U.S. patent application number 13/128800 was filed with the patent office on 2012-03-15 for inhibition of mammalian target of rapamycin.
This patent application is currently assigned to The Board of Regents of the University of Texas System. Invention is credited to Veronica Galvan, Salvatore Oddo, Zelton Dave Sharp, John R. Strong, Herbert G. Wheeler.
Application Number | 20120064143 13/128800 |
Document ID | / |
Family ID | 42170672 |
Filed Date | 2012-03-15 |
United States Patent
Application |
20120064143 |
Kind Code |
A1 |
Sharp; Zelton Dave ; et
al. |
March 15, 2012 |
INHIBITION OF MAMMALIAN TARGET OF RAPAMYCIN
Abstract
Disclosed are microcapsules that include an inhibitor of the
mammalian target of rapamycin (mTOR) within the microcapsules, and
pharmaceutical compositions and kits that include the
microcapsules. Also disclosed are methods for treating or
preventing an age-related disease, condition, or disorder in a
subject that involve administering to a subject a pharmaceutically
effective amount of microcapsules that includes an inhibitor of
mTOR within the microcapsules.
Inventors: |
Sharp; Zelton Dave; (San
Antonio, TX) ; Strong; John R.; (San Antonio, TX)
; Galvan; Veronica; (San Antonio, TX) ; Oddo;
Salvatore; (San Antonio, TX) ; Wheeler; Herbert
G.; (Boerne, TX) |
Assignee: |
The Board of Regents of the
University of Texas System
Austin
TX
|
Family ID: |
42170672 |
Appl. No.: |
13/128800 |
Filed: |
November 11, 2009 |
PCT Filed: |
November 11, 2009 |
PCT NO: |
PCT/US09/64044 |
371 Date: |
November 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61113481 |
Nov 11, 2008 |
|
|
|
Current U.S.
Class: |
424/439 ;
424/400; 514/291 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 9/5026 20130101; A61P 25/28 20180101; A61P 39/06 20180101;
A61P 35/00 20180101; A61K 9/1635 20130101; A61K 31/436 20130101;
A61K 31/00 20130101; A61K 31/436 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/439 ;
424/400; 514/291 |
International
Class: |
A61K 31/436 20060101
A61K031/436; A61P 25/28 20060101 A61P025/28; A61P 35/00 20060101
A61P035/00; A61K 9/00 20060101 A61K009/00 |
Goverment Interests
[0002] The United States government owns certain rights in the
present invention pursuant to grant numbers AG 029729 and AG022307
from the National Institutes of Health.
Claims
1-45. (canceled)
46. A method for orally administering rapamycin to a subject in
need thereof comprising administering to the subject a composition
comprising rapamycin in combination with a hydrophilic, swellable,
hydrogel forming material, wherein said composition is encased in a
coating that includes a water insoluble polymer and a hydrophilic
water permeable agent.
47. The method of claim 46, wherein the water insoluble polymer is
a methyl methacrylate-methacrylic acid copolymer.
48. The method of claim 46, wherein the subject is a mammal.
49. The method of claim 46, wherein the subject is a human.
50. The method of claim 46, wherein the subject in need thereof is
known or suspected to have an age-related disease.
51. The method of claim 50, wherein the age-related disease is
Alzheimer's disease or cancer.
52. The method of claim 50, wherein the subject is a human greater
than age 50.
53. The method of claim 46, wherein the composition reduces
age-related decline in cognition in the subject.
54. The method of claim 46, wherein the rapamycin containing
composition is comprised in a food or food additive.
55. The method of claim 46, wherein the rapamycin containing
composition comprises 25% to 60% by weight of rapamycin.
56. The method of claim 55, wherein the average blood level of
rapamycin in the subject is greater than 25 ng/ml after
administration of the composition.
57. A method for treating or delaying onset of an age-related
disease or prolonging the lifespan of a mammalian subject in need
thereof comprising administering to the subject an effective amount
of a pharmaceutical or nutraceutical composition comprising
rapamycin in combination with a hydrophilic, swellable, hydrogel
forming material, wherein the composition is encased in a coating
that includes a water insoluble polymer and a hydrophilic water
permeable agent, and wherein the age-related disease is treated,
the onset of age-related disease is delayed or the lifespan of the
subject is prolonged.
58. The method of claim 57, wherein the water insoluble polymer is
a methyl methacrylate-methacrylic acid copolymer.
59. The method of claim 57, wherein the subject in need thereof is
known or suspected to have an age-related disease.
60. The method of claim 57, wherein the subject in need thereof is
known to have an age-related disease.
61. The method of claim 60, wherein the age-related disease is
Alzheimer's disease or cancer.
62. The method of claim 57, wherein the composition comprises 25%
to 60% by weight of rapamycin, wherein the average blood level of
rapamycin in the subject is greater than 25 ng/ml after
administration of the composition, and wherein the subject is a
human greater than age 50.
63. A method for orally administering rapamycin to a subject in
need thereof comprising administering to the subject a composition
comprising 25% to 60% by weight of rapamycin in combination with a
hydrophilic, swellable, hydrogel forming material, wherein said
composition is encased in a coating that prevents release and
absorption of said composition until reaching the small intestines
of said subject, wherein said coating includes a water insoluble
polymer and a hydrophilic water permeable agent and the average
blood level of said rapamycin in said subject is greater than 25
ng/ml, and wherein said subject is a human greater than age 50.
64. A pharmaceutical or nutraceutical composition comprising
rapamycin in combination with a hydrophilic, swellable, hydrogel
forming material, wherein the composition is encased in a coating
that includes a water insoluble polymer and a hydrophilic water
permeable agent.
65. The pharmaceutical or nutraceutical composition of claim 64,
wherein the water insoluble polymer is a methyl
methacrylate-methacrylic acid copolymer.
66. The pharmaceutical or nutraceutical composition of claim 64,
wherein the composition comprises 25% to 60% by weight of rapamycin
or an analog thereof.
Description
[0001] The present application claims the benefit of the filing
date of U.S. Provisional Patent Application Ser. No. 61/113,481,
filed Nov. 11, 2008, the contents of which is herein incorporated
by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the fields of
pharmacology and the treatment and prevention of age-related
disorders. More specifically, the invention relates to
microcapsules that include an inhibitor of the mammalian target of
rapamycin (mTOR), and methods of treating or preventing age-related
diseases, disorders, and conditions in a subject using
microcapsules of the present invention.
[0005] 2. Description of Related Art
[0006] Because most deaths in developed nations result from
diseases whose incidences rise rapidly with age, interventions that
delay aging would benefit human health far more than would
preventive measures that affect only specific late-life diseases
such as heart disease, cancer or diabetes. There is intense
interest in the development of dietary additives that delay aging
and increase lifespans.
[0007] mTOR and cancer. Mammalian TOR is a critical effector in the
deregulated signaling pathways associated with cancer (Guertin and
Sabatini, 2007; Shaw and Cantley, 2006). Mutations in tsc1 or tsc2
genes, which lead to the hamartomatous syndrome tuberous sclerosis
complex (TSC), suggest a molecular connection between mTOR and
cancer. mTORC1 is the only known downstream effector common to two
of the major signaling pathways in cancer (Ras and PI3K), and which
is also integrated with nutrient signaling for regulation of cell
growth (mass) (Shaw and Cantley, 2006). Hyperactivated AKT
signaling likely mediates oncogenic transformation via mTOR (Skeen
et al., 2006).
[0008] It has been suggested that a major mTORC1 effector, S6
kinase 1 (S6K1), mediates deleterious effects such as insulin
resistance and type II diabetes (Patti and Kahn, 2004; Tremblay et
al., 2005b; Tremblay et al., 2005c; Um et al., 2006). Compared to
wild type, S6K1-deficient mice demonstrated a reduced rate of
growth including less white adipose tissue (WAT) due to smaller
cells (Shima et al., 1998). Interestingly, the phenotype of mice
deficient for S6K1 includes hypoinsulinemia coupled with increased
sensitivity to insulin (Um et al., 2004). Because of increased
lipolysis and metabolic rate, these mice appear to be resistant to
diet-induced obesity (Um et al., 2004). In muscle cells deficient
for S6K1 function, there is an increase in AMP and inorganic
phosphates, and a consequent increase in activated AMPK and
AMPK-dependent functions including mitochondrial biogenesis and
fatty acid -oxidation (Aguilar et al., 2007). Concomitant with this
response, there is also a decrease in lipid content of cells.
[0009] Rapamycin has been shown to act as a potent inhibitor of
adipocyte differentiation, an effect reversed by high FK506
concentrations, indicating an operative inhibitory effect mediated
by an immunophilin-rapamycin complex (Yeh et al., 1995). A model
for the critical role of mTOR and its kinase activity in 3T3-L1
preadipocyte differentiation has been proposed, wherein the mTOR
pathway and the phosphatidylinositol 3-kinase/Akt pathway act in
parallel during adipogenesis by mediating respectively nutrient
availability and insulin signals (Kim and Chen, 2004).
[0010] There is the need for more effective treatments of
age-related diseases and the need for a greater understanding of
agents that may increase lifespan and delay the appearance of
age-related disease.
SUMMARY OF THE INVENTION
[0011] The present invention is based in part of the finding that a
physiological state similar to food and/or growth factor
restriction, with retarded aging and reduced incidence of
age-related diseases, can be achieved in mammals, including humans,
by chronically blocking a central protein complex in the nutrient
sensing and growth factor-responding pathway called the mammalian
target of rapamycin (mTOR) by formulations of an inhibitor of mTOR
in a formulation that is encapsulated. For example, the inventors
have found that microencapsulated rapamycin fed late in life
extends lifespan in genetically heterogenous mice. Further,
microencapsulated rapamycin has been found to rescue cognition and
attenuate the pathology in mouse models of Alzheimer disease.
Microencapsulation improves therapeutic efficacy compared to
formulations that are not encapsulated. Chronic inhibition of mTOR
can be applied in improving the health and well being of
individuals, including mature adults, by ameliorating several major
categories of age-dependent diseases, thereby increasing the
quality and quantity of the productive years of life while
providing significant economic benefit.
[0012] Some embodiments of the present invention concern
microcapsules that include a core component that includes an
inhibitor of mTOR, wherein the core component is encased in a
coating. The inhibitor of mTOR may be an inhibitor of mammalian
target of rapamycin complex 1 (mTORC1) or an inhibitor of mammalian
target of rapamycin complex 2 (mTORC2). In particular embodiments,
the coating provides for delayed release of the inhibitor of mTOR
and/or preferential release of the therapeutic agent in the
intestinal tract of a subject (i.e., an enteric coating). The
enteric coating may be any such coating known to those of ordinary
skill in the art. Non-limiting examples of such coatings include
Eudragit S100, cellulose acetate phthalate (CAP), a methyl
acrylate-methacrylic acid copolymer, cellulose acetate succinate,
hydroxy propyl methyl cellulose phthalate, polyvinyl acetate
phthalate (PVAP), or a methyl methacrylate-methacrylic acid
copolymer. In some particular embodiments, the coating includes
Eudragit S100. The coating may include a mixture of one or more of
Eudragit S100, cellulose acetate phthalate (CAP), a methyl
acrylate-methacrylic acid copolymer, cellulose acetate succinate,
hydroxy propyl methyl cellulose phthalate, polyvinyl acetate
phthalate (PVAP), and a methyl methacrylate-methacrylic acid
copolymer
[0013] A "microcapsule" as used herein is defined as a vehicle for
delivery of a therapeutic agent to a subject which includes one or
more cores, where the core(s) are encased in a coating as set forth
above. In particular embodiments, the microcapsule includes a
single core that is encased in a coating. In further embodiments,
the microcapsule includes a plurality of cores encased in a coating
where the cores with surrounding coating are aggregated together to
form a single drug delivery structure. The core may be a solid or
it may be a liquid, and its state may depend upon ambient
temperature.
[0014] The microcapsules may be of any size or shape. Basic
geometrical shapes may be, for example, spheres, rods, cylinders,
cubes, cuboids, prism, pyramids, cones, truncated cones and
truncated pyramids. Star extrudates, cross extrudates, ribbed
extrudates and trilobes are furthermore suitable. Cavities, such as
incorporated tubes, may be incorporated into the microcapsule.
[0015] The microcapsules may be of regular shape or may have be
irregular in shape. The surface of the microcapsule may be smooth,
uneven, or jagged. They may be amorphous, spherical, or acicular in
shape, depending on the respective method of production. The
microcapsules may be formed using any method known to those of
ordinary skill in the art. Non-limiting examples of such methods
are discussed in greater detail below. In a single dosage that
includes microcapsules, the microcapsules may be of uniform size
and shape, or may be of variables sizes and shapes.
[0016] The microcapsules may be of any size. For example, the
maximum diameter of the microcapsule may be about 100 nm, 1 .mu.m,
10 .mu.m, 50 .mu.m, 100 .mu.m, 200 .mu.m, 300 .mu.m, 400 .mu.m, 500
.mu.m, 600 .mu.m, 700 .mu.m, 800 .mu.m, 900 .mu.m, 1 mm, 1.5 mm,
2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, 4.5 mm, 5.0 mm, 5.5 mm, 6.0
mm, 6.5 mm, 7.0 mm, 7.5 mm, 8.0 mm, 8.5 mm, 9.0 mm, 9.5 mm, 1.0 cm
or greater, or any range of maximum diameters derivable within the
aforementioned maximum diameters. For example, the maximum diameter
of the microcapsule may range from about 100 nm to about 1.0 cm. In
more particular embodiments, the mean diameter ranges from about
100 .mu.m to about 1 mm. In further embodiments, the mean diameter
ranges from about 100 .mu.m to about 0.1 mm.
[0017] The microcapsule may comprise at least 5%, at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 80%, at least 90%, at least 95%, at least
99% or more of an mTOR inhibitor by weight (w/w).
[0018] The inhibitor of mTOR may be rapamycin or a rapamycin
analog. In particular embodiments, the mTOR inhibitor is rapamycin.
In more particular embodiments, the mTORC1 is rapamycin and the
coating is Eudragit S100. In some embodiments, the inhibitor of
mTOR is a competitive inhibitor of the mTOR kinase. These interact
directly with the mTOR kinase and do not rely on an intracellular
receptor like FKBP12.
[0019] Any rapamycin analog known to those of ordinary skill in the
art is complated for inclusion in the microcapsules of the present
invention. Non-limiting examples of rapamycin analogs include
everolimus, tacrolimus, CCI-779, ABT-578, AP-23675, AP-23573,
AP-23841, 7-epi-rapamycin, 7-thiomethyl-rapamycin,
7-epi-trimethoxyphenyl-rapamycin, 7-epi-thiomethyl-rapamycin,
7-demethoxy-rapamycin, 32-demethoxy-rapamycin,
2-desmethyl-rapamycin, or 42-O-(2-hydroxy)ethyl rapamycin. Numerous
other examples of rapamycin analogs are discussed in the
specification below. The microcapsules of the present invention may
include rapamycin and one or more rapamycin analogs, or may include
more than one type of rapamycin analog.
