Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS OF IDENTIFYING BIOLOGICAL AGENT COMPOSITIONS
This application claims the benefit of U.S. provisional application no.
60/055,256
filed August 8, 1998.
FIELD OF THE INVENTION
The present invention relates to novel methods of identifying biological agent
compositions useful in pharmaceutical, biopharmaceutical, diagnostic, imaging,
immunology, veterinary, and agricultural applications.
BACKGROUND OF THE INVENTION
The conventional design of a new drug is very difficult. it demands design or
discovery of a new molecule which precisely matches its molecular target.
Moreover.
once such a molecule is discovered, the new drug candidate must be soluble,
bioavailable, resistant to metabolic enzymes, and be nontoxic to the patient.
Modifications of the new molecule, necessary to satisfy the above
requirements, too
often negatively affect its therapeutic efficacy. Due to enormous complexity,
producing a new drug takes a very long time and requires huge financial
resources.
Recent advances in combinatorial chemistry technologies have allowed for
faster
throughput in the design of new molecules. This development markedly reduces
the
time and cost in designing a desired molecule. However, the problem of
modifying
such a molecule so that it is soluble, bioavailable, resistant to metabolic
enzymes, and
capable of penetrating through membranes, often remains unsolved.
The drug delivery industry has addressed some of these problems, and as a
result
developed the ability to simplify new product development by incorporating
drugs
into a carrier. For drug delivery assisted products, the time of development
is
shortened to seven years, and the average cost is brought down significantly.
Unfortunately, most drug delivery systems have several serious limitations.
First,
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they are able to solve only a limited number of aforementioned problems, and
second
they are not applicable to many drugs.
SUMMARY OF THE INVENTION
This invention is in the area of "combinatorial drug delivery" or
"combinatorial
formulation". The invention provides for a method of identifying a biological
agent
composition that can be applied to pharmaceutics, biopharmaceutics,
diagnostics and
imaging, immunology, veterinary, agriculture, and other areas where the
properties of
biological agents exhibited during interaction with a living organism or cell
can be
improved. Many biological agents are suitable, including those useful for
diagnostics
or imaging, or those that can act on a cell, organ or organism to create a
change in the
functioning of the cell, organ or organism. This includes, but is not limited
to,
pharmaceutical agents, genes. vaccines, herbicides and the like.
The current invention provides a method of identifying a biological agent
composition of choice to create a complex that will render a target molecule
soluble,
bioavailable, resistant to metabolic enzymes, non-toxic, and freely traveling
through
membranes and into cells. By using segmented copolymers, such as for example,
block copolymers, and preparing libraries of biological agent compositions,
the
invention has the ability to rapidly complex and identify the compositions of
biological agents with desired biological properties. This invention can be
applied in
combination with high throughput screening of actual composition libraries,
and can
utilize mathematical concepts, which have been found to be beneficial in
Combinatorial Chemistry.
The invention reduces the time and cost for creating desired drug compounds,
which are not only immediately ready for clinical trials, but also possess a
number of
important characteristics increasing the probability of the ultimate success.
Unlike
combinatorial chemistry, the invention does not discover new drug structures
or alter
the desirable drug's characteristics, but instead provides optimal
compositions of a
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desired drug, solving the drug's problems of solubility, bioavailability,
resistance to
metabolic enzymes, toxicity, membrane transport, site specific delivery, and
the like.
Using a biological agent molecule as a starting point, the invention
identifies new
compositions with characteristics sought for the optimal performance of the
selected
molecule.
The invention thus relates to new methods of identifying biological agent
compositions. The process involves preparing a plurality of compositions
having
segmented copolymers, wherein these segmented copolymers differ in at least
one of
their segment lengths, and then screening these compositions for the desired
biological property or properties.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows identification of a biological agent composition with the
desired
(maximal) biological property (BP) using a library prepared based upon A"Bm
copolymers.
Figure 2 shows the relationship between CMC and pyrene partitioning
coefficient, P, for pluronic block copolymers having varying lengths of
ethylene
oxide and propylene oxide segments for Pluronic L 121, L 1 O 1, L 127, L 123,
L 104,
F108, L81, P85, P84, L61, L64, F87, L31, and F68 (37°C).
Figure 3 is a schematic of a high throughput screening procedure exemplifying
the relationship between computerized analysis ("computations") using a
virtual base
of polymer segments ("blocks") and segmented copolymers ("carrier molecules"),
chemical synthesis of new perspective carrier molecules. preparing library of
biological agent compositions ("complexing"), screening selected promising
compositions using physicochemical and biological analysis and identification
of a
biological agent composition with desired properties.
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Figure 4 shows a cycle of identifying a biological agent composition of choice
through the use of a parent database of Garners, computational analysis,
chemical
synthesis, preparation and testing of a real library Garners (base of
copolymers) and
biological agent compositions, and identification of a composition with
desired
properties ("optimized formulation").
The schematic presentations in Figure 3 and 4 serve only as examples of
possible
screening and identification procedures pursuant to the invention.
Particularly, the
elements and sequence of steps of these procedures can be varied to
accommodate the
properties of a certain biological agent, drug candidate and/or drug delivery
situation.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
~r~' : The review and classification of data obtained from testing the
compositions using high throughput screening (or otherwise) to draw
conclusions
from the classified data. Analysis identifies the compositions with desired
biological
properties answering to a set of criteria including but not limited to the
following: (i)
whether any of compositions are good enough to be a final product, and (ii)
whether
the data from the testing supports creation of a new library for a new testing
cycle.
~Architecture: Refers to copolymers having the same or similar formula. but
with different methods of joining each of the polymer segments.
~Basis of co~,~vmers: A plurality of segmented copolymers differing in at
least
one of their segment lengths, molecular architecture, or chemical structure.
~Biological agent: An agent that is useful for diagnosing or imaging or that
can
act on a cell, organ or organism, including but not limited to drugs (i.e.,
pharmaceuticals) to create a change in the functioning of the cell, organ or
organism.
Such agents can include, but are not limited to nucleic acids,
polynucleotides,
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antibacterial agents, antiviral agents, antifungal agents, anti-parasitic
agents,
tumoricidal or anti-cancer agents, proteins, toxins, enzymes, hormones,
neurotransmitters, glycoproteins, immunoglobulins, immunomodulators, dyes,
radiolabels, radio-opaque compounds, fluorescent compounds, polysaccharides.
cell
receptor binding molecules, anti-inflammatories, anti-glaucomic agents,
mydriatic
compounds, and local anesthetics.
~Biological ~uertv: Any property of a biological agent or biological agent
composition that affects the action of the biological agent or biological
agent
composition during interaction with a biological system. This includes
solubility,
stability, analysis of spectral properties, binding with plasma proteins, DNA,
RNA,
specific receptors. enzymes or other molecules, resistance to metabolic
enzymes,
chemical stability, toxicity, membrane transport, of transport into, out of,
within and
through target cells, tissues or organs, site specific delivery, specific
enzymatic
activities, activation or suppression of gene expression, total DNA, RNA and
protein
biosynthesis, cell proliferation and differentiation, apoptosis, hormone and
polypeptide secretion, bioavailability, phatmacokinetics, pharmacodynamics,
efficacy, toxicity, therapeutic index and the like.
~Canier: Segmented copolymers, and mixtures thereof including mixtures with
other segmented copolymers, homopolymers, biological agents. and surfactants.
~ omputational a_n_aly~: A computer program which analyzes structures of new
carriers in a virtual library, predicts their interaction with the drug
candidate, and
selects the most promising Garners.
~Drug candidate: A substance with biological activity potentially useful for
therapy. For the new composition development, the drug candidate can be used
as a
chemical substance or theoretical model defining the molecular structure and
known
properties including physicochemical properties and biological activity,
mechanism
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of action, disease target, initial screening results, and known or expected
problems
with pharmaceutical application.
~uigh ro ghput screeni~: Use of the set of analytical methods and procedures
to test the properties of the library of the biological agent compositions.
This includes
screening of the composition using a biological model, for example, cell,
animal or
plant, measurement of physicochemical property, computational analysis, and
the
like.
~Librarv of biological aEent com~o,~itions: A plurality of compositions of
biological agents with carriers.
~Parent database of carriers: A computer database containing information on
known drug carriers which includes (but is not limited to) at least one of the
following: structure of the carrier molecules (segmented copolymers),
structure and
properties of its building blocks (segments), molecular architecture, and
available data
on properties of compositions of these Garners with various molecules,
including
physicochemical properties and biological activity, mechanism of action,
disease
target, initial screening results, and known or expected problems with
pharmaceutical
application.
~p,;~aring com os~: Creation of compositions of a biological agent including
drug candidates with a carrier. This includes mixing of the biological agent
and the
carrier under specific conditions of solvent composition, concentration, pH,
temperature and the like as well as creation of computer database including
parent
database of carriers and information on biological agent including but not
limited to
chemical structure. This database can also contain available data on
properties of
biological agents, such as physicochemical properties, biological activity,
and
mechanism of action, disease target, initial screening results, and known or
expected
problems with pharmaceutical application.
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~Segmented copolymer: A conjugate of at least two different polymer segments.
~Surfactant: A surface active agent that is adsorbed at interface.
~Testing composition: Evaluation of the properties of composition using a
biological model, including but not limited to cell, animal or plant models,
measurement of physicochemical property of composition, and computational
analysis.
~Virtual library: A list of carriers potentially useful to the drug candidate.
The invention allows rapid selection and design of a carrier to meet specific
delivery and efficacy criteria. The invention may incorporate the use of, (i)
parental
databases having a large number of chemical templates, (ii) exploratory
virtual
libraries or carriers, (iii) computational analysis for predicting chemical
and physical
properties of the complexed compounds, (iv) validated solid-phase and solution-
phase
chemistries, and (v) testing.
In one embodiment, the invention relates to methods of identifying biological
agent compositions of choice comprising:
(a) preparing a plurality of segmented copolymers, the segmented copolymers
differing in at least one of the following, (i) at least one of their segment
lengths, (ii)
chemical structure, (iii) copolymer architecture;
(b) preparing compositions of the segmented copolymers with at least one
biological agent;
(c) testing at least one of the compositions of segmented copolymers with a
biological agent for biological properties using a cell, animal, plant or
other biological
model, or measurement of a chemical or physical property in a test tube, or a
theoretical model; and
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(d) identifying the compositions with desired biological properties.
In another preferred embodiment, the segmented copolymer has at least one
hydrophilic nonionic polymer and at least one hydrophobic nonionic segment. In
another preferred embodiment, the segmented polymers have at least one
cationic
segment and at least one nonionic segment. In yet another preferred
embodiment, the
segmented polymers have at least one anionic segment and at least one nonionic
segment. Also preferred are compositions where the segmented polymers have at
least one polynucleotide segment and at least one segment which is not a
nucleic acid.
Further preferred are compositions where the polymer segments comprise at
least one
polypeptide segment and at least one non-peptide polymer segment.
The biological agent is an agent which is useful for diagnostics or imaging or
that
can act on a cell, organ or organism to create a change in the functioning of
the cell,
organ or organism: This includes, but is not limited to, pharmaceutical
agents, genes,
vaccines, herbicides and the like.
The term "preparing" is used in the broad sense to include design of
theoretical
models for computational analysis. The invention does not require that all or
even
any of segmented copolymers are synthesized, or that all or any of
pharmaceutical
compositions are actually prepared. The plurality of segmented copolymers can
be
constructed "on paper" and then tested in a computer database. Similarly, the
compositions of the segmented copolymers with biological agents can be
presented as
a database.
The "testing" can be done using a variety of computational methods. so that
part
of or all process of the identification can be carried out or simulated
virtually (i. e.. by
computer).
The term "basis" refers to the plurality of segmented copolymers used in
accordance with the invention.
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The ability of segmented copolymers to form micelles, capture and release
biological agents, interact with various systems of cell and organism
affecting
biological properties or otherwise interact modifying the biological response
with
respect to a certain biological agent depends upon the lengths (number of
repeating
units) in the segmented copolymer.
Within a "basis" of segmented copolymers, it has been discovered that there
will
be at least one copolymer which will form a composition with a biological
agent
exhibiting the desired biological properties. Because of the complex
relationship
between the biological properties and biological agent compositions, the
discovery
and optimization of useful biological agent compositions is ordinarily a very
time-
consuming process, requiring a high number of trials. The present invention
provides
a rational combinatorial method for identifying a useful biological agent
composition
by determining which copolymer of the basis possesses the desired properties
with a
specific biological agent.
1 S In a preferred embodiment, the segmented copolymers have two segments
having
chemically different repeating units, designated "A" type and "B" type. The
length of
the A-type segment is designated as "n" and the length of B-type segment is
"m".
The length of the segment can be defined as its molecular mass or
polymerization
degree, or number of repeating units, atoms or the like. For example, if the
length of
the B-type segment is determined as a molecular mass of this segment. Mb, then
the
length of the A-type segment is calculated as follows:
_ 100-P
Ma Mb P
(1)
wherein P is the weight percentage of B-type segment in the block copolymer.
Assuming for simplicity that m and n designate the number of repeating units
of
the B- and A-type segments, and m and n can vary from 1 to N independently of
each
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other, this will produce a base of N2 segmented copolymers having a general
formula
A~Bm. This base can be presented as a plurality of points in the 2-dimensional
(2D)
coordinate system { n, m } .
In accordance with the present invention, the library of biological agent
compositions is tested for a useful biological property ("BP"). The BP can be
any
biological property including a drug therapeutic index, protein expression for
a gene,
immune response for a vaccine, or the like. The plurality of biological
properties of
the compositions within a composition library can be presented as a plurality
of points
in a three-dimensional space defined by a three-dimensional (3D) coordinate
system
{n, m, BP}. The mufti-dimensional space corresponding to the library of
biological
agent compositions and certain biological property is termed herein as "the
composition space." For example, using the base of A~Bm diblock copolymers,
the
plurality of biological properties represent a three-dimensional network. The
problem
of identifying the optimal (for example maximal) biological parameter by
testing the
composition library is in essence equivalent to the mathematical problem of
determining the extremum on a network (see Figure 1 ).
Computational and semi-computational methods have been developed to solve
such problems (see, for example, G.J. Borse, Numerical Methods With. Matlab: A
Resource for Scientists and Engineers (1997); A. Dolan, J. Aldous.. Networks
and
Algorithms.' An Introductory Approach ( 1994). These methods provide for
"rational
testing", so that the number of compositions that have to be tested to
identify the
desired biological property is substantially less that the total number of
compositions
in the library. For example, in the composition library on the base of A"Bm
copolymers the total number of compositions equals NZ. The number of tests to
be
performed with this library to identify optimal biological agent composition
approximates N. Using data from independent experiments, which are
collectively
called herein the "clues", one can further decrease the number of the tests to
be
performed.
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The segmented copolymers of the present invention can contain more than two
segments. The lengths of the copolymer segments and the BP will provide
variables
in the composition space. For a base of copolymers having k-segments, the
composition space is presented as follows: {nt, n2 ...nk, BP}. Similarly,
several
segmented copolymers with different architecture can be used in one library.
In this
case, the type of copolymer architecture can provide an additional variable in
the
composition space. For example, if j-types of the block copolymer
architectures
defined as r~ are used, then the composition space is as follows: {n,, n2
...nk, r~, BP}.
