Note: Descriptions are shown in the official language in which they were submitted.
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STILBAZIUM RESEARCH ASSAYS
RELATED APPLICATION DATA
This application claims the benefit of United States Provisional Patent
Application
Serial No. 60/669,615 filed on April 8, 2005, United States Provisional Patent
Application
Serial No. 60/734,518 filed on November 8, 2005 and United States Provisional
Patent
Application Serial No. 60/773,366 filed on February 13, 2006. The disclosures
of which are
incorporated herein by reference in their entireties.
FIELD OF THE INVENTION
The present invention relates to compositions, methods, and kits for use in
assays.
More particularly, the present invention relates to the use of stilbazium
compounds and
analogs thereof as fluorescents, stains or tags for use in assays.
BACKGROUND OF TIiE INVENTION
One of the most frequently used molecular biological techniques for detecting
homologous nucleic acid sequences is nucleic acid hybridization, i.e. DNA/DNA,
RNA/RNA
or RNA/DNA hybridization. In this technique, nucleic acid (DNA or RNA) used as
a probe is
labeled, and the labeled nucleic acid is hybridized to a nucleic acid (DNA or
RNA) to be
detected. When the nucleic acid used as a probe has liomology to the nucleic
acid to be
detected, each single-stranded nucleic acid hybridizes to its complementary
sequence so as to
form a double-stranded sequence, and then the double-stranded sequence is
detected by a
labeled probe.
The need to identify, mark, isolate, modify or otherwise determine analytes,
microorganisms, amino acids or nucleic acid sequences (for example multiple
pathogens or
multiple genes or multiple genetic variants) alone, in blood or in other
biological fluids has
become increasingly apparent in many branches of medicine. Most multi-analyte
assays, such
as assays that detect multiple nucleic acid sequences, involve multiple steps,
have poor
sensitivity, a limited dynamic range (typically on the order of 2 to 100-fold
differences)
and/or often utilize sophisticated instrumentation.
As the human genome is elucidated, there are numerous opportunities for
perfomling
assays to determine the presence of specific sequences, distinguishing between
alleles in
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homozygotes and heterozygotes, detemiining the presence of mutations,
evaluating cellular
expression patterns, etc. In many of these cases, one will wish to determine a
number of
different characteristics of the same sample in a single reaction. In many
assays, there is an
interest in determining the presence of specific sequences, whether genomic,
synthetic, or
eDNA. These sequences may be associated particularly with genes, regulatory
sequences,
repeats, multimeric regions, expression patterns, and the like. There is also
an interest in
determining the presence of one or more pathogens. The need to identify and
quantify a large
number of bases or sequences, potentially distributed over centimorgans of
DNA, offers a
major challenge. Ideally, the method is accurate, reasonably economical in
limiting the
amount of reagents required and/or provides for a highly multiplexed assay,
which allows for
differentiation and quantification of multiple genes, and/or single nucleotide
polymorphisms
(SNP) determination, and/or gene expression at the RNA or protein level.
Radioactivity has been the dominant read-out in early drug discovery assays.
However, the need for more information, higher throughput and miniaturization
has caused a
shift towards fluorescence detection. Fluorescence-based reagents can yield
more powerful,
multiple parameter assays that are higher in throughput and information
content and require
lower volumes of reagents and test compounds. Fluorescence is also safer and
less expensive
than radioactivity-based methods.
Another technique for detecting biological compounds is fluorescence in-situ
hybridization (FISH). Swiger et al. (1996) Environ. Mol. Mutagen. 27:245-254;
Raap (1998)
Mut. Res. 400:287-298; Nath et al. (1997) Biotechnic. Histol. 73:6-22. FISH
allows detection
of a predeternv.ned target oligonucleotide, e.g., DNA or RNA, within a
cellular or tissue
preparation by, for example, microscopic visualization. Thus, FISH is an
important tool in the
fields of, for example, molecular cytogenetics, pathology and immunology in
both clinical
and research laboratories. FISH involves the fluorescent tagging of an
oligonucleotide probe
to detect a specific complementary DNA or RNA sequence. Specifically, FISH
involves
incubating an oligonucleotide probe comprising an oligonucleotide that is
complementary to
at least a portion of the target oligonucleotide with a cellular or tissue
preparation containing
or suspected of containing the target oligonucleotide. A detectable label,
e.g., a fluorescent
dye molecule, is bound to the oligonucleotide probe. A fluorescence signal
generated at the
site of hybridization is typically visualized using an epi fluorescence
microscope. An
alternative approach is to use an oligonucleotide probe conjugated with an
antigen such as
biotin or digoxygenin and a fluorescently tagged antibody directed toward that
antigen to
visualize the hybridization of the probe to its DNA target. A variety of FISH
formats are
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known in the art. See, e.g., Dewald et al. (1993) Bone Marrow Transplantation
12:149-154;
Ward et al. (1993) Am. J. Hum. Genet. 52:854-865; Jalal et al. (1998) Mayo
Clin. Proc.
73:132-137; Zahed et al. (1992) Prenat. Diagn. 12:483-493; Kitadai et al.
(1995) Clin. Cancer
Res. 1:1095-1102; Neuhaus et al. (1999) Human Pathol. 30:81-86; Hack et al.,
eds., (1980)
Association of Cytogenetic Technologists Cytogenetics Laboratory Manual.
(Association of
Cytogenetic Technologists, San Francisco, Calif.); Buno et al. (1998) Blood
92:2315-2321;
Patterson et al. (1993) Science 260:976-979; Patterson et al. (1998) Cytometry
31:265-274;
Borzi et al. (1996) J. Immunol. Meth. 193:167-176; Wachtel et al. (1998)
Prenat. Diagn.
18:455-463; Bianchi (1998) J. Perinat. Med. 26:175-185; and Munne (1998) Mol.
Hum.
Reprod. 4:863-870.
Thus, an assay for the differentiation and/or quantification of single or
multiple genes,
and/or SNP determination andlor gene expression at the RNA or protein level,
that has higher
sensitivity, a large dynamic range, better fluorescence, a greater degree of
multiplexing
and/or fewer and more stable reagents would increase the simplicity and/or
reliability of
multianalyte assays, and may reduce their costs.
There is also a continuing need inthe assay art for labels with the following
features:
(i) high fluorescent intensity (for detection in small. quantities), (ii)
adequate separation
between the absorption and emission frequencies, (iii) desirable solubility,
(iv) ability to be
readily linked to other molecules, (v) stability towards harsh conditions and
high
temperatures, (vi) a symmetric, nearly Gaussian emission lineshape for
deconvolution of
multiple colors, and/or (vii) compatibility with automated analysis.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA through IL present confocal images of murine mammary carcinoma 4T1
cells incubated with a compound according to some embodiments of the present
invention
(Figures 1B, 1F and 1J) and co-stained with Hoechst (Figures 1A, 1E and lI,
blue) to stain
the nuclei and MitoTracker Deep Red (Figures 1C, 1G and 1K, appears green in
images) to
stain the mitochondria. Figure 1D presents an overlaid image of the images
presented in
Figures 1A through 1C. Figure.lH presents an overlaid image of the images
presented in
Figures lE through 1G. Figure 1L presents an overlaid image of the images
presented in
Figures lI through 1K.
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SUMMARY OF THE INVENTION
Embodiments of the invention include assays for determining the presence or
absence
of a cell, an analyte, a nucleic acid or a microorganism in a sample suspected
of containing a
cell, an analyte, a nucleic acid or a microorganism, said assay comprising
combining the
sample with a labeling reagent to form a labeled cell, nucleic acid or
microorganism, said
labeling reagent comprising a dye which directly stains the cell, analyte,
nucleic acid or
microorganism to provide a stained sample comprising a stained cell, analyte,
nucleic acid or
microorganism, wherein said dye is a compound represented by Formula I
RI ~ \ \ N ~R3
/N--- II-- x I ~ -----N
Rz ~ RS I !5~ R4
wherein for Formula I, the NR1R2 and NR3R4 moieties are in the ortho, meta or
para
positions; wherein X- is an anionic salt; wherein Rl, R2, R3, or R4 are
independently selected
from the group consisting of C1_lo alkyl (linear or branched), alkenes (linear
or branched), or
wherein Rl and R2 or R3 and R4 taken together with the nitrogen atom to which
they are
attached form pyrrolidino or piperidino rings; wherein R5 is a polyalkylene
glycol moiety, a
Ci-lo alkyl (linear or branched), an alkene (linear or branched), an alkyne, a
substituted and
unsubstitated aryl, a substituted and unsubstituted benzyl and/or an
organometallic moiety.
R5 may also be an organometallic compound such as organotin, organosilicon, or
organogermanium. Additionally, R5 may be (CH2),,-MR6, wherein n is a number
from 1 to 6,
M is an organometallic compound such as tin, silicon, or germanium, and
wherein R6 is a
selected from the group consisting of propyl, butyl, or any alkyl compound;
contacting the
stained sample; and observing the accumulation of the stained cell, analyte,
nucleic acid or
microorganism. The assays can include cellular, chemical and biological
assays. The dye
can be loaded into a lipid vesicle or suitable biomaterial. In particular, the
dye can be loaded
into a microcapsule, liposome, micelle and/or bicelle to form a
microencapsulated
formulation, a liposomal formulation, a micelle formulation and/or a bicelle
formulation,
respectively. Furtb.er, the dye can be pegylated.
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Embodiments of the present invention can include a probe comprising a ligand
or
antibody and a compound of Formula I. The probe can be used to detect cells,
analytes,
nucleic acids and microorganisms.
Embodiments of the present invention fizrther include methods of selecting an
analyte
that binds to a compound of Formula I.
Embodiments of the present invention include determining the presence or
absence of
one or more target compounds in a sample, wherein the compounds are
represented by
Formula I or a fluorescent molecule and the steps include providing a
plurality of
electrophoretic probes specific for the one or more target compounds, each
electrophoretic
probe having a target-binding moiety; combining with the sample the plurality
of
electrophoretic probes such that in the presence of a target compound a
complex is formed
between each target compound and one or more electrophoretic probes specific
therefor; and
separating and identifying the compounds to determine the presence or absence
of the one or
more target compounds.
Embodiments of the present invention further include kits for staining cells,
analytes,
nucleic acids andJor microorganisms.
DETAILED DESCRIPTION
The foregoing and other aspects of the present invention will now be described
in
more detail with respect to other embodiments described herein. It should be
appreciated that
the invention can be embodied in different forms and should not be construed
as limited to
the embodiments set forth herein. Rather, these embodiments are provided so
that this
disclosure will be thorough and complete, and will fully convey the scope of
the invention to
those skilled in the art.
The terminology used in the description of the invention herein is for the
purpose of
describing particular embodiments only and is not intended to be limiting of
the invention.
As used in the description of the embodiments of the invention and the
appended claims, the
singular forms "a", "an" and "the" are intended to include the plural forms as
well, unless the
context clearly indicates otherwise. Also, as used herein, "and/or" refers to
and encompasses
any and all possible combinations of one or more of the associated listed
items. As used
herein, phrases such as "between X and Y" and "between about X and Y" should
be
interpreted to include X and Y. As used herein, phrases such as "between about
X and Y"
mean "between about X and about Y." As used herein, phrases such as "from
about X to Y"
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mean "from about X to about Y." Unless otherwise defined, all terms, including
technical
and scientific terms used in the description, have the same meaning as
commonly understood
by one of ordinary skill in the art to which this invention belongs.
All publications, patent applications, patents and other references cited
herein are
incorporated by reference in their entireties for the teachings relevant to
the sentence and/or
paragraph in which the reference is presented.
"Alkyl" as used herein alone or as part of another group, refers to a straight
or
branched chain hydrocarbon containing from 1 to 10 carbon atoms.
Representative examples
of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl,
n-butyl, sec-butyl,
iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,
2,2-
dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and
the like.
