Methods and Compositions for the Treatment of Inflammatory Diseases - Patent application

Abstract

Compositions and methods for controlling chronic inflammation in a patient are provided herein. The compositions and methods use D6-arachidonic acid or its esters (D6-AA) as an anti-inflammatory agent that results in a significant reduction in the inflammatory process. In addition, the deuterated arachidonic acid or its ester compositions contain, on average, at least about 80% of the hydrogen atoms at each bis-allylic position replaced by deuterium atoms, and, on average, no more than about 30% of the hydrogen atoms at the mono-allylic positions replaced by deuterium atoms.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 292,388, filed December 21, 2021, and U.S. Provisional Patent Application No. 63 / 293,208, filed December 23, 2021, which are incorporated by reference in their entireties herein. [Background technology]

[0002] background Arachidonic acid is an essential n-6 polyunsaturated fatty acid (PUFA) present in the human diet. It is an essential PUFA and, moreover, a building block of phospholipids found in cell membranes, including neuronal cell membranes and neuronal organelles such as mitochondria and the endoplasmic reticulum, where it is the predominant PUFA. The structure of arachidonic acid contains four double bonds in a 1,4-polyene configuration, resulting in three bis-allylic positions and two mono-allylic positions flanking both ends of this configuration. The following structures provide examples of mono-allylic and bis-allylic positions in a 1,4-diene system: [ka]

[0003] Arachidonic acid is readily oxidized at the bis-allylic position as a substrate for several enzymes (such as COX1, COX2, various LOX enzymes, and cytochrome P450) that result in multiple proinflammatory and prothrombotic eicosanoid mediators, such as prostaglandins, leukotrienes, and thromboxanes, or by reactive oxygen species (ROS) leading to further proinflammatory metabolites. In either case, such oxidation leads to inflammation in all affected tissues, including the lungs.

[0004] Proinflammatory and prothrombotic eicosanoid mediators then initiate further inflammatory cascades. The role of arachidonic acid in the enzymatic generation of proinflammatory mediators is a key property of this PUFA. Indeed, replacement of arachidonic acid moieties in cell membranes by ad libitum consumption of fish rich in long-chain n-3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid was reported to reduce inflammation simply by a mass balance approach. See, e.g., Calder, Biochem. Soc. Trans., 33(part 2): 423-427 (2005).

[0005] In addition, the pathogenesis of many diseases involves increased levels of ROS, which in turn initiate a nonenzymatic chain reaction of arachidonic acid peroxidation in lipid membranes (LPO), leading to multiple toxic products, including other proinflammatory mediators. Although regulatory enzymes exist within the cell that neutralize the oxidized products resulting from LPO, the increased amount of ROS produced by the disease overwhelms these enzymes, leading to the accumulation of proinflammatory mediators.

[0006] Both enzymatic and nonenzymatic oxidative transformations occur at any of the three bis-allylic positions in arachidonic acid, since the carbon-hydrogen bond strength at these sites is the weakest in the molecule. The critical rate limiting step for both types of transformation is called hydrogen abstraction (C-H bond cleavage) and determines the rate of the entire process. Thus, disease progression and, by corollary, increased inflammation, are directly related to the rates of oxidation of arachidonic acid (enzymatic oxidation) and oxidation of PUFAs in general (LPO oxidation).

[0007] Oxidative processes involving arachidonic acid are key components in the development of inflammatory processes in vivo. As is well established, chronic inflammation has deleterious effects on the patient. For example, the presence of chronic vascular inflammation has been implicated as a causative factor in coronary artery disease. Similarly, chronic arthritic conditions are associated with varying levels of pain and disfigurement (especially of the fingers) that also result from uncontrolled and chronic inflammatory processes.

[0008] Chronic pain associated with arthritis can be controlled in many cases with monoclonal antibodies, such as TNF-alpha blockers. However, these antibodies carry substantial risks of invasive fungal, bacterial, and viral infections, as well as lymphomas and other malignancies, which can be fatal, especially in children and adolescents. For many patients, such risks are unacceptable.

[0009] Therefore, methods and compositions for controlling inflammation without the use of monoclonal antibodies would be of particular importance. Summary of the Invention

[0010] overview Provided herein are compositions and methods for controlling chronic inflammation in a patient. The compositions and methods use D6-arachidonic acid or its esters (D6-AA) as an anti-inflammatory agent that results in a significant reduction in the inflammatory process. In addition, D6-AA has a long half-life such that when therapeutic concentrations are achieved, the patient experiences long-term control of the chronic inflammatory process and long-term control of chronic pain.

[0011] In one embodiment, a method for controlling chronic inflammation in a patient is provided, comprising administering to the patient an effective amount of a composition comprising D6-AA for an effective period of time such that accumulated D6-AA in cell membranes attenuates the inflammatory process; thereby controlling chronic inflammation in the patient.

[0012] In one embodiment, the methods described herein for controlling chronic inflammation in a patient provide relief from chronic pain associated with chronic inflammation.

[0013] In some embodiments, most or all of the hydrogens at positions 7, 10, and 13 are replaced as shown below: [ka]

[0014] In another embodiment, the methods described herein include a composition designated D6-AA, which comprises D6-arachidonic acid or an ester thereof, in which, on average, at least about 80% of the hydrogen atoms at the bis-allylic positions are replaced by deuterium atoms, and, on average, no more than about 30% of the hydrogen atoms at the mono-allylic positions are replaced by deuterium atoms. For example, with an average of 85% deuteration at the three bis-allylic positions and 20% deuteration at the mono-allylic positions, the total amount of deuterium is (6×0.85)+(4×0.2)=5.9 (excluding naturally occurring deuterium) at each of the remaining methylene and methyl groups in the structure.

[0015] In one embodiment, a pharmaceutical composition is provided comprising a pharma- ceutically acceptable excipient and about 10 mg to about 2,000 mg of a composition of D6-arachidonic acid or an ester thereof, in which, on average, at least about 80% of the hydrogen atoms at each bis-allylic position are replaced by deuterium atoms and, on average, no more than about 30% of the hydrogen atoms at the mono-allylic positions are replaced by deuterium atoms.

[0016] In one embodiment, the chronic inflammation is vascular inflammation.

[0017] In one embodiment, the chronic inflammation is inflammation associated with arthritis. In one embodiment, the arthritis is rheumatoid arthritis. In one embodiment, the arthritis is osteoarthritis. In one embodiment, the chronic inflammation is neuroinflammation.

[0018] In one embodiment, the chronic inflammation is associated with a neurodegenerative disease.

[0019] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which there are shown and described embodiments of the present disclosure by way of example only. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. Incorporation by Reference

[0020] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that a publication, patent, or patent application incorporated by reference conflicts with a disclosure contained herein, it is intended that the specification supersede and / or take precedence over any such conflicting material.

[0021] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the nature and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (herein referred to as "drawings" or "figures"), in which: [Brief description of the drawings]

[0022] [Figure 1] The different bis-allylic positions may play different roles in the oxidation of arachidonic acid.

[0023] [Diagram 2] Arachidonic acid specifically isotopically modified at the bis-allylic positions. The bis-allylic positions are located at C7, C10, and C13. "D" stands for deuterium.

[0024] [Diagram 3]Groups of female (f) and male (m) mice fed a 6-week course of dietary (H)AA or a 6-week course of deuterated arachidonic acid (D6-ARA, 15 mg / day) were subsequently given a single intranasal dose of LPS. Control mice (norm) did not receive a single intranasal dose of LPS. Mice fed dietary AA showed increased alveolar lumen area (Fig. 3, median; Fig. 4, mean) and increased interalveolar septal thickness (Fig. 5, median; Fig. 6, mean), likely related to stronger edema and inflammatory infiltrates. Mice fed D6-ARA showed alveolar lumen area and thickness consistent with controls. Overall, the data for Figs. 3-6 show milder pulmonary inflammatory lesions after a course of D-type compared to H-type. Preliminary data (not shown) indicate that D6-ARA has the best protective effect compared to other deuterated PUFAs. [Figure 4] Groups of female (f) and male (m) mice fed a 6-week course of dietary (H)AA or a 6-week course of deuterated arachidonic acid (D6-ARA, 15 mg / day) were subsequently given a single intranasal dose of LPS. Control mice (norm) did not receive a single intranasal dose of LPS. Mice fed dietary AA showed increased alveolar lumen area (Fig. 3, median; Fig. 4, mean) and increased interalveolar septal thickness (Fig. 5, median; Fig. 6, mean), likely related to stronger edema and inflammatory infiltrates. Mice fed D6-ARA showed alveolar lumen area and thickness consistent with controls. Overall, the data for Figs. 3-6 show milder pulmonary inflammatory lesions after a course of D-type compared to H-type. Preliminary data (not shown) indicate that D6-ARA has the best protective effect compared to other deuterated PUFAs. [Diagram 5]Groups of female (f) and male (m) mice fed a 6-week course of dietary (H)AA or a 6-week course of deuterated arachidonic acid (D6-ARA, 15 mg / day) were subsequently given a single intranasal dose of LPS. Control mice (norm) did not receive a single intranasal dose of LPS. Mice fed dietary AA showed increased alveolar lumen area (Fig. 3, median; Fig. 4, mean) and increased interalveolar septal thickness (Fig. 5, median; Fig. 6, mean), likely related to stronger edema and inflammatory infiltrates. Mice fed D6-ARA showed alveolar lumen area and thickness consistent with controls. Overall, the data for Figs. 3-6 show milder pulmonary inflammatory lesions after a course of D-type compared to H-type. Preliminary data (not shown) indicate that D6-ARA has the best protective effect compared to other deuterated PUFAs. [Figure 6] Groups of female (f) and male (m) mice fed a 6-week course of dietary (H)AA or a 6-week course of deuterated arachidonic acid (D6-ARA, 15 mg / day) were subsequently given a single intranasal dose of LPS. Control mice (norm) did not receive a single intranasal dose of LPS. Mice fed dietary AA showed increased alveolar lumen area (Fig. 3, median; Fig. 4, mean) and increased interalveolar septal thickness (Fig. 5, median; Fig. 6, mean), likely related to stronger edema and inflammatory infiltrates. Mice fed D6-ARA showed alveolar lumen area and thickness consistent with controls. Overall, the data for Figs. 3-6 show milder pulmonary inflammatory lesions after a course of D-type compared to H-type. Preliminary data (not shown) indicate that D6-ARA has the best protective effect compared to other deuterated PUFAs.

[0025] [Figure 7A-7D] Figures 7A-D show various molecules and reactions related to PUFAs. Figure 7A shows various PUFAs. Figure 7B is a diagram showing hydrogen abstraction removing the bis-allylic hydrogen. Figure 7C shows the various products of nonenzymatic lipid peroxidation (LPO). Figure 7D shows the multiple products of enzymatic arachidonic acid oxidation.

[0026] [Figure 8] FIG. 8 shows the thickness of the interalveolar septa in BALB / c mice treated with PUFA.

[0027] [Figure 9A-9C] Figures 9A-9C show deuterated arachidonic acid as a percentage of total arachidonic acid at washout after 8 weeks of dosing followed by 9 weeks of H-AA feeding. Figure 9A shows graphs for viscera and skin. Figure 9B shows graphs for neural tissues: whole brain and whole eye. Figure 9C shows lung arachidonic acid and its conversion to longer chain PUFAs.

[0028] [Figure 10A-10C] 10A-C show histological assessment of the effects of intranasal administration of lipopolysaccharide in the lungs of treated male mice.

[0029] [Figure 11] FIG. 11 shows a graph of interalveolar septum thickness. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols typically refer to similar elements identically unless otherwise indicated. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the scope of the subject matter presented herein. It is readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the drawings, can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein.

[0031] Although specific embodiments and examples are disclosed below, the subject matter of the present invention extends beyond the specifically disclosed embodiments and / or uses to other alternative embodiments, as well as modifications and equivalents thereof. Thus, the claims appended hereto are not limited by any of the specific embodiments described below. For example, in any method or process disclosed herein, the activities or operations of the method or process may be performed in any suitable order and are not necessarily limited to any of the specific disclosed orders. Various operations may be described as multiple separate operations in an order that may aid in understanding a particular embodiment, however, the description instructions should not be construed to imply that these operations are order dependent. Furthermore, the structures, systems, and / or devices described herein may be implemented as integrated elements or separate elements.

