EP0518930A1 - Compositions a base de cyclodextrines - Google Patents

Compositions a base de cyclodextrines

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Publication number
EP0518930A1
EP0518930A1 EP91905452A EP91905452A EP0518930A1 EP 0518930 A1 EP0518930 A1 EP 0518930A1 EP 91905452 A EP91905452 A EP 91905452A EP 91905452 A EP91905452 A EP 91905452A EP 0518930 A1 EP0518930 A1 EP 0518930A1
Authority
EP
European Patent Office
Prior art keywords
cyclodextrin
substituted
group
cyclodextrin derivative
cyclodextrins
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP91905452A
Other languages
German (de)
English (en)
Other versions
EP0518930A4 (en
Inventor
John Hewlett Coates
Christopher John Easton
Stephen Frederick Lincoln
Stephen John Z K Kunststofflab. B L Van Eyk
Bruce Lindley May
Michael Lloyd Williams
Susan Elizabeth Brown
Angelo Lepore
Ming-Long Liao
Yin Luo
Vilma Macolino
Deborah Susanne Schiesser
Craig Bernard Whalland
Ian S. C. Mckenzie
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Australia Commercial Research and Development Ltd
Original Assignee
Australia Commercial Research and Development Ltd
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Publication date
Application filed by Australia Commercial Research and Development Ltd filed Critical Australia Commercial Research and Development Ltd
Publication of EP0518930A1 publication Critical patent/EP0518930A1/fr
Publication of EP0518930A4 publication Critical patent/EP0518930A4/en
Withdrawn legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof

Definitions

  • This invention concerns cyclodextrins, cyclodextrin derivatives, inclusion complexes and compositions containing same, methods for their preparation, and various uses thereof.
  • cyclodextrins For a detailed discussion of the background of cyclodextrins, the reader is invited to review the "Background of the Invention" section of the '359 Application and the publications cited therein. A brief review is also provided below.
  • Cyclodextrins (which are cyclic oligosaccharides) have been the subject of increasing interest for more than 25 years. This interest is primarily due to the ability of cyclodextrin molecules to form "inclusion complexes" with other molecules called “guests.”
  • a cyclodextrin molecule contains an apolar/hydrophobic cavity or annulus which can "include” an apolar/hydrophobic portion of a guest molecule. While a cyclodextrin' ⁇ cavity is hydrophobic, the remainder of the molecule is relatively hydrophilic, and thus a cyclodextrin has the potential to include a relatively hydrophobic -guest and render it soluble in water.
  • cyclodextrin derivatives including covalently linked cyclodextrins, which are capable of achieving inclusion complexes of greater stability.
  • the '359 Application provided, inter alia., many new cyclodextrin derivatives and processes, as well as synthetic pathways and intermediates for preparing such derivatives.
  • cyclodextrins to deliver peptides is disclosed in International Patent Application PCT US89/04099 to Cetus Corporation, which was published 19 April 1990 under International Publication No. WO 90/03784.
  • the disclosure of the Cetus Application is expressly incorporated herein.
  • the Cetus application discloses the use of various cyclodextrins includinghydroxypropyl cyclodextrins to solubilize and/or stabilize various polypeptides and especially proteins.
  • Such cyclodextrin compounds are limited in design and can, in some instances, present certain drawbacks with respect to solubility at low cyclodextrin concentration levels and/or low stability constants such that there remains a need for finding
  • Such other molecules include drugs such as, for example, amiodarone, a broad spectrum antiarrhythmic having several undesirable traits.
  • drugs such as, for example, amiodarone, a broad spectrum antiarrhythmic having several undesirable traits.
  • amiodarone HC1 was only administered orally, until very recently when an injectable solubilized by polysorbate 80 became available.
  • absorption is low and erratic and, therefore, its bioavailability is unpredictable.
  • the recommended administration is by slow infusion in a dilute solution. Only in extreme clinical emergency, may the drug be given as a IV bolus injection over one to two minutes, but this is generally met with poor tolerance.
  • the slow dissolution of the precipitate may also expose the patient to transient high drug levels, and subsequent reduction in blood pressure. Circulatory collapse may occur, and temporary hot flashes, sweating and nausea have also been reported.
  • Most of these adverse effects are also related to dosage and duration of therapy, concurrent use of other antiarrythymic agents, severity of the underlying disease state, and wide individual variation in the pharmokinetic profile of the drug. Accordingly, there is room for improvement in both oral and intravenous formulations of amiodarone.
  • many pharmaceutical agents can incite an immune response in a patient.
  • penicillin in some individuals is antigenic, giving rise to anti-penicillin antibodies. This can pose two adverse consequences. First, if penicillin is given to an allergic individual, a severe reaction may occur which can vary from a skin rash or asthma to anaphylaxis and death. Second, the drug does not react at its site of activity because it is sequestered by antibody.
  • Other pharmaceutical agents which may be potentially immunogenic are antibodies themselves, peptides or other synthetic or natural materials.
  • liposomes are a well known means of drug delivery.
  • problems can arise if a drug to be incorporated into the liposome has low solubility or wettability, or if it is susceptible to instability, e.g., upon
  • Difficulties with delivery of bioactive compounds are not unique to pharmaceuticals. For example, a continuing problem with applying some herbicides and pesticides to plants is that they evaporate too quickly after application to allow the bioactive molecule to be effective. Thus, there is a continuing need for enhanced delivery of other active agents such as herbicides, pesticides and agricultural agents such as fertilizers.
  • compositions and methods which are useful in preventing or decreasing the occurrence of certain products of enzyme-catalysis, or which can diminish the effective concentration of, or remove unwanted proteins or toxins from a patient.
  • this specification provides, inter alia, cyclodextrin derivatives, inclusion complexes, processes, syntheses and intermediates which are useful in the application of cyclodextrins to, inter alia, pharmaceutical (diagnostic and therapeutic) and industrial applications.
  • a first embodiment provides a method for designing cyclodextrin inclusion complexes comprising a useful agent and an otherwise substituted or unsubstituted cyclodextrin or two or more otherwise substituted or unsubstituted cyclodextrins linked by at least one linking group.
  • the process comprises first determining whether the useful agent possesses at least one group capable of non-covalent association. Next, the orientation of the agent in the cyclodextrin annulus and the relative position of the associable group is determined by considering the dipole moment of the agent and the position of hydrophobic or apolar groups on the agent.
  • the final step is to selectively substitute at least one substituent comprising a group which will associate with the associable group on the agent onto one or more C2, C3 or C6 positions of the cyclodextrin or linked cyclodextrins.
  • the substituent(s) should be configured in order to position its associable group(s) in the approximate vicinity of the associable group on the agent to promote association therebetween.
  • the process may further comprises the steps of: (1) determining the dipole moment of each individual cyclodextrin of the linked cyclodextrins; and (2) linking the cyclodextrins such that the dipole moments align where possible.
  • cyclodextrin derivatives and inclusion complexes including cyclodextrin derivatives comprising groups which are charged, polar or are capable of forming a non-covalent association with another group.
  • the inclusion complexes can comprise an active agent such as a pharmaceutical, herbicidal, pesticidal, agricultural, cosmetic or personal care agent. Synthetic procedures for preparing such derivatives are also provided.
  • SUBSTITUTESHEET Notable among such derivatives are those having the formula CD - W - R - L , wherein
  • CD represents an otherwise substituted or unsubstituted cyclodextrin
  • W represents a functional linking group, such as amino, amide, ester, thioether, thioamide, thioester or thiol,
  • R represents a substituted or unsubstituted group
  • L represents a group selected from reactive, charged, polar or associating groups such as amino, carboxyl, hydroxyl, sulfonate phosphate, acyloxy, alkyloxy or thiyl. Such compounds are useful both as delivery vehicles and as intermediates for preparing other compounds.
  • X represents - N - C - or - S - C -
  • R and R' represent substituted or unsubstituted groups, and R' can represent hydrogen
  • Q is a carboxylic acid group or a carboxylic acid group derivatized to undergo substitution.
  • Such compounds are very advantageous as delivery vehicles when Q is a carboxylic acid, and as intermediates when Q is a carboxylic acid group derivatized to undergo substitution.
  • cyclodextrin derivatives comprising first and second otherwise substituted or unsubstituted cyclodextrins covalently bonded together by at least one linking group which links a C2, C3 or C6 carbon on the first cyclodextrin to a C2, C3 or C6 carbon on the second cyclodextrin.
  • linking group which links a C2, C3 or C6 carbon on the first cyclodextrin to a C2, C3 or C6 carbon on the second cyclodextrin.
  • SUBSTITUTESHEET which an otherwise substituted or unsubstituted cyclodextrin, or two or more otherwise substituted or unsubstituted linked cyclodextrins, are covalently bound to a useful agent such that the covalent bond, when broken, will yield the agent in an active form.
  • cyclodextrin derivative comprising an otherwise substituted or unsubstituted cyclodextrin, or two or more otherwise substituted or unsubstituted linked cyclodextrins that are covalently bonded to a carrier which can target cells of interest within a patient.
  • carriers can comprise, for example, an antibody or fragment thereof, hormones or lymphokines.
  • inventions provide otherwise substituted or unsubstituted cyclodextrin, or two or more otherwise substituted or unsubstituted linked cyclodextrins covalently bonded, physically entrapped or encapsulated, or otherwise associated with a carrier useful for localized or prolonged delivery of a useful agent.
  • a carrier useful for localized or prolonged delivery of a useful agent.
  • Such carriers can include, for example, liposomes, solid matrices,etc.
  • cyclodextrins in accordance with this invention include processes in which cyclodextrins in accordance with this invention may be used. Included are processed for masking the taste of a pharmaceutical agent, for increasing the solubility of a pharmaceutical or other useful agent, for targeting a pharmaceutical agent to a selected group of cells, for encapsulating into a liposome a pharmaceutical agent of relatively low solubility or wettability, for improving the stability of a pharmaceutical agent that is encapsulated in a liposome, for decreasing or preventing an immunogenic reaction of a patient to a pharmaceutical agent, for the treatment of metabolic disorders associated with compounds produced in vivo through enzyme catalysis, and for treating a patient suffering from an excess of a toxin.
  • compositions including, inter alia advantageous composition for the delivery of cimetidine, peptides, amiodarone and piroxicam.
  • a useful agent such as a pharmaceutical is covalently bonded to a cyclodextrin, such that the covalent bond, when broken, will yield the agent in active form.
  • Yet another object of this invention comprises the formation of advantageous inclusion complexes and prodrugs comprising cyclodextrin derivatives and pharmaceutically active compounds such as cimetidine, polypeptides, amiodarone and piroxicam.
  • Yet another object of this invention is to provide many useful processes which can employ cyclodextrin derivatives in accordance with this invention.
  • Figure 1 illustrates the dipole moment of a cyclodextrin molecule
  • Figure 2 illustrates the dipole moment of the cyclodextrin host and guest molecule in an inclusion complex
  • Figure 3 illustrates the equilibrium between a cyclodextrin host and guest molecule
  • Figure 4 illustrates a cyclodextrin inclusion complex
  • Figure 5 illustrates a possible equilibrium for an inclusion complex comprising a guest molecule and cyclodextrins which are linked by their primary carbons;
  • Figure 6 illustrates a possible equilibrium for an inclusion complex comprising a guest molecule and linked cyclodextrins in which a primary carbon of one cyclodextrin is linked to a secondary carbon of another cyclodextrin;
  • FIGS 7A-7C illustrate additional types of inclusion complexes which can be prepared in accordance with this invention.
  • Figure 8 illustrates the hydrolysis of BTEE and BTEE + ⁇ - cyclodextrin by ⁇ -chymotrypsin
  • Figure 9 illustrates a semilogrithmic graph of the mean plasma concentration of amiodarone obtained after intravenous bolus injection of Cordarone X (open circle) and amiodarone- - CDNSc complex (close circle) ;
  • Figure 10 illustrates a semilogarithmic graph of the plasma concentration of amiodarone for dog 10 obtained after intravenous bolus injection of Cordarone X;
  • Figure 11 illustrates a semilogrithmic graph of the plasma concentration of amiodarone for dog 12 obtained after intravenous
  • Cyclodextrin - refers to ⁇ -, ⁇ - , y-, or ⁇ - cyclodextrins, which are those that are generally available. It will be appreciated, however, that if other cyclodextrins are discovered or become available in sufficient commercial quantities, such cyclodextrins shall also be encompassed by this invention.
  • Cyclodextrin Derivative - refers to a cyclodextrin- containing compound in which one or more atoms or groups of atoms are substituted for a C2, C3 or C6 hydroxyl or hydroxyl hydrogen, i.e. , "modified cyclodextrins.”
  • the term cyclodextrin derivative also encompasses "linked cyclodextrins" where two or more cyclodextrins are linked together, and compounds where a useful agent such as a pharmaceutical is covalently bonded to a cyclodextrin, such that the covalent bond, when broken will yield the agent in active form. This term also includes any salt or hydrate which can be formed from the cyclodextrin derivative.
  • Modified Cyclodextrin - refers to a species of cyclodextrin derivatives that contains one or more atoms or groups of atoms substituted for a C2, C3 or C6 hydroxyl or hydroxyl hydrogen.
  • modified cyclodextrin will not be meant to include compounds where two or more cyclodextrins are linked together, or compounds where a useful agent such as a pharmaceutical is covalently bound to a cyclodextrin. This term also includes any salt or hydrate which can be formed from the modified cyclodextrin.
  • Linked Cyclodextrins - refers to two or more cyclodextrins linked together by one or more bridging groups.
  • the bridging groups can link a C2, C3 or C6 position of one cyclodextrin to any one of the C2, C3, or C6 positions of the other cyclodextrin.
  • This term includes any salt or hydrate which can be formed from the linked cyclodextrins.
  • Prodrug - refers to a cyclodextrin derivative in which a pharmaceutical agent is covalently bonded to a substituted or unsubstituted cyclodextrin or to two or more linked cyclodextrins such that the covalent bond, when broken, yields the agent in active form.
  • the product formed by this reaction comprises the residue of the pharmaceutical linked to a cyclodextrin, or to a pendant arm substituted thereon, through a linking group that is formed by the reaction of a functional group on the pharmaceutical agent with a functional group on the cyclodextrin or pendant arm.
  • the residue of a pharmaceutical agent can be covalently bonded to the cyclodextrin through a linking group that is substituted for a C2, C3 or C6 hydroxyl or hydroxyl hydrogen, or it can be covalently bonded via a pendant arm that is substituted to a C2, C3 or C6 position.
  • This term also includes any salt or hydrate which can be formed from the prodrug.
  • Cyclodextrin Inclusion-Association Complex refers to an inclusion complex in which there are one or more associable groups or portions of a group located on a substituent that is substituted at a C2, C3 or C6 position of a cyclodextrin, which groups or portions form an association with one or more associable groups or portions of a guest atom or molecule.
  • the associable portions can include polar or charged groups or portions, or groups or portions capable of hydrogen bonding. This term also includes any salt or hydrate which can be formed from the inclusion-association complex.
  • Cyclodextrin Inclusion Salt - refers to an inclusion- association complex in which the associable group or portion of the cyclodextrin substituent carries a net positive or negative charge which causes it to associate with an oppositely charged
  • SUBS TITUTESHEET group or portion of a guest atom or molecule This term also includes any other salt or a hydrate which can be formed from the cyclodextrin inclusion salt.
  • Pharmaceutical Agent - refers to compounds or their salts or hydrates which have a pharmaceutically recognized use. Pharmaceutical shall also be taken to mean drug, isotope, toxin or any other molecule used either to detect, i.e., diagnose, or treat pathological lesion, e.g., cancer, or to prevent the occurrence of such a lesion. It will be appreciated that the term is not intended to cover compounds which have some type of bio-affecting activity, but which are not recognized as pharmaceutical agents by the medical community or drug regulatory agencies. For example, a dye is not considered to be a pharmaceutical agent, even though it might possess some type of bio-affecting activity.
