DOCOSAHEXAENOIC ACID AND EGG YOLK CONTAINING COMPOSITION SUITABLE FOR SICKLE CELL DISEASE TREATMENT

Abstract

A composition, including fatty acids, egg yolk and an acidic aqueous medium, that includes at least 20 percent DHA by weight (20 wt %). An oil in water emulsion is provided, such as a mayonnaise, paste or has a mayonnaise-like consistency, for example suitable to be pressed or squeezed from a flexible container or tube. Methods are provided for treating disease in which DHA is deficient, for example sickle cell disease, and a medical food product including the composition and a process for the preparation of a composition or medical food product.

Claims

1. A composition, comprising fatty acids, egg yolk and an acidic aqueous medium, wherein the composition comprises at least 20 percent docosahexaenoic acid (DHA) by weight (20 wt %).

2. The composition according to claim 1, wherein the composition comprises of consists of an oil-in-water emulsion.

3. The composition according to claim 1, wherein the composition is a mayonnaise, paste or has a mayonnaise-like consistency, for example suitable to be pressed or squeezed from a flexible container or tube.

4. The composition according to claim 1, wherein the DHA comprises or consists of a triglyceride ester of docosahexaenoic acid (DHATG) or other esters of DHA, such as ethyl esters.

5. The composition according to claim 1, wherein the fatty acids comprise or consist of omega-3 fatty acids and/or marine oil.

6-10. (canceled)

11. The composition according to claim 1, wherein the composition additionally comprises: one or more flavoring agents selected from the group consisting of salt, sugar, sweetening agents, pepper, lemon oil and d-limonene; one or more preservatives; and/or one or more vitamins selected from the group consisting of Vitamin C, Thiamin (B1), Riboflavin (B2), Niacin, Vitamin B and Vitamin E, wherein the acidic aqueous medium optionally comprises or consists of citric acid and/or vinegar.

12-15. (canceled)

16. The composition according to claim 1, wherein the composition comprises: DHA at 20-90 wt; other fatty acids at 1-50 wt; egg yolk at 0.5-25 wt %; and an acidic aqueous medium at 0.5-30 wt.

17. The composition according to claim 16, wherein the composition additionally comprises: lemon oil (or d-limonene) at 0.1-10 wt %; sugar at 0.5-20 wt %; salt at 0.01-5 wt %; citric acid at 0.1-10 wt %; water at 0.5-20 wt %; pepper at 0.01-5 wt %; and/or preservatives at 0.001-2 wt %.

18. The composition according to claim 1, wherein the composition comprises: omega-3 fish oil at 50-90 wt % comprising at least 20 wt % DHA, or DHA at 20-90 wt %; and other fatty acids at 1-50 wt %, water at 1-20 wt %, egg-yolk at 1-20 wt %, citric acid at 0.01-5, and optionally, one or more flavoring, stabilizing, and/or preserving agents.

19. The composition according to claim 1, wherein the composition additionally comprises: omega-3 fish oil at 50-90 wt % comprising at least 20 wt % DHA, or DHA at 20-90 wt %; and other fatty acids at 1-50 wt %; water at 1-20 wt %, pasteurized chicken egg yolk at 1-20 wt %, citric acid at 0.01-5 wt %; ascorbic acid at 0.01-10 wt %; sucralose at 0.001-1 wt %; sodium ascorbate at 0.01-10 wt %; orange flavor at 0.01-5 wt %; canola oil at 0.01-10 wt %; potassium sorbate at 0.01-1 wt %; sodium benzoate at 0.01-1 wt %; beta carotene beadlets at 0.001-10 wt %; xanthan gum at 0.001-10 wt %; and menthol at 0.001-10 wt %.

20. The composition according to claim 1, wherein the composition is essentially free of micelles having an average diameter of 1-10 μm, and/or wherein the composition is essentially free of surfactants.

21. (canceled)

22. A medical food product comprising the composition according to claim 1.

23. The medical food product according to claim 22 in the form of a mayonnaise, mayonnaise-like composition or paste.

24. The medical food product according to claim 23, packaged in a container formed of an airtight material in soft plastic, a tube or a static container with a piston or screw for dispensing.

25. (canceled)

26. The medical food product according to claim 24, wherein said plastic, tube or container is arranged to collapse during use proportionally to the amount of said composition remaining after a portion of said composition is dispensed from said container, wherein no air space forms in said container after said dispensing, and wherein no discernible oxidation occurs during the storage and use of said composition.

27. (canceled)

28. A method for treating a medical condition associated with pathologically low DHA levels in a subject, comprising administering the composition according to claim 1 to said subject.

