U.S. patent application number 10/742295 was filed with the patent office on 2004-12-16 for fatty acid phenolic conjugates.
Invention is credited to Siddiqui, Rafat, Stillwell, William, Zaloga, Gary P..
Application Number | 20040254357 10/742295 |
Document ID | / |
Family ID | 33513766 |
Filed Date | 2004-12-16 |
United States Patent
Application |
20040254357 |
Kind Code |
A1 |
Zaloga, Gary P. ; et
al. |
December 16, 2004 |
Fatty acid phenolic conjugates
Abstract
This invention relates to a biologically active formulation
containing a conjugate of a fatty acid and a complex phenol. The
fatty acid can be selected from a variety of fatty acids including
acids have between 12 and 24 carbon atoms. The phenol can be a
polynuclear phenol, a polyphenol or a polyfunctional phenol having
a variety of substituents. The formulation can include
pharmaceutically acceptable carrier, including diluent. The
formulation can be provided in an active dosage form suitable to
inhibit mammalian cell growth and/or metastasis of malignant cells.
The formulation can be used to induce cytotoxicity in mammalian
cells particularly tumor cells or to treat and prevent cellular
injury or dysfunction.
Inventors: |
Zaloga, Gary P.; (Fishers,
IN) ; Siddiqui, Rafat; (Carmel, IN) ;
Stillwell, William; (Indianapolis, IN) |
Correspondence
Address: |
WOODARD, EMHARDT, MORIARTY, MCNETT & HENRY LLP
BANK ONE CENTER/TOWER
111 MONUMENT CIRCLE, SUITE 3700
INDIANAPOLIS
IN
46204-5137
US
|
Family ID: |
33513766 |
Appl. No.: |
10/742295 |
Filed: |
December 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60435319 |
Dec 19, 2002 |
|
|
|
Current U.S.
Class: |
536/8 ; 549/403;
554/229 |
Current CPC
Class: |
C07C 69/587 20130101;
C07H 17/06 20130101; A61P 35/00 20180101 |
Class at
Publication: |
536/008 ;
549/403; 554/229 |
International
Class: |
C07H 017/06; C07D
37/78 |
Claims
What is claimed is:
1. A composition comprising: a phenol ester of a fatty acid, formed
of a phenol having greater than 11 carbons and a fatty acid having
greater than 12 carbon atoms; and a pharmaceutically acceptable
carrier, said ester provided in a dosage form sufficient to induce
cell cytotoxicity.
2. The composition of claim 1 wherein the fatty acid is a
polyunsaturated fatty acid.
3. The composition of claim 2 wherein the fatty acid is an
.omega.-3 long chain polyunsaturated fatty acid.
4. The composition of claim 1 wherein the fatty acid is selected
from the group consisting of: lauric acid, myristic acid, palmitic
acid, stearic acid, arachidic acid, behenic acid, lignoceric acid,
palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and
arachidonoic, docosahexanoic acid, and eicosopentenoic acid.
5. The composition of claim 1 wherein the phenol is a polyhydric
phenol or a polynuclear phenol.
6. The composition of claim 1 wherein the phenol is selected from
the group consisting of: resveratrol, quercetin, catechin,
propofol, and genistein.
7. The composition of claim 1 wherein the phenol is selected from
the group consisting of: resveratrol, quercetin, catechin,
propofol, and genistein.
8. A composition comprising: a phenol ester of a fatty acid, said
phenol having greater than 11 carbons and said fatty acid having
between 12 and 24 carbon atoms; and a pharmaceutically acceptable
carrier, said ester provided in a dosage form sufficient to induce
cell cytotoxicity.
9. The composition of claim 8 wherein the fatty acid is an
.omega.-3 long chain unsaturated or polyunsaturated fatty acid.
10. The composition of claim 8 wherein the phenol is a polyhydric
phenol or a polynuclear phenol.
11. The composition of claim 8 wherein the phenol is selected from
the group consisting of: resveratrol, quercetin, catechin,
propofol, and genistein.
12. The composition of claim 8 wherein the phenol is selected from
the group of phenols consisting of: resveratrol, catechins,
flavonoids, and alpha-tocopherol.
13. A method of treating mammalian cells, the method comprising
administering to the cells a pharmaceutical formulation comprising
a phenolic ester of a fatty acid in an amount effective to induce
cytotoxicity in at least a portion of the cells.
14. The method of claim 13 wherein the mammalian cells are tumor
cells.
15. The method of claim 13 wherein the fatty acid is .omega.-3 long
chain unsaturated or polyunsaturated fatty acid
16. The method of claim 13 wherein the fatty acid is selected from
the group of fatty acids consisting of: lauric acid, myristic acid,
palmitic acid, stearic acid, arachidic acid, behenic acid,
lignoceric acid, palmitoleic acid, oleic acid, linoleic acid,
linolenic acid, and arachidonoic, docosahexanoic acid, and
eicosopentenoic acid.
