U.S. patent application number 12/811859 was filed with the patent office on 2010-11-04 for methods and compositions for oral administration of insulin.
This patent application is currently assigned to OSHADI DRUG ADMINISTRATION LTD.. Invention is credited to Orna Gribova, Alexander Vol.
Application Number | 20100278922 12/811859 |
Document ID | / |
Family ID | 40326402 |
Filed Date | 2010-11-04 |
United States Patent
Application |
20100278922 |
Kind Code |
A1 |
Vol; Alexander ; et
al. |
November 4, 2010 |
METHODS AND COMPOSITIONS FOR ORAL ADMINISTRATION OF INSULIN
Abstract
The present invention provides a pharmaceutical composition
formulated for oral delivery of insulin, comprising a particulate
non-covalently associated mixture of pharmacologically inert silica
nanoparticles having a hydrophobic surface, a polysaccharide, and
insulin suspended in an oil. The present invention further provides
methods of manufacturing same and therapeutic methods utilizing
same for oral delivery of insulin.
Inventors: |
Vol; Alexander; (Rehovot,
IL) ; Gribova; Orna; (Rehovot, IL) |
Correspondence
Address: |
FENNEMORE CRAIG
3003 NORTH CENTRAL AVENUE, SUITE 2600
PHOENIX
AZ
85012
US
|
Assignee: |
OSHADI DRUG ADMINISTRATION
LTD.
Rehovot
IL
|
Family ID: |
40326402 |
Appl. No.: |
12/811859 |
Filed: |
January 8, 2009 |
PCT Filed: |
January 8, 2009 |
PCT NO: |
PCT/IL09/00037 |
371 Date: |
July 7, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61102020 |
Oct 2, 2008 |
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61080295 |
Jul 14, 2008 |
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Current U.S.
Class: |
424/489 ;
514/5.9 |
Current CPC
Class: |
A61K 38/17 20130101;
A61K 47/02 20130101; A61K 38/1841 20130101; A61K 38/27 20130101;
A61P 5/10 20180101; A61P 35/00 20180101; A61K 38/28 20130101; A61K
38/191 20130101; A61K 47/36 20130101; A61K 39/3955 20130101; A61K
9/4891 20130101; A61P 3/10 20180101; A61K 38/39 20130101; A61K
38/21 20130101; A61K 9/10 20130101; A61P 17/18 20180101; A61P 7/06
20180101; A61K 38/1816 20130101; A61K 38/16 20130101; A61K 38/465
20130101; A61K 9/0053 20130101; A61K 38/363 20130101; A61K 38/23
20130101; A61K 47/44 20130101 |
Class at
Publication: |
424/489 ;
514/5.9 |
International
Class: |
A61K 38/28 20060101
A61K038/28; A61K 9/14 20060101 A61K009/14; A61P 3/10 20060101
A61P003/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 8, 2008 |
IL |
188647 |
Claims
1-45. (canceled)
46. A pharmaceutical composition for oral use comprising an oil
having particulate matter suspended therein, wherein the
particulate matter comprises: a. a polysaccharide in intimate
non-covalent association with silica nanoparticles having a
hydrophobic surface, wherein the size of the silica nanoparticles
is between 1-100 nanometers; and b. an insulin protein
non-covalently associated with said silica nanoparticles and the
polysaccharide.
47. The pharmaceutical composition of claim 46, wherein the
polysaccharide comprises a branched polysaccharide.
48. The pharmaceutical composition of claim 46, wherein said
polysaccharide comprises amylopectin, starch, glycogen, chitin,
cellulose, amylose, beta glucan, and combinations and derivatives
thereof.
49. The pharmaceutical composition of claim 46, wherein said
composition is anhydrous.
50. The pharmaceutical composition of claim 46, wherein said size
of said silica nanoparticles is between 5-30 nanometers.
51. The pharmaceutical composition of claim 46, wherein said
hydrophobic surface of said silica nanoparticles comprises
hydrocarbon moieties.
52. The pharmaceutical composition of claim 46, further comprising
an additional biopolymer selected from the group consisting of a
polysaccharide and a high molecular weight structural protein,
wherein said additional biopolymer is a linear biopolymer.
53. The pharmaceutical composition of claim 52, wherein said
additional biopolymer is a high molecular weight structural protein
selected from the group consisting of elastin, collagen, keratin,
fibrinogen and combinations and derivatives thereof.
54. The pharmaceutical composition of claim 46, wherein said oil
comprises a mixture of oils selected from natural vegetable oils
and synthetic analogues thereof.
55. The pharmaceutical composition of claim 46, wherein said
composition further comprises an antioxidant.
56. The pharmaceutical composition of claim 46, further comprising
a wax.
57. The pharmaceutical composition of claim 46 for administering
insulin to a subject.
58. The pharmaceutical composition of claim 46 for treating
diabetes in a subject.
59. A method of manufacturing a pharmaceutical composition
formulated for oral delivery of insulin, said method comprising the
steps of: a. mixing silica nanoparticles having a hydrophobic
surface, wherein the size of said silica nanoparticles is between
1-100 nanometers, with a polysaccharide, whereby said silica
nanoparticles form an intimate non-covalent association with said
polysaccharide; b. mixing an insulin protein with an oil; and c.
mixing said silica nanoparticles and polysaccharide into the oil,
wherein said insulin forms an intimate non-covalent association
with said silica nanoparticles and said polysaccharide and wherein
said silica nanoparticles, said polysaccharide and said insulin
protein are dispersed in said oil.
60. The method of claim 59, wherein the polysaccharide comprises a
branched polysaccharide.
61. The method of claim 59, further comprising the step of adding
an additional oil component following the addition of the oil.
62. The method of claim 59, further comprising the step of adding a
wax following the addition of said oil.
63. The method of claim 59, further comprising the step of adding
an additional biopolymer to the mixture of silica nanoparticles and
polysaccharide, wherein said additional biopolymer is a linear
biopolymer.
64. A method of manufacturing a pharmaceutical composition
formulated for oral delivery of insulin, said method comprising the
steps of: a. mixing silica nanoparticles having a hydrophobic
surface, wherein the size of said silica nanoparticles is between
1-100 nanometers, with (a) a polysaccharide, and (b) an insulin
protein, whereby said silica nanoparticles form an intimate
non-covalent association with said polysaccharide and said insulin
protein; and b. mixing said silica nanoparticles, said
polysaccharide, and said insulin protein into an oil, wherein said
silica nanoparticles, said polysaccharide, and said insulin protein
are suspended in said oil.
65. The method of claim 64, wherein the polysaccharide comprises a
branched polysaccharide.
66. The method of claim 64, further comprising the step of adding
an additional oil component following the addition of the oil.
67. The method of claim 64, further comprising the step of adding a
wax following the addition of said oil.
68. The method of claim 64, further comprising the step of adding
an additional biopolymer selected from the group consisting of a
polysaccharide and a high molecular weight structural protein,
wherein said additional biopolymer is a linear biopolymer.
69. The method of claim 64, wherein said insulin is in a dry
lyophilized form.
Description
FIELD OF INVENTION
[0001] The present invention relates to pharmaceutical compositions
for oral delivery of insulin, comprising an intimate mixture of
solid dry particulate ingredients within an oil. Specifically the
pharmaceutical compositions comprise a particulate non-covalently
associated intimate mixture of pharmacologically inert silica
nanoparticles having a hydrophobic surface, a polysaccharide, and
insulin suspended or embedded in an oil or mixture of oils. The
present invention further provides methods of manufacturing same
and therapeutic methods utilizing same for oral delivery of
insulin.
BACKGROUND OF THE INVENTION
[0002] Oral delivery of active agents is a particularly desirable
route of administration, because of safety and convenience
considerations and because oral delivery replicates the physiologic
mode of insulin delivery. In addition, oral delivery provides for
more accurate dosing than multidose vials and can minimize or
eliminate the discomfort that often attends repeated hypodermic
injections.
[0003] There are many obstacles to successful oral delivery of
biological macromolecules. For example, biological macromolecules
are large and are amphipathic in nature. More importantly, the
active conformation of many biological macromolecules may be
sensitive to a variety of environmental factors, such as
temperature, oxidizing agents, pH, freezing, shaking and shear
stress. In planning oral delivery systems comprising biological
macromolecules as an active agent for drug development, these
complex structural and stability factors must be considered.
[0004] In addition, in general, for medical and therapeutic
applications, where a biological macromolecule is being
administered to a patient and is expected to perform its natural
biological function, delivery vehicles must be able to release
active molecules, at a rate that is consistent with the needs of
the particular patient or the disease process.
[0005] The hormone insulin, contributes to the normal regulation of
blood glucose levels through its release by the pancreas, more
specifically by the B-cells of a major type of pancreatic tissue
(the islets of Langerhans). Insulin secretion is a regulated
process, which, in normal subjects, provides stable concentrations
of glucose in blood during both fasting and feeding. Diabetes is a
disease state in which the pancreas does not release insulin at
levels capable of controlling glucose levels. Diabetes is
classified into two types. The first type is diabetes that is
insulin dependent and usually appears in young people. The islet
cells of the pancreas stop producing insulin mainly due to
autoimmune destruction and the patient must inject himself with the
missing hormone. These Type 1 diabetic patients are the minority of
total diabetic patients (up to 10% of the entire diabetic
population). The second type of diabetes (type 2) is non-insulin
dependent diabetes, which is caused by a combination of insulin
resistance and insufficient insulin secretion. This is the most
common type of diabetes in the Western world. Close to 8% of the
adult population of various countries around the world, including
the United States, have Type 2 diabetes, and about 30% of these
patients will need to use insulin at some point during their life
span due to secondary pancreas exhaustion.
[0006] The problem of providing bioavailable unmodified human
insulin, in a useful form, to the ever-increasing population of
diabetics has occupied physicians and scientists for almost 100
years. Many attempts have been made to solve some of the problems
of stability and biological delivery of this small protein.
Examples include: U.S. Pat. No. 7,455,830 which discloses bioactive
nanoparticles suitable for oral delivery of insulin which include a
shell substrate of chitosan, and a core substrate selected from the
group consisting of gamma-polyglutamic acid (PGA), alpha-PGA, water
soluble salts of PGA and metal salts of PGA; U.S. Pat. No.
7,470,663 which discloses a liquid solution formulated for oral
delivery, comprising a substantially monodispersed mixture of
conjugates, wherein each conjugate comprises human insulin
covalently coupled a carboxylic acid, which is covalently coupled
at the end distal to the carboxylic acid moiety to a methyl
terminated polyethylene glycol moiety. U.S. Pat. No. 7,384,914
which discloses a method of treating a mammal which has impaired
glucose tolerance by administering a therapeutically effective dose
of a pharmaceutical formulation comprising insulin and the delivery
agent 4-[(2-hydroxy-4-chlorobenzoyl)amino]butanoate (4-CNAB) in an
amount which facilitates absorption of the insulin from the
gastrointestinal tract of the treated mammal, and U.S. Pat. No.
6,656,922 which discloses a method for enhancing oral
administration of insulin by conjugating insulin to an hydrophobic
agent selected from the group consisting of bile acids, sterols,
alkanoic acids, and mixtures thereof to result in a hydrophobized
macromolecular agent.
[0007] As of today, most diabetic patients self-administer insulin
by daily subcutaneous injections. However, the limitations of
multiple daily injections, such as inconvenience, poor patient
acceptability, compliance and the difficulty of matching
postprandial insulin availability to postprandial requirements are
some of the better known shortcomings of insulin therapy.
[0008] A method of providing insulin without the need for
injections has been a goal in drug delivery. Insulin absorption in
the gastrointestinal tract is prevented by its large size and
enzymatic degradation. It would be desirable to create an oral
pharmaceutical formulation of a drug such as insulin (which is not
normally orally administrable due to, e.g., insufficient absorption
from the gastrointestinal tract), which formulation would provide
sufficient absorption and pharmacokinetic/pharmacodynamic
properties to provide the desired therapeutic effect.
[0009] Accordingly, there is a need for a method of administering
insulin to patients in need of insulin wherein those patients are
not subject to systemic hyperinsulinemia, which by itself can
increase the risk of vascular disease (that is normally associated
with such chronic insulin treatments, as discussed above). In other
words, it is desirable to provide compositions and methods for
treating diabetes without the drawbacks of systemic hyperglycemia
to decrease the incidence of vascular complications and other
detrimental effects.
Biopolymers and their Use in Delivering Active Proteins Such as
Insulin:
[0010] Biopolymers such as polysaccharides have been known for many
years. Polysaccharides are widely used as excipients in oral dosage
forms, as disclosed for example in U.S. Pat. No. 7,351,741 to
Weidner, U.S. Pat. No. 6,667,060 to Vandecruys and US patent
application 2004/0115264 to Blouquin. These references neither
disclose nor suggest use of biopolymers in combination with
nanoparticles and/or oil.
Nanoparticles and their Use in Delivering Active Proteins Such as
Insulin:
[0011] Silica nanoparticles are well known in the art as
pharmaceutical excipients and are their use is disclosed for
example in U.S. Pat. Nos. 6,322,765 to Muhlhofer and 6,698,247 to
Tennent, among many others. Coating of a nanoparticle-biopolymer
complex with oil, or utility of same in oral administration of
insulin are neither disclosed nor suggested.
[0012] Methods for imparting a hydrophobic surface to nanoparticles
are well known in the art and are described, for example, in Chung
et al. (Hydrophobic modification of silica nanoparticle by using
aerosol spray reactor. Colloids and Surfaces A: Physicochem. Eng.
Aspects 236 (2004) 73-79). Additional methods include the reverse
micelles method (Fu X, Qutubuddin S, Colloids Surf. A: Physicochem.
Eng. Aspects 179: 65, 2001), liquid precipitation method
(Krysztafkiewicz A, Jesionowski T, Binkowski S, Colloids Surf. A:
Physicochem. Eng. Aspects 173:73, 2000) and sol-gel method (Jean J,
Yang S, J. Am. Ceram. Soc. 83 (8):1928, 2000; Zhang J, Gao L,
Ceram. Int. 27: 143, 2001). Use of nanoparticles in combination
with a biopolymer and insulin, coating a nanoparticle-containing
complex with oil, or utility of same in oral administration of
insulin are neither disclosed nor suggested.
[0013] U.S. Pat. Nos. 7,105,229, 6,989,195, 6,482,517, 6,638,621,
6,458,387, 7,045,146, and 5,462,866 among many others disclose use
of nanoparticles or microparticles as excipients for proteins.
These references neither disclose nor suggest intimate non-covalent
association of nanoparticles with a biopolymer and insulin, or
embedding of a nanoparticle-biopolymer-insulin in an oil
coating.
[0014] US 2007/0154559 to Pai discloses an orally administrable
composition containing nanoparticles comprising a charged
water-soluble drug in complex with a counter-ion substance, a
lipid, a polymer, and an emulsifier. The compositions are formed by
(a) ionically bonding the drug with the counter-ion; (b) adding a
lipid, a polymer, and a solubilizing agent; dissolving the whole
mixture; and introducing the solution into an aqueous solution
containing an emulsifier; and (c) removing the solubilizing agent.
