U.S. patent application number 11/358976 was filed with the patent office on 2007-02-01 for linear cyclodextrin copolymers.
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 | 20070025952 11/358976 |
Document ID | / |
Family ID | 27376928 |
Filed Date | 2007-02-01 |
United States Patent
Application |
20070025952 |
Kind Code |
A1 |
Davis; Mark E. ; et
al. |
February 1, 2007 |
Linear cyclodextrin copolymers
Abstract
Linear cyclodextrin copolymers and linear oxidized cyclodextrin
copolymers containing an unoxidized and/or an oxidized cyclodextrin
moiety integrated into the polymer backbone are described. Methods
of preparing such copolymers are also described. The linear
cyclodextrin copolymer and linear oxidized cyclodextrin copolymer
of the invention may be used as a delivery vehicle of various
therapeutic agents.
Inventors: |
Davis; Mark E.; (Pasadena,
CA) ; Gonzalez; Hector; (San Francisco, CA) ;
Hwang; Suzie (Sue Jean); (Torrance, CA) |
Correspondence
Address: |
FISH & NEAVE IP GROUP;ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
California Institute of
Technology
Pasadena
CA
|
Family ID: |
27376928 |
Appl. No.: |
11/358976 |
Filed: |
February 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09339818 |
Jun 25, 1999 |
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11358976 |
Feb 21, 2006 |
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09203556 |
Dec 2, 1998 |
6509323 |
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09339818 |
Jun 25, 1999 |
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60091550 |
Jul 1, 1998 |
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Current U.S.
Class: |
424/78.3 ;
514/58; 525/54.2 |
Current CPC
Class: |
B82Y 5/00 20130101; C08G
69/48 20130101; C08G 69/40 20130101; C08G 75/06 20130101; C08B
37/0012 20130101; C08G 73/06 20130101; C08G 73/02 20130101; A61K
47/6951 20170801 |
Class at
Publication: |
424/078.3 ;
514/058; 525/054.2 |
International
Class: |
A61K 31/765 20060101
A61K031/765; A61K 31/724 20060101 A61K031/724; C08G 63/91 20060101
C08G063/91 |
Claims
1-45. (canceled)
46. A water-soluble, linear cyclodextrin copolymer having a linear
polymer backbone, the copolymer including a plurality of
cyclodextrin monomer moieties and linker moieties in the polymer
backbone, wherein, unless the cyclodextrin monomer moiety or the
linker moiety is present at the terminus of a polymer chain, each
of the cyclodextrin monomer moieties is attached to two of the
linker moieties, and each of the linker moieties covalently links
two cyclodextrin monomer moieties, and wherein the cyclodextrin
monomer moieties are unsubstituted or substituted by groups that do
not interfere with the copolymerization with the linker moiety.
47. The copolymer of claim 46, wherein the cyclodextrin monomer
moieties are the same or different throughout the polymer.
48. The polymer of claim 46, comprising repeating units of Formula
Ia, Ib, or a combination thereof: ##STR34## wherein C is a
substituted or unsubstituted cyclodextrin monomer moiety, and A is
a linker moiety bound to the cyclodextrin monomer moiety C.
49. The copolymer of claim 46, wherein the cyclodextrin monomer
moieties are substituted or unsubstituted .alpha.-, .beta.-, or
.gamma.-cyclodextrin, or a combination thereof.
50. The copolymer of claim 46, wherein the cyclodextrin monomer
moieties are independently selected from
6.sup.A,6.sup.B-deoxy-.alpha.-cyclodextrin,
6.sup.A,6.sup.C-deoxy-.alpha.-cyclodextrin,
6.sup.A,6.sup.D-deoxy-.alpha.-cyclodextrin,
6.sup.A,6.sup.B-deoxy-.beta.-cyclodextrin,
6.sup.A,6.sup.C-deoxy-.beta.-cyclodextrin,
6.sup.A,6.sup.D-deoxy-.beta.-cyclodextrin,
6.sup.A,6.sup.B-deoxy-.gamma.-cyclodextrin,
6.sup.A,6.sup.C-deoxy-.gamma.-cyclodextrin,
6.sup.A,6.sup.D-deoxy-.gamma.-cyclodextrin, and
6.sup.A,6.sup.E-deoxy-.gamma.-cyclodextrin.
51. The copolymer of claim 46, wherein the cyclodextrin monomer
moiety has the general Formula III: ##STR35## wherein p=5-7.
52. The cyclodextrin copolymer of claim 46, wherein the
cyclodextrin monomer moieties are, independently, selected from
2.sup.A,3.sup.A-deoxy-2.sup.A,3.sup.A-dihydro-.alpha.-cyclodextrin,
2.sup.A,3.sup.A-deoxy-2.sup.A,3.sup.A-dihydro-.beta.-cyclodextrin,
and
2.sup.A,3.sup.A-deoxy-2.sup.A,3.sup.A-dihydro-.gamma.-cyclodextrin.
53. The cyclodextrin copolymer of claim 46, wherein the linker
moiety is biodegradable or acid-labile.
54. The cyclodextrin copolymer of claim 46, wherein the
cyclodextrin copolymer is crosslinked to a polymer.
55. The cyclodextrin copolymer of claim 46, wherein at least one
ligand is bound to the linear cyclodextrin copolymer.
56. A composition comprising: a cyclodextrin copolymer as defined
in claim 46; and a therapeutic agent.
