WO2016041013A1 - Glycerolipids and uses therefor - Google Patents

Abstract

Disclosed are compositions and methods for treatment of atherosclerotic plaque. More particularly, the present invention relates to the use of plasmalogen level- enhancing compounds, including glycerolipids such as plasmalogens and acylglycerols, in compositions and methods for reducing the incidence of atherosclerosis, including the formation and progression of atherosclerosis, for preventing or delaying the onset of atherosclerosis and/or for treating atherosclerosis.

Description

TITLE OF THE INVENTION

"GLYCEROLIPIDS AND USES THEREFOR"

FIELD OF THE INVENTION

[0001] This application claims priority to Australian Provisional Application No. 2014903691 entitled "Glycerolipids and uses therefor", filed on 16 September 2014, the entire subject matter of which is hereby incorporated by reference herein.

[0002] This invention relates generally to the use of glycerolipids for treatment of atherosclerotic plaque. More particularly, the present invention relates to the use of plasmalogen level-enhancing compounds, including plasmalogens and acylglycerols, in compositions and methods for reducing the incidence of atherosclerosis, including the formation and progression of atherosclerosis, for preventing or delaying the onset of atherosclerosis and/or for treating atherosclerosis.

BACKGROUND OF THE INVENTION

[0003] Oxidative stress plays a contributing factor to the progression of atherosclerosis. GPx-1 is an anti-oxidant enzyme which is expressed ubiquitously in cells; it reduces and detoxifies hydrogen peroxide and lipid hydroperoxide [1]. Deficiency of GPx-1 in apoE-deficient mice (ApoE^/GPxl^"7") was demonstrated to result in a significant increase in atherosclerosis after 24 weeks of high-fat (21% fat, 0.15% cholesterol) feeding as compared to mice which were deficient in ApoE only (ΑροΕ^" ). This increase in atherosclerotic plaques was accompanied by elevation in the superoxide formation and protein nitration in the aorta [2] as well as increase in the expression of proinflammatory markers, vascular cellular adhesion molecular-1 (VCAM-1) and receptors for advanced glycation products (RAGE) [3].

[0004] Plasmalogens (alkenylphosphatidylcholine and

alkenylphosphatidylethanolamine) are subclasses of glycerophospholipids that are characterized by a cis vinyl ether bond linking an alkyl chain to the sn-1 position of the glycerol backbone. Plasmalogens are synthesized from the corresponding

alkylphospholipids (alkylphosphatidylcholine and alkylphosphatidylethanolamine) by the action of a desaturase. Plasmalogens have been proposed as a potential anti-oxidant because of three main characteristics: (1) an enhanced electron density and low bond dissociation of the vinyl ether linkage which makes them more susceptible to reactive oxygen species (ROS) attack than allylic and alkyl linkages [4]; (2) the position of plasmalogens in the hydrophilic domain of cell membranes makes them accessible to ROS attack [5]; and (3) the proposed slow propagation of the plasmalogen hemiacetal hydroperoxy radicals {i.e. , plasmalogen oxidative intermediate)[4]. Normalization of cellular plasmalogen levels by supplementation of alkylglycerol (precursor of plasmalogen synthesis) were shown to aid cellular cytotoxicity resistance of mutant Chinese Hamster Ovary cells and macrophage like cells, RAW.12 and RAW.108 against insults including long-wavelength UV light and ROS generators, respectively [6, 7].

SUMMARY OF THE INVENTION

[0005] The present invention is predicated in part on the determination that elevating plasmalogen levels attenuates formation of atherosclerosis in ApoE^" and

ApoE^VGPxl^"7" mice with a greater effect in the latter model. Plasmalogen elevation was also found to reduce weight gain, lower cholesterol and LDL-C levels, decrease

inflammation and reduce oxidative stress. Based on these determinations, the present inventors propose that increasing the level of plasmalogen is useful for reducing the incidence of atherosclerotic plaque and risk of cardiovascular disease and for treating atherosclerosis, as described hereafter.

[0006] Accordingly, in one aspect, the present invention provides methods for reducing the incidence of atherosclerotic plaque (e.g., in a blood vessel) in a subject. These methods generally comprise, consist or consist essentially of increasing the level of plasmalogen in the subject to thereby reduce the incidence of atherosclerotic plaque in the subject.

[0007] In another aspect, the present invention provides methods for inhibiting formation of atherosclerotic plaque (e.g., in a blood vessel) in a subject. These methods generally comprise, consist or consist essentially of increasing the level of plasmalogen in the subject to thereby inhibit formation of atherosclerotic plaque in the subject.

[0008] In yet another aspect, the present invention provides methods for regressing atherosclerotic plaque (e.g., in a blood vessel) in a subject. These methods generally comprise, consist or consist essentially of increasing the level of plasmalogen in the subject to thereby regress atherosclerotic plaque in the subject.

[0009] In still another aspect, the present invention provides methods for treating atherosclerosis in a subject. These methods generally comprise, consist or consist essentially of increasing the level of plasmalogen in the subject to thereby treat atherosclerosis in the subject.

[0010] In some embodiments, the methods comprise administering to the subject at least one (e.g. , 1, 2, 3, or more) plasmalogen level-enhancing compound selected from the group consisting of plasmalogen precursor lipids and heterologous plasmalogens, in an effective amount to increase the level of plasmalogen in the subject, and to thereby reduce the incidence and/or inhibit formation and/or to result in regression of atherosclerotic plaque and/or treat atherosclerosis in the subject. Suitably, the plasmalogen-precursor lipids are selected from alkylglycerols. [0011] In some embodiments, the methods further comprise identifying the subject as having atherosclerosis or being at risk of developing atherosclerosis prior to increasing the level of plasmalogen in the subject.

[0012] Suitably, the or each plasmalogen level-enhancing compound is derived from a natural source. Suitably, the or each plasmalogen level-enhancing compound is provided in the form of a dietary supplement, food additive or nutraceutical.

[0013] In some embodiments, the methods further comprising administering concurrently to the subject at least one ancillary agent for treating and/or preventing atherosclerosis.

[0014] In a related aspect, the present invention encompasses the use of at least one plasmalogen level-enhancing compound as broadly described above and elsewhere herein for reducing the incidence of atherosclerotic plaque, for regressing atherosclerotic plaque, for inhibiting formation of atherosclerotic plaque and/or for treating atherosclerosis. In some embodiments, the or each plasmalogen level-enhancing compound is prepared or manufactured as a medicament for any one or more of those applications.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a graphical representation showing weekly weight

measurements of mice on high fat diet supplemented with/without batyl alcohol across 12 weeks. Data represent weight ± SEM of C57/BL6 (circles), ApoE^" (triangles), and

ApoE^" /GPxl^v" (squares) mice on high fat diet supplemented with either 0% batyl alcohol (open symbols) or 2% batyl alcohol (closed symbols). Final weight on week 12 were compared between groups of each genotype using Student t-test; ** indicates P< 0.01 and *** indicates P<0.001.

[0016] Figure 2 is a graphical representation showing plasma concentration of alkenyl- and alkylphospholipids in mice following 12 weeks of high fat diet supplemented with/without batyl alcohol. Total plasma concentration of (A) alkenylphosphatidylcholine, (B) alkenylphosphatidylethanolamine, (C) alkylphosphatidylcholine, and (D)

alkylphosphatidylethanolamine in C57/BL6, ApoE^" , and ApoE^" /GPxl^v" mice on high fat diet supplemented with either 0% batyl alcohol (open bars) or 2% batyl alcohol (closed bars). Bars indicate median, whiskers indicate interquartiles. The two dietary groups of each genotype were analyzed using Student t-test; *** indicates P<0.001.

[0017] Figure 3 is a graphical representation showing the concentration of alkenyl- and alkylphospholipids in heart homogenates of mice following 12 weeks of high fat diet supplemented with/without batyl alcohol. Total plasma concentration of (A) alkenylphosphatidylcholine, (B) alkenylphosphatidylethanolamine, (C)

alkylphosphatidylcholine, and (D) alkylphosphatidylethanolamine in C57/BL6, ApoE^" , and ApoE^" /GPxl^v" mice on high fat diet supplemented with either 0% batyl alcohol (open bars) or 2% batyl alcohol (closed bars). Bars indicate median, whiskers indicate interquartile range. The two dietary groups of each genotype were analyzed using Student t-test; ** indicates P< 0.01, *** indicates P<0.001.

[0018] Figure 4 is photomicrographic representation showing aorta from mice fed a high fat diet supplemented with/without batyl alcohol via en face technique.

Photographs of aorta of C57/BL6, ApoE^" , and ApoE^" /GPxl^v" mice were compared within the genotype between the 0% batyl alcohol (top panels) and 2% batyl alcohol (bottom panels) dietary groups. Aorta was divided into three sections; aortic arch, thoracic, and abdominal. Red staining indicates atherosclerotic plaques.

[0019] Figure 5 is a graphical representation showing analyses of total and regional plaques in aorta from mice fed a high fat diet supplemented with/without batyl alcohol. Levels of lesions in the (A) whole aorta; (B) aortic arch; (C) thoracic aorta; (D) abdominal aorta. Levels of atherosclerotic plaques in the whole and regions of aorta of C57/BL6, ApoE^" , and ApoE^" /GPxl^v" mice were assessed and compared between 0% batyl alcohol (open bars) and 2% batyl alcohol (closed bars) dietary groups of each genotype. Data are represented as mean ± SEM, analyzed using Student t-tests; * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0020] Figure 6 is a photographic and graphical representation showing analyses of atherosclerotic lesions in the aortic sinus of mice fed a high fat diet supplemented with/without batyl alcohol. (A) Representative photomicrographs of aortic sinus of C57/BL6, ApoE^" , and ApoE^" /GPxl^v" mice fed with high fat diet containing either 0% batyl alcohol (top panels) or 2% batyl alcohol (bottom panels). Lesions were stained red using Oil red 0. Magnification of X40. (B) Analyses of atherosclerotic lesions in the aortic sinus of ApoE^" , and ApoE^" /GPxl^v" mice on high fat diet supplemented with 0% batyl alcohol (open bars) or 2% batyl alcohol (closed bars). Data are represented as mean ± SEM, and analyzed using Student t-tests; ** indicates P<0.01.

[0021] Figure 7 is a photographic and graphical representation showing analyses of VCAM-1 staining in the aortic sinus of mice fed a high fat diet supplemented with/without batyl alcohol. (A) Representative photomicrographs of aortic sinus of C57/BL6, ApoE^" , and ApoE^" /GPxl^v" mice fed with high fat diet containing either 0% batyl alcohol (top panels) or 2% batyl alcohol (bottom panels). Magnification of X40. VCAM-1 is detected as brown staining. (B) Analyses of VCAM-1 staining in the aortic sinus of ApoE^" , and ApoE^A7GPxl^"/_ mice on high fat diet supplemented with 0% batyl alcohol (open bars) or 2% batyl alcohol (closed bars). Data are represented as mean ± SEM, and analyzed using Student t-tests; * indicates P<0.05.

[0022] Figure 8 is a photographic and graphical representation showing analyses of nitrotyrosine staining in the aorta of mice fed a high fat diet supplemented with/without batyl alcohol. (A) Representative photomicrographs of aortic cross sections of C57/BL6, ApoE^" , and ApoE^' GPxl^"7" mice fed with high fat diet containing either 0% batyl alcohol (top panels) or 2% batyl alcohol (bottom panels). Magnification of X200. Nitrotyrosine is detected as brown staining. (B) Analyses of nitrotyrosine staining in the aortic cross sections of ApoE^" , and ApoE^" /GPxl^v" mice on high fat diet supplemented with 0% batyl alcohol (open bars) or 2% batyl alcohol (closed bars). Data are

represented as mean ± SEM, and analyzed using Student t-tests; *** indicates P<0.001.

[0023] Figure 9 is a graphical representation showing plasma alkyl and alkenyl phospholipids in mice treated with alkylglycerols. Eight week old C57/BL6 mice (n=8 per group) were placed on a high fat diet (22% fat, 0.15% cholesterol) and treated with a mixture of alkylglycerols (AKG, batyl alcohol (4.0 mg), chimyl alcohol (4.0 mg) and selachyl alcohol (2.0 mg) combined with an equal weight of lecithin (phosphatidylcholine, 10 mg), prepared as a 200 μΙ_ emulsion in water. The alkylglycerols were delivered by oral gavage daily for periods of 1, 2, 4, 8 and 12 weeks. Control animals were treated with the lecithin emulsion without alkylglycerol. Points show mean values, whiskers show standard deviation.

[0024] Figure 10 is a graphical representation showing plasma alkyl and alkenyl phospholipids in mice on chow or high fat diets, treated with alkylglycerols for four weeks. Eight week old C57/BL6 mice (n= 10 per group) were placed on a chow or high fat diet (22% fat, Specialty Feeds, WA, Australia) and treated with a mixture of

alkylglycerols (batyl alcohol (2.0 mg), chimyl alcohol (2.0 mg) and selachyl alcohol (1.0 mg) combined with an equal weight of lecithin (phosphatidylcholine, 5 mg), prepared as a 100 μΙ_ emulsion in water. The alkylglycerols were delivered by oral gavage daily for four weeks. Control animals were treated with the lecithin emulsion without alkylglycerol. Bars show mean values, whiskers show standard deviation. *^' indicates p< 0.05

[0025] Figure 11 is a graphical representation showing plasmalogen modulation is anti-inflammatory in C57BL6 mice. Eight week old C57/BL6 mice (n = 10 per group) were placed on a chow or high fat diet (22% fat, Specialty Feeds, WA, Australia) and treated with a mixture of alkylglycerols (batyl alcohol (2.0 mg), chimyl alcohol (2.0 mg) and selachyl alcohol (1.0 mg) combined with an equal weight of lecithin

(phosphatidylcholine, 5 mg), prepared as a 100 μΙ_ emulsion in water. The alkylglycerols were delivered by oral gavage daily for four weeks. Control animals were treated with the lecithin emulsion without alkylglycerol. Blood was taken and analyzed for monocyte activation (CDl lb) by FACS analysis. Bars show mean fluorescence intensity for each group, whiskers show standard error. indicates p<0.01. Ly6C-hi activation in HFD vs. HFD+AG showed p=0.08. DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0027] The articles "a" and "an" are used herein to refer to one or to more than one {i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0028] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word "about." In this manner, slight variations above and below the stated ranges (e.g., less than or equal to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%) can be used to achieve substantially the same results as values within the ranges. Also, the disclosure of these ranges is intended as a continuous range including every value between the minimum and maximum values.

[0029] As used herein, "acyl" refers to a group having a straight, branched, or cyclic configuration or a combination thereof, attached to the parent structure through a carbonyl functionality. Such groups may be saturated or unsaturated, aliphatic or aromatic, and carbocyclic or heterocyclic. Examples of a Ci-Ci₀acyl- group include acetyl- , benzoyl-, nicotinoyl-, propionyl-, isobutyryl-, oxalyl-, and the like. Lower-acyl refers to acyl groups containing one to four carbons. An acyl group can be unsubstituted or substituted with one or more of the following groups: halogen, H₂N— , (Ci- C₆alkyl)amino-, di(Ci-C₆alkyl)amino-, (Ci-C₆alkyl)C(0)N(Ci-C₃alkyl)-, (Ci- C₆alkyl)carboxyamido-, HC(0)NH-, H₂NC(0)-, (Ci-C₆alkyl)NHC(0)-, di(Ci- C₆alkyl)NC(0)— ,— CN, hydroxyl, Ci-C₆alkoxy-, d-C₆alkyl-, H0₂C— , (Ci- C₆alkoxy)carbonyl-, (Ci-C₆alkyl)C(0)— , (heteroaryl)C(O)-, (heteroaryl)C(0)NH-, C₆- Ci₄aryl-, Ci-Cgheteroaryl-, or C₃-C₈cycloalkyl-. Additional examples or generally applicable substituents are illustrated by the specific compounds described herein.

[0030] The terms "administration concurrently" or "administering concurrently" or "co-administering" and the like refer to the administration of a single composition containing two or more actives, or the administration of each active as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such actives are administered as a single composition. By "simultaneously" is meant that the active agents are administered at substantially the same time, and desirably together in the same formulation. By

"contemporaneously" it is meant that the active agents are administered closely in time, e.g., one agent is administered within from about one minute to within about one day before or after another. Any contemporaneous time is useful. However, it will often be the case that when not administered simultaneously, the agents will be administered within about one minute to within about eight hours and suitably within less than about one to about four hours. When administered contemporaneously, the agents are suitably administered at the same site on the subject. The term "same site" includes the exact location, but can be within about 0.5 to about 15 centimeters, preferably from within about 0.5 to about 5 centimeters. The term "separately" as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months. The active agents may be administered in either order. The term "sequentially" as used herein means that the agents are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the active agents may be administered in a regular repeating cycle.

[0031] The term "aliphatic" as used herein, includes saturated, unsaturated, straight chain {i.e., unbranched), or branched, aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. In some embodiments, the aliphatic may contain one or more function groups selected from double bond, triple bond or carbonyl group (C=0), or a combination thereof. As will be appreciated by one of ordinary skill in the art, "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, or acyl moieties. Thus, as used herein, the term "alkyl" includes straight, branched saturated groups. An analogous convention applies to other generic terms such as "alkenyl", "alkynyl", "acyl" and the like. Furthermore, as used herein, the terms "alkyl", "alkenyl", "alkynyl", "acyl" and the like encompass both substituted and unsubstituted groups. In some embodiments, the term "aliphatic" refers to— (Ci- C₂4)alkyl,— (C₂-C₂4)alkenyl,— (C₂-C₂₄)alkynyl,— (d-C₂₄)acyl. As understood by one skilled in the art, the range of carbon number indicated above encompasses individual number within the range.

[0032] As used herein, "alkenyl" refers to a straight or branched chain hydrocarbon containing from 2 to 30 carbons and containing at least one carbon-carbon double bond. In some embodiments, the alkenyl group contains 4 to 25, 4 to 24, 4 to 23, 4 to 22, 4 to 21, 4 to 20, 4 to 19, or 4 to 18 carbon atoms. In some embodiments, the alkenyl group contains 13, 14, 15, 16, 17, 18, 19, 20 to 21 carbon atoms. In other embodiments, the alkenyl group contains 13 to 15 carbon atoms. In still other embodiments, alkenyl groups contain 13, 14, 15, 16, 17, 18, 19, 20 or 21 carbon atoms. Representative examples of "alkenyl" include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-l- heptenyl, 3-decenyl, 3-undecenyl, 4-dodecenyl, 4-tridecenyl, 9-tetradecenyl, 8- pentadecenyl, 5-hexadecenyl, 8-heptadecenyl, 9-octadecenyl, 9-nonadecenyl and the like. Additional examples or generally applicable substituents are illustrated by the specific compounds described herein.

[0033] As used herein, the term "alkoxy" refers to a group represented by the formula :— 0— R', wherein R' is alkyl or alkenyl. In some embodiments the alkoxy group contains 1-10 carbon atoms. In other embodiments, the alkoxy group contains 1-9, 1-8,

1- 7, 1-6 or 1-5 carbon atoms. In some embodiments, the alkoxy group contains 2 to 0, 2 to 8, 2 to 7, 2 to 6, 2 to 5 carbon atoms. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, n- pentoxy, n-hexoxy, n-heptoxy and the like. Additional examples or generally applicable substituents are illustrated by the specific compounds described herein.

[0034] As used herein, "alkyl" refers to a straight or branched chain

hydrocarbon containing from 1 to 30 carbon atoms. In some embodiments, the alkyl group contains 4 to 25, 4 to 24, 4 to 23, 4 to 22, 4 to 21, 4 to 20, 4 to 19 or 4 to 18 carbon atoms. In one embodiment, the alkyl group contains 12, 13, 14, 15, 16, 17, 18, 19 or 20 to 21 carbon atoms. In some embodiments the alkyl group contains 12, 13, 14, 15, 16 or 17 to 21 carbon atoms. In some embodiments, the alkyl group contains 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 carbon atoms. In still other embodiments, alkyl group contains 1-5 carbon atoms, and in yet other embodiments, alkyl group contain 1-4 or 1-3 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n- octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n- hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl and the like. Additional examples or generally applicable substituents are illustrated by the specific compounds described herein.

[0035] As used herein, "alkynyl" refers to a straight or branched chain hydrocarbon containing from 2 to 30 carbons and containing at least one carbon-carbon triple bond. In some embodiments, the alkynyl group contains 4 to 25, 4 to 24, 4 to 23, 4 to 22, 4 to 21, 4 to 20, 4 to 19, or 4 to 18 carbon atoms. In some embodiments, the alkenyl group contains 13, 14, 15, 16, 17, 18, 19, 20 to 21 carbon atoms. In other embodiments, the alkynyl group contains 13 to 15 carbon atoms. In still other embodiments, alkynyl groups contain 13, 14, 15, 16, 17, 18, 19, 20 or 21 carbon atoms. Representative examples of "alkynyl" include, but are not limited to, ethynyl, 2-propynyl,

2- methyl-2-propynyl, 3-butynyl, 4-pentynyl, 5-hexynyl, 2-heptynyl, 2-methyl-l- heptynyl, 3-decynyl, 3-undecynyl, 4-dodecynyl, 4-tridecynyl, 9-tetradecynyl, 8- pentadecynyl, 5-hexadecynyl, 8-heptadecynyl, 9-octadecynyl, 9-nonadecynyl and the like. Additional examples or generally applicable substituents are illustrated by the specific compounds described herein.

