U.S. patent application number 16/125574 was filed with the patent office on 2019-08-08 for resveratrol esters.
The applicant listed for this patent is Cole Research & Design, LLC. Invention is credited to Jeptha N. Cole, Mahmoud A. ElSohly, Waseem Gul.
Application Number | 20190241498 16/125574 |
Document ID | / |
Family ID | 59088250 |
Filed Date | 2019-08-08 |
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United States Patent
Application |
20190241498 |
Kind Code |
A1 |
ElSohly; Mahmoud A. ; et
al. |
August 8, 2019 |
RESVERATROL ESTERS
Abstract
A resveratrol ester has the following structure: ##STR00001##
R.sup.1, R.sup.2 and R.sup.3 are H or ##STR00002## Each R.sup.4 is
independently a carbon chain of 2 to 4 carbon atoms comprising a
terminal carboxylic acid moiety, a carbon chain of 1 to 5 carbon
atoms comprising an amine moiety, or ##STR00003## R.sup.5 is a
carbon chain of 3 or 4 carbon atoms having a terminal carboxylic
acid moiety. At least one of R.sup.1, R.sup.2 and R.sup.3 is
##STR00004## Salts of resveratrol esters are also included.
Inventors: |
ElSohly; Mahmoud A.;
(Oxford, MS) ; Gul; Waseem; (Oxford, MS) ;
Cole; Jeptha N.; (Jackson, MS) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cole Research & Design, LLC |
Jackson |
MS |
US |
|
|
Family ID: |
59088250 |
Appl. No.: |
16/125574 |
Filed: |
September 7, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15389674 |
Dec 23, 2016 |
10099995 |
|
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16125574 |
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62387588 |
Dec 24, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07C 229/36 20130101;
A61P 17/02 20180101; C07C 67/08 20130101; C07C 69/40 20130101; C07C
229/08 20130101; C07C 69/42 20130101 |
International
Class: |
C07C 69/42 20060101
C07C069/42; A61P 17/02 20060101 A61P017/02; C07C 229/36 20060101
C07C229/36; C07C 67/08 20060101 C07C067/08; C07C 69/40 20060101
C07C069/40; C07C 229/08 20060101 C07C229/08 |
Claims
1. A resveratrol ester having the following structure: ##STR00015##
wherein R.sup.1, R.sup.2 and R.sup.3 are H or ##STR00016## each
R.sup.4 is independently a carbon chain of 2 to 4 carbon atoms
comprising a terminal carboxylic acid moiety, a carbon chain of 1
to 5 carbon atoms comprising an amine moiety,
--(CH.sub.2).sub.3(NH)(R.sup.6), or ##STR00017## R.sup.5 is a
carbon chain of 3 or 4 carbon atoms having a terminal carboxylic
acid moiety or a natural amino acid side chain, R.sup.6 is
--(CO)(CH.sub.2).sub.n(CO)(OH) and where n is 1 or 2, and at least
one of R.sup.1, R.sup.2 and R.sup.3 is ##STR00018## and salts
thereof.
2. The resveratrol ester of claim 1, wherein R.sup.4 is the carbon
chain of 2 to 4 carbon atoms comprising the terminal carboxylic
acid moiety, and salts thereof.
3. The resveratrol ester of claim 1, wherein R.sup.4 is the carbon
chain of 1 to 5 carbon atoms comprising the amine moiety, and salts
thereof.
4. The resveratrol ester of claim 1, wherein R.sup.4 is
##STR00019## and R.sup.5 is the carbon chain of 3 or 4 carbon atoms
having the terminal carboxylic acid moiety, and salts thereof.
5. The resveratrol ester of claim 2, wherein R.sup.4 is selected
from the group consisting of --(CH.sub.2)(CO)(OH),
--(CH.sub.2).sub.2(CO)(OH), and --(CH.sub.2).sub.3(CO)(OH).
6. The resveratrol ester of claim 5, wherein R.sup.4 is
--(CH.sub.2).sub.3(CO)(OH).
7. The resveratrol ester of claim 2, wherein R.sup.1, R.sup.2 and
R.sup.3 are ##STR00020##
8. The resveratrol ester of claim 7, wherein R.sup.4 is selected
from the group consisting of --(CH.sub.2)(CO)(OH),
--(CH.sub.2).sub.2(CO)(OH), and --(CH.sub.2).sub.3(CO)(OH).
9. The resveratrol ester of claim 8, wherein R.sup.4 is
--(CH.sub.2).sub.3(CO)(OH).
10. The resveratrol ester of claim 3, wherein R.sup.4 is selected
from the group consisting of --(NH.sub.2)CHCH.sub.3,
--CH(NH.sub.2)CH(CH.sub.3).sub.2,
--CH(NH.sub.2)CH.sub.2CH(CH.sub.3).sub.2,
--CH(NH.sub.2)CH(CH.sub.3)CH.sub.2CH.sub.3, --CH.sub.2NH.sub.2,
--(CH.sub.2).sub.3(C.sub.6H.sub.4)NH.sub.2,
--(CH.sub.2).sub.3NH.sub.2 and --(CH.sub.2).sub.5NH.sub.2.
11. The resveratrol ester of claim 10, wherein R.sup.4 is
--CH(NH.sub.2)CH(CH.sub.3).sub.2.
12. The resveratrol ester of claim 3, wherein R.sup.1, R.sup.2 and
R.sup.3 are ##STR00021##
13. The resveratrol ester of claim 12, wherein R.sup.4 is selected
from the group consisting of --(NH.sub.2)CHCH.sub.3,
--CH(NH.sub.2)CH(CH.sub.3).sub.2,
--CH(NH.sub.2)CH.sub.2CH(CH.sub.3).sub.2,
--CH(NH.sub.2)CH(CH.sub.3)CH.sub.2CH.sub.3, --CH.sub.2NH.sub.2,
--(CH.sub.2).sub.3(C.sub.6H.sub.4)NH.sub.2,
--(CH.sub.2).sub.3NH.sub.2 and --(CH.sub.2).sub.5NH.sub.2.
14. The resveratrol ester of claim 13, wherein R.sup.4 is
--CH(NH.sub.2)CH(CH.sub.3).sub.2.
