U.S. patent application number 14/761283 was filed with the patent office on 2015-12-17 for treating pulmonary conditions.
The applicant listed for this patent is NUSIRT SCIENCES, INC.. Invention is credited to Brooke BAGGETT, Antje BRUCKBAUER, Michael ZEMEL.
Application Number | 20150359771 14/761283 |
Document ID | / |
Family ID | 51210014 |
Filed Date | 2015-12-17 |
United States Patent
Application |
20150359771 |
Kind Code |
A1 |
ZEMEL; Michael ; et
al. |
December 17, 2015 |
TREATING PULMONARY CONDITIONS
Abstract
Compositions, methods, and kits useful for treating pulmonary
conditions are provided herein. Such compositions can contain
synergizing amounts of a non-specific phosphodiesterase inhibitor,
such as a methylxanthine, in combination with leucine and/or a
leucine metabolite, and resveratrol.
Inventors: |
ZEMEL; Michael; (Knoxville,
TN) ; BRUCKBAUER; Antje; (Knoxville, TN) ;
BAGGETT; Brooke; (Knoxville, TN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NUSIRT SCIENCES, INC. |
Nashville |
TN |
US |
|
|
Family ID: |
51210014 |
Appl. No.: |
14/761283 |
Filed: |
January 14, 2014 |
PCT Filed: |
January 14, 2014 |
PCT NO: |
PCT/US14/11531 |
371 Date: |
July 15, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61752909 |
Jan 15, 2013 |
|
|
|
Current U.S.
Class: |
514/263.34 ;
435/366; 435/375 |
Current CPC
Class: |
A61K 31/19 20130101;
A61K 31/05 20130101; A61K 31/522 20130101; A61P 11/06 20180101;
A61K 31/522 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61P 11/00 20180101; A61K 31/198 20130101;
A61K 31/198 20130101; A61K 31/05 20130101 |
International
Class: |
A61K 31/198 20060101
A61K031/198; A61K 31/05 20060101 A61K031/05; A61K 31/522 20060101
A61K031/522 |
Claims
1. A composition comprising: (a) leucine and/or one or more leucine
metabolites selected from the group consisting of keto-isocaproic
acid (KIC), alpha-hydroxy-isocaproic acid, and
hydroxymethylbutyrate (HMB); and (b) a methylxanthine; wherein the
composition comprises at least about 250 mg of leucine and/or at
least about 10 mg of the one or more leucine metabolites.
2. The composition of claim 1, further comprising resveratrol.
3. The composition of claim 1, wherein the composition comprises at
least about 500 mg of leucine.
4. The composition of claim 1, wherein the composition comprises
between about 250-1500 mg of leucine.
5. The composition of claim 1, wherein the composition comprises at
least about 200 mg of leucine metabolites.
6. The composition of claim 1, wherein the composition comprises
between about 10-750 mg of leucine metabolites.
7. The composition of claim 1, wherein the methylxanthine is
selected from the group consisting of theophylline and
theobromine.
8. The composition of claim 7, wherein the composition comprises at
least about 5 mg of theophylline.
9. The composition of claim 7, wherein the composition comprises
between about 1-100 or 5-50 mg of theophylline.
10. The composition of claim 1, wherein the composition comprises a
sub-therapeutic amount of the methylxanthine.
11. The composition of claim 7, wherein the composition comprises a
sub-therapeutic amount of theophylline.
12. The composition of claim 2, wherein the composition comprises
at least about 35 mg of resveratrol.
13. The composition of claim 2, wherein the composition comprises
between about 5-500 or 30-250 mg of resveratrol.
14. The composition of claim 1, wherein the composition is
substantially free of non-branched amino acids.
15. The composition of claim 1, wherein the amino acids in the
composition are substantially free of non-branched amino acids.
16. The composition of claim 1, wherein the percent of non-branched
amino acids relative to total amino acids in the composition is
less than about 0.1, 1, or 10%.
17. The composition of claim 1, wherein the composition is a unit
dosage.
18. The composition of claim 1, wherein the composition is
formulated for oral dosing, inhalation or intravenous delivery.
19. The composition of claim 1, wherein the composition is
formulated for sustained release to effect a circulating level of
greater than a desired circulating level over a time period of at
least about 4, 6, 12, 24, 36, or 48 hours.
20. The composition of claim 19, wherein the sustained release
formulation effects a circulating level of methylxanthine in a
subject that is greater than about 1 .mu.M for the time period.
21. A composition comprising: (a) leucine and/or one or more
leucine metabolites selected from the group consisting of
keto-isocaproic acid (KIC), alpha-hydroxy-isocaproic acid, and
hydroxymethylbutyrate (HMB); and (b) a methylxanthine; wherein the
mass ratio of (a) to (b) is at least about 15, 25, 50, 75, or 100,
and wherein the composition comprises at least about 5 mg of the
methylxanthine.
22. The composition of claim 20, further comprising at least about
10 mg of resveratrol.
23. The composition of claim 20, wherein the methylxanthine is
theophylline or theobromine.
24. A composition comprising: (a) leucine and/or one or more
leucine metabolites selected from the group consisting of
keto-isocaproic acid (KIC), alpha-hydroxy-isocaproic acid, and
hydroxymethylbutyrate (HMB); and (b) a methylxanthine; wherein the
molar ratio of (a) to (b) is at least about 400, 500, 750, or 1000,
and wherein the composition comprises at least about 0.05 .mu.g of
the methylxanthine.
25. The composition of claim 24, further comprising at least about
0.01 .mu.g of resveratrol.
26. The composition of claim 24, wherein the methylxanthine is
theophylline or theobromine.
27. The composition of claim 24, wherein the composition is
formulated as a unit dosage for inhalation.
28. The composition of claim 27, wherein the composition is
packaged in an inhaler.
29. The composition of claim 28, wherein the inhaler can comprise
at least about 20, 50, 200, or 1000 unit dosages of the
composition.
30. A composition comprising: (a) leucine; and (b) a
methylxanthine, wherein component (a) and (b) are present in an
amount effective to achieve a circulating level of about 0.3-2 mM
leucine and about 0.5-10 .mu.M methylxanthine in a subject.
31. The composition of claim 24, wherein the amounts of (a) and (b)
are selected to induce a circulating level of about 0.7-2 mM
leucine and about 0.7-3 .mu.M methylxanthine in a subject.
32. The composition of claim 24, wherein the subject is a human, a
domesticated animal, or a farm animal.
33. A composition comprising: (a) leucine; and (b) a
methylxanthine, wherein component (a) and (b) are present in an
amount that is effective in increasing expression level or
secretion of one or more inflammatory markers in a lung endothelial
cell selected from the group consisting of NF.kappa.B, eotaxin,
IL1-.beta., and IL6, or reducing expression level or secretion of
one or more anti-inflammatory markers in a lung endothelial cell
selected from the group consisting of adiponectin receptor 1 and
adiponectin receptor 2.
34. The composition of claim 33, wherein (b) is present in an
amount less than the amount required to achieve a circulating level
of about 40 .mu.M.
35. A method of treating pulmonary conditions in a subject in need
of treatment comprising administering to the subject a composition
of any one of the preceding claims.
36. A method of increasing expression level or secretion of an
inflammatory marker selected from the group consisting of
NF.kappa.B, eotaxin, IL1-.beta., and IL6, or reducing expression
level or secretion of an anti-inflammatory marker selected from the
group consisting of adiponectin receptor 1 and adiponectin receptor
2, the method comprising contacting a lung endothelial cell with a
composition of any one of the preceding claims to effect said
reduction and/or increase in expression level or secretion of said
inflammatory or said anti-inflammatory marker.
37. The method of claim 36, wherein the subject is a human, a
domesticated animal, or a farm animal.
38. The composition of claims 20, 24, or 33, wherein the
composition is formulated as a unit dosage.
39. A kit comprising a multi-day supply of unit dosages of the
composition of claims 1, 20, 24, or 33, and instructions directing
the administration of said multi-day supply over a period of
multiple days.
Description
CROSS-REFERENCE
[0001] This application claims benefit of priority to U.S.
Provisional Application No. 61/752,909, filed Jan. 15, 2013, which
is incorporated herein by reference in its entirety for all
purposes.
BACKGROUND OF THE INVENTION
[0002] Pulmonary diseases or conditions span a range of
lung-related diseases, including asthma and chronic obstructive
pulmonary disease (COPD), which affect millions of people
throughout the world.
[0003] For example, over 300 million people worldwide suffering
from asthma. It is predicted that the prevalence will increase to
about 400 million in the next decade. Asthma is a chronic airway
disorder identified by recurrent wheeze and intermittent air flow
limitation. It is characterized by airway inflammation, mucus
hypersecretion, and airway hyperresponsiveness (AHR). Studies have
shown that these clinical manifestations are, at least in part,
inflammatory responses mediated by T-helper type 2 (Th2) cells
together with mast cells, B cells and eosinophils, as well as a
number of inflammatory cytokines and chemokines.
[0004] Currently, there are three anti-inflammatory agents for
controlling asthma, which include inhaled steroids,
cysteinyl-leukotriene receptor antagonist and cromolyn. However,
the therapeutic efficacies of cysteinyl-leukotriene receptor
antagonist and cromolyn are highly variable and may be limited to
certain subgroup of patients. In addition, 5-10% of the asthmatics
are not well-controlled by current drug treatment and they require
oral steroids during exacerbation. Oral steroid usage is commonly
associated with a diversity of adverse effects, most notably
increases in appetite, stomach ulcers, difficulty sleeping
(insomnia), changes in mood and behavior, flushing (redness) of the
face, and short-term weight gain due to increased water retention.
If taken for long periods of time, steroid use may lead to
glaucoma, cataracts, high-blood pressure, heart disease, diabetes
mellitus, obesity, acid reflux/GERD, osteoporosis, myopathy,
increase in certain types of infections, and cushing syndrome.
[0005] COPD is a medical condition that is generally considered to
include one or both of chronic bronchitis and emphysema. Chronic
bronchitis is characterized by a persistent (such as more than one
year) productive cough that is not due to a medically defined cause
such as a microbial infection or carcinoma. Emphysema is an
abnormal permanent non-uniform enlargement of air spaces distal to
the terminal bronchioles, including destruction of the walls of the
air spaces. COPD is caused by noxious particles or gas, most
commonly from tobacco smoking, which triggers an abnormal
inflammatory response in the lung. The natural course of COPD is
characterized by occasional sudden worsening of symptoms called
acute exacerbations, most of which are caused by infections or air
pollution.
[0006] There is currently no cure for COPD and the only measures
that have been shown to reduce mortality are smoking cessation and
supplemental oxygen. COPD is treated with bronchodilators such as
beta-2 agonists and/or anticholinergics. Beta-2 agonist stimulate
beta-2 receptors while anticholinergics block stimulation from
cholinergic nerves both are medicines that relax smooth muscle
around the airways, increasing air flow. While these agents can
ameliorate certain symptoms to some degree, they are not effective
to halt progression of COPD.
[0007] Methylxanthines (a class of derivatives of xanthine and
alkaloids) have often been used as bronchodilators. Methylxanthines
relax smooth muscle, stimulate the central nervous system,
stimulate cardiac muscle, and act on the kidneys to promote
diuresis. Their usefulness in promoting relaxation of bronchial
smooth muscle is of benefit in the management of asthma.
[0008] Methylxanthine theophylline is an established medicament for
therapy of obstructive diseases of the respiratory tract.
Theophylline is a competitive but non-selective inhibitor of
several types of phosphodiesterases, the enzymes that degrade cAMP.
Increased concentrations of cAMP may mediate the observed
bronchodilation. Other proposed mechanisms of action of
theophylline include inhibition of the release of intracellular
calcium and competitive antagonism of the bronchoconstrictor
adenosine.
[0009] Theophylline is a relatively low-cost treatment and can be
administered in a sustained-release preparation that gives a
duration of around 12 hours. However, theophylline has a number of
side effects. The adverse gastrointestinal effects of theophylline
include nausea, vomiting, abdominal pain, cramping, and diarrhea.
Adverse central nervous system effects include insomnia, headache,
dizziness, nervousness, and seizures, which are often more severe
in children. Seizures may occur as the initial sign of theophylline
toxicity without any other preceding signs and symptoms. Increased
tremor in the patient's dominant hand has been reported.
Cardiovascular and pulmonary adverse effects include tachycardia,
arrhythmias, and tachypnea. Because of these toxicities,
theophylline is often used as a second or third line asthma
medication.
SUMMARY OF THE INVENTION
[0010] There remains a considerable need for low-cost therapy that
can effectively and safely treat pulmonary conditions. The present
invention addresses this need and provides related advantages as
well.
[0011] The subject application provides compositions, methods, and
kits useful for treating pulmonary conditions, including but not
limited to asthma and chronic obstructive pulmonary disease. The
subject compositions are particularly effective in reducing
expression and/or secretion of an inflammatory marker or increasing
the expression and/or secretion of an anti-inflammatory marker
associated with the inflammatory responses elicited during onset of
asthma or chronic obstructive pulmonary disease.
