Note: Descriptions are shown in the official language in which they were submitted.
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THERAPEUTIC DELIVERY COMPOSITIONS
AND METHODS OF USE THEREOF
Technical Field
The present invention relates to therapeutic
delivery compounds and to compositions comprising
therapeutic delivery compounds that kill or suppress the growth
of bacteria, viruses, fungi and protozoa, and methods of use
thereof. The compounds, compositions and methods are
effective for the delivery of drugs and other compounds to the
interior of cells and for controlling intracellular organisms.
Background of the Invention
Many new and potentially useful technologies are
being developed which may form the basis of future medical
cures and therapies. Examples of such technologies include,
gene replacement, antisense gene therapy, triplex gene therapy
and ribozyme-based therapy. However, to be successful, these
technologies require effective means for the delivery of the
therapeutic agent across cellular, nuclear and microorganismal
membranes.
The recent advent of technology, and. advances in
our ability to understand the structure and function of many
genes makes it possible to selectively turn off or modify the
activity of a given gene. Alteration of gene activity can be
accomplished many ways. For example, oligonucleotides that
are complementary to certain gene messages or viral sequences,
known as "antisense" compounds, have been shown to have an
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inhibitory effect ajaiust viruses. By creating an antisense
compound that hybridizes with the targeted RNA message of
cells or viruses the translation of the message into protein can be
interrupted or prevented. In this fashion gene activity can be
modulated.
The ability to deactivate specific genes provides
great therapeutic benefits. For example. it is theoretically
possible to fight viral diseases with antisense RNA and DNA
molecules that seek out and destroy viral gene products. In
tissue culture, antisense oligonucleotides have inhibited
infections by herpes-viruses, influenza viruses and the human
immunodeficiency virus that causes AIDS. It may also be
possible to target antisense oligonucleotides against mutated
oncogenes. Antisense technology also holds the potential for
regulating growth and development. However, in order for the
aene therapy to work, antisense therapeutic compounds must be
delivered across cellular plasma membranes to the cytosol.
Gene activity is also modified using sense DNA in
a technique known as gene therapy. 'Defective genes are
replaced or supplemented by the administration of "good" or
normal genes that are not subject to the defect. The
administered normal genes which insert into a chromosome, or
may be present in extracellular DNA, produce normal RNA,
which in turn leads to normal gene product. In this fashion
gene defects and deficiencies in the production of gene product
may be corrected. Still further gene therapy has the potential to
augment the normal genetic complement of a cell. For
example, it has been proposed that one way to combat HIV is to
introduce into an infected person's T cells a gene that makes the
cells resistant to HIV infection. This form of gene therapy is
sometimes called "intracellular immunization." Genetic
material such as polynucleotides may be administered to a
mammal to elicit an immune response against the gene product
of the administered nucleic acid sequence. Such gene vaccines
elicit anryirnmune. response in the following manner. First, the
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nucleic acid sequence is administered to a human or animal.
Next, the administered sequence is expressed to form gene
product within the human or animal. The gene product inside
the human or animal is recognized as foreign material and the
immune system of the human or animal mounts an
immunological response against the gene product. However,
this approach currently is not feasible due to a lack of effective
gene delivery systems that facilitate the delivery of genetic
material across both cellular and nuclear membranes.
Finally, gene therapy may be used as a method of
delivering drugs in vivo. For example, if genes that code for
therapeutic compounds can be delivered to endothelial cells, the
Qene products would have facilitated access to the blood stream.
Currently, genes are delivered to cells ex vivo and then
reintroduced to the animal.
Retroviral vectors can be used to deliver genes ex
vivo to isolated cells, which are then infused back into the
patient. However, retroviral vectors have some drawbacks,
such as being able to deliver genes only to dividing cells,
random integration of the gene to be delivered, potentially
causing unwanted genetic alterations, and possibly reverting
back to an infectious wild-type retroviral form. Another
drawback of antisense gene therapy is that it is effective at the
messenger RNA level, which means that antisense
oligonucleotides must be introduced in a quantity to interact
with all or a substantial number of the mRNA in the cytosol,
and that such treatment is only effective during active synthesis
of mRNA. Further, the oligonucleotides must be maintained at
this high quantity level throughout mRNA synthesis to be
effective over time.
Newly developed "triplex DNA" technology
represents an improvement in gene regulation. Triplex DNA
technology utilizes oligonucleotides and compounds that
specifically bind to particular regions of duplex DNA, thereby
inactivating the taryeted gene. An advantage of triplex DNA
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technology is that only a single copy of the oligonucleotide or
compound is required to alter gene expression because the
binding is at the DNA level, not the mRNA level. A drawback
of triplex DNA technology, however, is that the oligonucleotide
or compound must pass through not only the cellular
membrane, but also the microbial membrane in the case of
treating microbial infections, or the nuclear membrane in the
case of altering eukaryotic aene function or expression of
foreign DNA integrated into chromosomal DNA.
