Methods for assessing a patient's response to treatment of neurodegenerative diseases with deuterated arachidonic acid - Patent Application 20070229633

Abstract

A diagnostic method is disclosed for assessing the presence or absence of therapeutic levels of deuterated polyunsaturated fatty acids in diseased neurons during treatment of a patient with a neurodegenerative disease.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 17 / 169,271, filed February 5, 2021, U.S. Patent Application No. 17 / 391,909, filed August 2, 2021, and U.S. Provisional Patent Application No. 63 / 177,794, filed April 21, 2021, each of which is incorporated by reference in its entirety herein. FIELD OF THEINVENTION A diagnostic method is disclosed for assessing the presence or absence of therapeutic (effective) concentrations of deuterated polyunsaturated fatty acids in degenerating neurons during treatment of a patient with a neurodegenerative disease. [Background technology]

[0002] State of the art Lipid autooxidation of polyunsaturated fatty acids (PUFAs) in neurons has been implicated in the pathogenesis of many neurodegenerative diseases. Central to this oxidative pathway is the presence of a labile bis-allylic hydrogen atom found in arachidonic acid, the predominant PUFA found in neurons.

[0003] In cell membranes, arachidonic acid is stacked together and an oxidative process involving reactive oxygen species (ROS) acts as an initiator of autoxidation of those PUFAs by abstraction of bis-allylic hydrogen and formation of oxidative reactive species in the PUFAs. The initial oxidation at the first bis-allylic site then leads to the successive oxidation of additional PUFAs in the cell or mitochondrial membrane. The oxidative process starts with abstraction of hydrogen at the bis-allylic site on the first PUFA, then the next (second) PUFA, and so on in a sequential manner. At some point, the oxidative process damages or destroys the viability of neurons, which leads to the promotion of pathologies that cause the generation of excessive amounts of ROS.

[0004] It has been disclosed that deuteration of one or more bisallylic acid sites of arachidonic acid found in neurons can attenuate the progression of neurodegenerative diseases. The stability of deuterium-carbon bonds against such oxidation processes is significantly stronger (more stable) than that of hydrogen-carbon bonds. This means that carbon-deuterium bonds reduce the generation of oxidative species at bisallylic sites, thus inhibiting the autooxidation pathway. Termination of this pathway then enhances the survival of neurons, thereby attenuating the progression of diseases.

[0005] Clinical studies to date have shown that certain neurodegenerative diseases, including tauopathies, amyotrophic lateral sclerosis (ALS), infantile neuroaxonal dystrophy (INAD), and Friedreich's ataxia (FA), respond to such treatments. In addition, preclinical studies have demonstrated that certain other neurodegenerative diseases, including mitochondrial deficiencies associated with ROS, Alzheimer's disease, Huntington's disease, Parkinson's disease, schizophrenia, and bipolar disorder, to name a few, respond to such treatments as well.

[0006] Treatment of these neurodegenerative diseases with deuterated PUFAs is typically accomplished by administration of an active deuterated PUFA or a prodrug thereof. For example, in neurodegenerative diseases, the active deuterated PUFA is deuterated arachidonic acid. However, the disease can be treated by administration of either deuterated arachidonic acid or a prodrug thereof. In one case, the prodrug is a C1-C5 alkyl ester of deuterated arachidonic acid. In another case, the prodrug is a C1-C5 alkyl ester of deuterated linoleic acid or a C1-C5 alkyl ester thereof. In the case of esters of any of these PUFAs, they are easily removed by the conditions found in the stomach to provide the free acid or a salt thereof. In the case of deuterated linoleic acid, a portion of this compound is enzymatically converted to deuterated arachidonic acid, thereby providing the patient with a source of deuterated arachidonic acid.

[0007] While this treatment has been shown to be successful in clinical studies and animal models, it remains to be determined whether a given patient achieves or maintains (has) therapeutic concentrations of deuterated arachidonic acid in their neurons. Such concerns arise from a variety of difficulties in controlling complications. These include: PUFAs incorporated into cell membranes, including arachidonic acid, have a half-life determined by their turnover rate (the rate at which damaged PUFAs are removed and replaced by new PUFAs, combined with the rate at which arachidonic acid molecules are extracted (released) from the cell membrane as part of their bioconversion to prostaglandins, prostacyclin (PGI2), thromboxane A2 (TxA2), or a series of hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acids (DiHETEs), as well as numerous other breakdown products). The amount of PUFA (including deuterated PUFA) absorbed by humans depends on the total amount of PUFA consumed. Typically, the human metabolism cannot absorb the entire amount of consumed PUFA, and as PUFA consumption increases, the percentage of absorbed PUFA decreases. Since deuterated PUFA is included in the total amount of consumed PUFA, the percentage of PUFA that is not absorbed should include the same proportion of unabsorbed deuterated PUFA. Thus, if only 80% of the total PUFA consumed is absorbed, then only 80% of the deuterated PUFA will be absorbed. Furthermore, the amount absorbed is of course a variable that varies between individuals. The amount of PUFAs absorbed also depends on whether such absorption is hindered by digestive problems, such as diarrhea associated with C. difficile. In such cases, many nutrients, including PUFAs, consumed are not absorbed by the body but are simply "washed out." · For a given individual, their diet does not remain stagnant; therefore, PUFA consumption may vary over time, depending on absorption rates. Finally, the issue of patient compliance with the administration of deuterated PUFAs is a related factor regarding the amount of deuterated PUFA absorbed.

[0008] These concerns should all be evaluated to determine whether patients receiving deuterated PUFA therapy are achieving and / or maintaining therapeutic (effective) concentrations in their neurons.

[0009] Assessment of the concentration of deuterated arachidonic acid in neurons is further complicated because neurons are inaccessible and clinicians cannot simply extract samples of these neurons to assess the concentration. Administered deuterated arachidonic acid is taken up systemically into all cells. Data to date suggest that the body shunts a larger portion of deuterated arachidonic acid between neurons and the CSF compared to other parts of the body. Thus, the concentration of deuterated arachidonic acid in surrogate or reporter cells such as skin cells, red blood cells, etc. does not necessarily represent the concentration of deuterated arachidonic acid in neurons or in the CSF. Furthermore, spinal tap to extract CSF is not feasible as it requires a significant amount of time (e.g., as much as 30 minutes) and can cause significant discomfort to the patient, such as headaches and back pain that can last for days or weeks.

[0010] Therefore, there is a continuing need for a safe and effective diagnostic test that can determine whether a patient with a neurodegenerative disease treatable with deuterated PUFA has therapeutic or subtherapeutic levels of deuterated arachidonic acid in the patient's neurons, and also helps to evaluate whether the patient's metabolism is indicative of adequate absorption of deuterated arachidonic acid. Summary of the Invention

[0011] overview Provided herein is a diagnostic test for assessing whether therapeutic concentrations of deuterated arachidonic acid are present in neurons of a patient being treated for a neurodegenerative disease treatable with deuterated arachidonic acid or a prodrug thereof, without the need to access the patient's neurons or cerebrospinal fluid, based in part on defining reproducible correlations from separate clinical studies treating different treatable neurodegenerative diseases, all using the same deuterated arachidonic acid, esters thereof or prodrugs thereof.

[0012] The amount of deuterated arachidonic acid taken up by cells increases gradually over time. Thus, therapeutic concentrations of deuterated arachidonic acid are not achieved immediately. Rather, there is a lag time between the initiation of treatment and the appearance of therapeutic outcomes that may vary from patient to patient. At a certain time point after the initiation of treatment for a neurodegenerative disease, one or more assessments of the patient's physical response to the treatment are required to determine whether therapeutic outcomes have been achieved and whether the therapeutic effect is maintained. In one embodiment, such physical assessments are combined with obtaining samples of the patient's reporter cells, which are then utilized to determine the concentration of deuterated arachidonic acid in the cells. Alternatively, samples of the patient's reporter cells are obtained at regular intervals. First, the patient's therapeutic outcome is confirmed, and the clinician or statistician can correlate the concentration of deuterated arachidonic acid in the reporter cells at that time point as the minimum concentration required for therapy.

