AU2016363511A1 - Method for improving cognition - Google Patents

Method for improving cognition Download PDF

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AU2016363511A1
AU2016363511A1 AU2016363511A AU2016363511A AU2016363511A1 AU 2016363511 A1 AU2016363511 A1 AU 2016363511A1 AU 2016363511 A AU2016363511 A AU 2016363511A AU 2016363511 A AU2016363511 A AU 2016363511A AU 2016363511 A1 AU2016363511 A1 AU 2016363511A1
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Ellen Siobhan Mitchell
Amy Melissa POOLER
Claus Rieker
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Societe des Produits Nestle SA
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Nestle SA
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Abstract

A medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) for use in improving cognition which has been impaired by UBE3A deficiency.

Description

FIELD OF THE INVENTION
The present invention relates to the use of medium-chain triglycerides (MCTs) or mediumchain fatty acids (MCFAs) for improving cognition which has been impaired by UBE3A deficiency, for example in subjects with cognitive disorders or aging subjects. In particular, the invention relates to the use of MCTs for treating Angelman syndrome and/or Prader-Willi syndrome.
BACKGROUND TO THE INVENTION
UBE3A encodes a ubiquitin E3 ligase that targets specific proteins for degradation (de Bie, P. and Ciechanover A. (2011) Cell Death Differ. 18: 1393-402) and is essential for normal cognitive function due to its role as the major ubiquitin binding protein in the synapse. UBE3A’s ability to clear synaptic signalling proteins (e.g. Arc) enables homeostatic regulation of synaptic growth. Loss of UBE3A expression disrupts long term potentiation via the transcription factor Arc, and also gives rise to dysregulation of glutamate receptor activity. Over time an absence of UBE3A causes synaptic degradation and seizures.
UBE3A dysfunction has been linked to a reduction of cognitive function. In particular, it has been implicated as causative of certain disorders, such as Angelman syndrome.
Angelman syndrome is a genetic disorder that results in functionally severe developmental delay, which is characterised by severe speech impairment, movement or balance disorders and odd behaviour.
There are estimated to be between 1000 and 5000 Angelman syndrome cases in the US and Canada alone.
Angelman syndrome-associated seizures may sometimes be controlled using drugs or with the ketogenic diet, and a modified Atkins diet has also been tried in this context, however the success of these approaches is variable.
A recent study showed that certain cancer drugs may induce UBE3A, and use of a specific cancer drug in a mouse model of Angelman syndrome showed behavioural improvement (Huang, H.S. et al. (2011) Nature 481: 185-9). However, the drug is not brain bioavailable, and furthermore such cancer drugs inhibit cell division and are not appropriate for long term therapy of Angelman syndrome patients.
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Several clinical trials for the treatment of Angelman syndrome have been performed using antibiotics, methylating nutrients and antioxidants which have all shown little effect on cognition or behaviour. Accordingly, there are currently no reliable treatments for the disease.
Prader-Willi syndrome is a genetic disorder related to Angelman syndrome. Prader-Willi syndrome patients experience significant cognitive, neurological, endocrine and behavioural abnormalities. Furthermore, patients may have difficulty in feeding in infancy due to hypotonia, leading to growth retardation, however they become obese later in life due to an uncontrollable craving for food.
Approximately 400,000 patients worldwide have Prader-Willi syndrome.
Although behavioural therapy and other treatments may reduce the effects of Prader-Willi syndrome, there are no effective treatments for this disease.
Accordingly, there remains a significant need for methods for up-regulating UBE3A expression in the brain to improve cognitive function. Additionally, there remains a significant need for methods for the treatment of the diseases such as Angelman syndrome and Prader-Willi syndrome, in particular treatments that address their associated cognitive impairments.
SUMMARY OF THE INVENTION
The inventors have unexpectedly shown that a medium-chain triglyceride (MCT) diet increases UBE3A expression in the brain. Moreover, the inventors have shown that an MCT diet improves cognitive performance.
Specifically, the inventors found that a diet comprising C8 and/or C10 triglycerides increases UBE3A protein expression in the cortex and hippocampus compared to a control diet. The MCT diet was found to improve UBE3A expression levels in the aged subjects to levels that are comparable or superior to that found in young subjects.
Importantly, the inventors further found that the MCT diet gives rise to improved cognitive function in the rat models. Without wishing to be bound by theory, the observed improvements in cognition may result from the increased levels of UBE3A in the brain causing a downregulation of basal levels of the transcription factor Arc and/or improved control of Arc homeostasis.
Accordingly, in one aspect the invention provides a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) for use in improving cognition which has been impaired by UBE3A deficiency.
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In one aspect the invention provides a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) for use in improving cognition by up-regulating UBE3A expression.
The UBE3A deficiency may, for example, result from insufficient UBE3A activity in the brain, for example as a result of inadequate levels of expression of UBE3A or expression of an inactive form of the UBE3A enzyme. UBE3A deficiencies may arise with increasing age and/or may be readily assessed by the skilled person using, for example, molecular genetic testing that suggests deficient expression or function (e.g. as described herein).
In one embodiment, the cognition is improved through the use of the MCTs of the invention. In another embodiment, the cognition is improved through the use of the MCFAs of the invention.
In one embodiment, the MCT or MCFA improves cognition in a subject with a cognitive disorder. The cognitive disorder may be associated with or resulting from insufficient UBE3A activity in the brain.
In one embodiment, the MCT or MCFA improves cognition in a subject with Angelman syndrome or Prader-Willi syndrome.
In another embodiment, the MCT or MCFA improves cognition in an aging subject. The aging subject may, for example, be a human subject over the age of 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 years old.
In another aspect, the invention provides a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) for use in treating Angelman syndrome or Prader-Willi syndrome.
The MCT or MCFA may, for example, improve cognition in a subject with Angelman syndrome or Prader-Willi syndrome, and/or reduce the occurrence of seizures.
The MCT for use according to the invention may, for example, comprise three fatty acid moieties, each of which independently has between 6-12, 6-11,6-10, 7-12, 7-11, 7-10, 8-12, 8-11 or 8-10 carbon atoms. Preferably, the MCT for use according to the invention comprises three fatty acid moieties, each of which independently has between 8-10 carbon atoms.
The MCT for use according to the invention may, for example, comprise three fatty acid moieties, each of which independently has only 6, 7, 8, 9, 10, 11 or 12 carbon atoms. Preferably, the MCT for use according to the invention comprises three fatty acid moieties, each of which independently has only 8 or 10 carbon atoms, i.e. preferably, the MCT for use
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The MCT for use according to the invention may be a homotriglyceride (i.e. all of the fatty acid moieties of the MCT may be of the same identity, for example a C8 homotriglyceride may comprise 3 octanoic acid moieties).
In one embodiment, the MCT for use according to the invention comprises three fatty acid moieties with only 8 carbon atoms. In a preferred embodiment, all three fatty acid moieties are octanoic acid moieties.
In another embodiment, the MCT for use according to the invention comprises three fatty acid moieties with only 10 carbon atoms. In a preferred embodiment, all three fatty acid moieties are decanoic acid moieties.
The MCFAfor use according to the invention may, for example, have between 6-12, 6-11, 610, 7-12, 7-11,7-10, 8-12, 8-11 or 8-10 carbon atoms. Preferably, the MCFA for use according to the invention has between 8-10 carbon atoms.
