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WO2025153832A1 - Anti-inflammatory composition and use - Google Patents

Anti-inflammatory composition and use

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Publication number
WO2025153832A1
WO2025153832A1 PCT/GB2025/050088 GB2025050088W WO2025153832A1 WO 2025153832 A1 WO2025153832 A1 WO 2025153832A1 GB 2025050088 W GB2025050088 W GB 2025050088W WO 2025153832 A1 WO2025153832 A1 WO 2025153832A1
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WO
WIPO (PCT)
Prior art keywords
acid
dta
eta
docosa
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/GB2025/050088
Other languages
French (fr)
Inventor
Keith Coupland
David Andrew Parker
Andrew Neil BOA
Anbu Sellamuthu KOODUTHURAI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Northwood Consultants Ltd
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Northwood Consultants Ltd
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Publication date
Application filed by Northwood Consultants Ltd filed Critical Northwood Consultants Ltd
Publication of WO2025153832A1 publication Critical patent/WO2025153832A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • A61K31/232—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having three or more double bonds, e.g. etretinate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to compositions comprising one or more polyunsaturated fatty acids, use thereof as an anti-inflammatory agent, and in particular, to treat neurodegenerative disease.
  • Alzheimer’s disease is thought to be the key process leading to neuronal loss and cognitive decline. Cognitive decline is often a precursor to age-related neurodegenerative disease, the most common form being Alzheimer’s disease.
  • the World Alzheimer’s Report 2019 (Alzheimer’s Disease International - published September 2019) estimates that 50 million people are affected with Alzheimer’s disease worldwide and in the absence of an effective treatment the number is predicted to triple by 2050.
  • a hallmark of Alzheimer’s disease is the presence of senile plaque in the brain, together with the appearance of fibrillary tangles, composed of Tau-protein, inside brain cells.
  • Many clinical trials of drugs have been aimed at removing or preventing senile plaque formation.
  • Senile plaque is composed of toxic amyloid B (AB) peptide. None of the trials involving drugs, including long-chain polyunsaturated fatty acids (PUFAs), aimed at reducing build up or preventing AB peptide formation have been successful.
  • PUFAs long-chain polyunsaturated fatty acids
  • Eicosapentaenoic acid (2O:5co-3) (EPA) is a long-chain polyunsaturated fatty acid (PUFA) that has been extensively studied in many disease conditions and is recognized as having both anti-inflammatory and cardio-protective properties when included in the diet. EPA has also been shown to inhibit neuroinflammation in in vitro cultured BV-2 microglial cells (Moon, Dong-Oh et al, (2007), Inhibitory effects of eicosapentaenoic acid on lipopolysaccharide-induced activation in BV2 microglia, Int. Immunopharmacol. 7, 222-229).
  • PUFA polyunsaturated fatty acid
  • ETA 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (2O:4co-3)
  • ETA is a rare PUFA.
  • ETA is found in small quantities in some fish oils, about 1%, and in a mutant strain of the microorganism Mortierella alpina.
  • ETA is member of the omega-3 family of PUFAs derived from a-linolenic acid (18:3co-3) (ALA).
  • ALA is metabolized in vivo by a series of desaturations and elongations to higher molecular weight fatty acids with increasing degrees of unsaturation.
  • the present invention provides 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) for use in the treatment of neurodegenerative disease.
  • ETA eicosatetraenoic acid
  • DTA 14Z, 17Z-eicosatetraenoic acid
  • the present invention provides 8Z, 11Z, 14Z, 17Z- eicosatetraenoic acid (ETA) for use in the treatment of neurodegenerative disease.
  • ETA eicosatetraenoic acid
  • the present invention provides 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) for use in the treatment of neurodegenerative disease.
  • the invention provides a polyunsaturated fatty acid of the formula 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) in purified form.
  • composition comprising 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA).
  • According to one aspect of the invention is a method comprising administering 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA).
  • ETA eicosatetraenoic acid
  • a capsule comprising a core comprising 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA).
  • DTA 13, 16-trienoic acid
  • a capsule comprising a core comprising a combination of 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA).
  • Neurodegenerative disease refers to a condition having a pathophysiological component of neuronal death.
  • Neurodegeneration is the progressive loss of structure or function of neurons, including death of neurons.
  • diseases include Alzheimer's disease, impaired memory functions, amnesiac syndromes, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS) (including familial ALS and sporadic ALS), Pick's disease, dementia, depression, sleep disorders, psychoses, epilepsy, schizophrenia, paranoia, attention deficit hyperactivity disorder (ADHD), progressive supranuclear palsy, brain tumor, head trauma and Lyme disease.
  • ALS amyotrophic lateral sclerosis
  • ADHD attention deficit hyperactivity disorder
  • ETA 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (2O:4co-3) (ETA), used in the present invention, is a natural substance and is formed in the human body as an intermediate in the alpha-linolenic acid metabolic pathway (Crawford, M.A., (1983), Background to Essential Fatty Acids and Their Prostanoid Derivatives, British Medical Bulletin, 39, 3, 210-213).
  • ETA can be isolated from conventional fish oil containing approximately 1% ETA by transesterification in ethanol followed by isolation using solid phase chromatography. ETA can be synthesized by two successive C-l extensions of stearidonic acid (SDA) through conversion to fatty alcohols, then mesylates, chain elongation to the corresponding nitriles and finally methyl esters. These processes are described in Ghioni, C.
  • DTA 14-eicosatraenoic acid
  • DGLA 14-eicosatraenoic acid
  • ETA and/or DTA described and used herein are suitably in purified form.
  • the term "purified” used herein means separated from components that may occur with it in nature or in an artificially produced mixture.
  • ETA and/or DTA are purified when it is at least about 10% (e.g., at least 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, and 100%), by weight (excluding solvent), free from components that may occur with it in nature or in an artificially produced mixture.
  • Purity can be measured by any appropriate method, e.g. column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • omega-3 and/or omega-6 PUFAs Human consumption of some omega-3 and/or omega-6 PUFAs provide compounds with a range of downstream metabolites such as anti-inflammatory prostaglandins, lipoxins and the recently discovered specialized pro-resolving mediators (SPMs). These mediators include resolvins (short for resolution phase interaction products), maresins (short for macrophage mediators in resolving inflammation) and protectins (Serhan, C.N. and Levy, B.D., (2018), Resolvins in inflammation: emergence of the proresolving superfamily of mediators, J. Clin. Invest., 128 (7), 2657-2669.
  • SPMs pro-resolving mediators
  • ETA and DTA are capable of metabolising/conversion to many active derivatives, which can be a significant advantage in treating a multifactorial disease such as neurodegenerative disease, particularly Alzheimer’s disease, i.e. by providing a cascade of derived molecules capable of addressing many aspects of neuroinflammation.
  • ETA can metabolise to EPA (C2O:5co-3) and further to docosapentaenoic acid (C22:5co-3) (DPA).
  • EPA is the precursor to a range of SPMs including the E-series resolvins; resolvin El, E2 and E3.
  • ETA and/or DTA can act as a precursor to a series of lipoxygenase and cyclooxygenase derived novel metabolites with potential pro-resolving properties.
  • ETA and DTA on their own are significantly more effective in BV-2 microglial cells and/or RAW 264.7 cells and/or ‘freshly isolated ‘rat primary microglia cells at inhibiting the release of pro- inflammatory cytokines such as TNF-a and IL-6 than other omega-3 PUFAs, such as eicosapentaenoic acid (C2O:5co-3) (EPA) and docosahexaenoic acid (22:6co-3) (DHA).
  • pro- inflammatory cytokines such as TNF-a and IL-6 than other omega-3 PUFAs, such as eicosapentaenoic acid (C2O:5co-3) (EPA) and docosahexaenoic acid (22:6co-3) (DHA).
  • DTA was demonstrated to have a greater protective effect on neurons in relation to caspase 3/7 activation and neurite outgrowth than other omega- 3 PUFA’s such as EPA and DHA.
  • other omega- 3 PUFA’s such as EPA and DHA.
  • studies on the effects of externally added TNF-a to ‘freshly isolated’ cortical neurons have shown that DTA had a greater protective effect in relation to cell number, caspase 3/7 activation, mitochondrial integrity (TMRM) and neurite outgrowth than EPA and DHA.
  • TMRM mitochondrial integrity
  • ETA preferably in a composition, particularly a pharmaceutical composition, and used according to the present invention may be in the form of the free acid and/or any pharmaceutically acceptable salt and/or ester and/or amide thereof.
  • Suitable salts include alkali metal and/or ammonium salt, preferably sodium salt.
  • ETA is in lower alkyl ester form, preferably as the methyl, ethyl, propyl and/or butyl ester, more preferably methyl and/or ethyl ester, and particularly ethyl ester (ETA-EE).
  • DTA preferably in a composition, particularly a pharmaceutical composition, and used according to the present invention may be in the form of the free acid and/or any pharmaceutically acceptable salt and/or ester and/or amide thereof.
  • Suitable salts include alkali metal and/or ammonium salt, preferably sodium salt.
  • DTA is in lower alkyl ester form, preferably as the methyl, ethyl, propyl and/or butyl ester, more preferably methyl and/or ethyl ester, and particularly ethyl ester (DTA-EE).
  • ETA and DTA are each present in alkyl ester form
  • the alkyl ester for each of ETA and DTA may be the same or different.
  • ETA is preferably administered to/taken by a subject, preferably human adult, in an amount in the range from 1 to 10 g/day, more preferably 2 to 7 g/day, and particularly 3 to 5 g/day.
  • DTA is preferably administered to/taken by a subject, preferably human adult, in an amount in the range from 1 to 10 g/day, more preferably 2 to 7 g/day, and particularly 3 to 5 g/day.
  • the anti-neuroinflammatory effect of using ETA and/or DTA can be surprisingly, and preferably synergistically, increased by using ETA and/or DTA in combination with one or more other omega-3 and/or omega 6 PUFAs, suitably selected from the group consisting of EP A, DHA, stearidonic acid (6, 9, 12, 15-octadecatrienioc acid) (SDA), gamma linolenic acid (6, 9, 12-octadecatrienioc acid) (GLA), dihomo y linolenic acid (8, 11, 14-eicosatraenoic acid) (DGLA), and 7, 10, 13, 16, 19-docosapentaenoic acid (DPA); preferably selected from the group consisting of EP A, DHA, SDA and DGLA; more preferably selected from the group consisting of DHA and DGLA; and particularly DHA.
  • DPA omega-3 and/or omega 6 PUFAs
  • ETA and/or DTA and/or other omega-3 and/or omega 6 PUFAs defined herein may be chemically linked together in a single compound, e.g. as a mixed ester of a diol such as propylene glycol; and/or as a mixed amide of a diamine such as ethylenediamine and/or an amino alcohol such as tris-amino propanediol; and/or as a mixed ester/amide of an amino acid.
  • the molar ratio of ETA to EPA in a composition, preferably a pharmaceutical composition, used herein is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0:1.0, more preferably 0.5 to 2.0:1.0, particularly 0.7 to 1.4:1.0 and especially 0.9 to 1.1: 1.0.