Another emerging technology relates to the
therapeutic use of ribozymes for the treatr:.ent of genetic
disorders. Ribozymes are catalytic RNA molecules that consist
of a hybridizing region and an enzymatic region. Ribozymes
may in the future be en jineered so as to specifically bind to a
targeted region of nucleic acid sequence and cut or otherwise
enzymatically modify the sequence so as to alter its expression
or translation into gene product.
There is a great need, therefore, for improved
delivery systems for genetic material such as genes,
polynucleotides, and antisense oligonucleotides that can be used
in bene therapy. More specifically, there is a need for non-toxic
compositions'having surfactant properties that can facilitate the
transport of genetic compounds and other drugs and therapeutic
compounds across cellular membranes.
There is a particularly urgent need for an effective
treatment for Acquired Immune Deficiency Syndrome, or
AIDS, a disease thought to be caused by a human retrovirus, the
Human T Lymphotropic Virus III (HTLV-III) which is also
called human immunodeficiency virus or HIV. Like other
retroviruses, HIV has ribonucleic acid, or RNA, as its genetic
material. When the virus enters the host cell, a viral enzyme
called reverse transcriptase exploits the viral RNA as a template
to assemble a corresponding molecule of DNA. The DNA
travels through the cell nucleus and inserts itself among the host
chromosomes, where it provides the basis for viral replication.
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In the case of HIV, the host cell is often a T4 lymphocyte, a white blood cell
that
has a central and regulatory role in the immune system. Once it is inside a T4
cell, the virus
may remain latent until the lymphocyte is immunologically stimulated by a
secondary infection.
Then the virus reproducing itself rapidly killing or rendering ineffective the
host cell. The
resulting depletion of the T4 cells and loss of activity leaves the patient
vulnerable to
"opportunistic" infections by an agent that would not normally harm a healthy
person. The
virus damages the host by many other mechanisms as well.
Many therapies against AIDS infection that are currently being investigated.
Several of these therapies under investigation are based on interrupting the
reverse transcriptase
as it assembles the viral DNA destined to become the virus. The drugs used for
this purpose
are chemical analogs of the nucleic acids that form the sub-units of DNA. When
the analog
is supplied to an infected cell, reverse transcriptase will incorporate it
into a growing DNA
chain. Because the analog lacks the correct attachment point for the next sub
unit, however,
the chain is terminated. The truncated DNA cannot integrate itself into the
host chromosomes
or provide the basis for viral replication and so the spread of the infection
is halted. One of
the compounds that is thought to act by mimicking a nucleotide is
azidothymidine, or AZT.
However, AZT is known to have serious side effects and its efficacy in
mitigating the AIDS
disease has been questioned. The efficacy of AZT and other antiviral and
antimicrobial drugs
could be increased if improved means and methods for delivering therapeutic
agents to the site
of infection were available.
Summary of the Invention =
The present invention includes the use through delivery of therapeutic drugs
to
a human or animal for treating disease states such as, but not limited to,
bacterial infection and
infections caused by HIV and other DNA and RNA viruses. The present invention
relates
particularly to compositions and the use thereof for treating infectious
diseases and genetic
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disorders through gene therapy and intracellular delivery of antisense
oligonucleotides or other
nucleic acid sequences.
The present invention comprises a therapeutic delivery composition effective
for
treating a disease state comprising an administerable admixture of an
effective amount of a
therapeutic compound capable of altering nucleic acid sequence function and an
effective
amount of a surface active non-ionic block copolymer having the following
general formula:
HO(C2H40)b(C3H60)a(C2H40)bH
wherein a is an integer such thai: the hydrophobe or polyoxypropylene portion
of the copolymer
represented by (C3H60) has a molecular weight of approximately 750 and
approximately
15,000, preferably between approximately 2,250 and approximately 15,000, more
preferably
between approximately 3,250 and approximately 15,000 and b is an integer such
that the
hydrophile. or polyoxyethlene portion of the copolymer represented by (C2H40)
constitutes
approximately 1% to approximately 50% by weight of the compound, preferably
approximately
5% to approximately 20%.
A particularly useful composition is an admixture of a compound capable of
altering gene expression and/or protein translation, such as an antisense
oligonucleotide, a
triplex DNA compound, a ribozyme or other compound capable of altering nucleic
acid
sequence function and the above-described non-ionic block copolymer.
The composition of the present invention can be administered by a number of
routes including, but not limited topical, transdermal, oral, transmucosal,
subcutaneous injection,
intravenous injection, intraperitoneal injection and intramuscular injection.
Accordingly, an object of the invention is to7 provide a therapeutic drug
delivery
vehicle.
- ~.
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Another object of the present invention is to
provide compositions that facilitate delivery of one or more
therapeutic nucleic acid sequence function alterihg agents into
the interior of a cell, such as a phagocytic cell, when admixed
with a therapeutic agent.
