WO2015181224A1 - Novel water soluble 6-thioalkyl-cyclodextrins and uses thereof - Google Patents

Novel water soluble 6-thioalkyl-cyclodextrins and uses thereof Download PDF

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Publication number
WO2015181224A1
WO2015181224A1 PCT/EP2015/061686 EP2015061686W WO2015181224A1 WO 2015181224 A1 WO2015181224 A1 WO 2015181224A1 EP 2015061686 W EP2015061686 W EP 2015061686W WO 2015181224 A1 WO2015181224 A1 WO 2015181224A1
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Prior art keywords
compound
agents
cyclodextrin
formula
active agent
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PCT/EP2015/061686
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French (fr)
Inventor
Gerhard Wenz
Lisa Becker
Dennis SCHWARZ
Antje APPELT-MENZEL
Marco Metzger
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Universitaet Des Saarlandes
Julius-Maximilians-Universitaet Wuerzburg
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Publication of WO2015181224A1 publication Critical patent/WO2015181224A1/en

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    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
    • C08B37/0015Inclusion compounds, i.e. host-guest compounds, e.g. polyrotaxanes

Definitions

  • Cyciodextrins are cyclic oligomers of 6, 7, or 8 ( ⁇ -, ⁇ -, and y-CDs, respectively) D- glucopyranose units linked by ot-(l - 4) bonds. All CDs have an internal cavity that is mainly hydrophobic and that allows the binding of guest molecules. These guest molecules are typically of hydrophobic or amphiphilic nature, depending on their size and polarity pattern. The formation of a host-guest complex allows for the solubilization of compounds which are barely soluble or even insoluble in water, and may also improve their stabilization.
  • CDs Inclusion of active substanFLU14400PCTEPDlces in CDs (i.e. the formation of CD complexes or inclusion compounds) is particularly used for, e.g., the formulation of pharmaceutical drugs, and cosmetics, as well as for chromatographic separations.
  • Applications of CDs and CD derivatives in the pharmaceutical field are especially interesting since they allow solubilization of water-insoluble or volatile drugs in water.
  • the observed solubilization of an active pharmaceutical ingredient (API) is generally based on the complexation of the hydrophobic part of the API molecule in the CD cavity.
  • ⁇ -, ⁇ -, and y-CDs are commercially available. However, their application potential is still limited. CDs generally accept only host molecules of a limited size range in their cavity; and in particular many macromolecu!es are not complexed by CDs.
  • the Cyclodextrin : API molecule molar ratio is in general 1:1 or higher; i.e. no more than one guest molecule is complexed per molecule of cyclodextrin.
  • y-CDs the inclusion of two guest molecules is also known (W. Herrmann, S. Wehrle, and G. Wenz, Chem. Commun., pp. 1709-1710, 1997).
  • CDs have been chemically modified, e.g. through chemical functionalization of the hydroxyl groups, in order to synthesize CD derivatives.
  • Kawabata et al. Chem. Lett. 1986, 1933-1934 and Ling et al. Chem. Soc, Chem. Commun.
  • Cyclodextrin derivatives comprising one or more glycosyl or maltosyl substituents linked to the cyclodextrin by a sulphur atom are also described by V. Laine et al. in J. Chem. Soc, Perkin Trans., 2, 1995, pp. 1479-1487. These derivatives have been used in order to solubilize an anti-inflammatory agent such as prednisolone.
  • cyclodextrin derivatives known in the prior art are limited with regard to their applicability, in particular with respect to the active agents to be transported, the load capacity of active agents per mass unit of cyclodextrin derivative, their cost, their toxicity, their synthesis and/or their solubility in water.
  • the present invention provides novel cyclodextrin compounds having the formula
  • n 0, 1 or 2
  • Ri is independently selected from C x to Q-alkyl
  • R 2 is independently selected from C0-(NH-CH 2 -CH 2 ) o -NH 2 or (CH 2 -CH 2 -0) 0 -(CH 2 ) p -CH 3 ; wherein o is 1, 2, 3, 4, 5 or 6; p is 0 or 1,
  • R 2 is (CH 2 )i-X wherein X is NH 2 or OH, and wherein I is 2, 3, 4, 5 or 6;
  • R 2 is -CO-NH-R3, A ; R 3. or CO-(NR 3 ) 2 ,
  • R 3 and R 3 ' is independently from each other a hydrophilic branched or unbranched radical with 2 to 7, preferably 4 to 7 main chain atoms,
  • R 2 is CO-NH-(CH 2 ) q -R 4 , wherein q is 0, 1 or 2, and R 4 is selected from OH, CH 2 -OH, and a 5 or 6 membered substituted or unsubstituted heterocyclic ring system containing at least one N or one 0 atom,
  • R 2 is CO-N-R 5 , wherein R 5 is a 6 membered substituted or unsubstituted heterocyclic ring system and wherein N is part of the 6 membered heterocyclic ring,
  • R 2 is 0-CH 2 -CH 2 -NH-CH 2 -CH 2 -OH
  • the cyclodextrin compounds are preferably water soluble and/or do not form micelles in aqueous solution.
  • the invention provides a method of preparing the cyclodextrin compounds of the invention comprising reacting a compound of formula (X),
  • Ri is selected independently from C C 4 a Iky I;
  • Hal is halogen, o is 1, 2, 3, 4, 5, or 6, p is 0 or 1, or
  • R 3 and R 3 ' is a hydrophilic branched or unbranched radical with 2 to 7, preferably 4 to 7 main chain atoms; or
  • a compound of formula (E) NH-(CH 2 ) q -R 4 , wherein q is 1 or 2, and R 4 is selected from OH, CH 2 - OH, and a 5 or 6 membered substituted or unsubstituted heterocyclic ring system containing at least one N or one O atom; or with a compound of formula (F) HN-R 5 , wherein R 5 is a 6 membered substituted or unsubstituted heterocyclic ring system and wherein N is part of the 6 membered heterocyclic ring, wherein in the case of a reaction with the compound of formula (Z) (D), (E) or (F), the compound of formula (X) is first reacted with a coupling reagent.
  • compositions comprising the inventive cyclodextrin compounds and at least one active agent as specified hereinafter complexed by said cyclodextrin compound.
  • the present invention relates to the use of a cyclodextrin compound according to the invention as a host molecule for at least one active agent as specified hereinafter. Said use preferably results in an increase of solubility, prevention of aggregation, avoiding micellar formulations, reduction of injection pain for intravenous applications, reduction of vapor pressure and/or targeting of active agents to avoid side effects with respect to the active agent.
  • the present invention provides a method of encapsulating a pharmaceutically active agent by contacting a cyclodextrin compound of the invention with a pharmaceutically active agent under conditions suitable for forming a host-guest complex.
  • an interval which is defined as "(from) X to Y” equates with an interval which is defined as "between X and Y". Both intervals specifically include the upper limit and also the lower limit. This means that for example an interval of "2.5 mg/kg to 12.5 mg/kg” or "between 2.5 mg/kg to 12.5 mg/kg” includes a concentration of 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 11.5 and 12.5 as well as any given intermediate value.
  • Figure 1 Exemplary inventive cyclodextrins and overview of synthetic strategy.
  • FIG. 6 Cell viability test for the cyclodextrin compounds of the invention. The viability of cells exposed to cyclodextrin compounds was assessed using the CellTiter-Glo ® assay (Promega, #G7571) according to the manufacturer's manual on the colon tumor cell line Caco-2 (DSMZ #ACC-169; passage 6-9).
  • FIG. 7 Transport of midazolam through the Caco-2 barrier and empty inserts over 4 hours. Samples were taken after 5, 15, 30, 120 and 240 minutes. Transport behaviour of concentrations of 1.11 pg/mL and 5.55 pg/mL midazolam was investigated.
  • substitution at the primary OH groups of CD advantageously does not alter or even increases its binding potential.
  • substitution of all primary hydroxy I groups by thioether groups advantageously gave rise to very high binding potentials due to the higher hydrophobicity of sulfur compared to oxygen.
  • the generated cyclodextrin derivatives showed low ion strength in solution and no pH dependence was observed.
  • the inventive cyclodextrin derivatives can be obtained in well-defined stoichiometry, no statistic, randomly substituted products are generated, thereby simplifying quality control and analysis.
  • the CD thioethers of the invention are able to form highly stable complexes with active pharmaceutical ingredients, even with volatile APIs such as halothane, sevoflurane.
  • the CD derivatives of the invention can be used for solubilization and even transcellular delivery of APIs is conceivable.
  • the present inventors investigated the inclusion of volatile hydrophobic pharmaceutically active agents in the invented CD derivatives.
  • the anesthetic sevoflurane was already included in a-CD, ⁇ -CD and hydroxypropy! ⁇ -CD, but the complex in a-CD is nearly insoluble in water (WO2012/104730 Al) and the binding constants for ⁇ -CD and hydroxypropyl- -CD are rather poor.
  • the present invention thus provides water soluble CD derivatives with well-defined molecular structure and high binding affinities towards APIs and other active agents.
  • the present invention thus relates to a cyclodextrin compound having the formula
  • n 0, 1 or 2
  • Ri is independently selected from Ci to C 4 -alkyl
  • R 2 is independently selected from CO-(NH-CH 2 -CH 2 ) 0 -NH 2 or (CH 2 -CH 2 -0) 0 -(CH 2 ) p -CH 3 ; wherein o is 1, 2, 3, 4, 5 or 6; p is 0 or 1;
  • R 2 is -(CH 2 )
  • R 2 is -CO-NH-R3, ⁇ 3 or CO-(!MR 3 ) 2 , wherein R 3 and R 3 ' is independently from each other a hydrophilic branched or unbranched radical with 2 to 7 main chain atoms;
  • R 2 is CO-NH-(CH 2 ) q -R 4 , wherein q is 0, 1 or 2, and R 4 is selected from OH, CH 2 OH, and a 5 or 6 membered substituted or unsubstituted heterocyclic ring system containing at least one N or one O atom,
  • R 2 is CO-N-R5, wherein R 5 is a 6 membered substituted or unsubstituted heterocyclic ring system and wherein N is part of the 6 membered heterocyclic ring;
  • R 2 is 0-CH 2 -CH 2 -NH-CH 2 -CH 2 -OH.
  • cyclodextrin or "CD” is used herein to designate cyclic oligosaccharide compounds of 6, 7, or 8 ( ⁇ -, ⁇ -, and y-CDs, respectively) saccharide units having the general formula
  • n 6, 7 or 8.
  • cyclodextrin compounds and “cyclodextrin derivatives” are used interchangeably herein to designate compounds sharing the general cyclic oligosaccharide structure with cyclodextrins.
  • the cyclodextrin compounds of the present invention may comprise 6, 7 or 8 thioether glucopyranose units (i.e. can be ⁇ -, ⁇ - or y-cyclodextrin compounds, respectively); in particular 6-deoxy-6-thioether glucopyranose units.
  • the number of thioether glucopyranose units having an R 2 substituent at the secondary OH group is designated "m” herein.
  • the number of thioether glucopyranose units having no R 2 substituent is designated "n” herein.
  • all thioether glucopyranose units are comprise an R 2 substituent at the secondary OH-group.
  • unsubstituted thioether glucopyranose units are present in the inventive cyclodextrin compound.
  • the number of unsubstituted thioether glucopyranose units (n) can be 0, 1 or 2.
  • all cyclodextrin compounds envisaged herein are preferably (i) capable of complexing an active agent (ii) do not form micelles in aqueous solution and (iii) are nontoxic, comparable to the compounds tested in the appended examples.
  • suitable substituents depending on the above-mentioned desired properties and use of the cyclodextrin compound, e.g.
  • the cyclodextrin compounds of the present invention are hydrophilic and neutral or weakly charged, show low ion strength in solution and are scarcely pH dependent. It is also envisaged that some of the inventive cyclodextrin compounds may be capable of crossing cellular membranes, thus enabling their use as carriers for membranes or transcellular delivery.
  • Ri is independently selected from CI to C4-alkyl.
  • CI to C4-alkyl refers to straight and branched chain saturated acyclic hydrocarbon groups having from 1 to 4 carbon atoms such as, for example, methyl, ethyl, n-propyl, isopropyl (1-methylethyl), cyclopropyl, n-butyl, 2-butyl (2-methylpropyl), ier -butyl (1,1-dimethylethyl), or the like.
  • Ri is preferably methyl.
  • R 2 can be independently selected from CO-(NH-CH 2 -CH 2 ) 0 -NH 2 or (CH 2 -CH 2 -O) 0 -(CH 2 ) P -CH 3 ; wherein o is 1, 2, 3, 4, 5 or 6; p is 0 or l.
  • R 2 is CO-(NH-CH 2 -CH 2 ) 0 -NH 2 or (CH 2 -CH 2 -0)o-CH3, wherein o is 1, 2, 3 or 4, wherein o is preferably 3 or 4.
  • n > 2; R, is methyl; and R 2 is CO-(NH-CH 2 -CH 2 ) 0 -NH 2 ; wherein o is 1. It is further envisaged that o can be exactly 1.
  • R 2 can be -(CH 2 ) r X wherein X is H 2 or OH, and wherein I is 2, 3, 4, 5 or 6.
  • R 2 can be
  • R 3 and R 3 ' is independently from each other a hydrophilic branched or unbranched radical with 2 to 7 main chain atoms.
  • R 3 and R 3 ' can be independently from each other a hydrophilic branched or unbranched radical with 4 to 7 main chain atoms.
  • radical in general designates an aliphatic or aromatic substituted or unsubstituted hydrocarbon radical.
  • the radical is preferably an aliphatic radical which may be substituted or unsubstituted, saturated or unsaturated, branched or unbranched.
  • Exemplary radicals include alkyl-, alkenyl-, alkynyl- radicals with at least one terminal hydroxyl or amino group.
  • R 3 or R 3 ' is -CH 2 -CH 2 -OH. -CH 2 -CH 2 -NH 2
  • R 3 may comprise at least one terminal hydroxyl or amino group.
  • hydroxyl refers to a group of the general formula -OH.
  • amino refers to a group of the general formula -NR 2 , wherein R is hydrogen or a monovalent aliphatic or aromatic hydrocarbon substituent.
  • R 2 can be selected independently from CO-NH-(CH wherein q is 0, 1 or 2, and R is selected from OH, CH 2 -OH, and a 5 or 6 membered substituted or unsubstituted heterocyclic ring system containing at least one N or one 0 atom. In one embodiment, q is 1. In one embodiment, R 4 , when being a 5 or 6 membered heterocyclic ring system containing at least one N or one 0 atom, is an aromatic ring system.
  • substituted refers to any one or more hydrogen atoms on the indicated molecule being replaced with a selected group referred to herein as a "substituent".
  • a substituted group can have 1 to 5, preferably 1 to 3, and more preferably 1 independently selected substituents as defined herein.
