U.S. patent application number 12/176827 was filed with the patent office on 2008-11-20 for use of amino acids for making medicines for treating insulin resistance.
This patent application is currently assigned to Innodia Inc.. Invention is credited to Christophe Broca, Bernard Pau, Roger Pierre Petit, Gerard Ribes, Yves Sauvaire, Mohammed Taouis.
Application Number | 20080287344 12/176827 |
Document ID | / |
Family ID | 9549397 |
Filed Date | 2008-11-20 |
United States Patent
Application |
20080287344 |
Kind Code |
A1 |
Ribes; Gerard ; et
al. |
November 20, 2008 |
Use of Amino Acids for Making Medicines for Treating Insulin
Resistance
Abstract
The invention concerns the use of monohydroxy or polyhydroxy
amino acids, and the lactone forms thereof for making medicines
with insulin-analogue and/or insulin-sensitizing effects on
peripheral tissues targeted by insulin, and more particularly the
use thereof for making medicines for treating and preventing
insulin-resistance.
Inventors: |
Ribes; Gerard; (Montpellier,
FR) ; Taouis; Mohammed; (Tours, FR) ; Petit;
Roger Pierre; (Montpellier, FR) ; Broca;
Christophe; (Montpellier, FR) ; Sauvaire; Yves;
(Montferrier Sur Lez, FR) ; Pau; Bernard;
(Montpellier, FR) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Assignee: |
Innodia Inc.
Laval
CA
|
Family ID: |
9549397 |
Appl. No.: |
12/176827 |
Filed: |
July 21, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10069574 |
Aug 1, 2002 |
7402611 |
|
|
PCT/FR00/02361 |
Aug 23, 2000 |
|
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12176827 |
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Current U.S.
Class: |
514/1.1 ;
562/567 |
Current CPC
Class: |
A61P 3/04 20180101; A61P
3/10 20180101; A61K 31/195 20130101; A61P 35/00 20180101; A61K
31/198 20130101; A61P 5/50 20180101 |
Class at
Publication: |
514/3 ;
562/567 |
International
Class: |
A61K 38/28 20060101
A61K038/28; C07C 229/22 20060101 C07C229/22 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 27, 1999 |
FR |
99-10874 |
Claims
1) Use of a compound chosen from the group constituted by
mono-hydroxylated amino acids, poly-hydroxylated amino acids, and
the lactonic forms of these acids, for the manufacture of a
medicament with insulin mimetic and/or insulin-sensitizing
effects.
2) Use according to claim 1, characterised in that said medicament
is intended to exercise an insulin mimetic and/or
insulin-sensitizing effect at the level of the peripheral target
tissues of insulin.
3) Use according to claim 1 or 2, characterised in that said
compound reduces phosphatase activity associated with the
signalling route of the insulin receptor, and/or stimulates PI
3-kinase activity on IRS-1 and/or IRS-2.
4) Use according to any one of the previous claims, characterised
in that said compound is 4-hydroxyisoleucine of formula
##STR00002## and/or the lactonic form of this amino acid.
5) Use according to claim 4, characterised in that
4-hydroxyisoleucine is presented in the form of its 2S, 3R, 4S
isomer or the corresponding lactone.
6) Use according to any one of claims 1 to 5, for the manufacture
of a medicament intended for the treatment of insulin
resistance.
7) Use according to any one of claims 1 to 6, for the manufacture
of a medicament intended to combat the syndromes associated with
insulin resistance.
8) Use according to any one of claims 1 to 7, for the manufacture
of a medicament against hyperinsulinemia.
9) Use according to any of claims 1 to 7, for the manufacture of a
medicament against insulin resistance associated with ageing.
10) Use of any one of claims 1 to 7, for the manufacture of a
medicament against illnesses associated with obesity.
11) Use according to any one of claims 1 to 7, for the manufacture
of a medicament intended to inhibit the proliferation of cell lines
associated with the risk of the appearance of cancer.
12) Use according to any one of claims 1 to 5, for the manufacture
of a medicament intended for the prevention of insulin
resistance.
13) Use according to any one of claims 1-5, and 12, for the
manufacture of a medicament intended to reduce the need for
exogenic insulin.
