U.S. patent application number 16/584195 was filed with the patent office on 2020-01-16 for formulation and process for modulating wound healing.
This patent application is currently assigned to BioMendics, LLC. The applicant listed for this patent is BioMendics, LLC. Invention is credited to James M. Jamison, Karen M. McGuire, Jack L. Summers, Chun-che Tsai.
Application Number | 20200016096 16/584195 |
Document ID | / |
Family ID | 57073359 |
Filed Date | 2020-01-16 |
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United States Patent
Application |
20200016096 |
Kind Code |
A1 |
Tsai; Chun-che ; et
al. |
January 16, 2020 |
Formulation and Process for Modulating Wound Healing
Abstract
Methods and compounds are disclosed for wound healing by
modulating autophagy. A formulation for modulating autophagy
comprises a first modulating compound (FAM) selected from compounds
having the general structure (I): ##STR00001## wherein: L
represents a linker selected from --C.ident.C--, (a tolan),
--CH.dbd.CH-- (a stilbene, preferably trans); or
--CR.sub.a.dbd.CR.sub.b-- a stilbene derivative; where R.sub.a and
R.sub.b are independently H or phenyl optionally substituted with
--(R.sup.3).sub.p or --(R.sup.4).sub.q; R.sup.1 to R.sup.4 are
independent substituents at any available position of the phenyl
rings, preferably at 3, 3', 4, 4', and/or 5, 5'; and m, n, p, and q
are independently 0, 1, 2, or 3 representing the number of
substituents of the rings, respectively, but at least one of m or n
must be .gtoreq.1. Each R.sup.1 to R.sup.2 is independently
selected from substituents described herein, including but not
limited to hydroxyl, alkoxy, halo, halomethyl and glycosides. The
formulation may also include an auxiliary autophagy modulating
compound (AAM) as described herein. The formulation may include a
hydrogel formed by the compounds themselves or otherwise and may
include salts and/or complexes.
Inventors: |
Tsai; Chun-che; (Kent,
OH) ; McGuire; Karen M.; (Akron, OH) ;
Jamison; James M.; (Cleveland Heights, OH) ; Summers;
Jack L.; (Sun City Center, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioMendics, LLC |
Fairlawn |
OH |
US |
|
|
Assignee: |
BioMendics, LLC
Fairlawn
OH
|
Family ID: |
57073359 |
Appl. No.: |
16/584195 |
Filed: |
September 26, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16058437 |
Aug 8, 2018 |
10426742 |
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16584195 |
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15093146 |
Apr 7, 2016 |
10045950 |
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16058437 |
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62144539 |
Apr 8, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 8/733 20130101;
A61K 8/738 20130101; A61K 9/19 20130101; Y02A 50/401 20180101; A61P
17/14 20180101; A61K 8/33 20130101; A61P 1/16 20180101; A61P 9/10
20180101; A61P 17/00 20180101; A61Q 19/08 20130101; A61P 17/02
20180101; A61K 31/03 20130101; A61K 31/655 20130101; A61K 9/08
20130101; A61K 31/05 20130101; A61K 47/40 20130101; A61P 3/02
20180101; A61P 19/10 20180101; A61P 25/28 20180101; A61P 3/10
20180101; A61P 19/06 20180101; A61P 31/04 20180101; A61P 35/00
20180101; A61P 31/12 20180101; A61K 31/167 20130101; A61P 33/00
20180101; A61P 1/04 20180101; A61P 25/16 20180101; A61P 43/00
20180101; A61K 31/09 20130101; A61K 8/676 20130101; A61K 9/0019
20130101; A61K 8/70 20130101; A61P 27/02 20180101; A61P 11/06
20180101; A61P 15/00 20180101; A61K 47/36 20130101; A61P 19/02
20180101; A61Q 7/00 20130101; A61P 17/06 20180101; Y02A 50/30
20180101; A61K 9/06 20130101; A61K 8/347 20130101; A61K 31/7034
20130101; A61P 7/06 20180101; A61K 31/375 20130101; A61K 31/085
20130101; A61P 13/12 20180101; A61P 29/00 20180101; A61K 8/602
20130101; A61K 45/06 20130101; A61P 37/06 20180101; A61P 25/00
20180101; A61K 31/375 20130101; A61K 2300/00 20130101; A61K 31/05
20130101; A61K 2300/00 20130101; A61K 31/03 20130101; A61K 2300/00
20130101; A61K 31/7034 20130101; A61K 2300/00 20130101; A61K 31/085
20130101; A61K 2300/00 20130101; A61K 31/09 20130101; A61K 2300/00
20130101; A61K 31/167 20130101; A61K 2300/00 20130101; A61K 31/655
20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/05 20060101
A61K031/05; A61K 31/655 20060101 A61K031/655; A61K 31/375 20060101
A61K031/375; A61K 31/167 20060101 A61K031/167; A61K 31/03 20060101
A61K031/03; A61K 8/73 20060101 A61K008/73; A61K 8/70 20060101
A61K008/70; A61K 9/19 20060101 A61K009/19; A61K 9/08 20060101
A61K009/08; A61K 9/00 20060101 A61K009/00; A61K 8/67 20060101
A61K008/67; A61K 8/60 20060101 A61K008/60; A61K 8/33 20060101
A61K008/33; A61K 8/34 20060101 A61K008/34; A61Q 7/00 20060101
A61Q007/00; A61Q 19/08 20060101 A61Q019/08; A61K 47/40 20060101
A61K047/40; A61K 47/36 20060101 A61K047/36; A61K 9/06 20060101
A61K009/06; A61K 45/06 20060101 A61K045/06; A61K 31/7034 20060101
A61K031/7034; A61K 31/09 20060101 A61K031/09; A61K 31/085 20060101
A61K031/085 |
Claims
1. A method for promoting wound healing in a patient having a wound
or skin condition, comprising administering to a patient a
therapeutically effective amount of a formulation comprising: a
first autophagy modulating compound having the structure (I):
##STR00036## wherein L is a linker selected from the group
consisting of: --C.ident.C-- and --CR.sub.a.dbd.CR.sub.b--; R.sub.a
and R.sub.b are independently H or phenyl optionally substituted
with --(R.sup.3).sub.p or --(R.sup.4).sub.q; R.sup.1 to R.sup.4 are
independently substituents at any available position of the phenyl
rings; m, n, p, and q are, independently, 0, 1, 2, or 3,
representing the number of substituents on the rings, respectively,
and at least one of m or n must be .gtoreq.1; wherein each R.sup.1,
R.sup.2, R.sup.3, and R.sup.4 is independently selected from:
R.sup.5, wherein R.sup.5 is selected from (C.sub.1-C.sub.6)alkyl,
(C.sub.2-C.sub.6)alkenyl, or (C.sub.2-C.sub.6)alkynyl; optionally
substituted with 1 to 3 substituents selected from --OH, --SH,
-halo, --NH.sub.2, or NO.sub.2; YR.sup.6, wherein Y is O, S, or NH;
and R.sup.6 is selected from H or R.sup.5; ZR.sup.5, wherein Z is
--N(C.dbd.O)-- or --O(C.dbd.O)--; halo; NO.sub.2; SO.sub.3Na;
azide; and glycosides; and salts thereof; with the proviso that the
first autophagy modulating compound is not resveratrol or
4,4'-(ethyne-1,2-diyl)diphenol (TOLECINE, also known as
4,4'-dihydroxytolan).
2. The method of claim 1, wherein the first autophagy modulating
compound is a hydroxylated tolan, having from 1 to 4 hydroxyl
substituents.
3. The method of claim 1, wherein the first autophagy modulating
compound is selected from: 2,4,4'-trihydroxytolan;
4,3',5'-trihydroxytolan; 2,2',4,4'-tetrahydroxytolan;
3,3',5,5'-tetrahydroxytolan; 4-hydroxy-4'-(trifluoro)methyltolan;
4,4'-dihydroxy-3-methoxytolan;
2,4,4'-trihydroxytolan-beta-D-glucoside; and
4-hydroxy-4'-methoxytolan.
4. The method of claim 1, further comprising co-administering an
auxiliary autophagy modulating compound.
5. The method of claim 4, wherein the auxiliary autophagy modulator
compound is administered at the same time as the first autophagy
modulator compound.
6. The method of claim 4, wherein the auxiliary autophagy modulator
compound is administered prior to administering the first autophagy
modulator compound.
7. The method of claim 4, wherein the auxiliary autophagy
modulating compound is selected from the group consisting of:
substituted or unsubstituted parabenzoquinone, substituted or
unsubstituted orthobenzoquinone, substituted or unsubstituted
anthraquinone, an amino acid, an acidic monosaccharide, and a
vitamin or a salt thereof.
8. The method of claim 7, wherein the vitamin is an
oxygen-containing vitamin or an isoprenoid-containing vitamin.
9. The method of claim 1, wherein the first autophagy modulating
compound upregulates autophagy activity.
10. The method of claim 1, wherein the wound or skin condition is
one or more selected from aging, autoimmune diseases with
inflammation, avascular necrosis, bacterial infection, cancers,
diabetic neuropathies, endometriosis, fungal infection, gout,
hairloss, infectious arthritis, inflammation, inflammatory bowel,
ischemia, Lyme disease, organ/tissue transplant, parasitic
infection, psoriatic arthritis, psoriasis, pseudogout, rheumatoid
arthritis, scleraderma, scurvy, sepsis, skin diseases, surgical
scars, surgical adhesions, transfection procedures, ulcerative
colitis, ulcers, viral infection, warts, surgical wounds,
incisions, lacerations, cuts and scrapes, donor site wounds from
skin transplants, traumatic wounds, infectious wounds, ischemic
wounds, burns, bullous wounds, aseptic wounds, contused wounds,
incised wounds, lacerated wounds, non-penetrating wounds, open
wounds, penetrating wounds, perforating wounds, puncture wounds,
septic wounds, subcutaneous wounds, chronic ulcers, gastric ulcers,
skin ulcers, peptic ulcer, duodenal ulcer, gastric ulcer, gouty
ulcer, hypertensive ischemic ulcer, stasis ulcer, sublingual ulcer,
submucous ulcer, symptomatic ulcer, trophic ulcer, tropical ulcer,
veneral ulcer, hyperkeratosis, photo-aging, psoriasis, skin rashes,
sunburns, photoreactive processes, mouth sores and burns,
post-extraction wounds, endodontic wounds, ulcers and lesions of
bacterial or viral or autoimmunological origin, mechanical wounds,
chemical wounds, thermal wounds, infectious and lichenoid wounds,
herpes ulcers, stomatitis, aphthosa, acute necrotizing ulcerative
gingivitis, burning mouth syndrome, corneal ulcers, radial
keratotomy, corneal transplants, epikeratophakia, surgically
induced wounds in the eye, hemorrhoids, pruritus, proctitis, anal
fissures, dry cracked skin, seborrheic conditions, anthrax,
tetanus, gas gangrene, scalatina, erysipelas, sycosis barbae,
folliculitis, impetigo contagiosa, and impetigo bullosa.
11. The method of claim 1, wherein the first autophagy modulating
compound is dispersed in a hydrogel.
12. The method of claim 11, wherein the hydrogel is a liquid
crystalline hydrogel formed in part by the first autophagy
modulating compound.
13. The method of claim 11, wherein the hydrogel is an
alginate.
14. The method of claim 11, wherein the formulation comprises the
first autophagy modulating compound combined with a cyclodextrin in
a ratio of first autophagy modulating compound:cyclodextrin from
about 1:1 to about 1:10.
15. The method of claim 11, wherein the formulation comprises the
first autophagy modulating compound combined in a complex with
ascorbate and a cation.
16. A method for promoting wound healing in a patient having a
wound or skin condition, comprising administering to a patient a
therapeutically effective amount of a formulation comprising: a
first autophagy modulating compound having the structure (I):
##STR00037## wherein L is a linker comprising
--CR.sub.a.dbd.CR.sub.b--; R.sub.a and R.sub.b are independently H
or phenyl optionally substituted with --(R.sup.3).sub.p or
--(R.sup.4).sub.q; R.sup.1 to R.sup.4 are independently
substituents at any available position of the phenyl rings; m, n,
p, and q are, independently, 0, 1, 2, or 3, representing the number
of substituents on the rings, respectively, and at least one of m
or n must be .gtoreq.1; wherein each R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is independently selected from: R.sup.5, wherein R.sup.5 is
selected from (C.sub.1-C.sub.6)alkyl, (C.sub.2-C.sub.6)alkenyl, or
(C.sub.2-C.sub.6)alkynyl; optionally substituted with 1 to 3
substituents selected from --OH, --SH, -halo, --NH.sub.2, or
NO.sub.2; YR.sup.6, wherein Y is O, S, or NH; and R.sup.6 is
selected from H or R.sup.5; ZR.sup.5, wherein Z is --N(C.dbd.O)--
or --O(C.dbd.O)--; halo; NO.sub.2; SO.sub.3Na; azide; and
glycosides and salts thereof; with the proviso that the first
autophagy modulating compound is not resveratrol or
4,4'-(ethyne-1,2-diyl)diphenol (TOLECINE, also known as
4,4'-dihydroxytolan).
17. The method of claim 16, wherein the first autophagy modulating
compound is a trans-stilbene wherein L is --CH.dbd.CH--.
18. The method of claim 17, wherein the stilbene is a hydroxylated
stilbene, having from 1 to 4 hydroxyl substituents.
19. A method for modulating autophagy in a patient in need of
autophagy modulation, comprising administering to a patient a
therapeutically effective amount of a formulation comprising: a
first autophagy modulating compound having the structure (I):
##STR00038## wherein L is a linker selected from the group
consisting of: --C.ident.C-- and --CR.sub.a.dbd.CR.sub.b--; R.sub.a
and R.sub.b are independently H or phenyl optionally substituted
with --(R.sup.3).sub.p or --(R.sup.4).sub.q; R.sup.1 to R.sup.4 are
independently substituents at any available position of the phenyl
rings; m, n, p, and q are, independently, 0, 1, 2, or 3
representing the number of substituents on the rings, respectively,
and at least one of m or n must be .gtoreq.1; wherein each R.sup.1,
R.sup.2, R.sup.3, and R.sup.4 is independently selected from:
R.sup.5, wherein R.sup.5 is selected from (C.sub.1-C.sub.6)alkyl,
(C.sub.2-C.sub.6)alkenyl, or (C.sub.2-C.sub.6)alkynyl; optionally
substituted with 1 to 3 substituents selected from --OH, --SH,
-halo, --NH.sub.2, or NO.sub.2; YR.sup.6, wherein Y is O, S, or NH;
and R.sup.6 is selected from H or R.sup.5; ZR.sup.5, wherein Z is
--N(C.dbd.O)-- or --O(C.dbd.O)--; halo; NO.sub.2; SO.sub.3Na;
azide; and glycosides; and salts thereof.