[0013] Therapeutic concentrations were evaluated from clinical studies of different neurodegenerative diseases and the data supported the conclusion that there are equivalent therapeutic concentrations of deuterated arachidonic acid that provide for therapeutic outcome in each of the diseases. Because each of these diseases involves a different etiology, it was surprising that equivalent therapeutic concentrations of deuterated arachidonic acid were found to be therapeutic.

[0014] Thus, in one embodiment, there is provided a method for determining a minimum therapeutic concentration of deuterated arachidonic acid in neurons of a patient with a neurodegenerative disease treatable with deuterated arachidonic acid, without the need to access either the patient's neurons or cerebrospinal fluid, the method comprising the steps of: a) obtaining data from a representative number of human clinical studies of neurodegenerative diseases, each treatable with the same deuterated arachidonic acid or a prodrug thereof, the data comprising the concentration of deuterated arachidonic acid in accessible reporter cells at one or more time points after initiation of treatment; b) assess when patients show evidence of treatment outcomes; c) identifying the mean time from initiation of treatment to onset of therapeutic outcome and the mean concentration of deuterated arachidonic acid in reporter cells at such onset for each neurodegenerative disease; d) correlating the mean concentration of deuterated arachidonic acid in the reporter cell population with the mean time required for onset of a minimally required therapeutic outcome for each neurodegenerative disease as indicating evidence that the neurons have a minimally required therapeutic concentration of deuterated arachidonic acid for that disease; e) confirming that the minimum required therapeutic concentration of deuterated arachidonic acid and the mean time to onset of therapeutic results for each neurodegenerative disease are comparable to other neurodegenerative diseases; and f) based on said (therapeutic) equivalence, said mean minimum required therapeutic concentration and said mean time required for onset of therapeutic result are considered relative to other neurodegenerative diseases.

[0015] In some embodiments, a non-invasive method is provided for determining a concentration of deuterated arachidonic acid in reporter cells in a patient population suffering from a neurodegenerative disease treatable with deuterated arachidonic acid that correlates with onset of a therapeutic effect, the method comprising the steps of: a) obtaining data from a patient population having a first neurodegenerative disease during a treatment regimen with deuterated arachidonic acid or a prodrug thereof, the data comprising a concentration of deuterated arachidonic acid in a reporter cell population of the patients at one or more time points after initiation of the treatment; b) assess when patients show evidence of treatment outcomes; c) identifying the mean time from initiation of treatment to onset of therapeutic outcome and the mean concentration of deuterated arachidonic acid in reporter cells for each patient at the time of onset for each neurodegenerative disease; d) correlating the mean concentration of deuterated arachidonic acid in a reporter cell population of said patient as evidence that the patient's neurons have a minimum required therapeutic concentration of deuterated arachidonic acid for said neurodegenerative disease with the mean time required for onset of a minimum required therapeutic outcome for said neurodegenerative disease; e) repeating steps a)-d) for one or more additional neurodegenerative diseases; f) assessing the minimum required therapeutic concentration of deuterated arachidonic acid and the mean time to onset of therapeutic result for each of said one or more additional neurodegenerative diseases; and g) Obtaining the mean minimum required therapeutic concentration and the mean time required for onset of said therapeutic outcome for a set of neurodegenerative diseases.

[0016] In some embodiments, a method is provided for determining a patient's response to administration of a composition comprising deuterated arachidonic acid or a prodrug thereof during treatment of a treatable neurodegenerative disease, the method comprising the steps of: obtaining a population of red blood cells from said patient suffering from a neurodegenerative disease at a predefined time point after initiation of treatment when the outcome of the treatment should be evident (manifest) for the disease; assessing the concentration of said deuterated arachidonic acid in said red blood cells at that time, said concentration being relative to the amount of arachidonic acid present, including deuterated arachidonic acid; comparing said assessed concentration to a predefined concentration for a time point at which a therapeutic outcome is evident; determining whether the evaluation concentration is a therapeutic concentration or a sub-therapeutic concentration for a treatable neurodegenerative disease; Based on said determination, the amount of said deuterated arachidonic acid or prodrug thereof administered to said patient is optionally modified.

[0017] In one embodiment, a method is provided for determining whether a patient undergoing treatment for a treatable neurodegenerative disease with deuterated arachidonic acid or a prodrug thereof has reached an onset of therapeutic benefit or maintained a therapeutic concentration of said deuterated arachidonic acid in neurons of said patient, comprising the steps of: obtaining a population of reporter cells from said patient suffering from a neurodegenerative disease at a time when a treatment result is evident for the disease and at a predefined set of time points after initiation of the treatment; assessing the concentration of deuterated arachidonic acid in the reporter cells at that time; comparing said evaluated concentration to said given concentration for a time point at which a therapeutic result is evident; determining whether the evaluation concentration is a therapeutic concentration or a sub-therapeutic concentration for a treatable neurodegenerative disease; Based on said determination, the amount of said deuterated arachidonic acid or prodrug thereof administered to said patient is optionally modified.

[0018] In one embodiment, the method is repeated at a later time point to assess whether the patient has reached or maintained a therapeutic level.

[0019] In one embodiment, samples of reporter cells are taken at a set time about one month after the start of treatment, after which any subsequent samples can be taken at intervals such as one month thereafter, three months thereafter, semi-annually, or annually.

[0020] In one embodiment, the deuterated arachidonic acid is D2-arachidonic acid, D4-arachidonic acid or D6-arachidonic acid. Non-limiting examples of D2-arachidonic acid include 7,7-D2-arachidonic acid, 10,10-D2-arachidonic acid and 13,13-D2-arachidonic acid. Non-limiting examples of D4-arachidonic acid include 7,7,10,10-arachidonic acid, 7,7,13,13-D4-arachidonic acid and 10,10,13,13-D4-arachidonic acid. D6-arachidonic acid (as defined herein) includes 7,7,10,10,13,13-D6-arachidonic acid. Preferably, the deuterated arachidonic acid is D2-arachidonic acid or D6-arachidonic acid. In some embodiments, D6-arachidonic acid encompasses both 7,7,10,10,13,13-D6-arachidonic acid and compositions comprising deuterated arachidonic acid having at least one non-bis-allylic position in which at least 90% of the hydrogen atoms at positions 7,7,10,10,13,13 are replaced with deuterium and the level of deuterium replacement is above its natural abundance. In one embodiment, the non-bis-allylic positions are optionally substituted with deuterium at up to about 35%, preferably about 1-35%, and more preferably about 1-10% of the remaining hydrogen atoms.

[0021] In one embodiment, the reporter cells are any accessible cells in the body, preferably easily accessible without causing undue pain or inconvenience to the patient. Suitable reporter cells include, by way of example only, red blood cells, skin cells, fat cells, biopsy cells, epithelial cells, including those found in urine, and the like. In one embodiment, the reporter cells are preferably red blood cells.

[0022] In one embodiment, a method is provided for determining a patient's response to administration of a composition comprising deuterated arachidonic acid or a prodrug thereof during treatment of a treatable neurodegenerative disease, the method comprising the steps of: obtaining a population of red blood cells from a patient suffering from a neurodegenerative disease at a predefined time point after initiation of treatment that is a time point at which a therapeutic outcome is evident for that disease; assessing the concentration of deuterated arachidonic acid in the red blood cells at that time; comparing said evaluated concentration to a predefined concentration for a time point at which a therapeutic result is evident; determining whether the evaluation concentration is a therapeutic concentration or a sub-therapeutic concentration for a treatable neurodegenerative disease; and Based on the determination, the amount of the deuterated arachidonic acid or a prodrug thereof administered to the patient is optionally modified.