57. A method for preparing a water-soluble, linear cyclodextrin
copolymer comprising: (a) providing at least one cyclodextrin
comonomer precursor which is disubstituted with a leaving group.
(b) reacting the cyclodextrin monomer precursor with a linker
precursor comprising at least two functional groups through which a
linkage of the cyclodextrin monomer precursor can be achieved,
whereby a water-soluble, linear cyclodextrin copolymer comprising
alternating units of cyclodextrin monomer moieties and linker
moieties is prepared.
58. The method of claim 57, wherein at least one cyclodextrin
monomer moiety of the cyclodextrin copolymer has been modified with
at least one ligand.
59. The method of claim 58, wherein the ligand is selected from
vitamins, proteins, and polysaccharides.
60. The method of claim 57, comprising: polymerizing a cyclodextrin
monomer precursor, where the cyclodextrin monomer precursor is
disubstituted with the same or different leaving group, with a
linker A precursor capable of displacing the leaving groups to form
the water-soluble linear cyclodextrin copolymer having repeating
units of Formula Ia, Ib, or a combination thereof: ##STR36##
wherein C is a substituted or unsubstituted cyclodextrin monomer
moiety, and A is a linker moiety bound to the cyclodextrin
derivative C.
61. The method of any one of claims 57 or 60, wherein the
cyclodextrin monomer precursor is a diiodinated cyclodextrin
monomer precursor of Formula IVa, IVb, IVc, or a mixture thereof:
##STR37##
62. The method of claim 61, further comprising reacting the linear
cyclodextrin copolymer with a ligand to form a linear cyclodextrin
copolymer having at least one ligand bound to the copolymer.
63. The method of any one of claims 57 or 60, wherein the
cyclodextrin monomer moieties are derived from .alpha.-, .beta.-,
.gamma.-cyclodextrin, or a combination thereof.
64. The method of claim 61, further comprising: (a) aminating the
diiodinated cyclodextrin monomer precursor to form a diaminated
cyclodextrin monomer precursor; and (b) copolymerizing the
diaminated cyclodextrin monomer precursor to form the
water-soluble, linear cyclodextrin copolymer.
65. A method of producing a crosslinked cyclodextrin copolymer,
comprising: reacting at least one linear cyclodextrin copolymer
having a repeating unit of Formula Ia, Ib, or a combination
thereof: ##STR38## wherein C is a substituted or unsubstituted
cyclodextrin monomer moiety, and A is a linker moiety bound to the
cyclodextrin monomer moiety C, with a polymer in the presence of a
crosslinking agent.
66. The method of claim 65, wherein the polymer is a linear
cyclodextrin copolymer or a linear oxidized cyclodextrin
copolymer.
67. A copolymer prepared by the method of claim 57.
68. A composition comprising the copolymer of any one of claims 46
or 67 and an agriculturally biologically active compound.
69. The composition of claim 68, wherein the agriculturally
biologically active compound is selected from a fungicide,
herbicide, insecticide, and mildeweide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/339,818, filed Jun. 25, 1999, which is a
continuation-in-part of U.S. application Ser. No. 09/203,556, now
U.S. Pat. No. 6,509,323, filed Dec. 2, 1998, which claims the
benefit of U.S. Provisional Application Ser. No. 60/091,550, filed
Jul. 1, 1998, each of which is herein incorporated by reference in
its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to linear cyclodextrin copolymers and
linear oxidized cyclodextrin copolymers. These copolymers,
respectively, contain a cyclodextrin moiety, unoxidized or
oxidized, as a monomer unit integrated into the copolymer backbone.
The invention also relates methods of preparing linear cyclodextrin
copolymers and linear oxidized cyclodextrin copolymers. Such
cyclodextrin copolymers may be used as a delivery vehicle of
various therapeutic agents.
[0004] 2. Background of the Invention
[0005] Cyclodextrins are cyclic polysaccharides containing
naturally occurring D(+)-glucopyranose units in an .alpha.-(1,4)
linkage. The most common cyclodextrins which contain, respectively,
six, seven or eight glycopyranose 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: ##STR1## 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).
[0006] 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 cyclodextrin 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.
[0007] 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.
[0008] 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.
[0009] A biodegradable medicinal polymer assembly with
supermolecular structure is described in WO 96/09073 A1. 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.
[0010] However, there still exists a need in the art for linear
cyclodextrin polymers in which the cyclodextrin moiety is part of
the main chain and not a pendant moiety off the main chain and a
method for their preparation.
SUMMARY OF THE INVENTION
[0011] This invention answers this need by providing a linear
cyclodextrin copolymer. Such a linear cyclodextrin copolymer has a
repeating unit of formula Ia, Ib, or a combination thereof:
##STR2##
[0012] The invention also provides methods of preparing a linear
cyclodextrin copolymer. One method copolymerizes a cyclodextrin
monomer precursor disubstituted with the same or different leaving
group and a comonomer A precursor capable of displacing the leaving
group. Another such method involves iodinating a cyclodextrin
monomer precursor to form a diiodinated cyclodextrin monomer
precursor and then copolymerizing the diiodinated cyclodextrin
monomer precursor with a comonomer A precursor to produce the
linear cyclodextrin copolymer. Another method involves iodinating a
cyclodextrin monomer precursor to form a diiodinated cyclodextrin
monomer precursor, aminating the diiodinated cyclodextrin monomer
precursor to form a diaminated cyclodextrin monomer precursor and
then copolymerizing the diaminated cyclodextrin monomer precursor
with a comonomer A precursor to produce the linear cyclodextrin
copolymer. Yet another method involves the reduction of a linear
oxidized cyclodextrin copolymer to the linear cyclodextrin
copolymer.