20. The method of claim 19, wherein the patient suffers from a
condition in need of autophagy upregulation, said condition
comprising one or more of: wound healing, hair regrowth, bacterial
infections, inflammation, viral infection, Parkinson's disease,
neurodegenerative diseases, neuropathy, cardiovascular disease,
heat failure, heart disease, aging, Alzheimer's disease,
atherosclerosis, arterosclerosis, chronic obstructive pulmonary
disease (COPD), Crohn's disease, inflammatory bowel, colitis,
diabetes, diabetes type I or II, amyloidosis, bursitis, dermatitis,
angitis, autoimmune diseases with inflammation, blood diseases,
aplastic anemia, endometriosis, hepatitis, herpes, HIV, multiple
sclerosis, retinal detachment, age-related macular degeneration,
retinitis pigmentosa, Leber's congenital amaurosis, lysosomal
storage diseases, arthritis, psoriasis, osteopenia, osteoporosis,
surgical scars, surgical adhesions, space travel (bone density
disorder), tendonitis, ulcerative colitis, aging, cancer,
polycystic kidney and liver disease, kidney disease, liver disease,
asthma, diabetic retinopathy, fibromyalgia, ankylosing spondylitis,
celiac disease, Grave's disease, lupus, metabolic diseases,
nephritis, rheumatoid arthritis, osteolysis, ischemia-reperfusion
(I/R) injury, organ and tissue transplant, scleraderma, and sepsis.
Description
[0001] This application is a continuation application of U.S.
application Ser. No. 16/058,437, filed under 35 U.S.C. .sctn.
111(a) on Aug. 8, 2018, now allowed; which is a continuation
application of U.S. application Ser. No. 15/093,146, filed under 35
U.S.C. .sctn. 111(a) on Apr. 7, 2016, now U.S. Pat. No. 10,045,950;
which claims priority to U.S. Provisional Application No.
62/144,539, filed under 35 U.S.C. .sctn. 111(b) on Apr. 8, 2015.
The entire disclosures of all the aforementioned applications are
expressly incorporated herein by reference for all purposes.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to the field of
cellular biology and, more particularly, to compounds,
formulations, and methods for modulating autophagy to treat a wound
or skin related disease or condition. The name, "autophagy," also
known as autophagocytosis, is derived from the Greek words meaning
"eat" and "self." Autophagy is generally defined as
self-digestion.
[0003] Autophagy is primarily a lysosomal salvage or recycling
pathway that is commonly used by cells to perform homeostatic
functions by degrading aging proteins and organelles and
reabsorbing nucleotides, amino acids, and free fatty acids for new
molecule synthesis and ATP production. Autophagy may be
up-regulated in response to extra- or intracellular stress and
signals such as starvation, growth factor deprivation, ER stress,
and pathogen infection as a cellular-level self-preservation
mechanism. In some pathologic conditions, it may be desirable to
stimulate the autophagy process, while in other pathologic
conditions, such as wound healing, it may be desirable to suppress
or slow the autophagy process to reduce the destruction of cells.
Thus, modulation of autophagy may invoke either
enhancing/stimulating or suppressing/slowing the cell growth and
cell death process.
[0004] The most common form of autophagy involves (1) the formation
of an isolation membrane, which extends and the termini ultimately
(2) fuse to encompass the cellular contents within a
double-membrane vesicle known as an autophagosome. The
autophagosome then (3) fuses with a lysosome that provides enzymes
to (4) digest the contents of the autophagosome, which contents
then become available to the cell again as raw materials or
building block nutrients. Autophagy exhibits some similarities to
the parallel proteasome degradation of ubiquitin-tagged proteins,
but differs in that the autophagosome contains not only proteins,
but also cytoplasm, mitochondria, organelles and other cellular
structures. In this sense it is known as a bulk degradation
system.
[0005] Since the process of autophagy can be both beneficial and
detrimental to the cell depending on external factors and
conditions, the process must be tightly regulated. Both yeast and
mammalian systems have been studied and utilize up to 36 proteins.
FIG. 1 illustrates the process in mammals.
[0006] The yeast autophagy-related gene product (Atg8) has three
mammalian homologues: (1) LC3, (2) GABAA receptor-associated
protein (GABARAP), and (3) Golgi-associated ATPase enhancer
(GATE-16). Among them, LC3 is most actively studied and frequently
used as a mammalian autophagy marker. Shortly after translation
(proLC3), LC3 is processed at the C-terminus by Atg4A or Atg4B into
LC3-I. Upon induction or enhancement of autophagy, LC3-I is
conjugated to the substrate phosphatidylethanolamine (PE) via E1
(yeast Atg7) and E2 (yeast Atg3). The PE-LC3-I conjugate is
referred to as LC3-II. This conjugation occurs in the process at
the point of autophagosome formation and leads to the conversion of
soluble LC3-I to the autophagic vesicle associated LC3-II. This
lipid conjugation allows LC3-II to be used as a marker of autophagy
activity.
[0007] Wound progression is caused by many mechanisms including
local tissue hypo-perfusion, prolonged inflammation, free radical
damage, apoptosis, and necrosis. These are typically broken up into
three stages following the injury including; (i) the inflammation
phase, (ii) cell proliferation phase and (iii) remodeling phase.
Each one of these phases is linked to a biologically and
histologically unique fingerprint and autophagy plays a special
role at each phase. For example, during the inflammation stage,
autophagy is initially protective of tissue in that it attempts to
keep the wound edge cells from dying. In the proliferative stage,
during which involves cell multiplication and migration of multiple
cell types to close the wound, autophagy helps to regulate
.beta.1-integrins and other cell migration proteins to form a
controlled line of cells at the leading edge of the wound. In
remodeling, the direction of these migrating cells is often
controlled by the directionality and deposition of collagen that is
secreted by these transformed fibroblasts as pro-collagen. Collagen
along with other components such as fibronectin and lamin help
regenerate the basement membrane during the proliferation and
migration stages.
[0008] During the wound healing process collagen fibrils provide
the structural topology, rigidity and organization that allows for
proper cell migration. In chronic wounds and burns this environment
is subjected to a variety of factors including an altered pH,
chronic inflammation and often a loss in the basement membrane
including the essential collagen tracts that are used by migrating
wound cells (e.g. keratinocytes, fibroblasts, myofibroblasts) to
fill the wound and restore a layer of intact skin to protect the
body from invading microbes and environmental factors (e.g.
temperature regulation). During the natural healing process skin
cells secrete the soluble form of collagen known as pro-collagen
that can form higher order structures enzymatically or undergo an
entropy driven self-assembly process (Prockop, D. and D. Hulmes
(1994). Pro-collagen is secreted from the cell as a triple helix,
which can self-assemble into fibrils due to its liquid crystal
nature. This process is highly dependent on the pH, ionic strength,
temperature, and concentration. Fibrils then are formed into
individual collagen fibers, which are randomly assembled into
higher order 3-dimensional networks and structures.
[0009] U.S. Pat. Nos. 6,599,945 and 7,094,809 disclose several
hydroxytolan compounds and their use in inhibiting the formation of
infectious herpes virus particles or for treating gonorrhea caused
by Neisseria gonorrhoeae. WO2009/126700 discloses the use of
similar compounds for skin care, such as UV radiation, and cosmetic
uses. And U.S. Pat. No. 8,716,355 (WO2011/0130468) and 8,680,142
(WO2011/0160301) disclose similar hydroxytolans for use as
anti-tumor agents. However, the potential utility of these, or any
other hydroxytolans as autophagy-modulating compounds was unknown
until the making of the present invention. U.S. Pat. Nos.
6,008,260, 6,197,834 and 6,355,692 disclose certain hydroxylated
stilbenes, and specifically resveratrol. None of these references
disclose the use of such modified tolan or stilbene compounds as
autophagy modulating agents.
[0010] It would be advantageous if the process of autophagy could
be modulated, that is stimulated or enhanced in some conditions,
and slowed or suppressed in other conditions.
SUMMARY OF THE INVENTION
[0011] The present invention relates to compounds, formulations,
and methods for modulating autophagy, particularly for the
indication or purpose of promoting wound healing in a patient
having a chronic wound or skin condition. In one aspect, the
invention comprises a method of promoting wound healing comprising
administering at least one first autophagy modulating (FAM)
compound as described herein. In most embodiments for promotion of
wound healing, the autophagy modulation is directionally
upregulation of autophagy activity. In certain embodiments, the FAM
is administered with an auxiliary autophagy modulator (AAM)
compound. The FAM and AAM may be co-administered or administered
one prior to the other. If co-administered, they may be formulated
in the same dosage form or as two distinct dosages or drug
products. The AAM may modulate the effect of the FAM by either
stimulating or increasing its effect, or by depressing or
inhibiting its effect since many are hormetic compounds.
[0012] These autophagy modulators are liquid crystalline in nature
and act in specific combinations to treat a wound or skin related
disease or condition. The invention further describes the use of
these liquid crystals to create bioactive formulations such as
hydrogels and alginates that can also serve as bioactive membranes
to promote healing in a wound or skin related condition. Thus, in
some aspects, the invention comprises a formulation containing a
FAM and AAM. The formulation may be a hydrogel, such as an
alginate. The formulation may include a cationic salt of an FAM, or
a complex of an FAM and an AAM with a cation. In other
formulations, the FAM and/or AAM may be formulated with a
cyclodextrin.
[0013] The specific FAM may be a tolan or stilbene (including cis
and trans stilbenes) and may have any of a variety of substituents
as described herein. In some embodiments, the substituents are
hydroxyl or alkoxyl groups that increase solubility and polarity of
the molecule. Any FAM may be administered with any AAM. For
example, the FAM may be a tolan and the AAM may be a vitamin,
acidic sugar, amino acid, or quinolone. Likewise, the FAM may be a
stilbene and the AAM may be a vitamin, acidic sugar, amino acid, or
quinolone.
[0014] In some aspects, the method promotes wound healing in
specific indications, such as wherein the wound or skin condition
is one or more selected from: aging, autoimmune diseases with
inflammation, avascular necrosis, bacterial infection, cancers,
diabetic neuropathies, endometriosis, fungal infection, gout,
hairloss, infectious arthritis, inflammation, inflammatory bowel,
ischemia, Lyme disease, organ/tissue transplant, parasitic
infection, psoriatic arthritis, psoriasis, pseudogout, rheumatoid
arthritis, scleraderma, scurvy, sepsis, skin diseases, surgical
scars, surgical adhesions, transfection procedures, ulcerative
colitis, ulcers, viral infection, warts, surgical wounds,
incisions, lacerations, cuts and scrapes donor site wounds from
skin transplants, traumatic wounds, infectious wounds, ischemic
wounds, burns, bullous wounds, aseptic wounds, contused wounds,
incised wounds, lacerated wounds, non-penetrating wounds, open
wounds, penetrating wounds, perforating wounds, puncture wounds,
septic wounds, subcutaneous wounds, chronic ulcers, gastric ulcers,
skin ulcers, peptic ulcer, duodenal ulcer, gastric ulcer, gouty
ulcer, hypertensive ischemic ulcer, stasis ulcer, sublingual ulcer,
submucous ulcer, symptomatic ulcer, trophic ulcer, tropical ulcer
and veneral ulcer.
[0015] In some methods, the wound to be healed is dematological in
nature, such as cuts, abrasions, ulcers of many types and degrees,
aging, skin inelasticity, and the like. In other methods, the wound
may be ophthalmic or otic; in other methods, the wound may be oral
in nature, such as cancer sores, herpes viral infections, tooth
extraction wounds, gingivitis, or the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The accompanying drawings, incorporated herein and forming a
part of the specification, illustrate the present invention in its
several aspects and, together with the description, serve to
explain the principles of the invention. In the drawings, the
thickness of the lines, layers, and regions may be exaggerated for
clarity.
[0017] FIG. 1 is a schematic illustration showing the role of LC3
in autophagy.
[0018] FIG. 2 is a schematic illustration showing autophagy related
human diseases.
[0019] FIG. 3 is a bar graph of LC3-II fluorescent staining as
described in Example 5.
[0020] FIG. 4 is a bar graph of an LC3-II Western Blot as described
in Example 6.
[0021] FIG. 5 is a bar graph of the LC3-II/LC3-I ratio as described
in Example 6.
[0022] FIG. 6 is a graph of AKT signaling as described in Example
8.
[0023] FIG. 7 is a graph of Fibroblast Growth Factor as described
in Example 9.
[0024] The accompanying drawings, incorporated herein and forming a
part of the specification, illustrate the present invention in its
several aspects and, together with the description, serve to
explain the principles of the invention. In the drawings, the
thickness of the lines, layers, and regions may be exaggerated for
clarity.
DETAILED DESCRIPTION
[0025] Numerical ranges, measurements and parameters used to
characterize the invention--for example, angular degrees,
quantities of ingredients, polymer molecular weights, reaction
conditions (pH, temperatures, charge levels, etc.), physical
dimensions and so forth--are necessarily approximations; and, while
reported as precisely as possible, they inherently contain
imprecision derived from their respective measurements.
Consequently, all numbers expressing ranges of magnitudes as used
in the specification and claims are to be understood as being
modified in all instances by the term "about." All numerical ranges
are understood to include all possible incremental sub-ranges
within the outer boundaries of the range. Thus, a range of 30 to 90
units discloses, for example, 35 to 50 units, 45 to 85 units, and
40 to 80 units, etc. Unless otherwise defined, percentages are
weight/weight (wt/wt); although formulations are generally
weight/volume (w/v), in grams per 100 mL (which is equivalent to
wt/wt with aqueous solutions having a density of 1.0), and area of
wounds is expressed in cm.sup.2 as area/area (a/a).
[0026] All patents, published patent applications, and non-patent
literature references cited herein are incorporated herein by
reference in their entirety.
[0027] In some aspects, the invention comprises methods of
modulating autophagy comprising the administration of a first
autophagy modulator (FAM) compound and optionally an auxiliary
autophagy modulator (AAM) compound. The FAM and AAM compounds are
described in more detail in sections below. They may be given
sequentially or concomitantly. If given sequentially, the order may
be FAM first, then AMM, or AMM first, then FAM. If given
concomitantly, they may be given in separate, individual drug
products or a single drug product as a combination of ingredients.
The compounds may be administered from once daily up to about 6
times per day, depending on the formulation excipients.