Another object of the present invention is to
provide compositions that act synergistically with a delivered
agent once inside a cell.
Still another object of the invention-is to provide
nonionic block copolymers having surfactant properties that
facilitate the transmission and introduction across cellular
plasma. membranes of nucleic acid sequences and compounds
capable of altering nucleic acid sequence function.
A further object of the present invention is to
provide compositions and a method for treating genetic and
physiologic disorders using nucleic acid sequences and
antisense oligonucleotides in combination with nonionic block
copolymers.
Another object of the present invention is to
provide compositions and a method useful for manipulating the
expression of genes using triplex DNA compounds.
Yet another object of the invention is to provide
DNA vaccines.
It is an object of the present invention to provide
compositions which can be used to treat persons with infectious
diseases.
Yet another object of the present invention is to
provide a method of treating viral infections in humans or
animals.
Another object of the present invention is a
compound and method that is effective in inhibiting the
replication of viruses in both animals and humans.
Another object of the present invention is to
provide a compound and method that is effective in inhibiting
the replication of HIV and other RNA and DNA viruses.
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Yet another object of the present invention is to
provide a method of treating microbial infections in humans or
animals.
It is another object of the present invention to
inactivate virus in a blood product prior to infusion into a
person or animal.
These and other objects, features and advantages of
the present invention will become apparent after a review of the
following detailed description of the disclosed embodiment and
the appended claims.
Brief Description of the Drawings
Fig. 1 is a grid illustrating block copolymers by
molecular weight of hydrophobe and percent hydrophile.
Fig. 2 is a grid illustrating preferred therapeutic
delivery block copolymers by molecular weight of hydrophobe
and percent hydrophile.
Fig. 3 is a grid illustrating more preferred
therapeutic delivery block copolymers by molecular weight of
hydrophobe and percent hydrophile.
Detailed Description
The present invention includes gene therapy
compositions that are admixtures of a nonionic block
copolymer and nucleic acid sequences or compounds capable of
altering nucleic acid sequence function, and methods of
delivering these compositions to a human or animal in need
thereof for the intracellular alteration of gene expression and/or
protein translation. It has been unexpectedly found that high
molecular wei~ht surface active nonionic polyoxyethylene-
polyoxypropylene block copolymers having a low percentage of
-polyoxyethylene facilitate the transport of DNA and other
compounds into cells and thus are useful for the intracellular
delivery of therapeutic agents in vivo for. the treatment of
disease. It is believed that the block.cnpolymers are par:ticularly
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useful in helping to reseal membranes and thus increase the
percent survival of cells wherein nucleic acid sequences or
other compounds have been intracellularl'y introduced.
Surprisinaly, it has also been found. t-hat . compositions
comprising the nonionic block copolymers of the present
invention and nucleic acid sequences are less susceptible to the
degrad'uig effects of DNAase than nuc,deic acid sequences alone.
The present invention also comprises therapeutic
compositions and methods which kilL or inhibit the growth of
microorganisms and alter the expression or function nucleic
acid sequences. An example of the bacteria that the present
invention is effective a?ainst is mycobacteria species, such as
Mycobacteritcm tuberculosis, Mycobacterium avium, and
Mycobacteriitm leprae. Other microorganisms that the
invention is effective aaainst include, but are not limited to,
Chiamydia trachomatis, Chlanrydia pnectmoniae, Listeria
monocytogenes, Candida albicans, Cryptococcus neoformans,
Toxoplasma gondii, Pneumocystis carinii, Herpes simplex virus
type 1, Cytomegalovirus, influenza virus type A and B, and
respiratory syncytial virus.
. The present invention includes therapeutic
compositions and methods for treating DNA viruses and RNA
viruses, and infections and infectious diseases caused by such
viruses in a human or animal, including infections caused by
HIV or herpes or antigenically-related strains thereof.
Anti~enically-related strains are strains that cross react with
antibodies specific for HIV. One skilled in the art can readily
determine viral strains that are antigenically-related to HIV bv
conductin~ standard immunoassay tests usina anti-HIV
antibodies and the viral strain to be analyzed, and looking for
positive cross-reactivity. The therapeutic compositions
comprising the surface active copolymers disclosed herein are
effective in inhibiting or suppressing the replication of such
viruses in cells.
.v-__..4.._..___... _.