[0013] The invention further provides a linear oxidized
cyclodextrin copolymer. A linear oxidized cyclodextrin copolymer is
a linear cyclodextrin copolymer which contains at least one
oxidized cyclodextrin moiety of formula VIa or VIb: ##STR3## Each
cyclodextrin moiety of a linear cyclodextrin copolymer of the
invention may be oxidized so as to form a linear oxidized
cyclodextrin copolymer having a repeating unit of formula VIa, VIb,
or a combination thereof.
[0014] The invention also provides a method of preparing a linear
oxidized cyclodextrin copolymer. One method involves oxidizing a
linear cyclodextrin copolymer such that at least one cyclodextrin
monomer is oxidized. Other methods involve copolymerizing an
oxidized cyclodextrin monomer precursor with a comonomer A
precursor.
[0015] The invention still further provides a linear cyclodextrin
copolymer or linear oxidized cyclodextrin copolymer grafted onto a
substrate and a method of their preparation. The invention also
provides a linear cyclodextrin copolymer or linear oxidized
cyclodextrin copolymer crosslinked to another polymer and a method
of their preparation. A method of preparing crosslinked
cyclodextrin polymers involves reacting a linear or linear oxidized
cyclodextrin copolymer with a polymer in the presence of a
crosslinking agent.
[0016] The invention provides a linear cyclodextrin copolymer or
linear oxidized cyclodextrin copolymer having at least one ligand
bound to the cyclodextrin copolymer. The ligand may be bound to
either the cyclodextrin moiety or the comonomer A moiety of the
copolymer.
[0017] The invention also provides a cyclodextrin composition
containing at least one linear cyclodextrin copolymer of the
invention and at least one linear oxidized cyclodextrin copolymer
of the invention. The invention also provides therapeutic
compositions containing a therapeutic agent and a linear
cyclodextrin copolymer and/or a linear oxidized cyclodextrin
copolymer of the invention. A method of treatment by administering
a therapeutically effective amount of a therapeutic composition of
the invention is also described.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The following figures depict illustrative embodiments of the
invention. These depicted embodiments are to be understood as
illustrative of the invention and not as limiting in any way.
[0019] FIG. 1A shows transfection studies with plasmids encoding
Luciferase reporter gene particularly noting the transfection with
copolymer 16.
[0020] FIG. 1B shows transfection studies with plasmids encoding
Luciferase reporter gene particularly noting the toxicity of
copolymer 16 to BHK-21.
[0021] FIG. 2 shows the effect of copolymer 16/DNA charge ratio and
serum conditions on transfection efficiency (.circle-solid. and
.box-solid.) and cell survival ( and .tangle-solidup.) in BHK-21
cells. Result from transfection in 10% serum and serum-free media
are shown as, respectively, dotted and solid lines. Data are
reported at the mean+/-S.D. of three samples. Toxicity data are
presented as best fit lines.
[0022] FIG. 3 shows the effect of copolymer 16/DNA charge ratio and
serum conditions on transfection efficiency (.circle-solid. and
.box-solid.) and cell survival ( and .tangle-solidup.) in CHO-K1
cells. Results from transfection in 10% serum and serum-free media
are shown as, respectively, dotted and solid lines. Data are
reported at the mean+/-S.D. of three samples. Toxicity data are
presented as best fit lines.
[0023] FIG. 4A shows transfection studies with plasmids encoding
Luciferase reporter gene particularly noting the relative light
units.
[0024] FIG. 4B shows transfection studies with plasmids encoding
Luciferase reporter gene particularly noting the fraction cell
survival.
DETAILED DESCRIPTION OF THE INVENTION
[0025] One embodiment of the invention is a linear cyclodextrin
copolymer. 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.
[0026] According to the invention, a linear cyclodextrin copolymer
has a repeating unit of formula Ia, Ib, or a combination thereof:
##STR4## In formula 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 of the invention. Within a single linear
cyclodextrin copolymer of the invention, the cyclodextrin monomer C
unit may be the same or different and, likewise, the comonomer A
may be the same or different.
[0027] 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
[0028] 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. According to the invention, 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, thiophosphonic acid and sulfonic acids;
imidazole groups; and sulfate groups.
[0029] 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: ##STR5##
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
presursor 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: ##STR6##
[0030] In a preferred embodiment of a linear cyclodextrin copolymer
of the invention, the cyclodextrin monomer C has the following
general formula (II): ##STR7## 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-deoxy-.alpha.-cyclodextrin (n=0, m=4),
6.sup.A,6.sup.C-deoxy-.alpha.-cyclodextrin (n=1, m=3),
6.sup.A,6.sup.D-deoxy-.alpha.-cyclodextrin (n=2, m=2),
6.sup.A,6.sup.B-deoxy-.beta.-cyclodextrin (n=0, m=5),
6.sup.A,6.sup.C-deoxy-.beta.-cyclodextrin (n=1, m=4),
6.sup.A,6.sup.D-deoxy-.beta.-cyclodextrin (n==2, m=3),
6.sup.A,6.sup.D-deoxy-.gamma.-cyclodextrin (n=0, m=6),
6.sup.A,6.sup.C-deoxy-.gamma.-cyclodextrin (n=1, m=5),
6.sup.A,6.sup.D-deoxy-.gamma.cyclodextrin (n=2, m=4), and
6.sup.A,6.sup.E-deoxy-.gamma.cyclodextrin (n=3, m=3). In another
preferred embodiment of linear cyclodextrin copolymer of the
invention, a cyclodextrin monomer C unit has the following general
formula (III): ##STR8## where p=5-7. In formula (III), 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)
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-deoxy-2.sup.A,3.sup.A-dihydro-.alpha.-cyclodextrin,
2.sup.A,3.sup.A-deoxy-2.sup.A,3.sup.A-dihydro-.beta.-cyclodextrin
2.sup.A,3.sup.A-deoxy-2.sup.A,3.sup.A-dihydro-.gamma.cyclodextrin,
commonly referred to as, respectively,
2,3-deoxy-.alpha.-cyclodextrin 2,3-deoxy-.beta.-cyclodextrin and
2,3-deoxy-.gamma.-cyclodextrin.
