U.S. patent application number 13/968112 was filed with the patent office on 2014-07-17 for cyclodextrin-based materials compositions and uses related thereto.
This patent application is currently assigned to CERULEAN PHARMA INC.. The applicant listed for this patent is Nathalie C. Bellocq, Mark E. Davis, Suzie Hwang Pun. Invention is credited to Nathalie C. Bellocq, Mark E. Davis, Suzie Hwang Pun.
Application Number | 20140199370 13/968112 |
Document ID | / |
Family ID | 32094010 |
Filed Date | 2014-07-17 |
United States Patent
Application |
20140199370 |
Kind Code |
A1 |
Pun; Suzie Hwang ; et
al. |
July 17, 2014 |
CYCLODEXTRIN-BASED MATERIALS COMPOSITIONS AND USES RELATED
THERETO
Abstract
This application discloses cyclodextrin-modified materials for
carrying drugs and other active agents, such as nucleic acids.
Compositions are also disclosed of cyclodextrin-modified materials
that release such active agents under controlled conditions. The
invention also discloses compositions of cyclodextrin-modified
polymer carriers that are coupled to biorecognition molecules for
assisting the delivery of drugs to their site of action.
Inventors: |
Pun; Suzie Hwang; (Torrance,
CA) ; Bellocq; Nathalie C.; (Altadena, CA) ;
Davis; Mark E.; (Pasadena, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pun; Suzie Hwang
Bellocq; Nathalie C.
Davis; Mark E. |
Torrance
Altadena
Pasadena |
CA
CA
CA |
US
US
US |
|
|
Assignee: |
CERULEAN PHARMA INC.
Cambridge
MA
|
Family ID: |
32094010 |
Appl. No.: |
13/968112 |
Filed: |
August 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13707217 |
Dec 6, 2012 |
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13968112 |
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10681745 |
Oct 8, 2003 |
8357377 |
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13707217 |
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60417373 |
Oct 9, 2002 |
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Current U.S.
Class: |
424/450 ;
424/93.2; 435/375; 514/1.1; 514/44R; 525/54.24; 536/103 |
Current CPC
Class: |
A61L 15/44 20130101;
A61L 26/0023 20130101; B82Y 5/00 20130101; A61K 47/40 20130101;
A61K 47/645 20170801; A61L 2300/802 20130101; A61P 17/02 20180101;
A61L 2300/258 20130101; A61L 15/28 20130101; A61L 2300/252
20130101; A61P 43/00 20180101; A61L 2300/626 20130101; A61K 47/59
20170801; A61K 47/60 20170801; A61L 15/28 20130101; C08L 5/16
20130101; C08L 5/16 20130101; A61P 35/00 20180101; A61K 47/61
20170801; A61K 9/0019 20130101; A61K 9/0014 20130101; A61L 26/0023
20130101; A61L 26/0066 20130101; A61L 2300/622 20130101; A61K
47/6951 20170801; A61K 47/34 20130101 |
Class at
Publication: |
424/450 ;
536/103; 514/44.R; 424/93.2; 514/1.1; 525/54.24; 435/375 |
International
Class: |
A61K 47/48 20060101
A61K047/48 |
Claims
1. A polymer composition comprising a linear biocompatible polymer
including a plurality of inclusion hosts, and linking molecules,
each linking molecule comprising at least two moieties that form
inclusion complexes with the inclusion hosts, wherein the linking
molecules crosslink the polymer.
2. A polymer composition of claim 1, wherein the inclusion hosts
are cyclodextrin moieties.
3. A polymer composition of claim 1, further comprising at least
one biologically active compound or a prodrug form thereof.
4. A polymer composition of claim 3, wherein the biologically
active compound is a nucleic acid.
5. A polymer composition of claim 4, wherein the nucleic acid is
provided in a delivery system selected from a virus, a polymer, and
a liposome.
6. A method for transfecting a cell with a nucleic acid, comprising
contacting the cell with a polymer composition of claim 4.
7. A polymer composition of claim 3, wherein the compound is a
polypeptide or a small organic molecule.
8. A method for administering a biologically active compound to a
patient, comprising administering to the patient a polymer
composition of claim 3.
9. A polymer composition of claim 3, wherein the compound is a
protein or polypeptide.
10. A polymer composition of claim 3, wherein the biologically
active compound or prodrug form thereof is provided in a delivery
system.
11. A polymer composition of claim 10, wherein the delivery system
is selected from microparticles and liposomes.
12. A polymer composition of claim 3, wherein the biologically
active compound is covalently attached to a moiety that forms an
inclusion complex with the inclusion host.
13. A polymer composition of claim 1, further comprising at least
one adjuvant.
14. A polymer composition of claim 3, further comprising an
adjuvant that increases the effectiveness of the biologically
active agent.
15. A polymer composition comprising a biocompatible, biodegradable
polymer including a plurality of inclusion hosts, and linking
molecules, each linking molecule comprising at least two moieties
that form inclusion complexes with the inclusion hosts, wherein the
linking molecules crosslink the polymer.
16.-17. (canceled)
18. A therapeutic composition comprising a) a biocompatible polymer
including a plurality of inclusion hosts, b) linking molecules,
each linking molecule comprising at least two moieties that form
inclusion complexes with the inclusion hosts, and c) a therapeutic
agent, wherein the linking molecules crosslink the polymer.
19.-30. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/707,217, filed Dec. 6, 2012, which is a continuation of U.S.
application Ser. No. 10/681,745, filed Oct. 8, 2003, which claims
priority to U.S. Provisional Application No. 60/417,373, filed Oct.
9, 2002, the specification of which is hereby incorporated by
reference in its entirety herein.
BACKGROUND OF THE INVENTION
[0002] Polymers having pendant sugar moieties known as
"glycopolymers" (Bioconj. Chem., 3:256 (1992)) have attracted much
interest in recent years, largely as scaffolds for the multivalent
display of biologically important carbohydrate molecules. These
glycopolymers have been used as potent inhibitors of viral-host
cell attachment and leukocyte-endothelial cell adhesion (FEBS,
272:209 (1990); Can. J. Microbiol., 37:233 (1991); J. Am. Chem.
Soc., 119:3161 (1997)). Glycopolymers have also been explored as
vehicles for targeted drug and gene delivery (J. Hepatology, 21:806
(1994)), and as artificial substrates for cell adhesion (J. Cell
Biol., 115:485 (1991)). The suitability of glycopolymers as
biocompatible implant materials has been relatively unexplored and
is limited to a few examples described, for example, in Microbiol.
Chem. Phys., 195:3597 (1994).
[0003] For polymers used as biocompatible implant materials, their
properties, particularly the surface composition, are of great
importance. Efforts include introducing biocompatible components
into the bulk system and on their surface. Studies described, for
example, in J. Colloid Interface Sci., 149:84 (1992) have shown
that copolymers with a pendant glucose unit in the bulk or surfaces
with covalently bound neutral polysaccharides demonstrate the
reduction of platelet adhesion and protein adsorption.
[0004] Accordingly, biocompatible polymeric materials that are
easily prepared would be useful for drug delivery and other
biomedical uses.
SUMMARY OF THE INVENTION
[0005] This invention provides a material formed by crosslinking a
polymer bearing inclusion hosts (such as cyclodextrin) with a
linking molecule bearing at least two functional groups that, as
inclusion guests, form inclusion complexes with the inclusion host.
For polymers in which cyclodextrin is the inclusion host, exemplary
inclusion guests include naphthol, adamantane, cholesterol, and
derivatives thereof. In certain embodiments, the polymer bears
inclusion guests, and linking molecules bearing inclusion hosts
such as cyclodextrin are used to crosslink the polymer. Materials
according to the above description can be used to deliver
therapeutic agents, such as proteins, nucleic acids, and
pharmaceuticals.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 schematically depicts a crosslinked polymer matrix of
the present invention.
[0007] FIG. 2 illustrates a matrix of the invention including
therapeutic moieties.
[0008] FIG. 3 depicts molecular weights of CD-PEG.sub.3400 as a
function of polymerization times.
[0009] FIG. 4 presents results of transfection experiments.
[0010] FIG. 5 shows cell migration through a matrix as described
herein.
[0011] FIG. 6 shows a dynamic frequency sweep of CD-PEG.sub.3400
polymer without cross-linking. Concentration was 100 mg/ml in PBS,
temperature was 37.degree. C. and strain was 0.5%.
[0012] FIG. 7 presents a dynamic frequency sweep of CD-PEG.sub.3400
polymer without cross linker. Concentration was 100 mg/ml in PBS,
temperature was 20.degree. C. and strain was 0.5%.
[0013] FIG. 8 provides a dynamic frequency sweep of CD-PEG.sub.3400
polymer (100 mg/ml) with 36.5 mg/ml of di-Adamantane-PEG cross
linker. Temperature was 37.degree. C. and strain was 0.5%.
[0014] FIG. 9 is a dynamic frequency sweep of CD-PEG.sub.3400
polymer (100 mg/ml) with 36.5 mg/ml of di-Adamantane-PEG cross
linker. Temperature was 20.degree. C. and strain was 0.5%.
[0015] FIG. 10 shows a dynamic frequency sweep of bovine collagen
matrix at 2.4 mg/ml. Temperature was 37.degree. C. and strain was
0.5%.
[0016] FIG. 11 illustrates results of in vitro assays using a
matrix as described herein.
[0017] FIG. 12 portrays results of treating wounds with subject
compositions. (12A): ICR mouse treated with Matrix A (Left Wound)
and Matrix B (Right Wound). Animal shown at sacrifice 4 days post
surgery. The purple ink is markings from a surgical pen and not the
dye (dark blue/black in color) used in the matrices. Little dye
remains on the actual wound site; however, the remaining dye comes
from oozing and spreading of the matrices to the surrounding
unwounded tissues that occurred after applying the wound dressing.
(12B): ICR mouse treated with Matrix A (Left Wound) and Matrix B
(Right Wound). Animal shown at sacrifice 4 days post surgery. The
left and right wounds show that Matrix A and Matrix B respectively,
did not spread to surrounding tissues after injection.
[0018] FIG. 13 shows results of treating wounds with subject
compositions. (13A): ICR mouse treated with Matrix A (Left Wound)
and Matrix B (Right Wound). Animal shown at sacrifice 4 days post
surgery. Natural wound contraction can be seen in both left and
right wounds. A small amount of dye remaining from the original
injections can be seen; however, there is no dye present in the
surrounding unwounded tissues. (13B): ICR mouse treated with Matrix
A (Left Wound) and Matrix B (Right Wound). Animal shown at
sacrifice 4 days post surgery. The left and right wounds show that
Matrix A and Matrix B, respectively, did not spread to surrounding
tissues after injection.
[0019] FIG. 14 depicts PDGF-B expression levels within treated
sponges. QRT-PCR was performed on individual sponge samples from
each treatment group. The data is expressed as the average of 6
sponges assayed.+-.standard deviation.
[0020] FIG. 15 illustrates GAPDH RNA levels within treatment
groups.
[0021] FIG. 16 shows viral PDGF-B DNA within treated sponges. QPCR
assays were performed on sponge samples from each treatment group.
PCR primers targeted specific expression of human PDGF-B sequence
and do not cross react with rat PDGF-B DNA.
[0022] FIG. 17 portrays GAPDH DNA content within sponges.
[0023] FIG. 18 depicts viral hexon DNA within sponge treatment
groups.
[0024] FIG. 19 provides morphological analysis of PVA sponges.
DETAILED DESCRIPTION OF THE INVENTION
I. Overview
[0025] Cyclodextrins have a ring structure which has a basket-like
shape. This shape allows cyclodextrins to include many kinds of
molecules into their internal cavity. See, for example, Szejtli,
Cyclodextrins and Their Inclusion Complexes; Akademiai Klado,
Budapest, 1982; and Bender et al., Cyclodextrin Chemistry,
Springer-Verlag, Berlin, 1978. Cyclodextrins are able to form
inclusion complexes with an array of organic molecules including,
for example, drugs, pesticides, herbicides, and agents of war. See,
Tenjarla et al., J. Pharm. Sci., 87: 425-429 (1998); Zughul et al.,
Pharm. Dev. Technol., 3: 43-53 (1998); and Albers et al., Crit.
Rev. Ther. Drug Carrier Syst., 12: 311-337 (1995).
[0026] Linear cyclodextrin-based polymers (CDPs) have previously
been shown to have low toxicity both in vitro (in many different
cell lines) and in vivo (Gonzalez et al. 1999 Bioconjugate Chem
10:1068-1074; and Hwang et al. 2001 Bioconjugate Chem
12(2):280-290). The present invention relates, at least in part, to
biocompatible materials based on polymers that bear or include
cyclodextrin moieties, by crosslinking the linear strands with
linking molecules bearing two or more moieties that form inclusion
complexes with cyclodextrin, as shown in FIG. 1. Additionally,
those of skill in the art will recognize that this concept can
naturally be extended to polymers bearing or including inclusion
hosts other than cyclodextrin, in conjunction with linking
molecules that bear inclusion guests that form inclusion complexes
with those inclusion hosts, or, alternatively, polymers that bear
inclusion guests in conjunction with linking molecules that bear or
include inclusion hosts that form inclusion complexes with those
inclusion guests. Examples of inclusion hosts other than
cyclodextrins and related cycloamyloses include
perhydrotriphenylene (which forms inclusion complexes with
polyethylene), urea/thiourea (which form inclusion complexes with
fatty acids and related molecules as described in U.S. Pat. Nos.
4,776,984, 5,106,542, and 4,170,601), cyclophanes (such as those
described in U.S. Pat. No. 4,116,955), and those described in U.S.
Pat. Nos. 4,841,081, 4,367,072, and 4,898,654, all of which are
hereby incorporated by reference in their entireties.
[0027] In certain embodiments of the present invention, such as is
described below in Example 19, a polymer bears both inclusion hosts
and inclusion guests, and thus crosslinks with itself by forming
inclusion complexes between hosts and guests on the same polymer
chain and/or between hosts and guests on adjacent polymer chains.
Conditions under which the crosslinking is performed will influence
the balance between these two types of inclusion complexes. For
example, performing the complexation at high dilution will favor
the formation of intramolecular complexes, while performing the
complexation at high concentrations will favor the formation of
intermolecular complexes, including, in some cases, catenane- and
rotaxane-type structures. In certain such embodiments, a high
degree of intermolecular interaction increases the rigidity,
melting point, and strength of the material.
[0028] For purposes of the present application, polymers
`incorporate` inclusion hosts, such as cyclodextrin moieties, by
having inclusion hosts within the polymer chain, e.g., removing
inclusion hosts from the polymer would require severing the polymer
chain. Examples of such polymers are the linear cyclodextrin-based
polymers referred to above. Polymers that `bear` cyclodextrin
moieties have a polymer chain to which inclusion hosts are
attached, e.g., inclusion hosts are appended to a distinct polymer
chain. Polyethylenimine-CD polymers as described herein are
examples of this type of polymer. Polymers that `include` inclusion
hosts are those polymers that `bear` or `incorporate`, or both bear
and incorporate, inclusion hosts, or otherwise have covalently
bound inclusion hosts as part of the polymer chain. Any polymer
that includes inclusion hosts can be employed in the present
application. In certain embodiments, a polymer that incorporates
inclusion hosts is a linear (i.e., non-branched) polymer. In
certain embodiments, inclusion hosts, e.g., incorporated into or
borne on the polymer, are regularly spaced throughout or along the
polymer.
[0029] The physical properties of the resultant material can be
varied by selecting moieties that form inclusion complexes of
varying strength; the stronger the complex, the more durable and
rigid the resulting material. Similarly, using linking molecules
bearing more than two such moieties may increase the strength and
rigidity of the material by increasing the degree of crosslinking,
as will increasing the proportion of linking molecules to polymer
mass. Physical properties of the material can also be varied by
altering the flexibility of the linking molecules themselves, or by
altering the flexibility of linkers within the cyclodextrin polymer
itself.
[0030] Furthermore, the in vivo properties of the matrix may be
varied by using bonds in the matrix that are labile under
physiological conditions. For example, the polymer strands, the
crosslinking molecules, or both, may comprise bonds that are labile
under physiological conditions, such as ester and peptide bonds.
After placement in a physiological environment, these bonds will
gradually begin to cleave, resulting in a gradual degradation and
loss of structural integrity. A wide spectrum of properties can be
achieved by varying the frequency of such bonds in a polymer
strand, by combining labile and resistant crosslinking molecules in
varying proportions, or by selecting different labile bonds with
differing strengths. For example a peptide bond is generally more
resistant to cleavage than an ester bond, which is in turn less
labile than a thioester bond.
[0031] Compounds and materials such as therapeutic agents, viruses,
adjuvants, and the like, can be formulated with the polymer by
forming inclusion complexes, or by simple admixture or
encapsulation, without forming inclusion complexes, as is well
known in the art for ordinary biocompatible polymers.
[0032] Compounds increasing the therapeutic utility of the
material, such as signaling peptides, other moieties facilitating
cell migration, or adjuvants, may be incorporated into the
crosslinked material by conjugating an inclusion complex guest to
the entity of interest and including the conjugate in the material
as depicted in FIG. 2. The conjugate may be included before, during
or after the crosslinking process. Therapeutic compounds may also
be included in this fashion, preferably where the attachment
between the drug and the inclusion guest/host is labile under
physiological conditions, such as an ester bond. See U.S. Patent
Application Publication Nos. 20030008818 and 20030017972.
II. Definitions
[0033] For convenience, certain terms employed in the
specification, examples, and appended claims are collected
here.
[0034] An `adjuvant`, as the term is used herein, is a compound
that has little or no therapeutic value on its own, but increases
the effectiveness of a therapeutic agent. Exemplary adjuvants
include radiosensitizers, transfection-enhancing agents (such as
chloroquine and analogs thereof), chemotactic agents and
chemoattractants, peptides that modulate cell adhesion and/or cell
mobility, cell permeabilizing agents, inhibitors of multidrug
resistance and/or efflux pumps, etc.
[0035] The terms "biocompatible polymer" and "biocompatibility"
when used in relation to polymers are art-recognized. For example,
biocompatible polymers include polymers that are neither themselves
toxic to the host (e.g., an animal or human), nor degrade (if the
polymer degrades) at a rate that produces monomeric or oligomeric
subunits or other byproducts at toxic concentrations in the host.
In certain embodiments of the present invention, biodegradation
generally involves degradation of the polymer in an organism, e.g.,
into its monomeric subunits, which may be known to be effectively
non-toxic. Intermediate oligomeric products resulting from such
degradation may have different toxicological properties, however,
or biodegradation may involve oxidation or other biochemical
reactions that generate molecules other than monomeric subunits of
the polymer. Consequently, in certain embodiments, toxicology of a
biodegradable polymer intended for in vivo use, such as
implantation or injection into a patient, may be determined after
one or more toxicity analyses. It is not necessary that any subject
composition have a purity of 100% to be deemed biocompatible.
Hence, a subject composition may comprise 99%, 98%, 97%, 96%, 95%,
90% 85%, 80%, 75% or even less of biocompatible polymers, e.g.,
including polymers and other materials and excipients described
herein, and still be biocompatible.
[0036] To determine whether a polymer or other material is
biocompatible, it may be necessary to conduct a toxicity analysis.
Such assays are well known in the art. One example of such an assay
may be performed with live carcinoma cells, such as GT3TKB tumor
cells, in the following manner: the sample is degraded in 1 M NaOH
at 37.degree. C. until complete degradation is observed. The
solution is then neutralized with 1 M HCl. About 200 .mu.L of
various concentrations of the degraded sample products are placed
in 96-well tissue culture plates and seeded with human gastric
carcinoma cells (GT3TKB) at 104/well density. The degraded sample
products are incubated with the GT3TKB cells for 48 hours. The
results of the assay may be plotted as % relative growth vs.
concentration of degraded sample in the tissue-culture well. In
addition, polymers and formulations of the present invention may
also be evaluated by well-known in vivo tests, such as subcutaneous
implantations in rats to confirm that they do not cause significant
levels of irritation or inflammation at the subcutaneous
implantation sites.