The MCFA for use according to the invention may, for example, have only 6, 7, 8, 9, 10, 11 or 12 carbon atoms. Preferably, the MCFA for use according to the invention has only 8 or 10 carbon atoms, i.e. preferably, the MCFAfor use according to the invention is a C8 and/or C10 fatty acid.
In one embodiment, the MCFA for use according to the invention has only 8 carbon atoms. In a preferred embodiment, the MCFA is octanoic acid.
In another embodiment, the MCFA for use according to the invention has only 10 carbon atoms. In a preferred embodiment, the MCFA is decanoic acid.
The MCT or MCFA for use according to the invention may be administered enterally or parenterally. Preferably, the MCT or MCFA is administered enterally. For example, the MCT or MCFA may be administered in the form of a food stuff or a supplement.
The MCT or MCFA for use according to the present invention may, for example, be administered in a diet comprising about 1-10%, 2-10%, 3-10%, 4-10%, 5-10%, 6-10%, 7-10% or 8-10% (w/w) of the MCT and/or MCFA of the invention. The MCT or MCFAfor use according to the present invention may, for example, be administered in a diet comprising about 1.5-8% (w/w) of the MCT and/or MCFA of the invention. Preferably, the MCT or MCFA for use
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PCT/EP2016/078313 according to the present invention is administered in a diet comprising about 3-8% (w/w) of the MCT and/or MCFA of the invention.
In another aspect, the invention provides a composition comprising a medium-chain triglyceride (MCT) and/or medium-chain fatty acid (MCFA) of the invention for use in improving cognition which has been impaired by UBE3A deficiency.
The UBE3A deficiency may, for example, result from insufficient UBE3A activity in the brain, for example as a result of inadequate levels of expression of UBE3A or expression of an inactive form of the UBE3A enzyme.
In one embodiment, the composition comprises the MCTs of the invention. In another embodiment, the composition comprises the MCFAs of the invention.
In one embodiment, the composition improves cognition in a subject with a cognitive disorder. The cognitive disorder may be associated with or result from insufficient UBE3A activity in the brain.
Preferably, the composition improves cognition in a subject with Angelman syndrome or Prader-Willi syndrome.
In another embodiment, the composition improves cognition in an aging subject. The aging subject may, for example, be a human subject over the age of 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 years old.
In another aspect, the invention provides a composition comprising a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) of the invention for use in treating Angelman syndrome or Prader-Willi syndrome.
The composition may, for example, improve cognition in a subject with Angelman syndrome or Prader-Willi syndrome, and/or reduce the occurrence of seizures.
The MCT or MCFA may be as described herein.
In one embodiment, the composition of the invention comprises a homotriglyceride comprising C8 fatty acid moieties (i.e. fatty acid moieties with 8 carbon atoms), preferably octanoic acid moieties.
In one embodiment, the composition of the invention comprises a homotriglyceride comprising C10 fatty acid moieties (i.e. fatty acid moieties with 10 carbon atoms), preferably decanoic acid moieties.
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In one embodiment, the composition comprises a homotriglyceride comprising C8 fatty acid moieties (i.e. fatty acid moieties with 8 carbon atoms), preferably octanoic acid, and a homotriglyceride comprising C10 fatty acid moieties (i.e. fatty acid moieties with 10 carbon atoms), preferably decanoic acid.
In one embodiment, the ratio of C8:C10 fatty acid-comprising homotriglycerides is about 90:10 (w/w) to about 10:90 (w/w). In another embodiment, the ratio of C8:C10 fatty acid-comprising homotriglycerides is about 80:20 (w/w) to about 20:80 (w/w). In another embodiment, the ratio of C8:C10 fatty acid-comprising homotriglycerides is about 70:30 (w/w) to about 30:70 (w/w). In another embodiment, the ratio of C8:C10 fatty acid-comprising homotriglycerides is about 60:40 (w/w) to about 40:60 (w/w). In another embodiment, the ratio of C8:C10 fatty acidcomprising homotriglycerides is about 50:50 (w/w) to about 40:60 (w/w).
In one embodiment, the ratio of C8:C10 fatty acid-comprising homotriglycerides is about 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 or 10:90 (w/w). Preferably, the ratio of C8:C10 fatty acid-comprising homotriglycerides is about 50:50 or 40:60 (w/w).
The composition for use according to the present invention may, for example, comprise about 1-100%, 1-50%, 1-30%, 1-10%, 2-10%, 3-10%, 4-10%, 5-10%, 6-10%, 7-10% or 8-10% (w/w) of the MCT and/or MCFA of the invention. The composition for use according to the present invention may, for example, comprise about 1.5-8% (w/w) of the MCT and/or MCFA of the invention. Preferably, the composition for use according to the present invention comprises about 3-8% (w/w) of the MCT and/or MCFA of the invention.
In one embodiment, the composition for use according to the invention is in the form of a powder. The powder may, for example, be a spray-dried powder or a freeze-dried powder.
In one embodiment, the composition for use according to the invention is in the form of an emulsion. The emulsion may, for example, be an oil-in-water emulsion.
Preferably, the composition for use according to the invention is in the form of a food stuff. Preferably, the food stuff is a human food stuff. The food stuff may be enriched with the MCT and/or MCFA of the invention.
In one embodiment, the composition for use according to the invention is in the form of a beverage, mayonnaise, margarine, low fat spread, dairy product, cheese spread, processed cheese, dairy dessert, flavoured milk, cream, fermented milk product, cheese, butter, condensed milk product, ice cream mix, soya product, pasteurised liquid egg, bakery product, confectionary product, confectionary bar, chocolate bar, high fat bar, liquid emulsion, spray6
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In another aspect, the invention provides the use of a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) of the invention for the manufacture of a medicament or composition for improving cognition which has been impaired by UBE3A deficiency. As described herein, the improvement in cognition may be in a subject with a cognitive disorder; a subject with Angelman syndrome or Prader-Willi syndrome; or an aging subject.
In another aspect, the invention provides the use of a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) of the invention for the manufacture of a medicament or composition for treating Angelman syndrome or Prader-Willi syndrome.
In another aspect, the invention provides a method of improving cognition in a subject which has been impaired by UBE3A deficiency comprising administering an effective amount of a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) of the invention or a composition of the invention to the subject. As described herein, the improvement in cognition may be in a subject with a cognitive disorder; a subject with Angelman syndrome or PraderWilli syndrome; or an aging subject.
In another aspect, the invention provides a method of treating Angelman syndrome or PraderWilli syndrome in a subject comprising administering an effective amount of a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) of the invention or a composition of the invention to the subject.
In another aspect, the invention provides a method of increasing UBE3A expression in a subject comprising administering an effective amount of a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) of the invention or a composition of the invention to the subject.
The method may, for example, increase UBE3A expression in the brain, but not in other tissues, for example not in the periphery.
DESCRIPTION OF THE DRAWINGS
Figure 1
Social recognition at baseline and 8 weeks after chronic medium-chain triglyceride (MCT) supplemented diet (5% by weight) in 24 month-old rats.
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Cognitive performance was increased in MCT-treated groups compared to those fed a control diet (one way ANOVA analysis). F=10.56; **, p<0.01 MCT10 (capric triglyceride) vs control (CON); *, p<0.05 MCT8 (caprylic triglyceride) vs control (CON).
