Note: Descriptions are shown in the official language in which they were submitted.
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DIASTEREOISOMERS OF 4-HYDROXYISOLEUCINE AND USES THEREOF
BACKGROUND OF THE INVENTION
a) Field of the invention
The invention relates to isomers of 4-hydroxyisoleucine, and to lactones,
pharmaceutically acceptable salts and prodrugs thereof, to processes for their
preparation, to pharmaceutical compositions comprising the same and to their
use for
preventing and treating disorders of carbohydrate or lipid metabolism,
including
diabetes mellitus (type 1 and type 2 diabetes), pre-diabetes, and Metabolic
Syndrome.
b) Brief description of the related art
Diabetes mellitus is a disorder of carbohydrate metabolism, and develops
when the body cannot effectively control blood glucose levels. The disease is
characterized by inadequate secretion or utilization of insulin, high glucose
levels in
the blood and urine, and excessive thirst, hunger, weight loss, and urine
production.
It can lead to a number of serious complications, including cardiovascular
disease,
kidney disease, blindness, nerve damage, and limb ischemia.
Diabetes is divided into two types, 1 and 2, with the latter accounting for
about 90% of cases. In type I diabetes, the body destroys the insulin-
producing (3
cells of the pancreas, resulting in the inability of the body to produce
insulin. Type 1
diabetes typically occurs in children or young adults, and generally is
managed by
insulin administration, strict diet, and exercise. Type 1 diabetes is observed
as well in
older adults following therapeutic failure of type 2 diabetes. Type 2 diabetes
is
characterized by impaired insulin secretion due to altered (3 cell function,
as well as
decreased ability of normally insulin sensitive tissues (e.g., the liver and
muscle) to
respond to insulin. Type 2 diabetes generally develops in those over 45, but
is
recently also being detected in younger people. The disease is associated with
risk
factors such as age, family history, obesity, lack of regular exercise, high
blood
pressure, and hyperlipidemia. Treatment involves strict diet and exercise
regimens,
oral medications (e.g., medications that increase insulin secretion and/or
insulin
sensitivity), and, in some cases, insulin administration.
Type 2 diabetes is rapidly increasing in its importance as a major public
health concern in the Western world. While one hundred years ago it was a
relatively
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rare disease, today there are more than 200 million type 2 diabetics
worldwide, and
this number is estimated to increase to greater than about 300 million by the
year
2025. This dramatic increase in the incidence of type 2 diabetes parallels an
increase
in the prevalence of obesity in Western cultures. Further, as more cultures
adopt
Western dietary habits, it is likely that type 2 diabetes will reach epidemic
proportions
throughout the world. Given the seriousness of the complications associated
with this
disease, as well as its rapidly increasing incidence, the development of
effective
approaches to treatment is a primary concern in the field of medicine.
In 1973, Fowden et al., in Phytochemistry 12:1707-1711, 1973, reported the
presence of (2S,3R,4R)-4-hydroxy-3-methylpentanoic acid (4-hydroxyisoleucine
or 4-
OH) in the seeds of fenugreek (Trigonella foenum-graecum), an annual
herbaceous
plant that is widespread in regions of Asia, Africa, and Europe. Its absolute
configuration was subsequently restudied and corrected as being (2S,3R,4S) by
Alcock et al. in Phytochemistry 28:1835-1841, 1989. This unusual substance
represents about 0.6% of the seed weight and more that 85% of the free amino
acids
in the seeds, with two coexisting isomers: the (2S,3R,4S) isomer (-90%) and
the
(2R,3R,4S) isomer (-10%) (Sauvaire et al., Herbs, Botanicals and Teas (2000),
Edited by G. Mazza and B.P. Oomah, p. 107-129). It has been demonstrated that
the
(2S,3R,4S) isomer possesses insulinotropic and insulin sensitizing activities
(Broca
et al., Am. J. Physiol. 277:E617-E623, 1999; Broca et al., Eur. J. Pharmacol.
390:339-345, 2000; Broca et al., Am. J Physiol. Endocrinol. Metab. 287:E463-
E471,
2004) and that compound has since been developed for the treatment of diabetes
(PCT publication Nos. WO 97/32577 and WO 01/15689). On the other hand, it has
been reported that the (2R,3R,4S) isomer has no or much less biological
activity than
the (2S,3R,4S) isomer (Broca et al., Eur. J. Pharmaco. 390:339-345, 2000). 1
Notwithstanding the growing body of evidence on the positive activities of
4-hydroxyisoleucine for the treatment of diabetes, no one has ever
demonstrated that
configurational isomers of 4-hydroxyisoleucine, other than the (2S,3R,4S)
isomer,
could be useful for the prevention and/or treatment of metabolic diseases such
as
diabetes.
In view of the above, there is a need for alternative and improved compounds
for preventing and treating disorders of carbohydrate and lipid metabolism,
particularly diabetes.
There is also a need for pharmaceutical compositions and therapeutic
methods of stimulating glucose uptake and/or of stimulating insuling
secretion.
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The present invention provides such compounds along with methods for their
use. Accordingly, the present invention fulfils the above-mentioned needs and
also
other needs as it will be apparent to those skilled in the art upon reading
the following
specification.
SUMMARY OF THE INVENTION
The present inventors have found that configurational isomers of (2S,3R,4S)-
4-hydroxyisoleucine also exhibit insulinotropic activity in in vitro assays:
one that
monitors the glucose-dependent stimulation of insulin secretion in insulin
secreting
INS-1 cells and another that monitors glucose uptake in differentiated 3T3-L1
adipocytes.
Accordingly, the present invention features the use of isomers of
4-hydroxyisoleucine (4-OH) as defined herein, for therapeutic and/or
prophylactic
purposes. In preferred embodiments, the isomers according to the invention are
isomers of 2S,3R,4S 4-hydroxyisoleucine selected from the group consisting of:
OH CO2H OH CO2H
H3CjY__1 NH2 H3C' NH2
CH3 [8] (2S, 3R, 4R), CH3 [10]
(2S, 3S, 4S),
OH CO2H OH CO2H
H3C~NH2 H3C NHZ
CH3 [13] (2S,3S,4R), CH3 [17]
(2R, 3S, 4R),
OH CO2H OH CO2H
H3C" 2 H3C NH2
CH3 [20] (2R,3S,4S), CH3 [22]
(2R, 3R, 4R), and
OH COzH
H3C" Y ""'NH2
'CH3 [25] (2R, 3R, 4S).
Exemplary prodrugs include those compounds in which the carboxylate group
and the hydroxyl group are condensed to form one of the following lactones:
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H3C O O H3C O O H3C O O
H3O NH2 H Z3C NH2 H3C NH2
H3C00 H3C " OO H3C O O H3C O O
,
H3C 'NH2 1 H3C 'NH2 H3C NH2 or H3C NH2
Another aspect of the invention features the following
OH CO2H
H3C NH2
compound: CH3 [10] (2S,3S,4S), or its lactone
H3C "O O
prodrug. H3C NH2
In another aspect, the invention features pharmaceutical compositions
comprising one or more of such isomers, and a pharmaceutically acceptable
excipient.
In another aspect, the invention provides a method for treating a mammal
having a disorder of carbohydrate or lipid metabolism that includes
administering to
the mammal one or more isomer of 4-OH as defined herein. Preferably, the
disorder
is non-insulin dependent diabetes mellitus, more preferably type 2 diabetes
mellitus.
In one embodiment, the method can further include administering a second agent
to
the mammal, where the agent can be, for example, an antidiabetic agent.
According to another aspect, the invention is directed to a method of
treatment of disease in a mammal treatable by administration a compound
stimulating insulin secretion, which method comprises administration of a
therapeutically effective amount of a pharmaceutical composition comprising a
therapeutically effective amount of at least one isomer of 4-OH according to
the
invention, and a pharmaceutically acceptable carrier or excipient, either
alone or in
combination with other pharmacologically active agents
In another aspect, this invention is directed to a method for stimulating
glucose uptake by muscle cells and/or adipocytes, comprising contacting such
cells
with an effective amount of isomer(s) according to the invention.
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In another aspect, this invention is directed to a method for stimulating
insulin
secretion by beta-cells in the pancreatic islets, comprising contacting said
cells with
an effective amount of isomer(s) according to the invention.
In yet another aspect, this invention is directed to pharmaceutical
compositions and more particularly to the use of isomer(s) according to the
invention
in the preparation of a medicine for use in the treatment of a disorder of
carbohydrate
or lipid metabolism in which elevated circulating glucose levels are
problematic,
including but not limited to diabetes mellitus (type 1 and type 2 diabetes),
pre-
diabetes, Metabolic Syndrome, hyperglycemia, diabetic neuropathy and diabetic
nephropathy.
In a further aspect of the present invention there are provided processes for
the preparation of isomer(s) according to the invention.
An advantage of the invention is that it provides novel useful stimulators of
glucose uptake and stimulators of insulin secretion. The invention also
provides
compounds, compositions and methods for the unmet medical need of carbohydrate
or lipid metabolism, and more particularly type 2 diabetes.
Additional objects, advantages and features of the present invention will
become more apparent upon reading of the following non-restrictive description
of
preferred embodiments with reference to the accompanying drawings which are
exemplary and should not be interpreted as limiting the scope of the present
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a synthetic scheme showing the synthesis of each eight (8)
configurational isomers of 4-hydroxyisoleucine.