[0036] As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0037] As used herein, "aromatic" or "aryl" is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 atoms in each ring, wherein at least one ring is aromatic. Examples of such aryl elements include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl.

[0038] It will also be recognized that the compounds described herein may possess asymmetric centers and are therefore capable of existing in more than one stereoisomeric form. The invention thus also relates to compounds in substantially pure isomeric form at one or more asymmetric centers e.g., greater than 90% ee, such as 95% or 97% ee or greater than 99% ee, as well as mixtures, including racemic mixtures, thereof. Such isomers may be naturally occurring or may be prepared by asymmetric synthesis, for example using chiral intermediates, or by chiral resolution.

[0039] The term "atherosclerosis" refers to a form of arteriosclerosis

characterized by the deposition of atheromatous plaques (e.g., calcified plaques, non- calcified plaques, fibrocalcified plaques etc.) containing cholesterol and lipids on the innermost layer of the walls of large and medium-sized arteries. This deposition results in the progressive narrowing and hardening of arteries, and as such is a common type of cardiovascular disease. Atherosclerosis is associated with an increased incidence of hypertension, cardiac hypertrophy, myocardial infarction, congestive heart failure, stroke, and peripheral vascular disease.

[0040] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements. [0041] As used herein, "cycloalkyi" refers to a monovalent saturated cyclic or bicyclic hydrocarbon group of 3-12 carbons derived from a cycloalkane. In some embodiments, cycloalkyi contains 3 to 8 carbon atoms. In some embodiments, cycloalkyi contains 3 to 6 carbon atoms. Cycloalkyi groups may be optionally substituted with alkyl, alkoxy, halo, amino, thiol, or hydroxy substituents. Representative examples of cycloalkyi include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Additional examples of generally applicable substituents are illustrated by the specific compounds described herein.

[0042] As used herein, the term "dietary" denotes that which is naturally a part of the subjects diet.

[0043] By "effective amount", in the context of treating or preventing a condition is meant the administration of an amount of an agent or composition to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of that condition. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

[0044] The term "extracted" with reference to a particular composition or substance refers to a composition or substance extracted from a natural source, including organisms and parts thereof. For example, lipids or oils extracted from a natural source, refer to lipids or oil that have been separated from other cellular materials, such as the natural source in which the lipid or oil was synthesized. Extracted lipids or oils are obtained through a wide variety of methods, the simplest of which involves physical means alone. For example, mechanical crushing using various press configurations (e.g., screw, expeller, piston, bead beaters, etc.) can separate lipids or oils from cellular materials. Alternately, lipid or oil extraction can occur via treatment with various organic solvents (e.g., hexane), via enzymatic extraction, via osmotic shock, via ultrasonic extraction, via supercritical fluid extraction (e.g., C0₂ extraction), via saponification and via combinations of these methods. An extracted lipid or oil does not require that it is necessarily purified or further concentrated.

[0045] As used herein, the term "food additive" refers to a substantially-pure material which is added to food for a nutritional benefit.

[0046] The term "group" as applied to chemical species refers to a set of atoms that forms a portion of a molecule. In some instances, a group can include two or more atoms that are bonded to one another to form a portion of a molecule. A group can be monovalent or polyvalent (e.g. , bivalent) to allow bonding to one or more additional groups of a molecule. For example, a monovalent group can be envisioned as a molecule with one of its hydrogen atoms removed to allow bonding to another group of a molecule. A group can be positively or negatively charged. For example, a positively charged group can be envisioned as a neutral group with one or more protons {i.e., H⁺) added, and a negatively charged group can be envisioned as a neutral group with one or more protons removed. Non-limiting examples of groups include, but are not limited to, alkyl groups, alkylene groups, alkenyl groups, alkenylene groups, alkynyl groups, alkynylene groups, aryl groups, arylene groups, iminyl groups, iminylene groups, hydride groups, halo groups, hydroxy groups, alkoxy groups, carboxy groups, thio groups, alkylthio groups, disulfide groups, cyano groups, nitro groups, amino groups, alkylamino groups, dialkylamino groups, silyl groups, and siloxy groups. Groups such as alkyl, alkenyl, alkynyl, aryl, and heterocyclyl, whether used alone or in a compound word or in the definition of a group may be optionally substituted by one or more substituents. "Optionally substituted," as used herein, refers to a group may or may not be further substituted with one or more groups selected from alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, benzyloxy, haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, amino, alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino, diarylamino, phenylamino, diphenylamino, benzylamino, dibenzylamino, hydrazino, acyl, acylamino, diacylamino, acyloxy, heterocyclyl, heterocycloxy, heterocyclamino, haloheterocyclyl, carboxy ester, carboxy, carboxy amide, mercapto, alkylthio, benzylthio, acylthio and phosphorus-containing groups. As used herein, the term "optionally substituted" may also refer to the replacement of a CH₂ group with a carbonyl (C=0) group. Non-limiting examples of optional substituents include alkyl, preferably Ci_-8 alkyl {e.g., d-₆ alkyl such as methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), hydroxy Ci_-8 alkyl {e.g.,

hydroxymethyl, hydroxyethyl, hydroxypropyl), alkoxyalkyl {e.g., methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl, ethoxyethyl, ethoxypropyl etc.) Ci_-8 alkoxy, {e.g., Ci_-6 alkoxy such as methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy), halo (fluoro, chloro, bromo, iodo), monofluoromethyl, monochloromethyl, monobromomethyl, difluoromethyl, dichloromethyl, dibromomethyl, trifluoromethyl, trichloromethyl, tribromomethyl, hydroxy, phenyl, benzyl, phenoxy, benzyloxy, amino, Ci-8 alkylamino {e.g., Ci_-6 alkyl, such as methylamino, ethylamino, propylamino), di Ci_-8 alkylamino {e.g., Ci_-6 alkyl, such as dimethylamino, diethylamino, dipropylamino), acylamino {e.g., NHC(0)CH₃), phenylamino, nitro, formyl, -C(0)-Ci_-8 alkyl {e.g., Ci_-6 alkyl, such as acetyl), 0-C(0)-alkyl {e.g. , Ci_-6 alkyl, such as acetyloxy), benzoyl, replacement of CH₂ with C=0, C0₂H, C0₂ Ci_-8 alkyl {e.g., Ci_-6 alkyl such as methyl ester, ethyl ester, propyl ester, butyl ester), C0₂phenyl, CONH₂, CONHphenyl, CONHbenzyl, CONH Ci-₈ alkyl {e.g., Ci_-6 alkyl such as methyl amide, ethyl amide, propyl amide, butyl amide), CONH Ci_-8 alkylamine {e.g., Ci_-6 alkyl such as aminomethyl amide, aminoethyl amide, aminopropyl amide, aminobutyl amide), -C(0)heterocyclyl (e.g. , -C(0)- 1- piperidine, -C(0)- l-piperazine, -C(0)-4-morpholine), -C(0)heteroaryl (e.g. , -C(0)-1- pyridine, -C(0)- l-pyridazine, -C(0)- l-pyrimidine, -C(O)- l-pyrazine), CON Hdi Ci_-8 alkyl (e.g. , Ci-₆alkyl) .

[0047] "Heteroaralkyl" group means alkyl as defined above which is substituted with a heteroaryl group, e.g. ,-CH₂pyridinyl,-(CH2)2Pynmidinyl,-(CH₂)3imidazolyl, and the like, a nd derivatives thereof.

[0048] The term "heteroaryl" or "heteroaromatic", as used herein, represents a stable monocyclic or bicyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of 0, N and S. Heteroaryl groups within the scope of this definition include but are not limited to : acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl,

benzotriazolyl, furanyl, thienyl, benzothienyl, bezofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline. As with the definition of heterocycle below, "heteroaryl" is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl.

Further examples of "heterocyclyl" and "heteroaryl" include, but are not limited to, the following : benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazoyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1,4- dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,

dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,

dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof. Attachment of a heterocyclyl substituent can occur via a carbon atom or via a heteroatom .

[0049] The term "heterocycle", "heteroaliphatic" or "heterocyclyl" as used herein is intended to mean a 5-to 10-membered nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of 0, N and S, and includes bicyclic groups. [0050] The term "heterologous" as used herein with reference to plasmalogens refers to a plasmalogens that is made synthetically or isolated from another species or isolated from the same species as the subject but modified from either its original form or the form primarily expressed in the subject to be treated.

[0051] As used herein, the term "nutraceutical" refers to any substance thought to provide medical, health, or biological benefits. In some embodiments, nutraceuticals may prevent disease. In some embodiments, nutraceuticals may provide basic nutritional value. In some embodiments, a nutraceutical is a food or part of a food. In some embodiments, a nutraceutical agent may be a class of isolated nutrients, dietary supplements, vitamins, minerals, herbs, fortified foods, healing foods, genetically engineered foods, and processed foods. Nutraceuticals may also be known as

"phytochemical foods" or "functional foods."

[0052] The terms "patient," "subject," "host" or "individual" used

interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired.

Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates (e.g. , humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and/or rhesus monkeys {Macaca mulatta)) and baboon {Papio ursinus), as well as marmosets (species from the genus

Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g. , cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g. , dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish. In specific

embodiments, the subject is a primate such as a human. However, it will be understood that the aforementioned terms do not imply that symptoms are present.

[0053] By "pharmaceutically acceptable carrier" is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, transfection agents and the like.

[0054] "Phenylalkyl" means alkyl as defined above which is substituted with phenyl, e.g.,-CH₂phenyl,-(CH₂)₂Phenyl,-(CH₂)₃Phenyl, CH₃CH(CH₃)CH₂phenyl, and the like and derivatives thereof. Phenylalkyl is a subset of the aralkyl group. [0055] The term "polar group" as used herein refers to a group in which the nuclei of the atoms covalently bound to each other to form the group do not share the electrons of the covalent bond(s) joining them equally; that is the electron cloud is denser about one atom than another. This results in one end of the covalent bond(s) being relatively negative and the other end relatively positive; i.e., there is a negative pole and a positive pole. Examples of polar groups include, without limitation, hydroxy, alkoxy, carboxy, nitro, cyano, amino, ammonium, amido, ureido, sulfonamido, sulfinyl, sulfonyl, phosphono, morpholino, piperazinyl and tetrazolo or an ester thereof.

[0056] The term "pro-drug" is used in its broadest sense and encompasses those derivatives that are converted in vivo to the compounds of the invention. Such derivatives would readily occur to those skilled in the art, and include, for example, compounds where a free hydroxy group is converted into an ester derivative.

[0057] As used herein, the terms "prevent," "prevented," or "preventing," refer to a prophylactic treatment which increases the resistance of a subject to developing the disease or condition or, in other words, decreases the likelihood that the subject will develop the disease or condition as well as a treatment after the disease or condition has begun in order to reduce or eliminate it altogether or prevent it from becoming worse. These terms also include within their scope preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it.

[0058] As used herein, "racemate" refers to a mixture of enantiomers.

[0059] The terms "reduce," "inhibit," "diminish," "suppress," "decrease," and grammatical equivalents when used in reference to the level or incidence of

atherosclerotic plaque in a first sample relative to a second sample, mean that the level or incidence of atherosclerotic plaque in the first sample is lower than in the second sample by any amount that is statistically significant using any art-accepted statistical method of analysis. In one embodiment, the reduction may be determined objectively, for example when incidence or level of atherosclerotic plaque in a patient is lower than an earlier incidence or level of atherosclerotic plaque in the patient. In another embodiment, the reduction may be determined objectively, for example when incidence or level of atherosclerotic plaque in a patient is lower than the incidence or level of atherosclerotic plaque in a control patient (e.g., a patient not administered a

plasmalogen level-enhancing compound). In one embodiment, the incidence or level of atherosclerotic plaque in a first sample is at least 10% lower than the incidence or level of atherosclerotic plaque in a second sample. In another embodiment, the incidence or level of atherosclerotic plaque in a first sample is at least 25% lower than the incidence or level of atherosclerotic plaque in a second sample. In yet another embodiment, the incidence or level of atherosclerotic plaque in a first sample is at least 50% lower than the incidence or level of atherosclerotic plaque in a second sample. In a further embodiment, the incidence or level of atherosclerotic plaque in a first sample is at least 75% lower than incidence or level of atherosclerotic plaque in a second sample. In yet another embodiment, the incidence or level of atherosclerotic plaque in a first sample is at least 90% lower than the incidence or level of atherosclerotic plaque in a second sample. Alternatively, a difference may be expressed as an "n-fold" difference.

[0060] The term "reducing the incidence" refers to inhibiting the development and/or reducing the progression, or magnitude, of a condition or symptom thereof (e.g., atherosclerosis, atherosclerotic plaque etc.).

[0061] The terms "salts" and "prodrugs" includes any pharmaceutically acceptable salt, ester, hydrate, or any other compound which, upon administration to the recipient, is capable of providing (directly or indirectly) a compound of the invention, or an active metabolite or residue thereof. Suitable pharmaceutically acceptable salts include salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids, or salts of

pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulfonic, toluenesulfonic, benzenesulfonic, salicylic, sulfanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. Base salts include, but are not limited to, those formed with

pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium. Also, basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others. However, it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the invention since these may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts and prodrugs can be carried out by methods known in the art. For example, metal salts can be prepared by reaction of a compound of the invention with a metal hydroxide. An acid salt can be prepared by reacting an appropriate acid with a compound of the invention.

[0062] As used herein, "supplement" encompasses an herb, enzyme, vitamin, animal oil, naturally occurring hormone, medicinal plant, animal extract, or diet-oriented compound, or medicament such as those traditionally used in the treatment of a disease or a medical condition.

[0063] As used herein, the terms "treatment", "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disease or condition (e.g., a cardiovascular disease condition such as but not limited to atherosclerosis) and/or adverse affect attributable to the disease or condition. These terms also cover any treatment of a condition or disease in a mammal, particularly in a human, and include: (a) inhibiting the disease or condition, i.e., arresting its development; or (b) relieving the disease or condition, i.e., causing regression of the disease or condition.

[0064] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.

2. Plasmalogen level-enhancing compounds

[0065] It has been found that compounds that increase the level of

plasmalogens in vivo are effective for reducing the incidence and inhibiting the formation of atherosclerotic plaque in blood vessels of animals predisposed to the development of atherosclerosis. Plasmalogen level-enhancing compounds are thus useful for reducing the risk of developing atherosclerosis, delaying the onset of atherosclerosis, and/or reducing the risk of sequelae of atherosclerosis. Sequelae of atherosclerosis include angina, claudication, heart attack, ischemia, stroke, and others.

[0066] Numerous plasmalogen level-enhancing compounds are known, including plasmalogen precursor lipids and heterologous plasmalogens. For example, plasmalogen precursor lipids may be selected from alkylglycerols. Alkylglycerols are lipids with a glycerol backbone, to which fatty acid or fatty acid derivatives are coupled by means of an ether bond instead of the ester bond that characterizes most mono-, di- and triglycerols and related phospholipids (see, e.g., U.S. Pat. No. 6,121,245, which is incorporated herein by reference in its entirety). The glycerol ethers are widely distributed in animal tissues, although the liver of the elasmobranch fish remains the richest natural source. The naturally occurring alkylglycerols are in most cases etherified, and in some case esterified, with fatty acids of 16-18 C-atoms, sometimes unsaturated.

[0067] Suitably, the alkylglycerols are represented by formula (I) :

R₃ R₄

CH₂-0 CH₂ CH— CH R₅

CH— O R₂

CH₂-0 Ri

(I)

[0068] or pharmaceutically acceptable salts or prodrugs thereof,

[0069] wherein :

[0070] Ri and R₂ are the same or different and each is selected from the group consisting of hydrogen and aliphatic acyl groups of at most 24 carbon atoms; [0071] R₃ and R₄ are selected from the group consisting of hydrogen, or straight, branched, saturated and unsaturated alkoxy groups of at most 7 carbon atoms; and

[0072] R₅ is selected from the group consisting of straight chain and branched alkyl and alkenyl groups of 4 to 21 carbon atoms.

[0073] In some embodiments, R₃ and R₄ are each hydrogen . In other embodiments, one of R₃ and R₄ is hydrogen and the other is selected from the group consisting of straight, branched, saturated and unsaturated alkoxy groups of at most 7 carbon atoms.

[0074] In specific embodiments, Ri, R₂, R₃ and R₄ are each hydrogen and R₅ is a straight chain alkyl or alkenyl group of 13 to 15 carbon atoms.

[0075] Exemplary alkylglycerols are 1-0-alkylglycerols, which are suitably selected from the group consisting of 18 : 0 alkyl- l-glycerol, 18 : 1 alkyl- l-glycerol and 16: 0 alkyl- l-glycerol, or combination thereof. In specific embodiments, the 1-O- alkylglycerols are selected from the group consisting of 1-O-hexadecyl-sn-glycerol (chimyl alcohol), 1-O-octadecyl-sn-glycerol (batyl alcohol) and l-0-(9Z-octadecenyl)-sn- glycerol (selachyl alcohol) .

[0076] The compounds of formula (I) are capable of existing in stereoisomeric forms. It will be understood that the invention encompasses all optical isomers of the compounds of formula (I), and mixtures thereof. Hence, compounds of formula (I) that exist as diastereomers, racemates, enantiomers and geometric isomers are included within the scope of the present disclosure.

[0077] In specific embodiments, alkylglycerol compounds represented by formula (I) are selected from :

combinations thereof.

[0078] Alkylglycerols may be prepared synthetically. Synthesis of these compounds is well known in the art (see, for instance, Takaishi et al. , U. S. Pat. No. 4,465,869, UK Patent 1,029,610, and Magnusson et al. , Tetrahedron (2011) 67 1821el836, which are hereby incorporated by reference herein in their entirety). In addition, mono- and di-esters of alkylglycerols are well-known in the art and their syntheses have been described (see, e.g., Burgos et al. (1987), J. Org. Chem. 52: 4973- 4977; Hirth et a/. (1982) Helv. Chim. Acta 65: 1059-1084; and Hirth et a/. (1983) Helv. Chim. Acta 66: 1210-1240).

[0079] Alternatively, alkylglycerols are extracted from a natural source, illustrative examples of which include fish oils such as shark oils, and hematopoietic organs such as bone marrow and spleen. In specific embodiments, alkylglycerols are extracted from fish liver oils, particularly liver oils of elasmobranch fish such as sharks (e.g., Greenland shark, dogfish, ratfish, rabbitfish see, e.g., Hallgren et a/., U.S. Pat.

4,046,914, which is incorporated by reference herein in its entirety), rays, Seamouse etc. Shark liver oil may be obtained commercially (see, e.g., ALKYROL, Eurohealth, Inc., Parkside, Pa.). Common fatty alcohols found in shark liver oil are chimyl alcohol, batyl alcohol and selachyl alcohol. Non-limiting methods for extracting alkylglycerols are disclosed for example in Hallgren et a/, {supra) and Brohult et a/., International

Publication No. WO 1998/52550, which is incorporated by reference herein in its entirety).

[0080] Heterologous plasmalogens may be prepared from any suitable source. For example, they may be extracted from a natural source, such as but not limited to microorganisms and animals. Non-limiting examples of plasmalogen-producing

microorganisms anaerobic bacteria, suitably from the family Acidaminococcaceae, which are intestinal bacteria. Representative examples of plasmalogen-producing animals include birds, mammals, fishes, shellfishes, and the like. In some embodiments, the mammals are livestock mammals, representative examples of which include cow, pig, horse, sheep, goat, and the like. Suitable plasmalogen-containing mammalian tissues include skin, spinal cord, brain, intestines, heart, genitals, and the like. Examples of birds include chicken, domestic duck, quail, duck, pheasant, ostrich, turkey, and the like.

There is no particular limitation to an avian tissue to be used. For example, bird meat (in particular, bird's breast meat), bird skin, internal organs of birds, bird eggs etc., are suitably used. Two or more types of different tissues from one or more species of organisms may be used in combination. Methods for extracting plasmalogens are known in the art, non-limiting examples of which are described in Nishimukai et a/. (2003) Lipids 38(12) : 1227-1235, Horrmann et a/., U.S. Pat. No. 4,613,621 and Mawatari et a/., U.S. Publication No. 2013/0172293, which are incorporated herein by reference in their entirety.