29. The method according to claim 28, wherein the medical condition is a metabolic disease or a membranopathy.

30. The method according to claim 29, wherein the medical condition is sickle cell disease.

31. The method according to claim 30, wherein the subject is a pediatric subject.

32. The method according to claim 31, wherein the composition is administered in advance of sickle cell disease symptoms in a subject below about 9 months of age, to reduce the risk of, sickle cell disease occurring.

33. The method according to claim 29, wherein the membranopathy is selected from the list consisting of diabetes and ischemic disease.

34. The method according to claim 27 wherein the medical disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

35. A process for the preparation of a composition or medical food according to claim 1, comprising forming an oil-in-water emulsion comprising at least DHA, egg yolk and acidic aqueous medium, in an amount sufficient to provide a mayonnaise-type emulsion or paste; and completely filling said emulsion into a container of an airtight material, in plastic, a tube or container arranged to collapse during use proportionally to the amount of said composition remaining after a portion of said composition is dispensed from said container.

36-37. (canceled)

Description

FIGURES

[0195] The figures presented herein represent non-limiting, exemplary embodiments, in accordance with the invention, that provide support for carrying out the invention and demonstrating the beneficial effects described herein.

BRIEF DESCRIPTION OF THE FIGURES

[0196] FIG. 1: CRP levels from a trial in patients with sickle cell disease.

[0197] FIG. 2: Percentage change from baseline for DHA levels in red blood cell membranes from a trial in patients with sickle cell disease.

DETAILED DESCRIPTION OF THE FIGURES

[0198] FIG. 1: CRP levels from a trial in patients with sickle cell disease. Baseline, corresponding to CRP levels in patients at day 0, is represented by the “0” horizontal line. The CRP levels after a 28-day trial, indicate that C-Reactive Protein, a marker of inflammation, drops at various levels when using the formulation of the invention.

[0199] FIG. 2: Percentage change from baseline for DHA levels in red blood cell membranes from a trial in patients with sickle cell disease. Shown is a percentage change over a 28-day trial, compared to levels measured at day 0. The change of DHA in the plasma membrane of red blood cells increases substantially when compared to the same measurement made at the beginning of the study.

EXAMPLES

[0200] The following examples are non-limiting, exemplary embodiments, in accordance with the invention, that provide support for carrying out the invention and demonstrating the beneficial effects described herein.

[0201] The process is carried out as for the production of mayonnaise on a small or large scale, preferably in an inert atmosphere of N2 or CO2. In this way an oil-in-water emulsion can be produced with a high DHA content. The water used in the production process must be, as far as possible, preferably, without air and boiled under vacuum.

[0202] The emulsion is filled to the top into tubes and/or soft plastic containers that are air-resistant and collapse proportionally to the amount of preparation left after each dosage, no matter how many doses, so that no air space arises inside the container and no oxidation occurs during extended use. Alternatively, it can be filled into stiff containers, which, by using a screw or piston-like device, reduce in volume accordingly with each expressed dose of the emulsion.

[0203] The examples presented below disclose preferred precise compositions, in addition to wt % ranges for each of the components, which may be adjusted accordingly, depending on the needs of the subject.

Example Compositions

[0204]

TABLE-US-00001 preferred Example 1 grams % % range % range Docosahexaenoic Acid, 560 56.00%  20-90 .sup. 40-70 preferably as the triglyceride ester (DHATG) Other fatty acids 240 24.00% .sup. 1-50 .sup. 10-35 Egg yolk 50 5.00% 0.5-25   1-15 Lemon oil 10 1.00% 0.1-10 0.5-5 Sugar (or artificial 55 5.50% 0.5-20   2-10 sweetener) Salt 10 1.00% 0.01-5  0.1-3 Citric Acid 20 2.00% 0.1-10 .sup. 1-5 Water (air-free) 40 4.00% 0.5-20   1-10 Pepper 10 1.00% 0.01-5  0.1-2 Preservatives 5 0.50% 0.001-2   0.1-1 sum 1000

TABLE-US-00002 preferred Example 2 grams % range % range Docosahexaenoic Acid, 560 56.00%  20-90 .sup. 40-70 preferably as the triglyceride ester (DHATG) Other fatty acids 240 24.00% .sup. 1-50 .sup. 10-35 Egg yolk 50 5.00% 0.5-25   1-15 Lemon oil 5 0.50% 0.1-10 0.2-4 Sugar (or artificial 100 10.00% 0.5-20   5-15 sweetener) Salt 10 1.00% 0.01-5  0.1-3 Citric Acid 20 2.00% 0.1-10 .sup. 1-5 Water (air-free) 100 10.00% 0.5-20   5-15 Pepper 10 1.00% 0.01-5  0.1-2 Preservatives 5 0.50% 0.001-2   0.1-1 sum 1100

