U.S. patent application number 09/775011 was filed with the patent office on 2001-10-25 for cyclodextrin polymer compositions for use as drug carriers.
Invention is credited to Kosak, Kenneth M..
Application Number | 20010034333 09/775011 |
Document ID | / |
Family ID | 22834827 |
Filed Date | 2001-10-25 |
United States Patent
Application |
20010034333 |
Kind Code |
A1 |
Kosak, Kenneth M. |
October 25, 2001 |
Cyclodextrin polymer compositions for use as drug carriers
Abstract
This invention discloses compositions of cyclodextrin polymers
for carrying drugs and other active agents. Compositions are also
disclosed of cyclodextrin polymer carriers that release drugs under
controlled conditions. The invention also discloses compositions of
cyclodextrin polymer carriers that are coupled to biorecognition
molecules for targeting the delivery of drugs to their site of
action. The advantages of the water-soluble cyclodextrin polymer
carrier are: (1) Drugs can be used based on efficacy without
solubility or conjugation requirements. (2) Drugs can be delivered
as macromolecules and released within the cell. (3) Drugs can be
targeted by coupling the carrier to biorecognition molecules. (4)
Synthesis methods are independent of the drug to facilitate
multiple drug therapies.
Inventors: |
Kosak, Kenneth M.; (West
Valley City, UT) |
Correspondence
Address: |
KENNETH M. KOSAK
3194 S. 4400 W.
West Valley City
UT
84120
US
|
Family ID: |
22834827 |
Appl. No.: |
09/775011 |
Filed: |
February 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09775011 |
Feb 1, 2001 |
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PCT/US99/30820 |
Dec 27, 1999 |
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PCT/US99/30820 |
Dec 27, 1999 |
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09223055 |
Dec 30, 1998 |
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6048736 |
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Current U.S.
Class: |
514/44A ;
436/507; 436/536; 514/58 |
Current CPC
Class: |
A61K 47/6951 20170801;
A61K 9/1075 20130101; A61K 9/5192 20130101; A61K 9/5161 20130101;
B82Y 5/00 20130101; C08B 37/0012 20130101 |
Class at
Publication: |
514/44 ; 514/58;
436/536; 436/507 |
International
Class: |
A61K 048/00; A61K
031/724; G01N 033/536 |
Claims
What is claimed is:
1. A controlled release pharmaceutical composition comprising; a)
cyclodextrin molecules selected from the group consisting of
cyclodextrin derivatives, oxidized cyclodextrins, cyclodextrin
dimers, cyclodextrin trimers, and cyclodextrin polymers complexed
with; b) an active agent selected from the group consisting of
prodrugs, anticancer drugs, antineoplastic drugs, antifungal drugs,
antibacterial drugs, antiviral drugs, cardiac drugs, neurological
drugs, alkaloids, antibiotics, bioactive peptides, steroids,
steroid hormones, polypeptide hormones, interferons, interleukins,
narcotics, prostaglandins, purines, pyrimidines, anti-protozoan
drugs and anti-parasitic drugs wherein; c) said cyclodextrin
molecules are covalently cross-linked through a biocleavable
linkage to form a polymer that has entrapped the active agent and
wherein the cross-linking provides the function of controlled
release.
2. The composition of claim 1 wherein the biocleavable linkage is
selected from the group consisting of disulfide linkages, protected
disulfide linkages, ester bonds, aldehyde bonds, amide bonds,
polypeptide linkages and hydrazone linkages.
3. The composition of claim 1 further comprising a biorecognition
molecule coupled to the pharmaceutical composition.
4. The composition of claim 1 wherein said cyclodextrin dimers,
cyclodextrin trimers, and cyclodextrin polymers have been
derivatized to provide groups selected from the group consisting of
dialdehydes, sulfobutylethers, sulfopropylethers, hydroxyethyls,
hydroxypropyls, dihydroxy propyls, carboxylates and phosphates.
5. The composition of claim 1 wherein the active agent is selected
from the group consisting of ganciclovir, furosemide, indomethacin,
camptothecins, cyclosporins, chlorpromazine, methotrexate,
penicillin derivatives, anthracyclines, teramycins, tetracyclines,
chlorotetracyclines, clomocyclines, butoconazole, ellipticines,
guamecyclines, macrolides, filipins, fungichromins, nystatins,
5'-fluorouracil, 5'-fluoro-2'-deoxyuridine, allopurinol and
paclitaxe.
6. The composition of claim 1 wherein said cyclodextrin molecules
are coupled to an intermediate coupling substance selected from the
group consisting of serum albumins, glycoproteins, lipoproteins,
polysaccharides, lipopolysaccharides, amino polysaccharides,
polyacrylamides, lipids, glycolipids, N-(2-hydroxypropyl)
methacrylamides, poly cyanoacrylates, polyethylene glycols, poly
(D,L-lactic-coglycolic adds), dendrimers, poly
(D,L-lactide)-block-methox- ypolyethylene glycols and magnetic
particles.
7. A controlled release pharmaceutical composition comprising; a)
cyclodextrin molecules selected from the group consisting of
cyclodextrin derivatives, oxidized cyclodextrins, cyclodextrin
dimers, cyclodextrin trimers, and cyclodextrin polymers complexed
with; b) nucleic acid, wherein; c) said cyclodextrin molecules are
covalently cross-linked through a biocleavable linkage to form a
polymer that has entrapped the active agent and wherein the
cross-linking provides the function of controlled release.
8. The composition of claim 7 wherein the biocleavable linkage is
selected from the group consisting of disulfide linkages, protected
disulfide linkages, ester bonds, aldehyde bonds, amide bonds,
polypeptide linkages and hydrazone linkages.
9. The composition of claim 7 further comprising a biorecognition
molecule coupled to the pharmaceutical composition.
10. The composition of claim 7 wherein said cyclodextrin dimers,
cyclodextrin trimers, and cyclodextrin polymers have been
derivatized to provide groups selected from the group consisting of
dialdehydes, sulfobutylethers, sulfopropylethers, hydroxyethyls,
hydroxypropyls, dihydroxy propyls, carboxylates and phosphates.
11. The composition of claim 7 wherein the nucleic add is selected
from the group consisting of DNA, RNA, sense and antisense
oligonucleotides; sense and antisense oligodeoxynucleotides; sense
and antisense oligonucleotides and oligodeoxynucleotides containing
phosphodiesters, phosphorothioates, phosphorodithioates,
phosphoroamidates, alkyl phosphotriesters, methylphosphonates,
sulfamates, 3'-thioformacetals, methylene(methylimino)s,
3'-N-carbamates, and morpholino carbamates; synthetic nucleic add
polymers, phosphoric acid ester nucleic acids and peptide nucleic
acids.
12. The composition of claim 7 wherein said cyclodextrin molecules
are coupled to an intermediate coupling substance selected from the
group consisting of serum albumins, glycoproteins, lipoproteins,
polysaccharides, lipopolysaccharides, amino polysaccharides,
polyacrylamides, lipids, glycolipids, N-(2-hydroxypropyl)
methacrylamides, poly cyanoacrylates, polyethylene glycols, poly
(D,L-lactic-coglycolic acids), dendrimers, poly
(D,L-lactide)-block-metho- xypolyethylene glycols and magnetic
particles.
13. A controlled release pharmaceutical composition comprising; a)
cyclodextrin molecules selected from the group consisting of
cyclodextrin derivatives, oxidized cyclodextrins, cyclodextrin
dimers, cyclodextrin trimers, and cyclodextrin polymers complexed
with; b) toxin, wherein; c) said cyclodextrin molecules are
covalently cross-linked through a biocleavable linkage to form a
polymer that has entrapped the active agent and wherein the
cross-linking provides the function of controlled release.
14. The composition of claim 13 wherein the biocleavable linkage is
selected from the group consisting of disulfide linkages, protected
disulfide linkages, ester bonds, aldehyde bonds, amide bonds,
polypeptide linkages and hydrazone linkages.
15. The composition of claim 13 further comprising a biorecognition
molecule coupled to the pharmaceutical composition.
16. The composition of claim 13 wherein said cyclodextrin dimers,
cyclodextrin trimers, and cyclodextrin polymers have been
derivatized to provide groups selected from the group consisting of
dialdehydes, sulfobutylethers, sulfopropylethers, hydroxyethyls,
hydroxypropyls, dihydroxy propyls, carboxylates and phosphates.
17. The composition of claim 13 wherein the active agent is
selected from the group consisting of aflatoxins, ricins,
bungarotoxins, irinotecan, pesticides, cevadines, desatrines,
veratridine and cevine derivatives.
18. The composition of claim 13 wherein said cyclodextrin molecules
are coupled to an intermediate coupling substance selected from the
group consisting of serum albumins glycoproteins, lipoproteins,
polysaccharides, lipopolysaccharides, amino polysaccharides,
polyacrylamides, lipids, glycolipids, N-(2-hydroxypropyl)
methacrylamides, poly cyanoacrylates, polyethylene glycols, poly
(D,L-lactic-coglycolic acids), dendrimers, poly
(D,L-lactide)-block-metho- xypolyethylene glycols and magnetic
particles.
19. A pharmaceutical amylose composition comprising; a) amylose
selected from the group consisting of amylose segments, amylose
derivatives, oxidized amylose and amylose polymers complexed with;
b) an active agent, wherein; c) said amylose is covalently
cross-inked to form a polymer that has entrapped the active
agent.
20. A biocleavable crosslinking agent comprising; a) a compound
containing a biocleavable linkage selected from the group
consisting of polypeptide linkages and hydrazone linkages wherein;
b) said compound has terminal reactive coupling groups selected
from the group consisting of N-succinimidyls, N-maleimidyls,
p-nitrophenyl esters, iodoacetals, bromoacetals, oxiranes and
imidoesters.
21. A method for producing a cyclodextrin pharmaceutical
composition comprising combining cyclodextrin molecules selected
from the group consisting of cyclodextrin derivatives, cyclodextrin
dimers, cyclodextrin trimers, and cyclodextrin polymers with; a)
guest molecules coupled to a surface to form an inclusion complex
between the cyclodextrin molecules and the guest molecules on the
surface, and; b) covalently cross-linking the cyclodextrin
molecules to form a polymer.
22. A method for producing a cyclodextrin pharmaceutical
composition using a solid support comprising coupling a first
cyclodextrin molecule selected from the group consisting of
cyclodextrin derivatives, cyclodextrin dimers, cyclodextrin
trimers, and cyclodextrin polymers to a solid support through a
cleavable coupling agent and; a) coupling in succession, additional
cyclodextrin molecules to the first cyclodextrin molecule that is
coupled to the solid support to form a polymer and; b) cleaving the
first cyclodextrin molecule from the solid support.
23. A pharmaceutical catalytic agent composition comprising; a)
cyclodextrin molecules selected from the group consisting of
oxidized cyclodextrins, cyclodextrin dimers, cyclodextrin trimers,
and cyclodextrin polymers coupled with; b) a catalytic group
selected from the group consisting of carboxylates, imidazoles,
histamines, hydroxyls, amines, amides, aldehydes, ketones,
phosphates, sulfhydryls, halogens, amino acids, nucleic acids,
chelators, and metals.
Description
RELATED PATENT APPLICATIONS
[0001] This is a continuation-in-part of PCT/US99/30820, filed Dec.
27, 1999, which is a continuation-in-part of U.S. patent
application Ser. No. 09/223,055, filed Dec. 30, 1998, now U.S. Pat.
No. 6,048,736, issued Apr. 11, 2000. The contents of those
applications are incorporated herein.
TECHNICAL FIELD OF THE INVENTION
[0002] This invention discloses methods for preparing compositions
of cyclodextrin polymers for carrying drugs and other active agents
for therapeutic, medical or other uses. Methods are also disclosed
for preparing cyclodextrin polymer carriers that release drugs and
other active agents under controlled conditions. The invention also
discloses methods for preparing compositions of cyclodextrin
polymer carriers that are coupled to biorecognition molecules for
targeting the delivery of drugs and other active agents to their
site of action.
DESCRIPTION OF THE PRIOR ART
[0003] The pharmacokinetics of many anti-viral and anti-cancer
drugs and other active agents that penetrate cells are not easily
controlled. Therefore, there is a need for carriers of drugs that
facilitate their solubility, delivery and effectiveness. When drugs
are bound to polymers of the prior art they can be taken up at the
cell surface and transferred to the nucleus. This permits
modulation of drug uptake through cell surface properties. Also,
drug release can be controlled using specific enzymes and other
conditions within the cell.
[0004] Drugs and other active agents delivered as macromolecules
through polymer carriers have gained acceptance as a way for
improving nucleic acid therapies. Also, the prior art now employs
drug-polypeptide complexes to re-direct drugs to selected target
cells. However, because cyclodextrin polymers of the prior art lack
any specific release mechanism, many of the advantages of
cyclodextrins are limited.
[0005] The prior art of cyclodextrins has disclosed their use in
labeling materials for in vitro testing (Kosak, PCT WO 91/05605,
1991), and in drug preparations (fitai, et al, U.S. Pat. No.
4,523,031 and 4,523,037).
[0006] The preparation and use of individual cyclodextrins
conjugated to biorecognition molecules as drug carriers is
disclosed by Weinshenker, U.S. Pat. No. 5,068,227; 1991, where each
coupling site is limited to one drug molecule. However, Weinshenker
makes no disclosures or suggestions for any cyclodextrin polymers
and they cannot be made with the synthesis methods disclosed.
[0007] Review articles on the pharmaceutical applications of
cyclodextrins have identified many problems due to the high
turnover rate between inclusion complex formation and dissociation.
Stella, V. J., et al., Pharmaceut. Res. 14, 556-567 (1997), report
that even with the strongest theoretical binding constants, as soon
as the complex of free cyclodextrin and drug is diluted in the
bloodstream, over 30% is calculated to dissociate. Also, Rajewski,
R., et al, J. Pharm. Sci. 85, 1142-1169 (1996), solubilized the
anti-cancer drug Taxol with cyclodextrins. They reported on page
1145 that "any attempt to dilute the samples resulted in erratic
precipitation" due to competitive displacement factors found in
plasma. Because of these problems, cyclodextrins in the prior art
are used for solubilizing and stabilizing certain drugs before or
during administration but are not suitable for carrying and
delivering drugs in the bloodstream.
[0008] The cyclodextrin polymer carriers of the instant invention
overcome these problems and provide the new function of controlled
release of drugs, which is not disclosed or suggested by the prior
art.
SUMMARY DISCLOSURE OF THE INVENTION
[0009] It has been discovered that the water-soluble (or colloidal)
cyclodextrin polymer carriers of the instant invention overcome the
problems with individual (monomeric) cyclodextrins in the prior
art. The instant invention provides new properties and unexpected
advantages. In its simplest form, a cyclodextrin polymer carrier
comprises a cyclodextrin polymer that has a nucleic acid or other
active agent completely entrapped within it.
[0010] In one embodiment, the water-soluble (or colloidal)
cyclodextrin polymers of the instant invention overcome the problem
of low carrying capacity of individual cyclodextrins. Also, by
complete entrapment of the guest molecules, the problem of losing
drug or other active agent by diffusion when diluted in vivo, is
solved. In another embodiment, the invention also provides a means
for controlled release of the entrapped drug in vivo, which was not
possible in the prior art of cyclodextrins.
[0011] In another embodiment, the invention also provides a means
for targeting the cyclodextrin polymer carrier by coupling it to a
biorecognition molecule.
[0012] The advantages of the water-soluble cyclodextrin polymer
carrier are:
[0013] (1) Drugs can be used that are designed for efficacy without
solubility or conjugation requirements.
[0014] (2) Drugs can be delivered as macromolecules and released
within the cell.
[0015] (3) Drugs can be targeted by coupling the carrier to
biorecognition molecules.
[0016] These are new advantages and functions provided for drug
carrier technology that will also be useful for other drug delivery
applications. These compositions and methods are unanticipated or
suggested in the prior art.
INDUSTRIAL APPLICABILITY
[0017] These CD polymer carriers can be used in many fields of
medicine to deliver therapeutic drugs and other active agents
through a variety of routes including orally, nasally and
parenterally. Other routes include various applications for
delivery through ocular membranes and mucosal membranes, including
the use of electric charge as in iontophoresis.
DESCRIPTION OF THE BEST MODES FOR CARRYING OUT THE INVENTION
[0018] For the purposes of disclosing this invention, certain
words, phrases and terms used herein are defined as follows:
[0019] Active Agents.
[0020] Active agents function as the preferred guest molecules of
the instant invention. Active agents that are preferred in the
instant invention are chemicals and other substances that can form
an inclusion complex with a cyclodextrin or cyclodextrin polymer
and are inhibitory, antimetabolic, therapeutic or preventive toward
any disease (i.e. cancer, syphilis, gonorrhea, influenza and heart
disease) or inhibitory or toxic toward any disease causing agent.
Preferred active agents are any therapeutic drugs categorized in
The Merck Index, Eleventh Ed., Merck & Co. Inc., Rahway N.J.
(1989) and those listed by Cserhati, T., Anal.Biochem. 225(2),
328-332 (1995).
[0021] Active agents include but are not limited to therapeutic
drugs that include prodrugs, anticancer drugs, antineoplastic
drugs, antifungal drugs, antibacterial drugs, antiviral drugs,
cardiac drugs, neurological drugs, and drugs of abuse; alkaloids
(i.e. camptothecins), antibiotics, bioactive peptides, steroids,
steroid hormones, polypeptide hormones, interferons, interleukins,
narcotics, nucleic acids including antisense oligonucleotides,
pesticides and prostaglandins.
[0022] Active agents also include any toxins including aflatoxins,
ricins, bungarotoxins, iotecan, ganciclovir, furosemide,
indomethacin, chlorpromazine, methotrexate, cevine derivatives and
analogs including cevadines, desatrines, and veratridine, among
others. Also included but are not limited to, are;
[0023] various flavone derivatives and analogs including
dihydroxyflavones (chrysins), trihydroxyflavones (apigenins),
pentahydroxyflavones (morins), hexahydroxyflavones (myricetins),
flavyliums, quercetins, fisetins;
[0024] various antibiotics including derivatives and analogs such
as penicillin derivatives (i.e. ampicillin), anthracyclines (i.e.
doxorubicin, daunorubicin, mitoxantrone), butoconazole,
camptothecin, chalcomycin, chartreusin, chrysomicins (V and M),
chloramphenicol, chlorotetracyclines, clomocyclines, cyclosporins,
ellipticines, filipins, fungichromins, griseofulvin, griseoviridin,
guamecyclines, macrolides (i.e. amphotericins, chlorothricin),
methicillins, nystatins, chrymutasins, elsamicin, gilvocarin,
ravidomycin, lankacidin-group antibiotics (i.e. lankamycin),
mitomycin, teramycins, tetracyclines;
[0025] various anti-microbials including reserpine, spironolactone,
sulfacetarnide sodium, sulphonamide, thiamphenicols,
thiolutins;
[0026] various purine and pyrmidine derivatives and analogs
including 5'-fluorouracil, 5'-fluoro-2'-deoxyuridine, and
allopurinol;
[0027] various photosensitizer substances, especially those used
for singlet and triplet oxygen formation useful for photodynamic
therapy (van Lier, J. E. In "Photodynamic Therapy of Neoplastic
Disease"; Kessel D., Ed., CRC Press, Boca Raton, Fla., 1990, Vol.