14) Pharmaceutical composition or kit comprising both insulin and a
compound chosen from the group constituted by mono-hydroxylated
amino acids, poly-hydroxylated amino acids, and the lactonic forms
of these acids.
Description
[0001] This application is a divisional of, and claims priority
from, U.S. patent application Ser. No. 10/069,574, filed Aug. 1,
2002, which claims priority under 35 U.S.C. .sctn. 371 of
PCT/FR00/02361, filed Aug. 23, 2000, which claims priority under 35
U.S.C. .sctn. 119 from French patent application no. 99-10874,
filed Aug. 27, 1999.
[0002] The present application relates to the use of amino acids
for the manufacture of medicaments with an insulin mimetic and/or
insulin-sensitizing effects on the peripheral target tissues of
insulin, and more particularly the use of amino acids for the
manufacture of medicaments intended for the treatment and the
prevention of insulin resistance.
[0003] In the last few years, considerable progress has been made
in the understanding of the molecular mechanisms of how insulin
works. A diagram showing the main routes of transduction of the
insulin signal is given in FIG. 1. The insulin receptor is a
transmembrane receptor endowed with an intrinsic tyrosine kinase
activity (Combettes-Souverain M. and Issad T., Diab. Metab., 24,
477, 1998). The bonding of insulin to its receptor leads to the
autophosphorylation of the receptor on its tyrosine residues, which
stimulates its tyrosine kinase activity towards a certain number of
intracellular substrates such as Insulin Receptor Substrate 1
(IRS-1), Insulin Receptor Substrate 2 (IRS-2) and Src homology
collagen protein or Shc. This tyrosine kinase activity plays a
determining role in the transmission of the insulin signal, and it
is altered in numerous situations of insulin resistance (Ricort J.
M. et al., Diabetologia, 38, 1148, 1995). Overall the cellular
mechanisms of insulin resistance can be found at the bonding of the
hormone to its receptor or a more distal post-bonding stage to the
receptor. As the diagram in FIG. 1 shows, the phosphorylation on
the tyrosines of IRS and Shc by the insulin receptor allows the
activation of two main routes of cellular signalling, the MAP
kinase (MAPK) route and the phosphatidylinositol 3-kinase (PI 3-K)
route. Phosphotyrosine phosphatase (PTP) plays a role in the
regulation of these routes.
[0004] The inventors have previously reported the insulin secretor
effect that can be exercised in pancreatic cells by hydroxylated
amino acid extracts of fenugreek seeds (Trigonella foenum graecum
L.), and in particular by 4-hydroxyisoleucine (4-OH-Ile) and/or the
corresponding lactone (Patents FR 2 695 317 and 2 745 713 and
corresponding patents under priority).
[0005] Moreover the inventors have now shown that such compounds
are also able to act at the level of the insulin target cells,
namely the peripheral tissues such as the liver and muscle, by
exercising an activity at the level of the insulin receptor and/or
the cascade of signalling that the activation of this receptor
triggers. The effect observed in these peripheral tissues
corresponds overall to an insulin mimetic or insulin-sensitizing
effect.
[0006] The work carried out, as illustrated by the examples, has in
fact shown the effect of these compounds at the level of the
phosphorylation cascade set in motion under the receptor, notably
the increase of phosphorylation of the main proteins involved in
the transmission of the insulin signal. The PI 3-kinase
(Phosphatidylinositol 3-kinase) enzyme activated by phosphorylated
IRS-1 and playing an essential role in the translocation of the
glucose transporter GLUT 4, is also increased under the effect of
such compounds. Under the same conditions of use of these
compounds, the phosphatase activity associated with the signalling
route of the insulin receptor (PTP activity) is lowered, which is
consistent with the increase of phosphorylations mentioned above.
These compounds are therefore capable of acting on the signalling
routes which are normally triggered by insulin, whether it is by
activation of kinases, and/or inhibition of phophatases.
[0007] A subject of the present invention is to benefit from the
results obtained, and thus relates to any use of such amino acids
or their derivatives, as insulin mimetic or insulin-sensitizing
agents. It in particular relates to any use of such compounds for
the manufacture of medicaments with insulin mimetic and/or
insulin-sensitizing effects. These effects can be observed in the
peripheral target tissues of insulin. As these compounds act on the
signalling routes which are normally triggered by insulin, they can
in fact serve as substitutes, complements, potentializers and
sensitizers to insulin.