[0037] The term "biodegradable" is art-recognized, and includes
polymers, compositions and formulations, such as those described
herein, that are intended to degrade during use. Biodegradable
polymers typically differ from non-biodegradable polymers in that
the former may be degraded during use. In certain embodiments, such
use involves in vivo use, such as in vivo therapy, and in other
certain embodiments, such use involves in vitro use. In general,
degradation attributable to biodegradability involves the
degradation of a biodegradable polymer into its component subunits,
or digestion, e.g., by a biochemical process, of the polymer into
smaller, non-polymeric subunits. In certain embodiments, two
different types of biodegradation may generally be identified. For
example, one type of biodegradation may involve cleavage of bonds
(whether covalent or otherwise) in the polymer backbone. In such
biodegradation, monomers and oligomers typically result, and even
more typically, such biodegradation occurs by cleavage of a bond
connecting one or more of subunits of a polymer. In contrast,
another type of biodegradation may involve cleavage of a bond
(whether covalent or otherwise) internal to sidechain or that
connects a side chain to the polymer backbone. For example, a
therapeutic agent or other chemical moiety attached as a side chain
to the polymer backbone may be released by biodegradation. In
certain embodiments, one or the other or both general types of
biodegradation may occur during use of a polymer.
[0038] As used herein, the term "biodegradation" encompasses both
general types of biodegradation. The degradation rate of a
biodegradable polymer often depends in part on a variety of
factors, including the chemical identity of the linkage responsible
for any degradation, the molecular weight, crystallinity,
biostability, and degree of cross-linking of such polymer, the
physical characteristics (e.g., shape and size) of an implant, and
the mode and location of administration. For example, the greater
the molecular weight, the higher the degree of crystallinity,
and/or the greater the biostability, the biodegradation of any
biodegradable polymer is usually slower. The term "biodegradable"
is intended to cover materials and processes also termed
"bioerodible".
[0039] In certain embodiments wherein the biodegradable polymer
also has a therapeutic agent or other material associated with it,
the biodegradation rate of such polymer may be characterized by a
release rate of such materials. In such circumstances, the
biodegradation rate may depend on not only the chemical identity
and physical characteristics of the polymer, but also on the
identity of material(s) incorporated therein. Degradation of the
subject compositions includes not only the cleavage of
intramolecular bonds, e.g., by oxidation and/or hydrolysis, but
also the disruption of intermolecular bonds, such as dissociation
of host/guest complexes by competitive complex formation with
foreign inclusion hosts.
[0040] In certain embodiments, polymeric formulations of the
present invention biodegrade within a period that is acceptable in
the desired application. In certain embodiments, such as in vivo
therapy, such degradation occurs in a period usually less than
about five years, one year, six months, three months, one month,
fifteen days, five days, three days, or even one day on exposure to
a physiological solution with a pH between 6 and 8 having a
temperature of between 25 and 37.degree. C. In other embodiments,
the polymer degrades in a period of between about one hour and
several weeks, depending on the desired application.
[0041] A biohydrolyzable bond (e.g., ester, amide, carbonate,
carbamates, or imide) refers to a bond that is cleaved (e.g., an
ester is cleaved to form a hydroxyl and a carboxylic acid) under
physiological conditions. Physiological conditions include the
acidic and basic environments of the digestive tract (e.g.,
stomach, intestines, etc.), acidic environment of a tumor,
enzymatic cleavage, metabolism, and other biological processes, and
preferably refer to physiological conditions in a vertebrate, such
as a mammal.
[0042] The term "healthcare providers" refers to individuals or
organizations that provide healthcare services to a person,
community, etc. Examples of "healthcare providers" include doctors,
hospitals, continuing care retirement communities, skilled nursing
facilities, subacute care facilities, clinics, multispecialty
clinics, freestanding ambulatory centers, home health agencies, and
HMO's.
[0043] "Instruction(s)" as used herein means documents describing
relevant materials or methodologies pertaining to a kit. These
materials may include any combination of the following: background
information, list of components and their availability information
(purchase information, etc.), brief or detailed protocols for using
the kit, trouble-shooting, references, technical support, and any
other related documents. Instructions can be supplied with the kit
or as a separate member component, either as a paper form or an
electronic form which may be supplied on computer readable memory
device or downloaded from an internet website, or as recorded
presentation. Instructions can comprise one or multiple documents,
and are meant to include future updates.
[0044] "Kit" as used herein means a collection of at least two
components constituting the kit. Together, the components
constitute a functional unit for a given purpose. Individual member
components may be physically packaged together or separately. For
example, a kit comprising an instruction for using the kit may or
may not physically include the instruction with other individual
member components. Instead, the instruction can be supplied as a
separate member component, either in a paper form or an electronic
form which may be supplied on computer readable memory device or
downloaded from an internet website, or as recorded
presentation.
[0045] As used herein, the term `RNAi construct` is a generic term
including small interfering RNAs (siRNAs), hairpin RNAs, and other
RNA species which can be cleaved in vivo to form siRNAs. RNAi
constructs herein also include expression vectors (also referred to
as RNAi expression vectors) capable of giving rise to transcripts
which form dsRNAs or hairpin RNAs in cells, and/or transcripts
which can produce siRNAs in vivo.
[0046] An `effective amount` of a subject compound, with respect to
the subject method of treatment, refers to an amount of the
therapeutic in a preparation which, when applied as part of a
desired dosage regimen provides a benefit according to clinically
acceptable standards for the treatment or prophylaxis of a
particular disorder.
[0047] `Patients` or `subjects` to be treated by the subject method
are animals, preferably mammals, including humans.
[0048] The term "prophylactic or therapeutic" treatment is
art-recognized and includes administration to the host of one or
more of the subject compositions. If it is administered prior to
clinical manifestation of the unwanted condition (e.g., disease or
other unwanted state of the host animal) then the treatment is
prophylactic, i.e., it protects the host against developing the
unwanted condition, whereas if it is administered after
manifestation of the unwanted condition, the treatment is
therapeutic, (i.e., it is intended to diminish, ameliorate, or
stabilize the existing unwanted condition or side effects
thereof).
[0049] The term "preventing" is art-recognized, and when used in
relation to a condition, such as a local recurrence (e.g., pain), a
disease such as cancer, a syndrome complex such as heart failure or
any other medical condition, is well understood in the art, and
includes administration of a composition which reduces the
frequency of, or delays the onset of, symptoms of a medical
condition in a subject relative to a subject which does not receive
the composition. Thus, prevention of cancer includes, for example,
reducing the number of detectable cancerous growths in a population
of patients receiving a prophylactic treatment relative to an
untreated control population, and/or delaying the appearance of
detectable cancerous growths in a treated population versus an
untreated control population, e.g., by a statistically and/or
clinically significant amount. Prevention of an infection includes,
for example, reducing the number of diagnoses of the infection in a
treated population versus an untreated control population, and/or
delaying the onset of symptoms of the infection in a treated
population versus an untreated control population. Prevention of
pain includes, for example, reducing the frequency of, or
alternatively delaying, pain sensations experienced by subjects in
a treated population versus an untreated control population.
[0050] As used herein, the terms "therapeutic agent" include any
synthetic or naturally occurring biologically active compound or
composition of matter which, when administered to an organism
(human or nonhuman animal), induces a desired pharmacologic,
immunogenic, and/or physiologic effect by local and/or systemic
action. The term therefore encompasses those compounds or chemicals
traditionally regarded as drugs, vaccines, and biopharmaceuticals
including molecules such as proteins, peptides, hormones, nucleic
acids, gene constructs and the like. More particularly, the term
"therapeutic agent" includes compounds or compositions for use in
all of the major therapeutic areas including, but not limited to,
adjuvants; anti-infectives such as antibiotics and antiviral
agents; analgesics and analgesic combinations, anorexics,
anti-inflammatory agents, anti-epileptics, local and general
anesthetics, hypnotics, sedatives, antipsychotic agents,
neuroleptic agents, antidepressants, anxiolytics, antagonists,
neuron blocking agents, anticholinergic and cholinomimetic agents,
antimuscarinic and muscarinic agents, antiadrenergics,
antiarrhythmics, antihypertensive agents, hormones, and nutrients,
antiarthritics, antiasthmatic agents, anticonvulsants,
antihistamines, antinauseants, antineoplastics, antipruritics,
antipyretics; antispasmodics, cardiovascular preparations
(including calcium channel blockers, beta-blockers, beta-agonists
and antiarrythmics), antihypertensives, diuretics, vasodilators;
central nervous system stimulants; cough and cold preparations;
decongestants; diagnostics; hormones; bone growth stimulants and
bone resorption inhibitors; immunosuppressives; muscle relaxants;
psychostimulants; sedatives; tranquilizers; proteins, peptides, and
fragments thereof (whether naturally occurring, chemically
synthesized or recombinantly produced); and nucleic acid molecules
(polymeric forms of two or more nucleotides, either ribonucleotides
(RNA) or deoxyribonucleotides (DNA) including both double- and
single-stranded molecules, gene constructs, expression vectors,
antisense molecules and the like), small molecules (e.g.,
doxorubicin) and other biologically active macromolecules such as,
for example, proteins and enzymes. The agent may be a biologically
active agent used in medical, including veterinary, applications
and in agriculture, such as with plants, as well as other areas.
The term therapeutic agent also includes without limitation,
medicaments; vitamins; mineral supplements; substances used for the
treatment, prevention, diagnosis, cure or mitigation of disease or
illness; or substances which affect the structure or function of
the body; or pro-drugs, which become biologically active or more
active after they have been placed in a predetermined physiological
environment.
[0051] The term "therapeutic index" refers to the therapeutic index
of a drug defined as LD.sub.50/ED.sub.50.
[0052] `Acyl` refers to a group suitable for acylating a nitrogen
atom to form an amide or carbamate, a carbon atom to form a ketone,
a sulfur atom to form a thioester, or an oxygen atom to form an
ester group, e.g., a hydrocarbon attached to a --C(.dbd.O)--
moiety. Preferred acyl groups include benzoyl, acetyl, tert-butyl
acetyl, pivaloyl, and trifluoroacetyl. More preferred acyl groups
include acetyl and benzoyl. The most preferred acyl group is
acetyl.
[0053] The term `acylamino` is art-recognized and preferably refers
to a moiety that can be represented by the general formula:
##STR00001##
wherein R.sub.9 and R'.sub.11 each independently represent hydrogen
or a hydrocarbon substituent, such as alkyl, heteroalkyl, aryl,
heteroaryl, carbocyclic aliphatic, and heterocyclic aliphatic.
[0054] The terms `amine` and `amino` are art-recognized and refer
to both unsubstituted and substituted amines as well as ammonium
salts, e.g., as can be represented by the general formula:
##STR00002##
wherein R.sub.9, R.sub.10, and R'.sub.10 each independently
represent hydrogen or a hydrocarbon substituent, or R.sub.9 and
R.sub.10 taken together with the N atom to which they are attached
complete a heterocycle having from 4 to 8 atoms in the ring
structure. In preferred embodiments, none of R.sub.9, R.sub.10, and
R'.sub.10 is acyl, e.g., R.sub.9, R.sub.10, and R'.sub.10 are
selected from hydrogen, alkyl, heteroalkyl, aryl, heteroaryl,
carbocyclic aliphatic, and heterocyclic aliphatic. The term
`alkylamine` as used herein means an amine group, as defined above,
having at least one substituted or unsubstituted alkyl attached
thereto. Amino groups that are positively charged (e.g., R'.sub.10
is present) are referred to as `ammonium` groups. In amino groups
other than ammonium groups, the amine is preferably basic, e.g.,
its conjugate acid has a pK.sub.a above 7.
[0055] The terms `amido` and `amide` are art-recognized as an
amino-substituted carbonyl, such as a moiety that can be
represented by the general formula:
##STR00003##
wherein R.sub.9 and R.sub.10 are as defined above. In certain
embodiments, the amide will include imides.
[0056] `Alkyl` refers to a saturated or unsaturated hydrocarbon
chain having 1 to 18 carbon atoms, preferably 1 to 12, more
preferably 1 to 6, more preferably still 1 to 4 carbon atoms. Alkyl
chains may be straight (e.g., n-butyl) or branched (e.g.,
sec-butyl, isobutyl, or t-butyl). Preferred branched alkyls have
one or two branches, preferably one branch. Preferred alkyls are
saturated. Unsaturated alkyls have one or more double bonds and/or
one or more triple bonds. Preferred unsaturated alkyls have one or
two double bonds or one triple bond, more preferably one double
bond. Alkyl chains may be unsubstituted or substituted with from 1
to 4 substituents. Preferred alkyls are unsubstituted. Preferred
substituted alkyls are mono-, di-, or trisubstituted. Preferred
alkyl substituents include halo, haloalkyl, hydroxy, aryl (e.g.,
phenyl, tolyl, alkoxyphenyl, alkyloxycarbonylphenyl, halophenyl),
heterocyclyl, and heteroaryl.
[0057] The terms `alkenyl` and `alkynyl` refer to unsaturated
aliphatic groups analogous in length and possible substitution to
the alkyls described above, but that contain at least one double or
triple bond, respectively. When not otherwise indicated, the terms
alkenyl and alkynyl preferably refer to lower alkenyl and lower
alkynyl groups, respectively. When the term alkyl is present in a
list with the terms alkenyl and alkynyl, the term alkyl refers to
saturated alkyls exclusive of alkenyls and alkynyls.
[0058] The terms `alkoxyl` and `alkoxy` as used herein refer to an
--O-alkyl group. Representative alkoxyl groups include methoxy,
ethoxy, propyloxy, tert-butoxy, and the like. An `ether` is two
hydrocarbons covalently linked by an oxygen. Accordingly, the
substituent of a hydrocarbon that renders that hydrocarbon an ether
can be an alkoxyl, or another moiety such as --O-aryl,
--O-heteroaryl, --O-heteroalkyl, --O-aralkyl, --O-heteroaralkyl,
--O-carbocylic aliphatic, or --O-heterocyclic aliphatic.
[0059] The term `alkylthio` refers to an --S-alkyl group.
Representative alkylthio groups include methylthio, ethylthio, and
the like. `Thioether` refers to a sulfur atom bound to two
hydrocarbon substituents, e.g., an ether wherein the oxygen is
replaced by sulfur. Thus, a thioether substituent on a carbon atom
refers to a hydrocarbon-substituted sulfur atom substituent, such
as alkylthio or arylthio, etc.
[0060] The term `aralkyl`, as used herein, refers to an alkyl group
substituted with an aryl group.
[0061] `Aryl ring` refers to an aromatic hydrocarbon ring system.
Aromatic rings are monocyclic or fused bicyclic ring systems, such
as phenyl, naphthyl, etc. Monocyclic aromatic rings contain from
about 5 to about 10 carbon atoms, preferably from 5 to 7 carbon
atoms, and most preferably from 5 to 6 carbon atoms in the ring.
Bicyclic aromatic rings contain from 8 to 12 carbon atoms,
preferably 9 or 10 carbon atoms in the ring. The term `aryl` also
includes bicyclic ring systems wherein only one of the rings is
aromatic, e.g., the other ring is cycloalkyl, cycloalkenyl, or
heterocyclyl. Aromatic rings may be unsubstituted or substituted
with from 1 to about 5 substituents on the ring. Preferred aromatic
ring substituents include: halo, cyano, lower alkyl, heteroalkyl,
haloalkyl, phenyl, phenoxy, or any combination thereof. More
preferred substituents include lower alkyl, cyano, halo, and
haloalkyl.
[0062] `Carbocyclic aliphatic ring` refers to a saturated or
unsaturated hydrocarbon ring. Carbocyclic aliphatic rings are not
aromatic. Carbocyclic aliphatic rings are monocyclic, or are fused,
spiro, or bridged bicyclic ring systems. Monocyclic carbocyclic
aliphatic rings contain from about 4 to about 10 carbon atoms,
preferably from 4 to 7 carbon atoms, and most preferably from 5 to
6 carbon atoms in the ring. Bicyclic carbocyclic aliphatic rings
contain from 8 to 12 carbon atoms, preferably from 9 to 10 carbon
atoms in the ring. Carbocyclic aliphatic rings may be unsubstituted
or substituted with from 1 to 4 substituents on the ring. Preferred
carbocyclic aliphatic ring substituents include halo, cyano, alkyl,
heteroalkyl, haloalkyl, phenyl, phenoxy or any combination thereof.
More preferred substituents include halo and haloalkyl. Preferred
carbocyclic aliphatic rings include cyclopentyl, cyclohexyl,
cyclohexenyl, cycloheptyl, and cyclooctyl. More preferred
carbocyclic aliphatic rings include cyclohexyl, cycloheptyl, and
cyclooctyl.
[0063] The term `carbonyl` is art-recognized and includes such
moieties as can be represented by the general formula:
##STR00004##
wherein X is a bond or represents an oxygen or a sulfur, and
R.sub.11 represents a hydrogen, hydrocarbon substituent, or a
pharmaceutically acceptable salt, R.sub.11' represents a hydrogen
or hydrocarbon substituent. Where X is an oxygen and R.sub.11 or
R.sub.11' is not hydrogen, the formula represents an `ester`. Where
X is an oxygen, and R.sub.11 is as defined above, the moiety is
referred to herein as a carboxyl group, and particularly when
R.sub.11 is a hydrogen, the formula represents a `carboxylic acid`.
Where X is an oxygen, and R.sub.11' is hydrogen, the formula
represents a `formate`. In general, where the oxygen atom of the
above formula is replaced by sulfur, the formula represents a
`thiocarbonyl` group. Where X is a sulfur and R.sub.11 or R.sub.11'
is not hydrogen, the formula represents a `thioester.` Where X is a
sulfur and R.sub.11 is hydrogen, the formula represents a
`thiocarboxylic acid.` Where X is a sulfur and R.sub.11' is
hydrogen, the formula represents a `thioformate.` On the other
hand, where X is a bond, R.sub.11 is not hydrogen, and the carbonyl
is bound to a hydrocarbon, the above formula represents a `ketone`
group. Where X is a bond, R.sub.11 is hydrogen, and the carbonyl is
bound to a hydrocarbon, the above formula represents an `aldehyde`
or `formyl` group.
[0064] `Ci alkyl` is an alkyl chain having i member atoms. For
example, C4 alkyls contain four carbon member atoms. C4 alkyls
containing may be saturated or unsaturated with one or two double
bonds (cis or trans) or one triple bond. Preferred C4 alkyls are
saturated. Preferred unsaturated C4 alkyl have one double bond. C4
alkyl may be unsubstituted or substituted with one or two
substituents. Preferred substituents include lower alkyl, lower
heteroalkyl, cyano, halo, and haloalkyl.
[0065] `Halogen` refers to fluoro, chloro, bromo, or iodo
substituents. Preferred halo are fluoro, chloro and bromo; more
preferred are chloro and fluoro.
[0066] `Haloalkyl` refers to a straight, branched, or cyclic
hydrocarbon substituted with one or more halo substituents.
Preferred haloalkyl are C1-C12; more preferred are C1-C6; more
preferred still are C1-C3. Preferred halo substituents are fluoro
and chloro. The most preferred haloalkyl is trifluoromethyl.
