U.S. patent application number 11/819659 was filed with the patent office on 2008-02-28 for medicament for treatment of non-insulin dependent diabetes mellitus, hypertension and/or metabolic syndrome.
Invention is credited to Soren Gregersen, Kjeld Hermansen, Per Bendix Jeppesen.
Application Number | 20080051341 11/819659 |
Document ID | / |
Family ID | 8159060 |
Filed Date | 2008-02-28 |
United States Patent
Application |
20080051341 |
Kind Code |
A1 |
Hermansen; Kjeld ; et
al. |
February 28, 2008 |
Medicament for treatment of non-insulin dependent diabetes
mellitus, hypertension and/or metabolic syndrome
Abstract
A substance including the chemical structures of
bicyclo[3.2.1]octan or the chemical structures of kaurene for the
use in a dietary supplementation or as a constituent in a
medicament for the treatment of non-insulin dependent diabetes
mellitus, hypertension and/or the metabolic syndrome. The unique
chemical structures of bicyclo[3.2.1]octan alone or in a kaurene
structure provides the substances, such as e.g. steviol, isosteviol
and stevioside with the capability of enhancing or potentiating the
secretion of insulin in a plasma glucose dependent manner. The
substances including these unique chemical structures also have the
capability of reducing the glucagon concentration in the blood
and/or lowering the blood pressure thereby providing a
self-regulatory treatment system for non-insulin dependent diabetes
mellitus and/or hypertension. In a combination drug which also
comprise a soy protein, and/or soy fiber and/or at least one
isoflavone these substances act synergistically and such
combination drugs are highly useful both prophylacticly or directly
in the treatment of e.g. the metabolic syndrome and obesity and has
due to the self-regulatory effect a widespread applicability as a
dietary supplementation.
Inventors: |
Hermansen; Kjeld; (Ega,
DK) ; Gregersen; Soren; (Ega, DK) ; Jeppesen;
Per Bendix; (Ega, DK) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Family ID: |
8159060 |
Appl. No.: |
11/819659 |
Filed: |
June 28, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10933297 |
Sep 3, 2004 |
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11819659 |
Jun 28, 2007 |
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10210787 |
Jul 31, 2002 |
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10933297 |
Sep 3, 2004 |
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PCT/DK01/00075 |
Feb 1, 2001 |
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10210787 |
Jul 31, 2002 |
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Current U.S.
Class: |
424/401 ;
514/15.7; 514/34; 514/456; 514/557; 514/6.7; 514/6.9; 514/766 |
Current CPC
Class: |
A61K 36/00 20130101;
A61K 36/48 20130101; A61K 38/16 20130101; A61K 31/19 20130101; A61K
31/352 20130101; A61K 38/168 20130101; A61K 45/06 20130101; A61P
5/50 20180101; A61K 31/704 20130101; A61P 3/10 20180101; A61P 3/00
20180101; A61K 31/015 20130101; C07C 13/68 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; C07C 61/35
20130101; A61K 31/015 20130101; A61P 9/12 20180101; A61K 36/48
20130101; A61K 31/352 20130101; A61K 31/70 20130101; A61P 3/06
20180101; C07C 2603/86 20170501; A61P 3/04 20180101; C07H 15/256
20130101; A61K 31/192 20130101 |
Class at
Publication: |
514/012 ;
514/034; 514/456; 514/557; 514/766 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 31/015 20060101 A61K031/015; A61K 31/192 20060101
A61K031/192; A61P 3/00 20060101 A61P003/00; A61P 9/12 20060101
A61P009/12; A61P 3/10 20060101 A61P003/10; A61K 31/352 20060101
A61K031/352; A61K 31/70 20060101 A61K031/70 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 1, 2000 |
DK |
PA 2000 00163 |
Claims
1. A pharmaceutically acceptable composition comprising a substance
including a bicyclo[3.2.1]octan in a double ring system having a
basic chemical skeleton of kaurene structure with the structural
formula II ##STR4##
2. A composition according to claim 1 wherein the chemical
structure having the structural formula II provides the substance
with the capability of enhancing or potentiating the secretion of
insulin.
3. A composition according to claim 1 wherein the substance is
isolatable from a plant source.
4. A method of treating non-insulin dependent diabetes mellitus,
hypertension and/or the metabolic syndrome which comprises
administering to a subject in need thereof a therapeutically
effective amount of the composition of claim 1.
5. The method of claim 4, wherein the substance is selected from
the group consisting of steviol, isosteviol, glucosilsteviol,
gymnemic acid, steviolbioside, stevioside, Rebaudioside A,
Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E and
Dulcoside A.
6. The method of claim 4 wherein the subject is afflicted with
non-insulin dependent diabetes mellitus and wherein insulin
secretion is stimulated in the subject, the stimulation of the
insulin secretion being initiated by the presence of a plasma
glucose concentration of 6 mmol/l or larger.
7. A method of reducing the glucagon concentration in the blood
which comprises administering, to a subject in need thereof, a
glucagon-reducing amount of the composition of claim 1.
8. A method of lowering the blood pressure which comprises
administering, to a subject in need thereof, a blood pressure
reducing amount of the composition of claim 1.
9. The composition of claim 1, further comprising the claims at
least one soy protein.
10. The composition of claim 9, further comprising at least one
isoflavone.
11. The method of claim 4, said composition further comprising at
least one soy protein.
12. A method for the treatment of obesity, overweight or
dyslipidemia, which comprises administering to a subject in need
thereof a therapeutically effective amount of the composition of
claim 1.
13. A method of dietary supplementation which comprises
supplementing the diet of a subject with a composition comprising a
double ring system having a basic chemical skeleton of kaurene
structure with the structural formula II ##STR5##
14. The method of claim 13 wherein the dietary supplementation
further comprises supplementation with at least one soy protein
and/or at least one isoflavone.
15. The method of claim 13 wherein the substance is isolatable from
a plant source.