[0023] In one embodiment, separate clinical studies of treatable neurodegenerative diseases demonstrate that a concentration of about 3% or more deuterated 13,13-D2-arachidonic acid relative to the total amount of arachidonic acid, including deuterated arachidonic acid, in red blood cells acting as reporter cells correlates with the minimum concentration required to achieve a therapeutic outcome in those diseases, thus establishing a baseline for achieving a therapeutic outcome in those neurodegenerative diseases treatable with 13,13-D2-arachidonic acid and demonstrating that the concentration of 13,13-arachidonic acid in degenerating neurons is sufficient to provide treatment for the disease being treated.

[0024] In one embodiment, a diagnostic test is provided for determining whether a patient having a treatable neurodegenerative disease has a therapeutic or sub-therapeutic concentration of deuterated 13,13-D2-arachidonic acid without accessing the patient's neurons or spinal fluid, the test comprising the steps of: Obtaining a blood sample from a patient undergoing treatment for a neurodegenerative disease with 13,13-D2-arachidonic acid or its esters; Determine the concentration of 13,13-D2-arachidonic acid in red blood cells (RBCs); and The concentration is compared to a minimum therapeutic concentration of at least 3% 13,13-D2-arachidonic acid based on the total amount of arachidonic acid, including deuterated arachidonic acid, in the RBCs to determine whether the patient has therapeutic or subtherapeutic concentrations of D2-arachidonic acid in their neurons.

[0025] In one embodiment, higher concentrations of 13,13-D2-arachidonic acid are contemplated to provide enhanced therapeutic results, and the attending physician may conclude that such higher concentrations will be the target concentrations that will bring therapeutic results to a particular patient or group of patients.For example, the concentration of 13,13-D2-arachidonic acid can be set at 4%, 5%, 6%, 7%, 8%, 9% or even 10% as a specific therapeutic goal for a given patient, knowing that the concentration of 13,13-D2-arachidonic acid is the baseline for minimally required therapeutic results.The increase in the target concentration by the attending physician can be based on factors such as the age and weight of the patient, the condition of the patient, the progression of the disease, and other factors that are well known to clinicians.

[0026] Recognizing that deuteration at one or both of the remaining bis-allylic sites increases the stability of arachidonic acid against lipid autooxidation, the minimum required therapeutic concentration of D2-arachidonic acid described above can be correlated with the minimum therapeutic concentrations of D4-arachidonic acid or D6-arachidonic acid.

[0027] In one embodiment, a diagnostic test is provided for determining whether the concentration of deuterated D4-arachidonic acid in a patient having a treatable neurodegenerative disease is therapeutic or sub-therapeutic without accessing the patient's neurons or cerebrospinal fluid, the test comprising the steps of: Obtaining a blood sample from a patient undergoing treatment for a neurodegenerative disease with D4-arachidonic acid or an ester thereof; and The concentration of D4-arachidonic acid in red blood cells (RBCs) is determined and compared to a minimum therapeutic concentration of at least 1% D4-arachidonic acid based on the total amount of arachidonic acid, including deuterated arachidonic acid, to determine whether the patient has therapeutic or subtherapeutic concentrations of D4-arachidonic acid in neurons.

[0028] In one embodiment, a diagnostic test is provided for determining whether the concentration of deuterated D6-arachidonic acid in a patient having a treatable neurodegenerative disease is therapeutic or sub-therapeutic without accessing the patient's neurons or cerebrospinal fluid, the test comprising the steps of: Obtaining a blood sample from a patient undergoing treatment for a neurodegenerative disease with D4-arachidonic acid or an ester thereof; and The concentration of D6-arachidonic acid in red blood cells (RBCs) is determined and compared to a minimum therapeutic concentration of at least 0.5% D6-arachidonic acid, based on the total amount of arachidonic acid, including deuterated arachidonic acid, to determine whether the patient has therapeutic or subtherapeutic concentrations of D6-arachidonic acid in neurons.

[0029] In one embodiment, higher concentrations of either D4- or D6-arachidonic acid are provided for enhanced therapeutic results, and the attending physician can conclude that these concentrations are the minimum required target concentrations to produce a therapeutic result when enhanced results are set as target concentrations. For example, the concentrations of D4- and D6-arachidonic acid can be set as set forth in Table 1 below, where the concentrations are determined as described above:

[0030] [Table 1]

[0031] In one embodiment, a kit of parts is provided, including diagnostic materials for carrying out the method, as well as a correlation table correlating the concentration of deuterated arachidonic acid in red blood cells to that of human cerebrospinal fluid. In addition to the correlation table, the diagnostic materials can include one or more instructions as to when such tests should be performed, factors suggesting delaying such tests, etc. [Brief description of the drawings]

[0032] [Figure 1]FIG. 1 is a graph showing the percentage (%) of 13,13-D2-arachidonic acid in red blood cells (RBCs) and cerebrospinal fluid (SF) at the indicated time points after initiation of treatment with 11,11-D2-linoleic acid in adult patients. [Diagram 2] FIG. 2 is a graph showing the percentage (%) of 13,13-D2-arachidonic acid in red blood cells (RBC) and cerebrospinal fluid (SF) at the indicated time points after initiation of treatment with 11,11-D2-linoleic acid in young patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description Diagnostic methods or tests are provided for determining whether a patient undergoing treatment for a treatable neurodegenerative disease exhibits a minimum therapeutic concentration of deuterated arachidonic acid in the patient's neurons. Methods or tests are also provided for determining whether a patient undergoing treatment for a treatable neurodegenerative disease has reached or maintained a therapeutic concentration of deuterated arachidonic acid.

[0034] Before describing the invention in more detail, the following terms will first be defined. Terms not defined will be given a definition in context or their medically acceptable definition will be given.

[0035] 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.

[0036] As used herein, the term "optional" or "optionally" means that an event or circumstance described below may or may not occur, and that the description is meant to include cases where the event or circumstance occurs and cases where it does not occur.

[0037] As used herein, the term "about" when used in front of numerical designations such as temperature, time, amount, concentration, and other such designations, including ranges, indicates approximations that may vary by plus or minus (+ / -) 10%, 5%, 1%, or any sub-range or sub-value therebetween. Preferably, the term "about" when used in reference to the minimum therapeutic concentration means that the dose may vary by ±10%.

[0038] As used herein, the terms "comprising" or "comprising" mean that the compositions and methods include the recited elements, but do not exclude others.

[0039] As used herein, the term "consisting essentially of" when used in defining compositions and methods means excluding other elements of any essential significance 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.

[0040] As used herein, the term "consisting of" means excluding other ingredients and more than traces of substantial method steps. Embodiments defined by each of these transitional phrases are within the scope of this invention.

[0041] The term "linoleic acid" as used herein refers to the compound having the following formula, having the natural abundance of deuterium at each hydrogen atom, and pharma- ceutically acceptable salts thereof: [ka] Esters of linoleic acid are formed by replacing the -OH group with -OR. Such esters are as defined below.

[0042] As used herein and unless the context indicates otherwise, the term "deuterated linoleic acid or ester thereof" refers to a linoleic acid or ester compound containing one or two deuterium atoms at its 11-position. Specific compounds encompassed by this definition include, by way of example only, 11-D1-linoleic acid, 11,11-D2-linoleic acid, 8,11-D2-linoleic acid, 8,11,11-D3-linoleic acid and 8,8,11,11-D4-linoleic acid, and esters of any one of these compounds. Additional stabilization of the bis-allylic positions may also include the replacement of one or more of the bis-allylic carbon atoms with a heavy isotope, either alone or in combination with deuteration (or triteration). This is because isotope effects (IE), which result in stabilizing bonds with heavy isotopes, are additive to long-established and fundamental chemical theory (Westheimer, Chem. Rev. (1961) 61:265-273; Shchepinov, Rejunvenation Res, (2007), 10:47-59; Hill et al., Free Radic. Biol. Med. (2012) 53:893-906; Andreyev et al., Free Radic. Biol. Med. (2015) 82:63-72; Bigeleisen, J., “The validity of the use of tracers to follow chemical reactions.” Science (1949) 110: 14-16).