Figure 2 is a graph showing the effect of configurational isomers of
4-hydroxyisoleucine on the stimulation of glucose uptake by differentiated 3T3-
L1
adipocyte cells.
Figure 3 is a graph showing the glucose-dependent stimulation of insulin
secretion in INS-1 cells by configurational isomers of 4-hydroxyisoleucine.
DETAILED DESCRIPTION OF THE INVENTION
The invention features compounds, pharmaceutical compositions, and
methods that include isomers of (2S,3R,4S)-4-hydroxyisoleucine, a compound
that
has been shown to stimulate insulin secretion in a glucose dependent manner
and to
decrease insulin resistance (see, e.g., U.S. Patent No. 5,470,879; WO
01/15689;
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Broca et al., Am. J. Physiol. 277:E617-E623, 1999; and Broca et al., Am. J
Physiol.
Endocrinol. Metab. 287:E463-E471, 2004). More particularly, the invention
features
(2S,3S,4S)-4-hydroxyisoleucine and pharmaceutical compositions and methods
that
include one or more of the (2S,3S,4S), (2S,3R,4R), (2S,3S,4R), (2R,3S,4R),
(2R,3S,4S), (2R,3R,4R) or (2R,3R,4S) configurational isomers of 4-
hydroxyisoleucine for the treatment of mammals having a disorder of
carbohydrate or
lipid metabolism.
In order to provide an even clearer and more consistent understanding of the
specification and the claims, including the scope given herein to such terms,
the
following definitions are provided:
A) Definitions
The term "administration" or "administering" refers to a method of giving a
dosage of a pharmaceutical composition to a mammal, such as a human, where the
method is, e.g., oral, subcutaneous, topical, intravenous, intraperitoneal, or
intramuscular. The preferred method of administration can vary depending on
various
factors, e.g., the components of the pharmaceutical composition, site of the
potential
or actual disease, and severity of disease.
By "disorder of carbohydrate metabolism" is meant a metabolic disorder in
which the subject having the disorder cannot properly metabolize sugars.
Examples
of such disorders include, for example, diabetes mellitus (type 1 and type 2),
pre-
diabetes, hyperglycemia, impaired glucose tolerance, Metabolic Syndrome,
glucosuria, diabetic neuropathy and nephropathy, obesity, and eating
disorders.
By "disorder of lipid metabolism" is meant a metabolic disorder in which the
subject having the disorder cannot properly metabolize, distribute and/or
store fat.
Examples of such disorders include, but are not limited to type 2 diabetes,
pre-
diabetes, and Metabolic Syndrome.
By "effective amount" is meant the amount of a compound required to treat
or prevent a disorder of carbohydrate or lipid metabolism, such as, for
example,
diabetes and Metabolic Syndrome. The effective amount of active compound(s)
used
to practice the present invention for therapeutic or prophylactic treatment of
conditions caused by or contributed to by a disorder of carbohydrate or lipid
metabolism varies depending upon the manner of administration, and the age,
body
weight, and general health of the subject. Ultimately, the attending physician
or
veterinarian will decide the appropriate amount and dosage regimen. An
effective
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amount can also be that which provides some amelioration of one or more
symptoms
of the disorder or decreases the likelihood of incidence of the disorder.
Compounds that have the same molecular formula but differ in the nature or
sequence of bonding of their atoms or the arrangement of their atoms in space
are
termed "isomers". Isomers in which the connectivity between atoms is the same
but
which differ in the arrangement of their atoms in space are termed
"stereoisomers".
Stereoisomers that are not mirror images of one another are termed
"diastereomers" and those that are non-superimposable mirror images of each
other
are termed "enantiomers". When a compound has an asymmetric center, for
example, it is bonded to four different groups, a pair of enantiomers is
possible. An
enantiomer can be characterized by the absolute configuration of its
asymmetric
center and is described by the R- and S-sequencing rules of Cahn, Ingold, and
Prelog, or by the manner in which the molecule rotates the plane of polarized
light
and designated as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomers
respectively). A chiral compound can exist as either individual enantiomer or
as a
mixture thereof. A mixture containing equal proportions of the enantiomers is
called a
"racemic mixture".
Asymmetric or chiral centers may exist in the compounds of the present
invention. Unless indicated otherwise, the description or naming of a
particular
compound in the specification and claims is intended to include all individual
enantiomers and mixtures, racemic or otherwise, thereof. The methods for the
determination of stereochemistry and the separation of stereoisomers are well-
known
in the art (see discussion in Chapter 4 of "Advanced Organic Chemistry", 4th
edition
J. March, John Wiley and Sons, New York, 1992). Individual stereoisomers of
compounds or the present invention are prepared synthetically from
commercially
available starting materials that contain asymmetric or chiral centers or by
preparation of mixtures of enantiomeric compounds followed by resolution well-
known to those of ordinary skill in the art. These methods of resolution are
exemplified by (1) attachment of a racemic mixture of enantiomers, designated
(+I-),
to a chiral auxiliary, separation of the resulting diastereomers by
recrystallization or
chromatography and liberation of the optically pure product from the auxiliary
or (2)
direct separation of the mixture of optical enantiomers on chiral
chromatographic
columns. Enantiomers are designated herein by the symbols "R" or "S,"
depending
on the configuration of substituents around the chiral carbon atom, or are
drawn by
conventional means with a bolded line defining a substituent above the plane
of the
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page in three-dimensional space and a hashed or dashed line defining a
substituent
beneath the plane of the printed page in three-dimensional space.
As generally understood by those skilled in the art, an optically pure
compound is one that is enantiomerically pure. As used herein, the term
"optically
pure" is intended to mean a compound that comprises at least a sufficient
amount of
a single enantiomer to yield a compound having the desired pharmacological
activity.
Preferably, "optically pure" is intended to mean a compound that comprises at
least
90% of a single isomer (80% enantiomeric excess, i.e. "e.e."), preferably at
least 95%
(90% e.e.), more preferably at least 97.5% (95% e.e.), and most preferably at
least
99% (98% e.e.). Preferably, the compounds of the invention are optically pure.
The terms "isomer(s) of 4-hydroxyisoleucine", "isomer(s) of 4-OH",
"isomer(s) of the invention" or "compound(s) of the invention" as used herein,
refer to the diastereoisomers of (2S,3R,4S)-4-hydroxyisoleucine as defined
herein
and include pharmaceutically acceptable lactones, salts, crystal forms,
metabolites,
solvates, esters, and prodrugs thereof.
The term "pharmaceutically acceptable salt" as use herein, represents
those salts which are, within the scope of sound medical judgment, suitable
for use in
contact with the tissues of humans and animals without undue toxicity,
irritation,
allergic response and the like and are commensurate with a reasonable
benefit/risk
ratio. Pharmaceutically acceptable salts are well known in the art. For
example, S. M.
Berge et al. describe pharmaceutically acceptable salts in detail in J.
Pharmaceutical
Sciences 66:1-19, 1977. The salts can be prepared in situ during the final
isolation
and purification of the compounds of the invention or separately by reacting
the free
base group with a suitable organic acid. Representative acid addition salts
include
acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,
bisulfate, borate, butyrate, camphorate, camphersulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,
fumarate,
glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate,
hydrobromide, hydrochloride, h.ydroiodide, 2-hydroxy-ethanesulfonate,
lactobionate,
lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate,
2-
naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,
pamoate,
pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,
propionate,
stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate,
undecanoate,
valerate salts, and the like. Representative alkali or alkaline earth metal
salts include
sodium, lithium, potassium, calcium, magnesium and the like, as well as
nontoxic
ammonium, quaternary ammonium, and amine cations, including, but not limited
to
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ammonium, tetramethylammonium, tetraethylammonium, methylamine,
dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
The term "pharmaceutically acceptable ester" as used herein, represents
esters that hydrolyze in vivo and include those that break down readily in the
human
body to leave the parent compound or a salt thereof. Suitable ester groups
include,
for example, those derived from pharmaceutically acceptable aliphatic
carboxylic
acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids,
in which
each alkyl or alkenyl group preferably has not more than 6 carbon atoms.
Examples
of particular esters include formates, acetates, propionates, butyates,
acrylates and
ethylsuccinates.
The term "prodrug" as used herein, represents compounds that are rapidly
transformed in vivo to a parent compound of the above formula, for example, by
hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V.
Stella,
Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series,
Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American
Pharmaceutical Association and Pergamon Press, 1987, and Judkins et al.,
Synthetic
Communications 26(23):4351-4367, 1996, each of which is incorporated herein by
reference.
Prodrugs of isomers according to the invention are prepared by modifying
functional groups in such a way that the modifications may be cleaved in vivo
to
release the parent isomer. Prodrugs include modified isomers wherein a hydroxy
or
amino group in any of said isomer is bonded to any group that may be cleaved
in
vivo to regenerate the free hydroxyl or amino group, respectively. Examples of
prodrugs include, but are not limited to esters (e.g., acetate, formate, and
benzoate
derivatives), carbamates (e.g., N,N-dimethylaminocarbonyl) of hydroxy
functional
groups in compounds of Formulae (I), (II), or (III), and the like.
The term "pharmaceutically acceptable prodrugs" as used herein,
represents those prodrugs of the compounds of the present invention which are,
within the scope of sound medical judgment, suitable for use in contact with
the
tissues of humans and animals without undue toxicity, irritation, allergic
response,
and the like, commensurate with a reasonable benefit/risk ratio, and effective
for their
intended use, as well as the zwitterionic forms, where possible, of the
compounds of
the invention.
