Ether of phosphatidylcholine and phosphatidylethanolamine for use in treatment of diseases or conditions caused by plasmalogen deficiency, their hemolytic forms and mixtures thereof

Abstract

The present disclosure relates to certain compounds that can be consumed by a human subject to modulate plasmalogen levels in the subject, thereby providing improved health outcomes. The disclosure further relates to formulations of said compounds, compositions comprising at least one compound, and uses thereof.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 478,289, filed January 3, 2023, and U.S. Provisional Patent Application No. 63 / 589,539, filed October 11, 2023, the entire contents of each of which are hereby incorporated by reference in their entirety. TECHNICAL FIELD

[0003] The present disclosure relates to certain precursor compounds, such as lysolecithins (LPC(O)), which can be consumed by a human subject to increase the subject’s level of plasmalogens, thereby providing improved health outcomes. More particularly, the present disclosure relates to formulations of precursor compounds to provide optimal elevation of circulating and tissue plasmalogens, while maintaining optimal composition of plasmalogen species. BACKGROUND

[0004] As defined herein, plasmalogens comprise a group of plasmenyl phospholipids, which are major components of cell membranes. As part of the broader class of phospholipids, plasmanyl and / or plasmenyl phospholipids are a unique class of ether phospholipids that are major components of cell membranes. Their biophysical role in cell membranes has been studied, while knowledge about their biological role is an important new area of research. Plasmalogens are primarily present in the form of plasmenyl choline (PC) and plasmenyl ethanolamine (PE) species (also referred to as PC(P) and PE(P), respectively). They are characterized by a cis-vinyl ether linkage connecting an alkyl chain to the sn-1 carbon position of the glycerol backbone. They also optionally have an acyl-linked fatty acid ester bond in the sn-2 carbon position. Plasmalogens are typically esterified with polyunsaturated fatty acids such as arachidonic acid (20:4) and the omega-3 fatty acid docosahexaenoic acid (22:6, the major component of fish oil), while the vinyl ether-linked residue is typically saturated except for the vinyl ether (i.e., no other double bonds in the chain in addition to the vinyl ether group), or monounsaturated except for the vinyl ether (i.e., one double bond in the chain in addition to the vinyl ether group).

[0005] The biosynthesis of plasmalogens is a complex process that involves multiple enzymes within the peroxisome and endoplasmic reticulum. The rate-limiting step in this pathway is the formation of long-chain fatty alcohols by fatty acyl CoA reductases 1 and 2 (Far-1 / 2). By orally administering naturally occurring alkylglycerols (1-O-alkylglycerols or l-O-alkyl-2,3-diacylglycerols), the rate-limiting step in plasmalogen synthesis can be bypassed. These alkylglycerols can be directly incorporated into the phospholipid pathway and thus bypass the peroxisome. This results in an increase in circulating and tissue plasmalogens. Although alkylglycerols are present in our diet, the levels in a typical diet are insufficient to significantly increase human plasmalogen levels. Shark liver oil is rich in alkylglycerols and is currently used as a dietary supplement to reduce inflammation and improve immune function. Alkylglycerols can also be synthesized, providing a future avenue for an environmentally sustainable source of these compounds (Magnusson, C. D. et al., Tetrahedron (2011) 67: 1821-36; Shi, Y. et al., Green Chemistry (2010) 12(12)).

[0006] WO 2021 / 007623 Al to the Baker Heart and Diabetes Institute (this document is incorporated by reference herein in its entirety) generally relates to various compositions and methods for maintaining or modulating a mixture of ether lipid molecules in human tissue, for non-disease conditions and for treating certain disease conditions, including obesity, diabetes, fatty liver disease, cardiovascular disease, and Alzheimer’s disease.

[0007] There remains an unmet need for improved mixtures or compositions comprising plasmalogen precursor compounds that would make an important contribution to one or more fields including, but not limited to, the fields of medicine, pharmaceuticals, dietary supplements, and nutrition. SUMMARY

[0008] In one embodiment, a formulation is described, which can take the form of one or more of a medicament, a pharmaceutical, a dietary supplement, and a food product or other nutritional composition, comprising, consisting of, or consisting essentially of one or more ether lipid compounds. It has been discovered that ingestion of lysophosphalkylphosphatidylcholines (also known as LPC(O)) and phosphalkylphosphatidylcholines (also known as PC(O)) in krill oil results in an elevation of PE(P) and PC(P) species. This is not obvious because it is generally accepted that LPC(O) and PC(O) are dead ends on the plasmalogen biosynthesis pathway and there is no active pathway for conversion of LPC(O) and PC(O) to PE(P) and PC(P). Thus, it appears that LPC(O) / PC(O) can be introduced into the plasmalogen biosynthesis pathway and / or they can directly stimulate the natural biosynthesis pathway to increase plasmalogen synthesis.

[0009] The elevation of plasmalogen levels in circulation (plasma) following a given treatment of LPC(O) and PC(O) (krill oil) was compared to the elevation following a similar treatment of AKDAG (shark liver oil), identifying that LPC(O) and PC(O) have a higher bioavailability. That is, it was surprisingly found that a smaller dose of LPC(O) and PC(O) can result in an increase in plasmalogen circulating levels that is the same as that obtained by administration of a relatively larger dose of AKDAG (shark liver oil).

[0010] Notably, it was found that the phospholipids found in krill oil differ significantly from those in humans; however, it was expected that the LPC(O) levels would be related to plasmalogens or at least to the modulation of plasmalogens in humans.

[0011] Furthermore, it was recognized that the alkyl chain composition of LPC(O) and PC(O) in krill oil differs significantly from the alkenyl chain composition of plasmalogens in humans and, thus, can potentially affect the resulting plasmalogen composition, as previously reported for alkylglycerols (AKG) and alkyl diacylglycerols (AKDAG) (WO 2021 / 007623). This led to the development and disclosure of the present application for formulations of LPC(O) and prospective formulations of lysophosphalkylphosphatidylethanolamines (also known as LPE(O)) or lysophosphalkylphosphatidic acids (also known as LPA(O)) that match the plasmalogen composition known in human plasma as described herein. BRIEF DESCRIPTION OF DRAWINGS

[0012] Other aspects of the present disclosure will become apparent upon review of the detailed description of various embodiments of the disclosure described below when considered in connection with the accompanying drawings.

[0013] Figure 1The change in plasma ether lipid classes after KO, FO, or SLO supplementation relative to baseline / pre-treatment (% change) is described. The change in plasma ether lipid classes is grouped by supplement. Subjects showed significant percent changes in ether lipid classes after KO (measured at 15 and 30 days) and SLO treatment (measured at 21 days). FO supplementation had no significant effect on plasma ether lipid classes. A paired t-test was used to determine nominal significance of treatment effect (krill oil baseline vs. krill oil 15 days, krill oil baseline vs. krill oil 30 days, fish oil baseline vs. fish oil 15 days, fish oil baseline vs. fish oil 30 days; SLO pre-treatment vs. SLO 21 days); * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0014] Figure 2 The change in plasma ether lipid classes after KO, FO, or SLO supplementation relative to baseline / pre-treatment (% change) is described (grouped by lipid class). The effect of different supplements is grouped by plasma ether lipid class. Subjects showed significant percent changes in ether lipid classes after KO (measured at 15 and 30 days) and SLO treatment (measured at 21 days). FO supplementation had no significant effect on plasma ether lipid classes. A paired t-test was used to determine nominal significance of treatment effect (krill oil baseline vs. krill oil 15 days, krill oil baseline vs. krill oil 30 days, fish oil baseline vs. fish oil 15 days, fish oil baseline vs. fish oil 30 days; SLO pre-treatment vs. SLO 21 days); * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0015] Figure 3 The effect of KO supplementation on the alkenyl chain composition of PE plasmalogens is described. Data is presented as mean ± SD. KO supplementation had no significant effect on the alkenyl chain composition of PE plasmalogens. A repeated measures ANOVA followed by Tukey’s multiple comparison test was used to determine nominal significance of treatment effect; * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0016] Figure 4 The effect of FO supplementation on the alkenyl chain composition of PE plasmalogens is described. Data is presented as mean ± SD. FO supplementation had no significant effect on the alkenyl chain composition of PE plasmalogens. A repeated measures ANOVA followed by Tukey’s multiple comparison test was used to determine nominal significance of treatment effect; * indicates P < 0.05.

[0017] Figure 5The effect of SLO supplementation on the acyl chain composition of PE plasmalogens is described. Data are presented as mean ± SD. SLO supplementation decreased the proportion of PE plasmalogens containing 20:4 and increased the proportion of PE plasmalogens containing 18:1 and 22:6. Nominal significance of treatment effects was determined using paired t-tests; ** indicates P < 0.01, and *** indicates P < 0.001.

[0018] Figure 6 The effect of KO supplementation on the acyl chain composition of PE plasmalogens is described. Data are presented as mean ± SD. KO supplementation decreased the proportion of PE plasmalogens containing 18:1, 18:2, and 20:4 and increased the proportion of PE plasmalogens containing 20:5 and 22:6. Nominal significance of treatment effects was determined using repeated measures ANOVA followed by Tukey’s multiple comparison test; ** indicates P < 0.01, and *** indicates P < 0.001.

[0019] Figure 7 The effect of FO supplementation on the acyl chain composition of PE plasmalogens is described. Data are presented as mean ± SD. FO supplementation decreased the proportion of PE plasmalogens containing 18:1, 18:2, and 20:4 and increased the proportion of PE plasmalogens containing 20:5 and 22:6. Nominal significance of treatment effects was determined using repeated measures ANOVA followed by Tukey’s multiple comparison test; * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0020] Figure 8 The effect of SLO supplementation on the acyl chain composition of PE plasmalogens is described. Data are presented as mean ± SD. SLO supplementation decreased the proportion of PE plasmalogens containing 20:4 and increased the proportion of PE plasmalogens containing 18:1 and 22:6. Nominal significance of treatment effects was determined using paired t-tests; ** indicates P < 0.01, and *** indicates P < 0.001.

[0021] Figure 9 HepG2 cells are shown before and after plasmalogen precursor treatment. Cells were treated with 0.05% ethanol (vehicle control) or 20 mM of labeled plasmalogen precursors for 24 hours. Labeled precursors included alkylglycerol (O-16:0) d2 (AKG), LPC (O-16:0) d4 (LPC(O)), LPE (O-16:0) d5 (LPE(O)). Images were taken at 4x magnification using an Olympus CKX41 inverted microscope and analyzed using ImageJ. Scale bar for all images is 400 pm.

[0022] Figure 10 shows the labeled phosphatidyl ethanolamine acetal phospholipid (PE(P)) concentration after acetal phospholipid precursor treatment in HepG2 cells. Figure 10A A bar graph showing labeled PE(P) concentration for 16:0, 18:0, and 18:1 species is shown.

[0023] Figure 10B Labeled PE(P) / total label (%) is shown. Total label refers to the sum of labeled AKG, alkylacylglycerol (DG(O)), lysol alkylphosphatidylcholine (LPC(O)), lysophosphatidylcholine acetal phospholipid (LPC(P)), lysol alkylphosphatidylethanolamine (LPE(O)), lysophosphatidylethanolamine acetal phospholipid (LPE(P)), alkylphosphatidylcholine (PC(O)), phosphatidylcholine acetal phospholipid (PC(P)), alkylphosphatidylethanolamine (PE(O)), PE(P), monoalkyldiacylglycerol (TG(O)). Figure 10 shows mean ± standard deviation (n=3 per group), each symbol represents an individual sample. Figure 10B The control is not included because it divides the background value by the background value, which can be misleading. One-way ANOVA and Tukey post-hoc test were used. ns indicates p>0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001.

[0024] Figure 11 Labeled incorporation into lipid classes of the acetal phospholipid biosynthetic pathway after acetal phospholipid precursor treatment in HepG2 cells is shown. Cells were treated with 0.05% ethanol (vehicle control) or 20 mM of labeled acetal phospholipid precursors for 24 hours. Labeled precursors included alkylglycerol (O-16:0) d2 (AKG), LPC(O-16:0) d4 (LPC(O)), LPE(O-16:0) d5 (LPE(O)). Concentrations were normalized to phosphatidylcholine (PC) 34:1, one of the most abundant endogenous PCs. Figure 11 A bar graph showing labeled concentrations for AKG treatment is shown. Figure 11 B bar graph showing labeled concentrations for LPC(O) treatment is shown. Figure 11 C bar graph showing labeled concentrations for LPE(O) treatment is shown. Figure 11 Mean ± standard deviation (n=3 per group) is shown, each symbol represents an individual sample.

[0025] Figure 12 shows the distribution of label incorporation into the plasmenyl biosynthesis pathway in HepG2 cells following plasmenyl precursor treatment. Cells were treated with 0.05% ethanol (vehicle control) or 20 mM of labeled plasmenyl precursors for 24 hours. Label precursors included alkylglycerol (O-16:0) d2 (AKG), LPC (O-16:0) d4 (LPC(O)), LPE (O-16:0) d5 (LPE(O)). Figure 12A Label concentrations for each lipid class are shown divided by total label, expressed as a percentage of AKG treatment. Figure 12B Label concentrations for each lipid class are shown divided by total label, expressed as a percentage of LPE(O) treatment. Figure 12C Label concentrations for each lipid class are shown divided by total label, expressed as a percentage of LPE(O) treatment. Total label refers to the sum of label AKG, alkylacylglycerol (DG(O)), lysolkylphosphatidylcholine (LPC(O)), lysophosphatidylcholine plasmenyl (LPC(P)), lysolkylphosphatidylethanolamine (LPE(O)), lysophosphatidylethanolamine plasmenyl (LPE(P)), alkylphosphatidylcholine (PC(O)), phosphatidylcholine plasmenyl (PC(P)), alkylphosphatidylethanolamine (PE(O)), phosphatidylethanolamine plasmenyl (PE(P)), monoalkyldiacylglycerol (TG(O)).

[0026] Figure 13 Figure 13 shows 3T3 cells before and after plasmenyl precursor treatment. Cells were treated with 0.05% ethanol (vehicle control) or 20 mM of labeled plasmenyl precursors for 24 hours. Label precursors included alkylglycerol (O-16:0) d2 (AKG), LPC (O-16:0) d4 (LPC(O)), LPE (O-16:0) d5 (LPE(O)). Images were taken with an Olympus CKX41 inverted microscope at 4x magnification and analyzed using ImageJ. Scale bar for all images is 400 pm.

[0027] Figure 14 shows labeled phosphatidylethanolamine plasmenyl (PE(P)) concentrations in 3T3 cells following plasmenyl precursor treatment. Concentrations were normalized to phosphatidylcholine (PC) 34:1, one of the most abundant endogenous PCs. Figure 14A Bar graphs showing labeled PE(P) concentrations for 16:0, 18:0, and 18:1 species. Figure 14B Bar graphs showing log2 of labeled PE(P) concentrations for 16:0, 18:0, and 18:1 species. Figure 14CLabelled PE(P) / total label (%) is shown. Total label refers to the sum of labelled AKG, alkylacylglycerol (DG(O)), lysophosphatidylcholine (LPC(O)), lysophosphatidylcholine plasmalogen (LPC(P)), lysophosphatidylethanolamine (LPE(O)), lysophosphatidylethanolamine plasmalogen (LPE(P)), alkylphosphatidylcholine (PC(O)), phosphatidylcholine plasmalogen (PC(P)), alkylphosphatidylethanolamine (PE(O)), PE(P), monoalkyldiacylglycerol (TG(O)). Figure 14 shows mean ± standard deviation (n=3 per group), each symbol represents an individual sample. Figure 14C The control is not included because it divides the background value by the background value, which can be misleading. One-way ANOVA and Tukey post-hoc test were used. ns indicates p>0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001.

[0028] Figure 15 Labelled precursors of plasmalogens are shown to be incorporated into the plasmalogen biosynthetic pathway in 3T3 cells. Cells were treated with 0.05% ethanol (vehicle control) or 20 mM of labelled precursors of plasmalogens for 24 hours. Labelled precursors include alkylglycerol (O-16:0) d2 (AKG), LPC(O-16:0) d4 (LPC(O)), LPE(O-16:0) d5 (LPE(O)). Concentrations were normalised to phosphatidylcholine (PC) 34:1, one of the most abundant endogenous PCs. Figure 15 A shows a bar graph showing labelled precursor concentrations for AKG treatment. Figure 15 B shows a bar graph showing labelled precursor concentrations for LPC(O) treatment. Figure 15 C shows a bar graph showing labelled precursor concentrations for LPE(O) treatment. Figure 15 Mean ± standard deviation (n=3 per group) is shown, each symbol represents an individual sample.

[0029] Figure 16 shows the distribution of labelled precursor incorporation into the plasmalogen biosynthetic pathway in 3T3 cells after plasmalogen precursor treatment. Cells were treated with 0.05% ethanol (vehicle control) or 20 mM of labelled precursors of plasmalogens for 24 hours. Labelled precursors include alkylglycerol (O-16:0) d2 (AKG), LPC(O-16:0) d4 (LPC(O)), LPE(O-16:0) d5 (LPE(O)). Figure 16A Labelled precursor concentrations for each lipid class divided by total label are shown, expressed as a percentage of AKG treatment. Figure 16B Labelled precursor concentrations for each lipid class divided by total label are shown, expressed as a percentage of LPC(O) treatment.Figure 16C The marker concentration of each lipid class divided by the total marker is shown, expressed as a percentage of LPE(O) treatment.

[0030] Figure 17 A procedure for dosing a mixture of deuterated precursor molecules to 64 eight-week-old C57BL / 6 mice is described.

[0031] Figure 18 shows the concentration of labeled alkylglycerols (AKG) in mice following acylphospholipid precursor treatment at dose mixture A. Eight-week-old C57BL / 6 mice were administered a single dose of egg phospholipids (vehicle control), AKG mixture (AKG(O-16:0, 18:0, 18:1) d2), LPC(O) mixture (LPC(O-16:0, 18:0, 18:1) d2), LPE(O) mixture (LPE(O-16:0) d5 and unlabeled LPE(O-16:0, 18:0, 18:1)) via gavage. Blood was collected by tail tip bleeds prior to treatment and at 1, 4, 24, and 48 hours. Figure 18A A graph showing the concentration of labeled AKG O-16:0 species at time points 0, 1, 4, 24, and 48 hours of treatment at dose mixture A in female mice is shown. Figure 18B A bar graph showing the concentration of labeled AKG O-16:0 species at time points 0, 1, 4, 24, and 48 hours of treatment at dose mixture A in male mice is shown. Figure 18 shows the mean ± standard deviation (n = 4-5 per group), with each symbol representing an individual sample.

[0032] Figure 19 shows the concentration of labeled alkylphosphatidylcholines (PC(O)) in mice following acylphospholipid precursor treatment at dose mixture A. Eight-week-old C57BL / 6 mice were administered a single dose of egg phospholipids (vehicle control), AKG mixture (AKG(O-16:0, 18:0, 18:1) d2), LPC(O) mixture (LPC(O-16:0, 18:0, 18:1) d2), LPE(O) mixture (LPE(O-16:0) d5 and unlabeled LPE(O-16:0, 18:0, 18:1)) via gavage. Blood was collected by tail tip bleeds prior to treatment and at 1, 4, 24, and 48 hours. Figure 19A A graph showing the concentration of labeled PC(O) 16:0 species at time points 0, 1, 4, 24, and 48 hours of treatment at dose mixture A in female mice is shown. Figure 19B A bar graph showing the concentration of labeled PC(O) 16:0 species at time points 0, 1, 4, 24, and 48 hours of treatment at dose mixture A in male mice is shown. Figure 19 shows the mean ± standard deviation (n = 4-5 per group), with each symbol representing an individual sample.

[0033] Figure 20 shows the concentration of labeled alkenyl phosphatidyl ethanolamine (PE(O)) after treatment with the acetal phospholipid precursor of dose mix A in mice. Eight-week-old C57BL / 6 mice were administered a single dose of lecithin (vehicle control), AKG mix (AKG(O-16:0, 18:0, 18:1 )d2), LPC(O) mix (LPC(O-16:0, 18:0, 18:1 )d2), LPE(O) mix (LPE(O-16:0)d5 and unlabeled LPE(O-16:0, 18:0, 18:1 )) via gavage. Blood was collected by tail tip bleeds before treatment and at 1, 4, 24 and 48 hours. Figure 20A A plot showing the concentration of labeled PE(O) 16:0 species at the 0, 1, 4, 24 and 48 hour time points of treatment with dose mix A in female mice is shown. Figure 20B A plot showing the concentration of labeled PE(O) 16:0 species at the 0, 1, 4, 24 and 48 hour time points of treatment with dose mix A in male mice is shown. Figure 20 shows the mean ± standard deviation (n=4-5 per group), with each symbol representing an individual sample.

[0034] Figure 21 shows the concentration of labeled phosphatidyl ethanolamine acetal phospholipid (PE(P)) after treatment with the acetal phospholipid precursor of dose mix A in mice. Eight-week-old C57BL / 6 mice were administered a single dose of lecithin (vehicle control), AKG mix (AKG(O-16:0, 18:0, 18:1 )d2), LPC(O) mix (LPC(O-16:0, 18:0, 18:1 )d2), LPE(O) mix (LPE(O-16:0)d5 and unlabeled LPE(O-16:0, 18:0, 18:1 )) via gavage. Blood was collected by tail tip bleeds before treatment and at 1, 4, 24 and 48 hours. Figure 21A A plot showing the concentration of labeled PE(P) 16:0 species at the 0, 1, 4, 24 and 48 hour time points of treatment with dose mix A in female mice is shown. Figure 21B A plot showing the concentration of labeled PE(P) 16:0 species at the 0, 1, 4, 24 and 48 hour time points of treatment with dose mix A in male mice is shown. Figure 21 shows the mean ± standard deviation (n=4-5 per group), with each symbol representing an individual sample.

[0035] Figure 22 shows the concentration of labeled phosphatidyl ethanolamine plasmalogen (PE(P)) after plasmalogen precursor treatment at dose mix B in mice. Eight-week-old C57BL / 6 mice were administered a single dose of lecithin (vehicle control), AKG mix (AKG(O-16:0, 18:0, 18:1 )d2), LPC(O) mix (LPC(O-16:0, 18:0, 18:1 )d2), LPE(O) mix (LPE(O-16:0)d5 and unlabeled LPE(O-16:0, 18:0, 18:1 )) via gavage. Blood was collected by tail tip bleeds before treatment and at 1, 4, 24 and 48 hours. Figure 22A A bar graph showing the concentration of labeled PE(P) 16:0 species at time points 0, 1, 4, 24 and 48 hours after treatment with dose mix B in female mice is shown. Figure 22B A bar graph showing the concentration of labeled PE(P) 16:0 species at time points 0, 1, 4, 24 and 48 hours after treatment with dose mix B in male mice is shown. Figure 22 shows mean ± standard deviation (n=4 per group), each symbol represents an individual sample.

[0036] Figure 23 shows the maximum concentration (Cmax) of labeled phosphatidyl ethanolamine plasmalogen after plasmalogen precursor treatment in mice. max Eight-week-old C57BL / 6 mice were administered a single dose of lecithin (vehicle control), AKG mix (AKG(O-16:0, 18:0, 18:1 )d2), LPC(O) mix (LPC(O-16:0, 18:0, 18:1 )d2), LPE(O) mix (LPE(O-16:0)d5 and unlabeled LPE(O-16:0, 18:0, 18:1 )) via gavage. Blood was collected by tail tip bleeds before treatment and at 1, 4, 24 and 48 hours. Figure 23A A bar graph showing the concentration of labeled PE(P) 16:0 species at time points 0, 1, 4, 24 and 48 hours after treatment with dose mix B in female mice is shown. max A bar graph showing the concentration of labeled PE(P) 16:0 species at time points 0, 1, 4, 24 and 48 hours after treatment with dose mix B in female mice is shown. Figure 23B A bar graph showing the concentration of labeled PE(P) 16:0 species at time points 0, 1, 4, 24 and 48 hours after treatment with dose mix B in female mice is shown. max A bar graph showing the concentration of labeled PE(P) 16:0 species at time points 0, 1, 4, 24 and 48 hours after treatment with dose mix B in female mice is shown. Figure 23C A bar graph showing the concentration of labeled PE(P) 16:0 species at time points 0, 1, 4, 24 and 48 hours after treatment with dose mix B in female mice is shown. Figure 23D A bar graph showing the concentration of labeled PE(P) 16:0 species at time points 0, 1, 4, 24 and 48 hours after treatment with dose mix B in female mice is shown. max A bar graph showing the concentration of labeled PE(P) 16:0 species at time points 0, 1, 4, 24 and 48 hours after treatment with dose mix B in female mice is shown. Figure 23E A bar graph showing the concentration of labeled PE(P) 16:0 species at time points 0, 1, 4, 24 and 48 hours after treatment with dose mix B in female mice is shown. Figure 23F A bar graph showing the concentration of labeled PE(P) 16:0 species at time points 0, 1, 4, 24 and 48 hours after treatment with dose mix B in female mice is shown. max A bar graph showing the concentration of labeled PE(P) 16:0 species at time points 0, 1, 4, 24 and 48 hours after treatment with dose mix B in female mice is shown. Figure 23GA bar graph showing the treatment of dose mixture B in male mice is shown. Figure 23H A bar graph showing the log(2) AUC of the treatment of dose mixture B in male mice is shown. max Figure 23 shows the mean ± standard deviation (n = 4 per group), each symbol represents an individual sample. One-way ANOVA with Tukey’s post-hoc test was used. ns indicates p > 0.05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001.

[0037] Figure 24 shows the area under the curve (AUC) of labeled plasmalogen phosphatidylethanolamine plasmalogens after plasmalogen precursor treatment in mice. Eight-week-old C57BL / 6 mice were administered a single dose of lecithin (vehicle control), AKG mixture (AKG(O-16:0, 18:0, 18:1) d2), LPC(O) mixture (LPC(O-16:0, 18:0, 18:1) d2), LPE(O) mixture (LPE(O-16:0) d5 and unlabeled LPE(O-16:0, 18:0, 18:1)) via gavage. Blood was collected by tail tip bleeds before treatment and at 1, 4, 24, and 48 hours. Figure 24A A bar graph showing the AUC of the treatment of dose mixture A in female mice is shown. Figure 24B A bar graph showing the log(2) AUC of the treatment of dose mixture A in female mice is shown. Figure 24C A bar graph showing the AUC of the treatment of dose mixture A in male mice is shown. Figure 24D A bar graph showing the log(2) AUC of the treatment of dose mixture A in male mice is shown.

[0038] Figure 24E A bar graph showing the AUC of the treatment of dose mixture B in female mice is shown. Figure 24F A bar graph showing the log(2) AUC of the treatment of dose mixture B in female mice is shown. Figure 24G A bar graph showing the AUC of the treatment of dose mixture B in male mice is shown. Figure 24H A bar graph showing the log(2) AUC of the treatment of dose mixture B in male mice is shown. Figure 24 shows the mean ± standard deviation (n = 4 per group), each symbol represents an individual sample. One-way ANOVA with Tukey’s post-hoc test was used. ns indicates p > 0.05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001.

[0039] Figure 25 shows the area under the curve (AUC) of lipid classes of the plasmalogen biosynthetic pathway following plasmalogen precursor treatment of dose mix A in female mice. Eight-week-old C57BL / 6 mice were administered a single dose of egg phosphatidylcholine (vehicle control), AKG mix (AKG (0-16:0, 18:0, 18:1) d2), LPC(O) mix (LPC(O-16:0, 18:0, 18:1) d2), LPE(O) mix (LPE(O-16:0) d5 and unlabeled LPE(O-16:0, 18:0, 18:1)) via gavage. Blood was collected by tail tip bleeds prior to treatment and at 1, 4, 24, and 48 hours. Figure 25A A bar graph showing AUC of AKG treatment is shown. Figure 25B A bar graph showing AUC of LPC(O) treatment is shown. Figure 25C A bar graph showing AUC of LPE(O) treatment is shown. Figure 25 shows mean ± standard deviation (n=4 per group), with each symbol representing an individual sample.

[0040] Figure 26 shows the area under the curve (AUC) of lipid classes of the plasmalogen biosynthetic pathway following plasmalogen precursor treatment of dose mix A in male mice. Eight-week-old C57BL / 6 mice were administered a single dose of egg phosphatidylcholine (vehicle control), AKG mix (AKG (0-16:0, 18:0, 18:1) d2), LPC(O) mix (LPC(O-16:0, 18:0, 18:1) d2), LPE(O) mix (LPE(O-16:0) d5 and unlabeled LPE(O-16:0, 18:0, 18:1)) via gavage. Blood was collected by tail tip bleeds prior to treatment and at 1, 4, 24, and 48 hours. Figure 26A A bar graph showing AKG treatment is shown. Figure 26B A bar graph showing LPC(O) treatment is shown. Figure 26C A bar graph showing LPE(O) treatment is shown. Figure 26 shows mean ± standard deviation (n=4 per group), with each symbol representing an individual sample.