  • the term “pharmaceutical agent” does not include unreacted reactants, other products, or solvents (including water) that are present in the reaction mixture when synthesizing cyclodextrin derivatives or which are present when forming inclusion complexes.
  • Pesticidal, Herbicidal, Agricultural, Cosmetic or Personal Care Agent - refer to compounds or their salts or hydrates which have a generally recognized use in the fields.
  • a dye is not considered to be such an agent.
  • these terms do not include unreacted reactants, other products, or solvents (including water) that are present in the reaction mixture when synthesizing cyclodextrin derivatives or which are present when forming the inclusion complexes.
  • Useful Agent - includes pharmaceutical, pesticidal, herbicidal, agricultural, cosmetic or personal care agents or salts or hydrates thereof as defined above.
  • Carrier - refers to atoms or molecules to which a cyclodextrin, cyclodextrin derivative and/or inclusion complex
  • SUBSTITUTESHEET may be bound, entrapped or encapsulated, covalently or otherwise, and which may impart at least one advantageous property to the cyclodextrin, cyclodextrin derivative and/or inclusion complex which is not already possessed thereby.
  • the carrier or portions thereof generally will not be included in the cyclodextrin annulus, and will generally not comprise the useful agent for which delivery is sought by means of the cyclodextrin, cyclodextrin derivative and/or inclusion complex. Rather, the carrier generally will be employed to enhance delivery of the useful agent.
  • carriers can include targeting molecules for pharmaceutical applications such as antibodies or fragments thereof, hormones, cytokines, lymphokines, receptors in soluble form, other pharmaceutical agents, toxins or isotopes or any other agent where one molecule is bonded to the other.
  • the carrier may also comprise a matrix or physical encapsulant (e.g., liposome) with which the cyclodextrin, cyclodextrin derivative and/or inclusion complex may be physically associated.
  • Solubility - refers to solution in water or other aqueous-based media.
  • the '359 Application provided guidance in tailoring cyclodextrin derivatives to enhance the effect of substituents upon the inclusion complex.
  • the cyclodextrin can be substituted with associable groups or pendant arms containing associable groups, e.g., polar, charged or capable of forming other associations, which can exert a desired effect upon associable groups on the guest molecule.
  • associable groups e.g., polar, charged or capable of forming other associations
  • Other factors may also affect the orientation of the guest in the inclusion complex.
  • many molecules have a dipole moment; that is, their atoms and their electrons and nuclei are so arranged that one part of the molecule has a positive electrical charge while
  • an included species having a dipole moment will possess a tendency to orient itself so that the positive end of the included species will be in the vicinity of the secondary hydroxyls and the negative end will be in the vicinity of the primary hydroxyls.
  • This phenomenon is illustrated in Figure 2.
  • the skilled artisan will appreciate that the inclusion complex of Figure 2 is actually only part of the overall equilibrium equation illustrated in Figure 3, the amount of included species being dependent on the equilibrium or stability constant K.
  • the inclusion constant K is defined by the equation:
  • substitutions to the cyclodextrin can be tailored to achieve maximum effect.
  • This method of design can be particularly important in instances where the guest theoretically is capable of more than one orientation within the cyclodextrin annulus.
  • substitutions for the primary hydroxyls can be made so that they will exert a desired effect on the portion of the included molecule expected to be present in that vicinity.
  • substitutions for the secondary hydroxyls can also be made.
  • This method of inclusion complex design is generally illustrated in Figure 4. Whether to substitute for primary or secondary hydroxyls thus becomes a function of the molecule to
  • SUBSTITUTE SHEET be included, and the basic techniques for selectively achieving desired primary and secondary substitutions are provided in the '359 Application.
  • the cyclodextrin is negatively polarized in the vicinity of the secondary hydroxyls
  • This can thus enhance the attraction with the guest, and thereby enhance one or more properties of the inclusion complex such as stability.
  • FIG. 5 illustrates cyclodextrins linked by their primary carbons.
  • the dipole moments are in opposing directions, which leads to instability of the linked configuration. This can result in an equilibrium with a differently-configured species such as the staggered configuration (designated as B) . That equilibrium lessens the concentration of species A, which in turn decreases the concentration of the inclusion complex C.
  • the inclusion complex C can also have a somewhat unstable configuration because one end of the guest molecule can be in the vicinity of a cyclodextrin having a like charge.
  • Figure 6 illustrates a linked configuration which may ameliorate the aforementioned problem and thus lead to an inclusion complex having enhanced properties.
  • a primary carbon of a first cyclodextrin is linked to a secondary carbon of a second cyclodextrin, thereby creating a uniform dipole moment for the entire linked cyclodextrin species.
  • the cyclodextrins should thus remain aligned and facilitate inclusion of the polar molecule.
  • the dipole moment of the linked cyclodextrin can complement the dipole moment of the included guest.
  • SUBSTITUTE SHEET While the foregoing discussion is largely devoted to increased interaction between the host and guest, it should also be noted that the desired effect, in some instances, may be a decreased interaction, i.e., a less stable complex. For example, if a cyclodextrin is being used to mask the taste of a pharmaceutical, it may be desirable to provide an inclusion complex which is stable in the mouth but quickly dissociates in the stomach to release the pharmaceutical.
  • the same reasoning can also apply to cyclodextrin derivatives in which an agent is covalently linked to a cyclodextrin such that the covalent bond, when broken, yields the agent in active form, e.g., a prodrug. That is, the covalent bond can be designed to break relatively quickly to provide a fast delivery of the agent.
  • a bond that is susceptible to acid hydrolysis such as that in an ester or amide, would thus be susceptible to being broken in the acidic environment of the stomach.
  • ester linkages are preferred where quick hydrolysis is desired.
  • amide linkages which are part of a pendant arm can also provide relatively quick hydrolysis rates.
  • Amide linkages which are directly substituted onto the cyclodextrin generally posses long rates of hydrolysis and are thus more likely to be useful where release in the intestine is desired.
  • the bond could be susceptible to being broken by other normally occurring internal conditions in the stomach or intestine of a host.
  • the cyclodextrin derivative e.g., modified cyclodextrin, linked cyclodextrins or prodrug can be further substituted, either on the pendant arm, the linking group, or on another glucopyranosyl residue, with one or more groups which could assist in acid or base catalyzed hydrolysis of such ester or amide bonds.
  • Such reactions could thus be designed either to be pH-dependant reactions or to be catalyzed by, for example, endogenous enzymes or flora found in the particular areas of the body where release is desired.
  • the composition can contain multiple types of bonds and/or complexes designed to provide release at different rates and/or locations in the patient.
  • Figures 7A to C illustrate inclusion complexes comprising two cyclodextrins wherein each cyclodextrin has one or more substituents which can associate.
  • linked cyclodextrins contain associable groups X and Y, which represent charged or polar atoms or groups of atoms, or some other group capable of interaction (e.g., hydrogen bonding) .
  • the groups can be selected to provide desired release profiles.
  • the selected groups can be oppositely charged in the acidic environment of the stomach, but one or both could lose its charge (or take on opposite charges) in the relatively neutral environment of the intestines.
  • a histidine or imidazole group may be protonated (and thus positively charged) in the stomach but not in the intestine.
  • a negatively charged group such as sulfonate on another cyclodextrin
  • an association that could form in the stomach may essentially disappear in the intestine.
  • Such design provides a more stable complex in the stomach and a less stable complex in the intestine, resulting in more complexed drug passing through the stomach and more free drug being released in the intestine.
  • Figures 7A to C illustrate only complexes containing one drug molecule that is included through the secondary ends of the cyclodextrins, other configurations such as those shown in the '359 Application ( Figures 12D, F and G therein) , as well as other variations are certainly possible and are within the scope of this invention.
  • Figures 7B and C illustrate other variations of complexes wherein two otherwise substituted or unsubstituted cyclodextrins contain one or more associable groups. Of course, many other configurations are possible.
  • Modified cyclodextrins in accordance with this invention can comprise an otherwise substituted or unsubstituted cyclodextrin in which at least one C2, C3 or C6 hydroxyl is substituted with a group selected from -XR 1 , YR 3 , SiE'R'R 6 , and -R 7 , wherein X can R w and R 15 represent groups as defined by R ⁇ R 13 above, and represent
  • Y can represent
  • R 1 to R 11 can each represent the same or different groups selected from: the groups -XR 1 , YR 3 , SiR ⁇ 6 , and -R 7 are as defined above, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, and wherein any two or three groups bonded to the same substituent can be taken together to represent a single group multiply bonded to said same substituent, and wherein R 1 to R 11 may be further substituted by at least one -XR 1 , -YR 2 R 3 , -SiR 4 R 5 R 6 , -R 7 , halogen, and OR 12 , wherein R 12 is as defined for R 1 to R 11 .
  • Cyclodextrins in which one or more C2, C3 or C6 hydroxyls are selectively substituted by ether substituents are also encompassed.
  • ether substituents may be further substituted with any of the foregoing groups.
  • preferred groups include the substituted amino cyclodextrins, i.e., cyclodextrins wherein at least one substitution for said C2, C3 or C6 hydroxyl is of the formula -YR 2 R 3 , wherein Y is N, and R 2 and R 3 are as previously defined.
  • R 2 is hydrogen and R 3 represents amino, hydroxyl, carboxyl, sulfonate (S0 3 ") , phosphate (P0 4 " 3 ) , substituted alkyl, cycloalkyl, or aryl, or wherein R 2 and R 3 are taken together to represent a hereto substituted multiply bonded alkyl group.
  • inclusion complexes in which at least one pharmaceutical, pesticidal, herbicidal, agricultural, cosmetic, personal care or other useful agent is included in a modified cyclodextrin as described above.
  • cyclodextrins in accordance with this invention will possess one or more pendant arms as described in the '359 Application.
  • One general formula for preferred pendant arm cyclodextrin derivative are of the formula CD - W - R 13 - L, wherein
  • CD represents an otherwise substituted or unsubstituted cyclodextrin
  • W represents a functional linking group
  • R 13 represents a group defined the same as R ! -R 12 above, and
  • L represents a group selected from reactive, charged, polar or associating groups.
  • SUBSTITUTESHEET W represents an optional, functional linking group such as amino, amide, ester, thioether, thioamide, thioester, etc.
  • R 13 represents an optional arm such as substituted or unsubstituted: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and heterocyclyl, and
  • L represents an optional group selected from reactive, charged, polar or associating groups, e.g., amino, carboxyl, hydroxyl, sulfonate, phosphate, acyloxy, alkyloxy and thiyl.
  • each of the foregoing groups is optionally present.
  • the reactive, charged, polar or associating species can be bonded directly to the cyclodextrin or to the functional linking group.
  • a reactive, charged, polar or associating species may not always be desired.
  • such species can be anywhere on the arm, and there can be more than one such species on the arm, e.g, the arm could possess multiple groups that could associate with multiple groups on an included or associated molecule, such as biological molecules which may contain many repeating groups such as amino, carboxyl, and hydroxyl.
  • the arm can also contain other functional or reactive groups which, in turn, may be used to link yet other arms and charged, polar or associating species.
  • Preferred modified cyclodextrins of CD-W-R-L group also include those in which a carboxyl-substituted alkyl group is linked to a C2, C3 or C6 position of the cyclodextrin through an amino, ester, amide, thioether, thioester, thioamide or other functional linking group.
  • Alkyl groups of from 1-3, 1-6, and 1-10 carbons comprise preferred groups. Those of from 10-20 comprise another preferred group, and those of greater than 20 carbons comprise yet another preferred group.
  • SUBSTITUTESHEET can carry a net negative charge include hydroxyl, carboxyl, phosphate (P0 4 *3 ) or sulfonate (SOj" 1 ) .
  • Other groups having net negative charges will be readily apparent to those skilled in the art.
  • Preferred modified cyclodextrins of this group include those in which a carboxyl-substituted alkyl group is linked to a C2, C3 or C6 position of the cyclodextrin through an amino, ester, amide, thioether, thioester, thioamide or other functional linking group.
  • Alkyl groups of from 1-3, 1-6, and 1-10 carbons comprise preferred groups. Those of from 10-20 comprise another preferred group, and those of greater than 20 carbons comprise yet another preferred group.
  • 6 A -amino-6 A -deoxy-6 A -N-(3-carboxypropanoyl) - ⁇ -cyclodextrin hereinafter "j8-CDNSc"
  • j8-CDNSc 6 A -amino-6 A -deoxy-6 A -N-(3-carboxypropanoyl) - ⁇ -cyclodextrin
  • j8-CDNSc 6 A -amino-6 A -deoxy-6 A -N-(3-carboxypropanoyl) - ⁇ -cyclodextrin
  • modified cyclodextrin, /8-CDNSc is also an example of another preferred group of cyclodextrin derivatives having the formula CD - X - R 14 - Q, wherein:
  • X represents - N - C - or - S - C -
  • R 14 and R 15 represent groups as defined by R x -R 13 above, and Q is a carboxylic acid group or a carboxylic acid group derivatized to undergo substitution, e.g., acid chloride, acid anhydride or ester.
  • R 14 is alkyl comprise a preferred group.
  • those of from 1-3, 1-6, and 1-10 carbons comprise preferred groups.
  • those of from 10-20 comprise another preferred group, and those of greater than 20 carbons comprises yet another preferred group.
  • the compound can be used either for delivery of drugs such as amiodarone, or as an intermediate in the preparation of other cyclodextrin derivatives, including especially asymmetric linked cyclodextrins and prodrugs.
  • the carboxylic acid advantageously can be derivatized to undergo substitution, for example, by forming an acid chloride, acid anhydride or ester.
  • another preferred embodiment of this invention comprises at least two otherwise substituted or unsubstituted cyclodextrins covalently bonded to each other by at least one linking group.
  • the at least one linking group links a first cyclodextrin at a C2, C3 or C6 position to a second cyclodextrin at a C2, C3 or C6 position.
  • the cylodextrins can be substituted as described above.
  • that linking group is other than a disulfide that links the two cyclodextrins at the C6 position.
  • a preferred group of the linked cyclodextrins are those in which only two cyclodextrins are linked together. Further, from the discussion in Section II above, one can also see the importance of linked cyclodextrins in which a first otherwise substituted or unsubstituted cyclodextrin is linked through one of its primary (C6) carbons to a secondary (C2 or C3) carbon of a second otherwise substituted or unsubstituted cyclodextrin. Thus, otherwise substituted or unsubstituted cyclodextrins which are linked by C6-C3 or C6-C2 linkages comprise yet another preferred embodiment of this invention. As discussed below, preparation of such asymmetrical linked cyclodextrins is greatly facilitated by the above-described intermediates of the formula CD-X-R-Q.
  • the linked cyclodextrins are preferably linked by at least one linking group of the formula - X - R 16 - Y - or - R 17 - , wherein
  • X and Y can be the same or different, and represent functional linking groups, and of the formula - X - R 1 - Y - or - R 2 - , wherein
  • X and Y can be the same or different, and represent functional linking groups such as ether, thioether, ester, thioester, amide, thioamide, and amine, and
  • R 16 and R 17 represent groups as defined by R J -R 15 above, and are advantageously selected from substituted or unsubstituted: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and heterocyclyl.
  • the otherwise substituted or unsubstituted cyclodextrins which are linked may also be the same or different.
  • an ⁇ -cyclodextrin can be linked to a ⁇ - , y- or tf-cyclodextrin.
  • Such an asymmetrical configuration may be advantageous when the guest contains multiple hydrophobic groups of different size.
  • the length of the linking group may be varied to accommodate guest molecules of different sizes.
  • inclusion complexes in which at least one pharmaceutical, pesticidal, herbicidal, agricultural, cosmetic, personal care or other useful agent is included in a modified cyclodextrin or linked cyclodextrins as described above.
  • an otherwise substituted or unsubstituted cyclodextrin, or two or more otherwise substituted or unsubstituted linked cyclodextrins can be covalently bonded to a useful agent such as a pharmaceutical, herbicidal, pesticidal, agricultural, cosmetic or personal care agent, wherein the covalent bond, when broken, will yield the agent in an active form.