[0067] `Heteroalkyl` is a saturated or unsaturated chain of carbon
atoms and at least one heteroatom, wherein no two heteroatoms are
adjacent. Heteroalkyl chains contain from 1 to 18 member atoms
(carbon and heteroatoms) in the chain, preferably 1 to 12, more
preferably 1 to 6, more preferably still 1 to 4. Heteroalkyl chains
may be straight or branched. Preferred branched heteroalkyl have
one or two branches, preferably one branch. Preferred heteroalkyl
are saturated. Unsaturated heteroalkyl have one or more double
bonds and/or one or more triple bonds. Preferred unsaturated
heteroalkyl have one or two double bonds or one triple bond, more
preferably one double bond. Heteroalkyl chains may be unsubstituted
or substituted with from 1 to about 4 substituents unless otherwise
specified. Preferred heteroalkyl are unsubstituted. Preferred
heteroalkyl substituents include halo, aryl (e.g., phenyl, tolyl,
alkoxyphenyl, alkoxycarbonylphenyl, halophenyl), heterocyclyl,
heteroaryl. For example, alkyl chains substituted with the
following substituents are heteroalkyl: alkoxy (e.g., methoxy,
ethoxy, propoxy, butoxy, pentoxy), aryloxy (e.g., phenoxy,
chlorophenoxy, tolyloxy, methoxyphenoxy, benzyloxy,
alkoxycarbonylphenoxy, acyloxyphenoxy), acyloxy (e.g.,
propionyloxy, benzoyloxy, acetoxy), carbamoyloxy, carboxy,
mercapto, alkylthio, acylthio, arylthio (e.g., phenylthio,
chlorophenylthio, alkylphenylthio, alkoxyphenylthio, benzylthio,
alkoxycarbonylphenylthio), amino (e.g., amino, mono- and di-C1-C3
alkylamino, methylphenylamino, methylbenzylamino, C1-C3 alkylamido,
carbamamido, ureido, guanidino).
[0068] `Heteroatom` refers to a multivalent non-carbon atom, such
as a boron, phosphorous, silicon, nitrogen, sulfur, or oxygen atom,
preferably a nitrogen, sulfur, or oxygen atom. Groups containing
more than one heteroatom may contain different heteroatoms.
[0069] `Heteroaryl ring` refers to an aromatic ring system
containing carbon and from 1 to about 4 heteroatoms in the ring.
Heteroaromatic rings are monocyclic or fused bicyclic ring systems.
Monocyclic heteroaromatic rings contain from about 5 to about 10
member atoms (carbon and heteroatoms), preferably from 5 to 7, and
most preferably from 5 to 6 in the ring. Bicyclic heteroaromatic
rings contain from 8 to 12 member atoms, preferably 9 or 10 member
atoms in the ring. The term `heteroaryl` also includes bicyclic
ring systems wherein only one of the rings is aromatic, e.g., the
other ring is cycloalkyl, cycloalkenyl, or heterocyclyl.
Heteroaromatic rings may be unsubstituted or substituted with from
1 to about 4 substituents on the ring. Preferred heteroaromatic
ring substituents include halo, cyano, lower alkyl, heteroalkyl,
haloalkyl, phenyl, phenoxy or any combination thereof. Preferred
heteroaromatic rings include thienyl, thiazolyl, oxazolyl,
pyrrolyl, purinyl, pyrimidyl, pyridyl, and furanyl. More preferred
heteroaromatic rings include thienyl, furanyl, and pyridyl.
[0070] `Heterocyclic aliphatic ring` is a non-aromatic saturated or
unsaturated ring containing carbon and from 1 to about 4
heteroatoms in the ring, wherein no two heteroatoms are adjacent in
the ring and preferably no carbon in the ring attached to a
heteroatom also has a hydroxyl, amino, or thiol group attached to
it. Heterocyclic aliphatic rings are monocyclic, or are fused or
bridged bicyclic ring systems. Monocyclic heterocyclic aliphatic
rings contain from about 4 to about 10 member atoms (carbon and
heteroatoms), preferably from 4 to 7, and most preferably from 5 to
6 member atoms in the ring. Bicyclic heterocyclic aliphatic rings
contain from 8 to 12 member atoms, preferably 9 or 10 member atoms
in the ring. Heterocyclic aliphatic rings may be unsubstituted or
substituted with from 1 to about 4 substituents on the ring.
Preferred heterocyclic aliphatic ring substituents include halo,
cyano, lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or any
combination thereof. More preferred substituents include halo and
haloalkyl. Heterocyclyl groups include, for example, thiophene,
thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,
phenoxathin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,
pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,
indole, indazole, purine, quinolizine, isoquinoline, hydantoin,
oxazoline, imidazolinetrione, triazolinone, quinoline, phthalazine,
naphthyridine, quinoxaline, quinazoline, quinoline, pteridine,
carbazole, carboline, phenanthridine, acridine, phenanthroline,
phenazine, phenarsazine, phenothiazine, furazan, phenoxazine,
pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine,
morpholine, lactones, lactams such as azetidinones and
pyrrolidinones, sultams, sultones, and the like. Preferred
heterocyclic aliphatic rings include piperazyl, morpholinyl,
tetrahydrofuranyl, tetrahydropyranyl and piperidyl. Heterocycles
can also be polycycles.
[0071] The term `hydroxyl` means --OH.
[0072] `Lower alkyl` refers to an alkyl chain comprised of 1 to 5,
preferably 1 to 4 carbon member atoms, more preferably 1 or 2
carbon member atoms. Lower alkyls may be saturated or unsaturated.
Preferred lower alkyls are saturated. Lower alkyls may be
unsubstituted or substituted with one or about two substituents.
Preferred substituents on lower alkyl include cyano, halo,
trifluoromethyl, amino, and hydroxyl. Throughout the application,
preferred alkyl groups are lower alkyls. In preferred embodiments,
a substituent designated herein as alkyl is a lower alkyl.
Likewise, `lower alkenyl` and `lower alkynyl` have similar chain
lengths.
[0073] `Lower heteroalkyl` refers to a heteroalkyl chain comprised
of 1 to 4, preferably 1 to 3 member atoms, more preferably 1 to 2
member atoms. Lower heteroalkyl contain one or two non-adjacent
heteroatom member atoms. Preferred lower heteroalkyl contain one
heteroatom member atom. Lower heteroalkyl may be saturated or
unsaturated. Preferred lower heteroalkyl are saturated. Lower
heteroalkyl may be unsubstituted or substituted with one or about
two substituents. Preferred substituents on lower heteroalkyl
include cyano, halo, trifluoromethyl, and hydroxyl.
[0074] `Mi heteroalkyl` is a heteroalkyl chain having i member
atoms. For example, M4 heteroalkyls contain one or two non-adjacent
heteroatom member atoms. M4 heteroalkyls containing 1 heteroatom
member atom may be saturated or unsaturated with one double bond
(cis or trans) or one triple bond. Preferred M4 heteroalkyl
containing 2 heteroatom member atoms are saturated. Preferred
unsaturated M4 heteroalkyl have one double bond. M4 heteroalkyl may
be unsubstituted or substituted with one or two substituents.
Preferred substituents include lower alkyl, lower heteroalkyl,
cyano, halo, and haloalkyl.
[0075] `Isocyanate` refers to the group --NCO.
[0076] `Member atom` refers to a polyvalent atom (e.g., C, O, N, or
S atom) in a chain or ring system that constitutes a part of the
chain or ring. For example, in cresol, six carbon atoms are member
atoms of the ring and the oxygen atom and the carbon atom of the
methyl substituent are not member atoms of the ring.
[0077] As used herein, the term `nitro` means --NO.sub.2.
[0078] `Pharmaceutically acceptable salt` refers to a cationic salt
formed at any acidic (e.g., hydroxamic or carboxylic acid) group,
or an anionic salt formed at any basic (e.g., amino or guanidino)
group. Such salts are well known in the art. See e.g., World Patent
Publication 87/05297, Johnston et al., published Sep. 11, 1987,
incorporated herein by reference. Such salts are made by methods
known to one of ordinary skill in the art. It is recognized that
the skilled artisan may prefer one salt over another for improved
solubility, stability, formulation ease, price and the like.
Determination and optimization of such salts is within the purview
of the skilled artisan's practice. Preferred cations include the
alkali metals (such as sodium and potassium), and alkaline earth
metals (such as magnesium and calcium) and organic cations, such as
trimethylammonium, tetrabutylammonium, etc. Preferred anions
include halides (such as chloride), sulfonates, carboxylates,
phosphates, and the like. Clearly contemplated in such salts are
addition salts that may provide an optical center where once there
was none. For example, a chiral tartrate salt may be prepared from
the compounds of the invention. This definition includes such
chiral salts.
[0079] `Phenyl` is a six-membered monocyclic aromatic ring that may
or may not be substituted with from 1 to 5 substituents. The
substituents may be located at the ortho, meta or para position on
the phenyl ring, or any combination thereof. Preferred phenyl
substituents include: halo, cyano, lower alkyl, heteroalkyl,
haloalkyl, phenyl, phenoxy or any combination thereof. More
preferred substituents on the phenyl ring include halo and
haloalkyl. The most preferred substituent is halo.
[0080] The terms `polycyclyl` and `polycyclic group` refer to two
or more rings (e.g., cycloalkyls, cycloalkenyls, heteroaryls, aryls
and/or heterocyclyls) in which two or more member atoms of one ring
are member atoms of a second ring. Rings that are joined through
non-adjacent atoms are termed `bridged` rings, and rings that are
joined through adjacent atoms are `fused rings`.
[0081] The term `small organic molecule` refers to an organic
compound of less than about 2500 amu, preferably less than 1500
amu. The term encompasses most pharmaceuticals that are not
proteins or nucleic acids.
[0082] The term `sulfhydryl` means --SH, and the term `sulfonyl`
means --SO.sub.2--.
[0083] A `substitution` or `substituent` on a small organic
molecule generally refers to a position on a multi-valent atom
bound to a moiety other than hydrogen, e.g., a position on a chain
or ring exclusive of the member atoms of the chain or ring. Such
moieties include those defined herein and others as are known in
the art, for example, halogen, alkyl, alkenyl, alkynyl, azide,
haloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl,
formyl, ketone, or acyl), thiocarbonyl (such as thioester,
thioacetate, or thioformate), alkoxyl, phosphoryl, phosphonate,
phosphinate, amine, amide, amidine, imine, cyano, nitro, azido,
sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido,
sulfonyl, silyl, ether, cycloalkyl, heterocyclyl, heteroalkyl,
heteroalkenyl, and heteroalkynyl, heteroaralkyl, aralkyl, aryl or
heteroaryl. It will be understood by those skilled in the art that
certain substituents, such as aryl, heteroaryl, polycyclyl, alkoxy,
alkylamino, alkyl, cycloalkyl, heterocyclyl, alkenyl, alkynyl,
heteroalkyl, heteroalkenyl, and heteroalkynyl, can themselves be
substituted, if appropriate. This invention is not intended to be
limited in any manner by the permissible substituents of organic
compounds. It will be understood that `substitution` or
`substituted with` includes the implicit proviso that such
substitution is in accordance with permitted valence of the
substituted atom and the substituent, and that the substitution
results in a stable compound, e.g., which does not spontaneously
undergo transformation such as by rearrangement, cyclization,
elimination, hydrolysis, etc.
[0084] As used herein, the definition of each expression, e.g.,
alkyl, m, n, etc., when it occurs more than once in any structure,
is intended to be independent of its definition elsewhere in the
same structure.
[0085] The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent
methyl, ethyl, phenyl, trifluoromethanesulfonyl,
nonafluorobutanesulfonyl, p-toluenesulfonyl, and methanesulfonyl,
respectively. A more comprehensive list of the abbreviations
utilized by organic chemists of ordinary skill in the art appears
in the first issue of each volume of the Journal of Organic
Chemistry; this list is typically presented in a table entitled
Standard List of Abbreviations. The abbreviations contained in said
list, and all abbreviations utilized by organic chemists of
ordinary skill in the art are hereby incorporated by reference.
[0086] The terms ortho, meta and para apply to 1,2-, 1,3- and
1,4-disubstituted benzenes, respectively. For example, the names
1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.
[0087] The terms triflyl, tosyl, mesyl, and nonaflyl are
art-recognized and refer to trifluoromethanesulfonyl,
p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl
groups, respectively. The terms triflate, tosylate, mesylate, and
nonaflate are art-recognized and refer to trifluoromethanesulfonate
ester, p-toluenesulfonate ester, methanesulfonate ester, and
nonafluorobutanesulfonate ester functional groups and molecules
that contain said groups, respectively.
[0088] The phrase `protecting group` as used herein means temporary
substituents that protect a potentially reactive functional group
from undesired chemical transformations. Examples of such
protecting groups include esters of carboxylic acids, silyl ethers
of alcohols, and acetals and ketals of aldehydes and ketones,
respectively. The field of protecting group chemistry has been
reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in
Organic Synthesis, 2.sup.nd ed.; Wiley: New York, 1991; and
Kocienski, P. J. Protecting Groups, Georg Thieme Verlag: New York,
1994).
[0089] The term `prodrug` is intended to encompass compounds that,
under physiological conditions, are converted into the
therapeutically active agents of the present invention. A common
method for making a prodrug is to include selected moieties, such
as esters or ketals, that are hydrolyzed under physiological
conditions to reveal the desired molecule. In other embodiments,
the prodrug is converted by an enzymatic activity of the host
animal.
[0090] For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87,
inside cover. Also for purposes of this invention, the term
`hydrocarbon` is contemplated to include all permissible compounds
or moieties having at least one carbon-hydrogen bond. In a broad
aspect, the permissible hydrocarbons include acyclic and cyclic,
branched and unbranched, carbocyclic and heterocyclic, aromatic and
nonaromatic organic compounds which can be substituted or
unsubstituted.
[0091] Contemplated equivalents of the compounds described above
include compounds which otherwise correspond thereto, and which
have the same useful properties thereof, wherein one or more simple
variations of substituents are made which do not adversely affect
the efficacy of the compound. In general, the compounds of the
present invention may be prepared by the methods illustrated in the
general reaction schemes as, for example, described below, or by
modifications thereof, using readily available starting materials,
reagents and conventional synthesis procedures. In these reactions,
it is also possible to make use of variants that are in themselves
known, but are not mentioned here.
III. Exemplary Applications of Methods and Compositions
[0092] Therapeutic compositions according to the invention contain
a therapeutic agent and a material of the invention, such as a
cyclodextrin-based material. 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, proteins, polypeptides,
peptide fragments, small molecules (e.g., doxorubicin) and other
biologically active macromolecules such as, for example, proteins
and enzymes. In certain embodiments, the agent may be associated
with a delivery system, e.g., a nucleic acid may be contained in a
virus, or an agent may be carried within liposomes or microspheres,
and the delivery system is dispersed through the material.
[0093] A therapeutic composition of the invention may be prepared
by means known in the art. In a preferred embodiment, a material of
the invention, such as a cyclodextrin-based material, is mixed with
or prepared in the presence of a therapeutic agent, as described
above. According to the invention, the material acts as a delivery
vehicle for the therapeutic agent. The therapeutic agent (and/or an
adjuvant) and material may associate by means recognized by those
of skill in the art such as, for example, electrostatic
interaction, hydrogen bonding, hydrophobic interaction, formation
of inclusion complexes with the inclusion hosts, or covalent
attachment to the polymer, preferably by a reversible attachment
such as an ester or carbonate. In certain embodiments, the
therapeutic agent and/or adjuvant may be covalently attached,
optionally through a reversible linkage, to a moiety that forms an
inclusion complex with the inclusion hosts, e.g., cyclodextrin. 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
material 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)).
[0094] In certain embodiments, a therapeutic composition of the
invention is in a form suitable for injection, and in other
embodiments, the composition is suitable for topical application.
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, parenteral, intravenous, intranasal,
intraocular, intracranial or intraperitoneal injection.
[0095] 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). In other embodiments,
subject compositions can be used in the treatment of wounds, such
as incisions, diabetic ulcers, bedsores, lacerations, burns,
etc.
[0096] The therapeutic agent may range from a nucleic acid (such as
a vector, an RNAi construct, or an antisense oligonucleotide) or
protein to a small organic molecule. In certain embodiments, the
agent is an anti-cancer (such as camptothecin or related
derivatives), anti-fungal, anti-bacterial, anti-mycotic, or
anti-viral therapeutic. In certain embodiments, the agent is a
receptor agonist. In certain embodiments, the agent is a receptor
antagonist. In certain embodiments, the therapeutic agent is a
protease inhibitor. Furthermore, a polymer of the present invention
may contain one kind of therapeutic agent, or may contain more than
one kind of therapeutic agent. For instance, two or more different
cancer drugs, or a cancer drug and an immunosuppressant, or an
antibiotic and an anti-inflammatory agent may be included in the
composition.
[0097] 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).
[0098] In certain particular embodiments, compositions of the
invention may be used to treat wounds (i.e., to promote wound
healing. Although the matrix alone may be useful as a wound
sealant, such compositions may include, for example, PDGF-B or an
expression vector for producing PDGF-B in a target cell,
stimulators of cell proliferation or differentiation, stem cells or
progenitor cells, and/or other compounds known to be effective in
promoting healing, inhibiting infection, etc.
[0099] In other embodiments, compositions of the invention may be
used in the treatment of cancer. Such compositions may include a
chemotherapeutic agent, an angiogenesis-inhibiting agent, a cell
proliferation inhibitor, a radiosensitizer, and/or any other agent
useful in the treatment of cancer.
[0100] For example, compounds that may be formulated in a subject
composition for the treatment of cancer include: aminoglutethimide,
amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin,
buserelin, busulfan, campothecin, capecitabine, carboplatin,
carmustine, chlorambucil, cisplatin, cladribine, clodronate,
colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine,
dactinomycin, daunorubicin, dienestrol, diethylstilbestrol,
docetaxel, doxorubicin, epirubicin, estradiol, estramustine,
etoposide, exemestane, filgrastim, fludarabine, fludrocortisone,
fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein,
goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib,
interferon, irinotecan, ironotecan, letrozole, leucovorin,
leuprolide, levamisole, lomustine, mechlorethamine,
medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna,
methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide,
nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate,
pentostatin, plicamycin, porfimer, procarbazine, raltitrexed,
rituximab, streptozocin, suramin, tamoxifen, temozolomide,
teniposide, testosterone, thioguanine, thiotepa, titanocene
dichloride, topotecan, trastuzumab, tretinoin, vinblastine,
vincristine, vindesine, and vinorelbine.
[0101] These chemotherapeutic agents may be categorized by their
mechanism of action into, for example, following groups:
anti-metabolites/anti-cancer agents, such as pyrimidine analogs
(5-fluorouracil, floxuridine, capecitabine, gemcitabine and
cytarabine) and purine analogs, folate antagonists and related
inhibitors (mercaptopurine, thioguanine, pentostatin and
2-chlorodeoxyadeno sine (cladribine));
antiproliferative/antimitotic agents including natural products
such as vinca alkaloids (vinblastine, vincristine, and
vinorelbine), microtubule disruptors such as taxane (paclitaxel,
docetaxel), vincristin, vinblastin, nocodazole, epothilones and
navelbine, epidipodophyllotoxins (teniposide), DNA damaging agents
(actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,
camptothecin, carboplatin, chlorambucil, cisplatin,
cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin,
epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan,
merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,
procarbazine, teniposide, triethylenethiophosphoramide and
etoposide (VP16)); antibiotics such as dactinomycin (actinomycin
D), daunorubicin, doxorubicin (adriamycin), idarubicin,
anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin)
and mitomycin; enzymes (L-asparaginase which systemically
metabolizes L-asparagine and deprives cells which do not have the
capacity to synthesize their own asparagine); antiplatelet agents;
antiproliferative/antimitotic alkylating agents such as nitrogen
mustards (mechlorethamine, cyclophosphamide and analogs, melphalan,
chlorambucil), ethylenimines and methylmelamines
(hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan,
nitrosoureas (carmustine (BCNU) and analogs, streptozocin),
trazenes-dacarbazinine (DTIC); antiproliferative/antimitotic
antimetabolites such as folic acid analogs (methotrexate); platinum
coordination complexes (cisplatin, carboplatin), procarbazine,
hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs
(estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and
aromatase inhibitors (letrozole, anastrozole); anticoagulants
(heparin, synthetic heparin salts and other inhibitors of
thrombin); fibrinolytic agents (such as tissue plasminogen
activator, streptokinase and urokinase), aspirin, COX-2 inhibitors,
dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory
agents; antisecretory agents (breveldin); immunosuppressives
(cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin),
azathioprine, mycophenolate mofetil); anti-angiogenic compounds
(TNP-470, genistein) and growth factor inhibitors (vascular
endothelial growth factor (VEGF) inhibitors, fibroblast growth
factor (FGF) inhibitors); angiotensin receptor blocker; nitric
oxide donors; anti-sense oligonucleotides; antibodies
(trastuzumab); cell cycle inhibitors and differentiation inducers
(tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin
(adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin,
eniposide, epirubicin, etoposide, idarubicin and mitoxantrone,
topotecan, irinotecan), corticosteroids (cortisone, dexamethasone,
hydrocortisone, methylpednisolone, prednisone, and prenisolone);
growth factor signal transduction kinase inhibitors; mitochondrial
dysfunction inducers and caspase activators; chromatin
disruptors.