[0008] The present application thus relates to the use as an
insulin mimetic or insulin-sensitizing agent, of any amino acid or
amino acid derivative which exercises a reduction in the
phosphatase PTP activity and/or an increase of PI 3-kinase
activity, of an equivalent or even higher level to the reduction,
or increase respectively, caused by insulin. Any means which makes
it possible to note such an effect on the reduction in PTP activity
or increase in PI 3-kinase activity is appropriate. The following
examples give illustrations.
[0009] Such compounds correspond in a remarkable manner to amino
acids which do not recognise the insulin receptor on its binding
site, but which act at the post receptor level, under the
conditions mentioned in the examples for 4-OH-Ile, and also
correspond to the derivatives of these amino acids which have
retained, at least in kind, the properties of non-recognition of
the insulin receptor on its binding site and activity at
post-receptor level that the parent amino acid of said derivative
presents.
[0010] Advantageously, the use according to the invention is
characterised in that it relates to a compound chosen from the
group constituted by mono-hydroxylated amino acids,
polyhydroxylated amino acids, and lactonic forms of these mono- or
hydroxylated amino acids.
[0011] In particular the invention relates to the use of
4-hydroxyisoleucine of formula
##STR00001##
and/or its lactonic form.
[0012] In particular, the invention relates to the use of
4-hydroxyisoleucine (abbreviated to 4-OH-Ile) in the form of its
2S, 3R, 4S isomer, or the corresponding lactone.
[0013] Taking into account the effects observed, the medicaments
manufactured in accordance with the invention are particularly
suitable for treating insulin resistance, for combating or
preventing syndromes linked to insulin resistance, and for
preventing insulin resistance.
[0014] Moreover, it is known that excessive weight gain, lack of
exercise, poor diet and the ever increasing number of elderly
people are socioeconomic factors frequently encountered in western
countries. All contribute to the development of insulin resistance
and compensatory hyperinsulinemia often combined with obesity and
are potentially diabetogenic. Thus, the free fatty acids are
amongst the first candidates put forNard to attempt to explain the
close relationship between insulin resistance, obesity and
hyperinsulinism (Mac Garry J. D., J. Cell. Biochem., 555, 29,
1994).
[0015] Today this relationship has become a major phenomenon as
regards public health. A body of proof, both clinical and
epidemiological, linking hyperinsulinemia to the risk of future
cardiovascular diseases, athergenic or diabetogenic risk, has made
it possible to draw up the consistent Reaven's X syndrome table
(hyperinsulinemia, insulin resistance, increase of serum
triglycerides, arterial hypertension) and the morbid risks which
are associated with it (Reaven G. M., Diabetes, 37, 1595, 1988). In
addition, according to recent investigations, hyperinsulinemia by
encouraging the proliferation of certain epithelial cells (notably
those in the colon) appear to be associated with the risk of cancer
(Hu F. B. et al., J. Natl. Cancer Inst., 91, 542, 1999).
[0016] The present application also relates to any use of said
amino acids and derivatives for the manufacture of a medicament
intended to combat insulin resistance and insulin resistance
syndromes, in particular against hyperinsulinemia, insulin
resistance linked to ageing and against illnesses linked with
obesity.
[0017] The present application also relates to any use of said
amino acids and derivatives for the manufacture of a medicament
intended to prevent insulin resistances, and in particular for the
manufacture of a medicament intended to reduce the need for
exogenic insulin. Such medicaments, because of their insulin
mimetic and insulin-sensitizing properties, can in fact have the
effect of reducing the need for exogenic insulin that patients
whose endogenic insulin is deficient, even absent in the case of
type 1 diabetes present. Patients suffering from type 1 diabetes
suffer in fact from a total absence of the secretion of insulin
(destruction of producing cells) which restricts full exogenic
supplies of insulin. This situation, besides the intrinsic cost of
such administration, often leads to the development of insulin
resistance. In order to prevent and remedy these problems, for the
treatment of a deficit in endogenic insulin, and in particular an
absence of endogenic insulin such as type 1 diabetes, and for the
manufacture of a medicament intended for such treatments, the
present invention proposes to use at least one of said amino acids
or derivatives. Used in combination with insulin, they have the
advantage of reducing the need for exogenic insulin in the patient
(reduction of the necessary supply of insulin), and therefore
reduce the cost of treatment whilst preventing the development of
insulin resistance and its side effects.