Figure 2
UBE3a protein expression in the cortex of 24 month-old rats (one way ANOVA analysis). F=4.891; *, p<0.05 MCT8 or MCT10 compared to control (CTL).
Figure 3 mRNA expression of transcripts associated with UBE3A activity in the prefrontal cortex of 24 month-old rats in medium-chain triglyceride (MCT) treated and control (CON) groups; groups: MCT10 (capric triglyceride), MCT8 (caprylic triglyceride), “mix” (40:60 ratio of MCT8 and MCT10. For Arc hipp, the MCT 10 treated group decreased compared to the control group, p<0.05 (one way ANOVA). For Snrpn hipp, the “mix” MCT8 and MCT10 treated group increased compared to the control group, p<0.05 (one way ANOVA).
Figure 4
Object recognition after chronic (8 week) medium-chain triglyceride (MCT) treatment. Cognitive performance was increased in MCT-treated groups (compared to those fed a control diet) (one way ANOVA analysis). F=10.56; * p<0.05 MCT10 (capric triglyceride) vs control (con).
Figure 5
UBE3a protein expression in the cortex of 17 month-old rats. F=4.891; *, p<0.05 MCT10 compared to control (CTL). UBE3A protein expression in the MCT10-treated rats is comparable to that of young (6 month-old) rats (CTL young).
Figure 6
Comparison of UBE3a protein expression in the cortex and liver of 1 month-old mice which were neonatally exposed to MCT via maternal consumption of MCT-supplemented diet (via placental delivery of MCT and via breast milk). Student’s t-test analysis: *, p<0.05 compared to control (Con).
Figure 7
Morris Water Maze performance of 2 month-old mice which were neonatally exposed to MCT via maternal consumption of MCT-supplemented diet (via placental delivery of MCT and via 8
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PCT/EP2016/078313 breast milk). Time spent in the platform quadrant indicates level of memory retention after 4 days maze-training. Female C8-OTG (caprylic triglyceride) mice spent significantly more time in the Target quadrant (where the platform was) than 25% chance level t(11) = 3.533, p =0.005.
Figure 8
Fold change in UBE3Aand glutamate receptor, ionotropic, N-methyl D-aspartate 1 (GRIN1) levels in human iCell neurons following treatment with octanoic or decanoic acid, with respect to control (DMSO) treatment, *p<0.05 (one way ANOVA).
Figure 9
MCT C8/C10 significantly increased the expression of UBE3A (normalized to beta-actin) using t-test; ** = p<0.05.
Figure 10
Octanoic acid/C8 and decanoic acid/C10 significantly increased UBE3A expression levels in the absence of glucose in the media (normalized to the beta-actin). Decanoic acid/C10 at 5μΜ and 50μΜ significantly increased UBE3A expression in the presence of low glucose at 4mM (normalized to the beta-actin). One-way ANOVA; * = p<0.1; ** = p<0.05; *** = p<0.01
DETAILED DESCRIPTION OF THE INVENTION
Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O’D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical 9
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Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference.
In one aspect, the invention provides a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) for use in improving cognition which has been impaired by UBE3A deficiency.
In another aspect, the invention provides a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) for use in treating Angelman syndrome or Prader-Willi syndrome.
Medium-chain triglycerides (MCTs) and medium-chain fatty acids (MCFAs)
A triglyceride (also known as a triacylglycerol or a triacylglyceride) is an ester that is derived from glycerol and three fatty acids.
A medium-chain triglyceride (MCT) is a triglyceride in which all three fatty acid moieties are medium-chain fatty acid moieties. Medium-chain fatty acids (MCFAs) are fatty acids that have 6-12 carbon atoms. Preferably, the fatty acid moieties are aliphatic fatty acid moieties. Accordingly, a MCT is an ester formed from a glycerol molecule and three medium-chain fatty acid molecules.
The term “fatty acid moiety” refers to the part of the MCT that originates from a fatty acid in an esterification reaction with glycerol. For example, an esterification reaction between glycerol and only octanoic acid would result in a MCT with octanoic acid moieties.
The MCT for use according to the invention may, for example, comprise three fatty acid moieties, each of which independently has between 6-12, 6-11, 6-10, 7-12, 7-11,7-10, 8-12, 8-11 or 8-10 carbon atoms. Preferably, the MCT for use according to the invention comprises three fatty acid moieties, each of which independently has between 8-10 carbon atoms.
The MCT for use according to the invention may comprise three fatty acid moieties, each of which independently has only 6, 7, 8, 9, 10, 11 or 12 carbon atoms. Preferably, the MCT for use according to the invention comprises three fatty acid moieties, each of which independently has only 8 or 10 carbon atoms, i.e. preferably, the MCT for use according to the invention comprises only C8 and/or C10 fatty acid moieties. Preferably, the fatty acid moieties are octanoic acid and/or decanoic acid moieties.
The MCT for use according to the invention may be a homotriglyceride (i.e. all of the fatty acid moieties of the MCT may be of the same identity, for example a C8 homotriglyceride may comprise 3 octanoic acid moieties).
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Preferably, the MCT for use according to the invention comprises three fatty acid moieties with only 8 carbon atoms. In a preferred embodiment, all three fatty acid moieties are octanoic acid moieties.
Preferably, the MCT for use according to the invention comprises three fatty acid moieties with only 10 carbon atoms. In a preferred embodiment, all three fatty acid moieties are decanoic acid moieties.
Octanoic acid (also known as caprylic acid) is a saturated fatty acid of the formula CH3(CH2)6COOH.
Decanoic acid (also known as capric acid) is a saturated fatty acid of the formula CH3(CH2)8COOH.
MCTs are present in low amounts in palm oil and coconut oil, for example. Decanoic acid and octanoic acid form about 5-8% and 4-10% of the fatty acid composition of coconut oil, respectively.
UBE3A
UBE3A encodes a ubiquitin E3 ligase that targets specific proteins for degradation (de Bie, P. and Ciechanover A. (2011) Cell Death Differ. 18: 1393-402) and is essential for normal cognitive function due to its role as the major ubiquitin binding protein in the synapse.
UBE3A’s ability to clear synaptic signalling proteins (e.g. Arc) enables homeostatic regulation of synaptic growth. Loss of UBE3A expression disrupts long term potentiation via the transcription factor Arc, and also gives rise to dysregulation of glutamate receptor activity. Over time an absence of UBE3A causes synaptic degradation and seizures.
An example UBE3A sequence is deposited under NCBI Accession No. NP_000453.2.
