U.S. patent application number 11/392470 was filed with the patent office on 2006-11-30 for reduction in complement activation and inflammation during tissue injury by carotenoids, carotenoid analogs, or derivatives thereof.
Invention is credited to Samuel F. Lockwood, Geoff Nadolski.
Application Number | 20060270590 11/392470 |
Document ID | / |
Family ID | 37054093 |
Filed Date | 2006-11-30 |
United States Patent
Application |
20060270590 |
Kind Code |
A1 |
Lockwood; Samuel F. ; et
al. |
November 30, 2006 |
Reduction in complement activation and inflammation during tissue
injury by carotenoids, carotenoid analogs, or derivatives
thereof
Abstract
Administering water-soluble or dispersible synthetic analogs or
derivatives of astaxanthin, lutein, zeaxanthin, or lycophyll and/or
other carotenoids to a subject may reduce some of the adverse
effects of inflammation in a body organ or tissue. The analogs or
derivatives may be incorporated into pharmaceutical,
over-the-counter, or nutraceutical preparations. Administration of
the analogs or derivatives described herein may reduce deposition
of inflammatory mediators such as C-reactive protein, complement
system proteins or the membrane attack complex (MAC) in tissues.
Reduced deposition of these molecules in tissues may reduce cell
damage and/or lysis in the tissues.
Inventors: |
Lockwood; Samuel F.; (Lago
Vista, TX) ; Nadolski; Geoff; (Kailua, HI) |
Correspondence
Address: |
MEYERTONS, HOOD, KIVLIN, KOWERT & GOETZEL, P.C.
700 LAVACA, SUITE 800
AUSTIN
TX
78701
US
|
Family ID: |
37054093 |
Appl. No.: |
11/392470 |
Filed: |
March 29, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60666119 |
Mar 29, 2005 |
|
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|
Current U.S.
Class: |
514/165 ;
514/114; 514/171; 514/23; 514/406; 514/553; 514/561; 514/569;
514/570; 514/690; 514/763 |
Current CPC
Class: |
A61P 29/00 20180101;
A61K 31/195 20130101; A61K 38/16 20130101; A61P 9/10 20180101; A61K
45/06 20130101; A61K 31/573 20130101; A61K 31/7034 20130101; A61K
31/192 20130101; A61K 31/6615 20130101; A61K 31/341 20130101; A61K
31/225 20130101 |
Class at
Publication: |
514/002 ;
514/023; 514/114; 514/171; 514/553; 514/690; 514/569; 514/570;
514/406; 514/561; 514/763 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 31/7034 20060101 A61K031/7034; A61K 31/573
20060101 A61K031/573; A61K 31/192 20060101 A61K031/192; A61K 31/185
20060101 A61K031/185; A61K 31/195 20060101 A61K031/195 |
Claims
1. A method of preventing, inhibiting or reducing tissue damage
associated with an inflammatory response in a subject comprising
administering to a subject who would benefit from such treatment a
prophylactically or therapeutically effective amount of a
pharmaceutically acceptable formulation comprising a synthetic
carotenoid analog or a carotenoid derivative having the structure;
##STR81## where each R.sup.3 is independently hydrogen or methyl,
and where each R.sup.1 and R.sup.2 are independently: ##STR82##
where R.sup.4 is hydrogen or methyl; where each R.sup.5 is
independently hydrogen, --OH, or --OR.sup.6 wherein at least one
R.sup.5 group is --OR.sup.6; wherein each R.sup.6 is independently:
alkyl; aryl; -alkyl-N(R.sup.7).sub.2; -aryl-N(R.sup.7).sub.2;
-alkyl-N.sup.+ (R.sup.7).sub.3; -aryl-N.sup.+(R.sup.7).sub.3;
-alkyl-CO.sub.2R.sup.7; -aryl-CO.sub.2R.sup.7; -alkyl-CO.sub.2--;
-aryl-CO.sub.2.sup.-; --O--C(O)--R.sup.8; --P(O)(OR.sup.8).sub.2;
--S(O)(OR.sup.8).sub.2; an amino acid; a peptide, a carbohydrate;
--C(O)--(CH.sub.2).--CO.sub.2R.sup.9; a nucleoside reside, or a
co-antioxidant; where R.sup.7 is hydrogen, alkyl, or aryl; wherein
R.sup.8 is hydrogen, alkyl, aryl, benzyl or a co-antioxidant; where
R.sup.9 is hydrogen; alkyl; aryl; --P(O)(OR.sup.8).sub.2;
--S(O)(OR.sup.8).sub.2; an amino acid; a peptide, a carbohydrate; a
nucleoside, or a co-antioxidant; and where n is 1 to 9.
2. The method of claim 1, further comprising administering to the
subject at least one anti-inflammatory drug.
3. The method of claim 2, wherein at least one of the
anti-inflammatory drugs are selected from the list consisting of
cortisol; hydrocortisone; Prednisone; Prednisolone;
Methylprednisolone; Meprednisone; Triamcicolone; Paramethasone;
Fluprednisolone; Betamethasone; Dexamethasone; Fludrocortisone;
Aspirin; Diclofenac; Diflunisal; Etodolac; Fenoprofen;
Floctafenine; Flurbiprofen; Ibuprofen; Indomethacin; Ketorolac;
Ketoprofen; Meclofenamate; Mefenamic Acid; Meloxicam; Nabumetone;
Naproxen; Nimesulide; Oxaprozin; Phenylbutazone; Piroxicam;
Salsalate; Sulindac; Tenoxicam; Tiaprofenic Acid; Tolmetin;
Celecoxib; rofecoxib; etoricoxib; and valdecoxib.
4. The method of claim 1, wherein the formulation is administered
prior to the onset of an inflammatory response.
5. (canceled)
6. (canceled)
7. (canceled)
8. The method of claim 1, wherein the composition comprises one or
more carotenoid derivatives or analogs having the structure:
##STR83## where each R.sup.1 and R.sup.2 are independently:
##STR84## where each R.sup.5 is independently hydrogen, --OH, or
--OR.sup.6 wherein at least one R.sup.5 group is --OR.sup.6;
wherein each R.sup.6 is independently: alkyl; aryl;
-alkyl-N.sup.7).sub.2; -aryl-N(R.sup.7).sub.2;
-alkyl-N.sup.+(P.sup.7).sub.3; -aryl-N.sup.+(R.sup.7).sub.3;
-alkyl-CO.sub.2R.sup.7; -aryl-CO.sub.2R.sup.7; -alkyl-CO.sub.2--;
-aryl-CO.sub.2.sup.-; --O--C(O)--R.sup.8; --P(O)(OR.sup.8).sub.2;
--S(O)(OR.sup.8).sub.2; an amino acid; a peptide, a carbohydrate;
--C(O)--(CH.sub.2), --CO.sub.2R.sup.9; a nucleoside reside, or a
co-antioxidant; where R.sup.7 is hydrogen, alkyl, or aryl; wherein
R.sup.8 is hydrogen, alkyl, aryl, benzyl, or a co-antioxidant; and
where R.sup.9 is hydrogen; alkyl; aryl; --P(O)(OR.sup.8).sub.2;
--S(O)(OR.sup.8).sub.2; an amino acid; a peptide, a carbohydrate; a
nucleoside, or a co-antioxidant; and where n is 1 to 9.
9. The method of claim 1, wherein the composition comprises one or
more carotenoid derivatives or analogs having the structure:
##STR85## where each R.sup.1 and R.sup.2 are independently:
##STR86## where each R.sup.5 is independently hydrogen, --OH, or
--OR.sup.6 wherein at least one R.sup.5 group is --OR.sup.6;
wherein each R.sup.6 is independently: ##STR87## or a
co-antioxidant; wherein R.sup.8 is hydrogen, alkyl, aryl, benzyl,
Group IA metal, or a co-antioxidant; wherein R' is CH.sub.2; and
where n is 1 to 9.
10. The method of claim 1, wherein the composition comprises one or
more carotenoid derivatives or analogs having the structure:
##STR88## wherein each --OR.sup.6 is independently: ##STR89## or a
co-antioxidant; wherein R.sup.8 is hydrogen, alkyl, aryl, benzyl,
Group IA metal, or a co-antioxidant; wherein R' is CH.sub.2; and
where n is 1 to 9.
11. The method of claim 1, wherein the composition comprises one or
more carotenoid derivatives or analogs having the structure:
##STR90## wherein each --OR.sup.6 is independently: ##STR91## or a
co-antioxidant; wherein R.sup.8 is hydrogen, alkyl, aryl, benzyl,
Group IA metal, or a co-antioxidant; wherein R' is CH.sub.2; and
where n is 1 to 9.
12. The method of claim 1, wherein the composition comprises two or
more carotenoid derivatives or analogs having the structures:
##STR92## wherein each --OR.sup.6 is independently: ##STR93## or a
co-antioxidant; wherein R.sup.8 is hydrogen, alkyl, aryl, benzyl,
Group IA metal, or a co-antioxidant; wherein R' is CH.sub.2; and
where n is 1 to 9.
13. The method of claim 12, wherein each --OR.sup.6 independently
comprises: ##STR94## ##STR95## ##STR96## ##STR97## and, wherein R
is independently H, alkyl, aryl, benzyl, Group IA metal, or
co-antioxidant.
14. The method of claim 12, wherein each --OR.sup.6 independently
comprises: ##STR98## or a co-antioxidant; wherein R.sup.8 is
hydrogen, alkyl, aryl, benzyl, Group IA metal, or a co-antioxidant;
wherein R' is CH.sub.2; and where n is 1 to 9.
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. The method of claim 1, wherein the composition comprises one or
more carotenoid derivatives or analogs having the structures:
##STR99## where each R is independently H, alkyl, aryl, benzyl,
Group LX metal, or a co-antioxidant.
20. The method of claim 1, wherein the composition comprises one or
more carotenoid derivatives or analogs having the structures:
##STR100## where each R is independently H, alkyl, aryl, benzyl,
Group IA metal, or a co-antioxidant.
21. The method of claim 1, wherein the co-antioxidant comprises
Vitamin C, Vitamin C analogs, Vitamin C derivatives, Vitamin E,
Vitamin E analogs, Vitamin E derivatives, flavonoids, flavonoid
derivatives, or flavonoid analogs.
22. The method of claim 1, wherein the flavonoids comprise
quercetin, xanthohumol, isoxanthohumol, or genistein.
23. The method of claim 1, wherein the composition comprises one or
more carotenoid derivatives or analogs having the structures:
##STR101## where each R is independently H, alkyl, aryl, benzyl, or
a Group IA metal.
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
28. The method of claim 1, wherein the composition is administered
orally.
29. The method of claim 1, wherein the composition is administered
parenterally.
30. The method of claim 1, wherein the composition is administered
as an aqueous solution.
31. The method of claim 1, wherein the composition is administered
as an aqueous dispersion.
32. The method of claim 1, wherein the composition is administered
intravenously.
33. The method of claim 1, wherein the composition is administered
intravascularly.
34. The method of claim 1, wherein the composition is administered
by intramuscular injection.
35. The method of claim 1, wherein the composition is administered
subcutaneously.
36. The method of claim 1, wherein the composition is administered
transdermally.
37-47. (canceled)
48. A pharmaceutical composition comprising: a synthetic carotenoid
analog or a carotenoid derivative having the structure ##STR102##
where each R.sup.3 is independently hydrogen or methyl, and where
each R.sup.1 and R.sup.2 are independently: ##STR103## where
R.sup.4 is hydrogen or methyl; where each R.sup.5 is independently
hydrogen, --OH, or --OR.sup.6 wherein at least one R.sup.5 group is
--OR.sup.6; wherein each R.sup.6 is independently: alkyl; aryl;
-alkyl-N(R.sup.7).sub.2; -aryl-N(R.sup.7).sub.2;
-alkyl-N.sup.+(R.sup.7).sub.3; -aryl-N.sup.+(R.sup.7).sub.3;
-alkyl-CO.sub.2R.sup.7; -aryl-CO.sub.2R.sup.7; -alkyl-CO.sub.2--;
-aryl-CO.sub.2.sup.-; --O--C(O)--R.sup.8; --P(O)(OR.sup.8).sub.2;
--S(O)(OR.sup.8).sub.2; an amino acid; a peptide, a carbohydrate;
--C(O)--(CH.sub.2).sub.n--CO.sub.2R.sup.9; a nucleoside reside, or
a co-antioxidant; where R.sup.7 is hydrogen, alkyl, or aryl;
wherein R.sup.8 is hydrogen, alkyl, aryl, benzyl or a
co-antioxidant; where R.sup.9 is hydrogen; alkyl; aryl;
--P(O)(OR.sup.8).sub.2; --S(O)(OR.sup.8).sub.2; an amino acid; a
peptide, a carbohydrate; a nucleoside, or a co-antioxidant; and
where n is 1 to 9; and at least one pharmaceutically inactive
carrier; and at least one additional anti-inflammatory agent.
49-59. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 119(e) to Provisional Patent Application Ser. No.
60/666,119, filed Mar. 29, 2005, entitled "REDUCTION IN COMPLEMENT
ACTIVATION AND INFLAMMATION DURING TISSUE INJURY BY CAROTENOIDS,
CAROTENOID ANALOGS, OR DERIVATIVES THEREOF." The prior application
is commonly assigned with the present invention, and the contents
thereof are incorporated by reference in their entirety as though
fully set forth herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention generally relates to the fields of
medicinal and synthetic chemistry. Specifically, the invention
relates to the synthesis and use of water-soluble and
water-dispersible carotenoids, including analogs, derivatives, and
intermediates thereof, as therapeutic and/or prophylactic
anti-inflammatory and anti-oxidant agents that reduce tissue damage
associated with inflammation.
[0004] 2. Description of the Related Art
[0005] Inflammation plays an important role in the pathophysiology
of ischemic heart disease (Yeh et al., 2001). Elevated levels
(>2 mg/dl) of C-reactive protein concentration (CRP), commonly
used as a marker for an acute inflammatory response, are correlated
with increased mortality due to cardiovascular events (Lagrand et
al., 1999). This relationship holds true for asymptomatic
individuals (Ridker et al., 2000) and patients with unstable angina
(Lindahl et al., 2000) and acute myocardial infarction (Pietila et
al., 1996). It has been suggested that the epidemiological studies
relating CRP to the incidence and outcome of ischemic syndromes are
not simply due to CRP being a nonspecific marker of disease
susceptibility or inflammation but rather that CRP might be
involved directly in the pathogenesis of ischemic syndromes through
a proinflammatory effect mediated by complement activation
(Beranek, 1997). The primary evidence for this hypothesis is
derived from studies of autopsy specimens showing co-localization
of CRP with activated complement components in infarcted myocardial
tissue but not in healthy myocardium (Lagrand et al., 1997;
Yasojima et al., 1998b). Deposition of CRP also occurs in the
ischemic rabbit myocardium (Kushner et al., 1963) and is closely
correlated with the infiltration of polymorphonuclear leukocytes
(pro-inflammatory cells) to the ischemic tissue (du Clos et al.,
1981). Additionally, studies have shown that the endogenous
increase in plasma CRP secondary to a remote inflammatory lesion is
associated with an increase in myocardial tissue injury secondary
to regional ischemia and reperfusion. The myocardial injury occurs
via a complement-dependent mechanism, and can be ameliorated by
pretreatment with heparin, N-acetylheparin or can be prevented in
rabbits deficient in complement protein C6, which are incapable of
forming the membrane attack complex (Barrett et al., 2002). The
evolving paradigm suggests that in the normal, healthy adult any
elevations of CRP in the absence of acute infection or acute tissue
injury can potentially be deleterious; indeed, in umbilical cord
blood levels are very low (<0.01 mg/dl). For example,
cardiovascular patients at risk for inflammatory heart disease
benefit from lowering of circulating CRP levels, without evidence
of a "no effect level" for this marker (Ridker et al. 2005). Local
production of CRP by cells of other than hepatic origin has now
been convincingly demonstrated (Venugopal et al. 2005), suggesting
a tissue-specific role for this acute phase protein. Previous
studies have also shown that administering carotenoid analogs or
derivatives can reduce the serum concentration of CRP following
ischemic reperfusion injury (Publication No. US-2005-0009758 and
PCT International Application Number PCT/US2003/023706). Therapies
aimed at (1) reducing circulating levels of CRP in mammals; (2) in
the localized and/or systemic production of CRP by liver and other
tissues; and (3) the deposition of CRP (either with or without
other endogenous inflammatory mediators) in pathological injury
will have important therapeutic value (Ridker 2005).
[0006] Carotenoids are a group of natural pigments produced
principally by plants, yeast, and microalgae. The family of related
compounds now numbers greater than 700 described members, exclusive
of Z and E isomers. Humans and other animals cannot synthesize
carotenoids de novo and must obtain them from their diet. All
carotenoids share common chemical features, such as a
polyisoprenoid structure, a long polyene chain forming the
chromophore, and near symmetry around the central double bond.
Tail-to-tail linkage of two C.sub.20 geranyl-geranyl diphosphate
molecules produces the parent C.sub.40 carbon skeleton. Carotenoids
without oxygenated functional groups are called "carotenes",
reflecting their hydrocarbon nature; oxygenated carotenes are known
as "xanthophylls." Cyclization at one or both ends of the molecule
yields 7 identified end groups (illustrative structures shown in
FIG. 1). Examples of uses of carotenoid derivatives and analogs are
illustrated in U.S. patent application Ser. No. 10/793,671 filed on
Mar. 4, 2004, entitled "CAROTENOID ETHER ANALOGS OR DERIVATIVES FOR
THE INHIBITION AND AMELIORATION OF DISEASE" by Lockwood et al.
published on Jan. 13, 2005, as Publication No. US-2005-0009758 and
PCT International Application Number PCT/US2003/023706 filed on
Jul. 29, 2003, entitled "STRUCTURAL CAROTENOID ANALOGS FOR THE
INHIBITION AND AMELIORATION OF DISEASE" by Lockwood et al.
(International Publication Number WO 2004/011423 A2, published on
Feb. 5, 2004) both of which are incorporated by reference as though
fully set forth herein.
[0007] Documented carotenoid functions in nature include light
harvesting, photoprotection, and protective and sex-related
coloration in microscopic organisms, mammals, and birds,
respectively. A relatively recent observation has been the
protective role of carotenoids against age-related diseases in
humans as part of a complex antioxidant network within cells. This
role is dictated by the close relationship between the
physicochemical properties of individual carotenoids and their in
vivo functions in organisms. The long system of alternating double
and single bonds in the central part of the molecule (delocalizing
the .pi.-orbital electrons over the entire length of the polyene
chain) confers the distinctive molecular shape, chemical
reactivity, and light-absorbing properties of carotenoids.
Additionally, isomerism around C.dbd.C double bonds yields
distinctly different molecular structures that may be isolated as
separate compounds (known as Z ("cis") and E ("trans"), or
geometric, isomers). Of the more than 700 described carotenoids, an
even greater number of the theoretically possible mono-Z and poly-Z
isomers are sometimes encountered in nature. The presence of a Z
double bond creates greater steric hindrance between nearby
hydrogen atoms and/or methyl groups, so that Z isomers are
generally less stable thermodynamically, and more chemically
reactive, than the corresponding all-E form. The all-E
configuration is an extended, linear, and rigid molecule. Z-isomers
are, by contrast, not simple, linear molecules (the so-called
"bent-chain" isomers). The presence of any Z in the polyene chain
creates a bent-chain molecule. The tendency of Z-isomers to
crystallize or aggregate is much less than all-E, and Z isomers may
sometimes be more readily solubilized, absorbed, and transported in
vivo than their all-E counterparts. This has important implications
for enterable (e.g., oral) and parenteral (e.g., intravenous,
intra-arterial, intramuscular, intraperitoneal, intracoronary, and
subcutaneous) dosing in mammals.
[0008] Problems related to the use of some prior art carotenoids
and structural carotenoid analogs or derivatives include: (1) the
complex isomeric mixtures, including non-carotenoid contaminants,
provided in natural and synthetic sources leading to costly
increases in safety and efficacy tests required by such agencies as
the FDA; (2) limited bioavailability upon administration to a
subject; and (3) the differential induction of cytochrome P450
enzymes (this family of enzymes exhibits species-specific
differences which must be taken into account when extrapolating
animal work to human studies). Selection of the appropriate analog
or derivative and isomer composition for a particular application
increases the utility of carotenoid analogs or derivatives for the
uses defined herein.
[0009] New methods of reducing or inhibiting one or more of the
pathological complications associated with inflammation and/or
tissue injury associated with inflammation, including deposition of
pro-inflammatory molecules and protein complexes in a body tissue
of a subject would be useful therapeutic agents. Carotenoid analogs
or derivatives displaying properties of increased
water-dispersibility and bioavailability would be beneficial for
such applications.
SUMMARY OF THE INVENTION
[0010] Methods and pharmaceutical compositions for reducing or
inhibiting one or more of the pathological complications associated
with inflammation and/or tissue injury associated with
inflammation, including deposition of pro-inflammatory molecules
and protein complexes in a body tissue of a subject are provided
for herein. The methods and pharmaceutical compositions described
herein may be used to treat of prevent a myriad of pathologies
associated with inflammatory responses, including but not limited
to those affecting the respiratory, cardiovascular or nervous
systems, vision and hearing, dental tissues, smooth musculature,
and transplantation of cells and tissues. Such methods can be used
alone as the sole therapeutic regimen or in combination with one or
more other established protocols for addressing a particular
disease or condition. Carotenoid analogs or derivatives useful in
the treatment methods contemplated herein are characterized in
functioning as anti-inflammatory agents.
[0011] More specifically the presently disclosed treatment methods
and pharmaceutical compositions relate to preventing, reducing or
inhibiting one or more of the pathological complications associated
with inflammation and/or tissue injury associated with inflammation
caused, at least in part, by the deposition and accumulation of
pro-inflammatory molecules and protein complexes in a body tissue
of a subject. The treatment methods contemplated herein preferably
include administering a therapeutically effective amount of at
least one carotenoid analog or derivative which prevents, reduces
or inhibits activation of complement proteins, initiation of
complement-mediated cellular lysis, accumulation of the membrane
attack complex, or accumulation of C-reactive protein in a tissue,
such as for example a cardiovascular tissue, during an inflammatory
response.
