U.S. patent application number 13/032200 was filed with the patent office on 2011-06-16 for method of preparing a supramolecular complex containing a therapeutic agent and a multi-dimensional polymer network.
This patent application is currently assigned to CALIFORNIA INSTITUTE OF TECHNOLOGY. Invention is credited to Mark E. Davis, Hector Gonzalez, Suzie (Sue Jean) Hwang.
Application Number | 20110144190 13/032200 |
Document ID | / |
Family ID | 39387567 |
Filed Date | 2011-06-16 |
United States Patent
Application |
20110144190 |
Kind Code |
A1 |
Davis; Mark E. ; et
al. |
June 16, 2011 |
METHOD OF PREPARING A SUPRAMOLECULAR COMPLEX CONTAINING A
THERAPEUTIC AGENT AND A MULTI-DIMENSIONAL POLYMER NETWORK
Abstract
A method of preparing a supramolecular complex containing at
least one therapeutic agent and a multi-dimensional polymer network
is described. A supramolecular complex prepared by a method of the
invention is described. A method of treatment by administering a
therapeutically effective amount of a supramolecular complex of the
invention is also described. Such a supramolecular complex may be
used as a delivery vehicle for various therapeutic agents.
Inventors: |
Davis; Mark E.; (Pasadena,
CA) ; Gonzalez; Hector; (San Francisco, CA) ;
Hwang; Suzie (Sue Jean); (Torrance, CA) |
Assignee: |
CALIFORNIA INSTITUTE OF
TECHNOLOGY
Pasadena
CA
|
Family ID: |
39387567 |
Appl. No.: |
13/032200 |
Filed: |
February 22, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12075977 |
Mar 13, 2008 |
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13032200 |
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09453707 |
Dec 3, 1999 |
7375096 |
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12075977 |
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60127856 |
Apr 5, 1999 |
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60110847 |
Dec 4, 1998 |
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Current U.S.
Class: |
514/449 |
Current CPC
Class: |
A61K 31/724 20130101;
A61K 47/60 20170801; A61K 47/59 20170801; A61K 47/61 20170801; B82Y
5/00 20130101; A61K 47/6951 20170801; A61K 31/337 20130101; A61K
48/00 20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/449 |
International
Class: |
A61K 31/337 20060101
A61K031/337 |
Claims
1. A method of preparing a supramolecular complex comprising the
steps of: contacting at least one therapeutic agent and at least
one polymer to form a composite, and treating said polymer of said
composite under conditions sufficient to form a supramolecular
complex comprising said therapeutic agent and a multi-dimensional
polymer network
2-20. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/075,977, filed Mar. 13, 2008, which is a
continuation of U.S. patent application Ser. No. 09/453,707, filed
Dec. 3, 1999, now U.S. Pat. No. 7,375,096, which claims the benefit
of U.S. Provisional Application Nos. 60/110,847, filed Dec. 4,
1998, and 60/127,856, filed Apr. 5, 1999, all of which are
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to a method of preparing a
supramolecular complex containing at least one therapeutic agent
(e.g. DNA) and a multi-dimensional polymer network. Such a
supramolecular complex may be used as a delivery vehicle of a
therapeutic agent.
BACKGROUND OF THE INVENTION
[0003] Cyclodextrins are cyclic polysaccharides containing
naturally occurring D(+)-glucopyranose units in an .alpha.-(1,4)
linkage. The most common cyclodextrins are alpha
(.alpha.)-cyclodextrins, beta (.beta.)-cyclodextrins and gamma
(.gamma.)-cyclodextrins which contain, respectively, six, seven or
eight glucopyranose units. Structurally, the cyclic nature of a
cyclodextrin forms a torus or donut-like shape having an inner
apolar or hydrophobic cavity, the secondary hydroxyl groups
situated on one side of the cyclodextrin torus and the primary
hydroxyl groups situated on the other. Thus, using
(.beta.)-cyclodextrin as an example, a cyclodextrin is often
represented schematically as follows:
##STR00001##
The side on which the secondary hydroxyl groups are located has a
wider diameter than the side on which the primary hydroxyl groups
are located. The hydrophobic nature of the cyclodextrin inner
cavity allows for the inclusion of a variety of compounds.
(Comprehensive Supramolecular Chemistry, Volume 3, J. L. Atwood et
al., eds., Pergamon Press (1996); T. Cserhati, Analytical
Biochemistry, 225:328-332 (1995); Husain et al., Applied
Spectroscopy, 46:652-658 (1992); FR 2 665 169).
[0004] Cyclodextrins have been used as a delivery vehicle of
various therapeutic compounds by forming inclusion complexes with
various drugs that can fit into the hydrophobic cavity of the
cyclodextrin or by forming non-covalent association complexes with
other biologically active molecules such as oligonucleotides and
derivatives thereof. For example, U.S. Pat. No. 4,727,064 describes
pharmaceutical preparations consisting of a drug with substantially
low water solubility and an amorphous, water-soluble
cyclodextrin-based mixture. The drug forms an inclusion complex
with the cyclodextrins of the mixture. In U.S. Pat. No. 5,691,316,
a cyclodextrin cellular delivery system for oligonucleotides is
described. In such a system, an oligonucleotide is noncovalently
complexed with a cyclodextrin or, alternatively, the
oligonucleotide may be covalently bound to adamantine which in turn
is non-covalently associated with a cyclodextrin.
[0005] Various cyclodextrin containing polymers and methods of
their preparation are also known in the art. (Comprehensive
Supramolecular Chemistry, Volume 3, J. L. Atwood et al., eds.,
Pergamon Press (1996)). A process for producing a polymer
containing immobilized cyclodextrin is described in U.S. Pat. No.
5,608,015. According to the process, a cyclodextrin derivative is
reacted with either an acid halide monomer of an
.alpha.,.beta.-unsaturated acid or derivative thereof or with an
.alpha.,.beta.-unsaturated acid or derivative thereof having a
terminal isocyanate group or a derivative thereof. The cyclodextrin
derivative is obtained by reacting cyclodextrin with such compounds
as carbonyl halides and acid anhydrides. The resulting polymer
contains cyclodextrin units as side chains off a linear polymer
main chain.
[0006] U.S. Pat. No. 5,276,088 describes a method of synthesizing
cyclodextrin polymers by either reacting polyvinyl alcohol or
cellulose or derivatives thereof with cyclodextrin derivatives or
by copolymerization of a cyclodextrin derivative with vinyl acetate
or methyl methacrylate. Again, the resulting cyclodextrin polymer
contains a cyclodextrin moiety as a pendant moiety off the main
chain of the polymer.
[0007] A biodegradable medicinal polymer assembly with
supermolecular structure is described in WO 96/09073 A1 and U.S.
Pat. No. 5,855,900. The assembly comprises a number of
drug-carrying cyclic compounds prepared by binding a drug to an
.alpha., .beta. or .gamma.-cyclodextrin and then stringing the
drug/cyclodextrin compounds along a linear polymer with the
biodegradable moieties bound to both ends of the polymer. Such an
assembly is reportably capable of releasing a drug in response to a
specific biodegradation occurring in a disease. These assemblies
are commonly referred to as "necklace-type" cyclodextrin
polymers.
SUMMARY OF THE INVENTION
[0008] The invention provides a method of preparing a
supramolecular complex comprising at least one therapeutic agent
and a multi-dimensional polymer network. According to such a
method, at least one therapeutic agent is contacted with at least
one polymer to form a composite. The polymer of the composite is
then treated under conditions sufficient to form a supramolecular
complex containing the therapeutic agent and a multi-dimensional
polymer network.
[0009] The invention also provides a supramolecular complex
containing at least one therapeutic agent and a multi-dimensional
polymer network.
[0010] The invention further provides a method of treatment by
administering a therapeutically effective amount of a
supramolecular complex containing at least one therapeutic agent
and a multi-dimensional polymer network.
BRIEF DESCRIPTION OF DRAWINGS
[0011] FIG. 1. Agarose Gel of Reversible Crosslinking of Branched
PEI (25 kD) with DTBP.
[0012] FIG. 2A. Transfection with CD-DMS, CD-DMA, and CD-DMP.
[0013] FIG. 2B. Toxicity of CD-DMS, CD-DMA, and CD-DMP.
[0014] FIG. 2C. Transfection to BHK-21 Cells (serum free) with
CD-DMS and CD-DTBP.
[0015] FIG. 2D. Toxicity of CD-DMS and CD-DTBP with BHK-21 cells
(serum free).
[0016] FIG. 3A. Transfection of C6 and C9 Diamine-DMS
Copolymers.
[0017] FIG. 3B. Toxicity of C6 and C9 Diamine-DMS Copolymers.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The invention relates to a method of preparing a
supramolecular complex containing at least one therapeutic agent
and a multi-dimensional polymer network. According to a method of
the invention, at least one therapeutic agent is contacted with at
least one polymer to form a composite and then the polymer of the
composite is treated under conditions sufficient to form a
supramolecular complex containing the therapeutic agent and a
multi-dimensional polymer network.
[0019] A composite of at least one therapeutic agent and at least
one polymer may be defined as a combination or integration of at
least one therapeutic agent and at least one polymer, each as
described below. According to the invention, a "polymer" is defined
as either a single polymer molecule (e.g. a single polymer strand
or fragment) or as a group of two or more polymer molecules (e.g. a
group of two or more polymer strands or fragments). Thus, according
to the invention, a composite contains at least one single polymer
molecule; at least one group of two or more polymer molecules,
which may be the same or different; or a mixture of at least one
single polymer molecule and at least one group of two or more
polymer molecules, which may be the same or different. A polymer
molecule may be linear or branched. Accordingly, a group of two or
more polymer molecules may be linear, branched, or a mixture of
linear and branched polymers. According to the invention, prior to
formation of the composite, the polymer of the composite does not
exist as a substantially associated structure such as, for example,
a polymer gel. However, the polymer as part of the composite,
depending upon the nature of the polymers and the therapeutic
agent, may form such a substantially associated structure. Each
polymer of the composite may further contain or may be further
modified to contain at least one functional group through which
association of the polymers of the composite may be achieved, as
described below.
[0020] The composite may be prepared by any suitable means known in
the art. For example, the composite may be formed by simply
contacting, mixing or dispersing a therapeutic agent with a
polymer, each as described herein. A composite may also be prepared
by polymerizing monomers, which may be the same or different,
capable of forming a linear or branched polymer in the presence of
a therapeutic agent. In a preferred embodiment of the invention, a
composite may be prepared by polymerizing monomers, which may be
the same or different, capable of forming a linear or branched
polymer in the presence of a therapeutic agent where the
therapeutic agent acts as a template for the polymerization.
Trubetskoy et al., Nucleic Acids Research, Vol. 26, No. 18, pp.
4178-4185 (1998). The composite may be further modified with at
least one ligand, as described below. The ligand may be introduced
upon or after formation of the composite via ligand modification of
the therapeutic agent and/or the polymer of the composite, as
described herein. The composite may take any suitable form and,
preferably, is in the form of particles.