  • the molar ratio of ETA to DHA in a composition, preferably a pharmaceutical composition, used herein is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0:1.0, more preferably 0.5 to 2.0:1.0, particularly 0.7 to 1.4:1.0 and especially 0.9 to 1.1: 1.0.
  • the molar ratio of DTA to DPA in a composition, preferably a pharmaceutical composition, used herein is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0:1.0, more preferably 0.5 to 2.0:1.0, particularly 0.7 to 1.4:1.0 and especially 0.9 to 1.1: 1.0.
  • the total amount of ETA and/or DTA and/or other omega-3 and/or omega 6 PUFA is preferably in the range from 1 to 10 g/day, more preferably 2 to 7 g/day, and particularly 3 to 5 g/day.
  • the total amount of one or more antioxidants in a composition is suitably less than 0.50 wt%, preferably in the range from 0.001 to 0.30 wt%, more preferably 0.01 to 0.20 wt%, particularly 0.025 to 0.10 wt%, and especially 0.03 to 0.05 wt%, based on the total weight of ETA and/or DTA and any other omega 3 and/or omega 6 PUFA in the composition.
  • the antioxidant is not present in a therapeutically effective amount in the pharmaceutical composition.
  • the only components present in a therapeutically effective amount in a pharmaceutical composition defined and used herein, consist of ETA and/or DTA and/or other omega-3 and/or omega 6 PUFAs, suitably selected from the group consisting of EPA, DHA, SDA, GLA, DGLA, and DPA, preferably DHA.
  • therapeutically effective amount is meant the amount of a component or material that without causing significant negative or adverse side effects, is intended in relation to neurodegenerative disease, to (i) delay or prevent the onset thereof; (ii) slow down or stop the progression, aggravation, or deterioration of one or more symptoms; (iii) bring about amelioration of the symptoms; (iv) reduce the severity or incidence thereof; and/or (v) cure the condition or disorder.
  • the composition preferably a pharmaceutical composition, comprises at least 90.0 wt%, suitably at least 95.0 wt%, preferably at least 98.0 wt%, more preferably at least 99.0 wt%, particularly at least 99.5 wt%, and especially at least 99.9 wt%, of ETA, based on the total weight of the composition.
  • the composition preferably a pharmaceutical composition, comprises at least 90.0 wt%, suitably at least 95.0 wt%, preferably at least 98.0 wt%, more preferably at least 99.0 wt%, particularly at least 99.5 wt%, and especially at least 99.9 wt%, and up to 100 wt%, of ETA, based on the total weight of fatty acids (having a carbon chain length of 4 or greater) in the composition.
  • the composition preferably a pharmaceutical composition, comprises at least 90.0 wt%, suitably at least 95.0 wt%, preferably at least 98.0 wt%, more preferably at least 99.0 wt%, particularly at least 99.5 wt%, and especially at least 99.9 wt%, and up to 100 wt%, of DTA, based on the total weight of fatty acids (having a carbon chain length of 4 or greater) in the composition.
  • the composition preferably a pharmaceutical composition, comprises at least 90.0 wt%, suitably at least 95.0 wt%, preferably at least 98.0 wt%, more preferably at least 99.0 wt%, particularly at least 99.5 wt%, and especially at least 99.9 wt%, of ETA and/or DTA and at least one other omega 3 and/or omega 6 PUFA, suitably selected from the group consisting of EP A, DHA, SDA, GLA, DGLA, and DPA; preferably selected from the group consisting of EP A, DHA, SDA, and DGLA; more preferably selected from the group consisting of DHA and DGLA; and particularly DHA, based on the total weight of the composition.
  • the composition preferably a pharmaceutical composition, comprises at least 90.0 wt%, suitably at least 95.0 wt%, preferably at least 98.0 wt%, more preferably at least 99.0 wt%, particularly at least 99.5 wt%, and especially at least 99.9 wt%, of ETA and/or DTA and at least one other omega 3 and/or omega 6 PUFA, suitably selected from the group consisting of EP A, DHA, SDA, GLA, DGLA, and DPA; preferably selected from the group consisting of EP A, DHA, SDA, and DGLA; more preferably selected from the group consisting of DHA and DGLA; and particularly DHA, based on the total weight of fatty acids (having a carbon chain length of 4 or greater) in the composition.
  • the composition preferably a pharmaceutical composition, comprises less than 10.0 wt%, suitably less than 5.0 wt%, preferably less than 2.0 wt%, more preferably less than 1.0 wt%, particularly less than 0.5%, and especially less than 0.1 wt% of any omega-3 and/or omega 6 PUFA other than ETA, DTA, EP A, DHA, SDA, GLA, DGLA, and/or DPA, based on the total weight of the composition.
  • the composition preferably a pharmaceutical composition, comprises less than 10.0 wt%, suitably less than 5.0 wt%, preferably less than 2.0 wt%, more preferably less than 1.0 wt%, particularly less than 0.5%, and especially less than 0.1 wt% of any fatty acid (having a carbon chain length of 4 or greater) other than ETA, DTA, EP A, DHA, SDA, GLA, DGLA, and/or DPA, based on the total weight of the composition.
  • any fatty acid having a carbon chain length of 4 or greater
  • the composition preferably a pharmaceutical composition, comprises less than 10.0 wt%, suitably less than 5.0 wt%, preferably less than 2.0 wt%, more preferably less than 1.0 wt%, particularly less than 0.5%, and especially less than 0.1 wt% of any non-omega-3 and/or non-omega 6 PUFA, based on the total weight of the composition.
  • the composition preferably a pharmaceutical composition, comprises less than 10.0 wt%, suitably less than 5.0 wt%, preferably less than 2.0 wt%, more preferably less than 1.0 wt%, particularly less than 0.5%, and especially less than 0.1 wt% of any saturated fatty acid (having a carbon chain length of 4 or greater), based on the total weight of the composition.
  • the composition preferably a pharmaceutical composition, comprises less than 10.0 wt%, suitably less than 5.0 wt%, preferably less than 2.0 wt%, more preferably less than 1.0 wt%, particularly less than 0.5%, and especially less than 0.1 wt% of any hydrocarbon comprising at least one aromatic ring, based on the total weight of the composition.
  • the pharmaceutical composition of the invention may be administered systemically or locally.
  • the pharmaceutical composition may be administered orally, buccally, by injection, by percutaneous administration, parenterally, intrathecally, by endoscopy, topically, transdermally, transmucosally, nasally, by inhalation spray, rectally, vaginally, intratracheally, and via an implanted reservoir.
  • the pharmaceutical composition of the invention may be orally administered.
  • formulations adapted to oral administration include, but are not limited to, solid forms, liquid forms and gels.
  • solid forms adapted to oral administration include, but are not limited to, pill, tablet, capsule, soft gelatin capsule, hard gelatin capsule, dragees, granules, caplet, compressed tablet, cachet, wafer, sugar-coated pill, sugar coated tablet, or dispersing/or disintegrating tablet, powder, solid forms suitable for solution in, or suspension in, liquid prior to oral administration and effervescent tablet.
  • liquid form adapted to oral administration include, but are not limited to, solutions, suspensions, drinkable solutions, elixirs, sealed phial, potion, drench, syrup, liquor and sprays.
  • the pharmaceutical composition may be injected, preferably systemically injected.
  • formulations adapted to systemic injections include, but are not limited to, liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection.
  • the aim of this study was to investigate the effect of DTA, DHA and EPA in an in vitro model of CNS injury using a multiparametric cell-based protocol.
  • the assay was applied to “freshly isolated” rat primary microglia and cortical neurons.
  • Rat primary microglia were serum starved for 24 hours and subsequently pretreated with 50 pM DTA ethyl ester (DTA-EE, produced in Example 7 above), 50 pM DHA ethyl ester (DHA-EE) or 50 pM EPA ethyl ester (EPA-EE, ex BASF) for an additional 24 hours. Ethanol was used as vehicle control. After this, 500 ng ml’ 1 LPS was added and the cells were incubated for 6.5 hours. Medium was replaced and the cells were then exposed to 10 pM A (1-42) oligomers for 24 hours maintaining the presence of the same lipids that were used in the pretreatment and adding 0.2% DMSO to the vehicle control.
  • DTA-EE DTA ethyl ester
  • DHA-EE 50 pM DHA ethyl ester
  • EPA-EE pM EPA ethyl ester
  • cortical neurons were pretreated or not with 50 pM DTA-EE, 50 pM DHA-EE, or 50 pM EPA-EE for 24 hours (ethanol was also used as vehicle control).
  • the neurons were then incubated for 72 hours with the conditioned medium of the microglia that had been in contact with the Ap (1-42) oligomers (always maintaining paired the same pretreatment conditions in microglia and neurons). Samples of this conditioned medium were stored at -80°C to subsequently measure the amount of TNF-a by ELISA. Finally, cells were analysed for cell parameters associated with caspase 3/7 activation and neurite outgrowth.
  • Caspase 3/7 activation was determined using “The CellEvent Caspase-3/7 Green Detection Reagent” which is intrinsically a non-fluorescent peptide that inhibits the ability of the dye to bind to DNA. After activation of caspase-3/7 in apoptotic cells, the peptide was cleaved enabling the dye to bind to DNA and produce a bright, fluorogenic response. The cell was stained and measured at 488 nm/530 nm Ex/Em, enabling a direct quantification of apoptotic cells.
  • Neurite outgrowth was determined using -III-tubulin staining performed by immunofluorescence.
  • Cells were fixed with cold methanol for 10 minutes. After the fixation step, the samples were washed three times with Fetal Bovine Serum (FBS), stained with Hoechst 1:200 and blocked with PBS + 3% Bovine Serum Albumine (BSA) for 30 minutes. Finally, anti- -III-tubulin-FITC antibody was added at 1/150 in PBS + 0.5% BSA for 60 minutes at room temperature. Cells were then washed three times and analysed in a Cell Insight CX7 automated fluorescent microscope. To investigate the role of neurite extension, the geometric pattern of total length per neuron, neurite complexity and number of branch points were measured. a) Cytokine Production
  • DTA-EE was an order of magnitude more effective than DHA-EE or EPA-EE and afforded a 93% reduction in the inflammatory cytokine TNF-a.
  • DTA-EE provided a 3-fold recovery in neurite length compared with untreated cells and outperformed both DHA-EE and EPA-EE.
  • the aim of this study was to investigate the effect of mixtures of DTA, ETA, and DHA on the inhibition of TNF-a from LPS stimulated microglia.
  • the method used was the same as the rat primary microglia assay described in Example 8 except that the microglia were treated with 35 pM DTA-EE + 35 pM ETA-EE, 35 pM DTA-EE + 35 pM DHA-EE, or 25 pM DTA-EE + 25 pM ETA-EE + 25 pM DHA-EE.
  • the aim of this study was to determine the neuroprotective effect of pure lipids following treatment of neurons with externally added TNF-a.