[0031] 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 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'-dithiobispropionimidate (DTBP)).
Copolymerization of a comonomer A precursor with a cyclodextrin
monomer precursor leads to the formation of a linear cyclodextrin
copolymer of the invention containing comonomer A linkages of the
following general formulae: ##STR9## ##STR10##
[0032] 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.
[0033] A linear cyclodextrin copolymer of the invention 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
linear cyclodextrin copolymer. Preferably,
[0034] the ligand allows a linear 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 linear cyclodextrin copolymer of the
invention, 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. 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
ligand will 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. The ligand may be attached to a copolymer of
the invention by means known in the art.
[0035] Another embodiment of the invention is a method of preparing
a linear cyclodextrin copolymer. According to the invention, a
linear cyclodextrin copolymer of the invention 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 of the invention
iodinates a cyclodextrin monomer precursor as described above to
form a diiodinated cyclodextrin monomer precursor of formula IVa,
IVb, IVc or a mixture thereof. ##STR11## The diiodinated
cyclodextrin may be prepared by any means known in the art.
(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.
[0036] Also according to the invention, 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: ##STR12## The diaminated cyclodextrin monomer
precursor may be prepared by any means known in the art. (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, --SCH.sub.2CH.sub.2NH.sub.2, to form a
diaminated cyclodextrin monomer precursor of formula Vd, Ve, Vf or
a mixture thereof: ##STR13##
[0037] A linear cyclodextrin copolymer of the invention may also be
prepared by reducing a linear oxidized cyclodextrin copolymer of
the invention 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.
[0038] According to the invention, a linear cyclodextrin copolymer
of the invention 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 of the invention, 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: ##STR14## 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.
[0039] A linear oxidized cyclodextrin copolymer may be prepared by
oxidation of a linear cyclodextrin copolymer as discussed above.
Oxidation of a linear cyclodextrin copolymer of the invention 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 of the invention, a
linear oxidized copolymer of the invention may contain one oxidized
cyclodextrin monomer. In another embodiment, substantially all to
all cyclodextrin monomers of the copolymer would be oxidized.
[0040] Another method of preparing a linear oxidized cyclodextrin
copolymer of the invention 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: ##STR15## 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: ##STR16## 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: ##STR17## 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 --SCH.sub.2CH.sub.2NH.sub.2.
[0041] 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 of the invention.
[0042] A linear oxidized cyclodextrin copolymer may also be further
modified by attachment of at least one ligand to the copolymer. The
ligand is as described above.
[0043] 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 and
transfection efficiency.
[0044] 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 terminated with at
least one comonomer A precursor for further modification, each as
described herein.
[0045] 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.
[0046] 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 crossing agent may be any crosslinking
agent known in the art. Examples of crosslinking agents include
dihydrazides and disulfides. In a preferred embodiment, the
crosslinking agent is a labile group such that a crosslinked
copolymer may be uncrosslinked if desired.
[0047] 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.
[0048] The invention also provides a cyclodextrin composition
containing at least one linear cyclodextrin copolymer and at least
one linear oxidized cyclodextrin copolymer of the invention as
described above. Accordingly, either or both of the linear
cyclodextrin copolymer and linear oxidized cyclodextrin copolymer
may be crosslinked to another polymer and/or bound to a ligand as
described above. Therapeutic compositions according to the
invention contain a therapeutic agent and a linear cyclodextrin
copolymer or a linear oxidized cyclodextrin copolymer, including
crosslinked copolymers, of the invention. A linear cyclodextrin
copolymer, a linear oxidized cyclodextrin copolymer and their
crosslinked derivatives are as described above. The therapeutic
agent may be any 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) and other
biologically active macromolecules such as, for example, proteins
and enzymes.
[0049] A therapeutic composition of the invention may be prepared
by means known in the art. In a preferred embodiment, a copolymer
of the invention is mixed with a therapeutic agent, as described
above, and allowed to self-assemble. According to the invention,
the therapeutic agent and a linear cyclodextrin copolymer or a
linear oxidized cyclodextrin copolymer of the invention associate
with one another such that the copolymer acts as a delivery vehicle
for the therapeutic agent. The therapeutic agent and cyclodextrin
copolymer may associate by means recognized by those of skill in
the art such as, for example, electrostatic interaction and
hydrophobic interaction. 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 therapeutic
composition of the invention containing a copolymer of the
invention 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)).