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The present invention includes a therapeutic
composition useful for delivering antimicrobial drugs and
treating disease states comprising an admixture of a surface
active nonionic block copolymer, a compound capable of
altering nucleic acid sequence. function, and an antibiotic or
therapeutic drug. Examples of such compounds capable of
altering nucleic acid sequence function include genes,
oligonucleotides, antisense oligonucleotides, triplex DNA
compounds, and ribozymes. Drugs that can be used with the
nonionic copolymers of the present invention include, but are
not limited to, rifampin, isoniazid, ethambutol, gentamicin,
tetracycline, erythromycin, pyrazinamide, streptomycin,
clofazimine, rifabutin, fluoroquinolones such as ofloxacin and
sparfloxacin, azithromycin, clarithromycin, dapsone,
doxycyline, ciprofloxacin, ampicillin, amphotericin B,
fluconazole, ketoconazole, fluconazole, pyrimethamine,
sulfadiazine, clindamycin, azithromycin, paromycin, diclazaril,
clarithromycin, atovaquone, pentamidine, acyclovir,
trifluorouridine, AZT, DDI, DDC, and other antiviral
nucleoside analogs, foscornat, ganciclovir, viral protease
inhibitors, antisense and other modified oligonucleotides, and
ribavirin.
Preferred drugs to use for various infectious
microorganisms are listed in Table I.
Table I
rani m Drui!s
Bacteria
Mycobacterium tuberculosis Isoniazid, rifampin, ethambutol,
pyrazinamide, streptomycin,
clofazimine, rifabutin,
fluoroquinolones such as ofloxacin
and sparfloxacin
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Table I (continued)
r ni 5It DruLys
Mycobacterium avium Rifabutin, rifampin, azithromycin,
clarithromycin, fluoroquinolones
Mycobacterium leprae Dapsone
Chiamydia trachomatis Tetracycline, doxycyline,
erythromycin, ciprofloxacin
Chlamydia pneumoniae Doxycycline, erythromycin
Listeria nionocytogenes Ampicillin
F tn i
Candida albicans Amphotericin B, ketoconazole,
fluconazole
Cryptococcus neoformans Amphotericin B, ketoconazole,
fluconazole
Protozoa
Toxoplasma gondii Pyrimethamine, sulfadiazine,
clindamycin, azithromycin,
clarithromycin, atovaquone
Pneumocystis carinii Pentamidine, atovaquone
Cryptosporidium sp. Paromomycin, diciazaril
Vtr
Herpes simplex virus type 1 Acyclovir, trifluorouridine and other
and type 2 antiviral nucleoside analogs,
foscornat, antisense oligonucleotides,
and triplex-specific DNA sequences
Cytomegalovirus Foscarnet, ganciclovir
HIV AZT, DDI, DDC, foscarnat,
viral protease inhibitors, peptides,
antisense oligonucleotides, triplex
and other nucleic acid sequences
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Table I (continued)
Qrganism Dri.
Influenza virus type A and B Ribavirin
Respiratory syncytial virus Ribavirin
Varizella zoster virus Acyclovir
Optionally, surfactants and low molecular weight
alcohols are added to the therapeutic admixture of antimicrobial
drug and nonionic block copolymer. Examples of surfactants
useful in the present invention include Tween 80*and emulsions
with fatty acids such as phospholipids, cholate and amino acids.
The preferred surfactant is Tween 80. Surfactants are added to
the admixture at a concentration ranging from approximately
0.1% to approximately 5% v/v. The preferred surfactant
concentration is approximately 2%. The term "approximately"
as it applies to concentrations expressed herein means the stated
concentration plus or minus ten percent. The term "low
molecular weight alcohols" means alcohols having two to eight
carbons. An example of a low molecular weight alcohols useful
in the present invention is ethanol, which is the preferred low
molecular weight alcohol. Low molecular weight alcohols are
added to the admixture at a concentration ranging from
approximately 0.5% to approximately 5% v/v. The preferred
low molecular weight alcohol concentration is between
approximately 1% and approximately 3% v/v.
The present invention also includes compositions
and methods for immunizing animals or humans, otherwise
termed DNA vaccination. Immunization is accomplished by
administering a composition comprising the gene that codes for
the gene product to be immunized a~ainst contained in an
expression, in combination with a block copolymer that
promotes and facilitates uptake of 'enetic material across cell
* Trade Mark
1
2174'22
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membranes. The introduced gene is expressed, resulting in the production of an
antigenic gene
product.
Still further, compositions comprising non-ionic block copolymers and genes
that
code for compounds effective for killing, reducing, or retarding cancer, such
as lymphokines,
may be administered to humans or animals for the treatment of cancer.
The present invention comprises a surface active copolymer that is preferably
an ethylene oxide-propylene oxide condensation product with the following
general formula:
HO(CaHa0)b(C3H60)a(C2Ha0)bH
wherein a is an integer such that the hydrophobe or polyoxypropylene portion
of the copolymer
represented by (C3H60) has a molecular weight of between approximately 750 and
approximately 15,000 and b is an integer such that the hydrophile or
polyoxyethylene portion
of the copolymer represented by (C2H40) constitutes approximately 1% to
approximately 50%
by weight of the compound.