  • a useful agent such as a pharmaceutical, herbicidal, pesticidal, agricultural, cosmetic or personal care agent
  • SUBSTITUTESHEET cyclodextrins which can be used include those described above and in the '359 Application. Guidance in preparing such compounds is also provided in the '359 Application. Upon reading this disclosure, the skilled artisan will also appreciate that the intermediate CD-R-X-Q can be very helpful in preparing such derivatives.
  • a preferred embodiment of such derivatives comprises a pharmaceutical agent covalently bound to a cyclodextrin, i.e., a prodrug, wherein the covalent bond will be broken down in the normally occurring internal activity of a host animal.
  • carrier refers to atoms, molecules and other structure to which a cyclodextrin, cyclodextrin derivative and/or inclusion complex may be bound, associated or encapsulated, covalently or otherwise, and which may impart at least one advantageous property to the cyclodextrin, cyclodextrin derivative and/or inclusion complex which is not already possessed thereby.
  • Carriers can be categorized into several different groups.
  • a first group of carriers comprises those atoms or molecules which can be used to target cells of interest within a patient.
  • examples of such can include antibodies or fragments thereof which are specific to tumors for the therapy of cancer, hormones or interleukins for the therapy of infectious disease, cancer and for the treatment of immune deficiency.
  • the cyclodextrin derivative generally first would be conjugated to the carrier by a chemical reaction. Thereafter, the carrier- cyclodextrin conjugate would be exposed to the pharmaceutical agent which would form an inclusion complex with the cyclodextrin.
  • cyclodextrin prodrugs could be covalently attached to the targeting carrier.
  • using such carriers may provide other advantages such as: (a) the amount of drug which can be conjugated to the carrier
  • SUBSTITUTESHEET may be able to be increased
  • the drug may be in a "protected” form resistant to lysis by extra and intracellular enzymes and other materials;
  • the integrity of the compounds may be preserved, i.e. both the structure and function of both agent and carrier may be preserved as they will not be exposed to the stringent conditions often required for the conjugation procedures; moreover, cyclodextrins are likely to be resistant to such stringent conditions.
  • cyclodextrin derivatives disclosed in the '359 Application and herein lend themselves well to such applications since they may contain very precise substitutions which facilitate linking to the carrier.
  • a cyclodextrin derivative containing a thiol, amino or carboxylic acid group could be reacted with the complementary carboxylic acid amino, or thiol group to form an amide, thioamide, thioester or disulfide covalent bond.
  • Other reactive groups could be employed to form other bonds such as ester and amino.
  • the group on the cyclodextrin derivative could be activated to react easily with a complementary group on the antibody or other carrier.
  • a carboxylic acid group could be derivatized to undergo substitution by forming an acid chloride, acid anhydride or ester, which could then be reacted with an amino group on an antibody to form an amide bond.
  • the form of the pharmaceutical agent is not altered and can be targeted to the cells of interest in the protected form of the inclusion complex.
  • Another category of carriers includes atoms, molecules, matrices or encapsulants which are generally useful for localized or prolonged delivery.
  • examples of such carriers include solid matrices such as disks, and liposomes, although in some instances, liposomes can be targeting carriers as described above.
  • the cyclodextrin derivative could be covalently bonded or physically entrapped or encapsulated in the carrier, and the agent would then be included in the cyclodextrin (except where cyclodextrin prodrugs are employed) .
  • the inclusion complex may first be formed, and the inclusion complex could then be bonded, entrapped or encapsulated in the carrier.
  • the pharmaceutical agent to be incorporated into the liposomes generally can be included either in the buffer if is water soluble, or included in the organic solvent if it is hydrophobic.
  • cyclodextrins and derivatives thereof can be used to solubilize the compounds before they are incorporated into liposomes. Cyclodextrins and derivatives thereof can also help to improve the stability of agents in these formulations. This can be particularly important when lack of stability of the agent contained in the liposomes is a concern during prolonged storage in vivo or in vitro .
  • compositions may be in any pharmaceutically acceptable form for any type of administration, including topical, oral, rectal or parenteral administration. And because cyclodextrins and derivatives thereof can mask taste, different types of oral administrations may be possible, including chewable or effervescent tablets (e.g., for antacids), gargles, topical solutions and suspensions, pastes, ointments, etc. All forms of such compositions, including those which have been freeze-dried and spray dried are within the scope of this invention. Methods of treating a patient, including a human, will comprise administering therapeutically effective amounts of such compositions. Preparation and administration of such compositions will be within the skill of persons in such arts.
  • compositions may be administered, with or without carriers, inside of body cavities where the agent would be released more slowly than if it were in the form of the free drug.
  • examples of where such compositions could be employed include:
  • SUBSTITUTE SHEET (c) the pleural or pericardial spaces for the treatment of infection or cancer;
  • vagina for local treatment of cancer or infection, or for slow release of a contraceptive
  • cyclodextrins cyclodextrin derivatives and pharmaceutical compositions, including protecting the pharmaceutical agents from enzymes and acids of the gastrointestinal tract, lytic agents in the body such as in the saliva, enzymes in the lungs and in the body, particularly at the surface and within cells such as polymorphic nuclear leukocytes, macrophages and other cells, and possibly also from the destructive mechanisms found within the cytoplasm of most cells.
  • the cyclodextrin derivatives of this invention may also be used in other ways, for example, to encapsulate proteinase inhibitors.
  • Such complexes may be used therapeutically, e.g., for co-administration with peptides of therapeutic interest.
  • such complexes may be used for separating proteins, e.g., by chromatographic means.
  • Such compounds and compositions may also protect the patient from allergic reactions to the agent. This may be accomplished if the potential antigenic sites on the agent are included or otherwise shielded such that they are no longer immunogenic, and such that they are protected from attacking antibodies or lymphocytes. In such instances, the complex or prodrug likely would be administered parenterally in order to introduce the cyclodextrin into the bloodstream.
  • penicillin in some individuals is antigenic, giving rise to anti-penicillin antibodies.
  • sensitized is antigenic
  • penicillin compositions in accordance with this invention may well be "hidden” form the antibody and no such system anaphylaxis will occur. Moreover, if such penicillin compositions were initially administered, sensitivity may be prevented in the first instance.
  • Other substances which are potentially immunogenic include antibodies, peptides (discussed below) or other synthetic or natural materials.
  • compositions may prevent unwanted binding of an agent to a specific receptor on a cell, thereby preventing unwanted destruction by the cell. This could effectively prolong the half-life of the agent in circulation. It is noted that many agents act by binding to a receptor which then causes some appropriate function, and that hiding this site may decrease the therapeutic potential of the agent. There may be circumstances, however, where it is more desirable to have the agent act at a certain site (e.g. bacteria) and not bind to other cells which carry the receptor.
  • a certain site e.g. bacteria
  • another embodiment of this invention comprises an inclusion complex comprising cimetidine included in a cyclodextrin derivative, or a cyclodextrin prodrug comprising the residue of cimetidine covalently bonded to a cyclodextrin derivative as described herein or in the '359 Application, or a
  • SUBSTITUTESHEET pharmaceutical composition containing such inclusion complex or prodrug.
  • Any of the aforementioned forms of pharmaceutical compositions is envisioned, including those comprising carriers for targeted or prolonged delivery.
  • the cyclodextrin derivative is of the formula CD - W - R 13 - L, which is described above.
  • W represents an optional, functional linking group such as amino, amide, ester, thioether, thioamide, thioester, etc. ,
  • R 13 represents an optional arm such as substituted or unsubstituted: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and heterocyclyl, and
  • L represents an optional group selected from reactive, charged, polar or associating groups, e.g., amino, carboxyl, hydroxyl, sulfonate and phosphate.
  • One preferred group of compounds within the above formula comprises cyclodextrin derivatives in which R 13 represents alkyl groups of from 1-3, 1-6, and 1-10 carbons. Those of from 10-20 comprise another preferred group, and those of greater than 20 carbons comprises yet another preferred group. Yet another preferred group comprises cyclodextrin derivatives in which L represents a negatively charged group, for example carboxyl, hydroxyl, sulfonate and phosphate. Yet another preferred group comprises cyclodextrin derivatives in which W is amino, amide or ester.
  • Another preferred group useful in forming inclusion complexes with cimetidine comprises cyclodextrin derivatives having the formula CD - X - R 14 - Q, which is described above.
  • R is as described above, and preferably represents alkyl groups of from 1-3, 1-6, 1-10, 10-20 or greater than 20 carbons;
  • SUBSTITUTESHEET Q is a carboxylic acid group.
  • the present invention provides a compositions and methods for effectively stabilizing, solubilizing, and/or enhancing the delivery or activity in vivo or in vitro of polypeptides, especially proteins, by means of the cyclodextrin derivatives described herein and in the '359 Application.
  • Such compositions may be in the form of an aqueous solution.
  • the resulting compositions comprising the polypeptides are also within the scope of this invention. Lyophilized forms of such compositions are also within the scope of this invention.
  • the foregoing methods and compositions are directed toward improvements in biologically useful protein formulations.
  • formulations containing a combination of a therapeutic polypeptide and a cyclodextrin or modified cyclodextrin are suitable for oral administration. Partial encapsulation of the polypeptide in the cyclodextrin may prevent the enzyme-drug interaction and hence limit enzymatic degradation of the polypeptide. The rate of degradation of the polypeptide will of course only be reduced when the effective concentration of the polypeptide is below the K ⁇ of the enzyme. However, in vivo, most enzymes work below the 1 ⁇ so for purposes of drug delivery this condition will in most cases be fulfilled.
  • SUBSTITUTESHEET associated with compounds produced in vivo through enzyme catalysis For example, where a disorder results from the over ⁇ production of a physiologically active compound, and the production of that compound is enzyme-catalyzed, competitive binding of the substrate of the enzyme to a cyclodextrin can limit that over-production. Alternatively, encapsulation of the product of the enzyme-catalyzed reaction may limit the undesirable physiological effects of that compound. Other therapeutic benefit may be derived through maintaining a larger substrate pool by substrate binding to cyclodextrins.
  • Example 120 demonstrates this inhibition in an in vitro model system.
  • N-Benzoyl(l)-tyrosine ethyl ester is a substrate commonly used to determine the activity of ⁇ - chymotrypsin. It is shown that the rate of hydrolysis of BTEE by ⁇ !-chymotrypsin is slowed in the presence of j8-cyclodextrin ( ⁇ - CD) .
  • polypeptides intended for use according to the present invention are any polypeptides which are biologically or industrially useful, i.e., one which can be employed in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in man or animals.
  • SUBSTITUTESHEET diagnosis (in vivo or in vitro) are of particular interest.
  • Industrially useful polypeptides are those employed analytically, in production or which are otherwise useful in chemical industry.
  • Amino acids are organic compounds that have at least one amino (-NH 2 ) group and at least one carboxyl (-COOH) group.
  • Oligopeptides are amino acid oligomers which are formed by reacting amino and carboxyl groups from different amino acids to release a water molecule and form a peptide linkage.
  • Polypeptides are amino acid polymers which are formed in the same way. Generally, oligopeptides have between 2 and 20 peptide linkages whereas polypeptides have more. Proteins are very large polypeptides.
  • the amino acids which make up the polypeptides of the present invention may be natural or synthetic, may have the amino group attached to any non-carbonyl carbon atom (e.g., to the a , ⁇ , 7, ⁇ or e carbon as measured from the ⁇ -COOH group of the amino acid), may exist as enantiomers (e.g., D- or L-) or diasteriomers, may have blocked carboxyl or amino groups and may have side chains which are hydrophobic, hydrophilic, acidic, basic or neutral.
  • the synthetic polypeptides may contain reversed peptide linkages, in which case they must contain at least one diamine and one dicarboxylic acid monomer.
  • polypeptides intended for use in the methods and compositions of this invention include molecules to which nonpeptide prosthetic groups, such as carbohydrates, hemes and fatty acids, have been attached.
  • the polypeptides include molecules made by living organisms or cells, molecules made by synthetic organic chemistry and molecules which are synthetically modified biological products. They may have an amino acid sequence identical to that of a natural substance or one altered by techniques such as site-directed mutagenesis.
  • polypeptides may possess unique conformations (combinations of
  • polypeptides may have many biological functions. They may act as enzymes; enzyme inhibitors, antibodies; antigens; transporters of electrons, oxygen, metal ions, or small organic molecules; ionophores; antibiotics; mitogens; hormones; growth regulators; neurotransmitters; cell surface recognition proteins; cell chemotactic factors; and cytotoxins. They may also be receptors, agonists, antagonists of the following: ionophores, antibiotics, mitogens, hormones, neurotransmitters, growth regulators, cell surface recognition proteins, cell chemotactic factors and cytotoxins.
  • Uses for some of the preferred polypeptides of the invention include: immunization (as vaccine adjuvants) , in vitro diagnostics (to increase the solubility/stability or lower the nonspecific binding of antigens or antibodies) and in vivo diagnostics and therapeutics (to increase the solubility and/or stability of therapeutic and diagnostic polypeptides) .
  • Preferred therapeutic targets of these polypeptides are cancers, such as melanoma, renal cell carcinoma, myeloma, leukemia, breast cancer, colorectal cancer, lymphoma, neuroblastoma, astrocytoma and glioma; auto-immune diseases, such as diabetes mellitus, rheumatoid arthritis and multiple sclerosis; immunodeficiency diseases; and infectious diseases.
  • Anti-sera may include, for example, antirabies, antivenin (black widow spider venom) , hepatitis B immune globulin, tetanus immune globulin, intravenous immune globulin, pertussis immune globulin and rabies immune globulin.
  • Anti-toxins may include, for example, those for diphtheria and tetanus; Rho(D) immune globulin; serum components, such as 5% normal human serum albumin, 5% plasma protein fraction, 20% normal human serum albumin, 25% normal human serum albumin.
  • SUBSTITUTESHEET Also useful are factor II, factor VII, factor VIII, factor IX, factor X and Xa, antithrombin III, transferrin, haptoglobin, fibronectin, gamma globulins, protein C, protein S and thrombin; toxoids, such as diphtheria and tetanus; vaccines, including attenuated vaccines (such as those for cholera, influenza, meningitis, Yersinia pestis or plague, pneumonia, poliomyelitis, rabies, typhoid and staphylococcus) and live vaccines (such as those for poliomyelitis, measles, rubella and mumps) ; growth factors, hormones and like bioactive peptides, as illustrated by ⁇ -1-antitrypsin, atrial natriuretic factor (diuretic) , calcitonin, calmodulin, choriogonadotropin (or and ⁇ )
  • insulin insulin-like growth factor
  • an interferon typically ⁇ , ⁇ , 7
  • an interleukin typically 1, 2, 3, 4
  • lutropin lymphotoxin
  • macrophage derived growth factor typically 1, 2, 3, 4
  • macrophage inhibiting factor macrophage stimulating factor, megakaryocyte stimulating factor, nerve growth factor, pancreatic endorphin, parathyroid hormone, platelet derived growth factor, relaxin, secretin, skeletal growth factor, superoxide dismutase, thymic hormone factor, thymic factor, thymopoeitin, thyrotropin, tissue plasminogen activator, transforming growth factor ( ⁇ and ⁇ ) , tumor necrosis factor, tumor angiogenesis factor, vasoactive intestinal polypeptide and wound angiogenesis factor; immunosuppressives, such as RhO (D) ISG and IVGG's, thrombolytics such as urokinase, streptokinase and tissue
  • immunosuppressives such as
  • polypeptides contemplated by this invention are polypeptides specifically intended to veterinary use, including
  • SUBSTITUTE SHEET vaccines animal growth factors and bovine interferons and interleukin-2.