[0012] In one aspect of the invention, the subject composition
comprises leucine and/or one or more leucine metabolites selected
from the group consisting of keto-isocaproic acid (KIC),
alpha-hydroxy-isocaproic acid, and hydroxymethylbutyrate (HMB); and
a methylxanthine, wherein the composition comprises at least about
250 mg of leucine and/or at least about 100 mg of the one or more
leucine metabolites. In one aspect of the invention, the subject
composition comprises leucine and/or one or more leucine
metabolites selected from the group consisting of keto-isocaproic
acid (KIC), alpha-hydroxy-isocaproic acid, and
hydroxymethylbutyrate (HMB); and a methylxanthine, wherein the
composition comprises at least about 250 mg of leucine and/or at
least about 10 or 50 mg of the one or more leucine metabolites. The
composition can further comprise resveratrol.
[0013] In some embodiments, the composition comprises at least
about 500 mg of leucine. The composition can comprise between about
250-1500 mg of leucine. The composition can comprise at least about
200 mg of leucine metabolites. The composition can comprise between
about 100-750 mg of leucine metabolites. The composition can
comprise between about 10-750 or 50-750 mg of leucine
metabolites.
[0014] In another embodiment, the methylxanthine is selected from
the group consisting of theophylline and theobromine. The
methylxanthine may be present in a sub-therapeutic amount. The
composition may comprise at least about 5 mg of theophylline. The
composition can comprise between about 1-100 or 5-50 mg of
theophylline. The composition can comprise a sub-therapeutic amount
of theophylline.
[0015] In one embodiment, the composition comprises at least about
35 mg of resveratrol. The composition can comprise between about
5-500 or 30-250 mg of resveratrol.
[0016] In yet another embodiment, the composition is substantially
free of non-branched amino acids. The amino acids in the
composition can be substantially free of non-branched amino acids.
The percent of non-branched amino acids relative to total amino
acids in the composition can be less than about 0.1, 1, or 10%.
[0017] In some embodiments, the composition is a unit dosage. The
composition can be formulated for oral dosing, inhalation, or
intravenous delivery. The composition can be formulated for
sustained release over a period of at least about 1, 4, 6, 12, 24,
36, or 48 hours. The sustained release formulations may maintain a
desired circulating level of one or more components of the
composition over a period of at least about 1, 4, 6, 12, 24, 36, or
48 hours. The sustained release formulations may effect a
circulating level greater than a desired circulating level over a
period of at least about 4, 6, 12, 24, 36, or 48 hours. The desired
circulating level may be for any component of the compositions. The
component may be a methylxanthine, such as theophylline. In some
embodiments, the sustained release composition maintains the level
of the methylxanthine at greater than about 1 .mu.M for the
specified time period. The sustained release formulation can effect
a circulating level of methylxanthine in a subject that is greater
than about 1 .mu.M for the time period. The sustained release
formulation can effect a circulating level of methylxanthine in a
subject that is between about 1-10, 1-20, 1-30, or 1-40 .mu.M for
the time period.
[0018] In another aspect of the invention, the subject composition
comprises (a) leucine and/or one or more leucine metabolites
selected from the group consisting of keto-isocaproic acid (KIC),
alpha-hydroxy-isocaproic acid, and hydroxymethylbutyrate (HMB); and
(b) a methylxanthine, wherein the mass ratio of (a) to (b) is at
least about 15, 25, 50, 75, or 100, and wherein the composition
comprises at least about 5 mg of the methylxanthine. The
composition can further comprise at least about 10 mg of
resveratrol. In some embodiments, the methylxanthine is
theophylline or theobromine. The composition may be formulated as a
unit dosage.
[0019] In one aspect of the invention, the subject composition
comprises (a) leucine and/or one or more leucine metabolites
selected from the group consisting of keto-isocaproic acid (KIC),
alpha-hydroxy-isocaproic acid, and hydroxymethylbutyrate (HMB); and
(b) a methylxanthine, wherein the molar ratio of (a) to (b) is at
least about 400, 500, 750, or 1000, and wherein the composition
comprises at least about 0.05, 0.1, 0.5, 1, or 2 .mu.g of the
methylxanthine. The composition can comprise at least about 0.05,
0.1, 0.5, 1, or 2 mg of the methylxanthine. The composition can
further comprise at least about 15, 30, 50, 100, or 500 .mu.g of
leucine. The composition can further comprise at least about 5, 10,
15, 30, 50, 100, or 500 mg of leucine. The composition can further
comprise at least about 0.01, 0.05, 0.1, 0.5, or 1 .mu.g of
resveratrol. The composition can further comprise at least about
0.01, 0.05, 0.1, 0.5, or 1 mg of resveratrol. In some embodiments,
the methylxanthine is theophylline or theobromine. The composition
may be formulated as a unit dosage for inhalation. The composition
may be packaged in an inhaler. The inhaler can comprise at least
about 20, 50, 200, or 1000 unit dosages of the composition.
[0020] In yet another aspect of the invention, the subject
composition comprises (a) leucine; and (b) a methylxanthine,
wherein component (a) and (b) are present in an amount effective to
achieve a circulating level of about 0.3-2 mM leucine and about
0.5-10 .mu.M methylxanthine in a subject. The amounts of (a) and
(b) can be selected to induce a circulating level of about 0.7-2 mM
leucine and about 0.7-3 .mu.M methylxanthine in a subject. The
subject can be a human, a domesticated animal, or a farm
animal.
[0021] In another aspect, the subject composition comprises (a)
leucine; and (b) a methylxanthine, wherein component (a) and (b)
are present in an amount that is effective in improving a pulmonary
condition by reducing expression level or secretion of one or more
inflammatory markers in a lung endothelial cell selected from the
group consisting of NF.kappa.B, eotaxin, IL1-.beta., and IL6 or
increasing expression level or secretion of one or more
anti-inflammatory markers in a lung endothelial cell selected from
the group consisting of adiponectin receptor 1 and adiponectin
receptor 2.
[0022] In still another aspect, the subject composition comprises
(a) leucine; and (b) a methylxanthine, wherein component (a) and
(b) are present in an amount that is effective in reducing
expression level or secretion of one or more inflammatory markers
in a lung endothelial cell selected from the group consisting of
NF.kappa.B, eotaxin, IL1-.beta., and IL6, or increasing expression
level or secretion of one or more anti-inflammatory markers in a
lung endothelial cell selected from the group consisting of
adiponectin receptor 1 and adiponectin receptor 2. The amount of
(b) may be a sub-therapeutic amount. Alternatively, the amount of
(b) may be less than an amount required to achieve a circulating
level of about 40 .mu.M.
[0023] The invention also provides for a method of treating
pulmonary conditions in a subject in need of treatment comprising
administering to the subject a composition of any one of the
subject compositions.
[0024] In another aspect, the invention provides for a method of
reducing expression level or secretion of an inflammatory marker
selected from the group consisting of NF.kappa.B, eotaxin,
IL1-.beta., and IL6 or increasing expression level or secretion of
an anti-inflammatory marker selected from the group consisting of
adiponectin receptor 1 and adiponectin receptor 2, comprising
contacting a lung endothelial cell with a composition of any one of
the subject compositions described herein to effect said reduction
or increase in expression level or secretion of said inflammatory
marker or said anti-inflammatory marker. The subject can be a
human, a domesticated animal, or a farm animal.
[0025] In yet another aspect, the invention provides for a kit
comprising a multi-day supply of unit dosages of a subject
composition described herein, and instructions directing the
administration of said multi-day supply over a period of multiple
days.
INCORPORATION BY REFERENCE
[0026] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawing(s) of which:
[0028] FIG. 1 depicts the interactive effects of theophylline with
leucine and resveratrol on NF.kappa.B protein expression in mouse
lung endothelial cells. *p<0.02 vs. control.
[0029] FIG. 2 depicts the interactive effects of theophylline with
leucine and resveratrol (200 nM) conditioned adipocyte media on
NF.kappa.B protein expression in mouse lung endothelial cells.
[0030] FIG. 3 depicts the interactive effects of theophylline with
leucine and resveratrol conditioned adipocyte media on
phospho-NF.kappa.B protein expression in mouse lung endothelial
cells.
[0031] FIG. 4 depicts the interactive effects of theophylline with
leucine and resveratrol on IL1-.beta. from mouse lung endothelial
cells. *p<0.001 vs. all other treatments. TNF.alpha. used as a
positive control to stimulate secretion of the cytokine;
**indicates significant increase vs. all other treatments
(p<0.01).
[0032] FIG. 5 depicts the interactive effects of theophylline with
leucine and resveratrol on eotaxin secretion from mouse lung
endothelial cells. *p<0.05 vs. all other treatments. TNF.alpha.
used as a positive control to stimulate secretion of the
cytokine.
[0033] FIG. 6 depicts the interactive effects of theophylline with
leucine and resveratrol conditioned adipocyte media on eotaxin
secretion from mouse lung endothelial cells.
[0034] FIG. 7 depicts the interactive effects of theophylline with
leucine and resveratrol on cellular IL 6 content in mouse lung
endothelial cells. *p<0.02 vs. control.
[0035] FIG. 8 depicts the interactive effects of theophylline with
leucine and resveratrol on IL 6 secretion from mouse lung
endothelial cells. *p<0.0001 vs. all other treatments.
TNF.alpha. used as a positive control to stimulate secretion of the
cytokine; **indicates significant increase vs. all other
treatments.
[0036] FIG. 9 depicts the interactive effects of theophylline with
leucine and resveratrol conditioned adipocyte media on IL 6
secretion from mouse lung endothelial cells.
[0037] FIG. 10 depicts the interactive effects of theophylline with
leucine and resveratrol on adiponectin receptor 1 protein
expression in mouse lung endothelial cells.
[0038] FIG. 11 depicts the interactive effects of theophylline with
leucine and resveratrol on adiponectin receptor 2 protein
expression in mouse lung endothelial cells.
[0039] FIG. 12 depicts the interactive effects of theophylline with
leucine and resveratrol conditioned adipocyte media on adiponectin
receptor 2 protein expression in mouse lung endothelial cells.
*p=0.018 vs. all other treatments.
[0040] FIG. 13 depicts the interactive effects of theobromine with
leucine and resveratrol conditioned adipocyte media on adiponectin
receptor 1 protein expression in mouse lung endothelial cells.
*p=0.03 vs. all other treatments; **p=0.0005 vs. all other
treatments.
[0041] FIG. 14 depicts the interactive effects of theobromine with
leucine and resveratrol conditioned adipocyte media on adiponectin
receptor protein expression in mouse lung endothelial cells.
*p=0.0001 vs. all other treatments.
DETAILED DESCRIPTION OF THE INVENTION
[0042] Several aspects of the invention are described below with
reference to example applications for illustration. It should be
understood that numerous specific details, relationships, and
methods are set forth to provide a full understanding of the
invention. One having ordinary skill in the relevant art, however,
will readily recognize that the invention can be practiced without
one or more of the specific details or with other methods. Unless
stated otherwise, the present invention is not limited by the
illustrated ordering of acts or events, as some acts may occur in
different orders and/or concurrently with other acts or events.
Furthermore, not all illustrated acts or events are required to
implement a methodology in accordance with the present invention.
The concentration of various components in the disclosed
compositions are exemplary and not meant to be limited to the
recited concentration per se.
[0043] As used herein, the term "subject" or "individual" includes
mammals. Non-limiting examples of mammals include humans and mice,
including transgenic and non-transgenic mice. The methods described
herein can be useful in both human therapeutics, pre-clinical, and
veterinary applications. In some embodiments, the subject is a
mammal, and in some embodiments, the subject is human. Other
mammals include, and are not limited to, apes, chimpanzees,
orangutans, monkeys; domesticated animals (pets) such as dogs,
cats, guinea pigs, hamsters, mice, rats, rabbits, and ferrets;
domesticated farm animals such as cows, buffalo, bison, horses,
donkey, swine, sheep, and goats; or exotic animals typically found
in zoos, such as bear, lions, tigers, panthers, elephants,
hippopotamus, rhinoceros, giraffes, antelopes, sloth, gazelles,
zebras, wildebeests, prairie dogs, koala bears, kangaroo, pandas,
giant pandas, hyena, seals, sea lions, and elephant seals.
[0044] The terms "administer", "administered", "administers" and
"administering" are defined as the providing a composition to a
subject via a route known in the art, including but not limited to
intravenous, intraarterial, oral, nasal, inhaled, parenteral,
buccal, topical, transdermal, rectal, intramuscular, subcutaneous,
intraosseous, transmucosal, or intraperitoneal routes of
administration. In certain embodiments of the subject application,
oral routes of administering a composition may be preferred.
[0045] As used herein, "agent" or "biologically active agent"
refers to a biological, pharmaceutical, or chemical compound or
other moiety. Non-limiting examples include simple or complex
organic or inorganic molecule, a peptide, a protein, a peptide
nucleic acid (PNA), an oligonucleotide (including e.g., aptomer and
polynucleotides), an antibody, an antibody derivative, antibody
fragment, a vitamin derivative, a carbohydrate, a toxin, or a
chemotherapeutic compound. Various compounds can be synthesized,
for example, small molecules and oligomers (e.g., oligopeptides and
oligonucleotides), and synthetic organic compounds based on various
core structures. In addition, various natural sources can provide
compounds for screening, such as plant or animal extracts, and the
like. A skilled artisan can readily recognize that there is no
limit as to the structural nature of the agents of the present
invention.