[0032] For purposes of comparing various embodiments, certain aspects and advantages of those embodiments are described. It is not necessary that all such aspects or advantages be achieved by any particular embodiment. Thus, for example, various embodiments may be implemented in a manner that realizes or optimizes one advantage or group of advantages taught herein, without necessarily achieving other aspects or advantages that may also be taught or suggested herein.

[0033] A method of controlling inflammation in a patient is disclosed. In one embodiment, the inflammation results from the intracellular accumulation of pro-inflammatory oxidized PUFA products.

[0034] Before discussing the present invention in more detail, the following terms will first be defined. Terms not defined will be given their definition in the context or as medically accepted.

[0035] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the present invention. As used herein, the singular forms "a," "an," and "the" are intended to encompass the plural forms as well, unless the context clearly dictates otherwise.

[0036] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description encompasses instances in which the event or circumstance occurs as well as instances in which it does not occur.

[0037] As used herein, the term "about," when used prior to numerical designations, e.g., of temperatures, times, amounts, concentrations, and the like, including ranges, indicates approximations that may vary by (+) or (-) 15%, 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, the term "about," when used in reference to dosage, means that the dose may vary by + / - 10%.

[0038] As used herein, the terms "comprising" or "comprising" are intended to 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 to define compositions and methods, is intended to mean excluding any essential significance of other elements to the combination for the stated 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 characteristic(s) of the claimed invention.

[0040] As used herein, the term "consisting of" is intended to mean excluding more than trace elements of other components or substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

[0041] The term "D6-arachidonic acid" or "D6-AA" refers to a composition that contains, on average, at least about 4.8 to about 7.2 deuterium atoms in arachidonic acid (excluding its ester portion, if used). In one embodiment, this includes 7,7,10,10,13,13-D6-arachidonic acid or esters thereof. In another embodiment, this includes a deuterated arachidonic acid or ester composition in which, on average, at least about 80% of the hydrogen atoms at each bis-allylic position are replaced by deuterium atoms and, on average, no more than about 30% of the hydrogen atoms at the mono-allylic positions are replaced by deuterium atoms.

[0042] In some embodiments, the amount of deuterium replacing hydrogen at the bis-allylic positions (7, 10, and 13) and mono-allylic positions (4 and 16) of arachidonic acid is as follows: at least about 85% deuterium at the bis-allylic positions / not more than about 30% deuterium at the mono-allylic positions; at least about 85% deuterium at the bis-allylic positions / not more than about 25% deuterium at the mono-allylic positions; at least about 85% deuterium at the bis-allylic positions / not more than about 20% deuterium at the mono-allylic positions; at least about 85% deuterium at the bis-allylic positions / not more than about 1 ...6% deuterium at the mono-allylic positions; -It can be any one of about 5% or less deuterium at the allylic positions; at least about 90% at the bis-allylic positions / about 30% or less deuterium at the mono-allylic positions; at least about 90% at the bis-allylic positions / about 25% or less deuterium at the mono-allylic positions; at least about 90% at the bis-allylic positions / about 20% or less deuterium at the mono-allylic positions; at least about 90% at the bis-allylic positions / about 10% or less deuterium at the mono-allylic positions; at least about 90% at the bis-allylic positions / about 5% or less deuterium at the mono-allylic positions (all percentages are on an average basis).

[0043] As used herein, and unless the context indicates otherwise, the term "ester thereof" refers to C1-C6 alkyl esters, glycerol esters (including monoglycerides, diglycerides and triglycerides), sucrose esters, phosphate esters, and the like. The particular ester group used is not critical, provided that the ester is medicamentously acceptable (non-toxic and biocompatible). In one embodiment, the ester is a C1-C6 alkyl ester, preferably an ethyl ester.

[0044] As used herein, the term "phospholipid" refers to any or all phospholipids that are components of cell membranes or other lipid membranes within a cell. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin are included within the scope of this term. In motor neurons, cell membranes are rich in phospholipids that contain arachidonic acid.

[0045] The term "bis-allylic position" refers to the methylene groups (CH2) separating the two double bonds as shown above.

[0046] The term "mono-allylic position" refers to a methylene group having an adjacent double bond on one side and an additional methylene group on the opposite side, as shown above.

[0047] The term "cellular component" refers to any relevant structure found in a human cell, including organelles with lipid or phospholipid walls. Examples of such cellular components include, by way of example only, mitochondria, the endoplasmic reticulum, the Golgi apparatus, the nuclear membrane, etc. Cellular components also include the cell membrane (plasma membrane).

[0048] The term "regulatory enzymes", as it relates to the neutralization of oxidized arachidonic acid products, refers to those enzymes involved in removing, modifying, or destroying one or more oxidized arachidonic acid products from cells to prevent the accumulation of these oxidized products in the cells. Examples of regulatory enzymes include glutathione peroxidase (GPx) enzymes, particularly GPx4. When these enzymes are impaired, such impairment results in the accumulation of lipid peroxides that leads to loss of functionality, which is often followed by cell death. Gaschler, et al., Biochem. Biophys. Res. Commun., 482(3):419-425 (2017).

[0049] The term "oxidized PUFA products" refers to any oxidized form of polyunsaturated fatty acids, as well as any or all metabolic products formed from oxidized PUFAs, including reactive aldehydes, ketones, alcohols, and carboxyl derivatives that are either proinflammatory metabolites or precursors to proinflammatory metabolites.

[0050] The term "neutralization of oxidation products" refers to an enzymatic method that removes, modifies, or destroys toxic oxidized PUFA products from cells, preventing the accumulation of these oxidation products in cells. In healthy individuals, neutralization of oxidized PUFA products prevents the accumulation of these products, thereby ensuring that cellular dysfunction, especially neurodegenerative cells, is controlled.

[0051] As used herein, the term "disease pathology" refers to the causes, development, structural / functional changes, and natural history associated with the disease. Reduction in cellular function is included in disease pathology. The term "natural history" refers to the worsening of the disease in the absence of treatment by the methods described herein, e.g., prior to the initiation of treatment as described herein.

[0052] As used herein, the term "chronic inflammation" refers to known conditions in which inflammation plays an important role in the development and progression of chronic diseases, including, but not limited to, autoimmune diseases, neurodegenerative diseases, metabolic disorders such as atherosclerosis and obesity, fibrosis, and cancer, to name a few. Also, such diseases include asthma, arthritis (including rheumatoid arthritis, juvenile chronic arthritis, and osteoarthritis), myositis, Crohn's disease, gastritis, colitis, ulcerative colitis, inflammatory bowel disease, proctitis, pelvic inflammatory disease, systemic lupus, erythematosus, rhinitis, conjunctivitis, scleritis, chronic inflammatory polyneuropathy, Lyme disease, psoriasis, dermatitis, and eczema, neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, and multiple sclerosis, to name a few. Such diseases are considered to be chronic, where the condition involved is either lifelong (after diagnosis), such as autoimmune diseases, or takes a substantially long period of time to treat and resolve, such as obesity.

[0053] As used herein, the term "control" or "controlling", when it relates to chronic inflammation, means that the level of inflammation resulting from a chronic disease is reduced or attenuated over time, such that the level of discomfort or pain experienced by the patient is reduced. While the level of discomfort experienced by a patient is individual and different from other patients suffering from the same disease, the reduction in inflammation can be measured by the level of swelling, reduction in C-reactive protein (CRP), and other standard methods well known in the art. Preferably, a reduction of at least 15%, and preferably at least 35%, in CRP levels at the start of therapy and 45 or 90 days after the start of therapy is evidence of inflammation control.

[0054] The term "therapeutic concentration" refers to the in vivo concentration of deuterated arachidonic acid that controls chronic inflammation. In one embodiment, a concentration of D6-AA in red blood cells of at least about 1%, and preferably at least about 1.5% to about 6%, based on the total amount of arachidonic acid found therein (including deuterated arachidonic acid) is therapeutic.

[0055] 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 a prodrug of deuterated arachidonic acid.

[0056] As used herein, the term "pharmaceutical acceptable salts" of the compounds disclosed herein are within the scope of the methods described herein and include acid or base addition salts that possess 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, pharmaceutical acceptable salts are formed with inorganic acids (e.g., hydrochloric acid, boric acid, nitric acid, sulfuric acid, phosphoric acid, etc.), organic acids (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 acidic amino acids (e.g., aspartic acid and glutamic acid). When the compound has an acidic group, such as a carboxylic acid group, it can be formed with metals, such as alkali and alkaline earth metals (e.g., Na + , Li + , K + , Ca 2+ , Mg 2+ , Zn 2+Salts may be formed with organic amines (e.g., dicyclohexylamine, trimethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g., arginine, lysine, and ornithine). Such salts may 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, respectively, with a suitable acid or base and isolating the salt thus formed. inflammation

[0057] Inflammation-related disorders represent a heterogeneous group of diseases that share a common underlying inflammatory pathology, including, but not limited to, asthma, rheumatoid arthritis, juvenile chronic arthritis, osteoarthritis, myositis, Crohn's disease, gastritis, colitis, ulcerative colitis, inflammatory bowel disease, proctitis, pelvic inflammatory disease, systemic lupus erythematosus, rhinitis, conjunctivitis, scleritis, chronic inflammatory polyneuropathy, Lyme disease, psoriasis, dermatitis, eczema, autoimmune diseases, neurodegenerative disorders, and atherosclerosis.

[0058] Inflammation-related disorders are often accompanied by enhanced ROS (reactive oxygen species) production and lipid processing abnormalities (Winyard PG et al. Free radicals and inflammation. Burkhauser, Basel, 2000; Filippo et al., Journal of Alzheimer's Disease (2010); 20, S369-S379). The combination of ROS and lipid processing abnormalities leads to enhanced PUFA peroxidation that creates toxic reactive carbonyl compounds that inflict further damage at many other sites within the cell (Negre-Salvayre A et al., Br J Pharmacol 2008;153:6-20). As a result, both oxidatively damaged biomolecules and the detection of the presence of ROS in inflammatory conditions are associated with high oxidative stress levels.

[0059] Lipid peroxidation is important in the progression of atherosclerosis and the initial tissue injury caused by ischemic or traumatic brain injury. Atherosclerosis is a lipid storage disorder associated with an inflammatory response (Ross R, New Engl J Med. 1999;340:115-127).

[0060] Oxidation of PUFA-rich low-density lipoprotein (LDL) has been suggested to be a major risk factor for atherosclerosis and atherosclerosis-associated hypertension. Angiotensin II (Ang-II) is involved in this process and promotes macrophage lipid peroxidation both in vivo and in vitro (Keidar S. Life Sci. 1998;63:1-11). Despite the importance of oxidative stress in the pathogenesis of atherosclerosis, antioxidant therapies aimed at reducing lipid peroxidation have had limited success so far (Stocker R et al., Physiol Rev 2004;84:1381-1478).

[0061] Rheumatoid arthritis may be associated with inflammation and PUFA peroxidation through a reperfusion injury mechanism. The synovial cavity is normally under negative pressure. When the joint is in motion, blood vessels are maintained open, allowing nutrient absorption of the avascular cartilage. In rheumatoid synovitis, intracavity pressure is elevated, and with exercise, this pressure exceeds the capillary perfusion pressure and causes blood vessels to collapse. This leads to the occurrence of frequent episodes of "hypoxic reperfusion injury" and produces ROS that oxidize multiple targets, including, but not limited to, IgG, which triggers induced rheumatoid factor production; hyaluronan, which leads to hyaluronic acid fragmentation products that alter immune function; PUFAs, which produce reactive carbonyls that alter T cell / macrophage interactions; and lipoproteins, which lead to the production of monocyte chemotactic peptides. Progressive hypoxia alters immune function, primarily through calcium-mediated pathways (Mapp PI et al., Br Med Bull 1995;51:419-436).A variety of PUFA peroxidation products, including but not limited to conjugated dienes, isoprostanes, and reactive aldehydes such as HNE, can be detected in the plasma and synovial fluid of patients with rheumatoid arthritis and osteoarthritis (Selley ML Annals Rheum Disease 1992;51:481-484).