[0050] A therapeutic composition of the invention may be, for
example, a solid, liquid, suspension, or emulsion. Preferably a
therapeutic composition of the invention is in a form that can be
injected intravenously. Other modes of administration of a
therapeutic composition of the invention include, depending on the
state of the therapeutic composition, methods known in the art such
as, but not limited to, oral administration, topical application,
parenteral, intravenous, intranasal, intraocular, intracranial or
intraperitoneal injection.
[0051] Depending upon the type of therapeutic agent used, a
therapeutic composition 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 therapeutic composition 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.
[0052] Another embodiment of the invention is a composition
containing at least one biologically active compound having
agricultural utility and a linear cyclodextrin copolymer or a
linear oxidized cyclodextrin copolymer of the invention. 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.
[0053] 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
[0054] 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 and a
Progel-TSK G3000.sub.PWXL column using water as eluant at a 1.0 mL
min.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))
[0055] 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-Deoxy-.beta.-cyclodextrin, 2
(Tabushi et al. J. Am. Chem. 106, 4580-4584 (1984))
[0056] 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 centrifugation and the supernatant was decanted. 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-Deoxy-.beta.-cyclodextrin,
3 (Tabushi et al. Tetrahedron Lett. 18, 1527-1530 (1977))
[0057] 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 O-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-Deoxy-.beta.-cyclodextrin,
4 (Mungall et al., J. Org. Chem. 1659-1662 (1975))
[0058] 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 triterated
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.sup.+
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
[0059] 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
[0060] 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
[0061] 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 GP-C and appropriate fractions were lyophilized to
yield 67 mg (67%) of a colorless amorphous powder.
Example 8
.beta.-cyclodextrin-cystamine copolymer, 8
[0062] To a solution of 166.2 mg (7.38.times.10.sup.-5 mmol) of
cystamine dihydrochloride (Aldrich) in 15 mL of 0.1 N NaOH was
added 100 mg (7.38.times.10.sup.-5 mol) of 2 and 5 mL of
acetonitrile. The resulting homogeneous solution was heated at
80.degree. C. for 2 h before it was subjected to gel permeation
chromatography (GPC) on Toyopearl HW-40F resin. Fractions were
analyzed by GPC and appropriate fractions were lyophilized to yield
17.2 mg (19%) of a colorless amorphous powder.
Example 9
Polyethylene Glycol 600 Dihydride, 9
[0063] 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 10
Oxidation of .beta.-cyclodextrin-DSS copolymer, 10 (Hisamatsu et
al., Starch 44, 188-191 (1992))
[0064] 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 11
Polyethylene Glycol (PEG) 600 Diacid Chloride, 11
[0065] ##STR18##
[0066] 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 12
.beta.-cyclodextrin-PEG 600 copolymer, 12
[0067] ##STR19##
[0068] 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-amino-6.sup.A,6.sup.D-deoxy-.beta.-cyclodextrin 50
mL (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, 11. The resulting solution
was agitated with a Vortex mixer for 3 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 13
.beta.-cyclodextrin-DSP copolymer, 13
[0069] ##STR20##
[0070] 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-deoxy-.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 14
6.sup.A,6.sup.D-Bis-(2-aminoethylthio)-6.sup.A,6.sup.D-deoxy-.beta.-cyclod-
extrin, 14 (Tabushi, I: Shimokawa, K; Fugita, K. Tetrahedron Lett.
1977, 1527-1530)
[0071] ##STR21##
[0072] 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.; Fiester, 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-deoxy-.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 14 as a white powder.
Example 15
.beta.-cyclodextrin(cystamine)-DTBP copolymer, 15
[0073] ##STR22##
[0074] 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
14 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-2 HCl (DTBP. Pierce) 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 0.1 N 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 15 as a white powder.
Example 16
.beta.-cyclodextrin(cystamine)-DMS copolymer, 16
[0075] ##STR23##
[0076] 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 14, 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), 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 17
Folate Ligand Attachment to Cyclodextrin Polymer
1. Resin Coupling:
[0077] 50 mg of FMOC-PEG.sub.3400-NHS (Shearwater Polymers, Inc. of
Huntsville, Ala.) is dissolved in 1 mL of anhydrous
N,N-dimethylformamide (DMF) and is added to 10 equivalents of
hydrazide 2-chlorotrityl resin (Novabiochem USA of La Jolla,
Calif.) swelled in DMF. The mixture is stirred at 60.degree. C.
until all the polymer is coupled to the resin, as determined by a
GPC system equipped with a UV detector. The resin-polymer is then
transferred to a sintered glass column for all further
reactions.
2. Resin Capping:
[0078] The unreacted hydrazide groups on the resins are capped with
acetic anhydride and the acetic acid products are neutralized by
diisopropylethylamine.
3. Removal of Protecting Group:
[0079] The FMOC protecting group is removed by two washes with 20%
piperidine in DMF (1 mL total volume). The resin is then washed 10
times with 1 mL DMF and 5 times with 1 mL H.sub.2O.
4. Folic Acid Coupling:
[0080] 10 equivalents of folic acid and 1-(3
dimethylaminopropyl)-3-ethylcarbodiimide (EDC) is added to the
resin along with 1.5 mL H.sub.2O. 1N NaOH is added to the reaction
mixture until the folic acid is dissolved (around pH 10). The glass
column is then placed on a rotator and mixed overnight. The resin
is then washed 10 times with 1 mL NaOH (1N), 10 times with 1 mL of
50 mM sodium bicarbonate, and then 5 times each with water, THF,
and dichloromethane.