[0046] The term "effective amount" or "therapeutically effective
amount" refers to that amount of a compound described herein that
is sufficient to effect the intended application including but not
limited to disease treatment, as defined below. The therapeutically
effective amount may vary depending upon the intended application
(in vitro or in vivo), or the subject and disease condition being
treated, e.g., the weight and age of the subject, the severity of
the disease condition, the manner of administration and the like,
which can readily be determined by one of ordinary skill in the
art. The term also applies to a dose that will induce a particular
response in target cells, e.g., reduction of proliferation or down
regulation of activity of a target protein. The specific dose will
vary depending on the particular compounds chosen, the dosing
regimen to be followed, whether it is administered in combination
with other compounds, timing of administration, the tissue to which
it is administered, and the physical delivery system in which it is
carried.
[0047] The term "isolated", as applied to a component of a subject
composition, such component including, for example, a
methylxanthine PDE inhibitor (including but not limited to
theophylline and theobromine), leucine and leucine metabolites
(including HMB, KIC and alpha-hydroxy-isocaproic acid), and
resveratrol, refers to a preparation of the substance devoid of at
least some of the other components that may also be present where
the substance or a similar substance naturally occurs or is
initially obtained from. Leucine or its metabolite when used in the
subject composition is general in its free form and not as part of
a polypeptide or a biomolecule. An isolated substance may be
prepared by using a purification technique to enrich it from a
source mixture. Enrichment can be measured on an absolute basis,
such as weight per volume of solution, or it can be measured in
relation to a second, potentially interfering substance present in
the source mixture. Increasing enrichment of the embodiments of
this invention are increasingly more preferred. Thus, for example,
a 2-fold enrichment is preferred, 10-fold enrichment is more
preferred, 100-fold enrichment is more preferred, 1000-fold
enrichment is even more preferred. A substance can also be provided
in an isolated state by a process of artificial assembly, such as
by chemical synthesis.
[0048] A "modulator" of a pathway refers to a substance or agent
which modulates the activity or expression of one or more cellular
proteins mapped to the same specific signal transduction pathway. A
modulator may augment or suppress the activity and/or expression
level or pattern of a signaling molecule. A modulator can activate
a component in a pathway by directly binding to the component. A
modulator can also indirectly activate a component in a pathway by
interacting with one or more associated components. The output of
the pathway can be measured in terms of the expression or activity
level of proteins. The expression level of a protein in a pathway
can be reflected by levels of corresponding mRNA or related
transcription factors as well as the level of the protein in a
subcellular location. For instance, certain proteins are activated
by translocating in or out of a specific subcellular component,
including but not limited to nucleus, mitochondria, endosome,
lysosome or other membranous structure of a cell. The output of the
pathway can also be measured in terms of physiological effects,
such as mitochondrial biogenesis, fatty acid oxidation, or glucose
uptake.
[0049] An "activator" refers to a modulator that influences a
pathway in a manner that increases the pathway output. Activation
of a particular target may be direct (e.g. by interaction with the
target) or indirect (e.g. by interaction with a protein upstream of
the target in a signaling pathway including the target).
[0050] A "suppressor" can be a modulator that influences a pathway
in a manner that decreases pathway output.
[0051] The term "substantially free", as used herein, refers to
compositions that have less than about 10%, less than about 5%,
less than about 1%, less than about 0.5%, less than 0.1% or even
less of a specified component. For example a composition that is
substantially free of non-branched chain amino acids may have less
than about 1% of the non-branched chain amino acid lysine. The
percentage may be determined as a percent of the total composition
or a percent of a subset of the composition. For example, a
composition that is substantially free of non-branched chain amino
acids may have less than 1% of the non-branched chain amino acids
as a percent of the total composition, or as a percent of the amino
acids in the composition. The percentages may be mass, molar, or
volume percentages.
[0052] A "sub-therapeutic amount" of an agent, an activator or a
therapy is an amount less than the effective amount of that agent,
activator or therapy for an intended application, but when combined
with an effective or sub-therapeutic amount of another agent or
therapy can produce a desired result, due to, for example, synergy
in the resulting efficacious effects, and/or reduced side
effects.
[0053] A "synergistic" or "synergizing" effect can be such that the
one or more effects of the combination compositions are greater
than the one or more effects of each component alone, or they can
be greater than the sum of the one or more effects of each
component alone. The synergistic effect can be about, or greater
than about 10, 20, 30, 50, 75, 100, 110, 120, 150, 200, 250, 350,
or 500% or even more than the effect on a subject with one of the
components alone, or the additive effects of each of the components
when administered individually. The effect can be any of the
measurable effects described herein.
[0054] Compositions
[0055] The subject compositions comprise a combination of (i) a
non-specific PDE inhibitor, such as a methylxanthine, and (ii)
leucine and/or one or more leucine metabolites. The compositions
may further comprise resveratrol. The combination of these
components can be useful for treating pulmonary conditions,
including but not limited to asthma and chronic obstructive
pulmonary disease. The combination can be particularly effective in
reducing expression and/or secretion of an inflammatory marker or
increasing expression and/or secretion of an anti-inflammatory
marker associated with the inflammatory responses elicited during
onset of asthma or chronic obstructive pulmonary disease. In some
embodiments, the components are formulated to provide a synergistic
effect, including but not limited to reduction in dosing amounts
leading to reduced side effects to the subject and/or reduced cost
of treatment. In other embodiments, the synergistic effect can
allow for results that are not achievable through any other
conventional treatments.
[0056] In one embodiment, the subject composition comprises leucine
and/or one or more leucine metabolites selected from the group
consisting of keto-isocaproic acid (KIC), alpha-hydroxy-isocaproic
acid, and hydroxymethylbutyrate (HMB); and a methylxanthine,
wherein the composition comprises at least about 250 mg of leucine
and/or at least about 100 mg of the one or more leucine
metabolites.
[0057] In another embodiment, the subject composition comprises (a)
leucine and/or one or more leucine metabolites selected from the
group consisting of keto-isocaproic acid (KIC),
alpha-hydroxy-isocaproic acid, and hydroxymethylbutyrate (HMB); and
(b) a methylxanthine, wherein the mass ratio of (a) to (b) is at
least about 15, 25, 50, 75, or 100, and wherein the composition
comprises at least about 5 mg of the methylxanthine. As described
herein, a dosing of at least about 5 mg of methylxanthine can
provide a sub-therapeutic dosing that can be effective when
combined with a sufficient mass ratio of leucine or leucine
metabolite.
[0058] In one aspect of the invention, the subject composition
comprises (a) leucine and/or one or more leucine metabolites
selected from the group consisting of keto-isocaproic acid (KIC),
alpha-hydroxy-isocaproic acid, and hydroxymethylbutyrate (HMB); and
(b) a methylxanthine, wherein the molar ratio of (a) to (b) is at
least about 400, 500, 750, or 1000, and wherein the composition
comprises at least about 0.05, 0.1, 0.5, 1, or 2 .mu.g of the
methylxanthine. The composition can comprise at least about 0.05,
0.1, 0.5, 1, or 2 mg of the methylxanthine. The composition can
comprise at most about 0.001, 0.01, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 5,
10, or 20 grams of a methylxanthine, which may be theophylline
and/or theobromine. The composition can further comprise at least
about 15, 30, 50, 100, or 500 .mu.g of leucine. The composition can
further comprise at least about 5, 10, 15, 30, 50, 100, or 500 mg
of leucine. The composition can comprise at most about 0.001, 0.01,
0.1, 0.5, 1, 1.5, 2, 2.5, 3, 5, 10, or 20 grams of leucine and/or a
leucine metabolite. The composition can further comprise at least
about 0.01, 0.05, 0.1, 0.5, or 1 .mu.g of resveratrol. The
composition can further comprise at least about 0.01, 0.05, 0.1,
0.5, or 1 mg of resveratrol. In some embodiments, the
methylxanthine is theophylline or theobromine. The composition may
be formulated as a unit dosage for inhalation.
[0059] In some embodiments, the composition is held within an
inhaler designed to hold at least about 20, 50, 200, or 1000 unit
dosages. The inhaler can comprise at least about 0.05, 0.1, 0.5, 1,
or 2 .mu.g of a methylxanthine per unit dose. The inhaler can
comprise at least about 15, 30, 50, 100, or 500 of leucine per unit
dose. The inhaler can comprise at least about 0.01, 0.05, 0.1, 0.5,
or 1 .mu.g of resveratrol per unit dose.
[0060] In yet another embodiment, the subject composition comprises
(a) leucine; and (b) a methylxanthine, wherein component (a) and
(b) are present in an amount effective to achieve a circulating
level of about 0.3-2 mM leucine and about 0.5-10 .mu.M
methylxanthine in a subject. These targeted circulating levels
correspond to treatment concentrations described herein (see
Examples), which were shown to provide beneficial effects on
pulmonary conditions in a subject.
[0061] In still another embodiment, the subject composition
comprises (a) leucine; and (b) a methylxanthine, wherein component
(a) and (b) are present in an amount that is effective in reducing
expression level or secretion of one or more inflammatory markers
in a lung endothelial cell selected from the group consisting of
NF.kappa.B, eotaxin, IL1-.beta., and IL6, or in increase expression
level or secretion of one or more anti-inflammatory markers
selected from the group consisting of adiponectin receptor 1 and
adiponectin receptor 2. The amount of (b) may be a sub-therapeutic
amount. Alternatively, the amount of (b) may be less than an amount
required to achieve a circulating level of about 40 .mu.M. As
described in the Examples, the combination of leucine and a
methylxanthine can be combined to in amounts that have a beneficial
effect on reducing expression level or secretion of one or more
inflammatory markers in a lung endothelial cell such as NF.kappa.B,
eotaxin, IL1-.beta., and IL6, or increasing the expression level or
secretion of one or more anti-inflammatory markers selected from
the group consisting of adiponectin receptor 1 and adiponectin
receptor 2.
[0062] Phosphodiesterase Inhibitors
[0063] In some embodiments, the compositions can include a
phosphodiesterase (PDE) inhibitor, such as a non-selective PDE
inhibitor. PDE inhibitors can be naturally occurring or
non-naturally occurring (e.g. manufactured), and may be provided in
the form of a natural source comprising the PDE inhibitor, or an
extract thereof (e.g. purified). Examples of non-selective PDE
inhibitors include, but are not limited to, caffeine, theophylline,
theobromine, 3-isobutyl-1-methylxanthine (IBMX), pentoxifylline
(3,7-dihydro-3,7-dimethyl-1-(5oxohexyl)-1H-purine-2,6-dione),
aminophylline, paraxanthine, and salts, derivatives, metabolites,
catabolites, anabolites, precursors, and analogs thereof.
Non-limiting examples of natural sources of PDE inhibitors include
coffee, tea, guarana, verba mate, cocoa, and chocolate (e.g. dark
chocolate). In various embodiments, compositions are formulated
such that they do not contain (or exclude) one or more of the
following ingredients: caffeine, green tea extract or extracts from
guarana seed or guarana plants. Examples of phosphodiesterase
inhibitors that may be used in a subject compositions are described
in U.S. patent application Ser. No. 13/549,381, filed Jul. 13,
2012, which is herein incorporated by reference in its
entirety.
[0064] The PDE inhibitors may also be methylxanthines. Examples of
methylxanthines include caffeine, ephedrine, oxtriphylline,
aminophylline, paraxanthine, IBMX, pentoxifylline, theobromine, and
theophylline. Examples of aminophylline formulations include
aminophylline Boehringer (Boehringer Ingelheim GmbH). Examples of
ephedrine formulations include Bronkaid.RTM. (Bayer AG),
broncholate (Sanofi-Aventis), Primatene.RTM. (Wyeth), tedral
SA.RTM., and marax (Pfizer Inc). Examples of theophylline
formulations include euphyllin (Nycomed International Management
GmbH), and theo-dur (Pfizer Inc, Teva Pharmaceutical Industries
Ltd). Examples of oxtriphylline formulations include Choledyl SA
(Pfizer Inc).
[0065] Leucine and Leucine Metabolites
[0066] The invention provides for compositions that include leucine
and/or leucine metabolites. The leucine and/or leucine metabolites
can be used in free form. The term "free," as used herein in
reference to a component, indicates that the component is not
incorporated into a larger molecular complex. For example a
composition can include free leucine that is not incorporated in a
protein or free hydroxymethylbutyrate. The leucine can be
L-leucine.
[0067] Without being limited to theory, ingestion of branched chain
amino acids, such as leucine, can stimulate sirtuin signaling,
including Sirt1 and Sirt3, as well as AMPK signaling, one or more
of which can favorably modulate inflammatory cytokine patterns. In
some embodiments, any of the compositions described herein can
include salts, derivatives, metabolites, catabolites, anabolites,
precursors, and analogs of leucine. For example, the metabolites
can include hydroxymethylbutyrate (HMB), keto-isocaproic acid
(KIC), and keto isocaproate. The HMB can be in a variety of forms,
including calcium 3-hydroxy-3-methylbutyrate hydrate.
[0068] In certain embodiments of the invention, any of the
compositions disclosed herein can be formulated such that they do
not contain (or exclude) one or more amino acids selected from the
group consisting of lysine, glutamate, proline, arginine, valine,
isoleucine, aspartic acid, asparagine, glycine, threonine, serine,
phenylalanine, tyrosine, histidine, alanine, tryptophan,
methionine, glutamine, taurine, carnitine, cystine and
cysteine.