[0062] Oxidative damage is implicated in a variety of diseases, including mitochondrial diseases, neurodegenerative diseases, neurodegenerative muscular diseases, retinal diseases, energy processing disorders, kidney disease, liver disease, lipemia, cardiac disease, inflammation, and genetic disorders.

[0063] Oxidative stress at sites of chronic inflammation can also induce genetic changes. Reactive carbonyl products of PUFA peroxidation can react with DNA bases and alter their complementation patterns (Esterbauer H et al., Free Rad Biol Med 1991;11:81-128). The occurrence of mutations in the p53 tumor suppressor gene and other key regulatory genes can switch inflammation to chronic disease in rheumatoid arthritis and other inflammatory diseases (Tak PP et al., Immunology Today 2000;21:78-82).

[0064] Although the number of diseases associated with oxidative stress is large and diverse, it is well established that oxidative stress is caused by disturbances to the normal redox state within cells. An imbalance between the routine production and detoxification of reactive oxygen species ("ROS"), such as peroxides and free radicals, can result in oxidative damage to cellular structures and machinery. Under normal conditions, a potentially important source of ROS in aerobic organisms is the leakage of ROS from mitochondria during normal oxidative respiration. In addition, macrophages and enzymatic reactions are also known to contribute to the generation of ROS within cells. Since cells and their internal organelles are lipid membrane-bound, ROS can easily come into contact with membrane components and cause lipid oxidation. Ultimately, such oxidative damage can be transmitted to other biomolecules within the cell, such as DNA and proteins, via direct and indirect contact with ROS, components of oxidized membranes, or other oxidized cellular components. Thus, given the mobility of internal components and the interconnection of cellular pathways, the propagation of oxidative damage through cells can be easily envisioned.

[0065] PUFAs endow mitochondrial membranes with the appropriate fluidity required for optimal oxidative phosphorylation performance. PUFAs also play an important role in the initiation and propagation of oxidative stress. PUFAs react with ROS through a chain reaction that amplifies the original event (Sun M, Salomon RG, J. Am. Chem. Soc. 2004; 126:5699-5708). However, nonenzymatic formation of high levels of lipid hydroperoxides is known to result in several deleterious changes. Indeed, coenzyme Q10 has been implicated in increased PUFA toxicity through the peroxidation of PUFAs and the toxicity of the resulting products (Do TQ et al., PNAS USA 1996; 93:7534-7539). Such oxidation products adversely affect the fluidity and permeability of those membranes; they lead to the oxidation of membrane proteins; and they can be transformed into numerous highly reactive carbonyl compounds. The latter include reactive species such as acrolein, malonic dialdehyde, glyoxal, methylglyoxal, and others (Negre-Salvayre A, et al., Brit. J. Pharmacol. 2008; 153:6-20). However, the most prominent products of PUFA oxidation are α,β-unsaturated aldehydes such as 4-hydroxynon-2-enal (4-HNE; formed from n-6 PUFAs such as LA or AA), 4-hydroxyhex-2-enal (4-HHE; formed from n-3 PUFAs such as ALA or DHA), and the corresponding ketoaldehydes (Esterfbauer H, et al., Free Rad. Biol. Med. 1991; 11:81-128; Long EK, Picklo MJ. Free Rad. Biol. Med. 2010; 49:1-8). These reactive carbonyls cross-link (biomolecules) via Michael addition or Schiff base formation pathways and have been implicated in numerous pathological processes (such as those introduced above), age-related and oxidative stress-related conditions, and aging.Importantly, in some cases, PUFAs appear to oxidize at specific sites because the methylene groups in the 1,4-diene system (bis-allylic positions) are substantially less stable to ROS and enzymes such as cyclooxygenase, cytochrome and lipoxygenase than allylic methylenes.

[0066] Eicosanoids are important cell signaling molecules involved in an array of physiological and pathological processes, including various immune responses such as pro-inflammatory, anti-inflammatory, allergy, and fever. There are multiple oxidation and immune response-related pathways in the biosynthesis of eicosanoids from arachidonic acid.

[0067] In one pathway, the enzyme 5-lipoxygenase (5-LO or ALOX5) converts arachidonic acid (ARA) to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene A4 (LTA4), which is then converted by downstream enzymes to different leukotrienes (LTC4, LTD4, and LTE4). LTC4, LTD4, and LTE4 are known to be bronchoconstrictors and potent stimulators of mucus secretion in lung tissue and promote hyperresponsiveness in respiratory conditions such as asthma.

[0068] In another pathway leading to oxidation and inflammatory responses, 15-lipoxygenase (15-lipoxygenase 1, 15-LOX, 15-LOX1, or ALOX15) and 12-lipoxygenase (12-LOX or ALOX12) metabolize arachidonic acid to form 15(S)-hydroperoxyeicosatetraenoic acid (15(S)-HPETE) and 12(S)-hydroperoxyeicosatetraenoic acid (12(S)-HPETE). Both 15(S)-HPETE and 12(S)-HPETE are further reduced by cellular glutathione peroxidase to their corresponding hydroxy analogs, 15-hydroxyeicosatetraenoic acid (15(S)-HETE) and 12-hydroxyeicosatetraenoic acid (12(S)-HETE), respectively. 15(S)-HPETE and 15(S)-HETE are further metabolized to various bioactive products, such as lipoxins, hepoxilins, eoxins, 8(S),15(S)-diHETE, 5(S),15(S)-diHETE, and 15-oxo-eicosatetraenoic acid (15-oxo-ETE). 12 / 15-lipoxygenase metabolites have been shown to have both pro- and anti-inflammatory properties.

[0069] Yet another pathway, COX1 and COX2 (also known as prostaglandin-endoperoxide synthase-1 (PTGS1) and PTGS2, respectively), initiates the metabolism of arachidonic acid into a subclass of eicosanoids, including prostaglandins (e.g., prostacyclin) and thromboxanes. Prostaglandins are mediators of inflammatory and anaphylactic responses, and thromboxanes are mediators of vasoconstriction and platelet aggregation, and play a major role in blood clotting.

[0070] In another pathway, cytochrome P450 enzymes metabolize arachidonic acid to EETs (epoxyeicosatrienoic acids), which promote the cessation of inflammation by mediating a wide array of anti- and pro-inflammatory mechanisms.

[0071] Previous studies have examined the relative enzymatic activity of cyclooxygenase and lipoxygenase with isotopic species of arachidonic acid deuterated at the bis-allylic position, but these were mechanistic studies that did not use physiological or therapeutic amounts of deuterated arachidonic acid. One study tested the enzymatic activity of purified COX and LOX on different isotopic species of arachidonic acid, but did not test the activity in cells (Chistyakov, D., et al., Deuterated Arachidonic Acids Library for Regulation of Inflammation and Controlled Synthesis of Eicosanoids: An In Vitro Study, Molecules, 2018, 23:3331; which are incorporated herein by reference in their entirety). In another study, macrophage cell lines were incubated with D-AA or standard AA, and cell culture supernatants were analyzed for the amount of eicosanoids produced and secreted. However, this study used non-physiological conditions in which arachidonic acid was incubated with cells for 24 hours at a molar concentration of 25 micromolar, which resulted in up to 90% arachidonic acid incorporation in the cell membrane - a percentage that is several times unattainable under physiological conditions (Navratil, A. et al., Lipidomics Reveals Dramatic Kinetic Isotope Effects during the Enzymatic Oxygenation of Polyunsaturated Fatty Acids Ex Vivo). Therefore, neither study indicates whether compositions containing bis-allyl isotope modified essential PUFAs, including arachidonic acid, inhibit inflammation in vivo, which is an important consideration given the multiple metabolic pathways involving arachidonic acid and the potential pro- and anti-inflammatory effects that result therefrom.Furthermore, both the purified enzyme studies (Chistyakov et al.) and the cellular studies (Navratil et al.), whether in vitro or in vivo model systems, used very high concentrations of the specified AA species in the absence of other PUFAs and did not examine biologically relevant complex mixtures of fatty acids and their derived enzymatic reaction products that are present at low concentrations.

[0072] Apart from the proinflammatory enzymatic products of AA oxidation, the second major component of inflammation, which can be initiated by enzymes or small molecule ROS, is the nonenzymatic proinflammatory component, which is not susceptible to antioxidant approaches due to the structural aspects of the lipid bilayer that makes up the membrane. Inflammation-associated LPO is initiated through various mechanisms (Zhang et al., Myeloperoxidase Functions as a Major Enzymatic Catalyst for Initiation of Lipid Peroxidation at Sites of Inflammation, J Blot Chem 2002 doi: 10.1074 / jbc.M209124200).

[0073] Arachidonic acid is easily oxidized at the bis-allylic position as a substrate for several enzymes (such as COX1, COX2, various LOX enzymes, and cytochrome P450) that result in multiple proinflammatory and prothrombotic eicosanoid mediators, such as prostaglandins, leukotrienes, and thromboxanes, and then initiate further inflammatory cascades. Furthermore, oxidation can occur by reaction with reactive oxygen species (ROS), leading to further proinflammatory metabolites. In either case, such oxidation leads to inflammation in all affected tissues, including the lungs.

[0074] Inflammation increases the levels of reactive oxygen species (ROS), which in turn initiates a nonenzymatic chain reaction of AA peroxidation in lipid membranes (LPO), leading to multiple toxic products. In addition, the pathogenesis of many diseases involves increased levels of ROS, which in turn initiates a nonenzymatic chain reaction of arachidonic acid peroxidation in lipid membranes (LPO). This chain reaction leads to multiple toxic products, including other proinflammatory mediators. Although regulatory enzymes exist within the cell that neutralize the oxidized products resulting from LPO, the increased amount of ROS produced by the disease overwhelms these enzymes, leading to the accumulation of proinflammatory mediators.

[0075] Both enzymatic and nonenzymatic oxidative transformations occur at any of the three bis-allylic positions in the AA molecule, since the carbon-hydrogen bond strength at these sites is the weakest in the molecule. The critical rate limiting step for both types of transformation is called hydrogen abstraction (CH bond cleavage) and determines the rate of the entire process. That is, disease progression and, as a corollary, increased inflammation, are directly related to the rates of oxidation of arachidonic acid (enzymatic oxidation) and oxidation of PUFAs in general (LPO oxidation). Thus, the present disclosure provides a therapeutic method based on the concept that AA oxidation plays a pivotal role in multiple aspects of inflammatory diseases, including cytokine storm and thrombotic complications.

[0076] COX and LOX inhibitors are used to down-regulate this process with varying degrees of effectiveness.In contrast, the method of the present disclosure relies on the deuteration of three oxidation-prone sites in PUFA molecules, which substantially slows down the rate-limiting step of oxidation (both enzymatic and non-enzymatic) through isotope effect (IE).This D-PUFA, which is carried out in high yield through catalytic process, has been tested in a well-known lung inflammation model, the lipopolysaccharide (LPS)-induced lung inflammation model in mice (see Example 1).

