Indian Patents. 225253:A COMPOSITION COMPRISING A CRYSTALLINE COMPONENT OF MULTICOMPONENT MIXTURES.

Title of Invention	A COMPOSITION COMPRISING A CRYSTALLINE COMPONENT OF MULTICOMPONENT MIXTURES.
Abstract	A composition comprising a crystaline component of multicomponents mixtures of partial ethers of parent cyclodextrins characterized in that total ether subsitituents are less than two times the number of glucose residues, said crystaline phase being such that it contains, in a single crystal, a multitude of chemically individual compounds.

Full Text	APPLICATION FOR LETTERS PATENT TITLE: CRYSTALLINE MIXTURES OF PARTIAL METHYL ETHERS OF BETA-CYCLODEXTRIN AND RELATED COMPOUNDS Cross-reference to Related Applications This application takes priority from provisional patent application 60/164.949 November 12, 1999. Field of Invention This invention relates to mixtures of partial ethers of cyclodextrins that have the ability to crystallize and the use of these mixtures to solubilize and stabilize other compounds. Background of the Invention Cyclodextrins are a group of compounds consisting of, or derived from, alpha-, beta- and gamma-cyclodextrins, so called parent cyclodextrins. Alpha-, beta- and gamma- cyclodextrin are crystalline oligosaccharides consisting of six, seven or eight glucose residues, respectively, with residues connected by 1 i.o 4 alpha glycosidic bonds to form a macrocycle. Cyclodextrins have the ability to include other compounds into their macrocycles. No chemical bonds are formed in this process, called formation of inclusion complexes. Cyclodextrins function as hosts, while the included compounds are called guests. Inclusion complexes formed by cyclodextrins have been useful for solubiiizing and stabilizing guests such as drugs, ingredients of cosmetic and personal care products, dyes, agricultural chemicals and inorganic nanoparticles. To improve the ability to form inclusion comolexes, parent cyclodextrins have to be converted to derivatives. Each glucose residue of a cyclodextrin has one primary (OH- 6) and two secondary (OH-2 and OH-3) hydroxyls that can be substituted (for example, converted into ethers). The final product obtained by such reactions can be either chemically distinct cyclodextrin derivatives, or mixtures whose components are partially etherificd cyclodextrins that differ in number and position of substituents. Known mixtures of cyclodextrin derivatives exist in stable, noncrystalline (that is, amorphous) form. Partial methyl ethers of cyclodextrins synthesized under strongly basic conditions provide widely used amorphous mixtures (U.S. Patent 5,710,268; World Intellectual Property Organization 94/02516; U.S. Patent 4,746,734; German Patent 294,267). Compositions of matter containing partially methylated derivatives of cyclodextrins can be synthesized under conditions of very low basicity, (minimal effective basicity). (See U.S. Patent 5,681,828. U.S. Patent 5,935,941; U.S. Patent 6,001,821; World Intellectual Property Organization 92/22630.) The components of the latter mixtures are relatively few in number with substitution of the secondary hydroxyis predominating. Nevertheless, the mixtures prepared were found to exist in an amorphous state. Amorphous mixtures of partial 2-hydroxypropyl ethers of cyclodextrins (U.S. Patent 5,096,893; U.S. Patent 4,870,060) belong to this class and are produced using strong to moderately strong basicities. Many chemically individual (and crystalline) cyclodextrin derivatives are known (A. R. Khan, et al., Chemical Reviews, 98, 1977-1996, 1998; A. R. Hedges, Chemical Reviews, 98,2035-2044, 1998). Those of significant use include maltosyl-beta- cyclodextrin, which is produced industrially by enzymatic reaction (U.S. Patent 4,931,389), and heptakis(2,6-di-O-methyl)-beta-cyclodextrin produced by reaction in strongly basic medium in presence of barium salts (U.S. Patent 4,542,211). From the multitude of research generated compounds made by methods not suited for scale up, several chemically individual and crystalline methyl and 2-hydroxypropyl ethers of beta- cyclodextrin are known (K. Takeo et al., Die Starke 28,226-227, 1976; K. Takeo et al., Carbohydrate Research, 187,203-221, 1989; C. T. Rao et al., Journal of Organic Chemistry, 56,1327-1329,1991; J. Jindrich et al., Carbohydrate Research, 266, 75-80, 1995). It was believed that the crystallinity of these particular compounds was dependent on their being present as separate distinct entities, not as mixtures. The possibility of the existence of a crystalline lattice, which can accommodate a number of cyclodextrin derivatives of the kind described herein was not considered. Water-soluble cyclodextrin derivatives, after their preparation, have to be separated from water-soluble by-products, i.e., inorganic salts and small molecular weight organic compounds. This has been done by diffusion-based processes, by demineralization with ion exchange resins, or by precipitation with organic solvents (A. R. Hedges, Chemical Reviews, 98,2035-2044, 1998). In diffusion-based processes, the aqueous solution of product and by-products is brought into contact with a semi-permeable membrane which may, depending on the method of manufacture, permit only molecules that are smaller than those of the product to diffuse through. The other side of the membrane is in contact with water, or at least is covered by water. The smaller molecules of the by-product flow through the membrane to the aqueous side, where their concentration is lower. Molecules of water, since they obey the same physical law, flow in the opposite direction. This water flow leads to undesirable dilution of the desired product and can be prevented, or even reversed, by an increase of pressure on the product/by-product side. In the dialysis process, as used in the laboratory, the pressure on the product/by-product side of the membrane is not large enough to prevent fully the flow of water across the membrane into the desired product. Sturdy instrumentation that permits pressure large enough to prevent that flow or even reverse it, is known as reverse osmosis instrumentation. Use of reverse osmosis instrumentation to remove specific by-products, but not calcium salts, from cyclodextrin derivatives was disclosed in U.S. Patent 5,831,081. Summary of the Invention. The present invention provides means for making certain mixtures of cyclodextrin derivatives that can be separated into crystalline and noncrystalline (amorphous) components. The crystallization does not lead to separation of individual compounds. Rather, both the crystalline and noncrystalline components remain as mixtures of more than one compound. Crystalline and amorphous states are associated with different and distinct advantages. The present invention enables access to compositions having the advantages of both states from one reaction product. The data provided herein show that components of both crystalline and amorphous states may have similar numbers of substituents per cyclodextrin residue: it is the position of substituents that is critical for determining crystallization properties of particular cyclodextrin products. The invention provides compositions of matter comprising mixtures of partial ethers of parent cyclodextrins