U.S. patent application number 16/459039 was filed with the patent office on 2020-01-02 for block copolymers and uses thereof.
The applicant listed for this patent is Ecole Polytechnique Federale de Lausanne (EPFL). Invention is credited to James Brandon DIXON, Jeffrey A. Hubbell, Conlin P. O'Neil, Melody Swartz, Diana Velluto.
Application Number | 20200000936 16/459039 |
Document ID | / |
Family ID | 42243027 |
Filed Date | 2020-01-02 |
View All Diagrams
United States Patent
Application |
20200000936 |
Kind Code |
A1 |
DIXON; James Brandon ; et
al. |
January 2, 2020 |
BLOCK COPOLYMERS AND USES THEREOF
Abstract
An encoding/decoding apparatus and method using a low-density
parity-check code (LDPC code) is disclosed. Basic column group
information, serving as a set of information regarding positions of
rows with weight 1, is extracted from a reference column in each
column group of a predetermined parity-check matrix. Column group
information transforms the positions of rows with weight 1 into
positions whose lengths are within a required parity length. A
parity-check matrix is generated according to the generated column
group information. Data is enclosed or decoded based on the
generated parity-check matrix.
Inventors: |
DIXON; James Brandon;
(Marietta, GA) ; Hubbell; Jeffrey A.;
(Preverenges, CH) ; O'Neil; Conlin P.;
(Chavannes-renens, CH) ; Swartz; Melody;
(Preverenges, CH) ; Velluto; Diana; (London,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ecole Polytechnique Federale de Lausanne (EPFL) |
Lausanne |
|
CH |
|
|
Family ID: |
42243027 |
Appl. No.: |
16/459039 |
Filed: |
July 1, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15017028 |
Feb 5, 2016 |
10335499 |
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16459039 |
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13130892 |
May 24, 2011 |
9271929 |
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PCT/US2009/065693 |
Nov 24, 2009 |
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15017028 |
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61117892 |
Nov 25, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/5146 20130101;
C07K 16/00 20130101; Y02A 50/387 20180101; C08G 83/008 20130101;
C12N 15/115 20130101; C12N 15/113 20130101; C12N 2310/11 20130101;
A61P 43/00 20180101; C08L 2203/02 20130101; A61K 47/10 20130101;
C08G 65/329 20130101; A61K 31/573 20130101; Y10S 977/916 20130101;
A61K 47/34 20130101; Y10S 977/773 20130101; C08L 81/02 20130101;
C12N 2310/141 20130101; C12N 2320/32 20130101; C08L 71/02 20130101;
C08L 2205/05 20130101; C12N 2310/14 20130101; A61K 47/22 20130101;
A61K 48/0041 20130101; C08G 75/08 20130101; Y10S 977/906 20130101;
A61K 31/713 20130101; A61K 9/1075 20130101; Y02A 50/30 20180101;
C08G 65/3344 20130101; A61K 9/1273 20130101; A61K 31/337 20130101;
C12N 2310/16 20130101; C08L 71/02 20130101; C08L 81/00 20130101;
C08L 71/02 20130101; C08L 81/02 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 9/127 20060101 A61K009/127; A61K 9/51 20060101
A61K009/51; C08G 65/329 20060101 C08G065/329; C08G 65/334 20060101
C08G065/334; C08G 75/08 20060101 C08G075/08; C08L 71/02 20060101
C08L071/02; C08G 83/00 20060101 C08G083/00; A61K 9/107 20060101
A61K009/107; A61K 31/337 20060101 A61K031/337; A61K 31/573 20060101
A61K031/573; A61K 31/713 20060101 A61K031/713; A61K 47/10 20060101
A61K047/10; A61K 47/22 20060101 A61K047/22; A61K 47/34 20060101
A61K047/34; C07K 16/00 20060101 C07K016/00; C12N 15/113 20060101
C12N015/113; C12N 15/115 20060101 C12N015/115 |
Claims
1-46. (canceled)
47. A supramolecular structure comprising a (i) a block copolymer
comprising a positively charged block, and (ii) a nucleic acid,
wherein said supramolecular structure has a maximal diameter of 60
nm or less.
48. The supramolecular structure of claim 47, wherein said
supramolecular structure is a molecule or a vesicle.
49. The supramolecular structure of claim 47, wherein said block
copolymer further comprises a hydrophilic block or a hydrophobic
block.
50. The supramolecular structure of claim 49, wherein said block
copolymer further comprises a hydrophilic block and a hydrophobic
block.
51. The supramolecular structure of claim 47, wherein said maximal
diameter is 40 nm or less.
52. The supramolecular structure of claim 47, wherein said block
copolymer comprises PEG-PPS-PEI.
53. The supramolecular structure of claim 47, wherein said nucleic
acid comprises a single-stranded oligonucleotide, a short
interfering RNA, an aptamer, or plasmid DNA.
54. A method of transfecting a cell with a nucleic acid comprising
contacting said cell with the supramolecular structure of claim
47.
55. A block copolymer comprising PEG-PPS-PEI.
56. The block copolymer of claim 55, wherein the PPS block and the
PEI block are attached via a bond that is labile in an
endosome.
57. The block copolymer of claim 56, wherein said bond comprises a
disulfide bond, vinyl ether, orthoester, acyl hydrazone, or a
--N--PO.sub.3-- group.
58. The block copolymer of claim 55, further comprising a nucleic
acid complexed to said PEI.
59. A pharmaceutical composition comprising the block copolymer of
claim 55 and a pharmaceutically acceptable diluent.
60-78. (canceled)
79. The supramolecular structure of claim 47, wherein said block
copolymer further comprises a PPS block.
80. The supramolecular structure of claim 47, wherein said
positively charged block is PEI.
81. The supramolecular structure of claim 47, wherein said
supramolecular structure further comprises PEG-PPS.
Description
BACKGROUND OF THE INVENTION
[0001] The invention relates to the field of polymer chemistry.
[0002] In the field of pharmaceutical agent delivery there exists
an extensive interest in amphiphilic block copolymers that can
self-assemble in aqueous environments into stable supramolecular
structures. A variety of supramolecular structures can be generated
such as micellar and vesicular assemblies, both of which can be
important for pharmacological applications. Extensive
investigations have been conducted in poly(ethylene glycol)
(PEG)-containing block copolymers, such as copolymers with
poly(propylene glycol) and poly(ethylethylene). Such block
copolymers are generally prepared via ionic polymerization under
strictly anhydrous conditions, making it difficult to obtain
asymmetric block copolymers or to introduce biological
molecules.
[0003] Accordingly, there is a need for new block copolymers.
SUMMARY OF THE INVENTION
[0004] Block copolymers containing charged blocks or chemical
moieties sensitive to oxidation or hydrolysis have been developed.
We describe the use of such block copolymers in supramolecular
structures, e.g., micelles or vesicles, and pharmaceutical
compositions and in methods of preparing the supramolecular
structures and pharmaceutical compositions. The invention is
particularly useful for the delivery of pharmaceutical agents,
e.g., nucleic acids, to cells.
[0005] Accordingly, in one aspect, the invention features a block
copolymer including a hydrophilic block and a hydrophobic block
wherein at least one of the blocks is interrupted with a
hydrolysable or oxidation-sensitive chemical moiety. Desirably, the
block copolymer is capable of self-assembling into a supramolecular
structure, such as a micelle or vesicle. In certain embodiments,
the hydrolysable chemical moiety is an ester, amide, thioester,
anhydride, or ketal. In another embodiment, the hydrophilic block
is poly(ethylene glycol) (PEG), and the hydrophobic block is
poly(propylene sulfide) (PPS).
[0006] In another aspect, the invention features a supramolecular
structure, e.g., a micelle or a vesicle, containing (i) a block
copolymer of a hydrophilic block and a hydrophobic block and (ii)
an excipient. Desirably, the excipient is an amphipathic molecule.
In preferred embodiments, the excipient is
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or PEG of molecular weight
between 400 and 800. The supramolecular structure may further
contain a hydrophobic or hydrophilic pharmaceutical agent. The
pharmaceutical agent is, for example, a peptide, a nucleic acid, an
antibiotic, or a chemotherapeutic drug. In certain embodiments, the
pharmaceutical agent is selected from dexamethasone, paclitaxel,
cyclosporine A, sirolimus, everolimus, tacrolimus, amphotericin B,
or adriamycin. In other embodiments, the pharmaceutical agent is a
polypeptide, such as a protein or an antibody or an antigen-binding
fragment of an antibody. In preferred embodiments, the
pharmaceutical agent is encapsulated in the supramolecular
structure at an efficiency greater than 5%, 25%, 50%, 75%, 90%, or
even 95%. The supramolecular structure may be included in a
pharmaceutical composition with a pharmaceutically acceptable
diluent.
[0007] In a related aspect, the invention features a method of
encapsulating a pharmaceutical agent in a supramolecular structure,
e.g., a micelle or a vesicle. The method includes contacting the
pharmaceutical agent with an excipient and a block copolymer
containing a hydrophilic block and a hydrophobic block, applying
heat to homogenize the mixture of the pharmaceutical agent,
excipient, and block copolymer, and diluting the homogenized
mixture in an aqueous solution. In certain embodiments, the
excipient is DBU or PEG of molecular weight between 400 and
800.
[0008] The invention further features a method of making a vesicle
including forming micelles from a block copolymer containing a
hydrophilic block and a hydrophobic block, wherein a vesicle formed
by the block copolymer is thermodynamically more stable than a
micelle formed by the block copolymer, and heating the micelles to
form the vesicle. In certain embodiments, the vesicles formed by
the method are 70 to 800 nm in diameter. In other embodiments, the
micelles are suspended in a solution containing a pharmaceutical
agent, and the pharmaceutical agent is encapsulated in the vesicles
upon heating the solution. In another embodiment, the hydrophilic
block of the block copolymer contains PEG, and the hydrophobic
block of the block copolymer contains PPS. The invention also
provides a vesicle prepared by this method.
[0009] In another aspect, the invention features a dry formulation
containing micelles of a block copolymer having a hydrophilic block
and a hydrophobic block, wherein the water content of the
formulation is less than 5%, e.g., less than 2%. The dry
formulation may further contain a pharmaceutical agent. In certain
embodiments, the hydrophilic block of the block copolymer contains
PEG, and the hydrophobic block of the block copolymer contains
PPS.
[0010] The invention further features a supramolecular structure
containing a block copolymer containing a positively charged block
and a nucleic acid, wherein the supramolecular structure has a
maximal diameter of less than 60 nm. The block copolymer may
further include a hydrophilic block, e.g., PEG, and a hydrophobic
block, e.g., PPS. In one embodiment, the block copolymer contains
PPS, PEG, and polyethylene imine (PEI). In another embodiment, the
nucleic acid is a single-stranded oligonucleotide, a short
interfering RNA, an aptamer, or plasmid DNA. Desirably, the maximal
diameter of the supramolecular structure is less than 40 nm. In a
related aspect, the invention features a method of transfecting a
cell with a nucleic acid including contacting the cell with a
supramolecular structure containing a block copolymer containing a
positively charged block and the nucleic acid.
