WO2020202152A1 - Dunaliella alga preparation for prevention and/or treatment of a neurodegenerative disease, a disorder associated with protein misfolding and cognitive decline - Google Patents
Dunaliella alga preparation for prevention and/or treatment of a neurodegenerative disease, a disorder associated with protein misfolding and cognitive decline Download PDFInfo
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- WO2020202152A1 WO2020202152A1 PCT/IL2020/050393 IL2020050393W WO2020202152A1 WO 2020202152 A1 WO2020202152 A1 WO 2020202152A1 IL 2020050393 W IL2020050393 W IL 2020050393W WO 2020202152 A1 WO2020202152 A1 WO 2020202152A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/02—Algae
- A61K36/05—Chlorophycota or chlorophyta (green algae), e.g. Chlorella
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- 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
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- the invention relates to neuroprotective preparations. More specifically, the invention relates to Dunaliella alga preparations and uses thereof in the treatment and prophylaxis of neuiOdegenerative disorders, protein misfolding and cognitive decline.
- Citron M. et ah, Nature 1992, 360(6405): 672-4.
- AD Alzheimer's disease
- AD is a progressive neurodegenerative disease that is manifested, among others, in damage and eventually destruction of brain cells, leading to memory loss and changes in cognitive and other brain functions.
- AD occurs mainly among the elderly population (above 65 years) and it is the most common form of dementia, accounting for 60%-80% of dementia cases [1]
- AD The main histological features of AD are neurotic plaques (b-amyloid, Ab), neurofibrillary tangles (tau protein), neurovascular dysfunction and also loss of neurons and synapses in the neocortex, hippocampus and other subcortical regions of the brain.
- AD amyloid cascade hypothesis postulates that neurodegeneration in AD is caused by abnormal accumulation of Ab plaques in various areas of the brain. This neuritic accumulation of Ab in the brain has been hypothesized to result from an imbalance between Ab production and clearance [2].
- a small number of AD cases ⁇ 1 %), familial AD (early- onset), are linked to genetic mutations which are associated with increased Ab production [3 j.
- AD cases are sporadic (late-onset) cases that may be due to faulty clearance of Ab from the brain.
- RA Retinoic acid
- RXR retinoic X receptor
- RAR retinoic acid receptor
- RXR retinoic X receptor
- RXR is a ligand-activated nuclear receptor which for heterodimers with other nuclear receptors such as RAR, and binds to specific DNA sequences to regulate gene expression.
- the RXR/RAR heterodimer expressed particularly in AD vulnerable regions [8], induces expression of brain apolipoprotein E (apoE) and cholesterol-transporters (e.g.
- ATP- binding cassette transporter (member 1 of human transporter sub-family ABCA), also known as the cholesterol efflux regulatory protein (CERP) and ATP- binding cassette sub-family G member 1 (ABCG1)), acts as a cholesterol sensor, and decreases cellular uptake of amyloid b [9] in an apoE-dependent manner.
- retinoids signaling impacts the development of AD pathology by ligand activation of RAR and RXR [10].
- retinoids improved AD symptoms and reduced amyloid accumulation and tau hyperphosphorylation [11].
- the present disclosure provides a method for preventing, treating, ameliorating, reducing or delaying the onset of at least one of a neurodegenerative disease, a disorder associated with protein misfolding, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, comprising administering to the subject an effective amount of at least one Dunaliella algae preparation or any composition comprising thereof.
- the method according to the present disclosure results in amelioration or reduction of at least one symptom associated with at least one of a neurodegenerative disease, a disorder associated with protein misfolding and cognitive decline in a subject in need thereof.
- the method according to tire present disclosure is wherein the symptom is at least one of short term memory impairment, long tern memory impairment, impaired cognitive function, impaired learning function, b-amyloids deposition, anxiety, depression, or any combination thereof.
- the method according to the present disclosure results in improvement of at least one of cognitive function, short term memory, long term memory, acquisition time and clearance of b-amyloids in a subject in need thereof.
- the method according to the present disclosure is wherein the neurodegenerative disease is at least one of Alzheimer's disease, Parkinson's disease, Mild Cognitive Impairment (MCI), Parkinson's disease with MCI, Huntington's disease, Lewy body disease, Amyotrophic lateral sclerosis (ALS), Prion disease, Motor neuron disease (MND), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Friedreich's Ataxia and any other neurodegenerative -related dementia or ataxia.
- MCI Mild Cognitive Impairment
- ALS Amyotrophic lateral sclerosis
- MND Motor neuron disease
- SCA Spinocerebellar ataxia
- SMA Spinal muscular atrophy
- Friedreich's Ataxia any other neurodegenerative -related dementia or ataxia.
- the method according to the present disclosure is wherein the neurodegenerative disease is Alzheimer's disease.
- the method according to the present disclosure is for preventing, treating, ameliorating, reducing or delaying the onset of cognitive decline.
- the method according to the present disclosure is wherein the Dunaiieila algae is DunalieUa bardawil.
- the method according to the present disclosure is wherein the DunalieUa algae preparation is administered orally.
- the present disclosure provides at least one DunalieUa algae preparation or any composition comprising thereof for use in a method for preventing, treating, ameliorating, reducing or delaying the onset of at least one of a neurodegenerative disease, a disorder associated with protein misfoiding, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
- the at least one DunalieUa algae preparation or any composition comprising thereof for use according to the present disclosure is wherein the method results in amelioration or reduction of at least one symptom associated with at least one of a neurodegenerative disease, a disorder associated with protein misfoiding, and cognitive decline in a subject in need thereof.
- the at least one DunalieUa algae preparation or any composition comprising thereof for use according to the present disclosure is wherein the symptom is at least one of short term memory impairment, long term memory impairment, impaired cognitive function, impaired learning function, b-amyioids deposition, anxiety, depression or any combination thereof.
- the at least one DunalieUa algae preparation or any composition comprising thereof for use according to the present disclosure is wherein the method results in improvement of at least one of cognitive function, short term memory, long term memory, acquisition time and clearance of b-amyloids in a subject in need thereof.
- the at least one Dunaliella algae preparation or any composition comprising thereof for use according to the present disclosure is wherein the neurodegenerative disease is at least one of Alzheimer's disease, Parkinson's disease, Mild Cognitive Impairment (MCI), Parkinson's disease with MCI, Huntington's disease, Lewy body disease, Amyotrophic lateral sclerosis (ALS), Prion disease, Motor neuron disease (MND), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Friedreich's Ataxia and any other neurodegenerative-related dementia or ataxia.
- MCI Mild Cognitive Impairment
- ALS Amyotrophic lateral sclerosis
- ALS Amyotrophic lateral sclerosis
- MND Motor neuron disease
- SCA Spinocerebellar ataxia
- SMA Spinal muscular atrophy
- Friedreich's Ataxia any other neurodegenerative-related dementia or ataxia.
- the at least one Dunaliella algae preparation or any composition comprising thereof for use according to the present disclosure is wherein the neurodegenerative disease is Alzheimer's disease.
- the at least one Dunaliella algae preparation or any composition comprising thereof for use according to the present disclosure is for preventing, treating, ameliorating, reducing or delaying the onset of cognitive decline ⁇
- the at least one Dunaliella algae preparation or any composition compriing thereof for use according to the present disclosure is wherein the Dunaliella algae is Dunaliella bardawil.
- the at least one Dunaliella algae preparation or any composition comprising thereof for use according to the present disclosure is wherein the Dunaliella algae preparation is administered orally.
- the invention provides a method for improving at least one of short term memory impairment, long term memory impairment, impaired cognitive function, impaired learning function, b-amyloids deposition in a subject in need thereof.
- tire method of the invention comprises the step of administering to the subject an effective amount of at least one Dunaliella algae preparation or any composition comprising thereof.
- the method according to the present disclosure results in improvement of at least one of cognitive function, short term memory, long term memory, acquisition time and clearance of b-amy!oids in a subject in need thereof.
- the method according to the present disclosure is for preventing, treating, ameliorating, reducing or delaying the onset of cognitive decline.
- the method according to the present disclosure is wherein the Dunaliella algae is Dunaliella bardawil.
- the method according to the present disclosure is wherein the Dunaliella algae preparation is admi istered orally.
- the present disclosure further provides at least one Dunaliella algae preparation or any composition comprising thereof for use in a method for improving at least one of short term memory impairment, long term memory impairment impaired cognitive function, impaired learning function, b-amyloids deposition in a subject in need thereof.
- the present disclosure provides use of at least one Dunaliella algae preparation for the manufacture of a composition for preventing, treating, ameliorating, reducing or delaying the onset of at least one of a neurodegenerative disease, a disorder associated with protein misfolding, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
- the use according to the present disclosure is wherein the composition ameliorates or reduces at least one symptom associated with at least one of a neurodegenerative disease, a disorder associated with protein misfolding, and cognitive decline in a subject in need thereof.
- the use according to the present disclosure is wherein the symptom is at least one of short term memory impairment, long term memory impairment, impaired cognitive function, impaired learning function, b-amy!oids deposition, anxiety, depression or any combination thereof.
- the use according to the present disclosure is wherein the composition improves at least one of cognitive function, short term memory, long term memory, acquisition time and clearance of b-amyloids in a subject in need thereof.
- the use according to the present disclosure is wherein the neurodegenerative disease is at least one of Alzheimer's disease, Parkinson's disease, Mild Cognitive Impairment (MCI), Parkinson's disease with MCI, Huntington's disease, Lewy body disease, Amyotrophic lateral sclerosis (ALS), Prion disease, Motor neuron disease (MND), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Friedreich's Ataxia and any other neurodegenerative -related dementia or ataxia.
- MCI Mild Cognitive Impairment
- ALS Amyotrophic lateral sclerosis
- MND Motor neuron disease
- SCA Spinocerebellar ataxia
- SMA Spinal muscular atrophy
- Friedreich's Ataxia any other neurodegenerative -related dementia or ataxia.
- the use according to the present disclosure is wherein the neurodegenerative disease is Alzheimer's disease.
- the use according to the present disclosure is for preventing, treating, ameliorating, reducing or delaying the onset of cognitive decline.
- the use according to the present disclosure is wherein the Dunalielia algae is Dunalielia bardawil.
- the use according to the present disclosure is wherein the Dunalielia algae preparation is administered orally.
- Fig It Graphs showing the effect of treatment with the Dunalieiia algae preparations of the invention on the weight (gr.) of wiki type (WT) and Tg2576 (Tg) mice at the indicated time frame.
- a t-test showed significant difference between WT mice fed with the Dunaliella aigae preparations of the invention (WT Duna. Prep.) and WT mice fed with regular diet (WT control) (p ⁇ 0.05). Values are mean ⁇ S.E.M.
- Fig. 2.4 - Fig, 2D Bar graphs showing the effect of treatment with the Dunaliella algae preparations of the invention on behavioral parameters measured in WT and Tg2576 mice participating in an open field test.
- Fig. 2A is a bar graph representing total path. A two way ANOVA and post-hoc test showed significant effect of genotype (p ⁇ Q.05);
- Fig. 2B is a bar graph representing percentage of cell use;
- Fig. 2C is a bar graph representing percentage of mouse movement time in tire arena;
- Fig. 2D is a bar graph representing the time the mouse spent in the arena’s center.
- a two way ANOVA showed significant effect of genotype (p ⁇ 0.05). Values are mean ⁇ S.E.M.
- Fig. 4 A bar graph showing the effect of Dunaliella algae preparations treatment on spatial learning in WT and Tg2576 mice in a Barnes maze test. Black columns represent reference memory 24 hours after the last training trial (day 1) and white columns represent long term retention 7 days after the last training trial (day 7). A two way ANOVA and post-hoc test showed significant difference (p ⁇ Q 05) between Tg2576 Duna. Prep and Tg2576 control on day 7. Values are mean ⁇ S.E.M. Abbreviations: Tg, Tg2576; WT, wild type; Duna. Prep., Dunaliella algae preparations of the invention.