A "pharmaceutically acceptable active metabolite" is intended to mean a
pharmacologically active product produced through metabolism in the body of an
isomer of 4-OH.
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A"pharmaceuticafly acceptable solvate" is intended to mean a solvate that
retains the biological effectiveness and properties of the biologically active
components of isomers according to the invention. Examples of pharmaceutically
acceptable solvates include, but are not limited to water, isopropanol,
ethanol,
methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
B) Compounds according to the invention
As will be described in detail hereinafter, the inventors have prepared series
of isomers of 4-hydroxyisoleucine. According to preferred embodiments of the
invention, these isomers are active for stimulating glucose uptake and/or
stimulating
insulin secretion in mammals, and can therefore be useful for preventing
and/or
treating disorders in which elevated glucose levels are problematic.
Consequently,
providing such isomers is not only desirable for the treatment of diabetes,
but also for
the treatment of other disorders of carbohydrate or lipid metabolism.
According to a first aspect, the present invention features isomers of
2S,3R,4S 4-hydroxyisoleucine and pharmaceutically acceptable lactones, salts,
crystal forms, prodrugs, esters, metabolites, or solvates thereof. In certain
embodiments, the isomers of the present invention are selected from the group
consisting of:
OH CO2H OH CO2H
H3CAY-1-NH2 H3C 'NHZ
CH3 [8] (2S,3R,4R), CH3 [10]
(2S,3S,4S),
OH CO2H OH CO2H
H3C~NH2 H3C" v ""'NHZ
CH3 [13] (2S,3S,4R), CH3 [17]
(2R, 3S, 4R),
OH CO2H OH CO2H
H3C' O """1NH2 H3C NH2
CH3 [20] (2R, 3S, 4S), CH3 [221
(2R,3R,4R), and
OH COzH
H3C-y", NHZ
CH3 125] (2R,3R,4S).
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Exemplary prodrugs include those compounds in which the carboxylate group
and the hydroxyl group are condensed to form one of the following lactones:
H3C O O H3C ,.O O H3C O O
~ ' ~'
H3C NH2 H3C NH2 H3C NH2
H3C0 0 H3C .,0 H3C ' O O H3C ,O O
. . \i~ ~
H3C 'NHZ, H3C 'NH21 H3C NHZ or H3C NH2
In preferred embodiments, the isomers of the present invention are selected
from the group consisting of:
OH CO2H OH CO2H
H3C--'~NH2 H3C ~NH2
CH3 [8] (2S,3R,4R), CH3 [10]
(2S, 3S, 4S),
OH CO2H OH CO2H
H3C~NH2 H3C NH2
CH3 [13] (2S,3S,4R), CH3 [17]
(2R, 3S, 4R),
OH CO2H OH COZH
H3C" 2 H3C NH2
CH3 [20] (2R,3S,4S), and CH3 [22]
(2R, 3R, 4R).
According to a related aspect, the invention features the following compound:
OH CO2H
H3C' v 'NH2
CH3 [10] (2S,3S,4S),
and pharmaceutically acceptable lactones, salts, crystal forms, prodrugs,
esters,
metabolites, or solvates thereof. In a preferred embodiment, the lactone is a
lactone
prodrug having the following structure:
H3C "O O
H3C NH2
The isomers and compositions (see hereinafter) of the invention may be
prepared by employing techniques available in the art using starting materials
that
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are readily available. For instance, methods for the preparation of (2S,3R,4S)-
4-
hydroxyisoleucine have been described, see for example U.S. Patent Application
Publication No. US 2003/0219880; Rolland-Fulcrand et al., Eur. J. Org. Chem.
873-
877, 2004; and Wang et al., Eur. J. Org. Chem. 834-839, 2002. In addition,
this
compound can be isolated from the seeds of fenugreek (Trigonella foenum-
graecum). Methods for making additional configurational isomers of
4-hydroxyisoleucine, or prodrug thereof, have also been described in
PCT/FR2005/02805 filed Nov. 10, 2005 (WO 2006/ published on May
2006) which is incorporated herein by reference.
An additional aspect of the invention concerns new methods for the synthesis
of isomers according to the invention. Certain novel and exemplary methods of
preparing the inventive compounds are described in the Exemplification
section.
Such methods are within the scope of this invention.
C) Methods for stimulating glucose uptake and methods for stimulatina insulin
secretion
The isomers of the invention preferably stimulate glucose uptake by muscle
tissues or adipose tissues and/or stimulate insulin secretion by pancreatic (3-
cells.
The biological activity of the isomers of the invention may be measured by any
of the
methods available to those skilled in the art, including in vivo and in vitro
assays.
Some examples of suitable assays for such measurement are described herein in
the
Exemplification section. Additional examples of suitable art-recognized assays
for
such measurement are well known.
Accordingly, a related aspect, the invention provides a method of stimulating
glucose uptake by muscle and or adipose tissues, the method comprising:
- providing at least one isomer according to the invention as defined herein;
- providing a functional in vitro cell-based assay in which glucose uptake
stimulation is assessable; and
- introducing an effective amount of said isomer(s) into the assay for
stimulating
glucose uptake activity.
In one embodiment, the in vitro cell-based assay comprises 3T3-L1
adipocytes cells and is carried out in presence of about 10 pM 2-deoxy-D-
glucose
and about 16 pM 3H-deoxy-D-glucose.
Accordingly, a related aspect, the invention provides a method of stimulating
insulin secretion by (3-cells, the method comprising:
- providing at least one isomer according to the invention as defined herein;
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- providing a functional in vitro cell-based assay in which stimulation of
insulin
secretion is assessable; and
- introducing an effective amount of said isomer(s) into the assay for
stimulating
insulin secretion.
In one embodiment, the in vitro cell-based assay comprises INS-1 cells and is
carried out in presence of a glucose concentration of about 2 mM to about 10
mM.
D) Pharmaceutical Compositions and Therapeutic Applications
Without wishing to be bound by any particular theory, the inventors have
demonstrated that the isomers of the invention are suitable for stimulating
glucose
uptake, and/or stimulating insulin secretion. Therefore, present invention
pertains to
methods of using isomers of 4-OH and pharmaceutical compositions thereof for
treatment or prevention purposes. In preferred embodiments, the method
compromises administering any of the individual isomers described herein, or
any
combination thereof.
According to preferred embodiments of the invention, the mammal is a human
subject in need of treatment by the methods and/or isomers of the invention,
and is
selected for treatment based on this need. A human in need of treatment,
especially
when referring to type 2 diabetes is art-recognized and includes subjects that
have
been identified as having abnormally high blood glucose levels, a reduced
glucose
tolerance, a disregulation of fat metabolism, and may have a surplus ,of
weight (e.g.
the subject may be obese). Humans in need of treatment may also be at risk of
such
a disease or disorder, and would be expected based on diagnosis, e.g., medical
diagnosis, to benefit from treatment (e.g., curing, healing, preventing,
alleviating,
relieving, altering, remedying, ameliorating, improving, or affecting the
disease or
disorder, the symptom of the disease or disorder, or the risk of the disease
or
disorder).
Therefore, a related aspect of the invention concerns the use of isomers of
the
invention as an active ingredient in a pharmaceutical composition for
treatment or
prevention purposes. As used herein, "treating" or "treatment" is intended to
mean
at least the mitigation of a disease condition associated with a disorder of
carbohydrate or lipid metabolism, and more particularly type 2 diabetes in a
mammal,
such as a human, that is alleviated by a stimulation of insulin secretion
and/or by a
stimulation of glucose uptake, and includes curing, healing, inhibiting (e.g.,
arresting
or reducing the development of the disease or its clinical symptoms),
relieving from,
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improving and/or alleviating, in whole or in part, the disease condition
(e.g., causing
regression of the disease or its clinical symptoms).
As used herein, "prophylaxis" or "prevent" or "prevention" is intended to
mean at least the reduction of likelihood of a disease condition associated
with a
disorder of carbohydrate or lipid metabolism, and more particularly type 2
diabetes in
humans. Type 2 diabetes predisposing factors identified or proposed in the
scientific
literature include, among others, (i) a genetic predisposition to having the
disease
condition but not yet diagnosed as having it, (ii) being obese, (iii) having a
disregulation of fat metabolism and/or (iv) having a sedentary life style. For
example,
it is likely that one can prevent or treat type 2 diabetes in a human by
administering
an isomer of the invention or a composition comprising the same, when the
human is
at a pre-diabetic state, when the human is overweight, when the human shows
abnormally high blood glucose levels, and/or when the human exhibits a reduced
tolerance to glucose.
The subject may be a female human or a male human, and it may be a child,
a teenager, or an adult.
According to a specific aspect, the invention features a method for treating a
mammal, such as a human, having diabetes mellitus (type I or type 2 diabetes),
pre-
diabetes, or Metabolic Syndrome, that includes administering to the mammal an
isomer of the invention, and/or a composition comprising the same, in an
amount
sufficient to decrease its circulating glucose level.
According to certain embodiments, the isomers, compositions, and methods
of the invention are administered at a therapeutically effective dosage
sufficient to
reduce the glucose levels in a subject's plasma, from about at least 5, 10,
15, 20 25,
30, 40, 50, 75, or 100 percent, when compared to original levels prior to
treatment.