"Loweralkyl" as used herein, is a subset of alkyl, and refers to a straight or
branched
chain hydrocarbon group containing from 1 to 4 carbon atoms. Representative
examples of
lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-
propyl, n-butyl, iso-
butyl, tert-butyl, and the like.
Alkyl and loweralkyl groups may be unsubstituted or substituted one or more
times
with halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl,
aryl, arylalkyl,
heterocyclo, heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy,
haloalkoxy,
cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy,
heterocyclolalkyloxy, mercapto, alkyl-S(O),n, haloalkyl-S(O)m, alkenyl-S(O)n,,
alkynyl-
S(O),,,, cycloalkyl-S(O),,,, cycloalkylalkyl-S(O),,,, aryl-S(O),,,, arylalkyl-
S(O)n,, heterocyclo-
S(O)m, heterocycloalkyl-S(O)I,,, amino, alkylamino, alkenylamino,
alkynylamino,
haloalkylamino, cycloalkylamino, cycloalkylalkylami.no, arylamino,
arylalkylamino,
heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino,
acyloxy, ester,
amide, sulfonarnide, urea, alkoxyacylasnino, aminoacyloxy, nitro or cyano
where m=0,1 or 2.
"Alkoxy," as used herein alone or as part of another group, refers to an alkyl
group, as
defined herein, appended to the parent molecular moiety through an oxy group.
Representative examples of alkoxy include, but are not limited to, methoxy,
ethoxy, propoxy,
2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.
"Acyl" or "A.lkanoyl" as used herein alone or as part of another group, refers
to a -
C(O)R radical, where R is any suitable substituent such as alkyl, alkenyl,
alkynyl, aryl,
alkylaryl, etc.
"Cell" as used herein refers to a basic component of a living or fixed
organism and
includes organelles. Thus, detecting the presence of a cell, assaying a cell,
staining a cell, etc.
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can refer to a whole cell or at least one organelle of the cell. According to
embodiments of
the present invention, cells may be plant or animal cells. As recognized by
one sldlled in the
art, "organelles" as used herein refer to cellular components or structures
suspended in the
cytoplasm including those providing a boundary therefor and having specialized
functions.
Organelles include, but are not limited to, the nucleus, smooth and/or rough
endoplasmic
reticulum, centrosome, cytoskeleton, cell wall, cell membrane, flagella,
cilia, chloroplast,
mitochondria, golgi apparatus, ribosome, lysosome, centriole, acrosome,
glyoxysome,
secretory vesicle, peroxisome, vacuole, melanosome, myofibril and
parenthesome.
"Dye" as used herein refers to a substance that imparts color and/or
fluorescence
and/or is quantifiable or distinguishable. The color and/ox fluorescence can
be temporary,
semi-permanent or permanent.
"Analyte" as used herein refers to the substance or chemical constituent that
undergoes analysis. For example, an analyte can be a molecule, protein,
chemical substance,
etc. that can be detected as a result of biological, chemical or clinical
testing to evaluate the
same. Analytes can include, but are not limited to, ions; metabolites such as
glucose and
urea; trace metabolites such as hormones, drugs, steroid hormones; gases such
as respiratory
gases, anesthetic gases, toxic gases and flammable gases; toxic vapors;
proteins and nucleic
acids; antigens and antibodies and microorganisms.
"Nucleic acid" as used herein refers to an oligonucleotide, nucleotide, or
20. polynucleotide, and to DNA or RNA or chimeras thereof, single stranded or
double-stranded,
and can be fully or partially synthetic or naturally occurring. Nucleic acids
can include
modified nucleotides or nucleotide analogs. Further, the nucleic acids can be
from any
species of origin, including plant species or mammalian species such as human,
non-human
primate, mouse, rat, rabbit, cattle, goat, sheep, horse, pig, dog, cat, etc.
In some
embodiments, the nucleic acid is an isolated nucleic acid. As used herein, an
"isolated"
nucleic acid means a nucleic acid separated or substantially free from at
least some of the
other components of the naturally occurring organisnl, for example, the cell
structural
components or other polypeptides or nucleic acids commonly found associated
with the
nucleic acid.
"Lipid vesicle" as used herein refers to structures including amphiphiles, for
example,
surfactants or phospholipids, characterized by the presence of an internal
void. The internal
void can be filled with any appropriate material such as a liquid, aqueous
solution, gas, gel,
solid material or mixture thereof. Lipid vesicles include, but are not limited
to, liposomes,
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helices, discs, tubes, tori, hexagonal, phase structures, micelles, gel
phases, reverse micelles,
bicelles, microemulsions, emulsions and combinations thereof.
"Microorganism" as used herein refers to microscopic organisms that can exist
as a
single cell or cell clusters.
Embodiments of the present invention include a compound comprising Formula I:
R
,~ I N/ Rs
N---I-- I --N~
R I2 R5 R4
or a solvate thereof, wherein X- is an anionic salt, wherein Rl, R2, R3, or R4
are independently
selected from the group consisting of methyl, ethyl, Ci_lo alkyl (linear or
branched), alkenes
(linear or branched), or wherein when Rl and R2 or when R3 and R4 are taken
together with
the nitrogen atom to which they are attached, they form pyrrolidino or
piperidino rings. X"
can be selected from the group including fluoride, chloride, bromide, iodide
halide, mesylate,
tosylate, napthylate, nosylate, para-aminobenzoate, benzenesulfonate,
besylate, lauryl sulfate,
2,4-dihydroxy benzophenone, 2-(2-hydroxy-5'-methylphenyl) benzotriazole, ethyl
2-cyano-
3,3-diphenyl acrylate and 5-butyl phenyl salicylate. R5 is a polyalkylene
glycol moiety, a CI-
lo alkyl (linear or branched), an alkene (linear or branched), an alkyne, a
substituted and
unsubstituted aryl, a substituted and unsubstituted benzyl and/or an
organometallic moiety.
R5 may also be an organometallic compound such as organotin, organosilicon, or
organogermanium. Additionally, R5 may be (CH2)n-NIRb, wherein n is a number
from 1 to 6,
M is an organometallic compound such as tin, silicon, or germanium, and
wherein R6 is a
selected from the group consisting of propyl, butyl, or any alkyl compound.
The compound
may be a stilbazium compound.
In some embodiments of the present invention, the compound has the following
structure:
N+
~~_
CH3 N
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In other embodiments of the present invention, the compound has the following
structure:
N
In still other embodiments of the present invention, the compound has the
following
structure:
c,-
N N~~
J J
Stilbazium chloride as well as other stilbazium salts, analogs and homologs
thereof
may exist as bright red to dark red or other colored compounds that may
possess UV-visible
chromophores and may further exhibit characteristic strong fluorescence.
Stilbazium chloride as well as other stilbazium salts, analogs and homologs
exhibit
classic staining properties and can bond to cloth, paper, wood plastic, glass,
metal and other
substrates, as well as skin and related living tissues, while retaining its
red or pink or other
coloration. Stilbazium chloride as well as other stilbazium salts, analogs and
homologs may
stain biopolymers such as single stranded or double stranded deoxyribonucleic
acid (DNA)
and ribonucleic acid (RNA) by covalently bonding to nucleopliilic groups on
the DNA and
RNA chains.
One mechanism of action may include a strong covalent bond being formed
between
the nucleophile (Nu:) and one of the stilbazium chloride side chains. The
other side chain
retains the chromophore and fluorophore as shown in the scheme below.
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Scheme 1.
Nu:
I CI I z
N
Stilbazium
Chloride
Nu
N
CI'
W Intertn
ediate
Nu
Cl'
N+
N
Partially
covalently
bonded
Stilbazium
Chloride
The nucleophile may be a nucleic acid, a microorganism, an amino acid, a cell,
or an analyte.
Additionally, in particular embodiments, the nucleic acid, amino acid, cell,
or analyte may be
bound to a nucleophile such as OH, O", NH2, and the like before the
nucleophile is covalently
bonded to stilbazium chloride, salts, analogs and homologs thereof.
The compounds of the present invention are capable of existing as geometric
isomers.
All such isomers, individually and as m.ix-tures, are included within the
scope of the present
invention for their industrial uses. The E,E isomer is one configuration of
the invention, and
both the cisoid and transoid 2,6-conformations of the E,E-configuration are
possible.
Additionally, the ortho conformation of the structure can be formed in
addition to the para
and meta structures illustrated above. The ortho conformation structure can
include the same
salts and moieties as disclosed above and throughout the application.
The compounds of the present invention can be fast acting, staining the object
of
interest in less than 10 minutes, and in some embodiments, seconds. The
compounds of the
present invention may be visible in bright field and/or under fluorescence.
Moreover, the
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compounds of the present invention provide a non-toxic staining option for
viable cells and
can provide repeatable staining of living cells. Additionally, the compounds
of the present
invention can be used in multiple assays of the same cultures. Embodiments of
the present
invention can further provide detection of live cell mitotic division.
Compounds of the
present invention are safe to users and user-friendly. Additionally, compounds
of the present
invention may exhibit stability at room temperature for a period of time
sufficient to allow
appropriate assays to be performed.
Embodiments of the present invention can also include compounds represented by
Formula I that are encapsulated, formulated in lipid vesicles such as
liposomes, micelles and
bicelles and/or pegylated (PEG). As used herein, "encapsulated" refers to a
formulation of a
compound according to the present invention confined by a material or matter.
The material
or matter can be synthetic or of natural origin. Accordingly, a lipid vesicle
provides an
exemplary mechanism for encapsulation. Moreover, as understood by one skilled
in the art,
compounds of the present invention can further be encapsulated by application
of a coating
surrounding the compound. Such coatings can include biomaterials and further
include
materials discussed below in reference to microcapsules.
The amount of the compound encapsulated or formulated in lipid vesicles and/or
pegylated can be determined by one slcilled in the art based upon the method
for which the
compound is employed. In general, the compound can be present in an amount in
a range
from about 1 weight percent to about 50 weight percent or more of the
formulation. Such
encapsulated, lipid vesicle formulations and pegylated formulations can be
water soluble.
1. Microencapsulation
According to some embodiments, compounds of the present invention can be
encapsulated in microcapsules. As used herein, the term "microcapsules" is
intended to
contemplate single molecules, encapsulated discrete particulate,
multiparticulate, liquid
multicore and homogeneously dissolved active components. The encapsulation
method may
provide either a water soluble or oil soluble active component encapsulated in
a shell matrix
of either a water or oil soluble material. The microencapsulated active
component may be
protected from oxidation (e.g., W) and hydration, and may be released by
melting,
rupturing, biodegrading, or dissolving the surrounded shell matrix or by slow
diffusion of the
active component through the matrix. Microcapsules usually fall in the size
range of between
about 1 and 2000 microns, although smaller and larger sizes are known in the
art.
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Compounds of the present invention may be placed in a microcapsule or hollow
fiber
type used for distribution. They may also be dispersed in a polymeric material
or held as a
liquid.
The microcapsules can be made from a wide variety of materials, including
polyethylene, polypropylenes, polyesters, polyvinyl chloride, tristarch
acetates, polyethylene
oxides, polypropylene oxides, polyvinylidene chloride or fluoride, polyvinyl
alcohols,
polyvinyl acetates, urethanes, polycarbonates, and polylactones. Further
details on
microencapusulation are to be found in U.S. Patent Nos. 5,589,194 and
5,433,953.
Microcapsules suitable for use in the base materials of the present invention
have diameters
from about 1.0 to 2,000 microns.
No particular limitation is imposed on the shape for holding the active
ingredient. In
other words, there are various forms for holding the active ingredient by a
holding mixture.