US 2006/0177495 and 2003/0235619 to Allen disclose delivery
vehicles for delivering an active agent, comprising nanoparticles
composed of a biodegradable hydrophobic polymer forming a core and
an outer amphiphilic layer surrounding the polymer core and
containing a stabilizing lipid.
[0015] US 2006/0083781 to Shastri discloses nanoparticles
comprising a lipid and a polymer comprising an ionic or ionizable
moiety. These compositions as well differ significantly from those
of the present invention, inter alia in that (a) the polymer is not
outside the nanoparticles but rather forms a part of them; and (b)
the oil forms a part of the nanoparticles instead of coating the
nanoparticle-polymer mixture. In addition, the unique structures of
the matrix carrier compositions of the present invention is neither
disclosed nor suggested.
[0016] WO 96/37232 to Alonso Fernandez discloses methods for
preparation of colloidal systems through the formation of ionic
lipid-polysaccharide complexes. The colloidal systems are
stabilized through the formation of an ionic complex, at the
interface, comprised of a positively charged aminopolysaccharide
and a negatively charged phospholipid. These compositions as well
differ significantly from those of the present invention, inter
alia in that (a) the polymer is not outside the nanoparticles but
rather forms a part of them; and (b) the oil forms a part of the
nanoparticles instead of coating them. In addition, use of insulin
as an active agent is neither disclosed or suggested.
[0017] U.S. Pat. No. 6,548,264 to Tan et al. discloses
silica-coated nanoparticles and a process for producing
silica-coated nanoparticles. Silica-coated nanoparticles are
prepared by precipitating nano-sized cores from reagents dissolved
in the aqueous compartment of a water-in-oil microemulsion. A
reactive silicate is added to coat the cores with silica. The
silicate coating may further be derivatized with a protein. US
2007/0275969 to Gurny discloses pharmaceutical compositions for the
oral administration of pharmaceutical agents having low water
solubility. The pharmaceutical agents are solubilized with a
polymer, from which nanoparticles are formed.
[0018] In cosmetics formulations, it is common to use compositions
comprising water-in-oil emulsions containing an aqueous phase
dispersed in an oily phase. There are numerous examples in which
silica nanoparticles as well as polysaccharides are included in the
liquid fatty phase. U.S. Pat. No. 6,228,377 for example, discloses
water-in-oil emulsions containing a liquid fatty phase which
contains hydrophobic or hydrophilic fumed silica, a branched
polysaccharide alkyl ether, an emulsifying surfactant and oil.
These compositions differ significantly from those of the present
invention in that they include a water phase and surfactants that
serve as the most important structure forming factor of the
composition.
Additional Strategies
[0019] Methods for oral administration of insulin are the object of
extensive research efforts but have been proven generally
inefficient to date. A number of strategies for preventing
degradation of orally administered proteins have been suggested,
including use of core-shell particles (U.S. Pat. No. 7,090,868 to
Gower) and nano-tubes (U.S. Pat. No. 7,195,780 to Dennis).
Liposomes have been used as a carrier for orally administered
proteins, as well as aqueous emulsions and suspensions (U.S. Pat.
No. 7,316,818; WO 06/062544; U.S. Pat. No. 6,071,535; and U.S. Pat.
No. 5,874,105 to Watkins) and gas-filled liposomes (U.S. Pat. No.
6,551,576; U.S. Pat. No. 6,808,720; and U.S. Pat. No. 7,083,572 to
Unger et al.). Another composition comprises nanodroplets dispersed
in an aqueous medium (US 2007/0184076). Additional strategies are
found in WO 06/097793, WO 05/094785, and WO 03/066859 to Ben-Sason,
which describe matrix-carriers containing peptide-effectors that
provide penetration across biological barriers for administration
of hydrophobic proteins; and EP 0491114B1 to Guerrero Gomez-Pamo,
which describes preparation of non-covalent protein-polysaccharide
complexes for oral administration of biologically active
substances, stabilized by precipitates of organic salts. None of
these references discloses or suggests intimate non-covalent
association of nanoparticles with a biopolymer or a
nanoparticles-polymer matrix embedded in an oil coating.
[0020] In addition to the differences outlined above, none of the
above references discloses or suggests the enhanced bioavailability
of compositions of the present invention.
SUMMARY OF THE INVENTION
[0021] The present invention relates to pharmaceutical compositions
for oral delivery of insulin, comprising an intimate mixture of
solid dry particulate ingredients within an oil. Preferably the
compositions are anhydrous. Specifically the pharmaceutical
compositions comprise a particulate non-covalently associated
mixture of pharmacologically inert silica nanoparticles having a
hydrophobic surface, a polysaccharide, and insulin suspended in,
embedded in or dispersed in an oil or mixture of oils. The present
invention further provides methods of manufacturing same and
therapeutic methods utilizing same for oral delivery of
insulin.
[0022] According to the present invention it is now disclosed for
the first time that the compositions of the invention surprisingly
enable oral bioavailability of insulin. The present invention is
based in part on the surprising discovery that experimental
diabetic mice treated orally with a composition of the present
invention, maintained normal blood glucose levels (.about.100
mg/dL) for up to 12 hours after administration of the drug whereas
diabetic mice given the same amount of insulin by intravenous
injection could not maintain a normal blood glucose level for over
6 hours.
[0023] In one aspect, the present invention provides a
pharmaceutical composition for oral delivery of insulin comprising
an oil having particulate matter suspended therein, wherein the
particulate matter includes: (a) pharmacologically inert silica
nanoparticles having a hydrophobic surface, wherein the size of the
nanoparticles is between 1-100 nanometers, in intimate non-covalent
association with a polysaccharide; and (b) an insulin protein
attached to the silica nanoparticles and the polysaccharide via
non-covalent forces. In another embodiment, the insulin protein is
attached to the hydrophobic surfaces of the silica nanoparticles
and the polysaccharide via non-covalent forces (FIG. 1). In another
embodiment, the hydrophobic portion of the insulin protein is
attached to the hydrophobic surfaces of the silica nanoparticles
and the polysaccharide via non-covalent forces. In another
embodiment, the hydrophilic portion of the insulin protein is also
non-covalently attached to hydrophilic portion of the
polysaccharide. In another embodiment, the non-covalent forces
cause the nanoparticles, polysaccharide, and insulin to form an
intimate mixture. Each possibility represents a separate embodiment
of the present invention.
[0024] In one preferred embodiment of the present invention, the
polysaccharide comprises a branched polysaccharide. In another
embodiment, the branched polysaccharide is selected from the group
consisting of amylopectin, starch and glycogen. In another
embodiment, the branched polysaccharide is starch.
[0025] In another embodiment, the particulate matter including the
hydrophobic silica nanoparticles, the polysaccharide and the
insulin is dispersed in, embedded in or suspended within the oil
phase of the matrix composition. In another embodiment, the oil
phase is impregnated with the particulate matter. As provided
herein, the present invention provides compositions wherein the
particulate matter form a matrix that is impregnated and completely
surrounded by the oil phase. Preferably the weight of
polysaccharides will be greater than the weight of the silica. In
some embodiments the weight of the polysaccharides will be at least
twice that of the silica, in other embodiments the weight of the
polysaccharides will be 5 fold that of the silica in yet other
embodiments the polysaccharides will be at least 10 times greater
than the weight of silica nanoparticles. Each possibility
represents a separate embodiment of the present invention.
[0026] Reference to silica nanoparticles of the present invention
as having a "hydrophobic" surface encompasses silica nanoparticles
having a surface modified to be hydrophobic. In another embodiment,
the silica nanoparticles are modified by chemically coating the
surface with a hydrocarbon. In another embodiment, the coating
causes the silica nanoparticles to display hydrocarbon moieties on
their surface. Methods for imparting a hydrophobic surface to
silica nanoparticles are well known in the art, and are described
inter alia herein. Each possibility represents a separate
embodiment of the present invention.
[0027] In another embodiment, a pharmaceutical composition of the
present invention comprises a mixture of oils selected from natural
vegetable oils and synthetic analogues thereof.
[0028] In another embodiment, a pharmaceutical composition of the
present invention further comprises an additional oil component.
The term "additional oil component" encompasses an additional oil
or mixture of oils, as described elsewhere herein. In another
embodiment, the additional oil component comprises an antioxidant.
Each possibility represents a separate embodiment of the present
invention.
[0029] In another embodiment, a pharmaceutical composition of the
present invention further comprises a third oil or mixture of oils
in addition to the above-described additional oil or mixture of
oils. In another embodiment, the third oil component comprises an
antioxidant. Each possibility represents a separate embodiment of
the present invention.
[0030] In another embodiment, a matrix composition of the present
invention further comprises a wax.
[0031] In another embodiment, a matrix composition formulated for
oral administration of the present invention is in the form of a
tablet, capsule, or suspension.
[0032] In another embodiment, a pharmaceutical composition of the
present invention further comprises an additional biopolymer that
is a linear biopolymer. In another embodiment, the additional
biopolymer is a polysaccharide. In another embodiment, the
additional biopolymer is a linear polysaccharide. In another
embodiment, the additional biopolymer is a linear high molecular
weight structural protein. In another embodiment, the additional
biopolymer is selected from the group consisting of chitin,
cellulose, a linear alpha glucan, and a linear beta glucan. In
another embodiment, the additional biopolymer is selected from the
group consisting of chitin, amylose, cellulose, and beta glucan. In
another embodiment, a pharmaceutical composition of the present
invention comprises a branched polysaccharide and a linear
polysaccharide. Each possibility represents a separate embodiment
of the present invention.
[0033] In another embodiment, the additional biopolymer of methods
and compositions of the present invention is a fiber. In another
embodiment, the fiber is a dietary fiber. In another embodiment,
the dietary fiber is an insoluble fiber. In another embodiment, the
dietary fiber is a linear insoluble fiber. In another embodiment,
the dietary fiber is a soluble fiber. In another embodiment, the
dietary fiber is a linear soluble fiber. Each possibility
represents a separate embodiment of the present invention.
[0034] In another embodiment, a pharmaceutical composition of the
present invention comprises a branched biopolymer, a linear
polysaccharide, and an insoluble fiber. In another embodiment, a
composition of the present invention comprises a branched
polysaccharide, a linear polysaccharide, and an insoluble fiber. An
example of such is a composition comprising amylopectin, a branched
polysaccharide; chitin, a linear polysaccharide; and cellulose, an
insoluble fiber. Other branched and linear polysaccharides and
insoluble fibers disclosed herein are suitable as well. Each
possibility represents a separate embodiment of the present
invention.
[0035] In another embodiment, the present invention provides a
method of administering an insulin protein to a subject in need
thereof, comprising orally administering to the subject a
pharmaceutical composition of the present invention, thereby
administering an insulin protein to a subject.
[0036] In another embodiment, the present invention provides a
method of treating diabetes in a subject in need thereof,
comprising orally administering to the subject a pharmaceutical
composition of the present invention, thereby treating diabetes in
a subject. In another embodiment, the diabetes is an
insulin-dependent diabetes. In another embodiment, the diabetes is
a non-insulin-dependent diabetes. In another embodiment, the
diabetes is Type I diabetes. In another embodiment, the diabetes is
Type II diabetes. In another embodiment, the diabetes is juvenile
diabetes. In another embodiment, the diabetes is adolescent
diabetes. In another embodiment, the diabetes is adult diabetes. In
another embodiment, the diabetes is any other type of diabetes
known in the art. In another embodiment, a method of the present
invention is used to treat a complication of diabetes. Each
possibility represents a separate embodiment of the present
invention.
[0037] In another embodiment, the subject of a method of the
present invention is a human. In another embodiment, the subject is
a non-human mammal. Each possibility represents a separate
embodiment of the present invention.
[0038] As provided herein, oral administration of compositions of
the present invention lowers blood glucose levels for several hours
in animal (Example 6) and human (Example 7) subjects, without
causing glycemic instability or troublesome hypoglycemia symptoms.
Further, the compositions exhibit no detectable toxicity (Example
8).
[0039] In another embodiment, the present invention provides use of
a pharmaceutical composition of the present invention in the
preparation of a medicament for administering insulin to a
subject.
[0040] In another embodiment, the present invention provides use of
a pharmaceutical composition of the present invention in the
preparation of a medicament for treating diabetes in a subject.
[0041] In another embodiment, the present invention provides a
pharmaceutical composition of the present invention for
administering insulin to a subject.
[0042] In another embodiment, the present invention provides a
pharmaceutical composition of the present invention for treating
diabetes in a subject.
[0043] In certain embodiments, the insulin in a pharmaceutical
composition of the present invention is capable of reaching the
bloodstream of a subject, following oral administration, with over
20% of the biological activity intact, preferably over 30% of the
biological activity remains intact, more preferably at least 40% of
the biological activity remains intact, most preferably at least
50% of the biological activity remains intact. In another
embodiment, over 60% of the biological activity remains intact. In
another embodiment, over 70% of the biological activity remains
intact. In another embodiment, over 80% of the biological activity
remains intact. In another embodiment, over 90% of the biological
activity remains intact. Without wishing to be bound by any theory
or mechanism of action, these properties are believed to be due to
protection of the active agent from digestive enzymes and
mechanical forces in the intestines by the excipients of
pharmaceutical compositions of the present invention.
[0044] In another embodiment, a pharmaceutical composition of the
present invention is designed to provide short-term release.
"Short-term release", as used herein, refers to release over 8-12
hours, with maximal activity 4 hours after administration. In
another embodiment, a pharmaceutical composition of the present
invention is designed to provide medium-term release. "Medium-term
release", as used herein, refers to release over 12-18 hours, with
maximal activity 4-6 hours after administration. In another
embodiment, a pharmaceutical composition of the present invention
is designed to provide long-term release. "Long-term release", as
used herein, refers to release over 18-48 hours, with maximal
activity 4-8 hours after administration. In another embodiment, a
pharmaceutical composition of the present invention is designed to
provide very long-term release. "Very long-term release", as used
herein, refers to release over 18-72 hours, with maximal activity
6-8 hours after administration. In another embodiment, the longer
term-release compositions of the present invention exhibit a lower
peak with a longer tail following the peak activity. Each
possibility represents a separate embodiment of the present
invention.
[0045] In another aspect, the present invention provides a method
of manufacturing a pharmaceutical composition for oral delivery of
insulin, the method comprising the steps of: (a) blending
pharmacologically inert silica nanoparticles having a hydrophobic
surface, wherein the size of the nanoparticles is between 1-100
nanometers, with a polysaccharide, whereby the silica nanoparticles
form an intimate non-covalent association with the polysaccharide;
(b) mixing an insulin protein with an oil; and (c) mixing the
nanoparticles and polysaccharide into the oil. In another
embodiment, the silica nanoparticles, polysaccharide, and insulin
thereby form a matrix that becomes dispersed, embedded or suspended
in the oil. Preferably, the silica nanoparticles, polysaccharide,
and insulin form a complex. In another embodiment, the complex is
dispersed, embedded or suspended in the oil. In another embodiment,
the insulin protein is non-covalently attached to the hydrophobic
surfaces of the silica nanoparticles and to the hydrophilic and
hydrophobic portions, regions or patches of the surface of the
polysaccharide. In another embodiment, the particle size of the
pharmaceutical composition is between 100-500,000 nanometers (nm).