5. Cleavage from Resin:
[0081] 1% trifluoroacetic acid (TFA) in 1 ml DCM is added to the
resin twice for 1 minute each. The supernatant is collected and DCM
evaporated. The resulting oily film is rehydrated in H.sub.2O and
lyophilized, resulting in a light yellow powder. An NMR is taken to
confirm the presence of the PEG polymer.
6. Coupling to Polymer:
[0082] Folic acid-linker is reacted with 6 equivalents of a
cyclodextrin copolymer (oxidized as in Example 10) by mixing in 50
mmol borate (pH 8.5). The reaction mixture is analyzed and
conjugation polymer confirmed by a GPC system with a UV detection
at 285 nm. ##STR24## ##STR25##
Example 18
Folate Ligand Attachment to Cyclodextrin Polymer
1. Coupling:
[0083] 36 mg of t-butyl carbazate dissolved in 240 .mu.L of
DCM/ethyl acetate (1:1) was added to 260 mg of
FMOC-PEG.sub.3400-NHS (Shearwater Polymers) and mixed at room
temperature for 2 hours. The product was precipitated two times
from ethyl acetate/ether (1:1).
2. Removal of Protecting Group
[0084] FMOC protecting group was removed with 20% piperidine in
DMF. The solvent was removed in vacuo and product redissolved in
1.3 mL of DMSO.
3. Folic Acid Coupling:
[0085] 1.2 equivalents of folic acid and DCC and one drop of
pyridine was then added and the resulting solution stirred in the
dark at room temperature for 6 hours. DMSO was removed in vacuo and
conjugation of folio acid was confirmed by GPC with UV monitoring
at 285 nm.
4. Removal of Hydrazide Protecting Group:
[0086] Finally, the hydrazide was deprotected by stirring in 4M HCl
in dioxane for 1 hour before removing the solvent in vacuo. The
final product was purified by Toyopearl HW-40F column
chromatography.
5. Coupling to Polymer:
[0087] Folio acid-linker is reacted with 6 equivalents of a
cyclodextrin copolymer (oxidized as in Example 10) by mixing in 50
mmol borate (pH 8.5). The reaction mixture is analyzed and
conjugation polymer confirmed by a GPC system with a UV detection
at 285 nm. ##STR26##
Example 19
Transferrin Ligand Attachment to Cyclodextrin Polymer
1. Transferrin Oxidation
[0088] 500 mg of iron-free human transferrin (Sigma of St. Louis,
Mo.) is dissolved in 30 mM sodium acetate buffer and cooled to
0.degree. C. To this solution is added 20 mg of sodium periodate
dissolved in 4 .mu.L of 30 mM sodium acetate. The mixture is
stirred at 0.degree. C. overnight. Next 1 g of AG501-X8 resin
(Biorad) is added to remove salts before the solution is
lyophilized.
2. Resin Coupling:
[0089] 20 mg of FMOC-PEG.sub.3400-NHS (Shearwater Polymers, Inc. of
Huntsville, Ala.) was dissolved in 0.5 mL of anhydrous
N,N-dimethylformamide (DMF) and added to 10 equivalents of
hydrazide 2-chlorotrityl resin (Novabiochem USA of La Jolla,
Calif.) swelled in DMF. The mixture was stirred at 60.degree. C.
until all the polymer was coupled to the resin, as determined by a
GPC-system equipped with an ultraviolet (UV) detector. The
resin-polymer was then transferred to a sintered glass column for
all further reactions.
3. Resin Capping:
[0090] The unreacted hydrazide groups on the resins were capped
with acetic anhydride and the acetic acid products were neutralized
by diisopropylethylamine.
4. Removal of Protecting Group:
[0091] The FMOC protecting group was removed by two washes with 20%
piperidine in DMF (1 mL total volume). The resin was then washed 10
times with 1 mL DMF and 5 times with 1 mL H.sub.2O.
5. Transferrin Coupling:
[0092] To the resin is added 1.2 equivalents of transferrin
dissolved in 0.05 M sodium carbonate and 0.1 M sodium citrate
buffer, pH 9.5. 5 M cyanoborahydride in 1N NaOH is then added to
the solution. The glass column is placed on a rotator and mixed for
2 hours. The resin is then washed 15 times with water and 5 times
each with tetrahydrofuran (THF) and DCM.
6. Cleavage from Resin:
[0093] 1% trifluoroacetic acid (TFA) in 1 in mL DCM is added to the
resin twice for 1 minute each. The supernatant is then collected
and DCM evaporated. The resulting oily film is rehydrated in
H.sub.2O and lyophilized.
7. Coupling to Polymer:
[0094] Transferrin linker is reacted with 6 equivalents of a
cyclodextrin copolymer by reductive amination with sodium
cyanoborohydride: first, the copolymer is added to transferrin
linker dissolved in 0.05 M sodium carbonate and 0.1 M sodium
citrate buffer. 5 M cyanoborohydride in 1N NaOH is added and the
reaction is stirred for 2 hours at room temperature. Unreacted
aldehyde sites are blocked by adding ethanolamine and reacting for
15 minuted at room temperature. The resulting conjugate is purified
by dialysis. ##STR27## ##STR28##
Example 20
General Procedure for Cyclodextrin Copolymer Complexation with
Small Molecules
[0095] Cyclodextrin-based copolymer (CD-polymer) is dissolved in
water, buffer, or organic solvent at the appropriate concentration.