17. The method of claim 13 wherein the phenol is a polyhydric
phenol or a polynuclear phenol.
18. The method of claim 13 wherein the phenol is selected from the
group consisting of: resveratrol, quercetin, catechin, propofol,
and genistein.
19. The method of claim 13 wherein the phenol is selected from the
group of phenols consisting of: resveratrol, catechins, flavonoids,
and alpha-tocopherol.
20. A method of treating mammalian cells, the method comprising:
administering to the cells a pharmaceutical formulation comprising
a phenolic ester of a fatty acid in an amount effective to modulate
cell injury or cell dysfunction or both.
21. A method of inhibiting metastasis of malignant tumor cells or a
malignant growth in a mammal, said method comprising administering
to the mammal a pharmaceutical formulation comprising an effective
amount of a phenolic ester of a fatty acid in a pharmaceutically
acceptable carrier to inhibit the tumor cell or malignant growth
from metastasizing to a secondary tissue site.
22. The method of claim 21 wherein the fatty acid is a
polyunsaturated fatty acid.
23. The method of claim 21 wherein the fatty acid is an .omega.-3
long chain polyunsaturated fatty acid.
24. The method of claim 21 wherein the fatty acid is selected from
the group consisting of: lauric acid, myristic acid, palmitic acid,
stearic acid, arachidic acid, behenic acid, lignoceric acid,
palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and
arachidonoic, docosahexanoic acid, and eicosopentenoic acid.
25. The method of claim 21 wherein the phenol is a polyhydric
phenol or a polynuclear phenol.
26. The method of claim 21 wherein the phenol is selected from the
group consisting of: resveratrol, quercetin, catechin, propofol,
and genistein.
27. The method of claim 21 wherein the phenol is selected from the
group consisting of: resveratrol, quercetin, catechin, propofol,
and genistein.
28. The method of claim 21 wherein the pharmaceutical formulation
inhibits attachment of malignant cells to connective tissue at a
site secondary to an initial site of invasion within the
mammal.
29. A composition comprising: a histidyl conjugate of a fatty acid,
said hystidyl having greater than 6 carbons and said fatty acid
having between 12 and 24 carbon atoms; and a pharmaceutically
acceptable carrier, said ester provided in a dosage form sufficient
to induce cell cytotoxicity.
30. The composition of claim 28 wherein the fatty acid is an
.omega.-3 long chain polyunsaturated fatty acid.
31. A method of treating mammalian cells, the method comprising
administering to the cells a pharmaceutical formulation comprising
a histidyl conjugate of a fatty acid in an amount effective to
modulate cell injury or cell dysfunction or both.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application claims the benefit of U.S.
Provisional Application Ser. No. 60/435,319 filed on Dec. 19, 2002,
which is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] The present invention is directed to a method of modulating
cellular function and to a pharmaceutical composition containing a
phenolic ester of a fatty acid selected to modulate cellular
function.
[0003] Many drugs interact at the cell membrane surface by
combining with cell surface receptors, or alternatively are taken
into cells by specific transport systems. However, there are many
drugs which, while they act within the cells by modifying one of
many different functions such as DNA replication, actions of
intracellular enzymes, or the activity of systems such as lysosomes
or microtubules, are not able to penetrate cells very effectively.
For example, there may be inadequate numbers or types of cell
receptors and transport systems to which they can link or the
systems may transport the drug into the cell, the mitochondria, or
other nuclear membranes at less than optimum rates. This reduces or
masks the activity of many potential drugs.
[0004] In addition, the cell membrane contains many precursors to
bioactive substances, receptors, ion channels, and other proteins.
Functions of these compounds are dependent upon membrane structure
and properties. Substances, which alter membrane structure and
function can modify cellular function.
[0005] In the light of the above problems, there continues to be a
need for new and improved drugs to modulate cell functions, treat
specific diseases, and increase the distribution, absorption, and
biotransportation of drugs and other biologically active
substances. The present invention is such an improvement and
provides a wide variety of benefits and advantages.
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention relates to a method of modulating cell
function of mammalian cells, including modulation of cytotoxicity,
cell replication and repair, cell injury, and cell signaling. The
present invention also relates to a pharmaceutical formulation
selected to induce such cellular response. Certain forms and
features which are characteristic of the preferred embodiments are
disclosed herein and are described briefly below as follows.
[0007] In one aspect the present invention provides a
pharmaceutical formulation in dosage form selected to induce
modification of one or more cellular functions. The pharmaceutical
formulation includes a conjugate of a fatty acid and a phenol. More
preferably, the pharmaceutical formulation includes a phenolic
ester of a fatty acid. In preferred embodiments, the fatty acid is
selected to include fatty acids having between about 12 and 24
carbon atoms. The fatty acid includes a hydrocarbyl group that can
be a straight chain, a branched chain, or cyclic. Further, the
hydrocarbyl group can be saturated, mono-unsaturated, and
poly-unsaturated (conjugated or non-conjugated). The phenol can be
provided as a polynuclear phenol, a polyphenol, and a polyhydridic
phenol, each of which can be substituted with one or more
pharmaceutically acceptable substituents or functional groups.