  • Suitable substituents can be selected from the group including, sulfonate ( S0 3 ), sulfate (-S0 4 2 ), phosphate (-P0 4 3 ), phosphonate (-P0 3 2 ) sulfonyl, aminosulfonyl, sulfonylamino, carboxyamide, aminocarbonyl, carboxy, oxo, hydroxy, amino, nitro, cyano and ureido.
  • heterocyclic refers to a 5 or 6-membered ring containing at least one N or one 0 atom, and optionally one or more heteroatomic moieties selected from S, SO, S0 2 , O, N, or N-oxide.
  • Said heterocycle can be saturated or have one or more degrees of unsaturation.
  • the heterocycle may be optionally fused to one or more "heterocyclic" ring(s), aryl ring(s), heteroaryl ring(s), or carbocycle ring(s), each having optional substituents.
  • heterocyclic R moieties include, but are not limited to amino, 2-furyl, 3-furyl, 1-pyrrolyl, 2-pyrrolyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-picolyl or 3-picolyl.
  • heterocyclic R 4 moieties in accordance with the present invention include without limitation 1,4-dioxanyl, 1,3-dioxanyl, pyrrolidinyl, pyrrolidin-2-onyl, piperidinyl, imidazolidine-2,4-dionepiperidinyl, piperazinyl, piperazine-2,5-dionyl, morpholinyl, dihydropyranyl, dihydrocinnolinyl, 2,3-dihydrobenzo[l,4]dioxinyl, 3,4-dihydro-2H-benzo[b][l,4]-dioxepinyl, tetrahydropyranyl, 2,3-dihydrofuranyl, 2,3-dihydrobenzofuranyl, dihydroisoxazolyl, tetrahydrobenzodiazepinyl, tetrahydroquinolinyl, tetrahydro
  • aromatic ring system denotes fully unsaturated carbocycles and heterocycles in which at least one ring of a polycyclic ring system is aromatic.
  • R 2 can be CO-N-R 5 , wherein R 5 is a 6 membered substituted or unsubstituted heterocyclic ring system and wherein N is part of the 6 membered heterocyclic ring.
  • N-R 5 is selected from the group comprising morpholino having the general formula
  • Said heterocyclic ring may also be substituted, e.g. with one or more substituents selected from the group including sulfonate (-S0 3 ), sulfate (-S0 4 2 ), phosphate (-P0 4 3 ⁇ ), phosphonate (-P0 3 2 ), sulfonyl, aminosulfonyl, sulfonylamino, carboxyamide, aminocarbonyl, carboxy, oxo, hydroxy, amino, nitro, cyano and ureido.
  • the present invention provides, in a further aspect, cyclodextrin compounds as defined herein, which are water-soluble.
  • the water solubility of a substance is the saturation mass concentration of the substance in water at a given temperature. It is s expressed in mass of solute per volume of solution, i.e. kg/m 3 or g/L. It is for example envisaged that the invented cyclodextrin derivatives have a water-solubility of higher than 18 g/L at 25°C, such as 30 g/L Preferably, the inventive cyclodextrin derivatives have a water-solubility of more than 100 g/L.
  • the water-solubility can be assessed, e.g., using the column elution method or the flask method as described in OECD Guideline Nr. 105 (1995).
  • inventive cyclodextrin compounds do not form micelles in aqueous solution.
  • a micelle is an aggregate of surfactant molecules (usually amphiphilic organic molecules containing both hydrophobic groups ('tails') and hydrophilic groups (' heads') dispersed in a liquid colloid.
  • active ingredients e.g. APIs
  • micelle formation is problematic because the amount of active ingredient enclosed in the micelles cannot be controlled.
  • cyclodextrin compounds which do not form micelles in aqueous solution, but rather form complexes with active ingredients in a defined molar ratio (e.g., 1 molecule of CD complexes 0.05 to 2, or preferred 0.3 to 2 molecules of active ingredient).
  • Micelle formation can be assessed using various methods as described in Dominguez et al. J Chem Edu, 1997; 74(10): 1227- 1231 .
  • the UV-Absorption Spectroscopy Method is based on the tautomerism of ketones or (for example benzoylacetone (BZA), l-phenyl-l,3-butadione).
  • Keto-enol equilibria of BZA are extremely solvent sensitive and the proportion of the enolic form is much greater in nonpolar solvents, such as cyclohexane, than in polar or hydrogen-bond donor solvents, such as water or alcohols.
  • polar or hydrogen-bond donor solvents such as water or alcohols.
  • a surfactant is added to a water solution of BZA, the amount of enol increases abruptly when micelles, which provide a less polar solvent than the aqueous phase take up the enolic form.
  • Other methods for assessing micelle formation include the Fluorescence Spectroscopy Method based on the solvent dependence of vibrational band intensities in pyrene monomer fluorescence (M. Wilhelm, et al., Macromolecules, 1991; 24(5), 1033-1040) and the electrical conductivity method for anionic surfactants as well as dynamic light scattering.
  • the cyclodextrin compounds of the present invention may be neutral or hydrophilic or amphiphilic. It is contemplated that amphiphilic CD molecules can enter and cross cell membranes, and thus act as a carrier which supports an active ingredient, such as an API, in overcoming cellular barriers, such as the intestinal barrier or the blood-brain barrier (BBB).
  • an active ingredient such as an API
  • BBB blood-brain barrier
  • the present invention also provides a method of preparing a cyclodextrin compound as defined herein.
  • a halogenated cyclodextrin can be provided, e.g. following the protocol of Gadelle and Defaye, Angew. Chem. Int. Ed. Engl. 1991, 30: 78-79.
  • a cyclodextrin is reacted with a nucleophile (e.g., triphenylphosphane (PPh 3 )), and a halogen compound (e.g. /V-Bromosuccinimide, NBS) in a solvent such as DM F at high temperature (e.g. about 70°C).
  • a nucleophile e.g., triphenylphosphane (PPh 3 )
  • a halogen compound e.g. /V-Bromosuccinimide, NBS
  • TBAI tetra-n-butylammoniumiodide
  • a thioether cyclodextrin is obtained as described above, and reacted with a coupling reagent.
  • Suitable coupling reagents can be selected from the group of - carbonyldiimidazole, phosgene, diphosgene and triphosgene.
  • a compound of the formula (Z) (H 2 N-(CH 2 CH 2 -NH)q-CH 2 CH 2 -NH 2 ), wherein q is 0, 1, 2, 3, 4, or 5; or a compound of the formula (C) HO-(CH 2 )l-X, wherein X is NH 2 or OH, and wherein I is 2, 3, 4, 5 or 6;
  • R 3 is a hydrophilic branched or unbranched radical with 2 to 7 main chain atoms, in particular, wherein R 3 is a hydrophilic branched or unbranched radical with 4 to 7 main chain atoms; or a compound of formula (E) NH-(CH 2 )q-R 4 , wherein q is 1 or 2, and R 4 is selected from OH, CH 2 -OH, and a 5 or 6 membered substituted or unsubstituted heterocyclic ring system containing at least one N or one O atom, or a compound of formula (F) HN-R S , wherein R 5 is a 6 membered substituted or unsubstituted heterocyclic ring system and wherein N is part of the 6 membered heterocyclic ring.
  • the inventive cyclodextrin compounds as defined above also include the pharmaceutically acceptable salt(s) thereof.
  • pharmaceutically acceptable salt(s) means those salts of compounds of the invention that are safe and effective for the desired administration form.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • salt formation can be used to increase or decrease solubility, to improve stability or toxicity and to reduce hygroscopicity of a drug product.
  • any counterions used in pharmaceuticals must be considered safe, and several lists of pharmaceutically approved counterions exist, which vary depending on the source.
  • Approved salt formers can e.g. be found in the Handbook of Pharmaceutical Salts (Stahl PH, Wermuth CG, editors. 2002. Handbook of pharmaceutical salts: Properties, selection and use. Weinheim/Zurich: Wiley-VCH/VHCA.).
  • the present invention provides a composition comprising a cyclodextrin compound of the invention (as a host molecule) and at least one active agent complexed (as a guest molecule) with said cyclodextrin.
  • CD inclusion compounds are mainly nondirectional interactions such as hydrophobic and van der Waals interactions.
  • the dominance of solvatophobic interactions is evident in the fact that the inclusion of guests in CDs occurs preferentially in aqueous solution.
  • the magnitude of hydrophobic interactions is determined mainly by the hydrophobic surface area of the guest. Additional polar groups can be attached to the CD ring, e.g. to improve binding selectivity.
  • host : guest complex is used interchangeably with the terms "inclusion complex", and "CD complex” herein.
  • active agent is used interchangeably with the term “active ingredient” herein.
  • any active agent can be chosen which is capable of being complexed by the cyclodextrin compounds of the invention.
  • said active agent can be selected from the group consisting of a pesticide, herbicide, insecticide, antioxidant, plant growth instigator, sterilization agent, catalyst, chemical reagent, food product, nutrient, cosmetic, vitamin, sterility inhibitor, fertility instigator, microorganism, flavoring agent, sweetener, cleansing agent and pharmaceutically active agent.
  • pharmaceutically active agent is used interchangeably with the term “active pharmaceutical ingredient” or “API” herein and refers to a substance or substance combination used in manufacturing a drug product. It also refers to the active or central ingredient in the product which causes the direct effect on the disease diagnosis, prevention, treatment or cure.
  • Said pharmaceutically active agent can be selected from the group consisting of anesthetics, analgesics, steroids, cytostatic drug, antiviral agents, nutrients, nutritional agents, hematological agents, endocrine agents, metabolic agents, cardiovascular agents, renal agents, genitourinary agents, respiratory agents, central nervous system agents, gastrointestinal agents, anti-infective agents, biologic agents, immunological agents, dermatological agents, ophthalmic agents, antineoplastic agents, and diagnostic agents.
  • the anaesthetic and/or the analgesic is selected from the group consisting of midazolam, sevofiurane, halothane, propofol, xenon, fentanyl
  • the steroid is selected from the group consisting of hydrocortisone, testosterone, estradiol and contraceptives
  • the cytostatic drug is selected from the group consisting of camptothecin, paclitaxel, and anthracyclines, e.g.
  • the neurolepticum is selected from the group consisting of haloperidol, fluspirilen, and risperidon), and the antiviral agent is selected from the group consisting of lopinavir, ritonavir, atazanavir.
  • compositions of the present invention are used in the treatment of a subject.
  • treatment in all its grammatical forms includes therapeutic or prophylactic treatment.
  • a “therapeutic or prophylactic treatment” comprises prophylactic treatments aimed at the complete prevention of clinical and/or pathological manifestations or therapeutic treatment aimed at amelioration or remission of clinical and/or pathological manifestations.
  • treatment thus also includes the amelioration or prevention of diseases.
  • composition of the present invention A variety of routes are applicable for administration of the composition of the present invention, including, but not limited to, topically (e.g. epicutaneously, inhalationally, mucosally, intraocularly), enterally (e.g. orally, recta I ly), and parenterally (e.g. intravenously, intra-arterially, intraperitoneally, intramuscularly, subcutaneously, transdermally).
  • topically e.g. epicutaneously, inhalationally, mucosally, intraocularly
  • enterally e.g. orally, recta I ly
  • parenterally e.g. intravenously, intra-arterially, intraperitoneally, intramuscularly, subcutaneously, transdermally.
  • the pharmaceutical composition of the present invention is adapted for parenteral administration by infusion or injection.
  • infusion is meant a continuous administration over a certain period of time. For example such an administration may take in between 10 minutes to 4 days.
  • injection means is a transient infusion method of introducing fluid into the body of a subject. Injection(s) may e.g. be administered for 1, 2, 3 or 4 days.
  • composition according to the invention may be in solid, liquid or gaseous form and may be, inter alia, in the form of an ointment, a cream, transdermal patches, a gel, powder, a tablet, solution, an aerosol, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tincture or fluid extracts or in a form which is particularly suitable for the desired method of administration, in particular parenteral administration. It is however preferred that the inventive composition is in liquid form.
  • the present invention also envisages a method of diagnosing or treating a subject in need thereof, comprising administering the inventive composition, which preferably comprises a pharmaceutically effective amount of the pharmaceutically active agent.
  • the purposesubject is preferably a mammal such as a human, monkey, cat, dog, horse, pig, cattle, guinea pig, mouse or rat with human being preferred.
  • the exact administration dose of the inventive composition will depend on the purpose of the treatment (e.g. remission maintenance vs. acute flare of disease), and will be ascertainable by one skilled in the art using known techniques.
  • the molar ratio between the cyclodextrin compound and the active agent can be in any suitable range.
  • the cyclodextrin is present in a molar excess to ensure that the entire (amount) of active agent is complexed by the cyclodextrin.
  • the molar ratio between the cyclodextrin and the active agent can range from 10:1 to 1:1, e.g. it can be 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.
  • composition of the invention can further comprise one or more pharmaceutical excipient.
  • any pharmaceutical excipient is conceivable as long as it does not interfere with the ability of the inventive cyclodextrin compound to form a host : guest complex with the active agent.
  • compositions are typically inactive ingredients of a drug product that do not increase or affect the diagnostic, therapeutic, preventive, or curing action of the active ingredient.
  • pharmaceutically acceptable excipients are nontoxic to subjects to be treated at the dosages and concentrations employed.
  • pharmaceutically acceptable means approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.
  • compositions include, but are not limited to diluents (fillers, bulking agents, e.g. lactose, microcrystalline cellulose), disintegrants (e.g. sodium starch glycoiate, croscarmellose sodium), binders (e.g. PVP, HPMC), lubricants (e.g. magnesium stearate), glidants (e.g. colloidal Si0 2 ), solvents/co-solvents (e.g. aqueous vehicle, propylene glycol, glycerol), buffering agents (e.g. citrate, gluconates, lactates), preservatives (e.g.
  • diluents fillers, bulking agents, e.g. lactose, microcrystalline cellulose
  • disintegrants e.g. sodium starch glycoiate, croscarmellose sodium
  • binders e.g. PVP, HPMC
  • lubricants e.g. magnesium stearate
  • BHT e.g. BHT, BHA, Ascorbic acid
  • wetting agents e.g. polysorbates, sorbitan esters
  • anti-foaming agents e.g. Simethicone
  • thickening agents e.g. methylcellulose
  • a non-exhaustive list of exemplary pharmaceutically acceptable excipients includes without limitation buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl para ben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins;
  • the antibody preferably comprises the antibody at a concentration of between 5-200 mg/mL, preferably between 10-100 mg/mL.
  • the present invention relates to the use of a cyclodextrin compound as described herein as a host molecule for at least one active agent.
  • a cyclodextrin compound as described herein as a host molecule for at least one active agent.
