a healthy diet, for example, a diet low in low-density lipoprotein (LDL) and optionally high in high-density lipoprotein (HDL).

[0193] The exemplary combination therapy also includes surgical interventions, such as angioplasty, stent placement, or both.

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

[0195] In other cases, at the time of administration of the pharmaceutical composition of the present invention, or prior to such administration, the patient may be diagnosed with atherosclerosis or identified as having a risk of developing atherosclerosis.

[0196] In still other cases, the patient to be treated by the method of the present invention is selected based on one or more factors selected from the group consisting of age (e.g., 40, 45, 50, 55, 60, 65, 70, 75, or older than 80 years old), race, gender (male or female), exercise habits (e.g., regular exercise or not), other existing medical conditions (e.g., type II diabetes, hypertension, etc.), and current medical status (e.g., currently taking statin drugs, e.g., cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, etc., beta blockers, niacin, etc.).

[0197] In the following drawings, 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 + , e.g., -CH2CH(OH)CH2N(CH3)3Cl; SUCC: succinyl; DMSO: dimethyl sulfoxide.

Brief Description of the Drawings

[0198]

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DETAILED DESCRIPTION OF THE INVENTION

[0199] Definitions

[0200] Unless otherwise specified, 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 references to the plural unless the context clearly dictates otherwise.

[0202] Linker length. As used herein, the "linker length" or its synonym "length of the linker", when referring to the length of the linker that connects the two CD subunits of the cyclodextrin dimer, indicates the number of linker atoms along the shortest path through the linker. For clarity, the linker length does not include the oxygen atoms (or other atoms that may be present in place of such oxygen) of each CD subunit to which the linker is attached. For example, in Figure 3B, the linker length is 3 + n1 + n2, reflecting the shortest path through the triazole ring. When the linker is attached to one or both of the cyclodextrin monomers at multiple points, the linker length is the shortest path that connects 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 carbon and / or C3 carbon of each CD monomer and are connected 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 are connected through the small face (top) 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 linked at opposite ends, i.e., one monomer is linked from the small face (top), typically through the C6 carbon, and the other monomer is linked from the large face (bottom), typically through the C2 carbon and / or C3 carbon.

[0206] Degree of substitution (DS). As used herein, "degree of substitution" or "DS" refers to the number of a given sub-group attached to a monomer or dimer. For example, MeβCD (DS3) refers to βCD having on average three methyl R groups added to O2, O3, or O6 of CD, while HPβCD (DS3) indicates that the monomer or dimer has on average three hydroxypropyl groups added to O2, O3, or O6 of CD. When referring to a CD dimer, unless otherwise specified, DS is used to indicate 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 substituents and methyl substituents, all are included in the count). Terms such as "degree of substitution with substituent X" indicate the average number of the said substituent X per CD dimer, i.e., other substituents may be present but are not included in the count. DS can be measured by mass spectrometry (e.g., matrix-assisted laser desorption ionization method, "MALDI") or by NMR. MALDI is suitable for cyclodextrin derivatives having substituents that give a more typical Gaussian distribution of ions in the mass spectrum, as presented for methyl substituents, hydroxypropyl substituents, and sulfobutyl substituents in, for example, FIGS. 10G - 10I, 10P - 10Q, FIGS. 11C - 11G, FIG. 11I, FIG. 12E, and FIG. 12K. The average DS when identified by MALDI is calculated by averaging the peak heights of the peaks corresponding to each DS species of the CD in question. In other examples, there may be ion peak patterns that are not normally seen, for example, due to the formation of various adducts, fragmentation, cleavage products, etc. Other mass spectrometry techniques can be utilized to potentially avoid such problems. Alternatively, NMR can be used to identify the DS value, which was suitable for succinyl groups and quaternary ammonium groups considering the more complex MS spectra observed by MALDI. Then, to achieve the calculation of the average degree of substitution (DS), the peak corresponding to the proton of the nuclear dimer is identified, and first, the scale of the measurement values is determined such that the peak area corresponds to the known number of said protons in its structure. Then, the signals corresponding to the protons in the substituent are examined and appropriately scaled to obtain the average degree of substitution.In simpler cases, clearly resolved peaks corresponding to substituent protons are identified and have already been scaled as described above. Then, they are divided by the number of protons present in the peak to obtain the average number of substituents. For example, in the case of a hydroxypropyl substituent, a peak identified as corresponding to 14 protons in the nuclear structure (aromatic region of glucopyranose) is identified and normalized to 14 by signalization. Then, a peak corresponding to 3 protons of the methyl substituent is identified, and finally, the area of the peak is divided by 3 to obtain the average number of hydroxypropyl groups present per molecule. In other examples, the substituent peak and the cyclodextrin nuclear peak may be very close or overlapping. In this case, the number of contributing protons in the cyclodextrin nuclear structure is identified and then subtracted from the peak area (the peak area has already been 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 a methyl substituent (shown in FIGS. 11K - 11L), a collection of peaks was identified as corresponding to the 3 methyl hydrogens of the substituent and additionally, a population of 86 protons in the nuclear cyclodextrin dimer structure. Similar to the hydroxypropyl substituent example, a peak identified as corresponding to 14 protons in the nuclear structure (aromatic region of glucopyranose) is identified and normalized to 14 by identification and signalization; the area of the peak containing methyl hydrogens and nuclear cyclodextrin hydrogens is determined to be 92.77. Subtracting 86 protons of the nuclear cyclodextrin structure from this signal leaves 6.77; after division by the 3 protons of each methyl group, the average degree of substitution was estimated to be 2.26. For HP and ME substituted CDs, the integral value is divided by 3, for QA, the integral value is divided by 9, for SB, the integral value is divided by 2, and for SUCC, the integral value is divided by 4. The above calculations are straightforwardly applicable to other types of substituents based on the identification of peaks corresponding to protons in the substituent structure. The DS calculation using NMR is illustrated in FIGS. 10X - 10Y, 11L, 12H, 12N, 13I, and 14I.CD compositions, such as, for example, CD dimer compositions (defined below), may include 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 can fall between substituents having integer values. Unless otherwise specified, an integer DS value indicates a CD composition having a DS number when rounded to the nearest integer. For example, DS4 indicates that the DS value is 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, ignoring any substituents other than hydroxypropyl groups. Similarly, references to the average degree of substitution with a specified substituent are as defined above, but refer to the degree of substitution ignoring other types of substituents.

[0208] Hydroxypropyl (HP or Hp) -substituted cyclodextrin (CD). As used herein, the term "hydroxypropyl-substituted cyclodextrin" or "HP-substituted CD" refers to cyclodextrin linked to a hydroxypropyl group, i.e., -CH2-CH(OH)-CH3. Typically, the HP group is linked to an oxygen atom linked to the C2, C3, and / or C6 carbon atoms of the CD (most commonly, these sites of attachment are mixed).

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

[0210] Hydroxypropyl beta-cyclodextrin dimers 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 refer to hydroxypropyl beta-cyclodextrin dimers covalently linked using a linker L. Substituents can be present at specific average numbers. For example, DS4 indicates that on average, 4 HP groups are present. As further described herein, additional substituents can be present.

[0211] Similar conventions are used for other substituted cyclodextrins and cyclodextrin dimers, such as methyl (Me), quaternary ammonium (QA), succinyl (SUCC), sulfobutyl (SB), etc. For example, MeβCD indicates methyl beta-cyclodextrin. 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 using a linker L. Substituents can be present at specific average numbers. For example, DS4 indicates that on average, 4 Me groups are present. As further described herein, additional substituents can be present.

[0212] Cyclodextrin dimer composition. As used herein, the terms "cyclodextrin dimer composition" or "CD dimer composition" refer to a mixture of cyclodextrin dimers, for example, a mixture of CD dimers substituted with the same substituents but in different numbers. Typically, the CD dimer composition is characterized by having a specified degree of substitution with a specified substituent. Due to the symmetry of the CD molecules for the most part, the CD dimer composition can result from the synthesis process in which substituents are added to the CD dimers in a probabilistic manner such that individual CD molecules vary variously in the number and position of the substituents. Further, the CD dimer composition may or may not contain 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), and may have uniform 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, for example, by NMR and / or mass spectrometry as described above.

[0213] Terms such as "specifically binds" mean that a molecule, such as a cyclodextrin dimer of the present disclosure, forms a relatively stable complex with a binding partner, such as cholesterol (oxysterol, such as 7KC, etc.) under physiological conditions. Methods for determining 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, etc. In an exemplary embodiment, the cyclodextrin dimer of the present disclosure binds to cholesterol, oxysterol, or 7KC with a K of 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, 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 in binding.

[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., a cyclodextrin) has a greater solubilizing ability for 7KC than for cholesterol. The higher affinity can also be predicted by molecular docking, predicted by molecular dynamics simulations, or measured by calorimetry. In an exemplary embodiment, the cyclodextrin dimer has a binding affinity for 7KC that is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 30-fold, or at least 50-fold stronger than its binding affinity for cholesterol, which may optionally be determined by comparing the concentration at which 50% of the 7KC in suspension is solubilized, using, for example, the procedures described in the examples herein. In an exemplary embodiment, the cyclodextrin dimer has a binding affinity for 7KC that is at least 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold stronger than its binding affinity for cholesterol, which may optionally be determined by dividing the computer-calculated or measured binding affinity for cholesterol (K D ) by the computer-calculated binding affinity for 7KC.

[0215] A higher affinity for one compound over the other, e.g., a higher affinity for 7KC over cholesterol, can be identified using a "turbidity test" performed in an aqueous suspension containing 3% ethanol in PBS, 300 uM of sterol, and 1 mM of the cyclodextrin being tested. This single concentration of cyclodextrin is used for the purpose of normalizing the test results. To conduct the test, the sample is incubated at 37 °C for 30 minutes and then the absorbance at 350 nm is measured using, for example, 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, that 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 in the absence of certain surfactants or other solvents. Examples of 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, by way of illustration, dextromethorphan HBr (DXM), diphenhydramine HCl (DPH), lidocaine HCl (LDC), heparin, bendroflumethiazide, acyclovir, levaprazan, curcumin, and testosterone propionate (TP). The cyclodextrin dimers can be present in an amount sufficient to increase the solubility of the molecule and / or assist in better drug delivery. The molecular ratio of drug to cyclodextrin can be 1:1 or 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) that can solubilize the hydrophobic drug in an aqueous composition, such as phosphate buffered saline (PBS) or water. Solubilization can be measured by spectroscopic methods or other means known in the art. Solubilization can be measured at room temperature, physiological temperature (37 °C), or another appropriate temperature (e.g., 0 - 4 °C).

[0218] "Alkyl" consists of only carbon and hydrogen atoms and means a monovalent straight-chain or branched-chain saturated hydrocarbon moiety having from 1 to 12 carbon atoms.

[0219] "Lower alkyl" refers to an alkyl group having from 1 to 6 carbon atoms, such as 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, etc.

[0220] "Alkylene" means a divalent straight-chain or branched-chain saturated hydrocarbon radical having from 1 to 12 carbon atoms, or a divalent branched-chain saturated hydrocarbon having from 3 to 6 carbon atoms, such as methylene, ethylene, 2,2-dimethylethylene, propylene, 2-methylpropylene, butylene, pentylene, etc.

[0221] "Alkenyl" means a monovalent straight-chain hydrocarbon radical having from 2 to 12 carbon atoms and at least one double bond or a monovalent branched-chain hydrocarbon radical having from 3 to 12 carbon atoms and 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, etc.

[0222] "Alkoxyalkyl" means a moiety of the formula Ra-O-Rb-, wherein 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 -R-O-R'-O-R'', wherein R and R' are each alkylene and R'' is alkyl, as defined herein.

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

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

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

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

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

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

[0230] "Alkylsulfanyl" means a moiety of the formula -SR, wherein 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" means a group -NR'R'', wherein R' and R'' are each independently hydrogen or alkyl. Thus, "amino", as used herein, includes "alkylamino" and "dialkylamino".

[0234] "Alkylaminoalkyl" means a group -R-NHR', wherein R is alkylene and R' is alkyl. Examples of alkylaminoalkyl include methylaminomethyl, methylaminoethyl, methylaminopropyl, ethylaminoethyl, etc.

[0235] "Dialkylaminoalkyl" means a group -R-NR'R'', wherein R is alkylene and R' and R'' are alkyl, as defined herein. Examples of dialkylaminoalkyl include dimethylaminomethyl, dimethylaminoethyl, dimethylaminopropyl, N-methyl-N-ethylaminoethyl, etc.

[0236] "Aminoalkyl" means the group -R-R', where R' is amino and R is alkylene, as defined herein. Examples of "aminoalkyl" include aminomethyl, aminoethyl, 1-aminopropyl, 2-aminopropyl, etc.

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

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

[0239] "Aminocarbonylalkyloxy" or "carbamylalkyl" means the group -R-O-C(=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 the group of the formula -O-R-R', where R is alkylene and R' is alkynyl, as defined herein.