Administration routes include topical, transdermal, oral, nasal,
ophthalmic, otic, IV, IM, subcutaneous, rectal, and vaginal.
[0028] The use of pharmaceutical excipients in the preparation of
drug products is generally well understood from pharmaceutical
treatises such as Remington's Pharmaceutical Sciences, 18.sup.th
Edition (1990) and its subsequent editions, like Remingtons: The
Science and Practice of Pharmacy, 22.sup.nd edition (2012). Topical
formulations may be combined with solvents, emulsifiers,
emollients, solvents, etc. into solutions, suspensions, creams,
ointments and hydrogels, among others.
[0029] In certain embodiments, the invention involves a formulation
containing a FAM compound and an AAM compound. The relative amounts
of FAM to AAM in a formulation expressed as a molar ratio may range
from about 500:1 to about 1:500 (FAM:AAM), or, in certain
embodiments, from about 200:1 to 1:200. In liquid formulations, the
FAM may comprise from about 0.01% to about 40% (w/v) of the
formulation and the AMM may comprise from about 0.01% to about
99.9% (w/v) of the formulation. In certain embodiments, the FAM may
comprise from about from about 0.1% to about 40% (w/v) of the
formulation and the AMM may comprise from about 0.1% to about 60%
(w/v) of the formulation. Optimally the formulation concentration
of an FAM is between 0.5-15% (w/v).
Chemical and Biological Definitions
[0030] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described
herein. All references cited herein, including books, journal
articles, published U.S. or foreign patent applications, issued
U.S. or foreign patents, and any other references, are each
incorporated by reference in their entireties, including all data,
tables, figures, and text presented in the cited references.
[0031] The following terms used throughout this application have
the meanings ascribed below.
[0032] A "first autophagy modulator" or "FAM compound" or "FAM"
means a compound of formula I:
##STR00002##
[0033] wherein L is a linker selected from: --C.ident.C-- and
--CR.sub.a.dbd.CR.sub.b--; [0034] R.sub.a and R.sub.b are
independently H or phenyl optionally substituted with
--(R.sup.3).sub.p or --(R.sup.4).sub.q; [0035] R.sup.1 to R.sup.4
are independently substituents at any available position of the
phenyl rings; [0036] m, n, p and q are, independently, 0, 1, 2, or
3 representing the number of substituents on the rings,
respectively, and at least one of m or n must be .gtoreq.1;
[0037] wherein each R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is
independently selected from: [0038] R.sup.5, wherein R.sup.5 is
selected from (C.sub.1-C.sub.6)alkyl, (C.sub.2-C.sub.6)alkenyl, or
(C.sub.2-C.sub.6)alkynyl; optionally substituted with 1 to 3
substituents selected from --OH, --SH, -halo, --NH.sub.2, or
NO.sub.2; [0039] YR.sup.6, wherein Y is O, S, or NH; and R.sup.6 is
selected from H or R.sup.5; [0040] ZR.sup.5, wherein Z is
--N(C.dbd.O)-- or --O(C.dbd.O)--; [0041] halo; [0042] NO.sub.2;
[0043] SO.sub.3Na; [0044] azide; and [0045] glycosides [0046] and
salts thereof; [0047] with the proviso that the FAM is not
resveratrol or 4,4'-(ethyne-1,2-diyl)diphenol (TOLCINE).
[0048] An "auxiliary autophagy modulator" or "AAM compound" or
"AAM" means a compound as described herein that also has an
autophagy modulating effect. The effect may be stimulatory or
inhibitory depending on the compound. While not intending to be
bound by any particular theory, AAM compounds may have inhibitory
action by competing for a rate limiting step, such as cellular
uptake mechanisms. More specific AAM compounds are described in a
subsequent section.
[0049] Autophagy modulation refers to either up-regulation or
down-regulation of the process of autophagy in the cell. Depending
on the particular disease state or condition, it may be desirable
to achieve one or the other direction of regulation of autophagy,
as is described later. And, referring to the discussion of
hormesis, the dose of any particular FAM or AAM compound or
combination or complex may achieve up-regulation or down-regulation
or both.
[0050] As used herein, the term "--(C.sub.1-C.sub.6)alkyl" refers
to straight-chain and branched non-cyclic saturated hydrocarbons
having from 1 to 6 carbon atoms. Representative straight chain
--(C.sub.1-C.sub.6)alkyl groups include methyl, -ethyl, -n-propyl,
-n-butyl, -n-pentyl, and -n-hexyl. Representative branched-chain
--(C.sub.1-C.sub.6)alkyl groups include isopropyl, sec-butyl,
isobutyl, tert-butyl, isopentyl, neopentyl, 1-methylbutyl,
2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, and
1,2-dimethylpropyl, methylpentyl, 2-methylpentyl, 3-methylpentyl,
4-methylpentyl, 1-ethylbutyl, 2-ethylbutyl, 3-ethylbutyl,
1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl,
2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, and the
like. More generally, the subscript refers to the number of carbon
atoms in the chain. Thus, the term "--(C.sub.1-C.sub.4)alkyl"
refers to straight-chain and branched non-cyclic saturated
hydrocarbons having from 1 to 4 carbon atoms.
[0051] As used herein, the term "--(C.sub.2-C.sub.6)alkenyl" refers
to straight chain and branched non-cyclic hydrocarbons having from
2 to 6 carbon atoms and including at least one carbon-carbon double
bond. Representative straight chain and branched
--(C.sub.2-C.sub.6)alkenyl groups include -vinyl, allyl,
-1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl,
-3-methyl-1-butenyl, -2-methyl-2-butenyl, and the like.
[0052] As used herein, the term "--(C.sub.2-C.sub.6)alkynyl" refers
to straight chain and branched non-cyclic hydrocarbons having from
2 to 6 carbon atoms and including at least one carbon-carbon triple
bond. Representative straight chain and branched
--(C.sub.2-C.sub.6)alkynyl groups include -acetylenyl, -propynyl,
-1 butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl,
-3-methyl-1-butynyl, -4-pentynyl, and the like.
[0053] As used herein, "--(C.sub.1-C.sub.10)alkoxy" means a
straight chain or branched non-cyclic hydrocarbon having one or
more ether groups and from 1 to 10 carbon atoms. Representative
straight chain and branched (C.sub.1-C.sub.10)alkoxys include
-methoxy, -ethoxy, -propoxy, -butyloxy, -pentyloxy, -hexyloxy,
-heptyloxy, -methoxymethyl, -2-methoxyethyl, -5-methoxypentyl,
-3-ethoxybutyl and the like.
[0054] As used herein, "--(C.sub.1-C.sub.6)alkoxy" means a straight
chain or branched non-cyclic hydrocarbon having one or more ether
groups and from 1 to 6 carbon atoms. Representative straight chain
and branched (C.sub.1-C.sub.5)alkoxys include -methoxy, -ethoxy,
-propoxy, -butyloxy, -pentyloxy, -hexyloxy, -methoxymethyl,
-2-methoxyethyl, -5-methoxypentyl, -3-ethoxybutyl and the like.
[0055] As used herein, the term "--(C.sub.3-C.sub.12)cycloalkyl"
refers to cyclic saturated hydrocarbon having from 3 to 12 carbon
atoms. Representative (C.sub.3-C.sub.12)cycloalkyls include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclooctyl, and cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl,
and the like.
[0056] As used herein, the term "--(C.sub.4-C.sub.12)cycloalkenyl"
refers to a cyclic hydrocarbon having from 4 to 12 carbon atoms,
and including at least one carbon-carbon double bond.
Representative --(C.sub.3-C.sub.12)cycloalkenyls include
-cyclobutenyl, -cyclopentenyl, -cyclopentadienyl, -cyclohexenyl,
-cyclohexadienyl, -cycloheptenyl, -cycloheptadienyl,
-cycloheptatrienyl, -cyclooctenyl, -cyclooctadienyl,
-cyclooctatrienyl, -cyclooctatetraenyl, -cyclononenyl,
-cyclononadienyl, -cyclodecenyl, -cyclodecadienyl, -norbornenyl,
and the like.
[0057] As used herein a "-(7- to 12-membered)bicyclic aryl" means
an bicyclic aromatic carbocyclic ring containing 7 to 12 carbon
atoms. Representative -(7- to 12-membered) bicyclic aryl groups
include -indenyl, -naphthyl, and the like.
[0058] As used herein a "hydroxy(C.sub.1-C.sub.6)alkyl" means any
of the above-mentioned C.sub.1-6 alkyl groups substituted by one or
more hydroxy groups. Representative hydroxy(C.sub.1-C.sub.6)alkyl
groups include hydroxymethyl, hydroxyethyl, hydroxypropyl and
hydroxybutyl groups, and especially hydroxymethyl, 1-hydroxyethyl,
2-hydroxyethyl, 1,2-dihydroxyethyl, 2-hydroxypropyl,
3-hydroxypropyl, 3-hydroxybutyl, 4-hydroxybutyl,
2-hydroxy-1-methylpropyl, and 1,3-dihydroxyprop-2-yl.
[0059] Any of the groups defined above may be optionally
substituted. As used herein, the term "optionally substituted"
refers to a group that is either unsubstituted or substituted.
Optional substituents, when present and not otherwise indicated,
include 1, 2, or 3 groups each independently selected from the
group consisting of --(C.sub.1-C.sub.6)alkyl, OH, halo,
--C(halo).sub.3, --CH(halo).sub.2, --CH.sub.2(halo), NH.sub.2,
--NH(C.sub.1-C.sub.6)alkyl, CN, SH, phenyl, benzyl, (.dbd.O),
halo(C.sub.1-C.sub.6)alkyl-, hydroxy(C.sub.1-C.sub.6)alkyl-. Thus,
certain substituted embodiments include those named below.
[0060] As used herein a "dihydroxy(C.sub.1-C.sub.6)alkyl" means any
of the above-mentioned C.sub.1-6 alkyl groups substituted by two
hydroxy groups. Representative dihydroxy(C.sub.1-C.sub.6)alkyl
groups include dihydroxyethyl, dihydroxypropyl and dihydroxybutyl
groups, and especially 1,2-dihydroxyethyl, 1,3-dihydroxypropyl,
2,3-dihydroxypropyl, 1,3-dihydroxybutyl, 1,4-dihydroxybutyl, and
1,3-dihydroxyprop-2-yl.
[0061] As used herein, the terms "halo" and "halogen" refer to
fluoro, chloro, bromo or iodo.
[0062] As used herein, "--CH.sub.2(halo)" means a methyl group
where one of the hydrogens of the methyl group has been replaced
with a halogen. Representative --CH.sub.2(halo) groups include
--CH.sub.2F, --CH.sub.2Cl, --CH.sub.2Br, and --CH.sub.2I.
[0063] As used herein, "--CH(halo).sub.2" means a methyl group
where two of the hydrogens of the methyl group have been replaced
with a halogen. Representative --CH(halo).sub.2 groups include
--CHF.sub.2, --CHCl.sub.2, --CHBr.sub.2, --CHBrCl, --CHClI, and
--CHI.sub.2.
[0064] As used herein, "--C(halo).sub.3" means a methyl group where
each of the hydrogens of the methyl group has been replaced with a
halogen. Representative --C(halo).sub.3 groups include --CF.sub.3,
--CCl.sub.3, --CBr.sub.3, and --Cl.sub.3.
[0065] As used herein, "(halo).sub.p(C.sub.1-C.sub.6)alkyl-" means
a (C.sub.1-C.sub.6)alkyl chain substituted with halo in p
locations, where p is 1, 2 or 3. The halo substituents may be
substituted on the same or a different carbon in the
(C.sub.1-C.sub.6)alkyl. Representative
"(halo).sub.p(C.sub.1-C.sub.6)alkyl-" groups include, for example
--CH.sub.2CHF.sub.2, --CH.sub.2CH.sub.2CH.sub.2Cl, --CHBr.sub.2,
--CHBrCl, --CHClI, --CH.sub.2CHI.sub.2,
--CH.sub.2CH.sub.2CHClCH.sub.2Br, and the like.
[0066] As used herein, "azide" means a substituent of the formula
--N.dbd.N.dbd.N.
[0067] As used herein, "glycoside" means a 5- or 6-membered cyclic
sugar connected to the compound of Formula I. The bond is generally
a glycosidic bond from an anomeric carbon of the sugar, and may be
made via: (1) an oxygen atom, thus forming an "0-glycoside", (2) a
nitrogen atom, thus forming an "N-glycoside" or (3) a sulfur atom,
thus forming an "S-glycoside." Glycosides may be mono- or
di-saccharides, having one or two ring structures. Representative
glycosides formed from 6 membered sugars glucosides, galactosides,
mannosides, and altrosides; and glycosides formed from the 5
membered sugars, include ribosides, arabinosides, xylosides and
lyxosidees. The sugars may contain optional substituents, but many
embodiments contain only the native --H and --OH substituents that
define the respective sugars.
[0068] Overlap exists in the literature among use of the terms
"wound," "ulcer," and "sore" and, furthermore, the terms are often
used at random. Therefore, in the present context the term "wounds"
encompasses the term "ulcer", "lesion", "sore" and "infarction",
and the terms are used interchangeably unless otherwise indicated.
Wounds have been classified by many criteria, including size or
area (large or small), depth or layer involvement, causation,
difficulty in healing, etc. Some classify wounds by i) small tissue
loss due to surgical incisions, minor abrasions and minor bites, or
as ii) significant tissue loss, such as in ischemic ulcers,
pressure sores, fistulae, lacerations, severe bites, thermal burns
and donor site wounds (in soft and hard tissues) and infarctions.
All types and classification of wounds are encompassed by the
invention.
[0069] The term "skin" is used in a very broad sense embracing the
epidermal layer of the integument and, in those cases where the
skin surface is more or less injured, also the dermal layer
beneath. Apart from the stratum corneum, the epidermal layer of the
skin is the outer (epithelial) layer and the deeper connective
tissue layer of the skin is called the "dermis," which contains the
nerves and terminal organs of sensation.
[0070] A method that "promotes the healing of a wound" results in
the wound healing more quickly as a result of the treatment than a
similar wound heals in the absence of the treatment. "Promotion of
wound healing" can also mean that the method regulates the
proliferation and/or growth of, inter alia, keratinocytes, or that
the wound heals with less scarring, less wound contraction, less
collagen deposition and more superficial surface area. In certain
instances, "promotion of wound healing" can also mean that certain
methods of wound healing have improved success rates, (e.g., the
take rates of skin grafts) when used together with the method of
the present invention.