16. The method of claim 13 wherein the substance is selected from
the group consisting of steviol, isosteviol, glucosilsteviol,
gymnemic acid, steviolbioside, stevioside, Rebaudioside A,
Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E and
Dulcoside A.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 10/933,297 filed Sep. 3, 2004, which is a division of
10/210,787 filed Jul. 31, 2002, which is a continuation of
PCT/DK01/00075 filed Feb. 1, 2001. The prior applications set forth
above are hereby incorporated by reference in their entirety.
TECHNICAL FIELD
[0002] The present invention relates to a new medicament for the
treatment of non-insulin dependent diabetes mellitus, hypertension,
metabolic syndromes and other conditions in mammals.
BACKGROUND ART
[0003] Diabetes is a common disease that has a prevalence of 2-4%
in the population. Non-insulin dependent diabetes mellitus
comprises about 85% of diabetes most commonly occurring at the age
above 40 years. The incidence of non-insulin dependent diabetes
mellitus is increasing and is at a global level expected to surpass
200 million subjects at year 2010.
[0004] Diabetes is associated with increased morbidity and a
2-4-fold increase in mortality primarily due to cardiovascular
diseases and strokes.
[0005] Non-insulin dependent diabetes mellitus develops especially
in subjects with insulin resistance and a cluster of cardiovascular
risk factors such as obesity, hypertension and dyslipidemia, a
syndrome which first recently has been recognized and is named "The
metabolic syndrome" (Alberti K. G., Zimmet P. Z.; Definition,
diagnosis and classification of diabetes mellitus and its
complications". Part 1: Diagnosis and classification of diabetes
mellitus provisional report of a WHO consultation. Diabet. Med.
1998 July; 15(7), p. 539-53).
[0006] In accordance with the WHO-definition
(www.idi.org.au/whoreport.htm), a patient has metabolic syndrome if
insulin resistance and/or glucose intolerance is present together
with two or more of the following conditions: [0007] reduced
glucose tolerance or diabetes [0008] insulin sensivity (under
hyperinsulinaemic, euglycaemic conditions corresponding to a
glucose uptake below the lower quartile for the background
population) [0009] increased blood pressure (.gtoreq.140/90 mmHg)
[0010] increased plasma triglyceride (.gtoreq.1.7 mmol/l) and/or
low HDL cholesterol (<0.9 mmol/l for men; <1.0 mmol/l for
women) [0011] central adipositas (waist/hip ratio for men: >0.90
and for women >0.85) and/or Body Mass Index >30 kg/m.sup.2)
[0012] micro albuminuria (urine albumin excretion: .gtoreq.20 .mu.g
min.sup.-1 or albumin/creatinine ratio .gtoreq.2.0 mg/mmol.
[0013] It has become increasingly evident that the treatment should
aim at simultaneously normalizing blood glucose, blood pressure,
lipids and body weight to reduce the morbidity and mortality. Diet
treatment, exercise and avoiding smoking are the first treatment
modalities that should be started. However, it will often be
necessary to add pharmacological therapy but until today no single
drug that simultaneously attacks hyperglycaemia, hypertension and
dyslipidemia are available for patients with metabolic syndrome.
Instead, these patients may be treated with a combination of
several different drugs in addition to e.g., diet. This type of
treatment is difficult to adjust and administer to the patient and
such treatment may result in many unwanted adverse effects which in
themselves may need medical treatment.
[0014] Consequently there is a long felt need for a new and
combined medicament for the treatment of metabolic syndrome thereby
also preventing an increase in the number of persons developing the
non-insulin dependent diabetes mellitus.
[0015] Existing oral antidiabetic medicaments to be used in such
treatment include the classic insulinotropic agents sulphonylureas
(Lebovitz H. E. 1997. "The oral hypoglycemic agents". In: Ellenberg
and Rifkin's Diabetes Mellitus. D. J. Porte and R. S. Sherwin,
Editors: Appleton and Lange, p. 761-788). They act primarily by
stimulating the sulphonylurea-receptor on the insulin producing
beta-cells via closure of the K.sup.+.sub.ATP-sensitive channels.
However if such an action also affects the myocytes in the heart,
an increased risk of cardiac arrhythmias might be present. Also, it
is well know in the art that sulphonylureas can cause severe and
lifethreatening hypoglycemia, due to their continuous action as
long as they are present in the blood.
[0016] Consumption of soy protein rather than animal protein has
been found to lower blood cholesterol (Anderson J. W., Johnstone B.
M., Cook-Newell M. E.: Meta-analysis of the effects of soy protein
intake on serum lipids. N. Engl. J. Med. 1995; 333; p. 276-282). In
addition to this knowledge, recent research also provides evidence
that soy protein and/or isoflavones may improve endothelial
function and attenuate events leading to both lesion and thrombus
formation (Anderson J. W., Johnstone B. M., Cook-Newell M. E.:
"Meta-analysis of the effects of soy protein intake on serum
lipids"; N. Engl. J. Med. 1995; 333; p. 276-282; Potter S. M., Soy
protein and cardiovascular disease: "The impact of bioactive
components in soy". Nutrition Reviews 1998; 56, p. 231-235).
[0017] Several attempts to develop new antidiabetic agents and
drugs for the treatment or prophylacetic treatment of the syndrome
not having the adverse effects mentioned above, e.g. hypoglycemia
and potential harmful actions on the heart functions have been made
over the years. For this purpose, plants provide a vast resource of
compounds with the potential to become new antidiabetic agents.
[0018] For instance extracts of the leaves of Stevia rebaudiana
Bertoni, a herbaceous member of the Compositae family, have been
used for many years in the treatment of diabetes among Indians in
Paraguay and Brazil (Sakaguschi M., Kan P Aspesquisas japonesas com
Stevia rebaudiana (Bert) Bertoni e o estevioside. Cienc. Cultur.
34; p. 235-248, 1982; Oviedo C. A., Franciani G., Moreno R., et al.
"Action hipoglucemiante de la Stevia Rebaudiana Bertoni
(Kaa-he-e)". Excerpt. Med. 209, p. 92, 1979; Curi R., Alvarez M.,
Bazotte R. B., et al. Effect of Stevia rebaudiana on glucose
tolerance in normal adult humans. Braz. J. Med. Biol. Res., 19, p.