According to certain embodiments, the isomers, compositions, and methods
of the invention are administered at a therapeutically effective dosage
sufficient to
increase insulin levels in a subject's plasma from about at least 5, 10, 15,
20 25, 30,
40, 50, 75, or 100 percent, when compared to original levels prior to
treatment.
Typically, the isomers of the invention are given until glucose and/or insulin
levels go back to normal. Due to the nature of the disorders and conditions
targeted
by the isomers of the invention, it is likely that a chronic or lifetime
administration is
going to be required. In preferred embodiments, isomers and pharmaceutical
composition according to the invention are administered once to thrice a day.
The amount of glucose or insulin in the blood, or plasma of a subject can be
evaluated by using techniques and methods well known to those skilled in the
art,
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WO 2006/117696 PCT/IB2006/001758
including but not limited, to hand-held glucometer, enzymatic assays (e.g.,
glucose
oxidase or hexokinase bases assays), enzyme-linked immunosorbent assay
(ELISA),
quantitative immunoblotting test methods, and radiolabeled immunoassay (RIA).
Therefore, the present invention provides pharmaceutical compositions
comprising a therapeutically effective amount of an isomer of 4-OH as
described
herein in combination with a pharmaceutically acceptable carrier or excipient.
Suitable carriers or excipients include, but are not limited to, saline,
buffered saline,
dextrose, water, glycerol, ethanol, and combinations thereof. The
pharmaceutical
compositions may be administered in any effective, convenient manner
including, for
instance, administration by topical, parenteral, oral, anal, intravaginal,
intravenous,
intraperitoneal, intramuscular, intraocular, subcutaneous, intranasal,
intrabronchial,
or intradermal routes among others.
Acceptable methods of preparing suitable pharmaceutical forms of the
pharmaceutical compositions are known to those skilled in the art. For
example,
pharmaceutical preparations may be prepared following conventional techniques
of
the pharmaceutical chemist involving steps such as mixing, granulating, and
compressing when necessary for tablet forms, or mixing, filling, and
dissolving the
ingredients as appropriate, to give the desired products for various routes of
administration.
Toxicity and therapeutic efficacy of the isomers according to the invention
can
be evaluated by standard pharmaceutical procedures in cell cultures or
experimental
animals. The therapeutic efficacy of the isomers according to the invention
can be
evaluated in an animal model system that may be predictive of efficacy in
human
diseases. For instance, animal models for evaluating efficacy in glucose
uptake
include animal models for diabetes or other relevant animal models in which
glucose
infusion rate can be measured. Animal models for evaluating insulinotropic
efficacy
include animal models for diabetes or other relevant animal models in which
secretion of insulin can be measured. Examples of suitable animal models for
diabetes include, but are not limited to DIO mice, ob/ob mice, db/db mice, and
Zucker
fa/fa rats. Alternatively, the ability of an isomer can be evaluated in vitro,
by
examining the ability of the compound to stimulate glucose uptake using
differentiated 3T3-L1 adipocyte cells (see Example 2) or using L6 myocytes, by
examining the ability of the compound to stimulate insulin secretion using INS-
1 cells
(see Example 3) or using perfused pancreas. While agents that exhibit toxic
side
effects may be used, care should be taken to design a delivery system that
targets
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such agents to the site of affected tissue in order to minimize potential
damage to
unaffected cells and, thereby, reduce side effects, whenever possible.
A wide range of drugs can be used with the isomers, compositions, and
methods of the present invention. Such drugs may be selected from antidiabetic
agents, antihypertensive agents, anti-inflammatory agents, antiobesity agents,
etc.
A non-limiting list of useful antidiabetic agents that can be used in
combination with an isomer of the invention include insulin, biguanides, such
as, for
example metformin (Glucophage@, Bristol-Myers Squibb Company, U.S.; Stagid@),
Lipha Sante, Europe); sulfonylurea drugs, such as, for example, gliciazide
(Diamicronft glibenclamide, glipizide (GlucotrolO and Glucotrol XL , Pfizer),
glimepiride (Amaryl , Aventis), chlorpropamide (e.g., Diabinesee, Pfizer),
tolbutamide, and glyburide (e.g., Micronase@), Glynase@, and Diabetaft
glinides,
such as, for example, repaglinide (Prandin0 or NovoNorm ; Novo Nordisk),
ormitiglinide, nateglinide (Starlix ), senaglinide, and BTS-67582; insulin
sensitizing
agents, such as, for example, glitazones, a thiazolidinedione such as
rosiglitazone
maleate (Avandia@, Glaxo Smith Kline), pioglitazone (Actos , Eli Lilly,
Takeda),
troglitazone, ciglitazone, isaglitazone, darglitazone, englitazone, CS-011/CI-
1037, T
174, GI 262570, YM-440, MCC-555, JTT-501, AR-H039242, KRP-297, GW-409544,
CRE-16336, AR-H049020, LY510929, MBX-102, CLX-0940, GW-501516, and the
compounds described in WO 97/41097 (DRF-2344), WO 97/41119, WO 97/41120,
WO 98/45292, WO 99/19313 (NN622/DRF-2725), WO 00/23415, WO 00/23416,
WO 00/23417, WO 00/23425, WO 00/23445, WO 00/23451, WO 00/41121, WO
00/50414, WO 00/63153, WO 00/63189, WO 00/63190, WO 00/63191, WO
00/63192, WO 00/63193, WO 00/63196, and WO 00/63209; glucagon-like peptide 1
(GLP-1) receptor agonists, such as, for example, Exendin-4 (1-39) (Ex-4),
ByettaTM
(Amylin Pharmaceuticals Inc.), CJC-1131 (Conjuchem Inc.), NN-2211 (Scios
Inc.),
and those GLP-1 agonists described in WO 98/08871 and WO 00/42026; agents that
slow down carbohydrate absorption, such as, for example, a-glucosidase
inhibitors
(e.g., acarbose, miglitol, voglibose, and emiglitate); agents that inhibit
gastric
emptying, such as, for example, glucagon-Iike peptide 1, cholescystokinin,
amylin,
and pramlintide; glucagon antagonists, such as, for example, quinoxaline
derivatives
(e.g., 2-styryl-3-[3-(dimethylamino)propylmethylamino]-6,7-
dichloroquinoxaline;
Collins et al., Bioorganic and Medicinal Chemistry Letters 2(9):915-918,
1992), skyrin
and skyrin analogs (e.g., those described in WO 94/14426), 1-phenyl pyrazole
derivatives (e.g., those described in U.S. Patent No. 4,359,474), substituted
disilacyclohexanes (e.g., those described in U.S. Patent No. 4,374,130),
substituted
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pyridines and biphenyis (e.g., those described in WO 98/04528), substituted
pyridyl
pyrroles (e.g., those described in U.S. Patent No. 5,776,954), 2,4-diaryl-5-
pyridylimidazoles (e.g., those described in WO 98/21957, WO 98/22108, WO
98/22109, and U.S. Patent No. 5,880,139), 2,5-substituted aryl pyrroles (e.g.,
those
described in WO 97/16442 and U.S. Patent No. 5,837,719), substituted
pyrimidinone,
pyridone, and pyrimidine compounds (e.g., those described in WO 98/24780, WO
98/24782, WO 99/24404, and WO 99/32448), 2-(benzimidazol-2-ylthio)-1-(3,4-
dihydroxyphenyl)-1-ethanones (see Madsen et al., J. Med. Chem. 41:5151-5157,
1998), alkylidene hydrazides (e.g., those described in WO 99/01423 and WO
00/39088), and other compounds, such as those described in WO 00/69810, WO
02/00612, WO 02/40444, WO 02/40445, and WO 02/40446; and glucokinase
activators, such as, for example, those described in WO 00/58293, WO 01/44216,
WO 01183465, WO 01/83478, WO 01/85706, and WO 01185707.
Other examples of antidiabetic agents that can be used in combination with
one or more isomers according to the invention include imidazolines (e.g.,
efaroxan,
idazoxan, phentolamine, and 1-phenyl-2-(imidazolin-2-yl)benzimidazole);
glycogen
phosphorylase inhibitors (see, e.g., WO 97/09040); oxadiazolidinediones,
dipeptidyl
peptidase-IV (DPP-IV) inhibitors, protein tyrosine phosphatase (PTPase)
inhibitors,
inhibitors of hepatic enzymes involved in stimulation of gluconeogenesis
and/or
glycogenolysis, glucose uptake modulators, glycogen synthase kinase-3 (GSK-3)
inhibitors, compounds that modify lipid metabolism (e.g., antihyperlipidemic
agents
and antilipidemic agents), peroxisome proliferator-activated receptor (PPAR)
agonists or antagonists in general, retinoid X receptor (RXR) agonists (e.g.,
ALRT-
268, LG-1268, and LG-1069), and antihyperlipidemic agents or antilipidemic
agents
(e.g., cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin,
pravastatin,
simvastatin, probucol, and dextrothyroxine). Other suitable antidiabetic
agents are
listed in Table I provided elsewhere herein.
Examples of antihypertensive agents that can be used with the isomers of the
invention include (3-blockers (e.g., alprenolol, atenolol, timolol, pindolol,
propranolol,
and metoprolol), angiotensin converting enzyme (ACE) inhibitors (e.g.,
benazepril,
captopril, enalapril, fosinopril, lisinopril, quinapril, and ramipril),
calcium channel
blockers (e.g., nifedipine, felodipine, nicardipine, isradipine, nimodipine,
diltiazem,
and verapamil), and a-blockers (e.g., doxazosin, urapidil, prazosin, and
terazosin).