[0021] According to the invention, the polymer of the composite is
treated under conditions sufficient to form a supramolecular
complex comprising a therapeutic agent and a multi-dimensional
polymer network, each as described herein. "Treatment of the
polymer of the composite under conditions sufficient to form a
supramolecular complex" may be defined as any suitable reaction
condition(s), including the addition of additional agents or
reactants, that promote association of the polymer of the
composite. The polymer, as described above, may be associated via
interpolymer covalent bonds, noncovalent bonds (e.g. ionic bonds),
or noncovalent interactions (e.g. van der Waals interactions).
Association via intrapolymer covalent bonding, noncovalent bonding,
or noncovalent interactions of the polymer may occur as well. As a
result of such association, the polymer of the composite interacts
to form a multi-dimensional polymer network. Formation of a
multi-dimensional polymer network may be determined using
spectroscopy. A multi-dimensional polymer network exhibits
different spectrographic data (e.g. infrared spectroscopy, nuclear
magnetic resonance (NMR) spectroscopy) than the unassociated
polymer of the composite. In addition, a multi-dimensional network
of at least two polymers has an average molecular weight greater
than that of the individual polymers of the composite.
[0022] In a preferred embodiment of the invention, "treatment of
the polymer of the composite under conditions sufficient to form a
supramolecular complex" involves crosslinking reaction conditions.
For example, if the polymer of the composite is a single polymer
molecule, the polymer may be reacted with a molecule(s),
oligomer(s), or different polymer(s) that promotes crosslinking or
forms crosslinks such that intrapolymer crosslinking of or actual
crosslinking with the single polymer molecule of the composite
results. Similarly, if the polymer of the composite is a group of
two or more polymer molecules, the polymer may be reacted with a
molecule(s), oligomer(s), or different polymer(s) that promotes
crosslinking or forms crosslinks such that intrapolymer and/or
interpolymer, preferably interpolymer, crosslinking of or actual
crosslinking with the group of two or more polymer molecules of the
composite results.
[0023] The crosslinking agent may be any crosslinking agent known
in the art. The crosslinking agent may be any oligomer or polymer
(e.g. polyethylene glycol (PEG) polymer, polyethylene polymer)
capable of promoting crosslinking within or may be actually
crosslinking with the polymer of the composite. The crosslinking
oligomer or polymer may be the same or different as the polymer of
the composite. Likewise, the crosslinking agent may be any suitable
molecule capable of crosslinking with the polymer of the
composite.
[0024] Examples of crosslinking agents include dihydrazides and
dithiols. In a preferred embodiment, the crosslinking agent is a
labile group such that a crosslinked multi-dimensional polymer
network may be uncrosslinked as desired. A mixture of different
crosslinking agents may also be used. The different crosslinking
agents may exhibit varying degrees of lability. Accordingly, the
advantage of directed bioavailability (e.g. as in "timed release"
formulations) may be achieved. Examples of suitable crosslinking
agents include, but are not limited to, adipic acid dihydrazide,
polyethylene glycol 600 (PEG.sub.600) dihydrazide, dimethyl
3,3'-dithiobispropionimidate (DTBP), dithiobis(succinimidyl
propionate) (DSP), disuccinimidyl suberate (DSS), and
dimethylsuberimidate (DMS). The crosslinking agent may be further
modified with at least one ligand as described herein.
[0025] "Treatment of the polymer of the composite under conditions
sufficient to form a supramolecular complex" may also include
suitable reaction conditions that promote the crosslinking of
functional groups found on the polymer of the composite such that
association via a new bond or interaction, as described above,
results. The functional group may be any functional group known in
the art which forms a new bond or interaction, as described above,
under crosslinking reaction conditions. In a preferred embodiment
of the invention, the polymer of the composite is functionalized
with at least two thiol groups or may be modified to be
functionalized with at least two thiol groups, which under
appropriate oxidation conditions react to form a disulfide linkage.
A thiol-functionalized polymer may be prepared by means known in
the art including, for example, the addition of a thiolating
reagent (e.g. Traut's Reagent, commercially available from Pierce
Chemical Company, Rockford, Ill.). A thiol-functionalized polymer
may also be prepared by polymerization of a protected-thiol
monomer. After polymerization, the thiol groups may then be
deprotected to give free thiol groups which may then be reacted
under oxidation conditions to form a disulfide linkage(s). Suitable
oxidation conditions include, for example, air oxidation and the
use of an oxidizing reagent (e.g. ALDRITHIOL commercially available
from Aldrich Chemical Company, Inc., Milwaukee, Wis.).
[0026] The degree of association, as described above, of the
polymer of the composite forming the multi-dimensional polymer
network may vary from partial association to complete association.
By varying the degree of association of the polymer, a short chain
polymer may be made to exhibit the characteristics of a long chain
polymer while retaining the desired characteristics of a short
chain polymer upon disassociation. For example, long chain polymer
character promotes overall stability, i.e. resistance to
degradation, until the target cell is reached while short chain
polymer character promotes DNA release within the target cell. This
duality affords a supramolecular complex containing at least one
therapeutic agent and a multi-dimensional polymer network that
exhibits greater stability in both nonphysiological and
physiological conditions and greater shelf-life stability. Varying
the degree of association of the polymer of the supramolecular
complex also permits controlled release of the therapeutic
agent.
[0027] In a preferred embodiment of the invention, the polymer of
the composite is a substantially linear polymer. A substantially
linear polymer may be any suitable substantially linear polymer or
substantially linear copolymer known in the art capable as part of
a composite of associating, preferably crosslinking, to form a
multi-dimensional polymer network, as described above. According to
the invention, a substantially linear polymer may be prepared by
any means known in the art. Preferably, a substantially linear
polymer may be prepared by any suitable polymerization technique
known in the art including, but not limited to, those described in
Trubetskoy et al., Nucleic Acids Research, Vol. 26, No. 18, pp
4178-4185 (1998) (e.g. template polymerization, step
polymerization, chain polymerization). A substantially linear
polymer may be prepared from a suitable monomer. Examples of
suitable monomers for polymerization to form a substantially linear
polymer include monomers such as, for example,
bis(2-aminoethyl)-1,3-propanediamine (AEPD), and
N.sub.2,N.sub.2,N.sub.3,N.sub.3-(3'-PEG.sub.5000-aminopropane)-bis(2-amin-
oethyl)-1,3-propanediammonium di-trifluoroacetate (AEPD-PEG). The
substantially linear polymer may further contain or may be further
modified to contain a functional group (e.g. thiol group), as
described above. Preferably, the substantially linear polymer is
linear polyethyleneimine (PEI) or a linear cyclodextrin-containing
polymer, more preferably, a linear cyclodextrin-containing polymer.
A linear cyclodextrin-containing polymer may be any water-soluble
linear polymer containing at least one cyclodextrin moiety as part
of the polymer backbone. More preferably, the linear
cyclodextrin-containing polymer is a linear cyclodextrin copolymer
or a linear oxidized cyclodextrin copolymer, each as described
below.
[0028] A linear cyclodextrin copolymer is a polymer containing
cyclodextrin moieties as an integral part of its polymer backbone.
Previously, cyclodextrin moieties were not a part of the main
polymer chain but rather attached off a polymer backbone as pendant
moieties.
[0029] A linear cyclodextrin copolymer has a repeating unit of
formula Ia, Ib, or a combination thereof:
##STR00002##
In formulae Ia and Ib, C is a substituted or unsubstituted
cyclodextrin monomer and A is a comonomer bound, i.e. covalently
bound, to cyclodextrin C. Polymerization of a cyclodextrin monomer
C precursor with a comonomer A precursor results in a linear
cyclodextrin copolymer. Within a single linear cyclodextrin
copolymer, the cyclodextrin monomer C unit may be the same or
different and, likewise, the comonomer A may be the same or
different.
[0030] A cyclodextrin monomer precursor may be any cyclodextrin or
derivative thereof known in the art. As discussed above, a
cyclodextrin is defined as a cyclic polysaccharide most commonly
containing six to eight naturally occurring D(+)-glucopyranose
units in an .alpha.-(1,4) linkage. Preferably, the cyclodextrin
monomer precursor is a cyclodextrin having six, seven and eight
glucose units, i.e., respectively, an alpha (.alpha.)-cyclodextrin,
a beta (.beta.-cyclodextrin and a gamma (.gamma.)-cyclodextrin. A
cyclodextrin derivative may be any substituted cyclodextrin known
in the art where the substituent does not interfere with
copolymerization with comonomer A precursor as described below. A
cyclodextrin derivative may be neutral, cationic or anionic.
Examples of suitable substituents include, but are not limited to,
hydroxyalkyl groups, such as, for example, hydroxypropyl,
hydroxyethyl; ether groups, such as, for example, dihydroxypropyl
ethers, methyl-hydroxyethyl ethers, ethyl-hydroxyethyl ethers, and
ethyl-hydroxypropyl ethers; alkyl groups, such as, for example,
methyl; saccharides, such as, for example, glucosyl and maltosyl;
acid groups, such as, for example, carboxylic acids, phosphorous
acids, phosphinous acids, phosphonic acids, phosphoric acids,
thiophosphonic acids, and sulfonic acids; imidazole groups; sulfate
groups; and protected thiol groups.
[0031] A cyclodextrin monomer precursor may be further chemically
modified (e.g. halogenated, aminated) to facilitate or affect
copolymerization of the cyclodextrin monomer precursor with a
comonomer A precursor, as described below. Chemical modification of
a cyclodextrin monomer precursor allows for polymerization at only
two positions on each cyclodextrin moiety, i.e. the creation of a
bifunctional cyclodextrin moiety. The numbering scheme for the
C1-C6 positions of each glucopyranose ring is as follows:
##STR00003##
In a preferred embodiment, polymerization occurs at two of any C2,
C3 and C6 position, including combinations thereof, of the
cyclodextrin moiety. For example, one cyclodextrin monomer
precursor may be polymerized at two C6 positions while another
cyclodextrin monomer precursor may be polymerized at a C2 and a C6
position of the cyclodextrin moiety. Using .beta.-cyclodextrin as
an example, the lettering scheme for the relative position of each
glucopyranose ring in a cyclodextrin is as follows:
##STR00004##
[0032] In a preferred embodiment of a linear cyclodextrin
copolymer, the cyclodextrin monomer C has the following general
formula (II):
##STR00005##
In formula (II), n and m represent integers which, along with the
other two glucopyranose rings, define the total number of
glucopyranose units in the cyclodextrin monomer. Formula (II)
represents a cyclodextrin monomer which is capable of being
polymerized at two C6 positions on the cyclodextrin unit. Examples
of cyclodextrin monomers of formula (II) include, but are not
limited to, 6.sup.A,6.sup.B-dideoxy-.alpha.-cyclodextrin (n=0,
m=4), 6.sup.A,6.sup.C-dideoxy-.alpha.-cyclodextrin (n=1, m=3),
6.sup.A,6.sup.D-dideoxy-.alpha.-cyclodextrin (n=2, m=2),
6.sup.A,6.sup.B-dideoxy-.beta.-cyclodextrin (n=0, m=5),
6.sup.A,6.sup.C-dideoxy-.beta.-cyclodextrin (n=l, m=4),
6.sup.A,6.sup.D-dideoxy-.beta.-cyclodextrin (n=2, m=3),
6.sup.A,6.sup.B-dideoxy-.gamma.-cyclodextrin (n=0, m=6),
6.sup.A,6.sup.C-dideoxy-.gamma.-cyclodextrin (n=l, m=5),
6.sup.A,6.sup.D-dideoxy-.gamma.cyclodextrin (n=2, m=4), and
6.sup.A,6.sup.E-dideoxy-.gamma.-cyclodextrin (n=3, m=3). In another
preferred embodiment of a linear cyclodextrin copolymer, a
cyclodextrin monomer C unit has the following general formula
(III):
##STR00006##
where p=5-7. In formula (III), at least one of D(+)-glucopyranose
units of a cyclodextrin monomer has undergone ring opening to allow
for polymerization at a C2 and a C3 position of the cyclodextrin
unit. Cyclodextrin monomers of formula (III) such as, for example,
2.sup.A,3.sup.A-diamino-2.sup.A,3.sup.A-dideoxy-.beta.-cyclodextrin
and
2.sup.A,3.sup.A-dialdehyde-2.sup.A,3.sup.A-dideoxy-.beta.-cyclodextrin
are commercially available from Carbomer of Westborough, Mass.