[0043] As used herein, arachidonic acid has the numbering system set forth below: [ka] Here, each of the 7, 10 and 13 positions are bis-allylic positions within the structure.

[0044] As used herein and unless the context indicates otherwise, "deuterated arachidonic acid or ester thereof" refers to an arachidonic acid or ester compound having at least one deuterium atom at the bisallylic position and optionally additional deuterium atoms at other positions within the molecule. Such deuterated arachidonic acids include mono-bisallylic deuterated arachidonic acids having a deuterium atom at either the 7-, 10-, or 13-position, as well as those that are di-, tri-, tetra-, penta-, hexa-deuterated at the bisallylic site, and optionally those having deuteration at sites other than the bisallylic site at levels greater than the natural abundance of deuterium.

[0045] The term deuterated arachidonic acid includes 7,7-D2-arachidonic acid, 10,10-D2-arachidonic acid, and 13,13-D2-arachidonic acid. The term D4-arachidonic acid includes 7,7,10,10-D4-arachidonic acid, 7,7,13,13-D4-arachidonic acid, and 10,10,13,13-D4-arachidonic acid. The term D6-arachidonic acid includes 7,7,10,10,13,13-D6-arachidonic acid, as well as deuterated arachidonic acid in which at least 90% of the hydrogen atoms at the 7,7,10,10,13,13 positions are replaced with deuterium, and optionally up to 35% of the remaining hydrogen atoms at the non-bisallylic positions are replaced with deuterium.

[0046] The term "ester" as used herein means any pharma- ceutically acceptable ester of deuterated linoleic acid or deuterated arachidonic acid, such as C1-C6 alkyl esters, glycerol (including monoglycerides, diglycerides and triglycerides), sucrose esters, phosphate esters, etc. The particular ester used is not critical, so long as the ester is pharma- ceutically acceptable (non-toxic and biocompatible).

[0047] As used herein, the term "prodrug of deuterated arachidonic acid" refers to esters of deuterated arachidonic acid, e.g., C1-C5 alkyl esters, esters of deuterated linoleic acid, e.g., C1-C5 alkyl esters, etc. Both deuterated arachidonic acid and deuterated linoleic acid esters are readily converted to the corresponding acid / salt forms in the gastrointestinal tract after administration. A portion of the administered deuterated linoleic acid is enzymatically converted to deuterated arachidonic acid, and thus acts as a prodrug of such deuterated arachidonic acid. In one embodiment, 11,1l-D2-linoleic acid is converted to 13,13-D2-arachidonic acid. In one embodiment, 8,8,11,1l-D4-linoleic acid is converted to 10,10,13,13-D4-arachidonic acid.

[0048] The term "treatable neurodegenerative disease" or "neurodegenerative disease treatable with deuterated arachidonic acid" means that a particular neurodegenerative disease is recognized as treatable with deuterated arachidonic acid, as compared to a neurodegenerative disease that is not treatable with deuterated arachidonic acid. Neurodegenerative diseases treatable with deuterated arachidonic acid include Alzheimer's disease, mild cognitive impairment, frontal dementia, amyotrophic lateral sclerosis, multiple sclerosis, Friedreich's ataxia, Parkinson's disease, tauopathies (including PSP), and Huntington's disease. On the other hand, data to date suggest that the following diseases, including Tay-Sachs disease, GPX-4 deficiency, and neuronal membrane diseases, are not treatable with deuterated arachidonic acid and / or other deuterated polyunsaturated fatty acids.

[0049] As used herein, the term "etiology" of a disease refers to the cause of the disease. The terms "onset" or "pathology" refer to the onset, structural / functional changes, and natural history associated with the disease. The term "natural history" refers to the progression of a disease in the absence of treatment, or the progression of a disease in the absence of treatment with deuterated arachidonic acid or a prodrug thereof.

[0050] The term "reduction in disease progression rate" as used herein means that the disease progression rate is attenuated after the start of treatment, compared with the natural history of the patient.In one embodiment, the reduction in disease progression rate in ALS is determined by using ALSFRS-R score to determine the disease progression rate in natural history, and then again by measuring the disease progression rate during the interval from the start of treatment to the end of a set period (e.g., 6 months) thereafter.Then, both rates are annualized, and the reduction in disease progression rate brings about at least 30% change rate between the ALSFRS-R score before and after.

[0051] For PSP, this involves determining the rate of disease progression in the natural history by using the Progressive Supranuclear Palsy Rating Scale or the Unified Parkinson's Disease Rating Scale, and then measuring the score again at set intervals from the start of treatment until after a set time thereafter (e.g., at 1 month, and every 3 months thereafter). Both rates are then annualized, and a reduction in the rate of disease progression results in at least a 30% change in the later score compared to the earlier score.

[0052] Similarly, similar scales exist for determining the rate of disease progression in Alzheimer's disease, Parkinson's disease, Huntington's disease, Friedreich's ataxia, and the like.

[0053] As used herein, the term "minimal therapeutic outcome" means that patients undergoing treatment with deuterated arachidonic acid or a prodrug thereof show evidence of a reduction in the rate of disease progression of at least about 30% compared to the rate of disease progression determined during the natural history.

[0054] As used herein, the term "patient" refers to a human patient or a cohort of human patients suffering from a neurodegenerative disease. When multiple (two or more) patients are evaluated, the average disease progression of those patients is used.

[0055] As used herein, the term "pharmaceutically acceptable salt" of a compound disclosed herein is within the scope of the present invention and includes acid or base salts 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 a compound of the present invention has a basic group, such as an amino group, a pharmaceutically acceptable salt can be formed with an inorganic acid (e.g., hydrochloric acid, boric acid, nitric acid, sulfuric acid, phosphoric acid, etc.), an organic acid (e.g., alginic acid, 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 an acidic amino acid (e.g., aspartic acid, glutamic acid, etc.). When a compound of the present invention has an acidic group, such as a carboxylic acid group, it can be formed with an acid such as an alkali metal or alkaline earth metal (e.g., Na + , Li + , K + , Ca 2+ , Mg 2+ , Zn 2+ ), ammonia or organic amines (e.g., dicyclohexylamine, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g., arginine, lysine and ornithine). Such salts can be prepared in situ during the isolation and purification of the compounds, or by separately reacting the purified compounds in their free base or free acid form with the appropriate acid or base, respectively, and isolating the salt so formed.

[0056] compound The portion of deuterated linoleic acid or its ester administered to the patient is converted in vivo to deuterated arachidonic acid as a deesterified acid or its salt, and the portion of deuterated linoleic acid so converted acts as a prodrug of deuterated arachidonic acid. There are numerous deuterated linoleic acid compounds that are commercially available or known in the art. These include, by way of example only, 11-D1-linoleic acid, 11,11-D2-linoleic acid, 8,8,11,11-D4 linoleic acid (which upon enzymatic desaturation elongation produces 10,10,13,13-D4-arachidonic acid). Other deuterated linoleic acids are described in U.S. Pat. No. 10,052,299, which is incorporated herein by reference in its entirety. In addition, 11-D1-linoleic acid is commercially available from Cayman Chemical Company, Ann Arbor, Michigan 48108, USA.

[0057] In addition to deuterated linoleic acid or its ester, deuterated arachidonic acid can be administered to a patient to bypass the enzymatic conversion required for its prodrug. Such deuterated arachidonic acids include 7,7-D2-arachidonic acid, 10,10-D2-arachidonic acid, 13,13-D2-arachidonic acid, 7,7,10,10-D4-arachidonic acid, 7,7,13,13-D4-arachidonic acid, 10,10,13,13-D4-arachidonic acid, and 7,7,10,10,13,13-D6-arachidonic acid, as disclosed by Shchepinov et al., Molecules, 28(12):3331 et seq. (2018), which is incorporated herein by reference in its entirety. Other deuterated arachidonic acid compounds are known in the art.

