WO2002077000A2

WO2002077000A2 - Macrocyclic oligosaccharide derivatives which form manoscale assemblies

Info

Publication number: WO2002077000A2
Application number: PCT/IE2002/000038
Authority: WO
Inventors: Raphael Darcy; Bart Jan Ravoo
Original assignee: University College Dublin
Priority date: 2001-03-23
Filing date: 2002-03-25
Publication date: 2002-10-03
Also published as: WO2002077000A3; AU2002246302A1

Abstract

Macrocyclic oligosaccharide derivatives wherein the subunits making up the neurocycle are modified to enable the macrocycle to self assemble in a suitable solvent to form elongated assemblies comprising a plurality of macrocyclic units. These elongated assemblies have the ability to encapsulate guest molecules such as therapeutic drugs and can be used as hosts for the solubilisation of various compounds. Preferred embodiments of the present invention utilise cyclodextrin as the macrocyclic derivative to be modified.

Description

MACROCYCLIC OLIGOSACCHARIDE DERIVATIVES WHICH FORM NANOSCALE

ASSEMBLIES

INTRODUCTION

The present invention is directed towards the production of novel cyclic oligosaccharide derivatives of the type that self-assemble when in solution to form nanoscale structures.

Macrocyclic oligosaccharides are typified by cyclodextrinSj^" hich are cyclic oligosaccharides composed of D-glucose residues linked together by α-(1-4) bonds. The most common examples of cyclodextrins contain six, seven or eight α-(1-4)- linked D-glucopyranosyl units bonded together into cylinder-shaped molecules and are referred to as -, β-, and γ-cyclodextrins, respectively. As a consequence of the conformation of the glucopyranose units, all secondary hydroxyl groups are placed on one rim of the cylinder and all primary hydroxyl groups are placed on the other. The cylindrical interior (cavity) of the molecule is lined with hydrogen atoms and glycosidic oxygen atoms which cause it to be hydrophobic (lipophilic).

The cylindrical structures can be used as hosts for the inclusion of various compounds within their cavities, usually organic compounds, in the food, pharmaceutical and chemical industries. Cyclodextrins have been used to form inclusion complexes with hydrophobic molecules in which these molecules are encapsulated within the compatible hydrophobic cavity of the cyclodextrin macrocycle. This process of molecular,* encapsulation confers increased water solubility on the included molecule, as well as other properties such as increased stability and lowered volatility. It also allows control of the availability of the molecule, for example the bioavailability of a drug, (Uekama et al., in CRC Critical Reviews in Therapeutic Drug Carrier Systems, Vol. 3, 1-40 (1987)).

There are problems associated with the use of unmodified cyclodextrins to form inclusion complexes for the pharmaceutical industry. A limitation to the use of cyclodextrins as hosts for molecules, is that the hydrophobic molecules which can be included are limited by the size of the central cavity. Several attempts have been made to alter the cyclodextrin structures to enable them to encapsulate other molecules regardless of size. Cyclodextrins have been modified with lipophilic groups at the 2- and 3- positions (the secondary-hydroxyl side) of the glucose units, together with polar groups such as amino groups at the 6-positions (the primary- hydroxyl side), in order to confer amphiphilic character. Such derivatives are described in US Patent 5,718,905 (Skiba et al.) and form monolayers, nanoparticles, and mixed lyotropic (solution) phases with other amphiphiles.

Similar derivatives with lipophilic substitution on the secondary side have been described in various reports (P. Zhang et al., Journal of Physical Organic Chemistry

1992, 5, 518-528; A. Gulik et al., Langmuir 1998, 14, 1050-1057; D. Duchene and

D. Wouessidjewe, Proc. Int. Symp. Cyclodextrins, 8th, 1996, 423-430). Such derivatives are characterised by the formation of nanoparticulate aggregates which are able to trap hydrophobic or hydrophilic guest molecules to a greater or lesser extent. The entrapped guest is however instantaneously released upon contact of the nanoparticle with a solution medium (E. Lemos-Senna et al., Proc. Int. Symp.

Cyclodextrins, 8th, 1996, 431-434). These systems are capable of entrapping both water-soluble and water- insoluble drugs (M. Skiba et al., International Journal of

Pharmaceutics, 1996, 129, 113-121). The self-assembly properties of amphiphilic cyclodextrins have been reviewed by Coleman et al. in Molecular Engineering for

Advanced Materials, 1995, 77-97, Kluwer Academic Publishers (J. Becher and K.

Schaumberg eds).

US Patent 5,821,349 Djedaini-Pilard et al. describes cyclodextrins modified with alkylamino groups at the 6-positions for incorporation of included hydrophobic guest molecules only into other organised surfactant systems. However B. J. Ravoo and R. Darcy (Angewandte Chemie Int. Ed., 2000, 39, 4324-4326) have described amphiphilic cyclodextrins which can form bilayer vesicles capable of including either polar or lipophilic guest molecules.