[0077] Intranasal administration of the bacterial endotoxin lipopolysaccharide (LPS) has been shown to initiate a proinflammatory response in mouse lungs. For example, the mouse LPS model was used as a model of human acute lung injury (ALI) because mouse lung parenchyma is damaged by the production and release of proteases, reactive enzymes, and nitrogen species produced by activated pulmonary macrophages and migrating neutrophils in the interstitial and alveolar compartments. The end result is microvascular injury with intrapulmonary hemorrhage, edema, and fibrin deposition, as well as diffuse alveolar damage (Johnson and Ward, J. Clin. Invest., (1974), 54:349-357; Flierl et al., Med. Hypothesis Res., (2006), 3:727-738), which is also characteristic of patients with ALI and acute respiratory distress syndrome (ARDS) (Kabir et al., Shock, (2002), 17:300-303; Ward, Am. J. Pathol., (1996), 149:1081-1086). This LPS model recapitulates aspects of the inflammatory cascades involved in pulmonary inflammation associated with human lung disease, and is also useful as a screen for compounds that may disrupt these cascades and attenuate or halt the disease process. As discussed herein, in some embodiments, the D-PUFA of the present disclosure is administered to disrupt the inflammatory cascade of certain cytokines and chemokines that are known to induce and enhance pulmonary inflammation.Thus, for example, one skilled in the art can experimentally evaluate the effectiveness of D-PUFA by examining whether the level of inflammation-related markers is reduced.Two such markers, monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor alpha (TNF-α), have been found to be associated with early and chronic infection and inflammation in humans, and are reflected in rodent models of airway inflammation, including the mouse LPS pulmonary inflammation model.

[0078] In turn, proinflammatory and prothrombotic eicosanoid mediators initiate further inflammatory cascades. The role of arachidonic acid in the enzymatic production of proinflammatory mediators is an important property of this PUFA. In fact, it has been reported that replacing a portion of arachidonic acid in cell membranes by consuming fish rich in long-chain n-3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid reduces inflammation simply by a mass balance approach. See, for example, Calder, Biochem. Soc. Trans., 33(part 2):423-427 (2005).

[0079] Oxidative processes involving arachidonic acid are key components in the development of inflammatory processes in vivo. As is well established, chronic inflammation has deleterious effects on the patient. For example, the presence of chronic vascular inflammation has been implicated as a causative factor in coronary artery disease. Similarly, chronic arthritic conditions are associated with varying levels of pain and disfigurement (especially of the fingers) that also result from uncontrolled and chronic inflammatory processes. pathology

[0080] The pathology obtained for chronic inflammatory diseases is different from the underlying etiology of the disease. That is, what are the branching conditions that cause each of these diseases (etiology)?Once caused, the pathology of these diseases involves the accumulation of proinflammatory oxidized PUFA products, which are proinflammatory metabolites or precursors to proinflammatory metabolites.As the amount of oxidized products increases, the degree of inflammatory response is enhanced, and these oxidized PUFA products activate the biological cascade that leads to inflammation.

[0081] In accordance with the above, increasing concentrations of oxidized PUFA products in cells correlate with the progression of chronic diseases that affect these cells. The methods described herein provide for the in vivo delivery of deuterated arachidonic acid to these cells, thereby limiting the amount of oxidized PUFA products produced when both LPO and enzymatic oxidation of arachidonic acid are reduced. Taken together, these two mechanisms, alone or in combination, create oxidative stress on cells. Reducing oxidative stress has a positive impact on the treatment of disease as well as the underlying inflammation. Thus, unlike monoclonal antibodies and other steroidal or nonsteroidal anti-inflammatory products, the compositions of the present invention attenuate both disease inflammatory components as well as address the primary disease itself. This is because the ability of the disease to cause LPO and / or enzymatic oxidation of arachidonic acid is reduced by supplementing the cell membranes of high-risk cells with D6-AA. This, in turn, protects against cell lysis, a mechanism known to release proinflammatory cytokines and other intracellular components into the extracellular medium.

[0082] The origin of oxidative stress varies with the underlying etiology, but typically involves one or more different reactive oxygen species. Regardless of etiology, the production of oxidized PUFA products is evidence of an imbalance between the regular production of these oxidation products and their detoxification (neutralization). The lipid membranes of cells, as well as those of the endoplasmic reticulum and mitochondria of neurons, contain arachidonic acid, a 20-carbon chain polyunsaturated fatty acid ("PUFA") with four sites of cis-unsaturation. Three bis-arylmethylene groups separate each of these four sites. These groups are particularly susceptible to oxidative damage compared to arylmethylene and methylene groups. Apart from leading to further membrane damage, oxidation of arachidonic acid reduces the local concentration of arachidonic acid, which must be replaced. It is thus a double whammy: a beneficial bioactive membrane component is converted into a toxic membrane component.

[0083] Given that certain cells have a high concentration of arachidonic acid in their lipid membranes, the replacement of damaged or lost arachidonic acid in these membranes with deuterated arachidonic acid reinforces these structures of the cell and protects against the formation of oxidized lipid products. For example, once the bis-arylmethylene group of arachidonic acid is oxidized by ROS, a cascade of further oxidation of other arachidonic acid groups in the lipid membrane occurs. This results in the oxidation of the initial arachidonic acid component by a free radical mechanism, which in turn can oxidize adjacent arachidonic acid by the same free radical mechanism, with another adjacent arachidonic acid being oxidized once more in a process called lipid chain autoxidation. The resulting damage includes significantly more oxidized arachidonic acid products in the cell membrane and in the membranes of organelles. However, if oxidized arachidonic acid is adjacent to deuterated arachidonic acid, the deuterated arachidonic acid acts as a chain reaction terminator due to the much higher stability of the carbon-deuterium bond to oxidation compared to the carbon-hydrogen bond.

[0084] Oxidized arachidonic acid and other oxidized PUFA products adversely affect the fluidity and permeability of cell membranes of neurons in patients. In addition, they can lead to the oxidation of membrane proteins and can be converted into many highly reactive carbonyl compounds. The latter include reactive species such as acrolein, malonic dialdehyde, glyoxal, and methylglyoxal (Negre-Salvayre A, et al., Brit. J. Pharmacol. 2008; 153:6-20). The most prominent products of arachidonic acid oxidation are α,β-unsaturated aldehydes such as 4-hydroxynon-2-enal (4-HNE; formed from n-6 PUFAs such as LA or AA) and the corresponding ketoaldehydes (Esterfbauer H, et al., Free Rad. Biol. Med. 1991; 11:81-128). As mentioned above, these reactive carbonyls cross-link (biomolecules) through Michael addition or Schiff base formation pathways and perpetuate the underlying pathology of the disease. Each of these metabolic products derived from oxidized PUFAs is encompassed by the term "oxidized PUFA product(s)." Disease progression

[0085] Inflammation leads to an increase in inflammatory markers (CRP), such as C-reactive protein and creatine kinase (CK), including CK-BB (brain), CK-MB (heart), and CK-MM (skeletal muscle). When a patient is diagnosed with a particular chronic inflammatory disease, clinicians monitor the patient's CRP or CK kinase count to measure the effectiveness of the treatment. The treatment described herein provides a steady increase in the amount of deuterated arachidonic acid in the patient's cells over time until the concentration reaches an amount that reduces the amount of cellular oxidative stress that leads to inflammation. The replacement of about 1 to about 5 percent of the arachidonic acid content in the patient's red blood cells by D6-AA occurs over a period of twenty (20) to forty-five (45) days, during which a gradual decrease in the patient's CRP or CK kinase can be observed. Typically, around day 30, the patient's CRP or CK kinase levels are lower to significantly lower than before the start of the treatment, demonstrating the anti-inflammatory properties of the drug. As more D6-AA or its esters are administered to a patient, its intracellular concentration reaches about 5% to about 20% or more in the person's red blood cells. In all cases, the in vivo concentration of D6-AA is measured using red blood cells as a proxy for cells at high risk for or undergoing oxidative stress. The percentage of D6-AA given in these cells is measured based on the total weight of arachidonic acid in the red blood cells, including D6-AA.

[0086] Without being limited to any theory, the progression of chronic inflammatory disease correlates with the increase in cellular oxidative stress, including the accumulation of oxidized PUFA.Therapeutic intervention according to the methods described herein limits the further increase in oxidative stress, and then reduces these levels so as to control the inflammation associated therewith.Regardless of theoretical aspect, the examples provided below illustrate the significant reduction in inflammation achieved by D6-AA.

[0087] In one embodiment, the chronic inflammation is vascular inflammation.

[0088] In one embodiment, the chronic inflammation is inflammation associated with arthritis. In one embodiment, the arthritis is rheumatoid arthritis. In one embodiment, the arthritis is osteoarthritis. In one embodiment, the chronic inflammation is neuroinflammation. Preparation of compounds

[0089] Deuterated arachidonic acid is known in the art. For example, 7,7,10,10,13,13-D6-arachidonic acid is disclosed by Shchepinov, et al., Molecules, 28(12):31 et seq. (2018). Similarly, catalytic deuteration of arachidonic acid using a ruthenium catalyst, which results in about 80% or more deuteration of bis-allylic positions and about 30% or less deuteration of mono-allylic positions, is disclosed by Shchepinov et al., U.S. Pat. No. 10,730,821. Both of these references are incorporated herein by reference in their entirety. Still further, other deuterated arachidonic acid compounds are known in the art. The conversion of each of these PUFAs to their corresponding esters is well known in the art. method

[0090] In one embodiment, the method described herein includes administering D6-AA or its ester to a patient suffering from a chronic inflammatory disease. In vivo, D6-AA slowly accumulates in the cells of the body, including those prone to chronic inflammation. Because chronic inflammatory diseases cause higher CRP levels, and a reduction in inflammation corresponds to a reduction in CRP levels, clinicians can monitor the progress of therapy by measuring the amount of C-reactive protein (CRP) in patients. In addition, clinicians can correlate the increasing concentration of D6-AA in red blood cells over time with lower CRP levels. As the concentration of D6-AA in red blood cells increases, CRP levels are expected to decrease.

[0091] The methods described herein provide for rapid onset of therapeutic concentrations of deuterated arachidonic acid in vivo. In one embodiment, a method of reducing inflammation in an adult patient in a chronic inflammatory disease is provided, comprising administering to the patient periodically D6-AA or an ester thereof using a dosing regimen that includes a primer dose and a maintenance dose.

[0092] In some embodiments, the primer dose comprises periodic administration of deuterated arachidonic acid or an ester thereof. In some embodiments, the primer dose comprises at least about 10 milligrams of deuterated arachidonic acid or an ester thereof per day. In some embodiments, the primer dose comprises about 10 milligrams to about 2 grams of deuterated arachidonic acid or an ester thereof per day. In some embodiments, the primer dose comprises about 0.10 grams to about 1 gram. In some embodiments, the primer dose is continued for about 15 to about 50 days, or about 30 to about 45 days, for example, to rapidly achieve a therapeutic concentration of deuterated arachidonic acid in vivo, thereby slowing the rate of disease progression.

[0093] In some embodiments, a maintenance dose is administered periodically after completion of the primer dose. In some embodiments, the maintenance dose is administered at about 65% or less of the loading dose per day. In some embodiments, the maintenance dose is utilized to ensure that a therapeutic concentration of deuterated arachidonic acid is maintained in vivo to maintain the slowed disease progression.

[0094] In one embodiment, the periodic administration of the loading dose comprises administration of about 0.05 grams to about 2 grams of deuterated arachidonic acid or an ester thereof per day. In some embodiments, the loading dose is about 0.05 grams to about 1.5 grams per day. In some embodiments, the loading dose is about 0.10 grams to about 1.5 grams per day. In some embodiments, the loading dose is about 0.10 grams to about 1.25 grams per day. In some embodiments, the loading dose is about 0.10 grams to about 1 gram per day. In some embodiments, the loading dose is about 0.10 grams to about 0.5 grams per day. The loading dose can be any value or subrange within the recited ranges, including the endpoints.

[0095] In some embodiments, the loading dose is administered for at least 5 days per week, and preferably every day of the week.

[0096] In one embodiment, the regular daily administration of a maintenance dose of deuterated arachidonic acid or an ester thereof comprises 65% or less, or 55% or less of the loading dose. In an embodiment, the maintenance dose is administered on a daily basis, or at least 5 days per week, or at least once per week, or at least once per month. In another embodiment, the maintenance dose comprises 35% or less of the loading dose and is administered at least once per month.

[0097] In one embodiment, the periodic administration of the maintenance dose is calibrated to provide an amount of D6-AA or an ester thereof sufficient to replace the amount of deuterated arachidonic acid excreted from the body.

