Methods for monitoring a patient's response to treatment for retinal oxidative disease - Patents.com

Abstract

A method is disclosed for assessing the presence or absence of therapeutic concentrations of deuterated docosahexaenoic acid during treatment of a patient with a retinal oxidative disorder.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 309,468, filed February 11, 2022, and U.S. Provisional Patent Application No. 63 / 309,471, filed February 11, 2022, each of which is incorporated by reference in its entirety. [Background technology]

[0002] Many retinal diseases are mediated, at least in part, by lipid peroxidation of arachidonic acid or docosahexaenoic acid (DHA) found in the peripheral rods and cones of the retina, including, but not limited to, wet and dry age-related macular degeneration (including associated geographic atrophy), retinitis pigmentosa, diabetic retinopathy, cataracts, and Stargardt's disease.

[0003] The use of deuterated polyunsaturated fatty acids or esters thereof, including deuterated docosahexaenoic acid (D-DHA) or esters thereof, to treat these diseases, as disclosed in U.S. Patent No. 10,058,522 (incorporated herein by reference in its entirety). Specifically, the underlying pathology of these diseases involves lipid peroxidation at the bis-allylic position of polyunsaturated fatty acids (PUFAs) in the peripheral or outer segments of rods and cones found in the retina. These rods and cones contain significant amounts of DHA, constituting 30%-60% of the fatty acids in these outer segments.

[0004] Treatment of oxidative retinal diseases with D-DHA or its esters is complicated by the fact that it may take several weeks to months after the start of treatment to reach therapeutic concentration in the retina.In addition, rods and cones are inaccessible in living subjects, so monitoring the progress of patients to reach therapeutic concentration of drugs in the retina is not feasible.This can only be indirectly evaluated by periodically monitoring the retina for disease progression, assuming that any apparent reduction in disease progression is due to treatment.However, such an approach cannot consider whether such a lack of reduction is due to lack of efficacy of the drug or due to insufficient therapeutic levels of the drug in the target tissue, thereby making the appropriate D-DHA replacement level an important performance index of drug efficacy, i.e., the ratio of D-DHA to total DHA must reach a therapeutically effective ratio of the total DHA pool absorbed during daily dosing.

[0005] Regarding the latter, the uptake of D-DHA is controlled by the total amount of DHA ingested by the patient. In other words, the more naturally occurring DHA ingested during treatment, the more dilute the relative proportion of the administered D-DHA absorbed in the total DHA pool. Furthermore, an individual's diet rich in marine or fish oil (such as that found in some drugs) may reduce the relative D-DHA uptake of the drug into the body. Thus, each patient being treated with a fixed dose of a drug will absorb a different relative amount of the drug, and these amounts will vary from day to day and from patient to patient. This creates a challenge for clinicians as to how they can be sure that the patient is progressing in an appropriate manner to achieve therapeutic concentrations of the drug in rods and cones in an advantageous manner. This is particularly important because the longer it takes to achieve therapeutic concentrations, the greater the risk of retinal damage and additional vision loss.

[0006] Clearly, there is an unmet and much-needed need for a method that would allow clinicians to monitor patients to determine whether they are progressing appropriately to therapeutic concentrations of drug in the retina. Summary of the Invention

[0007] Methods are disclosed that allow clinicians to confirm that a patient's retinal uptake of D-DHA is proceeding appropriately by verifying that a certain steady-state concentration of the drug is identified in the patient's plasma and / or red blood cells. D-DHA absorption and tissue distribution follow first-order kinetics, and as shown in the examples below, a steady-state concentration of D-DHA in plasma occurs approximately 21-28 days after the start of treatment. Alternatively, a steady-state concentration of D-DHA in red blood cells occurs approximately 33-44 days after the start of treatment. When such a steady state is achieved, it proves that the patient has a maximum concentration of the drug in their blood and has reached a maximum D-DHA substitution level for total DHA. Since blood serves as a depot for delivering the drug to the retina, clinicians can verify that a steady-state concentration in the blood correlates with adequate uptake by the patient and maximum delivery of the drug to the retina. On the other hand, failure to timely achieve a steady-state concentration in the blood proves that the patient is ingesting either a food or drug rich in DHA. In either case, the patient may be required to adjust their diet and / or their medication may need to be increased.

[0008] As also shown in the following examples, the plasma steady-state concentration of D-DHA in plasma precedes that in the retina by about 7-10 weeks, while the steady-state concentration in erythrocytes precedes that in the retina by about 5-7 weeks. Furthermore, based on, for example, an average daily dietary intake of about 130 mg DHA per day (which represents, for example, the 90th percentile of the average normal DHA intake by men over 51 years old in the United States), in a 250 mg / day dosage regimen, the steady-state concentration of the drug in plasma, erythrocytes, and retina is about 65% of the total amount of DHA present, including D-DHA. In a 500 mg / day dosage regimen, the steady-state concentration of the drug in plasma and retina is about 80%, and in a 1,000 mg / day dosage regimen, the steady-state concentration of the drug in plasma and retina is about 88%. Thus, the time to reach steady-state relative D-DHA concentration remains the same at these three dosage levels, but the use of higher doses significantly increases the relative concentration of the drug at steady state.

[0009] Based on the above, dosages of D-DHA or esters thereof typically range from about 150 mg / day to about 1,000 mg / day, preferably from about 250 mg / day to about 500 mg / day. In a preferred embodiment, the dosage used is sufficient to achieve a steady state concentration of D-DHA of about 50% or greater based on the total amount of DHA present, including D-DHA.

[0010] Based on the finding that the steady-state concentration of D-DHA in either plasma or red blood cells correlates well with the steady-state concentration in retina, a standardized concentration curve can be created for each dose of this drug based on either plasma or red blood cells.Such standardized curves correlate with the concentration of the drug measured using different doses at various times from the start of treatment to the concentration that should reach steady state in plasma or red blood cells.Then, such standardized curves can be used to monitor and evaluate the overall response to treatment for a given patient.

[0011] Thus, in one embodiment, there is provided a method for monitoring a patient for uptake of D-DHA, comprising: administering to said patient periodically an effective dose of D-DHA or an ester thereof; obtaining one or more blood samples from the patient after initiation of treatment; assessing the amount of D-DHA in the sample relative to the total amount of DHA; comparing the assessed amount of D-DHA to a standard concentration curve, the curve being based on the particular dose of D-DHA or an ester thereof used, the blood constituent being assessed, and the length of time since the start of treatment; and determining whether the patient has achieved an adequate D-DHA replacement level based on the curve.

[0012] In one embodiment, the blood component being evaluated is plasma.

[0013] In one embodiment, the blood component being evaluated is a red blood cell.

[0014] In one embodiment, the length of time between initiation of treatment and testing is about 7 to about 45 days. In one embodiment, the length of time between initiation of treatment and testing is at least about 14 days. In another embodiment, the length of time between initiation of treatment and testing is at least about 30 days.

[0015] In one embodiment, a method for monitoring a patient for D-DHA uptake comprises: administering to the patient on a regular basis an effective dose of D-DHA or an ester thereof, the dose being about 250 mg / day; obtaining one or more plasma samples from the patient after initiation of treatment; assessing the amount of D-DHA in the sample relative to the total amount of DHA; comparing the assessed amount of D-DHA to a standard concentration curve, the curve being based on the length of time since the start of treatment; and determining whether the patient is adequately absorbing D-DHA based on the curve.

[0016] In one embodiment, a method for monitoring a patient for D-DHA uptake comprises: administering to the patient on a regular basis an effective dose of D-DHA or an ester thereof, the dose being about 500 mg / day; obtaining one or more red blood cell samples from the patient after initiation of treatment; assessing the amount of D-DHA in the sample relative to the total amount of DHA; comparing the assessed amount of D-DHA to a standard concentration curve, the curve being based on the length of time since the start of treatment; Based on the curve, a method is provided for monitoring a patient for D-DHA uptake, comprising determining whether the patient is adequately absorbing D-DHA.

[0017] In one embodiment, a method for monitoring a patient for D-DHA uptake comprises: administering to the patient on a regular basis an effective dose of D-DHA or an ester thereof, the dose being about 1000 mg / day; obtaining one or more red blood cell samples from the patient after initiation of treatment; assessing the amount of D-DHA in the sample relative to the total amount of DHA; comparing the assessed amount of D-DHA to a standard concentration curve, the curve being based on the length of time since the start of treatment; and determining whether the patient is adequately absorbing D-DHA based on the curve.

[0018] In one embodiment, if the concentration of deuterated docosahexaenoic acid is lower than that provided by the standardization curve, the clinician has the option of either prescribing a modification to the patient's diet to reduce the amount of naturally occurring DHA ingested per day and / or prescribing an increase in the amount of drug administered.

[0019] In one embodiment, the method includes comparing the amount of deuterated docosahexaenoic acid in the sample to a minimum therapeutic concentration of at least 50% of deuterated docosahexaenoic acid based on the total amount of docosahexaenoic acid, including deuterated docosahexaenoic acid, in the sample to determine whether the patient has a therapeutic or sub-therapeutic concentration of deuterated docosahexaenoic acid.

[0020] In one embodiment, the therapeutic concentration of deuterated docosahexaenoic acid is set at 60%, 70%, or even 80% of the therapeutic goal for a given patient.

[0021] In one embodiment, the method includes increasing the dose of deuterated DHA administered to the patient if the amount of deuterated docosahexaenoic acid in the sample is below the minimum therapeutic concentration.

[0022] In one embodiment, the method includes restricting the patient's dietary intake of docosahexaenoic acid during treatment with deuterated docosahexaenoic acid.

[0023] In one embodiment, the method involves limiting the patient's dietary docosahexaenoic acid intake (i.e., docosahexaenoic acid ingested by the patient, not including the amount of administered deuterated docosahexaenoic acid), to no more than about 132 mg per day. [Brief description of the drawings]

[0024] [Figure 1]Illustrates standardized curves showing increased concentrations of D-DHA using three different markers (plasma, red blood cells, and retina) at four different time points (8, 19, 38, and 78 days after initial D-DHA exposure) in a cohort of mice fed a customized rodent diet containing 0.5% w / w D-DHA.

[0025] [Diagram 2] Illustrates standardized curves showing the increase in concentration of D-DHA using different markers (plasma, red blood cells, and retina) for three different dosing regimens over the time course from treatment initiation to reaching steady state in patients with an average dietary intake of approximately 130 mg DHA per day. [Diagram 3] Illustrates standardized curves showing the increase in concentration of D-DHA using different markers (plasma, red blood cells, and retina) for three different dosing regimens over the time course from treatment initiation to reaching steady state in patients with an average dietary intake of approximately 130 mg DHA per day. [Figure 4] Illustrates standardized curves showing the increase in concentration of D-DHA using different markers (plasma, red blood cells, and retina) for three different dosing regimens over the time course from treatment initiation to reaching steady state in patients with an average dietary intake of approximately 130 mg DHA per day.

[0026] [Diagram 5]A shows a chemical diagram of the steps in iron-catalyzed lipid peroxidation of DHA-containing phospholipids and the formation of CEP. Iron catalyzes hydroxyl radical generation by the Fenton and Haber-Weiss reactions. B shows that ROS-driven hydrogen abstraction from the bis-allylic site generates a free radical that rapidly reacts with oxygen to form lipid peroxyl radicals. C shows that the newly formed ROS species abstracts a bis-allylic hydrogen atom from the adjacent PUFA, thus perpetuating the LPO chain reaction cycle. D shows that D-DHA was used in the examples. E shows that DHA peroxidation generates multiple oxidation products, including reactive carbonyls such as HHE and HOHA, which can give rise to protein modifications including OBA, CEP and MDA adducts. Substitution of hydrogen atoms with deuterium inhibits the rate-limiting step of ROS-driven abstraction from the bis-allylic site.