771-774, 1986; Hansson J. R., Oliveira B. H., "Stevioside and
related sweet diterpenoid glycoside". Nat. Prod. Rep. 21, p.
301-309, 1993).
[0019] Also, an antihyperglycemic effect has been found in rats
when supplementing the diet with dried S. rebaudiana leaves (Oviedo
C. A., Franciani G., Moreno R., et al. "Action hipoglucemiante de
la Stevia Rebaudiana Bertoni (Kaa-he-e)". Excerpt. Med. 209:92,
1979). Curi et al. found a slight suppression of plasma glucose
when extracts of Stevia rebaudiana leaves were taken orally during
a 3-day period. Furthermore, Oviedo et al. reported that tea
prepared from the leaves caused a 35% reduction in blood-glucose in
man.
[0020] A number of Stevia species have been examined and shown to
contain labdanes, clerodanes, kaurenes and beyerenes (Hansson J.
R., Oliveira B. H., "Stevioside and related sweet diterpenoid
glycoside". Nat. Prod. Rep. 21, p. 301-309, 1993). Any of these
substances as well as many others unidentified substances in the
leaves could be responsible for the reduction in blood glucose in
man.
[0021] In the work of Malaisse W. J. et al. (Malaisse W. J.,
Vanonderbergen A., Louchami K, Jijakli H. and Malaisse-Lagae F.,
"Effects of Artificial Sweeteners on Insulin Release and Cationic
Fluxes in Rat Pancreatic Islets", Cell. Signal. Vol 10, No. 10, p.
727-733, 1998) the effect of several artificial sweeteners,
including stevioside, on insulin release from isolated normal
pancreatic rat islets were studied. In this study it was reported
that in the presence of 7 mmol/l D-glucose, stevioside in a
concentration of 1.0 mmol/l caused a significant increase in
insulin output. Also the control group demonstrated a significant
increase in insulin output of about 16 times above the basal
release value in the presence of 20 mmol/l D-glucose increase. It
is therefore uncertain whether the insulin releasing effect is due
to the increased glucose level or the presence of stevioside. No
diabetic islet cells were studied and the skilled person within the
art will know that the mechanism for stimulating normal pancreatic
islet cells either not functions at its optimum or not functions at
all in the diabetic pancreatic cells, and that the study provided
no certain indication of the possible use of stevioside in the
treatment of non-insulin dependent diabetes mellitus, hypertension
and/or the metabolic syndrome.
[0022] In a Chinese study (Lin Qi-Xian, Cao Hai-Xing, Xie Dong, Li
Xing-Ming, Shang Ting-Lan, Chen Ya-Sen, Ju Rui-Fen, Dong Li-Li,
Wang Ye-Wen, Quian Bao-Gong, "Experiment of Extraction of
Stevioside", Chinese Journal og Pharmaceuticals 1991, No. 22, p
389-390) is indicated a method for extracting stevioside from
stevioside leafs from the origin of Bingzzhou in the Hunan
Province. The content of stevioside in the extract was determined
using HPLC although the article is silent of the purity of the
extract. The produced stevioside tablets were for no apparent
reason and medical indication applied to patients in the Wuhan
Second Hospital. No data on the influence of stevioside on blood
glucose, insulin and/or blood pressure is revealed. It is stated
that the tablets were effective to diabetes and hypertension during
preliminary clinical observations. However, total lack of data on
blood glucose, insulin and/or blood pressure i.e., lack of support
by test results and the missing information of which types of
diabetes that were treated, makes this an unsupported and
unconfirmed assertion.
[0023] Any detailed information of which substance or substances in
the leaves that might cause a possible anti-hyperglycemic effect
has not yet been disclosed for certainty, and the mechanism of how
and to which extent the plasma glucose is reduced is unknown. The
above mentioned articles and studies are concerned with the initial
discovery of the effects and provide no evidence of which specific
component(s) in the leaves that might be the active one(s).
[0024] The effect of intravenous stevioside on the blood pressure
was studied in spontaneously hypertensive rats ("The Effect of
Stevioside on Blood Pressure and Plasma Catecholamines in
Spontaneously Hypertensive Rats", Paul Chan, De-Yi Xu, Ju-Chi Liu,
yl-Jen Chen, Brian Tomlinson, Wen-Pin Huang, Juei-Tang Cheng, Life
Science, Vol. 63, No. 19, 1998, p. 1679-1684). The study showed
that during an intravenously administration of stevioside of 200
mg/kg the hypotensive effect was at a maximum, but although
reported as being significantly the fall in the systolic blood
pressure was only small. Neither the heart rate nor the plasma
catecholamines were significantly changed during the observation
period. This study indicated that stevioside advantageously could
be used for treating hypertension.
[0025] No reports of an effect on plasma glucagon level have
previously been reported. Glucagon, a pancreatic islet hormone,
acts as a diabetogenic hormone by increasing the hepatic glucose
output thereby elevating blood glucose.
[0026] Recent studies and tests made by the present inventors have
focused on especially the diterpenoid glycoside stevioside which is
a major constituent found in the leaves of Stevia rebaudiana where
it may occur in amounts of up to about 10% (Hansson J. R., Oliveira
B. H., "Stevioside and related sweet diterpenoid glycoside". Nat.
Prod. Rep. 21, p. 301-309, 1993; Bridel M., Lavielle R.,
Physiologie Vegetale: "Sur le principe sucre'du Kaa' he'e (Stevia
rebaudiana Bertoni): II Les produits d'hydrolyse diastasique du
stevioside, glucose et steviol". Acad. Sci. Paris 192, p.
1123-1125, 1931; Soejarto D. D., Kinghorn A. D., Farnsworth N. R.,
Potential sweetening agent of plant origin. III: "Organoleptic
evaluation of Stevia leaf herbarium samples for sweetness". J. Nat.
Prod. 45, p. 590-598, 1983; Mossettig E., Nes W. E. Stevioside. II:
"The structure of the aglucone"; J. Org. Chem. 20, p. 884-899,
1955; Kohda H., Hasai R., Yamasaki K. et al. "New sweet diterpene
glucosides from Stevia rebaudiana". Phytochemistry 15, p. 981-983,
1976).