[0071] The phenomenon of hormesis is commonly associated with
compounds that induce biologically opposite effects in a dose
dependent fashion, and is well described in the literature, for
example: Calabrese E J, Bachmann K A, Bailer A J, Bolger P M, Borak
J, et al. (2007), Biological stress response terminology:
Integrating the concepts of adaptive response and preconditioning
stress within a hormetic dose-response framework. Toxicol Appl
Pharmacol. 222:122-128; Penniston K L, Tanumihardjo S A. The acute
and chronic toxic effects of vitamin A. Am J Clin Nutr. 2006;
83:191-201; and Cook R, Calabrese E J. The importance of hormesis
to public health. Cien saude Colet. 2007; 12:955-963. Commonly
there is a stimulatory or beneficial effect at low doses and an
inhibitory or toxic effect at high doses. Hormesis has also been
characterized as autoprotection, adaptive response, and
preconditioning, among others; and by the shape of its dose
response curve including: e.g. .beta.-curve, biphasic, bell-shaped,
U-shaped or inverted-U shaped, bimodal, functional antagonism, and
dual response, among others. Known hormetic substances (or
"hormetins") include: vitamin A, ferulic acid, chalcone, rapamycin,
epigallocatechin-3-gallate and many others.
[0072] Hormesis can also represent adaptive responses where a
moderate stress is applied in order to provide the organism with
adaptive resistance when faced with a severe stressor.
Environmental stresses such as oxidative metabolic and thermal
stress serve as hormetins used to induce a specific response or
adaptive change (Mattson M P. Hormesis defined, Ageing Res Rev.
2008; 7:1-7). Hormetins have been shown to activate various stress
and detoxification pathways including; heat shock proteins,
antioxidant, protein chaperones, metabolism, calcium homeostasis
and growth factors (Mattson M P, Cheng A. Neurohormetic
phytochemicals: Low-dose toxins that induce adaptive neuronal
stress responses. Trends Neurosc. 2006; 29:632-639; and Mattson,
2008, noted above). These dose response effects have been analyzed
for a range of natural signaling molecules including nitric oxide,
adenosine, opioids, adrenergic agents, prostaglandins, estrogens,
androgens, 5-hydroxytryptamine and dopamine (Calabrese E J,
Bachmann K A, Bailer A J, Bolger P M, Borak J, et al. (2007),
Biological stress response terminology: Integrating the concepts of
adaptive response and preconditioning stress within a hormetic
dose-response framework. Toxicol Appl Pharmacol. 222:122-128 and
Hayes DP. Nutritional hormesis. Eur J Clin Nutr. 2007; 61:147-159)
Many of these hormetics are concentrated by bacteria, fungi,
viruses and plants to protect themselves from predatory species.
However, when many of these substances are utilized in lower
concentrations they can have beneficial effects.
[0073] A hydrogel is a dilute cross-linked aqueous system
comprising water and a gelling agent such as a polymeric plastic or
polysaccharide which can absorb and retain significant amounts of
water to form three-dimensional network structures. The hydrogel
structure is created by the interaction of water with hydrophilic
groups or domains present in the polymeric network upon hydration.
Hydrogels are categorized principally as weak or strong depending
on their flow behavior in steady-state.
[0074] Gelation occurs when the polymer concentration increases,
and disperses polymers begin to branch and form crosslinks. Once a
critical concentration is reached the sol becomes a gel and the
sol-gel transition occurs. Gels may be considered chemically linked
or physically linked. Physical gels can be subcategorized as strong
or weak, depending on the nature of the bonds, with strong physical
approaching chemical gels in linkage permanence. Strong physical
bonds include: lamellar microcrystals, glassy nodules or double and
triple helices; whereas weak physical gels include: hydrogen bonds,
block copolymers, micelles, and ionic associations.
[0075] Hydrogels, due to their significant water content possess a
degree of flexibility similar to natural tissue, and may exhibit
viscoelastic or pure elastic behavior, and stickiness. Properties
of a hydrogel may be modified by controlling the polarity, surface
properties, mechanical properties, and swelling behavior. Gels may
exhibit significant volume changes in response to small changes in
pH, ionic strength, temperature, electric field, and light.
Biodegradable hydrogels, containing labile bonds, are advantageous
in applications such as tissue engineering, wound healing and drug
delivery. These labile bonds can be present either in the polymer
backbone or in the cross-links used to prepare the hydrogel. The
labile bonds can be broken under physiological conditions either
enzymatically or chemically, in most of the cases by hydrolysis
(Bajpai, A. K., Shukla, S. K., Bhanu, S. & Kankane, S. (2008).
Responsive polymers in controlled drug delivery. Progress in
Polymer Science. 33: 1088-1118).
[0076] Ionic polymers having negatively charged groups at
physiological pH can be cross-linked by the addition of multivalent
cations; and even monovalent cations (e.g. K.sup.+, Na.sup.+, etc.)
may shield or screen the repulsion of negatively charged groups
(e.g. SO.sup.-.sub.3) to form stable gels. Examples include:
Na.sup.+alginate.sup.-; chitosan-polylysine; chitosan-glycerol
phosphate salt (Syed K. H. Gulrez.sup.1 and Saphwan Al-Assaf.
Progress in molecular and environmental bioengineering from
analysis and modeling to technology applications Chapter 5:
Hydrogels: Methods of preparation, characterization and
applications. Publisher InTech. Aug. 1, 2011(34)) and
chitosan-dextran hydrogels (Hennink, W. E. & Nostrum, C. F.
(2002). Novel crosslinking methods to design hydrogels. Advanced
drug delivery reviews. 54: 13).
[0077] Alginate is a naturally occurring anionic polysaccharide
typically obtained from brown seaweed, and has been extensively
investigated and used for many biomedical applications, due to its
biocompatibility, low toxicity, relatively low cost, and ability to
form hydrogels by addition of divalent cations such as Ca.sup.2+.
Alginate hydrogels have been used in a variety of applications
including; wound healing and drug delivery. Alginate wound
dressings maintain a moist wound environment, minimize bacterial
infection at the wound site, and facilitate wound healing. Drug
molecules can be released from alginate gels in a controlled
manner, depending on the cross-linker and cross-linking methods
employed. In addition, alginate gels can be orally administrated or
injected, making them extensively useful in the pharmaceutical
arena.
[0078] Alginates are polysaccharides constituted by variable
amounts of .beta.-D-mannuronic acid and its C5-epimer
.alpha.-L-guluronic acid linked by 1-4 glycosidic bonds. The
capability of alginate to confer viscosity in sol-gels is dependent
on its molecular mass (MM). The molecular mass (MM) of algal
alginates has been found to range from 48 to 186 kDa (38); whereas
some alginates isolated from A. vinelandii present MM in the range
of 80 to 4,000 kDa (Galindo, E.; Pena, C.; N nez, C.; Segura, D.
& Espin, G. (2007). Molecular and bioengineering strategies to
improve alginate and polyhydroxyalkanoate production by Azotobacter
vinelandii. Microbial Cell Factories, 6, 1-1). The saccharide
monomers are distributed in blocks of continuous mannuronate
residues (M), guluronate residues (G) or alternating residues (MG),
depending on the source species Smidsrod, 0. & Draget, K.
(1996). Chemistry and physical properties of alginates.
Carbohydrates European, 14, 6-12). The G-blocks of alginates
participate in intermolecular cross-linking with divalent cations
(e.g., Ca.sup.2+) to form hydrogels. The composition (i.e., M/G
ratio), sequence, G-block length, and molecular weight are critical
factors which alter the physical properties (e.g. increasing
G-block length increases ionic binding and the mechanical rigidity
of the gel) of alginate and alginate hydrogels. These same
properties control the stability of the gels along with the release
rate of alginates containing drugs. Alginates with a low M/G ratio
form strong and brittle gels, while alginates with a high M/G ratio
form weaker and softer, but more elastic gels. Finally, bacterial
alginates are acetylated to a variable extent at positions 0-2
and/or 0-3 of the mannuronate residues (Skjak-Braek, G.; Grasdalen,
H. & Larsen, B. (1986). Monomer sequence and acetylation
pattern in some bacterial alginates. Carbohydrates Research, 154,
239-250). The variability in molecular mass, monomer block
structure and acetylation all influence the physicochemical and
rheological characteristics of the gel polymer.
[0079] The majority of alginates use the divalent cation calcium or
monovalent ions such as Na.sup.+ or K.sup.+ while other ions such
as Mg.sup.2+ have been proposed but for the gelation process to
occur the concentration of magnesium ions required is 5-10 times
higher than that of calcium (Topuz, F., Henke, A., Richtering, W.
& Groll, J. (2012). Magnesium ions and alginate do form
hydrogels: a rheological study. Soft Matter. 8:4877-4881).
Alginates may have decreased water solubility such as alginic acid
or calcium alginate where the ion is shielded from ionization
resulting in insoluble alginates. Water soluble alginates can be
made simply by creating salts using monovalent anions such as
Na.sup.+ or K.sup.+ or a non polar group such as NH.sub.4 which is
generally water soluble.
First Autophagy Modulators (FAMs)
[0080] As noted, the first autophagy modulators are compounds of
Formula I, which comprises two phenyl rings joined by a linker, L,
and having at least one R.sup.1 or R.sup.2 attached to a phenyl
ring. In some embodiments, L is --C.ident.C-- and these FAM
compounds are known as "tolans," which are generally linear from
phenyl ring to phenyl ring. In other embodiments, L is
--CH.dbd.CH-- and these FAM compounds are known as "stilbenes"
which are isomeric in cis and trans forms about the double bond. In
some embodiments the FAM compound is a trans stilbene. In still
other embodiments, L is --CR.sub.a.dbd.CR.sub.b-- where R.sub.a
and/or R.sub.b may be a phenyl ring or H. These are also stilbenes,
in this case "phenyl stilbene derivatives" and they may also be
trans stilbenes or cis stilbenes.
[0081] There are two "primary" phenyl rings shown in the structure
and containing substituents R.sup.1 and R.sup.2, of which at least
one must be present (i.e. at least one of m or n is .gtoreq.1).
Optionally, there are also up to two "secondary" phenyl rings
within the options for R.sub.a and R.sub.b. On each phenyl ring (up
to four possible, two primary and two secondary) there may be from
zero to five of each substituent R.sup.1-4. In certain embodiments,
there are from one to three R.sup.1, and/or from one to three
R.sup.2 substituents on the primary phenyl rings. In some
embodiments, the position of R.sup.1 and/or R.sup.2 on the primary
phenyl rings is mostly at the para and meta positions, namely the
3, 4 or 5 position on one phenyl ring and the 3', 4' and 5'
positions on the other phenyl ring, although it is also possible to
have substituents in the ortho position (2, 2', 6 and 6'). There
may be one, two or three R.sup.1 substituents on the first phenyl
ring, and correspondingly, from zero to three R.sup.2 substituents
on the second phenyl ring. Conversely, there may be one, two or
three R.sup.2 substituents on the second phenyl ring, and
correspondingly, from zero to three R.sup.1 substituents on the
first phenyl ring. Additionally, in some embodiments, the secondary
phenyl rings may contain one to three substituents R.sup.3 and
R.sup.4. All permutations within these are possible, for example:
one R.sup.1 and one R.sup.2; two R.sup.1 and two R.sup.2; three
R.sup.1 and three R.sup.2; one R.sup.1 and two R.sup.2; one R.sup.1
and three R.sup.2; two R.sup.1 and one R.sup.2; two R.sup.1 and two
R.sup.2; two R.sup.1 and three R.sup.2; three R.sup.1 and one
R.sup.2; or three R.sup.1 and two R.sup.2. Likewise with R.sup.3
and R.sup.4. Each R.sup.14 is independently selected and if two or
more are present they may be the same or different. Examples of
some specific first autophagy modulators are given in Table A, the
tolans being analogues of the stilbenes so detailed structures are
not necessary for each individual compound.
TABLE-US-00001 TABLE A Certain Representative FAM compounds The
nature and position(s) of R.sup.1 and R.sup.2 substituents
Stilbenes Tolans ##STR00003## ##STR00004## Hydroxy ##STR00005##
3,5-dihydroxytolan, 3,4-dihydroxytolan, 3,4,5-trihydroxytolan,
3,3',4,5'-tetrahydroxytolan; and 3,3',4,4'-tetrahydroxytolan
3,5,4'-trihydroxytolan 3,4,4'-trihydroxytolan;
3,3',4'-trihydroxytolan; 2,4,4'-trihydroxytolan;
2,4,2',4'-tetrahydroxytolan 3,3',4,5'-tetrahydroxy-trans-stilbene
(aka piceatannol); and 3,3',4,4'- tetrahydroxy-trans-stilbene
3,4,4'-trihydroxy-trans-stilbene;
3,3',4'-trihydroxy-trans-stilbene; 4,4'-dihydroxy-trans-stilbene;
2,4,4'-trihydroxy-trans-stilbene;
2,4,2',4'-tetrahydroxy-trans-stilbene Alkyl and
3,5-dihydroxy-4-ethylstilbene, 3,5-dihydroxy-4-ethyltolan, mixed
3,5-dihydroxy-4'-ethylstilbene; 3,5-dihydroxy-4'-ethyltolan
3,5-dihydroxy-4-isopropyl-trans- 3,5-dihydroxy-4-isopropyltolan;
and stilbene; 3,5-dihydroxy-4-isopropyl-
3,5-dihydroxy-4-isopropyl-trans- trans-4'-hydroxystilbene; 3,5-
4'hydroxytolan; 3,5-dihydroxy-4- dihydroxy-4-isopropyl-trans-3',4'-
isopropyl-trans-3',4'-dihydroxytolan; dihydroxystilbene;
3,5-dihydroxy-4- 3,5-dihydroxy-4-isopropyl-trans-
isopropyl-trans-3',5'- 3',5'-dihydroxytolan; 3,5-dihydroxy-
dihydroxystilbene; 3,5-dihydroxy-4-
4-isopropyl-trans-3',4',5'-trihydroxytolan
isopropyl-trans-3',4',5'- 3,4-dihydroxy-4'-isopropyltolan;
trihydroxystilbene 3,5-dihydroxy-4'-isopropyltolan;
3,4-dihydroxy-4'-isopropyl-trans-stilbene;
3,4,5-trihydroxy-4'-isopropyltolan;
3,5-dihydroxy-4'-isopropyl-trans-stilbene;
4-hydroxy-4'-isopropyltolan
3,4,5-trihydroxy-4'-isopropyl-trans-stilbene;
4'-hydroxy-3,4-dimethyltolan,
4-hydroxy-4'-isopropyl-trans-stilbene;
4'-hydroxy-3,4,5-trimethyltolan,
4'-hydroxy-3,4-dimethyl-trans-stilbene,
4'-hydroxy-4,5-dimethyltolan.
