Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02847250 2014-03-20
THERAPEUTIC METHODS AND COMPOSITIONS FOR TREATING DIABETES
BACKGROUND OF THE INVENTION
[0001] Diabetes mellitus (DM), or simply diabetes, is a group of metabolic
diseases in which a
person has high blood sugar, either because the pancreas does not produce
enough insulin, or
because cells do not respond to the insulin that is produced. This high blood
sugar produces the
classical symptoms of polyuria (frequent urination),polydipsia (increased
thirst), and polyphagia
(increased hunger).
[0002] There are three main types of diabetes mellitus (DM). Type 1 DM results
from the body's
failure to produce insulin, and currently requires the person to inject
insulin or wear an insulin
pump. This form was previously referred to as "insulin-dependent diabetes
mellitus" (IDDM) or
"juvenile diabetes". Type 2 DM results from insulin resistance, a condition in
which cells fail to
use insulin properly, sometimes combined with an absolute insulin deficiency.
This form was
previously referred to as non insulin-dependent diabetes mellitus (NIDDM) or
"adult-onset
diabetes". The third main form, gestational diabetes, occurs when pregnant
women without a
previous diagnosis of diabetes develop a high blood glucose level. It may
precede development
of type 2 DM.
[0003] The classic symptoms of untreated diabetes are loss of weight, polyuria
(frequent
urination), polydipsia (increased thirst), and polyphagia (increased hunger).
Symptoms may
develop rapidly (weeks or months) in type 1 diabetes, while they usually
develop much more
slowly and may be subtle or absent in type 2 diabetes. Prolonged high blood
glucose can cause
glucose absorption in the lens of the eye, which leads to changes in its
shape, resulting in vision
changes. Blurred vision is a common complaint leading to a diabetes diagnosis.
A number of
skin rashes that can occur in diabetes are collectively known as diabetic
dermadromes.
[0004] Diabetes mellitus is a chronic disease, for which there is no known
cure except in very
specific situations. Management concentrates on keeping blood sugar levels as
close to normal
("euglycemia") as possible, without causing hypoglycemia. This can usually be
accomplished
with diet, exercise, and use of appropriate medications (insulin in the case
of type 1 diabetes; oral
medications, as well as possibly insulin, in type 2 diabetes). Metformin is
generally
recommended as a first line treatment for type 2 diabetes, as there is good
evidence that it
decreases mortality. Type 1 diabetes is typically treated with combinations of
regular and
-1-
CA 02847250 2014-03-20
NPH insulin, or synthetic insulin analogs. When insulin is used in type 2
diabetes, a long-acting
formulation is usually added initially, while continuing oral medications.
Doses of insulin are
then increased to effect.
[0005] There are several studies showing that diabetes is associated with
abnormal insulin
secretion and insulin sensitivity. Since insulin is the most important
substance in regulating
glucose metabolism, impaired insulin secretion results in an increase in
hepatic glucose
production and reduction of glucose uptake in muscle. On the other hand,
increased insulin
resistance is a key feature in T2DM. It is characterized with a remarkable
decrease in tissue
glucose utilization in response to insulin.
SUMMARY OF THE INVENTION
[0006] In one aspect provided herein are compositions for treating diabetes in
a subject
comprising an effective amount of borapetoside A or C, or a pharmaceutically
acceptable salt,
metabolite, solvate or prodrug thereof, and an insulin or insulin analog.
[0007] In another aspect provided herein are method for treating diabetes in a
subject comprising
administering an effective amount of borapetoside A or C, or a
pharmaceutically acceptable salt,
metabolite, solvate or prodrug thereof, with an insulin or insulin analog to
said subject.
INCORPORATION BY REFERENCE
[0008] All publications, patents, and patent applications mentioned in this
specification are
herein incorporated by reference to the same extent as if each individual
publication, patent, or
patent application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The novel features of the invention are set forth with particularity in
the appended claims.
A better understanding of the features and advantages of the present invention
will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in
which the principles of the invention are utilized, and the accompanying
drawings of which:
[0010] FIG. 1 shows chemical structures of borapetoside A and C.
[0011] FIG. 2A-C show illustrative effective results of Borapetoside A
enhancing glycogen
synthesis in C2C12 and Hep3B cells. Borapetoside A treatment increased the
glycogen content
-2-
CA 02847250 2014-03-20
in C2C12 (2A), IL-6-treated- C2C12 (2B), and Hep3B (2C). All data were
presented as mean
SEM with three independent experiments. (*, p < 0.05, compared with vehicle
control.)
[0012] FIG. 3A-C show illustrative hypoglycaemic effects of borapetoside A in
mice. The mice
were injected with borapetoside A, vehicle only, or positive drug
intraperitoneally. Sixty minutes
after injection, blood samples were collected from the mice and subjected to
plasma glucose
assay. The experiment was independently repeated for seven times, and the data
were presented
as mean SEM in the normal mice (3A), the mice with type 1 DM (3B), and the
mice with type
2 DM (3C). Asterisks indicate significant difference compared with vehicle
only (*, p <0.05).
[0013] FIG. 4A-D show effects of borapetoside A on plasma glucose
concentration and the AUC
in normal mice (4A/4B) and the mice with type 2 DM (4C/4D) that received an
IPGTT. The
blood samples were obtained before (at 0 min) and after glucose injection (at
30, 60, 120, and
150minutes) in the normal mice and the mice with type 2 DM (4A and 4C). The
AUC decreased
in the borapetoside A-treated group, as compared to the vehicle-treated group
in both normal and
T2DM mice (4B and 4D). Asterisk symbols indicate significant difference
between vehicle and
borapetoside A-treated groups at the same time point. Data were expressed as
mean SEM in
each group (n=6). *, p < 0.05, vs animals treated with vehicle.
[0014] FIG. 5A-C show illustrative effects of borapetoside A on glycogen
synthesis in skeletal
muscle in vivo. The normal mice (5A), the mice with type 1 DM (5B), and the
mice with type 2
DM (5C) were used in this study. Values expressed as mean SEM from six
animals in each
group. The average value of glycogen content of the vehicle-treated mice in
each group was seen
as 100.*, p < 0.05, compared animals treated with vehicle control.
[0015] FIG. 6A-C show illustrative effects of borapetoside A on glycogen
synthesis in liver in
vivo. The normal mice (6A), the mice with type 1 DM (6B), and the mice with
type 2 DM (6C)
were used in this study. Values expressed as mean SEM from six animals in
each group. The
average value of glycogen content of the vehicle-treated mice in each group
was seen as 100.*, p
<0.05, compared animals treated with vehicle control.
[0016] FIG. 7A-F show various protein expression levels in the liver of the
mice with type 1 DM
after 7-day treatment. The protein expression of IR-related signaling
mediators was analyzed by
immunoblot in the liver of the mice with type 1 DM after repeated
intraperitoneal
administrations of borapetoside A or insulin for 7 days (7A). The mice that
did not receive any
treatment were given the same volume of vehicle. The findings were reproduced
on 3 separate
-3-
CA 02847250 2014-03-20
experiments. The quantification protein levels expressed are expressed as mean
SEM in each
column, as shown in (7B) to (7F). (*, p < 0.05 compared with vehicle-treated.)
[0017] FIG. 8A-C show illustrative effect of borapetoside C on plasma glucose
concentration in
non-diabetic and T2DM mice during an oral glucose tolerance test (OGTT). OGTT
was
performed in the vehicle-treated (filled circles) and 5 mg/kg borapetoside C-
treated (open
circles) groups of both normal (8A) and T2DM (8B) mice. Values are expressed
as means+SEM
(n = 7). *p < 0.05 vs. the vehicle-treated group. The area under the curve
(AUC) decreased in the
borapetoside C-treated group as compared to the vehicle-treated group in both
normal and
T2DM mice (8C).
[0018] FIG. 9A-D show a representative immunoblot of protein expression of IR-
related
signaling mediators in the liver of Ti DM mice after repeated intraperitoneal
administrations of
borapetoside C (5 mg/kg twice per day) or insulin (0.5 IU/kg twice per day)
for 7 days. Mice
that did not receive any treatment were given the same volume of vehicle (2%
DMSO). The
findings were reproduced on 3 separate experiments. Quantification of the data
is shown in lower
panel. The quantification protein levels expressed are expressed as mean SEM
in each column.
p < 0.05 represents the levels of significance compared to the values of the
vehicle-treated
mice.
[0019] FIG. 10A-D show a representative immunoblot of protein expression of IR-
related
signaling mediators in the liver of Ti DM mice after repeated intraperitoneal
administration of
borapetoside C (0.1 mg/kg twice per day) or insulin (1.0 IU/kg twice per day)
for 7 days. Mice
that did not receive any treatment were given the same volume of vehicle (2%
DMSO). Findings
were reproduced on 3 separate experiments. The quantifications are shown in
the lower panel.
Quantified protein levels are expressed as mean SEM in each column. *p <0.05
represenst the
levels of significance for comparing the values of vehicle-treated mice; # p
<0.05 represents the
level of significance compared with the values with insulin-treated mice.
DETAILED DESCRIPTION OF THE INVENTION
[0020] In accordance with the practice of this invention, borapetoside A or C
is provided herein
as a pharmacological agent in combination with insulin for treatment of type 1
diabetes.
[0021] Herbal prescriptions have been recognized as potential remedy based on
the scientific
medical assessments. The hypoglycemic action and other medicinal uses of T
crispa extract
-4-
CA 02847250 2014-03-20
were reported in several previous studies. However, the active ingredients in
the extract are not
well investigated.
[0022] In accordance with the practice of this invention, single dose
administration of
borapetoside A decrease the serum glucose levels and increases the insulin
secretion in the
normal mice and Type 2 DM bearing mice. Furthermore, borapetoside A attenuated
the elevation
of plasma glucose induced by intraperitoneal glucose in the normal mice and
the mice with
diabetes. The continuous treatment with borapetoside A for 7 days induced the
phosphorylation
of IR, Akt, AS160, and GLUT2, and decreased the expression of
phosphoenolpyruvate
carboxykinase (PEPCK) in the liver of the Type 1 DM bearing mice.
[0023] Specifically, it was found that borapetoside A effectively increased
the glycogen content
in C2C12 skeletal muscle cells and human hepatocellular carcinoma cell lines
Hep3B at very low
concentration ranging from le to 1 e mol/L. Since glycogen content in the IL-6-
treated C2C12
cells was not increased with the insulin treatment, this result indicated that
insulin resistance in
C2C12 cells was induced after IL-6 treatment. In contrast, borapetoside A
treatment enhanced
the glycogen synthesis in IL-6-treated C2C12 cells (see e.g., FIG. 2). This
result revealed that
borapetoside A could still induce similar increase of glycogen content in
these insulin-resistant
C2C12 cells.