[0069] In some embodiments, the compositions may be substantially
free of one or more, or all of non-branched chain amino acids. For
example, the compositions can be free of alanine, arginine,
asparagine, aspartic acid, cysteine, glutamic acid, glutamine,
glycine, histidine, lysine, methionine, phenylalanine, proline,
serine, threonine, tryptophan, and/or tyrosine. In some
embodiments, the compositions may be substantially free of
isoleucine and/or valine. The compositions can be substantially
free of any non-branched chain amino acids. The mass or molar
amount of a non-branched chain amino acid can be less than about
0.01, 0.1, 0.5, 1, 2, 5, or 10% of the total composition or of the
total amino acids in the composition.
[0070] Pharmaceutically Active Agents
[0071] The subject compositions can further include one or more
pharmaceutically active agents other than a methylxanthine PDE
inhibitor. Examples of therapeutically active agents include
ibuprofen, aldoril, and gemfebrozil, verapamil, maxzide, diclofenac
and metrolol, maproltiline, triazolam and minoxidil. For example,
the combination compositions can comprise a pharmaceutically active
anti-diabetic agent, weight loss agent, or calcium regulation
agent. U.S. Pat. No. 7,109,198 and U.S. Patent Application No.
20090142336 describe a variety of pharmaceutically active agents or
therapeutically active agents suitable for inclusion in a
combination composition described herein. Examples of anti-diabetic
agents include biguanides (such as metformin), thiazoladinediones
and meglitinides (such as repaglinide, pioglitazone, and
rosiglitazone), alpha glucosidease inhibitors (such as acarbose),
sulfonylureas (such as tolbutamide, acetohexamide, tolazamide,
chlorpropamide, glipizide, glyburide, glimepiride, gliclazide),
incretins, ergot alkaloids (such as bromocriptine), and DPP
inhibitors (such as sitagliptin, vildagliptin, saxagliptin,
lingliptin, dutogliptin, gemigliptin, alogliptin, and berberine).
The anti-diabetic agent can be an oral anti-diabetic agent. The
anti-diabetic agent can also be injectable anti-diabetic drugs,
including insulin, amylin analogues (such as pramlintide), and
inretin mimetics (such as exenatide and liraglutide). Examples of
anti-obesity therapeutic agents include lipase inhibitors (such as
Orlistat), dopaminergic, noradrenergic, and serotoninergic
compounds, cannabinoid receptor antagonists (such as rimonabant),
exenatide, pramlintide, and CNS agents (such as topimerate). These
examples are provided for discussion purposes only, and are
intended to demonstrate the broad scope of applicability of the
invention to a wide variety of drugs. It is not meant to limit the
scope of the invention in any way.
[0072] In some embodiments, a methylxanthine PDE inhibitor can be
combined with a pair of pharmaceutically active agents as follow:
glipizide and metformin; glyburide and metformin; pioglitazone and
glimepiride; pioglitazone and metformin; repaglinide and metformin;
rosiglitazone and glimepiride; rosiglitazone and metformin; and
sitagliptin and metformin.
[0073] The amount of pharmaceutical agent, or any other component
used in a combination composition described herein, can be a used
in an amount that is sub-therapeutic. In some embodiments, using
sub-therapeutic amounts of an agent or component can reduce the
side-effects of the agent. Use of sub-therapeutic amounts can still
be effective, particularly when used in synergy with other agents
or components.
[0074] A sub-therapeutic amount of the agent or component can be
such that it is an amount below which would be considered
therapeutic. For example, FDA guidelines can suggest a specified
level of dosing to treat a particular condition, and a
sub-therapeutic amount would be any level that is below the FDA
suggested dosing level. The sub-therapeutic amount can be about 1,
5, 10, 15, 20, 25, 30, 35, 50, 75, 90, or 95% less than the amount
that is considered to be a therapeutic amount. The therapeutic
amount can be assessed for individual subjects, or for groups of
subjects. The group of subjects can be all potential subjects, or
subjects having a particular characteristic such as age, weight,
race, gender, or physical activity level.
[0075] In the case of metformin hydrochloride, the physician
suggested starting dose is 1000 mg daily, with subject specific
dosing having a range of 500 mg to a maximum of 2500 mg daily
(metformin hydrochloride extended-release tablets label
www.accessdata.fda.gov/drugsatfda_docs/label/2008/021574s0101b1.pdf).
The particular dosing for a subject can be determined by a
clinician by titrating the dose and measuring the therapeutic
response. The therapeutic dosing level can be determined by
measuring fasting plasma glucose levels and measuring glycosylated
hemoglobin. A sub-therapeutic amount can be any level that would be
below the recommended dosing of metformin. For example, if a
subject's therapeutic dosing level is determined to be 700 mg
daily, a dose of 600 mg would be a sub-therapeutic amount.
Alternatively, a sub-therapeutic amount can be determined relative
to a group of subjects rather than an individual subject. For
example, if the average therapeutic amount of metformin for
subjects with weights over 300 lbs is 2000 mg, then a
sub-therapeutic amount can be any amount below 2000 mg. In some
embodiments, the dosing can be recommended by a healthcare provider
including, but not limited to a patient's physician, nurse,
nutritionist, pharmacist, or other health care professional. A
health care professional may include a person or entity that is
associated with the health care system. Examples of health care
professionals may include surgeons, dentists, audiologists, speech
pathologists, physicians (including general practitioners and
specialists), physician assistants, nurses, midwives,
pharmaconomists/pharmacists, dietitians, therapists, psychologists,
physical therapists, phlebotomists, occupational therapists,
optometrists, chiropractors, clinical officers, emergency medical
technicians, paramedics, medical laboratory technicians,
radiographers, medical prosthetic technicians social workers, and a
wide variety of other human resources trained to provide some type
of health care service.
[0076] In the case of a methylxanthine, the therapeutically
effective level of the methylxanthine can be a circulating level
between about 44-111 .mu.M, which corresponds to about 10-20
.mu.g/mL. A sub-therapeutic level of the methylxanthine, such as
theophylline or theobromine, can be any circulating level below
about 110, 100, 90, 80, 70, 60, 50, 44, 40, 35, 30, 20, 10, 5, or 1
.mu.M or 10 .mu.g/mL. The sub-therapeutic level of the
methylxanthine, such as theophylline or theobromine, in a subject
composition formulated for administration can be less than about 1,
5, 10, 20, 30, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, or 250
mg of the methylxanthine.
[0077] Any of the components described herein, including leucine,
HMB, KIC, theophylline, theobromine, and resveratrol may be used in
a subject composition in free form, purified from a natural source,
and/or purified or prepared from a synthetic source. The natural
source can be an animal source or plant source. The components may
be pure to at least about 95, 97, 99, 99.5, 99.9, 99.99, or
99.999%.
[0078] Dosing Amounts
[0079] The invention provides for compositions that are
combinations of isolated components, such as leucine, metabolites
of leucine, such as HMB, methylxanthines, such as theophylline and
theobromine, and resveratrol, that have been isolated from one or
more sources. The invention provides for compositions that are
enriched in leucine, metabolites of leucine, such as HMB,
methylxanthines, such as theophylline and theobromine, and/or
resveratrol. The components can be isolated from natural sources or
created from synthetic sources and then enriched to increase the
purity of the components. For example, theophylline can be created
from a synthetic source and then enriched by one or more
purification methods. Additionally, leucine can be isolated from a
natural source and then enriched by one or more separations. The
isolated and enriched components, such as sildenafil and leucine,
can then be combined and formulated for administration to a
subject.
[0080] In some embodiments, a composition comprises an amount of a
methylxanthine or a PDE inhibitor (e.g., including but not limited
to theophylline or theobromine). The amount of a methylxanthine may
be a subtherapeutic amount, and/or an amount that is synergistic
with one or more other compounds in the composition or one or more
of the compounds administered simultaneously or in close temporal
proximity with the composition. In some embodiments, the
methylxanthine or PDE inhibitor is administered in a low dose, a
medium dose, or a high dose, which describes the relationship
between two doses, and generally do not define any particular dose
range. The compositions can be administered to a subject such that
the subject is administered a selected total daily dose of the
composition. The total daily dose can be determined by the sum of
doses administered over a 24 hour period.
[0081] A dose, which may be a unit dose, can comprise about, more
than about, or less than about 200, 250, 400, 500, 600, 700, 800,
900, 1000, 1100, 1250, or more mg of leucine. The leucine may be
free leucine. In some embodiments, a unit dose can comprise at
least about 1000 mg of free leucine. The composition may comprise
between about 10-1250, 200-1250, or 500-1250 mg of leucine. A dose,
which may be a unit dose, can comprise about, more than about, or
less than about 50, 100, 200, 250, 400, 500, 600, 700, 800, 900,
1000 or more mg of a leucine metabolite, such as HMB or KIC. The
leucine metabolite may be a free leucine metabolite. The
composition may comprise between about 10-900, 50-750, or 400-650
mg of the leucine metabolite, such as HMB or KIC. In some
embodiments, a unit dose can comprise at least about 400 mg of free
HMB.
[0082] In some embodiments, a daily dose of leucine can be about,
less than about, or more than about 0.5-3.0 g/day (e.g. 0.5, 0.75,
1, 1.25, 1.5, 1.75, 2, 2.5, 3, or more g/day). A daily dose of HMB
can be about, less than about, or more than about 0.20-3.0 g/day
(e.g. 0.2, 0.4, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, or more g/day). A
daily dose of KIC can be about, less than about, or more than about
0.2-3.0 g/day (e.g. 0.2, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2,
2.5, 3, or more g/day).
[0083] A dose, which may be a unit dose, can comprise a
methylxanthine PDE inhibitor, such as theophylline or theobromine,
that can be about, more than about, or less than about 0.01, 0.05,
0.1, 0.5, 1, 2, 5, 10, 20, 40, 60, 80, 100, 200, 400, 800, 1000, or
1500 mg of the methylxanthine PDE inhibitor. The composition can
comprise between about 1-100, 5-50, or 10-20 mg of the
methylxanthine, such as theophylline or theobromine. In some
embodiments, a unit dose can comprise at least about 20 mg of
theophylline or theobromine. In some embodiments, a unit dose can
comprise at least about 20 mg of theophylline.
[0084] In some embodiments, the composition comprises both
theophylline and theobromine, and the total amount of theophylline
and theobromine can be about, more than about, or less than about
0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 40, 60, 80, 100, 200, 400,
800, 1000, or 1500 mg.
[0085] In other embodiments, a daily dose of a methylxanthine PDE
inhibitor, such as theophylline or theobromine, can be about, more
than about, or less than about 0.0001 mg/kg (mg of methylxanthine
PDE inhibitor/kg of the subject receiving the dose), 0.005 mg/kg,
0.01 mg/kg, 0.5 mg/kg, 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10
mg/kg, 12.5 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 50 mg/kg, 75
mg/kg, 100 mg/kg, or more.
[0086] A dose, which may be a unit dose, can comprise about, less
than about, or more than about 1, 5, 10, 25, 35, 50, 51, 75, 100,
150, 200, 250, 300, 350, 400, 450, 500, or more mg of resveratrol.
The composition may comprise between about 5-500, 30-250, or 35-100
mg of resveratrol. In some embodiments, a unit dose can comprise at
least about 35 mg of resveratrol.
[0087] A daily low dose of resveratrol may comprise about, less
than about, or more than about 0.5 mg/kg (mg of resveratrol/kg of
the subject receiving the dose), 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5
mg/kg, 10 mg/kg, 12.5 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 50
mg/kg, 75 mg/kg, 100 mg/kg, or more; a daily medium dose of
resveratrol may comprise about, less than about, or more than about
20 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150
mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, or more; and a daily high
dose of resveratrol may comprise about, less than about, or more
than about 150 mg/kg, 175 mg/kg, 200 mg/kg, 225 mg/kg, 250 mg/kg,
300 mg/kg, 350 mg/kg, 400 mg/kg, or more.
[0088] In some embodiments, a composition, which may be formulated
as a unit dose, can comprise (a) at least about 250 mg of leucine
and/or at least about 100 mg of the one or more leucine metabolites
and (b) comprises at least about 5 mg of a methylxanthine, such as
theophylline or theobromine. The composition can further comprise
at least about 35 mg of resveratrol.
[0089] In other embodiments, a composition, formulated as a unit
dose, can comprise (a) between about 250-1500 mg of leucine or
100-750 mg of leucine metabolites and (b) 1-100 mg of a
methylxanthine, such as theophylline or theobromine. In other
embodiments, a composition, formulated as a unit dose, can comprise
(a) between about 250-1500 mg of leucine, 10-750 mg of leucine
metabolites, and/or 50-750 mg of leucine metabolites and (b) 1-100
mg of a methylxanthine, such as theophylline or theobromine. In
some embodiments, the methylxanthine is theophylline and the
composition comprises between about 5-50 mg of theophylline.
[0090] In some embodiments of the invention, the combination
compositions can have a specified ratio of leucine amino acids
and/or metabolites thereof to a methylxanthine PDE inhibitor. The
specified ratio can provide for effective and/or synergistic
treatment of pulmonary conditions, which, for example, may be
measured as a reduction in NF.kappa.B protein expression, reduction
in eotaxin secretion, reduction in IL1-.beta. secretion, reduction
in cellular IL6 content or secretion, reduction in adiponectin
receptor 1 protein expression, and reduction in adiponectin
receptor 2 protein expression. The ratio of leucine amino acids
and/or metabolites thereof to a selective PDE inhibitor activator
can be a mass ratio, a molar ratio, or a volume ratio.
