Note: Descriptions are shown in the official language in which they were submitted.
CA 02847286 2014-03-20
THERAPEUTIC METHODS AND COMPOSITIONS FOR TREATING DIABETES
UTILIZING DITERPENOID COMPOUNDS
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 I 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
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decreases mortality. Type 1 diabetes is typically treated with combinations of
regular and
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.
SUMMARY OF THE INVENTION
[0005] In one aspect provided herein are methods for treating insulin
resistant diabetes in a
subject comprising administering an effective amount of borapetoside A, or a
pharmaceutically
acceptable salt, metabolite, solvate or prodrug thereof, to said subject.
[0006] In another aspect provided herein are methods for treating diabetes in
a subject wherein
said subject is in a inflammatory condition comprising administering said
subject an effective
amount of borapetoside A, or a pharmaceutically acceptable salt, metabolite,
solvate or prodrug
thereof, to said subject.
INCORPORATION BY REFERENCE
[0007] 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
[0008] 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:
[0009] FIG. 1 shows chemical structure of borapetoside A.
[0010] 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
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.)
[0011] 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
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,
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).
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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
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.)
DETAILED DESCRIPTION OF THE INVENTION
[0016] In accordance with the practice of this invention, borapetoside A is
provided herein as a
pharmacological agent for treatment of diabetes.
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[0017] 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
were reported in several previous studies. However, the active ingredients in
the extract are not
well investigated.
[0018] 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.
[0019] 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 108 to10-7 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.
[0020] 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.
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[0021] Interleukin (IL)-6 is one of several proinflammatory cytokines that
have been associated
with insulin resistance and type 2 diabetes. A two- to threefold elevation of
circulating IL-6 has
been observed in these conditions. It has been established that IL-6 can
inhibit insulin receptor
(IR) signal transduction and insulin action in both primary mouse hepatocytes
and the human
hepatocarcinoma cell line, HepG2. This inhibition depends on duration of IL-6
exposure, with a
maximum effect at 1-1.5 h of pretreatment with IL-6 in both HepG2 cells and
primary
hepatocytes. The IL-6 effect is characterized by a decreased tyrosine
phosphorylation of IR
substrate (IRS)-1 and decreased association of the p85 subunit of
phosphatidylinositol 3-kinase
with IRS-1 in response to physiologic insulin levels. In addition, insulin-
dependent activation of
Akt, important in mediating insulin's downstream metabolic actions, is
markedly inhibited by
IL-6 treatment. Finally, a 1.5-h preincubation of primary hepatocytes with IL-
6 inhibits insulin-
induced glycogen synthesis by 75%. These data suggest that IL-6 plays a direct
role in insulin
resistance at the cellular level in both primary hepatocytes and HepG2 cell
lines and may
contribute to insulin resistance and type 2 diabetes.
[0022] 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
lowering effect of 10 mgkg-1 borapetoside A was similar to the effect of 300
mgkg-1 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-1
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.
[0023] 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 IPGIT test. Borapetoside A has notably
enhanced the glucose
uptake and utilization in peripheral tissues, which is a major site of glucose
disposal (Hollenbeck
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,
,
and Reaven, 1987), 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-I borapetoside A significantly
decreased the AUC.
[0024] 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).
[0025] 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
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).
[0026] 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
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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.
[0027] In some embodiments provide methods for treating insulin resistant
diabetes in a subject
comprising administering an effective amount of borapetoside A, or a
pharmaceutically
acceptable salt, metabolite, solvate or prodrug thereof, to said subject. In
some embodiments
provide methods of treating diabetes in a subject wherein said subject is in
an inflammatory
condition comprising administering said subject an effective amount of
borapetoside A, or a
pharmaceutically acceptable salt, metabolite, solvate or prodrug thereof, to
said subject. In some
embodiment, the inflammatory condition is induced by IL-6. In certain
embodiments,
borapetoside A induces increase of glycogen. In certain embodiments,
borapetoside A lowers
plasma glucose concentration in said subject. In some embodiments,
borapetoside A increases
the phosphorylation of IR, Akt, and AS160 in liver of said subject (e.g., a
subject with type 1
diabetes). In certain embodiments, borapetoside A decreases the expression of
PEPCK protein
in liver of the subject (e.g., a subject with type 1 diabetes). In certain
embodiments, borapetoside
A increases the insulin secretion of said subject (e.g., a subject with type 2
diabetes).
