U.S. patent application number 12/981261 was filed with the patent office on 2011-07-21 for oxylipins from long chain polyunsaturated fatty acids and methods of making and using the same.
This patent application is currently assigned to MARTEK BIOSCIENCES CORPORATION. Invention is credited to Linda Arterburn, William Barclay, Bindi Dangi, James Flatt, Jung Lee, Mary Van Elswyk.
Application Number | 20110178047 12/981261 |
Document ID | / |
Family ID | 36407879 |
Filed Date | 2011-07-21 |
United States Patent
Application |
20110178047 |
Kind Code |
A1 |
Arterburn; Linda ; et
al. |
July 21, 2011 |
Oxylipins From Long Chain Polyunsaturated Fatty Acids and Methods
of Making and Using the Same
Abstract
Disclosed are novel oxylipins, referred to herein as
docosanoids, that are derived from C22 polyunsaturated fatty acids,
and method of making and using such oxylipins. Also disclosed is
the use of docosapentaenoic acid (C22:5n-6) (DPAn-6),
docosapentaenoic acid (C22:5n-3) (DPAn-3), and docosatetraenoic
acid (DTAn-6: C22:4n-6) as substrates for the production of novel
oxylipins, and to the oxylipins produced thereby. Also disclosed is
the use of DPAn-6, DPAn-3, DTAn-6, and/or the oxylipins derived
therefrom, and/or novel docosanoids derived from the structures of
C22 fatty acids, in therapeutic and nutritional or cosmetic
applications, and particularly as anti-inflammatory or
anti-neurodegenerative compounds. The invention also relates to
novel ways of producing long chain polyunsaturated acid
(LCPUFA)-rich oils and compositions that contain enhanced and
effective amounts of LCPUFA-derived oxylipins, and particularly,
docosanoids.
Inventors: |
Arterburn; Linda; (Ellicott
City, MD) ; Barclay; William; (Boulder, CO) ;
Dangi; Bindi; (Elkridge, MD) ; Flatt; James;
(Colorado Springs, CO) ; Lee; Jung; (McLean,
VA) ; Van Elswyk; Mary; (Longmont, CO) |
Assignee: |
MARTEK BIOSCIENCES
CORPORATION
Columbia
MD
|
Family ID: |
36407879 |
Appl. No.: |
12/981261 |
Filed: |
December 29, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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|
11284790 |
Nov 21, 2005 |
7884131 |
|
|
12981261 |
|
|
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60729038 |
Oct 21, 2005 |
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60629842 |
Nov 19, 2004 |
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Current U.S.
Class: |
514/163 ;
514/560; 554/219 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 17/00 20180101; A61P 29/00 20180101; C12P 7/6427 20130101;
A61P 7/10 20180101; C07C 59/42 20130101; A61P 25/00 20180101; C07D
303/38 20130101; C12P 7/6472 20130101; A61P 25/28 20180101 |
Class at
Publication: |
514/163 ;
554/219; 514/560 |
International
Class: |
A61K 31/202 20060101
A61K031/202; C07C 59/42 20060101 C07C059/42; A61K 31/616 20060101
A61K031/616; A61P 29/00 20060101 A61P029/00; A61P 25/28 20060101
A61P025/28 |
Claims
1. (canceled)
2. An isolated docosanoid that is an R- or S-epimer of a docosanoid
selected from the group consisting of: monohydroxy derivatives of
DPAn-6, dihydroxy derivatives of DPAn-6, and tri-hydroxy
derivatives of DPAn-6.
3. The isolated docosanoid of claim 1, wherein the docosanoid is an
R- or S-epimer of a docosanoid selected from the group consisting
of: 7-hydroxy DPAn-6; 8-hydroxy DPAn-6; 10-hydroxy DPAn-6;
11-hydroxy DPAn-6; 13-hydroxy DPAn-6; 14-hydroxy DPAn-6; 17-hydroxy
DPAn-6; 7,17-dihydroxy DPAn-6; 10,17-dihydroxy DPAn-6;
13,17-dihydroxy DPAn-6; 7,14-dihydroxy DPAn-6; 8,14-dihydroxy
DPAn-6; 16,17-dihdroxy DPAn-6; 4,5-dihydroxy DPAn-6;
7,16,17-trihydroxy DPAn-6; and 4,5,17-trihydroxy DPAn-6.
4. (canceled)
5. A composition comprising at least one docosanoid of claim 2,
wherein the composition is a therapeutic composition, a nutritional
composition, or a cosmetic composition.
6-42. (canceled)
43. A method to prevent or reduce at least one symptom of
inflammation or neurodegeneration in an individual, comprising
administering to an individual at risk of, diagnosed with, or
suspected of having inflammation or neurodegeneration or a
condition or disease related thereto, an agent selected from the
group consisting of: DPAn-6, and an oxylipin derivative of DPAn-6,
to reduce at least one symptom of inflammation or neurodegeneration
in the individual.
44-54. (canceled)
55. The method of claim 43, wherein the oxylipin derivative is an
R- or S-epimer of a docosanoid selected from the group consisting
of: 7-hydroxy DPAn-6; 8-hydroxy DPAn-6; 10-hydroxy DPAn-6;
11-hydroxy DPAn-6; 13-hydroxy DPAn-6; 14-hydroxy DPAn-6; 17-hydroxy
DPAn-6; 7,17-dihydroxy DPAn-6; 10,17-dihydroxy DPAn-6;
13,17-dihydroxy DPAn-6; 7,14-dihydroxy DPAn-6; 8,14-dihydroxy
DPAn-6; 16,17-dihdroxy DPAn-6; 4,5-dihydroxy DPAn-6;
7,16,17-trihydroxy DPAn-6; and 4,5,17-trihydroxy DPAn-6.
56. (canceled)
57. The method of claim 43, wherein the agent is selected from the
group consisting of 17-hydroxy DPAn-6 and 10,17-dihydroxy
DPAn-6.
58. The method of claim 43, wherein the agent is 17-hydroxy
DPAn-6.
59. The method of claim 43, wherein the agent is 10,17-dihydroxy
DPAn-6.
60. The method of claim 43, wherein the agent is DPAn-6.
61. (canceled)
62. The method of claim 43, further comprising administering
aspirin to the individual.
63. The method of claim 43, further comprising administering at
least one agent selected from the group consisting of a statin, a
non-steroidal anti-inflammatory agent, an antioxidant, and a
neuroprotective agent, to the individual.
64-122. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority from U.S.
Provisional Application Ser. No. 60/629,842, filed Nov. 19, 2004,
and from U.S. Provisional Application Ser. No. 60/729,038, filed
Oct. 21, 2005. The entire disclosure of each of U.S. Provisional
Application Ser. No. 60/629,842, filed Nov. 19, 2004, and U.S.
Provisional Application Ser. No. 60/729,038 is incorporated herein
by reference.
FIELD OF THE INVENTION
[0002] This invention generally relates to the use of
docosapentaenoic acid (C22:5n-6) (DPAn-6), docosapentaenoic acid
(C22:5n-3) (DPAn-3), and docosatetraenoic acid (DTAn-6: C22:4n-6)
as substrates for the production of novel oxylipins, and to the
oxylipins produced thereby. The invention further relates to the
use of DPAn-6, DPAn-3, DTAn-6, and/or the oxylipins derived
therefrom, particularly as anti-inflammatory compounds. The
invention also relates to novel ways of producing long chain
polyunsaturated acid (LCPUFA)-rich oils and compositions that
contain enhanced and effective amounts of LCPUFA-derived oxylipins,
and particularly, docosanoids.
BACKGROUND OF THE INVENTION
[0003] Researchers in the 1990s identified hydroxy derivatives of
some fatty acids in macroalgae (seaweeds) and described the
possible role of these compounds in wound healing and cell
signaling in the organisms (Gerwick & Bernart 1993; Gerwick et
al 1993; Gerwick 1994). They recognized these compounds to be
similar to those produced in the human body through the
lipoxygenase pathway. These same researchers also attempted to
develop cell suspension cultures of these seaweeds to produce
eicosanoids and related oxylipins from C18 fatty acids (linoleic
acid, and linolenic acid) and arachidonic acid (C20:4n-6) (ARA) in
the red, brown and green seaweeds. However, production of seaweed
biomass in these cultures systems proved to be very poor (e.g.
about 0.6 to 1.0 g/L seaweed biomass after 15 days (Rorrer et al.
1996)) and even direct addition of key fatty acids to the cultures
only minimally increased production of oxylipins over that of
controls (Rorrer et al. 1997). Additionally, in some cases, the
added free fatty acids proved toxic to the cultures (Rorrer et al.
1997). Therefore these systems have only remained academically
interesting for producing oxygenated forms of these fatty acids,
and studies continue on the C18 and C20 oxylipins in these seaweeds
(e.g., Bouarab et al. 2004).
[0004] The oxylipins from the long chain omega-6 (n-6 or .omega.-6
or N6) fatty acid, ARA, have been well studied and are generally
considered to be proinflammatory in humans. Oxylipins from the long
chain omega-3 (n-3 or .omega.-3 or N3) fatty acids, however, have
generally been found to be anti-inflammatory. In the early 2000's,
Serhan and other researchers discovered that hydroxylated forms of
two long chain omega-3 polyunsaturated fatty acids (omega-3
LCPUFAs) (i.e., eicosapentaenoic acid (C20:5, n-3) (EPA) and
docosahexaenoic acid C22:6, n-3) (DHA)) were made in the human body
(Serhan et al. 2004a,b; Bannenberg et al. 2005a,b) They identified
pathways whereby the omega-3 (n-3 or .omega.-3) LCPUFAs, EPA and
DHA, were processed by cyclooxygenases, acetylated cyclooxygenase-2
or by lipoxygenase enzymes, resulting in production of novel mono-,
di- and tri-hydroxy derivatives of these fatty acids. The resulting
compounds, which were named "resolvins" (because they were involved
in the resolution phase of acute inflammation) or docosatrienes
(because they were made from docosahexaenoic acid and contain
conjugated double bonds), were determined to have strong
anti-inflammatory (Arita et al. 2005a,b,c; Flower & Perretti
2005; Hong et al. 2003; Marcjeselli et al. 2003; Aria et al. 2005),
antiproliferative, and neuroprotective (Bazan 2005a,b; Bazan et al.
2005; Belayev et al. 2005; Butovich et al. 2005; Chen & Bazan
2005; Lukiw et al. 2005; Mukherjee et al 2004) properties. These
compounds were also noted to have longer half-lives in the human
body as compared to other types of eicosanoids.
[0005] In the past few years, various patents and patent
application publications have described analogs of hydroxy
derivatives of ARA, DHA and EPA, the pathways by which they are
formed, methods for their synthesis in the laboratory via organic
synthetic means or through biogenesis using cyclooxygenase or
lipoxygenase enzymes, and use of these hydroxy derivatives as
pharmaceutical compounds for the treatment of inflammatory
diseases. These patents and publications are summarized briefly
below.
[0006] U.S. Pat. No. 4,560,514 describes the production of both
pro-inflammatory (LX-A) and anti-inflammatory tri-hydroxy lipoxins
(LX-B) derived from arachidonic acid (ARA). Use of these compounds
in both studying and preventing inflammation (as pharmaceutical
compounds) are also described.
[0007] U.S. Patent Application Publication No. 2003/0166716
describes the use of lipoxins (derived from ARA) and
aspirin-triggered lipoxins in the treatment of asthma and
inflammatory airway diseases. Chemical structures of various
anti-inflammatory lipoxin analogs are also taught.
[0008] U.S. Patent Application Publication No. 2003/0236423
discloses synthetic methods based on organic chemistry for
preparing trihydroxy polyunsaturated eicosanoids and their
structural analogs including methods for preparing derivatives of
these compounds. Uses for these compounds and their derivatives in
the treatment of inflammatory conditions or undesired cell
proliferation are also discussed.
[0009] PCT Publication No. WO 2004/078143 is directed to methods
for identifying receptors that interact with di- and tri-hydroxy
EPA resolving analogs.
[0010] U.S. Patent Application Publication No. 2004/0116408A1
discloses that the interaction of EPA or DHA in the human body with
cyclooxygenase-II (COX2) and an analgesic such as aspirin leads to
the formation of di- and tri-hydroxy EPA or DHA compounds with
beneficial effects relating to inflammation. It also teaches
methods of use and methods of preparing these compounds.
[0011] U.S. Patent Application Publication No. 2005/0075398A1
discloses that the docosatriene 10,17S-docosatriene (neuroprotectin
D1) appears to have neuroprotective effects in the human body.
[0012] PCT Publication No. WO 2005/089744A2 teaches that di- and
tri-hydroxy resolvin derivatives of EPA and DHA and stable analogs
thereof are beneficial in the treatment of airway diseases and
asthma.
[0013] While the references above describe lipoxins derived from
ARA and docosatrienes and resolvins derived from DHA and EPA, as
well as various applications of such compounds, there remains a
need in the art for alternative ways of delivering the
anti-inflammatory benefits and other benefits of these LCPUFA
oxylipins (and in particular docosanoids) to consumers other than
by providing consumers with combinations of LCPUFA oil and aspirin
or by chemically synthesizing these derivatives or their
analogs.
[0014] Moreover, none of the references above describe methods for
making these specific compounds in microbial cultures or plants,
nor do they describe methods for increasing the content of these
beneficial hydroxy fatty acid derivatives in edible oils. In
addition, none of these references describe any hydroxy derivatives
from other LCPUFAs, nor do any of these references suggest that
that there could be a beneficial role for hydroxy derivatives of
any LCPUFAs other than ARA, DHA and EPA.
SUMMARY OF THE INVENTION
[0015] One embodiment of the present invention generally relates to
an isolated docosanoid of docosapentaenoic acid (DPAn-6). Such a
docosanoid can include, but is not limited to, an R- or S-epimer of
a docosanoid selected from: monohydroxy derivatives of DPAn-6,
dihydroxy derivatives of DPAn-6, and tri-hydroxy derivatives of
DPAn-6. Such a docosanoid can more particularly include, but is not
limited to, an R- or S-epimer of a docosanoid selected from:
7-hydroxy DPAn-6; 8-hydroxy DPAn-6; 10-hydroxy DPAn-6; 11-hydroxy
DPAn-6; 13-hydroxy DPAn-6; 14-hydroxy DPAn-6; 17-hydroxy DPAn-6;
7,17-dihydroxy DPAn-6; 10,17-dihydroxy DPAn-6; 13,17-dihydroxy
DPAn-6; 7,14-dihydroxy DPAn-6; 8,14-dihydroxy DPAn-6;
16,17-dihdroxy DPAn-6; 4,5-dihydroxy DPAn-6; 7,16,17-trihydroxy
DPAn-6; and 4,5,17-trihydroxy DPAn-6; or an analog, derivative or
salt thereof.
[0016] Another embodiment of the present invention relates to an
isolated docosanoid of docosapentaenoic acid (DPAn-3). Such a
docosanoid can include, but is not limited to, an R- or S-epimer of
a docosanoid selected from: monohydroxy derivatives of DPAn-3,
dihydroxy derivatives of DPAn-3, and tri-hydroxy derivatives of
DPAn-3. Such a docosanoid can more particularly include, but is not
limited to, an R- or S-epimer of a docosanoid selected from:
7-hydroxy DPAn-3; 10-hydroxy DPAn-3; 11-hydroxy DPAn-3; 13-hydroxy
DPAn-3; 14-hydroxy DPAn-3; 16-hydroxy DPAn-3; 17-hydroxy DPAn-3;
7,17-dihydroxy DPAn-3; 10,17-dihydroxy DPAn-3; 8,14-dihydroxy
DPAn-3; 16,17-dihydroxy DPAn-3; 13,20-dihydroxy DPAn-3;
10,20-dihydroxy DPAn-3; and 7,16,17-trihydroxy DPAn-3; or an
analog, derivative or salt thereof.
[0017] Yet another embodiment of the present invention relates to
an isolated docosanoid of docosatetraenoic acid (DTAn-6). Such a
docosanoid can include, but is not limited to, an R- or S-epimer of
a docosanoid selected from: monohydroxy derivatives of DTAn-6,
dihydroxy derivatives of DTAn-6, and tri-hydroxy derivatives of
DTAn-6. Such a docosanoid can more particularly include, but is not
limited to, an R- or S-epimer of a docosanoid selected from:
7-hydroxy DTAn-6; 10-hydroxy DTAn-6; 13-hydroxy DTAn-6; 17-hydroxy
DTAn-6; 7,17-dihydroxy DTAn-6; 10,17-dihydroxy DTAn-6;
16,17-dihydroxy DTAn-6; and 7,16,17-trihydroxy DTAn-6; or an
analog, derivative or salt thereof.
[0018] Another embodiment of the present invention relates to an
isolated docosanoid of a C22 polyunsaturated fatty acid, wherein
the docosanoid is an R- or S-epimer of a docosanoid selected from:
4,5-epoxy-17-hydroxy DPA; 7,8-epoxy DHA; 10,11-epoxy DHA;
13,14-epoxy DHA; 19,20-epoxy DHA; 13,14-dihydroxy DHA;
16,17-dihydroxy DTAn-6; 7,16,17-trihydroxy DTAn-6;
4,5,17-trihydroxy DTAn-6; 7,16,17-trihydroxy DTAn-3;
16,17-dihydroxy DTAn-3; 16,17-dihydroxy DTRAn-6; 7,16,17-trihydroxy
DTRAn-6; 4,5-dihydroxy DTAn-6; and 10,16,17-trihydroxy DTRAn-6; or
an analog, derivative or salt thereof.
[0019] Another embodiment of the invention relates to a composition
comprising at least one of any of the above-described docosanoids.
The composition includes, but is not limited to, a therapeutic
composition, a nutritional composition or a cosmetic composition.
In one aspect, the composition further comprises aspirin. In
another aspect, the composition further comprises a compound
selected from: DPAn-6, DPAn-3, DTAn-6, DHA, EPA, an oxylipin
derivative of DHA and an oxylipin derivative of EPA. In another
aspect, the composition further comprises at least one agent
selected from: a statin, a non-steroidal anti-inflammatory agent,
an antioxidant, and a neuroprotective agent. In another aspect, the
composition further comprises a pharmaceutically acceptable
carrier. In yet another aspect, the composition comprises an oil
selected from: a microbial oil, a plant seed oil, and an aquatic
animal oil.
[0020] Yet another embodiment of the present invention relates to
an oil comprising at least about 10 .mu.g of docosanoid per gram of
oil. Other embodiments include an oil comprising at least about 20
.mu.g of docosanoid per gram of oil, at least about 50 .mu.g of
docosanoid per gram of oil, or at least about 100 .mu.g of
docosanoid per gram of oil. In one aspect, the docosanoid in the
above-identified oil is a polyunsaturated fatty acid selected from:
docosatetraenoic acid (DTAn-6), docosapentaenoic acid (DPAn-6),
docosapentaenoic acid (DPAn-3), docosahexaenoic acid (DHA), and
eicosapentaenoic acid (EPA). In another aspect, the docosanoid is
from a polyunsaturated fatty acid selected from: docosatetraenoic
acid (DTAn-6), docosapentaenoic acid (DPAn-6), and docosapentaenoic
acid (DPAn-3). In one aspect, the docosanoid is any of the
above-identified docosanoids. The oil can include, but is not
limited to, a microbial oil, a plant seed oil, and an aquatic
animal oil.
[0021] Another embodiment of the invention includes a composition
comprising any of the above-described oils, which can include, but
is not limited to, a therapeutic composition, a nutritional
composition or a cosmetic composition.
[0022] Yet another embodiment of the present invention relates to a
composition comprising a long chain polyunsaturated fatty acid
selected from: DPAn-6, DPAn-3, and DTAn-6 and a pharmaceutically or
nutritionally acceptable carrier. In one aspect, the composition
further comprises aspirin. In another aspect, the composition
further comprises an enzyme that catalyzes the production of the
docosanoids from DPAn-6, DTAn-6 or DPAn-3.
[0023] Another embodiment of the present invention relates to a
method to prevent or reduce at least one symptom of inflammation or
neurodegeneration in an individual. The method includes the step of
administering to an individual at risk of, diagnosed with, or
suspected of having inflammation or neurodegeneration or a
condition or disease related thereto, an agent selected from the
group consisting of: DPAn-6, DPAn-3, an oxylipin derivative of
DPAn-6, and an oxylipin derivative of DPAn-3, to reduce at least
one symptom of inflammation or neurodegeneration in the individual.
In one aspect, the agent is effective to reduce the production of
tumor necrosis factor-.alpha. (TNF-.alpha.) by T lymphocytes. In
another aspect, the agent is effective to reduce the migration of
neutrophils and macrophages into a site of inflammation. In another
aspect, the agent is effective to reduce interleukin-1.beta.
(IL-1.beta.) production in the individual. In yet another aspect,
the agent is effective to reduce macrophage chemotactic protein-1
(MCP-1) in the individual. The oxylipin derivative used in the
present method can include any of the above-identified docosanoids
of the present invention. In one preferred embodiment, the agent is
selected from: 17-hydroxy DPAn-6 and 10,17-dihydroxy DPAn-6, or a
derivative or analog or salt thereof. In another embodiment, the
agent is selected from: DPAn-6 and DPAn-3.
[0024] In one aspect, the method further includes administering at
least one long chain omega-3 fatty acid and/or oxylipin derivative
thereof to the individual. Such an omega-3 fatty acid can include,
but is not limited to, DHA and/or EPA.
[0025] In one aspect, the DPAn-6 or DPAn-3 is provided in one of
the following forms: as triglyceride containing DPAn-6 or DPAn-3,
as a phospholipid containing DPAn-6 or DPAn-3, as a free fatty
acid, as an ethyl or methyl ester of DPAn-6 or DPAn-3.
[0026] In another aspect, the DPAn-6, or DPAn-3, or oxylipin
derivative thereof is provided in the form of a microbial oil, an
animal oil, or from a plant oil that has been derived from an oil
seed plant that has been genetically modified to produce long chain
polyunsaturated fatty acids. In another aspect, the oxylipin
derivative is produced from an enzymatic conversion of DPAn-6 or
DPAn-3 to its oxylipin derivative. In yet another aspect, the
oxylipin derivative is chemically synthesized de novo.