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

[0243] "Arylalkyl" and "aralkyl" can be used synonymously and mean a 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 -O-R, where R is aryl as defined herein.

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

[0247] "Cyanoalkyl" means a moiety of the formula -R'-R'', wherein R' is an 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 is optionally substitutable 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 their partially unsaturated derivatives.

[0249] "Cycloalkenyl" means a monovalent unsaturated carbocyclic moiety consisting of a monocyclic or bicyclic ring and having at least one double bond. Cycloalkenyl is optionally substitutable 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, cycloheptenyl.

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

[0251] "Cycloalkylene" means a divalent saturated carbocyclic radical consisting of a monocyclic or bicyclic ring. Cycloalkylene is optionally substitutable with one or more substituents, each of which, unless specifically indicated otherwise, is independently hydroxy, alkyl, alkoxy, halo, haloalkyl, amino, monoalkylamino, or dialkylamino.

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

[0253] "Heteroalkyl" means an alkyl radical as defined herein, wherein one, two, or three hydrogen atoms are replaced by substituents independently selected from the group consisting of -ORa, -NRbRc, and -S(O)nRd, wherein n is an integer from 0 to 2, provided that the point of attachment of the heteroalkyl radical is through a carbon atom, wherein 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, etc.

[0254] "Heteroaryl" means a monocyclic or bicyclic radical having at least one aromatic ring with 5 to 12 ring atoms and 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 optionally be 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, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuryl, benzothiophenyl, benzothiopyranyl, benzimidazolyl, benzoxazolyl, benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl, benzopyranyl, indolyl, isoindolyl, triazolyl, triazinyl, quinoxalinyl, purinyl, quinazolinyl, quinolidinyl, naphthyridinyl, pteridinyl, carbazolyl, azepinyl, diazepinyl, acridinyl, etc., as well as their partially hydrogenated derivatives.

[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 -O-R, where R is heteroaryl as defined herein.

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

[0259] "Heterocyclyloxy" means a group of the formula -O-R-R', wherein R is alkylene and R' is heterocyclyl, as defined herein.

[0260] The terms "halo", "halogen", and "halide" are used synonymously and these terms denote the substituents fluoro, chloro, bromo, or iodo. In some embodiments, halo denotes a fluoro substituent.

[0261] "Haloalkyl" means an alkyl as defined herein, wherein one or more hydrogens are replaced by the same or different halogen. In some embodiments, haloalkyl is fluoroalkyl. In some embodiments, haloalkyl is perfluoroalkyl. Examples of haloalkyl include -CH2Cl, -CH2CF3, -CH2CCl3, perfluoroalkyl (e.g., -CF3), and the like.

[0262] "Haloalkoxy" means a moiety of the formula -OR, wherein R is a haloalkyl moiety as defined herein. In some embodiments, haloalkoxy is fluoroalkoxy. In some embodiments, haloalkoxy is perfluoroalkoxy. An example of 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 1 to 3 rings incorporating 1, 2, 3, or 4 heteroatoms (selected from nitrogen, oxygen, or sulfur). The heterocyclyl ring is optionally substitutable 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, benzazolidinyl, dihydrofuryl, tetrahydrofuryl, dihydropyranyl, tetrahydropyranyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, dihydroquinolinyl, dihydroisoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc.

[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', wherein R is hydrogen or alkyl and R' is hydroxyalkyl, as defined herein.

[0270] "Hydroxyalkylaminoalkyl" means a moiety of the formula -R-NR'-R'', wherein 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, wherein R is alkylene as defined herein.

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

[0274] "Hydroxyalkyl" means an alkyl moiety as defined herein, which is 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-(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 hydroxy substituents. Representative examples include, but are not limited to, 2-, 3-, or 4-hydroxycyclohexyl, etc.

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

[0277] "Carbamate" means a group of the formula -O-C(O)-NR'R'', wherein 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'', wherein 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, in front of a functional group, such as, for example, C1-12-alkyl or C5-12-heteroaryl, and integer values (i.e., 0, 1, 2, 12) are inserted for "n" and "m". This prefix indicates the number or range of carbon atoms present in the functional group. In the case of a ring system, this prefix indicates the number of ring atoms, or the range of the number of ring atoms, regardless of whether the ring atoms are carbon atoms or heteroatoms. When the functional group consists of a ring part and an acyclic part (i.e., in the case of "arylalkyl", consisting of an aryl part and an alkyl part), 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 examples of functional groups, there may be zero carbon atoms present; for example, in C0-aminosulfonyl (i.e., -SO2-NH2, where the possible R groups are both hydrogen), "0" indicates the absence of carbon atoms.

[0285] "Peptide" means an amide derivative resulting from two or more amino acids by the bonding of the amino group and carboxyl group of one amino acid. "Mono-peptide" means one amino acid, "di-peptide" means an amide compound containing two amino acids, "tri-peptide" means an amide compound containing three amino acids, and so on. The C-terminus of a "peptide" can be bonded to another moiety via an ester functional group.

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

[0287] "Leaving group" means a group having the meaning conventionally associated with this term in organic synthetic chemistry, i.e., an atom or group capable of being replaced 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, etc.

[0288] "Modulator" means a molecule that interacts with a target. Interactions include, but are not limited to, agonists, antagonists, etc. as defined herein.

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

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

[0291] "Inert organic solvent" or "inert solvent" means that the solvent is inert under the reaction conditions described in combination with the solvent, 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, and the like. Unless otherwise noted, the solvents used in the reactions of the present disclosure are inert solvents.

[0292] "Pharmaceutically acceptable" generally means safe, non-toxic, and useful for the preparation of pharmaceutical compositions that are not otherwise biologically or otherwise undesirable, and includes those acceptable not only for human pharmaceutical use but also for veterinary use.

[0293] A "pharmaceutically acceptable salt" of a compound means a pharmaceutically acceptable salt as defined herein that retains 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, etc.; or 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, mucic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, etc.; or salts formed when an acidic proton present in the parent compound is replaced by a metal ion such as an alkali metal ion, an alkaline earth metal ion, or an aluminum ion; or salts formed by coordination of an organic or inorganic base. Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, trimethylamine, tromethamine, etc. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide. Preferred 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. Reference to a pharmaceutically acceptable salt includes all solvate addition forms (solvates) or crystal polymorphs (polymorphs) of the acid addition salt as defined herein. Generally, when a particular salt is included in the formula herein, it is of course possible to replace it with other pharmaceutically acceptable salts within the scope of the present disclosure. For example, in the case of the quaternary ammonium salt of formula VIII, chloride or another negative ion or combinations thereof may be included. Similarly, in the case of the carboxymethyl sodium salt of formula IX, another positive ion may replace sodium as indicated.

[0294] "Protecting group" or "protective group" means a group that selectively blocks one reactive site in a polyfunctional compound such that a chemical reaction can occur selectively at another unprotected reactive site, in the meaning conventionally associated with this term in synthetic chemistry. Certain processes of the present disclosure rely on a protecting group to block reactive nitrogen atoms and / or oxygen atoms present in a reactant. For example, the terms "amino protecting group" and "nitrogen protecting group" are used synonymously herein, and these terms refer to organic groups that are intended to protect nitrogen atoms from unwanted 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. One of ordinary skill in the art will know how to select a group based on ease of removal and tolerance to subsequent reactions.

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

[0296] "Therapeutically effective amount" means an amount of a compound sufficient to exert the relevant treatment for a disease state when administered to a subject for treating the disease state. The "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 form 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 item incorporate by reference the broad definition of that variable item, and, if any, the preferred definition, the more preferred definition, and the particularly preferred definition.

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

[0299] Any unshared electron pair present on a carbon, oxygen, sulfur, or nitrogen atom in the structures herein is indicative of the presence of a hydrogen atom.

Examples

[0300] Example 1. Solubilization of Compounds by HPβCD

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

[0302] The inventors also tested variants of HPβCD by varying the number of hydroxypropyl groups on HPβCD. The inventors tested in the range of 3.7 - 21 (the maximum of possible substitution numbers). Although the data were noisy, the ability to solubilize 7KC and cholesterol decreased as the degree of substitution increased (Figures 2C - 2D). This was strongly supported by molecular docking of monomeric HPβCD with a wide range of substituents (Figure 2E). Monomers and sterols were designed in PyMOL based on known chemical properties. The most likely arrangement of each hydroxypropyl group was adopted, 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 cyclodextrin, that conformation was included in the calculation. HPβCD with a lower DS preferentially solubilized 7KC over cholesterol, suggesting that such HPβCD has specificity for 7KC. Without intending to be bound by theory, a possible explanation is that the maximum number of hydroxyl groups is available for hydrogen bonding with the 7-keto group at the 7-position of 7KC. However, this theory is not necessary for practicing the present invention.

[0303] Example 2. Computer Modeling of the Interaction between Cyclodextrin Monomers and Dimers and Cholesterol and 7KC

[0304] Overview

[0305] In this example, molecular modeling and computer simulation are described. This molecular modeling and computer simulation were performed to investigate the mechanism by which CD binds to sterols, predict the relative binding ability of cyclodextrin dimers to cholesterol and 7KC, and identify cyclodextrin dimers that are predicted to have a higher affinity for 7KC than for cholesterol. Perhaps the conformation in which the sterol is completely encapsulated by the CD or CD dimer shields the hydrophobic sterol from the hydrophilic solvent and thus allows the sterol to go into solution.

[0306] For the initial docking analysis (Figure 2E [monomer], Figure 4B [dimer]), computer modeling program PyMOL (PyMOL Molecular Graphics System, version 2.0, Schrodinger, LLC.) was used to construct HPβCD monomers and dimers at various substitution levels, and then the extended AutoDock Vina (Trott [et al.], J. Comput. Chem., 31(2):455 - 61.(2010)) developed at the Scripps Research Institute (La Jolla, CA, USA) was used to model the interaction of these putative CD molecules with 7KC or cholesterol. Autodock Vina is a molecular docking software that shows significant improvements in accuracy and speed compared to the previous Autodock4. This software uses a scoring function to approximate the standard chemical potential of the system, predicts the binding by non-covalent intermolecular bonds, and predicts the energetically favorable higher-order structure as well as the binding affinity. It was generally found that hydroxypropyl dimers and monomers with DS of about 2 - 6 showed the best specificity for 7KC.

[0307] For the binding of three derivatives of beta-cyclodextrin, native monomeric (DS0) beta-cyclodextrin (βCD), monomeric hydroxypropyl-beta-cyclodextrin (DS5, HPβCD), and dimeric 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) with either 7KC or cholesterol, in addition to docking simulations using AutoDock Vina, molecular dynamics simulations were performed using GROMACS 2018 (in particular, University of Groningen, Groningen, Netherlands; Bekker [et al.], World Scientific (1993); and Berendsen [et al.], Comp. Phys. Comm., 91:43 - 56. (1995)). Both of these ligands are asymmetric, and thus the simulations were performed both up and down with respect to the ligand orientation. These simulations were then repeated in the AMBER force field and at translated positions to establish which position / force field yielded the most beneficial data for these novel molecules (initial MD analysis, Figures 4D - 4MM). Since the GROMOS force field at the initial position was identified as being most effective in capturing the interaction between the CD dimer and the sterol, this force field and position were used for subsequent simplified MD simulations for other CD dimers (subsequent MD analysis, Figures 4NN - 4SS, Figures 5B - C, Figures 6B - 7B).

[0308] Generally, the addition of hydroxypropyl groups results in a more labile complex, but at the same time, it was found that some specificity for 7KC over cholesterol is brought about, which is different from that seen with native, unsubstituted βCD. This was observed because 7KC can form and reform a somewhat stable complex in both the up and down directions, while cholesterol is not able to form as stable a complex, possibly because cholesterol does not appear to be encapsulated by βCD as completely as 7KC, especially in the "down" direction. The dimerization of βCD resulted in a significantly higher affinity for sterol targets, such as 7KC and cholesterol. This was revealed by the formation of a stable dimer complex by strong energy interactions for all ligands and directions, where the ligand fits inside the hydrophobic core of the CD dimer, enabling the ligand to be solubilized in an aqueous solution.

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

[0310] Based on this extended computer - based computational analysis, the inventors believe that dimerization of βCD is optimal for forming strong and stable complexes with sterols, regardless of the type or position of the substituents or linkers used. Testing a wide variety of dimerized βCD molecules, those dimerized βCD molecules showed a much higher sterol affinity than monomeric βCD for many types of substituents and linkers, even if they are very chemically different from each other.

[0311] Computer - based computational methods

[0312] Initial docking simulation

[0313] The present inventors have developed a method for more rapidly and simply predicting by computer, using AutoDock Vina, the binding between cyclodextrin and various sterols without analyzing the entire trajectory. Analyzing the entire trajectory is very time-consuming and computationally expensive. By applying this technique to cyclodextrin systems, it has become possible to perform hundreds of docking simulations using the various cyclodextrins designed by the present inventors. This type of computer modeling presents the possible interactions that can occur between various cyclodextrins and various sterols, and provides both spatial information and binding affinity data.