containing a crystalline component and an amorphous component wherein the crystalline component is comprised of cyclodextrins wherein total ether substituents are less than two times the number of glucose residues. It is possible to obtain therefrom a crystalline component containing mixtures of partial ethers of cyclodextrins. These crystalline cyclodextrins can form new, useful inclusion complexes with active agents such as drugs, agricultural chemicals, reaction agents, cosmetics, dyes and catalysts. Detailed Description of the Invention During methylation of beta-cyclodextrin using calcium hydroxide (i.e., base of minimal effective basicity) and low temperature, it was observed that crystals started to separate from concentrated solutions of the product. The crystalline phase thus obtained contained, in a single crystal, a multitude of chemically individual compounds, which among other aspects, differed by the number of methyls per molecule. This set apart the present composition of matter from compositions of previous art, in which existence of the single crystalline phase was associated with chemical individuality. Using these crystals as a seed, crystallization of other mixtures of partial methyl ethers of beta-cyclodextrin made in conditions of minimal effective basicity was possible. Crystalline phases were eventually obtained, but with much lower yields, from the corresponding derivatives of alpha-and gamma-cyclodextrin and from similar mixtures of partial hydroxypropyl ethers of beta-cyclodextrin, partial carboxymethyl ethers cf beta-cyclodextrin and partial ethyl ethers of beta-cyclodextrin. In all of these cases, including partial methyl ethers of beta- cyclodextrin. reaction products contained both crystalline and amorphous phases. Crystalline mixtures of partial methyl ethers of cyclodcxtrins of the present invention can be made in forms which vary in extent of overall substitution, in ease of crystallization, in solubility in water and in capacity to act as hosts in formation of inclusion complexes. Noncrystalline components of the compositions of matter of the present invention can also vary in degree of substitution and in their capacity to act as hosts. The majority of cyclodextrin derivatives in use at present are amorphous mixtures*. From the user's point of view, the main advantage of amorphous character of a mixture is that it confers to its inclusion complexes high solubility. The principal disadvantage of the amorphous state of a mixture is that untoward properties of the cyclodextrin components are, given time, fully manifested. Components of amorphous mixtures of partial methyl and hydroxypropyl ethers of cyclodextrins, which have a high degree of substitution, are hygroscopic. This invariably leads to stickiness of the whole mixture and to difficulties in storage and mechanical processing. Another disadvantage is that the word "crystalline" nearly universally conveys feeling of the purity; the word "amorphous" conveys the very opposite. From the view of producer, the amorphous state requires that the crude reaction product must fulfill all the purity criteria required in final products. None of the purification methods for use on amorphous compositions are sufficiently cost effective for large-scale production. Crystalline materials, on the other hand, can be inexpensively recrystallized until the desired purity is achieved. The effectiveness of recrystallization is well recognized by regulatory agencies. (Manufacture of pharmaceutical grades of glucose and sucrose serve as examples.) In Example 1, a procedure is described for making the crystalline partial methyl ethers of cyclodextrin using calcium hydroxide. Results show that the level of methylation of the crystalline components of the mixture do not differ greatly in number of methyl groups per molecule from those of amorphous components left in mother liquors. The ease and rapidity with which crystallization of the purified muhicomponent mixture of partial methyl ethers of beta-cyclodextrin occurs suggests that its components are isomorphous. In Example 1, it also is shown that reverse osmosis instrumentation can be used effectively to isolate the product. In Example 2. it is shown that the crystalline phase can be isolated from many reaction products of cyclodextrins. The products of methylation of beta-cyclodextrin made using mild bases in addition to calcium hydroxide may also be used in compositions to provide crystalline products, albeit with lower yields. Results in Example 2 also show that a number of additional factors impact yields of the crystalline component of the mixtures. Yields of the crystalline component are improved when lower reaction temperatures and less alkylation agent are used. These factors are expected to impact crystallization potential since they diminish the number and variety of individual chemical compounds present in the reaction product. Surprisingly, there was a difference between alkylating agents, dimethyl sulfate giving higher yields than methyl iodide. Yields of the crystalline component were increased when barium ions were added to the reaction mixture. Partial j methyl ethers of beta-cyclodextrin were found to crystallize much better than those of alpha- and gamma-cyclodextrins. Changes in size of substituent (e.g., moving from the smaller methyl to the larger ethyl, carboxymethyl, 2-hydroxypropyl) substituents also decreased the ability of derivatives to form crystals. When 2-hydroxypropyl groups were tested, only components with two or fewer hydroxypropyl groups per cyclodextrin residue were detectable in crystals obtained. Results in Example 3 document the structure of components of the crystalline phase and described some of its properties. A single crystal of a methylation product of beta-cyclodextrin was grown large enough for all required analyses. That crystal contained 17% of water of crystallization. Analysis of the substitution pattern (J. Reuben, Carbohydrate Research, 1986, 157,201-213 and references therein) of this crystal revealed that the only methylation which occurred was that on OH-2 hydroxyls and not all of these were methylated. Mass spectrum of the same crystal documented that species from one to seven methyl groups per molecule were present. These results prove that the crystal is formed by isomorphous 2-O-oligomcthyl beta-cyclodextrins. The absence of methylation on OH-6 hydroxyls also was documented chemically as shown by ease of formation of tertiary butyl ethers. Average number of methyls per molecule of the crystalline product can be manipulated by changes in amount of methylation agent used in preparation. Increase in that number is accompanied by an increase in solubility in water. When amounts of methylation agent were increased substantially, species with more methyls than seven per molecule could be detected in crystalline phase by mass spectrometry. Results in Example 3 also show that the compositions of matter of the present invention are different from any partial methyl ethers of cyclodextrin of previous art. In Example 4, it is shown that both the crystalline and amorphous components of partial methyl ethers of cyclodextrin preparations are good solubilizers. Hydrocortisone, a drug that has commonly been