[0012] In some embodiments, methods of reducing, preventing or
inhibiting pathological complications associated with inflammation
and/or tissue injury associated with inflammation, including
deposition of pro-inflammatory molecules and protein complexes in a
body tissue of a subject may include administering to the cell,
group of cells or subject an effective amount of a pharmaceutically
acceptable formulation including a synthetic analog or derivative
of a carotenoid.
[0013] In some embodiments, methods of reducing, preventing or
inhibiting pathological complications associated with inflammation
and/or tissue injury associated with inflammation, including
deposition of pro-inflammatory molecules and protein complexes in a
body tissue of a subject may include administering to the cell,
group of cells or to a subject, an effective amount of a
pharmaceutically acceptable formulation including a synthetic
analog or derivative of a carotenoid.
[0014] In some embodiments, methods of reducing, preventing or
inhibiting pathological complications associated with inflammation
and/or ischemia/reperfusion injury in a body tissue of a subject
may include administering to the subject an effective amount of a
pharmaceutically acceptable formulation including a synthetic
analog or derivative of a carotenoid. In an embodiment, the
formulation may include diacid or diphosphate derivatives of a
carotenoid.
[0015] In some embodiments, methods of reducing, preventing or
inhibiting pathological complications associated with an
inflammatory response in a tissue of a subject may include
administering to the subject an effective amount of a
pharmaceutically acceptable formulation including a synthetic
analog or derivative of a carotenoid. In an embodiment, the
formulation may include diacid or diphosphate derivatives of a
carotenoid.
[0016] In some embodiments, methods of reducing, preventing or
inhibiting pathological complications associated with an
inflammatory response in a tissue of a subject may include
administering to the subject an effective amount of a
pharmaceutically acceptable formulation including a synthetic
analog or derivative of a carotenoid so as to reduce, prevent or
inhibit the activation of complement proteins (i.e., the
"complement cascade") in the subject. In an embodiment, the
formulation may include diacid or diphosphate derivatives of a
carotenoid.
[0017] In some embodiments, methods of reducing, preventing or
inhibiting pathological complications associated with an
inflammatory response in a tissue of a subject may include
administering to the subject an effective amount of a
pharmaceutically acceptable formulation including a synthetic
analog or derivative of a carotenoid so as to reduce, prevent or
inhibit the membrane attack complex (MAC) deposition in a tissue of
a subject. In an embodiment, the formulation may include diacid or
diphosphate derivatives of a carotenoid.
[0018] In some embodiments, methods of reducing, preventing or
inhibiting pathological complications associated with an
inflammatory response in a tissue of a subject may include
administering to the subject an effective amount of a
pharmaceutically acceptable formulation including a synthetic
analog or derivative of a carotenoid so as to reduce, prevent or
inhibit the accumulation/deposition of C-reactive protein (CRP) in
a tissue of a subject. In an embodiment, the formulation may
include diacid or diphosphate derivatives of a carotenoid.
[0019] In some embodiments, methods of methods of reducing,
preventing or inhibiting tissue injury associated with
ischemia/reperfusion of a tissue, especially a cardiovascular
tissue, of a subject may include administering to the subject an
effective amount of a pharmaceutically acceptable formulation
including a synthetic analog or derivative of a carotenoid so as to
reduce, prevent or inhibit the accumulation of the membrane attack
complex and/or C-reactive protein in the at the site of
ischemia/reperfusion. In an embodiment, the formulation may include
diacid or diphosphate derivatives of a carotenoid.
[0020] In some embodiments, methods of treating macular
degeneration (any age of onset) as well as Age-Related Macular
Degeneration (ARMD) in a subject may include administering to the
subject an effective amount of a pharmaceutically acceptable
formulation including a synthetic analog or derivative of a
carotenoid. The treatment may reduce tissue damage associated with
inflammation in the macula, and increase visual acuity or halt
progression of its deterioration. In an embodiment, the formulation
may include diacid or diphosphate derivatives of a carotenoid.
[0021] The presently described treatment methods, including the
administration of pharmaceutically acceptable formulations
containing synthetic carotenoid analogs or derivatives, may be
provided alone as a primary therapy, or may be provided in
conjunction with one more additional therapeutic agents (e.g.
anti-inflammatory medications). Such determination may be made by
an appropriate healthcare provider or practitioner of ordinary
skill in the art.
[0022] Administration of analogs or derivatives of carotenoids
according to the preceding embodiments may at least partially
prevent, reduce or inhibit one or more of the pathological
complications associated with inflammation and/or tissue injury.
Complications associated with inflammation and ischemia/reperfusion
injury that may be influenced according to some embodiments include
activation of complement proteins, deposition of activated
complement proteins and the membrane attack complex in tissues,
cellular and tissue damage caused by generation of reactive oxygen
species and other radicals, and deposition of C-reactive protein at
sites of inflammation. Reduction in the incidence and/or severity
of one or more of the aforementioned complications may reduce the
amount of tissue damage occurring at a site of-inflammation.
[0023] In some embodiments, the administration of structural
analogs or derivatives of carotenoids by one skilled in the
art--including consideration of the pharmacokinetics and
pharmacodynamics of therapeutic drug delivery--is expected to
inhibit and/or ameliorate disease conditions associated with
abnormal cell division. In some of the foregoing embodiments,
analogs or derivatives of carotenoids administered to cells may be
at least partially water-soluble.
[0024] "Water-soluble" structural carotenoid analogs or derivatives
are those analogs or derivatives that may be formulated in aqueous
solution, either alone or with one or more excipients.
Water-soluble carotenoid analogs or derivatives may include those
compounds and synthetic derivatives which form molecular
self-assemblies, and may be more properly termed "water
dispersible" carotenoid analogs or derivatives. Water-soluble
and/or "water-dispersible" carotenoid analogs or derivatives may be
preferred in some embodiments.
[0025] Water-soluble carotenoid analogs or derivatives may have a
water solubility of greater than about 1 mg/mL in some embodiments.
In certain embodiments, water-soluble carotenoid analogs or
derivatives may have a water solubility of greater than about 10
mg/mL. In certain embodiments, water-soluble carotenoid analogs or
derivatives may have a water solubility of greater than about 20
mg/mL. In certain embodiments, water-soluble carotenoid analogs or
derivatives may have a water solubility of greater than about 25
mg/mL. In some embodiments, water-soluble carotenoid analogs or
derivatives may have a water solubility of greater than about 50
mg/mL.
[0026] In some embodiments, water-soluble analogs or derivatives of
carotenoids may be administered to a cell, a group of cells or to a
subject alone or in combination with additional carotenoid analogs
or derivatives.
[0027] In some embodiments, a method to at least partially prevent,
reduce or inhibit one or more of the pathological complications
associated with inflammation and/or tissue injury may include
administering to the subject an effective amount of a
pharmaceutically acceptable formulation including a synthetic
analog or derivative of a carotenoid. The synthetic analog or
derivative of the carotenoid may have the structure ##STR1## where
each R.sup.3 is independently hydrogen or methyl, and where each
R.sup.1 and R.sup.2 are independently: ##STR2## ##STR3## where
R.sup.4 is hydrogen, methyl, or --CH.sub.2OH; and where each
R.sup.5 is independently hydrogen or --OH.
[0028] In some embodiments, a method to at least partially prevent,
reduce or inhibit one or more of the pathological complications
associated with inflammation and/or tissue injury may include
administering to the subject an effective amount of a
pharmaceutically acceptable formulation including a synthetic
analog or derivative of a carotenoid. The synthetic analog or
derivative of the carotenoid may have the structure ##STR4##
[0029] where each R.sup.3 is independently hydrogen or methyl, and
where each R.sup.1 and R.sup.2 are independently: ##STR5## where
R.sup.4 is hydrogen or methyl; where each R.sup.5 is independently
hydrogen, --OH, or --OR.sup.6 wherein at least one R.sup.5 group is
--OR.sup.6; wherein each R.sup.6 is independently: alkyl; aryl;
-alkyl-N(R.sup.7).sub.2; -aryl-N(R.sup.7).sub.2; -alkyl-CO.sub.2H;
-aryl-CO.sub.2H; --O--C(O)--R.sup.8; --P(O)(OR.sup.8).sub.2;
--S(O)(OR.sup.8).sub.2; an amino acid; a peptide, a carbohydrate;
--C(O)--(CH.sub.2).sub.n--CO.sub.2R.sup.9; --C(O)--OR.sup.9; a
nucleoside residue, or a co-antioxidant; where R.sup.7 is hydrogen,
alkyl, or aryl; wherein R.sup.9 is hydrogen, alkyl, aryl, benzyl,
or a co-antioxidant; and where R.sup.9 is hydrogen; alkyl; aryl;
--P(O)(OR.sup.8).sub.2; --S(O)(OR.sup.8).sub.2; an amino acid; a
peptide, a carbohydrate; a nucleoside, or a co-antioxidant; and
where n is 1 to 9. Pharmaceutically acceptable salts of any of the
above listed carotenoid derivatives may also be used to ameliorate
at least some of the pathological consequences associated with
inflammatory responses in a tissue.
[0030] Each co-antioxidant may be independently Vitamin C, Vitamin
C analogs, Vitamin C derivatives, Vitamin E, Vitamin E analogs,
Vitamin E derivatives, flavonoids, flavonoid derivatives, or
flavonoid analogs. Flavonoids include, but are not limited to,
quercetin, xanthohumol, isoxanthohumol, or genistein. Selection of
the co-antioxidant should not be seen as limiting for the
therapeutic application of the current invention.
[0031] The carotenoid analogs or derivatives for use in the
contemplated treatment methods and pharmaceutical compositions may
have one or more of the non-limiting structures ##STR6## Each R'
may be CH.sub.2 n may range from 1 to 9. Each R may be
independently H, alkyl, aryl, benzyl, a Group IA metal (e.g., Na,
K, Li or the like), or a co-antioxidant. Each co-antioxidant may be
independently Vitamin C, Vitamin C analogs, Vitamin C derivatives,
Vitamin E, Vitamin F analogs, Vitamin E derivatives, flavonoids,
flavonoid analogs, or flavonoid derivatives. Flavonoids may
include, for example, quercetin, xanthohumol, isoxanthohumol, or
genistein. In an embodiment, R' is CH.sub.2, n is I, and R is
sodium.
[0032] In some embodiments, the carotenoid analog or derivative may
have the structure ##STR7## Each R may be independently H, alkyl,
aryl, benzyl, a Group IA metal (e.g., Na, K, Li, or the like), or a
co-antioxidant. Each co-antioxidant may be independently Vitamin C,
Vitamin C analogs, Vitamin C derivatives, Vitamin E, Vitamin E
analogs, Vitamin E derivatives, flavonoids, flavonoid analogs, or
flavonoid derivatives. Flavonoids may include, for example,
quercetin, xanthohumol, isoxanthohumol, or genistein. In an
embodiment, R is sodium. When R includes Vitamin C, Vitamin C
analogs, or Vitamin C derivatives, some embodiments may include
carotenoid analogs or derivatives having the structure ##STR8##
Each R may be independently H, alkyl, aryl, benzyl, or a Group IA
metal.
[0033] In some embodiments, a pharmaceutical composition is
provided that may include one or more synthetic carotenoids ("a
co-formulation" strategy), or synthetic derivatives or analogs
thereof, in combination with one or more anti-inflammatory drugs.
Certain embodiments may further directed to pharmaceutical
compositions that include combinations of two or more carotenoids
or synthetic analogs or derivatives thereof. In an embodiment, a
pharmaceutical composition may include a chiral astaxanthin or a
synthetic derivative thereof in combination with one or more
additional anti-inflammatory drugs. In an embodiment, a
pharmaceutical composition may include a synthetic derivative of
lycophyll in combination with one or more additional
anti-inflammatory drugs. The pharmaceutical compositions may be
adapted to be administered orally, or by one or more parenteral
routes of administration. In an embodiment, the pharmaceutical
composition may be adapted such that at least a portion of the
dosage of carotenoid or synthetic derivative or analog thereof is
delivered prior to, during, or after at least a portion of the
additional anti-inflammatory drug(s) are delivered.
[0034] In some embodiments, separate pharmaceutical compositions
are provided, such that the one or more additional
anti-inflammatory drugs are delivered separately from carotenoid,
or synthetic derivatives or analogs thereof (sometimes referred to
in the art as a "co-administration" strategy). The pharmaceutical
compositions may be adapted to be administered orally, or by one or
more parenteral routes of administration. In an embodiment, the
pharmaceutical composition may be adapted such that at least a
portion of the dosage of the carotenoid or synthetic derivative or
analog thereof is delivered prior to, during, or after at least a
portion of the one or more additional anti-inflammatory drugs are
administered to the subject. The carotenoid, carotenoid analogs
and/or derivatives may also be administered alone.
[0035] Embodiments directed to pharmaceutical compositions may
further include appropriate vehicles for delivery of said
pharmaceutical composition to a desired site of action (i.e., the
site a subject's body where the biological effect of the
pharmaceutical composition is most desired). Pharmaceutical
compositions including xanthophyll carotenoids or analogs or
derivatives of astaxanthin, lutein, zeaxanthin, or lycophyll that
may be administered orally or intravenously may be particularly
advantageous for and suited to embodiments described herein. In yet
a further embodiment, an injectable astaxanthin formulation or a
structural analog or derivative may be administered with a
astaxanthin, zeaxanthin or lutein structural analog or derivative
and/or other carotenoid structural analogs or derivatives, or in
formulation with antioxidants and/or excipients that further the
intended purpose. In some embodiments, one or more of the
xanthophyll carotenoids or synthetic analogs or derivatives thereof
may be at least partially water-soluble.
[0036] Certain embodiments may further directed to pharmaceutical
compositions including combinations two or more structural
carotenoid analogs or derivatives. Pharmaceutical compositions
including injectable structural carotenoid analogs or derivatives
of lutein may be particularly advantageous for the methods
described herein. In yet a further embodiment, an injectable lutein
structural analog or derivative may be administered with a
zeaxanthin structural analog or derivative and/or other carotenoid
structural analogs or derivatives, or in formulation with
antioxidants and/or excipients that further the intended purpose.
In some embodiments, one or more of the lutein structural analogs
or derivatives are water-soluble.
[0037] In some embodiments, the administration of structural
analogs or derivatives of carotenoids by one skilled in the
art--including consideration of the pharmacokinetics and
pharmacodynamics of therapeutic drug delivery--is expected to
inhibit and/or ameliorate disease conditions associated with
elevated inflammation and elevated CRP. In some of the foregoing
embodiments, analogs or derivatives of carotenoids administered to
a subject may be at least partially water-soluble.
[0038] "Water-soluble" structural carotenoid analogs or derivatives
are those analogs or derivatives that may be formulated in aqueous
solution, either alone or with one or more excipients.
Water-soluble carotenoid analogs or derivatives may include those
compounds and synthetic derivatives that form molecular
self-assemblies, and may be more properly termed "water
dispersible" carotenoid analogs or derivatives. Water-soluble
and/or "water-dispersible" carotenoid analogs or derivatives may be
preferred in some embodiments.
[0039] Water-soluble carotenoid analogs or derivatives may have a
water solubility of greater than about 1 mg/mL in some embodiments.
In certain embodiments, water-soluble carotenoid analogs or
derivatives may have a water solubility of greater than about 5
mg/ml-10 mg/mL. In certain embodiments, water-soluble carotenoid
analogs or derivatives may have a water solubility of greater than
about 20 mg/mL. In certain embodiments, water-soluble carotenoid
analogs or derivatives may have a water solubility of greater than
about 25 mg/mL. In some embodiments, water-soluble carotenoid
analogs or derivatives may have a water solubility of greater than
about 50 mg/mL.
[0040] Certain embodiments may further directed to pharmaceutical
compositions including combinations two or more structural
carotenoid analogs or derivatives. Embodiments directed to
pharmaceutical compositions may further include appropriate
vehicles for delivery of said pharmaceutical composition to a
desired site of action (i.e., the site a subject's body where the
biological effect of the pharmaceutical composition is most
desired). Pharmaceutical compositions including injectable
structural carotenoid analogs or derivatives of astaxanthin, lutein
or zeaxanthin may be particularly advantageous for the methods
described herein. In yet a further embodiment, an injectable
astaxanthin structural analog or derivative may be administered
with a astaxanthin, zeaxanthin or lutein structural analog or
derivative and/or other carotenoid structural analogs or
derivatives, or in formulation with antioxidants and/or excipients
that further the intended purpose. In some embodiments, one or more
of the astaxanthin, lutein or zeaxanthin structural analogs or
derivatives are water-soluble.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The above brief description as well as further objects,
features and advantages of the methods and apparatus of the present
invention will be more fully appreciated by reference to the
following detailed description of presently preferred but
nonetheless illustrative embodiments in accordance with the present
invention when taken in conjunction with the accompanying
drawings.
[0042] FIG. 1 depicts a graphic representation of several examples
of "parent" carotenoid structures as found in nature.
[0043] FIG. 2 depicts a time series of the UV/Vis absorption
spectra of the disodium disuccinate derivative of natural source
lutein in water.
[0044] FIG. 3 depicts a UV/Vis absorption spectra of the disodium
disuccinate derivative of natural source lutein in water
(.lamda..sub.max=443 nm), ethanol (.lamda..sub.max=446 nm), and
DMSO (.lamda..sub.max4=461 nm).
[0045] FIG. 4 depicts a UV/Vis absorption spectra of the disodium
disuccinate derivative of natural source lutein in water
(.lamda..sub.max=442 nm) with increasing concentrations of
ethanol.
[0046] FIG. 5 depicts a time series of the UV/Vis is absorption
spectra of the disodium diphosphate derivative of natural source
lutein in water.
[0047] FIG. 6 depicts a UV/Vis absorption spectra of the disodium
diphosphate derivative of natural source lutein in 95% ethanol
(.lamda..sub.max=446 nm), 95% DMSO (.lamda..sub.max=459 nm), and
water (.lamda..sub.max=428 nm).
[0048] FIG. 7 depicts a UV/Vis absorption spectra of the disodium
diphosphate derivative of natural source lutein in water
(.lamda..sub.max=428 nm) with increasing concentrations of
ethanol.
[0049] FIG. 8 depicts a mean percent inhibition (.+-.SEM) of
superoxide anion signal as detected by DEPMPO spin-trap by the
disodium disuccinate derivative of natural source lutein (tested in
water).
[0050] FIG. 9 depicts a mean percent inhibition (.+-.SEM) of
superoxide anion signal as detected by DEPMPO spin-trap by the
disodium diphosphate derivative of natural source lutein (tested in
water).
[0051] FIG. 10 depicts the chemical structures of three synthetic
water-soluble carotenoid analogs or derivatives according to
certain non-limiting embodiments. (A) disuccinic acid astaxanthin
ester; (B) disodium disuccinic acid ester astaxanthin salt
(Cardax.TM.); and (C) divitamin C disuccinate astaxanthin
ester.
[0052] FIG. 11 depicts the effects of DDA (also known as
"Cardax.TM.") or saline on myocardial infarct size after 30 min of
left anterior descending coronary artery occlusion followed by 3 h
of reperfusion.
[0053] FIG. 12 Depicts the mean plasma and myocardial tissue
concentrations of non-esterified, free astaxanthin (nM) in rabbits
subjected to 30 min of left anterior descending coronary artery
occlusion and 3 h of reperfusion, following 4 daily intravenous
doses of DDA (50 mg/kg).
[0054] FIG. 13 Depicts the effect of DDA administration on serum
levels of a molecular marker of cardiac damage.
[0055] FIG. 14 shows representative fluorescent images of a heart
from a saline control rabbit (A and C) and a rabbit treated with
DDA (B and D) after 30 min of ischemia and 3 h of reperfusion.
[0056] FIG. 15 shows a complement-mediated red blood cell (RBC)
hemolysis assay conducted after DDA administration using human
erythrocytes as the target cell and rabbit plasma drawn after
reperfusion as the source of complement proteins.
[0057] While the invention is susceptible to various modifications
and alternative forms, specific embodiments thereof are shown by
way of example in the drawings and will herein be described in
detail. It should be understood, however, that the drawing and
detailed description thereto are not intended to limit the
invention to the particular form disclosed, but on the contrary,
the intention is to cover all modifications, equivalents and
alternatives falling within the spirit and scope of the present
invention as defined by the appended claims.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
[0058] The terms used throughout this specification generally have
their ordinary meanings in the art, within the context of the
invention, and in the specific context where each term is used.
Certain terms are discussed below, or elsewhere in the
specification, to provide additional guidance to the practitioner
in describing the devices and methods of the invention and how to
make and use them. It will be appreciated that the same thing can
be said in more than one way. Consequently, alternative language
and synonyms may be used for any one or more of the terms discussed
herein, nor is any special significance to be placed upon whether
or not a term is elaborated or discussed in greater detail herein.
Synonyms for certain terms are provided. A recital of one or more
synonyms does not exclude the use of other synonyms. The use of
examples anywhere in this specification, including examples of any
terms discussed herein, is illustrative only, and in no way limits
the scope and meaning of the invention or of any exemplified
term.
[0059] As used herein, the term "xanthophyll carotenoid" generally
refers to a naturally occurring or synthetic 40-carbon polyene
chain with a carotenoid structure that contains at least one
oxygen-containing functional group. The chain may include terminal
cyclic end groups. Exemplary, though non-limiting, xanthophyll
carotenoids include astaxanthin, zeaxanthin, lutein, echinenone,
lycophyll, canthaxanthin, and the like. Non-limiting examples of
carotenoids that are not xanthophyll carotenoids include
.beta.-carotene and lycopene.
[0060] As used herein, terms such as "carotenoid analog" and
"carotenoid derivative" generally refer to chemical compounds or
compositions derived from a naturally occurring or synthetic
carotenoid. Terms such as carotenoid analog and carotenoid
derivative may also generally refer to chemical compounds or
compositions that are synthetically derived from non-carotenoid
based parent compounds; however, which ultimately substantially
resemble a carotenoid derived analog. Non-limiting examples of
carotenoid analogs and derivatives that may be used according to
some of the embodiments described herein are depicted schematically
in FIG. 10.
