U.S. patent application number 13/137906 was filed with the patent office on 2012-03-22 for therapeutic compositions.
This patent application is currently assigned to BTG International Limited. Invention is credited to Richard L. Veech.
Application Number | 20120071548 13/137906 |
Document ID | / |
Family ID | 26717526 |
Filed Date | 2012-03-22 |
United States Patent
Application |
20120071548 |
Kind Code |
A1 |
Veech; Richard L. |
March 22, 2012 |
Therapeutic compositions
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 L.;
(Rockville, MD) |
Assignee: |
BTG International Limited
London
GB
|
Family ID: |
26717526 |
Appl. No.: |
13/137906 |
Filed: |
September 21, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10763393 |
Jan 26, 2004 |
8101653 |
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13137906 |
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10408667 |
Apr 8, 2003 |
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10763393 |
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10153873 |
May 24, 2002 |
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10408667 |
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09843694 |
Apr 30, 2001 |
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10153873 |
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09397100 |
Sep 16, 1999 |
6323237 |
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09843694 |
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PCT/US98/05072 |
Mar 17, 1998 |
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09397100 |
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60040858 |
Mar 17, 1997 |
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Current U.S.
Class: |
514/450 ;
514/557; 514/558; 560/180; 560/182 |
Current CPC
Class: |
A23L 33/10 20160801;
A61K 31/765 20130101; A23L 33/105 20160801; C07C 67/465 20130101;
C07C 67/46 20130101; C07C 69/72 20130101; A61P 9/00 20180101; A23V
2002/00 20130101; A61K 31/365 20130101; A61P 25/28 20180101; C07C
67/14 20130101; A61P 25/08 20180101; A61P 5/50 20180101; A61K 31/19
20130101; A61K 31/22 20130101; C07B 2200/07 20130101; A23L 33/30
20160801; A61P 3/10 20180101; C07C 69/675 20130101; A61P 25/00
20180101; A23L 27/66 20160801; C07D 323/00 20130101; A23V 2002/00
20130101; A23V 2250/30 20130101; C07C 67/14 20130101; C07C 69/72
20130101; C07C 67/46 20130101; C07C 69/72 20130101 |
Class at
Publication: |
514/450 ;
514/557; 514/558; 560/180; 560/182 |
International
Class: |
A61K 31/19 20060101
A61K031/19; A61P 25/00 20060101 A61P025/00; A61P 5/50 20060101
A61P005/50; A61P 25/08 20060101 A61P025/08; A61P 3/10 20060101
A61P003/10; C07C 67/465 20060101 C07C067/465; A61K 31/357 20060101
A61K031/357; C07C 69/72 20060101 C07C069/72; C07C 69/675 20060101
C07C069/675; C07C 67/46 20060101 C07C067/46; C07C 67/14 20060101
C07C067/14; A61P 25/28 20060101 A61P025/28; A61K 31/20 20060101
A61K031/20 |
Claims
1. A method of a treating a patient for a neuro-degenerative
disorder comprising administering to that patient a therapeutically
effective amount of one or more of D-.beta.-hydroxybutyric acid,
acetoacetate, or a metabolic precursor or physiologically
acceptable salt of D-.beta.-hydroxybutyric acid or acetoacetate,
such as to elevate the patient's blood level of ketone bodies,
defined as the sum total of D-.beta.-hydroxyburyric acid and
acetoacetate, to a therapeutic level effective to treat the
disorder wherein when a metabolic precursor is administered it is
not hydroxybutyryl carnitine.
2. A method of treating a patient in order to treat a
neuro-degenerative disorder comprising administering to that
patient a therapeutically effective amount of at least one of
D-.beta.-hydroxybutyric acid, acetoacetate, or a metabolic
precursor or physiologically acceptable salt of
D-.beta.-hydroxybutyric acid or acetoacetate, such as to elevate
the patient's blood level of ketone bodies, defined as the sum
total of D-.beta.-hydroxybutyric acid and acetoacetate, to a
therapeutic level effective to treat the disorder wherein the
patient's blood level is elevated to from 0.3 mM to 20 mM.
3. A method of treating a CNS cell, peripheral nerve cell, or
otherwise insulin insensitive cell in need of therapy for one or
more of neuro-degeneration, GABA preventable seizure, or
insufficient ability to metabolise glucose, comprising
administering to that cell one or more compounds selected from the
group consisting of D-.beta.-hydroxybutyric acid, acetoacetate,
compounds which are oligomers of D-.beta.-hydroxybutyric acid,
acetoacetyl esters of D-.beta.-hydroxybutyric acid and acetoacetyl
esters of oligomers of D-.beta.-hydroxybutyric acid, and
physiologically acceptable salts thereof.
4. A method of treating an patient for epilepsy, diabetes or an
insulin resistant state comprising administering to that patient a
therapeutically effective amount of one or more compounds selected
from the group consisting of D-.beta.-hydroxybutyric acid,
acetoacetate and metabolic precursors of D-.beta.-hydroxybutyric
acid or acetoacetate which comprise moieties selected from the
group consisting of R-1,3-butandiol, acetoacetyl and
D-.beta.-hydroxybutyryl moieties and physiologically acceptable
salts and esters thereof.
5. A method as claimed in claim 1 wherein on administration of the
compound to an unfasted patient in need of such therapy, the blood
level of ketone bodies, defined as the sum total of
D-3-hydroxybutyric acid and acetoacetate, is raised to between 0.3
and 20 mM.
6. A method as claimed in claim 1 wherein the neurodegenerative
disorder is selected from the group consisting of neurodegenerative
disorders involving inability to metabolise glucose, memory loss in
ageing, neurotoxic peptides or proteins, and genetic
abnormality.
7. A method as claimed in claim 6 wherein the neurodegenerative
disorder is selected from those involving neurotoxic protein
plaques.
8. A method as claimed in claim 1 wherein the metabolic precursor
is selected from the group consisting of Free Fatty Acids and
compounds comprising 1,3-butandiol, acetoacetyl or
D-.beta.-hydroxybutyryl moieties.
9. A method as claimed in claim 1, wherein the metabolic precursor
is a polymer or oligomer of D-.beta.-hydroxybutyrate.
10. A method as claimed in claim 9 wherein the metabolic precursor
is an aceroacetyl ester.
11. A method as claimed in claim 9 wherein metabolic precursor is
selected from the group consisting of compounds of general formulae
##STR00008## or physiologicial acceptable salts or esters thereof
wherein in each case n is selected such that the polymer or
oligomer is readily metabolised on administration to a human or
animal body to provide elevated ketone body levels in blood.
12. A method as claimed in claim 11 wherein n is an integer of 0 to
1,000.
13. A method as claimed in claim 11 wherein n is an integer of from
1 to 5.
14. A method as claimed in claim 1, wherein the level of ketone
bodies produced in the blood is in the ratio 1:1 to 20:1 of
D-.beta.-hydroxybutyrate to acetoacetate.
15. A method as claimed in claim 9 wherein the oligomer is a cyclic
oligorner of formula ##STR00009## where n is an integer of 1 or
more or a complex thereof with one or more cations or a salt
thereof
16. A method as claimed in claim 15 wherein the one or more cations
are selected from the group consisting of sodium, potassium,
magnesium and calcium.
17. A method as claimed in claim 15 wherein n is an integer from 1
to 20.
18. A method as claimed in claim 1 wherein it is
(R,R,R)-4,8,12-trimethyl-1,5,9-trioxadodeca-2,6,10-trione.