Examples of cyclodextrin monomers of formula (III) include, but are
not limited to, 2.sup.A,3.sup.A-dideoxy-2.sup.A,3.sup.A-dihydro
.alpha.-cyclodextrin,
2.sup.A,3.sup.A-dideoxy-2.sup.A,3.sup.A-dihydro-.beta.-cyclodextrin,
2.sup.A,3.sup.A-dideoxy-2.sup.A,3.sup.A-dihydro-.gamma.-cyclodextrin,
commonly referred to as, respectively,
2,3-dideoxy-.alpha.-cyclodextrin, 2,3-dideoxy-.beta.-cyclodextrin,
and 2,3-dideoxy-.gamma.-cyclodextrin.
[0033] A comonomer A precursor may be any straight chain or
branched, symmetric or asymmetric compound which upon reaction with
a cyclodextrin monomer precursor, as described above, links two
cyclodextrin monomers together. Preferably, a comonomer A precursor
is a compound containing at least two functional groups through
which reaction and thus linkage of the cyclodextrin monomers can be
achieved. Examples of possible functional groups, which may be the
same or different, terminal or internal, of each comonomer A
precursor include, but are not limited to, amino, acid, ester,
imidazole, and acyl halide groups and derivatives thereof. In a
preferred embodiment, the two functional groups are the same and
terminal. Upon copolymerization of a comonomer A precursor with a
cyclodextrin monomer precursor, two cyclodextrin monomers may be
linked together by joining the primary hydroxyl side of one
cyclodextrin monomer with the primary hydroxyl side of another
cyclodextrin monomer, by joining the secondary hydroxyl side of one
cyclodextrin monomer with the secondary hydroxyl side of another
cyclodextrin monomer, or by joining the primary hydroxyl side of
one cyclodextrin monomer with the secondary hydroxyl side of
another cyclodextrin monomer. Accordingly, combinations of such
linkages may exist in the final copolymer. Both the comonomer A
precursor and the comonomer A of the final copolymer may be
neutral, cationic (e.g. by containing protonated groups such as,
for example, quaternary ammonium groups) or anionic (e.g. by
containing deprotonated groups, such as, for example, sulfate,
phosphate or carboxylate anionic groups). The counterion of a
charged comonomer A precursor or comonomer A may be any suitable
counteranion or countercation (e.g. the counteranion of a cationic
comonomer A precursor or comonomer A may be a halide (e.g.
chloride) anion). The charge of comonomer A of the copolymer may be
adjusted by adjusting pH conditions. Examples of suitable comonomer
A precursors include, but are not limited to, cystamine,
1,6-diaminohexane, diimidazole, dithioimidazole, spermine,
dithiospermine, dihistidine, dithiohistidine, succinimide (e.g.
dithiobis(succinimidyl propionate) (DSP) and disuccinimidyl
suberate (DSS)), and imidates (e.g. dimethyl
3,3'-dithiobispropion-imidate (DTBP)). Copolymerization of a
comonomer A precursor with a cyclodextrin monomer precursor leads
to the formation of a linear cyclodextrin copolymer containing
comonomer A linkages of the following general formulae:
--HNC(O)(CH.sub.2).sub.xC(O)NH--,
--HNC(O)(CH.sub.2).sub.xSS(CH.sub.2).sub.xC(O)NH--,
--.sup.+H.sub.2N(CH.sub.2).sub.xSS(CH.sub.2).sub.xNH.sub.2.sup.+--,
--HNC(O)(CH.sub.2CH.sub.2O).sub.xCH.sub.2CH.sub.2C(O)NH--,
--HNNHC(O)(CH.sub.2CH.sub.2O).sub.xCH.sub.2CH.sub.2C(O)NHNH--,
--.sup.+H.sub.2NCH.sub.2(CH.sub.2CH.sub.2O).sub.xCH.sub.2CH.sub.2CH.sub.2-
NH.sub.2.sup.+--,
--HNC(O)(CH.sub.2CH.sub.2O).sub.xCH.sub.2CH.sub.2SS(CH.sub.2CH.sub.2O).su-
b.xCH.sub.2CH.sub.2C(O)NH--,
--HNC(NH.sub.2.sup.+)(CH.sub.2CH.sub.2O).sub.xCH.sub.2CH.sub.2C(NH.sub.2.-
sup.+)NH--,
--SCH.sub.2CH.sub.2NHC(NH.sub.2.sup.+)(CH.sub.2).sub.xC(NH.sub.2.sup.+)NH-
CH.sub.2CH.sub.2S--,
--SCH.sub.2CH.sub.2NHC(NH.sub.2.sup.+)(CH.sub.2).sub.xSS(CH.sub.2).sub.xC-
(NH.sub.2.sup.+)NHCH.sub.2CH.sub.2S--,
--SCH.sub.2CH.sub.2NHC(NH.sub.2.sup.+)CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2)-
.sub.xC(NH.sub.2.sup.+)NHCH.sub.2CH.sub.2CH.sub.2S--,
##STR00007## ##STR00008##
In the above formulae, x=1-50, and y+z=x. Preferably, x=1-30. More
preferably, x=1-20. In a preferred embodiment, comonomer A is
biodegradable or acid-labile. Also in a preferred embodiment, the
comonomer A precursor and hence the comonomer A may be selectively
chosen in order to achieve a desired application. For example, to
deliver small molecular therapeutic agents, a charged polymer may
not be necessary and the comonomer A may be a polyethylene glycol
group.
[0034] In a preferred embodiment of the invention, a linear
cyclodextrin copolymer may be prepared by copolymerizing a
cyclodextrin monomer precursor disubstituted with an appropriate
leaving group with a comonomer A precursor capable of displacing
the leaving groups. The leaving group, which may be the same or
different, may be any leaving group known in the art which may be
displaced upon copolymerization with a comonomer A precursor. In a
preferred embodiment, a linear cyclodextrin copolymer may be
prepared by iodinating a cyclodextrin monomer precursor to form a
diiodinated cyclodextrin monomer precursor and copolymerizing the
diiodinated cyclodextrin monomer precursor with a comonomer A
precursor to form a linear cyclodextrin copolymer having a
repeating unit of formula Ia, Ib, or a combination thereof, each as
described above. In a preferred embodiment, a method of preparing a
linear cyclodextrin iodinates a cyclodextrin monomer precursor as
described above to form a diiodinated cyclodextrin monomer
precursor of formula IVa, IVb, IVc or a mixture thereof:
##STR00009##
The diiodinated cyclodextrin may be prepared by any means known in
the art (see, e.g., Tabushi et al. J. Am. Chem. 106, 5267-5270
(1984); Tabushi et al. J. Am. Chem. 106, 4580-4584 (1984)). For
example, .beta.-cyclodextrin may be reacted with
biphenyl-4,4'-disulfonyl chloride in the presence of anhydrous
pyridine to form a biphenyl-4,4'-disulfonyl chloride capped
.beta.-cyclodextrin which may then be reacted with potassium iodide
to produce diiodo-.beta.-cyclodextrin. The cyclodextrin monomer
precursor is iodinated at only two positions. By copolymerizing the
diiodinated cyclodextrin monomer precursor with a comonomer A
precursor, as described above, a linear cyclodextrin polymer having
a repeating unit of formula Ia, Ib, or a combination thereof, also
as described above, may be prepared. If appropriate, the iodine or
iodo groups may be replaced with other known leaving groups.
[0035] The iodo groups or other appropriate leaving group may be
displaced with a group that permits reaction with a comonomer A
precursor, as described above. For example, a diiodinated
cyclodextrin monomer precursor of formula IVa, IVb, IVc or a
mixture thereof may be aminated to form a diaminated cyclodextrin
monomer precursor of formula Va, Vb, Vc or a mixture thereof:
##STR00010##
The diaminated cyclodextrin monomer precursor may be prepared by
any means known in the art (see, e.g., Tabushi et al. Tetrahedron
Lett. 18:1527-1530 (1977); Mungall et al., J. Org. Chem. 1659-1662
(1975)). For example, a diiodo-.beta.-cyclodextrin may be reacted
with sodium azide and then reduced to form a
diamino-.beta.-cyclodextrin. The cyclodextrin monomer precursor is
aminated at only two positions. The diaminated cyclodextrin monomer
precursor may then be copolymerized with a comonomer A precursor,
as described above, to produce a linear cyclodextrin copolymer
having a repeating unit of formula Ia, Ib, or a combination
thereof, also as described above. However, the amino functionality
of a diaminated cyclodextrin monomer precursor need not be directly
attached to the cyclodextrin moiety. Alternatively, the amino
functionality may be introduced by displacement of the iodo or
other appropriate leaving groups of a cyclodextrin monomer
precursor with amino group containing moieties such as, for
example, .sup.-SCH.sub.2CH.sub.2NH.sub.2, to form a diaminated
cyclodextrin monomer precursor of formula Vd, Ve, Vf or a mixture
thereof:
##STR00011##
[0036] A linear cyclodextrin copolymer may also be prepared by
reducing a linear oxidized cyclodextrin copolymer, as described
below. This method may be performed as long as the comonomer A does
not contain a reducible moiety or group such as, for example, a
disulfide linkage.
[0037] A linear cyclodextrin copolymer may be oxidized so as to
introduce at least one oxidized cyclodextrin monomer into the
copolymer such that the oxidized cyclodextrin monomer is an
integral part of the polymer backbone. A linear cyclodextrin
copolymer which contains at least one oxidized cyclodextrin monomer
is defined as a linear oxidized cyclodextrin copolymer. The
cyclodextrin monomer may be oxidized on either the secondary or
primary hydroxyl side of the cyclodextrin moiety. If more than one
oxidized cyclodextrin monomer is present in a linear oxidized
cyclodextrin copolymer, the same or different cyclodextrin monomers
oxidized on either the primary hydroxyl side, the secondary
hydroxyl side, or both may be present. For illustration purposes, a
linear oxidized cyclodextrin copolymer with oxidized secondary
hydroxyl groups has, for example, at least one unit of formula VIa
or VIb:
##STR00012##
In formulae VIa and VIb, C is a substituted or unsubstituted
oxidized cyclodextrin monomer and A is a comonomer bound, i.e.
covalently bound, to the oxidized cyclodextrin C. Also in formulae
VIa and VIb, oxidation of the secondary hydroxyl groups leads to
ring opening of the cyclodextrin moiety and the formation of
aldehyde groups.