[0061] As used herein, the term "cell or a group of cells" is meant
to include a single cell or group of cells that are isolated in
culture as well as those cells or groups of cells naturally
residing in a body or as part of a body organ or body tissue. The
term "organ", when used in reference to a part of the body of an
animal or of a human generally refers to the collection of cells,
tissues, connective tissues, fluids and structures that are part of
a structure in an animal or a human that is capable of performing
some specialized function. Groups of organs constitute one or more
specialized body systems. The specialized function performed by an
organ is typically essential to the life or the overall well-being
of the animal or human. Non-limiting examples of body organs
include the heart, lungs, kidney, ureter, urinary bladder, adrenal
glands, pituitary gland, skin, prostate, uterus, reproductive
organs (e.g., genitalia and accessory organs), liver, gall bladder,
brain, spinal cord, stomach, intestine, appendix, pancreas, lymph
nodes, breast, salivary glands, lacrimal glands, eyes, spleen,
thymus, bone marrow. Non-limiting examples of body systems include
the respiratory, circulatory, musculoskeletal, nervous, digestive,
endocrine, exocrine, hepato-biliary, reproductive, and urinary
systems. In animals the organs are generally made up of several
tissues, one of which usually predominates, and determines the
principal function of the organ. The term "tissue", when used in
reference to a part of a body or of an organ, generally refers to
an aggregation or collection of morphologically similar cells and
associated accessory cells and intercellular matter, including
extracellular matrix material and fluids, acting together to
perform specific functions in the body. There are generally four
basic types of tissue in animals and humans including muscle,
nerve, epithelial, and connective tissues.
[0062] As used herein, the term "organ", when used in reference to
a part of the body of an animal or of a human generally refers to
the collection of cells, tissues, connective tissues, fluids and
structures that are part of a structure in an animal or a human
that is capable of performing some specialized physiological
function. Groups of organs constitute one or more specialized body
systems. The specialized function performed by an organ is
typically essential to the life or to the overall well-being of the
animal or human. Non-limiting examples of body organs include the
heart, lungs, kidney, ureter, urinary bladder, adrenal glands,
pituitary gland, skin, prostate, uterus, reproductive organs (e.g.,
genitalia and accessory organs), liver, gall-bladder, brain, spinal
cord, stomach, intestine, appendix, pancreas, lymph nodes, breast,
salivary glands, lacrimal glands, eyes, spleen, thymus, bone
marrow. Non-limiting examples of body systems include the
respiratory, circulatory, cardiovascular, lymphatic, immune,
musculoskeletal, nervous, digestive, endocrine, exocrine,
hepato-biliary, reproductive, and urinary systems. In animals, the
organs are generally made up of several tissues, one of which
usually predominates, and determines the principal function of the
organ.
[0063] As used herein, the term "tissue", when used in reference to
a part of a body or of an organ, generally refers to an aggregation
or collection of morphologically similar cells and associated
accessory and support cells and intercellular matter, including
extracellular matrix material, vascular supply, and fluids, acting
together to perform specific functions in the body. There are
generally four basic types of tissue in animals and humans
including muscle, nerve, epithelial, and connective tissues.
[0064] As used herein, terms such as "deposition in a tissue,"
"tissue deposition," "tissue accumulation," or the like generally
refer to the accumulation of a particular factor or a group of
factors in tissue. The factor(s) deposited in the tissue may be
soluble or carried to the tissue as a suspended factor in plasma. A
factor may also be deposited by other cells. Once immobilized in
the tissue; a deposited factor may carry out any number of
physiological or pathological functions. Examples of factors that
may be deposited in tissues include acute phase proteins,
sediments, immune complexes, pathogens, hormones and the like.
[0065] As used herein the term "ischemia-reperfusion injury" is
generally defined as the pathology attributed to reoxygenation of
previously ischemic tissue (either chronically or acutely
ischemic), which includes atherosclerotic and thromboembolic
vascular disease and its related illnesses. In particular, major
diseases or processes including myocardial infarction, stroke,
peripheral vascular disease, venous or arterial occlusion and/or
restenosis, organ transplantation, coronary artery bypass graft
surgery, percutaneous transluminal coronary angioplasty, and
cardiovascular arrest and/or death are included, but are not seen
as limiting for other pathological processes which involve
reperfusion of ischemic tissue in their individual pathologies.
[0066] As used herein, terms such as "inflammation," "inflammatory
response," or the like, generally refer to an important biological
process that is a component of the immune system. Inflammation is
the first response of the immune system to infection or irritation
in a body tissue and may be referred to as the innate cascade.
Inflammation may generally be characterized as causing a tissue to
have one or more of the following charateristics: redness
("rubor"), heat ("calor"), swelling, pain ("dolor") and dysfunction
of the organs involved. Though inflammation is an important
component of innate immunity, if left unabated, it may result
severe and sometimes irreparable tissue damage. Low levels of
inflammation that persist through time without resolution
("chronic" or "smoldering" inflammation) are now recognized as an
important pathological component of many diseases, in particular
cardiovascular disease. Inflammation also contributes to the
pathophysiology of numerous disorders such as, for example, tissue
reperfusion injury following myocardial infacrtion, system lupus
erythematosis, Crohn's disease, and the like.
[0067] As used herein, the term "inflammatory disorder" generally
refers to inflammatory disorders that may be treated using the
methods comtemplated herein may include those disorders that are
characterized by aberrant or otherwise dysregulated, prolonged or
inappropriate inflammatory responses, such as, for example,
colorectal cancer; cardiovascular disease; ischemic reperfusion
injury; Rheumatoid arthritis; Osteoarthritis; Inflammatory
arthropathies (e.g., ankylosing spondylitis, psoriatic arthritis,
Reiter's syndrome); Acute gout; Dysmenorrhoea; Metastatic bone
pain; Headache and migraine; Postoperative pain; Mild-to-moderate
pain due to inflammation and tissue injury; Pyrexia; Renal colic. A
subset of inflammatory disorder may be due, at least in part, to
abberant activation of the complement system. The complement system
may contribute to the pathophysiology of certain diseases with an
immunological/inflammatory component, such as Alzheimer's disease,
asthma, systemic lupus erythematosus, diabetes mellitus,
glomerulonephritis, Crohn's disease, atherosclerosis, various forms
of arthritis (e.g. osteoarthritis and rheumatoid arthritis),
autoimmune heart disease, multiple sclerosis and Age-Related
Macular degeneration (ARMD). Deficiencies in terminal pathway
components predispose patients to certain autoimmune diseases and
infections (particularly meningitis).
[0068] As used herein, terms such Vas "complement," "complement
system, complement cascade," "complement pathway," or the like,
when used in reference to a group of immunologically active
polypeptides, generally refers to one or more of about 30 distinct
art-recognized plasma proteins that function together as a
component of the innate immune system of an organism. The terms may
also be used by the skilled artisan to refer to the biochemical
reactions that occur between various protein members of the
complement system to initiate one or more components of an innate
(e.g., formation of the membrane-attack complex or MAC) or cellular
immune response (e.g., opsonization).
[0069] As used herein, the term "membrane-attack complex" or "MAC"
generally refers to a multi-subunit macromolecular complex that is
formed on the membrane of a target cell by the multimerization of
terminal complement system components (in particular complement
proteins C5b through C9). Assembly of the components into a MAC on
the surface of a cell results in the formation of a
membrane-spanning hydrophilic pore, and ultimately in the lysis and
destruction of the cell on which the MAC formed. Although the
evolutionary function of the formation of the terminal MAC appears
to be protection against foreign invaders (i.e. infectious
disease), in a form of molecular mimicry, normal cells exposed to
this innate immune system can be destroyed (a form of auto-immune
disease).
[0070] As used herein, the term "complement-mediated lysis"
generally refers to the biological process described above, whereby
one or more activated complement system proteins ultimately
compromise the integrity of the plasma membrane of a cell.
[0071] As used herein, the term "C-reactive protein" or "CRP"
generally refers to an acute phase protein synthesized
predominantly in the liver, as well as in other cells locally such
as endothelial cells, in response to inflammation.
[0072] As used herein, the term "complement factor H," or "CFH"
generally refers to a roughly 155 kDa plasma glycoprotein that is a
key regulator of the complement system.
[0073] The term "modulate," as used herein, generally refers to a
change or an alteration in a biological parameter. Examples
biological parameters subject to modulation according to certain
embodiments described herein may include, by way of non-limiting
example only: inflammation, complement activation, MAC tissue
deposition, CRP tissue deposition, changes in protein or gene
expression, complement-mediated cellular lysis, tissue damage
associated with ischemia/reperfusion injury or the initiation or
progression of an inflammatory reaction, or the like. "Modulation"
may refer to a net increase or a net decrease in the biological
parameter.
[0074] As used herein the terms "inhibiting," "reducing,"
"ameliorating," and the like, when used in the context of
modulating a pathological or disease state, generally refers to the
prevention and/or reduction of at least a portion of the negative
consequences of the disease state. When used in the context of
biochemical pathway or of protein function, the term "inhibiting"
generally refers to a net reduction in the activity of the pathway
or function.
[0075] As used herein, the term "systemically," when used in the
context of a physiological parameter, generally refers to a
parameter that affects the entire body of a subject, or to a
particular body system.
[0076] As used herein the terms "administration," "administering,"
or the like, when used in the context of providing a pharmaceutical
or nutraceutical composition to a subject generally refers to
providing to the subject one or more pharmaceutical,
"over-the-counter" (OTC) or nutraceutical compositions in
combination with an appropriate delivery vehicle by any means such
that the administered compound achieves one or more of the intended
biological effects for which the compound was administered. By way
of non-limiting example, a composition may be administered
parenteral, subcutaneous, intravenous, intracoronary, rectal,
intramuscular, intra-peritoneal, transdermal, or buccal routes of
delivery. Alternatively, or concurrently, administration may be by
the oral route. The dosage administered will be dependent upon the
age, health, weight, and/or disease state of the recipient, kind of
concurrent treatment, if any, frequency of treatment, and/or the
nature of the effect desired. The dosage of pharmacologically
active compound that is administered will be dependent upon
multiple factors, such as the age, health, weight, and/or disease
state of the recipient, concurrent treatments, if any, the
frequency of treatment, and/or the nature and magnitude of the
biological effect that is desired.
[0077] As used herein, the term "treat" generally refers to an
action taken by a caregiver that involves substantially inhibiting,
slowing or reversing the progression of a disease, disorder or
condition, substantially ameliorating clinical symptoms of a
disease disorder or condition, or substantially preventing the
appearance of clinical symptoms of a disease, disorder or
condition.
[0078] As used herein, terms such as "pharmaceutical composition,"
"pharmaceutical formulation," "pharmaceutical preparation," or the
like, generally refer to formulations that are adapted to deliver a
prescribed dosage of one or more pharmacologically active compounds
to a cell, a group of cells, an organ or tissue, an animal or a
human. Methods of incorporating pharmacologically active compounds
into pharmaceutical preparations are widely known in the art. The
determination of an appropriate prescribed dosage of a
pharmacologically active compound to include in a pharmaceutical
composition in order to achieve a desired biological outcome is
within the skill level of an ordinary practitioner of the art. A
pharmaceutical composition may be provided as sustained-release or
timed-release formulations. Such formulations may release a bolus
of a compound from the formulation at a desired time, or may ensure
a relatively constant amount of the compound present in the dosage
is released over a given period of time. Terms such as "sustained
release" or "timed release" and the like are widely used in the
pharmaceutical arts and are readily understood by a practitioner of
ordinary skill in the art. Pharmaceutical preparations may be
prepared as solids, semi-solids, gels, hydrogels, liquids,
solutions, suspensions, emulsions, aerosols, powders, or
combinations thereof. Included in a pharmaceutical preparation may
be one or more carriers, preservatives, flavorings, excipients,
coatings, stabilizers, binders, solvents and/or auxiliaries that
are, typically, pharmacologically inert. It will be readily
appreciated by an ordinary practitioner of the art that,
pharmaceutical compositions, formulations and preparations may
include pharmaceutically acceptable salts of compounds. It will
further be appreciated by an ordinary practitioner of the art that
the term also encompasses those pharmaceutical compositions that
contain an admixture of two or more pharmacologically active
compounds, such compounds being administered, for example, as a
combination therapy.
[0079] The term "pharmaceutically acceptable salts" includes salts
prepared from by reacting pharmaceutically acceptable non-toxic
bases or acids, including inorganic or organic bases, with
inorganic or organic acids. Pharmaceutically acceptable salts may
include salts derived from inorganic bases include aluminum,
ammonium, calcium, copper, ferric, ferrous, lithium, magnesium,
manganic salts, manganous, potassium, sodium, zinc, etc. Examples
include the ammonium, calcium, magnesium, potassium, and sodium
salts. Salts derived from pharmaceutically acceptable organic
non-toxic bases include salts of primary, secondary, and tertiary
amines, substituted amines including naturally occurring
substituted amines, cyclic amines, and basic ion exchange resins,
such as arginine, betaine, caffeine, choline,
N,N'-dibenzylethylenediamine, diethylamine,
2-dibenzylethylenediamine, 2-diethylaminoethanol,
2-dimethylaminoethanol, ethanolamine, ethylenediamine,
N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,
histidine, hydrabamine, isopropylamine, lysine, methylglucamine,
morpholine, piperazine, piperidine, polyamine resins, procaine,
purines, theobromine, triethylamine, trimethylamine,
tripropylamine, tromethamine, etc.
[0080] As used herein the terms "subject" generally refers to a
mammal, and in particular to a human.
[0081] Terms such as "in need of treatment," "in need thereof,"
"benefit from such treatment," and the like, when used in the
context of a subject being administered a pharmacologically active
composition, generally refers to a judgment made by an appropriate
healthcare provider that an individual or animal requires or will
benefit from a specified treatment or medical intervention. Such
judgments may be made based on a variety of factors that are in the
realm of expertise of healthcare providers, but include knowledge
that the individual or animal is ill, will be ill, or is at risk of
becoming ill, as the result of a condition that may be ameliorated
or treated with the specified medical intervention.
[0082] As used herein, the term "additional anti-inflammatory
agent" generally refers to a pharmacologically active drug or
composition that may be co-administered with the subject carotenoid
analogs or derivatives, and whose primary biological function is to
inhibit, reduce or ameliorate at least a subset of symptoms
associated with inflammation. Anti-inflammatory drugs may generally
be divided into two broad categories; steroidal anti-inflammatory
drugs; and non-steroidal anti-inflammatory drugs. A "steroidal
anti-inflammatory drug" may generally refer to a
naturally-occurring glucocorticoid (e.g., cortisol; hydrocortisone)
or one or more synthetic glucocorticoids. Non-limiting examples of
glucocorticoids include Prednisone; Prednisolone;
Methylprednisolone; Meprednisone; Triamcicolone; Paramethasone;
Fluprednisolone; Betamethasone; Dexamethasone; and
Fludrocortisone.
[0083] Non-steroidal anti-inflammatory drugs, usually abbreviated
to NSAIDs, are drugs with analgesic, antipyretic and
anti-inflammatory effects--they reduce pain, fever and
inflammation. The term "non-steroidal" is used to distinguish these
drugs from steroids, which (amongst a broad range of other effects)
have a similar eicosanoid-depressing, anti-inflammatory action.
NSAIDs are sometimes also referred to as non-steroidal
anti-inflammatory agents/analgesics (NSAIAs). Most NSAIDs act as
non-selective inhibitors of the enzyme cyclooxygenase, inhibiting
both the cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2)
isoenzymes. Cyclooxygenase catalyses the formation of
prostaglandins and thromboxane from arachidonic acid (itself
derived from the cellular phospholipid bilayer by phospholipase
A.sub.2). Non-limiting examples of some NSAIDS used in certain
clinical setting for the treatment or reduction of sympatoms
associated with inflammation include, though are not limited to,
Aspirin; Diclofenac; Diflunisal; Etodolac; Fenoprofen;
Floctafenine; Flurbiprofen; Ibuprofen; Indomethacin; Ketorolac;
Ketoprofen; Meclofenamate; Mefenamic Acid; Meloxicam; Nabumetone;
Naproxen; Nimesulide; Oxaprozin; Phenylbutazone; Piroxicam;
Salsalate; Sulindac; Tenoxicam; Tiaprofenic Acid; Tolmetin;
Celecoxib; rofecoxib; etoricoxib; and valdecoxib.
[0084] By "therapeutically effective amount" is meant an amount of
a drug or pharmaceutical composition that will elicit at least one
desired biological or physiological response of a cell, a tissue, a
system, animal or human that is being sought by a researcher,
veterinarian, physician or other caregiver.
[0085] By "prophylactically effective amount" is meant an amount of
a pharmaceutical composition that will substantially prevent, delay
or reduce the risk of occurrence of the biological or physiological
event in a cell, a tissue, a system, animal or human that is being
sought by a researcher, veterinarian, physician or other
caregiver.
[0086] Terms such as "pharmaceutically inert," "pharmacologically
inert," or the like, as used herein, generally refers to a
compound, additive, binder, vehicle, and the like, that is
substantially free of any pharmacologic or "drug-like"
activity.
[0087] A "pharmaceutically or nutraceutically acceptable
formulation," as used herein, generally refers to a non-toxic
formulation containing a predetermined dosage of a pharmaceutical
and/or nutraceutical composition, wherein the dosage of the
pharmaceutical and/or nutraceutical composition is adequate to
achieve a desired biological outcome. The meaning of the term may
generally include an appropriate delivery vehicle that is suitable
for properly delivering the pharmaceutical composition in order to
achieve the desired biological outcome.
[0088] As used herein the term "antioxidant" may be generally
defined as any of various substances (such as beta-carotene,
vitamin C, and .alpha.-tocopherol) that inhibit oxidation or
reactions promoted by Reactive Oxygen Species (ROS) and other
radical and non-radical species.
[0089] As used herein the term "co-antioxidant" may be generally
defined as an antioxidant that is used and that acts in combination
with another antioxidant (e.g., two antioxidants that are
chemically and/or functionally coupled, or two antioxidants that
are combined and function with each another in a pharmaceutical
preparation). The effects of co-antioxidants may be additive (i.e.,
the anti-oxidative potential of one or more anti-oxidants acting
additively is approximately the sum of the oxidative potential of
each component anti-oxidant) or synergistic (i.e., the
anti-oxidative potential of one or more anti-oxidants acting
synergistically may be greater than the sum of the oxidative
potential of each component anti-oxidant).
[0090] The terms "Rn" in a chemical formula refer to hydrogen or a
functional, group, each independently selected, unless stated
otherwise. In some embodiments the functional group may be an
organic group. In some embodiments the functional group may be an
alkyl group. In some embodiments, the functional group may be a
hydrophobic or hydrophilic group.
[0091] Compounds described herein embrace isomers mixtures,
racemic, optically active, and optically inactive stereoisomers and
compounds.
The Complement System and Inflammation in Tissue Injury
[0092] The complement system plays an important role in host
defense mechanisms against infectious agents and in the
inflammatory response. Under normal physiological conditions,
complement proteins exist in body fluids in a latent, or inactive
state. In the presence of a pathogen or of an activating stimulus,
such as for example a localized inflammatory response, or the in
the presence of damaged or apoptotic cells, the proteins react with
one another and with surrounding molecules to activate the
complement system, resulting ultimately in the formation of the
membrane attack complex. It is generally known in the art that
three biochemical pathways may activate complement: the classical,
alternative, and mannose-binding lectin pathways. Complement
proteins C1-C9 are the major components of the classical activation
cascade which is most commonly initiated by binding of C1q to
initiator molecules. Regardless of the mechanism by which the
complement system is activated, all three pathways converge at the
formation of complement proteins C3 and C4. Activation of C3 and C4
ultimately results in the recruitment and activation of the
terminal complement proteins C5-C9 and formation of the MAC at the
site of the response Together, the 5-C9 and the MAC are highly
pro-inflammatory. It is believed that aberrant activation or
function of the complement system might contribute to the
pathophysiology of many diseases with an immune component, such as
Alzheimer's disease, asthma, systemic lupus erythematosus, diabetes
mellitus, glomerulonephritis, Crohn's disease, atherosclerosis,
various forms of arthritis (e.g. osteoarthritis and rheumatoid
arthritis), autoimmune heart disease, multiple sclerosis and Age
Related Macular degeneration, (ARMD). Deficiencies in terminal
pathway components predispose patients to certain autoimmune
diseases and infections (particularly meningitis).
[0093] CRP is known to be a highly sensitive, but nonspecific,
marker of inflammation. CRP was initially discovered due to its
ability to react with the C-polysaccharide of the pathogen
pneumococcus. In addition to C-protein, additional ligands such as
phosphocholine and other phospholipids, have been shown to target
CRP to sites of infection or pathology. In addition to interacting
with various phospholipid ligands, CRP associates with numerous
polypeptides. For example, it has been demonstrated that CRP can
activate the classical complement pathway by binding to complement
protein C1q, stimulate phagocytosis, and bind to immunoglobulin
receptors (Fc.gamma.R), through which it is though to affect the
humoral response to disease. It is thought that CRP facilitates
complement binding to invading foreign pathogens and to the surface
of damaged cells and tissues, thus targetting them for clearance by
the innate and adaptive immune system. Recent evidence suggests
that chronic elevated levels of circulating CRP are correlated with
an increased risk of developing cardiovascular disease later in
life.
[0094] Tissue injury following certain pathological insults such
as, for example, induction of an inflammatory response,
ischemia-reperfusion injury or other pathological processes that
result in localized tissue necrosis and cell damage may result in
the deposition of acute phase inflammatory molecules such as CRP
and activated complement proteins or the MAC at the site of injury.
Deposition of acute inflammatory mediators and complement system
proteins, particularly activated C3b and C5b-C9, at these sites may
accelerate tissue damage, at least in part by allowing the
formation of damaging immune complexes, recruiting leukocytes and
other pro-inflammatory cells to the site of injury, and worsening
cell membrane damage by promoting MAC formation on the surface of
the cell (reviewed in Black et al., 2004, J. Biol. Chem., Vol. 279,
pp 48487-48494, which is incorporated herein by reference). Under
normal physiological conditions, the CRP phospholipid ligands
phosphocholine, phosphatidylcholine, phosphatidylserine, and the
like are not exposed on the surface of cells. Following an
injurious insult to a tissue, such as for example a localized
inflammatory response, damaged or apoptotic cells expose these
phospholipids on their surface. The phospholipids ligands may then
be accessible to circulating CRP, which itself is expressed at high
levels during the acute phase of an inflammatory response,
resulting in the recruitment and deposition of ligand-bound CRP at
the site of injury. Ligand-bound or aggregated CRP efficiently
activates the classical complement pathway by interacting directly
with complement protein C1q. Formation of a biochemical complex
between ligand-bound CRP and C1q activates the complement cascade,
resulting ultimately in the formation of C3 and C4, which assembles
in a fashion similar to that initiated by antibody-antigen
complexes.