[0102] 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.
[0103] Another embodiment of the invention is a composition
containing at least one biologically active compound having
agricultural utility and a linear cyclodextrin-modified polymer or
a linear oxidized cyclodextrin-modified polymer 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.
IV. Exemplary Compositions
[0104] In certain embodiments, the underlying polymers are linear
cyclodextrin-containing polymers, e.g., the polymer backbone
includes cyclodextrin moieties. For example, the polymer may be a
water-soluble, linear cyclodextrin polymer produced by providing at
least one cyclodextrin derivative modified to bear one reactive
site at each of exactly two positions, and reacting the
cyclodextrin derivative with a linker having exactly two reactive
moieties capable of forming a covalent bond with the reactive sites
under polymerization conditions that promote reaction of the
reactive sites with the reactive moieties to form covalent bonds
between the linker and the cyclodextrin derivative, whereby a
linear polymer comprising alternating units of cyclodextrin
derivatives and linkers is produced. A variety of suitable polymers
are described in U.S. patent application 20020151523 and Ser. No.
10/656,838. Alternatively the polymer may be a water-soluble,
linear cyclodextrin polymer having a linear polymer backbone, which
polymer comprises a plurality of substituted or unsubstituted
cyclodextrin moieties and linker moieties in the linear polymer
backbone, wherein each of the cyclodextrin moieties, other than a
cyclodextrin moiety at the terminus of a polymer chain, is attached
to two of said linker moieties, each linker moiety covalently
linking two cyclodextrin moieties. In yet another embodiment, the
polymer is a water-soluble, linear cyclodextrin polymer comprising
a plurality of cyclodextrin moieties covalently linked together by
a plurality of linker moieties, wherein each cyclodextrin moiety,
other than a cyclodextrin moiety at the terminus of a polymer
chain, is attached to two linker moieties to form a linear
cyclodextrin polymer.
[0105] The linker group(s) may be an alkylene chain, a polyethylene
glycol (PEG) chain, polysuccinic anhydride, poly-L-glutamic acid,
poly(ethyleneimine), an oligosaccharide, an amino acid chain, or
any other suitable linkage. In certain embodiments, the linker
group itself can be stable under physiological conditions, such as
an alkylene chain, or it can be cleavable under physiological
conditions, such as by an enzyme (e.g., the linkage contains a
peptide sequence that is a substrate for a peptidase), or by
hydrolysis (e.g., the linkage contains a hydrolyzable group, such
as an ester or thioester). The linker groups can be biologically
inactive, such as a PEG, polyglycolic acid, or polylactic acid
chain, or can be biologically active, such as an oligo- or
polypeptide that, when cleaved from the moieties, binds a receptor,
deactivates an enzyme, etc. Various oligomeric linker groups that
are biologically compatible and/or bioerodible are known in the
art, and the selection of the linkage may influence the ultimate
properties of the material, such as whether it is durable when
implanted, whether it gradually deforms or shrinks after
implantation, or whether it gradually degrades and is absorbed by
the body. The linker group may be attached to the moieties by any
suitable bond or functional group, including carbon-carbon bonds,
esters, ethers, amides, amines, carbonates, carbamates,
sulfonamides, etc.
[0106] In exemplary embodiments, the cyclodextrin polymers used for
forming the material are copolymers of cyclodextrin and
polyethylene glycol (PEG). Such polymers can be prepared by
techniques well known in the art, such as the reaction exemplified
by the following equation:
##STR00005##
wherein the trapezoid represents a cyclodextrin moiety, as
described in greater detail below, and n represents an integer from
1 to 20, preferably from 2 to 12. The polymers may be modified,
e.g., by covalent attachment of therapeutic moieties, e.g., through
a linker that is cleavable under physiological conditions.
[0107] In such polymers, cyclodextrin moieties may represent at
least 2% of the weight of the copolymer, preferably at least 5% or
10%, or even as much as 20%, 40%, 50%, 60%, 80%, or even 90% of the
weight of the copolymer.
[0108] In certain embodiments, the cyclodextrin polymers used for
forming the material have a structure of the formula:
##STR00006##
[0109] wherein R represents, independently for each occurrence, H,
lower alkyl, a cyclodextrin moiety, or
##STR00007##
and
[0110] m, independently for each occurrence, represents an integer
from 2-10,000, preferably from 10 to 5,000, or from 100 to 1,000.
Suitable polymers of this type are discussed at greater length in
U.S. patent application Ser. No. 10/372,723 and PCT Application WO
03/072637.
[0111] In certain embodiments, R represents a cyclodextrin moiety
for at least about 1%, more preferably at least about 3%, and up to
about 5%, 10%, 20%, 35%, or even 50%, of the nitrogen atoms that
would be primary amines (i.e., bearing two occurrences of R that
represent H) but for the cyclodextrin moieties.
[0112] In certain embodiments, the cyclodextrin moieties make up at
least about 2%, 3%, or 4% by weight, up to 5%, 7%, or even 10% of
the cyclodextrin-modified polymer by weight.
[0113] In certain embodiments, at least about 2%, 3%, or 4% by
weight, up to 5%, 7%, or even 10% of the ethylenimine subunits in
the polymer are modified with a cyclodextrin moiety.
[0114] Copolymers of poly(ethylenimine) that bear nucleophilic
amino substituents susceptible to derivatization with cyclodextrin
moieties can also be used to prepare cyclodextrin-modified polymers
within the scope of the present invention.
[0115] Exemplary cyclodextrin moieties include cyclic structures
consisting essentially of from 6 to 9 saccharide moieties, such as
cyclodextrin and oxidized cyclodextrin. A cyclodextrin moiety
optionally comprises a linker moiety that forms a covalent linkage
between the cyclic structure and the polymer backbone, preferably
having from 1 to 20 atoms in the chain, such as alkyl chains,
including dicarboxylic acid derivatives (such as glutaric acid
derivatives, succinic acid derivatives, and the like), and
heteroalkyl chains, such as oligoethylene glycol chains.
Cyclodextrin moieties may further include one or more carbohydrate
moieties, preferably simple carbohydrate moieties such as
galactose, attached to the cyclic core, either directly (i.e., via
a carbohydrate linkage) or through a linker group.
[0116] 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.
##STR00008##
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). Additional methods
for modifying polymers are disclosed in Suh, J. and Noh, Y.,
Bioorg. Med. Chem. Lett. 1998, 8, 1327-1330.
[0117] 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, see U.S. Pat. Nos. 4,727,064,
5,608,015, 5,276,088, and 5,691,316. 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).
Cyclodextrin polymers have been produced by linking or
cross-linking cyclodextrins or mixtures of cyclodextrins and other
carbohydrates with polymerizing agents, e.g. epichlorhydrin,
diisocynanates, diepoxides (Insoluble cyclodextrin polymer beads,
Chem. Abstr. No. 222444m, 102: 94; Zsadon and Fenyvesi, 1st. Int.
Symp. on Cyclodextrins, J. Szejtli, ed., D. Reidel Publishing Co.,
Boston, pp. 327-336; Fenyvesi et al., 1979, Ann. Univ. Budapest,
Section Chim. 15: 13 22; and Wiedenhof et al., 1969, Die Stirke 21:
119-123). These polymerizing agents are capable of reacting with
the primary and secondary hydroxy groups on carbons 6, 2, and 3.
Cyclodextrin-modified polymer carriers can be coupled to
biorecognition molecules for targeting the delivery of drugs to
their site of action. See also U.S. Pat. Nos. 6,180,739, 5,981,740,
5,929,131, 5,910, 551, and 5,792,821 (disclosing polymerizable
cyclodextrin derivatives), U.S. Pat. No. 6,048,736 (discussing drug
delivery using cyclodextrin polymers), (disclosing polymerizable
cyclodextrin derivatives), U.S. Pat. No. 5,856,416 and U.S. Pat.
No. 5,593,768 (disclosing monomers and polymers that bear
cyclodextrin moieties), U.S. Pat. No. 5,608,015 (disclosing methods
for preparing polymerizable cyclodextrin derivatives), and U.S.
Pat. No. 5,516,766 (describing uses for cyclodextrin polymers).
[0118] The crosslinking molecules generally comprise two or more
moieties that form inclusion complexes with cyclodextrin or another
inclusion host, the moieties being covalently linked by a chain of
atoms that allows the moieties to be spaced from 0.5 to 50 nm
apart, preferably 1 to 30 nm apart. The moieties can be selected
from any molecules that form inclusion complexes with an inclusion
host such as cyclodextrin, e.g., phenyl rings, adamantane
polycycles, cholesterol, naphthol derivatives, etc. See, for
example, Szejtli, Cyclodextrins and Their Inclusion Complexes;
Akademiai Klado, Budapest, 1982; and Bender et al., Cyclodextrin
Chemistry, Springer-Verlag, Berlin, 1978. Cyclodextrins are able to
form inclusion complexes with an array of organic molecules
including, for example, drugs, pesticides, and herbicides. See,
Tenjarla et al., J. Pharm. Sci., 87: 425-429 (1998); Zughul et al.,
Pharm. Dev. Technol., 3: 43-53 (1998); and Albers et al., Crit.
Rev. Ther. Drug Carrier Syst., 12: 311-337 (1995). The moieties in
a crosslinking molecule may be the same or different, and different
crosslinking molecules may be used simultaneously in a single
material.
[0119] The covalent linkage between the moieties may be any
suitable linkage, such as an alkylene chain, a polyethylene glycol
(PEG) chain, poly(ethyleneimine), an oligosaccharide, an amino acid
chain, or any other suitable linkage. The linkage can be stable
under physiological conditions, such as an alkylene chain, or it
can be cleavable under physiological conditions, such as by an
enzyme (e.g., the linkage contains a peptide sequence that is a
substrate for a peptidase), or by hydrolysis (e.g., the linkage
contains a hydrolyzable group, such as an ester or thioester). The
linkage can be biologically inactive, such as a PEG, polyglycolic
acid, or polylactic acid chain, or can be biologically active, such
as an oligo- or polypeptide that, when cleaved from the moieties,
binds a receptor, deactivates an enzyme, etc. Various oligomeric
linkages that are biologically compatible and/or bioerodible are
known in the art, and the selection of the linkage may influence
the ultimate properties of the material, such as whether it is
durable when implanted, whether it gradually deforms or shrinks
after implantation, or whether it gradually degrades and is
absorbed by the body. The linkage may be attached to the moieties
by any suitable bond or functional group, including carbon-carbon
bonds, esters, ethers, amides, amines, carbonates, carbamates,
sulfonamides, etc.
[0120] In one exemplary embodiment, the crosslinking molecule
comprises moieties that are biologically active drugs joined by a
linkage that is biologically active and hydrolyzes under
physiological conditions. After implantation, the linkages are
gradually cleaved, releasing the active drugs and partially
degrading the structure of the material. If the polymer supporting
the cyclodextrin is also biodegradable, the material will gradually
disintegrate, slowly releasing the biologically active drugs
complexed within it. In such embodiments, additional pharmaceutical
agents may, but need not be, included in the material to obtain a
therapeutic effect.
[0121] Materials of the invention may further comprise one or more
biologically active agents. Such agents may also form inclusion
complexes with cyclodextrin, or may be merely dispersed through the
material. Exemplary agents include nucleic acids, viruses,
polypeptides, polyplexes, pharmaceuticals, small organic molecules,
antibodies, or any other therapeutic agents.
[0122] In certain embodiments, a subject composition comprises an
RNAi construct, e.g., for using RNA interference (RNAi) to effect
knockdown of a target gene. RNAi constructs comprise double
stranded RNA that can specifically block expression of a target
gene, and are suitable for delivery using the subject compositions.
RNAi provides a useful method of inhibiting gene expression in
vitro or in vivo. RNAi constructs can comprise either long
stretches of dsRNA identical or substantially identical to the
target nucleic acid sequence or short stretches of dsRNA identical
to substantially identical to only a region of the target nucleic
acid sequence.
[0123] Optionally, the RNAi constructs contain a nucleotide
sequence that hybridizes under physiologic conditions of the cell
to the nucleotide sequence of at least a portion of the mRNA
transcript for the gene to be inhibited (i.e., the "target" gene).
The double-stranded RNA need only be sufficiently similar to
natural RNA that it has the ability to mediate RNAi. Thus, the
invention has the advantage of being able to tolerate sequence
variations that might be expected due to genetic mutation, strain
polymorphism or evolutionary divergence. The number of tolerated
nucleotide mismatches between the target sequence and the RNAi
construct sequence is no more than 1 in 5 basepairs, or 1 in 10
basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs. Mismatches
in the center of the siRNA duplex are most critical and may
essentially abolish cleavage of the target RNA. In contrast,
nucleotides at the 3' end of the siRNA strand that is complementary
to the target RNA do not significantly contribute to specificity of
the target recognition. Sequence identity may be optimized by
sequence comparison and alignment algorithms known in the art (see
Gribskov and Devereux, Sequence Analysis Primer, Stockton Press,
1991, and references cited therein) and calculating the percent
difference between the nucleotide sequences by, for example, the
Smith-Waterman algorithm as implemented in the BESTFIT software
program using default parameters (e.g., University of Wisconsin
Genetic Computing Group). Greater than 90% sequence identity, or
even 100% sequence identity, between the inhibitory RNA and the
portion of the target gene is preferred. Alternatively, the duplex
region of the RNA may be defined functionally as a nucleotide
sequence that is capable of hybridizing with a portion of the
target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM
EDTA, 50.degree. C. or 70.degree. C. hybridization for 12-16 hours;
followed by washing).
[0124] The double-stranded structure may be formed by a single
self-complementary RNA strand or two complementary RNA strands. RNA
duplex formation may be initiated either inside or outside the
cell. The RNA may be introduced in an amount which allows delivery
of at least one copy per cell. Higher doses (e.g., at least 5, 10,
100, 500 or 1000 copies per cell) of double-stranded material may
yield more effective inhibition, while lower doses may also be
useful for specific applications. Inhibition is sequence-specific
in that nucleotide sequences corresponding to the duplex region of
the RNA are targeted for genetic inhibition.
[0125] The subject RNAi constructs can be "small interfering RNAs"
or "siRNAs." These nucleic acids are around 19-30 nucleotides in
length, and even more preferably 21-23 nucleotides in length. The
siRNAs are understood to recruit nuclease complexes and guide the
complexes to the target mRNA by pairing to the specific sequences.
As a result, the target mRNA is degraded by the nucleases in the
protein complex. In a particular embodiment, the 21-23 nucleotides
siRNA molecules comprise a 3' hydroxyl group. In certain
embodiments, the siRNA constructs can be generated by processing of
longer double-stranded RNAs, for example, in the presence of the
enzyme dicer. In one embodiment, the Drosophila in vitro system is
used. In this embodiment, dsRNA is combined with a soluble extract
derived from Drosophila embryo, thereby producing a combination.
The combination is maintained under conditions in which the dsRNA
is processed to RNA molecules of about 21 to about 23 nucleotides.
The siRNA molecules can be purified using a number of techniques
known to those of skill in the art. For example, gel
electrophoresis can be used to purify siRNAs. Alternatively,
non-denaturing methods, such as non-denaturing column
chromatography, can be used to purify the siRNA. In addition,
chromatography (e.g., size exclusion chromatography), glycerol
gradient centrifugation, affinity purification with antibody can be
used to purify siRNAs.
[0126] Production of RNAi constructs can be carried out by chemical
synthetic methods or by recombinant nucleic acid techniques.
Endogenous RNA polymerase of the treated cell may mediate
transcription in vivo, or cloned RNA polymerase can be used for
transcription in vitro. The RNAi constructs may include
modifications to either the phosphate-sugar backbone or the
nucleoside, e.g., to reduce susceptibility to cellular nucleases,
improve bioavailability, improve formulation characteristics,
and/or change other pharmacokinetic properties. For example, the
phosphodiester linkages of natural RNA may be modified to include
at least one of an nitrogen or sulfur heteroatom. Modifications in
RNA structure may be tailored to allow specific genetic inhibition
while avoiding a general response to dsRNA Likewise, bases may be
modified to block the activity of adenosine deaminase. The RNAi
construct may be produced enzymatically or by partial/total organic
synthesis, any modified ribonucleotide can be introduced by in
vitro enzymatic or organic synthesis. Methods of chemically
modifying RNA molecules can be adapted for modifying RNAi
constructs (see, e.g., Heidenreich et al. (1997) Nucleic Acids Res,
25:776-780; Wilson et al. (1994) J Mol Recog 7:89-98; Chen et al.
(1995) Nucleic Acids Res 23:2661-2668; Hirschbein et al. (1997)
Antisense Nucleic Acid Drug Dev 7:55-61). Merely to illustrate, the
backbone of an RNAi construct can be modified with
phosphorothioates, phosphoramidate, phosphodithioates, chimeric
methylphosphonate-phosphodiesters, peptide nucleic acids,
5-propynyl-pyrimidine containing oligomers or sugar modifications
(e.g., 2'-substituted ribonucleosides, a-configuration).
[0127] In some cases, at least one strand of the siRNA molecules
has a 3' overhang from about 1 to about 6 nucleotides in length,
though may be from 2 to 4 nucleotides in length. More preferably,
the 3' overhangs are 1-3 nucleotides in length. In certain
embodiments, one strand having a 3' overhang and the other strand
being blunt-ended or also having an overhang. The length of the
overhangs may be the same or different for each strand. In order to
further enhance the stability of the siRNA, the 3' overhangs can be
stabilized against degradation. In one embodiment, the RNA is
stabilized by including purine nucleotides, such as adenosine or
guanosine nucleotides. Alternatively, substitution of pyrimidine
nucleotides by modified analogues, e.g., substitution of uridine
nucleotide 3' overhangs by 2'-deoxythyinidine is tolerated and does
not affect the efficiency of RNAi. The absence of a 2' hydroxyl
significantly enhances the nuclease resistance of the overhang in
tissue culture medium and may be beneficial in vivo.
[0128] The RNAi construct can also be in the form of a long
double-stranded RNA. In certain embodiments, the RNAi construct is
at least 25, 50, 100, 200, 300 or 400 bases. In certain
embodiments, the RNAi construct is 400-800 bases in length. The
double-stranded RNAs are digested intracellularly, e.g., to produce
siRNA sequences in the cell. However, use of long double-stranded
RNAs in vivo is not always practical, presumably because of
deleterious effects which may be caused by the sequence-independent
dsRNA response. In such embodiments, the use of local delivery
systems and/or agents which reduce the effects of interferon or PKR
are preferred.
[0129] Alternatively, the RNAi construct is in the form of a
hairpin structure (named as hairpin RNA). The hairpin RNAs can be
synthesized exogenously or can be formed by transcribing from RNA
polymerase III promoters in vivo. Examples of making and using such
hairpin RNAs for gene silencing in mammalian cells are described
in, for example, Paddison et al., Genes Dev, 2002, 16:948-58;
McCaffrey et al., Nature, 2002, 418:38-9; McManus et al., RNA,
2002, 8:842-50; Yu et al., Proc Natl Acad Sci USA, 2002,
99:6047-52). Preferably, such hairpin RNAs are engineered in cells
or in an animal to ensure continuous and stable suppression of a
desired gene. It is known in the art that siRNAs can be produced by
processing a hairpin RNA in the cell.