[0008] In another aspect the present invention is directed to a
pharmaceutical formulation in dosage form that includes a conjugate
of a fatty acid and a histidyl moiety or an imidazyl moiety. As
listed above, the fatty acid can include a hydrocarbyl group that
can be a straight chain, a branched chain, or cyclic. The
hydrocarbyl group can be saturated, mono- or poly-unsaturated
(either conjugated or not), and the hydrocarbyl group can be
substituted.
[0009] In still yet another aspect, the present invention is
directed to a method of treating mammalian cells, the method
comprising administering to the cells a pharmaceutical formulation
comprising either a fatty acid conjugate of a phenyl moiety or a
fatty acid conjugate of a histidyl or imidazyl moiety in an amount
effective to induce modulation of at least one cellular function in
a portion of the treated cells. In a preferred embodiment, the
present invention provides a method for inducing cytotoxicity in
tumor cells including both benign and malignant cells. In other
preferred embodiments, the present invention provides a method of
modulating cellular replication and/or repair, and cell injury. In
still yet other preferred embodiments, the present invention
provides a method of altering cell signaling.
[0010] Further aspects, features, and advantages shall become
apparent from the description and drawings contained herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a scanned image of a TLC analysis of the
esterification reaction of resveratrol and docosahexaenoic
acid.
[0012] FIG. 2 is a scanned image of the UV spectrogram for the
resveratrol and docosahexaenoic acid conjugate prepared according
to the procedure described in Example 1.
[0013] FIG. 3 is a scanned image of a thin layer chromatography
plate used to analyze the reaction mixture containing propofol and
docosahexaenoic acid.
[0014] FIG. 4a is a UV spectrogram of propofol.
[0015] FIG. 4b is a UV spectrogram of the propofol-DHA conjugate
from the reaction described in Example 2.
[0016] FIG. 5a is a spectrogram from the gas chromatographic
analysis of propofol.
[0017] FIG. 5b is a spectrogram from the gas chromatographic
analysis of the propofol-DHA conjugate from the reaction described
in Example 2.
[0018] FIG. 6 is an infrared spectrogram of the propofol-DHA
conjugate from the reaction described in Example 2.
[0019] FIG. 7 is a mass spectrogram of the propofol-DHA conjugate
from the reaction described in Example 2.
[0020] FIG. 8 is a bar graph illustrating the effect of resveratrol
and docosahexaenoic acid on Jurkat cell growth.
[0021] FIG. 9 is a plot correlating the anti-oxidant properties and
the cytotoxic properties of several anti-oxidants.
[0022] FIGS. 10a-c are histograms illustrating the effect of the
resveratrol ester of docosahexaenoic acid on Jurkat leukemic cell
apoptosis.
[0023] FIG. 11 is a bar graph illustrating the induction of
apoptosis by DHA, EPA, alone or with propofol as additive or
synthetic conjugate.
[0024] FIGS. 12a-12r are scanned images of fluorescent microscopy
of apoptotic cells.
[0025] FIG. 13 is a bar graph illustrating the percent adherence of
cells from breast cell line MDA-MB-231 to vitronectin.
DETAILED DESCRIPTION OF THE INVENTION
[0026] For the purposes of promoting an understanding of the
principles of the invention, reference will now be made to the
embodiments illustrated herein and specific language will be used
to describe the same. It will nevertheless be understood that no
limitation of the scope of the invention is thereby intended. Any
alterations and further modifications in the described
formulations, treatment methods, and further applications of the
principles of the invention as described herein are contemplated as
would normally occur to one skilled in the art to which the
invention relates.
[0027] In preferred embodiments, the present invention provides
compounds and pharmaceutical formulations of a fatty acid conjugate
of a phenolic moiety. In preferred embodiments, the compounds and
pharmaceutical formulations of the present invention can be
selected to modulate cellular functions including, but not
restricted to: inducing cytotoxic activity, which can include
selective cytotoxic activity against specific cells, the induction
of apoptosis, and inhibition or reduction of tumor cell growth; and
modulating cell replication, cellular growth, and/or a cell's
response to injury. Additionally, preferred embodiments of the
present invention can alter cell signaling.
[0028] In one form, the present invention includes biologically
active compounds having a lipophilic component and a cytotoxic
component. The lipophile component can be derived from a fatty
acid, and the cytotoxic component can be derived from a phenolic
derivative. In selected embodiments, the biologically active
compound is a phenolic ester of a fatty acid. In other embodiments
the biologically active compound is a conjugate of a fatty acid
derivative and a phenol derivative connected via a bridge
group.
[0029] Selected embodiments of the present invention exhibit
increased transport or increased rate of transport of the
biologically active compounds across lipid membranes which may be
improved by linking them directly or indirectly to a lipid-soluble
component. Fatty acids are examples of lipid soluble components.