[0314] These structural predictions can be made as modeling for multiple different sterols and / or CD derivatives, and the features of potential mechanisms may be revealed. The present inventors are considering multiple binding hypotheses and would like to test them using computer calculation techniques. The present inventors have developed different models of HPβCD to test their binding theory:

[0315] Monomer-sterol association: The present inventors tested the monomer-sterol affinity for comparison with the dimer association for the purpose of assisting in determining whether the sterol is more likely to bind to the monomer or dimer of HPβCD, and which of 7KC and cholesterol the monomer shows specificity for (Figure 4A).

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

[0317] For the purpose of making the outputs 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 is of the head-to-head type (if possible) and whether the sterol is actually within the barrel-shaped HPβCD cavity. Then, this number of "complex-formed higher-order structures" (out of a maximum of 20 conformations) was added to the absolute value of the most favorable affinity. That is, an association that results in 15 / 20 conformations that form a complex with the sterol (head-to-head type and / or the sterol is within the CD cavity) and has a highest affinity of -10 kJ / mol has a score of 25 (|-10| + 15 = 25). In this computer calculation, if any atom of the ligand crosses the plane formed by the O4 atom of the CD, the ligand was considered to be in a complex regardless of the insertion angle or degree into the cavity. The resulting value is referred to as the "affinity score".

[0318] Next, this docking analysis was extended to determine whether 7KC specificity is affected by these factors by including a wide variety of substituents (including charged groups) and linkers. When the sulfobutyl substituent and methyl substituent were combined with the triazole linker and butyl linker and tested over the entire DS range of 0 to 20, a pattern similar to that of hydroxypropyl was shown, and in this case, the DS with the highest 7KC specificity was approximately 4 (Figures 5A and 6A). Therefore, for other cyclodextrins such as quaternary ammonium and carboxymethylation, testing was only performed at low DS (approximately 4).

[0319] Initial molecular dynamics simulations (Figures 4D - 4MM)

[0320] This initial simulation set was performed with both the GROMOS 54a7 and AMBER 99SB force fields using GROMACS 2018 (University of Groningen, Groningen, Netherlands), resulting in two reproductions of these simulations that helped to determine the consistency of the observed interactions. These two reproductions for each of the three CD molecules and for each orientation of the ligand were then repeated with different initial structures. In the repeats, 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 dimers were then analyzed using GROMACS tools.

[0321] Molecular dynamics, unlike docking, enables the simulation of molecules interacting in a time-dependent manner rather than just providing a snapshot of an energetically favorable higher-order structure as provided by docking. For each of the first three CD-sterol complexes, the simulation was extended up to 1 microsecond (a very long time for an MD simulation) to allow sufficient time for the complex to equilibrate. The output was then analyzed to identify the distance between the centroid consisting of all O4 oxygens (the center of the CD cavity for both the dimer and monomer) and the centroid 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), as well as both the Lennard-Jones and Coulomb energies of the interaction between the cyclodextrin and the ligand.

[0322] In this way, the distance indicates the proximity between the ligand and 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 (it is defined that the stronger the interaction, the larger the negative interaction energy). Figure 4C shows how useful the "angle" measurement is for determining how well the ligand is shielded from the surrounding water molecules: zero or 180 degrees indicates that the ligand is completely perpendicular to the plane of the cyclodextrin, while 90 degrees indicates that the ligand is parallel to the CD plane and thus is thought not to form a complex inside the cavity. In these simulations, the inventors selected that 30 degrees corresponds to the case where complex formation starts in the "up" configuration (the head of the sterol meets the bottom of the CD and the tail meets the top, and the whole ligand is inserted into the cavity of the CD), and 150 degrees corresponds to the case where complex formation first occurs in the "down" configuration (the tail of the sterol meets the bottom of the CD and the head meets the top, and the whole ligand is inserted into the cavity of the CD). For the dimer, only the plane of one CD monomer is considered for the angle between the CD and the ligand, but if the dimer is perfectly formed, this plane is a mirror image of the plane of the other monomer.

[0323] The number of water molecules within 3 Å of the ligand was determined over time to identify how well the CD shields the ligand from the surrounding solvent. Presumably, the more water molecules around the ligand, the less well the ligand is shielded from the surrounding water and thus the more likely it is not to be in solution. All of these simulations were extended up to 1 microsecond (1000 ns), which should be long enough to accurately describe the interaction between the CD and the sterol.

[0324] This long initial analysis provides proof that the simulations properly capture the interactions of the CD monomer and dimer with the sterol ligand and can thus be extended to other CD monomers and dimers without the need for such a laborious method.

[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 positions not translated by the GROMOS force field gave the best and most dynamic results for these complexes. This long initial analysis was important for establishing a precedent for modeling these new molecules, allowing for more focused simulations of other types of dimers over shorter timescales. That is, molecular dynamics analysis was extended using various promising linkers and substituents. First, docking calculations were performed for methyl (Figure 5A) and sulfobutyl (Figure 6A) βCD dimers with various DSs. This demonstrated that low DS (about 4) showed the most promising results in terms of dimers with the best 7KC specificity. Therefore, additional MD simulations were performed for DS4 βCD dimers with triazole and butyl linkers (Figures 4RR - 4SS, 5B - 5C, 6B - 6C, 7A - 7B). The inventors also performed simulations for DS0 βCD dimers (Figures 4NN - 4QQ). These simulations were run for 100 ns and analyzed only for angles and interaction energies to evaluate the main differences or similarities between these molecular interactions and those with the butyl-linked hydroxypropyl dimer.

[0327] Additional docking simulations

[0328] After the initial simulations demonstrated similar promise for a range of achievable substituents and linkers, additional screening was performed for more linkers, substituents, and even substitution positions using the same docking technique as above. This analysis helps show that the effectiveness of these molecules is largely (if not entirely) brought about by the actual dimerization of βCD and is independent of the type of linker or substituent.

[0329] Computer Calculation Results and Conclusions

[0330] Docking:

[0331] The inventors first examined whether HPβCD can bind to cholesterol and 7KC as a monomer (Figure 2E), and then examined whether HPβCD can bind to cholesterol and 7KC as a dimer (Figure 4B).

[0332] It was found that the HPβCD monomer (Figure 2E) has a high affinity for both cholesterol and 7KC at low degrees of substitution (DS), but as the DS increases, the affinity for both sterols appears to decrease. This is likely because the hydroxypropyl groups become crowded, preventing the sterol from entering the nucleus of the monomer. Additionally, there are fewer hydroxyl groups available to hydrogen bond with the carbonyl group of 7KC on the inner surface of the CD. The best specificity (not the best affinity) is seen as a spike at DS4, but the preference for 7KC extends from DS2 to DS6 and switches to cholesterol at DS7 and above. After DS10, little or no affinity is observed in these models.

[0333] The butyl-linked dimer shows a higher affinity for sterols compared to the monomeric CD, and the best affinity / specificity for 7KC was at dimerization DS10 and DS4 (Figure 4B). However, this specificity appears to exist only for dimers of specific DS for these calculations, and the changes between different DS shown in these calculations are significant. The triazole-linked dimer shows generally better specificity except at DS6 and has an affinity similar to that of the butyl-linked dimer. This specificity is hypothesized to be due to the formation of additional hydrogen bonds between the hydrogen-bond donating nitrogen and the hydrogen-bond accepting ketone of 7KC.

[0334] Initial Molecular Dynamics Analysis:

[0335] Figures 4D - 4O support the hypothesis that native (unsubstituted, DS0) monomeric βCD can form complexes with both 7KC and cholesterol in both the up and down directions, but that 7KC maintains more stable complex formation than cholesterol in the down direction and vice versa in the up direction. Cholesterol shows little variability throughout its trajectory in the up direction, indicating how cholesterol detaches from and re - associates with CD multiple times in the up direction (note that the angle changes significantly at about 150 ns, at which time cholesterol rotates around and re - associates in the opposite direction) (Figure 4D). This angle change indicates that the down direction is significantly more stable, so stable that cholesterol exits the cavity, rotates 180 degrees, and then re - associates, and the overall affinity of cholesterol is very high because this elaborate movement is possible to complete in the simulation.

[0336] On the other hand, 7KC does not re - associate once the complex dissociates in either direction, but the down direction is significantly more stable for more than half of the trajectory, supporting the hypothesis that the down direction is preferred for 7KC. This indicates that both 7KC and cholesterol prefer the down direction, in which case the head group associates with the upper base and the tail associates with the lower base. In this preferred higher - order structure, only cholesterol can actually detach from and re - associate with CD. This can explain why native CD is very good at solubilizing cholesterol and its derivatives but does not show specificity for 7KC. This slight preference of native, monomeric βCD for cholesterol is expected and 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 significantly less water than 7KC, especially in the "up" direction.

[0337] The AMBER force field (Figs. 4G - 4I) showed a significantly stronger interaction between native βCD and sterols. 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 stronger and longer interactions between the two molecules than the GROMOS force field, and the solubilization of sterols by native βCD in the AMBER force field appeared to be approximately the same in both the up and down directions between the two ligands. Despite this strong and stable interaction, the AMBER force field may not fully capture the interaction between βCD and sterols because the complex does not separate at all. Some movement is required to fully elucidate the interactions taking place, but 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 (Figs. 4J - 4O), the native complex was still effectively formed in both force fields, but again, the GROMOS was less consistent than AMBER in this case. The GROMOS force field showed a significant preference for the "up" direction for 7KC and the "down" direction for cholesterol, while AMBER only showed a strong interaction between both ligands and the CD. This indicates that 7KC and cholesterol interact with native βCD similarly and strongly, which is consistent with experimental data, although the ligand orientation appears to make a difference in the observed complex formation. The subtle differences in these trajectories are described in detail below.

[0339] The monomeric HPβCD of DS5 (Figs. 4P - 4AA) shows that the consistency of the interaction between CD and sterol is lower than that of natural CD in the GROMOS force field. At the same time, as seen in Fig. 4P, it was also found that the downward direction was preferred for 7KC. The AMBER force field (Figs. 4S, 4Y) also showed stronger and more consistent interactions here, but the stable complexes formed were still the same in both force fields. Overall, adding a hydroxypropyl group to the cyclodextrin monomer reduces the possibility of complex formation for both ligands in both force fields, but it can be seen that 7KC can consistently form and reform stable complexes more than cholesterol. Cholesterol generally has more water molecules that can approach it than 7KC in both force fields, so it seems that cholesterol cannot form a complex with HPβCD as easily as 7KC. This is clear in Fig. 4R, where 7KC forms and reforms the complex in the "down" direction, while cholesterol does not form a complex in the same way. Visualization of this trajectory also shows how strongly 7KC prefers the "up" direction and that it still forms a "down" - direction complex in about 500 ns.

[0340] When the ligand was translated, the deeper the ligand in the initial position was embedded by the cavity of CD, the more effectively HPβCD could form a complex with both sterols. In the case of this translational trajectory in GROMOS (Figure 4V), the preference for 7KC over cholesterol was even clearer than in the previous simulation, 7KC could form a stable complex in both directions, while cholesterol could form a stable complex only in the "down" direction. Furthermore, 7KC in the up direction was initially outside the cavity and could associate with the cavity, forming a very stable complex within 300 ns. The AMBER force field also showed significantly stronger interactions between HPβCD and the sterols, but still formed the same stable complex for both ligands and preferred the up direction, and there was generally slightly less water surrounding 7KC throughout the trajectory (see Figure 4T). This is probably due to the fact that the "down" direction shows the head group of the sterol protruding further outside the cavity than in the "up" direction. This is consistent with our experimental data (Figure 2) and shows that the HPβCD monomer has some specificity for 7KC while still forming a stable, seemingly solubilized complex with both KC and cholesterol. All of these simulations will be described in detail below.

[0341] The novel butyl-linked hydroxypropyl β-cyclodextrin dimer of DS5 of the present inventors was then modeled with 7KC and cholesterol in the GROMOS force field and the AMBER force field as seen in FIGS. 4BB to 4MM. The comparison between the plots of these trajectories and the plots for the monomeric form HPβCD and native βCD provides clear evidence that the dimerized version consistently binds the sterol significantly more surely than its corresponding monomeric version, whether hydroxypropylated or not. This is consistent with the experimental data of the present inventors (FIG. 16). The angles, distances, and energies, as well as the water molecules surrounding the ligand, are all much more stable than in the monomeric simulations and appear to be in a more solvated conformation. The GROMOS force field shows that when the complex is fully formed in the down direction, the distance between the centroid of the ligand and the CD is less than 5 Å (FIG. 4BB), while the monomers of the GROMOS force field consistently show that when the complex is formed, the intermolecular distance exceeds 5 to 10 Å. The AMBER force field also shows a very strong interaction between the sterol and the dimerized CD, with the interaction energy approaching -300 kJ / mol in the down direction compared to about -150 kJ / mol for the monomer (FIG. 4BB). This indicates that the dimer forms a very strong and stable complex with both ligands, especially in the down direction and particularly when compared to the monomeric βCD.