Specific examples include microcapsules in which the surface of the active
ingredient has
been covered with the holding mixture; and products processed into a desired
shape, each
being obtained by kneading the active ingredient in the holding mixture or
fonning a uniform
solution of the holding mixture and the active ingredient, dispersing the
active ingredient in
the holding mixture by the removal of the solvent or the like and then
processing the.
dispersion into a desired shape such as single molecule, molecular chain,
liquid, sphere,
sheet, film, rod, pipe, thread, tape or chip. In addition, these processed
products having a
surface covered with a barrier layer for controlli.ng the release of the
active ingredient and
those coated with an adhesive for improving applicability can be given as
examples. As
further examples, those obtained by filling the active ingredient in the
holding mixture
processed into a form of a capillary tube, heat sealing both ends of the
capillary tube and then
encapsulating the active ingredient therein; and those obtained by centrally
cutting the above-
mentioned capillary tube into two pieces, thereby having each one end as an
opening.
The container formed of a holding mixture which container has an active
ingredient
enclosed therein as a liquid phase to secure uniform release ability over a
long period of time.
As such shape, tube-, bottle- or bag-shaped container is used generally.
When the mixture is formed into a container, the sustained release layer
desirably has
a thickness of at least about 0.002 mm for effecting stable sustained release.
There occurs no
particular problem when the sustained release layer has a thickness not
smaller than about
0.002 mm, but that ranging from about 0.005 mm to 5 mm can be used. When it
exceeds
about 5 mm, the release amount of the compound tends to become too small.
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For solids, the release surface area of the sustained release preparation
formed of such
a container is desirably.001 cm2 or larger. A=range of from.01 em2 to 1 cm2
may be used.
When the active ingredient is enclosed and held in a container of the
sustained release
preparation, said container having been formed of a holding mixture, it may be
enclosed in
portions. The enclosed amount can be about 0.5mg to 5 mg, and may be about
lmg, 2mg,
3mg, or 4mg.
As the shape of the container formed of a holding mixture, a tube, bottle and
bag can
be used. In the case of the tube-shaped preparation, that having an internal
diameter of about
0.4 mm to 10 mm can be used. Internal diameters smaller than about 0.4 mm make
it difficult
to fill the active ingredient in the container, while those larger than about
10 mm make it
difficult to conduct encapsulation. The bottle-shaped preparation is formed by
blow molding
or injection molding and generally has an intemal volume of about 0.1 to 200
ml. The bottle
having an internal volume less than about 0.1 ml cannot be formed easily,
while that having
an internal volume greater than about 200 ml is not economical because there
is a large
difference between the amount of the active ingredient filled therein and the
internal volume.
In the case of a bag-shaped preparation, the amount of the active ingredient
filled in the bag is
desirably about 1 mg to 100 g:
In some embodiments, the entire microcapsule composition can include of about
40-
90 percent of liquid fill and about 10-40 percent of shell wall, the liquid
fill comprising about
5-60 percent of compound, about 25-50 percent of biological synergist and 20-
40 percent of a
water-immiscible organic solvent and the shell including as an integral part
thereof 0.5-20
percent of photostable ultraviolet light absorbent compound (all percentages
being based on
the weight of the entire microcapsule composition).
The compound remains inside the microcapsules while the composition is
packaged
and in storage, i.e., in a closed container due to the parrtial pressure of
the pyridinium salt
surrounding the microcapsules. The compound is chemically stable during
storage and after
application until it permeates the capsule walls.
Suitable fill stabilizers absorb ultraviolet radiation in the range of about
270-350
nanometers and convert it to a harmless form. They have a high absorption
coefficient in the
near ultraviolet portion of the spectrum (e.g. a log molar extinction
coefficient of from about
2 to 5) but only minimal absorption in the visible portion of the spectrum.
They do not exhibit
any substantial chemical reaction with the isocyanate groups and primary amine
groups of the
shell forming compounds during the microencapsulation process. Among the
compounds
which can be used as fill stabilizers are substituted benzophenones such as
2,4-dihydroxy
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benzophenone, 2-hydroxy-4-methoxy benzophenone, 2-hydroxy-4-octyloxy
benzophenone,
etc.; the benzotriazoles such as 2-(2-hydroxy-5'-methylphenyl) benzotriazole,
2-(3',5'-diallyl-
2'-hydroxylphenyl)benzotriazole, etc.; substituted acrylates such as ethyl 2-
cyano-3,3-
diphenyl acrylate, 2-ethylhexyl-2-cyano-3,3-diphenyl acetate, etc.;
salicylates such as phenyl
salicylates, 5-butyl phenyl salicylate, etc.; and nickel organic compounds
such as nickel bis
(octylphenol) sulfide, etc. Additional examples of each of these classes of
fill stabilizers may
be found in Kirk-Othmer, Encyclopedia of Chemical Technology. The fill
stabilizers may
comprise up to 5 percent, and are generally from about 0.01 to 2 percent, by
weight of the
microcapsule composition.
Another embodiment of the present invention may include heat sensitive
materials
that facilitate preservation stability, particularly in resistance to light,
and microcapsules
having an ultraviolet absorber enclosed therein, which are applicable to
various fields.
Desirable constituents that may be present in a base material include
materials that can absorb
heat and protect an underlying material from overheating. Thermal energy is
absorbed by the
phase change of such materials without causing an increase in the temperature
of these
materials. Suitable phase change materials include parafffnic hydrocarbons,
that is, straight
chain hydrocarbons represented by the formula Cr,Hõ+2, where ri can range from
13 to 28.
Other compounds that are suitable for phase change materials are a PABA salt,
2,2-dimethyl-
1,3-propane diol (DMP), 2-hydroxymethyl-2-methyl-1,3-propane diol (HMP) and
similar
compounds. Also useful are the fatty esters such as methyl palmitate. Phase
change materials
that can be used include paraffinic hydrocarbons.
Heat sensitive recording materials are well known which utilize a color
forming
reaction between a colorless or light-colored basic dye and an organic or
inorganic color
acceptor to obtain record images by thermally bringing the two chromogenic
substances into
contact with each other. Such heat sensitive recording materials are
relatively inexpensive,
are adapted for use with recording devices which are compact and easy to
maintain, and have
therefore found wide applications as recording media for facsimile systems,
various
computers, etc. In order to improve light resistance of heat sensitive
recording materials a
finely divided ultraviolet absorber or blocker can be added to the heat
sensitive recording
layer or protective layer.
Further embodiments of the present invention provide microcapsules that can
retain
an ultraviolet absorber, exhibit resistance to being ruptured at a usual
pressure and/or possess
suitable ultraviolet ray absorbing efficiency.
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Embodiments of the present invention can include a heat sensitive recording
material
comprising a substrate, a recording layer formed over the substrate and
containing.a colorless
or light-colored basic dye and a color acceptor, and a protective layer formed
over the
recording layer, the recording material being characterized in that
microcapsules having an
ultraviolet absorber enclosed therein and having substantially no color
forming ability are
incorporated in the protective layer.
Further, the present invention provides microcapsules having an ultraviolet
absorber
and as required an organic solvent enclosed therein, which have capsule wall
fihn of
synthetic resin and mean particle size of about 0.1 to 3 m.
The following are examples of ultraviolet absorbers that may be used in the
present
invention. Phenyl salicylate, p-tert-butylphenyl salicylate, p-octylphenyl
salicylate and like
salicylic acid type ultraviolet absorbers; 2,4-dihydroxybenzophenone, 2-
hydroxy-4-
methoxybenzophenone, 2-hydroxy-4-octyloxybenzophenone, 2-hydroxy-4-
dodecyloxybenzophenone, 2,2-dihydroxy-4-methoxybenzophenone, 2,2,'-dihydroxy-
4,4'-
dimethoxybenzophenone, 2-hydroxy-4-methoxy-5-sulfobenzophenone and like
benzophenone type ultraviolet absorbers; 2-ethylhexyl 2-cyano-3,3-diphenyt-
acrylate, ethyl
2-cyano-3,3-diphenylacrylate and like cyanoacrylate type ultraviolet
absorbers; bis(2,2,6,6-
tetramethyl-4-piperidyl) sebacate, bis(2,2,6,6-tetramethyl-4-piperidyl)
succinate,
bis(1,2,2,6,6-pentamethyl-4-piperidyl) 2-(3,5-di-tert-butyl-4-hydroxybenzyl)-2-
n-butyl
malonate and like hindered amine type ultraviolet absorbers; 2-(2'-
hydroxyphenyl)benzotriazole, 2-(2'-hydroxy-5'-methylphenyl)benzotriazole, 2-
(2'-hydroxy-5
-tert-butylphenyl)benzotriazole, 2- (2'-hydroxy-3',5'-di-tert-
butylphenyl)benzotriazole, 2- (2'-
hydroxy-3'-tert-butyl-5'-methylphenyl)-5-chlorobenzotriazole, 2-(2'-hydroxy-
3',5'-di-tert-
butylphenyl)-5-chlorobenzotriazole, 2-(2'-hydroxy-3',5'-di-tert-butylphenyl)-5-
tert-
butylbenzotriazole, 2-(2'-hydroxy-3',5'-di-tert-amylphenyl)benzotriazole, 2-
(2'-hydroxy-3',5'-
di-tert-amylphenyl)-5-tert-amylbenzotriazole, 2-(2'-hydroxy-3',5'-di-tert-
amylphenyl)-5-
methoxybenzotriazole, 2-[2'-hydroxy-3'-(3",4",5",6"-tetrahydrophthalimido-
methyl)-5'-
methylpheny 1)benzotriazole, 2-(2'-hydroxy-5'-tert-octylphenyl)benzotriazole,
2-(2'-hydroxy-
3'-sec-butyl-5'-tert-butylphenyl)benzotriazole, 2-(2'-hydroxy-3'-tert-amyl-5'-
phenoxyphenyl)-
5-methylbenzotriazole, 2-(2'-hydroxy-5'-n-dodecylphenyl)benzotriazole, 2-(2'-
hydroxy-5'-
sec-octyloxyphenyl)-5-phenylbenzotriazole, 2-(2'-hydroxy-3'-tert-amyl-5'-
phenylphenyl)-5-
methoxybenzotriazole, 2-[2'-hydroxy-3',5'-bis(c~tx-
dimethylbenzyl)phenyl]benzotri:azole and
like benzotriazole type ultraviolet absorbers which are solid at ordinary
temperature; 2-(2'-
Hydroxy-3'-dodecyl-5'-methylphenyl)-benzotriazole, 2-(2'-hydroxy-3'-undecyl-5'-
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methylphenyl)-benzotriazole, 2-(2'-hydroxy-3'-tridecyl-5'-methylphenyl)-
benzotriazole, 2-(2'-
hydroxy-3'-tetradecyl-5'-methylphenyl)-benzotriazole, 2-(2'-hydroxy-3'-
pentadecyl-5'-
methylphenyl)-benzotriazole, 2-(2'-hydroxy-3'-hexadecyl-5'-methylphenyl)-
benzotriazole, 2-
[2'-hydroxy-4'-(2"-ethylhexyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4'-(2"-
ethylheptyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4'-(2"-
ethyloctyl)oxyphenyl]-
benzotriazole, 2-[2'-hydroxy-4'-(2"-propyloctyl)oxyphenyl]-benzotriazole, 2-
[2'-hydroxy-4'-
(2"-propylheptyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4'-(2"-
propylhexyl)oxyphenyl]-
benzotriazole, 2-[2'-hydroxy-4'-(1"-ethylhexyl)oxyphenyl]-benzotriazole, 2-[2'-
hydroxy-4'-
(1 "-ethylheptyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4'-(1 "-
ethyloctyl)oxyphenyl]-
benzotriazole, 2-[2'-hydroxy-4'-(1 "-propyloctyl)oxyphenyl] -benzotriazole, 2-
[2'-hydroxy-4'-
(1 "-propylheptyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy4'-(1 "-
propylhexyl)oxyphenyl]-
benzotriazole, 2-(2'-hydroxy-3'-sec-butyl-5'-tert-butylphenyl-5-n-
butylbenzotriazole, 2-(2'-
hydroxy-3'-sec-butyl-5'-tert-butylphenyl) -5-tert-pentyl-benzotriazole, 2-(2'-
hydroxy-3-sec-
butyl-5'-tert-butylphenyl)-5-n-pentyl-benzotriazole, 2-(2'-hydroxy-3'-sec-
butyl-5'-tert-
pentylphenyl)-5-tert-butylbenzotriazole , 2-(2'-hydroxy-3'-sec-butyl-5'-tert-
pentylphenyl)-5-
n-butylbenzotriazole, 2-(2'-hydroxy-3',5'-di-tert-butylphenyl)-5-sec-
butylbenzotriazole, 2-(2'-
hydroxy-3',5'-di-tert-pentylphenyl)-5-sec-butylbenzotriazole, 2-(2'-hydroxy-3'-
tert-butyl-5'-
tert-pentylphenyl)-5-sec-butylbenzotriazole, 2-(2'-hydroxy-3',5'-di-sec-
butylphenyl)-5-
chlorobenzotriazole, 2-(2'-hydroxy-3',5'-di-sec-butylphenyl)-5-
methoxybenzotriazole, 2-(2'-
hydroxy-3',5'-di-sec-butylphenyl)-5-tert-butylbenzotriazole, 2-(2'-hydroxy-
3',5'-di-sec-
butylphenyl)-5-n-butylbenzotriazole, octyl 5-tert-butyl-3-(5-chloro-2H-
benzotriazole-2-yl)-4-
hydroxybenzene-propionate, condensate of methyl 3-[3-tert-butyl-5-(2H-
benzotriazole-2-yl)-
4-hydroxyphenyl]propionate and polyethylene glycol (molecular weight: about
300) and like
beiizotriazole type ultraviolet absorbers which are liquid at ordinary
temperature. Of course,
the ultraviolet absorber is not limited to thereabove and can be used as
required in a mixture
of at least two of them.