- Fig. 54 - Fig. 5C Schematic drawings of the in vitro blood brain barrier (BBB) cell culture system.
- the model consists of a monolayer of endothelial cells, forming tight junctions that are grown on a microporous membrane filter culture insert (luminal side) and glial cells seeded at the abluminal side of the filter.
- Low Density Lipoprotein (LDL, 100 m ⁇ 1 ,600 pg/ml) from a healthy volunteer is added to the luminal side. Cells are incubated for 24 hours and then samples are collected from the luminal and abluminal side and analyzed.
- LDL Low Density Lipoprotein
- Fig. 6A represents the carotenoids in the blood side of the barrier and Fig 6B represents the carotenoids in brain side of the barrier.
- Human LDL was added to the luminal side (blood) and incubated for 24 hours.
- Fig. 7 A - Fig. 7C Bar graphs showing the liver (Fig. 7 A), fat (Fig, 7B) and brain (Fig. 7C) 9-cis b-carotene and ail-trans b-carotene levels in the indicated mice tissues. Values are mean ⁇ S.E. Abbreviations: 9CBC, 9-cis b-carotene; WT, wild type, fiC, b-carotene.
- Fig. 8A- Fig. 8B Fig. 8A is a bar graph showing the level of insoluble beta amyloids (Ab 42) in Tg2576 control mice and Tg2576 mice fed on Dunaliella algae preparations diet.
- Fig. 8B is a bar graph showing the level of soluble beta amyloids (soluble Abeta) in Tg2576 control mice and Tg2576 mice fed on Dunaliella algae preparations diet.
- Duna. Prep., Dunaliella algae preparations of the invention Ab, beta amyloids.
- Fig. 9A- Fig. 9C Bar graphs showing counted RNA molecules of IL-la (Fig. 9A), IL- 1b (Fig. 9B) and TSPO (Fig. 9C) in WT and Tg2576 mice fed on Dunaliella algae preparations diet or on a control diet. Values are mean ⁇ S.E. Abbreviations: Duna. Prep., Dunaliella algae preparations of the invention.
- Fig. 11 A bar graph showing the cholesterol and triglyceride levels in the plasma of Tg2576 mice fed on Dunaliella algae preparations diet or on a control diet as well as of wild type fed as described above. Values are mean ⁇ S.E. Abbreviations: Duna. Prep., Dunaliella algae preparations of the invention, Choi, cholesterol; TG, triglyceride.
- Fig. 12 A schematic diagram showing the proposed effect of dietary 9-cis b-carotene after crossing the blood brain barrier (BBB).
- BBB blood brain barrier
- Fig. 13A - Fig. 13B High Performance Liquid Chromatography (HPLC) chromatograms of brain samples obtained from 5XFAD control mice fed on low fat chow diet (Fig. 13A) and of brain samples obtained from 5XFAD mice fed on Dunalieila algae preparation diet (Fig. 13B).
- HPLC High Performance Liquid Chromatography
- Fig. 14A - Fig. 14C Bar graphs showing the liver (Fig. 14A), fat (Fig. 14B) and brain (Fig. 14C) of 9-cis b-carotene quantity in the indicated tissue of 5xFAD m ice or wild type mice fed on Dunalieila algae preparation diet or the control diet. Values are mean ⁇ S.E. Abbreviations: Duna. Prep., Dunalieila algae preparations of the invention, 9CBC, 9-cis b-carotene, pC, b-carotene, WT, wild type, 9-cis, 9-cis b-carotene.
- Fig. 15 A bar graph showing cholesterol and triglyceride levels in the plasma of 5xFAB mice and wild type mice fed on Dunalieila algae preparation diet or the control diet. Values are mean ⁇ S.E. Abbreviations: Duna. Prep., Dunalieila algae preparations of the invention, Choi, cholesterol ; TG, triglyceride.
- Fig. 16A - Fig. 16C Bar graphs showing the effect of Dunalieila algae preparations treatment on short term/long term memory of 5XFAD mice.
- Fig. 16A is a bar graph showing new object recognition after 3 hours and
- Fig. 16B is a bar graph showing new' object recognition after 24 hours.
- Fig. 16C is a bar graph showing the latency period of 5XFAD mice fed on regular diet (control) and of 5XFAD mice fed on Dunalieila algae preparation diet (Duna. Prep.).
- Fig. 17 A bar graph showing recognition memory in the Y-maze test of SxFAD mice or wild type mice fed on Dunalieila algae preparation diet or the control diet.
- the graph presents the mice preference index, the ratio between the time the mice spent in the new' arm vs. the time they spent in both arms [PI- (time spent in new arm-time spent in old arm)/time spent in new + old arm].
- Fig. 18 A bar graph showing the results of a Barnes Maze test of SxFAD mice or wild type mice fed on Dunaliella algae preparation diet or the control diet. The graph presents latency time. Values fire mean ⁇ S.E. Abbreviations: Duna. Prep., Dunaliella algae preparations of the invention.
- the present disclosure is based on the surprising effect of a Dunaliella powder preparation on neurodegenerative disorders such as Alzheimer’s disease, D), disorders associated with protein misfolding and related conditions. More specifically, as a proof of concept, the invention demonstrated the effect of Dunaliella preparations on physiological as well as behavioral parameters that indicate cognitive function, using various AD like mouse models.
- Alzheimer’s disease is a devastating neurodegenerative disease accounting for 60-80 percent of dementia cases. Disease pathology worsens over time, leading to memory loss and changes in cognitive functions.
- the Inventors have found that 9-cis b-carotene (9b(1) and aii-trans BC crossed the blood brain barrier (BBB) and accumulated in the brain and that the Dunaliella. powder preparation affected plasma lipid levels.
- BBB blood brain barrier
- the Inventors have further found that the DunaUella powder preparation improved long term and short-term memory in the examined mouse models.
- the DunaUella powder preparation had a positive effect on AD neuropathology and affected gene and expression of genes involved in inflammation.
- the Tg2576 mouse model is one of the most common transgenic mouse models used in AD studies, containing the double Swedish mutation that cause Ab accumulation and cognitive impairment.
- the 5xFAD mouse mode! containing 5 familial mutations in the APP and PESN genes, is characterized by rapid and aggressive Ab accumulation and cognitive damage.
- Another aspect of the invention demonstrates the effect of the DunaUella powder preparation on Ab deposits in the mouse hippocampus.
- Tg2576 and 5xFAD mouse models are characterized by significant Ab accumulation caused by their mutations.
- Alzheimer’s disease brain contains soluble and insoluble Ab. While it was initially hypothesized that the Ab did not become toxic until the stage of insoluble, fibrillary forms of Ab, recent studies suggest that soluble Ab has a toxic effect on memory and strong correlation with dementia, including impair synapse structure and function and inhibited hippocampal LTP As shown by the following examples, hippocampus of treated and control transgenic mice were homogenized and their Ab level was measured using ELISA assay.
- TSPO protein level expression in the hippocampus of the mouse models was examined. The emphasis was on microglia and astrocyte activation markers, TSPO and GFAP, and on synaptic plasticity presynaptic protein, synaptophysin. As shown below, in Tg2576 fed on the DunaUella diet, a significant reduction in TSPO protein level was observed, compared to untreated mice.
- TSPO is a high affinity cholesterol transporter, mainly found on the outer mitochondrial membrane of steroid-synthesizing ceils. Under normal conditions, TSPO has lower expression level in microglia cells. However, in response to neuroi nfl animation or injury, TSPO expression is upregulated, which make it an adequate microglia activation marker
- AD model mice fed with the DunaUella preparation of the present disclosure experienced a variety of beneficial neuroprotective effects.
- Tg2576 mice fed with the DunaUella preparation had substantially higher survival rates as compared to Tg2576 mice fed with regular diet (Table 1).
- reduction in anxiety was observed in Tg2576 mice fed with DunaUella preparation as demonstrated by an open field behavioral test (Example 2).
- Improvement in the learning and memory capacities of Tg2576 mice fed with the DunaUella preparation were observed in an additional behavioral test, the Barnes maze test (Example 4).
- Example 13 beneficial effect on memory for the DunaUella preparation was also demonstrated in the 5xFAD mouse model, as shown in Example 13, which demonstrates the results obtained in a novel object recognition test. Further to the above beneficial effects, the insoluble b-amyloid levels were significantly reduced in Tg2576 mice that were fed with the Duncdiella algae preparation over the control group (fed on regular diet, as shown in Example 7).
- the present disclosure provides a method for preventing, treating, ameliorating, reducing or delaying the onset of at least one of a neurodegenerative disease, a disorder associated with protein misfolding, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof. More specifically, the method of the invention may comprise the steps of administering to the subject an effective amount of at least one Dunaliella algae preparation or any composition comprising thereof.
- Duncdiella as herein defined is a genus of the algae family Dunaliellaceae. Dunaliella is a motile, unicellular, rod to ovoid shaped (9-11 pm) green algae, which is common in marine waters. Some Dunaliella strains can accumulate very large amounts of b-carotene, which is a pro- vitamin A (retinol and other retinoids) and a health food. b-Carotene belongs to the carotenoids family, hydrophobic compounds which essentially consist of a C40 hydrocarbon and up to 15 double bonds.
- Carotenoids are classified into two types: carotenes composed of carbon and hydrogen only, such as b-carotene and lycopene; and xanthopylls, which also contain oxygen, such as lutein, canthaxanthin and zeaxanthin.
- the present disclosure encompasses any Dunaliella algae known in the art and any combinations thereof, for example hut not limited to Dunaliella bardawil, Dunaliella salina, Dunaliella acidophila, Dunaliella bioculata, Dunaliella lateralis, Dunaliella maritima, to name but few species known in the art.
- At least one Dunaliella algae preparation as used herein it is meant that at least one, two, three, four, five or more Dunaliella algae known in the art may be used for preparing the Duncdiella. algae preparation of the present disclosure, or any combinations thereof.
- the alga Dunalieiia bardawil is a halotolerant green alga and a natural source of beta carotene (fie) which can synthesize and accumulate c up to 10% of its dry weight.
- the Pc formed by the alga under high light intensity and high salinity is composed of approximately 50% all-trans j , 40% 9-cis Pc and 10% a-carotene. Therefore, this alga is the best-known source for 9-cis b € in nature. It is known that synthetic 9-cis C is extremely expensive and is not readily available for human use, since long-term toxicity trials should be performed with purified or synthetic 9-cis BC prior to approval for human use. It should be noted that further detailed preparation of Dunalieiia useful in the present aspect are described in more detail herein after.
- the Dunalieiia algae is Dunalieiia bardawil
- the method according to the present disclosure comprises the step of administering to the subject a therapeutic effective amount of Dunalieiia bardawil preparation or any composition thereof.
- the blood brain barrier is a highly selective barrier composed primarily of brain endothelial cells, astrocyte end-feet, pericytes, perivascular macrophages, and a basal membrane.
- BBB transporters that restrict the permeability of toxins as well as therapeutic agents, are the ABC transporters
- ABC transporters are trans- membrane protein that transport a wide variety of substrates, including lipids, sterols and drugs.
- the transporters are localized on the surface of brain endothelial cells of the BBB and brain parenchyma. Over 20 ABC proteins representing all sub-families have been associated with several human diseases, including AD and atherosclerosis [21].
- the transporter ABCA1 is transcriptionally regulated by Liver X Receptors (LXR) and Retinoic X Receptors (RXR).
- LXR Liver X Receptors
- RXR Retinoic X Receptors
- ABCA1 transports lipids onto apolipoproteins including apoE, which is the major cholesterol carrier in the brain and an established genetic risk factor for late-onset AD.
