U.S. patent application number 16/038513 was filed with the patent office on 2019-01-17 for hydroxybutyrate ester and medical use thereof.
The applicant listed for this patent is GOVERNMENT OF THE USA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH, OXFORD UNIVERSITY INNOVATION LIMITED, GOVERNMENT OF THE USA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH. Invention is credited to Kieran Clarke, Richard Lewis Veech.
Application Number | 20190014798 16/038513 |
Document ID | / |
Family ID | 44657154 |
Filed Date | 2019-01-17 |
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United States Patent
Application |
20190014798 |
Kind Code |
A1 |
Clarke; Kieran ; et
al. |
January 17, 2019 |
HYDROXYBUTYRATE ESTER AND MEDICAL USE THEREOF
Abstract
A compound which is 3-hydroxybutyl 3-hydroxybutyrate
enantiomerically enriched with respect to (3R)-hydroxybutyl
(3R)-hydroxybutyrate of formula (I) is an effective and palatable
precursor to the ketone body (3R)-hydroxybutyrate and may therefore
be used to treat a condition which is caused by, exacerbated by or
associated with elevated plasma levels of free fatty acids in a
human or animal subject, for instance a condition where weight loss
or weight gain is implicated, or to promote alertness or improve
cognitive function, or to treat, prevent or reduce the effects of
neurodegeneration, free radical toxicity, hypoxic conditions or
hyperglycaemia.
Inventors: |
Clarke; Kieran; (Oxford,
GB) ; Veech; Richard Lewis; (Rockville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GOVERNMENT OF THE USA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT
OF HEALTH
OXFORD UNIVERSITY INNOVATION LIMITED |
Bethesda
Oxford |
MD |
US
GB |
|
|
Family ID: |
44657154 |
Appl. No.: |
16/038513 |
Filed: |
July 18, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14101834 |
Dec 10, 2013 |
10051880 |
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16038513 |
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13031006 |
Feb 18, 2011 |
8642654 |
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14101834 |
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PCT/US2009/040766 |
Apr 16, 2009 |
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13031006 |
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61090751 |
Aug 21, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07B 2200/07 20130101;
A61P 25/16 20180101; A23L 33/10 20160801; A23L 2/52 20130101; A61P
25/28 20180101; A61P 3/00 20180101; C07C 69/675 20130101; A61K
31/22 20130101; A61P 21/00 20180101; A23V 2002/00 20130101; A23V
2002/00 20130101; A23V 2200/316 20130101; A23V 2200/322 20130101;
A23V 2200/328 20130101; A23V 2250/30 20130101 |
International
Class: |
A23L 2/52 20060101
A23L002/52; A23L 33/10 20060101 A23L033/10; A61K 31/22 20060101
A61K031/22; C07C 69/675 20060101 C07C069/675 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
No. W911NF-05-1-0479 awarded by ARMY/ARO. The government has
certain rights in this invention.
Claims
1. A compound which is (3R)-hydroxybutyl (3R)-hydroxybutyrate of
formula (I): ##STR00003##
2. An ingestible composition which comprises a compound as defined
in claim 1 and a dietetically or pharmaceutically acceptable
carrier.
3. A composition according to claim 2 which is a food product, a
beverage, a drink, a food supplement, a dietary supplement, a
functional food, a nutraceutical or a medicament.
4. (canceled)
5. A method of treating a condition which is caused by, exacerbated
by or associated with elevated plasma levels of free fatty acids in
a human or animal subject, which method comprises administering to
the subject a compound as defined in claim 1.
6. A method of treating a condition where weight loss or weight
gain is implicated, which method comprises administering to a
subject in need thereof a compound as defined in claim 1.
7. A method of suppressing appetite, treating obesity, promoting
weight loss, maintaining a healthy weight or decreasing the ratio
of fat to lean muscle, which method comprises administering to a
subject in need thereof a compound as defined in claim 1.
8. A method of preventing or treating a condition selected from
cognitive dysfunction, a neurodegenerative disease or disorder,
muscle impairment, fatigue and muscle fatigue, which method
comprises administering to a subject in need thereof a compound as
defined in claim 1.
9. A method of treating a patient suffering from a condition
selected from diabetes, hyperthyroidism, metabolic syndrome X, or
for treating a geriatric patient, which method comprises
administering thereto a compound as defined in claim 1.
10. A method of treating, preventing, or reducing the effects of,
neuro degeneration, free radical toxicity, hypoxic conditions or
hyperglycaemia which method comprises administering to a subject in
need thereof a compound as defined in claim 1.
11. A method according to claim 10, wherein the neurodegeneration
is caused by aging, trauma, anoxia or a neurodegenerative disease
or disorder.
12. A method of preventing or treating a neurodegenerative disease
or disorder selected from Alzheimer's disease, Parkinson's disease,
amyotrophic lateral sclerosis, epilepsy, astrocytoma, glioblastoma
and Huntington's chorea, which method comprises administering to a
subject in need thereof a compound as defined in claim 1.
13. A method of promoting alertness or improving cognitive function
in a subject, which method comprises administering to said subject
a compound as defined in claim 1.
14. The compound of claim 1, wherein the compound is
enantiomerically enriched to at least 90%.
15. The compound of claim 1, wherein the compound is
enantiomerically enriched to at least 95%.
16. The compound of claim 1, wherein the compound is
enantiomerically enriched to at least 97%.
17. The compound of claim 1, wherein the compound is
enantiomerically enriched to at least 98%.
18. The compound of claim 1, wherein the compound is
enantiomerically enriched to at least 99%.
19. A method of preventing or treating muscle impairment or muscle
fatigue in a subject in need thereof, comprising administering to
the subject a therapeutically effective amount of the compound of
claim 1.
Description
RELATED APPLICATIONS
[0001] This application is a 35 U.S.C. .sctn. 111(a) filing of
International Application Number PCT/US2009/040766 which was filed
on Apr. 16, 2009, which claims priority to U.S. Provisional
Application 61/090,751, which was filed on Aug. 21, 2008. The
entire contents of the aforementioned applications are hereby
incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to hydroxybutyrate esters
which elevate blood levels of ketone bodies, and to medical uses of
such esters.
BACKGROUND TO THE INVENTION
[0004] Ketone bodies are chemical compounds which are produced by
the liver from fatty acids released from adipose tissue. Ketone
bodies themselves can be used as a source of energy in most tissues
of the body. The intake of compounds that boost the levels of
ketone bodies in the blood can lead to various clinical benefits,
including an enhancement of physical and cognitive performance and
the treatment of cardiovascular conditions, diabetes,
neurodegenerative diseases and epilepsy.