I), including meso-chlorin e.sub.6 monoethylenediamine (Mce.sub.6),
phytalocyanine, porphyrins and their derivatives and analogs;
[0028] various steroidal compounds such as cortisones, estradiols,
hydrocortisone, testosterones, prednisolones, progesterones,
dexamethasones, beclomethasones and other methasone derivatives,
other steroid derivatives and analogs including cholesterols,
digitoxins, digoxins, digoxigenins;
[0029] various coumarin derivatives and analogs including
dihydroxycoumarins (esculetins), dicumarols, chrysarobins,
chrysophanic acids, emodins, secalonic acids;
[0030] various dopas, derivatives and analogs including dopas,
dopamines, epinephrines, and norepinephrines (arterenols);
[0031] various antineoplastic agents or cell growth inhibitors such
as cisplatins and taxanes including paclitaxel and docetaxel;
[0032] various barbiturates including phenobarbitone, amobarbital,
allobarbital, pentobarbital and other barbital derivatives;
[0033] various benzene derivatives including amino-benzoic acid,
bromobenzoic acid, benzocaine, benzodiazepines, benzothiazide,
butyl-p-aminobenzoate;
[0034] various polypeptide derivatives;
[0035] various carboxylic acid derivatives such as
bromoisovalerylurea, phenyl-butyric acid, phenyl-valeric acid;
[0036] Other active agents include, but are not limited to,
diphenyl hydantoin, adiphenine, anethole, aspirin, azopropazone,
bencyclane, chloralhydrate, chlorambucil, chlorpromazine,
chlorogenin, cinnamic acid, clofibrate, coenzyme A, cyclohexyl
anthranilate, diazepam, flufenamic acid, fluocinolone acetonide,
flurbiprofen, guaiazulene, ibuprofen, indican, indomethacin,
iodine, ketoprofen, mefanamic acid, menadione, metronidazole,
nitrazepam, phenytoin, propylparaben, proscillaridin, quinolone,
thalidomide, thiamine dilaurylsulphate, thiopental, triamcinolone,
vitamins A, D3, E, K3, and warfarin.
[0037] Other specific active agents are anti-viral drugs, nucleic
acids and other anti-viral substances including those against any
DNA and RNA viruses, AIDS, HIV and hepatitis viruses, adenoviruses,
alphaviruses, arenaviruses, coronaviruses, flaviviruses,
herpesviruses, myxoviruses, oncornaviruses, papovaviruses,
paramyxoviruses, parvoviruses, picornaviruses, poxviruses,
reoviruses, rhabdoviruses, rhinoviruses, togaviruses and viriods;
any anti-bacterial drugs, nucleic acids and other anti-bacterial
substances including those against gram-negative and gram-positive
bacteria, acinetobacter, achromobacter, bacteroides, clostridium,
chlamydia, enterobacteria, haemophilus, lactobacillus, neisseria,
staphyloccus, and streptoccocus; any anti-fungal drugs, nucleic
acids and other anti-fungal substances including those against
aspergillus, candida, coccidiodes, mycoses, phycomycetes, and
yeasts; any drugs, nucleic acids and other substances against
mycoplasma and rickettsia; any anti-protozoan drugs, nucleic acids
and other substances; any anti-parasitic drugs, nucleic acids and
other substances; any drugs, nucleic acids and other substances
against heart diseases, tumors, and virus infected cells, among
others.
[0038] Nucleic Acid Active Agents.
[0039] For the purposes of this invention, certain nucleic acids
are preferred as a specific class of active agents directed against
viral and other microbial diseases, against cancers, autoimmune and
genetic diseases. Specific nucleic acid active agents include any
anti-bacterial, anti-cancer, anti-fungal, anti-viral,
anti-parasitic and anti-protozoan nucleic acids. They also include
specific DNA sequences used for gene therapy.
[0040] Nucleic add active agents include all types of RNA
(including messenger RNA), all types of DNA, and oligonucleotides
including probes and primers used in the polymerase chain reaction
(PCR), hybridizations or DNA sequencing. Also preferred are
phosphodiester sense or antisense oligonudeotides, sense or
antisense oligodeoxynucleotides (ODN) and any sense or antisense
oligonucleotides or oligodeoxynucleotides where the sugar-phosphate
"backbone" has been derivatized or replaced with "backbone
analogues" or linkages such as with phosphorothioates,
phosphorodithioates, phosphoroamidates, alkyl phosphotriesters, or
methylphosphonates. Also preferred are any sense or antisense
oligonudeotides or oligodeoxynucleotides with non-phosphorous
backbone analogues or linkages such as sulfamates,
3'-thioformacetals, methylene(methylimino), 3'-N-carbamates, or
morpholino carbamates.
[0041] Some preferred examples of synthetic oligonucleotides and
ODNs are disclosed by J. F. Milligan, et al., J. Medicinal Chem.
36(14): 1923-1937 (1993) and Y. Shoji et al., Antimicrob. Agents
Chemotherapy, 40(7):1670-1675 (1996). Also included are synthetic
nucleic acid polymers and peptide nucleic acids (PNA) disclosed by
Egholm, et al, Nature 365:566-568(1993) and references therein,
including PNA damps (Nucleic Acids Res. 23:217(1995)). Also
included are nucleotide mimics or co-oligomers like phosphoric add
ester nucleic adds (PHONA), disclosed by Peyman, et al., Angew.
Chem. Int. Ed. Engl. 36:2809-2812 (1997). Also included are DNA
and/or RNA fragments, and derivatives from any tissue, cells,
nuclei, chromosomes, cytoplasm, mitochondria, ribosomes, and other
cellular sources.
[0042] Biocleavable Linkage or Bond.
[0043] For the instant invention, biocleavable linkages are defined
as types of specific chemical moieties or groups used within the
chemical substances that covalently or non-covalently couple and
cross-link the cyclodextrin polymer carriers. They are contained in
certain embodiments of the instant invention that provide the
function of controlled release of an entrapped drug or other active
agent. Biocleavable linkages or bonds are defined here under
distinct categories or types.
[0044] One category comprises the disulfide linkages and ester
bonds that are well known for covalently coupling drugs to
polymers. For drug delivery, they may be more useful for shorter
periods in vivo since they are cleaved in the bloodstream
relatively easily. Ester bonds include those between any acid and
alcohol, and imidoesters formed from alkyl imidates.
[0045] Another category comprises linkages or bonds that are more
specifically cleaved after entering the cell (intracellular
cleavage). The preferred linkages for release of drugs within the
cell are cleavable in acidic conditions like those found in
lysosomes. One example is an acid-sensitive (or acid-labile)
hydrazone linkage as described by Greenfield, et al, Cancer Res.
50, 6600-6607 (1990), and references therein. Also preferred are
certain natural or synthetic polypeptide linkages that contain
certain amino acid sequences (i.e. usually hydrophobic) that are
cleaved by specific enzymes such as cathepsins, found primarily
inside the cell. Using the convention of starting with the amino or
"N" terminus on the left and the carboxyl or "C" terminus on the
right, some examples are: any polypeptide linkages that contain the
sequence Phe-Leu, Leu-Phe or Phe-Phe, such as Gly-Phe-Leu,
Gly-Leu-Phe, Gly-Phe-Leu-Gly, Gly-Phe-Leu-Phe-Gly and
Gly-Phe-Phe-Gly, and others that have either of the Gly residues
substituted for one or more other peptides. Other linkage sequences
included are leucine enkephalin derivatives such as
Tyr-Gly-Gly-Phe-Leu, and the like.
[0046] Another preferred type of biocleavable linkage is any
"hindered" or "protected" disulfide bond that sterically inhibits
attack from thiolate ions. Examples of such protected disulfide
bonds are found in the coupling agents:
S-4-succinnimdyl-oxycarbonyl-.o slashed.-methyl benzyl thiosulfate
(SMBT) and 4succninidyloxycarbonyl-.o slashed.-methyl-.o
slashed.-(2-pyridyldithio) toluene (SMPT). Another useful coupling
agent resistant to reduction is SPDB disclosed by Worrell, et al.,
Anticancer Drug Design 1:179-188 (1986). Also included are certain
aryldithio thioimidates, substituted with a methyl or phenyl group
adjacent to the disulfide, which include ethyl S-acetyl
3-mercaptobutyrothioimidate (M-AMPT)and
3-(4-carboxyamidophenyldithio) proprionthioimidate (CDPT),
disclosed by S. Arpicco, et al., Bioconj. Chem. 8 (3):327-337
(1997). Another preferred category is certain linkages or bonds
subject to hydrolysis that include various aldehyde bonds with
amino or sulfhydryl groups. Also included are amide bonds such as
when N-hydroxysuccinimide ester (NHS ester) reacts with amines.
[0047] Biorecognition Molecules.
[0048] For the purposes of this invention, biorecognition molecules
are those that bind to a specific biological substance or site. The
biological substance or site is considered the "target" of the
biorecognition molecule that binds to it. In the prior art, many
drugs are "targeted" by coupling them to a biorecognition molecule
that has a specific binding affinity for the cells, tissue or
organism that the drug is intended for. For targeting a drug or
other active agent in this invention, a biorecognition molecule is
coupled to a cyclodextrin polymer carrier that is used to entrap
the drug. Examples of biorecognition molecules are described
below.
[0049] Ligand.
[0050] A ligand functions as a type of biorecognition molecule
defined as a selectively bindable material that has a selective (or
specific), affinity for another substance. The ligand is recognized
and bound by a usually, but not necessarily, larger specific
binding body or "binding partner", or "receptor". Examples of
ligands suitable for targeting are antigens, haptens, biotin,
biotin derivatives, lectins, galactosamine and fucosylamine
moieties, receptors, substrates, coenzymes and cofactors among
others.
[0051] When applied to the cyclodextrin polymers of this invention,
a ligand includes an antigen or hapten that is capable of being
bound by, or to, its corresponding antibody or fraction thereof.
Also included are viral antigens or hemagglutinins and
neuraminidases and nucleocapsids including those from any DNA and
RNA viruses, AIDS, HIV and hepatitis viruses, adenoviruses,
alphaviruses, arenaviruses, coronaviruses, flaviviruses,
herpesviruses, myxoviruses, oncornaviruses, papovaviruses,
paramyxoviruses, parvoviruses, picornaviruses, poxviruses,
reoviruses, rhabdoviruses, rhinoviruses, togaviruses and viroids;
any bacterial antigens including those of gram-negative and
gram-positive bacteria, acinetobacter, achromobacter, bacteroides,
clostridium, chlamydia, enterobacteria, haemophilus, lactobacillus,
neisseria, staphyloccus, and streptoccocus; any fungal antigens
including those of aspergillus, candida, coccidiodes, mycoses,
phycomycetes, and yeasts; any mycoplasma antigens; any rickettsial
antigens; any protozoan antigens; any parasite antigens; any human
antigens including those of blood cells, virus infected cells,
genetic markers, heart diseases, oncoproteins, plasma proteins,
complement factors, rheumatoid factors. Included are cancer and
tumor antigens such as alpha-fetoproteins, prostate specific
antigen (PSA) and CEA, cancer markers and oncoproteins, among
others.
[0052] Other substances that can function as ligands for
biorecognition are certain vitamins (i.e. folic acid, B.sub.12),
steroids, prostaglandins, carbohydrates, lipids, antibiotics,
drugs, digoxins, pesticides, narcotics, neuro-transmitters, and
substances used or modified such that they function as ligands.
Most preferred are certain proteins or protein fragments (i.e.
hormones, toxins), and synthetic or natural polypeptides with cell
affinity. Ligands also include various substances with selective
affinity for ligators that are produced through recombinant DNA,
genetic and molecular engineering. Except when stated otherwise,
ligands of the instant invention also include the ligands as
defined by K. E. Rubenstein, et al, U.S. Pat. No. 3,817,837
(1974).
[0053] Ligator.
[0054] A ligator functions as a type of biorecognition molecule
defined for this invention as a specific binding body or "partner"
or "receptor", that is usually, but not necessarily, larger than
the ligand it can bind to. For the purposes of this invention, it
is a specific substance or material or chemical or "reactant" that
is capable of selective affinity binding with a specific ligand. A
ligator can be a protein such as an antibody, a nonprotein binding
body or a "specific reactor."
[0055] When applied to this invention, a ligator includes an
antibody, which is defined to include all classes of antibodies,
monoclonal antibodies, chimeric antibodies, Fab fractions,
fragments and derivatives thereof. Under certain conditions, the
instant invention is also applicable to using other substances as
ligators. For instance, other ligators suitable for targeting
include naturally occurring receptors, any hemagglutinins and cell
membrane and nuclear derivatives that bind specifically to
hormones, vitamins, drugs, antibiotics, cancer markers, genetic
markers, viruses, and histocompatibility markers. Another group of
ligators includes any RNA and DNA binding substances such as
polyethylenimine (PEI) and polypeptides or proteins such as
histones and protamines.
[0056] Other ligators also include enzymes, especially cell surface
enzymes such as neuraminidases, plasma proteins, avidins,
streptavidins, chalones, cavitands, thyroglobulin, intrinsic
factor, globulins, chelators, surfactants, organometallic
substances, staphylococcal protein A, protein G, ribosomes,
bacteriophages, cytochromes, lectins, certain resins, and organic
polymers.
[0057] Preferred biorecognition molecules also include various
substances such as any proteins, protein fragments or polypeptides
with affinity for the surface of any cells, tissues or
microorganisms that are produced through recombinant DNA, genetic
and molecular engineering. For instance, any suitable membrane
transfer proteins such as TAT, from HIV virus.
[0058] Nucleic Acid Biorecognition Molecules.
[0059] For the purposes of this invention, certain nucleic acids
can function as biorecognition molecules. A nucleic acid
biorecognition molecule is defined as any nucleic acid sequence
from any source that is coupled to the cyclodextrin polymer carrier
for targeting a specific type of microbe, cell or tissue.
[0060] Preferred nucleic acid biorecognition molecules are
sequences that "recognize" or hybridize with a disease-specific
nucleic acid sequence (i.e. mRNA or DNA) found within a target
cell, as described by Z. Ma, et al., PNAS 97, 11159-11163 (2000). A
CD carrier containing a suitable active agent or, a CD catalytic
agent of this invention, would be coupled to a suitable nucleic
acid that recognized a disease-specific sequence in a cell.
[0061] Nucleic acid biorecognition molecules include all types of
RNA, all types of DNA, and oligonucleotides including probes and
primers used in the polymerase chain reaction (PCR), or DNA
sequencing. Also included are synthetic nucleic acid polymers and
peptide nucleic acids (PNA) disclosed by Egholm, et al, Nature
365:566-568(1993) and references therein, including PNA clamps
(Nucleic Acids Res. 23:217(1995)). Also included are DNA and/or RNA
fragments, and derivatives from any tissue, cells, nuclei,
chromosomes, cytoplasm, mitochondria, ribosomes, and other cellular
sources.
[0062] Cyclodextrin.
[0063] A cyclodextrin (CD), is an oligosaccharide composed of
glucose monomers coupled together to form a conical hollow molecule
with a hydrophobic interior or cavity. The cyclodextrins of the
instant invention can be any suitable cyclodextrin, including
alpha-, beta-, and gamma-cyclodextrins, and their combinations,
analogs, isomers, and derivatives. They function as components in
the synthesis of the cyclodextrin polymer carriers of the instant
invention.
[0064] In describing this invention, references to a cyclodextrin
"complex", means a noncovalent inclusion complex. An inclusion
complex is defined herein as a cyclodextrin functioning as a "host"
molecule, combined with one or more "guest" molecules that are
contained or bound, wholly or partially, within the hydrophobic
cavity of the cyclodextrin or its derivative.
[0065] Most preferred are cyclodextrin dimers, trimers and polymers
containing cyclodextrin derivatives such as carboxymethyl CD,
glucosyl CD, maltosyl CD, hydroxypropyl cyclodextrins (HPCD),
2-hydroxypropyl cyclodextrins, 2,3-dihydroxypropyl cyclodextrins
(DHPCD), sulfobutylether cyclodextrins (SBECD), ethylated and
methylated cyclodextrins.
[0066] Also preferred are oxidized cyclodextrins that provide
aldehydes and any oxidized forms of any cyclodextrn polymers or
derivatives that provide aldehydes. Some examples of suitable
derivatives are disclosed by Pitha, J., et al, J. Pharm. Sci. 75,
165-167 (1986) and Pitha, J., et al, Int. J. Pharmaceut. 29, 73-82
(1986).
[0067] Also preferred are any amphiphilic CD derivatives such as
those disclosed by Y. Kawabata, et al., Chem. Lett. p1933 (1986), K
Chmurski, et al., Langmuir 12, 4046 (1996), P. Zhang, et al., Tetr.
Lett. 32, No.24, 2769 (1991), P. Zhang, et al., J. Phys. Org. Chem.
5, 518 (1992), M. Tanaka, et al., Chem. Lett. p1307 (1987), S.
Taneva, et aL, Langmuir 5, 111 (1989), M. Weisser, et al., J. Phys.
Chem. 100, 17893 (1996), L. A. Godinez, et al., Langmuir 14, 137
(1998) and D. Duchene, "International Pharmaceut. Applic. of
Cyclodextrins Conference", Lawrence, Kans., USA, June 1997.
[0068] Also included are altered forms, such as crown ether-like
compounds prepared by Kandra, L., et al, J. Inclus. Phenom. 2,
869-875 (1984), and higher homologues of cyclodextrins, such as
those prepared by PulLey, et al, Biochem. Biophys. Res. Comm. 5, 11
(1961). Some useful reviews on cyclodextrins are: Atwood J. E. D.,
et al, Eds., "Inclusion Compounds", vols. 2 & 3, Academic
Press, NY (1984); Bender, M. L., et al, "Cyclodextnin Chemistry",
Springer-Verlag, Berlin, (1978) and Szejtli, J., "Cyclodextrins and
Their Inclusion Complexes", Akademiai Kiado, Budapest, Hungary
(1982). These references, including references contained therein,
are applicable to the synthesis of the preparations and components
of the instant invention and are hereby incorporated herein by
reference.
[0069] Cyclodextrin Dimers, Trimers and Polymers.
[0070] For this invention, individual cyclodextrin (CD-monomer)
derivatives function as the primary building structures, or
components, or units used to synthesize the water-soluble (or
colloidal) cyclodextrin polymer carriers. Also, certain preferred
CD dimers, and trimers of this invention are not used as building
units for polymers and can function as drug carriers or excipients
without additional crosslinking.
[0071] A cyclodextrin dimer is defined as two cyclodextrin
molecules covalently coupled or cross-linked together to enable
cooperative complexing with a guest molecule. Examples of some CD
dimers that can be derivatized and used in the drug carriers of
this invention, are described by; Breslow, R., et al, Amer. Chem.
Soc. 111, 8296-8297 (1989); Breslow, R., et a, Amer. Chem. Soc.
105, 1390 (1983) and Fujita, K., et al, J. Chem. Soc., Chem.
Commun., 1277 (1984).
[0072] A cyclodextrin trimer is defined as three cyclodextrin
molecules covalently coupled or cross-linked together to enable
cooperative complexing with a guest molecule. A cyclodextrin
polymer is defined as a unit of more than three cyclodextrin
molecules covalently coupled or cross-linked together to enable
cooperative complexing with several guest molecules.
[0073] A CD-block is defined as a CD dimer, trimer or polymer that
is used as the primary component, or unit (i.e. building block) for
additional crosslinking with other CD dimers, trimers or polymers
to synthesize a CD polymer carrier. Generally this involves at
least two crosslinking steps, where first the CD-blocks are
prepared by crosslinking CD monomers and derivatized or activated
for subsequent coupling. Then the CD-blocks are crosslinked in a
second reaction to entrap the active agent in the final CD carrier
composition. An example of this method is given below.
[0074] For this invention, preferred cyclodextrin dimer, trimer and
polymer units or blocks are synthesized by covalently coupling
through chemical groups such as through coupling agents generally
not to exceed 50 angstroms in spacer length. The synthesis of
preferred cyclodextrin dimer, trimer and polymer units or CD blocks
does not include the use of proteins or other "intermediate
coupling substances" (defined below), which can be incorporated
during final synthesis of the cyclodextrin polymer carrier.
Cooperative complexing means that in situations where the guest
molecule is large enough, the member cyclodextrins of the CD dimer,
trimer or polymer can each noncovalently complex with different
parts of the same guest molecule, or with smaller guests,
alternately complex with the same guest.
[0075] The prior art has disclosed dimers and polymers comprised of
cyclodextrins of the same size. An improved cyclodextrin dimer,
trimer or polymer comprises combinations of different sized
cyclodextrins to synthesize these units. These combinations may
more effectively complex with guest molecules that have
heterogeneous complexing sites. Combinations for this invention can
include the covalent coupling of an alpha CD with a beta CD, an
alpha CD with a gamma CD, a beta CD with a gamma CD and polymers
with various ratios of alpha, beta and gamma cyclodextrins.
[0076] Cyclodextrin Polymer Carrier.
[0077] A water-soluble (or colloidal) cyclodextrin polymer carrier
is a new composition provided by the instant invention. It is
defined herein as a polymer of cross-linked cyclodextrin
derivatives that has the distinguishing property of having
incorporated a drug or other active agent as a "captured guest".