In some preferred embodiments, the particle size is between
100-50,000 nm. Each possibility represents a separate embodiment of
the present invention.
[0046] In yet another aspect, the present invention provides a
method of manufacturing a pharmaceutical composition for oral
delivery of insulin, the method comprising the steps of: (a)
blending pharmacologically inert silica nanoparticles having a
hydrophobic surface, wherein the size of the nanoparticles is
between 1-100 nanometers, with (i) a polysaccharide, and (ii) an
insulin protein whereby the silica nanoparticles form an intimate
non-covalent association with the polysaccharide; and (b) mixing
the particulate matter (silica nanoparticles, polysaccharide, and
insulin protein) into an oil. In another embodiment, the silica
nanoparticles, polysaccharide, and insulin form a matrix that
becomes dispersed, embedded or suspended in the oil. Preferably,
the silica nanoparticles, polysaccharide, and insulin form a
complex. In another embodiment, the complex is dispersed, embedded
or suspended in the oil. In another embodiment, the insulin protein
is non-covalently attached to the hydrophobic surfaces of the
silica nanoparticles and to the hydrophilic and hydrophobic
portions, regions or patches of the surface of the polysaccharide.
In another embodiment, the particle size of the pharmaceutical
composition is between 100-500,000 nanometers. In some preferred
embodiments, the particle size is between 100-50,000 nanometers.
Each possibility represents a separate embodiment of the present
invention. As provided herein, methods have been developed to
formulate insulin in orally administrable form. In certain
preferred embodiments, the components are mixed in a particular
order in order to produce oil-coated matrix carrier compositions
that protect the insulin from digestive processes in the stomach
and small intestine. Further, without wishing to be bound by any
theory or mechanism of action, the polysaccharide, particularly
when branched, absorbs hydraulic and mechanical stresses
experienced during digestion. The oil coating constitutes a
physical barrier that provides additional protection against
digestive enzymes. The pharmaceutical compositions of the present
invention are converted in the digestive system to particles
smaller in size but similar in structure to the original
composition, which are absorbed similarly to chylomicrons and reach
the bloodstream without undergoing first-pass metabolism in the
liver. In another embodiment, the particles are broken down in
gastro-intestinal tract to particles having a characteristic size
between 30-1000 nanometers. In certain preferred embodiments, the
size of the particles after digestion is between 30-700 nm. While
the primary particles are in the nanometer to sub-micrometer range,
these may form conglomerates or agglomerates of larger dimensions
within the compositions of the present invention. The size of these
conglomerates or agglomerates ranges between 100-500,000
nanometers. In some preferred embodiments, the conglomerate or
agglomerate size is between 100-50,000 nanometers. In another
embodiment, the conglomerate or agglomerate size is between
100-5,000 nanometers. Each possibility represents a separate
embodiment of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1: Schematic view of a representative matrix-carrier
structure containing insulin, silica nanoparticles and a
polysaccharide. Top: Macrostructure containing branched fiber
structure of the polysaccharide impregnated with hydrophobic silica
nanoparticles. Bottom: microstructure depiction.
[0048] FIG. 2: Schematic view of the structure formed in the small
intestine due to joint action of hydrodynamic and enzymatic
processes.
[0049] FIG. 3: Light microscopy picture of insulin matrix carrier
Formulation IV (Example 5).
[0050] FIG. 4: A. Effect of oral administration of NovoRapid.TM.
insulin oral composition of the present invention (Formulation VI,
Example 4) on blood glucose levels (BGL) in diabetic (STZ-treated)
mice. Different symbols represent individual mice. The break
indicates the time of the insulin composition administration. B.
Effect of oral administration of Actrapid.TM. insulin compositions
(Formulation V, Example 4) on BGL in diabetic (STZ-treated) mice.
Different symbols represent individual mice.
[0051] FIG. 5: BGL levels in STZ-treated mice orally receiving 25
IU Insulin (by BIOCON) in PBS (gavage). Different symbols represent
individual mice.
[0052] FIG. 6: A. Dose response curve towards the insulin matrix
carrier compositions (Formulation IV, Example 5) of the present
invention on STZ mice (mean blood glucose concentrations are based
on the BGL of at least 5 mice). B-D. Data from individual STZ mice
administered (Formulation IV): 2 (B), 5 (C), and 10 (D) IU of
insulin. E. Effect of SC-injected insulin on STZ mice. F. Effect of
12 IU of insulin composition on normal mice. The breaks in figures
B-I indicate the time of administration. Different symbols
represent individual mice. G, H, I: Comparison of the effect of 10
(G), 5 (H), and 2 (I) IU of insulin on the BGL upon administration
of insulin by SC injection and orally using the matrix carrier
composition of the present invention. J-K. Comparison of the
pharmacodynamics of two different matrix carrier compositions for
oral delivery of insulin (formulation A versus formulation IV) --2
IU of insulin (J) and 7.5 IU of insulin (K).
[0053] FIG. 7: Efficacy of oral insulin compositions of the present
invention on healthy (A) and diabetic (B) human subjects. A. 30 IU
of the Actrapid.TM. relatively short-term release insulin matrix
carrier composition (Formulation II) was administered at time
12:00, as indicated by the stripe in the graph. B. Daily average
blood glucose levels. Gluco-Rite.TM. was administered on days 2-12.
Insulin matrix carrier composition (formulation V, example 4) was
first administered on the 13.sup.th day and continued for 14
days.
[0054] FIG. 8: Toxicity study of insulin oral compositions
(Formulation IV) of the present invention. Microscopic analysis of
the liver (A); kidney (B); and duodenum (C). In each case, left
panels are control samples and right panels are treated
samples.
[0055] FIG. 9: A-D Cryo SEM freeze fracture pictures of mice serum
taken 4 hours after oral administration (gavage) of 10 IU oral
insulin composition (Formulation IV).
DETAILED DESCRIPTION OF THE INVENTION
[0056] The present invention provides matrix carrier compositions
for oral delivery of insulin, comprising an intimate mixture of
solid dry particulate ingredients within an oil. Specifically the
pharmaceutical compositions comprise a particulate non-covalently
associated mixture of pharmacologically inert silica nanoparticles
having a hydrophobic surface, a branched polysaccharide and insulin
suspended, embedded or dispersed in an oil or mixture of oils. The
present invention further provides methods of manufacturing same
and therapeutic methods utilizing same for oral delivery of
insulin.
[0057] The oral insulin compositions of the present invention
provide an advantageous result over the subcutaneously administered
insulin, which is currently the state of the art, beyond the
benefit of ease of administration, pain-free administration, and
the potential for improved patient compliance. By administration of
the oral insulin compositions of the present invention, the blood
levels of insulin which occur upon the first (initial) phase of
insulin secretion by the pancreas can be simulated. The first phase
of insulin secretion, while of short duration, has an important
role in priming the liver to the metabolic events ahead (meal).
Because subcutaneously administered insulin does not undergo portal
circulation, this result is not possible with subcutaneously
administered insulin.
[0058] In another embodiment, the present invention provides a
pharmaceutical composition comprising: (a) pharmacologically inert
silica nanoparticles having a hydrophobic surface, wherein the
diameter of the nanoparticles is between 1-100 nanometers, in
intimate mixture with a polysaccharide; and (b) an insulin protein
non-covalently attached to the silica nanoparticles and the
polysaccharide; wherein the matrix formed by the silica
nanoparticles, polysaccharide, and insulin is suspended, embedded
or dispersed in oil. In another embodiment, the insulin is
non-covalently attached to the hydrophobic surfaces of the silica
nanoparticles and to the hydrophilic and hydrophobic portions,
regions or patches of the surface of the polysaccharide. In another
embodiment, the hydrophobic portion of the insulin protein is
attached to the hydrophobic surfaces of the silica nanoparticles
and the polysaccharide via non-covalent forces. In another
embodiment, the hydrophilic portion of the insulin protein is also
non-covalently attached to hydrophilic portion of the
polysaccharide. In another embodiment, the particle diameter of the
pharmaceutical composition following its formation, but prior to
ingestion is between 100-500,000 nm. In certain preferred
embodiments, the particle diameter is between 100-50,000
nanometers. In another embodiment, the particle diameter is between
100-5000 nm. Each possibility represents a separate embodiment of
the present invention.
[0059] Various components of pharmaceutical compositions of the
present invention, namely insulin, silica nanoparticles,
polysaccharides, high molecular weight structural proteins, and
oils, are described herein. Each embodiment thereof can be utilized
in methods of the present invention, and each such use represents a
separate embodiment of the present invention.
[0060] In another embodiment, the oil phase of the matrix carrier
composition comprises a plurality of oils.
[0061] In another embodiment, a pharmaceutical composition of the
present invention is held together by non-covalent forces (FIG. 1).
In another embodiment, without wishing to be bound by any theory or
mechanism of action, the non-covalent forces between the components
of the matrix composition enable the matrix composition to
self-assemble when the components are mixed together, as described
herein. In another embodiment, the non-covalent forces cause the
silica nanoparticles, polysaccharide and insulin to form an
intimate mixture. In another embodiment, the matrix composition
exhibits an ordered structure. In another embodiment, without
wishing to be bound by any theory or mechanism of action, the
matrix composition includes a solid phase containing at least two
solid pharmacologically inert materials (silica nanoparticles and
polysaccharides) with different properties. In another embodiment,
the silica nanoparticle/polysaccharide/insulin complex is
dispersed, embedded or suspended within the oil phase of the matrix
composition. In another embodiment, the oil phase is impregnated
with the silica nanoparticle/polysaccharide/insulin complex of the
matrix composition. As provided herein, the present invention
provides compositions wherein the silica nanoparticles,
polysaccharide, and insulin form a matrix that is impregnated and
completely surrounded by the oil phase. Each possibility represents
a separate embodiment of the present invention.
[0062] In another embodiment, a pharmaceutical composition of the
present invention further comprises an additional biopolymer that
is a linear biopolymer. In another embodiment, the additional
biopolymer is a linear polysaccharide. In another embodiment, the
additional biopolymer is a linear high molecular weight structural
protein. In another embodiment, the additional biopolymer is
selected from the group consisting of chitin, cellulose, a linear
alpha glucan, and a linear beta glucan. In another embodiment, the
additional biopolymer is selected from the group consisting of
chitin, amylose, cellulose, and beta glucan. Insulin compositions
are provided herein that comprise amylopectin, a branched
biopolymer, and chitin, a linear biopolymer (Example 5). Other
branched and linear biopolymers disclosed herein are suitable as
well. Each possibility represents a separate embodiment of the
present invention.
[0063] In another embodiment, the additional biopolymer of methods
and compositions of the present invention is a dietary fiber. In
another embodiment, the dietary fiber is an insoluble fiber. In
another embodiment, the dietary fiber is a linear insoluble fiber.
In another embodiment, the dietary fiber is a soluble fiber. In
another embodiment, the dietary fiber is a linear soluble
fiber.
[0064] In another embodiment, a composition of the present
invention comprises a branched biopolymer, a linear polysaccharide,
and an insoluble fiber. In another embodiment, a composition of the
present invention comprises a branched biopolymer, a polypeptide,
and an insoluble fiber. An example of such is a composition
comprising amylopectin, a branched polysaccharide; keratin, a
polypeptide; and cellulose, an insoluble fiber. Other branched
polysaccharides, polypeptides, and insoluble fibers disclosed
herein are suitable as well. In another embodiment, a composition
of the present invention comprises a branched polysaccharide, a
linear polysaccharide, and an insoluble fiber. An example of such
is a composition comprising amylopectin, a branched polysaccharide;
chitin, a linear polysaccharide; and cellulose, an insoluble fiber.
Other branched and linear polysaccharides and insoluble fibers
disclosed herein are suitable as well. Each possibility represents
a separate embodiment of the present invention.
[0065] Oil having particulate matter suspended therein, as used
herein, refers to particulate matter that is in contact with oil.
The composition as a whole need not be homogeneous with regard to
the distribution of the particulate matter. Rather, the particulate
matter is capable of being dispersed or suspended in the oil when
agitated. The particulate matter need not be completely
homogeneous, but rather is characterized by its containing the
ingredients specified herein and its intimate contact with the oil
of the present invention. Compositions wherein the particulate
matter is agglomerated fall within the scope of the present
invention.
Nanoparticles
[0066] The silica nanoparticles of methods and compositions of the
present invention are preferably pharmacologically inert. In
another embodiment, the silica nanoparticles are composed of
materials that are generally recognized as safe (GRAS). In another
embodiment, the silica nanoparticles are non-toxic. In another
embodiment, the silica nanoparticles are non-teratogenic. In
another embodiment, the silica nanoparticles are biologically
inert. Each possibility represents a separate embodiment of the
present invention.
[0067] In another embodiment, the nanoparticles are
silica-containing nanoparticles. "Silica-containing nanoparticles"
refers preferably to nanoparticles comprising silica, a silicate,
or a combination thereof. "Silica" refers to silicon dioxide.
Silica-containing nanoparticles are available commercially, e.g. as
99.99% pure finely ground silica. It will be understood by those
skilled in the art that lower grades of purity of silica are also
compatible with the present invention. "Silicate" refers to a
compound containing silicon and oxygen, e.g. in tetrahedral units
of SiO.sub.4. In another embodiment, the term refers to a compound
containing an anion in which one or more central silicon atoms are
surrounded by electronegative ligands. Non-limiting examples of
silicates are hexafluorosilicate, sodium silicate
(Na.sub.2SiO.sub.3), aluminum silicates, magnesium silicates, etc.
It is to be understood that the nanoparticles in structures of the
present invention can be either of a single type or of multiple
types, provided that, if multiple types are present, at least one
type is a silica-containing nanoparticles. In another embodiment,
essentially all the nanoparticles are silica-containing
nanoparticles. Silica is widely recognized as a safe food additive
(Thirteenth report of the Joint FAO/WHO Expert Committee on Food
Additives, FAO Nutrition Meetings Report Series; from the Joint
FAO/WHO Expert Committee on Food Additives meeting in Rome, May
27-Jun. 4, 1969). Each possibility represents a separate embodiment
of the present invention.
[0068] Reference to silica nanoparticles of the present invention
as having a "hydrophobic" surface indicates, in one embodiment,
that at least 40% of the silica nanoparticle surface is
hydrophobic. In another embodiment, at least 50% of the surface is
hydrophobic. In another embodiment, at least 60% of the surface is
hydrophobic. In another embodiment, at least 70% of the surface is
hydrophobic. In another embodiment, at least 80% of the surface is
hydrophobic. In another embodiment, at least 90% of the surface is
hydrophobic. In another embodiment, at least 95% of the surface is
hydrophobic. In another embodiment, 40-100% of the surface is
hydrophobic. In another embodiment, 50-100% of the surface is
hydrophobic. In another embodiment, 60-100% of the surface is
hydrophobic. In another embodiment, 70-100% of the surface is
hydrophobic. In another embodiment, 80-100% of the surface is
hydrophobic. In another embodiment, 90-100% of the surface is
hydrophobic. In another embodiment, 95-100% of the surface is
hydrophobic. In another embodiment, 40-60% of the surface is
hydrophobic. In another embodiment, 40-50% of the surface is
hydrophobic. In another embodiment, 40-70% of the surface is
hydrophobic. In another embodiment, 40-80% of the surface is
hydrophobic. Each possibility represents a separate embodiment of
the present invention.