[0342] The results of the AMBER force field (Figures 4EE, 4KK) support the findings from GROMOS force field simulations that dimerization of HPβCD creates a stronger and more stable interaction between the CD and the sterol, with a very small distance between the two molecules and a very large interaction energy. The dimerized CD also consistently shows that fewer than five water molecules surround the ligand, especially in the downward direction, while the monomeric CD shows that more than ten water molecules surround the ligand (Figure 4CC). Although this is sometimes also the case for the monomer with 7KC and cholesterol, the presence of water around the sterol was significantly reduced overall by dimerization. Dimerization of HPβCD also brought some specificity for 7KC, which remained constantly associated with at least one of the two linked CDs throughout the entire trajectory, regardless of the force field or translation, while cholesterol generally dissociated from both monomer pairs during at least a portion of the trajectory and even created a deformed head - tail dimer arrangement in which cholesterol could not be completely encapsulated by the dimer. These trajectories will be described in detail in the following section.

[0343] These simulations provide strong evidence that dimerization of HPβCD promotes complex formation with sterols by forming an encapsulation complex that shields the hydrophobic sterol from surrounding water molecules. The data imply that dimerized HPβCD has a much stronger sterol affinity overall than the monomer, and that dimerized HPβCD has a preference for 7KC as 7KC associates with at least one of the CDs significantly longer than cholesterol. The inventors can conclude from this that while strong complex formation with the AMBER force field is good evidence for the inventors' complex formation and stability justification, it is possible to gather more useful information from the GROMOS force field. This is because the GROMOS force field shows dynamic intermolecular interactions rather than just one very (sometimes unrealistically) stable complex as is the case with AMBER.

[0344] The details of 48 trajectories of hydroxypropyl-beta-cyclodextrin dimers, each with a length of 1 microsecond, will be described in detail below.

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

[0346] Native monomeric βCD and 7KC, upward direction, GROMOS force field:

[0347] In Figure 4F, 7KC starts with its head group inserted into the CD cavity and its tail extended outside the bottom. At 134 ns, the complex dissociates, 7KC moves towards the bottom, and rotates outside the cavity. Subsequently, although 7KC still associates with the bottom, it moves its head group in and out of the cavity. When the complex finally dissociates completely at 150 ns, 7KC moves around the box and re-associates with the upper bottom. 7KC continues to associate with and dissociate from the upper bottom, but does not enter the cavity again during the remainder of the trajectory.

[0348] Native monomeric βCD and cholesterol, upward direction, GROMOS force field:

[0349] Figure 4F shows that cholesterol (up) starts with its tail inserted into the CD cavity and its head group extended outside the lower base. At about 150 ns, the complex separates, and as seen by the large change in the "angle" of cholesterol, cholesterol rotates out of the cavity and towards the outside, becoming parallel to cyclodextrin, then re-associates in the opposite direction with its tail extended outside the lower base. Then, cholesterol re-inserts its head group and periodically repeats inserting the head group and becoming parallel to the CD for about 200 ns, which is visualized as the changes in the angle, energy, and distance of cholesterol (up) in Figure 4D. At about 300 ns, the complex completely separates (corresponding to the spike of cholesterol in Figure 4D), and cholesterol moves randomly around the CD molecule. The two molecules re-associate temporarily from 310 ns to about 1 nanosecond, at which time cholesterol lies parallel to the upper base of the CD. Then, cholesterol resumes random movement for about another 2 nanoseconds and finally re-associates with the lower base at 330 ns, and the tail of cholesterol is gently inserted into the CD cavity. Then, at about 400 ns, cholesterol reverses and associates its head group with the CD cavity, and this conformation remains relatively stable, with the head group regularly repeating association and dissociation, but the complex finally separates again at about 560 ns. At this point, cholesterol moves randomly around the CD temporarily and then associates its tail with the lower base of the CD. By 580 ns, the tail of cholesterol is perfectly inserted into the CD molecule and the head group extends from the lower base of the CD. Then, at 582 ns, the complex separates again, and finally at 610 ns the head group is inserted from the lower base to form the complex again. At about 680 ns, the complex separates again, is re-formed at 750 ns, then separates again at 880 ns, is re-formed at 920 ns, and continues to separate and re-form about every 10 ns until the end of the trajectory (always associating as seen at 920 ns). The fact that cholesterol completely exits the CD cavity and then re-associates within the simulation time indicates that this program was able to associate the two molecules by the molecules themselves and was independent of any external circumstances.This provides strong evidence that this interaction is reasonable, recurrent, and effectively captured by simulations.

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

[0351] In Figure 4F, 7KC starts with its tail inserted into the CD cavity and its head group extended outside the upper base. This complex maintains this higher-order structure, and 7KC moves back and forth and tilts within the cavity. The complex does not dissociate until 600 ns, but at 600 ns, 7KC quickly exits the cavity and wraps around the lower base. 7KC begins to float around the simulation box and periodically and temporarily associates with CD in a higher-order structure 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 enters the CD cavity, it is fixed there by the interaction force. This trajectory is quantifiable in Figure 4D because the plot of 7KC (up) remains relatively flat until approximately 600 ns. 600 ns is the point at which the complex dissociates and takes random movements.

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

[0353] In Figure 4F, cholesterol in the down position starts with the head group inside the CD cavity and the tail extending outside the bottom. This maintains stability until about 125 ns, but at about 125 ns, cholesterol rotates and exits the cavity. However, for the next 200 ns, cholesterol periodically continues to insert the head group from the bottom into the CD cavity. At about 340 ns, the complex completely separates, and cholesterol floats around the simulation box, but finally reassociates with the bottom in the same manner as before at about 560 ns. Then, about 30 ns later, cholesterol dissociates and reassociates parallel to the upper base. Then, for the remainder of the trajectory, cholesterol repeats like a pendulum between associating with the upper base in this way and floating randomly.

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

[0355] The interactions seen in the AMBER force field of Figure 4I support the strong solubilization of sterols by native monomeric βCD. Throughout the trajectory, both ligands remain inside the cyclodextrin ring in both directions, and there is 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, and the tail group slightly outside the upper base. 7KC remains exactly inside the CD cavity throughout the trajectory but oscillates slightly back and forth, which can be seen as slight fluctuations in the overall flat line in Figure 4G. This indicates that a stable higher-order structure is formed and does not separate. This is also consistent with the experimental data, but the AMBER force field shows stronger and longer intermolecular interactions than the GROMOS force field.

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

[0357] In Figure 4I, cholesterol (up) starts with the center of the molecule inside the CD cavity, the head group slightly protruding outside the lower base, while the tail group slightly protruding outside the upper base. This complex maintains stability throughout the trajectory. Cholesterol never exits the cavity, never changes direction, and just swings back and forth inside the cavity. Such small fluctuations in position correspond to the small protrusions, especially at the angular part, in Figure 4G.

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

[0359] In Figure 4I, 7KC (down) starts with the center of the molecule inside the CD cavity, the head group slightly protruding outside the upper base, while the tail group slightly protruding outside the lower base. This complex maintains stability throughout the trajectory. 7KC never exits the cavity, never changes direction, and just swings back and forth inside the cavity. Such small fluctuations in position correspond to the small protrusions, especially at the angular part, in Figure 4G.

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

[0361] Figure 4I shows that cholesterol (down) starts with the center of the molecule placed inside the CD cavity, the head group slightly protruding outside the upper base, while the tail group slightly protruding outside the lower base. This complex maintains stability throughout the trajectory. Cholesterol never exits the cavity, never changes direction, and just swings back and forth inside the cavity. Such small fluctuations in position correspond to the small protrusions, especially at the angular part, in Figure 4G.

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

[0363] In Figure 4L, 7KC starts with the molecule's center placed inside the CD cavity, the head group slightly protruding outside the lower base, while the tail group slightly protruding outside the upper base. The complex is stable until about 710 ns, but at about 710 ns, 7KC exits from the lower base, rotates, and associates parallel to the lower base. Then, at 715 ns, 7KC completely rotates and inserts the head group, the head group extends towards the upper base, and the tail extends outside the lower base. Next, 7KC associates with and dissociates from the CD cavity several times with the head group, and finally, at about 850 ns, the complex completely separates. The complex remains dissociated for the rest of the trajectory.

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

[0365] Figure 4L shows that cholesterol starts in a state where it is associated with CD, with the head group extending outside the lower base while the tail group extends from the upper base. At about 120 ns, the complex dissociates, at which time cholesterol moves to the upper base of CD, inserts the head group, and rotates inside and outside the cavity at the upper base, but finally completely dissociates again at about 160 ns. At about 163 ns, cholesterol reassociates with the lower base, but finally rotates back to the upper base 5 ns later. Then, cholesterol moves back and forth between association with the lower or upper base and random movement, and just at the very end of the trajectory, finally, in the last 3 nanoseconds, the complex is somewhat reformed. This continuous formation and deformation on the computer indicates that this has a strong tendency to form in reality.

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

[0367] Figure 4L shows that 7KC starts with the head group extending outside the upper base in the down position. At 40 ns, 7KC retreats, exits the cavity, meets parallel to the lower base, re-inserts the head group 2 ns later, and then retreats and exits again. At 45 ns, the complex completely separates. At this point, 7KC floats around the simulation box, meets the upper base again at 47 ns, temporarily inserts the head group, then rotates back parallel to the upper base, and finally the complex separates again at 51 ns. At 210 ns, the complex reforms, with the head group inserted from the lower base and the tail extending out as in the initial configuration. This complex remains stable until 268 ns, but at 268 ns, 7KC retreats again, exits the CD, and meets parallel to the lower base. The complex completely separates again but reforms temporarily at 360 ns. After that, 7KC sometimes meets parallel to one of the two planes with a higher-order structure similar to that at 710 ns but does not enter the CD cavity again. This trajectory is somewhat unclear because 7KC only meets the cavity for 100 ns, but the complex is still freely formed in the simulation. This indicates that the complex is likely to form in reality even though the interaction forces do not seem very consistent.

[0368] Translated native monomeric βCD and cholesterol, down direction, GROMOS force field:

[0369] In Figure 4L, cholesterol starts with the head group associated with the upper base and the tail extending outside the lower base. Cholesterol oscillates laterally within the cavity, but finally the complex separates at about 15 ns. At 17 ns, cholesterol re-inserts the head group and continues to rotate between a state parallel to the lower base of the CD and a state of inserting (always with the head group) into the cavity from the lower base until about 675 ns, when finally the complex completely dissociates. This shows that cholesterol has a strong interaction and tendency to form an apparently stable complex with native βCD, but once the complex completely dissociates from the lower base of the CD at 675 ns, it does not associate again.

[0370] Parallel-translated native monomeric βCD and 7KC, upward direction, AMBER force field:

[0371] In Fig. 4O, 7KC starts with its head group extending outside the lower base and its tail extending outside the upper base. This complex maintains stability throughout the trajectory, but 7KC exhibits more extreme bending than that seen in the downward direction. 7KC remains bent around the CD ring for a significant portion of the trajectory.

[0372] Parallel-translated native monomeric βCD and cholesterol, upward direction, AMBER force field:

[0373] Fig. 4O shows how cholesterol starts with its head group extending outside the lower base and its tail extending outside the upper base. This complex maintains stability throughout the trajectory. Cholesterol moves significantly back and forth within the cavity, but the angle inside the cavity remains relatively constant.

[0374] Parallel-translated native monomeric βCD and 7KC, downward direction, AMBER force field:

[0375] As seen in Fig. 4O, 7KC starts with its head group extending outside the upper base and its tail extending outside the lower base. This complex maintains stability throughout the trajectory, and 7KC shows no significant movement inside the CD cavity, as is evident from the horizontal and constant graph in Fig. 4M.

[0376] Parallel-translated native monomeric βCD and cholesterol, downward direction, AMBER force field:

[0377] Fig. 4O shows how cholesterol starts with its head group extending outside the upper base and its tail extending outside the lower base. This complex maintains stability throughout the trajectory, and cholesterol shows no significant movement inside the CD cavity, as is evident from the horizontal and constant graph in Fig. 4M.