[0020] In some embodiments, the microcapsules of the present
invention include one or more pharmaceutical or nutraceutical
agent. Non-limiting examples of such agents include a vitamin, an
herbal agent (such as ginkgo biloba or green tea), fish oil (omega
3 fatty acids), an antimicrobial agent, an antioxidant, a drug, or
an anti-inflammatory agent. For example, the core component may
include a second compound that is vitamin E, vitamin A, an
antibacterial antibiotic, an antioxidant, L-carnitine, lipoic acid,
metformine, resveratrol, leptine, a non-steroid anti-inflammatory
drug, a COX inhibitor, vitamin D, a mineral such as magnesium,
calcium, zinc or potassium, a trace element such as molybdenum or
iodine, a carotenoid (such as vitamin A), an enzyme such as lipase
or amylase, or an amino acid (such as lysine, arginine, taurine, or
proline). The drug may be an agent that is known or suspected to be
of benefit in treating or preventing an age-related disease,
disorder, or condition. For example, the drug may be an agent that
is known or suspected to be of benefit in the treatment or
prevention of a neurodegenerative disease, memory loss, abnormal
glucose metabolism, or cancer. Non-limiting examples of such agents
are discussed in the specification below. The core and/or coating
of the microcapsules set forth herein may include one or more
adjunct materials, such as carriers, binders, and the like that are
well-known to those of ordinary skill in the art.
[0021] In some embodiments, the microcapsule consists essentially
of a core componet that comprises rapamycin or a rapamycin analog,
wherein the core component is encased in a coating. The coating may
be any of the coatings discussed above, and the rapamycin analog
may be any of the rapamycin analogs discussed above. Non-limiting
examples of coatings include Eudragit S100, cellulose acetate
phthalate (CAP), a methyl acrylate-methacrylic acid copolymer,
cellulose acetate succinate, hydroxy propyl methyl cellulose
phthalate, polyvinyl acetate phthalate (PVAP), or a methyl
methacrylate-methacrylic acid copolymer. In particular embodiments,
the coating is Eudragit S100. In more particular embodiments, the
coating is Eudragit S100 and the core includes rapamycin. The
microcapsule may include one or more adjunct materials as discussed
above.
[0022] The core may include one or more additional components other
than one or more inhibitors of mTOR. For example, the core may
include the diluents are selected from the group comprising
mannitol, lactose, microcrystalline cellulose, dicalcium phosphate,
starch, pregelatinized starch, sorbitol or mixtures thereof. The
core may include a disintegrant such as sodium starch glycolate,
croscarmellose sodium, crospovidone, starch or mixtures thereof.
The core may include a binder such as hydroxypropyl cellulose,
hydroxy ethyl cellulose, ethyl cellulose, hydroxypropyl
methylcellulose, methylcellulose or mixtures thereof. The core may
include a lubricant such as calcium stearate, magnesium stearate,
sodium stearyl fumarate, talc, colloidal silicon dioxide or
mixtures thereof.
[0023] Other embodiments of the present invention concern
pharmaceutical or nutraceutical compositions for treating or
preventing an age-related disease, condition, or disorder that
include a microcapsule that includes a core component comprising an
inhibitor of mTOR, wherein the core component is encased in a
coating. The microcapsule may be any of the microcapsules of the
present invention. The pharmaceutical compositions set forth herein
may include one or more pharmaceutically acceptable agents, many of
which are well-known to those of ordinary skill in the art.
[0024] In some embodiments, the microcapsules are formulated with a
edible substance. The edible substance may be a food or food
additive. The composition may optionally include one or more
additional agents that can be applied in the treatment or
prevention of any disease, disorder, or health-related condition.
For example, the disease may be an age-related disease, such as a
neurodegenerative disease, abnormal glucose metabolism, or cancer.
The compositions may be formulated with one or more nutraceutical
agents, many of which are well-known to those of ordinary skill in
the art. For example, the nutraceutical agent may be a vitamin, a
nutritional supplement, or an agent derived from herbs or plants
that is known or suspected to be of benefit in promoting health and
well-being of a subject.
[0025] The present invention also concerns methods for treating or
preventing an age-related disease, condition, or disorder in a
subject, involving administering to a subject a pharmaceutically
effective amount of microcapsules of the present invention. The
present invention also concerns use of the microcapsules of the
present invention to treat or prevent an age-related disease,
condition, or disorder in a subject. The subject may be any
subject, such as a mammal. Non-limiting examples of mammals include
mice, rats, rabbits, dogs, cats, cows, sheep, horses, goats,
primates, and humans. In particular embodiments, the subject is a
human. The human may be a human who is known or suspected to have
an age-related disease. In some embodiments, the human is a human
greater than age 50, greater than age 55, greater than age 60,
greater than age 65, greater than age 70, greater than age 75, or
greater than age 80.
[0026] The age-related disease, condition, or disorder can be any
disease, condition, or disorder where the prevalence increases with
age. Non-limiting examples of age-related diseases include a
neurodegenerative disease, a disease associated with abnormal
glucose metabolism, and cancer. With respect to cancer,
non-limiting examples include breast cancer, lung cancer, prostate
cancer, ovarian cancer, brain cancer, liver cancer, cervical
cancer, colon cancer, renal cancer, skin cancer, head and neck
cancer, bone cancer, esophageal cancer, bladder cancer, uterine
cancer, lymphatic cancer, stomach cancer, pancreatic cancer,
testicular cancer, lymphoma, and leukemia. Non-limiting examples of
neurodegenerative diseases include Alzheimer disease, amyotrophic
lateral sclerosis (ALS), presenile dementia, senile dementia,
Parkinson's disease, Huntington's disease, and memory loss
associated with aging.
[0027] Other examples of age-related diseases, conditions, or
disorders contemplated for treatment or prevention using
microcapsules of the present invention include insulin resistance,
benign prostatic hyperplasia, hearing loss, osteoporosis,
age-related macular degeneration, a skin disease, or aging skin, a
skin disease, aging skin, sarcopenia, cardiovascular disease,
lipid/carbohydrate metabolism, cancer, and immune disease. The
microcapsules set forth herein may be administered to improve life
span, improve quality of life, reduce risk of oxidative damage and
cell senescence.
[0028] The subject may have an existing age-related disease,
condition or disorder, or the subject may be at risk of developing
an age-related disease, condition or disorder. The at-risk subject
may be a subject who has previously received treatment for an
age-related disease, condition, or disorder, where the disease,
condition, or disorder has previously been successfully treated.
The subject may be at risk because of other risk factors, such as
genetic risk factors or environmental risk factors.
[0029] The present invention also concerns a method of prolonging
the lifespan of a mammalian subject that involves administering to
a subject an effective amount of microcapsules of the present
invention, wherein lifespan is prolonged. Prolongation of lifespan
as used herein refers to a greater lifespan of the subject than the
subject would otherwise live in the absence of the microcapsules of
the present invention. An estimate of the lifespan the subject
would have otherwise lived in the absence of the microcapsules can
be obtained, for example, from demographic studies, Social Security
Administration Life Tables, and scientific literature concerning
lifespan. The present invention further concerns methods of
reducing the age-related decline in cognition in a mammalian
subject that involves administering to the subject an effective
amount of microcapsules of the present invention, wherein the
age-related decline in cognition is reduced. Reduction in
age-related decline of cognition may be assessed by comparing
cognition of the subject to a known index of cognition obtained
from a control subject or subjects.
[0030] The microcapsules may be administered using any method known
to those of ordinary skill in the art. Non-limiting examples of
routes of administration include orally, by nasogastric tube,
rectally, intraperitoneally, topically, subcutaneously,
intravenously, intraarterially, intramuscularly, via lavage, and
intrathecally. In some embodiments, the microcapsules are
administered by combining the microcapsules with a composition that
includes an edible substance.
[0031] The dose of microcapsules that is administered may be
determined by a practitioner using any method known to those of
ordinary skill in the art. In some embodiments, the dose of the
inhibitor of mTOR is about 1 microgram to about 100 mg per kg body
of the subject. Additional information concerning dosage regimens
is discussed in the specification below.
[0032] Other aspects of the present invention concern methods of
making a microcapsule that includes an inhibitor of mTOR that
involves applying a pharmaceutical coating to a core particle
comprising an inhibitor of mTOR, wherein the core particle becomes
coated with the coating. The coating may be an enteric coating. The
coating may be any of the coatings discussed above and elsewhere in
this specification. In specific embodiments, the coating is
Eudragit S100.
[0033] Any method known to those of ordinary skill in the art can
be used to apply the coating to the particle. In specific
embodiments, applying an enteric coating involves use of a spinning
disk atomizer, other methods may include pan coating,
air-suspension coating, centrifugal extrusion, vibrational nozzle,
spray-drying, interfacial polymerization, in situ polymerization,
matrix polymerization
[0034] Further aspects of the present invention concern kits that
include a first sealed container that includes a microcapsule or
microcapsules of the present invention. The kit may include a first
sealed container that includes any of the microcapsules of the
present invention. In some embodiments, the kit further includes
instructions for use of the microcapsules of the present invention.
In some embodiments, the kit further includes a second compound.
The second compound may be comprised in the first sealed container,
or may be comprised separately such as in a second sealed
container.
[0035] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
all modifications or alternatives equivalent thereto are within the
spirit and scope of the invention as set forth in the appended
claims. All references cited herein are incorporated by reference
in their entirety, for all purposes.
[0036] It is specifically contemplated that any limitation
discussed with respect to one embodiment of the invention may apply
to any other embodiment of the invention. Furthermore, any
composition of the invention may be used in any method of the
invention, and any method of the invention may be used to produce
or to utilize any composition of the invention.
[0037] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternative are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or."
[0038] Throughout this application, the term "about" is used to
indicate that a value includes the standard deviation of error for
the device and/or method being employed to determine the value.
[0039] As used herein the specification, "a" or "an" may mean one
or more, unless clearly indicated otherwise. As used herein in the
claim(s), when used in conjunction with the word "comprising," the
words "a" or "an" may mean one or more than one. As used herein
"another" may mean at least a second or more.
[0040] Any embodiment of any of the present medical devices,
perfusion systems, and kits may consist of or consist essentially
of--rather than comprise/include/contain/have--the described
features and/or steps. Thus, in any of the claims, the term
"consisting of" or "consisting essentially of" may be substituted
for any of the open-ended linking verbs recited above, in order to
change the scope of a given claim from what it would otherwise be
using the open-ended linking verb.
[0041] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0042] The following figures form part of the present specification
and are included to further demonstrate certain aspects of the
present invention. The invention may be better understood by
reference to one or more of these drawings in combination with the
detailed description of specific embodiments presented herein.
[0043] FIG. 1. Survival plots for male (left) and female (right)
mice, comparing control mice to those fed enalapril, CAPE or
rapamycin pooling across the three test sites. Enalapril and CAPE
were added to the diet at 4 months of age, and rapamycin at 20
months. P-values were calculated by the log-rank test.
[0044] FIG. 2. Survival plots for male and female mice, comparing
control mice to those fed rapamycin in the diet starting at 600
days of age, pooling across the three test sites. P values were
calculated by the log-rank test. Four percent of the control mice
and three percent of rapamycin-assigned mice were removed from the
experiment for technical reasons. Only five animals (three
controls, two rapamycin) were removed after the start of rapamycin
treatment at 600 days. Thus, there was no significant differences
between groups in censoring.
[0045] FIG. 3. Survival of control and rapamycin-treated mice for
males and females for each of the three test sites separately. P
values represent results of log-rank calculations. Vertical lines
at age 600 days indicate the age at which the mice were first
exposed to rapamycin.
[0046] FIG. 4A, 4B, 4C. Characterization of mice receiving
rapamycin from 270 days of age. A, Survival plots for male and
female mice, comparing control mice to rapamycin-treated mice of a
separate (Cohort 2006) population, in which mice were treated with
rapamycin from 270 days of age. Because at the time of the interim
analysis all live mice were between 800 and 995 days of age, only
limited information about the shape of the survival curve at ages
above 900 days, and the apparent change in slope at the oldest ages
(>990 days) reflects this experimental uncertainty. P values
were calculated by the log-rank test. B, Effects of dietary
rapamycin on an mTOR effector in the visceral fat pads from
750-day-old to 880-day-old male and female mice. Ribosomal subunit
protein S6 (rpS6) and its phosphorylation status (P-rpS6, double
arrow) were immunoassayed in tissue lysates prepared from mice
consuming microencapsulated rapamycin-containing or control diets.
Antibodies used are shown to the left. The ratio of intensity
values for P-rpS6/rpS6 is shown in the graphs for female and male
mice. Pan-actin was also immunoassayed in the blots to provide an
indication of protein loading for each lane. C, Whole blood
rapamycin content in 750-day-old to 880-day-old male and female
mice. In B and C, error bars show standard errors of the mean.
[0047] FIG. 5. Reduced P-rpS6(Ser240/244) in White Adipose
Tissue.
[0048] FIG. 6. Reduced P-rpS6(Ser240/244) in Liver.
[0049] FIG. 7. No detectable effects on P-rpS6(Ser240/244) in
brain.
[0050] FIG. 8. Increase in 4E-BP1 in male white adipose tissue.
[0051] FIG. 9. No detectable effect on 4E-BP1 in liver.
[0052] FIG. 10. Akt activation in male white adipose tissue.
[0053] FIG. 11. Akt activation in male liver.
[0054] FIG. 12. Akt activation in brain.
[0055] FIG. 13. Stability of rapamycin in food.
[0056] FIG. 14. Encapsulation of rapamycin improves stability in
laboratory chow. Rapamycin was added to commercially prepared lab
chow at 7 ppm and the food was then assayed for rapamycin content.
Rapamycin levels are less than expected, suggested that rapamycin
degraded during preparation or storage of the food (open bar).
Microencapsulation of the rapamycin reduced degradation (shaded
bar).
[0057] FIG. 15. Rapamycin is detectable in whole blood after
feeding diet containing encapsulated or unencapsulated rapamycin.
Encapsulated and unencapsulated rapamycin (7 ppm) was fed to mice
for 3 weeks and the blood assayed for rapamycin levels.
Encapsulation resulted in significantly higher blood levels of
rapamycin than observed using unencapsulated rapamycin.
[0058] FIG. 16. Microencapsulation.
[0059] FIG. 17. Levels of rapamycin in blood.
[0060] FIG. 18. Reduced mTOR signaling in calorie-restricted
mice.
[0061] FIG. 19. No effect of rapamycin on body weight.
[0062] FIG. 20. Rapamycin attenuates age-related decline in general
locomotor activity.
[0063] FIG. 21. No significant effect on adiposity in mice fed
rapamycin from 9 months of age.
[0064] FIG. 22. Effect of caloric restriction on lifespan.
[0065] FIG. 23. Visceral fat pad P-Ser473 Akt analysis: 20 months
of treatment.
[0066] FIG. 24. Gastrocnemius muscle P-Ser473 Akt analysis.
[0067] FIG. 25. No difference in body weight with or without
rapamycin in mice on a high fat diet 12 weeks of feeding.
[0068] FIG. 26. Rapamycin causes glucose intolerance in HET3 mice
fed a high fat diet.
[0069] FIG. 27. Effects of increasing dietary fat or calories on
rapamycin effects on glucose metabolism.
[0070] FIG. 28A, 28B, 28C, 28D. Rapamycin abrogates memory deficits
in the 3.times.Tg-AD and the hAPP(J20) mouse models of AD. A and C,
The mean latencies in reaching a hidden platform were significantly
decreased for rapamycin-fed 3.times.Tg-AD and hAPP(J20) mice with
respect to control-fed Tg groups (*P<0.044; and *P=0.036
respectively). Learning was effective in both hAPP(J20) and
3.times.Tg-AD groups [F(3,120)=10.29, P<0.0001 and
F(4,220)=16.95, P<0.0001 respectively]. No significant
interaction was observed between the day number and genotype; thus,
genotype had roughly the same effect at all times during training B
and D, Retention of the former platform site was impaired in
control-fed 3.times.Tg-AD and hAPP(J20) mice [P<0.01 and
P<0.001, Tukey's multiple comparisons test applied to a
significant effect of genotype (P=0.01 and P<0.0001
respectively) in one-way ANOVA], but was not significantly
different from that of non-Tg groups for rapamycin-fed
3.times.Tg-AD and hAPP(J20) animals. Data are mean.+-.SEM.