  • Illustrative vaccines include: bovine vaccines, for example those for anthrax, clostridium (multiple species) , pasteurella, leptospira pomona, bovine diarrhea, brucillosis, parainfluenza, 3-respiratory syncytial virus, tetanus, vesicular stomatitis and staphylococcus; canine vaccines, for example those for bordetella, coronavirus, distemper, parvovirus, parainfluenza and rabies; equine vaccines, for example those for anthrax, encephalomyelitis, influenza, tetanus, rabies and streptococcus- strangles; feline vaccines, such as those for leukemia, pneumonitis-chlamydia and rabies; ovine vaccines, for example those for anthrax, blackleg, bluetongue, enterotoxemia, tetanus and vibriosis
  • polypeptides contemplated by this invention have use in immunology. These include monoclonal antibodies, polyclonal antibodies (unconjugated) , second antibodies (alkaline phosphatase conjugated) , immunoglobulin screening and isotyping kits, protein A products and immunoassay reagents.
  • monoclonal antibodies are those approved for use in diagnostic kits, for example IgE, peroxidase-anti-peroxidase conjugated, human chorionic gonadotropin, T cell, ferritin conjugated, carcinogenic embryonic antigen (CEA) , OKT-II, anti-rabies, human growth hormone, Total T4, prolactin, 1251-IgE, UCG, thyroid stimulating hormone, chlamydia, gentamicin and rubella.
  • diagnostic kits for example IgE, peroxidase-anti-peroxidase conjugated, human chorionic gonadotropin, T cell, ferritin conjugated, carcinogenic embryonic antigen (CEA) , OKT-II, anti-rabies, human growth hormone, Total T4, prolactin, 1251-IgE, UCG, thyroid stimulating hormone, chlamydia, gentamicin and rubella.
  • IgE peroxidase-anti-peroxidase conjugated
  • monoclonal antibodies include monoclonal antibodies for human cell surface antigens, monoclonal antibodies for murine cell surface antigens, monoclonal antibodies to complement and blood proteins, monoclonal antibodies to immunoglobulins (human) , monoclonal antibodies to neurological antigens, monoclonal antibodies to tumor markers, monoclonal antibodies to cell components, Epstein Barr virus antigens, human lymphocyte antigen (HLA) typing, hematology antibodies, leucocyte antibodies,
  • SUBSTITUTESHEET bacterial antigens include parasitic antigens, T-cell lymphotropic virus (HIV-III) and cytoskeletal antibodies.
  • Useful polyclonal antibodies include affinity purified antibodies to immunoglobulins, antibodies to plant viruses, antisera to human isoenzymes and chromatographically purified antibodies.
  • Useful alkaline phosphatase conjugated second antibodies include affinity purified antibodies to immunoglobulins, antibodies to plant viruses, biotin-conjugated antibodies, fluorescein- isothiocyanate-conjugated antibodies (FITC) , gold-conjugated antibodies, peroxidase-conjugated antibodies, rhodamine conjugated antibodies and iodine-conjugated antibodies.
  • affinity purified antibodies to immunoglobulins antibodies to plant viruses
  • biotin-conjugated antibodies include fluorescein- isothiocyanate-conjugated antibodies (FITC) , gold-conjugated antibodies, peroxidase-conjugated antibodies, rhodamine conjugated antibodies and iodine-conjugated antibodies.
  • FITC fluorescein- isothiocyanate-conjugated antibodies
  • gold-conjugated antibodies gold-conjugated antibodies
  • peroxidase-conjugated antibodies include rh
  • Polypeptide immunoassay reagents include EIA grade enzymes, enzyme-antibody complexes, reagents for immunology, enzyme linked immunosorbent assays (ELISA) for use as standards or controls, immunoelectrophoresis (IEP) assays, radioim unoassays (RIA) for use as standards or controls, nephelometry for use as standards or controls, nuclear antigens and coatings for kit tubes and plate wells.
  • polypeptides contemplated by the present invention include polypeptides useful in cell biology/biochemistry, for example in serum-free cultures (as supplements and reagents for cell culture) , in glycoprotein and carbohydrate research (endoglycosidoses, exoglycosidoses, enzymes for carbohydrate research, enzymes for analysis of glycoprotein oligosaccharides) , as molecular weight markers (calibration proteins, e.g., for gel permeation chromatography, subunit proteins) , proteases (for use in blood research, protein sequencing, tissue digestion and cell harvest, total digestion of proteins and immobilized proteases) , cell surface recognition proteins (adenosine and analogs, cyclic nucleotides, phosphoinositides) , phospholipases and bioluminescence assay reagents.
  • glycoprotein and carbohydrate research endoglycosidoses, exoglycosidoses, enzymes for carbohydrate research, enzymes for
  • polypeptides contemplated for use herein are polypetides of particular interest in the field of molecular biology, including various enzymes and reagents.
  • the enzymes can include labelling enzymes, modifying enzymes, nucleases,
  • the reagents can include inhibitors, antibiotics and miscellaneous other reagents.
  • Preferred polypeptides for use in accord with the instant invention include growth regulators.
  • growth regulators are hematopoietic factors, which affect the maturation and proliferation of blood cells in lymphoid tissue and bone marrow; cytokines, which generally influence eukaryotic cell growth; and lymphokines, which affect lymphocyte growth.
  • Specific polypeptides which are growth regulators or lymphokines are: interleukin 1, 2, 3 and 4; a, ⁇ , and interferons; granulocyte colony stimulating factor (G-CSF) ; granulocyte - macrophage CSF (GM-CSF) ; macrophage CSF(m-CSF) ; megakaryocyte CSF; multi CSF or IL-3 (also known as BPA, HCGF, MCGF and PSF) ; erythropoietin; lymphotoxin; tumor necrosis factor (TNF, also known as cachectin) ; ⁇ and ⁇ transforming growth factor (TGF) ; platelet derived growth factor (PDGF) ; epidermal growth factor (EGF) ; nerve growth factor (NGF) ; insulin-like growth factor I and II (IGF I is also called somatomedin C) ; growth hormone (GF, also called somatotropin) ; and growth hormone releasing factor (GHRF, also called
  • Fusion proteins are covalently linked proteins or portions of proteins.
  • proteins or active fragments thereof having different purposes can be linked to provide a fused molecule having characteristics of both.
  • one of the proteins/protein fragments is a "seeker” and the other an “actor” or “destroyer”.
  • fusion proteins are pseudomonas exotoxin
  • SUBSTITUTESHEET linked to IL-2 diphtheria toxin linked to IL-2 or either toxin linked to other proteins, or linkages between other proteins such as those preferred proteins described in the preceding paragraph, or portions thereof.
  • SUBSTITUTESHEET linked to IL-2 diphtheria toxin linked to IL-2 or either toxin linked to other proteins, or linkages between other proteins such as those preferred proteins described in the preceding paragraph, or portions thereof.
  • Fusion proteins of IL-2 linked with toxin as described above are designed to kill cells with IL-2 receptors, thus find use in preventing graft-versus-host rejection.
  • Other fusion proteins have varying utilities, depending on the proteins or portions thereof which are combined.
  • Substance P and a toxin form a fusion protein which can be used to relieve chronic pain
  • ⁇ -melanocyte-stimulating hormone (MSH) and diphtheria toxin form a fusion protein designed to kill melanoma cells.
  • muteins which are mutationally altered proteins.
  • Preferred muteins are muteins of the preferred growth regulators and fusion proteins identified in the preceding paragraphs, and typically have the same purpose as the corresponding unaltered proteins.
  • Especially preferred muteins include IL-2 muteins, described for example in United States Patent Nos. 4,752,585 and 4,518,584, incorporated by reference herein in their entireties and relied upon; and muteins of ⁇ -interferon, described for example in United States Patent Nos. 4,737,462 and 4,588,585, incorporated by reference herein in their entireties and relied upon.
  • polypeptides may be used in the form of inclusion complexes with any of the cyclodextrin derivatives described herein in the '359 Application, or may be covalently bound to any such cyclodextrin derivative such that the covalent bond, when broken, will yield the polypeptide in useful form, e.g., a prodrug.
  • compositions comprising amiodarone have been formulated for both oral and intravenous administration.
  • a 1:4 (amiodarone: j8-CDNSc) molar ratio the solubility of amiodarone in aqueous solution was increased 35 times.
  • such compositions provided oral and intravenous delivery profiles which were improved in several respects.
  • another embodiment of this invention comprises an inclusion complex comprising amiodarone included in a cyclodextrin derivative, or a cyclodextrin prodrug comprising the residue of amiodarone covalently bonded to a cyclodextrin derivative as described herein or in the '359 Application, or a pharmaceutical composition containing such inclusion complex or prodrug.
  • a pharmaceutical composition containing such inclusion complex or prodrug Any of the aforementioned forms of pharmaceutical compositions is envisioned, including those comprising carriers for targeted or prolonged delivery.
  • the cyclodextrin derivative is of the formula CD - W - R 13 - L, or CD - X - R 14 - Q, which are described above, including preferred groups.
  • Another embodiment of this invention provides inclusion complexes and pharmaceutical compositions Piroxicam and related non-steroidal anti-inflammatory drugs with cyclodextrin derivatives as described herein and in the '359 Application.
  • Such compositions may improve the therapeutic performance of Piroxicam and related non-steroidal anti-inflammatory drugs, for example, through increases in their rates of dissolution and their solubilities as a consequence of their formulation with such cyclodextrin derivatives.
  • These inclusion complexes and pharmaceutical compositions may also substantially reduce the irritation of the gastro-intestinal tract associated with the oral administration of Piroxicam and related non-steroidal anti- inflammatory drugs. Bioavailability may also be improved.
  • Embodiments include compositions in which Piroxicam is included in a cyclodextrin derivative (e.g., ⁇ -amino cyclodextrin) using a 1:1, 1:2 or 1:4 molar ratio of Piroxicam to cyclodextrin derivative.
  • a cyclodextrin derivative e.g., ⁇ -amino cyclodextrin
  • Another embodiment comprises Piroxicam included in a linked cyclodextrin derivative using a 1:1 mole ratio of Piroxicam to cyclodextrin derivative.
  • there is formed a solid inclusion complex having a high wettability and solubility, and dissolves very rapidly to produce both the inclusion complex and free Piroxicam in solution.
  • Examples 121, 122 and 129 below illustrate such formulations.
  • Inclusion complexes, prodrugs and pharmaceutical compositions comprising many other pharmaceutical agents are also within the scope of this invention.
  • agents include Ketoprofen, lorazepam, propranolol hydrochloride, amikacin, nadolol, etoposide, captopril, iopamidol, fosinopril, cefuroxime sodium, beclomethasone, labetalol hydrochloride, ranitidine, ceftazidime, cefuroxime, cefaclor, dobuta ine, insulin, fluoxetine, nizatidine, diltiazem, terfenadine, nifedipine, glipizide labetalol, clotrimazole, betamethasone, flutamide, mometasone furoate, auranofin, cefonicid sodium, cimetidine, mupirocin, fenoldopam, amoxillin,
  • cyclodextrins are suitable for the control of treatment of metabolic disorders associated with compounds produced in vivo through enzyme catalysis.
  • a disorder results from the over ⁇ production of a physiologically active compound (e.g. , epilepsy)
  • a physiologically active compound e.g. , epilepsy
  • competitive binding of the substrate of the enzyme to a cyclodextrin can limit that over-production.
  • encapsulation of the product of the enzyme-catalyzed reaction may limit the undesirable physiological effects of that compound.
  • Other therapeutic benefit may be derived through maintaining a larger substrate pool by substrate binding to cyclodextrins.
  • cyclodextrins may encapsulate free drug, it is possible to intravenously administer cyclodextrins and derivatives thereof in accordance with this invention and the '359 Application intravenously to encapsulate or "mop up" drugs or toxins in the circulation and to release them at a slower rate.
  • the antineoplastic drug Melphalan which is known to be encapsulated by cyclodextrins and derivatives thereof, may be in too high a concentration in a patient. If cyclodextrins or derivatives thereof were given in large amounts intravenously, they could mop up excess Melphalan, free Melphalan in the circulation and potentially decrease the circulating amount of free material. This could lead to a step wherein then cyclodextrins or
  • SUBSTITUTESHEET derivatives thereof could be removed from the circulation.
  • cyclodextrin derivatives are of the formula CD-W-R-L, wherein
  • CD represents an otherwise substituted or unsubstituted cyclodextrin
  • W represents an optional, functional linking group such as amino, amide, ester, thioether, thioamide, thioester, etc. ,
  • R is as described above for R 13 and represents an optional arm such as substituted or unsubstituted: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl , cycloalkynyl, aryl, heteroaryl and heterocyclyl, and
  • L represents an optional group selected from reactive, charged, polar or associating groups, e.g., amino, carboxyl, hydroxyl, sulfonate and phosphate.
  • One preferred group of compounds within the above formula comprises cyclodextrin derivatives in which R represents alkyl groups of from 1-3, 1-6, and 1-10 carbons. Those of from 10-20 comprise another preferred group, and those of greater than 20 carbons comprises yet another preferred group. Yet another preferred group comprises cyclodextrin derivatives in which L represents a negatively charged group, for example carboxyl, hydroxyl, sulfonate and phosphate. Yet another preferred group comprises cyclodextrin derivatives in which W is amino, amide or ester.
  • SUBSTITUTESHEET Compounds of the formula CD-W-R-L have several uses. They can be used as hosts for advantageous inclusion complexes, such as those described above for amiodarone. They may also be used as intermediates for preparing other cyclodextrin derivatives including those in which a useful agent is covalently bound to the cyclodextrin such that the bond, when broken, will yield the agent in an active form. Generally, in such cases L will be a functional group such as amino, carboxyl or a sulfur-containing group.
  • CD-W-R-L genus having the formula CD-X-R-Q, wherein:
  • X represents - N - C - or - S - C - l 2 II II
  • R is as described above for R 14 , and advantageously represents a group such as substituted or unsubstituted: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and heterocyclyl, and
  • Q is a carboxylic acid group or a carboxylic acid group derivatized to undergo substitution, e.g., acid chloride, acid anhydride or ester.
  • Q is a carboxylic acid group
  • such compounds are particularly useful as hosts for inclusion complexes (e.g., with amiodarone)
  • Q is a carboxylic acid group derivatized to undergo substitution, e.g., acid chloride, acid anhydride or ester, then such compounds are highly reactive intermediates
  • SUBSTITUTE SHEET useful for preparing other pendant arm intermediates, and cyclodextrin derivatives in which a useful agent is covalently bound to the cyclodextrin such that the bond, when broken, will yield the agent in an active form (e.g., prodrugs). More so, however, this intermediate is extremely useful for preparing asymmetric linked cyclodextrins in which the cyclodextrins are different ( ⁇ , ⁇ , 7) and/or differently substituted, e.g., primary versus secondary. In such instances, the intermediate can then be reacted with a different amino- or thiol-substituted cyclodextrin to yield the asymmetrical linked cyclodextrins. This is particularly useful where it is desired to link a primary carbon of one cyclodextrin to a secondary carbon of another cyclodextrin.
  • the intermediate is highly reactive and should be either used directly or freeze dried and store crystalline in cold.
  • the intermediate can be hydrolyzed to the free acid which is stable indefinitely, and which can be reactivated if desired.
  • CD-X-R-Q Compounds of the formula CD-X-R-Q can be prepared by reacting an amino cyclodextrin (CD-NH 2 ) or a cyclodextrin thiol (CD-SH) with a dicarboxylic acid that has been derivatized to undergo substitution. To an extent, this process parallels the reaction for preparing linked cyclodextrins described in scheme 5 in subsection IV.J.
  • the reaction can be controlled such that the major products are either the succinate-linked cyclodextrins or the m-nitrophenyl ester of 6 A -amino-6 A -deoxy-6 A - N(3-carboxypropanoyl)-jS-cyclodextrin.
  • the linked product will be predominant when the molar ratio of amino cyclodextrin to derivatized dicarboxylic acid is at least about 2:1, and the reaction is allowed to proceed for several days at room temperature.
  • the molar ratio is about 1:1 or less, or the reaction is allowed to proceed for only a few hours, the predominant product will likely be the non-linked derivative.
  • Guidelines for preparing and optimizing yields of the linked and non-linked products are provided in Examples 58-65 below. Of course, a skilled artisan may also vary other parameters such as reaction temperature, pressure, etc. , and such modifications are well within the scope of this invention.
  • the free acid form i.e., where Q is the carboxylic acid
  • Q is the carboxylic acid
  • an anhydride such as succinic anhydride.