The identification of the useful biological agent composition of the present
invention
may require testing the compositions for more than one biological property. If
one
different biological property is to be tested. the composition space is as
follows: {n,,
n2 ...nk, r~, BPS, BP,... BP,}. Concentrations of segmented copolymers and
biological agents in the library of biological compositions can be also
varied. In this
case the concentrations of the copolymer, c~, biological agent, cb, provide
additional
variables in the composition space: {n,, n2 ...nk, r~, c~, cb, BP,. BP2...
BP,}.
In another preferred embodiment, the biological agent compositions contain
cationic, anionic or nonionic surfactants. In such cases. the concentrations
of
surfactant, (cs) provide additional variables in the composition space: {n,,
n2 ...nk, r~.
c~, cs, cb, BPI, BP,... BP,}. Instead of the concentration of the surfactant,
the ratio of
concentration of surfactants and segmented copolymers or similar dependent
parameters can be used. The library can be designed using a set of homologous
surfactants, for example, these differing in the length of hydrophobic groups,
so that
certain compositions in the library will differ from each other by type of the
surfactant
used. One example includes fatty acid soaps of saturated and unsaturated fatty
acids.
The block copolymer basis can be designed with fatty acids having varying
lengths of
the hydrocarbon tail (~), degree of saturation (O), and position of
unsaturated bonds
(y). All of these parameters wil! be additional variables in the composition
space: {n,,
n2 ...nk, h~, ~, O, Y, C~, CS, Cb, BPS, BPz... BPS}.
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The composition space can be designed using parameters that are dependent on
the parameters characterizing the copolymer base and composition library, such
as
(n~, n2 ...nk, r~, ~, D, y, c~, cs, cb). It its believed, for example, that
the critical micelle
concentration (CMC) of certain amphiphilic segmented copolymers, such as
polyethylene oxide)-block-polypropylene oxide)-block-polyethylene oxide) block
copolymers (also known under a name Pluronics'~, depends upon the length of
polymer segments (See Kabanov, et al., Macromolecules, 28:2303-2314, 1995).
Similarly, the partitioning coefficient of a drug in the block copolymer
micelles, P, in
certain cases depends on the length of the segments of this block copolymer
(Kabanov, et al., Macromolecules, 28:2303-2314, 1995). The relationship
between
CMC and P is shown in Figure 2.
In some cases, it is beneficial to use such dependent parameters as CMC and P
to
design the composition space. One example is a library of compositions of a
pharmaceutical drug (e.g., anticancer antibiotic, neuroleptic, anti-HIV drug
or the
like) with Pluronic block copolymers. In this case the copolymer
concentration, c,
serves as third useful parameter in the composition space. In the current
example the
concentration of Pluronic copolymers is varied from about 0.000001% (w/v) to
about
10% (w/v) or the solubility limit of the given copolymer at 37°C.
Therefore the
composition space can be presented as: {CMC, P, c, BP}, where BP is a useful
biological property (such as the therapeutic index).
Without wishing to be bound to a specific theory, one can exemplify the use of
CMC and P in the composition space using Pluronic block copolymers. Pluronic
micelles play an important role in certain biological agent compositions
(Kabanov et
al. FEBS Lett 258:343, 1989). Those micelles surround a drug with a
biologically
inert polymer shell, protect it while in the blood stream, deliver it to a
target cell and
dispense it to the target's intracellular compartment. In this way, the non-
targeted
cells are protected from the drug's potentially toxic effects (Kabanov et al.,
J. Contr.
Release, 22:141, 1992). At the same time, the single chains of Pluronic block
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copolymers (so-called "unimers") inhibit certain drug efflux mechanisms
resulting in
increased cytotoxic activity of the drugs against multiple drug resistant
(MDR)
tumors (Alakhov et al. Bioconjugate Chemistry 7: 209 (1996); Venne et al.,
Cancer
Research, 56:3626, ( 1996)). Similar mechanisms underlie the effects of
Pluronic
copolymers on drug transport across the blood-brain barrier and intestinal
epithelium.
By changing the lengths of the ethylene(oxide) and propylene(oxide) segments,
one
can select Pluronic copolymers which are more effective or less effective with
respect
to certain classes of cells and drug transport systems.
Therefore, the effects of block copolymers in biological agent compositions
are
two-fold. First, they form self assembled drug carriers masking the drug from
the
undesired interactions in the body and providing for site specific drug
delivery.
Second, they act as modifiers of biological response with respect to a drug by
affecting drug transport systems in the cell. These effects of block copolymer
are
related to the CMC and P.
Lower CMC and higher P indicate: (a) more stable micelles, (b) less block
copolymer unimers available in solution, and (c) stronger attachment of the
drug to
micelle. These conditions correspond to the highest degrees of protection of
the drug
by the micelle from degradation and elimination by the body defense
mechanisms.
Also, the metabolism of the drug is minimal under these conditions and the
drug toxic
effects are minimized. However, the release of the drug from the micelles in
the
disease site is also minimal, thereby decreasing drug therapeutic effect. The
concentration of the copolymer unimers is very low, which decreases the
effects of
the biological agent composition.
In contrast, when the CMCs are high and P is low, the micelles are very
unstable,
the concentration of unimers is high, and the drug is easily released in a
free form. At
the same time, high CMC is usually observed with hydrophilic block copolymers,
which are also not active with respect to the drug transport systems. Further,
since
the drug is mainly in freeform, it is also not protected from metabolic
degradation,
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and is more toxic than micelle-incorporated drugs. Therefore, with certain
applications (for example anticancer antibiotics), selection of the optimal
block
copolymer composition yields Pluronic copolymers with intermediate CMC and P
values. The CMC vs. P graph in Figure 2 provides a useful tool in the
identification
of such compositions with Pluronic block copolymers. It simplifies testing and
identification procedures by using CMC vs. P isotherm instead of
multidimensional
space with the lengths copolymer segments as the coordinates. The information
about
the effects of the unimers and the micelles in drug actions provides the clues
permitting to simplify the identification of the useful biological agent
composition.
Normally the clues are used to decrease the number of coordinates in the
composition
space as well as the number of compositions to be tested.
Segmented copolymers. The segmented copolymers of this invention are most
simply defined as conjugates of at least two different polymer segments (see
for
example, Tirrel, Interactions of Sur, f'actants with Polymers and Proteins,
Goddard and
Anantha-padmanabhan, Eds., pp. 59 et seq., CRC Press, Boca Raton, Ann Arbor,
London, Tokyo, 1992). Some segmented copolymer architectures are presented
below:
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Diblock Triblock
Multiblock
L
Graft
~ln~'~Aa_l~~~rblock
{The straight and wavy lines designate different polymer segments while the
circles designate the links between these segments.)
The simplest segmented copolymer architecture contains two segments joined at
their termini to give an A-B type diblock. Conjugation of more than two
segments by
their termini yields an A-B-A type triblock, ...ABAB... type multiblock, or
even
multisegment ...ABC... architectures. If a main chain in the segmented
copolymer
can be defined in which one or several repeating units are finked to different
polymer
segments, then the copolymer have a graft architecture, e.g., A(B)A type. More
complex architectures include for example (AB)" or A"Bm starblocks that have
more
than two polymer segments linked to a single center.
One method to produce segmented copolymers includes anionic polymerization
with sequential addition of two monomers. See for example, Schmolka J., Am.
Oil
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Chem. Soc., 1977, 54:110; Wilczek-Vera et al., Macromolecules, 1996, 29:4036.
This technique yields block copolymers with a narrow molecular mass
distribution of
the polymeric segments. Solid-phase synthesis of block copolymers has been
developed recently that permit controlling the growth of the polymer segments
with
very high precision (Vinogradov et al., Bioconjugate Chemistry, 7:3, 1996). In
some
cases the block copolymers are synthesized by initiating polymerization of a
polymer
segment on ends of another polymer segment (Katayose and Kataoka, Proc.
Intern.
Symp. Control. Rel. Bioact. Materials, 1996, 23:899) or by conjugation of
complete
polymer segments (Kabanov et al., Bioconjugate Chem., 1995, 6:639: Wolfert et
al.,
Human Gene Ther., 1996, 7:2123). Properties of block copolymers in relation to
this
invention are determined by ( 1 ) block copolymer architecture and (2)
properties of the
polymer segments. They are independent on the chemical structure of the links
used
for conjugation of these segments (see, e.g., Tirrel In Interactions of
Surfactants with
Polymers and Proteins, Goddard and Ananthapadmanabhan, Eds., pp. 59 et seq.,
CRC
I S Press, Boca Raton, Ann Arbor, London, Tokyo, 1992; Sperling, Introduction
to
Physical Polymer Science, 2d edn., p. 46 et seq., John Wiley & Sons, New York,
I 993).
Linking can be accomplished by a number of reactions, many of which have been
described generally in conjugate chemistry. These can involve a terminal
hydroxyl
Group on one polymer segment, e.g., RS-O-(C,H40)-H, in which RS is hydrogen or
a
blocking group such as alkyl, and an appropriate group on another polymer
segment.
the two being joined directly or indirectly; i.e., through a third component.
Alter-
natively, a terminal group can be converted to some other functional group,
for
example amino, which then is allowed to react either with the next polymer
segment
or another linking component. The linking group thus may be formed either by
reac-
tively involving a terminal group of a polymer segment or by replacing the
terminal
group. For example, a carboxylic acid group can be activated with N,N'-
dicyclohexylcarbodiimide and then allowed to react with an amino or hydroxy
group
to form an amide or ether respectively. Anhydrides and acid chlorides will
produce
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the same links with amines and alcohols. Alcohols can be activated by
carbonyldiimidazole and then linked to amines to produce urethane linkages or
activated to produce ethers or esters. Alkyl halides can be converted to
amines or
allowed to react with an amine, diamines, alcohols, or diol. A terminal
hydroxy
group can be oxidized to form the corresponding aldehyde or ketone. This
aldehyde
or ketone is then allowed to react with a precursor carrying a terminal amino
group to
form an imine which, in turn, is reduced, with (for example) sodium
borohydrate to
form the secondary amine. See Kabanov et al., J. Controlled Release, 22:141
(1992);
Meth. Enzymol., XLVII, Hirs & Timasheff, Eds., Acad. Press, 1977. The linkage
thereby formed is an -NH- group, replacing the terminal hydroxyl group of the
polymer segment.
Alternatively, a terminal hydroxyl group on the polymer can be allowed to
react
with bromoacetyl chloride to form a bromoacetyl ester which in turn is allowed
to
react with an amine precursor to form the -NH-CH2-C(O)- linkage. Immobilized
Enzymes, Berezin et al., Eds., MGU, Moscow, 1976, i.e., -NH-CH2-C(O)-. The
bromoacetyl ester of a polymer segment also can be allowed to react with a
diaminoalkane of the formula NHZ-CqH2q-NH2 which in turn is allowed to react
urith
an carboxy group on another polymer segment, or an activated derivative
thereof such
as an acid chloride or anhydride. The bromoacetyl ester also can be allowed to
react
with a cyanide salt to form a cyano intermediate. See, e.g., Sekiguchi et al.,
J.
Biochem., 85, 75 (1979); Tuengler et al., Biochem. Biophys. Acta, 484, 1
(1977);
Browne et al, BBRC, 67, 126 (1975); and Hunter et al., J.A.C.S., 84, 3491
{1962).
This cyano intermediate then can be converted to an imido ester, for instance
by
treatment with a solution of methanol and hydrogen chloride, which is allowed
to
reacted with a amine precursor to form a -NH-C(NH2+)CH2C(O)- linkage. A
terminal hydroxyl group also can be allowed to react with 1,1'-carbonyl-bis-
imidazole and this intermediate in turn allowed to react with an amino
precursor to
form a -NH-C(O)O- linkage. See Bartling et al., Nature, 243:342 (1973).
17
*rB
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A terminal hydroxyl also can be allowed to react with a cyclic anhydride such
as
succinic anhydride to yield a half ester which, in turn, is allowed to react
with a
precursor having terminal amonogroup using conventional condensation
techniques
for forming peptide bonds such as dicyclohexylcarbodiimide, diphenylchlorophos-
phonate, or 2-chloro-4,6-dimethoxy-1,3,5-triazine. See e.g., Means et al.,
Chemical
Modification of Proteins, Holder-Day (1971). Thus formed is the -NHC(O)-
(CH2)qC(O)O- linkage.
A terminal hydroxyl group also can be allowed to react with I,4-butanediol
diglycidyl ether to form an intermediate having a terminal epoxide function
linked to
the polymer through an ether bond. The terminal epoxide function, in turn, is
allowed
to react with an amino precursor. Pitha et al., Ezrr. J. Biochem., 94:11
{1979); Elling
and Kula, Biotech. Appl. Biochem., 13:354 ( 1991 ); Stark and Holmberg,
Biotech.
Bioeng., 34:942 (1989).
Halogenation of a terminal hydroxyl group permits subsequent, reaction with an
IS alkanediamine such as 1,6-hexanediamine. The resulting product then is
allowed to
react with carbon disulfide in the presence of potassium hydroxide, followed
by the
addition of proprionyl chloride to generate a isothiocyanate which in turn is
allowed
to react with an amino precursor to yield a -N-C(S)-N-{CH2)6-NH- linkage. See
Means et al., Chemical Modification of Proteins. Holder-Day (1971). The
polymer
chain terminating in an amino group also can be treated with phosgene and then
another polymer segment containing amino group to form a urea linkage. See
Means
et al., Chemical Modification of Proteins, Holder-Day (1971).
The polymer segment terminating in an amino group also can be treated with
dimethyl ester of an alkane dicarboxylic acid and the product allowed to react
with an
amino precursor to produce a -N-C(NH2+)-(CH2)4-C{NH2+)-N- linkage. See Lowe et
al., Affinity Chromatography, Wiley & Sons (1974). The polymer segment
terminating in an amino group can also be allowed to react with an alkanoic
acid or
fluorinated alkanoic acid, preferably an activated derivative thereof such as
an acid
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chloride or anhydride, to form a linking group -CONH-. Alternatively, an amino
precursor can be treated with an a.w-diisocyanoalkane to produce a -
NC(O)NH(CH2)6NHC(O)-N- linkage. See Means, Chemical Modification of
Proteins, Holden-Day ( 1971 ). Some linking groups thus can simply involve a
simple
functional group while others may have a spacer unit such as a polymethylene
chain
between two functional groups. When the linking group has such a polymethylene
chain, it can have as few as two methylene units but preferably contains more;
e.g.,
six or more methylene units. The above descriptions exemplify typical
strategies for
the formation of linkages between the segments of the copolymers. These
procedures
parallel those which are known to form conjugates of biologically active
agents and
other agents, including the general conjugation methods described by Means et
al.,
Chemical Modification of Proteins, Holden-Day ( 1971 ); Glazer et al.,
Chemical
Modification of Proteins, Elsevier, New York (1975); Immunotechnology Catalog
&
Handbook, Pierce Chemical Co.; and Polyethylene Glycol Derivatives Catalog,
Shearwater Polymers, Inc. ( 1994). It also will be appreciated that linkages
which are
not symmetrical, such as -CONH- or -NHCOO-, can be present in the reverse
orientation; e.g., -NHCO- and -OCONH-, respectively.
The polymeric segments of the copolymers can be nonionic water-soluble,
nonionic hydrophobic or poorly water soluble, cationic, anionic or
polyampholite,
such as a polypeptide. It is preferred that the degrees of polymerization of
these
polymer segments independently from each other are from about 3 to about
500,000
more preferably from about 5 to about 5000, still more preferably from about
20 to
about 500. If more than one segment of the same type has one segmented
copolymer,
then these segments may all have the same lengths or may have different
lengths.