4'-hydroxy-3,4,5-trimethyl-trans-stilbene,
4'-hydroxy-4,5-dimethyl-trans-stilbene Glycosides and mixed
##STR00006## E.g. 3,5'-dihydroxy-4'-methoxytolan 5-O-
.beta.-D-glucoside; 3',4'-dihydroxy-3-methoxytolan-5-
O-.beta.-D-glucoside; 3,4'-dihydroxytolan 5-O-.beta.-D- glucoside;
2,3-dihydroxy-4'-methoxytolan 5-O- .beta.-D-glucoside;
2,3,3'-trihydroxy-4'-methoxytolan 5- O-.beta.-D-glucoside; E.g.
3,5'-dihydroxy-4'-methoxystilbene- 5-O-.beta.-D-glucoside
3',4'-dihydroxy-3-methoxystilbene 5-O-.beta.-D-glucoside;
3,4'-dihydroxystilbene 5-O-.beta.-D-glucoside;
2,3-dihydroxy-4'-methoxystilbene 5-O-.beta.-D-glucoside;
2,3,3'-trihydroxy-4'.sup.1- methoxystilbene 5-O-.beta.-D-glucoside
Halo and mixed ##STR00007## 3,5-dihydroxy-4'-chlorotolan,
3,4-dihydroxy-4'-chlorotolan, or 4,5- dihydroxy-4'-chlorotolan,
3,4,5-dihydroxy-4'-chlorotolan 3,4-dihydroxy-4'-fluorotolan or 4,5-
dihydroxy-4'-fluorotolan, 3,4,5-trihydroxy-4'-fluorotolan,
3,5-dihydroxy-4'-fluorotolan 3,4-dihydroxy-4'-
(trifluoro)methyltolan 4,5-dihydroxy-4'-chlorostilbene,
3,4,5-dihydroxy-4'-chlorostilbene, and
2,3,4,5,6-penthydroxy-4'-chlorostilbene
3,4-dihydroxy-4'-fluorostilbene, or
4,5-dihydroxy-4'-fluorostilbene,
3,4,5-trihydroxy-4'-fluorostilbene,
3,5-dihydroxy-4'-fluorostilbene, ##STR00008##
3,4-dihydroxy-4'-(trifluoro)methylstilbene Thiol and
3,5-dihydroxy-4'-thiolstilbene, 3,5-dihydroxy-4'-thioltolan, mixed
3,4-dihydroxy-4'-thiolstilbene, or 3,4-dihydroxy-4'-thioltolan, or
4,5- 4,5-dihydroxy-4'-thiolstilbene, dihydroxy-4'-thioltolan,
3,4,5-dihydroxy-4'-thiolstilbene, 3,4,5-dihydroxy-4'-thioltolan,
3,4'-dithiol-trans-stilbene, 3,4'-dithiol-tolan,
4,4'-dithiol-trans-stilbene, 4,4'-dithiol-tolan, Alkoxy (O- alkyl)
and thioether (S-alkyl) and mixed ##STR00009##
3,5,4'-trimethoxytolan, 3,4,4'-trimethoxytolan, or 4,5,4'-
trimethoxytolan, 3,4,5,4'-tetramethoxytolan,
4'-hydroxy-3,5-dimethoxytolan, 4'-hydroxy-3,4-dimethoxytolan, or
4'-hydroxy-4,5-dimethoxytolan, 4'-hydroxy-3,4,5-trimethoxytolan,
3,5-dihydroxy-4'-methoxytolan, 4,4'-dimethoxytolan,
3,5-dimethoxymethoxy-4'- thiomethyltolan
3,5,3'-trihydroxy-4'-methoxytolan, 4,4'-dihydroxy-3-methoxytolan
3,4-dihydroxy-4'-methoxytolan 3,4-dimethoxytolan,
3,4'-dimethoxytolan, 3,5-dihydroxy-4'-methoxy-trans-stilbene,
4,4'-dimethoxy-trans-stilbene,
3,5-dimethoxy-4'-thiomethyl-trans-stilbene
3,5,3'-trihydroxy-4'-methoxy-trans-stilbene
4,4'-dihydroxy-3-methoxy-trans-stilbene
3,4-dihydroxy-4'-methoxy-trans-stilbene
3,4-dimethoxy-trans-stilbene, 3,4'-dimethoxy-trans-stilbene, Acyl
and 4'hydroxy-5-acetoxy-trans-stilbene, 4'hydroxy-5-acetoxy-tolan,
mixed 3,4'-dihydroxy-5-acetoxy-trans-stilbene,
3,4'-dihydroxy-5-acetoxy-tolan,
3,5-dihydroxy-4'-acetoxy-trans-stilbene,
3,5-dihydroxy-4'-acetoxy-tolan,
3,4'-dihydroxy-4-acetoxy-trans-stilbene,
3,4'-dihydroxy-4-acetoxytolan,
4,4'-dihydroxy-3-acetoxy-trans-stilbene,
4,4'-dihydroxy-3-acetoxytolan,
3,4-dihydroxy-4'-acetoxy-trans-stilbene
3,4-dihydroxy-4'-acetoxytolan
3,4,3'-trihydroxy-4'-acetoxy-trans-stilbene,
3,4,3'-trihydroxy-4'-acetoxytolan,
3,4,4'-trihydroxy-3'-acetoxy-trans-stilbene,
3,4,4'-trihydroxy-3'-acetoxytolan,
3,5,4'-trihydroxy-3'-acetoxy-trans-stilbene,
3,5,4'-trihydroxy-3'-acetoxytolan,
3,5,3'-trihydroxy-4'-acetoxy-trans-stilbene,
3,5,3'-trihydroxy-4'-acetoxytolan Amide and mixed ##STR00010##
3,5-dihydroxy-4'-acetamidetolan, 4,5-dihydyroxy-4'-acetamidetolan,
or 3,4-dihydyroxy-4'-acetamidetolan,
3,4,5-trihydyroxy-4'-acetamidetolan,
3,4,3'-trihydyroxy-4'-acetamidetolan,
3,4,5'-trihydyroxy-4'-acetamidetolan, 4'-hydroxy-5-acetamidetolan,
3,4'-dihydroxy-5-acetamidetolan, 4,4'-dihydroxy-3-acetamidetolan,
3,4-dihydroxy-4'-acetamidetolan, 3,4'-dihydroxy-4-acetamidetolan,
4,4'-dihydroxy-3-acetamidetolan, 3,4-dihydroxy-4'-acetamidetolan,
3,4,4'-trihydroxy-3'-acetamidetolan,
3,5,4'-trihydroxy-4-acetamidetolan,
3,4,3'-trihydroxy-4'-acetamidetolan,
3,4,4'-trihydroxy-3'acetamidetolan,
3,3'-dihydroxy-4'-acetamidetolan,
4,4'-dihydroxy-3-acetamide-trans-stilbene,
3,4-dihydroxy-4'-acetamide-trans-stilbene,
3,4'-dihydroxy-4-acetamide-trans-stilbene,
4,4'-dihydroxy-3-acetamide-trans-stilbene,
3,4-dihydroxy-4'-acetamide-trans-stilbene
3,4,4'-trihydroxy-3'-acetamide-trans-stilbene,
3,5,4'-trihydroxy-4-acetamide-trans-stilbene,
3,4,3'-trihydroxy-4'-acetamide-trans-stilbene,
3,4,4'-trihydroxy-3'acetamide-trans-stilbene,
3,3'-dihydroxy-4'-acetamide-trans-stilbene, Azides ##STR00011##
4-hydroxy-4'-azidotolan, 3,5-dihydroxy-4'-azidotolan,
4,5-dihydroxy-4'-azidotolan, or 3,4-dihydroxy-4'-azidotolan,
3,4,5-trihydroxy-4'-azidotolan, 4-hydroxy-4'-azidostilbene,
3,5-dihydroxy-4'-azidostilbene, 4,5-dihydroxy-4'-azidostilbene, or
3,4-dihydroxy-4'-azidostilbene, 3,4,5-trihydroxy-4'-azidostilbene,
Misc. ##STR00012## 4-hydroxy-4'-nitro-tolan,
3,5-dihydroxy-4'-nitro-tolan, 3,4-dihydroxy-4'-nitro-tolan, or
4,5-dihydroxy-4'-nitro-tolan, 3,4,5-trihydroxy-4'-nitro-tolan,
4-hydroxy-4'-nitro-trans-stilbene, ##STR00013##
3,5-dihydroxy-4'-nitro-trans-stilbene, ##STR00014##
3,4-dihydroxy-4'-nitro-trans-stilbene, or
4,5-dihydroxy-4'-nitro-trans-stilbene, ##STR00015##
3,4,5-trihydroxy-4'-nitro-trans-stilbene
[0082] Many of the stilbene compounds are well studied, naturally
occurring molecules and some are readily commercially available.
Others may be synthesized by routine methods, such as those
described by: Ali, M. A., Kondo, K. and Tsuda, Y. (1992). Synthesis
and Nematocidal activity of Hydroxystilbenes. Chem. Pharm. Bull.
40(5):1130-1136; and Thakkar, K., Geahlen, R. L. and Cushman, M.
(1993). Synthesis and Protein-tyrosine kinase inhibitory activity
of polyhydroxylated stilbene analogues of piceatannol. J. Med.
Chem. 36: 2950-2955). Phenyl stilbene derivatives, i.e. those in
which at least one R.sub.a or R.sub.b is a phenyl ring, and many
other stilbenes may be synthesized according to the methods shown
in the dissertation of Zhenlin Bai, Substituted Stilbenes and
1,2-Diaryl-1,2-diazidoethanes as Potential Anticancer Agents:
Syntheses and Estrogenic/Antiestrogenic Properties in MCF-7-2a
Cells, im Fachbereich Biologie, Chemie, Pharmazie der Freien
Universitat Berlin (2006). Glucoside derivatives may be obtained
according to procedures described in WO2007/020673 A1. Tolans may
be synthesized using the general procedures described in U.S. Pat.
No. 6,599,945 B2 of Docherty & Tsai.
[0083] It can be noted that the FAM compounds described above are
generally polar, and have certain electronegative substituents
(e.g. --OH, --OCH.sub.3, --NO.sub.2, -halo, --O(C.dbd.O)R, etc.) at
the respective ends. While this is not deemed essential, it may be
desirable to provide for a liquid crystal-like behavior for
molecules to assume a lyotrophic or partially ordered structure in
solution state.
[0084] FAM compounds also include salts of the compounds identified
above. FAM compounds, especially those mono or poly-hydroxylated
compounds, easily release one or more protons depending on pH to
form anions. Such anions may be combined with cations, such as the
mono-, di-, and tri-valaent cations to form salts. For monovalent
cations (M+) a single FAM is linked to form M.sup.+FAM.sup.- salts.
Similarly, for a divalent cation (M.sup.2+) two FAM molecules are
linked to form M.sup.2+(FAM.sup.-).sub.2 salts; and for a trivalent
cation (M.sup.3+) three FAM molecules are linked to form
M.sup.+3(FAM.sup.-).sub.3 salts. The salts are often readily
soluble in aqueous media, which may facilitate formulations.
Illustrative, but not limiting, cations for FAM salt formation
include: Na.sup.+ or K.sup.+, Mg.sup.2+, Mn.sup.2+, Zn.sup.2+,
Ca.sup.2+, Cu.sup.+, Cu.sup.2+Fe.sup.2+, and Fe.sup.3+.
Auxiliary Autophagy Modulators (AAMs) and Formulations
[0085] In certain embodiments, the FAM is used in combination with
an auxiliary autophagy modulator (AAM). The AMM, when used, may
take any of several forms described herein, falling into any of the
following classes: vitamins, amino acids, acidic sugars, and
quinine derivatives
[0086] Vitamins
[0087] Vitamins A, B, C, D, E, and K may all be useful as AAMs. By
convention, the term "vitamin" includes neither other essential
nutrients, such as dietary minerals, essential fatty acids, or
essential amino acids (which are needed in greater amounts than
vitamins), nor the great number of other nutrients that promote
health. Thirteen vitamins are universally recognized at present.
Vitamins are classified by their biological and chemical activity,
not their structure. Thus, each "vitamin" refers to a number of
"vitamer" compounds that all show the biological activity
associated with a particular vitamin. Such a set of chemicals is
grouped under an alphabetized vitamin "generic descriptor" title,
such as "vitamin A", which includes multiple compounds as described
below. Vitamers by definition are convertible to the active form of
the vitamin in the body, and are sometimes inter-convertible to one
another, as well. In certain embodiments the salt forms of the
vitamins, generally without long side aliphatic chains, are
excellent AAMs. In certain embodiments, the oxygen containing
vitamins are suitable AAMS.
[0088] Most vitamins will also form salts that are also within the
scope of AAMs. For example, vitamins may combine to form salts with
cationic elements like sodium, potassium, magnesium, manganese,
calcium, copper, zinc, or iron. Additionally vitamins can form a
diethanolamine salt, a 2-amino-2-ethyl-1,3-propanediol salt, a
triethanolamine salt, a morpholine salt, a piperazine salt, a
piperidine salt, an arginine salt, a lysine salt and a histidine
salt. Some vitamins form acetates, palmitates, oleates, linoleates,
stearates, lactates, succinates, maleates, citrates, and the
like.
[0089] Vitamin A refers to a group of lipid soluble, unsaturated,
isoprenoid compounds that includes but is not limited to retinol,
retinal, retinoic acid, carotenoids, retinyl acetate, retinyl
palmitate, .alpha.-carotene, .beta.-carotene, .gamma.-carotene,
.beta.-cryptoxanthin, xanthophyll, crytoxanthin, 13-cis retinoic
acid, 13-trans retinoic acid, tretinoin, ATRA (all trans retinoic
acid), lutin, 11-cis-retinal, 11-cis-retinol, 9-cis-retinal,
Lecithin, retinyl esters, 9-cis-.beta.-carotene, retinyl palmitate,
Acitretin, Vitamin A2 (3,4-dehydroretinol), A3(3-hydroxyretinol),
and salts thereof. All isomeric and stereochemical forms of these
isoprenoids are encompassed in the invention.