[0098] In some embodiments, the loading dose is administered for about 1 month to about 4 months. In some embodiments, the loading dose is administered for about 1 month to about 2 months. In some embodiments, the loading dose is administered for about 1 month. The length of time can be any value or subrange within the recited ranges, including the endpoints.

[0099] In some embodiments, the loading dose is administered during a meal, hi some embodiments, dosing is administered with a meal, e.g., with food.

[0100] In some embodiments, whether a patient has D6-AA incorporated into their cells at therapeutic levels is determined by simply measuring the patient's CRP level. In some embodiments, the amount of D6-AA is determined based on its concentration in red blood cells. In the event that the clinician determines that the patient is not responding, and the clinician predicts that, an increase in either the loading dose or the maintenance dose can be made.

[0101] In some embodiments, it is determined whether the patient has deuterated arachidonic acid incorporated into the patient's neurons at a therapeutic level. In some embodiments, the level of deuterated arachidonic acid is determined based on the amount of deuterated arachidonic acid in the patient's red blood cells. In some embodiments, the therapeutic goal is at least about 6% or at least about 12% deuterated arachidonic acid based on the total amount of arachidonic acid in red blood cells (e.g., in a blood sample). In some embodiments, the therapeutic goal is at least about 15%. In some embodiments, the therapeutic goal is at least about 20%. In some embodiments, the therapeutic goal is about 12% to about 30% deuterated arachidonic acid based on the total amount of arachidonic acid in red blood cells (e.g., in a blood sample). In some embodiments, the therapeutic goal is about 12% to about 25% deuterated arachidonic acid based on the total amount of arachidonic acid in red blood cells (e.g., in a blood sample). In some embodiments, the therapeutic goal is about 12% to about 20% deuterated arachidonic acid based on the total amount of arachidonic acid in red blood cells (e.g., in a blood sample). In some embodiments, the therapeutic goal is about 15% to about 25% deuterated arachidonic acid based on the total amount of arachidonic acid in red blood cells (e.g., in a blood sample). In some embodiments, the therapeutic goal is about 15% to about 20% deuterated arachidonic acid based on the total amount of arachidonic acid in red blood cells (e.g., in a blood sample). The amount can be any value or subrange within the recited range, including the endpoints.

[0102] In embodiments, D6-AA or its ester (e.g., ethyl ester) may be administered using any dosage regimen. For example, D6-AA or its ester may be administered daily, every 2 days, every 3 days, etc. In embodiments, D6-AA or its ester may be administered for a first period (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks or more), followed by a period (e.g., 1 day, 2 days, 1 week or more) during which D6-AA or its ester is not administered. In embodiments, D6-AA or its ester may be administered for a period until a therapeutic level is reached, and then administered less frequently. For example, D6-AA or its ester may be administered daily until a therapeutic level is reached, and then administered every 2, 3, 4, 5, 6, or 7 days to maintain the therapeutic level. Use in combination

[0103] The therapeutic methods provided herein can be used in combination with other therapeutic methods used to treat chronic inflammatory diseases. In one embodiment, D6-AA or its ester can be used alone. Alternatively, D6-AA or its ester can be used in combination with 11,11-D2-linoleic acid or its ester, and / or 8,8,11,11-D4-linoleic acid or its ester. In vivo, a portion of 11,11-D2-linoleic acid is converted to 13,13-D2-arachidonic acid, and a portion of 8,8,11,11-D4-linoleic acid is converted to 10,10,13,13-D4-arachidonic acid.

[0104] In another embodiment, the combination therapy can use drugs that act through mechanisms orthogonal to the methods described herein. Suitable drugs for use in combination include, but are not limited to, antioxidants such as edaravone, idebenone, mitoquinone, mitoquinol, vitamin C, or vitamin E, provided that the antioxidants do not interfere with the therapeutic action of deuterated linoleic acid or its esters. Pharmaceutical Compositions

[0105] The specific dosage of D6-AA or its esters can be achieved by various commonly accepted 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 is administered at least once per day, preferably once, twice or three times per day.

[0106] The present invention is not limited to any particular composition or pharmaceutical carrier, and therefore may vary.Generally, the compounds of the present invention are administered as pharmaceutical compositions by any of many known routes of administration.However, oral delivery is typically preferred using tablets, pills, capsules, etc.The particular form used for oral delivery is not critical, but due to the large amount of drug to be administered, preferably the daily or regular unit dose is divided into subunits having multiple tablets, pills, capsules, etc.

[0107] Pharmaceutical dosage forms of the compounds disclosed herein may be manufactured by any method known in the art, for example, conventional mixing, tabletting, encapsulation, etc. The compositions disclosed herein may include one or more physiologically acceptable inactive ingredients that facilitate processing of the active molecule into preparations for pharmaceutical use.

[0108] The composition may include the drug in combination with at least one pharma- ceutically acceptable excipient.Acceptable excipients are non-toxic, aid administration, and do not adversely affect the therapeutic effect of the claimed compound.Such excipients may be any solid, liquid, or semi-solid that is generally available to those skilled in the art.

[0109] 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).

[0110] The compositions disclosed herein may, if desired, be presented in a pack or dispenser device containing daily or regular unit dosages each containing the required number of subunits of drug. Such a pack or device may, for example, comprise metal or plastic foil, such as a blister pack, a vial, or other type of containment container. The pack or dispenser device may 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.

[0111] The amount of drug in the formulation will vary depending on the number of subunits required for daily or regular doses of the drug. Typically, the formulation will contain about 10-99 wt. % of drug based on the total formulation, with the balance of one or more suitable pharmaceutical excipients present, on a weight percent (wt. %) basis. Preferably, the compound is present at a level of about 50-99 wt. %.

[0112] In one embodiment, the drug is encapsulated in a capsule without the need for any excipients such as stabilizers, antioxidants, dyes, etc. This minimizes the number of capsules needed per day by maximizing the volume of drug in each capsule. EXAMPLES

[0113] The present disclosure will be further understood by reference to the following examples, which are intended to be purely exemplary of the present invention. The present disclosure is not limited in scope by the exemplified embodiments, which are intended only to be illustrative of one aspect of the disclosure. Any methods that are functionally equivalent are within the scope of this disclosure. Various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the above description and the accompanying drawings. Such modifications are 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. H-AA = non-deuterated arachidonic acid (with only deuterium at natural abundance) H-LA = non-deuterated linoleic acid (with only deuterium at natural abundance) D2-AA = 11,11-D2-arachidonic acid D6-AA = D-6 arachidonic acid with greater than 80% deuterium in the bis-allylic positions and less than or equal to 35% deuterium in the mono-allylic positions h=time kg = kilograms LPS = lipopolysaccharide D2-LA = 11,11-D2-linoleic acid (aka the "drug") LA= linoleic acid LPO = lipid peroxidation M = moles mg = milligrams ROS = reactive oxygen species μm = microns μm 2 = Square microns Example 1: Control of LPS-induced inflammation

[0114] LPS administration is known to promote inflammation through a variety of mechanisms, including the secretion of proinflammatory cytokines, eicosanoids, and induction of ROS. In this example, LPS was used to ascertain the extent of inflammation resulting from ROS-induced oxidation of H-AA versus D-AA in mouse lungs. Specifically, four groups of mice were used. The first group was control mice treated with H-LA control mice. The second group of mice was given a 6-week course of D-LA. It is understood that partial in vivo conversion of both H-LA and D-LA occurs to provide AA and 13,13-D2-AA, respectively. The third group of mice was given a 6-week course of H-AA. The fourth group of mice was given a 6-week course of D-AA.

[0115] All groups were then given a single intranasal dose of LPS to induce acute lung inflammation. The extent of the inflammatory response was based on the interalveolar septal distance, where the greater the septal distance, the greater the degree of inflammation. Animals were sacrificed and the interalveolar septal distance was measured. Table 1 provides the mean interalveolar septal spatial distance for all group results. [Table 1]

[0116] The above results evidence an approximately 25% reduction in the interalveolar septal clearance in mice treated with D-LA versus H-LA, however, there was an almost 60% reduction in the same clearance in mice treated with D-AA, demonstrating the benefit of D-AA in treating inflammation. Example 2: Control of LPS-induced inflammation - H-AA vs. D-AA

[0117] LPS administration was performed according to Example 1 above, except that four groups of mice were used. The first two groups (1 and 2) were control mice (male and female). The next two groups of mice (3 and 4) were given a six week course of H-AA (male and female). Finally, the last two groups of mice (5 and 6) were given a six week course of D6-AA (second group). Groups 3, 4, 5 and 6 were then given a single intranasal administration of LPS to induce acute pulmonary inflammation. The extent of the inflammatory response was determined according to Example 1. The results of this experiment are shown in Table 2. [Table 2]

[0118] The results of this experiment demonstrated a statistically significant reduction in inflammation in both male and female mice using D-AA compared to D-LA.

[0119] The height and distance of the interalveolar septa were then measured to provide the surface area of ​​inflammation. The results of this assessment are shown in Table 3. [Table 3]

[0120] The degree of alveolar lumen area change in both animal groups was sex-dependent. This index was significantly lower in females than in males, regardless of the acid form (H or D). However, for male mice fed the D form, the severity of the changes in the thickness of the interalveolar septum was significantly higher than that of females, highly correlated with the human case (females were less affected). Overall, the data obtained showed milder inflammation in lungs treated with D-6AA compared to H-AA, regardless of the sex of the mice, consistent with the data above.

[0121] The following examples, Examples 1a, 2a, and 3a, provide more complete details of Examples 1, 2, and 3, respectively. Example 1a: Administration of D-AA in a Mouse Model of Pneumonia

[0122] LPS treatment promotes inflammation through various mechanisms, including the secretion of proinflammatory cytokines, eicosanoids, and induction of ROS. Induction by intranasal administration of LPS is quickly followed by acute lung inflammation. In mice fed a 6-week course of dietary (H)AA followed by a single intranasal administration of LPS, the thickness of the interalveolar septa was significantly increased, which is likely related to the stronger edema and inflammatory infiltration. Such a marked inflammatory infiltration of the interalveolar septa contributes to their destruction and the more frequent formation of emphysematous foci in this group. At the same time, the lungs of mice fed type D were characterized by a reduction in the alveoli lumens, which may be related to the lower frequency (compared to type H) of emphysematous changes in the lungs (see Figures 3-6).

[0123] The degree of alveolar lumen area changes in both animal groups was sex-dependent. This index was significantly lower in females than in males, regardless of the form of acid (H or D). Moreover, among male mice fed with the D type, the severity of changes in the thickness of the interalveolar septum was significantly higher in males than in females, highly correlated with the human case (females being less affected). Overall, the data obtained show milder inflammatory lesions in the lungs after a course of the D type compared to the H type. Preliminary data (not shown) also show that D6-AA (7,7,10,10,13,13-D6-arachidonic acid) provides a better therapeutic effect compared to other deuterated PUFAs. Figure 4 below summarizes the data underlying Figures 3-6. [Table 4]

[0124] Oral data from human dosing in ongoing clinical trials shows an excellent safety record for analogous D-PUFA drugs that are metabolic precursors to AA. The expected safety of D-PUFA drugs combined with the convenience of oral dosing in D-PUFA gel capsules warrants further investigation of D-PUFAs as an approach to anti-inflammatory and potential preventative and therapeutic approaches against COVID-19-induced cytokine storm and thrombotic events. Other PUFA emulsions, such as IntraLipid, are formulated in emulsion form and safely dosed IV at several grams per day. One skilled in the art could imagine that such a formulation could allow for rapid bolus dosing of drug at the start of treatment followed by lipid gel capsule oral dosing of D-AA as a continuing therapy. These data provide a rationale for further investigation of deuterated PUFA technology as an approach to COVID inflammation-related treatment and other inflammation-related conditions. Example 2a: In vitro assay for inflammation

[0125] Cell-based in vitro assays of inflammation are well known in the art. These assays include examples such as e-selectin (also called endothelial leukocyte adhesion molecule or ELAM) and C-reactive protein (CRP). The ELAM assay measures the in vitro activity of test compounds by downregulation of ELAM in activated endothelial cells.