An example UBE3A sequence is:
MEKLHQCYWKSGEPQSDDIEASRMKRAAAKHLIERYYHQLTEGCGNEACTNEFCASCPTFLRMDNNAAAI KALELYKINAKLCDPHPSKKGASSAYLENSKGAPNNSCSEIKMNKKGARIDFKDVTYLTEEKVYEILELC REREDYSPLIRVIGRVFSSAEALVQSFRKVKQHTKEELKSLQAKDEDKDEDEKEKAACSAAAMEEDSEAS SSRIGDSSQGDNNLQKLGPDDVSVDIDAIRRVYTRLLSNEKIETAFLNALVYLSPNVECDLTYHNVYSRD PNYLNLFIIVMENRNLHSPEYLEMALPLFCKAMSKLPLAAQGKLIRLWSKYNADQIRRMMETFQQLITYK VISNEFNSRNLVNDDDAIVAASKCLKMVYYANWGGEVDTNHNEEDDEEPIPESSELTLQELLGEERRNK KGPRVDPLETELGVKTLDCRKPLIPFEEFINEPLNEVLEMDKDYTFFKVETENKFSFMTCPFILNAVTKN LGLYYDNRIRMYSERRITVLYSLVQGQQLNPYLRLKVRRDHIIDDALVRLEMIAMENPADLKKQLYVEFE GEQGVDEGGVSKEFFQLWEEIFNPDIGMFTYDESTKLFWFNPSSFETEGQFTLIGIVLGLAIYNNCILD VHFPMWYRKLMGKKGTFRDLGDSHPVLYQSLKDLLEYEGNVEDDMMITFQISQTDLFGNPMMYDLKENG DKIPITNENRKEFVNLYSDYILNKSVEKQFKAFRRGFHMVTNESPLKYLFRPEEIELLICGSRNLDFQAL EETTEYDGGYTRDSVLIREFWEIVHSFTDEQKRLFLQFTTGTDRAPVGGLGKLKMIIAKNGPDTERLPTS HTCFNVLLLPEYSSKEKLKERLLKAITYAKGFGML
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Cognition
The medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) and composition of the invention may be used for improving cognition. In particular, the MCT and composition of the invention may be used for improving cognition in a subject suffering from or at risk of suffering from a cognitive disorder, Angelman syndrome or Prader-Willi syndrome, and/or an aging subject.
The term “cognition” refers to the set of all mental abilities and processes, including knowledge, attention, long-term memory and working memory, judgment and evaluation, reasoning and “computation”, problem solving and decision making, comprehension and production of language.
Levels of and improvements in cognition can be readily assessed by the skilled person using any suitable neurological and cognitive tests that are known in the art, including cognitive tests designed to assess speed of information processing, executive function and memory. Suitable example tests include Mini Mental State Examination (MMSE), Cambridge Neuropsychological Test Automated Battery (CANTAB), Alzheimer's Disease Assessment Scale-cognitive test (ADAScog), Wisconsin Card Sorting Test, Verbal and Figural Fluency Test and Trail Making Test, electroencephalography (EEG), magnetoencephalography (MEG), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), Magnetic Resonance Imaging (MRI), functional Magnetic Resonance Imaging (fMRI), computerised tomography and long-term potentiation.
Cognitive and behavioural tests for assessing Angelman and Prader-Willi syndrome children include tests which measure attention, self-directed behaviour, motor ability and memory such as the Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III), Bayley, H. (2005); Preschool Language Scale, Fourth Edition (PLS-4),Zimmerman, I.L. et al. (2002); and Vineland Adaptive Behavior Scales, Second Edition (VABS-II), Sparrow, S.S. et al. (2005).
Improved cognition may, for example, be interpreted as a statistically significant difference from the baseline performance in a suitable test (e.g. based on the Bayley Scales of Infant and Toddler Development).
Genetic tests
Genetic test for Angelman syndrome
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Diagnosis of Angelman syndrome may be carried out via molecular genetic testing that suggests deficient expression or function of the maternally inherited UBE3A allele. Analysis of parent-specific DNA methylation imprints in the 15q11.2-q13 chromosome region detects approximately 80% of individuals with Angelman syndrome, including those with a deletion, uniparental disomy (UPD) or an imprinting defect (ID); fewer than 1% of individuals have a cytogenetically visible chromosome rearrangement (i.e. translocation or inversion). UBE3A sequence analysis detects pathogenic variants in an additional approximately 11% of individuals. Therefore, molecular genetic testing (e.g. methylation analysis and UBE3A sequence analysis) may identify alterations in approximately 90% of individuals.
Genetic test for Prader-Willi syndrome
A genetic test for Prader-Willi syndrome may assay the 5' CpG island of the SNRPN locus, which controls UBE3A transcription (via non-coding RNA encoded in the SNRPN transcript). The promoter, exon 1 and intron 1 region of SNRPN are un-methylated on the paternally expressed allele and methylated on the maternally repressed allele. Subjects with Prader-Willi syndrome have only the maternally methylated allele.
Physiological testing
EEG, a measure of electrical activity of the brain, is accomplished by placing electrodes on the scalp at various landmarks and recording greatly amplified brain signals. MEG is similar to EEG in that it measures the magnetic fields that are linked to electrical fields. MEG is used to measure spontaneous brain activity, including synchronous waves in the nervous system.
PET provides a measure of oxygen utilisation and glucose metabolism. In this technique, a radioactive positron-emitting tracer is administered, and tracer uptake by the brain is correlated with brain activity. These tracers emit gamma rays which are detected by sensors surrounding the head, resulting in a 3D map of brain activation. As soon as the tracer is taken up by the brain, the detected radioactivity occurs as a function of regional cerebral blood flow. During activation, an increase in cerebral blood flow and neuronal glucose metabolism can be detected within seconds.
Suitable analysis can also be based on neuropsychiatric testing, clinical examinations and individual complaints of loss of cognitive function (e.g. subjective memory loss).
Further suitable tests may be based on assessments of locomotion, memory and attention, seizure susceptibility, and social interaction and/or recognition. These tests are particularly
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Cognitive disorder
The medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) and composition of the invention may be used to improve cognition in a subject with a cognitive disorder.
The term “cognitive disorder” refers to disorders that give rise to impaired cognition, in particular disorders that primarily affect learning, memory, perception, and/or problem solving.
The medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) and composition of the invention may be used to improve cognition in a subject with Angelman syndrome or Prader-Willi syndrome.
The MCT, MCFA and composition of the invention may be used to treat Angelman syndrome or Prader-Willi syndrome.
Angelman syndrome
Angelman syndrome is a genetic disorder that results in functionally severe developmental delay, which is characterised by severe speech impairment, movement or balance disorders, intellectual disability, epilepsy and odd behaviour. Behavioural signatures of Angelman syndrome children include frequent laughter and/or smiling, and an easily excitable personality, often with hand flapping movements. The majority of Angelman syndrome patients experience seizures, with an onset usually before 3 years of age. Almost all show abnormal electroencephalograms (EEGs), which may be used during diagnosis. Patients with Angelman syndrome often exhibit deficient socialisation and communication in a manner that meets the diagnostic criteria for autism.
Angelman syndrome is caused by dysfunction of UBE3A, a gene located at the q11 -q 13 region of chromosome 15. A mutation in UBE3A can either arise spontaneously or be maternally inherited. In approximately 80% of patients with Angelman syndrome, the UBE3A region is deleted from the maternal chromosome, while the paternal chromosome is intact. UBE3A is transcribed from both chromosomes during gestation, but during the first year of life the paternal gene is epigenetically silenced in the brain. As a result, Angelman syndrome patients express UBE3A in the periphery, but show no UBE3A expression in the brain.
Prader-Willi syndrome
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Prader-Willi syndrome is a genetic disorder related to Angelman syndrome that is caused by lack of expression of a number of genes from the q11 -q 13 region of the paternally inherited chromosome 15.