19. A compound of formula ##STR00010## or physiologicial acceptable
salts or esters thereof. wherein n is an integer from 0 to 1000
20. A compound as defined in claim 19 wherein the ester is selected
from the group consisting of monohydric, dihydric or trihydric
alcohol esters.
21. A compound as claimed in claim 19 wherein the ester is of
(R)-1,3-butandiol.
22. A compound as claimed in claim 19 wherein n is selected from
the group of integers 0, 1, 2, 3 and 4.
23. A foodstuff comprising poly D-.beta.-hydroxybutyrate
characterised in that it is derived from a foodstuff generating
organism that has had a gene capable of producing
D-.beta.-hydroxybutyrate inserted therein.
24. A foodstuff characterised in that it comprises at least 5%
ketone bodies by weight.
25. A method for the synthesis of
D-.beta.-hydroxybutyryl-acetoacetate or poly or
oligo-D-.beta.-hydroxybutyryl-acetoacetate esters comprising the
reaction of acetoacetic acid halide with D-.beta.-hydroxybutyrate
or poly- or oligo-D-.beta.-hydroxybutyrate.
26. A method for synthesis of D-.beta.-hydroxybutyryl-acetoacetate
or oligo-D-.beta.-hydroxybutyryl-acetoacetate comprising reacting
D-.beta.-hydroxybutyryic acid with diketene.
27. A method of synthesising an oligomer of D-.beta.-hydroxybutyric
acid comprising heating a solution of D-.beta.-hydroxybutyric acid
in a solvent until an oligomer of a desired number of repeats is
produced.
28. Use of D-.beta.-hydroxybutyric acid, acetoacetate, or a
metabolic precursor or physiologically acceptable salt of
D-.beta.-hydroxybutyric acid or acetoacetate for the manufacture of
a medicament for the treatment of a disorder by a method as set out
in claim 1 provided that when the use is of a metabolic precursor
that is not racemic hydroxybutyryl carnitine.
29. A foodstuff as claimed in claim 23 for use in therapy.
30. Poly-D-.beta.-hydroxybutyrate for use in therapy.
31. A composition comprising a compound selected from those claimed
in claim 15 and poly D-.beta.-hydroxybutyrate together with a
physiologically acceptable carrier, in sterile and pyrogen free
form.
Description
[0001] This application is a continuation of application Ser. No.
10/763,393 filed Jan. 26, 2004, which is a continuation of
application Ser. No. 10/408,667, filed Apr. 8, 2003, which is a
continuation of application Ser. No. 10/153,873, filed May 24, 2002
(abandoned), which is a continuation of application Ser. No.
09/843,694, filed Apr. 30, 2001, which is a continuation of
application Ser. No. 09/397,100, filed Sep. 16, 1999 (now U.S. Pat.
No. 6,323,237), which is a CIP of PCT/US98/05072, filed Mar. 17,
1998, which claims benefit of provisional Application No.
60/040,853, filed Mar. 17, 1997, the entire contents of each of
which are hereby incorporated by reference.
THERAPEUTIC COMPOSITIONS
[0002] The present invention relates to compositions suitable for
administration to humans and animals 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 memory loss. The invention further provides methods of
treatment and novel esters and polymers for inclusion in the
compositions of the invention.
[0003] Abnormal elevation of blood sugar occurs not only in insulin
deficient and non insulin dependent diabetes but also in a variety
of other diseases. The hyperglycaemia of diabetes results from an
inability to metabolize and the over production of glucose. Both
types of diabetes are treated with diet; Type I diabetes almost
always requires additional insulin, whereas non-insulin dependent
diabetes, such as senile onset diabetes, may be treated with diet
and weight loss, although insulin is increasingly used to control
hyperglycaemia.
[0004] Increased sympathetic stimulation or elevated glucagon
levels, in addition to increasing glycogenolysis in liver, also
stimulate free fatty acid release from adipocytes. After acute
myocardial infarction or during heart failure, increased
sympathetic nervous-activity or administration of sympathomimetics
accelerate glycogenolysis, decrease release of insulin from P cells
of the pancreas and cause relative insulin resistance. While the
importance of diet, or substrate availability, is taken as a given
in the treatment of diabetes, the critical effects of substrate
choice in insulin resistant states has not been widely appreciated
or applied in clinical practice. Instead contemporary interest has
focused upon the complex signalling cascade which follows the
binding of insulin to its receptor. This increasingly complex
cascade of messages involving protein tyrosine kinases and
phosphatases, inositol and other phospholipids, while holding
promise for the ultimate understanding of non-insulin dependent
diabetes, has yet to provide significant new therapies for either
diabetes or insulin resistance.
[0005] Leaving aside the longer term effects of insulin on growth,
the acute metabolic effects of insulin have been thought to be
accounted for by action at three major enzymatic steps in the
conversion of glucose to CO.sub.2. Firstly insulin promotes the
translocation of the glucose transporter, Glut4, from endoplasmic
reticular to plasma membranes, thus increasing the transport of
glucose from the extra to intracellular phase.(see refs. 1 and 2).
Secondly, insulin increases the accumulation of glycogen. This has
been attributed to dephosphorylation of glycogen synthase (3) by
protein phosphatase 1. Thirdly, insulin stimulates the activity of
mitochondrial pyruvate dehydrogenase multi-enzyme complex (4 and 5)
through dephosphorylation by a Ca.sup.2+ sensitive (6)
intramitochondrial protein phosphosphatase.
[0006] An important, but poorly understood effect of insulin is its
use in cardiac disease where in combination with glucose, potassium
chloride and GIK, it improved electrocardiographic abnormalities
accompanying myocardial infarction (7 and 8), and improved cardiac
performance after post pump stunning (9). This treatment has been
advocated recently for a number of other serious cardiac diseases
(10 and 11). The beneficial effects of GIK infusion have been
attributed to its ability to decrease free fatty acid release and
improve membrane stability (12). However, other more recent work
suggests more fundamental reasons. In heart cells that are anoxic,
glucose is the only fuel capable of providing the ATP necessary to
maintain viability (13).
[0007] Administration of glucose plus insulin would increase the
availability of intracellular glucose providing a source of ATP
production in the absence of O.sub.2. While this would explain
certain beneficial effects, it would not account for the correction
of EKG abnormalities nor the improved cardiac index in hearts
treated with GIK because electrical activity and cardiac work
requires actively respiring cardiac cells, not ones which are
totally anoxic and therefore without electrical activity or the
ability to perform mechanical work.
[0008] Understanding the enzymatic sites of insulin's action does
not, by itself, define the effects of insulin deficiency upon the
cellular metabolism or physiological function. How insulin acts at
this larger level can best be understood by looking at the way
nature deals with insulin deficiency. The natural compensation for
decreased insulin during fasting is the accelerated hepatic
conversion of the free fatty acids to the ketone bodies raising
blood D-.beta.-hydroxybutyrate and acetoacetate to about 6 mM. At
these levels, ketones, rather than glucose, become the substrate
for most organs, including even the brain (14). Although mild
ketosis is the normal response to decreased insulin, physicians
fear ketone bodies because their massive overproduction can be life
threatening in diabetic ketoacidosis.
[0009] The present inventor has previously compared the effect of
physiological levels of ketone bodies to the metabolic and
physiological effects of insulin, particularly comparing the
insulin deficient working rat heart perfused with glucose alone, to
hearts to which was added either 4 mM D-.beta.-hydroxybutyrate/1 mM
acetoacetate, saturating doses of insulin or the combination and
has shown how provision of simple substrates can mimic the effects
of insulin in changing the concentrations of the intermediates of
both glycolysis and the TCA cycle and thereby controlling the flux
of glucose in this very specialised tissue. In addition he has
determined that a primary but previously unrecognized effect of
insulin or a ratio of ketones is to alter mitochondrial redox
states in such a way so as to increase the
.DELTA.G.sub.ATPhydrolysis and with that, the gradients of
inorganic ions between the various cellular phases and the
physiological performance of heart.