In a preferred embodiment, at least one segment of the copolymer is a nontoxic
and non-immunogenic polymer which is soluble in water. Such segments include
(but not are limited to) polyethers (e.g., polyethylene oxide),
polysaccharides (e.g.,
dextran), polyglycerol, homopolymers and copolymers of vinyl monomers (e.g.,
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polyacrylamide, polyacrylic esters (e.g., polyacryloyl morpholine),
polymethacrylamide, poly(N-(2-hydroxypropyl)methacrylamide, polyvinyl alcohol,
polyvinyl pyrrolidone, polyvinyltriazole, N-oxide of polyvinylpyridine,
copolymer of
vinylpyridine and vinylpyridine N-oxide) polyortho esters, polyaminoacids,
polyglycerols (e.g., poly-2-methyl-2-oxazoline, poly-2-ethyl-2-oxazoline) and
copolymers and derivatives thereof.
Preferred nonionic hydrophobic and poorly water soluble segments include
polypropylene oxide, copolymers of polyethylene oxide and polyethylene oxide,
polyalkylene oxide other than polyethylene oxide and polypropylene oxide,
homopolymers and copolymers of styrene (e.g., polystyrene), homopolymers and
copolymers isoprene (e.g., polyisoprene), homopolymers and copolymers
butadiene
(e.g., polybutadiene), homopolymers and copolymers propylene (e.g.,
polypropylene),
homopolymers and copolymers ethylene (e.~ , polyethylene), homopolymers and
copolymers of hydrophobic aminoacids and derivatives of aminoacids (e.g.,
alanine,
valine, isoleucine, leucine, norleucine, phenylalanine, tyrosine, tryptophan,
threonine,
proline, cistein, methionone, serine, glutamine, aparagine), homopolymers and
copolymers of nucleic acid and derivatives thereof.
Preferred polyanion segments include those such as polymethacrylic acid and
its
salts, polyacrylic acid and its salts. copolymers of methaerylic acid and its
salts,
copolymers of acrylic acid and its salts, heparin, polyphosphate, homopolymers
and
copolymers of anionic aminoacids (e.g., glutamic acid, aspartic acid),
polymalic acid,
polylactic acid, polynucleotides, carboxylated dextran, and the like.
Preferred polycation segments include polylysine, polyasparagine,
homopolymers and copolymers of cationic aminoacids (e.g., lysine, arginine.
histidine), alkanolamine esters of polymethacrylic acid {e.g., poly-
(dimethylammonioethyl methacrylate), polyamines (e.g:, spermine, polyspermine,
polyethyleneimine, polypropyleneimine, polybutileneimine, poolypentyleneimine,
polyhexyleneimine and copolymers thereof), copolymers of tertiary amines and
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secondary amines, partially or completely quaternized amines, polyvinyl
pyridine and
the quaternary ammonium salts of the polycation segments. These preferred
polycation segments also include aliphatic, heterocyclic or aromatic ionenes
(Rembaum et al., Polymer letters, 1968, 6;159; Tsutsui, T., In Development in
ionic
polymers -2, Wilson A.D. and Prosser, H.J. (eds.) Applied Science Publishers,
London, new York, vol. 2, pp. 167-187, 1986).
Additionally, dendrimers, for example, polyamidoamines of various generations
(Tomalia et al., Angew. Chem., Int. Ed. Engl. 1990, 29, 138) can be also used
as to
design the base of copolymers for combinatorial drug delivery in accordance
with the
current invention.
Particularly preferred are copolymers selected from the following polymer
groups:
(a) segmented copolymers having at least one hydrophilic nonionic polymer and
at least one hydrophobic nonionic segment ("hydrophilic-hydrophobic
copolymers'');
(b) segmented copolymers having at least one cationic segment and at least one
nonionic segment ("cationic copolymers");
(c) segmented copolymers having at least one anionic segment and at least one
nonionic segment ("anionic copolymers");
(d) segmented copolymers having at least one polypetide segment and at least
one non-peptide segment ("polypeptide copolymers");
(e) segmented copolymers having at least one polynucleotide segment and at
least one segment which is not a nucleic acid "polypeptide copolymers");
H,~philic- xdrophobic colz_l erg. Typical representatives of hydrophilic-
hydrophobic copolymers are the block copolymers of ethylene oxide and
propylene
oxide having the formulas:
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H3
HO CH2CH20 CHCH20 CH2CH20 H
x y z
(I)
H3
HO CH2CH20 CHCH20 H
x y
(II)
I H3 . ~H3
HO CHCH2 H2CH20 CHCH20 H
x y z
(III)
R1 R2 1 2
II
H[OCH2CH2]~- [OCHCH]~\ / [CHCHO]~- [CH'CH20]~ H
NCH2CH2N
H[OCH2CH2]~- [OCHCH]~/ \ [CHCHO]~- [CH2CH20]i H
1 ~ 2
(I~
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1112 1 2
H [CHCHO] ~ [CH2CH20]~ \ / [OCH2CH2] ~- [OCHCHJ~ H
NCH2CH2N
H [ H HO]~- [CH2CH20]~~ \ [OCH2CH2]~- [OCHCH]~H
R ~ R2 ~ 1 ~2
(IV-A)
in which x, y, z, i and j have values from about 2 to about 800, preferably
from
about S to about 200, more preferably from about 5 to about 80. and wherein
for each
R', Rz pair, one is hydrogen and the other is a methyl group.
Formulas (I) through (III) are oversimplified in that, in practice, the
orientation of
the isoprvpylene radicals within the B block will be random. This random
orientation
is indicated in formula (IV), which is more complete. Such poly(oxyethylene)-
poly(oxypropylene) compounds have been described by Santon, Am. Perfumer
Cosmet., 72(4):54-58 (1958); Schmolka, Loc. cit. 82(7):25 (1967); Schick, Non-
ionic
Surfactants, pp. 300-371 (Dekker, NY, 1967). A number of such compounds are
commercially available under such generic trade names as "poloxamers",
"pluronics''
and "synperonics." Platonic polymers within the B-A-B formula are often
referred to
1 S as "reversed" pluronics, "Platonic-R" or "meroxapol." The "polyoxamine"
polymer
of formula (XVII) is available from BASF (Wyandotte, MI) under the tradename
TetronicTM. The order of the polyoxyethylene and polyoxypropylene blocks
represented in formula (XVII) can be reversed, creating Tetronic-RTM, also
available
from BASF. See, Schmolka, J. Am. Oil Soc., 59:110 (1979). Polyoxypropylene-
polyoxyethylene block copolymers can also be designed with hydrophilic blocks
comprising a random mix of ethylene oxide and propylene oxide repeating units.
To
maintain the hydrophilic character of the block, ethylene oxide will
predominate.
Similarly, the hydrophobic block can be a mixture of ethylene oxide and
propylene
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WO 99/08112 PCT/US98/16300
oxide repeating units. Such block copolymers are available from BASF under the
tradename PluradotTM.
The diamine-linked pluronic of formula (IV) can also be a member of the family
of diamine-linked polyoxyethylene-polyoxypropylene polymers of formula:
R1 R2 3 4 RS 6
fifi Ifi
~ / CH2CH20 CH2CH20 CH2CH~0 H
'~
., N R N,
(V)
wherein the dashed lines represent symmetrical copies of the polyether
extending
off the second nitrogen, R* is an alkylene of 2 to 6 carbons, a cycloalkylene
of 5 to 8
carbons or phenylene, for R~ and R2, either (a) both are hydrogen or (b) one
is
hydrogen and the other is methyl, for R3 and R4 either (a) both are hydrogen
or (b)
one is hydrogen and the other is methyl, if both of R3 and R4 are hydrogen,
then one
RS and R6 is hydrogen and the other is methyl, and if one of R3 and Ra is
methyl, then
both of RS and R6 are hydrogen.
Those of ordinary skill in the art will recognize, in light of the discussion
herein,
1 S that even when the practice of the invention is confined for example, to
poly(oxyethylene)-poly(oxypropylene) compounds, the above exemplary formulas
are too coning. Thus, the units making up the first block need not consist
solely of
ethylene oxide. Similarly, not all of the B-type block need consist solely of
propylene
oxide units. Instead, the blocks can incorporate monomers other than those
defined in
formulas (I)-(V), so long as the parameters of the first embodiment are
maintained.
Thus, in the simplest of examples, at least one of the monomers in block A
might be
substituted with a side chain group as previously described.
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A variety of other examples of hydrophilic-hyrophobic block copolymers have
been synthesized that can be used in the present invention. These copolymers
have
the general formula A"Bm, wherein A is the hydrophilic homopolymer or
copolymer
segment, and B is a hydrophobic homopolymer or copolymer segment. Each of the
A
and B segments can be either straight chain or branched. Examples of block
copolymers that are particularly useful in the current invention include, but
are not
limited to polyethylene oxide)-b-poly(isoprene)-b-polyethylene oxide) triblock
copolymer (Morgan, et al., Biochem. Soc. Trans., 18:1021, 1990), polyethylene
oxide)-b-polystyrene) block copolymer (Dunn, et al., Pharm. Res., 11:1016,
1994),
polyethylene oxide)-b-poly(D,L-lactide) diblock copolymer (Hagan, et al.
Langmuir
12:2153, 1996), and polyethylene oxide)-b-poly((-benzyl L-aspartate) diblock
copolymer (Kwon. et al. Langmuir 12:945. 1993).
The hydrophilic homopolymer or copolymer A segments in hydrophilic-
hyrophobic block copolymers that can be used in the present invention will
contain at
least three monomeric units, each of which unit will have the same or
different
pendant group. Each pendant group will contain at least one atom selected from
the
group consisting of oxygen and nitrogen. Representative hydrophilic
homopolymers
or copolymers include but are not limited to polyethylene oxides, copolymers
of
ethylene oxide and propylene oxide, polysaccharides, polyacrylamides,
polygycerols,
polyvinylalcohols, poiyvinylpyrrolidones, polyvinylpyridine N-oxides,
copolymers of
vinylpyridine N-oxide and vinylpyridine, polyoxazolines, and
polyacroylmorpholines.
Preferably, the hydrophilic A segment is:
CHZ CH2 O
m
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a copolymer of
H3
CH2 CHz O and CHCH20 H
m _
CH2 CH
m
C=O
NHZ
CH2 CH
m
OH
CH2 CH
m
C=O
j
~O
CH2 CH
m
=O
N ~N
n~
N
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CH2 CH
m
C=O
N
C~
O
or
CH2 CH
m
C=O
N
O
in which each of m and j has a value of from 3 to 5000.
The hydrophobic B segments useful in this invention can also contain
fluorocarbon moieties including but not limited to fluoroalkyl segments, and
copolymers containing both fluorocarbon and hydrocarbon. One such example is
the
segmented block copolymers having the formula:
R~ _L ~ _ 1R2_L2_A ~ w_La_Ra_L3_R3
(VI)
in which:
either, (i) Ri is a monovalent fluorinated hydrocarbon of 2 to 50 carbon atoms
and R2 is a divalent hydrocarbon of 2 to 50 carbon atoms or (ii) Rl is a
monovalent
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WO 99/08112 PCT/US98/16300
hydrocarbon of 2 to 50 carbon atoms and R2 is a divalent fluorinated
hydrocarbon of
2 to SO carbon atoms;
R3 is, (i) hydrogen, (ii) a monovalent fluorinated hydrocarbon of 2 to 50
carbon
atoms, or (iii) a monovalent hydrocarbon of 2 to 50 carbon atoms;
R4 is, (i) a bond if R3 is hydrogen; (ii) a divalent hydrocarbon of 2 to 50
carbon
atoms if R3 is a fluorinated hydrocarbon, or (iii) a divalent fluorinated
hydrocarbon of
2 to 50 carbon atoms if R3 is a hydrocarbon;
each of L 1 and L2, independently of the other, is a linking group:
L3 and L4 taken together with R4, is a bond if R3 is hydrogen or if R3 is
other than
hydrogen each of L3 and L4, taken independently is a linking group;
A is a hydrophilic homopolymer or copolymer comprising at least three mono-
meric units each having the same or different pendant group containing at
least atom
selected from the group consisting of oxygen and nitrogen; and
w has a value of from 1 to 100.
The hydrophilic homopolymer or copolymer A will contain at least three mono-
meric units, each of which unit will have the same or different pendant group.
Each
pendant group will contain at least one atom selected from the group
consisting of
oxygen and nitrogen. Representative hydrophilic homopolymers or copolymers
include polyethylene oxides, copolymers of ethylene oxide and propylene oxide,
polysaccharides, polyacrylamides, polygycerols, polyvinylalcohols,
polyvinylpyrroli-
dones, polyvinylpyridine N-oxides, copolymers of vinylpyridine N-oxide and
vinylpyridine, polyoxazolines, and polyacroylmorpholines.
Cationic copol, ers. Useful segmented copolymers include a class of
copolymers in which at least one segment is a polycation. One example of these
structures is a basis of copolymers comprising a plurality of covalently bound
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polymer segments wherein the segments have (a) at least one polycation segment
which segment is a cationic homopolymer, copolymer, or block copolymer
comprising at least three aminoalkylene monomers, the monomers being selected
from the group consisting of at least one of the following:
(i) at least one tertiary amino monomer of the formula:
R NCR R4
'R3 Rs
A.
and the quaternary salts of the tertiary amino monomer, or (ii) at least one
secondary amino monomer of the formula:
R NH-R7 Rg
B.
and the acid addition and quaternary salts of the secondary amino monomer, in
which:
R' is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer;
i 5 each of R2 and R3, taken independently of the other, is the same or
different straight
or branched chain alkanediyl group of the formula:
-(CzH2z)"'-
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WO 99/08112 PCT/US98/16300
in which z has a value of from 2 to 8; R4 is hydrogen satisfying one bond of
the
depicted geminally bonded carbon atom; and RS is hydrogen, alkyl of 2 to 8
carbon
atoms, an A monomer, or a B monomer; R6 is hydrogen, alkyl of 2 to 8 carbon
atoms,
an A monomer, or a B monomer; R' is a straight or branched chain alkanediyl
group
of the formula:
-(CzHu~
in which z has a value of from 2 to 8; and R8 is hydrogen, alkyl of 2 to 8
carbon
atoms, an A monomer, or a B monomer; and
{b) at least one straight or branched nonionic hydrophilic segment A having
from
about 5 to about 1000 monomeric units which is defined above.
The polycationic segments in the copolymers of the invention can be branched.
For example, polyspermine-based copolymers are branched. The cationic segment
of
these copolymers was synthesized by condensation of 1,4-dibromobutane and N-(3-
aminopropyl)-1,3-propanediamine. This reaction yields highly branched polymer
products with primary, secondary, and tertiary amines.
An example of branched polycations are products of the condensation reactions
between polyamines containing at least 2 nitrogen atoms and alkyl halides
containing
at least 2 halide atoms (including bromide or chloride). In particular, the
branched
polycations are produced as a result of polycondensation. An example of this
reaction
is the reaction between N-(3-aminiopropyl)-1,3-propanediamine and 1,4-
dibromobutane, producing polyspermine.