[0028] In some embodiments provided herein are methods for the treatment of a
patient whose
diabetes is insulin resistant or in an inflammatory condition comprising
administering the patient
in need thereof with borapetoside A, or a pharmaceutically acceptable salt,
metabolite, solvate or
prodrug thereof.
[0029] In other embodiments, borapetoside A is isolated from the extracts of
Tinospora crispa.
Certain Pharmaceutical and Medical Terminology
[0030] 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
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used herein are for organizational purposes only and are not to be construed
as limiting the
subject matter described.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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,
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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
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.
[0037] 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 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
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.
[0038] The term "pharmaceutical composition" refers to a mixture of a compound
(i.e.,
borapetoside A 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.
[0039] 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.
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CA 02847286 2014-03-20
[0040] 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
[0041] 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.
[0042] In certain embodiments, borapetoside A as 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.
[0043] In some embodiments, borapetoside A, or a pharmaceutically acceptable
salt, metabolite,
solvate or prodrug thereof, is administered parenterally or intravenously. In
other embodiments,
borapetoside A, or a pharmaceutically acceptable salt, metabolite, solvate or
prodrug thereof, is
administered by injection. In some embodiments, borapetoside A, or a
pharmaceutically
acceptable salt, metabolite, solvate or prodrug thereof, is administered
orally.
[0044] 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
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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").
[0045] 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.
[0046] 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
[0047] In some embodiments provide pharmaceutical compositions comprising a
therapeutically
effective amount of borapetoside A; or a pharmaceutically acceptable salt,
metabolite, solvate or
prodrug thereof; and a pharmaceutically acceptable excipient.
[0048] In some embodiments, borapetoside A 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 pharmaceutical compositions
described herein:
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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).
[0049] Provided herein are pharmaceutical compositions comprising a compound
(i.e.,
borapetoside A) 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 described herein).
[0050] A pharmaceutical composition, as used herein, refers to a mixture of a
compound (i.e
borapetoside A 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 (i.e., borapetoside A 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.
[0051] In one embodiment, a compound (i.e., borapetoside A 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, such as Hank's solution,
Ringer's solution, or
physiological saline buffer. In other embodiments, a compound (i.e.,
borapetoside A 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
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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.
[0052] In another embodiment, compounds described herein are formulated for
oral
administration. Compounds described herein, including a compound (i.e.,
borapetoside A
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.
[0053] 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.
[0054] 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
purposes. Additionally, the dyestuffs and/or pigments are optionally utilized
to characterize
different combinations of active compound doses.
[0055] 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
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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.
100561 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
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.
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[0057] In one aspect, borapetoside A 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 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. Exemplary
injectors provide about 0.3 mL, 0.6mL, 1.0mL or other suitable volume of
solution at about a
concentration of 0.5 mg to 50 mg of a compound (i.e borapetoside A described
herein) per 1 mL
of solution. Each injector is capable of delivering only one dose of the
compound.
[0058] 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.
[0059] In yet other embodiments, borapetoside A 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 borapetoside A is
accomplished by means
of iontophoretic patches and the like. In certain embodiments, transdermal
patches provide
controlled delivery of borapetoside A. 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
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
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of the host at a controlled and predetermined rate over a prolonged period of
time, and means to
secure the device to the skin.
[0060] 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.
[0061] 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 (i.e., borapetoside A 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.
[0062] In other embodiments, the compounds (i.e., borapetoside A described
herein) 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 (i.e., borapetoside A 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.
[0063] 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
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reference. Formulations, which include a compound (i.e borapetoside A
described herein), which
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.
[0064] 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.
[0065] In still other embodiments, borapetoside A 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.
[0066] In certain embodiments, pharmaceutical compositions are formulated in
any conventional
manner using one or more physiologically acceptable carriers comprising
excipients and
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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 (i.e.,
borapetoside A 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.
[0067] Pharmaceutical compositions include at least one pharmaceutically
acceptable carrier,
diluent or excipient and at least one compound (i.e., borapetoside A described
herein) 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.
[0068] 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
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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.
[0069] In some embodiments, pharmaceutical composition comprising at least
compound (i.e.,
borapetoside A 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.
[0070] 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.