Several types of cyclodextrins have been prepared to date which show self assembling properties, however, these have only been shown to self-assemble into dimers. The largest class comprises amphiphilic molecules as described above in which one face of the cyclodextrin has been substituted with large hydrophobic groups while the other face is substituted with polar or ionic groups or left unsubstituted. These can form micelles or vesicles. Another class contains small hydrophobic substituents on one face and polar or ionic substituents or none on the other face. These molecules form dimers as mentioned previously in several solvents (B. Hamelin et al, Kim. Eur, J. 1999, 546-556). Also, cyclodextrins with polar or ionic substituents on one face of the molecule and no substituents on the

5 other face can form dimers in polar solvents through hydrogen bonding (A.R. Khan,

Supramoleular Cagm, 1995, 243-246) and even specific hetrodimers between cyclodextrins of opposite charge in water (B. Hamelin et al, Journal of Physical

Chemistry, 1995, No. 99, p. 17885 -17887; L. Jullien et al. Journal of Physical

Chemistry B, 1999, p.10866 - 10875; Peach Schwinte et al, Journal of Inclusion

10 Phenomena and Macrocyclic Chemistry, 1999, No.35, p. 657 - 662). All of above cyclodextrins form only diamonds due to the presence of multiple interacting groups only on one: side of the component sub unit. Heretofore, the cyclodextrins molecules of the prior art have been limited in their use as vehicles for guest molecules, for example, use in drug delivery by the size of the central cavity.

15. Attempts have been made to alter the , size of the cavity by modifying the cyclodextrin molecule. However, to date the size of the central cavity is still a limiting factor of the application of oligosaccharide macrocycles, such as cyclodextrin for use as vehicles for guest molecules.

0 Molecules of the prior art have not been synthesized which are capable of self- assembling into nanoscale aggregates in which the oligosaccharide assemblies may be used to include larger guest molecules than previously possible by virtue of the combined cavities of the assembled host molecules or other enclosures created by the shapes of the nanoscale structures. The preparation of such cyclodextrins is 5 not trivial since it requires the precise assembly and orientation of multiple groups which will attract each other. To our knowledge such an artificial self-assembling system has been devised for macrocyclic molecules only in the case of certain cycle peptides (J. D. Hartgerink et al, Journal of the American Chemical Society, 1996, 118, p.43).

30

A first object of the present invention is to provide novel cyclo-oligosaccharides, for example cyclodextrins, bearing multiple groups on one face and multiple groups on the second face having a cyclic structure which self-assembles in water or other suitable solvents by molecular stacking, to give molecular assemblies larger than

35 dimers, and usually some hundreds of nanometres in size; and which it is believed will enable retention of entrapped molecules even after dilution in a solution medium, with advantages for the delivery of therapeutic molecules.

A second object of the invention is to provide modified macrocyclic oligosaccharides bearing multiple groups which when present as a mixture in solution will assemble by molecular stacking of the different side groups.

A third object of the invention is to provide cyclic oligosaccharide structures that self-assemble in aqueous or other suitable solutions in a pH-dependent manner such that at neutral pH supramolecular nanoscale structures are the predominant structures.

STATEMENTS OF INVENTION

According to the present invention macrocyclic derivatives are provided characterised in that groups are attached to the faces of the macrocycle which enable continuous molecular stacking by attraction between the groups in separate molecules. Preferably macrocyclic oligosaccharide derivatives are provided capable of forming aggregates by self assembly comprising mono or disaccharide subunits, which subunjts comprise at least two chemically distinct sides, arid a central cavity and wherein the subunits are modified subunits bearing at least one side group on each side characterised in that at least one of the subunits making up the macro cycle is modified to enable continuous molecular stacking of the macro cycle by attraction between the side groups in separate macro cycles making up the aggregates. It will be appreciated that in a number of oliogasaccharide subunits, the chemically distinct sides will comprise primary and secondary hydroxyl sides. However in certain embodiments the oliogasaccharide subunits due to their stereochemistry may have at least two secondary sides or two primary sides, the important being that the sides are distinct from each other.

The advantage of these macrocyclic derivatives is that they spontaneously aggregate to form highly stable arrangements of the hydrophobic molecular cavities in which the cavities are in effect aligned in continuous channels with, improved potential for inclusion of large guest molecules or in which other cavities are formed by the three dimensional structure of the arrangements. For example the assemblies could form helical structures or looped or circular structures. The unique aggregation properties of the derivatives may be usefully employed in the encapsulation of drugs including biological macromolecules such as proteins and DNA in order to enhance delivery of these therapeutic entities to their respective sites of action.

In another embodiment, the aggregates of macrocyclic derivatives encapsulate other molecules.

In another embodiment, the aggregates of macrocyclic derivatives encapsulate

10 molecules for human or veterinary therapeutic use.

In another embodiment the subunits comprise side groups on the primary and secondary hydroxyl sides capable of forming intermolecular interactions with side groups on the primary or secondary hydroxyl side of an adjacent macrocycle unit.

;15

In another. embodiment, the macrocyclic derivatives are of the general formula:

M

20

in which n equals 2 - 11 or higher, and indicates the number of ring units making up the macrocycle, which may be the same or different, depending on the X-, A- and B-groups. These groups are designed to cause intermolecular attraction so as to

25 produce molecular assemblies of type (i), Figure 1. K, L, M independently are exemplified by: zero (thus providing a unit, as part of the macrocycle, which is an open chain rather than a ring); a simple chemical bond (thus providing a five-membered ring unit as in a furanose sugar); an atom or radical having a valency of at least 2.

Y, which may be the same or different, are groups which link the units making up the macrocycle, such as: oxygen, sulfur, selenium, nitrogen, phosphorus, carbon, or silicon radicals having a valency of 2-4; or OCH₂ as in (1-2)-linked fructofuranooligosaccharides; or OCH₂CH(OH) as in (1-6)-linked furanooligosaccharides; or OCH(CH₂OH) as in (1-5)-linked furanooligosaccharides.