  • This intermediate form is stable indefinitely, and can subsequently be activated, e.g., by making a p-nitrophenyl ester, to undergo substitution.
  • SUBSTITUTESHEET Purification of the linked product may be accomplished using Sephadex.
  • reaction rate for forming linked cyclodextrins also depends upon the cyclodextrin annulus size and the length of the linking group. This is believed to be due, at least in part, to the ability of the linking group to "fold" back and include in the cyclodextrin cavity, thereby obscuring the reactive group. Thus, it is believed that shorter linking groups will be less likely to fold back and include back into the annulus and obscure the reactive group. This correlates with the observations that cyclodextrins linked by oxalate have a faster reaction rate than if linked by will malonate, which is in turn faster than if linked by succinate, etc.
  • SUBSTITUTESHEET reagent can be found to substitute, with a leaving group, the cyclodextrin of choice at the carbon of choice, to prepare in optimal yield a suitably cyclodextrin intermediate.
  • This intermediate can be purified by the surprisingly highly efficient reverse phase chromatography procedure disclosed in the '359 Application.
  • Cyclodextrin manno epoxides can be prepared as described by the method of Breslow et al. J. Am. Chem. Soc. 1983, 105, 1390. For example, 2 A -m-nitrobenzenesufinyl- ⁇ -cyclodextrin is treated with base and heated to give ⁇ -cyclodextrin manno epoxide in good yield. Best results are obtained if the starting materials are pure. Purification of the epoxide is easily effected by Sephadex R chromatography.
  • the cyclodextrin manno epoxide need not be isolated.
  • Treatment of 2 A -m-nitrobenzenesulfinyl- ⁇ -cyclodextrin with ammonium hydroxide effects both the formation of the manno epoxide and the subsequent formation of the inverted 3 A -amino-3 A - deoxy-cyclodextrin.
  • 3 A -0-/5-naphthalenesulfinyl- ⁇ -cyclodextrin can be treated with base and heated to give /8-cyclodextrin allo epoxide.
  • This procedure is similar to that described above for preparing the manno epoxide.
  • Conditions and methods of purification are also similar.
  • Treatment of the epoxide with ammonium hydroxide or with ammonia leads to a 2 A -amino-2 A -deoxy- cyclodextrin where the stereochemistry at both C2 and C3 of the substituted glucose has been inverted from the normal cyclodextrin stereochemistry.
  • Thin layer chromatography (t.l.c.) was performed using Kieselgel 60 F ⁇ (Merck) on aluminium backing plates.
  • Running solvents used were: solvent A, 14:3:3 butanone-methanol-water; solvent B, 1:8:1 chloroformacetic acid-water; solvent C, 14:3:3 ethyl acetate-methanol-water.
  • Visualization was achieved by dipping the plate in either a solution of diphenylamine (0.1 g) , aniline (0.5 ml) and 85% phosphoric acid (1 ml) in acetone (10 ml), or a solution made up of 10:1 acetone - 15% sulfuric acid; and heating to char the spots.
  • Mass spectra were recorded on a VG ZAB 2HF mass spectrometer using the FAB technique with Xe or Ar as the collision gas.
  • the samples were dissolved in H 2 0 or DMSO and introduced into the spectrometer in a glycerol matrix.
  • TFA trifluoroacetic acid
  • l ⁇ N.m.r. spectra were recorded on a Varian T60 or Bruker CXP300 spectrometer and 13 C n.m.r. were recorded on a Bruker CXP300 or WP80 spectrometer. Solvents and internal standards are mentioned individually within the text. All ⁇ U n.m.r. samples were dried overnight in vacuo over P 2 0 5 . Melting points were determined using a Reichart melting point apparatus and are uncorrected.
  • ⁇ - Cyclodextrin ( ⁇ -CD) was supplied by Sigma Chemical Co. or Nihon Shokuhin Kako Co. and contained 3% water.
  • /S-Cyclodextrin (3-CD) was supplied by Nihon Shokuhin Kako Co. and contained 10% water.
  • j ⁇ -CD was stored in vacuo over phosphorus pentoxide to constant weight before use. It may also be preferred to dry ⁇ -CD before use in the same manner.
  • Other cyclodextrin derivatives, if required in an anhydrous form should be dried similarly.
  • the crude product (6.6 g) was dissolved in 30% aqueous methanol (100 ml) , filtered and loaded via the pump onto a 19 x 150 mm Cjg ⁇ -Bondapack HPLC column. This was eluted with 30% aqueous methanol at 15 ml.min "1 and gave pure fractions of ⁇ -CD (0 - 35 minutes) and ⁇ -CDOTs (1.83 g, 45 - 120 minutes). This separation procedure is unexpectedly very effective for up to 8 g quantities of the crude product.
  • the column was washed with several column volumes of methanol to elute multi-O-p- toluenesulfonyl substituted cyclodextrins.
  • Examples 12 and 13 illustrate alternative syntheses for preparing 6 A -amino-6 A -deoxy-o.-cyclodextrin .
  • EXAMPLE 12 6 A -Amino-6 A -deoxy- ⁇ -cyclodextrin ( ⁇ -CDNH 2 ) ⁇ -CDN 3 (3 g) was dissolved in water (90 ml) and palladium black (90 mg) was added. The mixture was shaken on a Parr hydrogenator under hydrogen (30 psi) at room temperature overnight. After venting the hydrogen, TCE (0.1 ml) was added to the mixture which was then shaken until an emulsion was obtained. After standing for 1 hour the solution was filtered (Whatman N* 1 filter paper) and the clear colorless solution was evaporated to dryness to give ⁇ -CDNH 2 (2.84 g) . T.l.c.
  • EXAMPLE 13 ⁇ -CDOTs (1.0 g) was dissolved in dry N,N-dimethylformamide (DMF, 50 ml) in a 400 ml pressure vessel. Condensed ammonia (100 ml) was added carefully and the vessel sealed. The pressure increased to 150 psi as the reaction mixture warmed from -33°C to 22°C (room temperature) . The reaction mixture was stirred at room temperature for 3 days. The ammonia was vented off and the DMF solution was dried in vacuo . The residue was dissolved in water (10 ml) and evaporated in vacuo to dryness twice to remove any residual DMF.
  • DMF dry N,N-dimethylformamide
  • EXAMPLE 14 6 A -o-p-Toluenesulfonyl-jS-cyclodextrin (/3-CDOTs) iS-CD (13 g) was dissolved in pyridine (100 ml) . p- Toluenesulfonyl chloride (1.7 g) was added over a period of 45 minutes with stirring, and the clear solution allowed to stand at room temperature overnight. The pyridine was removed in vacuo and the resulting oily residue triturated with acetone (100 ml) . The solid residue was separated by filtration and dissolved in boiling water (50 ml) .
  • EXAMPLE 15 6 A -Azido-6 A -deoxy-0-cyclodextrin ( ⁇ -CDK,) j ⁇ -CDOTs (2 g) and sodium azide (2 g) were dissolved in water (60 ml) and the solution heated on a boiling water bath for 90 minutes. The solution was concentrated in vacuo to approximately 10 ml. TCE (1 ml) was added and the mixture shaken to give a white precipitate. After standing for 60 minutes, the solid was collected by vacuum filtration, washed with small amounts of ice cold water (ca. 3 x 2 ml) and dried. The collected solid was heated in water (60 ml) until all of the product dissolved.
  • Examples 16 to 18 illustrate alternative syntheses for preparing 6 ⁇ -amino-6 ⁇ ---deoxy- ⁇ -cyclodextrin.
  • EXAMPLE 17 / 8-CDOTs (340 mg) was dissolved in 0.880 ammonia solution (10 ml) and left standing at room temperature for 2 weeks. After removing the ammonia in vacuo the residue was poured into acetone (50 ml) . The precipitate was collected by gravity filtration and dried in vacuo to give j8-CDNH 2 (301 mg). T.l.c. and HPLC showed a spot corresponding to ]8-CDNH 2 .
  • Anhydrous jS-CDOTs (1.0 g) was dissolved in dry DMF (50 ml) in a 400 ml pressure vessel. Condensed ammonia (100 ml) was added carefully and the vessel sealed. The pressure increased to 150 psi as the reaction mixture warmed from -33°C to 22°C (room temperature) . The reaction mixture was stirred at room temperature for 3 days. The ammonia was vented off and the DMF solution was dried in vacuo . The residue was dissolved in water (10 ml) and evaporated in vacuo to dryness twice to remove any residual DMF. The residue (0.895 g) was dissolved in 20% aqueous ammonia (10 ml) and dropped into acetone (200 ml) .
  • EXAMPLE 20 2 A -o-p-Toluenesulfonyl-/3-cyclodextrin (3-2CDOTs) Step 1: 3-Nitrophenyl p-toluenesulfonate (TsNP) can be prepared as follows. A solution of p-toluenesulfonyl chloride (1.91 g) and m-nitrophenol (1.39 g) in pyridine (10 ml) was stirred overnight. Ethyl acetate (50 ml) was added and the mixture washed with brine (2 x 50 ml) , dried over magnesium sulfate and concentrated in vacuo.
  • TsNP 3-Nitrophenyl p-toluenesulfonate
  • Step 2 Anhydrous j8-CD (7.02 g) was added to a solution of TsNP (1.81 g) in DMF (60 ml). Aqueous pH 11.5 carbonate buffer (0.2 M, 0.42 ml) was added and the mixture stirred at 60°C for 1 hour. The reaction mixture was neutralized with 1 N hydrochloric acid and unreacted 3-CD was precipitated by the addition of acetone (500 ml) . The mixture was filtered, the filtrate concentrated to a small volume (ca. 3 ml) and acetone (100 ml) was added. The precipitate was collected by filtration, washed with acetone and dried to give the crude product (1.19 g) .
  • EXAMPLE 22 2 A -0-(3-Nitrobenzenesulfonyl)- ⁇ -cyclodextrin ( ⁇ -2CD0Ns)
  • SUBSTITUTESHEET showed: Re (relative to ⁇ -CD), ⁇ -CDNH 2 , 0.7; ⁇ -CDNAc, 1.1.
  • HPLC of the crude product using a 75% CH 3 CN-H 2 0 eluant showed: t R (relative to ⁇ -CD), unknown, 0.6; ⁇ -CDNAc, 0.9; ⁇ -CDNH 2 , 1.2.
  • Examples 33 to 42 illustrate preparation of prodrugs in which Ibuprofen ( ⁇ -methyl-4--- (2-methylpropyl) -benzeneacetic acid) or Naproxen (6-methoxy- ⁇ -methyl-2-naphthaleneacetic acid) are covalently bonded directly to a cyclodextrin through ester linkages .
  • Step 1 3-Nitrophenyl ⁇ -methyl-4-(2-methylpropyl)- benzeneacetate (IbNP) can be prepared as follows. A mixture of ⁇ -methyl-4-(2-methylpropyl)-benzeneacetic acid (1.03 g) and 3- nitrophenol (0.70 g) were dissolved in dry ethyl acetate (100 ml) . The solution was cooled to 0°C, N,N'- dicyclohexylcarbodiimide (1.68 g,) was added and the reaction for one hour. The reaction mixture was allowed to warm to room temperature and stirred overnight.
  • EXAMPLE 34 2 A -o-( ⁇ -methyl-4-(2-methylpropyl) -benzeneacetyl) ) - ⁇ - cyclodextrin (0-2CDOIb) ⁇ -CD (1.14 g) was dissolved in a 1:1 mixture of aqueous NaOH (4 x 10"* M) - acetonitrile (100 ml). IbNP (Example 33, Step l; 0.323 g) in acetonitrile (10 ml) was gradually added with stirring and NaOH (0.1 M) was added until the reaction mixture reached pH 10. After being left to stir at room temperature for 100 minutes, hydrochloric acid (4 M) was added to the reaction mixture until it reached pH 2.5.
  • Step 1 3-Nitrophenyl (S)-6-methoxy- ⁇ -methyl-2- naphthaleneacetate (NpNP) can be prepared as follows. A mixture of (S)-6-methoxy- ⁇ -methyl-2-naphthaleneacetic acid (1.0 g) and 3-nitrophenol (0.6 g) were dissolved in dry ethyl acetate (100 ml). The solution was cooled to 0°C, DCC (1.0 g) was added and the reaction for one hour. The reaction mixture was allowed to warm to room temperature and stirred overnight. DCU was removed by filtration and the filtrate evaporated in vacuo to yield the crude ester as a solid.
  • Step 2 To a solution of /3-CD (1.13 g) in aqueous DMF (10 ml, 1:3) was added NpNP (0.351 g) and the reaction mixture was stirred at 100 - 110°C for 24 hours. After this time analysis of a portion of this mixture by HPLC showed: t R (relative to ⁇ - CD) , jS-2CDONp, 0.36; 3-CD, 1.0. The reaction was heated for a further 24 hours, the reaction mixture cooled and acetone was added until precipitation was complete. The precipitate was collected by gravity filtration (Whatman N 5 1 qualitative filter paper) and recrystallized from water to give a solid which was poorly soluble in water. HPLC using a 70% CH 3 CN-H 2 0 eluant showed: t R (relative to /3-CD), 0.36, 1.00.
  • SUBSTITUTESHEET H8 and H9 split into 2 quartets and 2 doublets respectively.
  • Horse liver acetone powder (0.89 g) was added to a suspension of IbOMe (0.89 g, 4.04 mmol) in 0.2 M phosphate buffer (36 ml). The reaction was followed by t.l.c. (5% acetic acid, 10% ethyl acetate, 85% hexane) . Conversion of the 50% of the IbOMe (R f 0.37) to Ibuprofen (R f 0.15) appeared complete after 11 hours. The reaction was quenched by adding 1 M hydrochloric acid until the solution reached pH 2. Ether (20 ml) was added to the reaction mixture and the two layers centrifuged (3000 r.p.m., 15 minutes) .
  • Step 1 To a solution of (R)- ⁇ -methyl-4-(2-methylpropyl)- benzeneacetic acid (Example 39, 80 mg, 0.4 mmol) in dry methanol (2 ml) was added cesium carbonate (70 mg, 0.22 mmol) in portions over a 5 minute period with stirring. The mixture was allowed to stir at room temperature for a further 55 minutes and then
  • Step 2 To a mixture of cesium (R)- ⁇ -methyl-4-(2- methylpropyl)benzeneacetate (0.127 g) in dry DMF (2 ml) was added jS-CDOTs (0.45 g,) . The mixture was stirred at 100°C for 20 hours and allowed to cool. Acetone (3 ml) was added to the solution until precipitation appeared complete. The resulting brown solid was collected by vacuum filtration and suspended in hot water (2 ml) . The white solid was collected by vacuum filtration and dried in vacuo over P 2 0 5 to give /3-CDOIb- in yield 188 mg. HPLC using a 70% CH 3 -H 2 0 eluant showed: t R (relative to j8-CD) , jS- CDOIbR, 0.32.
  • Examples 43- 50 illustrate preparation of prodrugs in which Ibuprofen ( ⁇ -methyl-4- (2-methylpropyl) -benzeneacetic acid) or Naproxen (6-methoxy- ⁇ -methyl-2-naphthaleneacetic acid) is covalently bonded directly to a cyclodextrin through amide linkages .
  • Step 1 ⁇ -Methyl-4-(2-methylpropyl)-benzeneacetic acid anhydride (Ib ) was prepared as follows. ⁇ -Methyl-4-(2- methylpropyl)-benzeneacetic acid (1500 mg) was dissolved in dry ether (60 ml) and DCC (750 mg) was added. Insoluble DCU forms
  • Step 2 ⁇ -CDNH 2 (600 mg) was dissolved in dry methanol (13 ml) and IbjO (1.3 g) added. The reaction was stirred at room temperature for 6 hours and water (20 ml) added. The mixture was stirred for a further 10 minutes and then filtered (Whatman * 1 filter paper) directly into acetone (200 ml) . The resulting precipitate was collected by gravity filtration and washed with acetone (2 x 20 ml) . The resultant solid was dried in vacuo to give both ⁇ -CDNIb and ⁇ -CDNH 2 (370 mg) .