[0091] In some embodiments, a composition can comprise (a) leucine
and/or metabolites thereof (including HMB) and (b) a methylxanthine
(including theophylline and theobromine), where the mass ratio of
(a) to (b) can be about, less than about, or greater than about
0.1, 0.5, 1, 2, 5, 10, 15, 25, 50, 75, 100, 200, 300, 350, 400,
450, 500, 550, 600, 650, 700, 750, or 800. In some embodiments, the
mass ratio of (a) to (b) is at least about 50. The composition can
also comprise a minimal amount of methylxanthine, such as 5, 10 or
50 mg of the methylxanthine or a range of methylxanthine amount,
such as 5-250 mg of methylxanthine.
[0092] In other embodiments, a composition can comprise (a) a
methylxanthine PDE inhibitor (including theophylline and
theobromine) and (b) resveratrol, where the mass ratio of (a) to
(b) can be about, less than about, or greater than about 0.01,
0.05, 0.1, 0.5, 1, 2, 5, 10, 50, 100, 200, 300, 350, 400, 450, 500,
550, 600, or 650.
[0093] In some embodiments, the composition can be formulated for
inhalation. The composition formulated for inhalation can be
formulated as a liquid. The composition formulated for inhalation
can be housed within an inhaler or nebulizer. The inhaler or
nebulizer can hold at least about 10, 20, 40, 100, 500, 1000, or
2000 unit doses. A unit dose of a subject composition can have a
volume of about or at least about 0.1, 0.25, 0.5, 1, or 5 mL. A
unit dose of a subject composition can have a volume of about 0.5-5
mL, which may be administered in about 1-10 inhalations.
[0094] The composition formulated for inhalation can be formulated
in a liquid form that comprises at least about, about, or less than
about 0.25, 0.5, 0.75, 1 mM or more of leucine.
[0095] The composition formulated for inhalation can be formulated
in a liquid form that comprises at least about, about, or less than
about 0.1, 0.25, 0.5, 0.75, 1, 10, 20, 40, 60 .mu.M or more of a
leucine metabolite (such as HMB). The composition formulated for
inhalation can be formulated in a liquid form that comprises at
least about, about, or less than about 0.25, 0.5, 0.75, 1 mM or
more of KIC.
[0096] The composition formulated for inhalation can be formulated
in a liquid form that comprises at least about, about, or less than
about 0.1, 0.25, 0.5, 0.75, 1, 10, 20, 40, 60, 80, 100, 120, 200,
or 400 .mu.M or more of the methylxanthine, such as theophylline or
theobromine.
[0097] In some embodiments, the dosing of leucine, any metabolites
of leucine, the PDE inhibitor (such as a methylxanthine), and
resveratrol can be designed to achieve a specified physiological
concentration or circulating level of leucine, metabolites of
leucine, a methylxanthine and/or resveratrol. The physiological
concentration can be a circulating level as measured in the blood
stream of a subject. The subject can be a human or an animal. A
selected dosing can be altered based on the characteristics of the
subject, such as weight, rate of energy metabolism, genetics,
ethnicity, height, or any other characteristic.
[0098] In some embodiments, a selected dose of a composition can be
administered to a subject such that the subject achieves a desired
circulating level of the composition. The desired circulating level
of a component may be either a therapeutically effective level or a
sub-therapeutic level.
[0099] The desired circulating level of the composition can be at
least about 0.25, 0.5, 0.75, 1 mM or more of leucine. The desired
circulating level of the composition can be at least about, less
than about, or more than about 0.1, 0.25, 0.5, 0.75, 1, 10, 20, 40,
60 .mu.M or more of a leucine metabolite (such as HMB). The desired
circulating level of the composition can be at least about 0.25,
0.5, 0.75, 1 mM or more of KIC.
[0100] The desired circulating level of the composition can be at
least about, less than about, or more than about 0.1, 0.25, 0.5,
0.75, 1, 10, 20, 40, 60, 80, 100, 120, 200, or 400 .mu.M or more of
the methylxanthine, such as theophylline or theobromine. The
therapeutically effective level of theophylline can be between
44-111 .mu.M, which corresponds to about 10-20 .mu.g/mL. A
sub-therapeutic level of theophylline can be any level below about
110, 100, 90, 80, 70, 60, 50, 44, 40, 35, 30, 20, 10, 5, or 1 .mu.M
or 10 .mu.g/mL.
[0101] The desired circulating level of the composition can be at
least about, less than about, or more than about 40, 60, 80, 100,
120, 150, 200, 300, 400, 800, 1600, 3000, or 5000 nM or more of the
resveratrol. The selected dose can be chosen based on the
characteristics of the subject, such as weight, height, ethnicity,
or genetics.
[0102] In some embodiments, a composition comprises leucine and a
methylxanthine in amounts that are effective to achieve a
circulating level of about 0.3-2 mM leucine and about 0.5-10 .mu.M
methylxanthine in a subject.
[0103] An oral dosing of about 1,125 mg leucine can achieve a
circulating level of leucine in a subject that is about 0.5 mM
leucine. An oral dosing of about 300 mg leucine can achieve a
circulating level of leucine in a subject that is about 0.25
mM.
[0104] An oral dosing of about 500 mg of HMB can achieve a
circulating level of HMB in a subject that is about 5 .mu.M HMB. An
oral dosing of about 100 mg of HMB can achieve a circulating level
of HMB in a subject that is about 0.8 .mu.M HMB.
[0105] An oral dosing of about 1000 mg of theophylline can achieve
a circulating level of theophylline in a subject that is about 110
.mu.M theophylline. An oral dosing of about 25-30 mg of
theophylline can achieve a circulating level of theophylline in a
subject that is about 1 .mu.M theophylline.
[0106] An oral dosing of about 1000 mg of theobromine can achieve a
circulating level of theobromine in a subject that is about 110
.mu.M theobromine. An oral dosing of about 25-30 mg of theobromine
can achieve a circulating level of theophylline in a subject that
is about 1 .mu.M theobromine.
[0107] An oral dosing of about 1100 mg of resveratrol can achieve a
circulating level of resveratrol in a subject that is about 0.5 mM
resveratrol. An oral dosing of about 50 mg of resveratrol can
achieve a circulating level of resveratrol in a subject that is
about 200 nM resveratrol.
[0108] A dosing prepared for inhalation, which may be in liquid
form, can be prepared at concentrations that are about 1, 1.5, 2,
2.5, or 3 times greater than the desired circulating concentration.
For example, a formulation for inhalation may comprise about 0.5,
1, or 0.5-1 mM leucine, which can achieve a circulating level of
leucine in the pulmonary tissue of a subject that is about 0.5 mM.
A formulation for inhalation may comprise about 200, 400, or
200-400 nM resveratrol, which can achieve a circulating level of
resveratrol in the pulmonary tissue of a subject that is about 200
nM. A formulation for inhalation may comprise about 1, 2, or 1-2
.mu.M methylxanthine, which can achieve a circulating level of
methylxanthine in the pulmonary tissue of a subject that is about 1
.mu.M.
[0109] In some embodiments, the compositions can be formulated to
achieve a desired circulating molar or mass ratios achieved after
administration one or more compositions to a subject. The
compositions can be a combination composition described herein. The
molar ratio can be adjusted to account for the bioavailability, the
uptake, and the metabolic processing of the one or more components
of a combination composition. For example, if the bioavailability
of a component is low, then the molar amount of a that component
can be increased relative to other components in the combination
composition. In some embodiments, the circulating molar or mass
ratio is achieved within about 0.1, 0.5, 0.75, 1, 3, 5, or 10, 12,
24, or 48 hours after administration. The circulating molar or mass
ratio can be maintained for a time period of about or greater than
about 0.1, 1, 2, 5, 10, 12, 18, 24, 36, 48, 72, or 96 hours.
[0110] In some embodiments, the circulating molar ratio of leucine
to a methylxanthine is about, less than about, or greater than
about 1, 5, 10, 50, 100, 500, 1000, 5000, or 10000. In some
embodiments, the circulating molar ratio of HMB to a methylxanthine
is about or greater than about, or less than about 0.01, 0.05, 0.1,
0.5, 1, 5, 10, 50, or 100. In some embodiments, the circulating
molar ratio of a methylxanthine to resveratrol is about, less than
about, or greater than about 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, or
100.
[0111] In some embodiments, a composition can comprise leucine and
a methylxanthine in an amount that is effective in improving
expression level or secretion of one or more inflammatory or
anti-inflammatory markers in a cell selected from the group
consisting of (a) NF.kappa.B protein expression, (b) eotaxin, (c)
IL1-.beta., and (d) cellular IL6 content or secretion, (e)
adiponectin receptor 1 protein expression, and (f) adiponectin
receptor 2 protein expression. The composition can have an amount
of methylxanthine that is less than the amount required to achieve
a circulating level of about 40 .mu.M. The composition can have an
amount of methylxanthine that is effective to achieve a circulating
level of about 0.1-40 .mu.M. The amount of methylxanthine can also
be greater than at least about 5 mg.
[0112] Dosing Forms
[0113] The compositions described herein can be compounded into a
variety of different dosage forms. It can be used orally as a
tablet, chewable tablet, caplets, capsule, soft gelatin capsules,
lozenges or solution. Alternatively, the compositions can be
formulated for inhalation or for intravenous delivery. The
compositions can also be formulated as a nasal spray or for
injection when in solution form. In some embodiments, the
composition may be a liquid composition suitable for oral
consumption.
[0114] Compositions formulated for inhalation may be packaged in an
inhaler using techniques known in the art. An inhaler may be
designed to dispense 0.25, 0.5, or 1 unit dose per inhalation. An
inhaler may have a canister that holds the subject composition
formulated for inhalation, a metering valve that allows for a
metered quantity of the formulation to be dispensed with each
actuation, and an actuator or mouthpiece that allows for the device
to be operated and direct the subject composition into the
subject's lungs. The formulated composition may include a liquefied
gas propellant and possibly stabilizing excipients. The actuator
may have a mating discharge nozzle that connects to the canister
and a dust cap to prevent contamination of the actuator. Upon
actuation, the subject composition may be volatized, which results
in the formation of droplets of the subject composition. The
droplets may rapidly evaporate resulting in micrometer-sized
particles that are then inhaled by the subject.
[0115] A protocol for treatment via inhalation can include
preparation of an aerosol having particle sizes of predetermined
mass medial aerodynamic diameter (MMAD) between 3 and 8 .mu.m
delivered predominantly to the conducting and central lungs with or
without overpressure using a jet, ultrasonic, electronic, vibrating
porous plate, vibrating mesh nebulizer or energized dry powder
inhaler. The jet or electronic nebulizers may further be combined
with airflow control and the aerosol may be administered with
overpressure. Nebulization devices and systems can allow for
individualization of a delivered volumetric flow and vaporized
aerosol together with a controlled airflow and with airflow
overpressure conditions into a treatment protocol suitable for
treatment of a variety of conditions including inflammatory
pulmonary diseases.
[0116] The nebulizing system can comprise components, such as a jet
or electronic nebulizer, a compressor and an electronic control
means that cumulatively have properties that enable control of the
breathing pattern by asserting a positive pressure (also called
NIPPV) during the inhalation. This pressure can reduce the need for
active breathing in COPD patients, which results in much more
effective and easier lung delivery of the drugs combination to COPD
patients having difficulty breathing or who are unable to breathe
without oxygen.
[0117] Inhalers and methods for formulating compositions for
inhalation are described in are described in U.S. Pat. Nos.
5,069,204, 7,870,856 and U.S. Patent Application No. 2010/0324002,
which are incorporated herein by reference in its entirety.
[0118] Compositions of the invention suitable for oral
administration can be presented as discrete dosage forms, such as
capsules, cachets, or tablets, or liquids or aerosol sprays each
containing a predetermined amount of an active ingredient as a
powder or in granules, a solution, or a suspension in an aqueous or
non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil
liquid emulsion, including liquid dosage forms (e.g., a suspension
or slurry), and oral solid dosage forms (e.g., a tablet or bulk
powder). Oral dosage forms may be formulated as tablets, pills,
dragees, capsules, emulsions, lipophilic and hydrophilic
suspensions, liquids, gels, syrups, slurries, suspensions and the
like, for oral ingestion by an individual or a patient to be
treated. Such dosage forms can be prepared by any of the methods of
formulation. For example, the active ingredients can be brought
into association with a carrier, which constitutes one or more
necessary ingredients. Capsules suitable for oral administration
include push-fit capsules made of gelatin, as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules can contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. Optionally, the inventive composition for
oral use can be obtained by mixing a composition a solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable excipients are, in
particular, fillers such as sugars, including lactose, sucrose,
mannitol, or sorbitol; cellulose preparations such as, for example,
maize starch, wheat starch, rice starch, potato starch, gelatin,
gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose,
sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
In general, the compositions are prepared by uniformly and
intimately admixing the active ingredient with liquid carriers or
finely divided solid carriers or both, and then, if necessary,
shaping the product into the desired presentation. For example, a
tablet can be prepared by compression or molding, optionally with
one or more accessory ingredients. Compressed tablets can be
prepared by compressing in a suitable machine the active ingredient
in a free-flowing form such as powder or granules, optionally mixed
with an excipient such as, but not limited to, a binder, a
lubricant, an inert diluent, and/or a surface active or dispersing
agent. Molded tablets can be made by molding in a suitable machine
a mixture of the powdered compound moistened with an inert liquid
diluent.