[0027] [Figure 6A]Charts and images demonstrating D-DHA protection against iron-induced retinal autofluorescence and degeneration are shown. Mice were fed D-DHA for 77 days, then switched to DHA for 73 days, for a total of 150 days. Figure 6A shows the % of D-DHA in the neural retina and RPE-choroid. Figure 6B shows a timeline of mice fed either D-DHA or DHA for 1, 2, or 4 weeks, starting at 2 months of age, and then given an intravitreal injection of iron in one eye and control saline in the other. Mice continued on their respective diets until final evaluation. Figure 6C shows retinal AF areas in BAF cSLO images from mice fed DHA or D-DHA for 4 weeks 1 week after iron injection (denoted 4+1 weeks). cSLO and OCT imaging was performed 1 week after IVT iron vs. saline injection. Figures 6D-G show representative BAF cSLO images in mice fed D-DHA or DHA for 1 week, given an IVT injection, and then euthanized 1 week later (1+1 weeks), or fed D-DHA for 2 weeks, given an IVT injection, and then euthanized 1 week later (2+1 weeks), etc. (d and e). Abbreviations used in the figures: SLO, scanning laser ophthalmoscopy; OCT, optical coherence tomography; BAF, blue autofluorescence; IRAF, infrared autofluorescence; ONL, outer nuclear layer. White lines indicate iron-induced high AF spots. White dashed arrows indicate blebs in damaged RPE cells. White lines indicate the position and orientation of the horizontal OCT b scan in panel e, and white asterisks indicate the vortex vein that was used as a landmark for the corresponding position of the OCT scan in the IRAF SLO images. In c, N = 3 mice / group; in b, and d-f, N = 10 mice / group. Error bars indicate mean ± SEM. (**P<0.01). [Figure 6B]Charts and images demonstrating D-DHA protection against iron-induced retinal autofluorescence and degeneration are shown. Mice were fed D-DHA for 77 days, then switched to DHA for 73 days, for a total of 150 days. Figure 6A shows the % of D-DHA in the neural retina and RPE-choroid. Figure 6B shows a timeline of mice fed either D-DHA or DHA for 1, 2, or 4 weeks, starting at 2 months of age, and then given an intravitreal injection of iron in one eye and control saline in the other. Mice continued on their respective diets until final evaluation. Figure 6C shows retinal AF areas in BAF cSLO images from mice fed DHA or D-DHA for 4 weeks 1 week after iron injection (denoted 4+1 weeks). cSLO and OCT imaging was performed 1 week after IVT iron vs. saline injection. Figures 6D-G show representative BAF cSLO images in mice fed D-DHA or DHA for 1 week, given an IVT injection, and then euthanized 1 week later (1+1 weeks), or fed D-DHA for 2 weeks, given an IVT injection, and then euthanized 1 week later (2+1 weeks), etc. (d and e). Abbreviations used in the figures: SLO, scanning laser ophthalmoscopy; OCT, optical coherence tomography; BAF, blue autofluorescence; IRAF, infrared autofluorescence; ONL, outer nuclear layer. White lines indicate iron-induced high AF spots. White dashed arrows indicate blebs in damaged RPE cells. White lines indicate the position and orientation of the horizontal OCT b scan in panel e, and white asterisks indicate the vortex vein that was used as a landmark for the corresponding position of the OCT scan in the IRAF SLO images. In c, N = 3 mice / group; in b, and d-f, N = 10 mice / group. Error bars indicate mean ± SEM. (**P<0.01). [Figure 6C]Charts and images demonstrating D-DHA protection against iron-induced retinal autofluorescence and degeneration are shown. Mice were fed D-DHA for 77 days, then switched to DHA for 73 days, for a total of 150 days. Figure 6A shows the % of D-DHA in the neural retina and RPE-choroid. Figure 6B shows a timeline of mice fed either D-DHA or DHA for 1, 2, or 4 weeks, starting at 2 months of age, and then given an intravitreal injection of iron in one eye and control saline in the other. Mice continued on their respective diets until final evaluation. Figure 6C shows retinal AF areas in BAF cSLO images from mice fed DHA or D-DHA for 4 weeks 1 week after iron injection (denoted 4+1 weeks). cSLO and OCT imaging was performed 1 week after IVT iron vs. saline injection. Figures 6D-G show representative BAF cSLO images in mice fed D-DHA or DHA for 1 week, given an IVT injection, and then euthanized 1 week later (1+1 weeks), or fed D-DHA for 2 weeks, given an IVT injection, and then euthanized 1 week later (2+1 weeks), etc. (d and e). Abbreviations used in the figures: SLO, scanning laser ophthalmoscopy; OCT, optical coherence tomography; BAF, blue autofluorescence; IRAF, infrared autofluorescence; ONL, outer nuclear layer. White lines indicate iron-induced high AF spots. White dashed arrows indicate blebs in damaged RPE cells. White lines indicate the position and orientation of the horizontal OCT b scan in panel e, and white asterisks indicate the vortex vein that was used as a landmark for the corresponding position of the OCT scan in the IRAF SLO images. In c, N = 3 mice / group; in b, and d-f, N = 10 mice / group. Error bars indicate mean ± SEM. (**P<0.01). [Figure 6D]Charts and images demonstrating D-DHA protection against iron-induced retinal autofluorescence and degeneration are shown. Mice were fed D-DHA for 77 days, then switched to DHA for 73 days, for a total of 150 days. Figure 6A shows the % of D-DHA in the neural retina and RPE-choroid. Figure 6B shows a timeline of mice fed either D-DHA or DHA for 1, 2, or 4 weeks, starting at 2 months of age, and then given an intravitreal injection of iron in one eye and control saline in the other. Mice continued on their respective diets until final evaluation. Figure 6C shows retinal AF areas in BAF cSLO images from mice fed DHA or D-DHA for 4 weeks 1 week after iron injection (denoted 4+1 weeks). cSLO and OCT imaging was performed 1 week after IVT iron vs. saline injection. Figures 6D-G show representative BAF cSLO images in mice fed D-DHA or DHA for 1 week, given an IVT injection, and then euthanized 1 week later (1+1 weeks), or fed D-DHA for 2 weeks, given an IVT injection, and then euthanized 1 week later (2+1 weeks), etc. (d and e). Abbreviations used in the figures: SLO, scanning laser ophthalmoscopy; OCT, optical coherence tomography; BAF, blue autofluorescence; IRAF, infrared autofluorescence; ONL, outer nuclear layer. White lines indicate iron-induced high AF spots. White dashed arrows indicate blebs in damaged RPE cells. White lines indicate the position and orientation of the horizontal OCT b scan in panel e, and white asterisks indicate the vortex vein that was used as a landmark for the corresponding position of the OCT scan in the IRAF SLO images. In c, N = 3 mice / group; in b, and d-f, N = 10 mice / group. Error bars indicate mean ± SEM. (**P<0.01). [Figure 6E]Charts and images demonstrating D-DHA protection against iron-induced retinal autofluorescence and degeneration are shown. Mice were fed D-DHA for 77 days, then switched to DHA for 73 days, for a total of 150 days. Figure 6A shows the % of D-DHA in the neural retina and RPE-choroid. Figure 6B shows a timeline of mice fed either D-DHA or DHA for 1, 2, or 4 weeks, starting at 2 months of age, and then given an intravitreal injection of iron in one eye and control saline in the other. Mice continued on their respective diets until final evaluation. Figure 6C shows retinal AF areas in BAF cSLO images from mice fed DHA or D-DHA for 4 weeks 1 week after iron injection (denoted 4+1 weeks). cSLO and OCT imaging was performed 1 week after IVT iron vs. saline injection. Figures 6D-G show representative BAF cSLO images in mice fed D-DHA or DHA for 1 week, given an IVT injection, and then euthanized 1 week later (1+1 weeks), or fed D-DHA for 2 weeks, given an IVT injection, and then euthanized 1 week later (2+1 weeks), etc. (d and e). Abbreviations used in the figures: SLO, scanning laser ophthalmoscopy; OCT, optical coherence tomography; BAF, blue autofluorescence; IRAF, infrared autofluorescence; ONL, outer nuclear layer. White lines indicate iron-induced high AF spots. White dashed arrows indicate blebs in damaged RPE cells. White lines indicate the position and orientation of the horizontal OCT b scan in panel e, and white asterisks indicate the vortex vein that was used as a landmark for the corresponding position of the OCT scan in the IRAF SLO images. In c, N = 3 mice / group; in b, and d-f, N = 10 mice / group. Error bars indicate mean ± SEM. (**P<0.01). [Figure 6F]Charts and images demonstrating D-DHA protection against iron-induced retinal autofluorescence and degeneration are shown. Mice were fed D-DHA for 77 days, then switched to DHA for 73 days, for a total of 150 days. Figure 6A shows the % of D-DHA in the neural retina and RPE-choroid. Figure 6B shows a timeline of mice fed either D-DHA or DHA for 1, 2, or 4 weeks, starting at 2 months of age, and then given an intravitreal injection of iron in one eye and control saline in the other. Mice continued on their respective diets until final evaluation. Figure 6C shows retinal AF areas in BAF cSLO images from mice fed DHA or D-DHA for 4 weeks 1 week after iron injection (denoted 4+1 weeks). cSLO and OCT imaging was performed 1 week after IVT iron vs. saline injection. Figures 6D-G show representative BAF cSLO images in mice fed D-DHA or DHA for 1 week, given an IVT injection, and then euthanized 1 week later (1+1 weeks), or fed D-DHA for 2 weeks, given an IVT injection, and then euthanized 1 week later (2+1 weeks), etc. (d and e). Abbreviations used in the figures: SLO, scanning laser ophthalmoscopy; OCT, optical coherence tomography; BAF, blue autofluorescence; IRAF, infrared autofluorescence; ONL, outer nuclear layer. White lines indicate iron-induced high AF spots. White dashed arrows indicate blebs in damaged RPE cells. White lines indicate the position and orientation of the horizontal OCT b scan in panel e, and white asterisks indicate the vortex vein that was used as a landmark for the corresponding position of the OCT scan in the IRAF SLO images. In c, N = 3 mice / group; in b, and d-f, N = 10 mice / group. Error bars indicate mean ± SEM. (**P<0.01). [Figure 6G]Charts and images demonstrating D-DHA protection against iron-induced retinal autofluorescence and degeneration are shown. Mice were fed D-DHA for 77 days, then switched to DHA for 73 days, for a total of 150 days. Figure 6A shows the % of D-DHA in the neural retina and RPE-choroid. Figure 6B shows a timeline of mice fed either D-DHA or DHA for 1, 2, or 4 weeks, starting at 2 months of age, and then given an intravitreal injection of iron in one eye and control saline in the other. Mice continued on their respective diets until final evaluation. Figure 6C shows retinal AF areas in BAF cSLO images from mice fed DHA or D-DHA for 4 weeks 1 week after iron injection (denoted 4+1 weeks). cSLO and OCT imaging was performed 1 week after IVT iron vs. saline injection. Figures 6D-G show representative BAF cSLO images in mice fed D-DHA or DHA for 1 week, given an IVT injection, and then euthanized 1 week later (1+1 weeks), or fed D-DHA for 2 weeks, given an IVT injection, and then euthanized 1 week later (2+1 weeks), etc. (d and e). Abbreviations used in the figures: SLO, scanning laser ophthalmoscopy; OCT, optical coherence tomography; BAF, blue autofluorescence; IRAF, infrared autofluorescence; ONL, outer nuclear layer. White lines indicate iron-induced high AF spots. White dashed arrows indicate blebs in damaged RPE cells. White lines indicate the position and orientation of the horizontal OCT b scan in panel e, and white asterisks indicate the vortex vein that was used as a landmark for the corresponding position of the OCT scan in the IRAF SLO images. In c, N = 3 mice / group; in b, and d-f, N = 10 mice / group. Error bars indicate mean ± SEM. (**P<0.01).

[0028] [Figure 7A]D-DHA protected retinal function and structure against iron injection. Figure 7A shows a graph showing the amplitude of electroretinogram after 4 weeks of dietary administration of either D-DHA or DHA. Figure 7B shows the amplitude of electroretinogram repeated 1 week after intravitreal injection of iron or saline. Figures 7C and 7D show images of toluidine blue staining performed on plastic sections prepared 1 week after injection. The enlarged images are from sections from mice fed the DHA diet for 4 weeks, then given IVT iron, and euthanized 1 week later. The black dashed arrows indicate atrophic RPE. The white dashed arrows indicate blebs in damaged RPE cells. The black solid arrows indicate infiltrating bone marrow cells. A two-sample t-test was performed to compare the total and outer retinal thickness between the DHA-Fe and D-DHA-Fe groups, respectively, at different locations. Figures 7E and 7F show spider graphs of the average thickness of each retinal layer. Error bars represent the mean ± SEM of total and outer retinal thickness (ONL to RPE) in the ventral (lower)-dorsal (upper) axis at the positions indicated on the x-axis. All statistical comparisons were performed using SAS v9.4 (SAS Institute Inc., Cary, NC). Due to the exploratory nature of this small study, no correction for multiple comparisons was performed. Error bars represent the mean ± SEM. *p<0.05. Scale bar=50 μM. For electroretinography, N=8-10 / group. For retinal thickness measurements, N=3 / group. [Figure 7B]D-DHA protected retinal function and structure against iron injection. Figure 7A shows a graph showing the amplitude of electroretinogram after 4 weeks of dietary administration of either D-DHA or DHA. Figure 7B shows the amplitude of electroretinogram repeated 1 week after intravitreal injection of iron or saline. Figures 7C and 7D show images of toluidine blue staining performed on plastic sections prepared 1 week after injection. The enlarged images are from sections from mice fed the DHA diet for 4 weeks, then given IVT iron, and euthanized 1 week later. The black dashed arrows indicate atrophic RPE. The white dashed arrows indicate blebs in damaged RPE cells. The black solid arrows indicate infiltrating bone marrow cells. A two-sample t-test was performed to compare the total and outer retinal thickness between the DHA-Fe and D-DHA-Fe groups, respectively, at different locations. Figures 7E and 7F show spider graphs of the average thickness of each retinal layer. Error bars represent the mean ± SEM of total and outer retinal thickness (ONL to RPE) in the ventral (lower)-dorsal (upper) axis at the positions indicated on the x-axis. All statistical comparisons were performed using SAS v9.4 (SAS Institute Inc., Cary, NC). Due to the exploratory nature of this small study, no correction for multiple comparisons was performed. Error bars represent the mean ± SEM. *p<0.05. Scale bar=50 μM. For electroretinography, N=8-10 / group. For retinal thickness measurements, N=3 / group. [Figure 7C]D-DHA protected retinal function and structure against iron injection. Figure 7A shows a graph showing the amplitude of electroretinogram after 4 weeks of dietary administration of either D-DHA or DHA. Figure 7B shows the amplitude of electroretinogram repeated 1 week after intravitreal injection of iron or saline. Figures 7C and 7D show images of toluidine blue staining performed on plastic sections prepared 1 week after injection. The enlarged images are from sections from mice fed the DHA diet for 4 weeks, then given IVT iron, and euthanized 1 week later. The black dashed arrows indicate atrophic RPE. The white dashed arrows indicate blebs in damaged RPE cells. The black solid arrows indicate infiltrating bone marrow cells. A two-sample t-test was performed to compare the total and outer retinal thickness between the DHA-Fe and D-DHA-Fe groups, respectively, at different locations. Figures 7E and 7F show spider graphs of the average thickness of each retinal layer. Error bars represent the mean ± SEM of total and outer retinal thickness (ONL to RPE) in the ventral (lower)-dorsal (upper) axis at the positions indicated on the x-axis. All statistical comparisons were performed using SAS v9.4 (SAS Institute Inc., Cary, NC). Due to the exploratory nature of this small study, no correction for multiple comparisons was performed. Error bars represent the mean ± SEM. *p<0.05. Scale bar=50 μM. For electroretinography, N=8-10 / group. For retinal thickness measurements, N=3 / group. [Figure 7D]D-DHA protected retinal function and structure against iron injection. Figure 7A shows a graph showing the amplitude of electroretinogram after 4 weeks of dietary administration of either D-DHA or DHA. Figure 7B shows the amplitude of electroretinogram repeated 1 week after intravitreal injection of iron or saline. Figures 7C and 7D show images of toluidine blue staining performed on plastic sections prepared 1 week after injection. The enlarged images are from sections from mice fed the DHA diet for 4 weeks, then given IVT iron, and euthanized 1 week later. The black dashed arrows indicate atrophic RPE. The white dashed arrows indicate blebs in damaged RPE cells. The black solid arrows indicate infiltrating bone marrow cells. A two-sample t-test was performed to compare the total and outer retinal thickness between the DHA-Fe and D-DHA-Fe groups, respectively, at different locations. Figures 7E and 7F show spider graphs of the average thickness of each retinal layer. Error bars represent the mean ± SEM of total and outer retinal thickness (ONL to RPE) in the ventral (lower)-dorsal (upper) axis at the positions indicated on the x-axis. All statistical comparisons were performed using SAS v9.4 (SAS Institute Inc., Cary, NC). Due to the exploratory nature of this small study, no correction for multiple comparisons was performed. Error bars represent the mean ± SEM. *p<0.05. Scale bar=50 μM. For electroretinography, N=8-10 / group. For retinal thickness measurements, N=3 / group. [Figure 7E]D-DHA protected retinal function and structure against iron injection. Figure 7A shows a graph showing the amplitude of electroretinogram after 4 weeks of dietary administration of either D-DHA or DHA. Figure 7B shows the amplitude of electroretinogram repeated 1 week after intravitreal injection of iron or saline. Figures 7C and 7D show images of toluidine blue staining performed on plastic sections prepared 1 week after injection. The enlarged images are from sections from mice fed the DHA diet for 4 weeks, then given IVT iron, and euthanized 1 week later. The black dashed arrows indicate atrophic RPE. The white dashed arrows indicate blebs in damaged RPE cells. The black solid arrows indicate infiltrating bone marrow cells. A two-sample t-test was performed to compare the total and outer retinal thickness between the DHA-Fe and D-DHA-Fe groups, respectively, at different locations. Figures 7E and 7F show spider graphs of the average thickness of each retinal layer. Error bars represent the mean ± SEM of total and outer retinal thickness (ONL to RPE) in the ventral (lower)-dorsal (upper) axis at the positions indicated on the x-axis. All statistical comparisons were performed using SAS v9.4 (SAS Institute Inc., Cary, NC). Due to the exploratory nature of this small study, no correction for multiple comparisons was performed. Error bars represent the mean ± SEM. *p<0.05. Scale bar=50 μM. For electroretinography, N=8-10 / group. For retinal thickness measurements, N=3 / group. [Figure 7F]D-DHA protected retinal function and structure against iron injection. Figure 7A shows a graph showing the amplitude of electroretinogram after 4 weeks of dietary administration of either D-DHA or DHA. Figure 7B shows the amplitude of electroretinogram repeated 1 week after intravitreal injection of iron or saline. Figures 7C and 7D show images of toluidine blue staining performed on plastic sections prepared 1 week after injection. The enlarged images are from sections from mice fed the DHA diet for 4 weeks, then given IVT iron, and euthanized 1 week later. The black dashed arrows indicate atrophic RPE. The white dashed arrows indicate blebs in damaged RPE cells. The black solid arrows indicate infiltrating bone marrow cells. A two-sample t-test was performed to compare the total and outer retinal thickness between the DHA-Fe and D-DHA-Fe groups, respectively, at different locations. Figures 7E and 7F show spider graphs of the average thickness of each retinal layer. Error bars represent the mean ± SEM of total and outer retinal thickness (ONL to RPE) in the ventral (lower)-dorsal (upper) axis at the positions indicated on the x-axis. All statistical comparisons were performed using SAS v9.4 (SAS Institute Inc., Cary, NC). Due to the exploratory nature of this small study, no correction for multiple comparisons was performed. Error bars represent the mean ± SEM. *p<0.05. Scale bar=50 μM. For electroretinography, N=8-10 / group. For retinal thickness measurements, N=3 / group.