[0041] Figure 27 A labeled plasmalogen phosphatidylethanolamine O-16:0 species in the brain following supplementation of labeled LPE(O) in mice is shown. Eight-week-old C57BL / 6 mice were administered a single dose of egg phosphatidylcholine (vehicle control) or LPE(O) mix (LPE(O-16:0, O-18:0, O-18:1, O-16:0-d5)) via gavage. Brain samples were collected 48 hours after gavage. Figure 27 A bar graph showing dose mix A treatment in female mice is shown. Figure 27B shows a bar graph showing the treatment of dose mixture A in male mice. Figure 27 C shows a bar graph showing the treatment of dose mixture B in female mice. Figure 27 D shows a bar graph showing the treatment of dose mixture B in male mice. Figure 27 The mean ± standard deviation is shown (n = 4 per group), each symbol represents an individual sample. Student's t-test was used to compare the mean differences between groups. *p < 0.05, ***p < 0.001;

[0042] Figure 28 shows the labeled phosphatidylethanolamine acetal phospholipids of the O-16:0 species in the brain following supplementation of the labeled AKG(O) or LPC(O) in mice. Eight-week-old C57BL / 6 mice were administered a single dose of lecithin (vehicle control), AKG mixture (AKG(O-16:0-d2, O-18:0-d2, O-18: l-d2)) or LPC(O) mixture (LPC(O-16:0-d2, O-18:0-d2) via gavage. Brain samples were collected 48 hours after gavage. Figure 28 A shows a bar graph showing the treatment of dose mixture A in female mice, Figure 28 B shows a bar graph showing the treatment of dose mixture A in male mice. Figure 28 C shows a bar graph showing the treatment of dose mixture B in female mice. Figure 28 D shows a bar graph showing the treatment of dose mixture B in male mice. Figure 28 The mean ± standard deviation is shown (n = 4 per group), each symbol represents an individual sample. One-way ANOVA was used to test the mean differences between groups.

[0043] Figure 29 shows the change in tracer alkyl diacylglycerol [TG(O)] concentration following administration of different precursor compounds. The precursor compounds are Figure 29A AKG-dose mixture C; Figure 29B AKDAG-oleic acid-dose mixture C; Figure 29C AKDAG-DHA-dose mixture C; Figure 29D LPC(O)-dose mixture C; Figure 29E AKG-dose mixture D; Figure 29F AKDAG-oleic acid-dose mixture E; Figure 29G AKDAG-DHA-dose mixture D; Figure 29H LPC(O)-dose mixture D.

[0044] Figure 30 shows the change in tracer lysophosphatidylcholine [LPC(O)] concentration following administration of different precursor compounds. The precursor compounds areFigure 30A AKG-dose mixture C; Figure 30B AKDAG-oleic acid-dose mixture C; Figure 30C AKDAG-DHA-dose mixture C; Figure 30D LPC(O)-dose mixture C; Figure 30E AKG-dose mixture D; Figure 30F AKDAG-oleic acid-dose mixture E; Figure 30G AKDAG-DHA-dose mixture D; Figure 30H LPC(O)-dose mixture D.

[0045] Figure 31 shows the change in tracer alkyl phosphatidylcholine [PC(O)] concentration following administration of different precursor compounds. The precursor compounds are Figure 31A AKG-dose mixture C; Figure 31B AKDAG-oleic acid-dose mixture C; Figure 31C AKDAG-DHA-dose mixture C; Figure 31D LPC(O)-dose mixture C; Figure 31E AKG-dose mixture D; Figure 31F AKDAG-oleic acid-dose mixture E; Figure 31G AKDAG-DHA-dose mixture D; Figure 31H LPC(O)-dose mixture D.

[0046] Figure 32 shows the change in tracer alkyl phosphatidylethanolamine [PE(O)] concentration following administration of different precursor compounds. The precursor compounds are Figure 32A AKG-dose mixture C; Figure 32B AKDAG-oleic acid-dose mixture C; Figure 32C AKDAG-DHA-dose mixture C; Figure 32D LPC(O)-dose mixture C; Figure 32E AKG-dose mixture D; Figure 32F AKDAG-oleic acid-dose mixture E; Figure 32G AKDAG-DHA-dose mixture D; Figure 32H LPC(O)-dose mixture D.

[0047] Figure 33 shows the change in tracer alkyl phosphatidylethanolamine [PE(P)] concentration following administration of different precursor compounds. The precursor compounds are Figure 33A AKG-dose mixture C; Figure 33B AKDAG-oleic acid-dose mixture C; Figure 33C AKDAG-DHA-dose mixture C; Figure 33DLPC(O)-dose mixture C; Figure 33E AKG-dose mixture D; Figure 33F AKDAG-oleic acid-dose mixture E; Figure 33G AKDAG-DHA-dose mixture D; Figure 33H LPC(O)-dose mixture D.

[0048] Figure 34 shows the change in tracer concentration of alkenyl phosphatidylcholine [PC(P)] after administration of different precursor compounds. The precursor compounds are Figure 34A AKG-dose mixture C; Figure 34B AKDAG-oleic acid-dose mixture C; Figure 34C AKDAG-DHA-dose mixture C; Figure 34D LPC(O)-dose mixture C; Figure 34E AKG-dose mixture D; Figure 34F AKDAG-oleic acid-dose mixture E; Figure 34G AKDAG-DHA-dose mixture D; Figure 34H LPC(O)-dose mixture D.

[0049] Figure 35 shows the maximum plasma concentration (Cmax) of major lipid classes after administration of different precursor compounds. The precursor compounds are Figure 35A TG(O); Figure 35B LPC(O); Figure 35C PC(O); Figure 35D PE(O); Figure 35E PE(P); Figure 35F PC(P).

[0050] Figure 36 shows the area under the curve (AUC) of major lipid classes after administration of precursor compounds. The precursor compounds are Figure 36A TG(O); Figure 36B LPC(O); Figure 36C PC(O); Figure 36D PE(O); Figure 36E PE(P); Figure 36F PC(P).

[0051] Figure 37 shows the effect of marker precursor supplementation on major ether lipid classes in the liver. The precursor compounds are Figure 37A TG(O); Figure 37B LPC(O); Figure 37C PC(O); Figure 37D PE(O); Figure 37E PE(P); Figure 37F PC(P).

[0052] Figure 38 shows the effect of marker precursor supplementation on major ether lipid classes in the spleen. The precursor compounds are Figure 38A TG(O); Figure 38B LPC(O); Figure 38C PC(O); Figure 38D PE(O); Figure 38E PE(P); Figure 38F PC(P).

[0053] Figure 39 shows the effect of marker precursor supplementation on major ether lipid classes in the brain. The precursor compounds are Figure 39A TG(O); Figure 39B LPC(O); Figure 39C PC(O); Figure 39D PE(O); Figure 39E PE(P); Figure 39F PC(P).

[0054] Figure 40 shows the effect of marker precursor supplementation on major ether lipid classes in the kidney. The precursor compounds are Figure 40A TG(O); Figure 40B LPC(O); Figure 40C PC(O); Figure 40D PE(O); Figure 40E PE(P); Figure 40F PC(P).

[0055] Figure 41 shows the effect of marker precursor supplementation on major ether lipid classes in visceral adipose tissue. The precursor compounds are Figure 41A TG(O); Figure 41B LPC(O); Figure 41C PC(O); Figure 41D PE(O); Figure 41E PE(P); Figure 41F PC(P).

[0056] Figure 42 shows the effect of marker precursor supplementation on major ether lipid classes in skeletal muscle. The precursor compounds are Figure 42A TG(O); Figure 42B LPC(O); Figure 42C PC(O); Figure 42D PE(O); Figure 42E PE(P); Figure 42F PC(P).

[0057] Figure 43 shows the effect of marker precursor supplementation on major ether lipid classes in the heart. The precursor compounds are Figure 43A TG(O); Figure 43B LPC(O); Figure 43CPC(O); Figure 43D PE(O); Figure 43E PE(P); Figure 43F PC(P).

[0058] Figure 44 The effect of LPC(O) supplementation on the level of plasmalogen in RAW 264.7 cells is shown. RAW 264.7 macrophages were treated with 20 μΜ LPC(O) with different SN1 composition for 24 hours, then harvested for lipidomics analysis. PE plasmalogen or PE(P) data were normalized to total cellular phosphatidylcholine (PC) level, and presented as mean ± SD (n = 3 / group). Each circle represents individual data points. Mean differences between groups were compared by one-way ANOVA followed by Fisher LSD test. * indicates P < 0.05, ** indicates P < 0.01, **** indicates P < 0.0001 compared to control.

[0059] Figure 45 The effect of LPE(O) supplementation on the level of plasmalogen in RAW 264.7 cells is shown. RAW 264.7 macrophages were treated with 20 μΜ LPE(O) with different SN1 composition for 24 hours, then harvested for lipidomics analysis. PE plasmalogen or PE(P) data were normalized to total cellular phosphatidylcholine (PC) level, and presented as mean ± SD (n = 3 / group). Each circle represents individual data points. Mean differences between groups were compared by one-way ANOVA followed by Fisher LSD test. **** indicates P < 0.0001 compared to control.

[0060] Figure 46 The effect of LPC(O) supplementation on the composition of plasmalogen in RAW 264.7 cells is shown. RAW 264.7 macrophages were treated with 20 μΜ LPC(O) with different SN1 composition for 24 hours, then harvested for lipidomics analysis. The relative proportion of PE(P) containing different SN1 was presented as mean ± SD (n = 3 / group).

[0061] Figure 47The effect of LPC(O) supplementation on the level of plasmenyl phospholipids in 3T3-L1 cells is shown. 3T3-L1 preadipocytes were treated with 20 mM LPC(O) with different SN1 composition for 24 hours, then harvested for lipidomics analysis. PE plasmenyl phospholipid or PE(P) data were normalized to total cellular phosphatidylcholine (PC) levels, and presented as mean ± SD (n=3 / group). Each circle represents an individual data point. Mean differences between groups were compared by one-way ANOVA followed by Fisher LSD test. * indicates P < 0.05, ** indicates P < 0.01, **** indicates P < 0.0001 compared to control.

[0062] Figure 48 The effect of LPE(O) supplementation on the level of plasmenyl phospholipids in 3T3-L1 cells is shown. 3T3-L1 preadipocytes were treated with 20 mM LPE(O) with different SN1 composition for 24 hours, then harvested for lipidomics analysis. PE plasmenyl phospholipid or PE(P) data were normalized to total cellular phosphatidylcholine (PC) levels, and presented as mean ± SD (n=3 / group). Each circle represents an individual data point. Mean differences between groups were compared by one-way ANOVA followed by Fisher LSD test. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001 compared to control.

[0063] Figure 49 The effect of LPC(O) supplementation on the composition of plasmenyl phospholipids in 3T3-L1 cells is shown. 3T3-L1 cells were treated with 20 mM LPC(O) with different SN1 composition for 24 hours, then harvested for lipidomics analysis. The relative proportion of PE(P) containing different SN1 was presented as mean ± SD (n=3 / group).

[0064] Figure 50 The effect of LPE(O) supplementation on the composition of plasmenyl phospholipids in 3T3-L1 cells is shown. 3T3-L1 cells were treated with 20 mM LPE(O) with different SN1 composition for 24 hours, then harvested for lipidomics analysis. The relative proportion of PE(P) containing different SN1 was presented as mean ± SD (n=3 / group).

[0065] Figure 51 shows the incorporation (area under the curve) of markers in lipid classes to the plasmenyl phospholipid biosynthetic pathway after low dose plasmenyl phospholipid precursor supplementation. Figure 51A Incorporation after AKG supplementation is shown; Figure 51B Incorporation after AKD AG-oleic acid supplementation is shown; Figure 51C Incorporation after AKD AG-DHA supplementation is shown;Figure 51D Incorporation after LPC(O) supplementation is shown.

[0066] Figure 52 shows the distribution of label incorporation into the plasmalogen biosynthetic pathway after plasmalogen precursor supplementation. Figure 52A Incorporation after AKG supplementation is shown. Figure 52B Incorporation after AKD AG-oleic acid supplementation is shown. Figure 52C Incorporation after AKD AG-DHA supplementation is shown. Figure 52D Incorporation after LPC(O) supplementation is shown.

[0067] Figure 53 shows the labeled ether lipids in feces after 24 hours of labeled precursor supplementation Figure 53A TG(O); Figure 53B LPC(O); Figure 53C PC(O); Figure 53D PE(O); Figure 53E PE(P); Figure 53F PC(P).

[0068] Figure 54 shows the labeled ether lipids in feces after 24 hours of labeled precursor supplementation Figure 54A TG(O); Figure 54B LPC(O); Figure 54C PC(O); Figure 54D PE(O); Figure 54E PE(P); Figure 54F PC(P).

[0069] Figure 55 All LPX(O) treatments inhibited the LPS-induced increase in TLR4 gene expression is shown. Data was normalized to control + LPS. Data is presented as mean ± SD (n = 3 / group). Mean differences between groups were compared by one-way ANOVA followed by Fisher’s LSD test. Asterisks (*) indicate P < 0.05 compared to control, and ^ indicates P < 0.05 compared to control + LPS.

[0070] Figure 56 All LPX(O) treatments inhibited the LPS-induced increase in TLR4 gene expression is shown. Data was normalized to control. Data is presented as mean ± SD (n = 3 / group). Mean differences between groups were compared by one-way ANOVA followed by Fisher’s LSD test. * indicates P < 0.05 compared to control, and ^ indicates P < 0.05 compared to control + LPS.

[0071] Table 1. List of abbreviations used in the disclosure.

[0072] Table 1. List of abbreviations used in the disclosure.

[0073]

[0074] DETAILED DESCRIPTION

[0075] The general structure of the phospholipids described herein is depicted by the following formula (I):

[0076]

[0077] In the compounds of formula (I), the sn-1 carbon is labeled as above. R 3 including phosphates and substituted phosphates. R 1 and R 2 may each independently be hydrogen, alkyl, alkenyl, acyl, and the like or substituted versions of these moieties, as described herein.

[0078] In Scheme I, some typical acetal phospholipids are shown in the top two structures, with the box highlighting the sn-1 substitution as derived from formula (I).

[0079]

[0080] Scheme I. Typical acetal phospholipids.

[0081] DEFINITIONS

[0082] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0083] The term “and / or,” e.g., “X and / or Y” shall be understood to mean either “X and Y” or, in the alternative, “X or Y” and shall be understood to provide explicit support for both meanings or for either meaning.

[0084] The terms “a” and “an,” as used herein, mean “one or more” and include the plural, unless the context clearly indicates otherwise.

[0085] The term “alkyl,” as used herein, refers to a saturated straight chain or branched chain hydrocarbon, such as a straight chain or branched chain group having 1-30, 1-18, 14-24, 14-18, 16, or 18 carbon atoms, which are referred to herein as C1-C30alkyl, C1-C18alkyl, C14-C24alkyl, C14-C18alkyl, C16alkyl, and C18alkyl, respectively. 30 alkyl, C1-C 18 alkyl, C 14 -C 24 alkyl, C 14 -C 18 alkyl, C 16 alkyl or C 18Alkyl. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, isopropyl, 2-methyl-l -propyl, 2-methyl-2-propyl, 2-methyl-l -butyl, 3-methyl-l -butyl, 2-methyl-3-butyl, 2,2-dimethyl-l -propyl, 2-methyl-l -pentyl, 3-methyl-l -pentyl, 4-methyl-l -pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-l -butyl, 3,3-dimethyl-l -butyl, 2-ethyl-l -butyl, isobutyl, t-butyl, isopentyl, neopentyl, and the like.

[0086] As used herein, the term "alkenyl" refers to unsaturated straight-chain or branched- chain hydrocarbons, such as straight-chain or branched-chain groups having 2-30, 2-18, 14-24, 14-18, 16, or 18 carbon atoms, respectively, herein referred to as C2-C30, C2-C18, C14-C24, C14-C18, C16, or C18 alkenyl. Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, isopropenyl, 2-methyl-l -butenyl, 3-methyl-l -butenyl, 2-methyl-3-butenyl, 2,2-dimethyl-l -propenyl, 2-methyl-l -pentenyl, 3-methyl-l -pentenyl, 4-methyl-l -pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 2,2-dimethyl-l -butenyl, 3,3-dimethyl-l -butenyl, 2-ethyl-l -butenyl, isobutenyl, t-butenyl, isopentenyl, neopentenyl, and the like. The term "alkenyl" can also be understood to mean an alkene having the structure C18:1, wherein the double bond is typically at the n7 or n9 position. The term "alkenyl" can also be understood to mean an alkene having the structure C18:1, wherein the double bond is between the first and second carbons adjacent to the ether linkage. The term "alkenyl" can also be understood to mean an alkene wherein one double bond is at the n7 or n9 position and a second double bond is between the first and second carbons. 30 Alkenyl, C2-C 18 Alkenyl, C 14 -C 24 Alkenyl, C 14 -C 18 Alkenyl, C 16 Alkenyl or C 18 Alkenyl. Multiple double bonds can be present in alkenyl compounds. Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, isopropenyl, 2-methyl-l -butenyl, 3-methyl-l -butenyl, 2-methyl-3-butenyl, 2,2-dimethyl-l -propenyl, 2-methyl-l -pentenyl, 3-methyl-l -pentenyl, 4-methyl-l -pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 2,2-dimethyl-l -butenyl, 3,3-dimethyl-l -butenyl, 2-ethyl-l -butenyl, isobutenyl, t-butenyl, isopentenyl, neopentenyl, and the like. The term "alkenyl" can also be understood to mean an alkene having the structure C18:1, wherein the double bond is typically at the n7 or n9 position. The term "alkenyl" can also be understood to mean an alkene having the structure C18:1, wherein the double bond is between the first and second carbons adjacent to the ether linkage. The term "alkenyl" can also be understood to mean an alkene wherein one double bond is at the n7 or n9 position and a second double bond is between the first and second carbons.

[0087] As used herein throughout, the term "acyl" refers to a radical having the general formula -C(O)R, where R is hydrogen or a saturated or unsaturated, straight-chained or branched hydrocarbon, such as a straight-chained or branched group having 1-30, 1-18, 14-24, 14-18, 16, or 18 carbon atoms, respectively, which are referred to herein as C1-C30, C1-C18, C14-C24, C14-C18, C16, or C18 acyl groups, respectively. 30 alkyl, C1-C 18 alkyl, C 14 -C 24 alkyl, C 14 -C 18 alkyl, C 16 alkyl, C 18 alkyl, or a straight-chained or branched group having 2-30, 2-18, 14-24, 14-18, 16, or 18 carbon atoms, respectively, which are referred to herein as C2-C30, C2-C18, C14-C24, C14-C18, C16, or C18 alkyl groups, respectively. 30 alkenyl, C2-C 18 alkenyl, C 14 -C 24 alkenyl, C 14 -C 18 alkenyl, C 16 alkenyl, or C 18 alkenyl. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, isopropyl, 2-methyl-l -propyl, 2-methyl-2-propyl, 2-methyl-l -butyl, 3-methyl-l -butyl, 2-methyl-3-butyl, 2,2-dimethyl-l -propyl, 2-methyl-l -pentyl, 3-methyl-l -pentyl, 4-methyl-l -pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-l -butyl, 3,3-dimethyl-l -butyl, 2-ethyl-l -butyl, isobutyl, t-butyl, isopentyl, neopentyl, and the like.

[0088] As used herein, the term "ether" refers to any organic compound having a structure similar to an ether functional group having an oxygen atom that links two alkyl or other organic groups. As used herein, the term "ether" can refer to a functional group having an oxygen atom that links a saturated, straight-chained or branched hydrocarbon, such as a straight-chained or branched group having 1-30, 1-18, 14-24, 14-18, 16, or 18 carbon atoms, respectively, which are referred to herein as C1-C30, C1-C18, C14-C24, C14-C18, C16, or C18 ether groups, respectively. 30 alkyl, C1-C 18 alkyl, C 14 -C 24 alkyl, C 14 -C 18 alkyl, C 16 alkyl, or C18 alkyl, and a second saturated linear or branched hydrocarbon. As used herein, the term "ether" can also refer to a functional group having an oxygen atom attached to a saturated linear or branched hydrocarbon, such as a linear or branched group having 1-30, 1-18, 14-24, 14-18, 16, or 18 carbon atoms, which are referred to herein as C1-C30, C1-C18, C14-C24, C14-C18, C16, or C18, respectively. 30 alkyl, C1-C 18 alkyl, C 14 -C 24 alkyl, C 14 -C 18 alkyl, C 16 alkyl, C 18 alkyl, and a second saturated linear or branched hydrocarbon. As used herein, the term "ether" can also refer to a functional group having an oxygen atom attached to a saturated linear or branched hydrocarbon, such as a linear or branched group having 1-30, 1-18, 14-24, 14-18, 16, or 18 carbon atoms, which are referred to herein as C1-C30, C1-C18, C14-C24, C14-C18, C16, or C18, respectively. 30 alkenyl, C2-C 18 alkenyl, C 14 -C 24 alkenyl, C 14 -C 18 alkenyl, C 16 alkenyl, or C 18 alkenyl. As used herein, the term "ether" can also refer to a functional group having an oxygen atom attached to an unsaturated linear or branched hydrocarbon, such as a linear or branched group having 2-30, 2-18, 14-24, 14-18, 16, or 18 carbon atoms, which are referred to herein as C2-C30, C2-C18, C14-C24, C14-C18, C16, or C18, respectively. 30 alkenyl, C2-C 18 alkenyl, C 14 -C 24 alkenyl, C 14 -C 18 alkenyl, C 16 alkenyl, or C 18 alkenyl, and a second unsaturated linear or branched hydrocarbon.

[0089] As used herein, the term "vinyl ether" refers to a group in which an alkene is adjacent to an ether linkage.

[0090] As used herein, the term "ester" refers to any organic compound having a structure similar to an ester functional group. As referred to herein, the term "ester" refers to any compound or functional group that can be represented by a radical having the general formula -RCOOR', where R is hydrogen or a saturated or unsaturated linear or branched hydrocarbon, such as a linear or branched group having 1-30, 1-18, 14-24, 14-18, 16, or 18 carbon atoms, which are referred to herein as C1-C30, C1-C18, C14-C24, C14-C18, C16, or C18, respectively. 30 alkyl, C1-C 18 alkyl, C14 -C 24 Alkyl, C 14 -C 18 Alkyl, C 16 Alkyl or C 18 an alkyl group, or a straight or branched chain group having 2-30, 2-18, 14-24, 14-18, 16 or 18 carbon atoms, which are referred to herein as C2-C 30 Alkenyl, C2-C 18 Alkenyl, C 14 -C 24 Alkenyl, C 14 -C 18 Alkenyl, C 16 Alkenyl or C 18 alkenyl, and R' is a saturated or unsaturated straight or branched chain hydrocarbon group, such as a straight or branched chain group having 1-30, 1-18, 14-24, 14-18, 16 or 18 carbon atoms, which are referred to herein as C1-C 30 Alkyl, C1-C 18 Alkyl, C 14 -C 24 Alkyl, C 14 -C 18 Alkyl, C 16 Alkyl or C 18 an alkyl group, or a straight or branched chain group having 2-30, 2-18, 14-24, 14-18, 16 or 18 carbon atoms, which are referred to herein as C2-C 30 Alkenyl, C2-C 18 Alkenyl, C 14 -C 24 Alkenyl, C 14 -C 18 Alkenyl, C 16 Alkenyl or C 18 Alkenyl.

[0091] The terms "substituted" and "optionally substituted" with respect to phosphate groups, alkyl groups, alkenyl groups or acyl groups mean that these groups are optionally substituted with additional moieties. In each case, the substituents or additional moieties may be independently selected from hydrogen, C 1-3 Alkyl and halogen (eg, Cl, F, Br, or I). In certain embodiments, a group is unsubstituted, ie, it is unsubstituted.

[0092] The lipid nomenclature used here follows the guidelines established by the Lipid Maps Consortium and the shorthand notation of Liebisch et al. [Liebisch et al., Fahy et al. (2009), Fahy et al. (2005)]. Alternatively, the nomenclature is based on Biochemical Nomenclature and Related Documents, 2nd Edition, Portland Press (London, 1992) Edited C Liebecq. [ISBN 1-85578-005-4], “Nomenclature of Lipids”. Phospholipids typically contain two fatty acid chains, and in the absence of detailed characterization, are denoted by the total composition of carbon atoms and double bonds (e.g., PC(38:6)). However, if the acyl chain composition has been determined, the nomenclature rules dictate this situation (e.g., PC(38:6) is changed to PC(16:0 / 22:6)).

[0093] The present disclosure relates to lipid molecules using a numbering system X:Y. The number X represents the number of carbon atoms present in the chain.

[0094] In the context of alkyl glycerols, alkyl acylglycerols, and alkyl diacylglycerols, the number Y represents the number of double bonds present in the chain. For example, an alkyl glycerol numbered 16:0 contains a hydrocarbon group with a 16-carbon chain and no double bonds. As a further example, an alkyl glycerol numbered 18:1 contains a hydrocarbon group with an 18-carbon chain and 1 double bond.

[0095] In the context of plasmalogens / plasmenyl phospholipids, the number Y in the first listed alkenyl chain (i.e., PE(P-X:Y / X:Y)) represents the number of double bonds present in the alkenyl chain in addition to the vinyl ether group. For example, in a plasmalogen numbered PE(P-16:0 / 20:4), the 16:0 alkenyl group contains a hydrocarbon group with a 16-carbon chain that has no double bonds in addition to the vinyl ether group (i.e., there is a double bond between the first 2 carbon atoms, and the remaining 14 carbons are saturated). As another example, in a plasmalogen numbered PE(P-18:1 / 20:4), the 18:1 alkenyl group contains a hydrocarbon group with an 18-carbon chain that has 1 double bond in addition to the vinyl ether group (i.e., there is a double bond between the first 2 carbon atoms, and there is also one between 2 of the remaining 16 carbons). As shown in Scheme I above, a plasmenyl PE plasmalogen is represented by the structure PE(P-16:0 / 22:6).

[0096] If the ether lipid contains one or more double bonds, these double bonds can be located at different positions in the hydrocarbon chain. For example, an alkyl glycerol numbered as 18:1 can contain, for example, a mixture of species with a cis-n1 and a cis-n9 double bond. As another example, an acetal phosphatidyl numbered as 18:1 (e.g., PE(P)) can contain, for example, a mixture of species with a cis-n1 and a cis-n9 double bond.

[0097] As used herein, the term “alkyl ether phosphatidyl” is understood to mean a phospholipid having an ether linkage with an alkyl group in the sn-1 position.

[0098] As used herein, the term “acetal phosphatidyl” is understood to mean a phospholipid having an ether linkage with an alkenyl group in the sn-1 position. Acetal phosphatidyl phospholipids are referred to as “acetal phospholipids.”

[0099] An acetal phospholipid having a “15:0” alkenyl group is a molecule having an ether linkage with a 15-carbon chain in the sn-1 position, which contains a double bond (typically a cis-vinyl ether group) between carbons 1 and 2, and which does not contain any other double bonds in the chain.

[0100] An acetal phospholipid having a “16:0” alkenyl group is a molecule having an ether linkage with a 16-carbon chain in the sn-1 position, which contains a double bond (typically a cis-vinyl ether group) between carbons 1 and 2, and which does not contain any other double bonds in the chain.

[0101] An acetal phospholipid having a “17:0” alkenyl group is a molecule having an ether linkage with a 17-carbon chain in the sn-1 position, which contains a double bond (typically a cis-vinyl ether group) between carbons 1 and 2, and which does not contain any other double bonds in the chain.

[0102] An acetal phospholipid having a “18:0” alkenyl group is a molecule having an ether linkage with an 18-carbon chain in the sn-1 position, which contains a double bond (typically a cis-vinyl ether group) between carbons 1 and 2, and which does not contain any other double bonds in the chain.

[0103] An acetal phospholipid having a “18:1” alkenyl group is a molecule having an ether linkage with an 18-carbon chain in the sn-1 position, which contains a double bond (typically a cis-vinyl ether group) between carbons 1 and 2, and typically has one additional double bond between carbons 7 and 8 (e.g., n7), between carbons 9 and 10 (e.g., n9), or between carbons 11 and 12 (e.g., n 11), and typically is a cis-double bond.

[0104] An alkenyl-acyl glycerophospholipid is a molecule having an ether linkage to a 18- carbon chain in the sn-2 position, which chain contains two double bonds, typically cis-double bonds, between carbons 9 and 10 and between carbons 11 and 12.

[0105] An alkenyl-acyl glycerophospholipid is a molecule having an ether linkage to a 18- carbon chain in the sn-2 position, which chain contains two double bonds, typically cis-double bonds, between carbons 9 and 10 and between carbons 11 and 12.

[0106] An alkenyl-acyl glycerophospholipid is a molecule having an ether linkage to a 18- carbon chain in the sn-2 position, which chain contains two double bonds, typically cis-double bonds, between carbons 9 and 10 and between carbons 11 and 12.

[0107] An alkenyl-acyl glycerophospholipid is a molecule having an ether linkage to a 18- carbon chain in the sn-2 position, which chain contains two double bonds, typically cis-double bonds, between carbons 9 and 10 and between carbons 11 and 12.

[0108] An alkenyl-acyl glycerophospholipid is a molecule having an ether linkage to a 18- carbon chain in the sn-2 position, which chain contains two double bonds, typically cis-double bonds, between carbons 9 and 10 and between carbons 11 and 12.

[0109] As used herein, "acylalkenyl" refers to a straight or branched chain hydrocarbon containing, for example, 2 to 30 carbons and containing at least one carbon-carbon double bond, covalently bound to an acyl group. For example, the nomenclature such as 22:6 or 18:2 used in the context of an acylalkenyl group refers to an acylalkenyl group having 22 carbons or 18 carbons and having 6 or 2 double bonds, respectively. Examples of acylalkenyl groups are:

[0110]

[0111] An acylalkenyl group can be present in species such as an alkylacylglycerol or an alkyl diacylglycerol (as an acyl group), or as an acyl group in an alkyl ether phosphatidyl or an alkylidene phosphatidyl phospholipid. Typically, when present in these species, there is no double bond between the alpha-carbon and the beta-carbon and the acyl group.