- ABCA1 deficiency exacerbates amyloidogenesis
- ABCAl overexpression diminishes amyloid load
- Other studies have demonstrated that lack of ABCA1 increases amyloid deposition and cognitive decline in different APP transgenic mouse model accompanied by a significant decrease in tire levels of soluble apoE [23].
- transgenic mice overexpressing ABCA1 in the brain have less amyloid plaques [24].
- Other studies have shown that seniors with lower levels of ABCB 1 in their brain epithelium had more plaque [25] and more vascular Ab [26] .
- ABC transporters apparently play a major role in amyloid b clearance and therefore understanding their function and the factors regulating them may lead to the development of new therapeutic strategies against AD.
- the neuroprotect ive effects demonstrated herein render the Dunaliella algae preparation of the present disclosure suitable for the treatment of various neurodegenerative diseases or disorders.
- neurodegenerative disease refers to a heterogeneous group of disorders that tire characterized by the progressive degeneration of the structure and function of the central nervous system or the peripheral nervous system.
- any known neurodegenerative disease is encompassed by the present disclosure.
- Alzheimer ’ s disease Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis (ALS), Friedreich's Ataxia, Lewy body disease, Spinal Muscle Atrophy, motor neuron disease, synucleopathies and tauopathies.
- ALS Amyotrophic lateral sclerosis
- Lewy body disease Lewy body disease
- Spinal Muscle Atrophy Spinal Muscle Atrophy
- motor neuron disease synucleopathies and tauopathies.
- the method according to the present disclosure may be specifically applicable for a neurodegenerative disease such as Alzheimer's disease, Parkinson’s disease, Mild Cognitive Impairment (MCI), Parkinson's disease with MCI, Huntington’s disease, Lewy body disease, Amyotrophic lateral sclerosis (ALS), Prion disease, Motor neuron disease (MND), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Friedreich's Ataxia and any other neurodegenerative -related dementia or ataxia.
- a neurodegenerative disease such as Alzheimer's disease, Parkinson’s disease, Mild Cognitive Impairment (MCI), Parkinson's disease with MCI, Huntington’s disease, Lewy body disease, Amyotrophic lateral sclerosis (ALS), Prion disease, Motor neuron disease (MND), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Friedreich's Ataxia and any other neurodegenerative -related dementia or ataxia.
- the inventors have demonstrated (among others) a variety of beneficial effects on cognitive functions in mice models of Alzheimer's disease. Therefore in some embodiments the method according to the present disclosure is specifically applicable for treating Alzheimer's disease
- AD Alzheimer's disease
- AD refers to a progressive neurodegenerative disease that is manifested, among others, in damage and eventually destruction of brain cells, leading to memory loss and changes in cognitive and other brain functions. More specifically, AD, refers to a disorder that involves deterioration of memory and other cognitive domains that leads to death within 3 to 9 years after diagnosis.
- the principal risk factor for Alzheimer’s disease is age. The incidence of the disease doubles every 5 years after 65 years of age, however, up to 5% of people with the disease have early onset AD (also known as younger-onset), that may appear at 40 or 50 years of age.
- Alzheimer’s disease may be pri arily a disorder of synaptic failure. Hippocampal synapses begin to decline in patients with mild cognitive impairment (a limited cognitive deficit often preceding dementia) in whom remaining synaptic profiles show compensatory increases in size. In mild Alzheimer’s disease, there is a reduction of about 25% in the presynaptic vesicle protein synaptophysin. With advancing disease, synapses are disproportionately lost relative to neurons, and this loss is the best correlate with dementia. Aging itself causes synaptic loss, which particularly affects tire dentate region of tire hippocampus.
- Disruptions of the release of presynaptic neurotransmitters and postsynaptic glutamatereceptor ion currents occur partially as a result of endocytosis of /V-methyl-D- aspartate (NMDA) surface receptors and endocytosis of a-ammo-3-hydroxy-5-methyl-4- isoxazole propionic acid surface receptors.
- NMDA /V-methyl-D- aspartate
- the latter further weakens synaptic activity by inducing a lasting reduction in currents after a high-frequency stimulus train.
- a similar shift in the balance between potentiation and depression in synapses occurs with normal aging. Intraneuronal Ab can trigger these synaptic deficits even earlier.
- Alzheimer's disease The normally high levels of neurotrophin receptors in cholinergic neurons in the basal forebrain are severely reduced in late-stage Alzheimer's disease.
- Ab is a potent mitochondrial poison, especially affecting the synaptic pool.
- In Alzheimer’s disease exposure to Ab inhibits key mitochondrial enzymes in the brain and in isolated mitochondria. Cytochrome c oxidase is specifically attacked. Consequently, electron transport, ATP production, oxygen consumption, and mitochondrial membrane potential all become impaired. The accumulation of Ab within structurally damaged mitochondria isolated from the brains of patients with Alzheimer's disease.
- AD Alzheimer’s disease
- AD Alzheimer's disease
- AD has no current cure, and the current treatments cannot stop AD from progressing.
- Diagnosis of AD may be performed by a skilled physician and include physical examinations and diagnostic tests (for example a Mini-Mental State Exam (MMSE)).
- MMSE Mini-Mental State Exam
- Early signs and symptoms of Alzheimer’s include among others memory impairment, difficulty concentrating, planning or problem-solving, problems with finishing daily tasks, confusion, language problems such as word-finding problems or reduced vocabulary in speech or writing, changes in mood, such as depression or other behavior and personality changes.
- Dunaliella algal preparations, compositions and methods of the invention are suitable for treating any stage of AD, at any age and for any conditions and symptoms associated therewith.
- plaques and tangles are involved with AD as well as in other age-related neurodegenerative processes.
- the invention further encompasses the use of the combined therapy disclosed herein for treating other age-related condi tions.
- Cognitive decline is among the most feared aspects of growing old. it is also the most costly, in terms of the financial, personal and societal burdens. It is important, because cognitive decline heralds dementia, illness and death.
- AD model mice fed with the Dunaliella algae preparation of the present disclosure experienced improvement in learning and memory capacities and improvement of memory capacity, being some of the symptoms associated with various neurodegenerative diseases (eg. AD and Huntington’s disease).
- the method according to the present disclosure is wherein the method results in amelioration and/or reduction of at least one symptom associated with at least one of a neurodegenerative disease, a disorder associated with protein misfoiding and cognitive decline in a subject in need thereof.
- amelioration it is meant to reduce, alleviate, lighten, improve or relieve by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
- Any symptom (or any conditions or symptom) associated with at least one of a neurodegenerative disease, a disorder associated with protein misfolding and cognitive decline in a subject in need thereof is encompassed by the present disclosure.
- symptoms include but are not l imited to decline in mental abilities (for example short-term memory and long-term memory decline, impaired learning function), behavioral and psychiatric problems, lack of coordination, unsteady gait, speech changes, tremor, confusion with time or place, decreased or poor judgment, changes in mood and personality.
- tire method according to the present disclosure is wherein the symptom is at least one of short term memory impairment, long term memory impairment, impaired cognitive function, impaired learning function, b-amyloids deposition, anxiety, depression, or any combination thereof.
- short term memory impairment is a system for temporarily storing and managing information required to carry out complex cognitive tasks such as learning, reasoning, and comprehension. Short-term memory is involved in the selection, initiation, and termination of information- processing functions such as encoding, storing, and retrieving data.
- short term memory impairment it is meant an impaired ability to form new episodic memories. Short term memory loss can have a substantial and negative impact on a person’s quality of life. The inability to form any new episodic memories renders a person to live in a perpetual“now” state, where new events are never encoded for later recall.
- the symptom herein defined may be long term memory impairment.
- NASH National Institutes of Health
- Long-term memories are formed when short-term memories, or non- permanent memories, are consolidated in the hippocampus, a brain structure located in the medial temporal lobe. Once the memories are consolidated, they are available independent from the hippocampus in the neocortex, where they can be retrieved. When a patient has long-term memory loss, the patient displays problems recalling stored memories, not creating new memories.
- the symptom herein defined may be impaired cognitive function.
- Impaired cognitive function! is when a person has trouble remembering, learning new' things, concentrating, or making decisions that affect their everyday life. Cognitive impairment ranges from mild to severe. With mild impairment, people may begin to notice changes in cognitive functions, but still be able to do their everyday activities. Severe levels of impairment can lead to losing the ability to understand the meaning or importance of something and the ability to talk or write, resulting in the inability to live independently.
- the symptom herein defined may be impaired learning function.
- impaired learning function means a decrease or reduction in the learning ability which affects acquisition, organization, retention, understanding or use of verbal or nonverbal information impaired learning function results from impairments in one or more processes related to perceiving, thinking, remembering or learning, and may also involve difficulties with organizational skills, social perception, social interaction and perspective taking.
- the invention provides methods for treating, inhibiting and reducing or in other words, improving impaired cognitive functions impaired as used herein is meant any reduced, damaged, retarded, decreased, or atenuated cogniti ve parameters, for example, learning and memory functions, by at least about 1%, 2%, 3%, 4%, 3%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%
- the symptom herein defined may be b- amyloids deposition.
- Beta-amyloid is a protein fragment that is deposited in the brain in the form of sticky, starch-like plaques, in an increased manner in individuals with AD.
- b- amyloids deposition means formation of beta-amyloid aggregates in the brain.
- Significant amyloid deposition is a characteristic feature of all patients with AD
- Amyloid beta denotes peptides of 36-43 amino acids that are crucially involved in Alzheimer’s disease as the main component of the amyloid plaques found in the brains of people with Alzheimer's disease.
- the peptides derive from the amyloid precursor protein (APP), which is cleaved by beta secretase and gamma secretase to yield Ab.
- APP amyloid precursor protein
- Ab molecules can aggregate to form flexible soluble oligomers which may exist in several forms.
- misfoided oligomers can induce other Ab molecules to also take the misfoided oligomeric form that is toxic to nerve cells. More specifically, Ab is a 4 kDa peptide (with 40- and 42-amino acid residue peptides as the predominant species) derived from proteolytic cleavage of a precursor protein termed amyloid precursor protein. Ab monomers readily aggregate in aqueous medium, giving rise to various types of assemblies including oligomers, protofibrils and amyloid fibrils.
- amyloid fibrils are larger and insoluble, and assemble into amyloid plaques, forming histological lesions that are characteristic of AD.
- the insoluble fibrillar beta amyloid lesions (known as neuritie plaques) do not necessarily correlate very well with disease progression, suggesting that are not directly causal. However, the soluble form of amyloid seems to better correlate with disease progression. It appears that high MW weight oligomers may cause synaptic loss and, ultimately, memory loss in AD.
- MCI Mild Cognitive Impairment
- the invention therefore in certain embodiments thereof, provides methods for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of age- associated mild cognitive impairment (MCI).
- MCI mild cognitive impairment
- Age-associated mild cognitive impairment is a condition that causes cognitive changes MCI that primarily affects memory may be classified as “amnestic MCI” where the subjects experience impairment in memorizing information that relate to recent events, appointments, conversations or recent events. MCI that affects thinking skills other than memory is known as “nonamnestic MCI”.
- thinking skills that may be affected by nonamnestic MCI include the ability to make sound decisions, judge the time or sequence of steps needed to complete a complex task, or visual perception.
- AMI age-related memory impairment
- AAMI age-associated memory impairment
- ACD age -associated cognitive decline
- the ability to encode new memories of events or facts and working memory shows decline in both cross-sectional and longitudinal studies.
- Studies comparing the effects of aging on episodic memory, semantic memory, short-term memory and priming find that episodic memory is especially impaired in normal aging; some types of short-term memory are also impaired.
- the deficits may be related to impairments seen in the ability to refresh recently processed information.
- the invention provides combined treatment for any cognitive decline, specifically cognitive decline associated with age, specifically, tire age of 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and more, years of age.
- the symptom or condition as herein defined is anxiety.