The fatty acids can absorb and/or pass through lipid-like materials
such as membranes, cellular membranes, and membranes surrounding
intracellular components such as a mitochondria and the nucleus.
Many fatty acids are naturally occurring. These fatty acids can be
combined (i.e., attached to a bioactive drug) and can transport the
bioactive compounds into the cell at a much higher rate than would
normally occur with the bioactive compounds in the absence of a
fatty acid. The conjugation of the biologically-active component
with a fatty acid can increase the cellular transport and/or
membrane association for a wide variety of bioactive compounds
including drugs, prodrugs, naturally occurring compounds, and their
derivatives. Additionally, selected fatty acids exhibit selected
bioactivity apart from their ability to facilitate binding
bioactive components to membranes or transporting them across the
membranes.
[0030] A number of diseases can be effected and treated by
modulating basic cell function. A non restrictive list of potential
diseases that can be treated according the present invention
include diseases that affect the central nervous system, including
Parkinson's disease and neuronal injuries, such as strokes; cardiac
diseases include myocardial infarction, heart failure, and
arrhythmia; and pulmonary diseases, including inflammatory lung
disease such as acute respiratory distress syndrome. Other
potential areas in which the present invention can contribute for
treatment and/or cure include arthritis, cancers and wound
repair.
[0031] The fatty acid component can include a long chain,
alkyl-substituted fatty acid. The fatty acid component preferably
includes greater than 12 carbon atoms and more preferably between
12 to 24 carbon atoms. The alkyl chain can be a straight chain, a
branched chain, or cyclic chain, all of which can be, mono- or
polyunsaturated, (conjugated or non-conjugated), and combinations
thereof.
[0032] In other preferred embodiments, the fatty acid includes
naturally occurring fatty acids; more specifically the .omega.-3
long chain polyunsaturated fatty acids. Referenced examples of
fatty acids for use in the present invention include, but are not
restricted to: lauric acid (n-dodecanoic acid), myristic acid
(n-tetradecanoic acid), palmitic acid (n-hexadecanoic acid),
stearic acid (n-octadecanoic acid), arachidic acid (n-eicosanoic
acid), behenic acid (n-docosanoic acid), lignoceric acid
(n-tetracosanoic acid), palmitoleic acid
(cis-.DELTA..sup.9-hexadecenoic acid), oleic acid
(cis-.DELTA..sup.9-octadecenic acid), linoleic acid (cis,
cis-.DELTA..sup.9, .DELTA..sup.12-octadecadienoic acid, cis,
trans-.DELTA..sup.9, .DELTA..sup.11-octadecadienoic acid, and
trans, cis-.DELTA..sup.10, .DELTA..sup.12-octadecadienoic acid),
linolenic acid (cis-.DELTA..sup.9, .DELTA..sup.12,
.DELTA..sup.15-octadecatrienoic acid, cis, trans,
cis-.DELTA..sup.9, .DELTA..sup.11, .DELTA..sup.13-octadecatri-
enoic acid, cis, trans, trans-.DELTA..sup.9, .DELTA..sup.11,
.DELTA..sup.13-octadecatrienoic acid, and trans, trans,
cis-.DELTA..sup.9, .DELTA..sup.11, .DELTA..sup.13-octadecatrienoic
acid), and arachidonoic (cis-.DELTA..sup.5, .DELTA..sup.8,
.DELTA..sup.11, .DELTA..sup.14-eicosatetraenoic acid),
docosahexanoic acid (DHA), and eicosopentenoic acid (EPA).
[0033] The phenolic component can be selected from a polynuclear
phenol, a polyphenol substituent, a polyhydridic phenol, and
polysubstituted phenol. Selected phenols exhibit biological
activities, especially cytotoxic activity against specific cell
lines, including anti-tumor activity against either benign or
malignant cells, anti-neoplastic activity, and anti-cell growth
activity.
[0034] Phenols are a class of chemical components that include an
aromatic ring with a hydroxyl group. Various phenols exhibit
wide-ranging biological activity. Specific phenols have been shown
to exhibit anti-oxidant and anti-carcinogenic activity and modulate
inflammatory responses such as sepsis, acute respiratory failure,
inflammatory bowel disease, arthritis, and other inflammatory
diseases. Some of these substances can exhibit cardio-protective
activities that include anti-arrhythmia, anti-arteriosclerotic
effects, and prevention of loss of cardiac contractile function in
patients with heart disease. Additionally, the phenolic derivatives
can exhibit cell protective effects including repair after
non-lethal cellular injury. The phenols can be classified into
different subcategories. The subcategories include simple phenols,
polynuclear phenolic species, polyphenol species, polyhydric
phenols, and other substituted phenols. Simple phenols include a
single aromatic ring with a hydroxyl substituent; a more complex
phenol includes additional substituents. Polynuclear phenols
include multiple (two or more) conjugated aromatic ring systems,
such as genistein and resveratrol.