[0024] Interleukin 6 (IL-6) is an interleukin that acts as both a pro-
inflammatory cytokine and an
anti-inflammatory myokine. In humans, it is encoded by the 1L6 gene. IL-6 is
an important
mediator of fever and of the acute phase response. It is capable of crossing
the blood-brain
barrier and initiating synthesis of PGE2 in the hypothalamus, thereby changing
the body's
temperature setpoint. In muscle and fatty tissue, IL-6 stimulates energy
mobilization that leads to
increased body temperature. IL-6 can be secreted by macrophages in response to
specific
microbial molecules, referred to aspathogen-associated molecular patterns
(PAMPs). These
PAMPs bind to an important group of detection molecules of the innate immune
system,
called pattern recognition receptors (PRRs), including Toll-like receptors
(TLRs). These are
present on the cell surface and intracellular compartments and induce
intracellular signaling
cascades that give rise to inflammatory cytokine production.
[0025] In accordance with the practice of this invention, it was found that
bolus intraperitoneal
injection of borapetoside A significantly lowers plasma glucose concentration
in a dose-
dependent manner in the normal mice, or the mice with diabetes (FIG. 3). The
plasma glucose
-5-
CA 02847250 2014-03-20
lowering effect of 10 mgkg-1 borapetoside A was similar to the effect of 300
mgkg4 metformin.
The increased plasma insulin level may contribute to the plasma glucose
lowering effect of
higher dose borapetoside A (3 and 10 mgkg-1) in the normal mice; however, the
plasma insulin in
the normal mice was not significantly increased with 0.3 and 1 mgkg-I
borapetoside A-treatment.
Furthermore, in the mice with type 1 DM, borapetodie A did not alter the
insulin level (Table 1)
but significantly decreased the plasma glucose level (FIG. 3). In some
embodiments there are
provided that borapetoside A exert the hypoglycemic action through insulin-
independent
mechanism both in normal and mice with diabetes. The effective dose of
borapetoside A (0.3 to
1 mgkg-1) to exert insulin-independent hypoglycemic action is consistent with
its effective
concentration to stimulate glycogen synthesis in insulin-resistant C2C12
cells.
[0026] Glucose tolerance test is one of the most critical criteria for
evaluating the therapeutic
efficacy of hypoglycemic drugs. Described herein, the effect of borapetoside A
on the glucose
utilization was further verified by IPGTT test. Borapetoside A has notably
enhanced the glucose
uptake and utilization in peripheral tissues, which is a major site of glucose
disposal, in the
normal mice and the mice with type 2 DM mice (FIG. 4). Even in the ineffective
dose of bolus
test, 0.1 mgkg-1 borapetoside A significantly decreased the AUC.
[0027] Glucose transport is the rate-limiting step in carbohydrate metabolism.
A family of
glucose transporters (GLUT) mediates the glucose transport across the cell
membrane. Both the
GLUT4 in skeletal muscle and GLUT2 in liver are the insulin sensitive glucose
transporters.
Relative to short-acting insulin, borapetodie A increased similar glycogen
synthesis in soleus
muscle in the mice with or without DM (FIG. 5). In addition to the skeletal
muscle, liver is
another important metabolic organ for glucose metabolism. Hepatic insulin
resistance is well
recognized as the primary leading cause for DM development. In this study,
borapetoside A also
increased the glycogen synthesis in liver in the mice with or without DM (FIG.
6). Insulin,
however, failed to enhance the glycogen synthesis in liver and soleus muscle
of the mice with
type 2 DM (FIG. 5C and 6C).
[0028] The pathways by which insulin regulates the glucose uptake and
carbohydrate
metabolism in muscle, fat, and liver tissues, are now well established. The
canonical pathway of
insulin signaling is via activation of phosphoinositide 3-kinase (PI3K) and
Akt, and accessory
pathways that contributed to specific insulin action. Insulin initiated the
signaling transduction
through IR, a large transmembrane protein. Then Akt plays a crucial role in
the hepatic insulin
-6-
CA 02847250 2014-03-20
signal transduction, in terms of glycogen synthesis. The current evidence
suggests that
phosphorylation of AS160 is a key regulators of insulin enhanced glucose
uptake. Moreover, Akt
substrate of 160 kDa, AS160, has strong implication in the regulation of
glucose transportation in
muscle, adipose tissue, and liver. As shown in FIG. 6, it was found that a
seven-day treatment
with borapetoside A increased the phosphorylation of IR. Akt, and AS160 as
well as the protein
expression of GLUT2 in liver in the mice with type 1 DM. Furthermore, the
expression of
PEPCK was significantly decreased in liver. PEPCK, is one of the key enzymes
of hepatic
carbohydrate metabolism and catalyzes a regulatory step in gluconeogenesis.
The insulin
deficiency is clearly associated with the changes in hepatic metabolism,
including increased
expression of PEPCK. The insulin levels of the mice with type 1 DM is limited,
meanwhile the
PEPCK gene is overexpressed in liver. Seven-day treatment of borapetoside A in
the mice with
type 1 DM decreased the expression of PEPCK protein in liver (FIG. 7).
[0029] Without binding to any particular theory of mechanism, these findings
suggest that
borapetoside A exerts an excellent glucose-lowering effect through an
enhancement of glucose
utilization of skeletal muscle and liver. The therapeutic efficacy of
borapetoside A is mediated
via the stimulation of IR/AKT/AK160/GLUT2 pathway, and the suppression of
PEPCK
expression which then contribute to the reduction of the hepatic
gluconeogenesis.
[0030] In the condition of insulin resistance, certain intracellular signaling
pathways become
more resistant to insulin stimulation. The normalization of insulin
sensitivity is important for the
body to ingest nutrients, in particular, dietary carbohydrates. To
characterize how borapetoside A
or C may regulate insulin sensitivity, the effect of borapetoside C on the
glucose utilization was
verified by OGTT test in the present study. The results show that borapetoside
A or C
significantly accelerated the glucose uptake and utilization in peripheral
tissues in both non-
diabetic and T2DM mice (FIG. 8A-C) after oral administration of glucose.
Moreover,
borapetoside C (5mg/kg) also increased glycogen content in skeletal muscle
(Table 2).
[0031] In addition to the skeletal muscle, liver is another important
metabolic organ for glucose
metabolism. Hepatic insulin resistance is well accepted to be the primary
leading cause for
developing DM. However, many of the cellular processes including glucose
homeostasis, fat
metabolism, and cell growth are regulated by the insulin signaling pathway.
Defects in cellular
processes mediated by insulin signaling pathways are central to the
development of obesity and
related diseases, such as insulin resistance, diabetes, and cancer. Although
the molecular events
-7-
CA 02847250 2014-03-20
are not fully elucidated yet, it is likely that Akt/PKB kinase activity plays
a crucial role in the
hepatic insulin action, at least in terms of glycogen synthesis (Eldar-
Finkelman et al., 1999;
Lavoie et al., 1999). Moreover, the diabetogenic action of STZ in the pancreas
is preceded by its
rapid selective uptake by pancreatic 13 cells through the low-affinity GLUT2.
Peripherally this
transporter is a component of the signaling pathway involved in glucose
sensing and regulation
of insulin secretion from pancreatic 13 cells. As shown in FIG. 9A-D, it was
found that
borapetoside C treatment increased phosphorylation of IR and Akt as well as
the protein levels of
GLUT2 in liver. In addition to the enhancement of insulin sensitivity,
borapetoside C could
induce IR phosphorylation and consequently lead to Akt phosphorylation and
GLUT2 expression
in 11DM mice. Compared with the effect of insulin, borapetoside C induced more
IR
phosphorylation but less Akt phosphorylation and GLUT2 expression than
insulin. This finding
indicates that borapetoside C may bind to different site of insulin receptor
and result in less
efficient activation of Akt/GLUT2 signaling.
[0032] The other method developed to evaluate insulin sensitivity in vivo
involved the use of the
insulin tolerance test, which is based on the change of plasma glucose level
after a bolus
injection of regular insulin, and its results reflected the insulin
sensitivity. It was found that a
bolus intraperitoneal injection of borapetoside C from 0.5 to 5 mg/kg
significantly lowered
plasma glucose concentrations in a dose-dependent manner in the non-diabetic,
11DM, and
12DM mice. In accordance with the practice of this invention, it was found
that Borapetoside C
at the ineffective single dose (0.1 mg/kg) combined with insulin (1.0 IU/kg)
enhance insulin-
induced lowering of plasma glucose (Table 3) and insulin-induced increase of
glycogen content
in skeletal muscle of diabetic mice and normal mice (Table 4). The increase of
insulin sensitivity
by borapetoside C is more prominent in diabetic mice than normal mice.
Similarly, Borapetoside
A at the ineffective single dose combined with insulin (1.0 IU/kg) would
enhance insulin-
induced lowering of plasma glucose. Corresponding to the enhancement of
insulin sensitivity,
insulin co-administered with low-dose borapetoside A or C enhances insulin-
induced IR
phosphorylation and consequent Akt phosphorylation and GLUT2 expression in
liver of Ti DM
mice (evidenced by FIG. 10A-D). These results suggest that borapetoside A or C
is not only a
hypoglycemic agent, but can also act as an adjuvant for insulin function.
[0033] Insulin is a peptide hormone, produced by beta cells in the pancreas,
and is central to
regulating carbohydrate and fat metabolism in the body. It causes cells in the
liver, skeletal
-8-
CA 02847250 2014-03-20
muscles, and fat tissue to absorb glucose from the blood. The human insulin
protein is composed
of 51 amino acids, and has a molecular weight of 5808 Da. Biosynthetic human
insulin (insulin
human rDNA, INN)for clinical use is manufactured byrecombinant DNA technology.
[0034] Several analogs of human insulin are available for clinical therapy.
These insulin analogs
are closely related to the human insulin structure, they were developed for
specific aspects of
glycemic control in terms of fast action (prandial insulins) and long action
(basal insulins).
Insulin is usually taken as subcutaneous injections by single-use syringes
with needles, via
an insulin pump, or by repeated-use insulin pens with needles.
[0035] An insulin analog is an altered form of insulin, different from any
occurring in nature, but
still available to the human body for performing the same action as human
insulin in terms of
glycemic control. Through genetic engineering of the underlying DNA, the amino
acid
sequence of insulin can be changed to alter its ADME (absorption,
distribution, metabolism, and
excretion) characteristics. Officially, the U.S. Food and Drug Administration
(FDA) refers to
these as "insulin receptor ligands", although they are more commonly referred
to as insulin
analogs. These modifications have been used to create two types of insulin
analogs: those that
are more readily absorbed from the injection site and therefore act faster
than natural insulin
injected subcutaneously, intended to supply the bolus level of insulin needed
at mealtime
(prandial insulin); and those that are released slowly over a period of
between 8 and 24 hours,
intended to supply the basal level of insulin during the day and particularly
at nighttime (basal
insulin). A few non-limited exemplary insulin analogs include NPH insulin,
Lispro, Aspart,
Glulisine, Glargine insulin, Detemir insulin, insulin degludec, and the like.