[0058] Additionally, deuterated arachidonic acid can be prepared by catalytic processes such as those described in U.S. Pat. No. 10,577,304, which is incorporated herein by reference in its entirety. These processes provide substantially complete deuteration at the bis-allylic sites, although some deuteration occurs at the non-bis-allylic sites, and the majority occurs at the mono-allylic sites. In general, deuteration at the three bis-allylic methylene sites converts six hydrogen atoms to deuterium with an efficiency of over 90%. This means that in a population of deuterated arachidonic acid, the six bis-allylic hydrogen atoms have an average of more than 5.4 deuterium atoms. Also, up to about 35% of the total number of hydrogen atoms in arachidonic acid (excluding the ester moiety) are replaced with deuterium. Since there are 32 hydrogen atoms in arachidonic acid, the percentage of deuterium in these compounds ranges from about 15% to as much as 35%. When the term "deuterated arachidonic acid" as defined refers to D6 arachidonic acid, it is understood that such arachidonic acid includes arachidonic acids having an average of more than 5.4 arachidonic acids at the bis-allylic site and having a range of deuteration from about 15% to as much as 35% based on the number of hydrogen atoms present in non-deuterated arachidonic acid containing protons on the carboxylic acid.

[0059] Esters of these deuterated fatty acids are prepared by conventional techniques well known in the art. Such esters are preferably derived from C1-C5 alkyl alcohols.

[0060] methodology Deuterated Linoleic Acid Some of the methods described herein utilize the enzymatic conversion of deuterated linoleic acid or an ester thereof to provide deuterated arachidonic acid. Specifically, it is well known that a portion of linoleic acid or an ester thereof consumed by an individual is converted in vivo to arachidonic acid, thereby acting as a prodrug of arachidonic acid.

[0061] In one embodiment, a patient treated with 11,11-D2-linoleic acid or an ester thereof will generate 13,13-D2-arachidonic acid in vivo. As noted above, a concentration of 13,13-D2-arachidonic acid in red blood cells is considered therapeutic when it reaches at least about 3% based on the total amount of arachidonic acid plus deuterated arachidonic acid found therein.

[0062] In one embodiment, a patient treated with 8,8,11,11-D4-linoleic acid or esters thereof will produce 10,10,13,13-D4-arachidonic acid in vivo. As noted above, a concentration of 10,10,13,13-D4-arachidonic acid in red blood cells is considered therapeutic when it reaches at least about 1% based on the total amount of arachidonic acid plus deuterated arachidonic acid found therein.

[0063] In one embodiment, a concentration of D6-arachidonic acid in red blood cells is considered therapeutic when it reaches at least about 1% based on the total amount of arachidonic acid plus deuterated arachidonic acid found therein.

[0064] Given that each disease has a distinct etiology and, in many cases, distinct pathology, it is surprising that each treatable neurodegenerative disease requires a similar threshold amount of deuterated arachidonic acid to achieve the minimal therapeutic concentration necessary to achieve the respective therapeutic outcome.

[0065] In one embodiment, deuterated linoleic acid, such as 11,11-D2-linoleic acid, is administered in a dosage regimen that includes a primer dose and a maintenance dose. In one embodiment, the primer dose includes regular administration of 11,11-D2-linoleic acid or its ester. In one embodiment, the primer dose includes at least about 7 grams of 11,11-D2-linoleic acid or its ester per day. In one embodiment, the primer dose is continued for at least about 30 days, for example, to rapidly achieve a therapeutic concentration of 13,13-D2-arachidonic acid in vivo by enzymatically converting a portion of the 11,11-D2-linoleic acid to 13,13-D2-arachidonic acid. In an embodiment, after completion of the primer dose, a maintenance dose is administered periodically, and the maintenance dose is at least about 3 grams of 11,11-D2-linoleic acid or its ester per day.

[0066] Deuterated Arachidonic Acid Some of the methods described herein utilize deuterated arachidonic acid or its esters. In such methods, it is preferred to use D2-arachidonic acid, D4-arachidonic acid or D6-arachidonic acid as the specific deuterated arachidonic acid. In the case of D6-arachidonic acid, the method includes deuteration produced by a catalytic process as described above. In any case, red blood cell concentrations of deuterated arachidonic acid that have been found to be therapeutic are listed in Table 1 above.

[0067] In one embodiment, deuterated arachidonic acid, such as 7,7,10,10,13,13-D6-arachidonic acid, is administered in a regimen that includes an initial dose and a maintenance dose. In one embodiment, the initial dose includes regular administration of 7,7,10,10,13,13-D6-arachidonic acid or an ester thereof. In one embodiment, the initial dose includes about 0.5 grams to about 5 grams of 7,7,10,10,13,13-D6-arachidonic acid or an ester thereof per day. In one embodiment, the initial dose is continued for about 24 days to about 45 days to rapidly achieve therapeutic concentrations of 7,7,10,10,13,13-D6-arachidonic acid in vivo. In one embodiment, a maintenance dose is administered periodically after completion of the initial dose. In one embodiment, about 70% or less of the starting daily dose of 7,7,10,10,13,13-D6-arachidonic acid or ester thereof is administered (as a maintenance dose). Specific Embodiments

[0023] [Embodiment 1] A non-invasive method for determining a concentration of deuterated arachidonic acid in a reporter cell of a patient population, said concentration correlating with onset of therapeutic benefit in a patient population suffering from a neurodegenerative disease treatable with deuterated arachidonic acid, comprising the steps of: a) obtaining data from a first population of neurodegenerative disease patients during a treatment regimen with deuterated arachidonic acid or a prodrug thereof, the data comprising a concentration of deuterated arachidonic acid in a reporter cell population of the patients at one or more time points after initiation of the treatment; b) assess when patients demonstrated clear evidence of treatment results; c) identifying the mean time from initiation of treatment to onset of treatment outcome in reporter cells for each patient, and the mean concentration of deuterated arachidonic acid at the time of onset of each neurodegenerative disease; d) correlating the mean concentration of deuterated arachidonic acid in a reporter cell population of the patient as evidence that the patient neurons have a minimum required therapeutic concentration of deuterated arachidonic acid for the disease with the mean time required for onset of a minimum required therapeutic outcome for the neurodegenerative disease; e) repeating steps a)-d) for one or more additional neurodegenerative diseases; f) assessing the minimum required therapeutic concentration of deuterated arachidonic acid and the mean time to onset of therapeutic result for each of said one or more additional neurodegenerative diseases; and g) obtaining the average minimum required therapeutic concentration and the average time required for onset of said therapeutic outcome for a set of neurodegenerative diseases; The method includes: [Embodiment 2] A method for determining a patient's response to administration of a composition comprising deuterated arachidonic acid or a prodrug thereof during treatment of a treatable neurodegenerative disease, comprising the steps of: obtaining a population of red blood cells from a patient suffering from a neurodegenerative disease at a predefined time point after initiation of treatment that is a time point at which a treatment result will be evident for that disease; assessing a concentration of said deuterated arachidonic acid in said red blood cells, said concentration being relative to an amount of arachidonic acid comprising deuterated arachidonic acid; comparing said evaluated concentration to said predefined concentration for a time point at which a therapeutic result is evident; determining whether the evaluation concentration is a therapeutic concentration or a sub-therapeutic concentration for a treatable neurodegenerative disease; Based on the determination, the dose of the deuterated arachidonic acid or a prodrug thereof administered to the patient is arbitrarily changed. The method includes: [Embodiment 3] Any of the above embodiments, wherein a reporter cell sample is obtained about one month after initiation of treatment. [Embodiment 4] Any of the above embodiments, wherein further samples of reporter cells are obtained at monthly, then three months, then semi-annual, or yearly intervals thereafter. [Embodiment 5] Any of the above embodiments, wherein the reporter cell is a red blood cell, a skin cell, an adipocyte, a biopsy cell, or an epithelial cell. [Embodiment 6] Any of the above embodiments, wherein the deuterated arachidonic acid is 13,13-D2-arachidonic acid. [Embodiment 7] Any of the above embodiments, wherein the deuterated arachidonic acid is D4-arachidonic acid. [Embodiment 8] Any of the above embodiments, wherein the deuterated arachidonic acid is D6-arachidonic acid, in which at least 90% of the hydrogen atoms at positions 7, 7, 10, 10, 13, and 13 are replaced with deuterium, and optionally up to 35% of the remaining hydrogen atoms at non-bis-allylic positions are replaced with deuterium. [Embodiment 9] A method for assessing a patient's response to administration of a composition comprising deuterated arachidonic acid or a prodrug thereof during treatment of a treatable neurodegenerative disease, comprising the steps of: assessing a concentration of deuterated arachidonic acid in the patient's red blood cells relative to a total abundance of arachidonic acid, including deuterated arachidonic acid; and comparing said concentration to a predefined minimum required therapeutic concentration of said deuterated arachidonic acid in said red blood cells; said patient's red blood cells are deemed to be sub-therapeutic when they have a concentration of said deuterated arachidonic acid that is less than said defined therapeutic concentration, and are deemed to be therapeutic when said concentration of deuterated arachidonic acid is equal to or greater than said defined therapeutic concentration; and Optionally, the method is repeated as necessary to evaluate whether the patient's concentration of deuterated arachidonic acid in the red blood cells has reached or remains at a therapeutic concentration. The method includes: [Embodiment 10] Embodiment 9, wherein a reporter cell sample is obtained about one month after the initiation of treatment.