used as comparison standard in this field, was solubilized more efficiently using compositions of the invention than by currently available cyclodextrin compositions. Hydrocortisone was shown to increase the solubility of crystalline partial methyl ethers of beta-cyclodextrin in water. Example 4 also describes methods that can be used to produce liquid or solid pharmaceutical formulations. The same methods are expected to have applications for preparation of personal care products and formulations used in agriculture. Results in Example 5 show that the crystalline mixtures containing partial methyl ethers of cyclodextrin are of low toxicity and that biological activity of guests is unaffected when using such cyclodextrin derivatives as hosts. Example 1 Preparation of Partly Crystalline Methyl Beta-Cvclodextrin Calcium oxide, freshly made from calcium hydroxide (23.9 g), was added to water (400 ml) while cooling. Beta-cyclodextrin hydrate (80 g) was then added while stirring, followed by dropwise addition of dimethyl sulfate (27.8 ml), which took about two hours. Stirring at room temperature was continued for four days. The still alkaline reaction mixture was then saturated with carbon dioxide until pH decreased to 5-6. Thereafter, the suspension was boiled for a few minutes to expel carbon dioxide and methanol. After cooling the suspended calcium carbonate was filtered off and the clear filtrate dialyzed against distilled water. In this particular dialysis, the mechanical pressure on the product/by-product side of the semipermeable membrane was not large enough to prevent inflow of water. Nevertheless, by a moderate increase of reverse pressure, it was possible to decrease inflow, while the removal of calcium salts was not impeded. It also was possible to obtain separation using a centrifugal filter unit equipped with an ultrafiltration membrane of nominal molecular weight cutoff of 3000 D. Although this cutoff was higher than the molecular weight of the product and washing washing was necessary, product was obtained. Consequently, use of commercially available instrumentation, would be appropriate for removal of calcium salts (e.g., methylsulfate, sulfate, bicarbonate and carbonate) which represent the majority of by-products present in the reaction product. The dialyzed solution of the product can be further purified by treatment with a solution of sodium carbonate until no calcium carbonate separated. The suspension was filtered and the filtrate was treated with demineralizing ion exchange resin, filtered again and evaporated in vacuo to yield 59.7 g of white mass. This white mass was dissolved in boiling water (60 ml) and left to crystallize. Crystals thus obtained were again recrystallized at 1:1 w/w (water to crystals), yielding 25.06 g of colorless needles. Mother liquors, when evaporated to dryness, yielded 30.19 g of white mass. Results of thin layer chromatography showed that crystals and mother liquors contain partial ethers of cyclodextrins that do not differ greatly in the number of methyl groups per molecule. Interestingly, crystals contained less of the beta-cyclodextrin and its monomethyl derivative than mother liquors. Neither the crystalline component, nor the component obtained by evaporating of the mother liquor, melted at less than 200 degrees centigrade. Example 2 Isolation of Crystalline Parts from Various Cvclodextrin Derivatives Prepared in Conditions of Minimal Effective Basicity Partial methyl ethers of beta-cyclodextrin (1 g), prepared in the conditions specified in Table 1, were dissolved in water (1 ml) at temperature of boiling water bath. If, upon cooling, crystals did not appear, the solution was seeded with crystals obtained in Example 1. After a week of standing at room temperature, the crystals were collected and weighed. Results in Table 1 show that the yield of crystals improves when methylation occurs at lower reaction temperatures and with use of less methylation agent. Mother liquors, when partially evaporated, yielded additional crystals. Beta W7 Ml.8, a commercial noncrystalline derivative produced by Wacker Biochem Corp., was used as a control. (Beta W7 Ml.8 is not prepared in conditions of minimal effective basicity.) Hydroxypropyl beta-cyclodextrin mixtures prepared using calcium hydroxide at room temperature yielded crystalline components that contained di(2-hydroxypropyl)-beta- cyclodextrins in addition to the starting beta-cyclodextrin and mono(2-hydroxypropyl)- beta- cyclodextrins. This was established by thin layer chromatography. Carboxymethyl substituents may also be present on cyclodextrins in the crystalline component. This was established by alkylating beta-cyclodextrin using chloroacetic acid, 2 moles per mole of glucose residue and using calcium hydroxide as a base. Large crystals in low yield were obtained from the product. Crystalline partial ethyl ethers were obtained by alkylation of beta-cyclodextrin with ethyl iodide under conditions similar to those used in preparation of P264 in Table 1. Products of methylation of alpha- and gamma-cyclodextrins were made using calcium hydroxide. The product, when processed as described above, yielded only small amounts of fine particles, possibly microcrystals. under these conditions. This failure to provide larger crystals was probably due to the very high water solubility. When methanol-propanol mixtures were used, crystals were obtained. The crystallization occurred slowh. In all the cases, except with partial methyl ethers of beta-cyclodextrin, the yields of crystals were low. Nevertheless, some crystals were obtained. When conditions of minimal effective basicity were not used, no crystals resulted. Example 3 Characterization of Crystalline Partial Methvl Ethers of Beta-Cvclodextrin The crystalline component of P264 (Tablel), after drying at 100 degrees centigrade in vacuo to remove all of its cystallization in water, was soluble in water at room temperature to the concentration 15%; at temperature of a boiling water bath, the solubility was in excess of 50%. The moderate solubility of the crystalline component of P264 in water at room temperature is not a barrier to achieving a higher total concentration of that composition in the presence of a suitable drug, as is shown in Example 4. Crystals did not melt at temperatures of up to 200 degrees centigrade. When a single crystal of P264 was hydrolyzed, only glucose and 2-O-methyl glucose were detected in hydrolyzate by I3C-N.M.R. (J. Reuben, Carbohydrate Research, 1986, 157. 201-213). Mass spectrum (FAB in positive ionization mode) of the same crystal documented the following composition: Beta cyclodextrin not detectable: less than 1% of molecules had one methyl, 3% two methyls, 1:5% three methyls, 41% four methyls, 25% five methyls, 11% six methyls, 3% seven methyls and less than 1% eight methyls. The absence of substitution on primary hydroxyls (OH-6) in P264 also was documented chemically. A sample of it was dissolved in trifluoracetic acid and saturated with isobutylene. This reaction is known to introduce tertiary butyl groups onto primary hydroxyls. The product isolated form this reaction mixture indeed contained tertiary butyl groups. The crystalline component of P258 (Table 1), after drying at 100 degrees centigrade in vacuo, was soluble in water at room temperature to the concentration 29%; at temperature of boiling water bath, the solubility was in excess of 50%. Crystals did not melt at temperatures of up to 200 degrees centigrade. Mass spectrum (FAB in positive ionization mode) showed that from di- to deca-methyl species were present with heptamethyl beta-cyclodextrin in the largest quantity. 