TABLE-US-00003 preferred Example 3 grams % range % range Docosahexaenoic Acid, 560 56.00%  20-90 40-70 preferably as the triglyceride ester (DHATG) Other fatty acids 240 24.00% .sup. 1-50 10-35 Egg yolk 50 5.00% 0.5-25  1-15 Sugar (or artificial 55 5.50% 0.5-20  2-10 sweetener) Salt 10 1.00% 0.01-5  0.1-3.sup.  Vinegar (eg 20%) 10 1.00% 0.01-5  0.1-3.sup.  Citric Acid 10 1.00% 0.1-10 0.5-5.sup.  Water (air-free) 25 2.50% 0.5-20  1-10 Pepper 5 0.50% 0.01-5  0.1-1.sup.  Preservatives 5 0.50% 0.001-2   0.1-1.sup.  sum 970 preferred General Composition grams % % range % range Docosahexaenoic Acid, 560 56.00%  20-90 40-70 preferably as the triglyceride ester (DHATG) Other fatty acids 240 24.00% .sup. 1-50 10-35 Egg yolk 50 5.00% 0.5-25  1-15 Acidic aqueous medium 150 15.00% 0.5-30 10-20 sum 1000

TABLE-US-00004 preferred Example 4 grams % % range % range Omega-3 fish oil 760 76.00%   70-80 75-78 (comprising 20 wt % or more DHA in the composition) Ascorbic acid USP 10 1.00%  0.1-5 1.0-2.5 Sucralose, FCC/JECFA 1 0.10% 0.001-1  0.01-0.1  Sodium Ascorbate, USP 5 0.50%  0.1-5 0.1-2.0 Water, process 100 10.00%    5-15  8.0-12.0 Egg yolk, pasteurized 100 10.00%    5-15  8.0-12.0 Orange flavor, natural 10 1.00%  0.1-4 1.0-3.0 Non-GMO Canola oil, FCC 5 0.50%  0.01-10 0.1-1.0 Citric acid anhydrous USP 5 0.50% 0.05-2 0.1-1.0 Potassium sorbate FCC 1 0.10% 0.01-1 0.05-0.1  Sodium benzoate FCC 1 0.10% 0.01-1 0.05-0.1  Beta Carotene beadlets 1 0.10% 0.001-10 0.01-1.0  (10% beta carotene), EP Xanthan gum, FCC 0.5 0.05% 0.001-10 0.01-1.0  L-Menthol, USP 0.5 0.05% 0.001-10 0.01-1.0  sum 1000

[0205] Clinical Tests:

[0206] In order to assess the properties of the inventive compositions, an open label, balanced, randomized, multicenter, phase II clinical trial is carried out to determine the effect of the compositions (medical food, in the form of a mayonnaise) enriched with docosahexaenoic acid (DHA, as a triglyceride ester) on the cell membrane fatty acid ratio of blood cells of sickle cell patients, ranging in age from 6-40 years of age.

[0207] Employed in the trial is a DHA enriched mayonnaise, for example as described above in Example 4.

[0208] Primary Objective: To determine the percentage change in the total blood cell membrane DHA and EPA concentration from baseline and after 4 weeks of treatment.

[0209] Secondary objective: To monitor the safety and tolerability of DHA enriched food in patients with sickle cell disease.

[0210] 15 Patients with sickle cell disease ranging in age from 6-40 years of age are enrolled in the study.

[0211] A single packet of the Omega 3 DHA enriched mayonnaise is administered once a day for 28 days at ambient temperature. A single packet of “Omega 3 DHA enriched Mayonnaise” is ingested orally (eaten) either directly or mixed with a small amount of yogurt. The investigational product is administered orally in the dosage as per this protocol for a period of 28 days at one packet per day. Each packet comprises 20 g mayonnaise. [0212] Visiting and Screening Procedure [0213] Visit 01: Screening and enrolment (Day −1) [0214] Visit 02: Baseline visit (Day 0) [0215] Visit 03: Follow up visit (Day 01) [0216] Visit 04: Follow up visit (Day 08) [0217] Visit 05: Follow up visit (Day 15) [0218] Visit 06: Follow up visit (Day 22) [0219] Visit 07: Primary end point & end of study visit (Day 28)

[0220] Haematology, Biochemistry and serology are assessed on each day of screening. At every visit, blood pressure, radial pulse rate, oral temperature and wellbeing status are assessed and recorded before start of treatment and after completion of treatment. At the beginning and end of the therapy visit, Lipid Profile test, fatty acid ratio (AA:Total Omega-3), DHA& EPA (Total Omega-3), AA (Arachidonic acid) and C-reactive protein (CRP) are measured. The sickle cell VOC pain is assessed through VAS (Visual Analogue Scale) at every visit. Monitoring for adverse events is conducted throughout the study period.

[0221] Efficacy is evaluated as follows:

[0222] Baseline visit—Day 0, and End of therapy visit—after 4 weeks treatment (i.e. 28th day).