[0018] The medicaments manufactured in accordance with the
invention could also be used to contribute to the inhibition of the
proliferation of certain cell lines associated with the risk of
cancer appearing.
[0019] The invention also relates to the use of said derivatives,
in particular 4-hydroxyisoleucine and/or its lactonic form to
manufacture medicaments which act by reducing the phosphatase
activity associated with the signalling route of the insulin
receptor, and/or by stimulating the PI 3-kinase activity on IRS-1
and/or IRS-2.
[0020] The present invention also relates to any pharmaceutical
composition, any pharmaceutical kit and any medicament comprising,
in a combined fashion, insulin and at least one of the amino acid
compounds or derivatives defined above. This combination can be
physical (insulin and amino acid or derivative are thus in the same
composition). Or alternatively it can correspond to a presentation
of insulin on one hand, and amino acid or derivative on the other
hand, in physically distinct compositions, but which are presented
as combined for combined use (kit-of-parts). This combined use can
be simultaneous or at different times.
[0021] The medicaments according to the invention can be
administered mainly by oral route, but also by intravenous or
intramuscular route, and contain excipients which are chosen
according to the adopted galenic form.
[0022] The dosage will be adapted according to the pathology to be
treated.
[0023] Other characteristics and advantages of the invention are
given, by way of illustration, in the examples which follow, in
which reference is made to FIGS. 2 to 11 (FIG. 1 was referred to
above and shows the diagram of the main routes of transduction of
the insulin signal), these FIGS. 2 to 11 represent:
[0024] FIG. 2, the effect of 4-OH-Ile on the phosphorylation of the
insulin receptor and its substrate in rat liver,
[0025] FIG. 3, the effect of 4-OH-Ile on the PI 3-kinase activity
of the liver,
[0026] FIG. 4, the bonding of insulin on its receptor in hepatic
LMH cell lines,
respectively,
[0027] FIGS. 5 to 11, the effect of 4-OH-Ile on, [0028] the PI
3-kinase activity of the muscle (FIG. 5), [0029] basal insulinemia
and glycemia in rats (FIG. 6), [0030] the PI 3-kinase activity of
the liver (FIG. 7) and of the muscle (FIG. 8) in type 2 diabetic
rats, [0031] the PI 3-kinase activity of the obese Zucker rat
(fa/fa) (FIG. 9), [0032] the PI 3-kinase activity associated with
the PDGF receptor or to the insulin receptor in the liver of a
normal rat (FIG. 10), and [0033] the phosphatase activity
associated with IRS-1 in the liver of a normal rat (FIG. 11).
Measurement of the PI 3-Kinase Activity
[0034] The PI 3-kinase activity was measured on immunoprecipitates
carried out with the anti-IRS-1 antibody. This made it possible to
determine the enzymatic activity associated with the action of
insulin and to compare it to the action induced by 4-OH-Ile.
EXPERIMENTAL PROTOCOL
[0035] Normal male Wistar rats (IFFA CREDO strain, France)
received, via intraperitoneal injection, either ordinary insulin on
its own (100 U/kg) or 4-OH-Ile on its own (18 mg/kg), or insulin
combined with 4-OH-Ile in the same doses. The control rats received
sodium chloride at 9 .Salinity. via intraperitoneal route. Fifteen
minutes after the injection, the animals were sacrificed and the
peripheral tissues (liver, muscle) were immediately removed and
frozen in liquid nitrogen.