[0112] As used herein, “acylalkyl” refers to a straight chain or branched hydrocarbon containing, for example, 1 to 30 carbons covalently bound to an acyl group. For example, the nomenclature 22:0 or 18:0 used in the context of acylalkyl groups refers to acylalkyl groups having 22 carbons or 18 carbons, respectively. Examples of acylalkyl groups are:

[0113]

[0114] It will also be recognized that the compounds described herein can have asymmetric centers and therefore exist in more than one stereoisomeric form. Accordingly, in at least some embodiments, the present disclosure is also directed to compounds in substantially pure isomeric form at one or more asymmetric centers, e.g., the enantiomeric purity of the compound is equal to or greater than 90% enantiomeric excess (“ee”), such as 95% ee, 97% ee, 99% ee, or greater than 99% ee. The present disclosure is also directed to compounds that exist as mixtures of stereoisomeric forms, including racemic, diastereomeric, or scalemic mixtures. Such isomers can be naturally occurring, or can be prepared by asymmetric synthesis, including but not limited to synthetic methods employing chiral intermediates, or by chiral resolution. Certain compounds contained in compositions of the present disclosure can exist in particular geometric or stereoisomeric forms. The present disclosure encompasses all such compounds within the scope of the disclosure, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)- isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof. Additional asymmetric carbon atoms can be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included within the scope of the present disclosure. If a particular enantiomer of a compound of the present disclosure is desired, it can be prepared either by asymmetric synthesis or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, if the molecule contains a basic functionality (such as an amino group) or an acidic functionality (such as a carboxyl group), diastereomeric salts are formed with an appropriate optically active acid or base, followed by resolution of the resultant diastereomeric mixture and cleavage of the auxiliary group to provide the pure desired enantiomer.

[0115] The present disclosure also relates to derivatives of glycerol. While glycerol is achiral, the derivatives are generally chiral. Typically, the glycerol employed will have a stereochemical configuration corresponding to that found in nature. In some embodiments, the glycerol derivative employed has a stereochemical configuration as shown in Formula (I) above.

[0116] As referred to herein, the term “alkyl glycerol” means a compound of Formula (I), wherein R 1group is a hydrocarbon chain, and R 2 and R 3 group is hydrogen. The term "alkyl glycerol" is additionally understood to mean a compound of formula (I-A) wherein R A is a hydrocarbon chain, and R x group is independently selected from hydrogen or C 1-3 alkyl. The term "alkyl glycerol" is additionally understood to mean a compound of formula (I-A1), (I-A2), and (I-A3). Although the term "alkyl glycerol" is used, it will be understood by those skilled in the art that the term encompasses species having a hydrocarbon group at the R 1 position that includes unsaturation of the hydrocarbon chain. However, as used herein, "alkyl glycerol" compounds do not contain a double bond between carbons 1 and 2 of the hydrocarbon chain (i.e., proximal to the ether linkage).

[0117] An alkyl glycerol having a "16:0" group is a molecule having an ether linkage in the sn-1 position with a 16-carbon saturated hydrocarbon chain and no double bonds in the chain, also known as chondrilline.

[0118] An alkyl glycerol having an "18:0" group is a molecule having an ether linkage in the sn-1 position with an 18-carbon saturated hydrocarbon chain and no double bonds in the chain, also known as batrachol.

[0119] An alkyl glycerol having an "18:1" group is a molecule having an ether linkage in the sn-1 position with an 18-carbon hydrocarbon chain that typically contains a double bond (typically a cis-double bond) between carbons 9 and 10, also known as batroxol.

[0120] As referred to herein, the term "alkyl acyl glycerol" means a compound of formula (I) wherein R 1 group is a hydrocarbon chain, R 2 and R 3 one of the R 2 and R 3 groups is an acyl group independently selected from acyl alkyl groups and acyl alkenyl groups. Although the term "alkyl acyl glycerol" is used, it will be understood by those skilled in the art that the term encompasses species having a hydrocarbon group at the R 1 position that includes unsaturation of the hydrocarbon chain. However, as used herein, "alkyl acyl glycerol" does not contain a double bond between carbons 1 and 2 of the R 1 hydrocarbon chain (i.e., proximal to the ether linkage).

[0121] As referred to herein, the term "alkyl diacyl glycerol" means a compound of formula (I) wherein R 1 group is a hydrocarbon chain, and R 2 and R 3The group is an acyl group independently selected from acylalkyl and acylalkenyl. Although the term "alkyl diacylglycerol" is used, one skilled in the art will understand that the term encompasses R 1 species having a hydrocarbon group at the position that includes unsaturation of the hydrocarbon chain. However, as used herein, "alkyl diacylglycerol" at R 1 does not contain a double bond between carbons 1 and 2 of the hydrocarbon chain (i.e., proximal to the ether linkage).

[0122] As used herein, the term "LPX(O)" refers to LPC(O) or LPE(O) as used elsewhere herein. In some embodiments, LPX(O) is selected from one or more of LysoPAF-C and LysoPAF-O. In some embodiments, LysoPAF-C is selected from one or more of LysoPAF-C16, LysoPAF-C18, and LysoPAF-C18:1.

[0123] The term "fatty acid" can refer to, for example, stearic acid, palmitic acid, or oleic acid.

[0124] As used herein, the terms "subject" and "patient" refer to a human or animal that is to be treated by the methods of the disclosure. In one embodiment, the subject is a human. In one embodiment, the subject is an animal, such as a domesticated animal, a working animal, or a farm animal. Domesticated animals include, but are not limited to, rabbits, birds, cats, dogs, fish, rats, turtles, reptiles (lizards, snakes), and the like. Working animals include, but are not limited to, cows, yaks, horses, and the like. Farm animals include, but are not limited to, sheep, pigs, cows, chickens, goats, geese, ducks, camels, and the like. In some embodiments, it is contemplated that the compositions described herein are formulated as an animal feed or dietary supplement for an animal. In some embodiments, the administration can be oral administration. In some embodiments, the administration can be via any appropriate route including liquid forms, capsules, tablets, or pastilles.

[0125] As used herein, the term "non-disease state" refers to a state in which the subject does not have an acetal phospholipid-associated disease or deficiency that requires treatment.

[0126] As used herein, the term "treating" includes any effect that results in the improvement of the condition, disease, disorder, etc., or an improvement in symptoms thereof, e.g., alleviation, reduction, increase, maintenance, modulation, amelioration, or elimination.

[0127] As used herein, the term "effective amount" refers to the amount of a compound (e.g., a compound of the disclosure) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

[0128] As used herein, the term “composition” refers to a product comprising the specified ingredients in the specified amounts, as well as any product directly or indirectly resulting from the combination of the specified ingredients in the specified amounts. In some embodiments, a composition comprises active and inactive ingredients. In some embodiments, a composition is a formulation. In some embodiments, a formulation is a composition that is suitable for administration to a subject (such as a human) and / or consumption by a subject (such as a human). A composition can be a pharmaceutical composition, and a formulation can be a pharmaceutical formulation. In some embodiments, a pharmaceutical composition or pharmaceutical formulation comprises an active agent in combination with a pharmaceutically acceptable carrier, diluent, excipient, solubilizer, or vehicle (inert or active). As used herein, the term “pharmaceutically acceptable” means that the carrier, diluent, excipient, solubilizer, or vehicle is compatible with the other ingredients of the formulation and is not toxic to the subject. Thus, it is understood that a “pharmaceutical composition” or “pharmaceutical formulation” is suitable for administration to a subject (such as a human) and / or consumption by a subject (such as a human), and can be, for example, approved by the U.S. Food and Drug Administration and / or the European Medicines Agency for such administration and / or consumption. It is also understood that a composition or formulation can not necessarily be, for example, approved by the U.S. Food and Drug Administration and / or the European Medicines Agency for administration to a subject (such as a human) and / or consumption by a subject (such as a human).

[0129] A pharmaceutical composition includes products comprising the active ingredients and inert ingredients that constitute carriers, and includes every product that can result from the combination, complexation or aggregation of two or more ingredients or the dissociation, other types of reactions or interactions of one or more ingredients. Thus, the pharmaceutical compositions of the present disclosure include every composition prepared by admixing at least one compound of the present disclosure with a pharmaceutically acceptable carrier, diluent, excipient, solubilizer, or vehicle.

[0130] In some embodiments, a formulation of the present disclosure is in the form of a beverage or food product. In some embodiments, a beverage or food product is formulated for general consumption, such as by being formulated as food grade. In some embodiments, a beverage or food product is formulated as a dietary supplement or other nutritional composition. In some embodiments, a beverage or food product is pharmaceutical grade. For the avoidance of doubt, it is understood that in some embodiments, a formulation of the present disclosure can be suitable for consumption by a subject (such as a human), but is not necessarily pharmaceutical grade (e.g., by being formulated as food grade or nutritional supplement grade, but not necessarily pharmaceutical grade).

[0131] In some embodiments, the formulation can contain one or more of a solubilizing agent, emulsifying agent, stabilizing agent, dispersing agent, antifoam agent, or diluent. The formulation can additionally contain one or more antioxidant compounds. It is contemplated that any antioxidant suitable for oral administration, such as vitamin A, vitamin E, vitamin C, retinol, tocopherol, L-ascorbyl palmitate, riboflavin, and carotenoids (including lutein, beta-carotene, zeaxanthin, and lycopene), and combinations thereof, can be formulated into the embodiments described herein. In some embodiments, the composition does not naturally undergo an oxidation reaction and no antioxidant compound need be added.

[0132] As used herein, the term “excipient” shall mean a non-active ingredient used as a vehicle (e.g., water, capsule shell, etc.), diluent, or component that makes up a dosage form or pharmaceutical composition that includes a drug, such as a therapeutic agent. The term also encompasses non-active ingredients that impart a cohesive function (e.g., binder), disintegrant function (e.g., disintegrant), lubricant function (e.g., lubricant), and / or other function (e.g., solvent, surfactant, etc.) to the composition.

[0133] As used herein, the term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound of the disclosure that, upon administration to a subject, is capable of providing a compound of this disclosure or an active metabolite or residue thereof. Pharmaceutically acceptable salts are described in detail in Berge et al., J. Pharmaceutical Sciences (1977) 66:1-19. As is known to those skilled in the art, “salts” of the compounds of the disclosure can be derived from inorganic or organic acids and bases. These salts can be prepared in situ during the final isolation and purification of the compounds of the disclosure, or separately by reacting the free base function with a suitable + a compound of formula (I), wherein each W is independently selected from H or C 1-4alkyl halides such as methyl, ethyl, propyl, and butyl chloride, bromide, and iodide; dialkyl sulfates such as dimethyl, diethyl, dibutyl, and diamyl sulfate; long chain alkyl halides such as decyl, dodecyl, myristyl, and stearyl chloride, bromide, and iodide; arylalkyl halides such as benzyl and phenethyl bromide; and the like. The resulting product has improved solubility or dispersibility.

[0134] Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, besylate, bicarbonate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Further examples of salts include the anion of the compounds of the present disclosure complexed with a suitable cation such as Na + , NH4 + , and NW4 + (wherein each W is independently selected from H or C 1-4 alkyl), and the like. For use in therapy, salts of the compounds of the present disclosure are considered to be pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable can also be used, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0135] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions (such as, for example, an oil / water or water / oil emulsion), and various types of wetting agents. The composition can also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, PA).

[0136] As used herein, in some embodiments, the term "carrier" refers to a pharmaceutically acceptable carrier. In some embodiments, the term "carrier" refers to any suitable carrier that can not be pharmaceutical grade. For example, in some embodiments, the carrier is food grade but can not be pharmaceutical grade.

[0137] As used herein, the term "day" is understood to mean a 24 hour period of time.

[0138] Generally, compositions specified as a percentage are by weight, unless otherwise specified.

[0139] Ether lipids and compositions

[0140] The present disclosure shows that ether lipids such as alkyl ether phosphatidyl phospholipids and acetal phosphatidyl phospholipids (acetal phospholipids) have in vivo characteristics associated with a healthy state and with conditions such as diabetes. It has also been found that administration of compositions containing ether lipids to a subject can affect in vivo ether lipid characteristics, for example, by supplementing phosphoester oil.

[0141] Formulations of the present disclosure can be used to maintain and / or modify in vivo ether lipid levels and / or ratios to levels and / or ratios associated with a natural, non-disease state (i.e., the natural state of a healthy human subject), and / or can be used to modify or regulate in vivo ether lipid levels to levels and / or ratios associated with a natural, non-disease state (i.e., the natural state of a healthy human subject). Exemplary, but non-limiting examples of the term "healthy human subject" are understood to mean a subject or patient who is not suffering from a disease or disorder associated with acetal phospholipid deficiency.

[0142] Examples of compounds that can be used to maintain and / or modify in vivo ether lipid levels and / or ratios thereof include alkyl glycerols, alkyl acyl glycerols (an ether compound that can be derived from a glycerol alcohol and an alkyl alcohol, and has an acyl group that can be derived from another glycerol alcohol and an acid), alkyl diacyl glycerols, and ether phospholipids, including but not limited to LPC(O), LPE(O), LPA(O), alkyl ether phosphatidyl phospholipids, and acetal phosphatidyl phospholipids. In some embodiments, formulations are used to maintain in vivo and / or modify in vivo alkyl ether phosphatidyl and / or acetal phosphatidyl phospholipid levels and / or ratios thereof. In some embodiments, alkyl ether phosphatidyl and / or acetal phosphatidyl phospholipids include those phospholipids having phosphatidylcholine and / or phosphatidylethanolamine groups. In some embodiments, formulations are used to maintain in vivo and / or modify in vivo acetal phosphatidyl phospholipid (acetal phospholipid) levels and / or ratios thereof. In some embodiments, formulations are used to maintain in vivo and / or modify in vivo phosphatidylethanolamine acetal phosphatidyl phospholipid (acetal phospholipid) levels and / or ratios thereof.

[0143] Table 2. Examples of alkyl ether phosphatidyl phospholipids, acetal phosphatidyl phospholipids, and related lipid species, and their abbreviations.

[0144] Table 2

[0145] Lipid Descriptions Abbreviations Alkenyl phosphatidylcholine = plasmalogen phosphatide PC(P) Alkenyl phosphatidylethanolamine = plasmalogen phosphatide PE(P) Alkyl phosphatidylcholine PC(O) Alkyl phosphatidylethanolamine PE(O) Lysolecithin LPC(O) Lysophosphatidylethanolamine LPE(O) Lysophosphatidic acid LPA(O) Alkyl glycerol AG, or AKG Diacylglycerol DG Triacylglycerol TG Alkyl diacylglycerol TG(O) or AKDAG Alkyl acylglycerol DG(O) Fatty acid FA

[0146] As described herein, healthy subjects tend to have alkyl ether phosphatidyl phospholipids and acylal phosphatidyl phospholipids profiles with certain alkenyl ether and alkyl ether groups present. For example, in the healthy subject group, a high proportion of ether lipids (e.g., alkyl ether phosphatidyl and / or acylal phosphatidyl phospholipids, including acylal phospholipids) with 18: 1 alkenyl ether groups, 18:0 alkyl ether groups, and 16:0 alkyl ether groups were found. It should be noted that the stated alkyl / alkenyl groups also contain a double bond between carbons 1 and 2 (cis-vinyl ether groups), as described above for several exemplary acylal phospholipid compounds.

[0147] In some embodiments, the formulation is for maintaining and / or modifying in vivo levels of ether lipids (e.g., alkyl ether phosphatidyl and / or acylal phosphatidyl phospholipids) with 18: 1 alkenyl ether groups, 18:0 alkyl ether groups, and 16:0 alkyl ether groups. In some embodiments, the formulation is for maintaining and / or modifying in vivo levels of acylal phospholipids with 18: 1 ether groups, 18:0 ether groups, and 16:0 ether groups. In some embodiments, the formulation is for maintaining in vivo or for modifying in vivo ether lipids (e.g., alkyl ether phosphatidyl and / or acylal phosphatidyl phospholipids) to a total ether lipid (e.g., alkyl ether phosphatidyl and / or acylal phosphatidyl phospholipids) profile in vivo, where the ether lipids (e.g., alkyl ether phosphatidyl and / or acylal phosphatidyl phospholipids) have a molar ratio of 18: 1 alkenyl ether groups to 18:0 alkyl ether groups to 16:0 alkyl ether groups of about 1 : 1.7: 1.4. In some embodiments, the formulation is for maintaining in vivo or for modifying in vivo ether lipids (e.g., alkyl ether phosphatidyl and / or acylal phosphatidyl phospholipids) to a total ether lipid (e.g., alkyl ether phosphatidyl and / or acylal phosphatidyl phospholipids) profile in vivo, where the ether lipids (e.g., alkyl ether phosphatidyl and / or acylal phosphatidyl phospholipids) have a molar percentage of 18: 1 alkenyl ether groups in the range of 18.6% to 27.9%, a molar percentage of 18:0 alkyl ether groups in the range of 32.6% to 45.8%, and a molar percentage of 16:0 alkyl ether groups in the range of 26.8% to 37.4%; or a molar percentage of 18: 1 alkenyl ether groups of about 23.3%, a molar percentage of 18:0 alkyl ether groups of about 39.2%, and a molar percentage of 16:0 alkyl ether groups of about 32.1%.

[0148] In some embodiments, the formulation is used to maintain or to modify in vivo the ether liposome to the in vivo vicinal acetal phospholipid lipid profile, wherein the ether lipid has a molar ratio of 18: 1 ether group to 18:0 ether group to 16:0 ether group of about 1 : 1.7: 1.4. In some embodiments, the formulation is used to maintain or to modify in vivo the ether liposome to the in vivo vicinal acetal phospholipid lipid profile, wherein the ether lipid has a molar percentage of 18: 1 ether group in the range of 18.6% to 27.9%, a molar percentage of 18:0 ether group in the range of 32.6% to 45.8%, and a molar percentage of 16:0 ether group in the range of 26.8% to 37.4%; or has a molar percentage of 18: 1 ether group of about 23.3%, a molar percentage of 18:0 ether group of about 39.2%, and a molar percentage of 16:0 ether group of about 32.1%.

[0149] In some embodiments, the composition comprises at least one isolated compound. In some embodiments, the purity of the at least one isolated compound is greater than 99%. In preferred embodiments, the purity of the at least one isolated compound is greater than 99.9%.

[0150] In some embodiments, the composition comprises a mixture of at least two compounds, wherein the % (w / v) of one compound is at least 90%.

[0151] In one aspect, dietary acetal phospholipids have been studied in humans after krill oil (KO) supplementation, as described in Sung et al., “Enrichment of n-3 containing ether phospholipids in plasma after 30 days of krill oil compared with fish oil supplementation,” Lipids (2022) 57: 115-124, which is incorporated by reference in its entirety. It has been recognized that ether phospholipids are a minor component in plasma and include alkyl or alkenyl phosphatidylcholines and phosphatidylethanolamines (the latter alkenyl class is referred to as acetal phospholipids). It is further noted that while acetal phospholipid levels are lower than diacyl lipids, even so, acetal phospholipids are still quite abundant. These have only recently been studied because techniques such as lipidomics have allowed detection and quantification of these and other minor lipid classes (see, e.g., Meikle et al., “Postprandial plasma phospholipids in men are influenced by the source of dietary fat,” J. Nutr. (2015) 145(9): 2012-2018). Methods of making certain ether lipid and / or acetal phospholipid compositions are described in WO 2021 / 007623 Al to the Beckman Heart and Diabetes Institute (inventors are Meikle), which is incorporated by reference. There are many protocols for synthesizing these ether lipid compounds using either chemical synthesis alone or a combination of chemical and enzymatic synthesis strategies. Synthesis of AKG compounds has been described in Carlos D. Magnusson et al., “Chemoenzymatic synthesis of a focused library of enantiopure structured 1-O-alkyl-2,3-diacyl-sn-glycerol type ether lipids,” Tetrahedron (2011) 67: 1821-1836; and Arnar Halldorsson et al., “Lipase catalysed kinetic resolution of 1-O-alkylglycerols by sequential transesterification,” Tetrahedron: Asymmetry (2004) 15: 2893-2899.Similarly, synthesis of lysophosphatidyl ethanolamine (LPE(P)) from commercially available 2,3-O-isopropylidene-sn-glycerol has been described in Guanghui Ni et al., “Synthesis and evaluation of immunostimulant plasmalogen lysophosphatidylethanolamine and analogues for natural killer T cells,” Bioorg. Med. Chem. (2014) 22(11): 2966-73. Gomes, MAGB, et al. describe the synthesis of a variety of alkyl ether lipids (Gomes MAGB, Bauduin A, Le Roux C, Fouinneteau R, Berthe W, Berchel M, Couthon H, Jaffrès PA. “Synthesis of ether lipids: natural compounds and analogues,” Beilstein J Org Chem. 2023 Sep 8; 19: 1299-1369) in the above-mentioned literature by Gomes. Figure 2 A synthetic route adapted to produce the alkyl LPC(O) compound of interest, which provides the R group for the sn-1 fatty acid of interest and stops at the lyso PAF step, is discussed. Those skilled in the art will appreciate that different protecting groups and / or deprotection protocols can also be used. Gomes’ synthesis of compound 2.1 of Figure 6 and the further references disclosed therein describe how to produce the starting compound 1-O-alkyl glycerol (i.e., Gomes’ compound 2.1). In addition, U.S. Patent No. 10,900,063 describes the production of LPC(O) using lipases, which is incorporated by reference herein in its entirety. Figure 2

[0152] LPC(O) synthesis route

[0153]

[0154] Scheme II. C18.0 LPC(O) synthesis

[0155] ​Starting material 1 (R-glycidyl acetone or R-(-)-2,3-O-isopropylidene-sn- glycerol; CAS 14347-78-5; available from CombiBlocks (98% purity) or Fluorochem (95% purity)) is condensed with an alkyl bromide or mesylate to form the corresponding alkyl substituted acetone compound (compound 2). TBAB shown in Scheme II above is tetrabutylammonium bromide, a commonly used phase transfer catalyst. The isopropylidene group can then be removed via acid hydrolysis (compound 3), and alternative protecting groups applied to give compound 6. Separately, 2-bromoethyl dichlorophosphate (compound 8) is prepared and then condensed with compound 6 to give compound 9. The terminal bromine atom is then replaced with trimethylamine, and finally an alcohol deprotection step is performed to provide compound 11.

[0156] As will be readily appreciated by one skilled in the art, the reagents and conditions suggested above are exemplary only and are not limiting. Other syntheses of SN-1 substituted LPOs can be achieved, for example, by providing appropriate alkyl bromides or alkyl mesylate compounds in step 1. For example, 1-bromooctadecane (CAS 112-89-0; available from TCI chemicals, >97.0% purity as determined by gas chromatography); oleyl mesylate (prepared by mesylation of oleyl alcohol: CAS 143-28-2; available from Sigma Aldrich, 85% purity as determined by gas chromatography); and 1-bromohexadecane (CAS 112-82-3; available from TCI chemicals, 96% purity as determined by gas chromatography) can be used for the synthesis of C18:0, C18:1 and C16:0 LPOs, respectively.

[0157] Suitable protecting groups for the alcohol moiety are well known in the art (see, for example, Greene's Protective Groups in Organic Synthesis, Wiley, DOI: 10.1002 / 9781118905074).

[0158] LPO synthesis route

[0159] Synthesis of the corresponding LPO compound can be carried out in a similar manner, using a substituted ammonium compound in place of trimethylamine in step 8. The ammonium compound can then be deprotected to leave a NH3 + group. Depending on the nature of the substituent and protecting group chosen, such deprotection can be carried out simultaneously with alcohol deprotection in step 9, or via an additional synthetic step. Alternative exemplary and non-limiting syntheses include:

[0160] (i) reduction of LPE(P) produced by the method of Ni et al. in the above-mentioned literature, either chemically (typically nickel) or enzymatically

[0161] saturating the vinyl double bond of the alkenyl compound, which provides an alternative synthesis of LPE(O); and

[0162] (ii) hydrolysis of the SN2 group of a commercially available alkyl PE compound, such as 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (catalog no. 878130) from, for example, Avanti Polar Lipids < avantilipids.com) using chemical or enzymatic means, such as a suitable lipase, to give C16.0 alkyl LPE(O).

[0163] In particular embodiments, combinations of lysolipid alkylphosphatidylcholines (LPC(O)) or lysolipid alkylphosphatidylethanolamines (LPE(O)) compounds are contemplated. Derivatives of the aforementioned compounds are contemplated. One exemplary embodiment includes a specific formulation of three species of lysolipid alkylphosphatidylcholines (LPC(O)). These species can include: LPC(O-18: l), shown as compound (I-A1); LPC(O-18:0), shown as compound (I-A2); and LPC(O-16:0), shown as compound (I-A3). For example, these ether lipids can have a molar ratio of R 1 The ratio can vary between different cell target populations and different subjects.

[0164] In this preferred embodiment, the compounds are depicted as follows, such as LPC(O-18: l):

[0165]

[0166]

[0167] Other compounds and combinations are also contemplated, such as combinations of lysolipid alkylphosphatidylethanolamines (LPE(O)), including: LPE(O-18: l), shown as compound (I-A4); LPE(O-18:0), shown as compound (I-A5); and LPE(O-16:0), shown as compound (I-A6). The molar ratio of R 1 The molar ratio, as well as the mole percent and weight percent, can vary.

[0168] In this embodiment, the compounds are depicted as follows, such as LPE(O-18: l):

[0169]

[0170] Other compounds and combinations are also contemplated, such as combinations of lysophosphoalkyl phosphatidic acids (LPA(O)), which are phosphoester compounds, including: LPA(O-18: l), shown as compound (I-A7); LPA(O-18:0), shown as compound (I-A8); and LPA(O-16:0), shown as compound (I-A9).R 1 The molar ratios, as well as the mole percent and weight percent, can vary.

[0171] In this example, the compounds are depicted as follows, such as LPA(O-18: l):

[0172]

[0173] When ingested, these phosphocholine species, particularly LPC(O), are absorbed in the gut and then metabolized into a series of ether phospholipids, including: alkylphosphatidylcholines (PC(O)), alkylphosphatidylethanolamines (PE(O)), alkenylphosphatidylcholines (plasmenyls, PC(P)), and alkenylphosphatidylethanolamines (plasmenyls, PE(P)). In studies, this resulted in increased levels of both PC(P) and PE(P) species, both of which are bioactive species with many desirable health properties. The bioavailability of LPC(O) and conversion into PC(P) and PE(P) is 5-12 times that of commonly used alkylglycerols (AKG) species, which are also used to increase plasmenyl levels. Without wishing to be bound by theory, it is believed that this means that lower doses can be used to achieve the same plasmenyl boost.

[0174] A further embodiment contemplated by the present disclosure is a formulation, which can be in the form of a food product, such as a dietary supplement, that when ingested results in increased levels of plasmenyls within the blood and tissues of a recipient, and thus ameliorates or overcomes any plasmenyl deficiency that can exist within the individual. Increasing plasmenyl levels can result in improved health outcomes. Such a formulation (e.g., an LPC(O) formulation) can also be incorporated into a range of food products for ease of delivery to a recipient. The intended recipients would be anyone who is deficient in plasmenyls and / or at risk for any of a range of metabolic diseases in which plasmenyls can play a protective role.

[0175] As used herein, the term "dietary supplement" refers to a food product intended to enhance the diet and thus improve nutrition of the subject. The dietary supplement can include only the composition described herein, or can include together other ingredients intended to supplement the diet, such as vitamins and minerals, fiber, herbs and other botanical extracts, including flower essences, homeopathic remedies, amino acids, enzymes and live microorganisms, probiotics, prebiotics, or any combination thereof. The dietary supplement can be formulated in a variety of ways, including oils, gummies, drops, capsules, fast melt formulations, lozenges, mouth sprays, chewing gums, gels, powders, pre-mixed beverages, meal replacement shakes or bars.

[0176] An example of a food in which a formulation as described herein can be incorporated is infant formula. Foods in which a formulation comprising, for example, LPC(O) can be incorporated include infant formula, follow-on formula, medical food, and special medical purpose food.

[0177] An example of a food in which a composition or formulation as described herein can be incorporated is any food that is formulated for human consumption.

[0178] As used herein, the terms "medical food" or "special medical purpose food" refer to a food that is specially formulated and intended for the dietary management of a disease, disorder, or condition that has unique nutritional needs that cannot be met by normal diet alone. Medical foods are intended to help patients who are temporarily or permanently unable to obtain sufficient nutritional intake from a normal diet or by altering a normal diet, as well as patients who are malnourished or at risk of becoming malnourished. Medical foods are to be used under medical supervision and can be administered orally or via tube feeding (e.g., nasogastric tube). As used herein, these terms are referenced in Regulation (EU) No 609 / 2013 and (Food and Drug Authority (FDA)) 21 CFR 101.9(j)(8)(ii). Thus, they are distinguished from dietary supplements, which can be consumed without medical supervision.

[0179] As used herein, the term "infant" is understood to mean a human of 12 months of age or younger. "Infant" is understood to also mean "preterm infant," i.e., a human of 12 months of age or younger and born prior to 36 weeks of gestation. As used herein, the term "toddler" is understood to mean a human of greater than one year of age to three years of age. As used herein, the term "child" refers to a human of greater than three years of age to 12 years of age. As used herein, unless otherwise specified, the term "infant formula" refers to liquid, semi-liquid, solid, and semi-solid human milk replacements or substitutes suitable for consumption by an infant. Synthetic formulas include components with semi-purified or purified sources. As used herein, "semi-purified" and "purified" refer to materials prepared by purifying natural materials or by synthesis. The term "infant formula" is understood to exclude unmodified human milk.