[0027] In any of the above aspects of this method of the invention,
the method can further include administering aspirin to the
individual. In one aspect, the method further includes
administering at least one agent selected from: a statin, a
non-steroidal anti-inflammatory agent, an antioxidant, and a
neuroprotective agent.
[0028] Another embodiment of the present invention relates to a
method to produce a docosanoid, comprising chemically synthesizing
any of the above-described docosanoids of the present
invention.
[0029] Yet another embodiment of the present invention relates to a
method to produce docosanoids, comprising catalytically producing
docosanoids by contacting a DPAn-6 substrate, a DTAn-6 substrate,
or a DPAn-3 substrate with an enzyme that catalyzes the production
of the docosanoids from said DPAn-6 substrate, said DTAn-6
substrate or said DPAn-3 substrate.
[0030] Yet another embodiment of the present invention relates to a
method to produce docosanoids, comprising culturing long chain
polyunsaturated fatty acid (LCPUFA)-producing microorganisms or
growing LCPUFA-producing plants that have been genetically modified
to overexpress an enzyme that catalyzes the production of the
docosanoids from a 22 carbon LCPUFA, to produce said
docosanoids.
[0031] Another method of the present invention relates to a method
to produce docosanoids, comprising contacting long chain
polyunsaturated fatty acids (LCPUFAs) produced by LCPUFA-producing
microorganisms, LCPUFA-producing plants, or LCPUFA-producing
animals, with an enzyme that catalyzes the conversion of said
LCPUFAs to docosanoids.
[0032] In one aspect of the above-described methods to produce
docosanoids, the enzyme is selected from the group consisting of a
lipoxygenase, a cyclooxygenase, and a cytochrome P450 enzyme. For
example, such enzymes include, but are not limited to:
12-lipoxygenase, 5-lipoxygenase, 15-lipoxygenase, cyclooxygenase-2,
hemoglobin alpha 1, hemoglobin beta, hemoglobin gamma A, CYP4A11,
CYP4B1, CYP4F11, CYP4F12, CYP4F2, CYP4F3, CYP4F8, CYP4V2, CYP4X1,
CYP41, CYP2J2, CYP2C8, thromboxane A synthase 1, prostaglandin I2
synthase, and prostacyclin synthase. In one aspect, the LCPUFA is
selected from: DPAn-6, DTAn-6 and DPAn-3.
[0033] In one aspect of the above-described methods, the
LCPUFA-producing microorganisms or LCPUFA-producing plants have
been genetically modified to produce LCPUFAs. In another aspect,
the LCPUFA-producing microorganisms endogenously produce LCPUFAs
(e.g., Thraustochytrids).
[0034] Yet another embodiment of the present invention relates to a
method to enrich an oil for the presence of at least one oxylipin
derived from an LCPUFA or stabilize said oxylipin in the oil. The
method includes culturing an LCPUFA-producing microorganism with a
compound that enhances the enzymatic activity of an enzyme that
catalyzes the conversion of LCPUFAs to oxylipins. In one aspect,
the compound stimulates expression of the enzyme. In another
aspect, the compound enhances or initiates autooxidation of the
LCPUFAs. In one preferred aspect, the compound is acetosalicylic
acid.
[0035] Another embodiment of the present invention relates to a
method to enrich an oil for the presence of at least one oxylipin
derived from an LCPUFA or stabilize said oxylipin in the oil. The
method includes rupturing microbes or plant oil seeds in the
presence of an enzyme that catalyzes the conversion of LCPUFAs to
oxylipins, wherein the microbes and plant oil seeds produce at
least one LCPUFA.
[0036] In one aspect of the above-described methods, the enzyme is
selected from the group consisting of a lipoxygenase, a
cyclooxygenase, and a cytochrome P450 enzyme. In another aspect,
the method further comprises recovering and purifying the
oxylipins. In this aspect, the oxylipins can also be further
processed and recovered as derivatives of the oxylipins or salts
thereof.
[0037] Another embodiment of the present invention relates to a
method to process an oil containing oxylipin derivatives of
LCPUFAs, comprising the steps of: (a) recovering an oil containing
oxylipin derivatives of LCPUFAs produced by a microbial, plant or
animal source; and (b) refining the oil using a process that
minimizes the removal of free fatty acids from the oil to produce
an oil that retains oxylipin derivatives of LCPUFAs. In one aspect,
the animal is an aquatic animal, including, but not limited to, a
fish. In one aspect, the plant is an oil seed plant. In one aspect,
the microbial source is a Thraustochytrid.
[0038] In the above-described method, in one aspect, the step of
refining comprises extraction of the oil with an alcohol, an
alcohol:water mixture, or organic solvent. In another aspect, the
step of refining comprises extraction of the oil with a non-polar
organic solvent. In yet another aspect, the step of refining
comprises extraction of the oil with an alcohol or an alcohol:water
mixture.
[0039] In the above-described method, the step of refining can
further comprise chill filtering, bleaching, further chill
filtering and deodorizing of the oil. In one aspect, the step of
refining further comprises bleaching and deodorizing the oil, in
the absence of chill filtering steps. In another aspect, the step
of refining further comprises deodorizing the oil, in the absence
of chill filtering or bleaching steps.
[0040] In the above-described method, the method can further
include a step of adding an antioxidant to the oil.
[0041] In the above-described method, the step of refining can
include preparing the oil as an emulsion.
[0042] In one aspect of the above-described method the oil is
further processed by contact with an enzyme that catalyzes the
conversion of LCPUFAs to oxylipins. Such an enzyme can include, but
is not limited to, a lipoxygenase, a cyclooxygenase, and a
cytochrome P450 enzyme. In one aspect, the enzyme is immobilized on
a substrate.
[0043] The above-described method can further include a step of
separating the LCPUFA oxylipin derivatives from LCPUFAs in the oil
by a technique including, but not limited to chromatography. This
step of separating can further include adding said separated LCPUFA
oxylipins to an oil or composition.
[0044] Yet another embodiment of the present invention relates to a
method to process an oil containing oxylipin derivatives of
LCPUFAs, comprising the steps of: (a) recovering an oil containing
oxylipin derivatives of LCPUFAs produced by a microbial, plant or
animal source; (b) refining the oil; and (c) separating LCPUFA
oxylipins from LCPUFAs in the oil. In one aspect, the method
further comprises, prior to step (c), a step of converting LCPUFAs
in the oil to LCPUFA oxylipins by a chemical or biological process.
In one aspect, the method further comprises adding said separated
LCPUFA oxylipins to a product.
[0045] Another embodiment of the present invention relates to a
method to prevent or reduce at least one symptom of inflammation or
neurodegeneration in an individual, comprising administering to a
patient at risk of, diagnosed with, or suspected of having
inflammation or neurodegeneration or a condition or disease related
thereto, an agent selected from: DTAn-6 and an oxylipin derivative
of DTAn-6, to reduce at least one symptom of inflammation or
neurodegeneration in the individual. In one aspect, the agent is an
R- or S-epimer of a docosanoid selected from the group consisting
of: monohydroxy derivatives of DTAn-6, dihydroxy derivatives of
DTAn-6, and tri-hydroxy derivatives of DTAn-6. In another aspect,
the agent is an R- or S-epimer of any of the above-described
docosanoids from DTAn-6, or an analog, derivative or salt
thereof.
[0046] Another embodiment of the present invention relates to an
organism comprising a PUFA PKS pathway, wherein the organism has
been genetically transformed to express an enzyme that converts an
LCPUFA to an oxylipin. In one aspect, the organism is selected from
the group consisting of plants and microorganisms. In another
aspect, the organism is an oil seed plant that has been genetically
modified to express a PUFA PKS pathway to produce long chain
polyunsaturated fatty acids. In yet another aspect, the organism is
a microorganism, including, but not limited to, a microorganism
comprising an endogenous PUFA PKS pathway. In one aspect, the
enzyme is selected from the group consisting of a lipoxygenase, a
cyclooxygenase, and a cytochrome P450 enzyme.
BRIEF DESCRIPTION OF THE FIGURES OF THE INVENTION
[0047] FIG. 1 is a graph showing the kinetics of 15-lipoxygenase
reactions with DHA, DPAn-6 and DPAn-3.
[0048] FIG. 2A shows the structure of 15-lipoxygenase products of
DHA.
[0049] FIG. 2B is a mass spectral analysis of 17-hydroxy DHA.
[0050] FIG. 2C is a mass spectral analysis of 10,17-dihydroxy
DHA.
[0051] FIG. 2D is a mass spectral analysis of 7,17-dihydroxy
DHA.
[0052] FIG. 3A shows the structure of 15-lipoxygenase products of
DPAn-6.
[0053] FIG. 3B is a mass spectral analysis of 17-hydroxy
DPAn-6.
[0054] FIG. 3C is a mass spectral analysis of 10,17-dihydroxy
DPAn-6.
[0055] FIG. 3D is a mass spectral analysis of 7,17-dihydroxy
DPAn-6.
[0056] FIG. 4A shows the structure of 15-lipoxygenase products of
DPAn-3.
[0057] FIG. 4B is a mass spectral analysis of 17-hydroxy
DPAn-3.
[0058] FIG. 4C is a mass spectral analysis of 10,17-dihydroxy
DPAn-3.
[0059] FIG. 4D is a mass spectral analysis of 7,17-dihydroxy
DPAn-3.
[0060] FIG. 5A shows the structure of 15-lipoxygenase products of
DTAn-6.
[0061] FIG. 5B is a mass spectral analysis of 17-hydroxy
DTAn-6.
[0062] FIG. 5C is a mass spectral analysis of 7,17-dihydroxy
DTAn-6.
[0063] FIG. 6 shows the major oxylipin products of DPAn-6 after
sequential treatment with 15-lipoxygenase followed by
hemoglobin.
[0064] FIG. 7 shows the major 5-lipoxygenase products of DHA.
[0065] FIG. 8 shows the major 5-lipoxygenase products of
DPAn-6.
[0066] FIG. 9 shows the major 15-lipoxygenase products of
DPAn-3.
[0067] FIG. 10 shows the major 5-lipoxygenase products of DHA.
[0068] FIG. 11 shows the major 5-lipoxygenase products of
DPAn-6.
[0069] FIG. 12 shows the major 5-lipoxygenase products of
DPAn-3.
[0070] FIG. 13 shows structures of EPA-derived oxylipins.
[0071] FIGS. 14A and 14B show structures of DHA-derived
oxylipins.
[0072] FIG. 15 shows structures of DPAn-6-derived oxylipins.
[0073] FIG. 16 shows structures of DPAn-3-derived oxylipins.
[0074] FIG. 17 shows structures of DTAn-6-derived oxylipins.
[0075] FIG. 18A is a mass spectral total ion chromatograph of mono-
and dihydroxy derivatives of DHA and DPAn-6 in algal DHA+DPAn-6
oil.
[0076] FIG. 18B shows MS/MS spectra of mono-hydroxy DPAn-6
derivatives in algal DHA+DPAn-6 oil.
[0077] FIG. 18C shows MS/MS spectra of dihydroxy DPAn-6 derivatives
in algal DHA+DPAn-6 oil.
[0078] FIG. 19 is a graph showing the effect of feeding LCPUFA oils
on paw edema in rats.
[0079] FIG. 20A is a graph showing the total cell migration into
air pouch exudates after administration of docosanoids derived from
DHA and DPAn-6 in the mouse dorsal air pouch model of
inflammation.
[0080] FIG. 20B is a graph showing IL-1.beta. concentrations in air
pouch exudates after administration of docosanoids derived from DHA
and DPAn-6 in the mouse dorsal air pouch model of inflammation.
[0081] FIG. 20C is a graph showing macrophage chemotactic protein 1
(MCP-1) concentrations in air pouch exudates after administration
of docosanoids derived from DHA and DPAn-6 in the mouse dorsal air
pouch model of inflammation.
[0082] FIG. 21 is a graph showing the effect of docosanoids on
TNF.alpha.-induced IL-1.beta. production in human glial cells.
[0083] FIG. 22 is a graph showing the effect of docosanoids on
TNF.alpha. secretion by human T lymphocytes.
[0084] FIG. 23 shows structures of additional, novel
C22-PUFA-derived oxylipins.
DETAILED DESCRIPTION OF THE INVENTION
[0085] Recognizing the need in the art for novel anti-inflammatory
compounds and for alternative ways of providing known
anti-inflammatory compounds, such as the lipoxins, resolvins and
docosatrienes described above, the present inventors have made
several interrelated discoveries that have resulted in the
provision of novel anti-inflammatory reagents and improved
compositions for use in anti-inflammation applications.
[0086] First, the present invention relates to the discovery by the
present inventors that the long chain omega-6 fatty acids,
docosapentaenoic acid (DPAn-6; C22:5n-6) and docosatetraenoic acid
(DTAn-6; C22:4n-6) (also called adrenic acid), as well as the
omega-3 counterpart of DPAn-6, docosapentaenoic acid (DPAn-3;
C22:5n-3), are substrates for the production of novel compounds
referred to generally herein as LCPUFA oxylipins, and more
particularly referred to as docosanoids (including mono-, di-,
tri-, tetra-, and penta-hydroxy derivatives of such docosanoids).
The terms "oxylipin" and "docosanoid" as used herein are defined
and described in detail below. The present inventors have
discovered that DPAn-6, DPAn-3, DTAn-6 and the oxylipin derivatives
thereof, can serve, like the long chain omega-3 fatty acids DHA and
EPA and their oxylipin derivatives, as potent anti-inflammatory
agents. Therefore, in one embodiment, the present invention
provides novel oxylipins derived from the omega-6 fatty acids
DPAn-6 and DTAn-6 and/or from the omega-3 fatty acid DPAn-3, and
derivatives and analogs thereof, as well as methods for the
production and use of such oxylipins as anti-inflammatory compounds
and nutritional/health supplements. The present invention also
provides the use of these LCPUFAs (DPAn-6, DTAn-6 and DPAn-3)
themselves as novel anti-inflammatory compounds (e.g., as a
precursor for the oxylipins or as an agent with intrinsic
anti-inflammatory activity).
[0087] Initially, the present inventors recognized that the
presence of DPAn-6 in a DHA oil substantially enhanced the
reduction in inflammation in patients (e.g., enhanced a reduction
in indicators or mediators of inflammation, such as
pro-inflammatory cytokine production and eicosanoid production) as
compared to a DHA oil that did not contain any other fatty acids.
From this discovery, the inventors have now discovered that the
unique structure of DPAn-6, DTAn-6, and DPAn-3 will allow these
LCPUFAs to serve as a substrate in an enzymatic reaction similar to
that which converts DHA to docosatrienes or resolvins, resulting in
the surprising discovery that DPAn-6, DTAn-6, and DPAn-3, and
oxylipin derivatives thereof are new, potent, anti-inflammatory
agents.
[0088] Prior to the present invention, it was not known that the
long chain omega-6 fatty acid, DPAn-6, could serve as a substrate
for producing novel oxylipins with anti-inflammatory properties
similar to or exceeding those of the previously described
docosatrienes and resolvins derived from EPA and DHA. Evidence
prior to this invention suggested that the presence of DPAn-6 in an
oil would lead to the production of pro-inflammatory compounds and
therefore decrease the overall anti-inflammatory effect of the
DHA-containing oil. For example, DPAn-6 can readily retroconvert to
arachidonic acid (ARA), which is generally considered to be
pro-inflammatory since it is a precursor to a variety of highly
potent pro-inflammatory eicosanoids, including leukotriene B4 and
prostaglandin E2. Indeed, most of the eicosanoids derived from the
omega-6 fatty acid ARA are pro-inflammatory (Gilroy et al, 2004;
Meydani et all, 1990; Simopoulos 2002), and consumption of ARA
reverses the anti-inflammatory effects of DHA (See Example 14
below). Therefore, prior to the present invention, it was generally
believed that DPAn-6 would be pro-inflammatory since it would feed
into the ARA metabolic pathway. Moreover, it was not recognized
prior to the present invention that docosapentaenoic acid (DPAn-6;
C22:5n-6), because of its unique structure, is an important
substrate for the production of novel oxylipins, or that novel
oxylipins could also be derived from docosapentaenoic acid (DPAn-3;
C22:5n-3) and docosatetraenoic acid (DTAn-6; C22:4n-6). Indeed, the
present inventors have found that DPAn-6 and DPAn-3 are superior
substrates in oxylipin-generating reactions as compared to DHA and
have found that DTAn-6 is also a substrate in oxylipin-generating
reactions. This is demonstrated with regard to the conversion of
each of DHA, DPAn-6 and DPAn-3 with 15-lipoxygenase in Example 1
below. Therefore, the production of docosanoids from DPAn-6 and
DPAn-3 is more efficient and will result in greater oxylipin
product levels than the production of docosanoids from DHA.
[0089] Additionally, it was not recognized that the oxylipins
synthesized from DPAn-6 and DPAn-3 have unique properties,
especially with regard to inflammation. In particular, and without
being bound by theory, the present inventors believe that DPAn-6
and DPAn-3 and oxylipin derivatives thereof, and particularly
DPAn-6 and oxylipin derivatives thereof, are equal to or even more
potent anti-inflammatory compounds than DHA, EPA, or the oxylipin
derivatives of those LCPUFAs. Without being bound by theory, the
present inventors also expect that DTAn-6 and oxylipin derivatives
thereof will have anti-inflammatory properties. Indeed,
combinations of DPAn-6 and DPAn-3 and/or oxylipin derivatives
thereof, and particularly DPAn-6 and/or oxylipin derivatives
thereof, with DHA or EPA and/or oxylipin derivatives thereof (and
particularly with DHA and/or oxylipin derivatives thereof) will
provide a greater benefit in nutritional applications (e.g., any
applications of the invention directed to the provision of
nutrients and nutritional agents to maintain, stabilize, enhance,
strengthen, or improve the health of an individual or the organic
process by which an organism assimilates and uses food and liquids
for functioning, growth and maintenance, and which includes
nutraceutical applications), therapeutic applications (e.g., any
applications of the invention directed to prevention, treatment,
management, healing, alleviation and/or cure of a disease or
condition that is a deviation from the health of an individual) and
other applications (e.g., cosmetic) than that provided by DHA, EPA
and/or oxylipin derivatives thereof alone.
[0090] More particularly, the present inventors have discovered
that consumption of an oil containing DPAn-6 in addition to the
omega-3 fatty acid, DHA, causes up to >90% reduction in
inflammatory cytokine production, while consuming DHA alone in an
oil facilitates reductions in inflammatory cytokine production of
only about 13-29%, even when the DHA dose is approximately three
times higher than in the DHA+DPAn-6 oil. Inflammatory eicosanoid
secretion is also significantly reduced by DPAn-6 as compared to
DHA alone. Therefore, the inventors discovered that an oil
containing DPAn-6 and its oxylipin derivatives has significant
anti-inflammatory properties. Furthermore, the inventors submit
that the presence of DPAn-6 and a long chain omega-3 fatty acid
(e.g., DHA), or the oxylipin derivatives thereof, jointly known as
docosanoids, in combination results in the production of
docosanoids (defined below) that have complementary
anti-inflammatory activities. Therefore, formulations containing
both a long chain omega-3 fatty acid such as DHA and DPAn-6 or
oxylipins thereof are significantly more potent anti-inflammatory
formulations than formulations containing omega-3 fatty acids
alone. Furthermore, DPAn-6 and its oxylipin derivatives represent
novel anti-inflammatory agents for use alone or in combination with
a variety of other agents. DPAn-3 and its oxylipin derivatives
and/or DTAn-6 and its oxylipin derivatives can also provide
advantages over the use of DHA alone.
[0091] The present inventors were the first to recognize that
DPAn-6 has anti-inflammatory properties and will enhance the
anti-inflammatory effect of long chain omega-3 fatty acids, such as
DHA. More particularly, the present inventors have recognized that
the most distal n-3 bond between carbons 19 and 20 in DHA is not
involved in the formation of the biologically important
docosatrienes or 17S-resolvins, and therefore, the absence of this
double bond in DPAn-6 would not hinder this fatty acid from being
metabolically converted to analogous oxylipins by biological
enzymes, such as the lipoxygenases. The inventors further
recognized the double bonds involved in the majority of enzymatic
conversions of DHA to oxylipins, particularly those compounds known
as resolvins (i.e., those double bonds between carbons 7 and 8,
carbons 10 and 11, carbons 13 and 14, and carbons 16 and 17 in
DHA), were also present in DPAn-6, DTAn-6 and DPAn-3, facilitating
their use as a substrate for the production of oxylipins. Without
being bound by theory, this is believed to account for the
differences in the data that were observed by the present inventors
in studies using oil containing DHA and DPAn-6 as compared to DHA
alone. The inventors have now demonstrated that the same enzymes
that convert DHA to docosanoids or the 17S-resolvins recognize any
(n-3) or (n-6) C-22 PUFA. Therefore, like DHA, DPAn-6, DTAn-6 and
DPAn-3 are substrates for novel oxylipins that can serve as potent
anti-inflammatory molecules. Additionally, these observations also
suggest that LCPUFA of 24 or more carbons and that have double
bonds located between carbons 7 and 8, carbons 10 and 11, carbons
13 and 14, and carbons 16 and 17, also serve as substrates for the
production of novel oxylipins, and can be produced or enhanced in
various oils and compositions using the methods outlined in the
present application.
[0092] The inventors were, therefore, the first to recognize that
the enzymes forming the oxylipins such as the previously described
docosatrienes and resolvins derived from DHA did not discriminate
between the (n-6) and (n-3) 22-carbon fatty acids as substrates
because of the presence of the particular double bonds in the same
location in these molecules. In fact, the inventors were the first
to discover that the C22n-6 fatty acids are preferred substrates
for these enzymes. The inventors were also the first to recognize
that oxylipins from DPAn-6 have strong anti-inflammatory activity,
and that a combination of oxylipins from both DHA and DPAn-6 has
more anti-inflammatory benefits than those from DHA alone.