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Another example of a branched polycation is polyethyleneimine represented by
the formula:
(NHCH2CH2)X [N(CH2CH2)CH2CH2)y
(VI)
One example of useful polyamine-based copolymers is the polymer of formula:
Ki_ Li_LG-L2_F_L3~1_ K2~
(VII)
in which:
F is a polyamine segment comprising a plurality of repeating units of formula -
NH-R°, wherein R° is an aliphatic group of 2 to 6 carbon
atoms, which may be
substituted;
G is polyethylene oxide or copolymer ethylene oxide and propylene oxide a
straight or branched nonionic segment defined above;
K' and K2 independently of the other, is hydrogen, hydroxy group, amonogroup,
G or F polymer segments;
and each of L~, L2 and L3, independently of the other, is a linking group or
chemical bond.
The amino groups of the polycationic segments can be quaternized to produce
ammonium salts. Examples include polyspermine and polyamines that are modified
with alkylhalides to produce tertiary and quaternized polyamines. Another
useful
type of cationic segments of well defined chemical structure are ionenes that
can be
aliphatic, heterocyclic or aromatic (Rembaum et al. Polymer Letters, 1968,
6:159;
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Tsutsui, T., Development in ionic polymers, Wilson, A.D. and Prosser, H.J.
(eds.),
Applied Science Publishers, London, New York, vol. 2, pp. 163-187, 1986).
Anionic co~ojvmers. Anionic copolymers contain at least one polyelectrolyte
segment that yields a polyanion in an aqueous environment. This includes both
strong polyacids having high ionization degrees in a broad range of pH, and
weak
polyacids characterized by pH-dependent ionization degrees. Anionic segments
normally have a plurality of pendant amino groups such as carobxylic groups,
sulfate
groups, sulfonate groups, phosphate groups, and the like. Examples of anionic
copolymers include but are not limited to polyoxyethylene-b-polymethacrylic
acid
(Wang, et al., J. Polym. Sci., Part A: Polym. Chem., 30:2251, 1992),
polystyrene-b-
polyacrylic acid (Zhong, et al. Macromolecules, 25:7160, 1992), polyacrylic
acid
grafted with polyoxyethylene-b-polyoxypropylene-b-polyoxyethylene (Bromberg
and
Levin, Macromol. Rapid Commun. 17:169 1996).
PolYpepti~ copolymers. Polypeptide copolymers have a plurality of covalently
bound polymer segments wherein the segments have at least one polypeptide
segment
and at least one non-peptide polymer segment. Polypeptide segments have a
plurality
of amino acid units or derivatives thereof.
Examples of useful segmented copolymers containing polypeptides include the
poly(oxyethylene)-poly-L-lysine) diblock copolymer of the following formula:
O
HO-(CH2CH20)i-C- (LYs)~ --
(XVIII)
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WO 99/08112 PCT/US98/16300
wherein i is an integer of from about 2 to about 500, and j is an integer from
about 4 to about 500. A second example is the poly(oxyethylene)-poly-(L-
alanine-L-
lysine) diblock copolymer of formula:
O
HO-(CH2CH20)i-C-(AlaLys)j -COOH
(XIX)
wherein i is an integer of from about 2 to about 500, and j is an integer from
about 2 to about 500.
The use of polypeptide copolymers in the invention allows for better control
of
the polypeptide segment lengths by using solid-phase and solution-phase
chemistries.
Segmented copolymers based on polypeptides with well defined chemical
structures
have been described in the literature, such as poly(amino acid)-b-poly(N,N-
dietylacrylamide)-b-poly(amino acid) (Bromberg and Levin, Bioconjugate Chem.
9:40, 1998). Further, the unit composition and sequence in polypeptides can be
varied including hydrophobic, hydrophilic, ionizable, hydrogen and chemical
bond
forming amino acids and derivatives thereof to produce broader variability in
the
basis of the segmented copolymers.
Polx~~leotide co~[vrners. Polynucleotide copolymers have a plurality of
covalently bound polymer segments wherein the segments have at least one
segment
containing at least three nucleic acid units or the derivatives thereof.
Similar to
polypeptide copolymers, the polynucleotide copolymers provide for better
control
over segment length and sequence by using solid-phase and solution-phase
chemistries. Segmented copolymers based on polynucleotides with well-defined
chemical structure have been described including, polyoxyethylene-b-
polynucleotide
copolymer , and polycation-b-polynucleotide copolymer (Vinogradov et al.,
Bioconjugate Chemistry, 7:3, 1996; See also U.S. Patent No. 5,656,611). As
with
polypeptide copolymers, polynucleotide copolymers permit variation of the unit
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WO 99/08112 PCT/US98/16300
composition and sequence in polynucleotide segments which is particularly
useful in
selecting proper biological agent compositions pursuant to this invention.
Greening a~savs and composition identification. The screening assays of this
invention are analytical tests which are useful in characterizing and
selecting
biological agent compositions by sorting them for "positive" and "negative"
compositions according to the initially defined criteria. Normally, one or
more
screening assays are required to identify a preferable biological agent
composition.
Depending upon the task, varieties of in vitro cell-free and cell-based, as
well as
in vivo screening assays can be used to select preferred biological agent
compositions. This includes, but is not limited to, physico-chemical tests
such as the
characterization of biological agent solubility and stability, analysis of
spectral
properties; characterization of biological agent binding with plasma proteins,
DNA,
RNA, specific receptors, enzymes or other molecules; transport-related tests
such as
analyses of transport into, out of, within, and through target cells, tissues
or organs;
functional tests such as analyses of specific enzymatic activities, activation
or
suppression of gene expression, total DNA, RNA and protein biosynthesis, cell
proliferation and differentiation assays, apoptosis analysis, hormone and
polypeptide
secretion assays; in vivo pharmacological tests, such as pharmacokineticts,
pharmacodynamics of biological agent, its efficacy, toxicity and therapeutic
index.
Since libraries of biological agent compositions are screened in this
invention,
high-throughput and ultra-high-throughput screening assays are preferred.
Depending
on the specific properties of the biological agent, and obstacles related to
the
properties that need to resolved by using present invention, various
combinations of
the screening assays can be used to identify a biological agent composition.
Many biological agents have limited solubility in aqueous solutions,
therefore,
they often cannot be administered at required doses in the body without
specifically
selected and optimized delivery systems for improving biological agent
solubility. In
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WO 99/08112 PCT/US98/16300
one embodiment, libraries of biological agent compositions with segmented
copolymer Garners are generated and screened to improve biological agent
solubility
in aqueous solutions. In the related screening system, solubility of the
biological
agent alone is determined and compared to that of a biological agent
formulated with
the selected carriers. A variety of methods can be used to determine the
biological
agent solubility including but not limiting light absorption, fluorescent,
spectrophothometry, circular dichroism, calorimetry, NMR, ESR, chromatography;
mass spectrometry and the like.
One of the most common problems related to the limited performance of
biological agents is their insufficient stability, which is often related to
their high
sensitivity to metabolic enzymes. Such enzymes, depending on the biological
agent
structure, include proteases, nucleases, redox enzymes, transferases, etc. In
one
preferred embodiment, libraries of biological agent compositions with
segmented
copolymer carriers are generated and screened to protect biological agent from
degradation by metabolic enzymes. The screening can include treatment of the
biological agent with isolated enzymes, their combinations or enzymatic
complexes
existing in isolated fractions of cells or tissues, followed by analysis of
the native
biological agent level in the analyzed sample. The screening can also be based
on the
biological agent administration in a whole organism followed by sampling and
analysis of the native biological agent level in the sample. or by continuous
monitoring of the native biological agent level in the body. Verity of methods
could
be used for detection of the native biological agent level including but not
limiting
HPLC, LC-MS, GC-MS, radioisotope methods, NMR, various bioassays, etc.
Another common obstacle that limits biological agent effectiveness is
insufficient
circulation time in the body due to clearance of the biological agent by the
reticuloendothelial system. According to the present invention, the libraries
of
biological agent compositions with segmented copolymer Garners can be prepared
and screened to reduce the biological agent clearance. The screening methods
can be
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based upon direct measurement of the biological agent binding with serum
proteins
such as albumin, low density and ultralow density lipopolyproteins, and the
like.
Also, these screening methods can use the analysis of the biological agent
phagocytosis by isolated cell populations such as macrophages,
polymorphonulear
cells, etc. The screening can also be based on biological agent administration
in a
whole organism followed by sampling and analysis of the native biological
agent
level in the sample, or by continuos monitoring of the native biological agent
level in
the body. Combinations of the above procedures can also be used.
Reduced efficacy of the biological agent is also often caused by its low
bioavailability. For example, most polypeptides and proteins, polynucleotides,
as
well as many low molecular weight pharmaceutical drugs are not effective when
administered orally. An important factor that limits oral bioavailability of
the above
pharmaceutical agents is their reduced adsorption through small intestinal
epithelial
tissue. According to the present invention, the libraries of such biological
agents with
segmented copolymer Garners can be prepared and screened to increase
biological
agent oral bioavailability. The screening methods include, but are not limited
to the
measurement of the biological agent transport across polarized epithelial cell
monolayers, for example, Caco-2 or Caco-4 cell monolayers. Another
bioavailability-related problem is low efficacy of central nervous system
agents,
which caused by limited transport of the agents across brain microvessel
endothelial
tissue that is also known as blood brain barrier (BBB). Libraries of
biological agent
compositions with segmented copolymer carriers can be generated and screened
for
compositions that increase biological agent transport across BBB. The
screening
methods for this assay can be based on the measurement of the biological agent
transport across polarized endothelial cell monolayers, such as primary bovine
brain
microvessel endothelial cells (BBMEC), human primary and immortalized brain
microvessel endothelial cells, etc. The bioavailability screening procedures
also
include administration of the biological agent in a whole organism followed by
sampling and analysis of the native biological agent level in the sample, or
by
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continuos monitoring of the native biological agent level in the body.
Combinations
of the above procedures can also be used. Depending on the nature of the
biological
agent, the efficacy of its transport can be measured by various methods
including but
not limited to fluorescence, absorption or other spectroscopy, radioisotope
methods,
various bio- or immunoassays, etc.
Biological activity of many biological agents is often significantly reduced
due to
insufficient efficacy of the biological agent transport through the cell
membrane. To
resolve this problem, libraries of biological agent compositions with
segmented
copolymer Garners can be generated and screened for compositions that can
improve
biological agent transmembraneous properties. Verity of screening methods can
be
used to evaluate the efficacy of biological agent transport through the
membrane.
These methods include but not limited by those based on analysis of the
biological
agent transport across artificial membranes such as lipid bilayers and
liposomes,
analysis of the biological agent uptake in cells including, cell lines,
primary cell
cultures, bacterial strains and isolates, etc. Depending on the biological
agent nature,
the efficacy of its transport can be measured by various methods including but
not
limited fluorescent, absorption or other spectroscopy, radioisotope methods,
various
bio- or immunoassays, etc.
The libraries of biological agent composition can be also screened using
direct
measurement of the biological agent biological activity. Depending on the
biological
agent properties, verity of screening methods can be used to analyze
biological agent
biological effect. For example, for many anticancer agents apoptosis assays,
such as
TONEL staining; proliferation assays, such as thymidine DNA incorporation rate
analysis, MTT, XTT and colony formation assays can be used to evaluate
efficacy of
the selected compositions. Cell adhesion based methods could be used for
evaluation
of immune modulating compositions.
Depending on the nature of the biological agent, more specific screening
methods
could be used to evaluate efficacy of the biological agent compositions
including but
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not limiting analyses of activity of specific enzymes that are known to be
direct or
indirect targets for particular biological agent; analysis of signal
transduction events
(such as tyrosine phosphorylation, association or dissociation of SH2 and/or
SH3
signaling proteins, changes in second messenger levels, etc.) that are
involved in the
biological agent mechanism of action; analyses of specific gene expression (by
using
hybridization, RT-PCR and/or protein expression assays) that known to involved
in
the biological agent mechanism of action.
In general, any assay that relates to biological activity, transport,
pharmacokinetics, stability of a biological agent compositions can be used to
screen
libraries of biological agent compositions with segmented copolymer carriers
for the
best performing composition with preselected biological agent.
The screening and identification of the biological agent compositions pursuant
this invention can use a virtual library. Without wishing to be limited to any
particular computational analysis, the use of the virtual library is
exemplified as
follows. The starting information for identifying the desired biological agent
composition includes, (i) the drug structure, (ii) the database of available
polymeric
segments for synthesis of segmented copolymers, and (iii) accumulated data on
segmented copolymer carriers, if available. The starting data also includes
the
selection of desirable biological properties, which identify the preferred
biological
agent composition for the given biological agent including drug candidates.
New
carrier molecules are virtually assembled by combining structures of polymeric
segments stored in computer database. The combination is subjected to the
rules of
the bond formation as if a new compound is synthesized as a result of a
chemical
reaction yielding a segmented copolymer of a specific molecular architecture.
A
plurality of virtually designed segmented copolymers is a virtual copolymer
base.
The physicochemical and biological properties of each copolymer of this base
is then
predicted in relation to its specific architecture using the properties of
separate blocks
and or model segmented copolymers that were actually synthesized and
characterized
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in the experiment. The properties of the isolated segments include but are not
limited
to chemical structures of the repeating units, lengths of polymeric segments,
QSAR
parameters derived from the structures and physicochemical and biological
parameters available from experiments or derived from experimental data.
S Exemplary properties of segments include molecular weight, volume, surface,
hydrophobicity, hydration energy, partitioning coefficients, ionization degree
(for
polyelectrolytes), etc. The prediction of the copolymer property can be based
on any
known or expected relationship between the properties of the segments and the
copolymer property.
Without wishing to be limited to a particulate theory, it is believed that
interpolation or extrapolation of any previously accumulated data on
properties of
copolymers gives the predicted values. At this stage, the data on the
experimentally
determined biological properties of the compositions of the biological agent,
for
example, drug candidate, and actually synthesized model segmented copolymers
are
used in the computerized analysis. The predicted properties of the copolymers
and
their compositions with biological agents are then compared to the desired
values, and
the score of fit is calculated. Finally, the virtual carnet molecules from the
base are
classified according their score, and the best compositions are identified.
Without wishing to be limited by a particular theory, it is believed that
possible
elements in the identification of a biological agent composition with desired
properties include but are not limited to parent databases comprising a large
variety of
chemical templates, exploratory virtual library of carriers, computational
analysis
predicting chemical and physical properties of biological agent compositions,
validated segmented copolymer chemistries (including solid-phase and solution-
phase
chemistries) and high throughput screening.
Biologjc~gents. The biological agents of the invention are agents useful for
diagnostics or imaging, or those that can act on a cell, organ or organism to
create a
change in the functioning of the cell, organ or organism, including but not
limited to
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pharmaceutical drugs, immunoadjuvants, vaccines genes, herbicides and the
like.
Such biological agents are used in e.g., diagnostics, therapy, immunization or
otherwise are applied to combat human and animal disease. Such agents include
but
are not limited to nucleic acids, polynucleotides, antibacterial agents,
antiviral agents,
antifungal agents, antiparasitic agents, tumoricidal or anti-cancer agents,
proteins,
toxins, enzymes, hormones, neurotransmitters, glycoproteins, immunoglobulins,
immunomodulators, dyes, radiolabels, radio-opaque compounds, fluorescent
compounds, polysaccharides, cell receptor binding molecules, anti-
inflammatories,
anti-glaucomic agents, mydriatic compounds and local anesthetics.
Special classes of biological agents that can be used in this invention
include
pharmaceutical drugs. Many drugs are rapidly cleared from the body or are
degraded
by the body's defense mechanisms. These problems, plus toxic side effects.
seriously
limit drug efficacy by reducing time available to the drug to reach its target
and by
limiting the amount of drug which can safely be given to the patient. In
addition,
many drugs do not readily penetrate tissues or effectively seek out and
concentrate in
appropriate cells to maximize their therapeutic effect. The use of the
biological agent
compositions pursuant to this invention permits to significantly improve
therapeutic
drugs by decreasing in their side effects, and increase in therapeutic action.