13C-N.M.R. data shows that about 76% of OH-2 were converted to methyl ether groups (calculated from intensities of C-l signals). These results show clear differentiation between the compositions of the present invention and of the previously known partial methyl ethers of cyclodextrins, both amorphous and crystalline. The compositions of matter of the present invention differ from all previously described amorphous materials, since a crystalline state exists therein and a phase containing the crystalline state can be obtained therefrom. It differs from the known crystalline heptakis(2,6-di-O-methyl)-beta-cyclodextrin, which contains 14 methyls per molecule, both in terms of number of methyls per moleucle and in effects of temperature on solubility in water (see Y. Kubota et al., Carbohydrate Research, 192, 159- 166, 1989). Example 4 Solubilizing Potency of Partial Methvl Ethers of Beta-Cvclodextrins and Their Use in Pharmaceutical Formulations Solubilization potency of cyclodextrin derivatives was compared through measurement of the solubility of hydrocortisone in 5%(w/w) aqueous solutions of the said derivatives, which in the case of the crystalline components of P264 and P258 (Table 1). were used without heat drying. The results are shown in Table 2. The concentration limits to which solubilization power of the crystalline component of P264 can be utilized were investigated in an experiment in which an excess of hydrocortisone was added to the aqueous system which contained more of the crystalline component of P264 than it could dissolve. The results in Table 3 show that, in these conditions, hydrocortisone solubilized additional amounts of the crystalline component of P264. In other words, the stability and solubility of the inclusion complex is high enough to lead to dissolution of all host available. Results in Table 3 also show that the amorphous component of P264, the mother liquors of P264, is a powerful solubilizer as well. A solution of a drug suitable for topical or parenteral uses was made by adjusting the hydrocortisone solution made as in Table 2 to iso;onicity by addition of sodium chloride. The solution thus made did not deteriorate during storage at room temperature for several months. The solutions of hydrocortisone and the crystalline components of P264 and P258, upon slow evaporation, yield glassy materials, which in some instances were bordered with traces of apparently crystalline phase products. The inclusion complex of crystalline partial methyl ethers of beta-cyclodextrin is very water soluble. However, with perseverance the crystalline state could be induced. Tablets suitable for oral or buccal use were made by direct compression of the residue after evaporation of the above solution of hydrocortisone inclusion complexes. These tablets dissolved clearly and fully within 2-3 minutes in water. Example 5 Bioeffects and Toxicity Issues The solutions of the crystalline partial methyl ethers of beta-cyclodextrin described above were nontoxic to mice when given as the only source of liquids at 2% concentration for two days. These solutions have a mildly sweet taste without aftertaste. When given by intraperitoneal injection, no toxic effects to mice were seen at doses to 6 g per kg; the first toxic effects were seen at 7 g per kg doses. To demonstrate that a drug included in crystalline partial methyl ethers of beta- cyclodextrin retains its potency, the blanching test was used (McKenzie, Archives of Dermatology, 86, 91-94,1962). An aqueous solution (25 microliters) containing hydrocortisone (8 mg/ml) and crystalline partial methyl ethers of beta-cyclodextrin (5%) was applied to the skin of the human forearm. After drying of the droplet by warm air, the test area was covered by an occlusive bandage overnight. The skin area to which the solution was applied was then evaluated for signs of blanching comparing the treated areas of both untreated skin (negative control) and skin to which a hydrocortisone solution (1%) in ethanol had been applied in a similar manner (positive control, known to produce blanching). The tested solution produced blanching surpassing that of the positive control. WE CLAIM: 1. A composition comprising a crystalline component of multicomponents mixtures of partial ethers of parent cyclodextrins characters in the that ether substituants are less than two times the number of clams residues, said crystaline phase being such that it contains, in a single crystal, a multitude of chemically individual compounds. 2. The composition as claimed in claim 1, wherein the ethers of cyclodextrings have formed inclusion complexes. 3. The composition as claimed in daim 2, wherein the ethers of cyclodextrins have formed inclusion complexes with a drug. 4. The composition as claimed In claim 2, wherein the ethers of cyclodextrins have formed inclusion complexes with an agricultural chemical. 5. The composition as claimed in claim 2, wherein the ethers of cydodextrins have formed inclusion complexes with a catalyst. 6. The composition as claimed in claim 2, wherein the ethers of cyclodextrins have formed inclusion complexes with a dye. 7. The composition as claimed in claim 1, compnsng multicomponent mixtures of partial ethers of parent cyclodextris containing a crystalline component and a non-crystaline component whersin the crystalline component is as described in claim 1. 8. The composition as claimed in claim 7, wherein the parent cydodextrin is a betacydodextrin wherein there are no more than 13 ether substituents replacing hydroxyl groups on the cydodextrin molecule. 9. The composition as claimed in daim 8, wherein the ether moieties are methyl ethers. 10.The composition as claimed in daim 7, wherein the ether moieties replacing the hydroxyi substituents on the parent cydodextrin are methyl ethers. 11. The composition as claimed in daim 10 wherein the parent cydodextrin is a betacyclodextrin. 12. The composition as claimed in claim 7, wherein the ethers of cyctodextrins have formed inclusion complexes. 13. The composition as claimed in claim 12, wherein the ethers of cydodextrins have formed inclusion complexes with a drug. 14. The composition as claimed in claim 7, wherein the ethers of cydodextrins have formed inclusion complexes with an agricultural chemical. 15. The composition as claimed in claim 12, wherein the ethers cydodextrins have formed inclusion complexes with a catalyst. 16. The composition as claimed in claim 12, wherein therinners of cydodextrins have formed inclusion complexes with a dye. A composition comprising a crystaline component of multicomponents mixtures of partial ethers of parent cyclodextrins characterized in that total ether subsitituents are less than two times the number of glucose residues, said crystaline phase being such that it contains, in a single crystal, a multitude of chemically individual compounds.