[0071] FIG. 29A, 29B, 30C, 29D, 29E, 29F, 29G, 29H, 29I. Rapamycin
decreases A.beta..sub.42 levels and deposition. A and B,
Representative Western blots from proteins extracted from brains of
3.times.Tg-AD and hAPP(J20) mice, respectively. C, D, and E,
Quantitative analyses of APP, C99 and C83 (normalized to
.beta.-actin levels) show that rapamycin had no significant effect
on APP processing in both transgenic lines. F and G, ELISA
measurements indicate that rapamycin did not alter As40 levels in
the brains of the 3.times.Tg-AD (f; P=0.89) or hAPP(J20) mice (G;
P=0.29). In contrast, rapamycin significantly decreased soluble
As42 levels in 3.times.Tg-AD and hAPP(J20) mice (P=0.02 and 0.04,
respectively). H and I, Representative microphotographs depicting
CA1 pyramidal neurons of the 3.times.Tg-AD mice stained with an
anti-As42 antibody. Statistical evaluations were conducted using a
two-tailed unpaired Student's t test.
[0072] FIG. 30A, 30B, 30C, 30D, 30E, 30F, 30G, 30H, 30I. Rapamycin
administration significantly decreases tau pathology in the
3.times.Tg-AD mice. A and B, Representative microphotographs of CA1
pyramidal neurons stained with the anti-tau antibody AT270, which
recognizes tau phosphorylated at Thr181, clearly indicate a
decrease in AT270 immunoreactivity in mice treated with rapamycin.
C and D, Higher magnification views of panels A and B respectively.
E and F, Serial sections to those shown above were stained with the
conformational-specific antitau antibody, MC1. While 8 month-old
3.times.Tg-AD mice begin to show MC1-positive inclusions in some
hippocampal neurons (E), we were unable to detect any MC1-positive
inclusions in brain of rapamycin-treated 3.times.Tg-AD mice. G,
Representative Western blots of protein extracted from brains of
3.times.Tg-AD mice and probed with the phospho-specific anti-tau
antibody, AT270 and with f -actin as a loading control. H,
Quantification analyses of the blots in panel G indicate that
rapamycin significantly reduced the steady-state levels of
phosphorylated tau at Thy181 (P=0.006). I, ELISA measurements show
that the levels of soluble tau were significantly reduced in the
brain of rapamycin-treated mice (P=0.01). No changes were detected
for insoluble tau levels (P>0.05). Statistical evaluations were
done using two-tailed unpaired Student's t-test and one-way ANOVA
for AT270 immunoreactivity levels and for ELISA determinations
respectively. Scale bar is 12.5 .mu.m for panels A, B, E and F; 100
.mu.m for panels C, D.
[0073] FIG. 31A, 31B, 31C, 31D, 31E, 31F. Rapamycin administration
increases autophagy in brain of hAPP(J20) and 3.times.Tg-AD mice.
A, Representative Western blots of proteins extracted from brains
of 3.times.Tg-AD mice. B, E, Quantification analyses (data are
normalized to s-actin) indicate that rapamycin significantly
increased the steady-state levels of ATG7 (B; P=0.03) and the
ATG5/ATG12 complex (C; P=0.04), indicating an increase in autophagy
levels in rapamycin-treated mice. While no significant changes were
observed in levels of LC3I (D; P>0.05), rapamycin significantly
increased brain levels of LC31I (E; P=0.03), further indicating an
increase in autophagy. E and F, Representative epifluorescent
images of hippocampal CA1 in brain of control-fed (E) and
rapamycin-fed (F) hAPP(J20) mice stained with an anti-LC3 antibody.
A marked increase in LC3-specific immunoreactivity was observed in
CA1 projections following rapamycin administration. Insets,
z-stacks of confocal images from the same region. Representative 2D
sections across the volumes are shown.
[0074] FIG. 32A, 32B. Akt activation in visceral fat of
rapamycin-treated UM-HET3 male mice treated for 5 weeks. A)
Immunoassay with antibodies used shown to the left of each blot
(P-Akt is specific for Ser 473). B) Data were quantified and shown
as graphs. Band intensities for female mouse #21 were eliminated
from statistical analysis since they were well outside the 95%
confidence limits of the mean.
[0075] FIG. 33. Reduction of Akt activation in visceral fat of
rapamycin-treated UM-HET3 male mice treated for 20 months with
rapa. Female data are also shown. Antibodies used are shown to the
left of each blot (P-Akt is specific for Ser 473). Data were
quantified and shown as graphs below the immunoblots.
[0076] FIG. 34. Short term versus long term treatment with
rapamycin in gastrocnemius muscle. Shown are graphs of quantified
intensity values P-473Ser Akt/Akt ratios. A) Five week treatment.
B) Twenty month treatment. Note that females show a significant
increase in Ser473 phosphorylation in females treated for 5 weeks
with rapamycin, with the same trend in males. In 20 month
treatment, there is no increase in Akt phosphorylation in females
or males.
[0077] FIG. 35. Immunoblot assay of S6K1 in liver tumors from
rapamycin (R)-treated and control (C) mice in cohort 3. These mice
were on rapa chow for 20 months. P-Thr(389)p70 is the signal from
phosphorylation-dependent antibody and p70 is the signal from the
phosphorylation-independent antibody.
[0078] FIG. 36A, 36B. Rapamycin decreases phosphorylation of p70
kinase. A, Quantitative analysis of p-P70 immunoreactivity in blots
of hippocampal lysates from control- or rapamycin-treated mice show
that rapamycin decreases phosphorylation of p70 kinase consistent
with inhibition of mTOR by rapamycin. b, Representative Western
blot from proteins extracted from hippocampi of control- or
rapamycin-treated hAPP(J20) mice (also known as PDAPP mice).
Statistical evaluations were conducted using a two-tailed unpaired
Student's t test.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0079] The present invention takes advantage of the recognition
that microencapsulation of an inhibitor of mTOR improves the
stability of the inhibitor, which thus improves the efficacy of the
inhibitor in reducing cell aging, organism longevity, and
age-related diseases of aging. For example, to improve stability of
the drug in the diet, the inventors have developed a
microencapsulation procedure which improves the fraction of
rapamycin that survives food preparation by 3 to 4-fold. Mice
consuming food with microencapsulated rapamycin has blood
concentrations approximately 10 fold higher than those that ate
non-encapsulated rapamycin-containing food. Microencapsulation of
rapamycin made this test financially feasible, as the estimated
costs for non-encapsulated rapamycin for the test was extremely
high. After at least 50% of the mice had died, mice in the
rapamycin group showed greater survival than controls (p<0001,
males and p<0.0007, females). These data strongly support the
concept that chronic inhibition of mTOR via any route of delivery
of rapamycin or other known or unknown mTOR inhibitors will
ameliorate age-related diseases such as cancer, metabolic syndromes
and neurodegenerative diseases, thereby improving overall health an
well being of mature adults.
A. mTOR INHIBITORS AND RAPAMYCIN
[0080] Any inhibitor of mTOR is contemplated for inclusion in the
present microcapsules and methods. In particular embodiments, the
inhibitor of mTOR is rapamycin or an analog of rapamycin. Rapamycin
(also known as sirolimus and marketed under the trade name
Rapamune.RTM.) is a known macrolide. The molecular formula of
rapamycin is C.sub.51H.sub.79NO.sub.13. The chemical name is
(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,-
21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[-
(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6-
,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohent-
ria-contine-1,5,11,28,29(4H,6H,31H)-pentone.
[0081] Rapamycin binds to a member of the FK binding protein (FKBP)
family, FKBP 12. The rapamycin/FKBP 12 complex binds to the protein
kinase mTOR to block the activity of signal transduction pathways.
Because the mTOR signaling network includes multiple tumor
suppressor genes, including PTEN, LKB1, TSC1, and TSC2, and
multiple proto-oncogenes including PI3K, Akt, and eEF4E, mTOR
signaling plays a central role in cell survival and proliferation.
Binding of the rapamycin/FKBP complex to mTOR causes arrest of the
cell cycle in the G1 phase (Janus et al., 2005).
[0082] mTOR inhibitors also include rapamycin analogs. Many
rapamycin analogs are known in the art. Non-limiting examples of
analogs of rapamycin include, but are not limited to, everolimus,
tacrolimus, CCI-779, ABT-578, AP-23675, AP-23573, AP-23841,
7-epi-rapamycin, 7-thiomethyl-rapamycin,
7-epi-trimethoxyphenyl-rapamycin, 7-epi-thiomethyl-rapamycin,
7-demethoxy-rapamycin, 32-demethoxy-rapamycin,
2-desmethyl-rapamycin, prerapamycin, temsirolimus, and
42-O-(2-hydroxy)ethyl rapamycin.
[0083] Other analogs of rapamycin include: rapamycin oximes (U.S.
Pat. No. 5,446,048); rapamycin aminoesters (U.S. Pat. No.
5,130,307); rapamycin dialdehydes (U.S. Pat. No. 6,680,330);
rapamycin 29-enols (U.S. Pat. No. 6,677,357); O-alkylated rapamycin
derivatives (U.S. Pat. No. 6,440,990); water soluble rapamycin
esters (U.S. Pat. No. 5,955,457); alkylated rapamycin derivatives
(U.S. Pat. No. 5,922,730); rapamycin amidino carbamates (U.S. Pat.
No. 5,637,590); biotin esters of rapamycin (U.S. Pat. No.
5,504,091); carbamates of rapamycin (U.S. Pat. No. 5,567,709);
rapamycin hydroxyesters (U.S. Pat. No. 5,362,718); rapamycin
42-sulfonates and 42-(N-carbalkoxy)sulfamates (U.S. Pat. No.
5,346,893); rapamycin oxepane isomers (U.S. Pat. No. 5,344,833);
imidazolidyl rapamycin derivatives (U.S. Pat. No. 5,310,903);
rapamycin alkoxyesters (U.S. Pat. No. 5,233,036); rapamycin
pyrazoles (U.S. Pat. No. 5,164,399); acyl derivatives of rapamycin
(U.S. Pat. No. 4,316,885); reduction products of rapamycin (U.S.
Pat. Nos. 5,102,876 and 5,138,051); rapamycin amide esters (U.S.
Pat. No. 5,118,677); rapamycin fluorinated esters (U.S. Pat. No.
5,100,883); rapamycin acetals (U.S. Pat. No. 5,151,413);
oxorapamycins (U.S. Pat. No. 6,399,625); and rapamycin silyl ethers
(U.S. Pat. No. 5,120,842), each of which is specifically
incorporated by reference.
[0084] Other analogs of rapamycin include those described in U.S.
Pat. Nos. 7,560,457; 7,538,119; 7,476,678; 7,470,682; 7,455,853;
7,446,111; 7,445,916; 7,282,505; 7,279,562; 7,273,874; 7,268,144;
7,241,771; 7,220,755; 7,160,867; 6,329,386; RE37,421; 6,200,985;
6,015,809; 6,004,973; 5,985,890; 5,955,457; 5,922,730; 5,912,253;
5,780,462; 5,665,772; 5,637,590; 5,567,709; 5,563,145; 5,559,122;
5,559,120; 5,559,119; 5,559,112; 5,550,133; 5,541,192; 5,541,191;
5,532,355; 5,530,121; 5,530,007; 5,525,610; 5,521,194; 5,519,031;
5,516,780; 5,508,399; 5,508,290; 5,508,286; 5,508,285; 5,504,291;
5,504,204; 5,491,231; 5,489,680; 5,489,595; 5,488,054; 5,486,524;
5,486,523; 5,486,522; 5,484,791; 5,484,790; 5,480,989; 5,480,988;
5,463,048; 5,446,048; 5,434,260; 5,411,967; 5,391,730; 5,389,639;
5,385,910; 5,385,909; 5,385,908; 5,378,836; 5,378,696; 5,373,014;
5,362,718; 5,358,944; 5,346,893; 5,344,833; 5,302,584; 5,262,424;
5,262,423; 5,260,300; 5,260,299; 5,233,036; 5,221,740; 5,221,670;
5,202,332; 5,194,447; 5,177,203; 5,169,851; 5,164,399; 5,162,333;
5,151,413; 5,138,051; 5,130,307; 5,120,842; 5,120,727; 5,120,726;
5,120,725; 5,118,678; 5,118,677; 5,100,883; 5,023,264; 5,023,263;
5,023,262; all of which are incorporated herein by reference.
Additional rapamycin analogs and derivatives can be found in the
following U.S. Patent Application Pub. Nos., all of which are
herein specifically incorporated by reference: 20080249123,
20080188511; 20080182867; 20080091008; 20080085880; 20080069797;
20070280992; 20070225313; 20070203172; 20070203171; 20070203170;
20070203169; 20070203168; 20070142423; 20060264453; and
20040010002.
[0085] Rapamycin or a rapamycin analog can be obtained from any
source known to those of ordinary skill in the art. The source may
be a commercial source, or natural source. Rapamycin or a rapamycin
analog may be chemically synthesized using any technique known to
those of ordinary skill in the art. Non-limiting examples of
information concerning rapamycin synthesis can be found in Schwecke
et al., 1995; Gregory et al., 2004; Gregory et al., 2006; Graziani,
2009.
B. PREPARATION OF MICROCAPSULES
[0086] The microcapsules of the present invention can be prepared
using any method known to those of ordinary skill in the field. Any
method known to those of ordinary skill in the art can be used to
obtain the core. The core is then coated using any method known to
those of ordinary skill in the art. In particular embodiments, the
coating is an enteric coating. Some examples of coating are
discussed below. In specific embodiments, applying an enteric
coating involves use of a spinning disk atomizer, other methods may
include pan coating, air-suspension coating, centrifugal extrusion,
vibrational nozzle, spray-drying, interfacial polymerization, in
situ polymerization, matrix polymerization.
[0087] Additional methods for preparing microcapsules are discussed
in the following U.S. Patent Application Pub. Nos.: 20080022965,
20080193653, 20070138673; 20070082829; 20060234053, 20060121122,
20050113282, 20040121155, 20040074089, and 20020009473, and the
following U.S. Pat. Nos. 7,576,903, 7,037,582, 6,936,644,
6,653,256, 6,592,916, 6,486,099, 4,460,722, each of which is herein
specifically incorporated by reference.
C. CORES
[0088] The core as used herein refers to that portion of the
microcapsule that includes the active agent, where the active agent
is encased in a coating. Active agents have been discussed above
and elsewhere in this specification.
[0089] The core may include any number of additional therapeutic
agents, or any number of additional adjunct ingredients. For
example, the core may further include at least one of an absorption
enhanced, a binder, a hardness enhancing agent, optionally a
disintegrant and another excipient. Examples of binders include
povidone (PVP: polyvinyl pyrrolidone), low molecular weight HPC
(hydroxypropyl cellulose), low molecular weight HPMC (hydroxypropyl
methylcellulose), low molecular weight carboxy methyl cellulose,
ethylcellulose, gelatin polyethylene oxide, acacia, dextrin,
magnesium aluminum silicate, starch, and polymethacrylates. The
core may include a stabilizer such as at least one of butyl
hydroxyanisole, ascorbic acid and citric acid. The core may include
a disintegrant selected from the group consisting of croscarmellose
sodium, crospovidone (cross-linked polyvinyl pyrolidone) sodium
carboxymethyl starch (sodium starch glycolate), cross-linked sodium
carboxymethyl cellulose (Croscarmellose), pregelatinized starch
(starch 1500), microcrystalline starch, water insoluble starch,
calcium carboxymethyl cellulose, magnesium aluminum silicate and a
combination thereof.