[0180] Infant formulas can include liquid and powdered dietary supplements, liquid and powdered human milk fortifiers, liquid and powdered preterm infant formulas, liquid and powdered infant formulas, liquid and powdered elemental and semi-elemental formulas, liquid and powdered toddler formulas, and powdered follow-on formulas suitable for use in infants and children. The compositions can be in any product form comprising the ingredients described herein, and which are safe and effective for oral administration.

[0181] Infant formulas can further include ingredients such as proteins, fats, carbohydrates, vitamins, minerals, anti-caking agents, emulsifiers, and the like. In some embodiments, formulas can contain purified cow's milk whey and / or casein as a protein source, a vegetable oil mixture as a fat source, lactose as a carbohydrate source, a vitamin mineral mixture, and other ingredients including, but not limited to, any antioxidant suitable for oral administration such as vitamin A, vitamin E, vitamin C, retinol, tocopherol, L-ascorbyl palmitate, riboflavin, and carotenoids including lutein, beta-carotene, zeaxanthin, and lycopene, and combinations thereof. Infant formulas can include oils, for example, vegetable oils, including, for example, high-oleic sunflower oil, coconut oil, rapeseed oil, sunflower oil, or fish oil, and combinations thereof. Infant formulas can include dairy products and dairy derivatives, including, for example, lactose, milk proteins, galacto-oligosaccharides, whey concentrates, fructo-oligosaccharides, and further compounds concentrated from milk secreted by mammals, including, but not limited to, humans, cows, and the like.

[0182] Infant formulas can include anti-caking agents such as tricalcium phosphate, potassium chloride, sodium and potassium citrate, magnesium hydrogen phosphate, and coagulants such as, for example, magnesium chloride, choline chloride, L-ascorbic acid, emulsifiers iron (II) sulfate, zinc sulfate.

[0183] The powder can be reconstituted with water prior to use to a caloric density customized to the nutritional needs of the end user, although in most cases the powder is reconstituted with water to form a composition comprising at least 19 kcal / fl oz (660 kcal / liter), more typically from about 20 kcal / fl oz (675-680 kcal / liter) to about 25 kcal / fl oz (820 kcal / liter), even more typically from about 20 kcal / fl oz (675-680 kcal / liter) to about 24 kcal / fl oz (800-810 kcal / liter). Generally, 22-24 kcal / fl oz formulas are more commonly used for preterm or low birth weight infants, and 20-21 kcal / fl oz (675-680 to 700 kcal / liter) formulas are more commonly used for term infants. In some embodiments, the reconstituted powder can have a caloric density of from about 50-100 kcal / liter to about 660 kcal / liter, including from about 150 kcal / liter to about 500 kcal / liter. In some particular embodiments, the emulsion can have a caloric density of 25, or 50, or 75, or 100 kcal / liter.

[0184] When the nutritional product is a powdered infant formula, the protein component is present in an amount of from about 5% to about 35%, including from about 8% to about 12%, and including from about 10% to about 12%, by weight of the infant formula; the fat component is present in an amount of from about 10% to about 35%, including from about 25% to about 30%, and including from about 26% to about 28%, by weight of the infant formula; and the carbohydrate component is present in an amount of from about 30% to about 85%, including from about 45% to about 60%, including from about 50% to about 55%, by weight of the infant formula.

[0185] The infant formula contemplated herein can be formulated to include at least one of fat, protein, and carbohydrate, and preferably also contains vitamins, minerals, and at least one compound of Formula (I), (I-A), (I-A1), (I-A2), (I-A3), (I-A4), (I-A5), and / or (I-A6), or combinations thereof.

[0186] There are many disease contexts in which plasmalogens have been demonstrated to exert protective effects. These include, but are not limited to: metabolic disorders (obesity, insulin resistance, type 2 diabetes; nonalcoholic fatty liver disease, nonalcoholic steatohepatitis); immune-related diseases (asthma, atopic dermatitis, type 1 diabetes, infections); cardiovascular diseases (atherosclerosis, cardiac remodeling, hypertension); neurological diseases (Alzheimer’s disease; Parkinson’s disease, autism, amyotrophic lateral sclerosis, frontotemporal dementia; multiple sclerosis; schizophrenia); cancer; myalgic encephalomyelitis / chronic fatigue syndrome; Barth syndrome; peroxisomal disorders (Zellweger spectrum disorders, rhizomelic chondrodysplasia punctata). These have been reviewed in recent papers such as Tremblay et al., “Plasmalogens and platelet-activating factor roles in chronic inflammatory diseases,” BioFactors (2022) 1-14; Schooneveldt et al., “Ether lipids in obesity: from cells to population studies,” Frontiers in Physiology (March 2022) 13:1-11; Boselli, Jr. et al., “Plasmalogen replacement therapy,” Membranes (2021) 11:838; and S. Paul, G.I. Lancaster, and P. Meikle, “Plasmalogens: a potential therapeutic target for neurodegenerative and cardiometabolic disease,” Progress Lipid Res. (2019) 74:186-195.

[0187] Formulations of the supplement are based on the ratio levels of the corresponding PE(P) species in circulation or in tissues, and thus, specific formulations can be used to target specific fluids (e.g., plasma), tissues (such as liver, heart, or adipose tissue), or specific cell types (such as immune cells). With respect to targeting plasma, contemplated embodiments of the formulation include ether lipids having about 1.0:1.7:1.4 18:1 alkenyl / alkyl R 1 groups to 18:0 alkyl R 1 groups to 16:0 alkyl R 1molar ratio of 18: 1 ether groups having a molar percentage in the range of 18.6% to 27.9%, 18:0 ether groups having a molar percentage in the range of 32.6% to 45.8%, or 16:0 ether groups having a molar percentage in the range of 26.8% to 37.4%.

[0188] With respect to targeting immune cells, contemplated embodiments of the formulation include ether lipids having a molar ratio of 18: 1 ether groups to 18:0 ether groups to 16:0 ether groups of about 1.0:3.4:3.3. 1 molar ratio of 18: 1 ether groups to 18:0 ether groups to 16:0 ether groups of about 1.0:3.4:3.3. 1 molar ratio of 18: 1 ether groups to 18:0 ether groups to 16:0 ether groups of about 1.0:3.4:3.3. 1 molar ratio of 18: 1 ether groups to 18:0 ether groups to 16:0 ether groups of about 1.0:3.4:3.3.

[0189] Embodiments of the contemplated formulation include at least one compound of Formula (I). In certain embodiments, the at least one compound of Formula (I) is a compound of Formula (I-A). In certain embodiments, the at least one compound of Formula (I-A) is one or more of Formula (I-A1), Formula (I-A2), and / or Formula (I-A3). In certain embodiments, the at least one compound of Formula (I) is a mixture of two compounds of Formula (I). In certain embodiments, the at least one compound of Formula (I) is a mixture of two compounds of Formula (I-A). In certain embodiments, the at least one compound of Formula (I-A) is a mixture of two compounds selected from Formula (I-A1), Formula (I-A2), and Formula (I-A3). In certain embodiments, the at least one compound of Formula (I-A) is a mixture of Formula (I-A1), Formula (I-A2), and Formula (I-A3).

[0190] In certain embodiments, the mixture of Formula (I-A1), Formula (I-A2), and Formula (I-A3) comprises at least 50% by molar percent of ether lipids. In certain embodiments, the mixture has a molar ratio of (I-A2) to (I-A1) of 1.2:1 to 2.5:1. In certain embodiments, the mixture has a molar ratio of (I-A2) to (I-A1) of 1.5:1 to 2.1:1. In certain embodiments, the mixture has a molar ratio of (I-A2) to (I-A1) of 1.7:1. In certain embodiments, the mixture has a molar ratio of (I-A2) to (I-A3) of 0.9:1 to 1.7:1. In certain embodiments, the mixture has a molar ratio of (I-A2) to (I-A3) of 1:1 to 1.5:1. In certain embodiments, the mixture has a molar ratio of (I-A2) to (I-A3) of 1.22:1. In certain embodiments, the mixture has a molar ratio of (I-A1) to (I-A3) of 0.5:1 to 1:1. In certain embodiments, the mixture has a molar ratio of (I-A1) to (I-A3) of 0.6:1 to 0.9:1. In certain embodiments, the mixture has a molar ratio of (I-A1) to (I-A3) of 0.72:1. In certain embodiments, the mixture has a molar percent of (I-A1) of 18.6% to 27.9%, a molar percent of (I-A2) of 32.6% to 45.8%, and a molar percent of (I-A3) of 26.8% to 37.4%. In certain embodiments, the mixture has a molar percent of (I-A1) of 23.3%, a molar percent of (I-A2) of 39.2%, and a molar percent of (I-A3) of 32.1%. In certain embodiments, the mixture has a molar ratio of (I-A1):(I-A2):(I-A3) of 1:1.7:1.4.

[0191] In certain embodiments, at least one compound of Formula (I), (I-A), (I-A1), (I-A2), and / or (I-A3) is converted to at least one plasmalogen in vivo.

[0192] Embodiments of contemplated formulations include at least one compound of Formula (I). In certain embodiments, the at least one compound of Formula (I) is a compound of Formula (I-A). In certain embodiments, the at least one compound of Formula (I-A) is one or more of Formula (I-A4), Formula (I-A5), and / or Formula (I-A6). In certain embodiments, the at least one compound of Formula (I) is a mixture of two compounds of Formula (I). In certain embodiments, the at least one compound of Formula (I) is a mixture of two compounds of Formula (I-A). In certain embodiments, the at least one compound of Formula (I-A) is a mixture of two compounds selected from Formula (I-A4), Formula (I-A5), and Formula (I-A6). In certain embodiments, the at least one compound of Formula (I-A) is a mixture of Formula (I-A4), Formula (I-A5), and Formula (I-A6).

[0193] In certain embodiments, the mixture of Formula (I-A4), Formula (I-A5), and Formula (I-A6) comprises at least 50% of the ether lipids in the composition by mole percent. In certain embodiments, the mixture has a molar ratio of (I-A5) to (I-A4) of 1.2:1 to 2.5:1. In certain embodiments, the mixture has a molar ratio of (I-A5) to (I-A4) of 1.5:1 to 2.1:1. In certain embodiments, the mixture has a molar ratio of (I-A5) to (I-A4) of 1.7:1. In certain embodiments, the mixture has a molar ratio of (I-A5) to (I-A6) of 0.9:1 to 1.7:1. In certain embodiments, the mixture has a molar ratio of (I-A5) to (I-A6) of 1:1 to 1.5:1. In certain embodiments, the mixture has a molar ratio of (I-A5) to (I-A6) of 1.29:1. In certain embodiments, the mixture has a molar ratio of (I-A4) to (I-A6) of 0.5:1 to 1:1. In certain embodiments, the mixture has a molar ratio of (I-A4) to (I-A6) of 0.6:1 to 0.9:1. In certain embodiments, the mixture has a molar ratio of (I-A4) to (I-A6) of 0.76:1. In certain embodiments, the mixture has a mole percent of (I-A4) of 18.6% to 27.9%, a mole percent of (I-A5) of 32.6% to 45.8%, and a mole percent of (I-A6) of 2.8% to 37.4%. In certain embodiments, the mixture has a mole percent of (I-A4) of 24.8%, a mole percent of (I-A5) of 42.4%, and a mole percent of (I-A6) of 32.8%. In certain embodiments, the mixture has a molar ratio of (I-A4):(I-A5):(I-A6) of 1:1.7:1.4.

[0194] In certain embodiments, the at least one compound of Formula (I), (I-A), (I-A4), (I-A5), and / or (I-A6) is converted in vivo to at least one plasmalogen.

[0195] In certain embodiments, the mixture of at least one compound of Formula (I) or (I-A) is a mixture of (I-A1), (I-A2), (I-A3), (I-A4), (I-A5), and / or (I-A6) compounds.

[0196] It is contemplated that the above disclosed ratios of the formulations will result in optimal ratios of plasmalogen PE(P) and PC(P) in the serum and tissues of a human subject to which the formulation is administered.

[0197] Other useful phosphatidyl classes can be prepared according to the principles herein, such as phosphatidylserine and phosphatidylinositol.

[0198] In further embodiments, LPC(O), LPE(O), and LPA(O) can be used in various advantageous combinations that can be effective to increase plasmalogen levels. The plasmalogens that are enhanced or modulated are generally represented by the plasmalogens listed above.

[0199] In certain embodiments, the formulations described above include: one or more liquid or gel-based carriers including, but not limited to, carriers selected from the group consisting of water and physiological saline solutions, urea, alcohols and derivatives thereof (e.g., methanol, ethanol, propanol, butanol), glycols (e.g., ethylene glycol, propylene glycol), and the like; natural or synthetic flavorants and food-grade colorants; thickening agents including, but not limited to, thickening agents selected from the group consisting of corn starch, guar gum, xanthan gum, and the like. In certain embodiments, one or more liquid or gel-based carriers can be added to the formulation in a weight / volume percentage of about 0.5% to about 95% weight / volume of the formulation. In certain embodiments, natural or synthetic flavorants can be added to the formulation in a weight / volume percentage of about 3.0% to about 10.0% weight / volume of the formulation. In certain embodiments, colorants can be added to the formulation in a weight / volume percentage of about 1.0% to about 10.0% weight / volume of the formulation. In certain embodiments, thickening agents can be added to the formulation in a weight / volume percentage of about 2% weight / volume of the formulation.

[0200] Delivery system

[0201] The formulations disclosed herein can be delivered via a dosage form including, but not limited to, tablets, capsules, solutions, suspensions, powders, chewing gum, and candy. The formulations disclosed herein can be delivered via a sublingual delivery system including, but not limited to, sublingual and lingual dissolvable tablets, droplets, and beverages. Alternatively, or in addition, an edible film, a hydrophilic polymer, an orally dissolvable film, or an orally dissolvable strip can be used.

[0202] For oral administration, the formulations disclosed herein can be further combined with one or more solid inactive ingredients for the manufacture of tablets, capsules, pills, powders, granules, or other suitable dosage forms. For example, the formulation components can be combined with at least one excipient including, but not limited to, an excipient selected from the group consisting of fillers, binders, humectants, disintegrants, dissolution retarders, absorption accelerators, wetting agents, absorbents, and lubricants. Other useful excipients include, but are not limited to, magnesium stearate, calcium stearate, mannitol, xylitol, sweeteners, starch, carboxymethylcellulose, microcrystalline cellulose, silica, gelatin, silicon dioxide, and the like. In some embodiments, the formulations according to the present disclosure can include one or more of beeswax (such as beeswax E901), carnauba wax (such as carnauba wax E903), shellac (such as shellac E904), candelilla wax (such as candelilla wax E902), microcrystalline wax (such as microcrystalline wax E905), paraffin wax, and diacylglycerol.

[0203] The components of the formulations administered according to the methods of the present disclosure can be administered in a variety of oral dosage forms. As will be apparent to those of ordinary skill in the art, in certain embodiments, a suitable dosage form can comprise one or more chemical compounds of the present disclosure and / or one or more pharmaceutically acceptable salts of the chemical compounds of the present disclosure.

[0204] To prepare a pharmaceutical formulation or composition to be administered according to the methods of the present disclosure, the pharmaceutically acceptable carrier can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, and cachets. A solid carrier can be one or more substances which also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

[0205] In powders, the carrier is a finely divided solid which is mixed with the finely divided active component. In tablets, one or more compounds of the present disclosure and / or pharmaceutically acceptable salts thereof, are mixed with one or more carrier materials having the requisite binding capacity, in the suitable proportions, and compressed into the desired shape and size.

[0206] In certain embodiments, the powders and tablets administered according to the methods of the present disclosure preferably can contain from about one-hundredth to about ninety-nine-hundredths (such as five or ten to about seventy percent) of the one or more compounds of the present disclosure and / or pharmaceutically acceptable salts thereof, collectively. Suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth gum, methylcellulose, sodium carboxymethylcellulose, low-melting wax, cocoa butter, and the like. The term "preparation" is intended to include formulations of the one or more compounds of the present disclosure and / or pharmaceutically acceptable salts thereof with encapsulating material as a carrier, thereby providing a capsule in which the one or more compounds of the present disclosure and / or pharmaceutically acceptable salts thereof (with or without the additional carrier) are surrounded by carrier which is in association with it. Similarly, sachets and tablets are included. Tablets, powders, capsules, pills, sachets, and tablets are included. Tablets, powders, capsules, pills, sachets, tablets, and fast melt formulations can be used as solid forms suitable for oral administration.

[0207] Capsules can be prepared in a manner that is additionally encapsulated for timed release. The encapsulation thickness can be modified to provide a delayed release of the capsule contents. Capsules can be prepared in a manner that targets the release of the capsule. In some embodiments, the capsule can be targeted to the stomach. In some embodiments, the capsule targeted for delivery to the stomach is encapsulated in a film coating. In some embodiments, the capsule can be targeted to the small intestine. In some embodiments, the capsule targeted for delivery to the small intestine is encapsulated in an enteric coating.

[0208] As used herein, the term "fast melt" refers to a pharmaceutical formulation that melts with little or no need for mastication when contacted with saliva.

[0209] Liquid formulations include, but are not limited to, solutions, suspensions, and emulsions, for example, aqueous or water-propylene glycol solutions. Formulated preparations can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the one or more compounds of the present disclosure and / or pharmaceutically acceptable salts thereof can be in powder form, such as a powder obtained by sterile isolation or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0210] Aqueous solutions suitable for oral use can be prepared by dissolving one or more compounds of the present disclosure and / or pharmaceutically acceptable salts thereof in water and adding suitable colorants, flavoring agents, stabilizers and thickening agents as desired.

[0211] Formulations suitable for buccal, oral, or sublingual administration include, but are not limited to, tablets, lozenges, troches, and pastilles, each containing a fixed dose of an active agent in an inert base such as gum arabic, acacia, cornstarch, potato starch, or other well-known pharmaceutically acceptable carriers. Lozenges can comprise the active agent in a flavored base, such as sucrose and acacia or traganth gum. Pastilles can comprise the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia. Additionally, pastilles can comprise the active agent in a suitable liquid carrier.

[0212] In some embodiments, the formulation comprises a solubilizing agent. As used herein, the term "solubilizing agent" refers to any agent that facilitates solubilization or dispersion of a composition when placed into a liquid. In some embodiments, a "solubilizing agent" can be a dispersing agent. Solubilizing agents can additionally improve the stability of the formulated composition. Suitable solubilizing agents include, but are not limited to, sodium carboxymethylcellulose, hypromellose, proline, xanthan gum, maltodextrin, alginate, wax, lipid, oil, alcohol, sugar, microcrystalline cellulose, starch, calcium phosphate, mannitol, sorbitol, erythritol, food grade solvents, phospholipids such as lecithin (including but not limited to egg yolk L-a-lecithin, such as egg yolk L-a-lecithin available from Sigma-Aldrich (St. Louis, MO, USA)), DMSO, ethanol, ethyl acetate, isopropyl alcohol, and the like. In some embodiments, solubilizing agents are used to improve the isolation of the compounds that make up the formulation and prevent them from precipitating or clumping in the formulation. In some embodiments, the formulation comprises a solubilizing agent and a suitable carrier. In some embodiments, such a formulation is a pharmaceutical grade product. In some embodiments, such a formulation is a food grade product.

[0213] Pharmaceutical formulations or preparations are preferably in unit dosage form. In such form, the formulation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these packaged forms.

[0214] Tablets, capsules, and lozenges for oral administration and liquid for oral use are preferred formulations.

[0215] Further details on formulation and administration techniques can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, PA).

[0216] Route of administration

[0217] The compounds of the present disclosure and / or their pharmaceutically acceptable salts can be administered by any route, including, but not limited to, oral, sublingual, buccal, or buccal spray.

[0218] The methods described above can be further understood in connection with the following examples. Furthermore, the following non-limiting examples are provided to illustrate the present disclosure. However, one skilled in the art will appreciate that it can be necessary to alter procedures for any given example of the present disclosure, e.g., altering the order or steps.

[0219] Numbered examples

[0220] 1. A composition comprising at least one compound of Formula (I) or a pharmaceutically acceptable salt thereof

[0221]

[0222] 2. The composition of Example 1, wherein R 3 is selected from the group consisting of a phosphate ester and a substituted phosphate ester.

[0223] 3. The composition of Example 2, wherein R 3 is a substituted phosphate ester.

[0224] 4. The composition of Example 3, wherein the substituted phosphate ester is substituted with an alkyl amine or an inositol.

[0225] 5. The composition of Example 4, wherein the alkyl amine is selected from the group consisting of

[0226] 6. The composition of any one of Examples 1-5, wherein R 1 and R 2 are each independently selected from the group consisting of hydrogen, an optionally substituted C 1-30 alkyl group, an optionally substituted C 2-30 alkenyl group, and an optionally substituted C 1-30 acyl group.

[0227] 7. The composition of Example 6, wherein R 1 and R 2 are each independently selected from the group consisting of an optionally substituted C 14-24 alkyl group, an optionally substituted C14-24 alkenyl group, or an optionally substituted C 14-24 acyl group.

[0228] 8. The composition according to embodiment 6, wherein if R 2 is hydrogen, then R 1 is an optionally substituted C 1-30 alkyl group, an optionally substituted C 2-30 alkenyl group, or an optionally substituted C 1-30 acyl group.

[0229] 9. The composition according to embodiment 8, wherein R 1 is an optionally substituted C 14-24 alkyl group, an optionally substituted C 14-24 alkenyl group, or an optionally substituted C 14-24 acyl group.

[0230] 10. The composition according to embodiment 9, wherein R 1 is an optionally substituted C 14-18 alkyl group, an optionally substituted C 14-18 alkenyl group, or an optionally substituted C 14-18 acyl group.

[0231] 11. The composition according to embodiment 10, wherein R 1 is an unsubstituted C 16 alkyl group.

[0232] 12. The composition according to embodiment 10, wherein R 1 is an unsubstituted C 18 alkyl group.

[0233] 13. The composition according to embodiment 10, wherein R 1 is an unsubstituted C 18 alkenyl group.

[0234] 14. The composition according to embodiment 1, wherein the at least one compound of formula (I) is a compound of formula (I-A) or a pharmaceutically acceptable salt thereof

[0235]

[0236] 15. The composition according to embodiment 14, wherein each R x is independently selected from hydrogen or C 1-3 alkyl.

[0237] 16. The composition according to embodiment 14 or 15, wherein n is 2 or 3.

[0238] 17. The composition of embodiment 14, wherein R x is methyl, and n is 3.

[0239] 18. The composition of any one of embodiments 14 to 17, wherein R A is an optionally substituted hydrocarbon chain containing 14 to 24 carbon atoms.

[0240] 19. The composition of embodiment 18, wherein R A is an optionally substituted hydrocarbon chain containing 16 to 18 carbon atoms.

[0241] 20. The composition of embodiment 19, wherein R A is an optionally substituted C 16-18 alkyl group, an optionally substituted C 16-18 alkenyl group, or an optionally substituted C 16-18 acyl group.

[0242] 21. The composition of embodiment 20, wherein R A is an optionally substituted C 16-18 alkyl group.

[0243] 22. The composition of embodiment 20, wherein R A is an optionally substituted C 16-18 alkenyl group.

[0244] 23. The composition of embodiment 19, wherein R A is an unsubstituted C 16 alkyl group.

[0245] 24. The composition of embodiment 19, wherein R A is an unsubstituted C 18 alkyl group.

[0246] 25. The composition of embodiment 19, wherein R A is an unsubstituted C 18 alkenyl group.

[0247] 26. The composition of any preceding embodiment, wherein the composition comprises a mixture of at least two compounds of formula (I) and / or (I-A).

[0248] 27. The composition of any preceding embodiment, wherein the composition comprises a mixture of three compounds of formula (I) and / or (I-A).

[0249] 28. The composition of embodiment 14, wherein the compound of formula (I-A) has structure (I-A1), (I-A2), or (I-A3)

[0250]

[0251] 29. The composition according to embodiment 28, wherein the composition comprises a mixture of at least two compounds selected from the group consisting of (I-A1), (I-A2), and (I-A3).

[0252] 30. The composition according to embodiment 28, wherein the composition comprises a mixture of (I-A1), (I-A2), and (I-A3).

[0253] 31. The composition according to embodiment 28, wherein the composition comprises a mixture of (I-A1), (I-A2), and (I-A3), wherein the mixture of (I-A1), (I-A2), and (I-A3) comprises at least 50% of the ether lipids in the composition by molar percentage.

[0254] 32. The composition according to embodiment 28, wherein the mixture has a molar ratio of (I-A2) to (I-A1) of 1.2:1 to 2.5:1.

[0255] 33. The composition according to embodiment 32, wherein the mixture has a molar ratio of (I-A2) to (I-A1) of 1.5:1 to 2.1:1.

[0256] 34. The composition according to embodiment 33, wherein the mixture has a molar ratio of (I-A2) to (I-A1) of 1.7:1.

[0257] 35. The composition according to embodiment 28, wherein the mixture has a molar ratio of (I-A2) to (I-A3) of 0.9:1 to 1.7:1.

[0258] 36. The composition according to embodiment 35, wherein the mixture has a molar ratio of (I-A2) to (I-A3) of 1:1 to 1.5:1.

[0259] 37. The composition according to embodiment 36, wherein the mixture has a molar ratio of (I-A2) to (I-A3) of 1.22:1.

[0260] 38. The composition according to embodiment 28, wherein the mixture has a molar ratio of (I-A1) to (I-A3) of 0.5:1 to 1:1.

[0261] 39. The composition according to embodiment 38, wherein the mixture has a molar ratio of (I-A1) to (I-A3) of 0.6:1 to 0.9:1.

[0262] 40. The composition of embodiment 39, wherein the mixture has a molar ratio of (I-A1) to (I-A3) of 0.72: 1.

[0263] 41. The composition of embodiment 30, wherein the mixture has a mole percent of (I-A1) of 18.6% to 27.9%, a mole percent of (I-A2) of 32.6% to 45.8%, and a mole percent of (I-A3) of 26.8% to 37.4%.

[0264] 42. The composition of embodiment 30, wherein the mixture has a mole percent of (I-A1) of 23.3%, a mole percent of (I-A2) of 39.2%, and a mole percent of (I-A3) of 32.1%.

[0265] 43. The composition of embodiment 30, wherein the mixture has a molar ratio of (I-A1):(I-A2):(I-A3) of 1: 1.7: 1.4.

[0266] 44. The composition of embodiment 14, wherein the compound of formula (I-A) has the structure (I-A4), (I-A5), or (I-A6)

[0267]

[0268] 45. The composition of embodiment 44, wherein the composition comprises a mixture of at least two compounds selected from the group consisting of (I-A4), (I-A5), and (I-A6).

[0269] 46. The composition of embodiment 44, wherein the composition comprises a mixture of (I-A4), (I-A5), and (I-A6).

[0270] 47. The composition of embodiment 44, wherein the composition comprises a mixture of (I-A4), (I-A5), and (I-A6), wherein the mixture of (I-A4), (I-A5), and (I-A6) comprises at least 50% of the ether lipids in the composition by mole percent.

[0271] 48. The composition of embodiment 44, wherein the mixture has a molar ratio of (I-A5) to (I-A4) of 1.2: 1 to 2.5: 1.

[0272] 49. The composition of embodiment 48, wherein the mixture has a molar ratio of (I-A5) to (I-A4) of 1.5: 1 to 2.1: 1.

[0273] 50. The composition of embodiment 49, wherein the mixture has a molar ratio of (I-A5) to (I-A4) of 1.7: 1.

[0274] 51. The composition of embodiment 44, wherein the mixture has a molar ratio of (I-A5) to (I-A6) of 0.9: 1 to 1.7: 1.

[0275] 52. The composition of embodiment 51, wherein the mixture has a molar ratio of (I-A5) to (I-A6) of 1: 1 to 1.5: 1.

[0276] 53. The composition of embodiment 52, wherein the mixture has a molar ratio of (I-A5) to (I-A6) of 1.29: 1.

[0277] 54. The composition of embodiment 44, wherein the mixture has a molar ratio of (I-A4) to (I-A6) of 0.5: 1 to 1: 1.

[0278] 55. The composition of embodiment 54, wherein the mixture has a molar ratio of (I-A4) to (I-A6) of 0.6: 1 to 0.9: 1.

[0279] 56. The composition of embodiment 55, wherein the mixture has a molar ratio of (I-A4) to (I-A6) of 0.76: 1.

[0280] 57. The composition of embodiment 46, wherein the mixture has a mole percent of (I-A4) of 18.6% to 27.9%, a mole percent of (I-A5) of 32.6% to 45.8%, and a mole percent of (I-A6) of 26.8% to 37.4%.

[0281] 58. The composition of embodiment 57, wherein the mixture has a mole percent of (I-A4) of 24.8%, a mole percent of (I-A5) of 42.4%, and a mole percent of (I-A6) of 32.8%.

[0282] 59. The composition of embodiment 46, wherein the mixture has a molar ratio of (I-A4):(I-A5):(I-A6) of 1: 1.7: 1.3.

[0283] 60. A formulation, such as a pharmaceutical formulation, comprising the composition of any one of embodiments 1 to 59 and at least one excipient.

[0284] 61. The formulation of embodiment 60, wherein the formulation is formulated for oral administration.

[0285] 62. The formulation according to embodiment 61, wherein the orally administered form is a tablet, capsule, solution, mouthwash, suspension, powder, chewing gum, candy, lozenge, sublingual delivery system, or fast dissolving formulation.

[0286] 63. The formulation according to any one of embodiments 60 to 62, wherein the pharmaceutical formulation is a food product.