[0046] ... [Embodiment 12] Any of the above embodiments, wherein the reporter cell is a red blood cell, a skin cell, an adipocyte, a biopsy cell, or an epithelial cell. [Embodiment 13] Any of the above embodiments, wherein the deuterated arachidonic acid is 13,13-D2-arachidonic acid. [Embodiment 14] Any of the above embodiments, wherein the deuterated arachidonic acid is D4-arachidonic acid. [Embodiment 15] Any of the above embodiments, wherein the deuterated arachidonic acid is D6-arachidonic acid, in which at least 90% of the hydrogen atoms at positions 7, 7, 10, 10, 13, and 13 in the deuterated D6-arachidonic acid are replaced with deuterium, and optionally up to 35% of the remaining hydrogen atoms at non-bis-allylic sites are replaced with deuterium. [Embodiment 16] A diagnostic method for determining whether the concentration of deuterated 13,13-D2-arachidonic acid in cerebrospinal fluid in a patient having a treatable neurodegenerative disease is at or below the therapeutic concentration without accessing the patient's neurons, comprising the steps of: Obtaining a blood sample from a patient undergoing treatment for a neurodegenerative disease with 13,13-D2-arachidonic acid or its esters; Determine the concentration of 13,13-D2-arachidonic acid in red blood cells (RBC); This concentration is compared to a minimum therapeutic concentration of 13,13-D2-arachidonic acid based on the total amount of arachidonic acid present in the RBCs, including deuterated arachidonic acid, to determine whether the patient has therapeutic or subtherapeutic levels of 13,13-D2-arachidonic acid in the neurons. Any of the above embodiments. [Embodiment 17] Embodiment 16, wherein the therapeutic concentration of 13,13-D2-arachidonic acid is set at 4%, 5%, 6%, 7%, 8%, 9%, or 10% as the therapeutic target for a given patient or cohort of patients. [Embodiment 18] Any of the above embodiments, wherein the reporter cell sample is obtained about one month after initiation of treatment. [Embodiment 19] Any of the above embodiments, wherein further samples of reporter cells are obtained at monthly, then three monthly, semi-annual, or yearly intervals thereafter. [Embodiment 20] A diagnostic method for determining whether the concentration of deuterated D4-arachidonic acid in a patient having a treatable neurodegenerative disease is at or below the therapeutic level without accessing the patient's neurons or cerebrospinal fluid, comprising the steps of: Obtaining a blood sample from a patient undergoing treatment for a neurodegenerative disease with D4-arachidonic acid or an ester thereof; and determining a concentration of D4-arachidonic acid in red blood cells (RBCs); and comparing this concentration to a minimum therapeutic concentration of at least 1% of D4-arachidonic acid based on the total amount of fatty acids (including D4-arachidonic acid) in said RBCs to determine whether said patient has a therapeutic or subtherapeutic concentration of D4-arachidonic acid in their neurons. The method includes: [Embodiment 21] Embodiment 20, wherein the therapeutic concentration of D4-arachidonic acid is set at 2%, 3% or 5% as a therapeutic target for a given patient or cohort of patients. [Embodiment 22] Any of the above embodiments, wherein the reporter cell sample is obtained about one month after initiation of treatment. [Embodiment 23] Any of the above embodiments, wherein further samples of reporter cells are obtained at monthly, then three monthly, semi-annual, or yearly intervals thereafter. [Embodiment 24] A diagnostic method for determining whether the concentration of deuterated D6-arachidonic acid in a patient having a treatable neurodegenerative disease is at or below the therapeutic level without accessing the patient's neurons or cerebrospinal fluid, comprising the steps of: Obtaining a blood sample from a patient undergoing treatment for a neurodegenerative disease with D4-arachidonic acid or an ester thereof; and determining a concentration of D6-arachidonic acid in red blood cells (RBCs); and comparing this concentration to a minimum therapeutic concentration of at least 0.5% D6-arachidonic acid based on the total amount of arachidonic acid (including deuterated arachidonic acid) in said RBCs to determine whether said patient has a therapeutic or subtherapeutic concentration of D6-arachidonic acid in their neurons. Any of the above embodiments. [Embodiment 25] Embodiment 24, wherein the therapeutic concentration of D6-arachidonic acid is set at 1%, 1.5%, 2%, 2.5%, or 3% as a therapeutic target for a given patient or cohort of patients. [Embodiment 26] Any of the above embodiments, wherein the reporter cell sample is obtained about one month after initiation of treatment. [Embodiment 27] Any of the above embodiments, wherein further samples of reporter cells are obtained at monthly, then three monthly, semi-annual, or yearly intervals thereafter. [Embodiment 28] Any of the above embodiments, wherein the deuterated arachidonic acid is D6-arachidonic acid, in which at least 90% of the hydrogen atoms at positions 7, 7, 10, 10, 13, and 13 are replaced with deuterium, and optionally up to 35% of the remaining hydrogen atoms at non-bis-allylic positions are replaced with deuterium. [Embodiment 29] Any of the above embodiments assembled into a kit of parts, including diagnostic materials for carrying out the method, as well as a correlation table correlating the concentration of deuterated arachidonic acid in red blood cells with the concentration of deuterated arachidonic acid in human cerebrospinal fluid. In addition to the correlation table, such diagnostic materials can include one or more instructions on when such tests should be performed, factors suggesting delaying such tests, etc. EXAMPLES

[0068] 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 exemplary embodiments, which are intended as illustrations of a single aspect of the present invention. Any method that is functionally equivalent is 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 the accompanying drawings. Such modifications fall within the scope of the appended claims. In these examples, the following terms are used herein and have the following meanings. Unless defined, abbreviations have their conventional medical meaning. D2-AA = 13,13-D2-arachidonic acid AA = arachidonic acid ALSFRS-R = ALS Functional Rating Scale-Revised BID = twice a day CNS = central nervous system D2-LA = 11,11-D2-linoleic acid LA = linoleic acid PK = pharmacokinetics PSP = progressive supranuclear palsy RBC = red blood cells SAE = serious adverse event. SF = cerebrospinal fluid TID = 3 times daily % Deuterated AA = the percentage of deuterated arachidonic acid based on the total amount of arachidonic acid (including deuterated arachidonic acid) present in the cells analyzed.