[0005] Ketone bodies include (R)-3-hydroxybutyrate and
acetoacetate. As discussed in WO2004/108740, these compounds could
in theory be administered directly to achieve elevated levels of
ketone bodies in a subject. However, direct administration of the
compounds is unpractical and potentially dangerous. For example,
direct administration of either (R)-3-hydroxybutyrate or
acetoacetate in its free acid form can result in significant
acidosis following rapid absorption from the gastrointestinal
tract. Administration of the sodium salt of these compounds in
unregulated amounts is also unsuitable due to a potentially
dangerous sodium overload that could accompany administration of
therapeutically relevant amounts of the compounds.
[0006] Against this background WO2004/108740 discloses derivatives
of (R)-3-hydroxybutyrate which serve as precursors to ketone bodies
such as acetoacetate and (R)-3-hydroxybutyrate and which therefore
elevate blood concentrations of ketone bodies when administered to
a subject. Examples of the derivatives include esters, for instance
esters derived from a variety of alcohols. WO2004/108740 further
discloses the use of these derivatives for treating metabolic
disorders such as insulin deficiency and insulin resistance, and as
nutritional supplements for increasing physical performance.
[0007] WO04/105742 teaches that compounds which reduce the level of
free fatty acids circulating in the plasma of a subject may be used
to treat muscle impairment or fatigue. Ketone bodies, such as
ketone body esters, are given examples of such compounds.
SUMMARY OF THE INVENTION
[0008] It has now been surprisingly found that one particular
enantiomer of one particular ester of 3-hydroxybutyrate is an
effective and palatable precursor to the ketone body
(3R)-hydroxybutyrate. Accordingly, the present invention provides a
compound which is 3-hydroxybutyl 3-hydroxybutyrate enantiomerically
enriched with respect to (3R)-hydroxybutyl (3R)-hydroxybutyrate of
formula (I):
##STR00001##
[0009] The invention further provides an ingestible composition
which comprises a compound of the invention as defined above and a
dietetically or pharmaceutically acceptable carrier.
[0010] (3R)-Hydroxybutyl (3R)-hydroxybutyrate reduces plasma levels
of fatty acids. The invention therefore further provides a compound
of the invention as defined above for use in treating a condition
which is caused by, exacerbated by or associated with elevated
plasma levels of free fatty acids in a human or animal subject.
[0011] In one embodiment, the invention further provides a compound
of the invention as defined above for use in treating a condition
where weight loss or weight gain is implicated. Thus, the compound
may be used to treat obesity or other overweight conditions, to
promote weight loss, to maintain a healthy weight, or to decrease
the ratio of fat to lean muscle in a subject (which may be a
healthy subject or a compromised subject). The compound may be used
as a dietary supplement.
[0012] The invention further provides a compound of the invention
as defined above for use in promoting alertness or improving
cognitive function, or in treating cognitive dysfunction.
[0013] The invention also provides a compound of the invention as
defined above for use in treating, preventing, or reducing the
effects of, neurodegeneration, free radical toxicity, hypoxic
conditions or hyperglycaemia.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 shows a plot of daily body weight (in grams/rat/day)
of rats fed a diet of carbohydrate, fat or the compound of the
invention ("ketone"), in the test described in Example 3.
[0015] FIG. 2 shows a plot of daily food intake (in grams/rat/day)
for rats fed a diet of carbohydrate, fat or the compound of the
invention ("ketone"), in the test described in Example 3.
[0016] FIG. 3 contains micrographs showing the levels of BDNF
positive cell bodies in the paraventricular nucleus (PVN) area of
the hypothalamus and in the Hippocampus of rats treated for 4 days
with a diet of fat, carbohydrate or the compound of the invention
("ketone"), in the test described in Example 5. A significantly
greater number of BDNF-positive cell bodies is seen in the PVN of
the rat treated with the ketone diet compared to fat and
carbohydrate diet-treated rats. A similar observation is made in
the hippocampus of rats treated with ketones.
[0017] FIG. 4 presents micrographs showing the levels of MC4R
positive cell bodies in the paraventricular nucleus (PVN) area of
the hypothalamus of rats treated for 14 days with a diet of fat,
carbohydrate or the compound of the invention ("ketone"), in the
test described in Example 5. Significantly greater numbers of MC4R
positive cell bodies are seen in the posterior magnocellular (pm)
and medial parvocellular (mpd) regions of the PVN in rats treated
with the ketone diet and the carbohydrate diet, than in rats
treated with the fat diet.
[0018] FIG. 5 shows magnifications of the micrographs in FIG. 5.
The micrographs show the presence of a significantly denser area of
MC4R positive cell bodies in the PVN of rats on the ketone diet or
carbohydrate (Cho) diet compared to rats on the fat diet.
[0019] FIG. 6 contains micrographs showing the levels of CART in
the posterior paraventricular nucleus (PVN) area of the
hypothalamus of rats, treated for 14 days with a diet of fat,
carbohydrate or the compound of the invention ("ketone"), in the
test described in Example 5. Significantly greater numbers of CART
positive cell bodies are seen in the PVNs of rats treated with the
ketone and carbohydrate diets, than in the rats treated with the
fat diet. The highest level of CART is seen in the PVN in the rats
treated with the ketone diet.
[0020] FIG. 7 presents micrographs showing the levels of CART in
the ventromedial nucleus (VMH), arcuate nucleus (ARC) and median
emminence (ME) areas of the hypothalamus of rats, treated for 14
days with a diet of fat, carbohydrate or the compound of the
invention ("ketone"), in the test described in Example 5. The
highest numbers of CART-positive cell bodies in these areas are
seen in the rats treated with the ketone diet.
[0021] FIG. 8 shows a magnification of each of the micrographs in
FIG. 7. The magnifications clearly show the presence of a
significantly greater number of CART positive cell bodies in the
PVN of rats on the ketone diet compared to rats on the fat and
carbohydrate diets.
[0022] FIG. 9 shows further magnifications of the micrographs in
FIGS. 7 and 9, showing that the PVN of the rats treated with the
ketone diet has the highest density of CART-positive cell
bodies.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The compound of the invention is 3-hydroxybutyl
3-hydroxybutyrate enantiomerically enriched with respect to the
(3R, 3R') enantiomer. The term "enriched" as employed herein means
that the level of the enriching isomer is higher than the level at
which that isomer would typically be produced in a racemic mixture.