[0126] For example, activated endothelial cells are generated by adding known activators, such as lipopolysaccharide, TNF, or IL-1 (3) to rat intestinal MVEC cells. The activated cells are cultured in a culture medium containing either deuterated PUFAs ("D-PUFAs") (0.01, 0.1, 1.0, 10.0, or 100 μm D-PUFAs and combinations of D-PUFAs at 1:1 or different ratios (e.g., D2-LA, D4-ALA, etc., see, e.g., species in formula (1) supra) or "H-PUFAs" (H-PUFAs are non-deuterated or tritiated). The cells are incubated with isotopically modified PUFAs such as deuterated LA, ALA, etc. (also referred to as deuterated) (0.01, 0.1, 1.0, 10.0, or 100 μM combinations of LA, ALA, etc., and corresponding ratios of the non-deuterated mixture) for 24, 48, and 72 hours. Activated cells are known to produce ELAMs, which can be measured, for example, using an E-selectin monoclonal antibody-based ELISA assay. D-PUFA-treated cells are expected to produce lower amounts of ELAMs compared to cells treated with H-PUFAs.

[0127] Similarly, a CRP assay can be used to measure the in vitro activity of test compounds in reducing the expression of CRP in human Hep3B epithelial cells. For example, activated epithelial cells are generated by adding known activators such as lipopolysaccharide, TNF, or IL-1 (3) to human Hep3B cells. The activated cells are incubated with D-PUFAs (0.01, 0.1, 1.0, 10.0, or 100 μM, different ratio combinations of different D-PUFAs) or H-PUFAs (0.01, 0.1, 1.0, 10.0, or 100 μM of the corresponding combinations of LA, ALA, and H-PUFAs) for 24, 48, and 72 hours. Activated cells are known to produce CRP, which can be measured using a CRP ELISA assay. D-PUFA-treated cells are expected to produce lower amounts of CRP compared to cells treated with H-PUFAs. Example 3a: In vivo assay for inflammation

[0128] In vivo assessment of anti-inflammatory activity can be measured by well-characterized assay methods that measure carrageenan-induced paw edema and by mouse ear inflammatory responses to topical arachidonic acid (see Gabor, M., Mouse Ear Inflammation Models and their Pharmacological Applications, 2000, which is incorporated herein by reference). Carrageenan-induced paw edema is a model of inflammation that measures the time-dependent formation of edema following administration of carrageenan to the intraplantar surface of the rat paw. Groups of 8-week-old Wistar albino rats (8-9 animals / group) are supplemented with D-PUFAs (e.g., see species in formula (1)) (0.01, 0.1, 1.0, 10.0, and 100 mg / kg) and different combinations of D-PUFAs (e.g., two types of D-PUFAs in a 1:1 ratio) or H-PUFAs (0.01, 0.1, 1.0, 10.0, and 100 mg / kg) and different combinations of H-PUFAs corresponding to the different combinations of D-PUFAs, as the sole PUFA source over a period of 8 weeks. Following the supplementation period, rats were lightly anesthetized under isoflurane and given a subplantar injection of 50 μL of saline containing 1% w / v carrageenan. Paw volumes were measured using a water volume measuring device and compared to paw volumes before carrageenan administration. Volumes are measured at 0.5, 1, 2, 3, 4, and 5 hours. Edema can be calculated as the increase in paw volume divided by the starting paw volume. Rats supplemented with D-PUFAs are expected to have lower levels of edema compared to rats supplemented with H-PUFAs.

[0129] Furthermore, application of arachidonic acid (AA) to the rat ear is known to produce an immediate vasodilatory response and erythema, followed by a sudden onset of edema, which should be maximal at 40-60 minutes. The onset of edema is thought to coincide with the extravasation of proteins and leukocytes. After one hour, the edema should rapidly subside, and by six hours, the inflammatory cells should leave the tissue such that the ear returns to near normal. Groups of 8-week-old male rats (8-9 animals / group) are supplemented with D-PUFAs (e.g., see species in formula (1)) (0.01, 0.1, 1.0, 10.0, and 100 mg / kg D-PUFAs) and different combinations of D-PUFAs (e.g., two types of D-PUFAs in a 1:1 ratio) or H-PUFAs (0.01, 0.1, 1.0, 10.0, and 100 mg / kg) and different combinations of H-PUFAs corresponding to the different combinations of D-PUFAs, as the sole source of PUFAs over a period of 8 weeks. Following the supplementation period, arachidonic acid is applied to the rats' ears and the vasodilatory response, erythema, and edema are measured as a function of time. Rats supplemented with D-PUFAs are expected to have lower levels of edema compared to rats supplemented with H-PUFAs. Example 4: Collagen-induced arthritis model

[0130] Groups of 8-10 week old DBA / 1 mice (8-9 animals / group) are supplemented with D-PUFAs (e.g., see species in formula (1)) (0.01, 0.1, 1.0, 10.0, and 100 mg / kg D-PUFAs) and different combinations of D-PUFAs (e.g., two types of D-PUFAs in a 1:1 ratio) or H-PUFAs (0.01, 0.1, 1.0, 10.0, and 100 mg / kg) and different combinations of H-PUFAs corresponding to different combinations of D-PUFAs, as the sole PUFA source over a period of 8 weeks. Mice are then injected intradermally at the base of the tail with 100 μg of bovine type II collagen in Freund's complete adjuvant (FCA) and observed by daily examination for the development of disease, which was recorded. It was predicted that D-PUFA treated mice would have a delayed onset of arthritic symptoms, or any onset, compared to H-PUFA treated mice.

[0131] Alternatively, 45 DBA / 1 mice aged 8-10 weeks were injected intradermally at the base of the tail with 100 μg of bovine type II collagen in Freund's complete adjuvant (FCA) and observed by daily examination for the development of disease, which was recorded. At the first appearance of clinical evidence of arthritis, mice were randomly assigned to one of three treatment groups: 1) control; 2) D-PUFA (0.01, 0.1, 1.0, 10.0, and 100 mg / kg) and 1:1 or different combinations of different species of D-PUFA) treatment; or 3) H-PUFA (0.01, 0.1, 1.0, 10.0, and 100 mg / kg) and 1:1 or different combinations of H-PUFAs corresponding in type to the D-PUFA combinations. The severity of arthritis in the affected paws is graded according to an established scoring system as follows: 0 (normal joints), 1 (mild / moderate visible edema and swelling), 2 (severe edema with distortion of the paw and joints), and 3 (deformed paw or joint with ankylosis). The sum of the scores for all four limbs of each mouse is used as an arthritis index (maximum score / mouse=12) that represents the overall disease severity and progression of the animal. Animals are clinically evaluated for disease 5 times per week until 10 weeks after disease onset, and paw measurements are obtained 3 times per week. Arthritic paws with no signs of disease at any time point after treatment are considered in remission. All mice are pre-bled before the start of the study, followed by at the onset of arthritis, 2 weeks after onset, 4 weeks after onset, and at the completion of the study. Serum obtained from each group is stored at -80°C until required. An ELISA assay is performed to measure total anti-collagen antibody levels in mouse CIA. It is expected that D-PUFA treated animals will have reduced arthritic symptoms compared to H-PUFA treated animals and control animals. Example 5: Deuterated arachidonic acid ameliorates inflammation-induced lung injury in lipopolysaccharide-treated mice

[0132] Excessive inflammatory damage to lung tissue during bacterial or viral infection occurs with the involvement of a dysregulated eicosanoid axis and impeded repair of complex components of cell membranes and secreted surfactant proteolipids. Acute respiratory distress syndrome (ARDS) further exacerbates the condition; ARDS is associated with aging contributing to as much as 40% of mortality. ARDS is often induced by bacterial or viral pneumonia. In rodents, ARDS is usually modeled as acute lung injury (ALI) by instillation of live bacteria (S. pneumoniae or P. aeruginosa) or non-specific damaging agents such as ozone. Bacterial ALI can be modeled by a sterile single challenge with E. coli lipopolysaccharide (LPS).

[0133] FIG. 7A shows the various PUFAs used: LA, linoleic acid; D2-LA, 11,11-D2-linoleic acid; AA, arachidonic acid; D2-AA, 13,13-D2-arachidonic acid (product of in vivo enzymatic elongation / extension of D2-LA); D6-AA, 7,7,10,10,13,13-D6-arachidonic acid. FIG. 7B is a schematic showing hydrogen abstraction of bis-allylic hydrogens, a major rate-limiting step of PUFA oxidation (both enzymatic and LPO), inhibited by deuteration. FIG. 7C shows the various products of nonenzymatic LPO, including reactive carbonyls such as HHE and HNE, which can covalently crosslink biomolecules. FIG. 7D shows that the numerous products of enzymatic AA oxidation are mostly proinflammatory and prothrombotic.

[0134] This approach uses deuteration of three oxidation-prone sites within the AA molecule to substantially slow down the rate-limiting step of oxidation (both enzymatic and nonenzymatic) via isotope effects (IE).Here, we show the effects of dietary arachidonic acid (H-AA) or its hexadeuterated form (D-AA) on the lungs and gastrointestinal tract of healthy and LPS-treated mice. Materials and Methods

[0135] Bis-allyl D-PUFAs were prepared by total synthesis and catalytic deuteration. In vivo model and sampling protocol

[0136] Mouse diets were fat-free AIN-93G-based Research Diets (New Brunswick, NJ) with lipids at 11.3% total fat by weight, plus either standard H-AA or D-AA at 0.25% each (Table 5). [Table 5]

[0137] Male and female BALB / c and C57BL / 6 mice were provided with standard environmental conditions, i.e., 22±1°C, 55±5% humidity, and a 12-h light / dark cycle, with food and water available ad libitum. At 6 weeks of age, animals were divided into eight groups (n=160 total, 20 of each sex) and continuously received ad libitum access to diets containing either H-AA or D-AA for 2, 4, 6, or 8 weeks, approximately 5 g of food / mouse per day, throughout the duration of the study. Two other groups of animals (n=40, 20 of each sex) served as controls without the PUFA-enriched diet and were not fed LPS. Body weight and food intake were measured once a week. After completing the appropriate diet course, half of the mice in each group (10 females and 10 males) were subjected to (i) a bronchoalveolar lavage (BAL) procedure to obtain cytological smears and (ii) tissue removal and collection of colonic contents (after humane euthanasia with a lethal dose of sodium thiopental, 100 mg / kg, intravenously). During the 9th week of this diet course, the other half of the group received 1 mg / kg LPS from Escherichia coli O111:B4 (Sigma Aldrich) intranasally (IN) 24 h before BAL and subsequent euthanasia. Forty D-AA diet animals (20 males, 20 females) were switched to the H-AA diet for a washout experiment. Collection of bronchoalveolar lavage (BAL) fluid

[0138] Briefly, under deep anesthesia (sodium thiopental), lungs were lavaged three times using three aliquots of 1 ml of 0.15 M NaCl. Each lavage consisted of slow injection of saline via the tracheal cannula and gentle aspiration. The three collected lavage fluids were combined. Samples were centrifuged in a microcentrifuge at 400 g and 4°C for 7 min using an F241.5P rotor (Beckman Coulter, USA). Supernatants were collected and stored frozen at -80°C for up to one month until further analysis. Differential cell counts in BAL

[0139] Differential cell counts were performed on smear slides from resuspended cell pellets of BAL using a commercially available May-Grunwald-Giemsa staining kit. All slides were evaluated under a light microscope (Optec BK5000) at a magnification of ×200. Counts of these cells, such as lymphocytes, neutrophils, plasma cells, and macrophages, were obtained from 10 fields of view on each slide. Histological analysis