[0287] 64. The formulation according to embodiment 63, wherein the formulation is a dietary supplement.

[0288] 65. The formulation according to embodiment 63, wherein the food product is a medical food product.

[0289] 66. The formulation according to embodiment 63, wherein the food product is an infant formula.

[0290] 67. The formulation according to any one of embodiments 60 to 66, for use in maintaining or modulating the level of plasmenyl phospholipids in a human subject in need thereof.

[0291] 68. The formulation according to any one of embodiments 60 to 66, for use in maintaining or modulating the level of plasmenyl phospholipids in an animal subject in need thereof.

[0292] 69. The formulation according to embodiment 68, wherein the animal is selected from the group consisting of a domestic animal, a work animal, and a farm animal.

[0293] 70. The formulation according to any one of embodiments 67 to 69, wherein the maintenance or modulation is needed to maintain or modulate the level of plasmenyl phospholipids at a level and / or ratio associated with a non-disease state.

[0294] 71. The composition according to any one of embodiments 1 to 59 or the formulation according to any one of embodiments 60 to 70, for use in increasing the level of plasmenyl phospholipid compounds in the blood or tissue of a subject.

[0295] 72. The composition for use according to embodiment 71, wherein the subject is a human.

[0296] 73. The composition for use according to embodiment 71, wherein the subject is an animal.

[0297] 74. The composition for use according to embodiment 73, wherein the animal subject is selected from the group consisting of a domestic animal, a work animal, and a farm animal.

[0298] 75. The composition according to any one of embodiments 1 to 59 or the formulation according to any one of embodiments 60 to 70, for use in therapy.

[0299] 76. The composition according to any one of embodiments 1 to 59 or the formulation according to any one of embodiments 60 to 70 for use in the treatment of a disease or disorder associated with an acylphospholipid deficiency.

[0300] 77. The composition or formulation for use according to embodiment 76, wherein the disease or disorder is a neurological disease.

[0301] 78. The composition or formulation according to embodiment 77, wherein the neurological disease is selected from the group consisting of Alzheimer’s disease, Parkinson’s disease, autism, amyotrophic lateral sclerosis, frontotemporal dementia, multiple sclerosis, and schizophrenia.

[0302] 79. The composition or formulation for use according to embodiment 76, wherein the disease or disorder is a metabolic disorder.

[0303] 80. The composition or formulation according to embodiment 79, wherein the metabolic disorder is selected from the group consisting of obesity, insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, an immune-related disorder, a cardiovascular disease, a neurological disease, cancer, myalgic encephalomyelitis / chronic fatigue syndrome, Barth syndrome, and a peroxisomal disorder.

[0304] 81. The composition or formulation according to embodiment 80, wherein the immune-related disorder is selected from the group consisting of asthma, atopic dermatitis, type 1 diabetes, and an infection.

[0305] 82. The composition or formulation according to embodiment 80, wherein the cardiovascular disease is selected from the group consisting of atherosclerosis, cardiac remodeling, and hypertension.

[0306] 83. The composition or formulation according to embodiment 80, wherein the peroxisomal disorder is Zellweger spectrum disorder or rhizomelic chondrodysplasia punctata.

[0307] 84. The composition or formulation for use according to any one of embodiments 71 to 83, wherein the compound of formula (I) or (I-A) is administered at a dose of 0.1 to 4000 mg per day.

[0308] 85. The composition or formulation for use according to embodiment 84, wherein the compound of formula (I) or (I-A) is administered at a dose of 0.1 to 2000 mg per day.

[0309] 86. The composition or formulation for use according to embodiment 85, wherein the compound of formula (I) or (I-A) is administered at a dose of 25 to 1600 mg per day.

[0310] 87. The composition or formulation for use according to embodiment 86, wherein the compound of Formula (I) or (I-A) is administered at a dose of 400 mg, 800 mg, or 1600 mg per day.

[0311] 88. The composition or formulation for use according to embodiment 86, wherein the compound of Formula (I) or (I-A) is administered at a dose of 200 mg per day.

[0312] 89. The composition or formulation for use according to embodiment 86, wherein the compound of Formula (I) or (I-A) is administered at a dose of 25 to 100 mg per day.

[0313] 90. A method of increasing the level of a plasmenylcholine compound in the blood or tissue of a subject in need thereof, the method comprising administering to the subject an effective amount of at least one compound of Formula (I) or a pharmaceutically acceptable salt thereof and / or at least one compound of Formula (I-A) or a pharmaceutically acceptable salt thereof according to embodiments 1 and 14, respectively.

[0314] 91. The method according to embodiment 90, wherein the subject is a human.

[0315] 92. The method according to embodiment 90, wherein the subject is an animal.

[0316] 93. The method according to embodiment 92, wherein the animal subject is selected from the group consisting of a domesticated animal, a working animal, and a farm animal.

[0317] 94. The method according to embodiment 90, wherein the bioavailability of a plasmenylcholine compound in the blood or tissue of the subject is improved upon administration of at least one compound of Formula (I) or a pharmaceutically acceptable salt thereof and / or at least one compound of Formula (I-A) or a pharmaceutically acceptable salt thereof relative to administration of AKG (alkyl glycerol).

[0318] 95. A method of treating a disease or disorder associated with plasmenylcholine deficiency, the method comprising administering to a subject in need thereof an effective amount of at least one compound of Formula (I) or a pharmaceutically acceptable salt thereof and / or at least one compound of Formula (I-A) or a pharmaceutically acceptable salt thereof according to embodiments 1 and 14, respectively.

[0319] 96. The method according to embodiment 95, wherein the subject is a human.

[0320] 97. The method according to embodiment 95, wherein the subject is an animal.

[0321] 98. The method according to embodiment 97, wherein the animal subject is selected from the group consisting of a domesticated animal, a working animal, and a farm animal.

[0322] 99. The method of embodiment 95, wherein the disease or disorder is a neurological disease.

[0323] 100. The method of embodiment 99, wherein the neurological disease is selected from the group consisting of Alzheimer’s disease, Parkinson’s disease, autism, amyotrophic lateral sclerosis, frontotemporal dementia, multiple sclerosis, and schizophrenia.

[0324] 101. The method of embodiment 95, wherein the disease or disorder is a metabolic disorder.

[0325] 102. The method of embodiment 101, wherein the metabolic disorder is selected from the group consisting of obesity, insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, an immune-related disorder, a cardiovascular disease, a neurological disease, cancer, myalgic encephalomyelitis / chronic fatigue syndrome, Beth syndrome, and a peroxisomal disorder.

[0326] 103. The method of embodiment 102, wherein the immune-related disorder is selected from the group consisting of asthma, atopic dermatitis, type 1 diabetes, and an infection.

[0327] 104. The method of embodiment 102, wherein the cardiovascular disease is selected from the group consisting of atherosclerosis, cardiac remodeling, and hypertension.

[0328] 105. The method of embodiment 102, wherein the peroxisomal disorder is Zellweger spectrum disorder or rhizomelic chondrodysplasia punctata.

[0329] 106. The method of any one of embodiments 90-105, wherein the compound of Formula (I) or (I-A) is administered at a dose of 0.1-4000 mg per day.

[0330] 107. The method of embodiment 106, wherein the compound of Formula (I) or (I-A) is administered at a dose of 0.1-2000 mg per day.

[0331] 108. The method of embodiment 107, wherein the compound of Formula (I) or (I-A) is administered at a dose of 25-1600 mg per day.

[0332] 109. The method of embodiment 108, wherein the compound of Formula (I) or (I-A) is administered at a dose of 400 mg, 800 mg, or 1600 mg per day.

[0333] 110. The method of any embodiment 108, wherein the compound of Formula (I) or (I-A) is administered at a dose of 200 mg per day.

[0334] 111. The method of embodiment 108, wherein the compound of Formula (I) or (I-A) is administered at a dose of 25 to 100 mg per day.

[0335] 112. The method of embodiment 111, wherein the compound of Formula (I) or (I-A) is administered at a dose of 25 mg, 50 mg, or 100 mg per day.

[0336] 113. Use of a composition according to any one of embodiments 1 to 59 or a formulation according to any one of embodiments 60 to 70 in the manufacture of a medicament for increasing the level of a plasmalogen compound in the blood or tissue of a subject in need thereof.

[0337] 114. Use of a composition according to any one of embodiments 1 to 59 or a formulation according to any one of embodiments 60 to 70 in the manufacture of a medicament for treating a disease or disorder associated with plasmalogen deficiency.

[0338] 115. The use of embodiment 114, wherein the disease or disorder is a neurological disease.

[0339] 116. The use of embodiment 115, wherein the neurological disease is selected from the group consisting of Alzheimer’s disease, Parkinson’s disease, autism, amyotrophic lateral sclerosis, frontotemporal dementia, multiple sclerosis, and schizophrenia.

[0340] 117. The use of embodiment 116, wherein the disease or disorder is a metabolic disorder.

[0341] 118. The use of embodiment 117, wherein the metabolic disorder is selected from obesity, insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, an immune-related disorder, a cardiovascular disease, a neurological disease, cancer, myalgic encephalomyelitis / chronic fatigue syndrome, Barth syndrome, and a peroxisomal disorder.

[0342] 119. The use of embodiment 118, wherein the immune-related disorder is selected from the group consisting of asthma, atopic dermatitis, type 1 diabetes, and an infection.

[0343] 120. The use of embodiment 118, wherein the cardiovascular disease is selected from the group consisting of atherosclerosis, cardiac remodeling, and hypertension.

[0344] 121. The use of embodiment 118, wherein the peroxisomal disorder is a Zellweger spectrum disorder or rhizomelic chondrodysplasia punctata.

[0345] 122. A kit for use in a method according to any of embodiments 90-112, the kit comprising at least one compound of Formula (I) or Formula (I-A), or a pharmaceutically acceptable salt thereof, according to any of embodiments 1 and 14, respectively.

[0346] 123. The composition according to embodiment 28, wherein the at least one compound is selected from a compound of Formula (I-A1), (I-A2), or (I-A3).

[0347] 124. The composition according to embodiment 29, wherein the mixture of at least two compounds is a 50:50 mixture.

[0348] 125. The composition according to embodiment 30, wherein the mixture of (I-A1), (I-A2), and (I-A3) is selected from:

[0349] LPC(O-16:0) 26.8% + LPC(O-18:0) 46.1% + LPC(O-18:1) 27.1%;

[0350] LPC(O-16:0) 46% + LPC(O-18:0) 21% + LPC(O-18:1) 33%;

[0351] LPC(O-16:0) 42% + LPC(O-18:0) 51% + LPC(O-18:1) 7%;

[0352] LPC(O-16:0) 62% + LPC(O-18:0) 23.9% + LPC(O-18:1) 14.1%;

[0353] LPC(O-16:0) 19.8% + LPC(O-18:0) 66% + LPC(O-18:1) 14.2%;

[0354] LPC(O-16:0) 32.5% + LPC(O-18:0) 39.6% + LPC(O-18:1) 27.9%; or

[0355] LPC(O-16:0) 34.1% + LPC(O-18:0) 41.5% + LPC(O-18:1) 24.4%.

[0356] 126. The composition according to embodiment 44, wherein the at least one compound is selected from a compound of Formula (I-A4), (I-A5), or (I-A6).

[0357] 127. The composition according to claim 45, wherein the mixture of at least two compounds is a 50:50 mixture.

[0358] 128. The composition of embodiment 46, wherein the mixture of (I-A4), (I-A5), and (I-A6) is selected from:

[0359] LPE(O-16:0) 26.8% + LPE(O-18:0) 46.1% + LPE(O-18:1) 27.1%;

[0360] LPE(O-16:0) 46% + LPE(O-18:0) 21% + LPE(O-18:1) 33%;

[0361] LPE(O-16:0) 42% + LPE(O-18:0) 51% + LPE(O-18:1) 7%;

[0362] LPE(O-16:0) 62% + LPE(O-18:0) 23.9% + LPE(O-18:1) 14.1%;

[0363] LPE(O-16:0) 19.8% + LPE(O-18:0) 66% + LPE(O-18:1) 14.2%;

[0364] LPE(O-16:0) 32.5% + LPE(O-18:0) 39.6% + LPE(O-18:1) 27.9%; or

[0365] LPE(O-16:0) 34.1% + LPE(O-18:0) 41.5% + LPE(O-18:1) 24.4%.

[0366] 129. The composition of any one of embodiments 1-59 or 123-128, wherein the composition maintains or modulates the ratio of the acetal phospholipid compounds observed in healthy human subjects.

[0367] 130. The composition of any one of embodiments 1-59 or 123-128, wherein the composition results in a reduction of inflammation or improvement or reduction of symptoms associated with an inflammatory disease.

[0368] 131. The composition of embodiment 130, wherein the reduction of inflammation is associated with a decrease in inflammatory cytokine levels.

[0369] 132. The composition of any one of embodiments 1-59 or 123-128, wherein the composition maintains or modulates inflammatory cytokine levels at levels observed in healthy human subjects.

[0370] 133. The composition of embodiment 132, wherein the inflammatory cytokine that is maintained or modulated is selected from the group consisting of IL-6, NFE2L2, TLR4.

[0371] 134. A formulation comprising the composition of any one of embodiments 1 to 59 or 123 to 128 and at least one excipient and / or solubilizer.

[0372] 135. The formulation of embodiment 134, wherein the formulation is formulated for oral administration.

[0373] 136. The formulation of embodiment 135, wherein the oral administration form is a beverage or a food product.

[0374] 137. The formulation of embodiment 135, wherein the oral administration form is a food product.

[0375] 138. The formulation of embodiment 135, wherein the oral administration form is an infant formula.

[0376] 139. The formulation of embodiment 134, wherein the formulation comprises a solubilizer.

[0377] 140. The formulation of embodiment 139, wherein the solubilizer is selected from the group consisting of sodium carboxymethylcellulose, hypromellose, proline, xanthan gum, maltodextrin, alginate, wax, lipid, oil, alcohol, sugar, microcrystalline cellulose, starch, calcium phosphate, mannitol, sorbitol, erythritol, food grade solvent, phospholipid, DMSO, ethanol, ethyl acetate, and isopropyl alcohol.

[0378] 141. The formulation of embodiment 139, wherein the solubilizer is lecithin.

[0379] 142. The formulation of embodiment 141, wherein the lecithin is purified egg yolk L-a- lecithin.

[0380] 143. The formulation of embodiment 139, wherein the formulation has improved stability compared to a formulation comprising the composition of any one of embodiments 1 to 59 or 123 to 128 and not comprising a solubilizer.

[0381] 144. The formulation of embodiment 139, wherein the formulation is less prone to degradation during freezing and thawing compared to a formulation comprising the composition of any one of embodiments 1 to 59 or 123 to 128 and not comprising a solubilizer.

[0382] 145. The composition of any one of embodiments 28 or 44, wherein the composition comprises a compound having a purity of at least 99.9%.

[0383] 146. A formulation comprising the composition of embodiment 145 and at least one excipient.

[0384] 147. The formulation of any one of embodiments 134-138 or 146, wherein the formulation comprises at least one solubilizing agent, emulsifying agent, stabilizing agent, dispersing agent, antifoaming agent, or diluent.

[0385] 148. The formulation of any one of embodiments 134-138 or 146-147, wherein the formulation additionally comprises at least one solubilizing agent.

[0386] 149. The formulation of embodiment 148, wherein the solubilizing agent is selected from the group consisting of sodium carboxymethylcellulose, hypromellose, proline, xanthan gum, maltodextrin, alginate, wax, lipid, oil, alcohol, sugar, microcrystalline cellulose, starch, calcium phosphate, mannitol, sorbitol, erythritol, food grade solvent, phospholipid, DMSO, ethanol, ethyl acetate, and isopropyl alcohol.

[0387] 150. The formulation of embodiment 149, wherein the solubilizing agent is selected from the group consisting of DMSO, ethanol, ethyl acetate, and isopropyl alcohol.

[0388] 151. The formulation of any one of embodiments 134-144 or 146-150, wherein the formulation additionally comprises at least one antioxidant compound.

[0389] 152. The formulation of any one of embodiments 134-144 or 146-151, wherein the formulation additionally comprises at least one antifoaming agent.

[0390] 153. The formulation of any one of embodiments 134-144 or 146-152, wherein the formulation is formulated for oral administration.

[0391] 154. The formulation of embodiment 153, wherein the oral administration form is a tablet, capsule, solution, mouthwash, suspension, powder, chewing gum, candy, lozenge, sublingual delivery system, or fast-dissolving formulation.

[0392] 155. The formulation of embodiment 154, wherein the oral administration form is a tablet.

[0393] 156. The composition of embodiment 30, wherein the mixture of (I-A1), (I-A2), and (I-A3) comprises 90% w / v (I-A1).

[0394] 157. The composition of embodiment 30, wherein the mixture of (I-A1), (I-A2), and (I-A3) comprises 90% w / v (I-A2).

[0395] 158. The composition of embodiment 30, wherein the mixture of (I-A1), (I-A2), and (I-A3) comprises 90% w / v (I-A3).

[0396] 159. The composition of embodiment 46, wherein the mixture of (I-A4), (I-A5), and (I-A6) comprises 90% w / v (I-A4).

[0397] 160. The composition of embodiment 46, wherein the mixture of (I-A4), (I-A5), and (I-A6) comprises 90% w / v (I-A5).

[0398] 161. The composition of embodiment 46, wherein the mixture of (I-A4), (I-A5), and (I-A6) comprises 90% w / v (I-A6).

[0399] The following non-limiting examples are shown below. Two studies were conducted to determine the effect of precursor compounds and various formulations of the precursor compounds on human plasmalogen levels.

[0400] Example 1

[0401] The first study shows the effect of krill oil and fish oil supplementation on plasmalogen levels in female subjects.

[0402] Details of the lipidomics analysis have been fully described in K. Huynh et al., “High-Throughput Plasma Lipidomics: Detailed Mapping of the Associations with Cardiometabolic Risk Factors,” Cell Chem. Biol. (2019) 26(1): 71-84, and H H. Sung et al., “Differential plasma postprandial lipidomic responses to krill oil and fish oil supplementations in women: A randomized crossover study,” Nutrition (2019) 65: 191-201. Briefly, plasma samples were extracted in CHCl3:MeOH (2:1) with an internal standard mixture containing non-physiological or stable isotope-labeled lipid standards, as previously described. Lipidomics analysis was performed by UHPLC ESI-MS / MS using an Agilent 1290 HPLC coupled with an Agilent 6490 triple quadrupole mass spectrometer. Chromatographic data were analyzed using Mass Hunter Quant, where the relative lipid abundance was calculated by correlating the chromatogram area of each lipid species with the corresponding internal standard. Correction factors were applied to adjust for different response factors, where these response factors are known. Chromatographically resolved species were labeled as such (e.g., PC(16:0 / 22:6) and PC(18:2 / 20:4)), while species that are mixed isomers were given standard phospholipid notation (e.g., PC(40:8) is a mixture of PC(20:4 / 20:4) and PC(18:2 / 22:6). Lipids were manually annotated to contain long-chain omega-3 components (i.e., 20:5 EPA, 22:5 DPA, and 22:6 DHA) where structural details were sufficient.

[0403] This study was a randomized crossover study in which krill oil (KO) and fish oil (FO) supplementation was for 30 days with a washout period of at least 4 weeks between supplementation. Participants were instructed to maintain their habitual diet and were asked not to consume any foods or supplements containing omega-3 PUFAs more than once per week during the study period. For the intervention, participants consumed 7 one-gram KO (Euphausia superba oil, Swisse Wellness Pty Ltd., Victoria, Australia) capsules containing 1.27 g LC omega-3 PUFAs (0.76 g EPA, 0.42 g DHA, 0.09 g DPA) or 5 one-gram FO (Natural FO, Swisse Wellness Pty Ltd., Victoria, Australia) capsules containing 1.44 g LC omega-3 PUFAs (0.79 g EPA, 0.47 g DHA, 0.18 g DPA) per day for 30 days. Participants were required to attend clinic three times for blood and data collection on day 0 (baseline), day 15 and day 30 of each supplementation period of blood sample collection. Prior to each clinic visit, participants consumed a low-fat evening meal and were advised to avoid alcohol and strenuous physical activity and to fast from 10 pm. On each study day, a standardized procedure was followed in which participants arrived at the clinic between 7 and 9 am and a fasting blood sample (10 mL) was collected via venipuncture by a qualified phlebotomist. Following blood sample collection, all participants completed a 24-h dietary recall and an electronic PUFA FFQ. The study protocol was approved by the Ethics Committee of Victoria University Human Research (HRE15-031). Informed consent was obtained from all participants prior to the study (ACTRN 12615000472572).

[0404] Example 2

[0405] A second study showed the effect of shark liver oil (SLO) supplementation on male subjects' plasmalogen levels.

[0406] This study was a double-blind, placebo-controlled crossover study in which participants (n=10) were overweight or obese (BMI between 28-40 kg / m2) and had a history of elevated blood pressure. Participants were randomized to receive either 1 g of SLO or placebo for 30 days with a washout period of at least 4 weeks between supplementation. Participants were instructed to maintain their habitual diet and were asked not to consume any foods or supplements containing omega-3 PUFAs more than once per week during the study period. For the intervention, participants consumed 1 g of SLO or placebo per day for 30 days. Participants were required to attend clinic three times for blood and data collection on day 0 (baseline), day 15 and day 30 of each supplementation period of blood sample collection. Prior to each clinic visit, participants consumed a low-fat evening meal and were advised to avoid alcohol and strenuous physical activity and to fast from 10 pm. On each study day, a standardized procedure was followed in which participants arrived at the clinic between 7 and 9 am and a fasting blood sample (10 mL) was collected via venipuncture by a qualified phlebotomist. Following blood sample collection, all participants completed a 24-h dietary recall and an electronic PUFA FFQ. The study protocol was approved by the Ethics Committee of Victoria University Human Research (HRE15-031). Informed consent was obtained from all participants prior to the study (ACTRN 12615000472572). 2Adult men (aged 25-60 years) without evidence of cardiovascular disease or diabetes were recruited. Written informed consent was obtained from all study participants prior to the start of the study. The study was conducted in accordance with the ethical principles set out in the Declaration of Helsinki and was approved by the Alfred Hospital Ethics Committee (approval number: 436 / 15). Participants were randomly allocated to either a placebo or treatment group and received either 4 g (purified SLO; Eurohealth, Ireland) or placebo (methylcellulose) for 3 weeks, followed by a 3-week washout period, and then crossed over to 3 weeks of alternative placebo treatment. Fasting blood samples were collected at the start and end of each intervention. Tables 3 and 4 show the Composition of alkyl diacylglycerols [TG(O)] and alkylglycerols (AKG) in shark liver oil.

[0407] Table 3. Composition of alkyl diacylglycerols [TG(O)] in shark liver oil.

[0408] Table 3

[0409] TG(O) Concentration (mM) Percentage (%) of total TG(O) TG (O-50: 1) 20.9 4.2 TG (O-52: 1) 20.3 3.5 TG (O-52: 2) 191.3 36.4 TG (O-53: 2) 12.3 2.1 TG (O-54: 1) 4.8 0.9 TG (O-54: 2) 61.8 10.7 TG (O-54: 3) 77.6 14.5 TG (O-56: 2) 31.7 5.7 TG (O-56: 3) 55.7 9.5 TG (O-58: 2) 15.8 2.8 TG (O-58: 3) 40.4 7.1 TG (O-60: 3) 14.7 2.6

[0410] Table 4. Composition of alkylglycerols (AKG) in shark liver oil.

[0411] Table 4

[0412] AKG Concentration (mM) Percentage of total AKG (%) AG (14:0) 6.2 1.1 AG (14:1) 1.4 0.2 AG (15:0) 3.2 0.6 AG (15:1) 5.1 0.9 AG (16:0) 83.2 14.6 AG (16:1) 51.1 9.0 AG (17:0) 10.3 1.8 AG (17:1) 14.4 2.5 AG (18:0) 33.1 5.8 AG (18:1) 341.6 59.9 AG (19:0) 2.4 0.4 AG (19:1) 5.3 0.9 AG (20:0) 1.3 0.2 AG (20:1) 12.0 2.1

[0413] The datasets of the studies of Example 1 and Example 2 were compared and the effect of these oil supplements on plasma ether lipid levels was inferred. Figure 1 and Figure 2 It is shown that both KO and SLO supplementation substantially increased the levels of different ether lipid classes in plasma, while FO supplementation had no great impact on these lipid classes. The increase in plasmalogens (PE(P) and PC(P)) was significantly higher for KO supplementation (30 days) compared to SLO supplementation (21 days).

[0414] The effect of supplementation on PE(P) composition was also inferred. Figure 3 and Figure 4 It is shown that KO and FO supplementation had no any significant impact on the alkenyl chain composition of PE plasmalogens, while Figure 5It is shown that SLO supplementation significantly altered this composition (increasing the proportion of PE plasmalogens containing 18:1 alkenyl chains and decreasing the proportion of PE plasmalogens containing 16:0 and 18:0 alkenyl chains). All of these supplements had a significant effect on the acyl chain composition of PE plasmalogens. Figure 6 and Figure 7 It is shown that both KO and FO supplementation decreased the proportion of PE plasmalogens containing 18:1, 18:2, and 20:4 and increased the proportion of PE plasmalogens containing 20:5 and 22:6, while Figure 8 It is shown that SLO supplementation decreased the proportion of PE plasmalogens containing 20:4 and increased the proportion of PE plasmalogens containing 18:1 and 22:6.

[0415] Without wishing to be bound by theory, it is believed that these differences can originate from the type and content of ether lipids found in KO and SLO. In KO, the ether lipids are primarily phosphatidylcholine ether lipids (PC(O) or LPC(O)), while the SLO ether lipids are monoalkyldiacylglycerols or TG(O)). It has been found that KO contains approximately 25 mg LPC(O) / g oil, while SLO contains approximately 200 mg alkyglycerol / g oil. Furthermore, the alky chain composition of KO and SLO is different. In SLO, the most abundant alky species is O-18:1 (Paul et al., Journal of Lipid Research (2021) 62:10009), while in KO, the most abundant alky species is O-16:0 (Table 4).

[0416] Table 5. Composition of lysophosphatidylcholine [LPC(O)] in krill oil. Several of the main components are presented in bold and underlined.

[0417] Table 5

[0418] Lipid species Percentage of total LPC(O) (%) LPC(O-16:0) 38.44 LPC(O-16:1) 22.65 LPC(O-14:0) 17.63 LPC(O-18:1) 7.46 LPC(O-15:0) 5.11 LPC(O-18:2) 3.19 LPC(O-18:0) 2.89 LPC(O-17:0) 1.56 LPC(O-20:1) 0.65 LPC(O-22:1) 0.22 LPC(O-20:0) 0.12 LPC(O-24:2) 0.03 LPC(O-22:0) 0.02 LPC(O-24:1) 0.02

[0419] In Table 6, the relative bioavailability of KO and SLO modulation of ether lipids is further compared. Considering the changes in ether lipid levels, KO LPC(O) appears to have significantly higher bioavailability than SLO alkyglycerol.

[0420] Table 6. Differences in bioavailability between krill oil and shark liver oil.

[0421] Table 6

[0422]

[0423] NC = not calculated as PC(P) levels were not increased after shark liver oil supplementation.

[0424] * Shark liver oil dose / day: 4 g shark liver oil = 800 mg alkylglycerol; the approximate molecular weight of alkylglycerol is 344 g / mol, which gives 2.33 mmol / 75 kg = 31 pmol / kg body weight. Treatment duration is 21 days.

[0425] ** Krill oil dose / day: 7 g krill oil = 175 mg LPC(O); the approximate molecular weight of LPC(O) is 480 g / mol, which gives 0.36 mmol / 75 kg = 4.38 pmol / kg body weight. Treatment duration is 30 days.

[0426] It was observed that KO provides a high twelve-fold change in PE(P) levels per micromole dose compared to SLO, and this effect is largely retained when adjusted for the treatment period (high eight-fold). There is a large improvement in bioavailability of LPC(O) / PC(O) in krill oil relative to alkyl diacylglycerols present in SLO.

[0427] Example 3

[0428] Animal experiments were performed by Product Safety labs (Dayton, NJ, USA). A total of 32 animals (all female) were used. Animals were selected based on adequate body weight gain, absence of clinical signs of illness or injury. Selected rats were stratified by body weight and randomly assigned to groups so that group mean body weights did not differ by more than 20% within gender across groups, and were assigned to the following test groups (Table 7). Animals had free access to food and water throughout the experiment. Each animal was dosed by oral gavage using a stainless steel ball-tipped gavage needle attached to an appropriate syringe. Dose administration was performed once on Day 1.

[0429] Table 7. Study design

[0430] Table 7

[0431]

[0432] Plasmalogen precursor compounds and formulations

[0433] Stable-isotope versions of four different lipid species were administered at 2 doses (C, D) (Table 8). All of these compounds were synthesized by Anthem Biosciences (Bangalore, India).

[0434] Table 8. Deuterium-labeled plasmalogen precursor compounds used in this study. “D2” indicates that two hydrogen atoms in the compound have been replaced with deuterium atoms (examples are provided in Scheme III below).