Where a percentage enrichment is referred to, the enriching isomer
constitutes that percentage of the total 3-hydroxybutyl
3-hydroxybutyrate present. Generally the 3-hydroxybutyl
3-hydroxybutyrate in the present invention is enantiomerically
enriched to at least 90%, preferably 95% with respect to
(3R)-hydroxybutyl (3R)-hydroxybutyrate. In other words, of the
total 3-hydroxybutyl 3-hydroxybutyrate present, at least 90% and
preferably 95% is the (3R)-hydroxybutyl (3R)-hydroxybutyrate
isomer, In a further embodiment the 3-hydroxybutyl
3-hydroxybutyrate may comprise at least 97%, for example 98%, or
99%, of the (3R,3R') enantiomer.
[0024] The compound of the invention as defined above may be
prepared by a process which comprises carrying out a
transesterification reaction between ethyl (3R)-hydroxybutyrate and
(3R)-1, 3-butanediol in the presence of a lipase enzyme. The
reaction is typically conducted at about 40.degree. C. for a period
of about 96 hours. An example of the process is described in
Example 1 which follows. The product of the reaction is typically
submitted to wiped film distillation (GMP). This comprises a
degassing pass, a second light cut pass to remove starting
materials and then a final pass. The conditions of the final pass
are typically 145.degree. C. at 1.8 Torr.
[0025] A sample of 3-hydroxybutyl 3-hydroxybutyrate enriched with
respect to the (3R, 3R') enantiomer gives measurably raised blood
levels of (3R)-hydroxybutyrate, a ketone body, when ingested
orally. The compound of the invention therefore represents an
effective means of delivering (3R)-hydroxybutyrate to a
subject.
[0026] Two particular advantages are associated with the invention.
First, the (3R, 3R') enantiomer is palatable and is less
bitter-tasting than other ketone bodies. It is therefore
particularly well-suited for oral administration. This contrasts
with many other ketone bodies, and their derivatives and
precursors, which are notoriously bad-tasting and thus difficult to
tolerate when taken orally. Second, the (3R, 3R') enantiomer is
cleaved in vivo to form (3R)-hydroxybutyrate and
(R)-1,3-butanediol. The (3R)-hydroxybutyrate is released
immediately, giving a rapid effect following ingestion. The
(R)-1,3-butanediol is converted in the liver to
(3R)-hydroxybutyrate which is then released into blood. Overall
this gives a favourable pharmacokinetic profile, since raised blood
levels of the desired (R)-3-hydroxybutyrate are both achieved
quickly and then sustained over a period of time following
ingestion of the compound of the invention.
[0027] The compound of the invention as defined above reduces
plasma levels of fatty acids. A compound of the invention may
therefore be used to reduce the level of free fatty acids
circulating in the plasma of a subject. As such it may be used to
treat a condition which is caused by, exacerbated by or associated
with elevated plasma levels of free fatty acids in a human or
animal subject. A human or animal subject may therefore be treated
by a method which comprises the administration thereto of a
compound of the invention as defined above. The condition of the
subject may thereby be improved or ameliorated.
[0028] Conditions which are caused by, exacerbated by or associated
with elevated plasma levels of free fatty acids include, but are
not limited to: neurodegenerative diseases or disorders, for
instance Alzheimer's disease, Parkinson's disease, Huntington's
chorea; hypoxic states, for instance angina pectoris, extreme
physical exertion, intermittent claudication, hypoxia, stroke and
myocardial infarction; insulin resistant states, for instance
infection, stress, obesity, diabetes and heart failure; and
inflammatory states including infection and autoimmune disease.
[0029] In addition to reducing plasma levels of fatty acids, a
compound of the invention acts on the appetite centres in the
brain. In particular, a compound of the invention increases the
levels of various anorexigenic neuropeptides (neuropeptides known
to be associated with decreased food intake and decreased appetite)
in the appetite centres of the brain and also induces higher levels
of malonyl CoA, a metabolite associated with decreased appetite and
food intake. The invention therefore further provides a compound of
the invention as defined above for use in treating a condition
where weight loss or weight gain is implicated. For example, the
compound may be used in suppressing appetite, treating obesity,
promoting weight loss, maintaining a healthy weight or decreasing
the ratio of fat to lean muscle in a subject. The subject in each
case may be a healthy subject or a compromised subject. A healthy
subject may be, for instance, an individual of healthy weight for
whom physical performance and/or physical appearance is important.
Examples include members of the military, athletes, body builders
and fashion models. A compromised subject may be an individual of
non-healthy weight, for instance an individual who is overweight,
clinically obese or clinically very obese. A compromised subject
may alternatively be an individual of healthy or non-healthy weight
who is suffering from a clinical condition, for instance a
condition listed below.
[0030] An individual of healthy weight has a body mass index (BMI)
of 18.5 to 24.9; an individual who is overweight has a body mass
index (BMI) of from 25 to 29.9; an individual who is clinically
obese has a body mass index of from 30 to 39.9; and an individual
who is clinically very obese has a body mass index of 40 or
more.
[0031] In addition to reducing plasma levels of fatty acids and
acting on the appetite centre in the brain, a compound of the
invention increases brain metabolic efficiency, by increasing brain
phosphorylation potential and the .DELTA.G' of ATP hydrolysis. A
compound of the invention thereby promotes improved cognitive
function and can be used to treat cognitive dysfunction or to
reduce the effects of neurodegeneration. A compound of the
invention also increases the level of the neuropeptide Brain
Derived Neurotropic Factor (BDNF) in both the paraventricular
nucleus (the appetite centre of the brain) and the hippocampus (a
part of the brain known to be important for memory). As well as
decreasing appetite, BDNF is known to prevent apoptosis and promote
neuronal growth in basal ganglia and other areas of interest, thus
the increased levels of BDNF produced by the compound of the
invention are expected to inhibit neurodegeneration, limit neural
tissue death after hypoxia or trauma and promote neural tissue
growth.
[0032] A compound of the invention also increases the level of the
anorexigenic neuropeptide Cocaine-and-Amphetamine Responsive
Transcript (CART). CART is known to promote alertness as well as to
decrease appetite. Thus, the increased levels of CART produced by
the compound of the invention are expected to improve cognitive
function.