  • Freshly isolated primary cortical neurons from 18-day embryonic rats were plated in two poly-D-lysine-coated M96 plates at a density of 40,000 cells/well in BrainPhys neuronal medium supplemented with 2% NeuroCult SMI and 10% heat-inactivated foetal bovine serum. Twenty-four hours after plating, the serum was removed. On day in vitro 2, cells were pretreated with 50 pM of DTA-EE, EPA-EE, or DHA-EE, in complete BrainPhys neuronal medium, in triplicate, and incubated for 24 hours (ethanol was used as the vehicle control, and 50 ng BDNF as the positive control).

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Abstract

8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa-10,l 3, 16-trienoic acid (DTA) have been shown to have anti-neuroinflammatory properties and suitable for use in the treatment of neurodegenerative disease, such as Alzheimer's disease. The anti-neuroinflammatory effect of using ETA and/or DTA can be surprisingly, and optionally synergistically increased by using ETA and/or DTA in combination with eicosapentaenoic acid (EPA),docosahexaenoic acid (DHA), stearidonic acid (6, 9, 12, 15 -octadecatrienioc acid) (SDA), gamma linolenic acid (6, 9, 12-octadecatrienioc acid) (GLA), dihomo γ linolenic acid (8, 11, 14-eicosatraenoic acid) (DGLA), and/or 7, 10, 13, 16, 19-docosapentaenoic acid (DPA), preferably docosahexaenoic acid (DHA).

Description

Anti-Inflammatory Composition and Use
Field of Invention
The present invention relates to compositions comprising one or more polyunsaturated fatty acids, use thereof as an anti-inflammatory agent, and in particular, to treat neurodegenerative disease.
Background
Neuroinflammation is thought to be the key process leading to neuronal loss and cognitive decline. Cognitive decline is often a precursor to age-related neurodegenerative disease, the most common form being Alzheimer’s disease. The World Alzheimer’s Report 2019 (Alzheimer’s Disease International - published September 2019) estimates that 50 million people are affected with Alzheimer’s disease worldwide and in the absence of an effective treatment the number is predicted to triple by 2050. Currently there is no cure for Alzheimer’s disease and without a suitable treatment the ensuing economic and social burden will be immense.
A hallmark of Alzheimer’s disease is the presence of senile plaque in the brain, together with the appearance of fibrillary tangles, composed of Tau-protein, inside brain cells. Many clinical trials of drugs have been aimed at removing or preventing senile plaque formation. Senile plaque is composed of toxic amyloid B (AB) peptide. None of the trials involving drugs, including long-chain polyunsaturated fatty acids (PUFAs), aimed at reducing build up or preventing AB peptide formation have been successful. Eicosapentaenoic acid (2O:5co-3) (EPA) is a long-chain polyunsaturated fatty acid (PUFA) that has been extensively studied in many disease conditions and is recognized as having both anti-inflammatory and cardio-protective properties when included in the diet. EPA has also been shown to inhibit neuroinflammation in in vitro cultured BV-2 microglial cells (Moon, Dong-Oh et al, (2007), Inhibitory effects of eicosapentaenoic acid on lipopolysaccharide-induced activation in BV2 microglia, Int. Immunopharmacol. 7, 222-229). Despite showing anti-inflammatory properties when evaluated in in vitro tests, in vivo clinical studies on dementia using EPA have been disappointing (Boston, P.F. et al, (2004), Ethyl-EPA in Alzheimers disease - a pilot study, PLEFA 71, 5, 341-346; Troesch, B. et al, (2020), Expert Opinion on Benefits of Long-Chain Omega-3 Fatty Acids (DHA and EPA), Aging and Clinical Nutrition, Nutrients 12, 2555).
8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (2O:4co-3) (ETA) [CAS 24880-40-8], also known as omega-3 arachidonic acid, is a rare PUFA. ETA is found in small quantities in some fish oils, about 1%, and in a mutant strain of the microorganism Mortierella alpina. ETA is member of the omega-3 family of PUFAs derived from a-linolenic acid (18:3co-3) (ALA). ALA is metabolized in vivo by a series of desaturations and elongations to higher molecular weight fatty acids with increasing degrees of unsaturation.
10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (22:3co-6) (DTA) is an extremely rare PUFA only found in small quantities in mammalian adrenal gland lipids as a cholesterol ester (Takayasu, K. et al, Fatty Acid Composition of Human and Rat Adrenal Lipids: Occurrence of Omega-6 Docosatrienoic Acid in Human Adrenal Cholesterol Ester.Lipids, 1970 Sep:5(9): 143-50). Biochemical synthesis of DTA is achieved by chain elongation with malonyl coenzyme A with rat liver microsomes (Christiansen K., et al, (1968) Chain Elongation of Alpha- and Gamma-Linolenic Acids and the Effect of Other Fatty Acids on Their Conversion in Vitro J Biol Chem 243 11, Issue of June 102969-2974).
There is a need for improved anti-inflammatory treatments for age-related neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease, and in particular Alzheimer’s disease. Such treatments may be the use of materials or drugs in isolation, or in combination. It is widely accepted that Alzheimer’s disease is an inflammatory disease and reducing inflammation provides an attractive target for drug/treatment development.
Summary of the Invention
We have surprisingly discovered that the use of ETA and/or DTA, compositions comprising ETA and/or DTA, and use thereof, overcomes or significantly reduces at least one of the aforementioned problems.
Accordingly, the present invention provides 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) for use in the treatment of neurodegenerative disease.
According to one aspect the present invention provides 8Z, 11Z, 14Z, 17Z- eicosatetraenoic acid (ETA) for use in the treatment of neurodegenerative disease. According to another aspect the present invention provides 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) for use in the treatment of neurodegenerative disease.
According to another aspect the present invention provides 8Z, 11Z, 14Z, 17Z- eicosatetraenoic acid (ETA) and 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) for use in the treatment of neurodegenerative disease.
According to one aspect the invention provides a polyunsaturated fatty acid of the formula 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) in purified form.
The invention also provides a composition comprising 8Z, 11Z, 14Z, 17Z- eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) in conjunction with a pharmaceutically acceptable adjuvant, excipient or carrier.
The invention further provides a composition comprising 8Z, 11Z, 14Z, 17Z- eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) for use in the treatment of neurodegenerative disease.
The invention yet further provides a composition comprising 8Z, 11Z, 14Z, 17Z- eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) and at least one fatty acid selected from the group consisting of eicosapentaenoic acid (EP A), docosahexaenoic acid (DHA), stearidonic acid (6, 9, 12, 15 -octadecatri enioc acid) (SDA), gamma linolenic acid (6, 9, 12-octadecatrienioc acid) (GLA), dihomo y linolenic acid (8, 11, 14-eicosatraenoic acid) (DGLA), and 7, 10, 13, 16, 19-docosapentaenoic acid (DPA), preferably docosahexaenoic acid (DHA); and combinations thereof.
According to one aspect of the invention is a composition comprising 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA).
According to another aspect of the invention is a composition comprising 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA).
According to another aspect of the invention is a composition comprising a combination of 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and 10Z, 13Z, 16Z- docosa-10, 13, 16-trienoic acid (DTA).
The invention still further provides a method of treating or preventing neurodegenerative disease comprising administering to a subject in need thereof a therapeutically effective amount of 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) or a composition comprising ETA and/or DTA.
According to one aspect of the invention is a method comprising administering 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA).
According to another aspect of the invention is a method comprising administering 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA). According to another aspect of the invention is a method comprising administering a combination of 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and 10Z, 13Z, 16Z- docosa-10, 13, 16-trienoic acid (DTA).
“Effective amount” means the amount of a compound that, when administered to a subject for the prophylaxis or treatment of a disease and/or condition, is sufficient to affect such prophylaxis or such treatment for the disease and/or condition. The "effective amount" can vary depending on the compound, the disease and/or condition and its severity, and the age, weight, etc., of the subject.
The invention even further provides a capsule comprising a shell and a core comprising 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z- docosa-10, 13, 16-trienoic acid (DTA) or a composition comprising ETA and/or DTA.
According to one aspect of the invention is a capsule comprising a core comprising 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA).
According to another aspect of the invention is a capsule comprising a core comprising 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA).
According to another aspect of the invention is a capsule comprising a core comprising a combination of 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA). Detailed Description
Neurodegenerative disease refers to a condition having a pathophysiological component of neuronal death. Neurodegeneration is the progressive loss of structure or function of neurons, including death of neurons. Examples of such diseases include Alzheimer's disease, impaired memory functions, amnesiac syndromes, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS) (including familial ALS and sporadic ALS), Pick's disease, dementia, depression, sleep disorders, psychoses, epilepsy, schizophrenia, paranoia, attention deficit hyperactivity disorder (ADHD), progressive supranuclear palsy, brain tumor, head trauma and Lyme disease.
8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (2O:4co-3) (ETA), used in the present invention, is a natural substance and is formed in the human body as an intermediate in the alpha-linolenic acid metabolic pathway (Crawford, M.A., (1983), Background to Essential Fatty Acids and Their Prostanoid Derivatives, British Medical Bulletin, 39, 3, 210-213).
ETA can be isolated from conventional fish oil containing approximately 1% ETA by transesterification in ethanol followed by isolation using solid phase chromatography. ETA can be synthesized by two successive C-l extensions of stearidonic acid (SDA) through conversion to fatty alcohols, then mesylates, chain elongation to the corresponding nitriles and finally methyl esters. These processes are described in Ghioni, C. et al, (2002), Metabolism of C18:4n-3 (stearidonic acid) and C20:4n-3 (ETA) in salmonid cells in culture and inhibition of the production of prostaglandin F2a (PGF2a) from C20:4n-6 (arachidonic acid), Fish Physiology and Biochemistry, 27, 81-96.
10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (22:3co-6) (DTA) used in the present invention can be synthesized by the C2 elongation of dihomo y linolenic acid (8, 11, 14-eicosatraenoic acid (C2O:3co-6) (DGLA) by conventional methods such as the malonic ester synthesis.
ETA and/or DTA described and used herein are suitably in purified form. The term "purified" used herein means separated from components that may occur with it in nature or in an artificially produced mixture. Typically, ETA and/or DTA are purified when it is at least about 10% (e.g., at least 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, and 100%), by weight (excluding solvent), free from components that may occur with it in nature or in an artificially produced mixture. Purity can be measured by any appropriate method, e.g. column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
Human consumption of some omega-3 and/or omega-6 PUFAs provide compounds with a range of downstream metabolites such as anti-inflammatory prostaglandins, lipoxins and the recently discovered specialized pro-resolving mediators (SPMs). These mediators include resolvins (short for resolution phase interaction products), maresins (short for macrophage mediators in resolving inflammation) and protectins (Serhan, C.N. and Levy, B.D., (2018), Resolvins in inflammation: emergence of the proresolving superfamily of mediators, J. Clin. Invest., 128 (7), 2657-2669. ETA and DTA are capable of metabolising/conversion to many active derivatives, which can be a significant advantage in treating a multifactorial disease such as neurodegenerative disease, particularly Alzheimer’s disease, i.e. by providing a cascade of derived molecules capable of addressing many aspects of neuroinflammation. ETA can metabolise to EPA (C2O:5co-3) and further to docosapentaenoic acid (C22:5co-3) (DPA). EPA is the precursor to a range of SPMs including the E-series resolvins; resolvin El, E2 and E3. ETA and/or DTA can act as a precursor to a series of lipoxygenase and cyclooxygenase derived novel metabolites with potential pro-resolving properties.
