U.S. patent application number 09/799124 was filed with the patent office on 2001-08-16 for therapeutic compositions (ii).
Invention is credited to Veech, Richard Lewis.
Application Number | 20010014696 09/799124 |
Document ID | / |
Family ID | 22279402 |
Filed Date | 2001-08-16 |
United States Patent
Application |
20010014696 |
Kind Code |
A1 |
Veech, Richard Lewis |
August 16, 2001 |
Therapeutic compositions (II)
Abstract
Compositions comprising ketone bodies and/or their metabolic
precursors are provided that are suitable for administration to
humans and animals and which have the properties of, inter alia,
(i) increasing cardiac efficiency, particularly efficiency in use
of glucose, (ii) for providing energy source, particularly in
diabetes and insulin resistant states and (iii) treating disorders
caused by damage to brain cells, particularly by retarding or
preventing brain damage in memory associated brain areas such as
found in Alzheimer's and similar conditions. These compositions may
be taken as nutritional aids, for example for athletes, or for the
treatment of medical conditions, particularly those associated with
poor cardiac efficiency, insulin resistance and neuronal damage.
The invention further provides methods of treatment and novel
esters and polymers for inclusion in the compositions of the
invention.
Inventors: |
Veech, Richard Lewis;
(Rockville, MD) |
Correspondence
Address: |
Nixon & Vanderhye
Eighth Floor
1100 North Glebe Road
Arlington
VA
22201-4714
US
|
Family ID: |
22279402 |
Appl. No.: |
09/799124 |
Filed: |
March 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09799124 |
Mar 6, 2001 |
|
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PCT/US99/21015 |
Sep 15, 1999 |
|
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60100371 |
Sep 15, 1998 |
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Current U.S.
Class: |
514/449 ;
549/267 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 3/10 20180101; A61P 25/00 20180101; C07D 323/00 20130101; A61P
21/04 20180101; A61P 25/08 20180101; A61P 9/10 20180101; A61P 25/28
20180101; A61P 25/16 20180101 |
Class at
Publication: |
514/449 ;
549/267 |
International
Class: |
A61K 031/335; C07D
321/00 |
Claims
1. A cyclic ester of (R)-3-hydroxybutyrate of formula (I) 4where n
is an integer of 1 or more or a complex thereof with one or more
cations or a salt thereof for use in therapy or nutrition.
2. A compound as claimed in claim 1 wherein the one or more cations
are selected from the group consisting of sodium, potassium,
magnesium and calcium or where the compound is a free uncomplexed
oligolide.
3. A compound as claimed in claim 1 or claim 2 wherein n is an
integer from 1 to to 20.
4. A compound as claimed in claim 1 wherein it is
(R,R,R)-4,8,12-trimethyl- -1,5,9-trioxadodeca-2,6,10-trione.
5. A method of treating a cell that is subject to malfunction due
to action of free radicals, toxic agents such as peptides and
proteins and genetic defects deleterious to cell metabolism,
insulin resistance or other glucose metabolism defects or defect
inducing states, ischemia, head trauma, and/or for increasing cell
efficiency characterised in that it comprises administration of a
cyclic oligomer of formula (I).
6. A method as claimed in claim 4 characterised in that the cyclic
oligomer of formula (I) acts as a neuronal stimulant e.g. capable
of stimulating axonal and/or dendritic growth in nerve cells, e.g.
in hippocampal or substantia nigral cells, in vivo or in vitro,
particularly in conditions where neuro-degeneration has serious
clinical consequences.
7. A method of accomplishing enteral or parenteral nutrition,
preferably oral route nutrition, comprising administration of a
cyclic oligomer of formula (I) in physiologically acceptable form,
optionally in a physiologically acceptable carrier.
8. A method of producing a physiologically acceptable ketosis in a
human or animal comprising oral administration of a cyclic oligomer
of formula (I).
9. A method as claimed in claim 8 wherein the human or animal is
fed less than 50% by weight of its caloric content of its diet as
fat.
10. A method as claimed in claim 8 wherein the human or animal is
fed from 0 to 25% by weight of its caloric content of its diet as
fat.
11. A method as claimed in claim 7 or 8 wherein the method is
performed on a patient needing therapy for one or more of
Alzheimer's, Parkinsonism, Amylotrophic lateral sclerosis,
Epilepsy, Free radical disease, Heart failure, Type II diabetes,
deficiency or blockage of pyruvate dehydrogenase, inability to
perform glycolysis in one or more cell types and Duchenne's
muscular dystrophy.
12. A method of providing a caloric substitute for carbohydrate for
the purpose of lowering blood glucose comprising administering a
composition comprising a cyclic oligomer of formula (I) to a human
or animal subject in need of such substitution.
13. A method of providing a caloric substitute for carbohydrate for
the purpose of body lipid content reduction comprising
administering a composition comprising a cyclic oligomer of formula
(I) to a human or animal subject in need of such reduction.
14. A method of increasing the efficiency of mitochondrial energy
production in a human or animal not suffering from a chronic or
acute metabolic disease comprising administering to the human or
animal an amount of a cyclic oligomer of formula (I) sufficient to
raise blood levels of (R)-3-hydroxybutyrate to from 0.5 to 20
mM.
15. A method as claimed in claim 14 wherein the level is raised to
from 1 to 10 mM.
16. The use of a cyclic ester formula I for the manufacture of a
medicament for the treatment of disease states mediated by free
radicals, toxic agents such as peptides and proteins, genetic
defects deleterious to cell metabolism, insulin resistance or other
glucose metabolism defects or defect inducing states, ischemia,
head trauma, or for increasing cell efficiency.
17. A composition characterised in that it comprises a cyclic
oligomer of formula (I) in physiologically acceptable form.
18. A composition as claimed in claim 11 characterised in that it
includes a physiologically acceptable carrier.
Description
[0001] The present invention relates to compositions suitable for
administration to humans and animals which have the properties of
increasing levels of (R)-3-hydroxybutyrate ((R)-3-hydroxybutyric
acid or D-.beta.-hydroxybutyrate) when so administered;
particularly when administered orally, topically, subcutaneously or
parenterally, but most advantageously orally.
[0002] Administration of (R)-3-hydroxybutyric acid has a number of
beneficial actions on the human and animal body. These include
inter alia, increasing cardiac efficiency, e.g. in heart failure,
provision of an alternative energy source to glucose, e.g. in
diabetes and insulin resistant states, and treating disorders
caused by damage to neuronal cells, e.g. CNS cells, particularly by
retarding or preventing brain damage such as found in Alzheimer's
and Parkinsonism and similar diseases and conditions.
[0003] Sodium hydroxybutyrate has been shown to increase cerebral
circulation and regional vasomotor reflexes by up to 40% (Biull.
Eksp. Biol. Med Vol 88 11, pp 555-557). EP 0780123 A1 further
teaches use of acetoacetate, .beta.-hydroxybutyrate, monohydric,
dihydric or trihydric alcohol esters of these or linear oligomers
of 2 to 10 repeats of .beta.-hydroxybutyrate for suppressing
cerebral edema, protecting cerebral function, rectifying cerebral
energy metabolism and reducing the extent of cerebral
infarction.