[0027] Also, its aglycone, steviol, has been found to be contained
in the leaves of Stevia rebaudiana as well as other sweet-tasting
glycosides e.g. Steviolbioside, Rebaudioside A, B, C, D and E, and
Dulcoside (Bridel M., Lavielle R., Physiologie Vegetale: "Sur le
principe sucre'du Kaa' he'e (Stevia rebaudiana Bertoni): II Les
produits d'hydrolyse diastasique du stevioside, glucose et
steviol". Acad. Sci. Paris 192, p. 1123-1125, 1931; Soejarto D. D.,
Kinghorn A. D., Farnsworth N. R., Potential sweetening agent of
plant origin. III: "Organoleptic evaluation of Stevia leaf
herbarium samples for sweetness". J. Nat. Prod. 45, p. 590-598,
1983; Mossettig E., Nes W. E. Stevioside. II: "The structure of the
aglucone"; J. Org. Chem. 20, p. 884-899, 1955; Mossettig E., Nes W.
E. Stevioside. II: "The structure of the aglucone"; J. Org. Chem.
20, p. 884-899, 1955; Kohda H., Hasai R., Yamasaki K. et al. "New
sweet diterpene glucosides from Stevia rebaudiana". Phytochemistry
15, p. 981-983, 1976).
[0028] The present inventors have already successfully proved that
both stevioside and steviol have an anti-hyperglycemic,
glucagonostatic and insulinotropic effect when administered
intravenously to rats and a stimulatory effect on the insulin
secretion from mouse islets in vitro.
[0029] No well defined, chemical stable, non-toxic, reliable and
non-adverse effects alternative to the sulphonylureas for the
treatment of non-insulin dependent diabetes mellitus is available
today, however, and these findings have given rise to further
studies and tests of analogues and derivates of these substances in
order to find improved and alternative drugs for a self-regulatory
treatment of diabetes, hypertension and especially metabolic
syndrome in mammals, and preferably in humans.
[0030] In order to prevent sequelae or to delay the developing of a
number of the above-mentioned metabolic and functional disorders in
humans, there is a need it for new and beneficial dietary
supplementations or new self-administrable non-prescription drugs
for prophylaxis. The present invention now satisfies this need.
SUMMARY OF THE INVENTION
[0031] Accordingly, the present invention relates to a selectively
responsive medicament composition comprising at least one substance
including a bicyclo[3.2.1]octan in a double ring system having a
basic chemical skeletal of a kaurene structure having the
structural formula II: ##STR1## or an analogue, derivative or
metabolite thereof, wherein the substance responds only at an
elevated plasma glucose concentrations. Generally, the response of
the substance is initiated by a plasma glucose concentration of 6
mmol/l or larger.
[0032] Preferably, the substance is selected from the group
consisting of steviol, isosteviol, glucosilsteviol, gymnemic acid,
steviolbioside, stevioside Rebaudioside A, Rebaudioside B,
Rebaudioside C, Rebaudioside D, Rebaudioside E and Dulcoside A,
their pharmaceutically acceptable analogues or their
pharmaceutically acceptable derivates. The substance can be
isolated from a plant source and can be used alone or in
combination with at least one soy protein alone or in combination
with at least one isoflavone.
[0033] The substance and composition can be used as a dietary
supplement or as a medicament for a mammal. As noted above the
substance or composition is responsive in the mammal only when the
mammal's plasma glucose concentrations are elevated. Thus, the
medicament can be used for treating the mammal for non-insulin
dependent diabetes mellitus, metabolic syndrome, to stimulate
insulin production, to reduce glucagon concentrations, to suppress
fasting plasma triglycerides or total cholesterol levels in the
mammal, or for treating hypertension in the mammal. Preferably, the
medicament is an oral medicament and is self-regulating.
[0034] The invention also relates to a method of making a
selectively responsive composition which comprises associating with
a carrier a bicyclo[3.2.1]octan in a double ring system having a
basic chemical skeletal of a kaurene structure having the
structural formula II, wherein the substance responds only at an
elevated plasma glucose concentrations. The composition that is
made can be used as a dietary supplement or as one of the
medicaments mentioned above.
[0035] The invention also relates to various treatment methods for
mammals, including treating non-insulin dependent diabetes
mellitus, treating metabolic syndrome, treating hypertension,
suppressing fasting plasma triglycerides, suppressing total
cholesterol level, or suppressing appetite.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] The invention is further illustrated by the following
examples and the accompanying drawings that are intended to
illustrate preferred features and properties of the invention,
wherein:
[0037] FIG. 1 shows the chemical structure of steviol, isosteviol
and stevioside,
[0038] FIG. 2a shows the effect of stevioside on blood glucose
during i.v. glucose tolerance test in normal Wistar rats,
[0039] FIG. 2b shows the effect of stevioside on blood glucose
during i.v. glucose tolerance test in GK rats,
[0040] FIG. 3a shows the effect of stevioside on glucose-induced
release during i.v. glucose tolerance test in normal Wistar
rats,
[0041] FIG. 3b shows the effect of stevioside on glucose-induced
release during i.v. glucose tolerance test in GK rats,
[0042] FIG. 4a shows the effect of stevioside on glucose-stimulated
insulin secretion from isolated mouse islets,
[0043] FIG. 4b shows the effect of steviol on glucose-stimulated
insulin secretion from isolated mouse islets,
[0044] FIG. 5a shows the effect of an i.v. bolus injection of
glucose on plasma glucagon levels during an intravenous glucose
tolerance test in GK rats,
[0045] FIG. 5b shows the effect of an i.v. bolus injection of
glucose and stevioside on plasma glucagon levels during a glucose
tolerance test in GK rats,
[0046] FIG. 6a shows the systolic blood pressure during 6 weeks
treatment of GK rats with stevioside,
[0047] FIG. 6b shows the diastolic blood pressure in GK rats
treated with stevioside.