[0095] In an embodiment, administering a carotenoid analog or
derivative to a subject may reduce the amount of CRP that is
deposited in a body tissue during an inflammatory response.
Reducing the amount of CRP recruited to inflamed or otherwise
damaged tissue by administering the carotenoid analogs or
derivatives embodied herein may at least partially inhibit aberrant
activation of terminal complement proteins C5b-C9 and/or formation
and deposition of the MAC in the damaged tissue. Reducing tissue
deposition of CRP and or terminal complement proteins C5b-C9 and
the MAC may prevent more severe or irreversible damage to the
tissue and may allow the subject's body to begin appropriate
regenerative and/or healing processes.
[0096] Age-related macular degeneration (ARMD) is thought to be the
result of a lifetime of oxidative insult that results in
photoreceptor death within the macula. Recent studies have
demonstrated the utility of lutein-based supplementation for the
clinical improvement of vision, reduction of ultraviolet (UV)-based
inflammation, and potentially the inhibition and/or amelioration of
age-related macular degeneration (ARMD).
[0097] Recent studies have also implicated the involvement of
complement system components and their regulator proteins in
contributing to the pathophysiology of macular degeneration,
including ARMD. Various components of the terminal complement
system, including complement proteins C5b-C9, have been identified
in deposits in the macula and surrounding tissues of patients with
ARMD, where the complexes were observed in Bruch's membrane, the
intercapillary pillars, and within drusen. The observation of
complement components in drusen and in the macula and in supporting
tissues in both humans and mice suggests that aberrant inflammatory
responses, including inappropriate activation of complement or its
regulatory components, contribute to the pathophysiology and
progression of macular degeneration, including ARMD.
[0098] CFH binds to numerous proteins in the serum and on the
surface of cells or in the interstitium, including C3b, CRP,
heparin, and sialic acid-rich polyanions. Under normal
physiological conditions, binding of CFH to activated complement
proteins, in particular to C3, on the surface of cells and in the
circulation serves to negatively regulate the activation and
activity of terminal complement components C5-C9. Additionally, CFH
is generally thought to attenuate CRP-dependent activation of the
classical complement pathway on the surface of ostensibly normal
cells or on cells that have not undergone irreversible damage. CFH
function, at least in part, by binding to and inhibiting soluble or
cell surface-bound complement protein C3b, thus inhibiting MAC
formation on the surface cells and preventing their lysis. Aberrant
CFH function has been implicated in contributing to the
pathophysiology of numerous inflammatory disorders including, but
not limited to, type-II diabetes mellitus, Alzheimer's disease,
rheumatoid arthritis, atherosclerosis, and human type II
membranoproliferative glomerulonephritis (MPGNII) (reviewed in de
Cordoba et al., Molecular Immunology Vol. 41, 2004, pp. 355-367,
which is incorporated herein by reference). More recently, a strong
association between the risk of developing ARMD and the presence of
a common polymorphism of the CFH gene has been identified. The most
prevalent ARMD risk allele identified encodes a CFH variant bearing
a Tyr402.fwdarw.His substitution (Klein et al., published online 10
Mar. 2005; 10.1126/science.1109557; Haines et al., published online
10 Mar. 2005; 10.1126/science.1110359; Edwards et al., published
online 10 Mar. 2005; 10.1126/science.1110189, all of which are
incorporated herein by reference). The amino acid 402 of CFH maps
to a region of the protein that interacts with CRP and heparin.
Substitution of the neutral amino acid Tyr with a positively charge
His residue is thought to affect the ability of CFH to bind to CRP
or heparin. Therefore, in subjects carrying the ARMD risk allele,
impaired binding of CFH to CRP may result in an inability of CFH to
attenuate MAC formation on the surface of damaged tissues of the
macular and surrounding areas, resulting in a net increase in the
tissue inflammation and damage. Therapeutic agents that affect
deposition of CRP or the MAC in tissues of the macula and
surrounding areas would therefore be useful to treat patients with
macular degeneration, including ARMD.
[0099] In an embodiment, administration of the carotenoid analogs
or derivatives described herein to a subject who is developing or
who is at risk of developing ARMD may reduce inflammation
associated with complement activation in the macula. Reducing
inflammation in the macula by administering carotenoid analogs or
derivatives may be associated with reduced deposition of CRP and/or
terminal complement proteins C5b-C9 and the MAC in the macula and
in the surrounding tissues of the in Bruch's membrane, the
intercapillary pillars, and within drusen. Reducing inflammation in
the macula by administering carotenoid analogs or derivatives may
be associated with a reduction in tissue damage and may improve
visual acuity and/or halt the deterioration of visual acuity.
[0100] Reduction in complement activation by administering the
carotenoid analogs or derivatives described herein is not limited
to that portion of complement that is acting on the surface of
cells in body tissues. On the contrary, the carotenoid analogs and
derivatives described herein may inhibit or reduce activation of
soluble complement components in the blood plasma or in other body
fluids of subjects who have been administered the compounds. Such
reduction or inhibition of complement system activity in subjects
may result in reduced complement-mediated lysis and cellular damage
in tissues or in cells suspended in fluids.
[0101] In some embodiments, carotenoid analogs or derivatives may
be employed in "self-formulating" aqueous solutions, in which the
compounds spontaneously self-assemble into macromolecular
complexes. These complexes may provide stable formulations in terms
of shelf life. The same formulations may be parenterally
administered, upon which the spontaneous self-assembly is overcome
by interactions with serum and/or tissue components in vivo.
[0102] Some specific embodiments may include phosphate derivatives,
succinate derivatives, co-antioxidant derivatives (e.g., Vitamin C,
Vitamin C analogs, Vitamin C derivatives, Vitamin E, Vitamin E
analogs, Vitamin E derivatives, flavonoids, flavonoid analogs, or
flavonoid derivatives), or combinations thereof derivatives or
analogs of carotenoids. Flavonoids may include, for example,
quercetin, xanthohumol, isoxanthohumol, or genistein. Derivatives
or analogs may be derived from any known carotenoid (naturally or
synthetically derived). Specific examples of naturally occurring
carotenoids which compounds described herein may be derived from
include for example zeaxanthin, lutein, lycophyll, astaxanthin, and
lycopene.
[0103] In some embodiments, one or more co-antioxidants may be
coupled to a carotenoid or carotenoid derivative or analog.
[0104] The synthesis of water-soluble and/or water-dispersible
carotenoids (e.g., C40) analogs or derivatives--as potential
parenteral agents for clinical applications may improve the
injectability of these compounds as therapeutic agents, a result
perhaps not achievable through other formulation methods. The
methodology may be extended to carotenoids with fewer than 40
carbon atoms in the molecular skeleton and differing ionic
character. The methodology may be extended to carotenoids with
greater than 40 carbon atoms in the molecular skeleton. The
methodology may be extended to non-symmetric carotenoids. The
aqueous dispersibility of these compounds allows proof-of-concept
studies in model systems (e.g. cell culture), where the high
lipophilicity of these compounds previously limited their
bioavailability and hence proper evaluation of efficacy.
Esterification or etherification may be useful to increase oral
bioavailability, a fortuitous side effect of the esterification
process, which can increase solubility in gastric mixed micelles.
The net overall effect is an improvement in potential clinical
utility for the lipophilic carotenoid compounds as therapeutic
agents.
[0105] In some embodiments, the principles of retrometabolic drug
design may be utilized to produce novel soft drugs from the
asymmetric parent carotenoid scaffold (e.g., RRR-lutein
(.beta.,.epsilon.-carotene-3,3'-diol)). For example, lutein
scaffold for derivatization was obtained commercially as purified
natural plant source material, and was primarily the
RRR-stereoisomer (one of 8 potential stereoisomers). Lutein (Scheme
1) possesses key characteristics--similar to starting material
astaxanthin--which make it an ideal starting platform for
retrometabolic syntheses: (1) synthetic handles (hydroxyl groups)
for conjugation, and (2) an excellent safety profile for the parent
compound. As stated above, lutein is available commercially from
multiple sources in bulk as primarily the RRR-stereoisomer, the
primary isomer in the human diet and human retinal tissue.
[0106] In some embodiments, carotenoid analogs or derivatives may
have increased water solubility and/or water dispersibility
relative to some or all known naturally occurring carotenoids.
Contradictory to previous research, improved results are obtained
with derivatized carotenoids relative to the base carotenoid,
wherein the base carotenoid is derivatized with substituents
including hydrophilic substituents and/or co-antioxidants.
[0107] In some embodiments, the carotenoid derivatives may include
compounds having a structure including a polyene chain (i.e.,
backbone of the molecule). The polyene chain may include between
about 5 and about 15 unsaturated bonds. In certain embodiments, the
polyene chain may include between about 7 and about 12 unsaturated
bonds. In some embodiments a carotenoid derivative may include 7 or
more conjugated double bonds to achieve acceptable antioxidant
properties.
[0108] In some embodiments, decreased antioxidant properties
associated with shorter polyene chains may be overcome by
increasing the dosage administered to a subject or patient.
[0109] In some embodiments, a chemical compound including a
carotenoid derivative or analog may have the general structure
(126): ##STR9## Each R.sup.11 may be independently hydrogen or
methyl. R.sup.9 and R.sup.10 may be independently H, an acyclic
alkene with one or more substituents, or a cyclic ring including
one or more substituents. y may be 5 to 12. In some embodiments, y
may be 3 to 15. In certain embodiments, the maximum value of y may
only be limited by the ultimate size of the chemical compound,
particularly as it relates to the size of the chemical compound and
the potential interference with the chemical compound's biological
availability as discussed herein. In some embodiments, substituents
may be at least partially hydrophilic. These carotenoid derivatives
may be included in a pharmaceutical composition.
[0110] In some embodiments, the carotenoid derivatives may include
compounds having the structure (128): ##STR10## Each R.sup.11 may
be independently hydrogen, methyl, alkyl, alkenyl, or aromatic
substituents. R.sup.9 and R.sup.10 may be independently H, an
acyclic alkene with at least one substituent, or a cyclic ring with
at least one substituent having general structure (130): ##STR11##
where n may be between 4 to 10 carbon atoms. W is the
substituent.
[0111] In some embodiments, each cyclic ring may be independently
two or more rings fused together to form a fused ring system (e.g.,
a bi-cyclic system). Each ring of the fused ring system may
independently contain one or more degrees of unsaturation. Each
ring of the fused ring system may be independently aromatic. Two or
more of the rings forming the fused ring system may form an
aromatic system.
[0112] In some embodiments, a chemical composition may include a
carotenoid derivative having the structure ##STR12## Each R.sup.3
may be independently hydrogen or methyl. R.sup.1 and R.sup.2 may be
a cyclic ring including at least one substituent. Each cyclic ring
may be independently: ##STR13## W is the substituent. In some
embodiments R.sup.1 and R.sup.2 may be an acyclic group including
at least one substituent. Each acyclic may be: ##STR14##
[0113] In some embodiments, a chemical composition may include a
carotenoid derivative having the structure ##STR15## R.sup.1 and
R.sup.2 may be a cyclic ring including at least one substituent.
Each cyclic ring may be independently: ##STR16## where W is the
substituent. In some embodiments R.sup.1 and R.sup.2 may be an
acyclic group including at least one substituent. Each acyclic
group may be: ##STR17##
[0114] In some embodiments, a method of treating or reducing tissue
damage associated with an inflammatory response may include
administering to the subject an effective amount of a
pharmaceutically acceptable formulation including a synthetic
analog or derivative of a carotenoid. The synthetic analog or
derivative of the carotenoid may have the structure ##STR18## At
least one substituent W may independently include ##STR19## or a
co-antioxidant. Each R' may be CH.sub.2. n may range from 1 to 9.
Each R may be independently H, alkyl, aryl, benzyl, Group IA metal,
or a co-antioxidant. Each co-antioxidant may be independently
Vitamin C, Vitamin C analogs, Vitamin C derivatives, Vitamin E,
Vitamin E analogs, Vitamin E derivatives, flavonoids, flavonoid
analogs, or flavonoid derivatives. Flavonoids may include, for
example, quercetin, xanthohumol, isoxanthohumol, or genistein.
[0115] Vitamin E may generally be divided into two categories
including tocopherols having a general structure ##STR20##
Alpha-tocopherol is used to designate when
R.sup.1.dbd.R.sup.2.dbd.CH.sub.3. Beta-tocopherol is used to
designate when R.sup.1.dbd.CH.sub.3 and R.sup.2.dbd.H.
Gamma-tocopherol is used to designate when R.sup.1.dbd.H and
R.sup.2.dbd.CH.sub.3. Delta-tocopherol is used to designate when
R.sup.1.dbd.R.sup.2.dbd.H.
[0116] The second category of Vitamin E may include tocotrienols
having a general structure ##STR21## Alpha-tocotrienol is used to
designate when R.sup.1.dbd.R.sup.2.dbd.CH.sub.3. Beta-tocotrienol
is used to designate when R.sup.1.dbd.CH.sub.3 and R.sup.2.dbd.H.
Gamma-tocotrienol is used to designate when R.sup.1.dbd.H and
R.sup.2.dbd.CH.sub.3. Delta-tocotrienol is used to designate when
R.sup.1.dbd.R.sup.2.dbd.H.
[0117] Quercetin, a flavonoid, may have the structure ##STR22##
[0118] In some embodiments, the carotenoid analog or derivative may
have the structure ##STR23## Each R may be independently H, alkyl,
aryl, benzyl, Group IA metal, or a co-antioxidant. Each
co-antioxidant may be independently Vitamin C, Vitamin C analogs,
Vitamin C derivatives, Vitamin E, Vitamin E analogs, Vitamin E
derivatives, flavonoids, flavonoid analogs, or flavonoid
derivatives. Flavonoids may include, for example, quercetin,
xanthohumol, isoxanthohumol, or genistein.
[0119] In some embodiments, the carotenoid analog or derivative may
have the structure ##STR24## Each R may be independently H, alkyl,
aryl, benzyl, Group IA metal (e.g., sodium), or a co-antioxidant.
Each co-antioxidant may be independently Vitamin C, Vitamin C
analogs, Vitamin C derivatives, Vitamin E, Vitamin E analogs,
Vitamin E derivatives, flavonoids, flavonoid analogs, or flavonoid
derivatives. Flavonoids may include, for example, quercetin,
xanthohumol, isoxanthohumol, or genistein. When R includes Vitamin
C, Vitamin C analogs, or Vitamin C derivatives, some embodiments
may include carotenoid analogs or derivatives having the structure
##STR25## Each R may be independently H, alkyl, aryl, benzyl, or
Group IA metal.
[0120] In some embodiments, a chemical compound including a
carotenoid derivative may have the general structure (132):
##STR26## Each R.sup.11 may be independently hydrogen or methyl.
Each R.sup.14 may be independently O or H.sub.2. Each R may be
independently OR.sup.12 or R.sup.12. Each R.sup.12 may be
independently-alkyl-NR.sup.13.sub.3.sup.+,
-aromatic-NR.sup.13.sub.3.sup.+, -alkyl-CO.sub.2,
-aromatic-CO.sub.2', -amino acid-NH.sub.3.sup.+, -phosphorylated
amino acid-NH.sub.3.sup.+, polyethylene glycol, dextran, H, alkyl,
co-antioxidant (e.g. Vitamin C, Vitamin C analogs, Vitamin C
derivatives, Vitamin E, Vitamin E analogs, Vitamin E derivatives,
flavonoids, flavonoid analogs, or flavonoid derivatives), or aryl.
Each R.sup.13 may be independently H, alkyl, or aryl. z may range
from 5 to 12. In some embodiments, z may range from about 3 to
about 15. In certain embodiments, the maximum value of z may only
be limited by the ultimate size of the chemical compound,
particularly as it relates to the size of the chemical compound and
the potential interference with the chemical compound's biological
availability as discussed herein. In some embodiments, substituents
may be at least partially hydrophilic. These carotenoid derivatives
may be used in a pharmaceutical composition.
[0121] In some embodiments, a chemical compound including a
carotenoid derivative may have the general structure (134):
##STR27## Each R.sup.11 may be independently hydrogen or methyl.
Each R.sup.14 may be independently O or H.sub.2. Each X may be
independently ##STR28## alkyl-NR.sup.12.sub.3.sup.+,
-aromatic-NR.sup.12.sub.3.sup.+, -alkyl-CO.sub.2.sup.-,
-aromatic-CO.sub.2.sup.-, -amino acid-NH.sub.3.sup.+,
-phosphorylated amino acid-NH.sub.3.sup.+, polyethylene glycol,
dextran, alkyl, Group IA metal, co-antioxidant (e.g. Vitamin C,
Vitamin C analogs, Vitamin C derivatives, Vitamin E, Vitamin E
analogs, Vitamin E derivatives, flavonoids, flavonoid analogs, or
flavonoid derivatives), or aryl. Each R.sup.12 is
independently-alkyl-N R.sup.13.sub.3.sup.+,
-aromatic-NR.sup.13.sub.3.sup.+, -alkyl-CO.sub.2.sup.-,
aromatic-CO.sub.2.sup.-, -amino acid-NH.sub.3.sup.+,
-phosphorylated amino acid-NH.sub.3.sup.+, polyethylene glycol,
dextran, H, alkyl, aryl, benzyl, Group IA metal, co-antioxidant
(e.g. Vitamin C, Vitamin C analogs, Vitamin C derivatives, Vitamin
E, Vitamin E analogs, Vitamin E derivatives, flavonoids, flavonoid
analogs, or flavonoid derivatives), or Group IA salt. Each R.sup.13
may be independently H, alkyl, or aryl. z may range from 5 to 12.
In some embodiments, z may range from about 3 to about 15. In
certain embodiments, the maximum value of z may only be limited by
the ultimate size of the chemical compound, particularly as it
relates to the size of the chemical compound and the potential
interference with the chemical compound's biological availability
as discussed herein. In some embodiments, substituents may be at
least partially hydrophilic. These carotenoid derivatives may be
used in a pharmaceutical composition.
[0122] In some non-limiting examples, five- and/or six-membered
ring carotenoid derivatives may be more easily synthesized.
Synthesis may come more easily due to, for example, the natural
stability of five- and six-membered rings. Synthesis of carotenoid
derivatives including five- and/or six-membered rings may be more
easily synthesized due to, for example, the availability of
naturally occurring carotenoids including five- and/or six-membered
rings. In some embodiments, five-membered rings may decrease steric
hindrance associated with rotation of the cyclic ring around the
molecular bond connecting the cyclic ring to the polyene chain.
Reducing steric hindrance may allow greater overlap of any
oribitals within a cyclic ring with the polyene chain, thereby
increasing the degree of conjugation and effective chromophore
length of the molecule. This may have the salutatory effect of
increasing antioxidant capacity of the carotenoid derivatives.
[0123] In some embodiments, a substituent (W) may be at least
partially hydrophilic. A hydrophilic substituent may assist in
increasing the water solubility of a carotenoid derivative. In some
embodiments, a carotenoid derivative may be at least partially
water-soluble. The cyclic ring may include at least one chiral
center. The acyclic alkene may include at least one chiral center.
The cyclic ring may include at least one degree of unsaturation. In
some cyclic ring embodiments, the cyclic ring may be aromatic. One
or more degrees of unsaturation within the ring may assist in
extending the conjugation of the carotenoid derivative. Extending
conjugation within the carotenoid derivative may have the
salutatory effect of increasing the antioxidant properties of the
carotenoid derivatives. In some embodiments, the substituent W may
include, for example, a carboxylic acid, an amino acid, an ester,
an alkanol, an amine, a phosphate, a succinate, a glycinate, an
ether, a glucoside, a sugar, or a carboxylate salt.
[0124] In some embodiments, each substituent --W may independently
include --XR. Each X may independently include O, N, or S. In some
embodiments, each substituent --W may independently comprises amino
acids, esters, carbamates, amides, carbonates, alcohol, phosphates,
or sulfonates. In some substituent embodiments, the substituent may
include, for example (d) through (uu): ##STR29## ##STR30##
##STR31## ##STR32## ##STR33## where each R is, for example,
independently-alkyl-NR.sup.12.sub.3.sup.+,
-aromatic-NR.sup.12.sub.3.sup.+, -alkyl-CO.sub.2.sup.-,
-aromatic-CO.sub.2.sup.-, -amino acid-NH.sub.3.sup.+,
-phosphorylated amino acid-NH.sub.3.sup.+, polyethylene glycol,
dextran, H, alkyl, Group IA metal, co-antioxidant (e.g. Vitamin C,
Vitamin C analogs, Vitamin C derivatives, Vitamin E, Vitamin E
analogs, Vitamin E derivatives, flavonoids, flavonoid analogs, or
flavonoid derivatives), or aryl. Each R' may be CH.sub.2. n may
range from 1 to 9. In some embodiments, substituents may include
any combination of (d) through (uu). In some embodiments,
negatively charged substituents may include Group IA metals, one
metal or a combination of different Group IA metals in an
embodiment with more than one negatively charged substituent, as
counter ions. Group IA metals may include, but are not limited to,
sodium, potassium, and/or lithium.
[0125] Water-soluble carotenoid analogs or derivatives may have a
water solubility of greater than about 1 mg/mL in some embodiments.
In certain embodiments, water-soluble carotenoid analogs or
derivatives may have a water solubility of greater than about 5
mg/mL. In certain embodiments, water-soluble carotenoid analogs or
derivatives may have a water solubility of greater than about 10
mg/mL. In certain embodiments, water-soluble carotenoid analogs or
derivatives may have a water solubility of greater than about 20
mg/mL. In some embodiments, water-soluble carotenoid analogs or
derivatives may have a water solubility of greater than about 50
mg/mL.
[0126] Naturally occurring carotenoids such as xanthophyll
carotenoids of the C40 series, which includes commercially
important compounds such as lutein, zeaxanthin, and astaxanthin,
have poor aqueous solubility in the native state. Varying the
chemical structure(s) of the esterified moieties may vastly
increase the aqueous solubility and/or dispersibility of
derivatized carotenoids.