[0004] Intravenous infusion of sodium salts of
(R)-3-hydroxybutyrate has been performed on normal human subjects
and patients for a number of conditions, e.g. those undergoing
treatment for severe sepsis in an intensive care unit and is found
to be non-toxic and capable of decreasing glucose free fatty acids
and glycerol concentration, but ineffective in decreasing leucine
oxidation.
[0005] The present inventor has further determined that compounds
and compositions that raise blood levels of (R)-3-hydroxybutyric
acid and/or acetoacetate also have utility in reducing free
radicals in vivo, and thus have a place in treatment of free
radical associated diseases.
[0006] (R)-3-hydroxybutyrate and acetoacetate, commonly referred to
as ketone bodies, provide a normal physiological alternative to the
usual energy producing substrates, glucose and fatty acids. During
prolonged fasting in man fatty acids are converted by liver to
(R)-3-hydroxybutyric acid and acetoacetate which can be utilized by
most major tissues of the body except liver. Under these
conditions, total blood ketone bodies are elevated to about 7 mM.
When these are modestly elevated in the blood, extrahepatic tissues
such as brain, heart and skeletal muscle utilize these ketone
bodies within the mitochondria to provide reducing power in the
form of NADH which is the primary substrate of the electron
transport system and generator of the energy required for the
synthesis of ATP. In turn, generation of mitochondrial NADH by
ketones, lowers the ratio of free mitochondrial [NAD.sup.+]/[NADH]
ratio and the cytosolic [NADP.sup.+]/[NADPH] ratio to which the
mitochondrial [NAD.sup.+]/[NADH] is linked. While the catabolism of
ketones reduces mitochondrial [NAD.sup.+]/[NADH] it oxidizes the
ratio of mitochondrial [ubiquinone]/[ubiquinol], [Q]/[QH.sub.2].
The semiquinone form of ubiquinol is the major source of the
generation by mitochondria of superoxide, O.sub.2.sup.-. By
decreasing the amount of the reduced form QH.sub.2, and its
semiquinone, one can decrease the generation of free radicals by
mitochondria while at the same time increasing the scavangers of
free radicals linked to the NADP system, such as glutathione.
[0007] The inventor has thus determined that free radical damage
resulting from excess reduced Q or inhibition of NADH
dehydrogenase, such as occurs in MPP induced toxicity, can be
reduced by administration of agents which elevate ketone body
levels in vivo.
[0008] A number of disease processes involve damage by free
radicals among which are the neurological diseases: Parkinson's
disease, amyotrophic lateral sclerosis, Alzheimer's disease and
cerebral ischemia. In addition excessive free radical damage has
been implicated as playing a role in coronary reperfusion, diabetic
angiopathy, inflammatory bowel disease and pancreatitis.
[0009] The inventor's copending WO 98/41201 discloses the
administration of linear esters of (R)-3-hydroxybutyric acid and/or
acetoacetate in producing elevated levels of the free compounds in
vivo. Oral administration of 4 mM solutions of the oligomer
tetra-(R)-3-hydroxybutyr- ate or its acetoacetyl ester was shown to
raise blood levels of ketone bodies such that (R)-3-hydroxybutyrate
levels could be measured to have increased by 1 to 2 mM for periods
in excess of 2 hours.
[0010] The inventor has now determined that unexpected advantages
are provided when the (R)-3-hydroxybutyric acid component of such
composition is administered as a cyclic oligomer. These advantages
may include, inter alia, (a) increased efficiency in raising blood
(R)-3-hydroxybutyric acid levels such that levels may be increased
by more than 2 mM, including attainment of near fasting levels and
beyond, (b) maintenance of elevated levels for periods of several
hours, (c) ability to be administered without counterion, such as
sodium or methylglucamine, where it is desirable not to increase a
patient's salt load or where significant dosing is envisaged and
(d) relative ease of manufacture of pure compound from polymeric
starting materials available through bioculture.
[0011] The present application particularly addresses the problem
of neurodegenerative diseases, particularly disease where neurons
are subject to neurotoxic effects of pathogenic agents such as
protein plaques and oxidative damage and further provides
compositions for use in treating these and the aforesaid
disorders.
[0012] In preferred embodiments the present invention provides
elevation of blood ketones necessary to correct the defects
described above and can be accomplished by parenteral or enteral
administration. Particularly it does not require the administration
of potentially toxic pharmacological agents. The present
invention's improved efficacy in raising levels, particularly blood
levels, of ketone bodies provides therapeutic effects of the
classical ketogenic diet, which is not itself found to be toxic in
children, with none of the side effects that render that unused
adults. Furthermore, the inventor has determined that with the
correction of the aforesaid metabolic and toxic defects, cytokine
responses and the increase in apoptotic peptides in degenerating
cells will decrease due to the increase in neuronal cell energy
status and the increased trophic stimulation resulting from
increased neurotransmitter, e.g. acetyl choline, synthesis.
[0013] The treatment that the present inventor provides goes beyond
ketone body effects on circulation, as it provides treatment for
cells that are unable to function due to neuro-degeneration and/or
metabolic defects, particularly in metabolism of glucose, e.g.
caused by neurotoxic agents such as peptides, proteins, free
radical damage and effect of genetic abnormality. The treatment
involves action of ketone bodies on the cells themselves and not
the flow of blood to them.
[0014] Thus in a first aspect of the present invention there is
provided a cyclic ester of (R)-3-hydroxybutyrate of formula (I)
1
[0015] where n is an integer of 1 or more
[0016] or a complex thereof with one or more cations or a salt
thereof for use in therapy or nutrition.
[0017] For oral delivery free cyclic oligomer may be preferred.
Where cations are present in a complex preferred cations are
sodium, potassium, magnesium and calcium, and are balanced by
physiologically acceptable counter-anion providing a salt
complex.
[0018] Examples of typical physiologically acceptable salts will be
selected from sodium, potassium, magnesium, L-Lysine and L-arginine
or e.g. more complex salts such as those of methyl glucamine
salts.
[0019] Preferably n is an integer from 1 to 200, more preferably
from 1 to 20, most preferably from 1 to 10 and particularly
conveniently is 1, i.e. (R, R,
R)-4,8,12-trimethyl-1,5,9-trioxadodeca-2,6,10-trione, 2,3,4 or
5.
[0020] The cyclic esters of the invention are preferably used in
the treatment of disease states mediated by free radicals, toxic
agents such as peptides and proteins, genetic defects detrimental
to nerve cell metabolism, insulin resistance or other glucose
metabolism defects or defect inducing states, ishemia, head trauma
and/or for increasing cell efficiency, e.g. cardiac cell efficiency
e.g. in heart failure.
[0021] A second aspect of the invention provides methods of
treating cells that are subject to malfunction due to action of
free radicals, toxic agents such as peptides and proteins, genetic
defects detrimental to cell metabolism, insulin resistance or other
glucose metabolism defects or defect inducing states, ischemia,
head trauma and/or for increasing cell efficiency characterised in
that it comprises administration of a cyclic oligomer of formula
(I). This may include treatment of such disease states in humans
and/or animals.