[0090] Vitamin B includes but is not limited to the following
compounds:
[0091] thiamine (B1); riboflavin (B2); niacin or niacinamide (forms
of B3); pantothenic acid, panthenol, pantothenol and calcium
pantothenate (forms of B5); pyridoxine, pyridoxine 5'-phosphate,
pyridoxal, pyridoxal phosphate, pyridoxal 5'-phosphate,
pyridoxamine, pyridoxamine 5'-phosphate, 4-pyridoxic acid (forms of
B6); biotin, vitamin H or coenzyme R (Forms of B7); Folic Acid,
folate, vitamin M, vitamin Bc, pteroyl-L-glutamic acid and
pteroyl-L-glutamate, (forms of B9); and Cobalamin, Cyanocobalamin,
Hydroxycobalamin, Methylcobalamin, Adenoxylcobalamin (forms of
B12); and salts thereof.
[0092] Vitamin C refers to ascorbic acid, its anion ascorbate, and
salts of ascorbate, as well as ascorbyl palmitate and salts thereof
(e.g. ascorbyl palmitate, magnesium ascorbyl palmitate, manganese
ascorbyl palmitate, calcium ascorbyl palmitate, zinc ascorbyl
palmitate, iron ascorbyl palmitate), benzyl ascorbate, and
2-ascorbyl phosphate.
[0093] Vitamin D refers to a group of lipid soluble secosteroid
molecules and includes but is not limited to: calcidiol, calcifero
(INN), Ergocalciferol and lumisterol (forms of D1); ergocalciferol,
fromergosterol, and 25-hydroxy vitamin D2 (forms of D2);
Cholecalciferol, 7-dehydrocholesterol, and
25-hydroxycholecalciferol (or 25-hydroxyvitamin D3, abbreviated
25(OH)D.sub.3, (forms of D3); 22-dihydroergocalciferol (D4);
Sitocalciferol, 7-dehydrositosterol (D5); 25-D-glucuronic acid,
25-D-hexuronic acid, 25-hydroxy vitamin D2-25-.beta.-D-glucuronide,
and salts thereof.
[0094] Vitamin E refers to a group of lipid soluble compounds that
are either tocopherols or tocotrienols, the most active of which is
.alpha.-tocopherol. Other tocopherols include, beta, gamma, delta.
Similarly, tocotrienols exist in alpha, beta, gamma and delta forms
as well. All isomeric and stereochemical forms of these tocopherols
and tocotrienols and their salts are encompassed in the invention.
For example, synthetic vitamin E is a mixture of eight isomeric
forms, usually labeled "all-rac" or "dl." Tocopherol and
tocotrienol derivatives include all R and all S stereoisomers of
tocopherols (RRR, RRS, RSR, SRR, RSS, SRS and SSS) and the two
stereoisomers of tocotrienols (e.g. R or S-.alpha.-tocotrienols).
Other examples include: Conjugated vitamin E molecules; vitamin E
or tocopherol or tocotrienol esters; alpha-tocopheryl acetate;
vitamin E esters (e.g. alpha-tocopheryl succinate) include a group
of compounds formed by esterifying a vitamin E molecule with a
carboxylic acid; d-.alpha.-tocopherol is often a mixture of two or
more enantiomers of other tocopherols
(.beta.,.gamma.,.delta.,.epsilon.,.zeta.,.eta.) or as tocotrienols,
n-propionate or linoleate such as vitamin E acetate or
alpha-tocopheryl acetate. Water soluble forms of vitamin E include:
Magnesium R-(+)-alpha lipoate,
6-hydroxy-2,5,7,8-tramethylchroman-2-carboxylic acid (trolox), or
salts of vitamin E
[0095] Vitamin K refers to a group of compounds having a
2-methyl-1,4,naphthoquinone core and a side chain at the 3
position. Vitamers K.sub.1 (phylloquinone, phytomenadione, or
phytonadione) and K.sub.2 (menaquinones) are naturally occurring.
In fact, K.sub.2 is not one, but a series of compounds having
varying-length isoprenoid side chains; and the menaquinone family
is sometimes designated MK-n, where n is the number of isoprenoid
groups, n=4 being the most common. In addition, some synthetic
vitamin K analogs have been made, including K.sub.3 (menadione)
which has no side chain, K.sub.4, K.sub.5
(2-methyl-4-amino-1-naphthol hydrochloride), vitamin K.sub.6
(2-methyl-1,4-naphthalenediamine) and K.sub.7. Many vitamin K
compounds form salts and the divalent salts are most useful as
AAMs. For example, salts of a cations may take the form:
M(Ki).sub.2 where M is a divalent cation or M(Ki).sub.3 where M is
a trivalent cation. In certain embodiments, useful AAMs include
salt dimers of vitamin K and a divalent cation like Ca or Mg.
[0096] Vitamin P (although a somewhat outdated term) refers to a
group of flavonoids, having the general structure of a 15-carbon
skeleton, which consists of two phenyl rings (A and B) and
heterocyclic ring (C). This carbon structure can be abbreviated
C6-C3-C6. Based on the nature of the substituents and position on
the skeleton, flavonoids fall into one of three chemical classes:
(1) flavonoids or bioflavonoids based on the flavone core
(2-phenyl-1,4-benzopyrone); (2) isoflavonoids based on the a
3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) core structure;
and (3) neoflavonoids, based on a 4-phenylcoumarine
(4-phenyl-1,2-benzopyrone) core structure.
[0097] Amino Acids
[0098] Certain amino acids and their derivatives are also useful as
AAMs. As is well known, and amino acid has the general formula
##STR00016##
where R is any of several well understood side chains. There are 20
amino acids coded by generic codes and humans synthesize 11 of
these, making the other 9 "essential" amino acids, which must be
consumed in the diet. The 20 are: alanine, arginine, asparagine,
aspartic acid, cysteine, glutamine, glutamic acid, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
proline, serine, threonine, tryptophan, tyrosine, and valine. All
isomeric and stereochemical forms of these amino acids, and salts
of these amino acids, are encompassed in the invention. In certain
embodiments, useful amino acids include but are not limited to:
tyrosine, phenylalanine, cysteine, serine, threonine, and
tryptophan. Additionally certain amino acid derivatives are useful,
for example lycopene and N-actylcysteine (NAC)
[0099] Acidic Sugars
[0100] Acidic sugars include mono-, and di-saccharides formed from
4 to 6-member aldoses and ketoses. They are generally acidic due to
the readiness with which a proton is released from the many
hydroxyl groups. Useful monosaccharides, include but are not
limited to erythrose, erythulose, threose, ribose, ribulose,
arabinose, xylose, xylulose, glucose, dextrose (or D-glucose),
mannose, galactose, fructose, and sorbose. Useful disaccharides
include but are not limited to maltose, sucrose, lactose,
cellobiose and trehalose. All isomeric and stereochemical forms of
these sugars are encompassed in the invention.
[0101] Quinone Derivatives
[0102] Quinone derivatives include those having 1, 2 or three
rings, therefore including 1,4-benzoquinones based on
##STR00017##
1,4-naphthoquinones based on
##STR00018##
9,10-anthraquinones based on
##STR00019##
and 1,3 indandiones based on
##STR00020##
These quinone derivatives may contain substituents at any position
other than the ketones, the substituents generally being selected
from hydroxyl, methoxy, methyl, ethyl, halo, and amino. From 1-4
hydroxyl substituents are particularly useful. For example, other
examples of hydroxy-1,4-benzoquinone derivatives include
2-hydroxy-1,4-benzoquinone, 2,3-dihydroxy-1,4-benzoquinone,
2,5-dihydroxy-1,4-benzoquinone, 2,6-dihydroxy-1,4-benzoquinone,
2,3,5-trihydroxy-1,4-benzoquinone, and
2,3,5,6-Tetrahydroxy-1,4-benzoquinone. Other 1,4-benzoquinone
derivatives include: 2,6-Dimethoxy-1,4-benzoquinone,
2,3,5,6-Tetramethyl-1,4-benzoquinone,
1,4-benzoquinonetetracarboxylic acid, blatellaquinone,
2,5-Dichloro-3,6-dihydroxybenzoquinone (chloranilic acid), and
2-Isopropyl-5-methylbenzo-1,4-quinone (thymoquinone).
[0103] Examples of mono-, di-, and
tetra-hydroxy-1,4-naphthoquinones include
2-hydroxy-1,4-naphthoquinone (lawsone),
5-hydroxy-1,4-naphthoquinone (juglone),
6-hydroxy-1,4-naphthoquinone, 2,3-dihydroxy-1,4-naphthoquinone,
2,5-dihydroxy-1,4-naphthoquinone, 2,6-dihydroxy-1,4-naphthoquinone,
2,7-dihydroxy-1,4-naphthoquinone, 2,8-dihydroxy-1,4-naphthoquinone,
5,6-dihydroxy-1,4-naphthoquinone, 5,7-dihydroxy-1,4-naphthoquinone,
5,8-dihydroxy-1,4-naphthoquinone (naphthazarin),
6,7-dihydroxy-1,4-naphthoquinone, and
2,3,5,7-tetrahydroxynaphthoquinone (spinochrome B). Other
1,4-naphthoqinone derivatives include menadione (sometimes referred
to as Vitamin K3 or 2-methyl-1,4-naphthoquinone).
[0104] Examples of 9,10 anthraquinolones include the dihydroxy
derivatives 1,2-Dihydroxyanthraquinone (alizarin),
1,3-Dihydroxyanthraquinone (purpuroxanthin, xantopurpurin),
1,4-Dihydroxyanthraquinone (quinizarin), 1,5-Dihydroxyanthraquinone
(anthrarufin), 1,6-Dihydroxyanthraquinone,
1,7-Dihydroxyanthraquinone, 1,8-Dihydroxyanthraquinone (dantron,
chrysazin). 2,3-Dihydroxyanthraquinone, 2,6-Dihydroxyanthraquinone,
and 2,7-Dihydroxyanthraquinone; the trihydroxy-derivatives
1,2,3-Trihydroxyanthraquinone (anthragallol),
1,2,4-Trihydroxyanthraquinone (purpurin),
1,2,5-Trihydroxyanthraquinone (oxyanthrarufin),
1,2,6-Trihydroxyanthraquinone (flavopurpurin),
1,2,7-Trihydroxyanthraquinone (isopurpurin, anthrapurpurin),
1,2,8-Trihydroxyanthraquinone (oxychrysazin),
1,3,5-Trihydroxyanthraquinone, 1,3,6-Trihydroxyanthraquinone,
1,3,7-Trihydroxyanthraquinone, 1,3,8-Trihydroxyanthraquinone,
1,4,5-Trihydroxyanthraquinone, 1,4,6-Trihydroxyanthraquinone,
1,6,7-Trihydroxyanthraquinone, and
2,3,6-Trihydroxyanthraquinone.
[0105] Salt complexes may be formed of FAM and/or AAM compounds as
has already been described. In addition, complexes may also be
formed between FAM compounds and certain AAM compounds. Such
FAM+AAM complexes include at least those with vitamins, such as
ascorbates and ascorbylpalmitates; those with amino acids, such as
arginates, lysinates, aspartates, glutamates; and those with acidic
sugars, such as glucosides, ribosides, galactosides, mannosides,
and the like. Example 4 provides a few concrete examples of both
salts and complexes of FAM and AAM compounds.
[0106] The nature of the FAM compounds as liquid crystals may
facilitate and/or mediate their role in wound healing. The polar
nature of the liquid crystals allows them to self assemble into
polymeric-like structures; they are thus capable of (i) generating
their own hydrogels, and/or, (ii) by addition of these molecules to
traditional hydrogels, enhancing the sol-gel transition state to
create a unique set of liquid crystal hydrogels. These gels may be
modified to create any variety of the aforementioned gel types from
strong to weak chemical bonding or the creation of a biodegradable
gel. Without wishing to be bound by any particular theory, it is
believed that these FAM molecules are useful in wound healing
applications, in part because the molecules themselves can behave
like the collagen fibrils that assemble higher order structures to
help with scaffolding and cell migration during wound healing.
Further, addition of these liquid crystal hydrogels may facilitate
proper collagen alignment and orientation reducing the risk of scar
or keloid formation during the wound healing process.
[0107] In addition to hydrogel formulations, another useful
formulation of FAM compounds is with cyclodextrins. A general
cylodextrin formula consists of an FAM with a ratio of FAM:
Cyclodextrin of about 1:1, 1.5:1, 1.5:2, 1.5:3, 1:3, 1.5:4, 1:4,
1.5:5, 1:5, 1.5:6, 1:6, 1.5:7, 1:7, 1.5:8, 1:8, 1.5:9, 1:9, 1.5:10
or 1:10. These ratios will allow for adequate dissolving of the FAM
in cyclodextrin. An AAM may be added from about 0.1% to about 99%
(w/v), e.g. 0.1-10%, 10-20%, 20-30%, 30-40%, 50-60%, 60-70%, and
80-90% (w/v) or higher.
Utility of FAMs and AAMs in Autophagy Modulation
[0108] In recent years, scientists have been studying the effects
of the regulation of the autophagy pathways as a way to treat a
variety of serious illnesses. In fact, dysregulation of autophagy
has been linked to major diseases including heart disease, cancer
and diabetes (See FIG. 2, taken from Klionsky D. J. (2010). The
Autophagy Connection. Developmental Cell. July 20; 19(1):11-2.)
This paper describes the link between the autophagy pathways and
major human diseases. While not naming individual "myopathies," the
ability to regulate this pathway is a major link to modulating
disease outcomes. Depending on the disease state it may be
beneficial to upregulate or downregulate cellular levels of
autophagy.
[0109] In some disease states, the pathological origins are related
to suppressed autophagic activity or the activation of autophagy
and its related signaling pathways will result in the suppression
of inflammation. Hence, diseases or conditions where it may be
beneficial to upregulate autophagy include: wound healing, promote
hair regrowth, bacterial infections, inflammation, viral infection,
Parkinson's disease, Alzheimers, neurodegenerative diseases,
neuropathy, cardiovascular disease, heat failure, heart disease,
aging, Alzheimer's disease, atherosclerosis, arterosclerosis,
chronic obstructive pulmonary disease (COPD), Crohn's disease,
inflammatory bowel, colitis, diabetes, diabetes type I and II,
amyloidosis, bursitis, dermatitis, angitis, autoimmune diseases
with inflammation, blood diseases, aplastic anemia, endometriosis,
hepatitis, herpes, HIV, multiple sclerosis, retinal detachment,
age-related macular degeneration, retinitis pigmentosa, and Leber's
congenital amaurosis, lysosomal storage diseases, arthritis,
psoriasis, osteopenia, osteoporosis, surgical scars, surgical
adhesions, space travel (bone density disorder), tendonitis, and
ulcerative colitis.