[0036] Thus an insulin analog described herein refers to any insulin analog
altered or modified
form of insulin, different from any occurring in nature, but still available
to the human body for
performing the same action as human insulin in terms of glycemic control.
[0037] In some embodiments provide composition for treating diabetes in a
subject comprising
an effective amount of borapetoside A or C, or a pharmaceutically acceptable
salt, metabolite,
solvate or prodrug thereof, and an insulin or insulin analog. In certain
embodiments, said
diabetes is type 1 diabetes. In certain embodiments, said diabetes is type 2
diabetes. In certain
embodiments, the composition comprises borapetoside A. In certain embodiments,
the
composition comprises borapetoside C. In certain embodiments, the composition
comprises
insulin. In certain embodiments, the composition comprises insulin analog. In
certain
-9-
CA 02847250 2014-03-20
embodiments, borapetoside A or C decreases serum glucose levels of said
subject. In certain
embodiments, borapetoside A or C induces increase of glycogen. In certain
embodiments,
borapetoside A or C increases insulin secretion in said subject.
[0038] In some embodiments provide methods for treating diabetes in a subject
comprising
administering an effective amount of borapetoside A or C, or a
pharmaceutically acceptable salt,
metabolite, solvate or prodrug thereof, with an insulin or insulin analog to
said subject. In
certain embodiments, said diabetes is type 1 diabetes. In certain embodiments,
said diabetes is
type 2 diabetes. In certain embodiments, said method comprises administering
borapetoside A.
In certain embodiments, said method comprises administering borapetoside C. In
certain
embodiments, said method comprises administering insulin. In certain
embodiments, said
method comprises administering an insulin analog. In certain embodiments, said
borapetoside A
or C, and said insulin or insulin analog is administered separately,
simultaneously or
sequentially. In certain embodiments, said borapetoside A or C, and said
insulin or insulin
analog are administered orally, parenterally intravenously or by injection. In
certain
embodiments, borapetoside A or C decreases serum glucose levels of said
subject. In certain
embodiments, borapetoside A or C induces increase of glycogen. In certain
embodiments,
borapetoside A or C increases insulin secretion in said subject.
[0039] In other embodiments, borapetoside A or C is isolated from the extracts
of Tinospora
crispa.
Certain Pharmaceutical and Medical Terminology
[0040] Unless otherwise stated, the following terms used in this application,
including the
specification and claims, have the definitions given below. It must be noted
that, as used in the
specification and the appended claims, the singular forms "a," "an" and "the"
include plural
referents unless the context clearly dictates otherwise. Unless otherwise
indicated, conventional
methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry,
recombinant
DNA techniques and pharmacology are employed. In this application, the use of
"or" or "and"
means "and/or" unless stated otherwise. Furthermore, use of the term
"including" as well as
other forms, such as "include", "includes," and "included," is not limiting.
The section headings
used herein are for organizational purposes only and are not to be construed
as limiting the
subject matter described.
-10-
CA 02847250 2014-03-20
[0041] The term "carrier," as used herein, refers to relatively nontoxic
chemical compounds or
agents that facilitate the incorporation of a compound into cells or tissues.
[0042] The terms "co-administration" or the like, as used herein, are meant to
encompass
administration of the selected therapeutic agents to a single patient, and are
intended to include
treatment regimens in which the agents are administered by the same or
different route of
administration or at the same or different time.
[0043] The term "diluent" refers to chemical compounds that are used to dilute
the compound of
interest prior to delivery. Diluents can also be used to stabilize compounds
because they can
provide a more stable environment. Salts dissolved in buffered solutions
(which also can provide
pH control or maintenance) are utilized as diluents in the art, including, but
not limited to a
phosphate buffered saline solution.
[0044] The terms "effective amount" or "therapeutically effective amount," as
used herein, refer
to a sufficient amount of an agent or a compound being administered which will
relieve to some
extent one or more of the symptoms of the disease or condition being treated.
The result can be
reduction and/or alleviation of the signs, symptoms, or causes of a disease,
or any other desired
alteration of a biological system. For example, an "effective amount" for
therapeutic uses is the
amount of the composition comprising a compound as disclosed herein required
to provide a
clinically significant decrease in disease symptoms. An appropriate
"effective" amount in any
individual case may be determined using techniques, such as a dose escalation
study.
[0045] The terms "enhance" or "enhancing," as used herein, means to increase
or prolong either
in potency or duration a desired effect. Thus, in regard to enhancing the
effect of therapeutic
agents, the term "enhancing" refers to the ability to increase or prolong,
either in potency or
duration, the effect of other therapeutic agents on a system. An "enhancing-
effective amount," as
used herein, refers to an amount adequate to enhance the effect of another
therapeutic agent in a
desired system.
[0046] A "metabolite" of a compound disclosed herein is a derivative of that
compound that is
formed when the compound is metabolized. The term "active metabolite" refers
to a biologically
active derivative of a compound that is formed when the compound is
metabolized. The term
"metabolized," as used herein, refers to the sum of the processes (including,
but not limited to,
hydrolysis reactions and reactions catalyzed by enzymes) by which a particular
substance is
changed by an organism. Thus, enzymes may produce specific structural
alterations to a
-11-
CA 02847250 2014-03-20
compound. For example, cytochrome P450 catalyzes a variety of oxidative and
reductive
reactions while uridine diphosphate glucuronyltransferases catalyze the
transfer of an activated
glucuronic-acid molecule to aromatic alcohols, aliphatic alcohols, carboxylic
acids, amines and
free sulphydryl groups. Metabolites of the compounds disclosed herein are
optionally identified
either by administration of compounds to a host and analysis of tissue samples
from the host, or
by incubation of compounds with hepatic cells in vitro and analysis of the
resulting compounds.
[0047] The term "pharmaceutical combination" as used herein, means a product
that results from
the mixing or combining of more than one active ingredient and includes both
fixed and non-
fixed combinations of the active ingredients. The term "fixed combination"
means that the active
ingredients, e.g. a compound (i.e., borapetoside A or C, insulin, insulin
analogs, described
herein) and a co-agent, are both administered to a patient simultaneously in
the form of a single
entity or dosage. The term "non-fixed combination" means that the active
ingredients, e.g. a
compound (i.e., borapetoside A or C, insulin, insulin analogs, described
herein) and a co-agent,
are administered to a patient as separate entities either simultaneously,
concurrently or
sequentially with no specific intervening time limits, wherein such
administration provides
effective levels of the two compounds in the body of the patient. The latter
also applies to
cocktail therapy, e.g. the administration of three or more active ingredients.
[0048] The term "pharmaceutical composition" refers to a mixture of a compound
(i.e.,
borapetoside A or C, insulin, insulin analogs, described herein) with other
chemical components,
such as carriers, stabilizers, diluents, dispersing agents, suspending agents,
thickening agents,
and/or excipients. The pharmaceutical composition facilitates administration
of the compound to
an organism. Multiple techniques of administering a compound exist in the art
including, but not
limited to: intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and
topical
administration.
[0049] The term "subject" or "patient" encompasses mammals. Examples of
mammals include,
but are not limited to, any member of the Mammalian class: humans, non-human
primates such
as chimpanzees, and other apes and monkey species; farm animals such as
cattle, horses, sheep,
goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory
animals including
rodents, such as rats, mice and guinea pigs, and the like. In one embodiment,
the mammal is a
human.
-12-
CA 02847250 2014-03-20
[0050] The terms "treat," "treating" or "treatment," as used herein, include
alleviating, abating or
ameliorating at least one symptom of a disease or condition, preventing
additional symptoms,
inhibiting the disease or condition, e.g., arresting the development of the
disease or condition,
relieving the disease or condition, causing regression of the disease or
condition, relieving a
condition caused by the disease or condition, or stopping the symptoms of the
disease or
condition either prophylactically and/or therapeutically.
Routes of Administration and Dosage
[0051] Suitable routes of administration include, but are not limited to,
oral, intravenous, rectal,
aerosol, parenteral, ophthalmic, pulmonary, transmucosal, transdermal,
vaginal, otic, nasal, and
topical administration. In addition, by way of example only, parenteral
delivery includes
intramuscular, subcutaneous, intravenous, intramedullary injections, as well
as intrathecal, direct
intraventricular, intraperitoneal, intralymphatic, and intranasal injections.
[0052] In certain embodiments, a compound (i.e., borapetoside A or C, insulin,
insulin analogs,
described herein) is administered in a local rather than systemic manner, for
example, via
injection of the compound directly into an organ, often in a depot preparation
or sustained release
formulation. In specific embodiments, long acting formulations are
administered by implantation
(for example subcutaneously or intramuscularly) or by intramuscular injection.
Furthermore, in
other embodiments, the drug is delivered in a targeted drug delivery system,
for example, in a
liposome coated with organ-specific antibody. In such embodiments, the
liposomes are targeted
to and taken up selectively by the organ. In yet other embodiments, the
compound as described
herein is provided in the form of a rapid release formulation, in the form of
an extended release
formulation, or in the form of an intermediate release formulation. In yet
other embodiments, the
compound described herein is administered topically.
[0053] In some embodiments, a compound (i.e., borapetoside A or C, insulin,
insulin analogs,
described herein) is administered parenterally or intravenously. In other
embodiments, a
compound (i.e., borapetoside A or C, insulin, insulin analogs, described
herein) is administered
by injection. In some embodiments, a compound (i.e., borapetoside A or C,
insulin, insulin
analogs, described herein) is administered orally.
[0054] In the case wherein the patient's condition does not improve, upon the
doctor's discretion
the administration of the compounds may be administered chronically, that is,
for an extended
period of time, including throughout the duration of the patient's life in
order to ameliorate or
-13-
CA 02847250 2014-03-20
otherwise control or limit the symptoms of the patient's disease or condition.
In the case
wherein the patient's status does improve, upon the doctor's discretion the
administration of the
compounds may be given continuously or temporarily suspended for a certain
length of time
(i.e., a "drug holiday").
[0055] The foregoing ranges are merely suggestive, as the number of variables
in regard to an
individual treatment regime is large, and considerable excursions from these
recommended
values are not uncommon. Such dosages may be altered depending on a number of
variables, not
limited to the activity of the compound used, the disease or condition to be
treated, the mode of
administration, the requirements of the individual subject, the severity of
the disease or condition
being treated, and the judgment of the practitioner.
[0056] Toxicity and therapeutic efficacy of such therapeutic regimens can be
determined by
standard pharmaceutical procedures in cell cultures or experimental animals,
including, but not
limited to, for determining the LD50 (the dose lethal to 50% of the
population) and the ED50 (the
dose therapeutically effective in 50% of the population). The dose ratio
between the toxic and
therapeutic effects is the therapeutic index and it can be expressed as the
ratio between LD50 and
ED50. Compounds exhibiting high therapeutic indices are preferred. The data
obtained from cell
culture assays and animal studies can be used in formulating a range of dosage
for use in human.
