U.S. patent application number 09/359086 was filed with the patent office on 2002-01-31 for nutritional and therapeutic uses of 3-hydroxyalkanoate oligomers.
Invention is credited to MARTIN, DAVID P., PEOPLES, OLIVER P., WILLIAMS, SIMON F., ZHONG, LUHUA.
Application Number | 20020013339 09/359086 |
Document ID | / |
Family ID | 22240563 |
Filed Date | 2002-01-31 |
United States Patent
Application |
20020013339 |
Kind Code |
A1 |
MARTIN, DAVID P. ; et
al. |
January 31, 2002 |
NUTRITIONAL AND THERAPEUTIC USES OF 3-HYDROXYALKANOATE
OLIGOMERS
Abstract
Nutritional or therapeutic compositions are provided for
increasing ketone body levels in the blood of mammals by providing
a source of ketone bodies in the form of linear or cyclic oligomers
and/or derivatives of 3-hydroxyacids. The 3-hydroxyacid can be in
the form of a linear oligomer of 3-hydroxyacids other than linear
homo-oligomers of 3-hydroxybutyric acid if administered in
combination with acetoacetate, cyclic oligomers of 3-hydroxyacids,
esters of the linear or cyclic oligomers, esters of 3-hydroxyacids
other than 3-hydroxybutyric acid, and combinations thereof. An
oligomer generally refers to a polymer of three or more
hydroxyacids. Preferred 3-hydroxyacids include 3-hydroxybutyrate,
3-hydroxyvalerate, 3-hydroxyhexanoate, and 3-hydroxyheptanoate.
Oligomers of odd-carbon number 3-hydroxyacids such as
3-hydroxyvalerate have advantages since they have a higher energy
content than oligomers of 3-hydroxyacids having an even-number of
carbons. The cyclic oligomers have advantageous properties since
they result in a sustained, and/or controlled, ketone blood level
over a period of hours. The compositions can be administered
orally, for example, as a nutritional or dietary supplement, or
intravenously. Increasing blood ketone levels is useful for seizure
control, metabolic disease control, reduction of protein
catabolism, appetite suppression, parenteral nutrition, increasing
cardiac efficiency, treatment of diabetes and insulin resistant
states, and treatment of effects of neurodegenerative disorders and
epilepsy.
Inventors: |
MARTIN, DAVID P.;
(ARLINGTON, MA) ; PEOPLES, OLIVER P.; (ARLINGTON,
MA) ; WILLIAMS, SIMON F.; (SHERBORN, MA) ;
ZHONG, LUHUA; (QUINCY, MA) |
Correspondence
Address: |
PATREA L. PABST
HOLLAND & KNIGHT LLP
ONE ATLANTIC CENTER
1201 WEST PEACHTREE STREET, SUITE 2000
ATLANTA
GA
30309-3400
US
|
Family ID: |
22240563 |
Appl. No.: |
09/359086 |
Filed: |
July 22, 1999 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60093760 |
Jul 22, 1998 |
|
|
|
Current U.S.
Class: |
514/310 |
Current CPC
Class: |
A61P 25/00 20180101;
A61K 31/00 20130101; A61P 25/28 20180101; A23K 20/105 20160501;
A61P 9/00 20180101; A61P 7/00 20180101; A61P 43/00 20180101; A61K
31/19 20130101; A23V 2002/00 20130101; A61P 3/04 20180101; A23V
2002/00 20130101; A61P 21/04 20180101; A61P 3/10 20180101; A23K
50/10 20160501; A61K 31/765 20130101; A61P 25/08 20180101; A61P
3/00 20180101; A23V 2002/00 20130101; A23V 2002/00 20130101; A23V
2250/206 20130101; A23L 33/10 20160801; A23V 2250/21 20130101; A23V
2250/30 20130101 |
Class at
Publication: |
514/310 |
International
Class: |
A61K 031/47; A01N
043/42 |
Claims
We claim:
1. A nutritional or therapeutic dietary composition comprising an
effective and biocompatible amount of 3-hydroxyacid to modulate
blood ketone body levels in mammals wherein the 3-hydroxyacid is in
a form selected from the group consisting of linear oligomers of
3-hydroxyacids other than linear homo-oligomers of 3-hydroxybutyric
acid in combination with acetoacetate, cyclic oligomers of
3-hydroxyacids, esters of 3-hydroxyacids other than
3-hydroxybutryic acid in combination with acetoacetates, esters of
3-hydroxyacid linear and cyclic oligomers other than linear
homo-oligomers of 3-hydroxybutyric acid in combination with
acetoacetate, and combinations thereof.
2. The composition of claim 1 wherein the hydroxyacid is not a
salt.
3. The composition of claim 1 wherein the 3-hydroxyacids are cyclic
oligomers.
4. The composition of claim 3 wherein the 3-hydroxyacid is
3-hydroxybutyric acid.
5. The composition of claim 1 wherein the 3-hydroxyacids are
selected from the group consisting of 3-hydroxyvalerate,
3-hydroxyhexanoate, 3-hydroxyheptanoate, 3-hydroxyoctanoate, and
combinations thereof.
6. The composition of claim 1 wherein the 3-hydroxyacid ester is a
cyclic ester.
7. The composition of claim 6 wherein the cyclic ester is the
triolide of 3 -hydroxybutyrate.
8. The composition of claim 3 wherein the cyclic oligomers comprise
the cyclic macrolide of R-3-hydroxyacids containing 3, 4, or 5
monomeric subunits.