Xϊ. X'ι_> X2, X2, X3. X'3. X4. X^*4, X5. Xβ are, independently zero or provide linking groups; these may be a simple covalent bond, or a dendrimeric group; other examples are: an atom or radical with a valency of at least two, CH₂, CH₂O, O, S, Se, N, P, carbonyl, ester, amido, amino, phosphate, sulfonyl, sulfoxide, a saturated or unsaturated aliphatic or aromatic carbon or silicon radical or a halogenated version of these. Where the X-group is a straight or branched aliphatic chain, n is preferably greater than one, and the number of carbons 2-18. The group of type X may be a cyclic aliphatic system such as hexyl or cholesteryl; examples of aromatic X-groups are benzyl and pyridyl.

Aι, AY Am, A' are, independently, zero or provide groups which are one of a pair of mutually attractive groups of types A and B. Examples of A-type are: groups capable of hydrogen-bond formation, a cation such as a protonated amino group, or an anion such as sulfate, sulfonato. Examples of B-type are: those listed above for A-type groups, with the provision that in any example of the embodiments described above, the A-type groups and the B-type groups constitute complementary pairs which are mutually attractive, for example: where A is a H- bond donor, B is a H-bond acceptor; where A is a H-bond acceptor, B is a H-bond donor; where A is a protonated amino group, B may be a carboxy anion or sulfonato anion or in general any anionic group which will attract the cationic group A; conversely, where B is a protonated amino group, A is an anion which will attract that group. Other examples of complementary group-A and -B pairs are: nucleobase pairs such as adenyl and cytidyl; urea and carboxylate; guanidinium and carboxylate; guanidinium and phosphate; amino acid pairs such as arginyl and aspartyl. In certain embodiments B₅, B₆ are groups which independently may or may not form a mutually attractive pair with an A-group, when B₅ and B₆ independently do not comprise a mutually attractive pair with an A group, either or both of B₅ or B₆ is preferably hydrogen.

A-type and B-type groups may be dendrimeric, and may include peptides, nucleotides, cyclic, straight-chain or branched oligosaccharides or polymeric groups such as poly(ethylenimine) (PEI), polyamides, polyaminoacids such as polylysine, polynucleotides and polysaccharides.

Provided that there are enough multiple groups of types A and B present to. cause molecular stacking, additional groups may be employed because of⁴ their non- immunogenic character, such as poly(ethylene glycol), or sialylGalGlcNAc; or antigenic groups such as antennary oligosaccharides which are intended to stimulate the production of antibodies; or groups such as Iactosyl which may be attached for the purpose of promoting adhesion of the amphiphile or of its complex with a guest molecule to specific cells or to specific proteins. Similarly other groups known in the art which are specific ligands for cellular receptors, such as folic acid, galactose, biotin, lipopolysaccharides, gangliosides, sialo-gangliosides, glycosphingolipids and the like may be attached to the polar face of the modified oligosaccharide or oligosaccharide analogue, thereby expressing a targeting ligand on the external surface of the micelles or vesicles of the invention. The groups may be clustered in order to promote 'recognition' by other molecules which involves multifunctional interactions. Where these groups are polymeric or dendrimeric they may be grafted onto the oligosaccharide for example by living polymerisation; or the cyclodextrin may be a copolymer, for example it may be cross-linked by means of difunctional or polyfunctional reagents such as activated diacids or diepoxides, or copolymerised within the matrix of a polylactic or glycolic acid. In another embodiment, the macrocyclic derivative has the formula:

in which n equals 3 - 11 or higher, and indicates the number of modified monosaccharide units in the macrocycle which may be the same or different, depending on the X-, A- and B-groups, and preferably are linked (1-4). In certain embodiments X₁₍ X₂, X₃ are linker groups; A^ is one of a pair of mutually attractive groups capable of interacting with the other of the pair in the B groups on a separate macrocyclic unit and B₂ and B₃ are independently one of a pair of mutually attractive groups capable of interacting with the other of the pair in the A groups on separate macrocyclic units.

These groups where appropriate are the same as those described in the embodiments above, and are designed to cause intermolecular attraction so as to produce molecular assemblies of type (i), Figure 1.

Preferably the macrocyclic oligosaccharide derivatives are those in which the modified units making up the macrocycle are, independently, aglycone derivatives of L-glucose, or of D- or L-hexoses such as mannose, galactose, altrose, idose, or rhamnose, or arabinose; or where the macrocycle is an oligomer of a disaccharide such as lactose or sucrose. Preferably the oligosaccharide is one in which at least two monocyclic units making up the macrocycle are derived from a (1-1)- or (1-2)- or (1-3)- or (1-6)-linked disaccharide, or from the disaccharide sucrose, or where at least one of the units (whether cyclic or open-chain) which make up the macrocycle is derived from fructose or a furanose sugar or sialic acid or from a carbohydrate analogue (defined for this purpose as a molecule which is not a natural carbohydrate nor a derivative thereof but which can usefully function either physically or pharmaceutically as a carbohydrate).

In a preferred embodiment of the invention, the macrocyclic derivative is a cyclodextrin derivative of the following formula:

in which n equals 5 - 11 or higher, and indicates the number of modified glucose units in the macrocycle which may be the same or different, depending on the X-, A- and B-groups. Groups of type X are linker groups, and groups of types A and B constitute mutually attractive pairs A, B. In certain embodiments X^ X₂, X₃ are linker groups; A1 is one of a pair of mutually attractive groups capable of interacting with the other of the pair in the B groups on a separate macrocyclic unit and B₂ and B₃ are independently one of a pair of mutually attractive groups capable of interacting with the other of the pair in the A groups on separate macrocyclic units. These groups are designed to cause intermolecular attraction so as to produce molecular assemblies of type (i), Figure 1.