Examples of anti-inflammatory agents that can be used with the isomers of
the invention include anti-histamines, and anti-TNFa.
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Examples of anti-obesity agents that can be used with the isomers of the
invention include XenicalT"" (Roche), MeridiaTM (Abbott), AcompliaTA (Sanofi-
Aventis), Pramlintide (Amylin), and sympathomimetic phentermine.
The isomers, compositions, and methods of the present invention may also
be used with analogs of 4-OH, such as those decribed in the PCT application
entitled
"ANALOGS OF 4-HYDROXYISOLEUCINE AND USES THEREOF" which claims
priority of US Provisional Application 60/654,342 filed February 18, 2005.
Accordingly, another aspect of relates to a pharmaceutical kit or
pharmaceutical composition that includes any of the isomers of 4-OH described
herein, or any combination thereof, and a second antidiabetic agent. The
pharmaceutical kit or composition can include a 4-hydroxyisoleucine isomer and
a
second antidiabetic agent that is formulated into a single composition, such
as, for
example, a tablet or a capsule. The invention also provides methods of
treating
diabetes (type 1 diabetes or type 2 diabetes), pre-diabetes, or Metabolic
Syndrome in
patients, which include administering to a patient one or more isomers of
4-hydroxyisoleucine such as those described herein, in combination with one or
more
antidiabetic agents. The combination of agents can be administered at or about
the
same time as one another or at different times.
The combinations of the invention provide several advantages. For example,
because the drug combinations described herein can be used to obtain an
improved
(e.g., additive or synergistic) effect, it is possible to consider
administering less of
each drug, leading to a decrease in the overall exposure of patients to drugs,
as well
as any untoward side effects of any of the drugs. In addition, greater control
of the
disease may be achieved, because the drugs can combat the disease through
different mechanisms.
Administration
With respect to the therapeutic methods of the invention, it is not intended
that the administration of compounds to a mammal be limited to a particular
mode of
administration, dosage, or frequency of dosing; the present invention includes
all
modes of administration, including oral, intraperitoneal, intramuscular,
intravenous,
intra-articular, intralesional, subcutaneous, by inhalation, or any other
route sufficient
to provide a dose adequate to prevent or treat diabetes (type 1 diabetes or
type 2
diabetes) and other disorders of carbohydrate or lipid metabolism, such as
those
described herein. One or more compounds may be administered to the mammal in a
single dose or multiple doses. When multiple doses are administered, the doses
may
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be separated from one another by, for example, several hours, one day, or one
week. It is to be understood that, for any particular subject, specific dosage
regimes
should be adjusted over time according to the individual need and the
professional
judgment of the person administering or supervising the administration of the
compositions. Exemplary mammals that can be treated using the isomers,
compositions, and methods of the invention include humans, primates such as
monkeys, animals of veterinary interest (e.g., cows, pigs, sheep, goats,
buffaloes,
and horses), and domestic pets (e.g., dogs and cats). The isomers and
compositions
of the invention can also be administered to rodents (e.g., mice, rats,
gerbils,
hamsters, guinea pigs, and rabbits) for treatment purposes and/or for
experimental
purposes (e.g., studying the compounds' mechanism(s) of action, screening and
testing efficacy of the isomers, structural design, etc.)
For clinical applications in therapy or as a prophylactic, isoniers or
compositions of the present invention can generally be administered, e.g.,
orally,
subcutaneously, parenterally, intravenously, intramuscularly, colonically,
nasally,
intraperitoneally, rectally, by inhalation, or buccally. Compositions
containing at least
one isomer of 4-hydroxyisoleucine according to the invention that is suitable
for use
in human or veterinary medicine can be presented in a form permitting
administration
by a suitable route. These compositions can be prepared according to customary
methods, using one or more pharmaceutically acceptable carriers or excipients.
The
carriers comprise, among other things, diluents, sterile aqueous media, and
various
non-toxic organic solvents. Acceptable carriers or diluents for therapeutic
use are
well known in the pharmaceutical field, and are described, for example, in
Remington: The Science and Practice of Pharmacy (20th ed.), ed. A.R. Gennaro,
Lippincott Williams & Wilkins, 2000, Philadelphia, and Encyclopedia of
Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999,
Marcel
Dekker, New York. The compositions can be presented in the form of tablets,
pills,
granules, powders, aqueous solutions or suspensions, injectable solutions,
elixirs, or
syrups, and the compositions may optionally contain one or more agents chosen
from the group comprising sweeteners, flavorings, colorings, and stabilizers
in order
to obtain pharmaceutically acceptable preparations.
The choice of vehicle and the content of active substance in the vehicle are
generally determined in accordance with the solubility and chemical properties
of the
product, the particular mode of administration, and the provisions to be
observed in
pharmaceutical practice. For example, excipients such as sodium citrate,
calcium
carbonate, and dicalcium phosphate and disintegrating agents such as starch,
alginic
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WO 2006/117696 PCT/IB2006/001758
acids, and certain complex silicates combined with lubricants (e.g., magnesium
stearate, sodium lauryl sulfate, and talc) can be used for preparing tablets.
To
prepare a capsule, it is advantageous to use high molecular weight
polyethylene
glycols. When aqueous suspensions are used, they can contain emulsifying
agents
that facilitate suspension. Diluents such as ethanol, polyethylene glycol,
propylene
glycol, glycerol, chloroform, or mixtures thereof can also be used. In
addition, low
calorie sweeteners, such as, for example, isomalt, sorbitol, xylitol, can be
used in a
formulation of the invention.
For parenteral administration, emulsions, suspensions, or solutions of the
compositions of the invention in vegetable oil (e.g., sesame oil, groundnut
oil, or olive
oil), aqueous-organic solutions (e.g., water and propylene glycol), injectable
organic
esters (e.g., ethyl oleate), or sterile aqueous solutions of the
pharmaceutically
acceptable salts can be used. The solutions of the salts of the compositions
of the
invention are especially useful for administration by intramuscular or
subcutaneous
injection. Aqueous solutions that include solutions of the salts in pure
distilled water
can be used for intravenous administration with the proviso that (i) their pH
is
adjusted suitably, (ii) they are appropriately buffered and rendered isotonic
with a
sufficient quantity of sodium chloride, and (iii) they are sterilized by
heating,
irradiation, or microfiltration. Suitable compositions containing the isomers
of the
invention can be dissolved or suspended in a suitable carrier for use in a
nebulizer or
a suspension or solution aerosol, or can be absorbed or adsorbed onto a
suitable
solid carrier for use in a dry powder inhaler. Solid compositions for rectal
administration include suppositories formulated in accordance with known
methods.
It is understood that the appropriate doses and concentrations of the agent(s)
in the
formulations (i.e., isomer(s) of 4-hydroxyisoleucine alone and/or in
combination with
other drug(s)) will vary, depending on a number of factors including the
dosages of
the agents to be administered, the route of administration, the nature of the
agent(s),
the frequency and mode of administration, the therapy desired, the form in
which the
agent(s) are administered, the potency of the agent(s), the sex, age, weight,
and
general condition of the subject to be treated, the nature and severity of the
condition
treated, any concomitant diseases to be treated, and other factors that will
be
apparent to those of skill in the art. A dose of the pharmaceutical
composition
contains at least a therapeutically effective amount of an isomer according to
the
invention and is preferably made up of one or more pharmaceutical dosage
units.
The selected dose can be administered to a human subject in need of treatment.
A
"therapeutically effective amount" is intended to mean that amount of
isomer(s) of
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the invention that confers a therapeutic effect on the subject treated. The
therapeutic
effect may be objective (i.e., measurable by some test or marker (e.g.,
insulin or
glucose levels)) or subjective (i.e., the subject gives an indication of or
feels an
effect).
A dose of the pharmaceutical composition contains at least a therapeutically
effective amount of an isomer according to the invention and is preferably
made up of
one or more pharmaceutical dosage units. The selected dose can be administered
to
a mammal, for example, a human patient, in need of treatment. As is noted
above, a
"therapeutically effective amount" is intended to mean that amount of
isomer(s)
according to the invention that, when administered to a subject for treating a
disease,
confers a therapeutic effect on the subject treated. The therapeutic effect
may be
objective (i.e., measurable by some test or marker (e.g., insulin or glucose
blood
levels)) or subjective (i.e., the subject gives an indication of or feels an
effect). For
instance, in one embodiment relating to type 2 diabetes, a "therapeutically
effective"
amount will increase glucose uptake by muscle and/or adipose tissues, and/or
it wi!l
stimulate insulin secretion by pancreatic (i-cells. In another embodiment
relating to
type 2 diabetes, a"therapeuticafly effective" amount reduces glucose levels
and/or
increase insulin levels in the subject's blood by, for example, at least about
20%, or
by at least about 40%, or even by at least about 60%, or by at least about 80%
relative to untreated subjects.
The amount that will correspond to a "therapeutically effective amount" will
vary depending upon factors such as the particular compound, the route of
administration, excipient usage, the disease condition and the severity
thereof, the
identity of the subject in need thereof, the age, weight, etc., of the subject
to be
treated and the possibility of co-usage with other agents for treating a
disease.
Nevertheless the therapeutically effective amount can be readily determined by
one
of skill in the art.