[0069] In another embodiment, reference to silica nanoparticles as
having a "hydrophobic" surface encompasses silica nanoparticles
having a surface chemically modified to be hydrophobic. In another
embodiment, the nanoparticles are chemically modified by coating
the surface with a hydrocarbon. In another embodiment, the coating
causes the nanoparticles to display hydrocarbon moieties on their
surface. In another embodiment, the hydrocarbon moieties are
selected from the group consisting of methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, T-butyl, pentyl, and iso-pentyl. In
another embodiment, the coating causes the nanoparticles to display
methyl moieties on their surface. Methods for imparting a
hydrophobic surface to nanoparticles are well known in the art, and
are described inter alia herein. As is known in the art it is
possible to chemically modify the surface of the fumed silica by
chemical reaction, generating a decrease in the number of silanol
groups. In particular, silanol groups can be substituted with
hydrophobic groups to obtain a hydrophobic silica. The hydrophobic
groups can be: trimethylsiloxy groups, which are obtained in
particular by treatment of fumed silica in the presence of
hexamethyldisilazane. Silicas thus treated are known as "silica
silylate" according to the CTFA (6th edition, 1995). They are sold,
for example, under the references "Aerosil R812.RTM." by the
company Degussa and "CAB-OSIL TS-530.RTM." by the company Cabot;
dimethylsilyloxy or polydimethylsiloxane groups, which are obtained
in particular by treatment of fumed silica in the presence of
polydimethylsiloxane or dimethyldichlorosilane. Silicas thus
treated are known as "silica dimethyl silylate" according to the
CTFA (6th edition, 1995). They are sold, for example, under the
references "Aerosil R972.RTM..", "Aerosil R974.RTM." by the company
Degussa, "CAB-O-SIL TS-610.RTM.." and "CAB-O-SIL TS-720.RTM.." by
the company Cabot. Each possibility represents a separate
embodiment of the present invention.
[0070] In another embodiment, nanoparticles of compositions of the
present invention are practically insoluble in water. "Practically
insoluble" refers, in another embodiment, to a substance having a
solubility of less than 100 parts per million weight/weight (ppm).
In another embodiment, the term refers to a solubility of less than
200 ppm. In another embodiment, the term refers to a solubility of
less than 80 ppm. In another embodiment, the term refers to a
solubility of less than 60 ppm. In another embodiment, the term
refers to a solubility of less than 50 ppm. In another embodiment,
the term refers to a solubility of less than 40 ppm. In another
embodiment, the term refers to a solubility of less than 30 ppm. In
another embodiment, the term refers to a solubility of less than 20
ppm. In another embodiment, the term refers to a solubility of less
than 15 ppm. In another embodiment, the term refers to a solubility
of less than 10 ppm. Each possibility represents a separate
embodiment of the present invention.
[0071] In another embodiment, the diameter of the silica
nanoparticles of methods and compositions of the present invention
is between 5-30 nanometers inclusive. In another embodiment, the
diameter is between 2-400 nanometers (nm) inclusive. In another
embodiment, the diameter is between 2-300 nm inclusive. In another
embodiment, the diameter is between 3-200 nm inclusive. In another
embodiment, the diameter is between 4-150 nm inclusive. In another
embodiment, the diameter is between 4-100 nm inclusive. In another
embodiment, the diameter is between 5-50 nm inclusive. In another
embodiment, the diameter is between 5-40 nm inclusive. In another
embodiment, the diameter is between 6-25 nm inclusive. In another
embodiment, the mean diameter of silica nanoparticles is 10-11
nm.
[0072] In another embodiment, the average diameter is about 5 nm.
In another embodiment, the average diameter is about 6 nm. In
another embodiment, the average diameter is about 7 nm. In another
embodiment, the average diameter is about 8 nm. In another
embodiment, the average diameter is about 9 nm. In another
embodiment, the average diameter is about 10 nm. In another
embodiment, the average diameter is about 12 nm. In another
embodiment, the average diameter is about 14 nm. In another
embodiment, the average diameter is about 16 nm. In another
embodiment, the average diameter is about 18 nm. In another
embodiment, the average diameter is about 20 nm. In another
embodiment, the average diameter is another diameter falling within
a range disclosed herein. Each possibility represents a separate
embodiment of the present invention.
[0073] In another embodiment, silica nanoparticles of the present
invention fall within a range of melting temperatures particularly
suitable for compositions of the present invention. In specific
embodiments, the silica nanoparticles have a melting temperature
(T.sub.m) of over 600.degree. C. In another embodiment, the T.sub.m
is between 600-4500.degree. C. In another embodiment, the T.sub.m
is another T.sub.m falling within a range disclosed herein. Each
possibility represents a separate embodiment of the present
invention.
Imparting a Hydrophobic Surface to a Nanoparticle
[0074] Methods for imparting a hydrophobic surface to nanoparticles
are well known in the art and are described, inter alia, in Chung
et al (Hydrophobic modification of silica nanoparticle by using
aerosol spray reactor. Colloids and Surfaces A: Physicochem. Eng.
Aspects 236 (2004) 73-79). Additional methods include the reverse
micelles method (Fu X, Qutubuddin S, Colloids Surf. A: Physicochem.
Eng. Aspects 179: 65, 2001), liquid precipitation method
(Krysztafkiewicz A, Jesionowski T, Binkowski S, Colloids Surf. A:
Physicochem. Eng. Aspects 173:73, 2000) and sol-gel method (Jean J,
Yang S, J. Am. Ceram. Soc. 83 (8):1928, 2000; Zhang J, Gao L,
Ceram. Int. 27: 143, 2001). US 2007/0172426 provides additional
methods of imparting a hydrophobic surface to a nanoparticle, by
combining them with a material having a first end that adsorbs to
the surface of the nanoparticle and a second end that extends away
from the nanoparticle and imparts hydrophobicity to the particles.
The material may be a generally aliphatic compound having a polar
end-group. The first end of each molecule of the compound may
include a carboxyl group, an amine group, a silane, etc., that
adsorbs to the surface of the particle. The second end of each
molecule of the compound may include alkane group that extends away
from the particle. Materials used to provide the hydrophobic
surface layer include saturated fatty acids such as lauric acid,
myristic acid, palmitic acid, and stearic acid, and unsaturated
variants thereof, such as palmitoleic acid, oleic acid, linoleic
acid, and linolenic acid. Silanes such as octadecyl trichlorosilane
can also be widely used to functionalize oxide surfaces. The
hydrophobic surface layer is provided by mixing the nanoparticles
into a volume of hydrophobic coating material suitable for coating
the particles. An excess of hydrophobic coating material is
generally used so that the nanoparticles form a suspension in the
hydrophobic coating material. Each nanoparticle then exhibits a
hydrophobic layer on its surface. Additional methods for utilizing
a hydrocarbon surfactant to coat nanoparticles are described in US
2006/0053971. Additional methods are described in US 2007/0098990.
The disclosed methods utilize multiple organic acids in which the
first acid is a low molecular weight organic carboxylic acid and
the second acid is a high molecular weight organic carboxylic acid.
The contents of each of the above patent applications are hereby
incorporated by reference.
Biopolymers
[0075] Methods and compositions of the present invention is
preferably comprise a branched biopolymer. "Branched" as used
herein encompasses both polymers that are naturally branched and
those engineered to be branched by physical treatment such as
thermal and/or ultrasound treatment. In general, branched polymers
are defined as polymers wherein a monomer subunit is covalently
bound to more than two monomer subunits. Such a monomer is the site
of a branch point, wherein multiple polymer chains converge. In
another embodiment, the branched biopolymer is a crosslinked
polymer. In another embodiment, the branched biopolymer is not
crosslinked. Non-limiting examples of branched polymers are
glycogen and amylopectin, forms of starch found in animals and
plants, respectively. Structures of amylopectin (CAS#9037-22-3) and
glycogen (CAS#9005-79-2) are depicted below:
##STR00001##
[0076] In another embodiment, the biopolymer is a fibrous
biopolymer. "Fibrous polymer" refers to a polymer in the form of a
network of discrete thread-shaped pieces. Non-limiting examples of
fibrous polymers are guar gum (found for example in Benefiber.TM.),
collagen, keratin, fibrin, and elastin. Biopolymers can be either
naturally fibrous or made fibrous by physical and chemical
treatment.
[0077] In another embodiment, the biopolymer is a fiber. "Fiber"
refers, in another embodiment, to an indigestible component that
acts as a bulking agent for feces. In another embodiment, the fiber
is an insoluble fiber. In another embodiment, the fiber is a
soluble fiber. Each possibility represents a separate embodiment of
the present invention. Each type of fiber and type of branched and
fibrous biopolymer represents a separate embodiment of the present
invention.
[0078] In another embodiment, the biopolymer is pharmacologically
inert. In another embodiment, the biopolymer is non-toxic. In
another embodiment, the biopolymer is non-teratogenic. In another
embodiment, the biopolymer is biologically inert. Each possibility
represents a separate embodiment of the present invention.
[0079] In another embodiment, the melting temperature of the
biopolymer falls within a range particularly suitable for
compositions of the present invention. In another embodiment, the
biopolymer has a melting temperature under 400.degree. C. In
another embodiment, the T.sub.m is below 350.degree. C. In another
embodiment, the T.sub.m is below 300.degree. C. In another
embodiment, the T.sub.m is below 250.degree. C. In another
embodiment, the T.sub.m is below 200.degree. C. In another
embodiment, the T.sub.m is below 150.degree. C. In another
embodiment, the T.sub.m is between 100-400.degree. C. In another
embodiment, the T.sub.m is any T.sub.m falling within a range
disclosed herein. Each possibility represents a separate embodiment
of the present invention.
[0080] Preferably, the biopolymer of methods and compositions of
the present invention is selected from the group consisting of a
polysaccharide and a structural protein.
Polysaccharides
[0081] "Saccharide" refers to any simple carbohydrate including
monosaccharides, monosaccharide derivatives, monosaccharide
analogs, sugars, including those, which form the individual units
in a polysaccharide. "Monosaccharide" refers to polyhydroxyaldehyde
(aldose) or polyhdroxyketone (ketose) and derivatives and analogs
thereof.
[0082] "Polysaccharide" refers to polymers formed from about 500
saccharide units linked to each other by hemiacetal or glycosidic
bonds. Typically, polysaccharides can contain as many as 100,000
saccharide units, and in some cases even more. The polysaccharide
may be either a straight chain, singly branched, or multiply
branched wherein each branch may have additional secondary
branches, and the monosaccharides may be standard D- or L-cyclic
sugars in the pyranose (6-membered ring) or furanose (5-membered
ring) forms such as D-fructose and D-galactose, respectively, or
they may be cyclic sugar derivatives, for example amino sugars such
as D-glucosamine, deoxy sugars such as D-fucose or L-rhamnose,
sugar phosphates such as D-ribose-5-phosphate, sugar acids such as
D-galacturonic acid, or multi-derivatized sugars such as
N-acetyl-D-glucosamine, N-acetylneuraminic acid (sialic acid), or
N-sulfato-D-glucosamine. When isolated from nature, polysaccharide
preparations comprise molecules that are heterogeneous in molecular
weight. Polysaccharides include, among other compounds,
galactomanans and galactomannan derivatives;
galacto-rhamnogalacturons and galacto-rhamnogalacturon derivatives,
and galacto-arabinogalacturon and galacto-arabinogalacturon
derivatives.
[0083] In another embodiment, the polysaccharide of methods and
compositions of the present invention is a naturally-occurring
polysaccharide. In another embodiment, the polysaccharide is a
synthetic polysaccharide. Non limiting examples of synthetic
polysaccharides can be found in U.S. Pat. No. 6,528,497 and in
Okada M. et al. Polymer journal, 15 (11); 821-26 (1983). In another
embodiment, the polysaccharide is a naturally-occurring branched
polysaccharide. In another embodiment, the polysaccharide is a
synthetic branched polysaccharide. Each possibility represents a
separate embodiment of the present invention.
[0084] In another embodiment, the polysaccharide is a branched
polysaccharide. This term is well understood to those skilled in
the art and can refer to any number and structure of branches in
the links between monosaccharide monomers. In another embodiment,
the polysaccharide is a naturally-occurring branched
polysaccharide. In another embodiment, the branched polysaccharide
is a starch. In another embodiment, the branched polysaccharide is
selected from the group consisting of amylopectin, glycogen, and a
branched alpha glucan. In another embodiment, the polysaccharide is
a synthetic branched polysaccharide. Each possibility represents a
separate embodiment of the present invention.
[0085] In another embodiment, the polysaccharide is an amphipathic
polysaccharide. This term is well understood to those skilled in
the art and refers to the existence of both hydrophobic and
hydrophilic regions on the polysaccharide. In another embodiment,
the polysaccharide is a naturally-occurring amphipathic
polysaccharide. Each possibility represents a separate embodiment
of the present invention.
[0086] In another embodiment, the average MW of the polysaccharide
is at least 100 kilodalton (kDa). In another embodiment, the
average MW is at least 150 kDa. In another embodiment, the average
MW is at least 200 kDa. In another embodiment, the average MW is at
least 300 kDa. In another embodiment, the average MW is at least
400 kDa. In another embodiment, the average MW is at least 500 kDa.
In another embodiment, the average MW is at least 600 kDa. In
another embodiment, the average MW is at least 800 kDa. In another
embodiment, the average MW is at least 1000 kDa. In another
embodiment, the average MW is between 100-1000 kDa. In another
embodiment, the average MW is between 150-1000 kDa. In another
embodiment, the average MW is between 200-1000 kDa. In another
embodiment, the average MW is between 100-800 kDa. In another
embodiment, the average MW is between 100-600 kDa. Each possibility
represents a separate embodiment of the present invention.
[0087] In another embodiment, the polysaccharide is selected from
the group consisting of starch, dextrin, cellulose, chitin, a
branched alpha glucan, a branched beta glucan and derivatives
thereof. Cellulose, dextrin, starch and glycogen are all polymers
of glucose and thus have the formula
(C.sub.6H.sub.10O.sub.5).sub.n.
[0088] In another embodiment, the polysaccharide is a starch, which
has the structure below. Non-limiting examples of starch are corn
starch, potato starch, rice starch, wheat starch, purum starch, and
starch from algae. In another embodiment, the starch is any other
starch known in the art. Each possibility represents a separate
embodiment of the present invention.
##STR00002##
[0089] In another embodiment, the polysaccharide is a dextrin.
"Dextrin" in another embodiment refers to a low-molecular-weight
carbohydrate produced by the hydrolysis of starch. In another
embodiment, the term refers to a linear .alpha.-(1,4)-linked
D-glucose polymer starting with an .alpha.-(1,6) bond or a mixture
of same. Dextrins are widely commercially available and can be
produced inter alia by digestion of branched amylopectin or
glycogen with .alpha.-amylase. A non-limiting example of a dextrin
is a maltodextrin having the structure below. In another
embodiment, the dextrin is any other dextrin known in the art. Each
possibility represents a separate embodiment of the present
invention.