[0378] Monomeric hydroxypropyl-β-CD and 7KC, upward direction, GROMOS force field:

[0379] 7KC in the up position (Figure 4R) starts with its tail inside the cavity of HPβCD and its head extended outside the bottom. At about 13 ns, 7KC rotates out from the bottom and associates parallel to the bottom. At 28 ns, the head group of 7KC rejoins the cavity but then rotates and returns outside several times. Up to about 47 ns, 7KC remains associated parallel to the bottom, but at about 47 ns the complex finally completely dissociates. Then, 7KC alternates 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-β-CD and cholesterol, upward direction, GROMOS force field:

[0381] In Figure 4R, cholesterol starts with its tail inserted into the CD cavity and its head group extended outside the bottom. This complex is stable up to about 3 ns, but at about 3 ns cholesterol finally rotates out from the bottom and becomes parallel to the CD. Then, by 7 ns, it rotates up to the top. Then, cholesterol moves randomly around the simulation box and sometimes associates parallel to either the top or the bottom, but never stabilizes inside the cavity except for a short time at about 300 ns. This lack of strong association is evident from the drastic fluctuations in Figure 4P and is supported by experimental evidence.

[0382] Monomeric hydroxypropyl-β-CD and 7KC, downward direction, GROMOS force field:

[0383] Figure 4R shows that 7KC starts somewhat outside the monomeric cavity and initially floats randomly around the simulation box. By 29 ns, 7KC associates the head group inside the cavity of HPβCD and extends the tail from the bottom. This remains stable until 35 ns, but at 35 ns the complex completely dissociates. The complex remains dissociated until 320 ns, but at 320 ns the complex finally reforms, with the head group again inside the cavity and the tail extending out from the bottom. The complex maintains association until about 470 ns, but finally dissociates again at about 470 ns and remains dissociated until the end of the trajectory.

[0384] Monomeric hydroxypropyl βCD and cholesterol, downward direction, GROMOS force field:

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

[0386] Monomeric hydroxypropyl βCD and 7KC, upward direction, AMBER force field:

[0387] 7KC (up) starts with the center of the molecule inside the CD cavity, the head group slightly protruding outside the lower base, while the tail group slightly protruding outside the upper base. Figure 4U shows how 7KC remains inside the cavity of HPβCD throughout the trajectory. 7KC moves slightly up and down but never exits far outside the cavity from either end. This complex does not separate.

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

[0389] The AMBER force field shows much more consistent interactions and much more stable complexes for both native and HPβCD than the GROMOS force field. In Figure 4U, cholesterol (up) starts with the center of the molecule inside the CD cavity, the head group slightly protruding outside the lower base, while the tail group slightly protruding outside the upper base. This complex maintains stability throughout the trajectory. Cholesterol never exits the cavity, never changes direction, and just sways back and forth inside the cavity. The most favorable higher-order structure occurs at 500 - 700 ns as seen in Figure 4S, but cholesterol and CD remain complexed throughout the entire trajectory. These small fluctuations in position correspond to the small bulges in Figure 4S, especially at the angular parts.

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

[0391] 7KC (down) starts with the center of the molecule inside the CD cavity, the head group slightly protruding outside the upper base, while the tail group slightly protruding outside the lower base. Figure 4U shows how much larger the head of 7KC protrudes from the cavity compared to the up direction, and also shows that the complex remains intact throughout the trajectory. This preference for the up direction is visually recognized in Figure 4S, where the "up" plot is much less variable than the "down" plot, but both are still significantly less variable than HPβCD with GROMOS.

[0392] Monomeric hydroxypropyl β-CD and cholesterol, down direction, AMBER force field:

[0393] Figure 4U shows that cholesterol (down) starts with the cholesterol inside the CD cavity, stretching the head group outside the upper base while stretching the tail group slightly outside the lower base. Noticeable is that the head group of cholesterol sometimes protrudes significantly more outside the cavity than in the case of the up direction, yet this complex still maintains stability throughout the trajectory. Cholesterol never completely exits the cavity and never changes direction. These small fluctuations in position correspond to the small bulges in Figure 4S, especially in the angular part. There are larger lateral movements and smaller radial rocking motions through the CD cavity that are visible compared to other complexes.

[0394] Translated monomeric hydroxypropyl β-CD and 7KC, up direction, GROMOS force field:

[0395] Figure 4X shows that the translated up-direction 7KC starts with the tail inserted into the CD cavity and the head group stretched outside the lower base. At about 105 ns, 7KC rotates and exits the cavity, then 7KC repeatedly swings like a pendulum between a state with the head group inserted into the CD and a state parallel to the CD about every 5 - 10 seconds, and it seems that the time spent in the higher-order structure with the head group inside the cavity is longer. At about 415 ns, the structure stabilizes and the head group is inserted, but at 700 ns the structure finally separates and dissociates completely. Thereafter, the complex remains dissociated for the rest of the trajectory except for a temporary re-association at 726 ns, and at 726 ns the head group of 7KC is inserted into the large face of the CD. The interaction energy here is, briefly speaking, comparable to that at 400 ns when the complex was formed. Since the complex seems to be able to form and separate easily, this interaction is realistic, strong, and highly likely to be captured by the simulation.

[0396] Translated monomeric hydroxypropyl βCD and cholesterol, upward direction, GROMOS force field:

[0397] Cholesterol starts with its head group inserted into the cavity and its tail extending outside the upper base. This complex remains stable for 60 ns, but then finally cholesterol rotates and exits from the lower base and associates parallel to the CD. Subsequently, cholesterol completely exits the CD and moves randomly around the simulation box, but finally at about 215 ns, it reunites its tail with the cavity of the CD and extends its head group from the lower base again. This is stable for about 30 ns, but then finally cholesterol exits the CD again, and then at 280 ns, it quickly reunites its head group with the cavity of the CD. At this time, the head group is inside the cavity and the tail extends from the lower base. This complex maintains its stability for the remainder of the trajectory. This indicates that the complex formed at the end of the trajectory, as seen in Figure 4X, is very stable and highly likely to form.

[0398] Translated monomeric hydroxypropyl βCD and 7KC, downward direction, GROMOS force field:

[0399] Translated downward 7KC starts with its head group inserted into the CD cavity and its tail extending outside the lower base. At about 105 ns, 7KC rotates and exits the cavity, and then 7KC repeats like a pendulum between the state with its head group inserted into the CD and the state parallel to the CD at about 5 - 10 ns intervals. It seems that the time spent in the higher-order structure with the head group inside the cavity is longer. At about 415 ns, the structure stabilizes and the head group is inserted, but finally at 700 ns, the structure separates again and completely dissociates. Subsequently, the complex remains dissociated for the remainder of the trajectory, as seen in Figure 4X.

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

[0401] For the case of cholesterol in the downward direction translated parallel to HPβCD, cholesterol starts with its tail inserted into the CD cavity and its head group extended outside the upper base. At 50 ns, the complex separates, but cholesterol maintains its association with the upper base, with its tail periodically entering and exiting the cavity, and then completely dissociates at 88 ns. The cholesterol molecule then associates with the lower base of the CD and then resumes random movement around the simulation box. The trajectory periodically alternates between association with one of the two faces and random movement, but finally, at 215 ns, and for the next 25 ns, the cholesterol tail re-enters the cavity from the upper base. Cholesterol then resumes random movement around the CD. At 275 nanoseconds, the cholesterol head group enters the cavity from the upper base and stays there, but finally the complex completely dissociates at about 410 ns. From this point until about 490 ns, cholesterol moves randomly around the simulation box, but at about 490 ns, cholesterol rotates towards the lower base and inserts its head group into the cavity. Until about 530 ns, the complex maintains this higher-order structure, but at about 530 ns, cholesterol moves out of the cavity, rotates, and then reinserts and returns its tail group from the lower base into the cavity. By 540 ns, cholesterol has resumed random movement. Cholesterol never re-enters the cavity again, but frequently associates closely with one of the faces of the CD. As is evident in Figure 4X, cholesterol never forms a stable complex with HPβCD no matter how long the time, so the interaction between HPβCD and cholesterol seems to be transient, even at the translated position, and not as strong as the interaction between HPβCD and 7KC.

[0402] Translated monomeric hydroxypropyl βCD and 7KC, upward direction, AMBER force field:

[0403] Figure 4AA shows that 7KC starts with the head group extending outside the upper base and the tail facing outward at the lower base, and the center of 7KC is located in the cavity of CD. This complex maintains stability throughout the trajectory, and 7KC shows no significant movement inside the CD cavity, as is evident from the flat graph in Figure 4Y.

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

[0405] Figure 4AA shows that cholesterol starts with the head group extending outside the upper base and the tail facing outward at the lower base, and the center of cholesterol is located in the cavity of CD. This complex maintains stability throughout the trajectory, and cholesterol shows no significant movement or displacement inside the CD cavity, as is evident from the flat graph in Figure 4Y.

[0406] Translated monomeric hydroxypropyl βCD and 7KC, downward direction, AMBER force field:

[0407] Figure 4AA shows that 7KC starts with the head group significantly extending outside the upper base and the tail directed outward from the lower base, but the tail is completely inside the cavity. This complex maintains stability throughout the trajectory, and 7KC shows no significant movement inside the CD cavity, as is evident from the flat graph in Figure 4Y. 7KC shows a greater lateral movement in this direction than in the upward direction.

[0408] Translated monomeric hydroxypropyl βCD and cholesterol, downward direction, AMBER force field:

[0409] Figure 4AA shows that cholesterol starts with the head group extending outside the upper base and the tail facing outward at the lower base, and the center of cholesterol is located in the cavity of the CD. This complex maintains stability throughout the trajectory, but cholesterol moves significantly inside the cavity, and often only the tails associate while the head groups protrude outside the CD. This can be seen in Figure 4Y, especially in terms of distance, where the variation in the downward direction is greater than that in the upward direction.

[0410] Dimerized hydroxypropyl βCD and 7KC, upward direction, GROMOS force field:

[0411] In Figure 4DD, 7KC starts inside the dimer and is well-confined. At about 100 ns, the dimer begins to stretch and contract, but 7KC remains inside the barrel structure between the two CDs despite this stretching and contracting. At 111 ns, the head groups dissociate from the monomers that were associated (in this context, the term "monomer" refers to the CD subunit regardless of whether it is part of a covalently linked dimer), while the tails remain associated with the cavity of the other monomer. After 5 ns, the head groups of 7KC progress to interact with the large face (not the cavity) of one monomer, while one tail remains tethered to the other. At 120 ns, the tail is released from the associated monomer, and the head groups enter the cavity of the other monomer. This conformation maintains stability, and the sterol-associated monomer oscillates around the empty monomer until the end of the trajectory.

[0412] Dimerized hydroxypropyl βCD and cholesterol, upward direction, GROMOS force field:

[0413] The trajectory of cholesterol (up) starts with cholesterol being encapsulated in the dimer. At about 22 ns, the dimer starts to move, and cholesterol moves with it and remains inside the dimer cavity. At about 200 ns, the monomer associated with the head group of cholesterol separates and dissociates from the dimer, but cholesterol remains associated with one of the monomers (the head group is aligned with the bottom and the tail is aligned with the top). This configuration is maintained, but at 355 ns, cholesterol finally completely dissociates from the cavity and rotates towards the bottom. Then, cholesterol sometimes continues to be between the two monomers while the head group loosely associates with one of the monomers, but finally completely separates and floats around the simulation box. As seen in Figure 4DD, cholesterol continues to interact intermittently with one of the CD monomers, but the dimer-cholesterol complex is never completely reformed.

[0414] Dimerized hydroxypropyl-β-CD and 7KC, down direction, GROMOS force field:

[0415] 7KC in the down position starts in a state confined inside the dimer as shown in Figure 4DD. The dimer does not start to deform until about 600 ns, and at about 600 ns, one monomer elongates and separates from the other, but 7KC continues to be between the two. At about 820 ns, 7KC dissociates the tail from one of the monomers, but the head group continues to be in the cavity of the other monomer. This conformation remains stable, and until the end of the trajectory, the sterol-associated monomer oscillates around the empty monomer.

[0416] Dimerized hydroxypropyl-β-CD and cholesterol, down direction, GROMOS force field:

[0417] Cholesterol in the down position (Figure 4DD) starts in a state inside the dimer cage. At about 50 ns, the complex begins to stretch, contract, and twist, but the cholesterol remains tethered inside the dimer throughout the trajectory. This is evident from the plot of the cholesterol angle in Figure 4BB being very flat and constant throughout the trajectory. This is the only complex that maintained intact throughout the trajectory in the GROMOS force field. Figure 4BB shows the molecular dynamics analysis of our novel butyl-linked hydroxypropyl β-cyclodextrin dimer (DS5) that forms a very stable complex with 7KC and cholesterol. The comparison of these graphs with those of the monomeric HPβCD provides clear evidence that the dimerized version consistently binds the sterol significantly more reliably than its monomeric counterpart. In the down direction, the energy, angle, and distance all remain very consistent with only minimal fluctuations, indicating that there is a stable apparent solubilized complex for both 7KC and cholesterol, and this complex does not change significantly over time. The same can be seen in the up direction, but there is some more variability, especially for cholesterol. This suggests that 7KC binds most effectively in the down direction and has a strong preference for the down direction as seen by the angle inversion at about 350 ns in the up direction. The angle inversion at about 350 ns is where 7KC exits the dimer and re-associates in the down direction. Cholesterol is similar, but the complex is not as stable as that formed with 7KC, indicating that while 7KC can form a more stable down complex from a less stable up complex, cholesterol does not have the same ability as 7KC.