[0048] FIG. 7a shows the effect of 10.sup.-3 mmol/l stevioside on
the insulin secretion from isolated mouse islets in the presence of
glucose ranging between 0 and 16.7 mmol/l,
[0049] FIG. 7b shows the effect of 10.sup.-6 mmol/l steviol on the
insulin secretion from isolated mouse islets in the presence of
glucose ranging between 0 and 16.7 mmol/l,
[0050] FIG. 8 a-d shows the acute effects of stevioside in type II
diabetic patients, and
[0051] FIG. 9a-g shows the effects of the action of the combination
of stevioside and soy based dietary supplementation in diabetic
GK-rats.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0052] Careful structural chemistry studies by the inventors have
revealed that all potential substances for stimulating the insulin
secretion extracted from the leaves of Stevia rebaudiana share the
common unique skeletal structure of bicyclo[3.2.1]octan of the
formula I: ##STR2## This bicyclo[3.2.1]octan can be found in e.g.
steviol, isosteviol and in stevioside. The formula I structure has
also been recognised in glucosilsteviol, gymnemic acid,
steviolbioside, Rebaudioside A, Rebaudioside B, Rebaudioside C,
Rebaudioside D, Rebaudioside E and Dulcoside A.
[0053] All these substances also share the common structure of
formula II: ##STR3## which is the basic structure in
kaur-16-en-18-oic acid.
[0054] These specific structures of the formula I or II are
recognized in several chemical compounds, which have been shown to
have a highly potent insulin stimulating effect on isolated mouse
pancreatic .beta.-cell, and these structures of formula I and II
are evidently the active parts of the molecules in causing the
stimulating task.
[0055] This assumption is further confirmed by the fact that tests
have shown that steviol having the smallest skeletal structure
stimulate the insulin secretion to a greater extent than e.g. the
glycoside stevioside having a much larger skeletal structure. Also,
the inventors of the present invention have succeeded in purifying
the different Rebaudiosides from Stevia rebaudiana and preclinical
animal studies indicate the same stimulatory effect on insulin
secretion.
[0056] Consequently this indicates that other compounds including
the structures of the formula I or II, such as e.g. analogues,
derivates and metabolites of the compounds mentioned above, can be
used alternatively.
[0057] Studies and tests on rats have disclosed that the insulin
stimulating effect of these substances is dependent on the
concentration of the plasma glucose.
[0058] The substances comprising the chemical structures, which
includes the formula I or II, did not cause an insulin release as
long as the plasma glucose concentration was below approximately 6
mmol/l. At plasma glucose concentration above 6 mmol/l, the
stimulating effect of the compounds provided an elevated plasma
insulin concentration resulting in an immediate suppression of
plasma glucose concentration thereby keeping this at a normal
level.
[0059] In addition to the above findings, the present inventors
have surprisingly found that the substances comprising the chemical
structures including the formula I or II also have the capabilities
of reducing the glucagon concentration in the blood.
[0060] This characteristic nature and qualities of the substances
make them an obvious choice as a component in a medicament for the
treatment of especially non-insulin dependent diabetes mellitus
(NIDDM).
[0061] The finding that e.g. intravenously administered stevioside
inhibited blood glucose responses to intravenous glucose in NIDDM
rats (GK rats) but not in normal rats supports this fact. This
finding is new and surprisingly has neither been expected nor
demonstrated in earlier studies that have only been concerned with
normal pancreatic islet cells.
[0062] As a further example of the unique action of the substances
according to the invention, stevioside infusion at normal blood
glucose did not cause any hypoglycemia irrespective of it being
given as a bolus or at a constant intravenous infusion.
[0063] Due to the insulin secretory stimulating effect induced by a
slightly elevated plasma glucose concentration, the simultaneous
plasma glucagon reducing effect and the inhibited blood glucose
response, these substances are able to control, regulate and adjust
the plasma glucose concentration of a NIDDM patient to a normal
level.
[0064] As a consequence of the glucose-dependency the substances
only act when needed, e.g. after the patient has increased blood
glucose after having eaten. In NIDDM patients treated with
medicaments including these substances hypoglycemia will not occur
and hypoglycemia will be counteracted.
[0065] Therefore, the substances provide a self-regulatory system
responding only at elevated plasma glucose concentration.
[0066] The substances are preferably used in medicaments for oral
medication. When taken orally, the glycosylated substances can be
partially metabolised but the basic skeletal structure of the
formula I or II will not be changed and the different
characteristic effects mentioned above will be preserved.
[0067] The treatment with a medicament including these substances
provides an attractive alternative to different types of drugs
available and presently used today for the treatment of NIDDM, such
drugs being drugs for stimulating the insulin secretion
(sulphonylureas or repaglinide), drugs for improving the insulin
sensivity (biguamides and thiazolidinediones) or drugs for
retarding gastrointestinal carbohydrate absorption
(.alpha.-glucosidase inhibitors).
[0068] The potential of these new substances has for the first time
also been tested in human NIDDM studies and the beneficial and
advantageously combined multiple effects in humans of a single
substance according to the invention has been demonstrated and will
be further described in the examples.
[0069] The above-mentioned human tests have been conducted by
orally administrating the substances, but within the scope of the
invention the substances can optionally be used in the preparation
of medicaments for intravenous, subcutaneous or intramuscular
medication.
[0070] The substances further bring along the blood pressure
reducing effect. In long-term experiments stevioside acutely
suppresses blood pressure in diabetic rat. This important discovery
is of the benefit to the diabetic patients that have developed
hypertension in relation to or besides their disease.
[0071] When at least one of the substances according to the
invention is combined in a medicament also comprising at least one
soy protein alone or in combination with at least one isoflavone,
it is possible to manufacture a combined preparation of a drug for
the treatment of patients with the metabolic syndrome in accordance
with the previously definition. Such a medicament may
advantageously be used in prophylacetic treatment of patient in a
risk group. For example, a slow-release drug on the basis
composition mentioned above provides a convenient treatment for the
patient with the metabolic syndrome.
[0072] The inventors of the present invention have demonstrated
that the combination of the substances according to the invention
and at least one soy protein have a new unexpected and surprisingly
synergistic effect surpassing the additive effect of the single
components of the medicament thereby providing a completely new and
very important medicament for therapeutic or prophylacetic
treatment of the metabolic syndrome.