[0435] Table 8

[0436]

[0437]

[0438] To facilitate administration of the precursor compounds, purified egg yolk L-a- phosphatidylcholine (Sigma-Aldrich, St. Louis, MO, USA) was used as a vehicle (14 mg for dose mix C and 47 mg for dose mix D). Stock solutions of individual precursors (100 mg / ml) were first prepared in chloroform:methanol (1 : 1). A stock solution of egg phospholipid (100 mg / ml; Sigma-Aldrich, St. Louis, MO, USA) was also prepared in chloroform:methanol (1 : 1). To prepare the precursor compound mixtures, appropriate volumes of precursor compound and egg phospholipid solutions were mixed, dried under a stream of nitrogen at 40 °C, then reconstituted in deionised water by vigorous vortexing, followed by sonication in a Soniclean water bath (Soniclean, Adelaide, SA, Australia) for 1 hr and further sonication using a Misonix S-4000 ultrasonicator (Thermo Fisher Scientific, Melbourne, VIC, Australia) at amplitude 25 for 2 x 30 s.

[0439] Blood sample collection

[0440] Blood samples were collected from all animals at 8 time points (pre-dose, 1 hr, 2 hr, 4 hr, 8 hr, 12 hr, 24 hr and 48 hr post-dose). Approximately 200 μΐ^of blood was collected sublingually into blood collection tubes containing K2EDTA under isoflurane anaesthesia and kept on ice until centrifugation. Following centrifugation, plasma was transferred to clean tubes and frozen at approximately -80 °C until analysis.

[0441] Terminal sacrifice and tissue collection

[0442] Fecal samples from the 24 hour and 48 hour periods were collected at terminal sacrifice and all surviving animals were euthanized using CO2asphyxiation. At 48 hr, brains, livers, spleens, kidneys, hearts, gastrocnemius muscles and abdominal adipose tissue were carefully dissected from all study animals, placed into fresh weighing boats and weighed, then snap-frozen within 5 minutes and stored at -80 °C. No other observations were made on any animals at necropsy.

[0443] Lipid extraction from plasma samples

[0444] Lipids were extracted from 10 μl of plasma samples using butanol:methanol (1 : 1) as previously described (Alshehry ZH. et al. 2015). Lipid extraction was performed in single batches, with quality control samples (pooled plasma QC, NIST QC and blank) included every 20 samples.

[0445] Tissue processing and extraction of lipids from tissue samples

[0446] Approximately 40-60 mg of tissue was homogenized in 400-600 μL of ice-cold phosphate buffered saline for 60 s using a TissueLyser II (Qiagen, USA) followed by 10-15 s of sonication at amplitude 25 using a Misonix S-4000 ultrasonicator (Thermo Fisher Scientific, Melbourne, Victoria, Australia). Protein content in the homogenate was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). The homogenate was then made up to a stock protein concentration of 5 mg / mL protein, with 10 μL aliquots from the stock solution containing 50 μg of protein used for lipid extraction. For adipose tissue, 10 μL aliquots from the stock homogenate were used for lipid extraction. Lipids were extracted following a single-phase chloroform:methanol (2: 1) extraction procedure as previously described (Miekle PJ. et al. 2011). Lipid extraction was performed in single batches for each tissue type, with liver and brain samples not extracted together, with quality control samples (pooled plasma QC, pooled tissue QC, NIST QC and blank) included approximately every 10 samples.

[0447] Liquid chromatography-mass spectrometry analysis

[0448] Lipidomics analysis was performed on an Agilent 1290 UHPLC system and Agilent 6495 triple quadrupole mass spectrometer with the column system set to 45 °C (ZORBAX eclipse plus C18 column: 2.1 x 100 mm, 1.8 mm, Agilent). Samples were placed in the autosampler tray (20 °C) and 1 pL was injected. Lipids were separated with a stepwise linear gradient of solvent A (50% water / 30% acetonitrile / 20% isopropanol) and solvent B (1% water / 9% acetonitrile / 90% isopropanol) at a flow rate of 0.4 mL / min, both solvents containing 10 mM ammonium formate. The solvent gradient started at 85% A, decreased to 50% in 2.5 min, decreased to 43% in 0.1 min, decreased to 30% in 6.4 min, decreased to 7% in 0.1 min, decreased to 4% in 1.9 min, then decreased to 0% in 0.1 min and held for 0.9 min, then increased back to 85% in 0.2 min and held for 3.8 min. The following mass spectrometer conditions were used; gas temperature 150 °C, gas flow 17 L / min, nebulizer 20 psi, sheath gas temperature 200 °C, capillary voltage 3500 V, and sheath gas flow 10 L / min.

[0449] A modified version of the previously reported MRM method (see K. Huynh et al., “High-Throughput Plasma Lipidomics: Detailed Mapping of the Associations with Cardiometabolic Risk Factors,” Cell Chem. Biol. (2019) 26(1): 71-84) was used to detect deuterium incorporation in lipid species of the ether phospholipid and ether glycerolipid classes: alkyl diacylglycerol [TG(O)], lysalkylphosphatidylcholine [LPC(O)], alkylphosphatidylcholine [PC(O)], alkylphosphatidylethanolamine [PE(O)], alkenylphosphatidylethanolamine [PE(P)] or PE plasmalogen, alkenylphosphatidylcholine [PC(P)] or PC plasmalogen. Both the deuterated and endogenous unlabelled versions of the lipids were measured. Lipids with deuterated isotopes are denoted as [+1] and [+2], while endogenous lipids are referred to as [+0]. Due to the location of the deuterium, precursors labelled with D2 will produce both the [+1] and [+2] lipid species, which makes them susceptible to removal by the Delta 1 desaturase (PEDS1) when producing plasmalogens. Since one deuterium is equivalent to 1 Da, the precursor mass of a lipid species labelled with D2 is shifted by 2 Da. Product ions were shifted based on the lipid class fragmentation pattern.

[0450] Use Agilent MassHunter quantitative analysis software to integrate peak value, and the area under the curve of every kind of lipid species is quantitatively carried out.In order to calculate the relative concentration of deuterium-labeled lipid in sample, the inventor first deducts the signal of lipid species in blank from sample.This removes any background signal that mass spectrometer has captured.Next, the inventor calculates the isotope ratio in 0hr sample (for tissue, the inventor uses the contrast male sample), and this isotope ratio is corresponding to the deuterium-labeled lipid area on the endogenous lipid area.This calculates the natural isotope ratio of every kind of lipid that should be removed.Then, the inventor multiplies the isotope ratio by the area deducting background of every kind of lipid, to obtain the endogenous signal of every kind of lipid.Then, from all deuterium-labeled lipid areas, deduct this value.Afterwards, the inventor divides this value by the internal standard area of ​​corresponding lipid class, and multiplies by the internal standard amount that adds in sample.This provides the relative concentration of deuterated lipid in sample.

[0451] Effects of labeled precursor supplementation on plasma ether lipids

[0452] Within 48 hours after oral administration of the precursor compound, the inventors observed different patterns in the concentrations of the various tracer lipids. Specifically, the concentration of TG(O) reached its highest point within 1-2 hours of administration and then declined rapidly in all treatment groups ( Figures 29A-29H ). In PC(O)( Figures 31A-31H ), except for the LPC(O)-dose mixture D( Figure 30H Interestingly, LPC(O) concentrations in all treatment groups took longer to reach their peak (12-24 hours) and then gradually declined ( Figures 30A-30H ). For PE(O)( Figures 32A-32H ), the trajectory of AKDAG-DHA treatment was higher than that of other treatments ( Figure 30C and Figure 30G ) is much steeper (rapid rise within 8 hours, followed by rapid fall). The trajectory of PE(P) is slower ( Figures 33A-33H ), it is noteworthy that LPC(O) showed a slower trajectory compared with the other groups, with its concentration peaking at the 24-hour mark and then gradually declining ( Figure 33D and Figure 33H ), however, a large amount of tracer PE(P) was shown at the 48 hour time point. In addition to LPC(O) ( Figure 34D and Figure 34H ), all groups had tracers PC(P)( Figures 34A-34H) concentrations peaked 24 hours after fluctuation at early time points and remained stable for up to 48 hours. For the LPC(O) group, tracer PC(P) concentrations gradually increased from 12 to 48 hours. These observations highlight the different timelines of peak and subsequent decline of lipid concentrations in each treatment group, revealing their different dynamics in terms of absorption and metabolism.

[0453] Next, the inventors compared the maximum plasma tracer concentrations (Cmax) of the main ether lipid classes in the different experimental groups. Figures 35A-35F ) other than TG(O) ( Figure 35A ) were consistently associated with the LPC(O) group. As expected, the inventors observed a dose-dependent increase in plasma LPC(O) levels after treatment with LPC(O) ( Figure 35B ). Furthermore, a similar dose-dependent pattern was observed for PC(O) ( Figure 35C ) and PE(O) ( Figure 35D ) within the LPC(O) group. In addition, the LPC(O) group exhibited significantly higher Cmax of PE(P) ( Figure 35E ) compared to the other groups. The dose mixture D LPC(O) group also showed an elevated Cmax of PC(P) ( Figure 35F ).

[0454] Although LPC(O) is much more efficient in PE(P) production than the other compounds, the actual conversion is low. LPC(O) dose mixture C treatment showed 1.4% conversion to PC(O) and present in plasma at Cmax ( Figure 35C ), but only 0.12% conversion to PE(P) and present in plasma at Cmax ( Figure 35F ).

[0455] Next, the inventors calculated the total exposure of the labeled precursor compounds as area under the curve (AUC) using the tracer lipid concentrations at different time points ( Figures 36A-36F ). In PE(P) production, the inventors did not observe significant differences in AUC between the AKG, oleic acid-containing AKDAG [AKDAG(OA)], and DHA-containing AKDAG [AKDAG(DHA)] groups ( Figure 36E ), particularly in the case of PE(P), where the dose mixture Cs of each compound was more pronounced. However, based on the comparison between the dose mixture C groups, LPC(O) administration ( Figure 36B ) resulted in approximately 5-fold increase in PE(P) relative to AKG and AKDAG. Interestingly, LPC(O) showed rapid metabolism to PC(O), resulting in a significant 16-fold increase in PC(O) levels ( Figure 36C), while PE(O) was moderately elevated by 3-fold ( Figure 36D ). This observation is interesting and indicates PC(O) as a metabolic sink for LPC(O). Unlike LPC(O), AKG and AKDAG(OA) treatments exhibited rapid metabolism to TG(O) and showed less conversion to PC(O), PE(O), and PE(P). On the other hand, AKDAG(DHA), especially at dose mixture D, demonstrated significant conversion to PE(P), although far less than LPC(O). Notably, PC(P) Figure 36F ) production remained limited in all treatments over the 2-day time period.

[0456] Effects of labeled precursor supplementation on tissue ether lipids

[0457] In addition to analyzing plasma lipids, the inventors also examined tissue ether lipids to evaluate the extent of labeled precursor incorporation into endogenous ether lipids. The inventors’ observations indicate that in the liver, spleen, brain, and kidney, labeled precursors were efficiently converted to newly synthesized LPC(O), PC(O), and PE(O), especially in the case of LPC(O) Figures 37A-43F ) administration. Within visceral adipose tissue, skeletal muscle, and heart, the inventors did not observe significant incorporation of labeled precursors into endogenous ether lipids Figures 41A-43F ).

[0458] Example 4

[0459] Aldehyde phospholipid precursor treatment

[0460] The acetal phospholipid precursors were used to treat cells and mice to compare their bioavailability and conversion to endogenous acetal phospholipids. Due to the availability of acetal phospholipid precursors, the inventors used precursors labeled with different numbers of deuterium atoms at different positions. The number of deuterium labeling sites is described with the letter “D” followed by a number. For example, “D2” refers to a precursor labeled with two deuterium atoms (see Scheme III). For example, cells were treated with AKG (0-16:0, D2) (Anthem Biosciences, Bangalore, India), LPC (0-16:0, D4) (Cayman Chemical, Ann Arbor, USA), and LPE (0-16:0, D5) (Avanti Polar Lipids, Birmingham, USA) (Table 9). These are deuterium-labeled acetal phospholipid precursors that contain a 16:0 alkenyl chain (Scheme III). In contrast, mice were administered a mixture of AKG (0-16:0, 0-18:0, 0-18:1, D2) (Anthem Biosciences, Bangalore, India), a mixture of LPC (0-16:0, 0-18:0, 0-18:1, D2) (Anthem Biosciences, Bangalore, India), and LPE (0-16:0, D5) (Table 9). The AKG and LPC (O) mixtures are deuterium-labeled and include 16:0, 18:0, and 18:1 alkenyl chains (Scheme III). LPE (O) contains a mixture of deuterium-labeled 16:0 alkenyl chains and unlabeled 16:0, 18:0, 18:1 alkenyl chains (Scheme III).

[0461] Table 9. Compositions of acetal phospholipid precursor treatments for HepG2 cells, 3T3 cells, and mice. Cells and mice were treated with controls, alkylglycerols (AKG), lysed alkylphosphatidylcholines (LPC(O)), and lysed alkylphosphatidylethanolamines (LPE(O)). Cell culture media included Dulbecco’s Modified Eagle Medium (DMEM).

[0462] Table 9

[0463]

[0464]

[0465] Scheme III. Deuterium-labeled acetal phospholipid precursors used in this study; alkylglycerols (AKG), lysed phosphatidylcholines (LPC(O)), and lysed phosphatidylethanolamines (LPE(O)).

[0466] Cell Culture

[0467] Cell line is selected based on the tissue that is usually affected in the disease associated with low plasmalogen level.Two cell lines are immortalized human liver cancer HepG2 and immortalized mouse fibroblast 3T3.Cell is cultivated in 37 ℃, 5% CO2 Dulbecco's modified Eagle medium (DMEM) (Gibco, the U.S.), and this DMEM is supplemented with 1% sodium pyruvate (Gibco, the U.S.) and 10% fetal bovine serum (Gibco, the U.S.) or 10% newborn calf serum (Gibco, New Zealand) for HepG2 and 3T3 respectively.HepG2 cells are passaged weekly, and every two to three days supplementary culture medium.After going down to posterity 30 times, these cells are discarded.3T3 cells are passaged every two to three days, and after going down to posterity 39 times, are discarded.Before inoculation occurs, cultivate two cell lines and continue to go down to posterity at least twice. HepG2 cells were seeded at 300,000 cells / ml of culture medium in 12-well plates to achieve 80% confluency, while 3T3 cells were brought to 100% confluency before the experiments.

[0468] deal with

[0469] To compare the uptake and incorporation of plasmalogen precursors into plasmalogens, cells were treated with deuterium-labeled AKG, LPC(O), and LPE(O) at a concentration of 20 μM (Table 9). Since AKG is soluble in ethanol, the control treatment was a medium containing 0.05% ethanol. Prior to treatment, images were acquired using an Olympus CKX41 inverted microscope and the cells were stained with a Ca-free medium. 2+ and Mg 2+ The cells were washed with phosphate-buffered saline (PBS). Figure 9 and Figure 13 Shown are images before and after the treatment of HEPG2 cells and 3T3 cells. Then, cells were treated with AKG, LPC (O), LPE (O) or a control, and incubated for 24 hours. After incubation, images were taken, and only adherent cells were harvested. For HepG2 cells, cells were incubated together with trypsin-ethylenediaminetetraacetic acid (EDTA) (0.05%), phenol red (Gibco, the U.S.) at 37°C and 5% CO2 for 5 minutes. Subsequently, centrifuged at 13,000g for 15 minutes. The supernatant was then removed, and Speedy Vac and Pump (Thermo Scientific) was used to dry the remaining sediment. For 3T3, cells were washed with PBS, and a cell scraper was used. Speedy Vac and Pump was then used to dry the cell suspension. After the drying process, the samples were stored at -80°C until lipid extraction was performed.

[0470] Animal experiments

[0471] To compare bioavailability and incorporation of the acetal phospholipid precursors into acetal phospholipids, mice were administered deuterium-labelled AKG mix, LPC(O) mix and LPE(O) (Table 9). Egg phospholipid was used as a vehicle to facilitate oral delivery and as a control treatment. Animal care and experiments were approved by the Alfred Health and Education Precinct Animal Ethics Committee (P8490). The study involved 64 approximately eight-week-old C57BL / 6 mice, consisting of 32 males and 32 females. Figure 9 A dosing schedule is shown for 64 eight-week-old C57BL / 6 mice treated with a single dose of deuterium-labelled precursors and control by oral gavage. The precursors include alkylglycerols (AKG), lysed alkylphosphatidylcholine (LPC(O)) and lysed alkylphosphatidylethanolamine (LPE(O)). Blood samples were collected by tail tip bleeds and cardiac puncture at 0, 1, 4, 24 and 48 hours. Mice were housed at the Precinct Animal Centre of the Baker Heart and Diabetes Institute, with a 12-hour light and dark cycle and given ad libitum access to a standard diet group (SF00-105) and water. There were eight treatment groups, each consisting of four females and four males, that received a single dose of egg phospholipid (vehicle control), AKG, LPC(O) and LPE(O) at either dose mix A or dose mix B (Table 10). Mice were randomly allocated to treatment groups based on body weight prior to treatment and fasted for 4 hours. Each mouse received 200 μl of its respective treatment by oral gavage. Blood was collected by tail tip bleeds prior to treatment and at 1, 4, 24 and 48 hours after treatment. At the end of the experiment, mice were anaesthetised by intraperitoneal injection of sodium pentobarbitone and blood was collected via cardiac puncture. Mice were then euthanased by cervical dislocation and tissues were collected and snap-frozen. Collected blood was placed into EDTA tubes and stored on ice. Plasma was isolated from blood via centrifugation at 1,710g, 20°C for 15 minutes. Plasma supernatant was stored at -80°C until lipid extraction.

[0472] Table 10. Dose mixtures for acetal phospholipid precursors and control treatments. The precursors include alkylglycerols (AKG), lysed alkylphosphatidylcholine (LPC(O)) and lysed alkylphosphatidylethanolamine (LPE(O)).

[0473] Table 10

[0474] Compound group Dose mix A (mg / mouse) Dose mix B (mg / mouse) Lecithin (vehicle control) 1.0 3.3 AKG 1.0 3.3 LPC(O) 1.5 5.0 LPE(O) 1.4 4.5

[0475] Lipid extraction

[0476] Lipid extraction was performed to collect lipids from cell and plasma samples for lipidomic analysis. As part of the extraction process, blank and pooled plasma quality control samples (PQCs) were extracted from the plasma of healthy individuals every 10-20 samples. In addition, the extraction of mouse plasma contained NIST quality controls (NIST QCs) and mouse plasma quality controls (mouse PQCs) which used plasma from two females and two males from each group. These quality control samples helped to identify any changes during the extraction process. Depending on the sample type, chloroform:methanol (2: 1) or butanol:methanol (BUME, 1: 1) were used to extract lipids based on previously reported methods (Alshehry Z.H. et al., Metabolites. 2015 Jun 17; 5(2): 389-403; Meikle PJ, Wong G, Tsorotes D, Barlow CK, Weir JM, Christopher MJ, et al. Plasma lipidomic analysis of stable and unstable coronary artery disease. Arteriosclerosis, Thrombosis, and Vascular Biology. 2011; 31(11): 2723-32). To calculate relative lipid concentrations, internal standards containing over 20 non-physiological lipids of known concentrations were mixed with chloroform:methanol or BUME and then used for extraction.

[0477] Extraction of HepG2 cells involved taking 10 mΐ of sample, blank, and PQC samples and mixing them with 200 mΐ of chloroform:methanol mixed with internal standards. The samples were then rotated on a rotating stirrer for 10 minutes, bath sonicated for 30 minutes, and left to stand at room temperature for 20 minutes. After that, the samples were centrifuged at 13,000 rpm for 10 minutes and dried using a Speedy Vac and Pump. The extracted lipids were then reconstituted with 50 mΐ of water-saturated butanol and sonicated for 10 minutes, followed by the addition of 50 mΐ of methanol. Finally, the samples were centrifuged at 4,000 rpm for 5 minutes and the supernatant was transferred to a glass vial (Agilent) with a Teflon glass insert (Agilent) and stored at -80 °C for lipidomic analysis.

[0478] Extraction of 3T3 cells involved taking 10 mΐ of sample, blank and PQC sample and mixing with 100 mΐ of BUME mixed with internal standards. The samples were then vortexed for 10 seconds, bath sonicated for 60 minutes and centrifuged at 13,000 rpm for 10 minutes. The supernatant was then transferred to a glass vial with a teflon insert for lipidomics analysis.

[0479] Mouse plasma extraction also used BUME but only 5 mΐ of sample was mixed with 5 mΐ of water. Samples included plasma supernatant, blank, PQC, NIST QC and mouse PQC. As only 5 mΐ of sample was used, half the concentration of internal standards was mixed with the BUME compared to the extraction of HepG2 and 3T3 cells.

[0480] To extract lipids from brain samples, approximately 50 mg of brain tissue was homogenised in 500 mΐ of ice-cold phosphate buffered saline for 60 s using a TissueLyser II (Qiagen, USA) followed by 10-15 s of sonication at amplitude 25 using a Misonix S-4000 sonicator (Thermo Fisher Scientific, Melbourne, Victoria, Australia). Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) was used to quantify the protein content of the homogenate. The homogenate was then made up to a stock protein concentration of 5 mg / mL protein, with 10 mΐ aliquots from the stock solution containing 50 pg of protein used for lipid extraction. Lipids were extracted following the single-phase chloroform:methanol (2: 1) extraction procedure as previously described (Meikle PJ, Wong G, Tsorotes D, Barlow CK, Weir JM, Christopher MJ, et al. Plasma lipidomic analysis of stable and unstable coronary artery disease. Arteriosclerosis, Thrombosis, and Vascular Biology. 2011; 31(11): 2723-32). Lipid extraction was performed in single batches with approximately every 10 samples including quality control samples (pooled plasma QC, pooled tissue QC, NIST QC and blank). TM BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) was used to quantify the protein content of the homogenate. The homogenate was then made up to a stock protein concentration of 5 mg / mL protein, with 10 mΐ aliquots from the stock solution containing 50 pg of protein used for lipid extraction. Lipids were extracted following the single-phase chloroform:methanol (2: 1) extraction procedure as previously described (Meikle PJ, Wong G, Tsorotes D, Barlow CK, Weir JM, Christopher MJ, et al. Plasma lipidomic analysis of stable and unstable coronary artery disease. Arteriosclerosis, Thrombosis, and Vascular Biology. 2011; 31(11): 2723-32). Lipid extraction was performed in single batches with approximately every 10 samples including quality control samples (pooled plasma QC, pooled tissue QC, NIST QC and blank).

[0481] Lipidomics analysis by UHPLC / MS / MS

[0482] Targeted lipidomics analysis of lipid extracts of HepG2 cells, 3T3 cells, and plasma samples was performed using ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC / MS / MS). Prior to analysis, lipid extracts were randomized, thawed at room temperature for one hour, and sonicated in a water bath for 15 minutes. Lipids were quantitatively analyzed using an Agilent 1290 series UHPLC system combined with an Agilent 6495C triple quadrupole mass spectrometer. First, samples were placed in the autosampler tray at 20 °C and 1 mΐ of cell sample or 2 mΐ of plasma sample was injected. Lipids were separated using a stepwise linear gradient of solvent A and solvent B under the liquid chromatography conditions outlined in Table 11 and Table 12. Then, lipids were identified based on their retention time, precursor ion, and product ion mass under the mass spectrometry conditions outlined in Table 13.

[0483] Table 11. Ultra-high performance liquid chromatography conditions for lipidomics analysis.

[0484] Table 11

[0485]

[0486] Table 12. Ultra-high performance liquid chromatography gradient conditions.

[0487] Table 12

[0488]

[0489] Table 13. Mass spectrometry conditions for lipidomics analysis.

[0490] Table 13

[0491] Conditions Gas temperature 150℃ Gas flow rate 17 l / min Sheath gas temperature 200℃ Sheath gas flow rate 10 l / min Capillary voltage 3500V Nebulizer 20 psi

[0492] A modified version of the previously reported MRM method was used to detect deuterium incorporation in lipid species of the glycerophospholipid and glycerolipid classes (Huynh, K., et al., 2019). Both the deuterated and endogenous unlabelled versions of the lipids were measured. Lipids with deuterated isotopes are denoted as [+1], [+2], [+4], and [+5], while endogenous lipids are referred to as [+0]. A precursor labelled with D4 produces a [+4] lipid species, while a precursor labelled with D5 produces a [+5] lipid species. However, due to the location of the deuterium, a precursor labelled with D2 will produce both a [+1] and a [+2] lipid species, which makes them susceptible to removal by the PEDS1 enzyme when producing aldehyde phosphatides. Since one deuterium is equivalent to 1 Da, the precursor mass of a lipid species labelled with D4 is shifted by 4 Da. Product ions were shifted based on the lipid class fragmentation pattern.

[0493] Agilent MassHunter Quantitative Analysis v10.0 software was used to integrate the peaks and quantify the area under the curve for each lipid. To calculate the relative concentration of deuterium labeled lipids in the cell samples, the inventors first subtracted the background signal from the blank samples. This removes any irrelevant background signal that the mass spectrometer has identified. Next, the inventors calculated the isotope ratio in the control sample, which corresponds to the deuterium labeled lipid area over the endogenous lipid area. This calculates the natural isotope of each lipid that should be removed. The inventors then multiplied the isotope ratio by the background subtracted area of each sample to get the endogenous signal of each lipid. This value is then subtracted from all the deuterium labeled lipid areas. After this, the inventors divide this value by the internal standard area of the corresponding lipid class and multiply by the amount of internal standard added. This provides the relative concentration of deuterium lipids in pmol / sample. Any variation in the lipid concentration in the samples, such as differences in cell number, can be removed by normalizing to one of the most abundant phospholipids, PC 34:1. The relative concentration of PC 34:1 is calculated by dividing the area of PC 34:1 by the area of its internal standard. This is then multiplied by 100 to give the concentration in pmol / sample. The relative concentration of deuterium lipids is then divided by the relative concentration of PC 34:1 to provide the concentration of PC 34:1 (pmol / pmol). The relative concentration of each lipid class is also calculated as the sum of the individual species within that class. When calculating the sum of TG(O), only the neutral loss species are considered.

[0494] The relative lipid concentration in mice was calculated by an additional step of multiplying the deuterium lipid concentration (pmol / sample) by 200 to give the concentration in plasma (pmol / ml) and then normalizing to PC 34:1. Furthermore, for the calculation of LPE(O), the concentration was divided by 0.65 due to the presence of both labeled and unlabeled precursors.

[0495] Effect of acetal phospholipid precursors on the acetal phospholipid biosynthetic pathway in HepG2 cells

[0496] To compare the uptake and incorporation of acetal phospholipid precursors into acetal phospholipids, HepG2 cells were treated with 20 mM of deuterium labeled acetal phospholipid precursors for 24 hours and the lipid concentrations were analyzed using LC-MS / MS. Images taken before and after treatment revealed that LPC(O) and LPE(O) resulted in floating cells and smaller adherent cells, while AKG did not affect cell morphology compared to the control Figure 9 ).

[0497] The labeled PE(P) levels of 16:0, 18:0 and 18:1 species were compared between the treatment groups. It was found that the concentration of the acetal phospholipid precursor groups was significantly higher compared to the control (p < 0.01, Figure 10A). This increase in labeled PE(P) was most pronounced in LPE(O), followed by LPC(O), and then AKG Figure 10A ) To better understand how efficient precursors are at being metabolized into plasmenyl phospholipids, the ratio of labeled PE(P) concentration to total labeled concentration was calculated. Here, total label refers to the sum of labeled AKG, alkylacylglycerols (DG(O)), LPC(O), lysophosphatidylcholine plasmenyls (LPC(P)), LPE(O), lysophosphatidylethanolamine plasmenyls (LPE(P)), alkylphosphatidylcholines (PC(O)), phosphatidylcholine plasmenyls (PC(P)), alkylphosphatidylethanolamines (PE(O)), PE(P), and monoalkyldiacylglycerols (TG(O)). This approach better expresses the efficiency of precursors because it takes into account the amount of precursor that is taken up by the cell. While some precursors can have greater uptake and result in higher PE(P) production, this does not necessarily mean that it is more efficient at producing PE(P) if it requires more precursor. Comparison between the plasmenyl phospholipid precursor groups shows that the ratio of LPE(O) is significantly greater than both AKG and LPC(O) (p < 0.0001, Figure 10B ) Additionally, the ratio of AKG is significantly increased compared to LPC(O) (p < 0.05, Figure 10B ) However, the difference between AKG and LPC(O) is less than the difference between LPE(O) and the other precursors.

[0498] Bar graphs were created to visualize the distribution of label between lipid classes in the plasmenyl phospholipid biosynthetic pathway Figure 11 ) After AKG treatment, a substantial proportion of label was incorporated into AKG and DG(O), representing 40% and 23% of total label, respectively Figure 11 A and Figure 12A ) In contrast, incorporation into TG(O), LPC(O), LPE(O), and PC(P) was minimal, and incorporation into PC(O), PE(O), and PE(P) was intermediate, with PE(P) containing 11% of total label Figure 11 A and Figure 12A ) After LPC(O) treatment, incorporation into LPC(O) was high and incorporation into PC(O) was greater, representing 53% of total label Figure 11 B and Figure 12B ) Incorporation into AKG, DG(O), and PE(P) was intermediate, with PE(P) consisting of 7% of total label Figure 11 B and Figure 12B ) The remaining lipid classes showed little incorporation Figure 11B). After LPE(O) treatment, incorporation into AKG, LPE(O), and PC(O) was moderate, and incorporation into PE(O) and PE(P) was higher Figure 11 C). Most of the label was incorporated into PE(P) and represented 35% of the total label Figure 12C ). In addition, incorporation into DG(O) was low, and incorporation into TG(O), LPC(O), and PC(P) was minimal Figure 11 C).