[0119] The liquid forms, in which the formulations disclosed herein
may be incorporated for administration orally or by injection,
include aqueous solution, suitably flavored syrups, aqueous or oil
suspensions, and flavored emulsions with edible oils such as
cottonseed oil, sesame oil, coconut oil, or peanut oil as well as
elixirs and similar pharmaceutical vehicles. Suitable dispersing or
suspending agents for aqueous suspensions include synthetic natural
gums, such as tragacanth, acacia, alginate, dextran, sodium
carboxymethyl cellulose, methylcellulose, polyvinylpyrrolidone or
gelatin.
[0120] A subject can be treated by combination of an injectable
composition and an orally ingested composition.
[0121] Liquid preparations for oral administration may take the
form of, for example, solutions, syrups or suspensions, or they may
be presented as a dry product for reconstitution with water or
other suitable vehicles before use. Such liquid preparations may be
prepared by conventional means with pharmaceutically acceptable
additives such as suspending agents (e.g., sorbitol syrup, methyl
cellulose or hydrogenated edible fats); emulsifying agents (e.g.,
lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily
esters or ethyl alcohol); preservatives (e.g., methyl or propyl
p-hydroxybenzoates or sorbic acid); and artificial or natural
colors and/or sweeteners.
[0122] The preparation of pharmaceutical compositions of this
invention, including oral and inhaled formulations, can be
conducted in accordance with generally accepted procedures for the
preparation of pharmaceutical preparations. See, for example,
Remington's Pharmaceutical Sciences 18th Edition (1990), E. W.
Martin ed., Mack Publishing Co., PA. Depending on the intended use
and mode of administration, it may be desirable to process the
magnesium-counter ion compound further in the preparation of
pharmaceutical compositions. Appropriate processing may include
mixing with appropriate non-toxic and non-interfering components,
sterilizing, dividing into dose units, and enclosing in a delivery
device.
[0123] This invention further encompasses anhydrous compositions
and dosage forms comprising an active ingredient, since water can
facilitate the degradation of some compounds. For example, water
may be added (e.g., 5%) in the arts as a means of simulating
long-term storage in order to determine characteristics such as
shelf-life or the stability of formulations over time Anhydrous
compositions and dosage forms of the invention can be prepared
using anhydrous or low moisture containing ingredients and low
moisture or low humidity conditions. Compositions and dosage forms
of the invention which contain lactose can be made anhydrous if
substantial contact with moisture and/or humidity during
manufacturing, packaging, and/or storage is expected. An anhydrous
composition may be prepared and stored such that its anhydrous
nature is maintained. Accordingly, anhydrous compositions may be
packaged using materials known to prevent exposure to water such
that they can be included in suitable formulary kits. Examples of
suitable packaging include, but are not limited to, hermetically
sealed foils, plastic or the like, unit dose containers, blister
packs, and strip packs.
[0124] An ingredient described herein can be combined in an
intimate admixture with a pharmaceutical carrier according to
conventional pharmaceutical compounding techniques. The carrier can
take a wide variety of forms depending on the form of preparation
desired for administration. In preparing the compositions for an
oral dosage form, any of the usual pharmaceutical media can be
employed as carriers, such as, for example, water, glycols, oils,
alcohols, flavoring agents, preservatives, coloring agents, and the
like in the case of oral liquid preparations (such as suspensions,
solutions, and elixirs) or aerosols; or carriers such as starches,
sugars, micro-crystalline cellulose, diluents, granulating agents,
lubricants, binders, and disintegrating agents can be used in the
case of oral solid preparations, in some embodiments without
employing the use of lactose. For example, suitable carriers
include powders, capsules, and tablets, with the solid oral
preparations. If desired, tablets can be coated by standard aqueous
or nonaqueous techniques.
[0125] Some examples of materials which may serve as
pharmaceutically acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0126] Binders suitable for use in dosage forms include, but are
not limited to, corn starch, potato starch, or other starches,
gelatin, natural and synthetic gums such as acacia, sodium
alginate, alginic acid, other alginates, powdered tragacanth, guar
gum, cellulose and its derivatives (e.g., ethyl cellulose,
cellulose acetate, carboxymethyl cellulose calcium, sodium
carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose,
pre-gelatinized starch, hydroxypropyl methyl cellulose,
microcrystalline cellulose, and mixtures thereof.
[0127] Lubricants which can be used to form compositions and dosage
forms of the invention include, but are not limited to, calcium
stearate, magnesium stearate, mineral oil, light mineral oil,
glycerin, sorbitol, mannitol, polyethylene glycol, other glycols,
stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable
oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil,
olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate,
ethylaureate, agar, or mixtures thereof. Additional lubricants
include, for example, a syloid silica gel, a coagulated aerosol of
synthetic silica, or mixtures thereof. A lubricant can optionally
be added, in an amount of less than about 1 weight percent of the
composition.
[0128] Lubricants can be also be used in conjunction with tissue
barriers which include, but are not limited to, polysaccharides,
polyglycans, seprafilm, interceed and hyaluronic acid.
[0129] Disintegrants may be used in the compositions of the
invention to provide tablets that disintegrate when exposed to an
aqueous environment. Too much of a disintegrant may produce tablets
which may disintegrate in the bottle. Too little may be
insufficient for disintegration to occur and may thus alter the
rate and extent of release of the active ingredient(s) from the
dosage form. Thus, a sufficient amount of disintegrant that is
neither too little nor too much to detrimentally alter the release
of the active ingredient(s) may be used to form the dosage forms of
the compounds disclosed herein. The amount of disintegrant used may
vary based upon the type of formulation and mode of administration,
and may be readily discernible to those of ordinary skill in the
art. About 0.5 to about 15 weight percent of disintegrant, or about
1 to about 5 weight percent of disintegrant, may be used in the
pharmaceutical composition. Disintegrants that can be used to form
compositions and dosage forms of the invention include, but are not
limited to, agar-agar, alginic acid, calcium carbonate,
microcrystalline cellulose, croscarmellose sodium, crospovidone,
polacrilin potassium, sodium starch glycolate, potato or tapioca
starch, other starches, pre-gelatinized starch, other starches,
clays, other algins, other celluloses, gums or mixtures
thereof.
[0130] Examples of suitable fillers for use in the compositions and
dosage forms disclosed herein include, but are not limited to,
talc, calcium carbonate (e.g., granules or powder),
microcrystalline cellulose, powdered cellulose, dextrates, kaolin,
mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch,
and mixtures thereof.
[0131] When aqueous suspensions and/or elixirs are desired for oral
administration, the active ingredient therein may be combined with
various sweetening or flavoring agents, coloring matter or dyes
and, if so desired, emulsifying and/or suspending agents, together
with such diluents as water, ethanol, propylene glycol, glycerin
and various combinations thereof.
[0132] The tablets can be uncoated or coated by known techniques to
delay disintegration and absorption in the gastrointestinal tract
and thereby provide a sustained action over a longer period. For
example, a time delay material such as glyceryl monostearate or
glyceryl distearate can be employed. Formulations for oral use can
also be presented as hard gelatin capsules wherein the active
ingredient is mixed with an inert solid diluent, for example,
calcium carbonate, calcium phosphate or kaolin, or as soft gelatin
capsules wherein the active ingredient is mixed with water or an
oil medium, for example, peanut oil, liquid paraffin or olive
oil.
[0133] In one embodiment, the composition may include a solubilizer
to ensure good solubilization and/or dissolution of the compound of
the present invention and to minimize precipitation of the compound
of the present invention. This can be especially important for
compositions for non-oral use, e.g., compositions for injection. A
solubilizer may also be added to increase the solubility of the
hydrophilic drug and/or other components, such as surfactants, or
to maintain the composition as a stable or homogeneous solution or
dispersion.
[0134] The composition can further include one or more
pharmaceutically acceptable additives and excipients. Such
additives and excipients include, without limitation, detackifiers,
anti-foaming agents, buffering agents, polymers, antioxidants,
preservatives, chelating agents, viscomodulators, tonicifiers,
flavorants, colorants, odorants, opacifiers, suspending agents,
binders, fillers, plasticizers, lubricants, and mixtures thereof. A
non-exhaustive list of examples of excipients includes
monoglycerides, magnesium stearate, modified food starch, gelatin,
microcrystalline cellulose, glycerin, stearic acid, silica, yellow
beeswax, lecithin, hydroxypropylcellulose, croscarmellose sodium,
and crospovidone.
[0135] The compositions described herein can also be formulated as
extended-release, sustained-release or time-release such that one
or more components are released over time. Delayed release can be
achieved by formulating the one or more components in a matrix of a
variety of materials or by microencapsulation. The compositions can
be formulated to release one or more components over a time period
of 1, 4, 6, 8, 12, 16, 20, 24, 36, or 48 hours. The release of the
one or more components can be at a constant or changing rate.
[0136] In some embodiments, a subject composition described herein
can be formulated in as matrix pellets in which particles of the
subject composition are embedded in a matrix of water-insoluble
plastic and which are enclosed by a membrane of water-insoluble
plastic containing embedded particles of lactose, produces and
maintains plasma levels of the subject composition within the
targeted therapeutic range. In other embodiments, a subject
composition can be formulated as a sustained release tablet
obtained by coating core granules composed mainly of the subject
composition with a layer of a coating film composed of a
hydrophobic material and a plastic excipient and optionally
containing an enteric polymer material to form coated granules and
then by compressing the coated granules together with a
disintegrating excipient. Sustained release formulations are
described in U.S. Pat. Nos. 4,803,080, and 6,426,091, which are
herein incorporated by reference in its entirety.
[0137] Using the controlled release dosage forms provided herein,
the one or more cofactors can be released in its dosage form at a
slower rate than observed for an immediate release formulation of
the same quantity of components. In some embodiments, the rate of
change in the biological sample measured as the change in
concentration over a defined time period from administration to
maximum concentration for an controlled release formulation is less
than about 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the rate of
the immediate release formulation. Furthermore, in some
embodiments, the rate of change in concentration over time is less
than about 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the rate
for the immediate release formulation.
[0138] In some embodiments, the rate of change of concentration
over time is reduced by increasing the time to maximum
concentration in a relatively proportional manner. For example, a
two-fold increase in the time to maximum concentration may reduce
the rate of change in concentration by approximately a factor of 2.
As a result, the one or more cofactors may be provided so that it
reaches its maximum concentration at a rate that is significantly
reduced over an immediate release dosage form. The compositions of
the present invention may be formulated to provide a shift in
maximum concentration by 24 hours, 16 hours, 8 hours, 4 hours, 2
hours, or at least 1 hour. The associated reduction in rate of
change in concentration may be by a factor of about 0.05, 0.10,
0.25, 0.5 or at least 0.8. In certain embodiments, this is
accomplished by releasing less than about 30%, 50%, 75%, 90%, or
95% of the one or more cofactors into the circulation within one
hour of such administration.
[0139] Optionally, the controlled release formulations exhibit
plasma concentration curves having initial (e.g., from 2 hours
after administration to 4 hours after administration) slopes less
than 75%, 50%, 40%, 30%, 20% or 10% of those for an immediate
release formulation of the same dosage of the same cofactor.
[0140] In some embodiments, the rate of release of the cofactor as
measured in dissolution studies is less than about 80%, 70%, 60%
50%, 40%, 30%, 20%, or 10% of the rate for an immediate release
formulation of the same cofactor over the first 1, 2, 4, 6, 8, 10,
or 12 hours.
[0141] The controlled release formulations provided herein can
adopt a variety of formats. In some embodiments, the formulation is
in an oral dosage form, including liquid dosage forms (e.g., a
suspension or slurry), and oral solid dosage forms (e.g., a tablet
or bulk powder), such as, but not limited to those, those described
herein.
[0142] The controlled release tablet of a formulation disclosed
herein can be of a matrix, reservoir or osmotic system. Although
any of the three systems is suitable, the latter two systems can
have more optimal capacity for encapsulating a relatively large
mass, such as for the inclusion of a large amount of a single
cofactor, or for inclusion of a plurality of cofactors, depending
on the genetic makeup of the individual. In some embodiments, the
slow-release tablet is based on a reservoir system, wherein the
core containing the one or more cofactors is encapsulated by a
porous membrane coating which, upon hydration, permits the one or
more cofactors to diffuse through. Because the combined mass of the
effective ingredients is generally in gram quantity, an efficient
delivery system can provide optimal results.
[0143] Thus, tablets or pills can also be coated or otherwise
compounded to provide a dosage form affording the advantage of
prolonged action. For example, the tablet or pill can comprise an
inner dosage an outer dosage component, the latter being in the
form of an envelope over the former. The two components can be
separated by an enteric layer which serves to resist disintegration
in the stomach and permits the inner component to pass intact into
the duodenum or to be delayed in release. A variety of materials
can be used for such enteric layers or coatings such materials
including a number of polymeric acids and mixtures of polymeric
acids with such materials as shellac, cetyl alcohol and cellulose
acetate. In some embodiments, a formulation comprising a plurality
of cofactors may have different cofactors released at different
rates or at different times. For example, there can be additional
layers of cofactors interspersed with enteric layers.