##STR00003##
[0090] In another embodiment, the polysaccharide is cellulose. A
non-limiting example of a cellulose is .alpha.-cellulose, which has
the structure below.
##STR00004##
[0091] In another embodiment, the polysaccharide is
.beta.-cellulose, a linear polymer of D-glucose linked by
.beta.(1.fwdarw.4) glycosidic bonds. In another embodiment, the
.beta.-cellulose has the structure below.
##STR00005##
In another embodiment, the cellulose is any other cellulose known
in the art. Each possibility represents a separate embodiment of
the present invention.
[0092] In another embodiment, the polysaccharide is chitin, a
long-chain polymer of N-acetylglucosamine, a derivative of glucose.
Typically, chitin has the molecular formula
(C.sub.8H.sub.13NO.sub.5).sub.n and the structure below. Each
possibility represents a separate embodiment of the present
invention.
##STR00006##
[0093] In another embodiment, the polysaccharide is an
alpha-glucan. Alpha-glucans of the present invention may be linear
or branched polymers of glucose with alpha 1-2, alpha 1-3, alpha
1-4, and/or alpha 1-6 glycosidic linkages. In another embodiment,
the alpha-glucan has unbranched linear glucose polymers with 1-4
glycosidic linkages, an example of which is alpha-amylose. In
another embodiment, the alpha-glucan has branched glucose polymers
with alpha 1-4 glycosidic linkages in the backbone and alpha 1-6
linkages at branch points, an example of which is amylopectin. In
another embodiment, the alpha-glucan is any other type of
alpha-glucan known in the art. Each possibility represents a
separate embodiment of the present invention.
[0094] In another embodiment, the polysaccharide is a beta-glucan.
"Beta-glucan" refers to polysaccharides containing D-glucopyranosyl
units linked together by (1.fwdarw.3) or (1.fwdarw.4)
beta-linkages. Beta-Glucans occur naturally in many cereal grains
such as oats and barley. The molecular weight of beta-glucan
molecules occurring in cereals is typically 200 to 2000 kDa; other
types contain up to about 250,000 glucose units. In another
embodiment, the beta-glucan is any other beta-glucan known in the
art. Each possibility represents a separate embodiment of the
present invention.
[0095] In another embodiment, a pharmaceutical composition of the
present invention comprises a branched polysaccharide and a linear
polysaccharide. In another embodiment, the linear polysaccharide is
selected from the group consisting of chitin, cellulose, amylose,
and beta glucan. In some preferred embodiments, the branched and
linear polysaccharides both have a melting temperature under
400.degree. C. Insulin compositions are provided herein that
comprise amylopectin, a branched polysaccharide, and chitin, a
linear polysaccharide (Example 5, Formulation IV). Other branched
polysaccharides and linear polysaccharides disclosed herein are
suitable as well.
[0096] In another embodiment, the additional biopolymer of methods
and compositions of the present invention is a fiber,
preferentially a dietary fiber. The definition of the term "fiber"
and "dietary fiber" as used herein includes unavailable
carbohydrates, indigestible residue, and plant cell polysaccharides
and lignin, all of which are resistant to hydrolysis by human
digestive enzymes. Preferred fibers are members selected from the
group consisting of guar gum, pectin, fructo-oligosaccharides and
derivatives thereof. Small amounts of other indigestible compounds,
such as phytates, tannins, saponins and cutin, may be included in
dietary fiber since these compounds are indigestible and associated
with dietary fiber polysaccharides. In another embodiment, the
dietary fiber is an insoluble fiber. In another embodiment, the
dietary fiber is a linear insoluble fiber. In another embodiment,
the dietary fiber is a soluble fiber. In another embodiment, the
dietary fiber is a linear soluble fiber. Each possibility
represents a separate embodiment of the present invention.
[0097] In another embodiment, the T.sub.m of a polysaccharide of a
composition of the present invention falls within a range of
melting temperatures particularly suitable for compositions of the
present invention. In another embodiment, the polysaccharide has a
T.sub.m under 400.degree. C. In another embodiment, the T.sub.m is
another T.sub.m or range of T.sub.m defined herein. Each
possibility represents a separate embodiment of the present
invention.
Structural Proteins
[0098] According to certain embodiments the dry solid particulate
ingredients of compositions may further comprise a structural
protein. the structural protein of methods and compositions of the
present invention is a high molecular weight (MW) structural
protein. In some embodiments, the structural protein comprises both
hydrophilic and hydrophobic residues that interact with the
hydrophobic and hydrophilic regions, respectively, of the
biologically active protein or peptide. In another embodiment, the
average MW of the structural protein is at least 100 kilodalton
(kDa). In another embodiment, the average MW is at least 150 kDa.
In another embodiment, the average MW is at least 200 kDa. In
another embodiment, the average MW is at least 300 kDa. In another
embodiment, the average MW is at least 400 kDa. In another
embodiment, the average MW is at least 500 kDa. In another
embodiment, the average MW is at least 600 kDa. In another
embodiment, the average MW is at least 800 kDa. In another
embodiment, the average MW is at least 1000 kDa. In another
embodiment, the average MW is between 100-1000 kDa. In another
embodiment, the average MW is between 150-1000 kDa. In another
embodiment, the average MW is between 200-1000 kDa. In another
embodiment, the average MW is between 100-800 kDa. In another
embodiment, the average MW is between 100-600 kDa. Each possibility
represents a separate embodiment of the present invention.
[0099] "Structural protein", in one embodiment, refers to a protein
included for the structure it confers to the matrix carrier
composition. In another embodiment, a structural protein of the
present invention lacks therapeutic activity. In another
embodiment, the term refers to a protein that confers structure to
a cell, cellular membrane, or extracellular membrane in vivo. In
another embodiment, the structural protein is a fibrous protein. In
another embodiment, the structural protein is a scleroprotein. In
another embodiment, the structural protein is selected from the
group consisting of elastin, collagen, keratin, and fibrinogen. In
another embodiment, the structural protein is any other fibrous
protein or scleroprotein known in the art. Each possibility
represents a separate embodiment of the present invention.
[0100] In another embodiment, the structural protein is elastin.
Non-limiting examples of elastin proteins are described, inter
alia, in GenBank Accession numbers NP.sub.--031951,
NP.sub.--786966, and AAC98394. In another embodiment, the elastin
is any other elastin known in the art. Each possibility represents
a separate embodiment of the present invention.
[0101] In another embodiment, the structural protein is collagen.
Non-limiting examples of collagen proteins include those encoded by
gene symbols COL3A1, COL14A1, COL11A2, COL5A2, COL11A1, COL5A1,
COL4A6, COL4A5, COL4A4, COL4A3, COL4A2, COL1A2, COL5A3, COL18A1,
COL12A1, COL19A1, COL24A1, COL4A1, and COL2A1. In another
embodiment, the collagen is any other collagen known in the art.
Each possibility represents a separate embodiment of the present
invention.
[0102] In another embodiment, the structural protein is keratin.
Non-limiting examples of keratin proteins include keratin 18,
keratin 14, keratin 3, and keratin 86 (GenBank Accession numbers
P05783, P02533, P12035, O43790, respectively. In another
embodiment, the keratin is any other keratin known in the art. Each
possibility represents a separate embodiment of the present
invention.
[0103] In another embodiment, the structural protein is fibrinogen.
Fibrinogen is a glycoprotein composed of three pairs of
polypeptides: two alpha, two beta, and two gamma chains.
Non-limiting examples of the fibrinogen alpha, beta, and gamma
chains are described, inter alia, in GenBank Accession numbers
P02671, P02675, and P02679. In another embodiment, the fibrinogen
is any other fibrinogen known in the art. Each possibility
represents a separate embodiment of the present invention.
[0104] In another embodiment, the T.sub.m of a structural protein
of a composition of the present invention falls within a range of
melting temperatures particularly suitable for compositions of the
present invention. In another embodiment, the structural protein
has a T.sub.m under 400.degree. C. Each possibility represents a
separate embodiment of the present invention.
Oils and Oil Coatings
[0105] The particulate matter of matrix compositions of the present
invention is surrounded by, suspended in, immersed in, embedded in
or dispersed in oil carrier. Typically, the oil phase, in addition
to coating the particulate matter, impregnates the particulate
matter, which is composed of the silica nanoparticles, branched
polysaccharide and insulin. Reference to an "oil," "oil layer,"
"oil phase," or "oil coating" does not preclude the presence of an
additional component or components useful in methods of the present
invention (e.g. a fat-soluble co-factor or anti-oxidant). Rather,
the term indicates that the oil, oil layer, oil phase, or coating
is composed primarily of a pharmaceutically acceptable oil carrier,
in which the other components are mixed and/or dissolved. The oil
carrier can be composed of either one or a plurality of types of
oils, as described further herein. In another embodiment, the
coating consists essentially of lipids and/or oils. In another
embodiment, the coating of the composition comprises a
pharmaceutically acceptable oil carrier. In another embodiment, the
oil carrier is a naturally occurring oil. In another embodiment,
the oil is a mixture of natural vegetable oils. In another
embodiment, the oil carrier is sesame oil. In another embodiment,
the oil carrier is olive oil. In another embodiment, the oil
carrier is linseed oil. In another embodiment, the oil carrier is
evening primrose oil. In another embodiment, the oil carrier is
silicone oil. In another embodiment, the oil carrier is sea
buckthorn oil. In another embodiment, the oil carrier is selected
from the group consisting of sesame oil, olive oil, linseed oil,
evening primrose oil, silicone oil, and sea buckthorn oil. In
another embodiment, the oil carrier includes, but is not limited
to, an oil selected from the group consisting of sunflower oil,
corn oil, soybean oil, jojoba oil, marrow oil, grapeseed oil,
hazelnut oil, apricot oil, macadamia oil and castor oil. In another
embodiment, the oil carrier is another suitable oil known in the
art. In another embodiment, the oil carrier is of animal origin,
such as lanolin. In another embodiment, the oil carrier is a
synthetic oil. In another embodiment, the oil carrier is a fatty
alcohol. In certain preferred embodiments, the oil carrier is
2-octyldodecanol. In certain other preferred embodiments, the oil
carrier is selected from the group consisting of a fatty acid ester
and a phenylsilicone. In certain more preferred embodiments, the
oil carrier is selected from the group consisting of a
phenyltrimethicone, a diphynyldimethicone, and a
poly-methylphenylsiloxane. Each possibility represents a separate
embodiment of the present invention.
[0106] In another embodiment, the oil consists essentially of
naturally-occurring lipids and/or oils. Each possibility represents
a separate embodiment of the present invention.
[0107] "Plurality of oils" refers, in another embodiment, to two or
more oils. In another embodiment, a composition of the present
invention comprises three or more oils. In another embodiment, a
composition of the present invention comprises four or more oils.
In another embodiment, a composition of the present invention
comprises more than four oils. In another embodiment, the oil phase
comprises a mixture of oils selected from natural vegetable oils.
Each possibility represents a separate embodiment of the present
invention.
[0108] In another embodiment, an oil component of the present
invention comprises a component capable of stimulating secretion of
bile salts or bile acids when ingested by a subject. In another
embodiment, the bile-stimulating component is an oil. In another
embodiment, the component is olive oil or an extract thereof. In
another embodiment, the component is any other bile salt/acid
stimulating lipid-soluble substance known in the art. In another
embodiment, the carrier is the bile salt/acid stimulating
substance. In another embodiment, the bile salt/acid stimulating
substance is a substance separate from the carrier. Each
possibility represents a separate embodiment of the present
invention.
[0109] In another embodiment, an oil component of the present
invention contains a significant quantity of one or more
anti-oxidants. For example, sea buckthorn (oblepicha) oil contains
a significant quantity of beta-carotene. In another embodiment, any
other oil enriched in one or more anti-oxidants may be used. Each
possibility represents a separate embodiment of the present
invention.
[0110] In another embodiment, an oil component of the present
invention comprises a component that has a melting temperature
(T.sub.m) of at least 10.degree. C. In another embodiment, the high
T.sub.m component is an oil. In another embodiment, the carrier is
the high T.sub.m component. In another embodiment, the high-T.sub.m
component is included in addition to the carrier. A non-limiting
example of a high-T.sub.m oil is jojoba oil. In another embodiment,
the high T.sub.m oil is any other high melting temperature oil
known in the art. In another embodiment, the high T.sub.m oil is
used as the oil carrier in the first oil component of a matrix
carrier of the present invention. Each possibility represents a
separate embodiment of the present invention.
[0111] In another embodiment, a matrix composition of the present
invention further comprises a third oil or mixture of oils. In
another embodiment, the third oil component comprises an
antioxidant. In another embodiment, the third oil component is
sesame oil. In another embodiment, the third oil component is
another suitable oil known in the art. In another embodiment, the
third oil, oil or mixture of oils has a higher viscosity than the
additional oil or mixture of oils. Each possibility represents a
separate embodiment of the present invention.
[0112] In another embodiment, a matrix composition of the present
invention further comprises an additional oil component. As
provided herein, mixing of multiple oil components of compositions
of the present invention in the correct order provides
self-ordering or self-organization of matrix structure, due to
competitive adsorption and minimization of the free energy. The
term "additional oil component" encompasses an oil or mixture of
oils, as described elsewhere herein. In another embodiment, the oil
carrier of the additional oil component is olive oil. In another
embodiment, the oil carrier is another suitable oil known in the
art. In another embodiment, the additional oil component comprises
an antioxidant. Each possibility represents a separate embodiment
of the present invention.
[0113] In another embodiment, the insulin protein is included in
the additional oil or mixture of oils, instead of in the
first-added oil or mixture of oils. In another embodiment, the
insulin protein is combined with an antioxidant and oil (the
first-added or additional oil or mixture of oils) prior to adding
to the solid phase. Each possibility represents a separate
embodiment of the present invention.
[0114] In another embodiment, the additional oil, oil or mixture of
oils has a higher viscosity than the first-added oil or mixture of
oils. In another embodiment, without wishing to be bound by any
theory or mechanism of action, the use of a higher viscosity oil or
oil mixture at this stage enables self-ordering or
self-organization of structure due to competitive adsorption and
minimization of the free energy. Each possibility represents a
separate embodiment of the present invention.
[0115] In another embodiment, a matrix composition of the present
invention further comprises a third oil or mixture of oils. In
another embodiment, the third oil component comprises an
antioxidant. In another embodiment, the oil carrier of the third
oil component is sesame oil. In another embodiment, the oil carrier
is another suitable oil known in the art. In another embodiment,
the third oil, oil or mixture of oils has a higher viscosity than
the additional oil or mixture of oils. Each possibility represents
a separate embodiment of the present invention.
[0116] In another embodiment, a highly penetrative oil carrier is
included in the outer oil or mixture of oils. Non-limiting examples
of highly penetrative oils are sesame oil, tea tree (Melaleuca)
oil, lavender oil, almond oil, and grape seed oil. In another
embodiment, the highly penetrative oil carrier promotes efficient
transport of the substances into the blood. Each possibility
represents a separate embodiment of the present invention.
[0117] In another embodiment, a matrix composition or
pharmaceutical composition of the present invention further
comprises a pharmaceutically acceptable wax. The term "wax" means a
lipophilic compound, which is solid at room temperature (25.degree.