The "capture" of the guest stabilizes the carrier complex and
overcomes the problem in the prior art of the CD host and guest
molecules separating by diffusion. Generally, the agent has also
formed a noncovalent "inclusion complex", or "inclusion compound"
with the cyclodextrins of the polymer.
[0078] Self-Assembled Cyclodextrin Polymer Carrier.
[0079] A self-assembled, or self-coupled or auto-assembled
cyclodextrin polymer carrier is a new composition provided by the
instant invention. It is defined herein as a water-soluble (or
colloidal) polymer of cross-linked cyclodextrin derivatives that
has the distinguishing property of having incorporated a drug or
other active agent as a "captured guest". The "capture" of the
guest also includes stabilizing the carrier complex and overcomes
the problem in the prior art of the CD host and guest molecules
separating by diffusion.
[0080] Captured Guest Cyclodextrin Polymer Carrier.
[0081] In a preferred embodiment the capturing is accomplished
through complete physical entrapment by the water-soluble (or
colloidal) CD polymer carrier. In this embodiment, "completely
entrapped" means that a captured guest is not covalently coupled to
the polymer but is physically entrapped by the covalently
cross-linked polymer of cyclodextrin molecules so that no
significant amount of active agent can leave the polymer by
diffusion or extraction. Completely entrapped smaller guest
molecules such as drugs and ligands are suitably "non-diffusable",
by being entrapped wholly within the polymer.
[0082] Completely entrapped larger guests such as proteins,
polypeptides, and nucleic acids (DNA, RNA, oligonucleotides) are
suitably non-diffusable by being entrapped wholly or partially so
that the guest and polymer still cannot separate by diffusion.
Completely entrapped guests cannot escape until the polymer itself
has been degraded or the covalent cross-link bonds are cleaved. In
this embodiment, essentially all possible exit routes for the guest
to leave the polymer have been closed by cross-linking. Therefore,
additional guest molecules (of that size or larger) cannot enter
the dosed polymer to be added to the cyclodextrin polymer
carrier.
[0083] This is made possible through the unique method for
synthesizing the cyclodextrin polymer carriers of the instant
invention. The distinguishing principal of the method is that the
guest molecules are completely entrapped during polymerization or
during the final cross-linking step of making the polymer carrier.
Initially, guest molecules are mixed with the "open" components of
the cyclodextrin polymer, which may comprise individual
cyclodextrins (or derivatives), cyclodextrin dimers, trimers or an
open cyclodextrin polymer. An open cyclodextrin polymer means that
the polymer is only partially cross-linked so that guest molecules
can enter or associate with the polymer by diffusion and form
complexes with member cyclodextrins. In the final synthesis step of
the polymer carrier, the polymer is closed by additional covalent
cross-linking which completely entraps the guests as defined
previously.
[0084] Controlled Release.
[0085] For this invention, controlled release is defined as the
release of a captured guest from the CD polymer carrier only by
cleavage of certain covalent linkages that were used to synthesize
the carrier. This definition specifically excludes release by
diffusion until said linkages are cleaved.
[0086] Targeted Cyclodextrin Polymer Carriers.
[0087] A targeted cyclodextrin (CD) polymer carrier is an
embodiment of this invention composed of a water-soluble (or
colloidal) cyclodextrin polymer carrier, or derivative described
herein, that has a biorecognition molecule covalently coupled to
its surface. However, the biorecognition molecule is not an
inclusion complex within the cyclodextrin carrier. The carrier is
thereby targeted through the specific binding properties of the
biorecognition molecule coupled to the surface.
[0088] During the coupling, the functions of the biorecognition
molecule and the targeted CD polymer carrier are not irreversibly
or adversely inhibited. Preferably, the biorecognition molecule
maintains specific binding properties that are functionally
identical or homologous to those it had before coupling.
Preferably, the biorecognition molecule is coupled through a
suitable spacer to avoid steric hindrance.
[0089] Targeted cyclodextrin polymer carriers coupled to avidin and
streptavidin are useful for subsequent noncovalent coupling to any
suitable biotinylated substance. Similarly, cyclodextrin polymer
carriers coupled to antibody can be noncovalently coupled to
another antibody, or to a nucleic acid or other suitable substance
that has the appropriate biorecognition properties. Another useful
cyclodextrin carrier comprises protein A, protein G, or any
suitable lectin or polypeptide that has been covalently coupled to
a cyclodextrin polymer carrier.
[0090] Biocleavable Cross-Linking Agent.
[0091] A biocleavable cross-linking agent comprises a new
composition for facilitating the synthesis of drug carriers with
controlled release. In one embodiment it is comprised of a
biocleavable sequence of amino acids between suitable compounds
that comprise or contain amino-reactive or thiol-reactive coupling
groups at each end for direct coupling to amino or sulfhydryl
groups on an active agent or polymer. Using the convention of
starting with the amino or "N" terminus on the left and the
carboxyl or "C" terminus on the right, some examples are: any
polypeptide compounds that contain the sequence Phe-Leu, Leu-Phe or
Phe-Phe, such as Gly-Phe-Leu, Gly-Leu-Phe, Gly-Phe-Leu-Gly,
Gly-Phe-Leu-Phe-Gly and Gly-Phe-Phe-Gly, Gly-Phe-Leu-Gly-Lys,
Lys-Gly-Phe-Leu-Gly-Lys and others that have either of the Gly
residues substituted for one or more other peptides. Also included
are leucine enkephalin derivatives such as Tyr-Gly-Gly-Phe-Leu. A
preferred embodiment comprises a polypeptide with a biocleavable
sequence as described, and also includes terminal compounds with
coupling groups such as N-succinimidyl N-maleimidyl, iodoacetal,
bromoacetal oxirane or imidoester coupling groups on each end.
[0092] For acid-labile biocleavable cross-linking agents, one
embodiment comprises a bifunctional coupling agent with a hydrazone
linkage incorporated into it. For instance, it would comprise a
hydrazone linkage between suitable compounds comprising aliphatic
chains or aromatic groups that have terminal N-succinimidyl,
N-maleimidyl, p-nitrophenyl ester (ONp), iodoacetal, bromoacetal
oxirane or imidoester coupling groups on each end. One example for
synthesizing an acid-labile biocleavable coupling agent is to first
react an excess of hydrazinobenzoic acid with glutaraldehyde to
couple one hydrazinobenzoic acid at each end of the dialdehyde.
This produces hydrazone linkages with terminal carboxyl groups at
each end. The terminal carboxyl groups are then converted to
N-succinimidyl ester groups.
[0093] Coupling.
[0094] For the instant invention, two distinct types of coupling
are defined. One type of coupling can be through noncovalent,
"attractive" binding as with a guest molecule and cyclodextrin,
antigen and antibody or biotin and avidin. Noncovalent coupling is
binding between substances through ionic or hydrogen bonding or van
der waals forces, and/or their hydrophobic or hydrophilic
properties.
[0095] Unless stated otherwise, the preferred coupling used in the
instant invention is through covalent, electron-pair bonds or
linkages. Many methods and agents for covalently coupling (or
crosslinking) cyclodextrins and cyclodextrin derivatives are known
and, with appropriate modification, can be used to couple the
desired substances through their "functional groups" for use in
this invention.
[0096] Where stability is desired, the preferred linkages are amide
bonds, peptide bonds, ether bonds, and thio ether bonds, among
others.
[0097] Functional Group.
[0098] A functional group is defined here as a potentially reactive
site on a substance where one or more atoms are available for
covalent coupling to some other substance. When needed, functional
groups can be added to various substances through derivatization or
substitution reactions.
[0099] Examples of functional groups are aldehydes, allyls, amines,
amides, azides, carboxyls, carbonyls, epoxys (oxiranes), ethynyls,
hydroxyls, ketones, certain metals, nitrenes, phosphates,
propargyls, sulfhydryls, sulfonyls, phenolic hydroxyls, indoles,
bromines, chlorines, iodines, and others. The prior art has shown
that most, if not all of these functional groups can be
incorporated into or added to cyclodextrins, biorecognition
molecules, drugs, nucleic acids and support materials.
[0100] Cross-Linking or Coupling Agent.
[0101] A coupling agent (or cross-linking agent), is defined as a
chemical substance that produces and/or reacts with functional
groups on a substance to produce covalent coupling, cross-linking,
or conjugation with that substance. Because of the stability of
covalent coupling, this is the preferred method. Depending on the
chemical makeup or functional group on the cyclodextrin, nucleic
acid, or biorecognition molecule, the appropriate coupling agent is
used to provide the necessary active functional group or to react
with the functional group. In certain preparations of the instant
invention, coupling agents are needed that provide a spacer between
cross-linked cyclodextrins or between cyclodextrin and a
biorecognition molecule to overcome steric hindrance. Preferably,
the spacer is a substance of 4 or more carbon atoms in length and
can include aliphatic, aromatic and heterocyclic structures.
[0102] With appropriate modifications by one skilled in the art,
the coupling methods referenced in U.S. Pat. No. 6,048,736 and
PCr/US99/30820, including references contained therein, are
applicable to the synthesis of the preparations and components of
the instant invention and are hereby incorporated by reference,
herein:
[0103] Examples of energy activated coupling or cross-linking
agents are ultraviolet (UV), visible and radioactive radiation that
can promote coupling or crosslinking of suitably derivatized
cyclodextrins. Examples are photochemical coupling agents disclosed
in U.S. Pat. No. 4,737,454, among others. Also useful in
synthesizing components of the instant invention are enzymes that
produce covalent coupling such as nucleic acid polymerases and
ligases, among others.
[0104] In some preferred aspects of this invention, cyclodextrin
dimers, trimers and polymers are first prepared for use as the
primary components, or CD-blocks to synthesize the cyclodextrin
polymer carriers. Useful derivatizing and/or coupling agents for
preparing CD-blocks are bifunctional, trifunctional or
polyfunctional crosslinking agents that will covalently couple to
the hydroxyl groups of cyclodextrin. Some preferred examples are
oxiranes such as epichlorohydrin, epoxides such as 1,4 butanediol
diglycidyl ether (BDE), glycerol diglycidyl ether (GDE),
trimethylolpropane triglycidyl ether (TMTE), glycerol propoxylate
triglycidyl ether (GPNI), 1,3-butadiene diepoxide,
triphenylolmethane triglycidyl ether, 4,4'-methylenebis
(N,N-diglycidylaniline), tetraphenylolethane glycidyl ether,
bisphenol A diglycidyl ether, bisphenol A propoxylate diglycidyl
ether, bisphenol F diglycidyl ether, cyclohexanedimethanol
diglycidyl ether, 2,2'-oxybis (6-oxabicyclo[3.1.0] hexane),
polyoxyethylene bis(glycidyl ether), resorcinol diglycidyl ether,
ethylene glycol diglycidyl ether (EGDE) and low molecular weight
forms of poly(ethylene glycol) diglycidyl ethers or polypropylene
glycol) diglycidyl ethers, among others.
[0105] Other preferred derivatizing and/or coupling agents for
hydroxyl groups are various disulfonyl compounds such as
benzene-1,3-disulfonyl chloride and 4,4'-biphenyl disulfonyl
chloride and also divinyl sulfone, among others.
[0106] Most preferred coupling agents are also chemical substances
that can provide the bio-compatible linkages for synthesizing the
cyclodextrin polymer carriers of the instant invention. Covalent
coupling or conjugation can be done through functional groups using
coupling agents such as glutaraldehyde, formaldehyde, cyanogen
bromide, azides, p-benzoquinone, maleic or succinic anhydrides,
carbodiimides, epichlorohydrin, ethyl chloroformate, dipyridyl
disulfide and polyaldehydes.
[0107] Also most preferred are derivatizing and/or coupling agents
that couple to thiol groups ("thiol-reactive") such as agents with
any maleimide, vinylsulfonyl, bromoacetal or iodoacetal groups,
including any bifunctional or polyfunctional forms. Examples are
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),
succinimidyl-4-(N-maleimidomethyl)cydohexane-1-carboxylate (SMCC),
succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB),
dithiobis-N-ethylmaleimide (DTEM), 1,1'-(methylenedi-4,1-phenylene)
bismaleimide (MPBM), o-phenylenebismaleimide, N-succinimidyl
iodoacetate (SIA), N-succinimidyl-(4-vinylsulfonyl) benzoate
(SVSB), and Tris-(2-maleimidoethyl) amine (TMEA) among others.
[0108] Other coupling groups or agents useful in the instant
invention are: p-nitrophenyl ester (ONp), bifunctional imidoesters
such as dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP),
dimethyl suberimidate (DMS), methyl 4-mercaptobutyrimidate,
dimethyl 3,3'-dithiobis-propionimid- ate (DTBP), and
2-iminothiolane (Traut's reagent);
[0109] bifunctional NHS esters such as disuccinimidyl suberate
(DSS), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES),
disuccinimidyl (N,N'-diacetylhomocystein) (DSAH), disuccinimidyl
tartrate (DST), dithiobis(succinimidyl propionate) (DSP), and
ethylene glycol bis(succinimidyl succinate) (EGS), including
various derivatives such as their sulfo-forms;
[0110] heterobifunctional reagents such as
N-5-azido-2-nitrobenzoyloxysucc- inimide (ANB-NOS), p-azidophenacyl
bromide, p-azidophenylglyoxal, 4-fluoro-3-nitrophenyl azide (FNPA),
N-hydroxysuccinimidyl-4azidobenzoate (HSAB),
methyl-4-azidobenzoimidate (MABI), p-nitrophenyl
2-diazo-3,3,3-trifluoropropionate,
N-succinimidyl-6(4'-azido-2'-nitrophen- ylamino) hexanoate
(Lomant's reagent II), N-succinimidyl
(4-azidophenyldithio)propionate (SADP),
N-succinimidyl-3-(2-pyridyldithio- )propionate (SPDP), and
N-(4-azidophenylthio)phthalimide (APTP), including various
derivatives such as their sulfo- forms;
[0111] homobifunctional reagents such as
1,5-difluoro-2,4-dinitrobenzene,
4,4'-difluoro-3,3'-dinitrophenylsulfone,
4,4'-diisothiocyano-2,2'-disulfo- nic acid stilbene (DIDS),
p-phenylenediisothiocyanate (DITC), carbonylbis(L-methionine
p-nitrophenyl ester), 4,4'-dithiobisphenylazide and
erythritolbiscarbonate, including various derivatives such as their
sulfo- forms;
[0112] photoactive coupling agents such as
N-5-azido-2-nitrobenzoylsuccini- mide (ANB-NOS), p-azidophenacyl
bromide (APB), p-azidophenyl glyoxal (APG),
N-(4-azidophenylthio)phthalimide (APTP), 4,4'-dithio-bis-phenylazi-
de (DTBPA), ethyl 4-azidophenyl-1,4-dithiobutyrimidate (EADB),
4-fluoro-3-nitrophenyl azide (FNPA),
N-hydroxysuccinimidyl-4-azidobenzoat- e (HSAB),
N-hydroxysuccinimidyl-4-azidosalicylic add (NHS-ASA),
methyl-4-azidobenzoimidate (MABI),
p-nitrophenyl-2-diazo-3,3,3-trifluorop- ropionate (PNP-DTP),
2-diazo-3,3,3-trifluoropropionyl chloride,
N-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH)
N-succinimidyl(4azidophenyl)1,3'-dithiopropionate (SADP),
sulfosuccinimidyl-2-(m-azido-o-nitobenzamido)-ethyl-1,3'-dithiopropionate
(SAND), sulfosuccinimidyl(4-azidophenyldithio)propionate
(Sulfo-SADP),
sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate
(Sulfo-SANPAH),
sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithi-
opropionate (SASD), and derivatives and analogs of these reagents,
among others. The structures and references for use are given for
many of these reagents in, "Pierce Handbook and General Catalog",
Pierce Chemical Co., Rockford, Ill., 61105.
[0113] Intermediate Coupling Substance.
[0114] In addition to covalently coupling directly through
functional groups of cyclodextrin derivatives to synthesize
water-soluble (or colloidal) polymers, it is also useful to include
an intermediate substance or "intermediate". By definition,
intermediate substances function as bio-compatible intermediates in
being suitably nonimmunogenic and nonallergenic. Although
intermediate substances may be degraded biologically, they are
"biologically neutral" in that they essentially lack specific
binding properties or biorecognition properties in their
application.
[0115] The intermediate can function as a "spacer" (e.g. "spacer
arm" of O'Carra, P., et al, FEBS Lett. 43, 169 (1974)), between the
cyclodextrin derivatives being covalently coupled to overcome
steric hindrance of subsequent binding reactions. The intermediate
can function as a polymer "backbone" to which many cyclodextrin
dimers, trimers or polymers are covalently coupled to form a larger
polymer. The intermediate can be included with cyclodextrin
derivatives as another monomer to be copolymerized with the
cyclodextrin derivatives (i.e. heteropolymer), to provide improved
structural properties, increase solubility or lower toxicity.
[0116] The intermediate substance may also provide the advantage of
additional coupling sites and thereby increase the number of
covalently coupled cyclodextrin derivatives within a polymer
carrier. The intermediate can also introduce certain other
desirable properties, such as a positive or negative net charge,
more efficient light energy transfer for photodynamic therapy. The
desired biorecognition molecule or other substance can be coupled
to the available sites on the intermediate substance and is thereby
coupled indirectly to the water-soluble cyclodextrin polymer
carrier of the instant invention.
[0117] Examples of such biologically neutral intermediate coupling
substances are certain proteins, polypeptides, polyamino acids,
serum albumins, glycoproteins, lipoproteins, nucleic acid polymers,
DNA, RNA, amino sugars, glucosamines, polysaccharides,
lipopolysaccharides, amino polysaccharides, polyglutamic acids,
polylysines, polyacrylamides, nylons, poly(allylamines), lipids,
glycolipids and suitable synthetic polymers, especially
biopolymers, resins and surfactants, as well as suitable
derivatives of these substances. Also included as suitable
intermediate coupling substances are the polymers disclosed in U.S.
Pat. No. 4,645,646. Also preferred as intermediates are
N-(2-hydroxypropyl)methacrylamide (HPMA), HPMA derivatives, poly
cyanoacrylates such as poly(butyl cyanoacrylate), poly(isobutyl or
isohexyl cyanoacrylate), polyethylene glycol (PEG), any PEG
derivatives, poly (D,L-lactic-coglycolic acid) (PLGA), PLGA
derivatives, dendrimers and poly
(D,L-lactide)-block-methoxy-polyethylene glycol (Diblock).
[0118] Various materials may be incorporated into the components of
the instant invention to produce new inventions with unexpected
properties for use in certain applications. For instance, the
addition of ferrous or magnetic particles may be used to give
cyclodextrin polymer carriers and other types of polymers (i.e.
HPMA, PEG), magnetic properties (Ithakissios, D. S., Clin. Chim.
Acta 84(1-2), 69-84, 1978). This would be useful for various in
vivo manipulations such as using magnetic fields to localize or
concentrate a magnetic polymer drug carrier in a specific part of
the body. Also, the magnetic particles may be used to trigger a
cytotoxic effect on cancer cells such as by vibrating them with
alternating magnetic fields.
[0119] CD Guest-Linked Agent.
[0120] A cyclodextrin guest-linked agent or "CD guest-linked agent"
comprises a new invention for facilitating the noncovalent coupling
of CD polymers with drugs, proteins, DNA, ODNs and other active
agents. In one embodiment a CD guest-linked agent or simply
"guest-linked agent" (GLA), is comprised of an active agent
covalently coupled to one or more molecules capable of forming an
inclusion complex ("complex") with a specific CD or CD polymer. The
inclusion complex between the CD and the GLA is preferably of
higher affinity than between the CD and the active agent alone. The
major advantage is that an active agent that is not easily
complexed with a cyclodextrin or CD polymer is thereby more easily
complexed or linked when it is a GLA. This then provides a unique
method for entrapping an active agent as a guest-linked agent in a
CD polymer.
[0121] Preferably the GLA includes guest molecules (the CD linker
or coupler), that have high affinity for a specific cyclodextrin.