[0090] The core may include a filler such as filler such as
monohydrate, microcrystalline cellulose, starch, lactitol, lactose,
a suitable inorganic calcium salt, sucrose, or a combination
thereof.
[0091] The core may include an antioxidant that is selected from
the group consisting of 4,4 (2,3 dimethyl tetramethylene
dipyrochatechol), Tocopherol-rich extract (natural vitamin E),
.alpha.-tocopherol (synthetic Vitamin E), .beta.-tocopherol,
.gamma.-tocopherol, .delta.-tocopherol, Butylhydroxinon, Butyl
hydroxyanisole (BHA), Butyl hydroxytoluene (BHT), Propyl Gallate,
Octyl gallate, Dodecyl Gallate, Tertiary butylhydroquinone (TBHQ),
Fumaric acid, Malic acid, Ascorbic acid (Vitamin C), Sodium
ascorbate, Calcium ascorbate, Potassium ascorbate, Ascorbyl
palmitate, Ascorbyl stearate, Citric acid, Sodium lactate,
Potassium lactate, Calcium lactate, Magnesium lactate, Anoxomer,
Erythorbic acid, Sodium erythorbate, Erythorbin acid, Sodium
erythorbin, Ethoxyquin, Glycine, Gum guaiac, Sodium citrates
(monosodium citrate, disodium citrate, trisodium citrate),
Potassium citrates (monopotassium citrate, tripotassium citrate),
Lecithin, Polyphosphate, Tartaric acid, Sodium tartrates
(monosodium tartrate, disodium tartrate), Potassium tartrates
(monopotassium tartrate, dipotassium tartrate), Sodium potassium
tartrate, Phosphoric acid, Sodium phosphates (monosodium phosphate,
disodium phosphate, trisodium phosphate), Potassium phosphates
(monopotassium phosphate, dipotassium phosphate, tripotassium
phosphate), Calcium disodium ethylene diamine tetra-acetate
(Calcium disodium EDTA), Lactic acid, Trihydroxy butyrophenone and
Thiodipropionic acid.
[0092] The core may include a chelating agent such as antioxidants,
dipotassium edentate, disodium edentate, edetate calcium disodium,
edetic acid, fumaric acid, malic acid, maltol, sodium edentate,
trisodium edetate.
[0093] The core may include a lubricant such as stearate salts;
stearic acid, corola oil, glyceryl palmitostearate, hydrogenated
vegetable oil, magnesium oxide, mineral oil, poloxamer,
polyethylene glycole, polyvinyl alcohol, magnesium stearate, sodium
benzoate, talc, sodium stearyl fumarate, compritol (glycerol
behenate), and sodium lauryl sulfate (SLS) or a combination ther A
preferred embodiment of the formulation according to the present
invention preferably features a core which contains a hydrophilic,
swellable, hydrogel-forming material, covered by a coating which
includes a water insoluble polymer and hydrophilic water permeable
agent, through which water enters the core. The swellable
hydrogel-forming material in the core then swells and bursts the
coating, after which the core more preferably disintegrates slowly
or otherwise releases the active ingredient. Another optional but
preferred embodiment relates to a release-controlling core with an
slow-erodible dry coating.
D. COATINGS
[0094] Many pharmaceutical dosage forms irritate the stomach due to
their chemical properties or are degraded by stomach acid thorugh
the action of enzymes, thus becoming less effective. The coating
may be an enteric coating, a coating that prevents release and
absorption of active ingredients until they reach the intestine.
"Enteric" refers to the small intestine, and therefore enteric
coatings facilitate delivery of agents to the small intestine. Some
enteric coatings facilitate delivery of agents to the colon. In
some embodiments, the enteric coating is a EUDRAGIT.RTM. coating.
Eudragit coatings include Eudragit L100-44 (for delivery to the
duodenum), Eudragit L 30 D-55 (for delivery to the duodenum),
Eudragit L 100 (for delivery to the jejunum), Eudragit S100 (for
delivery to the ileum), and Eudragit FS 30D (for colon delivery).
Other coatings include Eudragit RS, Eudragit RL, ethylcellulose,
and polyvinyl acetate. Benefits include pH-dependent drug release,
protection of active agents sensitive to gastric fluid, protection
of gastric mucosa from active agents, increase in drug
effectiveness, good storage stability, and GI and colon
targeting.
[0095] Some examples of enteric coating components include
cellulose acetate pthalate, methyl acrylate-methacrylic acid
copolymers, cellulose acetate succinate, hydroxy propyl methyl
cellulose phthalate, hydroxy propyl methyl cellulose acetate
succinate, polyvinyl acetate phthalate, methyl
methacrylate-methacrylic acid copolymers, sodium alginate, and
stearic acid. The coating may include suitable hydrophilic gelling
polymers including but not limited to cellulosic polymers, such as
methylcellulose, carboxymethylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose, hydroxyethylcellulose, and the like;
vinyl polymers, such as polyvinylpyrrolidone, polyvinyl alcohol,
and the like; acrylic polymers and copolymers, such as acrylic acid
polymer, methacrylic acid copolymers, ethyl acrylate-methyl
methacrylate copolymers, natural and synthetic gums, such as guar
gum, arabic gum, xanthan gum, gelatin, collagen, proteins,
polysaccharides, such as pectin, pectic acid, alginic acid, sodium
alginate, polyaminoacids, polyalcohols, polyglycols; and the like;
and mixtures thereof. Any other coating agent known to those of
ordinary skill in the art is contemplated for inclusion in the
coatings of the microcapsules set forth herein.
[0096] The coating may optionally comprises a plastisizer, such as
dibutyl sebacate, polyethylene glycol and polypropylene glycol,
dibutyl phthalate, diethyl phthalate, triethyl citrate, tributyl
citrate, acetylated monoglyceride, acetyl tributyl citrate,
triacetin, dimethyl phthalate, benzyl benzoate, butyl and/or glycol
esters of fatty acids, refined mineral oils, oleic acid, castor
oil, corn oil, camphor, glycerol and sorbitol or a combination
thereof. The coating may optionally include a gum. Non-limiting
examples of gums include homopolysaccharides such as locust bean
gum, galactans, mannans, vegetable gums such as alginates, gum
karaya, pectin, agar, tragacanth, accacia, carrageenan, tragacanth,
chitosan, agar, alginic acid, other polysaccharide gums (e.g.,
hydrocolloids), acacia catechu, salai guggal, indian bodellum,
copaiba gum, asafetida, cambi gum, Enterolobium cyclocarpum, mastic
gum, benzoin gum, sandarac, gambier gum, butea frondosa (Flame of
Forest Gum), myrrh, konjak mannan, guar gum, welan gum, gellan gum,
tara gum, locust bean gum, carageenan gum, glucomannan, galactan
gum, sodium alginate, tragacanth, chitosan, xanthan gum,
deacetylated xanthan gum, pectin, sodium polypectate, gluten,
karaya gum, tamarind gum, ghatti gum, Accaroid/Yacca/Red gum,
dammar gum, juniper gum, ester gum, ipil-ipil seed gum, gum talha
(acacia seyal), and cultured plant cell gums including those of the
plants of the genera: acacia, actinidia, aptenia, carbobrotus,
chickorium, cucumis, glycine, hibiscus, hordeum, letuca,
lycopersicon, malus, medicago, mesembryanthemum, oryza, panicum,
phalaris, phleum, poliathus, polycarbophil, sida, solanum,
trifolium, trigonella, Afzelia africana seed gum, Treculia africana
gum, detarium gum, cassia gum, carob gum, Prosopis africana gum,
Colocassia esulenta gum, Hakea gibbosa gum, khaya gum,
scleroglucan, zea, mixtures of any of the foregoing, and the
like.
E. APPLICATIONS
1. Definitions
[0097] "Treatment" and "treating" as used herein refer to
administration or application of a therapeutic agent to a subject
or performance of a procedure or modality on a subject for the
purpose of obtaining a therapeutic benefit of a disease or
health-related condition. For example, the microcapsules of the
present invention may be administered to a subject for the purpose
of treating a neurodegenerative disease in a subject. Treating as
used herein refers to cure of all signs and symptoms of the
disease, or reduction in the severity of signs or symptoms of a
disease.
[0098] The term "therapeutic benefit" or "therapeutically
effective" as used throughout this application refers to anything
that promotes or enhances the well-being of the subject with
respect to the medical treatment of this condition. This includes,
but is not limited to, a reduction in the frequency or severity of
the signs or symptoms of a disease. For example, administering
microcapsules of the present invention to reduce the signs and
symptoms of a neurodegenerative disease.
[0099] "Prevention" and "preventing" are used according to their
ordinary and plain meaning to mean "acting before" or such an act.
In the context of a particular disease or health-related condition,
those terms refer to administration or application of an agent,
drug, or remedy to a subject or performance of a procedure or
modality on a subject for the purpose of blocking the onset of a
disease or health-related condition. For example, administering the
microcapsules of the present invention for the purpose of blocking
the onset of a neurodegenerative disease in an elderly person.
2. Age-Related Diseases Associated with the TOR Pathway
[0100] The methods of the invention may be used to treat or prevent
age-related diseases, conditions, or disorders. Non-limiting
examples of age-related diseases, conditions, or disorders include
insulin resistance (i.e., impaired glucose tolerance), benign
prostatic hyperplasia, hearing loss, osteoporosis, age-related
macular degeneration, neurodegenerative diseases, a skin disease,
aging skin, or cancer. In one embodiment of the methods of the
invention, the age-related disease, condition, or disorder is a
skin disease. Examples of skin diseases for which the methods of
the invention may be used include seborreic keratosis, actinic
keratosis, keloid, psoriasis, and Kaposi's sarcoma.
[0101] Non-limiting examples of neurodegenerative diseases include
Alzheimer disease; epilepsy; Huntington's Disease; Parkinson's
Disease; stroke; spinal cord injury; traumatic brain injury; Lewy
body dementia; Pick's disease; Niewmann-Pick disease; amyloid
angiopathy; cerebral amyloid angiopathy; systemic amyloidosis;
hereditary cerebral hemorrhage with amyloidosis of the Dutch type;
inclusion body myositis; mild cognitive impairment; Down's
syndrome; and neuromuscular disorders including amyotrophic lateral
sclerosis (ALS), multiple sclerosis, and muscular dystrophies
including Duchenne dystrophy, Becker muscular dystrophy,
Facioscapulohumeral (Landouzy-Dejerine) muscular dystrophy, and
limb-girdle muscular dystrophy (LGMD). Also included is
neurodegenerative disease due to stroke, head trauma, spinal
injury, or other injuries to the brain, peripheral nervous, central
nervous, or neuromuscular system.
[0102] In another embodiment of the methods of the invention, the
age-related disease, condition, or disorder is an aging skin
condition. Examples of aging skin conditions for which the methods
of the invention may be used include age-related spots, pigment
spots, wrinkles, photo-aged skin, or angiogenic spots. In still
another embodiment of the methods of the invention, the inhibitor
of TOR is administered to extend an individual's healthy life
span.
[0103] The methods of the invention may be used to inhibit cellular
or organismal events. In one embodiment of the invention, the
cellular event being inhibited is cell aging. In another embodiment
of the invention the cellular event being inhibited is cell
hypertrophy. In still another embodiment of the invention, the
cellular event being inhibited is organism aging.
[0104] Other examples of age-related diseases for which mTOR
involvement has been demonstrated include the following: benign
prostatic hyperplasia, benign prostatic hyperplasia (BPH), benign
prostatic hypertrophy, benign enlargement of the prostrate (BEP),
metabolic syndrome including insulin resistance and its
complications, obesity (especially abdominal obesity), elevated
blood pressure, thrombosis, hypertension and atherosclerosis,
cardiac hypertrophy, and osteoporosis. With respect to specific
neurodegenerative diseases, the mTOR pathway has been shown to be
involved with Alzheimer's disease by increasing Tau protein
synthesis (Li et al., 2005). In addition, a correlation between
activated mTOR in blood lymphocytes and memory and cognitive
decline has been established in individuals suffering from
Alzheimer's disease (Paccalin et al., 2006).
[0105] With respect to cancer, non-limiting examples include breast
cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer,
liver cancer, cervical cancer, colon cancer, renal cancer, skin
cancer, head and neck cancer, bone cancer, esophageal cancer,
bladder cancer, uterine cancer, lymphatic cancer, stomach cancer,
pancreatic cancer, testicular cancer, lymphoma, or leukemia. Other
specific examples of cancer include squamous cell carcinoma, basal
cell carcinoma, adenoma, adenocarcinoma, linitis plastica,
insulinoma, glucagonoma, gastrinoma, vipoma, cholangiocarcinoma,
hepatocellular carcinoma, adenoid cystic carcinoma, carcinoid
tumor, prolactinoma, oncocytoma, hurthle cell adenoma, renal cell
carcinoma, endometrioid adenoma, cystadenoma, pseudomyxoma
peritonei, Warthin's tumor, thymoma, thecoma, granulosa cell tumor,
arrhenoblastoma, Sertoli-Leydig cell tumor, paraganglioma,
pheochromocytoma, glomus tumor, melanoma, soft tissue sarcoma,
desmoplastic small round cell tumor, fibroma, fibrosarcoma, myxoma,
lipoma, liposarcoma, leiomyoma, leiomyosarcoma, myoma, myosarcoma,
rhabdomyoma, rhabdomyosarcoma, pleomorphic adenoma, nephroblastoma,
brenner tumor, synovial sarcoma, mesothelioma, dysgerminoma, germ
cell tumors, embryonal carcinoma, yolk sac tumor, teratomas,
dermoid cysts, choriocarcinoma, mesonephromas, hemangioma, angioma,
hemangiosarcoma, angiosarcoma, hemangioendothelioma,
hemangioendothelioma, Kaposi's sarcoma, hemangiopericytoma,
lymphangioma, cystic lymphangioma, osteoma, osteosarcoma,
osteochondroma, cartilaginous exostosis, chondroma, chondrosarcoma,
giant cell tumors, Ewing's sarcoma, odontogenic tumors,
cementoblastoma, ameloblastoma, craniopharyngioma gliomas mixed
oligoastrocytomas, ependymoma, astrocytomas, glioblastomas,
oligodendrogliomas, neuroepitheliomatous neoplasms, neuroblastoma,
retinoblastoma, meningiomas, neurofibroma, neurofibromatosis,
schwannoma, neurinoma, neuromas, granular cell tumors, alveolar
soft part sarcomas, lymphomas, non-Hodgkin's lymphoma,
lymphosarcoma, Hodgkin's disease, small lymphocytic lymphoma,
lymphoplasmacytic lymphoma, mantle cell lymphoma, primary effusion
lymphoma, mediastinal (thymic) large cell lymphoma, diffuse large
B-cell lymphoma, intravascular large B-cell lymphoma, Burkitt
lymphoma, splenic marginal zone lymphoma, follicular lymphoma,
extranodal marginal zone B-cell lymphoma of mucosa-associated
lymphoid tissue (MALT-lymphoma), nodal marginal zone B-cell
lymphoma, mycosis fungoides, Sezary syndrome, peripheral T-cell
lymphoma, angioimmunoblastic T-cell lymphoma, subcutaneous
panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma,
hepatosplenic T-cell lymphoma, enteropathy type T-cell lymphoma,
lymphomatoid papulosis, primary cutaneous anaplastic large cell
lymphoma, extranodal NK/T cell lymphoma, blastic NK cell lymphoma,
plasmacytoma, multiple myeloma, mastocytoma, mast cell sarcoma,
mastocytosis, mast cell leukemia, langerhans cell histiocytosis,
histiocytic sarcoma, langerhans cell sarcoma dendritic cell
sarcoma, follicular dendritic cell sarcoma, Waldenstrom
macroglobulinemia, lymphomatoid granulomatosis, acute leukemia,
lymphocytic leukemia, acute lymphoblastic leukemia, acute
lymphocytic leukemia, chronic lymphocytic leukemia, adult T-cell
leukemia/lymphoma, plasma cell leukemia, T-cell large granular
lymphocytic leukemia, B-cell prolymphocytic leukemia, T-cell
prolymphocytic leukemia, pecursor B lymphoblastic leukemia,
precursor T lymphoblastic leukemia, acute erythroid leukemia,
lymphosarcoma cell leukemia, myeloid leukemia, myelogenous
leukemia, acute myelogenous leukemia, chronic myelogenous leukemia,
acute promyelocytic leukemia, acute promyelocytic leukemia, acute
myelomonocytic leukemia, basophilic leukemia, eosinophilic
leukemia, acute basophilic leukemia, acute myeloid leukemia,
chronic myelogenous leukemia, monocytic leukemia, acute monoblastic
and monocytic leukemia, acute megakaryoblastic leukemia, acute
myeloid leukemia and myelodysplastic syndrome, chloroma or myeloid
sarcoma, acute panmyelosis with myelofibrosis, hairy cell leukemia,
juvenile myelomonocytic leukemia, aggressive NK cell leukemia,
polycythemia vera, myeloproliferative disease, chronic idiopathic
myelofibrosis, essential thrombocytemia, chronic neutrophilic
leukemia, chronic eosinophilic leukemia/hypereosinophilic syndrome,
post-transplant lymphoproliferative disorder, chronic
myeloproliferative disease, myelodysplastic/myeloproliferative
diseases, chronic myelomonocytic leukemia and myelodysplastic
syndrome. In certain embodiments, the hyperproliferative lesion is
a disease that can affect the mouth of a subject. Examples include
leukoplakia, squamous cell hyperplastic lesions, premalignant
epithelial lesions, intraepithelial neoplastic lesions, focal
epithelial hyperplasia, and squamous carcinoma lesion.