C.), with a reversible solid/liquid change of state, having a
melting point of greater than or equal to 30.degree. C., which may
be up to 120.degree. C. By bringing the wax to the liquid state
(melting), it is possible to render it miscible with any oils
present and to form a microscopically homogeneous mixture, but on
returning the temperature of the mixture to room temperature,
recrystallization of the wax in the oils of the mixture is
obtained. The wax may be a natural wax, for example bees wax, a wax
derived from plant material, or a synthetic wax prepared by
esterification of a fatty acid and a long chain alcohol. Other
suitable waxes include petroleum waxes such as a paraffin wax. In
another embodiment, the wax stabilizes the matrix carrier
composition. In another embodiment, the inclusion of wax
facilitates formation of a tablet containing the matrix carrier
composition. Each possibility represents a separate embodiment of
the present invention.
Insulin Proteins
[0118] "Insulin protein" as used herein includes rapid-acting
insulin, very rapid-acting insulin, intermediate-acting insulin,
and long-acting insulin. Non-limiting examples of rapid-acting
insulin are lyspro insulin (Lysine-Proline insulin, sold by Eli
Lilly as Humalog.TM.), glu-lysine insulin (sold by Sanofi-Aventis
as Apidra.TM.), Actrapid.TM. and NovoRapid.TM. (both available from
Novo Nordisk), aspart insulin (sold by Novo Nordisk as
Novolog.TM.). A non-limiting example of very rapid-acting insulin
is Viaject.TM., marketed by Biodel. Non-limiting examples of
intermediate-acting insulin are NPH (Neutral Protamine Hagedorn)
and Lente insulin. A non-limiting example of long-acting insulin is
Lantus.TM. (insulin glargine). In some preferred embodiments, the
insulin is Insugen.TM. from Biocon.TM.. In another embodiment, the
insulin is a mixture of different types of insulin. Some
non-limiting examples of a such a mixture are Mixtard.RTM. 30,
Mixtard.RTM. 40, and Mixtard.RTM. 50, which are mixtures of
different proportions of short-acting insulin and NPH (intermediate
duration) insulin. In another embodiment, the insulin is any other
type of insulin known in the art. In another embodiment, the
insulin is naturally occurring insulin. In another embodiment, the
insulin is a modified form of insulin. It will be clear from the
present disclosure that methods and compositions of the present
invention are suitable for every type of natural and modified
insulin known in the art. Each possibility represents a separate
embodiment of the present invention.
Additional Components
[0119] In another embodiment, the composition further comprises an
antioxidant. In another embodiment, the antioxidant is a
pharmaceutically acceptable antioxidant. In another embodiment, the
antioxidant is selected from the group consisting of vitamin E,
superoxide dismutase (SOD), omega-3, and beta-carotene. Each
possibility represents a separate embodiment of the present
invention.
[0120] In another embodiment, the composition further comprises an
enhancer of the insulin protein. Non limiting examples of insulin
enhancers include: dodecylmaltoside, octylglucoside, and dioctyl
sodium sulphosuccinate. In another embodiment, the composition
further comprises a cofactor of the insulin protein. Non limiting
example of an insulin cofactor is chromium. Each possibility
represents a separate embodiment of the present invention.
[0121] In another embodiment, a composition of the present
invention further comprises a glucagon-like peptide or
glucagon-like peptide analogue. Glucagon-like peptides and their
analogues are well known in the art, and are described, inter alia,
in Eleftheriadou I. et al. (The effects of medications used for the
management of diabetes and obesity on postprandial lipid
metabolism. Curr Diabetes Rev 4 (4):340-56, 2008 and Vaidya H B et
al., Glucagon like peptides-1 modulators as newer target for
diabetes. Curr. Drug Targets 9 (10):911-20, 2008). Each possibility
represents a separate embodiment of the present invention.
[0122] In another embodiment, a composition of the present
invention further comprises a bioflavonoid. Bioflavonoids are well
known in the art, and are described, inter alia, in Ververidis F.
et al. (Biotechnology of flavonoids and other
phenylpropanoid-derived natural products. Part I: Chemical
diversity, impacts on plant biology and human health and Part II:
Reconstruction of multienzyme pathways in plants and microbes, 2
(10):1214-49, 2007). Each possibility represents a separate
embodiment of the present invention.
[0123] In another embodiment, a composition of the present
invention further comprises a pharmaceutical-grade surfactant.
Surfactants are well known in the art, and are described, inter
alia, in the Handbook of Pharmaceutical Excipients (eds. Raymond C.
Rowe, Paul J. Sheskey, and Sian C. Owen, copyright Pharmaceutical
Press, 2005). In another embodiment, the surfactant is any other
surfactant known in the art. Each possibility represents a separate
embodiment of the present invention.
[0124] In another embodiment, a composition of the present
invention further comprises pharmaceutical-grade emulsifier or
emulgator (emollient). Emulsifiers and emulgators are well known in
the art, and are described, inter alia, in the Handbook of
Pharmaceutical Excipients (ibid). Non-limiting examples of
emulsifiers and emulgators are eumulgin, Eumulgin B1 PH, Eumulgin
B2 PH, hydrogenated castor oil cetostearyl alcohol, and cetyl
alcohol. In another embodiment, the emulsifier or emulgator is any
other emulsifier or emulgator known in the art. Each possibility
represents a separate embodiment of the present invention.
[0125] In another embodiment, a composition of the present
invention further comprises pharmaceutical-grade stabilizer.
Stabilizers are well known in the art, and are described, inter
alia, in the Handbook of Pharmaceutical Excipients (ibid). In
another embodiment, the stabilizer is any other stabilizer known in
the art. Each possibility represents a separate embodiment of the
present invention.
[0126] In another embodiment, the weight of the particulate matter
of a composition of the present invention is not more than 33% of
the weight of the oil phase. The particulate matter is composed of
the silica nanoparticles, the branched polysaccharide, the insulin,
and any other solid components that may be incorporated into the
matrix. In another embodiment, the particulate matter is composed
of the silica nanoparticles, the branched polysaccharide, and the
insulin. The weight of the particulate matter is the weight of the
oil carrier plus additional oils mixed therewith and substances
dissolved therein, if any, for all the oil components combined. In
another embodiment, the weight of the particulate matter is not
more than 30% of the weight of the oil phase. In another
embodiment, the weight of the particulate matter is not more than
25% of the weight of the oil phase. In another embodiment, the
weight of the particulate matter is not more than 20% of the weight
of the oil phase. In another embodiment, the weight of the
particulate matter is not more than 15% of the weight of the oil
phase. In another embodiment, the weight of the particulate matter
is not more than 10% of the weight of the oil phase. In another
embodiment, the weight of the particulate matter is not more than
8% of the weight of the oil phase. In another embodiment, the
weight of the particulate matter is not more than 5% of the weight
of the oil phase. Each possibility represents a separate embodiment
of the present invention.
[0127] In another embodiment, the weight of the particulate matter
is not more than 75% of the total weight of the composition. In
another embodiment, the weight of the particulate matter is not
more than 50% of the total weight of the composition. In another
embodiment, the weight of the particulate matter is not more than
25% of the total weight of the composition. In another embodiment,
the weight of the particulate matter is not more than 30% of the
total weight of the composition. In another embodiment, the weight
of the particulate matter is not more than 20% of the total weight
of the composition. In another embodiment, the weight of the
particulate matter is not more than 15% of the total weight of the
composition. In another embodiment, the weight of the particulate
matter is not more than 10% of the total weight of the composition.
In another embodiment, the weight of the particulate matter is not
more than 8% of the total weight of the composition. In another
embodiment, the weight of the particulate matter is not more than
6% of the total weight of the composition. In another embodiment,
the weight of the particulate matter is not more than 5% of the
total weight of the composition. Each possibility represents a
separate embodiment of the present invention.
Methods of Administration
[0128] In another embodiment, the present invention provides a
method of administering an insulin protein to a subject in need
thereof, comprising orally administering to the subject a
pharmaceutical composition of the present invention, thereby
administering an insulin protein to a subject.
Formulation Methods
[0129] In another embodiment, the present invention provides a
method of manufacturing a pharmaceutical composition for oral
delivery of insulin, the method comprising the steps of: (a) dry
blending pharmacologically inert silica nanoparticles having a
hydrophobic surface, wherein the size of the silica nanoparticles
is between 1-100 nanometers, with at least one branched
polysaccharide, whereby the silica nanoparticles form an intimate
non-covalent association with the at least one branched
polysaccharide; (b) mixing or dissolving an insulin protein into an
oil; and (c) mixing the silica nanoparticles and at least one
branched polysaccharide into the oil, wherein the silica
nanoparticles, at least one branched polysaccharide, and insulin
are suspended in, embedded in or dispersed in the oil. Preferably,
the silica nanoparticles and at least one branched polysaccharide
form a complex. In another embodiment, the complex is suspended in,
embedded in or dispersed in the oil. In another embodiment, the
insulin protein is attached to the hydrophobic surfaces of the
silica nanoparticles and the at least one branched polysaccharide
via non-covalent forces. In another embodiment, the particle size
of the matrix carrier composition is between 100-500,000
nanometers. In some preferred embodiments, the particle size is
between 100-50,000 nanometers. In another embodiment, the particle
size is between 100-5,000 nm. Each possibility represents a
separate embodiment of the present invention.
[0130] Formulation methods of the present invention encompass
embodiments wherein additional components are present in step (a).
In another embodiment, more than one type of biopolymer is present
together with the silica nanoparticles. In another embodiment, a
branched polysaccharide and a dietary fiber are present together
with the silica nanoparticles. In another embodiment, a branched
polysaccharide and a linear polysaccharide are present together
with the silica nanoparticles. In another embodiment, a branched
biopolymer, a linear polysaccharide, and an insoluble fiber are
present together with the silica nanoparticles.
[0131] In another embodiment, the present invention provides a
method of manufacturing a pharmaceutical composition for oral
delivery of insulin, the method comprising the steps of: (a)
blending pharmacologically inert silica nanoparticles having a
hydrophobic surface, wherein the size of the silica nanoparticles
is between 1-100 nanometers, with (i) at least one branched
polysaccharide and (ii) an insulin protein whereby the silica
nanoparticles form an intimate non-covalent association with the at
least one branched polysaccharide; and (b) mixing the silica
nanoparticles, at least one branched polysaccharide, and insulin
protein into an oil. In certain embodiments, the insulin protein in
the form of a dry lyophilized powder is directly dissolved into the
oil of step (b). Preferably, the silica nanoparticles, at least one
branched polysaccharide, and insulin form a complex. In another
embodiment, the silica nanoparticles, branched polysaccharide, and
insulin form a matrix that becomes suspended in, embedded in or
dispersed in the oil. In another embodiment, the insulin protein is
non-covalently attached to the hydrophobic surfaces of the silica
nanoparticles and the at least one branched polysaccharide. In
another embodiment, the particle size of the pharmaceutical
composition is between 100-500,000 nanometers. In some preferred
embodiments, the particle size is between 100-50,000 nanometers. In
other embodiments, the particle size is between 100-5,000
nanometers. Each possibility represents a separate embodiment of
the present invention.
[0132] In another embodiment, the insulin is extracted from an
aqueous solution. In another embodiment, an aqueous insulin
solution is mixed with oil, resulting in extraction or dispersion
of the insulin directly into the oil phase of the resulting
emulsion. Methods for extracting active enzymes such as insulin
from an aqueous solution are well known in the art. In another
embodiment, a gel-forming water phase stabilizer is used to extract
the insulin. A non-limiting example of a gel-forming water phase
stabilizer is Silica 380, which is available as pharma-grade
hydrophilic nanoparticles. Each possibility represents a separate
embodiment of the present invention.
[0133] In another embodiment, step (b) of the above method
comprises the step of directly dissolving or dispersing a
lyophilized protein into the oil or oil mixture. In another
embodiment, a solution of the insulin protein is mixed with the oil
or oil mixture and the aqueous phase is then removed. In another
embodiment, a solution of the insulin protein is mixed with the oil
or oil mixture forming water-in-oil emulsion. Each possibility
represents a separate embodiment of the present invention.
[0134] The properties and classification of the silica
nanoparticles, branched polysaccharide, and insulin protein of the
above methods may be any of those described herein. Each
possibility represents a separate embodiment of the present
invention. "Oil" as referred to in methods of the present invention
can refer to either a single oil, a mixture of oils, or an oil
phase. As described herein, a mixture of oils or oil phase will
typically comprise an oil carrier. In another embodiment, the
mixture of oils or oil phase further comprises an additional oil or
oils or an additional component or components. Each possibility
represents a separate embodiment of the present invention.
[0135] In another embodiment, step (a) of a method of manufacturing
a pharmaceutical composition for oral delivery of insulin of the
present invention further comprises the step of confirming that the
silica nanoparticles and branched polysaccharide are properly
homogenized. In another embodiment, any of the following three
tests are utilized: (a) the mixture appears homogenous; (b) the
volume of the mixture is smaller than the sum of volumes of the 2
components; and (c) the mixture does not sink when placed on the
surface of a still body of water. In another embodiment, the
composition reaches a minimum volume that is not decreased upon
further mixing. Each possibility represents a separate embodiment
of the present invention.
[0136] The step of dry mixing is, in another embodiment, performed
using a high shear mixer. In another embodiment, the step of mixing
is performed using a high-speed mixer. In another embodiment, the
step of mixing is performed using any other means suitable for
generating a homogenous solid phase from silica nanoparticles and a
branched polysaccharide. Each possibility represents a separate
embodiment of the present invention.
[0137] In another embodiment, step (a) of a method of manufacturing
a pharmaceutical composition for oral delivery of insulin of the
present invention, i.e. the dry mixing step, further comprises
inclusion of an additional biopolymer that is a linear biopolymer.
In another embodiment, the additional biopolymer is a linear
polysaccharide. In another embodiment, the additional biopolymer is
a linear high molecular weight structural protein. In another
embodiment, the additional biopolymer is selected from the group
consisting of chitin, cellulose, a linear alpha glucan, and a
linear beta glucan. In another embodiment, the additional
biopolymer is selected from the group consisting of chitin,
amylose, cellulose, and beta glucan. In another embodiment, a
formulation method of the present invention comprises the steps of
including in the solid phase mixture both a branched polysaccharide
and a linear polysaccharide. Formulation methods are provided
herein that comprise inclusion of amylopectin, a branched
polysaccharide, and chitin, a linear polysaccharide (Example 5).
Other branched polysaccharides and linear polysaccharides disclosed
herein are suitable as well. Each possibility represents a separate
embodiment of the present invention.
[0138] In another embodiment, the additional biopolymer of
formulation methods of the present invention is a dietary fiber,
also known as "roughage." In another embodiment, the dietary fiber
is an insoluble fiber. In another embodiment, the dietary fiber is
a linear insoluble fiber. In another embodiment, the dietary fiber
is a soluble fiber. In another embodiment, the dietary fiber is a
linear soluble fiber.
[0139] In another embodiment, a method of manufacturing a
pharmaceutical composition for oral delivery of insulin of the
present invention comprises the steps of including a branched
biopolymer, a linear polysaccharide, and an insoluble fiber. In
another embodiment, the method comprises the steps of including in
the particulate matter mixture a branched polysaccharide, a linear
polysaccharide, and an insoluble fiber. An example of such is a
composition comprising amylopectin, a branched polysaccharide;
chitin, a linear polysaccharide; and cellulose, an insoluble fiber.