The biological agents with which the present compositions can be used include
but are not limited to non-steroidal anti-inflammatories such as indomethacin,
salicylic acid acetate, ibuprofen, sulindac, piroxicam, and naproxen,
antiglaucomic
agents such as timolol or pilocarpine, neurotransmitters such as
acetylcholine,
anesthetics such as dibucaine, neuroleptics such as the phenothiazines (for
example
compazine, thorazine, promazine, chlorpromazine, acepromazine, aminopromazine,
perazine, prochlorperazine, trifluoperazine, and thioproperazine), rauwolfia
alkaloids
(for example, resperine and deserpine), thioxanthenes (for example
chlorprothixene
and tiotixene), butyrophenones (for example haloperidol, moperone,
trifluoperidol,
timiperone, and droperidol), diphenylbutylpiperidines (for example pimozde),
and
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benzamides (for example sulpiride and tiapride); tranquilizers such as
glycerol
derivatives (for example mephenesin and methocarbamol), propanediols (for
example
meprobamate), diphenylmethane derivatives (for example orphenadrine,
benzotrapine, and hydroxyzine), and benzodiazepines (for example
chlordiazepoxide
and diazepam); hypnotics (for example zolpdem and butoctamide); beta-Mockers
(for
example propranolol, acebutonol, metoprolol, and pindolol); antidepressants
such as
dibenzazepines (for example, imipramine), dibenzocycloheptenes (for example,
amtiriptyline), and the tetracyclics (for example, mianserine); MAO inhibitors
(for
example phenelzine, iproniazid, and selegeline); psychostimulants such as
phenylehtylamine derivatives (for example amphetamines, dexamphetamines,
fenproporex, phentermine, amfeprramone, and pemoline) and
dimethylaminoethanols
(for example clofenciclan, cyprodenate, aminorex, and mazindol); GABA-mimetics
(for example, progabide); alkaloids (for example codergocrine,
dihydroergocristine,
and vincamine); anti-Parkinsonism agents (for example L-dopamine and
selegeline);
agents utilized in the treatment of Altzheimer's disease, cholinergics (for
example
citicoline and physostigmine); vasodilators {for example pentoxifyline); and
cerebro
active agents (for example pyritinol and meclofenoxate). These agents include
also
DNA topoisomerase inhibitors (including type I and type II), brain and tumor
imaging
agents, free radical scavenger drugs, anticoagulants, ionotropic drugs, and
neuropeptides such as endorphins.
The biological agent compositions also can be used advantageously with anti-
neoplastic agents such as paclitaxel, daunorubicin, doxorubicin, carminomycin,
4'-
epiadriamycin, 4-demethoxy-daunomycin, 11-deoxydaunorubicin, 13-
deoxydaunorubicin, adriamycin-14-benzoate, adriamycin-14-actanoate, adriamycin-
14-naphthaleneacetate, vinblastine, vincristine, mitomycin C, N-methyl
mitomycin C,
bleomycin A2, dideazatetrahydrofolic acid, aminopterin, methotrexate,
cholchicine
and cisplatin, antibacterial agents such as aminoglycosides including
gentamicin,
antiviral compounds such as rifampicin, 3'-azido-3'-deoxythymidine (AZT), and
acylovir; antifungal agents such as azoles including fluconazole, macrolides
such as
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amphotericin B, and candicidin; anti-parastic compounds such as antimonials.
These
biological agents include without limitation vinca alkaloids, such as
vincristine and
vinblastine, mitomycin-type antibiotics, such as mitomycin C and N-methyl
mitomycin, bleomycin-type antibiotics such as bleomycin A2, antifolates such
as
methotrexate, aminopterin, and dideaza-tetrahydrofolic acid, taxanes,
anthracycline
antibiotics and others.
The compositions also can utilize a variety of polypeptides such as
antibodies,
toxins such as diphtheria toxin, peptide hormones, such as colony stimulating
factor,
and tumor necrosis factors, neuropeptides, growth hormone, erythropoietin, and
thyroid hormone, lipoproteins such as ~,-lipoprotein, proteoglycans such as
hyaluronic
acid, glycoproteins such as gonadotropin hormone, immunomodulators or
cytokines
such as the interferons or interleukins, hormone receptors such as the
estrogen
receptor.
The compositions also can be used with enzyme inhibiting agents such as
reverse
transcriptase inhibitors, protease inhibitors, angiotensin converting enzymes,
5~-
reductase, and the like. Typical of these agents are peptide and nonpeptide
structures
such as finasteride, quinapril, ramipril, lisinopril, saquinavir, ritonavir,
indinavir,
nelfinavir, zidovudine, zalcitabine, allophenylnorstatine, kynostatin,
delaviridine, bis-
tetrahydrofuran ligands (see, for example Ghosh et al., J. Med. Chem. 1996,
39:3278), and didanosine. Such agents can be adminitered alone or in
combination
therapy; e.g., a combination therapy utilizing saquinavir, zalcitabine, and
didanosine,
zalcitabine, and zidovudine. See, for example, Collier et al., Antiviral Res.,
1996,
29:99.
The biological agent compositions can be used with nucleic acids such as
thymine, polynucleotides such as DNA or RNA polymers or synthetic
oligonucleotides, which may be derivatized by covalently modifying the 5'- or
the 3'-
end of the polynucleic acid molecule with hydrophobic substituents to
facilitate entry
into cells (see for example, Kabanov et al., FEBS Lett., 1990, 259, 327;
Kabanov and
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Alakhov, J. Contr. Rel., 1990, 28:15). Additionally, the phosphate backbone of
the
polynucleotides has been modified to remove the negative charge (see, for
example,
Agris et al., Biochemistry, 1968, 25:6268, Cazenave and Helene in Antisense
Nucleic
Acids and Proteins: Fundamentals and Applications, Mol and Van der Krol.,
Eds., p.
47 et seq., Marcel Dekker, New York, 1991 ), or the purine or pyrimidine bases
has
been modified, for example, to incorporate photo-induced crosslinking groups,
alkylating, groups, organometallic groups, intercalating groups, biotin,
fluorescent
and radioactive groups (see, for example, Antisense Nucleic Acids and
Proteins:
Fundamentals and Applications, Mol and Van der Krol, Eds., p. 47 et seq.,
Marcel
Dekker, New York, 1991; Milligan et al. In Gene Therapy for Neoplastic
Diseases,
Huber and Laso, Eds. P. 228 et seq., New York Academy of Sciences, New York,
1994). Such nucleic acid molecules can be among other things antisense nucleic
acid
molecules, phosphodiester, oligonucleotide (-anomers, ethylphospotriester
analogs,
phosphorothioates, phosphorodithioates, phosphoro-ethyletriesters,
methylphosphonates, and the like (see, for example, Crooke, Anti-Cancer Drug
Design 1991, 6:609; De Mesmaeker, et al., Acc. Chem. Res. 1995, 28:366). The
invention is used with antigene, ribozyme and aptamer nucleic acid drugs (see,
for
example. Stull and Szoka, Pharm. Res. 1995, 12:465).
Included among the suitable biological agents are viral genomes and viruses
(including the lipid and protein coat). This accounts for the possibility of
using a
variety of viral vectors in gene delivery (e.g., retroviruses, adenoviruses,
herpes-virus,
Pox-virus) used as complete viruses of their parts. See, for example. Hodgson,
Biotechnology, 1995, 13:222.
Suitable biological agents include oxygen transporters (e.g., porphines,
porphirines and their complexes with metal ions), coenzymes and vitamins
(e.g.,
NAD/NADH, vitamins B 12, chlorophylls), and the like.
Suitable biological agents further include agents used in diagnostics
visualization
methods, such as magnetic resonance imaging (e.g., gadolinium (III)
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diethylenetriamine penta-acetic acid), and may be a chelating group (e.g.,
diethylenetriamine penta-acetic acid, triethylenetriamine pentaacetic acid,
ethylenediamine-tetraacetic acid, 1,2-diaminocyclo-hexane-N,N,N',N'-tetra-
aceticacid, N,N'-di(2-hydroxybenzyl) ethylene diamine), N-(2-hydroxyethyl)
ethylene diamine triacetic acid and the like). Such biological agents may also
include
an alpha-, beta-, or gamma-emitting radionuclide (e.g., gallium 67, indium
111,
technetium 99). Suitable biological agents also include iodine containing
radiopaque
molecules.
The biological agent may also be a diagnostic agent, which may include a
paramagnetic or superparamagnetic element, or combination of paramagnetic
element
and radionuclide. The paramagnetic elements include but are not limited to,
gadolinium (III), dysporsium (III), holmium (III), europium (III) iron (III)
or
manganese (II).
The invention can be also used to identify useful fibrinolytic compositions
with
enzymes such as streptokinase, urokinase, tissue plasminogen activator or
other
fibrinolytic enzyme that is effective in dissolving blood clots and
reestablishing and
maintaining blood flow through trombosed coronary or other blood vessels. Also
this
invention is used to identify useful compositions for treating burns,
circulatory
diseases in which there is an acute impairment of circulation, in particular,
microcirculation, respiratory distress syndrome, as well as compositions for
reducing
tissue damage during angioplasty procedures. Further, the compositions
identified
using this invention including these to treat myocardial damage, ischemic
tissue,
tissue damaged by reperfusion injury, stroke, sickle cell anemia and
hypothermia.
These compositions are especially useful for treating vascular obstructions
caused by
abnormal cells which is an often complication during malaria and leukemia and
are
suitable as a perfusion medium for transplantation of organs. The invention is
also
suitable for identifying the compositions of antiinfective compounds, as well
as
modulators of immune response, and improved adjuvants, antigens and vaccines.
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Adjuvants suitable for use in this invention include but are not limited to
adjuvants of mineral, bacterial, plant, synthetic or host product origin.
Suitable
mineral adjuvants include aluminum compounds such as aluminum particles and
aluminum hydroxide. Suitable bacterial adjuvants include but are not limited
to,
muramyl dipeptides, lipid A, Bordetella pertussis, Freund's Complete Adjuvant,
lipopolysaccharides and its various derivatives, and the like. Suitable
adjuvants
include without limitation small immunogenes, such as sythetic peptide of
malaria.
polysaccharides, proteins, bacteria and viruses. Antigens that can be used in
the
present invention are compounds which, when introduced into a mammal will
result
in formation of antibodies. Suitable antigens include but are not limited to
natural,
recombinant, or synthetic products derived from viruses, bacteria, fungi,
parasites and
other infectious agents, as well as autoimmune disease, hormones or tumor
antigens
used in prophylactic or therapeutic vaccines. These antigens include
components
produced by enzymatic cleavage or can be compounds produced by recombinant
DNA technique. Suitable viral antigens include but are not limited to HIV,
rotavirus,
influenza, foot and mouth disease, herpes simplex, Epstein-Barr virus, Chicken
pox,
pseudorabies, rabies, hepatitis A, hepatitis B, hepatitis C, measles,
distemper,
Venezuelan equine encephalomyelitis, Rota virus, polyoma tumor virus, Feline
leukemia virus, reovirus, respiratory synticial virus, Lassa fever virus,
canine
parvovirus, bovine pappiloma virus, tick borne encephalitis, rinderpest, human
rhinovirus species, enterovirus species, Mengo virus, paramixovirus, avian
infectious
bronchitis virus. Suitable bacterial antigens include but are not limited to
Bordetella
pertussis, Brucella abortis, Escherichia coli, salmonella species, salmonella
typhi,
streptococci, cholera, shigella, pseudomonas, tuberculosis, leprosy and the
like. Also
suitable antigens include infections such as Rocky Mountain spotted fever and
thyphus, parasites such as malaria, schstosomes and trypanosomes, and fungus
such
as Cryptococcus neoformans. The protein and peptide antigens include subunits
of
recombinant proteins (such as herpes simplex, Epstein Barr virus, hepatitis B,
pseudorabies, flavivirus, Denge, yellow fever, Neissera gonorrhoeae, malaria,
trypanosome surface antigen, alphavirus, adenovirus and the like), proteins
(such as
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diphteria toxoid, tetanus toxoid, meningococcal outer membrane protein,
streptococcal M protein, hepatitis B, influenza hemagglutinin and the like),
synthetic
peptides (e.g. malaria, influenza, foot and mouth disease virus, hepatitis B,
hepatitis
C). Suitable polysaccharide and oligosaccharide antigens originate from
pneumococcus, haemphilis influenza, neisseria meningitides, Pseudomonas
aeruginosa, Klebsiella pneumoniae, pneumococcus.
Surfactants. Surfactants are defined herein in the most general sense as
surface
active agents that are adsorbed at interface (see, for example, Martin,
Physical
Pharmacy, 4th ed., p.370 et seq., Lea & Febiger, Philadelphia, London, 1993).
These
surface active agents in particular decrease the surface tension at the air-
water
interface in aqueous solutions (see, for example, Martin, Physical Pharmacy,
4th ed.,
p.370 et seq., Lea & Febiger, Philadelphia, London, 1993) and include without
limitation micelle forming amphiphiles, soaps, lipids, surface active drugs
and other
surface active biological agents, and the like (see, for example, Martin,
Physical
Pharmacy, 4th ed., Lea & Febiger, Philadelphia, London, 1993; Marcel Dekker,
New
York, Basel, 1979; Atwood and Florence, J. Pharm. Pharmacol., 1971, 23:2425;
Atwood and Florence, J. Pharm. Sci., 1974, 63:988; Florence and Attwood,
Physicochemical Principles of Pharmacy, 2d. edn., p.180 et seq., Chapman and
Hall,
New York, 1988; Hunter, Introduction to Modern Colloid Science, p.12 et seq.,
Oxford University Press. Oxford, 1993). '
Cationic surfactants that can be used in the present biological agent
compositions
include but are not limited to primary amines (e.g., hexylamine, heptylamine,
octylamine, decylamine, undecylamine, dodecylamine, pentadecyi amine,
hexadecyl
amine, oleylamine, stearylamine, diaminopropane, diaminobutane,
diaminopentane,
diaminohexane, diaminoheptane, diaminooctane, diaminononane, diaminodecane,
diaminododecane), secondary amines (e.g., N,N-distearylamine), tertiary amines
(e.g.,
N,N',N'-polyoxyethylene(10)-N-tallow-1,3-diaminopropane), quaternary amine
salts
(e.g., dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide,
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alkyltrimethylammonium bromide, tetradecyltrimethylammonium bromide,
benzalkonium chloride, benzethonium chloride, benzylonium bromide,
benzyldimethyldodecylammonium chloride, benzyldimethylhexadecylammonium
chloride, benzyltrimethylammonium methoxide, cetyldimethylethylammonium
bromide. dimethyldioctadecyl ammonium bromide, methylbenzethonium chloride,
decamethonium chloride, methyl mixed trialkyl ammonium chloride, methyl
trioctylammonium chloride), 1,2-diacyl-3-(trimethylammonio)propane (acyI group
=
dimyristoyl, dipalmitoyl; distearoyl, dioleoyl), 1,2-diacyl-3-(dimethy-
lammonio)propane {acyl group = dimyristoyl, dipalmitoyl, distearoyl,
dioleoyl), 1,2-
IO dioleoyl-3-(4'-trimethylammonio) butanoyl-sn-glycerol, 1,2-dioleoyl-3-
succinyl-sn-
glycerol choline ester, cholesteryl (4'-trimethylanunonio) butanoate),
heterocyclic
amines. imidazoles, thiazolium salts, N-alkyl pyridinium and quinaldinium
salts (e.g.,
cetylpyridinium halide), N-alkylpiperidinium salts, dialkyldimetylammonium
salts,
dicationic bolaform electrolytes (C,2Me6; C~2Bu6),
dialkylglycetylphosphorylcholine,
lysolecithin), cholesterol hemisuccinate choline ester, lipopolyamines (e.g.,
dioctadecylamidoglycylspermine (DOGS), dipalmitoyl phosphatidylethanol-
amidospermine (DPPES), N'-octadecylsperminecarboxamide hydroxytrifluoro-
acetate, N',N"-dioctadecylsperminecarboxamide hydroxytri-fluoroacetate, N'-
nonafluoropentadecylosperminecarboxamide hydroxytrifluoroacetate, N',N"-
dioctyl(sperminecarbonyl)glycinamide hydroxytrifluoroacetate, N'-
(heptadecafluoro-
decyl)-N'-(nonafluoropentadecyl)-sperminecarbonyl)glycinamede hydroxytrifluoro-
acetate. N'-[3,6,9-trioxa-7-(2'-oxaeicos-I 1'-enyl)heptaeicos-18-
enyl]sperminecarbox-
amide hydroxytrifluoroacetate, N'-(1,2-dioleoyl-sn-glycero-3-phosphoethanoyl)-
spermine carboxamide hydroxytrifluoroacetate) (see, for example, Behr et al.,
Proc.