Full Text

APPLICATION FOR LETTERS PATENT
TITLE: CRYSTALLINE MIXTURES OF PARTIAL METHYL ETHERS OF
BETA-CYCLODEXTRIN AND RELATED COMPOUNDS
Cross-reference to Related Applications
This application takes priority from provisional patent application 60/164.949
November 12, 1999.
Field of Invention
This invention relates to mixtures of partial ethers of cyclodextrins that have the
ability to crystallize and the use of these mixtures to solubilize and stabilize other
compounds.
Background of the Invention
Cyclodextrins are a group of compounds consisting of, or derived from, alpha-,
beta- and gamma-cyclodextrins, so called parent cyclodextrins. Alpha-, beta- and gamma-
cyclodextrin are crystalline oligosaccharides consisting of six, seven or eight glucose
residues, respectively, with residues connected by 1 i.o 4 alpha glycosidic bonds to form a
macrocycle. Cyclodextrins have the ability to include other compounds into their
macrocycles. No chemical bonds are formed in this process, called formation of inclusion
complexes. Cyclodextrins function as hosts, while the included compounds are called
guests. Inclusion complexes formed by cyclodextrins have been useful for solubiiizing
and stabilizing guests such as drugs, ingredients of cosmetic and personal care products,
dyes, agricultural chemicals and inorganic nanoparticles.
To improve the ability to form inclusion comolexes, parent cyclodextrins have to
be converted to derivatives. Each glucose residue of a cyclodextrin has one primary (OH-
6) and two secondary (OH-2 and OH-3) hydroxyls that can be substituted (for example,
converted into ethers). The final product obtained by such reactions can be either
chemically distinct cyclodextrin derivatives, or mixtures whose components are partially
etherificd cyclodextrins that differ in number and position of substituents.
Known mixtures of cyclodextrin derivatives exist in stable, noncrystalline (that is,
amorphous) form. Partial methyl ethers of cyclodextrins synthesized under strongly basic
conditions provide widely used amorphous mixtures (U.S. Patent 5,710,268; World
Intellectual Property Organization 94/02516; U.S. Patent 4,746,734; German Patent
294,267). Compositions of matter containing partially methylated derivatives of
cyclodextrins can be synthesized under conditions of very low basicity, (minimal effective
basicity). (See U.S. Patent 5,681,828. U.S. Patent 5,935,941; U.S. Patent 6,001,821;
World Intellectual Property Organization 92/22630.) The components of the latter
mixtures are relatively few in number with substitution of the secondary hydroxyis
predominating. Nevertheless, the mixtures prepared were found to exist in an amorphous
state. Amorphous mixtures of partial 2-hydroxypropyl ethers of cyclodextrins (U.S.
Patent 5,096,893; U.S. Patent 4,870,060) belong to this class and are produced using
strong to moderately strong basicities.
Many chemically individual (and crystalline) cyclodextrin derivatives are known
(A. R. Khan, et al., Chemical Reviews, 98, 1977-1996, 1998; A. R. Hedges, Chemical
Reviews, 98,2035-2044, 1998). Those of significant use include maltosyl-beta-
cyclodextrin, which is produced industrially by enzymatic reaction (U.S. Patent
4,931,389), and heptakis(2,6-di-O-methyl)-beta-cyclodextrin produced by reaction in
strongly basic medium in presence of barium salts (U.S. Patent 4,542,211). From the
multitude of research generated compounds made by methods not suited for scale up,
several chemically individual and crystalline methyl and 2-hydroxypropyl ethers of beta-
cyclodextrin are known (K. Takeo et al., Die Starke 28,226-227, 1976; K. Takeo et al.,
Carbohydrate Research, 187,203-221, 1989; C. T. Rao et al., Journal of Organic
Chemistry, 56,1327-1329,1991; J. Jindrich et al., Carbohydrate Research, 266, 75-80,
1995). It was believed that the crystallinity of these particular compounds was dependent
on their being present as separate distinct entities, not as mixtures. The possibility of the
existence of a crystalline lattice, which can accommodate a number of cyclodextrin
derivatives of the kind described herein was not considered.
Water-soluble cyclodextrin derivatives, after their preparation, have to be separated
from water-soluble by-products, i.e., inorganic salts and small molecular weight organic
compounds. This has been done by diffusion-based processes, by demineralization with
ion exchange resins, or by precipitation with organic solvents (A. R. Hedges, Chemical
Reviews, 98,2035-2044, 1998). In diffusion-based processes, the aqueous solution of
product and by-products is brought into contact with a semi-permeable membrane which
may, depending on the method of manufacture, permit only molecules that are smaller
than those of the product to diffuse through. The other side of the membrane is in contact
with water, or at least is covered by water. The smaller molecules of the by-product flow
through the membrane to the aqueous side, where their concentration is lower. Molecules
of water, since they obey the same physical law, flow in the opposite direction. This water
flow leads to undesirable dilution of the desired product and can be prevented, or even
reversed, by an increase of pressure on the product/by-product side. In the dialysis
process, as used in the laboratory, the pressure on the product/by-product side of the
membrane is not large enough to prevent fully the flow of water across the membrane into
the desired product. Sturdy instrumentation that permits pressure large enough to prevent
that flow or even reverse it, is known as reverse osmosis instrumentation. Use of reverse
osmosis instrumentation to remove specific by-products, but not calcium salts, from
cyclodextrin derivatives was disclosed in U.S. Patent 5,831,081.
Summary of the Invention.
The present invention provides means for making certain mixtures of cyclodextrin
derivatives that can be separated into crystalline and noncrystalline (amorphous)
components. The crystallization does not lead to separation of individual compounds.
Rather, both the crystalline and noncrystalline components remain as mixtures of more
than one compound. Crystalline and amorphous states are associated with different and
distinct advantages. The present invention enables access to compositions having the
advantages of both states from one reaction product. The data provided herein show that
components of both crystalline and amorphous states may have similar numbers of
substituents per cyclodextrin residue: it is the position of substituents that is critical for
determining crystallization properties of particular cyclodextrin products.
The invention provides compositions of matter comprising mixtures of partial
ethers of parent cyclodextrins containing a crystalline component and an amorphous
component wherein the crystalline component is comprised of cyclodextrins wherein total
ether substituents are less than two times the number of glucose residues. It is possible to
obtain therefrom a crystalline component containing mixtures of partial ethers of
cyclodextrins. These crystalline cyclodextrins can form new, useful inclusion complexes
with active agents such as drugs, agricultural chemicals, reaction agents, cosmetics, dyes
and catalysts.
Detailed Description of the Invention