Other branched and linear polysaccharides and insoluble fibers
disclosed herein are suitable as well. Each possibility represents
a separate embodiment of the present invention.
[0140] In another embodiment, a method of manufacturing a
pharmaceutical composition for oral delivery of insulin of the
present invention further comprises the step of adding an
additional oil following the addition of the first-added oil or
mixture of oils. The term "additional oil" encompasses an oil or
mixture of oils, as described elsewhere herein. In another
embodiment, the additional oil component comprises an antioxidant.
Each possibility represents a separate embodiment of the present
invention.
[0141] In another embodiment, the insulin protein is included in
the additional oil or mixture of oils, instead of in the
first-added oil or mixture of oils.
[0142] In another embodiment, the additional oil, oil or mixture of
oils has a higher viscosity than the first-added oil or mixture of
oils. In another embodiment, the use of a higher viscosity oil or
oil mixture at this stage enables formation of ordered structures
in the composition.
[0143] In another embodiment, a method of manufacturing a
pharmaceutical composition for oral delivery of insulin of the
present invention further comprises the step of adding a third oil
or mixture of oils after addition of the above-described additional
oil or mixture of oils. In another embodiment, the third oil
component comprises an antioxidant. Each possibility represents a
separate embodiment of the present invention.
[0144] In another embodiment, a highly penetrative oil carrier is
included in the outer oil or mixture of oils. In another
embodiment, the highly penetrative oil carrier promotes efficient
transport of the substances into the blood. Each possibility
represents a separate embodiment of the present invention.
[0145] In another embodiment, a method of manufacturing a
pharmaceutical composition for oral delivery of insulin of the
present invention further comprises the step of adding a
pharmaceutically acceptable wax following the addition of the
first-added oil or mixture of oils. In another embodiment, the wax
is a substance with properties similar to beeswax. In another
embodiment, the wax is a substance having the following properties:
(a) plastic (malleable) at normal ambient temperature; (b) having a
melting point above approximately 45.degree. C. (113.degree. F.);
(c) a low viscosity when melted, relative to a typical plastics;
(d) insoluble in water; and (e) hydrophobic. In certain preferred
embodiments, the wax is beeswax. In another embodiment, the wax
stabilizes the matrix carrier composition. In another embodiment,
the inclusion of wax facilitates formation of a tablet containing
the matrix carrier composition. Each possibility represents a
separate embodiment of the present invention.
[0146] In another embodiment, the wax is heated as part of a method
of the present invention. In another embodiment, the wax is
pulverized. In another embodiment, the wax is both heated and
pulverized. In another embodiment, the heating and/or pulverization
are performed prior to blending with the other components. In
another embodiment, the wax remains hot while blending with the
other components is begun. In another embodiment, the heating
and/or pulverization are performed during blending with the other
components. In another embodiment, the heating and/or pulverization
are performed both prior to and during blending with the other
components. Each possibility represents a separate embodiment of
the present invention.
[0147] In another embodiment, a composition of the present
invention further comprises one or more pharmaceutically acceptable
excipients, into which the matrix carrier composition is mixed. In
another embodiment, the excipients include one or more additional
polysaccharides. In another embodiment, the excipients include one
or more waxes. In another embodiment, the excipients provide a
desired taste to the composition. In another embodiment, the
excipients influence the drug consistency, and the final dosage
form such as a gel capsule or a hard gelatin capsule.
[0148] Non limiting examples of excipients include: Antifoaming
agents (dimethicone, simethicone); Antimicrobial preservatives
(benzalkonium chloride, benzelthonium chloride, butylparaben,
cetylpyridinium chloride, chlorobutanol, chlorocresol, cresol,
ethylparaben, methylparaben, methylparaben sodium, phenol,
phenylethyl alcohol, phenylmercuric acetate, phenylmercuric
nitrate, potassium benzoate, potassium sorbate, propylparaben,
propylparaben sodium, sodium benzoate, sodium dehydroacetate,
sodium propionate, sorbic acid, thimerosal, thymol); Chelating
agents (edetate disodium, ethylenediaminetetraacetic acid and
salts, edetic acid); Coating agents (sodium
carboxymethyl-cellulose, cellulose acetate, cellulose acetate
phthalate, ethylcellulose, gelatin, pharmaceutical glaze,
hydroxypropyl cellulose, hydroxypropyl methylcellulose,
hydroxypropyl methylcellulose phthalate, methacrylic acid
copolymer, methylcellulose, polyethylene glycol, polyvinyl acetate
phthalate, shellac, sucrose, titanium dioxide, carnauba wax,
microcrystalline wax, zein); Colorants (caramel, red, yellow, black
or blends, ferric oxide); Complexing agents
(ethylenediaminetetraacetic acid and salts (EDTA), edetic acid,
gentisic acid ethanolmaide, oxyquinoline sulfate); Desiccants
(calcium chloride, calcium sulfate, silicon dioxide); Emulsifying
and/or solubilizing agents (acacia, cholesterol, diethanolamine
(adjunct), glyceryl monostearate, lanolin alcohols, lecithin, mono-
and di-glycerides, monoethanolamine (adjunct), oleic acid
(adjunct), oleyl alcohol (stabilizer), poloxamer, polyoxyethylene
50 stearate, polyoxyl 35 caster oil, polyoxyl 40 hydrogenated
castor oil, polyoxyl 10 oleyl ether, polyoxyl 20 cetostearyl ether,
polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate
60, polysorbate 80, propylene glycol diacetate, propylene glycol
monostearate, sodium lauryl sulfate, sodium stearate, sorbitan
monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan
monostearate, stearic acid, trolamine, emulsifying wax); Flavors
and perfumes (anethole, benzaldehyde, ethyl vanillin, menthol,
methyl salicylate, monosodium glutamate, orange flower oil,
peppermint, peppermint oil, peppermint spirit, rose oil, stronger
rose water, thymol, tolu balsam tincture, vanilla, vanilla
tincture, vanillin); Humectants (glycerin, hexylene glycol,
propylene glycol, sorbitol); Polymers (e.g., cellulose acetate,
alkyl celluloses, hydroxyalkylcelluloses, acrylic polymers and
copolymers); Suspending and/or viscosity-increasing agents (acacia,
agar, alginic acid, aluminum monostearate, bentonite, purified
bentonite, magma bentonite, carbomer 934p, carboxymethylcellulose
calcium, carboxymethylcellulose sodium, carboxymethycellulose
sodium 12, carrageenan, microcrystalline and carboxymethylcellulose
sodium cellulose, dextrin, gelatin, guar gum, hydroxyethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,
magnesium aluminum silicate, methylcellulose, pectin, polyethylene
oxide, polyvinyl alcohol, povidone, propylene glycol alginate,
silicon dioxide, colloidal silicon dioxide, sodium alginate,
tragacanth, xanthan gum); Sweetening agents (aspartame, dextrates,
dextrose, excipient dextrose, fructose, mannitol, saccharin,
calcium saccharin, sodium saccharin, sorbitol, solution sorbitol,
sucrose, compressible sugar, confectioner's sugar, syrup); This
list is not meant to be exclusive, but instead merely
representative of the classes of excipients and the particular
excipients which may be used in oral dosage compositions of the
present invention. Each possibility represents a separate
embodiment of the present invention.
[0149] As provided herein, methods have been developed to formulate
insulin in orally administrable form. The components are, in some
preferred embodiments, mixed in a particular order in order to
produce oil-coated matrix carrier compositions that protect the
active ingredient from digestive processes in the stomach. Without
wishing to be bound by any theory or mechanism of action, the
biopolymer, particularly when branched, absorbs hydraulic and
mechanical stresses experienced during digestion. The oil coating
constitutes a physical barrier that provides additional protection
against digestive enzymes. Secretion of bile acids typically causes
dispersion of the oil suspension into smaller particles, which can
be absorbed in the small intestine. While the particle size is
reduced after traversing the stomach and entering the small
intestine, the particles remain in a size range of 30-1000 nm, too
large to be a substrate for lipases and peptidases, preserving the
protective effect of the composition. Advantageously, lipid-coating
particles of this size are absorbed to chylomicrons by lacteal
vessels, which are lymphatic vessels originating in the villi of
the small intestine. Particles absorbed in this manner can reach
the bloodstream without undergoing first-pass metabolism, largely
preserving the biological activity of the insulin.
[0150] Matrix carriers for any insulin protein can be designed
using the following principles: For purposes of illustration, the
following formulas may be utilized in practicing the invention:
[0151] 1. Quantify the R972 hydrophobic silica (specific area is
about 110 m.sup.2*g.sup.-1)
[0151] Si .gtoreq. 10 * D IU 27.5 ##EQU00001## wherein: D.sub.IU is
desired dosage of insulin per 1 ml in IU; and Si is the
concentration of silica mg*ml.sup.-1 [0152] Note: 27.5 IU*mg.sup.-1
is the specific activity of regular insulin; the formula should be
adjusted accordingly for other types of insulin. [0153] 2. The
specific weight of silica is about 2.4 g*cm.sup.-3, the
concentration of the branched polysaccharide in the medication must
therefore be at least 2.5 times of silica:
[0153] G.sub.AP.gtoreq.2.4*Si Wherein: G.sub.AP is the
concentration of amylopectin. [0154] 3. The concentration of the
linear polysaccharides (G.sub.LP) is estimated as:
[0154] 0.3 .ltoreq. G LP G AP .ltoreq. 0.7 ##EQU00002##
Note:
[0155] Using the above mentioned concentration ratios between the
silica nanoparticles and the polysaccharides, ensures the stability
of the pharmaceutical composition. [0156] 4. The chitin/fiber ratio
may be between 0-1. High ratios (>0.5) are used for the
formation of fast term release insulin compositions. [0157] 5.
Thickness of the protective oil layer is at least 10 times the
diameter of a globular molecule or the maximal branch size of an
elongated molecule. The thickness of the oil coating of the
pharmaceutical compositions of the present invention is determined
by the following properties of the oil or mixture of oils: (a) the
viscosity and melting temperature; (b) the acidity; and (c) the
concentration of polar groups. Typically, a first type of oil is
chosen, preferably having a relatively low viscosity and low
concentration of polar groups. Suitable examples are evening
primrose oil, sesame oil, and silicon oil. [0158] 6. The
concentration of insulin in the final formulation is determined,
based on its pharmacokinetics and pharmacodynamics.
[0159] The principles of the present invention are demonstrated by
means of the following non-limitative examples.
EXAMPLES
Example 1
Preparation of Insulin Composition
[0160] An insulin composition (Formulation I) was produced, using
the following ingredients:
[0161] Insulin Actrapid.TM., 9 ml
[0162] Olive oil, 11 ml
[0163] Benefiber.TM., 7 g
[0164] Silica R972, 1.2 g
[0165] Oblepicha, 9 ml
[0166] Sesame Oil up to 75 ml
Insulin was combined with sea buckthorn (oblepicha) oil and stirred
at 20 rpm for 2 min, and then at 50 rpm for 5 min. Benefiber.TM.
(Novartis Nutrition GmbH, Germany) and hydrophobic silica R972 were
placed into a beaker and mixed by vortexing at 900 rpm for 5 min.
Association between the Benefiber.TM. and the silica was determined
by the mixture's ability to float after being placed on the surface
of a water-filled beaker. The Benefiber.TM./silica mixture was
added to the oil-insulin solution and stirred for 25 minutes at 50
rpm. Olive oil was added, and the mixture was stirred at 50 rpm for
3 min. The volume was brought up to 75 ml with sesame oil, and the
mixture was stirred at 50 rpm for 20 min. The product was stored
refrigerated (3-8.degree. C.). The final insulin concentration was
12 IU/ml. For animal experiments, the composition was administered
by gavage. Other preparation of the product were packaged into
gelatin enteric covering capsules.
Example 2
Additional Actrapid.TM. Matrix Carrier Composition Formulated for
Short-Life
[0167] An additional Actrapid.TM. formulation (Formulation II)
using the following ingredients was designed for short-term insulin
release:
[0168] Insulin Actrapid.TM., 1 ml
[0169] Olive oil, 1.5 ml.
[0170] Ambrotose.TM., 0.7 g.
[0171] Silica R972, 0.1 g
[0172] Oblepicha oil, 1.5 ml
[0173] Evening primrose oil, 5 ml
[0174] 0.7 g of rice polysaccharides (Ambrotose.TM., Mannatech Inc,
Coppell, Tex. 75019, USA) was combined with 0.1 g hydrophobic fumed
silica R972 (Degussa Inc), and mixed by vortexing at 900 rpm for 5
min. Association between the Ambrotose.TM. and the silica was
determined by the mixture's ability to float after being placed on
the surface of a water-filled beaker 1 ml Actrapid.TM. insulin were
added and stirred for 15 minutes at 50 rpm. 1.5 ml of olive oil was
added and stirred for 2 minutes at 100 rpm with a magnetic stirrer.
Sea buckthorn (oblepicha) oil was added and stirred for 2 minutes
at 100 rpm with a magnetic stirrer. The volume was brought up to 5
ml with evening primrose oil and stirred at 50 rpm for 20 min. The
product was stored refrigerated (3-8.degree. C.). In a separate
preparation, the amount of ingredients used was doubled, yielding
identical results.
[0175] The final insulin concentration was 20 IU/ml. For human
administration, the product was packaged into 25 gelatin enteric
covering capsules.
[0176] In additional experiments, vitamin E was included in any one
of the oils used.
Example 3
Longterm Release Actrapid.TM. Matrix Carrier Composition
[0177] The following formulation (Formulation III) was manufactured
to provide longer-term Actrapid.TM. release:
[0178] Olive oil, 10 ml.
[0179] Benefiber.TM., 1.5 g.
[0180] Insulin Actrapid.TM., 2 ml
[0181] Silica R972, 0.7 g
[0182] Oblepicha oil, 10 ml
[0183] Evening primrose oil, 5 ml
[0184] Linseed oil, up to 40 ml.
[0185] Benefiber.TM. was combined with hydrophobic fumed silica
R972 and mixed by vortexing at 900 rpm for 5 minutes. Association
between the Benefiber.TM. and the silica was determined by the
mixture's ability to float after being placed on the surface of a
water-filled beaker. Actrapid.TM. insulin was added and stirred for
15 minutes at 50 rpm. Evening primrose oil was added and stirred
for 2 minutes at 100 rpm with a magnetic stirrer. Sea buckthorn
(oblepicha) oil was added and stirred for 2 minutes at 100 rpm with
a magnetic stirrer. Olive oil was added and stirred for 2 minutes
at 100 rpm with a magnetic stirrer. The volume was brought up to 40
ml with olive oil and stirred at 50 rpm for another 20 min. The
product was stored refrigerated (3-8.degree. C.).
[0186] The final insulin concentration was 5 IU/ml. For human
administration, the product was packaged into 25 gelatin enteric
covering capsules (commercially available from Shionogi and
Company, Ltd, Japan) containing 1.6 ml=7.8 IU insulin each.