15. A method of making the resveratrol ester of claim 1,
comprising: forming the resveratrol ester from resveratrol.
16. A composition, comprising: the resveratrol ester of claim 2,
and a pharmaceutically acceptable carrier.
17. (canceled)
18. A resveratrol ester selected from the group consisting of
resveratrol hemimalonate, resveratrol hemisuccinate, resveratrol
hemiglutarate, resveratrol 2-aminopropanoate, resveratrol
2-amino-3-methylbutanoate, resveratrol 2-amino-4-methylpentanoate,
resveratrol 2-amino-3-methylpentanoate, resveratrol aminoethanoate,
resveratrol 4-(4-aminophenyl)-butyrate, resveratrol
4-amino-butyrate, and resveratrol 6-amino-hexanoate.
19. The resveratrol ester of claim 18, wherein the resveratrol
ester is resveratrol hemiglutarate.
20. The resveratrol ester of claim 19, wherein the resveratrol
hemiglutarate is resveratrol trihemiglutarate.
21. (canceled)
22. A method of reducing scar formation, comprising: administering
an effective amount of the composition of claim 16, to a patient in
need thereof.
23. (canceled)
24. (canceled)
Description
BACKGROUND
[0001] Resveratrol (trans-3,4',5-trihydroxystilbene), a stilbenoid,
is a natural polyphenol present in various plants, some food
products, red wine and grapes. Resveratrol has the following
chemical structure:
##STR00005##
[0002] Resveratrol possesses anti-inflammatory, anti-carcinogenic
and anti-oxidant properties, and has been extensively studied. Huge
interest in resveratrol was created when it was discovered that it
was able to activate the SIRT1 gene, a gene implicated in the life
span extension associated with calorie-restricted diets. However,
beneficial effects have been challenging to observe in human
clinical studies.
[0003] It was recently discovered that application of resveratrol
to a wound through the layers of the epidermis can reduce scar
formation. Application of resveratrol to a wound before wound
formation or up to 24 hours after wound formation results in
re-epithelialization within 24 hours, resulting in an attenuated
scar. See U.S. Patent Application Publication No. 2015/0005391 to
Cole.
[0004] Although it is not known exactly how resveratrol reduces
scarring, resveratrol up-regulates and increases the expression of
a variety of agents which are involved in wound healing. One
possible explanation is that resveratrol causes the over-expression
of matrix metalloproteinase-9 (MMP-9), interleukin-8 (IL-8) and
SIRT1, and increases expression of epidermal growth factor receptor
(EGFR) on the keratinocyte membrane and nucleus. SIRT1 may then
promote differentiation, motility and proliferation of
keratinocytes, and deacetylation and inactivation of p53 protein,
inhibiting p53-dependent cell death from apoptosis in response to
stress in human tenocytes (fibroblast-like tendon cells). SIRT1 may
induce nitric oxide (NO) production, which inhibits Class I HDAC 2
from blocking growth factors including epithelial growth factor,
keratinocyte growth factor 2, fibroblast growth factor 10 (FGF-10)
and insulin-like growth factor 1 (IGF-1). SIRT1 may also decrease
inflammation and apoptosis through a variety of mechanisms. IL-8
has a direct and profound stimulatory effect on the migration of
keratinocytes, which is likely via the PLC-.gamma. pathway. IL-8
may also recruit neutrophils. MMP-9 degrades the Type IV collagen
of the basement membrane. EGFR may cause keratinocyte and
fibroblast migration and may protect and repair tissue through
nuclear DNA repair. Resveratrol may also inhibit
NF-.kappa.B-dependent pro-inflammatory and matrix-degrading gene
products induced by IL-1.beta. and nicotinamide.
SUMMARY
[0005] In a first aspect, the invention is a resveratrol ester
having the following structure:
##STR00006##
R.sup.1, R.sup.2 and R.sup.3 are H or
##STR00007##
Each R.sup.4 is independently a carbon chain of 2 to 4 carbon atoms
comprising a terminal carboxylic acid moiety, a carbon chain of 1
to 5 carbon atoms comprising an amine moiety, or
##STR00008##
R.sup.5 is a carbon chain of 3 or 4 carbon atoms having a terminal
carboxylic acid moiety. At least one of R.sup.1, R.sup.2 and
R.sup.3 is
##STR00009##
Salts of resveratrol esters are also included.
[0006] In a second aspect, the invention is a method of making a
resveratrol ester, comprising forming the resveratrol ester from
resveratrol.
[0007] In a third aspect, the invention is a composition comprising
a resveratrol ester and a pharmaceutically acceptable carrier.
[0008] In a fourth aspect, the invention is a resveratrol ester
selected from the group consisting of resveratrol hemimalonate,
resveratrol hemisuccinate, resveratrol hemiglutarate, resveratrol
2-aminopropanoate, resveratrol 2-amino-3-methylbutanoate,
resveratrol 2-amino-4-methylpentanoate, resveratrol
2-amino-3-methylpentanoate, resveratrol aminoethanoate, resveratrol
4-(4-aminophenyl)-butyrate, resveratrol 4-amino-butyrate, and
resveratrol 6-amino-hexanoate.
[0009] In a fifth aspect, the invention is resveratrol
trihemiglutarate.
[0010] In a sixth aspect, the invention is a method of reducing
scar formation, comprising administering an effective amount of a
composition comprising a resveratrol ester and a pharmaceutically
acceptable carrier to a patient in need thereof.
[0011] In a seventh aspect, the invention is a method of making a
composition comprising a resveratrol ester and a pharmaceutically
acceptable carrier. The method does not include a solvent
comprising alcohol.
Definitions
[0012] "Resveratrol esters" include resveratrol esters of
carboxylic acids, resveratrol esters of amino acids and amides
thereof with dicarboxylic acids. Species of resveratrol esters
contain the prefix mono-, di-, or tri- to indicate the number of
ester linkages present in the resveratrol ester. The absence of the
mono-, di-, or tri-prefix indicates a class containing the three
species. For example, resveratrol hemiglutarate refers to the class
of resveratrol esters containing the three species resveratrol
monohemiglutarate, resveratrol dihemiglutarate, and resveratrol
trihemiglutarate.