During methylation of beta-cyclodextrin using calcium hydroxide (i.e., base of
minimal effective basicity) and low temperature, it was observed that crystals started to
separate from concentrated solutions of the product. The crystalline phase thus obtained
contained, in a single crystal, a multitude of chemically individual compounds, which
among other aspects, differed by the number of methyls per molecule. This set apart the
present composition of matter from compositions of previous art, in which existence of the
single crystalline phase was associated with chemical individuality. Using these crystals
as a seed, crystallization of other mixtures of partial methyl ethers of beta-cyclodextrin
made in conditions of minimal effective basicity was possible. Crystalline phases were
eventually obtained, but with much lower yields, from the corresponding derivatives of
alpha-and gamma-cyclodextrin and from similar mixtures of partial hydroxypropyl ethers
of beta-cyclodextrin, partial carboxymethyl ethers cf beta-cyclodextrin and partial ethyl
ethers of beta-cyclodextrin. In all of these cases, including partial methyl ethers of beta-
cyclodextrin. reaction products contained both crystalline and amorphous phases.
Crystalline mixtures of partial methyl ethers of cyclodcxtrins of the present
invention can be made in forms which vary in extent of overall substitution, in ease of
crystallization, in solubility in water and in capacity to act as hosts in formation of
inclusion complexes. Noncrystalline components of the compositions of matter of the
present invention can also vary in degree of substitution and in their capacity to act as
hosts.
The majority of cyclodextrin derivatives in use at present are amorphous mixtures*.
From the user's point of view, the main advantage of amorphous character of a mixture is
that it confers to its inclusion complexes high solubility. The principal disadvantage of the
amorphous state of a mixture is that untoward properties of the cyclodextrin components
are, given time, fully manifested. Components of amorphous mixtures of partial methyl
and hydroxypropyl ethers of cyclodextrins, which have a high degree of substitution, are
hygroscopic. This invariably leads to stickiness of the whole mixture and to difficulties in
storage and mechanical processing. Another disadvantage is that the word "crystalline"
nearly universally conveys feeling of the purity; the word "amorphous" conveys the very
opposite.
From the view of producer, the amorphous state requires that the crude reaction
product must fulfill all the purity criteria required in final products. None of the
purification methods for use on amorphous compositions are sufficiently cost effective for
large-scale production. Crystalline materials, on the other hand, can be inexpensively
recrystallized until the desired purity is achieved. The effectiveness of recrystallization is
well recognized by regulatory agencies. (Manufacture of pharmaceutical grades of
glucose and sucrose serve as examples.)
In Example 1, a procedure is described for making the crystalline partial methyl
ethers of cyclodextrin using calcium hydroxide. Results show that the level of methylation
of the crystalline components of the mixture do not differ greatly in number of methyl
groups per molecule from those of amorphous components left in mother liquors. The
ease and rapidity with which crystallization of the purified muhicomponent mixture of
partial methyl ethers of beta-cyclodextrin occurs suggests that its components are
isomorphous. In Example 1, it also is shown that reverse osmosis instrumentation can be
used effectively to isolate the product.
In Example 2. it is shown that the crystalline phase can be isolated from many
reaction products of cyclodextrins. The products of methylation of beta-cyclodextrin made
using mild bases in addition to calcium hydroxide may also be used in compositions to
provide crystalline products, albeit with lower yields. Results in Example 2 also show that
a number of additional factors impact yields of the crystalline component of the mixtures.
Yields of the crystalline component are improved when lower reaction temperatures and
less alkylation agent are used. These factors are expected to impact crystallization
potential since they diminish the number and variety of individual chemical compounds
present in the reaction product. Surprisingly, there was a difference between alkylating
agents, dimethyl sulfate giving higher yields than methyl iodide. Yields of the crystalline
component were increased when barium ions were added to the reaction mixture. Partial j
methyl ethers of beta-cyclodextrin were found to crystallize much better than those of
alpha- and gamma-cyclodextrins. Changes in size of substituent (e.g., moving from the
smaller methyl to the larger ethyl, carboxymethyl, 2-hydroxypropyl) substituents also
decreased the ability of derivatives to form crystals. When 2-hydroxypropyl groups were
tested, only components with two or fewer hydroxypropyl groups per cyclodextrin residue
were detectable in crystals obtained.
Results in Example 3 document the structure of components of the crystalline
phase and described some of its properties. A single crystal of a methylation product of
beta-cyclodextrin was grown large enough for all required analyses. That crystal contained
17% of water of crystallization. Analysis of the substitution pattern (J. Reuben,
Carbohydrate Research, 1986, 157,201-213 and references therein) of this crystal revealed
that the only methylation which occurred was that on OH-2 hydroxyls and not all of these
were methylated. Mass spectrum of the same crystal documented that species from one to
seven methyl groups per molecule were present. These results prove that the crystal is
formed by isomorphous 2-O-oligomcthyl beta-cyclodextrins. The absence of methylation
on OH-6 hydroxyls also was documented chemically as shown by ease of formation of
tertiary butyl ethers. Average number of methyls per molecule of the crystalline product
can be manipulated by changes in amount of methylation agent used in preparation.
Increase in that number is accompanied by an increase in solubility in water. When
amounts of methylation agent were increased substantially, species with more methyls than
seven per molecule could be detected in crystalline phase by mass spectrometry. Results
in Example 3 also show that the compositions of matter of the present invention are
different from any partial methyl ethers of cyclodextrin of previous art.
In Example 4, it is shown that both the crystalline and amorphous components of
partial methyl ethers of cyclodextrin preparations are good solubilizers. Hydrocortisone, a
drug that has commonly been used as comparison standard in this field, was solubilized
more efficiently using compositions of the invention than by currently available
cyclodextrin compositions. Hydrocortisone was shown to increase the solubility of
crystalline partial methyl ethers of beta-cyclodextrin in water. Example 4 also describes
methods that can be used to produce liquid or solid pharmaceutical formulations. The
same methods are expected to have applications for preparation of personal care products
and formulations used in agriculture.
Results in Example 5 show that the crystalline mixtures containing partial methyl