Generally, these high affinity guest molecules ate those that fit
more snugly or closely within the hydrophobic cavity of either
alpha, beta or gamma cyclodextrin. The most preferred guest
molecules have association constants of 1.times.10.sup.3 M.sup.-1
or more. For example, preferred guest molecules for beta
cyclodextrin include any suitable adamantane analogs or derivatives
such as adamantane acetate (AAC), adamantane carboxylate (AC),
1-homoadamantanes (1-HAC), 3-homoadamantanes (3-HAC),
3-noradamantane carboxylate (NAC), norbornane acetate (NBA),
1-bicyclo[2.2.1]octanecarboxylate, 1-bicyclo-[2.2.1]heptane
carboxylate, 1-bicyclo[2.2.1]heptene carboxylate; any suitable
analogs or derivatives of cyclohexane such as
cyclohexanecarboxylate, cyclohexane acetic acid; any suitable
analogs or derivatives of cyclopentane such as
cyclopentanecarboxylate; any suitable analogs or derivatives of
benzene; any suitable analogs or derivatives of camphor; among
others.
[0122] For example, the GLA invention can be used to complex a drug
such as doxorubicin (DOX) with a CD polymer. The drug is first
derivatized to produce an active form with an amino-coupling group
such as NHS ester or p-nitrophenyl ester (ONp) as described by P.
Rejmanova, et al., Makromol. Chem. 178, 2159 (1977). DOX is
suitably derivatized to provide an ONOp group as described by V.
Omelyanenko, et al., J. Controlled Rel. 53, 25-37 (1998). A GLA is
then synthesized by covalently coupling the ONp-DOX derivative to a
suitable amino-derivatized adamantane such as 1-aminoadamantane,
1-adamantane methyamine, or 1-adamantane carboxamide, among
others.
[0123] Conversely, any suitable active agent can be derivatized to
produce an active form that provides an amino functional group for
coupling. A GLA is then synthesized by covalently coupling the
active agent through the amino group to an adamantane derivative
with an amino-coupling group on it. Such adamantane derivatives
include 1-adamantane carbonyl chloride, 1-adamantane
isothiocyanate, or adamantane derivatized to provide an NHS ester
or ONp, among others.
[0124] Also, any suitable active agent can be derivatized to
produce an active form that provides a sulfhydryl functional group
for coupling. A GLA is then synthesized by covalently coupling the
active agent through the sulfhydryl group to an adamantane
derivative with a thiol- or sulfhydryl-coupling group on it. Such
adamantane derivatives include 1-adamantane carbonyl chloride, iodo
adamantane, or adamantane derivatized to provide a maleimidyl or
bromoacetyl group. Also, suitable sulfhydryl-derivatized adamantane
can be used to couple to an active agent with available sulfhydryl
groups through disulfide coupling.
[0125] Also, when any suitable active agent is derivatized to
produce an active form that provides an amino, or sulfhydryl
functional group for coupling. A GLA is then synthesized by
covalently coupling the active agent to an adamantane derivative
with an amino, or thiol, or hydroxyl functional group available.
Such coupling is done through any suitable crosslinking agent
reactive with the functional groups available on the active agent
and the guest ("CD linker"). In any case, the newly synthesized CD
guest-linked agent can then be entrapped by mixing with the CD
dimers, CD trimers or CD polymer carriers described herein.
[0126] A new, preferred guest-linked agent has dimer, trimer or
small polymer adamantanes (or other suitable guests) coupled at
single sites on the drug, nucleic acid or other active agent. This
provides a new class of CD linkers that provides a higher number of
complexing guests than is possible to couple to smaller active
agents such as drugs and ODNs with fewer coupling sites. This new
CD linker is also more easily complexed with dimers, or trimers or
polymers of beta cyclodextrin, making the active agent more easily
solubilized and the inclusion complexes more stable than with
individual adamantanes coupled to the active agent.
[0127] Preferably, any of the coupling systems used to synthesize a
GLA also include a biocleavable linkage, described herein, to
provide for controlled release of the active agent if desired.
EXAMPLES OF THE BEST MODES FOR CARRYING OUT THE INVENTION
[0128] In the examples to follow, percentages are by weight unless
indicated otherwise. During the synthesis of the compositions of
the instant invention, it will be understood by those skilled in
the art of organic synthesis, that there are certain limitations
and conditions as to what compositions will comprise a suitable
carrier and may therefore be prepared mutatis mutandis. It will
also be understood in the art of cyclodextrins that there are
limitations as to which drugs and other agents can be used to form
inclusion complexes with certain cyclodextrins.
[0129] Specifically, it is known that smaller, alpha cyclodextrins
are preferably used to complex with the smaller drugs or active
agents. Whereas larger cyclodextrins are less limited, except that
a "close fit" is generally preferred for stronger complexing
affinity.
[0130] The terms "suitable" and "appropriate" refer to synthesis
methods known to those skilled in the art that are needed to
perform the described reaction or procedure. In the references to
follow, the methods are hereby incorporated herein by reference.
For example, organic synthesis reactions, including cited
references therein, that can be useful in the instant invention are
described in "The Merck Index", 9, pages ONR-1 to ONR-98, Merck
& Co., Rahway, N.J. (1976), and suitable protective methods are
described by T. W. Greene, "Protective Groups in Organic
Synthesis", Wiley-Interscience, NY (1981), among others. For
synthesis of nucleic acid probes, sequencing and hybridization
methods, see "Molecular Cloning", 2nd edition, T. Maniatis, et al,
Eds., Cold Spring Harbor Lab., Cold Spring Harbor, N.Y. (1989).
[0131] All reagents and substances listed, unless noted otherwise,
are commercially available from Aldrich Chemical Co., WI 53233;
Sigma Chemical Co., Mo. 63178; Pierce Chemical Co., IL. 61105;
Eastman Kodak Co., Rochester, N.Y.; Pharmatec Inc., Alachua Fla.
32615; and Research Organics, Cleveland, Ohio. Or, the substances
are available or can be synthesized through referenced methods,
including "The Merck Index", 9, Merck & Co., Rahway, N.J.
(1976).
[0132] Additional references cited in U.S. Pat. No. 6,048,736 and
PCT/US99/30820, are hereby incorporated herein by reference.
Cyclodextrin Polymer Carriers
[0133] The purpose is to provide a water-soluble (or colloidal)
cyclodextrin polymer carrier that has an active agent completely
entrapped. For synthesis, the general approach is; (1) to produce
or modify or protect, as needed, one or more functional or coupling
groups on the cyclodextrin components, consisting of cyclodextrins,
or open dimers, trimers or polymers; (2) combine under appropriate
conditions, a minimum of 2 of the cyclodextrin components with a
drug or active agent to produce a noncovalent inclusion complex and
(3) if needed, using various coupling methods cross-link the
cyclodextrin components to produce a polymer that completely
entraps the drug within the cyclodextrin polymer. In certain
applications, the complex between CD components and active agent
produced in step 2 will be suitable for carrying the active
agent.
[0134] Also, as described below, the cyclodextrin polymer carrier
may be suitably derivatized to include other useful substances
and/or chemical groups (e.g. biorecognition molecules, antenna, and
catalytic substances), to perform a particular function. Depending
on the requirements for chemical synthesis, the derivatization can
be done before entrapment or afterward, using suitable protection
and deprotection methods as needed.
[0135] Since cyclodextrins are composed of carbohydrates, they can
be suitably derivatized and coupled through well-known procedures
used for other carbohydrates, especially through available hydroxyl
groups. For instance, vicinal hydroxyl groups on the cyclodextrin
can be appropriately oxidized to produce aldehydes.
[0136] In addition, any functional group can be suitably added
through well-known methods while preserving the cyclodextrin
structure and complexing properties. Examples are: amidation,
esterification, acylation, N-alkylation, allylation, ethynylation,
oxidation, halogenation, hydrolysis, reactions with anhydrides, or
hydrazines and other amines, including the formation of acetals,
aldehydes, amides, imides, carbonyls, esters, isopropylidenes,
nitrenes, osazones, oximes, propargyls, sulfonates, sulfonyls,
sulfonamides, nitrates, carbonates, metal salts, hydrazones,
glycosones, mercaptals, and suitable combinations of these. The
functional groups are then available for the cross-linking of one
or more cyclodextrin molecules using a bifunctional reagent.
[0137] Additional examples of cyclodextrins, inclusion compounds
and catalytic groups including chemical methods for modifying
and/or derivatizing cyclodextrins that are useful in the instant
invention are described and referenced in U.S. Pat. No. 6,048,736
and PCT/US99/30820, which are incorporated herein by reference.
[0138] Bergeron, R. J., et al, Bioorgan. Chem. 5, 121-126
(1976)
[0139] Boger, J., et al, Helvet. Chim. Acta 61, 2190-2218
(1978)
[0140] Buckler, S. A., et al, U.S. Pat. No. 3,472,835 (1969)
[0141] Buckler, et al, U.S. Pat. No. 4,331,808 (1982)
[0142] Carlsson, J., et al, Eur. J. Biochem. 59, 567-572 (1975)
[0143] Case, L. C., U.S. Pat. No. 3,502,601 (1970) and U.S. Pat.
No. 3,510,471 (1970)
[0144] Cramer, F., et al, Chem. Ber. 103, 2138 (1970)
[0145] Cramer, F., et al, J. Amer. Chem. Soc. 89:1, 14-20
(1967)
[0146] Emert, J., et al, J. Amer. Chem. Soc. 97, 670 (1975)
[0147] Furue, M. A., et al, Polymer. Lett. 13, 357 (1975)
[0148] Gramera, R. E., et al, Fr. Demande 1, 584, 917 (1968)
[0149] Harada, A., et al, Macromolecules 9, 701 and 705 (1976)
[0150] Hatano, M., et al, Japan Kokai 77,71,583 (1977)
[0151] Hirai, H., J. Inclus. Phenom. 2, 455-466 (1984)
[0152] Ikeda, T., et al, J. Inclus. Phenom. 2, 669-674 (1984)
[0153] Ikeda, T., et al, J. Inclus. Phenom. 5, 93-98 (1987)
[0154] Iwakura, Y., et al, J. Amer. Chem. Soc. 97/15, 4432-4434
(1975)
[0155] Johnson, C. K. U.S. Pat. No. 3,654,261 (1972)
[0156] Kawaguchi Y., et al, Anal. Chem. 55, 1852-1857 (1983)
[0157] Klotz, I. M., et al, Arch. Biochem. Biophys. 96, 605-612
(1961)
[0158] Kobayashi, M., et al, Agric. Biol. Chem. 52, 2695-2702
(1988).
[0159] Lui, F-T., et al, Biochem. 18, 690-697 (1979)
[0160] Matsui, Y., et al, Chem. Lett., Oct., 1037-1040 (1976)
[0161] Ogata, N., Japan Kokai 77,121,096 (1977)
[0162] Parmerter, S. M., U.S. Pat. No. 3,426,011 (1969) and U.S.
Pat. No. 3,453,257 (1969)
[0163] Royer, G. P., et al, Biochem. Biophys. Res. Comm. 64,
478-484 (1975)
[0164] Szejtli, J., et al, Hung. Patent 19,626 (1978)
[0165] Tabushi, I., et al,J. Amer. Chem. Soc. 98/24, 7855-7856
(1976)
[0166] Tabushi I., et al, Tetrahed. Lett. No. 29, 2503-2506
(1977)
[0167] Tabushi, I., Acc. Chem. Res. 15, 66-72 (1982)
[0168] Traut, R. R., et al, Biochem. 12, 3266-3273 (1973)
[0169] Ueno, A., et al, J. Inclus. Phenom. 2, 555-563 (1984)
[0170] VanEtten, R. L., et al, J. Amer. Chem. Soc. 89/13 3242-3253
and 3253-3262 (1967)
[0171] Suitable coupling or cross-linking agents for preparing the
water-soluble (or colloidal) CD carriers of the instant invention
can be a variety of reagents previously described, including well
known crosslinkers used to polymerize CD's. Other suitable
crosslinkers or derivatizers are various epoxy compounds including
propylene oxide, 1,2-diethoxyethane, 1,2,7,8-diepoxyoctane,
2,3-epoxy-1-propanol (glycidol), glycerol propoxylate
triglycidylether and 1,4-butanediol diglycidyl ether (e.g. Gramera,
or Case, or Johnson, or Parmerter, supra). Also useful are methods
employing acrylic esters such as m-nitrophenyl acrylates, and
hexamethylenediamine and p-xylylenediamine complexes (e.g. Furue,
or Harada, or Hatano, or Ogata, supra), and aldehydes, ketones,
alkyl halides, acyl halides, silicon halides, isothiocyanates, and
epoxides (e.g. Buckler, supra).
Methods for Derivatizing Cyclodextrins
[0172] For this invention, individual cyclodextrin derivatives
(i.e. monomer) as well as dimers, trimers and polymers are the
primary components, or units used to synthesize the water-soluble
(or colloidal) cyclodextrin polymer carriers. Although native
cyclodextrins are useful for synthesizing the carriers, many other
useful properties can be incorporated into the carriers by first
derivatizing the cyclodextrin components before making the
polymers. Derivatizing is defined as the chemical modification of a
CD through addition of any functional or coupling group and/or
other substance. Generally, derivatized cyclodextrins can be used
to facilitate cross-linking reactions and introduce functional
groups for use during or after the carrier is prepared. Frequently,
an integral part of using derivatized cyclodextrins involves
protecting certain functional groups during certain cross-linking
steps and then deprotecting those groups for use in subsequent
steps.
[0173] A. Protected Hydroxyl Groups.
[0174] Primary and/or secondary hydroxyl groups on the cyclodextrin
(or derivatives), can be selectively protected and deprotected
using known methods during derivatizing and/or capping procedures,
to provide selective coupling at the primary or secondary end of
the CD molecule, as desired. For instance, formation of protective
esters (e.g. benzoates using benzoyl chloride), and selective
cleavage (deprotection), of primary esters using anhydrous
alcoholysis (e.g. Boyer, supra), provides mostly primary hydroxyls
for derivatization. After derivatization and/or coupling the
primary hydroxyls, the secondary hydroxyls can be deprotected for
additional derivatization, coupling and/or capping.
[0175] Preferred hydroxyl protection schemes include various
methods for silylation of the primary hydroxyls using
tert-butyldimethylsilyl chloride (TBDMS), (K. Takeo, et al.,
Carbohydrate Res. 187, 203 (1989)) for derivatization of the
secondary hydroxyls. Or, the use of sodium hydride with TBDMS (S.
Tian, et al., Tetrahedron Lett. 35, 9339 (1994)) to protect
secondary hydroxyls during derivatization of the primary hydroxyls.
The silyl groups are then removed by treatment with
tert-butylammonium fluoride.
[0176] B. Preparation of Sulfonylated Cyclodextrin
[0177] A variety of suitable methods are available for
sulfonylation of CD or CD polymer before or after protection of
specific hydroxyl groups (e.g. Bergeron, Boger or Ueno, supra),
and/or capping of the CD (e.g. Emert or Tabushi, supra). Suitably,
CD polymer (10 gm), is combined with a suitable sulfonylating
reagent (20 gm), such as p-toluenesulfonyl (tosyl) chloride,
mesitylenesulfonyl chloride or naphthalenesulfonyl chloride, among
others, in anhydrous pyridine, for 3-5 Hrs at room temperature
(RT).
[0178] C. Preparation of Oxidized Cyclodextrin for Dialdehyde
Cyclodextrin (Dial-CD).
[0179] A dialdehyde CD derivative (dial-CD) and dialdehyde
cyclodextrin polymer (dial-CD polymer) is prepared from oxidized
cyclodextrin or oxidized CD polymer by oxidation using known
methods (e.g. Royer or Kobayashii, supra), with sodium
metaperiodate in water or suitable buffer solution (e.g. 0.2 M
phosphate saline, pH 5-7), where one or more dialdehydes can be
produced per CD. For use in preparing cyclodextrin polymer
carriers, dial-CD can also include oxidized forms of HPCD, DHPCD
and SBE-CD.
[0180] D. Amino-Cyclodextrin (Amino-CD) Derivatives.
[0181] Amino groups can be introduced into CD polymer by reaction
of a sulfonylated CD polymer with azide compounds including
hydrazine, and 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone
(e.g. Ikeda, supra), or coupling to diamines as described by
Kawaguchi, or Matsui, supra.
[0182] Also, when desired, a "monoamino" CD, wherein one amino
group has been coupled, can be prepared through known methods,
including limited or sterically determined monosulfonylation,
and/or by specific protection and deprotection schemes. An amino-CD
or amino-CD polymer, is suitably protected and/or deprotected as
needed.
[0183] E. Diamino Derivatives.
[0184] A previously sulfonylated CD or CD polymer is suitably
iodinated so that it will couple to primary amino groups, using
known methods (e.g. Ikeda or Iwakura, supra). Suitably, 10 gm of
sulfonylated CD or CD polymer is combined with 12 gm of NaI on 200
ml of methanol, and mix at 70.degree. C. for 48-60 Hrs. The
iodinated CD product is collected by precipitation with acetone and
purified by column chromatography.
[0185] The iodinated CD or CD polymer is coupled through an amino
group to a suitable diamino substance. Suitable diamino substances
are; 1,4diaminobutane, 1,6-diaminohexane, 1,7-diaminoheptane,
1,8-diaminooctane, 1,10-diaminodecane, 1,12-diaminododecane, and
other aliphatic, or aromatic, or heterocyclic carboxylic acids with
two available amino groups for coupling. Coupling is done in a
suitable solvent such as dimethylformamide (DMF), mixing 10 gm of
iodinated CD polymer with a molar excess of the diamino substance
(e.g. 10-20 gm of 1,6-diaminohexane), at 100.degree. C. for 24 Hrs.
The product, amino-CD (or amino-CD polymer), is concentrated and
purified by column chromatography.
[0186] F. Protected Amino Groups.
[0187] The amino groups introduced by various methods can be
suitably protected by reaction with a halogenated alkylphthalimide
such as N-(4-bromobutyl)phthalimide. After other suitable
derivatizing, coupling and/or capping has been done, an amino group
is deprotected by reaction with hydrazine in suitable solvent.
[0188] Also, the diamino substances of various chain lengths can be
suitably derivatized before coupling. For instance, they can be
"half protected" as trifluoroacetamidoalkanes at one of the amino
ends, as described by Guilford, H., et al, Biochem. Soc. Trans. 3,
438 (1975), before coupling, and then suitably deprotected such as
by hydrolysis or alcoholysis. Yet another suitable method involves
the coupling of THP-protected amnino-alkynes, previously described,
to the iodinated CD or CD polymer and subsequent deprotection as
needed.
[0189] G. Sulfhydryl-Cyclodextrin (SH-CD) Derivatives.
[0190] A sulfhydryl group can be added to an amino-CD, suitably
prepared as described previously, by coupling the appropriate
thiolating agent to the available amino group. For instance,
thiolation of amino groups on amino-CD can be done by known methods
using S-acetylmercaptosuccinic anhydride (SAMSA), (e.g. Kiotz,
Rector, or Lui supra), SIAB, or 2-iminothiolane (e.g. Traut,
supra). The sulfhydryl is protected as a disulfide during
subsequent coupling reactions until it is exposed through disulfide
cleavage.
[0191] Sulfhydryls can also be introduced through reaction of
available hydroxyls with a suitable epoxy compound. For instance,
epichlorohydrin or a suitable diepoxy crosslinker previously
described, is coupled to a CD or CD polymer wherein free epoxy
groups are produced. Free epoxy groups are then reacted with sodium
thiosulfate to give thiosulfate esters (e.g. Carlsson, supra). The
thiosulfate esters are subsequently reduced to sulfhydryls with
dithiothreitol.
[0192] H. Cyclodextrin Dimer, Trimer and Polymer Derivatives.
[0193] Certain CD dimers, CD trimers and small CD polymers have
been derivatized and can function as carriers or excipients without
further crosslinking. Sulfate groups can be introduced by reacting
primary or secondary hydroxyl groups with various cyclic sultone
compounds to produce sulfoalkyl ether derivatives. For instance,
1,4-butane sultone reacts with the hydroxyl groups to produce a
sulfobutyl ether (SBE) group (Stella, et al. U.S. Pat. No.
5,134,127), or 1,3-propane sultone reacts with the hydroxyl groups
to produce a sulfopropyl ether group (Szejtli, supra).