[0011] In another aspect, the invention features a block copolymer
containing PPS, PEG, and PEI. In certain embodiments, the PPS block
and the PEI block are attached via a bond that is labile in an
endosome, e.g., a disulfide bond, vinyl ether, orthoester, acyl
hydrazone, or a --N--PO.sub.3-- group. In another embodiment, the
block copolymer includes a nucleic acid that is bound to the PEI
block. The block copolymer may be included in a pharmaceutical
composition containing a pharmaceutical agent and a
pharmaceutically acceptable diluent.
[0012] The invention further features a micelle between 10 and 60
nm in diameter containing two block copolymers, the first of which
contains a hydrophilic block and a hydrophobic block, the second of
which contains a hydrophilic block, a hydrophobic block, and a
positively charged block. In one particular embodiment, the first
block copolymer contains PEG and PPS, and the second block
copolymer contains PEG, PPS, and PEI. In other embodiments, the
micelle has a maximal diameter between 20 and 50 nm. The micelle
may be included in a pharmaceutical composition containing a
pharmaceutical agent and a pharmaceutically acceptable diluent.
[0013] In another aspect, the invention features a supramolecular
structure containing block copolymers containing a hydrophilic
block, e.g., PEG, and a hydrophobic block, e.g., PPS, wherein 5-25%
of the repeating units in the block copolymer have a charged
chemical moiety disposed at the outer surface of the supramolecular
structure. In certain embodiments, the charged chemical moiety is
carboxylic acid, sulfate, or sulfone. The supramolecular structure
may be included in a pharmaceutical composition containing a
pharmaceutical agent and a pharmaceutically acceptable diluent.
[0014] In any of the embodiments of the invention, the hydrophilic
block may contain poly(ethylene glycol), poly(ethylene
oxide)-co-poly(propylene oxide) di- or multiblock copolymers,
poly(ethylene oxide), poly(vinyl alcohol), poly(ethylene-co-vinyl
alcohol), poly(N-vinyl pyrrolidone), poly(acrylic acid),
poly(ethyloxazoline), poly(alkylacrylates), poly(acrylamide),
poly(N-alkylacrylamides), polypeptide, polysaccharide,
poly(N,N-dialkylacrylamides), hyaluronic acid, or poly
(N-acryloylmorpholine). The hydrophobic block may contain
poly(propylene sulfide), poly(propylene glycol), esterified
poly(acrylic acid), esterified poly(glutamic acid), esterified
poly(aspartic acid), or a polypeptide. In certain embodiments, the
charged block is PEI, a polypeptide, poly(amidoamine), poly(sodium
1-(N-acryloylpiperazin-1-yl)diazen-1-ium-1,2-diolate), poly(sodium
1-(N-acryloylhomopiperazin-1-yl)diazen-1-ium-1,2-diolate) or
poly(sodium
1-(N-acryloyl-2,5-dimethylpiperazin-1-yl)diazen-1-ium-1,2-diolate).
[0015] By a "block copolymer" is meant a compound containing at
least two blocks that each contain two or more repeating units of a
chemical moiety. The chemical moiety of one block is distinct from
a chemical moiety present in another block of the block copolymer.
For example, a block copolymer may contain a poly(ethyleneglycol)
(PEG) block and a poly(propylene sulfide) (PPS) block. Typically,
the number of repeating units in a block is between 4 and 250. An
exemplary hydrophilic block contains up to 250 repeating units, and
an exemplary hydrophobic block contains up to 100 repeating units.
Repeating units of a block may be interrupted or modified by a
group that confers a desirable functionality, e.g., the ability to
be hydrolyzed.
[0016] By a block that is "interrupted with" a hydrolysable
chemical moiety is meant a block of the same repeating unit that
includes within it the hydrolysable chemical moiety so that, when
the chemical moiety is hydrolyzed, the number of repeating units in
the block decreases. Upon hydrolysis, the block may decrease in
size by at least, e.g., 2, 4, 10, 15, 20, 30, 50, 75, 100, or 115
repeating units. Hydrolysable moieties include, e.g., esters,
amides, thioesters, anhydrides, and ketals. An exemplary block that
is interrupted with a hydrolysable chemical moiety is PEG.sub.46
esterified to PEG.sub.4.
[0017] By a "hydrolysable chemical moiety" is meant a chemical
moiety that is cleaved in aqueous solution with a half life of 1
year or less at pH 7.4 and 37.degree. C. Preferably, the half life
of the moiety at pH 7.4 and 37.degree. C. is one month or less.
[0018] By "nucleic acid" is meant any nucleobase oligomer. For the
purposes of this specification, modified oligonucleotides that do
not have a phosphorus atom in their internucleoside backbone are
also considered to be nucleobase oligomers. Non-limiting examples
of nucleic acids are antisense oligonucleotides, small interfering
RNAs (siRNAs), aptamers, and plasmid DNA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1A. Conversion of poly(ethylene glycol) monomethyl
ether (PEG MME) to PEG thioacetate (PEG TAc). PEG MME was dried
using a Dean Stark trap, and 2.5 eq of thionyl bromide was added.
The reaction was then refluxed for 4 h at 140.degree. C. The
toluene was evaporated, and the polymer was dissolved in
dichloromethane (DCM) and precipitated in cold diethyl ether. The
final conversion was accomplished by dissolving the PEG-Br in
dimethylformamide (DMF) with 5 eq K.sub.2CO.sub.3 and 5 eq
thiolacetic acid, and stirring overnight. The product was filtered,
and the DMF was evaporated and dissolved in DCM. The PEG-TAc was
purified over activated charcoal and precipitated in cold diethyl
ether.
[0020] FIG. 1B. PEG-TAc was converted to
PEG-ester-EG.sub.4-acrylate by reacting PEG-TAc with PEG(200)
diacrylate in excess. PEG-TAc was dissolved in tetrahydrofuran
(THF) and degassed thoroughly. Next, the solution was added to 50
eq of PEG diacrylate in THF with 1 eq of triethylamine as base. The
reaction was stirred overnight and purified by precipitation twice
in cold diethyl ether.
[0021] FIG. 1C. The final polymer was synthesized by forming the
PPS block initiated from benzenethiol, and using the PEG-acrylate
monomer as an end-capping reagent. This was accomplished by
dissolving DBU base into THF and degassing. The benzenethiol was
added under argon flow, and propylene sulfide monomer was added
through a gastight septum. After 1 hr, the
PEG-ester-EG.sub.4-ester-acrylate was added, dissolved in THF, and
degassed (0.5 eq). The reaction was allowed to stir overnight. The
final product was purified by dissolving the dried mixture in
toluene and filtering. After evaporating the toluene, the polymer
was dissolved in DCM and precipitated in cold diethyl ether.
[0022] FIG. 2A. Hydrolysis of PEG-PPS at 25.degree. C. over time at
various pH values. The polymer was prepared by solvent dispersion
from THF. The THF was removed under vacuum prior to the start of
the study. Degradation was quantified via gel permeation
chromatography, and the peaks for the free PPS and PEG blocks were
quantified. At elevated pH (8.4) degradation occurred more rapidly,
whereas at pH 5.4 no degredation was observed.
[0023] FIG. 2B. Degradation of the same polymer preparation as that
shown in FIG. 2A was quantified at 37.degree. C. Degradation was
greatest at high pH owing to rapid hydrolysis of the ester bonds
under these conditions. Unexpectedly, after 20 days degradation was
also observed at pH 5.4.
[0024] FIG. 3. Formulation excipients DBU and PEG600 were blended
with the block copolymer PEG-PPS at 95.degree. C. with
stirring.
[0025] FIG. 4. Dynamic scanning calorimetry of PEG-PPS blended with
DBU-HCl. The polymer and salt melted together creating a
homogeneous blend of the two materials.
[0026] FIG. 5. Characterization of PEG-PPS processing methods using
optical density. Samples were analyzed directly after preparation
at the same concentration (5 mg/mL). Legend represents solvent
dispersion (SD), thin film extrusion (TFE), and direct hydration
(DH).
[0027] FIG. 6. Gel permeation chromatograph of a formulation
incubated at 95.degree. C. for three hours versus control. The data
were acquired using a refractive index detector. For clarity, the
control is shown on the bottom, and the 180 min sample is offset by
5%. Both chromatograms were normalized.
[0028] FIG. 7. Degradation study of PEG-PPS at 95.degree. C. over
time. The bar graph represents the average number average molecular
weight (Mn) of the injected samples. From left to right are
unheated polymer control (Control); 15 min control heated without
salt (15 C); 15 and 30 min heated with salt (15, 30); 60 min heated
without salt (60 C); 60, 120, and 180 min heated with salt (60,
120, 180). Student's t-test comparing the unheated control with the
180 min at 95.degree. C. revealed the difference was not
statistically significant. Mp represents peak molecular weight, and
Mw represents weight average molecular weight. The PDI using gel
permeation chromatography (GPC) is calculated by Mw/Mn. The Mn=Sum
(NiMi)/Sum (Ni), and Mw=Sum (NiMi.sup.2)/Sum (NiMi).
[0029] FIG. 8. Degradation study analyzed using proton NMR.
Unheated, heated, and heated with salt preparations using the
polymer EG.sub.46-PS.sub.12 were compared. The polymer and salt
with polymer samples were heated at 95.degree. C. for three hours
prior to extraction in THF and precipitation in diethyl ether. The
purified fractions were measured via proton-NMR in chloroform-D
containing 0.1% TMS as an internal standard.
[0030] FIG. 9. Dynamic scanning calorimetry of the constituents
separately and mixed together. The mixture was prepared by mixing
EG.sub.46-PS.sub.12 50/50 wt/wt with DBU-HCl and heating at
95.degree. C. for 60 min. Samples were mixed thoroughly and
measured on the DSC. For comparison, the polymer and DBU-HCl salt
were also measured separately. The salt and polymer dissolved into
a molten state upon heating.
[0031] FIG. 10. Imaging vesicles formed via direct hydration using
cryogenic TEM. The formed lamellar phases were vitrified on a holey
carbon grid prior to imaging. Aggregates were extruded prior to
analysis using cryo-TEM.
[0032] FIG. 11A. Encapsulation efficiency of DBU-HCl formulation
compared to thin film hydration with extrusion. Ovalbumin was
reduced using TCEP and purified using Sephadex G50 and
freeze-dried. 26 mg of the reduced ovalbumin was dissolved in 500
.mu.l of distilled water and added to a preparation of PEG-PPS
blended with DBU-HCl at 95.degree. C. for 15 min. Above, 10 .mu.l
of this solution was added, mixed, and slowly diluted with
distilled water. Results were calculated from a standard curve made
with the same reduced ovalbumin sample. Thin film hydration with
extrusion results were taken from Jousma et al Int. J. Pharm. 35
(1987) 263-274.
[0033] FIG. 11B. Encapsulation efficiency of dexamethasone and
paclitaxel in PEG formulations. Dexamethasone or paclitaxel were
incubated with the indicated block copolymers and the indicated PEG
or control formulations according to the methods of the
invention.