ethers of cyclodextrin are of low toxicity and that biological activity of guests is unaffected
when using such cyclodextrin derivatives as hosts.
Example 1
Preparation of Partly Crystalline Methyl Beta-Cvclodextrin
Calcium oxide, freshly made from calcium hydroxide (23.9 g), was added to water
(400 ml) while cooling. Beta-cyclodextrin hydrate (80 g) was then added while stirring,
followed by dropwise addition of dimethyl sulfate (27.8 ml), which took about two hours.
Stirring at room temperature was continued for four days. The still alkaline reaction
mixture was then saturated with carbon dioxide until pH decreased to 5-6. Thereafter, the
suspension was boiled for a few minutes to expel carbon dioxide and methanol. After
cooling the suspended calcium carbonate was filtered off and the clear filtrate dialyzed
against distilled water. In this particular dialysis, the mechanical pressure on the
product/by-product side of the semipermeable membrane was not large enough to prevent
inflow of water. Nevertheless, by a moderate increase of reverse pressure, it was possible
to decrease inflow, while the removal of calcium salts was not impeded. It also was
possible to obtain separation using a centrifugal filter unit equipped with an ultrafiltration
membrane of nominal molecular weight cutoff of 3000 D. Although this cutoff was higher
than the molecular weight of the product and washing washing was necessary, product was
obtained. Consequently, use of commercially available instrumentation, would be
appropriate for removal of calcium salts (e.g., methylsulfate, sulfate, bicarbonate and
carbonate) which represent the majority of by-products present in the reaction product.
The dialyzed solution of the product can be further purified by treatment with a
solution of sodium carbonate until no calcium carbonate separated. The suspension was
filtered and the filtrate was treated with demineralizing ion exchange resin, filtered again
and evaporated in vacuo to yield 59.7 g of white mass. This white mass was dissolved in
boiling water (60 ml) and left to crystallize. Crystals thus obtained were again
recrystallized at 1:1 w/w (water to crystals), yielding 25.06 g of colorless needles. Mother
liquors, when evaporated to dryness, yielded 30.19 g of white mass. Results of thin layer
chromatography showed that crystals and mother liquors contain partial ethers of
cyclodextrins that do not differ greatly in the number of methyl groups per molecule.
Interestingly, crystals contained less of the beta-cyclodextrin and its monomethyl
derivative than mother liquors. Neither the crystalline component, nor the component
obtained by evaporating of the mother liquor, melted at less than 200 degrees centigrade.
Example 2
Isolation of Crystalline Parts from Various Cvclodextrin Derivatives Prepared in
Conditions of Minimal Effective Basicity
Partial methyl ethers of beta-cyclodextrin (1 g), prepared in the conditions
specified in Table 1, were dissolved in water (1 ml) at temperature of boiling water bath.
If, upon cooling, crystals did not appear, the solution was seeded with crystals obtained in
Example 1. After a week of standing at room temperature, the crystals were collected and
weighed. Results in Table 1 show that the yield of crystals improves when methylation
occurs at lower reaction temperatures and with use of less methylation agent. Mother
liquors, when partially evaporated, yielded additional crystals. Beta W7 Ml.8, a
commercial noncrystalline derivative produced by Wacker Biochem Corp., was used as a
control. (Beta W7 Ml.8 is not prepared in conditions of minimal effective basicity.)
Hydroxypropyl beta-cyclodextrin mixtures prepared using calcium hydroxide at room
temperature yielded crystalline components that contained di(2-hydroxypropyl)-beta-
cyclodextrins in addition to the starting beta-cyclodextrin and mono(2-hydroxypropyl)-
beta- cyclodextrins. This was established by thin layer chromatography. Carboxymethyl
substituents may also be present on cyclodextrins in the crystalline component. This was
established by alkylating beta-cyclodextrin using chloroacetic acid, 2 moles per mole of
glucose residue and using calcium hydroxide as a base. Large crystals in low yield were
obtained from the product. Crystalline partial ethyl ethers were obtained by alkylation of
beta-cyclodextrin with ethyl iodide under conditions similar to those used in preparation of
P264 in Table 1. Products of methylation of alpha- and gamma-cyclodextrins were made
using calcium hydroxide. The product, when processed as described above, yielded only
small amounts of fine particles, possibly microcrystals. under these conditions. This
failure to provide larger crystals was probably due to the very high water solubility. When
methanol-propanol mixtures were used, crystals were obtained. The crystallization
occurred slowh. In all the cases, except with partial methyl ethers of beta-cyclodextrin,
the yields of crystals were low. Nevertheless, some crystals were obtained. When
conditions of minimal effective basicity were not used, no crystals resulted.
Example 3
Characterization of Crystalline Partial Methvl Ethers of Beta-Cvclodextrin
The crystalline component of P264 (Tablel), after drying at 100 degrees centigrade
in vacuo to remove all of its cystallization in water, was soluble in water at room
temperature to the concentration 15%; at temperature of a boiling water bath, the solubility
was in excess of 50%. The moderate solubility of the crystalline component of P264 in
water at room temperature is not a barrier to achieving a higher total concentration of that
composition in the presence of a suitable drug, as is shown in Example 4. Crystals did not
melt at temperatures of up to 200 degrees centigrade.
When a single crystal of P264 was hydrolyzed, only glucose and 2-O-methyl
glucose were detected in hydrolyzate by I3C-N.M.R. (J. Reuben, Carbohydrate Research,
1986, 157. 201-213). Mass spectrum (FAB in positive ionization mode) of the same
crystal documented the following composition: Beta cyclodextrin not detectable: less than
1% of molecules had one methyl, 3% two methyls, 1:5% three methyls, 41% four methyls,
25% five methyls, 11% six methyls, 3% seven methyls and less than 1% eight methyls.
The absence of substitution on primary hydroxyls (OH-6) in P264 also was
documented chemically. A sample of it was dissolved in trifluoracetic acid and saturated
with isobutylene. This reaction is known to introduce tertiary butyl groups onto primary
hydroxyls. The product isolated form this reaction mixture indeed contained tertiary butyl
groups.
The crystalline component of P258 (Table 1), after drying at 100 degrees
centigrade in vacuo, was soluble in water at room temperature to the concentration 29%; at
temperature of boiling water bath, the solubility was in excess of 50%. Crystals did not
melt at temperatures of up to 200 degrees centigrade. Mass spectrum (FAB in positive
ionization mode) showed that from di- to deca-methyl species were present with
heptamethyl beta-cyclodextrin in the largest quantity. 13C-N.M.R. data shows that about
76% of OH-2 were converted to methyl ether groups (calculated from intensities of C-l
signals).
These results show clear differentiation between the compositions of the present