Example 4
Preparation of Additional Actrapid.TM. Matrix Carrier
Composition
[0187] An additional Actrapid.TM. matrix carrier composition
(Formulation V) was produced, using the following ingredients:
[0188] Olive oil, 11 ml
[0189] Benefiber.TM., 3 g
[0190] Insulin Actrapid.TM., 9 ml
[0191] Oblepicha oil, 9 ml
[0192] Hydrophobic silica R972, 1.2 g
[0193] Sesame Oil up to 75 ml.
[0194] Benefiber.TM. (Novartis Nutrition GmbH, Germany) and silica
were placed into a beaker and mixed by vortexing at 900 rpm for 5
minutes. Association between the Benefiber.TM. and the silica was
determined by the mixture's ability to float after being placed on
the surface of a water-filled beaker. Actrapid.TM. insulin was
added and stirred for 15 minutes at 50 rpm. Sesame oil and sea
buckthorn (oblepicha) oil were combined in a beaker and vortexed on
a low setting for 15 minutes. Olive oil was added to the oils and
stirred with a glass rod. The solid phase mixture and oil mixture
were combined and mixed at 100 rpm with a magnetic stirrer. The
volume was brought up to 75 ml with sesame oil and stirred with a
glass rod. The product contained 12 IU/ml insulin and was packaged
into gelatin enteric covering capsules.
[0195] An additional insulin matrix carrier composition
(Formulation VI) was prepared using NovoRapid.TM. insulin, using
the above protocol. In this case NovoRapid.TM. insulin was used
instead of Actrapid.TM. insulin.
Example 5
Additional Insulin Matrix Carrier Composition
[0196] An additional insulin matrix carrier composition
(Formulation IV) was prepared using BIOCON insulin, using the
following protocol and the ingredients set forth in Table 1, using
methods similar to those set forth in previous Examples. A light
microscopy picture of the composition is shown in FIG. 3.
1. Mix oblepicha+olive oil+1/3 of sesame oil. 2. Add Insugen.TM.
insulin powder (BIOCON) into the mixture of oils and mix. 3. Mix
fiber+chitin+amylopectin+silica. 4. Add the mixture of step 3 to
the mixture of oils and insulin of step 2 and mix. 5. Add the rest
of the sesame oil and mix.
TABLE-US-00001 TABLE 1 Ingredients for the preparation of the
BIOCON matrix carrier composition. Formulation IV Insulin Powder
(BIOCON), mg 36.4 109.2 182 Olive Oil, ml 33 33 33 Sea Buckthorn
Oil (Oblepicha), ml 42 42 42 Sesame Oil, ml up to 100 up to 100 up
to 100 Amylopectin, g 11.25 11.25 11.25 Chitin, g 1.9 1.9 1.9
Silica R972, g 2.5 2.5 2.5 Final concentration of insulin, IU/ml 10
30 50
[0197] Next, an additional short-term release insulin matrix
carrier composition (Formulation A) was prepared using BIOCON
insulin, using the ingredients set forth in Table 2.
TABLE-US-00002 TABLE 2 Ingredients for the preparation of an
insulin matrix carrier composition (Formulation A). Ingredient
Amount Olive oil 20 ml Ambrotose* 3 g Insulin powder 70 mg Silica
R972 0.6 g Sea buckthorn (oblepicha) oil 26 ml Sesame oil up to 70
ml Ambrotose .TM. powder contains Arabinogalactan (a gum from the
Larix decidua tree), Manapol, which is a gel extracted from the
inner leaf of aloe vera gel plant, gum ghatti, and gum tragacanth
or Manapol powder, oat fiber, brown macroalgae (Undaria
pinnatifida) sporophyll, vegetarian glucosamine-HCl, ghatti gum,
gum tragacanth and xylitol.
Example 6
Efficacy of the Oral Insulin Composition of the Present Invention
in Diabetic Mice
Materials and Experimental Methods
[0198] Streptozotocin (STZ)-induced diabetes treatment: Diabetes
was induced by 2 injections of 500 and 700 .mu.l of 1.5 mg/ml
streptozotocin separated by 48 hr, in male adult BALB/c mice (7-10
weeks old) of an average weight of 23-28 gr. Untreated mice of
approximately the same age and weight were used as control. Blood
glucose levels (BGL) were assessed 48 hours after STZ injection by
a standard FreeStyle.TM. glucometer (Abbot Diabetes Cere Inc,
Alameda, Calif.) from the tail vein blood samples.
[0199] Insulin compositions were administered orally to mice by
gavage (1 ml volume), without prior deprivation of food or water.
During the experiment mice were supplied with food and water as
usual.
Compositions: The first experiment utilized Formulations V and VI
described in Example 4. The second experiment utilized Formulation
IV described in Example 5. Blood insulin concentration. Blood
insulin concentrations were detected by ELISA (Human Insulin ELISA
kit, Linco).
Treatment Groups:
[0200] 1. Control group 1: no STZ treatment, no insulin
administration. 2. Control group 2: no STZ treatment, oral insulin
composition of the present invention administered by gavage 3.
Control group 3: STZ treated, no insulin administration. 4.
Diabetic (STZ treated) mice; insulin administered by SC injection.
5. Diabetic (STZ treated) mice; insulin was administered by gavage.
6. Diabetic (STZ treated) mice; insulin was administered using the
oral insulin composition of the present invention by gavage. 7.
Diabetic (STZ treated) mice; matrix carrier (without insulin)
administered by gavage as control.
Results
[0201] In a first experiment, diabetes was induced by
streptozotocin (STZ) in male adult BALB/c mice, followed by
administration of NovoRapid.TM. (9.5 IU) and Actrapid.TM. (12 IU)
based insulin compositions of the present invention. Both
compositions significantly reduced blood glucose levels (FIGS.
4A-B, respectively). By contrast, STZ-treated mice that received
empty matrix carrier compositions (lacking insulin), orally
administered Actrapid.TM. or NovoRapid.TM. insulin, or were given
25 IU Insulin (BIOCON) in PBS (gavage) (FIG. 5) did not exhibit
significant reduction in blood glucose levels. Normal mice (not
STZ-treated) that received insulin compositions exhibited no
significant reduction in blood glucose level. Normal and diabetic
mice injected with insulin, by contrast, exhibited hypoglycemia
symptoms that were in some cases fatal.
[0202] In a second experiment, an insulin composition of the
present invention was administered orally to STZ-treated mice by
gavage (1 ml volume) in dosages ranging from 2-10 IU. A
dose-responsive reduction in blood glucose levels was observed for
9-12 hours; however, levels rarely dropped below 100 mg/dL (FIG.
6A-D). The presence of human insulin in the blood following
administration of the insulin matrix carrier composition was
confirmed by ELISA. By contrast, subcutaneous injection of 10 IU of
insulin caused near-fatal hypoglycemia (FIG. 6E). Normal mice
receiving 2, 5, or 10 IU insulin compositions exhibited only a
slight reduction in blood glucose level (FIG. 6F), while those
receiving injected insulin experienced a precipitous and
occasionally fatal drop in glucose levels. As before, STZ-treated
mice that received empty matrix carrier compositions (lacking
insulin), orally administered insulin, or were left untreated did
not exhibit significant blood glucose level reduction.
[0203] In another experiment, direct comparison of 10, 5, or 2 IU
of insulin matrix carrier composition (Formulation IV) vs.
injection of the same amount of insulin solution (a standard
formulation) in 14-25 g mice reveled that mice treated with the
oral insulin composition of the present invention maintained normal
blood glucose levels for longer periods of time compared to the
insulin injected mice. These observation reflect on the increased
bioavailability of insulin when administered within the matrix
carrier composition of the present invention. In addition, mice
administered the matrix carrier compositions had no hypoglycemia,
while the injected mice exhibited severe hypoglycemia (FIGS. 6G, H,
I for 10, 5, and 2 IU, respectively).
[0204] FIGS. 6J-K: We have compared the pharmacodynamics of
Formulation A (Example 5) and. Formulation IV (Example 5). Results
are described in FIG. 6J (2 IU of insulin) and FIG. 6J (7.5 IU of
insulin) according to which mice administered Formulation IV showed
lower blood glucose levels for longer periods of time as opposed to
mice administered Formulation A.
[0205] Indication for the increased bioavailability of the insulin
administered orally using the compositions of the present invention
in comparison with injected insulin can be found by calculating the
"Effective Areas". "Effective Area" is defined as the sum of the
net changes in blood glucose level (BGL) values relative to the
basal level, along a defined period of time, calculated as
follows:
1. Obtain a baseline average of BGL for each time point. 2. For
each time point, subtract the BGL value in the treated groups (oral
insulin of the present invention and injected insulin) from the
baseline average. 3. Sum the values obtained in step 2 for all time
points. To obtain the values in the table, the "effective areas" of
the different treatments were then subtracted or divided. Results
are depicted in Table 3.
TABLE-US-00003 TABLE 3 Effective Areas of insulin oral matrix
carrier compositions versus injected insulin. Group Value 10 IU:
(injection- matrix carrier composition) -1042.7 mg/dL 5 IU:
(injection- matrix carrier composition) 340.29 mg/dL 2 IU:
(injection- matrix carrier composition) 834.4 mg/dL 10 IU:
injection/matrix carrier composition 0.64 5 IU: injection/matrix
carrier composition 1.32 2 IU: injection/matrix carrier composition
3.73 10 IU injection/5 IU injection 1.32 10 IU injection/2 IU
injection 1.61 10 IU matrix carrier composition/5 IU matrix 2.74
carrier composition 10 IU matrix carrier composition/2 IU matrix
9.45 carrier composition
[0206] As shown in Table 2, the relatively low 10 IU/5 IU and 10
IU/2 IU ratios for injected insulin indicate that these doses are
approaching the saturation dose for the mice. By contrast, the
relatively large ratios for the insulin matrix carrier composition
indicate that it is far from the saturation doses. Thus, matrix
carrier compositions of the present invention are more amenable to
accurate dosing within their therapeutic range, compared with
standard injected insulin formulations.
Example 7
The Efficacy of the Oral Insulin Compositions of the Present
Invention in Human Subjects
[0207] The effect of an insulin composition of the present
invention was tested on two human subjects, one healthy and one
diabetic. 30 IU of Actrapid.TM. matrix carrier composition
(Formulation II) reduced blood glucose levels in the healthy
subject from 105 to 80-90 mg/dL over a six-hour test period (FIG.
7A). The subject reported an unusual degree of hunger, but
otherwise no adverse reactions. By contrast, administration of
injected insulin to healthy subjects is known to cause
hypoglycemia, in some cases severe, with accompanying adverse
reactions.
[0208] 10 IU of the same formulation V (Example 4) were
administered 3 times per day over 14 days to a 67-year-old subject
having type I/II diabetes, who exhibited glucose levels of over 170
when untreated and 130-170 when receiving Gluco-Rite.TM.. Upon
taking the oral insulin composition of the present invention the
blood glucose levels dropped to an average of about 130 (FIG. 7B).
The subject reported feeling well during the entire period of
receiving the oral insulin composition of the present invention.
The subject has continued to take the compositions 3-4 times per
day, as needed, resulting in well-controlled glucose levels with no
adverse reactions. By contrast, the subject had a long-standing
history of intense sensations of dread and unease after receiving a
number of different injected insulin formulations.
[0209] The presence of elevated insulin levels in the diabetic
subject's blood following administration of the insulin composition
was confirmed by ELISA.
[0210] Thus, insulin compositions of the present invention are
capable of orally delivering various forms of insulin in a
biologically active form that can effectively treat diabetes. They
have the additional advantage of not inducing hypoglycemia in
either diabetic or normal subjects.
Example 8
Toxicity Study of Chronic Oral Administration of Insulin
Compositions
Materials and Experimental Methods
[0211] Fifteen 10 week-old Balb/C male mice were used. Mice were
administered daily 1 ml of insulin matrix carrier composition (25
IU/ml) (experimental group) or PBS (gavage control group) via
gavage over 15 days. On the 14th and 15th day, mice were
administered orally 100 ng of lipopolysaccharides (LPS) together
with the insulin matrix carrier composition or PBS. Negative
control mice were administered 1 ml PBS by gavage over 13 days, and
100 ng of LPS in 1 ml PBS by gavage on the 14th and 15th day.
Positive control mice were injected with 1 mg of LPS in 1 ml of
PBS. 3 h after LPS administration, mice were sacrificed, blood was
collected (for LPS detection) and gastro-system, liver and kidneys
were fixed in paraformaldehyde (PFA) 4% for histological
analysis.
Animal follow up and macroscopic analysis: Mice were weighed every
3 days, and their fur condition was detected daily. After
sacrifice, all organs or tissues were investigated for the presence
of pathological changes. Internal organs collection and fixation:
On day 15 mice were sacrificed, and their gastro-system, kidney,
and liver were collected from the abdominal cavity, weighed and
fixed in 10% formalin solution. Blood collection and plasma
preparation: Blood was collected from the heart of the mice into
tubes that contain EDTA. Plasma was be separated from the blood by
centrifugation; at first for 15 min at 3000.times.gmax followed by
15 min at 16,000.times.gmax. Supernatant was removed, placed in a
new tube and stored at -20.degree. C. Detection of LPS in mouse
serum: LPS in mouse serum was detected by HPLC. Sera taken on day
15 from the groups described above were prepared for HPLC analysis
by addition of 0.1M of EDTA.
Results
[0212] The toxicity of chronic administration of oral insulin
compositions of the present invention was investigated. No
pathological changes were observed in the animals' behavior. Their
fur was in normal condition, appearing smooth, clean, and bright.
No weight loss was detected; the mice gained weight normally.
Microscopic and Macroscopic Organ Analysis:
[0213] Microscopic analysis showed no evidence of pathology in
tissues of mice in all groups (liver--FIG. 8A; kidney--FIG. 8B;
duodenum--FIG. 8C). In addition, macroscopic analysis of internal
organs revealed no evidence of any pathology. Organs of all mice
were normally developed, had normal size, shape, appearance (bright
and smooth), and weight, were normally colored, and were in their
normal location. Livers were in all cases situated under diaphragm
and smooth and bright, and weights were about 1.6 g. Liver
parenchyma were dark colored. Kidneys exhibited a smooth surface.
Weight was consistently about 0.2 gr. Esophagus, stomach, and large
and small intestine exhibited normal size and location, and
pillories and duodenum were open.
[0214] Serum from the treated and untreated mice were also tested
for the presence of LPS. LPS was not present in serum of mice given
oral insulin composition of the present invention+LPS nor in serum
of mice given 1 ml PBS+LPS, showing that neither the matrix carrier
composition nor the gavage compromised the integrity of the mice's
gastrointestinal linings. Serum from mice injected 1 mg of LPS in 1
ml of PBS served as a positive control, and untreated mice served
as negative control.
[0215] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying current knowledge, readily modify and/or adapt for
various applications such specific embodiments without undue
experimentation and without departing from the generic concept,
and, therefore, such adaptations and modifications should and are
intended to be comprehended within the meaning and range of
equivalents of the disclosed embodiments. It is to be understood
that the phraseology or terminology employed herein is for the
purpose of description and not of limitation. The means, materials,
and steps for carrying out various disclosed functions may take a
variety of alternative forms without departing from the
invention.
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