As with Angelman syndrome, the chromosome 15q11-q13 region is subject to epigenetic silencing through imprinting. However, in the case of Prader-Willi syndrome, the gene(s) on the maternal chromosome are usually imprinted. Thus, if the paternal gene(s) are dysfunctional, this may give rise to the onset of disease.
Prader-Willi syndrome patients experience significant cognitive, neurological, endocrine and behavioural abnormalities. Furthermore, patients may have difficulty in feeding in infancy due to hypotonia, leading to growth retardation, however they become obese later in life due to an uncontrollable craving for food.
Composition
In another aspect, the invention provides a composition comprising a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) of the invention for use in improving cognition which has been impaired by UBE3A deficiency.
In another aspect, the invention provides a composition comprising a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) of the invention for use in treating Angelman syndrome or Prader-Willi syndrome.
The composition may comprise a plurality of MCTs and/or MCFAs as defined herein.
The composition may comprise a concentration of about 5 g/L to 150 g/L of the MCT and/or MCFA of the invention. The concentration may be about 5 g/L, 10 g/L, 15 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L, 110 g/L, 120 g/L, 130 g/L, 140 g/L, 150 g/L, 175 g/L, 200 g/L, 225 g/L or 250 g/L of the MCT and/or MCFA of the invention.
The composition may comprise a homotriglyceride comprising C8 fatty acid moieties (i.e. fatty acids with 8 carbon atoms), preferably octanoic acid moieties.
The composition may comprise a homotriglyceride comprising C10 fatty acid moieties (i.e. fatty acids with 10 carbon atoms), preferably decanoic acid moieties.
The composition may comprise a homotriglyceride comprising C8 fatty acid moieties (i.e. fatty acids with 8 carbon atoms), preferably octanoic acid, and a homotriglyceride comprising C10 fatty acid moieties (i.e. fatty acids with 10 carbon atoms), preferably decanoic acid.
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In one embodiment, the ratio of C8:C10 fatty acid-comprising homotriglycerides is about 90:10 (w/w) to about 10:90 (w/w). In another embodiment, the ratio of C8:C10 fatty acid-comprising homotriglycerides is about 80:20 (w/w) to about 20:80 (w/w). In another embodiment, the ratio of C8:C10 fatty acid-comprising homotriglycerides is about 70:30 (w/w) to about 30:70 (w/w). In another embodiment, the ratio of C8:C10 fatty acid-comprising homotriglycerides is about 60:40 (w/w) to about 40:60 (w/w). In another embodiment, the ratio of C8:C10 fatty acidcomprising homotriglycerides is about 50:50 (w/w) to about 40:60 (w/w).
In one embodiment, the ratio of C8:C10 fatty acid-comprising homotriglycerides is about 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 or 10:90 (w/w). Preferably, the ratio of C8:C10 fatty acid-comprising homotriglycerides is about 50:50 or 40:60 (w/w).
The composition may be free from or substantially free from mono- or poly-unsaturated fatty acids.
The composition may have a ketogenic ratio of 0.2 to 0.3:1. In other embodiments, the ratio may be 0.5:1 or 1:1 to 5:1. The term “ketogenic ratio” refers to the weight of lipid to the sum of the weights of carbohydrate and protein.
The composition may be in the form of a tablet, dragee, capsule, gel cap, powder, granule, solution, emulsion, suspension, coated particle, spray-dried particle or pill.
The composition may be in the form of a powder. The powder may, for example, be a spraydried powder or a freeze-dried powder.
The composition may be in the form of an emulsion. The emulsion may, for example, be an oil-in-water emulsion. The composition may be usable for reconstitution in water.
The composition may be inserted or mixed into a food substance. The composition may be in the form of a food stuff. Preferably, the food stuff is a human food stuff.
The composition may be in the form of a nutritional supplement. The term “nutritional supplement” refers to a product which is intended to supplement the general diet of a subject.
The composition may be in the form of a complete nutritional product. The term “complete nutritional product” refers to a product which is intended to be the sole item, meal or diet consumed by a subject.
The composition may be in the form of a beverage, mayonnaise, margarine, low fat spread, dairy product, cheese spread, processed cheese, dairy dessert, flavoured milk, cream, fermented milk product, cheese, butter, condensed milk product, ice cream mix, soya product, 16
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The composition may be an infant formula.
The composition may be enriched with a MCT and/or MCFA of the invention.
The term “enriched” means that the MCT and/or MCFA has been added to the composition. For example, the MCT and/or MCFA may be spiked (i.e. added within or into) the composition.
In one embodiment, where a composition, such as a food or food extract, natively contains a MCT and/or MCFA, enriched with a MCT or MCFA means that the enriched composition comprises a greater amount of the MCT or MCFA than occurs natively in the composition.
For example, an enriched composition, food or food extract may comprise at least 1.5-, at least 2-, at least 5-, at least 10-, at least 20-, at least 50- or at least 100-fold more MCT and/or MCFA of the invention than an equivalent native composition, food or food extract which has not been enriched.
The invention provides the use of a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) of the invention for the manufacture of a medicament or composition for improving cognition which has been impaired by UBE3A deficiency. The invention also provides the use of a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) of the invention for the manufacture of a medicament or composition for treating Angelman syndrome or Prader-Willi syndrome.
The term “medicament” includes a composition as described herein, including a nutritional supplement and a complete nutritional product. The medicament may comprise suitable pharmaceutically acceptable carriers, diluents and/or excipients.
Administration
The medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) and compositions of the invention may be administered enterally or parenterally. Preferably, the MCT, MCFA or composition is administered enterally. For example, the MCT or MCFA may be administered in the form of a food stuff or a supplement.
In general terms, administration of the MCT, MCFA or composition of the invention may, for example, be by an oral route or another route into the gastro-intestinal tract, for example the administration may also be by gastric tube feeding.
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Method of treatment
In certain aspects, the invention relates to the treatment of Angelman syndrome or PraderWilli syndrome. The treatment may, for example, improve cognition in a subject with Angelman syndrome or Prader-Willi syndrome, and/or reduce the occurrence of seizures.
It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment; although in the context of the invention references to preventing are more commonly associated with prophylactic treatment. Treatment may also include arresting progression in the severity of a disease.
The treatment of mammals, particularly humans, is preferred. However, both human and 10 veterinary treatments are within the scope of the invention.
The subject to be treated may, for example, be a child or an infant.
EXAMPLES
EXAMPLE 1
Materials and methods
Animals
Male Wistar rats, 21 months of age were ordered from Janvier Labs (France) and acclimated for 7 days to the facility. During this time, they were kept in pair-housed cages.
After the acclimatisation period, the rats were randomised into four groups according to cognition and body weight. Baseline cognition was tested via social recognition, using the procedure described below.
Rats were single-housed in standard cages with free access to food and water, and were kept at a 12 hour light cycle period, at a temperature between 20-24°C and a relative humidity between 50-60%. Rats were given a diet of 5% medium-chain triglycerides (MCTs): caprylic triglyceride (C8); capric triglyceride (C10); a 40:60 combination of C8 and C10 triglycerides;
or control (5% sunflower oil) via specially formulated animal chow for 8 weeks.