[0013] A "resveratrol precursor" or a "resveratrol prodrug" is a
compound that is converted to resveratrol by the body.
[0014] "Hydroxyl" (or hydroxy-) refers to an --OH moiety.
[0015] "Carboxylic acid" (or carboxy-) refers to a compound with at
least one --COOH moiety.
[0016] "Dicarboxylic acid" refers to a compound having two
carboxylic acid moieties (--COOH).
[0017] "Amino acid" refers to a compound having an amine moiety
(--NH.sub.2) and a carboxylic acid moiety (--COOH).
[0018] "Amide" refers to a compound with at least one --(CO)N--
moiety.
[0019] "Saturated" refers to a compound with no carbon-carbon
double or triple bonds.
[0020] A "carbonyl carbon" is a carbon atom that is double-bonded
to an oxygen atom.
[0021] An "ester linkage" refers to the oxygen-carbonyl bond in an
ester:
##STR00010##
[0022] All percentages (%) are weight/weight percentages, unless
stated otherwise.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The invention can be better understood with reference to the
following drawings and description.
[0024] FIG. 1 is the chemical structure of resveratrol
trihemiglutarate.
[0025] FIG. 2 is a mass spectrum of resveratrol trihemiglutarate
obtained by LC/MS.
[0026] FIG. 3A is a chromatogram of resveratrol obtained by HPLC in
units of millivolts (mV) using an evaporative light scattering
detector (ELSD).
[0027] FIG. 3B is a chromatogram of resveratrol obtained by HPLC in
units of milli absorbance units (mAU) using a UV detector.
[0028] FIG. 4A is a chromatogram of resveratrol trihemiglutarate
obtained by HPLC in units of millivolts (mV) using an ELSD.
[0029] FIG. 4B is a chromatogram of resveratrol trihemiglutarate
obtained by HPLC in units of milli absorbance units (mAU) using a
UV detector.
[0030] FIG. 5A is a microscopic image of untreated wound
tissue.
[0031] FIG. 56 is a microscopic image of wound tissue that has been
treated with a resveratrol ester.
[0032] FIG. 6 is a mass spectrum of resveratrol trihemisuccinate
obtained by LC/MS.
[0033] FIG. 7 is a mass spectrum of resveratrol tri-alaninate-boc
obtained by LC/MS.
[0034] FIG. 8 is a mass spectrum of resveratrol tri-alaninate HCl
obtained by LC/MS.
[0035] FIG. 9 is a mass spectrum of resveratrol tri-valinate-boc
obtained by LC/MS.
[0036] FIG. 10 is a mass spectrum of resveratrol di-valinate-boc
obtained by LC/MS.
[0037] FIG. 11 is a mass spectrum of resveratrol mono-valinate-boc
obtained by LC/MS.
[0038] FIG. 12 is a mass spectrum of resveratrol tri-valinate HCl
obtained by LC/MS.
[0039] FIG. 13 is a mass spectrum of resveratrol
tri-valinate-hemisuccinate obtained by LC/MS.
[0040] FIG. 14 is a mass spectrum of resveratrol
tri-phenylalaninate obtained by LC/MS.
DETAILED DESCRIPTION
[0041] It has been discovered that local administration of
resveratrol, such as into a wound, is challenging due to its low
solubility in water (0.03 g/L). As a result, a large amount of
resveratrol is typically required to deliver a therapeutically
effective amount to a target application site. The delivery of
resveratrol may be improved by administering a resveratrol
precursor having greater water solubility than resveratrol. By
increasing the solubility relative to resveratrol, a smaller amount
of the resveratrol precursor may be used to deliver a
therapeutically equivalent amount of resveratrol. However, if the
solubility of the resveratrol precursor is too high, the
resveratrol precursor will diffuse away from the target application
site and fail to deliver the resveratrol as intended. It is
important to avoid diffusion of the resveratrol precursor since
resveratrol is applied locally, preferably at the target
application site. There is a need for a resveratrol precursor, such
as a resveratrol ester, with sufficient water solubility to improve
local administration and bioavailability of resveratrol, but not so
water soluble so as to diffuse away from the application site.
[0042] Another challenge presented by the low solubility of
resveratrol is the difficulty in preparing aqueous compositions
containing resveratrol. Typically, the preparation of an aqueous
composition containing resveratrol involves a two-step process.
Resveratrol is first dissolved in an alcohol, such as ethanol.
Next, the resveratrol/alcohol solution is dissolved in water to
form an aqueous composition. Although this two-step process
overcomes the problems presented by the low solubility of
resveratrol in water, it is disfavored when the composition
containing resveratrol is to be used in the reduction of scarring
because alcohols are known fibrotic agents. The amount of alcohol
in the aqueous composition containing resveratrol may be reduced,
but cannot be completely eliminated. As a result, compositions
containing resveratrol prepared by a process involving dissolution
in alcohol will contain some amount of a fibrotic agent. There is a
need for a method of preparing an aqueous composition containing
resveratrol, or a resveratrol precursor, that excludes alcohol.
[0043] The present invention makes use of the discovery of
resveratrol precursors with greater water solubility and greater
bioavailability than resveratrol that are not so water soluble so
as to diffuse away from the application site. Resveratrol esters
were identified as promising precursor candidates for resveratrol
delivery because of the wide availability of esterases in vivo.
Applicants have surprisingly discovered that certain resveratrol
esters possess unexpected and superior efficacy and bioavailability
as compared to resveratrol. The resveratrol esters were found to
have increased water solubility while still allowing the
resveratrol molecules to enter cells and provide the intended
therapeutic benefits. The esters chosen provide increased polarity
without being so hydrophilic that the resveratrol precursors
diffuse away from the site of application.
[0044] The present invention also makes use of the discovery of an
improved method for producing aqueous compositions containing
resveratrol precursors, such as resveratrol esters. Resveratrol
esters have increased water solubility as compared to resveratrol,
which allows compositions containing resveratrol esters to be
prepared without first dissolving the resveratrol esters in
alcohol. The method eliminates the introduction of alcohol, a known
fibrotic agent, while also simplifying the production process.