[0038] A linear oxidized cyclodextrin copolymer may be prepared by
oxidation of a linear cyclodextrin copolymer as discussed above.
Oxidation of a linear cyclodextrin copolymer may be accomplished by
oxidation techniques known in the art. (Hisamatsu et al., Starch
44:188-191 (1992)). Preferably, an oxidant such as, for example,
sodium periodate is used. It would be understood by one of ordinary
skill in the art that under standard oxidation conditions that the
degree of oxidation may vary or be varied per copolymer. Thus in
one embodiment, a linear oxidized copolymer may contain one
oxidized cyclodextrin monomer. In another embodiment, substantially
all to all cyclodextrin monomers of the copolymer would be
oxidized.
[0039] Another method of preparing a linear oxidized cyclodextrin
copolymer involves the oxidation of a diiodinated or diaminated
cyclodextrin monomer precursor, as described above, to form an
oxidized diiodinated or diaminated cyclodextrin monomer precursor
and copolymerization of the oxidized diiodinated or diaminated
cyclodextrin monomer precursor with a comonomer A precursor. In a
preferred embodiment, an oxidized diiodinated cyclodextrin monomer
precursor of formula VIIa, VIIb, VIIc, or a mixture thereof:
##STR00013##
may be prepared by oxidation of a diiodinated cyclodextrin monomer
precursor of formulae IVa, IVb, IVc, or a mixture thereof, as
described above. In another preferred embodiment, an oxidized
diaminated cyclodextrin monomer precursor of formula VIIIa, VIIIb,
VIIIc or a mixture thereof:
##STR00014##
may be prepared by amination of an oxidized diiodinated
cyclodextrin monomer precursor of formulae VIIa, VIIb, VIIc, or a
mixture thereof, as described above. In still another preferred
embodiment, an oxidized diaminated cyclodextrin monomer precursor
of formula IXa, IXb, IXc or a mixture thereof:
##STR00015##
may be prepared by displacement of the iodo or other appropriate
leaving groups of an oxidized cyclodextrin monomer precursor
disubstituted with an iodo or other appropriate leaving group with
the amino group containing moiety
.sup.-SCH.sub.2CH.sub.2NH.sub.2.
[0040] Alternatively, an oxidized diiodinated or diaminated
cyclodextrin monomer precursor, as described above, may be prepared
by oxidizing a cyclodextrin monomer precursor to form an oxidized
cyclodextrin monomer precursor and then diiodinating and/or
diaminating the oxidized cyclodextrin monomer, as described above.
As discussed above, the cyclodextrin moiety may be modified with
other leaving groups other than iodo groups and other amino group
containing functionalities. The oxidized diiodinated or diaminated
cyclodextrin monomer precursor may then be copolymerized with a
comonomer A precursor, as described above, to form a linear
oxidized cyclodextrin copolymer.
[0041] In a preferred embodiment of the invention, a linear
cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer
terminates with at least one comonomer A precursor or hydrolyzed
product of the comonomer A precursor, each as described above. As a
result of termination of the cyclodextrin copolymer with at least
one comonomer A precursor, at least one free functional group, as
described above, exists per linear cyclodextrin copolymer or per
linear oxidized cyclodextrin copolymer. For example, the functional
group may be an acid group or a functional group that may be
hydrolyzed to an acid group. According to the invention, the
functional group may be further chemically modified as desired to
enhance the properties of the cyclodextrin copolymer, such as, for
example, colloidal stability and transfection efficiency. For
example, the functional group may be modified by reaction with PEG
to form a PEG terminated cyclodextrin copolymer to enhance
colloidal stability or with histidine to form an imidazolyl
terminated cyclodextrin copolymer to enhance intracellular (e.g.
endosomal release) and transfection efficiency.
[0042] Further chemistry may be performed on the cyclodextrin
copolymer through the modified functional group. For example, the
modified functional group may be used to extend a polymer chain by
linking a linear cyclodextrin copolymer or linear oxidized
cyclodextrin copolymer, as described herein, to the same or
different cyclodextrin copolymer or to a non-cyclodextrin polymer.
In a preferred embodiment of the invention, the polymer to be added
on is the same or different linear cyclodextrin copolymer or linear
oxidized cyclodextrin copolymer which may also terminate with at
least one comonomer A precursor for further modification, each as
described herein.
[0043] Alternatively, at least two of the same or different linear
cyclodextrin copolymers or linear oxidized cyclodextrin copolymers
containing a terminal functional group or a terminal modified
functional group, as described above, may be reacted and linked
together through the functional or modified functional group.
Preferably, upon reaction of the functional or modified functional
groups, a degradable moiety such as, for example, a disulfide
linkage is formed. For example, modification of the terminal
functional group with cysteine may be used to produce a linear
cyclodextrin copolymer or linear oxidized cyclodextrin copolymer
having at least one free thiol group. Reaction with the same or
different cyclodextrin copolymer also containing at least one free
thiol group will form a disulfide linkage between the two
copolymers. In a preferred embodiment of the invention, the
functional or modified functional groups may be selected to offer
linkages exhibiting different rates of degradation (e.g. via
enzymatic degradation) and thereby provide, if desired, a time
release system for a therapeutic agent. The resulting polymer may
be crosslinked, as described herein. A therapeutic agent, as
described herein, may be added prior to or post crosslinking of the
polymer. A ligand, as described herein, may also be bound through
the modified functional group.
[0044] According to the invention, a linear cyclodextrin copolymer
or linear oxidized cyclodextrin copolymer may be attached to or
grafted onto a substrate. The substrate may be any substrate as
recognized by those of ordinary skill in the art. In another
preferred embodiment of the invention, a linear cyclodextrin
copolymer or linear oxidized cyclodextrin copolymer may be
crosslinked to a polymer to form, respectively, a crosslinked
cyclodextrin copolymer or a crosslinked oxidized cyclodextrin
copolymer. The polymer may be any polymer capable of crosslinking
with a linear or linear oxidized cyclodextrin copolymer of the
invention (e.g. polyethylene glycol (PEG) polymer, polyethylene
polymer). The polymer may also be the same or different linear
cyclodextrin copolymer or linear oxidized cyclodextrin copolymer.
Thus, for example, a linear cyclodextrin copolymer may be
crosslinked to any polymer including, but not limited to, itself,
another linear cyclodextrin copolymer, and a linear oxidized
cyclodextrin copolymer. A crosslinked linear cyclodextrin copolymer
of the invention may be prepared by reacting a linear cyclodextrin
copolymer with a polymer in the presence of a crosslinking agent. A
crosslinked linear oxidized cyclodextrin copolymer of the invention
may be prepared by reacting a linear oxidized cyclodextrin
copolymer with a polymer in the presence of an appropriate
crosslinking agent. The crosslinking agent may be any crosslinking
agent known in the art. Examples of crosslinking agents include
dihydrazides and dithiols. In a preferred embodiment, the
crosslinking agent is a labile group such that a crosslinked
copolymer may be uncrosslinked if desired.
[0045] A linear cyclodextrin copolymer and a linear oxidized
cyclodextrin copolymer of the invention may be characterized by any
means known in the art. Such characterization methods or techniques
include, but are not limited to, gel permeation chromatography
(GPC), matrix assisted laser desorption ionization-time of flight
mass spectrometry (MALDI-TOF Mass spec), .sup.1H and .sup.13C NMR,
light scattering and titration.
[0046] In another preferred embodiment of the invention, the
polymer of the composite is a substantially branched polymer such
as, for example, branched polyethyleneimine (PEI) or a branched
cyclodextrin-containing polymer, preferably, a branched
cyclodextrin-containing polymer. A branched cyclodextrin-containing
polymer may be any water-soluble branched polymer containing at
least one cyclodextrin moiety which may be a part of the polymer
backbone and/or pendant from the polymer backbone. Preferably, a
branched cyclodextrin-containing polymer is a branched cyclodextrin
copolymer or a branched oxidized cyclodextrin copolymer. A branched
cyclodextrin copolymer or a branched oxidized cyclodextrin
copolymer is, respectively, a linear cyclodextrin copolymer or a
linear oxidized cyclodextrin copolymer, as described above, from
which a subordinate chain is branched. The branching subordinate
chain may be any saturated or unsaturated, linear or branched
hydrocarbon chain. The branching subordinate chain may further
contain various functional groups or substituents such as, for
example, hydroxyl, amino, acid, ester, amido, keto, formyl, and
nitro groups. The branching subordinate chain may also contain at
least one cyclodextrin moiety. The branching subordinate chain may
also be modified with a ligand, as described herein. Such ligand
modification includes, but is not limited to, attachment of a
ligand to a cyclodextrin moiety in the branching subordinate chain.
Preferably, the branched cyclodextrin-containing polymer is a
branched cyclodextrin copolymer or a branched oxidized cyclodextrin
copolymer, as defined above, of which the branching subordinate
chain contains at least one cyclodextrin moiety. According to the
invention, if the branching subordinate chain contains at least one
cyclodextrin moiety, the cyclodextrin moiety may facilitate
encapsulation of a therapeutic agent, each as described herein.
Preferably, a cyclodextrin moiety of a branching subordinate chain
facilitates encapsulation of a therapeutic agent in conjunction
with a cyclodextrin moiety in the polymer backbone. A branched
cyclodextrin-containing polymer may be prepared by any means known
in the art including, but not limited to, derivatization (e.g.
substitution) of a polymer (e.g. linear or branched PEI) with a
cyclodextrin monomer precursor, as defined above. A branched
cyclodextrin-containing polymer of the invention may be
characterized by any means known in the art. Such characterization
methods or techniques include, but are not limited to, gel
permeation chromatography (GPC), matrix assisted laser desorption
ionization-time of flight mass spectrometry (MALDI-TOF Mass spec),
.sup.1H and .sup.13C NMR, light scattering and titration.
[0047] According to the invention, a branched
cyclodextrin-containing polymer may be crosslinked under
crosslinking reaction conditions, each as described above. In a
preferred embodiment of the invention, a branched
cyclodextrin-containing polymer is crosslinked with itself. In
another preferred embodiment of the invention, a branched
cyclodextrin-containing polymer is crosslinked with a polymer. The
polymer may be the same or different branched
cyclodextrin-containing polymer, a substantially linear polymer, or
a substantially branched polymer, each as described above.
[0048] According to the invention, a substantially branched polymer
may be attached to or grafted onto a substrate, as described above.
Further chemistry may be performed on the substantially branched
polymer through a modified functional group, as described
above.
[0049] A poly(ethylenimine) (PEI) for use in the invention has a
weight average molecular weight of between about 800 and about
800,000 daltons, preferably, between about 2,000 and 100,000
daltons, more preferably, between about 2,000 and about 25,000
daltons. The PEI may be linear or branched. Suitable PEI compounds
are commercially available from many sources, including
polyethylenimine from Aldrich Chemical Company, polyethylenimine
from Polysciences, and POLYMIN poly(ethylenimine) and LUPASOL.TM.
poly(ethylenimine) available from BASF Corporation.