Social recognition
Rats were tested for behavioural effects of MCT via a social interaction test 3 days before sacrifice. This test involved introduction of an unfamiliar juvenile rat into the home cage of the test rat for 4 minutes. The amount of touching, sniffing and general exploration of the juvenile
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Western blots
NuPAGE Novex Midi Gels (4-12%; Life Technologies) were used for protein separation. The following antibodies were used: Synaptophysin (D35E4) XP® Rabbit mAb (Cell Signaling, Cat. #5461); Ube3a (Abgent, #346954); β-Tubulin Antibody (TUB 2.1, Santa Cruz, Cat. #sc10 58886); and β-Actin antibody (Sigma, Cat. #A1978).
After incubating with corresponding infrared fluorescent secondary antibodies from Li_COR Biosciences (#926-32213 IRDye® 800CW Donkey anti-Rabbit IgG and #926-68072 IRDye® 680RD Donkey anti-Mouse IgG), the signal of specific protein was detected and quantified on the Odyssey CLx imager (LI-COR).
Gene expression detection by PCR
Total RNA was extracted and purified with the RNAdvance tissue kit (Agencourt, Beverly, MA, USA). The quality of RNA samples was checked by using the Fragment Analyzer (Advanced Analytical Technologies, Inc, Ames, IA, USA). Reverse transcription was performed using PrimeScript Reagent kit from Katara Clontech (Cat. #RR037A) following the manufacturer’s instructions using 500 ng of each RNA sample. After adding LightCycler® 1536 DNA Green Master (Roche, Cat. #5573092001), gene expression was quantified on a Roche LightCycler 480. Primers for the following genes were used for the PCR reaction: Arc, snrpn, ube3a1, and beta-actin was used as an endogenous control.
Rat-Arc-F: acagacacagcagatccagc (SEQ ID NO: 2) ;
25 Rat-Arc-R: tgagtcatggagccgaagtc (SEQ ID NO: 3)
Rat-Actin-F: gtcgtaccactggcattgtg (SEQ ID NO: 4) ;
Rat-Actin-R: ctctcagctgtggtggtgaa (SEQ ID NO: 5)
Rat-Snrpn-F: TTGGTTCTGAGGAGTGATTTGC (SEQ ID NC ): 6)
Rat-Snrpn-R: CCTTGAATTCCACCACCTTG (SEQ ID NO: 7)
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Rat-Ube3a-F: ggcacccttgtttgaaactt (seq id no: 8)
Rat-Ube3a-R: gctcatgaccctgtcctttc (seq id no: 9)
Results
From a comparison of social recognition at baseline and 8 weeks after chronic MCT treatment, 5 cognitive performance was increased in MCT-treated groups compared to those fed a control diet (Figure 1).
Furthermore, UBE3a protein expression was observed to increase in the cortex of the 24 month-old rats of MCT-treated groups compared to those fed a control diet (Figure 2).
mRNA expression analysis showed that MCT treatment resulted in a down-regulation of the 10 transcription factor Arc, and a concomitant up-regulation of the non-coding RNAs snrpn and ube3a1, in comparison to control groups (Figure 3).
In summary, these studies showed cognitive improvement where UBE3A expression may have played a role via down-regulation of Arc. The increase in UBE3A expression may be due to changes in non-coding RNA, snrpn and ube3a1.
EXAMPLE 2
Materials and methods
Animals
Male Wistar rats, 15 months of age, and 6 month male rats were ordered from Janvier Labs (France) and acclimated for 7 days to the facility. During this time, they were kept in pair20 housed cages.
After the acclimatisation period, the rats were randomised into four groups according to cognition and body weight. Baseline cognition was tested via social recognition, using the procedure described for Example 1.
Rats were single-housed in standard cages with free access to food and water, and were kept 25 at a 12 hour light cycle period, at a temperature between 20-24°C and a relative humidity between 50-60%. Rats were given a diet of 5% medium-chain triglycerides (MCTs): caprylic triglyceride (C8); capric triglyceride (C10); a 50:50 combination of C8 and C10 triglycerides;
or control (sunflower oil) for 8 weeks.
Results
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From a comparison of object recognition at baseline and after chronic MCT treatment, cognitive performance was observed to increase in MCT-treated groups compared to those fed a control diet (Figure 4).
Furthermore, UBE3a protein expression was observed to increase in the cortex of the 17 month-old rats of MCT-treated groups compared to those fed a control diet (Figure 5).
EXAMPLE 3
UBE3A expression was analysed in immature mice that were exposed to a caprylic homotriglyceride (MCT8) diet during gestation and weaning (via nursing).
Materials and methods
Animals
Female C57 mice were mated and pregnancy tracked via vaginal smearing. MCT8 was mixed in animal chow (1.5%) and administered to pregnant mouse females between day 10 of gestation and postnatal day 28 while pregnant mice received chow supplemented with 1.5% sunflower oil (control).
At postnatal day 28, pup brains and livers were collected, fixed, frozen and processed for UBE3A expression.
In order to assess the cognitive performance effects of treatment, some mice were tested at postnatal day 60 in a Morris Water Maze task. The apparatus used for behaviour consisted of a gallon tank with a platform placed in one of the quadrants. The tank was filled with warm water (24°C), made opaque. A video camera recorded animal movement within the tank. Visual cues were placed in several quadrants of the tank.
Each mouse was given four trials per day to find the platform. Trials were given for four cumulative days and on the last day, a probe trial was performed (where the platform was removed) to assess spatial bias (mean % duration in the target quadrant, i.e. where the platform had originally been positioned).
Results
UBE3A expression was observed to increase in the brain following MCT8 treatment, but not in the liver (Figure 6).
These findings imply that the paternal UBE3A allele in the brain has been turned on by the MCT treatment.
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In the probe trial of the Morris Water Maze, female mice treated with MCT8 (C8-OTG) spent more time in the target quadrant than 25% chance level (where 25% is mean percentage duration expected if the mice did not remember the platform quadrant location) (Figure 7).
EXAMPLE 4
Materials and methods
Human iCell neurons (Cellular Dynamics International) were plated in 24 well plates and were grown to neuronal maturity by culturing for 2 weeks. Cells were then treated with octanoic or decanoic acid (100 μΜ) for 6 days, where fresh medium-chain fatty acid (MCFA) was added to the wells every 2 days. Cells were scraped and stored in Trizol, and mRNA extracted. qPCR was performed to probe UBE3Aand GRIN1 levels according to the protocol referred to above.
GRIN1 Forward: tctacaatggcacccacgtcatc (seq id no: 10)
GRIN1 Reverse: ggtgcagatcaccttcttgac (seq id no: ii)
Results
Both octanoic and decanoic acid were observed to increase expression of UBE3A and the glutamate receptor, ionotropic, N-methyl D-aspartate 1 (GRIN1) subunit (Figure 8), a marker of synaptic plasticity.
Example 5
Male Sprague-Dawley rats (8 MCT treated and 8 sunflower oil control) were acclimated for at least 5 days. Animals of all groups were provided with tap water ad libitum during the whole study. A standard diet (RM1, SDS Dietex) was provided also to the animals during the whole study. Oral administrations (P.O.)were performed on un-anaesthetized animals using an oral gavage probe.