  • the embodiments described in the context of the composition are also applicable to the use of the invention, mutatis mutandis.
  • the use comprises with respect to the at least one active agent an increase of solubility, prevention of aggregation, avoiding micellar formulations, reduction of injection pain for intravenous applications, reduction of vapor pressure of volatile active agents, targeting of active agents to avoid side effects.
  • the cyclodextrin compounds of the invention preferably increases solubility of otherwise insoluble or barely soluble active ingredients, e.g. in water, by complexing the active ingredient. Solubility of an active ingredient can be measured according to standard protocols as described in Thiele C. et al. J. Incl. Phenom. Macrocycl. Chem. 2011; 69:303-307. Shortly, solutions of CD compounds of different concentrations in water are stirred with an excess of the active ingredient. The resulting solutions are filtered and the concentration of the dissolved drug is determined by UV.
  • the cyclodextrin compounds of the invention may further prevent aggregation of active ingredients. It is thought that the cyclodextrin compounds will improve bioavailability of the ingredient due to dissolution of aggregates. [85] It has already been set out elsewhere herein that the cyclodextrin compounds according to the invention preferably avoid the formation of micellar formulations.
  • inventive cyclodextrin compounds reduce injection pain for intravenous application. Injection pain is typical for administration of emulsions such as state of the art propofol formulations.
  • the inventive compounds are envisaged to reduce the vapor pressure of said agents by complexing them.
  • the vapor pressure of an agent is typically quantified by gas chromatography applying the head space technique (S. Fourmentin, A. Ciobanu, D. Landy, G. Wenz, Beilstein J. Org. Chem. 2013, 9, 1185-1191).
  • inventive compounds may be further used for targeting active agents, in order to enable a fast and specific delivery of active agents. It is thought that the inventive compounds can be conjugated with cell specific ligands, such as folate, transferrin, or insulin.
  • the present invention provides a method of encapsulating a pharmaceutically active agent comprising contacting a cyclodextrin compound as defined herein with a pharmaceutically active agent under conditions suitable for forming a host-guest complex, thereby complexing the pharmaceutically active agent within the cyclodextrin compounds as described herein.
  • the term "encapsulating" in all its grammatical forms, designates the process of forming a host : guest complex between the inventive cyclodextrin derivative and the pharmaceutically active agent, i.e. the pharmaceutically active agent is complexed by the cyclodextrin derivative of the invention.
  • "encapsulating” does not involve the formation of micelles.
  • the ratio of cyclodextrin derivative and pharmaceutically active agent is between 4:1 and 1:1 (cyclodextrin: guest), i.e. 4:1, 3:1, 2:1 or 1:1 or any other desired molar ratio
  • the compound 3bj. for example, comprises seven glycopyranose units (i.e., is a ⁇ - cyclodextrin) and a thiomethyl group at the R 6 position.
  • Example 1 Heptakis[6-deoxy-6-methylsulfanyl-2-(2'-(2"- ⁇ 2" , -methoxyethoxy)- ethoxy)ethyl)]-P-cyclodextrin - 3b
  • H-8a 3.78-3.80 (m, 1H, H-5), 3.81 - 3.86 (m, 1H, H-3), 3.71-3.77 (m, 1H, H-8b), 3.50- 3.55 (m, 10H, H-10, 11, 12, 13) 3.43-3.45 (m, 1H, H-4), 3.41 - 3.42 (m, 2H, H-9), 3.34 - 3.37 (m, 1H, H-2), 3.23 (s, 3H, 0-CH 3 ), 3.05-3.08 (m, 1H, H-6a), 2.74-2.79 (m, 1H, H-6b), 2.07 (s, 3H, H-7).
  • the solvents were completely removed by vacuum distillation (bath temperature 70°C, 1 mbar) and the residue was dissolved in 50 mL of water and neutralized by addition of 1 M HCI.
  • the crude product was purified by Crossflow nanofiltration in water (1 kDa, Pall Minimate TFF Capsule) and a brown yellowish oil (0.277 g, 14 %) was obtained after lyophilization.
  • the solvents were completely removed by vacuum distillation (bath temperature 70°C, 1 mbar) and the residue was dissolved in 50 mL of water and neutralized by addition of 1 M HCI.
  • the crude product was purified by Crossflow nanofiltration in water (1 kDa, Pall Minimate TFF Capsule) and a brown yellowish oil (0.204 g, 10 %) was obtained after lyophilization.
  • C-NMR: ⁇ /ppm (DMSO-d 6 , 100 MHz) 100.2 (C-l), 85.9 (C-4), 80.1 (C-2), 72.8 (C-3), 71.3 (C- 9), 70.8 (C-8), 70.7 (C-5), 69.8-69.6 (C-10, C-ll, C-12, C-13, C-14, C-15), 58.1 (0-CH 3 ), 35.2 (C-6), 16.2 (C-7).
  • the corresponding binding constant K was calculated from the hyperbolic decay of the area A of the sevoflurane signal with the total concentration of the CD derivative [CD] 0 by non-linear regression according to Equation 1 given below.
  • the Henry constant was determined according to a known GC method according to Fourmentin et al. Beilstein J. Org. Chem. 2013, 9, 1185-1191 using the Henry constants at 37"C for sevoflurane.
  • the occupancy x of employed CD host by the guest was calculated by the law of mass action according to Equation 2 given below.
  • the binding constants of the a-CD derivatives 3a ! and 4a x were much lower than the ones of the corresponding ⁇ -CD derivatives 3bi and 4bx which can be attributed to the better space filling of the seven membered rings by sevoflurane.
  • the binding constant decreased with increasing lengths of the a Iky I substituents at the sulfur atoms as well as with the lengths of the oligoethylene oxide chains. This fact was attributed to an increasing loss of entropy upon complexation of the guests. The longer the substituents the higher the conformational freedom of the host leading to higher intrinsic entropy.
  • the filling degree (defined as the number of guest molecules per cyclodextrin host) is given both in molar as well as weight percentage based on the amount of the employed cyclodextrin derivative.
  • thiomethyi derivatives showed higher binding affinities to a certain guest than the corresponding thioethyl derivatives (Table 5).
  • the concentration range was for Caco-2: 130 ng/mL - 1.3 ⁇ . - 13 ⁇ g/mL - 130 ⁇ g/mL for primary fibroblasts: 13 ⁇ . - 130 ⁇ . - 1.3 mg/mL - 13 mg/mL
  • Both substances were each solved in standard basal medium (Caco-2: MEM, fibroblasts: DMEM, Gibco ® ) and human serum and cells were treated for 2 h and 24 h with the above-mentioned concentrations.
  • the test substances were removed by washing with PBS buffer (Sigma ® ), the cells in each well were overlaid with 100 ⁇ of basal medium and 100 ⁇ _ of CellTiter-Glo ® reagent and luminescence was measured after 2 minutes of shaking, and a 10-minute incubation at room temperature in the TECAN plate reader.
  • transepithelial electrical resistance transepithelial electrical resistance
  • the intestinal epithelial cells have - by the formation of cell adherence and tight junctions - formed a sufficient barrier and can be used to perform absorption studies.
  • the tested concentrations of active ingredient complex are based on the clinically relevant plasma concentrations of the anesthetic midazolam and are used in a 10 x and 50 x higher concentration for subsequent oral administration.
  • the filling degree of 3b t for midazolam is 7.4%.
  • the tested active ingredient or complex concentrations were as follows: concentration increase in concentration concentration concentration

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Abstract

The invention provides novel cyclodextrin compounds. Specifically, the invention relates to cyclodextrin derivatives with thioether groups at the primary hydroxy I positions and various substituents at the secondary hydroxy I groups. The invented cyclodextrin compounds are preferably water-soluble, show high binding potential of active ingredients and do not form micelles in water. Therefore, said compounds are envisaged for complexation and eventually solubilization of a variety of active agents, including pharmaceutically active ingredients. The invention further provides a method for preparing the inventive cyclodextrin compounds as well as composition comprising the cyclodextrin compounds of the invention.

Description

NOVEL WATER SOLUBLE 6-THIOALKYL-CYCLODEXTRINS AND USES
THEREOF
BACKGROUND
[1] Cyciodextrins (CDs) are cyclic oligomers of 6, 7, or 8 (α-, β-, and y-CDs, respectively) D- glucopyranose units linked by ot-(l - 4) bonds. All CDs have an internal cavity that is mainly hydrophobic and that allows the binding of guest molecules. These guest molecules are typically of hydrophobic or amphiphilic nature, depending on their size and polarity pattern. The formation of a host-guest complex allows for the solubilization of compounds which are barely soluble or even insoluble in water, and may also improve their stabilization.
[2] Inclusion of active substanFLU14400PCTEPDlces in CDs (i.e. the formation of CD complexes or inclusion compounds) is particularly used for, e.g., the formulation of pharmaceutical drugs, and cosmetics, as well as for chromatographic separations. Applications of CDs and CD derivatives in the pharmaceutical field are especially interesting since they allow solubilization of water-insoluble or volatile drugs in water. The observed solubilization of an active pharmaceutical ingredient (API) is generally based on the complexation of the hydrophobic part of the API molecule in the CD cavity. By so doing, CDs can, for example, increase the plasma-level or bioavailability of the complexed drugs and, in consequence, their therapeutic effect.
[3] α-, β-, and y-CDs are commercially available. However, their application potential is still limited. CDs generally accept only host molecules of a limited size range in their cavity; and in particular many macromolecu!es are not complexed by CDs. In addition, the Cyclodextrin : API molecule molar ratio is in general 1:1 or higher; i.e. no more than one guest molecule is complexed per molecule of cyclodextrin. In case of y-CDs the inclusion of two guest molecules is also known (W. Herrmann, S. Wehrle, and G. Wenz, Chem. Commun., pp. 1709-1710, 1997). The use of native CDs in pharmaceutical formulations is further limited by their low solubility in water (18 g/L or 25 mmol/L at 25°C for the most economically accessible β-CD) and their toxicity, in particular the nephrotoxicity of, e.g., β-CD in parenteral drug delivery (Frijlink et al. Pharm Res. 1991; 8(1):9-16). [4] CDs have been chemically modified, e.g. through chemical functionalization of the hydroxyl groups, in order to synthesize CD derivatives. E.g., Kawabata et al., Chem. Lett. 1986, 1933-1934 and Ling et al. Chem. Soc, Chem. Commun. 1993, 438-439 reported generation of hydrophobic β-CD derivatives by attaching long a Iky I chains (C4-C12) linkages to all primary positions via thioether or sulfoxide (WO2012/025937A1, De Boer, H. D.; van Egmond J. Br. J. Anaesth. 1996, 4, 473-9).
[5] Cyclodextrin derivatives comprising one or more glycosyl or maltosyl substituents linked to the cyclodextrin by a sulphur atom are also described by V. Laine et al. in J. Chem. Soc, Perkin Trans., 2, 1995, pp. 1479-1487. These derivatives have been used in order to solubilize an anti-inflammatory agent such as prednisolone.
[6] However, cyclodextrin derivatives known in the prior art are limited with regard to their applicability, in particular with respect to the active agents to be transported, the load capacity of active agents per mass unit of cyclodextrin derivative, their cost, their toxicity, their synthesis and/or their solubility in water.
[7] For example, statistical CD derivatives, such as hyd roxy pro pyl-CDs, methylated CDs and sulfonatobutyl CDs are indeed in use. However, quality control for such complex mixtures is a difficult issue. In addition, binding potentials of statistical CD derivatives are often lower than those of native CDs. Mazzaglia et al. J. Phys. Chem. C, 2008, 112 (17): 6764-6769 reported on amphiphilic β-CD derivatives with a Iky I chains (C2-C16) bound by the thioether linkages to the primary site and a statistical substitution with oligoethylene glycol at secondary sites. Becker and Ravoo Chem. Commun. (Camb). 2010; 46(24): 4369-71 (WO2011/039147A1) describe the synthesis of primary fluorinated cyclodextrin derivatives which exhibited amphiphilic properties after coupling to polar side chains at 2-/3-OH with different degrees of substitution. However, the authors generated only randomly substituted products; and in both cases lengths of the oligoethylene oxide side chains and locations of substitution have not been defined.
[8] Thus, there still exists a need in the prior art to provide improved CD derivatives that solve all of or some of the problems recited above. SUMMARY
[9] In a first aspect, the present invention provides novel cyclodextrin compounds having the formula
Figure imgf000004_0001
wherein
m + n is 6, 7 or 8; and n = 0, 1 or 2
Ri is independently selected from Cx to Q-alkyl;
R2 is independently selected from C0-(NH-CH2-CH2)o-NH2 or (CH2-CH2-0)0-(CH2)p-CH3; wherein o is 1, 2, 3, 4, 5 or 6; p is 0 or 1,
or R2 is (CH2)i-X wherein X is NH2 or OH, and wherein I is 2, 3, 4, 5 or 6;
O
-R3
or R2 is -CO-NH-R3, A ; R3. or CO-(NR3)2,
wherein R3 and R3' is independently from each other a hydrophilic branched or unbranched radical with 2 to 7, preferably 4 to 7 main chain atoms,
R2 is CO-NH-(CH2)q-R4, wherein q is 0, 1 or 2, and R4 is selected from OH, CH2-OH, and a 5 or 6 membered substituted or unsubstituted heterocyclic ring system containing at least one N or one 0 atom,
or R2 is CO-N-R5, wherein R5 is a 6 membered substituted or unsubstituted heterocyclic ring system and wherein N is part of the 6 membered heterocyclic ring,
or R2 is 0-CH2-CH2-NH-CH2-CH2-OH
or pharmaceutically acceptable salts thereof.
[10] It is envisaged that the cyclodextrin compounds are preferably water soluble and/or do not form micelles in aqueous solution.
[11] In further aspects the invention provides a method of preparing the cyclodextrin compounds of the invention comprising reacting a compound of formula (X),
Figure imgf000005_0001
wherein m + n is 6, 7 or 8;
Ri is selected independently from C C4 a Iky I;
with a compound of formula (Y) Hal-(CH2-CH2-0)0-(CH2)p-CH3,
wherein in formula (Y) Hal is halogen, o is 1, 2, 3, 4, 5, or 6, p is 0 or 1, or
with a compound of formula (Z) ( H2N-(CH2 CH2-NH)q-CH2 CH2-NH2),
wherein in formula (Z) q is 0, 1, 2, 3, 4, or 5, or
with a compound of formula (C) HO-(CH2)rX wherein X is NH2 or OH, and wherein I is 2, 3, 4, 5 or 6;
or with a compound of formula (D) selected from H2NR3 (formula Dl),
Figure imgf000005_0002
(formula D2) or HN(R3)2 (formula D3) wherein R3 and R3' is a hydrophilic branched or unbranched radical with 2 to 7, preferably 4 to 7 main chain atoms; or
a compound of formula (E) NH-(CH2)q-R4, wherein q is 1 or 2, and R4 is selected from OH, CH2- OH, and a 5 or 6 membered substituted or unsubstituted heterocyclic ring system containing at least one N or one O atom; or with a compound of formula (F) HN-R5, wherein R5 is a 6 membered substituted or unsubstituted heterocyclic ring system and wherein N is part of the 6 membered heterocyclic ring, wherein in the case of a reaction with the compound of formula (Z) (D), (E) or (F), the compound of formula (X) is first reacted with a coupling reagent.