Natl. Acad. Sci. 1989, 86:6982; Remy et al., Bioconjugate Chem. 1994, 5:647),
2,3-
dioleyl oxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtri-
fluoroacetate (DOSPA) (see, for example, Ciccarone et al., Focus 1993, 15:80),
N,N',N",N"'-tetramethyl-N,N',N",N"'-tetrapalmitylspermine (TM-TPS) (Lukow et
al., J. Virol.; 1993, 67:4566), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-
trimethylamonium
chloride (DOTMA) {see, for example, Felgner, et al., Proc. NatL Acad Sci., USA
47
CA 02299283 2000-02-07
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1987, 84:7413; Ciccarone et al., Focus 1993, 15:80), dimethyl
dioctadecylammonium
bromide (DDAB) (see, for example, Whitt et al., Focus 1991, 13:8), 1,2-
dioleoyl-3-
dimethyl-hydroxyethyl ammonium bromide (DORI) (see, for example, Felgner et
al.,
J. Biol. Chem., 1994, 269:2550), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl
ammonium bromide (DORIE) (see, for example, Felgner et al., J. Biol. Chem.
1994,
269:2550), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypropyl ammonium bromide
(DORIE-HP) (see, for example, Felgner et al., J. Biol. Chem., 1994, 269:2550),
1,2-
dioleyloxypropyl-3-dimethyl-hydroxybutyl ammonium bromide (DORIE-HB) (see,
for example, Felgner et al., J. Biol. Chem. 1994, 269:2550), 1,2-
dioleyloxypropyl-3-
dimethyl-hydroxypentyl ammonium bromide {DORIE-HPe) (see, for example,
Felgner et al., J. Biol. Chem., 1994, 269:2550), 1,2-dimyristyloxypropyl-3-
dimethyl-
hydroxyethyl ammonium bromide (DMRIE) {see, for example. Felgner et al., J.
Biol.
Chem., 1994, 269:2550), 1,2-dipalmitoyloxypropyl-3-dimethyl-hydroxyethyl
ammonium bromide (DPRIE) (see, for example, Felgner et al., J. Biol. Chem.,
1994,
269:2550), 1,2-distearoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide
(DSRIE) (see, for example, Felgner et al., J. Biol. Chem., 1994, 269:2550),
N,N-
dimethyl-N-[2-(2-methyl-4-( 1,1,3,3-tetramethylbutyl)-phenoxy]ethoxy)ethyl]-
benze-
nemethanaminium chloride (DEBDA), N-[1-(2,3-dioleyloxy)propyl]-N,N,N,-
trimethylammonium methylsulfate (DOTAB), lipopoly-L(or D)-lysine (see, for
example, Zhou, et al., Biochim. Biophys. Acta 1991, 1065:8), poly(L (or D)-
lysine
conjugated to N-glutarylphosphatidylethanolamine lysine (see. for example,
Zhou, et
al., Biochim. Biophys. Acta 1991, 1065:8), didodecyl glutamate ester with
pendant
amino group (Cl2GluPhCnN+) (see, for example, Behr, Bioconjugate Chem. 1994,
5:382), ditetradecyl glutamate ester with pendant amino group (C,4GluC"N~)
(see, foe
example, Behr, Bioconjugate Chem. 1994, 5:382), 9-{N',N"-dioctadecyl-
glycinamido)acridine (see, for example, Remy et al., Bioconjugate Chem. 1994,
5:647), ethyl 4-[[N-[3-bis(octadecylcarbamoyl)-2-oxapropylcarbonyl)-
glycinamido]pyrrole-2-carboxamido]-4-pyrrole-2-carboxylate (see, for example,
Remy et al., Bioconjugate Chem. 1994, 5:647), N',N'-
dioctadecylornithylglycinamide hydroptrifluoroacetate (see, for example, Remy
et al.,
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Bioconjugate Chem. 1994, 5:647), cationic derivatives of cholesterol (e.g.,
cholesteryl-3(-oxysuccinamidoethylenetrimethylammonium salt, cholesteryl-3(-
oxysuccinamidoethylenedimethylamine, cholesteryl-3 (-carboxyamido-
ethylenetrimethylammonium salt, cholesteryl-3(-carboxyamidoethylenedimethyl-
amine, 3([N-(N',N'-dimethylaminoetane-carbomoyl] cholesterol) (see, fcr
example,
Singhal and Huang, In Gene Therapeutics, Wolff, Ed., p.118 et seq.,
Birkhauser,
Boston, 1993), pH-sensitive cationic lipids (e.g., 4-(2,3-bis-palmitoyloxy-
propyl)-1-
methyl-1H-imidazole, 4-(2,3-bis-oleoyloxy-propyi)-1-methyl-1H-imidazole,
choleste-
rol-(3-imidazol-1-yl propyl) carbamate, 2,3-bis-palmitoyl-propyl-pyridin-4-yl-
amine)
and the like (see, for example, Budker, et al., Nature Biotechnology, 1996,
14:760).
Especially useful in the context of gene delivery and other applications are
compositions with mixtures of cationic surfactant and nonionic surfactants.
'This
includes, but is not limited to, dioloeoyl phosphatidylethanolamine (DOPE),
dioleoyl
phosphatidylcholine (DOPC) (see, for example, Felgner, et al., Proc. NatL
Acad. Sci.,
USA ( 1987); Singhal and Huang, Gene Therapeutics, Wolff, Ed., p. 118 et seg.,
Birkhauser, Boston, 1993). This also includes commercially available cationic
lipid
compositions including but not limited to LipofectAMINETM, Lipofectine~, DMRIE-
C, CelIFICTINTM, LipofectACETM Transfectam reagents (see, for example,
Ciccarone
et al., Focus 1993, 15:80; Lukow et al., J. Virol., 1993, 67:4566; Behr,
Bioconjugate
Chem. 1994, 5:382; Singhal and Huang, Gene Therapeutics. Wolff Ed., p. 118 et
seq., Birkhauser, Boston, 1993; GIBCO-BRL Co.; Promega Co., Sigma Co.) and
other cationic lipid compositions used for transfection of cells (see, for
example.
Felgner et al., J. Biol. Chem., 1994, 269:2550; Budker, et al., Nature
Biotechnology
1996, 14:760).
Anionic surfactants that can be used in the biological agent compositions
include
but are not limited to alkyl sulfates, alkyl sulfonates, fatty acid soap
including salts of
saturated and unsaturated fatty acids and derivatives (e.g., adrenic acid,
arachidonic
acid, 2-octenoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic
acid,
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unde celenic acid, lauric acid, myristoleic acid, myristic acid, palmitic
acid,
palmitoleic acid, heptadecanoic acid, stearic acid, nonanedecanoic acid,
heneicosanoic acid, docasanoic acid, tricosanoic acid, tetracosanoic acid, cis-
IS-
tetracosenoic acid, hexacosanoic acid, heptacosanoic acid, octacosanoic acid,
triocantanoic acid), salts of hydroxy-, hydroperoxy-, polyhydroxy-, epoxy-
fatty acids
(see, for example, Ingrain and Brash, Lipids 1988, 23:340; Honn et al.,
Prostaglandins 1992, 44:413; Yamamoto, Free Radic. Biod. Med., 1991, 10:149;
Fitzpatrick and Murphy, Pharmacol. Rev., 1989, 40:229; Muller et al.,
Prostaglandins
1989, 38:635; Falgueyret et al., FEBS Lett. 1990, 262:197; Cayman Chemical
Co.,
1994 Catalog, pp. 78-108), salts of carboxylic acids (e.g., valeric acid,
trans-2,4-
pentadienoic acid, hexanoic acid, trans-2-hexenoic acid, traps-3-hexenoic
acid, 2,6-
heptadienoic acid, 6-heptenoic acid. heptanoic acid, pimelic acid, suberic
acid,
sebacicic acid, azelaic acid, undecanedioic acid, decanedicarboxylic acid,
undecanedicarboxylic acid, dodecanedicarboxylic acid, hexadecanedioic acid,
docasenedioic acid, tetracosanedioic acid, prostanoic acid and its derivatives
(e.g.,
prostaglandins) (see, for example, Nelson et al., C&EN 1982, 30-44; Frolich,
Prostaglandins, 1984, 27:349; Cayman Chemical Co., 1994 Catalog, pp. 26-61 ),
leukotrienes and lipoxines (see for example, Samuelsson et al., Science 1987,
237:1171; Cayman Chemical Co., 1994 Catalog, pp. 64-75), alkyl phosphates, O-
phosphates (e.g., benfotiamine), alkyl phosphonates, natural and synthetic
lipids (e.g.,
dimethylallyl pyrophosphate ammonium salt, S-farnesylthioacetic acid, farnesyl
pyrophosphate, 2-hydroxymyristic acid, 2-fluorpalmitic acid,
inositoltrphosphates,
geranyl pyrophosphate, geranygeranyl pyrophosphate, (-hydroxyfarnesyl
phosphonic
acid, isopentyl pyrophoshate, phosphatidylserines, cardiolipines, phosphatidic
acid
and derivatives, lysophosphatidic acids, sphingolipids and like), synthetic
analogs of
lipids such as sodium-dialkyl sulfosuccinate (e.g., Aerosol OT~, n-alkyl
ethoxylated
sulfates, n-alkyl monothiocarbonates, alkyl- and arylsulfates (asaprol,
azosulfamide,
p-(benzylsulfonamideo)benzoic acid, cefonicid, CHAPS), mono- and dialkyl
dithiophosphates, N-alkanoyl-N-methylglucamine, perfluoroalcanoate, cholate
and
desoxycholate salts of bile acids, 4-chloroindoleacetic acid, cucurbic acid,
jasmonic
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acid, 7-epi jasmonic acid, 12-oxo-phytodienoic acid, traumatic acid, tuberonic
acid,
abscisic acid, acitertin, and the like.
Cationic and anionic surfactants that can be used in accordance with the
invention also include fluorocarbon and mixed fluorocarbon-hydrocarbon
surfactants.
See, for example, Mukerjee, P. Coll. Surfaces A: Physicochem. Engin. Asp.,
1994,
84: 1; Guo et al. J. Phys. Chem.1991, 95: 1829; Guo et al. J. Phys. Chem.1992,
96:
10068. The list of such surfactants that are useful in current inventions
includes but is
not limited to the salts of perfluorocarboxylic acids (e.g.,
pentafluoropropionic acid,
heptafluorobutyric acid, nonanfluoropentanoic acid, tridecafluoroheptanoic
acid,
pentadecafluorooctanoic acid, heptadecafluorononanoic acid.
nonadecafluorodecanoic
acid, perfluorododecanoic acid, perfluorotetradecanoic acid.
hexafluoroglutaric acid,
perfluoroadipic acid, perfluorosuberic acid, perfluorosebacicic acid), double
tail
hybrid surfactants (CmF2m+i){C"Hz"+OCH-OS03Na. See, for example, Guo et al.,
J.
Phys. Chem., 1992, 96: 6738, Guo et al., J. Phys. Chem., 1992, 96:10068; Guo
et al.,
.I. Phys. Chem., 1992, 96:10068), fluoroaliphatic phosphonates,
fluoroaliphatic
sulphates, and the like.
The biological agent compositions may additionally contain nonionic or
zwitterionic surfactants including but not limited to phospholipids (e.g.,
phosphatidylethanolamines, phosphatidylglycerols, phosphatidylinositols,
diacyl
phosphatidylcholines, di-O-alkyl phosphatidylcholines, platelet-activating
factors,
PAF agonists and PAF antagonists, lysophosphatidylcholines, lysophosphatidyl-
ethanolamines, lysophosphatidylglycerols, IysophosphatidyIinositols, lyso-
platelet-
activating factors and analogs, and the like), saturated and unsaturated fatty
acid
derivatives {e.g., ethyl esters, propyl esters, cholesteryl esters, coenzyme A
esters,
nitrophenyl esters, napthyl esters, monoglycerids, diglycerides, and
triglycerides, fatty
alcohols, fatty alcohol acetates, and the like), lipopolysaccharides, glyco-
and
shpingolipids (e.g., ceramides, cerebrosides, gaiactosyldiglycerides,
gangliosides,
lactocerebrosides, lysosulfatides, psychosines, shpingomyelins, sphingosines,
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WO 99/08112 PCTNS98/16300
sulfatides), chromophoric lipids (neutral lipids, phospholipids, cerebrosides,
sphingo-
myelins), cholesterol and cholesterol derivatives, Amphotericin B, abamectin,
acedi-
asulfone, n-alkylphenyl polyoxyethylene ether, n-alkyl poiyoxyethylene ethers
(e.g.,
TritonTM, sorbitan esters (e.g., SpanTM, polyglycol ether surfactants
(TergitolTM,
polyoxyethylenesorbitan (e.g., TweenTM, polysorbates, polyoxyethylated glycol
monoethers (e.g., BrijTM, polyoxylethylene 9 lauryl ether, polyoxylethylene 10
ether,
polyoxylethylene 10 tridecyl ether), lubrol, copolymers of ethylene oxide and
propylene oxide (e.g., PluronicTM, Pluronic-RTM, TeronicTM, PluradotTM, alkyl
aryl
polyether alcohol (TyloxapolTM, perfluoroalkyl polyoxylated amides, N,N-bis[3-
d-
gluconamidopropyl]cholamide, decanoyl-N-methylglucamide, n-decyl (-d-
glucopyranozide, n-decyl (-d-glucopyranozide, n-decyl (-d-maltopyranozide, n-
dodecyl (-d-glucopyranozide, n-undecyl (-d-glucopyranozide, n-heptyl (-d-
glucopyranozide, n-heptyl (-d-thioglucopyranozide, n-hexyl (-d-
glucopyranozide, n-
nonanoyl (-d-glucopyranozide 1-monooleyl-rac-glycerol, nonanoyl-N-methylgluca-
mide, n-dodecyl (-d-maltoside, n-dodecyl (-d-maltoside, N,N-bis[3-gluconamide-
propyl]deoxycholamide, diethylene glycol monopentyl ether, digitonin,
heptanoyl-N-
methylglucamide, heptanoyt-N-methylglucamide, octanoyl-N-methylglucamide, n-
octyl (-d-glucopyranozide, n-octyl (-d-glucopyranozide, n-octyl (-d-
thiogalactopyra-
nozide, n-octyl (-d-thioglucopyranozide, betaine (R'R2R3N+R'C02-, where
R'R2R3R'
hydrocarbon chains), sulfobetaine (R'R2R'N+R'S03-), phoshoplipids (e.g.
dialkyl
phosphatidylcholine), 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-
propanesulfonate, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate,
N-
decyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate, N-dodecyl-N,N-dimethyl-, i-
ammonio-1-propanesulfonate, N-hexadecyl-N,N-dimethyl-3-ammonio-1-propane-
sulfonate, N-octadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-octyl-N,N-
dimethyl-3-ammonio-1-propane-sulfonate, N-tetradecyl-N,N-dimethyl-3-ammonio-1-
propanesulfonate, dialkyl phosphatitidyl-ethanolamine.