Although the amount of ultraviolet absorber to be used is not limited
specifically, the
amount can be adjusted to 10 to 500 parts by weight, and generally from to 20
to 250 parts by
weight.
The microcapsules for use in the present invention can be prepared by various
known
methods. They are prepared generally by emulsifying and dispersing the core
material (oily
liquid) comprising an ultraviolet absorber and, if necessary, an organic
solvent in an aqueous
medium, and forming a wall film of high-molecular-weight substance around the
resulting
oily droplets.
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Examples of useful high-molecular-weight substances for formi.ng the wall fihn
of
microcapsules are polyurethane resin, polyurea resin, polyamide resin,
polyester resin,
polycarbonate resin, aminoaldehyde resin, melamine resin, polystyrene resin,
styrene-acrylate
copolymer resin, styrene-methacrylate copolymer resin, gelatin, polyvinyl
alcohol, etc.
Especially, microcapsules having a wall film of a synthetic resin,
particularly polyurea resin,
polyurethane resin and aminoaldehyde resin among other resins have excellent
retainability
of an ultraviolet absorber and high heat resistance and accordingly exhibit
the outstanding
additional effect to serve the function of a pigment which is to be
incorporated in the
protective layer for preventing sticking to the thermal head. Moreover,
microcapsules having
a wall fihn of polyurea resin or polyurethane resin are lower in refractive
index than
microcapsules with wall films of other materials and usual pigments, are
spherical in shape
and are therefore usable favorably because even if present in a large quantity
in the protective
layer, they are unlikely to reduce the density of record images (so-called
whitening) owing to
irregular reflection of light. Further, polyurea resin and polyurethane resin
are more elastic
than aminoaldehyde resin and therefore polyurea resin and polyurethane resin
are generally
used as a wall film for microcapsules that are used under a condition of high
pressure. On the
other hand, microcapsules having a wall film made,from aminoaldehyde resin
have a merit
that the wall film can be controlled in thickness without depending on
particle size of
emulsion because the microcapsules can be prepared by adding a wall-forming
material after
emulsification of a core material.
The present invention may also include an organic solvent together with an
ultraviolet
absorber. The organic solvent is not particularly linuted and various
hydrophobic solvents
can be used which are used in a field of pressure sensitive manifold papers.
Examples of
organic solvents are tricresyl phosphate, octyldiphenyl phosphate and like
phosphates, dibutyl
phthalate, dioctyl phthalate and like phthalates, butyl oleate and like
carboxylates, various
fatty acid amides, diethylene glycol dibenzoate, monoisopropylnaphthalene,
diisopropylnaphthalene and like alkylated naphthalenes, 1-methyl-l-phenyl-l-
tolylmethane,
1-methyl-l-phenyl-l-xylylmethane, 1-phenyl-l-tolylmethane and like alkylated
benzenes,
isopropylbiphenyl and like alkylated biphenyls, trimethylolpropane triacrylate
and like
acrylates, ester of polyols and unsaturated carboxylic acids, chlorinated
paraffin and
kerosene. These solvents can be used individually or in a mixture of at least
two of them.
Among these hydrophobic media having a high boiling point, tricresyl phosphate
and 1-
phenyl-l-tolyhnethane are desirable since they exhibit higli solubility in
connection with the
ultraviolet absorber to be used in the present invention. Generally, the lower
the viscosity of
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WO 2007/086900 PCT/US2006/012942
the core material, the smaller is the particle size resulting from
emulsification and the
narrower is the particle size distribution, so that a solvent having a low
boiling point is
conjointly usable to lower the viscosity of the core material. Examples of
such solvents
having a low boiling point are ethyl acetate, butyl acetate, methylene
chloride, etc.
The amount of organic solvent to be used should be suitably adjusted according
to the
kind and amount of ultraviolet absorber to be used and the kind of organic
solvent and is not
limited specifically. For example, in case of using an ultraviolet absorber
that is liquid at
ordinary temperature, an organic solvent is not necessarily used. However, in
case of using an
ultraviolet absorber which is solid at ordinary temperature, since it is
desired that the
ultraviolet absorber be in a fully dissolved state in the microcapsules, the
amount of organic
solvent, for example in case of microcapsules of polyurea resin or
polyurethane resin, is
adjusted generally from to usually about 10 to 60 wt. %, or from to about 20
to 60 wt. %,
based on the combined amount of organic solvent, ultraviolet absorber and wall-
forming
material. Further, in case of microcapsules of aminoaldehyde resin, the amount
of organic
solvent is adjusted to usually about 50 to 2000% by weight, generally from
about 100 to
1000% by weight of ultraviolet absorber.
Additionally, an absorber may be utilized. An absorber should be selected
which
reduces the sensitivity of the microcapsule in those portions of its spectral
sensitivity range
which interfere with the exposure of microcapsules at other wavelengths (its
inactive range)
without overly reducing the sensitivity of the microcapsule in those portions
of the spectral
sensitivity range in which the microcapsule is intended to be exposed (its
active range). In
some cases it may be necessary to balance the absorption characteristics of
the absorber in the
active range and the inactive range to achieve optimum exposure
characteristics. Generally,
absorbers having an extinction coefficient greater than about 100/M cm in the
inactive range
and less than about 100,000/M cm in the active range of the microcapsule are
used. When the
absorber is directly incorporated into the photosensitive composition,
ideally, it should not
inhibit free radical polymerization, and it should not generate free radicals
upon exposure.
The absorbers used in the present invention can be selected from among those
absorbers that are known in the photographic art. Examples of such compounds
include dyes
conventionally used as silver halide sensitizing dyes in color photography
(e.g., cyanine,
merocyanine, hemicyanine and styryl dyes) and ultraviolet absorbers. A number
of colored
dyes that absorb outside the desired sensitivity range of the microcapsules
and do not absorb
heavily within the range could also be used as absorbers in the present
invention. Among
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WO 2007/086900 PCT/US2006/012942
these, Sudart I, Sudan II, Sudan III, Sudan Orange G, Oil Red 0, Oil Blue N,
and Fast Garnet
GBC are examples of potentially useful compounds.
Additionally, ultraviolet absorbers that may be desirable include those
selected from =
hydroxybenzophenones, hydroxyphenylbenzo-triazoles and formamidines. The
absorbers
may be used alone or in combination to achieve the spectral sensitivity
characteristics that are
desired.
Representative examples of useful hydroxybenzophenones are 2-hydroxy-4-n-
octoxybenzophenone (UV-CHEK AM-300 from Ferro Chemical Division, Mark 1413
from
Argus Chemical Division, Witco Chem. Corp., and Cyasorb UV-531 Light Absorber
from
American Cyanamid), 4-dodecyl-2-hydroxybenzophenone (Eastman Inhibitor DOBP
from
Eastman Kodak), 2-hydroxy-4-methoxybenzophenone (Cyasorb UV-9 Light Absorber
from
American Cyanamid), and 2,2'-dihydroxy-4-methoxybenzophenone (Cyasorb UV-24
Light
Absorber from American Cyanamid). Representative examples of useful
hydroxybenzophenyl benzotriazoles are 2-(2'-hydroxy-5'-
methylphenyl)benzotriazole
(Tinuvin P from Ciba-Geigy Additives Dept.), 2-(3',5'-ditert-butyl-
2'hydroxyphenyl)-5-
chlorobenzotriazole (Tinuvin 327 from Ciba-Geigy), and 2-(2-hydroxy-5-t-
octylphenyl)benzotriazole (Cyasorb UV-5411 Light Absorber from American
Cyanamid).
Representative examples of useful formamidines are described in U.S. Patent
No. 4,021,471
and include N-(p-ethoxy-carbonylphenyl)-N'-ethyl-N'-phenylformamidine (Givsorb
UV-2
from Givaudan Corp.). The optimum absorber and concentration of absorber for a
particular
application depends on both the absorption maximum and extinction coefficient
of the
absorber candidates and the spectral sensitivity characteristics of the
associated
photoinitiators.
Additionally, the microcapsules, photosensitive compositions, image-forming
agents,
developers, and development techniques described in U.S. Patent Nos. 4,399,209
and
4,440,846.
Particularly, formulations to be applied in spraying forms such as water
dispersible
concentrates or wettable powders may contain surfactants such as wetting and
dispersing
agents, e.g. the condensation product of formaldehyde with naphthalene
sulphonate, an
alkylarylsulphonate, a lignin sulphonate, a fatty alkyl sulphate, and
ethoxylated alkylphenol
and an ethoxylated fatty alcohol.
2. Liposomal Formulations
As used herein, the term "liposome" refers to a structure including a lipid
bilayer
enclosing at least one aqueous comparhnent. The walls are prepared from lipid
molecules,
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which have the tendency both -to form bilayers and to minimize their surface
area. The lipid
molecules that comprise the liposome have hydrophilic and lipophilic portions.
Upon
exposure to water, the lipid molecules form a bilayer membrane wherein the
lipid ends of the
molecules in each layer are directed to the center of the membrane, and the
opposing polar
ends form the respective inner and outer surfaces of the bilayer membrane.
Thus, each side of
the membrane presents a hydrophilic surface while the interior of the membrane
comprises a
lipophilic medium.
Liposomes can be classified into several categories based on their overall
size and the
nature of the lamellar structure. The classifications include small
unilamellar vesicles (SUV),
multilamellar vesicles (1VII.,V), large unilamellar vesicles (LUV), and
oligolarnellar vesicles.
SUVs range in diameter from approximately about 20 to 50 nanometers and can
include a
single lipid bilayer surrounding an aqueous compartment. A characteristic of
SUVs is that a
large amount of the total lipid, about 70%, is located in the outer layer of
the bilayer. Where
SLTVs are single compartment vesicles of a fairly uniform size, MLVs vary
greatly in
diameter up to about 30,000 nanometers and are multicompartmental in their
structure
wherein the liposome bilayers can be typically organized as closed concentric
lamellae with
an aqueous layer separating each lamella from the- next. Large unilamellar
vesicles are, so
named because of their large diameter, which ranges from about 600 nanometers
to 30
microns. Oligolamellar vesicles are intermediate liposomes having a larger
aqueous space
than MLVs and a smaller aqueous space than LUVs. Oligolamellar vesicles have
more than
one internal compartment and possibly several concentric lamellae, but they
generally have
fewer lamellae than MLVs.