[0140] Lung, stomach, and colon samples were fixed with 4% formaldehyde (freshly prepared from paraform), paraffin embedded, and sectioned at 2-4 μm. All fixed tissue sections were stained with hematoxylin-eosin (H&E) for the given histology. All slides were evaluated in 10 fields each using a Genetic Pro Bino light microscope (A) and photographed with a digital camera (Delta Optical). Gastric and colon tissue sections were analyzed for signs of active and chronic inflammation, lymphoid follicle formation, and epithelial cell proliferation. The severity of lung injury was scored according to lung pathological changes, including interalveolar and perivascular hemorrhage, perivascular and peribronchial infiltration, perivascular edema, alveolar wall thickness, alveolar space value, and emphysematous changes. Lung tissue samples were further stained with a commercial Martius Scarlet Blue (MSB) staining kit (Avantik, USA) for fibrin visualization. The presence of yellow, pink or red homogeneous fibrin clots in pulmonary vessels was interpreted as thrombus clots. Microbiota analysis

[0141] Colonic contents of euthanized mice were collected for microbial community analysis, immediately frozen, and stored at -20°C until analysis. Samples were slowly thawed at +4°C, serial dilutions (10-3) were prepared, plated on specific media, and incubated at 37°C as follows: microaerophilic flora: Enterococcus - Enterococcal agar, 48 hours; Lactobacillus - Lactobac agar, 3 days; Bifidobacterium - Bifidum agar, 5 days at 37°C; aerobic bacteria: Staphylococcus - medium N10, 48 hours; coliform bacteria - Endo agar, 48 hours at 37°C; yeast-like fungi, Saburo agar, 48 hours. These specific media were purchased from GNC PMIB (Obolensk, Russia). At the end of the indicated incubation periods, colony forming units (CFU) were counted. Cytokine measurement using enzyme-linked immunosorbent assay (ELISA)

[0142] Cell-free BAL fluid supernatants, as well as colonic tissue samples, were analyzed using a commercially available mouse IL-1β ELISA kit (R&D systems, Lot P265854, USA) and a Biotek ELx-808 microplate reader (Biotek, USA) according to the instructions recommended by the manufacturer. AA isotope analysis (GC-MS / MS)

[0143] The relative ratio of H-AA to D-AA was determined by gas chromatography (GC) coupled to chemical ionization mass spectrometry. Briefly, freshly collected samples were homogenized and extracted by the Bligh and Dyer method, transferred to tubes, evaporated, blanketed with dry nitrogen, sealed, and shipped to Austin (TX) for further sample preparation and analysis. The dried samples were converted to fatty acid methyl esters (FAMEs) by a one-step method of hydrolysis and methylation. Solvent-mediated (SM) chemical ionization was performed using a Shimadzu GCMS-TQ8040 (Columbia, MD) instrument equipped with a BPX70 capillary column (25 m × 0.22 mm × 0.25 μm; Trajan, Pflugerville, TX) with CH3CN as the reagent. D-AA was separated from H-AA by chromatography. Total ion chromatograms were used to integrate the peak areas. statistical analysis

[0144] The experimental data were processed using the Statistica 10.0 software package. Data are shown in graphs as median and interquartile range (Me; Q25%; Q75%). For between-group comparisons, the non-parametric Kruskell-Wallace test was used with adjustment for multiple comparisons. Differences were considered statistically significant at p < 0.05. Effects of D-linoleic acid (D2-LIN) on the lungs of LPS-treated mice

[0145] In our first experiment, we evaluated the effect of linoleic acid (LA) supplementation on interalveolar septal thickness and AS in LPS-treated mice. In this experimental setting, mice (6 weeks old, both sexes) were fed the PUFA of interest once a day by oral gavage of 50 μL per mouse. At the completion of PUFA treatment, mice were intranasally administered 1 mg / kg LPS and sacrificed 24 h later for lung histology. The results (Figure 8) demonstrate a slight, but statistically significant, reduction in alveolar wall thickness in D2-LA mice compared to the H-LA group. This may be explained by a more pronounced hyperallergic response of the lung to H-LA compared to D2-LA. LPS treatment revealed focal perivascular and peribronchial lymphocytic infiltration along with the presence of serous edema, a sign of hyperplasia of the bronchial epithelium and its hypersecretion. Histologically, this effect was accompanied by inflammatory cell infiltration through the alveolar wall.

[0146] Because D2-LA is partially converted to D2-AA by the action of fatty acid desaturases 1 and 2 (FADS1 and FADS2) and fatty acid elongase 5 (ELOVL5), we hypothesized that the aforementioned effects of D-PUFA may be due to the deuteration of AA and that direct D6-AA supplementation would be much more effective than that with D2-LA.

[0147] Figure 8 shows the interalveolar septum thickness (μm) in BALB / c mice 6 weeks after a single intranasal administration of H- or D-linoleic acid (H-LA and D2-LA) followed by lipopolysaccharide. *-p≦0.05, comparing H-LA to D2-LA mice. Pharmacokinetic aspects of dietary supplementation with D-AA in healthy mice

[0148] As a first step to test this hypothesis, we studied the metabolic effects of such dietary supplementation. We measured the actual content of D-AA and then performed a washout experiment after long-term supplementation by switching from a D-AA diet to an H-AA diet by MS analysis of several mouse tissues in AA diet-fed mice. 12 weeks of D-AA dosing resulted in the incorporation of more than 60% of the deuterated form (about 90% of total AAs as D6-AA, except for the brain, which had 70% of total AAs as D6-AA) (Figure 9).

[0149] Figures 9A-9C show D-AA as % of total AAs at 8 weeks of dosing and washed out over the following 9 weeks with H-AA feeding. Figure 9A shows graphs for viscera and skin. Lungs took up approximately 45% of D-AA. Figure 9B shows graphs for neural tissues: whole brain and whole eye. Both took up approximately 50% of D-AA. Washout from the eye was more rapid than the brain. Figure 9C shows lung D-AA and its conversion by elongation to the long chain PUFA adrenic acid (22:4) and desaturation to docosapentaenoic acid (DPA, 22:5). All PUFAs followed similar washout kinetics.

[0150] Brain fatty acid metabolism, including overall turnover, tends to differ from that of organ tissues. Neuronal PUFAs are uniquely unsaturated, with approximately 40% of total brain fatty acids consisting of both AA and omega-3 docosahexaenoic acid (DHA), but less than 1% linoleic acid. Lung was similar to other tissues. Effects of H-AA and D-AA on Animal Health

[0151] H-AA and D-AA were well tolerated without apparent adverse reactions. Diet intake for 2, 4, 6 or 8 weeks did not lead to significant changes in the levels of IL-1β in BAL fluid or colon homogenates of conventional healthy mice, regardless of sex. Macroscopically, the lungs of all groups were normal at the time of euthanasia. Cellular infiltration in the BAL was not significantly affected by any of the AA-rich diets. After 8 weeks of AA-rich diet feeding, lymphocyte counts were increased compared to controls. Inflammatory cell counts in cytological smears of lavage did not differ significantly between H- and D-diet intakes.

[0152] The effect of the AA diet on the thickness of the interalveolar septum was dependent on duration and was affected by deuteration. The mean thickness of the interalveolar septum in mice after H-AA and D-AA diets fed for 2 or 4 weeks was not significantly different. On the other hand, the alveolar walls of healthy males and females after 8 weeks of H-AA intake were significantly (p≦0.05) thicker compared to those of the respective D-AA cohorts. No associated changes in the alveolar space were observed for the AA or D6-AA diets. Analysis of the composition of the gut microbiota and enzyme immunoassays of interleukin levels in lavage fluids and intestinal homogenates did not reveal any significant differences between mice on the diets, regardless of the duration of the diet course (data not shown). Effect of D-AA on the response to LPS

[0153] We challenged mice fed an AA diet for 6 weeks using LPS treatment. 24 hours after exposure to LPS, lung tissue showed intense perivascular, peribronchial, and septal inflammatory infiltrates with lymphocyte and macrophage predominance. Perivascular edema, signs of thrombus formation, alveolar wall thickening, irregular distribution of air spaces, and focal areas of alveolar hemorrhage were also observed. We preferred the more extreme intranasal administration to the intratracheal route, which induced IL-1β, which in turn amplified inflammation making it difficult to discern early and late causal relationships of LPS challenge. In fact, 24 hours after intranasal administration of LPS at a dose of 1 mg / kg, regardless of diet type or duration, there was no significant increase in IL-1β content in BAL fluid or colon homogenates compared to animals not treated with LPS. Intranasal LPS 24 hours after administration increased neutrophils in the BAL fluid in all treatment groups, regardless of sex or form of AA diet. The numbers of other inflammatory cells in LPS-treated animals changed similarly to those in untreated mice and were characterized by significantly higher numbers of lymphocytes after 8 weeks of ingestion of either H- or D-AA compared with a 2-week course of H-AA. We did not notice any statistically significant differences between the effects of H- and D-AA with regard to any of the analyzed inflammatory cell types, regardless of sex or LPS treatment.

[0154] In mice fed a 6-week course of dietary H-AA followed by a single intranasal dose of LPS, the thickness of the interalveolar septum was significantly increased, which is likely associated with stronger edema and higher inflammation severity with immune cell infiltration. At the same time, the lungs of some mice fed H-AA and challenged with LPS showed areas of emphysematous changes (Figures 10A-10C). Figures 10A-10C show histological evaluation of the effect of intranasal administration of lipopolysaccharide on the lungs of H-AA and D-AA treated male mice. Figure 10A corresponds to the lungs of a normal mouse; Figure 10B corresponds to the lungs of a mouse treated with LPS on the H-AA diet; Figure 10C corresponds to the lungs of a mouse treated with LPS on the D-AA diet. The arrows in the figures point to the emphysematous foci.

[0155] LPS-induced perivascular edema, as measured by alveolar wall thickness, in H-AA-fed mice was in contrast to the significantly milder effect in the male D-AA group and the almost negligible effect in D-AA females (Figure 11).

[0156] Figure 11 shows a graph of interalveolar septum thickness (μm) after a 6-week course of H- or D-AA with a single intranasal administration of LPS. Norm, male and female mice without LPS treatment. *-р≦0.05, H-AA female mice vs. control female; **-р≦0.05, H-AA male mice vs. control male; #-р≦0.05, H-AA female mice vs. D-AA female; ##-р≦0.05, H-AA male mice vs. D-AA male. Gender-dependent differences

[0157] It is well known that sex differences are important in models such as ALI. For example, female mice respond more strongly to P. aeruginosa. Estrogen is thought to confer a protective effect against LPS-induced acute lung inflammation and hemorrhagic shock-induced lung injury. Conversely, in an infection model with P. aeruginosa, females show a stronger lung inflammatory response and, at the same time, a higher bacterial load. This clearly indicates the complexity underlying sex differences in the pulmonary immune response. In our study, the degree of alveolar lumen area changes in both animal groups was sex-dependent: this index was significantly lower in females than in males, regardless of the AA form (H or D). Also, in D-AA mice, the severity of changes in the thickness of the interalveolar septum was significantly higher in males than in females, highly correlated with human cases. LPS model of ALI / ARDS

[0158] ARDS results from direct (e.g., pneumonia, acid aspiration) or indirect (e.g., pancreatitis, non-pulmonary sepsis) injury to the lung. Murine models of direct ALI include agents delivered intratracheally or intranasally. We induced ALI by intranasal administration of E. coli LPS, which is considered a common inducer of lung injury and inflammation. Dietary intake of H-AA before LPS exposure did not ameliorate, and even exacerbated, LPS-induced lung tissue injury, including alveolar wall thickening, depending on its duration. The damaging effect of LPS observed against the background of dietary D-AA was substantially mitigated. LPS-induced ALI is a well-established model of ARDS. LPS activates NF-κB, which in turn activates the NLRP3 inflammasome, leading to the secretion of IL-1β and IL-18, which induce inflammation by positive feedback. Various treatments have been reported to protect against the acute effects of LPS in mouse models, including ouabain and various other compounds such as xanthohumol and choririn, but the mechanisms of these protective effects are complex.