[0029] [Figure 8A]Figure 8A shows that D-DHA prevented the formation of CEP, an immunogenic protein adduct uniquely derived from DHA oxidation. Figure 8A shows epifluorescence micrographs of co-labeling for carboxyethylpyrrole (CEP-red) and rhodopsin (green) on frozen sections from mice fed D-DHA or DHA for 4 weeks 4 hours after intravitreal injection of iron or saline. Figure 8B shows immunolabeling for CEP 1 week after injection. Figure 8C shows a magnified image of co-labeling for CEP and rhodopsin corresponding to Figure 8B. Figure 8D shows a magnified image of immunolabeling for CEP corresponding to Figure 8B. Figure 8E shows immunolabeling for L-Ft 1 week after injection. Figures 8F and 8G show charts of quantification of pixel density of immunolabeling for CEP and L-Ft. White arrows indicate immunolabeling for CEP. Abbreviations used in the figures: CEP, carboxyethylpyrrole; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Representative images from N=4 mice / group are shown. Scale bar: 50 μm. Error bars indicate mean ± SEM. **P<0.01, ****P<0.0001). [Figure 8B] Figure 8A shows that D-DHA prevented the formation of CEP, an immunogenic protein adduct uniquely derived from DHA oxidation. Figure 8A shows epifluorescence micrographs of co-labeling for carboxyethylpyrrole (CEP-red) and rhodopsin (green) on frozen sections from mice fed D-DHA or DHA for 4 weeks 4 hours after intravitreal injection of iron or saline. Figure 8B shows immunolabeling for CEP 1 week after injection. Figure 8C shows a magnified image of co-labeling for CEP and rhodopsin corresponding to Figure 8B. Figure 8D shows a magnified image of immunolabeling for CEP corresponding to Figure 8B. Figure 8E shows immunolabeling for L-Ft 1 week after injection. Figures 8F and 8G show charts of quantification of pixel density of immunolabeling for CEP and L-Ft. White arrows indicate immunolabeling for CEP. Abbreviations used in the figures: CEP, carboxyethylpyrrole; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Representative images from N=4 mice / group are shown. Scale bar: 50 μm. Error bars indicate mean ± SEM. **P<0.01, ****P<0.0001). [Figure 8C] Figure 8A shows that D-DHA prevented the formation of CEP, an immunogenic protein adduct uniquely derived from DHA oxidation. Figure 8A shows epifluorescence micrographs of co-labeling for carboxyethylpyrrole (CEP-red) and rhodopsin (green) on frozen sections from mice fed D-DHA or DHA for 4 weeks 4 hours after intravitreal injection of iron or saline. Figure 8B shows immunolabeling for CEP 1 week after injection. Figure 8C shows a magnified image of co-labeling for CEP and rhodopsin corresponding to Figure 8B. Figure 8D shows a magnified image of immunolabeling for CEP corresponding to Figure 8B. Figure 8E shows immunolabeling for L-Ft 1 week after injection. Figures 8F and 8G show charts of quantification of pixel density of immunolabeling for CEP and L-Ft. White arrows indicate immunolabeling for CEP. Abbreviations used in the figures: CEP, carboxyethylpyrrole; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Representative images from N=4 mice / group are shown. Scale bar: 50 μm. Error bars indicate mean ± SEM. **P<0.01, ****P<0.0001). [Figure 8D]Figure 8A shows that D-DHA prevented the formation of CEP, an immunogenic protein adduct uniquely derived from DHA oxidation. Figure 8A shows epifluorescence micrographs of co-labeling for carboxyethylpyrrole (CEP-red) and rhodopsin (green) on frozen sections from mice fed D-DHA or DHA for 4 weeks 4 hours after intravitreal injection of iron or saline. Figure 8B shows immunolabeling for CEP 1 week after injection. Figure 8C shows a magnified image of co-labeling for CEP and rhodopsin corresponding to Figure 8B. Figure 8D shows a magnified image of immunolabeling for CEP corresponding to Figure 8B. Figure 8E shows immunolabeling for L-Ft 1 week after injection. Figures 8F and 8G show charts of quantification of pixel density of immunolabeling for CEP and L-Ft. White arrows indicate immunolabeling for CEP. Abbreviations used in the figures: CEP, carboxyethylpyrrole; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Representative images from N=4 mice / group are shown. Scale bar: 50 μm. Error bars indicate mean ± SEM. **P<0.01, ****P<0.0001). [Figure 8E] Figure 8A shows that D-DHA prevented the formation of CEP, an immunogenic protein adduct uniquely derived from DHA oxidation. Figure 8A shows epifluorescence micrographs of co-labeling for carboxyethylpyrrole (CEP-red) and rhodopsin (green) on frozen sections from mice fed D-DHA or DHA for 4 weeks 4 hours after intravitreal injection of iron or saline. Figure 8B shows immunolabeling for CEP 1 week after injection. Figure 8C shows a magnified image of co-labeling for CEP and rhodopsin corresponding to Figure 8B. Figure 8D shows a magnified image of immunolabeling for CEP corresponding to Figure 8B. Figure 8E shows immunolabeling for L-Ft 1 week after injection. Figures 8F and 8G show charts of quantification of pixel density of immunolabeling for CEP and L-Ft. White arrows indicate immunolabeling for CEP. Abbreviations used in the figures: CEP, carboxyethylpyrrole; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Representative images from N=4 mice / group are shown. Scale bar: 50 μm. Error bars indicate mean ± SEM. **P<0.01, ****P<0.0001). [Figure 8F] Figure 8A shows that D-DHA prevented the formation of CEP, an immunogenic protein adduct uniquely derived from DHA oxidation. Figure 8A shows epifluorescence micrographs of co-labeling for carboxyethylpyrrole (CEP-red) and rhodopsin (green) on frozen sections from mice fed D-DHA or DHA for 4 weeks 4 hours after intravitreal injection of iron or saline. Figure 8B shows immunolabeling for CEP 1 week after injection. Figure 8C shows a magnified image of co-labeling for CEP and rhodopsin corresponding to Figure 8B. Figure 8D shows a magnified image of immunolabeling for CEP corresponding to Figure 8B. Figure 8E shows immunolabeling for L-Ft 1 week after injection. Figures 8F and 8G show charts of quantification of pixel density of immunolabeling for CEP and L-Ft. White arrows indicate immunolabeling for CEP. Abbreviations used in the figures: CEP, carboxyethylpyrrole; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Representative images from N=4 mice / group are shown. Scale bar: 50 μm. Error bars indicate mean ± SEM. **P<0.01, ****P<0.0001). [Figure 8G]Figure 8A shows that D-DHA prevented the formation of CEP, an immunogenic protein adduct uniquely derived from DHA oxidation. Figure 8A shows epifluorescence micrographs of co-labeling for carboxyethylpyrrole (CEP-red) and rhodopsin (green) on frozen sections from mice fed D-DHA or DHA for 4 weeks 4 hours after intravitreal injection of iron or saline. Figure 8B shows immunolabeling for CEP 1 week after injection. Figure 8C shows a magnified image of co-labeling for CEP and rhodopsin corresponding to Figure 8B. Figure 8D shows a magnified image of immunolabeling for CEP corresponding to Figure 8B. Figure 8E shows immunolabeling for L-Ft 1 week after injection. Figures 8F and 8G show charts of quantification of pixel density of immunolabeling for CEP and L-Ft. White arrows indicate immunolabeling for CEP. Abbreviations used in the figures: CEP, carboxyethylpyrrole; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Representative images from N=4 mice / group are shown. Scale bar: 50 μm. Error bars indicate mean ± SEM. **P<0.01, ****P<0.0001).

[0030] [Figure 9] Figure 1 shows qPCR demonstrating that D-DHA protected against iron-induced oxidative stress, inflammation, and retinal cell death. Relative mRNA levels in the neural retina of the indicated genes from mice fed D-DHA or DHA for 4 weeks 1 week after iron or saline injection. Error bars indicate mean ± SEM. N=3-4 mice / group (*P<0.05, **P<0.01, ****P<0.0001, ****P<0.0001).

[0031] [Figure 10A]Figure 10 shows that D-DHA prevented iron-induced acute RPE atrophy and the development of progressive geographic atrophy. Mice were fed D-DHA or DHA for 4 weeks, after which one eye was intravitreally injected with iron and the other eye with control saline, and cSLO and OCT images were acquired 4 weeks after iron or saline injection. Figures 10A-C show representative BAF cSLO images (a), IRAF cSLO images (b), and OCT scans (c). White lines indicate the location of the horizontal OCT scan. Black arrows indicate high and low AF lesions in the IRAF cSLO images, corresponding to atrophic RPE in the OCT scan. White arrows indicate ONL thinning in the OCT scan. Figures 10D-E show toluidine blue staining performed on plastic sections prepared 4 weeks after injection. Black arrows indicate atrophic RPE and blebs within the RPE. White arrows indicate hypertrophic RPE. Representative images from N=4 mice / group are shown. Scale bar: 50 μm. [Figure 10B] Figure 10 shows that D-DHA prevented iron-induced acute RPE atrophy and the development of progressive geographic atrophy. Mice were fed D-DHA or DHA for 4 weeks, after which one eye was intravitreally injected with iron and the other eye with control saline, and cSLO and OCT images were acquired 4 weeks after iron or saline injection. Figures 10A-C show representative BAF cSLO images (a), IRAF cSLO images (b), and OCT scans (c). White lines indicate the location of the horizontal OCT scan. Black arrows indicate high and low AF lesions in the IRAF cSLO images, corresponding to atrophic RPE in the OCT scan. White arrows indicate ONL thinning in the OCT scan. Figures 10D-E show toluidine blue staining performed on plastic sections prepared 4 weeks after injection. Black arrows indicate atrophic RPE and blebs within the RPE. White arrows indicate hypertrophic RPE. Representative images from N=4 mice / group are shown. Scale bar: 50 μm. [Figure 10C]Figure 10 shows that D-DHA prevented iron-induced acute RPE atrophy and the development of progressive geographic atrophy. Mice were fed D-DHA or DHA for 4 weeks, after which one eye was intravitreally injected with iron and the other eye with control saline, and cSLO and OCT images were acquired 4 weeks after iron or saline injection. Figures 10A-C show representative BAF cSLO images (a), IRAF cSLO images (b), and OCT scans (c). White lines indicate the location of the horizontal OCT scan. Black arrows indicate high and low AF lesions in the IRAF cSLO images, corresponding to atrophic RPE in the OCT scan. White arrows indicate ONL thinning in the OCT scan. Figures 10D-E show toluidine blue staining performed on plastic sections prepared 4 weeks after injection. Black arrows indicate atrophic RPE and blebs within the RPE. White arrows indicate hypertrophic RPE. Representative images from N=4 mice / group are shown. Scale bar: 50 μm. [Figure 10D] Figure 10 shows that D-DHA prevented iron-induced acute RPE atrophy and the development of progressive geographic atrophy. Mice were fed D-DHA or DHA for 4 weeks, after which one eye was intravitreally injected with iron and the other eye with control saline, and cSLO and OCT images were acquired 4 weeks after iron or saline injection. Figures 10A-C show representative BAF cSLO images (a), IRAF cSLO images (b), and OCT scans (c). White lines indicate the location of the horizontal OCT scan. Black arrows indicate high and low AF lesions in the IRAF cSLO images, corresponding to atrophic RPE in the OCT scan. White arrows indicate ONL thinning in the OCT scan. Figures 10D-E show toluidine blue staining performed on plastic sections prepared 4 weeks after injection. Black arrows indicate atrophic RPE and blebs within the RPE. White arrows indicate hypertrophic RPE. Representative images from N=4 mice / group are shown. Scale bar: 50 μm. [Figure 10E]Figure 10 shows that D-DHA prevented iron-induced acute RPE atrophy and the development of progressive geographic atrophy. Mice were fed D-DHA or DHA for 4 weeks, after which one eye was intravitreally injected with iron and the other eye with control saline, and cSLO and OCT images were acquired 4 weeks after iron or saline injection. Figures 10A-C show representative BAF cSLO images (a), IRAF cSLO images (b), and OCT scans (c). White lines indicate the location of the horizontal OCT scan. Black arrows indicate high and low AF lesions in the IRAF cSLO images, corresponding to atrophic RPE in the OCT scan. White arrows indicate ONL thinning in the OCT scan. Figures 10D-E show toluidine blue staining performed on plastic sections prepared 4 weeks after injection. Black arrows indicate atrophic RPE and blebs within the RPE. White arrows indicate hypertrophic RPE. Representative images from N=4 mice / group are shown. Scale bar: 50 μm.