A variety of methods for preparing liposomes are known in the art, several of
which
are described in Liposome Techfaology (Gregoriadis, G., editor, three volumes,
CRC Press,
Boca Raton 1984) or have been described by Lichtenberg and Barenholz in
Methods of
Biochemical Analysis, Volume 33, 337-462 (1988). Further methods of preparing
liposomal
fornnulations can be found in U.S. Patent Nos. 7,022,336; 6,989,153;
6,726,924; 6,355,267;
6,110,491; 6,007,838; 5,094,785 and 4,515,736. Liposomes are also well
recognized as
useful for encapsulating biologically active materials. Preparation methods
particularly
involving the encapsulation of DNA by liposomes, and methods that have a
direct application
to liposome-mediated transfection, have been described by Hug and Sleight in
Biochimica
and Biophysica Acta, 1097, 1-17 (1991).
The liposomes of the present invention can be prepared from phospholipids, but
other
molecules of similar molecular shape and dimensions having both a hydrophobic
and a
CA 02603869 2007-10-03
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hydrophilic moiety can be used. For the purposes of the present invention, all
such suitable
liposome-forming molecules will be referred to herein as lipids. One or more
naturally
occurring and/or synthetic lipid compounds may be used in the preparation of
the liposomes.
Representative suitable phospholipids or lipid compounds for forming initial
liposomes useful in the present invention include, but are not limited to,
phospholipid-related
materials such as phosphatidylcholine (lecithin), lysolecithin,
lysophosphatidylethanol-amine,
phosphatidylserine, phosphatidylinositol, sphingomyelin,
phosphatidylethanolamine
(cephalin), cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate,
phosphatidylcholine, and dipahnitoyl-phosphatidylglycerol. Additional
nonphosphorous-
containing lipids include, but are not limited to, stearylamine, dodecylamine,
hexadecyl-
amine, acetyl palmitate, glycerol ricinoleate, hexadecyl sterate, isopropyl
myristate,
amphoteric acrylic polymers, fatty acid, fatty acid amides, cholesterol,
cholesterol ester,
diacylglycerol, diacylglycerolsuccinate, and the like.
As understood by one skilled in the art, different lipids can be used with
different
properties, cationic, anionic or neutral, but the preparation method can
remain the same
regardless of which lipid combination is used. More specifically, once lipids
have been
selected for use in the liposome, they can be dissolved in an organic solvent
to ensure
complete mixing. The organic solvent can be removed by evaporation followed by
drying and
a lipid film remains of the homogenous lipid mixture. The lipid mixture can be
frozen in
cakes and dried. The lipid cakes can be stored frozen until hydration.
The addition of an aqueous medium and agitation of the container hydrate the
lipid
cake. The resulting product is a large, multilamellar vesicle. This structure
can include
concentric rings of lipid bilayers separated by water. The large,
multilamellar vesicles can be
downsized by the application of energy, either in the form of ineclianical
energy in the
process of extrusion or by sonic energy in sonication. The hydrated lipid can
be forced
though a polycarbonate filter with progressively smaller pores to produce
particles with a
diameter of similar size to the pore. Before the final pore size is used, the
lipid suspension
may be subjected to several freeze-thaw cycles to ensure the final particles
are homogenous
in size. Final particle size is partly dependent on the lipid combination
used. The mean
particle size is reproducible from batch to batch. This process can produce
large, unilamellar
vesicles that can be reduced to small, unilamellar vesicles by the application
of sonic energy
from a sonicator. The particles in the test tube being sonicated can be
removed by
centrifugation. Mean size of the resulting vesicles can be influenced by
composition,
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concentration, volume and temperature of the lipid mixture and duration,
power, and tuning
of the sonicator. I
Specific liposome preparation methods include, but are not limited to, the
hand-
shaken method, sonication method, reverse-phase evaporation method, freeze-
dried
rehydration method, and the detergent depletion method. According to the hand-
shaken
method, in order to produce liposomes, lipid molecules are introduced into an
aqueous
environment. When dry lipid fihn is hydrated the lamellae swell and grow into
myelin
figures. Mechanical agitation, for example, vortexing, shaldng, swirling or
pipetting, causes
myelin figures (thin lipid tubules) to break and reseal the exposed
hydrophobic edges
resulting in the formation of liposomes.
The sonication method can be used to prepare small unilamellar vesicles. Two
exemplary sonication techniques include probe sonication and bath sonication.
During probe
sonication, the tip of a sonicator is directly immersed into the liposome
dispersion. The
dissipation of energy at the tip can result in local overheating, and
therefore, the vessel may
be immersed into an ice/water bath. During bath sonication, the liposome
dispersion in a
tube is placed into a bath sonicator. Material being sonicated can be kept in
a sterile
container, unlike the probe units, or under an iuiert atmosphere.
The reverse-phase evaporation method is based on the fonnation of inverted
micelles.
More specifically, inverted micelles are formed upon sonication of a mixt re
of a buffered
aqueous phase, which ineludes contains the water soluble molecules to be
encapsulated into
the liposomes and an organic phase in which the amphiphilic molecules are
solubilized. The
slow removal of the organic solvent leads to transformation of these inverted
micelles into a
gel-like and viscous state. During some point in this procedure, the gel state
collapses and
some of the inverted niicelles disintegrate. The excess of phospholipids in
the environnlent
contributes to the formation of a complete bilayer around the reinaining
micelles, which
results in formation of liposomes. Liposomes made by reverse phase evaporation
method can
be made from various lipid fonnulations and have a tendency to possess aqueous
volume-to-
lipid ratios that are four times higher than nZultilamellar liposomes or hand-
shalcen liposomes.
Duiing the freeze-dried rehydration method, freeze-dried liposomes are formed
from
prefomled liposomes. During dellydration, the lipid bilayers and the
inaterials to be
eneapsulated into the liposomes are brought into close contact. Upon
reswelling, the chances
for encapsulation of the adhered molecules increases. The aqueous phase is
generally added
in sniall portions to the dried materials. As a general rule, the total
volinne used for
rehydration is less than the starting volume of the liposome dispersion.
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The detergent depletion method can be used for preparation of a variety of
liposomes
and proteoliposome formulations. Detergents can be depleted from a mixed
detergent-lipid
inicelles by various techniques that lead to the formation of homogeneous
liposomes. In
practice, lipids below their phase transition temperature can be used with
this preparation
method. However, not all detergents are suited for this method. Exeinplary
detergents
include, but are not limited to, sodium cholate, alkyl(thio)glucoside, and
alkyloxypolyethylenes. Mixed micelles are prepared by adding the concentrated
detergent
solution to mtultilanlellar liposomes (the final concentration of the
detergent should be well
above the critical micelle concentration (CMC) of the detergent). The use of
different
detergents can result in different size distributions of the vesicles formed.
Faster depletion
rates can produce smaller size liposomes. The use of different detergents may
also result in
different ratios of large unilamellar vesicles/ oligolamellar
vesicles/multilamellar vesicles.
3. Micelles and Bicelles
As used herein, "micelle" refers to an aggregate of surfactant molecules
dispersed in
a liquid colloid. Micelles can be globular in shape, but may exist in other
shapes including,
but not limited to, ellipsoids, cylinders, bilayers, and vesicles. The shape
of a micelle can be
controlled largely by the molecular geometry of its surfactant molecules;
however, shape also
depends oli the conditions (such as temperature or pH, and the type and
concentration of any
added salt). In a micelle, the hydrophobic tails of several surfactant
molecules assemble into
an oil-like core that has less contact with water. In contrast, surfactant
monomers are
surrounded by water molecules that create a "cage" of molecules connected by
hydrogen
bonds. In a nonpolar solvent, the hydrophilic groups form the core of the
micelle, and the
hydrophobic groups remain on the surface of the micelle (so-called reverse
micelle).
Micelles may form when the concentration of surfactant is greater than the
critical
micelle concentration (CMC), and the temperature of the system is greater than
the critical
micelle temperature, or Krafft temperature. In water, the hydrophobic effect
is the driving
force for micelle formation, despite the fact that assembling surfactant
molecules together
reduces their entropy. Generally, above the CMC, the entropic penalty of
assembling the
surfactant molecules is less than the entropic penalty of the caging water
molecules.
As used herein, the term "bicelle" refers to a bilayered mixed micelle.
Bicelles can be
characterized as a mixture of long-chain bilayer forming phospholipids and
short-chain
micelle forming lipids of detergents.
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The preparation of micelles and bicelles are well known in the art. Further
details
regarding preparation of these structures can be found in US. Patent Nos.
6,897,297;
6,696,081; 6,586,559; 6,444,793;and 5,534,259.
4. PEGylation
Attachment of polyalkylene moieties as described herein can be employed to
increase
water solubility or solubility in aqueous solutions and/or extend the half-
life of the native
compounds discussed herein. Any conventional pegylation method can be
employed,
provided that the pegylated agent retains the desired activity. See also
Schacht, E.H. et al.
Poly(ethylene glycol) Chenzistry and Biological Applications, American
Chemical Society,
San Francisco, CA 297-315 (1997).
Polyalkylene glycol is a biocompatible polymer where, as used herein,
polyalkylene
glycol refers to straight or branched polyalkylene glycol polymers such as
polyethylene
glycol, polypropylene glycol, and polybutylene glycol, and further includes
the
monoalkylether of the polyalkylene glycol. In some embodiments of the present
invention,
the polyalkylene glycol polymer is a lower alkyl polyalkylene glycol moiety
such as a
polyethylene glycol moiety (PEG), a polypropylene glycol moiety, or a
polybutylene glycol
moiety. PEG has the formula -H(CH2CH2O)nH, where n can range from about I to
about
4000 or more. In some embodiments, n is I to 100, and in other embodiments, n
is 5 to 30.
PEG can range from average molecular weight of about 1 to about 22,000. For
example, an
average molecular weight of about 300 can correspond to n is 5, ati average
molecular weight
of about 2,300 can correspond to n is 50, an average molecular weight of about
13,300 can
correspond to n is 300 and an average molecular weight of about 22,000 can
correspond to n
is 500. In some embodiments, the PEG moiety can be linear or branched. In
further
embodiments, PEG can be attached to groups such as hydroxyl, alkyl, aryl, acyl
or ester. In
some embodiments, PEG can be an alkoxy PEG, such as methoxy-PEG (or mPEG),
where
one terminus is a relatively inert alkoxy group, while the other terminus is a
hydroxyl group.
PEG can be synthesized or is a commercially available product that can be
readily obtained.
According to some embodiments of the present invention, the pegylated
compounds
of the present invention can be water soluble, soluble in isopropyl alcohol
(IPA), ethanol
(ETOH), dimethyl sulfoxide (DMSO) and methanol (MTOH), less sensitive to UV
light than
a non-pegylated counterpart and/or economical to synthesize.
The compounds of the present invention can be pegylated at at least four sites
and/or
can be pegylated in many differing PEG lengths and molecular weights. In some
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embodiments, the PEG moiety is PEG200 through PEGsooo= Pegylated compounds of
the
present invention can further exhibit improved solubility, improved stability,
lower toxicity,
decreased degradation and chemical sensitivities and/or increased conjugation
potential to
like molecules and other molecules such as known drugs, biological tags,
labels, fluorescents,
radioisotopes and the like.
Embodiments of the present invention include assays in which a sample is
combined
with a labeling reagent. "Labeling reagents" include, but are not limited to,
luminescently
labeled macromolecules including fluorescent protein analogs and biosensors,
luminescent
macromolecular chimeras including those formed with the green fluorescent
protein and
mutants thereof, luminescently labeled primary or secondary antibodies that
react with
cellular antigens involved in a physiological response, luminescent stains,
dyes, and other
small molecules.