[0022] This aspect includes such use as a neuronal stimulant e.g.
capable of stimulating axonal and/or dendritic growth in nerve
cells, e.g. in hippocampal or substantia nigral cells, in vivo or
in vitro, particularly in conditions where neurodegeneration has
serious clinical consequences, through its elevating effect on
blood and plasma (R)-3-hydroxybutyrate and acetoacetate levels.
[0023] A third aspect of the invention provides a method of enteral
or parenteral nutrition, preferably oral route nutrition,
comprising administration of a cyclic oligomer of formula (I).
[0024] A fourth aspect of the invention provides the use of a
cyclic ester formula I for the manufacture of a medicament for the
treatment of disease states mediated by free radicals, toxic agents
such as peptides and proteins, genetic defects detrimental to cell
metabolism, insulin resistance or other glucose metabolism defects
or defect inducing states, ischemia, head trauma and/or for
increasing cell efficiency.
[0025] A fifth aspect of the invention provides composition
characterised in that it comprises a cyclic oligomer of formula (I)
in physiologically acceptable form e.g. with a physiologically
acceptable carrier.
[0026] Particularly the composition is suitable for parenteral or
enteral administration, particularly for oral administration. Where
the composition is for parenteral use it is sterile and pyrogen
free. For oral use the composition may include a foodstuff base and
may be in the form of an emulsion or mere admixture with solid
food.
[0027] Particularly the cyclic oligomer(s) comprise an effective
amount of the total composition, e.g. at least 2% or more, e.g. at
least 5%, of the composition by weight, more preferably 20% or more
and most preferably 50% to 100%. The composition may be adapted for
oral, parenteral or any other conventional form of
administration.
[0028] In preferred forms of all of the aspects of the invention
the compound of formula (I) is administered together with a
physiological ratio of acetoacetate or a metabolic precursor of
acetoacetate. The term metabolic precursor thereof particularly
relates to compounds that incorporate acetoacetyl moieties such as
acetoacetyl-1,3-butandiol, preferably
acetoacetyl-(R)-1,3-butandiol, acetoacetyl-(R)-3-hydroxybutyra- te,
and acetoacetylglycerol. Esters of any such compounds with
monohydric, dihydric or trihydric or higher, e.g. glucosyl,
alcohols are also envisaged.
[0029] In diabetic patients this use of the cyclic oligomers allows
maintenance of low blood sugar levels without fear of hypoglycemic
complications. In normal non-diabetic subjects the fasting blood
sugar is 80 to 90 mg % (4.4-5 mM) rising to 130 mg % (7.2 mM) after
a meal. In diabetics `tight control` of diabetes has long been
recommended as a method for retardation of vascular complications
but, in practice, physicians have found it difficult to keep blood
sugars tightly controlled below 150 mg % (8.3 mM) after eating
because of hypoglycaemic episodes. Hypoglycaemic coma occurs
regularly in normal subjects whose blood sugar drops to 2 mM. As
discussed earlier, (62, 63) in the presence of 5 mM blood ketones
there are no neurological symptoms when blood sugars fall to below
1 mM.
[0030] The present inventor has determined that supplementing type
II diabetics with cyclic oligomers of the invention will allow
better control of blood sugar, thus preventing the vascular changes
in eye and kidney which occur now after 20 years of diabetes and
which are the major cause of morbidity and mortality in
diabetics.
[0031] Where the therapy is aimed at seizure related disorders,
such as refractory epilepsy as is treated by the ketogenic diet,
therapy is improved by use of cyclic oligomers, due to the
reduction or elimination of both high lipid and carbohydrate
content. Such patients include those with genetic defects in the
brain glucose transporter system, in glycolysis or in PDH itself
such as in Leigh's syndrome, endotoxic shock or high stress
states.
[0032] Particular disorders treatable with these medicaments are
applicable to all conditions involving PDH blockage, including
those conditions occuring after head trauma, or involving reduction
or elimination of acetyl CoA supply to the mitochondrion such as
insulin coma and hypoglycaemia, defects in the glucose transporter
in the brain, or elsewhere (80), or in glycolytic enzyme steps.
[0033] Where the medicament or nutraceutical comprises acetoacetate
it is preferably not stored for a prolonged period or exposed to
temperatures in excess of 40.degree. C. Acetoacetate is unstable on
heating and decomposes violently at 100.degree. C. into acetone and
CO.sub.2. In such circumstances it is preferred that acetoacetate
is generated by the composition on contact with the bodies
metabolic processes. Preferably the composition comprises an ester
precursor of actetoacetate.
[0034] A sixth aspect of the invention provides a method of
treating a human or animal neuronal cell, e.g. brain cells, subject
to cell damage related disorder, particularly those which lead to
cell death, as referred to for the second to fourth aspects,
particularly a neurodegenerative disorder e.g. such as those
related to neurotoxic conditions such as presence of amyloid
protein, e.g. a memory or movement associated disorder such as
Alzheimer's or Parkinson's diseases, or epileptic seizures,
comprising administering to that person at least one of the
materials for use in the first to fifth aspects of the
invention.
[0035] The inventor has further determined that ketone bodies,
provided by administration of the cyclic oligomers of
(R)-3-hydroxybutyric acid in amounts sufficient to raise total
blood ketone body concentration to elevated levels result in more
than simple maintenance of cell viability but actually improve cell
function and growth beyond that of normal, i.e. control levels in a
manner unrelated to blood flow or nutrition. In this respect the
invention further provides use of the cyclic oligomers as agents
capable of producing neuronal stimulation, i.e. nerve growth factor
like activity, increase of metabolic rate and increase of extent of
functional features such as axons and dendrites. This aspect of the
present invention offers a mechanism for improvement of neuronal
function as well as mere retardation of degredation.
[0036] The recent work of Hoshi and collaborators (77, 78) strongly
suggests that a part of the amyloid protein whose accumulation is
the hallmark of Alzheimer's disease, A.beta..sub.1-42, acts to
stimulate mitochondrial histidine protein kinase which
phosphorylates and inactivates the pyruvate dehydrogenase
multienzyme complex. The PDH complex is a mitochondrial enzyme
responsible for the generation of acetyl CoA and NADH from the
pyruvate produced by glycolysis within the cytoplasm. The
mitochondrial acetyl CoA formed condenses with oxaloacetate to
start the Krebs TCA cycle completely combusting pyruvate to
CO.sub.2 while providing the mitochondria with the reducing power
which becomes the substrate for the electron transport system
through which the energy required for mitochondrial ATP synthesis
is generated
[0037] Ketone body utilization in brain is limited by the
transport, with lesser utilization occurring in the basal ganglion
at blood levels below 1 mM (76). However, at levels of 7.5 mM
achieved in normal man by prolonged fasting, the rate of ketone
body entry into brain is sufficient to take over the majority of
cerebral energy needs and to prevent hypoglycemic symptoms, even in
the face of blood sugar levels which would normally cause
convulsions or coma (63) .