We have surprisingly demonstrated herein that ETA and DTA are potently anti- neuroinflammatory on their own in in-vitro cultured BV-2 microglial cells and/or in mouse macrophage RAW 2647 cells and/or in ‘freshly isolated’ rat primary microglia cells. This is shown in experiments where BV-2 microglial cells and/or RAW 264.7 cells and/or ‘freshly isolated ‘rat primary microglia cells are stimulated with lipopolysaccharide (LPS) to release inflammatory cytokines such as tumour necrosis factor alpha (TNF-a) and Interleukin - 6 (IL-6). ETA and DTA on their own are significantly more effective in BV-2 microglial cells and/or RAW 264.7 cells and/or ‘freshly isolated ‘rat primary microglia cells at inhibiting the release of pro- inflammatory cytokines such as TNF-a and IL-6 than other omega-3 PUFAs, such as eicosapentaenoic acid (C2O:5co-3) (EPA) and docosahexaenoic acid (22:6co-3) (DHA). Moreover, in more advanced studies utilising ‘freshly isolated’ rat primary microglia and cortical neurons, DTA was demonstrated to have a greater protective effect on neurons in relation to caspase 3/7 activation and neurite outgrowth than other omega- 3 PUFA’s such as EPA and DHA. Also studies on the effects of externally added TNF-a to ‘freshly isolated’ cortical neurons have shown that DTA had a greater protective effect in relation to cell number, caspase 3/7 activation, mitochondrial integrity (TMRM) and neurite outgrowth than EPA and DHA.
ETA, preferably in a composition, particularly a pharmaceutical composition, and used according to the present invention may be in the form of the free acid and/or any pharmaceutically acceptable salt and/or ester and/or amide thereof. Suitable salts include alkali metal and/or ammonium salt, preferably sodium salt. In one embodiment, ETA is in lower alkyl ester form, preferably as the methyl, ethyl, propyl and/or butyl ester, more preferably methyl and/or ethyl ester, and particularly ethyl ester (ETA-EE).
DTA, preferably in a composition, particularly a pharmaceutical composition, and used according to the present invention may be in the form of the free acid and/or any pharmaceutically acceptable salt and/or ester and/or amide thereof. Suitable salts include alkali metal and/or ammonium salt, preferably sodium salt. In one embodiment, DTA is in lower alkyl ester form, preferably as the methyl, ethyl, propyl and/or butyl ester, more preferably methyl and/or ethyl ester, and particularly ethyl ester (DTA-EE).
When ETA and DTA are each present in alkyl ester form, the alkyl ester for each of ETA and DTA may be the same or different. In one embodiment, ETA is preferably administered to/taken by a subject, preferably human adult, in an amount in the range from 1 to 10 g/day, more preferably 2 to 7 g/day, and particularly 3 to 5 g/day.
In one embodiment, DTA is preferably administered to/taken by a subject, preferably human adult, in an amount in the range from 1 to 10 g/day, more preferably 2 to 7 g/day, and particularly 3 to 5 g/day.
The anti-neuroinflammatory effect of using ETA and/or DTA can be surprisingly, and preferably synergistically, increased by using ETA and/or DTA in combination with one or more other omega-3 and/or omega 6 PUFAs, suitably selected from the group consisting of EP A, DHA, stearidonic acid (6, 9, 12, 15-octadecatrienioc acid) (SDA), gamma linolenic acid (6, 9, 12-octadecatrienioc acid) (GLA), dihomo y linolenic acid (8, 11, 14-eicosatraenoic acid) (DGLA), and 7, 10, 13, 16, 19-docosapentaenoic acid (DPA); preferably selected from the group consisting of EP A, DHA, SDA and DGLA; more preferably selected from the group consisting of DHA and DGLA; and particularly DHA.
EP A, DHA, SDA, GLA, DGLA, and/or DPA, present in a composition, preferably a pharmaceutical composition, and used according to the present invention may be in the form of the free acid and/or any pharmaceutically acceptable salt and/or ester and/or amide thereof. Suitable salts include alkali metal and/or ammonium salt, preferably sodium salt. In one embodiment, the other omega-3 and/or omega 6 PUFA is used in lower alkyl ester form, prefer as the methyl, ethyl, propyl and/or butyl ester, more preferably methyl and/or ethyl ester, and particularly ethyl ester. In one embodiment, ETA and/or DTA and/or other omega-3 and/or omega 6 PUFAs defined herein may be chemically linked together in a single compound, e.g. as a mixed ester of a diol such as propylene glycol; and/or as a mixed amide of a diamine such as ethylenediamine and/or an amino alcohol such as tris-amino propanediol; and/or as a mixed ester/amide of an amino acid.
In one embodiment, the molar ratio of ETA to DTA in a composition, preferably a pharmaceutical composition, used herein according to the present invention, is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0:1.0, more preferably 0.5 to 2.0:1.0, particularly 0.7 to 1.4:1.0 and especially 0.9 to 1.1:1.0.
In one embodiment, the molar ratio of ETA and/or DTA to at least one other omega-3 and/or omega 6 PUFA, suitably selected from the group consisting of EP A, DHA, SDA, GLA, DGLA, and DPA in a composition, preferably a pharmaceutical composition, used herein according to the present invention, is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0: 1.0, more preferably 0.5 to 2.0: 1.0, particularly 0.7 to 1.4: 1.0 and especially 0.9 to 1.1:1.0.
In one embodiment, the molar ratio of ETA to EPA in a composition, preferably a pharmaceutical composition, used herein is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0:1.0, more preferably 0.5 to 2.0:1.0, particularly 0.7 to 1.4:1.0 and especially 0.9 to 1.1: 1.0.
In one embodiment, the molar ratio of DTA to EPA in a composition, preferably a pharmaceutical composition, used herein is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0:1.0, more preferably 0.5 to 2.0:1.0, particularly 0.7 to 1.4:1.0 and especially 0.9 to 1.1: 1.0.
In one embodiment, the molar ratio of ETA to DHA in a composition, preferably a pharmaceutical composition, used herein is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0:1.0, more preferably 0.5 to 2.0:1.0, particularly 0.7 to 1.4:1.0 and especially 0.9 to 1.1: 1.0.
In one embodiment, the molar ratio of DTA to DHA in a composition, preferably a pharmaceutical composition, used herein is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0: 1.0, more preferably 0.5 to 2.0: 1.0, particularly 0.7 to 1.4: 1.0 and especially 0.9 to 1.1: 1.0.
In one embodiment, the molar ratio of ETA to SDA in a composition, preferably a pharmaceutical composition, used herein is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0:1.0, more preferably 0.5 to 2.0:1.0, particularly 0.7 to 1.4:1.0 and especially 0.9 to 1.1: 1.0.
In one embodiment, the molar ratio of DTA to SDA in a composition, preferably a pharmaceutical composition, used herein is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0:1.0, more preferably 0.5 to 2.0:1.0, particularly 0.7 to 1.4:1.0 and especially 0.9 to 1.1: 1.0.
In one embodiment, the molar ratio of ETA to GLA in a composition, preferably a pharmaceutical composition, used herein is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0:1.0, more preferably 0.5 to 2.0:1.0, particularly 0.7 to 1.4:1.0 and especially 0.9 to 1.1: 1.0.
In one embodiment, the molar ratio of DTA to GLA in a composition, preferably a pharmaceutical composition, used herein is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0:1.0, more preferably 0.5 to 2.0:1.0, particularly 0.7 to 1.4:1.0 and especially 0.9 to 1.1: 1.0.
In one embodiment, the molar ratio of ETA to DGLA in a composition, preferably a pharmaceutical composition, used herein is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0:1.0, more preferably 0.5 to 2.0:1.0, particularly 0.7 to 1.4:1.0 and especially 0.9 to 1.1: 1.0.
In one embodiment, the molar ratio of DTA to DGLA in a composition, preferably a pharmaceutical composition, used herein is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0:1.0, more preferably 0.5 to 2.0:1.0, particularly 0.7 to 1.4:1.0 and especially 0.9 to 1.1: 1.0.
In one embodiment, the molar ratio of ETA to DPA in a composition, preferably a pharmaceutical composition, used herein is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0:1.0, more preferably 0.5 to 2.0:1.0, particularly 0.7 to 1.4:1.0 and especially 0.9 to 1.1: 1.0.
In one embodiment, the molar ratio of DTA to DPA in a composition, preferably a pharmaceutical composition, used herein is suitably in the range from 0.1 to 10.0:1.0, preferably 0.2 to 5.0:1.0, more preferably 0.5 to 2.0:1.0, particularly 0.7 to 1.4:1.0 and especially 0.9 to 1.1: 1.0.
In one embodiment, the total amount of ETA and/or DTA and/or other omega-3 and/or omega 6 PUFA, suitably selected from the group consisting of EP A, DHA, SDA, GLA, DGLA, and DPA, administered to/taken by a subject, preferably human adult, is preferably in the range from 1 to 10 g/day, more preferably 2 to 7 g/day, and particularly 3 to 5 g/day.
Since ETA and DTA and other omega-3 and/or omega 6 PUFAs defined herein are polyunsaturated fatty acids, they can be subject to oxidation, and any composition, particularly a pharmaceutical composition, comprising these materials may also contain an antioxidant. Suitable examples of antioxidants include a phenolic compound, a plant extract, or a sulphur-containing compound. The antioxidant may be ascorbic acid or a salt thereof, vitamin E, CoQIO, tocopherols, lecithin, citric acid, lipid soluble derivatives of more polar antioxidants such as ascobyl fatty acid esters (e.g. ascobyl palmitate), plant extracts (e.g. rosemary, sage and oregano oils, green tea extract), algal extracts, and synthetic antioxidants (e.g. butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), ethoxyquin, alkyl gallates, hydroquinones, tocotrienols), and combinations thereof. When present, the total amount of one or more antioxidants in a composition, preferably a pharmaceutical composition, is suitably less than 0.50 wt%, preferably in the range from 0.001 to 0.30 wt%, more preferably 0.01 to 0.20 wt%, particularly 0.025 to 0.10 wt%, and especially 0.03 to 0.05 wt%, based on the total weight of ETA and/or DTA and any other omega 3 and/or omega 6 PUFA in the composition. In one embodiment, the antioxidant is not present in a therapeutically effective amount in the pharmaceutical composition.
In one embodiment, the only components present in a therapeutically effective amount in a pharmaceutical composition defined and used herein, consist of ETA and/or DTA and/or other omega-3 and/or omega 6 PUFAs, suitably selected from the group consisting of EPA, DHA, SDA, GLA, DGLA, and DPA, preferably DHA.
By therapeutically effective amount is meant the amount of a component or material that without causing significant negative or adverse side effects, is intended in relation to neurodegenerative disease, to (i) delay or prevent the onset thereof; (ii) slow down or stop the progression, aggravation, or deterioration of one or more symptoms; (iii) bring about amelioration of the symptoms; (iv) reduce the severity or incidence thereof; and/or (v) cure the condition or disorder.