[0034] FIG. 11C. Bovine serum albumin and ovalbumin were prepared
at 50 mg/mL in distilled water. The formulations were prepared as
follows. Ten milligrams of PEG-PPS was heated with 10 mg of PEG500
dimethyl ether and heated at 95.degree. C. for 15 minutes. The melt
was mixed and allowed to cool to room temperature. After, a volume
of protein solution (5 .mu.L or 10 .mu.L) was added and slowly
diluted with distilled water up to 1 mL volume with mixing. To
calculate the encapsulation efficiencies, standard curves were
generated for both BSA (using fluorescamine) or ovalbumin (using
FITC-ovalbumin). The dispersed vesicle solutions were centrifuged
for 10 minutes at 10,000 g to sediment the vesicles, and we
measured the free protein in the supernatant.
[0035] FIG. 12. Optical density change over time with heating of
the polymer at 95.degree. C. PEG-PPS (EG.sub.46-PS.sub.64) micelles
were prepared from solvent dispersion in water using THF. After
removing the THF under vacuum for 1 h, the suspension at 10 mg/mL
was placed 1 mL each into 1.5 mL eppendorf tubes. To this aliquot,
200 .mu.L of THF was added. Initial samples for time=0 optical
density (OD) and dynamic light scattering (DLS) were removed, and
the tubes were placed into a pre-heated incubator at 95.degree. C.
Samples were drawn at specific time points, 100 .mu.l per sample
and aliquoted into a 96 well plate on ice. After the final sample
was removed, 50 .mu.l of each sample was added to a new 96 well
plate, and the optical density was measured at 400 nm using a plate
reader in absorbance mode.
[0036] FIG. 13. The samples for optical density (50 .mu.l) were
dispersed into 550 IA of double distilled water and measured using
DLS. Both the OD and DLS data clearly display a trend towards
larger particle size, and changing morphology.
[0037] FIG. 14. Cryogenic Transmission Electron Microscopy of the
thermal transition of PEG-PPS micelles into vesicles. The micelle
sample (left) displays small aggregates of PEG-PPS in good
agreement with the DLS results from FIG. 2. The 30 min sample
(right) shows the vesicles (polymersomes) created during
heating.
[0038] FIG. 15. The aggregation of PEG-PPS micelles during heating
captured using negative staining TEM. Here the 3 min sample from
the heating experiment was added to a 400 mesh carbon coated copper
grid which had been prepared by glow discharge. The sample was then
blotted off after 60 sec, and stained 30 sec with 2% uranyl
acetate. The image above was taken at 75,000.times.. The 2-D
surface vesicles form slowly into short worm like micelles.
[0039] FIG. 16A. Particle size distribution by dynamic light
scattering of PEG44-PPS20 cyclosporine A-loaded polymer
micelles.
[0040] FIG. 16B. Particle size distribution of the micelles of FIG.
16A after drying and rehydration.
[0041] FIG. 17A. Stability of PEG.sub.44-PPS.sub.20 after exposure
to gastric pH, as measured by gel permeation chromatography.
[0042] FIG. 17B. Stability of PEG.sub.44-PPS.sub.20 after exposure
to gastric pH, as measured by dynamic light scattering.
[0043] FIG. 18. A synthetic route to PEG-PPS-PEI. A disulfide link
between the PPS block and the PEI block allows destabilization of
the polymer after endocytosis.
[0044] FIG. 19. PEG-PPS-PEI was demonstrated to condense plasmid
DNA into nanoparticles of size distribution for transfection.
[0045] FIG. 20. PEG-PPS-PEI was demonstrated to transfect cells
very efficiently, even difficult-to-transfect cells such as 3T3
fibroblasts, shown here. Other cells were transfected at even
higher transfection efficiency, including 239T cells at 96% with
PEG2kDa-PPS.sub.27-PEI.sub.96.
[0046] FIG. 21. Cytotoxicity with PEG-PPS-PEI was much lower than
that with linear PEI of the same molecular weight at the same PEI
concentration.
[0047] FIG. 22. The size of the resulting gene pharmaceutical agent
complexes could be controlled by a number of means, including the
ratio of the polymers used to construct mixed micelles.
[0048] FIG. 23. PEG-PPS-based polymers were able to condense
oligonucleotide pharmaceutical agents here with an siRNA sequence,
for example to prevent gel migration. This demonstrates the
versatility of these polymers with oligonucleotides sequences as
well as plasmid DNA.
[0049] FIG. 24. PEG-PPS-based polymers were able to induce high
transfection efficiency with oligonucleotides pharmaceutical agents
here with siRNA knocking down expression of lamin A/C in HeLa
cells.
[0050] FIG. 25. Gene complexes formed with a 10:1 ratio of
PEG-PPS-PEI:PEG-PPS efficiently transfect cells. PEG-PPS was used
to reduce the size of PEG-PPS-PEI micelles resulting in smaller
complexes with gene-based pharmaceutical agents. Here, cells were
incubated with 30 nm complexes containing the green fluorescent
protein (GFP) gene sequence.
DETAILED DESCRIPTION OF THE INVENTION
[0051] The present invention provides various block copolymers
having at least two blocks, one hydrophilic and one hydrophobic.
Block copolymers may further include additional blocks, be
interrupted with hydrolysable chemical moieties, or be otherwise
modified. Each block of the copolymer is important for
self-assembly and biological function. The size of each block may
be determined independently of the other blocks, e.g., to tailor
the function of each block. Each block may be synthesized and bound
to the other blocks using methods known in the art, e.g., as
described in US 2003/0059906 and WO 2007/008300, which are hereby
incorporated by reference.
Block Copolymers
[0052] Hydrophilic Blocks.
[0053] The hydrophilic block, e.g., PEG, may be utilized to (i)
prevent non-specific nucleic acid/positively charged polymer
complex interactions with serum proteins, cells, and tissues in the
body, which allows for specific interactions to be designed via
incorporated ligands, and (ii) increase the solubility of the
complexes in aqueous milieu.
[0054] Polymers or molecules that are soluble or swell in an
aqueous environment will prevent protein absorption while still
enhancing the solubility of the particles. For example,
carbohydrate polymers such as hyaluronic acid (HA) may swell to
about 1000 times their volume and are used in nature to prevent
protein absorption. Other carbohydrate polymer or molecule
candidates are found in nature. Exemplary hydrophilic blocks
include poly(ethylene glycol), poly(ethylene
oxide)-co-poly(propylene oxide) di- or multiblock copolymers,
poly(ethylene oxide), poly(vinyl alcohol), poly(ethylene-co-vinyl
alcohol), poly(N-vinyl pyrrolidone), poly(acrylic acid),
poly(ethyloxazoline), poly(alkylacrylates), poly(acrylamide),
poly(N-alkylacrylamides), polypeptides, polysaccharides,
poly(N-acryloylmorpholine), or poly(N,N-dialkylacrylamides),
potentially bearing polar, ionic, or ionizable groups in the
aliphatic chains.
[0055] Hydrophilic blocks having molecular weights between 500 and
10,000 Da are practical and convenient, although higher molecular
weight hydrophilic blocks may be employed. For hydrophilic blocks,
a number of repeating units between about 10 and about 250 is
preferable because of the ease with which these materials may be
eliminated from the body by renal filtration. A PEG hydrophilic
block is preferably between 750 and 5500 Da, e.g., between 2 and 5
kDa (e.g., a block containing 115 units). Hydrophilic blocks with a
larger number of repeating units may also be cleared by the kidney
but at slower rates than hydrophilic polymers of lower number of
repeating units, which may place limits on doses that can be
applied.
[0056] Hydrophobic Blocks.
[0057] The hydrophobic block may include any polymer that is
hydrophobic in context. A preferred hydrophobic block is PPS.
Poly(propylene glycol) (PPG), a structural homolog of PPS with an
oxygen atom instead of a sulfur atom in the backbone, may also be
employed. Larger PPG chains may be required relative to the useful
length of PPS chains. In general, polymers that have low melting or
glass transition temperatures are most desirable because this
characteristic is most conducive to effective micellization.
[0058] Other polymers that are otherwise hydrophilic but are
derivatized with hydrophobic functionalities on their side chains
may be used in the hydrophobic block. Examples include esterified
poly(acrylic acid), esterified poly(glutamic acid) or poly(aspartic
acid), and hydrophobic peptides or peptoids (e.g., N-substituted
glycines). Hydrophobic blocks having molecular weights between 300
and 5000 Da are practical and convenient, although higher molecular
weight hydrophobic blocks may also be employed. For hydrophobic
blocks, the number of repeating units is, for example, between
about 4 and about 240, preferably between 4 and 70. For example, a
polypropylene sulfide hydrophobic block can vary from 150 to 16,000
Da, e.g., from 200 to 15,000 Da, depending on the initial
hydrophilic block (e.g., PEG) used and the desired application. A
higher number of repeating units may also be utilized; however,
self-assembled structures from such polymers may be far from their
equilibrium morphology.
[0059] Additional Blocks.
[0060] In certain embodiments, the copolymers of the invention have
only two polymeric blocks, although other chemical moieties, e.g.,
hydrolysable, charged, or biologically active moieties, may be
present. In other embodiments, the copolymers may include three or
more polymeric blocks. Such additional blocks may be employed, for
example, (i) to bind nucleic acids or other charged molecules via
electrostatic interactions, (ii) to help a self-assembled structure
enter the cell, and/or (iii) to execute another biological
function. When employed to bind charged molecules, e.g., a nucleic
acid, the additional block is preferably sized to produce
reversible binding with the charged molecule. For example, at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 95% of the
charged molecule may dissociate from the block under appropriate
cellular conditions, e.g., in the cytosol or within the nucleus. In
addition, short blocks may be employed to reduce or eliminate any
toxicity.
[0061] The use of a positively charged block in a block copolymer
is particularly advantageous for the formation of small complexes
with, e.g., nucleic acids. Exemplary positively charged blocks are
a polypeptide (e.g., polylysine), poly(ethyleneimine) (PEI),
poly(amidoamine), poly(sodium
1-(N-acryloylpiperazin-1-yl)diazen-1-ium-1,2-diolate), poly(sodium
1-(N-acryloylhomopiperazin-1-yl)diazen-1-ium-1,2-diolate) or
poly(sodium
1-(N-acryloyl-2,5-dimethylpiperazin-1-yl)diazen-1-ium-1,2-diolate).
Blocks having a positive charge between 5 and 20 at physiological
pH are practical and convenient, although blocks with a larger
number of positive charges may also be employed. Molecular weights
of a positively charged block between 500 and 10000 Da are
preferred. For polymers such as PEI, a number of repeating units
between about 10 and about 250 is preferred. The longer a charged
block becomes, in general the higher the corresponding
cytotoxicity.
[0062] Additional blocks may also be peptides. Peptides have been
extensively utilized in the field of pharmaceutical agent delivery
and other medical applications owing to the multitude of chemical
and biological functionalities they can embody. Examples of charged
peptides include the biologically active TAT peptide and
oligo(lysine) (e.g., Lys.sub.9) peptides. Both peptides are charged
and can bind to nucleic acids and other negatively charged
molecules. Additional examples of charged blocks include
oligo(histidine), oligo(arginine) (e.g., Arg.sub.9), and copolymers
of Lys, Arg, and His.
[0063] Furthermore, poly(amidoamine) (PAMAM) dendrimers have been
used to complex nucleic acids and may be included in a charged
block. PAMAM has been shown to efficiently escape the endosome,
allowing release of the complexed contents in the cytosol.