[0130] PCT application WO 01/77350 describes an exemplary vector
for bi-directional transcription of a transgene to yield both sense
and antisense RNA transcripts of the same transgene in a eukaryotic
cell. Accordingly, in certain embodiments, a subject composition
includes, for ultimate delivery, a recombinant vector having the
following unique characteristics: it comprises a viral replicon
having two overlapping transcription units arranged in an opposing
orientation and flanking a transgene for an RNAi construct of
interest, wherein the two overlapping transcription units yield
both sense and antisense RNA transcripts from the same transgene
fragment in a host cell.
V. Business Methods
[0131] Other aspects of the invention provides for certain methods
of doing business. In particular, practicing the methods of the
invention may enable novel therapeutic compositions and improved
formulations thereof. This technical step, when combined with one
or more additional steps, provides for novel approaches to
conducting a pharmaceutical, or preferably a life-science business.
For example, a therapeutic composition prepared by the method of
the invention may be tested for efficacy as therapeutics in a
variety of disease models, the potential therapeutic compositions
then tested for toxicity and other safety-profiling before
formulating, packaging and subsequently marketing the resulting
formulation for the treatment of disease. Alternatively, the rights
to develop and market such formulations or to conduct such steps
may be licensed to a third party for consideration.
[0132] Accordingly, in certain embodiments, the present invention
provides a method for conducting a pharmaceutical business,
comprising:
[0133] a. manufacturing a formulation or kit including a
pharmaceutical composition of any of the compositions disclosed
herein; and
[0134] b. marketing to healthcare providers the benefits of using
the formulation or kit in the treatment of a disease or
disorder.
[0135] In other embodiments, the present invention discloses a
method for conducting a pharmaceutical business, comprising:
[0136] a. providing a distribution network for selling a
pharmaceutical composition of any of the compositions disclosed
herein; and
[0137] b. providing instruction material to patients or physicians
for using the preparation in the treatment of a disease or
disorder.
[0138] In certain embodiments, the present invention provides a
method for conducting a pharmaceutical business, comprising:
[0139] a. determining an appropriate formulation and dosage of a
pharmaceutical composition of any of the compositions disclosed
herein;
[0140] b. conducting therapeutic profiling of formulations
identified in step (a), for efficacy and toxicity in animals;
and
[0141] c. providing a distribution network for selling a
preparation or preparations identified in step (b) as having an
acceptable therapeutic profile.
[0142] An additional step of the embodiment comprises providing a
sales group for marketing the preparation to healthcare
providers.
[0143] In still other embodiments, the present invention provides a
method for conducting a pharmaceutical business, comprising:
[0144] a. determining an appropriate formulation and dosage of a
pharmaceutical composition of any of the compositions disclosed
herein; and
[0145] b. licensing, to a third party, the rights for further
development and sale of the formulation.
EXEMPLIFICATION
[0146] The invention now being generally described, it will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention, and are not intended to
limit the invention.
Example 1
Synthesis of Polyethylenimine (PEI)-Cyclodextrin Conjugates
[0147] (Suh et al. 1992, J. Am. Chem. Soc. 114 7916-7917)
##STR00009##
[0148] Branched PEI (60 mg, Aldrich Mw 25,000) was dissolved in
DMSO (4 mL) and degassed water (6 mL) solvent mixture. Cyclodextrin
monotosylate (928 mg, Cyclodextrin Technologies Development, Inc.)
was added to the solution under argon. The cloudy solution turned
clear after the mixture was stirred at 70.degree. C. for about 1
hour. The solution turned slightly yellow after 48 hours at this
temperature under argon. The solution was transferred to a
Spectra/Por MWCO 25,000 membrane and dialyzed against water for 6
days. Water was then removed by lyophilization. A white powder was
obtained (134 mg) after the solution was lyophilized.
Cyclodextrin/PEI ratio was calculated based on the proton
integration of .sup.1H NMR.
Example 2
Synthesis of Hydrolysable Polyethylenimine (PEI)-Cyclodextrin
Conjugates
Ahn et al. 2002 Journal of Controlled Release 80, 273-282
##STR00010##
[0150] The hydrolysable PEI-PEG is synthesized as described in Ahn
et al. 2002 Journal of Controlled Release 80, 273-282 reference.
The resulting hydrolysable PEI polymer is dissolved in DMSO (4 mL)
and degassed water (6 mL) solvent mixture. Cyclodextrin
monotosylate (Cyclodextrin Technologies Development, Inc.) is added
to the solution under argon. The reaction mixture is then
precipitated into cold diethyl ether and the product is dried under
vacuum overnight. Cyclodextrin/PEI ratio is calculated based on the
proton integration of .sup.1H NMR.
Example 3
Synthesis of Linear Cyclodextrin-Based Polyethylene Glycol Polymers
(CD-PEG)
[0151] CD-PEG polymers were prepared by the polymerization of a
difunctionalized .beta.-cyclodextrin monomer (A) with a
difunctionalized polyethylene glycol comonomer (B) to give an ABAB
product. The synthesis procedure involves first the preparation of
the difunctionalized .beta.-cyclodextrin
(6A,6D-dideoxy-6A,6D-di(2-aminoethanethio)-.beta.-cyclodextrin
(denoted dicysteamine-.beta.-cyclodextrin) according to literature
procedures (Gonzalez et al. 1999 Bioconjugate Chem. 10:1068-1074;
and Hwang et al. 2001 Bioconjugate Chem. 12(2):280-290). The
polymerization step was carried using commercially available
difunctionalized polyethylene glycol. Three methods were
investigated.
Method I: Using Diacid-Polyethylene Glycol
##STR00011##
[0152] Synthesis:
[0153] Diacid-PEGs with various molecular weights (Mw=250, 600,
3000, 6000) were purchased from Fluka, Milwaukee, Wis. In a typical
experiment, PEG.sub.600-(COOH).sub.2 (0.096 g, 0.16 mmol) was
dissolved in 1 mL of a 25 mM MES buffer
(2-(N-morpholino)ethanesulfonic acid) at pH 6.5.
Dicysteamine-.beta.-cyclodextrin (0.2 g, 0.16 mmol) dissolved in 2
mL of a 25 mM MES buffer (pH 6.5) was added. Then,
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)
(0.612 g, 3.2 mmol, Aldrich, Milwaukee, Wis.) and
N-Hydroxysulfosuccinimide (Sulfo-NHS) (0.026 g, 0.12 mmol, Pierce,
Rockford, Ill.) were added. The resulting solution was allowed to
stir overnight at room temperature. The polymer solution was then
transferred to a Spectra/Por 7 MWCO 10,000 membrane (Spectrum,
Houston, Tex.) and dialyzed against water for 24 h. The solution
was then freeze-dried to dryness. This afforded 272 mg of white
solid (Yield: 93%).
Characterization:
[0154] Light Scattering and Molecular Weight Determination The
specific refractive index increment, dn/dc, was measured in
Phosphate Buffered Saline 1.times. (PBS) (Cellgro, Mediatech, Inc,
Herndon, Va.) using a Bausch & Lomb ABBE-3L refractive index.
Polymer samples were then analyzed on a Hitachi D6000 HPLC system
equipped with a ERC-7512 RI detector, a Precision Detectors
PD2020/DLS static light scattering detector and an PL aquagel-OH 30
(Polymer Laboratories, Amherst, Mass.) column using Phosphate
Buffered Saline 1.times. as eluant at a 0.7 mL/min flow rate. The
dn/dc, the weight average molecular weight Mw, and the
polydispersity index Mw/Mn determined for each polymer are reported
in the following table.
TABLE-US-00001 TABLE 1 Static Light Scattering and Molecular Weight
Determination of CD-PEG polymers. Polymer dn/dc Mw (Da)
Polydispersity CD-PEG.sub.6000 0.1267 21,560 1.69 CD-PEG.sub.3000
0.1296 10,490 1.35 CD-PEG.sub.600 0.1361 28,450 1.77 CD-PEG.sub.250
0.1359 21,150 1.06
Method II: Using Disuccinimidyl Propionate Polyethylene Glycol
##STR00012##
[0155] Synthesis:
[0156] Disuccinimidyl propionate polyethylene glycol
(PEG.sub.3400-(SPA).sub.2) (1.0854 g, 0.32 mmol, Shearwater
Polymers, Inc, Huntsville, Ala.) in solution in 5 mL of DMSO was
added to a solution of dicysteamine-.beta.-cyclodextrin (0.4 g,
0.32 mmol) dissolved in 2 mL of DMSO. A viscous solution was
immediately formed. The reaction mixture was then allowed to stir
overnight at room temperature under argon. Diethylether was added
to precipitate the polymer and then poured out with a pipet. The
residual ether was evaporated and the polymer was redissolved in
water. The resulting solution was then transferred to a 10,000 MWCO
Spectra/Por membrane and dialyzed against water for 24 h. The
solution was then freeze-dried to dryness. This afforded 1.329 g of
white solid (Yield: 95%).
Characterization: Light Scattering and Molecular Weight
Determination
[0157] This polymer was characterized using the same technique
presented above. The do/dc was calculated to be 0.1316 and the
weight average molecular weight Mw was determined to be 184,000 Da
with a polydispersity index Mw/Mn of 2.18.
Method III: Using Di-Benzotriazole Carbonate Polyethylene
Glycol
##STR00013##
[0158] Synthesis:
[0159] Di-benzotriazole carbonate polyethylene glycol
(PEG.sub.3400-(BTC).sub.2) (1 g, 0.32 mmol, Shearwater Polymers,
Inc, Huntsville, Ala.) in solution in 5 mL of DMSO was added to a
solution of dicysteamine-.beta.-cyclodextrin (0.4 g, 0.32 mmol)
dissolved in 2 mL of DMSO. A viscous solution was immediately
formed. The reaction mixture was then allowed to stir overnight at
room temperature under argon. Diethyl ether was added to
precipitate the polymer and then poured out with a pipette. The
residual ether was evaporated and the polymer was redissolved in
water. The resulting solution was then transferred to a 10,000 MWCO
Spectra/Por membrane and dialyzed against water for 24 h. The
solution was then freeze-dried to dryness. This afforded 1.3 g of
white solid (Yield: 95%).
Example 4
Molecular Weight Control of CD-PEG Polymers
[0160] Dicysteamine-.beta.-cyclodextrin.2HCl (0.577 g, 0.46 mmol)
was dried under vacuum at 100.degree. C. for 16 h. Disuccinimidyl
propionate polyethylene glycol (PEG.sub.3400-(SPA).sub.2) (1.565 g,
0.46 mmol, Shearwater Polymers, Inc, Huntsville, Ala.) was then
added. To the mixture was added dry DMSO (8 mL). After 10 min
stirring, DIEA (176 .mu.L, 1.01 mmol) was added under argon. A
portion of polymerization solution (1 mL) was removed at selected
times (15 min, 30 min, 60 min, 1 h, 2 h and 5 h). These samples
were then transferred to a 10,000 MWCO Spectra/Por membrane and
dialyzed against water for 24 h. The solutions were then
freeze-dried to dryness. MWs of the resulting solids were
determined as described above.
[0161] As shown in FIG. 3, the polymer Mw increased to around 80
kDa over a 5 h time course. The polymer Mw can be controlled
between 50 to 80 kDa.Example 5
Synthesis of Hydrolysable Linear Cyclodextrin-Based Polyethylene
Glycol Polymers (CD-PEG)
Method I: Using NHS-HBA-CM-PEG.sub.3400-CM-HBA-NHS
##STR00014##
[0163] NHS-HBA-CM-PEG.sub.3400-CM-HBA-NHS (0.2 g, 0.06 mmol,
Shearwater Polymers, Inc, Huntsville, Ala.) in solution in 1.5 mL
of DMSO is added to a solution of dicysteamine-.beta.-cyclodextrin
(0.074 g, 0.06 mmol) dissolved in 2 mL of DMSO. The reaction
mixture is then allowed to stir overnight at room temperature under
argon. The resulting polymer is precipitated with diethylether,
filtrated and dried under vacuum.
Method II: Using PEG Succinimidyl Succinate (PEG-(SS).sub.2
##STR00015##
[0164] Synthesis:
[0165] Disuccinimidyl succinate polyethylene glycol
(PEG.sub.3400-(SS).sub.2) (SunBio, Inc., 980-5 Kwan-yang Dong,
Anyang City, S. Korea) (0.3 mmol) in solution in 5 mL of DMSO is
added to a solution of dicysteamine-.beta.-cyclodextrin (0.3 mmol)
dissolved in 2 mL of DMSO. The reaction mixture is then allowed to
stir overnight at room temperature under argon. The resulting
polymer is precipitated with diethylether, filtrated and dried
under vacuum.
Example 6
Synthesis of Hydrolysable Linear Cyclodextrin-Based Polymer
##STR00016##
[0166] Synthesis:
[0167] Ethylene glycol bis[succinimidylsuccinate] (EGS) (0.2 mmol,
Pierce, Rockford, Ill.) in solution in 2 mL of DMSO is added to a
solution of dicysteamine-.beta.-cyclodextrin (0.2 mmol) dried
overnight at 100.degree. C. under vacuum and dissolved in 1.5 mL of
DMSO. The reaction mixture is then allowed to stir overnight at
room temperature under argon. The resulting mixture is precipitated
with Acetone, filtrated and dried under vacuum.
Example 7
Synthesis of Diadamantane Crosslinker:
Bis-(2(1-adamantyl)ethyl)phosphate
[0168] (Zhang et al. 1997, J. Am. Chem. Soc. 119(7):1676-1681)
##STR00017##
Synthesis:
[0169] Ahydrous pyridine (10 mL, Aldrich, Milwaukee, Wis.) was
cooled in an ice bath and methyl dichlorophosphate (1.488 g, 10
mmol, Aldrich, Milwaukee, Wis.) was added dropwise. The mixture was
kept cold for a further 15 min. During this period a precipitate of
N-methylpyridinium dichlorophosphate formed. 1-Adamantane ethanol
(4.758 g, 26.4 mmol, Aldrich, Milwaukee, Wis.) was added, and the
sealed mixture was stirred overnight at room temperature. It was
then poured into 10% NaHCO.sub.3 (50 mL) and the pyridine was
evaporated under vacuum. The slightly yellow solid was dissolved in
1 L of water and extracted with ether (three 150 mL portions). The
aqueous phase was acidified with 2 N HCl to pH 1, and then
extracted with three 150 mL portions of CHCl.sub.3:n-BuOH (7:3).
The combined organic layer (ether and CHCl.sub.3:n-BuOH) was washed
with water and a slightly yellow precipitate was formed in the
mixed solvents, at which point the solvents were evaporated under
vacuum. A slightly yellow solid was formed and was recrystallized
from acetone/hexane. The solid was dried under vacuum, yield
60%.
Characterization:
[0170] The product was characterized by .sup.1H NMR and .sup.13C
NMR. .sup.1H NMR (CDCl.sub.3): .delta. 1.45-1.75 (m, 28H,
--CH.sub.2--, adamantyl), 1.95 (m, 6H, C--H, adamantyl), 4.07 (q,
4H, --CH.sub.2--) and 8.60 (br, 1H, POOH). .sup.13C NMR
(CDCl.sub.3, 500 MHz): .delta. 28.59, 31.77, 37.02, 42.52, 43.96,
44.02, 64.21, 64.26. The product was also characterized by Mass
Spectroscopy: Electrospray Ionization: 421 [M-H].sup.-.
Example 8
Synthesis of Hydrolysable Adamantane Crosslinker
##STR00018##
[0171] Synthesis:
[0172] 1-Adamantanemethylamine (0.152 g, 0.92 mmol, Aldrich,
Milwaukee, Wis.) was added to Ethylene glycol
bis[succinimidylsuccinate] (EGS) (0.2 g, 0.43 mmol, Pierce,
Rockford, Ill.) in solution in 10 mL of anhydrous dichloromethane.
The resulting solution was stirred for 5 h at room temperature. It
was then acidified with 0.1 N HCl and extracted with
dichloromethane. The organic phase was dried with MgSO.sub.4 and
then concentrated to dryness under vacuum. This afforded 0.22 g of
solid (yield 90%).
Characterization:
[0173] The product was characterized by Mass Spectroscopy:
Electrospray Ionization: 557 [M+H].sup.+, 579 [M+Na].sup.+, 1135
[2M+Na].sup.+.
Example 9
Synthesis of Diadamantane Polyethylene Glycol Crosslinker
[0174] Various methods were investigated:
Method I: Using Diacid-Polyethylene Glycol
##STR00019##
[0176] 1-Adamantanemethylamine (0.64 mmol, Aldrich, Milwaukee,
Wis.) is added to PEG-diacid (0.32 mmol, Fluka, Milwaukee, Wis.)
dissolved in dichloromethane. 1,3-Dicyclohexylcarbodiimide (DCC)
(3.2 mmol, Aldrich, Milwaukee, Wis.) is added and the resulting
solution is stirred overnight at room temperature. The precipitate
(dicyclohexylisourea, DCU) is filtered off and the filtrate is
washed with 18% HCl. The organic phase is dried with MgSO.sub.4 and
then concentrated to dryness under vacuum. The resulting solid is
redissolved in water in order to precipitate the remaining DCC. DCC
is filtered off and the filtrate is freeze-dried.
Method II: Using Disuccinimidyl Propionate Polyethylene Glycol
##STR00020##
[0178] 1-Adamantanemethylamine (0.1 g, 0.60 mmol, Aldrich,
Milwaukee, Wis.) was added to Disuccinimidyl propionate
polyethylene glycol (PEG.sub.3400-(SPA).sub.2) (1.02 g, 0.30 mmol,
Shearwater Polymers, Inc, Huntsville, Ala.) dissolved in 10 mL of
dichloromethane. The resulting solution was stirred at room
temperature overnight. The solvent was evaporated under vacuum and
the residue was dissolved in water and centrifuged to remove excess
1-adamantanemethylamine. The supernatant was then transferred to a
1,000 MWCO Spectra/Por membrane and dialyzed against water for 24
h. The solution was then freeze-dried to dryness. This afforded
0.88 g of solid (yield 84%).
Method III: Using Polyethylene Glycol
(Sandier et al. 2000, Langmuir 16:1634-1642)
##STR00021##
[0180] Polyethylene glycol (Mw=1000) (1 g, 1 mmol, Aldrich,
Milwaukee, Wis.) was dried by heating under vacuum at 70.degree. C.
overnight. 1-Adamantyl isocyanate (0.39 g, 2.2 mmol, Aldrich,
Milwaukee, Wis.) was added to the dried polyethylene glycol after
its dissolution in anhydrous dichloromethane (25 mL). Two
catalysts, dibutyltin dilaurate (63.2 mg, 0.1 mmol, Aldrich,
Milwaukee, Wis.) and triethylamine (10.1 mg, 0.1 mmol, Aldrich,
Milwaukee, Wis.) were then added. The reaction mixture was heated
under reflux for 7 h. After removal of the solvent, the reaction
product was dissolved in distilled water. The aqueous solution was
purified by addition of activated carbon and successive
filtrations, and then freeze-dried. The resulting polymer was
recovered with a 70% yield and was characterized by .sup.1H
NMR.
Method IV: Using Di-Benzotriazole Carbonate Polyethylene Glycol
##STR00022##
[0182] 1-Adamantanemethylamine (0.1 g, 0.60 mmol, Aldrich,
Milwaukee, Wis.) is added to Dibenzotriazole carbonate polyethylene
glycol (PEG.sub.3400-(BTC).sub.2) (1.02 g, 0.30 mmol, Shearwater
Polymers, Inc, Huntsville, Ala.) dissolved in 10 mL of
dichloromethane. The resulting solution was stirred at room
temperature overnight. The solvent was evaporated under vacuum and
the residue was dissolved in water and centrifuged to remove excess
1-adamantanemethylamine. The supernatant was then transferred to a
1,000 MWCO Spectra/Por membrane and dialyzed against water for 24
h. The solution was then freeze-dried to dryness. This afforded
0.88 g of solid (yield 84%).