The present invention also comprises a therapeutic delivery composition useful
for altering gene expression and/or protein translation comprising an
administerable admixture
of an effective amount of an anti-sense oligonucleotide or other nucleic acid
sequence and an
effective amount of a non-ionic block copolymer having the following general
formula:
HO(C2H40)b(C3H60)a(C2H40)bH
wherein a is an integer such that the hydrophobe or polyoxypropylene portion
of the copolymer
represented by (C3H60) has a molecular weight of approximately 750 and
approximately
15,000, preferably between approximately 2,250 and approximately 15,000, more
preferably
between approximately 3,250 and approximately 15,000 and b is an integer such
that the
hydrophile or polyoxyethylene portion of the copolymer represented by (C2H40)
constitutes
approximately 1% to approximately 50% by weight of the compound, preferably
approximately
...
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5% to approximately 20%. The term admixture as used herein means any
combination of
therapeutic drug and non-ionic block 'copolymer, including solutions,
suspensions, or
encapsulations of drug in copolymer micelles. An effective amount is an amount
sufficient to
alter the activity and/or the amount of gene product produced by the gene or
genes sought to
be modulated in a human or animal.
The present invention also comprises a therapeutic delivery composition useful
for immunizing an animal or human against a particular gene product comprising
an
administerable admixture of an effective amount of an expression vector, the
gene that codes
for the gene product to be immunized against contained in the expression
vector and an
effective amount of a non-ionic block copolymer having, the following general
formula:
HO(C2H40)b(C3H60)a(C2H40)bH
wherein a is an integer such that the hydrophobe or polyoxypropylene portion
of the copolymer
represented by (C3H60) has a molecular weight of approximately 750 and
approximately
15,000, preferably between approximately 2,250 and approximately 15,000, more
preferably
between approximately 3,250 and approximately 15,000 and b is an integer such
that the
hydrophile or polyoxypropylene portion of the copolymer represented by (C2H40)
constitutes
approximately 1% to approximately 50% by weight of the compound, preferably
approximately
5% to approximately 20%. An effective amount is an amount sufficient to elicit
an
immunological response against the gene product of the nucleic acid sequence
administered to
the human or animal.
It should be understood that the molecular weight and percentage ranges that
are
described for the block copolyiner are to be considered outside ranges and
that any population
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of molecules that falls within the stated ranges is considered an embodiment
of the present
invention.
The entire block copolymer molecule is poorly soluble in water and is
substantially non-ionic. The steric configurations and physiochemical
properties of the
molecule, rather than the chemical nature of the constituent parts, are
believed to be largely
responsible for the anti-infective activity and therapeutic delivery activity.
Compositions of
the present invention include, but are not limited to aqueous solutions,
suspensions or
emulsions, such as oil-in-water emulsions.
The polymer blocks are formed by condensation of ethylene oxide and propylene
oxide, at elevated temperature and pressure, in the presence of a catalyst.
There is some
statistical variation in the number of monomer units which combine to form a
polymer chain
in each copolymer. The molecular weights given are approximations of the
average weight of
copolymer molecule in each preparation and are dependent on the assay
methodology and
calibration standards used. It is to be understood that the blocks of
propylene oxide and
ethylene oxide do not have to be pure. Small amounts of other materials can be
admixed so
long as the overall physical chemical properties are not substantially
changed. A more detailed
discussion of the preparation of these products is found in U.S. Patent No.
2,674,619, which
may be referred to for further details.
Ethylene oxide-propylene oxide condensation products which may be employed
in the present invention are summarized in Table II. It is to be understood
that these
compounds are merely representative of the compounds that can be used to
practice the present
invention and do not include all possible compounds that could be used to
practice the present
invention. The high molecular weight copolymers listed in Table II that do not
have a BASF
tradename are novel compositions that have never been synthesized before.
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Table II
Molecular
CRL # Poloxamer BASF Trade Weight % POE
Name of POP
122 L42 =1200 --20%
CRL-85171 181 L61 -=1750 =10%
CRL-85172 182 L62 ==1750 =20%
CRL-85173 183 L63 --=1750 =30%
CRL-85174 184 L64 =1750 =40%
CRL-85175 185 P65 =1750 =50%
CRL-85178 188 F68 =1750 =80%
CRL-85202 212 L72 =2050 =20%
CRL-85221 231 L81 =2250 =10%
CRL-8122 282 L92 ---2750 =20%
CRL-8131 331 L101 =3250 =10%
CRL-8133 333 P103 = 3250 --30%
CRL-8135 335 P105 =3250 =50%
CRL-9038 338 F108 =3250 =80%
CRL-8141 401 L121 =4000 =10%
CRL-8142 402 L122 =4000 --20%
CRL-8143 403 P123 =4000 =30%
CRL-8941 441 L141 =4400 =10%
CRL-8950 ----- ------ =6000 =5%
CRL-1235 ----- ------ =7500 =5%
CRL-1 190 ----- ------ --=10,000 =5%
CRL-336 ----- ------ =14,000 =5%
CRL-1183 ----- ----- =3750 =10%
CRL-1122 ----- ------ =5900 =12%
CRL-3362 ----- ------ =3900 =11 %
CRL-3632 ----- ------ =4740 =11%
CRL-9352 ----- ------ =7750 =15%
CRL-1187 ----- ------ =750 =25%
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A grid illustrating the range of copolymer
encompassed by the present invention based upon the molecular
weight of the hydrophobe portion and the percent hydrophile,
and showing selected nonionic block copolymers appears as
Figure 1._ The polymer blocks are formed by condensation, at
elevated temperature and pressure, of ethylene oxide and
propylene oxide in the presence of a catalyst. There is some
statistical variation in the number of monomer. units which
combine to form a polymer chain in each copolymer. The
molecular weights given are approximations of u'he average size
of copolymer molecules - in each preparation. A further
description of the preparation of these block copolymers is
found in U.S. Patent No. 2,674,619. (Also s e, "A Review of
Block Polymer Surfactants", Schmolka I.R., J. Am. Oil Chemist
Soc., 54:110-116 (1977) and Blo k and Graft
Copolymerization, Volume 2, edited by R.J. Ceresa, John Wiley
and Sons, New York, 1976.