[0064] Polymer Modifications.
[0065] Polymeric blocks in the copolymers of the invention may also
be modified. Such modifications include adding charged groups
(e.g., carboxylic acids groups, sulfates, sulfones, and amines),
hydrophilic groups (e.g., hydroxyl), hydrophobic groups (e.g.,
phenyl or methyl), hydrolysable groups (e.g., ester, amide,
thioester, anhydride, or ketal moieties), or groups sensitive to
oxidation. In particular, portions of a polymeric block that are
exposed to aqueous solution in supramolecular structures may be
modified to include one or more charged groups to allow for more
efficient uptake in vivo, as described herein. Blocks copolymers
may also be end capped with various groups as described herein and
known in the art.
[0066] One particular example of a block copolymer modified by a
hydrolysable chemical moiety is illustrated in Example 1. For such
a hydrolysable block copolymer, the PEG diacrylate preferably has a
molecular weight from 200 to 600 Da. Other hydrolysable blocks that
may be used include lactide or caprolactone groups.
[0067] Degradation In Vivo.
[0068] In order to avoid irreversible accumulation in the targeted
organs, the self-assembled carriers may demonstrate some form of
degradation in vivo. Polysulfides are known to readily undergo
oxidation to polysulfoxides and even to polysulfones, e.g., by the
action of mild oxidizing agents, such as hydrogen peroxide. Under
biological conditions, this oxidation can be performed
extracellularly, e.g., by macrophages, or intracellularly after
cellular uptake into an endosomal or lysosomal compartment. A
similar kind of reaction is used for oxidizing thioether-terminated
PEGs (used as emulsifiers in pulp and paper processing) in order to
break wastewater foams (see, e.g., U.S. Pat. No. 4,618,400).
[0069] The conversion of the polysulfides to polysulfoxides can
solubilize the block copolymers in water, allowing elimination
through excretion (Napoli A et al., Nature Materials, 2004. 3(3):
p. 183-189.). The conversion can trigger the instability of
self-assembled aggregates, e.g., the conversion of gels to micelles
or soluble polymers, the conversion of vesicles to micelles or
soluble polymers, or the conversion of micelles into micelles of
different size and shape or to soluble polymers. Destabilizing the
aggregate can also trigger the release of any encapsulated
pharmaceutical agents, e.g., a nucleic acid. The mechanisms of
clearance of soluble polymers are relatively well understood. The
most important such mechanism is clearance via renal filtration,
the effective molecular weight cutoff of which is approximately
30,000. Particles of size less than approximately 100 nm can be
cleared from the bloodstream in the liver. Lymphatic uptake also
may play a role in clearance.
[0070] Copolymers of the invention may also be synthesized such
that they respond to the changing environment of the endosome. For
example, a disulfide bond may be introduced into the copolymer so
that, as the environment of the endosome becomes reducing, the bond
is cleaved, thereby destabilizing the complex within the endosome.
An N--PO.sub.3 bond, which responds to low pH, e.g., as in an
endosome, may also be introduced into the structure. Additional
bonds that are sensitive to intracellular degradation, such as
vinyl ether, orthoester, and acyl hydrazone, may also be
employed.
[0071] Copolymers of the invention may also be synthesized so that
a hydrophobic or hydrophilic block is interrupted with a
hydrolysable chemical moiety, e.g., an ester or amide. Hydrolysis
of the moiety leads to a change in the relative amount of
hydrophobic or hydrophilic block, which in turn can lead to
destabilization of a supramolecular complex by changing its
favorable self-assembled morphology. In one embodiment, the
half-life of the hydrolysable bond is between 1 hour and 1 year in
an aqueous solution at pH 7.4 and 37.degree. C. Desirably, the
half-life is between 1 day and 9 months, more preferably between 2
days and 6 months, and most preferably between 4 days and 3 weeks.
In certain embodiments, a thioether or secondary amine is present
at the alpha or beta position relative to the hydrolysable
bond.
[0072] Self Assembly.
[0073] Amphiphilic block copolymers have long been used as
surfactants and dispersants in a wide variety of applications; the
formation of organized structures in a solvent that is selective
for one of the blocks is the basis of this behavior.
[0074] Well-defined self-assembled structures, such as spherical or
cylindrical micelles, lamellae, or vesicles (Booth et al.,
Macromol. Chem., Rapid Commun. 2000, 21, 501-527; Won, Science
1999, 283, 960-963; Discher et al., Science 1999, 284, 1143-1146;
and Eisenberg et al., Macromolecules 1998, 31, 3509) have been
observed in poly(oxyalkylene) block copolymers. The concentration
of the polymer solution and the temperature greatly influence the
kind of aggregates that can be formed: changing, e.g., from liquid
spherical micellar phases to cubic phases of spherical micelles and
finally to hexagonal phases of cylindrical micelles upon an
increase in temperature (Mortensen, Progr. Coll. Polym. Sci. 1993,
93, 72-75). The phase diagram and accessible structures of the
amphiphilic block copolymers exhibit a dependence on the block
length and number, i.e., basically, on the hydrophilic/lipophilic
balance.
[0075] Block copolymers of PEG with poly(ethylethylene) have shown
a propensity to form worm-like micelles at a ratio 55/45 between
hydrophilic and hydrophobic repeating units (total MW=4900), and to
form lamellar structures at a ratio 40:37 (total MW=3900).
[0076] This invention provides materials capable of generating a
wide variety of structures; for example, a material containing long
sequences of hydrophilic groups is able to form micelles, while a
high hydrophobic content facilitates the formation of lamellar
gels, and, under suitable conditions, vesicles.
[0077] The formation of vesicles can also be achieved by adding to
water a solution or colloidal suspension of the copolymer in an
organic solvent and subsequently removing the organic solvent.
[0078] Combinations of two or more block copolymers of the
invention may also be employed to form supramolecular structures.
Typically, PEG-PPS with Mw fractions (fPEG) of approximately
0.99-0.7 form micelles, whereas Mw fractions (fPEG) of
approximately 0.30 to 0.25 form vesicles.
[0079] Thermal Transitions of Block Copolymer Assemblies.
[0080] The invention also features a method of making micelles from
vesicles. In these embodiments, micelles are formed from block
copolymers that are thermodynamically disposed to form vesicles.
Upon heating the micelles, they spontaneously form vesicles. When
the micelles are in aqueous suspension with a dissolved compound,
e.g., pharmaceutical agent, the process of forming vesicles results
in encapsulation of the pharmaceutical agent in the interior of the
vesicles.
[0081] For example, polymer micelles are formed using a polymer
composition with an fPEG that thermodynamically favors vesicle
formation. In suspension, the micelles are metastable and can be
highly concentrated. Application of heat to the metastable micelles
induces spontaneous formation of vesicles, which are very small and
homogeneous in size distribution. Pharmaceutical agent incorporated
in the micelle suspension is loaded within the vesicles during
their formation. Micelles can be made from copolymers that would
otherwise form vesicles, i.e., under nonequilibrium conditions, by
means that include rapidly dissolving/dispersing the polymer
without heating, for example from powdered or lyophilizied polymer.
This rapid dissolution kinetically traps the polymer in a micellar
form. Addition of mobility provides the opportunity for the
nonequilibium form to approach equilibrium, here by forming the
more favored vesicle morphology.
[0082] We have shown that when heat is applied to a mixture
containing block copolymers and a salt, the mixture melts into a
homogeneous composition. Small molecule pharmaceutical agents also
melt in the formulation during heating. This process leads to
encapsulation of the pharmaceutical agents in the supramolecular
structures, e.g., micelles or vesicles, formed by the block
copolymers.
[0083] Drying and Rehydrating Block Copolymer Assemblies.
[0084] Supramolecular structures of the invention, e.g., vesicles,
may be dehydrated and made into dry formulations. Such dry
formulations may be rehydrated in vivo upon administration or in
vitro prior to administration or other use. Preferably, a dry
formulation includes less than 5% water by weight. The limit of
water content that is acceptable in a dry formulation may be
determined by measurement of storage lifetime by standard methods.
Dry formulations may be stable for greater than two weeks, one
month, six months, or one year. A pharmaceutical agent encapsulated
within the supramolecular structures, e.g., by a method described
herein, may be reconstituted to an active form upon rehydration.
The dried compositions may be rehydrated in any aqueous solution,
e.g., in a pharmaceutically acceptable diluent. In conventional
pharmaceutical processing by a number of drying methods, water
contents substantially less than 5% may be easily reached.
[0085] Pharmaceutical Compositions.
[0086] In suitable conditions for the generation of micelles, e.g.,
by treatment with heat, the block copolymers of the invention can
be used for the encapsulation of pharmaceutical agents such as
peptides, nucleic acids, antibiotics (e.g., ampicillin or
tetracycline), chemotherapeutics (e.g., doxorubicin), or other
small molecule pharmaceutical agents. When lamellar phases are to
be formed, vesicles can be generated from the lamellar structure
bending; in this way, water-dissolved pharmaceutical agents can be
entrapped in the internal cavity of the vesicle. Pharmaceutical
compositions may also employ excipients that increase the
encapsulation efficiency of one or more block copolymers for
pharmaceutical agents that are hydrophilic, hydrophobic, or
amphiphilic. The excipients may increase the compatibility of the
pharmaceutical agent with one or more blocks in the block
copolymer, e.g., by reducing repulsive forces or increasing
attractive forces between the pharmaceutical agent and one or more
blocks of the block copolymer.
[0087] Suitable exemplary excipients are
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and polyethylene glycol
(e.g., PEG 600). PEG having a molecular weight between 400 and 800
Da is effective as an excipient. Polyethylene glycols outside of
this range have not been effective. Other excipients that may be
used are PPS-PEG copolymers and hydrobromide or hydrochloride salts
of common organic bases such as triethanolamine, triethylamine, or
pyridine. The addition of an excipient to a mixture containing a
block copolymer and a pharmaceutical agent may increase the
efficiency of encapsulation of the pharmaceutical agent by greater
than 1.5-fold, 3-fold, 5-fold, 10-fold, or 50-fold. Examples of
improved pharmaceutical agent encapsulation in the presence of
excipients are provided herein.
[0088] The copolymers of the invention may be dispersed in a
pharmaceutically acceptable diluent. In addition, self-assembled
structures of the invention may include pharmaceutical agents or
biologically active compounds. In various embodiments, the
pharmaceutical composition includes about 1 ng to about 20 mg of
pharmaceutical agent, e.g., a nucleic acid or a hydrophobic
compound (e.g., paclitaxel or dexamethasone). In some embodiments,
the composition contains about 10 ng to about 10 mg, about 0.1 mg
to about 500 mg, about 1 mg to about 350 mg, about 25 mg to about
250 mg, or about 100 mg of pharmaceutical agents. Those of skill in
the art of clinical pharmacology can readily arrive at dosing
amounts using routine experimentation.