It will be understood by one skilled in the art that in certain aspacts, any one of the embodiments described by molecular formulae above may be used in admixture with at least one other, to give a molecular assembly of type (ii) as shown in Figure 1.

In another embodiment, any one of the embodiments described by the molecular formulae above may be used in admixture with at least one other, where terminal groups (as distinct from the linker groups of type X) on a fraction of the molecules in the mixture, preferably 50%, are all of the A-type, and the groups on the remaining molecules are all of the B-type, to give a molecular assembly of type (iii) as shown in Figure 1. In such an assembly, the molecule is constituted as a hetero- dimer of two oligosaccharide molecules.

The interacting macrocyclic units, when assembling, behave in the same manner as the monomer units of a normal polymer, so that the following possibilities are to be understood from Fig. 1: (a) the molecular 'stacks' illustrated may terminate in a molecule possessing no interacting groups A or B on one face (the face turned away from the stack) so that the stacking process is interrupted, thus terminating the stack; (b) the stacks may not necessarily exist individually, but may pack in extended alignment (see Figs. 4, 5), or in random coils, depending on the intermolecular interactions of the stacked molecules or their interactions with surrounding media; (c) the nanoscale or microscale assembly resulting from the stacking (as with normal linear polymers) may not only be extended in form (fibrous, see Figs. 4, 5) but can be, for example, of planar formation (as when the ends of the stacks are attached to or attracted to a planar surface and thereby arrayed indefinitely in two dimensions), or a three-dimensional formation (as when the ends of the stacks are attached to or attracted to a 3D surface such as that of a molecular aggregate, for example a micelle, vesicle, nanoparticle, nanocapsule or polymer bead); (d) the overall form of the nanoscale or microscale assembly may be decided by the arrangement or structure of the interacting groups A, B, for example a symmetrical dendrimeric arrangement may lead to a generally spherical assembly; (e) the inclusion of a guest molecule, such as a drug molecule, may take place by entrapment within the spaces or interstices created by such arrays as well as within the cyclodextrin cavities.

The coupling of nanoassemblies of the invention to antibodies may be an alternative route for targeting specific cell types. The synthetic procedures for antibody coupling are known in the art and may be applied to modified oligosaccharides or oligosaccharide analogues of the invention which, on the polar face provide either free amino groups for biotinylation, or free carboxylic groups for peptide coupling of an antibody via N-glutaryl detergent dialysis, or maleimide for sulfhydryl antibody coupling, or pyridyldithiopropionate for sulfhydryl and maleimide antibody coupling, or similar methods appreciated in the art. In another embodiment, certain of the groups of type X, or type A, or type B may be linked to each other, as independent sets, intramolecularly by reaction of their chemical precursor groups through catalysis (for example through irradiation), or by reaction of their chemical precursor groups with a polyfunctional linking agent.

In another embodiment, certain of the groups of type X, or type A, or type B may be linked to each other, as independent sets, intermoleculariy by reaction of their chemical precursor groups through catalysis, or by reaction of their chemical precursor groups with a polyfunctional linking reagent, to provide an oligomerised nanoassembly.

In another embodiment, the oligosaccharide molecules (of any of the molecular forms or embodiments described above) self-assemble in an aqueous solvent or alternatively non-aqueous solvent. After self-assembly, the resulting aggregates may be transferred by physical or chemical means from the aqueous solvent into another phase, such as an aqueous phase containing a proportion of an alcohol or other polar solvent for example dimethyl formamide, dimethyl sulfoxide, tetramethylurea, dimethyl carbonate , or a polymer, or into an emulsion, or gel-like matrix, or lyophilised suspension.

In a particularly preferred embodiment there is provided a nanoscale assembly comprising macrocyclic derivatives as described in the embodiments above.

In certain embodiments the assembly may be attached to surfaces or structures preferably these surfaces may include antibodies inert surfaces and other proteins.

In another embodiment, the assembly of oligosaccharide molecules may be composed of more than one of the molecular forms or embodiments described above, preferably to provide the molecular assembly with the complementary properties of the individual oligosaccharides, for example the property of cell- adhesion together with prodrug properties, or to modulate the colloidal stability of the assemblies.

In another embodiment, the derivatives or alternatively the assemblies of the invention may be mixed with other molecules, such as poly(ethylene glycol) or polyamines, to modulate the properties of their assemblies, for example to control their colloidal stability.

In another embodiment, the oligosaccharide derivatives or assembly forms a complex with a therapeutic molecule for its solubilisation or stabilisation, or in alternative embodiments for its formulation into pharmaceutical compositions useful for the treatment of human or animal diseases.

In another embodiment, the drugs that complex with the oligosaccharide derivatives or assemblies are of a lipophilic or polar nature. In certain embodiments the drugs which may be entrapped in the oligosaccharide assembly include but are not limited to: anti-neoplastic agents (paclitaxel, doxorubicin, cisplatin, etc); anti-inflammatory agents (diclofenac, rofecoxib, celecoxib, etc); antifungals such as amphotericin B; peptides, proteins and their analogues including those to. which nonpeptide groups such as carbohydrates, hemes and fatty acids are attached; oligosaccharides and their analogues such as Sialyl Lewis⁵* analogues; oligonucleotides and their analogues; plasmid DNA; and complexes of oligonucleotides or of DNA with gene delivery agents.