Example 10
Synthesis of Hydrolysable Diadamantane Polyethylene Glycol
Crosslinker
[0183] Various methods were investigated:
Method I: Using Polyethylene Glycol
##STR00023##
[0185] Polyethylene glycol (Mw=1000) (1 mmol, Aldrich, Milwaukee,
Wis.) is dried by heating under vacuum at 70.degree. C. overnight.
1-Adamantaneacetic acid (2.2 mmol, Aldrich, Milwaukee, Wis.) is
added to the dried polyethylene glycol after its dissolution in
anhydrous toluene (15 mL). p-Toluenesulfonic acid (Aldrich,
Milwaukee, Wis.) is then added in a catalytic amount. The resulting
mixture is azeotropically refluxed for 16 h using Dean-Stark
apparatus. After completion of the reaction, the solvent is removed
under vacuum and the resulting polymer is precipitated with
ether.
Method II: Using Disuccinimidyl Succinate Polyethylene Glycol
##STR00024##
[0187] 1-Adamantanemethylamine (0.1 g, 0.60 mmol, Aldrich,
Milwaukee, Wis.) was added to disuccinimidyl succinate polyethylene
glycol (PEG.sub.3400-(SS).sub.2) (SunBio, Inc., 980-5 Kwan-yang
Dong, Anyang City, S. Korea) (0.30 mmol) dissolved in 10 mL of
dichloromethane. The resulting solution was stirred at room
temperature overnight. The solvent was evaporated under vacuum and
the resulting polymer was precipitated with ether.
Example 11
Synthesis of Triadamantane Crosslinker
##STR00025##
[0189] 1-Adamantanemethylamine (0.212 g, 1.29 mmol, Aldrich,
Milwaukee, Wis.) was added to Tris-succinimidyl aminotriacetate
(TSAT) (0.2 g, 0.41 mmol, Pierce, Rockford, Ill.) dissolved in 10
mL of anhydrous dimethylformamide (Aldrich, Milwaukee, Wis.). The
resulting mixture was stirred at room temperature under argon for
14 h. The precipitated was filtered and characterized by mass
spectroscopy: Electrospray Ionization: 633.4 [M+H].sup.+, 655.6
[M+Na].sup.+, 1265.3 [2M+H].sup.+, 1287.1 [2M+Na].sup.+.
Example 12
Synthesis of Tetraadamantane Crosslinker
##STR00026##
[0191] 1-Adamantanemethylamine (0.212 g, 1.29 mmol, Aldrich,
Milwaukee, Wis.) was added to
Tetrakis-(N-succinimidylcarboxypropyl)pentaerythritol (NHS-4) (0.1
g, 0.12 mmol, Molecular Biosciences, Boulder, Colo.) dissolved in 5
mL of anhydrous dimethylformamide (Aldrich, Milwaukee, Wis.). The
resulting mixture was stirred at room temperature under argon for
14 h. The precipitated was filtered and characterized by Mass
Spectroscopy: Electrospray Ionization: 1013.7 [M+H].sup.+, 1035.8
[M+Na].sup.+.
Example 13
Synthesis of Tetra-Adamantane Polyethylene Glycol Crosslinker
Method I: Using Pentaerythritol Ethoxylate (15/4 EO/OH)
##STR00027##
[0193] Pentaerythritol ethoxylate (15/4 EO/OH) (Mn=797) (1 mmol,
Aldrich, Milwaukee, Wis.) is dried by heating under vacuum at
70.degree. C. overnight. 1-Adamantyl isocyanate (4.4 mmol, Aldrich,
Milwaukee, Wis.) is added to the dried polymer after its
dissolution in anhydrous dichloromethane (25 mL). Two catalysts,
dibutyltin dilaurate (0.1 mmol, Aldrich, Milwaukee, Wis.) and
triethylamine (0.1 mmol, Aldrich, Milwaukee, Wis.) are then added.
The reaction mixture is heated under reflux for 7 hours. After
removal of the solvent, the reaction product is dissolved in
distilled water. The aqueous solution is purified by addition of
activated carbon and successive filtrations, and then
freeze-dried.
Method II: Using 4 Arm PEG
##STR00028##
[0195] 4 arm PEG (Mn=10000) (1 mmol, Shearwater Polymers, Inc,
Huntsville, Ala.) is dried by heating under vacuum at 70.degree. C.
overnight. 1-Adamantyl isocyanate (4.4 mmol, Aldrich, Milwaukee,
Wis.) is added to the dried polymer after its dissolution in
anhydrous dichloromethane (25 mL). Two catalysts, dibutyltin
dilaurate (0.1 mmol, Aldrich, Milwaukee, Wis.) and triethylamine
(0.1 mmol, Aldrich, Milwaukee, Wis.) are then added. The reaction
mixture is heated under reflux for 7 hours. After removal of the
solvent, the reaction product is dissolved in distilled water. The
aqueous solution is purified by addition of activated carbon and
successive filtrations, and then freeze-dried.
Example 14
Synthesis of Hydrolysable Tetra-Adamantane Polyethylene Glycol
Crosslinker
Method I: Using Pentaerythritol Ethoxylate (15/4 EO/OH)
##STR00029##
[0197] Pentaerythritol ethoxylate (15/4 EO/OH) (Mn=797) (1 mmol,
Aldrich, Milwaukee, Wis.) is dried by heating under vacuum at
70.degree. C. overnight. 1-Adamantaneacetic acid (4.4 mmol,
Aldrich, Milwaukee, Wis.) is added to the dried polymer after its
dissolution in anhydrous toluene. p-Toluenesulfonic acid (Aldrich,
Milwaukee, Wis.) is then added in a catalytic amount. The resulting
mixture is azeotropically refluxed for 16 h using Dean-Stark
apparatus. After completion of the reaction, the solvent is removed
under vacuum and the resulting polymer is precipitated with
ether.
Method II: Using 4 Arm PEG
##STR00030##
[0199] 4 arm PEG (M.sub.n=10000) (1 mmol, Shearwater Polymers, Inc,
Huntsville, Ala.) is dried by heating under vacuum at 70.degree. C.
overnight. 1-Adamantaneacetic acid (4.4 mmol, Aldrich, Milwaukee,
Wis.) is added to the dried polymer after its dissolution in
anhydrous toluene. p-Toluenesulfonic acid (Aldrich, Milwaukee,
Wis.) is then added in a catalytic amount. The resulting mixture is
azeotropically refluxed for 16 h using Dean-Stark apparatus. After
completion of the reaction, the solvent is removed under vacuum and
the resulting polymer is precipitated with ether.
Method III: Using 4-Arm PEG-Succinimidyl Succinate
(PEG-SS).sub.4
##STR00031##
[0201] 1-Adamantanemethylamine (0.069 g, 0.44 mmol, Aldrich,
Milwaukee, Wis.) was added to 1 g of disuccinimidyl succinate
polyethylene glycol ((PEG.sub.iok-SS).sub.4) (SunBio, Inc., 980-5
Kwan-yang Dong, Anyang City, S. Korea) (0.1 mmol) previously
dissolved in 10 mL of dichloromethane. The resulting solution was
stirred at room temperature overnight. The solvent was evaporated
under vacuum and the resulting polymer was precipitated with
ether.
Example 15
Synthesis of Octa-Adamantane Polyethylene Glycol Crosslinker
##STR00032##
[0203] 8 arm PEG (Mn=10000) (1 mmol, Shearwater Polymers, Inc,
Huntsville, Ala.) is dried by heating under vacuum at 70.degree. C.
overnight. 1-Adamantyl isocyanate (4.4 mmol, Aldrich, Milwaukee,
Wis.) is added to the dried polymer after its dissolution in
anhydrous dichloromethane (25 mL). Two catalysts, dibutyltin
dilaurate (0.1 mmol, Aldrich, Milwaukee, Wis.) and triethylamine
(0.1 mmol, Aldrich, Milwaukee, Wis.) are then added. The reaction
mixture is heated under reflux for 7 hours. After removal of the
solvent, the reaction product is dissolved in distilled water. The
aqueous solution is purified by addition of activated carbon and
successive filtrations, and then freeze-dried.
Example 16
Synthesis of Hydrolysable Octa-Adamantane Polyethylene Glycol
Crosslinker
##STR00033##
[0205] 8 arm PEG (Mn=10000) (1 mmol, Shearwater Polymers, Inc,
Huntsville, Ala.) is dried by heating under vacuum at 70.degree. C.
overnight. 1-Adamantaneacetic acid (4.4 mmol, Aldrich, Milwaukee,
WI) is added to the dried polymer after its dissolution in
anhydrous toluene. p-Toluenesulfonic acid (Aldrich, Milwaukee,
Wis.) is then added in a catalytic amount. The resulting mixture is
azeotropically refluxed for 16 h using Dean-Stark apparatus. After
completion of the reaction, the solvent is removed under vacuum and
the resulting polymer is precipitated with ether.
Example 17
Synthesis of Multi Adamantane Crosslinker
Method I: Using Branched PEI
##STR00034##
[0207] 1-Adamantaneacetic acid (Aldrich, Milwaukee, Wis.) is added
to PEI (Aldrich, Milwaukee, Wis.) dissolved in MES buffer 25 mM, pH
6.5. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDC) (Aldrich, Milwaukee, Wis.) and N-Hydroxysuccinimide (NHS)
(Aldrich, Milwaukee, Wis.) are then added to the reaction mixture.
The reaction mixture is stirred at room temperature for 24 h and
then transferred to a 10000 MWCO Spectra/Por membrane and dialyzed
against water for 24 h. The solution was then freeze-dried to
dryness.
Method II: Using Pullulan
(Akiyoshi et al. 1993, Macromolecules 26:3062-3068)
##STR00035##
[0209] 1-Adamantyl isocyanate (Aldrich, Milwaukee, Wis.) is reacted
with Pullulan (Sigma-Aldrich, Milwaukee, Wis.) in anhydrous DMSO
containing pyridine at 80.degree. C. for 8 h. Ethanol is added to
the reaction mixture, and the resulting mixture is stored overnight
at 4.degree. C. The precipitates are separated, purified by
dialysis against water and lyophilized to dryness. The degree of
substitution of the adamantane group is determined by .sup.1H
NMR.
Example 18
Synthesis of Hydrolysable Multi Adamantane Crosslinker
(Sunamoto et al. 1992, Macromolecules 25:5665-5670)
##STR00036##
[0211] Pullulan (Sigma-Aldrich, Milwaukee, Wis.) is dissolved in
anhydrous DMF at 60.degree. C. 1-Adamantanecarbonyl chloride
(Aldrich, Milwaukee, Wis.) dissolved in anhydrous DMF and anhydrous
Pyridine are added. The resulting mixture is stirred at 60.degree.
C. for 2 h and another 1 h at room temperature. The mixture is
poured into ethanol. The precipitates are collected and washed with
ethanol and then, with diethyl ether. The solid is dried under
vacuum at 50.degree. C. for 2 h. The degree of substitution of the
adamantane group is determined by .sup.1H NMR.
Example 19
Synthesis of Self-Crosslinked Polymer
[0212] The synthesis of a polymer that contains cylodextrin and
adamantane functions is carried out in three steps.
1. Step 1: Synthesis of monomer
6.sup.A,6.sup.D-Bis-(2-amino-2-carboxylethylthio)-6.sup.A,6.sup.D-dideoxy-
-.beta.-cyclodextrin, (CD-BisCys)
##STR00037##
[0214] A 167 mL of 0.1 M sodium carbonate buffer was degassed for
45 minutes in a 500 mL 2-neck round bottom flask equipped with a
magnetic stir bar, a condenser and septa. To this solution was
added 1.96 g (16.2 mmol) of L-cysteine and 10.0 g (73.8 mmol) of
di-iodo-.beta.-cyclodextrin (Gonzalez et al. 1999 Bioconjugate
Chem. 10:1068-1074; and Hwang et al. 2001 Bioconjugate Chem. 12(2):
280-290; Gonzalez, H., Hwang, S. J., and Davis, M. E. (2000) Linear
Cyclodextrin Copolymers WO001734A1). The resulting suspension was
heated at a reflux temperature for 4.5 h. until the solution turned
clear colorless. Then, it was cooled to room temperature and
acidified to pH 3 using 1 N HCl. The product was crashed out by
slow addition of acetone (3 times weight ratio of the solution).
This afforded 9.0 g (90.0%) yield of CD-BisCys. The resulting solid
was subjected to anionic exchange column chromatography using a
gradient elution of 0-0.4 M ammonium bicarbonate solution. ESI/MS
(m/z): 1342 [M].sup.+, 1364 [M+Na].sup.+. Purity of CD-BisCys was
confirmed by HPLC.
2. Step 2: Synthesis of CD-BisCys-PEG.sub.3400 Copolymers
##STR00038##
[0216] CD-BisCys (2 g, 1.49 mmol) and SPA-PEG3400-SPA (5.07 g, 1.49
mmol, Shearwater Inc.) were dissolved in dry DMSO (40 mL). After 10
minutes diisopropylethylamine (DIEA, 0.571 mL, 2.2 eq, Aldrich) was
added under argon. The reaction mixture was stirred under argon for
2-6 days. An increase of viscosity was observed as a function of
polymerization time. Water (200 mL) was added to the polymerization
solution with vigorous stirring. The solution was then dialyzed in
25,000 MWCO Spectra/Por 7 membrane for 2.5 days at a concentration
of ca. 10 mg polymer/mL water. After lyophilization, a white fluffy
powder (6.2 g, 92% yield) was obtained.
Characterization: Light Scattering and Molecular Weight
Determination
[0217] The specific refractive index increment, dn/dc, was measured
in Phosphate Buffered Saline 1.times. (PBS) (Cellgro, Mediatech,
Inc, Herndon, Va.) using a Bausch & Lomb ABBE-3L refractive
index. Polymer samples were then analyzed on a Hitachi D6000 HPLC
system equipped with a ERC-7512 RI detector, a Precision Detectors
PD2020/DLS static light scattering detector and an PL aquagel-OH 30
(Polymer Laboratories, Amherst, Mass.) column using Phosphate
Buffered Saline 1.times. as eluant at a 0.7 mL/min flow rate. The
dn/dc was calculated to be 0.1348 and the weight average molecular
weight Mw was determined to be 103,500 Da with a polydispersity
index Mw/Mn of 1.71.
3. Adamantane Conjugation to CD-BisCys-PEG.sub.3400 Copolymers
##STR00039##
[0219] CD-BisCys-PEG.sub.3400 copolymer (0.32 mmol of repeat unit)
is dissolved in dry DMSO and stirred for 10 minutes.
1-Adamantanemethylamine (0.76 mmol, Aldrich, Milwaukee, Wis.), DIEA
(0.76 mmol), EDC (0.96 mmol) and NHS (0.71 mmol) are added to the
polymer solution. The mixture is stirred for about 16 hours. Water
is added to the resulting mixture to remove excess
1-Adamantanemethylamine. After filtration of the precipitates, the
solution is dialyzed against water with 10,000 MWCO Spectra/Por
membrane for 48 h and lyophilized to dryness. The degree of
substitution of the adamantane group is determined by .sup.1H
NMR.
Example 20
Synthesis of RGD-Modified Adamantane-PEG Derivative
##STR00040##
[0220] Step 1: Synthesis of VS-PEG.sub.5000-AD (2)
[0221] Vinylsulfone-PEG.sub.5000-NHS (1) (Shearwater Polymers,
0.147 mmol) was added to a round bottom flask equipped with a stir
bar and dissolved in 5 mL of DMSO. To this was added
Adamantanemethylamine (Aldrich, 0.147 mmol). The resulting solution
was stirred 1 h at room temperature. The resulting mixture was
dialyzed overnight against 3500 MWCO Membrane (Spectra Por). The
solution was then lyophilized to afford
Vinylsulfone-PEG.sub.5000-AD (2).
Step 2: RGDpep-PEG-AD Conjugate Synthesis
[0222] RGDpep-SH synthesized as described in Kok et al.
(Bioconjugate Chemistry (2002), 13(1), 128-135) is dissolved in PBS
(phosphate buffer saline) lx, pH 7.2. Vinylsulfone-PEG.sub.5000-AD
(2) is then added to the RGDpep-SH solution. The resulting solution
is stirred at room temperature for 2 hours. The polymer solution is
then transferred to a Spectra/Por 7 MWCO 3500 membrane (Spectrum,
Houston, Tex.) and dialyzed against water for 24 h. The solution is
then freeze-dried to dryness to afford RGDpep-PEG.sub.5000-AD
(3).
Example 21
Preparation of Material
[0223] The polymer bearing the inclusion host (Example 1-6) was
dissolved at 100 mg/mL in PBS (phosphate-buffered saline) lx, pH
7.2. The crosslinker (Example 7-18) (ratio:
Adamantane/cylodextrin:1/1 or 1/2) was then added. The resulting
mixture was mixed vigorously in order to get the crosslinker in
solution. An increase in the viscosity was observed in about 10
min.
Example 22
Preparation of Ionic Type Material
Using Multivalent Ion
[0224] The polymer bearing the inclusion host and carboxyl groups
(See Example 19, Step 2) was dissolved at 100 mg/mL in 0.1 M
CaCl.sub.2 aqueous solution and then added to a vial containing a
mixture of the crosslinker (Example 7-18) (ratio:
Adamantane/cyclodextrin:1/1 or 1/2). The resulting mixture was
mixed vigorously in order to get the crosslinker and the diamino
compound in solution. An increase in the viscosity was
observed.
Using Diamino Compound
[0225] The polymer bearing the inclusion host and carboxyl groups
(See Example 19, Step 2) was dissolved at 100 mg/mL in water and
then added to a vial containing a mixture of the crosslinker
(Example 7-18) (ratio: Adamantane/cyclodextrin:1/1 or 1/2) and a
diamino compound like PEG.sub.3400-(NH.sub.2).sub.2 (Shearwater
Polymers) or C.sub.nH.sub.2n--(NH.sub.2).sub.2 (ratio:
NH.sub.2/COO.sup.-: 1/1 or 1/2). The resulting mixture was mixed
vigorously in order to get the crosslinker and the diamino compound
in solution. An increase in the viscosity was observed.
Using Polycation
[0226] The polymer bearing the inclusion host and carboxyl groups
(See Example 19, Step 2) was dissolved at 100 mg/mL in water and
then added to a vial containing a mixture of the crosslinker
(Example 7-18) (ratio: Adamantane/cyclodextrin:1/1 or 1/2) and a
polycation like polylysine or polyethyleneimine. The resulting
mixture was mixed vigorously in order to get the crosslinker and
the polycation in solution. An increase in the viscosity was
observed.
Example 23
Preparation of Material Containing Virus or Protein
[0227] The polymer bearing the inclusion host (Example 1-6) was
dissolved in PBS (phosphate-buffered saline) 1.times., pH 7.2
containing the protein or virus at the desired concentration to
obtain a polymer solution at a final concentration of 100 mg/mL of
solution. The crosslinker (Example 7-18) (ratio:
Adamantane/cylodextrin:1/1 or 1/2) was then added. The resulting
mixture was mixed vigorously in order to get the crosslinker in
solution. An increase in the viscosity was observed in about 10
min.
Example 24
Preparation of Material Containing a Signaling Peptide
[0228] The polymer bearing the inclusion host (Example 1-6) is
dissolved in PBS (phosphate-buffered saline) 1.times., pH 7.2
(containing when desired proteins, virus, or other drugs or drug
delivery systems) at the desired concentration to obtain a polymer
solution at a final concentration of 100 mg/mL of solution. The
crosslinker (Example 7-18) (ratio: Adamantane/cylodextrin:1/2) and
the signaling peptide (Example 20) (ratio:
Adamantane/cyclodextrin:1/2) are then added. The resulting mixture
is mixed vigorously in order to get the crosslinker in solution. An
increase in the viscosity is observed in about 10 min.