[0010] The present application teaches that such ketone bodies can
also provide a therapeutic approach to the treatment of insulin
resistance where the normal insulin signalling pathway is
disordered and in conditions where the efficiency of cardiac
hydraulic work is decreased for metabolic reasons. The inventor has
determined that use of ketone bodies has great advantage over use
of insulin itself for reasons that will become evident from the
description below, not least of these being the elimination of
carbohydrate intake control otherwise necessary.
[0011] The present application further addresses the problem of
neurodegenerative diseases, particularly disease where neurons are
subject to neurotoxic effects of pathogenic agents such as protein
plaques and further provides compositions for use in treating these
and the aforesaid disorders.
[0012] Alzheimer's disease is a genetically heterogeneous group of
progressively fatal neurological diseases characterized
pathologically by accumulation of amyloid plaques in brain and
clinically by impairment of recent memory leading to dementia and
death. In addition to the cases of Alzheimer's disease linked to
genetic causes, sporadic cases, without an apparent family history
of the disease, also occur. For example pathological changes
characteristic of Alzheimer's disease occur after head trauma (73)
or after inflammatory diseases stimulating production of the
cytokine interleukin-1 (97).
[0013] The early symptom of the disease is loss of recent memory
associated with impairment and death of cell in the hippocampus
accounting for the early impairment of recent memory. Measurement
of the hippocampal volumes using magnetic resonance imaging (MRI)
shows that atrophy of hippocampus occurs prior to the clinical
onset of memory loss and progresses with a loss of volume of about
8% per year during the 2 years over which symptoms first appeared
(70).
[0014] The diagnosis of Alzheimer's disease is made clinically by
this impairment in recent memory, associated with lesions in the
hippocampal portion of the temporal lobe. Neuropathologically, the
diagnosis depends upon the finding of neurofibrillatory tangles
within the cells, amyloid or senile plaques in the extracellular
space and loss of neuronal number (61). The neurofibrillatory
tangles are comprised of paired hyperphosphorylated tau protein,
whose usual function in the cell, when not phosphorylated, is to
bind to and stabilize tubulin in its formation of microtubules
within the cell. Hyperphosphorylation of tau is catalysed by
glycogen synthase kinase 3.beta., among other kinases and
dephosphorylated by protein phosphatase 2A-1, 2B or 1(108).
[0015] However, there is not necessarily a clear, bright line
between the pathological brain changes and the memory deficits
which occur prematurely in Alzheimer's disease and the pathological
changes in brain anatomy and memory function which are found in the
"normal" aging population. Rather the difference is a quantitative
one dependent upon rate (94). Such changes in memory function in
the normal aged are also accompanied by a decreased glucose
tolerance signifying an inability to metabolize glucose. In such
situations, treatments aimed at rectifying the pathophysiological
processes of Alzheimer's disease, would be expected to be
applicable to the correction of the metabolic effects associated
with normal aging.
[0016] While Alzheimer's disease of the familial or the sporadic
type is the major dementia found in the aging population, other
types of dementia are also found. These include but are not limited
to: the fronto-temporal degeneration associated with Pick's
disease, vascular dementia, senile dementia of Lewy body type,
dementia of Parkinsonism with frontal atrophy, progressive
supranuclear palsy and corticobasal degeneration and Downs syndrome
associated Alzheimers'. Plaque formation is also seen in the
spongiform encephalopathies such as CJD, scrapie and BSE. The
present invention is directed to treatment of such
neurodegenerative diseases, particularly those involving neurotoxic
protein plaques, eg. amyloid plaques.
[0017] Many of these aforesaid apparently unrelated conditions have
the hyperphosphorylated tau proteins found in Alzheimer's disease
(69), opening up the possibility that the same kinase which
phosphorylated tau would also phosphorylate the PDH complex
producing a similar deficiency in mitochondrial energy production
and acetyl choline synthesis found in Alzheimer's disease but
involving other brain regions. The present inventor has determined
that in this respect treatments applicable to Alzheimer's disease
might be applied to these diseases as well. In addition, the
inventor has determined that such treatment will also be applicable
to peripheral neurological wasting diseases, such as myasthenia
gravis and muscular dystrophy.
[0018] At present there is no effective treatment for Alzheimer's
disease. Research efforts are focused on defining its genetic cause
but to date there has been no succesful gene therapy. Genetic
studies have linked Alzheimer's disease with Mongolism and in its
early onset form to locus on chromosome 21 causing accumulation of
amyloid precursor protein (APP) (73), a transmembrane glycoprotein
existing in 8 isoforms. Numerous fragments of this protein are
derived by proteolysis and the plaques characteristic of
Alzheimer's disease have been shown to contain accumulation of the
oligomer of .beta. amyloid protein (A .beta..sub.1-42). An early
onset autosomally dominant form of Alzheimer's disease has also
been related to a presenilin 1 locus on chromosome 14.
[0019] A late onset form of Alzheimer's disease is associated with
the type 4 allele of apolipoprotein E (69,98) on chromosome 19,
although other workers suggest that this apparent correlation may
be related instead of .alpha. 1 antichymotrypsin locus instead
(100). All transgenic mice expressing increased amounts of amyloid
precursor protein over 18 months of age showed hippocampal
degeneration with many of the pathological characteristics of
Alzheimer's disease (90).
[0020] The current status of knowledge on the defective genes and
gene products in Alzheimer's disease has recently been summarized
(Table 1 of ref. 96).
TABLE-US-00001 Chro- mosome Gene Defect Age of Onset A.beta.
Phenotype 21 .beta.APP mutations 50's Production of total A.beta.
peptides of A.beta..sub.1-42 19 apoE4 polymorphism 60's or >
density of A.beta. plaques and vascular deposits 14 Presenilin 1
mutations 40's & 50's production of A.beta..sub.1-42 1
Presenilin 2 mutations 50's production of A.beta..sub.1-42
[0021] It is clear from the above table that the common phenotype
associated with the genetic forms of Alzheimer's disease is the
accumulation of the amyloid peptide A.beta..sub.1-42 (96). It is
this A.beta..sub.1-42 which inactivates PDH thus impairing
mitochondrial energy and citrate production in normally obligate
glucose consuming tissue (95) and at the same time impairing
synthesis of the critical neurotransmitter, acetyl choline (67,68).
The application of A.beta..sub.1-42 to neuronal cells is associated
with the downregulation of the anti-apototic protein bcl-1 and
increases levels of bax, a protein known to be associated with cell
death (92). In addition to amyloid plaques comprised of
A.beta..sub.1-42, neurofibrillatory tangles comprised of
hyperphosphorylated tau protein, and decreased brain acetyl choline
levels, cell death is the fourth pathological characteristic of
Alzheimer's disease. These pathological characteristics can be
related, at least in part, to excess A.beta..sub.1-42 and its
inhibition of PDH.
[0022] Modest clinical improvement in symptoms can occur by
treatment with acetyl choline esterase inhibitors (57), presumably
by increasing cholinergic efferents originating in the septal
nuclei and traversing Broca's diagonal band to hippocampus in the
anterior portion of the limbic system of brain. However the
progress in the molecular biology of Alzheimer's disease has caused
the search for new therapies to concentrate upon four major areas
(96): (i) protease inhibitors that partially decrease the activity
of the enzymes (.beta. and .gamma. secretase) that cleave A.beta.