[0069] Example 1 - Determination of AA concentrations in RBCs and CSF / neurons of a single patient In this example, the relative concentrations of D2-AA in SF and RBC are determined based on the relative amounts of D2-AA to D2-LA to determine whether there is a 1:1 correlation between the two concentrations of D2-AA in SF and D2-AA in RBC. Specifically, the patient was continuously administered a daily dose of 9 grams of D2-LA ethyl ester over a period of about 6 months. Periodic samples of blood and SF were taken to measure the concentrations of both D2-LA and D2-AA in both RBC and SF. In all cases, D2-AA was obtained by deacylation of linoleic acid ethyl ester in the digestive tract followed by in vivo conversion of D2-LA to D2-AA. [Table 2] The results in the table show that the D2-AA concentration in the cerebrospinal fluid is already 8% based on the amount of arachidonic acid + deuterated arachidonic acid.

[0070] Next, Table 3 shows the concentrations of D2-LA and D2-AA in RBCs at 3 and 6 months for the same patients. [Table 3] Note that D2-AA concentrations in RBCs at 3 months were significantly lower than at 6 months, demonstrating a gradual increase in D2-AA over time. Furthermore, the ratio of D2-LA to D2-AA changed from 2.9:1 at 3 months to 2.1:1 at 6 months.

[0071] D2-AA can have a fairly linear rate of increase, since the amount of D2-AA increases in an incremental manner over time, based on the conversion of D2-LA, which limits the daily bioproduction of D2-AA. This is shown in Figure 1, where the solid line is defined by the D2-AA concentration at months 3 and 6, and then extrapolated to the start of treatment (month 0). From this linear relationship, the value of D2-AA at month 1 in RBCs is estimated. The amount at month 1 in SF is also provided (open circle).

[0072] Based on the above and Figure 1, it can be seen that the amount of D2-AA in RBCs at one month is about 3 percent (%) compared to 8% of the amount of D2-AA in SFs. Thus, this data suggests that in vivo concentrations of AA (including D2-AA) are more highly shunted into SFs (and therefore neurons) compared to RBCs and also other reporter cells.

[0073] Example 2: Determination of AA concentrations in RBCs and CSF / neurons in a cohort of 14 patients This example also determines the concentration of D2-AA in RBC.Specifically, a cohort of 14 patients was administered a daily dose of 3 grams of D2-LA ethyl ester for one month, followed by 2 grams of D2-LA ethyl ester for the remaining six months.All children except one were given blood samples at month 3, and all children were given blood samples at month 6.The concentration of D2-AA in RBC was measured.In all cases, D2-AA was obtained by deacylation of ethyl ester of linoleic acid in the digestive tract, followed by in vivo conversion of D2-LA to D2-AA.

[0074] At 3 months, the mean concentration of D2-AA in RBCs was 12% (min 6.8%, max 16.8%). At 6 months, the mean concentration of D2-AA in RBCs was measured to be 16.7% (min 12.0%, max 26.1%). Figure 2 depicts these results and shows the linear relationship of D2-AA accumulation in the body. Figure 2 includes the 1 month data for D2-AA in CSF as determined in Example 1.

[0075] As can be seen from Figures 1 and 2, the figures are virtually identical, strongly suggesting that administration of D2-LA to adult patients in Example 1 and to children in Example 2 maximized the conversion of D2-LA to D2-AA. The data further suggests that, once maximized, the amount of D2-AA produced over time is reproducible.

[0076] Example 3: Validation of Therapeutic Concentrations in ALS ALS patients were treated with D2-LA for a certain period of time. Patients were provided with different doses and different administration periods of D2-LA. All patients evaluated underwent ALSFRS-R analysis of their natural history before the start of treatment.

[0077] Blood samples from the patients were evaluated for D2-AA concentrations in their RBCs. PK sampling in 10 patients gave a mean D2-AA concentration in RBCs of 3.1% ± 2.0. Additionally, the functional scores (ALSFRS-R results) for each patient at the end of treatment were compared to the natural history scores at the start of treatment. Based on this comparison, the annualized rate changed from -14.2 ± 4.4 per year before treatment to -7.6 ± 1.4 during the treatment period, a 46% decrease (p = 0.07, two-tailed t-test for within-subject percentage change in slope).

[0078] The results of this study demonstrate an RBC D2-AA concentration of approximately 3%, based on the total amount of arachidonic acid plus deuterated arachidonic acid found in the RBCs, which is considered to be a therapeutic concentration that provides a therapeutic benefit in attenuating the rate of disease progression when the RBC D2-AA concentration is at least approximately 3%.

[0079] Example 4: Validation of therapeutic concentrations in PSP Three patients diagnosed with probable PSP (an example of a tauopathy) underwent baseline evaluation using the 28-item Progressive Supranuclear Palsy Rating Scale (PSPRS)

[19] and the Unified Parkinson's Disease Rating Scale (UPDRS). They were then treated with D2-Lin (2.88 g BID; 5.76 g total daily dose) and observed for disease progression. Scores on the above two assessment scales were determined every 3 months during the treatment period. Pharmacokinetic (PK) sampling was performed 3 times per month. These analytes included plasma and RBC membrane concentrations of (D2-LA) and its central active metabolite, D2-AA.

[0080] The three patients were two men (ages 66 and 73 years) and one woman (age 74 years), each of whom had had pretreatment syndrome for 6 and 3 years for the two men, respectively, and for 2 years for the one woman. Baseline PSPRS was 17 and 12 for the two men, respectively, and 13 for the one woman. Baseline UPDRS was 44 and 36 for the two men, respectively, and 21 for the one woman.

[0081] After 3 months of treatment, the slope of the PSPRS changed from a predicted score decline of 0.91 points / month to a mean decline of 0.16 points / month (±0.23 SEM).The slope of the UPDRS changed from a predicted increase of 0.95 points / month to a mean increase in score of 0.28 points / month (±0.41 SEM).

[0082] At month 3, the following data were collected: [Table 4]

[0083] At 12 months, the following data were collected: [Table 5]

[0084] The second male patient (Patient 2) received an increased dose after the first year of treatment (2.88 g TID; 8.64 g total daily dose) to further enhance the patient's treatment outcome.

[0085] The above results demonstrated that a percentage ratio of 13,13-D2-AA to (AA + 13,13-D2) in red blood cells of about 3% was required to obtain a therapeutic outcome. The data further demonstrated that a percentage ratio of 13,13-D2-AA to (AA + 13,13-D2-AA) in red blood cells of about 5% was preferred, with 6% or 8% being more preferred.

Claims

1. 1. A non-invasive method for determining a concentration of deuterated arachidonic acid in reporter cells of a patient population, said concentration correlating with onset of a therapeutic effect in a patient population suffering from a neurodegenerative disease treatable with said deuterated arachidonic acid, comprising the steps of: a) obtaining data from a population of patients having a first neurodegenerative disease during a treatment regimen with deuterated arachidonic acid or a prodrug thereof, the data comprising deuterated arachidonic acid concentrations in a reporter cell population of the patients at one or more time points after initiation of the treatment; b) assessing when the patient experienced visible results from treatment; c) determining the mean time from initiation of treatment to onset of therapeutic outcome and the mean concentration of deuterated arachidonic acid in reporter cells for each patient at the time of onset for each neurodegenerative disease; d) correlating the mean concentration of deuterated arachidonic acid in a reporter cell population of said patient as evidence that said patient's neurons have a minimum necessary therapeutic concentration of deuterated arachidonic acid for said disease with the mean time required for onset of a minimum necessary therapeutic outcome for said neurodegenerative disease; e) repeating steps a)-d) for one or more additional neurodegenerative diseases; f) assessing the minimum required therapeutic concentration of deuterated arachidonic acid and the mean time to onset of therapeutic result for each of said one or more additional neurodegenerative diseases; and g) Obtaining the mean minimum required therapeutic concentration and the mean time required for onset of said therapeutic outcome for a set of neurodegenerative diseases. The method includes:

2. 1. A method for determining a patient's response to administration of a composition comprising deuterated arachidonic acid or a prodrug thereof during treatment of a treatable neurodegenerative disease, comprising the steps of: providing a population of red blood cells from a patient suffering from a neurodegenerative disease at a predetermined time point after initiation of treatment that is a time point at which a therapeutic outcome is evident for the disease; assessing the concentration of deuterated arachidonic acid in the red blood cells, said concentration being relative to an amount of arachidonic acid comprising deuterated arachidonic acid; comparing said assessed concentration to said predefined concentration for a time point at which a therapeutic outcome is evident; determining whether the evaluation concentration is a therapeutic concentration or a sub-therapeutic concentration for a treatable neurodegenerative disease; Based on the determination, the dose of the deuterated arachidonic acid or a prodrug thereof administered to the patient is arbitrarily changed. The method includes:

3. 2. The method of claim 1, wherein a sample of reporter cells is obtained about one month after initiation of treatment.