[0038] In the copending application WO 98/41201, `Therapeutic
compositions`, it is the inventor's hypothesis that in Alzheimer's
disease, where there is a block at PDH which prevents the normal
energy production from glucose, if one can provide elevated, e.g.
normal fasting levels of ketones, one can bypass the PDH blockade
present in these patients thereby preventing cell death due to
energy depletion or lack of cholinergic stimulation and thus slow
the progression of the memory loss and dementia. Furthermore,
utilising the nerve growth/stimulatory effects of the ketone
bodies, particularly (R)-3-hydroxybutyrate or a physiological ratio
of this with acetoacetate, cells that are still viable can be
caused to improve beyond the state to which they have degenerated
and accordingly some improvement of function will be seen in
patients.
[0039] In fed animals and in man the liver content, which is
essentially that of blood, of acetoacetate is very low, e.g. 0.09
mM and (R)-3-hydroxybutyrate is 0.123 mM but rises after a 48 hour
fast to e.g. 0.65 mM acetoacetate and 1.8 mM (R)-3-hydroxybutyrate
(84). The ketone bodies rise in starvation because the fall in
insulin decreases the re-esterification of fatty acids to
triglyceride in adipose tissue causing the release of free fatty
acids into the blood stream. The released free fatty acids can then
be taken up and used as a source of energy by muscle, heart, kidney
and liver in the process of .beta.-oxidation. Liver, however, has
the capacity to convert the free fatty acids to a metabolic fuel,
ketones, for use by extra-hepatic organs, including the brain, as
an alternative to glucose during periods of fasting. The hepatic
synthesis of ketone bodies occurs from mitochondrial acetyl CoA
generated during the .beta.-oxidation of fatty acids by liver.
[0040] The ketone bodies enter extra-hepatic tissues on the same
carrier, where other monocarboxylates can act as competitive
inhibitors. Unphysiological isomers such as D-lactate or
(S)-3-hydroxybutyrate can also act as competitive inhibitors to
ketone body transport. Since ketone body transport across the blood
brain barrier is a limiting factor to ketone body utilization in
brain (76) every effort should be made to keep the blood
concentration of these unphysiological enantiomers at low levels
during ketogenic therapy. When blood ketone body concentrations are
elevated to levels found in starvation, heart, muscle, kidney and
brain utilize ketone bodies as the preferred energy substrate.
[0041] The present inventor has thus determined that the
mitochondrial acetyl CoA derived from ketone bodies as produced
using the cyclic oligomers taught by the present invention can thus
replace the acetyl CoA deficiency which occurs during inhibition of
PDH multienzyme complex in tissues dependent upon the metabolism of
glucose for their supply of metabolic energy. The mitochondrial
citrate supplied can also be transported to cytoplasm by the tri or
dicarboxcylic acid transporter where it can be converted to
cytoplasmic acetyl CoA required for the synthesis of acetyl
choline. The reactions of the Krebs cycle are shown in Scheme 1 to
help illustrate these concepts further.
[0042] Ketone bodies, in contrast to free fatty acids, cannot
produce acetyl CoA in liver. Since acetyl CoA is the essential
precursor of fatty acid, they cannot result in either increased
fatty acid or cholesterol synthesis in liver, which usually
accounts for over half of the body's synthesis of these two
potentially pathogenic materials. Liver is sensitive to the ratio
of acetoacetate/(R)-3-hydroxybutyrate presented to it and will
alter its mitochondrial free [NAD.sup.+]/[NADH], because of the
near equilibrium established by .beta.-hydroxybutyrate
dehydrogenase (EC 1.1.1.30) (31).
[0043] Inter alia, the aforementioned also indicates that one can
provide a method of increasing the efficiency of mitochondrial
energy production in a human or animal not suffering from a chronic
or acute metabolic disease comprising administering to the human or
animal an amount of a cyclic oligomer of formula (I) sufficient to
raise blood levels of (R)-3-hydroxybutyrate to from 0.5 to 20 mM.
2
[0044] The easiest way to increase blood ketones is starvation. On
prolonged fasting blood ketones reach levels of 7.5 mM (62, 63).
However, this option is not available on a long term basis, since
death routinely occurs after a 60 day fast.
[0045] The ketogenic diet, comprised mainly of lipid, has been used
since 1921 for the treatment of epilepsy in children, particularly
myoclonic and akinetic seizures (109) and has proven effective in
cases refractory to usual pharmacological means (71). Either oral
or parenteral administration of free fatty acids or triglycerides
can increase blood ketones, provided carbohydrate and insulin are
low to prevent re-esterification in adipose tissue. Rats fed diets
comprised of 70% corn oil, 20% casein hydrolysate, 5% cellulose, 5%
McCollum's salt mixture, develop blood ketones of about 2 mM.
Substitution of lard for corn oil raises blood ketones to almost 5
mM (Veech, unpublished).
[0046] In general the levels of ketone bodies achieved on such
diets are about 2 mM (R)-3-hydroxybutyrate and 1 mM acetoacetate
while the levels of free fatty acids are about 1 mM. Other
variations of composition have been tried including medium chain
length triglycerides. In general, compliance with such restricted
diets has been poor because of their unpalatability (56). High
lipid, low carbohydrate diets also have been tried as therapeutic
agents in cancer patients to reduce glucose availability to tumors
(88), as weight reducing diets in patients with and without
diabetes (74, 112) and to improve exercise tolerance (83).
[0047] The limitation of diets which rely upon lipid to raise blood
ketones to neurologically effective levels are many. Firstly,
levels of ketone bodies on lipid based diets tend to be below 3 mM,
significantly lower than the level of 7.5 mM achieved in overweight
humans during prolonged fasting. Secondly, unauthorized ingestion
of carbohydrate increases insulin secretion and causes a rapid
decrease in the hepatic conversion of free fatty acids to ketones
with a consequent drop in blood ketones and the diversion of lipid
to esterified to triglycerides by adipose tissue. Many anecdotal
reports relate the resumption of seizures in children who "broke
their diet with birthday cake". Thirdly their unpalatability and
the necessity to avoid carbohydrate to sustain high ketone body
levels makes such high lipid diets difficult to use in adults in an
out patient setting, particularly in societies where traditionally
high intake of refined sugars, bread, pasta, rice and potatoes
occurs. In practice, the traditional high ketone diet cannot be
enforced in patients, other than children beyond the age where all
food is prepared at home under strict supervision. Fourthly,
ingestion of such large amounts of lipid in the adult population
would lead to significant hypertriglyceridemia and
hypercholesterolemia with pathological sequelae of increased
vascular disease and sporadic hepatic and pancreatic disease, and
therefore could not be prescribed on medical grounds. Ingestion of
high lipid, low carbohydrate diets were popular in the 1970s for
weight reduction in the face of high caloric intake, provided that
carbohydrate intake was low. However, because of the increased
awareness of the relationship of elevated blood lipids to
atherosclerosis the popularity of this diet dropped abruptly.