"Biosensors" refer to macromolecules consisting of a biological functional
domain
and a luminescent probe or probes that report the environmental changes that
occur either
intemally or on their surface. A class of luminescently labeled macromolecules
designed to
sense and report these changes have been termed "fluorescent-protein
biosensors". The
protein component of the biosensor provides an evolved molecular recognition
moiety. A
fluorescent molecule attached to the protein component in the proximity of an
active site
transduces environmental changes into fluorescence signals that can be
detected using a
.20 system with an appropriate temporal and spatial resolution. Because the
modulation of native
protein activity within the living cell is reversible, and because fluorescent-
protein biosensors
can be designed to sense reversible changes in protein activity, these
biosensors are
essentially reusable.
"High content screening (HCS)" can be used to measure the effects of drugs on
complex molecular events such as signal transduction pathways, as well as cell
functions
including, but not limited to, apoptosis, cell division, cell adhesion,
locomotion, exocytosis,
cell growth and metabolism, protein synthesis, enzymatic fimctions, cell-cell
communication
and the detection of healthy and/or diseased cells. Multicolor fluorescence
permits multiple
targets and cell processes to be assayed in a single screen. Cross-correlation
of cellular
responses will yield a wealth of information required for target validation
and lead
optimization.
Methods of screening cells treated with dyes and fluorescent reagents is well
known
in the art. There is a considerable body of literature related to genetic
engineering of cells to
produce fluorescent proteins, such as modified green fluorescent protein
(GFP), as a reporter
CA 02603869 2007-10-03
WO 2007/086900 PCT/US2006/012942
molecule. Some properties of wild-type GFP are disclosed by Morise et al.
(Biochemistry 13
(1974), p. 2656-2662), and Ward et al. (Photochem. Photobiol. 31 (1980), p.
611-615). The
GFP of the jellyfish Aequorea Victoria has an excitation maximum at 395 nm and
an
emission maximum at 510 nm, and does not require an exogenous factor for
fluorescence
activity. Uses for GFP disclosed in the literature are widespread and include
the study of gene
expression and protein localization (Chalfie et al., Science 263 (1994), p.
12501-12504)), as a
tool for visualizing subcellular organelles (Rizzuto et al., Curr. Biology 5
(1995), p. 635-
642)), visualization of protein transport along the secretory pathway (Kaether
and Gerdes,
FEBS Letters 369 (1995), p. 267-271)), expression in plant cells (Hu and
Cheng, FEBS
Letters 369 (1995), p. 331-334)) and Drosophila embryos (Davis et al., Dev.
Biology 170
(1995), p. 726-729)), and as a reporter molecule fused to another protein of
interest (U.S.
Patent No. 5,491,084). Similarly, WO 96/23898 relates to methods of detecting
biologically
active substances affecting intracellular processes by utilizing a GFP
construct having a
protein kinase activation site. This patent, and all other patents referenced
in this application
are incorporated by reference in their entirety.
Numerous references are related to GFP proteins in,biological systems. For
example,
WO 96/09598 describes a system for isolating cells of interest utilizing the
expression of a
GFP like protein. WO 96/27675 describes the expression of GFP in plants. WO
95/21191
describes modified GFP protein expressed in transformed organisms to detect
mutagenesis.
U.S. Patent Nos. 5,401,629 and 5,436,128 describe assays and compositions for
detecting and
evaluating the intracellular transduction of an extracellular signal using
recombinant cells that
express cell surface receptors and contain reporter gene constructs that
include transcriptional
regulatory elements that are responsive to the activity of cell surface
receptors. There are
also numerous journals discussing the use of GFP proteins.
Performing a screen on many thousands of compounds requires parallel handling
and
processing of many compounds and assay component reagents. Standard high
throughput
screens ("HTS") use mixtures of compounds and biological reagents along with
some
indicator compound loaded into arrays of wells in standard microtiter plates
with 96 or 384
wells. The signal measured from each well, either fluorescence emission,
optical density, or
radioactivity, integrates the signal from all the material in the well giving
an overall
population average of all the molecules in the well.
Another system fluorescence imaging plate reader (FLIPR) uses a low angle
laser
scanning illumination and a mask to selectively excite fluorescence within
approximately 200
microns of the bottoms of the wells in standard 96 well plates in order to
reduce background
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WO 2007/086900 PCT/US2006/012942
when imaging cell monolayers. This system uses a charge couple device (CCD)
camera to
image the whole area of the plate bottom. Although this system measures
signals originating
from a cell monolayer at the bottom of the well, the signal measured is
averaged over the area
of the well and is therefore still considered a measurement of the average
response of a
population of cells. The image is analyzed to calculate the total fluorescence
per well for cell-
based assays. Fluid delivery devices have also been incorporated into cell
based screening
systems, such as the FLIPR system, in order to initiate a response, which is
then observed as
a whole cell population average response using a macro-imaging system.
In contrast to high throughput screens, various high-content screens (HCS)
have been
developed to address the need for more detailed information about the temporal-
spatial
dynamics of cell constituents and processes. High-content screens automate the
extraction of
multicolor fluorescence information derived from specific fluorescence-based
reagents
incorporated into cells (Giuliano and Taylor (1995), Curr. Op. Cell Biol. 7:4;
Giuliano et al.
(1995). Ann. Rev. Biophys. Biomol. Struct. 24:405). Cells are analyzed using
an optical
system that can measure spatial, as well as temporal dynamics. (Farkas et al.
(1993) Ann.
Rev. Physiol. 55:785; Giuliano et al. (1990) In Optical Microscopy for
Biology. B. Herman
and K. Jacobson (eds.), pp. 543-557. Wiley-Liss, New York; Hahn et al (1992)
Nature
359:736; Waggoner et al. (1996)'Hum. Pathol. 27:494). The concept is to treat
each cell as a
"well" that has spatial and temporal information on the activities of the
labeled constituents.
1 The types of biochemical and molecular information now accessible through
fluorescence-based reagents applied to cells include ion concentrations,
membrane potential,
specific translocations, enzyme activities, gene expression, as well as the
presence, amounts
and patterns of metabolites, proteins, lipids, carbohydrates, and nucleic acid
sequences
(DeBiasio et al., (1996) Mol. Biol. Cell. 7:1259; Giuliano et al., (1995) Ann.
Rev. Biophys.
Biomol. Struct. 24:405; Heim and Tsien, (1996) Curr. Biol. 6:178).
High-content screens can be performed on either fixed cells, using
fluorescently
labeled antibodies, biological ligands, and/or nucleic acid hybridization
probes, or live cells
using multicolor fluorescent indicators and "biosensors." The choice of fixed
or live cell
screens depends on the specific cell-based assay required.
As understood by one of skill in the art, any of the methods and screens
discussed
above can be utilized with the conlpounds of the present invention.
Generally, fixed cell assays are the simplest, since an array of initially
living cells in a
microtiter plate format can be treated with various compounds and doses, then
the cells can
be fixed, labeled with specific reagents, and quantified. No environmental
control of the cells
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WO 2007/086900 PCT/US2006/012942
is required after fixation. Spatial information is acquired, but only at one
time point. The
availability of thousands of antibodies, ligands and nucleic acid
hybridization probes that can
be applied to cells makes this an attractive approach for many types of cell-
based screens.
The fixation and labeling steps can be automated, allowing efficient
processing of assays.
Live cell assays are more sophisticated and powerful, since an array of living
cells
containing the desired reagents can be screened over time, as well as space.
Environmental
control of the cells (temperature, humidity, and carbon dioxide) should be
maintained during
measurement, since the physiological health of the cells should be maintained
for multiple
fluorescence measurements over time. There is a growing list of fluorescent
physiological
indicators and "biosensors" that can report changes in biochemical and
molecular activities
within cells (Giuliano et al., (1995) Ann. Rev. Biophys. Biomol. Struct.
24:405; Hahn et al.,
(1993) In Fluorescent and Luminescent Probes for Biological Activity. W. T.
Mason, (ed.),
pp. 349-359, Academic Press, San Diego).
The availability and use of fluorescence-based reagents has helped to advance
the
development of both fixed and live cell high-content screens. Advances in
instrumentation to
automatically extract multicolor, high-content information has made it
possible to develop
HCS into an automated-tool. An article by Taylor, et al. (American Scientist
80 (1992), p.
322-335) describes many of these methods and their applications. For example,
Proffitt et. al.
(Cytometry 24: 204-213 (1996)) describe a semi-automated fluorescence digital
imaging
system for quantifying relative cell numbers in situ in a variety of tissue
culture plate fonnats,
especially 96-well microtiter plates. The system consists of an
epifluorescence inverted
microscope with a motorized stage, video camera, image intensifier, and a
microcomputer
with a PC-Vision digitizer. Turbo Pascal software controls the stage and scans
the plate
taking multiple images per well. The software calculates total fluorescence
per well, provides
for daily calibration, and configures easily for a variety of tissue culture
plate formats.
Thresholding of digital images and reagents which fluoresce only when taken up
by living
cells are used to reduce background fluorescence without removing excess
fluorescent
reagent.
Scanning confocal microscope imaging (Go et al., (1997) Analytical
Biochemistry
247:210-215; Goldman et al., (1995) Experimental Cell Research 221:311-319)
and
multiphoton microscope imaging (Denk et al., (1990) Science 248:73; Gratton et
al., (1994)
Proc. of the Microscopical Society of America, pp. 154-155) are also well
established
methods for acquiring high resolution images of microscopic sainples. The
principle
advantage of these optical systems is the shallow depth of focus, which allows
features of
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limited axial extent to be resolved against the background. For example, it is
possible to
resolve internal cytoplasmic features of adherent cells from the features on
the cell surface.
Because scanning multiphoton imaging utilize short duration pulsed laser
systems to achieve
the high photon flux required, fluorescence lifetimes can also be measured in
these systems
(Lakowicz et al., (1992) Anal. Biochem. 202:316-330; Gerrittsen et al. (1997),
J. of
Fluorescence 7:11-15)), providing additional capability for different
detection modes. Small,
reliable and relatively inexpensive laser systems, such as laser diode pumped
lasers, are now
available to allow multiphoton confocal microscopy to be applied in a fairly
routine fashion.
A major component of the new drug discovery paradigm is a continually growing
family of fluorescent and luminescent reagents that are used to measure the
temporal and
spatial distribution, content, and activity of intracellular ions,
metabolites, macromolecules,
and organelles. Classes of these reagents include labeling reagents that
measure the
distribution and amount of molecules in living and fixed cells, environmental
indicators to
report signal transduction events in time and space, and fluorescent protein
biosensors to
measure target molecular activities within living cells. A multiparameter
approach that
combines several reagents in a single cell can be a powerful tool for drag
discovery.
Methods of the present invention include high affinity of fluorescent or
luminescent
stilbazium molecules and analogs for specific cellular components. The
affinity for specific
components is governed by forces such as ionic interactions, covalent bonding
(which
includes chimeric fusion with protein-based chromophores, fluorophores, and
lumiphores), as
well as hydrophobic interactions, electrical potential, and, in some cases,
simple entrapment
within a cellular component. Those skilled in this art will recognize a wide
variety of
fluorescent reporter molecules that can be used in addition to the compounds
of the present
invention for multiplexing, including, but not limited to, fluorescently
labeled biomolecules
such as proteins, phospholipids, cellular organelles and DNA hybridizing
probes.
The luminescent probes can be synthesized within the living cell or can be
transported
into the cell via several non-mechanical modes including diffusion,
facilitated or active
transport, signal-sequence-mediated transport, and endocytotic or pinocytotic
uptake.
Mechanical bulk loading methods, which are well lrnown in the art, can also be
used to load
luininescent probes into living cells (Barber et al. (1996), Neuroscience
Letters 207:17-20;
Bright et al. (1996), Cytometry 24:226-233; McNeil (1989) in Methods in Cell
Biology, Vol.