(.beta. amyloid fragments) from .beta.APP (.beta. amyloid precursor
proteins); (ii) compounds that bind to extracellular A.beta. that
prevent its cytotoxic effects; (iii) brain specific
anti-inflammatory drugs that block the microglial (brain
macrophages) activation, cytokine release, and acute phase response
that occur in affected brain regions; and (iv) compounds such as
antioxidants, neuronal calcium channel blocks, or antiapoptotic
agents that interfere with the mechanisms of A.beta. triggered
neurotoxicity.
[0023] The therapy which the present inventor now proposes differs
from the four approaches listed above in that it bypasses the block
in metabolic energy production resulting from inhibition of PDH by
A.beta..sub.1-42 by administering ketone bodies or their
precursors. Neuronal cells are capable of metabolizing such
compounds even in the presence of a deficiency of glucose, the
normal energy substrate for brain (63). Because ketones can
increase the .DELTA.G of ATP hydrolysis, the gradients of both
intracellular Na.sup.+ and Ca.sup.2+ will be increased, preventing
cell death associated with increased intracellular Ca2+.
Furthermore, the increase in citrate generation by the Krebs cycle
will provide, when translocated into cytoplasm, a source of
cytoplasmic acetyl CoA required to remedy the deficiency of acetyl
choline characteristic of Alzheimer's brains.
[0024] The elevation of blood ketones necessary to correct these
metabolic defects can be accomplished by parenteral, enteral means
or dietary means and does not require the administration of
potentially toxic pharmacological agents.
[0025] There has been long experience with ketogenic diets in
children treated for epilepsy. Such diets are however unsuitable
for use in adults due to adverse efects on the circulatory system.
The present inventions application of ketone bodies should provide
all the therapeutic effects of such diet, which is not itself found
to be toxic in children, with none of the side effects that render
it unused adults. Furthermore, the inventor has determined that
with the correction of the aforesaid metabolic 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 acetyl choline synthesis.
[0026] Since the priority date of this application, EP 0780123 A1
has been published which relates to use of acetoacetate,
.beta.-hydroxybutyrate, monhydric, dihydric or trihydric alcohol
esters of these or oligomers of .beta.-hydroxybutyrate for
suppressing cerebral edema, protecting cerebral function,
rectifying cerebral energy metabolism and reducing the extent of
cerebral infarction. It should be noted however, that it has been
known since 1979 that sodium hydroxybutyrate increases cerebral
circulation and regional vasomotor reflexes by up to 40% (Biull.
Eksp. Biol. Med Vol 88 11, pp 555-557). The treatment that the
present inventor now provides goes beyond such effects on
circulation as it provides treatment for cells that are unable to
function due to neurodegeneration, eg caused by neurotoxic agents
such as peptides and proteins, and genetic abnormality. The
treatment involves action of ketone bodies on the cells themselves
and not the flow of blood to them.
[0027] In reducing this invention to practice the inventor has
further determined that ketone bodies, provided by direct
adminsitration or by administration of their metabolic precursors
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, ie. control levels in a manner
unrelated to blood flow or nutrition. In this respect the invention
further provides use of ketone bodies as nerve stimulant factors,
ie. nerve growth factors and factors capable of stimulating
enhanced neuronal function, such as 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.
[0028] 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 as a
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. PDH thus stands at the
crossroads of the two major energy producing pathways of the cell,
glycolysis and the Krebs cycle, and clearly serves a critical
function in living cells.
[0029] There are two major consequences of the inhibition of PDH.
Firstly, in neuronal tissues, which under normal metabolic
conditions are totally dependent upon glucose for energy
production, inhibition of PDH results in a lowered efficiency of
energy production, a lowered energy of hydrolysis of ATP, a
decrease in both acetyl CoA and the metabolites of the first 1/3 of
the TCA cycle and a deficiency of mitochondrial NADH (95). A
decrease in the energy of ATP hydrolysis leads to increased
intracellular Na.sup.+ and Ca.sup.2+, loss of cellular K.sup.+ and
ultimately cell death (86). Hippocampal cells, critical for the
fixation of recent memories, are particularly sensitive to a number
of forms of injury, and the death of these cells is the hallmark
both clinically and pathologically of Alzheimer's disease.
[0030] A second major consequence of PDH inhibition is a deficiency
of mitochondrial citrate (95). Citrate, or one of its metabolites,
is exported to the cytoplasm from mitochondria where it is
converted to cytosolic acetyl CoA by ATP citrate lyase (EC 4.1.3.8)
in the reaction:
citrate.sup.3-+ATP.sup.4-+CoASH>acetyl
CoA+oxaloacetate.sup.2-+ADP.sup.3-+HPO.sub.4.sup.2-
The acetyl CoA then combines with choline through the action of
choline acetyl transferase
[0031] (EC 2.3.1.6) to form acetyl choline in the reaction:
choline.sup.++acetyl CoA>CoASH+acetyl choline.sup.+
Neuronal culture of septal cells exposed to 1 .mu.m
A.beta..sub.1-42 for 24 hours showed a decrease in acetyl choline
production of over five fold (78) with no decrease in the activity
of choline acetyl transferase. The inferred cause of this decreased
production was a deficiency of acetyl CoA due to inhibition of the
PDH complex caused by activation of the TPKI/GSK-3.beta. protein
kinase and subsequent phosphorylation of PDH (77).
[0032] As explained above isolated working hearts perfused with 10
mM glucose alone without insulin are inefficient and have impaired
mitochondrial energy production. This defect in cellular energy
production can be completely reversed by the provision of a
physiological ratio of ketone bodies consisting of 4 mM D-.beta.
hydroxybutyrate and 1 mM acetoacetate (95). Brain was thought to be
capable of using only glucose as its metabolic energy source and to
be insensitive to the actions of insulin. However, in a remarkable
clinical study performed in 1967, George Cahill and his
collaborators (47) showed that up to 60% of the brain's need for
metabolic energy could be met by ketone bodies in obese patients
undergoing prolonged fasting. Even more remarkably, Cahill showed
that administration of insulin to these patients in doses
sufficient to drop their blood sugar from 4 to under 2 mM was
associated with no impairment of mental functions in these patients
whose blood D-.beta. hydroxybutyrate was 5.5 mM and acetoacetate 2
mM (see FIG. 3 from ref 63). Clearly, when ketone bodies are
present in the blood at levels above 5 mM, they are able to
substitute for the brain's usual need for glucose and abolish the
hypoglycemic symptoms expected at blood glucose levels of 1.5
mM
[0033] 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) .
[0034] It is the inventors 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, eg. 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.
[0035] Furthermore, utilising the nerve growth/stimulatory effects
of the ketone bodies, particularly D-.beta.-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.
[0036] In fed animals and in man the liver content, which is
essentially that of blood, of acetoacetate is very low at 0.09 mM
and D-.beta. hydroxybutyrate is 0.123 mM but rises after a 48 hour
fast to 0.65 mM acetoacetate and 1.8 mM D-.beta. 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 extrahepatic 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 in
the following set of reactions:
##STR00001##
[0037] Once made in the liver, ketone bodies are transported out of
the liver into the blood stream by the monocarboxylate --H'
co-transporter (20) by the following reaction:
##STR00002##
[0038] 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
L-.beta.-hydroxybutyrate can also act as competitive inhibitors to
ketone body transport. Since ketone body transport across the blood
brain barrier is the 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:
##STR00003##
[0039] The present inventor has thus determined that the
mitochondrial acetyl CoA from ketone bodies 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 FIG. 3 to
help illustrate these concepts further.