[0045] Resveratrol esters may have an ester linkage at any of the
three hydroxyl moieties on resveratrol. Resveratrol esters may be
formed by any suitable chemical reaction, such as esterification
with a dicarboxylic acid or esterification with an amino acid.
Resveratrol esters include resveratrol with one, two, or three of
the hydroxyl moieties modified by an ester linkage. Preferably, the
resveratrol hydroxyl moieties have the same ester linkage when more
than one resveratrol hydroxyl moiety is modified.
[0046] Resveratrol esters of carboxylic acids have an ester linkage
between one or more of the resveratrol hydroxyl moieties oxygens
and the carbonyl carbon from the carboxylic acid moiety.
Preferably, the carboxylic acid used is a dicarboxylic acid.
Dicarboxylic acids are preferred because they retain a carboxylic
acid moiety after esterification at the other carboxylic acid
moiety. Retention of a carboxylic acid moiety increases the acidity
of the resveratrol esters, which in turn increases the solubility
of the resveratrol esters. Preferably, the dicarboxylic acid is a
linear saturated dicarboxylic acid containing up to 5 carbon atoms.
Suitable dicarboxylic acids include malonic acid (propanedioic
acid), succinic acid (butanedioic acid) and glutaric acid
(pentanedioic acid). A preferred dicarboxylic acid is glutaric
acid. It was surprisingly discovered that esters of resveratrol
with dicarboxylic acids having greater than 5 carbon atoms, such as
adipic acid (hexanedioic acid) ester, are too lipophilic for use in
resveratrol delivery. Similarly, esters of resveratrol with
monocarboxylic acids, such as acetates, propionates, and butyrates,
have lower water solubility than resveratrol itself and are too
lipophilic for use in resveratrol delivery. Preferred resveratrol
esters include hemimalonate [--(CO)(CH.sub.2)(CO)(OH)],
hemisuccinate [--(CO)(CH.sub.2).sub.2(CO)(OH)] and hemiglutarate
[--(CO)(CH.sub.2).sub.3(CO)(OH)]. A preferred hemiglutarate ester
of resveratrol is resveratrol trihemiglutarate. The structure of
resveratrol trihemiglutarate is shown in FIG. 1. Resveratrol esters
of dicarboxylic acids may be formulated as salts, for example, the
sodium, potassium, calcium, or magnesium salts.
[0047] Resveratrol esters of amino acids have an ester linkage
between one or more of the resveratrol hydroxyl moieties oxygens
and the carbonyl carbon from the carboxylic acid moiety of the
amino acid. If resveratrol esters of amino acids are formed by
esterification, the amine moiety must be protected before the
carboxylic acid moiety participates in esterification, such as with
the tert-butyloxycarbonyl protecting group (boc or t-boc). After
esterification, the amine moiety may optionally be de-protected.
Resveratrol esters of amino acids are often more stable than
resveratrol esters of dicarboxylic acids. Preferably, the amino
acid used has a low molecular weight. Suitable natural amino acids
include alanine (2-aminopropanoic acid), valine
(2-amino-3-methylbutanoic acid), leucine (2-amino-4-methylpentanoic
acid), isoleucine (2-amino-3-methylpentanoic acid), glycine
(aminoethanoic acid) and phenylalanine (2-amino-3-phenylpropanoic
acid). Suitable non-natural amino acids include
4-(4-aminophenyl)-butyric acid, 4-amino-butyric acid and
6-amino-hexanoic acid. A preferred amino acid is valine. Preferred
resveratrol esters formed from natural amino acids include
2-aminopropanoate (alaninate) [--(CO)(NH.sub.2)CHCH.sub.3],
2-amino-3-methylbutanoate (valinate)
[--(CO)CH(NH.sub.2)CH(CH.sub.3).sub.2], 2-amino-4-methylpentanoate
(leucinate) [--(CO)CH(NH.sub.2)CH.sub.2CH(CH.sub.3).sub.2],
2-amino-3-methylpentanoate (isoleucinate)
[--(CO)CH(NH.sub.2)CH(CH.sub.3)CH.sub.2CH.sub.3], aminoethanoate
(glycinate) [--(CO)CH.sub.2NH.sub.2] and 2-amino-3-phenylpropanoate
(phenylalaninate) [--(CO)(NH.sub.2)CHCH.sub.2C.sub.6H.sub.5].
Preferred resveratrol esters formed from non-natural amino acids
include 4-(4-aminophenyl)-butyrate
[--(CO)(CH.sub.2).sub.3(C.sub.6H.sub.4)NH.sub.2], 4-amino-butyrate
[--(CO)(CH.sub.2).sub.3NH.sub.2], and 6-amino-hexanoate
[--(CO)(CH.sub.2).sub.5NH.sub.2]. If a protecting group is used,
the resveratrol ester may be provided without removing the
protecting group, such as resveratrol tri-alaninate-boc.
Resveratrol esters of amino acids may be formulated as salts, for
example, the hydrochloride salt.
[0048] Resveratrol esters of amino acids also include resveratrol
esters of amides. Resveratrol esters of amides may be formed by
reacting the amine moiety of a resveratrol ester of an amino acid
with a dicarboxylic acid having a carbon chain of 3 or 4 carbon
atoms. Suitable dicarboxylic acids include malonic acid
(propanedioic acid) and succinic acid (butanedioic acid). Preferred
resveratrol esters of amides include N-hemimalonate
[--(CO)(CH.sub.2)(CO)(OH)] and N-hemisuccinate
[--(CO)(CH.sub.2).sub.2(CO)(OH)]. Resveratrol esters of amides may
be formulated as salts.
[0049] A resveratrol ester has the following general structure:
##STR00011##
R.sup.1, R.sup.2, and R.sup.3 may be a hydrogen atom (H) or
##STR00012##
Each R.sup.4 is independently a carbon chain of 2 to 4 carbon atoms
having a terminal carboxylic acid moiety, a carbon chain of 1 to 5
carbon atoms having an amine moiety, or
##STR00013##
R.sup.5 is a carbon chain of 3 or 4 carbon atoms having a terminal
carboxylic acid moiety. At least one of R.sup.1, R.sup.2, and
R.sup.3 is not H. Each R.sup.4 may be substituted or unsubstituted,
saturated or unsaturated, and straight or branched. Preferably,
each R.sup.4 is unsubstituted, saturated and linear. R.sup.1,
R.sup.2, and R.sup.3 may be the same, or may be different. The
resveratrol esters may optionally be formulated as salts.