It has been discovered that the copolymers
particularly effective as therapeutic delivery agents are shown
in Figures 2 and 3. As is apparent from Figures 2 and 3, the
copolymers most effective as therapeutic delivery agents are
high molecular weight and have low percentages of POE -
generally less than 20% POE
Non-ionic block copolymers form micelles above
their critical micelle concentration. The non-ionic copolymers
have negative thermal coefficients of solubility. In the cold, the
kinetic energy of water molecules is reduced and they form
weak hydrogen bonds with the oxygen of the POP blocks. This
hydration of the hydrophobe promotes solubility at low
temperatures. As the temperature rises, the "cloud point" is
reached; the increased kinetic energy of the water breaks the
hydrogen bonds, the polymer becomes insoluble and micelles
form.
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Thus, the copolymers, which are therapeutic
themselves, can form physical structures that can be combined
or loaded with -an additional, distinct therapeutic agent.
Consequently, the nonionic block copolymers of the present
invention can be used as therapeutic drug delivery vehicles.
Admixtures of therapeutic drugs with non-ionic block
copolymers have the advantage of synergistic activity of two
therapeutic agents. Further, copolymers having specific
characteristics can be selected for use with particular
therapeutic drugs. For example, CRL-8131, which is
hydrophobic, is an excellent carrier for hydrophobic antibiotics
such as rifampin. However, other agents which are not
distinctly hydrophobic can be used according to the present
invention.
A therapeutic delivery vehicle is prepared using
any of the surface active nonionic block copolymers of the
present invention in combination with any of a variety of
antimicrobial agents. In a one embodiment CRL-8131 is used
at a concentration of approximately 3% to approximately 5% to
construct a therapeutic delivery vehicle. Therapeutic delivery
vehicles made using copolymers that are more hydrophilic than
CRL-8131 normally require a higher concentration
(approximately 5% to approximately 10%) of the copolymer.
Using copolymer-based micelles as a therapeutic
drug del.ivery vehicle is particularly desirable because the
micelles are accumulated readily and are present for an
extended period of time, in macrophages, the site of HIV and
other viral infections and a major target for viral therapy.
Examples of such therapeutic copolymer-based therapeutic
compositions include CRL-8131 combined with 2% Tween 80*
and 1% ethanol, and CRL-8142 combined with 1% Tween 80
and 5% ethanol.
Nucleic acid sequences or other compounds
capable of altering nucleic acid sequence function are
administered to a human or animal to alter gene expression
* Trade Mark
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and/or modify the amount or activity of gene product. For
example, antisense oligonucleotides admixed with the above-
described nonionic block copolymers yield compositions useful
for delivery of the antisense oligonucleotides for the purpose of
altering or regulatinj gene expression and/or protein translation.
Further=, nucleic acid sequences such as genes can be
administered which incorporate into the chromosome replacing
or augmenting the defective gene. Alternatively, the
intraceL'lularly administered gene may reside in the cell and be
expressed in an extrachromosomal element.
The present invention also provides novel
compositions and methods for immunizing an animal or human.
The compositions comprise an expression vector, a gene that
codes for the gene product to be immunized against contained
in the expression vector, and a block copolymer effective for
transferring genetic material such as expression vectors across
the membrane of cells. The method of immunizing an animal
or human comprises administering of the expression vector-
containing copolymer composition to the animal or human. A
preferred mode of administration is by intraperitoneal injection.
This embodiment of the invention provides means for the
delivery of genetic sequences capable of expressing an
antigenic gene product directly into human or animal cells,
either in vivo or ex vivo with subsequent reintroduction into the
human or, animal. Once introduced into the cells the production
of antigenic gene product induces and maintains an immune
response by the human or animal against the introduced gene
product.