[0048] Substituting glucose in a liquid diet, where glucose
accounts for 47% of the caloric content, with racemic 1,3 butandiol
caused the blood ketone concentration to rise about 10 fold to 0.98
mM (R)-3-hydroxybutyrate and 0.33 mM acetoacetate (107). These
values are slightly less than obtained normally in a 48 hour fast
and far below the levels of 7.5 mM obtained in fasting man. Racemic
1,3 butandiol is converted by liver to acetoacetate and both the
unnatural L-.beta. and the natural D-.beta.-hydroxybutyrate
(respectively (S) 3-hydroxybutanoate and (R)-3-hydroxybutyrate).
Although racemic 1,3 butandiol has been extensively studied as a
cheap caloric source in animal food and has even been used
experimentally in human diets (81, 101) the production of the
unnatural L-isomer is likely in the long run to produce significant
toxicity as has been shown for the human use of the unnatural
D-lactate (64). One disadvantage of administering the unnatural L
isomer is that it competes for transport with the natural
(R)-3-hydroxybutyrate. Thus provision of the (R) 1,3 butandiol as a
precursor of ketone bodies is one possibility that avoids
unnecessary administration or production of the unnatural
isomer.
[0049] The mono and di-aceotacetyl esters of racemic 1,3 butandiol
have been suggested as a source of calories and tested in pigs
(67). Oral administration of a bolus of a diet containing 30% of
calories as the esters produced a brief peak of blood ketones to 5
mM. However, the use of racemic 1,3 butandiol with its production
of the abnormal (S) 3-hydroxybutanoate is not to be recommended for
the reasons stated above.
[0050] While use of racemic 1,3 butandiol in such formulations is
not recommended, the esters of (R) 1,3 butandiol can be used,
either alone or as the acetoacetate ester. Studies in rats have
shown that feeding racemic 1,3 butandiol caused liver cytosolic
[NAD.sup.+]/[NADH] to decrease from 1500 to about 1000 (87). By
comparison, administration of ethanol reduces hepatic [NAD-]/[NADH]
to around 200 (106).
[0051] Acetoacetate, when freshly prepared, can be used in infusion
solutions where it can be given in physiologically normal ratios
with (R)-3-hydroxybutyrate to optimum effect (95). Because of
manufacturing requirements which currently require long shelf life
and heat sterilized fluids, acetoacetate has frequently been given
in the form of an ester. This has been done to increase its shelf
life and increase its stability to heat during sterilization. In
the blood stream, esterase activity has been estimated to be about
0.1 mmol/min/ml and in liver about 15 mmol/min/g (68). In addition
to esters combining 1,3 butandiol and acetoacetate there has also
been extensive study of glycerol esters of acetoacetate in
parenteral (59) and enteral nutrition (82). Such preparations were
reported to decrease gut atrophy, due to the high uptake of
acetoacetate by gut cells and to be useful in treatment of bums
(85).
[0052] For preferred embodiments of the present invention, under
optimum conditions, a physiological ratio of ketones should be
produced through administration of cyclic oligomers and
acetoacetate. If it is not, in the whole animal, the liver will
adjust the ratio of ketones in accordance with its own
mitochondrial free [NAD.sup.+]/[NADH]. If an abnormal ratio of
ketones is given the liver will adjust the ratio, with coincident
changes in liver [NAD.sup.+]/[NADH]. In the working heart,
perfusion with acetoacetate as sole substrate, rapidly induces
heart failure (99) in contrast to rat hearts perfused with a
mixture of glucose, acetoacetate and (R)-3-hydroxybutyrate, where
cardiac efficiency was increased by a physiological ratio of ketone
bodies (95).
[0053] The cyclic oligomers for use in the present invention are
conveniently synthesized from the microorganism produced
polyesters. Natural polyesters of (R)-3-hydroxybutyrate are sold as
articles of commerce e.g. as polymers of 530,000 MW from
Alcaligenes eutrophus (Sigma Chemical Co. St. Louis) or as 250,000
MW polymers for sugar beets (Fluka, Switzerland). The bacteria
produce the polymer as a source of stored nutrient. The
fermentation of these polymers by bacteria was developed in the
1970s by ICI in the UK and Solvay et Cie in Belgium, as a
potentially biodegradable plastic for tampon covers and other uses.
The system responsible for the synthesis of the poly
(R)-3-hydroxybutyrate has now been cloned and variations in the
composition of the polymer produced, based on the substrates given
to the bacteria. The genes responsible for the synthesis of
polyalkanoates have been cloned and expressed in a number of
micro-organisms (93, 102, 113) allowing for production of this
material in a variety of organisms under extremely variable
conditions.
[0054] Preferred forms of cyclic oligomeric (R)-3-hydroxybutyrate
are, at least in part, readily digestable and/or metabolised by
humans or animals. These preferably are of 2 to 200 repeats,
typically 2 to 20 and most conveniently from 3 to 10 repeats long,
particularly of 3 repeats, i.e. the triolide. It will be realised
that mixtures of such oligomers may be employed with advantage that
a range of uptake characteristics might be obtained. Similarly
mixtures with the monomer or linear oligomers or polymers may be
provided in order to modify the blood level profile produced.
[0055] Cyclic oligomers for use in the invention may be provided,
inter alia, by methods described by Seebach et al. Helvetia Chimica
Acta Vol 71 (1988) pages 155-167, and Seebach et al. Helvetia
Chimica Acta, Vol 77 (1994) pages 2007 to 2033. For some
circumstances such cyclic oligomers of 5 to 7 or more
(R)-3-hydroxybutyrate units may be preferred as they may be more
easily broken down in vivo. The methods of synthesis of the
compounds described therein are incorporated herein by
reference.
[0056] Once the monomer is in the blood stream, and since liver is
incapable of metabolizing ketone bodies but can only alter the
ratio of (R)-3-hydroxybutyrate/acetoacetate, the ketone bodies are
transported to extrahepatic tissues where they can be utilized. The
blood levels of ketones achieved are not subject to variation
caused by noncompliant ingestion of carbohydrate, as is the case
with the present ketogenic diet. Rather, they would simply be an
additive to the normal diet, given in sufficient amounts to produce
a sustained blood level, typically of between 0.3 to 20 mM, more
preferably 2 to 7.5 mM, over a 24 hour period, depending upon the
condition being treated. In the case of resistant childhood
epilepsy, blood levels of 2 mM are currently thought to be
sufficient. In the case of Alzheimer's disease, attempts could even
be made to keep levels at 7.5 mM or more, as achieved in the
fasting man studies, in an effort to provide alternative energy and
acetyl CoA supplies to brain tissue in Alzheimer's patients where
PDH capacity is impaired because of excess amounts of
A.beta..sub.1-42 amyloid peptide (77, 78).
[0057] The determination by the inventor that (R)-3-hydroxybutyrate
and its mixtures with acetoactetate act as a nerve stimulant, e.g.
nerve growth stimulant and/or stimulant of axon and dendritic
growth, opens up the option of raising ketone body levels to lesser
degrees than required nutritionally in order to treat
neurodegeneration.
[0058] Compositions of the invention are preferably sterile and
pyrogen free, particularly endotoxin free. Secondly, they are
preferably formulated in such a way that they can be palatable when
given as an additive to a normal diet to improve compliance of the
patients in taking the supplements. The cyclic oligomers are
generally smell free. Formulations of the cyclic oligomers of
(R)-3-hydroxybutyrate and its mixtures with acetoacetate may be
coated with masking agents or may be targeted at the intestine by
enterically coating them or otherwise encapsulating them as is well
understood in the pharmaceuticals or nutraceuticals art.