[0035] Polyphenols, as this term is used herein, are considered to
include one or more phenols bonded together through one or more
linking groups. Examples of polyphenols include resveratrol,
catechins, flavonoids, and alpha-tocopherol, to name just a few. In
each of the above subcategories, the phenols can include additional
functional groups. For example, the phenols can include two or more
hydroxyl groups, i.e., polyhydridic phenols. More complex phenols
for use in the present invention include other substituents
including alkyl groups. The alkyl groups can be provided as a
straight chain, a branched chain, cyclic and/or aromatic; further,
the alkyl group can also be saturated or unsaturated, conjugated or
not. Additionally, the phenols and the alkyl groups can be
substituted with one or more of: amides (--C(O)NRR'), amino
(--NH.sub.2), secondary amino (--NRH), tertiary amino (--NRR'),
esters, ethers (--OR), halogens (i.e., fluoro (F.sup.-), chloro
(Cl.sup.-), bromo (Br.sup.-), iodo (I.sup.-)), hydroxyl (--OH),
oxygen, nitrogen, sulfonyl (--SO.sub.2--), thiols, (--S--),
thiolates, and thionyl (--SO--) substituents. Selected examples of
phenols for use in the present invention include: propofol
(2,6-diisopropylphenol), butylated hydroxytoluene, resveratrol, and
vitamin E.
[0036] The phenol-fatty acid conjugate can be prepared under
conditions suitable for ester formation. Various methods are known
to those skilled in the art. Preferred methods include forming a
more active intermediate (reactive ester, anhydride, or amide) of
the fatty acid and reacting that highly reactive intermediate with
the phenol in the presence of a non-nucleophilic base. The
resulting product can be isolated using techniques known to those
skilled in the art.
[0037] In another form, the present invention can include
pharmaceutical formulations having a lipophilic component and a
histidyl or imidazyl component. The lipophilic component is as
described above. The histidyl or imidazyl component can be
conjugated with the fatty acid through an amide linkage.
[0038] The pharmaceutical formulation for use in the present
invention can also include pharmaceutically-acceptable auxiliaries,
including diluents, carriers, excipients, buffering agents, wetting
agents, emulsifiers, lubricants, and antioxidants. Further, the
pharmaceutical formulation can include incorporating one or more
active components in a liposome or lipid vesicle.
[0039] As used herein, the term "pharmaceutically acceptable
carriers" means a non-toxic, inert solid, semi-solid or liquid
filler, diluent, encapsulating material or formulation auxiliary of
any type. Some examples of the materials that can serve as
pharmaceutically acceptable carriers are sugars, such as lactose,
glucose and sucrose; starches such as corn starch and potato
starch; cellulose and its derivatives such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; powdered
tragacanth; malt; gelatin; talc; excipients such as cocoa butter
and suppository waxes; oils such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
glycols, such as propylene glycol; polyols such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters such as ethyl
oleate and ethyl laurate; agar; buffering agents such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution, ethyl alcohol and phosphate
buffer solutions, as well as other non-toxic compatible substances
used in pharmaceutical formulations. Wetting agents, emulsifiers
and lubricants such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, releasing agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives
and antioxidants can also be present in the composition, according
to the judgement of the formulator. Examples of pharmaceutically
acceptable. antioxidants include--water soluble antioxidants such
as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium
metabisulfite, sodium sulfite, and the like; oil soluble
antioxidants such as ascorbyl palmitate, butylated hydroxyanisole
(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,
alpha-tocopherol and the like; and the metal chelating agents such
as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,
tartaric acid, phosphoric acid and the like.
[0040] By use of the "term effective amount" includes a sufficient
amount of the phenol ester of a fatty acid or conjugate of the
phenol and fatty acid to elicit the desired result, i.e., the
desired pharmacological or biochemical result over the amount in
which no result is observed. In preferred embodiments, the
effective amount of the phenol ester to induce cytotoxic activity
is an amount of greater than or equal to about 10 micromolar. More
preferably the effective amount to induce cytotoxic activity is
between about 25 and about 50 micromolar.
[0041] In other embodiments, the formulations of the present
invention can be used to inhibit metastasis of malignant cells or
malignant growths to a secondary invasion site. It is thought that
in one form, the formulations of the present invention can inhibit
malignant cells by disrupting the cell signaling mechanism or
effecting the detachment of the malignant cells from the primary
invasion site; circulating through the vascular system or once the
malignant cells are in the vascular system inhibiting these cells
from attaching to other tissue or other sites to propagate
secondary growths.
[0042] In one preferred embodiment, effective amount of the phenol
ester to inhibit metastasis is an amount of greater than or equal
to about 10 micromolar. More preferably the effective amount to
inhibit metastasis is between about 25 micromolar and about50
micromolar.
[0043] For the purpose of promoting further understanding and
appreciation of the present invention and its advantages, the
following examples are provided. It will be understood, however,
that these examples are illustrative and not limiting in any
fashion.