9. The composition of claim 8 wherein the 3-hydroxyacids are
selected from the group consisting of 3-hydroxybutyric acid,
3-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic
acid, and combinations thereof.
10. The composition of claim 1 wherein the 3-hydroxyacid oligomers
comprise R-3-hydroxyalkanoate oligomers terminated with an ester
linkage.
11. The composition of claim 10 wherein the oligomer is terminated
to 1,3 butanediol.
12. The composition of claim 1 in a formulation suitable for
intravenous administration to a mammal.
13. The composition of claim 1 wherein the 3-hydroxyacids are
derived from plant or bacterial biomass.
14. The composition of claim 1 in a dietary formulation for oral
ingestion.
15. The composition of claim 14 in a dietary formulation for
administration to livestock.
16. A method of modulating blood ketone levels in a mammal
comprising administering to the mammal an effective amount of a
nutritional or therapeutic dietary composition comprising an
effective and biocompatible amount of 3-hydroxyacid to modulate
blood ketone body levels in mammals wherein the 3-hydroxyacid is in
a form selected from the group consisting of linear oligomers of
3-hydroxyacids other than linear homo-oligomers of 3-hydroxybutyric
acid in combination with acetoacetate, cyclic oligomers of
3-hydroxyacids, esters of 3-hydroxyacids other than
3-hydroxybutryic acid in combination with acetoacetates, esters of
3-hydroxyacid linear and cyclic oligomers other than linear
homo-oligomers of 3-hydroxybutyric acid in combination with
acetoacetate, and combinations thereof.
17. The method of claim 16 wherein the blood ketone level is
effective to control seizures.
18. The method of claim 16 wherein the blood ketone level is
effective to control metabolic diseases relating to synthesis and
metabolism of ketone bodies.
19. The method of claim 16 wherein the blood ketone level is
effective to reduce protein catabolism in and/or suppress the
appetite of the mammal.
20. The method of claim 16 wherein the blood ketone level is
effective to increase the cardiac efficiency of the mammal.
21. The method of claim 16 wherein the blood ketone level is
effective to treat diseases selected from the group consisting of
diabetes and other insulin resistant states, neurodegenerative
disorders, and epilepsy.
22. The method of claim 21 wherein the neurodegenerative disorders
are selected from the group consisting of Alzheimer's disease,
fronto-temperal 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, Downs syndrome associated
Alzheimer's, myasthenia gravis, and muscular dystrophy.
23. The method of claim 16 wherein the mammal is a human or
livestock animal.
24. The method of claim 16 wherein the composition is administered
parenterally.
25. The method of claim 16 wherein the composition is administered
orally as a dietary or nutritional composition.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Priority is claimed to U.S. provisional application Serial
No. 60/093,760, filed Jul. 22, 1998.
BACKGROUND OF THE INVENTION
[0002] The present invention is generally in the field of
nutritional and therapeutic compositions for the modulation of
ketone levels in humans and other mammals.
[0003] There are a number of conditions in human and animals in
which it is desirable to increase the levels of ketone bodies in
the human or animal body. Examples include seizure control,
treatment of certain metabolic disorders, reduction of protein
catabolism, appetite suppression during weight loss, and parenteral
nutrition.
[0004] A number of treatments exist for seizure control in
epileptic patients. Anti-seizure medications are popular; however,
they are not always effective and can cause undesirable
side-effects. A ketogenic diet has been used since the turn of the
century, but lost favor with the development of anti-seizure
medications. The ketogenic diet recently has attracted new interest
for the treatment of certain forms of epilepsy, as well as other
medical conditions. The diet, which typically is carefully
controlled and doctor supervised, is very high in fat calories and
low in carbohydrates. The diet forces the body to metabolize fats
instead of carbohydrates for energy, thereby elevating the level of
acetoacetate and D-3-hydroxybutyrate in the blood. These compounds
are referred to as "ketone bodies," thus the term "ketogenic" is
used to describe the diet.
[0005] While the exact mechanism of action of the ketogenic diet is
not well understood, it is believed that the elevated blood levels
of ketone bodies have sedative effects which help to prevent
seizures. In order to be effective for this purpose, however, the
patient must strictly observe the diet. Vitamin and mineral
supplements are included in the diet to make it nutritionally
complete, since the diet is very high in fat, low in proteins, and
requires the near elimination of carbohydrates. Each patient's diet
is mathematically calculated based on the age, size, and activity
level of the patient. Patients normally follow the diet for one to
two years, at which time the patient is slowly weaned onto a normal
diet. The diet has been found to be particularly effective with
epileptic children. Major drawbacks are that the diet is not very
palatable and that patient compliance demands complete commitment
on the part of the patient and his or her family. Moreover, the
diet's high fat content can increase the risk of vascular diseases,
such as atherosclerosis.
[0006] Special diets are also used when a person urgently needs to
lose weight for health reasons, for example prior to surgery or due
to complications from obesity. In this situation, the doctor may
prescribe a diet greatly restricting the person's caloric intake.
With the caloric intake reduced, the body is forced to metabolize
storage reserves for energy. The body can derive energy from fat
and skeletal tissue, such as muscle and proteins. It is preferable,
however, that fat tissue be used rather than protein, since the
breakdown of proteins (i.e. "catabolism") can undesirably result in
muscular atrophy, immuno-suppression, and reduced wound healing.
Supplementation of the diet with hydroxybutyric acid has been shown
to reduce protein catabolism in subjects on low energy diets (Pawan
& Semple, Lancet 8:15 (1983)). It also has been reported that
3-hydroxybutyrate beneficially suppresses the appetite.