[0127] In some embodiments, highly water-dispersible C40 carotenoid
derivatives may include natural source RRR-lutein
(.beta.,.epsilon.-carotene-3,3'-diol) derivatives. Derivatives may
be synthesized by esterification with inorganic phosphate and
succinic acid, respectively, and subsequently converted to the
sodium salts. Deep orange, evenly colored aqueous suspensions were
obtained after addition of these derivatives to USP-purified water.
Aqueous dispersibility of the disuccinate sodium salt of natural
lutein was 2.85 mg/mL; the diphosphate salt demonstrated a
>10-fold increase in dispersibility at 29.27 mg/mL. Aqueous
suspensions may be obtained without the addition of heat,
detergents, co-solvents, or other additives.
[0128] The direct aqueous superoxide scavenging abilities of these
derivatives were subsequently evaluated by electron paramagnetic
resonance (EPR) spectroscopy in a well-characterized in vitro
isolated human neutrophil assay. The derivatives may be potent
(millimolar concentration) and nearly identical aqueous-phase
scavengers, demonstrating dose-dependent suppression of the
superoxide anion signal (as detected by spin-trap adducts of
DEPMPO) in the millimolar range. Evidence of card-pack aggregation
was obtained for the diphosphate derivative with UV-Vis
spectroscopy (discussed herein), whereas limited card-pack and/or
head-to-tail aggregation was noted for the disuccinate derivative.
These lutein-based soft drugs may find utility in those commercial
and clinical applications for which aqueous-phase singlet oxygen
quenching and direct radical scavenging may be required.
[0129] The absolute size of a carotenoid derivative (in 3
dimensions) is important when considering its use in biological
and/or medicinal applications. Some of the largest naturally
occurring carotenoids are no greater than about C.sub.50. This is
probably due to size limits imposed on molecules requiring
incorporation into and/or interaction with cellular membranes.
Cellular membranes may be particularly co-evolved with molecules of
a length of approximately 30 nm. In some embodiments, carotenoid
derivatives may be greater than or less than about 30 nm in size.
In certain embodiments, carotenoid derivatives may be able to
change conformation and/or otherwise assume an appropriate shape,
which effectively enables the carotenoid derivative to efficiently
interact with a cellular membrane.
[0130] Although the above structure, and subsequent structures,
depict alkenes in the E configuration this should not be seen as
limiting. Compounds discussed herein may include embodiments where
alkenes are in the Z configuration or include alkenes in a
combination of Z and E configurations within the same molecule. The
compounds depicted herein may naturally convert between the Z and E
configuration and/or exist in equilibrium between the two
configurations.
[0131] In an embodiment, a chemical compound may include a
carotenoid derivative having the structure (136) ##STR34## Each
R.sup.14 may be independently O or H.sub.2. Each R may be
independently OR.sup.12 or R.sup.12. Each R.sup.12 may be
independently -alkyl-NR.sup.13.sub.3.sup.+,
-aromatic-NR.sup.13.sub.3.sup.+, -alkyl-CO.sub.2.sup.-,
-aromatic-CO.sub.2.sup.-, -amino acid-NH.sub.3.sup.+,
-phosphorylated amino acid-NH.sub.3.sup.+, polyethylene glycol,
dextran, H, alkyl, peptides, poly-lysine, co-antioxidant (e.g.
Vitamin C, Vitamin C analogs, Vitamin C derivatives, Vitamin E,
Vitamin E analogs, Vitamin E derivatives, flavonoids, flavonoid
analogs, or flavonoid derivatives), or aryl. In addition, each
R.sup.13 may be independently H, alkyl, or aryl. The carotenoid
derivative may include at least one chiral center.
[0132] In a specific embodiment where R.sup.14 is H.sub.2, the
carotenoid derivative may have the structure (138) ##STR35##
[0133] In a specific embodiment where R.sup.14 is O, the carotenoid
derivative may have the structure (140) ##STR36##
[0134] In an embodiment, a chemical compound may include a
carotenoid derivative having the structure (142) ##STR37## Each
R.sup.14 may be independently O or H.sub.2. Each R may be
independently H, alkyl, benzyl, Group IA metal, co-antioxidant, or
aryl. The carotenoid derivative may include at least one chiral
center. In a specific embodiment R.sup.14 may be H.sub.2, the
carotenoid derivative having the structure (144) ##STR38##
[0135] In a specific embodiment where R.sup.14 is O, the carotenoid
derivative may have the structure (146) ##STR39##
[0136] In an embodiment, a chemical compound may include a
carotenoid derivative having the structure (148) ##STR40## Each
R.sup.14 may be independently O or H.sub.2. Each R' may be
CH.sub.2. n may range from 1 to 9. Each X may be independently
##STR41##
[0137] Group IA metal, or co-antioxidant (e.g. Vitamin C, Vitamin C
analogs, Vitamin C derivatives, Vitamin E, Vitamin E analogs,
Vitamin E derivatives, flavonoids, flavonoid analogs, or flavonoid
derivatives). Each R may be
independently-alkyl-NR.sup.12.sub.3.sup.+,
-aromatic-NR.sup.12.sub.3.sup.+, -alkyl-CO.sub.2.sup.-,
-aromatic-CO.sub.2.sup.-, -amino acid-NH.sub.3.sup.+,
-phosphorylated amino acid-NH.sub.3.sup.+, polyethylene glycol,
dextran, H, alkyl, Group IA metal, benzyl, co-antioxidant (e.g.
Vitamin C, Vitamin C analogs, Vitamin C derivatives, Vitamin E,
Vitamin E analogs, Vitamin E derivatives, flavonoids, flavonoid
analogs, or flavonoid derivatives), or aryl. Each R.sup.12 may be
independently H, alkyl, or aryl. The carotenoid derivative may
include at least one chiral center.
[0138] In a specific embodiment where R.sup.14 is H.sub.2, the
carotenoid derivative may have the structure (150) ##STR42## In a
specific embodiment where R.sup.14 is O, the carotenoid derivative
may have the structure (152) ##STR43##
[0139] In an embodiment, a chemical compound may include a
carotenoid derivative having the structure (148) ##STR44## Each
R.sup.14 may be independently O or H.sub.2. Each R' may be
CH.sub.2. n may range from 1 to 9. Each X may be independently
##STR45##
[0140] Group IA metal, or co-antioxidant (e.g. Vitamin C, Vitamin C
analogs, Vitamin C derivatives, Vitamin E, Vitamin E analogs,
Vitamin E derivatives, flavonoids, flavonoid analogs, or flavonoid
derivatives). Each R may be
independently-alkyl-NR.sup.12.sub.3.sup.+,
-aromatic-NR.sup.12.sub.3.sup.+, -alkyl-CO.sub.2.sup.-,
-aromatic-CO.sub.2.sup.-, -amino acid-NH.sub.3.sup.+,
-phosphorylated amino acid-NH.sub.3.sup.+, polyethylene glycol,
dextran, H, alkyl, Group IA metal, co-antioxidant (e.g. Vitamin C,
Vitamin C analogs, Vitamin C derivatives, Vitamin E, Vitamin E
analogs, Vitamin E derivatives, flavonoids, flavonoid analogs, or
flavonoid derivatives), or aryl. Each R.sup.12 may be independently
H, alkyl, or aryl. The carotenoid derivative may include at least
one chiral center.
[0141] In a specific embodiment where R.sup.14 is H.sub.2, the
carotenoid derivative may have the structure (150) ##STR46## In a
specific embodiment where R.sup.14 is O, the carotenoid derivative
may have the structure (152) ##STR47##
[0142] In an embodiment, a chemical compound may include a
carotenoid derivative having the structure (154) ##STR48## Each
R.sup.14 may be independently O or H.sub.2. The carotenoid
derivative may include at least one chiral center. In a specific
embodiment R.sup.14 may be H.sub.2, the carotenoid derivative
having the structure (156) ##STR49## In a specific embodiment where
R.sup.14 is O, the carotenoid derivative may have the structure
(158) ##STR50## In some embodiments, a chemical compound may
include a disuccinic acid ester carotenoid derivative having the
structure (160) ##STR51##
[0143] In some embodiments, a chemical compound may include a
disodium salt disuccinic acid ester carotenoid derivative having
the structure (162)+ ##STR52##
[0144] In some embodiments, a chemical compound may include a
carotenoid derivative with a co-antioxidant, in particular one or
more analogs or derivatives of vitamin C (i.e., L ascorbic acid)
coupled to a carotenoid. Some embodiments may include carboxylic
acid and/or carboxylate derivatives of vitamin C coupled to a
carotenoid (e.g., structure (164)) ##STR53## Carbohydr. Res. 1978,
60, 251-258,herein incorporated by reference, discloses oxidation
at C-6 of ascorbic acid as depicted in EQN. 5. ##STR54## Some
embodiments may include vitamin C and/or vitamin C analogs or
derivatives coupled to a carotenoid. Vitamin C may be coupled to
the carotenoid via an ether linkage (e.g., structure (166))
##STR55## Some embodiments may include vitamin C disuccinate
analogs or derivatives coupled to a carotenoid (e.g., structure
(168)) ##STR56## Some embodiments may include solutions or
pharmaceutical preparations of carotenoids and/or carotenoid
derivatives combined with co-antioxidants, in particular vitamin C
and/or vitamin C analogs or derivatives. Pharmaceutical
preparations may include about a 2:1 ratio of vitamin C to
carotenoid respectively.
[0145] In some embodiments, co-antioxidants (e.g., vitamin C) may
increase solubility of the chemical compound. In certain
embodiments, co-antioxidants (e.g., vitamin C) may decrease
toxicity associated with at least some carotenoid analogs or
derivatives. In certain embodiments, co-antioxidants (e.g., vitamin
C) may increase the potency of the chemical compound
synergistically. Co-antioxidants may be coupled (e.g., a covalent
bond) to the carotenoid derivative. Co-antioxidants may be included
as a part of a pharmaceutically acceptable formulation.
[0146] In some embodiments, a carotenoid (e.g., astaxanthin) may be
coupled to vitamin C forming an ether linkage. The ether linkage
may be formed using the Mitsunobu reaction as in EQN. 1.
##STR57##
[0147] In some embodiments, vitamin C may be selectively
esterified. Vitamin C may be selectively esterified at the C-3
position (e.g., EQN. 2). J. Org. Chem. 2000, 65, 911-913, herein
incorporated by reference, discloses selective esterification at
C-3 of unprotected ascorbic acid with primary alcohols.
##STR58##
[0148] In some embodiments, a carotenoid may be coupled to vitamin
C. Vitamin C may be coupled to the carotenoid at the C-6, C-5 diol
position as depicted in EQNS. 3 and 4 forming an acetal.
##STR59##
[0149] In some embodiments, a carotenoid may be coupled to a
water-soluble moiety (e.g., vitamin C) with a glyoxylate linker as
depicted in EQN. 6. Tetrahedron 1989, 22, 6987-6998, herein
incorporated by reference, discloses similar acetal formations.
##STR60##
[0150] In some embodiments, a carotenoid may be coupled to a
water-soluble moiety (e.g., vitamin C) with a glyoxylate linker as
depicted in EQN. 7. J. Med. Chem. 1988, 31, 1363-1368, herein
incorporated by reference, discloses the glyoxylic acid chloride.
##STR61##
[0151] In some embodiments, a carotenoid may be coupled to a
water-soluble moiety (e.g., vitamin C) with a phosphate linker as
depicted in EQN. 8. Carbohydr. Res. 1988, 176, 73-78, herein
incorporated by reference, discloses the L-ascorbate 6-phosphate.
##STR62##
[0152] In some embodiments, a carotenoid may be coupled to a
water-soluble moiety (e.g., vitamin C) with a phosphate linker as
depicted in EQN. 9. Carbohydr. Res. 1979, 68, 313-319, herein
incorporated by reference, discloses the 6-bromo derivative of
vitamin C. Carbohydr. Res. 1988, 176, 73-78, herein incorporated by
reference, discloses the 6-bromo derivative of vitamin C's reaction
with phosphates. ##STR63##
[0153] In some embodiments, a carotenoid may be coupled to a
water-soluble moiety (e.g., vitamin C) with a phosphate linker as
depicted in EQN. 10. J. Med. Chem. 2001, 44, 1749-1757 and J. Med.
Chem. 2001, 44, 3710-3720, herein incorporated by reference,
disclose the alkyl chloride derivative and its reaction with
nucleophiles, including phosphates, under mild basic conditions.
##STR64##
[0154] In some embodiments, a carotenoid may be coupled to a
water-soluble moiety (e.g., vitamin C) with a phosphate linker as
depicted in EQN. 11. Vitamin C may be coupled to the carotenoid
using selective esterification at C-3 of unprotected ascorbic acid
with primary alcohols. ##STR65##
[0155] In some embodiments, a carotenoid may be coupled to a
water-soluble moiety (e.g., vitamin C) with a phosphate linker as
in 242. Structure 242 may include one or more counterions (e.g.,
Group IA metals). ##STR66## EQN. 12 depicts an example of a
synthesis of a protected form of 242. ##STR67##
[0156] In some embodiments, a chemical compound may include a
carotenoid derivative including one or more amino acids (e.g.,
lysine) and/or amino acid analogs or derivatives (e.g., lysine
hydrochloric acid salt) coupled to a carotenoid (e.g., structure
(1170)). ##STR68##
[0157] In some embodiments, a carotenoid analog or derivative may
include: ##STR69## ##STR70## ##STR71## ##STR72## ##STR73##
##STR74## ##STR75##
[0158] In some embodiments, a chemical compound may include a
disuccinic acid ester carotenoid derivative having the structure
(160) ##STR76##
[0159] In some embodiments, a chemical compound may include a
disodium salt disuccinic acid ester carotenoid derivative having
the structure (162) ##STR77##
[0160] Compounds described herein embrace isomers mixtures,
racemic, optically active, and optically inactive stereoisomers and
compounds. Carotenoid analogs or derivatives may have increased
water solubility and/or water dispersibility relative to some or
all known naturally occurring carotenoids. In some embodiments, one
or more co-antioxidants may be coupled to a carotenoid or
carotenoid derivative or analog.
[0161] In some embodiments, carotenoid analogs or derivatives may
be employed in "self-formulating" aqueous solutions, in which the
compounds spontaneously self-assemble into macromolecular
complexes. These complexes may provide stable formulations in terms
of shelf life. The same formulations may be parenterally
administered, upon which the spontaneous self-assembly is overcome
by interactions with serum and/or tissue components in vivo.
[0162] Some specific embodiments may include phosphate, succinate,
co-antioxidant (e.g., Vitamin C, Vitamin C analogs, Vitamin C
derivatives, Vitamin E, Vitamin E analogs, Vitamin E derivatives,
or flavonoids), or combinations thereof derivatives or analogs of
carotenoids. Flavonoids may include, for example, quercetin,
xanthohumol, isoxanthohumol, or genistein. Derivatives or analogs
may be derived from any known carotenoid (naturally or
synthetically derived). Specific examples of naturally occurring
carotenoids which compounds described herein may be derived from
include for example zeaxanthin, lutein, lycophyll, astaxanthin, and
lycopene.
[0163] The synthesis of water-soluble and/or water-dispersible
carotenoids (e.g., C40) analogs or derivatives--as potential
parenteral agents for clinical applications may improve the
injectability of these compounds as therapeutic agents, a result
perhaps not achievable through other formulation methods. The
methodology may be extended to carotenoids with fewer than 40
carbon atoms in the molecular skeleton and differing ionic
character. The methodology may be extended to carotenoids with
greater than 40 carbon atoms in the molecular skeleton. The
methodology may be extended to non-symmetric carotenoids. The
aqueous dispersibility of these compounds allows proof-of-concept
studies in model systems (e.g. cell culture), where the high
lipophilicity of these compounds previously limited their
bioavailability and hence proper evaluation of efficacy.
Esterification or etherification may be useful to increase oral
bioavailability, a fortuitous side effect of the esterification
process, which can increase solubility in gastric mixed micelles.
The net overall effect is an improvement in potential clinical
utility for the lipophilic carotenoid compounds as therapeutic
agents.
[0164] In some embodiments, the principles of retrometabolic drug
design may be utilized to produce novel soft drugs from the
asymmetric parent carotenoid scaffold (e.g., RRR-lutein
(.beta.,.epsilon.-carotene-3,3'-diol)). For example, lutein
scaffold for derivatization was obtained commercially as purified
natural plant source material, and was primarily the
RRR-stereoisomer (one of 8 potential stereoisomers). Lutein (Scheme
1) possesses key characteristics-similar to starting material
astaxanthin which make it an ideal starting platform for
retrometabolic syntheses: (1) synthetic handles (hydroxyl groups)
for conjugation, and (2) an excellent safety profile for the parent
compound.
[0165] In some embodiments, carotenoid analogs or derivatives may
have increased water solubility and/or water dispersibility
relative to some or all known naturally occurring carotenoids.
[0166] In some embodiments, the carotenoid derivatives may include
compounds having a structure including a polyene chain (i.e.,
backbone of the molecule). The polyene chain may include between
about 5 and about 15 unsaturated bonds. In certain embodiments, the
polyene chain may include between about 7 and about 12 unsaturated
bonds. In some embodiments a carotenoid derivative may include 7 or
more conjugated double bonds to achieve acceptable antioxidant
properties.
[0167] In some embodiments, decreased antioxidant properties
associated with shorter polyene chains may be overcome by
increasing the dosage administered to a subject or patient.
[0168] In some embodiments, the carotenoid derivatives or analogs
may be synthesized from naturally-occurring carotenoids. In some
embodiments, the carotenoid derivatives may be synthesized from any
naturally-occurring carotenoid including one or more alcohol
substituents. In other embodiments, the carotenoid derivatives may
be synthesized from a derivative of a naturally-occurring
carotenoid including one or more alcohol substituents. The
synthesis may result in a single stereoisomer. The synthesis may
result in a single geometric isomer of the carotenoid derivative.
The synthesis/synthetic sequence may include any prior purification
or isolation steps carried out on the parent carotenoid.
[0169] In some embodiments, a synthesis may be a total synthesis
using methods described herein to synthesize carotenoid derivatives
and/or analogs. An example may include, but is not limited to, a
3S,3'S all-E carotenoid derivative, where the parent carotenoid is
astaxanthin. The synthetic sequence may include protecting and
subsequently deprotecting various functionalities of the carotenoid
and/or substituent precursor. When derivates or analogs are
prepared from alcohol functionalized carotenoids, a base catalyzed
reaction may be used to react the alcohol functional groups with
the substituent precursor. Substituent precursors include
precursors that include a functional group that may act as a
leaving group for a substitution reaction. The base may include any
non-nucleophilic base known to one skilled in the art such as, for
example, tertiary amines, pyridine, pyrrolidine, etc. The alcohol
may act as a nucleophile reacting with the substituent precursor,
displacing the leaving group. Leaving groups may include, but are
not limited to, I, Cl, Br, tosyl, brosyl, mesyl, or trifyl. These
are only a few examples of leaving groups that may be used, many
more are known and would be apparent to one skilled in the art. In
some embodiments, a base may be used to deprotonate the alcohol.
For example, reaction with alkyl lithium bases, alkali metal
hydroxide, or alkali metal alcohol salts may deprotonate a hydroxy
group of the carotenoid. In other examples the leaving group may be
internal and may subsequently be included in the final structure of
the carotenoid derivative, a non-limiting example may include
anhydrides or strained cyclic ethers. For example, the alcohol may
be reacted with succinic anhydride.
[0170] In an embodiment, the disuccinic acid ester of astaxanthin
may be further converted to the disodium salt. Examples of
synthetic sequences for the preparation of some of the specific
embodiments depicted are described in the Examples section. The
example depicted below is a generic non-limiting example of a
synthetic sequence for the preparation of astaxanthin carotenoid
derivatives. ##STR78##
[0171] In some embodiments, one or more of the conversions and/or
reactions discussed herein may be carried out within one reaction
vessel increasing the overall efficiency of the synthesis of the
final product. In some embodiments, a product of one reaction
during a total synthesis may not be fully worked up before
continuing on with the following reaction. In general, fully
working up a reaction implies completely isolating and purify the
product from a reaction. A reaction may instead only partially be
worked up. For example, solid impurities which fall out of solution
during the course of a reaction may be filtered off and the
filtrate washed with solvent to ensure all of the resulting product
is washed through and collected. In such a case the resulting
collected product still in solution may not be isolated, but may
then be combined with another reagent and further transformed. In
some cases multiple transformations may be carried out in a single
reaction flask simply by adding reagents one at a time without
working up intermediate products. These types of "shortcuts" will
improve the overall efficiency of a synthesis, especially when
dealing with larger quantity reactions (e.g., along the lines of
pilot plant scale and/or plant scale).
[0172] In some embodiments, an alcohol-functionalized carotenoid
may provide a skeleton with a useful handle with which to
appropriately derivatize a carotenoid based water dispersible end
product. The example depicted above is a generic nonlimiting
example; examples depicted in Schemes 1 and 2 provide more specific
examples of the synthesis of water-soluble and/or water-dispersible
carotenoid analogs or derivatives. Schemes 1 and 2 depict the
syntheses of two water-dispersible lutein derivatives, the sodium
salts of lutein disuccinate and lutein diphosphate. Derivatizing
hydrophobic carotenoids may impart water-dispersibility. ##STR79##
##STR80##
[0173] As seen in Scheme 1, the synthesis of disuccinate salt 103
began with succinylation of natural source lutein using succinic
anhydride and Hunig base (N,N'-diisopropylethylamine). Reactions
may be run in polar organic solvents. Disuccinylation of lutein was
optimized by running the reaction in a concentrated fashion and
using modest excesses of anhydride and base. Using high
concentrations of reagents may allow easier extraction of
impurities and side products once the reaction is complete. Aqueous
acidic workup yielded disuccinate 102, such that excess reagents
and reaction byproducts were removed by copiously extracting the
organic layer with dilute HCl. The resulting viscous, red-orange
oil was washed or slurried with hexanes to remove non-polar
impurities. A successfully functionalized carotenoid may be
transformed into an ionic salt derivative or analog in order to
increase the water solubility. A carotenoid may be transformed into
an ionic salt derivative or analog by reacting the carotenoid with
a base. Bases may include alkali metal hydroxides (e.g., sodium
hydroxide) or tertiary amines (e.g., triethylamine). In some
embodiments, bases, upon deprotonation of one or more moieties of
the carotenoid may result in by products which are easily removed
(e.g., removed under reduced pressure, extracted). The
water-dispersible derivative 103 was generated by treating compound
102 with methanolic sodium methoxide. The reaction was quenched
with water and the resulting red-orange aqueous layer was first
extracted with Et.sub.2O, then lyophilized to provide the sodium
salt in good yield.