[0040] The liver cannot utilize ketone bodies because it lacks the
3 Oxoacid CoA transferase necessary for the formation of
acetoacetyl CoA. Ketone bodies, in contrast to free fatty acids,
cannot produce acetyl CoA in liver. Since acetyl CoA is the
essential precursor of fatty acid synthesis through malonyl CoA and
cholesterol synthesis through cytosolic HMG CoA, ketone bodies
cannot result in either increased fatty acid or cholesterol
synthesis in liver, which usually accounts for over half of the
bodies synthesis of these two potentially pathogenic materials.
Liver is sensitive to the ratio of
actoacetate/D-.beta.-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) (55)
[0041] 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.
[0042] 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%
McCollums salt mixture, develop blood ketones of about 2 mM.
Substitution of lard for corn oil raises blood ketones to almost 5
mM (Veech, unpublished).
[0043] An example of a traditional 1500/day calorie ketogenic diet
recommended by the Marriott Corp. Health Care Services, Pediatric
Diet Manual, Revised August 1987 as suitable for a 4-6 year old
epileptic child contained from 3:1 to 4:1 g of fat for each g of
combined carbohydrate and protein. At each of 3 meals the patient
must eat 48 to 50 g fat, only 6 g protein and 10 to 6.5 g
carbohydrate. In practice this means that at each meal the child
must eat 32 g of margarine per day (about 1/4 stick) and drink 92 g
of heavy cream (about 100 ml), comprised mainly as medium chain
length triglycerides.
[0044] An example of a diet achieving a 3:1 ratio of fat to
combined carbohydrate and protein is given in Table 1 below.
TABLE-US-00002 TABLE 1 Sample 1500 calorie diet to achieve 3:1
lipid to carbohydrate + protein diet Amount (g) Fat (g) Protein (g)
CHO (g) Breakfast Egg 32 4 4 apple juice 70 7 margarine 11 10 heavy
cream 92 34 2 3 Total Breakfast 48 6 10 Lunch lean beef 12 1.75 3.5
cooked carrots 45 0.6 3 canned pears 40 4 margarine 14 12.5 heavy
cream 92 34 2 3 Total Lunch 48.25 6.1 10 Supper Frankfurter 22.5 6
3 Cooked broccoli 50 1 2 Watermelon 75 5 Margarine 8 7.5 Heavy
cream 92 34 2 3 Total Supper 47.5 6 10 Daily Total 143.75 18.1
30
[0045] In general the levels of ketone bodies achieved on such
diets are about 2 mM D-.beta. hydroxybutyrate and 1 mM acetoacetate
while the levels of free fatty acids 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) to improve exercise tolerance (83).
[0046] 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 normal
obese 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 the 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 with its
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.
[0047] Supplementing a liquid diet with 47% of its caloric content
with either glucose or racemic 1,3 butandiol caused the blood
ketone concentration to rise about 10 fold to 0.98 mM D-.beta.
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-hydroxybutanoate).
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 D-.beta.
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.
[0048] The mono and diester 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 brief peaks 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.
[0049] 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. (R) 1,3 butandiol may
easily be synthesized by reduction of the monomeric D-.beta.
hydroxybutyrate, with for example LiAlH.sub.4. (R) 1,3 butandiol is
subject to being oxidized in the liver to form D-.beta.
hydroxybutyrate without marked distortion of the hepatic redox
state. Studies in rats have shown that feeding racemic 1,3
butandiol caused liver cytosolic [NAD']/[NADH] to decrease from
1500 to about 1000 (87). By comparison, administration of ethanol
reduces hepatic [NAD-]/[NADH] to around 200 (106).
[0050] Acetoacetate, when freshly prepared, can be used in infusion
solutions where it can be given in physiologically normal ratios 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 burns (85).
[0051] However, neither 1,3 butandiol, which forms acetoacetate,
nor glycerol, which is a precursor of glucose, is part of the
normal redox couple, D-.beta. hydroxybutyrate/acetoacetate. For the
present invention, under optimum conditions, a physiological ratio
of ketones should be given. 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 pathological consequences are a distinct
possibility. 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
D-.beta. hydroxybutyrate, where cardiac efficiency was increased by
a physiological ratio of ketone bodies (95).
[0052] The best exogenous source of ketone bodies, which do not
require ingestion of large amounts of lipid nor the use of material
which produce the physiologically incompatible isomers
L-.beta.-hydroxybutyrate would be ketone bodies themselves. However
the present invention also provides alternatives for administration
in therapy.
[0053] A first alternative are polyesters of
D-.beta.-hydroxybutyrate. Natural polyesters of
D-.beta.-hydroxybutyrate are sold as articles of commerce at
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 D-.beta.-hydroxybutyrate has now been cloned and variations in
the composition of the polymer produced, based on the substrates
given to the bacteria demonstrated. However, these polymers failed
to be able to compete with petroleum based plastics. Nevertheless
the genes responsible for the synthesis of polyalkanoates has 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] Poly D-.beta.-hydroxybutyrate comes in a number of forms
from different biological sources as an insoluble white powder with
little taste and no odour and is suitable for incorporation into
compositions for oral or other means of administration. Esterases
capable of breaking the ester bonds of this material are ubiquitous
in plasma and most cells. These polymer are also easily split by
alkaline hydrolysis in vitro to make a series of polymers
culminating in the production of the monomer of MW 104, which is
transported from gut to portal vein by the normal monocarboxylate
transporter. Alternatively acid hydrolysis may be carried out using
the published method referred to in the Fluka promotional
material.
[0055] Preferred forms of D-.beta.-hydroxybutyrate polymer are
oligomers of that ketone body designed to be readily digestable
and/or metabolised by humans or animals. These preferably are of 2
to 100 repeats long, typically 2 to 20 and most conveniently from 3
to 10 repeats long. It will be realised that mixtures of such
oligomers may be employed with advantage that a range of uptake
characteristics might be obtained.
[0056] Particularly preferred are cyclic oligomers of
D-.beta.-hydroxybutyrate, known as oligolides, having formula
##STR00004## [0057] where n is an integer of 1 or more [0058] or a
complex thereof with one or more cations or a salt thereof
[0059] Preferred cations are sodium, potassium, magnesium and
calcium. Such cations are typically balanced by a physiologically
acceptable counter-anion such that a salt is provided.
[0060] Examples of typical physiologically acceptable salts will be
selected from sodium, potassium, magnesium, L-Lysine and L-arginine
or eg. more complex salts such as those of methyl glucamine
salts
[0061] 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, ie. (R, R, R)-4,8,12-trimethyl-1,
5,9-trioxadodeca-2,6,10-trione, 2, 3, 4 or 5.
[0062] 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.
[0063] In preferred forms of all of the aspects of the invention,
where the oligomer of of D-.beta.-hydroxybutyrate does not include
acetoacetyl groups it is optionally and preferably administered
together with a physiological ratio of acetoacetate or a metabolic
precursor of acetoacetate.
[0064] Once the monomer is in the blood stream, and since liver is
incapable of metabolizing ketone bodies but can only alter the
ratio of D-.beta.-hydroxybutyrate/acetoacetate, the ketone bodies
are transported to extrahepatic tissues where they can be utilized.
The blood levels of ketones achieved are not be 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 be
made to keep levels at 7.5 mM 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).
[0065] The determination by the inventor that
D-.beta.-hydroxybutyrate and its mixtures with acetoactetate act as
a nerve stimulant, eg. 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.