[12] In further aspects, the present invention provides compositions comprising the inventive cyclodextrin compounds and at least one active agent as specified hereinafter complexed by said cyclodextrin compound.
[13] In a further aspect, the present invention relates to the use of a cyclodextrin compound according to the invention as a host molecule for at least one active agent as specified hereinafter. Said use preferably results in an increase of solubility, prevention of aggregation, avoiding micellar formulations, reduction of injection pain for intravenous applications, reduction of vapor pressure and/or targeting of active agents to avoid side effects with respect to the active agent.
[14] In a further aspect, the present invention provides a method of encapsulating a pharmaceutically active agent by contacting a cyclodextrin compound of the invention with a pharmaceutically active agent under conditions suitable for forming a host-guest complex.
****
[15] It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
[16] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.
[17] The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".
[18] The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes, however, also the concrete number, e.g., about 20 includes 20.
[19] As used herein, an interval which is defined as "(from) X to Y" equates with an interval which is defined as "between X and Y". Both intervals specifically include the upper limit and also the lower limit. This means that for example an interval of "2.5 mg/kg to 12.5 mg/kg" or "between 2.5 mg/kg to 12.5 mg/kg" includes a concentration of 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 11.5 and 12.5 as well as any given intermediate value.
[20] The term "less than" or "greater than" includes the concrete number. For example, less than 20 means less than or equal to. Similarly, more than or greater than means more than or equal to, or greater than or equal to, respectively. [21] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having".
[22] When used herein "consisting of excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.
[23] In each instance herein any of the terms "comprising", "consisting essentially of and "consisting of" may be replaced with either of the other two terms.
[24] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
[25] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.
DESCRIPTION OF THE FIGURES
Figure 1. Exemplary inventive cyclodextrins and overview of synthetic strategy.
Figure 2. Decay of relative vapour pressure A/A0 as function of the host concentration 2bi measured by GC headspace; the curve was fitted according to Equation (1).
Figure 3. ESI-MS spectra of CD derivatives 2bt (A) and 3bt (B).
Figure 4. 1H-NMR spectra of the statistical CD derivatives 2bx (A) and the corresponding uniform derivative 3 j in D SO-d6 (B). Numbers in grey are the integrals of the respective signals.
Figure 5. Transmission (λ = 670 nm) of aqueous solutions (1.0 wt.%) of 2b and 3bi.
Figure 6. Cell viability test for the cyclodextrin compounds of the invention. The viability of cells exposed to cyclodextrin compounds was assessed using the CellTiter-Glo® assay (Promega, #G7571) according to the manufacturer's manual on the colon tumor cell line Caco-2 (DSMZ #ACC-169; passage 6-9).
Figure 7. Transport of midazolam through the Caco-2 barrier and empty inserts over 4 hours. Samples were taken after 5, 15, 30, 120 and 240 minutes. Transport behaviour of concentrations of 1.11 pg/mL and 5.55 pg/mL midazolam was investigated.
DETAILED DESCRIPTION
[26] The present inventors have unexpectedly found out that substitution at the primary OH groups of CD advantageously does not alter or even increases its binding potential. Substitution of all primary hydroxy I groups by thioether groups advantageously gave rise to very high binding potentials due to the higher hydrophobicity of sulfur compared to oxygen. In addition, the generated cyclodextrin derivatives showed low ion strength in solution and no pH dependence was observed. The inventive cyclodextrin derivatives can be obtained in well-defined stoichiometry, no statistic, randomly substituted products are generated, thereby simplifying quality control and analysis.
[27] The CD thioethers of the invention are able to form highly stable complexes with active pharmaceutical ingredients, even with volatile APIs such as halothane, sevoflurane. In addition, the CD derivatives of the invention can be used for solubilization and even transcellular delivery of APIs is conceivable. The present inventors investigated the inclusion of volatile hydrophobic pharmaceutically active agents in the invented CD derivatives. The anesthetic sevoflurane was already included in a-CD, β-CD and hydroxypropy!^-CD, but the complex in a-CD is nearly insoluble in water (WO2012/104730 Al) and the binding constants for β-CD and hydroxypropyl- -CD are rather poor. The present invention thus provides water soluble CD derivatives with well-defined molecular structure and high binding affinities towards APIs and other active agents.
[28] In a first aspect, the present invention thus relates to a cyclodextrin compound having the formula
Figure imgf000009_0001
wherein
m + n is 6, 7 or 8; and n = 0, 1 or 2
Ri is independently selected from Ci to C4-alkyl;
R2 is independently selected from CO-(NH-CH2-CH2)0-NH2 or (CH2-CH2-0)0-(CH2)p-CH3; wherein o is 1, 2, 3, 4, 5 or 6; p is 0 or 1;
or R2 is -(CH2)|-X wherein X is NH2 or OH, and wherein I is 2, 3, 4, 5 or 6; R '
or R2 is -CO-NH-R3, ^3 or CO-(!MR3)2, wherein R3 and R3' is independently from each other a hydrophilic branched or unbranched radical with 2 to 7 main chain atoms;
or R2 is CO-NH-(CH2)q-R4, wherein q is 0, 1 or 2, and R4 is selected from OH, CH2 OH, and a 5 or 6 membered substituted or unsubstituted heterocyclic ring system containing at least one N or one O atom,
or R2 is CO-N-R5, wherein R5 is a 6 membered substituted or unsubstituted heterocyclic ring system and wherein N is part of the 6 membered heterocyclic ring;
or R2 is 0-CH2-CH2-NH-CH2-CH2-OH.
[29] The term "cyclodextrin" or "CD" is used herein to designate cyclic oligosaccharide compounds of 6, 7, or 8 (α-, β-, and y-CDs, respectively) saccharide units having the general formula
Figure imgf000010_0001
n = 6, 7 or 8.
[30] The terms "cyclodextrin compounds" and "cyclodextrin derivatives" are used interchangeably herein to designate compounds sharing the general cyclic oligosaccharide structure with cyclodextrins.
[31] As stated above, the cyclodextrin compounds of the present invention may comprise 6, 7 or 8 thioether glucopyranose units (i.e. can be α-, β- or y-cyclodextrin compounds, respectively); in particular 6-deoxy-6-thioether glucopyranose units. The number of thioether glucopyranose units having an R2 substituent at the secondary OH group is designated "m" herein. The number of thioether glucopyranose units having no R2 substituent is designated "n" herein. Preferably, all thioether glucopyranose units are comprise an R2 substituent at the secondary OH-group. However, it is also envisaged that unsubstituted thioether glucopyranose units are present in the inventive cyclodextrin compound. The number of unsubstituted thioether glucopyranose units (n) can be 0, 1 or 2. [32] It is to be understood all cyclodextrin compounds envisaged herein are preferably (i) capable of complexing an active agent (ii) do not form micelles in aqueous solution and (iii) are nontoxic, comparable to the compounds tested in the appended examples. The person skilled in the art will readily be able to choose suitable substituents depending on the above-mentioned desired properties and use of the cyclodextrin compound, e.g. complexation of a hydrophobic pharmaceutically active ingredient. Advantageously and preferably, the cyclodextrin compounds of the present invention are hydrophilic and neutral or weakly charged, show low ion strength in solution and are scarcely pH dependent. It is also envisaged that some of the inventive cyclodextrin compounds may be capable of crossing cellular membranes, thus enabling their use as carriers for membranes or transcellular delivery.
[33] As stated above, Ri is independently selected from CI to C4-alkyl. The term "CI to C4-alkyl" as used herein refers to straight and branched chain saturated acyclic hydrocarbon groups having from 1 to 4 carbon atoms such as, for example, methyl, ethyl, n-propyl, isopropyl (1-methylethyl), cyclopropyl, n-butyl, 2-butyl (2-methylpropyl), ier -butyl (1,1-dimethylethyl), or the like. In some embodiments Ri is preferably methyl.
[34] As stated above, in the compounds of the present invention R2 can be independently selected from CO-(NH-CH2-CH2)0-NH2 or (CH2-CH2-O)0-(CH2)P-CH3; wherein o is 1, 2, 3, 4, 5 or 6; p is 0 or l.
[35] In one embodiment R2 is CO-(NH-CH2-CH2)0-NH2 or (CH2-CH2-0)o-CH3, wherein o is 1, 2, 3 or 4, wherein o is preferably 3 or 4.
[36] In one embodiment, m+n = 7; n = 0-2; Rn is methyl; and R2 is (CH2-CH2-0)0-CH3; wherein o is 3 or 4. It is further envisaged that o can be (exactly) 3 or 4.
[37] In another embodiment, m+n = 6; n = 0-2; Ri is methyl; and R2 is (CH2-CH2-0)o-CH3; wherein o is 3 or 4. It is further envisaged that o can be (exactly) 3 or 4.
[38] (10) In another embodiment, m+n = 8; n = 0-2; Ri is methyl; and R2 is (CH2-CH2-0)o-CH3; wherein o is 3. It is further envisaged that o can be (exactly) 3.
[39] (11) In another embodiment, m > 2; R, is methyl; and R2 is CO-(NH-CH2-CH2)0-NH2; wherein o is 1. It is further envisaged that o can be exactly 1.
[40] Alternatively, in the compounds of the present invention R2 can be -(CH2)rX wherein X is H2 or OH, and wherein I is 2, 3, 4, 5 or 6. [41] Alternatively, in the compounds of the present invention R2 can be
-CO-NH-R3,
Figure imgf000012_0001
or CO-(NR3)2, wherein R3 and R3' is independently from each other a hydrophilic branched or unbranched radical with 2 to 7 main chain atoms. In particular, R3 and R3' can be independently from each other a hydrophilic branched or unbranched radical with 4 to 7 main chain atoms.
[42] The term "radical" in general designates an aliphatic or aromatic substituted or unsubstituted hydrocarbon radical. In the context of the present invention, the radical is preferably an aliphatic radical which may be substituted or unsubstituted, saturated or unsaturated, branched or unbranched. Exemplary radicals include alkyl-, alkenyl-, alkynyl- radicals with at least one terminal hydroxyl or amino group. In one embodiment, R3 or R3' is -CH2-CH2-OH. -CH2-CH2-NH2
[43] R3 may comprise at least one terminal hydroxyl or amino group.
[44] The term "hydroxyl" refers to a group of the general formula -OH.
[45] The term "amino" refers to a group of the general formula -NR2, wherein R is hydrogen or a monovalent aliphatic or aromatic hydrocarbon substituent.
[46] Alternatively, R2 can be selected independently from CO-NH-(CH wherein q is 0, 1 or 2, and R is selected from OH, CH2-OH, and a 5 or 6 membered substituted or unsubstituted heterocyclic ring system containing at least one N or one 0 atom. In one embodiment, q is 1. In one embodiment, R4, when being a 5 or 6 membered heterocyclic ring system containing at least one N or one 0 atom, is an aromatic ring system.
[47] As used herein, the term "substituted" refers to any one or more hydrogen atoms on the indicated molecule being replaced with a selected group referred to herein as a "substituent". A substituted group can have 1 to 5, preferably 1 to 3, and more preferably 1 independently selected substituents as defined herein. Suitable substituents can be selected from the group including, sulfonate ( S03 ), sulfate (-S04 2 ), phosphate (-P04 3 ), phosphonate (-P03 2 ) sulfonyl, aminosulfonyl, sulfonylamino, carboxyamide, aminocarbonyl, carboxy, oxo, hydroxy, amino, nitro, cyano and ureido.
[48] As used herein and unless stated otherwise, the term "heterocyclic" or "heterocycle" refers to a 5 or 6-membered ring containing at least one N or one 0 atom, and optionally one or more heteroatomic moieties selected from S, SO, S02, O, N, or N-oxide. Said heterocycle can be saturated or have one or more degrees of unsaturation. The heterocycle may be optionally fused to one or more "heterocyclic" ring(s), aryl ring(s), heteroaryl ring(s), or carbocycle ring(s), each having optional substituents.
[49] Preferred examples of heterocyclic R moieties include, but are not limited to amino, 2-furyl, 3-furyl, 1-pyrrolyl, 2-pyrrolyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-picolyl or 3-picolyl.
[50] Other exemplary heterocyclic R4 moieties in accordance with the present invention include without limitation 1,4-dioxanyl, 1,3-dioxanyl, pyrrolidinyl, pyrrolidin-2-onyl, piperidinyl, imidazolidine-2,4-dionepiperidinyl, piperazinyl, piperazine-2,5-dionyl, morpholinyl, dihydropyranyl, dihydrocinnolinyl, 2,3-dihydrobenzo[l,4]dioxinyl, 3,4-dihydro-2H-benzo[b][l,4]-dioxepinyl, tetrahydropyranyl, 2,3-dihydrofuranyl, 2,3-dihydrobenzofuranyl, dihydroisoxazolyl, tetrahydrobenzodiazepinyl, tetrahydroquinolinyl, tetrahydrofuranyl, tetrahydronaphthyridinyl, tetrahydropurinyl, tetrahydrothiopyranyl, tetrahydrothiophenyl, tetrahydroquinoxalinyl, tetrahydropyridinyl, tetrahydrocarbolinyl, 4H-benzo[l,3]-dioxinyl, benzo[l,3]dioxonyl, 2,2- difluorobenzo-[l,3]-dioxonyl, 2,3-dihydro-phthalazine-l,4-dionyl, and isoindole-l,3-dionyl.
[51] The term "aromatic ring system" as used herein denotes fully unsaturated carbocycles and heterocycles in which at least one ring of a polycyclic ring system is aromatic.
[52] Alternatively, in the compounds of the present invention R2 can be CO-N-R5, wherein R5 is a 6 membered substituted or unsubstituted heterocyclic ring system and wherein N is part of the 6 membered heterocyclic ring.