[0174] In some embodiments, a carotenoid may be phosphorylated to
increase water solubility and/or dispersibility. In some
embodiments, a carotenoid may be diphosphorylated to increase water
solubility and/or dispersibility. Successful diphosphorylation of
lutein may be achieved using dimethyl phosphoroiodidate. Dimethyl
phosphoroiodidate may be formed in situ. Dimethyl phosphoroiodidate
may be formed by reacting commercially available trimethyl
phosphite with iodine. In some embodiments, a certain degree of
success in removing all four diphosphate methyl groups may be
realized when using bromotrimethylsilane in the presence of
N,O-bis(trimethylsilyl)acetamde. However, this deprotection
protocol may not be optimal in that methyl group dealkylation was
usually accompanied by the significant decomposition of lutein
phosphate.
[0175] In some embodiments, a three-step method to provide the
tetra-sodium salt of lutein diphosphate 109 may be achieved using
benzyl esters as protecting groups for the lutein phosphoric acids
(Scheme 2). Lutein (e.g., natural source) may be phosphorylated
using dibenzyl phosphoroiodidate. Dibenzyl phosphoroiodidate may be
formed in situ. Dibenzyl phosphoroiodidate may be formed by
reacting tribenzyl phosphite with iodine. As seen in Scheme 2,
tribenzyl phosphite may be prepared by the addition of benzyl
alcohol to phosphorus trichloride in the presence of triethylamine.
In some embodiments, silica gel chromatography of the crude
reaction mixture may yield tribenzyl phosphite in good yield.
Compound 106 was formed by treating lutein with freshly prepared
dibenzyl phosphoroiodidate in the presence of pyridine. Aqueous
workup of the reaction followed by the removal of pyridine by
azeotropic distillation using toluene may provide a crude red oil.
Contaminations, excess reagents, and reaction byproducts may be
removed during work up of the reaction or at a later time (e.g.,
after a subsequent reaction). Non-polar impurities may be removed
from the crude product mixture by alternately washing or slurrying
with hexanes and Et.sub.2O to give 106.
[0176] In some embodiments, dealkylation of one or more of the four
benzyl esters of the phosphoric acid moieties may occur during the
phosphorylation reaction. Dealkylation may occurr at the more
sensitive allylic 3' phosphate positions. As seen in Scheme 2, the
attempted removal of the phosphoric acid benzyl esters of 106 using
LiOH--H.sub.2O may result in the generation of a less polar product
versus compound 106, exhibiting a molecular ion of 828 as noted by
LC/MS analysis. Under these reaction conditions, dephosphorylation
at one of the two hydroxyls of the lutein derivative may occur
rather than the desired debenzylation to give compound 107. Such
data indirectly support compound 106's structure and thus the
occurrence of bis-dealkylation at one phosphate versus
mono-dealkylation at both phosphates as an additional result of the
phosphorylation of lutein. If mono-dealkylation at both phosphates
occurred during phosphorylation, then treatment of the resulting
product with LiOH--H.sub.2O would have produced a lutein derivative
possessing one phosphoric acid containing only one benzyl ester,
exhibiting a molecular ion of 738 upon LC/MS analysis.
[0177] In some embodiments, successful dealkylation of the
phosphate protecting groups of 106 may be achieved using
bromotrimethylsilane in the presence of
N,O-bis(trimethylsilyl)acetamide (see Scheme 2). A significant
amount of excess reagents and reaction byproducts may be removed
from the resulting red oil by alternately washing or slurrying the
crude mixture with ethyl acetate and CH.sub.2Cl.sub.2 to provide
diphosphate 108 as an orange oil.
[0178] In some embodiments, the sodium salt of lutein diphosphate
(109) may be generated by treating 108 with methanolic sodium
methoxide (see Scheme 2). The resulting crude orange solid may be
washed or slurried with methanol and then dissolved in water. The
aqueous layer may be extracted first with CH.sub.2Cl.sub.2, then
with ethyl acetate, and again with CH.sub.2Cl.sub.2. Lyophilization
of the red-orange aqueous solution may give the sodium salt as an
orange, hygroscopic solid. The phosphorylation process may provide
the desired water-dispersible lutein derivative 109 in good yield
over the three steps.
[0179] The synthetic preparation of carotenoid derivatives or
analogs such as disodium disuccinate astaxanthin 162 at multigram
scale (e.g., 200 g to 1 kg) is necessary if one wishes to produce
these molecules commercially. Synthetic modifications of
carotenoids, with the goal of increasing aqueous solubility and/or
dispersibility, have been sparingly reported in the literature. At
the time process development began, surveys of the peer-reviewed
and patent literature indicated that neither a synthetic sequence
nor an efficient process for the synthesis of 160 or 162 had been
reported. Therefore, the bench-scale synthetic sequence and later
the scale-up to multigram scale were optimized to improve both the
yield and purity of the desired compound. Examples of synthetic
preparation of carotenoids and carotenoid derivatives or analogs
are illustrated in U.S. Patent Application Ser. No. 60/615,032
filed on Oct. 1, 2004, entitled "METHODS FOR SYNTHESIS OF
CAROTENOIDS, INCLUDING ANALOGS, DERIVATIVES, AND SYNTHETIC AND
BIOLOGICAL INTERMEDIATES" to Lockwood et al. which is incorporated
by reference as if fully set forth herein.
[0180] The disodium disuccinate derivatives of synthetic
astaxanthin were successfully synthesized in gram amounts and at
high purity (>90%) area under the curve (AUC) by HPLC. The
compound in "racemic" form demonstrated water "dispersibility" of
8.64 mg/mL, a significant improvement over the parent compound
astaxanthin, which is insoluble in water. Initial biophysical
characterization demonstrated that Cardax.TM. derivatives (as both
the statistical mixture of stereoisomers and as individual
stereoisomers) were potent direct scavengers of superoxide anion in
the aqueous phase, the first such description in this model system
for a C40 carotenoid. Plasma-protein binding studies in vitro
revealed that the meso-(3R,3'S)-disodium disuccinate astaxanthin
derivative bound immediately and preferentially to human serum
albumin (HSA) at a binding site, suggesting that beneficial
ligand-binding associations might take place in vivo after
parenteral administration of the compound. The single- and
multiple-dose pharmacokinetics of an oral preparation of the
racemic compound (in lipophilic emulsion) were then investigated in
a murine model, and significant plasma and tissue levels of
nonesterified astaxanthin were achieved. Proof-of-concept studies
in ischemia-reperfusion injury performed in rodents subsequently
revealed that intravenous pretreatment with Cardax.TM. was
significantly cardioprotective and achieved myocardial salvage in
this experimental infarction model (e.g., up to 56% at the highest
dose tested). The test material for three of the studies described
above was obtained from a single pilot batch of compound (>200 g
single batch at >97% purity by HPLC).
[0181] In some embodiments, it may be advantageous to be able to
efficiently separate out individual stereoisomers of a racemic
mixture of a chemical compound. Efficiently separating out
individual stereoisomers on a relatively large scale may
advantageously increase availability of starting materials.
[0182] In some embodiments, chromatographic separation techniques
may be used to separate stereoisomers of a racemic mixture. In some
embodiments pure optically active stereoisomers may be reacted with
a mixture of stereoisomers of a chemical compound to form a mixture
of diastereomers. Diastereomers may have different physical
properties as opposed to stereoisomers, thus making it easier to
separate diastereomers.
[0183] For example it may be advantageous to separate out
stereoisomers from a racemic mixture of astaxanthin. In some
embodiments, astaxanthin may be coupled to an optically active
compound (e.g., dicamphanic acid). Coupling astaxanthin to
optically active compounds produces diastereomers with different
physical properties. The diastereomers produced may be separated
using chromatographic separation techniques as described
herein.
[0184] Bulk chromatographic separation of the diastereomeric
dicamphanic acid ester(s) of synthetic astaxanthin at preparative
chromatography scale was performed to subsequently make gram-scale
quantities of each stereoisomer of disodium disuccinate ester
astaxanthin.
[0185] As used herein the terms "structural carotenoid analogs or
derivatives" may be generally defined as carotenoids and the
biologically active structural analogs or derivatives thereof.
"Derivative" in the context of this application is generally
defined as a chemical substance derived from another substance
either directly or by modification or partial substitution.
"Analog" in the context of this application is generally defined as
a compound that resembles another in structure but is not
necessarily an isomer. Typical analogs or derivatives include
molecules which demonstrate equivalent or improved biologically
useful and relevant function, but which differ structurally from
the parent compounds. Parent carotenoids are selected from the more
than 700 naturally occurring carotenoids described in the
literature, and their stereo- and geometric isomers. Such analogs
or derivatives may include, but are not limited to, esters, ethers,
carbonates, amides, carbamates, phosphate esters and ethers,
sulfates, glycoside ethers, with or without spacers (linkers).
[0186] As used herein the terms "the synergistic combination of
more than one structural analog or derivative or synthetic
intermediate of carotenoids" may be generally defined as any
composition including one structural carotenoid analog or
derivative or synthetic intermediate combined with one or more
other structural carotenoid analogs or derivatives or synthetic
intermediate or co-antioxidants, either as derivatives or in
solutions and/or formulations.
[0187] As used herein the terms "subject" may be generally defined
as all mammals, in particular humans.
[0188] As used herein the terms "administration" may be generally
defined as the administration of the pharmaceutical or
over-the-counter (OTC) or nutraceutical compositions by any means
that achieves the intended purpose. For example, administration may
include parenteral, subcutaneous, intravenous, intracoronary,
rectal, intramuscular, intra-peritoneal, transdermal, or buccal
routes. Alternatively, or concurrently, administration may be by
the oral route. The dosage administered will be dependent upon the
age, health, weight, and/or disease state of the recipient, kind of
concurrent treatment, if any, frequency of treatment, and/or the
nature of the effect desired.
[0189] In some embodiments, techniques described herein may be
applied to the inhibition and/or amelioration of any disease or
disease state related to reactive oxygen species ("ROS") and other
radical and non-radical species.
[0190] In some embodiments, techniques described herein may be
applied to the inhibition and/or amelioration of inflammation,
including but not limited to ischemic reperfusion injury of a
tissue.
[0191] An embodiment may include the administration of structural
carotenoid analogs or derivatives or synthetic intermediates alone
or in combination to a subject such that the occurrence of
inflammation is thereby inhibited and/or ameliorated. The
structural carotenoid analogs or derivatives or synthetic
intermediates may be water-soluble and/or water dispersible
derivatives. The carotenoid derivatives may include any substituent
that substantially increases the water solubility of the naturally
occurring carotenoid. The carotenoid derivatives may retain and/or
improve the antioxidant properties of the parent carotenoid. The
carotenoid derivatives may retain the non-toxic properties of the
parent carotenoid. The carotenoid derivatives may have increased
bioavailability, relative to the parent carotenoid, upon
administration to a subject. The parent carotenoid may be naturally
occurring.
[0192] Another embodiments may include the administration of a
composition comprised of the synergistic combination of more than
one structural analog or derivative or synthetic intermediate of
carotenoids to a subject such that the occurrence of tissue damage
associated with an inflammatory response is thereby reduced. The
composition may be a "racemic" (i.e. mixture of the potential
stereoisomeric forms) mixture of carotenoid derivatives. Included
as well are pharmaceutical compositions comprised of structural
analogs or derivatives or synthetic intermediates of carotenoids in
combination with a pharmaceutically acceptable carrier. In one
embodiment, a pharmaceutically acceptable carrier may be serum
albumin. In one embodiment, structural analogs or derivatives or
synthetic intermediates of carotenoids may be complexed with human
serum albumin (i.e., HSA) in a solvent. HSA may act as a
pharmaceutically acceptable carrier.
[0193] In some embodiments, a single stereoisomer of a structural
analog or derivative or synthetic intermediate of carotenoids may
be administered to a human subject in order to ameliorate a
pathological condition. Administering a single stereoisomer of a
particular compound (e.g., as part of a pharmaceutical composition)
to a human subject may be advantageous (e.g., increasing the
potency of the pharmaceutical composition). Administering a single
stereoisomer may be advantageous due to the fact that only one
isomer of potentially many may be biologically active enough to
have the desired effect.
[0194] In some embodiments, compounds described herein may be
administered in the form of nutraceuticals. "Nutraceuticals" as
used herein, generally refers to dietary supplements, foods, or
medical foods that: 1. possess health benefits generally defined as
reducing the risk of a disease or health condition, including the
management of a disease or health condition or the improvement of
health; and 2. are safe for human consumption in such quantity, and
with such frequency, as required to realize such properties.
Generally a nutraceutical is any substance that is a food or a part
of a food and provides medical or health benefits, including the
prevention and treatment of disease. Such products may range from
isolated nutrients, dietary supplements and specific diets to
genetically engineered designer foods, herbal products, and
processed foods such as cereals, soups and beverages. It is
important to note that this definition applies to all categories of
food and parts of food, ranging from dietary supplements such as
folic acid, used for the prevention of spina bifida, to chicken
soup, taken to lessen the discomfort of the common cold. This
definition also includes a bio-engineered designer vegetable food,
rich in antioxidant ingredients, and a stimulant functional food or
pharmafood. Within the context of the description herein where the
composition, use and/or delivery of pharmaceuticals are described
nutraceuticals may also be composed, used, and/or delivered in a
similar manner where appropriate.
[0195] In some embodiments, compositions may include all
compositions of 1.0 gram or less of a particular structural
carotenoid analog, in combination with 1.0 gram or less of one or
more other structural carotenoid analogs or derivatives or
synthetic intermediates and/or co-antioxidants, in an amount which
is effective to achieve its intended purpose. While individual
subject needs vary, determination of optimal ranges of effective
amounts of each component is with the skill of the art. Typically,
a structural carotenoid analog or derivative or synthetic
intermediates may be administered to mammals, in particular humans,
orally at a dose of 5 to 100 mg per day referenced to the body
weight of the mammal or human being treated for a particular
disease. Typically, a structural carotenoid analog or derivative or
synthetic intermediate may be administered to mammals, in
particular humans, parenterally at a dose of between 5 to 1000 mg
per day referenced to the body weight of the mammal or human being
treated for a particular disease. In other embodiments, about 100
mg of a structural carotenoid analog or derivative or synthetic
intermediate is either orally or parenterally administered to treat
or prevent disease.
[0196] The unit oral dose may comprise from about 0.25 mg to about
1.0 gram, or about 5 to 25 mg, of a structural carotenoid analog.
The unit parenteral dose may include from about 25 mg to 1.0 gram,
or between 25 mg and 500 mg, of a structural carotenoid analog. The
unit intracoronary dose may include from about 25 mg to 1.0 gram,
or between 25 mg and 100 mg, of a structural carotenoid analog. The
unit doses may be administered one or more times daily, on
alternate days, in loading dose or bolus form, or titrated in a
parenteral solution to commonly accepted or novel biochemical
surrogate marker(s) or clinical endpoints as is with the skill of
the art.
[0197] In addition to administering a structural carotenoid analog
or derivative or synthetic intermediate as a raw chemical, the
compounds may be administered as part of a pharmaceutical
preparation containing suitable pharmaceutically acceptable
carriers, preservatives, excipients and auxiliaries which
facilitate processing of the structural carotenoid analog or
derivative or synthetic intermediates which may be used
pharmaceutically. The preparations, particularly those preparations
which may be administered orally and which may be used for the
preferred type of administration, such as tablets, softgels,
lozenges, dragees, and capsules, and also preparations which may be
administered rectally, such as suppositories, as well as suitable
solutions for administration by injection or orally or by
inhalation of aerosolized preparations, may be prepared in dose
ranges that provide similar bioavailability as described above,
together with the excipient. While individual needs may vary,
determination of the optimal ranges of effective amounts of each
component is within the skill of the art.
[0198] The pharmaceutical preparations may be manufactured in a
manner which is itself known to one skilled in the art, for
example, by means of conventional mixing, granulating,
dragee-making, softgel encapsulation, dissolving, extracting, or
lyophilizing processes. Thus, pharmaceutical preparations for oral
use may be obtained by combining the active compounds with solid
and semi-solid excipients and suitable preservatives, and/or
co-antioxidants. Optionally, the resulting mixture may be ground
and processed. The resulting mixture of granules may be used, after
adding suitable auxiliaries, if desired or necessary, to obtain
tablets, softgels, lozenges, capsules, or dragee cores.
[0199] Suitable excipients may be fillers such as saccharides
(e.g., lactose, sucrose, or mannose), sugar alcohols (e.g.,
mannitol or sorbitol), cellulose preparations and/or calcium
phosphates (e.g., tricalcium phosphate or calcium hydrogen
phosphate). In addition binders may be used such as starch paste
(e.g., maize or corn starch, wheat starch, rice starch, potato
starch, gelatin, tragacanth, methyl cellulose,
hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or
polyvinyl pyrrolidone). Disintegrating agents may be added (e.g.,
the above-mentioned starches) as well as carboxymethyl-starch,
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof (e.g., sodium alginate). Auxiliaries are, above all,
flow-regulating agents and lubricants (e.g., silica, talc, stearic
acid or salts thereof, such as magnesium stearate or calcium
stearate, and/or polyethylene glycol, or PEG). Dragee cores are
provided with suitable coatings, which, if desired, are resistant
to gastric juices. Softgelatin capsules ("softgels") are provided
with suitable coatings, which, typically, contain gelatin and/or
suitable edible dye(s). Animal component-free and kosher gelatin
capsules may be particularly suitable for the embodiments described
herein for wide availability of usage and consumption. For this
purpose, concentrated saccharide solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
polyethylene glycol (PEG) and/or titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures,
including dimethylsulfoxide (DMSO), tetrahydrofuran (THF), acetone,
ethanol, or other suitable solvents and co-solvents. In order to
produce coatings resistant to gastric juices, solutions of suitable
cellulose preparations such as acetylcellulose phthalate or
hydroxypropylmethyl-cellulose phthalate, may be used. Dye stuffs or
pigments may be added to the tablets or dragee coatings or
softgelatin capsules, for example, for identification or in order
to characterize combinations of active compound doses, or to
disguise the capsule contents for usage in clinical or other
studies.
[0200] Other pharmaceutical preparations that may be used orally
include push-fit capsules made of gelatin, as well as soft,
thermally sealed capsules made of gelatin and a plasticizer such as
glycerol or sorbitol. The push-fit capsules may contain the active
compounds in the form of granules that may be mixed with fillers
such as, for example, lactose, binders such as starches, and/or
lubricants such as talc or magnesium stearate and, optionally,
stabilizers and/or preservatives. In soft capsules, the active
compounds may be dissolved or suspended in suitable liquids, such
as fatty oils such as rice bran oil or peanut oil or palm oil, or
liquid paraffin. In some embodiments, stabilizers and preservatives
may be added.
[0201] In some embodiments, pulmonary administration of a
pharmaceutical preparation may be desirable. Pulmonary
administration may include, for example, inhalation of aerosolized
or nebulized liquid or solid particles of the pharmaceutically
active component dispersed in and surrounded by a gas.
[0202] Possible pharmaceutical preparations, which may be used
rectally, include, for example, suppositories, which consist of a
combination of the active compounds with a suppository base.
Suitable suppository bases are, for example, natural or synthetic
triglycerides, or paraffin hydrocarbons. In addition, it is also
possible to use gelatin rectal capsules that consist of a
combination of the active compounds with a base. Possible base
materials include, for example, liquid triglycerides, polyethylene
glycols, or paraffin hydrocarbons.
[0203] Suitable formulations for parenteral administration include,
but are not limited to, aqueous solutions of the active compounds
in water-soluble and/or water dispersible form, for example,
water-soluble salts, esters, carbonates, phosphate esters or
ethers, sulfates, glycoside ethers, together with spacers and/or
linkers. Suspensions of the active compounds as appropriate oily
injection suspensions may be administered, particularly suitable
for intramuscular injection. Suitable lipophilic solvents,
co-solvents (such as DMSO or ethanol), and/or vehicles including
fatty oils, for example, rice bran oil or peanut oil and/or palm
oil, or synthetic fatty acid esters, for example, ethyl oleate or
triglycerides, may be used. Aqueous injection suspensions may
contain substances that increase the viscosity of the suspension
including, for example, sodium carboxymethyl cellulose, sorbitol,
dextran, and/or cyclodextrins. Cyclodextrins (e.g.,
.beta.-cyclodextrin) may be used specifically to increase the water
solubility for parenteral injection of the structural carotenoid
analog. Liposomal formulations, in which mixtures of the structural
carotenoid analog or derivative with, for example, egg yolk
phosphotidylcholine (E-PC), may be made for injection. Optionally,
the suspension may contain stabilizers, for example, antioxidants
such as BHT, and/or preservatives, such as benzyl alcohol.
EXAMPLES
[0204] Having now described the invention, the same will be more
readily understood through reference to the following example(s),
which are provided by way of illustration, and are not intended to
be limiting of the present invention.
[0205] General. Natural source lutein (90%) was obtained from
ChemPacific, Inc. (Baltimore, Md.) as a red-orange solid and was
used without further purification. All other reagents and solvents
used were purchased from Acros (New Jersey, USA) and were used
without further purification. All reactions were performed under
N.sub.2 atmosphere. All flash chromatographic purifications were
performed on Natland International Corporation 230-400 mesh silica
gel using the indicated solvents. LC/MS (APCI) and LC/MS (ESI) were
recorded on an Agilent 1100 LC/MSD VL system; column: Zorbax
Eclipse XDB-C18 Rapid Resolution (4.6.times.75 mm, 3.5 .mu.m,
USUT002736); temperature: 25.degree. C.; starting pressure: 105
bar; flow rate: 1.0 mL/min; mobile phase (% A=0.025%
trifluoroacetic acid in H.sub.2O, % B=0.025% trifluoroacetic acid
in acetonitrile) Gradient program: 70% A/30% B (start), step
gradient to 50% B over 5 min, step gradient to 98% B over 8.30 min,
hold at 98% B over 25.20 min, step gradient to 30% B over 25.40
min; PDA Detector: 470 nm. The presence of trifluoroacetic acid in
the LC eluents acts to protonate synthesized lutein disuccinate and
diphosphate salts to give the free di-acid forms, yielding
M.sup.+=768 for the disuccinate salt sample and M.sup.+=728 for the
diphosphate salt sample in MS analyses. LRMS: +mode; ESI:
electrospray chemical ionization, ion collection using quadrapole;
APCI: atmospheric pressure chemical ionization, ion collection
using quadrapole. MS (ESI-IT) was recorded on a HCT plus Bruker
Daltonics Mass Spectrometer system, LRMS: +mode; ESI-IT:
electrospray chemical ionization, ion collection using ion trap.