Example 25
Studies Concerning Cell Mobility Through Matrix
[0229] a. Matrix (0.2 mL, composed of CD-PEG.sub.3400 and
bis-(2(1-adamantyl)ethyl)phosphate) was prepared as described in
Example 21 and added to the bottom of FluoroBlok inserts (for 24
well format, Falcon Catalog #351152). [0230] b. CCD cells were
rinsed with PBS and then exposed to 5 .mu.M of Calcein-AM, a
fluorescent marker, for 15-30 minutes. [0231] c. CCD cells were
then trypsinized and plated on top of the matrix in the insert at
10,000 cells/insert in 0.5 mL media. [0232] d. 1 mL of media
containing 10 ng/mL human PDGF protein was then added to the lower
chamber. [0233] e. Cell mobility through the matrix and into the
lower chamber was monitored for 3 days by fluorescence microscopy.
Successful migration through the matrix and past the insert was
demonstrated by the presence of cells in the lower chamber observed
by fluorescence microscopy 72 hours after plating as shown in FIG.
5.
Example 26
Transfection Studies to CCD Fibroblast Cells with CD-PEI Polyplexes
Formulated with Matrix
[0234] The well bottoms in 24-well plates were coated with
.about.0.2 mL of the following: [0235] a. No coating (control)
[0236] b. Matrix [0237] c. Matrix containing 1 .mu.g of luciferase
gene-containing plasmid. [0238] d. Matrix containing CDP polyplex.
[0239] e. Matrix containing CDPEI polyplex. [0240] f. No coating.
Free CDPEI polyplex (positive control I: traditional transfection
procedure) [0241] g. No coating. Free CDP polyplex (positive
control II: traditional transfection procedure).
[0242] CCD (fibroblast cells, ATCC) were plated in the 24 well
plates at 40,000 cells/well. 24 hours after plating the cells,
media was removed, cells rinsed with PBS, and lysed. The cell
lysates were analyzed for luciferase protein activity using
luciferase assay. Results are reported in RLU/well. This experiment
demonstrates that the cells are able to successfully migrate into
the matrix and be transfected by the polyplexes contained in the
matrix. Transfection efficiency is only slightly lower than
transfection by polyplexes free in media.
[0243] Note: the matrix is comprised of CD-PEG (see Example 3,
method II for synthesis procedure) crosslinked with a diadamantane
crosslinker (bis-(2(1-adamantyl)ethyl)phosphate, see Example 7 for
synthesis procedure). Matrix formulation procedure is described in
Example 21. When polyplexes are included in the matrix, the
polyplexes are prepared by adding a 10 .mu.L solution of polymer to
10 .mu.L solution of luciferase gene-containing plasmid (0.1 mg/mL)
at the optimum charge ratio. The polyplexes are included in the
buffer solution that is used to dissolve the CD-PEG component of
the matrix and matrix formulated as described in Example 21.
[0244] For positive control experiments, polyplexes are prepared as
described and added directly to the cell media.
Example 27
[0245] Materials can be prepared, as schematically depicted in FIG.
1, by the self-assembly of cyclodextrin (CD)-containing polymers
(A) and di- or multi-functional linkers (B) terminated with
molecules capable of forming inclusion complexes with the
cyclodextrin-containing polymers. The materials (P) may be
formulated to contain proteins, cells, viruses, polyplexes, or
other therapeutic agents or delivery systems containing therapeutic
agents.
[0246] An exemplary linker,
##STR00041##
can be prepared by following the protocols described in the Breslow
and Zhang, JACS (1996), 118, 8495-8496, and Zhang and Breslow, JACS
(1993), 115, 9353-9354. One of skill in the art can readily modify
this protocol, in light of techniques generally known in the art,
to arrive at a wide spectrum of different linkers that can be
employed as linking molecules as the term is used herein. The
difunctional linkers or cyclodextrin-containing polymers may
contain biodegradable linkages to facilitate the release of the
therapeutic agents and/or degradation of the material.
[0247] Compounds increasing the therapeutic utility of the
material, such as signaling peptides or other moieties facilitating
cell migration, may be incorporated into the material by
conjugating a inclusion complex guest to the entity of interest and
including the conjugate in the material as described in FIG. 2. The
conjugate may be included before, during or after the crosslinking
process.
[0248] For example, a polymer made of repeated subunits having the
formula:
##STR00042##
e.g., wherein the Mw of monomer is approximately 4800, can be
prepared as a solution of the polymer at 100 mg/mL in PBS
(phosphate-buffered saline) 1.times., pH 7.2. It may be desirable
to centrifuge the mixture and then agitate the solution to
solubilize the polymer. A 33.3 .mu.L aliquot of a solution of the
linking molecule
##STR00043##
at 132 mg/mL in dichloromethane, can be placed into a vial to let
the dichloromethane evaporate at room temperature or in an oven at
37.degree. C. 1 mL of the polymer solution can then be added to the
residual linker, optionally triturating to facilitate solution. The
solution may then be allowed to rest, and after a time, e.g., 10
min., the solution may become viscous. To include a foreign
substance, such as a therapeutic agent or viral particles, into the
crosslinked polymer, the substance may be present in the solution
prior to the final resting period. For example, the substance may
be present in the solvent used to dissolve the cyclodextrin polymer
or in the initial solution of the linking molecule, or may be added
to either component or to the final solution prior to the
crosslinking reaction.
Example 28
Methods and Results
[0249] Matrix 1 (60 kD polymer prepared according to Example 3,
method II and crosslinking agent prepared according to Example 9,
method II) and Matrix 2 (80 kD polymer prepared according to
Example 3, method II and crosslinking agent prepared according to
Example 9, method II) were used.
Application of 60 kD Matrix In Vivo.
[0250] 300 .mu.l of 60 kD matrix was drawn up into a 1 cc syringe
and all air bubbles were removed by moving the syringe plunger up
and down. A 23G needle was large enough for matrix to pass through
with some pressure required. A 20G needle was large enough to allow
the matrix to pass through easily.
[0251] Male Sprague-Dawley rats were sacrificed, shaved, and 8 mm
wounds were made on their dorsal regions. Using a 1 cc syringe with
20G needle, excess 60 kD matrix was expelled until 100 .mu.L of
volume remained. Matrix was then applied to an 8 mm wound punch
without any over-fill. The same procedure was performed except with
150 .mu.l of Matrix 1 and this over-filled an 8 mm punch wound in
rat dorsum skin.
[0252] The feasibility of applying matrix to an incisional wound
was explored. A full-thickness incision approximately 2 inches in
length was made on a rat dorsum. A syringe was filled with 200
.mu.L of 60 kD matrix and was successfully injected intra-dermally
at the incision site using a 20G needle.
Application of 80 kD Matrix
[0253] Two 8 mm full-thickness dermal punches were made on a rat
dorsum. One punch was treated with 100 .mu.L of 80 kD matrix using
a 1 cc syringe and a 20G needle. This volume filled the punch
evenly; however, more force was required to deliver 80 kD when
compared to 60 kD matrix. 150 .mu.L of 80 kD matrix overfilled an 8
mm dermal punch. 200 .mu.l of 80 kD matrix was successfully
delivered by intra-dermal injection although substantial force was
required to complete the injection.
Example 29
[0254] Studies were performed to acquire initial rheological data
of the CD-PEG.sub.3400 matrix (100 mg/ml) (Example 3, method II)
before and after crosslinking with di-Adamantane-PEG (36.5 mg/ml)
(Example 9, method II) These data were compared to collagen matrix
at 2.4 mg/ml, one of the standard formulations used in preclinical
model development.
[0255] Results:
[0256] The first series of experiments were performed with
CD-PEG.sub.3400 polymer at 100 mg/ml without cross linker.
Frequency sweeps at various constant strains were performed.
[0257] FIG. 7 shows a frequency sweep at 37.degree. C. G'' is
greater than G', resulting in a tan .delta. of approximately 3.2.
As expected, this polymer solution exhibits mostly viscous
behavior. This is confirmed by the slope of G' as a function of
frequency. For purely viscous fluids, a theoretical slope of 2 is
expected whereas for purely elastic fluids, a slope of 0 is
expected over a certain frequency range. On a structural level, the
interpretation of these results is that the polymer molecules
interact with each other by some sort of weak interaction. Energy
applied to the material can be stored by the molecules elastically
by moving nodes of interaction (rotation, entanglement). A large
part of this energy is then dissipated into heat. The measured
complex viscosity of this polymer solution was 0.27 Pa s at 10.0
rad/s.
[0258] FIG. 8 shows the same material at 20.degree. C. An increase
in viscosity to 1.46 Pa s at 10 rad/s was observed. This value is
approximately 7-fold higher than the viscosity at 37.degree. C.
[0259] FIG. 9 shows a frequency sweep for the CD-PEG.sub.3400
polymer crosslinked with 36.5 mg/ml of di-Adamantane-PEG. Addition
of the cross linker increased the viscosity of the matrix over 100
fold to approximately 33.1 Pa s. At the same time, tan .delta.
decreased approximately 3-fold to a value of 1.2 and the slope of
G' decreased to 0.58. These results are consistent with an increase
in elastic behavior. Structurally, the movement of the polymer
within the material is constrained and energy applied can be stored
in a more limited way between network points. This behavior is also
confirmed by the observation that the crosslinked matrix has a
tendency to draw long strings when an immersed object is pulled
away from the surface. Over all, viscous forces still are dominant
(tan .delta.>1) (see FIG. 11).
[0260] FIG. 10 shows the crosslinked polymer at 20.degree. C.
Viscosity increased further to approximately 50 Pa s while all the
other characteristics remained similar.
[0261] As a comparison, FIG. 11 shows a frequency sweep for the
collagen matrix at 2.4 mg/ml. The collagen matrix in its set state
(i.e., at 37.degree. C.) exhibits a typical gel behavior, with tan
.delta. at a low value of 0.14, a slope of G' of 0.07.
Structurally, this is an indication of a permanent and orderly
arranged network. Applied energy can only be stored to limited
extent between network nodes and is returned to a large extent. The
complex viscosity of this material was 12.8 Pa s.
Example 30
In Vitro Biocompatibility Experiment
Methods:
[0262] A matrix was prepared using 58 kD cyclodextrin (CD) polymer
prepared according to Example 3, method II, and crosslinking agent
from Example 9, method II. Upon formulation of the matrix, either
pEGFP DNA or GFCB virus was mixed together with the matrix. A low
and a high dose of DNA or virus were both formulated. The various
treatment groups are listed in Table 2. Final concentration of 58
kD cyclodextrin matrix was 100 mg/mL for all groups. Approximately
100 .mu.L of each respective reagent was pipetted into a 48-well
flat bottom plate. Each group was tested in duplicate. The 48-well
plate containing the test formulations was allowed to set at room
temperature for approximately 30 minutes. 2.5.times.10.sup.4
CCD-1074sk cells (human skin fibroblast) were placed on top of the
cross-linked matrix formulations in a volume of 200 .mu.L of DMEM
media containing 10% Fetal Bovine Serum (FBS). The CCD-1074sk cell
line was approximately 70% confluent at the time of cell counting
with cell viability greater than 95% via Trypan Blue staining. Each
well, containing groups 1-8, was examined daily under an inverted
microscope for cell viability and for GFP fluorescence signal. 200
.mu.L of fresh media was added (not replaced) to each of the wells
on days 4 and 8 after the start of the experiment. The plate was
incubated at 37.degree. C. with 5% CO.sub.2.
Synthesis of CD-1PEI
##STR00044##
[0264] Linear PEI.sub.25,000 (500 mg, Polysciences, Inc.) and
6-Monotosyl-.beta.-Cyclodextrin (3.868 g, Cyclodextrin Technologies
Development, Inc.) were dissolved in 36 mL of DMSO. The resulting
mixture was stirred at 70.degree. C. for 6 days. The solution
turned slightly yellow. The solution was then transferred to a
Spectra/Por MWCO 10,000 membrane and dialyzed against water for 6
days. Water was then removed by lyophilization to afford a slightly
colored solid. Cyclodextrin/PEI ratio was calculated based on the
proton integration of .sup.1H NMR (Varian 300 MHz, D.sub.2O)
.delta. 5.08 ppm (s br., C.sub.1H of CD), 3.3-4.1 ppm (m br.
C.sub.2H--C.sub.6H of CD), 2.5-3.2 ppm (m br. CH.sub.2 of PEI).
.beta. Cyclodextrin Polymers (CDP-Imidazole)
##STR00045##
[0266] .beta.CDP was synthesized according to previously described
procedures (Gonzalez, H., Hwang, S. & Davis, M. (1999)
Bioconjugate Chem. 10, 1068-74). Imidazole was conjugated to the
.beta.CDP polymer by amidation of the primary amines at the end of
the polymer with 4-imidazoleacetic acid (Aldrich, St. Louis, Mo.)
(Sehgal, D. & Vijay, I. (1994) Anal Biochem. 218, 87-91). In a
typical experiment, 200 mg (33.3 .mu.mol) of .beta.CDP were
dissolved in 800 .mu.L of 25 mM MES (pH 6.5) buffer to which was
added 4-imidazoleacetic acid, sodium salt hydrate (49.3 mg, 0.333
mmol). This solution was used to dissolve
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (0.128 g, 0.666
mmol). Then, N-hydroxysuccinimide (NHS) (3.83 mg, 33.3 .mu.mol)
dissolved in 200 .mu.L of 25 mM MES (pH 6.5) buffer was added
immediately to the polymer solution. The resulting solution was
stirred for 24 h at room temperature and then dialyzed against
water using a Spectra Por membrane MWCO 1000. The solution was
lyophilized to dryness. The imidazole content was determined by the
TNBS assay (Hermanson, G. T. (1996) Bioconjugate Techniques, pp.
132, Academic Press, Rockford, Ill.), followed by UV measurements
to quantify the amount of unreacted polymer end groups. The
imidazole conjugation was 73%.
TABLE-US-00002 TABLE 2 Treatment Groups/Well (48-well Flat Bottom
Plate) Group # Treatment 1 0.25 mg/mL pEGFP + CDP-Imidazole in 58
kD Cyclodextrin 2 0.025 mg/mL pEGFP + CDP-Imidazole in 58 kD
Cyclodextrin 3 0.25 mg/mL pEGFP + CD-1PEI in 58 kD Cyclodextrin 4
0.025 mg/mL pEGFP + CD-1PEI in 58 kD Cyclodextrin 5 1 .times.
10.sup.10 P/mL GFCB in 58 kD Cyclodextrin 6 1 .times. 10.sup.9 P/mL
GFCB in 58 kD Cyclodextrin 7 Empty Control (Cells Alone) 8 58 kD
Cyclodextrin Matrix Alone Control
Results:
[0267] Day 1: Groups 1-4 started dying. They were starting to lift
off the plate. CCD-1074sk cells were "rounded up" morphologically
and floating. Groups 5-8 looked healthy. The cells were attached to
the plate and "stretched out" morphologically (typical state of
fibroblasts); however, none of the groups had GFP expression at
this time. See FIG. 12.
[0268] Day 2: Groups 5-8 looked healthy. CCD-1074sk cells were
attached to the plate and "stretched out". Groups 5-6 (GFCB groups)
were positive for GFP expression. The higher dose of GFCB (Group 5)
had more GFP expression than the lower dose of GFCB (Group 6).
Groups 1, 3, and 4 looked to be completely dead at this time;
however, a couple cells had survived in group 2 (0.025 mg/mL
pEGFP+CDP-Imidazole in 58 kD Cyclodextrin). There was no detectable
GFP expression in groups 1-4.
[0269] Day 4: CCD-1074sk cells in groups 1-4 appeared to be dead.
Cells were floating. GFP expression was not detectable in any of
these groups. Groups 5-8 were healthy. Groups 5-6 were positive to
GFP expression, and expression appeared to be even brighter than
the day 2.
[0270] Day 5: CCD-1074sk cells in group 1-4 were dead and no GFP
expression was seen. Groups 5-8 were healthy. Groups 5-6 were
positive or GFP expression and once again the expression level
appeared to be brighter than the day before.
[0271] Day 6: CCD-1074sk cells in groups 1-4 were dead and no GFP
expression was seen. Cells were no longer monitored. Groups 5-8
were healthy. Groups 5-6 were still expressing GFP and the
expressions level appeared to still be brighter that the day
before.
[0272] Day 8: CCD-1074sk cells in groups 5-8 still looked healthy.
Morphologically, they did not look any different from day 6. They
were still expressing GFP and appeared to be at the brightest
observed intensity.
[0273] Day 12: CCD-1074sk cells in groups 5-8 were healthy. Cell
count seemed to be slightly more numerous than day 8; however, GFP
expression appeared to be decreasing.
[0274] Day 13: CCD-1074sk cells in groups 5-8 appeared healthy, but
GFP expression was starting to decrease.
Example 29
Methods
[0275] The components for Matrix A and Matrix B were formulated
following the written protocol from Insert Therapeutics, Inc.
Matrix A was 100 mg/mL of .beta.-Cyclodextrin-PEG.sub.3400 polymer
(Example 3, method II) in PBS at pH of 7.2 with 4.4 mg/mL of
di-Adamantane compound crosslinker (Example 9, method II). The two
components were aliquoted and kept separate until used. Matrix B
was 100 mg/mL of .beta.-Cyclodextrin-PEG.sub.3400 polymer in PBS at
a pH of 7.2 with 36.5 mg/mL of di-Adamantane PEG.sub.3400 compound
crosslinker. The components were aliquoted and kept separate.
Three different methods of delivery were tested: 1) Delivery of
Matrix a into a Wound Site Using a Surgical Dressing and Delivery
of Matrix B into a Wound Site Prior to Covering with a Surgical
Dressing:
[0276] An ICR mouse was anesthetized with ketamine and xylazine.
The dorsal region was shaved and two 8 mm dermal punches were made.
50 .mu.L of Matrix A (final adjusted volume) was mixed containing a
small amount of dye and then placed onto a small square piece of
OpSite dressing. The matrix was allowed to gel for about 2 minutes.
During this time, the left wound punch on the mouse was prepared
with Mastisol dressing adhesive. The OpSite dressing containing the
mixed Matrix A was then flipped over ("sticky" side down) and
placed over the left wound site. The right wound punch was prepared
with Mastisol adhesive dressing. Matrix B containing a small amount
of dye was also made up at a final adjusted volume of 50 .mu.L and
placed directly onto the right wound site. This was allowed to gel
for about 2 minutes then covered with the OpSite dressing. This
animal was allowed to recover from surgery and was sacrificed 4
days later.
2) Delivery of Matrix a with Pre-Mixing and Matrix B without
Pre-Mixing into a Wound Site Through a Previously Applied Surgical
Dressing:
[0277] An ICR mouse was anesthetized with ketamine and xylazine.
The dorsal region was shaved and two 8 mm dermal punches were made.
Two wound sites were prepared with Mastisol adhesive and 1 single
sheet of OpSite dressing was placed over the wounds. Matrix A was
prepared for a 50 .mu.L total injection volume containing a small
amount of dye. The matrix was drawn up directly into a 1 cc syringe
and a 23G needle was placed on the syringe. Excess matrix was
expelled from the syringe until approximately 50 .mu.L remained.
This material was then injected through the OpSite dressing onto
the left wound bed. For the right side wound, Matrix B was prepared
for .about.100 .mu.L total injection volume. A 1 cc syringe
containing a 23G needle was used to draw up approximately 82 .mu.L
of Matrix B. A small air pocket was then drawn into the syringe.
Approximately 18 .mu.L of the accompanying crosslinker was then
drawn into the same syringe needle. Another small air pocket was
then drawn into the syringe. Finally, a small amount (.about.2-4
.mu.L) of dye was drawn into the syringe. A 27G needle was placed
at one end of the right wound site through the dressing to vent air
from the covered wound site. The syringe with a 23G needle
containing the separated components of Matrix B was placed through
the dressing (without pre-mixing of the components) and the
materials were injected into the wound site. The 27G needle
provided venting of the small air pockets in the syringe. The
animal was allowed to recover after surgery and was sacrificed 4
days later.