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

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

[0420] Dimerized hydroxypropyl βCD and cholesterol, upward direction, AMBER force field:

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

[0422] Dimerized hydroxypropyl βCD and 7KC, downward direction, AMBER force field:

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

[0424] Dimerized hydroxypropyl βCD and cholesterol, downward direction, AMBER force field:

[0425] Figure 4GG shows that cholesterol remains located inside the cavity formed by two monomers throughout the trajectory. The complex shows some movement, and cholesterol moves slightly inside the cavity, but cholesterol remains complexed with the CD dimer throughout the trajectory.

[0426] Translated dimerized hydroxypropyl βCD and 7KC, upward direction, GROMOS force field:

[0427] Figure 4JJ shows that the dimerization complex starts with the parallel-translated up-direction 7KC fitting snugly into the cavities of both CD monomers. At about 140 ns, the complex elongates and causes the first fluctuation at this point in Figure 4GG, but quickly recovers. The complex continues to periodically repeat stretching and deformation as seen from the fluctuations in Figure 4GG, but 7KC remains inside both cavities and finally releases its tail from the monomer at about 700 ns. 7KC does not re-enter both cavities simultaneously for the remainder of the trajectory.

[0428] Parallel-translated dimerized hydroxypropyl-βCD and cholesterol, up direction, GROMOS force field:

[0429] Figure 4JJ shows that the parallel-translated up-direction cholesterol starts in a dimer and complex-formed state, and at about 100 ns, the complex starts 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 that was associated with the head of cholesterol through the lower base completely rotates 180 degrees with respect to the opposite base of the second monomer, and the somewhat deformed lower base (associated with cholesterol) associates with the somewhat deformed upper base of the second monomer, thereby generating a somewhat deformed head-tail dimer. This head-tail dimer never fully forms a complex with cholesterol, however, the head group of cholesterol maintains its association with the monomer it was initially associated with. This is the only trajectory that produces a head-tail dimer, and this conformation does not seem to effectively complex with cholesterol.

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

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

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

[0433] Cholesterol at the translated-down position forms a complex with the CD dimer for about 162 ns. At this point, the dimerized complex starts to stretch and deform, and then at 190 ns, the head group of cholesterol detaches from its monomer. By 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, but dissociates completely at 320 ns. As seen in Figure 4JJ, cholesterol does not re-enter either cavity for the rest of the trajectory, and the dimerized complex is never completely reformed, but cholesterol occasionally associates with the bottom of one monomer via the head group, similar to the conformation at 640 ns.

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

[0435] Figure 4MM shows in detail how 7KC remains located inside the cavity formed by the two monomers throughout the trajectory. The complex shows some movement around 7KC, but 7KC maintains approximately the same position throughout the trajectory.

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

[0437] Figure 4MM details how cholesterol continues to be located inside the cavity formed by two monomers throughout the trajectory. The complex and cholesterol move somewhat between trajectories, and in particular, the monomers associated with the cholesterol head move, but cholesterol never completely dissociates from any monomer. Cholesterol forms a complex with the CD dimer throughout the trajectory.

[0438] Translated dimeric hydroxypropyl-β-CD and 7KC, downward direction, AMBER force field:

[0439] Figure 4MM details how 7KC continues to be located inside the cavity formed by two monomers throughout the trajectory. The complex shows some movement around 7KC, but 7KC maintains almost exactly the same position throughout the trajectory. 7KC forms a complex with the CD dimer throughout the trajectory.

[0440] Translated dimeric hydroxypropyl-β-CD and cholesterol, downward direction, AMBER force field:

[0441] Figure 4MM details how cholesterol continues to be located inside the cavity formed by two monomers throughout the trajectory. The complex shows some movement around cholesterol, but cholesterol maintains almost exactly the same position throughout the trajectory. Cholesterol forms a complex with the CD dimer throughout the trajectory.

[0442] Furthermore, brief analyses were performed on both βCD dimers of DS0 with butyl linkers and triazole linkers (Figs. 4NN - 4QQ) as well as the hydroxypropyl dimer with a triazole linker (Figs. 4RR - 4SS). Simulations of DS0 indicate that while the triazole linker somewhat destabilizes the complex, it enables some additional specificity for 7KC. Slightly different yet still strong and favorable interactions are a good omen for both types of linkers.

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

[0444] Further MD analysis Further, shortened MD analyses were also performed on triazole - linked as well as butyl - linked methyl βCD, sulfobutyl βCD, and quaternary ammonium βCD. These were all DS4 (Figs. 5B - 5C, Figs. 6B - 6C, Figs. 7A - 7B). The methyl dimer with the butyl linker formed the most stable complex and seemed to prefer the up direction in both cases of linkers, but the interactions were very similar for the two methyl dimers tested. It is difficult to distinguish which is more specifically effective regarding the methyl substituents, but both linkers readily form complexes with both ligands. From the trajectories, it became clear that the head group of 7KC is not completely inside the cavity of the dimer but is stably maintained between the two sister monomers. The complex with 7KC in the down direction maintained association for about 50 ns, after which 7KC moved out of the cavity and only the head group maintained association with one monomer for the rest of the trajectory.

[0445] The negatively charged sulfobutyl dimer shows a similar pattern to the methyl and hydroxypropyl dimers. In this case, the triazole linker creates a slightly unstable complex but enables 7KC specificity. The charged and bulky sulfobutyl group appears to interact very favorably with both 7KC and cholesterol, but in both linker cases, the only separated complex is that of cholesterol. This indicates that the sulfobutyl dimer is likely to have very good specificity for 7KC compared to methyl and hydroxypropyl.

[0446] To further evaluate the use of charged substituents, MD simulations of the positively charged quaternary ammonium βCD of DS4 were performed. These trajectories revealed a strong binding between QAβCD and sterols as no released sterols were present 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 implies that the QAβCD of DS4 is well - suited to bind and solubilize sterols, much like other types of substituents.

[0447] In the last MD simulation, 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 maintained complex formation throughout all 100 ns. The interaction energy is slightly reduced in magnitude for the O - linked case compared to the butyl linkage, but the overall specificity appears to be better for linker O. This is because both cholesterol complexes dissociated by 100 ns. The interactions are similar to those of the butyl - linked dimer, but they appear to give slightly better 7KC specificity. This is clearly due to the nitrogen of the linker interacting with the carbonyl of 7KC.

[0448] Further docking screening

[0449] By docking simulation, many different candidate molecules can be rapidly modeled without the need for their synthesis. For this reason, using these docking techniques, a "screening" of many different substituent types, linker types, number of substitutions, and substitution positions was performed (Figs. 8 - 9). This screening allows one to determine whether a particular modification improves or worsens the specificity for 7KC.

[0450] Fig. 8E illustrates the evaluation of how the HPβCD dimers of the present inventors are affected by changes in the linker composition and attachment point, the site of hydroxypropylation, the linker length, and the chemical composition of the linker. Since the linker attachment site is not easily controlled during the chemical synthesis of cyclodextrin, it was tested by computer calculations. Docking calculations were performed for various sites of hydroxypropylation (Fig. 8A), carbon-only linkers of various lengths (chain lengths of 2 - 8 carbons, Fig. 8B) and triazole linkers (varying the n1 and n2 valences surrounding the triazole ring, Fig. 8C), as well as different attachment points to the O2 and / or O3 oxygen(s) of the dimerized HPβCD (Figs. 8F - 8G), and all different linker types (Fig. 9A). The results show that when the position of the hydroxypropyl group is changed, there is little effect on 7KC preference and a minimal effect on the overall sterol binding. The linker length of 3 - 5 carbons showed the greatest affinity and specificity for 7KC (Fig. 8B).

[0451] Various triazole linkers modeled with AutoDock are shown in Fig. 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 both sides of the ring are predicted to have the greatest affinity for 7KC.

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

[0453] The inventors also considered the fact that the linker can bind to the bottom of the cyclodextrin at either the C2 or C3 carbon. The inventors tested by molecular docking whether this affects the predicted affinity (FIG. 8F). The inventors also examined whether there are obvious differences in the affinity for sterols by linking with asymmetric linkers at various binding sites. These calculations show a tendency to bind to 7KC and cholesterol for all three possible linking sites that are present in approximately equal amounts in typical syntheses. These calculations show a tendency to bind to 7KC and cholesterol for all four possible linkages present in the synthesis of dimers linked by five different asymmetric linkers. Generally, no large difference was observed between the C2 and C3 binding sites.

[0454] The inventors' molecular modeling revealed differences in the level of 7KC specificity when the number of substituents is different. Of particular interest were the linked HPβCDs with three, four, or five hydroxypropyl groups, which showed the greatest 7KC specificity for any of the modeled butyl dimers (FIG. 4B). The inventors synthesized various butyl-linked and triazole-linked HPβCD dimers including DS about 3. Consistent with the inventors' prediction, HPβCD-butyl-DS3 and HPβCD-triazole-DS3 had higher 7KC specificity than cholesterol (FIGS. 16A - 16C).

[0455] When completing the docking analysis of hydroxypropyl CD dimers, docking against 7KC and cholesterol was performed on a variety of CD dimers with different linkers and varying degrees of substitution to examine how these factors affect 7KC and cholesterol binding (Figure 5A, Figure 6A, and Figure 9). Testing with butyl linkers and triazole linkers using methyl substituents and sulfobutyl substituents as DS1 - DS20 (Figure 5A, Figure 6A) led to results that were promising enough to prompt further molecular dynamics analysis and ultimately synthesis.

[0456] In Figures 5A and 6A, it has been observed that 7KC specificity is best at low DS (2 - 6) for both sulfobutyl and methyl substituents. MeβCD and SBβCD of DS4 behave most similarly to HPβCD of DS5, where 7KC is sufficiently solubilized but cholesterol is not. 7KC specificity appears to become less apparent with increasing DS for both linkers and all substituents. Since it appeared that approximately DS4 gave the maximum 7KC specificity for all the substituent types tested, only DS4 was tested for other linker types.

[0457] Substituents other than hydroxypropyl, methyl, or sulfobutyl were tested only at low DS using only butyl linkers, triazole linkers, linker O, and linker R (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 depends not on the type of linker or substituent, but on the number of substituents on the βCD ring. Although negative specificity was shown using a few substituent types and a few linker types, the average 7KC specificity was still well above zero for these 23 linkers and 7 substituent types at low DS (4).

[0458] By using molecular docking, it was possible to test how the length of the triazole linker or alkyl linker affects the 7KC specificity of cyclodextrin dimers having hydroxypropyl, methyl, and sulfobutyl substituents (Figs. 9B to 9C). It was shown that the specificity decreases as the linker length increases. Without wishing to be bound by theory, it is thought that the longer the linker length, the more the CD subunits can be separated at a longer distance, and thus the time spent in the higher-order structure that can effectively enclose a molecule of the size of 7KC or cholesterol is shortened. Based on these results, the inventors conclude that a dimer having a linker length that allows the guest (7KC or cholesterol) to fit into two CD subunits, for example, a linker length of 7 or fewer atoms, will exhibit better solubilization of the molecule.

[0459] The inventors also tested whether the specificity of the CD dimer for 7KC depends on the substitution position by creating a large number of different substitution patterns using sulfobutyl, hydroxypropyl, and methyl substituents, as well as combinations of these three (Figs. 9D - 9E). It was found that the 7KC specificity was largely maintained when the DS was approximately 4, whether a single substituent type or multiple substituent types were present on a single CD dimer. The type and position of these substituents did not significantly affect the 7KC specificity. The results of the docking simulations suggest that although the composition of both the linker and the substituent affects how well it can solubilize the guest for a given CD, the degree of specificity for 7KC most depends on the number of substituents on the CD ring. As seen in Figs. 4B and 5A - 5B, the butyl-linked dimer showed the highest 7KC specificity with a DS of approximately 2 - 5 for methyl, sulfobutyl, and hydroxypropyl substituents. This also holds true for the triazole linker, supporting the idea that multiple linker and substituent types may show similar specificity for 7KC at a substitution degree of 2 - 5. Furthermore, a wide range of substitution patterns / combinations consisting of 23 different linkers and 14 different substituents were docked to determine whether the linker or substituent pattern has an impact on 7KC specificity (Fig. 9A). Both of these analyses showed a change in the degree of 7KC specificity, but the average specificity still remained well above zero.

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

[0461] Since the methyl group, sulfobutyl group, and hydroxypropyl group are all very different from each other and the range of linkers tested included significant diversity, the inventors consider it not unreasonable to think that other substituent species with a linker length similar to that of the sterol guest will behave similarly to the butyl-linked CD dimer with a hydroxypropyl group. Although the type of substituent and linker may have some influence on other properties such as solubility and toxicity, the specificity for 7KC is predicted to be present similarly in other molecules of this class.