[0081] Alternatively, plasmalogens may be prepared synthetically. Synthesis of these compounds is well known in the art (see, for instance, Shin et al. (2003) J Org. Chem., 2003 68(17) : 6760-6766; Van den Bossche, et a/. (2007) J. Org. Chem. 72(13) : 5005-5007 and Khan et a/. , International Publication No. WO 2013/071418, which are incorporated herein by reference in their entirety) .

[0082] In specific embodiments, the heterologous plasmalogens are

represented by formula (II) :

CH₂-0 CH=CH— R₃

O II

CH— O C R₂

O II

CH₂-0 F> O R-i

O- (II)

[0083] or pharmaceutically acceptable salts or prodrugs thereof,

[0084] wherein :

[0085] Ri is a polar group; and

[0086] R₂ and R₃ are the same or different and each is selected from aliphatic hydrocarbon groups.

[0087] In some embodiments, Ri is selected from the group consisting of ethanolamine, choline, serine, inositol and glycerol. Suitably, Ri is an ethanolamine or a choline.

[0088] R² is generally an aliphatic hydrocarbon group, which is suitably derived from a fatty acid residue, illustrative examples of which include octadecadienoyl group, octadecatrienoyi group, icosatetraenoyi group, icosapentaenoyi group, docosatetraenoyi group, docosapentaenoyl group, docosahexaenoyl group, and the like.

[0089] R³ is generally an aliphatic hydrocarbon group having a carbon number of 1 to 24, representative examples of which include dodecyl group, tetradecyl group, hexadecyl group, octadecyl group, icosa nyl group, and the like.

[0090] In specific embodiments, the heterologous plasmalogen mainly includes an ethanolamine plasmalogen and/or a choline plasmalogen {i. e. , includes at least one member selected from the group consisting of an ethanolamine plasmalogen and a choline plasmalogen) . The mass ratio of ethanolamine plasmalogen to choline

plasmalogen (ethanolamine plasmalogen :choline plasmalogen) is suitably 1 : 5 to 1 : 0, 1 : 5 to 5 : 1, 1 : 3 to 3 : 1, 1 : 1 to 3 : 1, or 1 : 1 to 2 : 1.

[0091] The compounds of formula (II) are capable of existing in stereoisomeric forms. It will be understood that the invention encompasses all optical isomers of the compounds of formula (II), and mixtures thereof. Hence, compounds of formula (II) that exist as diastereomers, racemates, enantiomers and geometric isomers are included within the scope of the present disclosure. [0092] The plasmalogen level-enhancing compounds may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.

3. Compositions

[0093] In accordance with the present invention, it is proposed that

plasmalogen level-enhancing compounds are useful as actives for reducing the incidence of atherosclerotic plaque, for inhibiting formation of atherosclerotic plaque, for regressing atherosclerotic plaque and/or for treating atherosclerosis in a subject. The plasmalogen level-enhancing compounds may be administered either alone or with an acceptable carrier, which in some embodiments is a pharmaceutically acceptable carrier. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. Accordingly, the present invention contemplates compositions in which at least one plasmalogen level- enhancing compound is combined with an acceptable carrier to form a composition for use in the applications referenced above. Carriers will generally be selected in accordance with ordinary practice. Examples of pharmaceutically acceptable carriers and methods of manufacture for various compositions include, but are not limited to, those described in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa ., latest edition. The compositions may be prepared for administration via any suitably route including oral, rectal, transmucosal, intestinal, inhalational, sublingual, buccal or vaginal administration, or implanted reservoir; parenteral delivery, including intramuscular, subcutaneous, intra-articular, intra-synovial, intrasternal, intrathecal, intramedullary, intrathecal, intraventricular, intravenous, intradermal, intraperitoneal, intrahepatic, intralesional, intracranial, epidural, intranasal or intraocular delivery (e.g., injection or infusion).

[0094] In specific embodiments, the plasmalogen level-enhancing compounds are formulated using acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be

formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. These carriers may be selected from sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.

[0095] Compositions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as., for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or

polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more drugs as described above with the carrier, which constitutes one or more necessary ingredients. In general, the compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0096] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0097] In some embodiments, the compositions of the present invention that are suitable for oral administration are presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the plasmalogen level- enhancing compound(s); as a powder or granules; as solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water- in-oil liquid emulsion. The plasmalogen level-enhancing compound(s) may also be presented as a bolus, electuary or paste.

[0098] Compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

[0099] Dosage forms of the plasmalogen level-enhancing compounds of the invention may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of a plasmalogen level-enhancing compound may be achieved by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, controlled release may be achieved by using other polymer matrices, liposomes or microspheres.

[OIOO] Compositions for parenteral administration include aqueous solutions and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit- dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

[0101] In some embodiments, an acceptable oil is employed as a solvent or suspending medium in the compositions of the present invention. Fatty acids, such as oleic acid and its glyceride derivatives are suitably included in injectable formulations, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. The oil containing compositions of the present invention may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. The compositions suitably further comprise surfactants (such as non-ionic detergents including Tween™ or Span™) other emulsifying agents, or bioavailability enhancers.

[0102] Compositions suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0103] Compositions for rectal administration may be presented as a

suppository with a suitable base comprising for example cocoa butter or a salicylate.

[0104] Compositions suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns (including particle sizes in a range between 20 and 500 microns in increments of 5 microns such as 30 microns, 35 microns, etc.), which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable compositions in which the carrier is a liquid, for administration as for example a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol administration may be prepared according to conventional methods and may be delivered with other therapeutic agents such as pentamidine for treatment of Pneumocystis pneumonia .

[0105] Compositions suitable for vaginal administration may be presented as pessaries, rings, ta mpons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be

appropriate.

[0106] Compositions suitable for topical administration may be in the form of a topical solution, ointment, or cream in which the active component is suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Where the topica l formulation is in the form of an ointment or cream, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water. In some embodiments, the topical composition of the present invention is in the form of a spray.

[0107] The compositions of the present invention may also be administered by nasa l, aerosol or by inhalation administration routes. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents. In some embodiments, the nasal administration of the composition of the present invention is in the form of a spray. Any suitable carrier for spray application may be used in the present invention.

[0108] Alternatively, the compositions of the present invention may be in the form of a suppository for rectal administration. The suppositories can be prepared by mixing the agent with a suita ble non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug . Such materials include cocoa butter, beeswax and polyethylene glycols.

[0109] It should be understood that in addition to the ingredients particularly mentioned above the compositions of this invention may include other agents

conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

[0110] In some embodiments, the compositions of the present invention are formulated for oral administration. For oral administration to humans, the dosage range is 0.01 to 1000 mg/kg body weight in divided doses. In one embodiment the dosage range is 0.1 to 100 mg/kg body weight in divided doses. In another embodiment the dosage range is 0.5 to 20 mg/kg body weight in divided doses. For oral administration, the compositions may be provided in the form of tablets or capsules containing 1.0 to 1000 milligrams of the active ingredient, particularly, 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and 1000 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated .

[Olll] As specific embodiments, the plasmalogen level-enhancing compounds (e.g. , alkylglycerols and/or heterologous plasmalogens) may be administered at 10 to 100 mg, 100 to 200 mg, 300 to 600 mg, 600 to 1200 mg, 1200 to 1500 mg and 1500 to 2000 mg of plasmalogen level-enhancing compounds.

[0112] The frequency or schedule of administration of a composition described herein can vary with various factors, including, but not limited to, modes of

administration (e.g. , oral or intravenous administration), dosage, severity of the condition to be treated, treatment type (e.g. , preventive treatment or therapeutic treatment), and/or treatment regimen (e.g ., administration of the composition alone or in combination with other clinical treatments) . Accordingly, the time interval between any two consecutive administrations can vary, e.g ., between hours, days, weeks, or months. In some embodiments, the time interval between any two consecutive administrations can be at least about 4 hours, at least about 6 hours, at least a bout 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days or longer. In some embodiments, the time interval between any two consecutive administrations can be at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks or longer. In some embodiments, the time interval between any two consecutive administrations can be at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least a bout 5 months, at least about 6 months or longer.

[0113] The compositions may further include at least one ancillary agent for treating and/or preventing atherosclerosis. Non-limiting ancillary agents include statins (e.g. , lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, itavastatin, pitavastatin, ZD-4522 and rivastatin), cholesterol lowering agents (e.g. , HMG-CoA reductase inhibitors, sequestrants, nicotinyl alcohol, nicotinic acid and salts thereof, PPARa agonists, PPARay dual agonists, inhibitors of cholesterol absorption, acyl

CoA: cholesterol acyltransferase inhibitors, and anti-oxidants); DPP-IV inhibitors; insulin sensitizers (e.g. , PPAR agonists and biguanides) ; insulin and insulin mimetics;

sulfonylureas and other insulin secretagogues; a-glucosidase inhibitors; glucagon receptor antagonists; GLP-1, GLP- 1 mimetics, and GLP- 1 receptor agonists; GIP,GIP mimetics, and GIP receptor agonists; ACAP, PACAP mimetics, and PACAP receptor 3 agonists; PPAR5 agonists; antiobesity compounds; ileal bile acid transporter inhibitors; anti-inflammatory agents excluding glucocorticoids; protein tyrosine phosphatase- l B (PTP-1B) inhibitors; and antihypertensives (e.g., including those acting on the

angiotensin or renin systems, such as angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists or renin inhibitors, such as captopril, cilazapril, enalapril, fosinopril, lisinopril, quinapril, ramipril, zofenopril, candesartan, cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, and valsartan).

4. Applications

[0114] The present invention also extends to methods for reducing the incidence of atherosclerosis, including the formation and progression of atherosclerosis as well as the management of atherosclerosis in a subject. Accordingly, the present invention encompasses both prophylactic and therapeutic methods of treating a subject, e.g., a human, having or at risk of (or susceptible to) developing plaque rupture and/or myocardial infarction. The prophylactic methods may include identifying that a subject is at risk of developing atherosclerosis including sequelae such as ischemia. At-risk individuals include, but are not limited to, individuals with a family history of

atherosclerosis, hypercholesterolemia, low HDL levels, high LDL levels, hyperlipidemia, hypertriglyceridemia and dyslipidemia, EKG changes associated with risk of ischemia, sedentary lifestyle, angiographic evidence of partial coronary artery obstruction, echocardiographic evidence of myocardial damage, or any other evidence of a risk for a future or additional ischemic event (for example a myocardial ischemic event, such as a myocardial infarction (MI), or a neurovascular ischemia such as a cerebrovascular accident CVA), high blood pressure, smoking, insulin resistance including type 1 or type 2 diabetes, obesity, lack of physical activity, and individuals presenting any other clinical indicia suggesting that they have an increased likelihood of developing atherosclerosis and/or individuals who have previously been treated for atherosclerosis. Alternatively stated, an at-risk individual is any individual who is believed to be at a higher risk than the general population for developing atherosclerosis. In particular examples of the methods, individuals are selected for treatment who are at risk of future atherosclerosis, but who have no present evidence of atherosclerosis (such as electrocardiographic changes associated with ischemia (for example, peaked or inverted T-waves or ST segment elevations or depression in an appropriate clinical context), elevated CKMB, or clinical evidence of ischemia such as crushing sub-sternal chest pain or arm pain, shortness of breath and/or diaphoresis). The plasmalogen level-enhancing compounds also could be administered prior to procedures in which myocardial ischemia may occur, for example an angioplasty or surgery (such as a coronary artery bypass graft surgery). Also embraced is a method of administering a plasmalogen level-enhancing compound to an individual at demonstrated risk for atherosclerosis. The selection of an individual with such a status could be performed by a variety of methods, some of which are noted above. For example, an individual with one of more of an abnormal EKG not associated with active ischemia, prior history of myocardial infarction, elevated serum cholesterol, etc., would be at risk of atherosclerosis and its sequelae. Thus, an at-risk individual could be selected by physical testing or eliciting the potential subject's medical history to determine whether the subject has any indications of risk of atherosclerosis. If risk is demonstrated based on the indications discussed above, or any other indications that one skilled in the art would appreciate, then the individual would be considered at

demonstrated risk of atherosclerosis.

[0115] The subject to be treated is administered at least one plasmalogen level-enhancing compound of the invention for reducing the incidence of atherosclerosis, including the formation and progression of atherosclerosis, for preventing or delaying the onset of atherosclerosis and/or for treating atherosclerosis in the subject, or its sequelae. Treatment regimens may further include dietary restrictions (e.g. , limiting caloric intake) and exercise. In some embodiments, a treatment regimen will be administered in pharmaceutical (or veterinary) compositions together with a pharmaceutically acceptable carrier and in an effective amount to achieve their intended purpose. The dose of active compounds administered to a subject should be sufficient to achieve a beneficial response in the subject. The quantity of the pharmaceutically active compounds(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the active compound(s) for administration will depend on the judgment of the practitioner. In determining the effective amount of the active compound(s) to be administered for reducing the incidence of atherosclerosis, including the formation and progression of atherosclerosis, for preventing or delaying the onset of atherosclerosis and/or for treating atherosclerosis in the subject, or its sequelae, the medical practitioner or veterinarian may evaluate family history of atherosclerosis, HDL levels, LDL levels, EKG changes associated with risk of ischemia, sedentary lifestyle, angiographic evidence of partial coronary artery obstruction, echocardiographic evidence of myocardial damage, blood pressure, smoking, insulin resistance, obesity and/or lack of physical activity. In any event, those of skill in the art may readily determine suitable dosages of the therapeutic agents and suitable treatment regimens without undue experimentation.

[0116] Treatment regimens may further include administering concurrently with the plasmalogen level-enhancing compound(s) at least one ancillary agent for treating and/or preventing atherosclerosis, as described for example supra. These regimens may involve administering the plasmalogen level-enhancing compound(s) separately, simultaneously or sequentially with the ancillary agent(s). In some embodiments, this may be achieved by administering a single composition or pharmacological formulation that includes both types of agent, or by administering two separate compositions or formulations at the same time, wherein one composition includes the plasmalogen level- enhancing compound(s) and the other, the ancillary agent(s). In other embodiments, the treatment with the plasmalogen level-enhancing compound(s) may precede or follow the treatment with the ancillary agent(s) by intervals ranging from minutes to days. In embodiments where the plasmalogen level-enhancing compound(s) is(are) applied separately to the ancillary agent(s), one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the plasmalogen level-enhancing compound(s) would still be able to exert an advantageously combined effect on reducing the incidence of atherosclerosis, including the formation and progression of atherosclerosis. In such instances, it is contemplated that one would administer both modalities within about 1- 12 hours of each other and, more suitably, within about 2-6 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several hours (2, 3, 4, 5, 6 or 7) to several days (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0117] It is conceivable that more than one administration of either

plasmalogen level-enhancing compound(s) or the ancillary agent(s) will be desired .

Various combinations may be employed, where the plasmalogen level-enhancing compound(s) is(are) "A" and ancillary agent(s) is(are) "B", as exemplified below :

[0118] A/B/A B/A/B B/B/A A/ A/ B B/A/A A/ B/ B B/ B/ B/ A B/ B/ A/ B A/ A/ B/ B

A/B/A/B A/ B/B/A B/B/A/ A B/A/B/ A B/A/A/B B/ B/B/A A/A/A/B B/A/A/A A/B/A/ A A/ A/B/A A/B/B/B B/A/B/ B B/ B/A/B.

[0119] Other combinations are contemplated . Again, both agents are delivered to a subject in a combined amount effective to reduce the incidence of atherosclerosis, including the formation and progression of atherosclerosis, and desirably to prevent or delaying the onset of atherosclerosis and/or to treat atherosclerosis in the subject.

[0120] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

EXPERIMENTAL

EXAM PLE 1

ATTENUATION OF ATHEROSCLEROSIS IN APOE- AND APOE/GPXI -DEFICIENT M ICE

BY PLASMALOGEN MODULATION

Results

Phenotypic Assessment and Plasma Lipid Profile Resulting from Batyl Alcohol Administration

[0121] The present inventors first investigated whether supplementation of batyl alcohol caused any changes in phenotype and plasma clinical measurements of control (C57/BL6) mice as well as the ApoE^" , and ApoE^" /GPxl^v" mice. The general health of the mice was checked via whole body checking {i.e. , scruffing) thrice weekly. No adverse effects to the mice throughout the study were observed following batyl alcohol supplementation. All mice showed an increase in body weight across the study period (Figure 1). Both ApoE^" and ApoE^" /GPxl^v" mice experienced slight weight loss after the first week on batyl alcohol supplementation, after which the weight became normalized and started to increase weekly. In both the ApoE^" , and ApoE^" /GPxl^v" genotypes, mice fed a high fat diet without batyl alcohol supplementation had a higher body weight in the final week of the study as compared to those that received batyl alcohol supplementation; In ApoE^v" mice, there was 17.4% difference (P<0.001) in the final weight between the two dietary groups, whereas in ApoE^/GPxl^"7" mice, there was a 10.7% difference (P<0.01). Additionally, no obvious pathological changes were observed in the organs investigated due to batyl alcohol supplementation.

[0122] There was no significant alteration in the levels of plasma cholesterol and low density lipoprotein -cholesterol (LDL-C) in C57/BL6 and ApoE^" mice that were supplemented with batyl alcohol as compared to their corresponding controls {i.e. , 0% batyl alcohol groups). However, plasma cholesterol and LDL-C were reduced by 30.2% (P< 0.01) and 51.6% (P<0.001) respectively in ApoE^/GPxl^"7" mice (Table 1). The concentration of high density lipoprotein- cholesterol (HDL-C) was not significantly altered between the two dietary groups of C57/BL6 mice. The HDL-C concentration was decreased by 55.4% (P<0.001) and 67.2% (P<0.001) in ApoE^" and ApoE^/GPxl^"7" mice, respectively when they received the supplementation as compared to those that did not (Tablel). In contrast to ApoE^" and ApoE^"77GPxl^"7" mice, C57/BL6 mice had a significant increase in triglycerides, 26.8% (P<0.01) as compared to the groups that were not supplemented with batyl alcohol (Table 1). Batyl alcohol resulted in a drop in blood glucose in both the ApoE^" and ApoE^"77GPxl^"7" mice models (-34.6% and -30.2% respectively, Table 1) but had no effect on the C57/BL6 mice.

Alkyl- and Alkenylphospholipids in Plasma and Heart Homoqenates

[0123] The present inventors then investigated whether supplementation of batyl alcohol to mice over 12 weeks resulted in an increase in alkyl- and

alkenylphospholipids in plasma and cardiac tissue. Compared to the groups which did not receive batyl alcohol supplementation {i.e. , untreated), the total concentrations of alkenylphosphatidylcholine and alkenylphosphatidylethanolamine were significantly increased in plasma (Figure 2A and 2B) and hearts (Figure 3A and 3B) in C57/BL6, ApoE^" and ApoE^v7GPxl^v" mice which received the batyl alcohol supplementation.

Similarly the total concentrations of alkylphosphatidylcholine and

alkylphosphatidylethanolamine, which are precursors to alkenylphospholipids, were significantly increased in plasma (Figure 2C and 2D) and hearts (Figure 3C and 3D) of the treated groups as compared to their corresponding untreated groups. The

concentrations of circulating alkyl- and alkenylphospholipids, relative to

phosphatidylcholine in C57/BL6 mice that were not supplemented with batyl alcohol were lower as compared to that of ApoE^" and ApoE^"77GPxl^"7" mice (Table 2). In contrast, these levels were similar across the genotypes in the heart homogenates (Table 3). A greater difference in the relative concentration of the alkyl- and alkenylphospholipids was observed between the treated and untreated groups in plasma as compared to that in the heart. In plasma, the change in the levels of alkyl- and alkenylphospholipids ranged from 2.6 to 16.7 fold (Table 2), whereas in heart they ranged from 0.8 to 4.8 fold (Table 3).

[0124] Analyses of alkyl- and alkenylphospholipid species showed that supplementation of batyl alcohol increased some but not all species in plasma (Table 4) and heart (Table 5) while some species showed a lower level in mice treated with batyl alcohol. PC(P-40 : 5), PC(P-40: 6), PE(P-40 : 5), PE(P-40: 6), PC(0-36: 1), PC(0-36: 2),

PC(0-38:4), PC(O-40 : 5) and PE(O-38:4) were all at least 10-fold higher in the plasma of the batyl alcohol treated mice. These species were also elevated in the heart tissue (Table 5). While very few species were lower in plasma of the batyl alcohol treated mice, multiple species were significantly lower in the heart tissue of the batyl alcohol treated mice (Table 5), including PC(P-30:0), PC(P-32:0) PC(P-34:1), PC(P-34:2) PE(P-38:5) and PE(P-38:6) .