.sup.1H NMR analyses were attempted on Varian spectrometers (300
and 500 MHz). NMR analyses of natural source lutein as well as
synthesized lutein derivatives yielded only partially discernable
spectra, perhaps due to the presence of interfering impurities
(natural source lutein), or due to aggregation (natural source
lutein and derivatives). In attempts to circumvent the problems
associated with NMR analyses, samples were prepared using mixtures
of deuterated solvents including methanol/chloroform,
methanol/water, methyl sulfoxide/water, and
chloroform/methanol/water. However, such attempts failed to give
useful data.
[0206] Natural source lutein (.beta.,.epsilon.-carotene-3,3'-diol),
1. LC/MS (ESI): 9.95 min (2.78%), .lamda..sub.max 226 nm (17%), 425
nm (100%); 10.58 min (3.03%), .lamda..sub.max 225 nm (21%), 400 nm
(100%); 11.10 min (4.17%), .lamda..sub.max 225 nm (16%), 447 nm
(100%); 12.41 min (90.02%), .lamda..sub.max 269 nm (14%), 447 nm
(100%), m/z 568 M.sup.+ (69%), 551 [M-H.sub.2O+H].sup.+ (100%), 533
[M-2H.sub.2O+H].sup.+ (8%)
[0207] .beta.,.epsilon.-carotenyl 3,3'-disuccinate, 2. To a
solution of natural source lutein (1) (0.50 g, 0.879 mmol) in
CH.sub.2Cl.sub.2 (8 mL) was added N,N-diisopropylethylamine (3.1
mL, 17.58 mmol) and succinic anhydride (0.88 g, 8.79 mmol). The
solution was stirred at RT overnight and then diluted with
CH.sub.2Cl.sub.2 and quenched with water/1 M HCl (5/1). The aqueous
layer was extracted two times with CH.sub.2Cl.sub.2 and the
combined organic layer was washed three times with cold water/1 M
HCl (5/1), dried over Na.sub.2SO.sub.4, and concentrated. The
resulting red-orange oil was washed (slurried) three times with
hexanes to yield disuccinate 2 (0.433 g, 64%) as a red-orange
solid; LC/MS (APCI): 10.37 min (4.42%), .lamda..sub.max 227 nm
(56%), 448 nm (100%), m/z 769 [M+H].sup.+ (8%), 668
[M-C.sub.4O.sub.3H.sub.4].sup.+ (9%), 637 (36%), 138 (100%); 11.50
min (92.40%), .lamda..sub.max 269 nm (18%), 447 nm (100%), m/z 769
[M+H].sup.+ (7%), 668 [M-C.sub.4O.sub.3H.sub.4].sup.+ (9%), 651
(100%); 12.03 min (3.18%) .lamda..sub.max 227 nm (55%), 446 nm
(100%), m/z 668 [M-C.sub.4O.sub.3H.sub.4].sup.+ (15%), 550 (10%),
138 (100%)
[0208] .beta.,.epsilon.-carotenyl 3,3'-disuccinate sodium salt, 3.
To a solution of disuccinate 2 (0.32 g, 0.416 mmol) in
CH.sub.2Cl.sub.2/methanol (5 mL/1 mL) at 0.degree. C. was added
drop-wise sodium methoxide (25% wt in methanol; 0.170 mL, 0.748
mmol). The solution was stirred at RT overnight and then quenched
with water and stirred for 5 min. The solution was then
concentrated and the aqueous layer was washed four times with
Et.sub.2O. Lyophilization of the clear, red-orange aqueous solution
yielded 3 (0.278 g, 91%) as an orange, hygroscopic solid; LC/IMS
(APCI): 11.71 min (94.29%), .lamda..sub.max 269 nm (18%), 446 nm
(100%), m/z 769 [M-2Na+3H].sup.+ (8%), 668
[M-2Na+2H-C.sub.4O.sub.3H.sub.4].sup.+ (6%), 651 (100%); 12.74 min
(5.71%), .lamda..sub.max 227 nm (30%), 269 nm (18%), 332 nm (39%),
444 nm (100%), m/z 768 [M-2Na+2H].sup.+ (2%), 668
[M-2Na+2H-C.sub.4O.sub.3H.sub.4].sup.+ (3%), 651 (12%), 138
(100%)
[0209] Tribenzyl phosphite, 4. To a well-stirred solution of
phosphorus trichloride (1.7 mL, 19.4 mmol) in Et.sub.2O (430 mL) at
0.degree. C. was added dropwise a solution of triethylamine (8.4
mL, 60.3 mmol) in Et.sub.2O (20 mL), followed by a solution of
benzyl alcohol (8.1 mL, 77.8 mmol) in Et.sub.2O (20 mL). The
mixture was stirred at 0.degree. C. for 30 min and then at RT
overnight. The mixture was filtered and the filtrate concentrated
to give a colorless oil. Silica chromatography
(hexanes/Et.sub.2O/triethylamine, 4/1/1%) of the crude product
yielded 4 (5.68 g, 83%) as a clear, colorless oil that was stored
under N.sub.2 at -20.degree. C.; .sup.1H NMR: .delta. 7.38 (15H,
m), 4.90 (6H, d)
[0210] Dibenzyl phosphoroiodidate, 5. To a solution of tribenzyl
phosphite (5.43 g, 15.4 mmol) in CH.sub.2Cl.sub.2 (8 mL) at
0.degree. C. was added 1.sub.2 (3.76 g, 14.8 mmol). The mixture was
stirred at 0.degree. C. for 10 min or until the solution became
clear and colorless. The solution was then stirred at RT for 10 min
and used directly in the next step.
[0211] 3-(Bis
benzyl-phosphoryloxy)-3'-(phosphoryloxy)-.beta.,.epsilon.-carotene,
6. To a solution of natural source lutein (1) (0.842 g, 1.48 mmol)
in CH.sub.2Cl.sub.2 (8 mL) was added pyridine (4.8 mL, 59.2 mmol).
The solution was stirred at 0.degree. C. for 5 min and then freshly
prepared 5 (14.8 mmol) in CH.sub.2Cl.sub.2 (8 mL) was added
drop-wise to the mixture at 0.degree. C. The solution was stirred
at 0.degree. C. for 1 h and then diluted with CH.sub.2Cl.sub.2 and
quenched with brine. The aqueous layer was extracted twice with
CH.sub.2Cl.sub.2 and the combined organic layer was washed once
with brine, then dried over Na.sub.2SO.sub.4 and concentrated.
Pyridine was removed from the crude red oil by azeotropic
distillation using toluene. The crude product was alternately
washed (slurried) twice with hexanes and Et.sub.2O to yield 6 as a
red oil, used in the next step without further purification; LC/MS
(ESI): 9.93 min (44.78%), .lamda..sub.max 267 nm (33%), 444 nm
(100%), m/z 890 [M-H.sub.2O].sup.+ (8%), 811
[M-PO.sub.3H-H.sub.2O+H].sup.+ (73%), 533 (100%); 9.99 min (29.0%),
.lamda..sub.max 268 nm (24%), 446 nm (100%), m/z 890
[M-H.sub.2O].sup.+ (6%), 811 [M-PO.sub.3H-H.sub.2O+H].sup.+ (72%),
533 (100%); 10.06 min (26.23%), .lamda..sub.max 266 nm (15%), 332
nm (22%), 444 nm (100%), m/z 890 [M-H.sub.2O].sup.+ (5%), 811
[M-PO.sub.3H-H.sub.2O+H].sup.+ (90%), 533 (100%)
[0212] 3-(Bis
benzyl-phosphoryloxy)-3'-hydroxy-.beta.,.epsilon.-carotene, 7. To a
solution of 6 (0.033 mmol) in tetrahydrofuran/water (1 mL/0.5 mL)
at 0.degree. C. was added LiOH--H.sub.2O (0.003 g, 0.073 mmol). The
solution was stirred at RT for 1 h and then quenched with methanol.
The crude reaction mixture was analyzed by LC/MS; LC/MS (ESI):
10.02 min (40.60%), .lamda..sub.max 266 nm (12%), 333 nm (25%), 445
nm (100%), m/z 890 [M-H.sub.2O].sup.+ (33%), 811
[M-PO.sub.3H-H.sub.2O+H]+ (50%), 533 (100%); 16.37 min (49.56%)
.lamda..sub.max 267 nm (16%), 332 nm (27%), 446 nm (100%), m/z 828
M.sup.+ (55%), 550 (44%)
[0213] 3,3'-Diphosphoryloxy-.beta.,.epsilon.-carotene, 8. To a
solution of 6 (1.48 mmol) in CH.sub.2Cl.sub.2 (10 mL) at 0.degree.
C. was added drop-wise N,O-bis(trimethylsilyl)acetamide (3.7 mL,
14.8 mmol) and then bromotrimethylsilane (1.56 mL, 11.8 mmol). The
solution was stirred at 0.degree. C. for 1 h, quenched with
methanol, diluted with CH.sub.2Cl.sub.2, and then concentrated. The
resulting red oil was alternately washed (slurried) three times
with ethyl acetate and CH.sub.2Cl.sub.2 to yield crude phosphate 8
(2.23 g) as a dark orange oil, used in the next step without
further purification; LC/MS (ESI): 8.55 min (45.67%),
.lamda..sub.max 214 nm (25%), 268 nm (28%), 447 nm (100%), m/z 631
[M-PO.sub.3H-H.sub.2O+H].sup.+ (30%), 533 (18%), 279 (13%), 138
(87%); 8.95 min (35.0%), .lamda..sub.max 217 nm (14%), 268 nm
(23%), 448 nm (100%), m/z 631 [M-PO.sub.3H-H.sub.2O+H].sup.+ (26%),
533 (32%), 279 (18%), 138 (100%); 9.41 min (9.70%), .lamda..sub.max
225 nm (37%), 269 nm (23%), 335 nm (19%), 447 nm (100%), m/z 631
[M-PO.sub.3H-H.sub.2O+H].sup.+ (6%), 533 (18%), 279 (13%), 138
(100%)
[0214] 3,3'-Diphosphoryloxy-.beta.,.epsilon.-carotene sodium salt,
9. To a solution of crude 8 (ca 50%; 2.23 g, 3.06 mmol) in methanol
(20 mL) at 0.degree. C. was added drop-wise sodium methoxide (25%;
3.5 mL, 15.3 mmol). The solution was stirred at RT for 2 h and the
resulting orange solid was washed (slurried) three times with
methanol. Water was added to the moist solid and the resulting
aqueous layer was extracted with CH.sub.2Cl.sub.2, ethyl acetate,
and again with CH.sub.2Cl.sub.2. Lyophilization of the clear,
red-orange aqueous solution yielded 9 (0.956 g, 80% over 3 steps)
as an orange, hygroscopic solid; LC/MS (ESI): 7.81 min (22.34%),
.lamda..sub.max 215 nm (34%), 268 nm (30%), 448 nm (100%), m/z 711
[M-4Na-H.sub.2O+5H].sup.+ (9%), 533 (13%), 306 (100%); 8.33 min
(39.56%), .lamda..sub.max 217 nm (14%), 268 nm (20%), 448 nm
(100%), m/z 711 [M-4Na-H.sub.2O+5H].sup.+ (10%), 533 (11%), 306
(100%); 8.90 min (38.09%), .lamda..sub.max 223 nm (45%), 269 nm
(30%), 336 nm (26%), 448 nm (100%), m/z 711
[M-4Na-H.sub.2O+5H].sup.+ (8%), 631
[M-4Na-PO.sub.3H-H.sub.2O+5H].sup.+ (18%), 533 (20%), 306 (100%);
MS (ESI-IT): m/z 816 M.sup.+ (55%), 772 [M-2Na+2H].sup.+ (37%), 728
[M-4Na+4H].sup.+ (74%)
[0215] UV/Visible spectroscopy. For spectroscopic sample
preparations, 3 and 9 were dissolved in the appropriate solvent to
yield final concentrations of approximately 0.01 mM and 0.2 mM,
respectively. The solutions were then added to a rectangular
cuvette with 1 cm path length fitted with a glass stopper. The
absorption spectrum was subsequently registered between 250 and 750
nm. All spectra were accumulated one time with a bandwidth of 1.0
nm at a scan speed of 370 nm/min. For the aggregation time-series
measurements, spectra were obtained at baseline (immediately after
solvation; time zero) and then at the same intervals up to and
including 24 hours post-solvation (see FIG. 2-FIG. 7).
Concentration was held constant in the ethanolic titration of the
diphosphate lutein sodium salt, for which evidence of card-pack
aggregation was obtained (FIG. 5-FIG. 7).
[0216] Determination of aqueous solubility/dispersibility. 30.13 mg
of 3 was added to 1 mL of USP-purified water. The sample was
rotated for 2 hours, then centrifuged for 5 minutes. After
centrifuging, solid was visible in the bottom of the tube. A
125-.mu.L aliquot of the solution was then diluted to 25 mL. The
sample was analyzed by UVN is spectroscopy at 436 nm, and the
absorbance was compared to a standard curve compiled from 4
standards of known concentration. The concentration of the original
supernatant was calculated to be 2.85 mg/mL and the absorptivity
was 36.94 AU*mL/cm*mg. Slight error may have been introduced by the
small size of the original aliquot.
[0217] Next, 30.80 mg of 9 was added to 1 mL of USP-purified water.
The sample was rotated for 2 hours, then centrifuged for 5 minutes.
After centrifuging, solid was visible in the bottom of the tube. A
125-.mu.L aliquot of the solution was then diluted to 25 mL. The
sample was analyzed by UV/Vis spectroscopy at 411 nm, and the
absorbance was compared to a standard curve compiled from 4
standards of known concentration. The concentration of the original
supernatant was calculated to be 29.27 mg/mL and the absorptivity
was 2.90 AU*mL/cm*mg. Slight error may have been introduced by the
small size of the original aliquot.
[0218] Leukocyte Isolation and Preparation. Human polymorphonuclear
leukocytes (PMNs) were isolated from freshly sampled venous blood
of a single volunteer (S.F.L.) by Percoll density gradient
centrifugation as described previously. Briefly, each 10 mL of
whole blood was mixed with 0.8 mL of 0.1 M EDTA and 25 mL of
saline. The diluted blood was then layered over 9 mL of Percoll at
a specific density of 1.080 g/mL. After centrifugation at
400.times.g for 20 min at 20.degree. C., the plasma, mononuclear
cell, and Percoll layers were removed. Erythrocytes were
subsequently lysed by addition of 18 mL of ice-cold water for 30 s,
followed by 2 mL of 10.times. PIPES buffer (25 mM PIPES, 110 mM
NaCl, and 5 mM KCl, titrated to pH 7.4 with NaOH). Cells were then
pelleted at 4.degree. C., the supernatant was decanted, and the
procedure was repeated. After the second hypotonic cell lysis,
cells were washed twice with PAG buffer [PIPES buffer containing
0.003% human serum albumin (HSA) and 0.1% glucose]. Afterward, PMNs
were counted by light microscopy on a hemocytometer. The isolation
yielded PMNs with a purity of >95%. The final pellet was then
suspended in PAG-CM buffer (PAG buffer with 1 mM CaCl.sub.2 and 1
mM MgCl.sub.2). EPR Measurements. All EPR measurements were
performed using a Bruker ER 300 EPR spectrometer operating at
X-band with a TM.sub.110 cavity as previously described. The
microwave frequency was measured with a Model 575 microwave counter
(EIP Microwave, Inc., San Jose, Calif.). To measure superoxide
anion (O{overscore (.tangle-solidup.)}.sub.2) generation from
phorbol-ester (PMA)-stimulated PMNs, EPR spin-trapping studies were
performed using the spin trap DEPMPO (Oxis, Portland, Oreg.) at 10
mM. 1.times.10.sup.6 PMNs were stimulated with PMA (1 ng/mL) and
loaded into capillary tubes for EPR measurements. To determine the
radical scavenging ability of 3 and 9 in aqueous and ethanolic
formulations, PMNs were pre-incubated for 5 minutes with test
compound, followed by PMA stimulation.
[0219] Instrument settings used in the spin-trapping experiments
were as follows: modulation amplitude, 0.32 G; time constant, 0.16
s; scan time, 60 s; modulation frequency, 100 kHz; microwave power,
20 milliwatts; and microwave frequency, 9.76 GHz. The samples were
placed in a quartz EPR flat cell, and spectra were recorded. The
component signals in the spectra were identified and quantified as
reported previously.
UV/Vis Spectral Properties in Organic and Aqueous Solvents.
[0220] UV-Vis spectral evaluation of the disuccinate lutein sodium
salt is depicted in FIG. 2-FIG. 4. FIG. 2 depicts a time series of
the UV/Vis absorption spectra of the disodium disuccinate
derivative of natural source lutein in water. The .lamda..sub.max
(443 nm) obtained at time zero did not appreciably blue-shift over
the course of 24 hours, vibrational fine structure was maintained
(% III/II=35%), and the spectra became only slightly hypochromic
(i.e. decreased in absorbance intensity) over time, indicating
minimal time-dependent supramolecular assembly (aggregation) of the
card-pack type during this time period. Existence of head-to-tail
(J-type) aggregation in solution cannot be ruled out.
[0221] FIG. 3 depicts a UV/Vis absorption spectra of the disodium
disuccinate derivative of natural source lutein in water
(.lamda..sub.max=443 nm), ethanol (.lamda..sub.max=446 nm), and
DMSO (.lamda..sub.max=461 nm). Spectra were obtained at time zero.
A prominent cis peak is seen with a maximum at 282 nm in water. The
expected bathochromic shift of the spectrum in the more polarizable
solvent (DMSO) is seen (461 nm). Only a slight hypsochromic shift
is seen between the spectrum in water and that in ethanol,
reflecting minimal card-pack aggregation in aqueous solution.
Replacement of the main visible absorption band observed in EtOH by
an intense peak in the near UV region--narrow and displaying no
vibrational fine structure--is not observed in the aqueous solution
of this highly water-dispersible derivative, in comparison to the
spectrum of pure lutein in an organic/water mixture.
[0222] FIG. 4 depicts a UV/Vis absorption spectra of the disodium
disuccinate derivative of natural source lutein in water
(.lamda..sub.max=442 nm) with increasing concentrations of ethanol.
The .lamda..sub.max increases to 446 nm at an EtOH concentration of
44%, at which point no further shift of the absorption maximum
occurs (i.e. a molecular solution has been achieved), identical to
that obtained in 100% EtOH (See FIG. 3).
[0223] UV-Vis spectral evaluation of the diphosphate lutein sodium
salt is depicted in FIG. 5-FIG. 7. FIG. 5 depicts a time series of
the UV/Vis absorption spectra of the disodium diphosphate
derivative of natural source lutein in water. Loss of vibrational
fine structure (spectral distribution beginning to approach
unimodality) and the blue-shifted lambda max relative to the lutein
chromophore in EtOH suggested that card-pack aggregation was
present immediately upon solvation. The .lamda..sub.max (428 nm)
obtained at time zero did not appreciably blue-shift over the
course of 24 hours, and the spectra became slightly more
hypochromic over time (i.e. decreased in absorbance intensity),
indicating additional time-dependent supramolecular assembly
(aggregation) of the card-pack type during this time period. This
spectrum was essentially maintained over the course of 24 hours
(compare with FIG. 2, disuccinate lutein sodium salt).
[0224] FIG. 6 depicts a UV/Vis absorption spectra of the disodium
diphosphate derivative of natural source lutein in 95% ethanol
(.lamda..sub.max=446 nm), 95% DMSO (.lamda..sub.max=459 nm), and
water (.lamda..sub.max=428 nm). A red-shift was observed
(.lamda..sub.max to 446 nm), as was observed with the disuccinate
derivate. Wetting of the diphosphate lutein derivative with a small
amount of water was required to obtain appreciable solubility in
organic solvent (e.g. EtOH and DMSO). Spectra were obtained at time
zero. The expected bathochromic shift (in this case to 459 nm) of
the spectrum in the more polarizable solvent (95% DMSO) is seen.
Increased vibrational fine structure and red-shifting of the
spectra were observed in the organic solvents.
[0225] FIG. 7 depicts a UV/Vis absorption spectra of the disodium
diphosphate derivative of natural source lutein in water
(.lamda..sub.max=428 nm) with increasing concentrations of ethanol.
Concentration of the derivative was held constant for each
increased concentration of EtOH in solution. The .lamda..sub.max
increases to 448 nm at an EtOH concentration of 40%, at which no
further shift of the absorption maximum occurs (i.e. a molecular
solution is reached).
Direct Superoxide Anion Scavenging by EPR Spectroscopy
[0226] The mean percent inhibition of superoxide anion signal
(.+-.SEM) as detected by DEPMPO spin-trap by the disodium
disuccinate derivative of natural source lutein (tested in water)
is shown in FIG. 8. A 100 .mu.M formulation (0.1 mM) was also
tested in 40% EtOH, a concentration shown to produce a molecular
(i.e. non-aggregated) solution. As the concentration of the
derivative increased, inhibition of superoxide anion signal
increased in a dose-dependent manner. At 5 mM, approximately 3/4
(75%) of the superoxide anion signal was inhibited. No significant
scavenging (0% inhibition) was observed at 0.1 mM in water.
Addition of 40% EtOH to the derivative solution at 0.1 mM did not
significantly increase scavenging over that provided by the EtOH
vehicle alone (5% inhibition). The millimolar concentration
scavenging by the derivative was accomplished in water alone,
without the addition of organic co-solvent (e.g., acetone, EtOH),
heat, detergents, or other additives. This data suggested that
card-pack aggregation for this derivative was not occurring in
aqueous solution (and thus limiting the interaction of the
aggregated carotenoid derivative with aqueous superoxide
anion).