[0073] The present inventors have used the combination of the
substances according to the invention and at least one soy protein
as a dietary supplementation in human studies. The test results
significantly proved, as will be seen in the following examples,
that such combination has a beneficial impact on cardiovascular
risk markers in type II diabetic subjects.
[0074] Stevioside at a dose as high as 15 g/kg body weight was not
lethal to either mice, rats or hamsters (Toskulkao C., Chaturat L.,
Temcharoen P., Glinsukon T. "Acute toxicity of stevioside, a
natural sweetener, and its metabolite, steviol, in several animal
species". Drug Chem. Toxicol. 1997 February-May; 20(1-2), p.
31-44). In rats and mice, LD.sub.50 values of steviol were higher
than 15 g/kg body weight while the LD.sub.50 for hamsters were 5-6
g/kg body weight. The latter was accompanied with degeneration of
the proximal tubular cells, which correlated to increases in blood
urea nitrogen and creatinine. Stevioside is excreted by the urine
(Melis M. S. "Renal excretion of stevioside in rats". J. Nat. Prod.
1992 May; 55(5), p. 688-90) and is not metabolised in the isolated
perfused rat liver (Ishii-Iwamoto E. L., Bracht A. "Stevioside is
not metabolised in the isolated perfused rat liver". Res. Commun.
Mol. Pathol. Pharmacol. 1995 February; 87(2), p. 167-75).
[0075] Stevioside and steviol showed no mutagenic effect on a
number of Salmonella typhimurium strains (Klongpanichpak S.,
Temcharoen P., Toskulkao C., Apibal S., Glinsukon T. "Lack of
mutagenicity of stevioside and steviol in Salmonella typhimurium TA
98 and TA 100". J. Med. Assoc. That 1997 September; 80 Suppl. 1, p.
121-128; Suttajit M., Vinitketkaumnuen U., Meevatee U., Buddhasukh
D. "Mutagenicity and human chromosomal effect of stevioside, a
sweetener from Stevia rebaudiana Bertoni". Environ Health Perspect
1993 October; 101 Suppl. 3, p. 53-56). In another study, it was
confirmed that stevioside was not mutagenic whereas steviol,
however, produced dose-related positive responses in some
mutagenicity test (Matsui M., Matsui K., Kawasaki Y., Oda Y.,
Noguchi T., Kitagawa Y., Sawada M., Hayashi M., Nohmi T., Yoshihira
K., Ishidate M. Jr., Sofuni T. "Evaluation of the genotoxicity of
stevioside and steviol using six in vitro and one in vivo
mutagenicity assays". Mutagenesis 1996 November; 11(6), p.
573-579).
[0076] Stevioside is not carcinogenic in F344 rats (Toyoda K.,
Matsui H., Shoda T., Uneyama C., Takada K., Takahashi M.
"Assessment of the carcinogenicity of stevioside in F344 rats".
Food Chem. Toxicol. 1997 June; 35(6), p. 597-603). Doses as high as
2.5 g/kg body weight/day had no effect on growth or reproduction in
hamsters (Yodyingyuad V., Bunyawong S. "Effect of stevioside on
growth and reproduction". Hum. Reprod. 1991 January; 6(1), p.
158-165).
[0077] To the knowledge of the inventors, no observations or
reports showing potential toxic effects in humans have been
published.
[0078] It will be recognized by the skilled artisan that rearranged
structures of the formula II are within the scope of the invention,
and such rearrangements might occur naturally in the gastro
intestinal tract. As example can be mentioned that rearrangement
may occur at the C16 forming a double bond to the C15 and thereby
leaving a single bond open for substitution at position 17. A COOH
group at position 18 is open for a number of reactions such as
reaction with alcohol, as well as a number of substituents can be
provided at any point of the formula II structure. Also, other
substituents such as e.g. saccharides, at the various C-atoms and
the structures may be anticipated.
EXAMPLES
[0079] In the following examples, the type II diabetic
Goto-Kakizaki (GK) rats originated from Takeda Chemical Ind.,
Tokyo, Japan and were bred locally.
[0080] The normal wistar rats and the NMRI mice were available from
Bomholtgard Breeding and Research Centre Ltd., Ry, Denmark.
[0081] The rats had a weight of 300-350 g and the mice a weight of
22-25 g. The animals were kept on a standard pellet diet and tap
water ad libitum.
[0082] The stevioside is obtained from the Japanese company
WAKO-TriCHEM.
[0083] The abbreviation IAUC means Incremental Area Under the Curve
(above basal).
Example 1
[0084] As examples of the effects of a compound including the
chemical formulas II, stevioside was tested on normal Wistar rats
and on GK rats. 2.0 g glucose/kg body weight and 0.2 g
stevioside/kg body weight were dissolved in 0.9% saline and infused
intravenously. The plasma glucose and insulin levels were measured
over a period of 2 hours.
[0085] The results are shown in FIGS. 2a, 2b, 3a and 3b, were the
O-O series (n=6 for Wistar and n=14 for GK) illustrate glucose
infused alone and the {circle around (2)}-{circle around (2)}
series (n=6 for Wistar and n=12 for GK) illustrate the combined
glucose and stevioside infusion. Data are given as mean
.+-.SEM.
[0086] After administration of the glucose load, plasma glucose
raised immediately and plasma insulin raised abruptly. When
stevioside was added together with the glucose, a diminished
glucose response was found in the GK-rat and a significant decrease
was observed already after 30 min. In the GK rat, stevioside caused
a pronounced increase in the insulin response compared to the
Wistar rat. The stevioside-induced insulin response was delayed and
increased throughout the whole test. The insulin response was
monophasic.
[0087] This discovery of stevioside having a blood glucose reducing
effect in the type II diabetic rat indicates that stevioside and
compounds having a similar chemical structure can be used in a
medicament for the treatment of NIDDM in man.
Example 2
[0088] Islet from 6-10 NMRI mice were isolated and incubated in the
presence of 16.7 mmol/l and 10.sup.-9-10.sup.-3 mol/l stevioside or
10.sup.-9-10.sup.-3 mol/l steviol.