[0007] Total parenteral nutrition ("TPN") is used to provide
nutrients to patients who are unable to ingest food orally, such as
in the case of intestinal failure. Common causes of this condition
include inflammatory disorders of the gastrointestinal tract (e.g.,
Crohn's disease), radiation enteritis, and short bowel resulting
from surgical resection of necrotic or diseased bowel.
Approximately 22,000 outpatients and 150,000 inpatients currently
receive TPN in the United States alone (PR Newswire: Orphan Medical
Announcement, Jun. 9, 1995). Patients receive the nutrients, which
typically are concentrated fat emulsions, directly into their
veins. The nutrient compositions are described, for example, in
U.S. Pat. No. 4,563,354 to Chang et al.; EP 0321428 Al; U.S. Pat.
No. 5,093,044 to Wretlind et al.; PCT WO 88/08301; PCT WO 90/02548;
PCT WO 90/02549; and PCT WO 90/11753. Parenteral treatment with fat
emulsions, however, can have serious side effects, such as catheter
obstruction, hyperlipemia, thrombopathy, fat overload syndrome, and
fat embolism (Desrochers, et al., J. Nutr. Biochem. 6:111-18
(1995)). It would therefore be tremendously beneficial to develop
high energy, water soluble nutrients which can be used for
long-term intravenous feeding.
[0008] In principle, the ketone bodies R-3-hydroxybutyrate and
acetoacetate, which are natural constituents of human sera, could
be used for intravenous feeding in lieu of fat emulsions. These
compounds are good fuels for peripheral tissues, except during
prolonged starvation and diabetic ketoacidosis, and are ultimately
oxidized to carbon dioxide. Unfortunately, administration of these
compounds in their acid form can cause vein irritation, and
infusion of the compounds as sodium salts can result in a dangerous
sodium overload (Desrochers, et al., J. Nutr. Biochem., 6:111-18
(1995)). To overcome these problems, researchers have explored the
administration of R-3-hydroxybutyrate with other basic amino acid
salts (Beylot et al, Crit. Care Med. 22:1091-98 (1994); Lammerant,
et al., J. Mol. Cell. Cardiol. 17:421-33 (1985)). Such treatments,
however, may interfere with the transport of amino acids across the
blood-brain barrier and/or harm patients with hepatic or renal
pathologies (Desrochers, et al., J. Nutr. Biochem. 6:111-18
(1995)). Others have described the use of sodium salts of
3-hydroxybutyric acid oligomers as nutrients, in order to decrease
the ratio of salt to ketone body (Japanese Patent No. 94,321,778 to
Hiraide, et al.).
[0009] Another approach utilizing a ketone body as a nutrient
focuses on the synthesis of a glycerol monoester of acetoacetate,
which is hydrolyzed in plasma and tissues to glycerol and
acetoacetate (Birkhahn & Border, Am. J. Clin. Nutr. 31:436-41
(1978); Birkhahn, et al., J. Nutr. 109:1168-74 (1979)). This
composition was first to provide administration of large amounts of
a ketone body without a large sodium load.
[0010] Researchers also have explored using precursors to the
ketone bodies. For example, R, S-1,3-butanediol is a water soluble
precursor, which is metabolized in the liver to R,
S-3-hydroxybutyrate (Desrochers, et al., J. Nutr. Biochem. 6:111-18
(1995)). However, the diol is unsuitable for use as an intravenous
nutrient because it has a low caloric density per osmol, and
because its oxidation in the liver markedly increases the
[NADH]/[NAD.sup.+] ratio, which can induce alcoholic hypoglycemia.
One effort to address these problems has focused on using an
acetoacetate ester of R, S-1,3-butanediol, so that acetoacetate
liberated by esterases can trap the reducing equivalents generated
in the liver by the oxidation of the diol (Desrochers, et al., J.
Nutr. Biochem. 6:111-18 (1995)).
[0011] Modulating ketone body levels also is useful in the
production of animals for the meat industry. U.S. Pat. Nos.
4,329,359 and 4,423,072 to Stahly disclose feeding
dihydroxyalkanols and triglycerides to pregnant sows to improve the
metabolic stability of newborn pigs. These compositions function to
increase the ketone body levels in the sow. The ketone bodies then
are transferred across the placenta, providing a supplemental
energy source to the developing fetus.
[0012] PCT WO 98/41200 and PCT WO 98/41201 by British Technology
Group Ltd disclose the use of acetoacetate in combination with poly
D-.beta.-hydroxybutyrate or esters or oligomers thereof, and/or a
metabolic precursor or salt thereof in nutritional or therapeutic
compositions to elevate the levels of ketone bodies in the blood
for increasing cardiac efficiency, treatment of diabetes and
insulin resistant states, and treatment of effects of
neurodegenerative disorders and epilepsy. Although these
applications provide mechanisms by which the ketone levels can be
elevated for treatment of these disorders, the number of useful
composition is limited to acetoacetate in combination with either a
precursor of, or oligomer or ester of,
D-.beta.-hydroxybutyrate.
[0013] It is therefore an object of the present invention to
provide improved or alternative compositions for elevating ketone
levels in the body of humans and other mammals, which are suitable
for oral or parenteral administration.
[0014] It is a further object of the present invention to provide
compositions having better or longer bioavailability, or different
metabolic products, and methods of use thereof for seizure control,
metabolic disease control, reduction of protein catabolism,
appetite suppression, parenteral nutrition, increasing cardiac
efficiency, treatment of diabetes, treatment of effects of
neurodegenerative disorders or other conditions affecting or
effected by ketone level in humans and other mammals.