  • X H n.m.r. and 13 C n.m.r. indicate an isomeric mixture of the ⁇ -CDNIb X H n.m.r. ⁇ H 0.87, J 7 Hz, Me ⁇ , Me ⁇ ; 1.34, J 7 Hz, 1.39, J 7 Hz, ⁇ Me (2 isomers); 1.84, m, J 7 Hz, CH ⁇ ; 2.48, J 7Hz, (CH j Ji B ,,; 3.2 -5.1, 60H; 7.23, q, ArH. 13 C n.m.r.
  • EXAMPLE 48 6 A -Amino-6 A -deoxy-6 A -N- ( (S) -6-methoxy- ⁇ -methyl-2- naphthaleneacetyl)- ⁇ -cyclodextrin ( ⁇ -CDNNp)
  • Step 1 (S)-6-Methoxy- ⁇ -methyl-2-naphthaleneacetic acid anhydride (Np 2 0) was prepared as follows. (S)-6-Methoxy- ⁇ - methyl-2-naphthaleneacetic acid (250 mg) was dissolved in dry ether (10 ml) and DCC (130 mg) was added. Insoluble DCU formed immediately. After 1 hour the ether was removed in vacuo and ethyl acetate (100 ml) added to the residue. The product only
  • Step 2 ⁇ -CDNH 2 (28 mg) was dissolved in dry methanol (0.65 ml) and Np 2 0 (60 mg) added. The reaction was stirred at room temperature overnight. Acetone was added until precipitation appeared complete. The resulting precipitate was collected by gravity filtration (Whatman N 4 1 filter paper) and washed with acetone. The solid was collected and dried in vacuo. HPLC analysis showed two major and two minor products. The dried crude product was dissolved in 70% aqueous CH 3 CN (5 ml) and chromatographed on Sephadex eluting with the same solvent, ⁇ - CDNNp was isolated as a colorless solid (190 mg) . T.l.c. showed: Re (relative to ⁇ -CD), ⁇ -CDNNp, 1.4. HPLC using 70% CH 3 CN-H 2 0 showed: t R (relative to ⁇ -CD), ⁇ -CDNNp, 0.43.
  • CDN4N ⁇ -CDOTs (86 mg) and 1,4-diaminobutane (0.3 ml) were heated together at 70°C for 3 hours.
  • T.l.c. solvent A
  • Acetone (4 ml) was added and the resulting suspension centrifuged.
  • the solid was dissolved in water (0.1 ml), precipitated with acetone (4 ml) and centrifuged.
  • the solid was collected and dried in a vacuum desiccator over phosphorus pentoxide, to yield an off-white powder (84 mg) which still smelt of diaminobutane.
  • EXAMPLE 52 ⁇ -CDOTs (30 g) was dissolved in DMF (50 ml) with diaminobutane (5 g) and heated at 100°C for 3 hours. The mixture was poured into acetone (200 ml) and the solid was filtered off. Then the solid was dissolved in hot water (50 ml) and left to recrystallize. After standing for 2 days the solid was filtered off. Soxhlet extraction with ethanol overnight removed trapped diaminobutane. Recrystallization from hot water afforded pure ⁇ -CDN4N after prolonged standing.
  • Precipitation was effected by the addition of ethanol rather than acetone.
  • the solid was filtered off and Soxhlet extraction with ethanol removed most of the trapped diaminobutane. Recrystallization of the solid twice from hot water afforded ⁇ - CDN4N containing very large quantities of solvent, and loses up to 40% of it's weight upon drying.
  • Step 1 Bis-(3-nitrophenyl) succinate can be prepared as follows. Succinic acid (11.8 g) and 3-nitrophenol (27.8 g) were dissolved in dry ethyl acetate (1 1, 4A sieve) and the solution
  • the crystallized bis-(3-nitrophenyl) succinate was collected by vacuum filtration, rinsed with cold ethyl acetate (20 ml) and dried in vacuo to give bis-(3-nitrophenyl) succinate (21.34 g) .
  • the crude solid can alternately be purified by silica gel chromatography. M.p. 153- 154°C. ⁇ mn 1752 cm' 1 . *H n.m.r. (CDC1 3 ) ⁇ H 3.1, S, 4H; 7.2 - 8.6, ArH.
  • Step 2 Bis-(3-nitrophenyl) succinate (20 mg) was added in one portion to a stirred solution of ⁇ -3(2)CDNH 2 (100 mg) in DMF (3 ml) . The solution was left to stir at room temperature for two weeks, until t.l.c. showed no further reaction. The solution was poured into acetone (30 ml) and the resultant precipitate collected by filtration. The solid was rinsed with acetone and ether and air dried to give the crude product (93 mg) . T.l.c. (solvent B) of the product showed: Re (relative to ⁇ -CD), ⁇ -3CD 2 NSc, 0.75.
  • H 2 0 eluant showed: t R (relative to / 3-CD), 0.54, 0.77, 1.0, 2.90,
  • Step 2 To a stirred solution of dry
  • Step 1 Succinic acid (5.9 g, 0.05 mol) and 3-nitrophenol
  • Step 2 Bis-(3-nitrophenyl) succinate (160 mg) was added in portions to a solution of ⁇ -CDNH 2 (1 g) in dry pyridine with stirring over one hour. The reaction was followed by t.l.c. and appeared to be complete after 5 days. The pyridine was removed in vacuo and the solid dissolved in water. The water was removed in vacuo to remove traces of pyridine. The solid was dissolved in a minimum of water (8 ml) and added dropwise to ice-cold acetone (80 ml) and stirred rapidly for 10 minutes. The white powdery solid was collected by vacuum filtration and washed with
  • Examples 60 and 61 illustrate alternative methods for the preparation of N,N' -Bis- (6 A -deoxy-6 A - ⁇ -cyclodextrin) succinamide .
  • BioRex 70 The acid form of BioRex 70 was prepared by taking BioRex 70 (Na + form, as supplied, 100 ml) and batch rinsing in a scintered glass funnel as follows:-
  • the resin from above (- 50 ml) was suspended in Milli-Q® water (200 ml) with stirring. A solution of /8-CD 2 NSc (8 g) in Milli-Q® water (100 ml) was added and the mixture left to stir overnight at room temperature. The resin was collected by filtration in a scintered glass funnel and rinsed with water (5 x 100 ml) at 50°C. The combined filtrates were dried in vacuo to give 4.7 g of /S-CDjNSc. This was dissolved in water (20 ml) and filtered (0.22 ⁇ filter). Ethanol (- 20 ml) was added until a faint haze persisted.
  • the j8-CDNH 2 bound to the resin was isolated by rinsing the resin with a 20% ammonia solution (5 x 100 ml) warmed to 50°C. The filtrate was dried in vacuo to give 2.9 g of amine. HPLC analysis indicated there were two unidentified materials present with the amine. HPLC of the product using a 60% acetonitrile - water eluant showed: t R (relative to /S-CD) , 1.0, 2.43, 2.65, 3.06.
  • the crystallized product was collected by vacuum filtration, rinsed with cold ethyl acetate and dried to give bis-(3-nitrophenyl) glutarate as a cream coloured powder (10.81 g) . v ⁇ 1758 cm -1 .
  • Step 2 To a solution of /S-CDNH 2 (250 mg) in pyridine (3 ml) was added bis-(m-nitrophenyl) glutarate (45 mg) in small portions over 5 hours. The solution was stirred at room temperature for 5 days after which time t.l.c. showed one major new product and a small amount of
  • EXAMPLE 64 6 A -Amino-6 A -deoxy-6 A -N- (4-0-(3-nitrophenyl)- carboxypropanoy1)- ⁇ -cyclodextrin ( ⁇ -CDNScNP)
  • EXAMPLE 65 6 A -Amino-6 A -deoxy-6 A -N-(4-0-(3-nitro ⁇ henyl)- carboxypropanoyl)-/3-cyclodextrin (/3-CDNScNP)
  • SUBSTITUTE SHEET product showed: Re (relative to /8-CD) , /3-CDNScNP, 1.3.
  • Examples 67 and 68 illustrate alternative methods for the preparation of 6 A -Amino-6 A -deoxy-6 A -N- (3-carboxypropanoyl) - ⁇ - cyclodextrin.
  • EXAMPLE 70 6 A -Amino-6 A -deoxy-N- (3-aminocarbonylpropanoyl) - ⁇ - cyclodextrin (/3-CDNScN)
  • EXAMPLE 72 6 A -Amino-6 A -N-(2-N,2-N-(di-2-aminoethyl)-2-aminoethyl)-6 A - deoxy- ⁇ -cyclodextrin ( ⁇ -CDTren)
  • FAB MS M+H + requires 1102 found 1000, 1101, 1129, 1157. 13 C n.m.r (D 2 0) 31.0 5 , 36.9 3 , 38.8 0 , 46.3 8 , 47.5 4 , 50.8 4 , 53.6 9 , 54.7 3 , 55.0 5 , 61.6 6 , 67.7 9 , 72.0 7 , 72.9 X , 73.0 9 , 74.4 1# 82.4 7 , 85.0 i r 102.6 6 , 165.4 4 .
  • Step l A solution of dicyclohexylcarbodiimide (185.9 mg) in dichloromethane (2 mL) was added in one portion to a stirred solution of N-tBOC-L-glutamic acid- ⁇ - l butyl ester (538 mg) in dichloromethane (20 mL) . The mixture was left to stir at room temperature for 45 minutes, after which time the mixture was filtered to remove dicyclohexylurea and the clear filtrate was then dried in vacuo to give a yellowish oil. This oil was dissolved in dimethylformamide (10 mL) and added to a stirred solution of /3CDNH2 (1.057 g) in dimethylformamide (20 mL) .
  • SUBSTITUTESHEET Step 2 The product from above (447 mg) was dissolved in anhydrous trifluoroacetic acid (10 mL) and the solution was left to stand at room temperature for 18 hours. The solution was evaporated to dryness and acetone (30 mL) was added to the oily residue. A solution formed and this was evaporated to dryness. Water (10 mL) was added to the residue but none of the residue dissolved. On evaporation of the water, ethanol was added to obtain a white solid. The ethanol was evaporated and the residue was dissolved in water (10 mL) . This solution was filtered (0.22 ⁇ m) and dropped into acetone (150 mL) . The precipitate was collected by vacuum filtration, rinsed with acetone (10 mL) and ether (10 mL) and dried to give 448.7 mg of white powder (81% yield based on starting glutamic acid) .
  • Reagents used were of reagent grade unless otherwise stated.
  • Buffer systems (0.2 M carbonate buffers , pH 9.5, 10.0, 10.5, 11.0 and 11.5) were prepared by mixing calculated amounts of NaHC0 3 and Na 2 C0 3 in H 2 0. NaOH or HC1 (0.1 M) was used in pH adjustments of buffers with the aid of a Ross pH electrode (Model 81-03, Orion Research) and a pH meter (pHM64a, Radiometer A/S, Copenhagen) . The ionic strength of all buffer solutions was adjusted to 0.6 with potassium chloride.
  • the thermoregulator used consisted of a model 1419 Thermomix (B. Braun, W. Germany) and a Tecam incubator. Where a UV detector is mentioned a Waters
  • Table 3 gives the rate constants and half-lives for the hydrolysis of ⁇ -CDOIb at 3 alkaline pHs at 37°C.
  • ⁇ -CDOIb was found to be approximately 6-8 times more resistant to hydrolysis at a given alkaline pH than /8-CDOAc.
  • the hydrolysis in 0.1 M HC1 was followed for one week.
  • the hydrolysis rate observed was so slow that more and more interfering substances, presumably caused by the competing hydrolysis of the glucosidic linkage of the cyclodextrin moiety, accumulated in the mixture causing difficulties in the quantitization of released ⁇ -cyclodextrin.
  • Both of the diastereomeric esters hydrolysed at the same rate.
  • the ⁇ -CDOIb peak showed a large overlap with the buffer peak, and was therefore not applicable for kinetic measurement of the hydrolysis.
  • ⁇ -CDONp In contrast to the poor solubility of /3-CDONp, ⁇ -CDONp was readily soluble in cool water/acetonitrile (6:4) or water. However, the hydrolysis properties of ⁇ -CDONp (Table 4) are still very similar to those of /SCDONp in 0.1 M carbonate buffer at three pH's and 37°C.
  • Table 5 shows the rate constants and half-lives for hydrolysis of this compound at pH 11.0 and 11.5 at 37°C. The stability of this prodrug to hydrolysis was not much different from that of ⁇ -CDOIb, but was much greater than that of /8-CDOAc.
  • Table 6 shows the first-order rate constant (k ⁇ Jt ) , half-life (t 1/2 ) and coefficient of determination (r 2 ) obtained from linear regression based on peak area or peak height.
  • the half-life of /8-CD0Ib + under these conditions is greater than 10 hours.
  • a diastereomeric mixture of /8-CDOIb (1.4 g) was dissolved with heating in water (200 ml) and equilibrated to 37°C.
  • the hydrolysis was followed by HPLC using a 73:27 v/v acetonitrile - water mobile phase and a Waters model 441 UV absorbance detector.
  • the retention times for diastereomers A and B were 8.6 and 9.9 minutes respectively.
  • / 8-CDOIb" was selectively hydrolysed to release enriched (R)-Ibuprofen.
  • the reaction was terminated after two hours by adjusting the pH of the solution to 2.0 with 2N hydrochloric acid.
  • the solution was extracted with ether (3 x 200 ml) and the combined organic extracts were dried in vacuo to give white crystals.
  • the recovered (R)-Ibuprofen was converted to its correspondingmethyl ester and the optical purity measured by X H n.m.r. using Eu(hfc) 3 (a chiral shift reagent) .
  • SUBSTITUTESHEET was used as the mobile phase and that a Waters Model 441 UV Absorbance Detector at 254 nm was used for detection of the ester.
  • the retention time of the ester was 5.5 minutes.
  • the ester showed very rapid hydrolysis at pH 7.8 and 37°C as indicated by the yellow colour of the released 3-nitrophenol.
  • the rate constant (k lft ) was calculated to be 5.9542 hr" 1 which corresponds to a half-life of 7.0 minutes.
  • All cyclodextrins were stored in an evacuated dessicator over P 2 0 5 .
  • All drugs were stored in a dessicator over P 2 0 5 .
  • All dyes were stored in sealed containers to maintain a known water content determined by microanalysis. All weights were measured on a Mettler AE 160 balance. All spectra were recorded on a Zeiss DMR10 doublebea spectrophotometer with a thermostatted cell block ( ⁇ 0.1 K) at 298 K. Cells used for difference spectra experiments were two compartment QS 80 cells, with a pathlength of either 2 x 1.000 cm or 2 x 0.4375 cm. Cells used for straight spectra experiments were single compartment Ql cells of pathlength 0.201 cm.
  • SUBSTITUTESHEET 300-230 nm The concentration of this impurity varied with each cyclodextrin.
  • the significant absorbance of these impurities meant that the contribution of the cyclodextrin alone to the overall absorbance in the difference spectra could not be ignored, which made it necessary to measure the extinction coefficients of the cyclodextrin.
  • the inconsistency in the levels of impurities meant that the extinction coefficient needed to be re-measured for each cyclodextrin used.
  • the resultant spectrum must be due to the absorbance of the drug- cyclodextrin complex from the mixed drug-cyclodextrin solution.
  • This spectrum may have a magnitude large enough to measure accurately, and if so, a series of spectra could be obtained
  • Buffers were used as the solvent for all experiments.
  • a phosphate buffer of ionic strength 0.1 M was used. This was made by taking 4.4820 g of AR grade Na 2 HP0 4 .12H 2 0 and 1.7060 g of A.R. grade KH 2 P0 4 and making it up using Milli-Q® water to 500 ml in a volumetric flask. The density of phosphate buffer was determined to be 1.0029 g.cm" 3 at 298 K (Yin).
  • a tris buffer of ionic strength 0.2 M was used. This was made by taking 55.4 ml of 1.805 M HCl and 44.6859 g of A.R.