[0032] [Figure 11A]LC / MS analysis of lipids extracted from D-DHA-containing diets. The free fatty acid mixture obtained from sample extraction and saponification was dissolved in ethanol and injected in a 5 μl volume onto an Agilent XDB-C18 liquid chromatography column (1 mm × 150 mm) through which the running solvent was pumped at 100 μl / min. Solvent A was 70% CH3CN (v / v) and 0.1% formic acid (m / v). Solvent B was 0.1% formic acid (m / v) in pure CH3CN. The initial composition of the running solvent was 70% B for 5 min and increased to 100% B from 5 to 30 min. DHA eluted at 24.7 min. The column eluate was alkalized with 150 mM NH4OH and then analyzed by ESI-MS on a 4000 QTrap (Sciex) operating in enhanced negative mode over an m / z range of 320 to 345 and a scan speed of 250 / s. These procedures confirmed that the experimental rodent diet contained DHA but no detectable D-DHA, whereas the experimental D-DHA-supplemented diet contained only trace amounts of regular DHA (a). 13C correction applied to the DHA signal demonstrated that the peak at m / z 327.2 represented 78.4% of DHA and 13C-containing isotopologues. Peaks corresponding to DHA with 8, 9, 10, 11, 12, and 13 deuterium substitutions were readily identified in the experimental diets, as well as in the neural retina and RPE samples. The relative distribution of DHA isotopologues in the neural retina and RPE samples was indistinguishable from that in the experimental D-DHA-supplemented diet. After applying 13C correction to the integrated peaks, it was determined that the area of ​​the peak centered at 337.2 (corresponding to D10-DHA) constituted 45.6% of the area of ​​all deuterium-containing DHA isotopologue peaks. Fatty acids extracted from neural retina and RPE eluted as three peaks (b). The TIC shown was obtained from an enhanced negative mode scan of m / z 320-345 at 250 m / z / min. Mass spectrum of the three labeled peaks (c). Peak 1 shows DHA at 327.2, a C-containing isotopologue at 328.2, and isotopologues containing 8, 9, 10, 11, and 12 deuterium substitutions at corresponding m / z values. The relative peak areas were indistinguishable from those observed in the experimental diets.Peak 2 shows some DHA (tail from peak 1), a 329.2 / 330.2 peak indicative of DPA, and a set of peaks suggesting they represent D8-DPA, D9-DPA, D10-DPA, D11-DPA, and D12-DPA. As these species were not present in the feed, they likely represent D-DHA species reduced to D-DPA species. The relative peak areas of D8-DPA and D9-DPA are slightly larger than those of D8-DHA and D9-DHA, which may reflect a greater possibility of reduction to the corresponding DPA species when the degree of deuterium substitution is less. Peak 3, eluting at 27.9 min, most likely represents the n-6 series of docosatetraenoic acid (DTA), with no deuterium-substituted isotopologues observed. [Figure 11B]LC / MS analysis of lipids extracted from D-DHA-containing diets. The free fatty acid mixture obtained from sample extraction and saponification was dissolved in ethanol and injected in a 5 μl volume onto an Agilent XDB-C18 liquid chromatography column (1 mm × 150 mm) through which the running solvent was pumped at 100 μl / min. Solvent A was 70% CH3CN (v / v) and 0.1% formic acid (m / v). Solvent B was 0.1% formic acid (m / v) in pure CH3CN. The initial composition of the running solvent was 70% B for 5 min and increased to 100% B from 5 to 30 min. DHA eluted at 24.7 min. The column eluate was alkalized with 150 mM NH4OH and then analyzed by ESI-MS on a 4000 QTrap (Sciex) operating in enhanced negative mode over an m / z range of 320 to 345 and a scan speed of 250 / s. These procedures confirmed that the experimental rodent diet contained DHA but no detectable D-DHA, whereas the experimental D-DHA-supplemented diet contained only trace amounts of regular DHA (a). 13C correction applied to the DHA signal demonstrated that the peak at m / z 327.2 represented 78.4% of DHA and 13C-containing isotopologues. Peaks corresponding to DHA with 8, 9, 10, 11, 12, and 13 deuterium substitutions were readily identified in the experimental diets, as well as in the neural retina and RPE samples. The relative distribution of DHA isotopologues in the neural retina and RPE samples was indistinguishable from that in the experimental D-DHA-supplemented diet. After applying 13C correction to the integrated peaks, it was determined that the area of ​​the peak centered at 337.2 (corresponding to D10-DHA) constituted 45.6% of the area of ​​all deuterium-containing DHA isotopologue peaks. Fatty acids extracted from neural retina and RPE eluted as three peaks (b). The TIC shown was obtained from an enhanced negative mode scan of m / z 320-345 at 250 m / z / min. Mass spectrum of the three labeled peaks (c). Peak 1 shows DHA at 327.2, a C-containing isotopologue at 328.2, and isotopologues containing 8, 9, 10, 11, and 12 deuterium substitutions at corresponding m / z values. The relative peak areas were indistinguishable from those observed in the experimental diets.Peak 2 shows some DHA (tail from peak 1), a 329.2 / 330.2 peak indicative of DPA, and a set of peaks suggesting they represent D8-DPA, D9-DPA, D10-DPA, D11-DPA, and D12-DPA. As these species were not present in the feed, they likely represent D-DHA species reduced to D-DPA species. The relative peak areas of D8-DPA and D9-DPA are slightly larger than those of D8-DHA and D9-DHA, which may reflect a greater possibility of reduction to the corresponding DPA species when the degree of deuterium substitution is less. Peak 3, eluting at 27.9 min, most likely represents the n-6 series of docosatetraenoic acid (DTA), with no deuterium-substituted isotopologues observed. [Figure 11C]LC / MS analysis of lipids extracted from D-DHA-containing diets. The free fatty acid mixture obtained from sample extraction and saponification was dissolved in ethanol and injected in a 5 μl volume onto an Agilent XDB-C18 liquid chromatography column (1 mm × 150 mm) through which the running solvent was pumped at 100 μl / min. Solvent A was 70% CH3CN (v / v) and 0.1% formic acid (m / v). Solvent B was 0.1% formic acid (m / v) in pure CH3CN. The initial composition of the running solvent was 70% B for 5 min and increased to 100% B from 5 to 30 min. DHA eluted at 24.7 min. The column eluate was alkalized with 150 mM NH4OH and then analyzed by ESI-MS on a 4000 QTrap (Sciex) operating in enhanced negative mode over an m / z range of 320 to 345 and a scan speed of 250 / s. These procedures confirmed that the experimental rodent diet contained DHA but no detectable D-DHA, whereas the experimental D-DHA-supplemented diet contained only trace amounts of regular DHA (a). 13C correction applied to the DHA signal demonstrated that the peak at m / z 327.2 represented 78.4% of DHA and 13C-containing isotopologues. Peaks corresponding to DHA with 8, 9, 10, 11, 12, and 13 deuterium substitutions were readily identified in the experimental diets, as well as in the neural retina and RPE samples. The relative distribution of DHA isotopologues in the neural retina and RPE samples was indistinguishable from that in the experimental D-DHA-supplemented diet. After applying 13C correction to the integrated peaks, it was determined that the area of ​​the peak centered at 337.2 (corresponding to D10-DHA) constituted 45.6% of the area of ​​all deuterium-containing DHA isotopologue peaks. Fatty acids extracted from neural retina and RPE eluted as three peaks (b). The TIC shown was obtained from an enhanced negative mode scan of m / z 320-345 at 250 m / z / min. Mass spectrum of the three labeled peaks (c). Peak 1 shows DHA at 327.2, a C-containing isotopologue at 328.2, and isotopologues containing 8, 9, 10, 11, and 12 deuterium substitutions at corresponding m / z values. The relative peak areas were indistinguishable from those observed in the experimental diets.Peak 2 shows some DHA (tail from peak 1), a 329.2 / 330.2 peak indicative of DPA, and a set of peaks suggesting they represent D8-DPA, D9-DPA, D10-DPA, D11-DPA, and D12-DPA. As these species were not present in the feed, they likely represent D-DHA species reduced to D-DPA species. The relative peak areas of D8-DPA and D9-DPA are slightly larger than those of D8-DHA and D9-DHA, which may reflect a greater possibility of reduction to the corresponding DPA species when the degree of deuterium substitution is less. Peak 3, eluting at 27.9 min, most likely represents the n-6 series of docosatetraenoic acid (DTA), with no deuterium-substituted isotopologues observed.

[0033] [Figure 12A] Figure 1 shows that D-DHA showed a dose-dependent protective effect against iron-induced retinal AF. Starting at 2 months of age, mice were fed D-DHA or DHA for 1, 2, 3, and 4 weeks, after which one eye was intravitreal injected with iron and the other received control saline. One week after iron vs saline injection, BAF cSLO images were acquired from multiple mice fed D-DHA or DHA for 1 week (1+1wk) (a), 2 weeks (2+1wk) (b), 3 weeks (3+1wk) (c), and 4 weeks (4+1wk) (d) prior to injection. BAF cSLO pairs of images from the same mouse are shown in the same row, and images from different mice receiving the same treatment (iron or saline) are shown in the same column. [Figure 12B]Figure 1 shows that D-DHA showed a dose-dependent protective effect against iron-induced retinal AF. Starting at 2 months of age, mice were fed D-DHA or DHA for 1, 2, 3, and 4 weeks, after which one eye was intravitreal injected with iron and the other received control saline. One week after iron vs saline injection, BAF cSLO images were acquired from multiple mice fed D-DHA or DHA for 1 week (1+1wk) (a), 2 weeks (2+1wk) (b), 3 weeks (3+1wk) (c), and 4 weeks (4+1wk) (d) prior to injection. BAF cSLO pairs of images from the same mouse are shown in the same row, and images from different mice receiving the same treatment (iron or saline) are shown in the same column. [Figure 12C] Figure 1 shows that D-DHA showed a dose-dependent protective effect against iron-induced retinal AF. Starting at 2 months of age, mice were fed D-DHA or DHA for 1, 2, 3, and 4 weeks, after which one eye was intravitreal injected with iron and the other received control saline. One week after iron vs saline injection, BAF cSLO images were acquired from multiple mice fed D-DHA or DHA for 1 week (1+1wk) (a), 2 weeks (2+1wk) (b), 3 weeks (3+1wk) (c), and 4 weeks (4+1wk) (d) prior to injection. BAF cSLO pairs of images from the same mouse are shown in the same row, and images from different mice receiving the same treatment (iron or saline) are shown in the same column. [Figure 12D] Figure 1 shows that D-DHA showed a dose-dependent protective effect against iron-induced retinal AF. Starting at 2 months of age, mice were fed D-DHA or DHA for 1, 2, 3, and 4 weeks, after which one eye was intravitreal injected with iron and the other received control saline. One week after iron vs saline injection, BAF cSLO images were acquired from multiple mice fed D-DHA or DHA for 1 week (1+1wk) (a), 2 weeks (2+1wk) (b), 3 weeks (3+1wk) (c), and 4 weeks (4+1wk) (d) prior to injection. BAF cSLO pairs of images from the same mouse are shown in the same row, and images from different mice receiving the same treatment (iron or saline) are shown in the same column.

[0034] [Figure 13A] Figure 1 shows the long-term protective effect of D-DHA against the development of chronic geographic atrophy. Mice were fed D-DHA or DHA for 4 weeks, after which one eye was intravitreally injected with iron and the other eye with control saline. BAF cSLO images (a) and OCT scans (b) were acquired 4 weeks after injection. BAF / IRAF image pairs of images from the same mouse are shown in the same row, and images from different mice receiving the same treatment (iron or 781 saline) are shown in the same column. [Figure 13B] Figure 1 shows the long-term protective effect of D-DHA against the development of chronic geographic atrophy. Mice were fed D-DHA or DHA for 4 weeks, after which one eye was intravitreally injected with iron and the other eye with control saline. BAF cSLO images (a) and OCT scans (b) were acquired 4 weeks after injection. BAF / IRAF image pairs of images from the same mouse are shown in the same row, and images from different mice receiving the same treatment (iron or 781 saline) are shown in the same column.

[0035] [Figure 14] Dose response of D-DHA protection against iron-induced retinal damage. Representative BAF cSLO images (a) and OCT images (b) from animals fed the control DHA diet (left) and the D-DHA diet over an increasing period of time prior to intravitreal injection of FAC. Images were acquired 1 week after iron injection. Retinal D-DHA levels at 4+1 weeks were measured (see Table 1), and levels at other time points were extrapolated assuming first-order uptake kinetics as shown in Figure 2b. The appearance of autofluorescent spots served as a quantitative measure of damage or protection by D-DHA (a). OCT scans showed retinal thinning and destruction of the photoreceptor+RPE layer with absent or low levels of retinal D-DHA, with increasing preservation at higher D-DHA concentrations (b). Protective effect refers to the reduction (%) of AF area quantified by ImageJ software, N=3–5 / group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] A method for monitoring a patient's response to treatment of a retinal disease mediated at least in part by lipid peroxidation is disclosed.

[0037] A method for monitoring D-DHA uptake in patients being treated for oxidative retinal disease is disclosed. Before describing the invention in more detail, the following terms are defined. Terms not defined are given their definition in the context or are given their medically accepted definition.

[0038] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0039] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances when the event or circumstance occurs or does not occur.

[0040] As used herein, the term "about," when used before numerical designations, such as temperature, time, amounts, concentrations, and such others, including ranges, indicates an approximation that may vary by (+) or (-) 15%, 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, the term "about," when used in reference to a dose, means that the dose may vary by + / - 10%.

[0041] As used herein, the terms "comprising" or "comprises" are intended to mean that the compositions and methods include the recited elements, but do not exclude others.

[0042] As used herein, the term "consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements of any essential importance to the combination for the described purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0043] As used herein, the term "consisting of" is intended to mean excluding more than trace amounts of other ingredients and other substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

[0044] As used herein, unless the context requires otherwise, the term "ester thereof" means any of C1-C 10 These include alkyl esters, glycerol esters (as defined herein and including monoglycerides, diglycerides and triglycerides), sucrose esters, phosphate esters, and the like. The particular ester group used is not critical so long as the ester is pharma- ceutically acceptable (non-toxic and biocompatible). In one embodiment, the ester is a C1-C6 alkyl ester, preferably an ethyl ester.

[0045] As used herein, the term "deuterated DHA," "D-DHA," or "deuterated docosahexaenoic acid or ester thereof" refers to docosahexaenoic acid or an ester thereof having deuteration as described below. Prior to describing the deuteration, the structure of docosahexaenoic acid and specific sites therein are provided below in Formula A. [ka]

[0046] Deuteration is described as an average based on a population of such DHA compounds containing about 92 to about 96 percent total deuteration at the bis-allylic sites, where the total deuteration is presented as follows: a) about 87 percent to about 92 percent of the CD2 moieties at bis-allylic sites; b) greater than about 6 percent to about 12 percent CHD moieties in bis-allylic sites; and c) about 2 percent or less of CH moieties in bis-allylic sites; However, the total number of hydrogens and deuteriums at the bis-allylic positions is equal to 10.