Assessment of Atherosclerotic Lesions

[0125] The present inventors investigated the effect of elevated production of plasma and heart alkyl- and alkenylphospholipids to the progression of atherosclerosis in the aorta and aortic sinus of the mice. ApoE^" and ApoE^"7"/GPx ^7" mice fed on high fat diet without batyl alcohol supplementation for 12 weeks developed plaques throughout the aorta (Figure 4 and 5). This plaque deposition was reduced by 70.8% (P< 0.001) and 69.1% (P<0.001) in the whole aorta of the treated ApoE^" and ApoE^"77GPxl^"7" mice, respectively (Figure 4 and 5A). Similar levels of reduction in plaque deposition were observed in the aortic arch, thoracic, and abdominal regions of the aorta (Figure 5B - 5D).

[0126] The high fat diet feeding without batyl alcohol supplementation resulted in the development of 0.38mm² and 0.41mm² lesions in the aortic sinus of ApoE^" and ApoE^"7"/GPx ^7" mice, respectively. The lesion size was reduced by 12.3% (P = 0.177) and 40.3% (P<0.01) in the treated ApoE^" and ApoE^"77GPxl^"7" mice, respectively (Figure 6).

Pro-inflammatory and Oxidative Markers in Atherosclerosis

[0127] The anti-inflammatory effect of increased heart and circulating alkyl- and alkenylphospholipids were investigated by analyzing the expression of inflammatory marker VCAM-1 in an atherosclerosis prone site, the aortic sinus. High fat diet without batyl alcohol supplementation resulted in 22.4% and 37.1% of VCAM-1 staining in the aortic sinus of ApoE^" and ApoE^"77GPx ^7" mice, respectively. The VCAM-1 expression was reduced by 28.3% (P<0.05) in the treated ApoE^"77GPxl^"7" mice, but no significant changes was observed in the treated ApoE^" mice (Figure 7). [0128] The present inventors were interested in assessing the ability of alkyl- and alkenylphospholipids to attenuate levels of oxidative stress in the aortic tissues. Cross sections of the aorta were stained for nitrotyrosine. The results showed that nitrotyrosine levels were reduced by 78.3% (P<0.001) in the treated ApoE^"77GPxl^"7" mice. No change was observed in the ApoE^" mice (Figure 8).

Discussion

[0129] Treatment with batyl alcohol reduced weight gain in ApoE^~ and ApoE^^'/GPxl^{" "} mice: Treatment of C57/BL6, ApoE^" and ApoE^"77GPxl^"7" with batyl alcohol did not have any adverse effects on the mice. It was noted that while the

C57/BL6 mice showed no difference in weight gain relative to the control diet, the ApoE^" and ApoE^"77GPxl^"7" mice had a reduced weight gain on the batyl alcohol supplemented diet (Figure 1). In the ApoE^" mice this was primarily driven by an initial drop in weight upon commencement of the batyl alcohol diet, followed by normal weight gain for 6 weeks which then leveled out earlier than the control diet fed mice. The lower body weight in the ApoE^" mice on the batyl alcohol diet was not accompanied by any difference in total cholesterol, LDL-C or triglyceride, but was accompanied by a lower HDL-C level, which was reduced to the level found in the C57/BL6 control mice (Table 1). As circulating cholesterol is one of the primary drivers of atherosclerosis and HDL-C has a negative association with atherosclerosis, these plasma lipid measures indicate that the ApoE^" mice on the batyl alcohol diet had equal, or greater risk of atherosclerosis resulting from their plasma lipids relative to the ApoE^" mice on the control diet.

[0130] Similar to the ApoE^" mice, the ApoE^"77GPxl^"7" mice on the batyl alcohol diet also showed a lower weight at the 12 week time point which was driven primarily by a flattening of weight gain in the later weeks of the trial (Figure 1). However, in contrast to the ApoE^" mice, the ApoE^"77GPxl^"7" mice also showed a lower level of total cholesterol, LDL-C and HDL-C, potentially influencing atherosclerosis risk. The

mechanism whereby batyl alcohol may reduce weight gain on a high fat diet may involve modulation of MAPK and NF-κΒ signaling pathways as has recently been demonstrated in primary adipocytes treated with alkylglycerol [14]. Associated with the smaller weight gain of the ApoE^" and ApoE^"77GPxl^"7" mice relative to the C57/BL6 was a decrease in fasting blood glucose in these models which may indicate improved insulin sensitivity in these mice.

[0131] Treatment with batyl alcohol increases alkyl- and

alkenylphospholipid in plasma and heart: The circulating levels of alkyl- and alkenylphospholipid species were higher in ApoE^" and ApoE^"77GPxl^"7" relative to C57/BL6 control mice (Figure 2, Table 2) even when normalized to total phosphatidylcholine to allow for the higher lipoprotein levels in the ApoE^" and ApoE^"77GPxl^"7" mice. This suggests a higher proportional incorporation of these lipid classes into the lipoproteins in the ApoE^" and ApoE^"77GPxl^"7" mice. Dietary supplementation with batyl alcohol increased the level of alkyl- and alkenylphospholipids in all mouse strains although the relative increase was greatest in the ApoE^" mice with increases ranging from 4- to 16.7- fold, whereas the C57/BL6 control mice showed increases in the range of 2.5- to 6.7-fold. Phosphatidylethanolamine classes typically showed greater increases than the

phosphatidylcholine classes. However, when the individual species that were elevated were examined the present inventors observed that only species containing an 18 : 0 alkyl or alkenyl chain were elevated thus PC(P-40: 5) and PC(P-40: 6) which contain primarily PC(P-18 :0/22 : 5) , PC(P-18 : 0/22: 6) showed the greatest increase in the batyl alcohol treated animals relative to the control diet (24 to 45 fold in the ApoE^" mice), while PE(P- 40: 5) and PE(P-40 : 6) also showed similar increases in those species (25 to 36 fold). However, while species containing primarily an 18: 0 alkenyl chain represent

approximately 46% of the total PE(P) class they represent only 10% of the corresponding PC(P) class. Thus, while the upregulation of PC(P) and PE(P) species is comparable, the relative proportion of the total class leads to considerable differences between the upregulation at the class level. This raises an important consideration for the therapeutic potential of alkylglycerols to regulate plasmalogen synthesis as the alkyl chain present will influence the capacity to upregulate plasmalogen synthesis. Based on these results it is clear that by selecting a suitable formulation of alkylglycerol species it will be possible to modulate the level of plasmalogen and the specific profile of plasmalogens that are produced.

[0132] The analysis of the heart tissue showed a similar trend although the overall increase in plasmalogens was not as great with a 1.4 fold increase in PC(P) and 2.6 fold increase in PE(P). These effects were also driven primarily by plasmalogen species containing the 18 :0 alkenyl chain which here also made up a larger proportion of the PE(P) (43%) compared to the PC(P) (13%).

[0133] The alkyl species closely followed the trend of the plasmalogens in plasma, whereas in heart tissue a differential increase was observed in PC(P) and PE(P) but similar increases in PC(O) and PE(O), this also appears to relate to the relative abundance of the individual molecular species containing the alkyl 18: 0 chain relative to the total alkylphospholipid pools. While in plasma these are different between the PC and PE pools, in heart they are similar, corresponding to the similar fold increase observed in PC(O) and PE(O) classes in the batyl alcohol treated animals.

[0134] Mice treated with batyl alcohol have lower levels of

atherosclerotic plaque: Significant levels of plaque formation were observed in the aorta of both the ApoE^" and ApoE^/GPxl^"7" mice with higher levels in the ApoE^" compared to the ApoE^"7"/GPx ^7" mice. This is in contrast to previous reports where the ApoE^"7"/GPxl^"7" are reported to have higher plaque levels. However this may be due to the shorter dietary period of only 12 weeks in this instance compared to 20 weeks in previous studies. [0135] Plasmalogen enrichment via supplementation of batyl alcohol significantly reduced atherosclerotic plaque formation in the aorta of both the ApoE^" and ApoE^"77GPxl^"7" mice by up to 70% (Figures 4 and 5) . This was evident in the aortic arch as well as the thoracic and abdominal aorta regions.

[0136] In contrast to the overall lower level of plaque in the aorta of both treated mouse models a significantly lower level of plaque was observed in the aortic sinus of only the batyl alcohol treated ApoE^"77GPxl^"7" mice (Figure 6) and only a slightly (non-significant) lower level in the ApoE^" mice. The ApoE^"77GPxl^"7" mice are reported to have higher levels of oxidative stress and so the lower level of plaque in the aortic sinus may reflect ability of increased plasmalogens to more effectively reduce the level of oxidative stress in this model relative to the ApoE^" model . However, the lower level of plaque in the aortic sinus may also reflect the lower level of circulating cholesterol in the batyl alcohol treated ApoE^"77GPxl^"7" mice which may lead to a lower level of plaque formation.

[0137] Associated with the lower level of plaque in the aortic sinus of the batyl alcohol treated ApoE^"77GPxl^"7" mice, decreased inflammation was observed represented by VCAM staining in the aortic sinus (Figure 7) . Here also, the present inventors did not observe a significant effect in the ApoE^" model. This suggests that plasmalogen exerted its anti-inflammatory effect more effectively in an environment where there was a higher level of oxidative stress. This decrease in the VCAM- 1 expression, combined with lower levels of LDL-C may have reduced monocyte migration and adherence to the endothelial cells of the aorta, thus in turn, reducing the progression of atherosclerosis in the ApoE^"7" /GPxl^"7" mice, compared to ApoE^" mice.

[0138] Associated with the atherosclerotic plaque accumulation in the aorta of both untreated ApoE^" and ApoE^"77GPxl^"7" mice, the present inventors observed similar levels of nitrotyrosine, a product of superoxide and nitric oxide and a cytotoxin known to affect biomolecules in the endothelium vasculature [ 15] . The levels of nitrotyrosine in ApoE^"77GPxl^"7" mice were previously reported to be higher than that of ApoE^" [2, 3] . However, the similar levels observed in this study may be due to the shorter period of the dietary treatment as compared to that of the previous study ( 12 weeks vs. 20 weeks) [3] . The levels of nitrotyrosine were significantly reduced in the ApoE^"77GPxl^"7" mice, whereas a significant effect was not observed in the ApoE^"7" mice (Figure 8) . Previous studies on diabetes mice models [2] reported that the disease resulted in a significant increase in the GPxl mRNA in the ApoE^"7" model . Similarly they observed a significant increase in the mRNA levels of the GPxl isoform, GPx3, in the ApoE^"7" model, and significant increases in mRNA levels of GPx3, GPx4, catalase, Sod l and Sod2 (cytosolic and mitochondrial isoforms of the superoxide dismutase (SOD) family) in the ApoE^"7" /GPxl^"7" model. It was proposed that the expression of other antioxida nts was a compensatory mechanism for the disease progression in both models, and/or for the loss of GPxl in the ApoE^"7"/GPxl^"7" mice. In spite of this "compensatory mechanism", the levels of peroxynitrite in the ApoE^"7" did not change following the onset of the disease, whereas that of ApoE^"7"/GPxl^"7" model was significantly increased. It was proposed that GPxl might serve as a peroxynitrite reductase [2, 16]. The production of ROS has been shown to upregulate NADPH oxidase, which in turn, increases the production of superoxide, which is a substrate of the SOD enzyme family. SOD converts superoxide to hydrogen and lipid peroxide, which then feeds to the cyclic and initial production of ROS [2].

[0139] In the present study, no change in the level of nitrotyrosine was observed in the ApoE^" model, but a significant reduction of nitrotyrosine level was observed in the ApoE^"7"/GPxl^"7" model upon batyl alcohol supplementation (Figure 8). In light of the earlier findings by Lewis et al, it is proposed that the increase in SOD observed in the ApoE^"7"/GPxl^"7" model leads to a greater cyclic production of lipid peroxides and ROS in this model which ultimately feeds back to the production of peroxynitrite and then to nitrotyrosine. Whereas in the ApoE^"7" model the production of peroxynitrite and nitrotyrosine proceeds more directly from hydrogen peroxide and nitrite. When plasmalogens are increased in the ApoE^"77GPxl^"7" model, this serves to stall the cyclic production of ROS via SOD and lipid peroxides thereby reducing the

peroxynitrite and nitrotyrosine production. In the ApoE^"7" model, where hydrogen peroxide is the major source of peroxynitrite and nitrotyrosine [17], the increase in plasmalogens has less effect and so a decrease in nitrotyrosine was not observed.

Summary

[0140] Alkylglycerol dietary supplementation resulted in elevated levels of alkyl- and alkenylphospholipids in plasma and heart. The increase in plasmalogens (alkenylphospholipids) was associated with attenuation in the formation of

atherosclerosis in ApoE^" and ApoE^"7"/GPxl^"7" mice with a greater effect in the latter model. The increase in alkyl- and alkenylphospholipids appears to exert multiple effects leading to the reduction in atherosclerosis, these may include; reduction of weight gain, lowering of cholesterol and LDL-C levels, lowering of inflammation and reduction of oxidative stress. The treatment appears to have no adverse effects on organs and phenotype. The present inventors propose that plasmalogen enrichment represents a viable therapeutic strategy to prevent atherosclerosis and reduce cardiovascular disease risk.

Materials and Methods

Animal Groups and Diet Study

[0141] Six-week old C57/BL6 (control), ApoE^" (ARC, WA, Australia), and ApoE^" /GPxl^" (AM REP, VIC, Australia) mice were fed with high fat diet (22% fat, 0.15% cholesterol) (Specialty Feeds, WA, Australia), containing either 0% or 2% 1-O-octadecyl- rac-glycerol (batyl alcohol, Tokyo Chemical Industry, Astral Scientific) for 12 weeks (N = 10/group) . To generate a line of ΑροΕ^Λ"/ΰΡχ mice, GPxl-deficient mice on a C57/BL6 genetic background as described previously by de Haan et a/ [ 10] were mated with C57/BL6 ApoE^"7" mice [2] . The animals were housed in standard conditions with unrestricted access to food and water at the Precinct Animal Centre of the Baker IDI Heart and Diabetes Institute. They were maintained on a 12 hr light and dark cycle in a pathogen free environment. Food intake was measured weekly by food pellet

consumption . Body weights were determined weekly. After 12 weeks, animals had food withdrawn for 3 hr and blood was collected from the tail for blood glucose measurement using an Accu-Chek blood glucose monitor. M ice were then a naesthetized by Avertin (2,2,2-tribromoethanol) IP (0.3 ml. of 2.5% solution per 20 g mouse; Sigma Chemical Co, USA), and organs were rapidly dissected . The experiment was approved and conducted in accordance to the principles devised by the Alfred Medical Research and Education Precinct (AM REP) Animal Ethic Committee (ID : 3789) under guidelines laid down by the National Health and Medical Research of Council of Australia .

Clinical Measurements

[0142] Blood was obtained via direct heart puncture during organ dissection, and was collected into EDTA tubes. Plasma was separated from the blood via

centrifugation at 1,485 x g, room temperature for 10 min, and sucrose was added (final concentration of 0.6% (v/v)) as a cryoprotectant for lipoproteins [9, 10] prior to storage at -80°C.

[0143] Plasma was thawed slowly on ice and then concentrations of total cholesterol, triglycerides, LDL-cholesterol and HDL-cholesterol were measured using commercial enzymatic kits.

Tissue Homoqenization

[0144] M ice hearts were cut into two ha lves. The bottom half was snap frozen in liquid nitrogen and homogenized in ice cold PBS pH 7.6 containing lOOuM BHT using a Polytron electric homogenizer for 10 sec and then with a mini probe homogenizer for 15 sec at amplitude 23.

Lipid Extraction

[0145] Lipids were extracted as previously described [11] . Briefly, plasma or homogenized tissue were combined with internal standards and the lipids were extracted using 20 volumes of chloroform : methanol (2 : 1) . The extracted lipids were dried under a stream of nitrogen at 40°C and subsequently reconstituted in 1 : 1 mixture of water saturated butanol and methanol containing 5 mM ammonium formate. Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry

[0146] Lipids were quantified using multiple reaction monitoring (M RM) mode on a Agilent 1200 HPLC system and QTrap 4000 triple quadrupole mass spectrometer (Applied Biosystems) using methodology similar to that described previously [ 11] . LC separation was performed on a 2.1 x 100 mm C18 Poroshell column (Agilent, USA) at 300 μΙ/min . The following gradient conditions were used : 10 % B to 100 % B over 13 min, 100 % B over 3 min, and a return 10 % B over 1 min, followed by 10 % B over 3 min . Solvents A and B consisted of watenterahydrofuran : methanol in the ratio of 60 : 20 : 20 and 5 : 75 : 20 respectively, both containing 10 mM ammonium formate.

Quantification of Atherosclerotic Plaque

[0147] The entire aorta was cleaned of peripheral fat under a dissecting microscope (Olympus SZX9, Olympus Optical, Tokyo, Japan) . The aorta was then stained with Sudan IV-Herxheimer's solution (BDH, Poole UK) and the en face technique was employed to assess the tota l and regional (arch, thoracic, and abdominal) plaque area as previously described [ 12] . Digitized photographs of the opened aortas were obtained using a dissecting microscope equipped with a digital camera (Axiocam colour camera ; Carl Zeiss, North Ryde, NSW, Australia) . Plaque area was quantified as the proportion of aortic intimal surface area occupied by red-stained plaque using Abode Photoshop v 6.0.1 (Adobe Systems, Chatswood, NSW, Australia) .

[0148] Frozen sections of aortic sinus were assessed as previously described [2,

13] . Briefly, the sections were stained with Sudan IV-Herxheimer's solution and digitized photographs were taken using a light microscope (Olympus BX-50, Olympus Optical) equipped with the digital camera . The lesion area indicated by the red-stained plaque was quantified using Image-Pro Plus v.6.0 (Media Cybernetics, Rockville, USA) . All assessment was made in a blinded manner.

Immunohistochemistry

[0149] Immunohistochemical methods were employed as previously described [ 12] . Frozen sections of aortic sinus were stained for VCAM- 1 (rat monoclonal, BD Pharmigen ; 1 : 200) . Briefly, 4 pm frozen sections were fixed with cold acetone, air dried, and then quenched in 3% H₂0₂ in Tris buffered saline (TBS, pH 7.6) to prevent endogenous peroxidase activity. Sections were then blocked using 10% rabbit serum in TBS, and incubated with primary antibodies overnight in a humidified chamber at 4°C. Sections were then incubated with biotinylated secondary antibodies anti-rat IgG (raised in rabbit, Vector Laboratories; 1 : 200) for 10 min at room temperature, followed by horseradish peroxidase-conjugated streptavidin (Vectastain Elite ABC Staining Kit, Vector Laboratories) for 30 min at room temperature. Signals were visualized with 3,3'-diamino- benzidine tetrahydrochloride/H₂0₂ (DAB; Sigma-Aldrich, USA) . Subsequently, sections were counterstained in Mayer's hematoxylin, dehydrated, and coverslipped . [0150] Paraffin sections of the aorta were stained for nitrotyrosine (rabbit polyclonal, Millipore; 1 :200). In brief, 5 pm of the aortic sections were de-waxed, hydrated, and quenched in 3% H₂0₂ in TBS (pH 7.6). The sections were blocked using 10% horse serum in TBS, and then incubated with primary antibodies overnight in a humidified chamber at 4°C. The sections were incubated with biotinylated secondary anti- rabbit IgG antibodies (raised in goat, Vector Laboratories; 1 : 500), followed by

horseradish peroxidase-conjugated streptavidin as described above. The sections were subsequently processed in the same manner as the frozen sections as described above.

Data Analysis and Statistics

[0151] Mass spectrometric data which were non-normally distributed were analysed using Mann-Whitney U test, and were adjusted for multiple comparisons using Benjamini Hochberg. P<0.05 was considered significant. Data were expressed as median (interquartile range). The lipidomic data were normalized to the relative concentration of phosphatidylcholine (PC).

[0152] Data from the atherosclerotic plaque assessment and

immunohistochemistry were analyzed using Student t-test, comparing the control and treated groups per individual genotype. P<0.05 was considered significant. Data were expressed as mean ± SEM .

EXAMPLE 2

EFFECT OF MULTIPLE ALKYLGLYCEROLS AND TREATMENT TIME OF THE PLASMA LIPID PROFILE

IN C57/BL6 MICE ON A HIGH FAT DIET.

Results

Time course study

The time course study showed higher levels of alkyl and alkenyl phospholipid subclasses in animals treated with alkylglycerols compared to the control animals treated with carrier only (Table 6, Figure 9). The level of these lipid subclasses increased over the initial 2-4 weeks of the treatment and then levelled off until 8 weeks where there was a trend to decrease up to the 12 week time point. This response profile was reflected in most individual species within these subclasses (Table 7). Overall significant increases were also seen in LPC(P) and LPE(P) subclasses, although these were not evident at all time points (Table 6).