  • association constants at all wavelengths were not possible to calculate association constants at all wavelengths, as it depended on the shape of the spectrum as to whether or not a reasonable fit could be obtained at a particular wavelength.
  • An estimate of the association constant allowed initial calculation of the estimated equilibrium concentrations of drug or dye, cyclodextrin and complex.
  • An estimate of the extinction coefficient of the complex allowed calculation of an estimated absorbance of the sample/reference solution.
  • [AB] is the equilibrium concentra-tion of the complex AB K is the association constant.
  • [A] , [B] were expressed in terms of [AB], substituted in the expression for the equilibrium constant, and the resulting quadratic equation solved for [AB] , the concentration of drug- cyclodextrin complex at equilibrium.
  • c K[A] 0 [B] 0
  • Abs(reference cell) 0 for straight spectra method
  • K/E(AB) are estimated and iterated until convergence is SUBSTITUTESHEET found .
  • association constant should be constant within errors over all wavelengths.
  • the actual values for the association constant were found to vary with wavelength, and thus needed to be averaged to give a final association constant value. From the difference spectrum, regions were chosen which had a reasonable change in absorbance across the cyclodextrin concentration range.
  • the association constant values at these wavelengths were accepted as reasonable to include in the average, and were weighted according to the reciprocal of their error, and a mean value calculated. The root-mean-square deviation of the accepted values could then be calculated to give a final value, with error, for the association constant.
  • a stock solution of 8.27X1C 4 M Naproxen and a stock solution of 1.34X10 '1 M ⁇ -CD were made up in phosphate buffer at pH 6.9. A total of 17 spectra were run, both sample and reference solutions being made up by weight dilutions of the stocks.
  • a stock solution of 8.92x10 ⁇ M Naproxen and a stock solution of 1.58xl0" 2 M /3-CD were made up in phosphate buffer at pH 6.9.
  • a total of 16 spectra were run, both sample and reference solutions being made up by weight dilutions of the stocks.
  • a stock solution of 7.99x10"* M Naproxen and a stock solution of 1.2xl0" 2 M 7-CD were made up in phosphate buffer at pH 6.9. A total of 23 spectra were run, both sample and reference solutions being made up by weight dilutions of the stocks.
  • SUBSTITUTESHEET region 340-334 nm and 293-288 nm were averaged to give an association constant of 120 ⁇ 10.
  • a stock solution of 7.80 x 10 -4 M Naproxen and a stock solution of 4.23 x 10" 2 M /8-CDN4N were made up in tris buffer at pH 8.6. A total of 15 spectra were run, both sample and reference solutions being made up by weight dilutions of the stocks.
  • a stock solution of 7.00xl0 "3 M Ibuprofen and three stock solutions of 4.37 x 10' 2 M, 8.91xl0" 3 M and 4.47xl0" 3 M DIMEB were made up in phosphate buffer at pH 6.9.
  • a total of 16 spectra were run, both sample and reference solutions being made up by weight dilutions of the stocks.
  • a stock solution of 1.96x10"* M Piroxicam and a stock solution of 1.49X10" 1 M DIMEB were made up in phosphate buffer. A total of 20 spectra were run, both sample and reference solutions being made up by weight dilutions of the stocks.
  • a stock solution of 3.96x10"* M Panadol and a stock solution of 1.17X10" 2 M / 8-CD were made up in phosphate buffer at pH 6.9. A total of 18 spectra were run, both sample and reference solutions being made up by weight dilutions of the stocks.
  • SUBSTITUTESHEET recorded in 0.8750 cm pathlength cells over 300-220 nm, sampling at 1.0 nm intervals, using an integration time of 3.2 seconds per wavelength and a band width of 1.0 nm. Fitted values in the region 267-250 nm were averaged to give an association constant of 130 ⁇ 10.
  • a stock solution of 3.94x10"* M Panadol and a stock solution of 1.005xl0" 3 M DIMEB were made up in phosphate buffer at pH 6.9. A total of 19 spectra were run, both sample and reference solutions being made up by weight dilutions of the stocks.
  • each sample/reference solution pair was recorded in 0.8750 cm pathlength cells over 300-220 nm, sampling at 1.0 nm intervals, using an integration time of 3.2 seconds per wavelength and a band width of 1.0 nm. Fitted values in the region 259-255 nm were averaged to give an association constant of 83 ⁇ 3. When values in the region 261-260 nm, 254 nm and 232- 224 nm were included in the average the association constant was averaged to be 110 ⁇ 50.
  • a stock solution of 1.56X10 "4 M Crystal Violet and a stock solution of 1.60X10 "2 M /3-CD were made up in phosphate buffer at pH 6.9. A total of 16 spectra were run, both sample and reference solutions being made up by weight dilutions of the stocks.
  • a stock solution of 1.56x10 * * M Crystal Violet and a stock solution of 3.00xl0" 2 M /3-CDNH 2 were made up in phosphate buffer at pH 6.9. A total of 15 spectra were run, both sample and reference solutions being made up by weight dilutions of the stocks.
  • a stock solution of 1.05x10"* M Methyl Orange and a stock solution of 1.49X10 "2 M ⁇ -CO were made up in phosphate buffer at pH 6.0. A total of 16 spectra were run, both sample and reference solutions being made up by weight dilutions of the stocks.
  • a stock solution of 1.05x10"* M Methyl Orange and a stock solution of 2.8lxl0 *2 M / 8-CD were made up in phosphate buffer at pH 6.0. A total of 15 spectra were run, both sample and reference solutions being made up by weight dilutions of the stocks.
  • a stock solution of 6.09x10"* M Methyl Orange and a stock solutions of 2.99x10' ⁇ and 3.09xlO"*M ⁇ -CD 2 NSc were made up in phosphate buffer at pH 6.0. A total of 20 spectra were run, both sample and reference solutions being made up by weight dilutions of the stocks.
  • the glass titration vessel about 100 ml in volume, has three vertical holes for nitrogen purging tube, pH electrode and receiving solution from the burette respectively. It has a water jacket connected to a thermostatted water bath to keep the temperature constant during titration processes.
  • HCl and/or NaOH solutions are used as titrants in all the measurements of pKa's of cyclodextrin derivatives. They are prepared and standardized in the following way.
  • HCl solution was standardized as follows. Dilute from any higher concentration of HCl solution to about 0.005 M in a 500 ml volumetric flask using Milli-Q® water. Standardize the solution with Borax (disodium tetraborate, AR) to the precision of 0.001 millimolar. The end point is at about pH 5.1, for which Methyl red is a suitable indicator. However, since the color change is not sharp enough for standardizing such a diluted solution to the required precision, which is commonly ⁇ 0.1%, a pH electrode was used to determine the end point. Meanwhile, the initial volume of the Borax solution in the vessel has to be controlled with a pipet, and the weight of Borax samples for repetitions have to be within the accuracy of 0.001 g. The Milli-Q® water used to make Borax solution was bubbled with high purity nitrogen gas for one hour.
  • Borax sodium tetraborate
  • Carbon dioxide free NaOH was prepared as follows. Weigh about 5 grams AR grade NaOH into a small volumetric flask, add 5 ml Milli-Q® water, stand by for 24 hours. Decant the solution into 1 litre nitrogen bubbled Milli-Q® water. Standardize this carbon dioxide free NaOH solution in the normal way with potassium hydrogen phthalate.
  • a phosphate buffer solution (0.025 M disodium hydrogen ortho-phosphate and 0.025 M potassium dihydrogen phosphate) of pH -7 is used to standardize the pH meter since the iso pH of the ROSS electrode is 7.
  • SUBSTITUTESHEET can be used to determine the slope depending on the pH range of the measurement.
  • pH 4 buffer is preferred because of the interference of carbon dioxide at high pH.
  • the direct pH titrations for the cyclodextrin derivatives are performed in the normal way. 0.5 ml increments of the titrants produces sufficient number of data points for high precision data fitting.
  • the results of pKa and the fitted purities are usually checked with back titrations, in which acid or base of high concentration is added (normally not more than 10 ml) to reach the pH value of the final end point estimated from a direct titration before the recorded titration is performed in the normal manner. If the sample is a base, nitrogen purging for half an hour follows the acidification before the back titration with the NaOH. The temperature was held at 25.0 °C for all the experiments reported below.
  • the relationship between the volume of the added titrant and the resulted pH value can be derived from the acid dissociation constant expressions and the charge and mass balance equations.
  • the theoretical titrant volume is expressed as a non-linear function of the corresponding concentration of free acid (converted from the measured pH value) , with the total amount of sample used (in millimole) and the initial volume (millilitre) of the solution as the fixed parameters and the estimated acid dissociation constants as the variable parameters.
  • the calculated volumes are compared to the experimental values using a non-linear least squares fitting computer program.
  • the new values are calculated via reestimation of the unknown acid dissociation constants in continuous iteration until convergence (within the specified limit of error) is found between the measured and calculated titrant volumes. This process allows the establishment of the best-fit values for the acid dissociation constants of the chemicals titrated.
  • the measured pH values Prior to the fitting process, the measured pH values are corrected for electrode error.
  • SUBSTITUTESHEET used to titrate the sample solution from pH 3.6 to pH 10.29.
  • the fitted pKa is 4.70 ⁇ 0.01.
  • /8-CDN4N (0.1192 g) and potassium chloride (1.67 g) were dissolved in 0.004945 M hydrochloric acid (40.0 ml).
  • the molar ratio of /8-CDN4N to hydrochloric acid is 1:1 on the basis of 100% purity for / 8-CDN4N.
  • 0.005765 M sodium hydroxide containing 0.5 M potassium chloride was used to titrate the sample solution from pH 5.5 to pH 10.1.
  • the fitted pKaj is 8.56 ⁇ 0.02
  • pKa 2 is 10.33 ⁇ 0.01 at 25°C and an ionic strength of 0.5.
  • a Perkin-Elmer 3000 Fluorescence Spectrometer is used in all the experiments of fluorescence measurement.
  • the sample cuvette, 1 cm 2 is not thermostatted.
  • the excitation wavelength is 366 nm and a cut off filter at 390 nm is installed in the emission light path.
  • the intensities of the fluorescence of all samples are measured at the peak wavelength which should be searched between 400 nm to
  • ⁇ CD and /8-CD were suplied by Nihon Shokuhin Kako Co..
  • the fluorescing probe molecule used in the following experiments is essentially non-fluorescent in water, but is highly fluorescent when dissolved in nonpolar solvents or when bound to macromole ⁇ ules. It has been used as a microprobe for conformation changes in the proteins to which it binds.
  • TNS can be included in the annuli of some cyclodextrins or cyclodextrin polymers in which cases the fluorescence of TNS in aqueous solution is enhanced to different extend depending upon the including molecules and their concentrations in the solution [A. Harada et.al. Macromolecules 10, 676-681 (1977)]. This observation allows the measurement for the stability constant of the inclusion by varying the concentration of one cyclodextrin while keeping the TNS concentration constant.
  • the measured fluorescence intensities at the presence of various cyclodextrins can be approximated to be proportional to the concentration of the included TNS molecules, e.g., [CD-TNS] in the case of 1:1 inclusion, or [CD 2 -TNS] in the case of 2:1 inclusion, etc.
  • I I** K[CD] / ( 1+K[CD] )
  • the double reciprical form of the above equation i.e., 1/1 as a linear function of I/[CD] is sometimes applied to comfirm the 1:1 ratio of the inclusion.
  • the stability constant can also be calculated from the fitted slope of the straight line.
  • Kj is the first stability constant for the 1:1 inclusion at lower cyclodextrin concentration
  • K 2 is the second stability constant for the 2:1 inclusion at higher cyclodextrin concentration
  • ⁇ is the ratio of the molar fluorescence enhancement by the 1:1 complex (CD-TNS) to the molar enhancement by the 2:1 complex (CD 2 -TNS) .
  • CD-TNS 1:1 complex
  • CD 2 -TNS 2:1 complex
  • the non-linear fitting subroutine used in all the data analysis for the fluorescence experiments is DATAFIT by Dr T. Kurucsev, interfaced by a main program called FLUO.NLF.F.
  • sample solutions 2.0 ml of each, were prepared by mixing the appropriate amount of /8-CD 2 NSc stock and the buffer solutions with 0.040 ml of 5.007xl0" 5 M TNS stock solution so that the [TNS] in all samples were l.OxlO" 6 M, and [CD]s were in 10:1, 15:1, 20:1, ..., 100:1, ..., 1000:1 ratio to [TNS] .
  • the peak of the fluorescence was found to be at 438 nm. The fluorescence intensities at this wavelength, increasing with the increase of [CD], were measured for all the sample solutions.
  • the peak of the fluorescence was found to be at 442 nm. The fluorescence intensities at this wavelength, increasing with the increase of [CD], were measured for all the sample solutions.
  • SUBSTITUTE SHEET chemicals used to measure association constants of drugs by liquid chromatography in Example 119 is as follows .
  • HPLC HPLC was carried out using an ICI LC1500 pump connected to a Waters Lambda-Max 481 detector. Samples were injected using an ICI LC1600 autosampler controlled by an ICI DP800 Data Station. The column used was a Merck Hibar Lichrosorb Diol column (4 x 250mm). Solutions of ⁇ -CD (55.7 mg) , ⁇ -CD 2 NSc (56.5 mg) , /8-CD (45.5 mg) and /8-CD 2 NSc (54.4 mg) in 0.1 M phosphate buffer pH 7.4 (25 ml) were prepared and filtered (0.22 ⁇ m) before use.
  • the column is equilibrated and eluted at 1ml/min with successive concentrations of drug in 0.1 M phosphate buffer (pH 7.4).
  • 0.1 M phosphate buffer pH 7.4
  • a 50 ⁇ L sample of pure buffer is run as a blank, followed by 50 ⁇ L samples of each of the cyclodextrin solutions. Peaks are detected over the range 300 - 380 nm.
  • the shorter wavelengths are used for increased sensitivity at the lower drug concentrations and the longer wavelengths are used to avoid detector overload at higher drug concentrations.
  • the negative peak which occurs at about 6 minutes corresponds to a depletion of drug in the eluant due to both dilution (calculated from the injection of pure buffer) and inclusion of the drug by the cyclodextrin.
  • the included drug elutes with the cyclodextrin at about 2 minutes and gives rise to a positive peak.
  • r is a measure of the relative abilities of the cyclodextrin derivatives to bind a particular drug.
  • the concentrations of indomethacin used to elute the column were 1.003 x 10"* M, 2.006 x 10"* M, 4.012 X 10"* M and 1.003 x 10' 3 M and were prepared by dilution of a stock solution of indomethacin (359.0 g, 1.003xl0 "3 mole) in buffer (1L) . All solutions were passed through a 0.22 ⁇ m filter before use.
  • EXAMPLE 120 Effect of cyclodextrins on the hydrolysis of N-Benzoyl- (1)-tyrosine ethyl ester (BTEE) by ⁇ -Chymotrypsin
  • N-Benzoyl-(l)-tyrosine ethyl ester (BTEE) is a substrate commonly used to determine the activity of ⁇ -chymotrypsin. It is shown that the rate of hydrolysis of BTEE by ⁇ -chymotrypsin is slowed in the presence of /8-cyclodextrin (/3-CD) .
  • the solution was diluted to 1 1 with Milli-Q® water and dry methanol (432 ml) added.
  • Substrate solution. BTEE (14.8 mg) was dissolved with sonication in the buffer solution (100 ml) .
  • UV measurements volumes were measured with a 1000 ⁇ l or a 200 ⁇ l variable pippetor. Where 3 ml volumes were required 3 x 1000 ⁇ l samples were used. Where 150 ⁇ l was required a single 150 ⁇ l sample was used. All solutions were placed in the spectrophotometer room for three hours before measurements were taken to allow equilibration of the solutions to room temperature. Measurements were made in a matched pair of 1 cm quartz cells the absorption was measured over time at 256 nm.
  • the reference cell was filled with substrate solution (3 ml) and water (150 ⁇ l) and mixed to ensure homogeneity.