- anxiety it is meant an abnormal and overwhelming sense of apprehension and fear often marked by physical signs (such as tension, sweating, and increased pulse rate), by doubt concerning the reality and nature of the threat, and by self-doubt about one's capacity to cope with it.
- the symptom or condition as herein defined is depression.
- depression means a state of low mood and aversion to activity that can affect a person's thoughts, behavior, tendencies, feelings, and sense of well-being.
- a depressed mood is a normal temporary reaction to life events - such as loss of a loved one. It is also a symptom of some physical diseases and a side effect of some drugs and medical treatments. Depressed mood may also be a symptom of some mood disorders such as major depressive disorder or dysthymia.
- AD model mice fed with tire Dunaliella algae preparation of the present disclosure experienced, inter alia, reduction in anxiety and improvement in the learning and memory capacities.
- the Inventors have shown that in the AD model m ice fed with tire Dunaliella algae preparation of the present disclosure the level of insoluble b- amyloid was significantly reduced as compared to the control group (fed with regular diet).
- the method according to the present disclosure is wherein said method results in improvement of at least one of cognitive function, short term memory, long term memory, acquisition time and clearance of b-amyloids in a subject in need thereof.
- improvement it is meant any recovery, advance or enhancement by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,
- the present invention further provides improvement of cognitive function as discussed above, specifically, learning function, short term and long term memory, and reduction in anxiety and depression, for subjects that do not necessarily suffer from neurodegenerative disorders, specifically, the invention provides methods for improving cognitive function for healthy subjects.
- the term“ irnprovemenf’ as used herein it is meant any recovery, advance or enhancement as indicated above of at least one of short term memory impairment, long term memory impairment impaired cognitive function, impaired learning function, b-amyloids deposition in a subject in need thereof.
- Dunaiiella algae preparation for use and compositions and uses as herein defined are also useful for the preventing, reducing or treating cognitive decline.
- the method according to the present disclosure is for preventing, treating, ameliorating, reducing or delaying the onset of cognitive decline.
- cognitive decline as used herein it is meant impairment of cognitive function of an individual to the point where normal functioning is impossible without treatment. Some of the common signs of cognitive decline are confusion, poor motor coordination, loss of short-term or long-term memory, identity confusion and impaired judgement.
- insoluble b-amyloid levels were significantly reduced in Tg2576 mice that were fed with tire Dunaliella algae preparation of the present disclosure over the control group (on regular diet).
- various human degenerative conditions including Alzheimer’s disease, light- chain amyloidosis and the spongiform encephalopathies, are associated with the deposition in tissue of proteinaceous aggregates (which are misfolded proteins) known as “amyloid fibrils” or“plaques”.
- the methods, uses and Dunaliella algae preparation for use as herein defined are for preventing, treating, ameliorating, reducing or delaying the onset of a disorder associated with protein misfolding comprising administering to said subject an effective amount of at least one Dunaliella algae preparation or any composition comprising thereof.
- Protein misfolding and aggregation as used herein, relates to an impaired physical process by which a protein chain acquires its native three-dimensional structure, a conformation that is usually biologically functional, in an expeditious and reproducible manner.
- Protein folding is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil. Each protein exists as an unfolded polypeptide or random coil when translated from a sequence of triRNA to a linear chain of amino acids. Amino acids interact with each other to produce a well-defined three-dimensional structure, the folded protein, known as the native state. The correct three-dimensional structure is essential to function, although some parts of functional proteins may remain unfolded.
- misfolded proteins have modified or toxic functionality.
- Several neurodegenerative and other diseases are believed to result from the accumulation of amyloid fibrils formed by misfolded proteins. More specifically, under some conditions, proteins may not ibid into their biochemically functional forms resulting in protein denaturation. A fully denatured protein lacks both tertiary and secondary structure, and exists as a so-called random coil. Under certain conditions some proteins can refold; however, in many cases, denaturation is irreversible. Cells may protect their proteins against the denaturing influence of heat with enzymes known as chaperones or heat shock proteins, which assist other proteins both in folding and in remaining folded.
- the term“ disorder associated with protein misfolding” as used herein it is referred to a disease or disorder directly or indirectly resulting from accumulation of misfolded aggregates of proteins in organs or tissues. More specifically, aggregated proteins are associated with prion-related illnesses such as Creutzfeldt-Jakob disease, bovine spongiform encephalopathy (mad cow disease), amyloid-related illnesses such as Alzheimer's disease and familial amyloid cardiomyopathy or polyneuropathy, as well as intracytoplasmic aggregation diseases such as Huntington’s and Parkinson’s disease.
- prion-related illnesses such as Creutzfeldt-Jakob disease, bovine spongiform encephalopathy (mad cow disease)
- amyloid-related illnesses such as Alzheimer's disease and familial amyloid cardiomyopathy or polyneuropathy
- intracytoplasmic aggregation diseases such as Huntington’s and Parkinson’s disease.
- disorder associated with protein misfolding is a group of disorders associated with beta-amyloid protein aggregation that includes Alzheimer's disease (AD), where deposits of a protein precursor called beta-amyloid build up (termed plaques) in the spaces between nerve cells and twisted fibers of tau protein build up (termed tangles) inside the cells.
- AD Alzheimer's disease
- Beta-amyloid protein aggregations as used herein relates to cerebral plaques laden with b-amyloid peptide (Ab) and dystrophic neurites in neocortical terminal fields as well as prominent neurofibrillary tangles in medial temporal-lobe structures, which are important pathological features of Alzheimer’s disease. Subsequently, loss of neurons and white matter congophilic (amyloid) angiopathy are also present.
- Ab peptides are natural products of metabolism consisting of 36 to 43 amino acids. Monomers of Ab40 are much more prevalent than the aggregation-prone and damaging Ab42 species b-amyloid peptides originate from proteolysis of the amyloid precursor protein by the sequential enzymatic actions of beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1), a b-secretase, and g-secretase, a protein complex with presenilin 1 at its catalytic core.
- BACE-1 beta-site amyloid precursor protein cleaving enzyme 1
- An imbalance between production and clearance and aggregation of peptides causes Ab to accumulate, and this excess may be the initiating factor in Alzheimer’s disease.
- b-amyloid can also grow into fibrils, which arrange themselves Into b-pleated sheets to form the insoluble fibers of advanced amyloid plaques.
- Soluble oligomers and intermediate amyloid are the most neurotoxic forms of Ab.
- dimers and trimers of Ab are toxic to synapses.
- Experimental evidence indicates that Ab accumulation precedes and drives tau protein aggregation.
- Tau protein refers to neurofibrillary tangles, which are filamentous inclusions in pyramidal neurons, characteristic for Alzheimer’s disease and other neurodegenerative disorders termed tauopathies. Elucidation of the mechanisms of their formation may provide targets for future therapies. Accumulation of hyperphosphorylated Tau protein as paired helical filaments in pyramidal neurons is a major hallmark of Alzheimer disease (AD). Besides hyperphosphorylation, other modifications of the Tau protein, such as cross-linking, are likely to contribute to the characteristic features of paired helical filaments, including their insolubility and resistance against proteolytic degradation. These neurofibrillary tangles, consist of hyperphosphorylated and aggregated forms of the microtubule-associated protein tau.
- tau is a developmental! y regulated phosphoprotein that promotes assembly and stability of microtubules and is thus involved in axonal transport.
- tau proteins aggregate and form fibrillar insoluble intracellular inclusions, so-called neurofibrillary tangles it has been suggested that ionic interactions and covalent cross-linking contribute to pathological Tau aggregation and tangle formation.
- Reactive carbonyl compounds which are increased under conditions of oxidative stress and in aging have been proposed as potential compounds responsible for tau aggregation.
- Alpha-synuclein pathology disorders are disorders characterized by the presence of a specific intracellular protein aggregates (inclusion bodies) known as Lewy bodies that contain mainly alpha-synuclein protein.
- Alpha-synuclein protein consists of 140 amino acids and is found naturally as an unfolded cytoplasmic protein in neuronal synaptic areas.
- Alpha-synuclein interrupts normal cell functions and leads to decreases in neurite outgrowth and cell adhesion.
- Alpha-synuclein aggregates comprising monomeric, oligomeric intermediate, or fibrillar forms are thought to be involved in a critical step in the pathogenesis of Parkin on’s disease (PD) and in other alpha- synucleinopathies, such as multiple system atrophy (MSA) and dementia with Lewy bodies (DLB).
- MSA multiple system atrophy
- LLB dementia with Lewy bodies
- These chronic neurodegenerative diseases of the CNS are characterized by the development of Lewy bodies containing alpha-synuclein protein.
- Oligomeric and monomeric alpha-synuclein have both been detected in cerebrospinal fluid and plasma samples from PD patients, suggesting that small aggregates of alpha-synuclein access the extracellular space.
- the invention provides Dunaliella algal preparations, compositions, kits and methods applicable in protecting against any neurodegeneration, or any neuronal damage as discussed herein.
- Neurodegeneration is a common theme of many nervous system diseases and disorders, such as Parkinson's disease, Alzheimer's disease, ALS, head trauma and epilepsy.
- a common theme of these diseases and disorders is the loss of neural cell functions and/or neural cell death or damage.
- the Inventors disclose Dunaliella algal preparations, composition and methods involving exposing neural cells to Dunaliella algal preparations, whether directly or through administration to a patient, for neuro-protection or protection from any neuronal damage or injury and thereby prevention and treatment of pathologies which cause neural cell function deterioration and death.
- the term "damage" or injury relates to any disruption of physiological cell functions or cell death.
- disruption of physiological cell functions include: oxidative stress (for example, lipid peroxidation, DNA and RNA oxidation and protein oxidation), non-specific g!ycation, protein misfolding, DNA mutation, loss of any cellular structure integrity, metabolic stress, ionizing and non-ionizing radiation damage and chemical stress (for example, exposure to acid or basic substances).
- the expression“ protection from neural cell function deterioration and death” means either preventing or decreasing neural death, or preventing or decreasing the deterioration in neural function (as exemplified for instance by secretion of neurotransmitters, dendrite and axonal growth, transfer of electrical impulses, response to stimuli, maintaining structural integrity of myelin sheaths and Ranvier's nodes, etc.)
- neural cell function relates to any norma! physiological cellular activity, depending on the specific cell type.
- Non-limiting examples of such functions include cell viability, secretion of neurotransmitters, dendrite and axonal growth, transfer of electrical impulses and response to stimuli in neurons, maintaining structural integrity of myelin sheaths and Ranvier's nodes in oligodendrocytes and Schwann cells, and supplying nutrients and oxygen, and recycling neurotransmitters in astrocytes.
- neural cell relates to ceils that may he any one of centra! nervous system neurons and glia! cells, astrocyte, neuron ceils, oligodendrocyte, Schwann cells, satellite cells, spindle cells, neuronauditory inner hair cells of organ of Corti, auditory outer hair ceils of organ of Corti, basal cells of olfactory epithelium, cold-sensitive primary sensory neurons, heat-sensitive primary sensory neurons, Merkel cells of epidermis, olfactory receptor neurons, pain-sensitive primary sensory neurons, photoreceptor rod cells, photoreceptor blue-sensitive cone cells of eye, photoreceptor green-sensiti ve cone ceils of eye, photoreceptor red-sensitive cone ceils of eye, proprioceptive primary sensory neurons, touch-sensitive primary sensory neurons, type I carotid body ceils, type II carotid body cells, type I hair ceils of vestibular apparatus of ear, type II hair cells
- neural cell function relates to any normal physiological cellular activity, depending on the specific cell type.
- Non-limiting examples of such functions include cell viability, secretion of neurotransmitters, dendrite and axonal growth, transfer of electrical impulses and response to stimuli in neurons, maintaining structural integrity of myelin sheaths and Ranvier's nodes in oligodendrocytes and Schwann cells, and supplying nutrients and oxygen, and recycling neurotransmitters in astrocytes.