Summary Of The Invention
[0015] Nutritional or therapeutic compositions are provided for
increasing ketone body levels in the blood of mammals by providing
a source of ketone bodies in the form of linear or cyclic oligomers
and/or derivatives of 3-hydroxyacids. The 3-hydroxyacid can be in
the form of a linear oligomer of 3-hydroxyacids other than linear
homo-oligomers of 3-hydroxybutyric acid if administered in
combination with acetoacetate, cyclic oligomers of 3-hydroxyacids,
esters of the linear or cyclic oligomers, esters of 3-hydroxyacids
other than 3-hydroxybutyric acid, and combinations thereof. An
oligomer generally refers to a polymer of three or more
hydroxyacids. Preferred 3-hydroxyacids include 3-hydroxybutyrate,
3-hydroxyvalerate, 3-hydroxyhexanoate, and 3-hydroxyheptanoate.
Oligomers of odd-carbon number 3-hydroxyacids such as
3-hydroxyvalerate have advantages since they have a higher energy
content than oligomers of 3-hydroxyacids having an even-number of
carbons. The cyclic oligomers have advantageous properties since
they result in a sustained, and/or controlled, ketone blood level
over a period of hours.
[0016] The compositions can be administered orally, for example, as
a nutritional or dietary supplement, or intravenously. Increasing
blood ketone levels is useful for seizure control, metabolic
disease control, reduction of protein catabolism, appetite
suppression, parenteral nutrition, increasing cardiac efficiency,
treatment of diabetes and insulin resistant states, and treatment
of effects of neurodegenerative disorders and epilepsy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a graph of plasma total ketone bodies (nm) from a
dog given a single oral bolus of triolide at 5% of the daily
caloric requirement, over time in minutes, for R-BHB (squares),
acetoacetate (diamonds, control), and total ketone bodies
(triangles).
DETAILED DESCRIPTION OF THE INVENTION
[0018] It was discovered that certain hydroxyacids, derivatives,
oligomers and esters thereof, can provide a source of ketone bodies
to modulate ketone body levels in the blood of mammals, and that
biologically produced polyhydroxyalkanoates are an excellent source
for these hydroxyacids. These oligomers and/or derivatives of
3-hydroxyacids can be readily adapted to produce a variety of
nutritional and therapeutic compositions, without the drawbacks
associated with known methods and compositions for elevating ketone
levels.
[0019] I. Nutritional and Therapeutic 3-Hydroxyacids
Compositions
[0020] The compositions include 3-hydroxyacids, linear or cyclic
oligomers thereof, esters of the 3-hydroxyacids or oligomers,
derivatives of 3-hydroxyacids, and combinations thereof. In one
preferred embodiment, the compositions include the cyclic macrolide
of R-3-hydroxyacids containing 3, 4, or 5 monomeric subunits.
Preferred 3-hydroxyacids include 3-hydroxybutyric acid,
3-hydroxyvaleric acid, 3-hydroxyhexanoic acid and
3-hydroxyheptanoic acid. The preferred length of the oligomer must
be such that the derivative has a suitable digestion rate for
sustained release of monomer. In another preferred embodiment, the
cyclic trimer (triolide) is used in a combination with other cyclic
oligolides or linear esters and/or mixtures of both.
[0021] The general formula for 3-hydroxyacids is: 1
[0022] Where:
[0023] R.sub.1 is selected from hydrogen, methyl, alkyl, alkenyl,
aryl, arylalkyl, heteroalkyl, heteroaryl, thiol, disulfide, ether,
thiolether, amine, amide, halogen,
[0024] R.sub.2 and R.sub.3 are independently selected from
hydrogen, methyl, alkyl, alkenyl, aryl, arylalkyl, heteroalkyl,
heteroaryl, thiol, disulfide, ether, thiolether, amine, amide,
halogen, hydroxy, ester, nitrogen-substituted radicals, and/or
oxygen-substituted radicals.
[0025] R.sub.4 is selected from hydrogen, alkyl, alkenyl, aryl,
arylalkyl, heteroalkyl, heteroaryl, thiol, disulfide, ether,
thiolether, amine, amide, halogen, hydroxy, ester,
nitrogen-substituted radicals, and/or oxygen-substituted
radicals.
[0026] further, when R.sub.4 is not hydrogen or a halogen, R.sub.3
can be a direct bond to R.sub.4 and R.sub.4 can be methyl.
[0027] The following definitions may be used through the
specification.
[0028] The term "alkyl" refers to C.sub.2-15 straight, branched or
cyclic alkyl groups.
[0029] The term "alkenyl" refers to a branched or straight chain
C.sub.2-C.sub15) hydrocarbon which also comprises one or more
carbon-carbon double bonds. rbon which also comprises one or
more
[0030] The term "aryl" refers to a group a group containing one or
more aromatic rings. Aryl groups can be unsubstituted or
substituted with substituents independently selected from alkyl,
haloalkyl, alkoxy, amino, alkyl amino, dialkylamino, hydroxy, halo,
and nitro.
[0031] The term "arylalkyl" refers to an alkyl group (as defined
above) to which is appended an aryl group.
[0032] The term "heteroalkyl" refers to an alkyl group (as defined
above) wherein one or more of the carbon atoms is replaced with a
non-carbon atom (such as, for example, oxygen, nitrogen,
sulfur).
[0033] The term "heteroaryl" refers to a group containing one or
more aromatic rings wherein at least one of the atoms in an
aromatic ring in not carbon. Heteroaryl groups can be unsubstituted
or substituted with substituents independently selected from alkyl,
haloalkyl, alkoxy, amino, alkylamino, dialkylamino, hydroxy, halo
and nitro.