[0194] New, more useful excipients with higher binding affinities
can be prepared from CD dimers, trimers or polymers than
derivatives of single CD molecules. These new excipients are
synthesized by first preparing CD dimers, trimers or polymers of
cyclodextrin by crosslinking monomer cyclodextrins by various
means. For instance crosslinking is done using bifunctional or
multifunctional epoxy crosslinkers such as epichlorohydrin, 2,3
epoxy-1,4-butanedione, glycerol diglycidyl ether, or glycerol
propoxylate triglycidyl ether, among others.
[0195] Then the crosslinked CD products are derivatized with a
cyclic sultone such as 1,4-butane to provide sulfobutyl groups or
1,3-propane sultone to provide sulfopropyl groups in basic
conditions such as 1-50% NaOH in water.
[0196] Also, such CD dimers, CD trimers and small CD polymers can
be derivatized to provide hydroxyethyl, hydroxypropyl or dihydroxy
propyl groups by derivatizing them with ethylene oxide, propylene
oxide or glycidol. Other useful derivatives include CD dimers,
trimers or polymers with carboxylate groups using methods disclosed
or referenced herein.
[0197] Also, phosphate groups can be added to CD dimers, trimers or
polymers by several known methods. For example, E. Tarelli, et al.,
Carbohydrate Res. 302(1-2), 27-34 (1997) describes reacting
cyclodextrins with inorganic metaphosphates in aqueous solution at
pH 4, drying and warming to produce monophosphate esters.
[0198] Other useful derivatives include CD dimers, trimers or
polymers that have been oxidized, such as with NaIO.sub.4, to
produce dialdehyde groups. The dialdehydes are then coupled to any
suitable amino-containing or sulfhydryl-containing compound to
provide the desired derivative.
[0199] The resulting derivatives are generally more soluble that
the initial crosslinked CD dimers, CD trimers and CD polymers and
would be suitable for use as drug or other active agent carriers or
as excipients. They are usually more ionic to allow migration in an
electric field for applications such as iontophoresis.
[0200] I. Preparation of Carboxylic Acid CD Derivatives.
[0201] A preferred method for adding carboxylate groups is to
couple glutaric or succinic anhydride to a hydroxyl group on the
CD, or CD dimer, trimer or polymer. This produces a terminal
carboxylate, which can then be protected by esterification as
needed. Also, carboxylates can be derivatized to an NHS ester using
N-hydroxysuccinimide and carbodiimide such as dicyclohexyl
carbodiimide.
[0202] Alternatively, a previously sulfonylated CD or CD polymer
can be suitably iodinated as previously described for diamino
groups. An iodinated CD polymer or a dial-CD polymer is coupled
through the amino group to a suitable amino-carboxylic acid to
provide the desired length of spacer. Suitable amino-carboxylic
acids are; 4-aminobutyric acid, 6-aminohexanoic acid,
7-amninoheptanoic acid, 8-aminocaprylic acid, 12-aminododecanoic
acid, and other aliphatic, or aromatic, or heterocyclic carboxylic
acids with an available amino group for coupling.
[0203] Coupling of amino-carboxylic acid to iodinated CD or CD
polymer is done in a suitable solvent such as dimethylformamide
(DMF), mixing 10 gm of iodinated CD polymer with a molar excess of
amino-carboxylic acid (e.g. 10 gm of 6-aminohexanoic acid), at
100.degree. C. for 24 Hrs. The product, CD-carboxylic acid, is
concentrated and purified by column chromatography.
[0204] Coupling of amino-carboxylic acid to dial-CD or dial-CD
polymer is done by reductive alkylation. In a suitable buffer (e.g.
0.1 M borate, pH 7.5-8.5, 0.1-0.5 M triethanolamine), 10 gm of dial
CD polymer is mixed with a molar excess of amino-carboxylic acid
(e.g. 10 gm of 12-aminodecanoic acid), at RT for 1-2 Hrs. The
Schiffs base coupling is stabilized by suitable reduction with
NaBH.sub.4 (e.g. 0.1-1 mg/ml), for 1-12 Hrs. The product,
CD-carboxylic acid, is concentrated and purified by column
chromatography and dried for subsequent reactions as needed.
[0205] J. Capping Cyclodextrins.
[0206] Capping is a type of derivatizing defined herein as coupling
any suitable chemical "capping substance" to two or more sites on
the CD molecule so that the substance spans the area between the
coupled sites. Preferably, the capping substance spans across one
of the end openings of the CD molecule and thereby stops the
passage of a guest molecule through the capped CD molecule.
[0207] It is well known that capping with disulfonyl chloride
compounds is also useful for synthesizing bifunctional derivatives
of cyclodextrins. For instance, when the CD has been capped with a
suitable disulfonyl compound, it is coupled at two of the available
hydroxyl groups. These two coupled sites can then be disubstituted
to introduce various thiol or amino groups through nucleophilic
displacement (Tabushi, supra). For instance, displacement with
ammonia gives amino groups, displacement with hydrogen sulfide
gives thiol groups.
[0208] The CD's used herein can be suitably complexed with one or
more guest molecules and/or derivatized and/or capped before,
during or after their incorporation into the water-soluble CD
polymer carrier of the instant invention. In addition, the
derivatizing and/or capping can be a done to produce CD's with the
desired substances coupled to specific locations on the CD
molecule. In the preparation of CD derivatives for use as hosts for
drugs or other agents, modifications that increase affinity between
the host CD and guest(s) are preferred. For instance, the host CD's
of this invention are preferably derivatized (e.g. methylated or
benzylated), and/or capped by various means to increase host-guest
affinity.
[0209] K. Derivatizing and Capping Substances.
[0210] Preferably, the capping substance is coupled at the primary
or secondary "end" of the CD molecule, forming a bridge across
either (or both) opening(s) that includes suitable hydrophobic
groups in the capping substance. The capping substances can be
coupled directly to available hydroxyls on the CD, or they can be
coupled to suitable functional groups such as; diamino (or
triamino), compounds to iodinated CD, or azido compounds to
sulfonylated hydroxyls, and/or through "spacers" added to the
CD.
[0211] Suitable disulfonyl capping substances are
biphenyl-4,4'-disulfonyl chloride, 1,3-benzene disulfonyl chloride,
2,4-mesitylene disulfonyl chloride, 2,6-naphthalene disulfonyl
chloride, 4,4'-oxybis(benzene sulfonyl chloride), 4,4'-methylene
bis(benzene sulfonyl chloride), m,m'-benzophenone-disulfonyl
chloride, p,p'-stilbene-disulfonyl chloride, and
diphenylmethane-p,p'-disulfonyl chloride, among others. Other
suitable capping substances are imidazoles, 6-methylamino-deoxy and
6-methylamino-6-deoxy derivatives transformed to the corresponding
N-formyl compounds, terephthaloyl chloride, dianhydrides such as
3,3',4,4'-benzophenonetetracarboxylic dianhydride and
3,4,9,10-perylenetetracarboxylic anhydride, azido compounds such as
2,6-bis(4-azidobenzylidene)-4methylcyclohexanone, and derivatives
of aurintricarboxylic acid (e.g. thionyl chloride derivatives,
triammonium salts "aluminons"), among others (e.g. Szejtli, Emert,
Tabushi, or Cramer, supra).
Preparation I
Cyclodextrin Polymer Carrier with Completely Entrapped
Anthracene
[0212] The purpose is to prepare a water-soluble (or colloidal)
cyclodextrin polymer carrier with completely entrapped anthracene.
In this preparation, beta cyclodextrin was cross-linked while
complexed with anthracene at a molar ratio of 4:1. The procedure
was to combine 10 ml of water containing 0.0002 moles of
cyclodextrin with 1 ml of chloroform containing 0.00005 moles of
anthracene. After about 15 minutes of mixing at about 20,000 rpm
with a stainless steel impeller and Dremel motor, most of the
solvent had evaporated. While still mixing, 0.4 ml of
epichlorohydrin and 0.2 ml of 2 N NaOH was added. After about 20
minutes, the reaction was stopped by adding 0.4 ml of ethanolamine.
The resulting solution was allowed to settle and examined over UV
illumination.
[0213] The turbid solution had a greenish-yellow, fluorescent top
layer indicating unincorporated anthracene. However, the aqueous
phase of the solution showed a distinct blue fluorescence,
indicating that some anthracene was complexed in the cross-linked
cyclodextrin polymer suspended in the aqueous phase.
[0214] The preparation in the aqueous phase was separated and
concentrated by evaporation and then extracted 3 times with 4 ml of
fresh chloroform by mixing, settling and drawing off the solvent
phase. The preparation was resuspended in water and produced a
turbid suspension that was still blue fluorescent. Since chloroform
extraction did not remove the anthracene, it showed that the
anthracene was completely entrapped within the cyclodextrin
polymer.
Preparation II
Cyclodextrin Polymer Carrier with Completely Entrapped 2AA
[0215] The purpose is to prepare a water-soluble (or colloidal)
cyclodextrin polymer carrier with completely entrapped
2-aminoanthracene (2AA). The procedure was to combine 0.5 ml of
4.4% beta cyclodextrin in water, with 0.02 ml of solution
containing 80% 1,4 butanediol diglycidyl ether (BDE), 10% of 0.1 M
guest molecule, 2-aminoanthracene in dimethylformamide, and 2.4% 2
N NaOH while mixing vigorously and incubating at 60.degree. C.
[0216] After about 1 hour, 0.2 ml of 0.01 M K.sub.2HPO.sub.4 (K2
buffer, pH 8.6), and 0.02 ml more of BDE was added and mixed to
continue the crosslinking. After about one half hour more, the
mixture was mixed with 0.1 ml of 1M lysine for about 2.5 hours
more. The preparation was then centrifuged for 8 minutes at 2500
rpm and 0.55 ml of supernatant was fractionated on a column of
Sephadex.RTM. G-25 (14.times.0.8 cm) equilibrated with K2
buffer.
[0217] The 0.5 ml fractions were then collected and examined for
color to indicate the presence of the guest molecule 2AA. Fractions
were also tested for carbohydrate to indicate cyclodextrin polymer.
To a 50 .mu.l aliquot of polymer fraction in water was added 1 drop
of test reagent (3 gm potassium dichromate, 10 ml conc.
H.sub.2SO.sub.4 and 290 ml water). The mixture was heated gently to
oxidize the samples. The intensity of the dark residue was graded
on a scale of 1 to 10.
[0218] The carbohydrate test showed that the polymerized
cyclodextrin was in fractions 4 through 8, which was in the area of
the void volume determined previously with a blue dextran control
sample. Also, yellow color was seen in corresponding fractions 4
through 6, showing that guest molecule 2AA could not be separated
from the polymer on the column. The carbohydrate (cyclodextrin)
test and yellow color test results for the column fractions are
shown in Table A below (Exper. Nov. 14, 1989).
1TABLE A CD Polymer Fraction 1 2 3 4 5 6 7 8 9 Relative
Carbohydrate 0 0 5 9 10 10 10 9 5 Yellow Color No No No Yes Yes Yes
No No No
Preparation III
Cyclodextrin Polymer Carrier with Tethered Guest
[0219] The purpose is to first prepare a water-soluble (or
colloidal) cyclodextrin polymer using 1,4 butanediol diglycidyl
ether (BDE) to crosslink with the cyclodextrin hydroxyl groups.
Additional BDE molecules are allowed to randomly couple at only one
end before excess lysine is added. The lysine is covalently
incorporated by covalently coupling to free ends of the BDE
previously coupled to the cyclodextrin. The combination of BDE and
lysine functions as a spacer group on the cyclodextrin polymer. The
fluorophore 2-aminoanthracene is then covalently tethered as a
captured guest to the cyclodextrin polymer through the amino group
on the BDElysine spacer using glutaraldehyde.
[0220] A. Preparation of Cyclodextrin Polymer and Incorporated
Lysine.
[0221] The procedure was to combine 2 ml of 4.4% beta cyclodextrin
in water, 0.1 ml of 2 N NaOH and 0.116 ml of 1,4 butanediol
diglycidyl ether (BDP) while mixing and incubating at 50.degree. C.
The molar ratio of BDE to cyclodextrin was about 5:1. After about 4
hours, a 0.5 ml aliquot of the mixture was mixed with 0.2 ml of
lysine (0.8 M in water, neutralized) for about 1.5 hours. The CD
polymer was then fractionated on a column of Sephadex.RTM. G-25
(21.times.0.8 cm) equilibrated with distilled H.sub.2O and
pre-calibrated with free cyclodextrin.
[0222] The 1 ml fractions were then collected and tested for
carbohydrate to demonstrate cyclodextrin polymer. To a 50 .mu.1
aliquot of polymer fraction in water was added 1 drop of test
reagent (3 gm potassium dichromate, 10 ml conc. H.sub.2SO.sub.4 and
290 ml water). The mixture was heated gently to oxidize the
samples. The intensity of the dark residue was graded on a scale of
1-10. The polymerized cyclodextrin was in the fractions (3-4)
containing a carbohydrate peak that eluted well ahead of the free
cyclodextrin control (which peaked at fraction 9). The CD polymer
fractions (3,4) were pooled.
[0223] B. Preparation of CD Polymer with Tethered Fluorophore
(FL-CD).
[0224] The CD polymer with lysine was then coupled through the
lysine groups to the guest molecule 2-aminoanthracene by a two step
glutaraldehyde method based on Guesdon, J-L, et al, J of Histochem.
Cytochem. 27, 1131-1139 (1979). The procedure was to combine 0.9 ml
of the CD polymer with 0.1 ml of 25% glutaraldehyde (in water) and
0.02 ml 2 N NaOH (starting pH 12), and mix for about 25 minutes.
The mixture was fractionated to remove excess glutaraldehyde on a
column of Sephadex.RTM. G-25 (9.times.0.8 cm) equilibrated with
distilled H.sub.2O, collecting 0.3 ml fractions. The polymer
fractions were pooled in a 1.4 ml volume. The 2-aminoanthracene was
then coupled by mixing in a total of 0.06 ml of 5 mM
2-aminoanthracene in methanol:chloroform (4:1) and 0.01 ml 2 N NaOH
(starting pH 12). This was reacted for 4 hours then blocked with
0.1 ml of ethanolamine. The Schiff base coupling was stabilized by
adding 0.01 gm of NaBH.sub.4 and incubating overnight.
[0225] The mixture was then neutralized with 1 N HCl and excess
2-aminoanthracene was removed by fractionating on a column of
Sephadex.RTM. G-25 (9.times.0.8 cm) equilibrated with distilled
H.sub.2O, collecting 0.5 ml fractions. The fractions were then
tested for carbohydrate as described previously and those with
carbohydrate were also tested for guest molecule using
chemiluminescence (CL). The CL procedure was to activate the
2-aminoanthracene using oxidation of bis(2,4,6-trichlorophenyl)
oxalate ester (TCPO). Into an FL-CD sample (0.02 ml in 0.1 ml of
0.1 M K.sub.2HPO.sub.4), was added 0.01 ml 0.22% TCPO in ethyl
acetate. After placing the sample into a dark chamber in the
luminometer, 1 ml of 0.4 M H.sub.2O.sub.2 was injected and the
light emission recorded on a chart recorder. The carbohydrate
(cyclodextrin) test and CL test results for the fractions are shown
in Table B below Exper. CD/1).
2TABLE B FL-CD Polymer Fraction 1 2 3 4 5 6 7 8 9 Relative 5 7 9 9
10 8 6 5 NT Cyclodextrin Relative .25 .30 3.33 10.0 >10 3.6 1.1
.40 .28 CL Emission
[0226] These data show that the carbohydrate peak also corresponds
to the most fluorophore CL activity. This CL activity shows that
the guest molecule 2-aminoanthracene is coupled to the CD polymer
and could not be separated by column chromatography.
Preparation IV
Cyclodextrin Polymer Carrier Targeted with Antibody Protein
[0227] The purpose is to synthesize a targeted cyclodextrin polymer
carrier by covalently coupling a biorecognition molecule to a
cyclodextrin polymer carrier. In this example, the carrier was
prepared as in Preparation III, and the biorecognition molecule is
antibody protein.
[0228] A. Preparation of FL-CD Polymer with Coupled
N-Hydroxysuccinimidyl (NHS) Ester.
[0229] In this step a cyclodextrin polymer carrier is covalently
coupled with NHS ester to form a NHS-CD. FL-CD polymer carrier
(with tethered 2-aminoanthracene) was prepared as above and
fractionated by column chromatography using Sephacryl.RTM. S200 in
a 1.5.times.18.5 cm column equilibrated with water (Exper. CD/8).
The purified FL-CD was collected in 1 ml fractions #8-19, and
pooled to give a greenish-yellow fluorescent solution. The solution
was dried at 60.degree. C. to give about 0.36 gm. The product was
dissolved in water and titrated to pH 6 with 6 N HCl giving 0.144
gm FL-CD polymer carrier per ml.
[0230] The procedure is to form NHS esters with the carboxylic acid
groups on the lysine that is incorporated into the FLCD polymer
carrier. To 1 ml of dissolved FL-CD polymer carrier was added 0.1
gin of N,N'-dicyclohexylcarbodiimide PCC) and mixed to dissolve.
Then 0.1 gm of N-hydroxysuccinimide was added with mixing. After
about 1.5 hours, 0.05 ml of glacial acetic acid was added and mixed
about 25 minutes. To the mixture was added about 4 ml of anhydrous
methanol, then it was mixed, centrifuged and the light yellow
supernatant was collected. The resulting solution of FL-CD polymer
with coupled NHS ester groups was concentrated by evaporation and
stored in the refrigerator.
[0231] B. Coupling of Gamma Globulin with the FL-CD Polymer
Carrier.
[0232] In this step the purpose is to covalently couple antibody
protein (human gamma globulin) to cyclodextrin polymer carrier with
tethered guest 2-aminoanthracene.
[0233] To a glass test tube was added 0.2 ml of 0.1 M
K.sub.2PO.sub.4, pH 8.5 in water, 0.1 ml 1.6% human gamma globulin
and 0.2 ml of 50% methanol containing about 0.09 gm/ml of FL-CD
polymer carrier with coupled NHS ester. The pH of the mixture was
adjusted to about pH 7 with 2 N NaOH and incubated about 2 days at
RT. The labeled protein was recovered by precipitation by adding
1.5 ml of 52% (NH.sub.4).sub.2SO.sub.4 in water to the mixture and
centrifuging to collect the precipitate. The precipitate was
dialyzed against distilled water to remove (NH.sub.4).sub.2SO.sub.4
and concentrated to a final volume of 0.11 ml.
[0234] Aliquots of the targeted cyclodextrin polymer carrier were
tested for CL activity using TCPO as described previously. The peak
height of CL activity of the carrier was low but the CL activity
continued for a longer time when compared to the FL-CD polymer
carrier alone and to control gamma globulin. The CL activity showed
that the gamma globulin biorecognition molecule was coupled to the
FL-CD polymer carrier.
Preparation V
Cyclodextrin Polymer Carrier with Completely Entrapped Paclitaxel
(Taxol)
[0235] The purpose is to synthesize a water-soluble (or colloidal)
cyclodextrin polymer carrier that contains completely entrapped
paclitaxel (PTX) and the polymer includes acid-labile hydrazone
linkages that provide controlled release. The following Table C is
a schematic of the reactions employed.
3TABLE C 1 2 3 Cyclodextrin Hydrazone Linkages Cross-linked
Aldehydes with Terminal Amines Polymer
[0236] A. Preparation of Dialdehyde CD Using Oxidation.
[0237] The purpose is to produce oxidized cyclodextrin (CD) to
provide dialdehydes that can subsequently be reacted with hydrazine
to form an acid-labile hydrazone linkage. The hydrazone linkages on
each cyclodextrin will also have terminal amino groups for
subsequent crosslinking to make the polymer carrier.
[0238] The oxidation procedure is based on published methods used
to oxidize other polysaccharides and specifically cyclodextrins
(Kobayashi supra). This method introduces dialdehyde groups at the
C-2, C-3-trans-diol position of the cyclodextrin glucose
residues.
[0239] The procedure was to add sodium m-periodate (NaIO.sub.4) to
30 mM cyclodextrn in 100 ml of water while mixing at 30.degree. C.