[0089] The results of these tests are illustrated in FIGS. 4a and
4b where each column represents mean .+-.SEM from 24 incubations of
single islets. Black bars in FIG. 4a indicate that stevioside is
present and hatched bars indicate that stevioside is absent.
[0090] Black bars in FIG. 4b indicate that steviol is present and
hatched bars indicate that steviol is absent.
[0091] The figures show that stevioside and steviol are capable of
potentiating glucose-stimulated insulin secretion. Further tests
confirmed that a stimulatory effect was found already at a very low
concentration (above 0.1 nM).
Example 3
[0092] During a glucose tolerance test, an intravenous bolus of
stevioside of 0.2 g/kg body weight was injected in GK rats (the
{circle around (2)}-{circle around (2)} series (n=6)). GK rats
receiving 0.9% saline intravenously served as controls (the O-O
series (n=6)). Glucose 2.0 g/kg body weight was administered as a
bolus at timepoint 0 min. The plasma glucagon responses are shown
as mean .+-.SEM in FIGS. 5a (control) and 5b (GK). The plasma
glucagon was suppressed in the stevioside treated GK rat.
Example 4
[0093] GK rats were treated with stevioside 0.025 g/kg body
weight/24 h for 6 weeks. Stevioside was administered in the
drinking water. GK rats receiving drinking water with 0.111 g
D-glucose/kg body weight/24 h served as controls. Systolic (FIG.
6a, control: O-O series, stevioside-treated: {circle around
(2)}-{circle around (2)} series) and diastolic (FIG. 6b, control:
O-O series, stevioside-treated: {circle around (2)}-{circle around
(2)} series) blood pressures were measured on the tail.
[0094] The figures show a 10-15% decrease in the blood pressure
detectable after 2 weeks of treatment and the effect hereafter was
stable and consistent during the study period.
Example 5
[0095] The influence of the maximal stimulatory doses of 10.sup.-3
mol/l stevioside and 10.sup.-6 mol/l steviol was studied in NMRI
mouse islets over a range between 0 and 16.7 mmol/l glucose. Both
stevioside (FIG. 7a) and steviol (FIG. 7b) potentiated insulin
secretion at and above 8.3 mmol/l and indicated that the initiating
level for stimulating insulin secretion was between 3.3 mmol/l and
8.3 mmol/l of glucose. Black bars in FIG. 7a indicate that
stevioside is present and hatched bars indicate that stevioside is
absent. Black bars in FIG. 7b indicate that steviol is present and
hatched bars indicate that steviol is absent.
Example 6
[0096] Twenty type II diabetic patients (6 female/14 males) with a
mean age of 63.6.+-.7.5 years participated in a controlled
randomised double blind crossover trial. They were supplemented for
6 weeks with soy protein for (50 g/day) with high levels of
isoflavones (minimum 165 mg/day) and cotyledon fibers (20 g/day) or
placebo (casein 5 .mu.g/day) and cellulose (20 g/day) separated by
a 3 week wash-out period.
[0097] This dietary supplement significantly reduced
LDL-Cholesterol by 10% (p<0.05), LDL/HDL ratio by 12%
(p<0.05), Apo B-100 by 30% (p<0.01), triglycerides by 22%
(p<0.05) and homocystein by 14% (p<0.01). No change was
observed in HDL-Cholesterol, Factor VIIc, von Willebrandt factor,
fibrinogen, PAI-1, HbAlc or 24 hour blood pressure.
[0098] The results indicate beneficial effects of dietary
supplementation with soy protein on cardiovascular risk markers in
type II diabetic subjects. The improvement is also seen in
individuals with near-normal lipid values. Ingestion of soy product
has been shown to further improve the effectiveness of low-fat
diets in non-diabetic subjects and the dietary supplementation in
type II diabetic patients may provide an acceptable and effective
option for blood lipid control, thereby postponing or even
preventing drug therapy.
Example 7
[0099] Twelve type II diabetic patients (4 female/8 males) with a
mean age of 65.8.+-.1.6 years, a diabetes duration of 6.0.+-.1.3
years, a mean body mass index of 28.5.+-.1.0, and a mean glycated
hemoglobin HbAlc of 7.4.+-.0.4 percent were included in the
study.
[0100] The experiment was an acute, paired, cross-over study in
which two test meals were served during the experiments (A:
Standard meal supplemented with 1 g of stevioside given orally; B:
Standard meal given together with 1 g of gelatine (placebo) given
orally. The total energy content of the test meals was 1725 kJ
(protein 16 E %, fat 30 E %, carbohydrate 54 E %).
[0101] Blood samples were drawn from an antecubital vein 30 minutes
before and 240 minutes after ingestion of the test meal. The
arterial blood pressure was continuously monitored during the
experiment. Students paired t-test was used for comparing the
effects of stevioside with placebo on the parameters measured. Data
are given as mean .+-.SEM.
[0102] Stevioside reduced the postprandial blood glucose response
by 18.+-.5% (p<0.004) compared to placebo (absolute IAUC
638.+-.55 vs. 522.+-.64 mmol/l.times.240 min; p<0.02) as seen in
FIG. 8a. Stevioside tended to stimulate the insulin response in
type II diabetic patients (enhance the area under the insulin
response curve (IAUC)), however the difference did not reach
statistical significance (p=0.09) (FIG. 8b).
[0103] Stevioside significantly reduced the postprandial glucagon
levels compared to placebo (348.+-.46 vs. 281.+-.33; p=0.02) (FIG.
8c).
[0104] Stevioside significantly reduced the postprandial glucagon
like peptide-1 (GLP-1) levels compared to placebo (2208.+-.253 vs.
1529.+-.296; p<0.045) (FIG. 8d).