In one embodiment, the composition, preferably a pharmaceutical composition, comprises at least 90.0 wt%, suitably at least 95.0 wt%, preferably at least 98.0 wt%, more preferably at least 99.0 wt%, particularly at least 99.5 wt%, and especially at least 99.9 wt%, of ETA, based on the total weight of the composition.
In one embodiment, the composition, preferably a pharmaceutical composition, comprises at least 90.0 wt%, suitably at least 95.0 wt%, preferably at least 98.0 wt%, more preferably at least 99.0 wt%, particularly at least 99.5 wt%, and especially at least 99.9 wt%, of DTA, based on the total weight of the composition. In one embodiment, the composition, preferably a pharmaceutical composition, comprises at least 90.0 wt%, suitably at least 95.0 wt%, preferably at least 98.0 wt%, more preferably at least 99.0 wt%, particularly at least 99.5 wt%, and especially at least 99.9 wt%, and up to 100 wt%, of ETA, based on the total weight of fatty acids (having a carbon chain length of 4 or greater) in the composition.
In one embodiment, the composition, preferably a pharmaceutical composition, comprises at least 90.0 wt%, suitably at least 95.0 wt%, preferably at least 98.0 wt%, more preferably at least 99.0 wt%, particularly at least 99.5 wt%, and especially at least 99.9 wt%, and up to 100 wt%, of DTA, based on the total weight of fatty acids (having a carbon chain length of 4 or greater) in the composition.
In one embodiment, the composition, preferably a pharmaceutical composition, comprises at least 90.0 wt%, suitably at least 95.0 wt%, preferably at least 98.0 wt%, more preferably at least 99.0 wt%, particularly at least 99.5 wt%, and especially at least 99.9 wt%, of ETA and/or DTA and at least one other omega 3 and/or omega 6 PUFA, suitably selected from the group consisting of EP A, DHA, SDA, GLA, DGLA, and DPA; preferably selected from the group consisting of EP A, DHA, SDA, and DGLA; more preferably selected from the group consisting of DHA and DGLA; and particularly DHA, based on the total weight of the composition.
In one embodiment, the composition, preferably a pharmaceutical composition, comprises at least 90.0 wt%, suitably at least 95.0 wt%, preferably at least 98.0 wt%, more preferably at least 99.0 wt%, particularly at least 99.5 wt%, and especially at least 99.9 wt%, of ETA and/or DTA and at least one other omega 3 and/or omega 6 PUFA, suitably selected from the group consisting of EP A, DHA, SDA, GLA, DGLA, and DPA; preferably selected from the group consisting of EP A, DHA, SDA, and DGLA; more preferably selected from the group consisting of DHA and DGLA; and particularly DHA, based on the total weight of fatty acids (having a carbon chain length of 4 or greater) in the composition.
In one embodiment, the composition, preferably a pharmaceutical composition, comprises less than 10.0 wt%, suitably less than 5.0 wt%, preferably less than 2.0 wt%, more preferably less than 1.0 wt%, particularly less than 0.5%, and especially less than 0.1 wt% of any omega-3 and/or omega 6 PUFA other than ETA, DTA, EP A, DHA, SDA, GLA, DGLA, and/or DPA, based on the total weight of the composition.
In one embodiment, the composition, preferably a pharmaceutical composition, comprises less than 10.0 wt%, suitably less than 5.0 wt%, preferably less than 2.0 wt%, more preferably less than 1.0 wt%, particularly less than 0.5%, and especially less than 0.1 wt% of any fatty acid (having a carbon chain length of 4 or greater) other than ETA, DTA, EP A, DHA, SDA, GLA, DGLA, and/or DPA, based on the total weight of the composition.
In one embodiment, the composition, preferably a pharmaceutical composition, comprises less than 10.0 wt%, suitably less than 5.0 wt%, preferably less than 2.0 wt%, more preferably less than 1.0 wt%, particularly less than 0.5%, and especially less than 0.1 wt% of any non-omega-3 and/or non-omega 6 PUFA, based on the total weight of the composition. In one embodiment, the composition, preferably a pharmaceutical composition, comprises less than 10.0 wt%, suitably less than 5.0 wt%, preferably less than 2.0 wt%, more preferably less than 1.0 wt%, particularly less than 0.5%, and especially less than 0.1 wt% of any saturated fatty acid (having a carbon chain length of 4 or greater), based on the total weight of the composition.
In one embodiment, the composition, preferably a pharmaceutical composition, comprises less than 10.0 wt%, suitably less than 5.0 wt%, preferably less than 2.0 wt%, more preferably less than 1.0 wt%, particularly less than 0.5%, and especially less than 0.1 wt% of any hydrocarbon comprising at least one aromatic ring, based on the total weight of the composition.
In one embodiment, the pharmaceutical composition of the invention may be administered systemically or locally. The pharmaceutical composition may be administered orally, buccally, by injection, by percutaneous administration, parenterally, intrathecally, by endoscopy, topically, transdermally, transmucosally, nasally, by inhalation spray, rectally, vaginally, intratracheally, and via an implanted reservoir.
In one embodiment, the pharmaceutical composition of the invention may be orally administered. Examples of formulations adapted to oral administration include, but are not limited to, solid forms, liquid forms and gels. Examples of solid forms adapted to oral administration include, but are not limited to, pill, tablet, capsule, soft gelatin capsule, hard gelatin capsule, dragees, granules, caplet, compressed tablet, cachet, wafer, sugar-coated pill, sugar coated tablet, or dispersing/or disintegrating tablet, powder, solid forms suitable for solution in, or suspension in, liquid prior to oral administration and effervescent tablet. Examples of liquid form adapted to oral administration include, but are not limited to, solutions, suspensions, drinkable solutions, elixirs, sealed phial, potion, drench, syrup, liquor and sprays.
In one embodiment, the pharmaceutical composition may be injected, preferably systemically injected. Examples of formulations adapted to systemic injections include, but are not limited to, liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection.
Examples of systemic injections include, but are not limited to, intravenous, intratumoral, intracranial, intralymphatic, intraperitoneal, intramuscular, subcutaneous, intradermal, intraarticular, intrasynovial, intrastemal, intrathecal, intravesical, intrahepatic, intralesional, infusion techniques and perfusion. In another embodiment, when injected, the composition, the pharmaceutical composition or the medicament of the invention is sterile. Methods for obtaining a sterile pharmaceutical composition include, but are not limited to, GMP ("good manufacturing practice") synthesis.
In one embodiment, ETA and/or DTA and optionally other omega-3 and/or omega 6 PUFAs defined herein are preferably taken orally, suitably in an emulsion, tablet or in an encapsulated form, preferably in the core of a capsule shell, such as a gelatin capsule. In one embodiment, the capsule shell is suitably a gelatin capsule and may be a hard or soft gel capsule, preferably a soft gel capsule. In addition to gelatin, the capsule shell may also comprise a plasticizer, preferably a pharmaceutically approved plasticiser. Suitable plasticisers include glycerin, propylene glycol, polyethylene glycol, sorbitol, vegetable oil and derivatives (including castor oil, diacylated monoglycerides), triacetin, tributyl citrate, triethyl citrate, acetyl tributyl citrate, acetyl triethyl citrate, dibutyl sebacate, and mixtures thereof.
The capsule shell may also comprise other minor components such as a preservative, opacifier and/or colourant. The capsules defined herein may comprise further coatings in the shell, for example enteric polymer or wax coatings.
In one embodiment, the concentration in the capsule shell of (i) gelatin is suitably in the range from 35 to 99.9 wt%, preferably 45 to 95 wt%, more preferably 50 to 85 wt%, particularly 55 to 75 wt% and especially 60 to 70 wt%; and/or (ii) plasticizer, preferably glycerin, is suitably in the range from 0.1 to 65 wt%, preferably 5 to 55 wt%, more preferably 15 to 50 wt%, particularly 25 to 45 wt% and especially 30 to 40 wt%, both based on the total weight of the capsule shell.
In one embodiment, the core of the capsule shell comprises, consists essentially of, or consists of ETA. In one embodiment, the core of the capsule shell comprises, consists essentially of, or consists of DTA. In one embodiment, the core of the capsule shell comprises, consists essentially of, or consists of ETA and DTA. The core of the capsule can be a composition, preferably a pharmaceutical composition, defined hereinabove. In one embodiment, the core of the capsule comprises, consists essentially of, or consists of ETA and/or DTA and at least one other omega-3 and/or omega 6 PUFA, preferably selected from the group consisting of EP A, DHA, SDA, GLA, DGLA, and DPA; more preferably selected from the group consisting of EPA, DHA, SDA, and DGLA; particularly selected from the group consisting of DHA and DGLA; and especially DHA. The ETA and/or DTA to other omega-3 and/or omega 6 PUFA molar ratios defined hereinabove for a composition, preferably a pharmaceutical composition, also apply to the core of the capsule.
In one embodiment, the core of the capsule comprises at least 90.0 wt%, suitably at least 95.0 wt%, preferably at least 98.0 wt%, more preferably at least 99.0 wt%, particularly at least 99.5 wt%, and especially at least 99.9 wt%, of ETA and/or DTA, based on the total weight of core.
In one embodiment, the core of the capsule comprises at least 90.0 wt%, suitably at least 95.0 wt%, preferably at least 98.0 wt%, more preferably at least 99.0 wt%, particularly at least 99.5 wt%, and especially at least 99.9 wt%, and up to 100 wt%, of ETA and/or DTA, based on the total weight of fatty acids (having a carbon chain length of 4 or greater) in the core.
In one embodiment, the core of the capsule comprises at least 90.0 wt%, suitably at least 95.0 wt%, preferably at least 98.0 wt%, more preferably at least 99.0 wt%, particularly at least 99.5 wt%, and especially at least 99.9 wt%, of ETA and/or DTA and at least one other omega 3 and/or omega 6 PUFA, suitably selected from the group consisting of EPA, DHA, SDA, GLA, DGLA, and DPA; preferably selected from the group consisting of EP A, DHA and DGLA; more preferably selected from the group consisting of DHA and DGLA; and particularly DHA, based on the total weight of core.
In one embodiment, the core of the capsule comprises at least 90.0 wt%, suitably at least 95.0 wt%, preferably at least 98.0 wt%, more preferably at least 99.0 wt%, particularly at least 99.5 wt%, and especially at least 99.9 wt%, of ETA and/or DTA and at least one other omega 3 and/or omega 6 PUFA, suitably selected from the group consisting of EP A, DHA, SDA, GLA, DGLA, and DPA; preferably selected from the group consisting of EP A, DHA, SDA, and DGLA; more preferably selected from the group consisting of DHA and DGLA; and particularly DHA, based on the total weight of fatty acids (having a carbon chain length of 4 or greater) in the core.
In one embodiment, the core of the capsule comprises less than 10.0 wt%, suitably less than 5.0 wt%, preferably less than 2.0 wt%, more preferably less than 1.0 wt%, particularly less than 0.5%, and especially less than 0.1 wt% of any omega-3 and/or omega 6 PUFA other than ETA, DTA, EP A, DHA, SDA, GLA, DGLA, and/or DPA, based on the total weight of the core.