The molar ratio of NaIO.sub.4 to cyclodextrin was 2:1, to give 1 to
2 dialdehydes per CD molecule. The reaction was continued in the
dark for 6 to 8 hours. Remaining NaIO.sub.4 was consumed with a
molar excess of ethylene glycol. The resulting dialdehyde
cyclodextrin (dial-CD) was fractionated using gel filtration on a
Sephadex.TM. G-25 column. The more open dial-CD molecules have been
found to elute ahead of the native CD. The fractions were
concentrated by evaporation under vacuum.
[0240] The amount of CD (mw 1135) as carbohydrate in each fraction
is monitored by a colorimetric test for carbohydrates. To 2 ml of
water containing diluted dial-CD fraction (0.01-0.05 mg) is added
0.05 ml of 80% phenol. Then 5 ml of concentrated sulfuric acid is
added rapidly to mix. Color is allowed to develop 20 minutes at
25-30.degree. C. and the absorbance is read at 490 nm. The
absorbance is compared to a series of identically treated CD
standards at 0.005, 0.01, 0.02, 0.04, 0.08 and 0.1 mg per m
H.sub.2O.
[0241] B. Preparation of Hydrazone Linkages on the CD.
[0242] This reaction involves a condensation reaction of the
hydrazine with available aldehydes to produce a hydrazone linkage.
The objective is to react dial-CD with enough hydrazine so that
ideally each available aldehyde is coupled to a single hydrazine
with minimal cross-linking. The dial-CD preparation is dissolved in
water to give starting concentrations of 30 mM. While stirring the
solution at room temperature, a 3 to 4-fold molar excess of
hydrazine (Sigma) is added with continued stirring for 2 hours. The
resulting hydrazone cyclodextrin (Hz-CD) is fractionated on a
Sephadex.TM. G-15 column and the fractions dried to constant weight
by vacuum evaporation.
[0243] The number of amino groups is determined colorimetrically
using a Blue G-250 assay reagent for protein (Reagent Kit Cat
#23200, Pierce, Rockford Ill.) with the absorbance read at 595 nm.
To ensure that enough amino groups are available, the Hz-CD
fractions with at least 2 free amino groups available per mole are
used in the next step.
[0244] C. Preparation of CD Polymer with Completely Entrapped
Drug.
[0245] The purpose is to cross-link Hz-CD to form a water-soluble
(or colloidal) cyclodextrin polymer carrier that is acid-labile.
The polymers preferably have molecular weights of 20,000-50,000,
although higher or lower molecular weights can be used. In this
procedure the Hz-CD monomers are cross-linked through the terminal
amino groups on the hydrazine derivatives.
[0246] In order to entrap the drug, the paclitaxel (PTX) is
dissolved in a solvent and mixed with the Hz-CD to form inclusion
complexes. Then the Hz-CD is cross-linked to form the polymer and
completely entrap the drug in polymer aggregates.
[0247] Preparations can be made with molecular ratios between 1:1
and 1:8 of PTX to Hz-CD. A near saturated suspension of Hz-CD is
prepared in 0.05 M phosphate buffer, pH 7.5 (PB). The FIX (about 2
mM) in methanol is added with vigorous mixing (20,000 rpm
impeller). While mixing, the drug is exposed to the aqueous phase
to allow complexes to form between the PTX and Hz-CD. Mixing is
continued for 15 minutes to one hour. The cross-linking reagent is
then be added while continuing to mix.
[0248] At this point, a variety of amino-reactive, bifunctional
cleavable or noncleavable agents with different spacer lengths can
be used to cross-link amino groups. For this example, cross-linking
is done with a bifunctional cleavable coupling agent Tech. Bull.
#0544, Pierce Chem. Co., Rockford Ill.), dithiobis(succinimidyl
propionate) (DSP, spacer length 12 angstroms). During
polymerization, the objective is to completely entrap the drug in
polymer aggregates that are soluble (or colloidal). The
cross-liking reaction is run for about 3 hours or just before
insoluble polymers form.
[0249] The resulting PTX-loaded CD polymer (PT-CD) is then
fractionated by gel exclusion chromatography on pre-calibrated
columns of Sephacryl.RTM. S200-HR (40.times.5 cm) equilibrated with
PB. Pre-calibration is done using various molecular weight dextrans
(i.e. 15,000 to 60,000, Sigma) in separate runs.
[0250] The PIX-CD fractions that elute in molecular weight ranges
between 20,000 and 50,000 are vacuum dried at 50.degree. C. and
weighed. For some procedures, fractions may be concentrated by
centrifugal filtration using suitable molecular weight cutoff
filter tubes (Micron Separations Inc., Westboro Mass.). Other
fractions of higher or lower molecular weight may also be suitable.
The approximate moles of product are calculated as total grams of
dried carrier divided by the apparent molecular weight.
[0251] Alternatively, suitable derivatives of cyclodextrin can be
used to prepare the carrier including CD-blocks described
previously. Preferred CD derivatives are hydroxypropyl cyclodextrin
(HPCD) and 2,3-dihydroxypropyl cyclodextrin (DHPCD). For oxidation
to dialdehyde, a preferred form of HPCD or DHPCD is one with 3-4
degrees of substitution with propylene oxide or glycidol (Pitha,
supra).
[0252] Alternatively, other cross-linkers that provide longer
spacer lengths to avoid steric hindrance can be used. One example
is ethylene glycol bis(succinimidylsuccinate) (EGS, 16.1 angstrom
spacer). EGS is cleavable with hydroxylamine
[0253] Also, the Hz-CD can be polymerized using the water-soluble
bifunctional reagent dimethyl adipimate (DMA, 8.6 angstrom spacer,
Technical Bull #0438, Pierce). The parameters of molar ratios and
reaction times for cross-linking Hz-CD with DMA are suitably
optimized for the desired polymer size. A near saturated suspension
of Hz-CD and PTX in methanol is prepared in PB with vigorous mixing
as described previously. As the solvent evaporates, the drug is
forced into the aqueous phase to allow complexes to form between
the PITX and Hz-CD. The DMA is added and mixed for 2-6 hours. The
resulting PTX-CD is fractionated by gel exclusion chromatography as
described previously. The hydrazone linkages provide controlled
release when hydrolyzed to release free drug maximally at pH
4-5.
Preparation VI
Targeted Cyclodextrin Polymer Carrier with Entrapped
Doxorubicin
[0254] A. Preparation of CD Polymer with Incorporated Amino
Groups.
[0255] The purpose is to cross-link gamma cyclodextrin (Mol. Wt.
1297), to form a water-soluble (or colloidal) cyclodextrin polymer
carrier that has completely entrapped doxorubicin (DOX).
Cyclodextrins are crosslinked through their hydroxyl groups to each
other using 1,4 butanediol diglycidyl ether (BDE, Mol. Wt.
202.2).
[0256] In order to entrap the drug, the DOX is dissolved in a
solvent and mixed with the CD to form inclusion complexes. Then the
CD is cross-linked to form the polymer and completely entrap the
drug in polymer aggregates.
[0257] A preparation is made to give a molecular ratio between 1:2
and 1:8 of DOX to CD. While mixing, the drug is exposed to the CD
to allow complexes to form between the DOX and CD. The procedure is
to combine near saturated DOX with 100 ml of 4.0% cyclodextrin in
0-20% (v/v) dimethylformamide (DMF) in water, with 5 ml of 2 N NaOH
(starting pH 13), with vigorous mixing (20,000 rpm impeller). The
cross-linking reaction is initiated by adding 10 ml of 95% BDE
while ring and incubating at 60.degree. C.
[0258] The reaction is conducted for 2-6 hours followed by the
addition of a molar excess of lysine (0.75 ml of 4 M lysine in
water, adjusted to pH 8). Lysine is incorporated into the polymer
as the BDE cross-links the lysine through one of its amino groups
to the cyclodextrin. The excess lysine also couples to and blocks
any remaining free BDE. Mixing is continued for one more hour and
the mixture is then neutralized with 1 N HCl.
[0259] Aliquots of the drug-loaded CD polymer carrier are then
fractionated by gel exclusion chromatography on pre-calibrated
columns of Sephacryl.RTM. S200-HR (40.times.5 cm) equilibrated with
distilled H.sub.2O. Pre-calibration is done using various molecular
weight dextrans (i.e. 15,000 to 60,000, Sigma) in separate
runs.
[0260] The carrier fractions that elute in molecular weight ranges
between 20,000 and 50,000 are taken to the next step. Other
fractions of higher or lower molecular weight may also be suitable.
The fractions are then vacuum dried at 50.degree. C. and weighed.
The approximate moles of product are calculated as total grams of
dried carrier divided by the apparent molecular weight. The
relative amount of CD in the fractions can be monitored by a
calorimetric test as described previously. The CD polymer fractions
can also be tested for the presence of amino groups as described
previously.
[0261] If needed, additional lysine molecules can be added to the
PolyCD. The procedure is to again treat the carrier with BDE as
described above, but for only 20-30 minutes. Additional amino
groups are then introduced with the excess lysine treatment and the
product is fractionated on Sephacryl.RTM. as described.
[0262] Alternatively, a selective derivatization procedure is used
that takes advantage of the more reactive primary hydroxyls. The
procedure is to first "tosylate" two or more primary hydroxyls on
each cyclodextrin and then replace the tosyl groups with amino
groups. The cyclodextrins are then complexed with the DOX and
polymerized by cross-linking through the aminos using a
bifunctional cross-linking agent.
[0263] The tosylation step is done by reacting 12 grams of
cyclodextrin with 9 grams of p-toluenesulfonyl chloride (tosyl
chloride) in 100 ml of anhydrous pyridine solvent. The tosyl
chloride is added in 3 gram aliquots over a 36 hour period with
constant stirring of the mixture for a total of 48 hours. The
reaction is stopped with 20 ml of methanol. The product is
precipitated, filtered and washed with 200 ml aliquots of
chloroform, then dried.
[0264] The tosyl groups are substituted for azide by dissolving 1.3
gm of the tosylated cyclodextrin in 100 ml of dimethylformamide
(DMF) and adding 1 gm of sodium azide. The mixture is heated with
siring to 100.degree. C. for 2 hours and the product is collected
from dried supernatant. The product is dissolved in 10 ml of water,
precipitated with acetone and dried.
[0265] The azide cyclodextrin is reduced to the amine by dissolving
1 gm in 100 ml of 20% methanol/water containing 0.4 gm of palladium
black catalyst (Sigma). The mixture is stirred 1 hour under
H.sub.2, then filter through Celite. The amino-derivatized
cyclodextrin is collected by drying.
[0266] The resulting amino-cyclodextrin (amino-CD), can then be
complexed with DOX and polymerized using any water-soluble
bifunctional reagent such as dimethyl suberimidate (DMS, Mol. Wt
273.2), which is routinely used to selectively couple amino groups
Technical Bull., Pierce).
[0267] The parameters of molar ratios and reaction times for
cross-linking amino-CD with DMS are optimized for the desired
polymer carrier. Typically, to 100 ml of 4.0% amino-CD previously
complexed with DOX in 0.2 M triethanolamine HCl, pH 8.5 in water,
is added 4 gm of DMS and me at 60.degree. C. for 2-6 hours. The
reaction is stopped by the addition of a molar excess of lysine (4
M, adjusted to pH 8). Lysine is incorporated into the polymer as
the DMS cross-links the lysine and additional amino groups are
available for coupling to antibody. The excess lysine also couples
to and blocks any remaining free DMS. The mixing is continued for
one more hour and the mixture is then neutralized with 1 N HCl. The
resulting polymer is fractionated by gel exclusion chromatography
as described previously. Alternatively, any suitable biorecognition
molecule with an available amino group can be used in place of the
lysine such as antibodies or other proteins, polypeptides, or
amino-sugars. Also, other anticancer drugs can be used in place of
DOX such as daunomycin, puromycin or ellipticine.
[0268] B. Introduction of Sulfhydryl Groups by Thiolating Amines on
the CD Carrier.
[0269] The CD polymer carrier is thiolated by modifying the lysine
residues using 2-iminothiolane (FW 137.6), based on the technical
bulletin from Pierce Chem. Co. The number of available amino groups
on the carrier can be determined as described previously. The molar
ratio that is used between the carrier and 2-iminothiolane is about
1:10.
[0270] The reaction is carried out by combining 0.4 mmoles of
carrier dissolved in 0.16 M borate buffer (pH 8.0), and 4 mmoles of
2-iminothiolane. The mixture is mixed for about 2 hours at room
temperature. The resulting thiolated carrier is separated by gel
chromatography using a Sephadex.RTM. G15 column equilibrated with
0.05 M phosphate buffer, pH 7.5.
[0271] Aliquots of the thiolated carrier can be tested for the
presence of sulfhydryl groups. The test for sulfhydryl is a
standard test that employs 5,5'-dithiobis(2-nitrobenzoic acid,
DTNB). The procedure is to combine 0.1 ml of thio-polyCD samples
diluted in water with 1 ml of deoxygenated 0.2 M Tris buffer, pH
8.2, and add 0.1 ml of 0.01 M DTNB in deoxygenated methanol. Color
is allowed to develop for 30 minutes and the absorbance is read at
412 nm on a spectrophotometer. The results are compared to a
standard curve of identically tested dilutions of
2-mercaptoethanol. The goal is to introduce an average of at least
three sulfhydryl groups (2-mercaptoethanol molar equivalents), for
each mole of thiolated carrier.
[0272] C. Conjugation of the Thiolated CD Carrier to
Maleimide-Activated Antibody.
[0273] The molar ratio of thiolated carrier to antibody is about
4:1. For instance, 0.02 mmoles of the 20,000 molecular weight
fraction of thiolated carrier is conjugated with 0.005 mmoles of
antibody previously coupled through amino groups to
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS-antibody) (MW
140,000). Other molar ratios of thiolated carrier to antibody can
be used during conjugation.
[0274] The conjugation reaction is to combine freshly prepared
MBS-antibody with thiolated carrier in 0.05 M phosphate buffer, pH
7.5, and stir for 2 hours at room temperature. The conjugate is
fractionated by gel exclusion chromatography using bovine IgG
calibrated Sephacryl.RTM. S200-HR column equilibrated with the same
buffer. The fractionated conjugate is collected in a fraction
collector equipped with an ultraviolet monitor set at 280 nm to
detect the IgG. The conjugate is in the fractions containing IgG
and corresponding to molecular weights greater than 200,000.
[0275] The conjugate fractions can be tested for protein content
using the Bradford calorimetric method and tested for carbohydrate
as described previously. Fractions greater than 200,000 molecular
weight are pooled and concentrated by centrifugation filtration
using 100,000 molecular weight cutoff filter tubes (Micron
Separations Inc., Westboro Mass.) or by precipitation with ammonium
sulfate and dialysis against PB.
Preparation VII
Oxidized Cyclodextrin Polymer Carrier with Entrapped
Doxorubicin
[0276] (CD41B) The purpose is to prepare oxidize cyclodextrin (CD)
to provide dialdehydes that are then reacted with a diamino
compound such as 1,6 hexanediamine to provide terminal amino groups
for subsequent crosslinking to make the polymer carrier.
Alternatively, CD monomers, dimers or polymers can be derivatized
using methods previously described for introducing active groups
for coupling with amino groups. For instance, the CD (or CD-block)
can be suitably tosylated, or treated with various bifunctional
epoxy compounds such as BDE or GDE before coupling to the amino
compound.
[0277] (CD52) In some preparations, CD-block dimers, trimers and
polymers were first synthesized by crosslinking the CD with epoxy
coupling agents such as BDE, GDE or TMTE. For GDE, the procedure
was to combine about 4.5 gm of CD dissolved in about 60 ml of
60.degree. C. water with about 0.1 ml of 10 N KOH and about 0.5 ml
of GDE. The solution is mixed vigorously at 60.degree. C. for 2-4
hours or until the GDE is consumed. At this point the mixture can
be oxidized to produce dial-CD blocks, or the CD-blocks can be
treated with glycidol before oxidation to produce "polyaldehyde-CD"
for use in the next step. Alternatively, the un-oxidized CD-blocks
can be derivatized with amino or thiol groups for subsequent
crosslinking in CD carrier synthesis.
[0278] A. Preparation of Dialdehyde CD Using Oxidation.
[0279] The oxidation procedure is similar to that of Preparation V.
The procedure was to add 3.42 gm of sodium m-periodate (NaIO.sub.4)
to 4.546 gm of beta cyclodextrin dissolved in 60 ml of water and
0.5 ml of 10 N KOH at 70-80.degree. C. The molar ratio of
NaIO.sub.4 to cyclodextrin was 4:1. After one hour, the pH of the
solution was adjusted up to 14 with 0.03 ml of 10 N KOH and the
reaction was continued in the dark overnight.
[0280] The pH was adjusted to 7 with 0.02 ml of 10 N KOH and the
remaining NaIO.sub.4 was consumed with a 2.times. molar excess of
ethylene glycol (0.5 ml). The resulting dialdehyde cyclodextrin
(dial-CD) was clarified by filtration through a 0.2 micron filter
and fractionated on a Sephadex.TM. G-25 column. The leading
fractions containing dial-CD were pooled.
[0281] In some preparations, the dial-CD was subsequently
crosslinked using various epoxides such as BDE or GDE, which
produced CD-block dimers, trimers and polymers containing
aldehydes. Alternatively, the CD (or CD-block) has been treated
with glycidol (2-7.times. molar excess, pH 7-10) before oxidation,
which produces 2,3-dihydroxypropyl cyclodextrin (DHPCD) with
additional diols at the primary and secondary sides of the
molecule. Subsequent oxidation then converts the diols to aldehydes
to produce a "polyaldehyde-CD", which can then be coupled with
amino groups.
[0282] B. Preparation of 1,6 Hexanediamine-Coupled CD Monomer.
[0283] This reaction involves the reaction of the amino groups on
1,6 hexanediamine (HXDA) with available aldehydes on the dial-CD
(or poly-CD) to form aldehyde bonds with amino groups so that
ideally each aldehyde is coupled to a single HFA molecule with
minimal cross-linking. About a 4.times. molar excess of HXDA was
added to about 0.0014 moles of the dial-CD preparation in water
while stirring at room temperature. The pH was adjusted to 7 with
about 1.3 ml of 6 N HCl, and reacted overnight. The resulting 1,6
hexanediamine-coupled cyclodextrin monomer (HXDA-CD) was
concentrated by evaporation to about 15 ml. The entire solution was
applied to a Sephadex.TM. G-25 column in water. The fractions were
tested colorimetrically for carbohydrate content and amino groups.
The front fractions containing carbohydrate and amino groups were
pooled and HXDA-CD monomer was concentrated by evaporation to about
20 ml.
[0284] Alternatively, other amino compounds have been coupled to
the oxidized CD such as hydrazine, adipic add dihydrazide, glutamic
acid, beta-phenylethylamine, laurylamine and cystamine. Many other
useful amino compounds can be coupled to the oxidized CD or
CD-block such as polypeptides, 6-amino-N-hexanoic acid, arginine,
protamines, N-(2-aminoethyl)-1,3-propanediamine (AEPD),
polyethylenimine (PEI) and nucleic acids.
[0285] C. Preparation of HXDA-CD Polymer with Completely Entrapped
DOX.
[0286] (CD49) The purpose is to cross-link HXDA-CD monomer through
the terminal amino groups to form a water-soluble (or colloidal)
cyclodextrin polymer carrier. The cattier is prepared by combining
HXDA-CD monomer with DOX, then adding an "activated" HXDA-CD
monomer that polymerizes with the HXDA-CD and entraps the DOX in
situ. The "activated" monomer can be any CD monomer (or CD-block)
that has been treated to provide active coupling groups that will
crosslink with the amino groups on the other CD monomer or
CD-block. Monomers can be activated by treating them with a variety
of bifunctional coupling agents listed previously. Also, dial-CD
can be used as the activated monomer where the aldehyde groups can
couple to the amino groups of the HXDA-CD.
[0287] In this case, the HXDA-CD is treated with glutaraldehyde to
form aldehyde bonds with amino groups before combining it with the
other monomer and drug. Activated monomer was prepared by combining
about 0.126 gm of HXDA-CD in 2.1 ml water with 0.35 ml of 1 N NaOH,
and then adding 0.35 ml of 20% glutaraldehyde in water.