[0050] The resveratrol esters may be combined with pharmaceutically
acceptable excipients or carriers to form compositions containing
resveratrol esters that may be applied therapeutically. Preferably,
the compositions containing resveratrol esters are administered by
injection or topically. For example, a composition containing
resveratrol esters may be administered topically as a lotion,
ointment, cream, gel, paste, foam, suspension, topical solution or
other suitable topical form. Preferably, the compositions
containing resveratrol esters are sterile.
[0051] Compositions containing resveratrol esters are preferably
prepared without first dissolving the resveratrol esters in
alcohol. Resveratrol esters may be dissolved in emulsifiers and
solubilizers that do not contain alcohol. Suitable solvents include
emulsifiers and solubilizers in the KOLLIPHOR.RTM. portfolio
produced by BASF. A preferred solvent is KOLLIPHOR.RTM. ELP.
[0052] Compositions containing resveratrol esters may optionally
contain agents that do not materially affect the basic and novel
characteristics of the resveratrol esters. For example,
compositions containing resveratrol esters may optionally include
agents such as stabilizers, preservatives or pH adjusters. If the
compositions containing resveratrol esters are administered
topically, the pH of the compositions must be carefully chosen to
deliver the ester in its intended form without being irritating to
the skin or tissue. Preferably, the pH of compositions containing
resveratrol esters that are administered topically is 4.0-7.0 to
closely match the pH of normal skin.
[0053] Preferably, the resveratrol esters are present in a
composition at a concentration of at least 0.1 micromoles/liter,
more preferably at a concentration of at least 1.0
micromoles/liter, and most preferably at a concentration of at
least 5.0 micromoles/liter. Preferably, the resveratrol esters are
present in those compositions at a concentration of at most 1000
micromoles/liter. Examples include 7.5, 8.0, 9.0, 10, 12.5, 15, 16,
17, 18, 19, 20, 21, 21.9, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32.5,
35, 37.5, 40, 42.5, 45, 47.5, 50, 55, 60, 65, 70, 75, 80, 90, 100,
150, 200, 250, 300, 350, 400, 450 and 500 micromoles/liter.
[0054] Premeasured amounts of the compositions containing
resveratrol esters may also be used. These are referred to as unit
dosage forms, since each premeasured amount is intended to be used
on a single patient for one or more application, all used at the
same time. Examples include prefilled syringes, pouches, packets
and tubes. Another example is a tube or dispenser which may be used
to form foam of its contents just prior to application, for example
by shaking or using a foaming agent. A self-foaming tablet, which
forms foam when placed into water, could also be used. The volume
of material present in these unit dosage forms may be 0.1 to 100
mL, or 1 to 50 mL, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 20, 25, 30, 35, 40 and 45 mL.
[0055] A list of exemplary resveratrol esters is given in Table
A:
TABLE-US-00001 TABLE A resveratrol monohemimalonate resveratrol
dihemimalonate resveratrol trihemimalonate resveratrol
monohemisuccinate resveratrol dihemisuccinate resveratrol
trihemisuccinate resveratrol monohemiglutarate resveratrol
dihemiglutarate resveratrol trihemiglutarate resveratrol
mono-2-aminopropanoate resveratrol di-2-amino-3-methylbutanoate
resveratrol tri-2-amino-4-methylpentanoate resveratrol
mono-2-amino-3-methylpentanoate resveratrol di-aminoethanoate
resveratrol tri-4-(4-aminophenyl)-butyrate resveratrol
mono-4-amino-butyrate resveratrol di-6-amino-hexanoate
EXAMPLES
Example 1--Resveratrol Trihemiglutarate Synthesis
[0056] The following scheme depicts the process of preparing the
trihemiglutarate ester of resveratrol:
##STR00014##
[0057] Resveratrol (1) 2 g, (Combi-Blocks, Inc., CR-1053, batch#
A83528) was dissolved in 50 mL tetrahydrofuran (THF). 75 mg of
4-dimethylaminopyridine (DMAP) was added to the solution while
stirring at room temperature. 0.8 mL triethylamine (Et.sub.3N)
(Sigma-Aldrich, T0886, batch #126K07554) and 3.32 g glutaric
anhydride (Sigma-Aldrich, G3806, batch #0418JB) were added to the
stirring solution of resveratrol. The reaction was allowed to stir
overnight. In the morning TLC (using 50% ethyl acetate in hexanes
as the solvent system with a spray reagent composed of 5% sulfuric
acid in methanol, heat) against starting material indicated
disappearance of starting material and appearance of a new polar
spot. The solvent of the reaction mixture was evaporated to produce
a semi-solid gum. This semi-solid gum was separated by
chromatography on silica gel (60% ethyl acetate in hexanes with
0.1% trifloroacetic acid) and pure resveratrol trihemiglutarate (6)
was obtained after combining the pure fractions (2.12 g).
[0058] The mass of the resveratrol trihemiglutarate product (6) was
confirmed by LC/MS. The mass spectrum is shown in FIG. 2.
[0059] The resveratrol starting material (1) and the resveratrol
trihemiglutarate product (6) were analyzed by HPLC. Chromatograms
were obtained using an evaporative light scattering detector (ELSD)
and a UV detector. FIG. 3A is a chromatogram of resveratrol (1)
obtained using an ELSD. FIG. 3B is a chromatogram of resveratrol
(1) obtained using a UV detector. FIG. 4A is a chromatogram of
resveratrol trihemiglutarate (6) obtained using an ELSD. FIG. 4B is
a chromatogram of resveratrol trihemiglutarate (6) obtained using a
UV detector.
Example 2--Resveratrol Trihemisuccinate Synthesis
[0060] Resveratrol was dissolved in tetrahydrofuran (THF).