[0034] The term "thiol" refers to RSH where R is alkyl, alkenyl,
aryl, arylalkyl, heteroalkyl, or heteroaryl (as defined above).
[0035] The term "disulfide" refers to groups containing a
sulfur-sulfur bond.
[0036] The term "ether" refers to groups containing a C-O-C
unit.
Hydroxyacid Oligomers
[0037] In one preferred embodiment, the compositions include linear
oligomers of 3-hydroxyacids having from 5 to 10 carbon atoms. As
used herein, the term "oligomer" means a polymer having a weight
average molecular weight of less than about 2000 g/mol, preferably
less than about 1000 g/mol, or having less than about 100 monomeric
subunits. Representative examples include oligomers of
3-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyheptanoate,
3-hydroxyoctanoate, and combinations thereof. As used herein, a
homo-oligomer includes only one type of 3-hydroxyacid, while an
oligomer can refer to either a homo-oligomer or hetero-oligomer
including more than one type of 3-hydroxyacid.
[0038] In another preferred embodiment, the compositions include
3-hydroxyacids having an odd number of carbons, which have a higher
caloric value than 3-hydroxyacids having an even number of carbons.
For example, oligomeric esters of 3-hydroxyvalerate (alone or mixed
with other hydroxyalkanoates) can be used to deliver the odd
numbered hydroxyacid 3-hydroxyvalerate.
[0039] In still another preferred embodiment, the compositions
include cyclic oligomers of 3-hydroxyalkanoic acids or
3-hydroxyalkanoate oligomer esters, including 3-hydroxyacids of
from 4 to 10 carbon atoms. The hydroxyacids are liberated as a
result of digestion or metabolism of the ester form. By providing
the hydroxyacids in ester form, these compositions can eliminate
complications caused by delivery of the acid or salt forms of
hydroxyalkanoic acids.
[0040] As demonstrated by the following examples, cyclic oligomers
have the advantage that the ketone body levels remain elevated for
a prolonged period of time of at least several hours after
ingestion. For example, cyclic esters of 3-hydroxybutyrate, such as
the triolide of 3-hydroxybutyrate, can provide sustained release of
ketone bodies. Slow release provides a major advantage over prior
art compositions, since the slow release of monomers provides a
more constant level of ketone bodies, such as 3-hydroxybutyrate, to
the body over a prolonged period of time. This release profile
reduces the frequency of doses required to maintain a specific
ketone body concentration, which is especially important during
periods, such as during sleep, when it is difficult to administer
the material.
Derivatized HydroxyAcids
[0041] Since the family of PHAs contains a large variety of
hydroxyacids with varying side chain substituents, judicious
selection of the type of 3-hydroxyacids provides a means to
increase the caloric density on a per acid basis or to provide
acids with odd number chain lengths. Preferred derivatives are
where the R groups on the formula shown above are ethyl or
methyl.
Esters of 3-Hydroxyacids or Oligomers
[0042] The compositions also can include esters of 3-hydroxyacids
or esters of either linear or cyclic 3-hydroxyacid oligomers. In
another preferred embodiment, the compositions include
R-3-hydroxyalkanoate oligomers terminated with an ester linkage,
for example, to 1,3-butanediol. The length of the oligomer
preferably is such that the derivative has a solubility suitable
for intravenous administration. The 1,3-butanediol may be coupled
to the hydroxyacid oligomer by the primary alcohol, the secondary
alcohol, and/or mixtures of both. Following parenteral (e.g.,
intravenous) administration of the oligomer esters, the
non-R-3-hydroxyacid units should be readily tolerated and
metabolized in the body after it is released from the oligomer
derivative.
[0043] The hydroxyacid oligomer also preferably is selected to
include desirable physical and nutritional properties, such as
water solubility and calorific benefits.
Sources of the Hydroxyacid Compositions
[0044] A useful source of hydroxyacids and hydroxyacid oligomers is
the family of microbial storage polyesters, the
polyhydroxyalkanoates, which can be accumulated intracellularly by
numerous microorganisms. Poly [(R)-3-hydroxyalkanoates] (PHAs) are
biodegradable and biocompatible thermoplastic materials, produced
from renewable resources, with a broad range of industrial and
biomedical applications (Williams & Peoples, CHEMTECH 26:38-44
(1996)).
[0045] In recent years, the PHA biopolymers have emerged from what
was originally considered to be a single homopolymer,
poly-3-hydroxybutyrate (PHB), into a broad class of polyesters with
different monomer compositions. To date around 100 different
monomers have been incorporated into the PHA polymers (Steinbuchel
& Valentin, FEMS Microbiol. Lett. 128:219-28 (1995)). As
described herein, these naturally occurring polyesters can be
converted into derivatives suitable for nutritional and therapeutic
uses.
Methods for Making the Hydroxyacid Oligomers and Derivatives
[0046] Representative methods for preparing the hydroxyacid
oligomer derivatives described herein include direct degradation of
polyhydroxyalkanoates to oligomeric derivatives; ring-opening of
cyclic oligomers of 3-hydroxyalkanoates; polymerization of
hydroxyalkanoates or derivatives thereof; and, stepwise synthesis
hydroxyalkanoate oligomers beginning or ending with modification of
a terminal hydroxyalkanoate unit. Such syntheses can be readily
carried out using methods known in the art. In a preferred
embodiment of the methods for synthesis of hydroxyacid oligomers
terminated with an ester linkage to an alcohol, the process
includes direct degradation of polyhydroxyalkanoate with the
alcohol; ring-opening of a cyclic oligomer of hydroxyalkanoate with
an alcohol; and, stepwise synthesis of hydroxyalkanoate oligomers
beginning or ending with esterification of a terminal
hydroxyalkanoate unit by an alcohol. Such syntheses can be carried
out using methods known in the art.