The small molecule is dissolved in a solvent miscible with the
solvent of the CD-polymer solution and is added to the CD-polymer
solution. The mixture is then stirred for 1/2 hour and then allowed
to come to equilibrium overnight.
Example 21
Cyclodextrin-Copolymer Complexation with Doxorubicin
[0096] Doxorubicin and CD-polymer were dissolved at various
concentrations in PBS (phosphate buffered saline, pH 7.2). The
association constant between the CD and doxorubicin was determined
by measuring the extent of doxorubicin's fluorescence increase upon
complexation with the CD. (The hydrophobic interaction between the
CD and doxorubicin enhances the fluorescence intensity).
Association constant was approximately 200 M.sup.-1 at pH 7.1.
Addition of .beta.-CD consistently enhanced doxorubicin
fluorescence, indicating complexation between the CD-polymer and
doxorubicin. Husain et al., Applied Spectroscopy Vol. 46, No. 4,
652-658 (1992) found the association constant between .beta.-CD and
doxorubicin to be 210 M.sup.-1 at pH 7.1.
Example 22
Small Molecule Delivery to Cultured Cells
[0097] Media containing doxorubicin and doxorubicin/CD-polymer
complexes at various concentrations were applied to cultured cell
lines. After 5 hours, the media was removed and replaced with fresh
media. Doxorubicin effect on cell survival was determined by the
MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium)
toxicity assay. (R. Ian Feshney, "Culture of Animal Cells", 3rd
ed., Wiley-Liss:New York (1994)). The results are illustrated in
the table below. Copolymer 15 or 16 (138 .mu.M equivalent of CD
monomer) was not toxic to KB or KB-VI (a multidrug resistant
derivative of KB) cell lines in the absence of doxorubicin. For
receptor-mediated delivery, a ligand such a folate is covalently
attached to the CD-polymer used for doxorubicin complexation.
TABLE-US-00001 IC.sub.50 (.mu.M of Cell Line CD-polymer
doxorubicin) KB none .about.0.1 KB-VI (multidrug resistant) none
.about.10 KB-VI copolymer 15 or 16 (138 .mu.M .about.2-3 equivalent
of CD monomer)
Example 23
Fixed Permanent Charged Copolymer Complexation with Plasmid
[0098] 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.
[0099] 1 .mu.g of DNA at a concentration of 0.2 .mu.g/.mu.L in
distilled water was mixed with 10 .mu.L of copolymer 15 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.TAB 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 (15) retarded DNA at charge ratios of 6 and
above, indicating complexation under these conditions.
Example 24
Crosslinking Copolymer Complexation with Plasmid
[0100] Copolymer 15 or copolymer 16 is oxidized as in Example 10.
Oxidized copolymer 15 or 16 is then complexed with a DNA plasmid as
in Examples 23 and 26. A crosslinking agent (for example,
PEG.sub.600-Dihydrazide) is then added to encapsulate the DNA.
Encapsulation success is determined by light scattering and
visualized by electron microscopy.
Example 25
Variably Charged (pH-Sensitive) Copolymer Complexation with
Plasmid
[0101] Equal volumes of a CD-polymer and DNA plasmid solutions in
water are mixed in appropriate polymer/plasmid charge ratios. The
pH of the mixture is adjusted to form a charged CD-polymer. The
mixture is then allowed to equilibrate and self-assemble at room
temperature for 30 minutes. A crosslinking agent (for example,
PEG.sub.600-Dihydrazide) is then added to encapsulate the DNA. A
concentrated buffer solution is then added to render the pH and
thus the CD-polymer neutral. Encapsulation success is determined by
light scattering and visualized by electron microscopy.
Example 26
Transfection Studies with Plasmids Encoding Luciferase Reporter
Gene
[0102] 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 encapsulated by the CD-polymer as
in Examples 23 or 25 such that the DNA/polymer complexes were
assembled at polymer amine: DNA phosphate charge ratios of 6, 12,
24, 36, and 60 as described in DNA binding studies of Example 23.
Media solution containing the DNA/polymer complexes was added to
cultured cells and replaced with fresh media after 5 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. The
results are shown in FIG. 1A. DNA/polymer complexes successfully
transfected BHK-21 cells at a charge ratios of 6, 12, and 24. 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)). The results are shown in FIG. 1B. Maximum
toxicity was seen at a polymer amine: DNA phosphate charge ratios
of 36 and 60 with 91% cell survival.
Example 27
Transfection Studies with Plasmids Encoding Luciferase Reporter
Gene
[0103] 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 encapsulated by the CD-polymer as
in Example 23 except copolymer 15 was replaced with copolymer 16
and that the DNA/polymer complexes successfully transfected BHK-21
cells at charge ratios of 10, 20, 30, and 40 with maximum
transfection at polymer amine:DNA phosphate charge ratio of 20.
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. The
results are illustrated below and shown in FIG. 1A. DNA/polymer
complexes successfully transfected BHK-21 cells at a charge ratios
of 6, 12, and 24. 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)). The results are
illustrated below and shown in FIG. 1B. Maximum toxicity was seen
at a polymer amine: DNA phosphate charge ratios of 40 and 50 with
33% cell survival.