Monocyte activation study

[0153] Mice treated with alkylglycerol for four weeks showed significantly higher levels of alkyl and alkenylphospholipids compared to the control animals (Figure 10). The mice on a high fat diet showed higher levels of both CDl lb positive Ly6C-hi and CDl lb positive Ly6C-lo monocytes. These were attenuated in the alkylglycerol treated mice (Figure 11). Discussion

[0154] These studies highlight two important aspects of alkylglycerol supplementation :

[0155] First, the present inventors have demonstrated that the use of a combination or alkylglycerol species (batyl alcohol (C18 : 0), chimyl alcohol (C 16 : 0) and selachyl alcohol ( 18 : 1) provides for the upregulation of a wide range of a lkyl and alkenyl (plasmalogen) phospholipids. This is clearly demonstrated in Table 7 where 14/17 PC(O), 7/ 12 PC(P), 9/ 10 PE(O) and 17/27 PE(P) species were significantly elevated resulting in between a 61% a nd a 173% higher level of these lipid subclasses compared to the untreated mice (Table 6) . This was achieved with a dose of alkylglycerol of only 10 mg/day per mouse.

[0156] Secondly, the present inventors have been able to demonstrate that alkylglycerol treatment is able to suppress the activation of monocytes in high fat fed mice. This suggests that one mechanism by which plasma logen upregulation may attenuate atherosclerosis onset and progression is to suppress monocyte activation and thereby limit movement of monocytes across the arterial wall into the intima .

Materials and Methods

Time Course Study:

[0157] Eight week old C57/BL6 mice (n= 8 per group) were placed on a high fat diet (22% fat, 0. 15% cholesterol, Specialty Feeds, WA, Australia) and treated with a mixture of alkylglycerols (batyl alcohol (4.0 mg), chimyl alcohol (4.0 mg) and selachyl alcohol (2.0 mg) combined with an equal weight of lecithin (phosphatidylcholine, 10 mg), prepared as a 200 μΙ_ emulsion in water. The alkylglycerols were delivered by oral gavage daily for periods of 1, 2, 4, 8 and 12 weeks. Control animals were treated with the lecithin emulsion without alkylglycerol .

[0158] The animals were housed in standard conditions with unrestricted access to food and water at the Precinct Animal Centre of the Baker IDI Heart and Diabetes Institute. They were maintained on a 12 hr light and dark cycle in a pathogen free environment. Body weights were determined weekly. After the designated time periods, animals had food withdrawn for 3 hr. and blood then anaesthetized by Avertin (2,2,2- tribromoethanol) IP (0.3 mL of 2.5% solution per 20 g mouse; Sigma Chemical Co, USA) . Blood was collected by heart puncture into EDTA tubes and plasma prepared by centrifugation at l,485xg, room temperature for 10 min. The experiment was approved and conducted in accordance to the principles devised by the Alfred Medical Research and Education Precinct (AM REP) Animal Ethic Committee (ID : 3789) under guidelines laid down by the National Health and Medical Research of Council of Australia . [0159] Plasma lipids were analysed by mass spectrometry as described in Example 1.

Monocyte Activation Study:

[0160] Eight week old C57/BL6 mice (n= 10 per group) were placed on a chow or a high fat diet (22% fat, Specialty Feeds, WA, Australia) and treated with a mixture of alkylglycerols (batyl alcohol (2.0 mg), chimyl alcohol (2.0 mg) and selachyl alcohol ( 1.0 mg) combined with an equal weight of lecithin (phosphatidylcholine, 5 mg), prepared as a 100 μΙ_ emulsion in water. The alkylglycerols were delivered by oral gavage daily for four weeks. Control animals were treated with the lecithin emulsion without alkylglycerol.

[0161] The animals were housed in standard conditions with unrestricted access to food and water at the Precinct Animal Centre of the Baker IDI Heart and Diabetes Institute. They were maintained on a 12-hr. light and dark cycle in a pathogen free environment. Body weights were determined weekly. After the designated time periods, animals had food withdrawn for 3 hr. and blood then anaesthetized by Avertin (2,2,2- tribromoethanol) IP (0.3 mL of 2.5% solution per 20 g mouse; Sigma Chemical Co, USA) . Blood was collected by heart puncture into EDTA tubes and plasma prepared by centrifugation at l,485xg, room temperature for 10 min. The experiment was approved and conducted in accordance to the principles devised by the Alfred Medical Research and Education Precinct (AM REP) Animal Ethic Committee (ID : 3789) under guidelines laid down by the National Health and Medical Resea rch of Council of Australia .

[0162] Plasma lipids were analysed by mass spectrometry as described in Example 1.

[0163] The distribution a nd reactivity of monocytes isolated from the treated mice was assessed by FACS analysis. Briefly, monocytes were identified using CD45 and CD 115, then classified as Ly6-C^hl and Ly6-C'°, activation was determined by staining for the integrin marker CD l lb.

[0164] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0165] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application .

Throughout the specification the aim has been to describe the preferred

embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims. Table 1. Plasma clinical measurements of mice on high fat diet supplemented with/without 2% batyl alcohol.

Parameter³ C57/BL6 ApoE ApoE ^■' /GPxl -'-

0% BA 2% BA 0% BA 2% BA 0% BA 2% BA

Cholesterol 4.19 ± 0.31 4.05 ± 0.11 31.56 ± 3.92 38.00 ± 3.21 38.88 ± 2.47 27.13 ± 1.96

LDL-C 0.64 ± 0.07 0.49 ± 0.03 32.41 ± 3.98 26.28 ± 1.99 36.51 ± 1.87 17.66 ± 1.55

HDL-C 4.34 ± 0.32 4.33 ± 0.1 9.45 ± 0.70 4.21 ± 0.26 10.12 ± 1.12 3.32 ± 0.2

Triglyceride 0.41 ± 0.02 0.52 ± 0.03 1.36 ± 0.28 1.62 ± 0.17 1.97 ± 0.19 1.48 ± 0.19

Fasting blood 11.18 ± 0.76 11.78 ± 0.58 10.94 ± 0.92 7.15 ± 0.28 10.36 ± 1.17 7.23 ± 0.68

glucose

a parameters are expressed in mmol/L. Data represent mean ± SEM . The two dietary groups (0% and 2% batyl alcohol) were analysed using Student t- the values in bold indicate statistical significance (P<0.05) .

Table 2. Changes in total concentration of alkenyl- and alkylphospholipids in plasma of mice on high fat diet supplemented with/without batyl alcohol.

Lipid species³ C57/BL6 ApoE^" ApoE ^/GPxl^"

Median Fold difference with Median concentration Fold difference with Median Fold difference with concentration for 2% BA group^c for 0% BA group 2% BA group^c concentration for 2% BA group^c

0% BA group (pmol/pmol PC) 0% BA group

(pmol/μιηοΙ PC) (pmol/pmol PC)

Total PC(P) 6465 3.11 14754 3.98 14350 4.30

Total PC(O) 10559 2.63 45030 4.98 43478 5.93

Total PE(P) 16540 6.00 33990 16.18 37790 16.65

Total PE(O) 1485 6.71 4796 12.40 4504 14.25 ^a PC(P) - alkenylphosphatidylcholine, PC(O) - alkylphosphatidylcholine, PE(P) - alkenylphosphatidylethanolamine, PE(O) - alkylphosphatidylethanolamine ^bMedian concentration of lipids in mice which were on high fat diet supplemented with 0% batyl alcohol.

cFold difference of median concentration of lipids in mice fed with high fat diet containing 2% batyl alcohol as compared to those fed with the diet contai 0% batyl alcohol. Data were analyzed using Whitney U test, corrected for multiple comparisons by Benjamini Hochberg. Values in bold indicate statistical significance (P<0.05).

Table 3. Changes in total concentration of alkenyl- and alkylphospholipids in heart homogenates of mice on high fat diet supplemented with/without batyl alcohol.

Lipid species³ C57/BL6 ApoE -/- ApoE^"/ /GPxl -'-

Median Fold difference with Median Fold difference with Median Fold difference with concentration for 2% BA group^c concentration for 2% BA group^c concentration for 2% BA group^c

0% BA group 0% BA group 0% BA group

(pmol/μιηοΙ PC) (pmol/pmol PC) (pmol/pmol PC)

Total PC(P) 31913 0.77 26167 1.41 28944 1.25

Total PC(O) 12277 2.41 20483 4.33 20250 4.84

Total PE(P) 146330 2.03 172510 2.55 175310 2.94

Total PE(O) 4965 2.78 6880 3.28 6592 3.93 ^a PC(P) - alkenylphosphatidylcholine, PC(O) - alkylphosphatidylcholine, PE(P) - alkenylphosphatidylethanolamine, PE(O) - alkylphosphatidylethanolamine ^bMedian concentration of lipids in mice which were on high fat diet supplemented with 0% batyl alcohol.

cFold difference of median concentration of lipids in mice fed with high fat diet containing 2% batyl alcohol as compared to those fed with the diet contai 0% batyl alcohol. Data were analyzed using Whitney U test, corrected for multiple comparisons by Benjamini Hochberg. Values in bold indicate statistical significance (P<0.05).

Table 4. Changes in concentration of alkenyl- and alkylphospholipids in plasma of mice on high fat diet supplemented with/without batyl alcohol.

Lipid species³ C57/BL6 ApoE -/- ApoE ' /GPxl^■'^■

Median Fold difference with Median Fold difference with Median concentration Fold difference with concentration for 0% 2% BA group^c concentration for 2% BA group^c for 0% BA group 2% BA group^c BA group 0% BA group (pmol/pmol PC)

(pmol/μιηοΙ PC) (pmol/pmol PC)

PC(P-30:0) 298 0.90 1835 1.67 2178 1.23

PC(P-32:0) 89 0.52 881 0.57 799 0.57

PC(P-32: 1) 50 0.86 268 0.88 220 1.14

PC(P-34: 1) 146 1.24 670 1.15 616 1.32

PC(P-34:2) 137 1.12 560 1.01 442 1.77

PC(P-34:3) 14 1.07 76 0.83 66 1.05

PC(P-36:2) 146 2.57 176 6.20 169 6.33

PC(P-36:4) 3390 1.12 3869 1.15 3652 1.26

PC(P-36:5) 61 1.23 211 1.20 219 1.21

PC(P-38:4) 0 n/a 71 37.11 82 27.38

PC(P-38:5) 1517 1.02 4069 1.68 3982 1.80

PC(P-38:6) 220 0.91 558 1.15 558 1.30

PC(P-40:5) 254 36.87 1093 24.47 1079 27.22

PC(P-40:6) 78 50.06 236 44.61 243 40.97

PE(P-34: 1) 250 1.76 1230 1.33 1180 1.42

PE(P-34:2) 0 n/a 300 0.53 270 1.26

PE(P-36: 1) 170 3.94 1240 3.49 1010 4.10

PE(P-36:2) 150 4.53 840 2.13 480 4.23

PE(P-36:4) 390 0.62 600 1.02 680 1.07

PE(P-38:4) 230 7.22 640 11.53 800 8.14

PE(P-38:5) 5750 0.77 10490 1.80 10850 2.16

PE(P-38:6) 1320 0.70 2400 1.23 2660 1.11

PE(P-40:4) 540 2.50 1780 6.56 1580 7.82

Lipid species³ C57/BL6 ApoE ApoE' /GPxl^■'^■

Median Fold difference with Median Fold difference with Median concentration Fold difference with concentration for 0% 2% BA group^c concentration for 2% BA group^c for 0% BA group 2% BA group^c BA group 0% BA group (pmol/pmol PC)

(pmol/pmol PC) (pmol/pmol PC)

PE(P-40:5) 7230 10.48 12340 36.62 16130 32.55

PE(P-40:6) 670 19.15 1740 25.83 2220 19.27

PC(O-32:0) 506 1.19 5222 1.17 4645 1.47

PC(0-32:1) 290 1.18 1605 1.71 1625 1.93

PC(0-32:2) 22 1.32 115 1.43 101 1.60

PC(0-34:1) 1680 1.60 10165 1.49 10139 1.76

PC(0-34:2) 417 1.47 2340 1.49 1847 2.12

PC(0-34:3) 44 1.41 200 1.81 171 2.22

PC(0-34:4) 39 0.87 109 1.24 138 0.89

PC(0-35:4) 98 1.39 338 1.02 257 1.37

PC(O-36:0) 14 6.50 533 2.66 389 3.58

PC(0-36:1) 222 29.43 2112 32.54 1967 38.52

PC(0-36:2) 127 23.50 1807 13.57 1686 17.32

PC(0-36:3) 1983 1.31 3722 2.26 3847 2.27

PC(0-36:4) 1633 1.21 6895 1.59 6457 1.75

PC(0-36:5) 412 0.78 1179 0.92 1148 0.91

PC(0-38:4) 344 13.73 1737 32.32 1963 30.88

PC(0-38:5) 1400 1.03 4068 1.40 4160 1.45

PC(O-40:5) 211 11.53 986 21.42 999 25.35

PC(O-40:6) 220 0.45 613 1.77 598 1.94

PC(O-40:7) 840 0.94 1382 1.85 1574 1.51

PE(0-34:1) 207 3.89 1111 4.08 944 6.04

PE(0-34:2) 45 1.29 193 1.63 117 3.95

PE(0-36:2) 184 8.09 808 13.89 674 18.29

PE(0-36:3) 41 7.83 131 16.95 94 24.73

Lipid species³ C57/BL6 ApoE -/- ApoE ' /GPxl^■'^■

Median Fold difference with Median Fold difference with Median concentration Fold difference with concentration for 0% 2% BA group^c concentration for 2% BA group^c for 0% BA group 2% BA group^c

BA group 0% BA group (pmol/pmol PC)

(pmol/μιηοΙ PC) (pmol/pmol PC)

PE(0-36:4) 80 1.44 145 3.80 150 4.37

PE(0-36:5) 25 2.24 51 5.31 61 6.26

PE(0-38:4) 298 13.23 570 38.33 605 33.91

PE(0-38:5) 334 6.17 979 12.80 911 13.33

PE(O-40:5) 181 5.75 678 9.06 610 11.62

PE(O-40:6) 136 1.17 43 0.33 41 0.56

PE(O-40:7) 62 1.74 104 3.38 139 1.94 ^a PC(P) - alkenylphosphatidylcholine, PC(O) - alkylphosphatidylcholine, PE(P) - alkenylphosphatidylethanolamine, PE(O) - alkylphosphatidylethanolamine ^bMedian concentration of lipids in mice which were on high fat diet supplemented with 0% batyl alcohol.

cFold difference of median concentration of lipids in mice fed with high fat diet containing 2% batyl alcohol as compared to those fed with the diet contai 0% batyl alcohol. Data were analyzed using Whitney U test, corrected for multiple comparisons by Benjamini Hochberg. Values in bold indicate stati significance (P<0.05).

Table 5. Changes in concentration of alkenyl- and alkylphospholipids in heart homogenates of mice on high fat diet supplemented with/without batyl alcohol.

Lipid C57/BL6 ApoE -/- ApoE ' /GPxl^■'^■ species³

Median concentration Fold difference with Median Fold difference with Median concentration Fold difference with 2% for 0% BA group 2% BA group^c concentration for 2% BA group^c for 0% BA group BA group^c

(pmol/pmol PC) 0% BA group (pmol/pmol PC)

(pmol/pmol PC)

PC(P-30:0) 49 0.45 82 79 0.10

0.13

PC(P-32:0) 240 0.38 282 322 0.24

0.28

PC(P-32:1) 66 0.58 85 86 0.59

0.61

PC(P-34:1) 851 0.50 709 707 0.46

0.46

PC(P-34:2) 401 0.56 468 453 0.44

0.37

PC(P-34:3) 17 0.71 24 24 0.50

0.54

PC(P-36:2) 131 1.15 199 258 1.45

1.77

PC(P-36:4) 6015 0.79 5884 6608 0.70

0.73

PC(P-36:5) 131 0.74 133 172 0.40

0.44

PC(P-38:4) 975 1.93 1265 1260 2.71

2.62

PC(P-38:5) 11446 0.57 8700 9628 0.50

0.60

PC(P-38:6) 9772 0.36 6314 6596 0.29

0.36

PC(P-40:5) 857 5.47 1 157 1257 13.06

13.29

PC(P-40:6) 807 2.77 704 878 4.77

6.82

PE(P-34:1) 900 0.71 980 0.67 880 1.00

PE(P-34:2) 770 1.09 1010 0.00 900 0.00

PE(P-36:1) 530 13.64 1650 12.47 1690 15.65

PE(P-36:2) 870 3.51 1520 3.12 1430 4.26

PE(P-36:4) 6240 0.75 6590 0.55 8000 0.54

PE(P-38:4) 4270 4.32 5070 6.21 7120 5.24

PE(P-38:5) 37390 0.74 40740 0.48 42120 0.57

PE(P-38:6) 53480 0.63 47290 0.45 49340 0.39

Lipid C57/BL6 ApoE -/- ApoE ' /GPxl^■'^■ species³

Median concentration Fold difference with Median Fold difference with Median concentration Fold difference with 2% for 0% BA group 2% BA group^c concentration for 2% BA group^c for 0% BA group BA group^c

(pmol/pmol PC) 0% BA group (pmol/pmol PC)

(pmol/pmol PC)

PE(P-40:4) 7270 6.18 11560 5.75 12160 6.68

PE(P-40:5) 15930 4.90 24990 6.09 26370 7.06

PE(P-40:6) 22020 3.26 23560 5.38 27280 4.42

PC(O-32:0) 649 2.21 2845 0.67 2382 0.92

PC(0-32:1) 209 2.64 938 1.46 840 2.12

PC(0-32:2) 13 1.46 47 0.91 33 1.42

PC(0-34:1) 1 105 2.85 2768 2.51 2544 3.34

PC(0-34:2) 21 1 1.78 657 1.30 569 1.91

PC(0-34:3) 18 2.44 81 1.42 75 1.79

PC(0-34:4) 15 0.80 24 1.00 23 1.04

PC(0-35:4) 19 0.79 26 0.96 32 0.66

PC(O-36:0) 289 2.47 387 5.86 388 6.36

PC(0-36:1) 126 33.23 828 22.24 706 26.99

PC(0-36:2) 133 11.79 438 14.46 342 21.96

PC(0-36:3) 456 1.26 568 1.03 611 1.20

PC(0-36:4) 1502 0.91 2329 1.04 2712 0.93

PC(0-36:5) 122 1.36 441 0.86 465 0.79

PC(0-38:4) 314 10.53 695 24.30 991 17.16

PC(0-38:5) 4381 0.98 4530 1.59 5027 1.51

PC(O-40:5) 263 8.93 520 15.30 597 14.68

PC(O-40:6) 857 5.47 1 157 13.29 1257 13.06

PC(O-40:7) 1311 0.65 792 1.28 1104 0.76

PE(0-34:1) 99 3.14 219 2.29 210 3.29

PE(0-34:2) 35 1.80 97 0.55 66 1.21

PE(0-36:2) 37 3.03 63 3.35 59 0.00

Lipid C57/BL6 ApoE -/- ApoE ' /GPxl^■'^■

species³

Median concentration Fold difference with Median Fold difference with Median concentration Fold difference with 2%

for 0% BA group 2% BA group^c concentration for 2% BA group^c for 0% BA group BA group^c

(pmol/μιηοΙ PC) 0% BA group (pmol/pmol PC)

(pmol/pmol PC)

PE(0-36:3) 23 6.83 54 5.76 38 11.45

PE(0-36:4) 251 1.15 483 0.74 447 1.04

PE(0-36:5) 37 1.95 154 0.48 103 0.93

PE(0-36:6) 56 0.68 63 0.46 75 0.37

PE(0-38:4) 97 46.72 659 14.71 544 20.67

PE(0-38:5) 1681 1.24 2182 1.61 2281 1.93

PE(O-40:5) 712 6.31 1202 5.48 1244 6.46

PE(O-40:6) 1 199 0.80 1 138 0.53 1103 0.67

PE(O-40:7) 647 0.80 480 0.67 584 0.54 ^a PC(P) - alkenylphosphatidylcholine, PC(O) - alkylphosphatidylcholine, PE(P) - alkenylphosphatidylethanolamine, PE(O) - alkylphosphatidylethanolamine ^bMedian concentration of lipids in mice which were on high fat diet supplemented with 0% batyl alcohol.

cFold difference of median concentration of lipids in mice fed with high fat diet containing 2% batyl alcohol as compared to those fed with the diet contai 0% batyl alcohol. Data were analyzed using Whitney U test, corrected for multiple comparisons by Benjamini Hochberg. Values in bold indicate statis significance (P<0.05).

Table 6. Plasma lipid classes and subclasses in mice treated with alkylglycerols.