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

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

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

[0465] For DS measurement, 1H and 2D NMR spectra were recorded on a Varian VXR-600 at 600 MHz using the residual solvent signal as the internal reference. To elucidate the structure, the sample was dissolved in DMSO-d6 / D2O. At least 16 scans were performed to record the FID signal so as to obtain a spectral window covering at least 0 ppm to +10 ppm. The calculation of the degree of substitution (DS) can be achieved by setting the integral value of the anomeric region to 14 (14 is the number of anomeric protons of the β-cyclodextrin dimer) and dividing the integral value of the alkyl region by 3 (see Figure 10J).

[0466] General description of synthesis and characterization

[0467] HP(βCD-but-βCD)

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

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

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

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

[0472] Hydroxypropylation of the βCD dimer was achieved by using sodium hydroxide as the base in aqueous conditions at room temperature. Purification of the hydroxypropyl-βCD dimer (HP(βCD-but-βCD)) utilized ion exchange resin treatment, activated carbon clarification, and large-scale dialysis. The identity and structure of the product were confirmed by MALDI analysis and TLC analysis of the compound (Figures 10G - 10N).

[0473] HP(βCD-triazole-βCD)

[0474] The preparation of a hydroxypropyl-β-cyclodextrin dimer connected through one triazole moiety across the bottom 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) as this reagent is not commercially available. The second step is to prepare two βCD monomers, 2-O-prop-2-ynyl-β-CD and 2-O-(3-azidopropyl)-βCD respectively. The third step of the synthesis is the construction of the dimer core by copper-catalyzed azide-alkyne cycloaddition, and the final step is the preparation of a series of 2-hydroxypropyl triazole-linked dimers by a classical alkylation approach.

[0475] Specifically, 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. 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 the selective base for deprotecting the lower rim. Specifically, this approach activates only the hydroxyl group located at C2. As a result, the monomers prepared by this method are substituted only with O2 (these are a single type of isomer). The two monomers are characterized by NMR spectroscopy, MALDI, and TLC (Figures 10S - 10U).

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

[0478] The 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 - 10Q).

[0479] Detailed description of the synthesis (HP(βCD-but-βCD))

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

[0481] Under an inert atmosphere, dry TBDMS-βCD (10 g, 5.17 mmol) was dissolved in THF (400 mL), and sodium hydride (2.5 g, 50 mmol) was added slowly (over 30 minutes) with caution. The addition of sodium hydride caused the formation of hydrogen, and the suspension foamed vigorously. After stirring for 15 minutes, the reaction mixture gelled, making stirring difficult. For the purpose of breaking the gel, the reaction mixture was heated until gentle reflux occurred and reflux was maintained for 30 minutes. The yellowish heterogeneous suspension became more stirrable than before, and the gel-like structure disappeared. The reaction mixture was cooled to room temperature in a water bath. When the alkylating agent 1,4-dibromobutane (1.25 mL, 2.25 g, 10.5 mmol) was added dropwise (15 minutes), the color of the reaction mixture turned dark orange.

[0482] The brownish suspension was stirred overnight under an inert atmosphere. Since the conversion rate was estimated by TLC to be 10 - 15% (eluent: chloroform: methanol: water = 50:10:1, v / v / v, see Figure 10C), it was judged that post-treatment could be carried out.

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

[0484] The crude reaction product was purified by chromatography, and based on TLC analysis (Figure 10C), the fractions containing the product were collected and evaporated under reduced pressure until dry to obtain a white substance. This was dried in a drying box in the presence of KOH and P2O5 until a constant weight was obtained (TBDMS-βCD-but-βCD-TBDMS, 3.5 g).

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

[0486] Under an inert atmosphere, dry TBDMS-βCD-but-βCD-TBDMS (3.5 g, 0.89 mmol) was dissolved in THF (250 mL). To the yellowish solution, tetrabutylammonium fluoride (8.75 g, 33.47 mmol) was added all at once. After stirring at room temperature for 30 minutes, the color of the reaction mixture turned dark green. The reaction mixture was stirred overnight at room temperature. 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 gently until refluxing and refluxed for 2 hours. At this stage, the reaction conversion was complete as no starting material was detectable by TLC. The reaction mixture was cooled to room temperature and concentrated under reduced pressure (to approximately 10 mL), methanol (200 mL) was added, and a white precipitate was obtained. The solid was filtered off, analyzed by TLC, and dried in a drying box in the presence of KOH and P2O5 until a constant weight was achieved (1.2 g). TLC analysis showed that this substance contained a negligible (≤3%) amount of tetrabutylammonium fluoride. The mother liquor was concentrated under reduced pressure (to approximately 10 mL), purified by chromatography (eluent: 1,4-dioxane:NH3 = 10:7 v / v), the fractions containing the product were collected, evaporated to dryness under reduced pressure, and a white substance was obtained. This was dried in a drying box in the presence of KOH and P2O5 until a constant weight was achieved (βCD-but-βCD, 0.55 g).

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

[0488] β-CD-but-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 turned into 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 all at once. The reaction vessel was flushed with argon, sealed, and stirred at room temperature for 2 days. The reaction mixture was concentrated under reduced pressure until it became a viscous syrup, and acetone (50 mL) was added thereto for precipitation. The white solid was filtered through a sintered glass filter and washed thoroughly with acetone (3 × 15 mL). This substance was dissolved in water (50 mL), treated with an ion-exchange resin (to remove salts), clarified with activated carbon, membrane-filtered, and dialyzed against purified water for 1 day. The residue was evaporated to dryness under reduced pressure to obtain a white solid (0.8 g).

[0489] Detailed description of synthesis (HP(βCD-triazole-βCD))

[0490] Step 1: Preparation of azide-linker

[0491] While stirring vigorously, dissolve 1,3-dibromopropane (10 mL, 20.18 g, 0.1 mol) in 40 mL of DMSO. Prepare a solution of sodium azide (6.7 g, 0.1 mol) in DMSO (240 mL) and add this dropwise (over 2 hours) to the dihalopropane solution. Stir the solution overnight at room temperature. Then extract the crude reaction product with n-hexane (3 × 100 mL), back-extract the collected n-hexane phase with water (3 × 50 mL), and carefully evaporate the organic phase under reduced pressure (strictly at 40 °C, 400 mbar; otherwise, the target compound may be distilled off). Purify the oily residue by chromatography (eluent: n-hexane-EtAc = 98:2, isocratic solvent elution). Collect the appropriate fractions, concentrate under reduced pressure, and obtain the target compound as a viscous oil (this can be stored in a light-protected refrigerated container under an inert atmosphere). Visualize the compound by dipping the TLC plate in a dichloromethane solution of triphenylphosphine (10%) for about 15 seconds, drying the TLC plate below 60 °C, dipping the TLC plate in a ninhydrin ethanol solution (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-propynyl-βCD

[0493] To a solution of β-cyclodextrin (20 g, 17.62 mmol) in dry DMSO (300 mL) is added lithium hydride (212 mg, 26.432 mmol). The resulting suspension is stirred at room temperature under N2 until it becomes clear (12 - 24 hours). Then, propargyl bromide (1.964 mL, 17.62 mmol) and a catalytic amount of lithium iodide (about 20 mg) are added, and the mixture is stirred at 55 °C for 5 hours under light protection. When the product is characterized using TLC (10:5:2 CH3CN:H2O:25% v / v aqueous NH3 solution), spots corresponding to monopropargylated and non-propargylated β-cyclodextrin are seen respectively. The solution is poured into acetone (3.2 L), the precipitate is filtered off 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. This crude mixture is placed on top of a silica column (25 × 6 cm) and chromatography is carried out (10:5:2 CH3CN:H2O:25% v / v aqueous NH3 solution), and after lyophilization, 2-O-propargyl-β-CD is obtained as a solid. 2-O-propargyl-β-CD is analyzed by MALDI and NMR (Figures 10T and 10U).

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

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

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

[0497] While stirring vigorously, dissolve 2-O-prop-2-ynyl-β-CD and 2-O-(3-azidopropyl)-β-CD in water (300 mL) (each at a concentration of about 8 - 12 mM). To completely dissolve the heterogeneous mixture, add dimethylformamide (DMF) (about 300 mL) to this suspension (the addition of DMF is a slightly exothermic process). Add copper(II) bromide (2 g, 13.49 mmol) to the solution. Stir the suspension at room temperature for 1 hour. The reaction is monitored by TLC and is expected to react in about 1 hour (eluent: CH3CN:H2O:NH3 = 10:5:2). Filter the crude reaction product and concentrate the mother liquor under reduced pressure (60 °C). Dilute the gel-like substance with water and add silica (15 g). Concentrate the heterogeneous mixture under reduced pressure until dry. Load this crude mixture onto the top of a silica column and perform chromatography (10:5:2 CH3CN:H2O:25% v / v aqueous NH3 solution), and after drying, obtain the BCD-(triazole)1-BCD dimer. The preparation of the BCD-(triazole)1-BCD dimer was characterized by NMR (Figure 10V).

[0498] Step 4: HP(βCD-triazole-βCD)

[0499] β-CD-(triazole)1-β-CD dimer, which may be obtained by the above steps 1 to 3 or by other methods. This (1 g, 0.418 mmol) 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. The mixture became 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 all at once. 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, and the viscous syrup 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 substance was dissolved in water (50 mL), treated with an ion exchange resin (for the purpose of removing salts), clarified with activated carbon, membrane filtered, and dialyzed against purified water for 1 day. The residue was evaporated to dryness under reduced pressure to obtain a white solid (0.8 g). The HP(βCD-triazole-βCD) products were analyzed by NMR (Figures 10W, 10X, and 10Y), and their degrees of substitution were calculated as shown in the figures respectively.

[0500] Example 4. Synthesis of Methyl-Substituted Cyclodextrin Dimer

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

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

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

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

[0505] Synthesis

[0506] While stirring vigorously, βCD-(triazole)1-βCD dimer nucleus (1.1 g, 0.46 mmol) was suspended in deionized H2O (100 mL), and sodium hydroxide (0.35 g, 8.8 mmol) was added. The resulting slightly yellow suspension was stirred for 30 minutes until completely dissolved. When the temperature of the yellowish transparent solution stabilized at about 20 °C, methyl iodide (0.5 mL, 1.14 g, 8.03 mmol) was added all at once while stirring vigorously (Note: Methyl iodide is immiscible with the reaction mixture, so it was stirred vigorously for efficiency). The reaction mixture was stirred at room temperature for 24 hours and then treated with ion exchange resin (H+ resin (6 g) and OH− (6 g) resin were added to the solution, stirred for 15 minutes, and filtered off (the resin was washed 3 times with 15 mL of deionized water each)). The resulting filtrate (final pH = 7) was clarified with activated carbon (while stirring vigorously, activated carbon (0.2 g) was added to the solution, stirred for 30 minutes, and filtered off (the activated carbon pad was washed 3 times with 15 mL of deionized water each)). The colorless solution was evaporated under reduced pressure (40 °C), and the title compound was obtained as a white powder (about 1 g).

[0507] Characterization

[0508] The reaction process was monitored by TLC (Figure 11B), and the resulting substance was characterized by MALDI-TOF and NMR analysis as shown in Figures 11C to 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 (low DS SB)

[0514] While vigorously stirring, βCD-(triazole)1-βCD dimer nucleus (1.2 g, 0.5 mmol) was suspended in deionized H2O (60 mL). Sodium hydroxide (0.39 g, 9.75 mmol) was added to the mixture, and the resulting solution was heated to 60 °C. Butanesultone (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 hours. Then, to decompose the remaining butanesultone, the reaction was heated at 90 °C for an additional 1 hour. The reaction mixture was cooled and treated with ion exchange resins. To the solution, cation exchange resin (H+ resin, 2 g) and anion exchange resin (OH− resin, 2 g) were added, stirred for 15 minutes, and filtered off (the resins were washed 3 times with 15 mL of deionized water each). The resulting filtrate (final pH = 7) was clarified with activated carbon (while vigorously stirring, activated carbon (0.3 g) was added to the solution, stirred for 30 minutes, and filtered off (the activated carbon pad was washed 3 times with 15 mL of deionized water each)).

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

[0516] Characterization

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

[0518] Synthesis (high DS)

[0519] While stirring vigorously, a dimer nucleus of (βCD-(triazole)1-βCD) (1.2 g, 0.5 mmol) was suspended in deionized H2O (60 mL). Sodium hydroxide (1.22 g, 30.5 mmol) was added to the mixture, and the resulting solution was heated to 60 °C. Butanesultone (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 hours. Then, to decompose the remaining butanesultone, the reaction mixture was further heated at 90 °C for 1 hour. The reaction mixture was cooled and treated with ion exchange resin. To the solution, a cation exchange resin (H+ resin, 4 g) and an anion exchange resin (OH- resin, 4 g) were added, stirred for 15 minutes, and filtered off (the resin was washed three times with 15 mL of deionized water each). The resulting filtrate (final pH = 7) was clarified with activated carbon (while stirring vigorously, 0.5 g of activated carbon was added to the solution, stirred for 30 minutes, and filtered (the activated carbon pad was washed three times with 15 mL of deionized water each)). The colorless solution was evaporated under reduced pressure (40 °C) to obtain a white powder (1.51 g).