[0089] Suitable diluents include, but are not limited to, saline,
buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. The composition can be adapted for the mode
of administration and can be in the form of, for example, a pill,
tablet, capsule, spray, powder, or liquid. In some embodiments, the
pharmaceutical composition contains one or more pharmaceutically
acceptable additives suitable for the selected route and mode of
administration. These compositions may be administered by, without
limitation, any parenteral route including intravenous,
intra-arterial, intramuscular, subcutaneous, intradermal,
intraperitoneal, intrathecal, as well as topically, orally, and by
mucosal routes of delivery such as intranasal, inhalation, rectal,
vaginal, buccal, and sublingual. In some embodiments, the
pharmaceutical compositions of the invention are prepared for
administration to vertebrate (e.g., mammalian) subjects in the form
of liquids, including sterile, non-pyrogenic liquids for injection,
emulsions, powders, aerosols, tablets, capsules, enteric coated
tablets, or suppositories. Conventional procedures and ingredients
for the selection and preparation of suitable formulations are
described, for example, in Remington's Pharmaceutical Sciences
(2003-20th edition) and in The United States Pharmacopeia: The
National Formulary (USP 24 NF19), published in 1999.
[0090] Pharmaceutical agents may be hydrophilic, hydrophobic, or
amphoteric. The type of supramolecular structure employed to
encapsulate an agent will depend on the solubility characteristics
of the agent and the copolymer. Typically, hydrophilic agents will
be encapsulated in the interior of vesicles, and hydrophobic agents
will be encapsulated in the interior of micelles. Agents that may
be employed with copolymers of the invention include but are not
limited to natural and synthetic compounds, e.g., a nucleic acid,
having the following therapeutic activities: anti-arthritic,
anti-arrhythmic, anti-bacterial, anticholinergic, anticancer,
anticoagulant, antidiuretic, antidote, antiepileptic, antifungal,
anti-inflammatory, antimetabolic, antimigraine, antineoplastic,
antiparasitic, antipyretic, antiseizure, antisera, antispasmodic,
analgesic, anesthetic, beta-blocking, biological response
modifying, bone metabolism regulating, cardiovascular, diuretic,
enzymatic, fertility enhancing, growth-promoting, hemostatic,
hormonal, hormonal suppressing, hypercalcemic alleviating,
hypocalcemic alleviating, hypoglycemic alleviating, hyperglycemic
alleviating, immunosuppressive, immunoenhancing, muscle relaxing,
neurotransmitting, parasympathomimetic, sympathominetric plasma
extending, plasma expanding, psychotropic, thrombolytic,
chemotherapeutic, and vasodilating.
[0091] Nucleic Acids.
[0092] In certain embodiments of the invention, the block copolymer
composition contains a nucleic acid. The nucleic acid may associate
with one or more blocks of a copolymer and may be incorporated into
supramolecular structures containing block copolymers, e.g.,
micelles or vesicles. In preferred embodiments, the nucleic acid is
present in a therapeutically effective amount in a pharmaceutical
composition containing one or more block copolymers. Antisense
oligonucleotides, small interfering RNAs (siRNAs), aptamers, and
plasmid DNA are examples.
[0093] Oligonucleotides containing modified backbones or
non-natural internucleoside linkages may be employed. Nucleobase
oligomers that have modified oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity, wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Various salts, mixed salts and free acid forms are also
included. Representative United States patents that teach the
preparation of the above phosphorus-containing linkages include,
but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863;
4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;
5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;
5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;
5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of
which is herein incorporated by reference.
[0094] Nucleobase oligomers having modified oligonucleotide
backbones that do not include a phosphorus atom therein have
backbones that are formed by short chain alkyl or cycloalkyl
internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl
internucleoside linkages, or one or more short chain heteroatomic
or heterocyclic internucleoside linkages. These include those
having morpholino linkages (formed in part from the sugar portion
of a nucleoside); siloxane backbones; sulfide, sulfoxide and
sulfone backbones; formacetyl and thioformacetyl backbones;
methylene formacetyl and thioformacetyl backbones; alkene
containing backbones; sulfamate backbones; methyleneimino and
methylenehydrazino backbones; sulfonate and sulfonamide backbones;
amide backbones; and others having mixed N, O, S and CH.sub.2
components. Representative United States patents that teach the
preparation of the above oligonucleotides include, but are not
limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and
5,677,439, each of which is herein incorporated by reference.
[0095] In other nucleobase oligomers, both the sugar and the
internucleoside linkage, i.e., the backbone, are replaced with
novel groups. One such nucleobase oligomer, is referred to as a
Peptide Nucleic Acid (PNA). In PNA compounds, the sugar-backbone of
an oligonucleotide is replaced with an amide containing backbone,
in particular an aminoethylglycine backbone. The nucleobases are
retained and are bound directly or indirectly to aza nitrogen atoms
of the amide portion of the backbone. Methods for making and using
these nucleobase oligomers are described, for example, in "Peptide
Nucleic Acids: Protocols and Applications" Ed. P. E. Nielsen,
Horizon Press, Norfolk, United Kingdom, 1999. Representative United
States patents that teach the preparation of PNAs include, but are
not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262,
each of which is herein incorporated by reference. Further teaching
of PNA compounds can be found in Nielsen et al., Science, 1991,
254, 1497-1500.
[0096] In particular embodiments of the invention, the nucleobase
oligomers have phosphorothioate backbones and nucleosides with
heteroatom backbones, and in particular
--CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- (known as a methylene
(methylimino) or MMI backbone),
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2--, and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2--. In other embodiments, the
oligonucleotides have morpholino backbone structures described in
U.S. Pat. No. 5,034,506.
[0097] Nucleobase oligomers may also contain one or more
substituted sugar moieties. Nucleobase oligomers comprise one of
the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-,
S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein
the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted
C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and
alkynyl. Particularly preferred are
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCHO].sub.2, where n and m are
from 1 to about 10. Other preferred nucleobase oligomers include
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or
O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3,
SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of a nucleobase oligomer, a group for
improving the pharmacodynamic properties of a nucleobase oligomer,
or other substituents having similar properties. Preferred
modifications are 2'-O-methyl and 2'-methoxyethoxy
(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE). Another desirable modification is
2'-dimethylaminooxyethoxy (i.e.,
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2), also known as 2'-DMAOE. Other
modifications include, 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (2'-F).
Similar modifications may also be made at other positions on an
oligonucleotide or other nucleobase oligomer, particularly the 3'
position of the sugar of the 3' terminal nucleotide or in 2'-5'
linked oligonucleotides and the 5' position of the 5' terminal
nucleotide. Nucleobase oligomers may also have sugar mimetics such
as cyclobutyl moieties in place of the pentofuranosyl sugar.
Representative United States patents that teach the preparation of
such modified sugar structures include, but are not limited to,
U.S. Pat. Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044,
5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811,
5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873,
5,646,265, 5,658,873, 5,670,633, and 5,700,920, each of which is
herein incorporated by reference in its entirety.
[0098] Nucleobase oligomers may also include nucleobase
modifications or substitutions. As used herein, "unmodified" or
"natural" nucleobases include the purine bases adenine (A) and
guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and
uracil (U). Modified nucleobases include other synthetic and
natural nucleobases, such as 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil,
cytosine, 5-propynyl uracil, 6-azo uracil, thymine, 5-uracil
(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,
8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other
5-substituted uracils and cytosines, 7-methylguanine,
7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine,
7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Further
nucleobases include those disclosed in U.S. Pat. No. 3,687,808,
those disclosed in The Concise Encyclopedia of Polymer Science and
Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley &
Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie,
International Edition, 1991, 30, 613, and those disclosed by
Sanghvi, Y. S., Chapter 15, Antisense Research and Applications,
pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993.
Certain of these nucleobases are particularly useful for increasing
the binding affinity of an antisense or (partially) complementary
oligonucleotide of the invention to a target nucleic acid. These
include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6,
and O-6 substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and
Lebleu, B., eds., Antisense Research and Applications, CRC Press,
Boca Raton, 1993, pp. 276-278) and are desirable base
substitutions, even more particularly when combined with
2'-O-methoxyethyl or 2'-O-methyl sugar modifications.
Representative United States patents that teach the preparation of
certain of the above-noted modified nucleobases as well as other
modified nucleobases include U.S. Pat. Nos. 4,845,205, 5,130,302,
5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255,
5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,594,121,
5,596,091, 5,614,617, 5,681,941, and 5,750,692, each of which is
herein incorporated by reference.
[0099] Another modification of a nucleobase oligomer of the
invention involves chemically linking to the nucleobase oligomer
one or more moieties or conjugates that enhance the activity,
cellular distribution, or cellular uptake of the oligonucleotide.
Such moieties include but are not limited to lipid moieties such as
a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,
86:6553-6556, 1989), cholic acid (Manoharan et al., Bioorg. Med.
Chem. Let, 4:1053-1060, 1994), a thioether, e.g.,
hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci.,
660:306-309, 1992 Manoharan et al., Bioorg. Med. Chem. Let.,
3:2765-2770, 1993), a thiocholesterol (Oberhauser et al., Nucl.
Acids Res., 20:533-538, 1992), an aliphatic chain, e.g.,
dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,
10:1111-1118, 1991; Kabanov et al., FEBS Lett., 259:327-330, 1990;
Svinarchuk et al., Biochimie, 75:49-54, 1993), a phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 36:3651-3654, 1995; Shea et al., Nucl. Acids
Res., 18:3777-3783, 1990), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 14:969-973,
1995), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 36:3651-3654, 1995), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1264:229-237, 1995), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 277:923-937, 1996). Representative United
States patents that teach the preparation of such nucleobase
oligomer conjugates include U.S. Pat. Nos. 4,587,044, 4,605,735,
4,667,025, 4,762,779, 4,789,737, 4,824,941, 4,828,979, 4,835,263,
4,876,335, 4,904,582, 4,948,882, 4,958,013, 5,082,830, 5,109,124,
5,112,963, 5,118,802, 5,138,045, 5,214,136, 5,218,105, 5,245,022,
5,254,469, 5,258,506, 5,262,536, 5,272,250, 5,292,873, 5,317,098,
5,371,241, 5,391,723, 5,414,077, 5,416,203, 5,451,463, 5,486,603,
5,510,475, 5,512,439, 5,512,667, 5,514,785, 5,525,465, 5,541,313,
5,545,730, 5,552,538, 5,565,552, 5,567,810, 5,574,142, 5,578,717,
5,578,718, 5,580,731, 5,585,481, 5,587,371, 5,591,584, 5,595,726,
5,597,696, 5,599,923, 5,599,928, 5,608,046, and 5,688,941, each of
which is herein incorporated by reference.
[0100] The present invention also includes nucleobase oligomers
that are chimeric compounds. "Chimeric" nucleobase oligomers are
nucleobase oligomers, particularly oligonucleotides, that contain
two or more chemically distinct regions, each made up of at least
one monomer unit, i.e., a nucleotide in the case of an
oligonucleotide. A chimeric nucleobase oligomer may contain one or
more regions to confer increased resistance to nuclease
degradation, increased cellular uptake, and/or increased binding
affinity for the target nucleic acid.
[0101] Chimeric nucleobase oligomers may be formed as composite
structures of two or more nucleobase oligomers as described above.