[0227] The mean percent inhibition of superoxide anion signal
(.+-.SEM) as detected by DEPMPO spin-trap by the disodium
diphosphate derivative of natural source lutein (tested in water)
is shown in FIG. 9. A 100 .mu.M formulation (0.1 mM) was also
tested in 40% EtOH, a concentration also shown to produce a
molecular (i.e. non-aggregated) solution of this derivative. As the
concentration of the derivative increased, inhibition of the
superoxide anion signal increased in a dose-dependent manner. At 5
mM, slightly more than 90% of the superoxide anion signal was
inhibited (versus 75% for the disuccinate lutein sodium salt). As
for the disuccinate lutein sodium salt, no apparent scavenging (0%
inhibition) was observed at 0.1 mM in water. However, a significant
increase over background scavenging by the EtOH vehicle (5%) was
observed after the addition of 40% EtOH, resulting in a mean 18%
inhibition of superoxide anion signal. This suggested that
disaggregation of the compound lead to an increase in scavenging
ability by this derivative, pointing to slightly increased
scavenging ability of molecular solutions of the more
water-dispersible diphosphate derivative relative to the
disuccinate derivative. Again, the millimolar concentration
scavenging by the derivative was accomplished in water alone,
without the addition of organic co-solvent (e.g., acetone, EtOH),
heat, detergents, or other additives. TABLE-US-00001 TABLE 1
Descriptive statistics of mean % inhibition of superoxide anion
signal for aqueous and ethanolic (40%) formulations of disodium
disuccinate derivatives of natural source lutein tested in the
current study. Mean (% Sample Solvent Concentration N inhibition)
S.D. SEM Min Max Range Lutein 40% 0.1 mM 3 5.0 4.4 2.5 0 8 8
Disuccinate EtOH Sodium Salt Lutein Water 0.1 mM 1 0.0 ND ND 0 0 0
Disuccinate Sodium Salt Lutein Water 1.0 mM 3 13.0 5.6 3.2 8 19 11
Disuccinate Sodium Salt Lutein Water 3.0 mM 3 61.7 4.0 2.3 58 66 8
Disuccinate Sodium Salt Lutein Water 5.0 mM 3 74.7 4.5 2.6 70 79 9
Disuccinate Sodium Salt Sample sizes of 3 were evaluated for each
formulation, with the exception of natural source lutein in 40%
EtOH stock solution (N = 1). Mean % inhibition did not increase
over background levels until sample concentration reached 1 mM in
water; likewise, addition of 40% EtOH at the 0.1 mM concentration
did not increase scavenging over background levels attributable to
the EtOH vehicle (mean = 5% inhibition).
[0228] TABLE-US-00002 TABLE 2 Descriptive statistics of mean %
inhibition of superoxide anion signal for aqueous and ethanolic
(40%) formulations of disodium diphosphate derivatives of natural
source lutein tested in the current study. Mean (% Sample Solvent
Concentration N inhibition) S.D. SEM Min Max Range Lutein
(PO.sub.4).sub.2 40% 0.1 mM 3 18.0 7.0 4.0 11 25 14 Na Salt EtOH
Lutein (PO.sub.4).sub.2 Water 0.1 mM 1 0.0 ND ND 0 0 0 Na Salt
Lutein (PO.sub.4).sub.2 Water 1.0 mM 3 9.3 3.5 2.0 6 13 7 Na Salt
Lutein (PO.sub.4).sub.2 Water 3.0 mM 3 72.3 3.1 1.8 69 75 6 Na Salt
Lutein (PO.sub.4).sub.2 Water 5.0 mM 3 91.0 2.6 1.5 88 93 5 Na Salt
Sample sizes of 3 were evaluated for each formulation, with the
exception of lutein diphosphate in water at 100 .mu.M (0.1 mM)
where N = 1. Mean % inhibition of superoxide anion signal increased
in a dose-dependent manner as the concentration of lutein
diphosphate was increased in the test assay. At 100 .mu.M in water,
no inhibition of scavenging was seen. The molecular solution in 40%
EtOH (mean % inhibition = 18%) was increased above background
scavenging (5%) by the ethanolic vehicle, suggesting that
disaggregation increased scavenging at that concentration. Slightly
increased # scavenging (on a molar basis) may have been obtained
with the diphosphate derivative in comparison to disuccinate
derivative (see Table 1 and FIG. 8).
[0229] In the current study, facile preparations of the disodium
disuccinate and tetrasodium phosphate esters of natural source
(RRR) lutein are described. These asymmetric C40 carotenoid
derivatives exhibited aqueous dispersibility of 2.85 and 29.27
mg/mL, respectively. Evidence for both card-pack (H-type) and
head-to-tail (J-type) supramolecular assembly was obtained with
UV-Vis spectroscopy for the aqueous solutions of these compounds.
Electronic paramagnetic spectroscopy of direct aqueous superoxide
scavenging by these derivatives demonstrated nearly identical
dose-dependent scavenging profiles, with slightly increased
scavenging noted for the diphosphate derivative. In each case,
scavenging in the millimolar range was observed. These results show
that as parenteral soft drugs with aqueous radical scavenging
activity, both compounds are useful in those clinical applications
in which rapid and/or intravenous delivery is desired for the
desired therapeutic effect(s).
Experimental Methods
[0230] Preparation of stock solutions of Cardax.TM. (DDA) and
placebo for injection. DDA was from a lot previously characterized
in detail (Frey et al. 2004). The crystalline material was
dissolved directly in sterile-filtered (0.2 micron Millipore.RTM.
filter) deionized water. The maximum aqueous dispersibility of DDA
is slightly greater than 10 mM (8.64 mg/ml). Sterile sodium
chloride solution (0.9%) for injection was used as the treatment
(placebo) for the control group. DDA or placebo solution was
administered by slow ear vein injection using an infusion pump set
at 1 ml/min.
[0231] Dosing schedule. Male New Zealand white rabbits (2.3-2.6 kg)
were assigned randomly to two separate groups. Each animal received
DDA aqueous formulation (50 mg/kg), or an equal volume of sterile
NaCl solution, once per day intravenously. The dose of DDA was
selected based on the findings of previous investigations in which
it was determined that a dosing regimen over four days produced
statistically significant myocardial salvage in Sprague-Dawley rats
(41% mean salvage at 50 mg/kg) and mongrel canines (68% mean
salvage at 50 mg/kg) after ischemia and reperfusion (Gross and
Lockwood, 2004; Gross and Lockwood, In Press). The animals in each
group received the respective treatments on each of four
consecutive days, with the experimental protocol being initiated on
fifth day.
[0232] Surgical preparation and experimental occlusion. One day
after the last treatment (DDA or placebo), rabbits were
anesthetized with a combination of xylazine (3.0 mg/kg) and
ketamine (35 mg/kg) administered intramuscularly, followed by an
intravenous injection of sodium pentobarbital (15 mg/kg). An
endotracheal tube was inserted and the animals were placed on a
positive pressure ventilator (Harvard Apparatus, Cambridge, Mass.).
The right jugular vein was cannulated for blood sampling and the
right carotid artery was instrumented with a Millar catheter
micro-tip pressure transducer (Millar Instruments Inc., Houston,
Tex.). The Millar catheter transducer was positioned immediately
above the aortic valves to monitor aortic blood pressure. The lead
II electrocardiogram was monitored throughout the protocol. A left
thoracotomy and pericardiotomy were performed, followed by
identification of the left anterior descending coronary artery. A
silk suture (3-0; Genzyme Corporation, Fall River, Mass.) was
passed under the artery and around a short length of polyethylene
tubing. Simultaneous downward displacement of the polyethylene
tubing while applying upward traction on the suture resulted in
occlusion of the coronary artery and cessation of regional blood
flow. Coronary artery occlusion was maintained for 30 min after
which time reperfusion was initiated by withdrawing the
polyethylene tubing. Regional myocardial ischemia was verified by
the presence of a zone of cyanosis in the area of distribution of
the occluded vessel and by changes in the electrocardiogram
consistent with the presence of transmural regional myocardial
ischemia (ST-segment elevation). Experimental protocol. The animals
were allowed to stabilize for 15 min before beginning the protocol
that involved both a vehicle control and a DDA-treated group.
Cessation of coronary blood flow was maintained for 30 minutes
after which the ligature was removed and the heart was allowed to
reperfuse for a period of three hours before terminating the
study.
[0233] Tetrazolium method to determine infarct size. At the
completion of the 3 hr reperfusion period, the hearts were removed,
the aorta was cannulated, and the coronary vascular bed was
perfused on a Langendorff apparatus with Krebs-Henseleit buffer at
a constant flow of 30 to 32 ml/min. The hearts were perfused with
buffer for 10 min to clear the vascular compartment of plasma and
blood cellular elements. Fifty milliliters of a 1% solution of
triphenyltetrazolium chloride (TTC, Sigma, St. Louis, Mo.) in
phosphate buffer (pH 7.4, 37.degree. C.) was perfused through the
heart. TTC demarcates the non-infarcted myocardium within the area
at risk with a brick red color, indicating the presence of a
formazan precipitate resulting from reduction of TTC by
dehydrogenases present in viable myocardial tissue. Irreversibly
injured tissue, lacking cytosolic dehydrogenases, is unable to form
the formazan precipitate and appears pale yellow. Upon completion
of the TTC infusion, the left anterior descending coronary artery
was ligated at the site identical to that ligated during the
induction of regional myocardial ischemia. The perfusion pump was
stopped, and 3 ml of a 0.25% solution of Evan's Blue was injected
slowly through a side-arm port connected to the aortic cannula. The
dye was passed through the heart for 15 sec to ensure its uniform
tissue distribution. The presence of Evan's Blue was used to
demarcate the left ventricular tissue that was not subjected to
regional ischemia, as opposed to the risk region. The heart was
removed from the perfusion apparatus and cut into transverse
sections at right angles to the vertical axis. The right ventricle,
apex, and atrial tissue were discarded. Both surfaces of each
tissue section were traced onto clear acetate sheets. The images
were photocopied and enlarged, then digitized using a flatbed
scanner. The areas of the normal left ventricle non-risk region,
area at risk, and infarct region were determined by calculating the
number of pixels occupying each area using Adobe PhotoShop software
(Adobe Systems, Seattle, Wash.). Total area at risk is expressed as
the percentage of the left ventricle. Infarct size is expressed as
the percentage of the area at risk.
[0234] Plasma and tissue concentrations of non-esterified free
astaxanthin. To determine the plasma and tissue concentrations of
non-esterified, free astaxanthin in blood and organs, samples were
taken at the end of reperfusion in selected rabbits (n=5) treated
with DDA, and determined by methods previously described (Osterlie
et al., 2000). Non-esterified, free astaxanthin, in vivo, is
generated after cleavage of the water-dispersible disuccinate
diester to monosuccinate, and subsequently to non-esterified, free
astaxanthin by the intrinsic esterase activity of serum albumin
(Curry et al., 1999), or by non-specific esterase activity in
plasma and solid organs (Jensen et al., 1999). Non-esterified, free
astaxanthin then accumulates in myocardium and other tissues after
plasma clearance in a dose-dependent manner after both oral
(Showalter et al., 2004) and intravenous administration (Gross and
Lockwood 2004a,b).
[0235] Measurement of cardiac-specific troponin 1. Whole blood was
drawn at baseline (pre-ischemia) and at the end of reperfusion for
the determination of cardiac-specific troponin I (cTnl). Serum
levels of the proteins were measured using a commercially available
ELISA kit. Collected plasma samples were prepared from whole blood
and were snap frozen in liquid nitrogen. The samples were stored at
-80.degree. C. until the day of the assay when they were thawed
over ice and diluted appropriately with the sample diluent supplied
with each assay kit. Determination of the target protein using a
protein standard curve was performed according to standard
procedure in the art.
[0236] Analysis of MAC and CRP deposition in tissues by indirect
immunofluorescence. The immunofluorescent method for detection of
CRP was performed essentially as described previously (Lauver et
al., 2005). Briefly, tissue samples used for infarct size
determination were fixed in 10% buffered formalin immediately after
the completion of the experimental protocol. The tissue samples
were embedded in paraffin blocks and cut into sections of 2 .mu.m
in thickness, which were then mounted on glass slides. Two
consecutive sections (mirror images) from a single heart slice were
mounted on each slide. The slides were deparaffinized and subjected
to antigen unmasking using a commercially available kit for this
purpose (Vector Laboratories, Burlingame, Calif.). After blocking
for 30 minutes, primary antibodies were incubated at room
temperature in a humidity chamber for 45 minutes. One section per
slide was incubated with a chicken anti-rabbit CRP antibody (1.5
.mu.g/ml final concentration, Strategic BioSolutions, Newark, Del.)
and the other section was incubated with a chicken anti-rabbit MAC
antibody (1:2500 final dilution, developed in conjunction with
Lampire Biological Laboratories, Pipersville, Pa.). Both sections
were incubated with a biotinylated goat anti-chicken secondary
antibody (1.5 .mu.g/ml final concentration, Vector Laboratories)
for 30 minutes. The slides were incubated with Fluorescein and
Texas Red (CRP and MAC sections, respectively)-labeled streptavidin
(Fluorescent Streptavidin Kit, Vector Laboratories) to visualize
the proteins. ProLong Gold antifade mounting medium (Molecular
Probes, Eugene, Oreg.) and coverslips were used to preserve the
sections. For comparison, digital images were captured using a
digital camera (Sony DKC5000; Sony Corporation of America, New
York, N.Y.) connected to a Leica fluorescent stereoscope (Leica MZ
FLIII) and the accompanying software (Leica Microsystems Inc.,
Bannockburn, Ill.). Images were analyzed using 1P Lab (Scanalytics,
Inc., Fairfax, Va.) software to determine mean fluorescence
intensity per heart section. The sections were normalized to the
amount of background on each slide. The mean intensities for three
hearts in each treatment group were averaged and compared.
[0237] Assessment of complement inhibition. A red blood cell (RBC)
lysis assay was used to determine whether the pretreatment with DDA
compared to placebo-treated animals was able to inhibit the rabbit
complement system. The ex vivo analysis of complement activity is
based on the C5b-9-dependent lysis of human red blood cells upon
exposure to rabbit plasma. Complement-mediated RBC hemolysis was
assessed by a turbidometric method described previously (Pascual et
al., 1990). The hemolysis assay is an accepted method of assessing
the complement titer of plasma or serum samples (Whaley, 1985).
Rabbit plasma was obtained from whole blood samples drawn from
rabbits that were pretreated with DDA (50 mg/kg, 4 days, n=5) or
sterile 0.9% sodium chloride solution (4 days, n=5). After
obtaining informed consent, human whole blood for the isolation of
red blood cells was obtained by venipuncture of the forearm vein of
a healthy, male donor who had not been exposed to any medication
for the past seven days. The cells were washed three times in 10 ml
phosphate buffered saline (PBS, pH 7.4) and diluted in PBS to
achieve a final RBC concentration of 1.times.10.sup.8 cells/ml. The
assay was initiated by the addition of 15 .mu.l of diluted human
RBCs to 185 .mu.l of rabbit plasma, and the light transmittance was
monitored for 5 min. The final assay volume was 200 .mu.l. One
hundred percent light transmittance was set with RBCs lysed with a
1:1 mixture of rabbit plasma and deionized H.sub.2O.
[0238] Statistical Analysis. Results are expressed as the mean
values.+-.S.E.M. Parameters between the two groups were compared
using the Student's t test for unpaired comparisons. P values of
<0.05 and <0.01 are regarded as significant and denoted by an
asterisk and double asterisk, respectively.
Example 1
[0239] Determination of test animal vital signs. No differences in
heart rate, blood pressure, or blood gases at baseline or
throughout the experimental protocol performed on day 5 between the
two groups was observed (data not shown). Turning to FIG. 11, no
significant differences in areas at risk were observed between the
animals treated with DDA or with saline, indicating that both
groups were subjected to similar degrees of ischemia.
Example 2
[0240] Effect of DDA on Myocardial Infarct Size. Remaining with
FIG. 11, each treatment group consisted of 9 animals in which
either DDA or saline placebo was administered for 4 days before
commencing the experimental protocol involving myocardial
ischemia/reperfusion. The mean size of the area at risk expressed
as a percentage of the total left ventricle was similar in both
groups. Rabbits treated with DDA (50 mg/kg/day) exhibited
significantly smaller mean infarcts expressed as a percentage of
the area at risk (25.8.+-.4.2%) compared with rabbits treated with
placebo (52.5.+-.7.5%, **p<0.01). This represented mean
myocardial salvage of 51%. These results therefore demonstrate that
disodium disuccinate astaxanthin treatment can significantly reduce
the size of an infarct relative to the area of myocardium at risk
in rabbits subjected to 30 minutes of coronary artery occlusion
followed by a three hour period of reperfusion. DDA produced a mean
myocardial salvage of approximately 51% when the rabbits were dosed
with 50 mg/kg daily for four consecutive days. This level of
salvage at the 50 mg/kg subchronic intravenous dose is intermediate
between that obtained in rats (41% salvage) and mongrel dogs (68%),
demonstrating appropriate pharmacokinetic scaling across several
species of mammals (Gross and Lockwood, 2004); Gross and Lockwood
2004b).
Example 3
[0241] Plasma and tissue levels of non-esterified, free
astaxanthin. Turning to FIG. 12, the mean plasma concentration of
non-esterified, free astaxanthin at the end of 3 hours of
reperfusion is presented. Pretreatment with DDA at 50 mg/kg for 4
days resulted in a mean plasma concentration of 222.+-.51 nM.
However, the mean myocardial tissue concentration of DDA was
several orders of magnitude greater than that observed in the
plasma (FIG. 12), revealing highly favorable mean myocardium/serum
ratios in the rabbit after intravenous subchronic administration.
We were able to achieve plasma concentrations of non-esterified
astaxanthin that were roughly equal to those previously found in
other species using the same intravenous dosage regimen (Gross and
Lockwood, 2004; Gross and Lockwood, In Press). We also observed a
marked accumulation of non-esterified astaxanthin in the myocardium
(mean>10 .mu.M) in the rabbits utilized in this study. Rapid
plasma clearance of free astaxanthin, and excellent myocardium- and
hepatic/serum ratios had previously been demonstrated after oral
admistration of this compound to black mice (Showalter et al.
2004). The current results further demonstrate the favorable
pharmacokinetic profile of DDA in mammals.
Example 4
[0242] Serum levels of cardiac-specific troponin 1. Turning to FIG.
13, the mean serum concentrations of cTnI were similar at baseline
(pre-ischemia) in both treatment groups. DDA treated rabbits
exhibited a lower mean cTnI concentration at the end of reperfusion
as compared with vehicle controls. These results therefore
demonstrate that treatment of a subject with disodium disuccinate
astaxanthin can result in a mean reduction in the circulating
concentration of the biochemical injury marker, cardiac-specific
troponin I. Although the results did not achieve statistical
significance, clear evidence of a downward trend in this serum
marker of irreversible myocardial tissue injury was obtained. The
reduced statistical power observed in this study versus those
obtained in prior studies for this marker (Lauver et al. 2005) may
have been due to the curtailed period of reperfusion in the current
study. In other words, it is likely that statistically significant
differences in peak plasma cTnl may be achieved by prolonging
cardiac reperfusion time.
Example 5
[0243] Immunofluorescence. Along with the generation of reactive
oxygen species, the activation of the complement system serves an
integral role in myocardial reperfusion injury (Lucchesi, 1994).
Therefore we sought to investigate the effects of DDA on the tissue
deposition of CRP and the terminal complex (C5b-9; MAC), both of
which are recruited to an deposited on tissue undergoing
ischemia/reperfusion-associated inflammation. Left ventricular
tissue sections obtained from hearts that had been treated with
saline or with DDA, then subjected to 30 min of regional ischemia,
followed by 3 h of reperfusion were subjected to indirect
immunofluorescence analysis to the detect tissue deposition of CRP
and the MAC. Turning to FIG. 14, heart sections taken from the
infarct region in animals treated with saline (panels A and C,
respectively) demonstrated bright fluorescence with both anti-CRP
(green) and anti-MAC (red) antibodies, indicating the deposition of
both proteins in the area of infarction. Conversely, hearts treated
with DDA (panels B and D, respectively) exhibited significantly
reduced fluorescence, indicative of a reduction in the deposition
of CRP and MAC in the infarct region. The mean intensity of
fluorescence (panel E) in heart sections obtained after treatment
with DDA was significantly (*p<0.05) lower in tissue sections
stained for either CRP or MAC. These results therefore demonstrate
that treatment of a subject with disodium disuccinate astaxanthin
can significantly reduce the deposition of CRP and the MAC in
damaged tissue.
Example 5
[0244] Inhibition of complement activation. The erythrocyte
hemolysis assay was used to determine the ability of DDA to inhibit
the activation of the complement system (FIG. 15). DDA
significantly attenuated complement-mediated erythrocyte lysis
after the 3 h reperfusion period. The hemolytic response was
followed for 300 seconds. Values are expressed as mean.+-.S.E.M.;
saline group, n=5 (white bars); DDA group, n=5 (black bars); **
p<0.01 versus saline. Pretreatment with DDA (50 mg/kg, 4 days)
significantly reduced (**p<0.01) mean rabbit plasma-induced
human erythrocyte hemolysis compared to plasma from placebo treated
rabbits.
[0245] In this patent, certain U.S. patents, U.S. patent
applications, and other materials (e.g., articles) have been
incorporated by reference. The text of such U.S. patents, U.S.
patent applications, and other materials is, however, only
incorporated by reference to the extent that no conflict exists
between such text and the other statements and drawings set forth
herein. In the event of such conflict, then any such conflicting
text in such incorporated by reference U.S. patents, U.S. patent
applications, and other materials is specifically not incorporated
by reference in this patent.
[0246] Further modifications and alternative embodiments of various
aspects of the invention may be apparent to those skilled in the
art in view of this description. Accordingly, this description is
to be construed as illustrative only and is for the purpose of
teaching those skilled in the art the general manner of carrying
out the invention. It is to be understood that the forms of the
invention shown and described herein are to be taken as the
presently preferred embodiments. Elements and materials may be
substituted for those illustrated and described herein, parts and
processes may be reversed, and certain features of the invention
may be utilized independently, all as would be apparent to one
skilled in the art after having the benefit of this description to
the invention. Changes may be made in the elements described herein
without departing from the spirit and scope of the invention as
described in the following claims. In addition, it is to be
understood that features described herein independently may, in
certain embodiments, be combined.
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