[0059] Since ketone bodies contain from about 4 to 6 calories/g,
there is preferably a compensatory decrease in the amounts of the
other nutrients taken to avoid obesity.
[0060] Particular advantages of using the cyclic oligomers taught
in the present invention are:
[0061] 1) they can be eaten with a normal dietary load of
carbohydrate without decreasing blood ketone body levels which
decrease would impair the effects of the treatment,
[0062] 2) they will not raise blood VLDL and cholesterol, as with
current cream and margarine containing diets, thus eliminating the
risk of accelerated vascular disease, fatty liver and
pancreatitis,
[0063] 3) they will have a wider range of use in a greater variety
of patients, including but not limited to: type II diabetes to
prevent hypoglycemic seizures and coma, in Alzheimer's disease and
other neurodegenerative states to prevent death of nerve cells e.g.
hippocampal cells, and in refractory epilepsy due to either
decreases in cerebral glucose transporters, defects in glycolysis,
or so called Leigh's syndromes with congenital defects in PDH.
[0064] The cyclic oligomers of the invention can be used in oral
and parenteral use in emulsions, whereas acetoacetate, in the
unesterified state, is less preferred as it is subject to
spontaneous decarboxylation to acetone with a half time at room
temperature of about 30 days. Where the compositions of the
invention do include acetoacetate this may be in the form of a
precursor. Acetoacetate may conveniently be provided as
(R)-3-hydroxybutyrate esters as provided by the copending
`therapeutic compositions` application.
[0065] Treatment may comprise provision of a significant portion of
the caloric intake of patients with the cyclic
(R)-3-hydroxybutyrate oligomer or oligomers formulated to give
retarded release, so as to maintain blood ketones in the elevated
range, e.g. 0.5 to 20 mM, preferably 2-7.5 mM range, over a 24 hour
period. Release of the ketone bodies into the blood may be
restricted by application of a variety of techniques such as
microencapsulation, adsorption and the like which is currently
practised in the oral administration of a number of pharmaceutical
agents. Enterically coated forms targeting delivery post stomach
may be particularly used where the material does not require, or is
not susceptible to, hydrolysis in acid environment. Where some such
hydrolysis is desired uncoated forms may be used. Some forms may
include enzymes capable of cleaving the esters to release the
ketone bodies such as those referred to in Doi. Microbial
Polyesters
[0066] Preferred cyclic oligomers, e.g. the triolide, may be merely
added as such to foodstuffs and/or may be supplemented in a
treatment regime by other ketone body generators of different
release profile such as the monomeric (R)-3-hydroxybutyrate. The
latter can be provided as an aqueous solution, e.g. as a salt, e.g.
sodium, potassium, magnesium or calcium salt
[0067] For a 1500 calorie diet, the human adult patient could
consume 198 g of cyclic esters of the present invention per day.
For a 2000 calorie diet of the same proportions, one could consume
264 g of ketones per day. On the ketogenic lipid diet blood ketones
are elevated to about 2 mM, which proves to be effective to some
degree at least in over 60% of children treated. On the ketone
diet, ketone levels should be higher because ketones have been
substituted at the caloric equivalent of fat, that is 1.5 g of
ketone/ g of fat. Accordingly, blood ketones should be
approximately 3 mM, an effective level in children, but still below
the level achieved in fasting man of 7.5 mM.
[0068] The advantage of using compounds which directly raised
ketone body levels, including the present cyclic oligomers which
raise blood levels of ketone bodies themselves are several.
Firstly, provision of ketone bodies themselves does not require the
limitation of carbohydrate, thus increasing the palatability of the
dietary formulations, particularly in cultures where high
carbohydrate diets are common. Secondly, ketone bodies can be
metabolised by muscle, heart and brain tissue, but not liver. Hence
the fatty liver, which may be an untoward side effect of the
ketogenic diet, is avoided. Thirdly, the ability to include
carbohydrate in the dietary formulations increases the chance of
compliance and opens up practical therapeutic approaches to type II
diabetics where insulin is high, making the known ketogenic diet
unworkable.
[0069] The present inventor has determined that, while any
elevation of ketone bodies may be desirable, a preferred amount of
cyclic ester to be administered will be sufficient, with any
acetoacetyl component, to elevate blood ketone body levels to the
0.5 to 20 mM level, preferably to the 2 mM to 7.5 mM level and
above, particularly when attempting to arrest the death of brain
cells in diseases such as Alzheimer's and Parkinsonism. While dead
cells cannot be restored, arrest of further deterioration and at
least some restoration of function is to be anticipated.
[0070] The total amount of ketone bodies used in treatment of
neurodegeneration such as Alzheimer's and Parkinsonism will
preferably elevate blood levels of ketone bodies by from 0.5 mM to
20 mM. The present inventor estimates that 200 to 300 g (0.5
pounds) of ketone bodies equivalent per patient per day might be
required to achieve this. Where the treatment is through
maintenance of cells against the effects of neurotoxin this may be
at a level sufficient to act as a significant caloric source, e.g.
2 to 7.5 mM in blood. Where it relies on the nerve stimulatory
factor effect of the (R)-3-hydroxybutyrate so produced, the amount
administered may be lower, e.g. to provide 0.2 to 4 mM increase,
but can of course be more for this or other disease.
[0071] It will be realised that treatment for neurodegenerative
diseases such as Alzheimer's or Parkinsonism will most effectively
be given soon after identifying patient's with a predisposition to
develop the disease. Thus treatment for Alzheimers' most
effectively follows a positive test result for one or more
conditions selected from the group (i) mutations in the amyloid
precursor protein gene on chromosome 21, (ii) mutations in the
presenilin gene on chromosome 14, (iii) presence of isoforms of
apolipoprotein E. Other tests shown to be indicative of Alzheimer's
will of course be applicable.
[0072] Following such a positive test result it will be appropriate
to prevent the development of memory loss and/or other neurological
dysfunction by elevation of the total sum of the concentrations of
the ketone bodies (R)-3-hydroxybutyrate and/or acetoacetate in the
patient's blood or plasma to say between 1.5 and 10 mM, more
preferably 2 to 8 mM, by one of several means. Preferably the
patient is fed a diet of sufficient quantities of compound of
formula (I), optionally parenterally but preferably and
advantageously enterally.
[0073] It will be realised that hypoglycemic brain dysfunction will
also be treatable using the treatments and compositions and
compounds of the present invention. A further property associated
with the present treatment will be general improvement in muscle
performance.
[0074] The provision of cyclic oligomer based foodstuffs and
medicaments of the invention is faciliated by the ready
availability of a number of relatively cheap, or potentially cheap,
starting materials from which cyclic (R)-3-hydroxybutyric acid may
be derived (see Microbial Polyesters Yoshiharu Doi. ISBN
0-89573-746-9 Chapters 1.1, 3.2 and 8). The availability of genes
capable of insertion into foodstuff generating organisms provides a
means for creating products such as yoghurts and cheese that are
enriched in the cyclic oligomer-(R)-3-hydroxybutyric acid or, after
breakdown with enzymes capable of cleaving such polymers, with the
monomeric substance itself (see Doi. Chapter 8).