EXAMPLE 1
Synthesis Of Resveratrol Ester Of Docosahexaenoic Acid
[0044] The synthesis of the resveratrol ester of docosahexaenoic
acid was performed in two steps. In the first step, the
docosahexaenoic-anhydride was synthesized, and in a second step
this anhydride was coupled to resveratrol.
[0045] For the initial step to form the anhydride, docosahexaenoic
acid (DHA, 0.19 .mu.M), dicyclohexylcarbodiimide (DCC, 0.145
.mu.M), and the anti-oxidant butylated hydroxytoluene (BHT, 1.5
.mu.M) were dissolved in 4 mls of dimethylformamide (DMF) and mixed
for one hour at room temperature under an atmosphere of nitrogen.
In the second step, resveratrol (RVT) (0.095, .mu.M) and
4-dimethylaminopyridine (DMAP, 0.095 .mu.M) were then added to the
DHA-anhydride mixture. The resulting solution was stirred at room
temperature under an atmosphere of nitrogen for about 17 hours. The
reaction product was filtered, washed with chloroform, and then
separated by analytical thin layer chromatography (TLC). The TLC
plate was developed with a methylene chloride/ethyl ether/acetic
acid (80/20/1, v/v/v). The reaction products were visualized on the
TLC plate using iodine and ultraviolet (UV) absorbence. Although
there were five possible reaction products (RVT includes three
hydroxyl substituents), the initial analysis of the developed TLC
plate revealed that only two products were produced in substantial
quantities. (See FIG. 1.) The two products had Rfs of about 0.50
and 0.55 using the eluent system described above. The species
having an Rf equal to about 0.50 was isolated from the TLC plate
and analyzed using UV spectroscopy. The UV spectra is illustrated
in FIG. 2 and indicates the presence of both DHA and RVT in the
isolated product. The formation of the DHA-RVT ester was further
verified by hydrolyzing the previously isolated product with a
sodium hydroxide. The hydrolysis yielded 2 products: one was
identified as DHA and the other was identified as RVT based upon
their Rf values on a TLC plate. The molecular weight of the
reaction product was roughly estimated based upon the UV spectra.
This reaction product was further tested on Jurkat cells.
EXAMPLE 2
Synthesis, Purification and Characterization of 2,6
Diisopropylphenyldocosahexaenoate (Propofol-DHA)
[0046] Synthesis
[0047] To minimize auto-oxidation, all procedures were performed in
reduced light and under nitrogen. The reaction was carried out in
two steps: synthesis of docosahexaenoic acid anhydride
(DHA-anhydride), subsequently followed by the esterification of DHA
by 2,6 diisopropylphenol (propofol).
[0048] DHA (100 mg, 0.305 mmol), a coupling reagent,
N,N'-dicyclohexylcarbodiimide (94 mg, 0.450 mmol), and an
anti-oxidant, 2,6 di-tert-butyl-4 methylphenol (BHT) (5 mg) were
dissolved in 5 ml of chloroform. The reaction was stirred for 60
min at room temperature. propofol (49.8 mg, 0.28 mmol) and
4-(dimethyl amino) pyridine (18.5 mg, 0.152 mmol were then added to
the reaction. The resulting mixture was stirred for a period of 12
hours; the reaction suspension was then filtered and washed with
petroleum ether and subjected to purification.
[0049] Purification
[0050] The product of the reaction was purified on an analytical
thin layer plate (Silica Gel, 60 A, 0.2 mm thickness); the plate
was developed in a solvent mixture of Petroleum Ether/Ethyl Acetate
(92/8, v/v), and the products were visualized by iodine vapor. The
result was compared to the control, which contained the entire
reaction mixture without propofol.
[0051] As compared to the control, a new product was formed (See
FIG. 3, illustrating the thin layer chromatographic analysis of the
reaction mixture) with a Rf value=0.90. In FIG. 3, lane 1 is the
reaction mixture without propofol; lane 2 is the entire reaction
mixture; and lane 3 is unreacted DHA and propofol. The band
corresponding to the new compound was scraped off the TLC plate,
suspended with chloroform/methanol (20/80, v/v), passed through a
glass filter, and subjected to characterization.
[0052] Characterization
[0053] The characterization of the compound was performed by the
combination of techniques described bellow:
[0054] a) Absorption Spectra
[0055] The propofol spectra showed two absorption peaks at 219 nm
and 274 nm (FIG. 4a). These peaks were shifted to 214 nm and 260 nm
(FIG. 4b) respectively for the new compound (propofol-DHA).
[0056] b) Gas Chromatography Analysis
[0057] The propofol-DHA conjugate was hydrolyzed and then
methylated for analysis by Gas Chromatography. The results
illustrated in FIGS. 5a and 5b demonstrate that the commercially
available propofol (Adrich-Sigma Chemical Co.) contained 97% of
pure compound (2,6 diisopropylphenol) with a retention time of 4.89
min and 3% of other propofol isomers (See FIG. 5a). Analysis of the
hydrolyzed product of propofol-DHA conjugate resulted in 50% yield
of DHA (retention time 26.10), a 40% yield (retention time 4.89) of
propofol (2,6 diisopropylphenol), and remaining a 10% yield of
other isoforms of propofol (See FIG. 5b).