[0093] In another embodiment of the invention, the present
inventors have also discovered novel ways of producing LCPUFA-rich
oils that also contain enhanced and effective amounts of LCPUFA
oxylipins (and in particular docosanoids), including the novel
oxylipins of the present invention, as well as oxylipins that had
been previously described. These LCPUFA-rich oils can be used in
nutritional (including nutraceutical), cosmetic and/or
pharmaceutical (including therapeutic) applications to deliver the
immediate anti-inflammatory/neuroprotective action(s) of the
hydroxy-LCPUFA derivatives along with the inherent long-term
benefits of the LCPUFAs themselves.
[0094] The present inventors have also discovered that conventional
sources of LCPUFAs, such as algal oils and fish oils, have only
extremely small amounts of the hydroxyl-derivatives of LCPUFAs, and
therefore, of the LCPUFA oxylipins, particularly docosanoids (e.g.,
from about 1 ng/g oil to about 10 .mu.g/g oil). This is in part due
to genetic and environmental factors associated with the production
organisms (e.g., algae, fish), and is also due to the methods used
to process LCPUFA oils from these organisms. Realizing that the
provision of oils enriched in LCPUFA oxylipins would be of great
benefit to human nutrition and health and would provide an
alternative to the provision of chemically synthesized oxylipin
analogs or to oils containing inadequate amounts of LCPUFA
oxylipins, the present inventors have discovered alternative ways
to produce these LCPUFA oils so that they are enriched in LCPUFA
oxylipins (and in particular docosanoids), as well as alternative
ways to process the LCPUFA oils to further enrich and enhance the
LCPUFA oxylipin (and in particular docosanoid) content of the oils,
thereby significantly enhancing their LCPUFA oxylipin (and in
particular docosanoid) levels over those found in conventionally
produced/processed LCPUFA oils.
[0095] In addition, the present inventors have discovered the
oxylipins that are produced from DPAn-6, DTAn-6 and DPAn-3, and
these oxylipins can now be chemically or biogenically produced and
used as crude, semi-pure or pure compounds in a variety of
compositions and formulations, or even added to oils, such as
LCPUFA- or LCPUFA-oxylipin-containing oils, to enhance or
supplement the natural oxylipins in such oils. Such compounds can
also serve as lead compounds for the production of additional
active analogs of these oxylipins in the design and production of
nutritional agents and therapeutic drugs.
GENERAL DEFINITIONS
[0096] For the purposes of this application, long chain
polyunsaturated fatty acids (LCPUFAs) are defined as fatty acids of
18 and more carbon chain length, and are preferably fatty acids of
20 or more carbon chain length, containing 3 or more double bonds.
LCPUFAs of the omega-6 series include: di-homo-gammalinoleic acid
(C20:3n-6), arachidonic acid (C20:4n-6), docosatetraenoic acid or
adrenic acid (C22:4n-6), and docosapentaenoic acid (C22:5n-6). The
LCPUFAs of the omega-3 series include: eicosatrienoic acid
(C20:3n-3), eicosatetraenoic acid (C20:4n-3), eicosapentaenoic acid
(C20:5n-3), docosapentaenoic acid (C22:5n-3), and docosahexaenoic
acid (C22:6n-3). The LCPUFAs also include fatty acids with greater
than 22 carbons and 4 or more double bonds including, but not
limited to, C24:6(n-3) and C28:8(n-3).
[0097] The terms "polyunsaturated fatty acid" and "PUFA" include
not only the free fatty acid form, but other forms as well, such as
the triacylglycerol (TAG) form, the phospholipid (PL) form and
other esterified forms.
[0098] As used herein, the term "lipid" includes phospholipids;
free fatty acids; esters of fatty acids; triacylglycerols;
diacylglycerides; monoacylglycerides; lysophospholipids; soaps;
phosphatides; sterols and sterol esters; carotenoids; xanthophylls
(e.g., oxycarotenoids); hydrocarbons; and other lipids known to one
of ordinary skill in the art.
[0099] For the purposes of this application, "oxylipins" are
defined as biologically active, oxygenated derivatives of
polyunsaturated fatty acids, formed by oxidative metabolism of
polyunsaturated fatty acids. Oxylipins that are formed via the
lipoxygenase pathway are called lipoxins. Oxylipins that are formed
via the cyclooxygenase pathway are called prostanoids. Oxylipins
formed from 20 carbon fatty acids (arachidonic acid and
eicosapentaenoic acid) are called eicosanoids. Eicosanoids include
prostaglandins, leukotrienes and thromboxanes. They are formed
either via the lipoxygenase pathway (leukotrienes) or via the
cyclooxygenase pathway (prostaglandins, prostacyclin,
thromboxanes). Oxylipins formed from 22 carbon fatty acids
(docosapentaenoic acid (n-6 or n-3), docosahexaenoic acid and
docosatetraenoic acid) are called docosanoids. Specific examples of
these compounds are described below. General reference to an
oxylipin described herein is intended to encompass the derivatives
and analogs of a specified oxylipin compound.
[0100] As used herein, the term "analog" refers to a chemical
compound that is structurally similar to another compound but
differs slightly in composition (as in the replacement of one atom
by an atom of a different element or in the presence of a
particular functional group, or the replacement of one functional
group by another functional group) (see detailed discussion of
analogs of the present invention below).
[0101] As used herein, the term "derivative", when used to describe
a compound of the present invention, means that at least one
hydrogen bound to the unsubstituted compound is replaced with a
different atom or a chemical moiety (see detailed discussion of
derivatives of the present invention below).
[0102] In general, the term "biologically active" indicates that a
compound has at least one detectable activity that has an effect on
the metabolic or other processes of a cell or organism, as measured
or observed in vivo (i.e., in a natural physiological environment)
or in vitro (i.e., under laboratory conditions).
[0103] The oxygenated derivatives of long chain polyunsaturated
fatty acids (LCPUFAs) include mono-, di-, tri-, tetra-, and
penta-hydroxy derivatives of the LCPUFAs, and also include the
free, esterified, peroxy and epoxy forms of these derivatives.
These mono-, di-, tri-, tetra-, and penta-hydroxy derivatives of
LCPUFAs are those derivatives that contain 3, 4 or more double
bonds, generally at least two of which are conjugated, and one or
more non-carboxy, hydroxyl groups. Preferably, these derivatives
contain 4-6 double bonds and at least 1-3 non-carboxy, hydroxyl
groups, and more preferably, 2 or more non-carboxy, hydroxyl
groups.
[0104] Oxygenated derivatives of the omega-3 fatty acids EPA and
DHA, catalyzed by lipoxygenase or cyclo-oxygenase enzymes,
including acetylated forms of cyclooxygenase 2 (COX2), which are
capable of down regulating or resolving inflammatory processes, are
commonly referred to as "resolvins", which is a coined term
(neologism) that is functional in nature. The "docosatrienes" are a
subclass of oxylipins derived from DHA and contain three conjugated
double bonds. "Protectin" is another coined functional term for
hydroxy derivatives of the omega-3 fatty acid DHA that have a
neuroprotective effect.
[0105] According to the present invention, the term "docosanoid"
specifically refers to any oxygenated derivatives (oxylipins) of
any 22-carbon LCPUFA (e.g., DHA, DPAn-6, DPAn-3, or DTAn-6). The
structures of such derivatives are described in detail below. It is
noted that while the present inventors recognize that the novel
oxylipin derivatives (docosanoids) of the present invention that
are derived from DPAn-6, DPAn-3 and DTAn-6 might also be considered
to be "resolvins" or "protectins" based on similar functional
attributes of such oxylipins, for the purposes of this invention,
it is preferred that the novel oxylipins of the present invention
be generally referenced using the term "docosanoid", which provides
a clear structural definition of such compounds. The docosanoids
from DPAn-6, DPAn-3 and DTAn-6 have never before been described, to
the best of the present inventors' knowledge.
Oxylipins Useful in the Present Invention
[0106] One embodiment of the present invention relates to novel
oxylipins derived from DPAn-6, DPAn-3, or DTAn-6, and any analogs
or derivatives of such oxylipins, including any compositions or
formulations or products containing such oxylipins or analogs or
derivatives thereof, as well as oils or other compositions or
formulations or products that have been enriched by any method for
any LCPUFA oxylipin or analogs or derivatives thereof, and
particularly for any oxylipin derived from DHA, EPA, DPAn-6, DPAn-3
or DTAn-6, and more particularly, for any docosanoid, and even more
particularly, for any oxylipin derived from DPAn-6, DPAn-3 or
DTAn-6. The present invention also relates to any oils or other
compositions or formulations or products in which such oxylipins
(any oxylipin derived from DHA, EPA, DPAn-6, DPAn-3 or DTAn-6, and
more particularly, any docosanoid) are stabilized or retained in
the oils or compositions to improve the quantity, quality or
stability of the oxylipin in the oil or composition, and/or to
improve the absorption, bioavailability, and/or efficacy of the
oxylipins contained in oils or compositions.
[0107] As discussed above, a variety of DHA- and EPA-derived
oxylipins having anti-inflammatory activity, anti-proliferative
activity, antioxidant activity, neuroprotective or vasoregulatory
activity (Ye et al, 2002) are known, which have been more commonly
referred to as resolvins or protectins. Such oxylipins are
referenced as being encompassed by the present invention,
particularly in embodiments where such oxylipins are enriched in
oils and compositions, preferably using the methods and processing
steps of the present invention. In addition, the present invention
provides novel oxylipins derived from DPAn-6, DPAn-3, and DTAn-6,
including analogs or derivatives thereof, which can also be
enriched in various oils and compositions, preferably using the
methods and processes of the invention, or which can be produced
and if desired, isolated or purified, by a variety of biological or
chemical methods, including by de novo production, for use in any
therapeutic, nutritional (including nutraceutical), cosmetic, or
other application as described herein. Therefore, the present
invention encompasses isolated, semi-purified and purified
oxylipins as described herein, as well as sources of oxylipins
including synthesized and natural sources (e.g., oils or plants and
portions thereof), and includes any source that has been enriched
for the presence of an oxylipin useful in the present invention by
genetic, biological or chemical methods, or by processing steps as
described herein.
[0108] In general, oxylipins can have either pro-inflammatory or
anti-inflammatory properties. According to the present invention,
pro-inflammatory properties are properties (characteristics,
activities, functions) that enhance inflammation in a cell, tissue
or organism, and anti-inflammatory properties are properties that
inhibit such inflammation. Inflammation in cells, tissues and/or
organisms can be identified by a variety of characteristics
including, but not limited to, the production of "proinflammatory"
cytokines (e.g., interleukin-1.alpha. (IL-1.alpha.), IL-1.beta.,
tumor necrosis factor-.alpha.(TNF.alpha.), IL-6, IL-8, IL-12,
macrophage inflammatory protein-1.alpha. (MIP-1.alpha.), macrophage
chemotactic protein-1 (MCP-1; also known as macrophage/monocyte
chemotactic and activating factor or monocyte chemoattractant
protein-1) and interferon-.gamma. (IFN-.gamma.)), eicosanoid
production, histamine production, bradykinin production,
prostaglandin production, leukotriene production, fever, edema or
other swelling, and accumulation of cellular mediators (e.g.,
neutrophils, macrophages, lymphocytes, etc.) at the site of
inflammation.
[0109] In one embodiment, oxylipins useful in the present invention
are those having anti-inflammatory properties, such as those
derived from DHA, EPA, DPAn-6, DPAn-3 and DTAn-6 (described in
detail below). Other important bioactive properties of oxylipins
include, but are not limited to, anti-proliferative activity,
antioxidant activity, neuroprotective and/or vasoregulatory
activity. These properties are also preferred properties of
oxylipins useful in the present invention, and are preferably
characteristic of oxylipins derived from DHA, EPA, DPAn-6, DTAn-6
and DPAn-3. In another embodiment, oxylipins of the present
invention include any oxylipins derived from DPAn-6 or DPAn-3 or
DTAn-6, regardless of the particular functional properties of the
oxylipin. Preferred oxylipins derived from DPAn-6 or DPAn-3 or
DTAn-6 include those that provide a nutritional and/or therapeutic
benefit, and more preferably, have anti-inflammatory activity,
anti-proliferative activity, antioxidant activity, and/or
neuroprotective activity.
EPA-Derived Oxylipins
[0110] Oxylipins derived from EPA that are useful in the present
invention include, but are not limited to: 15-epi-lipoxin A4
(5S,6R,15R-trihydroxy eicosatetraenoic acid) and its intermediate
15R-hydroxy eicosapentaenoic acid (15R-HEPE); Resolvin E1
(5,12,18-trihydroxy EPA) and its intermediates
5,6-epoxy,18R-hydroxy-EPE, and 5S-hydro(peroxy),18R-hydroxy-EPE,
and 18R-hydroxy-EPE (18R-HEPE); and Resolvin E2
(5S,18R-dihydroxy-EPE or 5S,18R-diHEPE) and its intermediates. See
FIG. 13 below for structures of these EPA derivatives. EPA-derived
oxylipins are described in detail in Serhan (2005), which is
incorporated herein by reference in its entirety.
DHA-Derived Oxylipins
[0111] Oxylipins derived from DHA that are useful in the present
invention include, but are not limited to: Resolvin D1
(7,8,17R-trihydroxy DHA) and Resolvin D2 (7,16,17R-trihydroxy DHA)
along with their S-epimers and their intermediates including:
17S/R-hydroperoxy DHA, and 7S-hydroperoxy,17S/R-OH-DHA, and
7(8)-epoxy-17S/R-OH-DHA; Resolvin D4 (4,5,17R-trihydroxy DHA) and
Resolvin D3 (4,11,17R trihydroxy DHA) along with their S-epimers
and their intermediates including 17S/R-hydroperoxy DHA, and
4S-hydroperoxy,17S/R-OH DHA and 4(5)-epoxy-17S/R-OH DHA; and
Neuroprotectin D1 (10,17S-docosatriene, protectin D1) along with
its R epimer and their intermediates including the dihydroxy
product 16,17-epoxy-docosatriene (16,17-epoxy-DT) and the
hydroperoxy product 17S-hydroperoxy DHA; Resolvin D5
(7S,17S-dihydroxy DHA) and Resolvin D6 and their hydroxyl
containing intermediates; and epoxide derivatives 7,8 epoxy DPA,
10,11-expoxy DPA, 13,14-epoxy DPA, and 19,20-epoxy DPA and
dihydroxy derivative 13,14-dihydroxy docosapentaenoic acid; other
mono-hydroxy DHA derivatives, including the R and S epimers of
7-hydroxy DHA, 10-hydroxy DHA, 11-hydroxy DHA, 13-hydroxy DHA,
14-hydroxy DHA, 16-hydroxy DHA and 17-hydroxy DHA; and other
dihydroxy DHA derivatives, including the R and S epimers of
10,20-dihydroxy DHA, 7,14-dihydroxy DHA and 8,14-dihydroxy DHA. See
Examples 2, 7, and 10, and FIGS. 2A-2D, FIG. 7, FIG. 10 and FIGS.
14A and B below for descriptions and structures of these DHA
derivatives. DHA-derived oxylipins are described in detail in
Serhan (2005) and Ye et al (2002), which are incorporated herein by
reference in its entirety.
DPAn-6-, DTAn-6- and DPAn-3-Derived Oxylipins and Other Novel
Docosanoids from C22 Fatty Acids
[0112] One embodiment of the present invention relates to novel
oxylipins that are derived from DPAn-6, DTAn-6, or DPA-n-3. Another
embodiment of the invention relates to novel docosanoids that can
be derived from C22 PUFAs. Specifically, the present inventors
describe herein novel docosanoids, the structures of which were
designed de novo from C22 fatty acid structures. Oxylipins
encompassed by the present invention include any oxylipins derived
from DPAn-6, DTAn-6, or DPAn-3, or generally from C22 fatty acids,
and more particularly described herein as docosanoids. Novel
docosanoids include any oxygenated derivative of DPAn-6, DTAn-6,
DPAn-3, or any other novel oxygenated derivatives of C22 fatty
acids (e.g., see FIG. 23), including any derivatives or analogs
thereof. In particular, docosanoids of the present invention
include, but are not limited to, any R- or S-epimer of any
monohydroxy, dihydroxy, or trihydroxy derivative of any of DPAn-6,
DTAn-6 or DPAn-3 or an C22 fatty acids, and can include
derivatizations at any carbon that forms a carbon-carbon double
bond in the reference LCPUFA. Docosanoids of the present invention
also include any product of an enzyme reaction that uses DPAn-6,
DTAn-6, or DPAn-3 as a substrate and that is catalyzed by an
oxylipin-generating enzyme including, but not limited to
lipoxygenases, cyclooxygenases, cytochrome P450 enzymes and other
heme-containing enzymes, such as those described in Table 1 (see
below). Table 1 provides sufficient information to identify the
listed known enzymes, including official names, official symbols,
aliases, organisms, and/or sequence database accession numbers for
the enzymes.
Table 1. Lipoxygenase (LOX), cyclooxygenase (COX), cytochrome P450
(CYP) enzymes and other heme-containing enzymes that can be used to
process LCPUFA oils and fatty acids to produce their hydroxyl fatty
acid derivatives by methods described herein.
Lipoxygenase Type Enzymes
ALOX12
[0113] Official Symbol: ALOX12 and Name: arachidonate
12-lipoxygenase [Homo sapiens]
Other Aliases: HGNC:429, LOG12
[0114] Other Designations: 12(S)-lipoxygenase; platelet-type
12-lipoxygenase/arachidonate 12-lipoxygenase
Chromosome: 17; Location: 17p13.1GeneID: 239
Alox5
[0115] Official Symbol Alox5 and Name: arachidonate 5-lipoxygenase
[Rattus norvegicus]
Other Aliases: RGD:2096, LOX5A
[0116] Other Designations: 5-Lipoxygenase; 5-lipoxygenase
Chromosome: 4; Location: 4q42GeneID: 25290
ALOXE3
[0117] Official Symbol: ALOXE3 and Name: arachidonate lipoxygenase
3 [Homo sapiens]
Other Aliases: HGNC:13743
[0118] Other Designations: epidermal lipoxygenase;
lipoxygenase-3
Chromosome: 17; Location: 17p13.1GeneID: 59344
LOC425997
[0119] similar to arachidonate lipoxygenase 3; epidermal
lipoxygenase; lipoxygenase-3 [Gallus gallus]
Chromosome: UnGeneID: 425997
LOC489486
[0120] similar to Arachidonate 12-lipoxygenase, 12R type
(Epidermis-type lipoxygenase 12) (12R-lipoxygenase) (12R-LOX)
[Canis familiaris]
Chromosome: 5GeneID: 489486
LOC584973
[0121] similar to Arachidonate 12-lipoxygenase, 12R type
(Epidermis-type lipoxygenase 12) (12R-lipoxygenase) (12R-LOX)
[Strongylocentrotus purpuratus]
Chromosome:UnGeneID: 584973
LOC583202
[0122] similar to Arachidonate 12-lipoxygenase, 12R type
(Epidermis-type lipoxygenase 12) (12R-lipoxygenase) (12R-LOX)
[Strongylocentrotus purpuratus]
Chromosome:UnGeneID: 583202
LOC579368
[0123] similar to Arachidonate 12-lipoxygenase, 12R type
(Epidermis-type lipoxygenase 12) (12R-lipoxygenase) (12R-LOX)
[Strongylocentrotus purpuratus]
Chromosome: UnGeneID: 579368
LOC504803
[0124] similar to Arachidonate 12-lipoxygenase, 12R type
(Epidermis-type lipoxygenase 12) (12R-lipoxygenase) (12R-LOX) [Bos
taurus]
Chromosome: UnGeneID: 504803
ALOX5
[0125] Official Symbol: ALOX5 and Name: arachidonate 5-lipoxygenase
[Homo sapiens]Other Aliases: HGNC:435, 5-LO, 5LPG, LOG5Other
Designations: arachidonic acid 5-lipoxygenase; leukotriene A4
synthaseChromosome: 10;
Location: 10q11.2GeneID:240
OSJNBa0057G07.
[0126] lipoxygenase L-2; lipoxygenase [Oryza sativa (japonica
cultivar-group)]GeneID:3044798
Alox15b
[0127] Official Symbol Alox15b and Name: arachidonate
15-lipoxygenase, second type [Mus musculus]
Other Aliases: MGI:1098228, 8-LOX, 8S-LOX, Alox8
Other Designations: 8S-lipoxygenase
Chromosome: 11; Location: 11 B4GeneID: 11688
ALOX5AP
[0128] Official Symbol: ALOX5AP and Name: arachidonate
5-lipoxygenase-activating protein [Homo sapiens]
Other Aliases: HGNC:436, FLAP
[0129] Other Designations:MK-886-binding protein; five-lipoxygenase
activating protein Chromosome: 13; Location: 13q12GeneID: 241
LOC489485
[0130] similar to Arachidonate 15-lipoxygenase, type II (15-LOX-2)
(8S-lipoxygenase) (8S-LOX) [Canis familiaris]
Chromosome: 5GeneID: 489485
LOC557523
[0131] similar to Arachidonate 5-lipoxygenase (5-lipoxygenase)
(5-LO) [Danio rerio]
Chromosome: 15GeneID: 557523
Alox5ap
[0132] Official Symbol Alox5ap and Name: arachidonate
5-lipoxygenase activating protein [Mus musculus]
Other Aliases: MGI:107505, Flap
[0133] Other Designations:arachidonate 5 lipoxygenase activating
protein
Chromosome: 5GeneID: 11690
LOC562561
[0134] similar to Arachidonate 5-lipoxygenase (5-lipoxygenase)
(5-LO) [Danio rerio]
Chromosome: UnGeneID: 562561
LOC423769
[0135] similar to Arachidonate 5-lipoxygenase (5-lipoxygenase)
(5-LO) [Gallus gallus]
Chromosome: 6GeneID: 423769
LOC573013
[0136] similar to Arachidonate 5-lipoxygenase (5-lipoxygenase)
(5-LO) [Danio rerio]
Chromosome: UnGeneID: 573013
LOC584481
[0137] similar to Arachidonate 5-lipoxygenase (5-lipoxygenase)
(5-LO) [Strongylocentrotus purpuratus]
Chromosome: UnGeneID: 584481
5LOX-potato
[0138] AAD04258. Reports 5-lipoxygenase [S . . . [gi:2789652]
15-LOX Soybean
[0139] P08170. Reports Seed lipoxygenase . . . [gi:126398]
12-LOX-porcine
[0140] D10621. Reports Sus scrofa gene f . . . [gi:60391233]
B) Cyclooxygenase Enzymes
[0141] COX2-human AAN87129. Reports prostaglandin syn . . .