MCT C8/C10 at a ratio of 60/40 (1.8 g/kg bodyweight) or regular sunflower oil (1.8g/kg bodyweight) was given once daily for 5 days. After 5 days animals were sacrificed and brains collected and stored at -80°C. RNA was extracted from dissected brain tissue (pre-frontal cortex/PFC) and processed for real-time PCR. The MCT C8/C10 combination significantly increased the expression of UBE3A (normalized to beta-actin) according to t-test; ** = p<0.05 as shown in Figure 9.
Example 6
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Human iCell neurons (Cellular Dynamics International) were plated in 24-well plates and were cultured for 2 weeks. Cells were then treated with octanoic (C8) or decanoic acid (C10) at different concentrations (C8: 0.25μΜ, 2.5μΜ, 25μΜ; C10: 0.5μΜ, 5μΜ, 50μΜ) at 3 different glucose concentrations (25mM, 4mM, OmM) for 3 days.
Cells were harvested and processed for real-time PCR. The following probes were used: UBE3Aand beta-actin.
Octanoic acid/C8 and decanoic acid/C10 significantly increased UBE3A expression levels in the abscence of glucose in the media (normalized to the beta-actin).
Decanoic acid/C10 at 5μΜ and 50μΜ significantly increased UBE3A expression in the presence of low glucose at 4mM (normalized to the beta-actin9 as measured using One-way ANOVA; * = p<0.1; ** = p<0.05; *** = p<0.01 shown in Figure 10.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described compositions or uses of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention, which are obvious to those skilled in biochemistry and biotechnology or related fields, are intended to be within the scope of the following claims.
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Claims (10)

1. A medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) for use in improving cognition which has been impaired by UBE3A deficiency.
2. The MCT or MCFA for use according to claim 1, wherein the MCT or MCFA improves
5 cognition in a subject with a cognitive disorder and/or an aging subject.
3. The MCT or MCFA for use according to claim 1 or 2, wherein the MCT or MCFA improves cognition in a subject with Angelman syndrome or Prader-Willi syndrome.
4. A medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) for use in treating Angelman syndrome or Prader-Willi syndrome or for treating a reduction in
10 cognition associated with ageing.
5. The MCT or MCFA for use according to any one of the preceding claims, wherein the MCT comprises three fatty acid moieties, each of which independently has only 8 or 10 carbon atoms, preferably the fatty acid moieties are octanoic acid and/or decanoic acid moieties, or wherein the MCFA is octanoic or decanoic acid.
15 6. The MCT for use according to any one of the preceding claims, wherein the MCT is a homotriglyceride.
7. The MCT or MCFA for use according to any one of the preceding claims, wherein the MCT or MCFA is administered enterally or parenterally.
8. A composition comprising a medium-chain triglyceride (MCT) or medium-chain fatty
20 acid (MCFA) for use in improving cognition which has been impaired by UBE3A deficiency.
9. The composition for use according to claim 8, wherein the composition improves cognition in a subject with a cognitive disorder and/or an aging subject.
10. The composition for use according to claim 8 or 9, wherein the composition improves
25 cognition in a subject with Angelman syndrome or Prader-Willi syndrome.
11. A composition comprising a medium-chain triglyceride (MCT) or medium-chain fatty acid (MCFA) for use in treating Angelman syndrome or Prader-Willi syndrome or for treating a reduction in cognition associated with ageing...
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12. The composition for use according to any one of claims 8-11, wherein the composition comprises a homotriglyceride comprising C8 fatty acids, preferably octanoic acid, and/or a homotriglyceride comprising C10 fatty acids, preferably decanoic acid.
13. The composition for use according to claim 12, wherein the ratio of C8:C10 fatty acid5 comprising homotriglycerides is about 50:50 (w/w) to about 40:60 (w/w).
14. The composition for use according to any one of claims 8-13, wherein the composition is in the form of a food stuff.
15. The composition for use according to any one of claims 8-14, wherein the composition is in the form of a beverage, mayonnaise, margarine, low fat spread, dairy product,
10 cheese spread, processed cheese, dairy dessert, flavoured milk, cream, fermented milk product, cheese, butter, condensed milk product, ice cream mix, soya product, pasteurised liquid egg, bakery product, confectionary product, confectionary bar, chocolate bar, high fat bar, liquid emulsion, spray-dried powder, freeze-dried powder, UHT pudding, pasteurised pudding, gel, jelly, yoghurt, or a food with a fat-based or
15 water-containing filling.
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1/10
DRAWINGS novel vs total exploration baseline after 8 wk treatment
Figure 1
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2/10
Figure 2
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Ford change
Arc Wpp
CON mix MCT8 MCT10
Snrpn hipp
CON mix MCT8 MCT10
Ube3a1 hipp
CON mix MCT8 MCT10
Figure 3
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4/10 ratio novel vs total exploration
Figure 4
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5/10
UBE3A hippocampus
UBE3E/b-actin
Figure 5
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6/10 brain liver *
Figure 6
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Mean Duration % In each quadrant during the probe trial (♦/- 1SEM)
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Figure 7
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8/10 *
iCell Neurons-Ube3a iCell neurons-GRIN1 *
Fold change
Figure 8 iCell treatment
ΛΛΛΛΛΛΛΛ
ΛΛΛΛΛΛΛΛ
ΛΛΛΛΛΛΛΛ
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□)
C re .c o
Ό
ΛΛΛΛΛΛΛΛ
ΛΛΛΛΛΛΛΛ
w.v.w.
G* iCell treatment
WO 2017/093060
PCT/EP2016/078313
9/10
U B E3A/b-actin
Ctrl C8/C10
5 Figure 9
WO 2017/093060
PCT/EP2016/078313
10/10
U BE3A/ b-aetin UBE3A/ b-actin
25mM glucose 4mM glucose Omtt glucose
25 m M glucose 4mM glucose OmM glucose
Figure 10 eolf-seql SEQUENCE LISTING <110> Nestec S.A.