Drug-complexed monomer was prepared by combining about 0.021 gm of
H A-CD in 0.35 ml of water with about 3.25 mg of DOX in 0.28 ml of
water.
[0288] After about 20 minutes, drug-loaded carrier was prepared by
combining one half of the activated monomer (0.063 gm in 1.4 ml)
with the drug-complexed monomer preparation. A drug-free control
carrier was also prepared by combining 1.4 ml of the same activated
monomer solution with 0.35 ml of water containing 0.021 gm of
untreated monomer only. Both mixtures were allowed to crosslink for
about 1.5 hours, then dialyzed for about 1 hour in 12,400 molecular
weight cutoff (MWCO), cellulose tubing against 70% isopropanol in
water. Dialysis was continued for about three more hours against
distilled water.
[0289] After no detectable DOX was found in the dialysate, the
preparations were analyzed for DOX. DOX was measured by diluting
0.02 ml aliquots of sample into 0.18 ml of 1 N NaOH, reading the
absorbance at 620 nm and comparing the absorbances to a standard
curve of DOX. Aliquots of the preparations were also dried to
determine the weight % DOX.
[0290] The preparations were then tested in a cytotoxicity assay
using Daudi cells from human Burkitt lymphoma. The test is a
colorimetric assay based on the ability of metabolically active
cells to reduce thiazolyl blue (MTT) to a blue formazan product
(Alley, et al., Cancer Res. 48:589 (1988)).
[0291] The procedure was to incubate growth phase cells in RPMI
media with 10% fetal bovine serum and containing different
concentrations of each carrier preparation for 48 hours.
Cytotoxicity was determined as a function of the concentration of
carrier needed to inhibit growth by 50%. This was measured by a
reduction in the amount of colored product compared to untreated
control cells. With the control cell value taken as zero
cytotoxicity, there was a five-fold increase in cytotoxicity of the
DOX-loaded carrier vs. the control after 48 hours. Results are
presented in the following Table D.
4TABLE D Weight % Preparation DOX Conc. Total Dry Wt. % DOX
Cytotoxicity DOX-loaded 1.80 mg/ml 25.0 mg/ml 7.2 60.5 Control
Carrier 0 27.0 mg/ml 0 10.0 The cytotoxicity results show that the
drug was released to kill the cells.
Preparation VIII
Beta CD Polymer Carrier Crosslinked Through Sulfhydryls with
Entrapped Doxorubicin
[0292] (CD45) The purpose is to crosslink thiolated cyclodextrin
monomers through their sulfhydryl groups while they are complexed
with drug to completely entrap the drug in the cattier.
[0293] A. Preparation of Thiolated CD Monomer.
[0294] (CD41b) HXDA-CD was prepared as described previously. The
HXDA-CD was thiolated by slowly combining and mixing about 0.6 gm
of HXDA-CD in 10 ml of 0.1 M K.sub.2HPO.sub.4 buffer, pH 8.5, in
water with 0.33 gm of iminothiolane (2-IT). After about 2 hours,
the mixture was fractionated on a Sephadex.TM. G25 column in water.
The leading fractions containing the carbohydrate peak were pooled
and concentrated by evaporation to give about 0.076 gm per ml.
[0295] B. DOX-Loaded Carrier Prepared by Dithiol Crosslinking of
Thiolated CD Monomer.
[0296] In duplicate preparations, a 0.2 ml aliquot of the thiolated
CD monomer (about 0.012 mmoles) was combined with about 0.0002
mmoles of doxorubicin and mixed to allow complexing. Crosslinking
through dithiol linkages was initiated by the addition of 0.05 ml
of 30% H.sub.2O.sub.2 and heating over boiling water. Through
oxidation and coupling of the sulfhydryl groups to form dithiols,
the monomer becomes crosslinked. A polymer carrier formed that
entrapped the DOX in the carrier and formed a red particulate
suspension that was precipitated by centrifugation. The carrier
preparations were resuspended and dialyzed overnight in 12,400 MWCO
cellulose tubing against 10% isopropanol. The recovered dialysates
were again centrifuged and produced red pellets indicating that DOX
was still entrapped in the carrier.
Preparation IX
Coupling Methods for Targeting Cyclodextrin Polymer Carriers
[0297] These are methods for synthesizing cyclodextrin polymer
carriers wherein a coupling group is included in the composition to
provide for coupling to any suitable biorecognition molecule with a
suitable functional group. The biorecognition molecule can be a
suitable protein, including antibodies and avidins or streptavidin,
or ligands, or nucleic acids.
[0298] A. Preparation of NHS-CD Polymer Carriers.
[0299] In a suitable anhydrous solvent such as DMF, the
CD-carboxylic acid polymer is combined with N-hydroxysuccinimide
and an aromatic carbodiimide such as N,N-dicyclohexylcarbodiimide,
at approximately equimolar ratios and reacted at RT for 1-3 Hrs.
The product, N-hydroxysuccinimide cyclodextrin (NHS-CD), is
separated in the filtrate from precipitated dicyclohexylurea,
collected by evaporation and purified by chromatography.
[0300] Under appropriate conditions, NHS-CD polymer derivatives can
be prepared by coupling NHS esters directly to amino-CD or amino-CD
polymer. Preferably, the NHS ester is a bifunctional NHS coupling
agent with a suitable spacer. Suitable NHS coupling agents for use
in this invention have been previously described, including DSS,
bis(sulfosuccinimidyl)sube- rate (BS.sup.3), DSP, DTSSP, SPDP,
BSOCOES, DSAH, DST, and EGS, among others.
[0301] B. Preparation of Sulfhydryl-CD Polymer Carriers.
[0302] Sulfhydryls on polymer carriers can be used for disulfide
coupling to other available sulfhydryls on the desired
biorecognition molecule such as antibodies, or avidins, or
streptavidin, or ligands, or nucleic acids. If needed, the
available sulfhydryls can be introduced by thiolation of the
biorecognition molecule before coupling. Alternatively, a
sulfhydryl-containing CD polymer carrier is coupled to any
maleimide derivative of protein, ligand, nucleic acid or biotin,
(e.g. biotin-maleimide) or iodoacetyl derivatives such as
N-iodoacetyl-N'biotinylhexylenediamine.
[0303] C. Maleimido-Cyclodextrin Polymer Carriers and
Iodo-Cyclodextrin Polymer Carriers.
[0304] The maleimido-cyclodextrin polymer carriers (Mal-CD), of
this invention are suitable for coupling to native or introduced
sulfhydryls on the desired biorecognition molecule.
[0305] A maleimido group is added to an amino-CD polymer carrier
suitably prepared as described previously, by coupling a suitable
hetero-bifunctional coupling agent to the available amino group.
The hetero-bifunctional coupling agent consists of a suitable
spacer with a maleimide group at one end and an NHS ester at the
other end. Examples are previously described and include MBS, SMCC,
SMPB, SPDP, among others. The reaction is carried out so that the
NHS ester couples to the available amino group on the CD polymer
carrier, leaving the maleimide group free for subsequent coupling
to an available sulfhydryl on a biorecognition molecule.
[0306] Under appropriate conditions, Iodo-Cyclodextrin (Iodo-CD)
polymer carriers can be prepared for coupling to sulfhydryl groups.
For instance, NHS esters of iodoacids can be coupled to the
amino-CD polymers. Suitable iodoacids for use in this invention are
iodopropionic acid, iodobutyric acid, iodohexanoic acid,
iodohippuric acid, 3-iodotyrosine, among others. Before coupling to
the amino-CD polymer, the appropriate Iodo-NHS ester is prepared by
known methods (e.g. Rector, supra). For instance, equinolar amounts
of iodopropionic acid and N-hydroxysuccinimide are mixed, with
suitable carbodiimide, in anhydrous dioxane at RT for 1-2 Hrs, the
precipitate removed by filtration, and the NHS iodopropionic acid
ester is collected in the filtrate. The NHS iodopropionic acid
ester is then coupled to the amino-CD polymer carrier.
Preparation X
Biotinylated Cyclodextrin Polymer Carriers
[0307] Biotinylated CD polymer carriers can be produced by a
variety of known biotinylation methods suitably modified for use
with CD's. For instance, combining an amino-CD polymer derivative
with a known N-hydroxysuccinimide derivative of biotin in
appropriate buffer such as 0.1 M phosphate, pH 8.0, reacting for up
to 1 hour at room temperature. Examples of biotin derivatives that
can be used are, biotin-N-hydroxysuccinimide, biotinamidocaproate
N-hydroxysuccinimide ester or sulfosuccinimidyl
2-(biotinamino)ethyl-1,3'-dithiopropionate, among others.
[0308] Through the use of suitable protection and deprotection
schemes, as needed, any CD polymer carrier of the instant invention
can be coupled to biotin or a suitable derivative thereof, through
any suitable coupling group. For instance, biocytin can be coupled
through an available amino group to any NHS-CD label described
herein. Likewise, thiolated biotin can be coupled to any mal-CD
polymer carrier.
[0309] The biotinylated CD polymer carrier can be used to couple a
plurality of carriers to an intermediate. For instance, by
combining dilute solutions of the biotinylated CD carrier with
avidin or streptavidin in the appropriate molar ratio, 1, 2 or 3
biotinylated CD carriers will couple to the avidin or streptavidin
and produce a complex with one or more biotin-binding sites still
available.
Preparation XI
A Cyclodextrin Polymer Carrier Prepared on a Solid Support
[0310] Another embodiment for a water-soluble (or colloidal)
cyclodextrin polymer carrier can be synthesized with a more
predictable number of CD molecules, giving new advantages of
uniform structure and chemical properties. The synthesis method is
to couple an initial CD molecule (or derivative) to a solid support
using a cleavable coupling agent. Then additional CD molecules (or
CD derivatives with or without protected groups) are attached to
the first CD in a controlled, step-wise manner. Alternatively, a
suitable intermediate substance (i.e. amino derivatized PEG or
HPMA) can be initially coupled to the solid support and CD
molecules coupled to it. After the desired number of CD molecules
have been linked together to form an open polymer, the carrier is
then cleaved from the solid support. The desired drug or other
active agent is then allowed to complex with the polymer. Also, the
polymer can be further cross-linked to close the polymer and
completely entrap the active agent.
[0311] The CD molecules used in this procedure can include tethered
guests and antenna substances, and be suitably derivatized and/or
capped before coupling to incorporate other desirable features.
However, it is preferred that each CD molecule (or dimer, or
trimer), that is coupled, has a well-defined structure to
facilitate the production of CD polymer carriers with uniform and
consistent properties.
[0312] A variety of suitable materials, such as those used in
chromatography (e.g. Smokova-Keulemansova, supra), can be used for
a solid support. The solid support can be in the form of particles,
beads, fibers, plates, and tubing walls, and composed of styrenes,
acrylamides, silica gels, solid or porous glass, dextrans, and
celluloses, among others that are suitably derivatized as needed
and compatible with the reactions used.
[0313] The initial coupling agent used to couple the initial CD or
intermediate substance to the support is preferably one that is
readily cleaved when desired. Suitably, the initial coupling agent
is a bifunctional, amino-reactive reagent such as those with a
cleavable disulfide group, including DTBP, DSP, DTSSP and
photoactive couplers like BASED, SADP, SAND, and SASD. Other
suitable initial amino-reactive coupling agents are periodate
cleavable, such as DST and sulfo-DST, or hydroxylamine cleavable at
the ethyl ester linkage, such as EGS and sulfo-EGS.
[0314] The coupling agents used to couple subsequent CD's to make
an open polymer are preferably noncleavable, or biocleavable, or
cleavable by a different mechanism than the initial coupling agent
When coupling through amino-derivatized CD molecules,
amino-reactive, bifunctional coupling agents such as DMA, DMP, DMS,
DSS and DSG would be used. When coupling through
sulfhydryl-derivatized CD molecules, sulfhydryl-reactive,
bifunctional coupling agents would be used such as MBS. Diepoxy
coupling agents such as BDE can be used to couple through amino,
sulfhydryl or hydroxyl functional groups.
[0315] In another embodiment, when the polymer is cleaved from the
support after synthesis, it can leave a suitable functional group
for targeting by subsequently coupling a biorecognition molecule to
the polymer. Or, the remaining functional group can be converted to
an NHS ester by various known means for subsequent coupling to an
amino group on a biorecognition molecule.
CD Polymer Carrier Synthesis
[0316] A suitable method for synthesizing water-soluble (or
colloidal) cyclodextrin polymer carriers of the instant invention
is as follows;
[0317] 1. A suitable amino-derivatized solid support is prepared.
For instance, porous glass beads or predried silica gel is
amino-derivatized with (3-aminopropyl)trimethoxysilane. The solid
support is then treated for coupling (activated), with a
bifunctional, cleavable disulfide coupling agent, DSP. The
uncoupled reagents are removed.
[0318] 2. To the support is added for coupling, an excess of
amino-CD derivative, or amino-2, 3 dihydroxypropyl beta
cyclodextrin (amino-DHPCD) or amino-derivatized HPMA (amino-HPMA).
The uncoupled reagents are removed.
[0319] 3. The initial CD or intermediate substance is then treated
for additional coupling (activated) with a suitable bifunctional
coupling agent that will react with the initial CD and subsequently
couple to additional CD molecules. For instance, with amino-DHPCD
or amino-HPMA, an amino-reactive agent such as DSS is used. Or, the
initial CD or intermediate substance can be treated with a diepoxy
such as BDE. The unreacted coupling agent is then removed.
[0320] 4. An excess of amino-DHPCD molecules (which may include
some halogenated alkylphalimide protected amino groups) are then
added to covalently couple to the initial CD or intermediate
substance. The unreacted amino-DHPCD molecules are then
removed.
[0321] 5. The immobilized preparation is then treated again for
additional coupling with a suitable bifunctional coupling agent
such as DSS or BDE. The unreacted coupling agent is then
removed.
[0322] 6. Another cycle of excess amino-DHPCD molecules are then
added as before to covalently couple to the preparation and
unreacted reagent is removed.
[0323] 7. Depending on how large an open polymer is desired, the
steps are repeated of activating the preparation again with
coupling agent, removing the unreacted agent, adding excess
amino-DHPCD molecules for coupling and removing the unreacted CD
molecules. After the last cycle of DHPCD molecules have been
coupled to the preparation, unreacted molecules are removed.
[0324] 8. The cyclodextrin polymer carrier is then cleaved from the
solid support by reduction of the disulfide bond in the initial
coupling agent.
[0325] 9. To the open polymer preparation the desired drug or other
active agent is added and allowed to complex with the polymer. In
this example, puromycin is added to allow inclusion complexes to
form with the polymer.
[0326] 10. If desired, the drug-loaded polymer can be further
cross-linked to dose the polymer and completely entrap the active
agent In this example, the amino-reactive coupling agent DSS is
used to cross-link the available amino groups. If employed,
previously protected amino groups are made available by a
deprotection step before final cross-linking.
[0327] Other modifications can be included before final cleavage.
For instance, acid-labile linkages can be incorporated into the
final cross-linking to provide controlled release of entrapped
active agent. In one embodiment, vicinal hydroxyls on the DHPCD
molecules of the open polymer can be oxidized to dialdehydes using
Na metaperiodate. The dialdehydes are then coupled to hydrazine to
provide acid-labile hydrazone linkages with terminal amino
groups.
[0328] The open polymer is then loaded with drug as before and then
closed by cross-linking the terminal amino groups. The final
cross-linking is done using a bifunctional, amino-reactive coupling
agent such as DSS or BDE. Also, the drug-loaded carrier can be
treated with acetic or succinic anhydride to give carboxylates that
are converted to NHS esters through reaction with carbodiimides and
N-hydroxysuccinimide. The carrier can also be targeted by coupling
it to a suitable biorecognition molecule.
Preparation XII
A Cyclodextrin Polymer Carrier Prepared From a Cyclodextrin
Monolayer
[0329] Another embodiment for a water-soluble (or colloidal)
cyclodextrin polymer carrier can be synthesized wherein the
cyclodextrin monomers are first cross-linked to form an open
polymer that is in the form of a sheet or layer of cross-linked
cyclodextrin molecules. This embodiment can provide new advantages
of organized structure and chemical properties.
[0330] In the first step of the synthesis method, CD molecules (or
CD derivatives such as HPCD or DHPCD) are positioned on a surface
so that their primary or secondary ends are facing the surface and
their edges are within coupling distance of each other. Ideally,
all of the CD molecules are oriented in the same direction. One way
of accomplishing this is to prepare a surface (i.e. a solid support
or flexible surface) onto which guest molecules have been
covalently coupled (i.e. through spacer groups) so that each guest
is available to form an inclusion complex with a cyclodextrin.
Suitably, guest molecules are used that force the cyclodextrin
molecules to bind to them in only one orientation. For instance, if
the guest molecules are just big enough, they will form the
strongest binding inclusion complex by only entering the larger,
secondary end of the cyclodextrin molecule and not the smaller
primary end. Examples of the most preferable inclusion compounds,
especially with aromatic compounds, are well known for alpha CD,
beta CD and gamma CD. For instance, adamantane and anthracene
derivatives bind primarily through the secondary end of beta CD and
pyrene derivatives bind primarily through the secondary end of
gamma CD. In the following example, 2-aminoanthracene can be
replaced with a suitable amino-derivatized adamantane such as
1-aminoadamantane, 1-adamantane methyamine, or 1-adamantane
carboxamide.
[0331] In this example, 2-aminoanthracene is coupled to
amino-derivatized glass beads using a bifunctional NHS coupling
agent such as DSS. The 2-aminoanthracene is immobilized in very
high density so that many molecules are within one beta CD
diameter's distance apart (i.e. about 6 angstroms). Then in
suitable solvent or aqueous buffer, beta CD is mixed with the beads
to form inclusion complexes with the immobilized 2-aminoanthracene
on the bead surface. The excess CD may be removed.
[0332] Then the complexed CD molecules are cross-linked with a
diepoxy such as BDE, or a triepoxy such as glycerol propoxylate
triglycidylether, so that every CD molecule is coupled to at least
two (preferably 3 or 4) of its neighbor CD molecules.
Alternatively, derivatized CD molecules can be used such as
amino-CD or amino-HPCD or amino-DHPCD, and then cross-linked using
a cleavable, bifunctional coupling agent such as DSP, DST or EGS.
Also, amino-CD molecules can be cross-linked using a suitable
biocleavable coupling agent described herein.
[0333] The resulting open polymer is a sheet or monolayer of CD
molecules (or CD derivatives with or without protected groups). The
resulting CD monolayer is then removed from the immobilized
2-aminoanthracene molecules by using a suitable competing solvent
or surfactant to cause dissociation.
[0334] The CD monolayer is then mixed, in suitable solvent or
aqueous buffer, with a drug or other active agent (i.e. paclitaxel)
to allow the monolayer to form inclusion complexes with the drug.
The drug-loaded CD monolayer is then further cross-linked to close
the polymer and completely entrap the drug. The resulting CD
monolayer carrier can also be targeted by coupling it to a
biorecognition molecule. The CD monolayer carrier can be further
derivatized to provide functional groups that are then used for
coupling to the biorecognition molecule.
Preparation XIII
CD Polymer Carriers with Antenna Substances
[0335] A new water-soluble (or colloidal) CD polymer carrier with
potentially greater cytotoxic or catalytic efficiency can be
synthesized by incorporating antenna substances. An antenna
substance is defined as certain light and/or energy collecting
substances that transfer the energy to a catalyst or energy emitter
in the carrier. The various antenna substances of the invention can
be conjugated and/or noncovalently coupled in "close proxnity" so
that they will cooperatively participate in an energy transfer
reaction resulting in the emission of energy or a product. The most
preferred application is in photodynamic therapy where photoactive
agents are used to kill cancer cells.
[0336] The antenna substances can be coupled to the carrier in
various ways to promote the most efficient cytotoxic or catalytic
activity. For instance, the antennas can be covalently coupled to
the CD derivative or CD polymer, to the guest photoactive agent, or
to an intermediate substance that is part of the CD carrier.
Certain photosynthetic antenna substances (e.g. chlorophylls,
pigments) can also be coupled noncovalently to the CD carrier
through binding to certain polypeptides (e.g. from photosynthetic
plants, algae and bacteria), which are then covalently bound to the
CD carrier. Examples of photosynthetic substances are described by
H. Zuber, TIBS 11, 414-419, October, 1986, and J. Deisenhofer, et
al, Science 245, 1463-1473 (1989), the contents of which are
incorporated herein by reference.