[gi:27151898]
C) Hemoglobin Containing Enzymes
HBA1
[0142] Official Symbol: HBA1 and Name: hemoglobin, alpha 1 [Homo
sapiens]
Other Aliases: HGNC:4823, CD31
[0143] Other Designations: alpha 1 globin; alpha one globin;
alpha-1 globin; alpha-1-globin; alpha-2 globin; alpha-2-globin;
hemoglobin alpha 1 globin chain; hemoglobin alpha 2; hemoglobin
alpha-1 chain; hemoglobin alpha-2
Chromosome: 16; Location: 16p13.3GeneID: 3039
HBB
[0144] Official Symbol: HBB and Name: hemoglobin, beta [Homo
sapiens] Other Aliases:HGNC:4827, CD113t-C, HBD, hemoglobin Other
Designations: beta globin; beta globin chain; haemoglobin A beta
chain; hemoglobin beta chain; hemoglobin delta Etolia variant
Chromosome: 11; Location: 11p15.5GeneID: 3043
HBG1
[0145] Official Symbol: HBG1 and Name: hemoglobin, gamma A [Homo
sapiens]
Other Aliases: HGNC:4831, HBGA, HBGR, HSGGL1, PRO2979
[0146] Other Designations: A-gamma globin; gamma A hemoglobin;
gamma globin; hemoglobin gamma-a chain; hemoglobin, gamma,
regulator of Chromosome: 11; Location: 11p15.5GeneID: 3047
D) Cytochrome P450 Type Enzymes
[0147] (Gene, Organism, Gene Database: SwissProt, Gene database:
EMBL/Genbank/DDBJ) CYP4A11, Homo sapiens, CP4AB HUMAN, L04751
D26481 S67580 S67581 AF525488 AY369778 X71480 CYP4A4, Oryctolagus
cuniculus, CP4A4 RABIT, L04758 J02818 CYP4A5, Oryctolagus
cuniculus, CP4A5 RABIT, M28655 X57209 CYP4A6, Oryctolaqus
cuniculus, CP4A6 RABIT, M28656 M29531 CYP4A7, Oryctolaqus
cuniculus, CP4A7 RABIT, M28657 M29530 CYP4B1, Homo sapiens, CP4B1
HUMAN, J02871 X16699 AF491285 AY064485 AY064486 CYP4B1, Oryctolaqus
cuniculus, CP4B1 RABIT, M29852 AF176914 AF332576 CYP4C1, Blaberus
discoidalis, CP4C1 BLAD1, M63798 CYP4C21, Blattella germanica,
CP4CU BLAGE, AF275641 CYP4E4, Drosophila melanogaster, C4AE1 DROME,
AE003423 AL009194 AY058450 U34331 CYP4F11, Homo sapiens, CP4FB
HUMAN, AF236085 BC016853 AC005336 CYP4F12, Homo sapiens, CP4FC
HUMAN, AY008841 AB035130 AB035131 AY358977 CYP4F2, Homo sapiens,
CP4F2 HUMAN, D26480 U02388 AB015306 AF467894 AC005336 BC067437
BC067439 BC067440 AF221943 CYP4F3 Homo sapiens CP4F3 HUMAN, D12620
D12621 AB002454 AB002461 AF054821 AY792513 CYP4F8 Homo sapiens
CP4F8 HUMAN, AF133298 CYP4V2 Homo sapiens CP4V2 HUMAN, AY422002
AK122600 AK126473 BC060857 CYP4V2, Pongo pygmaeus CP4V2 PONPY,
CR858234 CYP4X1, Homo sapiens CP4X1 HUMAN, AY358537 AK098065
BC028102 CYP4Z1, Homo sapiens CP4Z1 HUMAN, AY262056 AY358631
Cyp4a1, Rattus norvegicus CP4A1 RAT, M14972 X07259 M57718 Cyp4a2,
Rattus norvegicus CP4A2 RAT, M57719 BC078684 Cyp4a3, Rattus
norvegicus CP4A3 RAT, M33936 Cyp4a8, Rattus norvegicus CP4A8 RAT,
M37828 Cyp4aa1, Drosophila melanogaster, C4AA1 DROME AE003808
Cyp4ac1, Drosophila melanogaster, C4AC1 DROME AE003609 AY051602
Cyp4ac2, Drosophila melanogaster, C4AC2 DROME, AE003609 Cyp4ac3,
Drosophila melanogaster, C4AC3 DROME, AE003609 AY061002 Cyp4ad1,
Drosophila melanogaster, C4AD1 DROME, AE003837 AY061058 Cyp4b1, Mus
musculus, CP4B1 MOUSE, D50834 BC008996 Cyp4b1 Rattus norvegicus
CP4B1 RAT, M29853 BC074012 Cyp4c3, Drosophila melanogaster, CP4C3
DROME, AE003775 BT010108 U34323 Cyp4d1, Drosophila melanogaster,
CP4D1 DROME, X67645 AF016992 AF016993 AF016994 AF016995 AF016996
AF016997 AF016998 AF016999 AF017000 AF017001 AF017002 AF017003
AF017004 AE003423 AE003423 Z98269 Cyp4d1, Drosophila simulans,
CP4D1 DROSI, AF017005 Cyp4d10, Drosophila mettleri, C4D10 DROMT,
U91634 Cyp4d14, Drosophila melanogaster, C4D14 DROME, AE003423
AL009194 Cyp4d2, Drosophila melanogaster, CP4D2 DROME, X75955
Z23005 AE003423 AL009194 AY118763 AF017006 AF017007 AF017008
AF017009 AF017010 AF017011 AF017012 AF017013 AF017014 AF017015
AF017016 AF017017 AF017018-Cyp4d20, Drosophila melanogaster, C4D20
DROME, AE003475 Cyp4d21, Drosophila melanogaster, C4D21 DROME,
AE003618 Cyp4d8, Drosophila melanogaster, CP4D8 DROME, AE003558
AY058442 U34329 Cyp4e1, Drosophila melanogaster, CP4E1 DROME,
AE003837 AY118793 Cyp4e2, Drosophila melanogaster, CP4E2 DROME,
U56957 AE003837 AY058518 X86076 U34332 Cyp4e3, Drosophila
melanogaster, CP4E3 DROME, AE003626 U34330 Cyp4e5, Drosophila
mettleri, CP4E5 DROMT, U78486 Cyp4f1, Rattus norvegicus, CP4F1 RAT,
M94548 AF200361 Cyp4f14, Mus musculus, CP4FE MOUSE, AB037541
AB037540 AF233644 AK005007 AK018676 BC011228 Cyp4f4, Rattus
norvegicus, CP4F4 RAT, U39206 Cyp4f5, Rattus norvegicus, CP4F5 RAT,
U39207 Cyp4f6, Rattus norvegicus, CP4F6 RAT, U39208 Cyp4g1,
Drosophila melanogaster, CP4G1 DROME, AE003417 AL009188 U34328
Cyp4g15, Drosophila melanogaster, C4G15 DROME, AF159624 AE003486
AY060719 Cyp4p1, Drosophila melanogaster, CP4P1 DROME, AE003834
AY071584 U34327 Cyp4p2, Drosophila melanogaster, CP4P2 DROME,
AE003834 AY051564 Cyp4p3, Drosophila melanogaster, CP4P3 DROME,
AE003834 AY075201 Cyp4s3, Drosophila melanogaster, CP4S3 DROME
AE003498 Cyp4v3, Mus musculus, CP4V3 MOUSE, AB056457 AK004724
Cyp4X1, Rattus norvegicus, CP4X1 RAT, AF439343 CYP2 Family of
Cytochrome P450 Enzymes (Sequences from Genbank) CYP2J2 sequences
from GenBank
NM.sub.--000775
[0148] Homo sapiens cytochrome P450, family 2, subfamily J,
polypeptide 2 (CYP2J2)
gi|18491007|ref|NM.sub.--000775.2|[18491007]
NM.sub.--000770
[0149] Homo sapiens cytochrome P450, family 2, subfamily C,
polypeptide 8 (CYP2C8), transcript variant Hp1-1, mRNA
gi|13787188|ref|NM.sub.--000770.2|[13787188]
NM.sub.--030878
[0150] Homo sapiens cytochrome P450, family 2, subfamily C,
polypeptide 8 (CYP2C8), transcript variant Hp1-2, mRNA
gi|13787186|ref|NM.sub.--030878.1|[13787186]
NM.sub.--023025
[0151] Rattus norvegicus cytochrome P450, family 2, subfamily J,
polypeptide 4 (Cyp2j4), mRNA
gi|61889087|ref|NM.sub.--023025.2|[61889087]
DN992115
[0152] TC119679 Human adult whole brain, large insert, pCMV
expression library Homo sapiens cDNA clone TC119679 5' similar to
Homo sapiens cytochrome P450, family 2, subfamily J, polypeptide 2
(CYP2J2), mRNA sequence gi|66251946|gb|DN992115.1|[66251946]
Z84061
[0153] SSZ84061 Porcine small intestine cDNA library Sus scrofa
cDNA clone c13d09 5' similar to cytochrome P450 monooxygenase
CYP2J2, mRNA sequence gi|1806390|emb|Z84061.1|[1806390]
BC091149
[0154] Rattus norvegicus cytochrome P450, family 2, subfamily J,
polypeptide 4, mRNA (cDNA clone MGC:108684 IMAGE:7323516), complete
cds gi|60688166|gb|BC091149.1|[60688166]
NW.sub.--380169
[0155] Bos taurus chromosome Un genomic contig, whole genome
shotgun sequence
gi|61630302|ref|NW.sub.--380169.1|BtUn_WGA215002.sub.--1[6163030-
2]
BC032594
[0156] Homo sapiens cytochrome P450, family 2, subfamily J,
polypeptide 2, mRNA (cDNA clone MGC:44831 IMAGE:5527808), complete
cds gi|21595666|gb|BC032594.1|[21595666]
NT.sub.--086582
[0157] Homo sapiens chromosome 1 genomic contig, alternate assembly
gi|51460368|ref|NT.sub.--086582.1|Hs1.sub.--86277[51460368]
NT.sub.--032977
[0158] Homo sapiens chromosome 1 genomic contig
gi|51458674|ref|NT.sub.--032977.7|Hs1.sub.--33153[51458674]
CO581852
[0159] ILLUMIGEN_MCQ.sub.--46633 Katze_MMJJ Macaca mulatta cDNA
clone IBIUW:17960 5' similar to Bases 384 to 953 highly similar to
human CYP2J2 (Hs.152096), mRNA sequence
gi|50413382|gb|CO581852.1|[50413382]
AY410198
[0160] Mus musculus CYP2J2 gene, VIRTUAL TRANSCRIPT, partial
sequence, genomic survey sequence
gi|39766166|gb|AY410198.1|[39766166]
AY410197
[0161] Pan troglodytes CYP2J2 gene, VIRTUAL TRANSCRIPT, partial
sequence, genomic survey sequence
gi|39766165|gb|AY410197.1|[39766165]
AY410196
[0162] Homo sapiens CYP2J2 gene, VIRTUAL TRANSCRIPT, partial
sequence, genomic survey sequence
gi|39766164|gb|AY410196.1|[39766164]
AY426985
[0163] Homo sapiens cytochrome P450, family 2, subfamily J,
polypeptide 2 (CYP2J2) gene, complete cds
gi|37574503|gb|AY426985.1|[37574503]
AB080265
[0164] Homo sapiens CYP2J2 mRNA for cytochrome P450 2J2, complete
cds gi|18874076|dbj|AB080265.1|[18874076]
AF272142
[0165] Homo sapiens cytochrome P450 (CYP2J2) gene, complete cds
gi|21262185|gb|AF272142.1|[21262185]
U37143
[0166] Homo sapiens cytochrome P450 monooxygenase CYP2J2 mRNA,
complete cds gi|18254512|gb|U37143.2|HSU37143[18254512]
AF039089
[0167] Homo sapiens cytochrome P450 (CYP2J2) gene, partial cds
gi|14486567|gb|AF039089.1|AF039089[14486567] CYP5 Family of
Cytochrome P450 Enzymes (Sequences from Genbank)
NM.sub.--011539
[0168] Mus musculus thromboxane A synthase 1, platelet (Tbxas1),
mRNA gi|31981465|ref|NM.sub.--011539.2|[31981465]
NM.sub.--030984
[0169] Homo sapiens thromboxane A synthase 1 (platelet, cytochrome
P450, family 5, subfamily A) (TBXAS1), transcript variant TXS-II,
mRNA gi|13699839|ref|NM.sub.--030984.1|[13699839]
NM.sub.--001061
[0170] Homo sapiens thromboxane A synthase 1 (platelet, cytochrome
P450, family 5, subfamily A) (TBXAS1), transcript variant TXS-I,
mRNA gi|13699838|ref|NM.sub.--001061.2|[13699838]
BC041157
[0171] Homo sapiens thromboxane A synthase 1 (platelet, cytochrome
P450, family 5, subfamily A), transcript variant TXS-I, mRNA (cDNA
clone MGC:48726 IMAGE:5755195), complete cds
gi|27371225|gb|BC041157.1|[27371225] CYP8 Family of Cytochrome P450
Enzymes (sequences from Genbank)
NM.sub.--000961
[0172] Homo sapiens prostaglandin I2 (prostacyclin) synthase
(PTGIS), mRNA gi|61676177|ref|NM.sub.--000961.3|[61676177]
NM.sub.--008968
[0173] Mus musculus prostaglandin 12 (prostacyclin) synthase
(Ptgis), mRNA gi|31982083|ref|NM.sub.--008968.2|[31982083]
D83402
[0174] Homo sapiens PTGIS(CYP8) gene for prostacyclin synthase,
complete cds gi|60683846|dbj|D83402.2|[60683846]
BC062151
[0175] Mus musculus prostaglandin 12 (prostacyclin) synthase, mRNA
(cDNA clone MGC:70035 IMAGE:6512164), complete cds
gi|38328177|gb|BC062151.1|[38328177]
[0176] a) DPAn-6-Derived Oxylipins
[0177] DPAn-6-derived oxylipins (also referred to as oxylipins, or
more particularly, docosanoids, from DPAn-6) include but are not
limited to, any R- or S-epimer of any monohydroxy, dihydroxy,
trihydroxy, or multi-hydroxy derivative of DPAn-6, and can include
hydroxy derivatizations at any carbon that forms a carbon-carbon
double bond in DPAn-6. Some exemplary, novel DPAn-6 derived
oxylipins of the present invention include, but are not limited to:
the R- and S-epimers of the monohydroxy products of DPAn-6,
including 7-hydroxy DPAn-6, 8-hydroxy DPAn-6, 10-hydroxy DPAn-6,
11-hydroxy DPAn-6, 13-hydroxy DPAn-6, 14-hydroxy DPAn-6, and
17-hydroxy DPAn-6 (most particularly 17-hydroxy DPAn-6); the R and
S epimers of the dihydroxy derivatives of DPAn-6, including
7,17-dihydroxy DPAn-6, 10,17-dihydroxy DPAn-6, 13,17-dihydroxy
DPAn-6, 7,14-dihydroxy DPAn-6, 8,14-dihydroxy DPAn-6,
16,17-dihdroxy DPAn-6, and 4,5-dihydroxy DPAn-6 (most particularly
10,17-dihydroxy DPAn-6); and tri-hydroxy derivatives of DPAn-6,
including R and S epimers of 7,16,17-trihydroxy DPAn-6 and
4,5,17-trihydroxy DPAn-6. Structures of the DPAn-6 oxylipins are
described and/or shown in Examples 3, 6, 8, and 11 and in FIGS.
3A-3D, FIG. 6, FIG. 8, FIG. 11 and FIG. 15.
[0178] The structures of various docosanoid products of enzymatic
(15-lipoxygenase, 5-lipoxygenase, 12-lipoxygenase and hemoglobin)
conversion of DPAn-6 are shown in Examples 3, 6, 8, and 11. These
DPAn-6 derivatives are structurally analogous to those produced
from DHA (Examples 2, 7 and 10) and DPAn-3 (Examples. 4, 9, and 12)
when the same enzymes are used.
[0179] Examples 3-12 demonstrate the production of docosanoid
products from DPAn-6, as well as DHA, DPAn-3 DTAn-6, and Example 13
describes the oxylipin (docosanoid) products found in a DHA/DPAn-6
LCPUFA oil.
[0180] b) DPAn-3-Derived Oxylipins
[0181] DPAn-3-derived oxylipins (also referred to as oxylipins, or
more particularly, docosanoids, from DPAn-3) include but are not
limited to, any R- or S-epimer of any monohydroxy, dihydroxy,
trihydroxy, or multi-hydroxy derivative of DPAn-3, and can include
hydroxy derivatizations at any carbon that forms a carbon-carbon
double bond in DPAn-3. Some exemplary, novel DPAn-3 derived
oxylipins of the present invention include, but are not limited to:
the R- and S-epimers of the monohydroxy products of DPAn-3,
including 7-hydroxy DPAn-3, 10-hydroxy DPAn-3, 11-hydroxy DPAn-3,
13-hydroxy DPAn-3, 14-hydroxy DPAn-3, 16-hydroxy DPAn-3, and
17-hydroxy DPAn-3; the R and S epimers of the dihydroxy derivatives
of DPAn-3, including 7,17-dihydroxy DPAn-3, 10,17-dihydroxy DPAn-3,
8,14-dihydroxy DPAn-3, 16,17-dihydroxy DPAn-3, 13,20-dihydroxy
DPAn-3, and 10,20-dihydroxy DPAn-3; and tri-hydroxy derivatives of
DPAn-3, including R and S epimers of 7,16,17-trihydroxy DPAn-3.
Structures of the DPAn-3 oxylipins are described and/or shown in
Examples 4, 9, and 12 and in FIGS. 4A-4D, FIG. 9, FIG. 12 and FIG.
16.
[0182] c) DTAn-6-Derived Oxylipins
[0183] DTAn-6-derived oxylipins (also referred to as oxylipins, or
more particularly, docosanoids, from DTAn-6) include but are not
limited to, any R- or S-epimer of any monohydroxy, dihydroxy,
trihydroxy, or multi-hydroxy derivative of DTAn-6, and can include
hydroxy derivatizations at any carbon that forms a carbon-carbon
double bond in DTAn-6. Some exemplary, novel DTAn-6 derived
oxylipins of the present invention include, but are not limited to:
the R- and S-epimers of the monohydroxy products of DTAn-6,
including 7-hydroxy DTAn-6, 10-hydroxy DTAn-6, 13-hydroxy DTAn-6,
and 17-hydroxy DTAn-6; the R and S epimers of the dihydroxy
derivatives of DTAn-6, including 7,17-dihydroxy DTAn-6,
10,17-dihydroxy DTAn-6, and 16,17-dihydroxy DTAn-6; and tri-hydroxy
derivatives of DTAn-6, including R and S epimers of
7,16,17-trihydroxy DTAn-6. Structures of the DTAn-6 oxylipins are
described and/or shown in Example 5 and in FIGS. 5A-5C and FIG.
17.
[0184] d) Novel C22-PUFA-Derived Oxylipins
[0185] Other novel C22-PUFA-derived oxylipins (also referred to as
oxylipins, or more particularly, docosanoids, from a C22-PUFA)
include but are not limited to, any R- or S-epimer of any
monohydroxy, dihydroxy, trihydroxy, or multi-hydroxy derivative of
C22-PUFAs, and can include hydroxy derivatizations at any carbon
that forms a carbon-carbon double bond in the C22-PUFAs. Some
exemplary, novel docosanoids that are encompassed by the present
invention include, but are not limited to 4,5-epoxy-17-hydroxy DPA,
7,8-epoxy DHA, 10,11-epoxy DHA, 13,14-epoxy DHA, 19,20-epoxy DHA,
13,14-dihydroxy DHA, 16,17-dihydroxy DTAn-6, 7,16,17-trihydroxy
DTAn-6, 4,5,17-trihydroxy DTAn-6, 7,16,17-trihydroxy DTAn-3,
16,17-dihydroxy DTAn-3, 16,17-dihydroxy DTRAn-6, 7,16,17-trihydroxy
DTRAn-6, 4,5-dihydroxy DTAn-6, and 10,16,17-trihydroxy DTRAn-6.
Structures of these C22-PUFA-derived docosanoids are shown in FIG.
23.
[0186] DPAn-6-, DTAn-6- and DPAn-3-derived oxylipins, or other
C22-PUFA-derived oxylipins of the present invention, as well as
analogs or derivatives of any of such oxylipins of the present
invention, can be produced by chemical synthesis or biological
synthesis, including by de novo synthesis or enzymatic conversion
of a substrate. Alternatively, such oxylipins can be produced by
isolation, enrichment and/or conversion of substrates from natural
sources (described below). According to the present invention,
reference to an oxylipin "derived from" a specific LCPUFA, such as
a "DPAn-6-derived oxylipin" or a "DPAn-6 oxylipin derivative", or a
"DPAn-6 oxylipin analog" by way of example, refers to an oxylipin
that has been produced by any method, using the knowledge of the
structure of an oxylipin that can be produced using DPAn-6 as a
substrate. Such an oxylipin need not be produced by an enzymatic
reaction or biological system, but, as mentioned above, can
alternatively be chemically synthesized de novo. In addition,
analogs or derivatives of naturally occurring DPAn-6 oxylipins may
be designed based on the structure of the naturally occurring
DPAn-6 oxylipins, but which differ from the naturally occurring
DPAn-6 oxylipin by at least one modification. Such analogs may also
be synthesized de novo using chemical synthesis methods or using by
modifications of biological production methods (e.g., enzyme
reactions). Methods of producing oxylipins according to the present
invention, including methods of enriching natural sources of such
oxylipins, and by enzymatic conversion of substrates are described
herein. Chemical synthesis methods for compounds such as oxylipins
are also known in the art and can readily be applied to the novel
oxylipin compounds of the present invention. Such methods are also
described herein.