The dosage of such compounds lies preferably within a range of circulating
concentrations that
include the ED50 with minimal toxicity. The dosage may vary within this range
depending upon
the dosage form employed and the route of administration utilized.
Pharmaceutical Formulation
[0057] In some embodiments provide pharmaceutical compositions comprising a
therapeutically
effective amount of a compound (i.e., borapetoside A or C, insulin, insulin
analogs, described
herein) and a pharmaceutically acceptable excipient.
[0058] In some embodiments, compounds (i.e., borapetoside A or C, insulin,
insulin analogs,
described herein) are formulated into pharmaceutical compositions. In specific
embodiments,
pharmaceutical compositions are formulated in a conventional manner using one
or more
physiologically acceptable carriers comprising excipients and auxiliaries
which facilitate
processing of the active compounds into preparations which can be used
pharmaceutically.
Proper formulation is dependent upon the route of administration chosen. Any
pharmaceutically
acceptable techniques, carriers, and excipients are used as suitable to
formulate the
-14-
CA 02847250 2014-03-20
pharmaceutical compositions described herein: Remington: The Science and
Practice of
Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover,
John E.,
Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pennsylvania 1975;
Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel
Decker, New
York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems,
Seventh Ed.
(Lippincott Williams & Wilkins1999).
[0059] Provided herein are pharmaceutical compositions comprising a compound
(i.e.,
borapetoside A or C, insulin, insulin analogs, described herein) and a
pharmaceutically
acceptable diluent(s), excipient(s), or carrier(s). In certain embodiments,
the compounds
described are administered as pharmaceutical compositions in which a compound
(i.e.,
borapetoside A described herein) is mixed with other active ingredients, as in
combination
therapy. Encompassed herein are all combinations of actives set forth in the
combination
therapies section below and throughout this disclosure. In specific
embodiments, the
pharmaceutical compositions include one or more compounds (i.e., borapetoside
A or C, insulin,
insulin analogs, described herein).
[0060] A pharmaceutical composition, as used herein, refers to a mixture of a
compound a
compound (i.e., borapetoside A or C, insulin, insulin analogs, described
herein) with other
chemical components, such as carriers, stabilizers, diluents, dispersing
agents, suspending
agents, thickening agents, and/or excipients. In certain embodiments, the
pharmaceutical
composition facilitates administration of the compound to an organism. In some
embodiments,
practicing the methods of treatment or use provided herein, therapeutically
effective amounts of
compounds a compound (i.e., borapetoside A or C, insulin, insulin analogs,
described herein) are
administered in a pharmaceutical composition to a mammal having a disease or
condition to be
treated. In specific embodiments, the mammal is a human. In certain
embodiments,
therapeutically effective amounts vary depending on the severity of the
disease, the age and
relative health of the subject, the potency of the compound used and other
factors. The
compounds described herein are used singly or in combination with one or more
therapeutic
agents as components of mixtures.
[0061] In one embodiment, a compound a compound (i.e., borapetoside A or C,
insulin, insulin
analogs, described herein) is formulated in an aqueous solution. In specific
embodiments, the
aqueous solution is selected from, by way of example only, a physiologically
compatible buffer,
-15-
CA 02847250 2014-03-20
such as Hank's solution, Ringer's solution, or physiological saline buffer. In
other embodiments,
a compound a compound (i.e., borapetoside A or C, insulin, insulin analogs,
described herein) is
formulated for transmucosal administration. In specific embodiments,
transmucosal formulations
include penetrants that are appropriate to the barrier to be permeated. In
still other embodiments
wherein the compounds described herein are formulated for other parenteral
injections,
appropriate formulations include aqueous or nonaqueous solutions. In specific
embodiments,
such solutions include physiologically compatible buffers and/or excipients.
[0062] In another embodiment, compounds described herein are formulated for
oral
administration. Compounds described herein, including compounds (i.e.,
borapetoside A or C,
insulin, insulin analogs, described herein) are formulated by combining the
active compounds
with, e.g., pharmaceutically acceptable carriers or excipients. In various
embodiments, the
compounds described herein are formulated in oral dosage forms that include,
by way of
example only, tablets, powders, pills, dragees, capsules, liquids, gels,
syrups, elixirs, slurries,
suspensions and the like.
[0063] In certain embodiments, pharmaceutical preparations for oral use are
obtained by mixing
one or more solid excipients with one or more of the compounds described
herein, optionally
grinding the resulting mixture, and processing the mixture of granules, after
adding suitable
auxiliaries, if desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol;
cellulose preparations
such as: for example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum
tragacanth, methylcellulose, microcrystalline cellulose,
hydroxypropylmethylcellulose, sodium
carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or
povidone) or calcium
phosphate. In specific embodiments, disintegrating agents are optionally
added. Disintegrating
agents include, by way of example only, cross-linked croscarmellose sodium,
polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0064] In one embodiment, dosage forms, such as dragee cores and tablets, are
provided with
one or more suitable coating. In specific embodiments, concentrated sugar
solutions are used for
coating the dosage form. The sugar solutions, optionally contain additional
components, such as
by way of example only, gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene
glycol, and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent
mixtures. Dyestuffs and/or pigments are also optionally added to the coatings
for identification
-16-
CA 02847250 2014-03-20
purposes. Additionally, the dyestuffs and/or pigments are optionally utilized
to characterize
different combinations of active compound doses.
[0065] In certain embodiments, therapeutically effective amounts of at least
one of the
compounds described herein are formulated into other oral dosage forms. Oral
dosage forms
include push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a
plasticizer, such as glycerol or sorbitol. In specific embodiments, push-fit
capsules contain the
active ingredients in admixture with one or more filler. Fillers include, by
way of example only,
lactose, binders such as starches, and/or lubricants such as talc or magnesium
stearate and,
optionally, stabilizers. In other embodiments, soft capsules, contain one or
more active
compound that is dissolved or suspended in a suitable liquid. Suitable liquids
include, by way of
example only, one or more fatty oil, liquid paraffin, or liquid polyethylene
glycol. In addition,
stabilizers are optionally added.
[0066] In other embodiments, therapeutically effective amounts of at least one
of the compounds
described herein are formulated for buccal or sublingual administration.
Formulations suitable
for buccal or sublingual administration include, by way of example only,
tablets, lozenges, or
gels. In still other embodiments, the compounds described herein are
formulated for parental
injection, including formulations suitable for bolus injection or continuous
infusion. In specific
embodiments, formulations for injection are presented in unit dosage form
(e.g., in ampoules) or
in multi-dose containers. Preservatives are, optionally, added to the
injection formulations. In
still other embodiments, the pharmaceutical compositions of a compound (i.e.,
borapetoside A
described herein) are formulated in a form suitable for parenteral injection
as a sterile
suspensions, solutions or emulsions in oily or aqueous vehicles. Parenteral
injection formulations
optionally contain formulatory agents such as suspending, stabilizing and/or
dispersing agents. In
specific embodiments, pharmaceutical formulations for parenteral
administration include
aqueous solutions of the active compounds in water-soluble form. In additional
embodiments,
suspensions of the active compounds are prepared as appropriate oily injection
suspensions.
Suitable lipophilic solvents or vehicles for use in the pharmaceutical
compositions described
herein include, by way of example only, fatty oils such as sesame oil, or
synthetic fatty acid
esters, such as ethyl oleate or triglycerides, or liposomes. In certain
specific embodiments,
aqueous injection suspensions contain substances which increase the viscosity
of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the
suspension
- I 7-
CA 02847250 2014-03-20
,
=
contains suitable stabilizers or agents which increase the solubility of the
compounds to allow for
the preparation of highly concentrated solutions. Alternatively, in other
embodiments, the active
ingredient is in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free
water, before use.
[0067] In one aspect, borapetoside A or C, insulin or insulin analogs, as
described herein is
prepared as solutions for parenteral injection as described herein or known in
the art and
administered with an automatic injector. Automatic injectors, such as those
disclosed in U.S.
Patent Nos. 4,031,893, 5,358,489; 5,540,664; 5,665,071, 5,695,472 and
WO/2005/087297 (each
of which are incorporated herein by reference for such disclosure) are known.
In general, all
automatic injectors contain a volume of solution that includes a compound
(i.e., borapetoside A
or C, insulin, insulin analogs, described herein) to be injected. In general,
automatic injectors
include a reservoir for holding the solution, which is in fluid communication
with a needle for
delivering the drug, as well as a mechanism for automatically deploying the
needle, inserting the
needle into the patient and delivering the dose into the patient. Each
injector is capable of
delivering only one dose of the compound.
[0068] In still other embodiments, borapetoside A is administered topically.
The compounds
described herein are formulated into a variety of topically administrable
compositions, such as
solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams
or ointments. Such
pharmaceutical compositions optionally contain solubilizers, stabilizers,
tonicity enhancing
agents, buffers and preservatives.
[0069] In yet other embodiments, any compound described herein is formulated
for transdermal
administration. In specific embodiments, transdermal formulations employ
transdermal delivery
devices and transdermal delivery patches and can be lipophilic emulsions or
buffered, aqueous
solutions, dissolved and/or dispersed in a polymer or an adhesive. In various
embodiments, such
patches are constructed for continuous, pulsatile, or on demand delivery of
pharmaceutical
agents. In additional embodiments, the transdermal delivery of any compound
described herein is
accomplished by means of iontophoretic patches and the like. In specific
embodiments, the rate
of absorption is slowed by using rate-controlling membranes or by trapping the
compound within
a polymer matrix or gel. In alternative embodiments, absorption enhancers are
used to increase
absorption. Absorption enhancers or carriers include absorbable
pharmaceutically acceptable
solvents that assist passage through the skin. For example, in one embodiment,
transdermal
-18-
CA 02847250 2014-03-20
devices are in the form of a bandage comprising a backing member, a reservoir
containing the
compound optionally with carriers, optionally a rate controlling barrier to
deliver the compound
to the skin of the host at a controlled and predetermined rate over a
prolonged period of time, and
means to secure the device to the skin.
[0070] Transdermal formulations described herein may be administered using a
variety of
devices which have been described in the art. For example, such devices
include, but are not
limited to, U.S. Pat. Nos. 3,598,122, 3,598,123, 3,710,795, 3,731,683,
3,742,951, 3,814,097,
3,921,636, 3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894, 4,060,084,
4,069,307,
4,077,407, 4,201,211, 4,230,105, 4,292,299, 4,292,303, 5,336,168, 5,665,378,
5,837,280,
5,869,090, 6,923,983, 6,929,801 and 6,946,144.
[0071] The transdermal dosage forms described herein may incorporate certain
pharmaceutically
acceptable excipients which are conventional in the art. In one embodiment,
the transdermal
formulations described herein include at least three components: (1) a
formulation of a
compound described herein; (2) a penetration enhancer; and (3) an aqueous
adjuvant. In addition,
transdermal formulations can include additional components such as, but not
limited to, gelling
agents, creams and ointment bases, and the like. In some embodiments, the
transdermal
formulations further include a woven or non-woven backing material to enhance
absorption and
prevent the removal of the transdermal formulation from the skin. In other
embodiments, the
transdermal formulations described herein maintain a saturated or
supersaturated state to
promote diffusion into the skin.
[0072] In other embodiments, the compounds are formulated for administration
by inhalation.
Various forms suitable for administration by inhalation include, but are not
limited to, aerosols,
mists or powders. Pharmaceutical compositions of a compound described herein
are
conveniently delivered in the form of an aerosol spray presentation from
pressurized packs or a
nebuliser, with the use of a suitable propellant (e.g.,
dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other
suitable gas). In
specific embodiments, the dosage unit of a pressurized aerosol is determined
by providing a
valve to deliver a metered amount. In certain embodiments, capsules and
cartridges of, such as,
by way of example only, gelatins for use in an inhaler or insufflator are
formulated containing a
powder mix of the compound and a suitable powder base such as lactose or
starch.
-19-
CA 02847250 2014-03-20
[0073] Intranasal formulations are known in the art and are described in, for
example, U.S. Pat.
Nos. 4,476,116, 5,116,817 and 6,391,452, each of which is specifically
incorporated herein by
reference. Formulations, which include a compound described herein, are
prepared according to
these and other techniques well-known in the art are prepared as solutions in
saline, employing
benzyl alcohol or other suitable preservatives, fluorocarbons, and/or other
solubilizing or
dispersing agents known in the art. See, for example, Ansel, H. C. et al.,
Pharmaceutical Dosage
Forms and Drug Delivery Systems, Sixth Ed. (1995). Preferably these
compositions and
formulations are prepared with suitable nontoxic pharmaceutically acceptable
ingredients. These
ingredients are found in sources such as REMINGTON: THE SCIENCE AND PRACTICE
OF
PHARMACY, 21st edition, 2005, a standard reference in the field. The choice of
suitable
carriers is highly dependent upon the exact nature of the nasal dosage form
desired, e.g.,
solutions, suspensions, ointments, or gels. Nasal dosage forms generally
contain large amounts
of water in addition to the active ingredient. Minor amounts of other
ingredients such as pH
adjusters, emulsifiers or dispersing agents, preservatives, surfactants,
gelling agents, or buffering
and other stabilizing and solubilizing agents may also be present. Preferably,
the nasal dosage
form should be isotonic with nasal secretions.
[0074] For administration by inhalation, the compounds described herein, may
be in a form as an
aerosol, a mist or a powder. Pharmaceutical compositions described herein are
conveniently
delivered in the form of an aerosol spray presentation from pressurized packs
or a nebuliser, with
the use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case
of a pressurized
aerosol, the dosage unit may be determined by providing a valve to deliver a
metered amount.
Capsules and cartridges of, such as, by way of example only, gelatin for use
in an inhaler or
insufflator may be formulated containing a powder mix of the compound
described herein and a
suitable powder base such as lactose or starch.
[0075] In still other embodiments, any compound described herein is formulated
in rectal
compositions such as enemas, rectal gels, rectal foams, rectal aerosols,
suppositories, jelly
suppositories, or retention enemas, containing conventional suppository bases
such as cocoa
butter or other glycerides, as well as synthetic polymers such as
polyvinylpyrrolidone, PEG, and
the like. In suppository forms of the compositions, a low-melting wax such as,
but not limited to,
a mixture of fatty acid glycerides, optionally in combination with cocoa
butter is first melted.
-20-
CA 02847250 2014-03-20
,
[0076] In certain embodiments, pharmaceutical compositions are formulated in
any conventional
manner using one or more physiologically acceptable carriers comprising
excipients and
auxiliaries which facilitate processing of the active compounds into
preparations which can be
used pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
Any pharmaceutically acceptable techniques, carriers, and excipients is
optionally used as
suitable and as understood in the art. Pharmaceutical compositions comprising
a compound
described herein may be manufactured in a conventional manner, such as, by way
of example
only, by means of conventional mixing, dissolving, granulating, dragee-making,
levigating,
emulsifying, encapsulating, entrapping or compression processes.
[0077] Pharmaceutical compositions include at least one pharmaceutically
acceptable carrier,
diluent or excipient and at least one compound described herein as an active
ingredient. The
active ingredient is in free-acid or free-base form, or in a pharmaceutically
acceptable salt form.
In addition, the methods and pharmaceutical compositions described herein
include the use
crystalline forms (also known as polymorphs), as well as active metabolites of
these compounds
having the same type of activity. All tautomers of the compounds described
herein are included
within the scope of the compounds presented herein. Additionally, the
compounds described
herein encompass unsolvated as well as solvated forms with pharmaceutically
acceptable
solvents such as water, ethanol, and the like. The solvated forms of the
compounds presented
herein are also considered to be disclosed herein. In addition, the
pharmaceutical compositions
optionally include other medicinal or pharmaceutical agents, carriers,
adjuvants, such as
preserving, stabilizing, wetting or emulsifying agents, solution promoters,
salts for regulating the
osmotic pressure, buffers, and/or other therapeutically valuable substances.
[0078] Methods for the preparation of compositions comprising the compounds
described herein
include formulating the compounds with one or more inert, pharmaceutically
acceptable
excipients or carriers to form a solid, semi-solid or liquid. Solid
compositions include, but are not
limited to, powders, tablets, dispersible granules, capsules, cachets, and
suppositories. Liquid
compositions include solutions in which a compound is dissolved, emulsions
comprising a
compound, or a solution containing liposomes, micelles, or nanoparticles
comprising a
compound as disclosed herein. Semi-solid compositions include, but are not
limited to, gels,
suspensions and creams. The form of the pharmaceutical compositions described
herein include
liquid solutions or suspensions, solid forms suitable for solution or
suspension in a liquid prior to
-21-
CA 02847250 2014-03-20
use, or as emulsions. These compositions also optionally contain minor amounts
of nontoxic,
auxiliary substances, such as wetting or emulsifying agents, pH buffering
agents, and so forth.
[0079] In some embodiments, pharmaceutical composition comprising at least
compounds (i.e.,
borapetoside A or C, and insulin or insulin analog described herein)
illustratively takes the form
of a liquid where the agents are present in solution, in suspension or both.
Typically when the
composition is administered as a solution or suspension a first portion of the
agent is present in
solution and a second portion of the agent is present in particulate form, in
suspension in a liquid
matrix. In some embodiments, a liquid composition includes a gel formulation.
In other
embodiments, the liquid composition is aqueous.
[0080] In certain embodiments, pharmaceutical aqueous suspensions include one
or more
polymers as suspending agents. Polymers include water-soluble polymers such as
cellulosic
polymers, e.g., hydroxypropyl methylcellulose, and water-insoluble polymers
such as cross-
linked carboxyl-containing polymers. Certain pharmaceutical compositions
described herein
include a mucoadhesive polymer, selected from, for example,
carboxymethylcellulose, carbomer
(acrylic acid polymer), poly(methylmethacrylate), polyacrylamide,
polycarbophil, acrylic
acid/butyl acrylate copolymer, sodium alginate and dextran.
[0081] Pharmaceutical compositions also, optionally include solubilizing
agents to aid in the
solubility of the compounds described herein. The term "solubilizing agent"
generally includes
agents that result in formation of a micellar solution or a true solution of
the agent. Certain
acceptable nonionic surfactants, for example polysorbate 80, are useful as
solubilizing agents, as
can ophthalmically acceptable glycols, polyglycols, e.g., polyethylene glycol
400, and glycol
ethers.
[0082] Furthermore, pharmaceutical compositions optionally include one or more
pH adjusting
agents or buffering agents, including acids such as acetic, boric, citric,
lactic, phosphoric and
hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium
borate, sodium
citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane;
and buffers such
as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids,
bases and buffers
are included in an amount required to maintain pH of the composition in an
acceptable range.
[0083] Additionally, pharmaceutical compositions optionally include one or
more salts in an
amount required to bring osmolality of the composition into an acceptable
range. Such salts
include those having sodium, potassium or ammonium cations and chloride,
citrate, ascorbate,
-22-
CA 02847250 2014-03-20
,
borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions;
suitable salts include
sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and
ammonium
sulfate.
[0084] Other pharmaceutical compositions optionally include one or more
preservatives to
inhibit microbial activity. Suitable preservatives include mercury-containing
substances such as
merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium
compounds such
as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium
chloride.
[0085] Still other pharmaceutical compositions include one or more surfactants
to enhance
physical stability or for other purposes. Suitable nonionic surfactants
include polyoxyethylene
fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60)
hydrogenated castor oil; and
polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,
octoxynol 40.
[0086] Still other pharmaceutical compositions may include one or more
antioxidants to enhance
chemical stability where required. Suitable antioxidants include, by way of
example only,
ascorbic acid and sodium metabisulfite.
[0087] In certain embodiments, pharmaceutical aqueous suspension compositions
are packaged
in single-dose non-reclosable containers. Alternatively, multiple-dose
reclosable containers are
used, in which case it is typical to include a preservative in the
composition.
[0088] In alternative embodiments, other delivery systems for hydrophobic
pharmaceutical
compounds are employed. Liposomes and emulsions are examples of delivery
vehicles or
carriers herein. In certain embodiments, organic solvents such as N-
methylpyrrolidone are also
employed. In additional embodiments, the compounds described herein are
delivered using a
sustained-release system, such as semipermeable matrices of solid hydrophobic
polymers
containing the therapeutic agent. Various sustained-release materials are
useful herein. In some
embodiments, sustained-release capsules release the compounds for a few hours
up to over 24
hours. Depending on the chemical nature and the biological stability of the
therapeutic reagent,
additional strategies for protein stabilization may be employed.
[0089] In certain embodiments, the formulations described herein include one
or more
antioxidants, metal chelating agents, thiol containing compounds and/or other
general stabilizing
agents. Examples of such stabilizing agents, include, but are not limited to:
(a) about 0.5% to
about 2% w/v glycerol, (b) about 0.1% to about 1% w/v methionine, (c) about
0.1% to about 2%
w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to
about 2% w/v
-23-
CA 02847250 2014-03-20
,
,
ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate 80, (g) 0.001% to
about 0.05% w/v.
polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k)
cyclodextrins, (1) pentosan
polysulfate and other heparinoids, (m) divalent cations such as magnesium and
zinc; or (n)
combinations thereof.
General Consideration for Combination Treatments
[0090] In general, the compositions described herein and, in embodiments where
combinational
therapy is employed based on the mode of action described herein, other agents
do not have to be
administered in the same pharmaceutical composition, and in some embodiments,
because of
different physical and chemical characteristics, are administered by different
routes. In some
embodiments, the initial administration is made according to established
protocols, and then,
based upon the observed effects, the dosage, modes of administration and times
of administration
is modified by the skilled clinician.
[0091] In some embodiments, therapeutically-effective dosages vary when the
drugs are used in
treatment combinations. Combination treatment further includes periodic
treatments that start
and stop at various times to assist with the clinical management of the
patient. For combination
therapies described herein, dosages of the co-administered compounds vary
depending on the
type of co-drug employed, on the specific drug employed, on the disease,
disorder, or condition
being treated and so forth.
[0092] It is understood that in some embodiments, the dosage regimen to treat,
prevent, or
ameliorate the condition(s) for which relief is sought, is modified in
accordance with a variety of
factors. These factors include the disorder from which the subject suffers, as
well as the age,
weight, sex, diet, and medical condition of the subject. Thus, in other
embodiments, the dosage
regimen actually employed varies widely and therefore deviates from the dosage
regimens set
forth herein.
[0093] It is understood that in some embodiments, the dosage regimen to treat,
prevent, or
ameliorate the condition(s) for which relief is sought, is modified in
accordance with a variety of
factors. These factors include the disorder from which the subject suffers, as
well as the age,
weight, sex, diet, and medical condition of the subject. Thus, in other
embodiments, the dosage
regimen actually employed varies widely and therefore deviates from the dosage
regimens set
forth herein.
-24-
CA 02847250 2014-03-20
Examples
Example 1. Plant material and preparation of plant extracts
[0094] The vines of Tinospora crispa Miers (Menispermaceae) were collected and
grounded.
Borapetoside A (see FIG. 1) was extracted by the known method (Lam et al.
2012). Alternative
methods may be used to prepare Borapetoside A.
Example 2. Cell lines and cell culture preparation
[0095] The C2C12 skeletal muscle cells were cultured in Dulbecco's modified
Eagle's medium
(DMEM; Gibco/Invitrogen, Carlsbad, CA), and the human hepatocellular carcinoma
cell line,
Hep3B, was cultured in Roswell Park Memorial Institute medium 1640 (RPMI 1640
medium;
Gibco/Invitrogen, Carlsbad, CA). Cells were cultured at 37 C in the humidified
incubator
supplied with 5% CO2. Culture mediums were supplemented with 4.5 mgmL-1
glucose, penicillin
100 IUmL-1, streptomycin 100 ligmL-1 and 10% fetal bovine serum
(Gibco/Invitrogen, Carlsbad,
CA). Only C2C12 cells were then switched to 2% horse serum for 3 days after
reaching the 70%
confluence. Myotubes formation was achieved after 4 days of incubation, and
the cells were used
for subsequent experiments. Culture mediums were replaced by serum free DMEM
or RPMI for
24h before experiments (see e.g., procedure described in Sultan et al., 2006).
Example 3: Glycogen content assay in cultured cells
[0096] C2C12 and Hep3B cells were treated with 1 nM insulin, 100 [tM
metformin, and the
assigned concentration of borapetoside A for 30 minutes. To develop the
insulin resistance state
of cells, C2C12 cells were incubated with IL-6 at 20 ngmL-1 for 1 hour. Later,
the cells were
treated with 1 nM insulin, 100 M metformin, and the assigned concentration of
borapetoside A
for 30 minutes. After the assigned treatment, cells were washed twice with
phosphate buffered
saline (PBS). Glycogen content was measured as previously described, with some
modifications
(see e.g., Savage et al., 2008). The protein concentrations of the cell
lysates were determined by
using Pierce BCA Protein Assay Kit (Thermo-Scientific, Rockford, IL). The
glycogen content of
each sample was normalized by total protein.
Example 4: Animal study of Borapetoside A Treatment re Plasma Glucose and
Insulin Level
[0097] The 4-week-old male ICR mice were purchased from the Animal Center of
the College of
Medicine, National Taiwan University (Taipei, Taiwan) with delicate humane
care. This animal
study was conducted by following the University ethical guidelines and the
'Guide for the Care
and Use of Laboratory Animals ' published by the US National Institutes of
Health (NIH
-25-
CA 02847250 2014-03-20
publication no. 85-23, revised 1996). The animal experiments were approved by
the Institutional
Animal Care and Use Committee (IACUC) of the National Taiwan University (IACUC
No.
20110073). Animals were housed in a room at the constant temperature, 22 1 C,
with 12 hours
light and dark cycles.
[0098] After three days of acclimatization, the mice had free access to a
standard rodent chow
(normal mice), a standard rodent chow after single 250mgkg-1 streptozotocin
(STZ)
intraperitoneal injection (T1DM mice), and a fat-rich chow diet and fructose-
sweetened water
(T2DM mice) for 4 weeks. The induction of Type 1 DM was assessed and confirmed
when the
mice had plasma glucose levels? 350 mgdL-1, accompanied with polyuria,
hyperphagia and
decreased body weight. The induction Type 2 DM in the mice was assessed by
measuring fasting
plasma glucose levels and confirmed when plasma glucose level was? 150mgdL-1
after the 4-
week induction. The animals were assigned randomly into three groups, with
seven animals in
each group. In group I, the mice were treated with vehicle, 2% DMSO in normal
saline, as the
negative control. In group IT, the mice were intraperitoneally treated with
borapetoside A (0.1-10
mgkg1). In group III, the positive control, the mice received
intraperitoneally metformin
(300mgkg-1; Sigma Chemical Co., St. Louis, MO, U.S.A.) or insulin (0.5 IUkg-1;
Insulin
Actrapid0 HM; Novo Nordisk, Denmark). In the acute study, mice under
anesthesia received a
single intraperitoneal administration of borapetoside A, negative control, or
positive control. In
the subacute study, mice received intraperitoneal administrations of
borapetoside A, vehicle, or
insulin twice a day for seven days.
[0099] Blood samples were collected from the orbital vascular plexus of the
mice under
anesthesia. Blood samples were then centrifuged at 13000 rpm for 5 min, and
the plasma
samples were collected and kept on ice prior to the assay. The plasma glucose
concentration was
measured by Glucose Kit Reagent (Biosystems S.A., Barcelona, Spain) following
the
manufacturer's instructions, and the insulin levels were estimated using mouse
insulin ELISA kit
(Mercodia AB, Uppsala, Sweden).
[00100] The mice were maintained under anesthesia throughout the
procedure. Glucose
(2.0 gkg-1) was administered intraperitoneally. Blood samples were collected
from the orbital
vascular plexus at 0, 30, 60, 120 min. The concentrations of the plasma
glucose were measured,
and the values of AUC were calculated accordantly.
-26-
CA 02847250 2014-03-20
. .
[001011 The mice were injected with borapetoside A (10mgke,
interperitoneal) for 60
minutes or insulin (0.5 IUke, interperitoneal) for 30 minutes. After the
treatment, the anesthetic
mice were sacrificed by cervical dislocation to collect the soleus muscle and
liver. About 40 mg
of each sample was dissolved in 1 mol/L KOH at 75 C for 30 min. The dissolved
homogenate
was neutralized by glacial acetic acid and then incubated overnight in acetate
buffer (0.3 mol/L
sodium acetate, pH 4.8) containing amyloglucosidase (Sigma, St. Louis, MO).
The mixture was
then neutralized with 1 mol/L NaOH to stop the reaction. The glycogen contents
in the tissue
samples were determined as 1.tg of glucose per mg of tissue (wet weight).
Western blot assay and analysis
[00102] Western blot analysis was carried out by known methods
with a slight
modification. Briefly, tissues were homogenized in T-PER Tissue Protein
Extraction Reagent
(Pierce Biotechnology, Rockford, IL) with HaltTM Protease Inhibitor Single-Use
Cocktail (Pierce
Biotechnology, Rockford, IL). Liver homogenates were prepared by mechanical
homogenization
(Polytron PT3100, Luzernerstrasse, Switzerland). After centrifuging the
homogenates at 10,000
g for 30 minutes, the protein concentrations were determined by using a BCA
Protein Assay Kit
(Pierce Biotechnology, Rockford, IL). Later, 60iig of protein preparations
were applied on 10%
sodium dodecyl sulfate-polyacrylamide gel for electrophoresis and then
transferred to
polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes
were analysed
by using the antibodies against phospho-insulin receptor 13 (IRf3) (Tyr1345),
phospho-Akt (Ser473),
phosphor-AS160 (Thr642), AS160 (Cell Signaling Technology, Beverly, MA), IRI3,
Akt, 13-
Actin(Santa Cruz Biotechnology, Santa Cruz, California), GLUT2 (Abeam,
Cambridge, UK),
and PEPCK (a gift from Professor DK Granner), respectively. Chemiluminescence
detection and
image analysis was performed with UVP-Biochimie Bioimager and Lab Works
software (UVP,
Upland, CA). The intensity of each band was quantified with ImageQuant.
Statistical analysis of the data
[00103] The results are presented as mean the standard error of
the mean (SEM).
Statistical difference between the means of the various groups were analyzed
using one way
analysis of variance (ANOVA), followed by Turkey's multiple test with Prism
5.0 demo
software (GraphPad Software Inc., CA). Results were considered statistical
significant at the
95% confidence interval (i.e., p < 0.05).
Results And Analysis
-27-
CA 02847250 2014-03-20
[00104] To investigate the influence of borapetoside A treatment on the
uptake of glucose
in metabolic organs and tissues, such as the liver and skeletal muscles, the
C2C12 myotube and
Hep3B hepatocytes were exposed to borapetoside A at the assigned
concentrations. As shown in
FIG. 2A, when differentiated C2C12 myotubes were stimulated with 10-8, 10-7,
and 10-6 mol/L
borapetoside A, there were 1.53+0.10-, 1.28+0.04-, 1.49+0.15-fold increase of
glycogen,
respectively. In the IL-6-induced insulin resistant model, glycogen content
was not increased by
insulin treatment. However, the glycogen contents of the IL-6-treated cells
were increased by
1.35+0.08-, 1.38+0.06-, 1.64+0.05-fold while receiving the 10-8, 10-7,and 10-6
mol/L
borapetoside A treatment, respectively (FIG. 2B). Hepatocyte Hep3B stimulated
with 10-7, 10-6
and 10-5 mol/L borapetoside A showed a 1.46+0.05-, 1.39+0.12-, 1.54 0.11-fold
increase of
glycogen content (FIG. 2C).
[00105] Thus it is clearly shown that Borapetoside A induced glycogen
accumulation in
C212 and Hep3B and thus enhances glycogen synthesis.
[00106] The effects of borapetoside A on blood glucose and insulin levels
were measured
and analyzed. In FIG. 3, the antihyperglycemic effects of borapetoside A and
the reference drug,
metformin, was analyzed at 60 min after a single dose administration in the
normal, type 1 DM,
and type 2 DM animal models, respectively. Borapetoside A was administered to
the normal
mice, the mice with type 1 DM, and the mice with type 2 DM at dosages of 0.1,
0.3, 1.0, 3.0, and
10.0 mgkg-1. The plasma glucose level of the treated mice was examined at 0
and 60 minutes
after the treatment. Borapetoside A lowered the plasma glucose level in normal
mice, the type 1
DM and type 2 DM bearing mice in a dose-dependent manner. A significant
decrease of plasma
glucose level and the mice with type 2 DM were observed when the dose of
borapetoside A was
higher than 0.3 mgkg-I. In the normal mice, the effect was not observed until
the treatment with 1
mgkg-1 of borapetoside A. As such, it is clearly shown that Borapetoside A
could stimulate
insulin release in the normal mice and the mice with type 2 DM but not in
those with type 1 DM
(Table 1). The plasma insulin level was dose-dependently increased at the dose
range between
0.3 and 10 mgkg-I by borapetoside A in the normal mice. In contrast, in the
mice with type 2 DM
group, the concentration of plasma insulin was increased only by 3.0 and 10
mgkg-I of
borapetoside A, However, in the mice with type 1 DM, the insulin-secretion
deficient model
animals, neither the borapetoside A nor metformin could rescue the plasma
insulin level.
-28-
CA 02847250 2014-03-20
Table 1
The plasma insulin levels in normal and diabetic mice before and after
treatment.
Groups Plasma insulin (pmo1/1)
Before-treatment After-treatment
Normal mice vehicle borapetoside A 66.8 w 2.4 67.8 w 3,0
0.1 mg kg-1 61.0 w 4.6 68.3 w 4.4
0.3 mg kg-1 63.4 w 3,9 129.4 w 22.0'
1 mg kg 66.1 w 5.4 118,8 w 10.1'
3 mg kg-1 64.4w 3.6 121.2 w 14.2'
mgkg-1 68.9w 1.3 108.2 w 2.9'
Metformin 66.6 w 1,3 72 w 3.1
TI DM mice vehicle borapetoside A 22.8 w 0,2 22.8 w 0.1
0.1 mg kg-1 22.9 w 0.1 22.7 w 0,2
0.3 mg kg-1 22.9 w 0.1 22.7 w 0.3
1 mg kg-1 23.1 w 0.2 23.0 w 0,2
3 mg kg-1 23.1 0.2 23.0 0.0
10mgkg-1 211 w 0.1 23,0 0.1
Metformin 22.4 w 0.4 22,7 w 0.3
T2DM mice vehicle borapetoside A 139.1 2.1 141.7w 2,3
0.1 mg kg-1 140.5 4,1 134.5 w 9.3
0.3 mg kg 133.0 4.2 149.0 w 9.5
1 mg kg-1 126.8 w 5,7 123.7 9.6
3 mg kg-1 138.3 w 3,6 184.3 w 14.6'
10mgkg-1 134.9 w 3.5 205.3 w 22.2'
Metformin 148.1 w 15.2 182.0 27.7
Values were expressed as meanw SEM from six animals in each group.
* p-< 0.05 from vehicle group vs drug-treated.
[00107] The influence of borapetoside A on glucose tolerance test were
measured and
analyzed. The influence of borapetoside A on plasma glucose levels in the
normal mice and the
mice with type 2 DM was first investigated. As shown in FIG. 4A, the
concentrations of plasma
glucose increased at 30 min after the administration of dextrose in all groups
and decreased
thereafter in both the normal mice and the mice with type 2 DM. The increase
of plasma glucose
level at 30 min was significantly suppressed in borapetoside A-treated (0.1,
1, and 10 mgkg-1)
and metformin-treated groups, compared to the vehicle group. The area under
the curve (AUC)
of borapetoside A (0.1, 1, and 10mgkg-1) also dose-dependently decreased to
78.8 3.0%,
69.9 7.1%, and 68.9 3.5% of vehicle control in normal mice, and 74.9 4.5, 71.1
3.2, and
68.9 1.9% of vehicle control in type 2 DM bearing mice respectively (FIG. 4B
and D). These
data indicated that glucose tolerance was improved in the borapetoside A-
treated group.
[00108] The influence of borapetoside A on glycogen synthesis in the
isolated soleus
muscle and the liver was measured and analyzed. To further characterize the
effects of
-29-
CA 02847250 2014-03-20
borapetoside A treatment on glycogen metabolism, glycogen content of each
treated groups was
assessed. All three groups, the normal mice, the mice with type 1 DM, and the
mice with type 2
DM, were intraeritoneally injected with borapetoside A, Actrapid or metformin.
Their glycogen
contents in liver and soleus muscle were determined 60 minutes after the
injections. As shown in
FIG. 5A-C, in soleus muscle, the glycogen content was significantly increased
to 116.5 4.2%,
155.6 8.6%, and 171.8 12.0% of vehicle control in the borapetoside A treated
normal mice,
mice with type 1 DM, and mice with type 2 DM, respectively. Furthermore,
regarding to major
metabolic organ, liver, borapetoside A treatment significantly resulted in an
increase of glycogen
content to 257.8 15.8%, 196.9 15.6%, and 146.1 12.0% of vehicle control in the
normal mice,
the mice with type 1 DM, and the mice with type 2 DM (FIG. 6A-C).
[00109] The influence of borapetoside A on the insulin signaling pathway
in the livers of
the mice with type 1 DM was assessed. To characterize the molecular effects of
borapetoside A
on glucose utilization in liver, the phosphorylation status of IR, Akt/PKB,
and AS160 and the
expression profile of GLUT2 and PEPCK in liver tissues was further examined
via western blot
analysis. The liver tissues were collected from the mice with type 1 DM
receiving borapetoside
A treatment at a dosage of 10 mgkg-1 or receiving insulin treatment twice a
day for 7 days. The
results showed that borapetoside A treatment increased the phosphorylated IR,
Akt, and AS160,
and it also increased the expression of GLUT2 in the liver tissues of the mice
with type 1 DM
(FIG. 7A). Moreover, the significant decrease of PEPCK expression was observed
in the liver
tissue. The density ratios of phosphor-Tyr1345-IR to IR, phosphor-Ser473-Akt
to Akt, phosphor-
Thr642-AS160 to AS160, GLUT2 to I3-actin and PEPCK to 13-actin were calculated
and plotted in
the FIG. 7B-7F.
Example 5: Insulin Sensitivity Study with borapetoside C
Animals and treatment protocol
[00110] This study was conducted following the University ethical
guidelines on animal
experimentation and complied with the Guide for the Care and Use of Laboratory
Animals
published by the US National Institutes of Health (NIH publication no. 85-23,
revised 1996). The
animal facility was well controlled for temperature (22 1 C), and humidity (60
5%) and a 12
h/12 h light-dark cycle was maintained with access to food and water ad
libitum. Four-week-old
male ICR mice were acquired from BioLasco Taiwan Co., Ltd. and maintained at
College of
-30-
CA 02847250 2014-03-20
Medicine Experimental Animal Center, National Taiwan University. The study was
conducted
on 8-10 week-old male ICR mice.
[00111] T1DM mice were induced by a known method. In brief, an
intraperitoneal
injection of streptozotocin (STZ; Sigma Chemical Co.; St. Louis, MO) at 150
mg/kg was
performed in mice that were fasted for 48h. The induction of T1DM was assessed
and confirmed
when the mice had plasma glucose levels > 350 mg/dL, accompanied with
polyuria, hyperphagia
and decreased body weight. The control mice group received an injection of
vehicle and then
carried out for 4 weeks. T2DM mice were induced by maintaining on a fat-rich
chow diet and
fructose-sweetened water for 4 weeks from the age of 4-5 weeks according to
previous methods.
The induction T2DM mice were assessed by measuring fasting plasma glucose
levels and
confirmed when plasma glucose level was > 150mg/dL after a 4-week induction.
[00112] Blood samples were collected from the orbital vascular plexus of
mice under
anesthesia with sodium pentobarbital (80 mg/kg, intraperitoneal, Sigma
Chemical Co., St. Louis,
MO, USA). Blood samples were then centrifuged at 13000 rpm for 5 min, and the
plasma was
kept on ice prior to the assay. The plasma glucose concentration was measured
using commercial
kits following manufacturer's instructions (BioSystems S.A., Barcelona,
Spain).
[00113] To perform OGTT, mice were fasted overnight, divided into 2 groups
and then
administered a vehicle control and 5 mg/kg borapetoside C. This was followed
by administration
of a glucose solution at 2 g/kg via tube feeding. Blood samples were withdrawn
from the orbital
vascular plexus at intervals of 30, 60, 120, and 150 min after glucose
administration. For the
ITT, mice were fasted for 3 h. Human insulin (Insulin Actrapidt HM; Novo
Nordisk, Denmark)
was injected intraperitoneally after an intraperitoneal administration of
0.1mg/kg borapetoside C
for 30 min. Blood samples were collected from the orbital vascular plexus at
the timed intervals
mentioned above.
Glycogen content assay
[00114] Glycogen content of skeletal muscles was measured by a known
method. In brief,
mice were injected with borapetoside C (5.0mg/kg, interperitoneal) for 60 min
or insulin (0.5
IU/kg, interperitoneal.) for 30 min, and the soleus muscle was isolated from
anesthetized mice.
About 40 mg of muscle sample was dissolved in 1 N KOH at 75 C for 30 min. The
dissolved
homogenate was neutralized by glacial acetic acid and then incubated overnight
in acetate buffer
(0.3 M sodium acetate, pH 4.8) containing amyloglucosidase (Sigma, St. Louis,
MO). The
-31-
CA 02847250 2014-03-20
mixture was then neutralized with 1 N NaOH to stop the reaction. The glycogen
contents in the
tissue samples were determined as ng of glucose per mg of tissue (wet weight).
Collection of liver tissue
[00115] After treatment, the mice were sacrificed by cervical dislocation
under anesthesia.
The liver was immediately frozen in liquid nitrogen. The liver tissue was
preserved at -80 C
before they were used for further assays.
Western blot analysis
[00116] Tissues were homogenized in T-PER Tissue Protein Extraction
Reagent (Pierce
Biotechnology, Rockford, IL) with HaltTM Protease Inhibitor Single-Use
Cocktail (Pierce
Biotechnology, Rockford, IL). Liver homogenates were prepared by mechanical
homogenization
(Polytron PT3100, Luzernerstrasse, Switzerland). After centrifuging the
homogenates at 10,000
g for 30min, the supernatants were collected and frozen at -80 C for further
use. The protein
concentrations were determined by using a BCA Protein Assay Kit (Pierce
Biotechnology,
Rockford, IL). For western blot analysis, about 60pg of protein preparations
were applied on
10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then
transferred to
polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes
were blocked
with 5% (w/v) non-fat dry milk in phosphate buffered saline (PBS) containing
0.1% Tween 20
(PBS-T). After blocking, the blotted membrane was incubated with anti-phospho-
insulin receptor
13 (IRI3) (Tyr1345), phospho-Akt (Ser473) (Cell Signaling Technology, Beverly,
MA), anti- IR13,
Akt, 13-Actin (Santa Cruz Biotechnology, Santa Cruz, California), and anti-
GLUT2 antibodies
(Abcam, Cambridge, UK) in presence of 3% bovine serum albumin (BSA) in PBS-T
buffer.