[0106] The microcapsules of the present invention can be applied in
the treatment of any disease for with use of an inhibitor of mTOR
is contemplated. The following U.S. patents disclose various
properties and uses of rapamycin and are herein incorporated by
reference. U.S. Pat. No. 5,100,899 discloses inhibition of
transplant rejection by rapamycin; U.S. Pat. No. 3,993,749
discloses rapamycin antifungal properties; U.S. Pat. No. 4,885,171
discloses antitumor activity of rapamycin against lymphatic
leukemia, colon and mammary cancers, melanocarcinoma and
ependymoblastoma; U.S. Pat. No. 5,206,018 discloses rapamycin
treatment of malignant mammary and skin carcinomas, and central
nervous system neoplasms; U.S. Pat. No. 4,401,653 discloses the use
of rapamycin in combination with picibanil in the treatment of
tumors; U.S. Pat. No. 5,078,999 discloses a method of treating
systemic lupus erythematosus with rapamycin; U.S. Pat. No.
5,080,899 discloses a method of treating pulmonary inflammation
with rapamycin that is useful in the symptomatic relief of diseases
in which pulmonary inflammation is a component, i.e., asthma,
chronic obstructive pulmonary disease, emphysema, bronchitis, and
acute respiratory distress syndrome; U.S. Pat. No. 6,670,355
discloses the use of rapamycin in treating cardiovascular, cerebral
vascular, or peripheral vascular disease; U.S. Pat. No. 5,561,138
discloses the use of rapamycin in treating immune related anemia;
U.S. Pat. No. 5,288,711 discloses a method of preventing or
treating hyperproliferative vascular disease including intimal
smooth muscle cell hyperplasia, restenosis, and vascular occlusion
with rapamycin; and U.S. Pat. No. 5,321,009 discloses the use of
rapamycin in treating insulin dependent diabetes mellitus. In
general, any disease which may be ameliorated, treated, cured or
prevented by administration of rapamycin or a rapamycin derivative
may be treated by administration of the microcapsules described
herein. Non-limiting examples of such diseases include--organ or
tissue transplant rejection, graft-versus-host disease, autoimmune
disease and inflammatory conditions, arthritis (for example
rheumatoid arthritis, arthritis chronica progrediente and arthritis
deformans) and rheumatic diseases, autoimmune diseases, autoimmune
hematological disorders, systemic lupus erythematosus, sclerodoma,
Wegener granulamatosis, dermatomyositis, chronic active hepatitis,
myasthenia gravis, psoriasis, Steven-Johnson syndrome, idiopathic
sprue, autoimmune inflammatory bowel disease (including ulcerative
colitis and Crohn's disease), endocrine opthalmopathy, Graves
disease, sarcoidosis, multiple sclerosis, primary biliary
cirrhosis, juvenile diabetes uveitis, keratoconjunctivitis sicca,
vernal keratoconjunctivitis, interstitial lung fibrosis, psoriatic
arthritis, glomerulonephritis, autosomal-dominant polycystic kidney
disease, juvenile dermatomyositis, asthma, chronic obstructive
pulmonary disease, emphysema, bronchitis, and acute respiratory
distress syndrome, tumors, hyperproliferative skin disorders,
fungal infections, dry eye, vascular disease, diabetes, and ocular
disease (such as neovascularization of the eye due to age-related
macular degeneration).
3. Preventive Therapies
[0107] Certain embodiments of the methods set forth herein pertain
to methods of preventing a disease or health-related condition in a
subject. Preventive strategies are of key importance in medicine
today.
[0108] The quantity of pharmaceutical composition to be
administered, according to dose, number of treatments and duration
of treatments, depends on the subject to be treated, the state of
the subject, the nature of the disease to be prevented and the
protection desired. Precise amounts of the therapeutic composition
also depend on the judgment of the practitioner and are peculiar to
each individual. For example, the frequency of application of the
composition can be once a day, twice a day, once a week, twice a
week, or once a month. Duration of treatment may range from one
month to one year or longer. Again, the precise preventive regimen
will be highly dependent on the subject, the nature of the risk
factor, and the judgment of the practitioner.
F. COMPOSITIONS
[0109] Certain of the methods set forth herein pertain to methods
involving the administration of a composition comprising the
microcapsules of the present invention.
[0110] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
surfactants, antioxidants, preservatives (e.g., antibacterial
agents, antifungal agents), isotonic agents, absorption delaying
agents, salts, preservatives, drugs, drug stabilizers, gels,
binders, excipients, disintegration agents, lubricants, sweetening
agents, flavoring agents, dyes, such like materials and
combinations thereof, as would be known to one of ordinary skill in
the art (Remington's, 1990). Except insofar as any conventional
carrier is incompatible with the active ingredient, its use in the
therapeutic or pharmaceutical compositions is contemplated. The
compositions used in the present invention may comprise different
types of carriers depending on whether it is to be administered in
solid, liquid or aerosol form, and whether it need to be sterile
for such routes of administration as injection.
[0111] The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions, and these are discussed in
greater detail below. For human administration, preparations should
meet sterility, pyrogenicity, general safety and purity standards
as required by FDA Office of Biologics standards.
[0112] The formulation may vary depending upon the route of
administration. For parenteral administration in an aqueous
solution, for example, the solution should be suitably buffered if
necessary and the liquid diluent first rendered isotonic with
sufficient saline or glucose. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure.
[0113] In certain embodiments, pharmaceutical composition includes
at least about 0.1% by weight of the active compound. In other
embodiments, the pharmaceutical composition includes about 2% to
about 75% of the weight of the composition, or between about 25% to
about 60% by weight of the composition, for example, and any range
derivable therein.
[0114] The pharmaceutical composition of the present invention may
comprise various antioxidants to retard oxidation of one or more
component. Additionally, the prevention of the action of
microorganisms can be brought about by preservatives such as
various antibacterial and antifungal agents, including but not
limited to parabens (e.g., methylparabens, propylparabens),
chlorobutanol, phenol, sorbic acid, thimerosal or combinations
thereof. The composition must be stable under the conditions of
manufacture and storage, and preserved against the contaminating
action of microorganisms, such as bacteria and fungi.
[0115] 1. Routes of Administration
[0116] The microcapsules can be administered to the subject using
any method known to those of ordinary skill in the art. For
example, a pharmaceutically effective amount of the composition may
be administered in a composition including an aqueous media that is
administered intravenously, intracerebrally, intracranially,
intrathecally, into the substantia nigra or the region of the
substantia nigra, intradermally, intraarterially,
intraperitoneally, intralesionally, intratracheally, intranasally,
topically, intramuscularly, intraperitoneally, subcutaneously,
orally, topically, locally, inhalation (e.g., aerosol inhalation),
injection, infusion, continuous infusion, localized perfusion
bathing target cells directly, via a catheter, via a lavage, in
cremes, in lipid compositions (e.g., liposomes), or by other method
or any combination of the forgoing as would be known to one of
ordinary skill in the art (Remington's, 1990). Solid compositions
of microcapsules may be administered orally.
[0117] In particular embodiments, the composition is administered
to a subject using a drug delivery device. Any drug delivery device
is contemplated for use in delivering a pharmaceutically effective
amount of the inhibitor of mTOR.
[0118] 2. Dosage
[0119] A pharmaceutically effective amount of an inhibitor of mTOR
is determined based on the intended goal. The quantity to be
administered, both according to number of treatments and dose,
depends on the subject to be treated, the state of the subject, the
protection desired, and the route of administration. Precise
amounts of the therapeutic agent also depend on the judgment of the
practitioner and are peculiar to each individual.
[0120] The amount of rapamycin or rapamycin analog to be
administered will depend upon the disease to be treated, the length
of duration desired and the bioavailability profile of the implant,
and the site of administration. Generally, the effective amount
will be within the discretion and wisdom of the patient's attending
physician. Guidelines for administration include dose ranges of
from about 0.01 mg to about 500 mg of rapamycin or rapamycin
analog.
[0121] For example, a dose of the inhibitor of mTOR may be about
0.0001 milligrams to about 1.0 milligrams, or about 0.001
milligrams to about 0.1 milligrams, or about 0.1 milligrams to
about 1.0 milligrams, or even about 10 milligrams per dose or so.
Multiple doses can also be administered. In some embodiments, a
dose is at least about 0.0001 milligrams. In further embodiments, a
dose is at least about 0.001 milligrams. In still further
embodiments, a dose is at least 0.01 milligrams. In still further
embodiments, a dose is at least about 0.1 milligrams. In more
particular embodiments, a dose may be at least 1.0 milligrams. In
even more particular embodiments, a dose may be at least 10
milligrams. In further embodiments, a dose is at least 100
milligrams or higher.
[0122] In other non-limiting examples, a dose may also comprise
from about 1 microgram/kg/body weight, about 5 microgram/kg/body
weight, about 10 microgram/kg/body weight, about 50
microgram/kg/body weight, about 100 microgram/kg/body weight, about
200 microgram/kg/body weight, about 350 microgram/kg/body weight,
about 500 microgram/kg/body weight, about 1 milligram/kg/body
weight, about 5 milligram/kg/body weight, about 10
milligram/kg/body weight, about 50 milligram/kg/body weight, about
100 milligram/kg/body weight, about 200 milligram/kg/body weight,
about 350 milligram/kg/body weight, about 500 milligram/kg/body
weight, to about 1000 mg/kg/body weight or more per administration,
and any range derivable therein. In non-limiting examples of a
derivable range from the numbers listed herein, a range of about 5
mg/kg/body weight to about 100 mg/kg/body weight, about 5
microgram/kg/body weight to about 500 milligram/kg/body weight,
etc., can be administered, based on the numbers described
above.
[0123] The dose can be repeated as needed as determined by those of
ordinary skill in the art. Thus, in some embodiments of the methods
set forth herein, a single dose is contemplated. In other
embodiments, two or more doses are contemplated. Where more than
one dose is administered to a subject, the time interval between
doses can be any time interval as determined by those of ordinary
skill in the art. For example, the time interval between doses may
be about 1 hour to about 2 hours, about 2 hours to about 6 hours,
about 6 hours to about 10 hours, about 10 hours to about 24 hours,
about 1 day to about 2 days, about 1 week to about 2 weeks, or
longer, or any time interval derivable within any of these recited
ranges.
[0124] In certain embodiments, it may be desirable to provide a
continuous supply of a pharmaceutical composition to the patient.
This could be accomplished by catheterization, followed by
continuous administration of the therapeutic agent. The
administration could be intra-operative or post-operative.
[0125] 3. Secondary Treatment
[0126] Certain embodiments of the present invention provide for the
administration or application of one or more secondary forms of
therapies. The type of therapy is dependent upon the type of
disease that is being treated or prevented. The secondary form of
therapy may be administration of one or more secondary
pharmacological agents that can be applied in the treatment or
prevention of a disease associated with aging, including any of the
diseases set forth above.
[0127] If the secondary therapy is a pharmacological agent, it may
be administered prior to, concurrently, or following administration
of the inhibitor of mTOR.
[0128] The interval between the inhibitor of mTOR and the secondary
therapy may be any interval as determined by those of ordinary
skill in the art. For example, the interval may be minutes to
weeks. In embodiments where the agents are separately administered,
one would generally ensure that a significant period of time did
not expire between the time of each delivery, such that each
therapeutic agent would still be able to exert an advantageously
combined effect on the subject. For example, the interval between
therapeutic agents may be about 12 h to about 24 h of each other
and, more preferably, within about 6 hours to about 12 h of each
other. In some situations, it may be desirable to extend the time
period for treatment significantly, however, where several d (2, 3,
4, 5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse
between the respective administrations. In some embodiments, the
timing of administration of a secondary therapeutic agent is
determined based on the response of the subject to the inhibitor of
mTOR.
G. EXAMPLES
[0129] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Lifespan Extension by Rapamycin Fed to Genetically Heterogeneous
Mice from 20 Months of Age
Methods
[0130] Mouse production, maintenance, and estimation of lifespan.
Mice were produced at each of the three test sites by mating CB6F1
females with C3D2F1 males to produce a genetically heterogeneous
population. Details of the methods used for health monitoring were
provided previously (Miller et al., 2007); in brief, each of the
three colonies was evaluated four times each year for infectious
agents, including pinworm. All such tests were negative throughout
the entire study period. Each test site enrolled approximately
equal numbers of 19-21 day-old weanlings each month over a six
month period, housing 3 males or 4 females/cage. Each site used
diets that the manufacturer claimed were based on the NIH-31
standard for breeding cages and the period between weaning and the
initiation of experimental diets, as follows: For breeding cages,
UM used Purina 5008, UT used Teklad 7912, and TJL used Purina 5K52.
For weanlings prior to 4 months of age, UM used Purina 5008, UT
used Teklad 7912, and TJL used Purina 5LG6. Starting when 4 months
old, mice in the Control, Enalapril, and CAPE groups received
Purina 5LG6 at all three sites, without additives (control group)
or with the test agent. Mice in the Rapa group remained on the
weanling diet until they began to receive rapamycin, in Purina
5LG6, at 600 days of age. Separate cohorts of control and
rapamycin-treated mice were established in the same way one year
later, again at each test site, but with rapamycin initiated at 270
days rather than at 600 days of age. Additional husbandry details,
including accounts of tests for T cell subset distribution and
activity administered to a subset of each group, are provided
elsewhere (Nadon et al., 2008). The principal endpoint was age at
death (for mice found dead at daily inspections) or age at
euthanasia (for mice deemed unlikely to survive for more than an
additional 48 h).