Such nucleobase oligomers, when oligonucleotides, have also been
referred to in the art as hybrids or gapmers. Representative United
States patents that teach the preparation of such hybrid structures
include U.S. Pat. Nos. 5,013,830, 5,149,797, 5,220,007, 5,256,775,
5,366,878, 5,403,711, 5,491,133, 5,565,350, 5,623,065, 5,652,355,
5,652,356, and 5,700,922, each of which is herein incorporated by
reference in its entirety.
[0102] Locked nucleic acids (LNAs) are nucleobase oligomers that
can be employed in the present invention. LNAs contain a 2'O, 4'-C
methylene bridge that restrict the flexibility of the ribofuranose
ring of the nucleotide analog and locks it into the rigid bicyclic
N-type conformation. LNAs show improved resistance to certain exo-
and endonucleases and activate RNAse H, and can be incorporated
into almost any nucleobase oligomer. Moreover, LNA-containing
nucleobase oligomers can be prepared using standard phosphoramidite
synthesis protocols. Additional details regarding LNAs can be found
in PCT publication No. WO 99/14226 and U.S. Patent Application
Publication No. US 2002/0094555 A1, each of which is hereby
incorporated by reference.
[0103] Arabinonucleic acids (ANAs) can also be employed in the
present invention. ANAs are nucleobase oligomers based on
D-arabinose sugars instead of the natural D-2'-deoxyribose sugars.
Underivatized ANA analogs have similar binding affinity for RNA as
do phosphorothioates. When the arabinose sugar is derivatized with
fluorine (2' F-ANA), an enhancement in binding affinity results,
and selective hydrolysis of bound RNA occurs efficiently in the
resulting ANA/RNA and F-ANA/RNA duplexes. These analogs can be made
stable in cellular media by a derivatization at their termini with
simple L sugars. The use of ANAs in therapy is discussed, for
example, in Damha et al., Nucleosides Nucleotides & Nucleic
Acids 20: 429-440, 2001.
[0104] Polypeptides
[0105] Polypeptides may be employed in embodiments of the
invention. The amino acids of the polypeptide may be natural,
non-natural, or a mixture thereof. The polypeptide may be produced
by recombinant genetic technology or chemical synthesis using
methods known in the art. Examples of polypeptides are a
single-chain peptide fragment (e.g., a polypeptide of 5-20 amino
acids joined by conventional peptide bonds), a naturally occurring
protein, and an antibody or antigen-binding fragment thereof.
Methods for Internalization and Therapeutic Methods
[0106] The copolymers may utilize biological pathways for both
delivery and therapeutic action. In one embodiment, a block
copolymer that self-assembles in aqueous environments into
nanoscale micelles or vesicles may be employed for the delivery of
pharmaceutical agents, such as siRNA or other nucleic acids.
Moreover, a block copolymer of the invention can exploit changing
intracellular environments, e.g., the reductive environment of the
endosome, for efficient delivery of the pharmaceutical agent and a
biological pathway for therapeutic action, e.g., the activation of
the RNAi pathway for gene silencing. The development of
biologically responsive materials to induce release of a
therapeutic agent within the early endosome and destabilize it
holds promise for the development of delivery systems that can
overcome limitations of current delivery systems.
[0107] Colloidal particles such as nanospheres, liposomes, and
micelles have been studied extensively for site-specific
pharmaceutical agent delivery. Unless the reticuloendothelial
system (RES) is a target, the particles must escape capture by the
RES of the liver and the filtration activity of the lungs.
Prolonged survival of colloidal systems in the blood has been
obtained by the use of PEG-containing amphiphiles (Lasic et al.,
Ed. Stealth Liposomes; CRC Press: Boca Raton, Fla., 1995). By
virtue of marked reduction of opsonization by plasma proteins, the
macrophages clearance of PEG-based liposomes has been drastically
reduced (Torchilin et al., Biochim Biophys Acta 1994, 1195,
11-20).
[0108] A variety of internalization agents, i.e., compounds or
species that enhance the internalization of the copolymers of the
invention, such as antibodies, growth factors, cytokines, adhesion
factors, oligonucleotide sequences and nuclear localization
sequences has served to enhance the delivery capabilities of
PEG-coated liposomes, and it has been demonstrated that the maximal
activity is shown by ligands tethered to the distal end of PEG
chains (Blume et al., Biochim. Biophys. Acta 1993, 1149, 180-184;
Zalipsky et al., Bioconjugate Chem. 1995, 6, 705-708; Zalipsky, J.
Controlled Release 1996, 39, 153-161; Gabizon, Bioconjugate Chem.
1999, 10, 289-298). This approach can be employed with the polymers
of the invention. Some internalization agents can lead to very
efficient cellular uptake, such as the use of growth factors, for
example, fibroblast growth factor to effect cellular uptake of DNA
formulations. Other internalization agents can lead to very
efficient intracellular trafficking, such as nuclear localization
sequences, and these may be used in the present invention.
Additional internalization agents include transferrin, folate, a
lectin, growth factor, an RGD peptide, and a mannose-containing
glycopeptide.
[0109] The copolymers of the present invention are useful for any
application in the controlled release, e.g., in the cytosol or
nucleus, of a pharmaceutical agent, e.g., nucleic acid. The release
of the contents, e.g., the nucleic acid, of the self-assembled
aggregate, such as a micelle or vesicle, may be achieved through
sensitivity of the aggregate to the environment, such as triggering
a release based on the lowering of pH, increase in the extent of
oxidation, and increase in the concentration of proteases during
the process of intracellular trafficking from the endosome to the
lysosome. Excipients may also be incorporated along with the
pharmaceutical agent to help it in reaching its final biological
target, such as incorporation of agents that assist in
destabilizing or permeabilizing biological membranes, such as the
endosomal or lysosomal membranes, to enhance transport of the
nucleic acid into the cytoplasm or ultimately into the nucleus.
[0110] The polymers may also be employed to deliver mixtures of
pharmaceutical agents, e.g., two or more different nucleic acids or
a nucleic acid and a pharmaceutical agent, such as an
antibiotic.
[0111] Gene-Based Pharmaceutical Agents.
[0112] The block copolymers of the invention may be used to deliver
nucleic acids for the up- or down-regulation of genes. Examples of
nucleic acids include siRNA, ODN (antisense), and pDNA, including
pDNA encoding therapeutic proteins.
[0113] The internalization of DNA/positively charged polymer
complexes can be enhanced by the covalent attachment of ligands,
such as transferrin, folate, lectins, epidermal growth factor
(EGF), RGD peptides, and mannose-containing species such as
mannose-containing glycopeptides to bind to the mannose receptor
(Kircheis, R., et al., Gene Ther, 1997. 4(5): p. 409-18;
Gottschalk, S., et al., Gene Ther, 1994. 1(3): p. 185-91; Erbacher,
P., et al., Hum Gene Ther, 1996. 7(6): p. 721-9; Blessing, T., et
al., Bioconjug Chem, 2001. 12(4): p. 529-37; Harbottle, R. P., et
al., Hum Gene Ther, 1998. 9(7): p. 1037-47; East L, Isacke C M.
Biochimica et Biophysica Acta, 2002 1572: p. 364-386.). The ligand
functions to direct the DNA complex to the cell surface by
specifically binding to a receptor, and mediating endocytosis.
Fusogenic peptides and other functional groups have been attached
to enhance endosomal escape of the DNA complex (Carlisle, R. C.,
Curr Opin Mol Ther, 2002. 4(4): p. 306-12; Bell, P. C., et al., J
Am Chem Soc, 2003. 125(6): p. 1551-8).
[0114] There exists a parallel need for delivery of other
gene-based pharmaceutical agents, including ODN and pDNA. Here, the
agent must also be delivered to the cell and its cytoplasm, and
eventually to the nucleus. With ODNs, the need is even more acute,
since they function by stoichiometric competition. With plasmids,
the challenge is even higher, since the large size of the plasmid
greatly inhibits its passage through the membranes of the cell,
e.g., the plasma membrane and the endosomal membranes.
[0115] Methods for Delivering Pharmaceutical Agents.
[0116] The invention provides methods for delivering a
pharmaceutical agent, e.g., a nucleic acid, to a cell or an animal,
e.g., a mammal, or plant by contacting the cell or administering to
the animal a pharmaceutical composition of the invention. The
delivery may reduce or inhibit the expression of a target gene in a
cell (e.g., a eukaryotic cell, a plant cell, an animal cell, an
invertebrate cell, a vertebrate cell, such as a mammalian or human
cell, or a pathogen cell) or may treat the animal or cell by any
mechanism specific to the pharmaceutical agent contained in the
pharmaceutical composition. The method may be used to treat
infection by a pathogen or to treat a nonpathogenic disease, e.g.,
cancer, postsurgical adhesions, scar formation, or restenosis after
removal of arterial block (e.g., via balloon angioplasty or
stenting). Typically, a nucleic acid internalized in the cell
specifically reduces or inhibits the expression of a target gene,
e.g., one associated with the disease (e.g., all or a region of a
gene, a gene promoter, or a portion of a gene and its promoter).
Exemplary pathogens include bacteria, protozoan, yeast, and fungi.
In some embodiments, the nucleic acid or other molecule inhibits
the expression of an endogenous gene in a vertebrate cell or a
pathogen cell (e.g., a bacterial, a yeast cell, or a fungal cell),
or inhibits the expression of a pathogen gene in a cell infected
with the pathogen (e.g., a plant or animal cell). The nucleic acid
or other molecule may also reduce or inhibit the expression of an
endogenous gene, e.g., in a cancer cell or in cells that produce
undesirable effects, e.g., restenosis, scar formation, and
postsurgical adhesions. In some embodiments, the target gene is a
gene associated with cancer, such as an oncogene, or a gene
encoding a protein associated with a disease, such as a mutant
protein, a dominant negative protein, or an overexpressed
protein.
[0117] Alternatively, the nucleic acid or other pharmaceutical
agent delivered may increase the expression of a gene. For example,
the copolymer of the invention may be used to deliver a plasmid or
other gene vector to the nucleus where one or more genes contained
on the plasmid may be expressed. Such a system may be employed to
enable expression of gene products that are not expressed
endogenously, to increase expression of endogenous gene products,
and to replace gene products that are mutated or otherwise
non-functional. In some cases, local expression of these genes is
mostly desired, as with, without limitation, vascular endothelial
growth factor, transforming growth factor beta, platelet derived
growth factor, fibroblast growth factor, insulin-like growth
factor, bone morphogenetic protein, growth and differentiation
factor, nerve growth factor, neurotrophin, cytokines, and
transcription factors, such as hif-1alpha, runx2, and sox-9.
[0118] The nucleic acid or other pharmaceutical agent may reduce,
inhibit, or increase expression of a target gene by at least 20,
40, 60, 80, 90, 95, or 100%. The methods of the invention may also
be used to simultaneously reduce or inhibit the expression of one
or more target genes while increasing the expression of one or more
other target genes.
[0119] Treatment of Disease.
[0120] The compositions of the inventions may be used to treat a
disease, e.g., cancer, in an animal, e.g., a human. Exemplary
cancers that can be treated using the methods described herein
include prostate cancers, breast cancers, ovarian cancers,
pancreatic cancers, gastric cancers, bladder cancers, salivary
gland carcinomas, gastrointestinal cancers, lung cancers, colon
cancers, melanomas, brain tumors, leukemias, lymphomas, and
carcinomas. Benign tumors may also be treated or prevented using
the methods of the present invention. Other cancers and cancer
related genes that may be targeted are known in the art.