3) Injection of Matrix A and B into PVA Sponges Using Various Gauge
Needles:
[0278] An HSD rat was anesthetized with ketamine and xylazine and
PVA sponges were subcutaneously implanted onto the ventral side of
the rat. Incisions were closed with wound clips and the animal was
allowed to recover for 4 days. Matrix A was formulated at an
adjusted injection volume of .about.200 .mu.L with dye (.about.2-4
.mu.L). The matrix mixture was drawn into a 1 cc syringe through a
22G needle and mixed in the syringe. Attempts were made to remove
excess air bubbles. The mixed matrix was injected into the center
of a PVA sponge on a sacrificed animal. After a few minutes, the
wound site was opened and the sponge area was examined.
[0279] Matrix B was prepared for .about.200 .mu.L injection. The
matrix contained a small amount of dye and was drawn into a 1 cc
syringe using a 22G needle, which was removed and replaced with a
shorter 23G needle for injection. The materials were mixed in the
syringe and an attempt to remove excess air bubbles was made.
Matrix was injected into the center of a PVA sponge, allowed to sit
for a few minutes, and then the animal was opened for examination
of the injected sponge.
Results:
[0280] 1) Delivery of Matrix A into a Wound Site Using a Surgical
Dressing and Delivery of Matrix B into a Wound Site Prior to
Covering with a Surgical Dressing:
[0281] 50 .mu.L of Matrix A (final adjusted volume) was mixed with
a small amount of dye, placed onto a small square piece of OpSite
dressing, allowed to gel for a short period of time (.about.2
minutes), and then flipped over ("sticky" side down) on the left
wound site. Upon sacrifice of the animal 4 days after surgery, the
wound appeared to be dry and the animal was observed to have
tolerated Matrix A without difficulties. Matrix appeared to stay in
its original position at the time of delivery, i.e., on top of the
wound site including some of the surrounding unwounded tissue. The
matrix that remained in the wound site was superficial and less
abundant than the day of surgery. No gross appearance of
inflammation was seen at the wound site. See FIGS. 12A-12B.
[0282] Matrix B containing a small amount of dye was also made up
at a final adjusted volume of 50 .mu.L and placed directly onto the
right wound site and then covered with OpSite dressing. When the
animal was sacrificed 4 days later, the wound appeared to be dry
and the animal seemed to have tolerated Matrix B well. No
inflammation or any other adverse effects were grossly seen around
the wound site. The matrix did not disperse onto surrounding tissue
after surgery, but rather stayed in the original wound site. The
matrix that remained in the wound site at the time of sacrifice was
superficial and less abundant than at the day of surgery. See FIGS.
12A-12B.
2) Delivery of Matrix A with Pre-Mixing and Matrix B without
Pre-Mixing into a Wound Site Through a Previously Applied Surgical
Dressing:
[0283] 50 .mu.L of Matrix A (final adjusted volume) was mixed with
a small amount of dye and then injected directly under the OpSite
dressing and on the left wound site. The matrix remained localized
in the wound site and did not disperse to the surrounding unwounded
tissue. Due to air bubbles in the syringe, it was not possible to
make precise measurements of the dosing volume. Upon sacrifice of
the animal 4 days after surgery, the wound appeared to be dry and
the animal tolerated Matrix A without any observable adverse
effects. The matrix stayed in its original position at delivery,
i.e., at the wound site including some surrounding unwounded
tissue. The matrix did not disperse onto other surrounding tissue
after the surgery; however, the matrix was only on the surface of
the wound and less abundant than at the time of surgery. Upon gross
examination, no inflammation was seen at the wound site. See FIGS.
13A-13B.
[0284] 100 .mu.L final volume of Matrix B containing a small amount
of dye was drawn into a syringe. The various components of the
matrix (polymer, crosslinker, and dye) were drawn individually into
the syringe with air pockets to separate the individual components.
A venting needle to eliminate the air pockets in the syringe was
placed in one end of the wound through the dressing and the
separated components of Matrix B were injected directly onto the
right wound site through the OpSite dressing without any prior
mixing. This method required the least amount of pressure to
successfully inject the matrix into the wound site, and the venting
needle worked to eliminate air pockets under the skin. Once all the
components of Matrix B were delivered into the wound site, the
matrix gelled quickly by visual elimination and touch, and no
matrix leaked out of the dressing. The matrix remained localized to
the wound bed and did not grossly spread to surrounding tissues.
When the animal was sacrificed 4 days later, the wound appeared to
be dry and the animal seemed to have tolerated Matrix B without
gross adverse effects. No inflammation or any other adverse effects
were seen around the wound site, and the matrix appeared to stay in
the local wound area. The matrix was localized to the surface of
the wound and at lower amounts than at the original time of
injection. See FIGS. 13A-13B.
3) Injection of Matrix A and B into PVA Sponges Using Various Gauge
Needles:
[0285] Matrix A was prepared with a small amount of dye and drawn
into a 1 cc syringe through a 22G needle. Approximately 200 .mu.L
of the matrix was injected into a PVA sponge, allowed to sit for a
few minutes, and then the animal was opened for examination. Upon
examination of the sponge, the matrix appeared to remain localized
and had minimal dispersion to the surrounding areas.
[0286] Matrix B was prepared, mixed with a small amount of dye, and
drawn into a 1 cc syringe with a 22G needle. The 22G needle (11/2
inches) was replaced with a shorter 23G needle (1 inch).
Approximately 200 .mu.L of the matrix was injected into a PVA
sponge, allowed to sit for a few minutes, and then the animal was
opened for examination. Upon further examination of the sponge, the
matrix appeared to remain localized and had minimal dispersion to
the surrounding areas.
Conclusion:
[0287] Matrix A and Matrix B both gelled extremely quickly and were
very viscous and thick. Even though it is possible to inject the
matrices subcutaneously and into PVA sponges, the easiest method of
delivery was to draw the various components of the matrix
separately (separated by a small pocket of air) into a syringe and
then inject directly through a covered dermal punch using a 23G
needle. In this method, a second smaller gauge needle can assist in
venting excess air from under the wound dressing.
Example 30
[0288] Platelet derived growth factor B (PDGF-B) has been shown to
stimulate fibroblast proliferation and the synthesis of
extracellular matrix. PDGF-B expression was compared by RT-PCR for
cyclodextrin and collagen matrices in the rat PVA sponge model.
TABLE-US-00003 Animal # 200 .mu.L Total Injection Volume/Sponge Grp
# Sponge Injection Contents 6 1: Collagen alone (N = 6 2: 58 kD
Cyclodextrin(CD)-Adamantane Matrix Sponges/Group) alone 3: 2
.times. 10.sup.10 PN PGCB in Collagen 4: 2 .times. 10.sup.10 PN
PGCB in CD-Adamantane Matrix 5: 50 .mu.g pCTK-PD (plasmid DNA
encoding PDGF-B) + L-PEI in CD-Adamantane Matrix 6: 50 .mu.g
pCTK-PD + CD.sub.Imidazole in CD-Adamantane Matrix
Methods
[0289] Six small incisions (0.5 cm) were performed and polyvinyl
alcohol (PVA, M-PACT.RTM.) sponges were subcutaneously implanted on
the ventral side of male Harlan Sprague-Dawley rats. Four days
after implantation, test and control materials were diluted with
vPBS and liquid collagen (Cohesion Technologies.RTM.) or 58 kD
Cyclodextrin (CD)-Adamantane matrix as described above and injected
into PVA sponges. Final collagen formulation was 1.8 mg/mL, pHed
with NaOH, and brought up to volume with vPBS. Final 58 kD
Cyclodextrin (CD)-Adamantane matrix formulation was 100 mg/mL. 200
.mu.L total volume of test reagent was delivered to each sponge.
Animals were sacrificed and sponges were removed 2 days after
injections. Sponges were cut into three pieces and the majority of
the sponge (.about.70-80%) was frozen in liquid nitrogen and stored
at -80.degree. C. for QPCR and QRT-PCR analysis. A small portion of
the sponge was fixed in 4% PFA at 4.degree. C. for 18 hours,
paraffin embedded, and sectioned (5 .mu.m). Sections were stained
using Masson's Trichrome (cells=black, vasculature=red, and
collagen=blue) and Hematoxylin-Eosin methods. Tissue sections were
analyzed microscopically for gross histology by two separate
investigators.
Results:
[0290] Implanted PVA sponges were treated with either collagen,
cyclodextrin matrix (CD), 2.times.10.sup.10 PN of PGCB in Collagen,
2.times.10.sup.10 PN of PGCB in CD, 50 .mu.g of pCTK-PD+L-PEI in
CD, or 50 .mu.g pCTK-PD+CD.sub.Imidazole in CD. Sponges were
removed 48 hours after treatment and assayed for human PDGF-B RNA
and viral DNA by QRT-PCR and QPCR methods, respectively. A portion
of each sponge was also fixed, embedded, sectioned, and stained
using Masson's Trichrome for gross morphological examination.
[0291] QRT-PCR (quantitative RT-PCR) analysis for human PDGF-B RNA
expression showed that sponges treated with 2.times.10.sup.10 PN of
PGCB in CD had the highest average RNA content of
7.0.times.10.sup.8 MEQ of PDGF-B/assayed sponge (FIG. 14). Sponges
treated with 2.times.10.sup.10 PN of PGCB in Collagen had similar
average levels of PDGF-B RNA at 3.2.times.10.sup.8 MEQ of
PDGF-B/assayed sponge. Sponges treated with 50 .mu.g of
pCTK-PD+L-PEI in CD and 50 .mu.g pCTK-PD CD.sub.Imidazole in CD had
PDGF-B RNA levels of 6.8.times.10.sup.5 and 9.1.times.10.sup.4 MEQ
of PDGF-B/assayed sponge, respectively. Finally, sponges treated
with either Collagen or Cyclodextrin showed no detectable levels of
PDGF-B RNA (FIG. 14, Table 3). A Student's t-test analysis showed
that all sponge treatment groups were statistically different
(p.gtoreq.0.05) except for the sponge comparisons between
2.times.10.sup.10 PN of PGCB in collagen and 2.times.10.sup.10 PN
of PGCB in CD (Table 4). GAPDH QRT-PCR control assays were also
performed to monitor RNA quality and total RNA per sample. The
results showed a consistent amount of RNA ranging from
1.9.times.10.sup.7 to 6.9.times.10.sup.7 MEQ of GAPDH/assayed
sponge for all treatment groups (FIG. 15).
TABLE-US-00004 TABLE 3 QRT-PCR Results for hPDGF-B RNA Detection.
Group Count (n) Mean Std. Dev. Std. Err. Collagen 6 BQL .cndot.
.cndot. 58 kD CD 5 BQL .cndot. .cndot. 2 .times. 10.sup.10 PN PGCB
in Collagen 6 3.2E+8 2.0E8 8.2E+7 2 .times. 10.sup.10 PN PGCB in CD
6 7.0E+8 4.1E+8 1.7E+8 50 .mu.g pCTK-PD + L-PEI in CD 6 6.9E+5
2.9E+5 1.2E+5 50 .mu.g pCTK-PD + CD.sub.Imidazole 6 9.1E+4 1.7E+5
7.1E+4 in CD BQL = Below Quantitation Levels (<2 .times.
10.sup.3 MEQ of PDGF-B/Assayed Sponge) MEQ = Molecular Equivalent
is an arbitrarily assigned number based on the dilution factor of a
positive control.
TABLE-US-00005 TABLE 4 Statistical Analysis of hPDGF-B RNA between
Treatment Groups. Unpaired Comparison of Treatment Groups p-Value 2
.times. 10.sup.10 PN PGCB in Collagen vs. 0.0699 2 x 10.sup.10 PN
PGCB in CD 2 .times. 10.sup.10 PN PGCB in Collagen vs. 0.0030 50
.mu.g pCTK-PD + L-PEI in CD 2 .times. 10.sup.10 PN PGCB in Collagen
vs. 0.0030 50 .mu.g pCTK-PD + CD.sub.Imidazole in CD 2 .times.
10.sup.10 PN PGCB in CD vs. 0.0020 50 .mu.g pCTK-PD + L-PEI in CD 2
.times. 10.sup.10 PN PGCB in CD vs. 0.0020 50 .mu.g pCTK-PD +
CD.sub.Imidazole in CD 50 .mu.g pCTK-PD + L-PEI in CD vs. 0.0014 50
.mu.g pCTK-PD + CD.sub.Imidazole in CD Note: Statistical analyses
could not be performed for collagen and 58 kD CD treatment
groups.
[0292] QPCR (quantitative PCR) analysis for human PDGF-B DNA showed
that sponges treated with 50 .mu.g of pCTK-PD+L-PEI in CD and 50
.mu.g pCTK-PD+CD.sub.Imidazole in CD had the highest average levels
at 2.4.times.10.sup.12 and 1.7.times.10.sup.12 MEQ of
PDGF-B/assayed sponge, respectively (FIG. 16, Table 5). Sponges
treated with 2.times.10.sup.10 PN of PGCB in Collagen and
2.times.10.sup.10 PN of PGCB in CD had average human PDGF-B DNA
levels of 4.5.times.10.sup.10 and 4.1.times.10.sup.10 MEQ of
PDGF-B/assayed sponge, respectively. However, sponges treated with
collagen or cyclodextrin also had positive PDGF-B DNA levels at
9.8.times.10.sup.8 and 1.3.times.10.sup.8 MEQ of PDGF-B/assayed
sponge, respectively. A Student's t-test analysis showed that all
groups were statistically different (p 0.05) except for sponge
comparisons between collagen and cyclodextrin and between
2.times.10.sup.10 PN of PGCB in collagen and 2.times.10.sup.10 PN
of PGCB in CD treatment groups (Table 6). Mouse GAPDH
quantification was performed to ensure the quality of DNA and equal
amount of DNA input, and the results ranged from 9.4.times.10.sup.8
to 2.8.times.10.sup.9 MEQ of GAPDH/assayed sponge (FIG. 17).
TABLE-US-00006 TABLE 5 PCR Results for Viral PDGF-B Detection
within Sponges Group Count (n) Mean Std. Dev. Std. Err. Collagen 6
9.8E+8 1.5E+9 6.1E+8 58 kD CD 5 1.3E+8 2.1E+8 9.2E+7 2 .times.
10.sup.10 PN PGCB in Collagen 6 4.5E+10 3.9E+10 1.6E+10 2 .times.
10.sup.10 PN PGCB in CD 6 4.1E+10 2.3E+10 9.2E+9 50 .mu.g pCTK-PD +
L-PEI in CD 6 2.4E+12 1.1E+11 4.6E+10 50 .mu.g pCTK-PD + 6 1.7E+12
4.1E+11 1.7E+11 CD.sub.Imidazole in CD
TABLE-US-00007 TABLE 6 Statistical Analysis of human PDGF-B DNA
Content within Sponges Unpaired Comparison of Treatment Groups
p-Value Collagen vs. 58 kD CD 0.2382 Collagen vs. 2 .times.
10.sup.10 PN PGCB in Collagen 0.0207 Collagen vs. 2 .times.
10.sup.10 PN PGCB in CD 0.0015 Collagen vs. 50 .mu.g pCTK-PD +
L-PEI in CD <0.0001 Collagen vs. 50 .mu.g pCTK-PD +
CD.sub.Imidazole in CD <0.0001 58 kD CD vs. 2 x 10.sup.10 PN
PGCB in Collagen 0.0321 58 kD CD vs. 2 x 10.sup.10 PN PGCB in CD
0.0030 58 kD CD vs. 50 .mu.g pCTK-PD + L-PEI in CD <0.0001 58 kD
CD vs. 50 .mu.g pCTK-PD + CD.sub.Imidazole in CD <0.0001 2
.times. 10.sup.10 PN PGCB in Collagen vs. 0.8197 2 x 10.sup.10 PN
PGCB in CD 2 .times. 10.sup.10 PN PGCB in Collagen vs. <0.0001
50 .mu.g pCTK-PD + L-PEI in CD 2 .times. 10.sup.10 PN PGCB in
Collagen vs. <0.0001 50 .mu.g pCTK-PD + CD.sub.Imidazole in CD 2
.times. 10.sup.10 PN PGCB in CD vs. <0.0001 50 .mu.g pCTK-PD +
L-PEI in CD 2 .times. 10.sup.10 PN PGCB in CD vs. <0.0001 50
.mu.g pCTK-PD + CD.sub.Imidazole in CD 50 .mu.g pCTK-PD + L-PEI in
CD vs. 0.0018 50 .mu.g pCTK-PD + CD.sub.Imidazole in CD
[0293] QPCR results showed significant levels of PDGF-B DNA
detected in collagen and cyclodextrin control sponges. The source
of the contribution for this observation has not been completely
determined; however, hexon QPCR results, which will only detect
hexon sequence contained in adenovirus, indicated that DNA detected
in control sponges derived from PCTK-PD plasmid (FIG. 18). Since
PDGF-B RNA was not detected in control sponges, this suggests that
contamination most likely occurred at or after the time of sponge
harvest.
TABLE-US-00008 TABLE 7 Summary PVA Sponge Morphological Analysis.
Granu- Capsule Immune lation Collagen Vascu- Group Size Response
Tissue Deposition lature Collagen Thin Minimal Minimal Minimal
Minimal 58 kD CD Thin to Light to Minimal Minimal Light to Moderate
Moderate to Light Moderate 2 .times. 10.sup.10 PN PGCB Thick
Moderate Minimal Light Moderate in Collagen to Light to Heavy 2
.times. 10.sup.10 PN PGCB Very Severe Minimal Light Moderate in CD
Thick to Light 50 .mu.g pCTK-PD + Thin to Light to Minimal Minimal
Light to L-PEI in CD Moderate Moderate to Light Moderate 50 .mu.g
pCTK-PD + Thin to Light to Minimal Minimal Light to
CD.sub.Imidazole in CD Moderate Moderate to Light Moderate
[0294] 5 .mu.m, Masson's Trichrome stained paraffin sections were
analyzed by two separate observers. Sponge characteristics were
qualitatively evaluated by the following criteria:
Minimal<Light<Moderate<Heavy<Severe for all categories
except Capsule Size. The following criteria were used for
evaluation of Capsule Size: Thin<Moderate<Thick<Very
Thick.
[0295] Morphological analysis of the sponges was performed to
evaluate host immune responses. Although this study was designed
with gene expression and inflammation as primary endpoints,
granulation tissue formation, collagen deposition, and
neo-vascularization were also analyzed when present. Upon harvest,
sponges treated with 2.times.10.sup.10 PN of PGCB in CD had an
observable and elevated amount of exudate within and surrounding
the sponges (Table 7, FIG. 19). These sponges were also much larger
in size than other groups. Gross histological examination revealed
that sponges treated with 2.times.10.sup.10 PN of PGCB in CD had a
severe immune response characterized by a thick capsule and the
presence of cellular infiltrate. Granulation tissue, collagen, and
neo-vascularization were also observed. Sponges treated with
2.times.10.sup.10 PN of PGCB in collagen also showed granulation
tissue formation, collagen deposition, and neo-vascularization;
however, the immune response was not as severe as sponges treated
with PGCB in CD. Sponges treated with cyclodextrin alone, 50 .mu.g
of pCTK-PD+L-PEI in CD, or 50 .mu.g pCTK-PD+CD.sub.Imidazole in CD
all had similar results with minimal amounts of granulation tissue.
The overall immune response found in these treatments groups was
similar to sponges treated with PGCB in collagen. Finally, sponges
treated with the collagen alone were found to have minimal amounts
of tissue, collagen, or vasculature as well as a minimal immune
response.
[0296] All of the above-cited references and publications are
hereby incorporated by reference.
[0297] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *