U.S. patent application number 12/918037 was filed with the patent office on 2011-01-20 for treatment of energy utilization disease.
This patent application is currently assigned to SUMMIT CORPORATION PLC. Invention is credited to Graeme Horne, Robert James Nash, Francis Xavier Wilson.
Application Number | 20110015226 12/918037 |
Document ID | / |
Family ID | 40589554 |
Filed Date | 2011-01-20 |
United States Patent
Application |
20110015226 |
Kind Code |
A1 |
Nash; Robert James ; et
al. |
January 20, 2011 |
Treatment of Energy Utilization Disease
Abstract
Described are compositions comprising imino sugar acids for the
treatment of energy utilization disease (e.g. metabolic syndrome,
including any disease or disorder associated therewith, for example
central obesity, elevated levels of triglycerides and diabetes,
including type 1 diabetes, type 2 diabetes and insulin resistance),
processes for producing said compositions from various plant
sources, together with various products, compounds, compositions,
medical uses and methods based thereon.
Inventors: |
Nash; Robert James;
(Abingdon, GB) ; Wilson; Francis Xavier;
(Abingdon, GB) ; Horne; Graeme; (Abingdon,
GB) |
Correspondence
Address: |
HESLIN ROTHENBERG FARLEY & MESITI PC
5 COLUMBIA CIRCLE
ALBANY
NY
12203
US
|
Assignee: |
SUMMIT CORPORATION PLC
Abingdon
GB
|
Family ID: |
40589554 |
Appl. No.: |
12/918037 |
Filed: |
February 17, 2009 |
PCT Filed: |
February 17, 2009 |
PCT NO: |
PCT/GB09/00417 |
371 Date: |
August 17, 2010 |
Current U.S.
Class: |
514/315 |
Current CPC
Class: |
A61K 31/445 20130101;
A61K 36/42 20130101; A61K 36/752 20130101; A61P 27/02 20180101;
A61P 1/00 20180101; A61K 36/815 20130101; A61P 3/00 20180101; A61P
3/04 20180101; A61P 3/06 20180101; A61P 3/10 20180101; A61P 13/12
20180101; A61P 19/00 20180101; A61P 19/02 20180101; A61K 36/19
20130101; A61P 15/00 20180101; A61P 15/08 20180101; A61P 21/00
20180101; A61K 36/27 20130101; A61K 36/28 20130101; A61K 36/48
20130101; A61K 31/40 20130101; A61K 45/06 20130101; A61P 19/10
20180101; A61P 27/12 20180101; A61P 9/10 20180101; A61P 9/12
20180101; A61P 7/00 20180101 |
Class at
Publication: |
514/315 |
International
Class: |
A61K 31/445 20060101
A61K031/445; A61P 3/00 20060101 A61P003/00; A61P 3/10 20060101
A61P003/10; A61P 9/12 20060101 A61P009/12; A61P 19/02 20060101
A61P019/02; A61P 15/00 20060101 A61P015/00; A61P 1/00 20060101
A61P001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 18, 2008 |
GB |
0802903 |
Feb 18, 2008 |
GB |
0802904 |
Feb 18, 2008 |
GB |
0802907 |
Claims
1-37. (canceled)
38. A method of treating diabetes, obesity or disorders associated
with metabolic syndrome in a patient, the method comprising the
administration to the patient of an isolated imino sugar acid,
wherein the imino sugar acid does not inhibit disaccharidase
activity, exhibiting an IC.sub.50 value in the .mu.M or mM range or
greater.
39. A method of treating diabetes, obesity or disorders associated
with metabolic syndrome in a patient, which method comprises the
administration to the patient of an imino sugar acid in combination
with a dipeptidyl peptidase-4 (DPP-4) inhibitor.
40. A method according to claim 39 wherein: (i) the imino sugar
acid is admixed, or physically associated within a single unit
dose, with the dipeptidyl peptidase-4 (DPP-4) inhibitor; or (ii)
the imino sugar acid is co-packaged with the dipeptidyl peptidase-4
(DPP-4) inhibitor; or (iii) the imino sugar acid is co-administered
with the dipeptidyl peptidase-4 (DPP-4) inhibitor.
41. The method of claim 38 wherein the imino sugar acid is of a
structural class selected from piperidine, pyrrolidine,
pyrrolizidine, indolizidine and nortropane.
42. The method of claim 38 wherein the imino sugar acid is
polyhydroxylated.
43. The method of claim 38 wherein the imino sugar acid is a
piperidine imino sugar acid.
44. The method of claim 43 wherein the imino sugar acid is: (a) a
pipecolic or hydroxypipecolic acid; (b) a piperidine ISA having at
least 3 free hydroxyl (or hydroxyalkyl) groups on the ring system
nucleus; or (c) N-acid derivatives of (a) or (b); or (d) a
polyhydroxypipecolic acid having at least two (e.g. 3) free
hydroxyl (or hydroxyalkyl) groups on the ring system nucleus.
45. The method of claim 38 wherein the imino sugar or imino sugar
acid is of formula (I): ##STR00044## wherein R.sub.1 is absent or
is C.sub.1-C.sub.6 alkyl; the location of the carboxyl group may be
switched from the ring carbon to the ring nitrogen to produce an
N-acid analogue of the compound of formula (I); or an acyl (e.g.
O-acyl) derivative or dehydroxylated analogue in which one or more
ring hydroxyl(s) are absent; or a pharmaceutically acceptable salt
or derivative thereof.
46. The method of claim 45 wherein the imino sugar or imino sugar
acid is selected from the structures shown below: ##STR00045## or
(a) N-acid analogues of the foregoing structures (G1)-(G4) in which
the location of the carboxyl group is switched from the ring carbon
to the ring nitrogen; (b) acyl (e.g. O-acyl) derivatives of the
foregoing structures (or of the N-acid analogues of (a)); (c)
dehydroxylated analogues of the foregoing structures (or of the
N-acid analogues of (a) and (b)) in which one or more ring
hydroxyl(s) are absent.
47. A composition comprising an imino sugar acid combined with a
dipeptidyl peptidase-4 (DPP-4) inhibitor.
48. The composition of claim 47 wherein the imino sugar acid is of
a structural class selected from piperidine, pyrrolidine,
pyrrolizidine, indolizidine and nortropane.
49. The composition of claim 47 wherein the imino sugar acid is
polyhydroxylated.
50. The composition of claim 48 wherein the imino sugar acid is a
piperidine imino sugar acid.
51. The composition of claim 50 wherein the imino sugar acid is:
(a) a pipecolic or hydroxypipecolic acid; (b) a piperidine ISA
having at least 3 free hydroxyl (or hydroxyalkyl) groups on the
ring system nucleus; or (c) N-acid derivatives of (a) or (b); or
(d) a polyhydroxypipecolic acid having at least two (e.g. 3) free
hydroxyl (or hydroxyalkyl) groups on the ring system nucleus.
52. The composition of claim 47 wherein the imino sugar or imino
sugar acid is of formula (I): ##STR00046## wherein R.sub.1 is
absent or is C.sub.1-C.sub.6 alkyl; the location of the carboxyl
group may be switched from the ring carbon to the ring nitrogen to
produce an N-acid analogue of the compound of formula (I); or an
acyl (e.g. O-acyl) derivative or dehydroxylated analogue in which
one or more ring hydroxyl(s) are absent;
53. The composition of claim 52 wherein the imino sugar or imino
sugar acid is selected from the structures shown below:
##STR00047## or (a) N-acid analogues of the foregoing structures
(G1)-(G4) in which the location of the carboxyl group is switched
from the ring carbon to the ring nitrogen; (b) acyl (e.g. O-acyl)
derivatives of the foregoing structures (or of the N-acid analogues
of (a)); (c) dehydroxylated analogues of the foregoing structures
(or of the N-acid analogues of (a) and (b)) in which one or more
ring hydroxyl(s) are absent.
54. A method of treating diabetes, obesity or disorders associated
with metabolic syndrome in a patient, which method comprises
administering to the patient the composition of claim 47.
55. A method of treating hyperglycaemia, glucose intolerance,
hyperinsulinaemia, glucosuria, metabolic acidosis, cataracts,
diabetic neuropathy, diabetic nephropathy, diabetic retinopathy,
macular degeneration, glomerulosclerosis, diabetic cardiomyopathy,
insulin resistance, impaired glucose metabolism, arthritis,
hypertension, hyperlipidemia, osteoporosis, osteopenia, bone loss,
brittle bone syndromes, acute coronary syndrome, infertility, short
bowel syndrome, chronic fatigue, eating disorders or intestinal
motility dysfunction in a patient, which method comprises
administering to the patient an imino sugar acid as defined in
claim 38.
56. A method of treating or preventing type 2 diabetes in a
patient, which method comprises administering to the patient the
imino sugar acid of claim 38.
57. A method according to claim 39 wherein the imino acid sugar
acid is selected from the structures shown below: ##STR00048## or
(a) N-acid analogues of the foregoing structures (G1)-(G4) in which
the location of the carboxyl group is switched from the ring carbon
to the ring nitrogen; (b) acyl (e.g. O-acyl) derivatives of the
foregoing structures (or of the N-acid analogues of (a)); (c)
dehydroxylated analogues of the foregoing structures (or of the
N-acid analogues of (a) and (b)) in which one or more ring
hydroxyl(s) are absent.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions for the
treatment of energy utilization disease, for example metabolic
syndrome (including any disease or disorder associated therewith,
for example central obesity, elevated levels of triglycerides and
diabetes, including type 1 diabetes, type 2 diabetes and insulin
resistance), to processes for producing said compositions from
various plant sources, together with various products, compounds,
compositions, medical uses and methods based thereon.
[0002] The invention also relates to methods for monitoring the
quality of a herbal food additive, to processes for producing a
herbal food additive as well as to herbal food additives, foods and
beverages obtainable by such processes.
BACKGROUND TO THE INVENTION
[0003] Imino sugar acids (ISAs) constitute a subclass of the more
widely distributed class of phytochemicals known as imino sugars.
Many known ISAs are phytochemicals, present as secondary
metabolites in plant tissues (where they may play a role in
defence). Structurally, ISAs exhibit great diversity. Many ISAs are
small molecules, with molecular weights below 250 Daltons. The
skeletons are derived from sugar acids that can be classified
structurally on the basis of the configuration of the
N-heterocycle: Watson et al. (2001) Phytochemistry 56: 265-295 have
classified a comprehensive range of polyhydroxylated alkaloids
inter alfa as piperidine, pyrroline, pyrrolidine, pyrrolizidine,
indolizidine and nortropanes ISAs (see FIGS. 1-7 of Watson et al.
(2001), the disclosure of which is incorporated herein by
reference).
[0004] Although imino sugars are widely distributed in plants
(Watson et al., 2001), the imino sugar acids are much less widely
distributed. As described herein, the present inventors have
discovered that the botanical distribution of imino sugar acids
correlates with medicinal plants used for the control of energy
utilization disease, including diabetes, obesity and other
disorders associated with metabolic syndrome.
Energy Utilization Diseases
[0005] Energy utilization diseases encompass a wide range of
diseases and include, for example, disorders of homeostasis,
metabolic diseases, dysfunction of sugar metabolism and appetite
disorders.
[0006] Examples of energy utilization diseases therefore include
insulin resistance, various forms of diabetes, metabolic syndrome,
obesity, wasting syndromes (for example, cancer associated
cachexia), myopathies, gastrointestinal disease, growth
retardation, hypercholesterolemia, atherosclerosis and
age-associated metabolic dysfunction.
[0007] Energy utilization diseases also include conditions
associated with metabolic syndrome, obesity and/or diabetes,
including for example hyperglycaemia, glucose intolerance,
hyperinsulinaemia, glucosuria, metabolic acidosis, cataracts,
diabetic neuropathy, diabetic nephropathy, diabetic retinopatiy,
macular degeneration, glomerulosclerosis, diabetic cardiomyopathy,
insulin resistance, impaired glucose metabolism, arthritis,
hypertension, hyperlipidemia, osteoporosis, osteopenia, bone loss,
brittle bone syndromes, acute coronary syndrome, infertility, short
bowel syndrome, chronic fatigue, eating disorders and intestinal
motility dysfunction.
Insulin Resistance, Metabolic Syndrome and Diabetes
[0008] In healthy individuals, blood glucose levels are maintained
within a narrow range by two pancreatic hormones: insulin (produced
by pancreatic .beta.-cells) and glucagon (produced by pancreatic
.alpha.-cells). Pancreatic .beta.-cells sense increases in blood
glucose levels and respond by secreting insulin. Insulin promotes
glucose uptake by tissues of the body, thereby restoring blood
glucose concentration to the physiological range. Glucagon acts
reciprocally, increasing blood glucose levels under fasting
conditions, primarily by stimulating glucose production in the
liver.
[0009] Insulin resistance is characterized by a reduced action of
insulin in skeletal muscle, adipocytes and hepatocytes so that
normal amounts of insulin become inadequate to produce a normal
insulin response from the cells of these tissues. In adipocytes,
insulin resistance results in hydrolysis of stored triglycerides,
leading to elevated free fatty acids in the blood plasma. In
muscle, insulin resistance reduces glucose uptake while in
hepatocytes it reduces glucose storage. In both of the latter cases
an elevation of blood glucose concentrations results.
[0010] High plasma levels of insulin and glucose due to insulin
resistance often progresses to metabolic syndrome and type 2
diabetes.
[0011] Metabolic syndrome is a constellation of abnormalities and
disorders that increase the risk of cardiovascular disease and
diabetes. The incidence is very high in many developed countries:
some studies indicate prevalence in the USA of up to 25% of the
population. The disorder is also known as (metabolic) syndrome X,
insulin resistance syndrome, Reaven's syndrome and CHAOS. Metabolic
syndrome may be diagnosed by the presence of three or more of the
following symptoms: central obesity (waist measurement of more than
40 inches for men and more than 35 inches for women); high levels
of triglycerides (150 mg/dL or higher); low levels of HDL (below 40
mg/dL for men and below 50 mg/dL for women) and high blood pressure
(130/85 mm Hg or higher). Associated diseases and signs are: fatty
liver (often progressing to non-alcoholic fatty liver disease),
polycystic ovarian syndrome, hemochromatosis (iron overload) and
acanthosis nigricans (dark skin patches).
[0012] The first line treatment of metabolic syndrome is change of
lifestyle (caloric restriction and physical activity). However,
drug treatment is frequently required. Generally, the individual
diseases that comprise the metabolic syndrome are treated
separately (e.g. diuretics and ACE inhibitors for hypertension).
Cholesterol drugs may be used to lower LDL cholesterol and
triglyceride levels, if they are elevated, and to raise HDL levels
if they are low. Use of drugs that decrease insulin resistance
(e.g. metformin and thiazolidinediones is controversial). A recent
study indicated that cardiovascular exercise was therapeutic in
less than 31% of cases, the most probable benefit was to
triglyceride levels, with 43% showing improvement; conversely 91%
of test subjects did not exhibit a decrease in fasting plasma
glucose or insulin resistance.
[0013] Type 2 diabetes is a chronic disease that is characterised
by persistently elevated blood glucose levels (hyperglycaemia).
Insulin resistance together with impaired insulin secretion from
the pancreatic .beta.-cells characterizes the disease. The
progression of insulin resistance to type 2 diabetes is marked by
the development of hyperglycaemia after eating when pancreatic
.beta.-cells become unable to produce adequate insulin to maintain
normal blood sugar levels (euglycemia)).
[0014] The most important drug currently used to treat type 2
diabetes is metformin (Glucophage, Diabex, Diaformin, Fortamet,
Riomet, Glumetza, Cidophage and others). Metformin is of the
biguanide class of oral antihyperglycaemic agents. Other biguanides
include phenformin and buformin (now withdrawn). Metformin works
primarily by reducing liver release of blood glucose from glycogen
stores, but also has some effect in increasing the uptake of
glucose. Other widely used drug classes include those of the
sulfonylurea group (including glibenclamide and gliclazide). These
drugs increase glucose stimulated insulin secretion by the
pancreas. Newer drug classes include thiazolidinediones (e.g.
rosiglitazone, pioglitazone, and troglitazone), which act by
binding to PPARs (peroxisome proliferator-activated receptors), a
group of receptor molecules inside the cell nucleus. Other classes
include .alpha.-glucosidase inhibitors (acarbose and miglitol),
meglitinides (which stimulate insulin release and include
nateglinide, repaglinide and their analogues), peptide analogs
(e.g. incretin mimetics, which act as insulin secretagogues,
glucagon-like peptide analogues (e.g. exenatide), dipeptidyl
peptidase-4 (DPP-4) inhibitors (which increase incretin levels
(e.g. sitagliptin) and amylin agonist analogues (which slow gastric
emptying and suppress glucagon (e.g. pramlintide).
[0015] However, no existing therapies for the different forms of
type 2 diabetes seem to improve function of key intrinsic factors
in the .beta.-cells and all existing therapies fail to arrest
progression of the disease and, over time, also fail to normalise
glucose levels and/or prevent subsequent complications. The
existing therapies are also associated with undesirable side
effects. For example, insulin secretagogues and insulin injections
may cause hypoglycaemia and weight gain. Patients may also become
unresponsive to insulin secretagogues over time. Metformin and
.alpha.-glucosidase inhibitors often lead to gastrointestinal
problems and PPAR agonts tend to cause increased weight gain and
oedema. Exenatide is also reported to cause nausea and
vomiting.
[0016] Type 1 diabetes (or insulin dependent diabetes) is
characterized by loss of the insulin-producing beta cells of the
islets of Langerhans in the pancreas, leading to a deficiency of
insulin. The main cause of this beta cell loss is a T-cell mediated
autoimmune attack. There is no known preventative measure that can
be taken against type 1 diabetes, which comprises up to 10% of
diabetes mellitus cases in North America and Europe. Most affected
people are otherwise healthy and of a healthy weight when onset
occurs. Sensitivity and responsiveness to insulin are usually
normal, especially in the early stages.
[0017] The principal treatment of type 1 diabetes, even from the
earliest stages, is replacement of insulin combined with careful
monitoring of blood glucose levels using blood-testing monitors.
Without insulin, ketosis and diabetic ketoacidosis can develop and
coma or death will result. Apart from the common subcutaneous
injections, it is also possible to deliver insulin by a pump, which
allows continuous infusion of insulin 24 hours a day at preset
levels, and the ability to program doses (a bolus) of insulin as
needed at meal times. An inhaled form of insulin, Exubera, has
recently been approved by the FDA.
[0018] The treatment of type 1 diabetes must be continued
indefinitely. While treatment does not impair normal activities,
great awareness, appropriate care, and discipline in testing and
medication must be observed.
[0019] Thus, new and/or alternative antidiabetic drug treatments,
particularly those that are able to restore .beta.-cell function,
are required. In particular, there is a real and substantial unmet
clinical need for an effective drug that is capable of treating
both type 2 and type 1 diabetes and associated conditions with
fewer side effects than existing drug therapies as well as
treatments for metabolic syndrome are required, particularly
treatments which are effective against obesity and/or elevated
triglyceride levels.
Herbal Food Additives and Remedies
[0020] There is presently great interest in the use of herbal
remedies and supplements and a growing acceptance from food
manufacturers, healthcare companies and the medical profession that
herbal products have value and can complement established
formulations and treatments. Herbal food additives and supplements
are now widely used. In particular, demand for low-carbohydrate,
low-sugar food alternatives has led to a growing interest in
natural sweeteners and steviol glycosides (responsible for the
sweet taste of the leaves of the stevia plant (Stevia rebaudiana))
are being developed by Coca Cola as natural sweeteners.
[0021] However, quality control of herbal food additives is
difficult due to the complex nature and inherent non-uniformity of
plant materials. The materials used in herbal and plant-based food
additive are usually whole plants or parts or extracts thereof.
Since plant materials contain many different chemical components
the materials are complex mixtures. This makes it very difficult to
standardize and control the quality of the materials. Moreover,
many herbal food additives are mixtures of two or more plant-based
components and are therefore mixtures of mixtures, so introducing a
further level of complexity. Furthermore, the recipes and methods
of manufacture used are often not uniform and may remain
undisclosed. These factors make it very difficult to ensure that
two samples of a given product, obtained from disparate sources and
ostensibly identical, do in fact contain the same mixture of
ingredients. This problem, which leads to difficulties in
controlling the quality of such materials, has limited the use of
certain herbal extracts even amongst herbal practitioners.
[0022] Such problems may be particularly acute in the case of
Stevia-derived products, since certain steviosides have been
reported to have possible mutagenic activity and hence
fractionating Stevia-derived plant material to remove such material
may be of particular importance. Other problems arise from the fact
that the plants used in the practice of herbal food additive are
frequently unavailable locally and therefore need to be obtained
from sources which are remote from the end user. However, the
supply of such plants from remote locations can be erratic and
inaccurate, particularly because no detailed monographs including
identity and quality standards exist for many such plants. The
complex mixture of ingredients found in medicinal plants varies
widely in type and concentration depending on many factors
including the botanical source, the location where the plant is
grown, what other plants or microorganisms are growing near it, the
time of year when the plant is harvested, the conditions under
which the material is stored and processed and the extraction
procedure used.
[0023] There is therefore a need for sensitive processes which can
profile herbal products and so establish a standard specification
for a medicinal plant material which can be related to activity, so
permitting quality control in the production of herbal food
additives and ideally quantifying the components known or likely to
be active.
[0024] The imino sugar acids are analogues of sugar acids in which
the ring oxygen is replaced by a nitrogen. Although imino sugars
are widely distributed in plants (Watson et al. (2001)
Phytochemistry 56: 265-295), the imino sugar acids are much less
widely distributed. The present inventors have now discovered that
the botanical distribution of imino sugar acids correlates with
medicinal plants used for the control of diabetes and obesity.
Imino sugar acids have not hitherto been reported from these
plants, possibly because carbohydrate-like compounds are difficult
to analyse using the conventional HPLC analytical systems and
compounds affecting sugar perception in these plants have attracted
more interest. Thus, qualitative and/or quantitative analysis of
herbal material for imino sugar acids can form the basis of quality
control procedures during the sourcing, preparation and processing
of herbal food additives and foods/beverages based thereon,
particularly those foods and beverages formulated to form part of a
low-calorie or low-sugar diet. In some applications it may be
important to ensure that imino sugar acids are absent from the
herbal material in order to ensure that inappropriate or
undesirable pharmaceutical activities are eliminated from the
supplemented foodstuff or beverage.
SUMMARY OF THE INVENTION
[0025] The present invention is based, at least in part, on the
surprising discovery that the botanical distribution of imino sugar
acids correlates with medicinal plants used for the control of
diabetes, obesity and other disorders associated with metabolic
syndrome. Thus, for the first time ISAs have been identified as
important bioactive principles in established anti-obesity and
anti-diabetic herbal medicines.
[0026] Thus, according to the invention there is provided an
isolated imino sugar acid for the treatment of an energy
utilization disease (for example metabolic syndrome, including type
1, type 2 diabetes and insulin resistance) as well as a
nutraceutical or pharmaceutical composition comprising an isolated
imino sugar acid.
[0027] Thus, according to the invention there is provided a process
for the production of a composition for the treatment of energy
utilization disease (for example metabolic syndrome (including type
1, type 2 diabetes and insulin resistance) comprising the steps of:
[0028] (a) providing plant material; [0029] (b) isolating one or
more imino sugar acid(s) from said plant material (and optionally
removing or reducing potential toxins); and then [0030] (c)
formulating said isolated imino sugar acid(s) with a pharmaceutical
excipient to produce an anti-metabolic syndrome composition in
which the amount and concentration of the isolated imino sugar
acid(s) is sufficient to treat the energy utilization disease in a
human subject.
[0031] The invention contemplates synthetic analogues of the
naturally-occurring ISAs described herein. Such synthetic analogues
may be produced by a process comprising the steps of: (a) isolating
one or more imino sugar acid(s) from plant material; (b)
determining the structure of said ISA; and then (c) synthesising
said synthetic ISA analogue. Thus, the invention contemplates a
process for the production of an ISA for the treatment of metabolic
syndrome and/or diabetes (including type 1 and type 2 diabetes and
insulin resistance) comprising the steps of: (a) isolating one or
more imino sugar acid(s) from plant material; (b) determining the
structure of said ISA; and then (c) synthesising said ISA to
produce a synthetic ISA for use in the treatment of metabolic
syndrome and/or diabetes (including type 1 and type 2 diabetes and
insulin resistance).
[0032] The invention contemplates synthetic derivatives of the
naturally occurring ISAs described herein. Such synthetic
derivatives may be produced by a process comprising the steps of:
(a) isolating one or more imino sugar acid(s) from plant material;
and then (b) derivatizing said isolated ISA (e.g. chemically or
enzymatically) to produce a synthetic ISA derivative. Thus, the
invention contemplates a process for the production of an ISA for
the treatment of metabolic syndrome and/or diabetes (including type
1 and type 2 diabetes and insulin resistance) comprising the steps
of: (a) isolating one or more imino sugar acid(s) from plant
material; and then (b) derivatizing said isolated ISA (e.g.
chemically or enzymatically) to produce a synthetic ISA
derivative.
[0033] The plant material used as starting material in step (a) is
preferably derived from a botanical source selected from: (a)
Stevia spp. (e.g. S. rebaudiana); (b) Gymnema spp. (e.g. G.
sylvestre); (c) Andrographis spp. (e.g. A. paniculata); (d)
leguminous species (e.g. Aspalanthus linearis (Rooibos), Baphia
spp., Glycine max (soya), Alexa spp.), Castanospermum australe),
Lotus spp.; (e) plants of the family Rutaceae (for example Citrus
spp., e.g. C. aurantium), (f) plants of the Cucurbitaceae (for
example Asian Pumpkin, Cucurbita ficifolia and Momordica charantia)
or (g) Solanaceae (e.g. Lycium barbarum, Goji).
[0034] The imino sugar acid is preferably of a structural class
selected from piperidine, pyrrolidine, pyrroline, pyrrolizidine,
indolidine and nortropane ISAs.
[0035] In another aspect, the invention contemplates an
anti-metabolic syndrome and/or anti-diabetes (including type 1 and
type 2 diabetes and insulin resistance) composition obtainable by
the process of any one of the preceding claims for use as an
anti-metabolic syndrome and/or anti-diabetes (including type 1 and
type 2 diabetes and insulin resistance) agent.
[0036] In another aspect, the invention contemplates an
anti-metabolic syndrome and/or anti-diabetes (including type 1 and
type 2 diabetes and insulin resistance) composition obtained by the
process of the invention.
[0037] In another aspect, the invention contemplates a process for
producing a pharmaceutical composition comprising the step of
monitoring the quality of said composition by detecting the
presence or absence or measuring the amount of an imino sugar acid
in a sample of said composition. Such embodiments find particular
application in processes for the production of anti-diabetic drugs
based on purification from natural plant sources.
[0038] In another aspect, the invention contemplates a method for
monitoring the quality of a pharmaceutical composition comprising
the steps of: (a) providing a sample of the composition; and (b)
detecting the presence or absence or measuring the amount of an
imino sugar acid in said sample. Again such embodiments find
particular application in processes for the production of
anti-diabetic drugs based on purification from natural plant
sources.
[0039] The latter two embodiments find particular application in
the production of anti-diabetic drugs based on isolation from
Gymnema spp. (e.g. from G. sylvestre, Aspalanthus, Glycine max,
Lycium, Momordica or Cucurbita species).
[0040] Certain ISAs described herein are novel. According to the
invention, we also provide those novel ISAs as products per se,
together with processes for their preparation, compositions
containing them, as well as their use as pharmaceuticals. Some of
the ISAs described herein are known, as such, but not as
pharmaceuticals. According to the invention, we claim as
pharmaceuticals per se such ISAs as are known in the art but which
are not previously described for use as pharmaceuticals.
Herbal Quality Monitoring Aspects of the Invention
[0041] According to another aspect of the present invention there
is provided a process for producing a herbal food additive
comprising the step of monitoring the quality of said herbal food
additive by detecting the presence or absence or measuring the
amount of an imino sugar acid in a sample of said herbal food
additive.
[0042] In another aspect, the invention provides a method for
monitoring the quality of a herbal food additive comprising the
steps of: (a) providing a sample of the herbal food additive; and
(b) detecting the presence or absence or measuring the amount of an
imino sugar acid in said sample.
[0043] In this context, the term quality is used to define the
overall fitness of the herbal food additive for its intended use,
and may include for example the presence or absence of one or more
phytochemicals (at an appropriate concentration) which indicates
the use of a particular source, condition, purity and an acceptable
or unacceptable degree of contamination with undesirable
supplements and/or contaminants.
[0044] In circumstances where a desirable biological activity is
associated with the imino sugar acid (e.g. anti-obesity or
anti-diabetic activity), the invention preferably comprises the
step of monitoring the quality of said herbal food additive by
detecting the presence or measuring the amount of an imino sugar
acid in a sample of said herbal food additive.
[0045] However, in circumstances where a biological activity is
associated with the imino sugar acid (e.g. glycosidase inhibitory
activity, anti-obesity or anti-diabetic activity) but it is desired
to eliminate such activity from the food additive, the process of
the invention preferably comprises the step of monitoring the
quality of said herbal food additive by detecting the absence of an
imino sugar acid in a sample of said herbal food additive. Such
embodiments of the invention may find particular application in
circumstances where: (a) the herbal food additive is derived from a
plant source which is known to contain imino sugar acids together
with other phytochemicals and (b) where the other phytochemicals
but not the imino sugar acids are required. Such circumstances may
arise, for example, in cases where the herbal food additive
comprises Stevia-derived material containing both imino sugar acids
and steviol glycosides and where a food additive comprising steviol
glycosides free of imino sugar acids is required (e.g. to serve as
a natural sweetener with no additional biological activity).
[0046] In the latter embodiments, the step of monitoring the
quality of said herbal food additive by detecting the absence of an
imino sugar acid in a sample of said herbal food additive typically
takes the form of an analytical step which yields an upper limit
value for the concentration of the imino sugar acid analyte and
need not be such as to confirm absolute absence of imino sugar
acid. In many cases, the step is such as to yield a value which can
be used to confirm that the concentration of imino sugar acid(s) in
the sample falls below a particular threshold value. In such
circumstances the threshold value will depend on the use to which
the herbal food additive is to be put. In most cases it will
reflect a concentration below which the biological effects of the
imino sugar acids are acceptable (e.g. undetectable) when the
herbal additive is in use.
[0047] In a further aspect, the invention provides a process for
producing a supplemented foodstuff or beverage comprising the steps
of: [0048] (a) providing a herbal food additive; [0049] (b)
monitoring the quality of said herbal food additive of step (a)
according to the method of the invention; and [0050] (c) adding the
herbal food additive to a foodstuff or beverage to produce said
supplemented foodstuff or beverage.
[0051] This aspect of the invention finds broad utility in the
production of any supplemented foodstuff or beverage and any
foodstuff or beverage may be used, including chilled foods and
beverages, hot foods and beverages, sweetened foods and beverages,
carbonated beverages, alcoholic beverages and non-alcoholic
beverages. The supplemented foodstuff or beverage is preferably a
carbonated beverage.
[0052] Particularly preferred are beverages or foodstuffs which are
sweetened with one or more steviol glycosides. In such embodiments,
the steviol glycosides are preferably selected from: (a)
stevioside; (b) rebaudioside; and (c) dulcoside A. For example, the
steviol glycoside preferably comprise or consist essentially of
isolated rebaudioside A, B, C, D and/or E. Particularly preferred
are beverages or foodstuffs consisting essentially of rebaudioside
A.
[0053] Any herbal food additive may be used according to the
invention, but preferred are herbal food additives derived from
Stevia spp., Gymnema spp. (for example Gymnema sylvestre), Citrus
spp., Aspalanthus linearis (Rooibos), Glycine max (Soya), Pumpkin
(Cucurbita ficifolia and other Cucubitaceae species, e.g. Momordica
charantia), Lycium barbarum (Goji) or mixtures thereof.
[0054] The processes and methods of the invention preferably
further comprise the step of detecting the presence or absence or
measuring the amount of an imino sugar in said sample of herbal
food additive. In such embodiments, the imino sugar and/or imino
sugar acid may be of a structural class selected from piperidine,
pyrrolidine, pyrrolizidine, indolizidine and nortropane. In
preferred embodiments, the imino sugar is a glycosidase inhibitor
(e.g. a glucosidase or glucuronidase inhibitor).
[0055] In embodiments where the presence or absence of imino sugars
or imino sugar acids are detected according to the invention,
preferred are processes in which the presence or absence of
pyrrolidine imino sugars or imino sugar acids is detected.
Particularly preferred is
2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP) or derivatives
thereof. The latter embodiment is particularly preferred in cases
where the herbal food additive comprises Stevia-derived material
containing both DMDP and derivatives and steviol glycosides and
where a food additive comprising steviol glycosides free of DMDP or
derivatives is required (e.g. to serve as a natural sweetener with
no additional biological activity).
##STR00001##
[0056] Examples of imino sugars (deoxynojirimycin, DNJ at top left;
DMDP at bottom left) and imino sugar acids derived from them
[0057] The imino sugar acid used as analyte in the processes and
methods of the invention may be any imino sugar acid, including
piperidine, pyrroline, pyrrolidine, pyrrolizidine, indolizidine and
nortropane imino sugar acids. Preferred are piperidine imino sugar
acids (e.g. pipecolic acids).
[0058] The imino sugar and/or imino sugar acid used as analyte in
the processes and methods of the invention is preferably
polyhydroxylated.
[0059] The herbal food additive is preferably plant material or one
or more phytochemicals derived from Stevia spp., for example Stevia
rebaudiana.
[0060] The invention also contemplates a herbal food additive
obtainable by the methods and processes of the invention.
DETAILED DESCRIPTION OF THE INVENTION
Definitions and General Preferences
[0061] Where used herein and unless specifically indicated
otherwise, the following terms are intended to have the following
meanings in addition to any broader (or narrower) meanings the
terms might enjoy in the art:
[0062] As used herein, the term "energy utilization disease"
encompasses any disease or disorder arising from abnormal energy
utilization. The term therefore covers disorders and diseases of
homeostasis, metabolic disease, dysfunction of sugar metabolism and
appetite disorders. The term therefore includes insulin resistance,
various forms of diabetes, metabolic syndrome, obesity, wasting
syndromes (for example, cancer associated cachexia), myopathies,
gastrointestinal disease, growth retardation, hypercholesterolemia,
atherosclerosis and age-associated metabolic dysfunction. The term
also covers conditions associated with metabolic syndrome, obesity
and/or diabetes, including for example hyperglycaemia, glucose
intolerance, hyperinsulinaemia, glucosuria, metabolic acidosis,
cataracts, diabetic neuropathy, diabetic nephropathy, diabetic
retinopathy, macular degeneration, glomerulosclerosis, diabetic
cardiomyopathy, insulin resistance, impaired glucose metabolism,
arthritis, hypertension, hyperlipidemia, osteoporosis, osteopenia,
bone loss, brittle bone syndromes, acute coronary syndrome,
infertility, short bowel syndrome, chronic fatigue, eating
disorders and intestinal motility dysfunction.
[0063] References herein to the treatment of metabolic syndrome are
to be interpreted to include the treatment of any or all of the
disorders associated with metabolic syndrome, including in
particular obesity (e.g. central obesity), and elevated serum
triglycerides and diabetes (including type 1 and type 2 diabetes
and insulin resistance).
[0064] The term imino sugar acid defines a sugar acid analogue in
which the ring oxygen is replaced by a nitrogen. The term N-acid
ISA defines an imino sugar acid in which the carboxylic acid group
is located on the ring nitrogen. The term N-acid derivative defines
imino sugar analogues in which the ring nitrogen is substituted
with a carboxylic acid group.
[0065] Preferred ISAs are selected from the following structural
classes: piperidine (including (poly)hydroxypipecolic acids);
pyrroline; pyrrolidine (including (poly)hydroxyprolines);
pyrrolizidine; indolizidine and nortropane.
[0066] As used herein, the term polyhydroxylated as applied to
imino sugar acids defines an ISA having at least 2 (preferably at
least 3) free hydroxyl (or hydroxyalkyl) groups on the ring system
nucleus.
[0067] The term isolated as applied to the ISAs of the invention is
used herein to indicate that the ISA exists in a physical milieu
distinct from that in which it occurs in nature (or in the case of
synthetic, non-naturally-occurring ISAs, is purified). For example,
the isolated material may be substantially isolated (for example
purified) with respect to the complex cellular milieu in which it
naturally occurs.
[0068] When the isolated material (e.g. synthetic, non-naturally
occurring ISA) is purified, the absolute level of purity is not
critical and those skilled in the art can readily determine
appropriate levels of purity according to the use to which the
material is to be put. Preferred, however, are purity levels of 90%
w/w, 99% w/w or higher. In some circumstances, the isolated ISA
forms part of a composition (for example a more or less crude
extract containing many other substances) or buffer system, which
may for example contain other components. In other circumstances,
the isolated ISA may be purified to essential homogeneity, for
example as determined spectrophotometrically, by NMR or by
chromatography (for example GC-MS of the
trimethylsilyl-derivatives).
[0069] The term phytochemical is used herein in a broad sense to
encompass any chemical constituent of a plant, including
macromolecules and small molecules. Important examples include
alkaloids (for example imino sugars and imino sugars acids, e.g.
selected from the structural classes pyrrolidines, piperidines,
pyrrolizidine, indolizidines, tropanes and nortropanes),
carbohydrate analogues, phenolic compounds, terpenoids, enzyme
inhibitors, glycosides, nucleotides, amino acids, lipids and
sugars.
[0070] The terms derivative and pharmaceutically acceptable
derivative as applied to the ISAs of the invention define ISAs
which are obtained (or obtainable) by chemical derivatization of
the parent ISAs of the invention. The pharmaceutically acceptable
derivatives are suitable for administration to or use in contact
with the tissues of humans without undue toxicity, irritation or
allergic response (i.e. commensurate with a reasonable benefit/risk
ratio). Preferred derivatives are base obtained (or obtainable) by
alkylation, esterification or acylation of the parent ISAs of the
invention. Particularly preferred are acyl (e.g. butyl)
derivatives, for example O-acyl (e.g. O-butyl) derivatives. N-acid
imino sugar acids are also preferred.
[0071] The derivatives may be antidiabetic per se, or may be
inactive until processed in vivo. In the latter case, the
derivatives of the invention act as pro-drugs. Particularly
preferred pro-drugs are ester derivatives which are esterified at
one or more of the free hydroxyls and which are activated by
hydrolysis in vivo. The pharmaceutically acceptable derivatives of
the invention retain some or all of the anti-diabetic activity of
the parent ISA. In some cases, the activity is increased by
derivatization. Derivatization may also augment other biological
activities of the ISA, for example bioavailability and/or
glycosidase inhibitory activity and/or glycosidase inhibitory
profile. For example, derivatization may decrease glycosidase
inhibitory potency and/or increase specificity.
[0072] Imino sugar acids of the invention which do not inhibit
glucosidase activity (or do not inhibit glucosidase activity to a
clinically-significant extent) may exhibit no detectable inhibitory
activity or may be poor inhibitors, for example exhibiting
IC.sub.50 values in the .mu.M or mM ranges or greater. Typically,
clinically significant glucosidase inhibition arises only when
compounds have IC.sub.50 values in the submicromolar range. In such
embodiments, the glucosidase may be a mammalian
.alpha.-glucosidase, for example an .alpha.-glucosidase and/or a
digestive .alpha.-glucosidase (for example, a disaccharidase such
as saccharose), so that the imino sugar acid may exhibit no
detectable inhibitory activity (or may exhibit IC.sub.50 values in
the .mu.M or mM ranges or greater) in respect of these enzyme
classes.
[0073] Imino sugar acids of the invention which do not inhibit
glucosidase activity (or do not inhibit glucosidase activity to a
clinically-significant extent) may spare the activity of desirable
glucosidase activity in vivo, and in particular may spare digestive
glucosidase activity to the extent that adverse gastric
side-effects seen with the use of known anti-diabetic imino sugar
agents (such as miglitol) are reduced or eliminated.
[0074] The term pharmaceutically acceptable salt as applied to the
ISAs of the invention defines any non-toxic organic or inorganic
acid addition salt of the free base compounds which are suitable
for use in contact with the tissues of humans and lower animals
without undue toxicity, irritation, allergic response and which are
commensurate with a reasonable benefit/risk ratio. Suitable
pharmaceutically acceptable salts are well known in the art.
Examples are the salts with inorganic acids (for example
hydrochloric, hydrobromic, sulphuric and phosphoric acids), organic
carboxylic acids (for example acetic, propionic, glycolic, lactic,
pyruvic, malonic, succinic, fumaric, malic, tartaric, citric,
ascorbic, maleic, hydroxymaleic, dihydroxymaleic, benzoic,
phenylacetic, 4-aminobenzoic, 4-hydroxybenzoic, anthranilic,
cinnamic, salicylic, 2-phenoxybenzoic, 2-acetoxybenzoic and
mandelic acid) and organic sulfonic acids (for example
methanesulfonic acid and p-toluenesulfonic acid). The ISA drugs of
the invention may also be converted into salts by reaction with an
alkali metal halide, for example sodium chloride, sodium iodide or
lithium iodide. Preferably, the ISAs of the invention are converted
into their salts by reaction with a stoichiometric amount of sodium
chloride in the presence of a solvent such as acetone.
[0075] These salts and the free base compounds can exist in either
a hydrated or a substantially anhydrous form. Crystalline forms of
the compounds of the invention are also contemplated and in general
the acid addition salts of the ISAs of the invention are
crystalline materials which are soluble in water and various
hydrophilic organic solvents and which in comparison to their free
base forms, demonstrate higher melting points and an increased
solubility.
[0076] The term herbal medicine is used herein to define a
pharmaceutical composition in which at least one active principle
is not chemically synthesized and is a phytochemical constituent of
a plant. In most cases, this non-synthetic active principle is not
isolated (as defined herein), but present together with other
phytochemicals with which it is associated in the source plant. In
some cases, however, the plant-derived bioactive principle(s) may
be in a concentrated fraction or isolated (sometimes involving high
degrees of purification). In many cases, however, the herbal
medicine comprises a more or less crude extract, infusion or
fraction of a plant or even an unprocessed whole plant (or part
thereof), though in such cases the plant (or plant part) is usually
at least dried and/or milled.
[0077] The term herbal food additive is used herein to define a
composition in which at least one component is not chemically
synthesized but rather is a phytochemical constituent of a plant.
In most cases, this non-synthetic component is not purified, but
present together with other phytochemicals with which it is
associated in the source plant. In some cases, however, the
plant-derived component(s) may be in a concentrated fraction or
isolated (sometimes to high degrees of purity). In many cases,
however, the herbal food additive comprises a more or less crude
extract, infusion or fraction of a plant or even an unprocessed
whole plant (or part thereof), though in such cases the plant (or
plant part) is usually at least dried and/or milled.
[0078] The term bioactive principle is used herein to define a
phytochemical which is necessary or sufficient for the
pharmaceutical efficacy of the herbal medicament in which it is
comprised. In the case of the present invention, the bioactive
principle comprises the antidiabetic ISA of the invention (e.g. a
compound of formula (G2)).
[0079] The term standard specification is used herein to define a
characteristic, or a phytochemical profile, which is correlated
with an acceptable quality of the herbal medicine. In this context,
the term quality is used to define the overall fitness of the
herbal medicament for its intended use, and includes the presence
of one or more of the bioactive principles (at an appropriate
concentration) described above or else the presence of one or more
bioactive markers or a phytochemical profile which correlates with
the presence of one or more of the bioactive principles (at an
appropriate concentration).
[0080] The term phytochemical profile is used herein to define a
set of characteristics relating to different phytochemical
constituents.
[0081] In its broadest aspect, the present invention contemplates
all optical isomers, racemic forms and diastereomers of the ISAs of
the invention. Those skilled in the art will appreciate that, owing
to the asymmetrically substituted carbon atoms present in the ISAs
of the invention, the ISAs of the invention may exist and be
synthesised and/or isolated in optically active and racemic forms.
Thus, references to the ISAs of the present invention encompass the
ISAs as a mixture of diastereomers, as individual diastereomers, as
a mixture of enantiomers as well as in the form of individual
enantiomers.
[0082] Therefore, the present invention contemplates all optical
isomers and racemic forms thereof of the ISAs of the invention, and
unless indicated otherwise (e.g. by use of dash-wedge structural
formulae) the compounds shown herein are intended to encompass all
possible optical isomers of the compounds so depicted. In cases
where the stereochemical form of the ISA is important for
pharmaceutical utility, the invention contemplates use of an
isolated eutomer. Diastereoisomers may be separated using
conventional techniques, e.g. chromatography or fractional
crystallisation. The various optical isomers may be isolated by
separation of a racemic or other mixture of the compounds using
conventional, e.g. fractional crystallisation or HPLC, techniques.
Alternatively the desired optical isomers may be made by reaction
of the appropriate optically active starting materials under
conditions which will not cause racemisation.
Imino Sugar Acids of the Invention
[0083] The imino sugar acids (ISAs) are analogues of sugar acids in
which the ring oxygen is replaced by a nitrogen. Although imino
sugars are widely distributed in plants (Watson et al. (2001)
Phytochemistry 56: 265-295), the imino sugar acids are much less
widely distributed.
[0084] Imino sugar acids can be classified structurally on the
basis of the configuration of the N-heterocycle. Examples include
piperidine, pyrroline, pyrrolidine, pyrrolizidine, indolizidine and
nortropanes imino sugar acids (see FIGS. 1-7 of Watson et al.
(2001), the disclosure of which is incorporated herein by
reference).
[0085] Particularly preferred are imino sugar acids selected from
the following structural classes: [0086] (a) piperidine ISAs
(including (poly)hydroxypipecolic acids); [0087] (b) pyrroline
ISAs; [0088] (c) pyrrolidine ISAs (including
(poly)hydroxyprolines); [0089] (d) pyrrolizidine ISAs; [0090] (e)
indolizidine ISAs; and [0091] (f) nortropane ISAs.
[0092] However, ISA mixtures or combinations containing two or more
different ISAs representative of one or more of the classes listed
above may also be used.
[0093] Preferred are polyhydroxylated ISAs. Particularly preferred
are ISAs having a small molecular weight, since these may exhibit
desirable pharmacokinetics. Thus, the ISA may have a molecular
weight of 100 to 400 Daltons, preferably 150 to 300 Daltons and
most preferably 200 to 250 Daltons.
[0094] Also preferred are ISAs, which are analogues of
hydroxymethyl-substituted imino sugars in which one or more
hydroxymethyl groups are replaced with carboxyl groups.
Exemplary Piperidine Imino Sugar Acids
[0095] The ISA of the invention may be a piperidine ISA having at
least 3 free hydroxyl (or hydroxyalkyl) groups on the ring system
nucleus. Exemplary piperidine ISAs are hydroxypipecolic acids.
Particularly preferred hydroxypipecolic acids are
polyhydroxypipecolic acids having at least two (e.g. 3) free
hydroxyl (or hydroxyalkyl) groups on the ring system nucleus. Also
contemplated are N-acid derivatives of the foregoing and N-acid
derivatives of piperidine imino sugars such as
1-deoxynojirimycin.
[0096] Exemplary piperidine ISAs according to the invention are
compounds of formula (I):
##STR00002##
wherein [0097] R.sub.1 is absent or is C.sub.1-C.sub.6 alkyl;
[0098] the location of the carboxyl group may be switched from the
ring carbon to the ring nitrogen to produce an N-acid analogue of
the compound of formula (I); [0099] or an acyl (e.g. O-acyl)
derivative or dehydroxylated analogue in which one or more ring
hydroxyl(s) are absent; or a pharmaceutically acceptable salt or
derivative thereof.
[0100] Thus, exemplary piperidine ISAs according to the invention
may be selected from the structures shown below:
##STR00003## ##STR00004##
[0101] Also specifically contemplated are: (a) N-acid analogues of
the foregoing structures (G1)-(G4) in which the location of the
carboxyl group is switched from the ring carbon to the ring
nitrogen; (b) acyl (e.g. O-acyl) derivatives of the foregoing
structures (or of the N-acid analogues of (a)); (c) dehydroxylated
analogues of the foregoing structures (or of the N-acid analogues
of (a) and (b)) in which one or more ring hydroxyl(s) are
absent.
[0102] The compound of formula (G2) was first isolated from the
legume Baphia racemosa (Fabaceae) (see Booth et al., 2007:
(2R,3R,4R,5S)-3,4,5-trihydroxypipecolic acid dihydrate
[(2S,3R,4R,5S-trihydroxypiperidine-2-carboxylic acid dihydrate Acta
Crystalographica Section E63, 3783-3784 and references therein) but
the present inventors have now discovered that it is a major
component of Gymnema sylvestre (Asclepiadaceae). Although G.
sylvestre is widely claimed to have anti-diabetic and weight
control activity, most attention has focused on gymnemic acids
which can temporarily block sugar taste. These gymnemic acids and
other glycosides (saponins) appear to have some of the
anti-diabetic activity of the plant when tested in animal models.
The activity of these compounds appears to be due to affects on
membrane permeability in vitro although due to similar chemical
properties, imino sugar acids may well unknowingly also have been
present; it is not clear in our opinion that this effect on
membrane permeability would affect the pancreas in vivo (Persaud et
al., 1999, J. Endocrinol.: 163:207-12).
[0103] Piperidine ISAs for use according to the invention may be
isolated for example from Gymnema spp. (e.g. G. sylvestre) (see
Example 1, below).
Exemplary Pyrrolidine Imino Sugar Acids
[0104] The ISA of the invention may be pyrrolidine ISAs having at
least 1 (preferably at least 2 or 3) free hydroxyl (or
hydroxyalkyl) groups on the ring system nucleus. Preferred
pyrrolidine ISAs are hydroxyprolines. Particularly preferred
hydroxyprolines are polyhydroxyprolines having at least two (e.g.
at least 3) free hydroxyl (or hydroxyalkyl) groups on the ring
system nucleus. Also contemplated are N-acid derivatives of the
foregoing.
[0105] Exemplary pyrrolidine ISAs according to the invention may be
selected from the structures shown below:
##STR00005## ##STR00006##
[0106] Also specifically contemplated are: (a) N-acid analogues of
the foregoing structures S3 and S4 in which the location of the
carboxyl group is switched from the ring carbon to the ring
nitrogen, e.g. T4; (b) acyl (e.g. O-acyl) derivatives of the
foregoing structures (or of the N-acid analogues of (a)); and (c)
dehydroxylated analogues of the foregoing structures (or of the
N-acid analogues of (a) and (b)) in which one or more ring
hydroxyl(s) are absent (except that dehydroxylated analogues of the
monohydroxylated S3 and S4 are not contemplated).
[0107] Pyrrolidine ISAs for use according to the invention may be
isolated for example from Stevia spp. (e.g. S. rebaudiana) (see
Example 2, below).
Exemplary Pyrrolizidine Imino Sugar Acids
[0108] The ISA of the invention may be a pyrrolizidine ISA having
at least 2 (preferably at least 3, 4 or 5) free hydroxyl (or
hydroxyalkyl) groups on the ring system nucleus.
[0109] Exemplary pyrrolizidine ISAs according to the invention may
be selected from the structures shown below:
##STR00007##
[0110] Also specifically contemplated are: (a) acyl (e.g. O-acyl)
derivatives of the foregoing structures, (b) dehydroxylated
analogues of the foregoing structures in which one or more ring
hydroxyl(s) are absent.
[0111] Pyrrolizidine ISAs for use according to the invention may be
isolated for example from plants of the family Rutaceae (for
example Citrus spp., e.g. C. aurantium) (see Example 3, below).
Exemplary Indolizidine Imino Sugar Acids
[0112] The ISA of the invention may be an indolizidine ISA having
at least 2 (preferably at least 3, 4 or 5) free hydroxyl (or
hydroxyalkyl) groups on the ring system nucleus.
[0113] Exemplary indolizidine ISAs according to the invention may
be selected from the structures shown below:
##STR00008##
[0114] Also specifically contemplated are: (a) acyl (e.g. O-acyl)
derivatives of the foregoing structures and (b) dehydroxylated
analogues of the foregoing structures in which one or more ring
hydroxyl(s) are absent.
[0115] Indolizidine ISAs for use according to the invention may be
isolated for example from Citrus species (Rutaceae), Lotus species,
Castanospemium and Alexa species (Fabaceae); Eugenia and Syzygium
species (Myrtaceae) and Cucurbita species (Cucurbitaceae).
Exemplary Nortropane Imino Sugar Acids
[0116] The ISA of the invention may be a nortropane ISA having at
least 2 (preferably at least 3) free hydroxyl (or hydroxyalkyl)
groups on the ring system nucleus. Also contemplated are N-acid
derivatives of the foregoing. An exemplary nortropane ISA according
to the invention has the structure shown below:
[0117] Nor-tropane ISAs for use according to the invention may be
isolated for example from plants in the Solanaceae (e.g. Solanum
and Lycium species) and Moraceae (Mulberry).
##STR00009##
Exemplary Ring-Open Imino Sugars
[0118] Also considered are amino sugars acids formed by the opening
of the imino ring such as compound P1 and P2 (found in Cucurbita
species) and P3. Such compounds may also be the biological
precursors of the imino sugar acids.
##STR00010##
Biological Activities and Functional Attributes of the ISAs of the
Invention
[0119] The ISAs of the invention preferably do not inhibit
glucosidase activity (or do not inhibit glucosidase activity to a
clinically-significant extent). Without wishing to be bound by any
theory, it is thought that the ISAs of the invention may stimulate,
directly or indirectly, pancreatic .beta.-cell activity and/or
regeneration in vivo. Thus, preferred ISAs for use according to the
invention stimulate, directly or indirectly, pancreatic .beta.-cell
activity and/or regeneration in vivo and improve insulin response.
In such embodiments, the ISAs find particular application in the
treatment of type 1 (or insulin-dependent) diabetes, since the ISA
may promote functional regeneration of pancreatic .beta.-cells (as
reported for Gymnema extracts by Shanmugasundaram et al. (1990) Use
of Gymnema sylvestre leaf extract in the control of blood glucose
in insulin-dependent diabetes mellitus. J Ethnopharmacol. 1990
October; 30(3): 281-94).
[0120] Without wishing to be bound by any theory, the compounds may
inhibit glucuronidases, iduronidase, sialidase or hexosaminidases.
Reducing glucuronidase activity may for example improve beta cell
function, directly or indirectly, via improved removal of toxins as
glucuronides.
[0121] Compounds which stimulate pancreatic .beta.-cell activity
and/or regeneration in vivo may be readily identified by various
methods known in the art, including both in vivo and ex vivo
cell-based assays. For example, insulin release from isolated
pancreatic beta-islet cells may be used as an index of stimulatory
activity and measured according to the following method.
[0122] Spague Dawley rats are killed by cervical dislocation then
the branch of the bile duct leading to the liver and the duodenal
end of the duct in the pancreas clamped. Collagenase solution (type
V, 50 mg/ml) is then injected into the bile duct, distending the
pancreas, which is then removed and incubated for 12 minutes at
37.degree. C. 10 ml of cold Hank's buffer is added and the
suspension agitated vigorously for 1 min. After 5 minutes on ice,
settled islets are washed three times using ice-cold Hank's buffer
and good sized islets selected under microscrope for transfer to a
perifusion apparatus (Dickinson et al. 1997. Eur J Pharmacol., 339,
69-76). Islets from several rats were pooled then twenty selected
for each perifusion chamber containing oxygenated (95% O2/5% CO2)
Gey & Gey buffer with 1 mg/ml bovine serum albumin and glucose.
Islets are perifused for one hour in medium containing 4 mM glucose
to equilibrate then perifusate collected at two minute intervals
(the first five fractions being used to establish baseline insulin
levels). The media in the perifusion container is then changed to
one containing the test concentration of glucose and test compound
with subsequent fractions being collected for a further hour.
Levels of insulin released in the perifusant are then assayed using
a 96-well ELISA.
[0123] Compounds for use according to the invention which improve
insulin response may be readily identified by various methods known
in the art, including both in vivo and ex vivo cell-based assays.
For example, the ability of compounds of the invention to modulate
carbohydrate tolerance can be determined by the following in vivo
assay in lean mice as a model.
[0124] Male ddy (29-33 g) or C57BL/6J (4-6 weeks) mice are fasted
overnight and then used for acute carbohydrate loading tests.
Glucose (2.5 g/kg body weight), maltose (2.5 g/kg body weight),
sucrose (2.5 g/kg body weight), isosucrose (2.5 g/kg body weight)
or starch (1 g/kg body weight) as well as the test compounds were
dissolved in 0.9% NaCl solution and administered to mouse via
stomach tube. A control group is loaded with saline only. Blood
samples are taken from the tail vein into lithium heparinised tubes
and plasma separated by centrifugation both preloading and at
various times post loading. Plasma glucose and insulin are measured
using commercially available kits.
[0125] Compounds having particular utility in the treatment of
obesity may be readily identified by various methods known in the
art, including both in vivo and ex vivo cell-based assays. For
example, the effect of compounds of the invention in diet-induced
obese mice may be determined as follows.
[0126] Male C57BL/6J (4-6 weeks) mice are allowed free access to a
high fat diet (45% kcal obtained from fat, Research Diets USA) and
water. After 7 days acclimatization, mice were dosed in groups of
ten mice orally with different concentrations of test compound or
vehicle for 32 days. Both a fed and fasted carbohydrate test using
glucose is performed at 28 days with plasma glucose and insulin
measured. All animals are then returned to a normal diet. At the
end of the experiment, plasma glucose, insulin, triglycerides,
cholesterol is measured. Body fat, protein, water and ash levels of
the carcasses are also measured using standard chemical analysis
techniques (Dickinson et al 2001. Physiology and Behaviour 74,
425-433).
[0127] Compounds having particular utility in the treatment of
metabolic dysfunction may be readily identified by various methods
known in the art, including both in vivo and ex vivo cell-based
assays. For example, db/db mice can be used as a chronic model of
metabolic dysfunction, as described below.
[0128] db/db mice obtained from Jackson Labs were allowed 14 days
acclimatisation with access to standard laboratory chow and water
where body weight is monitored prior to experimentation. Basel
levels of plasma glucose and insulin as well as HbA1c are measured
to allow group assignment and then animals are dosed in groups of
twelve mice orally with different concentrations of test compound
or vehicle for 6 weeks. Daily measurements of food and water Wake
as well as bodyweight are taken with HbA1c monitored weekly. Both a
fed and fasted carbohydrate test using glucose is performed at
different points in the experiment with plasma glucose and insulin
measured.
Chemical Synthesis
[0129] The ISAs described herein may be made by conventional
methods. Methods of making heteroaromatic ring systems are well
known in the art. In particular, methods of synthesis are discussed
in Comprehensive Heterocyclic Chemistry, Vol. 1 (Eds.: A R
Katritzky, C W Rees), Pergamon Press, Oxford, 1984 and
Comprehensive Heterocyclic Chemistry II: A Review of the Literature
1982-1995 The Structure, Reactions, Synthesis, and Uses of
Heterocyclic Compounds, Alan R. Katritzky (Editor), Charles W. Rees
(Editor), E. F. V. Scriven (Editor), Pergamon Pr, June 1996. Other
general resources which would aid synthesis of the compounds of
interest include March's Advanced Organic Chemistry: Reactions,
Mechanisms, and Structure, Wiley-Interscience; 5th edition (Jan.
15, 2001). Some exemplary synthetic schemes for producing ISAs for
use according to the invention are shown below:
##STR00011##
[0130] Further relevant information may be found in the following
references, the content of which is incorporated herein by
reference:
[0131] Synthesis of 2S-carboxy-3R,4R,5S-trihydroxypiperidine, a
naturally occurring inhibitor of .beta.-D-glucuronidase. Bernotas,
Ronald C.; Ganem, Bruce. Dep. Chem., Cornell Univ., Ithaca, N.Y.,
USA. Tetrahedron Letters (1985), 26(41), 4981-2
[0132] Enantiospecific syntheses of 2S,3R,4R,5S-trihydroxypipecolic
acid, 2R,3R,4R,5S-trihydroxypipecolic acid,
2S,4S,5S-dihydroxypipecolic acid, and bulgecinine from
D-glucuronolactone. Bashyal, B. P.; Chow, H. F.; Fleet, G. W. J.
Dyson Perrins Lab., Oxford Univ., Oxford, UK. Tetrahedron Letters
(1986), 27(27), 3205-8.
[0133] The synthesis of polyhydroxylated amino acids from
glucuronolactone: enantiospecific syntheses of
2S,3R,4R,5S-trihydroxypipecolic acid,
2R,3R,4R,5S-trihydroxypipecolic acid and 2R,3R,4R-dihydroxyproline.
Bashyal, Bharat P.; Chow, Hak Fun; Fellows, Linda E.; Fleet, George
W. J. Dyson Perrins Lab., Oxford Univ., Oxford, UK. Tetrahedron
(1987), 43(2), 415-22.
[0134] Synthesis of deoxymannojirimycin, fagomine, and
deoxynojirimycin, 2-acetamido-1,5-imino-1,2,5-trideoxy-D-mannitol,
2-acetamido-1,5-imino-1,2,5-trideoxy-D-glucitol,
2S,3R,4R,5R-trihydroxypipecolic acid and
2S,3R,4R,5S-trihydroxypipecolic acid from methyl
3-O-benzyl-2,6-dideoxy-2,6-imino-.alpha.-D-mannofuranoside. Fleet,
George W. J.; Fellows, L. E.; Smith, Paul W. Dyson Perrins Lab.,
Oxford Univ., Oxford, UK. Tetrahedron (1987), 43(5), 979-90.
[0135] Preparation of 2-carboxy-3,4,5-trihydroxypiperidines as
allergy inhibitors, antiarthritics, and for control of mucous
production. Lockhoff, Oswald; Hayauchi, Yutaka. (Bayer A.-G., Fed.
Rep. Ger.). Ger. Offen. (1988), 16 pp. CODEN: GWXXBX DE 3628486 A1
19880225 Patent written in German.
[0136] Synthesis of aza sugars as potent inhibitors of
glycosidases. Le Merrer, Yves; Poitout, Lydie; Depezay,
Jean-Claude; Dosbaa, Isabelle; Geoffroy, Sabine; Foglietti,
Marie-Jose. Laboratoire de Chimie et Biochimie Pharmacologiques et
Toxicologiques, Universite Rene Descartes, associe au CNRS, Paris,
Fr. Bioorganic & Medicinal Chemistry (1997), 5(3), 519-533.
[0137] A new asymmetric synthesis of
(2S,3R,4R,5S)-trihydroxypipecolic acid. Tsimilaza, Andriamihamina;
Tite, Tony; Boutefnouchet, Sabrina; Lallemand, Marie-Christine;
Tillequin, Francois; Husson, Henri-Philippe. Laboratoire de
Pharmacognosie, Faculte des Sciences Pharmaceutiques et
Biologiques, UMR 8638 Associee au CNRS, l'Universite
Paris-Descartes, Paris, Fr, Tetrahedron: Asymmetry (2007), 18(13),
1585-1588.
[0138] Synthesis of both enantiomers of hydroxypipecolic acid
derivatives equivalent to 5-azapyranuronic acids and evaluation of
their inhibitory activities against glycosidases. Yoshimura,
Yuichi; Ohara, Chiaki; Imahori, Tatsushi; Saito, Yukako; Kato,
Atsushi; Miyauchi, Saori; Adachi, Isao; Takahata, Hiroki. Faculty
of Pharmaceutical Sciences, 4-4-1, Komatsushima, Tohoku
Pharmaceutical University, Aoba-ku, Miyagi, Sendai, Japan.
Bioorganic & Medicinal Chemistry (2008), 16(17), 8273-8286.
Purification from Botanic Sources
[0139] The ISAs described herein may be isolated from natural
sources. For example, plant material from the following botanic
sources may be used as starting material for the isolation and
purification of the ISAs for use according to the invention:
Stevia, Gymnema, Citrus, Andrographis paniculata, Lycium species,
leguminous spp. e.g. Aspalanthus linearis (Rooibos), Glycine max,
Lotus species and Castanospermum australe (Fabaceae) and
Cucurbitaceae species. The ISAs of the invention are water-soluble
and can be concentrated by anion exchange chromatography or cation
exchange chromatography. Size exclusion methods can also be used to
concentrate them. Thus, it will be appreciated that those skilled
in the art can readily purify and isolate the ISAs of the invention
using standard techniques.
Medical Applications
[0140] The invention finds broad application in the treatment of
any energy utilization disease.
[0141] Thus, diseases which may be treated according to the
invention include, for example, disorders of homeostasis, metabolic
diseases, dysfunction of sugar metabolism and appetite
disorders.
[0142] In preferred embodiments, the invention finds application in
the treatment of insulin resistance, various forms of diabetes,
metabolic syndrome, obesity, wasting syndromes (for example, cancer
associated cachexia), myopathies, gastrointestinal disease, growth
retardation, hypercholesterolemia, atherosclerosis and
age-associated metabolic dysfunction.
[0143] The invention may also be used for the treatment of
conditions associated with metabolic syndrome, obesity and/or
diabetes, including for example hyperglycaemia, glucose
intolerance, hyperinsulinaemia, glucosuria, metabolic acidosis,
cataracts, diabetic neuropathy, diabetic nephropathy, diabetic
retinopathy, macular degeneration, glomerulosclerosis, diabetic
cardiomyopathy, insulin resistance, impaired glucose metabolism,
arthritis, hypertension, hyperlipidemia, osteoporosis, osteopenia,
bone loss, brittle bone syndromes, acute coronary syndrome,
infertility, short bowel syndrome, chronic fatigue, eating
disorders, intestinal motility dysfunction and sugar metabolism
dysfunction.
[0144] The invention may also be used to suppress appetite.
[0145] Particularly preferred is the treatment of insulin
resistance, metabolic syndrome, obesity and diabetes (particularly
type 2 diabetes).
Insulin Resistance, Metabolic Syndrome and Diabetes
[0146] The invention finds application in the treatment of insulin
resistance. Insulin resistance is characterized by a reduced action
of insulin in skeletal muscle, adipocytes and hepatocytes so that
normal amounts of insulin become inadequate to produce a normal
insulin response from the cells of these tissues. In adipocytes,
insulin resistance results in hydrolysis of stored triglycerides,
leading to elevated free fatty acids in the blood plasma. In
muscle, insulin resistance reduces glucose uptake while in
hepatocytes it reduces glucose storage. In both of the latter cases
an elevation of blood glucose concentrations results. High plasma
levels of insulin and glucose due to insulin resistance often
progresses to metabolic syndrome and type 2 diabetes.
[0147] The invention finds application in the treatment of
metabolic syndrome (as herein defined). The disorder is also known
as (metabolic) syndrome X, insulin resistance syndrome, Reaven's
syndrome and CHAOS.
[0148] The invention finds application in the treatment of diseases
associated with metabolic syndrome, including for example: fatty
liver (often progressing to non-alcoholic fatty liver disease),
polycystic ovarian syndrome, hemochromatosis (iron overload) and
acanthosis nigricans (dark skin patches).
[0149] The invention finds application in the treatment of Type 2
diabetes. Type 2 diabetes is a chronic disease that is
characterised by persistently elevated blood glucose levels
(hyperglycaemia). Insulin resistance together with impaired insulin
secretion from the pancreatic .beta.-cells characterizes the
disease. The progression of insulin resistance to type 2 diabetes
is marked by the development of hyperglycaemia after eating when
pancreatic .beta.-cells become unable to produce adequate insulin
to maintain normal blood sugar levels (euglycemia)).
[0150] The invention finds application in the treatment of Type 1
diabetes (or insulin dependent diabetes). Type 1 diabetes is
characterized by loss of the insulin-producing beta cells of the
islets of Langerhans in the pancreas, leading to a deficiency of
insulin. The main cause of this beta cell loss is a T-cell mediated
autoimmune attack. There is no known preventative measure that can
be taken against type 1 diabetes, which comprises up to 10% of
diabetes mellitus cases in North America and Europe. Most affected
people are otherwise healthy and of a healthy weight when onset
occurs. Sensitivity and responsiveness to insulin are usually
normal, especially in the early stages.
[0151] Thus, the invention finds broad application in the treatment
and/or prophylaxis of metabolic syndrome and/or diabetes (including
type 1 and type 2 diabetes and insulin resistance), including in
particular obesity (especially central obesity) and elevated serum
triglyceride levels.
[0152] These medical applications may be applied to any
warm-blooded animal, including humans. The applications include
veterinary applications, wherein the ISAs are administered to
non-human animals, including primates, dogs, cats, horses, cattle
and sheep.
Herbal Quality Control Aspects
Food Additive Samples
[0153] The food additive samples used in the methods of the present
invention may be dried plant material or aliquots of the herbal
food additive in the form in which it is added to foodstuffs and
beverages. Alternatively, the samples may be pre-processed in any
of a wide variety of ways prior to characterization. Pre-processing
may involve physical or chemical pre-processing, for example
powdering, grinding, freezing, evaporation, filtration, pressing,
spray drying, extrusion, supercritical solvent extraction and
tincture production.
[0154] Preferably, the food additive sample is fractionated prior
to characterization. Any suitable method of fractionation may be
employed, including solvent extraction(s). In a preferred
embodiment the sample is fractionated by: (a) ion-exchange
chromatography to produce an extract enriched in polar compounds
and a non-polar residue; and then (b) chromatographic fractionation
of the enriched extract of step (a) to yield one or more polar
fractions comprising one or more polar phytochemical(s). In such
embodiments the chromatographic fractionation preferably comprises
gas-liquid chromatography (GC), for example GC-MS. When GC is used,
the enriched extract may be derivatized prior to
chromatography.
[0155] In cases where the herbal food additive is administered or
sold in the form of a whole plant (or part thereof), the plant
material may be dried prior to use. Any convenient form of drying
may be used, including freeze-drying, spray drying or
air-drying.
Detection of Imino Sugar Acids and Imino Sugars
[0156] Any suitable form of characterization of the food additive
sample may be employed, including without limitation functional
and/or physical and/or chemical characterization, sufficient to
detect the presence or absence or measure the amount of imino sugar
acid/imino sugar in the sample.
[0157] Where the samples are physically characterized, the
characterization may be selected from: (a) quantification of the
phytochemical component(s); and/or (b) measurement of the purity of
the constituents; and/or (c) determination of molecular weight (or
molecular weight distribution or various statistical functions
thereof in the case of fractions which comprise a plurality of
different phytochemical constituents); and/or (d) determination of
the molecular formula (e) (e.g. by nuclear magnetic resonance);
and/or (e) spectral analysis.
[0158] Spectral analysis is particularly preferred, and may produce
any or all of the following spectra: [0159] (a) mass spectra (e.g.
the mass to charge (m/z) value versus abundance), and/or [0160] (b)
chromatographic data (e.g. spectra, column retention times, elution
profiles etc), and/or [0161] (c) photodiode array (PDA) spectra
(e.g. in both UV and visible ranges), and/or [0162] (d)
electrochemical detection [0163] (e) nuclear magnetic resonance
(NMR) spectra (e.g. spectral data sets obtained via .sup.1H and/or
.sup.13C NMR).
[0164] When used according to the invention, the spectral analysis
may be coupled with fractionation of the sample, for example by use
of GC-MS and/or HPLC-PDA-MS.
[0165] Particularly preferred is the use of GC-MS to detect the
presence or absence or measure the amount of imino sugar acid/imino
sugar in the sample.
[0166] Where the samples are chemically characterized, the
characterization may be selected from measurements of the chemical
reactivity of phytochemical constituent(s), the solubility of
phytochemical constituent(s), the stability and melting point of
phytochemical constituent(s) or any combination thereof.
[0167] Where the samples are functionally characterized, the
characterization may comprise a biological assay, for example
selected from in vivo or in vitro assays, enzyme inhibition assays
(for example glycosidase and/or lipase inhibition), receptor
binding assays, cellular assays (e.g. cell replication,
cell-pathogen, cell-cell interaction and cell secretion assays),
immunoassays, anti-microbial activity (e.g. bacterial and viral
cell-binding and/or replication) assays, toxicity assays (e.g.
LD.sub.50 assays) or any combination thereof.
Solvent Extractions
[0168] Suitable polar solvents for use in the process of the
invention include without limitation organic solvents such as
organic alcohols. Preferred are ethanol and methanol, as well as
ethanol/water or methanol/water mixtures. Preferably, the polar
solvent is selected from 51 to 80% ethanol/water, 31 to 50%
ethanol/water, and up to 30% ethanol/water. Particularly preferred
is a polar solvent which is approximately 50% ethanol/water.
Suitable non-polar solvents for use in the process of the invention
include without limitation organic solvents such as hexane and
dichloromethane (DCM) or chloroform. Particularly preferred is
dichloromethane. The conditions (time, temperature, degree of
agitation etc.) under which the extraction(s) are performed can be
readily determined empirically and vary according to the nature of
the sample, the nature of any pre-processing and the solvent system
selected.
Chromatographic Fractionation
[0169] Chromatographic fractionation may comprise gas-liquid
chromatography. Gas-liquid chromatography is a process whereby a
complex mixture of volatile substances is separated into its
constituents by partitioning the sample between an inert gas under
pressure and a thin layer of non-volatile liquid coated on an inert
support inside a heated column. In order to achieve a good
separation of specific compounds in a mixture, it is crucial to use
a column with the correct characteristics. The nature of the solid
support, type and amount of liquid phase, method of packing,
overall length and column temperature are important factors.
[0170] Those skilled in the art, by routine trial and error and by
using common general knowledge, will be able readily to determine
the appropriate column characteristics according to the
circumstances, including inter alia the extract under study and the
nature of the solvent used in the extraction and the types of
chemicals expected in those solvents. Particularly preferred, and
useful in many circumstances, are capillary columns coated with a
non-polar liquid phase (25 m.times.0.22 mm id.times.0.25 .mu.m BPX5
stationary phase, produced by SGE Ltd., or equivalents
thereof).
[0171] Many compounds are unsuitable for direct injection into a
gas chromatograph because of their high polarity, low volatility or
thermal instability. Compounds that are highly hydroxylated are
difficult to vapourise because of inter-molecular hydrogen bonding.
However, by replacing the hydroxyl hydrogens with other chemical
groups, they can be made sufficiently volatile for GC analysis. The
two most popular means of derivatising hydroxyl groups are
acetylation and silylation, where acetylates [CH.sub.3CO--O--R] or
silyl ethers, e.g. trimethylsilyl (TMS) ethers
[(CH.sub.3).sub.3Si--O--R] are formed. Thus, in embodiments where
the enriched extract is chromatographically fractionated on an
analytical scale the phytochemical constituents of the enriched
extract are preferably derivatized, for example by acylation or
silylation. Particularly preferred is trimethyl silyl (TMS)
derivatization.
[0172] Chromatographic fractionation may also comprise ion exchange
chromatography. Ion-exchange chromatography partially purifies
ionic species to concentrate them and remove contaminating
substances. Those skilled in the art, by routine trial and error
and using common general knowledge, will be able readily to
identify suitable column packing materials and mobile phase(s),
which will depend inter alia on the quantities to be fractionated,
the extracts under study and the nature of the solvent used in the
extraction. Particularly preferred in the methods of the present
invention are strongly acidic cation exchange resins which can be
used in either the free acid or hydrogen (H.sup.+) form or in the
ammonium (NH.sub.4.sup.+) salt form). These forms adsorb cations
from solution and release an equivalent number of counter-ions back
into solution (either H.sup.+ or NH.sub.4.sup.+ ions, depending on
the form used). Also preferred are strongly basic anion exchange
resins which when used in the hydroxide form (OH-) will strongly
bind imino sugar acids. The imino sugar acids can then be released
by the use of acids such as acetic acid (e.g. a 2M solution).
Fraction Characterization
[0173] The form the characterization takes depends on the nature of
the herbal food additive under study and the characterization
techniques employed. In general, any or all of the following
approaches may be used:
(a) Functional Characterization
[0174] The functional characterization may comprise a biological
assay. Biological assays may be carried out in vivo or in vitro,
and may include enzyme inhibition assays (for example glycosidase
and/or lipase inhibition). Other biological assays include receptor
binding assays, cellular assays (including cell replication,
cell-pathogen and cell-cell interaction and cell secretion assays),
immunoassays, anti-microbial activity (e.g. bacterial and viral
cell-binding and/or replication) assays and toxicity assays (e.g.
LD.sub.50 assays).
[0175] Functional characterization may also be carried out
indirectly by a form of characterization which permits the
identification of one or more indices of biological activity.
(b) Physical Characterization
[0176] This can take the form of quantification of the
phytochemical component(s) present in any given fraction or at any
other stage in the process, measurement of the purity of the
constituents, determination of molecular weight (or molecular
weight distribution or various statistical functions thereof in the
case of fractions which comprise a plurality of different
phytochemical constituents), determination of the molecular formula
(e) (e.g. by nuclear magnetic resonance) and various spectral
analyses.
[0177] Particularly useful spectral characteristics include: [0178]
Mass spectra (e.g. the mass to charge (m/z) value versus
abundance), and/or [0179] Chromatographic data (e.g. spectra,
column retention times, elution profiles etc), and/or [0180]
Photodiode array (PDA) spectra (e.g. in both UV and visible
ranges), and/or [0181] Nuclear magnetic resonance (NMR) spectra
(including spectral data sets obtained via .sup.1H and/or .sup.13C
NMR).
[0182] Spectral characterization can be coupled with the
fractionation step. For example, GC-MS and HPLC-PDA-MS can be used
(as described herein) to couple the fractionation with the
obtention of mass spectral, UV-visible spectral and chromatographic
spectral data.
[0183] Any or all of the above characteristics can be used to
define a "chemical fingerprint" for any given sample (or any
fraction or phytochemical constituent thereof).
(c) Chemical Characterization
[0184] This can take the form of measurements inter alia of the
chemical reactivity of phytochemical constituent(s), their
solubility, stability and melting point.
Posology
[0185] The ISAs of the present invention can be administered by
oral or parenteral routes, including intravenous, intramuscular,
intraperitoneal, subcutaneous, transdermal, airway (aerosol),
rectal, vaginal and topical (including buccal and sublingual)
administration. Preferred is oral administration.
[0186] The amount of the ISA administered can vary widely according
to the particular dosage unit employed, the period of treatment,
the age and sex of the patient treated, the nature and extent of
the disorder treated, and the particular ISA selected.
[0187] Moreover, the ISAs of the invention can be used in
conjunction with other agents known to be useful in the treatment
of metabolic syndrome and/or diabetes (including type 1 and type 2
diabetes and insulin resistance) and in such embodiments the dose
may be adjusted accordingly.
[0188] In general, the effective amount of the ISA administered
will generally range from about 0.01 mg/kg to 500 mg/kg daily. A
unit dosage may contain from 0.05 to 500 mg of the ISA, and can be
taken one or more times per day. The ISA can be administered with a
pharmaceutical carrier using conventional dosage unit forms either
orally, parenterally, or topically, as described below.
[0189] The preferred route of administration is oral
administration. In general a suitable dose will be in the range of
0.01 to 500 mg per kilogram body weight of the recipient per day,
preferably in the range of 0.1 to 50 mg per kilogram body weight
per day and most preferably in the range 1 to 5 mg per kilogram
body weight per day.
[0190] The desired dose is preferably presented as a single dose
for daily administration. However, two, three, four, five or six or
more sub-doses administered at appropriate intervals throughout the
day may also be employed. These sub-doses may be administered in
unit dosage forms, for example, containing 0.001 to 100 mg,
preferably 0.01 to 10 mg, and most preferably 0.5 to 1.0 mg of
active ingredient per unit dosage form.
Formulation
[0191] The compositions of the invention comprise the ISA of the
invention, optionally together with a pharmaceutically acceptable
excipient.
[0192] The ISA of the invention may take any form. It may be
synthetic, purified or isolated from natural sources (for example
from any of the botanic sources identified herein, including for
example a botanical source selected from: (a) Stevia spp. (e.g. S.
rebaudiana); (b) Gymnema spp. (e.g. G. sylvestre); (c) Andrographis
spp. (e.g. A. paniculata); (d) leguminous species (e.g. Aspalanthus
spp., Baphia spp., Glycine max, Alexa spp. Castanospermum
australe), Lotus spp.; (e) plants of the family Rutaceae (for
example Citrus spp., e.g. C. aurantium); (f) Lycium barbarum (Goji)
and (g) plants of the Cucurbitaceae (e.g. C. ficifolia, Siam
Pumpkin and Momordica charantia).
[0193] When isolated from a natural source, the ISA of the
invention may be purified. However, the compositions of the
invention may take the form of herbal medicines, as hereinbefore
defined. Such herbal medicines preferably are analysed to determine
whether they meet a standard specification prior to use.
[0194] The herbal medicines for use according to the invention may
be dried plant material. Alternatively, the herbal medicine may be
processed plant material, the processing involving physical or
chemical pre-processing, for example powdering, grinding, freezing,
evaporation, filtration, pressing, spray drying, extrusion,
supercritical solvent extraction and tincture production. In cases
where the herbal medicine is administered or sold in the form of a
whole plant (or part thereof), the plant material may be dried
prior to use. Any convenient form of drying may be used, including
freeze-drying, spray drying or air-drying.
[0195] In embodiments where the ISA of the invention is formulated
together with a pharmaceutically acceptable excipient, any suitable
excipient may be used, including for example inert diluents,
disintegrating agents, binding agents, lubricating agents,
sweetening agents, flavouring agents, colouring agent and
preservatives. Suitable inert diluents include sodium and calcium
carbonate, sodium and calcium phosphate, and lactose, while
cornstarch and alginic acid are suitable disintegrating agents.
Binding agents may include starch and gelatin, while the
lubricating agent, if present, will generally be magnesium
stearate, stearic acid or talc.
[0196] The pharmaceutical compositions may take any suitable form,
and include for example tablets, elixirs, capsules, solutions,
suspensions, powders, granules and aerosols.
[0197] The pharmaceutical composition may take the form of a kit of
parts. The kit may comprise the composition of the invention
together with instructions for use and/or a plurality of different
components in unit dosage form.
[0198] Tablets for oral use may include the ISA of the invention,
either alone or together with other plant material associated with
the botanical source(s) (in the case of herbal medicine
embodiments). The tablets may contain the ISA of the invention
mixed with pharmaceutically acceptable excipients, such as inert
diluents, disintegrating agents, binding agents, lubricating
agents, sweetening agents, flavouring agents, colouring agents and
preservatives. Suitable inert diluents include sodium and calcium
carbonate, sodium and calcium phosphate, and lactose, while
cornstarch and alginic acid are suitable disintegrating agents.
Binding agents may include starch and gelatin, while the
lubricating agent, if present, will generally be magnesium
stearate, stearic acid or talc. If desired, the tablets may be
coated with a material such as glyceryl monostearate or glyceryl
distearate, to delay absorption in the gastrointestinal tract.
[0199] Capsules for oral use include hard gelatin capsules in which
the ISA of the invention is mixed with a solid diluent, and soft
gelatin capsules wherein the active ingredient is mixed with water
or an oil such as peanut oil, liquid paraffin or olive oil. For
oral administration the ISA of the invention can be formulated into
solid or liquid preparations such as capsules, pills, tablets,
troches, lozenges, melts, powders, granules, solutions,
suspensions, dispersions or emulsions (which solutions, suspensions
dispersions or emulsions may be aqueous or non-aqueous). The solid
unit dosage forms can be a capsule which can be of the ordinary
hard- or soft-shelled gelatin type containing, for example,
surfactants, lubricants, and inert fillers such as lactose,
sucrose, calcium phosphate, and cornstarch. In another embodiment,
the ISAs of the invention are tableted with conventional tablet
bases such as lactose, sucrose, and cornstarch in combination with
binders such as acacia, cornstarch, or gelatin, disintegrating
agents intended to assist the break-up and dissolution of the
tablet following administration such as potato starch, alginic
acid, corn starch, and guar gum, lubricants intended to improve the
flow of tablet granulations and to prevent the adhesion of tablet
material to the surfaces of the tablet dies and punches, for
example, talc, stearic acid, or magnesium, calcium, or zinc
stearate, dyes, colouring agent, and flavouring agent intended to
enhance the aesthetic qualities of the tablets and make them more
acceptable to the patient. Suitable excipients for use in oral
liquid dosage forms include diluents such as water and alcohols,
for example, ethanol, benzyl alcohol, and the polyethylene
alcohols, either with or without the addition of a pharmaceutically
acceptably surfactant, suspending agent or emulsifying agent.
[0200] The ISAs of the invention may also be administered
parenterally, that is, subcutaneously, intravenously,
intramuscularly, or interperitoneally.
[0201] In such embodiments, the ISA is provided as injectable doses
in a physiologically acceptable diluent together with a
pharmaceutical carrier (which can be a sterile liquid or mixture of
liquids). Suitable liquids include water, saline, aqueous dextrose
and related sugar solutions, an alcohol (such as ethanol,
isopropanol, or hexadecyl alcohol), glycols (such as propylene
glycol or polyethylene glycol), glycerol ketals (such as
2,2-dimethyl-1,3-dioxolane-4-methanol), ethers (such as
poly(ethylene-glycol) 400), an oil, a fatty acid, a fatty acid
ester or glyceride, or an acetylated fatty acid glyceride with or
without the addition of a pharmaceutically acceptable surfactant
(such as a soap or a detergent), suspending agent (such as pectin,
carhomers, methylcellulose, hydroxypropylmethylcellulose, or
carboxymethylcellulose), or emulsifying agent and other
pharmaceutically adjuvants. Suitable oils which can be used in the
parenteral formulations of this invention are those of petroleum,
animal, vegetable, or synthetic origin, for example, peanut oil,
soybean oil, sesame oil, cottonseed oil, corn oil, olive oil,
petrolatum, and mineral oil.
[0202] Suitable fatty acids include oleic acid, stearic acid, and
isostearic acid. Suitable fatty acid esters are, for example, ethyl
oleate and isopropyl myristate.
[0203] Suitable soaps include fatty alkali metal, ammonium, and
triethanolamine salts and suitable detergents include cationic
detergents, for example, dimethyl dialkyl ammonium halides, alkyl
pyridinium halides, and alkylamines acetates; anionic detergents,
for example, alkyl, aryl, and olefin sulphonates, alkyl, olefin,
ether, and monoglyceride sulphates, and sulphosuccinates; nonionic
detergents, for example, fatty amine oxides, fatty acid
alkanolamides, and polyoxyethylenepolypropylene copolymers; and
amphoteric detergents, for example, alkyl-beta-aminopropionates,
and 2-alkylimidazoline quarternary ammonium salts, as well as
mixtures.
[0204] The parenteral compositions of this invention will typically
contain from about 0.5 to about 25% by weight of the ISA of the
invention in solution. Preservatives and buffers may also be used.
In order to minimize or eliminate irritation at the site of
injection, such compositions may contain a non-ionic surfactant
having a hydrophile-lipophile balance (HLB) of from about 12 to
about 17. The quantity of surfactant in such formulations ranges
from about 5 to about 15% by weight. The surfactant can be a single
component having the above HLB or can be a mixture of two or more
components having the desired HLB. Illustrative of surfactants used
in parenteral formulations are the class of polyethylene sorbitan
fatty acid esters, for example, sorbitan monooleate and the high
molecular weight adducts of ethylene oxide with a hydrophobic base,
formed by the condensation of propylene oxide with propylene
glycol.
[0205] When used adjunctively, the ISAs of the invention may be
formulated for use with one or more other drug(s). In particular,
the ISAs of the invention may be used in combination with
anti-diabetic agents (as herein described).
[0206] For example, the ISAs of the invention may be used with
anti-diabetic agents selected from the following drug classes:
biguanide, sulfonylurea, thiazolidinediones, .alpha.-glucosidase
inhibitors, meglitinides, peptide analogs, dipeptidyl peptidase-4
(DPP-4) inhibitors and amylin agonist. Adjunctive use may be
reflected in a specific unit dosage designed to be compatible (or
to synergize) with the other drug(s), or in formulations in which
the ISA is admixed with one or more anti-diabetic agent(s) (or else
physically associated with the other agent(s) within a single unit
dose). Adjunctive uses of the ISAs of the invention may also be
reflected in the composition of the pharmaceutical kits of the
invention, in which the ISA of the invention is co-packaged (e.g.
as part of an array of unit doses) with the anti-diabetic drug(s).
Adjunctive use may also be reflected in information and/or
instructions relating to the co-administration of the ISA with
anti-diabetic drug(s).
[0207] The ISAs of the invention may also be formulated as
nutraceuticals forming part of a beverage or foodstuff.
EXEMPLIFICATION
[0208] The invention will now be described with reference to
specific Examples. These are merely exemplary and for illustrative
purposes only: they are not intended to be limiting in any way to
the scope of the monopoly claimed or to the invention described.
These examples constitute the best mode currently contemplated for
practicing the invention.
Example 1
Detection of the Compound of Formula (G2) in Gymnema
[0209] Gymnema sylvestre is a liana or climbing plant with stems up
to 8 m in length. It grows in open woods and bushland at an
altitude of 100-1000 m in India, China, Indonesia, Japan, Malaysia,
Sri Lanka, Vietnam and South Africa: Both the leaf and root are
used in Ayurvedic medicine. Because of its property of abolishing
the taste of sugar it was given the Hindi names of Gurmar and
Madhunashini meaning `sugar destroying` The herb is traditionally
used for the treatment of metabolic syndrome and Gymnema extracts
are sold in Japan for the control of obesity.
[0210] A controlled study on insulin-dependent diabetics found that
a water-soluble Gymnema extract (400 mg/day) reduced insulin
requirements (by about 50%) (Shanmugasundaram et al. (1990), J
Ethnopharmacol. 30:281-294). Over the duration of treatment Gymnema
lowered fasting mean blood glucose (by about 35%), glycosylated
haemoglobin and glycosylated plasma protein levels from baseline
values. Cholesterol was significantly reduced and brought to near
normal levels. Triglycerides, free fatty acids and serum amylase
were also lowered. The treatment period ranged from 6-30 months.
The significant decrease in glycosylated haemoglobin occurred after
6-8 months of Gymnema treatment but remained significantly higher
than normal values. None of these reductions was observed in
control patents on insulin therapy alone who were studied over a
period of 10-12 months. The authors suggested that Gymnema enhanced
endogenous insulin production, possibly by pancreatic regeneration,
as levels of C-peptide, a by-product of the conversion of
proinsulin to insulin, were apparently raised (in comparison to
both the insulin alone group and normal subjects).
[0211] A second study by the same research group found that the
same Gymnema preparation (400 mg/day) produced similar results for
non-insulin-dependent diabetics (Baskaran et al. (1990) J
Ethnopharmacol. 30:295-300). Fasting blood glucose, glycosylated
haemoglobin and glycosylated plasma protein were significantly
reduced compared to baseline values (p<0.001) after 18-20 months
of treatment. None of these reductions was observed in patients
receiving conventional therapy alone who were studied over a period
of 10-12 months. By the end of the treatment period cholesterol,
triglycerides, phospholipids and free fatty acid levels were also
significantly reduced compared to baseline values in those
receiving Gymnema (p<0.001). Control patients receiving only
conventional therapy achieved reductions in cholesterol,
triglycerides and free fatty acids (p<0.05-p<0.001). Fasting
and post-prandial serum insulin levels were significantly increased
in the Gymnema group compared to those taking only conventional
drugs (p<0.01). Twenty-one of the 22 patients were able to
reduce their intake of hypoglycaemic drugs; 5 of these discontinued
hypoglycaemic drugs entirely and maintained their blood glucose
homeostasis with Gymnema extract alone. The authors' suggestion of
beta cell regeneration or repair facilitated by Gymnema was
supported by the higher insulin levels in the serum of patients
after Gymnema supplementation. Gymnema administration to healthy
volunteers did not produce any acute reduction in fasting blood
glucose level.
[0212] The trace shown in FIG. 1(a) is the GC-MS chromatogram of
Gymnema water extract showing the compound of formula (G2)
(trimethylsilyl derivative) as a major component at 9.26 minutes
after removal of sugars.
Example 2
Uptake of G2 after Oral Administration of Gymnema sylvestre
[0213] In a preliminary experiment on one male volunteer to
determine if G2 was readily absorbed from the gastrointestinal
tract, a water extract of Gymnema leaves obtained commercially
containing 7 mg of G2 was drunk and urine monitored for G2 over 4
hours (0-2 hours and 2-4 hours). An internal standard of 1 mg of
castanospermine was added to the two samples of urine prior to
applying to a cation exchange resin (IR120 in the H.sup.+ form).
After washing the resin with copious water, the bound material was
displaced wing excess 2M NH.sub.4.sup.+ solution and dried for
GC-MS analysis. The samples were derivatised using Pierce Tri-Sil
to produce trimethyl-silyl-derivatives of the imino sugars and
imino sugar acids. GC-MS was carried out on a Perkin Elmer
TurboMass Gold mass spectrometer, with a quadrupole ion filter
system, which was run at 250.degree. C. constantly during analysis.
The detector mass range was set to 100 to 650 amu. The temperature
of the transfer line (GC to MS) was held at 250.degree. C. The GC
column was a high polarity fused-silica column (Varian `Factor
Four` VF-5 ms column, 25 m.times.0.25 mm i.d., 0.25 .mu.m phase
thickness). The carrier gas (helium) flow rate was 1 ml min-1. G2
gives a characteristic mass spectrum as the tms derivative and was
well resolved allowing quantification.
[0214] The G2 was detected in the urine at both time periods and
the 7 mg appeared to be recovered within the four hours.
[0215] The characteristic mass spectrum (tms) of imino sugar acid
G2 is shown in FIG. 1(b).
Example 3
Detection of ISAs in Stevia rebaudiana
[0216] Stevia rebaudiana (Asteraceae) is well known to contain
sweeteners called steviosides and related compounds. Stevia has
shown promise in medical research for treating such conditions as
obesity and high blood pressure. Steviol glycosides have been
reported to have a negligible effect on blood glucose (e.g. see
Barriocanal L A et al., 2008, Regul. Toxicol. Pharmacol. 51:37-41).
Stevioside has been shown to induce antihyperglycaemic,
insulinotropic and glucagonostatic effects in vivo in rats in
several studies. However, the steviosides themselves have been
reported to have possible mutagenic activity and hence
fractionating Stevia-derived material to remove steviosides could
improve the products for anti-diabetic or obesity use.
[0217] The trace shown in FIG. 1(c) is the GC-MS chromatogram of
water extract of Stevia rebaudiana after removal of sugars showing
several imino sugar acids and one imino sugar at 7.29 minutes
(DMDP).
[0218] The major imino sugar acids in S. rebaudiana are novel
pyrrolidines such as those shown below. The acid carbon chain
length on the nitrogen is also observed to be longer than that
shown with addition of 14 mass units (CH.sub.2) noted in mass
spectra of the parent compounds identified. Also present are
piperidines structurally related to 1-deoxynojirimycin such as
shown in structures T1 and G6. O-butyl forms of the compounds also
exist in Stevia although these carbon chain lengths might also
vary. The imino sugar DMDP is the compound S2 without the acid on
the nitrogen.
##STR00012##
Example 4
Detection of ISAs in Citrus spp.
[0219] Citrus species (Rutaceae) contain piperidine imino sugar
acids related to the compound of formula (G2) with novel N- and
O-butyl-derivatives but also appear to always contain pyrrolizidine
acids such as the compound of formula (C1) shown above
(epialexaflorine). This ISA was first isolated from Alexa species
(Leguminosae) (Pereira et al. (1991): Isolation of
7a-epialexaflorine from leaves of Alexa grandiflora--a unique
pyrrolizidine amino acid with a carboxylic acid substituent at C-3.
Tetrahedron 47 (29): 5637-5640). The pyrrolizidine acid appears to
be ubiquitous in Citrus species. Citrus spp. contain low levels of
the imino sugar acids in comparison to Gymnema and Stevia. Bitter
orange (Citrus aurantium) in China and grapefruit have anecdotal
claims for antidiabetic or weight control uses. The ISA S9 was
found to be a more major component of Bitter Oranges than C1.
Example 5
Inhibition of Glycosidases by ISAs
[0220] The results tabulated below were obtained using commercially
available glycosidases and p-nitrophenyl-substrates using standard
methods described for example by Watson et al (1997) Phytochemistry
46 255-259. All enzymes and substrates were bought from Sigma.
Enzyme and substrate solutions were made using sodium phosphate
buffers at the pH optima values. The enzymes used were
.alpha.-D-glucosidase Saccharomyces cerevisiae, Bacillus
stearothermophilus, Oryzae sativa), .beta.-D-glucosidase (Almond),
.alpha.-D-mannosidase (Jack bean), .alpha.-D-galactosidase (Green
coffee beans), .beta.-D-galactosidase (Bovine liver),
.alpha.-L-fucosidase (Bovine kidney),
N-acetyl-.beta.-D-glucosaminidase (Bovine kidney, Jack bean,
Aspergillus oryzae), Naringinase (Penicillium decumbens) and
amyloglucosidase (Aspergillus niger). All enzymes were used with
the appropriate p-nitrophenyl substrates (5 mM). Amyloglucosidase
activity was measured using amylopectin (0.1%) (Merck) mixed with
sodium phosphate buffer, pH 4.5 in a glass bottle, and healed in a
boiling water bath for 20 minutes to dissolve with released glucose
measured using `Trinder` glucose detection solution (Sigma).
[0221] Of particular interest is the ability of acids of the imino
sugar glucosidase inhibitors such as DAB
(1,4-dideoxy-1,4-imino-D-arabinitol) and DNJ (1-deoxynojirimycin)
to inhibit the mammalian hexosaminidase which the parent compounds
do not. Elevated levels of this enzyme activity has been shown to
occur in urine of diabetic patients (Yamanouchi et al. (1998)
Diabetes care 21: 619-624) but it may be that the inhibition of the
enzyme activity helps to control metabolic disturbances seen in
diabetics. The inhibition of the alpha-glucosidases is also reduced
by addition of the acid substituent to the imino sugars (DMDP, DAB
and DNJ).
TABLE-US-00001 N- N- N- ethanoic- propanoic- N-butyl-2- N-
ethanoic- N- DMDP DNJ butylester hydroxyethyl- DAB ethanoic- Assay
S2 T1 of G2 T2 G2 T3 T4 DNJ G6 .alpha.-D- NI NI NI NI NI NI
glucosidase (Yeast) .alpha.-D- 92.7 NI NI NI NI NI glucosidase 51.7
uM (Bacillus) .alpha.-D- NI 99.1 NI 67.2 NI 99.7 glucosidase 2.4 uM
259 uM 1.0 uM (Rice) .beta.-D- 55 NI NI NI NI NI glucosidase
(Almond) .alpha.-D- NI NI NI NI NI NI galactosidase Green coffee
bean .beta.-D- 60 NI NI NI NI NI galactosidase Bovine liver
.alpha.-L-fucosidase NI NI NI NI NI NI Bovine kidney .alpha.-D- NI
NI NI NI NI NI mannosidase Jack bean .beta.-D- NI NI NI NI NI NI
mannosidase Cellullomonas fimi Naringinase NI NI NI NI NI NI
Penicillium decumbens N-acetyl-.beta.-D- NI 86.1 NI 82.0 81.9 83.6
glucosaminidase 32.2 uM 12.2 uM 39.6 uM 10.4 uM (Bovine kidney)
N-acetyl-.beta.-D- NI NI NI NI NI NI glucosaminidase (Jack bean)
N-acetyl-.beta.-D- NI NI NI NI NI NI glucosaminidase (A. oryzae)
Amyloglucosidase NI 53 NI NI NI NI Aspergillus niger .beta.- 507 uM
50 uM 280 uM 60 uM 160 uM 80 uM Glucuronidase Bovine liver
[0222] Of particular interest was the ability of imino sugar acids
to inhibit .beta.-Glucuronidase. The assay involved the use of
Bovine liver .beta.-glucuronidase using p-nitrophenyl
.beta.-D-glucuronide purchased from Sigma. The enzyme was assayed
at 27.degree. C. in 0.1M citric acid/0.2M disodium hydrogen
phosphate buffer at pH 5.0. The incubation mixture consisted of 10
.mu.l of 3000 units/ml enzyme solution, 10 .mu.l of 1 mg/ml aqueous
inhibitor solution and 50 .mu.l of 5 mM para-nitrophenyl
.beta.-D-glucuronide made up in buffer at pH 5.0. The reactions
were stopped by adding 70 .mu.l of 0.4M glycine (pH 10.4) during
the exponential phase of the reaction, which had been determined at
the beginning using uninhibited assays in which water replaced
inhibitor. Final absorbances were read at 405 nm using a Versamax
microplate reader (Molecular Devices). Assays were carried out in
triplicate, and the values given were means of the three replicates
per assay. Results are expressed above as IC.sub.50 values (the
concentration of the compound giving 50% inhibition of the enzyme
activity. The controls contained water in place of the
inhibitor.
##STR00013##
Example 6
Inhibition of Glycosidase Enzymes by the Compound of Formula
(G2)
[0223] DNJ is a potent inhibitor of a wide range of
.alpha.-glucosidases and inhibits digestive glucosidases with Ki
values in the low or sub-.mu.M range (Watson et al., 2001).
Anti-diabetic drugs (Glyset and Miglitol) were derived from DNJ by
Bayer and these function by reducing uptake of glucose into the
blood but they also have side effects such as disturbance of the
digestive tact. In contrast G2 is a very weak inhibitor of
.alpha.-glucosidases only just reaching 50% inhibition at nearly mM
concentration and so is unlikely to function in the same way. G2
has also been reported to be a weak inhibitor of glucuronidase and
iduronidase (Booth et al. (2007) Acta Crystalographica Section E63,
o3783-o3784 and references therein). It is not clear if inhibition
of these enzymes could be involved in weight control or control of
metabolic syndrome. The imino sugar fagomine has been shown to
potentiate insulin release but the mechanism is unknown and might
be via glucosidase inhibition (Taniguchi et al. (1998) Norm. Metab.
Res. 30: 679-683). Although there is interest in compounds such as
DNJ and fagomine as potential anti-diabetic agents, it may well be
that the glucosidase inhibition is in fact not important for some
of the in vivo anti-diabetic activity of these imino sugars;
formation of small amounts of the acids in vivo may result in
compounds showing glucuronidase inhibition. Without wishing to be
bound by any theory, inhibition of the glucuronidase activity
elevated in metabolic syndrome may aid removal of toxins and
improve regulation of metabolism generally and specifically beta
cell function and growth.
TABLE-US-00002 Cmpd of formula Assay DNJ (G2) .alpha.-D-glucosidase
36 NI (Yeast) .alpha.-D-glucosidase 100 49 (Bacillus) 2 uM
.alpha.-D-glucosidase 100 55 (Rice) 0.9 uM .beta.-D-glucosidase NI
NI (Almond) .alpha.-D-galactosidase NI NI Green coffee bean
.beta.-D-galactosidase NI NI Bovine liver .alpha.-L-fucosidase NI
NI Bovine kidney .alpha.-D-mannosidase 26 29 Jack bean
.beta.-D-mannosidase NI NI Cellullomonas fimi Naringinase 20 NI
Penicillium decumbens N-acetyl-.beta.-D- NI NI glucosaminidase
(Bovine kidney) N-acetyl-.beta.-D- NI 10 glucosaminidase (Jack
bean) N-acetyl-.beta.-D- NI NI glucosaminidase (A. oryzae)
Amyloglucosidase 32 NI Aspergillus niger .beta.-Glucuronidase NI 90
Bovine liver 107 uM % inhibition of compounds tested at 0.8 mM
Italics = IC.sub.50 NI = no inhibition
Example 7
Increase in Plasma Insulin Levels In Vivo by the Compound of
Formula (G2)
[0224] The study started with 42 male ob/ob mice, 10 weeks of age.
The mice were fed a normal chow diet during the whole study. After
1 week of acclimatization, mice were matched on basis of body
weight, plasma glucose and insulin (after 4 h-fasting) and divided
in 3 groups of 10 animals (t=0 days). For the next 6 days, mice
received vehicle (control group 1) or 5 mg/kg/day (group 2) or 50
mg/kg/day (group 3) of the compound of formula (G2) (test compound)
around 12.00 h by gavage (5 ml/kg mouse). On the 7th day, mice were
fasted at 8.00 h and received the last gavage (vehicle for group 1
or 5 mg/kg/day, test compound for group 2 or 50 mg/kg/day test
compound for group 3) at 12.00 h. At 13.00 h the mice subsequently
received a glucose bolus (2 g/kg mouse, 5 ml/kg mouse) by gavage as
start of the oral glucose tolerance test (OGTT). Measurement of
blood glucose and plasma collection was at t=0 (just before gavage
of glucose) and at t=5, 15, 30, 45, 60 and 120 min after glucose
bolus. After the OGTT, mice were sacrificed with CO.sub.2 and
additional blood was collected in heparin-coated tubes via heart
puncture (>100 .mu.l plasma was obtained) and heparin-plasma
samples were analysed.
[0225] On day 0 mice were randomized on body weight and 4 h fasted
plasma glucose and insulin levels (table 3.1.a). Twelve out of 42
mice were excluded, to create more homogenous groups with respect
to body weight, plasma glucose and insulin.
[0226] Both body weight (FIG. 3.2.1) and food intake (FIG. 3.2.2)
were not significantly changed after treatment with 5 mg/kg/day or
50 mg/kg/day G2, when compared to the control (vehicle) group.
[0227] When compared to the control group, plasma insulin levels at
0 min (just before the oral load of glucose) seemed to be reduced
somewhat in both test compound treatment groups, although this
reduction was not significant. No significant changes were seen
between 3 groups at 15, 30, 45 and 60 min. Two hours after the
glucose bolus, plasma insulin concentrations were significantly
increased in the 5 mg/kg and the 50 mg/kg test compound treated
groups when compared to the control group.
[0228] The data showed that plasma insulin levels were
significantly increased in both test compound treatment groups 120
min after the oral bolus of glucose, possibly as a result of an
improved pancreatic .beta.-cell function.
Example 8
Properties of the Compound of Formula (G2)
Chemical Properties
[0229] The compound of formula (G2) is an ISA of molecular weight
177. It is freely soluble in water. The compound is stable under
all normal laboratory storage conditions.
Occurrence and Exposure Data
[0230] The compound of formula (G2) is a natural product that
occurs in polar extracts of a range of plants that are known in
Ayurveda and the European plant pharmacopoeia. The present
inventors have detected the compound at concentrations ca. 0.2
mg/mL in several herbal medicinal products used in the management
of obesity and diabetes in humans. Such products are generally
regarded as safe and, at typical doses, exposure to the compound of
formula (G2) from their consumption is around 1 mg/day.
[0231] The anti-diabetic and anti-obesity effects of one herbal
formulation has been verified in an experimental animal model, in
which hyperlipidaemic Wistar rats received either a single dose
equivalent to 0.6 mg compound formula (G2)/Kg or 10 daily doses up
to 0.4 mg/Kg. No toxicity was reported and the results indicated
the herbal formulation's ability to reduce body weight gain, and
lower concentrations of plasma triglycerides, and fasting and
postprandial bloodglucose in animals on a high energy diet. In
other studies, herbal extracts have been administered daily to rats
at doses estimated to be equivalent to 5-50 mg/Kg for up to 52
weeks with no observable toxic effect.
Predictive Toxicology Screen
[0232] The compound of formula (G2) was screened for toxicity
liabilities using a validated Acute Toxicity assay. Fertilized eggs
were obtained from breeding pairs of adult Tuebingen (Tu) zebrafish
and arrayed at the 2-4 cell stage of development into 24-well
culture plates containing fresh 0.3.times. Danieau's solution.
Plates were incubated at 28.5.degree. C. in a humidity controlled
environment prior to assessment. Stock concentrations of Compound
were produced by serial dilution in 100% DMSO (final concentration
exposed to larvae, 0.5%). Screening was performed at seven doses
(1, 5, 25, 50, 100, 200 and 500 mM) alongside VASTox internal
controls and vehicle. Dosing of Compound took place at 72 h post
fertilisation (hpf; at which point embryogenesis is complete) with
visual assessment of lethality and gross morphology at 96 hpf (24 h
incubation). 14 larvae were exposed to each dose of Compound giving
a total of 84 larvae assessed (excluding controls). At 96 hpf,
larvae were observed and screened using a dissecting
stereomicroscope for the presence or absence of: (1) heartbeat (2)
circulation; (3) necrosis; and (4) motility (touch response). If
all four criteria were satisfied, a larva would be classified as
dead. The compound was screened blind.
[0233] Results obtained at concentrations of 1 mM, 5 mM, 25 mM, 50
mM, 100 mM, 200 mM, 500 mM showed that the compound of formula (G2)
did not cause acute toxicity to zebrafish larvae when exposed via
an aqueous dose.
Example 9
Quality Control Process
[0234] 10 g of dried herbal food additive is put into a 250 ml
conical flask then enough 50% ethanol/water added to soak the plant
material, allowing 2 cm extra solvent on top. This is left for 15
hours or overnight to extract. The extract is filtered using a
Buchner funnel. The less polar components may be extracted for HPLC
analysis for example. The plant material is either discarded or
kept for sequential extraction with dichloromethane (DCM).
Preferably fresh material is used for the DCM step but if
insufficient is available, a sequential extraction can be performed
or might be used to further characterize the components). HP20
resin may be used to clean the extracts to make them more suitable
for HPLC analysis.
[0235] Dowex 50 resin (50-100 mesh) (or equivalent such as
Amberlite IR120) is prepared by adding excess 2M HCl and soaking
for a minimum of 15 minutes. The resin is then washed with excess
deionized water to pH 7. The prepared resin is poured into
10.times.1 cm columns and reservoirs attached. The columns are
washed with 25 ml of 50% aqueous ethanol to equilibrate the resin
with the same solvent as used to prepare the plant samples. For
each column, the reservoir is filled with the extract which is
allowed to pass slowly through the resin.
[0236] The pH of the eluent is monitored which should be around 1
or 2. If it rises to 6 or 7 then the resin is exhausted. If this
should happen, a little more resin is added to the top of the
column and if necessary the whole sample is applied to the column
again to ensure binding of al of the ionic components. After all of
the sample has been applied to the column, it is washed with 75 ml
of 50% aqueous ethanol followed by 75 ml of water. These washings
are discarded normally for ISA analysis but can be analysed for
unretained components such as flavonoids and sugars. The water is
used to remove the alcohol and other unretained components prior to
eluting the bound constituents.
[0237] The column is eluted with 100 ml of 2M ammonium hydroxide
and this is collected in a 250 ml round bottom flask. This is
evaporated to 3-5 ml on a rotary evaporator at less than 40.degree.
C. and transferred to a weighed 7 ml vial. The drying is completed
by blowing down with nitrogen and/or freeze-drying. Care is taken
to dry the samples on the same day and not to leave them sitting in
the ammonia solution longer than necessary (typically less than 15
minutes) as compound degradation could otherwise occur. 1-3 mg of
each dried sample is placed in GC vials and freeze dried again
prior to derivatisation for analysis.
Notes
(a) HP-20 Resin
[0238] Diaion HP-20 (manufactured by Sumitomo Ltd) is a
styrene-divinylbenzene polymer resin. It is hydrophobic and adsorbs
lipophilic compounds and weak acids. The synthetic adsorbent HP and
SP series are insoluble three-dimensional crosslinked polymers with
macropores. They do not possess ion exchange or other functional
groups, however they have a large surface area and are able to
absorb a variety of organic substances by means of van der Waals'
forces. The polymer matrix can be classified as either the aromatic
(styrene-divinylbenzene) type or the acrylic (methacrylic)
type.
[0239] Once compounds are adsorbed they can be washed off the resin
by the application of a suitable solvent. HP-20 is used in the
following manner to remove excessive amounts of fats and
chlorophyll from dichloromethane (DCM) extracts of plants.
[0240] The solubilised extract is dried under vacuum onto the
resin. The resin is eluted with methanol containing increasing
amounts of acetone (up to 30% acetone). This is enough to wash off
all compounds of interest whilst leaving fats and chlorophylls
adsorbed onto the HP-20 resin. The HP-20 resin is cleaned for
re-use by washing with acetone and hexane. This washes off all
unwanted compounds and the resin can be used once again after a
final wash with methanol.
(b) Ion Exchange Chromatography
[0241] Samples are initially processed by extraction using
approximately 50% aqueous alcohol, which separates the polar
constituents from the more non-polar components of each plant and
denatures any proteins that may be present in the extract. The
extracts are then processed by ion exchange chromatography which
separates and concentrates the ionic compounds in each extract
(predominantly alkaloids, amino acids and small amines) from the
non-ionic compounds which wood also be present in the extracts
(mainly sugars, fats and most of the phenolic compounds). The
samples are then analysed in enzyme assays, by GC-MS or HPLC.
[0242] The filtered extracts are loaded onto Dowex 50W-X8 resin,
which is a polystyrene resin cross-linked with divinylbenzene. It
is a strongly acidic cation exchanger which can be used in either
the free acid or hydrogen (H.sup.+) form or in the salt form e.g.
ammonium (NH.sub.4.sup.+) salt. Both forms of the resin adsorb
cations from solution and release an equivalent number of
counter-ions back into solution (either H.sup.+ or NH.sub.4.sup.+
ions, depending on the form of the resin used). In the H.sup.+
form, Dowex 50W-X8 resin adsorbs all ionic compounds from solution
(except very strong acids), regardless of their charge, and this is
the preferred form.
[0243] On adsorption of cations from the extract, protons are
displaced from the resin causing the pH of the eluate to fall from
pH 6.0 (the pH of the distilled water used to rinse the resin prior
to use) to approximately pH 2.0, depending on the concentration of
the sample. The more dilute the sample, the smaller the drop in pH.
However, once the resin capacity has been reached, continued sample
loading causes the pH to rise to that of the crude extract
itself.
[0244] The Dowex 50W-X8 resin (50-100 mesh size) is prepared for
use by washing with 2M HCl to ensure complete conversion to the
H.sup.+ form. The excess acid is removed by extensive rinsing with
distilled water. After the crude extract has been loaded onto the
resin, the column is washed with distilled water to remove any
unbound material until the pH of the eluate rises to that of the
water itself. The bound compounds are eluted with a 2M solution of
ammonium hydroxide (NH.sub.4.sup.+OH.sup.-). The column is washed
to pH 6.0 with water and the ammonia is removed from the sample by
evaporation under reduced pressure at 40.degree. C. using a rotary
evaporator.
[0245] ISAs of the invention can be further purified by binding
them to anion exchange resin such as Amberlite CG400 in the
hydroxide form. The ISAs can be displaced with acids such as 1M
acetic acid and dried. The resin is prepared for use by soaking for
1 hour in 1M NaOH prior to washing with water to pH 8.
(c) Gas Chromatography-Mass Spectrometry (GC-MS)
[0246] Gas-liquid chromatography is a process whereby a complex
mixture of volatile substances is separated into its constituents
by partitioning the sample between an inert gas under pressure and
a thin layer of non-volatile liquid coated on an inert support
inside a heated column. In order to achieve a good separation of
specific compounds in a mixture, it is crucial to use a column with
the correct characteristics. The nature of the solid support, type
and amount of liquid phase, method of packing, overall length and
column temperature are important factors. Preferably capillary
columns coated with a non-polar liquid phase (25 m.times.0.22 mm
id.times.0.25 .mu.m BPX5 stationary phase, produced by SGE Ltd.) or
equivalents thereof are used.
[0247] Many compounds are unsuitable for direct injection into a
gas chromatograph because of either their high polarity, low
volatility or thermal instability. Compounds that are highly
hydroxylated are difficult to vapourise because of inter-molecular
hydrogen bonding. However, by replacing the hydroxyl hydrogens with
other chemical groups, they can be made sufficiently volatile for
GC analysis. The two most popular means of derivatising hydroxyl
groups are acetylation and silylation, where acetylates
[CH.sub.3CO--O--R] or silyl ethers, e.g. trimethylsilyl (TMS)
ethers [(CH.sub.3).sub.3Si--O--R] are formed. Preferred is the
silylation of samples prior to analysis using Sigma Sil A (a
mixture of trimethylchlorosilane, hexamethyldisilazane and pyridine
1:3:9) produced by the Sigma Chemical Company. An alternative is
Pierce Tri-Sil. Derivatisation is achieved by the addition of 100
.mu.l of the trimethylsilylation reagent to each mg of dried
material in a sealed vial (the reagent degrades in the presence of
water) and the reaction is completed by heating the samples at
60.degree. C. for 15 minutes.
[0248] The trimethylsilyl ethers in each derivatised sample are
separated on the column using a temperature programme. A
temperature programme is used as this allows the rapid separation
of compounds of a very wide boiling range.
[0249] In electron impact mass spectrometry the effluent from the
gas chromatograph, which contains the separated and vaporised
compounds, is passed into the ion chamber of the mass spectrometer
which is under a high vacuum. The molecules are bombarded by a beam
of electrons accelerated from a filament which ionises and
fragments them. Initially, one electron is removed from each
molecule to form a positively charged molecular ion (M.sup.+, i.e.
a radical cation). Breakage of bonds relative to bond strength
occurs rapidly in the molecular ion to generate fragment ions. The
manner in which molecules fragment is highly characteristic and can
be used as a form of `fingerprint` identification. The various ions
are accelerated into the analyser portion of the mass spectrometer
where they are sorted according to their mass to charge ratios (m/z
values) which are equivalent to the molecular weights of the
fragments. The ion signal is amplified by an electron multiplier
and the mass spectrum is plotted from low to high mass. The m/z
values are plotted against relative abundance of the ions to give
the visual `fingerprint`.
(d) HPLC-PDA/MS/ELS (Evaporative Light Scattering Detection)
[0250] With this technique, samples are dissolved in a suitable
solvent and separated on a column using a solvent mixture that is
pumped under pressure through the column. Three detectors are used;
a mass spectrometer, as described above, and a photodiode array
system that measures whether the compounds absorb light at
wavelengths in both the UV and visible ranges and a light
scattering detector (ELS). The ELS is particularly well suited to
detect the imino sugar acids and imino sugars that usually lack a
chromophore.
[0251] A Waters Integrity.TM. HPLC-PDA/MS system fitted with a
reverse phase C.sub.8 HPLC column (50 mm.times.2.1 mm id.times.3.5
.mu.m, Waters) was used to analyse non-polar compounds extracted by
DCM and cleaned using HP20 resin. The rate of solvent flow through
the column was 0.35 ml/min and a linear gradient starting at 90%
water and 10% acetonitrile (containing 0.01% trifluoroacetic acid)
was used, rising to 100% acetonitrile over 6 minutes and held for a
further 6.5 minutes.
[0252] Absorbance (photodiode array--PDA) data was collected from
200-600 nm and mass spectral data collected between 71 and 600 m/z.
Imino sugar acids are not well resolved or detected by such HPLC
systems but many other groups of phytochemicals are that may
co-occur with imino sugar acids in herbal preparations. ELS
detection allows the imino sugars and imino sugar acids to be
observed. However, other forms of HPLC are available that are
better suited to detection of carbohydrates and imino sugars and
imino sugar acids; examples are the Dionex carbohydrate HPLC
systems and HILIC (hydrophilic interaction chromatography) coupled
for example to electrochemical detectors, mass spectrometers or ELS
detectors.
[0253] An example of a HILIC system suited to imino sugars and
imino sugar acids is an HPLC fitted with a SeQuant HPLC column
(ZIC-HILIC packing, 150.times.4.6 mm, 3.5 um particle size, 200 A
pore size) with a mobile phase of 40:55:5 of Water:Acetonitrile:100
mM Ammonium Acetate pH5.7 and a flow rate of 0.5 ml/min. Detection
using a Polymer Laboratories PL-ELS 1000 (light scattering
detector) gave good resolution of imino sugars and imino sugar
acids.
Table Showing Examples of Separation of Amino Acids, Imino Sugars
and Imino Sugar Acids Using a HILIC HPLC System
TABLE-US-00003 [0254] Retention Time Compound Name in Minutes
Aspartic Acid 3.53 Adenine 4.00 Phenylalanine 4.20 Tyrosine 4.72
Valine 4.88 Imino sugar acid S9 4.97 Proline 5.20 Imino sugar acid
G2 5.27 Alanine 5.37 Asparagine 5.59 6-epi-castanospermine 6.49
Castanospermine 6.55 DNJ 11.23 Casuarine 11.61 Swainsonine 14.33
DMDP 14.64 3,7-diepicasuarine 14.81 Australine 16.26 DAB 17.19
##STR00014##
[0255] A Dionex HPLC system used for separating and detecting imino
sugars and imino sugar acids consisted of a Dionex ION PAC CS10
4.times.250 mm column with an ION PAC CG10 4.times.50 mm guard
column and a mobile phase of Methane Sulphonic Acid diluted to 80
mM and water. The pump was a Dionex P680, to deliver eluent A (80
mM MSA) and eluent B (WATER, ultrapure) as required, at a total
flow rate of 1 ml/min. The injector was a Rheodyne manual injector
and the oven, a Dionex LC30, was used to maintain column and guard
at 30 C. A pneumatic controller (Dionex PC10, (He gas, .about.1.5
ml/min NaOH at 60 psi) was used to add 300 mM NaOH to the eluent
flow between the column and the detector. The detector was a Dionex
ED40 Electrochemical Detector and the data analysed using Dionex
Chromeleon software.
Example 10
Lack of Inhibition of Digestive Glucosidases by Compound G2
[0256] Deoxynojirimycin (DNJ), N-butyl-DNJ (Zavesca.RTM.) and the
N-hydroxymethyl-derivative (Miglitol, Glyset.RTM.) developed by
Bayer for treatment of Diabetes type 2 are potent inhibitors of
glucosidases (see Watson et al., 2001, Phytochemistry 56: 265-295).
The inhibition of glucosidases is responsible both for
gastrointestinal disturbance (side effects of the drugs) and the
benefits to diabetic patients of taking Glyset.RTM. which functions
through slowing of glucose release and uptake in the GI tract, thus
controlling post-prandial blood glucose levels. The iminosugar
acids of the invention such as G2 have the advantage of being able
to control blood glucose levels through a mechanism not involving
inhibition of digestive glucosidases and, therefore, avoid the side
effects of DNJ and drugs derived from it.
TABLE-US-00004 Inhibitory effect of G2 and DNJ on rat intestinal
glycosidases IC.sub.50 value .mu.M G2 DNJ* maltase LI 0.36
isomaltase NI 0.3 sucrase NI 0.21 cellobiase NI ND lactase NI ND
trehalase NI ND ND = not determined; *from Yasuda et al., 2002, J.
Nat. Prod. 65, 198-202. LI = inhibition not reaching 50% at 1 mM;
NI = no inhibition at 1 mM
Enzyme activities were prepared from brush border membranes of rat
small intestine using disaccharides as substrates as described by
Yasuda et al., 2002.
[0257] Acarbose, another glucosidase inhibitor used for diabetes
type 2 which reduces glucose release and uptake, is also reported
to be a potent inhibitor of rat small intestine maltase (IC.sub.50
0.16 .mu.M) and sucrase (IC.sub.50 2.9 .mu.M) (Kato et al., 2008,
J. Agric. Food Chem. 56, 8206-8211).
[0258] A comparison of the activity on rabbit digestive
disaccharidases of compounds of the invention G2, S2, T1 and T2 and
DNJ and Zavesca have also been made. The results of this comparison
are shown in the table below. G2, S2 and T2 showed no inhibition of
the disaccharidases at 0.8 mM whereas T1 showed very weak
inhibition of maltase and sucrase in comparison to DNJ and Zavesca.
Isomaltase inhibition was not determined for S2 and T1.
TABLE-US-00005 Inhibitory effect of compounds of the invention and
DNJ and Zavesca on rabbit intestinal glycosidases sucrase maltase
isomaltase IC50 (uM) IC50 (uM) IC50 (uM) Zavesca 0.22 0.97 0.19 DNJ
0.09 0.18 0.02 G2 NI NI NI S2 NI NI ND T1 6.1 19 ND T2 NI NI NI
Rabbit Disaccharidase Inhibition Assay Method
[0259] Sucrose (8.3 mM), maltose (5 mM) and isomaltose (6.3 mM)
substrates were made using 0.2 M Macllvaine (citrate-phosphate)
buffer, pH 6.0. The glucose detection reagent was prepared
according to the supplier's instructions (Megazyme Ltd). Linearity
of the timecourse of the reaction between the glucose detection
reagent and glucose was tested using a series of glucose
dilutions.
Mammalian Disaccharidase Preparation
[0260] The method for preparation of mammalian small intestine
mucosal disaccharidases was based on Griffiths (1998), omitting
ammonium sulphate fractionation steps. The small intestine was
dissected out of a female wild rabbit, and opened longitudinally
using a scalpel. The mucosal layer was scraped off using the edge
of a clean microscope slide, and placed into 3 ml of dH.sub.2O. The
mucosal layer suspension was centrifuged at around 1000 rpm for 30
seconds to sediment tissue debris. The supernatant (6 ml) was
removed, and centrifuged again at 3500 rcf for 1 minute to remove
fine particulates from suspension. The supernatant was diluted to
give 0.2-0.25 mg/ml protein and stored at -30.degree. C.
[0261] Enzyme assays were carried out using 5-15 .mu.l of enzyme
preparation, 5 .mu.l of test compound (or dH.sub.2O for controls)
and 15-25 .mu.l of substrate solution. Reactions were covered using
a sheet of plate sealing film, and incubated at 37.degree. C. for
60 minutes. Glucose (from disaccharide hydrolysis) was quantified
by adding 200 .mu.l of glucose detection reagent and incubating the
reactions for a further twenty-five minutes. Absorbance was
measured at 510 nm against control blanks.
[0262] In this way enzyme inhibition of mammalian small intestine
mucosal sucrase, maltase and isomaltase could be determined.
Example 11
Correlation of the Presence of Iminosugar Acids in Plants Claimed
to Have Anti-Diabetic Activity
[0263] The imino sugar acids are rare in plants but they have also
been identified in Lotus species (Fabaceae), Pumpkin (Cucurbita
species), Aspalanthus linearis (Rooibos), Alexa species and
Castanospermum australe (Fabaceae), Eugenia and Syzygium species
(Myrtaceae), Lycium barbarum (Goji, Solanaceae) and Andrographis
paniculata (Acanthaceae).
[0264] Gymnema sylvestre is a plant claimed to have anti-diabetic
or anti-obesity activity and has clinical evidence of activity
(e.g. see Baskaran et al., 1990, Antidiabetic effect of a leaf
extract from Gymnema sylvestre in non-insulin-dependent diabetes
mellitus patients. J Ethnopharmacol. 30:295-300 and
Shanmugasundaram et al., 1990, Use of Gymnema sylvestre leaf
extract in the control of blood glucose in insulin-dependent
diabetes mellitus J Ethnopharmacol. 30:281-94). The present
inventors have now discovered that Gymnema sylvestre leaves, seeds
and stems contain the iminosugar acid G2. Products sold
commercially for weight loss such as Floressance "Citrus, Garcinia,
Gymnema concentre" capsules also contain G2 (2.4 mg per capsule in
Lot 8205CC). The presence of G2 or other iminosugar acids in
Gymnema has not been reported by any other researchers. To date
published research has concentrated on other components of Gymnema
that affect sweet taste such as Gymnemic acids, which unlike G2 are
not shown to be orally available.
[0265] We have also discovered that the plant Gurana (also called
Guarana) Paullinia cupana contains an N-methyl-3-deoxy-form of G2
also reported as Glabrin from Pongamia glabra (this structure had
no stereochemistry reported). Gurana has claims for weight loss,
e.g. see Anderson T and Foght J, 2001, Weight loss and delayed
gastric emptying following a South Amercian herbal preparation in
overweight patients. J. Hum. Nutr. Diet 14, 243. Although Gurana is
well known to contain caffeine which is claimed to give some
protection from development of diabetes type 2, iminosugar acids
related to compound G2 are key components of Gurana with
anti-diabetic and anti-obesity activity. Pongamia is orally toxic
due to other components.
[0266] The present inventors have also isolated iminosugar acids of
the pyrrolidine structure from other plant species claimed to have
anti-diabetic activity such as Bitter Orange which contains various
stachydrines as examples shown below.
##STR00015##
[0267] The present inventors have also recently isolated from
Citrus a compound identified as an acid of a pyrrolizidine alkaloid
and related to epialexaflorine (see Watson et al., 2001) shown
below. It also contains compounds which are N- and
O-butyl-derivatives of G2 and O-butyl-N-acid-derivatives of
Deoxynojirimycin-like compounds (e.g. T3) proven by semi-synthesis
and GC-MS analysis of the compounds and the Citrus extracts as
trimethylsilyl-derivatives.
##STR00016##
[0268] Chinese pumpkin (or Siam Pumpkin) is claimed to increase
beta cells in diabetic rats, Xia, T., 2007, Journal of the Science
and Food and Agriculture 87, 1753-1757. The present inventors have
shown that open chain iminosugar acid compounds (e.g. example P3)
are present in seeds and fruit of the European pumpkin (Cucurbita
pepo). Several Cucurbitaceae species have claims for anti-diabetic
activity including Bitter melon (Momordica charantia) which shows
several potential iminosugar acids in the CG400 OH-bound fraction
analysed by GC-MS and by characteristic activity in glucuronidase
and hexosaminidase assays.
[0269] The therapeutic association of iminosugar acids and diabetes
or weight control is also shown by the example of Stevia rebaudiana
which has been shown to control blood glucose levels and to
modulate insulin levels. While research effort by others has
focused on steviosides and rebaudiosides and related glycosides
these appear to have no pharmacological activity, e.g. see
Barriocanal L A et al., 2008, Apparent lack of pharmacological
effect of steviol glycosides used as sweeteners in humans. Regul.
Toxicol. Pharmacol. 51:37-41. The plant, however, has proven
anti-glycaemic activity and was used traditionally to treat
diabetes in South America, e.g. see Ferreira E B et al., 2006,
Comparative effects of Stevia rebaudiana leaves and stevioside on
glycaemia and hepatic gluconeogenesis. Planta Med. 72 :691. The
discovery of iminosugar acids such as S2 in Stevia therefore
suggests that removal of these compounds eliminates the
anti-diabetic activity of Stevia.
Example 12
Measurement of Glycosidase Activities of Iminosugar Acids in Plant
Claimed to Have Anti-Diabetic or Anti-Obesity Activity
[0270] Iminosugar acids can show distinctive inhibition of
glucuronidase and/or hexosaminidase activities which are not shown
by other iminosugars such as deoxynojirimycin (DNJ), Miglitol and
DAB (1,4-dideoxy-1,4-imino-D-arabinitol); these iminosugar
compounds appear to exhibit potential therapeutic activity in
diabetes or obesity through inhibition of glucosidases and glycogen
phosphorylase but therefore also have side effects caused by
inhibition of digestive disaccharidase activity.
[0271] Compound G2 of the invention has an IC.sub.50 against
mammalian glucuronidase of 107 .mu.M while the
N-hydroxyethyl-derivative of G2 is more potent against the
glucuronidase (IC.sub.50 60 .mu.M) and mammalian
6-N-acetylglucosaminidase (IC.sub.50 12.2 .mu.M). DNJ is not
inhibitory to either enzyme activity at 0.8 mM but N-propionic DNJ
is inhibitory to both glucuronidase (IC.sub.50 50 .mu.M) and the
hexosaminidase (IC.sub.50 32 .mu.M).
Table showing Comparison of the Glycosidase Inhibition Profile of
the Iminosugars DNJ and Miglitol with Three Iminosugar Acids of the
Invention (Inhibition Shown as % Inhibition at 0.8 mM--IC.sub.50
Values in Italics)
TABLE-US-00006 Assay DNJ Miglitol G2 S2 T3 .alpha.-D-glucosidase 36
NI NI NI NI (Yeast) .alpha.-D-glucosidase 100 83 49 51.7 uM NI
(Bacillus) .alpha.-D-glucosidase 100 100 55 NI 259 uM (Rice)
.beta.-D-glucosidase 64 99 NI 55 NI (Almond)
.alpha.-D-galactosidase NI 19 NI NI NI Green coffee bean
.beta.-D-galactosidase NI 87 NI 60 NI Bovine liver
.alpha.-L-fucosidase 6 -5 NI NI NI Bovine kidney
.alpha.-D-mannosidase 27 7 29 NI NI Jack bean .beta.-D-mannosidase
-19 NI NI NI NI Cellullomonas fimi Naringinase 21 26 NI NI NI
Penicillium decumbens N-acetyl-.beta.-D- NI NI NI NI 12.2 uM
glucosaminidase (Bovine kidney) N-acetyl-.beta.-D- NI NI 10 NI NI
glucosaminidase (Jack bean) N-acetyl-.beta.-D- NI NI NI NI NI
glucosaminidase (A. oryzae) Amyloglucosidase 32 78 NI NI NI
Aspergillus niger .beta.-Glucuronidase NI NI 107 uM 507 uM 60 uM
Bovine liver
[0272] Several plants claimed to have anti-diabetic activity were
assessed for the ability to inhibit mammalian glucuronidase or
hexosaminidase activity. Plants contain many aromatic components
such as flavonoids and polyphenols that can have non-specific
protein binding activity or have strong colours making the use of
standard colorimetric glycosidase assays impossible. The aqueous
ethanol extracts of plants were therefore fractionated using a
combination of cation and anion exchange methods to purify the
iminosugar acid components. The methods are familiar to those
experienced in the art and involve binding the amino acids and
alkaloids to a strongly acidic cation exchange resin (e.g. IR120 in
the H.sup.+ form), washing with water, displacing the bound
components with 2M ammonia solution, concentrating under reduced
pressure to remove the ammonia and then binding the iminosugar
acids to a strongly basic anion exchange resin (e.g. Amberlite
CG400 in the OH form) and after washing with water, displacing the
iminosugar acids with 1M acetic acid. After removal of excess
acetate these fractions are suitable for glycosidase assays.
Glycosidase Assay Methods
[0273] All enzymes and para-nitrophenyl substrates were purchased
from Sigma, with the exception of beta-mannosidase which came from
Megazyme. Enzymes were assayed at 27.degree. C. in 0.1M citric
acid/0.2M disodium hydrogen phosphate buffers at the optimum pH for
the enzyme. The incubation mixture consisted of 10 .mu.l enzyme
solution, 10 .mu.l of 1 mg/ml aqueous solution of extract and 50
.mu.l of the appropriate 5 mM para-nitrophenyl substrate made up in
buffer at the optimum pH for the enzyme. The reactions were stopped
by addition of 70 .mu.l 0.4M glycine (pH 10.4) during the
exponential phase of the reaction, which had been determined at the
beginning using uninhibited assays in which water replaced
inhibitor. Final absorbances were read at 405 nm using a Versamax
microplate reader (Molecular Devices). Assays were carried out in
triplicate, and the values given are means of the three replicates
per assay. (See Watson, A. A., Nash, R. J., Wormald, M. R., Harvey,
D. J., Dealler, S., Lees, E., Asano, N., Kizu, H., Kato, A.,
Griffiths, R. C., Cairns, A. J. and Fleet, G. W. J. (1997).
Glycosidase-inhibiting pyrrolidine alkaloids from Hyacinthoides
non-scripta. Phytochemistry 46 (2): 255-259.)
[0274] The table below gives details of the enzymes used and the
conditions of the individual assays.
TABLE-US-00007 [Enzyme] working Reaction stock conditions Enzyme
Source Substrate (5 mM) pH (U/ml) (mins) .alpha.-D- Saccharomyces
PNP-.alpha.-D- 6.8 0.5 15 glucosidase cerevisiae glucopyranoside
.alpha.-D- Bacillus PNP-.alpha.-D- 6.8 0.25 15 glucosidase
sterothermophilus glucopyranoside .alpha.-D- Rice PNP-.alpha.-D-
4.0 7.5 20 glucosidase glucopyranoside .beta.-D- Almond
PNP-.beta.-D- 5.0 0.125 15 glucosidase glucopyranoside .alpha.-D-
Green coffee bean PNP-.alpha.-D- 6.5 0.2 15 galactosidase
galactopyranoside .beta.-D- Bovine liver PNP-.beta.-D- 7.3 0.1 15
galactosidase galactopyranoside .alpha.-L- Bovine kidney
PNP-.alpha.-L- 5.5 0.4 15 fucosidase fucopyranoside .alpha.-D- Jack
bean PNP-.alpha.-D- 4.5 0.4 10 mannosidase mannopyranoside
.beta.-D- Cellullomonas fimi PNP-.beta.-D- 6.5 1 15 mannosidase
mannopyranoside Naringinase Penicillium PNP-.alpha.-L- 4.0 0.05 15
decumbens rhamnopyranoside N-acetyl-.beta.-D- Bovine kidney
PNP-N-acetyl-.beta.-D- 4.25 0.35 15 glucosaminidase glucosaminide
N-acetyl-.beta.-D- Jack bean PNP-N-acetyl-.beta.-D- 5.0 0.25 20
glucosaminidase glucosaminide N-acetyl-.beta.-D- Aspergillus oryzae
PNP-N-acetyl-.beta.-D- 5.0 0.125 15 hexosaminidase glucosaminide
Amyloglucosidase Aspergillus niger PNP-.alpha.-D- 4.5 20 30@32
glucopyranoside .beta.-D- Bovine liver PNP-.beta.-D-glucuronide 5.0
3000 20 glucuronidase
Results
[0275] The anion exchange resin (CG400 OH.sup.-) bound fractions of
the plants Stevia rebaudiana and Gymnema sylvestre give complete
inhibition of glucuronidase activity at 1.43 mg/ml. This inhibition
is probably due to the high concentration of compound G2 in Gymnema
and compounds such as S2 in Stevia.
[0276] Examples of other plants studied with claims for
anti-diabetic and or anti-obesity activity are Goji fruits (Lycium
barbarum), Theobroma cacao nibs, Aspalanthus linearis (Rooibos
tea), Hoodia gordonii, Korean Ginseng (Panax ginseng) and Soya bean
(Glycine max). These were all fractionated into CG400 OH-- bound
fractions prior to assays. The results in the two tables below are
expressed as % inhibition by 1.43 mg/ml except for caffeine which
was run at 143 ug/ml as a control compound for Cacao nibs.
TABLE-US-00008 Mean % Inhibition shown by iminosugar acid fraction
of cacao nibs and caffeine Caffeine Cacao nibs Enzyme Source 143
ug/ml DT0181/121/2 N-acetyl-.beta.-D- Bovine kidney 1.3 19.6
glucosaminidase .beta.-glucuronidase Bovine liver -2.4 30.5
TABLE-US-00009 Mean % Inhibition shown by iminosugar acid fractions
of plants claimed to have anti-diabetic or weight control
properties Hoodia Korean Korean tablets ginseng ginseng DT0181/
AM0241/ DT Soya Enzyme Source 143/1 119/2 XCD01 Goji Rooibos beans
N-acetyl- Bovine 73.8 -16.1 7.8 25 5 88 .beta.-D-glucosaminidase
kidney .beta.- Bovine 41.2 72.9 36 3 27 87 glucuronidase liver
[0277] It can be seen from the results obtained with the plants
above that they show some inhibition of either bovine glucuronidase
or hexosaminidase. The GC-MS analysis of the CG400 OH-- (anion
exchange resin) bound fractions showed that levels of possible
iminosugar acids in these samples were not as high as in Stevia or
Gymnema and there were many other major components such as protein
amino acids which will have diluted the inhibitors in such complex
mixtures. However, soya beans for example show surprisingly strong
inhibition of both enzyme activities. Rooibos and Goji berries show
a relatively low level of inhibition of the enzymes but both show
an activity. Korean Ginseng shows a strong inhibition of the
glucuronidase activity in two different samples tested, while
Hoodia gives inhibition of both enzyme activities. Further
purification and isolation work is needed to fully characterize the
components in these iminosugar acid fractions giving the
inhibitions. Once purified the strength of the inhibition should
increase greatly.
Example 13
In Vivo Activity of Compound G2--Enhancement of Insulin Levels in
ddy Mice
Method
[0278] 7 week old ddy mice (weight 30-31 g) were divided into 4
groups (groups 1 to 4). Each group had 3-4 mice. All test mouse
were fasted for 16 hours before being given the test compounds.
[0279] The aim of this experiment was to determine if G2 (here
coded BR1) can stimulate insulin release.
Group 1: Control. un-treated mice (without glucose and G2). Group
2: Oral administration of glucose alone. Group 3: Oral
administration of G2 alone. Group 4: Oral administration of G2 with
glucose.
[0280] Serum insulin levels were measured after 30 minutes (see
FIG. 2).
Results
[0281] Comparing Groups 1 (un-treated) and Group 3 (oral
administration of G2 alone) it is clear that G2 did not affect the
insulin level. However, combination of glucose and G2 is much
effective than glucose alone. These results suggest that G2 does
not affect insulin release itself but it appears to enhance
levels.
[0282] FIG. 3 Effects of G2 (BR1) (100 mg/kg body weight) on serum
insulin levels.
[0283] Serum insulin concentrations of male ddy mouse after 30
minutes later an oral load with or without D-glucose (2.5 g/kg body
weight). Each value represents the mean.+-.SEM (n=3-4). *
P<0.05, ** P<0.01.
Example 14
In Vivo Activity of Compound G2--Control of Blood Glucose Levels in
ddy Mice
Method
[0284] 7 weeks old ddy mice (weight 30-31 g) were divided into 4
groups (from group 1 to 4). Each group had 5 mouse. All test mouse
were fasted for 16 hours for this experiment.
[0285] The aim of this experiment was to show if G2 (here coded as
BR1) could suppress a hyperglycemia after a meal. Thus, maltose was
chosen as the loading sugar as used for Bayer's glucosidase
inhibitor Glucobay. Either maltose (2.5 g/kg) or Surose (2.5 g/kg)
or starch (1.0 g/kg) can be used but maltose is most popular.
[0286] The hypothesis was that if G2 can suppress the blood glucose
level it may:
1) inhibit maltase (inhibit to change from maltose to glucose). 2)
inhibit glucose transport. 3) stimulate insulin release. 4) inhibit
glycogenphosphorylase. 5) another activity
[0287] Thus, there were 4 groups (see FIG. 4).
Group 1: Control. Maltose-loading only (without G2 condition).
Group 2: oral administration of G2 with maltose (same time). Group
3: intraperitoneal injection of G2 and oral administration of
maltose (same time). Group 4: oral administration of G2 30 minutes
before maltose-loading. (Glucobay is taken 30 minutes before
meals).
Results
[0288] It was shown that G2 can suppress hyperglycemia in every
group.
Curve of group 2 (p.o.) and group 3 (i.p.) is almost same. This
result is very important because if inhibition mechanism of G2 is
"inhibition of maltase" and/or "inhibition of glucose transporter",
the i.p. route would not inhibit hyperglycemia. Thus, the site of
action of G2 is not in the intestinal tract and maybe the pancreas.
Furthermore, this result showed that G2 is absorbed into the blood
very quickly.
[0289] See FIG. 5 for the effects of G2 (BR1) on blood glucose
levels.
Example 15
Inhibition of Mammalian .beta.-Glucuronidase and
.beta.-N-acetylglucosaminidase by Iminosugar Acids
[0290] The following tables show comparative % inhibitions of
commercially available (Sigma) bovine .beta.-glucuronidase (liver)
and .beta.-N-acetylglucosaminidase (kidney) by iminosugar acids and
amino acids at approximately 0.8 mM. Where strong inhibition was
observed IC.sub.50 values were calculated and are shown as .mu.M
next to the % inhibition values. It can be seen that the ability to
inhibit the glucuronidase and hexosaminidase activities is not
found in many compounds of this group other than iminosugar acids.
The assays were conducted using p-nitrophenyl-substrates as
described previously.
TABLE-US-00010 % Glucuronidase % Nacetyl glucosaminidase inhibition
at 0.8 mM inhibition at 0.8 mM Structure Bovine Liver Bovine kidney
##STR00017## -2.2 7.1 ##STR00018## -2.3 5.1 ##STR00019## 3.9 49.4
##STR00020## -3.7 -4.7 ##STR00021## -4.4 -0.6 ##STR00022## 88.9 107
uM 2.4 ##STR00023## 3.8 8 ##STR00024## 58.6 531 uM 4.8 ##STR00025##
-3.2 -4.2 ##STR00026## -1.7 2.3 ##STR00027## -1.6 0.8 ##STR00028##
1.4 2.9 ##STR00029## -2 17 ##STR00030## 85.3 81 uM 90.1 5.1 uM
##STR00031## 0.8 8 ##STR00032## 0.6 7.6 ##STR00033## 13.8 6.6
##STR00034## 3.3 25.8 ##STR00035## 4 82.3 137 uM ##STR00036## 97.9
7.3 uM 57.9 ##STR00037## 99.6 0.04 uM -5.2
Examples 16 to 21
Synthesis of Imino Sugar Acids
General Alkylation Procedure
[0291] Alkyl halide (0.46 mmol) and K.sub.2CO.sub.3 (0.61 mmol)
were added to a solution of iminosugar (0.31 mmol) in DMF (2 mL)
and the resulting reaction mixture was stirred at 60.degree. C. for
16 h. The solvent was removed under reduced pressure and the
residue purified by ion exchange chromatography using a combination
of Dowex-50WX8-400 and SPE cartridges such as POH or isolute
SCX-2.
[0292] The following compounds were prepared using the general
alkylation procedure:
2-((2R,3R,4R,6S)-3,4,5-Trihydroxy-2-(hydroxymethyl)piperidin-1-yl)acetic
acid (G6)
##STR00038##
[0294] .sup.1H-NMR (D.sub.2O): 3.78 (1H, dd, J=2.7 Hz, J=13.2 Hz).
3.72 (1H, dd, J=2.4 Hz, J=13.2 Hz), 3.59-3.51 (1H, m), 3.41 (1H, t,
J=9.5 Hz), 3.33 (2H, s), 3.23 (1H, t, J=9.3 Hz), 3.05 (1H, dd,
J=5.0 Hz, J=11.6 Hz), 2.70-2.58 (2H, m); .sup.13C-NMR (D.sub.2O):
176.6, 77.8, 69.0, 68.2, 65.6, 56.8, 56.7, 56.4; MS (M+H.sup.+):
222.3
2-((2R,3R,4R,5S)-3,4,6-Trihydroxy-2-(hydroxymethyl)piperidin-1-yl)propanoi-
c acid (T1)
##STR00039##
[0296] .sup.1H-NMR (D.sub.2O): 3.92 (2H, s), 3.63-3.59 (1H, m),
3.49 (1H, t, J=5.9 Hz), 3.35-3.29 (3H, m), 3.12 (1H, m), 2.79 (1H,
m), 2.70 (1H, m), 2.49-2.46 (2H, m); .sup.13C-NMR (D.sub.2O): 76.6,
68.1, 67.0, 65.3, 54.9, 53.6, 49.2, 31.0; MS (M+H.sup.+): 236.0
(2S,3R,4R,5S)-Butyl
1-butyl-3,4,5-trihydroxypiperidine-2-carboxylate (T2)
##STR00040##
[0298] .sup.1H-NMR (D.sub.2O): 4.20-4.07 (2H, m), 3.54-3.48 (1H,
m), 3.47 (1H, t, J=5.8 Hz), 3.19 (1H, t, J=5.6 Hz), 3.04 (1H, dd,
J=2.9 Hz, J=6.9 Hz), 2.91 (1H, d, J=5.9 Hz), 2.39 (1H, dt, J=3.2
Hz, J=6.8 Hz), 2.25 (1H, m), 2.07 (1H, t, J=6.6 Hz), 1.57 (2H, m),
1.43 (1H, m), 1.30 (3H, m), 1.15 (2H, m), 0.81 (3H, t, J=4.5 Hz),
0.77 (3H, J=4.4 Hz); .sup.13C-NMR (D.sub.2O): 77.1, 72.2, 71.2,
68.8, 66.2 (2.times.), 54.8, 29.8, 26.4, 19.9, 18.5, 13.0
(2.times.); MS (M+H.sup.+): 290.3
(2S,3R,4R,5S)-3,4,5-Trihydroxy-1-(2-hydroxyethyl)piperidine-2-carboxylic
acid (T3)
##STR00041##
[0300] .sup.1H-NMR (D.sub.2O): 3.80-3.67 (3H, m), 3.55 (1H, t,
J=5.8 Hz), 3.40 (1H, m), 3.32 (1H, t, J=5.6 Hz), 3.10 (1H, m), 3.03
(1H, m), 2.84 (1H, m), 2.59 (1H, m); MS (M+H.sup.+): 222.5
2-((2R,3R,4R,5R)-3,4-Dihydroxy-2,5-bis(hydroxymethyl)pyrrolidin-1-yl)aceti-
c acid (S2)
##STR00042##
[0302] .sup.1H-NMR (D.sub.2O): 4.13 (2H, dd, J=0.9 Hz, J=3.2 Hz),
3.95 (1H, d, J=9.8 Hz), 3.91 (5H, m), 3.66 (2H, bs). .sup.13C-NMR
(D.sub.2O): 74.2 (2.times.), 69.8 (2.times.), 56.6 (2.times.),
52.1; MS (M+H.sup.+): 222.3.
2-((2R,3R,4R)-3,4-Dihydroxy-2-(hydroxymethyl)pyrrolidin-1-yl)acetic
acid (T4)
##STR00043##
[0304] .sup.1H-NMR (D.sub.2O): 4.27 (1H, m), 4.05 (1H, m), 3.69
(1H, d, J=9.8 Hz), 3.91 (2H, m), 3.76 (1H, d, J=9.8 Hz), 3.72 (1H,
m), 3.55-3.52 (2H, m). .sup.13C-NMR (D.sub.2O): 76.0, 73.6
(.times.2), 59.7, 57.8, 57.6; MS (M+H.sup.+): 192.3.
EQUIVALENTS
[0305] The foregoing description details presently preferred
embodiments of the present invention. Numerous modifications and
variations in practice thereof are expected to occur to those
skilled in the art upon consideration of these descriptions. Those
modifications and variations are intended to be encompassed within
the claims appended hereto.
* * * * *