[0110] In other disease states the pathological origins are related
to overexpression of autophagic activity and the activation of
autophagy and its related signaling pathways. Hence, diseases or
conditions where it is beneficial to downregulate autophagy
include: Aging, Cancer, polycystic kidney and liver disease, kidney
disease, liver disease, asthma, diabetic retinopathy, fibromyalgia,
ankylosing spondylitis, celiac disease, Grave's disease, lupus,
metabolic diseases, nephritis, rheumatoid arthritis, osteolysis,
ischemia-reperfusion (I/R) injury, organ and tissue transplant,
scleraderma, and sepsis.
[0111] In wound healing, increasing autophagy levels assists in
tissue protection, decreases inflammation and promotes the
synthesis of procollagen, hyaluronan and elastin. As shown herein
FAM and AAM compounds have been used alone and in combination to
promote wound healing, hair growth, and skin repair following
damage from UV radiation exposure.
[0112] In bacterial infections various types of bacteria attempt to
interfere with the autophagy pathway to prevent the cellular uptake
of the bacteria ultimately leading to autophagolysosme degradation
of the bacteria. Two clinically important skin pathogens,
Streptococcus sp. and Staphylococcus aureus interfere with the
autophagy pathway (see, I. Nakagawa, et al, Autophagy defends cells
against invading group A Streptococcus, Science 306, 1037-1040
(2004); and Schnaith, et al, Staphylococcus aureus subvert
autophagy for induction of caspase-independent host cell death, J
Biol Chem 282, 2695-2706 (2007). Invasive skin infections with
group A Streptococcus are characterized by the prevention of
cellular uptake of bacteria due to encapsulation; and if bacteria
are taken up by keratinocytes, the majority of streptococci are
killed within a few hours (H. M. Schrager, J. G. Rheinwald and M.
R. Wessels: Hyaluronic acid capsule and the role of streptococcal
entry into keratinocytes in invasive skin infection, J Clin Invest
98, 1954-1958 (1996)). Nakagawa et al. (cited above) showed that
autophagy is responsible for the killing activity. Although, some
bacteria survive, the reduction of the number of extracellular
streptococci is likely to have a partially protective effect. As
the mechanistic studies of the action of autophagy against group A
Streptococcus have not been performed in keratinocytes, additional
studies will be necessary to understand the relevance and
efficiency of this putative antibacterial strategy in the skin. S.
aureus induces autophagy via its alpha-toxin (Schnaith et al,
above, and M. B. Mestre, C. M. Fader, C. Sola and M. I. Colombo:
Alpha-hemolysin is required for the activation of the autophagic
pathway in Staphylococcus aureus-infected cells, Autophagy 6,
110-125 (2010)). Pore-forming toxins cause a drop in nutrient and
energy levels that trigger autophagy as a rescue mechanism to
re-establish cellular homoeostasis (N. Kloft, et al: Pro-autophagic
signal induction by bacterial pore forming toxins, Med Microbiol
Immunol 199, 299-309 (2010)). Whether autophagy suppresses or
enhances S. aureus infection in the skin in vivo remains to be
determined.
EXAMPLES
Example 1: Synthesis of Certain FAMs of the Invention: The
Following Compounds Were Prepared and Given Identifying Numbers as
Shown in Table B
TABLE-US-00002 [0113] TABLE B Stilbene and Tolan compounds useful
in the invention Identifier Compound Name Structure BM2201
4,4'-dihydroxy-trans-stilbene ##STR00021## BM2301
3,5,4'-trihydroxy-trans-stilbene ##STR00022## BM2401
3,3',5,5'-tetrahydroxy-trans- stilbene ##STR00023## BM2213
4-hydroxy-4'-(trifluoro)methyl- trans-stilbene ##STR00024## BM3103
4-hydoxy-4'-methoxytolan ##STR00025## BM3302 2,4,4'-trihydroxytolan
##STR00026## BM3032 2,4,4'-trimethoxytolan ##STR00027## BM3203
4,4'-dihydroxy-3-methoxytolan ##STR00028## BM3402
2,4,2',4'-tetrahydroxy-tolan ##STR00029## BM3206
4,4'-dihydroxytolan-2-O-.beta.-D- glucoside ##STR00030## BM3301
3,5,4'-trihydroxytolan ##STR00031## BM3401
3,3',5,5'-tetrahydroxytolan ##STR00032## BM3213 4-hydroxy-4'-
(trifluoro)methyltolan ##STR00033##
[0114] Stilbene compounds (BM2xxx series) were synthesized/obtained
according to the procedures described in Ali, M et al 1992, and
Thakkar, K. et al 1993, noted above. Tolan compounds (BM3xxx
series) were synthesized according to the procedures described in
U.S. Pat. No. 6,599,945 B2 of Docherty & Tsai.
Example 2: Simplified Synthesis Procedures for
4-hydroxy-4'methoxytolan
##STR00034##
[0116] Synthesis Procedure for
4-Hydroxy-4'-methoxytolan(4-((4'-methoxyphenyl)ethynyl)phenol) was
a Heck-type reaction modified from (Pavia, M. R.; Cohen, M. P.;
Dilley, G. J.; Dubuc, G. R.; Durgin, T. L.; Forman, F. W.; Hediger,
M. E.; Milot, G.; Powers, T. S.; Sucholeiki, I.; Zhou, S.;
Hangauer, D. G. The design and synthesis of substituted biphenyl
libraries. Bioorg. Med. Chem. 1996, 4, 659-666. Jeffery, T.
Heck-type reactions in water. Tetrahedron Lett, 1994, 35,
3051-3054, Jeffery, T.; Galland, J. C. Tetraalkylammonium salts in
heck-type reactions using an alkali metal hydrogen carbonate or an
alkali metal acetate as the base. Tetrahedron Lett, 1994, 35,
4103-4106, and Schmidt-Radde, R. H.; Vollhardt, K.; Peter C. The
total synthesis of angular [4]- and [5] phenylene J Am Chem Soc,
1992, 114, 9713-9715). The resulting product was a yellowish powder
and was verified using .sup.1HNMR (CDCl.sub.3, 300 MHz): .delta.
ppm: 7.44 (d, 4H, J=8.7, Ar-H), 6.89 (d, 2H, J=8.7, Ar-H), 6.82 (d,
2H, J=8.7, Ar-H), 4.89 (s, 1H, OH), 3.85 (s, 3H, CH.sub.3O). The
resulting product was 98.2% pure and was used for all subsequent
testing.
Example 3: Synthesis Procedure Outline for 2,4,4'-trimethoxytolan:
Synthesis was a Heck-Type Reaction Analogous to that of Example
2
##STR00035##
[0117] The resulting product was an off white powder and was
verified using .sup.1HNMR (CDCl.sub.3, 400 MHz): .delta. ppm: 7.47
(d, 2H, J=6.4, Ar-H), 7.40 (s, 1H, Ar-H), 6.85 (d, 2H, J=6.8,
Ar-H), 6.47 (dd, 2H, J=2.4, Ar-H), 3.89 (s, 3H, CH.sub.3O), 3.82
(s, 6H, 2CH.sub.30) and found to be 99.3% pure.
Example 4: Melting Points of FAM Salts and FAM and AAM Salt
Complexes
[0118] All salts were made by combining sufficient quantities of
each stilbene, tolan or combination with magnesium hydroxide, zinc
oxide, ascorbic acid or ascorbyl palmitate to generate a salt
solution. Each solution was then dried in 20 mL scintillation vials
using a rotary evaporator (Centrifan, Harvard Biosciences) set to
40.degree. C. and evaporated with a mixture of ethanol and ice to
allow for a slower evaporation. Once evaporated and dried
completely the salts were ground into a fine powder and their
melting points were used to confirm the formation of the salt.
Melting points (Table C, below) were determined using a Meltemp II
apparatus outfitted with a temperature probe and thermal couple to
provide a digital read out. Apparatus was calibrated and compounds
with known melting points were tested to confirm calibration prior
to analysis of unknowns.
TABLE-US-00003 TABLE C Melting points Melting Point FAM's
4-hydroxy-4'-methoxytolan 140-144.degree. C. 3,5,4' trihydroxytolan
208-214.degree. C. 3,5,3',5'tetrahydroxystilbene 319-322.degree. C.
2,4,4' trimethoxytolan 68-73.degree. C. FAM complexes
Mg-4-hydroxy-4'-methoxytolan 150-152.degree. C. Mg-3,5,4'
trihydroxytolan 222-232.degree. C. Mg-3,5,3',5'
tetrahydroxystilbene 324-337.degree. C. Mg-2,4,4' trimethoxytolan
71-76.degree. C. Zn-3,5,4' trihydroxytolan 161-164.degree. C.
Zn-3,5,3',5'tetrahydroxystilbene 217-221.degree. C. Zn-2,4,4'
trimethoxytolan 69-73.degree. C. Zn-4-hydroxy-4'-methoxytolan
168-173.degree. C. FAM + AAM complexes Mg-Ascorbate-4-hydroxy-4'-
173-183.degree. C. methoxytolan Zn-Ascorbate-4-hydroxy-4'-
168-175.degree. C. methoxytolan AAM's Ascorbic Acid 190-192.degree.
C. Mg(OH).sub.2 350.degree. C. ZnO 1975.degree. C. Ascorbyl
Palmitate 115-116.degree. C.
Example 5: LC3-II (Microtubule Associated Protein 1 Light Chain 3)
Staining in Human Dermal Fibroblasts
[0119] Staining method was modified from procedures described by:
Furuta, S, (2000) Ras is involved in the negative control of
autophagy through the class I PI3-kinase, Oncogene. 23: 3898-3904;
Ge, J. N. et al, (2008) Effect of starvation-induced autophagy on
cell cycle of tumor cells, Chinese Journal of Cancer 27:8 102-108;
and Settembre, C. et al (2011) TFEB Links autophagy to lysosomal
biogenesis, Science 332:17 1429-1433. HDFn (Human dermal
fibroblasts neonatal, ThermoFisher) cells were seeded at 5,000
cells/well into a black well clear bottom plate (Coning, Corning,
N.Y.). Cells were then treated with media alone, FAM, AAM or
combination thereof for 8 hrs. Following treatment time(s) the
cells were fixed with 4% (w/v) PFA (paraformaldehyde) at room
temperature for 15 min. Cells were then washed with PBS (phosphate
buffered saline, pH 7.4) and blocked with 5% (w/v) BSA in PBS for 1
hr. Cell were washed again with PBS and incubated in primary
antibody for LC3-IIB (rabbit monoclonal antibody, Thermo-Fisher)
for 3 hrs. Cells were rinsed again in PBS and the secondary
fluorescent antibody (Alexafluor 488 rabbit anti goat,
ThermoFisher) was added for 30 minutes. The plates were then imaged
using the SpecraMax i3X (Molecular Devices, Sunnyvale, Calif.) and
SoftMax Pro 6.5.1 software was used to determine the number of
LC3-II positive cells. Results are shown in FIG. 3
Example 6: Autophagy Modulation and Hormesis
[0120] LC3-II Western blot: Human dermal fibroblasts were plated at
a density of 1.times.10.sup.6 cells per T25 tissue culture treated
flasks. Cells were then treated with an FAM, AAM or combination for
8 hrs. Cells were then trypsinized, washed with 1.times.PBS and
lysed with RIPA buffer+protease inhibitor on ice. Cells were
sonicated and a BCA protein assay (Pierce Scientific) was run to
determine protein concentrations. Concentrations were then
normalized to 100 ug per sample and run on a 12% (w/v)
polyacrylamide gel with loading dye and appropriate molecular
weight markers (All reagents were purchased from National
Diagnostics). The gel was then transferred, blocked and a primary
monoclonal antibody to MAPLC3-2 (Thermo-Fisher) was added to the
membrane and allowed to incubate overnight. Following appropriate
washing a Eu-labeled secondary antibody (Molecular Devices) was
added and the membrane was imaged using the Spectramax i3x equipped
with the Scan Later module. The gel was subsequently stripped and
re-probed for actin to confirm appropriate loading. SoftMax Pro
6.5.1 software was used to determine changes in average band
intensity. Results are shown in FIGS. 4 & 5.
Example 7: Autophagy, Wound Healing and the Skin
[0121] Very low levels of autophagy are present in the skin,
functioning to degrade protein aggregates, damaged organelles and
to effect skin color through an FGF-PI3K-AKT-MTOR signaling pathway
in the melanosomes (Belleudi, et al. The receptor tyrosine kinase
FGFR2b/KGFR controls early differentiation of human keratinocytes,
PLoS One 2011; 6:e24194; PMID:21957444;
http://dx.doi.org/10.1371/journal.pone.0024194; and Belleudi, et
al, Expression and signaling of the tyrosine kinase FGFR2b/KGFR
regulates phagocytosis and melanosome uptake in human
keratinocytes. FASEB J 2011; 25:170-81; PMID:20844240;
http://dx.doi.org/10.1096/fj.10-162156.) The induction of autophagy
in human kertinocytes negatively regulates p62, preventing
excessive inflammation and induction of cathelicidin (found in the
lysosomes of macrophage and PMN's). (Lee, et al Autophagy
Negatively Regulates Keratinocyte Inflammatory Responses via
Scaffolding Protein p62/SQSTM1. J Immunol. published online 15 Dec.
2010). In a deep wound second degree burn model the autophagy
inducer rapamycin was shown to enhance autophagic vesicle
formation, improve wound reepithelization times and decreased IL-8,
methane dicarboxylic aldehyde (MDA an indicator of oxidative
stress) and myeloperoxidase (an indicator of the production of
hypochlorous acid (HOCl), hydrogen peroxide (H.sub.2O.sub.2) and
chloride anions (Cl--)) levels. (Xiao et al, (2013) Rapamycin
reduces burn wound progression by enhancing autophagy in deep
second-degree burn in rats. Wound Rep. Reg. 21: 852-859). This
indicates that the induction of autophagy in the skin creates an
anti-inflammatory effect. Rapamycin is a known hormetic chemical
that, when used in a dose dependent fashion, can prevent or treat a
variety of diseases.
[0122] The formulations described in this patent are hormetic
substances that have been shown to induce autophagy in a dose
dependent fashion for the treatment of a variety of diseases and
medical conditions, including increased wound closure and
re-epitheliazation, as shown below.
Example 8: Protein Kinase B (AKT)
[0123] HDFn cells were plated in T-25 flasks at a density of
1.times.10.sup.6 cells per flasks and allowed to attach and spread
overnight. Cells were then treated for 8 hrs with Control media,
FAM, AAM or combination. Cells were removed using trypsin, followed
by trypsin neutralizer and spun down to collect cell pellet. Cells
were washed in ice cold 1.times.PBS and then lysed with RIM
buffer+protease inhibitor on ice. Samples were spun down, aliquoted
and frozen until they could be assayed using an ELISA. Cells were
sonicated and diluted 1:5 as recommended in kit instructions.