[0036] For the determination of the PI 3-kinase activity, the
tissues are ground in a buffer containing protease and phosphatase
inhibitors as well as a solubilising agent (Triton), as described
by Taouis et al., J. Biol. Chem., 269, 14912, 1994. After
solubilisation, the supernatants are immunoprecipitated with
anti-IRS-1 and the PI 3-kinase activity is measured in the
immunoprecipitate. In fact, the reaction is initiated by the
addition of an artificial substrate of the enzyme:
phosphatidylinositol (PI) and (.sup.33P) gamma ATP. The product of
the reaction is subjected to thin layer chromatography (TLC plate)
and the levels of phosphorylation of PI are measured by a STORM
phospho-imager (Molecular Dynamics). The activity is expressed in
arbitrary units given by the apparatus (conversion of the
radioactivity to luminescence by means of a laser beam).
Measurement of the Phosphatase Activity Associated with the Insulin
Signaling Route
[0037] The phosphatase activity was measured on the
immunoprecipitates carried out with the anti-IRS-1 antibody and
insulin anti-receptor.
[0038] The solubilisation protocol is the same as that for PI
3-kinase following the methodology described by Taouis et al. (J.
Biol. Chem., 269, 14912, 1994). After immunoprecipitation, the
phosphatase activity was measured according to the method described
by Chen. et al. (J. Biol. Chem., 272, 8026, 1997).
Example 1
Comparison of the Effects of Insulin and 4-OH-Ile on the
Phosphorylation of the Insulin Receptor and IRS-1 in the Liver of
Normal Rats
[0039] The results in FIG. 2 clearly show that the treatment of
animals by a single injection of 4-OH-Ile (200 .mu.g/kg I.P.)
induces the activation of the insulin receptor (IR) and its
substrate (IRS-1) in vivo. The effect of 4-OH-Ile (40H) is
comparable to that of insulin (Ins).
[0040] These results show the insulin mimetic effects of 4-OH-Ile
in the activation of the phosphorylation of the insulin receptor
and IRS-1 the phosphorylation of which is indispensable for the
activation of effector proteins such as PI 3-kinase.
Example 2
Comparison of the Effects of Insulin and 4-OH-Ile on the PI
3-Kinase Activity of the Liver
[0041] The results in histograms are given in FIG. 3. In this
diagram, it can be seen that insulin but also 4-OH-Ile on their
own, significantly stimulate (p<0.05) the hepatic PI 3-kinase
activity. When the two substances are administered together, a much
greater effect is apparent.
[0042] These observations demonstrate that 4-OH-Ile has insulin
mimetic effects in the liver. In addition, the greater effect
objectivized when the two substances are injected jointly
encourages synergetic activation mechanisms.
[0043] This is confirmed by the comparative study of the effects of
insulin and 4-hydroxyisoleucine on the insulin membrane receptor of
the hepatocyte: the 4-hydroxyisoleucine has no bond with this
receptor.
[0044] In FIG. 4 the comparative results concerning the bonding of
isoleucine and 4-OH-Ile are given. The LMH cells used in these
trials are from chicken hepatocarcinoma (Kawaguchi T. et al.,
Cancer Res., 47, 4460, 1987).
Example 3
Comparison of the Effects of Insulin and 4-OH-Ile on the Pi
3-Kinase Activity of Muscle
[0045] The Results in Histograms are Shown in FIG. 5. In This
Diagram, it can be noted that not only insulin clearly stimulates
the PI 3-kinase activity, but also that 4-OH-Ile has a comparable
effect. Thus, the insulin mimetic effects of 40H-Ile observed at
hepatic level are confirmed at muscular level.
[0046] It is noted therefore in the examination of the results of
Examples 1 and 2 that at liver and muscle level,
4-hydroxyisoleucine stimulates, independently from insulin, the
IRS-1/PI 3-kinase route which is the major route in the control of
metabolic and mitogenic actions of insulin. In addition,
4-hydroxyisoleucine does not recognise the insulin receptor, or at
least does not enter into competition with insulin in binding to
the receptor.
Example 4
[0047] The beneficial effect of 4-hydroxyisoleucine against
relative hyperinsulinemia has been researched in vivo during
chronic administration (for one month) of plant amino acid (25
mg/kg/day, by intraperitoneal route) on rats made non insulin
dependant diabetic (type 2 diabetes) by joint injections of
nicotinamide and streptozotocin (Masiello et al., Diabetes, 47,
224, 1998). Regular blood samples are taken from the caudal vein of
the rats made it possible to evaluate the level of plasmatic
insulin using a radio-immunological method (Herbert et al., J.