52
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The following examples will serve to further typify the nature of the
invention
but should not be construed as a limitation on the scope thereof, which is def
ned
solely by the appended claims.
~XAMPLF;~.1
Testing a library of oligonucleotidP ctompo i ion
A. GOAL: Identify the composition of antisense oligonucleotide against ICAM-1
designed using a base of polyamine-polyether copolymers with the following
characteristics: at least 50% inhibition of ICAM-1 in IFN-y stimulated marine
endothelial cells Bend.3.
B. The 20-mer phosphorothioate antisense oligonucleotide IP-3082 (Strepkowski.
et
al., J. Immunol. 1994) was synthesized using a DNA synthesator as previously
described.
C. A base of polyamine-polyether copolymers was synthesized as previously
described (Kabanov et al., Bioconjugate Chem, 6:639-643, 1995). This base
includes,
1) polyspenmine (10)-b-polyoxyethylene (34) {"PS915"); 2) polyspermine (10)-b-
polyoxyethylene(104) ("PS946"); 3) polyspermine(10)-b-polyoxyethylene(182)
("PS980"); 4) polyspermine(10)-b-polyoxy-ethylene(27)-b-polyoxypropylene(40)-b-
polyoxyethylene(27) (PSP85); 3,4-ionene-b- polyoxy-ethylene(182) ("PEG I3.4");
polyethyleneimine(40) (PEI); polyethyleneimine(500) 25% grafted with
polyoxyethylene(114) ("PSOMPEI/25"); polyethyleneimine(500) SS% grafted with
polyoxyethylene(114) ("PSOMPEI/50"); polyethyleneimine(500) 75% grafted with
polyoxyethylene( 114) ("PSOMPEI/75"); polyoxyethylene( 182)-
polyethyleneimine(40)-b-polyoxy-ethylene(182) (P80PEI/1);
polyethyleneimine(500)
25 % grafted with polyoxyethylene(182) ("P80MPEI/2/25"). Here the number of
repeating units of each is presented in brackets. The copolymers were
formulated
with 2*-mer phosphorothioate antisense oligonucleotide IP-3082 (Strepkowski,
et al.,
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WO 99/08112 PCT/US98/16300
J. Immunol., 1994) against ICAM-1 at polymer to oligonucleotide ratio (w/w)
l:l,
3:1, 5:1, 6:1, 10:1, 17:1 and 20:1 to form a composition library.
D. The biological activity of the library of oligonucleotide compositions was
tested
in vitro using a cell model. The test included assay of inhibition of
expression of
ICAM-1 using marine endothelial cells Bend.3, stimulated by IFN-( to increase
expression of ICAM-1 (Strepkowski, et al. J. Immunol. 1994). Briefly, Bend.3
cells
were maintained in culture flasks with RPMI1640 medium supplemented with 10%
FBS, 1% Penicillin-Streptomycin and 1% Hepes at 37°C, 5% C02. Plated
Bend.3
cells were treated with IP-3082 alone or with the compositions of the
composition
library in RPMI1640 medium without serum at a final concentration of
oligonucleotide 2(M. Free oligonucleotide reveals no activity under these
conditions.
After 4 hours incubation at 37°C, cells were washed twice with PBS, and
25 U/ml
IFN-( in RPMI1640 medium supplemented with 10% serum was added to cells
followed by incubation at 37°C for 16 hours. ICAM-1 expression was
evaluated by
Flow Cytometry using an biotinylated antibody against marine ICAM-1 (YN1) that
was revealed after with Streptavidin-Phycoerythrin conjugate. The optimal
composition as determined in this test is P80PEI/2/25 at nitrogen to phosphate
ratio
20:1. The results were as follows.
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WO 99/08112 PCT/US98/16300
Composition Inhibition of ICAM-1 Opohymer to
at the oligonucleotide ratio
optional nitrogen (w/w) j
to phosphate
ratio
PS915 0 At any ratio
PS980 15% 1:1
~
PS946 0 At any ratio
PSP85 0 At any ratio
PEG I,3,4 0 At any ratio
PEI 27% 3:1
P50MPEI/25 40% 5:1
PSOMPEI/50 40% 10:1
PSOMPEI/75 33% 17:1
P80PEI/1 33% 6:1
P80PEI/2/25 60% ~ 20:1
~
' ~ Mean values of triplicates are presented, SEM is less than 10% (p<0.05)
10 A. GOAL: Identify the compositions of expression vector pAPR-ICAM-1
encoding
the marine ICAM-1 under the control of Cytomegalovirus (CMV) promoter from a
library of cationic segmented copolymers with the following characteristics:
transfection efficacy in COS-7 cells exceeds that of Lipofectamine.
B. Plasmid pAPR-ICAM-1 containing marine ICAM-1 gene under the control of the
CMV promoter was propagated in E. coli DHSa strain and purified using the
Flexiprep kit from Pharmacia.
C. A base of cationic, segmented copolymers consists of polyamine-polyether
copolymers listed in Example 1C, polyethyleneimine(500)-b-polyoxyethylene(550)
("PE250200/2"), and polylysine-polyether conjugates synthesized as previously
described (US Patent 5656611): polylysine(150) 25% grafted with
CA 02299283 2000-02-07
WO 99/08112 PCT/US98/16300
polyoxyethylene(114) ("PL180S0/20"), polylysine(1S0) SO% grafted with
polyoxyethylene(114) ("PL180S0/SO"), polylysine(1S0) 7S% grafted with
polyoxyethylene(114) ("PL180S0/7S"), polylysine(19)-b-polyoxyethylene(114)
("PL40/SO"), polylysine(19)-b-polyoxyethylene(41)-b-polyoxypropylene(16)-b-
S polyoxyethylene(41) ("PL40/F38'~, polylysine-(19)-b-polyoxyethylene(27)-b-
polyoxypropylene(40)-b-polyoxyethylene-(27) {"PL40/P8S"), polylysine-(19)-b-
polyoxyethylene( 17)-b-polyoxypropylene(64)-b-polyoxy-ethylene( 17)
("PL40/P123"), polylysine(100)-b- polyoxyethylene{114) ("PL120S0"). Here, the
number of repeating units of each is presented in brackets. The copolymers
were
formulated with the expression vector pAPR-ICAM-1 at polymer to DNA ratio
(wlw)
1:1, 3:1, 40:1 and 6:1, 85:1 to form a composition library.
D. Screening protocol. COS-7 cells were maintained in culture flasks with DMEM
medium supplemented with 10% FBS, 1 % Penicillin-Streptomycin and 1 % Hepes,
at
1 S 37°C, S% C02. Compositions of polymer with plasmid pAPR-ICAM-1 were
prepared
by mixing DNA plasmid with various polymer in the medium for 20 min. at
37°C.
Lipofectamine was used at the concentration of 1 S mg/ml. Plated Bend.3 cells
were
treated with DNA alone or with the compositions in DMEM medium without serum
at the final DNA concentration of 2 (g/ml. After 4 hours incubation at
37°C, cells
were washed twice with PBS and DMEM medium supplemented with 10% serum
was added to cells followed by an incubation at 37°C for 16 hours. ICAM-
1
expression was evaluated by Flow Cytometry using an biotinylated antibody
against
marine ICAM-1 (YN1) that was revealed after with Streptavidin-Phycoerythrin
conjugate. The results of testing are shown in the table. The desired
composition as
2S determined in this test is PE250200/2 at nitrogen to phosphate ratio 1:1.
This
composition surpasses Lipofectamine:
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CA 02299283 2000-02-07
WO 99/08112 PCTNS98/16300
Composition ICAM-1 expression,Number of Optimal polymer
tested of Lipofectamineexpression, to DNA ratio
% of (w/w)
Lipofectamine
PS915 0 0 At any ratio
PS946 0 0 At any ratio
PS980 0 0 At any ratio
PSP85 0 0 At any ratio
PEG 13,4 0 0 At any ratio
PL18050/25 0 0 At any ratio
PL18050/50 0 0 At any ratio
PL18050/75 0 0 At any ratio
PL40/50 0 0 At any ratio
PL40/F38 0 0 At any ratio
PL401P85 0 0 At any ratio
PL40/P 123 0 0 At any ratio
PL12050 0 0 At any ratio
PEI_ 100% 0 3:1
PSOMPEI/25 19% 50% 40:1
PSOMPEI/50 18% 50% 85:1
P80PEI/1 0 0 At any ratio
PSOMPEI/75 0 0 At any ratio
P80PEI/2/25 0 0 At any ratio
PE250200/2 120% 138% 1:1
A. Goal: To identify Pluronic copolymer forming most stable micelles in
aqueous
solutions.
B. Pluronic block copolymers were obtained from BASF Co. (Parispany, NJ) The
base of Pluronic block copolymers was designed including: polyoxyethylene( 1 )-
b-
polyoxypropylene(16)-b-polyoxyethylene(1) (Pluronic L31), polyoxyethylene(2)-b-
polyoxypropylene(31)-b-polyoxyethylene(2) (Pluronic L61), polyoxyethyiene(13)-
b-
polyoxypropylene(31)-b-polyoxyethylene(13) (Pluronic L64), polyoxyethylene(80)-
b-polyoxypropylene(31)-b-polyoxyethylene(80) (Pluronic F68),
polyoxyethylene(3)-
b-polyoxypropylene(40)-b-polyoxyethylene(3) Pluronic L81, polyoxyethylene(17)-
b-
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polyoxypropylene(40)-b-polyoxyethylene(17) (Platonic P84), polyoxyethylene(27)-
b-
polyoxypropylene(40)-b-polyoxyethylene(27) (Platonic P85), Platonic F87,
polyoxyethylene(4)-b-polyoxypropylene(53)-b-polyoxyethylene(4) (Platonic
L101),
polyoxyethylene(23)-b-polyoxypropylene(53)-b-polyoxyethylene(23) (Platonic
L104), polyoxyethylene(136)-b-polyoxypropylene(53)-b-polyoxyethylene(136)
(Platonic F108), polyoxyethylene(5)-b-polyoxypropylene (64)-b-
polyoxyethylene(S)
(Platonic L 121 ), polyoxyethylene( 17)-b-polyoxypropylene(64)-b-
polyoxyethylene{ 17) (Platonic L 123) and polyoxyethylene(95)-b-
polyoxypropylene(64)-b-polyoxyethylene(95) (Platonic L 127). Here the number
of
repeating units of each is presented in brackets.
C. The critical micelle concentrations (CMCs) of Platonic copolymers of the
base B
were determined at 37°C, phosphate-buffered saline, pH 7.4 using pyrene
fluorescent
probe as previously described by Kabanov et al. (Macromolecules 28:2303,
1995).
The results are presented in the table. Result: Platonic L 12I forms the most
stable
micelles (lowest CMC).
_ Platonic copolymer ~~ CMC
, M
L31 _
_
~~ 1.2E-02
L61 1.1 E-04
L64 4.8E-04
F68 3.OE-04
L81 2.3E-OS
P84 1.7E-04
P85 6.SE-OS
F87 9.OE-OS
L101 2.1E-06
L 104 3.3E-06
F 108 2.OE-OS
L121 9.OE-07
L123 4.3E-06
F 127 3.OE-06
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A. Goal: To identify Pluronic copolymer having the highest capacity for
solubilizing
a model drug, pyrene.
B. Same base of Pluronic block copolymers is used as in Example 3B.
C. The partitioning coefficients (P) of pyrene in the micelles of Pluronic
copolymers
were determined at 3 7°C, phosphate-buffered saline, pH 7.4 using
pyrene fluorescent
probe as previously described by Kabanov et al. (Macromolecules 28:2303,
1995).
The results are presented in the table. Result: Pluronic forms the highest
capacity for
solubilizing pyrene (highest P).
Pluronic copolymer P
__ L31 __
~ 225
L61 4193
L64 444
F68 171
L81 2400
P84 533
P85 2000
F87 229
LI01 14286
L104 6000
F108 2000
L121 20000
L123 9524
F127 9090
A. Goal: To predict CMC and P for polyoxyethylene(9)-b-polyoxypropylene{21 )-b-
polyoxyethylene(9) (Pluronic L44) using the experimental data obtained in
Examples
3 and 4.
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B. CMC and partitioning coefficient of pyrene (P) were related to the
structure of
Pluronic block copolymers. The structure was described by variables i and j,
where i
equals the molecular mass of polyoxypropylene segment divided by 300 and j
equals
weight percentage of polyoxyethylene segments divided by 10. For convenience,
Loglo(CMC) and Loglo(P) are used in the instead of CMC and P. Visual
inspection of
Loglo(CMC) and Loglo(P) values shows their nearly linear dependence on i and
j.
Therefore the following second order equations were tried:
Loglo(CMC)=(al*i2+bl*i+cl)*(ml*j2+nl*j+ol) (1)
Loglo(P) _ (a2*i2+b2*i+c2)*(m2*j2+n2*j+o2) (2)
The parameters a,b,c,m,n,o for each of the equations were calculated by least
squares method from the experimental data of CMC and P measured for 14
pluronics.
(The sum of squares of deviation (E8z) was minimized by adjusting
a,b;c,m,n,o). The
calculations were performed using the Solver function in MS Excel.
C. The CMC calculations for the Pluronic base are as follows:
Pluronici j Logo Logo Deviation8' CMC deviation
(CMC) (CMC) 8 Calculated
From Calculated
experiment
L31 3 1 -1.9E+00-1.96395652-3.6E-021.3E-031.09E-021.9
L61 6 I -4.OE+p0-3.783623441.7E-O13.1E-021.65E-04-4.4
L64 6 4 -3.3E+00-336511356-4.6E-022.1E-034.31E-041.4
F68 6 8 -3.SE+00-3.359228781.6E-Ol2.7E-024.37E-04-4.6
L81 8 1 -4.6E+00-4.75516172-I.IE-011.3E-021.76E-OS2.5
P84 8 4 -3.8E+00-:1.22918915-4.6E-012.1 5.90E-OS12.1
E-01
P85 8 5 -4.2E+00-.1.152993673.3E-02l.lE-037.03E-OS-0.8
F87 8 7 -4.OE+00-4.14929574-1.OE-O1l.lE-027.09E-052.6
L101 10 1 -5.7E+00-5.533441611.4E-O12.1E-022.93E-06-2.5
LI04 10 4 -S.SE+00-4.921382835.6E-013.1E-01I.20E-OS-10.2
F108 10 8 -4.7E+00-4.9127765-2.1E-O14.6E-021.22E-OS4.6
L121 12 1 -6.OE+00-6.1184631-7.3E-025.3E-037.63E-071.2
L123 12 3 -5.4E+00-5.60350968-2.4E-015.6E-022.49E-064.4
F127 12 7 -S.SE+p0-5.33895791.8E-O13.4E-024.58E-06-3.3
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D. The parameters in equation (1) are as follows : al = 0.034129633; bt = -
1.16411354; c1= 0.41047985; ml - 0.003688849; n1 - -0.04454141; of -
0.748662669. Final equation for determining CMC is as follows:
Logl°(CMC) _ (0.03413*i2 - 1.1641*i - 0.4105) * ( 0.003689*j2 -
0.04454*j +
0.7487) (3)
E. The mean standard deviation of Log1°(CMC) is 0.24 (i.e., 5.3%
deviation from the
original values). The CMC value predicted for Pluronic L44 is 4.69*10-
°3 M
(Logs°(CMC)= -2.3291). The CMC value determined for Pluronic L44 in
experiment
using pyrene fluorescent probe as previously described by Kabanov et al.