- neurode generation is the umbrella term for the progressive loss of structure or function of neurons, including death of neurons.
- Many neurodegenerative diseases including Parkinson’s, Alzheimer’s, ALS and Huntington’s occur as a result of neurodegenerative processes.
- Other examples of neurodegeneration include Friedreich’s ataxia, Lewy body disease, spinal muscular atrophy, multiple sclerosis, frontotemporal dementia, corticobasal degeneration, progressive supranuclear palsy, multiple system atrophy, hereditary spastic paraparesis, amyloidoses and Charcot Marie Tooth it should not be overlooked that normal aging processes include progressive neurodegeneration.
- neuro-pathological condition relates to any pathological condition caused by, or which causes, or is associated with neural cell disorders, such as any deterioration of the neural cell functions or viability.
- such conditions may be neurodegenerative disorders, brain traumas, metabolic disorders which affect the nervous system, such as phenylketonuria, immunological disorders which affect the brain, such as Hashimoto's Thyroiditis, genetic diseases which affects neural cells, such as Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, Fabry disease, Gaucher disease, Father disease, and Niemann-Piek disease, nutrient deficiencies such as vitamin Be, and D deficiencies, and any sequelae which affects the nervous system.
- Dunaliella algal preparations, methods and compositions of the invention may be applicable for treating neuro-pathological and neurodegenerative disorders or any pathologic condition associated therewith.
- association linked
- related when referring to pathologies herein described, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, co-exist at a higher than coincidental frequency, or where at least one disease, disorder condition or pathology causes the second disease, disorder, condition or pathology.
- Such conditions may include for example, Parkinson’s disease, Alzheimer's disease, amyotrophic lateral sclerosis, head trauma, epilepsy, stroke, neuroinyotonia/Isaacs syndrome, lower motor neuron lesion, Werdnig-Hoffman disease, amyotrophic lateral sclerosis, Kennedy disease, organophosphate poisoning, benzodiazepine withdrawal, magnesium deficiency, myalgie encephalomyelitis, dehydration, fatigue, lyrne disease, myasthenia gravis, rabies, fibromyalgia, subarachnoid hemorrhage, intracerebral hemorrhage, occlusion and stenosis of precerebral arteries, occlusion and stenosis of basilar artery, occlusion and stenosis of carotid artery, occlusion and stenosis of vertebral artery, occlusion of cerebral arteries, cerebral thrombosis
- disease refers to a state in which there is a disturbance of normal functioning.
- condition refers to a state in which there is a disturbance of normal functioning.
- a beneficial (therapeutic) effect may be achieved in at least one, for example 2, 3, 4, 5 or more diseases, disorders or conditions as herein defined, by the Dunalielia algae preparation of the present disclosure.
- treat, treating, treatment mean ameliorating or reducing one or more clinical indicia of disease activity in a subject having a disease or disorder as herein defined. Amel i ration or reduction in the clinical indicia of disease may be subtle or significant.
- preventing or delaying the onset of at least one of a neurodegenerative disease, a disorder associated with protein misfolding, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof comprising administering to said subject an effective amount of at least one Dunalielia algae preparation or any composition comprising thereof are also encompassed by the present disclosure.
- preventing it is meant to provide a "preventive treatment” or “prophylactic treatment”, namely acting in a protective manner, defending against or preventing something, especially a condition or disease as herein defined.
- the disease, disorder or condition as herein defined often begin subtly hut progress until they significantly impede the affected individual’s quality of life. Factors such as age, genetics and lack of proper nutrients contribute to the development of the disease.
- delaying the onset in the context of the disorder, disease or condition as defined herein, it is meant any postponement, suspension, impediment or retardation of the manifestation of the disease or symptoms associated therewith as herein defined.
- Subject in need thereof means warm-blooded animals (such as for example humans, rats, mice, dogs, cats, guinea pigs and primates).
- the subject is diagnosed with the disease, disorder or condition herein defined. Diagnosis of the disease, disorder or condition herein defined may be performed by a skilled physician, as known in the art.
- the "Dunaiiella algae preparation” or " Dunaliella preparation” of the present disclosure may be prepared by any known method.
- the Dunaliella algae preparation is prepared as described by the Examples below and is a Dunaliella algae powder preparation.
- the Dunaliella preparation of the present disclosure is prepared as an extract.
- extract it is meant any substance or a mixture of substances extracted from Dunaliella, using enzymes, organic solvents or hydrophilic solvents for extraction.
- the term extract encompasses substances obtained by using either organic solvents such as, for example, alcohols (e.g. ethanol), hexane, ethyl- cetate or isopropyl-alcohol, or by hydrophilic solvents such as water.
- an extract may be prepared by any physical extraction such as cutting, mincing, grinding, either fresh, frozen or dried Dunaiiella material. The extracts may be dried after said extraction and may be further processed (extracted) by any extraction method, independently from previous extraction steps.
- Such steps may be repeated independently.
- other extraction techniques may be employed, non-limiting examples of which include chromatography, including size-exclusion, hydrophobic interaction, and anion and cation exchangers, differential centrifugation, differential precipitation (for example, using ammonium sulfate), differential filtration and dialysis.
- fresh, frozen, dried or evaporated Dunaliella material may be used for any of the above preparation procedures.
- the Dunaliella preparation of the present disclosure is a powder preparation.
- the "powder preparation ' as used herein may be prepared by any method known in the art.
- the powder preparation is as disclosed by US Patent 8,722,057. More specifically, different methods of preparation will produce powders with different properties. In order to prepare powders consisting of particles having a particular size and shape, careful selection of the preparation technique is necessary. Grinding, the thermal decomposition of solids and the deposition of solids from the liquid or vapor phase are the commonest techniques used for the preparation of powders. Any pharmaceutically compatible binding agents, excipients and/or adjuvant materials can be included as part of the powder preparation as herein defined.
- a raw material to be used for carrying out a method for producing the present invention is microalgae, and preferably the algae belonging to the genus Dunaliella as one type of the green algae.
- the algae belonging to the genus Dunaliella fire known to produce and store a large amount of b-carotene in the alga body.
- Dunaliella bardawil and Dunaliella salina store a large amount of b-carotene in the alga bodies, they are further preferable to be used.
- the algae belonging to the genus Dunaliella are cultured in a culture device such as a culture tank and a culture pool outside or inside for a predetermined time, and then pumped out from such a culture facility by using a pumping means such as a pump.
- the culture solution pumped out is filtered through a predetermined mesh net so as to remove foreign substances contaminated in the culture device.
- the culture solution from which foreign substances are removed is dehydrated by a centrifuge so that the solid part in the culture solution is concentrated to a predetermined concentration.
- the concentration of the solid part in the culture solution after centrifugation is preferably 10 to 30% by weight from the viewpoint that the culture solution has fluidity although it is concentrated.
- the centrifuge is preferably an apparatus capable of carrying out centrifugation of the culture solution in a batch or continuous manner, and more preferably an apparatus capable of carrying out centrifugation continuously from the viewpoint of workability and productivity.
- a centrifuge generally available centrifuges are used, and the rotation rate of a rotor of the centrifuge is not particularly limited but it is set for each centrifuge used so as to have the above-mentioned concentration of the solid part of the culture solution.
- a pH adjusting step is carried out in which a culture solution concentrated to a predetermined concentration is treated in a basic state.
- a basic compound, its aqueous solution or the like is added to the culture solution concentrated to a predetermined concentration, and the culture solution is preferably stirred and mixed with a stirring device such as a stirrer in a highly basic state in which the hydrogen ion exponent, i.e., pH is 9.5 or higher at a temperature of about 25° C, more preferably stirred and mixed in a highly basic state in which pH is 10.0 or higher, and most preferably stirred and mixed in a highly basic state in which pH is 11.0 or higher.
- a stirring device such as a stirrer in a highly basic state in which the hydrogen ion exponent, i.e., pH is 9.5 or higher at a temperature of about 25° C, more preferably stirred and mixed in a highly basic state in which pH is 10.0 or higher, and most preferably stirred and mixed in a highly basic state in
- pH is less than 9.5 because it is difficult to stably control Dunaliella powder so as to have a total pheophorbide amount of 160 mg % or less and an existing pheophorbide amount of 100 mg % or less throughout the year.
- Preferable examples of basic compounds to be used in the pH adjusting step include lithium hydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, tetramethylammonium hydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide, thallium hydroxide, and guanidine. More preferable examples include widely used sodium hydroxide, potassium hydroxide, and calcium hydroxide. Furthermore, two or more thereof may be used together. In addition, an aqueous solution thereof having an arbitrary' ⁇ concentration can he used.
- a neutralization treatment step is carried out in order that the liquid property is made to he in a neutral range around pH 7 at a temperature of 25° C. This step will be necessary in the case where it is difficult to distribute Dunaliella powder at such a high pH when, for example, the obtained Dunaliella powder is sold as health foods or processed foods. Note here that if another neutralization treatment step is carried out when processed foods are produced using the Dunaliella powder, the neutralization treatment step is not necessarily carried out in the present invention.
- an inorganic acid or an organic acid is used as compounds to be used in the neutralization treatment step.
- the inorganic acid include hydrochloric acid, phosphoric acid, sulfuric acid, and nitric acid.
- the organic acid include formic acid, acetic acid, citric acid, and oxalic acid. Furthermore, two or more thereof may be used together. In addition, aqueous solutions thereof having an arbitrary concentration can be used.
- a desalting treatment step can be carried out.
- well-known methods can he used.
- desalting treatment using a chitosan solution which is described in Japanese Patent Laid-Open No. 1995-000147, can be used.
- a heat treatment step for further decreasing pheophorbide harmful to a human body, which is contained in a culture solution subjected to desalting treatment and concentrated to a predetermined concentration, or for killing general bacteria, a heat treatment step may be carried out at a predetermined temperature for a predetermined time.
- the heat treatment step is carried out preferably in a temperature range from 70° C. to 140° C., and more preferably in a temperature range from 80° C. to 130° C. It is not preferable that heat treatment temperature is carried out at a temperature of less than 70° C.
- time necessary for the heat treatment step is preferably in the range from 2 to 80 min, and more preferably in the range from 5 to 60 min.
- heat treatment time is less than 2 min because various pheophorbide amounts cannot be decreased or sterilization sufficiently for selling as health foods and the like cannot be performed it is not preferable that heat treatment time is longer than 80 min because b-carotene cannot be obtained at a high content due to oxidati ve degradation.
- the heat treatment step is not necessarily carried out after the desalting treatment step, but it may be earned out in arbitrary orders, for example, it is carried out before tire pH adjusting treatment step.
- paste which has been obtained after a neutralization treatment step or heat treatment step if necessary, is formed into a dried powder product by removing water from the paste by well-known methods such as spray drying, or lyophilization under decreased pressure.
- the Dunalieila powder obtained in the above-mentioned series of methods has a total pheophorbide amount of 160 nig % or less and an existing pheophorbide amount of 100 mg % or less, and contains 3 to 20 g of b-carotene in 100 g of the Dunalieila powder. Furthermore, the amount of b-carotene contained in 100 g of the Dunalieila powder differs depending upon the algae belonging to the genus Dunalieila to be used as a raw material, but the amount is more preferably 5 to 15 g, and most preferably 6 to 10 g.
- the amount of b-carotene contained in 100 g of the Dunalieila powder is less than 3 g because a commercial value thereof is lowered. Furthermore, in order to achieve a content of 20 g or higher, the algae belonging to the genus Dunalieila as the raw material is required to contain more b-carotene.
- the Dunaliella preparation of the present disclosure is based on Dunaliella bardawi! prepared as detailed below. It should be noted that the family Dunaliellaceae, and specifically, tire genus Dunaliella is a single-celled, photosynthetic green alga, that is characteristic for its ability to outcompete other organisms and thrive in hypersaline environments.