[53] E.g., it is envisaged that N-R5 is selected from the group comprising morpholino having the general formula
Figure imgf000013_0001
[54] or piperazine having the general formula
Figure imgf000014_0001
[55] Said heterocyclic ring may also be substituted, e.g. with one or more substituents selected from the group including sulfonate (-S03 ), sulfate (-S04 2 ), phosphate (-P04 3~), phosphonate (-P03 2 ), sulfonyl, aminosulfonyl, sulfonylamino, carboxyamide, aminocarbonyl, carboxy, oxo, hydroxy, amino, nitro, cyano and ureido. The present invention provides, in a further aspect, cyclodextrin compounds as defined herein, which are water-soluble. The water solubility of a substance is the saturation mass concentration of the substance in water at a given temperature. It is s expressed in mass of solute per volume of solution, i.e. kg/m3 or g/L. It is for example envisaged that the invented cyclodextrin derivatives have a water-solubility of higher than 18 g/L at 25°C, such as 30 g/L Preferably, the inventive cyclodextrin derivatives have a water-solubility of more than 100 g/L. The water-solubility can be assessed, e.g., using the column elution method or the flask method as described in OECD Guideline Nr. 105 (1995).
[56] It is in particular envisaged that the inventive cyclodextrin compounds do not form micelles in aqueous solution. A micelle is an aggregate of surfactant molecules (usually amphiphilic organic molecules containing both hydrophobic groups ('tails') and hydrophilic groups (' heads') dispersed in a liquid colloid. For the transport of active ingredients, e.g. APIs, micelle formation is problematic because the amount of active ingredient enclosed in the micelles cannot be controlled. Therefore, it is preferred to provide cyclodextrin compounds which do not form micelles in aqueous solution, but rather form complexes with active ingredients in a defined molar ratio (e.g., 1 molecule of CD complexes 0.05 to 2, or preferred 0.3 to 2 molecules of active ingredient). Micelle formation can be assessed using various methods as described in Dominguez et al. J Chem Edu, 1997; 74(10): 1227- 1231 . E.g., the UV-Absorption Spectroscopy Method is based on the tautomerism of ketones or (for example benzoylacetone (BZA), l-phenyl-l,3-butadione). Keto-enol equilibria of BZA are extremely solvent sensitive and the proportion of the enolic form is much greater in nonpolar solvents, such as cyclohexane, than in polar or hydrogen-bond donor solvents, such as water or alcohols. When a surfactant is added to a water solution of BZA, the amount of enol increases abruptly when micelles, which provide a less polar solvent than the aqueous phase take up the enolic form. Other methods for assessing micelle formation include the Fluorescence Spectroscopy Method based on the solvent dependence of vibrational band intensities in pyrene monomer fluorescence (M. Wilhelm, et al., Macromolecules, 1991; 24(5), 1033-1040) and the electrical conductivity method for anionic surfactants as well as dynamic light scattering.
[57] The cyclodextrin compounds of the present invention may be neutral or hydrophilic or amphiphilic. It is contemplated that amphiphilic CD molecules can enter and cross cell membranes, and thus act as a carrier which supports an active ingredient, such as an API, in overcoming cellular barriers, such as the intestinal barrier or the blood-brain barrier (BBB).
Synthesis
[58] The present invention also provides a method of preparing a cyclodextrin compound as defined herein.
[59] First, a halogenated cyclodextrin can be provided, e.g. following the protocol of Gadelle and Defaye, Angew. Chem. Int. Ed. Engl. 1991, 30: 78-79. A cyclodextrin is reacted with a nucleophile (e.g., triphenylphosphane (PPh3)), and a halogen compound (e.g. /V-Bromosuccinimide, NBS) in a solvent such as DM F at high temperature (e.g. about 70°C). Next, the thioethers are prepared by reaction of the halogenated cyclodextrin with linear thiolates NaSR (R =CnH2n, n = 1, 2, 3 or 4) by nucieophilic displacement reactions in a suitable solvent such as DM F. Then, the thioether cyclodextrin is reacted with a compound of formula (Y) Hal-(CH2-CH2-0)0-(CH2)p-CH3, wherein Hal is halogen, o is 1, 2, 3, 4, 5, or 6, p is 0 or 1, under basic conditions (e.g. in the presence of NaH and tetra-n-butylammoniumiodide (TBAI)). The reaction mixture is quenched by addition of ethanol or methanol, the solvents are removed by vacuum distillation and the residue is dissolved in water and neutralized by addition of HCI.
[60] An exemplary reaction scheme is depicted below:
Figure imgf000015_0001
[61] Alternatively, a thioether cyclodextrin is obtained as described above, and reacted with a coupling reagent. Suitable coupling reagents can be selected from the group of - carbonyldiimidazole, phosgene, diphosgene and triphosgene. Subsequently, there is added, depending on the cyclodextrin compound to be prepared, a compound of the formula (Z) (H2N-(CH2CH2-NH)q-CH2CH2-NH2), wherein q is 0, 1, 2, 3, 4, or 5; or a compound of the formula (C) HO-(CH2)l-X, wherein X is NH2 or OH, and wherein I is 2, 3, 4, 5 or 6;
or a compound of formula (D) selected form H2N-R3 (formula Dl),
Figure imgf000016_0001
(formula D2) or HN(R3)2
(formula D3), wherein R3 is a hydrophilic branched or unbranched radical with 2 to 7 main chain atoms, in particular, wherein R3 is a hydrophilic branched or unbranched radical with 4 to 7 main chain atoms; or a compound of formula (E) NH-(CH2)q-R4, wherein q is 1 or 2, and R4 is selected from OH, CH2-OH, and a 5 or 6 membered substituted or unsubstituted heterocyclic ring system containing at least one N or one O atom, or a compound of formula (F) HN-RS, wherein R5 is a 6 membered substituted or unsubstituted heterocyclic ring system and wherein N is part of the 6 membered heterocyclic ring.
Pharmaceutically acceptable salts
[62] For the purpose of the invention, the inventive cyclodextrin compounds as defined above also include the pharmaceutically acceptable salt(s) thereof. The phrase "pharmaceutically acceptable salt(s)", as used herein, means those salts of compounds of the invention that are safe and effective for the desired administration form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[63] The use of salt formation as a means of varying the properties of pharmaceutical compounds is well known and well documented. Salt formation can be used to increase or decrease solubility, to improve stability or toxicity and to reduce hygroscopicity of a drug product. There are a wide range of chemically diverse acids and bases, with a range of pKa values, molecular weights, solubilities and other properties, used for this purpose. Of course, any counterions used in pharmaceuticals must be considered safe, and several lists of pharmaceutically approved counterions exist, which vary depending on the source. Approved salt formers can e.g. be found in the Handbook of Pharmaceutical Salts (Stahl PH, Wermuth CG, editors. 2002. Handbook of pharmaceutical salts: Properties, selection and use. Weinheim/Zurich: Wiley-VCH/VHCA.). Compositions
[64] In a further aspect, the present invention provides a composition comprising a cyclodextrin compound of the invention (as a host molecule) and at least one active agent complexed (as a guest molecule) with said cyclodextrin.
[65] Without wishing to be bound by theory, it is thought that the driving forces for the formation of CD inclusion compounds are mainly nondirectional interactions such as hydrophobic and van der Waals interactions. The dominance of solvatophobic interactions is evident in the fact that the inclusion of guests in CDs occurs preferentially in aqueous solution. The magnitude of hydrophobic interactions is determined mainly by the hydrophobic surface area of the guest. Additional polar groups can be attached to the CD ring, e.g. to improve binding selectivity. The term "host : guest complex" is used interchangeably with the terms "inclusion complex", and "CD complex" herein.
[66] The term "active agent" is used interchangeably with the term "active ingredient" herein. Generally, any active agent can be chosen which is capable of being complexed by the cyclodextrin compounds of the invention. In particular, said active agent can be selected from the group consisting of a pesticide, herbicide, insecticide, antioxidant, plant growth instigator, sterilization agent, catalyst, chemical reagent, food product, nutrient, cosmetic, vitamin, sterility inhibitor, fertility instigator, microorganism, flavoring agent, sweetener, cleansing agent and pharmaceutically active agent.
[67] The term "pharmaceutically active agent" is used interchangeably with the term "active pharmaceutical ingredient" or "API" herein and refers to a substance or substance combination used in manufacturing a drug product. It also refers to the active or central ingredient in the product which causes the direct effect on the disease diagnosis, prevention, treatment or cure. (18) Said pharmaceutically active agent can be selected from the group consisting of anesthetics, analgesics, steroids, cytostatic drug, antiviral agents, nutrients, nutritional agents, hematological agents, endocrine agents, metabolic agents, cardiovascular agents, renal agents, genitourinary agents, respiratory agents, central nervous system agents, gastrointestinal agents, anti-infective agents, biologic agents, immunological agents, dermatological agents, ophthalmic agents, antineoplastic agents, and diagnostic agents.
[68] It is in particular envisaged that the anaesthetic and/or the analgesic is selected from the group consisting of midazolam, sevofiurane, halothane, propofol, xenon, fentanyl, the steroid is selected from the group consisting of hydrocortisone, testosterone, estradiol and contraceptives, the cytostatic drug is selected from the group consisting of camptothecin, paclitaxel, and anthracyclines, e.g. idarubicine, the neurolepticum is selected from the group consisting of haloperidol, fluspirilen, and risperidon), and the antiviral agent is selected from the group consisting of lopinavir, ritonavir, atazanavir.
[69] It is thus envisaged that the compositions of the present invention are used in the treatment of a subject. The term "treatment" in all its grammatical forms includes therapeutic or prophylactic treatment. A "therapeutic or prophylactic treatment" comprises prophylactic treatments aimed at the complete prevention of clinical and/or pathological manifestations or therapeutic treatment aimed at amelioration or remission of clinical and/or pathological manifestations. The term "treatment" thus also includes the amelioration or prevention of diseases.
[70] A variety of routes are applicable for administration of the composition of the present invention, including, but not limited to, topically (e.g. epicutaneously, inhalationally, mucosally, intraocularly), enterally (e.g. orally, recta I ly), and parenterally (e.g. intravenously, intra-arterially, intraperitoneally, intramuscularly, subcutaneously, transdermally).
[71] Preferably, the pharmaceutical composition of the present invention is adapted for parenteral administration by infusion or injection. With an "infusion" is meant a continuous administration over a certain period of time. For example such an administration may take in between 10 minutes to 4 days. An "injection" means is a transient infusion method of introducing fluid into the body of a subject. Injection(s) may e.g. be administered for 1, 2, 3 or 4 days.
[72] The composition according to the invention may be in solid, liquid or gaseous form and may be, inter alia, in the form of an ointment, a cream, transdermal patches, a gel, powder, a tablet, solution, an aerosol, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tincture or fluid extracts or in a form which is particularly suitable for the desired method of administration, in particular parenteral administration. It is however preferred that the inventive composition is in liquid form.
[73] In accordance with the foregoing, the present invention also envisages a method of diagnosing or treating a subject in need thereof, comprising administering the inventive composition, which preferably comprises a pharmaceutically effective amount of the pharmaceutically active agent.
[74] The„subject" is preferably a mammal such as a human, monkey, cat, dog, horse, pig, cattle, guinea pig, mouse or rat with human being preferred. [75] The exact administration dose of the inventive composition will depend on the purpose of the treatment (e.g. remission maintenance vs. acute flare of disease), and will be ascertainable by one skilled in the art using known techniques.
[76] In the compositions of the invention, the molar ratio between the cyclodextrin compound and the active agent can be in any suitable range. Typically, the cyclodextrin is present in a molar excess to ensure that the entire (amount) of active agent is complexed by the cyclodextrin. In illustrative examples, the molar ratio between the cyclodextrin and the active agent can range from 10:1 to 1:1, e.g. it can be 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.
Excipient
[77] A composition of the invention can further comprise one or more pharmaceutical excipient. In general, any pharmaceutical excipient is conceivable as long as it does not interfere with the ability of the inventive cyclodextrin compound to form a host : guest complex with the active agent.
[78] Pharmaceutical excipients are typically inactive ingredients of a drug product that do not increase or affect the diagnostic, therapeutic, preventive, or curing action of the active ingredient. Generally, "pharmaceutically acceptable excipients" are nontoxic to subjects to be treated at the dosages and concentrations employed. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.
[79] Pharmaceutically acceptable excipients include, but are not limited to diluents (fillers, bulking agents, e.g. lactose, microcrystalline cellulose), disintegrants (e.g. sodium starch glycoiate, croscarmellose sodium), binders (e.g. PVP, HPMC), lubricants (e.g. magnesium stearate), glidants (e.g. colloidal Si02), solvents/co-solvents (e.g. aqueous vehicle, propylene glycol, glycerol), buffering agents (e.g. citrate, gluconates, lactates), preservatives (e.g. Na benzoate, parabens (Me, Pr and Bu), BKC), anti-oxidants (e.g. BHT, BHA, Ascorbic acid), wetting agents (e.g. polysorbates, sorbitan esters), anti-foaming agents (e.g. Simethicone), thickening agents (e.g. methylcellulose or hydroxyethylcellulose), sweetening agents (e.g. sorbitol, saccharin, aspartame, acesulfame), flavouring agents (e.g. peppermint, lemon oils, butterscotch, etc.), humectants (e.g. propylene, glycol, glycerol, sorbitol). The person skilled in the art will readily be able to choose suitable pharmaceutically acceptable excipients, depending, e.g. on the formulation and administration route of the composition. [80] A non-exhaustive list of exemplary pharmaceutically acceptable excipients includes without limitation buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl para ben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; tonicifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactant such as polysorbate; salt-forming counter- ions such as sodium; metal complexes (e. g. Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM, PLU ONICS™ or polyethylene glycol (PEG). The antibody preferably comprises the antibody at a concentration of between 5-200 mg/mL, preferably between 10-100 mg/mL.
Use
[81] In a further aspect, the present invention relates to the use of a cyclodextrin compound as described herein as a host molecule for at least one active agent. The embodiments described in the context of the composition are also applicable to the use of the invention, mutatis mutandis.
[82] It is in particular envisaged that the use comprises with respect to the at least one active agent an increase of solubility, prevention of aggregation, avoiding micellar formulations, reduction of injection pain for intravenous applications, reduction of vapor pressure of volatile active agents, targeting of active agents to avoid side effects.
[83] The cyclodextrin compounds of the invention preferably increases solubility of otherwise insoluble or barely soluble active ingredients, e.g. in water, by complexing the active ingredient. Solubility of an active ingredient can be measured according to standard protocols as described in Thiele C. et al. J. Incl. Phenom. Macrocycl. Chem. 2011; 69:303-307. Shortly, solutions of CD compounds of different concentrations in water are stirred with an excess of the active ingredient. The resulting solutions are filtered and the concentration of the dissolved drug is determined by UV.
[84] The cyclodextrin compounds of the invention may further prevent aggregation of active ingredients. It is thought that the cyclodextrin compounds will improve bioavailability of the ingredient due to dissolution of aggregates. [85] It has already been set out elsewhere herein that the cyclodextrin compounds according to the invention preferably avoid the formation of micellar formulations.
[86] In addition, it is envisaged that the inventive cyclodextrin compounds reduce injection pain for intravenous application. Injection pain is typical for administration of emulsions such as state of the art propofol formulations.