[0159] In contrast, D-PUFAs may have a simpler mechanism of action to reduce inflammation. We previously demonstrated the inhibitory effect of bis-allylic deuteration of AA on the formation of various proinflammatory eicosanoids, while simultaneously inhibiting LPO. This combination of useful features prompted us to test D-PUFAs in animal models of inflammation. D2-LA

[0160] In vivo, a portion of ingested LA is incorporated into most tissues, with notable exceptions including the retina and brain. Depending on the tissue, a variable fraction of LA is converted to AA by enzymatic elongation and desaturation. Neural tissue contains AA but not LA, whereas in adipose tissue, the predominant n-6 species is LA. AA derived from LA by enzymatic elongation and desaturation are then incorporated into virtually every tissue. Thus, administration of D2-LA to mammals would lead to tissue uptake of 13,13-D2-AA (Figures 7A-7D). Deuteration at C13 would lead to inhibition of COX1 / 2 and 15-LOX, and reduce the levels of some proinflammatory species as well as LPO. Indeed, our preliminary experiments revealed that although LPS-induced changes in alveolar morphology were seen in both mice receiving H- or D2-LA for 6 weeks, the thickness of the interalveolar septum was significantly higher in mice treated with H-LA compared to the D2-LA group, regardless of sex. Ingestion of D-AA for 6 weeks led to a better protection against LPS-induced alveolar wall thickening. Moreover, the difference in the effect on the alveoli between the H-AA and D-AA diets was even more pronounced compared to that of the LA isoform. Based on our prior findings, a greater inhibitory effect would be expected from fully deuterated D6-AA compared to D2-AA. D6-ARA

[0161] AA is more efficiently incorporated into tissues when ingested as preformed AA than as LA, with subsequent metabolic conversion to AA. Nevertheless, within tissues or pools (e.g., plasma phospholipids), AA levels tend to be relatively tightly regulated. One effect of dietary AA is increased tissue AA turnover.

[0162] LPS activates phospholipase A2, leading to the release of AA into the surrounding tissues and blood, regulating global inflammation at the organism level, including sepsis and survival. Thus, fatty acid metabolism may be important for the regulation of acute LPS-induced lung injury and other conditions. Exogenous oxidized phospholipids induce hyperinflammatory responses in vitro. ω-3 PUFAs downregulate inflammation by competing with AA for LOX and COX. Moreover, some oxidation products of long-chain PUFAs (e.g., isoprostanes and neuroprostanes) have interesting biological activities. In mice infected with P. aeruginosa, survival was improved by fish oil supplementation. Surprisingly, these effects were also seen with AA, whereby a single AA bolus resulted in increased superoxide dismutase (SOD), decreased activity of myeloperoxidase (MPO), decreased malondialdehyde (MDA), decreased lactate dehydrogenase (LDH), attenuated PQ-induced histological damage, and induction of proinflammatory cytokines, indicating multiple mechanisms of acute-chronic responses possibly involving the proinflammatory actions of some AA oxidation products, such as PGE2 and asthma-related proinflammatory mediators (SPMs) like LXB4.

[0163] In bacterial infections, the situation is more complicated because some microorganisms, such as the common opportunistic pathogen P. aeruginosa, produce secreted forms of lipoxygenase that oxidize AA.

[0164] AA is the major PUFA involved in inflammation. For example, peripheral blood lymphocytes and monocytes were reported to have an average AA content of 16-20% of total fatty acids. AA is cleaved and released by phospholipase A2 (PLA2), which acts as a substrate for COX, LOX and cytochrome P450, to obtain proinflammatory eicosanoid mediators and regulators such as prostaglandins, leukotrienes and thromboxanes. Patients suffering from acute COPD have increased AA content in sputum. It has been suggested that the severity of emphysematous changes may also be associated in such patients with high AA levels and COX2 transducing mediators. In our study, dietary intake of D-AA for 6 or 8 weeks significantly reduced endotoxin-induced peribronchial infiltration and improved both interalveolar space and interalveolar wall thickness compared to those in mice fed H-AA. The inflammation and emphysema observed in the lungs of mice fed H-AA was substantially reduced in mice dosed with D-AA, suggesting that: i) the formation of eicosanoids from dietary H-AA was increased; and ii) better replacement of n-6 PUFAs and surfactant PL by D-AA (more stable against both enzymatic and nonenzymatic oxidation) in the lungs leads to reduced production of COX- and LOX-transducing mediators and reduced stimulation of phospholipase A2 by oxidative stress. The role of surfactants

[0165] Pulmonary surfactant covers a significant alveolar surface in all mammalian species, stabilizing the alveoli and preventing them from collapsing. It is composed of 10% protein and 90% lipid with a predominance of phospholipids (PL). Within the PL, both acyl chains can be saturated (no double bonds), monounsaturated (one double bond), or polyunsaturated (i.e., two or more double bonds, such as in ARA). The structure of surfactant PL allows it to regulate host-pathogen interactions as well as to respond to environmentally induced oxidative stress. Alterations in the pulmonary surfactant system have long been documented in ARDS, or more generally in any pulmonary disease with evident alveolar inflammation. Most surfactants collected from patients suffering from ARDS (including humans associated with pulmonary infections) or pneumonia are characterized by a significant decrease in the percentage of palmitic acid, while the relative amount of unsaturated species (including ARA) in PL is significantly increased. The multiple double bonds of polyunsaturated fatty acids make them highly susceptible to enzymatic and nonenzymatic oxidation and enhance oxidative stress. Although the intraalveolar microenvironment is often considered to have no direct physical contact with the plasma pool, there is some evidence suggesting that systemic metabolic conditions, as well as dietary perturbations, can modulate surfactant Pl. Continuous enteral feeding of a fish oil-enriched formulation promotes significant modulation of the PUFA composition of rat surfactant phospholipids within 72 hours. Thus, n-6 PUFAs (i.e., 18:2 n-6 and 20:4 n-6, precursors of eicosanoids and leukotrienes) were replaced by long-chain n-3 PUFAs. The ability of enteral feeding to rapidly modulate surfactant PUFA composition may facilitate timely attenuation of eicosanoid-driven inflammatory processes and reduce the risk of ARDS development. AA, the yin and yang of inflammation

[0166] Excess AA is proinflammatory under certain conditions of viral and bacterial infections that can be modeled by LPS. LPS treatment promotes inflammation through various mechanisms, including secretion of proinflammatory cytokines, eicosanoids, and induction of ROS. Acute lung inflammation follows soon after intranasal administration of LPS. The COVID-19 pandemic has caused significant morbidity and mortality worldwide. Elderly people and people with other pre-existing conditions have been responsible for higher mortality among COVID-19 patients. Many viruses have high affinity for receptors found in the lungs. COVID-19, SARS, MERS, and influenza (as well as some non-infectious diseases) trigger an immune response and attract immune cells to the area to attack the virus, resulting in local inflammation. In some cases, this can lead to a “cytokine storm” involving excessive and unregulated production of proinflammatory cytokines / chemokines, including IL-6, TNF-α, IFN-γ, IL-2, and IL-7, resulting in hyperinflammation that can be fatal. Because the immune system of young people relies more on innate mechanisms, it usually produces lower levels of inflammatory-driving cytokines. Furthermore, COVID patients, even with thromboprophylaxis, demonstrate an increased incidence of thrombotic complications (almost half of patients admitted to the ICU), which further contributes to the high mortality from COVID due to pulmonary embolism, stroke, and heart attack. In fact, AA as well as its oxidation products are elevated in COVID patients.

[0167] There is a complex involvement of the microbiota in various inflammation-related responses. It is known that the gut microbiota is important for the severity of pulmonary infections. However, we did not observe any significant differences here - this fact indicates the importance of molecular mechanisms intrinsic to mammalian cells and tissues. Overview of dietary D-AA as a drug

[0168] Traditional approaches to alleviate the deleterious effects of eicosanoid-mediated hyperinflammatory responses rely on small molecule inhibitors of COX and LOX enzymes. However, this leads to exacerbation of pulmonary symptoms, such as in the case of aspirin-exacerbated respiratory disease, where dysregulated AA metabolism plays a key role. Thus, D-AA may represent a milder approach for the treatment of inflammatory pulmonary pathologies.

[0169] Oral data from human dosing in ongoing clinical trials shows an excellent safety record for D2-LA, the metabolic precursor to 13,13-D2-AA. The expected safety of D-PUFA drugs combined with the convenience of oral dosing in D-PUFA gel capsules warrants further investigation of D-PUFAs as an approach to anti-inflammation and potential preventatives, treatments, and therapy for COVID-19-induced cytokine storm and thrombotic events. Other PUFA emulsions, such as IntraLipid, are formulated in emulsion form and safely dosed IV at several grams per day. One skilled in the art could imagine that such a formulation could allow for rapid bolus dosing of drug at the start of treatment followed by lipid gel capsule oral dosing of D-AA as a continuing therapy. These data provide a rationale for further investigation of D-PUFA technology as an approach to COVID inflammation-related treatment.

[0170] D-AA is less prone to either enzymatic or nonenzymatic oxidation and is therefore more potent in regulatory interactions that do not require chemical conversion. In high-fat fed mice, AA reduces LPS-induced inflammation in macrophages and septic death in mice by binding to MD2. Thus, in addition to its enhanced stability, D-AA may also be beneficial due to its improved metabolism. D-AA may be a viable therapeutic agent for inflammatory conditions involving enzymatic and nonenzymatic oxidation.

[0171] Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the disclosure be limited by the specific examples provided therein. The present disclosure has been described with reference to the above specification, but the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. Furthermore, it should be understood that all aspects of the present disclosure are not limited to the specific depictions, shapes, or relative proportions set forth herein, which depend upon various conditions and variables. It should be understood that various alternatives to the embodiments of the disclosure described herein may be utilized in implementing the present disclosure. It is therefore contemplated that the present invention should also encompass all such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present disclosure, and that methods and structures within the scope of these claims, and equivalents thereof, are covered thereby.

Claims

1. A method for controlling chronic inflammation in a patient, comprising administering to the patient a composition comprising an effective amount of D6-arachidonic acid for a period of time effective to treat the chronic inflammation in the patient.

2. 2. The method of claim 1, wherein the chronic inflammation is associated with chronic pain in the patient, and controlling the chronic inflammation reduces the level of the associated chronic pain.

3. 2. The method of claim 1, wherein the D6-arachidonic acid or ester thereof has a formula in which some or all of the hydrogen atoms at positions 7, 10, and 13 are replaced with deuterium.

4. The D6-arachidonic acid has the following formula: 【Chemistry 1】 4. The method of claim 3, having the following structure:

5. The method of claim 3, wherein the D6-arachidonic acid is a composition comprising arachidonic acid or an ester thereof in which, on average, at least about 80% of the hydrogen atoms at each bis-allylic position are replaced by deuterium atoms, and, on average, no more than about 35% of the hydrogen atoms at the mono-allylic positions are replaced by deuterium atoms.

6. The method of claim 1, wherein the amount of D6-arachidonic acid or its ester administered to the patient is sufficient to provide a D6-arachidonic acid concentration in the patient's red blood cells of at least about 12%, based on the total concentration of arachidonic acid in the red blood cells, including D6-arachidonic acid.

7. A pharmaceutical composition comprising a pharma- ceutically acceptable excipient and about 100 mg to about 2,000 mg of a composition of D6-arachidonic acid or an ester thereof, wherein, on average, at least about 80% of the hydrogen atoms at each bis-allylic position are replaced by deuterium atoms and, on average, no more than about 35% of the hydrogen atoms at the mono-allylic positions are replaced by deuterium atoms.

8. 8. The pharmaceutical composition of claim 7, wherein the composition comprises about 100 mg to about 1,000 mg of D6-arachidonic acid or an ester thereof, wherein, on average, at least about 80% of the hydrogen atoms at each bis-allylic position are replaced by deuterium atoms, and, on average, no more than about 35% of the hydrogen atoms at the mono-allylic positions are replaced by deuterium atoms.

9. 8. The pharmaceutical composition of claim 7, wherein the composition comprises about 100 mg to about 750 mg of D6-arachidonic acid or an ester thereof, wherein, on average, at least about 80% of the hydrogen atoms at each bis-allylic position are replaced by deuterium atoms, and, on average, no more than about 35% of the hydrogen atoms at the mono-allylic positions are replaced by deuterium atoms.

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