[0187] According to the present invention, the language
"docosanoid-like compounds" or "docosanoid analogs" or "docosanoid
derivatives" is intended to include analogs of any docosanoids
described herein, including any of the novel docosanoids of the
present invention that include a C.sub.22 fatty acid having at
least three olefinic groups (carbon-carbon double bonds). Similar
language can also be used to more generally describe analogs and
derivatives of any oxylipins as described herein (e.g.,
oxylipin-like compounds, oxylipin analogs, oxylipin
derivatives).
[0188] As used herein, the term "analog" refers to a chemical
compound that is structurally similar to another compound but
differs slightly in composition (as in the replacement of one atom
by an atom of a different element or in the presence of a
particular functional group, or the replacement of one functional
group by another functional group). Thus, an analog is a compound
that is similar or comparable in function and appearance, but not
in structure or origin to the reference compound. For example, the
reference compound can be a reference docosanoid such as any
docosanoid derived from DHA, DPAn-6, DPAn-3 or DTAn-6, and an
analog is a substance possessing a chemical structure or chemical
properties similar to those of the reference docosanoid.
[0189] The terms "substituted", "substituted derivative" and
"derivative", when used to describe a compound of the present
invention, means that at least one hydrogen bound to the
unsubstituted compound is replaced with a different atom or a
chemical moiety. Examples of substituents include, but are not
limited to, hydroxy, alkyl, halogen, nitro, cyano, heterocycle,
aryl, heteroaryl, amino, amide, ester, ether, carboxylic acid,
thiol, thioester, thioether, sulfoxide, sulfone, carbamate,
peptidyl, PO.sub.3H.sub.2, and mixtures thereof.
[0190] Although a derivative has a similar physical structure to
the parent compound, the derivative may have different chemical
and/or biological properties than the parent compound. Such
properties can include, but are not limited to, increased or
decreased activity of the parent compound, new activity as compared
to the parent compound, enhanced or decreased bioavailability,
enhanced or decreased efficacy, enhanced or decreased stability in
vitro and/or in vivo, and/or enhanced or decreased absorbtion
properties.
[0191] It will be appreciated by those skilled in the art that
compounds of the invention having a chiral center may exist in and
be isolated in optically active and racemic forms. Some compounds
may exhibit polymorphism. It is to be understood that the present
invention encompasses any racemic, optically-active, polymorphic,
or stereoisomeric form, or mixtures thereof, of a compound of the
invention, which possess the useful properties described herein, it
being well known in the art how to prepare optically active forms
(for example, by resolution of the racemic form by
recrystallization techniques, by synthesis from optically-active
starting materials, by chiral synthesis, or by chromatographic
separation using a chiral stationary phase) and how to determine
anti-inflammatory activity, for example, using standard tests
described herein, or using other similar tests which are well known
in the art.
[0192] Prodrugs of any of the oxylipins described herein, and
particularly, any of the docosanoids described herein, and even
more particularly, any specific docosanoids as shown, for example,
in any of FIGS. 2A-2D, 3A-3D, 4A-4D, 5A-5C, 6-17, 18A-18C and 23,
may be identified using routine techniques known in the art.
Various forms of prodrugs are known in the art. For examples of
such prodrug derivatives, see, for example, a) Design of Prodrugs,
edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology,
Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press,
1985); b) A Textbook of Drug Design and Development, edited by
Krogsgaard-Larsen and H. Bundgaard, Chapter 5 "Design and
Application of Prodrugs," by H. Bundgaard p. 113-191 (1991); c) H.
Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992); d) H.
Bundgaard, et al., Journal of Pharmaceutical Sciences, 77:285
(1988); and e) N. Kakeya, et al., Chem. Pharm. Bull., 32: 692
(1984), each of which is specifically incorporated herein by
reference.
[0193] In addition, the invention also includes solvates,
metabolites, and salts (preferably pharmaceutically acceptable
salts) of compounds of any of the oxylipins described herein, and
particularly, any of the docosanoids described herein, and even
more particularly, any specific docosanoids as shown, for example,
in any of FIGS. 2A-2D, 3A-3D, 4A-4D, 5A-5C, 6-17, 18A-18C and
23.
[0194] The term "solvate" refers to an aggregate of a molecule with
one or more solvent molecules. A "metabolite" is a
pharmacologically active product produced through in vivo
metabolism in the body or organism of a specified compound or salt
thereof. Such products may result for example from the oxidation,
reduction, hydrolysis, amidation, deamidation, esterification,
deesterification, enzymatic cleavage, and the like, of the
administered or produced compound. Accordingly, the invention
includes metabolites of compounds of any of the oxylipins described
herein, and particularly, any of the docosanoids described herein,
and even more particularly, any specific docosanoids as shown, for
example, in any of FIGS. 2A-2D, 3A-3D, 4A-4D, 5A-5C, 6-17, 18A-18C
and 23, including compounds produced by a process comprising
contacting a compound of this invention with an organism for a
period of time sufficient to yield a metabolic product thereof.
[0195] A "pharmaceutically acceptable salt" or "salt" as used
herein, includes salts that retain the biological effectiveness of
the free acids and bases of the specified compound and that are not
biologically or otherwise undesirable. A compound of the invention
may possess a sufficiently acidic, a sufficiently basic, or both
functional groups, and accordingly react with any of a number of
inorganic or organic bases, and inorganic and organic acids, to
form a pharmaceutically acceptable sale. Examples of
pharmaceutically acceptable salts include those salts prepared by
reaction of the compounds of the present invention with a mineral
or organic acid or an inorganic base, such salts including
sulfates, pyrosulfates, bisulfates, sulfites, bisulfites,
phosphates, monohydrogenphosphates, dihydrogenphosphates,
metaphosphates, pyrophosphates, chlorides, bromides, iodides,
acetates, propionates, decanoates, caprylates, acrylates, formates,
isobutyrates, caproates, heptanoates, propiolates, oxalates,
malonates, succinates, suberates, sebacates, fumarates, maleates,
butyn-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates,
methylbenzoates, dinitromenzoates, hydroxybenzoates,
methoxybenzoates, phthalates, sulfonates, xylenesulfonates,
pheylacetates, phenylpropionates, phenylbutyrates, citrates,
lactates, .gamma.-hydroxybutyrates, glycollates, tartrates,
methanesulfonates, propanesulfonates, naphthalene-1-sulfonates,
naphthalene-2-sulfonates, and mandelates. Since a single compound
of the present invention may include more than one acidic or basic
moieties, the compounds of the present invention may include mono,
di or tri-salts in a single compound.
[0196] If the inventive compound is a base, the desired
pharmaceutically acceptable salt may be prepared by any suitable
method available in the art, for example, treatment of the free
base with an acidic compound, particularly an inorganic acid, such
as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid and the like, or with an organic acid, such as
acetic acid, maleic acid, succinic acid, mandelic acid, fumaric
acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid,
salicylic acid, a pyranosidyl acid, such as glucuronic acid or
galacturonic acid, an alphahydroxy acid, such as citric acid or
tartaric acid, an amino acid, such as aspartic acid or glutamic
acid, an aromatic acid, such as benzoic acid or cinnamic acid, a
sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic
acid, or the like.
[0197] If the inventive compound is an acid, the desired
pharmaceutically acceptable salt may be prepared by any suitable
method, for example, treatment of the free acid with an inorganic
or organic base. Preferred inorganic salts are those formed with
alkali and alkaline earth metals such as lithium, sodium,
potassium, barium and calcium. Preferred organic base salts
include, for example, ammonium, dibenzylammonium, benzylammonium,
2-hydroxyethylammonium, bis(2-hydroxyethyl)ammonium,
phenylethylbenzylamine, dibenzylethylenediamine, and the like
salts. Other salts of acidic moieties may include, for example,
those salts formed with procaine, quinine and N-methylglusoamine,
plus salts formed with basic amino acids such as glycine,
ornithine, histidine, phenylglycine, lysine and arginine.
Oils, Compositions, Formulations or Products Containing DPAn-6,
DPAn-3 DTAn-6, Other C22-LCPUFAs, Other LCPUFAs and/or Oxylipins
Derived Therefrom
[0198] The present invention includes oils, compositions,
formulations and products comprising LCPUFAs and/or LCPUFA
oxylipins described herein. According to the present invention, the
term "product" can be used to generally or generically describe any
oil, composition, or formulation of the present invention, although
one term might be preferred over another depending on the context
of use of the product. In one embodiment of the invention, oils,
compositions, and formulations include at least DPAn-6, DTAn-6 or
DPAn-3, or oxylipins derived therefrom, or any combinations
thereof, and may additionally include any other LCPUFAs and/or any
oxylipins derived therefrom. Such oxylipins can be produced by any
chemical or biological (biogenic) method, including de novo
synthesis, enzymatic conversion from any source (e.g., by enzymes
including lipoxygenases, cyclooxygenases, cytochrome P450 enzymes
and other heme-containing enzymes), purification from any source,
and production from any biological source (e.g., microbial, plant,
animal sources).
[0199] In one embodiment of the invention, oils are enriched for
the presence of any LCPUFA-derived oxylipin (also known as an
LCPUFA oxylipin), including any oxylipin derived from DHA, EPA,
DPAn-6, DTAn-6, and/or DPAn-3, with LCPUFA-derived docosanoids
being preferred, and oxylipins derived from DPAn-6, DTAn-6, or
DPAn-3 being particularly preferred. In another embodiment, oils,
compositions or formulations containing any LCPUFA-derived oxylipin
are produced, processed or treated to retain, and/or improve the
stability, absorption, bioactivity, bioavailability or efficacy of
the LCPUFA oxylipins in the oil, compositions or formulations.
Various methods of producing, processing and supplementing oils,
compositions or formulations are described below.
Sources of LCPUFAs and LCPUFA-Derived Oxylipins for Use in the
Present Invention
[0200] Any source of LCPUFA can be used to produce the LCPUFAs,
oxylipins, oils, compositions or formulations of the present
invention, including, for example, animal (invertebrates and
vertebrates), plant and microbial sources.
[0201] Examples of animal sources include aquatic animals (e.g.,
fish, marine mammals, and crustaceans such as krill and other
euphausids) and lipids extracted from animal tissues (e.g., brain,
liver, eyes, etc.).
[0202] More preferred sources include microorganisms and plants.
Preferred microbial sources of LCPUFAs include algae, fungi
(including yeast and filamentous fungi of the genus Mortierella),
protists and bacteria. The use of a microorganism source, such as
algae, can provide organoleptic advantages, i.e., fatty acids from
a microorganism source may not have the fishy taste and smell that
fatty acids from a fish source tend to have. However, fish oils are
also included in the present invention. While fish oils may
naturally undergo oxidation processes that produce aldehydes and
ketones that impart bad odors and tastes to such fish oils, the
present invention takes advantage of "directed" or "targeted"
oxidation of specific compounds to produce docosanoids or mixtures
of docosanoids that provide a beneficial quality to the oils
containing such docosanoids, including fish oils. In a preferred
embodiment, fish oils containing DHA and/or EPA, and DPAn-6, DTAn-6
and/or DPAn-3, are utilized in the invention.
[0203] Examples of bacterial sources include marine bacterial
sources, such as members of the genus Shewanella and Vibrio.
[0204] Most preferably, the LCPUFA source comprises algae or
protists. Preferred algal and protist genera are members of the
kingdom Stramenopila, and more preferably, are members of the algal
groups: dinoflagellates, diatoms, chrysophytes or
thraustochytrids.
[0205] Preferably, dinoflagellates are members of the genus
Crypthecodinium and even more preferably, members of the species
Crypthecodinium cohnii.
[0206] Developments have resulted in frequent revision of the
taxonomy of the Thraustochytrids (thraustochytrids). Taxonomic
theorists generally place Thraustochytrids with the algae or
algae-like protists. However, because of taxonomic uncertainty, it
would be best for the purposes of the present invention to consider
the strains described in the present invention as Thraustochytrids
to include the following organisms: Order: Thraustochytriales;
Family: Thraustochytriaceae (Genera: Thraustochytrium (which for
this application, includes Ulkenia, although some consider it to be
a separate genus), Schizochytrium, Japonochytrium, Aplanochytrium,
or Elina) or Labyrinthulaceae (Genera: Labyrinthula,
Labyrinthuloides, or Labyrinthomyxa). Also, the following genera
are sometimes included in either family Thraustochytriaceae or
Labyrinthulaceae: Althomia, Corallochytrium, Diplophyrys, and
Pyrrhosorus), and for the purposes of this invention are
encompassed by reference to a Thraustochytrid or a member of the
order Thraustochytriales. It is recognized that at the time of this
invention, revision in the taxonomy of Thraustochytrids places the
genus Labyrinthuloides in the family of Labyrinthulaceae and
confirms the placement of the two families Thraustochytriaceae and
Labyrinthulaceae within the Stramenopile lineage. It is noted that
the Labyrinthulaceae are sometimes commonly called labyrinthulids
or labyrinthula, or labyrinthuloides and the Thraustochytriaceae
are commonly called thraustochytrids, although, as discussed above,
for the purposes of clarity of this invention, reference to
Thraustochytrids encompasses any member of the order
Thraustochytriales and/or includes members of both
Thraustochytriaceae and Labyrinthulaceae. Information regarding
such algae can be found, for example, in U.S. Pat. Nos. 5,407,957,
5,130,242 and 5,340,594, which are incorporated herein by reference
in their entirety.
[0207] Particularly preferred LCPUFA and oxylipin sources for use
in the present invention include microorganisms from a genus
including, but not limited to: Thraustochytrium, Japonochytrium,
Aplanochytrium, Elina and Schizochytrium within the
Thraustochytriaceae, and Labyrinthula, Labyrinthuloides, and
Labyrinthomyxa within the Labyrinthulaceae. Preferred species
within these genera include, but are not limited to: any species
within Labyrinthula, including Labyrinthula sp., Labyrinthula
algeriensis, Labyrinthula cienkowskii, Labyrinthula chattonii,
Labyrinthula coenocystis, Labyrinthula macrocystis, Labyrinthula
macrocystis atlantica, Labyrinthula macrocystis macrocystis,
Labyrinthula magnifica, Labyrinthula minuta, Labyrinthula
roscoffensis, Labyrinthula valkanovii, Labyrinthula vitellina,
Labyrinthula vitellina pacifica, Labyrinthula vitellina vitellina,
Labyrinthula zopfii; any Labyrinthuloides species, including
Labyrinthuloides sp., Labyrinthuloides minuta, Labyrinthuloides
schizochytrops; any Labyrinthomyxa species, including
Labyrinthomyxa sp., Labyrinthomyxa pohlia, Labyrinthomyxa
sauvageaui, any Aplanochytrium species, including Aplanochytrium
sp. and Aplanochytrium kerguelensis; any Elina species, including
Elina sp., Elina marisalba, Elina sinorifica; any Japonochytrium
species, including Japonochytrium sp., Japonochytrium marinum; any
Schizochytrium species, including Schizochytrium sp.,
Schizochytrium aggregatum, Schizochytrium limacinum, Schizochytrium
minutum, Schizochytrium octosporum; and any Thraustochytrium
species, including Thraustochytrium sp., Thraustochytrium
aggregatum, Thraustochytrium arudimentale, Thraustochytrium
aureurn, Thraustochytrium benthicola, Thraustochytrium globosum,
Thraustochytrium kinnei, Thraustochytrium motivum, Thraustochytrium
pachydermum, Thraustochytrium proliferum, Thraustochytrium roseum,
Thraustochytrium striatum, Ulkenia sp., Ulkenia minuta, Ulkenia
profunda, Ulkenia radiate, Ulkenia sarkariana, and Ulkenia
visurgensis. Particularly preferred species within these genera
include, but are not limited to: any Schizochytrium species,
including Schizochytrium aggregatum, Schizochytrium limacinum,
Schizochytrium minutum; or any Thraustochytrium species (including
former Ulkenia species such as U. visurgensis, U. amoeboida, U.
sarkariana, U. profunda, U. radiata, U. minuta and Ulkenia sp.
BP-5601), and including Thraustochytrium striatum, Thraustochytrium
aureum, Thraustochytrium roseum; and any Japonochytrium species.
Particularly preferred strains of Thraustochytriales include, but
are not limited to: Schizochytrium sp. (S31)(ATCC 20888);
Schizochytrium sp. (S8)(ATCC 20889); Schizochytrium sp.
(LC-RM)(ATCC 18915); Schizochytrium sp. (SR21); Schizochytrium
aggregatum (Goldstein et Belsky)(ATCC 28209); Schizochytrium
limacinum (Honda et Yokochi)(IFO 32693); Thraustochytrium sp.
(23B)(ATCC 20892); Thraustochytrium striatum (Schneider)(ATCC
24473); Thraustochytrium aureum (Goldstein)(ATCC 34304);
Thraustochytrium roseum (Goldstein)(ATCC 28210); Japonochytrium sp.
(L1)(ATCC 28207); Thraustochytrium sp. 12B (ATCC 20890);
Thraustochytrium sp. U42-2 (ATCC 20891); and Labyrinthula
(labyrinthulid) strain L59 (Kumon) (IPOD AIST No. FERM
P-19897).
[0208] In one aspect, the organism-sources of oils are genetically
engineered to enhance the production of LCPUFAs and/or LCPUFA
oxylipins. The more preferred sources are microorganisms (which can
be grown in fermentors), or oilseed crops. For example,
microorganisms and plants can be genetically engineered to express
genes that produce LCPUFAs. Such genes can include genes encoding
proteins involved in the classical fatty acid synthase pathways, or
genes encoding proteins involved in the PUFA polyketide synthase
(PKS) pathway. The genes and proteins involved in the classical
fatty acid synthase pathways, and genetically modified organisms,
such as plants, transformed with such genes, are described, for
example, in Napier and Sayanova, Proceedings of the Nutrition
Society (2005), 64:387-393; Robert et al., Functional Plant Biology
(2005) 32:473-479; or U.S. Patent Application Publication
2004/0172682. The PUFA PKS pathway, genes and proteins included in
this pathway, and genetically modified microorganisms and plants
transformed with such genes for the expression and production of
PUFAs are described in detail in: U.S. Pat. No. 6,566,583; U.S.
Patent Application Publication No. 20020194641, U.S. Patent
Application Publication No. 20040235127 A1, and U.S. Patent
Application Publication No. 20050100995A1, each of which is
incorporated herein by reference in its entirety.
[0209] Preferred oilseed crops include soybeans, corn, safflower,
sunflower, canola, flax, or rapeseed, linseed, and tobacco that
have been genetically modified to produce LCPUFA as described
above. More preferably, the oilseed crops also possess, or can be
modified to possess (e.g., by genetic engineering), enzyme systems
for converting the LCPUFA to its hydroxy derivative forms (i.e.,
oxylipin). Such enzymes are well known in the art and are
described, for example, in Table 1.
[0210] Genetic transformation techniques for microorganisms and
plants are well-known in the art. It is an embodiment of the
present invention that the nucleic acid molecules encoding any one
or more enzymes for converting an LCPUFA to its hydroxy-derivative
form (and, if required, cofactors therefor) can be used to
transform plants or microorganisms to initiate, improve and/or
alter (modify, change) the oxylipin production capabilities of such
plants or microorganisms. Transformation techniques for
microorganisms are well known in the art and are discussed, for
example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Labs Press. A general technique for
transformation of dinoflagellates, which can be adapted for use
with Crypthecodinium cohnii, is described in detail in Lohuis and
Miller, The Plant Journal (1998) 13(3): 427-435. A general
technique for genetic transformation of Thraustochytrids is
described in detail U.S. Patent Application Publication No.
20030166207, published Sep. 4, 2003.
[0211] Methods for the genetic engineering of plants are also well
known in the art. For instance, numerous methods for plant
transformation have been developed, including biological and
physical transformation protocols. See, for example, Mild et al.,
"Procedures for Introducing Foreign DNA into Plants" in Methods in
Plant Molecular Biology and Biotechnology, Glick, B. R. and
Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 67-88.
In addition, vectors and in vitro culture methods for plant cell or
tissue transformation and regeneration of plants are available.
See, for example, Gruber et al., "Vectors for Plant Transformation"
in Methods in Plant Molecular Biology and Biotechnology, Glick, B.
R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp.
89-119. See also, Horsch et al., Science 227:1229 (1985); Kado, C.
I., Crit. Rev. Plant. Sci. 10:1 (1991); Moloney et al., Plant Cell
Reports 8:238 (1989); U.S. Pat. No. 4,940,838; U.S. Pat. No.
5,464,763; Sanford et al., Part. Sci. Technol. 5:27 (1987);
Sanford, J. C., Trends Biotech. 6:299 (1988); Sanford, J. C.,
Physiol. Plant 79:206 (1990); Klein et al., Biotechnology 10:268
(1992); Zhang et al., Bio/Technology 9:996 (1991); Deshayes et al.,
EMBO J., 4:2731 (1985); Christou et al., Proc Natl. Acad. Sci. USA
84:3962 (1987); Hain et al., Mol. Gen. Genet. 199:161 (1985);
Draper et al., Plant Cell Physiol. 23:451 (1982); Donn et al., In
Abstracts of VIIth International Congress on Plant Cell and Tissue
Culture IAPTC, A2-38, p. 53 (1990); D'Halluin et al., Plant Cell
4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol. 24:51-61
(1994).
[0212] Preferably, microorganisms or oilseed plants useful as
sources of LCPUFAs and oxylipins derived therefrom are
microorganisms or plants that produce PUFAs (either naturally or by
genetic engineering) having C20 or greater polyunsaturated fatty
acids. Preferably, the LCPUFAs produced by the microorganism or
plants have 3, 4 or more double bonds. Even more preferably, the
microorganisms or plants produce C20 or greater LCPUFAs with 5 or
more double bonds. Even more preferably, the microorganisms or
plants produce C20 or greater LCPUFAs including, but not limited
to:EPA (20:5n-3), DHA (C22:6n-3), DPAn-3(22:5n-3), DPAn-6(22:5n-6),
DTAn-6 (22:4n-6) or combinations of these LCPUFAs.