[0047] Cyclic oligolides of (R)-3-hydroxybutyric acid can be
prepared by a number of known methods, which are described, for
example, in Seebach, et al., Angew. Chem. Int. Eng. Ed., 4:434-35
(1992); Seebach, et al., Helv. Chim. Acta., 71:155-67 (1988);
Seebach, et al., Helv. Chim. Acta. 72:1704-17 (1989); and Mueller,
et al., Chimia 45:376 (1991). These methods involve conversion from
the bacterially-derived polyester, poly-(R)-3-hydroxybutyrate
(PHB), or macrolide formation from the constituent acid
(R)-3-hydroxybutyrate or esters thereof. The most direct route is
degradation of PHB under acid catalyzed conditions to a mixture of
linear oligomers and cyclic oligolides. Oligolides and oligomers
can be isolated from the crude mixture via conventional washing,
extraction, and distillation steps to yield purified materials.
[0048] II. Nutritional and Dietary Compositions
[0049] The compositions can be adapted for enteral or parenteral
administration, for example, by combining the composition with the
appropriate delivery vehicle. For enteral administration, the
compositions can be added to food or drink, for example, as a
dietary supplement. Alternatively, the compositions can be
delivered parenterally, for example, by dissolving in a
physiological saline solution for injection. Using genetic
engineering techniques, plants can be engineered to express the
appropriate 3-hydroxyacids or oligomers of 3-hydroxyacids. Suitable
means and methods are described in WO97/15681 and PCT/US99/04999 by
Metabolix.
[0050] The hydroxyacid formulations can be administered alone, in
dry or powdered form, in solution in a carrier such as water,
normal saline, or phosphate buffered saline, or mixed with other
materials which will elevate blood ketones, such as free fatty
acids, triglycerides alone or in combination with protein or
carbohydrate. Traditional ketogenic diets, such as the diet
recommended by the Marriott Corp. Health Care Services, Pediatric
Diet Manual, Revised August 1987, contains from 3:1 to 4:1 g of fat
for each g of combined carbohydrate and protein. Since the fat is
metabolized to yield 3-hydroxyacid and acetoacetate, and desired
levels are in the range of at least about 1 to 2 mM up to a maximum
of about 7.5 mM (achieved during prolonged fasting of obese
individuals), although ranges can be from 0.3 to 20 mM, the
compositions containing the 3-hydroxyacids can be formulated to
yield similar values to those of the traditional ketogenic diets,
recognizing that the yield will be more efficient when the
3-hydroxy acids are administered directly.
[0051] These compositions can be mixed with meat or carbohydrate,
as demonstrated in the examples, preferably maintaining an excess
of 3-hydroxy acid relative to the amount of carbohydrate or
protein.
[0052] III. Applications of the Compositions
[0053] The compositions described herein can readily be used in a
variety of nutritional and therapeutic applications. One of skill
in the art can readily select the appropriate hydroxyacid oligomer
or derivative, as well as amounts thereof, for administration. The
particular composition used will depend on the target ketone blood
levels (required for a particular patient), as well as the route
and frequency of administration. In all cases, the digestion and
metabolism of these compounds advantageously provides for the slow
release of ketone bodies.
[0054] Representative uses for the compositions described herein
are provided below:
[0055] Using the hydroxyacid oligomer derivatives described herein,
it is possible to sustain ketosis while overcoming drawbacks of the
ketogenic diet. During normal digestion and metabolism of these
compounds, ketone bodies (such as 3-hydroxybutyrate and
acetoacetate) are released into the blood. The blood level of
ketone bodies can be maintained at a level necessary to produce
ketosis and reduce seizures, which for example, are associated with
epilepsy. The hydroxyacid oligomer derivatives described herein can
also be administered to maintain the blood level of ketone bodies
at a level necessary to reduce protein catabolism and provide
appetite suppression, to aid in weight loss. Thus, addition of
these ketogenic compounds to the diet functions to mimic some
effects of a ketogenic diet. Preferred blood levels to be obtained
are in the range of 2 to 3 mM 3-hydroxyacid. The caloric value of
the ketone bodies is approximately 1.5 g of ketone/e g of fat. The
hydroxyacid oligomer derivatives described herein can be
administered parenterally to a mammal, typically a human, to
maintain the blood level of ketone bodies at a level necessary to
provide nutrients to the body. The compositions should be
particularly useful to patients who are unable to digest food
orally or otherwise require total parenteral nutrition. The
compositions can be formulated to provide high energy, water
soluble nutrients, suitable for long-term intravenous feeding.
[0056] The hydroxyacid oligomer derivatives described herein can be
administered to maintain the blood level of ketone bodies at a
level necessary to overcome deficiencies caused by metabolic
disorders, such as insulin deficiencies or insulin resistant
states. The hydroxyacid oligomer derivatives described herein can
be administered to maintain the blood level of ketone bodies at a
level necessary to treat insulin resistance in which the normal
insulin signaling pathways is disordered and in conditions in which
cardiac (hydraulic work) efficiency is reduced due to metabolic
reasons, as described in PCT WO 98/41200 and PCT WO 98/41201, which
are incorporated herein by reference.