[0050] According to the invention, a polymer of the composite, or
one of the monomers which form a polymer of the composite, may be
modified with at least one ligand such that the resulting composite
or supramolecular complex is associated with at least one ligand,
each as described herein. Alternatively, according to a method of
the invention, once a composite or a supramolecular complex is
formed, it may then be contacted with a ligand such that the
composite or supramolecular complex is modified with at least one
ligand in such a way that the ligand is associated with the
composite or supramolecular complex, each as described herein. The
ligand of such a ligand-containing composite or ligand-containing
supramolecular complex allows for targeting and/or binding to a
desired cell. If more than one ligand is attached, the ligand may
be the same or different. Examples of suitable ligands include, but
are not limited to, vitamins (e.g. folic acid), proteins (e.g.
transferrin, and monoclonal antibodies) and polysaccharides. The
choice of ligand may vary depending upon the type of delivery
desired. For example, receptor-mediated delivery may by achieved
by, but not limited to, the use of a folic acid ligand while
antisense oligo delivery may be achieved by, but not limited to,
use of a transferrin ligand.
[0051] The ligand may be associated with the composite or
supramolecular complex by means known in the art. For example, a
linear cyclodextrin copolymer or linear oxidized cyclodextrin
copolymer may be modified with at least one ligand attached to the
cyclodextrin copolymer. The ligand may be attached to the
cyclodextrin copolymer through the cyclodextrin monomer C or
comonomer A. Preferably, the ligand is attached to at least one
cyclodextrin moiety of the cyclodextrin copolymer. Preferably, the
ligand allows a cyclodextrin copolymer to target and bind to a
cell. If more than one ligand, which may be the same or different,
is attached to a cyclodextrin copolymer, the additional ligand or
ligands may be bound to the same or different cyclodextrin moiety
or the same or different comonomer A of the copolymer. A
cyclodextrin copolymer may also be further modified to contain a
functional group to promote association of the cyclodextrin
copolymer with the therapeutic agent and/or other polymer(s) of the
composite.
[0052] According to a method of the invention, upon formation of
the supramolecular complex, the therapeutic agent becomes
encapsulated in the multi-dimensional polymer network created from
the polymer of a composite, as described above. Encapsulation is
defined as any means by which, the therapeutic agent associates
(e.g. electrostatic interaction, hydrophobic interaction, actual
encapsulation) with the multi-dimensional polymer network. The
degree of association may be determined by techniques known in the
art including, for example, fluorescence studies, DNA mobility
studies, light scattering, electron microscopy, and will vary
depending upon the therapeutic agent. As a mode of delivery, for
example, a supramolecular complex containing a multi-dimensional
polymer network created from the polymer of a composite, as
described above, and DNA may be used to aid in transfection, i.e.
the uptake of DNA into an animal (e.g. human) cell. (Boussif, O.
Proceedings of the National Academy of Sciences, 92:7297-7301
(1995); Zanta et al. Bioconjugate Chemistry, 8:839-844 (1997)).
[0053] The therapeutic agent is not an integral part of the
multi-dimensional polymer network of the supramolecular complex.
Upon encapsulation, the therapeutic agent may or may not retain its
biological or therapeutic activity. Regardless, upon decomplexation
or uncrosslinking of the supramolecular complex, specifically, of
the multi-dimensional polymer network, the activity of the
therapeutic agent is restored. Accordingly, encapsulation of the
therapeutic agent affords, advantageously, protection against loss
of activity due to, for example, degradation and offers enhanced
bioavailability. Encapsulation of a lipophilic therapeutic agent
offers enhanced, if not complete, solubility of the lipophilic
therapeutic agent. The therapeutic agent may be further modified
with at least one ligand prior to or after composite or
supramolecular complex formation, as described above.
[0054] The therapeutic agent may be any lipophilic or hydrophilic,
synthetic or naturally occurring biologically active therapeutic
agent including those known in the art. Examples of suitable
therapeutic agents include, but are not limited to, antibiotics,
steroids, polynucleotides (e.g. genomic DNA, cDNA, mRNA and
antisense oligonucleotides), plasmids, peptides, peptide fragments,
small molecules (e.g. doxorubicin), chelating agents (e.g.
deferoxamine (DESFERAL), ethylenediaminetetraacetic acid (EDTA)),
natural products (e.g. Taxol, Amphotericin), and other biologically
active macromolecules such as, for example, proteins and
enzymes.
[0055] A supramolecular complex of the invention may be, for
example, a solid, liquid, suspension, or emulsion. Preferably a
supramolecular complex of the invention is in a form that can be
injected intravenously. Other modes of administration of a
supramolecular complex of the invention include, depending on the
state of the supramolecular complex, methods known in the art such
as, but not limited to, oral administration, topical application,
parenteral, intravenous, intranasal, intraocular, intracranial or
intraperitoneal injection. Prior to administration, a
supramolecular complex may be isolated and purified by any means
known in the art including, for example, centrifugation, dialysis
and/or lyophilization.
[0056] Depending upon the type of therapeutic agent used, a
supramolecular complex of the invention may be used in a variety of
therapeutic methods (e.g. DNA vaccines, antibiotics, antiviral
agents) for the treatment of inherited or acquired disorders such
as, for example, cystic fibrosis, Gaucher's disease, muscular
dystrophy, AIDS, cancers (e.g., multiple myeloma, leukemia,
melanoma, and ovarian carcinoma), cardiovascular conditions (e.g.,
progressive heart failure, restenosis, and hemophilia), and
neurological conditions (e.g., brain trauma). According to the
invention, a method of treatment administers a therapeutically
effective amount of a supramolecular complex as prepared by a
method of the invention. A therapeutically effective amount, as
recognized by those of skill in the art, will be determined on a
case by case basis. Factors to be considered include, but are not
limited to, the disorder to be treated and the physical
characteristics of the one suffering from the disorder.
[0057] In another embodiment of the invention, the therapeutic
agent is at least one biologically active compound having
agricultural utility. The agriculturally biologically active
compounds include those known in the art. For example, suitable
agriculturally biologically active compounds include, but are not
limited to, fungicides, herbicides, insecticides, and
mildewcides.
[0058] The following examples are given to illustrate the
invention. It should be understood, however, that the invention is
not to be limited to the specific conditions or details described
in these examples.
EXAMPLES
[0059] Materials. .beta.-cyclodextrin (Cerestar USA, Inc. of
Hammond, Ind.) was dried in vacuo (<0.1 mTorr) at 120.degree. C.
for 12 h before use. Biphenyl-4,4'-disulfonyl chloride (Aldrich
Chemical Company, Inc. of Milwaukee, Wis.) was recrystallized from
chloroform/hexanes. Potassium iodide was powdered with a mortar and
pestle and dried in an oven at 200.degree. C. All other reagents
were obtained from commercial suppliers and were used as received
without further purification. Polymer samples were analyzed on a
Hitachi HPLC system equipped with an Anspec RI detector, a
Precision Detectors DLS detector, and a Progel-TSK G3000.sub.PWXL
column using 0.3 M NaCl or water as eluant at a 1.0 mLmin.sup.-1
flow rate.
Example 1
Biphenyl-4,4'-disulfonyl-A,D-Capped .beta.-Cyclodextrin, 1 (Tabushi
et al. J. Am. Chem. Soc. 106, 5267-5270 (1984))
[0060] A 500 mL round bottom flask equipped with a magnetic
stirbar, a Schlenk adapter and a septum was charged with 7.92 g
(6.98 mmol) of dry .beta.-cyclodextrin and 250 mL of anhydrous
pyridine (Aldrich Chemical Company, Inc.). The resulting solution
was stirred at 50.degree. C. under nitrogen while 2.204 g (6.28
mmol) of biphenyl-4,4'-disulfonyl chloride was added in four equal
portions at 15 min intervals. After stirring at 50.degree. C. for
an additional 3 h, the solvent was removed in vacuo and the residue
was subjected to reversed-phase column chromatography using a
gradient elution of 0-40% acetonitrile in water. Fractions were
analyzed by high performance liquid chromatography (HPLC) and the
appropriate fractions were combined. After removing the bulk of the
acetonitrile on a rotary evaporator, the resulting aqueous
suspension was lyophilized to dryness. This afforded 3.39 g (38%)
of 1 as a colorless solid.
Example 2
6.sup.A,6.sup.D-Diiodo-6.sup.A,6.sup.D-Dideoxy-.beta.-cyclodextrin,
2 (Tabushi et al. J. Am. Chem. 106, 4580-4584 (1984))
[0061] A 40 mL centrifuge tube equipped with a magnetic stirbar, a
Schlenk adapter and a septum was charged with 1.02 g (7.2 mmol) of
1, 3.54 g (21.3 mmol) of dry, powdered potassium iodide (Aldrich)
and 15 mL of anhydrous N,N-dimethylformamide (DMF) (Aldrich). The
resulting suspension was stirred at 80.degree. C. under nitrogen
for 2 h. After cooling to room temperature, the solids were
separated by filtration and the supernatant was collected. The
solid precipitate was washed with a second portion of anhydrous DMF
and the supernatants were combined and concentrated in vacuo. The
residue was then dissolved in 14 mL of water and cooled in an ice
bath before 0.75 mL (7.3 mmol) of tetrachloroethylene (Aldrich) was
added with rapid stirring. The precipitated inclusion complex was
filtered on a medium glass frit and washed with a small portion of
acetone before it was dried under vacuum over P.sub.2O.sub.5 for 14
h. This afforded 0.90 g (92%) of 2 as a white solid.
Example 3
6.sup.A,6.sup.D-Diazido-6.sup.A,6.sup.D-Dideoxy-.beta.-cyclodextrin,
3 (Tabushi et al. Tetrahedron Lett. 18, 1527-1530 (1977))
[0062] A 100 mL round bottom flask equipped with a magnetic
stirbar, a Schlenk adapter and a septum was charged with 1.704 g
(1.25 mmol) of .beta.-cyclodextrin diiodide, 0.49 g (7.53 mmol) of
sodium azide (EM Science of Gibbstown, N.J.) and 10 mL of anhydrous
N,N-dimethylformamide (DMF). The resulting suspension was stirred
at 60.degree. C. under nitrogen for 14 h. The solvent was then
removed in vacuo. The resulting residue was dissolved in enough
water to make a 0.2 M solution in salt and then passed through 11.3
g of Biorad AG501-X8(D) resin to remove residual salts. The eluant
was then lyophilized to dryness yielding 1.232 g (83%) of 3 as a
white amorphous solid which was carried on to the next step without
further purification.
Example 4
6.sup.A,6.sup.D-Diamino-6.sup.A,6.sup.D-Dideoxy-.beta.-cyclodextrin,
4 (Mungall et al., J. Org. Chem. 1659-1662 (1975))
[0063] A 250 mL round bottom flask equipped with a magnetic stirbar
and a septum was charged with 1.232 g (1.04 mmol) of
.beta.-cyclodextrin bisazide and 50 mL of anhydrous pyridine
(Aldrich). To this stirring suspension was added 0.898 g (3.42
mmol) of triphenylphosphine. The resulting suspension was stirred
for 1 h at ambient temperature before 10 mL of concentrated aqueous
ammonia was added. The addition of ammonia was accompanied by a
rapid gas evolution and the solution became homogeneous. After 14
h, the solvent was removed in vacuo and the residue was triturated
with 50 mL of water. The solids were filtered off and the filtrate
was made acidic (pH<4) with 10% HCl before it was applied to an
ion exchange column containing Toyopearl SP-650M (NH.sub.4+ form)
resin. The product 4 was eluted with a gradient of 0-0.5 M ammonium
bicarbonate. Appropriate fractions were combined and lyophilized to
yield 0.832 g (71%) of the product 4 as the bis(hydrogen carbonate)
salt.