29, Taylor and Wang (eds.), pp. 153-173). These methods include
electroporation and other
mechanical methods such as scrape-loading, bead-loading, impact-loading,
syringe-loading,
hypertonic and hypotonic loading. Additionally, cells can be genetically
engineered to
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express reporter molecules, such as GFP, coupled to a protein of interest as
previously
described (Chalfie and Prasher U.S. Pat. No. 5,491,084; Cubitt et al. (1995),
Trends in
Biochemical Science 20:448-455).
Furthermore, certain cell types within an organism may contain components that
can
be specifically labeled with a formulation of stilbazium or an analog thereof
that may not
occur in other cell types. For example, epithelial cells often contain
polarized membrane
components. That is, these cells asymmetrically distribute macromolecules
along their plasma
membrane. Connective or supporting tissue cells often contain granules in
which are trapped
molecules specific to that cell type (e.g., heparin, histamine, serotonin,
etc.). Most muscular
tissue cells contain a sarcoplasmic reticulum, a specialized organelle whose
function is to
regulate the concentration of calcium ions within the cell cytoplasm. Many
nervous tissue
cells contain secretory granules and vesicles in which are trapped
neurohormones or
neurotransmitters. Therefore, fluorescent molecules can be designed to label
not only specific
components within specific cells, but also specific cells within a population
of mixed cell
types.
Those skilled in the art will recognize a wide variety of ways to measure
fluorescence.
For example, some fluorescent reporter molecules exhibit a change in
excitation or emission
spectra, some exhibit resonance energy transfer where one fluorescent reporter
loses
fluorescence, while a second gains in fluorescence, some exhibit a loss
(quenching) or
appearance of fluorescence, while some report rotational movements (Giuliano
et al. (1995),
Ann. Rev. of Biophysics and Biomol. Structure 24:405-434; Giuliano et al.
(1995), Methods
inNeuroscience 27:1-16).
In biochemistry, molecular biology and medical diagnostics, it is often
desirable to
add a fluorescent label to a protein so that the protein can be easily tracked
and quantified.
The normal procedures for labeling requires that the protein be covalently
reacted in vitro
with fluorescent dyes, then repurified to remove excess dye and any damaged
protein. If the
labeled protein is to be used inside cells, it is usually microinjected;
however, this can be a
difficult and/or time-consuming operation that may not be realistically
performed on large
numbers of cells.
The compounds of the present invention can be utilized as staining agents for
DNA/RNA in their isolated state or for isolated cells and organelles. Also,
the compounds
may be utilized as nuclear staining agents for cell based assays. Further
applications include
using the compounds of the present invention to stain tissues and/or organs in
whole animals
as well as embryos, larvae, nematodes, insects and other parasites. In some
embodiments,
CA 02603869 2007-10-03
WO 2007/086900 PCT/US2006/012942
compounds of the present invention could be complexed with specific antibodies
for imaging
of specific organ tissues in the whole animal model systems. The term
"antibody" or
"antibody molecule" in the various grammatical forms as used herein refers to
an
immunoglobulin molecule (including IgG, IgE, IgA, IgM, IgD) and/or
immunologically
active portions of irnmunoglobulin molecules, i.e., molecules that contain an
antibody
combining site or paratope and can bind antigen. An "antibody combining site"
or "antigen
binding site" is that structural portion of an antibody molecule comprised of
heavy and light
chain variable and hypervariable (CDR) regions that specifically binds
antigen. As is known
in the art, particular properties of antibodies relate to immunoglobulin
isotype. In
representative embodiments, the antibody or antigen-binding fragment is an
IgG2a, an IgGl
or an IgG2b isotype molecule. The antibody or fragment can further be from any
species of
origin including avian (e.g., chicken, turkey, duck, geese, quail, etc.) and
mammalian (e.g.,
human, non-human primate, mouse, rat, rabbit, cattle, goat, sheep, horse, pig,
dog, cat, etc.)
species.
The present invention can provide systems, methods, and screens that combine
high
throughput screening (HTS) and high content screening (HCS) that may improve
target
validation and candidate optimization at least by combining many cell
screening formats with
fluorescence-based molecular reagents and computer-based feature extraction,
data analysis,
and automation, resulting in increased quantity and speed of data collection,
shortened cycle
times, and, ultimately, faster evaluation.
Accordingly, in some embodiments, the invention features a technique for
determining the presence, location, or quantity of a cell, and thus, cellular
organelles such as
the nucleus, smooth and/or rough endoplasmic reticulum, centrosome,
cytoskeleton, cell wall,
cell membrane, flagella, cilia, chloroplast, mitochondria, golgi apparatus,
ribosome,
lysosome, centriole, acrosome, glyoxysome, secretory vesicle, peroxisome,
vacuole,
melanosome, myofibril and parenthesome. In other embodiments, the invention
features a
technique for determining the presence, location, quantity and/or health of
analytes and
microorganisms.
In other embodiments, the invention features a technique for determ;ning the
presence, location, or quantity of an RNA molecule of interest in a cell or an
in vitro sample.
This method involves (a) expressing, in the cell or sample, a fusion RNA
molecule including
the RNA molecule of interest covalently linked to an RNA aptamer, (b)
contacting the cell or
sample with a fluorophore under conditions that allow the RNA aptamer to bind
the
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fluorophore and thereby increase or decrease its fluorescence, and (c)
visualizing the
fluorescence of the fluorophore.
In still other embodiments, the invention provides methods for determining the
presence, location, or quantity of a DNA molecule of interest in a cell or an
in vitro sample.
This method involves (a) expressing, in the cell or the sample, a fusion DNA
molecule
including the DNA molecule of interest covalently linked to a DNA aptamer, (b)
contacting
the cell or the sample with a fluorophore under conditions that allow the DNA
aptamer to
bind the fluorophore and thereby increase or decrease its fluorescence, and
(c) visualizing the
fluorescence of the fluorophore.
Embodiments of the invention also include assays for detennining the presence
or
absence of a cell, analyte, nucleic acid or microorganism in a sample, such as
a fluid or
aqueous sample, suspected of containing a microorganism, said assay comprising
combining
the sample with a labeling reagent to form a labeled cell, nucleic acid or
microorganism, said
labeling reagent comprising a dye which directly stains the cell, analyte,
nucleic acid or
microorganism to provide a stained sample comprising a stained cell, analyte,
nucleic acid or
microorganism, wherein said dye is a compound represented by Formula I
= . . (
R,\ ~- \ \ N/ ~R3
/N -- I N
----- / X I
R5 ~
R2 Rq
wherein for Formula I, the NR1R2 and NR3R4 moieties are in the ortho, meta or
para
positions; wherein X- is an anionic salt and X- can be selected from the group
including
fluoride, chloride, bromide, iodide halide, mesylate, tosylate, napthylate,
nosylate, para-
aminobenzoate, benzenesulfonate, besylate, lauryl sulfate, 2,4-dihydroxy
benzophenone, 2-
(2-hydroxy-5'-methylphenyl) benzotriazole, ethyl 2-cyano-3,3-diphenyl acrylate
and 5-butyl
phenyl salicylate.; wherein RI, R2, R3, or R4 are independently selected from
the group
consisting of methyl, ethyl, C1-10 alkyl (linear or branched), alkenes (linear
or branched), or
wherein Rl and R2 or R3 and R4 taken together with the nitrogen atom to which
they are
attached form pyrrolidino or piperidino rings; wherein R5 is a polyalkylene
glycol moiety, a
C1_10 alkyl (linear or branched), an alkene (linear or branched), an alkyne, a
substituted and
unsubstituted aryl, a substituted and unsubstituted benzyl and/or an
organometallic moiety.
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RS may also be an organometallic compound such as organotin, organosilicon, or
organogermanium. Additionally, RS may be (CH2)n MRG6, wherein n is a number
from 1 to 6,
M is an organometallic compound such as tin, silicon, or germanium, and
wherein R6 is a
selected from the group consisting of propyl, butyl, or any alkyl compound;
contacting the
stained sample; and observing the accumulation of the stained cell, analyte,
nucleic acid or
microorganism. The dye can be encapsulated. In some embodiments, the dye can
be loaded
into a microcapsule or a lipid vesicle such as a liposome, micelle and/or
bicelle, to form a
microencapsulated formulation or a lipid vesicle formulation, respectively.
The dye can also
be pegylated.
In various embodiments of the invention, a cell may be bound by one of the
compounds according to Formula I. In some embodiments, the cell is a
prokaryotic cell, such
as a gram-negative or gram-positive bacterial cell. In other embodiments, the
cell is a
eukaryotic cell. For example, the cell may be a yeast, Caenorhabditis,
Xenopus, Drosophila,
zebrafish, squid, plant, mammalian, embryonic, or human cell. In yet other
embodiments, the
cell or the sample is contacted with the fluorophore by incubating the cell or
the sample with
the fluorophore. In still other embodiments, the fluorophore is injected into
the cell or,
administered to a plant, embryo, mammal, transgenic animal, or human including
the cell.
In other embodiments, the population of nucleic acids contains more than one
DNA
molecule or more than one RNA molecule. The nucleic acids may have naturally-
occurring
or non-naturally-occurring polynucleotide sequences. Regions of the nucleic
acids; such as all
or part of a loop, tetraloop, or helix; contain random sequences that differ
between some or
all of the members of the population. In other embodiments, the sequence of a
loop, tetraloop,
or helix is the same in all of the members of the population. The lengths of
any of the loops
or helices may be the same or may differ between members of the population.
The
populations of nucleic acids may contain any number of unique molecules. For
example, the
population may contain as few as about 10, 102, 109, or 1011 unique molecules
or as many as
about 1013, 1015, 1020, or more unique molecules. In some embodiments, at
least one of the
polynucleotide sequences is a non-naturally-occurring sequence. The nucleic
acids may either
all have the same length or some of the molecules may differ in length.
By "fluorophore" is meant a compound that is capable of emitting a fluorescent
signal. As described herein, fluorophores of use in the invention have a
higher fluorescence
intensity when bound to a nucleic acid or protein than when unbound in
solution. The
fluorescence intensity of the bound fluorophore can be at least about 1, 5,
10, 50, 100, 500, or
1000 times that of the unbound fluorophore in an aqueous solution. Examples of
conditions
33
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that may enhance the fluorescence of the bound fluorophore include
rigidification,
conformational restriction, and sequestration from solvent. In one embodiment,
the
fluorophore does not covalently bind the aptamer.
Other fluorophores have a lower fluorescence intensity when bound to a nucleic
acid
or protein than when unbound in solution. The fluorescence intensity of the
bound
fluorophore can be at least about 2, 5, 10, 50, 100, 500, or 1000 less than
that of the unbound
fluorophore in an aqueous solution. An example of a condition that may
decrease the
fluorescence of the bound fluorophore is a change in the conformation of the
fluorophore that
decreases its fluorescence intensity. In one embodiment, the fluorophore does
not covalently
bind the aptamer.
Other fluorophores for use in multiplexing, such as calcium-sensing dyes, may
adopt
at least two different conformational states that result in different
fluorescence intensities. An
aptamer of the invention may modulate the fluorescence of the fluorophore by
increasing the
percentage of the fluorophore in a particular conformational state with
increased or decreased
fluorescence.
The fluorophore is soluble in an aqueous solution at a concentration of about
0.11M,
l M, 10 M, and 50 M. Incubating a cell with these concentrations of the
fluorophore may
not effect the viability of the cell. In another embodiment, incubating a cell
with - the
fluorophore at these. concentrations for as few as about I or 2 hours to as
many as about 8, 12,
24, 36, or more hours does not require the presence of another compound to
prevent toxic
effects of the fluorophore, such as the inactivation of proteins in the cell;
inhibition of
replication, transcription, or translation; or the induction of cell death. By
"cell permeable" is
meant capable of migrating through a cell membrane or cell wall into the
cytoplasm or
periplasm of a cell by active or passive diffusion. The fluorophore can
migrate through both
the outer and inner membranes of gram-negative bacteria or both the cell wall
and plasma
membrane of plant cells. Additionally, the fluorophore can be used to
visualize a cell, analyte
and/or nucleic acids in an in vitro sample.