<120> Method for improving cognition <130> 14969 <160> 11
<170> PatentIn version 3.5 <210> 1 <211> 875 <212> PRT <213> Homo sap ens <400> 1 Met Glu Lys Leu His Gln Cys Tyr Trp Lys Ser Gly Glu Pro Gln Ser 1 5 10 15 Asp Asp Ile Glu Ala Ser Arg Met Lys Arg Ala Ala Ala Lys His Leu 20 25 30 Ile Glu Arg Tyr Tyr His Gln Leu Thr Glu Gly Cys Gly Asn Glu Ala 35 40 45 Cys Thr Asn Glu Phe Cys Ala Ser Cys Pro Thr Phe Leu Arg Met Asp 50 55 60 Asn Asn Ala Ala Ala Ile Lys Ala Leu Glu Leu Tyr Lys Ile Asn Ala 65 70 75 80 Lys Leu Cys Asp Pro His Pro Ser Lys Lys Gly Ala Ser Ser Ala Tyr 85 90 95 Leu Glu Asn Ser Lys Gly Ala Pro Asn Asn Ser Cys Ser Glu Ile Lys 100 105 110 Met Asn Lys Lys Gly Ala Arg Ile Asp Phe Lys Asp Val Thr Tyr Leu 115 120 125 Thr Glu Glu Lys Val Tyr Glu Ile Leu Glu Leu Cys Arg Glu Arg Glu 130 135 140 Asp Tyr Ser Pro Leu Ile Arg Val Ile Gly Arg Val Phe Ser Ser Ala 145 150 155 160 Glu Ala Leu Val Gln Ser Phe Arg Lys Val Lys Gln His Thr Lys Glu 165 170 175 Glu Leu Lys Ser Leu Gln Ala Lys Asp Glu Asp Lys Asp Glu Asp Glu 180 185 190 Lys Glu Lys Ala Ala Cys Ser Ala Ala Ala Met Glu Glu Asp Ser Glu
Page 1 eolf-seql
195 200 205 Ala Ser Ser Ser Arg Ile Gly Asp Ser Ser Gln Gly Asp Asn Asn Leu 210 215 220 Gln Lys Leu Gly Pro Asp Asp Val Ser Val Asp Ile Asp Ala Ile Arg 225 230 235 240 Arg Val Tyr Thr Arg Leu Leu Ser Asn Glu Lys Ile Glu Thr Ala Phe 245 250 255 Leu Asn Ala Leu Val Tyr Leu Ser Pro Asn Val Glu Cys Asp Leu Thr 260 265 270 Tyr His Asn Val Tyr Ser Arg Asp Pro Asn Tyr Leu Asn Leu Phe Ile 275 280 285 Ile Val Met Glu Asn Arg Asn Leu His Ser Pro Glu Tyr Leu Glu Met 290 295 300 Ala Leu Pro Leu Phe Cys Lys Ala Met Ser Lys Leu Pro Leu Ala Ala 305 310 315 320 Gln Gly Lys Leu Ile Arg Leu Trp Ser Lys Tyr Asn Ala Asp Gln Ile 325 330 335 Arg Arg Met Met Glu Thr Phe Gln Gln Leu Ile Thr Tyr Lys Val Ile 340 345 350 Ser Asn Glu Phe Asn Ser Arg Asn Leu Val Asn Asp Asp Asp Ala Ile 355 360 365 Val Ala Ala Ser Lys Cys Leu Lys Met Val Tyr Tyr Ala Asn Val Val 370 375 380 Gly Gly Glu Val Asp Thr Asn His Asn Glu Glu Asp Asp Glu Glu Pro 385 390 395 400 Ile Pro Glu Ser Ser Glu Leu Thr Leu Gln Glu Leu Leu Gly Glu Glu 405 410 415 Arg Arg Asn Lys Lys Gly Pro Arg Val Asp Pro Leu Glu Thr Glu Leu 420 425 430 Gly Val Lys Thr Leu Asp Cys Arg Lys Pro Leu Ile Pro Phe Glu Glu 435 440 445 Phe Ile Asn Glu Pro Leu Asn Glu Val Leu Glu Met Asp Lys Asp Tyr 450 455 460 Thr Phe Phe Lys Val Glu Thr Glu Asn Lys Phe Ser Phe Met Thr Cys Pa ge 2
eolf-seql
465 470 475 480 Pro Phe Ile Leu Asn Ala Val Thr Lys Asn Leu Gly Leu Tyr Tyr Asp 485 490 495 Asn Arg Ile Arg Met Tyr Ser Glu Arg Arg Ile Thr Val Leu Tyr Ser 500 505 510 Leu Val Gln Gly Gln Gln Leu Asn Pro Tyr Leu Arg Leu Lys Val Arg 515 520 525 Arg Asp His Ile Ile Asp Asp Ala Leu Val Arg Leu Glu Met Ile Ala 530 535 540 Met Glu Asn Pro Ala Asp Leu Lys Lys Gln Leu Tyr Val Glu Phe Glu 545 550 555 560 Gly Glu Gln Gly Val Asp Glu Gly Gly Val Ser Lys Glu Phe Phe Gln 565 570 575 Leu Val Val Glu Glu Ile Phe Asn Pro Asp Ile Gly Met Phe Thr Tyr 580 585 590 Asp Glu Ser Thr Lys Leu Phe Trp Phe Asn Pro Ser Ser Phe Glu Thr 595 600 605 Glu Gly Gln Phe Thr Leu Ile Gly Ile Val Leu Gly Leu Ala Ile Tyr 610 615 620 Asn Asn Cys Ile Leu Asp Val His Phe Pro Met Val Val Tyr Arg Lys 625 630 635 640 Leu Met Gly Lys Lys Gly Thr Phe Arg Asp Leu Gly Asp Ser His Pro 645 650 655 Val Leu Tyr Gln Ser Leu Lys Asp Leu Leu Glu Tyr Glu Gly Asn Val 660 665 670 Glu Asp Asp Met Met Ile Thr Phe Gln Ile Ser Gln Thr Asp Leu Phe 675 680 685 Gly Asn Pro Met Met Tyr Asp Leu Lys Glu Asn Gly Asp Lys Ile Pro 690 695 700 Ile Thr Asn Glu Asn Arg Lys Glu Phe Val Asn Leu Tyr Ser Asp Tyr 705 710 715 720 Ile Leu Asn Lys Ser Val Glu Lys Gln Phe Lys Ala Phe Arg Arg Gly 725 730 735 Phe His Met Val Thr Asn Glu Ser Pro Leu Lys Tyr Leu Phe Arg Pro
Page 3 eolf-seql
740
745
750
Glu Glu Ile 755 Glu Leu Leu Ile Cys Gly Ser Arg Asn 760 Leu 765 Asp Phe Gln Ala Leu Glu Glu Thr Thr Glu Tyr Asp Gly Gly Tyr Thr Arg Asp Ser 770 775 780 Val Leu Ile Arg Glu Phe Trp Glu Ile Val His Ser Phe Thr Asp Glu 785 790 795 800 Gln Lys Arg Leu Phe Leu Gln Phe Thr Thr Gly Thr Asp Arg Ala Pro 805 810 815 Val Gly Gly Leu Gly Lys Leu Lys Met Ile Ile Ala Lys Asn Gly Pro 820 825 830 Asp Thr Glu Arg Leu Pro Thr Ser His Thr Cys Phe Asn Val Leu Leu 835 840 845 Leu Pro Glu Tyr Ser Ser Lys Glu Lys Leu Lys Glu Arg Leu Leu Lys 850 855 860 Ala Ile Thr Tyr Ala Lys Gly Phe Gly Met Leu 865 870 875
<210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220>
<223> Primer Rat-Arc-F <400> 2 acagacacag cagatccagc <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220>
<223> Primer Rat-Arc-R <400> 3 tgagtcatgg agccgaagtc <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220>
<223> Primer Rat-Actin-F <400> 4
Page 4 eolf-seql gtcgtaccac tggcattgtg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220>
<223> Primer Rat-Actin-R <400> 5 ctctcagctg tggtggtgaa 20 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220>
<223> Primer Rat-Snrpn-F <400> 6 ttggttctga ggagtgattt gc 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220>
<223> Primer Rat-Snrpn-R <400> 7 ccttgaattc caccaccttg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220>
<223> Primer Rat-Ube3a-F <400> 8 ggcacccttg tttgaaactt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220>
<223> Primer Rat-Ube3a-R <400> 9 gctcatgacc ctgtcctttc 20 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence
Page 5
<220> <223> Primer GRIN1 Forward <400> 10 tctacaatgg cacccacgtc atc eolf-seql 23 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer GRIN1 Reverse <400> 11 ggtgcagatc accttcttga c 21
Page 6
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