4. 4. The method of claim 3, wherein further samples of reporter cells are obtained at monthly, three monthly, semi-annual, or yearly intervals thereafter.

5. 5. The method of claim 1, 3 or 4, wherein the reporter cell is a red blood cell, a skin cell, an adipocyte, a biopsy cell, or an epithelial cell.

6. The method of claim 2 or 5, wherein the deuterated arachidonic acid is 13,13-D2-arachidonic acid.

7. The method of claim 2 or 5, wherein the deuterated arachidonic acid is D4-arachidonic acid.

8. The method of claims 2 or 5, wherein the deuterated arachidonic acid is D6-arachidonic acid, in which at least 90% of the hydrogen atoms at positions 7,7,10,10,13,13 are replaced with deuterium, and optionally up to 35% of the remaining hydrogen atoms at non-bis-allylic positions are replaced with deuterium.

9. 1. A method for assessing a patient's response to administration of a composition comprising deuterated arachidonic acid or a prodrug thereof during treatment of a treatable neurodegenerative disease, comprising the steps of: assessing the concentration of deuterated arachidonic acid in the patient's red blood cells relative to the total amount of arachidonic acid present, including deuterated arachidonic acid; comparing said concentration to a predefined minimum required therapeutic concentration of said deuterated arachidonic acid in said red blood cells; the patient's red blood cells are deemed to be sub-therapeutic when they have a concentration of said deuterated arachidonic acid that is less than said determined therapeutic concentration, and are deemed to be therapeutic when said concentration of deuterated arachidonic acid is equal to or greater than said determined therapeutic concentration; and Optionally, the method is repeated as needed to assess whether the patient's concentration of deuterated arachidonic acid in the red blood cells has reached or remains at a therapeutic concentration. The method includes:

10. 10. The method of claim 9, wherein the reporter cell sample is obtained about one month after initiation of treatment.

11. 11. The method of claim 10, wherein further samples of reporter cells are obtained at monthly, three monthly, semi-annual, or yearly intervals thereafter.

12. 12. The method of claim 9, 10 or 11, wherein the reporter cell is a red blood cell, a skin cell, an adipocyte, a biopsy cell, or an epithelial cell.

13. 10. The method of claim 9, wherein the deuterated arachidonic acid is 13,13-D2-arachidonic acid.

14. 10. The method of claim 9, wherein the deuterated arachidonic acid is D4-arachidonic acid.

15. 10. The method of claim 9, wherein the deuterated arachidonic acid is D6-arachidonic acid, wherein at least 90% of the hydrogen atoms at positions 7,7,10,10,13,13 are replaced with deuterium, and optionally up to 35% of the remaining hydrogen atoms at non-bis-allylic positions are replaced with deuterium.

16. 1. A method for determining whether a concentration of deuterated 13,13-D2-arachidonic acid in a patient having a treatable neurodegenerative disease is at or below therapeutic levels without accessing the patient's neurons or cerebrospinal fluid, comprising the steps of: providing a blood sample from a patient undergoing treatment for a neurodegenerative disease with 13,13-D2-arachidonic acid or an ester thereof; determining the concentration of 13,13-D2-arachidonic acid in red blood cells (RBC); This concentration is compared to the minimum therapeutic concentration of 13,13-D2-arachidonic acid based on the total amount of arachidonic acid, including deuterated arachidonic acid, present in the RBCs to determine whether the patient has therapeutic or subtherapeutic levels of 13,13-D2-arachidonic acid in their neurons. The method includes:

17. 17. The method of claim 16, wherein the therapeutic concentration of 13,13-D2-arachidonic acid is set at 4%, 5%, 6%, 7%, 8%, 9%, or 10% as a therapeutic target for a given patient or cohort of patients.

18. 17. The method of claim 16, wherein the reporter cell sample is obtained about one month after initiation of treatment.

19. 17. The method of claim 16, wherein further samples of reporter cells are obtained at monthly, three monthly, semi-annual, or yearly intervals thereafter.

20. 1. A method for determining whether the concentration of deuterated D4-arachidonic acid in a patient having a treatable neurodegenerative disease is at or below the therapeutic level without accessing the patient's neurons or cerebrospinal fluid, comprising the steps of: Providing a blood sample from a patient undergoing treatment for a neurodegenerative disease with D4-arachidonic acid or an ester thereof; and determining the concentration of D4-arachidonic acid in red blood cells (RBCs); and comparing this concentration to a minimum therapeutic concentration of at least 1% of D4-arachidonic acid based on the total amount of fatty acids (including D4-arachidonic acid) in said RBCs to determine whether said patient has therapeutic or subtherapeutic levels of D4-arachidonic acid in their neurons. The method includes:

21. 21. The method of claim 20, wherein the therapeutic concentration of D4-arachidonic acid is set at 2%, 3% or 5% as a therapeutic target for a given patient or cohort of patients.

22. 21. The method of claim 20, wherein the reporter cell sample is obtained about one month after initiation of treatment.

23. 21. The method of claim 20, wherein further samples of reporter cells are taken at monthly, quarterly, semi-annual or annual intervals thereafter.

24. 1. A method for determining whether the concentration of deuterated D6-arachidonic acid in a patient having a treatable neurodegenerative disease is at or below the therapeutic level without accessing the patient's neurons or cerebrospinal fluid, comprising the steps of: Providing a blood sample from a patient undergoing treatment for a neurodegenerative disease with D4-arachidonic acid or an ester thereof; and determining the concentration of D6-arachidonic acid in red blood cells (RBCs); and comparing this concentration to a minimum therapeutic concentration of at least 0.5% D6-arachidonic acid based on the total amount of arachidonic acid (including deuterated arachidonic acid) in the RBCs to determine whether the patient has a therapeutic or subtherapeutic level of D6-arachidonic acid in neurons. The method includes:

25. 25. The method of claim 24, wherein the therapeutic concentration of D6-arachidonic acid is set at 1%, 1.5%, 2%, 2.5%, or 3% as a therapeutic target for a given patient or cohort of patients.

26. 25. The method of claim 24, wherein the reporter cell sample is obtained about one month after initiation of treatment.

27. 25. The method of claim 24, wherein further samples of reporter cells are obtained at monthly, three monthly, semi-annual, or yearly intervals thereafter.

28. 25. The method of claim 24, wherein the deuterated arachidonic acid is D6-arachidonic acid, wherein at least 90% of the hydrogen atoms at positions 7,7,10,10,13,13 are replaced with deuterium in the deuterated D6-arachidonic acid, and optionally up to 35% of the remaining hydrogen atoms at non-bis-allylic positions are replaced with deuterium.

29. A kit of parts including diagnostic materials for carrying out said method, as well as a correlation table correlating the concentration of deuterated arachidonic acid in red blood cells with that of human cerebrospinal fluid.

30. The kit of claim 29, wherein the diagnostic material includes one or more instructions as to when the test should be performed and / or factors suggesting delaying the test.

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