[0213] In another embodiment, it is preferred that the
microorganism or plant sources of LCPUFAs naturally express enzymes
such as cyclooxygenases, lipoxygenases, cytochrome P450 enzymes
(including hydroxylases, peroxidases, and oxygenases), and/or other
heme-containing enzymes for biochemical conversion of LCPUFAs to
oxylipins (e.g., to the hydroxy, peroxide, or epoxide derivatives
of LCPUFAs). The invention also includes organisms (e.g., plants or
microorganisms) that have been naturally selected or genetically
engineered to express these enzymes and/or to have enhanced
activity of these enzymes in the organism. Organisms can be
genetically engineered to express or target any enzyme that
catalyzes the biochemical conversion of LCPUFAs to oxylipins such
as cyclooxygenases, lipoxygenases, cytochrome P450 enzymes
(including hydroxylases, peroxidases, and oxygenases), and/or other
heme-containing enzymes for biochemical conversion of LCPUFAs to
oxylipins.
[0214] Numerous examples of such enzymes are known in the art and
are listed in Table 1, although the invention is not limited to
these particular enzymes. The enzymes in Table 1 are described by
their name, official symbols, aliases, organisms, and/or by
reference to the database accession number in the National Center
for Biotechnology Information that contains the sequence
information for the enzymes and genes encoding such enzymes. All of
the information included in each of the database accession numbers
is incorporated herein by reference. These enzymes and the genes
encoding such enzymes, or homologues (including natural variants)
thereof, can be used to genetically engineer an organism that
produces LCPUFAs to express the enzyme or to target the an
endogenous form of the enzyme to initiate, increase or enhance the
activity of the enzyme in the organism. Optionally, these enzymes
can be targeted to a particular compartment (e.g., plastids in
plants), which is separated from compartments containing LCPUFAs,
regulating the potential for formation and degradation of oxylipins
produced in vivo. The enzymes (endogenous or recombinant) may be
placed under the control of an inducible promoter, so that the
production of oxylipins from LCPUFAs can be controlled in the
organism. For example, in a plant, oxylipins can be formed during
post-harvest processing in which the oilseeds are disrupted to
allow contact of the LCPUFAs and oxygenase enzymes.
[0215] Microbial or plant cell sources of LCPUFAs useful in the
present invention preferably include those microorganisms or plant
cells that can be grown in a fermentor or photobioreactor. More
preferably, microbial or plant cell sources of LCPUFAs useful in
the present invention preferably include those microorganisms or
plant cells that can be grown heterotrophically in fermentors.
Unique Characteristics of Oils Produced by the Present
Invention
[0216] Oils containing oxylipins of LCPUFAs described herein have
unique characteristics as compared to oxylipins that are chemically
synthesized or produced by enzymatic conversion in vitro as
described prior to the present invention. The LCPUFA oxylipins, and
in particular, the docosanoids, are present in the oils in their
free and/or esterifed forms. In the esterified form, the LCPUFA
oxylipins, and in particular, the docosanoids, can be present in
the triglyceride, diglyceride, monoglyceride, phospholipid, sterol
ester and/or wax ester forms. Since the oxylipins have only been
described previously in the free fatty acid form, the esterified
forms represent novel forms of oxylipins, the presence of which can
be enhanced, stabilized or retained in oils or compositions of the
present invention. Without being bound by theory, the present
inventors believe that once the LCPUFA oxylipins, and in
particular, the docosanoids, are formed in the free fatty acid
form, they can be re-esterified into one of the esterifed forms.
Alternatively, the fatty acid molecules can be converted to
oxylipins while they are still in an esterifed form.
[0217] The LCPUFA oil processed by the methods described according
to the present invention (see below) will have total LCPUFA
oxylipin concentrations, and in particular total docosanoid
concentrations, that are at least 2.times., at least 3.times., at
least 4.times., at least 5.times., at least 10.times., at least
20.times., at least 50.times., at least 100.times., at least
200.times., at least 400.times., at least 1,000.times., or at least
5,000.times. higher (including any other increment of 1.times.,
e.g., 20.times., 21.times., 22.times., etc.) than the trace
concentrations normally found in LCPUFA oils that have been through
the standard refining, bleaching, and deodorization process
commonly used for edible oils. LCPUFA oils produced by the
processes outlined according to the present invention will
preferably contain at least 1 .mu.g, at least 5 .mu.g, at least 10
.mu.g, at least 15 .mu.g, at least 20 .mu.g, at least 30 .mu.g, at
least 50 .mu.g, at least 100 .mu.g, at least 200 .mu.g, at least
500 .mu.g, at least 1,000 .mu.g, at least 2,000 .mu.g, at least
5,000 .mu.g, at least 10,000 .mu.g, or at least 50,000 .mu.g or
more of at least one or more LCPUFA oxylipins, and in particular,
docosanoids, per gram of oil (including any other increment in 0.1
.mu.g increments). It is noted that through processing and
purification of oils or compositions, the LCFUA oxylipin
concentrations could actually be much higher (e.g., approaching
100%) during the production phase, although the oils and
compositions would typically be diluted or titrated to the amounts
described above prior to being used in a nutritional, therapeutic,
or other process.
[0218] The oils produced from the present invention are enriched
preferably with hydroxyl forms of DHA, and/or EPA and/or DPAn-3
and/or DPAn-6, and/or DTAn-6. LCPUFA hydroxy derivative-rich oils
from this invention can be enriched with hydroxy forms of LCPUFA,
including derivatives from just one LCPUFA (e.g. from DHA or EPA or
DPAn-6 or DPAn-3 or DTAn-6), or from a combination of LCPUFAs (for
example, DHA plus DPA (n-6 and/or n-3), DTAn-6, or EPA).
DPAn-6 or DPAn-3 or DTAn-6 Oils, Compositions and Formulations
[0219] One embodiment of the present invention includes the use of
the LCPUFAs themselves, and particularly, DPAn-6 and/or DPAn-3, as
anti-inflammatory or neuroprotective agents (i.e., the LCPUFAs are
provided, alone or in combination with oxylipin metabolites
thereof). DPAn-6 and/or DPAn-3 can be provided alone or in
combination with other LCPUFAs, and preferably DHA and/or EPA.
DTAn-6 having anti-inflammatory or neuroprotective properties is
also encompassed by the present invention. Preferably, DPAn-6,
DPAn-3 or DTAn-6 used in the present invention is provided in one
of the following forms: as triglyceride containing DPAn-6, DTAn-6
and/or DPAn-3, as a phospholipid containing DPAn-6, DTAn-6 and/or
DPAn-3, as a free fatty acid, as an ethyl or methyl ester of
DPAn-6, DTAn-6 and/or DPAn-3.
[0220] In a preferred embodiment, the DPAn-6, DTAn-6 and/or DPAn-3
is provided in the form of an oil, and preferably a microbial oil
(wild-type or genetically modified) or a plant oil from an oil seed
plant that has been modified with genes that catalyze the
production of LCPUFAs. Preferred microbial and oilseed sources have
been described in detail above. Preferably, the DPAn-6, DTAn-6 or
DPAn-3 to be used in the present invention, including oils or
compositions containing such LCPUFAS and/or oxylipin-derivatives
thereof, contains one or more of the following additional LCPUFAs
or oxylipin-derivatives thereof: DHA or EPA. Most preferably, the
additional LCPUFA is DHA.
[0221] DPAn-6 is the longest chain fatty acid in the omega-6
series. Docosapentaenoic acid (n-6) is found in numerous human
foods and human breast milk at levels from 0.0 to 2.4% (Taber et
al. 1998) and represents approximately 0.1% of total fatty acids
(Koletzko et al. 1992), respectively. Major sources of DPAn-6 in
the diet for adults and children are poultry (meat and eggs) and
seafood (Taber et al. 1998, Nichols et al. 1998). DPAn-6 is
typically a component of tissues in the human body, including the
heart (Rocquelin et al. 1989), brain (Svennerholm et al. 1978,
O'Brien et al. 1965), liver (Salem 1989), red blood cells (Sanders
et al. 1978, Sanders et al. 1979) and adipose tissue (Clandinin et
al. 1981).
[0222] Oils, compositions, or formulations (or any products) useful
in the present invention preferably comprise DPAn-6, DPAn-3 and/or
DTAn-6 in an amount that is at least about 2 weight percent, or at
least about 5 weight percent, or at least about 10 weight percent,
or at least about 15 weight percent, or at least about 20 weight
percent, or at least about 25 weight percent, or at least about 30
weight percent, or at least about 35 weight percent, or at least
about 40 weight percent, or at least about 45 weight percent, or at
least about 50 weight percent, and so on, in increments of 1 weight
percent (i.e., 2, 3, 4, 5, . . . ) up to or at least about 95
weight percent or higher of the total lipids in the oil,
composition of formulation. DHA and/or EPA can also be included in
an amount that is at least about 2 weight percent, or at least
about 5 weight percent, or at least about 10 weight percent, or at
least about 15 weight percent, or at least about 20 weight percent,
or at least about 25 weight percent, or at least about 30 weight
percent, or at least about 35 weight percent, or at least about 40
weight percent, or at least about 45 weight percent, or at least
about 50 weight percent, and so on, in increments of 1 weight
percent (i.e., 2, 3, 4, 5, . . . ) up to or at least about 95
weight percent or higher of the total lipids in the oil,
composition, formulation or other product.
[0223] In another preferred embodiment, the oil, composition,
formulation or other product comprises about 30 weight percent or
more, about 35 weight percent or more, about 40 weight percent or
more, about 45 weight percent or more, about 50 weight percent or
more, about 55 weight percent or more, about 60 weight percent or
more, about 65 weight percent or more, about 70 weight percent or
more, about 75 weight percent or more, or about 80 weight percent
or more, or about 85 weight percent or more, or about 90 weight
percent or more, or about 95 weight percent or more of a
combination of DPAn-6 and DHA. Preferably, the ratio of DHA to DPA
(n-6) in the oil, composition, formulation or other product is
between about 1:10 to about 10:1, or any ratio between 1:10 and
10:1.
Forms of Provision of LCPUFAs and Oxylipins
[0224] In accordance with the present invention, the LCPUFAs and/or
oxylipin derivatives thereof that are used in oils, supplements,
cosmetics, therapeutic compositions, and other formulations or
products described herein are provided in a variety of forms. For
example, such forms include, but are not limited to: an algal oil
comprising the LCPUFAs and/or oxylipin derivatives thereof,
preferably produced as described herein; a plant oil comprising the
PUFA and/or oxylipin derivatives thereof, preferably produced as
described herein; triglyceride oil comprising the PUFA;
phospholipids comprising the PUFA; a combination of protein,
triglyceride and/or phospholipid comprising the PUFA; dried marine
microalgae comprising the PUFA; sphingolipids comprising the PUFA;
esters of the PUFA; free fatty acid; a conjugate of the PUFA with
another bioactive molecule; and combinations thereof. Long chain
fatty acids can be provided in amounts and/or ratios that are
different from the amounts or ratios that occur in the natural
source of the fatty acids, such as by blending, purification,
enrichment (e.g., through culture and/or processing techniques) and
genetic engineering of the source. Bioactive molecules can include
any suitable molecule, including, but not limited to, a protein, an
amino acid (e.g. naturally occurring amino acids such as
DHA-glycine, DHA-lysine, or amino acid analogs), a drug, and a
carbohydrate. The forms outlined herein allow flexibility in the
formulation of foods with high sensory quality, dietary or
nutritional supplements, and pharmaceutical agents.
[0225] In one embodiment of the invention, a source of the desired
phospholipids includes purified phospholipids from eggs, plant
oils, and animal organs prepared via extraction by polar solvents
(including alcohol or acetone) such as the Friolex process and
phospholipid extraction process (PEP) (or related processes) for
the preparation of oils or compositions (nutritional supplements,
cosmetics, therapeutic formulations) rich in DPAn-6 and/or DPAn-6
or docosanoids derived therefrom, alone or in combination with DHA
and/or EPA and/or oxylipins derived therefrom. The Friolex and
related processes are described in greater detail in PCT Patent
Nos. PCT/IB01/00841, entitled "Method for the Fractionation of Oil
and Polar Lipid-Containing Native Raw Materials", filed Apr. 12,
2001, published as WO 01/76715 on Oct. 18, 2001; PCT/IB01/00963,
entitled "Method for the Fractionation of Oil and Polar
Lipid-Containing Native Raw Materials Using Alcohol and
Centrifugation", filed Apr. 12, 2001, published as WO 01/76385 on
Oct. 18, 2001; and PCT/DE95/01065 entitled "Process For Extracting
Native Products Which Are Not Water-Soluble From Native Substance
Mixtures By Centrifugal Force", filed Aug. 12, 1995, published as
WO 96/05278 on Feb. 22, 1996; each of which is incorporated herein
by reference in its entirety.
[0226] Any biologically acceptable dosage forms, and combinations
thereof, are contemplated by the inventive subject matter. Examples
of such dosage forms include, without limitation, chewable tablets,
quick dissolve tablets, effervescent tablets, reconstitutable
powders, elixirs, liquids, solutions, suspensions, emulsions,
tablets, multi-layer tablets, bi-layer tablets, capsules, soft
gelatin capsules, hard gelatin capsules, caplets, lozenges,
chewable lozenges, beads, powders, granules, particles,
microparticles, dispersible granules, cachets, douches,
suppositories, creams, topicals, inhalants, aerosol inhalants,
patches, particle inhalants, implants, depot implants, ingestibles,
injectables, infusions, health bars, confections, cereals, cereal
coatings, foods, nutritive foods, functional foods and combinations
thereof. The preparations of the above dosage forms are well known
to persons of ordinary skill in the art. Preferably, a food (food
product) that is enriched with the desired LCPUFAs and/or oxylipin
derivatives thereof is selected from the group including, but not
limited to: baked goods and mixes; chewing gum; breakfast cereals;
cheese products; nuts and nut-based products; gelatins, pudding,
and fillings; frozen dairy products; milk products; dairy product
analogs; hard or soft candy; soups and soup mixes; snack foods;
processed fruit juice; processed vegetable juice; fats and oils;
fish products; plant protein products; poultry products; and meat
products.
[0227] More particularly, oils containing LCPUFAs and oxylipin
derivatives thereof, and particularly, enhanced levels of LCPUFA
oxylipins (and in particular docosanoids), will be useful as
dietary supplements in the form of oil-filled capsules or through
fortification of foods, beverages or infant formula to enhance the
anti-inflammatory benefits of these products and/or promote more
balanced immune function over that achieved by an LCPUFA oil with
low or no LCPUFA oxylipin (and in particular docosanoid) content.
For example, LCPUFA oxylipin (and in particular
docosanoid)-enriched LCPUFA oils capsules, and preferably gelatin
capsules for protection against oxidation, are provided for
delivery of both the LCPUFA(s) and enhanced LCPUFA oxylipin (and in
particular docosanoid) content in a single dietary supplement. In
another application, foods and beverages, including but not limited
to dairy products and dairy analogs, bakery products and
confectionaries, processed meats and meat analogs, grain products
and cereals, liquid and powered beverages, including juices and
juice drinks, carbonated and processed beverage products or infant
formulas would be fortified with LCPUFA oils with enhanced levels
of LCPUFA oxylipins (and in particular docosanoids) and thereby
increase the LCPUFA oxylipin (and in particular docosanoid) intake
over the non-LCPUFA oxylipin (and in particular
docosanoid)-enriched LCPUFA oils alone. In another example, LCPUFA
oxylipin (and in particular docosanoid)-enriched LCPUFA oils could
be microencapsulated prior to fortification of the foods, beverages
or formulas to reduce oxidation/degradation of the LCPUFA oxylipins
(and in particular docosanoids) and/or LCPUFA and improve
organoleptic properties and shelf-life of the fortified
food/beverage or infant formula products. In another example,
LCPUFA oxylipin (and in particular docosanoid)-enriched oils could
be formulated into a cream or emulsion for topical applications for
reduction of inflammation, or the LCPUFA oxylipin (and in
particular docosanoid)-enriched oils could be formulated into sun
screens or cosmetics, such as face or hand creams, moisturizers,
foundations, eye gels or shaving creams, to reduce skin irritation
or redness, allergic reactions, or puffiness/edema. In another
example, more highly enriched or purified forms of the LCPUFA
oxylipins (and in particular docosanoids) or LCPUFA oxylipin (and
in particular docosanoid)-rich oils could be used in pharmaceutical
formulations to prevent or reduce symptoms of conditions or
diseases associated with local, systemic, chronic or acute
inflammatory reactions or processes.
Additional Components
[0228] In one embodiment of the present invention, any of the
sources of LCPUFAs and/or oxylipin derivatives thereof, including
any oils or compositions or formulations containing such LCPUFAs or
oxylipin derivatives thereof, can be provided with one or more
additional components that may be useful in a method of the
invention. Such additional components include, but are not limited
to, any additional anti-inflammatory agent, nutritional supplement
(e.g., vitamins, minerals and other nutritional agents, including
nutraceutical agents), a therapeutic agent, or a pharmaceutical or
a nutritional carrier (e.g., any excipient, diluent, delivery
vehicle or carrier compounds and formulations that can be used in
conjunction with pharmaceutical (including therapeutic)
compositions or nutritional compositions).
[0229] In one preferred embodiment, the LCPUFAs and/or oxylipin
derivatives thereof are provided along with acetosalicylic acid
(ASA), or aspirin or any other anti-inflammatory agent.
Methods to Produce and Optimize Production of LCPUFAs and
LCPUFA-Derived Oxylipins
[0230] Methods for producing LCPUFA-containing oils (including DHA
and DPAn-6) using microbial technology have been taught in the art.
U.S. Pat. No. 5,130,242 and U.S. Pat. No. 5,340,594 teach methods
for producing DHA and DPA rich lipids via fermentation using
Schizochytrium spp. or Thraustochytrium spp. U.S. Patent
Application Publication No. 2003/0161866 describes a process for
preparing oils containing DHA and DPAn-6 by cultivating a
microorganism belonging to the presumptive genus Ulkenia.
[0231] Methods for producing LCPUFA-containing plants and plant
seed oils have been described in, for example, U.S. Pat. No.
6,566,583; U.S. Patent Application Publication No. 20020194641,
U.S. Patent Application Publication No. 20040235127A1, and U.S.
Patent Application Publication No. 20050100995A1, as well as Napier
and Sayanova, Proceedings of the Nutrition Society (2005),
64:387-393; Robert et al., Functional Plant Biology (2005)
32:473-479; or U.S. Patent Application Publication
2004/0172682.
[0232] As discussed above, oxylipins useful in the present
invention can be produced through chemical synthesis using LCPUFA
precursors or can be synthesized completely de novo. Chemical
synthesis methods for oxylipin compounds are known in the art
(e.g., see Rodriguez and Spur (2004); Rodriguez and Spur, 2005;
Guilford et al. (2004)). In addition, general chemical synthesis
methods are well known in the art. For example, the compounds of
present invention may be prepared by both conventional and solid
phase synthetic techniques known to those skilled in the art.
Useful conventional techniques include those disclosed by U.S. Pat.
Nos. 5,569,769 and 5,242,940, and PCT publication No. WO 96/37476,
all of which are incorporated herein in their entirety by this
reference. Combinatorial synthetic techniques, however, may be
particularly useful for the synthesis of the compounds of the
present invention. See, e.g., Brown, Contemporary Organic
Synthesis, 1997, 216; Felder and Poppinger, Adv. Drug Res., 1997,
30, 111; Balkenhohl et al., Angew. Chem. Int. Ed. Engl., 1996, 35,
2288; Hermkens et al., Tetrahedron, 1996, 52, 4527; Hermkens et
al., Tetrahedron, 1997, 53, 5643; Thompson et al., Chem. Rev.,
1996, 96, 555; and Nefzi et al., Chem. Rev., 1997, 2, 449-472.
[0233] The compounds of the present invention can be synthesized
from readily available starting materials. Various substituents on
the compounds of the present invention can be present in the
starting compounds, added to any one of the intermediates or added
after formation of the final products by known methods of
substitution or conversion reactions. If the substituents
themselves are reactive, then the substituents can themselves be
protected according to the techniques known in the art. A variety
of protecting groups are known in the art, and can be employed.
Examples of many of the possible groups can be found in "Protective
Groups in Organic Synthesis" by T. W. Green, John Wiley and Sons,
1981, which is incorporated herein in its entirety. For example,
nitro groups can be added by nitration and the nitro group can be
converted to other groups, such as amino by reduction, and halogen
by diazotization of the amino group and replacement of the diazo
group with halogen. Acyl groups can be added by Friedel-Crafts
acylation. The acyl groups can then be transformed to the
corresponding alkyl groups by various methods, including the
Wolff-Kishner reduction and Clemmenson reduction Amino groups can
be alkylated to form mono-and di-alkylamino groups; and mercapto
and hydroxy groups can be alkylated to form corresponding ethers.
Primary alcohols can be oxidized by oxidizing agents known in the
art to form carboxylic acids or aldehydes, and secondary alcohols
can be oxidized to form ketones. Thus, substitution or alteration
reactions can be employed to provide a variety of substituents
throughout the molecule of the starting material, intermediates, or
the final product, including isolated products.
[0234] Since the compounds of the present invention can have
certain substituents which are necessarily present, the
introduction of each substituent is, of course, dependent on the
specific substituents involved and the chemistry necessary for
their formation. Thus, consideration of how one substituent would
be affected by a chemical reaction when forming a second
substituent would involve techniques familiar to one of ordinary
skill in the art. This would further be dependent upon the ring
involved.
[0235] Alternatively, the oxylipins are catalytically produced via
an enzyme-based technology using LCPUFAs as the substrate. In one
embodiment, enzymes such as lipoxygenases, cyclooxygenases,
cytochrome P450 enzymes and other heme-containing enzymes, such as
those described in Table 1 (e.g., provided as recombinant or
isolated/immobilized enzyme preparations) are contacted in vitro
with the LCPUFAs produced by an organism, such as during extraction
or post-harvest processing of a microorganism biomass or plant or
oilseed or animal, whereby LCPUFAs produced by the organism are
converted to oxylipins. The oxylipin derivatives of LCPUFAs can
also be produced by microorganisms in a fermentor and recovered and
purified for use. Preferred methods of production and recovery of
oxylipins which are believed to enhance the quantity, quality and
stability of the compounds are described below. The oxylipins
produced by any of the above production technologies, can be
further processed and recovered as derivatives of the oxylipins or
salts thereof to aid in the recoverability, stability, absorption,
bioavailability and/or efficacy, if desired. In addition, the
oxylipins produced by any of the technologies described herein can
be used to supplement other sources of oxylipins (e.g., a refined
LCPUFA oil) or provided in the form of any composition or
formulation for use in any application described herein.