[0033] The compounds of the invention are therefore useful for (a)
promoting alertness and improved cognitive function, and (b)
inhibiting neurodegeneration. The invention therefore further
provides a compound of the invention as defined above for use in
promoting alertness or improving cognitive function, or in treating
cognitive dysfunction.
[0034] The invention also provides a compound of the invention as
defined above for use in treating, preventing, or reducing the
effects of, neurodegeneration, free radical toxicity, hypoxic
conditions or hyperglycaemia.
[0035] In one embodiment, the compound of the invention as defined
above is for use in treating, preventing, or reducing the effects
of, neurodegeneration. A compound of the invention may be used to
treat, prevent, or reduce the effects of neurodegeneration arising
from any particular cause. The neurodegeneration may for instance
be caused by a neurodegenerative disease or disorder, or may be
caused by aging, trauma, anoxia and the like. Examples of
neurodegenerative diseases or disorders that can be treated using a
compound of the invention include, but are not limited to
Alzheimer's disease, Parkinson's disease, amyotrophic lateral
sclerosis, epilepsy, astrocytoma, glioblastoma and Huntington's
chorea.
[0036] Further examples of conditions which a compound of the
invention may be used to prevent or treat include muscle
impairment, fatigue and muscle fatigue. Muscle impairment and
muscle fatigue may be prevented or treated in a healthy or
compromised subject. A compromised subject may be, for instance, an
individual suffering from myalgic encephalopathy (ME, or chronic
fatigue syndrome) or the symptoms thereof. A compound of the
invention may also be used to treat a patient suffering from a
condition such as diabetes, metabolic syndrome X or
hyperthyroidism, or a geriatric patient.
[0037] The aforementioned conditions are further examples of
conditions which are caused by, exacerbated by or associated with
elevated plasma levels of free fatty acids; the monoester compound
of the invention can therefore be used to treat these
conditions.
[0038] In another embodiment, a compound of the invention is used
to treat a patient suffering from a condition selected from
diabetes, hyperpyrexia, hyperthyroidism, metabolic syndrome X,
fever and infection, or a geriatric patient.
[0039] A compound of the invention may be administered in
combination with one or more additional agents, for instance an
agent selected from micronutrients and medicaments. The compound of
the invention and the additional agent may be formulated together
in a single composition for ingestion. Alternatively the compound
of the invention and the additional agent may be formulated
separately for separate, simultaneous or sequential
administration.
[0040] When the additional agent is a medicament it may be, for
instance, a standard therapy for a condition from which the subject
is suffering. For instance, a compound of the invention may be
administered in combination with conventional anti-diabetic agents
to a subject suffering from diabetes. Conventional anti-diabetic
agents include insulin sensitisers such as the thiazolidinediones,
insulin secretogogues such as sulphonylureas, biguanide
antihyperglycemic agents such as metformin, and combinations
thereof.
[0041] When the additional agent is a micronutrient it may be, for
instance, a mineral or a vitamin. Examples include iron, calcium,
magnesium, vitamin A, the B vitamins, vitamin C, vitamin D and
vitamin E.
[0042] Ketone bodies act on niacin receptors. A compound of the
invention may therefore advantageously be administered in
combination with niacin (vitamin B3) as both ketone bodies and
niacin act on adipose tissue to inhibit free fatty acid
release.
[0043] The compound of the invention as defined above, namely
3-hydroxybutyl 3-hydroxybutyrate enantiomerically enriched with
respect to the (3R, 3R') enantiomer, can be formulated into an
ingestible composition which further comprises a dietetically or
pharmaceutically acceptable carrier. The compositions may be food
products, beverages, drinks, supplements, dietary supplements,
functional foods, nutraceuticals or medicaments.
[0044] The concentration of the compound of the invention in the
ingestible composition depends on a variety of factors, including
the particular format of the composition, the intended use of the
composition and the target population. Generally the composition
will contain the compound of the invention in an amount effective
to reduce plasma levels of free fatty acids. Typically the amount
is that required to achieve a circulating concentration of
beta-hydroxybutyrate (bHB) and/or acetoacetate of from 10 .mu.M to
20 mM, preferably from 50 .mu.M to 10 mM, more preferably from 100
.mu.M to 5 mM, in a subject who ingests the composition. In one
embodiment, an amount is used to achieve a circulating
concentration of from 0.7 mM to 5 mM, for example from 1 mM to 5
mM.
[0045] The subject of the present invention is hydrolysed rapidly
into two natural products, beta-hydroxybutyrate (bHB) and
(R)-1,3-butanediol, and is therefore a natural calorie source which
can be classified as a food and can form part of a food
product.
[0046] A food product is an edible material composed primarily of
one or more of the macronutrients protein, carbohydrate and fat,
which is used in the body of an organism to sustain growth, repair
damage, aid vital processes or furnish energy. A food product may
also contain one or more micronutrients such as vitamins or
minerals, or additional dietary ingredients such as flavourants and
colourants. Examples of food products into which the compound of
the invention may be incorporated as an additive include snack
bars, meal replacement bars, cereals, confectionery and probiotic
formulations including yoghurts.
[0047] Examples of beverages and drinks include soft beverages,
energy drinks, dry drink mixes, nutritional beverages, meal or food
replacement drinks, compositions for rehydration (for instance
during or after exercise) and herbal teas for infusion or herbal
blends for decoction in water.
[0048] A composition for rehydration typically comprises water, a
sugar carbohydrate and the compound of the invention. The
composition may also comprise suitable flavourings, colourants and
preservatives, as will be appreciated by the skilled person. The
carbohydrate sugar is present as an energy source, and suitable
sugars are known, including glucose and trehalose. A meal or food
replacement drink may be of the type commonly advocated for use in
weight loss regimens. Such drink formulations typically comprise
appropriate quantities of one or more macronutrients, i.e. sources
of protein, fat and/or carbohydrate, together with optional
additional ingredients such as solubilising agents, preservatives,
sweetening agents, flavouring agents and colourants.
[0049] A nutraceutical is a food ingredient, food supplement or
food product which is considered to provide a medical or health
benefit, including the prevention and treatment of disease. In
general a nutraceutical is specifically adapted to confer a
particular health benefit on the consumer. A nutraceutical
typically comprises a micronutrient such as a vitamin, mineral,
herb or phytochemical at a higher level than would be found in a
corresponding regular food product. That level is typically
selected to optimise the intended health benefit of the
nutraceutical when taken either as a single serving or as part of a
diet regimen or course of nutritional therapy. In the present case
the level would be a level effective to reduce plasma levels of
fatty acids,
[0050] A functional food is a food that is marketed as providing a
health benefit beyond that of supplying pure nutrition to the
consumer. A functional food typically incorporates an ingredient
such as a micronutrient as mentioned above, which confers a
specific medical or physiological benefit other than a nutritional
effect. A functional food typically carries a health claim on the
packaging.