4-dimethylaminopyridine (DMAP) was added to the solution while
stirring. Succinic anhydride and triethylamine (Et.sub.3N) were
added to the stirring solution of resveratrol to produce crude
resveratrol trihemisuccinate. The resveratrol trihemisuccinate was
loaded onto a silica gel column packed in hexanes. The product was
brought down beginning with 10% ethyl acetate (EtOAc)/90% hexane
with 0.1% trifluoroacetic acid (TFA). Fractions were collected and
tested by thin layer chromatography for purity. Similar fractions
containing pure product were combined.
[0061] The mass of the resveratrol trihemisuccinate product was
confirmed by LC/MS. The mass spectrum is shown in FIG. 6.
Example 3--Resveratrol Tri-Alaninate-Boc Synthesis
[0062] Resveratrol was dissolved in dichloromethane (DCM).
4-dimethylaminopyridine (DMAP) was added to the solution while
stirring. In a separate flask, boc-8-alanine hydroxide and
dicyclohexylcarbodiimide (DCC) were dissolved in dichloromethane.
The resveratrol solution and the alanine solution were combined
while stirring to produce crude resveratrol tri-alaninate-boc. The
crude resveratrol tri-alaninate-boc was dissolved in
dichloromethane/ethyl acetate and loaded onto a silica gel column
packed in hexanes. The product was brought down with a step
gradient beginning at 100% hexane and increasing ethyl acetate
until reaching 50% hexane/50% ethyl acetate. Fractions were
collected and tested by thin layer chromatography for purity.
Similar fractions containing pure product were combined.
[0063] The mass of the resveratrol tri-alaninate-boc product was
confirmed by LC/MS. The mass spectrum is shown in FIG. 7.
Example 4--Resveratrol Tri-Alaninate HCl Synthesis
[0064] Resveratrol tri-alaninate-boc was prepared according to
Example 3. The resveratrol tri-alaninate-boc was then dissolved in
tetrahydrofuran (THF). Hydrochloric acid (HCl) gas was bubbled
through the solution at room temperature while stirring. A white
precipitate was formed, resveratrol tri-alaninate HCl, and was
filtered to produce a pure product.
[0065] The mass of the resveratrol tri-alaninate HCl product was
confirmed by LC/MS. The mass spectrum is shown in FIG. 8.
Example 5--Resveratrol Mono-, Di-, and Tri-Valinate-Boc
Synthesis
[0066] Resveratrol was dissolved in tetrahydrofuran (THF).
4-dimethylaminopyridine (DMAP) was added to the solution while
stirring. In a separate flask, boc-valine hydroxide and
dicyclohexylcarbodiimide (DCC) were dissolved in tetrahydrofuran
while stirring. The resveratrol solution and the valine solution
were combined while stirring to produce crude resveratrol
valine-boc. The crude resveratrol valine-boc was dissolved in
dichloromethane/hexane and loaded onto a silica gel column packed
in hexanes. The product was brought down with a step gradient
beginning at 100% hexane (with 0.1% trifluoroacetic acid (TFA)) and
increasing ethyl acetate until reaching 60% hexane/40% ethyl
acetate (with 0.1% TFA). Fractions were collected and tested by
thin layer chromatography (TLC) for purity. Optimal separation on
TLC plates occurred in 40% ethyl acetate/60% hexane with 0.1% TFA.
TLC showed three products: Resveratrol mono-valinate-boc,
resveratrol di-valinate-boc, and resveratrol tri-valinate-boc.
Similar fractions containing pure product were combined. Some
fractions containing mixtures of products were ran on additional
silica gel columns under the same conditions.
[0067] The mass of the resveratrol tri-valinate-boc product was
confirmed by LC/MS. The mass spectrum is shown in FIG. 9.
[0068] The mass of the resveratrol di-valinate-boc product was
confirmed by LC/MS.
[0069] The mass spectrum is shown in FIG. 10.
[0070] [.sup.76] The mass of the resveratrol mono-valinate-boc
product was confirmed by LC/MS. The mass spectrum is shown in FIG.
11.
Example 6--Resveratrol Tri-Valinate HCl Synthesis
[0071] Resveratrol tri-valinate-boc was prepared according to
Example 5. The resveratrol tri-valinate-boc was then dissolved in
tetrahydrofuran (THF). Hydrochloric acid (HCl) gas was bubbled
through the solution at room temperature while stirring. A white
precipitate was formed, resveratrol tri-valinate HCl, and was
filtered to produce a pure product.
[0072] The mass of the resveratrol tri-valinate HCl product was
confirmed by LC/MS. The mass spectrum is shown in FIG. 12.
Example 7--Resveratrol Tri-Valinate-Hemisuccinate Synthesis
[0073] Resveratrol tri-valinate was dissolved in dichloromethane
(DCM). 4-dimethylaminopyridine (DMAP) was added to the solution
while stirring. Succinic anhydride and triethylamine (Et.sub.3N)
were added to the stirring solution of resveratrol to produce crude
resveratrol tri-valinate-hemisuccinate. The crude resveratrol
tri-valinate-hemisuccinate was loaded (in DCM) onto a silica gel
column packed in hexanes. The product was brought down in step
gradient beginning with 30% ethyl acetate (EtOAc)/70% hexane,
slowly increasing to 100% ethyl acetate, then adding 5%
acetonitrile/95% ethyl acetate and increasing to 80%
acetonitrile/20% ethyl acetate. 0.1% trifluoroacetic acid was added
in the solvent system for the entire separation. Fractions were
collected and tested by thin layer chromatography for purity.
Similar fractions containing pure product were combined.
[0074] The mass of the resveratrol tri-valinate-hemisuccinate
product was confirmed by LC/MS. The mass spectrum is shown in FIG.
13.
Example 8--Resveratrol Tri-Phenylalaninate Synthesis
[0075] Resveratrol and 4-dimethylaminopyridine (DMAP) were
dissolved in tetrahydrofuran (THF) while stirring. In a separate
flask, boc-phenylalanine and dicyclohexylcarbodiimide (DCC) were
dissolved in tetrahydrofuran while stirring. The resveratrol
solution and the phenylalanine solution were combined while
stirring. The reaction formed a precipitate containing crude
resveratrol tri-phenylalaninate-boc, which was vacuum filtered. The
filtered product was loaded in dichloromethane onto a silica gel
column packed with hexane. The product was brought down in step
gradient beginning with 2% ethyl acetate (EtOAc)/98% hexane, slowly
increasing to 15% ethyl acetate/85% hexane. Fractions were tested
by thin layer chromatography for purity. Similar fractions
containing pure product were combined. The sample containing pure
fractions of resveratrol tri-phenylalaninate-boc was dissolved in
THF while stirring. Hydrochloric acid (HCl) gas was then bubbled
through the solution while stirring. A white precipitate was
formed, which was vacuum filtered after completion of the reaction.