[0071] Pharmaceutical compositions also, optionally include solubilizing
agents to aid in the
solubility of a compound (i.e., borapetoside A 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.
[0072] 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.
[0073] 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,
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borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions;
suitable salts include
sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and
ammonium
sulfate.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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
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CA 02847286 2014-03-20
ascorbic acid, (0 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.
[0080] 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.
Examples
Example 1. Plant material and preparation of plant extracts
[0081] 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
[0082] 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-I
glucose, penicillin
100 IUmL-1, streptomycin 100 pgmL-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
[0083] C2C12 and Hep3B cells were treated with 1 nM insulin, 100 ,M 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
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CA 02847286 2014-03-20
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
[0084] 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
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.
[0085] After three days of acclimatization, the mice had free access to a
standard rodent chow
(normal mice), a standard rodent chow after single 250mgkg4 streptozotocin
(STZ)
intraperitoneal injection (Ti DM 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 II, the mice were intraperitoneally treated with
borapetoside A (0.1-10
mgkg-1). 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.
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CA 02847286 2014-03-20
[0086] 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).
[0087] 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.
[0088] The mice were injected with borapetoside A (10mgkg-1, interperitoneal)
for 60 minutes or
insulin (0.5 IUkg-1, 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.g of glucose per mg of tissue (wet weight).
Western blot assay and analysis
[0089] 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 Ha1tTM 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, 601.ig 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 p (1R13) (Tyr1345),
phospho-Akt (Ser473),
phosphor-AS160 (Thr642), AS160 (Cell Signaling Technology, Beverly, MA), IRO,
Akt, [3-
Actin(Santa Cruz Biotechnology, Santa Cruz, California), GLUT2 (Abeam,
Cambridge, UK),
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CA 02847286 2014-03-20
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
[0090] 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
[0091] 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).
[0092] Thus it is clearly shown that Borapetoside A induced glycogen
accumulation in C212 and
Hep3B and thus enhances glycogen synthesis.
[0093] 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-I. 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
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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
mgke 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-1 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
mgke 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.
Table 1
The plasma insulin levels in normal and diabetic mice before and after
treatment.
Groups Plasma insulin (pmolil)
Before-treatment After-treatment
Normal mice vehicle borapetoside A 6E8 2.4 67.8 3.0
0.1 mg kg-1 61.0 4,6 68.3 4,4
0.3 mg kg-1 63.4 3.9 129.4 22,0*
1 mg kg-1 66.1 5.4 118.8 10.1*
3 mg kg-1 64.4 3.6 121,2 14.2*
10mgkg-1 68.9 1.3 108.2 2.9*
Metformin 66.6 1.3 72 3.1
TI DM mice vehicle borapetoside A 22.8 0.2 22.8 0.1
0.1 mg kg-1 22.9 0.1 22.7 0.2
0.2 mg kg-1 22.9 0.1 22.7 0,3
1 mg kg-1 23.1 0,2 23.0 0,2
3 mg kg-1 23.1 0,2 23.0 0.0
mg kg-1 23.1 0,1 23.0 0.1
Metformin 22.4 0.4 22,7 0.3
T2DIVI mice vehicle borapetoside A 139.1 2.1 141,7 2.3
0.1 mg kg-1 140.5 4.1 134.5 9,
0.3 mg kg 133.0 4,2 149.0 9,5
1 mg kg-1 126.8 5.7 123.7 9.6
3 mg kg-1 138.3 3.6 184.3 14.6*
10mgkg-1 134.9 3,5 205.3 22.2*
Metformin 148.1 15,2 182.0 27.7
Values were expressed as mean SEM from six animals in each group.
0.05 from vehicle group vs drug-treated.
[0094] 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
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CA 02847286 2014-03-20
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.
[0095] 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
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).
[0096] 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 13-actin and PEPCK to f3-actin were calculated
and plotted in
the FIG. 7B-7F.
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CA 02847286 2014-03-20
,
,
Example 5: Oral Formulation
[0097] To prepare a pharmaceutical composition for oral delivery, 100 mg of an
exemplary
borapetoside A 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.
[0098] In some instances, 100 mg of borapetoside A 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
[0099] 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
[00100] 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%
sodium chloride solution. The mixture is incorporated into an inhalation
delivery unit, such as a
nebulizer, which is suitable for inhalation administration.
1001011 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.
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