In another embodiment the oligosaccharide derivative or assembly is complexed with a molecule or atom used for analysis or diagnosis, for example a peptide antigen or an antibody; or a molecule used as a radiation sensitiser, for example a porphyrin.

In another embodiment the oligosaccharide or assembly is complexed with a molecule which functions as a prodrug, for example a precursor of nitric oxide.

In another embodiment, the oligosaccharide derivative or assembly complex may be attached covalently to a polymer; the polymer may be grafted onto the oligosaccharide molecules of the complex for example by living polymerisation; or the oligosaccharide may be a copolymer, for example the oligosaccharide may be cross-linked by means of difunctional or polyfunctional reagents such as activated diacids or diepoxides, or copolymerised within the matrix of a polylactic or polyglycolic acid. In another embodiment, the guest molecule is attached covalently to the central cavity of the oligosaccharide derivative or assembly so as to provide a precursor of the active form of the gdest molecule, for example to provide a prodrug which may be biodegraded to release an active form of the drug.

In another embodiment, the oligosaccharide-drug or alternatively the assembly complex is prepared by sonication. The advantage of this is that the complex forms smaller particles, which are easily absorbed.

In a preferred embodiment, the average particle diameter of the aggregate formed by the oligosaccharides of the invention is in the range of 50 - 500 nrh. In further embodiments the length of the assembly is in the range 5nm to 5 micrometres and preferably in the range 5nm to 1000nm.

In another embodiment, the oligosaccharide derivative or the asembly is present as a pharmaceutical formulation with any pharmaceutically acceptable ingredient such as a diluent, carrier, preservative (including anti-oxidant), binder, excipient, flavouring agent, thickener, lubricant, dispersing, wetting, surface active or isotonic agent which is compatible with the oligosaccharide or complex or assembly of same.

In another embodiment, ,the derivative or assembly formulated as a pharmacuetical formulation is dispersed in a suitable solvent, buffer, isotonic solution, emulsion, gel or lyophilised suspension.

The derivative or assembly formulated as pharmaceutical formulation is preferably administered parenterally, but may also be administered by alternative routes such as oral, topical, intranasal, intraocular, vaginal, rectal or by inhalation spray in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes percutaneous injections, intravenous, intramuscular, intrasteral, intrathecal, intraperitoneal injection or infusion techniques.

The present invention also provides the derivative or assemblies jn pharmaceutical formulations exhibiting sustained release of a drug. Such formulations are generally known and include devices made of inert polymers or of biodegradable^" polyacids or polyesters in which the active ingredient (the present oligosaccharide or its complex) is either dispersed, covalently linked via labile bonds, or stored as a reservoir between polymer membranes. Sustained release is achieved through diffusion of the active ingredient through the polymer matrix or hydrolysis of any covalent linkages present. Sustained release may also be attained by delivery of the active ingredient via osmotic pumps, in which the oligosaccharide may also act as an osmotic driving agent providing potential for the influx of water.

DETAILED DESCRIPTION OF THE INVENTION

The invention will be more easily understood from the following description of some examples, given by way of reference to the accompanying figures:

Fig. 1: Arrangements of multiple mutually-attracting group pairs A and B, A' and B'etc. on two faces of cyclic oligosaccharides which will produce molecular stacking.

Fig. 2: Formula of β-cyclodextrin.

Fig. 3: Formula of heptakis(6-amino-2-O-carboxymethyl-6-deoxy)-β-cyclodextrin.

Fig. 4: Electron micrograph of heptakis(6-amino-2-O-carboxymethyl-6-deoxy)-β- cyclodextrin in HEPES buffer (pH 7.4). Negative staining with 1% uranyl acetate.

Fig. 5: Electron micrograph of heptakis(6-amino-2-O-carboxymethyl-6-deoxy)-β- cyclodextrin in HEPES buffer (pH 7.4). Negative staining with 1 % phosphotungstic acid.

Example 1 illustrates the preparation of intermediate products and a final oligosaccharide product, heptakis(6-amino-2-O-carboxymethyl-6-deoxy)-β- cyclodextrin. Example 2 illustrates the characterisation of the molecular aggregates of this oligosaccharide. EXAMPLE 1:

Preparation of heptakis(6-amino-2-O-carboxymethyl-6-deoxy)-β-cyclodextrin

Heptakis (6-azido-6-deoxy)-β-cyclodextrin (prepared by the method of H. Parrot- Lopez et al., J. Am Chem. Soc, 1992, 114, 5479-5480) was reacted with sodium iodoacetate in the presence of potassium t-butoxide at 80 °C in DMF to yield heptakis(6-azido-2-O-carboxymethyl-6-deoxy)-β-cyclodextrin. This product was isolated by precipitation from water at pH 2. The ¹H. NMR (300 MHz, d₆-DMSO) showed a singlet for the methylene protons of the carboxymethyl group at_.4.35 ppm and separate signals for HI' at 5.15 and H1 at 4.92 ppm. According to NMR, a reproducible average of five carboxymethyl groups was introduced. This; product was then reduced to heptakis(6-amino-2-O-carboxymethyl-6-deoxy)-β-cyclodextrin by conventional phosphine reduction. The cyclodextrin was isolated by precipitation in water at pH 2 and purified by size exclusion chromatography. The final product retained traces of phosphines. The structure (with five carboxymethyl groups on O- 2) was confirmed by NMR. The ¹H NMR (300 MHz, D₂O) spectrum shows a singlet for the methylene protons of the carboxymethyl group at 4.228 ppm and signals for H1' at 5.07 as well as H1 at 5.30 ppm. The ¹³C NMR (500 MHz, D₂O) spectrum shows the carbonyl and the methylene carbon of the carboxymethyl group at 174.4 ppm and 69.1 ppm respectively, as well as C2' at 80.0 and C2 at 68.0 ppm. No shift was observed for C3' relative to C3, confirming substitution at O-2 instead of O-3, as anticipated for this type of reaction at the secondary side of cyclodextrins.