  • the sample cell was filled with substrate solution (3 ml) and enzyme solution (150 ⁇ l) was added smoothly to the cell. Five seconds after the beginning of the enzymes addition the solution was stirred for 10 seconds. Thirty seconds after the start of the enzymes addition the data collection was started. Data was collected for 10 minutes, by which time the hydrolysis was apparently quite complete. Data were printed out during collection on chart paper and were also collected on disc.
  • the sample solutions were prepared separately three times and data collected to ensure the reproducibility of the results.
  • the reference cell was filled with substrate, /8-cyclodextrin solution (3 ml) and water (150 ⁇ l) and mixed to ensure homogeneity.
  • the sample cell was filled with substrate, /3-cyclodextrin solution (3 ml) and enzyme solution (150 ⁇ l) was added smoothly to the cell. Five seconds after the beginning of the enzymes addition the solution was stirred for 10 seconds. Thirty seconds after the start of the enzymes addition the data collection was started. Data was collected for 10 minutes, by which time the hydrolysis was apparently quite complete. Data were printed out during collection on chart paper and were also collected on disc.
  • Figure 1 shows a typical plot of the experiments. Note the rate of hydrolysis of BTEE in the presence of /8-CD is slower than without /8-CD.
  • the rate of the enzyme catalysed hydrolysis is proportional to the effective (or available) [BTEE] at concentrations below K,,,.
  • the effective [BTEE] equals the total [BTEE], therefore:
  • [BTEE] eff , 3.98 x 10" 5 mol.l" 1 corresponding to a slope of 0.025 units/3 seconds.
  • [/8-CD] [/8-CD] 0 - [/8-CD.BTEE]
  • [BTEE] [BTEE] ⁇ .,.,,,
  • EXAMPLE 132 Naproxen with 6 A -amino-6 A -deoxy-/8-cyclodextrin 1:1 complex
  • a solution of /8-CDNH 2 250 mg, 0.22 mmol
  • Milli-Q® water 10 ml
  • solid Naproxen 50 mg, 0.22 mmol
  • the resulting suspension was stirred at room temperature for 4 hours during which time a clear colorless solution was obtained.
  • Filtration and evaporation to dryness in vacuo resulted in the isolation of a glassy material. Drying to constant weight in vacuo over phosphorus pentoxide gave a yellow powder (268 mg) which was the Naproxen modified cyclodextrin formulation.
  • Amiodarone.HCl (Sigma, 0.0200 g, 0.0293 mmol) and /8-CDNSc (0.0763 g, 0.0618 mmol) were mixed in 40 ml Milli-Q® water and stirred with a magnetic stirrer for 24 hours. The resulting solution contained only a slight haze of undissolved material. The solution was filtered (0.2 ⁇ m filter) and dried in vacuo. The collected crystals, which were almost transparent, readily dissolved in pure water but not in phosphate buffer of pH 7.
  • Amiodarone.HCl has a maximum absorbance at 242 nm in ethanol, pure water and ethanol/water mixture (4:1 ratio by weight) .
  • the extinction coefficient of amiodarone.HCl in ethanol/water mixture solution (4:1 ratio by weight) was measured using a series of solutions of amiodarone.HCl in this solvent. These were prepared by diluting an amiodarone.HCl solution of known concentration in pure ethanol with the appropriate amount of ethanol and water.
  • the extinction coeffecient of amiodarone.HCl at 242 nm in such a mixed solvent is 46,000 (g amiodarone.HCl / g solvent)" 1 .cm" 1 .
  • the saturated solution of amiodarone.HCl in pure water was prepared by adding an excess of amiodarone.HCl (0.1 g) to 10 ml Milli-Q® water and shaking the sample bottle in a water bath thermostatted at 25.0°C for at least 3 days.
  • the clear solution (0.4 g) was mixed with 1.6 g ethanol.
  • the concentration of the solution was calculated from the measured absorbance at 242 nm and the known extinction coefficient, which led to the determination of the solubility of amiodarone.HCl in pure water, 200 mg.l" 1 . Same procedure was repeated 3 days after to assure
  • EXAMPLE 136 Composition and solubility of the amiodarone//3-CDNSc solid Anhydrous amiodarone//S-CDNSc complex (0.0364 g) was dissolved in 1.0089 g Milli-Q® water. The resulted solution was very viscous and seemed to be close to its saturation. The solution was diluted 100 fold with water for taking UV spectra.
  • the concentration of the original solution in terms of pure amiodarone.HCl was calculated, using the measured absorbance at 242 nm and the extinction coefficient 46,000 (g amiodarone.HCl/g solvent)" 1 .cm" 1 , as 6.9 g.l" 1 , which should present the lower bound of the solubility of amiodarone.HCl in water in the presence of /S-CDNSc. It is -35 times higher than that in pure water without /8-CDNSc. Such a concentration can not be achieved by dissolving corresponding amount of amiodarone.HCl and /8-CDNSc into water directly.
  • the extinction coefficient of /3-CDNSc is 77 M ⁇ .cm" 1 and its contribution to the absorbance at 242 nm of the 100 fold diluted amiodarone.HCl//3-CDNSc solution above would be only 0.003, negligibly small compared to the total of -0.6 absorbance unit.
  • Amiodarone HCl was obtained from Sigma chemical Company Ltd. (Dorset, England) and / 8-CDNSc was prepared as described above. All other chemicals used in the study were of analytical grade.
  • Dogs were administered amiodarone HCl as a single injection into the right cephalic vein, using an indwelling catheter, at a dose of 5 mg kg "1 .
  • Amiodarone HCl was given with and without
  • Blood (ca 5 ml) was then sampled from either jugular vein of all animals at 0 (predose) , 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours. The blood was collected into heparinized glass vacutainers and centrifuged (1500 g for 10 min. at 18°C) to prepare plasma within 30 min. of collection. Plasma samples were stored at -20°C pending analysis. Plasma concentration of amiodarone HCl in the samples was measured by the high performance liquid chromatography procedure described by Kess et al.
  • SUBSTITUTESHEET erratic fluctuation of plasma drug levels in the first four hours after injection ( Figures 10 and 11) . Such phenomenon was not observed in animals treated with amiodarone HCl-/8-CDNSc.
  • plasma amiodarone levels fell substantially in the first 10 to 15 minutes and then declined slowly with a long elimination half-life.
  • SUBSTITUTE SHEET improvement in the pharmacokinetic profile of the drug may allow a more accurate prediction of drug levels from the doses administered and amiodarone therapy for patients can be individually tailored.
  • EXAMPLE 138 Stability Constants for the inclusion of Cimetidine by ⁇ - Cyclodextrin and 6 A -amino-6 A -deoxy-6 A -N-(3-carboxypropanoyl)- ⁇ - cyclodextrin
  • a 50 ⁇ l sample of pure buffer was run as a blank, followed by 50 ⁇ l samples of each of the cyclodextrin solutions, injected via an autosampler.
  • the negative peak which occurs at about 6 minutes corresponds to a depletion of Cimetidine in the eluent due to both dilution (calculated from the injection of pure buffer) and the inclusion of cimetidine by the cyclodextrin.
  • the included cimetidine elutes with the cyclodextrin at about 2 minutes and gives rise to a positive peak.
  • Cimetidine (0.08 g) and 6 A -amino-6 A -deoxy-6 A -N-(3- carboxypropanoyl)- ⁇ -cyclodextrin (0.43g) were dissolved in water (10ml) .
  • the solution was evaporated to dryness under reduced pressure and the resulting solid was dried in a dessicator over P 2 0 5 .
  • the 1:1 formulation of cimetidine with 6 A -amino-6 A -deoxy- 6 A -N-(3-carboxypropanoyl)- ⁇ -cyclodextrin prepared in this way dissolved completely in water (0.5ml) to give a clear solution.
  • cyclodextrin derivatives and inclusion complexes in accordance with this invention.
  • uses can include therapeutic and diagnostic drug delivery, such as multiple routes of administration (i.v. , p.o. , ophthalmic, transdermal, etc.) , improved bioavailability, reduction of irritating drug effects, quantitative reliability, reduced dosing volume, elimination of organic solvents, stable, convenient storage and handling, and previously insoluble or unstable drugs may now be considered for development which enhances removal of lipophilic substances from blood.
  • diagnostic kits include improved low end sensitivity, reduced reaction times, more stable liquid components, greater recovered bioactivity in lyophilized components, reduced effect of interfering substances in serum, plasma and urine specimens and enhanced spectrophotometric response.
  • SUBSTITUTESHEET Bioprocessing enables improved down-line recovery (quantity) , improved bioactivity (quality) , and greater handling convenience. Also, with regard to cell culture, serum free formulations and enhanced productivity of enzymatic reactions are provided.
  • This invention is also applicable to food products, cosmetics, toiletries, taste/smell masking, stable, convenient forms, timed release, homogeneous reaction mixtures, catalyzed reactions, and greater precision and reproducibility of laborator techniques.

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Abstract

On décrit des dérivés de cyclodextrines et des composés les renfermant, ainsi que des intermédiaires et des synthèses pour leur production. On décrit également des procédés permettant de concevoir des composés afin d'obtenir des propriétés améliorées. L'invention concerne aussi des compositions pharmaceutiques, entre autres la cimétidine, l'amiodarone, les polypeptides et le piroxicame, ainsi que de nombreux procédés dans lesquels on peut utiliser les dérivés de cyclodextrines.
EP19910905452 1990-03-02 1991-03-01 Cyclodextrin compositions and methods for pharmaceutical and industrial applications Withdrawn EP0518930A4 (en)

Applications Claiming Priority (26)

Application Number Priority Date Filing Date Title
AU8899/90 1990-03-02
AUPJ889990 1990-03-02
AUPJ899390 1990-03-08
AU8993/90 1990-03-08
AU9344/90 1990-03-28
AUPJ934490 1990-03-28
AUPJ937390 1990-03-29
AU9373/90 1990-03-29
AU9756/90 1990-04-23
AUPJ975690 1990-04-23
AU1538/90 1990-08-03
AUPK153890 1990-08-03
AUPK175590 1990-08-16
AU1755/90 1990-08-16
AUPK226990 1990-09-12
AU2269/90 1990-09-12
AUPK359690 1990-11-29
AU3596/90 1990-11-29
AU3624/90 1990-11-30
AUPK362490 1990-11-30
AUPK428491 1991-01-21
AU4284/91 1991-01-21
AUPK460391 1991-02-14
AU4603/91 1991-02-14
AU4856/91 1991-02-27
AUPK485691 1991-02-27

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EP0518930A1 true EP0518930A1 (fr) 1992-12-23
EP0518930A4 EP0518930A4 (en) 1993-09-15

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AT399718B (de) * 1992-04-16 1995-07-25 Lek Tovarna Farmacevtskih Inklusionskomplexe von optisch aktiven und racemischen 1,4-dihydropyridinen mit methyl-beta- cyclodextrin oder anderen cyclodextrinderivaten, ein verfahren zur herstellung von optisch aktiven dihydropyridinen und deren inklusionskomplexen und diese komplexe enthaltende pharmazeutische formulierungen
RU2143896C1 (ru) * 1993-03-05 2000-01-10 Хексаль Аг Кристаллический комплекс циклодекстрина с гидрохлоридом ранитидина, способ его получения и содержащие его фармацевтические составы
FR2705350B1 (fr) * 1993-05-13 1995-07-07 Oreal Nouveaux dérivés de mono(6-amino 6-désoxy) cyclodextrine substituée en position 6 par un reste d'alpha-aminoacide, leur procédé de préparation et leurs utilisations.
DE4338508A1 (de) * 1993-11-11 1995-05-18 Asta Medica Ag Arzneimittelzubereitungen enthaltend Thioctsäure oder Dihydroliponsäure in Form von Einschlußverbindungen mit Cyclodextrinen oder Cyclodextrinderivaten und in Form von Granulaten, Kau- oder Brausetabletten
FR2714067B1 (fr) 1993-12-22 1996-01-12 Commissariat Energie Atomique Nouveaux dérivés de cyclodextrines, utilisables en particulier pour solubiliser des composés chimiques hydrophobes tels que des médicaments, et leur procédé de préparation.
FR2713934B1 (fr) * 1993-12-22 1996-01-12 Commissariat Energie Atomique Utilisation de cyclodextrines aminées pour la solubilisation aqueuse de composés hydrophobes, en particulier de molécules pharmaceutiquement actives.
BE1008978A5 (fr) * 1994-12-27 1996-10-01 Solvay Adjuvants pour vaccins.
HUP9700632A3 (en) * 1997-03-24 1999-10-28 Cyclolab Ciklodextrin Kutato F Pharmaceutical compositions containing propylamine derivative and cyclodextrine and process for producing the same
US6642214B1 (en) 1997-11-11 2003-11-04 Ceramoptec Industries, Inc. Detoxication of active pharmaceutical substances using cyclodextrine oligomers
KR100247558B1 (ko) * 1997-11-24 2000-05-01 김충섭 사이클로덱스트린을 이용한 이온토포레시스 개선방법
ATE279445T1 (de) * 1999-03-05 2004-10-15 Wolff Walsrode Ag Regioselektiv substituierte ester von oligo- und polysacchariden und verfahren zu ihrer herstellung
DE10018617A1 (de) * 2000-01-13 2001-10-31 Joerg G Moser Cyclodextrin-Dimere mit Peptid-Spacerstrukturen zur Entgiftung von pharmazeutischen Wirkstoffen hohen Nebenwirkungspotentials
US6869939B2 (en) * 2002-05-04 2005-03-22 Cydex, Inc. Formulations containing amiodarone and sulfoalkyl ether cyclodextrin
ES2286177T3 (es) * 2002-06-17 2007-12-01 Chiesi Farmaceutici S.P.A. Procedimiento para la preparacion de piroxicam: compuestos de inclusion de betaciclodextrina.
PT2277551E (pt) 2002-09-06 2013-08-22 Cerulean Pharma Inc Polímeros à base de ciclodextrina para a administração de medicamentos ligados por ligação covalente
US20070020299A1 (en) 2003-12-31 2007-01-25 Pipkin James D Inhalant formulation containing sulfoalkyl ether cyclodextrin and corticosteroid
JP5050191B2 (ja) * 2005-11-01 2012-10-17 国立大学法人三重大学 シクロデキストリン誘導体の単離精製方法
JP2010516625A (ja) 2007-01-24 2010-05-20 インサート セラピューティクス， インコーポレイテッド 制御された薬物送達のためのテザー基を有するポリマー−薬物コンジュゲート
US11020363B2 (en) 2009-05-29 2021-06-01 Cydex Pharmaceuticals, Inc. Injectable nitrogen mustard compositions comprising a cyclodextrin derivative and methods of making and using the same
PL2434886T3 (pl) 2009-05-29 2020-05-18 Cydex Pharmaceuticals, Inc. Kompozycje do wstrzykiwania melfalanu zawierające pochodną cyklodekstryny i sposoby ich wytwarzania i stosowania
US8492538B1 (en) 2009-06-04 2013-07-23 Jose R. Matos Cyclodextrin derivative salts
JP2011190341A (ja) * 2010-03-15 2011-09-29 Nano Dex Kk シクロデキストリン化合物
WO2014055493A1 (fr) 2012-10-02 2014-04-10 Cerulean Pharma Inc. Procédés et systèmes s'appliquant à la précipitation de polymères et à la génération de particules
GB201608936D0 (en) 2016-05-20 2016-07-06 Polytherics Ltd Novel conjugates and novel conjugating reagents
EP3906263A4 (fr) 2019-01-03 2022-08-31 Underdog Pharmaceuticals, Inc. Dimères de cyclodextrine, leurs compositions et leurs utilisations
CA3183207A1 (fr) 2020-06-18 2021-12-23 Daniel Joseph Fitzgerald Complexes d'acides gras de proteine de spicule de coronavirus et leur utilisation
WO2022029334A1 (fr) 2020-08-07 2022-02-10 Gbiotech S.À.R.L. Polythérapies pour le traitement d'une infection à coronavirus

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