[87] When used for complexation of volatile active agents, such as volatile anaesthetics, the inventive compounds are envisaged to reduce the vapor pressure of said agents by complexing them. The vapor pressure of an agent is typically quantified by gas chromatography applying the head space technique (S. Fourmentin, A. Ciobanu, D. Landy, G. Wenz, Beilstein J. Org. Chem. 2013, 9, 1185-1191).
[88] The inventive compounds may be further used for targeting active agents, in order to enable a fast and specific delivery of active agents. It is thought that the inventive compounds can be conjugated with cell specific ligands, such as folate, transferrin, or insulin.
Method
[89] In a further aspect, the present invention provides a method of encapsulating a pharmaceutically active agent comprising contacting a cyclodextrin compound as defined herein with a pharmaceutically active agent under conditions suitable for forming a host-guest complex, thereby complexing the pharmaceutically active agent within the cyclodextrin compounds as described herein.
[90] The term "encapsulating" in all its grammatical forms, designates the process of forming a host : guest complex between the inventive cyclodextrin derivative and the pharmaceutically active agent, i.e. the pharmaceutically active agent is complexed by the cyclodextrin derivative of the invention. Preferably, "encapsulating" does not involve the formation of micelles. In one preferred embodiment, the ratio of cyclodextrin derivative and pharmaceutically active agent is between 4:1 and 1:1 (cyclodextrin: guest), i.e. 4:1, 3:1, 2:1 or 1:1 or any other desired molar ratio
[91] A better understanding of the present invention and of its advantages will be had from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way. EXAMPLES
[92] Materials. All chemicals (except CDs) were purchased from Sigma Aldrich, Merck, Acros Organ ics, Fisher Scientific or TCI Europe and were used without further purification, α-, β- and γ-CD were provided by Wacker Chemie AG, Munich, Germany and were used after drying overnight at 60°C under reduced pressure. Human serum was kindly provided by University Hospital of Wiirzburg. All measurements were performed in saline HEPES-buffer solution (pH=7.4) with a NaCI concentration of 0.9 wt.%.
[93] Some products were purified by cross-flow nanofiltration using a membrane called Minimate TPP Capsule from Pall, Crailsheim, Germany, further a membrane called Omega with a Cut-off of 650 Da was used. Freeze drying was carried out with a lyophi!izer Lyophille Alpha 1-4 produced by Christ, Osterode am Harz, Germany.
[94] Throughout the description and in the following examples, a systemized nomenclature is used to designate the compounds of the invention which is derivable from Figure 1 and the following matrix.
[95]
Group General structure No. glyopyranose units R6
l =Thiomethyl, SMei
2 =Thioethyl, SEt2 e glycol
ne glycol
Figure imgf000022_0001
Figure imgf000023_0001
Accordingly, the compound 3bj., for example, comprises seven glycopyranose units (i.e., is a β- cyclodextrin) and a thiomethyl group at the R6 position.
Example 1 : Heptakis[6-deoxy-6-methylsulfanyl-2-(2'-(2"-<2",-methoxyethoxy)- ethoxy)ethyl)]-P-cyclodextrin - 3b
Figure imgf000024_0001
[96] 2.60 g (65 mmol) NaH (60 wt.% dispersion in mineral oil, Sigma-Aldrich) was washed twice with 25 mL of n-pentane under N2 and stirred at rt for 1 h. After addition of 6.25 g (4.64 mmol) heptakis(6-deoxy-6-methylsulfanyl)^-cyclodextrin dissolved in 130 mL of DMF, 17.8 g (65 mmol) 2- (2-(2-methoxyethoxy)ethoxy)ethyl iodide and 17.5 mg (0.05 mmol) tetra-n-butylammonium iodide were added and the resulting reaction mixture was stirred at 60°C under N2 for 6 d. The reaction was quenched by addition of 50 mL of ethanol and stirred at rt for further 30 min. The solvents were completely removed by vacuum distillation (bath temperature 70°C, 1 mbar) and the residue was dissolved in 200 mL of water and neutralized by addition of 1 M HCI. The crude product was isolated by extraction with ethyl acetate at 70°C using a Kutscher-Steudel extractor. The organic phase was concentrated in vacuo and the remaining residue was fractionized by column chromatography over 1.0 kg of silica (60 A, 70-230 mesh, Fluka) with an ethyl acetate / methanol gradient (100/0→ 90/10 → 0/100 v/v) as eluent. The product (7.5 g, 68%) was obtained as a yellowish oil after complete removal of the eluent by vacuum distillation and drying at 60°C in vacuo (0.03 mbar) for 3 d.
TLC: Rf (EtOAc/MeOH 9/1 v/v) = 0.06.
Rf ( eOH) = 0.57.
1H-NMR δ/ppm (DMSO-d6, 400 MHz) - 5.03 (d, 1H, H-l, 3J= 3.3 Hz), 4.89 (s, 1H, OH-3), 4.01-3.96
(m, 1H, H-8a), 3.79-3.69 (m, 3H, H-3, H-5, H-8b), 3.53 (s, 8H, H-8, H-9) 3.50 (m, 1H, H- 4), 3.44-3.38 (m, 3H, H-2, H-9), 3.24 (s, 3H, 0-CH3), 3.10-3.07 (m, 1H, H-6a), 2.75 (dd, 1H, H-6b, 3J= 14.1 Hz, 7.8 Hz), 2.08 (s, 3H, H-7). 13C-NMR: δ/ppm (DMSO-d6, 100 MHz) = 100.5 (C-l), 85.5 (C-4), 71.3 (C-2, C-3, C-5), 69.8-69.6 (C-8, C-9), 58.0 (C-10), 35.0 (C-6), 16.0 (C-7).
ESI-MS: m/z = 2390.90 [M+Na]+.
Example 2: Hexakis[6-deoxy-6-methylsulfanyl-2-(2'-{2"-<2<"-methoxyethoxy)- ethoxy)ethyl)]-a-cyclodextrin - 3a1
Figure imgf000026_0001
[97] 0.421 g (10.5 mmol) NaH (60 wt.% dispersion in mineral oil, Sigma-Aldrich) was washed twice with 10 mL of n-pentane under N2 and stirred at rt for 1 h. After addition of 0.97 g (0.84 mmol) hexakis(6-deoxy-6-methylsulfanyl)-a-cyclodextrin dissolved in 50 mL of D F, 2.87 g (10.5 mmol) 2- (2-(2-methoxyethoxy)ethoxy)ethyl iodide and 3 mg (0.008 mmol) tetra-n-butylammonium iodide were added and the resulting reaction mixture was stirred at 60°C under N2 for 6 d. The reaction was quenched by addition of 10 mL of methanol and stirred at rt for further 30 min. The solvents were completely removed by vacuum distillation (bath temperature 70°C, 1 mbar) and the residue was dissolved in 40 mL of water and neutralized by addition of 1 M HCI. The crude product was purified by Crossflow nanofiltration in water (650 Da, Pall Minimate TFF Capsule) and a yellowish oil (95 mg, 13 %) was obtained after lyophilization.
1H-NIVI δ/ppm (DMSO-d6, 400 MHz) = 5.00 (bs, 1H, H-l), 4.61 (bs, 1H, OH-3), 3.89-3.95 (m, 1H,
H-8a), 3.78-3.80 (m, 1H, H-5), 3.81 - 3.86 (m, 1H, H-3), 3.71-3.77 (m, 1H, H-8b), 3.50- 3.55 (m, 10H, H-10, 11, 12, 13) 3.43-3.45 (m, 1H, H-4), 3.41 - 3.42 (m, 2H, H-9), 3.34 - 3.37 (m, 1H, H-2), 3.23 (s, 3H, 0-CH3), 3.05-3.08 (m, 1H, H-6a), 2.74-2.79 (m, 1H, H-6b), 2.07 (s, 3H, H-7).
13C-NMR: δ/ppm (DMSO-d5( 100 MHz) = 100.2 (C-l), 85.9 (C-4), 80.2 (C-2), 72.8 (C-3), 71.3 (C-9),
70.8 (C-8) 70.7 (C-5), 69.5-69.9 (C-10, C-ll, C-12, C-13), 58.1 (0-CH3), 35.1 (C-6), 16.2 (C-7). Example 3: Octakis[6-deoxy-6-methylsulfanyl-2-(2'-(2"-{2<<<-methoxyethoxy)- ethoxy)ethyl)]-Y-cyclodextrin - 3c
Figure imgf000027_0001
[98] 0.418 g (10.4 mmol) NaH (60 wt.% dispersion in mineral oil, Sigma-Aldrich) was washed twice with 10 mL of n-pentane under N2 and stirred at rt for 1 h. After addition of 1.01 g (0.65 mmol) octakis(6-deoxy-6-methylsu!fanyl)-y-cyclodextrin dissolved in 50 mL of DMF, 2.90 g (10.6 mmol) 2-(2- (2-methoxyethoxy)ethoxy)ethyl iodide and 3 mg (0.008 mmol) tetra-n-butylammonium iodide were added and the resulting reaction mixture was stirred at 60°C under N2 for 4 d. The reaction was quenched by addition of 10 mL of methanol and stirred at rt for further 30 min. The solvents were completely removed by vacuum distillation (bath temperature 70°C, 1 mbar) and the residue was dissolved in 50 mL of water and neutralized by addition of 1 m HCI. The crude product was purified by Crossflow nanofiltration in water (1 kDa, Pall inimate TFF Capsule) and a yellowish oil (1.57 g, 89 %) was obtained after lyophilization.
TLC: Rf (EE: OH:NH4OH:H20 7:7:5:2 v/v) = 0.82.
1H-NMR δ/ppm (DMSO-d6, 400 MHz) = 5.10 (d, 1H, H-l, 3J =3.0 Hz), 4.84 (bs, 1H, OH-3), 3.92- 3.96 (m, 1H, H-8a), 3.76-3.80 (m, 1H, H-8b), 3.73-3.76 (m, 1H, H-5) 3.68-3.70 (m, 1H, H-3), 3.50-3.53 (m, 8H, H-10, 11, 12, 13) 3.41-3.44 (m, 1H, H-9), 3.38-3.41 (m, 2H, H-2), 3.35-3.38 (m, 1H, H-4), 3.23 (s, 3H, 0-CH3), 3.05-3.10 (m, 1H, H-6a), 2.71-2.76 (m, 1H, H-6b), 2.09 (s, 3H, H-7).
13C-NMR: δ/ppm (DMSO-d6, 100 MHz) = 99.8 (C-l), 84.1 (C-4), 80.7 (C-2), 72.3 (C-3), 71.3 (C-9),
71.0 (C-8) 70.7 (C-5), 69.5-69.9 (C-10, C-ll, C-12, C-13), 58.0 (0-CH3), 35.1 (C-6), 16.1 (C-7).
ESI-MS: m/z = 2729.32 [M+Na]+. Example 4: Heptakis[6-deoxy-6-methylsulfanyl-2-(2'-(2"-(2"'-{2""-methoxy- ethoxy)ethoxy)ethoxy)ethyl)]-P-cyclodextrin - 4bi
Figure imgf000028_0001
[99] 0.36 g (8.92 mmol) NaH (60 wt.% dispersion in mineral oil, Sigma-Aldrich) was washed twice with 10 mL of n-pentane under N2 and stirred at rt for 1 h. After addition of 1.01 g (0.74 mmol) heptakis(6-deoxy-6-methylsulfanyl)^-cyclodextrin dissolved in 50 mL of DMF, 2.84 g (8.92 mmol) 2- (2-(2-(2-methoxyethoxy)ethoxy)ethoxy)ethyl iodide and 3 mg (0.008 mmol) tetra-n-butylammonium iodide were added and the resulting reaction mixture was stirred at 80°C under N2 for 7 d. The reaction was quenched by addition of 10 mL of methanol and stirred at rt for further 30 min. The solvents were completely removed by vacuum distillation (bath temperature 70°C, 1 mbar) and the residue was dissolved in 50 mL of water and neutralized by addition of 1 M HCI. The crude product was purified by Crossflow nanofiltration in water (1 kDa, Pall Minimate TFF Capsule) and a brown yellowish oil (0.277 g, 14 %) was obtained after lyophilization.
1H-NMR δ/ppm (DMSO-d6, 400 MHz) = 5.03 (d, 1H, H-l, 3J= 3.0 Hz), 4.89 (s, 1H, OH-3), 4.00-3.94
(m, 1H, H-8a), 3.78-3.70 (m, 1H, H-8b), 3.53-3.50 (m, 12H, H-9, H-10, H-ll, H-12, H- 13, H-14), 3.44-3.38 (m, 4H, H-2, H-4, H-15), 3.24 (s, 3H, 0-CH3), 3.10-3.06 (m, 1H, H- 6a), 2.78-2.69 (m, 1H, H-6b), 2.08 (s, 3H, H-7).
13C-NMR: δ/ppm (DMSO-d6, 100 MHz) = 100.6 (C-l), 85.6 (C-4), 80.6 (C-2), 72.6 (C-3), 71.3 (C- 9), 70.8 (C-8), 70.7 (C-5), 69.8-69.6 (C-10, C-ll, C-12, C-13, C-14, C-15), 58.1 (0-CH3), 35.1 (C-6), 16.0 (C-7).
ESI-MS: m/z = 2699.55 [M+Na]+. Example 5: Hexakis[6-deoxy-6-meihylsulfanyl-2-(2'-(2"-(2'"-C2""-methoxy- ethoxy)ethoxy)ethoxy)ethyl)]-a-cyclodextrin - 4a
Figure imgf000029_0001
[100] 0.42 g (10.4 mmol) NaH (60 wt.% dispersion in mineral oil, Sigma-Aldrich) was washed twice with 10 mL of n-pentane under N2 and stirred at rt for 1 h. After addition of 1.00 g (0.87 mmol) hexakis(6-deoxy-6-methylsulfanyl)-a-cyclodextrin dissolved in 50 mL of DMF, 2.25 g (10.4 mmol) 2- (2-(2-(2-methoxyethoxy)ethoxy)ethoxy)ethyl iodide and 3 mg (0.008 mmol) tetra-n-butylammonium iodide were added and the resulting reaction mixture was stirred at 80°C under N2 for 7 d. The reaction was quenched by addition of 10 mL of methanol and stirred at rt for further 30 min. The solvents were completely removed by vacuum distillation (bath temperature 70°C, 1 mbar) and the residue was dissolved in 50 mL of water and neutralized by addition of 1 M HCI. The crude product was purified by Crossflow nanofiltration in water (1 kDa, Pall Minimate TFF Capsule) and a brown yellowish oil (0.204 g, 10 %) was obtained after lyophilization.
TLC: Rf (ΕΕ:'ΡΓΟΗ:ΝΗ4ΟΗ:Η20 7:7:5:2 v/v) = 0.58.