invention and of the previously known partial methyl ethers of cyclodextrins, both
amorphous and crystalline. The compositions of matter of the present invention differ
from all previously described amorphous materials, since a crystalline state exists therein
and a phase containing the crystalline state can be obtained therefrom. It differs from the
known crystalline heptakis(2,6-di-O-methyl)-beta-cyclodextrin, which contains 14 methyls
per molecule, both in terms of number of methyls per moleucle and in effects of
temperature on solubility in water (see Y. Kubota et al., Carbohydrate Research, 192, 159-
166, 1989).
Example 4
Solubilizing Potency of Partial Methvl Ethers of Beta-Cvclodextrins and Their Use in
Pharmaceutical Formulations
Solubilization potency of cyclodextrin derivatives was compared through
measurement of the solubility of hydrocortisone in 5%(w/w) aqueous solutions of the said
derivatives, which in the case of the crystalline components of P264 and P258 (Table 1).
were used without heat drying. The results are shown in Table 2.
The concentration limits to which solubilization power of the crystalline
component of P264 can be utilized were investigated in an experiment in which an excess
of hydrocortisone was added to the aqueous system which contained more of the
crystalline component of P264 than it could dissolve. The results in Table 3 show that, in
these conditions, hydrocortisone solubilized additional amounts of the crystalline
component of P264. In other words, the stability and solubility of the inclusion complex is
high enough to lead to dissolution of all host available. Results in Table 3 also show that
the amorphous component of P264, the mother liquors of P264, is a powerful solubilizer as
well.
A solution of a drug suitable for topical or parenteral uses was made by adjusting
the hydrocortisone solution made as in Table 2 to iso;onicity by addition of sodium
chloride. The solution thus made did not deteriorate during storage at room temperature
for several months.
The solutions of hydrocortisone and the crystalline components of P264 and P258,
upon slow evaporation, yield glassy materials, which in some instances were bordered with
traces of apparently crystalline phase products. The inclusion complex of crystalline
partial methyl ethers of beta-cyclodextrin is very water soluble. However, with
perseverance the crystalline state could be induced. Tablets suitable for oral or buccal use
were made by direct compression of the residue after evaporation of the above solution of
hydrocortisone inclusion complexes. These tablets dissolved clearly and fully within 2-3
minutes in water.
Example 5
Bioeffects and Toxicity Issues
The solutions of the crystalline partial methyl ethers of beta-cyclodextrin described
above were nontoxic to mice when given as the only source of liquids at 2% concentration
for two days. These solutions have a mildly sweet taste without aftertaste. When given by
intraperitoneal injection, no toxic effects to mice were seen at doses to 6 g per kg; the first
toxic effects were seen at 7 g per kg doses.
To demonstrate that a drug included in crystalline partial methyl ethers of beta-
cyclodextrin retains its potency, the blanching test was used (McKenzie, Archives of
Dermatology, 86, 91-94,1962). An aqueous solution (25 microliters) containing
hydrocortisone (8 mg/ml) and crystalline partial methyl ethers of beta-cyclodextrin (5%)
was applied to the skin of the human forearm. After drying of the droplet by warm air, the
test area was covered by an occlusive bandage overnight. The skin area to which the
solution was applied was then evaluated for signs of blanching comparing the treated areas
of both untreated skin (negative control) and skin to which a hydrocortisone solution (1%)
in ethanol had been applied in a similar manner (positive control, known to produce
blanching). The tested solution produced blanching surpassing that of the positive control.
WE CLAIM:
1. A composition comprising a crystalline component of multicomponents
mixtures of partial ethers of parent cyclodextrins characters in the that
ether substituants are less than two times the number of clams residues,
said crystaline phase being such that it contains, in a single crystal, a
multitude of chemically individual compounds.
2. The composition as claimed in claim 1, wherein the ethers of cyclodextrings
have formed inclusion complexes.
3. The composition as claimed in daim 2, wherein the ethers of cyclodextrins
have formed inclusion complexes with a drug.
4. The composition as claimed In claim 2, wherein the ethers of cyclodextrins
have formed inclusion complexes with an agricultural chemical.
5. The composition as claimed in claim 2, wherein the ethers of cydodextrins
have formed inclusion complexes with a catalyst.
6. The composition as claimed in claim 2, wherein the ethers of cyclodextrins
have formed inclusion complexes with a dye.
7. The composition as claimed in claim 1, compnsng multicomponent
mixtures of partial ethers of parent cyclodextris containing a crystalline
component and a non-crystaline component whersin the crystalline
component is as described in claim 1.
8. The composition as claimed in claim 7, wherein the parent cydodextrin is
a betacydodextrin wherein there are no more than 13 ether substituents
replacing hydroxyl groups on the cydodextrin molecule.
9. The composition as claimed in daim 8, wherein the ether moieties are
methyl ethers.
10.The composition as claimed in daim 7, wherein the ether moieties
replacing the hydroxyi substituents on the parent cydodextrin are methyl
ethers.
11. The composition as claimed in daim 10 wherein the parent cydodextrin is
a betacyclodextrin.
12. The composition as claimed in claim 7, wherein the ethers of cyctodextrins
have formed inclusion complexes.
13. The composition as claimed in claim 12, wherein the ethers of
cydodextrins have formed inclusion complexes with a drug.
14. The composition as claimed in claim 7, wherein the ethers of cydodextrins
have formed inclusion complexes with an agricultural chemical.
15. The composition as claimed in claim 12, wherein the ethers
cydodextrins have formed inclusion complexes with a catalyst.
16. The composition as claimed in claim 12, wherein therinners of
cydodextrins have formed inclusion complexes with a dye.
A composition comprising a crystaline component of multicomponents mixtures of
partial ethers of parent cyclodextrins characterized in that total ether subsitituents
are less than two times the number of glucose residues, said crystaline phase
being such that it contains, in a single crystal, a multitude of chemically individual
compounds.

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Patent Number

225253

Indian Patent Application Number

IN/PCT/2002/00643/KOL

PG Journal Number

45/2008

Publication Date

07-Nov-2008

Grant Date

05-Nov-2008

Date of Filing

13-May-2002

Name of Patentee

ROQUETTE FRERES

Applicant Address

62132 LESTREM

Inventors:

#	Inventor's Name	Inventor's Address
1	PITHA JOSEF	P.O. BOX 127 DEAL ISLAND, MD 21821

PCT International Classification Number

C08B 37/16,C08L 5/16

PCT International Application Number

PCT/US2000/42064

PCT International Filing date

2000-11-09

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/164,949	1999-11-12	U.S.A.