[0058] c) Infrared Spectra
[0059] The infrared absorption spectra of the propofol-DHA
conjugate (See FIG. 6) showed two strong absorptions for
characteristic ester group: a C.dbd.O stretch (1750 cm.sup.-1) and
a C--C--O docosahexaenoate stretch (1250 cm.sup.-1). The spectra
also show absorptions at 3000-3070 em.sup.-1, which are
characteristic of the aromatic C--H stretch. These results further
confirm formation of a propofol-DHA conjugate.
[0060] d) Mass Spectra
[0061] Mass spectroscopy was used to analyze the propofol-DHA
conjugate (See FIG. 7). The mass spectra revealed a compound in the
reaction mixture having a molecular weight of 489.3, which appeared
to be very close to the calculated molecular weight of 488.85. A
slight discrepancy in the molecular weight is due to protonization
of product during mass spectrum analysis.
[0062] The analytical results described above indicate that
esterification of DHA with propofol results in a pure propofol-DHA
conjugated product.
EXAMPLE 3
Synthesis, Purification and Characterization of
2,6-Diisopropylphenyl Eicosopentenoate (Propofol-EPA)
[0063] A propofol-EPA conjugate was also synthesized and
characterized using similar synthetic and analytical approaches as
those described above for the propofol-DHA conjugate.
EXAMPLE 4
Effect of DHA and RVT on Jurkat Leukemic Cell Growth
[0064] In order to evaluate the anti-proliferative activity and
synergism between DHA and RVT, the two compounds were added
separately to cell cultures containing Jurkat leukemic cells. In a
separate experiment, a mixture of DHA and RVT components were added
to the Jurkat leukemic cell cultures. The results are illustrated
in FIG. 8 as a bar graph. Jurkat cells (Jurkat clone E6-1 from
American Type Culture Collection of Manassas, Va.
(5.times.10.sup.4/well)) were incubated with either DHA or RVT, or
a combination as indicated in a 96-well plate for 24 hours at
37.degree. C. using a 5% CO.sub.2 in RPMI media containing 2%
serum. Cell growth and viability were determined using a WST-1 Cell
Proliferation Kit (sold by Roche Molecular Biochemicals of
Indianapolis, Ind.). The control was a "ethanol-treated" control
group. For the results illustrated in FIG. 8, the average was taken
of three separate experiments. For the combination of DHA and RVT,
a student t test provided a P<0.001 relative to the
"ethanol-treated" control group. The results indicate that neither
the DHA (up to 30 .mu.M) nor the RVT (10 .mu.M) alone had any
significant effect on Jurkat cell growth. However, when the two
components were combined into a single formulation, a synergistic
inhibition of Jurkat cell growth was observed. The combination of
DHA (30 .mu.M) and RVT (10 .mu.M) inhibited cell growth by about
60%. This is indicative of a synergistic interaction between DHA
and RVT on cancer cell growth. Furthermore, increasing the RVT
concentration further enhanced the cytotoxic effect of the DHA.
EXAMPLE 5
Evaluation of Cytotoxicity of Polyphenolic Anti-Oxidants
[0065] Resveratrol (RVT) discussed above is a polyphenolic
anti-oxidant. A number of different anti-oxidants were evaluated to
consider whether there was a correlation of the anti-oxidation
properties of these components and cell growth inhibition. The
different anti-oxidants were selected to include those with and
those without polyphenolic ring structures. The results of the
evaluation are illustrated in FIG. 9.
[0066] The Jurkat cells were incubated in a 96-well plate
(5.times.10.sup.4 cells/well). The wells were then treated with one
of the following phenolic compounds: propofol (PPF, 25 .mu.M),
butylated hydroxytoluene (BHT, 10 .mu.M), resveratrol (RVT 25
.mu.M), vitamin E (VTE, 25 .mu.M), and vitamin C (VTC, 25 .mu.M)
both in the presence and then in the absence of DHA (5 .mu.M) for 2
hours at 37.degree. C. at 5% CO.sub.2 in a serum-free RPMI medium.
The phenols PPF, BHT, RVT, and VTE were suspended in DMSO while the
RTC was dissolved in the RPMI media. Control cells were treated
with equal amounts of the appropriate vehicles (DMSO, ethanol, or
both, or the RPMI media). Cell viability was determined using a
WST-1 Cell Proliferation Kit (sold by Roche Molecular Biochemicals
of Indianapolis, Ind.). The anti-oxidation index for each of the
phenols was determined using 2,7-dichlorodihydrofluorescein
diacetate (5 .mu.M) according to the procedure described in Sanchez
et al. Anal. Biochem. 1990, 187, 129-132 and LeBel et al. Chem.