Control Alkylglycerol % % % % % %

Predictor (pmol/mL) (pmol/mL) difference p-value difference p-value difference p-value difference p-value difference p-value difference

Total d Cer 167 187 -11 6.22E-01 16 3.11E-01 15 6.72E-01 -4 9.58E-01 -17 8.35E-01 -39

Total Cer 6515 7411 -12 5.81E-01 3 7.26E-01 2 9.26E-01 4 8.54E-01 -13 8.86E-01 -37

Total MHC 2₄391 25227 -3 8.12E-01 -5 7.03E-01 -15 6.61E-01 6 8.31E-01 7 8.64E-01 -13

Total DHC 654 699 -6 5.81E-01 -5 5.54E-01 -7 6.74E-01 -4 8.31E-01 -2 9.47E-01 -14

Total THC 78 74 6 5.81E-01 10 4.02E-01 30 1.50E-01 -10 8.31E-01 13 4.62E-01 -10

Total GM3 193 187 4 6.46E-01 7 3.95E-01 1 9.78E-01 0 9.58E-01 20 1.40E-01 -10

Total SM 63065 60203 5 6.22E-01 8 4.02E-01 6 8.07E-01 9 4.38E-01 14 4.62E-01 -14

Total PC 1925208 1977221 -3 8.12E-01 -5 5.85E-01 -2 9.74E-01 9 4.94E-01 10 8.13E-01 -22

Total PC(O) 23796 14486 64 4 S-ii- ! O 14 7.90E-02 50 83 106 !il!!!l 59

Total PC(P) 7614 5187 47 mmmm 12 2.21E-01 25 1.82E-01 61 83 48

Total LPC [snl] 318146 337770 -6 5.31E-01 -4 6.00E-01 -8 6.88E-01 -1 9.58E-01 -4 8.98E-01 -14

Total LPC [sn2] 95321 100120 -5 6.22E-01 -2 8.31E-01 -8 7.87E-01 15 2.39E-01 -4 9.47E-01 -24

Total LPC(O) 5603 5392 4 1.44E-01 0 8.90E-01 4 6.61E-01 6 2.21E-01 7 3.77E-01 1

Total LPC(P) 42 36 16 5 3.18E-01 15 1.82E-01 13 2.92E-01 32 .-. . ^: F. - \ 16

Total PE 9736 10064 -3 8.12E-01 -7 5.07E-01 -3 9.55E-01 0 9.58E-01 42 -35

Total PE(O) 1960 897 119 4 40ί:· : ;. 45 ii!!ii 128 ■ 20 .- > 2 173 ; .'.-.?;·.· C 126 i!!I!I 99

Total PE(P) 15330 9184 67 mmmm 38 63 7.17E-02 87 mmmm 92 50

Total LPE [snl] 15953 16398 -3 6.22E-01 -2 6.96E-01 -10 6.61E-01 0 9.98E-01 9 8.13E-01 -9

Total LPE [sn2] 8250 7879 5 7.10E-01 13 2.86E-01 -7 8.07E-01 36 1.33E-01 6 8.86E-01 -17

Total LPE(P) 355 309 15 l!!iii 21 :¾:;. 12 2.63E-01 19 18 5

Total PI 91558 92489 -1 8.12E-01 -1 9.34E-01 -7 7.57E-01 4 8.31E-01 11 8.13E-01 -13

Total LPI [snl] 1508 1606 -6 5.81E-01 -2 8.15E-01 -13 6.61E-01 -5 8.54E-01 2 8.86E-01 -14

Total LPI [sn2] 413 382 8 6.22E-01 34 1 9.78E-01 48 1.73E-01 -9 8.64E-01 -22

Total PS 253 261 -3 9.42E-01 144 1 8.62E-01 80 2.21E-01 -22 3.77E-01 -75

Total COH 1382351 1471693 -6 6.22E-01 -11 3.11E-01 -7 8.07E-01 13 4.96E-01 6 8.86E-01 -26

Total CE 3168567 3053556 4 6.22E-01 4 6.07E-01 -3 9.46E-01 3 8.47E-01 14 3.77E-01 1

Total DG 16429 19560 -16 6.22E-01 4 7.23E-01 2 9.74E-01 7 8.54E-01 3 8.86E-01 -57

Total TG 129979 172657 -25 5.81E-01 -22 2.99E-01 6 9.74E-01 -34 4.38E-01 -11 8.98E-01 -52

Total oxPC 19 19 0 9.42E-01 5 8.38E-01 35 4.30E-01 28 5.69E-01 2 8.98E-01 -46

¹ All time points represent the average of all measurements across the 12 weeks study.

² Represents the % difference between the control and alkylglycerol treated mice at each time point, p values were calculated by Stud t-test of log transformed values and have been corrected for multiple comparison by the method of Benjamini Hochberg.

Table 7. Plasma lipid species in mice treated with alkylglycerols.

all time points¹ all time points¹ 1 week² 2 weeks² 4 weeks² 8 weeks² 12 wee

Control Alkylglycerol % % % % % %

Predictor (pmol/mL) (pmol/mL) difference p-value difference p-value difference p-value difference p-value difference p-value difference

Cer(dl8 0/22:0) 65 54 20 8.38E-01 8 5.71E-01 35 7.77E-01 -4 9.53E-01 -2 9.81E-01 -35

Cer(dl8 0/24:0) 47 41 13 9.99E-01 41 7.53E-02 46 7.86E-01 0 9.08E-01 -36 2.32E-01 -31

Cer(dl8 0/24:1) 55 68 -19 3.65E-01 2 8.88E-01 -2 9.87E-01 -6 9.08E-01 -13 8.70E-01 -47

Cer(dl8 1/16:0) 4186 4820 -13 6.52E-01 4 7.47E-01 0 9.87E-01 10 7.13E-01 -15 9.32E-01 -40

Cer(dl8 1/18:0) 35 32 9 9.77E-01 9 8.83E-01 80 9.29E-01 5 5.20E-01 -7 9.81E-01 -38

Cer(dl8 1/20:0) 156 164 -5 8.38E-01 -3 7.17E-01 -5 8.97E-01 14 3.20E-01 -5 9.81E-01 -16

Cer(dl8 1/22:0) 733 784 -7 8.37E-01 6 6.96E-01 11 9.16E-01 4 8.05E-01 -6 9.49E-01 -34

Cer(dl8 1/24:0) 562 651 -14 1.02E-01 -3 7.53E-01 10 9.56E-01 -19 '-.βζ^'α- ^' -15 4.62E-01 -38

Cer(dl8 1/24:1) 836 946 -12 3.47E-01 0 9.38E-01 -2 9.87E-01 -8 5.63E-01 -8 9.18E-01 -30

HexCer(dl8 1/16:0) 2214 2380 -7 7.08E-01 -4 7.02E-01 -16 3.17E-01 2 8.79E-01 -3 9.81E-01 -15

HexCer(dl8 1/18:0) 369 386 -5 8.86E-01 -3 7.76E-01 -19 1.73E-01 1 9.08E-01 11 6.86E-01 -12

HexCer(dl8 1/20:0) 1727 1600 8 6.40E-01 -2 9.37E-01 -9 9.80E-01 33 3.41E-01 17 6.46E-01 -3

HexCer(dl8 1/22:0) 10007 9907 1 9.86E-01 -6 7.84E-01 -12 9.84E-01 15 5.35E-01 13 8.13E-01 -11

HexCer(dl8 1/24:0) 2830 3027 -6 5.92E-01 -3 8.07E-01 -16 6.97E-01 -7 8.46E-01 6 8.13E-01 -15

HexCer(dl8 1/24:1) 7243 7928 -9 6.06E-01 -7 5.46E-01 -19 2.41E-01 -4 9.08E-01 1 9.36E-01 -16

Hex2Cer(dl8 1/16:0) 186 94 98 9.37E-01 -1 9.05E-01 -4 9.87E-01 -8 5.20E-01 56 9.97E-01 -15

Hex2Cer(dl8 1/22:0) 50 52 -3 7.52E-01 4 6.90E-01 -2 9.87E-01 10 4.76E-01 7 8.13E-01 -32

Hex2Cer(dl8 1/24:0) 295 327 -10 3.65E-01 -8 3.80E-01 -16 1.80E-01 -6 7.28E-01 -6 9.59E-01 -15

00 Hex2Cer(dl8 1/24:1) 155 156 -1 9.99E-01 -6 6.29E-01 5 9.60E-01 -1 8.63E-01 10 7.50E-01 -13

Hex3Cer(dl8 1/16:0) 78 74 6 5.41E-01 10 4.02E-01 30 1.68E-01 -10 7.41E-01 13 5.45E-01 -10

GM3(dl8:l/16:0) 125 120 4 7.36E-01 14 9.78E-02 0 9.87E-01 -4 6.93E-01 15 3.48E-01 -5

GM3(dl8:l/18:0) 69 67 3 8.38E-01 -4 7.20E-01 2 9.80E-01 8 3.96E-01 29 1.93E-01 -21

SM(31:1) 93 94 -2 9.03E-01 -5 5.16E-01 -8 8.31E-01 4 7.11E-01 7 7.09E-01 -12

SM(32:0) 39 39 1 9.09E-01 9 3.22E-01 -5 9.80E-01 5 7.12E-01 2 9.27E-01 -10

SM(32:1) 1652 1641 1 9.86E-01 2 8.60E-01 -5 9.80E-01 9 4.06E-01 8 6.94E-01 -15

SM(32:2) 252 253 0 9.99E-01 -3 8.12E-01 -12 8.31E-01 9 5.20E-01 10 6.29E-01 -13

SM(33:1) 1939 1937 0 9.77E-01 2 7.94E-01 0 9.87E-01 14 2.78E-01 3 8.70E-01 -18

SM(34:0) 1054 1023 3 6.96E-01 14 .ίΟίν .; 9 7.67E-01 4 7.04E-01 7 7.26E-01 -18

SM(34:1) 28424 27378 4 6.59E-01 10 2.37E-01 3 9.80E-01 6 5.79E-01 12 6.29E-01 -13

SM(34:2) 6381 6414 -1 9.62E-01 3 7.89E-01 -3 9.87E-01 1 9.08E-01 12 5.78E-01 -17

SM(35:1) 463 447 4 7.00E-01 4 6.63E-01 6 8.79E-01 6 5.38E-01 13 5.24E-01 -12

SM(35:2) 140 134 5 7.13E-01 2 8.80E-01 -2 9.87E-01 10 3.52E-01 13 4.19E-01 -6

SM(36:1) 2949 2849 4 7.49E-01 13 2.27E-01 4 9.87E-01 5 6.82E-01 12 6.73E-01 -15

SM(36:2) 1609 1569 3 8.38E-01 6 5.88E-01 0 l.OOE+00 6 5.95E-01 15 4.62E-01 -16

SM(36:3) 370 356 4 7.94E-01 1 9.20E-01 -2 9.87E-01 12 2.94E-01 20 1.91E-01 -15

SM(38:1) 3508 3219 9 3.81E-01 0 9.99E-01 17 8.14E-01 23 1.49E-01 21 4.40E-01 -13

all time points' all time points' 1 week² 2 weeks² 4 weeks² 8 weeks² 12 wee

Control Alkylglycerol % % % % % %

Predictor (pmol/mL) (pmol/mL) difference p-value difference p-value difference p-value difference p-value difference p-value difference

SM(38:2) 1268 1186 7 5.78E-01 -1 9.45E-01 17 8.28E-01 19 2.81E-01 17 4.62E-01 -16

SM(39:1) 2466 1724 43 13 5.06E-01 55 4.23E-01 61 ^■ 04?:· 02 68 14

SM(41:1) 3651 3323 10 3.65E-01 9 5.27E-01 12 9.32E-01 19 2.59E-01 21 4.26E-01 -13

SM(41:2) 2209 2036 8 3.77E-01 10 3.66E-01 9 8.05E-01 16 1.83E-01 18 1.92E-01 -14

SM(42:1) 4599 4580 0 9.69E-01 11 5.43E-01 6 9.87E-01 0 9.08E-01 11 7.75E-01 -27

PC(28:0) 123 142 -13 2 οβίΓ ο:· -11 1.42E-01 -9 8.31E-01 -13 2.59E-01 -1 9.88E-01 -32

PC(30:0) 1775 2081 -15 mm M -3 6.32E-01 -12 4.66E-01 -16 7.85E-02 -4 8.70E-01 -36

PC(31:0) 1027 1156 -11 1.64E-01 5 6.83E-01 -8 7.86E-01 -16 7.28E-02 2 9.36E-01 -33

PC(31:1) 574 673 -15 4.28E-01 -19 1.09E-01 -1 9.87E-01 -6 8.22E-01 14 7.09E-01 -48

PC(32:0) 7436 8289 -10 1.92E-01 3 6.86E-01 -10 5.67E-01 -2 9.08E-01 1 9.49E-01 -35

PC(32:1) 35842 41945 -15 4.53E-01 -21 6.74E-02 -9 9.87E-01 2 8.11E-01 13 7.79E-01 -43

PC(32:2) 4322 4794 -10 7.11E-01 -14 1.55E-01 -1 9.91E-01 7 5.92E-01 24 5.96E-01 -46

PC(33:0) 1650 1654 0 9.99E-01 3 7.43E-01 -4 9.80E-01 2 7.66E-01 19 2.75E-01 -19

PC(33:1) 14166 15500 -9 5.39E-01 -12 2.61E-01 -3 9.97E-01 -5 7.47E-01 10 7.75E-01 -30

PC(33:2) 3102 3487 -11 3.65E-01 -17 1.42E-01 5 9.87E-01 -8 6.02E-01 15 7.02E-01 -41

PC(34:0) 3160 3590 -12 2.28E-01 8 5.43E-01 -12 7.67E-01 -5 6.95E-01 -9 8.63E-01 -33

PC(34:1) 411941 424105 -3 8.93E-01 -6 4.86E-01 -2 l.OOE+00 8 5.20E-01 10 7.50E-01 -21

PC(34:2) 320709 328853 -2 8.86E-01 -4 7.44E-01 7 9.80E-01 5 6.52E-01 17 7.09E-01 -30

PC(34:3) 23713 25941 -9 5.68E-01 -10 3.34E-01 0 9.87E-01 2 7.93E-01 12 7.50E-01 -38

PC(34:4) 738 787 -6 5.69E-01 -7 3.69E-01 -10 8.39E-01 2 7.55E-01 16 6.29E-01 -28

PC(34:5) 115 122 -6 8.38E-01 -7 6.27E-01 -9 9.87E-01 0 9.08E-01 25 5.06E-01 -36

PC(35:1) 20178 21723 -7 5.56E-01 -5 4.65E-01 -5 9.87E-01 -5 7.00E-01 1 9.32E-01 -22

PC(35:2) 17236 17839 -3 7.94E-01 -6 4.11E-01 1 9.87E-01 5 5.95E-01 7 7.75E-01 -24

PC(35:3) 2910 3057 -5 6.56E-01 -8 3.80E-01 1 9.87E-01 -1 9.67E-01 13 6.31E-01 -28

PC(35:4) 705 781 -10 2.98E-01 -16 1.54E-01 -8 8.24E-01 8 5.20E-01 8 7.50E-01 -32

PC(35:5) 239 256 -7 7.65E-01 -8 6.10E-01 -3 9.87E-01 -2 9.84E-01 22 5.78E-01 -38

PC(36:0) 568 610 -7 6.59E-01 14 4.23E-01 -5 9.87E-01 1 9.08E-01 -12 8.70E-01 -27

PC(36:1) 151084 162213 -7 7.08E-01 0 9.87E-01 -5 9.87E-01 0 9.10E-01 -9 9.38E-01 -19

PC(36:2) 183803 188679 -3 8.38E-01 0 9.90E-01 8 8.96E-01 4 6.59E-01 0 9.91E-01 -23

PC(36:3) 163569 163567 0 9.86E-01 -3 6.75E-01 -3 9.87E-01 14 2.58E-01 12 7.21E-01 -19

PC(18:1_18:3) 10245 10586 -3 8.38E-01 -7 5.71E-01 -2 9.96E-01 4 7.66E-01 16 7.30E-01 -26

PC(16:0_20:4) 86574 90590 -4 7.94E-01 -10 3.60E-01 -13 5.36E-01 10 4.79E-01 20 5.79E-01 -22

PC(36:5) 32282 32831 -2 9.99E-01 -5 7.15E-01 -7 9.87E-01 12 4.79E-01 20 6.29E-01 -26

PC(36:6) 2046 2077 -2 9.60E-01 -8 5.38E-01 -13 8.79E-01 7 6.02E-01 29 4.27E-01 -24

PC(37:4) 4647 4722 -2 8.93E-01 -4 6.15E-01 -11 5.36E-01 10 4.00E-01 12 5.78E-01 -15

PC(37:6) 1510 1514 0 9.77E-01 -15 2.25E-01 -4 9.80E-01 2 8.22E-01 29 2.39E-01 -11

PC(38:2) 4504 4536 -1 9.85E-01 7 7.44E-01 -20 9.80E-01 -10 8.25E-01 47 4.00E-01 -21

PC(38:3) 30646 30067 2 8.14E-01 -2 8.36E-01 -1 9.87E-01 27 1.83E-01 7 8.13E-01 -17

all time points¹ all time points¹ 1 week² 2 weeks² 4 weeks² 8 weeks² 12 wee

Control Alkylglycerol % % % % % %

Predictor (pmol/mL) (pmol/mL) difference p-value difference p-value difference p-value difference p-value difference p-value difference

PC(38 4) 44892 47275 -5 8.14E-01 -3 7.82E-01 -12 5.67E-01 14 3.59E-01 7 8.13E-01 -23

PC(38 5) 52130 52546 -1 9.86E-01 -5 6.57E-01 -13 7.12E-01 14 3.51E-01 15 6.39E-01 -16

PC(18 2_20:4) 11954 11880 1 9.99E-01 -12 3.51E-01 -17 7.99E-01 11 5.20E-01 27 4.08E-01 -13

PC(16 0_22:6) 162977 160308 2 9.37E-01 -10 3.88E-01 -8 8.31E-01 17 4.26E-01 19 6.29E-01 -9

PC(38 7) 8662 8165 6 8.05E-01 -13 3.30E-01 -15 6.91E-01 23 2.94E-01 35 3.28E-01 -6

PC(39 5) 268 428 -37 5.68E-01 1 9.60E-01 105 7.06E-01 1 9.08E-01 8 7.94E-01 -11

PC(39 6) 6350 6272 1 9.86E-01 -11 3.00E-01 -15 5.79E-01 3 9.08E-01 20 3.34E-01 1

PC(40 4) 1445 1473 -2 9.85E-01 -3 8.45E-01 -27 5.36E-01 36 1.61E-01 -2 9.81E-01 -13

PC(40 5) 4174 4238 -2 9.60E-01 -10 4.77E-01 -13 8.12E-01 7 6.75E-01 15 7.14E-01 -12

PC(40 6) 44256 43448 2 8.38E-01 -5 7.38E-01 -4 9.80E-01 21 3.61E-01 10 7.75E-01 -11

PC(40 7) 39963 37670 6 7.94E-01 -11 2.67E-01 -17 2.72E-01 26 2.94E-01 24 4.62E-01 -2

PC(40 8) 4846 4753 2 9.60E-01 -10 3.56E 01 -12 7.06E-01 14 4.47E-01 16 5.78E-01 -4

PC(O-32:0) 1876 952 97 mmmm 40 w 77 mmmm 115 mmmm 161 88

PC(0-32:1) 946 483 96 mm 34 74 m mwm 113 mmmm 139 ^■ .¾2ϊ.-¾ 85

PC(0-34:1) 5061 2692 88 IliSilil 29 mmmm 74 mmm 113 I1111SI 130 mmmm 76

PC(0-34:2) 1635 572 186 :.!¾?;· 5.3 42 W 182 W W % 214 3 ;.ίί:·0ί 245 mmmm 200

PC(0-34:4) 59 59 -1 9.17E-01 -1 9.04E-01 -15 3.99E-01 7 3.59E-01 5 6.73E-01 0

PC(0-35:4) 117 120 -3 6.36E-01 -13 •i.6i:.^:- Z -22 1.26E-01 8 5.95E-01 22 l.llE-01 -11

PC(O-36:0) 38 24 59 50 j.. ,^':r;-03 50 m mm 80 66 mmmm 43

PC(0-36:1) 1103 387 185 65 ^■ .8^'??:· 03 157 mmmm, 218 mmmm 263 ^■■i i m ? 185

PC(0-36:2) 871 152 472 mmmm 80 278 m 273 i.oi m 368 ^■ .¾2ϊ.-¾ 202

PC(0-36:4) 3184 2524 26 -2 8.60E-01 13 5.36E-01 44 52 .. ί.ίίνθ.^': 21

PC(0-36:5) 511 349 47 J..i>:¾-C3 3 6.94E-01 34 1.71E-01 61 74 iiiiii 45

....