(Macromolecules 28:2303, 1995) is 6* 10~ M.
F. The P calculations for the Pluronic base are as follows:
Pluronici j Log,a Logo Deviation8' CMC deviation
(CMC) (CMC) 8 Calculated
From Calculated
experiment
L31 3 1 2.4E+00 2.4073893S.SE-023.OE-03255.4 2.3
L61 6 l 3.6E+00 3.08349 -5.4E-012.9E-011211.9-14.9
L64 6 4 2.6E+00 2.69039054.3E-021.8E-03490.2 1.6
F68 6 8 2.2E+00 2.42387681.9E-OI3.6E-02265.3 8.5
L81 8 I 3.4E+00 3.35370621.7E-O13.OE-023578.55.1
P84 8 4 2.7E+00 3.1006613.7E01 1.4E-O11260.813.7
P85 8 5 3.3E+p0 2.9920609-3.1E-019.SE-02981.8 -9.4
F87 8 7 2.4E+00 2.83848314.8E-O12.3E-O1689.4 20.3
L101 10 1 4.2E+00 4.0395083-1.2E-O11.3E-0210952.3-2.8
L104 10 4 3.8E+00 3.5245305-2.SE-O16.4E-023346.0-6.7
F108 10 8 3.3E+00 3.1753838-1.3E-O11.6E-021497.5-3.8
L121 12 t 4.3E+00 4.54089622.4E-O15.8E-0234745.35.6
L123 12 3 4.OE+00 4.1278659I.SE-012.2E-0213423.53.7
F127 12 7 4.OE+00 3.6269901-3.3E-OII.IE-O14236.3-8.4
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D. The .parameters in equation (2) are as follows: a2 = 0.00101044; b2 =
0.107791158; c2 = 0.916109753; m2 = 0.005753186; n2 = -0.11070097; 02 =
2.033053894. Final equation for determining P is as follows:
Loglo(P) _ (0.0010104*i2 - 0.10779*i - 0.9161) * (0.005753*j2 -0.1107*j +
2.033))
(4)
E. The mean standard deviation of Loglo(P) is 0.29 (i.e., 9.3% deviation from
the
original values). The P value predicted for Pluronic L44 is 196.7 (Loglo(P)= -
2.2937).
The P value determined for Pluronic L44 in experiment using pyrene as
previously
described by Kabanov et al. (Macromolecules, 28:2303, 1995) is 150.
E~sA~EL~~
Combinatorial approach to identification of a bio_l,~gi al ,gent composition
using
peptide block co~vmer library
A. Goal: A procedure is designed for selection of segmented copolymers (2-20
kD)
that have the following characteristics:
1 ) amphiphilic block copolymers capable to form micelles in aqueous
solutions;
2) solubilize a given drug molecule;
3) nontoxic;
4) available for GMP chemical synthesis.
More specifically, desired macromolecules should be linear block copolymers
A'X(B~A~)"B~y consisting of chemically joined hydrophilic Aa and hydrophobic
Bb
blocks, x = 0 or 1, n ( 0. Contained blocks) may specifically bind the drug
molecule.
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B. General method - iterative combinatorial approach, applying virtual and
synthetic
libraries:
Starting point
Step 0. Having a parent database of macromolecules. The database holds (for
each
entry):
(i) molecular structure type {pattern); (ii) parameters of blocks
(experimental and
calculated), (iii) physicochemical parameters (including data on affinity to
specific
drugs).
Iteration cycle
Step 1. Generation of virtual library of macromolecules. This includes:
estimation of global parameters for generated molecules. The estimation is
based on
the data in the parent database.
Step 2. Searching the virtual library for best candidates for solubilization
of the
given drug.
Step 3. Chemical synthesis of library of compounds. This includes
combinatorial
alteration of best candidates(s) selected in step 2.
Step 4. Acquiring experimental data on the synthetic compounds. including data
on the interaction with the drug(s).
Steps. Extending the parent library.
The iterations should be repeated until no improvement is obtained after last
iteration.
C. Block copolymers. General formula: Block copolymers A'X(B~Ak)"B~y consist
of
chemically joined hydrophilic Aa and hydrophobic Bb blocks, x = 0 or 1, n >_
0. The
junction can be: peptide bond -CO-NH-, ester bond -CO-O-, ether bond -O-, etc.
Each
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block must be at least 1 kD. The block copolymer segments consist of one or
more
building blocks, e.g. amino acid residues, oxo acid residues, alkoxy residues
etc.
Peptide bond joining amino acid residues will be used for routine work.
D. Building blocks. The hydrophilic building blocks block are simple
hydrophilic
amino acids. The hydrophobic building blocks are amino acids with a
constrained
backbone including cycloaliphatic or aromatic ring. The N-terminal building
block
should be an acyl residue instead amino acid to keep the molecule neutral.
E. Computational methods. All computations are performed on a Windows NT
workstation.
F. The database. Programmable relational database capable to cooperate with
third
party software. ISIS-BASE and ORACLE should be considered. MS EXCEL,
VISUAL BASIC and/or VISUAL C++ can be applied for data exchange.
G. Parameters of blocks. Any QSAR parameters available, including charge,
dipole
moments, volume, surface, Loge and related values. These parameters may be
calculated using ACD/LABS and HYPERCHEM/CHEM+ software.
H. Chemical synthesis. The macromolecules will be assembled by means of solid
phase peptide synthesis, with classic Fmoc-type chemistry. All the building
blocks
will be introduced as Fmoc-derivatives of respective amino acid residues. To
avoid
charges, C-terminal amide resin and N-terminal aryl will be used. The final
product
should subject to very simple purification procedure, e.g. extraction on C18
cartridges.
I. Experimental data. The experimental parameters: CMC in various conditions
and
related parameters, partition coefficients, etc. A simple system for rapid
measurement
of CMC and partitioning coefficients for various small molecules must be set
up.
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K. The phases of the project: (i) preliminary (obtaining or synthesis and
characterization of some basic building blocks, e.g., H2N-PEG1500-COOH, Fmoc-
PEG1500-COOH; example syntheses; selection of the QSAR approach (parameter
sets and functions); (ii) development (setting up parallel synthesis and/or
synthesis of
libraries; setting up the database with computational QSAR procedures; setting
up
assay systems; extending the set of available building blocks; application).
L. Synthesis of building blocks. Necessary materials: Rink amide AM resin; -
Fmoc-
PEG1500-COOH; Fmoc-NH-(CH2)5-COOH; other reagents: PyBOP, DIPEA,
piperidin, TFA, triisopropylsilane, DMAP; solvents: DMF, acetonitrile.
Testing P-gp i h~ibit'irng effects of Platonic co~olxmer compo i ion
A. Goal: To identify Platonic composition having the highest activity in
inhibition of
glycoprotein P (P-gp) efflux system in a multiple drug resistant (MDR) cell
line. The
efficacy of P-gp inhibition to be characterized using the rhodamine 123 uptake
test
(Miller et al. Bioconjugate Chemistry 8: 649, 1997).
B. Materials: Rhodamine 123 was purchased from Sigma (St Louis, IL). Platonic
polymers were obtained from BASF Corp. (Parispany, NJ). The compositions of
the
Platonic copolymers with rhodamine 123 were prepared to obtain a library of
compositions. The following compositions are studied: 0.02%, 0.05% and 0.1
Platonic L61, Ø02%, 0.05%, 0.1% and 0.5% Platonic L64, 0.05%, 0.1%, 0.5%,
1%,
and 2% Platonic L44.
C. Cell line: Human breast carcinoma MCF-7 cells (ATCC HTB22) and their MDR
MCF-7 ADR cell subline, derided from the parental cells by selection with Dox
(Batist et al. 1986), were kindly presented by YI,, Lee (Willian Beaumont
Hospital,
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WO 99/08112 PCTNS98/16300
Royal Oak, MI). The cells were maintained in vitro as a monolayer culture in D-
MEM supplemented with 10% FBS at 37°C in a humidified atmosphere
with 5%
C02.
D. Drug uptake assay: The cells in suspension (2.5 x 105 cells/tube) were pre-
incubated for 10 minutes at 37°C prior to drug addition. Free rhodamine
123 (0.5(M)
or rhodamine 123 with Pluronic copolymers of the library of compositions were
added to the cells and incubated for 45 minutes under the culture conditions
as
described above. The cells were then placed in an ice-water slurry to stop the
incubation and washed two time with cold D-PBS. The cell fluorescence was
analyzed by flow cytometry on a Coutter Epics XL cytometer, excitation 488 nm
(argon laser), using a 525-nm filter. A minimum of 10,000 events were analyzed
for
each point. The experimental values of cell fluorescence were normalized
according
to the cell size by dividing the mean channel fluorescence value by the
forward light
scatter value. The experiments were performed in triplicates.
E. The results are presented in the table as the enhancement in rhodamine 123
uptake
in the composition compared to the free rhodamine 123 uptake MCF-7 and MCF-7
Adr cells. The most active composition is Pluronic L61 (0.1 %).
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Composition Increase in rhodamine
123 uptake
MCF-7 MCF-7 ADR
L61 (0.02%) N/d 37
L61 (0.05%) 2.8 44
L61 (0.1%) N/d 51
L64 (0.01%) -1.4 2
L64 (0.02%) -1.3 3.3
L64 (0.05%) 1 11.5
L64 (0.1%) 1.1 31
L64 (0.5%) 1.3 40
L44 (0.05%) 1.3 1.5
L44 (0.1%) -1.3 2.6
L44 (0.5%) -- 1 29.5
L44 (1%) 1.4 26
L44 (2%) 1.6 25
EXAMPLE 8
T ing P-gp inhibiting, effects of Platonic co~ymer compositions
A. Goal: To identify Platonic composition having the highest activity in
inhibition of
glycoprotein P (P-gp) efflux system in a multiple drug resistant (MDR) cell
line. The
efficacy of P-gp inhibition to be characterized using the rhodamine 123 uptake
test
(Miller et al. Bioconjugate Chemistry 8: 649, 1997).
B. Compared to Example 7, the broader base of Platonic copolymers is used
including Platonic L31, polyoxyethylene(41)-b-polyoxypropylene(16)-b-
polyoxyethylene(41) (Platonic F38), Platonic L44, Platonic L61, Platonic L64,
Platonic F68, Platonic L81, Platonic P84, Platonic P85, Platonic F87,
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polyoxyethylene(123)-b-polyoxypropylene(48)-b-polyoxyethylene(123) (Platonic
98), Platonic L101, Platonic L104, polyoxyethylene(34)-b-polyoxypropylene(53)-
b-
polyoxyethylene(34) (Platonic 105), Platonic F 108, Platonic L 121, Platonic L
123
and Platonic L 127. The concentration of Platonic copolymers are 0.0001 %,
0.001 %,
0.01 %, 0.1 % and 1 %.
C. The drug uptake test is performed as described in example 7.
D. The results are presented in the table as the enhancement in rhodamine 123
uptake
in the composition at the optimal Platonic concentration compared to the free
rhodamine 123 uptake in MCF-7 Adr cells. The most active composition is
Platonic
L61 - i.e., the result is the same as in example 7 using a narrower library of
composition. This demonstrates that the desired biological agent composition
can be
identified using very limited number of compositions in the library.
Composition Increase in rhodamine
123
uptake
L31 b0
L38 14
L44 12
L61 90
L64 60
L68 35
L81 60
P84 40
P85 65
F87 14
F98 17
L101 43
L104 60
P105 14
F108 18
L121 18
L123 30
F127 12
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A. Goal: To identify composition of Doxorubicin (Dox) having the highest
cytotoxic
activity against an MDR cell line.
B. Materials: Dox was purchased from Sigma (St Louis, IL). Pluronic polymers
were
obtained from BASF Corp. (Parispany, NJ). The compositions of the Pluronic
copolymers with Dox were prepared to obtain a library of compositions. The
I0 following compositions are studied: 0.02%, 0.025% and 0.1% Pluronic L6I,
0.02%,
0.05% and 0.1% Pluronic L64, 0.02%, 0.05%, 0.1% and 0.5% Pluronic L44, 0.008%,
0.04%, 0.2% and I % Pluronic P85, 1 % Pluronic F 108, 0.2 % Pluronic F 127,
mixture
of 0.025 % Pluronic L61 and 0.2 % Pluronic F 127.
I S C. Cytotoxicity assay: The cells were placed (3000 cells per well) in a 96-
well plate
and allowed to reattaching overnight. Dox or Dox-formulated with polymers was
incubated with the cells for 2 h at 37°C with 5% C02. The cells were
washed three
times and cultured for 4 days. The drug cytotoxicity was determined by a
standard
2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-carboxanilide inner
salt
20 (XTT) assay (Scudievo et al., 1988). The absorbance at (450 was determined
using a
microplate reader. All the experiments were carried out in triplicates. SEM
values
were less than 10% (p < 0.05).
D. The results are presented in the table as the resistance reversion index,
i.e. ratio of
25 ICso in free drug to ICso of the drug-copolymer composition. The most
active
composition is Pluronic L61 (0.1 %).
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Composition Increase in rhodamine
123 uptake
MCF-7 MCF-7 ADR
L61 (0.02%) N/d 37
L61 (0.05%) 2.8 44
L61 (0.1%) N/d 51
L64 (0.01%) -1.4 2
L64 (0.02%) -1.3 3.3
L64 (0.05%} 1 11.5
L64 (0.1 %) 1.1 31
L64 (0.5%) I.3 40
L44 (0.05%) 1.3 1.5
L44 (0.1%) -1.3 2.6
L44 (0.5%) 1 29.5
L44 { 1 %) 1.4 26
L44 (2%) 1.6 25
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Composition Resistance reversion
index
MCF-7 MCF-7 ADR
L61 (0.02%) 1 125
L61 (0.1%) 1.1 740
L64 (0.02%) 1 40
L64 (0.05%) 1 72
L64 (0.1%) 1 529
L44 (0.02%) 1 3.3
L44 (0.05%) -1.4 4
L44 (0.1%) -1.3 11.8
L44 (0.5%) -1.3 25
P85 (0.008%) 1 2.8
P85 (0.04%) -. i 6.4
P85 (0.2%) 1 9.3
P85 (1%) 1 9.4
F108 (1%) 1 3
L61 (0.025%) 1.9 115
F 127 (0.2%) 1 1.3
L61/F127 -..1.3 129
(0.025%/0.2%)
71