The precipitate was washed with THF, leaving pure resveratrol
tri-phenylalaninate.
[0076] The mass of the resveratrol tri-phenylalaninate product was
confirmed by LC/MS. The mass spectrum is shown in FIG. 14.
Example 9--Comparative Solubility
[0077] The solubility of resveratrol, resveratrol trihemiglutarate,
resveratrol trihemisuccinate, resveratrol
tri-valinate-hemisuccinate, resveratrol tri-valinate, resveratrol
di-valinate and resveratrol tri-phenylalaninate was compared. Two 4
mg samples of each compound were weighed.
[0078] The first sample was used to determine solubility. The
sample was mixed with 1 mL of pH 7.4 phosphate buffered saline
(PBS) buffer solution to produce a 4 mg/mL solution. The solution
was vortexed and filtered to remove undissolved particles. The
filtered solution was then diluted in pH 7.4 buffer in triplicate
and stored in a refrigerator to reduce hydrolysis. The solutions
were allowed to sit at room temperature for 5 minutes and then were
analyzed by HPLC.
[0079] The second sample was used to prepare a calibration curve in
ethanol. Calibrators were made based on expected solubility. After
visually inspecting solubility in the buffer, the calibration curve
was determined in order to include points above and below the
expected concentration. The calibrators were stored in a
refrigerator until ready for analysis by HPLC.
[0080] The results of the solubility study are as follows:
TABLE-US-00002 Compound Solubility Resveratrol 0.0048 mg/mL
Resveratrol trihemiglutarate 2.06 mg/mL Resveratrol
trihemisuccinate 0.291 mg/mL Resveratrol tri-valinate-hemisuccinate
0.201 mg/mL Resveratrol tri-valinate Not soluble Resveratrol
di-valinate Not soluble Resveratrol tri-phenylalaninate Not
soluble
[0081] Several of the compounds hydrolyzed too quickly to be
analyzed by HPLC. Resveratrol trihemiglutarate and resveratrol
tri-valinate-hemisuccinate slightly hydrolyzed to the mono- and
di-substituted compounds. Resveratrol trihemisuccinate experienced
quick hydrolysis and was quantitated without resveratrol.
[0082] Resveratrol trihemiglutarate was found to have the highest
solubility and was approximately 400 times more soluble than
resveratrol. Resveratrol trihemisuccinate and resveratrol
tri-valinate-hemisuccinate also showed an improved solubility as
compared to resveratrol. Although resveratrol tri-valinate,
resveratrol di-valinate and resveratrol tri-phenylalaninate were
insoluble at pH 7.4, these compounds were expected to be freely
soluble at pH 4.0 or less.
Example 10--General Synthesis of Resveratrol Esters Formed from
Amino Acids
[0083] Resveratrol is dissolved in THF. Di-tert-butyl dicarbonate
is added to an amino acid under aqueous conditions to protect the
amine group. DMAP is added to the resveratrol while stirring at
room temperature. Triethylamine and the protected amino acid are
added to the stirring solution of resveratrol and allowed to stir
overnight. In the morning, the amine group is de-protected with a
strong acid. Thin-layer chromatography is used to verify
disappearance of starting material and appearance of a new polar
spot. The solvent of the reaction mixture is evaporated. This
product is separated by chromatography on silica gel to isolate the
desired amino acid ester product.
Example 11--Preparation of a Composition Containing 100 .mu.M
Resveratrol Trihemiglutarate
[0084] A composition for topical administration was prepared by
mixing the following ingredients:
[0085] Resveratrol trihemiglutarate--100 .mu.M
[0086] Calcium chloride--0.3 mM
[0087] Magnesium chloride--3.3 mM
[0088] Hydroxypropyl methylcellulose (HPMC) Gel 8%
[0089] The composition delivered resveratrol to a wound and
improved the healing process in a subject.
Example 12--Preparation of 150 .mu.M Resveratrol Ester Solution
[0090] A 150 .mu.M solution of resveratrol trihemiglutarate was
prepared. First, 9.975 mg of resveratrol trihemiglutarate powder
was accurately weighed. Next, the 9.975 mg of resveratrol
trihemiglutarate powder was dissolved in 5 mL of a 25%
KOLLIPHOR.RTM. ELP solution. The resveratrol trihemiglutarate
solution was then filtered into a sterile vial.
Example 13--In Vivo Application of Various Compositions Containing
Resveratrol or Resveratrol Esters in a Rat Model
[0091] 12 Sprague-Dawley rats were placed randomly into 6 different
groups, Study Groups 1-6, resulting in 2 animals per study group.
Animals were anesthetized and the dorsal subscapular areas were
shaved of hair with electric trimmers. Demarcations were made in a
gently widening parallel approximately 1 cm subscapular and 2.2 cm
in length. The left subscapular incision served as a control and
the right subscapular incision served as the treatment site. The
subscapular areas were incised with a #15 blade though the skin and
subcutaneous panniculus carnosus muscle. The composition mixture
corresponding to each group was then instilled into the right
subscapular wound and the incision closed with 5-0 nylon
interrupted fashion. Additional mixture was applied to the surface
of the treatment site incision. Three biopsies were taken of normal
skin at the time of incision and prior to any mixture instillation.
Each site was then monitored and photographed daily. Wound gross
morphology was noted daily. Sutures were removed and biopsies were
taken at day 8 of the study (postoperative day #7) and sent to
pathology for histology review. Histology was reviewed
independently by two dermatopathologists.