Methods to Optimize Production of LCPUFA Oxylipin Concentrations in
Oils Produced by Organisms
[0236] The production or fermentation conditions can be optimized
to enhance production of the LCPUFA oxylipins (and in particular
docosanoids) and/or to stabilize them once they have been produced.
These methods include selecting culture conditions that enhance
activity and/or expression of the enzymes producing these
compounds. For example, any culture condition that alters the cell
concentration and/or specific growth rate of the culture can
potentially alter the cellular composition. Culture conditions that
are known to modify the production of metabolites or secondary
metabolites in microorganisms include but are not limited to the
following: hypoosmotic or hyperosomotic salinity stress, nutrient
limitation stress (such as nitrogen, phosphorus, carbon, and/or
trace metals), temperature stress (higher or lower than customary),
elevated or reduced levels of oxygen and/or carbon dioxide, and
physical stresses such as shear. In addition, the level of
metabolites or secondary metabolites in cells can vary with phase
of growth (exponential vs stationary), and by providing various
precursor molecules for bioconversion by the microorganism.
[0237] These methods also include use of additives, both organic
and inorganic, which enhance this enzymatic activity, or
alternatively, directly enhance auto-oxidation of the LCPUFAs to
these compounds and/or stabilize the LCPUFA oxylipins (and in
particular docosanoids) once they are produced. For example,
compounds that modify or acetylate COX2 (such as one of the many
forms of acetylsalicylic acid) or compounds which stimulate
expression or activity of COX2, lipoxygenase, cytochrome P450
enzymes (including hydroxylases, peroxidases, and oxygenases)
and/or other heme-containing enzymes, can be added to the culture
medium. Examples of compounds that may enhance the expression or
activity of lipoxygenases, cyclooxygenases, cytochrome P450 and
other heme-containing enzymes in culture include, but are not
limited to: ATP, cytokines (e.g., interleukin-4, interleukin-13, or
granulocyte-macrophage colony-stimulating factor), hormones (e.g.,
bradykinin or 1,25-dihydroxyvitamin D.sub.3), cationic metals
(e.g., Ca.sup.2+), phospholipids (e.g., phosphatidyl serine), fatty
acids (e.g., DHA), preformed hydroperoxides, glucocorticoids (e.g.,
dexamethasone), nonsteroidal anti-inflammatory compounds (e.g.,
acetosalicylic acid or aspirin), and other inducers of cytochrome
P450 activities (e.g., ethanol, fibrates and other peroxisome
proliferators, phenobarbital, steroids, and rifampicin).
Additionally, compounds or conditions that lead to autooxidation of
the LCPUFAs in the microorganism resulting in formation of the
mono- thru penta-hydroxy derivatives of these LCPUFA are also
preferred. For example, such compounds or conditions that can
promote autooxidation of LCPUFAs include, but are not limited to,
metals (including transition metals such as iron, copper or zinc,
and alkali earth metals such as magnesium), peroxides, lipid
radicals, and high oxygen conditions.
Improved Oil Extraction Processes that Enhance LCPUFA Oxylipin
Content or Retention
[0238] As enzymes play an important role in the formation of
hydroxy derivatives of LCPUFAs, there are preferable methods for
enhancing contact between these enzymes and the LCPUFAs to enhance
formation of the hydroxy derivatives. In one preferred process, the
microbial cells or oilseeds are ruptured (e.g., via homogenization
for the microbial cells or by crushing for the oilseeds) and the
resulting oil and biomass mixture is allowed to incubate for a
period of time under optimal conditions (e.g., temperature, pH,
residual water activity, ion concentration and presence of any
necessary cofactors) to allow the enzymes liberated in the biomass
to react directly with the LCPUFAs. Similarly, auto-oxidation
processes can be facilitated in this manner.
Modification of Oil Processing Conditions
[0239] Preferred oil processing methods include methods that are
focused on minimally processing the oil. Processes used in
conventional oilseed processing tend to remove free fatty acids or
free fatty acid-like compounds and thereby remove the fatty
acid-like hydroxy derivatives of LCPUFAs. In particular, caustic
treatments of the oils focused on removal of free fatty acids
(commonly referred to as refining the oil), should be avoided.
Preferably the oil is extracted with an alcohol (e.g. isopropyl
alcohol) or other organic solvent (e.g. hexane), or mixtures
thereof, or supercritical fluids (e.g. carbon dioxide) and the
resulting oil is chill filtered, bleached, chill filtered again and
then deodorized. In a more preferable method the chill filtration
steps are eliminated and the oil is simply bleached and deodorized
after extraction. In an even more preferable method, the only
processing step after extraction of the oil is limited to
deodorization of the oil. In the above extractions, alcohols or
alcohol water mixtures are preferable for use in extracting the oil
rather than using organic solvents such as hexane. As an
alternative to chemical extraction, oils may be separated from the
biomass through expeller pressing, or disruption followed by
centrifugation, using a separating processing aid such as a primary
alcohol or carrier oil. These crude oils may be purified and
stabilized through one or more of the methods described above.
Methods for Further Processing LCPUFA Oil (Microbial, Plant, Fish)
to Enhance and/or Stabilize LCPUFA Oxylipin Content
[0240] In one preferred method, once the oils have been extracted
and processed by the methods described above or by any other
suitable method, antioxidants can be added to the oil to help
stabilize the LCPUFA oxylipins (and in particular docosanoids) in
the oil. In another preferred method, antioxidants may be added at
one or more points in the extraction and purification process to
minimize potential oxidative degradation of oxylipins and/or
LCPUFAs. In addition, the oxylipins will become more polar
molecules as more hydroxy groups are incorporated into them, the
oil can be prepared in an emulsion form to enhance
content/solubility/stability of both polar and less polar forms of
the LCPUFA oxylipins (and in particular docosanoids) and facilitate
their use in, e.g., a wider variety of food and pharmaceutical
applications than those available to use of an oil ingredient form
alone.
[0241] In a preferable downstream process, an LCPUFA-rich oil
(microbial-, plant- or animal (including fish)-based) or hydrolyzed
or saponified form of the oil can be processed in an enzyme-based
reaction system (e.g. column or stirred tank reactor) to facilitate
the enzymatic production of the LCPUFA oxylipins (and in particular
docosanoids) in the oil. The enzymes can be present in either free
or immobilized forms in these systems. Exemplary enzymes (including
lipoxygenases, cyclooxygenases, cytochrome P450 enzymes and other
heme-containing enzymes) that can be utilized in these systems are
listed in Table 1. Reaction conditions, such as temperature, pH,
residual water activity, ion concentration and presence of
cofactors, can be chosen to maximize the rate and extent of
conversion of PUFAs to lipoxins. The oil can be processed through
the column/reactor either in the oil form or as hydrolyzed free
fatty acids, which are produced by hydrolyzing the PUFA-containing
triglycerides in the oil to convert the PUFAs from an esterified to
a free acid form.
[0242] In one embodiment of the invention, any of the oils produced
by any of the methods described herein can be further processed to
separate or purify the LCPUFA oxylipins from the LCPUFAs in the
oil. This process can be performed on oils that have been processed
by any refinement process, including oils or products thereof that
have been treated to convert LCPUFAs in the oil to oxylipin
derivatives. For example, LCPUFA oxylipins can be separated from
LCPUFAs by any suitable technique, such as any chromatography
technique, including, but not limited to, silica gel liquid
chromatography. In one embodiment, LCPUFA oxylipins produced,
enriched or purified by the processes of the present invention
(including any of the production/processing methods described
herein and/or de novo synthesis) can be added back to (titrated
into) another oil, such as an LCPUFA oil produced by any method,
and/or can be added to any composition or formulation or other
product.
[0243] After the oils/fatty acids have been processed in this
manner, the oil/fatty acids can be used directly in food,
pharmaceutical or cosmetic applications or can be used to add (by
blending) to LCPUFA or non-LCPUFA-containing oils to enhance their
content of LCPUFA oxylipins (and in particular docosanoids). In
this manner, a consistent LCPUFA oxylipin (and in particular
docosanoid) content of the final oil product can be achieved.
[0244] When using lipoxygenase enzymes in these types of systems,
up to 100% of the target LCPUFA can be transformed into their
hydroxy derivatives. An example of such a system would be an
immobilized enzyme column containing immobilized 15-lipoxygenase.
When DPAn-6 is processed thru this system, the DPAn-6 is
transformed to the hydroperoxides 17-hydroperoxyxy DPAn-6 and
10,17-di-hydroperoxy DPAn-6, which can then be transformed into the
hydroxy derivatives 17-hydroxy DPAn-6 and 10,17-di-hyroxy DPAn-6,
following reduction with an agent such as NaBH.sub.4. This
concentrated form of LCPUFA oxylipins (and in particular
docosanoids) can then be titrated into an appropriate edible oil to
achieve the desired LCPUFA oxylipin (and in particular docosanoid)
content in the final oil.
Applications of DPAn-6, DPAn-3, and/or DTAn-6 LCPUFA Oxylipins and
Oils or Compositions Comprising DPAn-6, DPAn-3, and/or DTAn-6
and/or any Other LCPUFA Oxylipins
[0245] The present invention is based on the use of LCPUFAs
comprising DPAn-6, DTAn-6, and/or DPAn-3 and/or the oxylipin
derivatives thereof, and/or various oils that have been enriched
for oxylipin derivatives of C20 and greater PUFAs, and particularly
for docosanoids, to provide anti-inflammatory, anti-proliferative,
neuroprotective and/or vasoregulatory effects in humans and other
animals. Such effects are useful for enhancing the general health
of an individual, as well as in treating or preventing a variety of
diseases and conditions in an individual. For example, the
invention includes methods for treating metabolic imbalances and
disease states that could benefit from the modulation of
inflammation provided by the LCPUFA and oxylipin, and particularly,
docosanoid, containing compositions and oils described herein.
[0246] Additional applications encompassed by the present invention
for the use of any of the LCPUFA and/or oxylipin-containing oils,
compositions or formulations described herein (preferably including
DPAn-6, DPAn-3 or oxylipin derivatives thereof, and as applicable
DTAn-6 or oxylipin derivatives thereof, as well as oils and
products produced with such oils that are enriched for oxylipin
derivatives), include, but are not limited to, the following: (1)
Rh.sup.+ incompatibility during pregnancy; (2) inflammatory
diseases of the bowel and gastrointestinal tract (e.g. Crohn's,
inflammatory bowel disease, colitis, and necrotizing enterocolitis
in infants); (3) autoimmune diseases (e.g. insulin-dependent
diabetes mellitus (Type I diabetes), multiple sclerosis, rheumatoid
arthritis, systemic lupus erythematosus, myasthenia gravis, celiac
disease, autoimmune thyroiditis, Addison's disease, Graves' disease
and rheumatic carditis); (4) chronic adult-onset diseases that
involve inflammation (e.g. cardiovascular disease, Type II
diabetes, age-related macular degeneration, atopic diseases,
metabolic syndrome, Alzheimer's disease, cystic fibrosis, colon
cancer, etc.); (5) inflammatory diseases of the skin (e.g.,
dermatitis (any form), eczema, psoriasis, rosacea, acne, pyoderma
gangrenosum, urticaria, etc.); (6) inflammatory diseases of the
eye; and (7) inflammation due to infectious diseases (bacteria,
fungal, viral, parasitic, etc.). Many of these are diseases in
which patients may not want to be on steroids or non-specific
anti-inflammatory drugs because of negative side effects.
[0247] Accordingly, one embodiment of the present invention relates
to the use of: (1) DPAn-6, DPAn-3 and/or an oxylipin derivative
(docosanoid) thereof and, in some embodiments, DTAn-6 and/or an
oxylipin derivative thereof, alone or in combination with each
other and/or with other LCPUFAs and/or oxylipin derivatives thereof
(preferably DHA or EPA, and most preferably, DHA); and/or (2) an
oil or product produced using such oil, wherein the oil has been
enriched in quantity, quality and/or stability of the LCPUFA
oxylipins contained therein, and preferably the docosanoids. The
use of these compositions is typically provided by an oil or
product using such oil, a nutritional supplement, cosmetic
formulation or pharmaceutical composition (medicament or medicine).
Such oils, supplements, compositions and formulations can be used
for the reduction of inflammation in a patient that has or is at
risk of developing inflammation or a disease or condition
associated with inflammation. Such oils, supplements, compositions
and formulations can also be used for the reduction of any symptoms
related to neurodegeneration or a disease associated with
neurodegeneration in a patient that has or is at risk of developing
a neurodegenerative condition or disease. In particular, the
patient to be treated using the composition of the invention has
inflammation associated with the production of eicosanoids and/or
what are generally termed in the art as "proinflammatory"
cytokines. Such cytokines include, but are not limited to,
interleukin-1.alpha. (IL-1.alpha.), IL-1.beta., tumor necrosis
factor-.alpha. (TNF.alpha.), IL-6, IL-8, IL-12, macrophage
inflammatory protein-1.alpha. (MIP-1.alpha.), macrophage
chemotactic protein-1 (MCP-1) and interferon-.gamma. (IFN-.gamma.).
The patient is administered a composition comprising an amount of
such LCPUFAs and/or oxylipin derivatives thereof in an amount
effective to reduce at least one symptom of inflammation or
neurodegeneration in the patient.
[0248] Symptoms of inflammation include both physiological and
biological symptoms including, but are not limited to, cytokine
production, eicosanoid production, histamine production, bradykinin
production, prostaglandin production, leukotriene production,
fever, edema or other swelling, pain (e.g., headaches, muscle
aches, cramps, joint aches), chills, fatigue/loss of energy, loss
of appetite, muscle or joint stiffness, redness of tissues, fluid
retention, and accumulation of cellular mediators (e.g.,
neutrophils, macrophages, lymphocytes, etc.) at the site of
inflammation. Diseases associated with inflammation include, but
are not limited to, conditions associated with infection by
infectious agents (e.g., bacteria, viruses), shock, ischemia,
cardiopulmonary diseases, autoimmune diseases, neurodegenerative
conditions, and allergic inflammatory conditions, and various other
diseases detailed previously herein. The Examples describe the use
of docosanoids of the present invention to reduce inflammation in
vivo and in vitro, as measured by multiple parameters of an
inflammatory response.
[0249] Symptoms associated with neurodegeneration include both
physiological and biological symptoms including, but not limited
to: neurodegeneration, intellectual decline, behavioral disorders,
sleep disorders, common medical complications, dementia, psychosis,
anxiety, depression, inflammation, pain, and dysphagia.
Neurodegenerative diseases that may be treated using the oxylipin
derivatives and compositions of the invention include, but are not
limited to: schizophrenia, bipolar disorder, dyslexia, dyspraxia,
attention deficit hyperactivity disorder (ADHD), epilepsy, autism,
Alzheimer's Disease, Parkinson's Disease, senile dementia,
peroxisomal proliferator activation disorder (PPAR), multiple
sclerosis, diabetes-induced neuropathy, macular degeneration,
retinopathy of prematurity, Huntington's Disease, amyotrophic
lateral sclerosis (ALS), retinitis pigmentosa, cerebral palsy,
muscular dystrophy, cancer, cystic fibrosis, neural tube defects,
depression, Zellweger syndrome, Lissencepahly, Down's Syndrome,
Muscle-Eye-Brain Disease, Walker-Warburg Syndrome,
Charoct-Marie-Tooth Disease, inclusion body myositis (IBM) and
Aniridia.
[0250] In one embodiment of the present invention, the novel
docosanoids of the invention, and/or oils or compositions
containing such docosanoids are used to selectively target the
particular proinflammatory cytokines and conditions or diseases
associated with the production of these cytokines. Based on the
observation by the present inventors that particular docosanoids of
the invention may selectively inhibit certain cytokines, the
inventors propose that such docosanoids can be used in particular
conditions or diseases to provide a more selective treatment of an
individual and avoid side effects that may be associated with more
global inhibition of inflammatory processes. For example, the
present inventors have shown that the DPAn-6 docosanoids,
17-hydroxy DPAn-6 and 10,17-dihydroxy DPAn-6, significantly reduced
secretion of the potent pro-inflammatory cytokine IL-1.beta., with
the reduction produced by 10,17-dihydroxy DPAn-6 being
significantly larger than with that produced by either the DHA
oxylipin derivative or the general anti-inflammatory agent,
indomethacin. Even more striking were the observed differences
between the activities of two different oxylipin derivatives of
DPAn-6. As shown in Examples 20 and 21, while both 17-HDPAn-6 and
10,17-dihydroxy DPAn-6 are demonstrated to be potent
anti-inflammatory agents, there were differences between the
activity of these two DPAn-6 oxylipins in their effect on cytokine
production (e.g., IL-1.beta.), indicating that one compound may be
more suitable than the other for specific applications (e.g.,
sepsis versus swelling). Specifically, 17-HDPAn-6 was more potent
than the DHA-derived oxylipin for inhibiting cell migration, and
10,17-dihydroxy DPAn-6 was more potent than the DHA oxylipin for
reduction in IL-1.beta. secretion. Therefore, one of skill in the
art can select docosanoids of the present invention for specific
uses, and reduce the potential side effects of a treatment as
compared to using more pan-specific or generic anti-inflammatory
agents.
[0251] The compositions and method of the present invention
preferably protect the patient from inflammation, or a condition or
disease associated with inflammation. As used herein, the phrase
"protected from a disease" (or symptom or condition) refers to
reducing the symptoms of the disease; reducing the occurrence of
the disease, and/or reducing the severity of the disease.
Protecting a patient can refer to the ability of a nutritional or
therapeutic composition of the present invention, when administered
to the patient, to prevent inflammation from occurring and/or to
cure or to alleviate inflammation and/or disease/condition
symptoms, signs or causes. As such, to protect a patient from a
disease or condition includes both preventing occurrence of the
disease or condition (prophylactic treatment) and treating a
patient that has a disease or condition or that is experiencing
initial symptoms of a disease or condition (therapeutic treatment).
The term, "disease" or "condition" refers to any deviation from the
normal health of an animal and includes a state when disease
symptoms are present, as well as conditions in which a deviation
(e.g., infection, gene mutation, genetic defect, etc.) has
occurred, but symptoms are not yet manifested.
[0252] According to the present invention, the oxylipins (or
analogs or derivatives thereof), compositions comprising such
oxylipins, and methods of the invention, are suitable for use in
any individual (subject) that is a member of the Vertebrate class,
Mammalia, including, without limitation, primates, livestock and
domestic pets (e.g., a companion animal). Most typically, an
individual will be a human. According to the present invention, the
terms "patient", "individual" and "subject" can be used
interchangeably, and do not necessarily refer to an animal or
person who is ill or sick (i.e., the terms can reference a healthy
individual or an individual who is not experiencing any symptoms of
a disease or condition). In one embodiment, an individual to which
an oxylipin(s) or composition or formulation or oil of the present
invention can be administered includes an individual who is at risk
of, diagnosed with, or suspected of having inflammation or
neurodegeneration or a condition or disease related thereto.
Individuals can also be healthy individuals, wherein oxylipins or
compositions of the invention are used to enhance, maintain or
stabilize the health of the individual.
[0253] The amount of an LCPUFA or oxylipin derivative thereof to be
administered to a individual can be any amount suitable to provide
the desired result of reducing at least one symptom of inflammation
or neurodegeneration or protecting the individual from a condition
or disease associated with such inflammation or neurodegeneration.
In one embodiment, an LCPUFA such as DPAn-6 is administered in a
dosage of from about 0.5 mg of the PUFA per kg body weight of the
individual to about 200 mg of the PUFA per kg body weight of the
individual, although dosages are not limited to these amounts. An
LCPUFA oxylipin derivative or mixture of oxylipin derivatives is
administered in a dosage of from about 0.2 ug of the oxylipin per
kg body weight of the individual to about 50 mg of the oxylipin per
kg body weight of the individual, although dosages are not limited
to these amounts.
[0254] Although compositions and formulations of the invention can
be administered topically or as an injectable, the most preferred
route of administration is oral administration. Preferably, the
compositions and formulations used herein are administered to
subjects in the form of nutritional supplements and/or foods
(including food products) and/or pharmaceutical formulations and/or
beverages, more preferably foods, beverages, and/or nutritional
supplements, more preferably, foods and beverages, more preferably
foods.
[0255] As discussed above, a variety of additional agents can be
included in the compositions when administered or provided to the
subject, such as other anti-inflammatory agents, vitamins,
minerals, carriers, excipients, and other therapeutic agents. A
preferred additional agent is aspirin, or another suitable
anti-inflammatory agent.
[0256] The oxylipins (or analogs or derivatives or salts thereof),
compositions comprising such oxylipins, and methods of the
invention, are also suitable for use as feed ingredients,
nutritional supplements or therapeutic agents in aquaculture
applications in any individual (subject) that is a member of the
Vertebrate class such as fish or for invertebrates such as
shrimp.
[0257] The following experimental results are provided for purposes
of illustration and are not intended to limit the scope of the
invention.
EXAMPLES
Example 1
[0258] The following example demonstrates that DPAn-6 can be
completely converted to a mono-hydroxy diene derivative by
15-lipoxygenase, and is more efficiently converted than either of
DPAn-3 or DHA.
[0259] Soybean 15-lipoxygenase (Sigma-Aldrich, St. Louis, Mo.) at a
final concentration of 4 .mu.g/ml was mixed into 100 .mu.M
solutions of DHA, DPAn-6, or DPAn-3 (NuChek Prep, Elysian, Minn.)
in 0.05M sodium borate buffer, pH 9.0, and the reaction mixtures
were incubated at 0.degree. C. Appearance of the mono-hydroxy
conjugated diene derivatives of the fatty acids was monitored
through absorbance at 238 nm. Conjugated diene products were
quantified using an extinction coefficient of 28,000 M.sup.-1
cm.sup.-1 (Shimizu et al; Methods in Enzymology, 1990 Vol 187,
296-306). As shown in Example. 1, 100% of the DPAn-6 was
efficiently converted to its conjugated diene derivative under
these conditions, whereas about 85% of DPAn-3 and 50% of DHA were
converted to their respective conjugated diene (mono-hydroxy)
derivative by the 15-lipoxygenase. No appreciable accumulation of
the dihydroxy derivatives occurred under these reaction
conditions.