1H-NMR δ/ppm (DMSO-d6, 400 MHz) = 5.02 (d, 1H, H-l, 3J= 3.0 Hz), 4.64 (s, 1H, OH-3), 3.98-3.94
(m, 1H, H-8a), 3.85-3.81 (m, 1H, H-3), 3.81-3.77 (m, 1H, H-5), 3.77-3.74 (m, 1H, H-8b), 3.53-3.52 (m, 12H, H-9, H-10, H-ll, H-12, H-13, H-14), 3.45-3.42 (m, 2H, H-15), 3.36- 3.35 (m, 1H, H-2), 3.25 (s, 3H, 0-CH3), 3.10-3.07 (m, 1H, H-6a), 2.81-2.75 (m, 1H, H- 6b), 2.09 (s, 3H, H-7).
"C-NMR: δ/ppm (DMSO-d6, 100 MHz) = 100.2 (C-l), 85.9 (C-4), 80.1 (C-2), 72.8 (C-3), 71.3 (C- 9), 70.8 (C-8), 70.7 (C-5), 69.8-69.6 (C-10, C-ll, C-12, C-13, C-14, C-15), 58.1 (0-CH3), 35.2 (C-6), 16.2 (C-7).
ESI-MS: m/z = 2316.49 [M+Na]+. Example 6: Heptakis[6-deoxy-6-methylsulfanyl-2-(2'-aminoethyl)carbamoyl)]- - cyclodextrin
Figure imgf000030_0001
[101] 0.5 g (0.38 mmol) Heptakis(6-deoxy-6-methylsulfanyl)^-cyclodextrin were dissolved in 20 mL of DM F [Sigma Aldrich, 648531) and 0.845 g (5.21 mmol) Ι,Ι'-carbonyldiimidazole (Sigma Aldrich, 115533) were added. The solution was stirred under N2 at rt for 2 hrs. Then 0.313 g (5.21 mmol) ethylenediamine (Sigma Aldrich, 391085) were added and the solution was stirred at 60 °C for 3 d. After cooling to rt, the crude product was precipitated in ice-water (150 mL) and the pH-value was adjusted to 3 by addition of 1 M HCI. The solution was filtered and purified by Crossfiow nanofiltration in water (650 Da, Pall Minimate TFF Capsule). The product (95 mg, 13 %) was obtained after lyophilization as a colorless solid.
TLC: R, (EE:'PrOH:NH4OH:H20 7:7:5:2 v/v) = 0.41.
1H-NMR: δ/ppm DMSO-d6, 400 MHz) = 5.99 (bs, 2H, OH-3/NH), 4.90 (s, 1H, H-l), 3.80 (bs, 1H, H- 5), 3.62 (d, 1H, H-3, 3J= 7.0 Hz), 3.34 (bs, 2H, H-2/4), 3.14 (d, 1H, H-6a, 3J= 14.1 Hz), 2.77-2.68 (m, 1 H, H-6b), 2.09 (d, 3H, H-7, 4J= 1.5 Hz), 1.23-1.03 (m, 2H, H-8), 0.92-0.75 (m, 2H, H-9).
ESI-MS: m/z = 1540.06 [M+Na]*.
!R: vm cm_1 = 3276 (OH), 1707 (C=0), 1524 (C-N-H) 1253 (C-N and/or N-H). Example 7: Influence of the Ring Size on the Inclusion Properties
[102] The inclusion properties of the host molecules were assessed by measurement of the vapour pressure of the guest by head space gas chromatography as a function of the host concentration as described by Lantz et a!. Anal. Bioanal. Chem. 2005; 383(2):160-6 and Fourmentin et al. J. Org. Chem. 2013, 9, 1185-1191. For this purpose, a Shimadzu GC-17A GC equipped with a head space unit from Shimadzu, Kyoto, Japan was used. Vials of 5 mL volume were used, the ratio between gas (V = 3.2 mL) and aqueous (V = 1.8 mL) phase was f = 1.77.
[103] As shown in Figure 2, the vapour pressure of the guest sevoflurane significantly drops due to complexation by the cyclodextrin host molecule of the invention 3bx (the respective vapour pressure behaviour is not illustrated by such a graphical respresentation for other cyclodextrins of the invention).
[104] The corresponding binding constant K was calculated from the hyperbolic decay of the area A of the sevoflurane signal with the total concentration of the CD derivative [CD]0 by non-linear regression according to Equation 1 given below. The Henry constant was determined according to a known GC method according to Fourmentin et al. Beilstein J. Org. Chem. 2013, 9, 1185-1191 using the Henry constants
Figure imgf000031_0001
at 37"C for sevoflurane. The occupancy x of employed CD host by the guest was calculated by the law of mass action according to Equation 2 given below. The solubility of the free guest sevoflurane in water [G]-5A mM at 25°C was calculated by the ideal gas law from its Henry constant and vapour pressure p = 263 mbar according to the following Equation 3:
•n 1 + Ύ
Figure imgf000031_0002
'.C\ =
R ika [105] Commercially available native CDs and β-CD derivatives (DIMEB, TRIMEB, HPpCD) showed rather poor affinities to sevoflurane, as shown in Table 1. Among the native CDs β-CD had far the highest binding constant which was attributed to the best space filling of this host by sevoflurane.
[106] It was found indeed an even higher binding constant for heptakis-2,6-di-0-methyl^-CD (DIMEB), but medical applications remain highly questionable for this host because of its known high toxicity. On the other hand, heptakis-2,3,6-tri-0-methyl^-CD (TRIMEB) and the less toxic derivative hydroxypropyl^-CD performed much worse. The low binding potential of TRIMEB was already found for other guests and can be attributed to the lack of intramolecular hydrogen bonds stabilizing the CD framework.
Table 1. Binding data for sevoflurane in native CDs and commercial CD derivatives at 25°C.
Host K Occupancy x
[L/mol] [mol%]
-CD 18 9
β-CD 150 45
γ-CD 9 5
DIMEB 713 79
TRIMEB 27 13
ΗΡ-β-CD 163 47
[107] The new hydroxyethylated CD thioether compounds, listed in Table 2, generally showed higher binding constants than the respective native CDs.
[108] The binding constants of the a-CD derivatives 3a! and 4ax were much lower than the ones of the corresponding β-CD derivatives 3bi and 4bx which can be attributed to the better space filling of the seven membered rings by sevoflurane. The binding constant decreased with increasing lengths of the a Iky I substituents at the sulfur atoms as well as with the lengths of the oligoethylene oxide chains. This fact was attributed to an increasing loss of entropy upon complexation of the guests. The longer the substituents the higher the conformational freedom of the host leading to higher intrinsic entropy. Also the two statistical derivatives, 2bi and 2b2 showed somewhat lower binding constants than the regioselectively modified derivatives 3, which might be due to a smaller amount of residual secondary hydroxy I groups known to stabilize the CD framework by intramolecular hydrogen bonds. Among the CD thioethers 3bx performed best reaching occupancies close to 100%. Table 2. Binding data for sevoflurane in the new CD thioethers at 25°C.
Host K Occupancy
[L/mol] [mol%]
3ax 64 26
43l 9 5
Figure imgf000033_0001
2b2 263 59
Figure imgf000033_0002
3b2 286 61
Figure imgf000033_0003
[109] Despite all binding measurements were performed under physiological pH and ionic strength, the inventors were interested in the binding potential of the best host 3bi approaching in vivo conditions to estimate the performance of this CD derivative for the delivery in the bodies of animals or humans. As expected, the binding constant slightly dropped in 5 wt. % albumin solution and further dropped in human serum (Table 3). At 37°C a further decrease of K was observed, but it still remained rather high. The occupancy of 3bi was still 87 mol% in human serum at body temperature. Therefore this compound is well suited for the delivery of sevoflurane. Potentially oral aqueous dosage forms can be developed for both anaesthesia and the treatment of pain. 3bi is also able to complex other hydrofluoric anaesthetics such has halothane for which the binding constant K = 9090 L/mol (occupancy of the host 98%) was even higher than for sevoflurane.
Table 3. Binding data for sevoflurane in 3bi for various media and temperatures.
Medium Temperature K Occupancy x
rc] [L/mol] [mol%]
albumin* 25 2175 92
human serum 25 1802 90
Water 37 1427 88
albumin* 37 1382 88
human serum 37 1331 87
*! 5 w %
[110] Inclusion properties were further investigated by the phase solubility method according to
Higuchi and Connors, Adv. Anal. Chem. Inst rum. 1965, 4, 117-210 using HPLC equipped with a UV- detector for the determination of the concentration of the guest. The filling degree (defined as the number of guest molecules per cyclodextrin host) is given both in molar as well as weight percentage based on the amount of the employed cyclodextrin derivative.
[Ill] The β-CD derivative of the invention show much higher solubilities and stronger affinities to the active ingredients than native β-CD (Table 4).
Table 4: R6=SMe, c
Host filling degree K [L/mol] guest
3aj [ct-CD] 17 mol% 1.6 wt. % 57 sevoflurane0
3ba [β-CD] 89 mol% 7.5 wt.% 2325 sevoflurane"
3bx [β-CD] 53 mol% 7.2 wt.% 27715 midazolam''
3bi [β-CD] 1 mol% (< 1 wt.%) 7 propofol0
3bi [β-CD] 97 mol% 8.2 wt.%) 8208 halothane0
3d [y-CDl 29 mol% 3.5 wt.%) 10422 midazolam6
°GC Headspace, °HPLC measurements
Surprisingly, thiomethyi derivatives showed higher binding affinities to a certain guest than the corresponding thioethyl derivatives (Table 5).
Table 5: Influence of the primary substitution pattern.
Host 6 n (EG) filling degree K [L/mol] Guest
2bx [β-CD] SMe 1-3 88 mol% 8.7 g% 2050 sevoflurane0
2b2 [β-CD] Set 1-3 45 mol% 4.3 g% 238 sevoflurane0
3b! [β-CD] SMe 3 89 mol% 7.5 g% 2325 sevoflurane"
3bi [β-CD] SMe 3 97 mol% 8.2 G% 8208 halothane0
3bi [β-CD] SMe 3 53 mol% 7.2 g% 27715 midazolam0
3b2 [β-CD] Set 3 88 mol% 7.2 g% 2169 sevoflurane0
3b2 [β-CD] Set 3 92 mol% 7.5 g% 3444 halothane0
3b2 [β-CD] Set 3 39 mol% 5.2 g% 16050 midazolam0
^GC~Headspace, *HPLC measurements The influence of secondary substitution is shown in Table 6, using sevoflurane as an exemplary guest molecule. A triethylene glycol chain seems to have the optimum chain length with a filling degree of 89 mol% and a binding strength of 2325 L/mol.
Table 6: Influence of the secondary substitution pattern, R6 = SMe.
Host n (EG) filling degree K [L/mol] Guest
2bi [β-CD] 1-3 88 mol% 8.7 g% 2050 sevoflurane0
3bi [β-CD] 3 89 moi% 7.5 g% 2325 sevoflurane0 bi [β-CD] 4 69 mol% 5.2 g% 724 sevoflurane0
GC Headspace, HPLC measurements
The data in Table 7 show that neither temperature nor medium significantly influence the complexation behavior of the tested cyclodextrins.
Table 7: Influence of temperature and medium on filling degree/K for 3bi, guest = sevoflurane".
Medium T [°C] filling degree K [L/mol]
Isotonic HEPES 21 90 mol% 7.6 g% 2530
Isotonic HEPES 25 89 mol% 7.5 g% 2325
Serum 25 85 mol% 7.2 g% 1628
5 % Albumin 23 87 mol% 7.4 g% 1964
Isotonic HEPES 37 76 mol% 6.4 g% 914
5 % Albumin 37 75 mol% 6.4 g% 886
Serum 37 75 mol% 6.3 g% 853
"GC Headspace
Example 8: Characterization of the products by NMR- and ESI- S spectroscopy
[112] All NMR spectra including H, 13C, H/H-COSY and C/H-COSY were measured at room temperature by a Bruker BioSpin spectrometer Magnet System 400 MHz Ultra shield plus (1H: 400 MHz, 13C: 100.6 MHz). The chemical shifts are given in parts per million (ppmj in relation to the corresponding solvent signal. The data analysis was performed with SpecManager included in ACDLabs 10.0 from Advanced Chemistry Development Inc., Toronto, Ontario, Canada. The proton and carbon atoms of the glucose units were marked with 1, 2, 3 etc. starting from the anomeric proton/carbon. The multiplicities were assigned as follows: s for singlet, d for doublet, t for triplet, bs for broad signal and m for multiplet. Mass spectra were recorded by a LC MS spectrometer ZQ-4000 from Waters GmbH, Eschborn, Germany, operated in ESI+ and ESI- mode.
[113] The ESI-MS of 2th (Figure 3A) showed a rather broad molecular weight distribution typical for CD derivatives with statistical substitution pattern. On the other hand, nearly uniform CD derivatives were synthesized by regioselective deprotonation of all 2-OH positions with NaH in DMF solution according to Tian and D'Souza, Tetrahedron Lett. 1994, 35, 9339-9342 and subsequent complete alkylation with l-(CH2-CH2-0)n-CH3(n = 3,4) for 4-7 d at 60-80 . The resulting derivatives 3 and 4 were isolated by liquid-liquid extraction at 50°C with a Kutscher-Steudel extractor and subsequent column chromatography. Yields were high as shown in Table 8. The ESI-MS of 3bi(Figure 3B) showed a significantly lower polydispersity than Zbt.
[114] Also the 1H-NMR spectrum of 3bi was much better resolved than the one of the statistical derivative 2bx due to its homogenous substitution pattern and uniform lengths of the oligoethylene oxide groups (Figure 4).
Example 9: Determination of lower critical solution temperature (LCST)
[115] The LCST transitions were recorded with a UV-Vis spectrometer Evolution 220 from Thermo Scientific, Waltham, MA, USA, equipped with a heating device from Harrick, Pleasantville, New York.
[116] All β-CD derivatives 2, 3 and 4 were indeed highly soluble in water at 25°C but upon heating the clear solutions turned turbid at a certain temperature and the compounds precipitated. The observed phase separation at the so-called lower critical solution temperature (LCST) is typical for uncharged polymeric amphiphiles, such as methyl cellulose poly(/V-isopropylacryl amide) (pNIPAM) and also for methylated CDs and CDs completely modified with oligoethylene glycol units. While the LCST transition of the statistical derivative 2 x was within a rather broad temperature range (30 - 40°C), the uniform derivative 3bj, showed a sharp transition at 42"C (Figure 5).
Table 8: Lower critical solution temperatures of CD derivatives.