[0144] Methods of making sustained release tablets are known in the
art, e.g., see U.S. Patent Publications 2006/051416 and
2007/0065512, or other references disclosed herein. Methods such as
described in U.S. Pat. Nos. 4,606,909, 4,769,027, 4,897,268, and
5,395,626 can be used to prepare sustained release formulations of
the one or more cofactors determined by the genetic makeup of an
individual. In some embodiments, the formulation is prepared using
OROS.RTM. technology, such as described in U.S. Pat. Nos.
6,919,373, 6,923,800, 6,929,803, and 6,939,556. Other methods, such
as described in U.S. Pat. Nos. 6,797,283, 6,764,697, and 6,635,268,
can also be used to prepare the formulations disclosed herein.
[0145] In some embodiments, the compositions can be formulated in a
food composition. For example, the compositions can be a beverage
or other liquids, solid food, semi-solid food, with or without a
food carrier. For example, the compositions can include a black tea
supplemented with any of the compositions described herein. The
composition can be a dairy product supplemented any of the
compositions described herein. In some embodiments, the
compositions can be formulated in a food composition. For example,
the compositions can comprise a beverage, solid food, semi-solid
food, or a food carrier.
[0146] In some embodiments, liquid food carriers, such as in the
form of beverages, such as supplemented juices, coffees, teas,
sodas, flavored waters, and the like can be used. For example, the
beverage can comprise the formulation as well as a liquid
component, such as various deodorant or natural carbohydrates
present in conventional beverages. Examples of natural
carbohydrates include, but are not limited to, monosaccharides such
as, glucose and fructose; disaccharides such as maltose and
sucrose; conventional sugars, such as dextrin and cyclodextrin; and
sugar alcohols, such as xylitol and erythritol. Natural deodorant
such as taumatin, stevia extract, levaudioside A, glycyrrhizin, and
synthetic deodorant such as saccharin and aspartame may also be
used. Agents such as flavoring agents, coloring agents, and others
can also be used. For example, pectic acid and the salt thereof,
alginic acid and the salt thereof, organic acid, protective
colloidal adhesive, pH controlling agent, stabilizer, a
preservative, glycerin, alcohol, or carbonizing agents can also be
used. Fruit and vegetables can also be used in preparing foods or
beverages comprising the formulations discussed herein.
[0147] Alternatively, the compositions can be a snack bar
supplemented with any of the compositions described herein. For
example, the snack bar can be a chocolate bar, a granola bar, or a
trail mix bar. In yet another embodiment, the present dietary
supplement or food compositions are formulated to have suitable and
desirable taste, texture, and viscosity for consumption. Any
suitable food carrier can be used in the present food compositions.
Food carriers of the present invention include practically any food
product. Examples of such food carriers include, but are not
limited to food bars (granola bars, protein bars, candy bars,
etc.), cereal products (oatmeal, breakfast cereals, granola, etc.),
bakery products (bread, donuts, crackers, bagels, pastries, cakes,
etc.), beverages (milk-based beverage, sports drinks, fruit juices,
alcoholic beverages, bottled waters), pastas, grains (rice, corn,
oats, rye, wheat, flour, etc.), egg products, snacks (candy, chips,
gum, chocolate, etc.), meats, fruits, and vegetables. In an
embodiment, food carriers employed herein can mask the undesirable
taste (e.g., bitterness). Where desired, the food composition
presented herein exhibit more desirable textures and aromas than
that of any of the components described herein. For example, liquid
food carriers may be used according to the invention to obtain the
present food compositions in the form of beverages, such as
supplemented juices, coffees, teas, and the like. In other
embodiments, solid food carriers may be used according to the
invention to obtain the present food compositions in the form of
meal replacements, such as supplemented snack bars, pasta, breads,
and the like. In yet other embodiments, semi-solid food carriers
may be used according to the invention to obtain the present food
compositions in the form of gums, chewy candies or snacks, and the
like.
[0148] The dosing of the combination compositions can be
administered about, less than about, or more than about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10 or more times a daily. A subject can receive
dosing for a period of about, less than about, or greater than
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days,
weeks or months. A unit dose can be a fraction of the daily dose,
such as the daily dose divided by the number of unit doses to be
administered per day. A unit dose can be a fraction of the daily
dose that is the daily dose divided by the number of unit doses to
be administered per day and further divided by the number of unit
doses (e.g. tablets) per administration. The number of unit doses
per administration may be about, less than about, or more than
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The number of doses
per day may be about, less than about, or more than about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, or more. The number of unit doses per day may
be determined by dividing the daily dose by the unit dose, and may
be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, 20, or more unit doses
per day. For example, a unit dose can be about 1/2, 1/3, 1/4, 1/5,
1/6, 1/7, 1/8, 1/9, 1/10. A unit dose can be about one-third of the
daily amount and administered to the subject three times daily. A
unit dose can be about one-half of the daily amount and
administered to the subject twice daily. A unit dose can be about
one-fourth of the daily amount with two unit doses administered to
the subject twice daily. In some embodiments, a unit dose comprises
about, less than about, or more than about 50 mg resveratrol. In
some embodiments, a unit dose comprises about, less than about, or
more than about 550 mg leucine. In some embodiments, a unit dose
comprises about, less than about, or more than about 200 mg of one
or more leucine metabolites.
[0149] In some embodiments, a unit dose (e.g. a unit dose
comprising one or more leucine metabolites, such as HMB) is
administered as one unit dose two timer per day. A unit dose may
comprise more than one capsule, tablet, vial, or entity.
[0150] Compositions disclosed herein can further comprise a
flavorant and can be a solid, liquid, gel or emulsion.
[0151] Methods
[0152] The subject composition is particularly useful for
ameliorating inflammatory responses elicited during onset of a
pulmonary condition. In one embodiment, the invention provides for
methods for reducing expression level or secretion of an
inflammatory marker including but not limited to NF.kappa.B,
eotaxin, IL1-.beta., and IL6, or increasing expression level or
secretion of an anti-inflammatory marker including but not limited
to adiponectin receptor 1 and adiponectin receptor 2, comprising
contacting a lung endothelial cell with any of the subject
compositions.
[0153] In various embodiments of the invention, a composition is
administered to the subject in an amount that delivers synergizing
amounts of leucine and/or a metabolite thereof, a methylxanthine
PDE inhibitor, and/or resveratrol sufficient treat pulmonary
conditions of the subject.
[0154] In some embodiments, the amounts of the methylxanthine, such
as theophylline or theobromine, in the composition, if administered
to a subject alone and without leucine, a leucine metabolite, or
resveratrol, would cause no therapeutic effect in the subject.
Additionally, the amounts of leucine, a leucine metabolite, or
resveratrol, if administered to the subject without the
methylxanthine, would have no therapeutic effect on the subject.
However, when the methylxanthine is administered in conjunction
with either leucine, a leucine metabolite, or resveratrol, a
therapeutic effect is observed.
[0155] Accordingly, the invention provides a method for
administering a composition comprising (a) leucine and/or one or
more metabolites thereof and (b) a methylxanthine present in a
sub-therapeutic amount, wherein the composition is effective in
increasing treating pulmonary conditions by at least about 5 fold
as compared to that of component (b) when it is used alone. The
amount of leucine in the composition may also be a sub-therapeutic
amount.
[0156] The invention also provides a method for administering a
composition comprising (a) leucine and/or one or more metabolites
thereof, (b) a methylxanthine present in a sub-therapeutic amount,
and (c) resveratrol wherein the composition is effective in
increasing treating pulmonary conditions by at least about 5 fold
as compared to that of component (b) when it is being used
alone.
[0157] Quantification of the therapeutic effect can show that the
effect of a composition that comprises (a) a methylxanthine and (b)
leucine or a leucine metabolite is greater than the predicted
effect of administering (a) or (b) alone, assuming simple additive
effects of (a) and (b), and thus the effect is synergistic. The
synergistic effect can be quantified as the measured effect above
the predicted simple additive effect of the components of the
composition. For example, if administration of component (a) alone
yields an effect of 10% relative to control, administration of
component (b) alone yields an effect of 15% relative to control,
and administration of a composition comprising both (a) and (b)
yields an effect of 60% relative to control, the synergistic effect
would be 60%-(15%+10%), or 35%.
[0158] At an in vitro level, the beneficial effects of the
compositions can be measured on cultured lung endothelial cells,
such as mouse primary lung endothelial cells, treated with
corresponding concentrations of the compositions described herein.
Analysis of the cells can allow for quantification of the
beneficial effects. For example, western blots or ELISA assays can
be performed to measure the quantity of (a) reduction in NF.kappa.B
protein expression, (b) eotaxin secretion, (c) reduction in
IL1-.beta. secretion, (d) reduction in cellular IL6 content or
secretion, (e) increase in adiponectin receptor 1 protein
expression, and/or (f) increase in adiponectin receptor 2 protein
expression in the cell as a result of treatment with a composition
described herein.
[0159] Accordingly, the multi-component compositions described
herein (such as theophylline/leucine,
theophylline/leucine/resveratrol, theobromine/leucine, and
theobromine/leucine/resveratrol) may have a synergistic effect on
(a) reduction in NF.kappa.B protein expression, (b) eotaxin
secretion, (c) reduction in IL1-.beta. secretion, (d) reduction in
cellular IL6 content or secretion, (e) increase in adiponectin
receptor 1 protein expression, and/or (f) increase in adiponectin
receptor 2 protein expression that is at least about 10, 20, 50,
100, 200, or 300%.
[0160] The output of the pathways and beneficial effects achieved
in a subject can be measured using one or more methods, disclosed
herein and/or known in the art. For example, measurements of COPD
and asthma may be made using spirometry, refinements of spirometry,
such as measurement of FEV6 and inspiratory capacity; measurements
of functional outcomes, such as dyspnea indexes and exercise tests;
and measurements of global-clinical outcomes, such as quality of
life questionnaires and assessment of frequency and severity of
acute exacerbations. In some embodiments, administration of the
compositions described herein to a subject can effect spirometry
values, lung capacity, and functional outcome measurements in the
subject that are improvements of at least about 10, 30, 50, 100,
200 or 300% greater relative to no administration of the
compositions or prior to administration of the compositions. The
effects may also be synergistic effects of at least about 10, 20,
50, 100, 200, or 300%.
[0161] The compositions can be administered to a subject orally or
by any other methods. Methods of oral administration include
administering the composition as a liquid, a solid, or a semi-solid
that can be taken in the form of a dietary supplement or a food
stuff.
[0162] The compositions can be administered periodically. For
example, the compositions can be administered one, two, three, four
times a day, or even more frequent. The subject can be administered
every 1, 2, 3, 4, 5, 6 or 7 days. In some embodiments, the
compositions are administered three times daily. The administration
can be concurrent with meal time of a subject. The period of
treatment or diet supplementation can be for about 1, 2, 3, 4, 5,
6, 7, 8, or 9 days, 2 weeks, 1-11 months, or 1 year, 2 years, 5
years or even longer. In some embodiments of the invention, the
dosages that are administered to a subject can change or remain
constant over the period of treatment. For example, the daily
dosing amounts can increase or decrease over the period of
administration.
[0163] The length of the period of administration and/or the dosing
amounts can be determined by a physician or any other type of
clinician. The physician or clinician can observe the subject's
response to the administered compositions and adjust the dosing
based on the subject's performance. For example, dosing for
subjects that show reduced effects in energy regulation can be
increased to achieve desired results.
[0164] In some embodiments, the compositions administered to a
subject can be optimized for a given subject. For example, the
ratio of branched chain amino acids to a sirtuin pathway activator
or the particular components in a combination composition can be
adjusted. The ratio and/or particular components can be selected
after evaluation of the subject after being administered one or
more compositions with varying ratios of branched chain amino acids
to a sirtuin pathway activator or varying combination composition
components.
[0165] Another aspect of the invention provides for achieving
desired effects in one or more subjects after administration of a
combination composition described herein for a specified time
period. For example, the beneficial effects of the compositions
described herein can be observed after administration of the
compositions to the subject for 1, 2, 3, 4, 6, 8, 10, 12, 24, or 52
weeks.
[0166] The invention provides for a method of treating subjects,
comprising identifying a pool of subjects amenable to treatment.
The identifying step can include one or more screening tests or
assays. For example, subjects that are identified as asthmatic,
diabetic or that have above average or significantly greater than
average body mass indices and/or weight can be selected for
treatment. The identifying step can include a genetic test that
identifies one or more genetic variants that suggest that the
subject is amenable to treatment. The identified subjects can then
be treated with one or more compositions described herein. For
example, they may be treated with a combination composition
comprising a sirtuin pathway activator and a branched-chain amino
acid.
[0167] The invention also provides for methods of manufacturing the
compositions described herein. In some embodiments, the manufacture
of a composition described herein comprises mixing or combining two
or more components. These components can include a PDE inhibitor or
sirtuin or AMPK pathway activator (such as a polyphenol or
polyphenol precursor like resveratrol, or a methylxanthine), and
leucine or metabolites thereof (such as HMB, or KIC). The amount or
ratio of components can be that as described herein. For example,
the mass ratio of leucine compared with resveratrol can be greater
than about 80.