Example 5
.beta.-Cyclodextrin-DSP copolymer, 5
[0064] A 20 mL scintillation vial was charged with a solution of
92.6 mg (7.65.times.10.sup.-5 mol) of the bis(hydrogen carbonate)
salt of 4 in 1 mL of water. The pH of the solution was adjusted to
10 with 1 M NaOH before a solution of 30.9 mg (7.65.times.10.sup.-5
mol) of dithiobis(succinimidyl propionate) (DSP, Pierce Chemical
Co. of Rockford, Ill.) in 1 mL of chloroform was added. The
resulting biphasic mixture was agitated with a Vortex mixer for 0.5
h. The aqueous layer was then decanted and extracted with 3.times.1
mL of fresh chloroform. The aqueous polymer solution was then
subjected to gel permeation chromatography (GPC) on Toyopearl
HW-40F resin using water as eluant. Fractions were analyzed by GPC
and appropriate fractions were lyophilized to yield 85 mg (85%) as
a colorless amorphous powder.
Example 6
.beta.-cyclodextrin-DSS copolymer, 6
[0065] A .beta.-cyclodextrin-DSS copolymer, 6, was synthesized in a
manner analogous to the DSP polymer, 5, except that disuccinimidyl
suberate (DSS, Pierce Chemical Co. of Rockford, Ill.) was
substituted for the DSP reagent. Compound 6 was obtained in 67%
yield.
Example 7
.beta.-Cyclodextrin-DTBP copolymer, 7
[0066] A 20 mL scintillation vial was charged with a solution of
91.2 mg (7.26.times.10.sup.-5 mol) of the bis(hydrogen carbonate)
salt of 4 in 1 mL of water. The pH of the solution was adjusted to
10 with 1 M NaOH before 22.4 mg (7.26.times.10.sup.-5 mol) of
dimethyl 3,3'-dithiobis(propionimidate) 2 HCl (DTBP, Pierce
Chemical Co of Rockford, Ill.) was added. The resulting homogeneous
solution was agitated with a Vortex mixer for 0.5 h. The aqueous
polymer solution was then subjected to gel permeation
chromatography (GPC) on Toyopearl HW-40F resin. Fractions were
analyzed by GPC and appropriate fractions were lyophilized to yield
67 mg (67%) of a colorless amorphous powder.
Example 8
Polyethylene glycol 600 dihydrazide, 8
[0067] A 100 mL round bottom flask equipped with a magnetic stirbar
and a reflux condenser was charged with 1.82 g (3.0 mmol) of
polyethylene glycol 600 (Fluka Chemical Corp of Milwaukee, Wis.),
40 mL of absolute ethanol (Quantum Chemicals Pty Ltd of Tuscola,
Ill.) and a few drops of sulfuric acid. The resulting solution was
heated to reflux for 14 h. Solid sodium carbonate was added to
quench the reaction and the solution of the PEG diester was
transferred under nitrogen to an addition funnel. This solution was
then added dropwise to a solution of 0.6 mL (9.0 mmol) of hydrazine
hydrate (Aldrich) in 10 mL of absolute ethanol. A small amount of a
cloudy precipitate formed. The resulting solution was heated to
reflux for 1 h before it was filtered and concentrated. GPC
analysis revealed a higher molecular weight impurity contaminating
the product. Gel permeation chromatography on Toyopearl HW-40 resin
enabled a partial purification of this material to approximately
85% purity.
Example 9
Oxidation of .beta.-cyclodextrin-DSS copolymer, 9 (Hisamatsu et
al., Starch 44, 188-191 (1992))
[0068] The .beta.-cyclodextrin-DSS copolymer 6 (92.8 mg,
7.3.times.10.sup.-5 mol) was dissolved in 1.0 mL of water and
cooled in an ice bath before 14.8 mg (7.3.times.10.sup.-5 mol) of
sodium periodate was added. The solution immediately turned bright
yellow and was allowed to stir in the dark at 0.degree. C. for 14
h. The solution was then subjected to gel permeation chromatography
(GPC) on Toyopearl HW-40 resin using water as eluant. Fractions
were analyzed by GPC. Appropriate fractions were combined and
lyophilized to dryness to yield 84.2 mg (91%) of a light brown
amorphous solid.
Example 10
Polyethylene glycol (PEG) 600 diacid chloride, 10
##STR00016##
[0070] A 50 mL round bottom flask equipped with a magnetic stirbar
and a reflux condenser was charged with 5.07 g (ca. 8.4 mmol) of
polyethylene glycol 600 diacid (Fluka Chemical Corp of Milwaukee,
Wis.) and 10 mL of anhydrous chloroform (Aldrich). To this stirring
solution was added 3.9 mL (53.4 mmol) of thionyl chloride (Aldrich)
and the resulting solution was heated to reflux for 1 h, during
which time gas evolution was evident. The resulting solution was
allowed to cool to room temperature before the solvent and excess
thionyl chloride were removed in vacuo. The resulting oil was
stored in a dry box and used without purification.
Example 11
.beta.-Cyclodextrin-PEG 600 copolymer, 11
##STR00017##
[0072] A 20 mL scintillation vial was charged with a solution of
112.5 mg (8.95.times.10.sup.-5 mol) of the bis(hydrogen carbonate)
salt of
6.sup.A,6.sup.D-diamino-6.sup.A,6.sup.D-dideoxy-.beta.-cyclodextrin,
50 .mu.L (3.6.times.10.sup.-4 mol) of triethylamine (Aldrich), and
5 mL of anhydrous N,N-dimethylacetamide (DMAc, Aldrich). The
resulting suspension was then treated with 58 mg
(9.1.times.10.sup.-5 mol) of polyethylene glycol 600 diacid
chloride, 10. The resulting solution was agitated with a Vortex
mixer for 5 minutes and then allowed to stand at 25.degree. C. for
1 h during which time it became homogeneous. The solvent was
removed in vacuo and the residue was subjected to gel permeation
chromatography on Toyopearl HW-40F resin using water as eluant.
Fractions were analyzed by GPC and appropriate fractions were
lyophilized to dryness to yield 115 mg (75%) of a colorless
amorphous powder.
Example 12
.beta.-Cyclodextrin-DSP copolymer, 12
##STR00018##
[0074] A 8 mL vial was charged with a solution of 102.3 mg
(8.80.times.10.sup.-5 mol) of
2.sup.A,3.sup.A-diamino-2.sup.A,3.sup.A-dideoxy-.beta.-cyclodextrin
in 1 mL of water. The pH of the solution was adjusted to 10 with 1
M NaOH before a solution of 36.4 mg (8.80.times.10.sup.-5 mol) of
dithiobis(succinimidyl propionate) (DSP, Pierce Chemical Co. of
Rockford, Ill.) in 1 mL of chloroform was added. The resulting
biphasic mixture was agitated with a Vortex mixer for 0.5 h. The
aqueous layer was then decanted and extracted with 3.times.1 mL of
fresh chloroform. The aqueous polymer solution was then subjected
to gel permeation chromatography.
Example 13
6.sup.A,6.sup.D-Bis-(2-aminoethylthio)-6.sup.A,6.sup.D-dideoxy-.beta.-cycl-
odextrin, 13 (Tabushi, I: Shimokawa, K; Fugita, K. Tetrahedron
Lett. 1977, 1527-1530)
##STR00019##
[0076] A 25 mL Schlenk flask equipped with a magnetic stirbar and a
septum was charged with 0.91 mL (7.37 mmol) of a 0.81 M solution of
sodium 2-aminoethylthiolate in ethanol. (Fieser, L. F.; Fieser, M.
Reagents for Organic Synthesis; Wiley: New York, 1967; Vol. 3, pp.
265-266). The solution was evaporated to dryness and the solid was
redissolved in 5 mL of anhydrous DMF (Aldrich).
6.sup.A,6.sup.D-Diiodo-6.sup.A,6.sup.D-dideoxy-.beta.-cyclodextrin
(100 mg, 7.38.times.10.sup.-5 mol) was added and the resulting
suspension was stirred at 60.degree. C. under nitrogen for 2 h.
After cooling to room temperature, the solution was concentrated in
vacuo and the residue was redissolved in water. After acidifying
with 0.1 N HCl, the solution was applied to a Toyopearl SP-650M
ion-exchange column (NH.sub.4.sup.+form) and the product was eluted
with a 0 to 0.4 M ammonium bicarbonate gradient. Appropriate
fractions were combined and lyophilized to dryness. This afforded
80 mg (79%) of 13 as a white powder.
Example 14
.beta.-Cyclodextrin(cystamine)-DTBP copolymer, 14
##STR00020##
[0078] A 4 mL vial was charged with a solution of 19.6 mg
(1.42.times.10.sup.-5 mol) of the bis(hydrogen carbonate) salt of
13 in 0.5 mL of 0.1 M NaHCO.sub.3. The solution was cooled in an
ice bath before 4.4 mg (1.4.times.10.sup.-5 mol) of dimethyl
3,3'-dithiobispropionimidate-2HCl (DTBP, Pierce Chemical Co. of
Rockford, Ill.) was added. The resulting solution was then agitated
with a Vortex mixer and allowed to stand at 0.degree. C. for 1 h.
The reaction was quenched with 1M Tris-HCl before it was acidified
to pH 4 with 0.1N HCl. The aqueous polymer solution was then
subjected to gel permeation chromatography on Toyopearl HW-40F
resin. Fractions were analyzed by GPC and appropriate fractions
were lyophilized to dryness. This afforded 21.3 mg (100%) of 14 as
a white powder.
Example 15
.beta.-Cyclodextrin(cystamine)-DMS copolymer, 15
##STR00021##
[0080] A 10 mL Schlenk flask equipped with a magnetic stirbar and a
septum was charged with 200 mg (1.60.times.10.sup.-4 mol) of 13, 44
.mu.L (3.2.times.10.sup.-4 mol) of triethylamine (Aldrich Chemical
Co., Milwaukee, Wis.), 43.6 mg (1.60.times.10.sup.-4 mol) of
dimethylsuberimidate.2HCl (DMS, Pierce Chemical Co. of Rockford,
Ill.), and 3 mL of anhydrous DMF (Aldrich Chemical Co., Milwaukee,
Wis.). The resulting slurry was heated to 80.degree. C. for 18
hours under a steady stream of nitrogen during which time most of
the solvent had evaporated. The residue which remained was
redissolved in 10 mL of water and the resulting solution was then
acidified with 10% HCl to pH 4. This solution was then passed
through an Amicon Centricon Plus-20 5,000 NMWL centrifugal filter.
After washing with 2.times.10 mL portions of water, the polymer
solution was lyophilized to dryness yielding 41.4 mg (18%) of an
off-white amorphous solid.