[0051] In accordance with the present invention a nutraceutical or
functional food product typically contains the compound of the
invention as defined above in an amount effective to lower plasma
levels of free fatty acids in a subject. More typically the
nutraceutical or functional food product contains the compound in
an amount effective to suppress appetite, treat obesity or promote
weight loss in a subject.
[0052] A dietary supplement is a product that is intended to
supplement the normal diet of a human subject and which contains a
dietary ingredient such as a vitamin, mineral, herb or other
botanical product, or amino acid. A dietary supplement is typically
presented in unit dosage format and is designed for consumption
with, before or after food but not in place of food. A dietary
supplement is thus often presented as a tablet or capsule, or as
dried powder or granules for sprinkling over food or adding to
water or a beverage.
[0053] A compound of the invention as defined above may be
formulated into a medicament or a dietary supplement by mixing with
a dietetically or pharmaceutically acceptable carrier or excipient.
Such a earner or excipient may be a solvent, dispersion medium,
coating, isotonic or absorption delaying agent, sweetener or the
like. These include any and all solvents, dispersion media,
coatings, isotonic and absorption delaying agents, sweeteners and
the like. Suitable carriers may be prepared from a wide range of
materials including, but not limited to, diluents, binders and
adhesives, lubricants, disintegrants, colouring agents, bulking
agents, flavouring agents, sweetening agents and miscellaneous
materials such as buffers and adsorbents that may be needed in
order to prepare a particular dosage form. The use of such media
and agents for pharmaceutically active substances is well known in
the art.
[0054] For example, the solid oral forms may contain, together with
the active compound, diluents such as lactose, dextrose,
saccharose, cellulose, corn starch or potato starch; lubricants
such as silica, talc, stearic acid, magnesium or calcium stearate
and/or polyethylene glycols; binding agents such as starches,
arabic gums, gelatin, methyicellulose, carboxymethylcellulose, or
polyvinyl pyrrolidone; disintegrating agents such as starch,
alginic acid, alginates or sodium starch glycolate; effervescing
mixtures; dyestuffs, sweeteners; wetting agents such as lecithin,
polysorbates, lauryl sulphates. Such preparations may be
manufactured in known manners, for example by means of mixing,
granulating, tabletting, sugar coating, or film-coating
processes.
[0055] Liquid dispersions for oral administration may be syrups,
emulsions and suspensions. The syrups may contain as carrier, for
example, saccharose or saccharose with glycerol and/or mannitol
and/or sorbitol. In particular, a syrup for diabetic patients can
contain as carriers only products, for example sorbitol, which do
not metabolise to glucose or which only metabolise a very small
amount to glucose. The suspensions and the emulsions may contain as
carrier, for example, a natural gum, agar, sodium alginate, pectin,
methyicellulose, carboxymethylcellulose or polyvinyl alcohol.
[0056] A compound of the invention as defined above is also
suitably formulated into granules or a powder. In this form it can
be readily dispersed in water or other liquid such as tea or a soft
drink for human subjects to drink, for instance a beverage or drink
as described above. It may also be encapsulated, tabletted or
formulated with a physiologically acceptable vehicle into unit
dosage forms. A unit dosage can comprise a therapeutically
effective amount of the extract for a single daily administration,
or it can be formulated into smaller quantities to provide for
multiple doses in a day. The composition may thus, for instance, be
formulated into tablets, capsules, syrups, elixirs, enteral
formulations or any other orally administrable form.
[0057] Examples of physiologically acceptable carriers include
water, oil, emulsions, alcohol or any other suitable material.
The invention will be further described in the Examples which
follow.
Example 1 Synthesis of (3R)-hydroxybutyl (3R)-hydroxybutyrate
##STR00002##
[0059] The ethyl (3R)-hydroxybutyrate (ca. 3 kg),
(R)-1,3-butanediol (ca. 1.5 kg), and solid-supported Candida
antarctica lipase B (ca. 300 g) are combined in a 20 litre rotary
evaporator flask and placed on a large-scale Buchi evaporator. The
system is evacuated to 8-10 torr with rotation at 40-45 C until the
diol is consumed (as analysed by .sup.1H NMR spectroscopy; ca. 3
days). The crude material is filtered (neat) to separate the enzyme
and excess ethyl (3R)-hydroxybutyrate is removed by evaporation (to
a final pressure and temperature of 2-3 torr and 80-85 C).
Throughout, chilled water is circulated [-5 C during the reaction,
+5 C during removal of ethyl (3R)-hydroxybutyrate]. Activated
carbon (8 large spatula measures) is added, mixing on the rotary
evaporator is continued for 15 min and then the neat mixture is
filtered through a Celite.RTM. plug, the product (filtrate) being
decanted directly into plastic vessels for storage. The Celite.RTM.
plug is washed with ether (ca. 500 mL), the solvent removed from
the washings in vacuo, and the residue added to the bulk for
storage.
Example 2 In Vivo Testing of (3R)-hydroxybutyl
(3R)-hydroxybutyrate--Calorie-Controlled Diets
[0060] Young adult male Wistar rats (starting weight 70 g) (Harlan
UK Limited) (n=50) were housed at approximately 20.degree. C. on a
12 h:12 h reverse light:dark photoperiod. Rats were fed standard
laboratory chow (Chow) (SDS, Essex, UK) prior to the starting the
experimental diets: (a) normal "Western" diet (Western) in which
34% of kilocalories came from added palmitate (n=20), (b)
high-carbohydrate (CHO) in which 70% of kilocalories came from
added corn starch (n=10) or (c) (3R)-hydroxybutyl
(3R)-hydroxybutyrate diet (monoester) in which 30% of kilocalories
came from (3R)-hydroxybutyl (3R).about.hydroxybutyrate (n=20).
[0061] The macronutrient compositions of the three diets are shown
below. All diets contained the same energy in kCal/g but had
different macronutrients.