For administration to mammals, and particularly humans, it is expected that in
the treatment of an adult dosages from about 0.1 mg to about 50 mg (e.g.,
about 5
mg to about 100 mg, about 1 mg to about 50 mg, or about 5 mg to about 25 mg)
of
each active compound per kg body weight per day can be used. A typical oral
dosage can be, for example, in the range of from about 50 mg to about 5 g per
day,
(e.g., about 100 mg to about 4 g, 250 mg to 3 g, or 500 mg to 2 g),
administered in
one or more dosages, such as I to 3 dosages. Dosages can be increased or
decreased as needed, as can readily be determined by those of skill in the
art. For
example, the amount of a particular agent can be decreased when used in
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combination with another agent, if determined to be appropriate. In addition,
reference can be made to standard amounts and approaches that are used to
administer the agents mentioned herein.
Examples of dosages for antidiabetic agents mentioned herein are provided
in Table 1, below. The antidiabetic agents can be used in these dosages when
combined with a configurational isomer of 4-hydroxyisoleucine, which generally
is
administered in an amount in the range of, for example, 250 mg - 1 g/day
(e.g., 350-
900, 450-800, or 550-700 mg/day). Alternatively, due to the potential additive
or
synergistic effects obtained when using drug combinations of the invention,
the
amounts in Table I and/or the amount of isomer administered can be decreased
(by,
e.g., about 10-70%, 20-60%, 30-50%, or 35-45%), as determined to be
appropriate
by those of skill in this art.
The physician in any event will determine the actual dosage that will be most
suitable for an individual. The above dosages are exemplary of the average
case.
There can, of course, be individual instances where higher or lower dosage
ranges
are merited, and such are within the scope of this invention.
As for dosing, it is understood that duration of a treatment using any of the
compounds or compositions of the invention will vary depending on several
factors,
such as those listed herein before for dosing. Nevertheless, appropriate
duration of
administration can be readily determined by one of skill in the art. According
to
certain embodiments, the compounds of the invention are administered on a
daily,
weekly or on a continuous basis.
Table 1: List of well-known antidiabetic agents
Antidiabetic agent Recommended dosage and/or administration
Insulin 400 IU per vial - 40 IU per day (mean value)
Gliciazide (Diamicron) 80 mg/tablet - 1 to 4 tablets per day
5 mg/tablet - 1 to 3 tablets per day (Glibenclamide);
Glibenciamide (Daonil) or Glyburide 1.25 to 6 mg/tablet - 1 to 2 tablets per
day
(Micronase, Glynase, Diabeta) (Glyburide)
Glipizide (Glucotrol, Glibenese) 5 mg/tablet - 1 to 4 tablets per day
Glimepiride (Amaryl, Amarel) 1 to 4 mg/tablet - 6 mg per day maximum
Chlorpropamide (Diabinese) 250 mg/tablet - 125 to 1000 mg per day per day
Tolbutamide 500 mg/tablet - 1 to 4 tablets per day
Repaglinide (Prandin) 0.5 to 16 mg per day
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Antidiabetic agent Recommended dosage and/or administration
Nateglinide, Senaglinide (Starlix) 60 to 120 mg/tablet - 3 tablets per day
Tolazamide 100 to 500 mg/tablet
Rosiglitazone 2 to 8 mg/tablet - 8 mg per day maximum
Pioglitazone 15 to 45 mg/tablet - 15 to 45 mg per day
Troglitazone 200 to 400 mg/tablet - 200 to 600 mg per day
Ciglitazone 0.1 mg/tablet
Exenatide (Amylin) 0.09 to 0.270 mg per day
Acarbose 50 to 100 mg/tablet - 150 to 600 mg per day
Miglitol 50 to 100 mg/tablet - 150 to 300 mg per day
Voglibose 0.1 to 0.9 mg per day
Phentolamine 50 mg - 4 to 6 times per day
Cholestyramine (Colestipol) 4 g/unit - 12 to 16 g per day
Clofibrate 500 mg/capsule - 1 to 4 capsules per day
Gemfibrozil (Lipur) 450 mg/tablet - 2 tablets per day
Lovastatin 10 and 20 mg/tablet
Pravastatin 20 mg/tablet - 10 to 40 mg per day
Simvastatin (Zocor, Lodales) 5 and 20 mg/tablet - 5 to 40 mg per day
Probucol 250 mg/tablet - 1 g per day
Dextrothyroxine 2 to 6 mg per day
Alprenolol 50 mg/tablet - 4 to 8 tablets per day
Atenolol 50 to 100 mg / tablet - 100 to 200 mg per day
Timolol 10 mg/tablet - 10 to 20 mg per day
Pindolol 5 and 15 mg/tablet - 5 to 60 mg per day
Propranolol 40 mg/tablet - 80 to 160 mg per day
Metoprolol 100 and 200 mg/tablet - 50 to 200 mg per day
Captopril 25 and 50 mg/tablet - 12.5 to 150 mg per day
Enalapril 5 and 20 mg/tablet - 5 to 40 mg per day
Nifedipine 10 mg/capsule - 30 to 60 mg per day
Diltiazem 60 mg/tablet - 3 to 6 tablets per day
Verapamil 120 and 240 mg/capsule - 240 to 360 mg per day
Doxazosin 2 to 8 mg per day
Prazozin 2.5 and 5 mg/tablet - 2.5 to 20 mg per day
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The isomers and compositions of the invention are conceived to be effective
primarily in the treatment of disorders of carbohydrate metabolism,
particularly type 2
diabetes. However, it is conceivable that the isomers and compositions
according to
the present invention may also be useful in connection with disorders of fat
metabolism, including but not limited to lipodystrophy associated with HIV and
lipidemia, because they may influence fat distribution.
It is also conceivable to use isomers of the invention for others related or
unrelated applications. For instance, it might be useful to provide in-
dwelling devices
such as catheters coated with the isomers of the invention, for improving
cardiovascular functions.
EXAMPLES
The Examples set forth herein below provide exemplary syntheses of the
compounds of the invention. Also provided are exemplary methods for assaying
the
compounds of the invention for their activity as stimulators of glucose uptake
and as
stimulators of insulin secretion. These examples are given to enable those
skilled in
the art to more closely understand and to practice the present invention and
are not
intended to either define or limit its scope.
Example 1: General procedure for the preparation of isomers of
4-hydroxyisoleucine
A) General Experimental Procedures
A scheme for the synthesis of the eight different configurational isomers
(SRS, SRR, SSS, SSR, RSR, RSS, RRR, and RRS) of 4-hydroxyisoleucine is given
in Figure 1. Imine intermediate I was prepared from p-anisidine and ethyl
glyoxalate
(Cordova et al., A highly enantioselective amino acid-catalyzed route to
functionalized alpha-amino acids, J. Am. Chem. Soc. 124:1842-43, 2002). The
reaction of imine I with 2-butanone in the presence of L-proline as a catalyst
followed
by silica gel chromatography yielded 2S,3S isomer 2. Epimerization at C-3 was
achieved with 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) to yield 2S,3R isomer 3.
The
(2S,3R,4S); (2S,3R,4R); (2S,3S,4S); and (2S,3S,4R) isomers of 4-
hydroxyisoleucine
are obtained from either 2 or 3 as follows:
Deprotection of amine moiety of 3 (removal of p-methoxyphenyl group) with
ceric ammonium nitrate (CAN) and subsequent reduction with KBH4 in water and
concomitant cyclization provided lactone 4, which upon base hydrolysis with
lithium
hydroxide and recrystallization from absolute ethanol gave pure (2S,3R,4S)-4-
24
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hydroxyisoleucine 5. Alternatively, deprotection of the amine moiety of 3 with
CAN
was followed by isolation of amine intermediate 6, which was subsequently
reduced
with potassium borohydride in methanol to give the lactone intermediate 7,
which
upon base hydrolysis with lithium hydroxide and recrystallization from ethanol
gave
(2S,3R,4R) 4-hydroxyisoleucine (compound 8). Further purification of compound
8
was carried out using preparative HPLC.
Similar reactions starting from compound 2, using sodium borohydride
instead of potassium borohydride for preparation of lactone 12 from
aminoketone 11
lead to the isolation of (2S,3S,4S) 4-hydroxyisoleucine (compound 10) and
(2S,3S,4R) 4-hydroxyisoleucine (compound 13).
When compound I was reacted with 2-butanone in the presence of a catalytic
amount of D-proline, compound 14, which is the enantiomer of compound 2, was
formed. As above, epimerization of the C-3 of compound 14 was achieved with
1,5-
diazabicyclo[4.3.0]non-5-ene (DBN) to yield 2R,3S isomer 15. By reaction
sequences
identical to those used for the preparation of compounds 5, 8, 10, and 13, the
(2R, 3S, 4R); (2R, 3S, 4S); (2R, 3R, 4R); and (2R, 3R, 4S) isomers (compounds
17, 20,
22, and 25, respectively) were obtained from compounds 14 and 15.
Detailed reaction cdnditions used in the preparation of compounds I through
are as follows. 'H and 13C NMR spectra are of D20 solutions, and chemical
shifts
20 are reported in ppm using methanol (6 3.34 for'H and 6 49.50 for'3C) as the
internal
standard.