Example 28
Transfection Studies with Plasmids Encoding GFP Reporter Gene
[0104] Plasmids encoding the green fluorescent protein are
encapsulated by the CD-polymer as in Examples 23 or 25. Media
solution containing tire DNA/polymer complexes is added to cultured
cells and replaced with fresh media after 5 hours of incubation at
37.degree. C. The cells are detached from the surface with trypsin,
washed, and resuspended in Hanks Balanced Salt Solution with
propidium iodide. The cells are then analyzed by fluorescence
activated cell sorting (FACS). Cell viability is determined by cell
size and propidium iodide exclusion, and transfection success by
GFP protein fluorescence.
Example 29
Polymer Complexation with Oligos
[0105] Complexation with antisense oligos is accomplished following
the procedures for plasmid complexation of Examples 23 or 25.
Example 30
Transfection Studies with Oligos
[0106] Antisense oligos directed against the luciferase gene are
encapsulated by the CD-polymer as described in Example 29. Media
solution containing the oligo/polymer complexes is added to HeLa
X1/5 cells (HeLa cells that constitutively express the luciferase
gene, donated by CLONTECH) and replaced with fresh media after 5
hours of incubation at 37.degree. C. Cells are lysed 48 hours after
transfection and appropriate substrates for the luciferase assay
are added to the lysates. Luciferase activity, measured in terms of
fight units produced, is quantified by a luminometer. Transfection
success is determined by knockout of luciferase activity.
Example 31
Toxicity of .beta.-cyclodextrin(cystamine)-DTBP copolymer, 15
[0107] The acute toxicity of copolymer 15 was investigated using
Swiss-Webster "white mice." A total of 48 mice were used as
described in the table below. Single intravenous (i.v.) or
intraperitoneal (i.p.) injections of sterile saline solutions or of
copolymer 15 were given to the mice. The animals were followed for
five days after which they sacrificed and groww necropsy performed.
No mortality and no toxicity was observed. TABLE-US-00002 Con- Dose
Group #/Sex centration Volume Dose Treatment No. (M/F) CoPolymer
(mg/mL) (mL) (mg) Regimen 1 3/3 CoPolymer 15 0.5275 0.1 0.05 i.v.,
once 2 3/3 CoPolymer 15 5.275 0.1 0.53 i.v., once 3 3/3 CoPolymer
15 52.75 0.1 5.28 i.v., once 4 3/3 CoPolymer 15 0.5275 0.1 0.05
i.p., once 5 3/3 CoPolymer 15 5.275 0.1 0.53 i.p., once 6 3/3
CoPolymer 15 52.75 0.1 5.28 i.p., once 7 3/3 0.9% saline 0.000 0.1
0.00 i.v., once 8 3/3 0.9% saline 0.000 0.1 0.00 i.p., once
Example 32
Transfection Studies with Plasmids Encoding Luciferase Reporter
Gene
[0108] Plasmids encoding the luciferase gene were encapsulated by
the CD-polymer as in Example 23 except copolymer 15 was replaced
with copolymer 16. The DNA/polymer complexes were used to
successfully transfect BHK-21 or CHO-K1 cells, each plated in 24
well plates at a cell density of 60,000 cells/well 24 hours before
transfection, at various charge ratios in 10% serum and serum-free
conditions following the procedure outlined in Example 27. 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 (i.e., relative light units (RLU)), was quanitified by a
luminometer. 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)). Toxicity was measured by
determining total cellular protein in the wells 48 hours after
transfection. The transfection and cell survival results in 10%
serum and serum free media are illustrated below and shown in FIGS.
2 and 3.
[0109] Luciferase protein activity in BHK-21 cells transfected in
serum-free conditions reached a stable maximum at 30+/- with
.about.5.times.10.sup.7 RLUs. The presence of 10% serum in the
transfection media decreased luciferase activity at all charge
ratios except 70+/-. With CHO-K1 cells, increasing charge ratio
also enhanced the transfection for all conditions tested.
Additionally, transfection in serum decreased light units by an
order of magnitude.
[0110] Copolymer 16 showed toxicity only to BHK-21 cells for
transfections in the absence of serum. Toxicity was minimized with
the presence of 10% serum during transfection. No noticeable
toxicity was observed from transfections to CHO-K1 cells.
[0111] The effect of copolymer 16/DNA charge ratio and serum
conditions on transfection efficiency (.circle-solid. and
.box-solid.) and cell survival ( and .tangle-solidup.) in BHK-21
cells. Results from transfection in 10% serum and serum-free media
are shown as, respectively, dotted and solid lies. Data are
reported as the mean+/-S.D. of three samples. Toxicity data are
presented as best fit lines.
[0112] The effect of copolymer 16/DNA charge ratio and serum
conditions on transfection efficiency (.circle-solid. and
.box-solid.) and cell survival ( and .tangle-solidup.) in CHO-K1
cells. Results from transfection in 10% serum and serum-free media
are shown as, respectively, dotted and solid lines. Data are
reported as the mean+/-S.D. of three samples. Toxicity data are
presented as best fit lines.
Comparative Example 1
Transfection Studies with Plasmids Encoding Luciferase Reporter
Gene
[0113] Following the procedure of Example 32, transfection
efficiency and toxicity of various non-viral vectors with BHK-21
and CHO-K1 cells were studied and compared against those achieved
with DNA/copolymer 16 complexes. The CHK-21 and CHO-K1 cells were
transfected at a range of charge ratios and starting cell densities
for all vectors in serum-free media. The results are illustrated
below and shown in FIGS. 4A and 4B and illustrate the optimum
transfection conditions found for each vector.
[0114] 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. ##STR29## ##STR30##
##STR31## ##STR32## ##STR33##
* * * * *