Clin. Endocr., 25. 1375, 1965), 15 hours after injection of the
product which took place at 5.30 pm. In these same samples, the
plasmatic glucose level was measured using an enzymatic method
(Trinder P., J. Clin. Pathol. 22, 158, 1969).
[0048] The results obtained are shown in FIG. 6.
[0049] It is observed that the daily administration of
4-hydroxyisoleucine has the effect of significantly lowering
(p>0.05) the insulinemia of the animals treated.
[0050] In addition, after cessation of the treatment, it can be
noted that the plasmatic insulin again rises to regain values close
to those observed before the treatment. Under these conditions, a
slight reduction in glycemia was observed at the end of the
treatment.
[0051] The lowering of insulinemia observed in the rat after a
chronic treatment with 4-hydroxyisoleucine confirms in vivo the
insulin mimetic and/or insulin-sensitizing effects observed during
experiments in vitro.
Example 5
Comparison of the Effects of Insulin and Those of 4-OH-Ile on the
PI 3-Kinase Activity of the Muscle and the Liver Removed from Rats
with Type 2 Diabetes (Masiello et al., Diabetes 47, 224, 1998)
[0052] The results in histograms are shown in FIGS. 7 and 8. It is
noted that, if insulin activates hepatic PI 3-kinase (FIG. 7) and
muscular PI 3-kinase (FIG. 8), it is the same for the 4-OH-Ile.
4-OH-Ile in this pathological situation induces the same effect as
insulin with the same intensity. In addition, it is noticed that
4-OH-Ile significantly stimulates the IRS-1/PI 3-kinase route more
clearly in the muscle (p<0.01) than in the liver
(p<0.05).
Example 6
[0053] During chronic administration for 4 weeks of 4-OH-Ile (50
mg/kg by intraperitoneal route) in obese Zucker rats (fa/fa), the
PI 3-kinase activity of the liver was measured at the end of the
treatment. The results in histograms are shown in FIG. 9. They show
that the basal activity of PI 3-kinase (animals sacrificed 17 hours
after the last administration of 4-OH-Ile) is increased in the
animals treated.
Example 7
[0054] The 4-OH-Ile activates the PI 3-kinase associated with the
insulin receptor but not that associated with the PDGF receptor
(Platelet Derived Growth Factor).
[0055] The PI 3-kinase activities of the PDGF receptor and of the
insulin receptor in the presence or the absence of 4-OH-Ile (18
mg/kg I.P.) were compared following the protocol already
described.
[0056] The results in histograms are shown in FIG. 10: they clearly
show the specificity of the route of action of 4-OH-Ile at the
level of the rat liver, that is to say that the PI 3-kinase
activity associated with the insulin receptor is the only one
increased (p<0.05).
Example 8
Effect of 4-OH-Ile on the Phosphatase Activity Associated with the
Signaling Route of the Insulin Receptor
[0057] With the aim of better locating the action site of the
4-OH-Ile, its impact on phosphatase activity was studied. The
phosphatase activity associated with IRS-1 was measured.
[0058] FIG. 11 shows that 4-OH-Ile (200 .mu.mol/1) significantly
inhibits (p<0.05) this activity in the liver of a normal
rat.
Example 9
[0059] A medicament according to the invention is particularly
suitable for the symptomatic treatment of insulin resistant states,
in particular insulin resistant states combined with obesity. It
can be prepared from a mono- or polyhydroxylated amino acid and/or
its lactonic forms using any appropriate technique known to a
person skilled in the art. It can, in particular, be made from
4-hydroxyisoleucine. This product being hydrosoluble, such a
medicament can easily be made in the form of a solution (in
physiological serum for example), or in solid galenic form such as
tablets or capsules. The pathologies targeted being chronic,
administration by oral route appeared to be the most suitable. Such
medicaments can thus be easily administered in multiple daily
doses, adapted to the individual case of the patient involved, for
example in the order of 2 to 3 times per day. A medicament
according to the invention can also comprise insulin.
* * * * *