B) Detailed Experimental Procedures
Synthesis of compound 1
25 To a stirred solution of p-anisidine (50 g, 406 mmol) in toluene (400 mL)
in a 1
liter, round-bottomed flask was added sodium sulfate (200 g, -2.5 eq). Ethyl
glyoxalate (82 mL, 50% in toluene, 406 mmol) was added slowly to the above
reaction mixture, and the mixture was stirred for 30 min. After this time, the
sodium
sufate was filtered off using celite and toluene was removed under reduced
pressure.
Compound 1 (80 g, 95%) was isolated after drying and used as is for the next
reaction.
Synthesis of compound 2
A mixture of 2-butanone (800 mL, 22 eq) and L-proline (15.8 g, 0.35 eq) in dry
DMF (600 mL) was stirred at room temperature under nitrogen. To this reaction
mixture was slowly added a solution of compound I in dry DMF (200 mL) and Et3N
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(22.4 mL, 0.40 eq). After stirring the reaction mixture at room temperature
for 8 h, L-
proline was filtered off, excess 2-butanone was removed under reduced
pressure,
and DMF was removed in vacuo at 50 C. The crude amine (compound 2) was
purified by column chromatography (Si02, 85:15 hexanes/EtOAc).
Synthesis of compound 3
Compound 2 was dissolved in t-BuOMe (15 mL) and to the stirred reaction
mixture was added 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) (1 mL, -0.04 eq). The
reaction mixture was stirred under nitrogen for 2 h. A solid cake was obtained
after
overnight evaporation of the solvent at room temperature, which upon
recrystallization from hot ethanol gave compound 3 (48 g, 43% yield).
Synthesis of (2S,3R,4S)-4-Hydroxyisoieucine (compound 5)
To a solution of compound 3 (11.6 g, 40 mmol) in CH3CN (20 mL) was added
a solution of ammonium cerium (IV) nitrate (CAN) (65.6 g, 3 eq) in water (120
mL)
with stirring at 0 C. The color gradually changed from blue to green upon
addition of
CAN. The reaction mixture was stirred for 2.5 h, and the progress of the
reaction
followed by TLC analysis. After completion, the reaction mixture was extracted
with
EtOAc (4 x 150 mL) and the aqueous phase used for the next step.
The aqueous phase was neutralised to pH 7 with saturated Na2CO3, and
cooled to -15 C and stirred. After cooling for 30 min, KBH4 (3.2 g, 60 mmol,
1.5 eq)
was added to the reaction mixture. The reaction was allowed to warm to 0 C for
about 45 min and followed by TLC. The reaction mixture was then made basic
with 2
N Na2CO3 to a pH of 8-9 and extracted with CH2CI2 (5 x 400 mL). The organic
phase
was washed with water, dried over Na2SO4 and evaporated under reduced pressure
to obtain a 90:10 mixture of lactones (compound 4 (3S,4R,5S) to compound 7
(3S,4R,5R); 3.73 g, 62.6%).
To a solution of the 90:10 lactone mixture in water (96 mL, 0.3 M) was added
LiOH (1.1 g, 43.3 mmol, 1.5 eq), and the mixture was stirred at room
temperature for
2 h. After the reaction was complete, it was acidified by careful addition of
AcOH
(43.3 mmol, 2.4 mL). The reaction mixture was concentrated under reduced
pressure
and last traces of water were removed by repeated addition and removal of
ethanol.
The crude product was crystallised from absolute EtOH to give 1.56 g of 98%
pure
(2S,3R,4S) 4-hydroxyisoleucine (compound 5). Further purification by
preparative
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HPLC gave compound 5 as white shiny powder: mp 215-222 (subl.); [a]pH2O +30.7
(c,1); 'H NMR (200 MHz) S 3.90 (m, 1 H), 3.84 (m, 1 H), 1.91(m, 1 H), 1.23 (d,
J= 5.6
Hz, 3H) 0.95 (d, J = 6.6 Hz, 3H); 13C NMR (75 MHz) (5 174.32, 70.46, 57.54,
41.90,
21.30, 12.70.
Synthesis of (2S,3R,4R)-4-Hydroxyisoleucine (compound 8)
To a solution of compound 3 (11.6 g, 40 mmol) in CH3CN (20 mL) was added
a solution of ceric ammonium nitrate (CAN) (65.6 g, 3 eq) in water (120 mL)
with
stirring at 0 C. The color gradually changed from blue to green upon addition
of CAN.
The reaction mixture was stirred for 45 min, and the progress of the reaction
followed
by TLC. After completion, the reaction mixture was extracted with EtOAc (4 x
150
mL) and the aqueous phase was carefully neutralised with saturated Na2CO3
solution
to slightly basic pH (-8). The aqueous phase was extracted with CH2CI2 (4 x
150 mL)
and organic extracts were combined, washed with brine, dried over anhydrous
Na2SO4 and concentrated under reduced pressure to yield 5.52 g (79.7%) of
compound 6 as a brownish oil.
To a solution of compound 6 in methanol (15 mL), cooled to 0 C, was quickly,
added KBH4 (2.58 g, 47.8 mmol). The reaction mixture was stirred at 0 C for 45
min
and then gradually warmed to room temperature. The solvent was removed in
vacuo,
and the mixture was diluted with water. The aqueous phase was extracted with
CH2CI2 (4 x 150 mL). The organic phase was washed with brine, dried over
anhydrous Na2SO4 and evaporated in vacuum to give a 75:25 mixture of compound
7
(3S,4R,5R) to compound 4 (3S,4R,5S) (2.9 g, 70.2%).
The solution of compound 7/compound 4 mixture in water (100 mL) was
treated with LiOH (805 mg, 33.7 mmol) and stirred at room temperature for 1 h
before carefully acidifying with AcOH (1.91 mL, 33.72 mmol). After
concentrating
under reduced pressure, the traces of water were removed by repeated addition
and
removal of absolute ethanol. A crude greyish solid was obtained from a cold
solution
of 90% ethanol. Further recrystallization from 90% ethanol yielded 1.4 g of
75:25
diastereomeric ratio of compound 8 to compound 5. Repeated crystallisations
improved the purity of compound 8 to 90%, and further purification using
preparative
HPLC gave pure (2S,3R,4R) 4-hydroxyisoleucine (compound 8) as a white shiny
material: mp 202-204 C (subl.); [a ]oHZO - 21.6 (c, 0.5); 1H-NMR (300 MHz) b
4.05 (m,
1 H), 3.80 (d, J = 4.2 Hz, 1 H), 2.13 (m, 1 H) 1.20 (d, J = 6.3 Hz, 3H), 1.05
(d, J = 7.2
Hz, 3H);13C NMR (75 MHz) (5 174.49, 69.13, 59.97, 39.12, 20.71, 9.38.
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Synthesis of (2S,3S,4S)-4-Hydroxyisoleucine (compound 10)
Compound 2 (5.6 g, 20 mmol) was dissolved in acetonitrile (10 mL), and to
this was added a solution of ceric ammonium nitrate (CAN) (33 g, 60 mmol) in
water
(60 mL) with stirring at 0 C. The reaction mixture color gradually changed
from blue
to green upon addition of CAN. The reaction mixture was stirred for 45 min and
extracted with ethyl acetate (4 x 150 mL). The aqueous phase was neutralized
with
saturated Na2CO3 and pH was carefully adjusted to 7. After cooling the
reaction
mixture to -15 C for 90 min, KBH4 (1.6 g, 30 mmol, 1.5 eq) was added. The
reaction
was allowed to warm up to 0 C for about 45 min and then treated with 2 N
Na2CO3 to
a pH of 8-9, followed by extraction with CH2CI2 (5 x 400 mL). The organic
phase was
washed with water, dried over anhydrous Na2SO4 and evaporated under reduced
pressure to obtain 1.42 g of a 75:25 mixture of lactones (compound 9
(3S,4S,5S) to
compound 12 (3S,4S,5R)).
To the mixture of lactones in water (35 mL) was added LiOH (395 mg, 16.5
mmol, 1.5 eq) and the mixture was stirred at room temperature for 2 h. After
this
time, the reaction mixture was carefully acidified with AcOH (16.5 mmol, 0.9
mL). The
solvent was removed under vacuum, and repeated addition and removal of
absolute
ethanol led to complete removal of water. The crude material obtained was
dissolved
in 90% EtOH and left overnight. The separated white solid was filtered and
washed
several times with EtOH, and recrystallized from 90% EtOH to obtain white
crystals
of (2S,3S,4S)-4-hydroxyisoleucine (compound 10, 500 mg). Further purification
using
preparative HPLC led to pure shiny material: mp 253-255 C; [a ]o"2O +28 (c,
0.25);'H
NMR (300 MHz) 5 4.11 (m, 1 H), 3.87 (d, J= 2.7 Hz, 1 H), 2.21 (m, 1 H), 1.23
(d, J=
6.3 Hz, 3H), 0.92 (d, J = 7.5 Hz, 3H); 13C NMR (75 MHz) (5 174.64, 71.39,
60.39,
38.97, 21.11, 6.19.
Synthesis of (2S,3S,4R)-4-Hydroxyisoleucine (compound 13)
To a solution of compound 2 (11.6 g, 40 mmol) in acetonitrile (20 mL) was
added a solution of ammonium cerium (IV) nitrate (CAN) (65.6g, 120 mmol) in
water
(120 mL) with stirring at 0 C. The reaction mixture color gradually changed
from blue
to green upon addition of CAN. The reaction mixture was stirred for 45 min and
extracted with ethyl acetate (4 x 150 mL). The aqueous phase was carefully
neutralised with saturated Na2CO3 solution to a pH of 8, followed by
extraction with
CH2CI2 (4 x 150 mL). The combined organic extracts were washed with brine,
dried
over anhydrous Na2SO4 and concentrated under reduced pressure to yield 4 g of
compound 11 as brown oil.