EXAMPLE 2:

Physicochemical characterisation of aggregate formation by heptakis(6-amino-2-O- carboxymethyl-6-deoxy)-β-cyclodextrin.

Heptakis(6-amino-2-O-carboxymethyl-6-deoxy)-β-cyclodextrin readily dissolves in 10 mM HEPES buffer (pH 7.4) upon gentle agitation at room temperature. The solution (0.1-1.0 mg/mL) is not completely transparent. Using transmission electron microscopy (with either uranyl acetate or phosphotungstic acid as negative stain), it was observed that cyclodextrin 3 self-assembles into elongated tape-like structures (Figure 4 and Figure 5). The tapes are between 50 and 300 nanometer wide and up to 20 micrometer long, yet only several nanometer thick. They seem quite flexible, since multiple twists, bends and folds are observed. However, the tapes are also rather fragile, and readily break into smaller fragments during the preparation of samples for electron microscopy. A significant fraction of the cyclodextrin is present as amorphous aggregates. As expected, no tapes but only amorphous aggregates are observed when the pH is either reduced from 7.4 to 4, or increased from 7.4 to 10, or when 0.5 M NaCI is added to the cyclodextrin solution. At high and low pH, and also in the presence of more than 0.1 M NaCI, the solution of cyclodextrin 3 becomes completely transparent. Clearly, the elongated tapes at neutral pH and low ionic strength scatter visible light much more than the smaller, amorphous aggregates present at high and low pH, and at high ionic strength. The intensity of the scattered light at 400 nm as a function of NaCI concentration is constant up to 40 mM of added NaCI, and rapidly decreases at higher ionic strength. This demonstrates that the elongated tapes only form in aqueous solution of low ionic strength.

It is suggested that the elongated tapes result from stacking of the catanionic cyclodextrin heptakis(6-amino-2-O-carboxymethyl-6-deoxy)-β-cyclodextrin. At neutral pH and low ionic strength, these molecules stack face to face, similar to the heterodimers described in the literature, except that here each molecule has oppositely charged faces, so that the molecular stacking can continue indefinitely in two directions. The cyclodextrins can form an efficient intermolecular hydrogen bond network, with each molecule providing multiple NH hydrogen bond donor sites and CO hydrogen bond acceptor sites. In this way, they imitate the stacking of certain cyclic peptides (J. D. Hartgerink et al., J. Am. Chem. Soc, 1996, 118, 43- 50) to form nanotubes in water, although here the protonated amino and carboxylate groups operate each on one side of the molecule, rather than alternately on the same side as do the hydrogen bonding NH and CO groups in the case of the peptides.

The examples show the use of cyclodextrin, i.e. a macrocycle comprising glucose subunits, as the macrocycle modified to enable continuous molecular stacking or supramolecular polymerization. It will be obvious to one skilled in the art that other saccharide subunits have similar properties to glucose which enable them to form a macrocycle that can be utilized in the present invention. Other saccharide subunits that may be employed include, as mentioned previously, the mono-saccharides such as the tetroses; for example furans; pentoses, for example fructose and ribose; hexoses, for example galactose and glucose; and heptoses and further monosaccharides in the series; and disaccharides which, as will be obvious to the skilled person, are two monosaccharide units linked together. The important features of the saccharides being the ability to form macrocyclic units; to have at least two distinct regions bearing side groups available for modification. In this case the modification of the side groups provides groups which are one of a pair of mutually attractive groups, the other of the pair being borne on a separate macrocycle unit.

Another important feature of the macrocycle is the presence of a central cavity defined by the component saccharide subunit. The size of the cavity will of course depend on the number and type of subunits utilized in the macrocycle. Of course it will be obvious to one skilled in the art that the use of any of the saccharide subunits described above will provide an oligosaccharide macrocycle capable of modification to enable continuous molecular stacking. In particular any saccharide whether they be monosaccharide or disaccharide capable of forming macrocyclic ring structures bearing at least one side group on both of the distinct sides and which side groups can be modified to provide mutually attractive groups may be utilized. Of course it is appreciated that the saccharides form a large group of compounds, however, for the purposes defined in this invention, the above mentioned characteristic features provided by the glucose subunits of the cyclodextrin macrocycle can be found in all appropriate saccharides and thus macrocycles can be provided which have saccharide subunits which may be of the same type as in the glucose subunits of cyclodextrin or macrocycles may be provided in which the subunit comprise different saccharide subunit type. For example, a macrocycle comprising glucose subunits with fructose subunit.