[0499] To validate the findings in HepG2 cells, 3T3 cells were treated with 20 μΜ of deuterium labeled plasmenyl phospholipid precursors for 24 hours, and lipid concentrations were analyzed using LC-MS / MS. Unlike HepG2 cells, plasmenyl phospholipid precursor treatment in 3T3 cells did not result in changes in cell morphology compared to control Figure 13 ). By comparing the labeled PE(P) concentration between treatment groups, it was found that the concentration significantly increased after plasmenyl phospholipid precursor treatment compared to control (p < 0.01, Figure 14A and Figure 14B ). This increase was most pronounced in AKG, followed by LPE(O), and then LPC(O) Figure 14A ). However, when comparing the ratio of labeled PE(P) concentration to total label concentration, it was observed that LPE(O) was significantly higher compared to both AKG and LPC(O), which is consistent with the results from HepG2 cells Figure 14C ). Although the ratio of LPC(O) was higher than AKG, this difference was much smaller than the difference between LPE(O) and AKG Figure 14C

[0500] Bar graphs were similarly generated to assess the label distribution within the plasmenyl phospholipid biosynthetic pathway Figure 15 ). After AKG treatment, a high proportion of the label was incorporated into AKG, representing 55% of the total label Figure 15 A and Figure 16A ). This finding is consistent with the results observed in HepG2 cells. In addition, incorporation into PC(O) and PE(O) was moderate, and incorporation into the remaining lipid classes was minimal, with only 11% of the total label incorporated into PE(P) Figure 15 A and Figure 16A ). When comparing the effects of AKG treatment between cell lines, it was found that a lower proportion of the label was incorporated into PE(P) and DG(O) in 3T3 cells compared to HepG2 cells Figure 11 A and Figure 15 A). After LPC(O) treatment, incorporation into LPC(O) was high and incorporation into PC(O) was moderate, which is in contrast to the results observed in HepG2 cells​Figure 11 B and Figure 15 B). Incorporation into the remaining lipid classes was minimal, with only 4% of total label incorporated into PE(P) ( Figure 15 B and Figure 16B ). After LPE(O) treatment, incorporation into LPE(O) was moderate, and incorporation into PC(O), PE(O), and PE(P) was higher ( Figure 15 C). While a substantial proportion of label was incorporated into PE(P) in HepG2 cells, in 3T3 cells the majority of label was incorporated into PE(O) and represented 34% of total label ( Figure 12C and Figure 16C ). Incorporation into PE(P) was also high, representing 29% of total label ( Figure 16C ). The remaining lipid classes showed little to moderate incorporation ( Figure 15 C).

[0501] To compare bioavailability and incorporation of acetal phospholipid precursors into acetal phospholipids, mice were treated with a single dose of deuterium-labeled acetal phospholipid precursors ( Figure 17 ). LC-MS / MS was used to analyze plasma lipid concentrations from multiple time points. Unlike the cell lines, lipidomic analysis in mice only considered 16:0 lipid species, as LPE(O) treatment only used the labeled 16:0 compared to AKG and LPC(O) with 16:0, 18:0, and 18:1. Although all treated mice had their lipid concentrations measured, the data reported here focus on mice treated with dose mixture A, as mice treated with dose mixture B exhibited similar trends.

[0502] The levels of labeled AKG were compared between treatment groups to estimate the rate at which AKG was taken up into the circulation. In females, the labeled AKG concentrations were high 1 hour after treatment for all precursors ( Figure 18A , Figure 19A , Figure 20A ). However, in males, AKG treatment resulted in a significant increase in labeled AKG 1 hour after treatment compared to LPC(O) and LPE(O) ( Figure 18B , Figure 19B and Figure 20B ). Furthermore, male AKG treatment resulted in approximately twice the amount of labeled AKG compared to females ( Figure 18A and Figure 18B ).

[0503] Since acetal phospholipid precursors are first converted into these lipids before acetal phospholipids are produced, the levels of labeled PC(O) and PE(O) were measured. For all acetal phospholipid precursors, the highest concentrations of labeled PC(O) occurred 4 hours after treatment ( Figure 19Aand Figure 19B ). Furthermore, LPE(O) treatment resulted in the highest increase in labelled PC(O), followed by LPC(O) and then AKG Figure 19A and Figure 19B ). Similar to PC(O), the labelled PE(O) concentration of all plasmalogen precursors reached a maximum after 4 hours of treatment Figure 20A and Figure 20B ). LPE(O) treatment resulted in the highest increase in labelled PE(O), followed by LPC(O) and then AKG.

[0504] When examining the labelled PE(P) concentration, it was revealed that LPE(O) treatment resulted in the highest increase, followed by LPC(O) and then AKG Figure 21A , Figure 21B , Figure 22A , Figure 22B ). This was observed after 24 hours of treatment and was consistent in both female and male mice receiving dose mixture A Figure 21A , Figure 21B ) or dose mixture B Figure 22A , Figure 22B ).

[0505] The Cmaxof PE(P) was compared between treatment groups max To assess the bioavailability of plasmalogen precursors, the Cmaxof PE(P) was compared between treatment groups max (p < 0.01, Figure 23A , Figure 23B and Figure 23C , Figure 23D ). Furthermore, LPE(O) treatment resulted in a significantly higher Cmaxof PE(P) compared to LPC(O) max (p < 0.001, Figure 23A and Figure 23C ). Males treated with LPE(O) treatment produced a Cmaxof PE(P) max 1.45 times that of females Figure 23A and Figure 23C ). Similar trends were evident in mice treated with dose mixture B Figure 23E , Figure 23F and Figure 23G , Figure 23H ). In both males and females, LPE(O) treatment resulted in a significant increase in Cmaxof PE(P) max (p < 0.05, Figure 23E and Figure 23G ). Furthermore, in males, LPE(O) treatment resulted in a significantly higher Cmaxof PE(P) compared to LPC(O) maxsignificantly greater, and this difference was only observed as a trend in females (p < 0.0001, Figure 23E and Figure 23G ). LPC(O) treatment resulted in a greater C max but was only considered a trend in mice treated with dose mixture B compared to mice treated with dose mixture A ( Figures 23A-23H ). When comparing mice treated with different doses, it was found that in females, dose composition B of LPE(O) resulted in 1.77-fold greater C max ( Figure 23A and Figure 23E ). In contrast, in males, dose composition B of LPE(O) resulted in 1.55-fold greater C max ( Figure 23C and Figure 23G ).

[0506] The bioavailability of the plasmalogen precursors was further assessed by comparing the AUC of PE(P) between groups. Similar to C max Results, both males and females treated with dose mixture A of LPC(O) or LPE(O) showed significantly higher AUC compared to control or AKG (p < 0.001, Figure 24A , Figure 24B and Figure 24C and Figure 24D ). Furthermore, LPE(O) treatment resulted in a significantly greater AUC compared to LPC(O) (p < 0.0001, Figure 24A and Figure 24C ). It was further observed that LPE(O) treatment resulted in 1.3-fold greater AUC in males compared to females ( Figure 24A and Figure 24C ). Again, these trends were reflected in mice treated with dose mixture B. In both males and females, LPE(O) treatment resulted in a greater AUC compared to LPC(O), AKG, or control (p < 0.05, Figure 24E , Figure 24F and Figure 24G and Figure 24H ). LPC(O) treatment resulted in a greater AUC compared to AKG ( Figure 24E and Figure 24G ). When comparing mice treated with different doses, it was observed that in both females and males, dose composition B of LPE(O) resulted in 1.5-fold greater AUC compared to dose composition A ( Figures 24A-24H ).

[0507] AUC bar graphs were created for females and males treated with dose mixture A to visualize the distribution of markers in the plasmalogen biosynthesis pathway (Figures 25 and 26). In females, AKG treatment resulted in high incorporation into DG(O) and TG(O), and moderate incorporation into PC(O) and PE(O). Figure 25A ). There is little or no incorporation into AKG, LPC(O), LPE(O), PE(P) and PC(P) ( Figure 25A In males, a similar trend was evident after AKG treatment ( Figure 26A In contrast, in females, LPC(O) treatment resulted in moderate incorporation into DG(O), TG(O), LPC(O), and PE(P), and high incorporation into PC(O) and PE(O) ( Figure 25B ). Almost no incorporation ( Figure 25B Although LPC(O) treatment resulted in a greater increase in TG(O) in males, other lipid classes showed similar trends to those in females ( Figure 25B and Figure 26B In both females and males, incorporation into PE(O) was high, into PC(O) and PE(P) was moderate, and into the remaining lipid classes was almost non-existent after LPE(O) treatment ( Figure 25C and Figure 26C ).

[0508] Organizational Analysis

[0509] The conversion of labeled precursor compounds to brain PE plasmalogen was tested, and it was found that LPE(O) supplementation resulted in a small but significant increase in labeled PE plasmalogen in the brain ( Figure 27 ), whereas the increase in marker PE(P) with AKG and LPC(O) supplementation was quite variable ( Figure 28 ).

[0510] Statistical analysis

[0511] Lipidomic analysis was performed on four replicates of HepG2 and 3T3 cells and plasma samples. However, the samples collected from males treated with the dose mixture A of lecithin had five replicates at 0 hours after treatment. This was because one mouse had already died before treatment but was still included in the analysis. Graphs were generated using Microsoft Excel and presented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 9.5.1. One-way ANOVA was performed to compare the treatment groups, followed by Tukey’s post-hoc test to determine which groups were significantly different. Significance was indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. p < 0.05 was considered statistically significant.

[0512] Example 5

[0513] This example relates to the modulation of the level and composition of plasmalogen by LPC(O) and LPE(O) supplementation in RAW 264.7 cells using different SN-1 alkyl composition.

[0514] Cell culture methods

[0515] The mouse macrophage cell line RAW 264.7 was used in this study. Cells were cultured at 37°C with 5% CO2 in Roswell Park Memorial Institute (RPMI) 1640 medium containing L-glutamine with 1% sodium pyruvate (Gibco, USA) and 10% fetal bovine serum (Gibco, USA). Cells were passaged weekly and the medium was replenished every two to three days until passage 30, at which point the cells were discarded. Cells were seeded in 12-well plates at a concentration of 300,000 cells / ml of medium and allowed to grow until they reached 80% confluence.

[0516] Supplementation of plasmalogen precursors in cells

[0517] To evaluate the incorporation of the plasmalogen precursors into cellular plasmalogens, cells were supplemented with compounds having different SN1 composition, i.e. different ratios of O-16:0, O-18:0 and O-18:1 at a concentration of 20 mM of LPC(O) or LPE(O) (Table 14). The plasmalogen precursors LPC(O) and LPE(O) were dissolved in chloroform:methanol (2:1), evaporated under a stream of nitrogen at 40 °C and then reconstituted in RPMI medium containing 2% bovine serum albumin (Sigma-Aldrich, USA). Control treatment of cells consisted of RPMI containing 2% bovine serum albumin. Once ready, the medium containing the precursors was vortexed and sonicated in an ultrasonic water bath (Soniclean, Adelaide, South Australia, Australia) for 15 minutes.

[0518] Table 14. List of plasmalogen precursor compounds used in this study and their molar percentage (%).

[0519] Table 14

[0520] Group Number Compound Group 1 Vehicle Control 2 LPC(O-16:0) 100% 3 LPC(O-18:0) 100% 4 LPC(O-18:1) 100% 5 LPC(O-16:0) 50% + LPC(O-18:0) 50% 6 LPC(O-18:0) 50% + LPC(O-18:1) 50% 7 LPC(O-16:0) 50% + LPC(O-18:1) 50% 8 LPC(O-16:0) 26.8% + LPC(O-18:0) 46.1% + LPC(O-18:1) 27.1% 9 LPC(O-16:0) 46% + LPC(O-18:0) 21% + LPC(O-18:1) 33% 10 LPC(O-16:0) 42% + LPC(O-18:0) 51% + LPC(O-18:1) 7% 11 LPC(O-16:0) 62% + LPC(O-18:0) 23.9% + LPC(O-18:1) 14.1% 12 LPC(O-16:0) 19.8% + LPC(O-18:0) 66% + LPC(O-18:1) 14.2% 13 LPC(O-16:0) 32.5% + LPC(O-18:0) 39.6% + LPC(O-18:1) 27.9% 14 LPC(O-16:0) 34.1% + LPC(O-18:0) 41.5% + LPC(O-18:1) 24.4% 15 LPE(O-16:0) 100% 16 LPE(O-18:0) 100% 17 LPE(O-18:1) 100% 18 LPE(O-16:0) 50% + LPE(O-18:0) 50% 19 LPE(O-18:0) 50% + LPE(O-18:1) 50% 20 LPE(O-16:0) 50% + LPE(O-18:1) 50% 21 LPE(O-16:0) 26.8% + LPE(O-18:0) 46.1% + LPE(O-18:1) 27.1% 22 LPE(O-16:0) 46% + LPE(O-18:0) 21% + LPE(O-18:1) 33% 23 LPE(O-16:0) 42% + LPE(O-18:0) 51% + LPE(O-18:1) 7% 24 LPE(O-16:0) 62% + LPE(O-18:0) 23.9% + LPE(O-18:1) 14.1% 25 LPE(O-16:0) 19.8% + LPE(O-18:0) 66% + LPE(O-18:1) 14.2% 26 LPE (O-16:0) 32.5% + LPE (O-18:0) 39.6% + LPE (O-18:1) 27.9% 27 LPE (O-16:0) 34.1% + LPE (O-18:0) 41.5% + LPE (O-18:1) 24.4%

[0521] Lipid extraction

[0522] Lipids were extracted from cells using the butanol-methanol method following established protocols. Each lyophilized cell sample received 10 pl of MilliQ water and 100 pl of a butanol-methanol mixture (1 : 1) containing a specific set of internal standards. Thereafter, each sample was vortexed for a few seconds and placed in an ultrasonic water bath for 1 hour. Subsequently, samples were centrifuged at 13,000 g for 15 minutes and the resulting supernatant was carefully transferred to a mass spectrometry vial with an insert. These vials were then frozen at -80 °C until they were ready for mass spectrometry analysis.

[0523] Lipidomics analysis

[0524] A targeted lipidomics protocol using LC-MS / MS (Huynh et al., “High- Throughput Plasma Lipidomics: Detailed Mapping of the Associations with Cardiometabolic Risk Factors,” Cell Chem. Biol., January 17, 2019; 26(1): 71-84.e4, doi:10.1016 / j.chembiol.2018.10.008) was used for samples from the present study, following previously published protocols. The method employed liquid chromatography (Agilent 1290 Infinity) coupled with tandem mass spectrometry (Agilent 6495C), operating in dynamic multiple reaction monitoring (MRM) mode. Multiple lipid species belonging to different lipid classes were measured, including phosphatidylcholines (PC), alkylphosphatidylcholines [PC(O)], alkenylphosphatidylcholines [PC(P)], phosphatidylethanolamines (PE), alkylphosphatidylethanolamines [PE(O)], alkenylphosphatidylethanolamines [PE(P)] or PE plasmalogens, lysophosphatidylcholines (LPC), lysophosphatidylcholines [LPC(O)], lysophosphatidylcholines [LPC(P)], lysophosphatidylethanolamines (LPE), lysophosphatidylethanolamines [LPE(O)], lysophosphatidylethanolamines [LPE(P)], triacylglycerols (TG), and alkyl diacylglycerols [TG(O)]. The lipid species measured in the present study are listed in Supplementary Table 1, which is included at the end of the present example.

[0525] The chromatographic analysis gradient used in this analysis involved a series of steps: starting at a flow rate of 0.4 mL / min at 15% B, increasing to 50% B in 2.5 min, followed by an increment to 57% in 0.1 min, an increment to 70% in 6.4 min, an increment to 93% in 0.1 min, and finally an increment to 96% in 1.9 min, ending at 100% B in 0.1 min. The solvent was held at 100% B for 0.9 min, for a total run time of 12.0 min. Equilibration was initiated by reducing the solvent from 100% B to 15% B in 0.2 min, and was maintained for a total of 16 min.

[0526] For data processing, the vendor software Qualitative Analysis B.07.00 and QQQQuantitative Analysis B.10.0.0 were used. Relative quantification of the lipid species was determined by comparison of the lipid species to the corresponding internal standard. The total concentration of a lipid class was determined by summing the concentrations of the individual lipid species within each respective lipid class.

[0527] Statistical analysis

[0528] Lipidomic analysis was performed on triplicate cell samples. Images were generated using Microsoft Excel and presented as mean values with standard deviation error bars. Statistical analysis was performed using GraphPad Prism 9.5.1. Initially, one-way analysis of variance (ANOVA) was applied to compare different treatment groups. Then, post-hoc analysis was performed using Fisher LSD test to identify significant differences between groups. An AP value less than 0.05 was considered statistically significant.

[0529] Prior to the start of treatment, cells were washed with phosphate buffered saline (PBS) without Ca 2+ and Mg 2+ . After 24 hours of treatment with precursors, cells were harvested via initial PBS wash followed by cell scraping. Cell suspensions were then dried overnight using a SPD121P SpeedVac concentrator. After drying, samples were stored at -80 °C until lipid extraction was performed.

[0530] Results

[0531] Supplementation of both LPC(O) and LPE(O) resulted in a significant increase in total cellular PE(P) levels, albeit to different extents (7-114% increase relative to control group) Figure 44 and Figure 45 ). Notably, LPC(O-16:0) 100% exhibited greater efficacy in increasing endogenous PE(P) levels compared to LPC(O-18:0) 100% and LPC(O-18:1) 100% ( Figure 44 ). Conversely, both LPE(O-16:0) 100% and LPE(O-18:0) 100% showed equal efficacy in increasing PE(P) levels, while LPE(O-18:1) 100% was relatively poor ( Figure 45 ). While different combinations of O-16:0, O-18:0, and O-18:1 significantly increased PE(P) levels, none of the combinations matched the efficacy of the 100% O-16:0 species in increasing PE(P) levels (Figures 43-44).

[0532] Different combinations of O-16:0, O-18:0, and O-18:1 species also significantly affected the composition of alkylacylphospholipids in cells Figure 46 and Figure 47 ). Within LPC(O) treatment, both single and double species supplementation resulted in changes in the relative proportions of 16:0, 18:0, and 18:1 PE(P) in cells Figure 45). However, specific mixtures, particularly LPC(O-16:0) 34.1% + LPC(O-18:0) 41.5% + LPC(O-18:1) 24.4%, were able to maintain these ratios while increasing total PE(P) levels ( Figure 46 ) In the LPE(O) treatment, only one combination (i.e., LPE(O-16:0) 62%, LPE(O-18:0) 23.9%, and LPE(O-18:1) 14.1%) successfully maintained the relative ratios of 16:0, 18:0, and 18:1 PE(P) while increasing total PE(P) levels ( Figure 46 ) However, all other combinations increased total PE(P) levels while altering the relative ratios of these species.

[0533] Without wishing to be bound by theory, it is believed that these results demonstrate the efficacy of the novel acyltransferase substrates LPC(O) and LPE(O) in significantly increasing cellular PE(P) levels within RAW 264.7 macrophages. Nonetheless, the extent of this increase exhibited significant variation among the different SN1 compositions for both LPC(O) and LPE(O) compounds. Notably, 100% O-16:0 was shown to be the most potent in increasing endogenous PE(P) levels compared to the O-18:0 and O-18:1 species. This apparent discrepancy highlights the different effects of precursor species with different SN1 compositions on cellular acyltransferase levels, which is potentially attributed to variations in their metabolic processes or their different effects on the de novo acyltransferase synthesis pathway.

[0534] Without wishing to be bound by theory, the ubiquity of the 16:0 species as the most abundant PE(P) species within these cells suggests that there can be a greater propensity to accumulate more 16:0 species upon supplementation. Notably, it is known that changes in specific PE(P) species can have negative effects on other species. Thus, supplementation with O-18:0 or O-18:1 precursors to increase 18:0 or 18:1 PE(P) species, respectively, can trigger stronger feedback inhibition of de novo synthesis of 16:0 PE(P), resulting in a significant decrease in the overall increase in total cellular PE(P) levels.

[0535] These findings exemplify the significant effects of precursor compounds with different SN1 compositions on the acyltransferase composition within cells. Specific mixtures within the LPC(O) and LPE(O) treatments were able to maintain the relative ratios of 16:0, 18:0, and 18:1 PE(P) species while increasing total PE(P) levels, which indicates the existence of a potential pathway to modulate cellular acyltransferase composition without disrupting the balance of individual species.

[0536] Supplementary Table 1: Acquisition details of dynamic multiple reaction monitoring transitions used in this study.

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543]

[0544]

[0545]

[0546] For all transitions, the polarity was positive, with a fragmentation voltage set to 166 and an accelerator voltage set to 5.

[0547] LPC: lysophosphatidylcholine; LPC(O): lysophosphatidylcholine (O); LPC(P): lysophosphatidylcholine (P); LPE: lysophosphatidylethanolamine; LPE(O): lysophosphatidylethanolamine (O); LPE(P): lysophosphatidylethanolamine (P); PC: phosphatidylcholine; PC(O): phosphatidylcholine (O); PC(P): phosphatidylcholine (P); PE: phosphatidylethanolamine; PE(O): phosphatidylethanolamine (O); PE(P): phosphatidylethanolamine (P); TG: triacylglycerol; TG(O): triacylglycerol (O); ISTD: internal standard; NL: neutral loss; SIM: single ion monitoring.

[0548] Example 6

[0549] This example relates to the modulation of the level and composition of plasmalogens by LPC(O) and LPE(O) supplementation in 3T3-L1 cells using different SN-1 composition.

[0550] The methods employed in this study were the same as described in Example 5, except for the cell line and media used. For this study, the mouse fibroblast / pre-adipocyte cell line 3T3-L1 was used and was cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, USA) supplemented with 1% sodium pyruvate (Gibco, USA) and 10% newborn calf serum (Gibco, New Zealand).

[0551] In 3T3-L1 preadipocytes, none of the plasmalogen precursor treatments resulted in a significant increase in intracellular PE(P) levels; rather, some treatments, particularly 100% LPC(O-18:1) and 100% LPE(O-18:1), appeared to decrease these levels Figures 47 to 48 ). Exploring the effects within both the LPC(O) and LPE(O) treatments, single species supplementation revealed that the relative proportions of 16:0, 18:0, and 18:1 PE(P) changed subtly, i.e., supplementation of a particular SN1 species increased the proportion of the corresponding PE(P) species while decreasing the proportion of other PE(P) species Figures 49 to 50

[0552] In 3T3-L1 cells, no increase in cellular PE(P) levels was observed following supplementation of LPC(O) or LPE(O) under the conditions tested. These cells already exhibited high PE(P) baseline levels, which likely accounts for the lack of further increase in PE(P) levels following precursor supplementation. Nonetheless, the studies revealed that precursor compounds with different SN1 compositions had a significant impact on cellular PE(P) composition. Without wishing to be bound by theory, it is believed that these results underscore the role of SN1 / alkyl chain composition in plasmalogen precursor supplements for maintaining the natural plasmalogen composition of healthy subjects.

[0553] Example 7

[0554] The mouse macrophage cell line RAW 264.7 was used for additional studies to determine whether the observed increases in plasmalogen levels / composition changes by feeding LPC(O) and LPE(O) ether lipids to cells could measure any functional outcomes / phenotypic outcomes.

[0555] Based on the ratios that appear to maintain the most frequently observed plasmalogen compound ratios in healthy human subjects, a subset of the Table 14 compound mixtures with different SN-1 fatty acids was selected. The compound mixtures tested were:

[0556] Vehicle control

[0557] LPC(O-18:1) 100%

[0558] LPC(O-16:0) 34.1% + LPC(O-18:0) 41.5% + LPC(O-18:1) 24.4%

[0559] LPC(O-16:0) 62% + LPC(O-18:0) 23.9% + LPC(O-18:1) 14.1%

[0560] LPE(O-18:1) 100%

[0561] ​LPE (O-16:0) 34.1% + LPE (O-18:0) 41.5% + LPE (O-18:1) 24.4%

[0562] LPE (O-16:0) 62% + LPE (O-18:0) 23.9% + LPE (O-18:1) 14.1%

[0563] Cells were pre-treated with 20 mM of the precursor compound mixture for 24 hours, followed by LPS (1 nM) treatment for 6 hours to stimulate the immune response. Following LPS treatment, cells were harvested for gene expression analysis using qPCR. Total RNA was isolated from cells using TRIzol™ reagent (Invitrogen, Thermo Fisher Scientific, USA). 500 mΐ of TRIzol was added to each well of the cell culture plate, followed by scraping the cells from the culture plate with a scraper. The resulting cell lysate was stored at -80 °C until subsequent processing. On the day of RNA isolation, the cell lysate was thawed and 100 mΐ of chloroform was added to each sample. Each sample tube was then mixed vigorously for 20 seconds, followed by incubation at room temperature for 5 minutes. Subsequently, the samples were centrifuged at 12000 g for 15 minutes at 4 °C.

[0564] The aqueous phase was carefully transferred to a fresh tube. 500 mΐ of isopropanol was added to each sample and mixed thoroughly, followed by incubation at room temperature for 30 minutes. The samples were then centrifuged at 17000 g for 15 minutes at 4 °C. The supernatant was decanted, leaving the RNA pellet.

[0565] To the RNA pellet, 1 ml of 75% molecular grade ethanol (Sigma-Aldrich, USA) was added, vortexed briefly for 5 seconds, and centrifuged at 7500 g for 5 minutes at 4 °C. The supernatant was then discarded. Subsequently, an additional 1 ml of 75% ice-cold ethanol was added to the pellet, followed by centrifugation at 7500 g for 5 minutes at 4 °C. This washing process was repeated once, and after the final centrifugation, the supernatant was discarded, leaving a clean RNA pellet.

[0566] The tube containing the pellet was placed on a heating block at 55 °C to dry for 10 minutes. After drying, 20 mΐ of molecular grade water (Sigma-Aldrich, USA) was added to dissolve the RNA pellet. The quantity and quality of the isolated RNA were determined using a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA), and the RNA samples were stored at -80 °C until further analysis.

[0567] Complementary DNA was synthesized by reverse transcription using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, ThermoFisher Scientific, USA) according to the manufacturer’s recommendations.

[0568] Quantitative real-time PCR was performed using Taqman™ assays (Il6: Mm00446190_m1, Nfe2l2: Mm00477784_m1, Tlr4: Mm00445273_m1, Acox1: Mm00443579_m1, Cpt1a: Mm00550438_m1, Sod1: Mm01344233_g1, Pex16: Mm00455021_m1, Hnrnpab: Mm01288699_m1) and TaqMan TM Fast Advanced Master Mix (Applied Biosystems, Thermo Fisher Scientific, USA) and amplified on an Applied Biosystem Quant 7 Real-Time PCR instrument (Life Technologies, ThermoFisher Scientific, USA) according to the manufacturer’s recommendations. Target gene expression was normalized to housekeeping gene [Heterogeneous nuclear ribonucleoprotein (Hnrnpab)] expression and reference group (control or control + LPS) using the 2 - AACtquantification method and expressed as relative values (Livak KJ, Schmittgen TD. Methods. Vol. 25. San Diego, CA: 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method; pp. 402-408).

[0569] Data are presented as mean ± SD (n = 3 / group). Mean differences between groups were compared by one-way ANOVA followed by Fisher LSD test. Asterisks (*) indicate P < 0.05 compared to control and ^ indicates P < 0.05 compared to control + LPS.

[0570] While none of the tested compounds seemed to affect TNF a levels (as measured by ELISA, data not shown), at the gene expression level, many markers of inflammatory / immune response were affected.

[0571] LPC(O-18:1) supplementation inhibited the LPS-induced increase in IL6 gene expression, as did all LPE(O) treatments. All LPX(O) treatments decreased NFE2L2 gene expression, although only 4 of the 6 (mixtures 4-7 above, including all LPE(O)) were statistically significant in this assay.

[0572] All LPX(O) treatments inhibited the LPS-induced increase in TLR4 gene expression Figure 55 ). Reductions in the range of 11-20% were noted compared to the induction following LPS treatment, minus the control vehicle level.

[0573] These data suggest that there can be an anti-inflammatory activity associated with LPX(O) supplementation, particularly LPE(O), in RAW 264.7 cells (Xing, X. et al. IL-6 is an anti-inflammatory cytokine required for controlling local or systemic acute inflammatory responses. J Clin Invest. 1998; 15: 311-320; He, F. et al. 2020 NRF2, a transcription factor for stress response and beyond. Int. J. Mol. Sci 2020: 21(13), 4777; Saleh et al. The anti-inflammatory properties of phytochemicals and their effects on epigenetic mechanisms involved in TLR4 / NF-kB-mediated inflammation. Front. Immunol. 2021: 12.)

[0574] RAW 264.7 macrophages were treated with 20 mM LPC(O) or LPE(O) with different SN1 composition for 24 hours and further treated with LPS (1 nM) for 6 hours before being harvested for gene expression analysis. Gene expression data were normalized to Hnrnpab expression and presented as mean ± SD (n=3 / group). Mean differences between groups were compared by one-way ANOVA followed by Fisher LSD test. Results are depicted in Figure 56 where * indicates P<0.05 compared to control and ^ indicates P<0.05 compared to control + LPS.

[0575] Example 8

[0576] Formulations of LPX(O) compounds.

[0577] Certain LPX(O) compounds and compositions are formulated for stable aqueous delivery. By mixing the LPX(O) compounds and compositions with a solubilizing agent, formulations of emulsified LPX(O) compounds can be obtained that can withstand freeze-thaw processes and / or are suitable for delivery to animals for pharmacokinetic studies.

[0578] Preparation of alkyl glycerols, alkyl diacylglycerols and lysoalkylphosphatidylcholine formulations.

[0579] 1. Stock solutions (100 mg / ml) of individual precursor compounds were prepared in chloroform:methanol (1 : 1) solution.