[0075] Methods of preparing poly (R)-3-hydroxybutyrate are not
specifically claimed as these are known in the art. For example
Shang et al, (1994) Appli. Environ. Microbiol. 60: 1198-1205. This
polymer is available commercially from Fluka Chemical Co. P1082,
cat#81329, 1993-94, 980. Second St. Ronkonkoma N.Y. 11779-7238, 800
358 5287.
[0076] The present invention will now be described further by way
of illustration only by reference to the following Figures and
experimental examples. Further embodiments falling within the scope
of the invention will occur to those skilled in the art in the
light of these.
FIGURE
[0077] FIG. 1 is a graph showing blood (R)-3-hydroxybutyrate level
produced after time after feeding rats with the triolide of
(R)-3-hydroxybutyrate, a cyclic oligomer produced in Example 1 in
yoghurt and controls fed yoghurt alone.
EXAMPLES
Example 1
Preparation of
(R,R,R)-4,8,12-trimethyl-1,5,9-trioxadodeca-2,6,10-trione: Triolide
of (R)-3-hydroxybutyric Acid
[0078] 3
[0079] Synthesis was as described in Angew. Chem. Int. Ed. Engl.
(1992), 31, 434. A mixture of poly[(R)-3-hydroxybutyric acid] (50
g) and toluene4-sulphonic acid monohydrate (21.5 g, 0.113 mole) in
toluene (840 ml) and 1,2-dichloroethane (210 ml) was stirred and
heated to reflux for 20 hours. The water was removed by Dean-stark
trap for 15 hours whereafter the brown solution was cooled to room
temperature and washed first with a half saturated solution of
sodium carbonate then with saturated sodium chloride, dried over
magnesium sulphate and evacuated in vacuo. The brown semi-solid
residue was distilled using a Kugelrohr apparatus to yield a white
solid (18.1 g) at 120-130.degree. C./0.15 mmHg. Above 130.degree.
C. a waxy solid began to distill-- distillation being stopped at
this point. The distilled material had mp 100-102.degree. C.
(literature mp 110-110.5.degree. C.). Recrystallisation from hexane
gave colourless crystals in yield 15.3 g. Mp=107-108.degree. C.;
[.alpha.].sub.D-35.1 (c=1.005, CHCl.sub.3), (lit.=-33.9). .sup.1H
NMR (300 MHz, CDCl.sub.3): .delta.=1.30 (d, 9H, CH.sub.3); 2.4-2.6
(m, 6H; CH.sub.2); 5.31-5.39 (M, 3H; HC-O). .sup.13C NMR
(CDCl.sub.3) .delta.=20.86 (CH.sub.3), 42.21 (CH.sub.2), 68.92
(CH), 170.12 (CO). Elemental analysis: calculated for
C.sub.12H.sub.18O.sub.6: C, 55.81; H 7.02; Found: C, 55.67; H,
7.15.
Comparative Example 1
Preparation of Oligomers of (R)-3-hydroxybutyric Acid
(R)-3-hydroxybutyrate)
[0080] (R)-3-hydroxybutyric acid (Fluka-5.0 g: 0.048 mole),
p-toluene sulphonic acid (0.025 g) and benzene (100 ml) were
stirred under reflux within a Dean-Stark trap arrangement for 24
hours. The reaction mixture was cooled and the benzene evaporated
in vacuo (0.5 mm Hg). 4.4 g of colourless oil was obtained of which
a 20 mg sample was converted to the methyl ester for analysis of
number of monomer repeats using NMR. These studies show that the
product is a mixture of oligomers of D-.beta.-hydroxybutyrate of
average number of repeats 3.75, being mainly a mixture of trimers,
tetramers and pentamers with the single most abundant material
being the tetramer. The product mixture was soluble in 1 equivalent
of sodium hydroxide.
Comparative Example 2
Preparation of Acetoacetyl Ester of Oligomeric (R)-3-hydroxybutyric
Acid
[0081] A further batch of the colourless oil product from Example 1
(4.5 g) was heated for 1 hour at 60.degree. C. with diketene (3.8
g) and sodium actetate (0.045 g) under nitrogen. Further diketene
(3.8 g) was added and the reaction heated for a further hour,
cooled and diluted with ether, washed with water and then extracted
with saturated sodium bicarbonate (5.times.100 ml). Combined
extract was washed with ether then acidified with concentrated HCl
(added dropwise). Ethyl acetate extraction (3.times.50 ml) was
followed by drying over magnesium sulphate and evaporation in
vacua. A yellow solid/oil mixture was obtained (7.6 g) which was
chromatographed on a silica column using dichloromethane/methanol
(98:2) to give a light amber oil product. Faster moving impurities
were isolated (1.6 g) and after recolumning
carbontetrachloride/methanol (99:1) 0.8 g of oil was recovered
which was shown by NMR and Mass spectrometry to be the desired
mixture of acetoacetylated oligomers of (R)-3-hydroxybutyrate. The
product mixture had an Rf of 0.44 in dichloromethane/methanol
(90:1) and was soluble in 1 equivalent of sodium hydroxide. Both
products of Comparative Examples 1 and 2 are amenable to separation
of individual components by preparative HPLC.
EXAMPLE 2
Oral Administration of Triolide of (R)-3-hydroxybutyrate of Example
1 to Rats
[0082] The ability of orally administered triolide to raise blood
ketone levels was investigated as follows. The day before the
experiment commenced, 12 Wistar rats weighing 316.+-.10 g were
placed in separate cages. They had no access to food for 15 hours
prior to presentation with triolide containing compositions, but
water was provided ad libitum.
[0083] On the morning of the experiment 0.64 g of triolide was
mixed with 5 g Co-op brand Black Cherry yoghurt in separate feeding
bowls for 9 of the rats. The remaining 3 rats were given 5 g of the
yoghurt without the triolide as controls. The yoghurt containing
bowls were placed in the cages and the rats timed while they ate.
Two of the three control rats ate all the yoghurt and four of the
six triolide yoghurt rats ate approximately half the provided
amount. The remaining six rats slept.
[0084] Control rats (n=2) were killed at 60 and 180 minutes after
ingestion of yoghurt while triolide fed rats were killed at 80,
140, 150 and 155 minutes. Blood samples were taken for assay of
(R)-3-hydroxybutyrate. Brains were funnel frozen and later
extracted in perchloric acid and extracts neutralised and assayed.
Blood levels of (R)-3-hydroxybutyrate were measured using a
NAD.sup.+/EDTA assay of Anal. Biochem (1983) 131, p 478-482. 1.0 ml
of a solution made up from 2-amino-2-methyl-1-propanol (100 mM pH
9.9, 0.094 g/10 ml), NAD.sup.+ (30 mM, 0.199 g/10 ml) and EDTA (4
mM, 0.015 g/10 ml) was added to each of a number of cuvettes and 4
.mu.l sample or (R)-3-hydroxybutyrate control.