TABLE-US-00001 TABLE 1 Energy Carbohydrate Diet (kCal/g) Fat
Protein (% kCal) Monoester Western 1.76 34 27 39 0 Carbohydrate
1.76 4 26 70 0 Monoester 1.76 4 27 39 30
[0062] Diets and the monoester were manufactured at the University
of Oxford. Water was provided ad libitum. This research project was
approved by Oxford Animal Ethics Review Committees and the Home
Office.
[0063] Rats were individually housed one week prior to the start of
the experiments, so that they were accustomed to living in a
solitary environment by the time the study started. They consumed
standard laboratory chow ad libitum until they were placed on their
experimental diet. Rats fed the Western and carbohydrate diets were
fed the same number of calories as those consumed by the
monoester-fed rats the previous day.
[0064] All rats were fed for 66 days. After this period the rat
body, heart and fat pad weights were determined. The results are
shown in Table 2:
TABLE-US-00002 TABLE 2 Physical characteristic of Western
Carbohydrate Monoester diet rat diet (n = 20) diet (n = 10) (n =
20) Final body weight (g) 226 .+-. 5 213 .+-. 8 213 .+-. 5 Heart
weight (g) 0.69 .+-. 0.02 0.65 .+-. 0.02 0.64 .+-. 0.02 Heart to
body weight (g) 0.31 .+-. 0.01 0.31 .+-. 0.02 0.29 .+-. 0.01
Epididymal fat (g) 2.49 .+-. 0.3 1.99 .+-. 0.2 1.48 .+-. 0.2*
Epididymal fat to body 1.08 .+-. 0.2 0.93 .+-. 0.2 0.69 .+-. 0.1*
weight *P < 0.05
[0065] The results in Table 2 show that the fat pad weight was
significantly lower at the end of the 66-day test in the rats that
had been fed the monoester diet than in the rats fed either the
Western (i.e. high fat) diet or the carbohydrate diet. The fat to
body weight was also significantly lower for the rats fed
monoester.
Example 3: In Vivo Testing of (3R)-hydroxybutyl
(3R)-hydroxybutyrate--Meal-Fed Diets
[0066] Example 2 was repeated using the same foods as shown in
Table 1 but where the rats were meal-fed. Rats in this example
therefore had a free choice of how much food to eat at each meal,
rather than being calorifically-controlled as in Example 2.
[0067] The daily body weight (in grams per rat per day) was plotted
against time for rats in each of the three diet groups over the
first six days of the test. The resulting graph is shown in FIG. 1.
One-way analysis of variance with Tukey-Kramer multiple comparison
test was used (n=8 per group, **p<0.001). Significantly reduced
body weight is seen from the 3.sup.rd to 6.sup.th days in rats fed
the monoester diet. The body weight of rats in the group fed the
carbohydrate diet remained high throughout the feeding process.
[0068] Meal-fed rats on the monoester diet ate less food and lost
more weight than rats on the other two diets. The daily food intake
(in grams per rat per day) was plotted against time for rats in
each of the three diet groups over the first seven days of the
test. The resulting graph is shown in FIG. 2. Again, one-way
analysis of variance with Tukey-Kramer multiple comparison test was
used (n=8 per group, ***p<0.001). Rats on the monoester diet
displayed a consistently reduced daily food intake throughout the
period compared with rats on the carbohydrate and Western
diets.
Example 4: In Vivo Testing of (3R)-hydroxybutyl
(3R)-hydroxybutyrate--Isonitrogenous and Isocaloric Diets
[0069] In a 28-day study, male and female Wistar rats weighing
approximately 350 g were randomized to one of three diet groups
(n=10 males and 10 females/group) and administered either a
carbohydrate diet (CHOD), a normal human diet (NHD), or a ketone
diet (KD). The diets were isonitrogenous and isocaloric, differing
only in their relative amounts of carbohydrate, fat, and ketones.
In the KD, the ketone used was the ketone monoester
(3R)-hydroxybutyl (3R)-hydroxybutyrate diet (i.e. the compound of
the invention). In the KD, approximately 1/3 of the energy was
derived from the ketone ester, while in the NHD and CHOD, 1/3 of
the energy was derived from palmitate and starch, respectively. The
compositions of the experimental diets (expressed in terms of % of
calories) are summarized in Table 3.
TABLE-US-00003 TABLE 3 Normal Human Carbohydrate Ketone Diet (KD)
Diet (NHD) Diet (CHOD) Carbohydrate 38.5 38.6 70.2 Protein 26.9
27.0 26.2 Fat 3.7 34.3 3.7 Ketone monoester 31 0 0 Total 100 100
100
[0070] The compositions of the experimental diets (g/100 g) are
summarized in Table 4 below.
TABLE-US-00004 TABLE 4 Ketone Normal Human Diet Carbohydrate Diet
Diet (NHD) (CHOD) Rodent chow 25.7 25.7 25.7 Sugar-free jelly 13.4
13.4 13.4 Water 49.6 55.1 46.3 Palm oil 0 5.8 0 Corn flour 0 0 14.5
Ketone monoester 11.4 0 0 Total 100 100 100
[0071] On day 28 of the study, male rats in the KD group weighed
significantly less than male rats in the CHOD and NHD groups
(390.+-.26 g vs. 418.+-.15 g and 413.+-.16 g, respectively). The
total amount of weight gained by male rats in the KD group was
significantly less than the total amount of weight gained by the
CHOD and NHD groups. Feed consumption was significantly lower in
males fed the KD compared with males fed the CHOD and NHD diets
(feed intake during days 22 to 29: 239.+-.17 g vs. 269.+-.7 g and
269.+-.7 g, respectively). The average intake of the ketone
monoester in males was approximately 11 g/kg body weight/day.
[0072] On days 15, 22, and 28 of the study, female rats in the KD
group weighed significantly less than female rats in the CHOD and
NHD groups (on day 28: 240.+-.13 g vs. 253.+-.12 g and 258.+-.13 g,
respectively). The total amount of weight gained by female rats in
the KD group was significantly less than the total amount of weight
gained by the CHOD and NHD groups. Feed consumption was
significantly lower in females fed the KD compared with females fed
the CHOD and NHD diets (feed intake during days 22 to 29: 175.+-.12
g vs. 191.+-.5 g and 194 f 7 g, respectively). The average intake
of the ketone monoester in females was approximately 13.0 g/kg body
weight/day.