[0131] Removal of mice from the longevity population. The Cohort
2005 study population, distributed almost equally among the three
test sites, consisted initially of 1960 mice, of which 674 were
assigned to the control group and 317 to 328 to each of the four
treatment groups. Of these, 51 mice were removed from the study
because of fighting (31 mice), accidental death (such as chip
implantation or cage flooding; 13 mice), or because of technical
error (error in gender assignment or diet selection; 7 mice). For
survival analyses, mice were treated as alive at the date of their
removal from the protocol, and lost to follow-up thereafter. These
censored mice were not included in calculations of median
longevity.
[0132] Estimation of age at death (lifespan). Mice were examined at
least daily for signs of ill health, and were euthanized for humane
reasons if they were so severely moribund that they were
considered, by an experienced technician, unlikely to survive for
more than an additional 48 hrs. A mouse was considered severely
moribund if it exhibited more than one of the following clinical
signs: (a) inability to eat or to drink; (b) severe lethargy, as
indicated by a lack of response such as a reluctance to move when
gently prodded with a forceps; (c) severe balance or gait
disturbance; (d) rapid weight loss over a period of one week or
more; or (e) a severely ulcerated or bleeding tumor. The age at
which a moribund mouse was euthanized was taken as the best
available estimate of its natural lifespan. Mice found dead were
also noted at each daily inspection. Bodies were saved for later
analysis, to be reported elsewhere.
[0133] Control and experimental diets. TestDiet, Inc. (Richmond,
Ind.) prepared batches of Purina 5LG6 food containing each of the
test substances, as well as control diet batches, at intervals of
approximately 120 days, and shipped each batch of food at the same
time to each of the three test sites. Enalapril was purchased from
Sigma (catalogue E6888-5G) and used at 120 mg per kg food; on the
assumption that the average mouse weighs 30 gm and consumes 5 gm of
food/day, this dose supplies 20 mg enalapril per kg body
weight/day. CAPE, i.e. caffeic acid phenethyl ester, was purchased
from Cayman (Ann Arbor, Mich.; Catalogue 70750), and used at either
of two doses: the high dose was 300 mg/kg food (50 mg/kg body
weight/day), and the low dose was 30 mg/kg food (5 mg/kg body
weight/day). Enalapril was tested because in aged humans and in
rodent models of hypertension, obesity, diabetes, and congestive
heart failure, it has been reported to improve many of these
conditions. CAPE was tested because this agent has been reported to
possess antioxidant, anti-inflammatory, and immunomodulatory
capabilities, as well as specific toxicity to transformed and tumor
cells. Lifespans of mice given enalapril or CAPE are compared with
controls and those given rapamycin in FIG. 1. Rapamycin was
purchased from LC Labs (Woburn, Mass.). The rapamycin was
microencapsulated by Southwest Research Institute (San Antonio,
Tex.), using a spinning disk atomization coating process with the
enteric coating material Eudragit S100 ((Rohm Pharma, Germany).
This methacrylate polymer is stable at pH levels below 7 and thus
protects the rapamycin from the acidic conditions of the stomach;
the protective coating dissolves in the small intestine, permitting
absorption of the active agent. This thermoplastic coating material
increased the fraction of rapamycin that survived the food
preparation process by 3 to 4-fold. Because the coating material is
water soluble only in non-acidic conditions, the encapsulated
rapamycin is released in the small intestine rather than in the
stomach. A pilot study showed that encapsulated rapamycin led to
blood concentrations approximately 10-fold higher than achieved by
equivalent doses of non-encapsulated rapamycin. The encapsulated
rapamycin was administered at 14 mg/kg food (2.24 mg of rapamycin
per kg body weight/day). Encapsulated rapamycin was then
incorporated into 5LG6 mouse chow and distributed to all three test
sites.
[0134] Measurement of Rapamycin. Rapamycin was obtained from LC
Laboratories (Woburn, Mass.). 32-desmethoxyrapamycin (32-RPM) was
obtained from Sigma Chemical Company (St. Louis, Mo.). HPLC grade
methanol and acetonitrile were purchased from Fisher (Fair Lawn,
N.J.). All other reagents were purchased from Sigma Chemical
Company (St. Louis, Mo.). Milli-Q water was used for preparation of
all solutions. The HPLC system consisted of a Waters 510 HPLC pump,
Waters 717 autosampler, Waters 2487 UV detector, and Waters Empower
chromatographic software (Waters, Milford, Mass.). The HPLC
analytical column was a Grace Alltima C18 (4.6.times.150 mm, 5
micron) purchased from Alltech (Deerfield, Ill.). The mobile phase
was 64% (v/v) acetonitrile, and 36% water. The flow rate of the
mobile phase was 1.5 ml/min and the wavelength of absorbance was
278 nm. The temperature of the HPLC analytical column was
maintained at 70.degree. C. during the chromatographic runs using
an Eppendorf CH-30 column heater. Rapamycin and 32-RPM powder were
dissolved in methanol at a concentration of 1 mg/ml and stored in
aliquots at -80.degree. C. A working stock solution was prepared
each day from the methanol stock solutions at a concentration of 1
.mu.g/ml and used to spike the calibrators. Calibrator samples were
prepared daily by spiking either whole blood or mouse food with
stock solutions to achieve final concentrations of 0, 4, 8, 12, 24,
100, and 200 ng/ml.
[0135] Rapamycin was quantified in mouse blood using HPLC with UV
detection. Briefly, 0.5 mL of calibrators and unknown samples were
mixed with 75 .mu.L of 1.0 .mu.g/mL 32-desmethoxy rapamycin
(internal standard), 1.0 mL ZnSO4 (50 g/L) and 1.0 mL of acetone.
The samples were vortexed vigorously for 20 sec, then centrifuged
at 2600 g at 23.degree. C. temperature for 5 min (subsequent
centrifugations were performed under the same conditions).
Supernatants were transferred to clean test tubes, then 200 .mu.L
of 100 mM NaOH was added, followed by vortexing. Then, 2 mL of
1-chlorobutane was added and the samples were capped, vortexed (1
min), and centrifuged. The supernatants were transferred to 10 mL
glass tubes and dried to residue under a stream of nitrogen at
ambient temperature. The dried extracts were dissolved in 750 .mu.L
of mobile phase and then 2 mL of hexane was added to each tube. The
tubes were capped, vortexed for 30 sec, and centrifuged for 2 min.
The hexane layers were removed and discarded. The remaining
extracts were dried under nitrogen and reconstituted in 250 .mu.L
of mobile phase, and then 200 .mu.L of the final extracts were
injected into the HPLC. The ratio of the peak area of rapamycin to
that of the internal standard (response ratio) for each unknown
sample was compared against a linear regression of calibrator
response ratios to quantify rapamycin. The concentration of
rapamycin was expressed as ng/mL whole blood.
[0136] Rapamycin content of mouse chow was verified using HPLC with
UV detection. Briefly, 100 mg of chow for spiked calibrators and
unknown samples were crushed with a mortar and pestle, then
vortexed vigorously with 20 .mu.L of 100 .mu.g/mL 32-RPM (internal
standard) and 0.5 mL methanol. The samples were then mechanically
shaken for 10 min. Next, 0.5 mL of Millipore water was added and
the samples were vortexed vigorously for 20 sec. The samples were
centrifuged for 10 min and then 40 .mu.L were injected into the
HPLC. The ratio of the peak area of rapamycin to that of the
internal standard (response ratio) was compared against a linear
regression of calibrator response ratios at rapamycin
concentrations of 0, 2, 4, 8, 10, and 20 ng/mg of food to quantify
rapamycin. The concentration of rapamycin in food was expressed as
ng/mg food (parts per million).
[0137] Rapamycin effectiveness. To assay for the status of an
mTORC1 downstream effector, phosphorylation of ribosomal protein S6
(Ser240/244), a substrate of S6 kinase 1, was measured in visceral
adipose tissue lysates in mice fed an encapsulated rapamycin diet
for 420 days or a control diet with empty microcapsules. Tissues
were dissected and snap frozen in liquid nitrogen for storage at
-80.degree. C., ground into powder under liquid nitrogen and
dissolved in 10 volumes of buffer (50 mM Tris-HCl (pH 7.5), 120 mM
NaCl, 1% NP-40, 1 mM EDTA, 50 mM NaF, 40 mM 2-glycerophosphate, 0.1
mM Na orthovanadate (pH 10), 1 mM benzamidine, and 1.times.
Complete protease inhibitor cocktail (Roche). After sonication and
microcentrifugation, lysates were quantified, 30. 40 .mu.g of
soluble protein from each extract was loaded on a 4-12% gradient
PAGE and electrophoresed overnight at 5V. Gels were then
transferred to nitrocellulose membranes (dry procedure), blocked
and incubated with the primary antibodies [S6 Ribosomal Protein
(5G10) Rabbit mAb cat. #2217; Phospho-S6 Ribosomal Protein
(Ser235/236) Antibody cat. #2215; and Cat. #4968 Pan-Actin
Antibody; Cell Signaling Technologies, Danvers Mass.], followed by
secondary antibody [Anti-rabbit IgG, (H+L), Peroxidase Conjugated
Antibody, cat. #31460 Pierce, Rockford Ill.] for detection by
chemiluminescence. Signal intensities for each immunoblot were
captured using a Kodak Image Station, which were analysed using
Kodak 1D image analysis software.
Results
[0138] In male and female mice at each of three collaborating
research sites, median and maximum life-span of mice were extended
by feeding encapsulated rapamycin starting at 600 days of age (FIG.
2). The data set was analyzed with 2% (38 of 1,901) of mice still
alive. For data pooled across sites, a log-rank test rejected the
null hypothesis that treatment and control groups did not differ
(P<0.0001); mice fed rapamycin were longer lived than controls
(P<0.0001) in both males and females. Expressed as mean
lifespan, the effect sizes were 9% for males and 13% for females in
the pooled data set. Expressed as life expectancy at 600 days (the
age of first exposure to rapamycin), the effect sizes were 28% for
males and 38% for females. Mice treated with other agents
(enalapril and CAPE (caffeic acid phenethyl ester)) evaluated in
parallel did not differ from controls at the doses used (FIG.
1).
[0139] Rapamycin-fed and control mice were then compared separately
for each combination of site and gender. Rapamycin had a consistent
benefit, compared with controls, with P values ranging from 0.03 to
0.0001 (FIG. 3). Female mice at all three sites had improved
survival after rapamycin feeding (FIG. 3). Mean lifespan increases
for females were 15%, 16% and 7% (TJL, UM and UT, respectively),
and life expectancy at 600 days increased by 45%, 48% and 22% for
females at the three sites. Median lifespan estimates of control
females were consistent across sites (881-895 days), and were
similar to values noted in Cohort 2004, which ranged from 858 to
909 days (Miller et al., 2007). Thus, the improvement in survival
seen in the rapamycin-fed females is not an artifact of low
survival for the control females. Male mice at all three sites also
had improved survival after rapamycin feeding (FIG. 3). Mean
lifespan increases for males were 5%, 8% and 15% (TJL, UM and UT,
respectively), and male life expectancy at 600 days increased by
16%, 23% and 52%. Interpretation is complicated by differences
among sites in survival of control males, and because mice assigned
to the rapamycin-fed group at UT and perhaps at UM had lower
mortality before 600 days than controls. Control mice at UT and UM
differed from those fed rapamycin not only in exposure to rapamycin
from 600 days of age but also in specific formulation of the mouse
chows (all based on the NIH-31 standard) used between weaning and
600 days. Thus, one cannot rule out the possibility that improved
survival among males in the rapamycin group, at UT and at UM, might
reflect differences in nutritional or health status between control
and rapamycin groups before 600 days, rather than solely the
effects of rapamycin. Notably, the significant benefits of
rapamycin on male (and female) survival at TJL could not have been
affected by diet before drug administration, because at TJL both
control and rapamycin-fed mice received the same chow (Purina 5LG6)
throughout this period. Maximum lifespan was increased by rapamycin
feeding. Table 1 shows the ages at the 90th percentile for control
and rapamycintreated mice, along with the 95% upper confidence
bound for the controls.
TABLE-US-00001 TABLE 1 The effect of rapamycin on maximum lifespan
Age in days at 90th Age in days at 90th percentile for percentile
for controls (upper rapamycin-treated Percentage Comparison Sites
confidence limit)* mice increase Females Rapamycin versus controls
All sites 1,094 (1,136) 1,245 14 Rapamycin versus controls TJL
1,100 (1,165) 1,282 17 Rapamycin versus controls UM 1,094 (1,149)
1,250 14 Rapamycin versus controls UT 1,089 (1,159) 1,179 8 Males
Rapamycin versus controls All sites 1,078 (1,111) 1,179 9 Rapamycin
versus controls TJL 1,035 (1,091) 1,142 10 Rapamycin versus
controls UM 1,141 (1,177) 1,188 4 Rapamycin versus controls UT
1,020 (1,101) 1,179 16 Rapamycin versus controls All sites 1,078
(1,111) 1,179 9 *The upper limit of the 95% confidence interval for
control mice is indicated in parentheses. For example, in the top
row, for females pooled across sites, the 95% confidence interval
for controls goes up to 1,136 days, and the estimate for 90th
percentile survival for the rapamycin-treated mice is 1,245 days.
This gives good evidence that the 90th percentile survival for
rapamycin-treated mice (1,245) is substantially above that for
controls (1,094).
For each site and sex, the 90th percentile age for
rapamycin-treated mice is higher than the upper limit for the
corresponding control group, showing that rapamycin increases the
age for 90.sup.th percentile survival.
[0140] To determine whether increases in maximal lifespan due to
rapamycin feeding are statistically significant, the proportion of
living mice in each group after 90% had died in the joint life
table (Wang et al., 2004) were compared (Table 2).
TABLE-US-00002 TABLE 2 Details of calculation for comparison of
surviving proportion of mice at the 90.sup.th percentile age. Age
for Youngest 90.sup.th Number Number live Site Sex percentile Group
alive dead Total % Live mouse p-value TJL F 1167 Controls 4 91 95
4.2% 1192 P = Rapa 11 37 48 22.9% 1192 0.0006 UM F 1162 Controls 2
93 95 2.1% 1187 P = Rapa 13 35 48 27.1% 1147 0.0001 UT F 1123
Controls 8 91 99 8.1% 1180 P = Rapa 7 41 48 14.6% 1189 0.22 Pooled
F Controls 14 275 289 4.8% P < Rapa 31 113 144 21.5% 0.0001 TJL
M 1088 Controls 8 118 126 6.3% 1146 P = Rapa 11 46 57 19.3% 1243
0.008 UM M 1154 Controls 9 103 112 8.0% 1161 P = Rapa 9 42 51 17.6%
1228 0.07 UT M 1112 Controls 4 115 119 3.4% 1157 P = Rapa 14 48 60
23.3% 1156 0.0001 Pooled M Controls 21 336 357 5.9% P < Rapa 34
134 168 20.2% 0.0001 The table lists, for each combination of site,
gender, and treatment group, the number of mice that were alive
(and number dead) at the age (column 3) at which 90% of the joint
distribution (control plus rapamycin for the site/gender
combination) had died. For example, for females at TJL, 4.2% of the
controls (4/95) and 22.9% of the rapamycin-treated mice (11/48)
were still alive at the age of 1167 days. At the time of analysis
(Feb. 1, 2009), there were no live control mice at ages below the
90th percentile age in any of the groups. The was one live female,
at UM, at an age below the 90.sup.th percentile threshold, but this
mouse was in the rapamycin group, and its age at death would
therefore not have a major effect on the statistics and pvalues
listed in the table.