[0223] Primary Efficacy Evaluation: Patients are evaluated by total blood cell membrane DHA, EPA and AA (Arachidonic acid) concentration at Baseline and end of therapy visit.

[0224] Secondary Efficacy Evaluation: Patients are evaluated by fatty acid ratio (AA:Total Omega-3), DHA& EPA (Total omega-3) and AA (Arachidonic acid) at end of therapy visit. C-reactive protein (CRP) levels will be evaluated at Day 0 and Day 28.

[0225] Primary end points: The percentage change in the total blood cell membrane DHA and EPA concentration from baseline after 4 weeks of treatment. To increase the DHA level above baseline and a lower ratio between AA (Arachidonic acid) and Total Omega-3.

[0226] Secondary end points: Monitoring for Safety and tolerability is carried out throughout the study period, to decrease CRP over a 28-day period, and to determine the percentage change of Lipid profile from baseline to day 28.

[0227] Systemic Bioavailability: When Omega 3 fatty acids (DHATG) are administered under fasted condition, on average the peak (Cmax) and total (AUC0-72 h) exposure were lower by up to 20 to 80-fold, respectively, for total plasma EPA, and lower by up to 2 to 4-fold, respectively, for total plasma DHA, in comparison to those observed under fed condition (high-fat high-calorie meal). Therefore, Omega 3 fatty acids (DHATG) are preferably taken with food. In healthy volunteers and in patients with hypertriglyceridemia, EPA and DHA are absorbed when administered as ethyl esters orally. Omega-3-acids administered as ethyl esters induce significant, dose-dependent increases in serum phospholipid EPA content.

[0228] Methods for determining CRP levels in blood are known to a skilled person and carried out accordingly. C-reactive protein (CRP) is an acute-phase reactant synthesized by the liver and is used as a marker of inflammation. The blood (serum) CRP concentration can increase by up to 1,000-fold after infection, trauma, surgery, and other acute inflammatory events, and is elevated in subjects with chronic inflammation, such as in sickle cell subjects. Immunoassays for determining CRP levels are established in the art.

[0229] Methods for quantitatively determining fatty acids in red blood cells are known to a skilled person and carried out accordingly. For example, blood is collected tubes containing EDTA. Within 48 h of collection, RBCs are separated from the blood samples, washed with phosphate-buffered saline and stored in a −80° freezer until analysis. RBC membrane levels of e.g. EPA, DHA levels, and AA are determined using mass spectrometry analysis, for example by capillary column-negative ion mass spectrometry, and can expressed as percentage of total fatty acids. Method descriptions are, for example, available from Lagerstedt et al. (Mol. Genet. Metab. 2001; 73:38-45).

[0230] The prophetic data presented in FIG. 1 show expected CRP levels to be obtained from the trial in patients with sickle cell disease. The study may or may not employ a placebo control, for example patients receiving a mayonnaise without omega 3 or DHA enrichment. Baseline, represented by the “0” horizontal line in FIG. 1 indicates CRP levels at day 0. The figure shows that C-Reactive Protein, a marker of inflammation, will drop at various levels of supplementation with the formulation used in the trial. The expected data from the trial reflects the pooled data and shows a drop from baseline (black horizontal line). The CRP levels in patients are therefore reduced, indicating a therapeutic effect.

[0231] The prophetic data presented in FIG. 2 shows the % change (at 28 days) from baseline (measured at day 0) for DHA levels in red blood cell membranes. The study may or may not employ a placebo control, for example patients receiving a mayonnaise without omega 3 or DHA enrichment, but the change of DHA in the cell membrane is expected to increase substantially, similar to Dose 1 at the very least, when compared to the same measurement made at the beginning of the study.

[0232] Thus, DHA levels enable measurement of DNA being incorporated into the red blood cell membranes and replenishing the fatty acid created by the metabolic disorder. DHA can therefore—via administration in the inventive composition—reestablish the proper balance of this essential polyunsaturated fatty acid. By doing this, the red blood cell becomes flexible, does not break, and reduces overall inflammation (noted in FIG. 1) and consequently reduces disease and/or disease symptoms. Reduced inflammation drops d-dimer levels and reduces aggregation of the red blood cells, which is a cause of the painful crisis seen in sickle cell patients.

[0233] Preliminary results from the trial, which is ongoing, are positive. At present, no patient enrolled in the trial has experienced an attack (painful crisis seen in sickle cell patients), indicating that the DHA levels are corrected, and inflammation and disease symptoms are reduced. No patients have discontinued the study, and no complaints were evident regarding administration difficulties, with respect to smell or taste of the compositions. The compositions and formulations of the invention therefore enable surprisingly enhanced DHA bioavailability in patients, in addition to providing an easy-to-administer formulation that indicates good patient compliance due to reduced unwanted smell or taste typically associated with fish oil preparations.