Following the incubation, the membranes were washed 3 times with PBS-T for 15
min each and
then incubated with the appropriate peroxidase-conjugated secondary antibodies
(Santa Cruz
Biotechnology, USA) in PBS-T. After removal of the secondary antibody, blots
were washed
and developed using the enhanced chemiluminescenece (ECL) western blotting
system
(Millipore, Billerica, MA). The density of the protein bands were quantified
using ImageQuant.
Statistical analysis
[00117] Results were presented as mean SEM for the number (n) of animals
in the group
as indicated in the tables and figures. Statistical difference between the
means of the various
groups were analyzed using one way analysis of variance (ANOVA) followed by
Turkey's
-32-
CA 02847250 2014-03-20
,
multiple test with Prism 5.0 demo software (GraphPad Software Inc., La Jolla,
CA). Data were
considered statistically significant at *p <0.05.
Results and Analysis
[00118] To determine the effect of borapetoside C on glucose tolerance,
OGTTs were
performed in non-diabetic and T2DM mice. In non-diabetic mice, the basal
plasma glucose
concentrations in the borapetoside C-treated and vehicle-treated groups were
115 3.5 mg/dL
and 121.8 1.5 mg/dL respectively as shown in FIG. 8A. At 60 min after oral
administration of
glucose, the plasma glucose concentration was elevated to 406.8 28.0 mg/dL
in the vehicle-
treated mice, and to 312.8 21.3 mg/dL in the borapetoside C-treated mice.
The plasma glucose
level of borapetoside C-treated mice was significantly lower than that of
vehicle-treated mice
after oral administration of glucose. The plasma glucose levels of
borapetoside C-treated mice
were maintained significantly lower than those of the vehicle-treated mice at
120 min, and 150
min after treatment. In T2DM mice (FIG. 8B), the basal plasma glucose
concentrations of the
vehicle- and borapetoside C-treated groups were 200.0 6.8 mg/dL and 206.8
19.0 mg/dL
respectively. Sixty min after oral glucose administration, the plasma glucose
concentration
elevated to 411.1 11.7 mg/dL in vehicle-treated mice, and to 338.2 24.5
mg/dL in
borapetoside C-treated mice. The plasma glucose levels in borapetoside C-
treated mice at 60,
120, and 150 min after oral administration of glucose were significantly lower
than those of
vehicle-treated mice at the same time points. These results indicate that
borapetoside C
significantly enhanced glucose utilization in T2DM mice. Borapetoside C also
decreased the area
under the curve (AUC) by 22% and 16% of that of the vehicle-treated group in
normal and
T2DM mice, respectively (FIG. 8C). These data indicate that glucose tolerance
improved in the
borapetoside C-treated group.
[00119] The glycogen content in skeletal muscle of the normal, T1DM, and
T2DM mice,
were determined at 30 min after intraeritoneal injection with borapetoside C
(5 mg/kg) and
Actrapid (0.5 IU/kg; a short-acting insulin provided by Novo Nordisk). As
shown in Table 2,
borapetoside C significantly increased glycogen synthesis in both normal and
diabetic mice.
Relative to the insulin effect, borapetoside C caused a more dramatic increase
in glycogen
content in T2DM mice, but a less dramatic change in T1DM mice.
[00120] Table 2. Glycogen synthesis in skeletal muscle in normal and
diabetic mice.
-33-
CA 02847250 2014-03-20
Vehicle Borapetoside C Insulin
Normal mice 21.5+0.4 25.1+1.3* 30.2+2.0***
T1DM mice 16.7+0.9 23.2+0.7*** 30.0+1.0***
T2DM mice 192+0.8 23.3+0.4*** 20.2+0.6
Values expressed as mean SEM from six animals in each group. T1DM mice, T2DM
and normal mice treated with vehicle at the same volume. *p<0.05 , **p<0.01
vs.
***p<0.005 represents the level of significance compared with the value with
vehicle-treated mice.
[00121] To characterize the mechanistic effect of borapetoside C on
glucose utilization of
liver, western blot analyses for the levels of IR and Akt/PKB phosphorylation
states and GLUT2
expression in the liver of T1DM mice were examined after a 7-day treatment of
borapetoside C
at 5.0 mg/kg or insulin at 0.5 IU/kg twice daily were performed. The results
showed that
borapetoside C treatment increased the levels of phosphorylated IR,
phosphorylated Akt and the
expression of GLUT2 in the liver of T1DM mice (FIG 9). Compared with the
effect of insulin,
borapetoside C induced more phosphorylation of IR, but less phosphorylation of
Akt and
GLUT2 expresiion than insulin. The density ratios of phosphor-Tyr1345-IR to
IR, phosphor-
Ser473-Akt to Akt and GLUT2 to 13-actin were calculated and plotted in the
lower panel of FIG. 9.
[00122] In a previous study, borapetoside C showed a hypoglycemic effect
beyond the
dose of 0.1 mg/kg both in normal, T1DM, and T2DM mice. Therefore, the effects
of
borapetoside C on insulin tolerance were tested in this study. The non-
diabetic, T1DM, and
T2DM mice were injected with insulin at various doses (i.e., 0.1 IU/kg, 0.5
IU/kg, and 1.0 IU/kg)
in conjunction with either borapetoside C (0.1 mg/kg) or a vehicle control
(Table 3), and their
plasma glucose levels were examined before and 30 mm after insulin injection.
The activity of
lowering plasma glucose levels (AL) was calculated by the formula: (G,-
Gt)/G1x100%, where the
G, was the initial glucose concentration and Gt was the plasma glucose
concentration after 30 -
min treatment with insulin. Insulin reduced the plasma glucose levels in both
normal and diabetic
mice, and the glucose levels were further decreased when insulin was co-
administered with
borapetoside C to the mice. Since the dose of borapetoside C at 0.1mg/kg did
not alter the
-34-
CA 02847250 2014-03-20
plasma glucose levels, these results indicate that borapetoside C
significantly increased the
sensitivity of diabetic mice to exogenous insulin.
[00123] Table 3. Plasma glucose reduction in mice treated with insulin and
borapetoside
C.
AL (%)*
Insulin 0.1 IU/kg 0.5 IU/kg 1.0
IU/kg
Normal mice Vehicle 14.6+0.8
37.5+1.1 56.8+1.9
Borapetoside C 17.2+1.2
45.7+3.7 64.7+1.8*
T1DM mice Vehicle 8.7+1.6 11.5+0.6
24.0+1.3
Borapetoside C 10.5+1.4
18.9+2.1 32.2+2.1**
T2DM mice Vehicle 19.5+1.5 27.5+2.6
32.0+1.1
Borapetoside C 24.0+1.1
31.6+1.6 42.5+0.8***
[00124] Both
non-diabetic and diabetic mice were subjected to various doses of insulin
injections at 30 min after intraperitoneal injections with borapetoside C (0.1
mg/kg) or with the
vehicle. The glycogen contents in skeletal muscles were then determined at 30
min after insulin
injections (Table 4). The ratios of increased glycogen content in the other
groups were
normalized to those of vehicle-treated group. The ratio was 119.8%, 125.3%,
and 108.5% in
normal, T1DM, and T2DM mice of insulin-treated group. In borapetoside C
combined with
insulin-treated group, it was 130.2%, 162.8%, and 122.6% in normal, T1DM, and
T2DM mice.
Administration of borapetoside C followed by insulin injection significantly
enhanced glycogen
synthesis in normal mice, T1DM mice, and T2DM mice by 52.5%, 148.2%, and
165.9% as
compared to that in mice injected only with insulin. The increase of insulin
sensitivity by
borapetoside C is more prominent in diabetic mice than normal mice. Similarly,
plasma glucose
reduction in mice treated with insulin and borapetoside A are tested and
measured.
[00125] Table 4. Glycogen content in skeletal muscle in mice treated with
insulin and
borapetoside C.
-35-
CA 02847250 2014-03-20
Vehicle BoC Insulin BoC +Insulin
Normal mice 31.8 0.4 32.7 0.7 38.1 0.3*** ...###
41.4 0.6 '
T1DM mice 30.4 1.6 30.0 1.2 38.1 2.3****#
49.5 3.5
T2DM mice 32.7 1.0 30.7 1.3 35.5 I.1 40.1 1.4**'4
[00126] Effect of combining borapetoside C and insulin on the protein
level in the liver of
T1DM mice. After a continuous 7-day treatment, the degree of tyrosine
phosphorylation of IR,
serine phosphorylation of Akt and GLUT2 in the liver was not significantly
increased in the
borapetoside C-treated (0.1 mg/kg twice daily) group. However, under the
combination of
insulin stimulation (1.0 IU/kg twice daily), the level of tyrosine
phosphorylation of IR, serine
phosphorylation of Akt and GLUT2 were elevated to 1.4, 3.0,and 1.3-fold of
their vehicle-treated
counterparts (FIG. 10). Similarly, glycogen content in skeletal muscle in mice
treated with
insulin and borapetoside A are tested and measured.
[00127] As demonstrated by Examples 1-3, borapetoside A can be used in
combination
with insulin to treat type 1 diabetes.
Example 5: Oral Formulation
[00128] To prepare a pharmaceutical composition for oral delivery, 100 mg
of an
exemplary borapetoside A or C was mixed with 100 mg of corn oil. The mixture
was
incorporated into an oral dosage unit in a capsule, which is suitable for oral
administration.
[00129] In some instances, 100 mg of borapetoside A or C is mixed with 750
mg of starch.
The mixture is incorporated into an oral dosage unit for, such as a hard
gelatin capsule, which is
suitable for oral administration.
Example 6: Sublingual (Hard Lozenge) Formulation
[00130] To prepare a pharmaceutical composition for buccal delivery, such
as a hard
lozenge, mix 100 mg of a compound described herein, with 420 mg of powdered
sugar mixed,
with 1.6 mL of light corn syrup, 2.4 mL distilled water, and 0.42 mL mint
extract. The mixture is
gently blended and poured into a mold to form a lozenge suitable for buccal
administration.
Example 7: Inhalation Composition
[00131] To prepare a pharmaceutical composition for inhalation delivery,
20 mg of a
compound described herein is mixed with 50 mg of anhydrous citric acid and 100
mL of 0.9%
-36-
CA 02847250 2014-03-20
sodium chloride solution. The mixture is incorporated into an inhalation
delivery unit, such as a
nebulizer, which is suitable for inhalation administration.
[00132] While preferred embodiments of the present invention have been
shown and
described herein, it will be obvious to those skilled in the art that such
embodiments are provided
by way of example only. Numerous variations, changes, and substitutions will
now occur to
those skilled in the art without departing from the invention. It should be
understood that various
alternatives to the embodiments of the invention described herein may be
employed in practicing
the invention. It is intended that the following claims define the scope of
the invention and that
methods and structures within the scope of these claims and their equivalents
be covered thereby.
-37-