Example 16
Fixed Permanent Charged Copolymer Complexation with Plasmid
[0081] In general, equal volumes of fixed charged CD-polymer and
DNA plasmid solutions in water are mixed at appropriate
polymer/plasmid charge ratios. The mixture is then allowed to
equilibrate and self-assemble at room temperature overnight.
Complexation success is monitored by transferring a small aliquot
of the mixture to 0.6% agarose gel and checking for DNA mobility.
Free DNA travels under an applied voltage, whereas complexed DNA is
retarded at the well.
[0082] 1 .mu.g of DNA at a concentration of 0.1 .mu.g/.mu.L in
distilled water was mixed with 10 .mu.L of copolymer 14 at polymer
amine: DNA phosphate charge ratios of 2.4, 6, 12, 24, 36, 60, and
120. The solution was mixed manually by a micropipette and then
gently mixed overnight on a lab rotator. 1 .mu.g/.mu.L of loading
buffer (40% sucrose, 0.25% bromophenol blue, and 200 mM
Tris-Acetate buffer containing 5 mM EDTA (Gao et al., Biochemistry
35:1027-1036 (1996)) was added to each solution the following
morning. Each DNA/polymer sample was loaded on a 0.6% agarose
electrophoresis gel containing 6 .mu.g of EtBr/100 mL in
1.times.TAE buffer (40 mM Tris-acetate/1 mM EDTA) and 40V was
applied to the gel for 1 hour. The extent of DNA/polymer
complexation was indicated by DNA retardation in the gel migration
pattern. The polymer (14) retarded DNA at charge ratios of 6 and
above, indicating complexation under these conditions.
Example 17
Crosslinking of Copolymer-Plasmid Complex
##STR00022##
[0084] Copolymer 14 or copolymer 15 was oxidized as in Example 9.
Oxidized copolymer 14 or 15 was then complexed with a DNA plasmid
as in Example 16. The solution was buffered with a borate buffer to
pH 8.5 and a crosslinking agent, PEG.sub.600-Dihydrazide, was then
added and the supramolecular complex formed was analyzed by light
scattering, zeta potential, and electron microscopy. Using oxidized
copolymer 15, the polymer-plasmid DNA composite gave an average
particle size of 90 nm by light scattering and had a surface charge
of 40 mV as determined by zeta potential measurement. Upon addition
of PEG.sub.600-Dihydrazide, the supramolecular complex had an
average size of 120 nm and a surface charge of 17 mV. Electron
microscopy showed the composite to be uniform in size while the
supramolecular complex revealed some dispersion in size.
Example 18
Transfection Studies with Plasmids Encoding Luciferase Reporter
Gene
[0085] BHK-21 cells were plated in 24 well plates at a cell density
of 60,000 cells/well 24 hours before transfection. Plasmids
encoding the luciferase gene were mixed with the CD-polymer as in
Example 16 except copolymer 14 was replaced with copolymer 15.
Media solution containing the DNA/polymer complexes was added to
cultured cells and replaced with fresh media after 24 hours of
incubation at 37.degree. C. The cells were lysed 48 hours after
transfection. Appropriate substrates for the luciferase light assay
were added to the cell lysate. Luciferase activity, measured in
terms of light units produced, was quantified by a luminometer.
DNA/polymer complexes successfully transfected BHK-21 cells at a
charge ratios of 10, 20, 30, and 40 with maximum transfection at
polymer amine:DNA phosphate charge ratio of 40. Cell lysate was
also used to determine cell viability by the Lowry protein assay.
(Lowry et al., Journal of Biological Chemistry, Vol. 193, 265-275
(1951)). No toxicity was observed up to charge ratios of 40.
Example 19
Transfection Studies with a Supramolecular Complex
[0086] The supramolecular complex formed in Example 17 was used to
transfect BHK-21 cells following the procedure of Example 18. No
transfection was observed.
Example 20
Crosslinking of Polyethyleneimine-Plasmid Complex
[0087] Polyethyleneimine (PEI) is complexed with a DNA plasmid as
in Example 16. A crosslinking agent (for example, dimethyl
3,3'-dithiobispropionimidate (DTBP, commercially available from
Pierce Chemical Co. of Rockford, Ill.); dithiobis(succinimidyl
propionate) (DSP, commercially available from Pierce Chemical Co.
of Rockford, Ill.) for biodegradable crosslinking; and
disuccinimidyl suberate (DSS, commercially available from Pierce
Chemical Co.) or dimethylsuberimidate (DMS, commercially available
from Pierce Chemical Co.) for less biodegradable crosslinking) is
then added and the supramolecular complex formed is analyzed by
light scattering, zeta potential, and electron microscopy.
Example 21
Crosslinking Polymers Formed from DNA Template Polymerization
[0088] Template polymerization using DNA as the template is
accomplished as described by Trubetskoy et al. Nucleic Acids
Research, Vol. 26, No. 18, pp 4178-4185 (1998).
[0089] DNA is contacted with AEPD and comonomers A. The resultant
composite of substantially linear polymer and DNA is crosslinked by
adding suitable crosslinking agents (for example, DTBP, DSP, DSS,
DMS) and the supramolecular complex formed is analyzed by light
scattering, zeta potential, and electron microscopy.
Example 22
Crosslinking Polymers Formed from DNA Template Polymerization
[0090] Template polymerization using DNA as the template is
accomplished as described by Trubetskoy et al. Nucleic Acids
Research, Vol. 26, No. 18, pp 4178-4185 (1998).
[0091] DNA is contacted with oxidized cyclodextrin diamines (for
example, IXa, IXb, IXc) and comonomers A. The resultant composite
of substantially linear polymer and DNA is crosslinked by adding
suitable crosslinking agents (for example, adipic acid dihydrazide,
polyethylene glycol 600 dihydrazide 8 of Example 8) and the
supramolecular complex formed is analyzed by light scattering, zeta
potential, and electron microscopy.
Example 23
Thiolation of Cyclodextrin (CD) Polymer with Traut's Reagent
[0092] Under nitrogen, 10.1 mg (7.34.times.10.sup.-5 mol) of
Traut's reagent (Pierce Chemical Co. of Rockford, Ill.) was added
to 1.00 mL of a 5.0 mM solution of .beta.-CD(cystamine)-DMS
copolymer 15 in 0.1 M Na.sub.2CO.sub.3 (pH 10.0) containing 1.0 mM
EDTA. The resulting solution was allowed to stand under nitrogen,
N2, at ambient temperature for 2 hours. The solution was then
opened to air and filtered through an Amicon 5,000 NMWL centrifugal
filter after which the supernatant was diluted with 10.0 mL of
water and filtered a second time. The supernatant solution was then
diluted to a 1.00 mL volume in water and stored under nitrogen. An
aliquot was titrated with Ellman's reagent (Hermanson, G. T.,
Bioconjugate Techniques; Academic: New York, p. 89 (1996)) to yield
a thiol content of 1.56.times.10.sup.-6 mol, corresponding to thiol
functionalization of 31% of the polymer cyclodextrin moieties.
Example 24
Air Oxidation of Thiolated Cyclodextrin (CD) Polymer
[0093] Five (5) 9 .mu.L aliquots (total of 45 .mu.L) of 3 mM
thiolated CD polymer of Example 23 was added to 20 .mu.L of plasmid
DNA (0.24 .mu.g/.mu.L) at 10 minute intervals. The resulting
solution was allowed to oxidize in air overnight. Electron
microscopy showed the resulting supramolecular complex to be
uniform in size.
Example 25
Oxidation of Thiolated Cyclodextrin (CD) Polymer with
Aldrithiol
[0094] Five (5) 9 .mu.L aliquots (total of 45 .mu.L of 3 mM
thiolated CD polymer of Example 23 was added to 20 .mu.L of plasmid
DNA (0.24 .mu.g/.mu.L) at 10 minute intervals. Two equivalents of
oxidizing reagent ALDRITHIOL (commercially available from Aldrich
Chemical Company, Inc., Milwaukee, Wis.) based on the thiolated CD
polymer was immediately added to the solution and gently mixed by
pipetting. Electron microscopy showed the resulting supramolecular
complex to be uniform in size.
Example 26
Synthesis of .beta.-cyclodextrin(cystamine)-DMA copolymer, 16
##STR00023##
[0096] A 20 mL scintillation vial equipped with a magnetic stirbar
was charged with 180 mg (0.131 mmol) of 13 and 32 mg of dimethyl
adipimidate (DMA, Pierce Chemical Co. of Rockford, Ill.). To this
was added 500 .mu.L of 0.5 M Na.sub.2CO.sub.3. The resulting
solution was covered with foil and stirred overnight. The mixture
was acidified with 0.1 N HCl and dialyzed with Spectrapor MWCO
3,500 membrane for 2 days and lyophilized to afford 41 mg of a
white amorphous solid with Mw=14 kD, as determined by light
scattering.
Example 27
Synthesis of .beta.-cyclodextrin(cystamine)-DMP copolymer, 17
##STR00024##
[0098] A 20 mL scintillation vial equipped with a magnetic stirbar
was charged with 160 mg (0.116 mmol) of 13 and 30.1 mg of dimethyl
pimelimidate (DMP, Pierce Chemical Co. of Rockford, Ill.). To this
was added 500 .mu.l of 0.5 M Na.sub.2CO.sub.3. The resulting
solution was covered with foil and stirred overnight. The mixture
was then acidified with 0.1 N HCl and dialyzed with Spectrapor MWCO
3,500 membrane for 2 days and lyophilized to afford 22 mg of a
white amorphous solid with Mw=14 kD, as determined by light
scattering.
Example 28
Transfection Studies with Plasmids Encoding Luciferase Reporter
Gene
##STR00025##
[0100] BHK-21 cells were plated in 24-well plates at cell density
of 60,000 cells/well in 1 mL media. Plasmids encoding the
luciferase gene were mixed with the CD-polymer as in Example 16
using copolymers 14, 15, 16, or 17. After 24 hours, media was
removed and transfection mixture (200 .mu.L of Optimem with 20
.mu.L of polymer/DNA solution) was added to each well. After 5
hours, 800 .mu.L of complete media (DMEM+10% BS, Gibco) was added
to each well. 24 hours after transfection, media was replaced with
1 mL complete media. After another 24 hours, media was removed and
cells were washed with 1 mL PBS. Cells were then lysed with 0.050
mL of Cell Culture Lysis Buffer (Promega) by one freeze-thaw cycle.
4 .mu.L of cell lysate was used for luciferase assay, measured in
terms of light units produced, and 10 .mu.L, was used for Biorad's
protein DC assay. The transfection and toxicity results are
illustrated in FIGS. 2A, 2B, 2C and 2D.
Example 29
Effect of Heparan Sulfate on PEI/DNA Particle
[0101] Various concentrations of linear polyethyleneimine (IPEI) 2
kD were mixed with DNA for 30 minutes. Heparan sulfate (75.times.
of DNA) was added to the PEI/DNA solution for 30 minutes. An
agarose gel was run to examine the results. At low PEI/DNA ratio,
heparan sulfate was able to strip PEI away from DNA. However, at a
higher concentration of PEI, PEI remained associated with the DNA
even with the addition of heparan sulfate.