In one embodiment, the core of the capsule comprises less than 10.0 wt%, suitably less than 5.0 wt%, preferably less than 2.0 wt%, more preferably less than 1.0 wt%, particularly less than 0.5%, and especially less than 0.1 wt% of any fatty acid (having a carbon chain of 4 or greater) other than ETA, DTA, EPA, DHA, SDA, GLA, DGLA, and/or DPA, based on the total weight of the core. In one embodiment, the core of the capsule comprises less than 10.0 wt%, suitably less than 5.0 wt%, preferably less than 2.0 wt%, more preferably less than 1.0 wt%, particularly less than 0.5%, and especially less than 0.1 wt% of any non-omega-3 and/or non-omega 6 PUFA, based on the total weight of the core.
In one embodiment, the core of the capsule comprises less than 10.0 wt%, suitably less than 5.0 wt%, preferably less than 2.0 wt%, more preferably less than 1.0 wt%, particularly less than 0.5%, and especially less than 0.1 wt% of any saturated fatty acid (having a carbon chain length of 4 or greater), based on the total weight of the core.
In one embodiment, the composition, preferably a pharmaceutical composition, comprising ETA and/or DTA, and/or other omega-3 and/or omega 6 PUFA, suitably selected from the group consisting of EP A, DHA, SDA, GLA, DGLA, and DPA, is an oil-in-water or water-in-oil, preferably oil-in-water, emulsion. The molar ratios of ETA and/or DTA to other omega-3 and/or omega 6 PUFA defined hereinabove for a composition, preferably for a pharmaceutical composition, also apply to the emulsion.
The emulsion according to the present invention may also contain an antioxidant. The antioxidant may be present in the aqueous and/or the oil phase. Suitable examples of antioxidants include one or more of those listed hereinabove. One or more antioxidants, if desired, are present in the emulsion by a total amount, preferably in the range from 0.001 wt% to 0.50 wt%, more preferably 0.01 wt% to 0.20 wt%, particularly 0.025 wt% to 0.10 wt%, and especially 0.03 wt% to 0.05 wt%, based on the total weight of ETA and/or DTA and any other omega 3 and/or omega 6 PUFA in the emulsion.
The composition, preferably a pharmaceutical composition, defined herein may also contain other materials such as excipients that are soluble in water, in particular nonionic, anionic, cationic surfactants, salts, pH adjusters, hydrating agents, chelates, metal ions, polymers, dispersing agents, colorants, preservatives and hydrotropes.
The composition, preferably a pharmaceutical composition, comprising ETA and/or DTA and optionally any other omega-3 and/or omega 6 PUFA defined herein may be administered to any warm-blooded animal, preferably a human, a pet or livestock. Pets include dogs, cats, guinea pigs, and rabbits; and livestock includes pigs, cattle, sheep, goats, and horses.
The present invention further relates to a nutraceutical composition comprising ETA and/or DTA and optionally any other omega-3 and/or omega 6 PUFA, preferably selected from the group consisting of EP A, DHA, SDA, GLA, DGLA, and DPA, preferably DHA, and a nutraceutically acceptable excipient.
In one embodiment, the nutraceutical composition can be used as a dietary supplement to food and/or beverages. Moreover, a multi-vitamin and mineral supplement may be added to the nutraceutical composition to obtain an adequate amount of an essential nutrient, which is missing in some diets. The multi-vitamin and mineral supplement may also be useful for disease prevention and protection against nutritional losses and deficiencies due to lifestyle patterns. In one embodiment, the nutraceutical composition is preferably used, in a subject in need thereof, for (i) reducing or preventing impaired memory functions; and/or (ii) reducing or preventing amnesic syndromes; and/or (iii) for enhancing, boosting and/or restoring cognitive functions; and/or (iv) reducing or preventing the decline of cognitive functions; and/or (v) reducing or preventing a cognitive dysfunction, i.e. a state wherein a cognitive function is deteriorated or is lower than typical.
In one embodiment, the nutraceutical composition is preferably used for enhancing, boosting and/or restoring memory in a subject in need thereof. In one embodiment, the nutraceutical composition is preferably used for preventing the decline of memory in a subject in need thereof.
Examples of cognitive functions include, but are not limited to, memory, learning, language, attention, perception, motor skills, visual and spatial processing and executive functions.
All of the features described herein may be combined with any of the above aspects, in any combination.
Brief Description of the Drawings
The invention will now be described in a non-limiting way by reference to the following Figures, in which:
Figure 1 is a JH NMR spectral profile of DTA-EE (DTA ethyl ester) recorded in CDC13; Figure 2 is a 13C NMR spectral profile of DTA-EE (DTA ethyl ester) recorded in CDCf; and
Figure 3 is a 13C DEPT-135 NMR spectral profile of DTA-EE (DTA ethyl ester) recorded in CDC13.
Examples
Example 1
The aim of this study was to investigate the effect of ETA and EPA in LPS-induced TNF-a production in mouse microglia BV-2 cells. TNF-a is produced in response to a stimulus such as invading microbes (LPS is a recognisable outer coating of many microbial organisms). TNF-a is a representative cytokine among many such cytokines but is considered most important in neurodegenerative diseases such as Alzheimer’s disease.
BV-2 microglial cells were seeded in 96-well plates at a density of 20,000 cells per well in Roswell Park Memorial Institute Medium 1640 (RPMI) and 10% Fetal Bovine Serum (FBS). The next day, the cells were washed once with Dulbecco’s Phosphate Buffered Saline (DPBS) and the serum was starved for 24 hours in 150 pl of RPMI. The following day, the cells were treated with 150 pl of 50 pM of EPA ethyl ester (EPA-EE, ex BASF) or 50 pM ETA ethyl ester (ETA-EE, ex AK Scientific, Palo Alto CA, USA). Appropriate controls (0.5% ethanol) were also included in the assay. After 24 hours, the cells were incubated for 6 hours with 10 pl of 7,500 ng ml’1 lipopolysaccharide (LPS) to reach a final concentration of 500 ng ml’1. After the incubation period, the supernatants were collected and frozen at -80°C until the enzyme-linked immunosorbent assay (ELISA) was performed to measure TNF-a concentration. To determine the viability of the cells after the treatments, a water- soluble tetrazolium salt (WST-8) assay was carried out. Cell counting kit (CCK)-8 reagent (WST-8, ex Sigma- Aldrich) was diluted 1: 10 in RPMI media and 100 pl was added to each well and the plate was incubated at 37°C. After 1 hour, the absorbance was measured at 450 nm using a Synergy II microplate reader. WST-8 was bioreduced by cellular dehydrogenases to an orange formazan product that is soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of living cells. A DuoSet ELISA Ancillary Reagent Kit2 (ex R&D Systems) was used following the manufacturer’s instructions. All the experiments were carried out in triplicate. a) Cell Viability (WST-8)
The results were normalised to the LPS + 0.5% EtOH which was set as 100% of cell viability.
The results show that both ETA-EE and EPA-EE did not reduce cell viability. b) TNF-a Production (ELISA)
ETA-EE showed a 27% reduction in TNF-a levels compared with a 22% reduction with EPA-EE.
Example 2
The procedure of Example 1 was repeated except that 30 pM DHA ethyl ester (DHA- EE) was also used, and the amount of ETA-EE and EPA-EE was 30 pM instead of 50 pM. a) Cell Viability (WST-8)
The results were normalised to the LPS + 0.5% EtOH which was set as 100% of cell viability. The results show that ETA-EE, EPA-EE and DHA-EE did not reduce cell viability. b) TNF-a Production (ELISA)
ETA-EE showed a 31.4% reduction in TNF-a levels compared with a 25.8% reduction with EPA-EE and a 21.9% reduction with DHA-EE.
Example 3
The procedure of Example 1 was repeated except that 50 pM dihomo y linolenic acid ethyl ester (DGLA-EE) was also used. a) Cell viability (WST-8)
The results were normalised to the LPS + 0.5% EtOH which was set as 100% of cell viability.
The results show that ETA-EE, EPA-EE, and DGLA-EE did not reduce cell viability. b) TNF-a Production (ELISA)
ETA-EE showed a 46% reduction in TNF-a levels compared with a 33% reduction with EPA-EE and a 40% reduction with DGLA-EE.
Example 4
The procedure of Example 1 was repeated, except that the cells were treated with a mixture of 30 pM ETA-EE and 30 pM DHA-EE. The concentration of the EtOH vehicle used was increased to 1.0%. a) Cell Viability (WST-8)
The results were normalised to the LPS + 1.0% EtOH which was set as 100% of cell viability. The combined treatment of ETA-EE and DHA-EE showed no significant effect on cell viability. b) TNF-a Production (ELISA)
Treatment with ETA-EE + DHA-EE showed a 56% reduction in TNF-a levels.
Example 5
The aim of this study was to investigate the effect of ETA and EPA in LPS-induced interleukin-6 (IL-6) production in mouse microglia BV-2 cells. IL-6 is an important inflammatory cytokine implicated in neuroinflammation and neurodegenerative disease.
BV-2 microglia cells were seeded in 96 well plates at a density of 20,000 cells per well. On day 2, cells were serum starved for 24 hours in DMEM. Then, cells were pre-treated with 50 pM ETA ethyl ester (ETA-EE, ex-Cayman Chemical, Ann Arbor Michigan 48108 USA) or 50 pM EPA ethyl ester (EPA-EE, ex BASF). Appropriate vehicle control was included, i.e. 0.5% ethanol. On day 4, cells were LPS-induced (500 ng ml’1) for 24 hours and supernatants were collected and used for an ELISA assay to measure IL-6 levels. A WST-8 assay was performed to measure cell viability. All the experiments were carried out in triplicate. a) Cell Viability (WST-8)
The results were normalised to the LPS + 0.5% EtOH which was set as 100% of cell viability.
The results show that both ETA-EE and EPA-EE did not reduce cell viability. b) IL-6 Production (ELISA)
ETA-EE showed a 65% reduction in IL-6 levels compared with a 49% reduction with
EPA-EE.
Example 6 The aim of this study was to investigate the effect of ETA and EPA in LPS-induced
IL-6 production in mouse macrophage RAW264.7 cells.
RAW264.7 cells were seeded in 96 well plates at a density of 30,000 cells/well. On day 2, cells were serum starved for 24 hours in DMEM. Then, cells were pre-treated with 50 pM of either ETA ethyl ester (ETA-EE, ex-Cayman Chemical, Ann Arbor Michigan 48108 USA) or 50 pM EPA ethyl ester (EPA-EE, ex BASF). Appropriate vehicle control was included, i.e. 0.5% ethanol. On day 4, cells were LPS-induced (500 ng mF1) for 6 hours and supernatants were collected and used for an ELISA assay to measure IL-6 levels. A WST-8 assay was performed to measure cell viability. a) Cell Viability (WST-8) The results were normalised to the LPS + 0.5% EtOH which was set as 100% of cell viability.