The following specific examples illustrate various
aspects of the invention, such as compositions and methods of
the invention useful for gene therapy, and compositions and
methods of the invention useful for gene-mediated
immunization. It should be appreciated that other embodiments
and uses will be apparent to those skilled in the art and that the
inventioil is not limited to these specific illustrative examples.
-20- 21 ~7 41 2 Z
Example I
A therapeutic delivery vehicle is prepared by combining any of the surface
active
non-ionic block copolymers, such as CRL-8131 with any of a variety of
compounds capable.
of altering nucleic acid sequence function. For CRL-8131 a concentration of
three to five
percent weight per volume is desirable to construct the therapeutic vehicle.
For more
hydrophilic copolymer a five to ten percent weight per volume.
300 milligrams of CRL-8131 was added to 10 ml of 0.9% NaCI and the mixture
is solubilized by storage at temperatures of 2 - 4 C until a clear solution is
formed. An
appropriate amount of a compound capable of altering nucleic acid gene
function is added to
the mixture and micelles associating the copolymer and the compound are formed
by raising
the temperature above 5 C and allowing the suspension of micelles to
equilibrate. The
equilibrated suspension is suitable for administration.
For example, an antisense oligonucleotide sequence, such as one of those
disclosed by Matsukara, M. et al Proc. Natl. Acad. Sci. USA 84:7706 - 7710
(1987), which
may be referred to for further details, is combined with the copolymer to form
a micelle
composition.
Briefly, phosphorothioate or methylphosphonate derivatives of a sequence
complementary to regions of the art/trs genes of HIV having the sequence
5'-TCGTCGCTGTCTCG-3' are prepared according to the method of Matsukura et al
300
milligrams of CRL-8131 is added to 10 ml of 0.9% NaCl and the mixture is
solubilized by
storage at temperatures of 2 - 4 C until a clear solution is formed. The
desired antisense oligonucleotide subsequently is mixed with the copolymer
solution to provide a concentration effective in inhibiting viral activity
when administered to a patient infected with the HIV virus. Generally the
effective amount of antisense compound will be such that the final
concentration in the blood
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is in the range of 1 M to 100 M, although other effective
amounts of antisense compounds outside this range may be
found for specific antisense compounds. One skilled in the art
can readily test the relative effectiveness of any particular
antisense oligonucleotide according to the in vivo test of
Matsukura, et al.
An average person has approximately 6.25 liters of
blood. Thus, oligonucleotide concentrations of approximately 6
mM to 600 mM are required in the composition when 1 ml
injections are to be administered. Lower oligonucleotide
compositions can be used with larger adrninistration volumes.
Example II
The antiinfective antisense oligonucleotide
composition of Example I is administered to HIV patients by
any route effective to reduce viral activity. The preferred route
of administration is by intravenous injection. The antisense
composition may be administered multiple times a day to
ensure that an effective amount of the antisense oligonucleotide
is maintained.
Example III
A gene therapy composition for treating an animal
or human suffering from the effects of a defective or missing
gene is made by combining a copolymer, such as CRL-8131
with a normal copy of the defective gene. For example, for
patients suffering from adenosine deaminase (ADA) deficiency
a gene therapy composition is made that contains a normal copy
of the adenosine deaminase gene. The gene therapy
composition is made by mixing a copolymer prepared as
described above in Example I with the desired gene, removing
blood from the human or animal, transfecting blood cells with
the ADA gene-containinj composition, and reintroducing the
transfected blood cells into the human or animal. The
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-
introduced gene is expressed in vivo, alleviating the effects of
the original gene deficiency.
Example IV
Similarly, the gene therapy composition of
Example III is combined with isolated T-lymphocytes to form
T-lymphocytes containing the ADA gene. The ADA gene-
containing T-lymphocytes are subsequently administered, for
example by injection, into the patient suffering from adenosine
deaminase deficiency. The administered cells express the ADA
and produce adenosine deaminase, thus augmenting the supply
of the enzyme in the patient and correcting the deficiency.
Example V
DNA vaccination is carried out essentially as
described for gene therapy in Examples I]T or IV, except that
the gene that is introduced into the host expresses an antigenic
gene product that is recognized as foreign by the host animal,
thus eliciting an immune response.
Example VI
A composition comprising copolymer CRL-8131
and an expression vector containing the gD gene of Herpes
simplex virus type-1 was used in a transfection experiment.
DNA transfection normally is performed using standard
calcium chloride and DEAE dextran precipitation techniques.
DEAE dextran is used to rough up the cell membrane and
calcium is used to precipitate DNA onto the cell surface,
facilitating DNA uptake into the cells. This procedure is
generally toxic to the cells, however, and causes substantial
cellular mortality.
A new transfection system was discovered using
the block copolymer of the present invention in place of
calcium chloride. In fact, it was surprisingly discovered that
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copolymer assisted transfection occurs even in the absence of
DEAE dextran.