[0085] The two control rats ate 5.2.+-.0.1 g yoghurt and their
plasma (R)-3-hydroxybutyrate concentrations were about 0.45 mM at
60 minutes and 180 minutes. The four triolide fed rats ate
0.39.+-.0.03 g of the triolide and 2.6.+-.0.2 g of yoghurt. Their
plasma (R)-3-hydroxybutyrate concentrations were 0.8 mM after 80
minutes and 1.1 mM for the group sacrificed at about 150 minutes.
All rats displayed no ill effects from ingestion of triolide. Thus
serum (R)-3-hydroxybutyrate was found to be elevated by 0.65 mM by
feeding of only 0.4 g triolide. Note, as the rats had been fasted,
the initial levels of (R)-3-hydroxybutyrate were elevated from the
0.1 mM fed state to about 0.45 mM.
[0086] The test rats thus showed increase in plasma
(R)-3-hydroxybutyrate over at least 3 hours with no ill effects. It
should be noted that two other rats fed approximately 1.5 g
triolide each in `Hob-Nob` biscuit showed no ill effects after two
weeks.
[0087] It should be noted that the increased levels of
(R)-3-hydroxybutyrate will also be mirrored in acetoacetate levels,
not measured here, as there is a rapid establishment of equilibrium
between the two in vivo such that acetoacetate levels will be
between 40 and 100% of the (R)-3-hydroxybutyrate levels.
Comparative Example 3
Oral Administration of (R)-3-hydroxybutyrate Oligomers and
Acetoacetyl (R)-3-hydroxybutyrate Oligomers to Rats
[0088] The ability of orally administered (R)-3-hydroxybutyrate and
the linear oligomers of Comparative examples 1 and 2 to raise blood
ketone body levels was investigated as follows. Rats were fasted
overnight and then gavaged with 100 .mu.l/100 g bodyweight of 4M
(R)-3-hydroxybutyrate brought to pH 7.4 using methyl glucamine.
Plasma levels of (R)-3-hydroxybutyrate were measured at 0.62 mM
after 30 minutes as compared to 3 mM when 9M (R)-3-hydroxybutyrate
is used.
[0089] This procedure was repeated with 2M solutions of the
mixtures (R)-3-hydroxybutyrate oligomers and their acetoacetyl
esters described in Comparative Examples 1 and 2. The
(R)-3-hydroxybutyrate oligomer (19/1) and the acetoacetyl ester
(20/4) were both brought to pH 7.6 with methyl glucamine and the
blood (R)-3-hydroxybutyrate level monitored using the aforesaid
assay procedure. Increases in serum (R)-3-hydroxybutyrate were
shown to be of 0.2 mM to 0.5 mM at 60 and 120 minutes after
gavaging.
EXAMPLE 5
[0090]
1TABLE 2 Sample 1500 calorie ketogenic diet using cyclic oligomer
(I) of invention. The cyclic oligomer is assumed to contain 6
kcal/g fats, 9 kcal/g carbohydrate and 4 kcal/g protein. Oligomers
have been substituted to give equilvalent calories Protein Cyclic
Amount (g) Fat (g) (g) CHO (g) (I) (g) Breakfast Egg 32 4 4 apple
juice 70 7 ketones 66 66 skim milk 92 0 2 3 Total Breakfast 4 6 10
66 Lunch lean beef 12 1.75 3.5 cooked carrots 45 0.6 3 canned pears
40 4 ketones 69.75 69.75 skim milk 92 2 3 Total Lunch 1.75 6.1 10
69.75 Supper Frankfurter 22.5 6 3 cooked broccoli 50 1 2 watermelon
75 5 ketones 62.25 62.25 skim milk 92 2 3 Total Supper 6 6 10 62.25
Daily Total 11.75 18.1 30 198
EXAMPLE 6
Effect of (R)-3-hydroxybutyrate on Hippocampal Cells
Methods
[0091] Culture Medium and Chemicals
[0092] The serum free medium used from 0 to day 4 contained
Neurobasal medium with B27 supplement diluted 50 fold (Life
Technology, Gaithersburg, Md.) to which was added: 0.5 mM
L-glutamine, 25 .mu.M Na L-glutamate, 100 U/ml penicillin and 100
.mu.g/ml streptomycin. After day 4, DMEM/F12 medium containing 5
.mu.M insulin, 30 nM 1-thyroxine, 20 nM progesterone, 30 nM Na
selenite 100 U/ml penicillin and 100 .mu.g/ml streptomycin were
used.
[0093] Hippocampal Microisland Cultures
[0094] The primary hippocampal cultures were removed from Wistar
embryos on day 18 and dispersed by gentle agitation in a pipette.
The suspension was centrifuged at 1,500.times.g for 10 min and the
supernatant discarded. The pellet was resuspended in new media to a
final cell count of 0.4-0.5.times.10.sup.6 cells/ml. Ten .mu.l of
this suspension was pipetted into the center of poly D-lysine
coated culture wells and the plates incubated at 38.degree. C. for
4 hrs and then 400 .mu.l of fresh Neurobasal media was added. After
2 days of incubation, half of the media was exchanged for fresh
media and the incubation continued for 2 more days. After day 4,
the medium was changed with DMEM/F12 medium containing 5 .mu.M
insulin, 30 nM 1-thyroxine, 20 nM progesterone, 30 nM Na selenite
100 U/ml penicillin and 100 .mu.g/ml streptomycin. The wells were
divided into 4 groups: half the wells received
(R)-3-hydroxybutyrate to a final concentration of 8 mM while and
half of the wells received 5 nM amyloid .beta..sub.1-42 (Sigma).
These media were exchanged 2 days later (day 8) and the cells were
fixed on day 10 and stained with anti MAP2 (Boehringer Manheim,
Indianapolis, Ind.) to visualize neurons and vimentin and GFAP
(Boehringer) to visualize glial cells.
Results
[0095] Cell Counts
[0096] Addition of (R)-3-hydroxybutyrate to the incubation resulted
in an increase in the neuronal cell number per microisland from a
mean of 30 to a mean of 70 cells per microisland. Addition of 5 nM
amyloid .beta..sub.1-42 to the cultures reduced the cell numbers
from 70 to 30 cells per microisland, confirming the previous
observations of Hoshi et al, that amyloid .beta..sub.1-42 is toxic
to hippocampal neurons. Addition of (R)-3-hydroxybutyrate to
cultures containing amyloid .beta..sub.1-42 increased the cell
number from a mean of 30 to 70 cells per microisland. From these
data we conclude that addition of substrate level quantities of
(R)-3-hydroxybutyrate, to media whose major nutrients are glucose,
pyruvate and L-glutamine, slows the rate of cell death in culture.
It is further concluded that (R)-3-hydroxybutyrate can decrease the
increased rate of hippocampal cell death caused by the addition of
amyloid .beta..sub.1-42 in culture.
[0097] The number of dendritic outgrowths and the length of axons
were both observed to have increased with presence
of(R)-3-hydroxybutyrate, whether .beta..sub.1-42 was present or
not. This is indicative of nerve growth factor like behaviour.
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