- Certain species of this genus can accumulate relatively large amounts of b-carotenoids and glycerol in very harsh growth conditions consisting of high light intensities, high salt concentrations, and limited oxygen and nitrogen levels.
- Dunaliella bardawi! is well-known microalgae accumulating high levels of beta-carotene under growth-limiting conditions, that is primarily composed of the isomers 9-cis and all-trans.
- the Dunaliella used by the invention is grown in any growth conditions for example, any salinity conditions, as well as any light conditions and any temperature conditions.
- salinity conditions include salt concentrations of 1M, 2M, 3M or more NaCl, up to 4M.
- Dunaliella preparations used in the present invention are prepared from any Dunaliella species, strains and isolates.
- the Dunaliella preparations as used herein are prepared from Dunaliella Bardawi!.
- Dunaliella bardawi ⁇ as used herein is the Ben-Amotz and Avion, isolated from salt pond near Bardawil Lagoon, North Yale, , 1976.
- the Dunaliella bardawil is as used herein as denoted by ATCC ® 30861 TM It should be understood that tire invention further encompasses the use of any progeny, strain, isolate, mutant or variant of the Dunaliella bardawil as denoted by ATCC ® 30861, for any of the aspects described by the invention.
- Dunaliella bardawil (hereinafter "Dh") was grown and cultivated in large body open salt water ponds of 50,000 m to obtain algae comprising approximately 8% by weight of b- carotene (hereinafter “BC") at an approximately 1 : 1 (by weight) ratio of 9-cis and all- trans isomers of BC, or greater than 1 : 1 ratio of 9-cis and all-trans isomers of BC.
- BC b- carotene
- the algae were harvested by dislodging centrifuges into a concentrated paste. The paste was washed to remove salt and sterilized, and then spray dried to yield Db powder comprising approximately 8% BC and less than 5% moisture.
- the powder was packaged in capsules of 250-300 mg algae containing 15-20 mg of BC each together with all of the natural components of the algae.
- the BC of the capsules retains the original ratio of isomers.
- the capsules were packaged in vacuum closed blisters which have a shelf life of up to three years.
- the Dunaliella preparation of the present disclosure is encapsulated.
- Encapsulation is the process used to entrap one substance (termed core material or active agent) within another (coating, shell, or carrier/wall material). More specifically, a dried powder of Dunaliella algae, a tablet obtained by compressing and hardening the dried powder of Dunaliella algae, or a capsule obtained by encapsulating the dried powder of Dunaliella algae are known. In any states, firstly, it is necessary to dry a culture solution of Dunaliella algae and to form it into dried powder.
- Japanese Patent Laid-Open No. 1997-203 discloses that a dried powder product of the algae belonging to the genus Dunaliella is obtained by previously decreasing the water content of a culture solution of cultured Dunaliella alga body to. preferably, about 50% for easy drying, followed by being subjected to nebulizalion drying, vacuum drying or freeze drying.
- Dried powder products of the algae belonging to d e genus Dunaliella are sold as foods. It is necessary to carry out a step of decreasing compounds that may be harmful to a human body in a step of producing a dried powder product from the harvested algae belonging to the genus Dunaliella in order to satisfy a predetermined safety standard.
- the Dunaliella algae preparation of the present disclosure may be a Dunaliella bardawii preparation.
- the Dunaliella algae preparation of the present disclosure may be adapted for add-on to a beverage, solid, semi-solid or liquid food, food additive, food supplement, medical food, botanical drug, drug and/or a pharmaceutical compound.
- the Dunaliella algae preparation of the present disclosure is used as a functional food.
- functional foods it is meant whole, fortified, enriched or enhanced foods that provide health benefits beyond the provision of essential nutrients (e.g., vitamins and minerals), when they are consumed at efficacious levels as part of a varied diet on a regular basis.
- the Dunaliella preparation of the present disclosure is used as a food supplement.
- a food supplement the term coined by the European Commission for Food and Feed Safety, or a dietary supplement, an analogous term adopted by the US Food and Drug Administration (FDA), relates to any kind of substances, natural or synthetic, with a nutritional or physiological effect whose purpose is to supplement the normal diet.
- FDA US Food and Drug Administration
- dietary supplement is defined as a product (other than tobacco) intended to supplement the diet that bears or contains one or more of the following dietary ingredients: a vitamin, a mineral, an herb or other botanical, an amino acid, a dietary substance for use by man to supplement the diet by increasing the total dietary intake, or a concentrate, metabolite, constituent, extract, or combination of any of the aforementioned ingredients.
- dietary supplements Under food or dietary supplements is meant those marketed in a form of pills, capsules, powders, drinks, and energy bars and other dose forms.
- European and the US laws regulate dietary supplements under a different set of regulations than those covering "conventional" foods and drug products. According thereto, a dietary supplement must be labeled as such and be intended for ingestion and must not be represented for use as conventional food or as a sole item of a meal or a diet.
- the Dunaliella algae preparation of the present disclosure may be used as an add-on to medical foods.
- medical foods it is meant foods that are specially formulated and intended for the dietary management of a disease that has distinctive nutritional needs that cannot be met by normal diet alone.
- medical food as defined in the FDA's 1988 Orphan Drug Act Amendments is a food which is formulated to be consumed or administered entirely under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation.
- botanical drags are also pertinent to the present context.
- the Dunaliella algae preparation of the present disclosure may be an add-on to a botanical drug.
- botanical drug refers to products that are intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease in humans.
- a botanical drug product consists of vegetable materials, which may include plant m aterials, algae, macroscopic fungi, or combinations thereof.
- a botanical drug product may be available as (but not limited to) a solution (e.g., tea), powder, tablet, capsule, elixir, topical, or injection.
- Botanical drug products often have unique features, for example, complex mixtures, lack of a distinct active ingredient, and substantial prior human use. Fermentation products and highly purified or chemically modified botanical substances are not considered botanical drag products.
- a botanical product may be a food (including a dietary supplement), a drug (including a biological drug), a medical device (e.g., gutta-percha), or a cosmetic.
- botanical drags may include botanical ingredients in combination with either a synthetic or highly purified drug or a biotechnology derived or other naturally derived drag.
- botanical drugs may also contain animals or animal parts (e.g., insects, annelids, shark cartilage) and/or minerals or a combination thereof.
- the method according to the present disclosure comprises administering to the subject an effective amount of at least one Dunaliella algae preparation or any composition comprising thereof.
- the term“ effective amount” means an amount necessary to achieve a selected result.
- the effective amount is determined by tire severity and type of the disease or condition in conjunction with the preventive or therapeutic objectives, the route of administration and the patient’s general condition (age, sex, weight and other considerations known to the attending physician).
- the effective amount may be determined based on animal models, such as these presented in the Examples.
- the Dunaliella algae preparation of the present disclosure is comprised in a composition.
- the composition comprising the Dunaliella algae preparation of the present disclosure may be prepared according to any method known in the art.
- composition comprising the Dunaliella algae preparation of the present disclosure is a pharmaceutical composition.
- compositions comprising the Dunaliella aigae preparation of the present disclosure generally comprise a buffering agent, an agent which adjusts the osmolarity thereof, and optionally, one or more pharmaceutically acceptable carriers, excipients and/or additives as known in the art.
- Supplementary active ingredients can also he incorporated into the compositions.
- the carrier can he solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
- the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
- compositions of the invention as well as all Dunaliella algal preparations described above may be applicable for any of the neurodegenerative disorders discussed above, specifically , any conditions associated with aggregation of beta- amyloid, any of the tauopathies mentioned above and/or any early signs or symptoms associated therewith.
- Administering the Dunaliella aigae preparation of the present disclosure or any composition comprising the same may be performed by any route known in the art, enteral or parenteral. In some embodiment the method according to the present disclosure is wherein said Dunaliella aigae preparation is administered orally.
- the Dunaliella algal preparations of the invention or any composition thereof may be administered by oral, intravenous, intramuscular, subcutaneous, intraperitoneai, parenteral, transdermai, intravaginal, intranasal, mucosal, sublingual, topical, rectal or subcutaneous administration, or any combination thereof.
- the composition of the invention may be particularly suitable for oral or mucosal administration use.
- the usefulness of an oral formulation requires that the active agent or preparations of the invention be bio-available. Bioavailability of orally administered drugs can be affected by a number of factors, such as drug absorption throughout the gastrointestinal tract, stability of the drug in the gastrointestinal tract, and the first pass effect.
- an active agent or combination requires that the active agent have sufficient stability in the stomach and intestinal lumen to pass through the intestinal wall.
- Many drugs tend to degrade quickly in the intestinal tract or have poor absorption in the intestinal tract so that oral administration is not an effective method for administering the drug.
- Dunaliella algal preparations and composition of the invention may be suitable for mucosal administration, for example, pulmonary, buccal, nasal, intranasal, sublingual, rectal, vaginal administration and any combination thereof.
- compositions suitable for oral administration are typically solid dosage forms (e.g., tablets) or liquid preparations (e.g., solutions, suspensions, or elixirs).
- Solid dosage forms are desirable for ease of determining and administering dosage of active ingredient, and ease of administration, particularly administration by the subject at home.
- Liquid dosage forms also allow subjects to easily take the required dose of active ingredient.
- Liquid preparations can be prepared as a drink, or to be administered, for example by a nasal-gastric tube (NG tube).
- NG tube nasal-gastric tube
- Liquid oral pharmaceutical compositions generally require a suitable solvent or carrier system in which to dissolve or disperse the active agent, thus enabling the composition to be administered to a subject.
- a suitable solvent system is compatible with the active agent and non-toxic to the subject.
- liquid oral formulations use a water-based or an oil-based solvent.
- compositions of the invention can also optionally be formulated to reduce or avoid the degradation, decomposition, or deactivation of the active agents by the gastrointestinal system, e.g., by gastric fluid in the stomach.
- the compositions can optionally be formulated to pass through the stomach unaltered and to dissolve in the intestines, i.e., enteric coated compositions.
- Oral compositions can also be prepared using an excipient.
- Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
- Oral dosage forms comprising Dunaliella algal preparations are provided, wherein the dosage forms, upon oral administration, provide a therapeutically effective blood level of Dunaliella algal preparations to a subject. Also provided are dosage forms comprising said Dunaliella algal preparations wherein the dosage forms, upon administration, provide a therapeutically effective blood level of the Dunaliella algal preparations to a subject.
- the active combined compounds e.g., Dunaliella algal preparations
- the active combined compounds can be incorporated with excipients or carriers suitable for administration by inhalation or absorption, e.g., via nasal sprays or drops, or rectal or vaginal suppositories.
- the method according to the present disclosure is wherein said Dunaliella algae preparation is administered in combination with at least one additional agent.
- the methods of the present disclosure encompass combination therapy with at least one additional therapeutic agent, the type of additional therapeutic agent depending on the type of the disease or condition being treated.
- concurrent administration can mean one dosage form in which the two or more agents are contained whereas consecutive administration can mean separate dosage forms administered to the subject at different times and optionally by different routes of administration.
- the present disclosure further provides at least one Dunaliella algae preparation or any composition comprising thereof for use in a method for preventing, treating, ameliorating, reducing or delaying the onset of at least one of a neurodegenerative disease, a disorder associated with protein misfolding, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
- the at least one Dunaliella algae preparation or any composition comprising thereof for use according to the present disclosure is wherein said method results in amelioration or reduction of at least one symptom associated with at least one of a neurodegenerative disease, a disorder associated with protein misfolding, and cognitive decline in a subject in need thereof.
- the at least one Dunaliella algae preparation or any composition comprising thereof for use according to the present disclosure is wherein said symptom is at least one of short term memory impairment, long term memory impairment, impaired cognitive function, impaired learning function b -amyloids deposition, anxiety, depression or any combination thereof.
- the at least one Dunaliella algae preparation or any composition comprising thereof for use according to the present disclosure is wherein said method results in improvement of at least one of cognitive function, short term memory, long term memory, acquisition time and clearance of b-amyloids in a subject in need thereof.