Vero cells were incubated in DEAE dextran for 30
seconds. A mixture of copolymer and an expression vector
containing glycoprotein gD DNA of Herpes simplex virus type-
1 was added to Vero cells immediately after the removal of
DEAE dextran. It was found that up to 40% of the cells were
effectively transfected with the gD gene.
Surprisingly, in two out of four experiments
copolymers were able to transfect Vero cells at a lower than
40% efficiency even when the DEAE dextran incubation step is
omitted.
Example VII
Other studies have also demonstrated that block
copolymers are effective in transferring genetic material across
cellular membranes in vivo. DNA vaccine-induced
immunization was successful when an expression vector
containing the gD gene of Herpes simplex virus type-1
combined with copolymer was injected intraperitoneally into
rabbits every two weeks. Sera was collected and tested for the
presence of anti-gD antibody. Low levels of anti-gD antibody
were detected after 4 weeks of inoculation in this fashion.
These results demonstrate that genetic material administered
intraperitoneally with a copolymer delivery vehicle is taken up
by cells in vivo and expressed to give the gene product in
quantities sufficient to elicit an immune response.
Example VIII
DNAse protection experiments. Five different
compounds (CRL 1122, 3362, 3632, 9352, and 8131) were used
in experiments to test the degree of protection. DNA was
mixed with compounds at 4 C, and after 15 min. at 37 C
DNAse I(1 l of 10 mJmi solution) was added. After 30 min.
of incubation at 37 C, DNAse I was removed by treatment with
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proteinase K(3 l.t.l of 10 ma/mi solution). Controls were:
DNAse I in the absence of nonionic block copolymer and DNA
alone without any D4Ase I treatment.
DNA was protected from DNAse I dearadation in
all samples in which nonionic block copolymers were present.
The best protection of DNA was achieved with CRL-3362 and
8131. DNA copolymer compositions did not migrate in
horizorital agarose electrophoresis and remained within the
wells (stained with ethidium bromide). Effective protection
against DNAse I action was achieved in solutions of i volume
DNA solution (1 b/ml) to 5 volumes of nonionic block
copolymer (30 8/ml). The estimated amount of protection
varied from experiment to experiment and was estimated to be
within 15-40% of total DNA.
Additional experiments showed that DNA-
copolymer compounds failed to transform E. coli competent
cells via the calcium method. Phenol also failed to dissolve
nonionic block copolymer away from DNA. DNA bound to
NBC can be precipitated by addina 5 volumes of isopropyl
alcohol.
Example IX
Transfection experiments. Typical transfection
experiments for transient expression of herpes viral
glycoprotein aenes and other genes of interest involved the
followirij procedure. Cells such as COS (African monkey
kidney cells; CV 1) are seeded on 6-well plates. Transfection is
performed when cells are 50-80% confluent (still in log growth
phase). Cells are first washed with PBS buffer, they are
incubated with 0.5 ml of DEAE-Dextran solution (500 ma/m1)
for 1-2 minutes, this solution is aspirated and DNA precipitate
is added to cells. DNA to be transfected is mixed for 30 min. at
room temperature with CaC12 at controlled pH conditions to
form a fine precipitate. This solution is mixed with I ml of
growth medium (DMEM) and put onto cells for 4 hours at
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37 C. At this time, the cells are shocked with 15~1o ~iycerol and
subsequently washed with PBS. This osmotic shock facilitates
the taking up of CaCI2-DNA precipitate into cells. Cells are
then washed a,aain with PBS, and incubated with growth media
at 37 C for 48 hours.
Gene expression is detected in most cases using
specific monoclonal antibodies directed against the expressed
proteins usin; indirect immunofluorescence. The expressed
proteins can be also labeled with radioactive tracers and
immunoprecipitated or detected in westerns.
25 l of DNA (7 a) and 25 l rf nonionic block
copolymer (30 ?/ml) were used. Additionally, mixing of
nonionic block copolymer with DNA on ice, and addition of
mixture into the cells produced similar results as when they
were added separately (DNA added first and nonionic block
copolymer second).
Copolymers 1183, 1187, 8131, 1235, 8950AQ and
1190AQ (where AQ indicates that the nonionic block
copolymers were diluted 1:10 and 25 l were used). Typical
results are as follows. Transfection with DNA alone, dextran
alone, copolymer alone, and DNA plus dextran had negiiaible
transfection of less than 0.2%. In contrast, the positive control
of DNA plus dextran plus glycerol has transfection of 2% while
various copolymers plus DNA were successful in transfectina
DNA into cells up to 2.5 times better than the control, as shown
in Table III:
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Table III
Copolymer Percent
Transfection
1183 2%
1187 5%
8131 2%
1235 3%
8950AQ 4%
1190AQ 5%
There was no copolymer associated toxicity
except mild toxicity with 1187. The others were toxic
especially after alycerol treatment.
It should be understood that the foregoing relates
only to preferred embodiments of the present invention and that
numerous modifications and alterations may be made without
departing from the spirit and scope of the invention as set forth
in the appended claims.