Summing across the three sites, 4.8% of the female control mice
were alive at these ages, compared with 21.5% of the
rapamycin-treated females (P<0.0001). For males, the
corresponding values were 5.9% of controls and 20.2% of
rapamycin-treated mice (P<0.0001). The site-specific
calculations documented a significant effect on females at both TJL
(P<0.0006) and UM (P<0.0001); for males, a significant effect
at both TJL (P50.008) and UT (P50.0001) was noted, with a marginal
effect at UM (P50.07). Rapamycin feeding initiated at 600 days of
age thus leads to a significant increase in maximal lifespan.
[0141] To test if the spectrum of lesions was altered by dietary
rapamycin, complete necropsies were conducted on 31 control and 40
rapamycin fed mice that were either found dead or killed when
moribund (Table 3). Although rapamycin postpones death, it did not
change the distribution of presumptive causes of death.
TABLE-US-00003 TABLE 3 Lesions in rapamycin-treated mice and in
controls at the time of death. Cause of Death Controls Rapamycin
Abscesses 1 1 Adrenal tumor 1 Carcinoma (GI) 1 Carcinoma (renal) 1
Cardiac degeneration 1 Cardiomyopathy 1 Fibrosarcoma 2 Gastric
ulcer 1 Heart failure 2 1 Heart fibrosis Hemangiosarcoma 3 5
Hepatocarcinoma 3 3 Leiomyosarcoma 1 Lymphoma 10 15 Mammary
adenocarcinoma 1 Myocardial infarct 1 Pleuritis 1 Prostatitis 2
Pulmonary tumor 4 7 Septicemia 1 Diagnosable cases 31 40 Autolysis
17 12 Unknown 2 1 Grand Total 50 53 The mean age at death was 977
for controls (N = 31) and 1005 days for rapamycin-treated (N = 40)
mice, among those animals for which a presumptive cause of death
could be determined. Cause of death was inferred, where possible,
based on gross evaluation, followed by histopathologic examination
of a standard set of tissues from each mouse by an experienced
veterinary pathologist. Tumors were deemed the cause of death based
on tumor type, size, number, and distribution. Cause of death for
mice with inflammatory or degenerative lesions was based on the
location and severity of the lesions and the likelihood that such
lesions were severe enough to cause morbidity and mortality. Many
animals had small, localized tumors and various degenerative
lesions, which were deemed unlikely to have contributed to their
death. Autolysis precluded diagnosis in 29 cases, and the cause of
death could not be determined in three other cases as
indicated.
[0142] A separate group of mice was used to evaluate the effects of
encapsulated rapamycin initiated at 270 days of age (FIG. 4A). At
the time of analysis, 51% of the females and 68% of the males had
died, and a stratified log-rank test showed significantly lower
mortality risk in the rapamycin-treated mice compared to controls,
pooling across the three test sites (P=0.0002 for males and
P<0.0001 for females). When each site was evaluated separately,
the beneficial effect of rapamycin for females was significant at
each site (P<0.005); for males, the effect was significant
(P<0.025) at UM and UT, but not at TJL. Rapamycin seems to
reduce mid-life mortality risk when started at 270 days of age, but
additional data are needed to provide an accurate estimate of
effect size, and to evaluate effects on maximal longevity.
[0143] To document biochemical effects of rapamycin at the dose
used for the lifespan studies, the phosphorylation status of
ribosomal protein subunit S6 (rpS6)--a target substrate of S6
kinase 1 in the mTOR signalling pathway20--was evaluated in
visceral white adipose tissue (a sensitive indicator of mTOR
inhibition by rapamycin treatment in vivo). FIG. 4B shows that
rapamycin feeding reduced the levels of phosphorylated rpS6
4-5-fold when fed from 270 to about 800 days of age. Blood levels
of rapamycin in the treated mice were equivalent in males and
females, between 60 and 70 ng/ml.
[0144] Initial evidence that reduced TOR function can extend
longevity came primarily from studies in yeast (Kaeberlein et al.,
2005; Powers et al., 2006) and invertebrates (Jia et al., 2005;
Kapahi et al., 2004; Vellai et al., 2003). Beneficial effects of
diet restriction (Masoro, 2005) and dwarf mutations, both of which
extend lifespan in rodents, may, to some degree, result from
repression of the mTOR complex 1 (mTORC1) pathway (Sharp and
Bartke, 2005; Hsieh and Papaconstantinou, 2004).
[0145] It is not yet known to what extent inhibition of mTOR will
recapitulate other aspects of the phenotypes associated with diet
restriction or dwarf mutations. The demonstration that rapamycin
feeding increases lifespan even when started late in life, as well
as the absence of changes in body weight, distinguishes these
results from studies using diet restriction: in all cases diet
restriction reduces body weight, and in most reports (Mason),
2005), although not all (Dhahbi et al., 2004), diet restriction
produces little, if any, benefit if started after about 550 days of
age.
[0146] To illustrate biochemical effects of the dose of rapamycin
used in this study, the phosphorylation status of ribosomal protein
subunit S6 (rpS6), a target substrate of S6 kinase 1 in the mTOR
signaling pathway (Petroulakis et al., 2007) was evaluated, in
white adipose tissue (WAT) in a separate group of young adult
UM-HET3 mice fed rapamycin-containing food for 5 weeks.
Phosphorylated-rpS6 is greatly reduced, becoming barely detectable
in rapamycin-fed mice, relative to total rpS6 (FIG. 5). While most
of the control mice have a robust signal for phosphorylated rpS6,
some have very little of this modification. Importantly, all mice
fed rapamycin have very little phosphorylated rpS6.
[0147] Liver and brain were assayed to determine if this dose of
rapamycin in food affected rpS6 phosphorylation in other organ
systems. FIG. 6 shows immunoblot assays of rpS6 phosphorylation in
liver. Quantification of the ratio of phosphorylated rpS6 to total
rpS6 protein at this dose of rapamycin (see graphs for females,
males and both) is more pronounced in males than females, the
latter of which reach statistical significance in this assay.
Analysis of combined male and female phosphorylated rpS6 showed
significantly lower levels in treated mice. Our conclusion for S6
kinase 1 activity in liver is that both sexes are responding at
this dose of rapamycin, with males being more responsive.
[0148] FIG. 7 shows an analysis of S6K1 activity in the brain from
rapamycin treated and untreated UM-Het3 mice. The effect on
mTOR/S6K1 as measured by this assay is much less pronounced in
brain compared to WAT and liver. Since rapamycin readily crosses
the blood brain barrier (Pong and Zaleska, 2003), this response is
interesting and could be biomedically relevant.
[0149] When nutrients, energy and growth factor inputs are
favorable for activation of mTORC1 kinase activity, another of its
target substrates is 4E-BP1, a repressor of cap-dependent
translation (Gingras et al., 2001). Phosphorylation of 4E-BP1
inhibits its repressor function. Rapamycin inhibits mTORC1-mediated
phosphorylation of 4E-BP1. Analysis of 4E-BP1 in WAT in UM-Het3
mice chronically treated with rapamycin revealed that the ratio of
phosphorylated 4E-BP1 was no different in a combined analysis of
males and females (FIG. 8). There is a significant increase in
total 4E-BP1 proteins, relative to .beta.-actin, in fat from
rapamycin-consuming male mice. WAT from females treated chronically
with rapamycin showed no difference in the ratio of phosphorylated
4E-BP1 compared to total 4E-BP1, or in levels of 4EBP1 protein
compared to .beta.-actin. There is an increased sensitivity of
males to rapamycin treatment; ratios of phosphorylated 4E-BP1 to
total protein are statistically different relative to controls.
Also there is an increase in 4E-BP1 total proteins compared to
.beta.-actin. These data are consistent with cell-based studies
showing a differential inhibition of S6K1 and 4E-BP1, which is
cell-type-specific (Choo et al., 2008.). While rapamycin inhibits
S6K1 activity over the course of their experiments (24-48 hours),
4E-BP1 phosphorylation recovers within 6 hours.
[0150] FIG. 9 shows immunoblot assays of 4E-BP1 phosphorylation in
liver from mice chronically treated with rapamycin. Again,
consistent with cell-based experiments, there was no statistical
difference in the ratio 4E-BP1 phosphorylation in males or females,
in fact phosphorylation increased modestly in the five females
assayed in this experiment. Since .beta.-actin was not assayed in
these experiments, 4E-BP1 levels were not analyzed.
[0151] Note that 4E-BP2 is the dominant form of 4E-BP proteins
expressed in the brain (Banko et al., 2005). The immunological
reagents used above are specific for 4E-BP1, thus an analysis of
these translation repressors in the brain is pending development of
4E-BP2-specific antibodies.
[0152] In vivo evidence indicates that activation of S6K1 acts to
suppress insulin signaling through modulation of IRS1 (Um et al.,
2006). This predicts that rapamycin treatment de-represses this
signaling, leading to an increase in Akt phosphorylation.
Immunoblot results of an analysis of Akt phosphorylation in WAT
obtained from mice consuming rapamycin-containing food showed that
in females there is no difference in the level of phosphorylation
of Akt in response to rapamycin treatment. The results in males in
FIG. 10 a clear increase in phosphorylation of Akt compared to
controls. When combined, data on males and females is highly
significant.
[0153] FIG. 11 shows immunoassay data for Akt activation in liver
of UM-Het3 mice consuming food that contains rapamycin. As
documented in WAT above, we observe a significant increase in Akt
phosphorylation in male, but not female, liver.
[0154] FIG. 12 shows immunoassay data for Akt activation in brain
of UM-Het3 mice consuming food that contains rapamycin.
Interestingly, there appears to be a significant increase in Akt
phosphorylation in the rapamycin-treated mice, both males and
females.
Summarizing these immunoassays to determine the organ-specific
effects of chronic exposure to dietary rapamycin, all of the organs
tested show evidence of expected effects on down stream and
upstream mTORC1 effectors. For S6K1 activity, WAT appears to be
hypersensitive at the 7-ppm dose compared to liver. Male WAT
appears to be more sensitive than female. Brain S6K1 activity was
no different in rapamycin-treated mice compared to controls. For
4E-BP1 phosphorylation, there were little effects documented in any
tissue assayed, consistent with cell-based experiments showing
recovery of 4E-BP1 phosphorylation after 6-24 hours of treatment.
An unexpected increase in the levels of 4E-BP1 in male WAT was
documented. Akt activation was observed in male, but not female WAT
and liver. Brain Akt was elevated by rapamycin in both male and
females. Thus, there appears to be organ- and sex-specific
responses to the level of rapamycin tested, which is again
consistent with cell-based analyses of rapamycin effects. Based on
these results, it is concluded that dietary rapamycin is having the
expected biological effects on target organs tested.
Example 2
Studies to Examine Rapamycin Stability in Food
[0155] Studies were conducted to examine the stability of rapamycin
in food. Rapamycin was sent to the Southwest Research Institute
(San Antonio) for microencapsulation by dissolving the rapamycin in
an organic solvent containing a dissolved enteric coating, Eudragit
S100. This polymer is stable at pH levels below 7, as discussed in
Example 1. Samples of encapsulated and unencapsulated rapamycin
were incorporated into commercial mouse chow at a concentration of
0.7, 7, and 70 ppm and the levels of rapamycin in the food were
assayed (FIG. 13-14). The encapsulated rapamycin survived the
process of incorporation into the chow better than the
unencapsulated rapamycin, as demonstrated by the 3-fold higher
concentration of rapamycin detected in the diet made with
encapsulated rapamycin than in the diet made with unencapsulated
rapamycin. Diets made from encapsulated and unencapsulated
rapamycin were fed to mice for 4-5 weeks and concentrations of
rapamycin in 200 .mu.l of whole blood samples were determined using
HPLC with UV detection. The average blood level observed after
feeding the encapsulated rapamycin was greater than 25 ng/ml, which
compares favorably with therapeutic levels in human treatment
protocols of at least 12 ng/ml (FIG. 15). By contrast, mice fed the
diet prepared with unencapsulated rapamycin had less than 2.5
ng/ml, which is the detection limit of the assay. As a result, the
dose was increased to 14 ppm in the diet for the longevity studies
of Example 1.
Example 3
Rapamycin Rescues Cognition and Attenuates Pathology in Mouse
Models of Alzheimer Disease
Methods
[0156] Mice. Rapamycin administration and behavioral experiments
involving hAPP(J20) mice were conducted at the Buck Institute.
Experimental groups were: control-fed non-Tg, n=10; rapamycin-fed
non-Tg, n=10; control-fed Tg, n=12; rapamycin-fed Tg, n=12, all
animals were males and 7 mo. Rapamycin administration and
behavioral experiments involving 3.times.Tg-AD mice were conducted
at the UTHSCSA and experimental groups were: control-fed non-Tg,
n=13; rapamycin-fed non-Tg, n=14; control-fed 3.times.Tg-AD, n=14;
rapamycin-fed 3.times.Tg-AD, n=16; males and females were included
in equal proportions. The derivation and characterization of the
3.times.Tg-AD and hAPP(J20) mice have been described elsewhere
(Hsia et al., 1999; Mucke et al., 2000; Oddo et al., 2003). The
hAPP(J20) mice were maintained by heterozygous crossed with
C57BL/6J mice (Jackson Laboratories, Bar Harbor, Me.). The
hAPP(J20) mice were heterozygous with respect to the transgene.
Non-Tg littermates were used as controls. The 3.times.Tg-AD mice
were homozygous for the APP and tau transgenes and for the M146V
mutation knocked into the PS1 gene.
[0157] Rapamycin treatment. Mice were fed chow containing either
microencapsulated rapamycin at 2.24 mg/kg or a control diet as
described in Example 1. For the duration of the treatment, all mice
were given ad libitum access to rapamycin or control food and
water.
[0158] Behavioural testing. The MWM (protocol detailed in
Supplementary Information) was used to test spatial learning and
memory. The Morris water maze (MWM) (Morris, 1984) was used to test
spatial memory. All animals showed no deficiencies in swimming
abilities, directional swimming or climbing onto a cued platform
during pre-training and had no sensorimotor deficits as determined
with a battery of neurobehavioral tasks performed prior to testing.
All groups were assessed for swimming ability with a straight water
alley (15 by 200 cm) containing a submerged (1 cm) 12.times.12 cm
platform 2 days before testing. The procedure described by Morris
et al., 2006 was followed as described (Galvan et al., 2006; Galvan
et al., 2008). Briefly, the J20 mice were given a series of six
trials, one hour apart in a light-colored tank filled with opaque
water whitened by the addition of non-toxic paint at a temperature
of 24.0.+-.1.0.degree. C. In the visible portion of the protocol,
which tests non-spatial learning, animals were trained to find a
12.times.12-cm submerged platform (1 cm below water surface) that
was marked with a colored pole that served as a landmark and which
was placed in different quadrants of the pool. The animals were
lowered into the pool facing the pool wall and were released at
different locations in each trial. Each animal was given a maximum
of 60 seconds to find the submerged platform. If it did not find
the platform in that time, the animal was gently guided to it.