Ring Yield LCST
R6 R2
Size [%] PC]
3ai 6 SMe (CH2CH20)3Me 70% 43 aj 6 SMe (CH2CH20)4Me 10 %* 65
3b! 7 SMe (CH2CH20)3Me 68 % 42 3b2 7 SEt (CH2CH20)3Me 71 % 61
4b! 7 SMe (CH2CH20)4Me 14 %* 54
3Ci 8 SMe (CH2CH20)3Me 89 % 49
*) loss of compound during ultrafiltration
[117] The LCST was only scarcely dependent on the ring size of CD but increased with the length of the hydrophilic oligoethylene oxide chain, as listed in Table 8.
Example 10: Cytotoxicity assays
[118] The effect of test substances on the cell viability was assessed using the CellTiter-Glo® assay (Promega, #G7571) according to the manufacturer's manual on human primary dermal fibroblasts (passage 3-7) and the colon tumor cell line Caco-2 (DSMZ #ACC-169; passage 6-9). The testing was carried out after reaching of about 80 % confluence (~2-4 days) and a seeding density of 5xl03 cells per well in 96-well plates. The measuring principle of the CellTiter-Glo® luminescent cell viability assay is based on the determination of the number of metabolically active cells through the quantification of cell ATP. This is consumed by the Ultra-Glo™ recombinant luciferase to convert Beetle Luciferin into oxyluciferin. The luminescence generated in this reaction is measured in a plate reader (TECAN infinite SV1200) at an emission wavelength of 537 nm. The tested concentrations of the excipients 3bi, 3b2, 2bi and 2bz were based on experimentally determined degrees of filling of the two anesthetics midazolam and sevoflurane (both <10 wt.%) and their plasma concentrations in clinical use. A first series of tests was performed with Caco-2 cells in the relevant concentration range, i.e. lOx lower and up to about lOOx above the clinically relevant concentration. For primary fibroblasts, this range was increased by two orders of magnitude.
[119] Thus, the concentration range was for Caco-2: 130 ng/mL - 1.3 μ^ηηΙ. - 13 μg/mL - 130 μg/mL for primary fibroblasts: 13 μ^ιηΙ. - 130 μ^ηηΙ. - 1.3 mg/mL - 13 mg/mL
[120] Both substances were each solved in standard basal medium (Caco-2: MEM, fibroblasts: DMEM, Gibco®) and human serum and cells were treated for 2 h and 24 h with the above-mentioned concentrations. For viability assessment, the test substances were removed by washing with PBS buffer (Sigma®), the cells in each well were overlaid with 100 μί of basal medium and 100 μΙ_ of CellTiter-Glo® reagent and luminescence was measured after 2 minutes of shaking, and a 10-minute incubation at room temperature in the TECAN plate reader.
[121] According to DIN EN ISO 10993-5, a more than 30% deviation of measurement values of treated cells compared to the untreated control was defined as cytotoxic. After treatment of the Caco-2 cell line, cytotoxic effects could be detected at none of the tested concentrations of 3bt and 2b1( both after 2 h and after 24 h of incubation (Figure 6 A-D). Similarly, 3b2 exhibited no cytotoxic effects up to a concentration of 13 mg/mL in basal medium and was non-toxic at 130 mg/mL in serum. For primary fibroblasts, no cytotoxic effects occurred below a concentration of 1.3 mg/mL in basal medium of 3bi and 2b2 after 24 h incubation, which is still 10 times higher than the clinically relevant range. Similar observations could be made for the substance 2bx. Here, too, no cytotoxic effects were detected up to a concentration of 1.3 mg/mL in basal medium after 24 h incubation. Serum had a protective effect on the cells.
[122] All results presented are based on at least three independent biological tests, i.e. for each experiment primary cells of another donor or cell lines of another passage were. The graphs show the arithmetic mean with standard deviation. Statistical significance was determined by analysis of variance p < 0.01 (**) and p <0.001 (***).
Example 11 : Transport studies
[123] The transport behaviour of the active ingredient midazolam complexed by the cyclodextrin 3 j (adjuvant) was investigated. For this purpose a model of the intestinal epithelial barrier employing the colon cancer cell line Caco-2 was used. The cells were used up to a maximum number of 25 passages. The Caco-2 cells were seeded at a seeding density of 8xl04 cells/cm2 onto PET 12-well inserts (Greiner, #667610) with a pore diameter of 1 μιη, which had previously been coated with collagen I (100 ^ηηί in 0,1% acetic acid). To generate functional models of the intestinal barrier, cells were cultivated for 14 days in an incubator (37 °C, 5% C02, 95% relative humidity) and supplied three times a week with 1 mL of Caco-2 medium ( EM-Earle with stable glutamine (Life Technologies, #41090-028) + 20% FCS + 1% Na-pyruvate + 1% non-essential amino acids) apically and 2 mL basolaterally. To test barrier integrity, transepithelial electrical resistance (TEER = transepithelial electrical resistance) was measured regularly with the TEER Millicell ERS-2 epithelial volt-ohm meter by Millipore as well as by measuring permeability using FITC-dextran molecules of 4 kDa in size. Starting from a TEER > 200 Ω/cm2, the intestinal epithelial cells have - by the formation of cell adherence and tight junctions - formed a sufficient barrier and can be used to perform absorption studies. [124] The tested concentrations of active ingredient complex are based on the clinically relevant plasma concentrations of the anesthetic midazolam and are used in a 10 x and 50 x higher concentration for subsequent oral administration. The filling degree of 3bt for midazolam is 7.4%. Thus, the tested active ingredient or complex concentrations were as follows: concentration increase in concentration concentration
midazolam in compared to clinically complex ^g/mL]
complex ^g/mL] relevant concentration concentration 1 15 1.11 10 x
concentration 2 75 5.55 50 x
[125] Transport behaviour of midazolam complexed with 3bi and dissolved in MEM basal medium (without FCS) from the apical to the basolateral side (blood side, simulated by human serum) of the intestinal models was studied over 4 h. Samples were obtained on the acceptor side after every 5, 15, 30, 120 and 240 minutes. Here, 100 μί. of sample volume were removed and replaced with fresh serum. For accounting, samples were also taken on the donor side at the beginning t=0 minutes and at the end of the study after 240 minutes. Quantitative analysis of midazolam concentration in the test samples (serum or MEM basal medium) was determined using an HPLC-ESI-MS/MS method by Dr. Katalin Papai by the company Sapiotec.
[126] As can be seen in Figure 7, directional transport of the drug midazolam, complexed by the cyclodextrin 3bi (adjuvant), could be observed from the apical to the basolateral side through the intestinal epithelial models. Compared to the inserts populated with cells (Caco-2 model), passage of the test substance midazolam through the non-populated empty cell inserts is effected by diffusion at a higher speed. Also, transport of midazolam at the higher concentration of 5.55 g/mL took place at a higher rate than at the lower concentration of 1.11 g/mL. A transport across the intestinal barrier of 3bi could not be observed (results not shown).
[127] All results are based on at least three independent biological experiments, at any attempt another passage cell lines were used. The graphs show the arithmetic mean with standard deviation.

Claims

Claims
A cyclodextrin compound having the formula
Figure imgf000040_0001
wherein
m + n is 6, 7 or 8; and n = 0, 1 or 2
Ri is independently selected from Ci to C4-alkyl;
R2 is independently selected from CO-(NH-CH2-CH2)0-NH2 or (CH2-CH2-0)0-(CH2)p-CH3; wherein o is 1, 2, 3, 4, 5 or 6; p is 0 or 1;
or R2 is -(CH2)|-X wherein X is NH2 or OH, and wherein I is 2, 3, 4, 5 or 6;
or R2 is -CO-NH-R3,
Figure imgf000040_0002
CO-(NR3)2>
wherein R3 and R3' is independently from each other a hydrophilic branched or unbranched radical with 2 to 7, preferably 4 to 7 main chain atoms;
or R2 is CO-NH-(CH2)q-R4, wherein q is 0, 1 or 2, and R4 is selected from OH, CH2-OH, and a 5 or 6 membered substituted or unsubstituted heterocyclic ring system containing at least one N or one 0 atom;
or R2 is CO-N-Rs, wherein R5 is a 6 membered substituted or unsubstituted heterocyclic ring system and wherein N is part of the 6 membered heterocyclic ring;
or R2 is 0-CH2-CH2-NH-CH2-CH2-OH; or pharmaceutically acceptable salts thereof.
The compound of claim 1, wherein is methyl, ethyl, n-propyl, isopropyl cyclopropyl, butyl, 2-butyl, or rerf. -butyl, wherein Ri is preferably methyl.
The compound of claim 1 or claim 2, wherein R4, when being a 5 or 6 membered heterocyclic ring system containing at least one N or one O atom, is an aromatic ring system.
The compound of claim 3, wherein q is 1.
The compound of claim 4, wherein R4 is amino, 2-furyl, 3-fury 1, 1-pyrrolyl, 2-pyrrolyl, 2-picolyl or 3-picolyl.
6. The compound of claim 1, wherein N-R5 is morpholino or piperazine.
7. The compound of claim 1, wherein R2 is CO-(NH-CH2-CH2)0-NH2 or (CH2-CH2-0)0-CH3< wherein o is 1, 2, 3 or 4, wherein o is preferably 3 or 4.
8. The compound of claim 1, wherein R3 or R3' comprises at least one terminal hydroxy I or amino group.
9. The compound of claim 1, wherein
m+n = 7; n is 0-2;
Rn is methyl; and R2 is (CH2-CH2-0)0-CH3;
wherein o is 3 or 4;
or pharmaceutically acceptable salts thereof.
10. The compound of claim 1 or 2, wherein
m + n = 6; n is 0-2;
i is methyl; and
R2 is (CH2-CH2-0)0-CH3;
wherein o is 3 or 4;
or pharmaceutically acceptable salts thereof.
11. The compound of claim 1 or 2, wherein
m + n = 8; n = 0-2
i is methyl; and
R2 is (CH2-CH2-0)o-CH3;
wherein o is 3;
or pharmaceutically acceptable salts thereof.
12. The compound of claim 1 or 2,
wherein m is > 2;
Ri is methyl; and
R2 is CO-(NH-CH2-CH2)o-NH2;
wherein o is 1;
or pharmaceutically acceptable salts thereof.
13. A cyclodextrin compound as defined in any of the foregoing claims, which is water soluble.
14. The cyclodextrin compound of claim 13, which does not form micelles in aqueous solution.
15. A method of preparing a cyclodextrin compound as defined in any of claims 1-14, comprising
Reacting a compound of formula (X),
Figure imgf000042_0001
wherein m + n is 6, 7 or 8;
Ri is selected independently from C C4 a Iky I;
with a compound of formula (Y) Hal-(CH2-CH2-0)0-(CH2)p-CH3,
wherein in formula (Y) Hal is halogen, o is 1, 2, 3, 4, 5, or 6, p is 0 or 1;
or with a compound of formula (Z) (H2N-(CH2CH2-NH)q-CH2CH2-NH2),
wherein in formula (Z) q is 0, 1, 2, 3, 4, or 5;
or with a compound of formula (C) HO-(CH2)rX wherein X is NH2 or OH, and wherein I is 2, 3, 4, 5 or 6;
or with a compound of formula (D) selected form H2NR3 (formula Dl),
Figure imgf000042_0002
(formula D2) or HN(R3)2 (formula D3),
wherein R3 and R3 is a hydrophilic branched or unbranched radical with 2 to 7, preferably 4 to 7 main chain atoms;
or with a compound of formula (E) NH-(CH2)q-R4,
wherein q is 1 or 2, and R is selected from OH, CH2-OH, and a 5 or 6 membered atom; or with a compound of formula (F) HN-R5, wherein R5 is a 6 membered substituted or unsubstituted heterocyclic ring system and wherein N is part of the 6 membered heterocyclic ring;
wherein in the case of a reaction with the compound of formula (Z) (D), (E) or (F), the compound of formula (X) is first reacted with a coupling reagent.
The method of claim 15, wherein the coupling reagent is selected from the group consisting of Ι, -carbony!diimidazole, phosgen, diphosgen and triphosgen.
17. A composition comprising a cyclodextrin compound of any of claims 1-14 and at least one active agent complexed (as a guest molecule) with this cyclodextrin.
18. The composition of claim 17, wherein the active agent is selected from the group consisting of a pesticide, herbicide, insecticide, antioxidant, plant growth instigator, sterilization agent, catalyst, chemical reagent, food product, nutrient, cosmetic, vitamin, sterility inhibitor, fertility instigator, microorganism, flavoring agent, sweetener, cleansing agent and pharmaceutically active agent.
19. The composition of claim 18, wherein the pharmaceutically active agent is selected from the group of anesthetics, analgesics, steroids, cytostatic drug, antiviral agents, nutrients, nutritional agents, hematological agents, endocrine agents, metabolic agents, cardiovascular agents, renal agents, genitourinary agents, respiratory agents, central nervous system agents, gastrointestinal agents, anti-infective agents, biologic agents, immunological agents, dermatological agents, ophthalmic agents, antineoplastic agents, and diagnostic agents.
20. The composition of claim 19, wherein the anaesthetic and/or the analgesic is selected from the group consisting of midazolam, sevoflurane, halothane, propofol, xenon, fentanyl, the steroid is selected from the group consisting of hydrocortisone, testosterone, estradiol and contraceptives, the cytostatic drug is selected from the group consisting of camptothecin, paclitaxel, and anthracyclines, e.g. idarubicine, the neurolepticum is selected from the group consisting of haloperidol, f!uspirilen, and risperidon), and the antiviral agent is selected from the group consisting of lopinavir, ritonavir, atazanavir.
21. The composition of any of claims 17 to 20, wherein the molar ratio between the compound and the active agent is between 10:1 to 1:1.
22. The composition of any of claim 17 to 21, further comprising one or more pharmaceutically acceptable excipients.
23. Use of a cyclodextrin compound of any of claims 1-14 as a host molecule for at least one active agent.
24. The use of claim 23, wherein the use comprises with respect to the at least one active agent an increase of solubility, prevention of aggregation, avoiding micellar formulations, reduction of injection pain for intravenous applications, reduction of vapor pressure of volatile active agents, targeting of active agents to avoid side effects.
25. The use of claim 23, wherein the active agent is selected from is selected from the group consisting of a pesticide, herbicide, insecticide, antioxidant, plant growth instigator, sterilization agent, catalyst, chemical reagent, food product, nutrient, cosmetic, vitamin, sterility inhibitor, fertility instigator, microorganism, flavoring agent, sweetener, cleansing agent and pharmaceutically active agent. A method of encapsulating a pharmaceutically active agent, comprising contacting a cyclodextrin compound as defined in any of claims 1-14 with a pharmaceutically active agent under conditions suitable for forming a host-guest complex, thereby complexing the pharmaceutically active agent within the cyclodextrin compound of any of claims 1-14.
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