It is believed that one skilled in the art can, based on the description herein, utilise the present invention to its fullest extent. The above specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Claims

1. Macrocyclic oliogosaccharide derivatives capable of forming aggregates by self assembly comprising mono or disaccharide subunits, which subunits comprise at least two chemically distinct sides, and a central cavity and wherein the subunits are modified subunits bearing at least one side group on each side characterised in that at least one of the subunits making up the macro cycle is modified to enable continuous molecular stacking of the macro cycle by attraction between the side groups in separate macro cycles making up the aggregates.

2. A derivative as claimed in claim 1 wherein the subunits comprise side, groups on the primary and secondary hydroxyl sides capable of forming, intermolecular interactions with side groups on the primary or secondary hydroxyl side of an adjacent macrocycle unit.

3. A derivative as claimed in claim 1 or 2 having the general formula:

>

in which: n equals 2 - 11 or higher and indicates the number of ring units making up the macrocycle which may be the same or different depending on the X-, A-, and B- groups, if any one of K, L, M are zero, the remaining are independently one or more of;

a simple chemical bond; an atom or radical having a valency of at least 2 and can be in any position not occupied by a moiety involved in linking adjacent subunits forming the macrocycle,

Y, which may be the same or different are groups which link the subunits making up the macrocycle;

^' Xi, X'i, X₂, X'_2| X₃, X3, X₄, X'₄, X₅, Xe are independently zero or are linking groups;

Aι, A'ι, A_t, A'_4ι are independently zero or provide groups which are one of a pair of mutually attractive groups of types A and B,

BL B'_I, B_2> B'₂, B_3> B'₃, are independently zero or are groups which are one of a pair of mutually attractive groups of types A and B,

B₅ and B₆ are groups which may or may not form a mutually attractive pair with an A group.

4. A derivative as claimed in claim 3 wherein the Y group is selected from the group comprising:

oxygen, sulphur, selenium, nitrogen, phosphorous, carbon or silicon radicals having a valency of 2-4; or OCH₂ as in (1-2) - linked fructofuranoligosaccharides; or OCH₂CH(OH) as in (1-6)- linked furanooligosaccharides; or

OCH₂(CH₂OH) as in (1-5)- linked furanooligosaccharides.

5. A derivative as claimed in claim 3 or 4 wherein X_{1 (} X'-j, X₂, X'₂, X₃, X'_3ι X , X'₄, X₅, X₆ independently may comprise: a dendrimeric group, or

an atom or radical with a valency of at least 2.

6. A derivative as claimed in claim 5 wherein the radical is selected from the group comprising: CH_2ι CH₂ O, O, S, Se, N, P, carbonyl, ester, amido, amino, phosphate, sulfonyl, sulfonoxide, saturated or unsaturated aliphatic or aromatic carbon or silicon radical or a halogenated version of these.

7. A derivative as claimed in claim 5 wherein the dendrimeric group may be selected from the group comprising: -

peptides, nucleotides, cyclic, straight chain or branched oligosaccharides or polymeric groups.

8. A derivative as claimed in claim 7 wherein the polymeric groups may be selected from the group comprising:

polethylenimine (PEI), polyamides, polyamino acids, polynucleotides and polysaccharides.

9. A derivative as claimed in any of claims 3 to 8 wherein A₁₍ A A₄, A'₄, are independently chosen from:

groups capable of hydrogen bond formation, a cation or an anion, and dendrimeric groups .

10. A derivative as claimed in claim 9 wherein the cation is a protonated amino group.

11. A derivative as claimed in claim 9 or 10 wherein the anion is a carboxylate anion.

12. A derivative as claimed in any of claims 3 to 11 wherein the/or each of the B type groups comprise one of a complimentary pair capable of attracting the other of the complimentary pair in the A type groups of claims 3 to 11.

13. A derivative as claimed in claim 12 wherein the B type groups independently are chosen from: groups capable of H-bond formation, a cation or an anion, and dendrimeric groups.

14. A derivative as claimed in claim 13 wherein the cation is a protonated amino group.

15. A derivative as claimed in claims 13 or 14 wherein the anion is a sulphate group.

16. A derivative as claimed in claim 3 wherein when B₅ and B₆ independently do not comprise a mutually attractive pair with an A group of claims 3 to 11, either or both of B₅ or B₆ is hydrogen.

17. A derivative as claimed in claim 3 to 16 wherein B₅ and B₆ comprise independently non-immunogenic groups.

18. A derivative as claimed in claim 17 wherein the non immunogenic groups comprise groups selected from one or more of: poly(ethylene glycol), sialyl

GalGalcMAc.

19. A derivative as claimed in claim 3 wherein B₅ and B₆ comprise independently antigenic groups.

20. A derivative as claimed in claim 3 wherein B₅ and B₆ independently are groups for promoting adhesion to specific cells, proteins, antibodies or inert surfaces.

21. A derivative as claimed in claim 20 wherein the adhesion promoting groups are ligands for recognition by specific cell receptors.

22. A derivative as claimed in claims 3 to 21 having the formula

in which n equals 3 - 11 or higher and indicates the number of modified monosaccharide subunits in the macrocycle which may be the same or different depending on the X- A- and B- groups,

Xi. X2. X3 are linker groups;

A₁ is one of a pair of mutually attractive groups capable of interacting with the other of the pair in the B groups on a separate macrocyclic unit

B₂ and B₃ are independently one of a pair of mutually attractive groups capable of interacting with the other of the pair in the A groups on separate macrocyclic units.