[0066] 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 oligomers and polymers are
generally taste and smell free. Formulations of
D-.beta.-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 art.
[0067] Since ketone bodies contain about 6 calories/g, there is
preferably a compensatory decrease in the amounts of the other
nutrients taken to avoid obesity.
[0068] Particular advantages of using the ketone bodies or
precursors such as poly or oligo-D-.beta.-hydroxybutyrate or
acetoacetate esters are:
[0069] 1) they can be eaten with a normal dietary load of
carbohydrate without impairing its effects,
[0070] 2) they will not raise blood VLDL, as with current cream and
margarine containing diets, thus eliminating the risk of
accelerated vascular disease, fatty liver and pancreatitis,
[0071] 3) they will have a wider range of use in a greater variety
of patients, including: type II diabetes to prevent hypoglycemic
seizures and coma, in Alzheimer's disease and other
neurodegenerative states to prevent death of nerve cells eg.
hippocampal cells, and in refractory epilepsy due to either
decreases in cerebral glucose transporters, defects in glycolysis,
or so called
[0072] Leigh's syndromes with congenital defects in PDH.
[0073] The second group of particular alternatives are acetoacetate
esters of D-.beta.-hydroxybutyrate. Esters which provide a
physiological ratio of acetoacetate to D-.beta.-hydroxybutyrate are
preferred eg. from 1:1 to 1:20, more preferably from 1:1 to 1:10.
The tetramer of D-.beta.-hydroxybutyrate with a terminal
acetoacetate residue is particularly preferred. Such materials have
the added virtue of having a physiological ratio of
D-.beta.-hydroxybutyrate/acetoacetate moieties, thus removing the
burden on liver of having to adjust the redox state of the
administered nutrient without inducing abnormal reduction of
hepatic [NAD.sup.+]/[NADH] as occurs with excessive alcohol
consumption. The polymeric esters, depending upon their length,
have decreasing water solubility, but are heat stable. Such
polymers can for example 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.
[0074] Examples of poly D-.beta.-hydroxybutyrate or terminally
oxidized poly D-.beta.-hydroxybutyrate esters useable as ketone
body precursors are given below.
Poly (R) 3-Hydroxybutyric Acid
##STR00005##
[0075] Oxidized Poly (R) 3-Hydroxybutyric Acid
##STR00006##
[0077] In each case n is selected such that the polymer or oligomer
is readily metabolised on administration to a human or animal body
to provide elevated ketone body levels in blood. Preferred values
of n are inetegers of 0 to 1,000, more preferably 0 to 200, still
more preferably 1 to 50 most preferably 1 to 20 particularly
conveniently being from 3 to 5.
[0078] A number of variations of this material, including the
polyester D-.beta.-hydroxybutyrate itself can also be tried for
suitable manufacturing characteristics. The material is a tasteless
white powder. After partial alkaline hydrolysis, a mixture of
varying chain length polymers would be provided, which would tend
to smooth gut absorption and maintain high sustained levels of
ketone over a 24 hour period.
[0079] Treatment may comprise provision of a significant portion of
the caloric intake of patients with the D-.beta.-hydroxybutyrate
polyester formulated to give retarded release, so as to maintain
blood ketones in the elevated range, eg. 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. Enetrically coated forms targeting
delivery post stomach may be particularly used where the material
does not require 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 sucha s those referred to in Doi. Microbial
Polyesters.
[0080] Intravenous infusion of sodium salts of
D-.beta.-hydroxybutyrate has been performed on normal human
subjects and patients for a number of conditions, eg. those
undergoing treatment for severe sepsis in an intensive care unit.
It was found to be non-toxic and capable of decreasing glucose free
fatty acids and glycerol concentration, but ineffective in
decreasing leucine oxidation.
[0081] The monomer of D-.beta.-hydroxybutyrate is a white,
odourless crystal with a slightly tart or acid taste which is less
in intensity in comparison to vinegar or lemon juice. It can be
formulated into most foodstuffs, eg. drinks, puddings, mashed
vegetables or inert fillers. The acid forms of
D-.beta.-hydroxybutyrate are suitable for use orally as they have a
pKa of 4.4. This is less acid than citric acid with pKal of 3.1 and
pKa2 of 4.8 and slightly more acidic than acetic acid with a pKa of
4.7.
[0082] Preferably, only the natural D- or (R) isomer is used in
this formulation. Since in practice it is not possible to achieve
absolute isomeric purity, the article of commerce currently sold by
Sigma, St. Louis Mo. or Fluka, Ronkonkoma, N.Y. is the most
suitable for this purpose. The optical rotation of the commercially
available D-.beta.-hydroxybutyratic acid is -25.degree..+-.1 at the
wavelength of Na and its melting point 43-46.degree. C. The optical
rotation of the Na salt of D-.beta.-hydroxybutyrate is
-14.5.degree. and its melting point 149-153.degree. C. Both can be
assayed by standard enzymatic analysis using
D-.beta.-hydroxybutyrate dehydrogenase (EC 1.1.1.30)(5).
Acetoacetate can be determined using the same enzyme (56). The
unphysiological (S) isomer is not measurable with enzymatic
analysis but can be measured using GC mass spec (13).
[0083] For a 1500 calorie diet, the human adult patient could
consume 198 g of ketones 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.
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/1 g of fat. Accordingly, blood ketones should be
approximately 3 mM, but still below the level achieved in fasting
man of 7.5 mM.
[0084] The advantage of using 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
metabolized 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.
[0085] The present inventor has determined that, while any
elevation of ketone bodies may be desirable, a preferred amount of
ketone bodies to be administered will be sufficient to elevate
blood 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. While dead
cells cannot be restored, arrest of further deterioration and at
least some restoration of function is to be anticipated.
[0086] Thus in a first aspect of the present invention there is
provided the use acetoacetate, D-.beta.-hydroxybutyrate or a
metabolic precursor of either in the manufacture of a medicament or
nutritional aid (i) for increasing cardiac efficiency, particularly
efficiency in use of glucose (ii) for providing energy source,
particularly in treating diabetes and insulin resistant states or
by increasing the response of a body to insulin (iii) for
reversing, retarding or preventing nerve cell damage or death
related disorders, particularly neurodegenerative disorders such as
memory associated disorders such as Alzheimer's, seizure and
related states such as encepalophies such as CJD and BSE.
[0087] The term metabolic precursor thereof particularly relates to
compounds that comprise 1,3-butandiol, acetoacetyl or
D-.beta.-hydroxybutyrate moieties such as
acetoacetyl-1,3-butandiol, acetoacetyl-D-.beta.-hydroxybutyrate,
and acetoacetylglycerol. Esters of any such compounds with
monohydric, dihydric or trihydric alcohols is also envisaged.
[0088] This aspect includes such use as a neuronal stimulant eg
capable of stimulating axonal and/or dendritic growth in nerve
cells, eg. in Hippocampus or Substantia nigra particularly in
diseases where neurogeneration has serious clinical
consequences.
[0089] In diabetic patients this use of these compounds 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.
[0090] The present inventor has determined that supplementing type
II diabetics with ketone bodies would 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.
[0091] 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 ketone bodies, their polymers or
esters or precursors such as butandiol compounds, due to the
reduction or elimination of 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.
[0092] 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 eleimination of acetyl CoA supply to the mitochondrion such as
insulin coma and hypoglycaemia, defects in the glucose transporter
in the brain or in glycolytic enzyme steps or in pyruvate
transport.