[0580] 2. Similarly, stock solutions (100 mg / ml) of purified egg yolk L-a-phosphatidylcholine (Sigma-Aldrich, St. Louis, MO, USA) were prepared in chloroform:methanol (1 : 1) solution.

[0581] 3. Precursor compound mixtures were prepared by combining appropriate volumes of precursor compound stocks (Table 15).

[0582] 4. The mixture of precursor compounds and phosphatidylcholine stocks were then aliquoted into scintillation vials (Table 15).

[0583] 5. Subsequently, the mixture of precursor compounds and phosphatidylcholine was dried under a stream of nitrogen at 40 °C.

[0584] 6. After complete evaporation of the chloroform and methanol, the dried lipids were reconstituted in 3 ml of deionized water by vigorous vortexing, followed by 1 hour of sonication in an ultrasonic bath (Soniclean, Adelaide, South Australia, Australia) and an additional 2 x 30 seconds of sonication using a Misonix S-4000 ultrasonicator (Thermo Fisher Scientific, Melbourne, Victoria, Australia) at amplitude 25.

[0585] Table 15. Preparation of alkyl glycerols, alkyl diacylglycerols and lysoalkylphosphatidylcholine formulations.

[0586]

[0587] Preparation of lysoalkylphosphatidylethanolamine formulations

[0588] 1. Stock solutions (10 mg / ml) of individual precursor compounds were prepared in chloroform:methanol (2: 1) solution*.

[0589] 2. Similarly, a stock solution (100 mg / ml) of purified egg yolk L-a-phosphatidylcholine (Sigma-Aldrich, St. Louis, MO, USA) was prepared in a chloroform:methanol (2:1) solution.

[0590] 3. The precursor compound mixture (Table 16) was prepared by combining appropriate volumes of the precursor compound stocks.

[0591] 4. The mixture of precursor compounds and phospholipid stock was then aliquoted into glass tubes (Table 16).

[0592] 5. Subsequently, the mixture of precursor compounds and phospholipid was dried under a stream of nitrogen at 40 °C.

[0593] 6. After complete evaporation of the chloroform and methanol, the dried lipid was reconstituted in 3 ml of deionized water by vigorous vortexing, followed by 1 hour of sonication in an ultrasonic bath (Soniclean, Adelaide, South Australia, Australia) and additional sonication using a Misonix S-4000 ultrasonicator (Thermo Fisher Scientific, Melbourne, Victoria, Australia) at amplitude 25 for 2 x 30 seconds.

[0594] * The LPE(O-16:0), LPE(O-16:0)-d5 and LPE(O-18:0) solutions were briefly heated in a water bath at approximately 40 °C.

[0595] Table 16. Preparation of lysocompound formulations.

[0596] Table 16

[0597]

[0598] Without wishing to be bound by theory, it is believed that the analysis of the formulations described above shows that purified egg yolk L-a-phosphatidylcholine is a suitable solubilizer for use in the preparation of stable aqueous formulations suitable for, for example, delivery to animals and capable of withstanding freezing and thawing. Without wishing to be bound by theory, it is believed that formulations comprising said solubilizer have improved stability and / or are better able to withstand freezing and thawing, such as not being susceptible to degradation during freezing and thawing, compared to similar formulations that do not contain a solubilizer, such as purified egg yolk L-a-phosphatidylcholine.

[0599] In view of the improved bioavailability of LPC(O), various combinations of LPC(O), LPE(O) and LPA(O) have been considered for use in pharmaceutical formulations and dietary supplements. The dosage and serving size can vary depending on the age, height and weight of the subject.

[0600] In embodiments, a useful human dose is 2-3 mg / kg, equivalent to 100-300 mg LPC(O) equivalents per person per day. In further embodiments, a useful human dose is 200 mg LPC(O) equivalents per person per day.

[0601] In another embodiment, the maintenance dose can be 0.2-2 mg / kg, 25-100 mg LPC(O) equivalents per person per day. That is, for some people, the maintenance dose can be 25 mg, 50 mg, or 100 mg LPC(O) equivalents per person per day.

[0602] In another embodiment, if a large increase in plasmalogen or a rapid increase thereof is desired, the dose can consist of 3-25 mg / kg, 300-2000 mg LPC(O) equivalents per person per day. That is, if a large increase in plasmalogen or a rapid increase thereof is desired, the dose can consist of 400 mg, 800 mg, or 1600 mg LPC(O) equivalents per person per day.

[0603] In another embodiment, the dose can consist of 0.1-4000 mg LPC(O) equivalents per person per day.

[0604] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The use of the term “about” is intended to describe values that are within the ordinary error for the quantifying device or method, and / or the precision of the device or method. In other embodiments, the value can be within about 10% of the recited value; in other embodiments, the value can be within about 5% of the recited value; in other embodiments, the value can be within about 2% of the recited value; in other embodiments, the value can be within about 1% of the recited value. The foregoing ranges are intended to encompass all values and subranges within the specified ranges. Unless otherwise stated, all methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0605] While the disclosure has been described in the foregoing specification with a certain degree of particularity, those skilled in the art will appreciate that the disclosure is susceptible to other embodiments and that certain details of the disclosure can be varied considerably without departing from the basic principles of the disclosure.

[0606] All references cited herein are incorporated by reference in their entirety. The disclosure can be embodied in other specific forms without departing from the spirit or essential attributes of the disclosure, and therefore, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the disclosure.

Claims

1. A composition comprising at least one compound of Formula (I) or a pharmaceutically acceptable salt thereof 2. The composition according to claim 1, wherein R 3 Selected from phosphates and substituted phosphates.

3. The composition of claim 2, wherein R 3 is a substituted phosphate ester.

4. The composition of claim 3, wherein the substituted phosphate ester is substituted with an alkyl amine or an inositol.

5. The composition of claim 4, wherein the alkyl amine is selected from 6. The composition of any one of claims 1 to 5, wherein R 1 and R 2 are each independently selected from hydrogen, an optionally substituted C 1-30 alkyl group, an optionally substituted C 2-30 alkenyl group, and an optionally substituted C 1-30 acyl group.

7. The composition of claim 6, wherein R 1 and R 2 are each independently selected from the group consisting of optionally substituted C 14-24 alkyl groups, optionally substituted C 14-24 alkenyl groups, and optionally substituted C 14-24 acyl groups.

8. The composition of claim 6, wherein if R 2 is hydrogen, then R 1 is an optionally substituted C 1-30 alkyl group, an optionally substituted C 2-30 alkenyl group, or an optionally substituted C 1-30 acyl group.

9. The composition of claim 8, wherein R 1 is an optionally substituted C 14-24 alkyl group, an optionally substituted C 14-24 alkenyl group, or an optionally substituted C 14-24 acyl group.

10. The composition of claim 9, wherein R 1 is an optionally substituted C 14-18 alkyl group, an optionally substituted C 14-18 alkenyl group, or an optionally substituted C 14-18 acyl group.

11. The composition of claim 10, wherein R 1 is an unsubstituted C 16 alkyl group.

12. The composition of claim 10, wherein R 1 is an unsubstituted C 18 alkyl group.

13. The composition of claim 10, wherein R 1 is an unsubstituted C 18 alkenyl group.

14. The composition of claim 1, wherein the at least one compound of Formula (I) is a compound of Formula (I-A) or a pharmaceutically acceptable salt thereof 15. The composition of claim 14, wherein each R x is independently selected from hydrogen or C 1-3 alkyl.

16. The composition of claim 14 or 15, wherein n is 2 or 3.

17. The composition of claim 14, wherein R x is methyl, and n is 3.

18. The composition according to any one of claims 14 to 17, wherein R A is an optionally substituted hydrocarbon chain containing from 14 to 24 carbon atoms.

19. The composition of claim 18, wherein R A is an optionally substituted hydrocarbon chain containing 16 to 18 carbon atoms.

20. The composition of claim 19, wherein R A is an optionally substituted C 16-18 alkyl group, an optionally substituted C 16-18 alkenyl group, or an optionally substituted C 16-18 acyl group.

21. The composition of claim 20, wherein R A is an optionally substituted C 16-18 alkyl group.

22. The composition of claim 20, wherein R A is an optionally substituted C 16-18 alkenyl group.

23. The composition of claim 19, wherein R A is an unsubstituted C 16 alkyl group.

24. The composition of claim 19, wherein R A is an unsubstituted C 18 alkyl group.

25. The composition of claim 19, wherein R A is an unsubstituted C 18 alkenyl group.

26. The composition of any preceding claim, wherein the composition comprises a mixture of at least two compounds of Formula (I) and / or (I-A).

27. The composition of any preceding claim, wherein the composition comprises a mixture of three compounds of Formula (I) and / or (I-A).

28. The composition of claim 14, wherein the compound of Formula (I-A) has the structure (I-A1), (I-A2), or (I-A3) 29. The composition of claim 28, wherein the composition comprises a mixture of at least two compounds selected from the group consisting of (I-A1), (I-A2), and (I-A3).

30. The composition of claim 28, wherein the composition comprises a mixture of (I-A1), (I-A2), and (I-A3).

31. The composition of claim 28, wherein the composition comprises a mixture of (I-A1), (I-A2), and (I-A3), wherein the mixture of (I-A1), (I-A2), and (I-A3) comprises at least 50% of the ether lipids in the composition by mole percent.

32. The composition of claim 28, wherein the mixture has a molar ratio of (I-A2) to (I-A1) of 1.2:1 to 2.5:

1.

33. The composition of claim 32, wherein the mixture has a molar ratio of (I-A2) to (I-A1) of 1.5:1 to 2.1:

1.

34. The composition of claim 33, wherein the mixture has a molar ratio of (I-A2) to (I-A1) of 1.7:

1.

35. The composition of claim 28, wherein the mixture has a molar ratio of (I-A2) to (I-A3) of 0.9:1 to 1.7:

1.

36. The composition of claim 35, wherein the mixture has a molar ratio of (I-A2) to (I-A3) of 1:1 to 1.5:

1.

37. The composition of claim 36, wherein the mixture has a molar ratio of (I-A2) to (I-A3) of 1.22:

1.

38. The composition of claim 28, wherein the mixture has a molar ratio of (I-A1) to (I-A3) of 0.5:1 to 1:

1.

39. The composition of claim 38, wherein the mixture has a molar ratio of (I-A1) to (I-A3) of 0.6:1 to 0.9:

1.

40. The composition of claim 39, wherein the mixture has a molar ratio of (I-A1) to (I-A3) of 0.72:

1.

41. The composition of claim 30, wherein the mixture has a mole percent of (I-A1) of 18.6% to 27.9%, a mole percent of (I-A2) of 32.6% to 45.8%, and a mole percent of (I-A3) of 26.8% to 37.4%.

42. The composition of claim 30, wherein the mixture has a mole percent of (I-A1) of 23.3%, a mole percent of (I-A2) of 39.2%, and a mole percent of (I-A3) of 32.1%.

43. The composition of claim 30, wherein the mixture has a molar ratio of (I-A1):(I-A2):(I-A3) of 1: 1.7: 1.

4.

44. The composition of claim 14, wherein the compound of Formula (I-A) has the structure (I-A4), (I-A5), or (I-A6) 45. The composition of claim 44, wherein the composition comprises a mixture of at least two compounds selected from the group consisting of (I-A4), (I-A5), and (I-A6).

46. The composition of claim 44, wherein the composition comprises a mixture of (I-A4), (I-A5), and (I-A6).

47. The composition of claim 44, wherein the composition comprises a mixture of (I-A4), (I-A5), and (I-A6), wherein the mixture of (I-A4), (I-A5), and (I-A6) comprises at least 50% of the ether lipids in the composition by mole percent.

48. The composition of claim 44, wherein the mixture has a molar ratio of (I-A5) to (I-A4) of 1.2: 1 to 2.5:

1.

49. The composition of claim 48, wherein the mixture has a molar ratio of (I-A5) to (I-A4) of 1.5: 1 to 2.1:

1.

50. The composition of claim 49, wherein the mixture has a molar ratio of (I-A5) to (I-A4) of 1.7:

1.

51. The composition of claim 44, wherein the mixture has a molar ratio of (I-A5) to (I-A6) of 0.9: 1 to 1.7:

1.

52. The composition of claim 51, wherein the mixture has a molar ratio of (I-A5) to (I-A6) of 1: 1 to 1.5:

1.

53. The composition of claim 52, wherein the mixture has a molar ratio of (I-A5) to (I-A6) of 1.29:

1.

54. The composition of claim 44, wherein the mixture has a molar ratio of (I-A4) to (I-A6) of 0.5: 1 to 1:

1.

55. The composition of claim 54, wherein the mixture has a molar ratio of (I-A4) to (I-A6) of 0.6: 1 to 0.9:

1.

56. The composition of claim 55, wherein the mixture has a molar ratio of (I-A4) to (I-A6) of 0.76:

1.

57. The composition of claim 46, wherein the mixture has a mole percent of (I-A4) of 18.6% to 27.9%, a mole percent of (I-A5) of 32.6% to 45.8%, and a mole percent of (I-A6) of 26.8% to 37.4%.

58. The composition of claim 57, wherein the mixture has a mole percent of (I-A4) of 24.8%, a mole percent of (I-A5) of 42.4%, and a mole percent of (I-A6) of 32.8%.

59. The composition of claim 46, wherein the mixture has a molar ratio of (I-A4):(I-A5):(I-A6) of 1: 1.7: 1.

3.

60. A formulation, such as a pharmaceutical formulation, comprising the composition of any one of claims 1 to 59 and at least one excipient.

61. The formulation of claim 60, wherein the formulation is formulated for oral administration.

62. The formulation of claim 61, wherein the oral administration form is a tablet, capsule, solution, mouthwash, suspension, powder, chewing gum, candy, lozenge, sublingual delivery system, or fast-dissolve formulation.

63. The formulation of any one of claims 60 to 62, wherein the formulation is a food product.

64. The formulation of claim 63, wherein the formulation is a dietary supplement.

65. The formulation of claim 63, wherein the food product is a medical food product.

66. The formulation of claim 63, wherein the food product is an infant formula.

67. The formulation of any one of claims 60 to 66, for use in maintaining or modulating the level of a plasmenyl phospholipid in a human subject in need thereof.

68. The formulation of any one of claims 60 to 66, for use in maintaining or modulating the level of a plasmenyl phospholipid in an animal subject in need thereof.

69. The formulation of claim 68, wherein the animal is selected from the group consisting of a domestic animal, a work animal, and a farm animal.

70. The formulation of any one of claims 67 to 69, wherein the maintenance or modulation is needed to maintain or modulate the level of a plasmenyl phospholipid at a level and / or ratio associated with a non-disease state.

71. The composition of any one of claims 1 to 59 or the formulation of any one of claims 60 to 70, for use in increasing the level of a plasmenyl phospholipid compound in the blood or tissue of a subject.

72. The composition for use of claim 71, wherein the subject is a human.

73. The composition for use of claim 71, wherein the subject is an animal.

74. The composition for use of claim 73, wherein the animal subject is selected from a domesticated animal, a working animal, and a farm animal.

75. The composition of any one of claims 1 to 59 or the formulation of any one of claims 60 to 70 for use in therapy.

76. The composition of any one of claims 1 to 59 or the formulation of any one of claims 60 to 70 for use in treating a disease or disorder associated with plasmalogen deficiency.

77. The composition or formulation for use of claim 76, wherein the disease or disorder is a neurological disease.

78. The composition or formulation of claim 77, wherein the neurological disease is selected from Alzheimer’s disease, Parkinson’s disease, autism, amyotrophic lateral sclerosis, frontotemporal dementia, multiple sclerosis, and schizophrenia.

79. The composition or formulation for use of claim 76, wherein the disease or disorder is a metabolic disorder.

80. The composition or formulation of claim 79, wherein the metabolic disorder is selected from the group consisting of obesity, insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, an immune-related disorder, a cardiovascular disease, a neurological disease, cancer, myalgic encephalomyelitis / chronic fatigue syndrome, Barth syndrome, and a peroxisomal disorder.

81. The composition or formulation of claim 80, wherein the immune-related disorder is selected from the group consisting of asthma, atopic dermatitis, type 1 diabetes, and an infection.

82. The composition or formulation of claim 80, wherein the cardiovascular disease is selected from the group consisting of atherosclerosis, cardiac remodeling, and hypertension.

83. The composition or formulation of claim 80, wherein the peroxisomal disorder is Zellweger spectrum disorder or rhizomelic chondrodysplasia punctata.

84. The composition or formulation for use of any one of claims 71 to 83, wherein the compound of Formula (I) or (I-A) is administered at a dose of 0.1 to 4000 mg per day.

85. The composition or formulation for use of claim 84, wherein the compound of Formula (I) or (I-A) is administered at a dose of 0.1 to 2000 mg per day.

86. The composition or formulation for use of claim 85, wherein the compound of Formula (I) or (I-A) is administered at a dose of 25 to 1600 mg per day.

87. The composition or formulation for use of claim 86, wherein the compound of Formula (I) or (I-A) is administered at a dose of 400 mg, 800 mg, or 1600 mg per day.

88. The composition or formulation for use of claim 86, wherein the compound of Formula (I) or (I-A) is administered at a dose of 200 mg per day.

89. The composition or formulation for use of claim 86, wherein the compound of Formula (I) or (I-A) is administered at a dose of 25 to 100 mg per day.

90. A method of increasing levels of a plasmalogen compound in blood or tissue of a subject in need thereof, the method comprising administering to the subject an effective amount of at least one compound of Formula (I) or a pharmaceutically acceptable salt thereof and / or at least one compound of Formula (I-A) or a pharmaceutically acceptable salt thereof according to claims 1 and 14, respectively.

91. The method of claim 90, wherein the subject is a human.

92. The method of claim 90, wherein the subject is an animal.

93. The method of claim 92, wherein the animal subject is selected from the group consisting of a domesticated animal, a working animal, and a farm animal.

94. The method of claim 90, wherein bioavailability of a plasmalogen compound in blood or tissue of the subject is improved following administration of at least one compound of Formula (I) or a pharmaceutically acceptable salt thereof and / or at least one compound of Formula (I-A) or a pharmaceutically acceptable salt thereof relative to administration of AKG (alkyl glycerol).

95. A method of treating a disease or disorder associated with plasmalogen deficiency, the method comprising administering to a subject in need thereof an effective amount of at least one compound of Formula (I) or a pharmaceutically acceptable salt thereof and / or at least one compound of Formula (I-A) or a pharmaceutically acceptable salt thereof according to claims 1 and 14, respectively.

96. The method of claim 95, wherein the subject is a human.

97. The method of claim 95, wherein the subject is an animal.

98. The method of claim 97, wherein the animal subject is selected from the group consisting of a domesticated animal, a working animal, and a farm animal.

99. The method of claim 95, wherein the disease or disorder is a neurological disease.

100. The method of claim 99, wherein the neurological disease is selected from the group consisting of Alzheimer’s disease, Parkinson’s disease, autism, amyotrophic lateral sclerosis, frontotemporal dementia, multiple sclerosis, and schizophrenia.

101. The method of claim 95, wherein the disease or disorder is a metabolic disorder.

102. The method of claim 101, wherein the metabolic disorder is selected from the group consisting of obesity, insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, an immune-related disorder, a cardiovascular disease, a neurological disease, cancer, myalgic encephalomyelitis / chronic fatigue syndrome, Beth syndrome, and a peroxisomal disorder.

103. The method of claim 102, wherein the immune-related disorder is selected from the group consisting of asthma, atopic dermatitis, type 1 diabetes, and an infection.

104. The method of claim 102, wherein the cardiovascular disease is selected from the group consisting of atherosclerosis, cardiac remodeling, and hypertension.

105. The method of claim 102, wherein the peroxisomal disorder is a Zellweger spectrum disorder or rhizomelic chondrodysplasia punctata.

106. The method of any one of claims 90-105, wherein the compound of Formula (I) or (I-A) is administered at a dose of 0.1-4000 mg per day.

107. The method of claim 106, wherein the compound of Formula (I) or (I-A) is administered at a dose of 0.1-2000 mg per day.

108. The method of claim 107, wherein the compound of Formula (I) or (I-A) is administered at a dose of 25-1600 mg per day.

109. The method of claim 108, wherein the compound of Formula (I) or (I-A) is administered at a dose of 400 mg, 800 mg, or 1600 mg per day.

110. The method of any claim 108, wherein the compound of Formula (I) or (I-A) is administered at a dose of 200 mg per day.

111. The method of claim 108, wherein the compound of Formula (I) or (I-A) is administered at a dose of 25-100 mg per day.

112. The method of claim 111, wherein the compound of Formula (I) or (I-A) is administered at a dose of 25 mg, 50 mg, or 100 mg per day.

113. Use of a composition of any one of claims 1-59 or a formulation of any one of claims 60-70 in the manufacture of a medicament for increasing the level of a plasmalogen compound in blood or tissue of a subject in need thereof.

114. Use of a composition of any one of claims 1-59 or a formulation of any one of claims 60-70 in the manufacture of a medicament for treating a disease or disorder associated with plasmalogen deficiency.

115. The use of claim 114, wherein the disease or disorder is a neurological disease.

116. The use of claim 115, wherein the neurological disease is selected from the group consisting of Alzheimer’s disease, Parkinson’s disease, autism, amyotrophic lateral sclerosis, frontotemporal dementia, multiple sclerosis, and schizophrenia.

117. The use of claim 116, wherein the disease or disorder is a metabolic disorder.

118. The use of claim 117, wherein the metabolic disorder is selected from obesity, insulin resistance, type 2 diabetes, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, an immune-related disorder, a cardiovascular disease, a neurological disease, cancer, myalgic encephalomyelitis / chronic fatigue syndrome, Barth syndrome, and a peroxisomal disorder.

119. The use of claim 118, wherein the immune-related disorder is selected from the group consisting of asthma, atopic dermatitis, type 1 diabetes, and infection.

120. The use of claim 118, wherein the cardiovascular disease is selected from the group consisting of atherosclerosis, cardiac remodeling, and hypertension.

121. The use of claim 118, wherein the peroxisomal disorder is a Zellweger Spectrum Disorder or rhizomelic chondrodysplasia punctata.

122. A kit for use in the method of any one of claims 90 to 112, comprising at least one compound of Formula (I) or Formula (I-A), or a pharmaceutically acceptable salt thereof, according to any one of claims 1 and 14, respectively.

123. The composition of claim 28, wherein the at least one compound is selected from a compound of Formula (I-A1), (I-A2), or (I-A3).

124. The composition of claim 29, wherein the mixture of at least two compounds is a 50:50 mixture.

125. The composition of claim 30, wherein the mixture of (I-A1), (I-A2), and (I-A3) is selected from: LPC(O-16:0) 26.8% + LPC(O-18:0) 46.1% + LPC(O-18:1) 27.1%; LPC(O-16:0) 46% + LPC(O-18:0) 21% + LPC(O-18:1) 33%; LPC(O-16:0) 42% + LPC(O-18:0) 51% + LPC(O-18:1) 7%; LPC(O-16:0) 62% + LPC(O-18:0) 23.9% + LPC(O-18:1) 14.1%; LPC(O-16:0) 19.8% + LPC(O-18:0) 66% + LPC(O-18:1) 14.2%; LPC(O-16:0) 32.5% + LPC(O-18:0) 39.6% + LPC(O-18:1) 27.9%; or LPC(O-16:0) 34.1% + LPC(O-18:0) 41.5% + LPC(O-18:1) 24.4%.

126. The composition of claim 44, wherein the at least one compound is selected from a compound of Formula (I-A4), (I-A5), or (I-A6).

127. The composition of claim 45, wherein the mixture of at least two compounds is a 50:50 mixture.

128. The composition of claim 46, wherein the mixture of (I-A4), (I-A5), and (I-A6) is selected from: LPE(O-16:0) 26.8% + LPE(O-18:0) 46.1% + LPE(O-18:1) 27.1%; LPE(O-16:0) 46% + LPE(O-18:0) 21% + LPE(O-18:1) 33%; LPE(O-16:0) 42% + LPE(O-18:0) 51% + LPE(O-18:1) 7%; LPE(O-16:0) 62% + LPE(O-18:0) 23.9% + LPE(O-18:1) 14.1%; LPE(O-16:0) 19.8% + LPE(O-18:0) 66% + LPE(O-18:1) 14.2%; LPE(O-16:0) 32.5% + LPE(O-18:0) 39.6% + LPE(O-18:1) 27.9%; or LPE(O-16:0) 34.1% + LPE(O-18:0) 41.5% + LPE(O-18:1) 24.4%. LPE (O-16:0) 19.8% + LPE (O-18:0) 66% + LPE (O-18:1) 14.2%; LPE (O-16:0) 32.5% + LPE (O-18:0) 39.6% + LPE (O-18:1) 27.9%; or LPE (O-16:0) 34.1% + LPE (O-18:0) 41.5% + LPE (O-18:1) 24.4%.

129. The composition of any one of claims 1-59 or 123-128, wherein the composition maintains or modulates the ratio of acetal phospholipid compounds observed in healthy human subjects.

130. The composition of any one of claims 1-59 or 123-128, wherein the composition results in a reduction of inflammation or improvement or reduction of symptoms associated with an inflammatory disease.

131. The composition of claim 130, wherein the reduction of inflammation is associated with a decrease in inflammatory cytokine levels.

132. The composition of any one of claims 1-59 or 123-128, wherein the composition maintains or modulates inflammatory cytokine levels at levels observed in healthy human subjects.

133. The composition of claim 132, wherein the inflammatory cytokine that is maintained or modulated is selected from the group consisting of IL-6, NFE2L2, TLR4.

134. A formulation comprising the composition of any one of claims 1-59 or 123-128 and at least one excipient and / or solubilizer.

135. The formulation of claim 134, wherein the formulation is formulated for oral administration.

136. The formulation of claim 135, wherein the oral administration form is a beverage or a food product.

137. The formulation of claim 135, wherein the oral administration form is a food product.

138. The formulation of claim 135, wherein the oral administration form is an infant formula.

139. The formulation of claim 134, wherein the formulation comprises a solubilizer.

140. The formulation of claim 139, wherein the solubilizer is selected from the group consisting of sodium carboxymethylcellulose, hypromellose, proline, xanthan gum, maltodextrin, alginate, wax, lipid, oil, alcohol, sugar, microcrystalline cellulose, starch, calcium phosphate, mannitol, sorbitol, erythritol, food grade solvent, phospholipid, DMSO, ethanol, ethyl acetate, and isopropyl alcohol.

141. The formulation of claim 139, wherein the solubilizer is an egg phospholipid.

142. The formulation of claim 141, wherein the egg phospholipid is purified egg yolk L-α- phosphatidylcholine.

143. The formulation of claim 139, wherein the formulation has improved stability compared to a formulation comprising the composition of any one of claims 1-59 or 123-128 and not comprising a solubilizer.

144. The formulation of claim 139, wherein the formulation is not susceptible to degradation during freezing and thawing compared to a formulation comprising the composition of any one of claims 1 to 59 or 123 to 128 and not comprising a solubilizing agent.

145. The composition of any one of claim 28 or claim 44, wherein the composition comprises a compound having a purity of at least 99.9%.

146. A formulation comprising the composition of claim 145 and at least one excipient.

147. The formulation of any one of claims 134 to 138 or claim 146, wherein the formulation comprises at least one of a solubilizing agent, an emulsifying agent, a stabilizing agent, a dispersing agent, an antifoaming agent, or a diluent.

148. The formulation of any one of claims 134 to 138 or claims 146 to 147, wherein the formulation additionally comprises at least one solubilizing agent.

149. The formulation of claim 148, wherein the solubilizing agent is selected from the group consisting of sodium carboxymethylcellulose, hypromellose, proline, xanthan gum, maltodextrin, alginate, wax, lipid, oil, alcohol, sugar, microcrystalline cellulose, starch, calcium phosphate, mannitol, sorbitol, erythritol, food grade solvent, phospholipid, DMSO, ethanol, ethyl acetate, and isopropyl alcohol.

150. The formulation of claim 149, wherein the solubilizing agent is selected from the group consisting of DMSO, ethanol, ethyl acetate, and isopropyl alcohol.

151. The formulation of any one of claims 134 to 144 or claims 146 to 150, wherein the formulation additionally comprises at least one antioxidant compound.

152. The formulation of any one of claims 134 to 144 or claims 146 to 151, wherein the formulation additionally comprises at least one antifoaming agent.

153. The formulation of any one of claims 134 to 144 or claims 146 to 152, wherein the formulation is formulated for oral administration.

154. The formulation of claim 153, wherein the oral administration form is a tablet, a capsule, a solution, a mouthwash, a suspension, a powder, a chewing gum, a candy, a lozenge, a sublingual delivery system, or a fast-dissolving formulation.

155. The formulation of claim 154, wherein the oral administration form is a tablet.

156. The composition of claim 30, wherein the mixture of (I-A1), (I-A2), and (I-A3) comprises 90% w / v (I-A1).

157. The composition of claim 30, wherein the mixture of (I-A1), (I-A2), and (I-A3) comprises 90% w / v (I-A2).

158. The composition of claim 30, wherein the mixture of (I-A1), (I-A2), and (I-A3) comprises 90% w / v (I-A3).

159. The composition of claim 46, wherein the mixture of (I-A4), (I-A5), and (I-A6) comprises 90% w / v (I-A4).

160. The composition of claim 46, wherein the mixture of (I-A4), (I-A5), and (I-A6) comprises 90% w / v (I-A5).

161. The composition of claim 46, wherein the mixture of (I-A4), (I-A5), and (I-A6) comprises 90% w / v (I-A6).

Images