PC(0-38:4) 1159 675 72 ! . I.: 21 49 m mmm 104 SI sr.- S 124 mw m 67 :

PC(0-38:5) 3702 3309 12 2.78E-01 -9 2.90E-01 4 9.80E-01 23 1.25E-01 36 6.24E-02 4

PC(O-40:5) 592 396 50 11 2.10E-01 39 .V/^" £ -:2 61 m 92 -,\4.-< £;^■ :-^■<¾ 42

PC(O-40:6) 1424 477 199 mmmm 35 !i!!il 195 W W:§ : 234 mmmm 294 ^■■i i m ? 223

PC(O-40:7) 1518 1231 23 -1 8.87E-01 10 6.80E-01 28 1.23E-01 51 23

PC(P-30:0) 24 28 -13 10 3.93E-01 -28 .fi::·.- _~ -15 2.94E-01 1 9.36E-01 -33

PC(P-16:0/16:0) 349 353 -1 9.99E-01 -7 6.44E-01 5 9.87E-01 -15 5.95E-01 28 1.86E-01 -9

PC(P-32:1) 91 78 15 7.51E-02 14 1.90E-01 5 9.80E-01 11 2.99E-01 38 9.78E-02 7

PC(P-16:0/18:1) 338 285 19 ; ., ^■. ^■ :. 13 2.14E-01 3 9.80E-01 16 1.05E-01 41 6.24E-02 17

PC(P-16:0/18:2) 281 229 22 i ^'-ysi- z 22 5.67E-02 5 9.84E-01 21 7.69E-02 31 2.16E-01 33

PC(P-16:0/18:3) 23 24 -3 9.41E-01 -8 5.46E-01 -16 8.31E 01 16 4.06E-01 30 4.26E-01 -35

PC(P-18:0/18:2) 102 30 242 .: .:·. .. 51 1.04E-01 137 263 Of; 341 ^■ 81 sr.- S 372

PC(P-16:0/20:4) 775 688 13 2.48E-01 9 4.59E-01 0 9.87E-01 21 2.99E-01 35 2.07E-01 0

PC(P-16:0/20:5) 116 103 13 3.25E-01 13 3.13E-01 -11 9.03E-01 16 2.49E-01 31 1.82E-01 10

PC(P-18:0/20:4) 187 97 92 09 7 6.31E-01 61 6.70E-02 154 130 116

all time points¹ all time points¹ 1 week² 2 weeks² 4 weeks² 8 weeks² 12 wee

Control Alkylglycerol % % % % % %

Predictor (pmol/mL) (pmol/mL) difference p-value difference p-value difference p-value difference p-value difference p-value difference

PC(P-38:5) 3700 2658 39 13 1.79E-01 17 5.08E-01 53 !iiiiii 76 liilllil 36

PC(P-16:0/22:6) 945 472 100 mmmm. 19 1.46E-01 67 2.<?^■££^■02 126 -·'· of. 162 :.(^■■.^■:. 114

PC(P-18:0/22:6) 683 140 387 47 5.03E-02 239 635 ^'. ^{■ "}i ^■ .^': 512 473

PC(14 0/0:0) 1556 1780 -13 -1 9.51E-01 -15 4.08E-01 -22 -15 l.OlE-01 -11

PC(15 0/0:0) 1182 1297 -9 9.30E-02 -4 3.91E-01 -4 9.80E-01 -18 -7 7.01E-01 -14

PC(16 0/0:0) 109349 114556 -5 5.44E-01 0 9.92E-01 -6 9.02E-01 0 9.08E-01 -3 9.88E-01 -14

PC(16 1/0:0) 12653 13878 -9 3.65E-01 -13 2.72E-01 -20 6.43E-01 -6 6.65E-01 -5 9.36E-01 -8

PC(17 0/0:0) 1540 1641 -6 3.42E-01 3 7.55E-01 -1 9.87E-01 -6 5.85E-01 -12 7.08E-01 -17

PC(17 1/0:0) 2067 2309 -10 3.03E-01 -11 2.03E-01 -14 8.97E-01 -11 2.99E-01 -8 8.70E-01 -12

PC(18 0/0:0) 38853 40367 -4 6.52E-01 6 5.30E-01 -3 9.87E-01 2 9.08E-01 -8 8.70E-01 -16

PC(18 1/0:0) 73896 79873 -7 3.65E-01 -11 2.00E-01 -13 8.05E-01 -1 9.12E-01 -4 9.49E-01 -13

PC(18 2/0:0) 37071 39437 -6 3.65E-01 -5 5.34E-01 -3 9.87E-01 -4 7.03E-01 -4 9.33E-01 -17

PC(18 3/0:0) 1462 1786 -18 m: m -1 9.50E-01 -11 9.80E-01 -27 3.:S£-02 -24 1.55E-01 -27

PC(20 0/0:0) 864 886 -3 8.05E-01 -7 6.47E-01 7 9.80E-01 1 9.08E-01 0 9.97E-01 -17

PC(20 1/0:0) 2337 2481 -6 6.17E-01 -16 9.17E-02 -19 5.52E-01 4 8.11E-01 3 9.36E-01 -10

PC(20 2/0:0) 1405 1539 -9 5.41E-01 -16 2.46E-01 -26 4.08E-01 0 9.79E-01 -2 9.86E-01 -11

PC(20 3/0:0) 7550 7820 -3 7.08E-01 -6 5.39E-01 -10 7.67E-01 13 3.10E-01 -3 9.91E-01 -15

PC(20 4/0:0) 10336 11171 -7 1.64E-01 -4 6.24E-01 -15 3.68E-01 -2 8.46E-01 -4 9.18E-01 -13

PC(20 5/0:0) 3230 3489 -7 5.40E-01 0 9.85E-01 -17 8.72E-01 -4 8.44E-01 -5 9.81E-01 -17

PC(22 5/0:0) 793 862 -8 3.65E-01 -1 9.57E-01 -10 8.96E-01 -11 4.07E-01 -5 9.38E-01 -17

171

PC(22 6/0:0) 11995 12592 -5 5.40E-01 -7 4.43E-01 -11 5.67E-01 1 9.33E-01 0 9.91E-01 -9

PC(26 0/0:0) 5 4 21 7.86E-01 7 7.11E-01 -16 7.86E-01 0 9.08E-01 1 9.91E-01 -9

PC(0:0/14:0) 348 397 -13 j.. ; :^"i: 0¾ -5 6.48E-01 -15 3.68E-01 -19 2.v?£~-;2 -13 1.33E-01 -13

PC(0:0/15:0) 365 424 -14 -9 1.46E-01 -11 7.12E-01 -25 ^'. ^{■ "}i ^■ .^': -12 2.55E-01 -15

PC(0:0/16:0) 23715 25254 -6 4.28E-01 -7 3.62E-01 -10 7.08E-01 -7 5.95E-01 3 9.33E-01 -12

PC(0:0/16:1) 2975 3322 -10 4.07E-01 -14 1.88E-01 -19 7.12E-01 10 4.79E-01 -3 9.81E-01 -28

PC(0:0/17:0) 3754 4095 -8 1.95E-01 1 8.64E-01 -8 8.92E-01 -8 3.81E-01 -12 6.37E-01 -15

PC(0:0/17:1) 556 619 -10 4.43E-01 -8 3.74E-01 -14 9.45E-01 12 4.47E-01 -10 8.49E-01 -30

PC(0:0/18:0) 8759 9367 -6 5.37E-01 -3 7.82E-01 -6 9.35E-01 -9 5.95E-01 -2 9.81E-01 -16

PC(0:0/18:1) 25976 27225 -5 7.65E-01 -2 8.19E-01 -11 9.73E-01 26 1.44E-01 -6 9.38E-01 -26

PC(0:0/18:2) 12771 13155 -3 8.38E-01 4 7.01E-01 2 9.87E-01 30 2.17E-01 -5 9.27E-01 -35

PC(0:0/18:3) 278 321 -13 4.38E-01 10 3.78E-01 -9 9.87E-01 13 8.22E-01 -44 6.24E-02 -34

PC(0:0/20:0) 193 211 -8 5.02E-01 -26 0 9.87E-01 -12 6.93E-01 11 7.28E-01 -15

PC(0:0/20:1) 649 658 -1 8.38E-01 -14 2.49E-01 -22 6.68E-01 31 1.25E-01 0 9.81E-01 -14

PC(0:0/20:2) 602 620 -3 8.08E-01 -7 6.54E-01 -22 6.32E-01 42 1.05E-01 -11 8.99E-01 -20

PC(0:0/20:3) 2964 2808 6 8.38E-01 7 6.07E-01 2 9.91E-01 73 -6 9.38E-01 -30

PC(0:0/20:4) 4115 4262 -3 7.58E-01 8 4.15E-01 -12 6.97E-01 34 1.52E-01 -10 8.10E-01 -27

PC(0:0/20:5) 1052 1116 -6 8.86E-01 13 3.82E-01 -16 9.80E-01 35 2.58E-01 -7 9.76E-01 -39

all time points' all time points' 1 week² 2 weeks² 4 weeks² 8 weeks² 12 wee

Control Alkylglycerol % % % % % %

Predictor (pmol/mL) (pmol/mL) difference p-value difference p-value difference p-value difference p-value difference p-value difference

PC(0:0/22:5) 924 949 -3 7.68E-01 2 8 71E-01 -9 8 28E-01 15 2.99E-01 -4 9.36E-01 -17

PC(0:0/22:6) 5326 5316 0 9 86E-01 10 4.62E-01 -6 9 45E-01 51 1.25E-01 -2 9.48E-01 -33

LPC(O-16:0) 714 538 33 i ^'.'.A- i 10 5.88E-02 31 ;; >.l. 111 35 ,o 53 31

LPC(O-18:0) 216 105 105 wmmm 30 111 :¾ 118 156 mmmrn 114

LPC(0-18:1) 305 281 9 3.65E-01 1 8 58E-01 12 7.67E-01 6 7.66E-01 12 7.66E-01 10

LPC(O-20:0) 4317 4416 -2 3.65E-01 -2 6 23E-01 -2 9 32E-01 -1 8.63E-01 -2 8.51E-01 -5

LPC(O-22:0) 17 16 6 5.68E-01 20 ?>¾ ;^■; -5 8 28E-01 10 6.82E-01 15 5.45E-01 -9

LPC(0-22:1) 13 14 -4 6 60E-01 10 3 13E-01 -6 9 16E-01 -4 7.11E-01 -5 8.20E-01 -12

LPC(O-2₄:0) 8 8 3 8 86E-01 32 5 93E-02 -14 5.36E-01 7 6.92E-01 12 8.10E-01 -17

LPC(0-2₄:1) 13 14 -6 4.28E-01 1 9 41E-01 -17 1.68E-01 -2 9.08E-01 -3 9.76E-01 -8

LPC(P-16:0) 32 30 8 1.92E-01 3 4 70E-01 9 5.53E-01 0 9.79E-01 22 1.92E-01 7

LPC(P-18:0) 7 4 85 i¾ mmm 13 9 61E-02 41 2 ΰ^;.^:·ί 91 5.39E-02 110 ?.ύίϊ- : 55

LPC(P-18:1) 3 2 92 6 6 37E-01 40 3.68E -01 31 1.70E-01 32 2.70E-01 -6

PE(32:1) 79 104 -24 2.88E-01 -22 1 03E-01 -9 9 80E -01 -25 3.10E-01 32 3.63E-01 -65

PE(34:1) 439 539 -19 3.42E-01 -11 2 07E-01 -2 9 87E -01 -17 4.93E-01 16 6.00E-01 -56

PE(34:2) 394 414 -5 8 86E-01 -3 8 51E-01 16 9 29E -01 5 7.03E-01 50 -57

PE(36:1) 382 426 -10 3.65E-01 11 2 18E-01 -1 9 87E -01 -17 4.26E-01 2 9.36E-01 -38

PE(36:2) 765 781 -2 8 20E-01 -1 9 14E-01 24 5.21E -01 -10 7.24E-01 8 8.13E-01 -31

PE(36:3) 492 489 1 9 99E-01 -16 9 90E-02 9 8 90E -01 -3 9.08E-01 51 -32

PE(36:4) 1134 1213 -7 7.36E-01 -10 3 83E-01 -13 7.86E -01 -1 9.08E-01 45 ■ νΐΐ,ω. -37

PE(38:3) 122 121 1 9 41E-01 4 7 87E-01 3 9 87E -01 8 6.93E-01 38 9.78E-02 -36

PE(38:4) 1575 1615 -2 9 86E-01 1 9 69E-01 5 9 84E -01 5 6.93E-01 33 9.78E-02 -39

PE(38:5) 894 889 1 9 14E-01 -14 1 38E-01 -13 7.06E -01 15 4.06E-01 54 '; ^:;;,^:-·:;< -27

PE(38:6) 2292 2339 -2 9 77E-01 -14 3 32E-01 -14 8 28E -01 -2 9.08E-01 64 ;...Τ.-.ί.^;■';. -27

PE(40:4) 19 21 -10 7.94E-01 14 4 89E-01 3 9 87E -01 -1 8.23E-01 -3 9.27E-01 -44

PE(40:5) 19 19 -2 9 99E-01 27 2 54E-01 -4 9 87E -01 -31 1.18E-01 36 1.92E-01 -31

PE(40:6) 577 569 1 8 63E-01 0 9 98E-01 7 9 87E -01 7 6.77E-01 43 l.llE-01 -34

PE(40:7) 551 525 5 7.26E-01 -10 4 82E-01 -8 9 80E -01 11 5.28E-01 71 -26

PE(0-34:1) 386 147 162 llilil 44 166 :¾ -·._': 209 I1111SI 196 164

PE(0-34:2) 112 24 364 mil 49 489 1. 503 Z.viZ 0':; 356 368

PE(0-36:4) 147 54 171 ^■r??;- 5.5 81 185 ¾ 311 175 :.:::^■-^■■: ■ 115

PE(0-36:5) 50 17 184 ¾smmm 67 229 3 311 wmmm 164 ^■■: 132

PE(0-36:6) 6 2 259 7.94E-01 294 3.33E-01 -16 7.86E-01 24 7.81E-01 8 9.49E-01 -39

PE(0-38:4) 570 264 115 ¾: ¾!:-·;^■:^■ 44 si mmm 125 i I 180 !l!!ii 110 ^■ c :- ;: 101

PE(0-38:5) 85 39 118 mmm 67 1 153 :i 146 140 !i!!ll 81

PE(O-40:5) 446 283 57 :8§i£¾:¾:§: 29 8 31E-02 50 3.68E-01 87 wmmm 68 9.78E-02 41

PE(O-40:6) 26 10 160 mmm 81 Y3i.- 02 243 % 155 217 84

PE(O-40:7) 134 52 160 mmm 56 34? 195 6 00E-02 248 144 117

all time points' all time points' 1 week² 2 weeks² 4 weeks² 8 weeks² 12 wee

PE P-18 1/20:5) [FA] 1 1 91 9 14E-01 91 7.48E-01 103 4.08E-01 -33 8.11E-01 71 6.82E-01 -46

PE P-18 1/22:4) [FA] 7 7 -1 9 99E-01 16 4.92E-01 18 8.31E-01 -10 7.85E-01 -5 8.13E-01 -21

PE P-18 1/22:5)a [FA] 89 75 19 1 26E-01 28 5.79E-02 38 7.06E-01 40 mm- m 23 5.89E-01 -18

(jO PE P-18 1/22:6) [FA] 1237 756 64 GC'ir -C 25 5.85E-02 77 9.26E-02 85 mmm i 91 m 38

PE P-20 0/18:1) [FA] 68 81 -16 6 23E-02 31 ..:;. i.--y^;. -26 3.68E-01 -22 2.05E-01 -10 7.09E-01 -38

PE P-20 0/18:2) [FA] 67 70 -4 6 96E-01 32 m -13 8.00E-01 -8 5.95E-01 -3 9.33E-01 -25

PE P-20 0/20:4) [FA] 132 140 -5 7 OlE-01 32 6 14E-02 -12 7.06E-01 -5 6.75E-01 -1 9.91E-01 -27

PE P-20 0/22:6) [FA] 340 298 14 1 59E-01 36 :2 ¾-i:.^:- Z -3 9.80E-01 19 3.52E-01 37 2.07E-01 -6

PE P-20 1/20:4) [FA] 18 18 -2 9 16E-01 22 3 06E-01 -18 7.67E-01 4 7.96E-01 7 9.81E-01 -21

PE P-20 1/22:6) [FA] 32 28 15 3 81E-01 11 5 70E-01 -1 9.80E-01 33 2.81E-01 36 2.71E-01 2

PE 16:0/0:0) 2567 2680 -4 5 68E-01 -6 4 37E-01 -12 6.12E-01 -1 9.64E-01 21 2.61E-01 -19

PE 18:0/0:0) 2329 2396 -3 6 96E-01 7 5 13E-01 2 9.87E-01 2 8.22E-01 0 9.86E-01 -27

PE 18:1/0:0) 2962 3152 -6 3 55E-01 -6 3 96E-01 -16 4.97E-01 -6 6.02E-01 1 9.36E-01 -6

PE 18:2/0:0) 2225 2296 -3 7 75E-01 0 9 97E-01 2 9.87E-01 -4 8.63E-01 -2 9.91E-01 -14

PE 20:4/0:0) 2272 2319 -2 9 37E-01 -3 6 07E-01 -16 4.17E-01 4 7.91E-01 10 7.50E-01 -2

PE 22:6/0:0) 3598 3556 1 9 62E-01 -4 6 40E-01 -15 6.80E-01 3 8.22E-01 20 4.62E-01 6

PE 0:0/16:0) 683 726 -6 3 65E-01 -11 1 06E-01 -17 3.79E-01 -9 5.85E-01 20 2.00E-01 -8

PE 0:0/18:0) 624 661 -6 4 53E-01 -2 7 82E-01 -2 9.87E-01 -13 4.42E-01 6 8.20E-01 -19

PE 0:0/18:1) 1068 1082 -1 8 42E-01 9 1 65E-01 -11 8.31E-01 16 4.07E-01 0 9.81E-01 -18

PE 0:0/18:2) 1141 1082 5 8 37E-01 19 2 41E-01 13 8.79E-01 46 2.07E-01 -9 8.62E-01 -31

PE 0:0/20:4) 1405 1290 9 6 59E-01 20 2 37E-01 -8 9.32E-01 60 8.99E-02 0 9.81E-01 -15

all time points' all time points' 1 week² 2 weeks² 4 weeks² 8 weeks² 12 wee

Control Alkylglycerol % % % % % %

Predictor (pmol/mL) (pmol/mL) difference p-value difference p-value difference p-value difference p-value difference p-value difference

PE(0:0/22:6) 3328 3038 10 5.80E-01 19 2.80E-01 -10 8.97E-01 54 9.35E-02 15 7.50E-01 -15

LPE(P-16:0) 192 174 10 ^■mmmm-. 19 m&mm 8 7.06E-01 12 1.25E-01 13 1.84E-01 1

LPE(P-18:0) 112 89 26 22 mm m 24 6.84E-02 34 mm m 35 mmm 17

LPE(P-18:1) 51 47 9 2.24E-01 26 •i. 'i:.^:- Z 5 9.80E-01 11 3.41E-01 9 5.78E-01 -1

PI(32:0) 96 125 -23 18 2.08E-01 -15 8.31E-01 -37 7.87E-02 -34 1.19E-01 -46

PI(32:1) 892 1013 -12 2.52E-01 7 4.21E-01 -6 9.87E-01 -24 2.08E-01 -10 8.13E-01 -30

PI(34:0) 52 62 -17 :. 6 6.62E-01 -1 9.87E-01 -34 ί..'ί.-.ί.^; ';. -16 5.28E-01 -36