The results show that ETA-EE and EPA-EE did not affect cell viability. b) IL-6 Production (ELISA)
ETA-EE showed a 73% reduction in IL-6 levels compared with a 53% reduction with the conventional anti-inflammatory lipid, EPA-EE.
Example 7
DTA ethyl ester (DTA-EE) was synthesised using the following reaction stages. 1) Esterification of DGLA to DGLA-EE
DGLA was esterified to produce DGLA ethyl ester (DGLA-EE) using Novazyme 435, diethyl carbonate, and triethyl orthoformate in EtOH according to a literature protocol (R. Morrone, N. D’Antona, D. Biondi, D. Lambusta, G. Nicolosi, Journal of Molecular Catalysis B: Enzymatic 84 (2012) 173-176).
An equimolar amount of diethyl carbonate (2.36 g (d = 0.975 g ml'1), 2.42 ml, 20 mmol) and triethyl orthoformate (2.96 g (d = 0.891 g ml'1, 20 mmol), 3.32 ml) was added to DGLA (6.12 g, 20 mmol) in a 50 ml RB flask. Novozyme 435 (300 mg) and 30 pl dry EtOH were added and stirred at 60°C overnight. The reaction was monitored by (AgNCL coated SiO2) TLC in hexane:ethyl acetate (v/v, 1:1), Rf = 0.47.
(Yield: 7.8 g). Confirmed by GCMS, Rt = 13.23 min. The reaction product was purified by passing through an Ag-impregnated SiO2 column (4 cm) using 1 : 1 (v/v) hexane: ethyl acetate, and a pale-yellow oily product isolated.
Novozyme 435
1 . Diethyl Carbonate
2. Triethyl orthoformate
3. EtOH DGLA- EE Mol. Wt.: 334.5359
2) Reduction of DGLA-EE to DGLA-OH
Pellets of LiAlH4 (0.91 g, 23 mmol, 2.4 equivalents) were ground into powder and suspended in dry THF (100 ml) in a 500 ml three-necked RB flask and cooled down to 0-5°C in an ice bath. DGLA-EE (3.34 g, 10 mmol) in dry THF (10 ml) was added dropwise to the above mixture over 30-40 minutes under a stream of N2. The mixture was stirred at 8-12°C for 1 hour and 12-18°C for 2 hours under N2. After cooling the reaction mixture to 3-5°C, a solution of water (0.4 ml) and THF (12.5 ml) was added under a stream of N2 over 15-20 minutes. The aqueous solution of 2M NaOH (11.4 ml) was then added over 10-15 minutes. The mixture was stirred at 10-15°C overnight, and the anhydrous Na2SC>4 was added and stirred for 30 minutes. The sticky (inorganic) solid materials were removed by filtration and washed several times with THF. The filtrate was concentrated by rotavap and high vacuum, a colourless oily product isolated and used in the next step without further purification. Yield: 2.83 g (97%).
3) Mesylation of DGLA-OH to DGLA-OMs
Both DGLA-OH (2.83 g, 9.67 mmol) and Et3N (2.84 ml, 20.31 mmol, 2.1 equivalents) were added to dry DCM (100 ml) in a three-necked RB flask (250 ml) under constant stirring. The mixture was cooled down to 0-3 °C in an ice bath, and a DCM solution (10 ml) of methane sulfonyl chloride (0.82 ml, 10.64 mmol, 1.1 equivalents) was added slowly to the above mixture with constant stirring under a stream of N2. After stirring the above mixture for 1 hour at 1°C, it was made acidic by IM HC1. The mixture was transferred into a separation funnel extracted with DCM and dried under anhydrous MgSCL. The solvent was removed by rotavap and high vacuum, and the resulting mesylate compound was used for the next step without further purification. Yield: 2.87 g (74%).
DGLA-OH DGLA-OMs
C20H36O C2iHsgO3S
Mol. Wt.: 292.4992 Mol. Wt.: 370.5896 4) Bromination of DGLA-Oms to DGLA-Br
A mixture of DMF (2 ml) and LiBr (1.28 g, 15.5 mmol, 2 equivalents) was stirred at 50°C for 1 hour. The temperature was raised to 40°C, and DGLA-OMs (2.87 g, 7.75 mmol) in DMF (3 ml) were added and stirred at 70°C for 3 hours a under a nitrogen atmosphere. The solution was then cooled down to room temperature and stirred overnight. Diethyl ether was added, the mixture separated, the ether layer was washed with water, 10% aq HC1 (x2), 5% NaHCOs solution, and finally again with a large amount of water. The resulting organic solution was dried over anhydrous MgSCL and filtered, and the solvent was evaporated by rotavap and a high vacuum. Yield: 2.2 g (86%).
5) Alkylation of DGLA-Br to Diethyl Malonate Derivative of DGLA (DGLA- DEM)
NaH (0.291 g, 12.12 mmol, 2 equivalents) in dry DMF (5 ml) was stirred at room temperature under N2 atm for a few minutes. DMF solution (5 ml portions) of diethyl malonate (0.971 g, 6.06 mmol) was dripped, followed by DGLA-Br (2.155 g, 6.06 mmol) in DMF (5 ml) added dropwise to the above mixture. The reaction mixture was heated at 110°C for 4 hours under N2, and IM HC1 was added to bring the reaction mixture to acidic, and transferred into a separation funnel and extracted with hexane. The organic phase was washed with water several times and dried under anhydrous Na2SO4. The resulting yellow oil was isolated, yielding 2.60 g (98%). The crude product was purified by Ag/SiCF column chromatography using 0-10% (v/v) EtOAc - hexane) gradient. The first two fractions (10 ml) in hexane were discarded.
A dark yellow solution in the third fraction (100 ml) in hexane alone contained the reaction product. Yield: 2.365 g (91%).
6) Ester Hydrolysis/Decarboxylation of DGLA-DEM to DTA
DGLA-DEM (2.35 g, 5.4 mmol) was heated at reflux with aq. NaOH (1.081, 27 mmol, 5 equivalents) in water (9.3 ml) and EtOH (19.3 ml) for 2 hours under a nitrogen atmosphere. The temperature was cooled down to 50°C and the oil phase was separated after adjusting the pH of the mixture to pH 2-3 with 6M H2SO4. The aqueous phase was extracted with diethyl ether (x 2). The combined organic phases were washed several times with water until the aqueous phase was pH 6. The organic (ether) phase was dried over anhydrous Na2SO4 and filtered, and the solvent was evaporated by rotavap and a high vacuum overnight. Yield: 1.839 g (64%).
DGLA-DEM DGLA-Malonate
Mol WL: 4346517 Mol WL: 378 5454
After hydrolysis, the DTA was purified by the Ag/SiCE column. The first fraction (10 ml) of hexane was discarded. A yellow oily product was isolated from the second fraction in hexane (10 ml) and stored overnight at -20°C. The product was then washed with (ice) cold hexane and heated at 150-160°C under vacuum for 3 hours. The resulting dark (brown) oily residue (Yield: 1.58 g, 97%) was purified by Ag/SiCh column chromatography using (0-50% (v/v) EtOAc-hexane) gradient. Yield: 0.652 g (67%).
7) Esterification of DTA to DTA-EE
DTA was esterified using Novazyme 435, diethyl carbonate, and triethyl orthoformate in EtOH according to a literature protocol (R. Morrone, N. D’Antona, D. Biondi, D. Lambusta, G. Nicolosi, Journal of Molecular Catalysis B: Enzymatic 84 (2012) 173— 176).
An equimolar amount of diethyl carbonate (0.078 g (d = 0.975 g/ml), 80 pl, 0.66 mmol) and tri ethyl orthoformate (0.097 g, 109 pl, 0.66 mmol) was added to DTA (0.22 g, 0.66 mmol) in a 5 ml RB flask. Novozyme 435 (11 mg) and 1.0 pl of dry EtOH were added and stirred at 60°C overnight. The reaction was monitored by (AgNOs coated SiO2) TLC in hexane:ethyl acetate (v/v, 1:1), Rf = 0.51. (Yield: 0.22 g). Confirmed by GCMS, Rt = 14.25 min. The recti on product was purified by passing through an Ag-impregnated SiO2 column (4 cm) using 1:1 (v/v) hexane:ethyl acetate, isolated a yellow oily product DTA-EE. Figures 1, 2 and 3 show the NMR spectral profiles of DTA-EE. ’H NMR (CDC13, 400 MHz) 6 (ppm): 5.35 (m, 6H), 4.11 (q, 2H, J = 7.2 Hz), 2.79 (t, 4H, J = 5.9 Hz), 2.27 (t, 2H, J = 7.6 Hz), 2.04 (q, 4H,
J = 7.0 Hz), 1.60 (t, 2H, J = 7.3 Hz), 1.27 (m, 19H), 0.87 (t, 3H, J = 6.7 Hz). 13C NMR (CDC13, 100 MHz) 6 (ppm): 14.16, 14.34, 14.66, 22.66, 25.06, 25.71, 27.30, 29.22, 29.33, 29.43, 29.71, 31.61, 60.25, 127.71, 127.76, 128.32, 128.35, 130.40, 130.48, 174.02.
Example 8
The aim of this study was to investigate the effect of DTA, DHA and EPA in an in vitro model of CNS injury using a multiparametric cell-based protocol. The assay was applied to “freshly isolated” rat primary microglia and cortical neurons.
Rat primary microglia were serum starved for 24 hours and subsequently pretreated with 50 pM DTA ethyl ester (DTA-EE, produced in Example 7 above), 50 pM DHA ethyl ester (DHA-EE) or 50 pM EPA ethyl ester (EPA-EE, ex BASF) for an additional 24 hours. Ethanol was used as vehicle control. After this, 500 ng ml’1 LPS was added and the cells were incubated for 6.5 hours. Medium was replaced and the cells were then exposed to 10 pM A (1-42) oligomers for 24 hours maintaining the presence of the same lipids that were used in the pretreatment and adding 0.2% DMSO to the vehicle control. In parallel, freshly isolated cortical neurons were pretreated or not with 50 pM DTA-EE, 50 pM DHA-EE, or 50 pM EPA-EE for 24 hours (ethanol was also used as vehicle control). The neurons were then incubated for 72 hours with the conditioned medium of the microglia that had been in contact with the Ap (1-42) oligomers (always maintaining paired the same pretreatment conditions in microglia and neurons). Samples of this conditioned medium were stored at -80°C to subsequently measure the amount of TNF-a by ELISA. Finally, cells were analysed for cell parameters associated with caspase 3/7 activation and neurite outgrowth.
Caspase 3/7 activation was determined using “The CellEvent Caspase-3/7 Green Detection Reagent” which is intrinsically a non-fluorescent peptide that inhibits the ability of the dye to bind to DNA. After activation of caspase-3/7 in apoptotic cells, the peptide was cleaved enabling the dye to bind to DNA and produce a bright, fluorogenic response. The cell was stained and measured at 488 nm/530 nm Ex/Em, enabling a direct quantification of apoptotic cells.