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To a solution of 11 in MeOH (15 mL) at 0 C was quickly added NaBH4 (962
mg, 1.1 eq, 25.43 mmol). The reaction mixture was vigorously stirred at 0 C
for 45
min and gradually warmed to room temperature. The solvent was removed under
reduced pressure, the residue diluted with water, and the aqueous phase
extracted
with CH2CI2 (4 x 150 mL). The combined organic phases were washed with brine,
dried over anhydrous Na2SO4 and evaporated in vacuum to give 2 g of a mixture
of
compound 12 (3S,4S,5R) and compound 9 (3S,4S,5S).
The mixture was dissolved in water (40 mL) and LiOH (556.9 mg, 18.6 mmol)
was added. The reaction mixture was stirred at room temperature for 1 h and
carefully acidified with AcOH (1.31 mL). The solvent was removed under vacuum.
The crude product was dissolved in a minimum amount of water and the compound
was loaded on a column packed with dowex 50w x 8(H+) resin (50 g). The column
was first eluted with water 4 x 50 mL and then fractions were collected by
eluting with
2 M NH4OH. The isolated product was dissolved in 90% EtOH and left standing
over
night. The separated solid (250 mg) was filtered, washed with cold EtOH, and
recrystalised from 90% EtOH to obtain a mixture of diastereoisomers.
This diastereoisomer mixture of compounds 10 and 13 was purified by
preparative HPLC to produce (2S,3S,4R) 4-Hydroxyisoleucine (compound 13) as a
white shiny powder: mp 173-175 C; [a ]p 2O + 6.0 (c, 0.25); 'H NMR (300 MHz) 5
4.02 (d, J= 3 Hz, 1 H), 3.81 (m, 1 H), 2.12 (m, 1 H) 1.28 (d, J= 6.6 Hz, 3H),
0.97 (d, J
= 7.2 Hz, 3H);13C NMR (75 MHz) S 174.93, 70.18, 56.34, 40.46, 21.24, 12.15.
Syntheses of (2R,3S,4R)-4-Hydroxyisoleucine (compound 17), (2R,3S,4S)-4-
Hydroxyisoleucine (compound 20), (2R,3R,4R)-4-Hydroxyisoleucine (compound 22),
and (2R,3R,4S)-4-Hydroxyisoleucine (compound 25)
The procedures used in the syntheses of compounds 17, 20, 22, and 26 were
identical to those used for compounds 5, 8, 10, and 13, except that compound I
was
reacted with 2-butanone in the presence of D-proline to produce compound 14
(the
antipode of compound 12). The physical and NMR data of compounds 17, 20, 22,
and 25 are as follows:
(2R,3S,4R)-4-Hydroxyisoleucine (compound 17): mp 217-225 C (subl.); [a ]p 20 -
31
(c, 1); 'H NMR (200 MHz) b 3.89 (m, 1 H), 3.84 (m, 1 H), 1.90 (m, 1 H) 1.23
(d, J= 6.4
Hz, 3H), 0.95 (d, J =7 Hz, 3H); 13C NMR (50 MHz) (5 174.36, 70.43, 57.51,
41.91,
21.30, 12.6.
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2R,3S,4S)-4-Hydroxyisoleucine (compound 20): mp 200-204 C (subl.); [a ]p"Z
+22
(c, 0.5); ' H NMR (200 MHz) (5 4.04 (m, 1 H), 3.80 (m, 1 H), 2.12 (m, 1 H),
1.19 (d, J=
6.2 Hz, 3H) 1.05 (d, J = 7.2 Hz, 3H); 13C NMR (50 MHz) 5 174.55, 69.12, 59.97,
39.12, 20.73, 9.40.
(2R,3R,4R)-4-Hydroxyisoleucine (compound 22): mp 250-254 C; [a ]0 2O -30 (c,
0.25); 'H-NMR (200 MHz) S 4.10 (m, 1 H), 3.87 (d, J= 2.6 Hz 1 H), 2.23 (m, 1
H) 1.23
(d, J = 6.6 Hz, 3H), 0.92 (d, J = 7.2 Hz,3H); 13C NMR (50 MHz) (5 174.64,
71.29,
60.35, 38.96, 21.12, 6.22.
(2R,3R,4S)-4-Hydroxyisoleucine (compound 25): mp 173 C; [a ]p"2O -5.6 (c,
0.25); 'H
NMR (300 MHz) b 4.01 (d, J= 2.7 Hz, 1 H), 3.80 (m, 1 H), 2.11 (m, 1 H) 1.27
(d, J=
6.3 Hz, 3H), 0.97 (d, J = 7.2 Hz, 3H); 13C NMR (75 MHz) 5 174.96, 70.18,
56.35,
40.44, 21.23, 12.10.
Example 2: Stimulation of glucose uptake by differentiated 3T3-L1 adipocyte
cells by configurational isomers of (2S,3R,4S)-4-hydroxyisoleucine
3T3-L1 adipocyte cells (ATCC; CI-173) were cultured in 12 well tissue culture
plates for 3 days in order to reach confluence (Lakshmanan et al., Analysis of
insulin-
stimulated glucose uptake in differentiated 3T3-L1 adipocytes. Diabetes
Mellitus:
Methods and Protocols, Saire Ozcna, Ed., Humana Press Inc., Tonowa, New Jersey
97-103, 2003). The culture medium was removed and replaced with
differentiation
medium (Green and Meuth, Cell 3:127-133, 1974; Madsen et al., Biochem. J.
375:539-549, 2003) and the cells were incubated for an additional 9 days. The
state
of differentiation was confirmed by visual examination. Cell starvation was
conducted
for 5 hours by replacing the differentiation medium with one lacking fetal
calf serum.
During the last 30 minutes of the starvation period, the cells were exposed to
each of
the configurational isomers of 4-hydroxyisoleucine (compounds 5, 8, 10, 13,
17, 20,
22, and 25) at a concentration of 0.5 mM. Cells exposed to insulin (0.0167
U/mL;
Sigma; Cat. No. 15534) for the last 30 minutes of the starvation period were
used as
a positive control and cells exposed to 0.5 mM isoleucine were used as a
control for
background uptake. All treatments were performed in quadruplicate. Cells were
washed, then fresh medium containing 16 pM 3H-deoxy-D-glucose (0.5 pCi/mL) and
10 pM 2-deoxy-D-glucose was added and the cells were incubated for 10 min.
Glucose uptake was stopped by washing the cells with ice cold PBS. The cells
were
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lysed and specific activity in the lysate was determined relative to
background uptake
of 3H-deoxy-glucose. Results, which are shown in Figure 2, were standardized
on
the basis of protein content per well.
As expected, insulin strongly promoted glucose uptake while isoleucine did
not. All configurational isomers of (2S,3R,4S)-4-hydroxyisoleucine (compound
5)
showed good stimulation of glucose uptake, with compounds 8, 13, 20, and 25
having the best activity. Accordingly, the conformational isomers of
(2S,3R,4S)-4-
hydroxyisoleucine have the potential to be therapeutic agents for the
treatment of
diabetes and related conditions.
Example 3: Glucose-dependent stimulation of insulin secretion in INS-1 cells
by configurational isomers of (2S,3R,4S)-4-hydroxyisoleucine
The configurational isomers of 4-hydroxyisoleucine were tested in a blinded
fashion for insulinotropic effect on INS-1 cells. Briefly, the cells were
plated at a
density of 2 x 105 in 12 well plates and incubated for 2 days in RPMI with 10%
fetal
calf serum and 11 mM glucose. The medium was removed on the third day post-
plating and replaced with RPMI containing 3 mM glucose with 10% fetal calf
serum.
The cells were incubated for an additional 24 hours. On the fourth day post-
plating,
the medium was removed and replaced with Krebs-Ringer bicarbonate buffer
containing 2 mM glucose. The cells were incubated for 30 min and the buffer
was
removed and replaced with Krebs-Ringer bicarbonate buffer with 4.5 mM glucose
containing an optical isomer at a concentration of 0.5 mM. The cells were
incubated
for 1 hour. Basal insulin secretion was determined by incubating the cells in
the
presence of buffer with 2 mM glucose. The presence of glucose at 4.5 and 10 mM
stimulated insulin secretion and served as the reference control and positive
control,
respectively. A positive stimulatory response by a tested 4-hydroxyisoleucine
configurational isomer was taken as the response above that elicited by 4.5 mM
glucose. As shown in Figure 3, all of the configurational isomers of 4-
hydroxyisoleucine except compound 13 (the 2S,3S,4R isomer) exhibited
insulinotropic activity, with compounds 8, 10, and 20 as active as compound 5
(the
2S,3R,4S isomer known to have insulinotropic activity, see Broca et al., 4-
Hydroxyisoleucine: effects of synthetic and natural analogues on insulin
excretion,
Eur. J. PharmacoL 390:339-345, 2000; and Sauvaire et al., Diabetes 47:206-210,
1998).
It is understood that the examples and embodiments described herein are for
illustrative purposes only and that various modifications or changes in light
thereof
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will be suggested to persons skilled in the art and are to be included within
the spirit
and purview of this application and scope of the appended claims.
32