Example 5: Effect of (3R)-hydroxybutyl (3R)-hvdroxybutyrate on
Neuropeptide Levels, Levels of Krebs Cycle and CoA Intermediates,
and Levels of Free Nucleotides in the Brain
[0073] In a 14-day study, Wistar rats weighing about 250 g were
randomized to one of three diet groups (n=6 rats/group) and
administered either a carbohydrate ("starch") diet, a normal human
("fat") diet or a ketone ester diet, in which the ketone ester used
was the ketone monoester (3R)-hydroxybutyl (3R)-hydroxybutyrate
diet (i.e. the compound of the invention). The three diets were
eaten in pair fed meals for 3 hours per day. The composition of the
diets expressed in terms of g/100 g are summarised in Table 5 and
the compositions expressed in terms of % of calories are summarised
in Table 6.
TABLE-US-00005 TABLE 5 Component Starch Fat Ketone Ester Chow 25.7
25.7 25.7 Sugar Free Jello 13.4 13.4 13.4 Water 46.3 55.1 49.6 Palm
Oil 0 5.8 0 Corn Starch 14.5 0 0 Ketone Ester 0 0 11.4 Total 100
100 100
TABLE-US-00006 TABLE 6 Component Starch Fat Ketone Ester
Carbohydrate 70.2 38.6 38.5 Protein 26.2 27 26.9 Fat 3.7 34.3 3.7
Ketone Ester 0 0 31 Total 100 100 100
[0074] The rats on the ketone ester diet were found to consume less
food and gain less weight than those eating the diets supplemented
by starch (carbohydrate) or fat. The results are consistent with
the results in Examples 2 to 4.
[0075] After eating the 3 diets in pair fed meals for 3 hours per
day for 14 days, the levels of various Krebs cycle and CoA
intermediates in the brain were measured enzymatically and by mass
spectrometry using standard techniques. The results are shown in
Table 7.
[0076] As can be seen in Table 7, the ketone ester fed rats were
found to have higher malonyl CoA levels in the brain (bold font in
the table). Malonyl CoA is a metabolite known to be associated with
decreased food intake; it is known to decrease appetite (Wolfgang,
M. J. and Lane, M. D. (2006) J. Biol. Chem. 281, 37265-37269).
These data are consistent with the use of a diet comprising the
ketone compound of the invention to decrease appetite. The values
in Table 7 are means, in .mu.moles/g wet weight, .+-.SEM with n=6
to 8. CoA's are given in nmol/g wet weight.
TABLE-US-00007 TABLE 7 BRAIN KREBS CYCLE AND COA INTERMEDIATES
Starch Fat Ketone Ester Citrate 0.199 .+-. 0.006 0.205 .+-. 0.005
0.222 .+-. 0.010 Isocitrate 0.0080 .+-. 0.0006 0.0086 .+-. 0.0004
0.0093 .+-. 0.008 .alpha.-ketoglutarate 0.128 .+-. 0.007 0.138 .+-.
0.008 0.140 .+-. 0.008 Succinyl CoA 0.831 .+-. 0.075 0.777 .+-.
0.158 0.910 .+-. 0.207 (.mu.M) Succinate 0.0800 .+-. 0.0022 0.0822
.+-. 0.0034 0.0864 .+-. 0.0033 Fumarate 0.0728 .+-. 0.0042 0.0801
.+-. 0.0055 0.0745 .+-. 0.0058 L-Malate 0.179 .+-. 0.011 0.192 .+-.
0.012 0.181 .+-. 0.013 Calc 0.0026 .+-. 0.0003 0.0021 .+-. 0.0001
0.0026 .+-. 0.0004 Oxaloacetate Acetyl CoA 7.87 .+-. 1.32 8.20 .+-.
1.02 6.43 .+-. 0.53 (.mu.M) Malonyl CoA 0.954 .+-. 0.061 1.02 .+-.
0.14 .sup. 1.26 .+-. 0.13.sup.a (.mu.M) .sup.ap < 0.05 between
ketone ester and starch
[0077] After the rats were fed on the diets for 14 days, the ratios
of free nucleotide and the free nucleotide concentrations were
determined. Measurements were performed on the freeze blown brain
and metabolite ratios of the rats calculated as previously
described (Veech, R. L. et al, J. Biol. Chem. 254: 6538-47, 1979).
The results are shown in Table 8. The values in Table 8 are given
as means.+-.SEM (n=6 to 8). Cytosolic pH was assumed to be 7.2.
TABLE-US-00008 TABLE 8 CALCULATED FREE NUCLEOTIDE RATIOS AND
CALCULATED FREE NUCLEOTIDE CONCENTRATIONS Starch Fat Ketone Ester
Free [NAD.sup.+]/[NADH] 319 .+-. 23 256 .+-. 29.sup.a,b 357 .+-. 27
cytosol from Lactate/Pyruvate Free [NAD.sup.+]/[NADH] 0.62 .+-.
0.08 0.66 .+-. 0.06 0.80 .+-. 0.17 mitochochondria from
.alpha.KGxNH.sub.4.sup.+/Glut Free [NAD.sup.+]/[NADPH] 0.028 .+-.
0.002 0.028 .+-. 0.002 0.026 .+-. 0.003 cytosol from
.alpha.KGxCO.sub.2/IsoCit E.sub.hCoQ/CoQH.sub.2 mV from 29.6 .+-.
0.5 30.5 .+-. 0.8 28.9 .+-. 0.6 Fum/Suce Free [Mg.sup.2+] mM from
1.5 .+-. 0.26 1.5 .+-. 0.11 1.5 .+-. 0.13 [Cit]/[Isocit]
Phosphorylation Potential 27,100 .+-. 4,020 26,800 .+-. 2,700
38,100 .+-. 3,390.sup.a,b from Kg + g (Eqn 1) M.sup.-1 .DELTA.G'
ATP kJ/mol -58.6 .+-. 0.4 -58.6 .+-. 0.2 -59.6 .+-. 0.2.sup.a,b
Free [ADP] cytosol .mu.M/g 0.027 .+-. 0.001 0.027 .+-. 0.001 0.027
.+-. 0.001 from PCr/Cr Free [AMP] cytosol .mu.M/g 0.0004 .+-.
0.00003 0.0004 .+-. 0.00003 0.0004 .+-. 0.00003 from
K.sub.myokinase .sup.ap < 0.05 between ketone ester and starch,
.sup.bp < 0.05 between ketone ester and fat, both as judged by
Mann-Whitney U test.