[0047] In one embodiment, the population of deuterated DHA has about 93 to about 96 percent total deuteration at the bis-allylic sites, where the total deuteration is represented as follows: a) about 87 to about 92 percent of the CD2 moiety at the bis-allylic site; b) about 6.5 to about 12 percent CHD moieties at bis-allylic sites; and c) about 1.5 percent or less of CH moieties in bis-allylic sites; However, the total number of deuterium and hydrogen atoms at the bis-allylic positions is equal to 10.

[0048] In one embodiment, the population of deuterated DHA is characterized as having about 92 to about 95 percent total deuteration at bis-allylic sites, where the total deuteration is represented as follows: a) about 88-92 percent of the CD2 moiety in bis-allylic sites; b) about 6.5 to 12 percent CHD moieties at bis-allylic sites; c) about 1.5 percent or less of CH2 moieties in bis-allylic sites; and d) total deuteration at both mono- and allylic sites of less than 25% in total on average; However, the total number of hydrogens and deuteriums at the bis-allylic positions is equal to 10.

[0049] The degree of deuteration at the two mono-allylic sites differs due to steric hindrance imparted by the carboxyl or carboxyl ester. In one embodiment, the degree of deuteration at the proximal mono-allylic site is about 0.5% to about 5%. In another embodiment, the degree of deuteration at the proximal mono-allylic site is about 1% to about 5%. In other words, on average, only about 0.5% to about 5% or 1% to about 5% of the hydrogen atoms found at the proximal mono-allylic site of a composition comprising a population of deuterated DHA are replaced by deuterium.

[0050] In one embodiment, the level of deuteration at the distal mono-allylic site is about 10% to about 20%. In another embodiment, the level of deuteration at the distal mono-allylic site is about 12% to about 18%. Stated another way, on average, only about 10% to about 20% or 12% to about 18% of the hydrogen atoms found at the distal mono-allylic site of a composition comprising multiple deuterated DHA are replaced by deuterium.

[0051] In one embodiment, the deuterated docosahexaenoic acid or ester thereof comprises a population of compounds of formula I: [ka] In the formula, R is hydrogen or C1-C 10 is alkyl; each X is independently hydrogen or deuterium, and the sum of the amounts of deuterium defined by both X groups is such that, on average, the total amount of deuteration on carbon atoms is less than about 5%; each X 1 are independently hydrogen or deuterium, and both X 1 The total amount of deuterium, defined by 1 an amount such that less than about 25% of the groups are deuterium, the remainder being hydrogen; Each Y is independently hydrogen or deuterium, and a particular value of each Y is, on average: a) about 87 to about 92 percent of the Y groups on each carbon atom are deuterium; b) greater than about 5 percent to about 12 percent of the Y groups on each carbon atom are substituted with a single hydrogen and a single deuterium; and c) less than about 2 percent of the carbon atoms of the Y groups are replaced with two hydrogen atoms; However, the sum of all Y groups equals 10.

[0052] In one embodiment, compositions comprising a compound of formula I have less than 1.5 percent of the carbon atoms in the bis-allylic moiety replaced with two hydrogen atoms.

[0053] In one embodiment, the compositions described herein do not replace hydrogen with deuterium at any site other than the mono-allylic and bis-allylic sites, and therefore the level of deuterium found at the remaining sites of DHA is its natural abundance.

[0054] In one embodiment, the deuterated DHA is provided in a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and an effective amount of the deuterated DHA described herein.

[0055] The population is useful in treating retinal diseases mediated at least in part by lipid peroxidation of DHA found in the outer rods and cones of the retina. Such methods include administering a composition comprising the population of deuterated DHA described herein to a patient in need thereof. In one embodiment, the composition comprising the population of deuterated DHA is administered in a pharma- ceutically acceptable formulation.

[0056] When describing ex vivo populations, the term "D-DHA or drug" refers to deuterated docosahexaenoic acid or its esters: When describing in vivo populations, the esters are hydrolyzed in the gastrointestinal tract, and in the retinal environment, docosahexaenoic acid is incorporated into glycerol esters such as cardiolipin, plasmalogens, and phospholipids, including those of formula II. [ka] (In the formula, R 1 is a fatty acid residue or a docosahexaenoic acid residue, R2 is the docosahexaenoic acid residue, and R 3 (R is choline, ethanolamine, serine, inositol, or hydrogen, monovalent or divalent salts.) Unlike fatty acids found elsewhere in the body, the retina contains 1 and R 2 and R can contain a residue of deuterated docosahexaenoic acid. 1 is selected from the residue of a saturated fatty acid or the residue of docosahexaenoic acid, R 2 is a residue of docosahexaenoic acid. With respect to the terms "residue of a fatty acid" or "residue of docosahexaenoic acid," each of these refers to an ester bond formed between a carboxyl group and a hydroxyl group of glycerol combined with the elimination of water.

[0057] In one embodiment, deuteration at other sites on the docosahexaenoic acid or esters thereof is unaffected, and thus the level of deuteration at sites other than the bis-allylic and mono-allylic sites is at natural abundance.

[0058] The term "naturally occurring docosahexaenoic acid" refers to any and all sources of DHA, where the deuterium abundance is based on its natural abundance.

[0059] As used herein, the term "phospholipid" refers to any and all phospholipids that are components of cell membranes. This term includes phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. In motor neurons, cell membranes are rich in phospholipids that contain arachidonic acid.

[0060] The term "bis-allylic moiety" refers to a methylene group (CH2) separating two double bonds.

[0061] The term "mono-allylic moiety" refers to a methylene group having an adjacent double bond on one side and an additional methylene group on the other side.

[0062] The term "retinal disease" refers to any and all retinal diseases that are mediated at least in part by reactive oxygen species (ROS), including, by way of example only, wet or dry age-related macular degeneration (AMD), retinitis pigmentosa (RP), Stargardt's disease (SD), diabetic retinopathy (DR), cataracts, and the like.

[0063] The term "oxidized PUFA products" refers to any oxidized form of polyunsaturated fatty acids, as well as any and all metabolic products formed from oxidized PUFAs, including reactive aldehydes, ketones, alcohols, and carboxyl derivatives that are toxic to cells when found in phospholipids, lipid bilayers, or as enzyme substrates.

[0064] As used herein, the term "disease pathology" refers to the causes, development, structural / functional changes, and natural history associated with the disease. The disease pathology includes the loss of cellular function.

[0065] The term "therapeutic concentration" refers to a concentration of deuterated DHA that reduces the rate of oxidative retinal disease. Such a concentration is based on replacing at least about 20%, preferably at least about 50%, preferably at least about 60%, more preferably at least about 70%, and most preferably at least about 80% of the DHA in the outer segments of the rods and cones of the retina with deuterated DHA as described herein. To achieve this level of replacement level, administration of DHA over a period of time (weeks to months) is required, since deuterated DHA is slowly exchanged in the rods of the cones and has limited uptake. Generally, about 0.1 to 1 gram of deuterated DHA is administered daily. Preferably, the administration of deuterated DHA is either 250 mg / day or 500 mg / day. Deuterated DHA is preferably delivered in a pharma- ceutically acceptable manner, including (optionally) the use of pharma- ceutically acceptable excipients. Over a period of at least two or four weeks, sufficient deuterated DHA is taken up into the rods and cones to provide therapy.

[0066] As used herein, the term "patient" refers to a human patient or a cohort of human patients suffering from a neurodegenerative disease treatable by administration of deuterated DHA. The term "subject" refers to a mammalian subject.

[0067] As used herein, the term "maintenance dose" refers to a dose of deuterated DHA that is less than the initial dose and is sufficient to maintain a therapeutic concentration of deuterated DHA in the outer rods and cones of retinal cells. In one embodiment, the maintenance dose of deuterated DHA is about 30 to about 70% of the initial dose of deuterated DHA. It is understood that the initial dose is intended to increase the concentration of deuterated DHA in the outer rods and cones of the retina until a therapeutic concentration is achieved. At that point, and at the discretion of the attending physician, it may be advantageous to lower the dose of deuterated DHA so that the maintenance dose is sufficient to maintain a therapeutic concentration without further increase in intraretinal concentration.

[0068] As used herein, the term "regular dosing" refers to a dosing schedule that is substantially compatible with the dosing described herein. In other words, regular dosing includes patients that are at least 75 percent, preferably at least 80 percent, compliant with the dosing regimen described herein over a 30-day period. In an embodiment, the dosing schedule includes scheduled dosing breaks. For example, a dosing schedule that provides dosing 6 days per week is one form of regular dosing. Another example is allowing a patient to take a break from dosing for about 3 or 7 days or more (e.g., for personal reasons), provided that the patient is otherwise at least 75 percent compliant. Also, for patients who transition from a loading dose to a maintenance dose, compliance is confirmed by both the loading dose and the maintenance dose.

[0069] As used herein, the term "pharmaceutically acceptable salts" of the compounds disclosed herein includes acid or base addition salts that are within the scope of the methods described herein and that retain the desired pharmacological activity and are not biologically undesirable (e.g., the salts are not overly toxic, allergenic, or irritating and are bioavailable). When the compound has a basic group, such as an amino group, pharmaceutically acceptable salts can be formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (e.g., alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and p-toluenesulfonic acid), or acidic amino acids (e.g., aspartic acid and glutamic acid). When the compound has an acidic group, such as a carboxylic acid ... alkali and alkaline earth metals (e.g., Na + , Li + , K + , Ca 2+ , Mg 2+ , Zn 2+ ), ammonia or organic amines (e.g., dicyclohexylamine, trimethylamine, trimethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g., arginine, lysine, and ornithine, etc.). Such salts can be prepared in situ during the isolation and purification of the compound, or by separately reacting the purified compound in its free base or free acid form with the appropriate acid or base, respectively, and isolating the salt thus formed.

[0070] Compound synthesis The reduced DHA compositions described herein are obtained in one synthetic step from docosahexaenoic acid ethyl ester (Et-DHA) by direct H / D exchange with deuterium oxide (DO) catalyzed by the complex [CpRu(CHCN)PF], as shown in Scheme 1. [ka] In the formula, R, X, X 1 , and Y are as defined above.

[0071] With respect to Scheme 1, the reaction can be carried out in a suitable inert solvent using docosahexaenoic acid ethyl ester (or any other suitable ester), compound 1, a stoichiometric excess of deuterium oxide, in the presence of a ruthenium catalyst, as described in U.S. Pat. No. 10,577,304, the entirety of which is incorporated herein by reference.

[0072] As described herein, the synthetic method used limits the formation of the thermodynamic products produced. In scheme 1, this is achieved by adjusting one or more of the reaction conditions. In one embodiment, the reaction time is shortened. In one embodiment, the reaction temperature is reduced. In one embodiment, the amount of catalyst used is limited. Preferably, a combination of two or three of these embodiments is combined to minimize the amount of thermodynamic products formed.

[0073] Generally, the amount of deuterium oxide used is generally about 150-200 equivalents per equivalent of compound 1. The deuterium oxide is added to an inert solvent such as acetone. The amount of catalyst used is generally about 1-2.5% by weight based on the amount of compound 1 used. The inert solvent is used in an amount sufficient to make the catalyst and deuterium oxide miscible in the resulting solution and to dissolve compound 1. The reaction is carried out at about 15°C to about 26°C, preferably about 19°C to about 23°C, for a time sufficient to achieve sufficient deuteration of compound 1 while limiting the amount of thermodynamic products formed. Typically, this is about 5-7 hours, preferably 5-6 hours.

[0074] After the reaction is complete, the resulting mixture is first treated with benzene, toluene, etc. to deactivate the catalyst, whereupon the destroyed catalyst is removed by charcoal, titanium dioxide, imidazole, carboxyimidazole, benzimidazole, 2-carboxybenzimidazole, 4-carboxybenzimidazole, 5-carboxybenzimidazole, 6-carboxybenzimidazole, thiazole, 2-carboxythiazole, 4-carboxythiazole, 5-carboxythiazole, cysteine, mercaptonicotinic acid, salicylic acid, 2-thiolbenzoic acid, 2-aminobenzoic acid, EDTA, combinations of two (or more) of the above, etc. The solvent and deuterium oxide are removed under vacuum to provide the resulting product.

[0075] As shown in Example 1 and the accompanying Appendix (incorporated herein by reference in its entirety), the total amount of deuteration at the bis-allylic moiety ranges from about 92 to about 97 percent. In other words, after deuteration, the 10 hydrogen atoms at the bis-allylic moiety are replaced with, on average, about 9.2 to about 9.7 deuterium atoms, leaving only about 0.3 to 0.8 hydrogen atoms. Furthermore, high field NMR establishes that, on average, about 87 to about 92 percent of the carbon atoms at the bis-allylic moiety have two deuterium atoms, and more than about 5 to about 12 percent of the carbon atoms at the bis-allylic moiety have one hydrogen and one deuterium substitution, with the remainder being CH2 moieties.

[0076] In view of the above, it has been determined that even if complete deuteration of the bis-allylic sites is not achieved, the presence of CHD groups at these sites confers greater stability against lipid peroxidation than CH2 groups. By limiting the reaction conditions so that, on average, no more than about 2% of the carbon atoms at the bis-allylic sites are CH2 groups, the resulting composition still provides superior control over LPO in vivo.

[0077] pathology The resulting pathology of each oxidative retinal disease is different from the underlying etiology of the disease. That is, whatever the various conditions that cause each of these oxidative retinal diseases (etiology), once caused, the pathology of these diseases involves the accumulation of oxidized DHA products. By limiting oxidative damage, the pathology of the disease is addressed. In the case of AMD as an example, animal studies demonstrate that the loss of vision is significantly limited by treating animals with deuterated DHA compared to untreated animals.

[0078] Without wishing to be bound by any theory, incorporation of deuterated DHA into the outer segments of the retina's rods and cones, as well as the surrounding retinal tissue, limits the extent of oxidation by reactive oxygen species, which in turn protects cells in the retina from the damage and destruction typical of AMD.

[0079] methodology In one embodiment, the method described herein includes administration of deuterated DHA to a patient suffering from an oxidative retinal disease. The drug is delivered to the patient at a dose prescribed by the attending physician. Typically, such a dose is about 0.1 to about 1.0 grams per day. Accumulation of deuterated DHA in the body can be monitored, for example, by blood tests, to ensure that the patient is accumulating deuterated DHA consistent with the achievement of a therapeutic outcome. If blood tests demonstrate insufficient levels of deuterated DHA, the clinician can determine whether dietary intake of DHA should be adjusted, dosing should be increased, or the change from a loading dose to a maintenance dose can be delayed. Specific examples of methods for administering DHA are described in U.S. Provisional Patent Application Nos. 63 / 224,674, 63 / 224,679, and 63 / 224,690, each of which is incorporated by reference in its entirety.

[0080] The methods described herein may include administering deuterated DHA or an ester thereof to a patient to accumulate therapeutic levels of deuterated DHA for use in the methods described herein.