[0093] Preferably the hydroxybutyrate is in the form of non-racemic
D-.beta.-hydroxybutyrate and more preferably it is administered in
a form which also supplies acetoacetate. Preferably the metabolic
precursor is one which when administered to a human or animal body
is metabolised, eg. by liver, to produce one or both of
D-.beta.-hydroxybutyrate and acetoacetate, more preferably in a
physiological ratio. Particularly preferred are
poly-D-.beta.-hydroxybutyric acid or
acetoacetoyl-.beta.-hydroxybutyrate oligomers or an ester of one or
both of these. Lower alkyl esters such as C.sub.1-4 alkyl esters
may be employed but more preferably are more physiologically
acceptable esters such as the respective 1,3-butandiol esters,
particularly employing (R)-1,3-butandiol. Most preferred are the
acetoacetyl-tri-, tetra- and penta-D-.beta.-hydroxybutyrate esters.
Ester precursors will include esters of 1,3-butandiol, preferably
(R) form and particularly acetoacetate esters such as acetoacetyl
glycerol.
[0094] Preferred poly D-.beta.-hydroxybutyrate esters are those
which are esters of the preferred oligomers of 2-100 repeats, eg.
2-20 repeats most preferably 2-10 repeats.
[0095] Where the medicament or nutritional product of the invention
is for use without prolonged storage it is convenient to use it in
the form of a liquid or solid composition comprising the hydroxy
substituted carboxylic acid and/or the ketone, preferably
comprising both and where these are the D-.beta.-hydroxybutyrate
acids and acetoacetate together preferably in the ratio of about
3:1 to 5:1, more preferably about 4:1.
[0096] Where the medicament or aid 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. For example, the ethyl ester of
acetoacetate is relatively stable with a boiling point of
180.8.degree. C.
[0097] Still more preferably, the medicament or aid comprises an
acetoacetyl ester of D-.beta.-hydroxybutyrate or such an ester of
an oligomer of D-.beta.-hydroxybutyrate as described. This may be
supplemented with D-.beta.-hydroxybutyrate or one of the polymers
of that, e.g. oligo-D-.beta.-hydroxybutyrate, in order to bring
about the preferred ratio of the two components. Such a composition
will provide the two preferred components when the ester and
polymer are metabolised in the stomach or in the plasma of the
human or animal which has consumed them. Again an (R) 1,3-butandiol
ester of the acetoacetyl-D-.beta.-hydroxybutyrate may be most
preferred as it will be more lipophilic until metabolised or
otherwise deesterified and all its components are convereted to the
desired ketone bodies.
[0098] A second aspect of the invention provides novel esters of
acetoacetate for use in therapy or as a nutritional aid. Such
esters may include C.sub.1-4 alkyl esters but most preferred are
the D-.beta.-hydroxybutyryl-acetoacetate esters referred to
above.
[0099] A third aspect of the present invention provides a
poly-D-.beta.-hydroxybutyrate for use in therapy, particularly
where this is in a form selected for its ability to be degraded in
acid conditions of the stomach or by esterases in vivo.
[0100] A fourth aspect of the invention provides a method for the
synthesis of D-.beta.-hydroxybutyryl-acetoacetate esters comprising
the reaction of acetoacetic acid halide, e.g. acetoacetyl chloride,
with D-.beta.-hydroxybutyrate. Preferably this is achieved by
reacting acetoacetic acid with an activating agent, such as thionyl
chloride, to produce the acid chloride.
[0101] A fifth aspect of the present invention provides a method
for the synthesis of D-.beta.-hydroxybutyryl-acetoacetate esters
comprising the reaction of D-.beta.-halobutyrate or its oligomers
with acetoacetic acid, activated forms thereof or diketene .
[0102] A sixth aspect of the present invention provides a
D-.beta.-hydroxybutyryl-acetoacetate ester per se, a
physiologically acceptable salt or short or mdium chain mono, di or
trihydric alcohol or 1,3-butandiol estsr thereof.
[0103] A seventh aspect of the present invention provides
poly-D-.beta.-hydroxybutyrate together with a pharmaceutically or
physiologically acceptable carrier.
[0104] An eighth aspect of the present invention provides a
composition comprising D-.beta.-hydroxybutyrate and acetoacetic
acid in a ratio of from 1;1 to 20:1, more preferably 2:1 to 10:1
and most preferably from 3:1 to 5:1 together with a
pharmaceutically or physiologically acceptable carrier. Preferably
the ratio of these components is about 4:1. Such a composition does
not consist of plasma, serum or animal or plant tissue already used
as a medicament or foodstuff, thus it is that which is preferably
sterile and pyrogen free. Particularly the ketione bodies comprise
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.
[0105] A ninth aspect of the present invention comprises a method
of treating a human or animal in order to increase their cardiac
efficiency comprising administering to that person at least one of
a materials for use in the first to eight aspects of the
invention.
[0106] A tenth aspect of the present invention comprises a method
of treating a human or animal in order to increase their the
response to insulin comprising administering to that person at
least one of a materials for use in the first to eight aspects of
the invention.
[0107] An eleventh aspect of the present invention comprises a
method of treating a human or animal in order to treat an insulin
resistant state comprising administering to that person least one
at least one of a materials for use in the first to eight aspects
of the invention.
[0108] By insulin resistant state herein is included forms of
diabetes, particularly those that do not respond fully to
insulin.
[0109] A twelvth aspect of the invention provides a method of
treating a human or animal in order to treat a nerve cell, eg.
brain cell, death or damage related disorder as referred to for the
first aspect, particularly a neurodegenerative disorder eg. such as
those related to neurotoxic conditions such as presence of amyloid
protein, eg. a memory associated disorder such as Alzheimer's
disease, or epileptic seizures, comprising administering to that
person least one at least one of a materials for use in the first
to eight aspects of the invention.
[0110] Preferred methods of the ninth to twelvth aspects of the
invention use the preferred ketones and polyacids and acid esters
of the invention.
[0111] Methods of preparing poly D-.beta.-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.
[0112] Particular advantages of use of the biologically available
polymers of the invention include the reduction in the amount of
counter ions such as sodium that have to be coadministered with
them. This reduction in sodium load is advantageous particularly in
ill health. By biologically available is meant those materials
which can be used by the body to produce the least one of a
D-.beta.-hydroxybutyrate, acetoacetate and a mixture of these in
physiological ratio as described above
[0113] The amount of ketone bodies used in treatment of
neurodegeneration such as Alzheimer's and Parkinsonism will
preferably elevate blood levels to 0.5 mM to 20 mM, eg 2 mM to 7.5
mM as described above. The present inventor estimates that 200 to
300 g (0.5 pounds) of ketone bodies per patient per day mignt be
required to achieve this. Where the treatment is through
maintenance of cells against the effects of neurotox in this may be
at a higher level, eg. 2 to 7.5 mM in blood. Where it relies on the
nerve stimulatory factor effect of the D-.beta.-hydroxybutyrate so
produced the amount administered may be lower, eg. to provide 0.2
to 4 mM, but can of course be more for this or other disease.
[0114] It will be realised that treatment for neurodegenerative
diseases such as Alzheimer's 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.
[0115] 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 D-.beta.-hydroxybutyrate and 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
D-.beta.-hydroxybutyrate, its metabolisable polymers, its
acetoacetate esters or their precursors (R)-1,3-butandiol and its
acetoacetate esters, eg. acetoacetyl glycerol, or its administered
intravenously or intrarterially the ketone bodies
D-.beta.-hydroxybutyrate and acetoacetic acid. All of the organic
materials referred to above are optionally in salt or ester form.