[0337] Suitable antenna substances are any aliphatic, aromatic or
heterocyclic compounds that are capable of collecting light energy
or photons. The most preferred antenna substances for use in
photodynamic therapy are those that absorb infrared and far
infrared light Examples include carotenoids, folic acids, retinols,
retinals, rhodopsins, viologens, chlorophylls,
bacteriochlorophylls, phycobiliproteins, phycoerythrins,
porphyrins, MCe.sub.6, open chain tetrapyrroles (bilins),
tryptophan and/or tyrosine-containing substances (e.g.
polypeptides), Rose Bengal, fluorophores, scintillators, and
various derivatives, analogs and precursors of the antenna
substances.
Preparation XIV
Cyclodextrin Catalyst Agent
[0338] A cyclodextrin catalyst agent is a new invention defined
herein as a cyclodextrin derivative of an individual cyclodextrin,
or dimer, trimer or polymer, wherein the CD derivative host
functions as an "artificial enzyme", and certain guest molecules
function as chemical substrates. When the chemical substrate comes
in contact with the cyclodextrin catalyst agent under appropriate
conditions, it is modified to produce a product that is inhibitory
or toxic to certain cells, microbes or parasites. In one
embodiment, the CD catalyst agent is coupled to a biorecognition
molecule for targeting specific infected cells, cancer cells,
tissues or disease organisms.
[0339] With suitable derivatization, the CD catalyst agents can be
synthesized to bind and modify prodrugs into active drugs and
modify other specific substrates. The CD catalyst agent can also
catalyze specific reactions such as generate free radicals,
including singlet or triplet oxygen that directly kills infected
cells, cancer cells, or disease organisms.
[0340] Preferred CD catalyst agents include various photosensitizer
substances, especially those used for singlet and triplet oxygen
formation useful for photodynamic therapy (van Lier, J. E. In
"Photodynamic Therapy of Neoplastic Disease"; Kessel, D., Ed., CRC
Press, Boca Raton, Fla., 1990, Vol. I), including meso-chlorin
e.sub.6 monoethylenediamine (Mce.sub.6), phytalocyanine, porphyrins
and their derivatives and analogs.
[0341] Suitably, the CD catalyst agent requires derivatives that
provide a "recognition site" and one or more "catalytic groups" on
the CD agent (e.g. references; Ikeda, VanEtten, Hirai or Tabushi).
Depending on the CD molecule used, the substrate to be catalyzed,
and the reaction intended, the recognition site and catalytic
groups can be provided through one or several derivatives, as
needed. The recognition site generally involves the hydrophobic
cavity of the CD molecule, and provides a means for specifically
binding and/or orienting the substrate of interest with the CD
catalytic agent.
[0342] The catalytic groups are generally organic and/or inorganic
chemical residues, functional groups and ionic species that provide
a suitable chemical environment for promoting the catalytic
reaction. The catalytic groups can be any known chemical residue or
species that provides the desired catalytic reaction, including
carboxylates, imidazoles, histamines, hydroxyls, amines, amides,
aldehydes, ketones, phosphates, sulfhydryls, halogens, amino adds,
nucleic acids, chelators, and metals. Additional examples of
suitable catalytic groups useful in the instant invention can be
found in the art of derivatizing CD's and derivatizing or "genetic
engineering" of antibodies for use as enzymes. Other suitable
references are; M. L. Bender, I. Tabushi E. Baldwin, P. G. Schultz
(below).
[0343] In addition, an improved CD catalyst agent can be
synthesized wherein the recognition site and/or catalytic groups is
provided or augmented through the use of one or more suitable
captured guests, described herein, that interact (e.g. binding,
alignment and/or excimer formation), with the substrate being
catalyzed. In this case, the captured guest is preferably coupled
to the CD molecule by a suitable spacer group to allow interaction
with the substrate, and can be any suitable aliphatic, aromatic, or
heterocyclic compound, including any suitable inclusion compounds
described herein.
[0344] Suitable CD catalyst agent reactions include hydrolysis
(e.g. of any suitable ester or amide containing substrates),
oxidation, dephosphorylation, acid-base catalysis, formylation,
dichloromethylation, carboxylation, rearrangement, substitution,
allylation, and halogenation, among others. In any case, the
catalyst CD agents can be prepared so that the catalyst CD product
is inhibitory or toxic to certain cells, microbes or parasites. The
cyclodextrin catalyst agents can also be coupled to a variety of
substances, such as biorecognition molecules, ligands, antigens,
antibodies, nucleotides, nucleic acids, and liposomes, as well as
to a variety of support materials including magnetic particles for
use in assays and chemical processes.
[0345] An improved CD catalyst agent comprises the direct or
indirect coupling of any suitable antenna substance described
herein, to the CD molecule, for collection of light energy that is
transferred to the CD catalyst and thereby accelerates the desired
reaction such as in photodynamic therapy.
Preparation XV
Amylose Polymer Carriers for Active Agents
[0346] The helical segments of amyloses, can be suitably
polymerized, derivatized and/or capped to produce a carrier for
drugs, nucleic acids and other active agents wherein suitable
functional and/or coupling groups are included. Yet another
composition includes the use of "self assembly" substances coupled
to the amylose.
[0347] Suitably, these amylose polymers have the necessary
properties to form an inclusion complex with all or part of a
nucleic acid, especially those with little or no net charge such as
certain sense and antisense ODNs. Or, suitably, these amylose
polymers have primary, secondary or tertiary amine groups to
provide the necessary cationic charge for complexing with all or
part of a suitable nucleic acid.
[0348] Preferred substances are soluble or colloidal polymers of
helical segments of amyloses. Especially useful are helical amylose
molecules of more than 5 and less than 120 glucose units, that
favor formation of rigid linear helixes.
[0349] In one preferred embodiment, a suitable amylose polymer or
amylose segments are derivatized by various methods described
herein for cyclodextrins, to provide cationic amine groups along
the "edges" of the amylose chain. Then, a suitable anionic nucleic
acid is mixed with the derivatized amylose to allow complexing to
form between the cationic amylose and the anionic nucleic acid.
[0350] In another preferred embodiment, the amylose segments are
first derivatized before including a nucleic acid. For instance,
some or all of the available hydroxyl groups are suitably thiolated
by various methods described herein for thiolating cyclodextrins,
to provide sulfhydryl groups along the "edges" of the amylose
chain.
[0351] Sulfhydryls are introduced through reaction of available
hydroxyls with a suitable epoxy compound. For instance,
epichlorohydrin or a suitable diepoxy crosslinker previously
described, is coupled to a CD or CD polymer wherein free epoxy
groups are produced. Free epoxy groups are then reacted with sodium
thiosulfate to give thiosulfate esters (e.g. Carlsson, supra). The
thiosulfate esters are subsequently reduced to sulfhydryls with
dithiothreitol.
[0352] Then, a nucleic acid is mixed with the derivatized amylose
to allow inclusion complexes to form under mild reducing
conditions. After the nucleic acid is wholly or partially
incorporated into the amylose, the complex is exposed to mild
oxidation, which causes the sulfhydryl groups to crosslink and
entrap the nucleic acid in the amylose.
[0353] In another preferred embodiment, the amylose segments are
derivatized by various methods described herein for cyclodextrins,
to provide amino groups along the "edges" of the amylose chain.
Then, a nucleic acid or other active agent is mixed with the
derivatized amylose to allow inclusion complexes to form. After the
active agent is wholly or partially incorporated into the amylose,
the complex is exposed to any suitable amino-specific crosslinking
agent, which entraps the active agent within the amylose.
[0354] Another preferred embodiment has incorporated cross-links
that contain biocleavable linkages between the sulfhydryl or amino
groups as described previously.
[0355] Helical amylose polymers can be targeted by coupling them to
biorecognition molecules such as proteins, polypeptides, lipids,
lipoproteins, nucleic adds, surfactants, virus coat proteins, and
organic molecules. They can include intermediate substances of
acrylamides (HPMA), PEG, nylons, polystyrenes, resins, metals and
celluloses, and their combinations.
Preparation XVI
Micelle Polymer Carriers with Controlled Release
[0356] A micelle polymer carrier is a new invention defined herein
as a water-soluble (or colloidal) micelle that has been suitably
polymerized so that it completely entraps a nucleic acid or other
active agent. The formation of micelles for carrying drugs, nucleic
acids and other active agents is well known. However, micelle
carriers of the prior art suffer from uncontrolled loss of the drug
due to diffusion. This invention solves that problem through
cross-linking the micelle components to completely entrap the drug
or other active agent until it is delivered to the site of
action.
[0357] For this invention, any suitable technology now used for
preparing drug-carrying micelles is applicable to the synthesis of
this invention including disclosures of reagents for preparing
liposomes and those of Alkan-Onyuksel, H., Pharmaceutical Res. 11,
206-212 (1994). A distinguishing property of this invention is that
the micelle-forming components must have suitable functional groups
available on their hydrophilic "heads" to permit cross-linking
after the micelle has been formed with a drug inside.
[0358] In one preferred embodiment, suitable micelles are formed
that contain a drug. Then the head groups in the hydrophilic
surface are suitably cross-linked using various bifunctional
cross-linking agents so that the micelle cannot release the
entrapped drug. Another preferred embodiment has incorporated
cross-links that contain biocleavable linkages as described
previously. Also, this carrier can be suitably targeted by coupling
suitable biorecognition molecules to the surface.
Preparation XVII
Amphiphilic Cyclodextrin Dimers, Trimers and Polymers
[0359] The purpose is to prepare a cure of water-soluble (or
colloidal) amphiphilic cyclodextrin dimers, trimers and polymers
with alkyl carbon chains attached. The cyclodextrins are
cross-linked through hydroxyl groups using 1,4 butanediol
diglycidyl ether (BDE) at low pH to favor reaction at the primary
hydroxyl groups. Excess BDE molecules coupled at one end to the CD
provide terminal oxirane groups that are subsequently thiolated by
reaction with thiosulfate and reduction. Alkyl carbon chains ate
coupled to the CD derivatives using a "long chain epoxy" that
couples to other available hydroxyl groups (CD88).
[0360] A. Cross-Linking with BDE.
[0361] Into 125 ml of hot water (70-80.degree. C.) adjusted to pH
4.5-5 with 0.05 ml 6 N HCl, was dissolved 2.84 gm of beta
cyclodextrin (0.0025 moles). To this solution 4.1 ml of BDE (about
0.0125 moles) was added with mixing and continued heating for about
2 hours.
[0362] B. Coupling with a Long Chain Epoxy.
[0363] The mixture was adjusted to pH>10 with KOH and 1.28 gm
(about 0.005 moles) of dodecyl/tetradecyl glycidyl ether (DTGE) was
added and mixed vigorously. The solution was periodically mixed for
about 1.5 hours, heated for about 3 hours and then left at room
temperature (rt) overnight The resulting solution was light yellow
and turbid.
[0364] C. Coupling with Na Thiosulfate.
[0365] To the reheated mixture, 6 gm (about 0.025 moles) of sodium
thiosulfate was added and mixed. After about 1 hour, the pH was
adjusted to 7 with KOH and the solution was heated for about 3.5
hours more. Excess DTGE was removed by chilling to solidify the
DTGE and the solution was decanted.
[0366] D. Dialysis.
[0367] The mixture was dialyzed against a continuous flow of
distilled water in 500 mwco tubing (Spectra/Por CE) for about 40
hours. The solution was concentrated by evaporation to 8 ml to give
a clear, light yellow solution.
[0368] E. Reduction with Dithiothreitol to Provide Thiol
Groups.
[0369] To the mixture, 8 ml of water and 0.96 gm (about 0.0062
moles) of dithiothreitol (DTT) was added, mixed and left overnight
The turbid solution was then dialyzed against a continuous flow of
distilled water in 500 mwco tubing (Spectra/Por CE) for about 40
hours. The solution was concentrated by evaporation to 3.7 ml to
give a clear, yellow solution. Total yield based on dry weight was
2.276 gm of thiolated, amphiphilic cyclodextrin polymer.
[0370] F. Column Chromatography and Testing.
[0371] The mixture was fractionated on a Sephadex.TM. G15 column
(2.5.times.47 cm) in water. The fractions were tested for relative
carbohydrate and thiol concentration.
[0372] Carbohydrate was tested for by combining: 0.012 ml of
sample, 0.01 ml of 1.5% 1-naphthol in methanol and finally 0.1 ml
of 36N H.sub.2SO.sub.4 to produce a color reaction. The absorbance
was read at 620 nm and sample carbohydrate concentration was
calculated by linear regression using values from a cyclodextrin
standard curve.
[0373] Thiol groups were tested for by combining: 0.008 ml of
sample and 0.1 ml of 0.0125% 2,2'-dithio-bis(5-nitropyridine)
(DTNP) in 62.5% isopropanol, pH 6 to produce a color reaction. The
absorbance was read at 405 nm and sample thiol concentration was
calculated by linear regression using values from a cysteine
standard curve.
[0374] Fractions with corresponding peak concentrations of
carbohydrate and thiol were pooled and concentrated by evaporation.
The final volume was 2.2 ml and the total yield based on dry weight
was 1.144 gm.
[0375] The resulting mixture of thiolated amphiphilic CD dimers,
trimers and polymers was highly water soluble and amorphous
(glassy) when dried.
Preparation XVIII
Cyclodextrin Dimers, Trimers and Polymers with Biocleavable
Polypeptide Linkages
[0376] The purpose is to prepare cyclodextrin dimers, trimers and
polymers crosslinked through biocleavable polypeptide linkages.
This reaction employs the hetero-bifunctional crosslinking agent,
m-maliemidobenzoyl-N-hydroxysuccinimide ester (MBS) to crosslink an
amino-containing cationic substance with the thiolated CD dimers,
trimers and polymers of Preparation XVII (CD95). In this example,
the biocleavable linkage of leucine enkephalinamide, which contains
the peptide sequence: Tyr-Gly-Gly-Phe-Leu (Sigma) is coupled to the
cyclodextrin dimers, trimers and polymers.
[0377] To about 0.1 ml of 50% dimethylformamide (DMF) and phosphate
buffered water, pH 6.5, containing about 0.0044 grams of leucine
enkephalinamide, was added about 0.0036 gm of MBS dissolved in 0.1
ml DMF. This was mixed and allowed to react for about 30 minutes,
which produced a clear solution. Then about 0.08 ml of water
containing about 0.028 gm of the thiolated mixture of CD dimers,
trimers and polymers (Prep. XVII) was added and mixed. After about
1 hour, the solution was clear but had turned brownish-orange, it
was left overnight. The clear, brownish-orange mixture was
exhaustively dialyzed first against 70%, then 35%, then 23%
isopropanol, then finally distilled water in 2000 mwco dialysis
tubing (Sigma). During the dialysis, the clear brownish-orange
solution had turned very turbid with a precipitate, indicating a
crosslinked product was produced.
Preparation XIX
Cationic Cyclodextrin Dimers, Trimers and Polymers
[0378] The purpose is to prepare cyclodextrin dimers, trimers and
polymers with cationic groups attached. This reaction employs the
hetero-bifunctional crosslinking agent,
m-maliemido-benzoyl-N-hydroxysucc- inimide ester (MBS) to crosslink
an amino-containing cationic substance with the thiolated CD
dimers, trimers and polymers of Preparation XVII (CD95).
[0379] To about 0.1 ml of 50% dimethylformamide (DMF) and phosphate
buffered water, pH 6.5, containing about 0.003 mmoles of
neutralized polyethylenimine, mw about 800 (PEI800) was added about
0.0018 gm of MBS dissolved in 0.05 ml DMF. This was mixed and
allowed to react for about 20 minutes, which produced a turbid
solution. Then about 0.08 ml of water containing about 0.028 gm of
the thiolated mixture of CD dimers, trimers and polymers (Prep.
XVII was added, mixed, and left overnight.
[0380] The mixture was exhaustively dialyzed first against 70%,
then 35%, then 23% isopropanol, then finally distilled water in
2000 mwco dialysis tubing (Sigma). The product was tested for
binding to DNA (Promega DNA ladder) by first mixing 0.008 ml of
3-fold serially diluted aqueous solutions of the cationic CD
polymer preparation with equal volumes containing about 1 microgram
of DNA. These mixtures were then run on 2% agarose gel
electrophoresis in Tris-borate EDTA buffer (with ethidium bromide
dye) at 60 volts, 36 milliamps for about 1 hour. The results were
viewed and photographed over UV illumination. Results showed that
the DNA bands of the mixture diluted out 81-fold were significantly
diminished compared to DNA alone. This indicates that the cationic
mixture of amphiphilic CD dimers, trimers and polymers bound to the
DNA and inhibited migration through the gel.
Preparation XX
Phosphoramidite-Adamantane
[0381] Using suitable synthesis methods based on those described
and referenced, phosphoramidite groups can be coupled to adamantane
or other suitable guests for use as CD linkers.
[0382] A new CD linker comprises phosphoramidite or other suitable
nucleotide analog coupled to an adamantane dimer, adamantane trimer
or small adamantane polymer (poly adamantane). This new linker is
synthesized to allow incorporation into nucleic acids without
adversely affecting their function and may also include suitable
protective groups such as FMOC as needed. The resulting
phosphoramidite-adamantane can then be used in DNA synthesizing
machines for production of guest-linked oligonucleotides or ODNs or
primers for PCR, with dimer or poly adamantane incorporated into
their structure.
Preparation XXI
Amino- and Thiol-Reactive Adamantane Dimers
[0383] A. Preparation of NHS-Adamantane and NHS-Adamantane
Dimers.
[0384] In a suitable anhydrous solvent such as DMF,
adamantanecarboxyate is combined with N-hydroxysuccinimide and an
aromatic carbodiimide such as N,N-dicyclohexylcarbodiimide, at
approximately equimolar ratios and reacted at RT for 1-3 Hrs. The
product, N-hydroxysuccinimide adamantane (NHS-Adamantane), is
separated in the filtrate from precipitated dicyclohexylurea,
collected by evaporation and purified by column chromatography.
[0385] An adamantane dimer (or polymer) with an NHS ester group for
coupling is made by first coupling the adamantanes to a suitable
substance with two (or more) available amino groups (suitably
temporarily protected if needed) and a carboxylate group. Some
examples are 3,5-diaminobenzoate, diaminopentanoic add (ornithine),
lysine and carboxylated low molecular weight polyethylenimine,
among others. For instance 1-adamantane carbonyl chloride is first
coupled to the amino groups of 3,5-diaminobenzoate (or the
temporarily protected ethyl ester if needed) in anhydrous
conditions. The unprotected carboxylate group of the resulting
3,5-di-adamantane benzoate is then derivatized to an NHS ester as
described for NHS-Adamantane, above. The NHS ester-adamantane dimer
can then be coupled to any suitable drug nucleic acid or other
active agent with an available amino group.
[0386] B. Preparation of Thiol-Reactive Adamantane.
[0387] Alternatively, the 3,5-di-adamantane benzoate of A., above,
is derivatized to a maleimido ester instead of an NHS ester, for
sulfhydryl coupling.
[0388] To prepare 2-bromo-N-acetamide adamantane, compounds
1-hydroxy adamantane or 1-adamantane methanol are derivatized based
on the procedure of B. Frisch, et al., Bioconj. Chem. 7,180-186
(1996).
[0389] To prepare maleimido adamantane, the compound
1-aminoadamantane is coupled through the amino group to
m-maleimidobenzoyl-N-hydroxysuccinimid- e ester, leaving the
maleimido group available for coupling to a thiol group. To prepare
iso-maleimido adamantane, 1-aminoadamantane in suitable anhydrous
solvent, is coupled through the amino group to maleic anhydride to
give the maleamic acid derivative. The maleamic acid derivative is
then dehydrated in suitable anhydrous solvent using an appropriate
dehydrating agent such as trifluoroacetic anhydride to produce the
iso-maleimido adamantane available for coupling to an amino or
thiol group.
[0390] While the invention has been described with reference to
certain specific embodiments, it is understood that changes may be
made by one skilled in the art and it would not thereby depart from
the spirit and scope of the invention, which is limited only by the
claims appended hereto.
* * * * *