[0081] In one embodiment, deuterated DHA or an ester thereof is administered to a patient in an amount sufficient to result in a concentration of deuterated DHA in the patient (e.g., in red blood cells, plasma, and / or retinal cells) of at least about 50%, preferably at least about 60%, more preferably at least about 70%, and most preferably at least about 80%, based on the total amount of DHA, including deuterated DHA, found therein. In embodiments, the percentage of deuterated DHA compared to total DHA in the patient (e.g., in red blood cells, plasma, and / or retinal cells) can be about 50% to about 80%, about 50% to about 70%, or about 50% to about 60%.

[0082] combination The treatment provided herein can be combined with any other treatment used with oxidative retinal disease, provided that such treatment does not interfere with the treatment described herein.In the case of macular degeneration, drugs such as bevacizumab, ranibizumab, aflibercept, and brolucizumab are all prescribed to attenuate disease progression, and can be used in combination with the therapy described herein.

[0083] In another embodiment, the combination therapy can use drugs that act through an orthogonal mechanism of action to the methods described herein.Drugs suitable for combination use include, but are not limited to, antioxidants such as edaravone, idebenone, mitoquinone, mitoquinol, vitamin C, or vitamin E, riluzole, which preferentially blocks TTX-sensitive sodium channels, traditional pain relievers, and the like.

[0084] Pharmaceutical Compositions The specific dosage of deuterated DHA (drug) can be achieved by any number of acceptable modes of administration.As mentioned above, the actual amount of drug (i.e., active ingredient) used in the daily or regular dose according to the method of the present invention is described in detail above.The drug can be administered at least once a day, preferably once or twice or three times a day.

[0085] The present invention is not limited to any particular composition or pharmaceutical carrier, which itself can vary.Generally, the compounds of the present invention are administered as pharmaceutical compositions by any of a number of known administration routes.However, oral delivery is typically preferred, using tablets, pills, capsules, etc.The particular form used for oral delivery is not critical.

[0086] Pharmaceutical dosage forms of the compounds disclosed herein can be prepared by any of the methods known in the art, such as conventional mixing, tableting, encapsulation, etc. The compositions disclosed herein can include one or more physiologically acceptable inactive ingredients that facilitate processing of the active molecules into preparations for pharmaceutical use.

[0087] The composition may include a drug in combination with at least one pharma- ceutically acceptable excipient. Acceptable excipients are non-toxic, aid in administration, and do not adversely affect the therapeutic utility of the claimed compounds. Such excipients may be any solid, liquid, or semi-solid that is generally available to those skilled in the art. One such excipient is edible oils such as oleic acid (e.g., olive oil), canola oil, and other well-known edible oils. Such oils may also contain emulsifiers, sweeteners, colorants, preservatives, and other well-known auxiliary substances.

[0088] Solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, etc. Other suitable pharmaceutical excipients and their formulations are described in Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990).

[0089] The compositions disclosed herein can be provided in a pack or dispenser device, if desired, containing a daily or regular unit dose, each containing a drug in the required number of subunits. Such a pack or device can, for example, comprise a metal or plastic foil, such as a blister pack, a vial, or any other type of container. The pack or dispenser device can be accompanied by instructions for administration, including, for example, instructions for taking all of the subunits that make up the daily or regular dose contained therein.

[0090] The amount of drug in the formulation may vary depending on the number of subunits required for a daily or periodic dose of the drug. Typically, the formulation contains, on a weight percent (wt%) basis, about 10-100 weight percent of the drug based on the total formulation, with the remainder, if present, being one or more suitable pharmaceutical excipients. Preferably, the compound is present at a level of about 50-99 weight percent.

[0091] In a preferred embodiment, the drug is encapsulated in a capsule without the need for pharmaceutical excipients such as stabilizers, antioxidants, colorants, etc. This minimizes the number of capsules required per day by maximizing the volume of drug in each capsule.

[0092] Testing Protocol Once administered, the attending physician needs to monitor the absorption rate of deuterated DHA into the retina. Because physical access to the retina is not feasible, the method described in the examples shows that either plasma or red blood cells (RBCs) can be used as a surrogate to evaluate whether absorption is proceeding properly. This is because both plasma and RBCs reach a steady-state concentration, allowing the clinician to determine whether the patient has reached a steady-state concentration, even though this occurs at different times after the start of treatment. When the steady-state concentration is reached, the clinician is confident that the maximum concentration of deuterated DHA is found in the blood supplying the retina, and therefore the retina is receiving an adequate amount of deuterated DHA.

[0093] Testing plasma or RBC for individual components contained therein is well established in the art.The following example demonstrates one method for carrying out such evaluation.However, the test protocol used is not critical as long as the test analysis provides the ratio of deuterated DHA in blood or plasma based on the total amount of DHA present in the sample, which contains both non-deuterated DHA and deuterated DHA.

[0094] However, because each patient has a different absorption rate of deuterated PUFAs in general, and deuterated DHA in particular, based on their diet and their unique physiological function, as demonstrated in the examples, it is necessary to test the patient for proper incorporation of deuterated DHA. Furthermore, such testing can evaluate whether the patient is following the dosing instructions provided by the attending physician. In one embodiment, the method includes limiting the patient's dietary DHA intake to 132 mg / day or less during treatment with deuterated DHA. In one embodiment, the method includes limiting the patient's dietary DHA intake to 71 mg / day or less during treatment with deuterated DHA. In one embodiment, the method includes limiting the patient's dietary DHA intake to 62 mg / day or less during treatment with deuterated DHA. In one embodiment, the method includes limiting the patient's dietary DHA intake to 54 mg / day or less during treatment with deuterated DHA. In one embodiment, dietary DHA intake is limited to about 54 mg to about 132 mg per day, about 62 mg to about 132 mg per day, or about 71 mg to about 132 mg per day.

[0095] The method of testing is not critical, so long as the test analysis provides the percentage of deuterated DHA in the blood or plasma based on the total amount of DHA present in the sample, which contains both non-deuterated DHA and deuterated DHA.

[0096] In addition to the above, standardization curves can be created for each dose of DHA to establish the timing and concentration of blood steady state for different dosing regimens of deuterated DHA.As shown in the examples, standardized curves for determining the time between the start of treatment and steady state concentration are provided for two different markers (plasma and RBC) using two different dosing regimens.Using this procedure, standardization curves for any dosing concentration of deuterated DHA can be established. EXAMPLES

[0097] The present invention will be further understood by reference to the following examples, which are intended to be purely exemplary of the present invention. The present invention is not limited in scope by the illustrated embodiments, which are intended only as illustrations of single aspects of the invention. Any methods that are functionally equivalent are within the scope of the present invention. In addition to those described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications fall within the scope of the appended claims.

[0098] As used herein, the following abbreviations have the following definitions. Terms not defined have their accepted scientific definitions.

[0099] Example 1: Synthesis of D-PUFA Combine 200 grams of docosahexaenoic acid ethyl ester, 1.9 kg of DO (approximately 170 equivalents), and approximately 3.8 to approximately 4 grams of [CpRu(CH3CN)3]PF6 with a sufficient amount of acetone to homogenize the reaction mixture (the total volume of the reaction mixture is approximately 9 liters).

[0100] The isotope exchange process is maintained at a temperature of about 19 °C to about 23 °C for about 5 to 7 hours. The reaction mixture is then treated to deactivate the catalyst and the solution is worked up. The recovered products can be evaluated by conventional techniques including HPLC, NMR, MS, etc.

[0101] Hydrogen atoms of bis-allylic moieties 1 The H NMR peak is 2.8 ppm downfield from TMS. In the absence of catalytic deuteration, the natural abundance of deuterium oxide is negligible, so the integration of this peak corresponds to 10 protons. After catalytic deuteration, this peak can be integrated and correlated with the proton peaks of the starting DHA to estimate the degree of deuteration at the bis-allylic sites.

[0102] 1 H NMR spectra can also be used to estimate the degree of deuteration from the two monoallylic positions and to obtain data for each of these positions, since they give signals at different values ​​of chemical shift due to their proximity or distance from the carboxylate.

[0103] Example 2: Determination of D-DHA replacement rate in plasma, red blood cells and retina Mature adult C57BL / 6J mice were fed a customized rodent diet containing 0.5% w / w D-DHA and no native DHA for 78 days. Animals were sacrificed and plasma, red blood cells and retinal tissues were dissected on study days 8, 19, 38 and 78 (6 animals per time point, 3 males + 3 females). D-DHA+DHA was extracted from samples and derivatized to methyl esters with a mixture of heptane / toluene (63:37 by volume) and methanol / dimethoxypropane / sulfuric acid (85:11:4 by volume) by gentle shaking at 80°C for 2 hours, followed by separation of the organic phase and drying under nitrogen. D-DHA+DHA methyl esters were structurally identified and quantified by gas chromatography coupled with a tandem mass spectrometry detector, and D-DHA substitution levels (percentage of total DHA) were calculated. The results are presented in Table 1 and graphed in Figure 1. The measured data revealed that the D-DHA replacement rate follows first-order kinetics, i.e., it doubles at regular intervals in each sample type (e.g., about every 6-7 days in plasma, about every 10 days in red blood cells, and about every 20-22 days in retina, such that the maximum concentration (steady state) is reached sooner in plasma and red blood cells (accessible in human subjects by simple blood sampling) than in the retina (not accessible in living human subjects). With the known doubling times, the steady state concentration can be calculated by single-point and / or multipoint measurements without the need to wait until the steady state is actually reached. [Table 1]

[0104] Example 3: Prediction of retinal D-DHA replacement rate from plasma and red blood cell replacement rates In contrast to controlled experimental diets, naturally occurring DHA is ingested by patients treated with D-DHA, which may dilute the relative proportion of administered D-DHA absorbed with the total DHA pool and its final concentration at steady state in blood and ultimately in target tissues such as the retina. The data and standard curve from Example 1 allow the retinal steady state D-DHA concentration to be determined in advance with reasonable accuracy by calculating the D-DHA / total DHA ratio in plasma and / or red blood cells. Table 2 illustrates this using an average daily dietary intake of about 130 mg DHA per day (which represents the 90th percentile of the average normal DHA intake by men over 51 years of age in the United States). Figures 2-4 illustrate how measurements of plasma and red blood cell D-DHA concentrations can be used to predict expected retinal steady state concentrations. With a known fixed daily dose and measured replacement rate of D-DHA at steady state, the average dietary DHA intake of an individual patient can be estimated and monitored, allowing either timed adjustment of the daily D-DHA dose or dietary intervention to reduce natural DHA intake until the desired therapeutic D-DHA replacement level can be reached, preferably about 50% or more. [Table 2]

[0105] Example 4: Deuterated docosahexaenoic acid protects against oxidative stress and geographic atrophy-like retinal degeneration in a mouse model with iron overload Oxidative stress plays a major role in the pathogenesis of neurodegenerative and retinal diseases. The retina is particularly susceptible to oxidative damage due to its high content of polyunsaturated fatty acids (PUFAs), photooxidation, high oxygen tension supplied by the choriocapillaris, and abundant mitochondria. PUFAs are essential components of cell and mitochondrial membranes and are essential for optimal metabolism. PUFAs are vulnerable to oxidative stress and react with reactive oxygen species (ROS) via the lipid peroxidation (LPO) chain reaction. However, antioxidant therapy cannot prevent LPO or neutralize secondary products of LPO for stoichiometric reasons. Moreover, ROS may also regulate cell signaling, so completely eradicating ROS can be harmful. Bis-allylic hydrogen abstraction is the rate-limiting step of ROS-driven PUFA oxidation. Substituting hydrogen atoms at the bis-allylic site with deuterium atoms can slow down the LPO chain reaction through the isotope effect (Figure 5). PUFAs cannot be synthesized de novo in the human body from carbon sources such as acetate. Typically, linoleic acid and α-linolenic acid serve in the diet as the primary precursors for the biosynthesis of all n-6 and n-3 PUFAs, respectively. D-PUFAs can be incorporated into mitochondrial and cellular membranes after oral administration, replacing a fraction of the PUFAs naturally present in the membranes and conferring resistance to oxidative stress and LPO. D-PUFAs have been studied in multiple pathologies involving oxidative stress and LPO. A deuterated version of linoleic acid (11,11-D2-Lin; RT0001) inhibited LPO and rescued cell death in both animal models and clinical trials in several neurodegenerative diseases, including Friedreich's ataxia (FRDA), infantile neuroaxonal dystrophy (INAD), and progressive supranuclear palsy (PSP). D-PUFAs also reduce LPO and hold therapeutic potential in preclinical trials for Alzheimer's, Parkinson's, and Huntington's diseases. Oxidative stress is involved in several retinal diseases, including age-related macular degeneration (AMD), light-induced damage, iron-associated retinal degeneration, Leber's hereditary optic neuropathy, and retinitis pigmentosa.Docosahexaenoic acid (cervonic acid; DHA, C22:6, n-3) is the most abundant PUFA in the retina, accounting for up to 40% of the total fatty acids in human rod photoreceptor outer segments. DHA is important for the integrity of photoreceptors and visual function. Although the intake of DHA-rich fatty fish is associated with a lower risk of AMD, supplementation with n-3 PUFA has not shown any appreciable benefit in patients with AMD or retinitis pigmentosa. Furthermore, high doses of DHA may increase the risk in conditions involving oxidative stress due to its high susceptibility to oxidation.

[0106] Addition of DHA to the human RPE cell line ARPE-19 increased oxidative stress and LPO under high-intensity light exposure. Levels of carboxyethylpyrrole (CEP), an immunogenic protein adduct derived from the oxidation of DHA, are elevated in retinal tissue and plasma from AND patients. Furthermore, immunization of mice with CEP adducts resulted in AMD-like retinal degeneration. These evidences suggest that nonenzymatic oxidation of DHA in the retina may play an important role in the pathogenesis of retinal disorders, including oxidative stress.

[0107] In this example, we study the effect of deuterated DHA on oxidative stress and LPO in mice with iron-induced oxidative stress in the retina. Previously analyzed mouse models that received intravitreal (IVT) injections of iron found retinal pathology similar to human AMD, including increased oxidative stress and CEP in the retina, followed by geographic atrophy of the RPE. Here, mice were fed a diet containing D-DHA isotopologues, the most commonly used being 6,6,9,9,12,12,15,15,18,18-D10-(4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoic acid ethyl ester envelopes, for 11 weeks, followed by a washout period in a pharmacokinetic study to establish a dosing regimen for efficient retinal uptake. To determine the protective effect of D-DHA against LPO, mice were fed a diet containing D-DHA for 1-4 weeks and then given an IVT injection of iron or control saline. In mice fed D-DHA for 4 weeks, >50% replacement of DHA with D-DHA in the neural retina was observed.

[0108] This regimen provided near-complete protection against iron-induced retinal damage by inhibiting oxidative stress and DHA oxidation.