[0092] The Compositions of Study Groups 1-6 were: (1)
Ca.sup.++/Mg.sup.++/siRNA (MCP-1 inhibitor)/hyaluronic acid
tetramer in 8% hydroxypropyl methyl cellulose gel; (2) 100 .mu.M
resveratrol trihemiglutarate in 8% hydroxypropyl methyl cellulose
gel; (3) 400 .mu.M resveratrol trihemiglutarate in 8% hydroxypropyl
methyl cellulose gel; (4) 100 .mu.M
resveratrol/Ca.sup.++/Mg.sup.++/siRNA (MCP-1 inhibitor)/hyaluronic
acid tetramer in 8% hydroxypropyl methyl cellulose gel; (5) 100
.mu.M resveratrol trihemiglutarate/Ca.sup.++/Mg.sup.++/siRNA (MCP-1
inhibitor)/hyaluronic acid tetramer in 8% hydroxypropyl methyl
cellulose gel; and (6) 200 .mu.M resveratrol
trihemiglutarate/Ca.sup.++/Mg.sup.++/siRNA (MCP-1
inhibitor)/hyaluronic acid tetramer in 8% hydroxypropyl methyl
cellulose gel. A siRNA (MCP-1 inhibitor)
[0093] The specimens in Group 1 demonstrated poor
re-epithelialization. One specimen experienced dehiscence at the
superior pole of the treatment site as well as increased crusting
until day 5 with resulting depression at the treatment site. The
results suggest that resveratrol is necessary for rapid
mobilization of the epithelial keratinocytes. Histology review
indicated the largest treatment site dermis fibrosis compared to
the control. Mononuclear dermis infiltration was 25% higher in the
treatment site. There was moderate to severe evidence of
trichogranuloma in all histology specimens.
[0094] The specimens in Groups 2 and 3 were compared to previous
studies that involved treatment with natural resveratrol. Similar
results were noted for 100 .mu.M resveratrol trihemiglutarate as
compared to 100 .mu.M natural resveratrol but the resveratrol
trihemiglutarate demonstrated mild evidence of trichogranuloma in
each of the histology specimens. Earlier studies of 400 .mu.M
natural resveratrol showed inflammation and erythema at the
treatment site 48 hours post-incision and a similar response was
seen in the 400 .mu.M resveratrol trihemiglutarate. There were no
differences in histology between control and treatment site at
either dosage. The study showed no appreciable difference in
treatment between resveratrol trihemiglutarate alone as compared to
natural resveratrol alone.
[0095] A comparison of Group 4 treatment sites and control sites
showed no difference in fibrosis or monocytes at 3 days. However,
one specimen died 36 hours postoperatively due to pulmonary
embolism. The remaining specimen showed an increase in dermis
fibrosis in the resveratrol treatment at 8 days. The death of one
specimen limited a full comparison.
[0096] A comparison of Group 5 treatment sites and control sites
showed no significant difference in re-epithelialization rate or
dermal mononuclear infiltrates. The histology comparison noted a
25% increase in panniculus mononuclear infiltrates in the treatment
site. As compared to Group 4, Group 5 showed a slightly increased
dermal mononuclear infiltrate.
[0097] Group 6 demonstrated the most rapid re-epithelialization
with 30-50% treatment site re-epithelialization noted at 24 hours
compared to 10% in the control sites. The treatment sites showed
significantly better re-epithelialization until the 3.sup.rd
postoperative day. Histological comparison revealed a visible
decrease in monocytes and fibroblasts in the healing junction. FIG.
5A is a microscopic image of an untreated control incision. FIG. 5B
is a microscopic image of an incision treated with a resveratrol
ester. The treated incision showed notably uniform epidermal repair
as compared to the deep indention of the epidermis seen in the
untreated control incision. The differences in fibrosis and
mononuclear dermal infiltrates were not considered significant.
Moderate to severe trichogranuloma formation was noted in the
specimens.
[0098] It was postulated that the results may have been different
if rat siRNA (MCP-1 inhibitor) had been used instead of human siRNA
(MCP-1 inhibitor). It was also suspected that the observed
trichogranulomas were caused by rat hair inadvertently entering the
incision site during the procedure.
Example 14--In Vivo Application of Various Compositions Containing
Resveratrol Esters in a Rat Model (Prophetic)
[0099] 15 Sprague-Dawley Rats, 6-8 weeks old, will be placed
randomly into 5 different groups, Study Groups 1-5, resulting in 3
animals per study group. An incision, 2 cm in length, will be made
on both the right and left shoulder of each rat: the left side will
be an untreated control, while the right side will be treated with
the Compositions 1-5, with the Study Group number corresponding to
the Composition number.
[0100] Compositions 1-5 will be: (1) 0.5 g resveratrol
trihemiglutarate in 1.0 cc aqueous hydroxypropyl methyl cellulose
gel (resveratrol trihemiglutarate concentration=2.19
micromoles/liter); (2) 0.5 g resveratrol trihemiglutarate in 1.0 cc
aqueous high molecular weight hyaluronic acid gel (resveratrol
trihemiglutarate concentration=2.19 micromoles/liter); (3) 0.5 g
resveratrol trihemiglutarate and 0.5 g tretinoin in 1.0 cc aqueous
hydroxypropyl methyl cellulose gel (resveratrol trihemiglutarate
concentration=2.19 micromoles/liter); (4) 0.5 g resveratrol
trihemiglutarate and 0.5 g luteolin in 1.0 cc aqueous hydroxypropyl
methyl cellulose gel (resveratrol trihemiglutarate
concentration=2.19 micromoles/liter); and (5) resveratrol
trihemiglutarate powder.
[0101] After each incision is made, the resveratrol
trihemiglutarate containing composition will be applied to the
right incision just prior to closure using interrupted 5-0 nylon
sutures. The left incision will also be closed using interrupted
5-0 nylon sutures. Each incision will be photographed and
measurements will be taken, each day for 7 days. On the 4.sup.th
day, serum blood samples will be taken for systemic absorption
assay. On the 7.sup.th day, a punch biopsy will be taken from each
test and control incision.
[0102] Since each skin flap of the incisions will be very close
together, when the composition containing resveratrol
trihemiglutarate is applied soon after the incision is made, the
incision on the right shoulder will heal before fibroplasia begins,
so no scar is expected to form. This is in contrast to the
otherwise identical incision on the left side, where no resveratrol
trihemiglutarate will be applied, which is expected to display a
typical scar.
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