23. A derivative as claimed in claim 22 wherein the monosaccharide subunits making up the macrocycle independently are chosen from: aglycone derivatives of L-glucose, D-hexoses, L-hexoses or disaccharide units, pentoses, furanose sugars, sialic acid and carbohydrate analogs.

24. A derivative as claimed in claim 23 wherein the disaccharide units may be (1-1)- or (1-2)- or (1-3)- or (1-6)- linked disaccharides.

25. A derivative as claimed in claims 22 to 24 wherein the macrocyclic derivative is a cyclodextrin derivative having the formula:

in which n equals 5-11 or higher and indicates the number of modified glucose subunits in the macrocycle which may be the same or different depending on the X, A and B groups,

Xi, X2. X3 are linker groups;

A1 is one of a pair of mutually attractive groups capable of interacting with the other of the pair in the B groups on a separate macrocyclic unit

26. A derivative as claimed in claims 3 to 25 wherein the X groups, A or B groups of macrocyclic units are capable of linking to the X groups, A or B groups of separate macrocyclic units by reaction of their chemical precursor group, catalysis or reaction of the chemical precursor group with a polyfunctional linking agent to provide an oligomerised nanoassembly.

27. A derivative as claimed in any preceding claim wherein the macrocycle units self assemble to form elongated nanoassemblies in an aqueous solvent.

28. A derivative as claimed in claims 1 to 26 wherein the macrocyclic units self- assemble to form elongated nanoassemblies in a non-aqueous solvent.

29. A derivative as claimed in claims 27 or 28 wherein the solvents also comprise molecules to modulate the assembly of the macrocycle.

30. A derivative as claimed in claim 29 wherein the molecules are poly(ethylene glycol) or polyamines.

31. A derivative as claimed in any preceding claim wherein the derivative has means for attachment to inert surfaces.

32. A derivative as claimed in any preceding claim complexed with a guest molecule, within the central cavity of the macrocycle.

33. A derivative as claimed in claim 32 wherein the guest molecule is covalently attached within the central cavity.

34. A derivative as claimed in claims 32 or 33 wherein the therapeutic molecule is selected from the group comprising anti-neo plastic agents, anti- inflammatory agents, anti-fungals, peptides, proteins and their analogs, oligosaccharides and their analogs, oligonucleotides and their analogs, plasmid DNA and complexes of oligonucleotide and of DNA with gene delivery agents.

35. A derivative as claimed in claims 32 or 33 wherein the derivative is complexed with a molecule or atom used for analysis or diagnosis.

36. A derivative as claimed in claims 32 or 33 wherein the derivative is complexed with a molecule which functions as a prodrug.

37. A nanoscale assembly comprising oligosaccharide macrocyclic derivatives as claimed in any preceding claim.

38. An assembly as claimed in claim 37 wherein the length of the assembly is in the range 5nm to 5 /m.

39. An assembly as claimed in claim 38 wherein the length of the assembly is in the range 5nm to 1000nm

40. An assembly as claimed in claims 37 to 39 wherein a guest molecule is complexed in a central channel defined by the aligned central cavities of each macrocycle unit making up the assembly.

41. An assembly as claimed in claims 37 to 40 wherein a guest molecule is complexed in or on cavities formed by the three dimensional structure of the assembly.

42. An assembly as claimed in claim 40 or 41 wherein the guest molecule is a therapeutic molecule.

43. An assembly as claimed in claim 42 wherein the therapeutic molecule is ■ selected from the group comprising anti-neoplastic agents, anti- inflammatory agents, anti-immunogenic agents, anti-fungals, peptides, proteins and their analogs, oligosaccharides and their analogs, oligonucleotides and their analogs, plasmid DNA and complexes of oligonucleotide and of DNA with gene delivery agents.

44. An assembly as claimed in claims 40 or 41 wherein the guest molecule is a molecule or atom used for analysis or diagnosis.

45. An assembly as claimed in claims 40 or 41 wherein the guest molecule functions as a prodrug.

46. An assembly as claimed in claims 37 to 45 wherein the assembly has means for attachment to any one of the following: antibody; inert surface; protein.

47. An assembly as claimed in claims 37 to 46 wherein the assembly includes at least two different types of macrocycle units.

48. An assembly as claimed in claims 37 to 47 wherein the different component macrocycles have complementary properties.

49. An assembly as claimed in claims 37 to 46 wherein the component macrocycle units are all of the same type.

50. A pharmaceutical formulation for use in the treatment of human or animals comprising derivatives of claims 1 to 36.

51. A pharmaceutical formulation for use in the treatment of humans or animals comprising an assembly of claims 37 to 49.

52. A pharmaceutical formulation as claimed in claims 50 or 51 also comprising one or more pharmaceutically acceptable ingredients selected from the group comprising: diluent, carrier, preservative, binder, antioxidant,. excipient, flavouring agent, thickener, lubricant, dispersing agent, wetting agent, surface active agent, isotonic agent, and adjuvants.

53. A pharmaceutical formulation as claimed in claims 50 to 52 herein the assembly or derivative is dispersed in one or a combination of members of the group comprising a suitable solvent buffer, isotonic solution, emulsion, gel or lyophilised suspension.

54. A pharmaceutical formulation as claimed in claims 50 to 53 for administration by one of the following routes comprising parenteral, oral, topical, intranasal, intraocular, vaginal, rectal or by inhalation spray in dosage unit formulation.

55. A pharmaceutical formulation as claimed in claims 50 to 54 wherein the formulation comprises means for sustained release of the guest molecule.