[0109] Dietary D-DHA was efficiently taken up by neural retina and RPE cells. To determine the pharmacokinetics of ocular D-DHA uptake, uptake and elimination from the neural retina and RPE / choroid / sclera, 12-week-old C57BL / 6J mice were fed a 0.5% D-DHA-containing diet (0.5 g D-DHA / 100 g food, Table 3) for 77 days, followed by an additional 73-day washout period with DHA. At week 4, >55% of the DHA in the retina was D-DHA, rising to >60% at week 5 (Figure 6A). At similar time points, D-DHA in the RPE / choroid / sclera was >80%. Washout in the RPE / choroid / sclera was also more rapid than the retina. Uptake and elimination followed classical first-order kinetics. Based on the accumulation and excretion data, 2-month-old mice were fed diets containing either D-DHA or natural DHA control for 1, 2, 3, and 4 weeks before IVT injection of iron and saline control (Figure 6B). To more closely resemble the typical DHA dose in human formula omega-3 supplements (e.g., 1500 mg / day DHA in Lovaza), D-DHA and DHA experimental mouse diets were adjusted to 0.25% instead of 0.5% for the majority of the study. With 0.25% D-DHA diet, retinal D-DHA levels exceeded 50% at 5 weeks (52.2±1.5% and 55% of total DHA by our GC and LC-based MS methods, respectively), regardless of whether the eye was injected with iron or saline control. Table 3 shows the D-DHA content as a percentage of D-DHA+DHA in the neural retina and RPE from mice fed D-DHA or DHA at week 4, given an IVT injection, and then continued on the D-DHA diet for an additional week. [Table 3]

[0110] D-DHA protected against iron-induced retinal autofluorescence (AF) and degeneration In a previous study, IVT iron was shown to induce retinal AF and degeneration. To evaluate the protective effect of 0.25% D-DHA diet, confocal laser scanning ophthalmoscopy (cSLO) and optical coherence tomography (OCT) were used for in vivo imaging 1 week after injection. For cSLO imaging, both blue autofluorescence (BAF) and near-infrared autofluorescence (IRAF) were performed. One week after iron injection, BAF images of mice fed natural DHA showed undulations of the photoreceptor layer, as well as intense hyperautofluorescent spots representing autofluorescent RPE and bone marrow cells (Figure 6D and Figure 6E). These same retinas imaged with IRAF showed high autofluorescence and low autofluorescence in the superior retina. BAF and IRAF images of mice fed D-DHA revealed a dose-dependent reduction in iron-induced retinal AF in mice fed D-DHA 1, 2, 3, and 4 weeks before iron injection (Figure 6D and Figure 6E, Figure 12). Optical coherence tomography (OCT) scans of mice fed natural DHA showed a marked thinning of the outer nuclear layer in the superior retina 1 week after iron injection (Figure 6G). In contrast, mice fed D-DHA for 4 weeks showed complete preservation of retinal structure in OCT scans (Figure 6G).

[0111] The extent to which D-DHA replaced natural DHA was determined by LC / MS by feeding experimental diets containing 0.25% D-DHA for increasing periods of time. Microdissected samples of neural retina and RPE from mice fed the experimental diets for 4 weeks were analyzed after receiving IVT iron or control saline, and then the experimental diets were continued for an additional week. 22.6% of the DHA in the control diet was found to be at natural abundance. 13 It was confirmed that the diet was composed of isotopomers with 327.2 / 283.2 and did not contain D-DHA. Thus, the signal from the 327.2 / 283.2 transition represents 78.4% of the total DHA. The experimental diet did not contain detectable native DHA, but rather a distribution of deuterium-substituted DHA isotopologues that are a result of the method of D-DHA preparation (Figure 11A-C). Natural abundance 13After applying a correction for C, it was determined that the D10-DHA isotopologue constituted 45.6% of the D-DHA species in the analyzed samples, with the D8-DHA, D9-DHA, D11-DHA, and D12-DHA isotopologues making up the remainder. Thus, the signal from the 337.2 / 293.2 transition represented 45.6% of the D-DHA. Applying these corrections, the D-DHA isotopologues as a percentage of total DHA (i.e., D-DHA + DHA) were determined to be 59.3-60.8% in isolated RPE and 55.0% in neural retina. There were no significant differences between iron-treated and saline control eyes (Table 3).

[0112] D-DHA protected retinal function and histology. To assess retinal function, electroretinograms were performed on mice fed D-DHA or native DHA for 4 weeks. In mice that were not given an IVT injection, administration of D-DHA did not cause significant differences in the amplitude of rod-b, rod-a, and cone-b waves compared to mice fed native DHA. Thus, using this measure, ingestion of D-DHA did not affect retinal function (Figure 7A). In mice fed control DHA, one week after iron injection, the amplitude of rod b, rod a, and cone b waves was significantly reduced in iron-injected eyes compared to saline controls, consistent with iron-induced retinal damage. Iron-injected eyes from mice with 50% or more retinal D-DHA had a significant preservation of rod b, rod a, and cone b-wave amplitudes compared to the DHA + iron-injected group (Figure 7B). There was no significant difference between saline- and iron-injected eyes from mice fed the D-DHA diet, indicating complete anatomical and functional protection. Toluidine blue staining was performed on plastic sections to examine retinal histology. One week after iron injection, there was thinning of the outer nuclear layer (ONL) in the superior retina of DHA-fed mice, intracellular vesicles in degenerated RPE cells (white dashed arrows), and bone marrow cells infiltrating between the neural retina and the RPE layer (black solid arrows) (Figures 7C and 7D). Mice fed D-DHA showed complete protection of retinal structure against the toxicity of iron injection (Figures 7C and 7D). Quantification of total and outer retinal thickness in IVT iron-injected eyes from DHA-fed mice showed a reduction in the superior retina. In contrast, IVT iron-injected eyes from D-DHA-fed mice were significantly protected and did not differ from saline-injected eyes (Figures 7E and 7F). In summary, >50% retinal D-DHA replacement resulted in complete protection of retinal function and structure against iron-induced injury.

[0113] D-DHA prevented the formation of CEP, a specific oxidation product of DHA. Iron-catalyzed peroxidation of DHA-containing phospholipids results in a unique carboxyethylpyrrole (CEP) adduct that is not formed from any other PUFA. CEP has been detected by IHC in human AMD eyes and mouse retinas, including those from mice that received IVT iron. To test whether D-DHA can protect against iron-induced CEP formation, mice were fed D-DHA or DHA for 4 weeks and then IVT-injected with iron or control saline.

[0114] Frozen sections were prepared 4 hours and 1 week after injection. Co-labeling for CEP and rhodopsin was performed to evaluate and localize CEP. Four hours after injection, increased immunolabeling for CEP was present in rhodopsin-colabeled photoreceptor outer segments in IVT iron-injected eyes of DHA-fed mice, but not in IVT iron-injected eyes of D-DHA-fed mice (Figure 8A). One week after injection, CEP immunolabeling was localized to the RPE and infiltrating bone marrow cells in IVT iron-injected eyes of DHA-fed mice, likely resulting from phagocytosed and oxidized photoreceptor outer segments (Figure 8B). CEP immunolabeling was not detected in D-DHA-fed mice. These results indicate that iron induced CEP accumulation and that D-DHA in >50% retinal replacement prevented CEP accumulation by inhibiting DHA oxidation. Ferritin light chain (L-Ft) immunolabeling was performed to assess retinal iron levels and localization, since L-Ft protein levels increase in response to elevated intracellular iron. One week after saline injection, L-Ft weakly labeled the ganglion cell layer, outer plexiform layer, and inner segment layer (Figure 8E). Increased L-Ft staining was observed in the inner plexiform layer, outer plexiform layer, and inner segment in both DHA / IVT iron and D-DHA / IVT iron mice (Figure 8E). These two groups did not differ from each other, indicating that D-DHA did not prevent IVT iron-induced iron accumulation in retinal cells, but instead blocked its downstream toxic effects. Quantification of pixel density for CEP and L-Ft labeling was performed using ImageJ software (Figures 8F and 8G) to quantitatively verify the above results.

[0115] D-DHA protected against iron-induced retinal cell death, oxidative stress, and mRNA changes indicative of inflammation Quantitative PCR was used to assess mRNA changes in the neural retina of mice fed D-DHA or DHA for 4 weeks. Cell type-specific, iron-regulated, antioxidant, and inflammation-related genes were assessed 1 week after iron or saline injection. The mRNA levels of the rod-specific gene rhodopsin (Rho), and the cone-specific genes cone opsin 1 midwave and shortwave sensitivity (Opn1mw and Opn1sw) were measured to assess rod and cone photoreceptor stress and differentiation. The mRNA levels of Rho, Opn1sw, and were significantly reduced in the neural retina of DHA-fed mice receiving IVT iron compared to IVT saline controls. In contrast, there was no change in these mRNAs in the neural retina of D-DHA / IVT iron mice compared to IVT saline controls (Figure 9). The mRNA levels of transferrin receptor (Tfrc), which is inversely proportional to intracellular iron levels, can be used as an indicator of intracellular iron levels. One week after iron injection, Tfrc mRNA levels in the neural retina were significantly decreased in DHA / IVT-iron and D-DHA / IVT-iron mice, indicating iron loading in the neural retina of both groups. These two groups had slightly different Tfrc levels, possibly as a result of the loss of some photoreceptors in the DHA / IVT-iron group (Figure 9). The mRNA abundance of antioxidant solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), glutathione S-transferase isoform m1 (GSTm1), glutathione synthesis (GSS), catalase (Cat), heme oxidase 1 (Hmox1), and superoxide dismutase 1 (Sod1) was measured to examine oxidative stress. The mRNA levels of Slc7a11, Gpx4, GSTm1, Cat, Hmox1 and Sod1 were significantly increased in the neural retina of DHA / IVT iron compared to saline-injected eyes. This antioxidant upregulation was prevented in D-DHA / IVT iron eyes, and there was no significant difference between iron- and saline-injected eyes in mice fed D-DHA. Retinal inflammation was examined by detecting the mRNA levels of IL1β, IL6 and cluster of differentiation 68 (Cd68).The mRNA levels of IL1β and Cd68 were significantly increased in DHA / IVT iron retinas compared to saline controls, but not in D-DHA / IVT iron retinas. The mRNA levels of glutathione synthase (GSS) and IL6 were not increased by IVT iron in mice on either diet (Figure 9). In summary, 250% retinal D-DHA can significantly protect against iron-induced oxidative stress, photoreceptor cell damage, and inflammation in the neural retina.

[0116] D-DHA prevented the development of iron-induced geographic atrophy Mice fed D-DHA for 4 weeks showed complete retinal protection 1 week after iron injection (Figure 6). Our previous study showed geographic atrophy in the superior retina within 1 month of IVT iron injection. To evaluate whether D-DHA could protect against geographic atrophy in this model, mice were continued on the respective diets for 4 weeks after iron or saline injection. At this time point, BAF and IRAF images showed low AF in the superior retina of DHA-fed mice, as well as geographic atrophy (Figures 10A and 10B). This corresponded to photoreceptor (white arrow) and RPE degeneration (black arrow) in OCT scans (Figure 10C). D-DHA completely protected against the development of geographic atrophy (Figures 10A-C). cSLO and OCT scans were obtained from multiple mice, all showing the protective effect of D-DHA against chronic retinal degeneration (Figures 13A-B). Taken together, 250% retinal D-DHA provided long-term protection against the development of iron-induced geographic atrophy.

[0117] Consideration In this example, we analyzed whether inhibition of DHA oxidation could prevent oxidative stress and retinal degeneration in a mouse model with retinal iron overload. We observed that IVT iron induces retinal AF, oxidative stress, accumulation of carboxyethylpyrrole (CEP), a DHA-specific oxidation product, and photoreceptor degeneration followed by progressive geographic atrophy, which recapitulates the characteristics of human AMD. In this example, we demonstrated that administration of D-DHA completely protects against all of these iron-induced retinal changes.

[0118] Mice fed D-DHA 1, 2, and 3 weeks prior to iron injection showed a dose-dependent reduction in iron-induced retinal AF and retinal degeneration, with a >50% protective effect already observed at >30% retinal D-DHA replacement levels (Figure 14). In mice fed D-DHA 4 weeks prior to iron injection, a complete protection of retinal structure and function was observed after D-DHA reached 50% retinal replacement levels.

[0119] D-DHA inhibited oxidative stress and LPO, which are particularly harmful due to its autocatalytic radical chain reaction cycle and nonenzymatic nature. Hydrogen abstraction at the bis-allylic site is the rate-limiting step of LPO. PUFAs deuterated at the bis-allylic position inhibit this step by isotope effect. D-DHA prevented oxidative stress-induced increases in the mRNA levels of antioxidants GSTm1, catalase, Sod1, Hmox1, Gpx4, and Slc7a11. Furthermore, immunolabeling of CEP was not detected in the retinas of mice with retinal D-DHA substitution levels of ≥50% before iron injection, suggesting that deuteration may inhibit the oxidation of DHA and the accumulation of its toxic derivative CEP, contributing to retinal protection. CEP (Figure 5E) is a DHA-specific adduct-forming oxidation product. CEP adducts have been found to be increased in drusen deposits and plasma of AMD patients and elevated in rodent retinas after exposure to strong light. Mice immunized with CEP adducts accumulated complement component-3 in Bruch's membrane and drusen deposits under the RPE, characteristic of dry AMD, and exhibited RPE degeneration.CEP adducts also stimulated neovascularization in vivo through a VEGF-independent pathway.

[0120] LPO has been shown to be harmful to cells in multiple ways. For example, it can make lipid bilayers leaky and rigid. At the chemical level, LPO can generate low molecular weight species such as lipid peroxides, prostaglandin-like isoprostanes, and isoketals, which have primarily detrimental effects. Another group of LPO products that have been implicated in numerous pathologies include activated carbonyls, including malondialdehyde, 4-HNE (derived from n-6 PUFAs; lipid soluble), and 4-HHE (derived from n-3 PUFAs; water soluble). These are highly reactive (Figure 5E) and can irreversibly crosslink phospholipids, proteins, and cause DNA transversion. By inhibiting LPO, D-PUFAs reduce the levels of these compounds. Furthermore, D-PUFAs can cross-protect various H-PUFAs. D-PUFAs incorporated into membranes protect other PUFAs in this membrane by terminating the LPO chain reaction. For example, the presence of the n-6 PUFA D2-linoleic acid in lipid bilayers downregulated not only 4-HNE but also 4-HHE formation.