Examples of typical physiologically acceptable salts will be
selected from sodium, potassium, magnesium, L-Lysine and L-arginine
or eg. more complex salts such as those of methyl glucamine salts.
Esters will be those as described previously for other aspects of
the invention.
[0116] A still further aspect of the invention provides the ketone
bodies of the invention by suitable control of diet. Thus this
aspect provides a method of treatment of a human or animal for a
disorder of one or more of the ninth to the twelvth aspects of the
invention comprising one of (i) total fasting of the individual and
(ii) feeding the individual a ketogenic diet eg. of 60-80% lipid
with carbohydrate content 20% or less by weight.
[0117] For the purpose of treaing seizures, eg. in epilepsy, a diet
may involve ad lib ingestion of carbohydrate by oral or enteral
route or of the compounds specified above.
[0118] In all these treatments other than the ketogenic diet there
is the improvement that a method of avoiding drop in blood ketones
which accompanies the ingestion of excess carbohydrate and a method
which avoids feeding of excess lipid which accelerates the
synthesis by liver of fatty acids and cholesterol which would
otherwise contribute to vascular disease.
[0119] 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.
[0120] The provision of ketone body 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 (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
generating products such as yoghurts and cheese that are enriched
in either poly-(R)-3-hydroxybutyric acid or, after breakdown with
enzymes capable of cleaving such polymers, with the monomeric
substance itself (see Doi. Chapter 8).
[0121] 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.
FIGURES
[0122] FIG. 1 is a graph showing blood (R)-3-hydroxybutyrate level
produced after time after gavage of (R)-3-hydroxybutyrate, an
oligomer of this as produced in Example 1 and an acetoacetyl
monomer therof as produced in Example 2;
[0123] FIG. 2 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; and
[0124] FIG. 3 shows the reactions of the Krebs cycle.
EXAMPLES
Example 1
Preparation of Oligomers of (R)-3-Hydroxybutyric Acid
(D-.beta.-Hydroxybutyrate)
[0125] (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 withn 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
avaerage 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.
Example 2
Preparation of Acetoacetyl Ester of Oligomeric (R)-3-Hydroxybutyric
Acid
[0126] 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
vacuo. 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 D-.beta.-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 Example 1 and Example 2 are susceptible to separation
of individual components by preparative HPLC.
Example 3
Oral Administration of D-.beta.-Hydroxybutyrate, Oligomers and
Acetoacetyl D-.beta.-Hydroxybutyrate Oligomers to Rats
[0127] The ability of orally administered D-.beta.-hydroxybutyrate
and the oligomers of Examples 1 and 2 to raise blood ketone body
levels was investigated as follows. Rats were starved overnight and
then gavaged with 100 .mu.l/100 g bodyweight of 4M
D-.beta.-hydroxybutyrate brought to pH 7.74 using methyl glucamine.
Blood levels of D-.beta.-hydroxybutyrate measured using and
NAD+/EDTA assay of Anal. Biochem. 131, p 478-482 (1983). 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+ (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 D-.beta.-hydroxybutyrate control.
[0128] As the rats had been fasted the initial levels of
D-.beta.-hydroxybutyrate were elevated from the 0.1 mM fed state.
However, consistent serum increases of D-.beta.-hydroxybutyrate,
between 1 and 3.2 mM increase in each case, were provided.
[0129] This procedure was repeated with 2M solutions of the
mixtures of D-.beta.-hydroxybutyrate oligomers and their
acetoacetyl esters described in Examples 1 and 2. The
D-.beta.-hydroxybutyrate oligomer (19/1 in FIG. 1) and the
acetoacetyl ester (20/4 in FIG. 1) were both brought to pH 7.6 with
methyl glucamine and the blood D-.beta.-hydroxybutyrate level
monitored using the aforesaid assay procedure. Increases in serum
D-.beta.-hydroxybutyrate were shown to be of 0.5 to 1.2 mM at 60
and 120 minutes after gavaging. These results demonstrate the
efficacy of orally administered D-.beta.-hydroxybutyrate and its
metabolic precursors of the invention in raising blood levels
significantly for a period of hours after intake.
[0130] It was noted that the oligomeric esters 19/1 and 20/4, while
not elevating the blood ketone body level as high as the monomer
itself, did result in elevation for a much longer period of time
and thus are suited to adminsitration less frequently than the
monomer.
Example 4
TABLE-US-00003 [0131] TABLE 2 Sample 1500 calorie ketogenic diet
using ketone bodies, their esters or polymers. The ketones were
assumed to contain 6 kcal/g, fats 9 kcal/g, carbohydrate and
protein 4 kcal/g. Ketones have been substituted to give equivalent
calories. Amount Fat Protein CHO Ketones (g) (g) (g) (g) (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 5
Effect of Increased Blood D-.beta.-Hydroxybutyrate Levels on Whole
Brain GABA Levels
[0132] To assess the effect of D-.beta.-hydroxybutyrate on whole
brain GABA levels, and thus provide an indication of antiepileptic
effect of ketone body or precursor treatment aimed at increasing
blood ketone body levels, whole rat brain was frozen at set times
after administration of D-.beta.-hydroxybutyrate as described in
Example 3. GABA was assayed using standard HPLC technique and
related to protein content using standard protein assay. At t=0
GABA levels were 191 pmoles/.mu.g protein while at 120 minutes this
was elevated at 466 pmoles/.mu.gprotein, demonstrating antepileptic
potential.
Example 6
Effect of D-.beta.-Hydroxybutyrate on .beta.-Amyloid Toxicity to
Hippocampal Cells in Vitro
Culture Medium and Chemicals
[0133] 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.
Hippocampal Microisland Cultures
[0134] The primary hippocampal cultures were removed from Wistar
embryos on day 18 and dispersed by gentle aggitation in a pipette.
The suspension was centrifuge at 1,500.times.g for 10 min and the
supernatant discarded. New media was make 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
[0135] 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 Na D-.beta.-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 visual neurons
and vimentin and GFAP (Boehringer) to visualize glial cells.
Results
Cell Counts
[0136] Addition of D-.beta.-hydroxybutyrate to the incubation
resulted in an increase in the neuronal cell number per microisland
from a mean of 30 to 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 D-.beta.-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
D-.beta.-hydroxybutyrate, to media whose major nutrients are
glucose, pyruvate and L-glutamine, slows the rate of cell death in
culture. We further conclude that D-.beta.-hydroxybutyrate can
decrease the increased rate of hippocampal cell death caused by the
addition of amyloid .beta..sub.1-42 in culture.
[0137] The number of dendritic outgrowths and the length of axons
were both observed to have increased with presence of
D-.beta.-hydroxybutyrate, whether .beta..sub.1-42 was present or
not. This is indicative of nerve growth factor like behaviour.
Example 7
Preparation of
(R,R,R)-4,8,12-trimethyl-1,5,9-trioxadodeca-2,6,10-trione: triolide
of (R)-3-hydroxybutyric acid
##STR00007##
[0139] Synthesis was as described in Angew. Chem. Int. Ed. Engl.
(1992), 31, 434. A mixture of poly[(R)-3-hydroxybutyric acid] (50
g) and toluene-4-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.
Example 8
Oral Administration of Triolide of D-.beta.-Hydroxybutyrate of
Example 1 to Rats
[0140] 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.
[0141] 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.
[0142] 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
D-.beta.-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 D-.beta.-hydroxybutyrate control.
[0143] 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 D-.beta.-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.
[0144] The test rats thus showed increase in plasma
D-.beta.-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.
[0145] For all examples given above, 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.
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