Embodiments of the present invention may include nucleic acid probes
comprising a
single-stranded oligonucleotide and a compound represented by Formula I
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R, N + Rs
/N___~_ ~ I I -- N\
R2 RS R4
wherein for Formula I, the NR1R2 and NR3R4 moieties are in the ortho, meta or
para
positions;
wherein X- is an anionic salt;
wherein Rl, R2, R3, or R4 are independently selected from the group consisting
of
methyl, ethyl, CI_lo a1ky1(linear or branched), alkenes (linear or branched),
or wherein Rl and
R2 or R3 and R4 taken together with the nitrogen atom to which they are
attached form
pyrrolidino or piperidino rings;
wherein R5 is a polyalkylene glycol moiety, a Cl-lo alkyl (linear or
branched), an
alkene (linear or branched), an alkyne, a substituted and unsubstituted aryl,
a substituted and
unsubstituted benzyl and/or an organometallic moiety. R5 may also be an
organometallic
compound such as' organotin, organosilicon, or organogermanium. Additionally,
R5 may be
(CHZ)I,-MRb, wherein n is a number from I to 6, M is an organometallic
corimpound such as
tin, silicon, or germanium, and wherein R6 is a selected from the group
consisting of propyl,
butyl, or any alkyl compound.
The single-stranded oligonucleotide can be a DNA oligomer. A phosphorus atom
in
the DNA oligomer can be linked by the chemical bond via a linker. The single-
stranded
oligonucleotide can have a sequence complementary to a specific sequence in a
target nucleic
acid containing the specific sequence
Embodiments of the present invention may include methods of selecting a
nucleic
acid molecule which binds to Formula I, wherein said binding increases the
fluorescence
intensity of said Formula I, said method comprising the steps of:
(a) providing a population of candidate nucleic acid molecules;
(b) selecting said candidate nucleic acid molecules which bind said Formula I;
(c) contacting said candidate nucleic acid molecules which bind said Formula I
with
said Formula I; and
(d) selecting said nucleic acid molecules which, upon binding said Formula I
or,
increase its fluorescence intensity.
The nucleic acid can be a DNA or an RNA.
CA 02603869 2007-10-03
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Another embodiment of the present invention includes methods of determining
the
presence, location, or quantity of a nucleic acid of interest in a cell or an
in vitro sample, said
method comprising the steps of: (a) expressing in said cell or said sample a
nucleic acid of
interest; (b) contacting said cell or said sample with Formula I; whereby said
compound binds
to said Formula I and increases its fluorescence intensity; and (c)
visualizing or measuring the
fluorescence of said Formula I, thereby determining the presence, location, or
quantity of said
nucleic acid of interest in said cell or said in vitro sample.
Embodiments of the present invention include methods of determining whether
Formula I is capable of modulating the transcription of a nucleic acid of
interest, said method
further comprising the steps of: (a) expressing in a cell or an in vitro
sarnple a nucleic acid of
interest; (b) contacting said cell or said sample with said Formula I or with
said Formula I
alone, whereby said compound binds to said Formula I and increases its
fluorescence
intensity; and (c) measuring said fluorescence intensity in the presence and
absence of said
compound, whereby said compound is determined to modulate said transcription
if said
compound effects a change in said fluorescence intensity.
Other embodiments include kits for staining nucleic or amino acids in a
sarnple,
comprising: (a) a staining mixture that contains one or more dyes to form a
combined
mixture; wherein each dye independently has the Formula I; b) instructions for
combining
said dye or dyes with a sample containing or thought to contain nucleic or
amino acids, said
instructions comprising i) combining a sample that is thought to contain
nucleic or amino
acids with a staining mixture that contains said dye or dyes to form a
combined mixture; and
ii) incubating the combined mixture for a time sufficient for the dye in the
staining mixture to
associate with the nucleic or amino acids in the sample mixture to form a dye-
amino acid or
dye-nucleic acid complex that gives a detectable optical response upon
illumination.
Additional embodiments include methods of detecting a target analyte in a
sample
containing or suspected of containing one or more analytes, comprising the
steps of: (a)
providing the sample on a solid support wherein the analyte is a nucleic acid
molecule; (b)
combining with said sample a specific-binding molecule, wherein (i) said
specific-binding
molecule is a polymerase chain reaction amplification product comprising
biotin as a
detectable label, and (ii) said combining is performed under conditions that
allow formation
of a first complex comprising said specific-binding molecule and said analyte,
when present;
(c) removing any unbound specific-binding molecule; (d) providing a compound
having the
Formula I; and (e) detecting an optical response based upon the binding of the
compound.
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Embodiments of the present invention include methods for analyzing cells
comprising
providing an array of locations which contain multiple cells wherein the cells
contain one or
more fluorescent reporter molecules; scanning multiple cells in each of the
locations
containing cells to obtain fluorescent signals from the fluorescent reporter
molecule in the
cells; converting the fluorescent signals into digital data; and utilizing the
digital data to
determine the distribution, environment or activity of the fluorescent
reporter molecule within
the cells.
Embodiments of the present invention may also be utilized within cell arrays.
Cell
arrays are used for screening large numbers of compounds for activity with
respect to a
particular biological function and involves preparing arrays of cells for
parallel handling of
cells and reagents. Standard 96 well microtiter plates which are 86 mm by 129
mm, with 6
nun diameter wells on a 9 nm pitch, are used for compatibility with current
automated
loading and robotic handling systems. The microplate is typically 20 mm by 30
mm, with cell
locations that are 100-200 microns in dimension on a pitch of about 500
microns. Methods
for making microplates are described in U.S. Patent No. 6,103,479. Microplates
may consist
of coplanar layers ~of materials to which cells adhere, patterned with
materials to which cells
will not adhere, or etched 3-dimensional surfaces of similarly pattered
materials. The terms
"well" and "znicrowell" refer to a location in an array of any construction to
which cells
adhere and within which the cells are imaged. Microplates may also include
fluid delivery
channels in the spaces between the wells. The smaller format of a microplate
increases the
overall efficiency of the system by minimizing the quantities of the reagents,
storage and
handling during preparation and the overall movement required for the scanning
operation. In
addition, the whole area of the microplate can be imaged more efficiently,
allowing a second
mode of operation for the microplate reader as described later in this
document.
As described above, according to the present invention, it is possible to
provide a
novel compound which is an unfamiliar fluorescent dye which may show a large
fluorescent
enhancement upon intercalation into a double-stranded nucleic acid when used
in detection of
the nucleic acid, and shows a great difference between the excitation
wavelength and the
emission wavelength (i.e., has a large Stokes shift). The compound is used for
conventional
nucleic acid assays by contacting it with a double-stranded nucleic acid or by
linking it with a
single-stranded oligonucleotide to from a nucleic acid probe.
The compounds of the present invention may be further characterized in that
their
fluorescent spectram does not overlap with that of any known fluorescent
intercalative dye.
Therefore, combined use of at least two nucleic acid probes using the
compounds of the
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present invention and conventionally laiown fluorescent intercalative dye(s)
makes it possible
to measure the amplification products from at least two target nucleic acids
in a sample in a
closed vessel without separation while amplifying the target nucleic acids,
i.e., measure at
least target nucleic acids simultaneously.
The present invention is explained in greater detail in the Example that
follows. This
example is intended as illustrative of the invention and is not to be taken
are limiting thereof.
EXAMPLE
Spectra were obtained on solutions at ambient temperature (22 C) in
methacrylate
cuvettes with a lcm path length in a Beclan.an DU-640 spectrometer (Beckman-
Coulter, Palo
Alto CA). The scan rate was l0nm/min from 800nm to 250nm. Blank scans were run
in the
same diluent as used for the analyte and subtracted from the compound spectra.
Both stilbazium chloride (as a red powder) and a liposomal formulation contain
stilbazium (as a turbid, colored solution) were provided. Ethidium bromide,
DMSO and
Ethanol were purchased from Sigma-Aldrich (St. Louis, Mo). Methacrylate, 1.5
mL cuvettes
were purchased from Fisher Scientific. The molar absorption (E) was calculated
by dividing
the observed optical density by the number of moles of analytes
([molar]*volume) and
presents the theoretical absorption of 1.0 mole of the test substance.
Stilbazium chloride, when dissolved in absolute ethanol, resulted in a deep
red-
colored solution. A second dilution in either absolute or 70% (aq.) ethanol
was performed
prior to perfonning the analyses. In both cases, a single, broad peak of
absorption was
observed with an apparent maxima of 509-511 nm and a single, broad peak with a
molar
extinction coefficient, E = 6638400.
The molecule was similarly soluble in DMSO with deep, red coloration at the
4.1
mg/mL initial concentration. Spectra were obtained after a secondary dilution
in the same
solvent to a final concentration of 50 M which yielded a single, broad peak
with E_
4401574 at 507nm.
The stilbazium chloride was found to be mostly insoluble in water. Thus,
spectra
were performed in solutions of ethanol and DMSO. Among the 2 solvents, greater
absorption
was observed for the ethanolic solutions than for compound dissolved in DMSO
(E =
6638400 Vs. 4401574). Secondary dilution into 70% aqueous ethanol was
identical to the
results with absolute ethanol.
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The liposomal formulation provided as a yellow to pink colored solution was
added
directly to a cuvette containing water (50 L in 1.0 mL) yielded apparent light
scattering
turbidity. The resulting spectrum had a maxima at 585nrn with E= 302400. When
formulated as a water soluble liposome complex, the formu]ation was slightly
turbid on
addition to water in a 1:20 dilution. The resulting spectrum showed a shift in
the wavelength
with maximal optical density at 585 nm and E= 302400. The reduced molar
absorption is not
surprising since the analyte is contained in the liposome suspension. Light,
scattering on the
surface of the liposomes likely hindered interaction with the compound.
For comparison, the DNA binding dye, ethidium bromide, dissolved in water, had
its
maximal optical density at 284 nm with E= 6602400 and a much smaller peak at
478 nm E
700460.
Compounds of the present invention can fluoresce in a range from about 400 nm
to
700 nm.
Cells Staining
Murine mammary carcinoma 4T1 cell populations were individually incubated with
three compounds according to the present invention for about 30 minutes, and
subsequently
co-stained with HOECHST (blue) and MitoTracker Deep Red (green). Confocal
images
were taken with a Zeiss Meta 510 LSM. The dyes were excited by laser at a
wavelength of
540nm (red).
Figure lA through 1L present confocal images of 4T1 cells incubated with a
compound according to some embodiments of the present invention (Figures 1B,
1F and 1J)
and co-stained with Hoechst (Figures 1A, 1E and lI, blue) to stain the nuclei
and
MitoTracker Deep Red (Figures 1C, 1G and 1K, appears green in images) to stain
the
mitochondria. Figure 1D presents an overlaid image of the images presented in
Figures 1A
through 1C. Figure 1H presents an overlaid image of the images presented in
Figures lE
through 1G. Figure 1L presents an overlaid image of the images presented in
Figures lI
through 1K. The overlaid images indicate that the compounds of the present
invention may
stain cells, in particular, the mitochondria, in a viable or fixed cell
population.
The foregoing is illustrative of the present invention and is not to be
construed as
limiting thereof. Although exemplary embodiments of this invention have been
described,
those skilled in the art will readily appreciate that many modifications are
possible in the
exemplary embodiments without materially departing from the novel teachings
and
advantages of this invention. Accordingly, all such modifications are intended
to be included
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within the scope of this invention as defined in the claims. The invention is
defined by the
following claims, with equivalents of the claims to be included therein.