Example 2
[0260] The following example describes the major 15-lipoxygenase
products of DHA.
[0261] DHA (100 .mu.M, NuChek Prep, Elysian, Minn.) was incubated
with 15-LOX (4 .mu.g/ml) in 0.05M sodium borate buffer, pH 9.0, at
4.degree. C. with vigorous stiffing for 30 mM Reaction products
were reduced with NaBH.sub.4 (0.45 mg/ml) and then extracted on a
solid phase C-18 cartridge (Supelco Discovery DSC-19) using
anhydrous ethanol for elution. Reaction products were analyzed by
LC/MS/MS using an Agilent 1100 Series High Performance Liquid
Chromatography (HPLC) Instrument (San Paulo, Calif. USA) interfaced
with an Esquire 3000 ion trap mass spectrometer equipped with
electrospray ionization source (Bruker Daltonics, Billerica Mass.
USA). The HPLC was carried out on a LUNA C18(2) column
(250.times.4.6 mm, 5 micron, Phenomenex, Torrance Calif., USA)
using a mobile phase consisting of 100 mM ammonium acetate in 30%
methanol in water with an acetonitrile gradient increasing from 48
to 90% over 50 mM (0.4 ml/min flow rate). The mass spectrometer was
operated in the negative ion detection mode. Nitrogen was used as
nebulizing and drying gas with nebulizer pressure at 20 psi and
drying gas flow rate of 7 L/min. The interface temperature was
maintained at 330 C.
[0262] FIG. 2A depicts the structures of the mono- and dihydroxy
products of this DHA reaction. FIG. 2B depicts MS/MS spectrum of
the mono-hydroxy product showing the molecular ion (m/z of 343) and
the characteristic fragments of 17-hydroxy DHA. Inset shows the UV
spectrum of this compound with the expected peak at 237 nm,
characteristic of a conjugated diene. FIGS. 2C and 2D depict MS/MS
spectra of the two dihydroxy products with molecular ions (m/z of
359) and characteristic fragments of 10,17-hydroxy DHA (2C) and
7,17-dihydroxy DHA (2D) indicated. The UV spectrum insets show the
expected triplet peaks at 270 nm characteristic of a conjugated
triene for 10,17-dihydroxy DHA and a single peak at 242
characteristic of two pairs of conjugated dienes separated by a
methylene group for 7,17-dihydroxy DHA.
Example 3
[0263] The following example indicates the major 15-lipoxygenase
products of DPAn-6 and demonstrates production of mono- and
dihydroxy derivatives analogous to those produced from DHA (see
Example 2).
[0264] DPAn-6 was treated with 15-lipoxygenase and analyzed by
LC/MS/MS under the conditions described in Example 2. FIG. 3A
depicts the structures of the mono- and dihydroxy reaction products
of this DPAn-6/15-LOX reaction. FIG. 3B depicts MS/MS spectrum of
the mono-hydroxy product showing molecular ion (m/z of 345) and
fragments characteristic of 17-hydroxy DPAn-6. The inset shows the
UV spectrum of this compound with the expected peak at 237 nm
characteristic of a conjugated diene. FIGS. 3C and 3D depict MS/MS
spectra of the two dihydroxy products with molecular ions (m/z of
361) and fragments characteristic of 10,17-hydroxy DPAn-6 (3C) and
7,17-dihydroxy DPAn-6 (3D) indicated. The UV spectrum insets show
the expected triplet peaks at 270 nm characteristic of a conjugated
triene for 10,17-dihydroxy DPAn-6 and a single peak at 242
characteristic of two pairs of conjugated dienes separated by a
methylene group for 7,17-dihydroxy DPAn-6.
Example 4
[0265] The following example indicates the major 15-lipoxygenase
products of DPAn-3 and demonstrates production of mono- and
dihydroxy derivatives analogous to those produced from DHA (Example
2) and DPAn-6 (Example 3).
[0266] DPAn-3 was treated with 15-lipoxygenase and analyzed by
LC/MS/MS under conditions described in Example 2. FIG. 4A depicts
the structures of the mono- and dihydroxy reaction products of this
DPAn-3/15-LOX reaction. FIG. 4B depicts LC/MS spectrum of the
monohydroxy product showing molecular ion (m/z of 345) and
fragments characteristic of 17-hydroxy DPAn-3. Inset shows UV
spectrum of this compound with the expected peak at 237 nm,
characteristic of a conjugated diene. FIGS. 4C and 4D depict MS/MS
spectra of the two dihydroxy products with molecular ions (m/z of
361) with fragments characteristic of 10,17-hydroxy DPAn-3 (4C) and
7,17-dihydroxy DPAn-3 (4D) indicated. The UV spectrum insets show
the expected triplet peaks at 270 nm characteristic of a conjugated
triene for 10,17-dihydroxy DPAn-3 and a single peak at 242
characteristic of two pairs of conjugated dienes separated by a
methylene group for 7,17-dihydroxy DPAn-3.
Example 5
[0267] The following example indicates the major 15-lipoxygenase
products of DTAn-6 and demonstrates production of a mono-hydroxy
and a dihydroxy derivative analogous to those formed from DHA
(Example 2), DPAn-6 (Example 3) and DPAn-3 (Example 4).
[0268] DTAn-6 was mixed with 15-lipoxygenase and analyzed by
LC/MS/MS under conditions described in Example 2. FIG. 5A depicts
the structure of the mono-hydroxy reaction product. FIG. 5B depicts
an LC/MS spectrum of the mono-hydroxy product showing molecular ion
(m/z of 347) and fragments characteristic of 17-hydroxy DTAn-6.
Inset shows UV spectrum indicating the expected peak at 237 nm,
characteristic of a conjugated diene. FIG. 5C depicts an LC/MS
spectra of the dihydroxy product with molecular ion (m/z of 361)
and fragments characteristic of 7,17-hydroxy DTAn-6 indicated. The
UV spectrum inset shows the expected peak at 242, characteristic of
two pairs of conjugated dienes separated by a methylene group.
Example 6
[0269] The following example shows the structure of the enzymatic
oxylipin products produced from DPAn-6 after sequential treatment
with 15-lipoxygenase followed by hemoglobin.
[0270] DPAn-6 (at a concentration of 100 .mu.M) was mixed with
soybean 15-lipoxygenase (20 .mu.g/ml final concentration) with
vigorous stiffing at 4.degree. C. Products were immediately
extracted on Supelco Discovery DSC-19 cartridges using anhydrous
ethanol for final elution. The hydroperoxy derivatives thus
obtained were concentrated to 1.5 mM and were used for subsequent
hemoglobin catalyzed reactions. The hydroperoxy derivatives (final
reaction concentration, 30 .mu.g/ml) were mixed with human
hemoglobin (300 .mu.g/ml, Sigma-Aldrich) in Dulbecco's phosphate
buffered saline at 37.degree. C. for 15 minutes. The reaction was
acidified to pH 3 with glacial acetic acid and products purified by
solid phase extraction. Reaction products were analyzed by
LC-MS/MS. FIG. 6 illustrates the docosanoid products of the
enzymatic reaction as deduced from the mass spectra (not
shown).
Example 7
[0271] The following example indicates of the major 5-lipoxygenase
products of DHA.
[0272] To a 10-ml reaction mixture containing 100 .mu.M DHA (NuChek
Prep, Elysian, Minn.) in 0.05M NaMES buffer, pH 6.3, 100 .mu.M SDS
and 0.02% C.sub.12E.sub.10, was added 200 .mu.l of 10 U/.mu.l
potato 5-lipoxygenase (Caymen Chemicals, Minneapolis, Minn.). The
reaction proceeded for 30 min at 4.degree. C., and reaction
products were reduced by addition of 1 ml of 0.5 mg/ml NaBH.sub.4.
Reaction products were extracted using solid phase C-18 cartridges
and analyzed by LC/MS/MS as described in Example 2. Major reaction
products as determined by tandem mass spectroscopy along with the
diagnostic molecular ion and fragments are shown (FIG. 7).
Example 8
[0273] The following example indicates the major 5-lipoxygenase
product of DPAn-6 and indicates production of a mono-hydroxy
derivative analogous to the 5-LOX products of DHA (Example 7).
[0274] DPAn-6 (100 .mu.M) was treated with 5-lipoxygenase as
described in Example 7. Reaction products were analyzed by LC/MS/MS
as in Example 2. Major reaction products as determined by tandem
mass spectroscopy along with the diagnostic molecular ion and
fragments are shown (FIG. 8).
Example 9
[0275] The following example indicates the major 5-lipoxygenase
products of DPAn-3 and indicates production of mono- and dihydroxy
derivatives analogous to the 5-LOX products of DHA (Example 7).
[0276] DPAn-3 (100 .mu.M) was treated with 5-lipoxygenase as
described in Example 7. Reaction products were analyzed by LC/MS/MS
as in Example 2. Major reaction products as determined by tandem
mass spectroscopy along with the diagnostic molecular ion and
fragments are shown (FIG. 9).
Example 10
[0277] The following example indicates the major 12-lipoxygenase
products of DHA.
[0278] For the enzyme reaction, 100 .mu.l of 0.75 U/.mu.l porcine
leukocyte-derived 12-lipoxygenase (Caymen Chemicals, Minneapolis,
Minn.) was added to a 10-ml solution containing 100 .mu.M DHA
(NuChek Prep, Elysian, Minn.) in 0.1M Tris-HCl, pH 7.5, 5 mM EDTA
and 0.03% Tween-20. The reaction continued for 30 mM at 4.degree.
C. and reaction products were reduced by adding 1 ml of 0.5 mg/ml
NaBH.sub.4. Reaction products were extracted using solid phase C-18
cartridges and analyzed by LC/MS/MS as described in Example 2.
Major reaction products as determined by tandem mass spectroscopy,
along with the diagnostic molecular ion and fragments, are shown
(FIG. 10).
Example 11
[0279] The following example indicates the major 12-lipoxygenase
products of DPAn-6 and indicates production of mono- and dihydroxy
derivatives analogous to those from the DHA/12-LOX reaction
(Example 10).
[0280] DPAn-6 (100 .mu.M) was treated with 12-lipoxygenase as
described in Example 10. Reaction products were analyzed by
LC/MS/MS as in Example 2. Major reaction products as determined by
tandem mass spectroscopy, along with the diagnostic molecular ion
and fragments, are shown (FIG. 11).
Example 12
[0281] The following example indicates the major 12-lipoxygenase
products of DPAn-3 and indicates production of mono- and dihydroxy
derivatives analogous to those produced from the DHA/12-LOX
reaction (Example 10) and the DPAn-6/12-LOX reaction (Example
11).
[0282] DPAn-3 (100 .mu.M) was treated with 12-lipoxygenase as
described in Example 10. Reaction products were analyzed by
LC/MS/MS as in Example 2. Major reaction products as determined by
tandem mass spectroscopy along with the diagnostic molecular ion
and fragments are shown (FIG. 12).
Example 13
[0283] The following example describes a mass spectral analysis of
oxylipins in algal DHA/DPAn-6 LCPUFA oil.
[0284] Algal-derived DHA+DPAn-6 oil (0.5 g) diluted in 1.5 ml
hexane was run through a 15 mm.times.200 mm silica gel column,
using increasing concentrations of ethyl acetate in hexane to elute
the various lipid classes. Fractions containing lipids were
identified by thin layer chromatography (TLC) using petroleum
ether: ethyl ether: acetic acid (80:20:1) as the mobile phase and
then further screened for mono- and dihydroxy docosanoids (m/z of
343, 345, 359, or 361) using LC/MS on a Hewlett Packard model 1100
liquid chromatograph equipped with electro spray ionization (ESI)
and a Hewlett Packard model 1100 mass selective detector (MSD).
Fractions containing hydroxyl docosanoid products were pooled,
concentrated, and further analyzed by tandem mass spectroscopy
(MS/MS) on a Applied Biosystems API QSTAR.RTM. Pulsar i Hybrid
triple quadrapole-time of flight hybrid LC/MS/MS (Colorado
University mass spectroscopy facility). The sample was introduced
using direct infusion into an ESI source utilizing negative
ionization.
[0285] FIG. 18A depicts an MS total ion chromatograph (TIC) of the
docosanoid fraction, indicating the presence of mono-hydroxy DPA
(HDPA) and dihydroxy DPA (di-HDPA) ([M-H] of 345 and 361 m/z,
respectively) and mono-hydroxy DHA (HDHA, [M-H] of 343 m/z) along
with fragments corresponding to [M-H]--H.sub.2O, [M-H]-CO.sub.2 and
[M-H]--H.sub.2O/CO.sub.2 that are characteristic fragments of these
compounds.
[0286] FIG. 18B depicts an MS/MS spectra of mono-hydroxy DPAn-6
([M-H] 345 m/z) showing characteristic [M-H]--H.sub.2O,
[M-H]--CO.sub.2 and [M-H]--H.sub.2O/CO.sub.2 fragments along with
m/z 245 and 201 fragments indicating the presence of 17-HDPAn-6 in
the oil.
[0287] FIG. 18C depicts an MS/MS of dihydroxy-DPAn-6 with
characteristic fragments corresponding to [M-H]--H2O (m/z 343),
[M-H]--CO.sub.2 (m/z 317) and [M-H]--H.sub.2O/CO.sub.2 (m/z 299),
[M-H]-2 H.sub.2O/CO.sub.2 (m/z 281) and fragments indicating the
presence of 10,17-dihydroxyDPAn-6 (m/z 261-H.sub.2O/CO.sub.2;
153).
Example 14
[0288] The following example shows the results of a rat paw edema
study in which various combinations of LCPUFAs were fed to the
animals
[0289] Adult, male, Sprague Dawley rats (n=10/treatment group) were
fed modified AlN-76 diets formulated to include 1.2% DHA, 1.2%
DHA+0.44% DPAn-6, or 1.2% DHA+0.46% arachidonic acid (ARA) for 4
weeks. Carrageenan (1%) was used to induce hind paw edema on Day 14
(left paw) and Day 28 (right paw) of feeding. Edema was measured
plethysmographically using water displacement 3 hours
post-injection. Day 28 means (.+-.stdev) are shown in FIG. 19.
Similar results were obtained on day 14. *p.ltoreq.0.05.
[0290] FIG. 19 shows that the oil containing a combination of DHA
and DPAn-6 produced a statistically significantly better reduction
in edema volume than DHA alone or DHA and ARA. The omega-6 fatty
acid ARA reversed the anti-inflammatory activity of DHA in this
model.
Example 15
[0291] The following example demonstrates the potent
anti-inflammatory effect of the DPAn-6-derived oxylipins 17-hydroxy
DPAn-6 and 10,17-dihydroxy-DPAn-6 in a mouse dorsal air pouch
model.
[0292] Pure 17R-hydroxy DHA (17R-HDHA) was purchased from Caymen
Chemicals (Ann Arbor, Mich.). Docosanoids 17-hydroxy-DPAn-6
(17-HDPAn-6) and 10,17-dihydroxyDPAn-6 (10,17-HDPAn-6) were
synthesized biogenically from DPAn-6 (NuChek Prep, Elysian, Minn.)
using soybean 15-lipoxygenase (Sigma-Aldrich) and purified by HPLC
as described in Example 2. Organic solvents were evaporated and the
compounds were re-dissolved in phosphate buffered saline (PBS),
filter sterilized and concentrations were adjusted to 1000 ng/ml
using molar extinction coefficients of 28,000 and 40,000 M.sup.-1
cm.sup.-1 for the mono- and dihydroxy docosanoids, respectively.
Female C57/B16 mice (n=10 mice per group) were injected with
sterile air subcutaneously in the back to initiate dorsal air
pouches. Six days later, 0.9 ml sterile PBS followed by 100 ng
docosanoid in 0.1 ml PBS or PBS alone were administered by
intra-pouch injection. This injection was followed within 5 min by
intra-pouch injection of 100 ng of mouse recombinant TNF.alpha.
(Peprotech, Inc, NJ, USA) in 0.1 ml PBS. Control animals received
no TNF.alpha.. As a positive control, 2 mg/kg indomethacin
(Calbiochem, San Diego, Calif.) was administered intraperitoneally
30 min prior to administration of TNF.alpha.. Four hours after
TNF.alpha. administration, air pouch exudates were removed and
cells were stained with Turk's solution and counted. Exudates were
frozen for later cytokine analyses using commercial ELISA kits.
Bars represent group (n=10) means (.+-.stdev). Groups were compared
using Student's t test, with p values indicated.
[0293] FIG. 20A shows the total cell migration into air pouch
exudates. 17-hydroxy DPAn-6 and 10,17-dihydroxy DPAn-6 resulted in
significant reductions in the total number of cells in the pouch,
due to reductions in both the number of neutrophils and macrophages
(not shown). 17-hydroxy DPAn-6 was more potent than both
17R-hydroxy DHA and indomethacin in reducing cell infiltration.
[0294] FIG. 20B shows the IL-1.beta. concentrations in air pouch
exudates. Both 17-hydroxy DPAn-6 and 10,17-dihydroxy DPAn-6
resulted in significant reductions in the secretion of the potent
pro-inflammatory cytokine IL-1.beta., with the reduction produced
by 10,17-dihydroxy DPAn-6 significantly larger than with that
produced by either the DHA oxylipin derivative or indomethacin.
[0295] FIG. 20C shows the macrophage chemotactic protein-1 (MCP-1)
concentrations in air pouch exudates. Both 17-hydroxy DPAn-6 and
10,17-dihydroxy DPAn-6 resulted in significant reductions in the
secretion of this chemoattractant cytokine, and both compounds
resulted in a larger inhibition of MCP-1 secretion than
indomethacin.
[0296] FIGS. 20A-C indicate that the two DPAn-6 oxylipin
derivatives 17-hydroxy DPAn-6 and 10,17-dihydroxy DPAn-6 are potent
anti-inflammatory agents, resulting in reduced immune cell
migration in this inflammation model. A reduction in key
pro-inflammatory cytokines may contribute to this anti-inflammatory
activity. Notably, there are differences between the activity of
these two DPAn-6 oxylipins in their effect on cytokine production
(e.g., IL-1.beta.), suggesting that one compound may be more
suitable than the other for specific applications (e.g., sepsis vs
swelling). 17-hydroxy DPAn-6 is more potent than the DHA-derived
oxylipin for inhibiting cell migration and 10,17-dihydroxy DPAn-6
is more potent than the DHA oxylipin for reduction in IL-1.beta.
secretion.
Example 16
[0297] The following example shows the anti-inflammatory effect of
DHA and DPAn-6-derived docosanoids in cell culture.
[0298] Effect of Docosanoids on TNF.alpha.-induced IL-1.beta.
Production by Glial Cell: Human glial cells (DBGTRG-05MG, ATCC,
Manassas, Va.) were cultured for 24 hrs in 96-well culture dishes
(10.sup.5 cells per well) in 0.2 ml RPMI-medium containing
supplements and serum (as specified by ATCC) after which the medium
was replaced with fresh medium containing docosanoids or vehicle
(PBS) followed within 5 minutes by addition of human recombinant
TNF.alpha. (Sigma-Aldrich, St. Louis, Mo.) at a final concentration
of 100 ng/ml. Cells were incubated for 17 hrs before supernatants
were removed and cells were lysed with 0.2% Triton-X100 in PBS.
Cell lysates were assayed for IL-1.beta. using a commercial ELISA
kit (R&D Systems, Minneapolis, Minn.) (FIG. 21). Bars represent
means (n=3).+-.stdev. *p=0.06 compared to control using t-sided
Student's t test. 17-HDHA: 17R-hydroxy DHA; 17HDPAn-6: 17-hydroxy
DPAn-6; 10,17-diHDPAn-6: 10,17-dihydroxy DPAn-6.
Example 17
[0299] The following example further illustrates the
anti-inflammatory effect of 10,17-dihydroxy DPAn-6 on human
lymphocytes in culture and demonstrates that the dihydroxy DPAn-6
compound is more potent than the DHA analog (10,17,dihydroxy DHA)
in reducing TNF.alpha. secretion by T lymphocytes stimulated with
anti-CD3/anti-CD28 antibodies.
[0300] FIG. 22A: Effect of Docosanoids on TNF.alpha. Secretion by
Human T Lymphocytes. The assay was performed essentially as
described in Ariel et al, 2005. Briefly, human peripheral blood
mononuclear cells were isolated from venous blood by
Ficoll-Paque.TM. Plus (Amersham biosciences) gradient. T
lymphocytes were isolated using a human T cell enrichment column
(R&D Systems) per manufacturer's instructions. Purified T cells
were treated with 10,17-dihydroxy DPAn-6 or 10,17-dihydroxy DHA or
vehicle (0.05% ethanol) in RPMI-1640 media containing 10% heat
inactivated fetal bovine serum for 6 hrs at 37.degree. C.
Lymphocytes (200,000 cells in 200 .mu.l media per well) were then
transferred to 96-well plates coated with both anti-CD3 antibody
and anti-CD28 antibody (100 .mu.l of 2 .mu.g/ml of each antibody
overnight to coat wells) and incubated for 41 hrs. TNF.alpha.
concentrations were determined in cell supernatants by ELISA
(R&D Systems). Group means.+-.stdev (n=4) were compared by
Student's t test. *p<0.05 and **p<0.01 compared to control; #
indicates statistical difference (p=0.037) between the groups
treated with 10,17-dihydroxy DPAn-6 and 10,17-dihydroxy DHA at the
10 nM concentrations.
[0301] FIG. 22B shows the TNF.alpha. concentration in supernatants
from lymphocytes not treated with docosanoids that were cultured in
uncoated wells or in wells coated with anti-CD3 antibody only, with
anti-CD28 antibody only or with a combination of the two
antibodies.
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[0334] Each reference described or cited herein is incorporated
herein by reference in its entirety.
[0335] While various embodiments of the present invention have been
described in detail, it is apparent that modifications and
adaptations of those embodiments will occur to those skilled in the
art. It is to be expressly understood, however, that such
modifications and adaptations are within the scope of the present
invention, as set forth in the following claims.
* * * * *