[0078] The results of Table show 8 that, after 14 days of diet,
brain phosphorylation potential and .DELTA.G' of ATP hydrolysis
were significantly higher in the ketone ester fed rats than in the
rats fed with the carbohydrate and fat diets. The only change in
the brain of the ketone-fed rats was increased energy:
phosphorylation and .DELTA.G. This increased energy is consistent
with the effects of ketones in the perfused working rat heart
(Sato, K., Kashiwaya, Y., Keon, C. A., Tsuchiya, N., King, M. T.,
Radda, G. K., Chance, B., Clarke, K., and Veech, R. L. (1995) FASEB
J. 9, 651-658). The huge redox changes that are observed in the
heart with ketone perfusion are however not observed.
[0079] These data are consistent with the use of a diet comprising
the ketone compound of the invention to increase brain metabolic
efficiency and thereby promote improved cognitive function, treat
or reduce the effects of neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease and Huntington's chorea
or, for instance, protect the brain and central nervous system
against neurodegeneration due to aging, trauma, anoxia and the
like.
[0080] Effects of the Ketone Compound of the Invention on
Neuropeptide Signalling in the Brain
[0081] After 14 days of feeding with the fat, carbohydrate or
ketone ester diet, the levels of various neuropeptides known to be
associated with decreased food intake and decreased appetite
("anorexigenic" peptides) were surveyed in the paraventricular
nucleus (PVN) area of the hypothalamus and in the hippocampus of
the rat brain. The neuropeptide levels were measured using standard
antibody techniques performed on sectioned brains of the rats. The
results are shown in the micrographs of FIGS. 3 to 9.
[0082] The peptides measured were Brain Derived Neurotropic Factor
(BDNF), melanocyte-stimulating hormone receptor 4 (MC4R), and
Cocaine-and-Amphetamine Responsive Transcript (CART). As well as
being anorexigenic these peptides have other important actions,
thus: [0083] BDNF decreases appetite and is also known to prevent
apoptosis in basal ganglia and other areas of interest, thus
increased levels of BDNF are expected to inhibit neurodegeneration
as well as decrease appetite; [0084] CART is known to promote
alertness and decrease appetite, in a manner similar to caffeine or
modafinil (a mood-brightening and memory-enhancing stimulant drug),
thus increased levels of CART are expected to improve cognitive
function as well as decrease appetite; [0085] MC4R facilitates the
break down of a large peptide into various hormones including
Melanocyte stimulating hormone, which in turn regulates appetite.
Mutations in MC4R are known to cause obesity.
[0086] These peptides are therefore of central importance in many
of the important therapeutic aspects of ketone ester feeding in
addition to the suppression of appetite. Most particularly in a)
the promotion of alterness and improved cognitive function, and b)
the inhibition of neurodegeneration from variety of causes such as
aging, trauma, anoxia and the like.
[0087] The results in FIG. 3 show significantly more BDNF-positive
cell bodies in the PVN of the rats treated with the ketone diet
compared to fat and carbohydrate diet-treated rats. A similar
observation is made in the hippocampus of rats treated with
ketones. The PVN is a part of the brain which is known to control
appetite, whereas the hippocampus is known to be important for
memory. The results therefore support that a diet comprising the
ketone compound of the invention can be used to decrease appetite,
inhibit neurodegeneration and promote improved cognitive
function.
[0088] The micrographs in FIGS. 4 and 5 show a significantly higher
density of MC4R positive cell bodies in the posterior magnocellular
(pm) and medial parvocellular (mpd) regions of the PVN in rats
treated with the ketone or carbohydrate (Cho) diet, compared to
rats treated with the fat diet. This supports that a diet
comprising the ketone compound of the invention can be used to
decrease appetite and promote weight loss.
[0089] FIGS. 6, 8 and 9 show the levels of CART in the posterior
paraventricular nucleus (PVN) area of the hypothalamus of rats,
treated for 14 days with the fat diet, carbohydrate diet or the
diet comprising the ketone compound of the invention. FIG. 7 shows
the levels of CART in the ventromedial nucleus (VMH), arcuate
nucleus (ARC) and median emminence (ME) areas of the hypothalamus.
Significantly greater numbers of CART positive cell bodies are seen
in the PVNs of rats treated with the ketone and carbohydrate diets,
than in the rats treated with the fat diet. The highest level of
CART is seen in the PVN in the rats treated with the ketone diet.
Furthermore, the rats treated with the ketone diet contain the
highest number of CART-positive cell bodies in the ventromedial
nucleus (VMH), arcuate nucleus
[0090] (ARC) and median emminence (ME) areas of the hypothalamus.
These results further support that a diet comprising the ketone
compound of the invention can be used to decrease appetite, inhibit
neurodegeneration and promote improved cognitive function. In
summary, the micrographs in FIGS. 3 to 9 show that the ketone diet
produces [0091] more BDNF in the paraventricular nucleus and
hippocampus (FIG. 3); [0092] more CART in the paraventricular
nucleus (FIGS. 6 to 9); and [0093] more MC4R activity in the
paraventricular nucleus (FIGS. 4 and 5).
[0094] These data are consistent with the use of a diet comprising
the ketone compound of the invention to decrease appetite, inhibit
neurodegeneration and promote improved cognitive function.
Example 6: Tablet Composition
[0095] Tablets, each weighing 0.15 g and containing 25 mg of a
compound of the invention were manufactured as follows:
Composition for 10,000 tablets
[0096] Compound of the invention (250 g)
[0097] Lactose (800 g)
[0098] Corn starch (415 g)
[0099] Talc powder (30 g)
[0100] Magnesium stearate (5 g)
[0101] The compound of the invention, lactose and half of the corn
starch were mixed. The mixture was then forced through a sieve 0.5
mm mesh size. Corn starch (10 g) is suspended in warm water (90
ml). The resulting paste was used to granulate the powder. The
granulate was dried and broken up into small fragments on a sieve
of 1.4 mm mesh size. The remaining quantity of starch, talc and
magnesium was added, carefully mixed and processed into
tablets.
Example 7: Syrup Formulation
TABLE-US-00009 [0102] Compound of invention 250 mg Sorbitol
Solution 1.50 g Glycerol 2.00 g Sodium benzoate 0.005 g Flavour
0.0125 ml Purified Water q.s. to 5.00 ml
[0103] The compound of the invention was dissolved in a mixture of
the glycerol and most of the purified water. An aqueous solution of
the sodium benzoate was then added to the solution, followed by
addition of the sorbital solution and finally the flavour. The
volume was made up with purified water and mixed well.
* * * * *