PI(34:1) 5156 5846 -12 2.88E-01 14 1.05E-01 -2 9.87E-01 -28 1.44E-01 -16 6.29E-01 -29

PI(36:1) 3173 3657 -13 2.74E-01 16 2.17E-01 -7 9.96E-01 -27 1.59E-01 -20 4.08E-01 -26

PI(36:2) 4144 4555 -9 4.53E-01 14 2.66E-01 -5 9.87E-01 -17 2.94E-01 -14 7.41E-01 -23

PI(36:3) 4157 4220 -1 9.04E-01 3 8.03E-01 4 9.87E-01 -8 6.95E-01 11 8.13E-01 -20

PI(36:4) 11472 11554 -1 9.03E-01 -4 7.17E-01 -10 8.28E-01 6 6.82E-01 18 7.09E-01 -14

PI(38:2) 670 678 -1 8.00E-01 -22 5.09E-02 -37 1.68E-01 10 4.86E-01 20 3.68E-01 6

PI(38:3) 4395 4263 3 7.85E-01 1 9.55E-01 -4 9.87E-01 10 4.26E-01 19 6.29E-01 -11

PI(38:4) 39982 39720 1 9.86E-01 -4 5.95E-01 -10 7.06E-01 12 4.00E-01 16 5.15E-01 -10

PI(38:5) 13578 12939 5 7.20E-01 -6 3.73E-01 -5 9.03E-01 21 7.28E-02 16 5.78E-01 -5

PI(38:6) 1544 1584 -3 8.86E-01 7 6.61E-01 0 9.91E-01 -9 6.02E-01 13 7.50E-01 -23

PI(40:4) 329 335 -2 9.39E-01 -16 7.40E-02 -17 7.12E-01 5 8.06E-01 13 7.20E-01 -2

PI(40:5) 302 287 5 4.53E-01 3 8.31E-01 -1 9.96E-01 14 5.85E-01 28 6.29E-01 -15

PI(40:6) 1617 1650 -2 9.60E-01 10 5.19E-01 -5 9.87E-01 -5 7.85E-01 13 7.84E-01 -19

PI(18:0/0:0) 294 303 -3 9.60E-01 -7 4.34E-01 -8 8.72E-01 6 7.47E-01 30 1.92E-01 -29

PI(18:l/0:0) 270 307 -12 3.65E-01 1 9.33E-01 -5 9.87E-01 -22 4.07E-01 -8 8.94E-01 -25

PI(18:2/0:0) 141 153 -8 3.81E-01 13 2.38E-01 -10 9.80E-01 -12 6.30E-01 -12 4.82E-01 -21

PI(20:4/0:0) 803 843 -5 6.70E-01 -5 6.45E-01 -17 5.29E-01 -1 9.08E-01 -2 9.91E-01 -1

PI(0:0/18:0) 27 29 -7 5.68E-01 -27 9.42E-02 -15 7.67E-01 -11 7.11E-01 30 2.97E-01 -21

PI(0:0/18:1) 60 62 -3 8.38E-01 46 -1 9.87E-01 12 6.93E-01 -26 2.06E-01 -33

PI(0:0/18:2) 30 29 4 9.99E-01 35 5.80E-02 5 9.84E-01 46 1.41E-01 -23 3.34E-01 -34

PI(0:0/20:4) 296 262 13 5.68E-01 38 6.00E-02 3 9.96E-01 63 1.41E-01 -8 9.18E-01 -19

PS(36:1) 631 102 521 9.99E-01 155 ^■: ., ^■. :. 16 8.00E-01 110 3.22E-01 -21 2.56E-01 -62

PS(38:4) 136 159 -15 8.83E-01 134 8 9.87E-01 58 1.88E-01 -22 6.29E-01 -80

Cholesterol 1382351 1471693 -6 7.01E-01 -11 3.11E-01 -7 9.56E-01 13 4.86E-01 6 9.27E-01 -26

CE(14:0) 62273 69362 -10 1.95E-01 -5 6.26E-01 -18 1.47E-01 -16 1.05E-01 8 7.08E-01 -13

CE(15:0) 45358 45400 0 9.97E-01 0 9.94E-01 -15 7.67E-01 -1 9.84E-01 14 6.29E-01 2

CE(16:0) 120847 124361 -3 6.59E-01 -5 4.12E-01 -6 7.86E-01 -3 7.66E-01 10 4.29E-01 -7

CE(16:1) 574840 598623 -4 5.93E-01 -7 2.78E-01 -4 9.80E-01 -7 4.79E-01 12 4.62E-01 -12

CE(16:2) 14055 13815 2 8.38E-01 8 4.51E-01 -6 9.87E-01 0 9.33E-01 13 6.29E-01 -8

CE(17:0) 11335 13343 -15 1.62E-01 -2 9.06E-01 -6 9.84E-01 -16 2.94E-01 -17 6.00E-01 -37

CE(18:0) 5638 5959 -5 6.46E-01 14 6.04E-01 35 8.28E-01 -11 6.21E-01 -16 6.00E-01 -41

all time points' all time points' 1 week² 2 weeks² 4 weeks² 8 weeks² 12 wee

Control Alkylglycerol % % % % % %

Predictor (pmol/mL) (pmol/mL) difference p-value difference p-value difference p-value difference p-value difference p-value difference

CE(18:1) 520557 505097 3 6 62E-01 0 9.74E-01 3 9.87E-01 2 7.96E-01 11 3.34E-01 1

CE(18:2) 491850 455832 8 2 59E-01 8 2.97E-01 9 7.67E-01 4 6.93E-01 14 2.10E-01 6

CE(18:3) 145284 146275 -1 8 38E-01 15 2.03E-01 -12 9.80E-01 -7 5.92E-01 6 8.10E-01 -2

CE(20:4) 588125 552966 6 4 53E-01 8 4.93E-01 -5 8.88E-01 6 6.95E-01 15 4.62E-01 9

CE(20:5) 264021 242488 9 5 54E-01 24 3.49E-01 -14 9.80E-01 11 6.51E-01 19 6.81E-01 2

CE(22:5) 6788 6525 4 6 57E-01 8 4.69E-01 -13 7.12E-01 -4 9.08E-01 30 2.49E-01 3

CE(22:6) 317595 273509 16 3 38E-01 10 6.14E-01 -6 9.80E-01 26 4.51E-01 28 4.95E-01 17

DG(14:0_16 0) 306 333 -8 5 83E-01 22 1.29E-01 -5 9.84E-01 17 6.02E-01 -22 5.28E-01 -37

DG(14:0_18 1) 247 313 -21 7 75E-01 -4 7.99E-01 12 8.97E-01 52 3.59E-01 -19 9.27E-01 -64

DG(16:0_16 0) 2071 2149 -4 6 87E-01 13 7.35E-02 -1 9.87E-01 5 6.93E-01 -5 8.10E-01 -25

DG(16:0_18 0) 2856 2823 1 9 99E-01 16 ; 7 7.67E-01 1 9.08E-01 -3 8.13E-01 -13

DG(16:0_18 1) 2372 3683 -36 5 68E-01 -5 7.70E-01 3 9.87E-01 11 9.08E-01 8 8.80E-01 -79

DG(16:0_18 2) 391 475 -18 6 57E-01 -3 8.28E-01 15 9.80E-01 23 6.02E-01 13 8.99E-01 -67

DG(16:0_22 6) 198 300 -34 4 53E-01 0 9.76E-01 -2 9.87E-01 18 6.77E-01 -12 8.63E-01 -74

DG(16:1_18 1) 1392 1836 -24 7 65E-01 -7 6.83E-01 -2 9.87E-01 15 7.15E-01 4 8.20E-01 -67

DG(18:0_18 0) 1092 1058 3 8 70E-01 24 4 7 7.67E-01 5 7.66E-01 -5 7.01E-01 -14

DG(18:0_18 1) 546 622 -12 8 38E-01 7 6.93E- 01 15 9.03E-01 10 8.39E-01 -3 9.89E-01 -52

DG(18:1_18 1) 3781 4620 -18 7 94E-01 -4 8.41E- 01 0 9.87E-01 4 8.79E-01 10 7.50E-01 -60

DG(18:1_18 2) 881 1007 -13 6 81E-01 -2 9.18E- 01 0 9.87E-01 5 8.79E-01 11 8.13E-01 -55

DG(18:1_20 3) 297 339 -12 3 81E-01 -4 8.05E- 01 -16 7.12E-01 -7 7.15E-01 -4 9.86E-01 -33

TG(14:0_16 0_18 1) 2768 4248 -35 2 88E-01 -29 1.60E- 01 12 9.25E-01 -18 5.20E-01 -52 6.53E-01 -42

TG(14:0_16 0_18 2) 2320 3183 -27 3 14E-01 -26 1.18E- 01 11 9.87E-01 -39 2.80E-01 -16 9.76E-01 -52

TG(14:0_16 1_18 1) 2032 2813 -28 3 35E-01 -29 1.03E- 01 15 9.29E-01 -28 3.81E-01 -32 8.13E-01 -43

TG(14:0_16 1_18 2) 367 490 -25 4 46E-01 -10 5.98E- 01 25 8.28E-01 -28 3.52E-01 -39 7.75E-01 -41

TG(14:0_17 0_18 1) 350 566 -38 2 19E-01 -35 9.09E- 02 14 9.80E-01 -20 4.92E-01 -52 6.63E-01 -49

TG(14:0_18 0_18 1) 405 196 107 9 86E-01 -38 1.12E- 01 11 9.80E-01 -47 5.20E-01 -54 7.75E-01 -50

TG(14:0_18 2_18 2) 82 102 -20 5 07E-01 0 9.99E- 01 36 8.31E-01 -32 4.30E-01 -38 8.13E-01 -41

TG(14:1_16 0_18 1) 288 415 -31 7 51E-02 -20 1.49E- 01 -4 9.87E-01 -41 1.62E-01 -27 7.50E-01 -51

TG(14:1_16 1_18 0) 3549 4929 -28 2 83E-01 -27 1.51E- 01 0 9.87E-01 -38 2.94E-01 -14 9.81E-01 -52

TG(14:1_18 0_18 2) 315 403 -22 2 79E-01 -21 1.84E- 01 7 9.87E-01 -32 2.94E-01 -15 9.38E-01 -44

TG(14:1_18 1_18 1) 4039 5210 -22 4 03E-01 -27 1.31E- 01 7 9.87E-01 -35 3.54E-01 -8 9.76E-01 -46

TG(15:0_16 0_18 1) 1209 1721 -30 3 55E-01 -21 3.71E- 01 4 9.87E-01 -37 2.84E-01 -26 9.36E-01 -53

TG(15:0_18 1_18 1) 342 458 -25 3 95E-01 -18 4.41E- 01 22 9.29E-01 -37 4.00E-01 -21 9.36E-01 -51

TG(16:0_16 0_16 0) 628 1016 -38 2 61E-01 -29 1.03E- 01 1 9.87E-01 -14 6.82E-01 -56 7.01E-01 -46

TG(16:0_16 0_18 0) 754 1098 -31 1 64E-01 -30 1.28E- 01 3 9.87E-01 -10 7.81E-01 -55 5.49E-01 -31

TG(16:0_16 0_18 1) 7835 11912 -34 3 65E-01 -25 2.03E- 01 4 9.87E-01 -34 3.52E-01 -31 9.59E-01 -57

TG(16:0_16 0_18 2) 827 1117 -26 3 42E-01 -15 2.98E- 01 19 9.36E-01 -33 2.58E-01 -29 8.63E-01 -49

TG(16:0_16 1_17 0) 425 670 -37 5 92E-01 -26 1.56E- 01 10 9.87E-01 -20 8.63E-01 -46 7.30E-01 -55

all time points' all time points' 1 week² 2 weeks² 4 weeks² 8 weeks² 12 wee

Control Alkylglycerol % % % % % %

Predictor (pmol/mL) (pmol/mL) difference p-value difference p-value difference p-value difference p-value difference p-value difference

TG(16 0_16 1_18 1) 21337 28114 -24 3.81E-01 -23 2.31E-01 -1 9.87E-01 -36 3.52E-01 -9 9.70E-01 -48

TG(16 0_17 0_18 1) 362 542 -33 2.61E-01 -21 1.57E-01 -1 l.OOE+00 -24 3.54E-01 -43 7.20E-01 -48

TG(16 0_17 0_18 2) 654 909 -28 3.65E-01 -20 3.70E-01 0 9.87E-01 -38 4.15E-01 -17 9.97E-01 -53

TG(16 0_18 0_18 1) 1335 2055 -35 3.65E-01 -24 2.22E-01 13 9.62E-01 -14 6.66E-01 -49 7.25E-01 -51

TG(16 0_18 1_18 1) 35717 48395 -26 5.76E-01 -20 4.79E-01 4 9.87E-01 -41 5.20E-01 3 8.13E-01 -61

TG(16 0_18 1_18 2) 7406 8747 -15 4.63E-01 -12 5.50E-01 17 9.87E-01 -37 2.94E-01 2 8.88E-01 -45

TG(16 0_18 2_18 2) 715 775 -8 6.57E-01 11 7.09E-01 36 9.32E-01 -41 2.14E-01 3 8.77E-01 -46

TG(16 1_16 1_16 1) 515 637 -19 6.62E-01 -26 1.38E-01 6 9.87E-01 -34 4.00E-01 -6 9.33E-01 -39

TG(16 1_16 1_18 0) 221 310 -29 4.63E-01 -25 1.84E-01 32 7.08E-01 -11 7.03E-01 -42 6.46E-01 -45

TG(16 1_16 1_18 1) 4438 5316 -17 5.83E-01 -27 1.73E-01 4 9.87E-01 -30 4.88E-01 7 7.75E-01 -43

TG(16 1_17 0_18 1) 4287 5889 -27 3.65E-01 -20 3.52E-01 8 9.87E-01 -34 3.58E-01 -25 9.36E-01 -48

TG(16 1_18 1_18 1) 7531 9217 -18 7.01E-01 -25 2.35E-01 3 9.87E-01 -27 6.38E-01 3 8.10E-01 -46

TG(16 1_18 1_18 2) 3302 3813 -13 6.52E-01 -16 4.28E-01 20 9.80E-01 -32 4.07E-01 4 8.20E-01 -42

TG(17 0_18 1_18 1) 655 885 -26 3.42E-01 -21 2.70E-01 -2 9.87E-01 -37 2.53E-01 -16 9.39E-01 -48

TG(18 0_18 0_18 1) 201 296 -32 2.61E-01 -30 7.78E-02 12 9.62E-01 -19 4.79E-01 -46 6.73E-01 -44

TG(18 0_18 1_18 1) 1420 1926 -26 5.41E-01 -17 3.09E-01 22 8.53E-01 -13 6.82E-01 -33 8.23E-01 -49

TG(18 0_18 2_18 2) 100 109 -8 9.03E-01 2 9.03E-01 54 6.38E-01 -36 2.99E-01 -14 9.59E-01 -19

TG(18 1_18 1_18 1) 5980 7531 -21 7.36E-01 -25 3.89E-01 13 9.87E-01 -27 7.13E-01 -3 8.13E-01 -53

TG(18 1_18 1_18 2) 3358 4156 -19 5.39E-01 -21 3.17E-01 18 9.60E-01 -32 3.81E-01 -11 9.86E-01 -43

TG(18 1_18 1_20 4) 154 174 -11 4.43E-01 2 9.23E-01 2 9.87E-01 -35 1.65E-01 -7 9.36E-01 -25

TG(18 1_18 1_22 6) 1025 1036 -1 9.77E-01 -2 9.33E-01 5 9.87E-01 -11 9.08E-01 5 9.33E-01 -11

TG(18 1_18 2_18 2) 690 766 -10 6.51E-01 1 9.68E-01 21 9.80E-01 -31 4.06E-01 -10 9.86E-01 -33

TG(18 2_18 2_18 2) 41 40 1 9.60E-01 18 5.65E-01 51 6.32E-01 -30 4.07E-01 -11 9.76E-01 -18

OX PC 34:3 + 20 19 19 0 9.86E-01 5 8.38E-01 35 5.36E-01 28 5.45E-01 2 9.36E-01 -46

SM(34:1) 28424 27378 4 6.59E-01 10 2.37E-01 3 9.80E-01 6 5.79E-01 12 6.29E-01 -13 3

All time points represent the average of all measurements across the 12 weeks study.

Represents the % difference between the control and alkylglycerol treated mice at each time point, p values were calculated by Stud test of log transformed values and have been corrected for multiple comparison by the method of Benjamini Hochberg.

BIBLIOGRAPHY

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Claims

WHAT IS CLAIMED IS:

1. A method of reducing the incidence of atherosclerotic plaque in a subject, the method comprising, consisting or consisting essentially of: increasing the level of plasmalogen in the subject to thereby reduce the incidence of atherosclerotic plaque in the subject.

2. A method of inhibiting formation of atherosclerotic plaque in a subject, the method comprising, consisting or consisting essentially of: increasing the level of plasmalogen in the subject to thereby inhibit formation of atherosclerotic plaque in the subject.

3. A method of regressing atherosclerotic plaque in a subject, the method comprising, consisting or consisting essentially of: increasing the level of plasmalogen in the subject to thereby regress atherosclerotic plaque in the subject.

4. A method of treating atherosclerosis in a subject, the method comprising, consisting or consisting essentially of: increasing the level of plasmalogen in the subject to thereby treat atherosclerosis in the subject.

5. A method according to any one of claims 1 to 4, comprising administering to the subject at least one (e.g., 1, 2, 3, or more) plasmalogen level-enhancing compound selected from the group consisting of plasmalogen precursor lipids and heterologous plasmalogens, in an effective amount to increase the level of plasmalogen in the subject, and to thereby reduce the incidence and/or inhibit formation and/or to result in regression of atherosclerotic plaque and/or treat atherosclerosis in the subject.

6. A method according to any one of claims 1 to 5, further comprising identifying the subject as having atherosclerosis or being at risk of developing atherosclerosis prior to increasing the level of plasmalogen in the subject.

7. A method according to claim 5 or claim 6, wherein the plasmalogen precursor lipids are selected from alkylglycerols.

8. A method according to claim 4, wherein the alkylglycerols are represented by formula (I) :

R₃ R₄

CH₂-0 CH₂ CH— CH R₅

CH— O R₂

CH₂-0 Ri or pharmaceutically acceptable salts or prodrugs thereof, wherein :

Ri and R₂ are the same or different and each is selected from the group consisting of hydrogen and aliphatic acyl groups of at most 24 carbon atoms; R₃ and R₄ are selected from the group consisting of hydrogen, or straight, branched, saturated and unsaturated alkoxy groups of at most 7 carbon atoms, and

R₅ is selected from the group consisting of straight chain and branched alkyl and alkenyl groups of 4 to 21 carbon atoms.

9. A method according to claim 8, wherein R₃ and R₄ are each hydrogen.

10. A method according to claim 8, wherein one of R₃ and R₄ is hydrogen and the other is selected from the group consisting of straight, branched, saturated and unsaturated alkoxy groups of at most 7 carbon atoms.

11. A method according to claim 8, wherein Ri, R₂, R₃ and R₄ are each hydrogen and R₅ is a straight chain alkyl or alkenyl group of 13 to 15 carbon atoms.

12. A method according to claim 8, wherein the alkylglycerols are l-O- alkylglycerols.

13. A method according to claim 12, wherein the 1-O-alkylglycerols are selected from the group consisting of 18: 0 alkyl-l-glycerol, 18 : 1 alkyl-l-glycerol and 16: 0 alkyl- l-glycerol, or combination thereof.

14. A method according to claim 13, wherein the alkylglycerols are selected from the group consisting of 1-O-hexadecyl-sn-glycerol (chimyl alcohol), 1-0-octadecyl-sn- glycerol (batyl alcohol) and l-0-(9Z-octadecenyl)-sn-glycerol (selachyl alcohol).

15. A method according to any one of claims 1 to 14, wherein the subject is administered at least two alkylglycerols selected from the group consisting of 1-O- hexadecyl-sn-glycerol (chimyl alcohol), 1-0-octadecyl-sn-glycerol (batyl alcohol) and 1- 0-(9Z-octadecenyl)-sn-glycerol (selachyl alcohol).

16. A method according to any one of claims 1 to 14, wherein the subject is administered at least three alkylglycerols including 1-O-hexadecyl-sn-glycerol (chimyl alcohol), 1-0-octadecyl-sn-glycerol (batyl alcohol) and l-0-(9Z-octadecenyl)-sn-glycerol (selachyl alcohol).

17. A method according to any one of claims 8 to 16, wherein the alkylglycerols are extracted from a natural source.

18. A method according to claim 17, wherein the natural source is a fish liver oil.

19. A method according to claim 18, wherein the fish is an elasmobranch fish.

20. A method according to claim 19, wherein the elasmobranch fish is a shark.

21. A method according to claim 5 or claim 6, wherein the heterologous plasmalogens are represented by formula (II) :

CH₂-0 CH=CH— R₃

O II

CH— O C R₂

O II

CH₂-0 F> O R-i

O^" (II) or pharmaceutically acceptable salts or prodrugs thereof,

wherein :

Ri is a polar group; and

R₂ and R₃ are the same or different and each is selected from aliphatic

hydrocarbon groups.

22. A method according to claim 21, wherein Ri is selected from the group consisting of ethanolamine, choline, serine, inositol and glycerol .

23. A method according to claim 21, wherein Ri is an ethanolamine or a choline.

24. A method according to claim 21, wherein R² is an aliphatic hydrocarbon group, which is suitably derived from a fatty acid residue.

25. A method according to claim 24, wherein the aliphatic hydrocarbon group is selected from the group consisting of octadecadienoyl group, octadecatrienoyl group, icosatetraenoyi group, icosapentaenoyi group, docosatetraenoyi group, docosapentaenoyi group and docosahexaenoyl group.

26. A method according to claim 21, wherein R³ is an aliphatic hydrocarbon group having a carbon number of 1 to 24.

27. A method according to claim 26, wherein the aliphatic hydrocarbon group is selected from the group consisting of dodecyl group, tetradecyl group, hexadecyl group, octadecyl group, icosanyl group.

28. A method according to any preceding claim, further comprising administering concurrently to the subject at least one ancillary agent for treating and/or preventing atherosclerosis.

29. Use of at least one plasmalogen level-enhancing compound as defined in any preceding claim for reducing the incidence of atherosclerotic plaque, for regressing atherosclerotic plaque, for inhibiting formation of atherosclerotic plaque and/or for treating atherosclerosis.

30. Use of at least one plasmalogen level-enhancing compound as defined in any preceding claim in the manufacture of a medicament for reducing the incidence of atherosclerotic plaque, for regressing atherosclerotic plaque, for inhibiting formation of atherosclerotic plaque and/or for treating atherosclerosis.