After remaining on the platform for 20 seconds, the animal was
removed and placed in a dry cage. Twenty minutes later, each animal
was given a second trial, using a different release position. This
process was repeated a total of 6 times for each mouse, with each
trial about 20 minutes apart. In the non-cued part of the protocol,
the water tank was surrounded by opaque dark panels at
approximately 30 cm from the edge of the pool. Four rectangular
drawings with geometric designs in black and white were evenly
spaced on the panels to serve as distal cues. The animals were
trained to find the submerged platform by swimming 6 times every
day for 2 days following the same procedure described for the cued
training above. These 6 trials were then followed by a probe trial
for which the platform was removed from the pool. In the probe
trial, each animal was allowed to swim for 30 seconds before being
removed. The percent of time spent in the area previously
containing the platform, as well as the number of times that each
animal crossed the previous platform location were determined as a
measure of platform location retention. Because rodents are good
swimmers and are monitored while in the water, they never drown and
do not suffer significant adverse effects from this test. During
the course of testing, animals were monitored daily, and their
weights are recorded weekly. Performance in all tasks was recorded
by a computer-based video tracking system (Water2020, HVS Image,
U.K). Data were analyzed offline by using HVS Image and processed
with Microsoft Excel. The MWM testing for the 3.times.Tg-AD mice
was conducted in a circular tank of 1.5 meters in diameter located
in a room with extra maze cues. The location of the platform (14 cm
in diameter) was kept constant for each mouse during training and
was 1.5 cm beneath the surface of the water, which was maintained
at 25.degree. C. throughout the duration of the testing. During
training, the mice received four trials a day that were alternated
among four pseudorandom starting points with a 25-second
intertribal interval. If a mouse failed to find the platform within
60 seconds, it was guided to the platform by the researcher and
kept there for 10 seconds. Probe trials were conducted twenty-four
hours after the last training trial. During the probe trials, the
platform was removed and mice were free to swim in the tank for
sixty seconds. The training and probe trials were recorded by a
video camera mounted on the ceiling and data were analyzed using
the EthoVisioXT tracking system.
[0159] Western blotting, A.beta. determinations and
immunohistochemistry. Tissue was processed and analyzed as
described previously 13, 25, 26 and is described in detail in
Supplementary Information. A.beta. and tau were measured using
specific ELISAs.
[0160] 3.times.Tg-AD mice were sacrificed by CO2 asphyxiation. The
brains were extracted and cut in-half sagitally and tissue was
processed as described (Oddo et al., 2008). The hAPP(J20) mice were
euthanized by isoflurane overdose. Hemibrains were flash frozen.
One hemibrain was homogeneized in liquid N2 while the other was
used in immunohistochemical determinations. For Western blot
analyses, proteins from both hAPP(J20) and 3.times.Tg-AD soluble
fractions were resolved by SDS/PAGE (Invitrogen, Temecula, Calif.)
under reducing conditions and transferred to a nitrocellulose or
PVDF membrane. The membrane was incubated in a 5% solution of
non-fat milk or in 5% BSA for 1 hour at 20.degree. C. After
overnight incubation at 4.degree. C. with the appropriate primary
antibody, the blots were washed in Tween 20-TBS (T-TBS) (0.02%
Tween 20, 100 mM Tris pH 7.5; 150 nM NaCl) for 20 minutes and
incubated at 20.degree. C. with secondary antibody. The blots were
then washed in T-TBS 3 times for 20 minutes each and then incubated
for 5 minutes with Super Signal (Pierce, Rockford, Ill.), washed
again and exposed to film. A.beta.40 and A.beta.42 levels were
measured from the soluble and insoluble fractions using a sandwich
ELISA protocol as described previously (Oddo et al., 2005).
A.beta.40 and A.beta.42 in hAPP(J20) mice were quantitated in
guanidine homogenates of Tg hAPP(J20) hemibrains as described
(Galvan et al., 2006) using specific ELISA assays (Invitrogen,
Carlsbad, Calif.).
[0161] Concerning immunohistochemistry, ten-micrometer cryosections
from snap-frozen brains were post-fixed in 4% paraformaldehyde and
stained with LC3-specific antibodies (10 .mu.g/ml, Nous, Littleton,
Colo.) followed by AlexaFluor488-conjugated donkey anti-rabbit IgG
(1:500, Molecular Probes, Invitrogen, CA), and imaged with a
epifluorescence microscope (Nikon Eclipse E800 with a FITC cube)
and with a laser scanning confocal microscope (Zeiss LSM 510) using
a 488 Argon laser and a 505 long pass filter. Images were obtained
using 20.times. and 60.times. objectives. The z-stacks of confocal
images were processed using LSM Viewer software (Zeiss). A.beta.
and tau immunohistochemistry was performed in 50 .mu.m thick
sections obtained using a vibratome slicing system and standard
protocols. Images were obtained with a digital Zeiss camera and
analyzed with ImageJ.
[0162] Statistical analyses. Statistical analyses were performed
using GraphPad Prism (GraphPad, San Diego, Calif.) and StatView. In
two-variable experiments, two-way ANOVA followed by Bonferroni's
post-hoc tests were used to evaluate the significance of
differences between group means. When analyzing one-variable
experiments with more than 2 groups, significance of differences
among means was evaluated using oneway ANOVA followed by Tukey's
post-hoc test. Evaluation of differences between two groups was
evaluated using Student's t test. Values of P<0.05 were
considered significant.
Results
[0163] Studies were conducted to determine whether rapamycin
prevents or delays age-associated disease such as AD. A
rapamycin-supplemented diet, which was identical to the diet that
extended lifespan in mice (as set forth in Example 1), or a control
chow was fed to the 3.times.Tg-AD and hAPP(J20) mice. Functional
and biochemical outcomes in two independent laboratories at
separate locations were measured. The 3.times.Tg-AD and hAPP(J20)
mice and the appropriate non-transgenic controls were treated for
10 and 12 weeks starting at 6.5 and 7 months of age, respectively.
At the end of treatment, learning and memory were tested using the
Morris water maze (MWM). Significant deficits in learning and
memory were observed in control-fed Tg animals (FIG. 28),
consistent with previous observations in both mouse models (Oddo et
al., 2008; Galvan et al., 2006; Galvan et al., 2008; Saganich et
al., 2006; Billings et al., 2005). Rapamycin-fed Tg mice, however,
showed improved learning and memory (FIG. 28). Remarkably, in the
rapamycin-fed Tg mice, retention of the former location of the
escape platform was restored to levels indistinguishable from those
of non-Tg mice in both mouse models (FIG. 28B, 28D). Taken
together, these data indicate that rapamycin treatment can
ameliorate learning deficits and abolish memory impairments in two
independent mouse models of AD. At the end of the behavioral
assessment, all mice were euthanized and their brains were isolated
and processed for neuropathological or biochemical evaluation. To
elucidate the mechanism underlying the improvement in learning and
memory in the transgenic mice treated with rapamycin, APP
processing by Western blots was analyzed. The levels of full-length
APP from transgenic mice on the rapamycin or control diet using
22C11 (an N-terminal specific APP antibody) was first measured. It
was found that APP steady-state levels were not significantly
altered by rapamycin administration (FIG. 29A, 29C). To investigate
the steady-state levels of the major C-terminal derivatives,
protein extracts were probed with a C terminal-specific APP
antibody. The results indicate that the levels of C99 and C83 were
unchanged after rapamycin administration in both transgenic lines
(FIG. 29A, 29B, 29D, 29E). These results indicate that rapamycin
administration did not alter APP steady-state levels or its
processing in either transgenic mouse. At the end of treatment,
3.times.Tg-AD and hAPP(J20) mice were 8 and 7 months old,
respectively. At this age both transgenic mice show an increase in
soluble A.beta. levels with 3.times.Tg-AD mice also showing an
accumulation of intraneuronal A.beta.8, 12, 17. Previous studies
have shown that extracellular A.beta. deposits are not apparent at
this age in either transgenic line (Hsia et al., 1999; Oddo et al.,
2003). While A.beta.40 levels remained unchanged, it was found that
rapamycin significantly decreased soluble A.beta.42 levels by
32.78.+-.6.68% in brains of 3.times.Tg-AD mice and by
52.35.+-.13.14% in brains of hAPP(J20) mice (FIG. 29F, 29G, 29H).
The levels of insoluble A.beta.40 and A.beta.42 were below
detection in both transgenic mouse models, consistent with previous
reports (Hsia et al., 1999; Mucke et al., 2000; Oddo et al., 2003).
To determine whether intracellular A.beta. accumulation was
affected by rapamycin, hippocampal sections from treated and
untreated 3.times.Tg-AD brains were immunostained with an
A.beta.-specific antibody. The results indicate a significant
decrease in the number of the A.beta.-positive neurons in the
hippomampi of rapamycin-treated 3.times.Tg-AD mice as compared to
control-fed 3.times.Tg-AD mice (FIG. 29H, 29I). In addition to
A.beta. accumulation, 3.times.Tg-AD mice develop an age-dependent
accumulation of phosphorylated and aggregated tau (Oddo et al.,
2003a; Oddo et al., 2003b; Oddo et al., 2007). At 8 months of age,
3.times.Tg-AD mice showed somatodendritic accumulation of soluble
tau species that are phosphorylated at different epitopes in CA1
pyramidal neurons. Following rapamycin administration, a marked
reduction in tau immunoreactivity was observed using the anti-tau
antibodies AT270 and MC-1, which recognize tau phosphorylated at
Thr181 and a conformational change in tau, respectively (FIG. 30A,
30B, 30C, 30D). These changes in tau are thought to occur early in
the disease process. While MC1-positive neurons become apparent at
this age in the hippocampi of 3.times.Tg-AD mice (FIG. 30E), no
MC1-positive neurons were detected in rapamycin-treated mice (FIG.
30E, 30F). The immunohistochemical data were also confirmed by
Western blot analysis (FIG. 30G, 30H). To better quantify the
changes in tau we measured soluble and insoluble tau levels by
sandwich ELISA and found that rapamycin selectively decreased
soluble tau levels (FIG. 30I). Taken together, these data indicate
that early tau pathology in 8-month-old 3.times.Tg-AD mice is
significantly decreased after rapamycin administration. The
decrease in A.beta. and tau pathology may be due to a decrease in
their production or to an increase in their degradation. The data
presented here indicate that the rapamycin-mediated reduction in
A.beta. and tau levels is not due to changes in production because
the steady-state levels of C99/C83 (resulting from cleavage of APP
by .beta.- and .alpha.-secretase respectively) as well as the tau
transgene were not altered.
[0164] To better understand the mechanism underlying the
rapamycin-mediated reduction in A.beta. and tau pathology,
autophagy, a major cellular degradation pathway, was measured.
While the specific mechanisms underlying autophagy induction are
still being investigated, the current data indicate a series of
proteins known as autophagy-related proteins (Atg) (Mizushima et
al., 1998). The formation of a covalent complex between two
autophagy-related proteins, Atg5 and Atg12 appears to be essential
for autophagy induction (Mizushima et al., 1998; Suzuki et al.,
2001). The formation of this complex is regulated by Atg7 and
Atg10. Autophagy induction can also be monitored by measuring the
levels of light chain 3 II (LC3-II), which is incorporated in the
autophagosome membrane during its formation (Kabeya et al., 2000).
It was found that the levels of Atg7 and the Atg5/Atg12 complex
were significantly increased in rapamycin-treated transgenic mice
compared to mice on the control diet (FIG. 31A, 31B, 31C, 31D,
31E), indicating a rapamycin-mediated increase in autophagy. The
increase in autophagy was further confirmed by a significant
increase in the total levels of LC3-II, as determined by
Western blots and by an increase in LC3 immunoreactivity in
hippocampal sections (FIG. 31A, 31E, 31F). While we cannot exclude
other mechanisms that may be involved in the rapamycin-mediated
decrease in A.beta. and tau levels, these data support the
involvement of autophagy in the amelioration of the AD-like
neuropathological phenotype in both animal models.
[0165] A decrease in A.beta. levels may also contribute to the
observed amelioration in tau pathology in 3.times.Tg-AD mice
because it has been shown that lowering A.beta. reduces tau
pathology (Oddo et al., 2008; Oddo et al., 2006; Oddo et al.,
2004). These data are consistent with a recent report in transgenic
mice showing that decreasing autophagy increases A.beta. levels
while increasing autophagy decreases A.beta. levels (Pickford et
al., 2008). These results, obtained from two independent
laboratories, show that rapamycin has a robust protective effect on
the development of AD-like neuropathology and rescues the loss of
memory in two very different transgenic mouse models of AD. These
data show that rapamycin, at a dose that extended lifespan in mice,
increases autophagy and reduces AD pathology.
Example 4
Delayed Onset or Less Severe Cancer Contributes to Extended
Longevity in Het3 Mice Chronically Treated with Enterically
Delivered Rapamycin
[0166] New data on mTORC1 signaling in Het3 mice chronically
treated with enterically delivered rapamycin that is consistent
with delayed onset or less severe cancer as one mechanisms
contributing to extended longevity.
[0167] Since cancer primarily strikes people with a median age of
68 (Edwards et al., 2002), elderly individuals are at greater risk
for this disease. In light of this demographic, it is significant
that chronic treatment with rapamycin beginning at 20 months of age
(60 in human years) extended the life span in the genetically
heterogenous mice tested; the primary cause of death was cancer as
set forth in Example 1. Thus technology for the prevention of
clinically manifested cancer in this population is a goal of cancer
research worldwide.
[0168] For clinical applications, a major concern is that chronic
application of rapamycin or rapalogs in a cancer prevention
protocol may result in an increase in Akt Ser463 phosphorylation,
which, as a pro growth stimulus (reviewed in Guertin and Sabatini
2009; Lane and Breuleux, 2009), would counteract any repressive
effect. Recent immunoblot data from our lab indicates that this
does not happen in normal fat and skeletal muscle in a long-term
treatment setting. To illustrate, FIG. 32 shows immunoblot assays
of visceral fat dissected from mice consuming food with rapamycin
for 5 weeks. There was a significant induction in Akt Ser473
phosphorylation in response to this relatively short treatment.
[0169] In contrast, visceral fat from mice treated with rapamycin
for 20 months does not show this activation, and in males is
significantly reduced (FIG. 33). The same trend is seen in skeletal
muscle (FIG. 34).
[0170] These data suggest that chronic treatment with enterically
delivered rapamycin does not enhance tumor promoting activation of
Akt, in somatic tissues but rather may reduce it.
[0171] If chronic treatment with enteric rapamycin delays cancer or
reduces its severity so that it does not present symptomatically
until very late in life, one prediction is that the
growth-promoting potential of mTORC1 signaling should be repressed
in treated mice. In two of monitor mice from cohort 3, two males
each with hepatocellular carcinoma were analyzed, one
rapamycin-treated the other a control.
[0172] FIG. 35 shows immunoassay data from these two tumors, which
indicate that chronic enteric rapamycin is significantly repressing
the phosphorylation of Thr389 by mTORC1. Thus inhibition of this
mTORC1 effector strongly suggests that delayed onset of or less
severe cancer is a major mechanism of extended lifespan in mice
consuming rapamycin chow. This is also consistent with tumor
responses to in calorically and growth factor (dwarf mice)
restricted mice. In sum, these data strongly support the concept
that prevention of cancer presentation in moderately elderly people
by enterically-delivered rapamycin is feasible.
[0173] All of the microcapsules, methods, and kits disclosed and
claimed herein can be made and executed without undue
experimentation in light of the present disclosure. While the
microcapsules, methods, and kits of this invention have been
described in terms of preferred embodiments, it will be apparent to
those of skill in the art that variations may be applied without
departing from the concept, spirit and scope of the invention. More
specifically, it will be apparent that certain agents which are
both chemically and physiologically related may be substituted for
the agents described herein while the same or similar results would
be achieved. All such similar substitutes and modifications
apparent to those skilled in the art are deemed to be within the
spirit, scope and concept of the invention as defined by the
appended claims.
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