[0057] The hydroxyacid oligomer derivatives described herein can
also be administered to a mammal, typically a human, to maintain
the blood level of ketone bodies at a level necessary to treat a
variety of neurodegenerative diseases, particularly those involving
neurotoxic plaques, such as amyloid plaques. Examples of
neurodegenerative diseases which the compositions described herein
may aid in treating include Alzheimer's disease, fronto-temperal
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, Downs syndrome associated Alzheimer's, myasthenia
gravis, and muscular dystrophy. See, for example, PCT WO 98/41200
and PCT WO 98/41201 by British Technology Group, Ltd., which
discloses that elevated levels of ketone bodies can improve nerve
cell function and growth, at least in part by enhancing cellular
energy production. The preferred ketone blood level for treatment
of neurodegenerative disorders is greater than for diet or
seizures, more typically in the range of 7.5 mM.
Supplemental Energy Source for Livestock
[0058] The hydroxyacid oligomer derivatives described herein can be
administered to animals, such as pigs, particularly pregnant sows,
to provide a supplemental energy source and to possibly improve the
metabolic stability of newborn animals. For example, by increasing
the ketone body levels in a pregnant sow, ketone bodies are
transferred across the placenta, providing a supplemental energy
source to the developing fetus.
[0059] The compositions and methods described herein are further
described by the following non-limiting examples.
EXAMPLE 1
Preparation of
(R,R,R)-4,8,12-Trimethyl-1,5,9-Trioxadodeca-2,6,10-Trione or
Triolide of (R)-3-Hydroxybutyric Acid
[0060] PHB (20 g) was dissolved in dioxane (700 mL) containing
p-toluene sulfonic acid monohydrate (4 g) and concentrated sulfuric
acid (5 mL). After refluxing for 4 days, the reaction had achieved
40% conversion to the triolide, as determined by gas chromatography
(GC) analysis (Riis & Mai, J. Chromatography 445:285-89
(1988)). The reaction mixture was cooled to room temperature and
quenched with saturated sodium bicarbonate solution. Dioxane was
removed by rotary evaporation. The residue was extracted into ethyl
acetate (400 mL), washed with brine, and concentrated to an oil.
Vacuum distillation yielded purified triolide (4 g).
EXAMPLE 2
Use of 3-Hydroxyalkanoic Acid Oligolide for Enteral Nutrition
[0061] A mongrel dog (21 kg) was fasted overnight and given an oral
bolus of triolide
((R,R,R)-4,8,12-trimethyl-1,5,9-trioxadodeca-2,6,10-trione, 10 g)
in gelatin. This amount of triolide is equivalent to 5% of the
daily caloric requirement. Blood was sampled at 0, 15, 30, 45, and
60 minutes and every half hour thereafter for a total of six
hours.
[0062] The blood samples were analyzed for glucose via enzymatic
assay, and for acetoacetate and 3-hydroxybutyrate via GC-mass
spectrometry (GC-MS) assay. As shown by FIG. 1, within 90 minutes,
the blood concentrations of 3-hydroxybutyrate and acetoacetate
reached 0.3 and 0.05 mM, and the total ketone bodies in the blood
were 0.36 mM. After the fourth hour, the total ketone body
concentration remained elevated at 0.24 mM. Glucose concentration
in the blood dropped from 6.5 mM to 5 mM during the experiment.
[0063] These results show that an oral dose of a 3-hydroxyalkanoic
oligolide can elevate the ketone body concentration in the blood. A
significant finding is that the ketone body concentration remains
elevated several hours after administration, demonstrating that the
triolide is useful for the slow release of ketone bodies.
EXAMPLE 3
Use of 3-Hydroxyalkanoic Acid Oligolide for Enteral Nutrition
[0064] A mongrel dog (25.5 kg) was fasted overnight and fed a
mixture of meat (111 g) and triolide
((R,R,R)-4,8,12-trimethyl-1,5,9-trioxadodeca-2,- 6,10-trione, 23.5
g). This amount of triolide is equivalent to 10% of the daily
caloric requirement. Identical amounts of meat and triolide were
given at 0, 120, 360 and 540 minutes. Blood was sampled at regular
intervals for 12 hours.
[0065] Throughout the experiment, the dog exhibited no signs of
distress; unusual behavior; or abnormal bodily functions, such as
diarrhea, nausea, vomiting, or frequent urination. The blood
samples were analyzed for acetoacetate and 3-hydroxybutyrate.
Within 30 minutes, the concentrations of 3-hydroxybutyrate and
acetoacetate reached 0.85 and 0.15 mM, respectively. The total
ketone bodies in the blood were 1.0 mM. After the third feeding of
triolide, total ketone body concentration remained elevated and
steady at about 0.6 mM. Glucose concentration in the blood remained
within the normal range of 3.1 to 5.9 mM. Other clinical chemistry
profiles remained normal throughout the experiment. By the next
morning, the ketone body concentration in the blood had returned to
the normal value of 0.02 mM.
[0066] These results show that triolide is digested by the dog,
resulting in a sustained increased in blood ketone body
concentration. Significantly, the ketone body concentration is
within the range achieved by the ketogenic diet used in the
nutritional treatment of intractable epilepsy. Furthermore, the
triolide was found to be well accepted by the dog, which showed no
sign of distress and no perturbation of clinical chemistry
parameters. These results further demonstrate that the triolide is
useful for the slow release of ketone bodies.