- the at least one Dunaliella algae preparation or any composition comprising thereof for use according to the present disclosure is wherein said neurodegenerative disease is at least one of Alzheimer's disease, Parkinson's disease, Mild Cognitive impairment (MCI), Parkinson's disease with MCI, Huntington's disease, Lewy body disease, Amyotrophic lateral sclerosis (ALS), Prion disease, Motor neuron disease (MND), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Friedreich's Ataxia and any other neurodegenerative-related dementia or ataxia.
- MCI Mild Cognitive impairment
- ALS Amyotrophic lateral sclerosis
- MND Motor neuron disease
- SCA Spinocerebellar ataxia
- SMA Spinal muscular atrophy
- Friedreich's Ataxia any other neurodegenerative-related dementia or ataxia.
- the at least one Dunaliella algae preparation or any composition comprising thereof for use according to the present disclosure is wherein said neurodegenerati ve disease is Alzheimer’s disease.
- the at least one Dunaliella algae preparation or any composition comprising thereof for use according to the present disclosure is for preventing, treating, ameliorating, reducing or delaying the onset of cognitive decline, Specifically, as defined in connection with other aspects of the invention.
- the at least one Dimaiiella algae preparation or any composition comprising thereof for use according to the present disclosure is wherein said method further comprises administration of at least one additional agent.
- the at least one Dunaliella algae preparation or any composition comprising thereof for use according to the present disclosure is wherein said Dunaliella algae is Dunaliella bardawil.
- the at least one Dunaliella algae preparation or any composition comprising thereof for use according to the present disclosure is wherein said Dunaliella algae preparation is administered orally.
- the present disclosure provides the use of at least one Dunaliella algae preparation for the manufacture of a composition for preventing, treating, ameliorating, reducing or delaying the onset of at least one of a neurodegenerative disease, a disorder associated with protein misfolding, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
- the use according to the present disclosure is wherein said composition ameliorates or reduces at least one symptom associated with at least one of a neurodegenerative disease, a disorder associated with protein misfolding, and cognitive decline in a subject in need thereof.
- the use according to the present disclosure is wherein said symptom is at least one of short term memory impairment, long term memory impairment, impaired cognitive function, impaired learning function, b-amyloids deposition, anxiety, depression or any combination thereof.
- the use according to the present disclosure is wherein said composition improves at least one of cognitive function, short term memory, long term memory, acquisition time and clearance of b-amyloids in a subject in need thereof.
- said neurodegenerative disease is at least one of Alzheimer's disease, Pa rkinson's disease, Mild Cognitive Impairment (MCI), Parkinson's disease with MCI, Huntington’s disease Lewy body disease, Amyotrophic lateral sclerosis (ALS), Prion disease, Motor neuron disease (MND), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Friedreich’s Ataxia and any other neurodegenerative -related dementia or ataxia.
- MCI Mild Cognitive Impairment
- ALS Amyotrophic lateral sclerosis
- MND Motor neuron disease
- SCA Spinocerebellar ataxia
- SMA Spinal muscular atrophy
- the use according to the present disclosure is wherein said neurodegenerative disease is Alzheimer's disease.
- the use according to the present disclosure is for preventing, treating, ameliorating, reducing or delaying the onset of cognitive decline, specifically, as described herein in connection with other aspects of the invention.
- the use according to the present disclosure is wherein said Dunalieila algae preparation is administered in combination with at least one additional agent.
- Dunalieila algae is Dunalieila bardawil.
- the use according to the present disclosure is wherein said Dunalieila algae preparation is administered orally.
- the invention provides at least one Dunalieila algae preparation or any composition comprising thereof for use in a method for improving at least one of short term memory impairment, long term memory impairment, impaired cognitive function, impaired learning function, b-amyloids deposition in a subject in need thereof.
- the invention provides a method for improving at least one of short term memory impairment, long term memory impairment, impaired cognitive function, impaired learning function, b-amyloids deposition in a subject in need thereof.
- the method of the invention comprises the step of administering to the subject an effective amount of at least one Dunaliella algae preparation or any composition comprising thereof.
- the method according to the present disclosure results in improvement of at least one of cognitive function, short term memory, long term memory, acquisition time and clearance of b-amyloids in a subject in need thereof.
- the method according to the present disclosure is for preventing, treating, ameliorating, reducing or delaying the onset of cognitive decline.
- the method according to the present disclosure is wherein the Dunaliella algae is Dunaliella bardawil.
- the method according to the present disclosure is wherein the Dunaliella algae preparation is administered orally.
- compositions comprising “, “includes”, “including” , “ having “ and their conjugates mean “including but not limited to”. This term encompasses the terms “consisting of” and “consisting essentially of”.
- Consisting essentially of means that the composition or method may include additional ingredients and/or steps, hut only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method.
- range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should he considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to specifically disclose sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
- mice The following mice models were used:
- Tg2576 mice (Taconic Biosciences, Inc.) [27] and age-, sex-, and strain-matched C57B16 wild type (WT) ice.
- Tg2576 mice were self-bread in the Inventors’ animal facility.
- Tg2576 mice express the human 695-aa isoform of the amyloid precursor protein (APP) containing the Swedish double mutation (APPswe) driven by a hamster prion promoter.
- APP amyloid precursor protein
- APPswe Swedish double mutation driven by a hamster prion promoter.
- the Tg2576 model has been chosen since it is well-characterized. Furthermore, in this model, mice pathology develops relatively slowly, but rapidly enough for obtaining substantive findings, and thus this model is more pertinent to future human applications.
- mice have a relatively high survival rate (it has been previously shown that by 12 months approximately 75% of the mice survive). Since Tg2576 mice tend to he aggressive, animals were housed one animal per cage at the SPF (specific pathogen free) approved Sheba Animal Facility.
- mice 5XFAD mice. These mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). 5XFAD mice overexpress mutant human amyloid precursor protein (APP) with the Swedish (K670N, M671L), Florida (I716V), and London (V717I) mutations along with mutant human presenilin 1 (PS1) with two FAD mutations (M146L and L286V).
- APP human amyloid precursor protein
- PS1 mutant human presenilin 1
- Tg2576 mice were treated for 10 months (from 2 to 12 months of age). Weight was obtain every 2 months. Two weeks before sacrifice, behavioral tests were performed (including open field, measuring general locomotors activity, anxiety and willingness to explore, Y- maze spontaneous alternation, measuring exploratory and spatial working memory and Barnes maze, measuring long term memory). Animals were sacrificed at the age of 12 months.
- mice 5XFAD mice were treated for 6 months (from approximately 1 to 6 months of age). Weight was monitored monthly. Two weeks before sacrifice, behavioral tests were performed(including open field, measuring general locomotors activity, anxiety and willingness to explore, Y-maze and Novel object recognition, measuring exploratory and spatial working memory Novel object recognition and Barnes maze, measuring long term memory). Animals were sacrificed at the age of 7 months.
- Tg2576 and WT control mice were randomly allocated into two groups each (12 mice in each group) and were fed for 10 months on 8% Dunaliella algae powder diet (also referred to herein as the“ Dunaliella dief) or on control diet.
- Low-fat chow diet (18% protein, 5% fat; TD2018, Harlan Teklad) was used as basic (control) diet.
- To prepare the food 750 ml distilled hot water were mixed with 28 gram gelatin until the solution was clear. Then, 1 ki logram powder of the low-fat chow- diet (control) or low-fat chow diet with Dunaliella algae powder (80 g/kg feed) were well mixed with the warm gelatin solution.
- the food was divided into tablets and stored in -80°C (thereby, except for the Dunaliella algae powder content, the two diets had essentially the same content and texture). Feed was replaced every two days to mini mize oxidation and degradation of the ingredients.
- Dunaliella bardawil (hereinafter "Db", Nikken Sohonsha Corporation) was grown and cultivated in large body open salt water ponds of 50,000 m 2 to obtain algae comprising approximately 5-8% by weight of b-carotene (hereinafter "BC") at an approximately 1 :1 (by weight) ratio of 9-cis and all-trans isomers of BC, or greater than 1 :1 ratio of 9-cis and all-trans isomers of BC.
- BC b-carotene
- the algae were harvested by dislodging centrifuges into a concentrated paste. The paste was washed to remove the salt and sterilized, and then spray dried to yield Dunaliella bardawil powder comprising approximately 5-8% BC and less than 5% moisture.
- the powder was packaged in capsules of 250-500 mg algae containing BC (5-8%) together with all of the other natural components of the algae (e.g. protein, lipids, carbohydrates).
- the BC of the capsules retains the original ratio of isomers.
- the capsules were packaged in vacuum closed blisters which have a shelf life of up to three years.
- the Dunaliella algae powder preparation used herein contains about 7% b-carotene composed of 40%-50% 9-cis b carotene (9 C) and 50%-60% all trans b carotene.
- the model consists of a monolayer of endothelial cells, obtained from primary cultures of endothelial cells established from freshly collected porcine brain forming tight junctions that are grown on a microporous membrane filter culture insert (the“Luminal side” in Figure 6) and of a monolayer of glial ceils extracted from new' horn rats’ cortex that are seeded at the abluminal side of the filter (the“Abluminal side” in Figure 6).
- Low density lipoprotein (LDL, 100 m ⁇ , 1,600 pg/ml from a healthy volunteer) was added to the luminal side. Cells were then incubated for 24 hours and thereafter samples were collected and analyzed using HPLC.
- the model was prepared by first seeding the glial cells on the abluminal side of a filter, a week later, the above -described endothelial cells were seeded on the luminal side of the filter and two days later LDL w'as added to the luminal side of the filter as indicated above.
- b- earotene in order to examine whether b- earotene can be converted into retinol in the brain different cells are isolated from the brain (hippocampal neurons and astrocytes) and the expression (mRNA and protein) of b-carotene 15,15’-monooxygenase 1 (BCMOl) and its activity (retinol formation) in the cells are measured.
- BCMOl b-carotene 15,15’-monooxygenase 1
- LDL was obtained from healthy volunteers by sequential ultracentrifugation (density, 1.063 g/ml), and the concentration was determined by the Lowry method.
- the Barnes maze is a spatial- learning task that allows ani als to use spatial cues to locate a means of escape from a mildly aversive environment [29]. Both the Barnes maze and the Morris water maze (MWM) examine spatial memory, but unlike the MWM, the Bames maze lack the stress induced by swimming.
- mice are placed in a cylindrical dark chamber at the center of a circular table containing 18 holes around the edge. After 10 seconds the chamber is lifted and the animal receive negative reinforcements, such as bright light, loud buzzer, an exposed environment, and air jets [30, 31], in order to motivate them to find the escape hole leading to a drawer underneath one of the holes. The animal explores the maze until it finds and enters the escape box in 180 seconds.
- the mouse fails to enter the escape box within 180 seconds it is picked up gently by the base of the tail, placed in the palm of the hand and let down at the side of the escape hole.
- the mouse enters the escape box and remains there for an additional 60 seconds before it is removed and taken to its home cage. After each trial, the maze and escape box are cleaned thoroughly with a 10% alcohol solution to remove odors.
- the escape latency is the duration of time between removal of the cylinder and the animal's entry into the escape box.
- the animals will be subjected to 4 trials per day for 4 days. On the fifth day a recall test will be performed. The escape box will be removed and the same parameters will be measured. A second recall test will be performed on day 12 to assess long term retention, of where the target escape box was located.
- the Y maze spontaneous alternation is a behavioral test for measuring the willingness of rodents to explore new environments.
- Y-maze test allows assessment of spatial working memory that is dependent upon the hippocampus [32].
- the mouse at the age of 12 months is placed at the end of one arm of the Y -maze and allowed to freely explore the maze for 6 minutes.
- Alternation will be determined from successive entries into the three arms on overlapping triplet sets in which three different arms are entered.