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

[0521] Example 6. Synthesis of Quaternary Ammonium Substituted Cyclodextrin Dimer

[0522] The molecules to be synthesized are illustrated in Figures 3I and 13G.

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

[0524] Quaternary Ammonium (βCD-(triazole)1-βCD) Dimer (Synthesis Example)

[0525] The preparation of the QA dimer was achieved in a one-step reaction (see Figure 13A). The βCD-(triazole)1-βCD dimer nucleus was prepared by the synthetic strategy described in Example 2 above.

[0526] Synthesis

[0527] While stirring vigorously, (BCD-(triazole)1-BCD) dimer nucleus (1.2 g, 0.5 mmol) was suspended in deionized H2O (100 mL), and sodium hydroxide (0.39 g, 9.8 mmol) was added. The resulting slightly yellow suspension was stirred for 30 minutes until completely dissolved. When the temperature of the yellowish transparent solution stabilized at 5 - 10 °C, glycidyltrimethylammonium chloride (1.17 mL, 1.32 g, 8.7 mmol) was added all at once while stirring vigorously. The reaction mixture was stirred at room temperature for 24 hours, and then, when the temperature of the solution stabilized at 5 - 10 °C, the second portion of glycidyltrimethylammonium chloride (0.4 mL, 0.45 g, 3 mmol) was added while stirring vigorously. The reaction mixture was heated at 50 °C for 3 hours, then cooled and treated with ion exchange resin (H+ resin (6 g) and OH- (6 g) resin were added to the solution, stirred for 15 minutes, and filtered off (the resin was washed 3 times with 15 mL of deionized water each)). The resulting filtrate (final pH = 7) was clarified with activated carbon (while stirring vigorously, activated carbon (0.2 g) was added to the solution, stirred for 30 minutes, and filtered off (the activated carbon pad was washed 3 times with 15 mL of deionized water each)). The colorless solution was evaporated under reduced pressure (40 °C), and the title compound was obtained as a white powder (about 800 mg).

[0528] Characterization

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

[0530] In the case of the QA-BCD derivative, the typical Gaussian distribution with a regular pattern observed in the random substitution derivatives is lost, while an irregular pattern of fragmentation is detected. Identification / assignment of these irregular peaks is extremely difficult because a simple pattern of fragmentation cannot be predicted. The most likely reason for the irregular pattern observed in the MALDI spectrum is that the trimethylammonium moiety is unstable under the experimental conditions. Specifically, the elimination product (see Figure 2) is the result of the cleavage of the trimethylammonium moiety, while the demethylation product (see Figure 2) is the result of the progressive cleavage of the methyl group from the cationic side chain. Since uninformative peaks are generated during laser desorption, it is reasonable to conclude that the MALDI conditions are not appropriate for specifying the DS of the QA-BCD derivative. However, the DS of the QA-BCD derivative can be specified by NMR (Figure 13I), which was estimated to be approximately 2.1.

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

[0532] Figure 3G and Figure 14G illustrate the molecule to be synthesized. The preparation of the succinyl-substituted dimer (Succ dimer) was achieved in a one-step reaction (see Figure 14A).

[0533] Synthesis

[0534] Under an inert atmosphere, while vigorously stirring, a dimer nucleus of (βCD-(triazole)1-βCD) (1.2 g, 0.5 mmol) was suspended in pyridine (23 mL). For the purpose of increasing the solubility of the (βCD-(triazole)1-βCD) dimer, the suspension was heated at 40 °C for 1 hour, but it could not be completely dissolved. 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 hours. The crude reaction product was concentrated under reduced pressure, dissolved in water (50 mL) (a clear solution was not obtained), and treated with ion-exchange resins (H+ resin (2 g) and OH− (2 g) resins were added to the solution, stirred for 15 minutes, and filtered (the resins were washed 3 times with 15 mL of deionized water each)). The resulting filtrate (final pH = 7) was clarified with activated carbon (while vigorously stirring, activated carbon (0.5 g) was added to the solution, stirred for 30 minutes, and filtered (the activated carbon pad was washed 3 times with 15 mL of deionized water each)). The colorless solution was evaporated under reduced pressure (40 °C), and the title compound was obtained as a white powder (about 900 mg).

[0535] Characterization

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

[0537] Similar to the case of the QA dimer, MALDI analysis was found to be inappropriate for DS determination. DS was estimated to be about 2.1 by NMR (Figure 14I).

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

[0539] Method

[0540] Blood was drawn from healthy providers by a qualified phlebotomist. Test substance or PBS only (negative control) was added to whole blood at various concentrations and incubated at 37 °C for 3 hours. The blood was then centrifuged and serum was collected. The serum was frozen and then processed for mass spectrometry.

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

[0542] To 50 μL of plasma sample, 0.5 ng of d7-7-ketocholesterol (Toronto Research Chemicals, North York, Ontario, CA), an internal standard, was added. d7-7-ketocholesterol was prepared as a 0.1 ng / μL ethanol solution. The sample was treated with 250 μL of acetonitrile, vortex mixed, and centrifuged at 12,000×g for 10 minutes to remove proteins. The supernatant was dried under vacuum and then treated with 75 μL of QAO reagent. The 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. The stock solution was then diluted 1:4 with methanol containing 5% acetic acid to a final working concentration of 2.5 mg / mL. The mixture was reacted at room temperature for 2 days and then analyzed by LC-MS / MS.

[0543] The standard substance of 7-ketocholesterol (Toronto Research Chemicals, North York, Ontario, CA) was prepared at 1 - 100 ng / ml in charcoal-stripped plasma, SP1070, (Golden West Biological, Temecula, CA, USA) and in phosphate-buffered saline. Since there was residual 7-ketocholesterol detected in the charcoal-stripped plasma, the standard substance prepared from PBS was used.

[0544] The QAO-7-ketocholesterol derivative was analyzed by electrospray ionization (ESI) in positive mode using a 4000 Q-TRAP hybrid / triple quadrupole linear ion trap mass spectrometer (SCIEX, Framingham, MA, USA). The mass spectrometer was connected to 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: source voltage 4500 kV, GS1 50, GS2 50, CUR 20, TEM 550, and CAD gas medium. The compounds were quantified using multiple reaction monitoring (MRM) and transitions optimized by injection of the pure derivatized compounds as shown in Table 1 below. The transitions in bold were used for quantification.

Table 1

[0546] Separation was achieved using a Gemini 3μ C6-phenyl 110Å, 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 containing 0.1% formic acid, B: acetonitrile containing 0.1% formic acid. The initial concentration of solvent B was 20%, which then increased linearly to 60% B in 10 minutes, then to 95% B in 0.1 minute, maintained at that level for 3 minutes, decreased to the initial 20% B in 0.1 minute, and maintained at that level for 4 minutes. The retention time of 7-ketocholesterol was 8.46 minutes.

[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 by comparing the standard curve generated from the peak area ratio of the analyte to the internal standard and the 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 precision (relative standard deviation) of 8.5% at this time. The signal-to-noise (S / N) was 19:1. The accuracy at a concentration of 100 ng / mL was 98% with a precision of 0.5% and an S / N of 24:1.

[0548] Results

[0549] Figures 15A and 15B demonstrate that the HPβCD dimer (DS is approximately 8 as identified by both MALDI and NMR, see FIGS. 10I and 10J) can remove 7KC from blood cells (whole blood) much more efficiently than the HPβCD monomer. This is an ex vivo assay in human subjects, but it allows for obtaining results that can predict the effect in human patients much more accurately than experiments in non-human animals. Figure 15C demonstrates that the HPβCD dimer has no appreciable effect on plasma cholesterol levels. This implies that the HPβCD dimer does not remove large amounts of cholesterol from blood cells. Removing too large an amount of cholesterol from cells potentially causes disruption of cell membranes and organelle membranes and may lead to cell death. The inventors wanted to dire...

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 method for making a cyclodextrin dimer comprising the steps of: (a) reacting a top-protected β-cyclodextrin with a dialkylating agent, thereby producing a top-protected βCD dimer linked through a bottom base, and optionally purifying said top-protected βCD dimer; (b) deprotecting the top-bottom protected βCD dimer, thereby producing a deprotected βCD dimer, and optionally purifying the deprotected βCD dimer; and (c) ionizing said deprotected βCD dimer with one or more of hydroxypropyl, methyl, succinyl, sulfobutyl, and / or quaternary ammonium, or —CH ２ CH(OH)CH ２ N (CH ３ ) ３ ＋ linking said cyclodextrin dimer to a cyclodextrin group, thereby producing said cyclodextrin dimer, and optionally purifying said cyclodextrin dimer; wherein said CD dimer has an average degree of substitution of 1 to 40 and contains up to 28 methyl groups.

2. (i) the capped β-cyclodextrin comprises heptakis(6-O-tert-butyldimethylsilyl)-β-cyclodextrin; (ii) the dialkylating agent comprises a dibromoalkane or 1,4-dibromobutane; (iii) step (a) is carried out under anhydrous conditions and / or using sodium hydride as a base; (iv) the purification in step (a) comprises normal phase chromatography with isocratic elution; (v) step (b) is carried out using tetrabutylammonium fluoride in tetrahydrofuran (THF); (vi) the purification in step (b) comprises normal phase chromatography with isocratic elution; or (vii) any combination of (i) to (vi); The method of claim 1, wherein

3. (i) step (c) comprises reacting the deprotected βCD dimer with a hydroxypropylating agent, propylene oxide, a methylating reagent, methyl iodide, a succinylating reagent, succinic anhydride, a sulfobutylating reagent, 1,4-butane sultone, and / or a quaternary ammonium linking reagent, or glycidyltrimethylammonium chloride; (ii) step (c) is carried out in aqueous conditions, optionally including sodium hydroxide as a base; (iii) said purification in step (c) comprises one or more of ion exchange resin treatment, activated charcoal clarification, and dialysis; or (iv) any combination of (i) to (iii); The method according to claim 1 or 2,

4. (a) reacting 2-O-(n-azidoalkyl)-βCD with 2-O-(n-alkyne)-βCD, thereby forming a βCD-triazole-βCD dimer; wherein the βCD-triazole-βCD dimer has the following structure: 【Chemistry 2】 (Formula XII), wherein n1 is 1-8 and / or n2 is 1-8, wherein n1 and n2 are each 1-4, wherein n1 is 1 and n2 is 3, or wherein the length of the triazole linker is 5-8; and 2. The method of claim 1, comprising: (b) hydroxypropylating the βCD-triazole-βCD dimer with a hydroxypropylating agent, propylene oxide, a methylating reagent, methyl iodide, a succinylating reagent, succinic anhydride, a sulfobutylating reagent, 1,4-butane sultone, and / or a quaternary ammonium linking reagent, or glycidyl trimethyl ammonium chloride, thereby producing a cyclodextrin dimer having an average degree of substitution of 1 to 40 and containing up to 28 methyl groups.

5. (i) step (a) is carried out using a copper(I) catalyst, optionally 15 mM copper(I); (ii) step (a) is carried out in an aqueous solution; (iii) the aqueous solution contains dimethylformamide (DMF), or 50% DMF (v / v); (iv) the process further comprises, after step (a), purifying the cyclodextrin dimer by silica gel chromatography; or (v) any combination of (i) to (iv); The method according to claim 4, wherein

6. moreover, (i) prior to step (a), reacting an n-azido-1-bromo-alkane with β-cyclodextrin, optionally using a catalytic amount of lithium iodide, to produce said 2-O-(n-azidoalkyl)-βCD; (ii) purifying the 2-O-(n-azidoalkyl)-βCD by silica gel chromatography; (iii) before step (a), reacting an n-azido-1-bromo-alkane with β-cyclodextrin using a catalytic amount of lithium iodide to produce said 2-O-(n-azidoalkyl)-βCD; (iv) purifying the 2-O-(n-azidoalkyl)-βCD by silica gel chromatography; or (v) any combination of (i) to (iv); 6. The method of claim 4 or 5, comprising:

7. 6. The method of claim 5, wherein step (i) is carried out in dry DMSO, and wherein step (i) further comprises the addition of lithium hydride.

8. 5. The method of claim 4, wherein step (b) is carried out in aqueous conditions, optionally including sodium hydroxide as a base, and / or the method further comprises purifying the cyclodextrin dimer after step (b) using one or more of ion exchange resin treatment, activated charcoal clarification, membrane filtration, and dialysis.

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