[0121] A threshold protective effect on liposome stability under oxidative stress has been shown for various D-PUFAs, revealing a strong protective effect of D-DHA, which efficiently inhibits LPO even when present at a fraction of 1% of total PUFAs in lipid membranes in vitro. In this example, mice with 50% or more D-DHA incorporated into neural retina and RPE cells showed a complete protective effect against iron-induced oxidative damage. This is probably due to the high total content of DHA in the retina and the rod outer segment membrane phospholipids (supraenoic phospholipids with more than six double bonds) that have up to 30 mol% twin DHA acyl chains. This unique feature of photoreceptor outer segments may require a D-DHA level of ≧50% to completely isolate the proximal unprotected supraenoic DHA chains from each other. The high levels of hydroxyl radicals generated by Fe via the Fenton reaction and subsequent LPO cycle in this dramatic model may require high concentrations of retinal D-DHA, although lower levels may be sufficient in less severe oxidative conditions. D-DHA inhibited oxidative damage-associated inflammation in the neural retina. One week after injection, the mRNA levels of IL1β and Cd68 had no significant difference between iron-injected neural retina from mice fed D-DHA and saline-injected neural retina, which were significantly increased in iron-injected neural retina from mice fed DHA. Oxidative stress and lipid peroxidation can induce inflammatory responses, including infiltration and activation of microglia and macrophages, and secretion of proinflammatory cytokines such as IL1β, IL-6, and TNF-α. Overall, the results suggest that D-DHA can prevent neuroinflammation induced by iron. D-DHA showed long-term protection against geographic atrophy. Mice with retinal D-DHA levels ≥50% were completely protected from chronic development of geographic atrophy in the superior retina compared to mice fed DHA. Furthermore, it was found that IVT iron-induced damage appeared to be less severe with the DHA diet than in a previous study (LabDiet 5001) in mice fed a "normal diet" that did not contain DHA.It was previously observed that iron-induced retinal AF throughout the retina in mice fed LabDiet 5001 occurred 1 week after iron injection, followed by the "kidney bean"-shaped geographic atrophy that typically occurs 4 weeks after iron injection. In this example, we observed that iron-induced AF was more restricted to the upper retina in mice fed DHA for 4 weeks 1 week after iron injection (Figure 12), and that complete "kidney bean"-shaped geographic atrophy occurred in only some, but not all, mice (Figure 13), which correlated with the amount of upper area AF 1 week after iron injection. Further studies can be performed to determine the basis for the more severe iron-induced retinal degeneration in mice on LabDiet 5001 (which is different from the DHA control diet used in this study). Since the only difference between the control DHA diet and the D-DHA diet used herein is whether DHA is deuterated or not, these results suggest that D-DHA provides long-term protection against photoreceptor and RPE degeneration induced by iron overload. D-PUFA has been reported to inhibit LPO in several mouse models of neurological diseases associated with oxidative stress, including Parkinson's disease, Alzheimer's disease, Huntington's disease, and childhood neuroaxonal dystrophy. RT001 (D2-Lin ethyl ester) inhibits LPO in Friedreich's ataxia. 8 It has been tested in clinical trials for AMD and infantile neuroaxonal dystrophy, showing remarkable safety and tolerability. Here, we report the protective effect of D-DHA in retinal disease in vivo using a mouse model that mimics the characteristics of human AMD. The results show that D-DHA can prevent iron-induced retinal degeneration by inhibiting the oxidation of DHA. D-DHA may be a viable therapeutic agent for retinal pathogenesis involving oxidative stress and lipid peroxidation.

[0122] Materials and Methods D-DHA synthesis D-DHA was synthesized as previously described (e.g. AV Smarun, M Petkovic, MS Shchepinov, D Vidovic. Site-Specific Deuteration of Polyunsaturated Alkenes. The Journal of organic chemistry. Dec 15 2017;82(24):13115-13120). Catalytic exchange results in an assortment of D-DHA isotopologues from D6 to D12, centered around D10, which typically represents 30-40% of all bisallylic isotopologues. At least 90% of D-DHA is enriched with two Ds at all bisallylic carbons, and the remaining 10% is enriched with at least one D at each of the bisallylic positions.

[0123] Ocular D-DHA Deposition and Removal Eleven-week-old C57BL / 6J mice were purchased from Jackson Laboratory (Bar Harbor, ME) and housed at the Dean McGee Eye Institute Animal Research Facility, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, under a 12-h day / night cycle and 20-25 lux light conditions. After 1 week of acclimation to the animal care facility with free access to laboratory rodent chow and water, animals were assigned to experimental groups. To determine ocular D-DHA deposition, mice were fed AIN93G diet instead of laboratory rodent chow. 51The mice were switched to an experimental D-DHA supplemented diet (Research Diet, Inc. New Brunswick, NJ) containing 0.5% D-DHA + 6.5% high oleic soybean oil in the diet. The diet was vacuum packaged and stored at -20°C. Diet was replaced three times a week ad libitum with fresh diet stored at 4°C after harvesting from -20°C. Based on previously estimated retinal attachment kinetics, eyecups including retina, optic nerve, and sclera, retinal pigment epithelium-choroid (RPE-choroid) were excised from six mice (three females and three males) at different time points during 11 weeks of D-DHA feeding. Tissues were snap frozen in liquid nitrogen and stored at -80°C until fatty acid analysis. After 11 weeks (77 days) on the D-DHA diet, animals were switched to a washout diet containing 0.5% DHA + 6.5% high oleic soybean oil (w / w) in AIN93G and maintained on this diet until euthanasia at three different time points up to 73 days, at which time tissues were harvested for fatty acid analysis. All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center.

[0124] Iron-induced acute RPE atrophy Adult male wild-type C57BL / 6J mice (stock no. 000664, Jackson Labs, Bar Harbor, ME, USA) were housed under standard conditions under cyclic lighting (12 h:12 h light / dark cycle). Mice had free access to water and food. Starting at 2 months of age, mice were fed the above AIN93G diet supplemented with 0.25% D-DHA or DHA (control diet) for 1, 2, 3, or 4 weeks prior to intravitreal injection. The complete composition of the DHA and D-DHA-containing diets is shown in Table 4. Mice were fed 1 μl of 0.5 mM ammonium ferric citrate or 1 μl of ammonium ferric citrate diluted in 0.9% NaCl (saline) (MP Biomedicals LLC, Santa Ana, CA).

[0125] As a control, 1 μl of saline was injected intravitreally. Intravitreal injections were performed as previously described. 54Mice were maintained on their respective diets until final evaluation. All housing and procedures were performed in accordance with the NIH Guide for the Care and Use of Experimental Animals and approved by the University of Pennsylvania Animal Care and Use Committee. [Table 4-1] [Table 4-2]

[0126] mass spectrometry To confirm the reproducibility of the results, three types of mass spectrometry were performed. First, lipids were extracted from the retina by a modified Folch method (CHCl3 / CH3OH, 2:1), derivatized to fatty acid methyl esters (FAMEs), and analyzed by high-resolution capillary gas chromatography and specific chemical ionization tandem mass spectrometry. Baseline resolved DHA and D-DHA total ion signals were integrated and the ratio of D-DHA / total DHA was calculated. The second type of mass spectrometry was performed on control and experimental diet samples, as well as on microdissected neural retina and RPE from animals fed the experimental diets. Lipids were extracted, saponified, and analyzed by ESI-LC / MS on a 4000 QTrap (Sciex) operating in enhanced negative mode over an m / z range of 320-345 and a scan speed of 250 / s. This analysis confirmed that the control diet contained DHA but no detectable D-DHA, while the experimental diet contained a range of D-DHA isotopologues but only trace amounts of native DHA (Figure 11). Peaks corresponding to DHA with 8, 9, 10, 11, 12, and 13 deuterium substitutions were readily identified in the experimental diets, as well as in the neural retina and RPE samples. The relative distribution of DHA isotopologues in the neural retina and RPE samples was indistinguishable from that in the experimental D-DHA supplemented diets.

[0127] A third type of mass spectrometry was performed on microdissected neural retina and RPE from animals fed the two diets using the same extraction and chromatographic procedures. However, ESI-LC / MS analysis was performed in negative multiple reaction monitoring mode for the transitions 327.2 / 283.2 (corresponding to 78.4% of normal DHA) and 337.2 / 293.2 (corresponding to 45.6% of deuterated DHA). Results for D-DHA are reported as a percentage of total DHA (i.e., D-DHA+DHA).

[0128] In vivo imaging system Mice were given general anesthesia and placed on a platform. Pupils were dilated with 1% tropicamide saline (Akorn, Inc., Lake Forest, IL). Optical coherence tomography (OCT) imaging was performed for visualization of retinal structures by using Bioptigen®.

[0129] Envisu (R2200, Bioptigen Inc., Durham, NC, USA) was coupled to a broadband LED light source (T870-HP, Superlum Diodes, Ltd, Ireland). Confocal laser scanning ophthalmoscopy (cSLO) (Spectralis HRA, Heidelberg Engineering, Franklin, MA, USA) was used for visualization of retinal AF using BluePeak™ or simply blue AF (488 nm excitation) and near-infrared AF (787 nm excitation) imaging modes.

[0130] Electroretinography Mice were dark-adapted overnight and anesthetized using the same procedure. Electroretinograms were recorded with an Espion E3 system (Diagnosys LLC, Lowell, MA) equipped with a Ganzfeld Color Dome stimulator as previously described. All electroretinograms were performed at the same time of day.

[0131] Tissue preparation and immunofluorescence Immunofluorescence on frozen sections was performed as previously described. Primary antibodies used: mouse anti-CEP (1:200, (gift from John Crabb, Cleveland Clinic, OH); rabbit anti-rhodopsin (1:200; Abcam); rabbit anti-L-FT (1:1000, gift from Maura Poli and Paolo Arosio, University of Brescia, Italy). Images were acquired with a drop fluorescence microscope (Nikon 80i microscope, BN Nikon, Tokyo, Japan) and analyzed using NIS-Elements (Nikon).

[0132] Plastic sections and toluidine blue staining Plastic sections (3 μm) were cut in the sagittal plane. The third eyelid was used for orientation during eyecup implantation. Toluidine blue staining on plastic sections was used to assess retinal morphology.

[0133] RNA extraction and quantitative RT-PCR Neural retinal tissue was isolated as previously described (e.g., M Hadziahmetovic et al. Age-dependent retinal iron accumulation and degeneration in hepcidin knockout mice. Investigative ophthalmology & visual science. Jan 5 2011;52(1):109-18). Changes in gene expression were assessed in neural retina and purified RPE cells. Gapdh was used as an endogenous control. Taqman probes (ABI, Grand The following proteins were used (Mm00441941), Slc7a11 (Mm00442530), Gpx4 (Mm00515041), GSS (Mm00515065), GSTm1 (Mm00833915), Cat (Mm00437992), Sod1 (Mm01700393), Hmox1 (Mm00516005), Cd68 (Mm03047343), IL-1β (Mm00434228), and IL-6 (Mm00446190). The amount of target mRNA was compared between groups of interest. All reactions were performed in technical triplicates (three reactions per eye) and biological replicates (three to five mice per genotype).

[0134] statistical analysis Statistical analysis was performed using GraphPad Prism 6.0 (San Diego, CA). One-way analysis of variance (ANOVA) was performed, and post-hoc analysis was performed using the Tukey-Kramer test when differences were observed in the ANOVA test (p<0.05). Mean values ​​± SEM were calculated for each group.

Claims

1. 1. A method for assessing deuterated docosahexaenoic acid used to monitor a patient for uptake of deuterated docosahexaenoic acid, comprising: using one or more blood samples obtained after initiation of treatment from said patient who has been administered an effective dose of deuterated docosahexaenoic acid or an ester thereof on a regular basis; obtaining the amount of deuterated docosahexaenoic acid and the total amount of docosahexaenoic acid measured in the one or more blood samples; Evaluating the amount of the measured deuterated docosahexaenoic acid relative to the total amount of docosahexaenoic acid; comparing the assessed amount of deuterated docosahexaenoic acid to a standard concentration curve, wherein the curve is based on the specific dose of deuterated docosahexaenoic acid or its ester used, the blood constituent being assessed, and the length of time since the start of treatment; and providing the results of said comparison for determining whether said patient is adequately absorbing deuterated docosahexaenoic acid.

2. The method of claim 1 , wherein the blood component being evaluated is plasma.

3. The method of claim 1 , wherein the blood component being evaluated is a red blood cell.

4. 10. The method of claim 1, wherein the length of time between initiation of treatment and testing is from about 3 to about 45 days.

5. 5. The method of claim 4, wherein the length of time between initiation of treatment and testing is at least about 14 days.

6. 5. The method of claim 4, wherein the length of time between initiation of treatment and testing is at least about 30 days.

7. 1. A method for assessing deuterated docosahexaenoic acid used to monitor a patient for uptake of deuterated docosahexaenoic acid, comprising: using one or more plasma samples obtained after initiation of treatment from the patient receiving regular administration of an effective dose of deuterated docosahexaenoic acid or an ester thereof, wherein the dose is about 250 mg / day; obtaining the amount of deuterated docosahexaenoic acid and the total amount of docosahexaenoic acid measured in the one or more blood samples; Evaluating the amount of the measured deuterated docosahexaenoic acid relative to the total amount of docosahexaenoic acid; comparing the estimated amount of deuterated docosahexaenoic acid to a standard concentration curve, wherein the curve is based on the length of time from the start of treatment; and providing the results of said comparison for determining whether said patient is adequately absorbing deuterated docosahexaenoic acid.

8. 1. A method for assessing deuterated docosahexaenoic acid for use in monitoring a patient for deuterated docosahexaenoic acid uptake, comprising: using one or more blood samples obtained after initiation of treatment from the patient receiving regular administration of an effective dose of deuterated docosahexaenoic acid or an ester thereof, wherein the dose is about 500 mg / day; obtaining the amount of deuterated docosahexaenoic acid and the total amount of docosahexaenoic acid measured in the one or more blood samples; Evaluating the amount of the measured deuterated docosahexaenoic acid relative to the total amount of docosahexaenoic acid; comparing the estimated amount of deuterated docosahexaenoic acid to a standard concentration curve, wherein the curve is based on the length of time from the start of treatment; and providing the results of said comparison for determining whether said patient is adequately absorbing deuterated docosahexaenoic acid.

9. 1. A method for assessing deuterated docosahexaenoic acid used to monitor a patient for uptake of deuterated docosahexaenoic acid, comprising: using one or more plasma samples obtained after initiation of treatment from the patient receiving regular administration of an effective dose of deuterated docosahexaenoic acid or an ester thereof, wherein the dose is about 1,000 mg / day; obtaining the amount of deuterated docosahexaenoic acid and the total amount of docosahexaenoic acid measured in the one or more blood samples; Evaluating the amount of the measured deuterated docosahexaenoic acid relative to the total amount of docosahexaenoic acid; comparing the estimated amount of deuterated docosahexaenoic acid to a standard concentration curve, wherein the curve is based on the length of time from the start of treatment; and providing the results of said comparison for determining whether said patient is adequately absorbing deuterated docosahexaenoic acid.

10. 2. The method of claim 1, wherein if the concentration of deuterated docosahexaenoic acid is lower than the concentration provided by the standardized curve, the result is provided as an indication to adopt one of the following options: improving the patient's diet to reduce the amount of DHA-containing fat consumed per day and / or increasing the amount of drug administered.

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