Example 8
[0105] Four test diets (A: Standard carbohydrate rich laboratory
animal diet (Altromin); n=12 (Alt). B: Altromin supplemented with
stevioside (Altromin+Stevioside); n=12; (Alt+Ste). C: Soy plus 20%
Altromin; n=12; (Soy). D: Soy plus 20% Altromin plus stevioside;
n=12; (Soy+Ste)) were administered for four weeks to four groups of
adult rats. Each experimental group consisted of twelve female
Goto-Kakizaki with an age of 9 weeks. The rats received the
stevioside (0.025 g/kg body weight/day) with the drinking water. By
the end of the third experimental week intra-arterial catheters
were implanted into the carotid artery thereby enabling blood
sampling during a 240 minutes glucose-tolerance test which was
carried out by the end of the experiment at week 4. Blood samples
were drawn after a bolus infusion of 2.0 g D-glucose/kg body
weight. Plasma concentrations of glucose, insulin, and glucagon
were measured during the glucose tolerance test. Immediately before
the glucose tolerance test fasting levels of triglycerides and
cholesterol were determined. Concomitantly, the systolic blood
pressure was measured using a tail cuff.
Effects on Plasma-Glucose:
[0106] As seen at FIG. 8 and in Table I below stevioside reduced
the incremental area (IAUC) under the glucose response curve during
the glucose tolerance testing both in the Altromin (p<0.05) and
in the soy+20% Altromin group (Soy) (p<0.001). The relative
effect of stevioside was more pronounced in the group receiving
soy+20% Altromin group compared to the group receiving Altromin.
The combination of soy and stevioside synergistically reduced the
area under the glucose response curve compared to the Altromin
group (p<0.0001) (FIG. 9a.).
(Plasma glucose was measured using MPR 3, 166 391, Glucose/GOD-PAP
Method from Boehringer Mannheim)
Effects on Plasma Insulin:
[0107] The group receiving soy+stevioside (Soy+Ste) has reduced
incremental area under the insulin response curve compared to the
Altromin+stevioside group (Alt+Ste) as seen in FIG. 9 and in Table
I below. Considering the concomitant blood glucose responses this
indicates that soy increases the insulin sensitivity. Stevioside
did not alter the insulin responses in the Altromin and soy diets
when studying the total response curve from 0 to 240 minutes.
However, in both groups supplementation of the diets with
stevioside significantly improved the first phase insulin
responses--which is subdued as a characteristic feature of type II
diabetes. The combination of soy+stevioside synergistically
improved the first phase insulin response (p<0.05) (FIG.
9b).
(Plasma insulin was measured using Sensitive Rat Insulin RIA, Cat #
SRI-13K from Linco)
Effects on Plasma Glucagon:
[0108] Stevioside significantly reduced the area under the
plasma-glucagon response curve during the glucose tolerance test in
both the groups receiving Altromin (p<0.003) and soy (p<0.01)
(see FIG. 9c and Table I below).
(Plasma glucagon was measured using Glucagon RIA, Cat # GL-32K from
Linco)
Effects on Blood Pressure:
[0109] A marked significant suppression of the systolic blood
pressure (p<0.05) (Table I) is elicited by stevioside in
combination with either Altromin (.DELTA.=-28 mmHg) or soy
(.DELTA.=-21 mmHg) as depicted in FIG. 9d.
(Blood pressure was measured using TSE Non-Invasive Blood Pressure
Monitoring System from Technical Scientific Equipment GmbH)
Effects on Body Weight:
[0110] The initial weights in the four groups did not differ (FIG.
5). Apparently the combination of soy and stevioside prevented
weight gain as seen in FIG. 9e.
Effects on Triglyceride and Cholesterol:
[0111] Stevioside causes a significant suppression of the fasting
triglyceride levels in combination with either Altromin (p<0.05)
or soy (p<0.02) (Table I). Soy significantly reduced the fasting
triglyceride levels with or without supplementation of stevioside
(p<0.05 and p<0.002, respectively) (Table I). Stevioside
given in combination with soy synergistically reduced the fasting
total cholesterol levels compared to diets containing Altromin
alone (p<0.0001). Soy alone also reduced the total cholesterol
levels compared to Altromin alone (p<0.002) (FIG. 9f. and FIG.
9g) (Table I).
(Plasma cholesterol was measured GOD-PAP from Roche and
triglycerides was measured using GHOD-PAP from Roche)
[0112] Stevioside exerts beneficial effects in type II diabetes
i.e. reduces blood glucose, suppresses glucagon and improve first
phase insulin secretion. The results also indicates that soy
improves insulin sensitivity, a characteristic feature of the
metabolic syndrome. Stevioside exerts a pronounced blood pressure
reduction both with as well as without the presence of soy. The
combination of stevioside and soy has a synergistic suppressive
effect on blood glucose levels, enhances first phase insulin
secretion, suppresses fasting plasma triglycerides and total
cholesterol and the combination of soy and stevioside seems to
prevent weight gain. The combination of stevioside and soy appears
to possess the potential of an effective treatment of a number of
the characteristic features of the metabolic syndrome i.e. type II
diabetes, hypertension, dyslipidemia and obesity. TABLE-US-00001
TABLE I Table I: Areas under the p-glucose, -insulin and -glucagon
response curves during the glucose tolerance test in the four
experimental groups. Change in systolic blood pressure at start and
at end of the study period. Fasting plasma- triglyceride and -total
cholesterol concentrations by the end of the study. IAUC IAUC IAUC
IAUC p-glucose p-insulin p-insulin p-glucagon Change in blood (mM
.times. (ng/ml .times. (ng/ml .times. (pg/ml .times. pressure
(mmHg) Triglycerides Cholesterol Group 240 min) 240 min) 30 min)
240 min) From week 0 to 4 (mM) (mM) Altromin 991 .+-. 96 317 .+-.
55 11 .+-. 4 21918 .+-. 1467 5 .+-. 4 0.72 .+-. 0.10 2.51 .+-. 007
Altromin + 757 .+-. 53 375 .+-. 42 19 .+-. 4 17023 .+-. 1449 -23
.+-. 6 0.50 .+-. 0.04 2.28 .+-. 0.18 Stevioside Soy + 20% Altromin
820 .+-. 75 218 .+-. 22 9 .+-. 2 26200 .+-. 2410 8 .+-. 3 0.49 .+-.
0.04 2.13 .+-. 0.08 Soy + 20% Altromin + 439 .+-. 56 248 .+-. 27 24
.+-. 5 17229 .+-. 1819 -13 .+-. 5 0.37 .+-. 0.02 1.84 .+-. 0.06
Stevioside
* * * * *