Res. Toxicol. 1992, 5, 227-231. The fluorescence intensity was due
to the interaction of the free oxygen radicals with the dye and was
measured at .lambda..sub.ex 495 nm and .lambda..sub.em 529 nm in a
96 well plate fluorescence reader. The cytotoxic index was
calculated as the percentage of dead cells compared to the control.
The anti-oxidation index was calculated as the percentage of
fluorescence intensity reduction to that of the controls (vehicle
treated cells). Review of the data in FIG. 9 indicates that there
is not a strong correlation between the anti-oxidation properties
and the cyctoxicity of the tested phenols. Consequently, it is
suggested that the anti-oxidation property is not necessarily
responsible for the DHA-induced cytotoxic effects.
EXAMPLE 6
Effect of the Resveratrol Ester of Docosahexaenoic Acid on Jurkat
Leukemic Cells Apoptosis
[0067] The DHA-RVT ester prepared according to Example 1 was
dissolved in DMSO. This solution was used to treat Jurkat cells
labeled with YO-PRO-1 (sold by Molecular Probes of Oregon), which
is a green fluorescent dye used in flagging apoptotic cells. The
labeled cells were analyzed using a FACStar-plus flow cytometer
equipped with a water-cooled argon laser emitting at a frequency of
488 nm. Detection of the fluorescent probe was ascertained using a
530.+-.30 band-pass filter. The data depicted in FIGS. 10a-c are
illustrated in the form of a histogram. The fluorescent intensity
is indicative of apoptotic expression. The unfilled histogram
represents the labeled, non-treated cells (1); the filled histogram
represents the treated cells (2). The results indicate that a
concentration of 10 .mu.M of the DHA-RVT ester (FIG. 10a) produced
apoptosis in nearly all of the Jurkat cells. At approximately 1
.mu.M concentration, the DHA-RVT ester induced apoptosis in
approximately 50% of the Jurkat cells (FIG. 10b) while at a
concentration of 0.1 .mu.M the ester had little effect on the
Jurkat cells (FIG. 10c). Concentrations of resveratrol (10 .mu.M)
plus DHA (10 .mu.M) have no effect on Jurkat cell growth.
EXAMPLE 7
Biological Effects of Propofol-DHA and Propofol-EPA Conjugates
[0068] DHA and EPA conjugates of propofol are used to test their
activities against MDA-231-MB breast cancer cells. Induction of
apoptosis was assayed by incubating cells with DHA, EPA, or
propofol alone or DHA, EPA with propofol as well as DHA-, and EPA
as propofol conjugates for 4 hours at 37.degree. C. After this
incubation, a specific fluorescent caspase 3 inhibitor
(FITC-DEVD-FMK) that binds specifically to activated caspase 3 was
added, and FITC positive fluorescence was observed under the
microscope. Results indicate that 25 .mu.M DHA-propofol or
EPA-propofol effectively induced apoptosis in these cells whereas
similar concentrations of DHA, EPA or propofol or propofol with DHA
or EPA were not very effective (FIG. 11). Representative pictures
indicating apoptotic positive cells are shown in FIGS. 12a-12r.
EXAMPLE 8
Cell Adhesion Assay
[0069] The cell adhesion assay was performed according to the
procedure described in Deryugina, E. I., Ratnikov, B., Postnova, T.
I., Rozanov, D. V., Strongin, A. Y. (2002) J. Biol. Chem. 277,
9749-9756. The cell adhesion assay was preformed using cyctomatrix
of human vitronectin in a 96-well plate (Chemicon International
Incorp. of Temecula, Calif.). Each well was incubated with 100
.mu.L of breast carcinoma MDA-MB-231 cells (1.times.10.sup.5
cells/ml) at 37.degree. C. for 45 min in a CO.sub.2 incubator.
Individual wells were treated with one of the following: (a)
individual agents: DHA, EPA, and propofol; or (b) the combined
agents: DHA and propofol, and EPA and propofol; or (c) the
conjugates DHA-propofol, or EPA propofol (prepared as described
above in Examples 2 and 3). After incubation, the wells were washed
three times with PBS, and the adhered cells were stained with
crystal violet in 10% ethanol for 5 min at room temperature. The
excess stain was subsequently removed by washing the wells six
times with PBS. The stained cells were dissolved in 100 microliters
(.mu.L) of solublizing buffer (1:1 mixture of 0.1M
NaH.sub.2PO.sub.4 at a pH of 4.5 in 50% ethanol) and the absorbance
of each of the resulting samples was read at 540 nm. The absorbance
of dye in the control (vehicle treated cells) was regarded as 100%,
and the percent adherence of treated cells was calculated in
comparison to that control. The results are graphically displayed
in FIG. 13.
[0070] The results illustrated in the bar graph of FIG. 13 indicate
that the two conjugates, DHA-propofol and EPA-propofol possess
unique biological properties including the inhibition of adherence
of cancerous cells to connective tissue, vitronectin, that is not
displayed by either the individual agents, DHA, EPA or propofol or
by the simple combination of these agents.
* * * * *