[0168] In some embodiments, the compositions can be combined or
mixed with a pharmaceutically active agent, a carrier, and/or an
excipient. Examples of such components are described herein. The
combined compositions can be formed into a unit dosage as tablets,
capsules, gel capsules, slow-release tablets, or the like.
[0169] In some embodiments, the composition is prepared such that a
solid composition containing a substantially homogeneous mixture of
the one or more components is achieved, such that the one or more
components are dispersed evenly throughout the composition so that
the composition may be readily subdivided into equally effective
unit dosage forms such as tablets, pills and capsules.
[0170] Kits
[0171] The invention also provides kits. The kits include one or
more compositions described herein, in suitable packaging, and may
further comprise written material that can include instructions for
use, discussion of clinical studies, listing of side effects, and
the like. Such kits may also include information, such as
scientific literature references, package insert materials,
clinical trial results, and/or summaries of these and the like,
which indicate or establish the activities and/or advantages of the
composition, and/or which describe dosing, administration, side
effects, drug interactions, or other information useful to the
health care provider. Such information may be based on the results
of various studies, for example, studies using experimental animals
involving in vivo models and studies based on human clinical
trials. A kit may comprise one or more unit doses described herein.
In some embodiments, a kit comprises about, less than about, or
more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 30, 31, 60, 90, 120, 150, 180, 210, or more
unit doses. Instructions for use can comprise dosing instructions,
such as instructions to take 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
unit doses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times per day.
For example, a kit may comprise a unit dose supplied as a tablet,
with each tablet package separately, multiples of tablets packaged
separately according to the number of unit doses per administration
(e.g. pairs of tablets), or all tablets packaged together (e.g. in
a bottle). As a further example, a kit may comprise a unit dose
supplied as a bottled drink, the kit comprising 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 24, 28, 36, 48, 72, or more
bottles.
[0172] The kit may further contain another agent. In some
embodiments, the compound of the present invention and the agent
are provided as separate compositions in separate containers within
the kit. In some embodiments, the compound of the present invention
and the agent are provided as a single composition within a
container in the kit. Suitable packaging and additional articles
for use (e.g., measuring cup for liquid preparations, foil wrapping
to minimize exposure to air, and the like) are known in the art and
may be included in the kit. Kits described herein can be provided,
marketed and/or promoted to health providers, including physicians,
nurses, pharmacists, formulary officials, and the like. Kits may
also, in some embodiments, be marketed directly to the
consumer.
EXAMPLES
Example 1
Effects of Leucine, Theophylline, Theobromine and Resveratrol on
Indicators of Pulmonary Conditions
[0173] Techniques
[0174] Cell Culture:
[0175] Mouse primary lung endothelial cells (MLEC) were obtained
from Cell Biologics (Chicago, Ill.). Cells were grown to confluence
in T75 culture flasks precoated with 0.2% gelatin. Cells were grown
in Dulbecco's modified eagle's medium (DMEM) containing a growth
factor supplement (Cell Biologics # M1 166) which included 5% fetal
bovine serum (FBS), heparin, EGF, hydrocortisone, L-glutamine and
antibiotics at 37.degree. C. in 5% CO.sub.2.
[0176] Adipocyte Conditioned Media Experiments:
[0177] 3T3L1 adipocytes (passage 11 to 13) were grown and
differentiated on 6-well plates and treated for 48 h with the
treatments indicated in "Results". Media was then collected and
pooled together for each treatment group. Pooled media was then
used to treat confluent MLEC for 24 h in lieu of direct treatments.
The MLEC media from each cell replicate was collected, aliquoted
and stored in -20.degree. C. for further experiments. The cells
(MLEC and adipocytes) were washed once with ice-cold HBSS, then
ice-cold RIPA buffer plus Protease and Phosphatase Inhibitors
(Sigma) was added. The cell extract was then incubated on ice for
10 min, scraped, transferred to a new microcentrifuge tube,
homogenized for 5 sec and then centrifuged for 10 min at
12,000.times.g at 4.degree. C. Then the clear supernatant from each
cell replicate was aliquoted and stored at -20.degree. C. for
further experiments, as indicated below.
[0178] Western Blot:
[0179] NF.kappa.B and phospho-NF.kappa.B antibodies were obtained
from Cell Signaling (Danvers, Mass.). Cells were treated as
indicated in results and the cellular fractions were prepared using
standard methods. Protein was measured by BCA kit (Thermo
Scientific). For Western blot, 2 .mu.g (for NF.kappa.B) or 4 .mu.g
(for phospho-NF.kappa.B) of protein from the cell lysate was
resolved on 10% Tris/HCL polyacrylamide gels (Criterion precast
gel, Bio-Rad Laboratories, Hercules, Calif.), transferred to PVDF
membranes (NF.kappa.B and phospho-NF.kappa.B), incubated in
blocking buffer (3% BSA in TBS) and then incubated with primary
antibody, washed and incubated with secondary horseradish
peroxidase-conjugated antibody. Visualization and chemiluminescent
detection was conducted using BioRad ChemiDoc instrumentation and
software (Bio-Rad Laboratories, Hercules, Calif.) and band
intensity was assessed using Image Lab 4.0 (Bio-Rad Laboratories,
Hercules, Calif.), with correction for background and loading
controls. NF.kappa.B was detected at 60 kDA, and phospho-NF.kappa.B
at 52-59 kDA.
[0180] Cytokines:
[0181] Interleukin 1-.beta., eotaxin, and interleukin 6 were all
measured via enzyme-linked immunosorbent assay using specific
antibodies for each cytokine (Abcam, Cambridge, Mass.) and a
horseradish peroxidase-based second antibody detection system, with
chromogenic detection at 450 nm using a microplate reader (Synergy
HT, BioTek Instruments, Winooski, Vt.).
[0182] Results
[0183] Mouse primary lung endothelial cells were treated with
combinations of theophylline (1 .mu.M), theobromine (1 .mu.M),
leucine (0.5 mM), and resveratrol (200 nM). Each of theophylline,
theobromine, leucine, and resveratrol may be purchased from
appropriate vendors in the form indicated or salts of the forms
indicated. Following treatment, western blots or ELISA assays were
performed on the MLEC to determine the level of various indicators
of airway diseases, such as NF.kappa.B protein expression, eotaxin
secretion, IL1-.beta. secretion, cellular IL6 content or secretion,
adiponectin receptor 1 protein expression, and adiponectin receptor
2 protein expression.
[0184] Treatment with 0.5 mM leucine corresponds to a circulating
level of the same molarity achieved by orally administering about
1,125 mg of leucine to a human subject. Treatment with 0.25 mM
leucine corresponds to a circulating level of the same molarity
achieved by orally administering about 300 mg of leucine to a human
subject.
[0185] Treatment with 110 .mu.M theophylline corresponds to a
circulating level of the same molarity achieved by orally
administering about 1000 mg of theophylline to a human subject.
Treatment with 1 .mu.M theophylline corresponds to a circulating
level of the same molarity achieved by orally administering about
25-30 mg of theophylline to a human subject.
[0186] Treatment with 0.5 mM resveratrol corresponds to a
circulating level of the same molarity achieved by orally
administering about 1100 mg of resveratrol to a human subject.
Treatment with 200 nM resveratrol corresponds to a circulating
level of the same molarity achieved by orally administering about
50 mg of resveratrol to a human subject.
[0187] Theophylline exerted no independent effect on NF.kappa.B
protein expression relative to control (FIG. 1), but the
theophylline/leucine and theophylline/leucine/resveratrol
combinations resulted in significant reductions in NF.kappa.B
(p<0.02); there was no significant difference between
theophylline/leucine and theophylline/leucine/resveratrol
treatments. Treatment of MLEC with a combination of resveratrol and
leucine had no effect on the NF.kappa.B levels relative to control.
Phospho-NF.kappa.B exhibited similar trends. As shown by this data,
the combination of theophylline with leucine and theophylline with
leucine and resveratrol has a synergistic effect because treatment
with alone with (a) theophylline or (b) leucine and resveratrol had
no effect on NF.kappa.B or phospho-NF.kappa.B levels. The
synergistic effect of combining theophylline with leucine or
leucine and resveratrol was an improvement of at least about 22%
relative to baseline (treatment with theophylline alone or leucine
and resveratrol alone).
[0188] These effects on NF.kappa.B and phospho-NF.kappa.B were not
recapitulated in the conditioned media experiments (FIG. 2 and FIG.
3), indicating that these are direct effects on MLEC and not
mediated by alterations in cytokine secretion by adipocytes. The
conditioned media experiments utilized media prepared from
adipocytes treated with a control, theophylline,
theophylline/leucine, resveratrol/leucine, and
theophylline/resveratrol/leucine at the concentrations indicated
above.
[0189] In contrast, although this low concentration of theophylline
exerted no significant independent effect on any of the
inflammatory mediators studied, the leucine/theophylline and
leucine/theophylline/resveratrol combinations resulted in marked
reductions (.about.70%), relative to control, in IL1-.beta.
secretion (p<0.001; FIG. 4), with a smaller reduction found with
the leucine/resveratrol combination in the absence of theophylline
(p<0.01). TNF.alpha. used as a positive control to stimulate
secretion of the cytokine, and an increase in IL1-.beta. secretion
was observed when MLEC was treated with TNF.alpha. (p<0.01).
[0190] As can be seen in FIG. 4, the combined treatment of
theophylline with (a) leucine or (b) leucine and resveratrol
yielded a synergistic effect on IL1-.beta. secretion. Treatment
with theophylline alone had no effect relative to the control and
treatment with leucine/resveratrol had an effect of about 20%
relative to control. In comparison, the combined treatment of
theophylline with (a) leucine or (b) leucine and resveratrol had an
improvement of about 70% relative to baseline. In this case, the
synergistic effect is at least about 50% relative to the
control.
[0191] Similarly, the leucine/theophylline combination elicited a
significant 44% decrease in the secretion of the chemokine eotaxin
into the media, relative to control, (p<0.05; FIG. 5), while the
other treatments (theophylline alone,
theophylline/leucine/resveratrol, leucine resveratrol, and
TNF.alpha.) exerted no significant effect. As can be seen here, the
combined treatment of theophylline/leucine had a synergistic effect
of at least about 70% on eotaxin secretion, relative to the
control.
[0192] The effects shown in FIG. 5 are a direct effect on MLEC, as
the conditioned media experiments produced no significant effect on
eotaxin (FIG. 6).
[0193] Cellular expression of IL 6 was unaffected by theophylline,
but the theophylline/leucine and theophylline/leucine/resveratrol
treatments caused modest, significant decreases (p<0.02; FIG.
7).
[0194] Similarly, IL 6 secretion into the media was decreased by
.about.50% by both the theophylline/leucine and
theophylline/leucine/resveratrol treatments (p<0.0001; FIG.
8).
[0195] These direct effects were not recapitulated in the
conditioned media experiments (FIG. 9), demonstrating that this
anti-inflammatory effect is not mediated by alterations in
adipocyte cytokine production.
[0196] There was no direct effect of any of the treatments on MLEC
expression of either adiponectin receptor-1 (FIG. 10) or
adiponectin receptor 2 (FIG. 11).
[0197] However, treatment of adipocytes with the
theophylline/leucine/resveratrol combination produced conditioned
media that, when applied to MLEC, elicited a marked increase in the
expression of the anti-inflammatory adiponectin-2 receptor in MLEC
(p=0.0018, FIG. 12). As can be seen in FIG. 12, only treatment with
theophylline/resveratrol/leucine yielded an improvement relative to
control, suggesting a synergistic effect between all three
components. For this combination, the synergistic effect was about
100%.
[0198] Similarly, when the theophylline was replaced with
theobromine, another methylxanthine/non-specific PDE inhibitor, the
theobromine/leucine and leucine/resveratrol combinations stimulated
modest, significant increases in adiponectin receptor 1 (p<0.03,
FIG. 13), with a further, more robust increase with the
theobromine/leucine/resveratrol combination (p<0.0005, FIG.
13).
[0199] Both the theobromine/leucine and
theobromine/leucine/resveratrol combinations stimulated robust
increases in adiponectin receptor 2 expression (p<0.0001, FIG.
14). As seen in FIG. 14, the combination of theobromine with either
leucine or leucine/resveratrol yielded a synergistic effect of
about 100%.
[0200] These data demonstrate significant synergy between the
non-specific PDE inhibitors theophylline and theobromine with
leucine in attenuating the inflammatory responses that result in
airway diseases, including asthma and promoting anti-inflammatory
responses. Notably, these effects are achieved at otherwise
subtherapeutic doses of theophylline (1-2% of therapeutically
effective levels).
[0201] It should be understood from the foregoing that, while
particular implementations have been illustrated and described,
various modifications can be made thereto and are contemplated
herein. It is also not intended that the invention be limited by
the specific examples provided within the specification. While the
invention has been described with reference to the aforementioned
specification, the descriptions and illustrations of the preferable
embodiments herein are not meant to be construed in a limiting
sense. Furthermore, it shall be understood that all aspects of the
invention are not limited to the specific depictions,
configurations or relative proportions set forth herein which
depend upon a variety of conditions and variables. Various
modifications in form and detail of the embodiments of the
invention will be apparent to a person skilled in the art. It is
therefore contemplated that the invention shall also cover any such
modifications, variations and equivalents.
* * * * *
References