EXAMPLE 4
Synthesis of an Alkyl Ester Terminated 3-Hydroxyalkanoate
Oligomer
[0067] Oligomeric (R)-3-hydroxybutyrate was prepared via
condensation reaction of methyl (R)-3-hydroxybutyrate.
Specifically, methyl (R)-3-hydroxybutyrate (250 .mu.l) was heated
with dibutyltin oxide (2 mg) at 110.degree. C. for 72 hours. The
reaction vial was left open to the atmosphere to permit removal of
methanol. After cooling, the reaction formed a crystalline, white
solid material, which was washed with methanol and allowed to air
dry. NMR analysis showed formation of oligomeric
(R)-3-hydroxybutyrate having an approximate molecular weight of
1,700 g/mol. Gel permeation chromatography (GPC) analysis confirmed
the Mw at about 2,000 g/mol. NMR analysis also demonstrated the
presence of a terminal methyl ester.
EXAMPLE 5
Synthesis of a Butanediol Ester Terminated 3-Hydroxyalkanoate
Oligomer
[0068] Oligomeric (R)-3-hydroxybutyrate butanediol ester was
prepared via controlled transesterification of the microbial
polyester, poly[(R)-3-hydroxybutyrate] with 1,3-butanediol.
Specifically, PHB (10 g, Mw 600,000) was dissolved with heating in
200 mL of dioxane and 1,3-Butanediol (2.1 mL). After dissolution,
the reaction mixture was cooled and concentrated sulfuric acid (1
mL) was slowly added. The reaction mixture was heated at reflux for
48 hours. Samples were removed periodically and precipitated into
water. After 6 hours, 95% of the product was recovered, having a Mw
of 4,300 Da according to GPC analysis. After 45 hours, 52% of the
product was recovered, having a Mw of 2,000 Da according to GPC
analysis. NMR analysis demonstrated a 3-hydroxybutyrate oligomer of
approximately 1,000 g/mol and demonstrated the presence of a
terminal 1,3-butanediol ester.
EXAMPLE 6
Synthesis of a 3-Hydroxyalkanoate Oligomer
[0069] Oligomeric (R)-3-hydroxybutyrate was prepared via controlled
hydrolysis of the microbial polyester, poly[(R)-3-hydroxybutyrate].
Specifically, PHB (150 g) was dissolved with heating in 2 L of
glacial acetic acid. Water (350 ml) was slowly added to the viscous
solution to form a single phase. The reaction mixture was heated at
reflux for 18 hours. After cooling to about 55.degree. C., the
mixture was poured with rapid stirring into 9 L of water. The white
precipitate was collected and washed with water to yield 92 g of
3-hydroxybutyrate oligomer after drying. NMR analysis demonstrated
a 3-hydroxybutyrate oligomer of approximately 1,000 g/mol, with no
terminal crotonization. GPC analysis confirmed a molecular weight
of 1,000 g/mol.
[0070] A similar process was used, but with the addition of
hydrochloric acid, to produce 3-hydroxybutyrate oligomer of lower
molecular mass (approximately 200 g/mol). Oligomeric
(R)-3-hydroxyvalerate can be prepared using the same approach from
poly(3-hydroxyvalerate) which can be obtained by fermentation using
Chromobacter violaceum (Steinbuchel, et. al, Appl. Microbiol.
Biotechnol. 39:443-49 (1993)).
EXAMPLE 7
Use of 3-Hydroxyalkanoic Oligomers for Enteral Nutrition
[0071] Sprague-Dawley rats were fed commercial rat chow for 10 days
and then switched to a control diet containing 75% of the calories
from starch, 20% as casein, and 5% as polyunsaturated oil, plus
mineral mix and liver extract supplements. After 15 days, two
groups of rats were fed an experimental diet containing 25% of the
calories from a 3-hydroxybutyrate oligomer. Two different
oligomers, short and medium, were used with molecular masses of
either the 200 g/mol or 1000 g/mol, respectively. A control group
was kept on the control diet without oligomer.
[0072] The weight of each rat was measured daily. Urine samples
were collected daily and analyzed for 3-hydroxybutyrate by GC-MS.
After 5 days on the experimental diet, the rats were euthanized,
and a blood sample was collected and analyzed for 3-hydroxybutyrate
and acetoacetate by GC-MS.
[0073] The weight of the control group increased uniformly
throughout the experiment, as did the weight of rats fed the
experimental diet containing the medium HB oligomer. The weight of
rats fed the experimental diet containing short HB oligomer
decreased slightly while on the experimental diet.
[0074] The concentration of ketone bodies in the rat blood plasma
collected at time of euthanasia was measured by GC-MS. The control
group showed normal concentrations of 3-hydroxybutyrate and
acetoacetate, 0.07 and 0.02 mM, respectively. Rats fed the short HB
oligomer had 3-hydroxybutyrate and acetoacetate concentrations of
0.65 and 0.05 mM, respectively, while rats fed the medium oligomer
had concentrations of 0.15 and 0.04 mM, respectively. These result
show that the rats fed 3-hydroxybutyrate oligomers had increased
levels of ketone bodies in their blood.
[0075] The concentration of 3-hydroxybutyrate in the urine of rats
fed short and medium oligomers was determined by GC-MS to be
approximately 3.5 and 1.0 mM, respectively. 3-Hydroxybutyrate was
undetectable in the urine of the control rats. These results show
that an oral dose of 3-hydroxybutyrate oligomers elevates the
ketone body concentration in the blood and in the urine.
[0076] Modifications and variations of the present invention will
be obvious to those of skill in the art from the foregoing detailed
description. Such modifications and variations are intended to come
within the scope of the following claims.
* * * * *