Samples were then assayed using an AKT ELISA (Thermo-Fisher) and
concentrations were determined using an AKT standard curve. Results
are shown in FIG. 6.
Example 9: Fibroblast Growth Factor (FGF)
[0124] HDFn cells were plated in a 6 well tissue culture treated
plate and allowed to reach 70-80% (cells/area) confluency. A cell
scraper was then used to create a "scratch" down the middle of the
plate to simulate injury to the cell monolayer. Dishes were then
washed with media to remove any cellular debris. Cells were then
treated with Control media, FAM, AAM or combination and samples
were then take at 3, 8, and 24 hrs. Samples were spun down,
aliquoted and frozen until they could be assayed using an ELISA.
FGF Streptavidin-HRP ELISA kit was used to determine concentration
of EGF in each sample as compared to a known standard curve.
Results are shown in FIG. 7.
Example 10: In Vivo Evaluation of the LCB's for Wound Healing
[0125] Twenty, 5-6
[0126] week old Balbc/J (stock #000651) male mice were transferred
to Jackson Labs in vivo research laboratory in Sacramento, Calif.
The mice were ear notched for identification and housed in
individually and positively ventilated polycarbonate cages with
HEPA filtered air. Bed-o-cob corn cob bedding was used and cages
were changed every two weeks. The animal room is lighted entirely
with artificial fluorescent lighting, with a controlled 12 h
light/dark cycle. The normal temperature and relative humidity
ranges in the animal rooms are 22.+-.4.degree. C. and 50.+-.15%,
respectively. The animal rooms were set to have 15 air exchanges
per hour. Filtered tap water, acidified to a pH of 2.8 to 3.1, and
rodent chow was provided ad libitum. Following a 5-7 days
acclimation, mice were randomized by body weight into 2 cohorts of
10 mice each. On study day 0, mice were anesthetized and two full
thickness excision wounds (.about.6 mm) were made on the dorsum
(backs) of mice. One of the wounds was covered using a
semi-occlusive polyurethane dressing (Tegaderm.TM.). Dressings
covered the wounds for 5 consecutive (5) days from the day of
wounding (d 0). The doses of 4-hydroxy-4'-methoxytolan were based
on those that promoted lesion healing in mice in previous herpes
virus studies. Wound measurements were made on day 5, 7, 9 and 11.
Digital images of wounds were taken of each mouse. Test agents were
a viscous paste and wounds were surrounded with a saddle glued in
place to eliminate cross contamination and wound closure due to
contraction. Therefore, changes in wound area are due to
re-epithelization. During the five days all wounds are covered with
glycerin (control) or glycerin and test agent. At day 5, test
agents were removed and changes in wound area were measured (as
area/area %) in an effort to gauge the persistence of the compounds
in the wound site. By day 5, glycerin produced 26.1% wound closure,
whereas 4-hydroxy-4'-methoxytolan (2.5%) closed produced 92.8%
wound closure and 5% 4-hydroxy-4'-methoxytolan produced 91.4% wound
closure. Prior to statistical analysis a test for Normality was run
and a Z-score computed to confirm a normally distributed area for
each wound on day 1 for all mice across groups, r.sup.2=0.989.
Statistics are computed based One-Way ANOVA's comparing changes in
wound area on day 5. All mice tolerated the treatments and all
weekly clinical observations report bright, alert, responsive, and
hydrated mice.
TABLE-US-00004 TABLE D Wound size reduction Day Average Area
(cm.sup.2) Wound Area (%)Closed Control Animals 0 2.11 0 5 1.56
26.1 7 0.63 70.2 9 0.55 73.9 11 0.49 76.8 4-hydroxy-4'- 0 1.98 0
methoxytolan 5 0.17 91.4 5% (w/v) 7 0.08 100 9 0.00 100.0 11 0.00
100.0 Day Average Area (cm.sup.2) Wound Area (%) 4-hydroxy-4'- 0
2.07 0 methoxytolan 5 0.15 92.8 2.5% (w/v) 7 0.04 100 9 0.00 100.0
11 0.00 100.0
Example 11: Excised Skin Samples Treated with a Control or with 5%
(w/v) 4-hydroxy-4'-methoxytolan
[0127] H&E staining of control treated cells revealed a
keratinocyte layer of 1-2 cells thick on top of a loosely organized
layer of myofibroblasts, connective tissue with a few deep
sebaceous glands beginning to form. 4-hydroxy-4'-methoxytolan
treated skin revealed-extensive keratinocyte proliferation with
cell layers 7-8 cells thick. The dermis showed regular and
proliferative myofibroblasts and connective tissue surrounding
highly proliferative and prevalent sebaceous glands extending to
the surface to reestablish hair follicles.
Example 12: Autophagy and Hair Re-Growth in Skin Appendages (Hair
Loss)
[0128] Autophagosome-like structures have been detected by electron
microscopy of hair and sebaceous glands. While the physiological
relevance of autophagy in skin appendages is not well understood at
present, current existing data suggests induction of autophagy may
prevent hairloss through a Wnt1 dependent cell rejuvenating process
where damaged cells undergo cell death and hair stem cell are
stimulated to generate hair growth (Castilho R. M., et al. (2009)
mTOR mediates Wnt-induced epidermal stem cell exhaustion and aging,
Cell Stem Cell 5, 279-289; and Vishnyakova, et al. (2013) Possible
Role of Autophagy Activation in Stimulation of Regeneration,
Molecular Biology. 47(5): 692-700).
Example 13: UV Radiation Damage and Anti-Aging
[0129] The skin is the largest organ in the human body and is in
contact with the environment. As such it is constantly subjected to
damage, both from outside and from the inside, which threatens its
balance and alters its appearance. This damage is often manifested
as chronic low levels of inflammation. It is known for example that
excessive exposure to UV is reflected by various cutaneous
manifestations, such as actinic erythemas, solar elastosis, or else
the premature appearance of the effects of cutaneous aging: the
skin becomes loose, deeply wrinkled, rough, dry, sprinkled with
hypopigmented or hyperpigmented spots and dilated vessels. These
manifestations of UV exposure, which reflect profound structural
changes in the cutaneous tissue, are unsightly and ugly, and many
people have a tendency to want to smooth them out. TEM and IF
microscopy analysis of cultured dermal fibroblasts from women of
different ages revealed an impaired autophagic flux. When young
dermal fibroblasts were treated with lysosomal protease inhibitors
to mimic the condition of aged dermal fibroblasts the reduced
autophagic activity, altered the fibroblast content of type I
procollagen, hyaluronan and elastin, and caused a breakdown of
collagen fibrils. Together these findings suggest that impaired
autophagic induction leads to deterioration of dermal integrity and
skin fragility (Tashiro K, et al. (2014) Age-related disruption of
autophagy in dermal fibroblasts modulates extracellular matrix
components, Biochem Biophys Res Commun. 443(1): 167-172).
[0130] Autophagy ensures that damaged cellular organelles and
protein aggregates are degraded properly and do not accumulate
causing cellular dysfunction. Enhanced autophagic activity is also
seen in response to caloric restriction (CR) which has shown to
prolong life expectancy.
Example 14: A 40% (w/v) solution of
Hydroxypropyl-.beta.-cyclodextrin
[0131] Solution is prepared by adding into a sterile graduated
beaker 40.0 g of Hydroxypropyl-.beta.-cyclodextrin to 70 mL of
water and mixed thoroughly. Once the solution is clear QS to 100
mL. Weigh out 1.0 g of the liquid crystal compound (FAM) and
transfer into a sterile glass bottle. Add 1.5 mL of ethanol to the
bottle and dissolve completely. Slowly add 1 mL of cyclodextrin
while stirring to ensure drug remains in solution. Add 5 mL of
water while stirring to ensure drug stays in solution. Sonicate if
necessary. Formulation should be a clear solution. Filter using a
0.2 um filter. The suspension is frozen below -40.degree. C. and is
lyophilized. The lyophilized cake maybe reconstituted with sterile
water prior to use.
Example 15: Preparation of an Injectable Liquid Crystal Formulation
FAM Cyclodetrin Formulation
[0132] 100 mg of a 4,4'-dihydroxytolan compound are weighed and
placed in a 5 ml scintillation tube. 1.5 ml of absolute ethanol is
added to the tube and shaken until the 4,4'-dihydroxytolan is
completely dissolved. 5 grams of pyrogen free
hydroxypropyl-.beta.-cyclodextrin (Sigma) are weighed on an
analytical scale and placed in a graduated cylinder. Water is added
with shaking until the volume reaches 90 ml. The above ethanolic
solution of FAM is added to the aqueous solution containing
hydroxypropyl-.beta.-cyclodextrin with stirring. Water is added to
the clear solution to make the total volume 100 ml. The solution is
sterile filtered through a 0.22 micron filter. The suspension is
frozen below -40.degree. C. and is lyophilized. The lyophilized
cake is reconstituted with sterile water for injection prior to
use.
Example 16: Preparation of Drop Solution
[0133] The solution is compounded from the ingredients;
FAM-cyclodextrin 0.625 parts; saccharin sodium 0.3 parts; sorbic
acid 0.1 parts; ethanol 30.0 parts; flavoring 1.0 parts; distilled
water q.s. ad 100.0 parts. The FAM-cyclodextrin complex and the
flavoring are dissolved in the ethanol, and the sorbic acid and the
saccharin are dissolved in the distilled water. The two solutions
are uniformly admixed with each other, and the mixed solution is
filtered until free from suspended matter: 1 ml of the filtrate
contains the FAM and is an oral dosage unit composition with
effective therapeutic action.
Example 17: Preparation of Micronized Drug and Drug Suspensions
[0134] 16 grams of micronized FAM is milled with a 4 inch mill size
and compressed nitrogen gas/compressed air (dew point
>40.degree. C.) as milling gas. The material is manually fed
into the hopper and placed on top of the feed tray. The material is
drawn into a confined, circular chamber by way of a pressurized
milling gas. The powder becomes suspended in a high velocity stream
in the milling chamber. Particle size distribution is measured on a
particle size analyzer. The milling conditions are then adjusted to
give material with an acceptable micron size.
Example 18: An FAM or AAM Modulator-Cyclodextrin Complex Gel
[0135] 100 mg of an FAM is weighed and placed in a sterile test
tube. The FAM is dissolved in 2-3 ml of purified absolute ethanol.
50 ml of a 10-50% (w/v) solution of
hydroxypropyl-.beta.-cyclodextrin (other cyclodextrans may also be
used based on the need for water absorption such as
.alpha.-cyclodextrins, .gamma.-cyclodextrins and certain modified
.beta.-cyclodextrins) is prepared in a 150 ml sterile beaker and
the solution is heated to 70-80.degree. C. while stirring on a hot
plate. The ethanolic solution of FAM is slowly added to the beaker
with stirring. At this stage the AAM may be added from 1-25% (w/v).
The addition of the FAM will start the gel-sol transition and if
desired a gelling molecule such as sodium pectate dissolved in
deionized water can be added to further enhance gelation. Other gel
enhancers include the monovalent or divalent cation FAM or AAM
salts which will form ionic cross linkages to enhance gel
formation.
[0136] The use of cations can be selected based on the desire to
increase or decrease solubility in water. In order to enhance the
gelation process an FAM or its magnesium dimer is added to alginate
copolymers during mixing and the M/G ratio is adjusted to create
stabile FAM:Mg.sup.2+:Alginate biodegradable sheets. The FAM cation
interacts through hydrogen bonding with the pocket created by the G
form alginate copolymers (see FIG. 15). All ratios can be adjusted
to optimize FAM concentration and polymerization. When necessary in
addition to Mg.sup.2+ other ions such as Ca.sup.2+, K.sup.+ or
Zn.sup.2+ may be added further enhance the gelling process.
[0137] Alginates G, M or G/M copolymers through the addition of
divalent cations such as calcium form calcium alginate sheets.
These sheets can be created in a sterile environment and are
non-irritating, non-sensitizing and biodegradable. This makes
liquid crystal FAM molecules ideal molecules to promote alginate
gelation, polymerization, control copolymer block structure and
alter acetylation to influence the physicochemical and rheological
characteristics of the polymer. In addition to adjusting the
molecular mass of the individual alginate monomers the selected
FAM's may also alter gel viscosity.
[0138] A variety of polymeric sugar molecules that when combined
with liquid crystal FAM and AAM molecules described in this
application can create unique hydrogels, alginates and drug
delivery systems that can be used in creating novel wound care
products. Examples include but are not limited to chitosan,
hyaluronic acid, pectin, heparin, alginate, chondroitin sulfate A,
D &E, PEG (polyethylene glycol), PLA (polylactic acid) and
polymers thereof and polyphosphazene.
[0139] In certain instances an additional FAM or AAM may be added
to any of the above formulations to improve solubility, adjust pH,
balance cation or anion concentration, improve adherence to the
skin, increase or decrease solubility in water, create a
concentration gradient, improve gel-sol transition, increase or
decrease electrical conductivity, increase or decrease capacitance,
adjust overall resistance or impedance.
[0140] The formulations described in this patent are liquid crystal
hormetic substances that have been shown to induce autophagy in a
dose dependent fashion for the treatment of a variety of diseases
and medical conditions, including increased wound closure and
re-epitheliazation.
Example 19: An FAM or AAM Modulator-Alginate Complex
[0141] One gram of sodium alginate is dissolved in deionized water
in a 500 mL beaker and to that is added if desired a selected AAM
(1-25% w/v) is added while mixing. To this solution is added a
therapeutically effective amount of an FAM that was previously
dissolved in ethanol to a desired concentration 1-50% (w/v) and if
necessary the addition of a monovalent or divalent cationic salt is
added until the gel has reached desired consistency. For salts of
FAM's the previous step is not required. The liquid crystal nature
of the FAM molecules creates unique co-block polymers that
associate through hydrogen, electrostatic and ionic bonding. This
solution can then be used to coat woven cotton or other fibers to
create alginate bandages. This same solution can also be used to
create biodegradable sheets, films, beads or gels.
[0142] The foregoing description of the various aspects and
embodiments of the present invention has been presented for
purposes of illustration and description. It is not intended to be
exhaustive of all embodiments or to limit the invention to the
specific aspects disclosed. Obvious modifications or variations are
possible in light of the above teachings and such modifications and
variations may well fall within the scope of the invention as
determined by the appended claims when interpreted in accordance
with the breadth to which they are fairly, legally and equitably
entitled.
* * * * *
References