- An actual alternation will be defined as entries into all three arms consecutively (i.e., ABC, CAB, or BCA but not BAB).
- An entry will be defined as placing all four paws within the boundaries of the arm.
- the maze arms will be cleaned with 30% ' ethanol between tasks to remove residual odors.
- the open field test is a commonly used qualitative and quantitative measure of general locomotor activity, anxiety and willingness to explore in rodents.
- the mouse is placed at the comer of the test box and allowed to freely explore the area for 5 minutes.
- Four measurements are recorded: (a) total path, which indicates general activity and exploratory behavior; (b). percentage of ceil used, which indicates general activity and exploratory behavior; (c). percentage of time moving, which indicates anxiety and general activity; and (d). sum center, which is the sum of the time the mouse spends in the arena's center that indicates anxiety.
- the test is recorded and analyzed. Higher scores in each of these measurements reflect lower anxiety and higher locomotor activity.
- the mouse is presented with two identical objects for 5 minutes, 3 hours later for short term memory, 24 hours for long term memory; one of the objects is replaced by a different one.
- the amount of time taken to explore the new object provides an index of recognition memory.
- the soluble fraction was designated "Ab8qG.
- the latter (insoluble) fraction was designated "Ab ⁇ hbo ⁇ ”.
- Ab ⁇ 42 was measured by sandwich ELISA (WAKO, Osaka, Japan) according to the manufacturer’s instructions.
- RNA 70 ng was used to assess the expression of 43 mouse genes that were possibly Alzheimer's' (AD) related and RXR affected, divided to different pathways: Inflammation, lipidation, BBB and synaptic plasticity. Inclusion criteria: Values with Ratio higher than 1.4 or lower than 0.7 and p ⁇ 0.05 were considered significant and used for statistical analysis.
- mice hippocampus were lysed using PARISTM Kit (invitrogenTM) or RIPA buffer.
- the protein lysis was denatured for 5 minutes at 95 °C in SDS-PAGE sample buffer and separated by 12% SDS-PAGE. Proteins were transferred onto nitrocellulose membranes.
- Membranes were incubated with blocking buffer for 1 hour prior to incubation with primary antibodies as required: anti-GFAP ( Abeam ab53554, 1 :500), anti-Synaptophysin (abeam [YE269] ab 32127, 1 : 10000), PBR (Santa Cruz, EL- 169: sc-20120, 1 : 100), a Tubulin (Santa Cruze, B-7:se-5286, 1: 100), GAPDH (1 :150).
- Proteins were visualized using IRDye® 680CW Goat anti-Rahbit (1 : 15000) and IRDye® 680CW Donkey anti- Mouse (1 : 15000), IRDye®800CW Donkey anti-Goat (1 : 15000), IRDye® 800CW Goat anti-Mouse (1 : 15000) secondary antibodies. Images were captured with the Odyssey system (CLX). The proteins were quantified using Image Studio Ver 5.2 software.
- mice Brain tissue were homogenized with 2 mL ethanol containing 10 mM butylated hydroxy toluene which was followed by the addition of 2 mL hexane and 1 mL of Double- distilled water (DDW). The samples were mixed and centrifuged for 5 minutes at lOOOx g. The hexane layer was separated and dried under a stream of N2.
- DDW Double- distilled water
- Dried samples were suspended in 100 pL methyl-tert-butyl -ether, and pc concentrations were determined by reverse phase HPLC on a YMC C30 column (GT995031546QT, 150 x 4.6, 3 pm particle size; YMC Inc., Allentown, PA, USA) with methanol/methyl-tert-butyi-ether/water 1.5% ammonium acetate as the mobile phase at a flow rate of 1 mL/min. bo was detected by monitoring its absorbance at 450 nm and by comparison with the retention times of authentic standards.
- Tg2576 mice and thirty C57B1/6 WT mice were randomly allocated at the age of two months into two groups each and placed for TO months on two different diet types: regular chow diet and Dunaliella algae powder preparation diet (80 gr of 9-cis b -carotene rich Dunaliella algae powder in 1 Kg chow diet feed) in a 2 (mice type) by 2 (diet type) design.
- Tg2576 mice had a lower survival rate in comparison to WT mice.
- BBB blood brain barrier
- glial and endothelial cells were incubated for 24 hours in the presence of LDL (inherently containing ail-trans and 9-cis b-carotene) and then samples were collected and carotenoids were extracted and analyzed using HPLC.
- LDL inherently containing ail-trans and 9-cis b-carotene
- the "BBB in-vitro model" described above is also used for assessment of crossing the BBB by carotenes in LDL extracted from a healthy volunteer that are administered with the Dunaiieila algae diet described herein.
- mice brain carotenoids were extracted and then measured using HPLC. As shown in Figure 7, while in the control diet there were negligible tissue levels of 9-cis b Carotene, in mice fed with the Dunaiieila diet there were much higher levels of both a!i-trans and 9-cis b Carotene. In particular, in the brain of the group fed on Dunaiieila diet, the carotenoid level was significantly higher than in the control groups (Fig. 7C). The results suggest that exposure to a diet rich in carotenoids lead to its accumulation in body tissues, specifically, liver (Fig. 7A), fat (Fig. 7B), including the brain(Fig. 7C), and thus may be a source for brain carotenoids which in turn may be a source for brain retinoids. EXAMPLE 7
- AD Alzheimer's disease
- Amyloid beta, b- amyloids neurotic plaques
- retinol and b-carotene potentially inhibit amyloid b formation.
- the Inventors next examined the effect of the Dunaliella algae powder preparation diet on .46 peptides levels (insoluble and soluble) in the hippocampus of the assayed Tg2576 mouse. For quantitati ve assessment of hippocampal formation of A6 peptides, total A61-42 was measured by sandwich ELISA as detailed above.
- the treatment described herein may affect Alzheimer’s disease via three possible (hypothetical) pathways: via ACB transporters and lipids, BBB, plasticity and inflammation.
- RXR and downstream genes namely apoE, ABCA1 and ABCG1 were assessed in the hippocampus of animals fed on the Dunaliella algae powder preparation rich diet or control diet, and in addition, expression of the above genes was assayed in vitro , in hippocampal neurons culture.
- Total RNA was extracted and analyzed by RT-PCR. Analysis of protein expression was measured by quantitative Western blotting.
- dietary 9-cis b-carotene can cross the BBB and may be converted to retinoids within the brain cells by enzymatic activity (e.g. of the enzyme BCMOl).
- a schematic diagram showing a proposed mechanism of action of dietary 9-cis b-carotene is shown in Figure 12. Without wishing to be bound by theory, increased levels of retinoids intensify clearance of Ab and consequently improve cognitive function.
- the Dunaliella algae preparation described herein may be available rich source for 9-cis b-carotene and other isomers thereof or substances.
- Novel object recognition in 5XFAD mice fed on Dunaliella algae powder preparation In the test described herein, the mouse is presented with two identical objects for 5 minutes, and 3 hours later (for short term memory) and 24 hours late one of the objects is replaced by a different one (for long term memory). The length of time taken to explore the new object provides an index of recognition memory. This test was performed in 5XFAD mice fed on Dunaliella algae powder preparation, as described above, aiming to explore the effect of the Dunaliella diet on memory.
- the Dunaliella algae powder preparation diet improved short term memory in 5XFAD mice as deduced from the higher percentage of new objection recognition in the 5XFAD mice fed on the Dunaliella algae powder preparation diet (5XFAD Duna. Prep.).
- the Dunaliella algae powder preparation diet also improved long term memory in 5XFAD mice.
- Amyloid b was extracted from 5xFAD ice hippocampus as described in the method section above, in order to evaluate the level thereof and to examine the possible effect of the Dunaliella algae powder preparation.
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- Alternative & Traditional Medicine (AREA)
- Microbiology (AREA)
- Hospice & Palliative Care (AREA)
- Medical Informatics (AREA)
- Mycology (AREA)
- Psychiatry (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
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Abstract
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US17/594,084 US20220175860A1 (en) | 2019-04-01 | 2020-04-01 | Dunaliella alga preparation for prevention and/or treatment of a neurodegenerative disease, a disorder associated with protein misfolding and cognitive decline |
EP20785151.0A EP3946398A4 (en) | 2019-04-01 | 2020-04-01 | Dunaliella alga preparation for prevention and/or treatment of a neurodegenerative disease, a disorder associated with protein misfolding and cognitive decline |
CN202080040585.3A CN113905750A (en) | 2019-04-01 | 2020-04-01 | Dunaliella algae preparations for preventing and/or treating neurodegenerative diseases, diseases associated with protein misfolding, and cognitive decline |
JP2021560202A JP2022533900A (en) | 2019-04-01 | 2020-04-01 | Dunaliella algal preparations for the prevention and/or treatment of neurodegenerative diseases, protein misfolding related disorders and cognitive decline |
IL286860A IL286860A (en) | 2019-04-01 | 2021-09-30 | Dunaliella alga preparation for prevention and/or treatment of a neurodegenerative disease, a disorder associated with protein misfolding and cognitive decline |
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US201962827308P | 2019-04-01 | 2019-04-01 | |
US62/827,308 | 2019-04-01 |
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WO2020202152A1 true WO2020202152A1 (en) | 2020-10-08 |
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PCT/IL2020/050393 WO2020202152A1 (en) | 2019-04-01 | 2020-04-01 | Dunaliella alga preparation for prevention and/or treatment of a neurodegenerative disease, a disorder associated with protein misfolding and cognitive decline |
Country Status (6)
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US (1) | US20220175860A1 (en) |
EP (1) | EP3946398A4 (en) |
JP (1) | JP2022533900A (en) |
CN (1) | CN113905750A (en) |
IL (1) | IL286860A (en) |
WO (1) | WO2020202152A1 (en) |
Citations (1)
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US20050063991A1 (en) * | 2003-09-24 | 2005-03-24 | Aviv Shaish | Therapeutic uses of dunaliella powder |
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WO2009133552A2 (en) * | 2008-04-29 | 2009-11-05 | Nikken Sohonsha Corporation | Methods of treating ophthalmic disorders |
DE212015000033U1 (en) * | 2014-03-19 | 2016-08-26 | U.S. Nutraceuticals, Llc D/B/A Valensa International | Therapeutic astaxanthin and phospholipid composition |
CN104095897A (en) * | 2014-06-24 | 2014-10-15 | 伏思思 | Maca dunaliella salina compound preparation |
CN106071768A (en) * | 2016-07-12 | 2016-11-09 | 淳安县禾宸农产品有限公司 | A kind of marine active substance and fruit and vegerable mixed type cake |
-
2020
- 2020-04-01 JP JP2021560202A patent/JP2022533900A/en active Pending
- 2020-04-01 WO PCT/IL2020/050393 patent/WO2020202152A1/en active Application Filing
- 2020-04-01 CN CN202080040585.3A patent/CN113905750A/en active Pending
- 2020-04-01 EP EP20785151.0A patent/EP3946398A4/en active Pending
- 2020-04-01 US US17/594,084 patent/US20220175860A1/en active Pending
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2021
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050063991A1 (en) * | 2003-09-24 | 2005-03-24 | Aviv Shaish | Therapeutic uses of dunaliella powder |
Non-Patent Citations (1)
Title |
---|
OLASEHINDE ET AL.: "Therapeutic potentials of microalgae in the treatment of Alzheimer's disease", MOLECULES, vol. 22, 18 March 2017 (2017-03-18), pages 480, XP055642392 * |
Also Published As
Publication number | Publication date |
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JP2022533900A (en) | 2022-07-27 |
EP3946398A1 (en) | 2022-02-09 |
IL286860A (en) | 2021-12-01 |
EP3946398A4 (en) | 2023-01-18 |
CN113905750A (en) | 2022-01-07 |
US20220175860A1 (en) | 2022-06-09 |
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