Neurite outgrowth was determined using -III-tubulin staining performed by immunofluorescence. Cells were fixed with cold methanol for 10 minutes. After the fixation step, the samples were washed three times with Fetal Bovine Serum (FBS), stained with Hoechst 1:200 and blocked with PBS + 3% Bovine Serum Albumine (BSA) for 30 minutes. Finally, anti- -III-tubulin-FITC antibody was added at 1/150 in PBS + 0.5% BSA for 60 minutes at room temperature. Cells were then washed three times and analysed in a Cell Insight CX7 automated fluorescent microscope. To investigate the role of neurite extension, the geometric pattern of total length per neuron, neurite complexity and number of branch points were measured. a) Cytokine Production
TNF-a generated from lipid treatment of microglia following stimulation with LPS.
DTA-EE was an order of magnitude more effective than DHA-EE or EPA-EE and afforded a 93% reduction in the inflammatory cytokine TNF-a. b) Caspase 3/7Activation
Effect of lipid treatment of microglia and neurons on pro-apoptotic caspases. Pretreatment of both microglia and neurons with DTA-EE produced a reduction in caspase activation of 57%, outperforming both DHA-EE and EPA-EE. c) Neurite Outgrowth
Effect of lipid treatment of microglia and neurons on neurite outgrowth.
The exposure of neurons to treated microglia conditioned medium produced a loss of more than 80% of neurites. DTA-EE provided a 3-fold recovery in neurite length compared with untreated cells and outperformed both DHA-EE and EPA-EE. Example 9
The aim of this study was to investigate the effect of mixtures of DTA, ETA, and DHA on the inhibition of TNF-a from LPS stimulated microglia. The method used was the same as the rat primary microglia assay described in Example 8 except that the microglia were treated with 35 pM DTA-EE + 35 pM ETA-EE, 35 pM DTA-EE + 35 pM DHA-EE, or 25 pM DTA-EE + 25 pM ETA-EE + 25 pM DHA-EE. TNF-a Production (ELISA)
All 3 mixtures afforded a significant reduction in TNF-a levels.
Example 10
The aim of this study was to determine the neuroprotective effect of pure lipids following treatment of neurons with externally added TNF-a.
Freshly isolated primary cortical neurons from 18-day embryonic rats were plated in two poly-D-lysine-coated M96 plates at a density of 40,000 cells/well in BrainPhys neuronal medium supplemented with 2% NeuroCult SMI and 10% heat-inactivated foetal bovine serum. Twenty-four hours after plating, the serum was removed. On day in vitro 2, cells were pretreated with 50 pM of DTA-EE, EPA-EE, or DHA-EE, in complete BrainPhys neuronal medium, in triplicate, and incubated for 24 hours (ethanol was used as the vehicle control, and 50 ng BDNF as the positive control). After pretreatment, the neurons were incubated or not with 100 ng ml’1 TNF-a in Neurobasal medium supplemented with 1% B-27 for 96 hours (performing a medium change after the first 72 hours with a second dose of 100 ng ml’1 TNF-a). During the incubation with TNF-a, the same test compounds were kept present in the culture medium. Finally, cells were simultaneously loaded with several fluorescent dyes and analysed with Cell Insight CX7 (Thermoscientific). Cell parameters associated with cell number, caspase 3/7 activation, mitochondrial integrity (TMRM) and neurite outgrowth, all indicative of prelethal cytotoxic effects, were measured.
Caspase 3/7 activation and neurite outgrowth were determined as described in Example 8. Mitochondrial health status was determined by detecting changes in mitochondrial membrane potential. MitoProbe™ TMRM detects changes in mitochondrial membrane potential. In a healthy cell with active mitochondria, the TMRM was easily isolated, emitting a red-orange fluorescent signal. When apoptosis was induced, the mitochondrial membrane depolarized and the TMRM signal decreased. A Hoechst 33342 nucleic acid stain was used to determine cell number. Cells were stained and measured at 380 nm/460 nm Ex/Em. This dye allowed a sensitive cell number determination by fluorescence microscopy. a) Neurite Outgrowth
The TNF-a challenge resulted in a loss of 60% of the neurites. DTA-EE afforded the highest recovery of the neurites. b) Mitochondrial Function (Integrity) The TNF-a challenge resulted in a 52% reduction in mitochondrial function. DTA-
EE treatment resulted a complete recovery of mitochondrial function. c) Cell Number
The TNF-a challenge resulted in a reduction in the number of nuclei of 44%. DTA-
EE effectively prevented this reduction in cell number.
d) Caspase Activation
TNF-a increased caspase activation, reaching 11% of cells with active pro-apoptotic caspases which represents a 6-fold increase over baseline levels. DTA-EE was more effective than other lipids, limiting caspase activation to 5% of cells.
The above examples illustrate the improved properties of ETA and/or DTA, and uses thereof according to the present invention.
0738P.WO.Spec(2)

Claims

1. 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa- 10, 13, 16-trienoic acid (DTA) for use in the treatment of neurodegenerative disease.
2. 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) according to claim 1 for use in the treatment of neurodegenerative disease.
3. 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) according to claim 1 for use in the treatment of neurodegenerative disease.
4. 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) according to claim 1 for use in the treatment of neurodegenerative disease.
5. 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa- 10, 13, 16-trienoic acid (DTA) for use according to any one of the preceding claims wherein the neurodegenerative disease is Alzheimer’s disease.
6. 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa- 10, 13, 16-trienoic acid (DTA) according to any one of the preceding claims wherein the ETA and/or DTA are each in lower alkyl ester form.
7. 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa-
10, 13, 16-trienoic acid (DTA) according to claim 6 wherein the lower alkyl ester form is the methyl and/or ethyl ester.
8. A composition comprising 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) in conjunction with a pharmaceutically acceptable adjuvant, excipient or carrier.
9. The composition according to claim 8 wherein ETA or DTA is the only material present in a therapeutically effective amount.
10. The composition according to claim 8 comprising 8Z, 11Z, 14Z, 17Z- eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) and at least one fatty acid selected from the group consisting of eicosapentaenoic acid (EP A), docosahexaenoic acid (DHA), stearidonic acid (6, 9, 12, 15 -octadecatri enioc acid) (SDA), gamma linolenic acid (6, 9, 12-octadecatrienioc acid) (GLA), dihomo y linolenic acid (8, 11, 14-eicosatraenoic acid) (DGLA), and 7,
10. 13, 16, 19-docosapentaenoic acid (DPA); and combinations thereof.
11. The composition according to claim 10 wherein the at least one fatty acid comprises docosahexaenoic acid (DHA.
12. The composition according to claim 8 comprising 8Z, 11Z, 14Z, 17Z- eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid
(DTA) for use in the treatment of neurodegenerative disease.
13. The composition according to claim 12 wherein the neurodegenerative disease is Alzheimer’s disease.
14. The composition according to either one of claims 12 or 13 wherein ETA or DTA is the only material present in a therapeutically effective amount.
15. The composition according to either one of claims 12 or 13 comprising at least one fatty acid selected from eicosapentaenoic acid (EP A), docosahexaenoic acid (DHA), stearidonic acid (6, 9, 12, 15 -octadecatri enioc acid) (SDA), gamma linolenic acid (6, 9, 12-octadecatri enioc acid) (GLA), dihomo y linolenic acid (8, 11, 14- eicosatraenoic acid) (DGLA), and/or 7, 10, 13, 16, 19-docosapentaenoic acid (DPA); and combinations thereof.
16. The composition according to claim 15 wherein the at least one fatty acid comprises docosahexaenoic acid (DHA).
17. The composition according to any one of claims 8 to 16 wherein the composition comprises 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA).
18. The composition according to any one of claims 8 to 16 wherein the composition comprises 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA).
19. The composition according to any one of claims 8 to 16 wherein the composition comprises a combination of 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid
(ETA) and 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA).
20. The composition according to any one of claims 8 to 19 wherein the ETA and/or DTA are each in lower alkyl ester form.
21. The composition according to claim 20 wherein the lower alkyl ester form is methyl and/or ethyl ester.
22. A method of treating or preventing neurodegenerative disease comprising administering to a subject in need thereof a therapeutically effective amount of 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa-10, 13, 16- trienoic acid (DTA) or a composition comprising ETA and/or DTA.
23. The method according to claim 22 wherein the neurodegenerative disease is Alzheimer’s disease.
24. The method according to either one of claims 22 or 23 wherein ETA or DTA is the only material administered in a therapeutically effective amount.
25. The method according to either one of claims 22 or 23 comprising administering a therapeutically effective amount to the subject of at least one fatty acid selected from eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), stearidonic acid (6, 9, 12, 15-octadecatrienioc acid) (SDA), gamma linolenic acid (6, 9, 12-octadecatrienioc acid) (GLA), dihomo y linolenic acid (8, 11, 14-eicosatraenoic acid) (DGLA), and/or 7, 10, 13, 16, 19-docosapentaenoic acid (DPA); and combinations thereof.
26. The method according to claim 25 wherein the at least one fatty acid comprises docosahexaenoic acid (DHA).
27. The method according to any one of claims 22 to 26 wherein the composition comprises 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA).
28. The method according to any one of claims 22 to 26 wherein the composition comprises 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA).
29. The method according to any one of claims 22 to 26 wherein the composition comprises a combination of 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid (ETA) and 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA).
30. The method according to any one of claims 22 to 29 wherein the ETA and/or DTA are each in lower alkyl ester form.
31. The method according to claim 30 wherein the lower alkyl ester form is methyl and/or ethyl ester.
32. A capsule comprising a shell and a core comprising 8Z, 11Z, 14Z, 17Z- eicosatetraenoic acid (ETA) and/or 10Z, 13Z, 16Z-docosa-10, 13, 16-trienoic acid (DTA) or a composition comprising ETA and/or DTA.
33. The capsule according to claim 32 wherein the core comprises at least one fatty acid selected from eicosapentaenoic acid (EP A), docosahexaenoic acid (DHA), stearidonic acid (6, 9, 12, 15-octadecatrienioc acid) (SDA), gamma linolenic acid (6, 9, 12-octadecatrienioc acid) (GLA), dihomo y linolenic acid (8, 11, 14- eicosatetraenoic acid) (DGLA), and/or 7, 10, 13, 16, 19-docosapentaenoic acid (DPA); and combinations thereof.
34. The capsule according to claim 33 wherein the at least one fatty acid comprises docosahexaenoic acid (DHA).
35. The capsule according to any one of claims 32 to 34 wherein the shell comprises gelatin.
36. A polyunsaturated fatty acid of the formula 10Z, 13Z, 16Z-docosa-10,l 3, 16- trienoic acid (DTA) in purified form.
37. The polyunsaturated fatty acid of the formula 10Z, 13Z, 16Z-docosa-10,l 3, 16-trienoic acid (DTA) according to claim 36 wherein DTA is in lower alkyl ester form.
38. The polyunsaturated fatty acid according to claim 37 wherein the lower alkyl ester form is methyl and/or ethyl ester.
39. A polyunsaturated fatty acid according to any one of claims 36 to 38 wherein the purified form of 10Z, 13Z, 16Z-docosa-10,l 3, 16-trienoic acid (DTA) is at least about 10% by weight (excluding solvent), free from components that may occur with it in nature or in an artificially produced mixture.
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