[0121] Exemplary endogenous proteins that may be associated with
disease include ANA (anti-nuclear antibody) found in SLE (systemic
lupus erythematosis), abnormal immunoglobulins including IgG and
IgA, Bence Jones protein associated with various multiple myelomas,
and abnormal amyloid proteins in various amyloidoses including
hereditary amyloidosis and Alzheimer's disease. In Huntington's
Disease, a genetic abnormality in the HD (huntingtin) gene results
in an expanded tract of repeated glutamine residues. In addition to
this mutant gene, HD patients have a copy of chromosome 4 which has
a normal sized CAG repeat. Thus, methods of the invention can be
used to silence the abnormal gene, but not the normal gene.
[0122] Exemplary diseases that may be treated with the methods
include infection by pathogens, such as a virus, a bacterium, a
yeast, a fungus, a protozoan, or a parasite. The nucleic acid may
be delivered to the pathogen or to a cell infected with the
pathogen. The pathogen may be an intracellular or extracellular
pathogen. The target nucleic acid sequence is, for example, a gene
of the pathogen that is necessary for replication and/or
pathogenesis, or a gene encoding a cellular receptor necessary for
a cell to be infected with the pathogen. Such methods may be
employed prior to, concurrent with, or following the administration
of the in-dwelling device to a patient to prevent infections.
In-dwelling devices include, but are not limited to, surgical
implants, prosthetic devices, and catheters, i.e., devices that are
introduced to the body of an individual and remain in position for
an extended time. Such devices include, for example, artificial
joints, heart valves, pacemakers, vascular grafts, vascular
catheters, cerebrospinal fluid shunts, urinary catheters, and
continuous ambulatory peritoneal dialysis (CAPD) catheters.
[0123] A bacterial infection may be due to one or more of the
following bacteria: Chlamydophila pneumoniae, C. psittaci, C.
abortus, Chlamydia trachomatis, Simkania negevensis, Parachlamydia
acanthamoebae, Pseudomonas aeruginosa, P. alcaligenes, P.
chlororaphis, P. fluorescens, P. luteola, P. mendocina, P.
monteilii, P. oryzihabitans, P. pertocinogena, P. pseudalcaligenes,
P. putida, P. stutzeri, Burkholderia cepacia, Aeromonas
hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella
typhimurium, S. typhi, S. paratyphi, S. enteritidis, Shigella
dysenteriae, S. flexneri, S. sonnei, Enterobacter cloacae, E.
aerogenes, Klebsiella pneumoniae, K oxytoca, Serratia marcescens,
Francisella tularensis, Morganella morganii, Proteus mirabilis,
Proteus vulgaris, Providencia alcalifaciens, P. rettgeri, P.
stuartii, Acinetobacter calcoaceticus, A. haemolyticus, Yersinia
enterocolitica, Y. pestis, Y. pseudotuberculosis, E intermedia,
Bordetella pertussis, B. parapertussis, B. bronchiseptica,
Haemophilus influenzae, H. parainfluenzae, H. haemolyticus, H.
parahaemolyticus, H. ducreyi, Pasteurella multocida, P.
haemolytica, Branhamella catarrhalis, Helicobacter pylori,
Campylobacter fetus, C. jejuni, C. coli, Borrelia burgdorferi, V.
cholerae, V. parahaemolyticus, Legionella pneumophila, Listeria
monocytogenes, Neisseria gonorrhea, N. meningitidis, Kingella
dentrificans, K. kingae, K. oxalis, Moraxella catarrhalis, M.
atlantae, M. lacunata, M. nonliquefaciens, M. osloensis, M.
phenylpyruvica, Gardnerella vaginalis, Bacteroides fragilis,
Bacteroides distasonis, Bacteroides 3452A homology group,
Bacteroides vulgatus, B. ovalus, B. thetaiotaomicron, B. uniformis,
B. eggerthii, B. splanchnicus, Clostridium difficile, Mycobacterium
tuberculosis, M. avium, M. intracellulare, M. leprae, C.
diphtheriae, C. ulcerans, C. accolens, C. afermentans, C.
amycolatum, C. argentorense, C. auris, C. bovis, C. confusum, C.
coyleae, C. durum, C. falsenii, C. glucuronolyticum, C. imitans, C.
jeikeium, C. kutscheri, C. kroppenstedtii, C. lipophilum, C.
macginleyi, C. matruchoti, C. mucifaciens, C. pilosum, C.
propinquum, C. renale, C. riegelii, C. sanguinis, C. singulare, C.
striatum, C. sundsvallense, C. thomssenii, C. urealyticum, C.
xerosis, Streptococcus pneumoniae, S. agalactiae, S. pyogenes,
Enterococcus avium, E. casseliflavus, E. cecorum, E. dispar, E.
durans, E. faecalis, E. faecium, E. flavescens, E. gallinarum, E.
hirae, E. malodoratus, E. mundtii, E. pseudoavium, E. raffinosus,
E. solitarius, Staphylococcus aureus, S. epidermidis, S.
saprophyticus, S. intermedius, S. hyicus, S. haemolyticus, S.
hominis, and/or S. saccharolyticus.
[0124] A viral infection may be due to one or more of the following
viruses: Hepatitis B, Hepatitis C, picornarirus, polio, HIV,
coxsacchie, herpes simplex virus Type I and 2, St. Louis
encephalitis, Epstein-Barr, myxoviruses, JC, coxsakieviruses B,
togaviruses, measles, paramyxoviruses, echoviruses, bunyaviruses,
cytomegaloviruses, varicella-zoster, mumps, equine encephalitis,
lymphocytic choriomeningitis, rhabodoviruses including rabies,
simian virus 40, human polyoma virus, parvoviruses, papilloma
viruses, primate adenoviruses, coronaviruses, retroviruses, Dengue,
yellow fever, Japanese encephalitis virus, BK, Retrovirus,
Herpesvirus, Hepadenovirus, Poxvirus, Parvovirus, Papillornavirus,
and Papovavirus. The target viral nucleic acid sequence is, for
example, necessary for replication and/or pathogenesis of the virus
in an infected cell. Such viral target genes are necessary for the
propagation of the virus and include, e.g., the HIV gag, env, and
pol genes, the HPV6 LI and E2 genes, the HPV I I LI and E2 genes,
the HPV 16 E6 and E7 genes, the BPV 18 E6 and E7 genes, the HBV
surface antigens, the HBV core antigen, HBV reverse transcriptase,
the HSV gD gene, the HSVvp 16 gene, the HSV gC, gH, gL and gB
genes, the HSV ICPO, ICP4 and ICP6 genes, Varicella zoster gB, gC
and gH genes, and the BCR-abl chromosomal sequences, and non-coding
viral polynucleotide sequences which provide regulatory functions
necessary for transfer of the infection from cell to cell, e.g.,
the HIV LTR, and other viral promoter sequences, such as HSV vp 16
promoter, HSV-ICPO promoter, HSV-ICP4, ICP6 and gD promoters, the
HBV surface antigen promoter, the HBV pre-genomic promoter, among
others.
[0125] The copolymers of the invention can be used to treat
subjects already infected with a virus, such as HIV, in order to
shut down or inhibit a viral gene function essential to virus
replication and/or pathogenesis, such as HIV gag. Alternatively,
this method can be employed to inhibit the functions of viruses,
which exist in mammals as latent viruses, e.g., Varicella zoster
virus, the causative agent of shingles. Similarly, diseases such as
atherosclerosis, ulcers, chronic fatigue syndrome, and autoimmune
disorders, recurrences of HSV-I and HSV-2, HPV persistent
infection, e.g., genital warts, and chronic BBV infection among
others, which have been shown to be caused, at least in part, by
viruses, bacteria, or another pathogen, can be treated according to
this method by targeting certain viral polynucleotide sequences
essential to viral replication and/or pathogenesis in the mammalian
subject.
[0126] Preferably, the nucleic acid or other molecule is
administered in an amount sufficient to treat the disease or
condition, e.g., to prevent, stabilize, or inhibit the growth of
the pathogen or to kill the pathogen or to increase or decrease the
expression of an endogenous gene whose under- or overexpression
results in a disease.
EXAMPLES
Example 1. Block Copolymer Functionality can be Designed to Enable
Hydrolytic and Oxidative Sensitivity
[0127] PPS serves as a useful hydrophobic block in micelle and
vesicle formation, as it can be oxidized to form the hydrophilic
sulfone and sulfoxide products, after which it can presumably be
excreted from the body by renal filtration. It is possible to build
into the block copolymeric amphiphile other degradable segments,
for example hydrolyzable chemical moieties. Such moieties may be
placed within the hydrophilic domain of the block copolymer, so
that moieties' accessibility to water does not become a limiting
factor in hydrolysis rate and thus release rate. One route by which
to accomplish this is shown in FIGS. 1A-1C.
[0128] The effect of the synthesis shown in FIGS. 1A-1C is to
insert a hydrolytic link within the PEG chain, from which a PEG-PPS
block copolymer is made, i.e., the PEG block is interrupted with a
hydrolysable chemical moiety. As hydrolysis ensues, the fraction of
the block copolymer comprised by the hydrophile (fPEG) changes
dramatically, thus changing the nature of the assembly that forms,
releasing the contents of a vesicle, for example, formed from the
block copolymer. This instability is illustrated in FIGS. 2A and
2B.
Example 2. Excipients can be Used to Enhance Encapsulation
Efficiency of Hydrophobic Pharmaceutical Agents in Micelles
[0129] Encapsulation of hydrophobic molecules within polymer
micelles during micellization can be a difficult challenge. We have
demonstrated a novel method for formation of polymer micelles that
involves the use of an excipient in the encapsulation process that
is both soluble in the polymer and soluble in water, considering
the organic base DBU and the polymer PEG as examples, as shown in
FIG. 3. FIG. 4, with Tables 1 and 2, demonstrate that the
excipients can be used to obtain very high encapsulation efficiency
of agents for which encapsulation is difficult, using paclitaxel
and dexamethasone as examples.
TABLE-US-00001 TABLE 1 Dynamic Light Scattering of pharmaceutical
agent loaded PEG-PPS micelles. 100 .mu.l of sample was dispersed
into 900 .mu.l of distilled water before performing the DLS
measurement. PDI represents the polydispersity index. PDI for DLS
measurements is defined in ISO 13321:1996. The PDI is a
dimensionless parameter defined as the broadness of the size
distribution, which is defined as: PDI = .mu. 2 ( .GAMMA. ) 2
##EQU00001## Pharmaceutical Z- Excipient agent Avg(nm) Volume(nm)
PDI PEG600 Paclitaxel 66.36 24.54 0.292 DBU- Paclitaxel 52.02 22.55
0.610 HCl PEG600 Dexamethasone 48.59 19.26 0.440 DBU- Dexamethasone
29.79 20.67 0.295 HCl
Where .mu..sub.2 is the second cumulant and .GAMMA. is the decay
rate. In this case, the decay rate is representative of the
Gaussian distribution of decay rates observed in the sample.