Example 30
Crosslinking Experiment Using Branched Polyethyleneimine (bPEI) 25
kDa with Varying Concentration of Heparan Sulfate
[0102] 10 .mu.L (1 .mu.g) of DNA and 10 .mu.L of polyethyleneimine
(PEI, 1.41 mM, 5+/-charge ratio) was mixed together for 30 minutes.
Then crosslinker dithiobispropionimidate (DTBP) or
dimethylsuperimidate (DMS) was added to the DNA/PEI solution. After
90 minutes, different concentration of heparan sulfate (HS) was
added for competitive binding with PEI. The agarose gel was run
after 20 minutes to examine the effect of crosslinker on the
binding of PEI to DNA. For 0.1-/+HS, HS could not bind to PEI to
cause PEI to dissociate from DNA. Higher concentrations of HS could
dissociate PEI from DNA only in the absence of DTBP (or DMS). Thus,
with the presence of crosslinker on PEI, PEI has a higher affinity
to DNA. However, at 3-/+HS, the concentration is high enough such
that HS dissociated PEI from DNA even with the presence of
crosslinker.
Example 31
Crosslinking Experiment with Pentalysine Using Crosslinker DTBP and
Reducing Agent Tris(2-carboxyethyl)phosphine (TCEP)
[0103] Pentalysine was added to DNA for 15 minutes. Crosslinker
DTBP was then added to the solution mixture for over 60 minutes.
TCEP was added and an agarose gel was run after 30 minutes.
Pentalysine itself was not strong enough to bind to the DNA.
However, with the addition of crosslinker DTBP, crosslinked
pentalysine bound to the DNA. When reducing agent TCEP was added,
pentalysine once again dissociated from DNA. Thus, crosslinking
with DTBP increased the affinity of pentalysine to DNA.
Example 32
Reversible Crosslinking of Branched PEI (bPEI) (25 kDa) with
DTBP
[0104] 1 ug of DNA plasmid (.about.5 kpb) was complexed with bPEI
(25 kDa) at a 5+/-charge ratio for 30 minutes. Crosslinker DTBP
(dithiobispropionimidate, Pierce Chemical Co. of Rockford, Ill.)
was then added and allowed to react with the primary amines on PEI
for 90 minutes. After the reaction, some of the solutions were
treated with a reducing agent, TCEP (Tris2-carboxyethyl)phosphine)
for 25 minutes. Heparan sulfate was then added to the mixture at a
2:1 charge ratio with respect to PEI to dissociate the
particles.
[0105] Heparan sulfate was unable to dissociate crosslinked PEI
from the DNA. However, after reduction of the crosslinking agent
with TCEP, heparan sulfate was able to dissociate the PEI from DNA.
Thus crosslinking DTBP is able to stabilize PEI/DNA composites.
This stabilization is reversible under reductive conditions. The
results are illustrated in the agarose gel of FIG. 1.
Example 33
.beta.-Cyclodextrin(cystamine)-PEG600 Copolymer, 18
##STR00026##
[0107] A 100 mL round-bottom flask equipped with a magnetic
stirbar, a Schlenk adapter and a septum was charged with 1.564 g
(1.25 mmol) of 13 and 25 mL of freshly distilled dimethylacetamide
(DMAc, Aldrich). To the slurry was added 0.7 mL (4 eq) of
triethylamine (Aldrich) and a solution of 10 in 5 mL of DMAc. The
resulting mixture was heated to 80.degree. C. for 2 hours. After
this time, the reaction was allowed to cool to ambient temperature
and the solvent was removed in vacuo. The residue was then taken up
into 50 mL of water and the resulting solution dialyzed against
water in a Spectra/Por 7 MWCO 3,500 membrane. The resulting
solution was lyophilized to dryness to afford 1.515 g (66%) of an
off-white amorphous solid with Mw=25,000, as determined by light
scattering.
Example 34
Synthesis of .beta.-Cyclodextrin-Tosylate, 19 (Melton, L. D., and
Slessor, K. N., Carbohydrate Research, 18, p 29 (1971))
##STR00027##
[0109] A 500 mL round-bottom flask equipped with a magnetic
stirbar, a vacuum adapter and a septum was charged with a solution
of dry .beta.-cyclodextrin (8.530 g, 7.51 mmol) and 200 mL of dry
pyridine. The solution was cooled to 0.degree. C. before 1.29 g
(6.76 mmol) of tosyl chloride was added. The resulting solution was
allowed to warm to room temperature overnight. The pyridine was
removed as much as possible in vacuo. The resulting residue was
then recrystallized twice from 40 mL of hot water to yield 7.54
(88%) of a white crystalline solid.
Example 35
Synthesis of .beta.-cyclodextrin-thiol, 20 (K. Fujita, et al.,
Bioorg. Chem., Vol. 11, p 72 (1982) and K. Fujita, et al., Bioorg.
Chem., Vol. 11, p. 108 (1982))
##STR00028##
[0111] A 50 mL round bottom flask with a magnetic stirbar and a
Schlenk adapter was charged with 1.00 g (0.776 mmol) of 19, 0.59 g
(7.75 mmol) of thiourea (Aldrich) and 7.8 mL of 0.1N NaOH solution.
The resulting mixture was heated at 80.degree. C. for 6 hours under
nitrogen. Next, 0.62 g (15.5 mmol) of sodium hydroxide was added
and the reaction mixture was heated at 80.degree. C. under nitrogen
for another hour. The reaction was allowed to cool to room
temperature before it was brought to pH 4.0 with 10% HCl. The total
solution volume was brought to 20 mL and then was cooled in an ice
bath before 0.8 mL of tetrachloroethylene was added. The reaction
mixture was stirred vigorously at 0.degree. C. for 0.5 h before the
precipitated solid was collected in a fine glass frit. The solid
was pumped down overnight to yield 0.60 g (67%) of a white
amorphous solid.
Example 36
Synthesis of .beta.-cyclodextrin-iodide, 21
##STR00029##
[0113] A round bottom flask with a magnetic stirbar and a Schlenk
adapter is charged with 19, 15 equivalents of potassium iodide, and
DMF. The resulting mixture is heated at 80.degree. C. for 3 hours,
after which the reaction is allowed to cool to room temperature.
The mixture is then filtered to remove the precipitate and the
filtrate evaporated to dryness and redissolved in water at
0.degree. C. Tetrachloroethylene is added and the resulting slurry
stirred vigorously at 0.degree. C. for 20 minutes. The solid is
collected on a medium glass frit, triterated with acetone and
stored over P.sub.2O.sub.5.
Example 37
Synthesis of .beta.-cyclodextrin-thiol-PEG Appended Polymer, 22
##STR00030##
[0115] A 100 mL round-bottom flask equipped with a magnetic stirbar
and a reflux condensor was charged with 2.433 g (2.11 mmol) of 20
and 0.650 g of functionalized PEG (PEG with pendant olefins,
received from Yoshiyuki Koyama of Otsuma Women's University, Tokyo,
Japan). The resulting mixture was heated at reflux for 12 hours,
during which time 20 dissolved. The reaction mixture was allowed to
cool to room temperature and precipitated solid was removed by
centrifugation. The supernatant was dialyzed against water in a
Spectra/Por 7 MWCO 1,000 membrane. The solution was lyophilized to
give an amorphous white solid.
Example 38
Synthesis of branched PEI-cyclodextrin polymer, 23
[0116] A 20 mL scintillation vial equipped with a magnetic stirbar
is charged with branched PEI (25 kD, Aldrich) and 22. To this is
added degassed sodium carbonate buffer. The resulting solution
stirred at 80.degree. C. for 4 hours. The mixture is acidified with
0.1 N HCl and dialyzed with Spectra/Por MWCO 3,500 membrane for 2
days and lyophilized.
Example 39
Synthesis of hexamethylenediamine-DMS copolymer, 24
##STR00031##
[0118] A 20 mL scintillation vial equipped with a magnetic stirbar
was charged with 80 mg (0.690 mmol) of hexamethylenediamine
(Aldrich) and 195 mg of dimethyl suberimidate (DMS, Pierce Chemical
Co. of Rockford, Ill.). To this was added 250 .mu.l of 0.5 M
Na.sub.2CO.sub.3. The resulting solution was covered with foil and
stirred overnight. The mixture was then acidified with 0.1 N HCl
and dialyzed with Spectrapor MWCO 3,500 membrane for 2 days and
lyophilized to afford 30 mg of a white amorphous solid.
Example 40
Synthesis of 1,9-diaminononane-DMS copolymer, 25
##STR00032##
[0120] A 20 mL scintillation vial equipped with a magnetic stirbar
was charged with 85 mg (0.537 mmol) of 1,9-diaminononane (Aldrich)
and 146 mg of dimethyl suberimidate (DMS, Pierce Chemical Co. of
Rockford, Ill.). To this was added 250 .mu.l of 0.5 M
Na.sub.2CO.sub.3. The resulting solution was covered with foil and
stirred overnight. The mixture was then acidified with 0.1 N HCl
and dialyzed with Spectrapor MWCO 3,500 membrane for 2 days and
lyophilized to afford 41.7 mg of a white amorphous solid.
Example 41
Transfections with diamine-DMS copolymers 24 and 25
[0121] Transfections were conducted as described in Example 26,
except copolymers 24 and 25 replaced copolymers 14, 15, 16, and 17.
Transfection and toxicity results are illustrated in FIG. 3A and
FIG. 3B, respectively. Removal of cyclodextrin from the polymer
backbone results in polymers with high toxicity.
Example 42
Solubilization of Taxol with 18
[0122] Excess amounts of paclitaxel was added to an 18% solution of
18. The solutions were agitated, vortexed, and then filtered by a 2
.mu.M nylon filter to remove any undissolved paclitaxel. The
filtered solution was then injected into an HPLC equipped with an
Altima C8 reverse phase column. Paclitaxel was detected by UV
absorption at 227 nm, and concentration of paclitaxel determined by
peak integration. Calibration plots of paclitaxel concentration vs.
peak area showed a linear relationship up to 25 .mu.g/mL. The
presence of 18% solution of 18 clearly enhanced solubility of
paclitaxel greater than 30 times.
Example 43
Delivery of paclitaxel with 18 or 22
[0123] Cells are counted on a hemocytometer and plated at a density
of 4,000 cells/well in 96 well plates. Paclitaxel is mixed with
polymer 18 conjugated with a ligand or polymer 22 conjugated with a
ligand for targeted delivery. The solutions are allowed to mix for
at least 30 minutes, after which, the drug/polymer solutions are
added to the cells with serial dilution. The culture plates are
incubated at 37.degree. C. After two days, the IC.sub.50 of the
paclitaxel to the cells is determined by MTT assay. The culture
medium is removed, and the cells are washed with PBS. Next, 50
.mu.L/well MTT is added, followed by 150 .mu.L/well media. After 4
hours of incubation at 37.degree. C., the MTT solution is removed
and the formazan is solubilized by the addition of 200 .mu.L/well
DMSO. The absorbance of the formazan is read at 562 nm by a
microtiter plate reader.
[0124] It should be understood that the foregoing discussion and
examples merely present a detailed description of certain preferred
embodiments. It will be apparent to those of ordinary skill in the
art that various modifications and equivalents can be made without
departing from the spirit and scope of the invention. All the
patents, journal articles and other documents discussed or cited
above are herein incorporated by reference in their entirety.
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