TABLE-US-00002 TABLE 2 Encapsulation efficiency (EE) of small
molecule pharmaceutical agents using excipient formulations.
Pharmaceutical Excipient agent EE None Dexamethasone 5.5% PEG600
Paclitaxel 75.9% DBU-HCl Paclitaxel 40.6% PEG600 Dexamethasone
37.7% DBU-HCl Dexamethasone 46.1%
[0130] Samples used in Table 2 were prepared as follows. 10 mg of
PEG-PPS was added to a 1.5 mL centrifuge tube with either 90 mg of
PEG600 or DBU-HCl, and 2 mg of either paclitaxel or dexamethasone.
This was heated at 95.degree. C. for 15 min and mixed thoroughly.
After cooling to RT, the blend was slowly diluted to 1 mL with
distilled water. The free pharmaceutical agent was pelleted via
centrifugation for 10 minutes at 10,000 g, and the pellet and
supernatant were separately freeze dried, and analyzed in THF via
gel permeation chromatography. Dexamethasone and paclitaxel results
were quantified using a standard curve.
[0131] Dexamethasone and amphotericin B were more efficiently
encapsulated via solvent dispersion than via the method of the
invention. In contrast, paclitaxel was more efficiently
encapsulated via the method of the invention. Other pharmaceutical
agents such as sirolimus and everolimus were also efficiently
encapsulated using the methods of the invention. The encapsulation
efficiency depends on the structure of the pharmaceutical agent.
The flexibility of the PEG-PPS system to encapsulate pharmaceutical
agents is very large because the system accommodates a variety of
techniques to encapsulate most pharmaceutical agents at high
efficiencies.
Example 3. Excipients can be Used to Enhance Encapsulation
Efficiency of Hydrophilic Pharmaceutical Agents in Vesicles
[0132] Polymeric vesicles represent very powerful tools for
protection and delivery of hydrophilic pharmaceutical agents, such
as peptides, proteins, nucleic acids, and genes; however, they are
difficult to load. We have developed novel mechanisms to load
vesicles at very high loading efficiency. One method is to dissolve
in the polymer an excipient that is soluble both in the polymer and
in water, such as DBU or PEGs, as illustrated above in the
formation of polymer micelles. An aqueous solution of the
pharmaceutical agent to be encapsulated is added to the polymer
mixture with the excipient (the so-called direct hydration method).
Typical results are illustrated in Table 3 and FIGS. 5-10 and
11A-11C.
TABLE-US-00003 TABLE 3 Particle size determination using dynamic
light scattering. Values are reported as zeta-size. The processing
methods are solvent dispersion using tetrahydrofuran (SD), thin
film extrusion (TFE), direct hydration (DH), and direct hydration
with extrusion (DHE). fPEG SD TFE DH DHE 0.16 83.3 240.8 416.8
281.0 0.18 74.9 224.3 531.8 227.8 0.23 121.9 230.3 1417.0 125.5
0.33 102.9 196.4 458.3 139.4 0.39 43.4 315.7 695.4 117.7 0.49 20.0
159.4 278.6 97.8 0.60 15.9 46.5 113.6 46.4 0.68 18.4 55.5 244.3
77.5 0.72 25.2 42.7 225.1 83.6 0.87 14.6 56.4 558.4 114.0
Example 4. Thermal Transitions can Induce Vesicle Formation from
Micelles
[0133] As mentioned above, vesicles are powerful tools with which
to encapsulate hydrophilic pharmaceutical agents, to modulate their
release, to target their release, and to protect them from
biological clearance and degradation mechanisms. We have developed
a method to form polymer micelles involving application of heat.
Polymer micelles are formed, using a polymer composition with an
fPEG that would thermodynamically form vesicles instead. In
suspension, the micelles can be metastable and can be concentrated
to a high degree. Application of heat to the metastable micelles
induces spontaneous formation of vesicles, which can be very small
and homogeneous in size distribution. Pharmaceutical agent
incorporated in the micelle suspension will be loaded within the
vesicles during their formation. The approach is illustrated in
FIGS. 12-15.
[0134] The ultrasmall size of the polymer vesicles formed by this
method may be particularly useful in some applications. For
example, in targeting tumors from the bloodstream via the enhanced
permeation and retention effect, smaller particles are more
effective than larger particles in penetration of the fenestrated
endothelium in the tumor microcirculation. Smaller particles are
more effective than larger ones in penetration of the arterial wall
under physiological pressure or mild overpressure, in penetration
of mucosal surfaces and targeting cells beneath, such as dendritic
cells, in permeation of the interstitium to target lymph nodes
draining the tissue site, and in targeting the lymphatics in the
gut.
Example 5. PEG-PPS Vesicle Formulations can be Stable Upon Drying
and Rehydration
[0135] Formulations that can solubilize hydrophobic pharmaceutical
agents and can be administered in dry form are useful in a number
of pharmaceutical applications. We have demonstrated that PEG-PPS
micelles can be dried into a tablet and subsequently resuspended
rapidly, to the same size distribution, without loss of
encapsulated pharmaceutical agent. For example,
PEG.sub.44-PPS.sub.20 micelles were formed with size mean of 21 nm
(FIG. 16A), loaded with cyclosporine A. The suspension was dried,
and then the dried sample was placed in water to allow brief
rehydration. The measured size distribution showed a mean of 20.3
nm (FIG. 16B). Throughout the process, high encapsulation
efficiency was maintained (Table 7). The particles, being primarily
sensitive to oxidation, are stable at gastric pH (FIGS. 17A and
17B).
TABLE-US-00004 TABLE 7 High loading of cyclosporine A (CsA) was
obtained in PEG.sub.44-PPS.sub.20 micelles, and this loading was
maintained after the micelle suspension was dried at 80.degree. C.
and rehydrated in water. CsA CsA Encapsulation Mean added loading
efficiency aggregate Polymer (mg) (mg/mg) (%) sixe (nm)
PEG.sub.44-PPS.sub.20 4 0.130 64 21 PEG.sub.44-PPS.sub.20 4 0.123
62 20 after rehydration
Example 6. PEG-PPS-PEI Copolymers can Efficiently Deliver
Gene-Based Pharmaceutical Agents
[0136] Previous work described, among other embodiments,
PEG-PPS-polycation triblock copolymers, including the case where
the polycation blocks were based on peptides. We have developed
easier synthetic routes to analogous polymers, including
PEG-PPS-PEI block copolymers (FIG. 18). These copolymers achieve
high transfection efficiency, both with plasmid DNA and also with
siRNA. The polymers also demonstrated much lower cytotoxicity than
did linear PEI of the same PEI molecular weight at the same PEI
concentration. These results are shown in FIGS. 19-21.
Example 7. Blends of PEG-PPS and PPS-PEI can Form Very Small
Complexes with Gene-Based Pharmaceutical Agents
[0137] It is sometimes particularly desirable to obtain
nanoparticles with gene-based pharmaceutical agents that are very
small. This was possible by using mixed micelles of PEG-PPS and
PPS-PEI to obtain very small complexes. Results with this approach
are shown in Table 8 and FIGS. 22-24. It is also possible to
incorporate hydrophobic agents within the PPS domains of these
complexes to induce an additional effect, such as present a
bioactive agent or deliver an agent that enhances transfection
efficiency.
[0138] As mentioned above, the ultrasmall size of the polymer
micelles and other particles may be important; the sizes formed by
this method may be particularly useful in some applications. For
example, in targeting tumors from the bloodstream via the enhanced
permeation and retention effect, in penetration of the arterial
wall under physiological pressure or mild overpressure, in
penetration of the fenestrated endothelium in the tumor
microcirculation, in penetration of mucosal surfaces and targeting
cells beneath, such as dendritic cells, and in targeting the
lymphatics in the gut, smaller particles are more effective than
larger particles.
TABLE-US-00005 TABLE 8 PEG-PPS/PPS-PEI mixed micelles condense
gene-based pharmaceutical agents into very small nanoparticles.
Size Size of Size of of the the particles the particles particles
complexed with complexed Polymer alone siRNA with DNA
PEG.sub.44-PPS.sub.27- ~160 nm ~150 nm ~180 nm PEI.sub.96
PEG.sub.44-PPS.sub.20- ~170 nm ~160 nm -- PEI.sub.70
PEG.sub.44-PPS.sub.20/ ~60 nm ~40 nm ~45 nm
PPS.sub.10-PEI.sub.50
Example 8. Blends of PEG-PPS and PEG-PPS-PEI can Form Very Small
Complexes with Gene-Based Pharmaceutical Agents
[0139] In many situations of gene and gene-based pharmaceutical
agent delivery (plasmid DNA, siRNA, antisense oligonucleotides,
aptamers, or microRNA, for example), the size of the particle is
critical for effective delivery. Small particles penetrate tissues
better than larger particles and may also lead to higher
cytoplasmic delivery and transfection efficiency. For example,
complex delivery to tumor beds is more effective with very small
complexes. The gene complexes formed from PEG-PPS-PEI can be
substantially reduced in size by incorporation of PEG-PPS in the
micelle into which the gene is incorporated. As such, the
gene-non-binding PEG-PPS drives the formation of smaller size
micelles from PEG-PPS-PEI. FIG. 25 demonstrates that ca. 30 nm
plasmid DNA complexes can be formed from a 9:1 ratio of PEG-PPS and
PEG-PPS-PEI.
Example 9. Surface Modified Nanoparticles
[0140] Given that PEG-PPS nanoparticles can survive harsh
conditions such as gastric pH, we contemplated their transport in
the gut lymphatics, determining their potential to be processed by
mechanisms related to fat uptake in the gut. This was measured
using a coculture model of caco-2 cells (enterocytes) and lymphatic
endothelial cells (LECs), allowing full characterization of uptake,
packaging, and transit (collectively referred to as transport). It
was determined that the surface characteristics of PPS-based
nanoparticles can be engineered to provide for effective transport:
for example, particles on which about 10% of the terminal chain
groups were substituted with a carboxyl functionality were
transported at a level 5-fold higher than that of equivalent
particles lacking a surface charge. Other surface charging
moieties, such as sulfates and sulfones, are similarly effective
after optimization.
[0141] The uptake of PEG-PPS nanoparticles is biospecific, as the
control macromolecule dextran was not well transported, and the
transport was blocked at cold temperatures.
[0142] Other surface characteristics also lead to enhanced
transport. Particles formed with a terminal hydroxyl group were
about 10-fold better transported than analogous particles with
terminal methoxy groups. Thus, PPS nanoparticles with 90%
--OCH.sub.3 and 10% COOH are actively transported across LECs
(5.times. better than others). Moreover, PPS nanoparticles with 90%
--OH and 10% COOH are actively transported across Caco-2 cells
(10.times. better than others).
OTHER EMBODIMENTS
[0143] The description of the specific embodiments of the invention
is presented for the purposes of illustration. It is not intended
to be exhaustive nor to limit the scope of the invention to the
specific forms described herein. Although the invention has been
described with reference to several embodiments, it will be
understood by one of ordinary skill in the art that various
modifications can be made without departing from the spirit and the
scope of the invention, as set forth in the claims. All patents,
patent applications, and publications referenced herein are hereby
incorporated by reference.
[0144] Other embodiments are within the claims.
* * * * *