U.S. patent application number 12/517149 was filed with the patent office on 2010-07-08 for peptide nanoparticles and uses therefor.
This patent application is currently assigned to ANTERIOS, INC.. Invention is credited to Jonathan Edelson, Timothy Kotyla.
Application Number | 20100172943 12/517149 |
Document ID | / |
Family ID | 40002812 |
Filed Date | 2010-07-08 |
United States Patent
Application |
20100172943 |
Kind Code |
A1 |
Edelson; Jonathan ; et
al. |
July 8, 2010 |
PEPTIDE NANOPARTICLES AND USES THEREFOR
Abstract
The present invention provides nanoparticle compositions
including one or more peptides. The present invention achieves
transdermal delivery of such peptides without the need for peptide
modification, or for use of chemical or mechanical abrasion or
disruption of skin.
Inventors: |
Edelson; Jonathan;
(Scarsdale, NY) ; Kotyla; Timothy; (Lowell,
MA) |
Correspondence
Address: |
CHOATE, HALL & STEWART LLP
TWO INTERNATIONAL PLACE
BOSTON
MA
02110
US
|
Assignee: |
ANTERIOS, INC.
New York
NY
|
Family ID: |
40002812 |
Appl. No.: |
12/517149 |
Filed: |
November 30, 2007 |
PCT Filed: |
November 30, 2007 |
PCT NO: |
PCT/US07/86040 |
371 Date: |
March 1, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60872206 |
Dec 1, 2006 |
|
|
|
Current U.S.
Class: |
424/401 ;
424/489; 424/59; 435/29; 514/1.1; 977/773 |
Current CPC
Class: |
A61P 17/18 20180101;
A61K 38/2207 20130101; A61K 8/0241 20130101; A61Q 19/00 20130101;
A61K 8/11 20130101; A61Q 1/06 20130101; A61K 8/044 20130101; A61P
43/00 20180101; A61Q 19/08 20130101; A61Q 17/04 20130101; A61K
8/064 20130101; A61P 25/00 20180101; A61K 9/14 20130101; A61P 31/12
20180101; A61K 8/64 20130101; A61P 1/14 20180101; A61K 2800/413
20130101; A61K 8/068 20130101; A61K 8/92 20130101; A61K 38/08
20130101; B82Y 5/00 20130101; A61P 17/02 20180101; A61P 17/00
20180101 |
Class at
Publication: |
424/401 ; 514/19;
514/18; 514/17; 514/16; 514/15; 514/14; 514/13; 514/12; 424/489;
424/59; 435/29; 977/773 |
International
Class: |
A61K 8/64 20060101
A61K008/64; A61K 9/14 20060101 A61K009/14; A61K 38/05 20060101
A61K038/05; A61K 38/06 20060101 A61K038/06; A61K 38/07 20060101
A61K038/07; A61K 38/08 20060101 A61K038/08; A61K 38/10 20060101
A61K038/10; A61K 38/16 20060101 A61K038/16; A61P 17/02 20060101
A61P017/02; A61Q 1/06 20060101 A61Q001/06; A61Q 17/04 20060101
A61Q017/04; A61Q 19/00 20060101 A61Q019/00; A61Q 19/08 20060101
A61Q019/08; A61K 8/04 20060101 A61K008/04; C12Q 1/02 20060101
C12Q001/02 |
Claims
1. A suspension or dispersion of nanoparticles comprising a
population of particles, wherein the majority of particles have
diameters between approximately 10 and approximately 300
nanometers, and wherein said nanoparticles comprises at least one
unmodified peptide of length between 2 and 30 amino acids that has
biological activity in the skin, subcutaneous tissue or contiguous
muscles.
2. The suspension or dispersion of claim 1, wherein the majority of
particles have a range of diameters between approximately 10 and
approximately 250 nanometers.
3. The suspension or dispersion of claim 1, wherein the majority of
particles have a range of diameters between approximately 10 and
approximately 200 nanometers.
4. The suspension or dispersion of claim 1, wherein the majority of
particles have a range of diameters between approximately 10 and
approximately 150 nanometers.
5. The suspension or dispersion of claim 1, wherein the majority of
particles have a range of diameters between approximately 10 and
approximately 120 nanometers.
6. The suspension or dispersion of claim 1, wherein the majority of
particles have a range of diameters between approximately 10 and
approximately 100 nanometers.
7. The suspension or dispersion of claim 1, wherein the majority of
particles have a range of diameters between approximately 10 and
approximately 50 nanometers.
8. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises KTTKS.
9. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises EYKTTKSSRL.
10. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises VIEYKTTK.
11. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises KTTK.
12. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises GKTVIEYKTTKS.
13. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises GKTVIEYKTTKSSRL.
14. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises WGKTVIEYKTTKSSRLPIID.
15. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises CTSHTGAWGKTVIEYKTTKS.
16. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises TTKS.
17. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises EEMQRR.
18. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises gastrin-releasing peptide.
19. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises VGVAPG.
20. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises YYRADA.
21. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises GHK.
22. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises TTKS.
23. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises interferon.
24. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises interferon inducer.
25. The suspension or dispersion of claim 1, wherein said
nanoparticles comprise an unmodified peptide whose amino acid
sequence comprises P144.
26. The suspension or dispersion of claim 1, wherein the
nanoparticle is a nanoemulsion.
27. The suspension or dispersion of claim 26, wherein the
nanoemulsion comprises at least one oil, at least one surfactant,
water, and the unmodified peptide.
28. The suspension or dispersion of claim 26, wherein the
nanoemulsion comprises a single oil, a single surfactant, water,
and the unmodified peptide.
29. (canceled)
30. The suspension or dispersion of claim 1, further comprising an
oil, and wherein the oil is selected from the group consisting of
almond, apricot kernel, avocado, babassu, bergamot, black current
seed, borage, cade, camomile, canola, caraway, carnauba, castor,
cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton
seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol,
gourd, grape seed, hazel nut, hyssop, jojoba, kukui nut, lavandin,
lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed,
meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm,
palm kernel, peach kernel, peanut, poppy seed, pumpkin seed,
rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana,
savoury, sea buckthorn, sesame, shea butter, silicone, soybean,
sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat
germ oils; butyl stearate; caprylic triglyceride; capric
triglyceride; cyclomethicone; diethyl sebacate; dimethicone 360;
isopropyl myristate; mineral oil; octyldodecanol; oleyl alcohol;
silicone oil; short chain triglycerides; medium chain
triglycerides; Labrafac WL 1349; long chain triglycerides;
saturated oils thereof; and unsaturated oils thereof.
31. The suspension or dispersion of claim 30, wherein the oil is
soy oil.
32. The suspension or dispersion of claim 30, wherein the oil is a
medium chain triglyceride.
33. The suspension or dispersion of claim 30, wherein the oil is
Labrafac WL 1349.
34. The suspension or dispersion of claim 1, further comprising a
surfactant, wherein the surfactant is selected from the group
consisting of phosphoglycerides; phosphatidylcholines; dipalmitoyl
phosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine
(DOPE); dioleyloxypropyltriethylammonium (DOTMA);
dioleoylphosphatidylcholine; cholesterol; cholesterol ester;
diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol
(DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol
(PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid,
such as palmitic acid or oleic acid; fatty acids; fatty acid
monoglycerides; fatty acid diglycerides; fatty acid amides;
sorbitan trioleate (Span 85) glycocholate; sorbitan monolaurate
(Span 20); polysorbate 20 (Tween-20); polysorbate 60 (Tween-60);
polysorbate 65 (Tween-65); polysorbate 80 (Tween-80); polysorbate
85 (Tween-85); polyoxyethylene monostearate; surfactin; a
poloxomer; a sorbitan fatty acid ester such as sorbitan trioleate;
lecithin; lysolecithin; phosphatidylserine; phosphatidylinositol;
sphingomyelin; phosphatidylethanolamine (cephalin); cardiolipin;
phosphatidic acid; cerebrosides; dicetylphosphate;
dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine;
hexadecyl-amine; acetyl palmitate; glycerol ricinoleate; hexadecyl
sterate; isopropyl myristate; tyloxapol; poly(ethylene
glycol)5000-phosphatidylethanolamine; poly(ethylene
glycol)400-monostearate; phospholipids; synthetic and/or natural
detergents having high surfactant properties; deoxycholates;
cyclodextrins; chaotropic salts; and ion pairing agents.
35. The suspension or dispersion of claim 34, wherein the
surfactant is Tween.
36. The suspension or dispersion of claim 1, further comprising an
oil and a surfactant, wherein the ratio of surfactant to oil ranges
between 0.5:1 and approximately 2:1 by weight.
37. The suspension or dispersion of claim 1, further comprising an
oil and a surfactant, wherein the ratio of surfactant to oil is
approximately 0.25:1 by weight.
38. The suspension or dispersion of claim 1, further comprising an
oil and a surfactant, wherein the ratio of surfactant to oil is
approximately 0:5:1 by weight.
39. The suspension or dispersion of claim 1, further comprising an
oil and a surfactant, wherein the ratio of surfactant to oil is
approximately 1:1 by weight.
40. The suspension or dispersion of claim 1, further comprising an
oil and a surfactant, wherein the ratio of surfactant to oil is
approximately 2:1 by weight.
41. The suspension or dispersion of claim 1, further comprising an
oil and a surfactant, wherein the ratio of surfactant to oil is
approximately 3:1 by weight.
42. The suspension or dispersion of claim 1, wherein the
nanoparticle is a nanomicelle.
43. The suspension or dispersion of claim 1, further comprising at
least one additional component that, together with the suspension
or dispersion, creates a cream, oil, ointment, gel, spray,
lipstick, or sunscreen.
44. A method, comprising steps of: providing a subject; and
administering an amount of the suspension or dispersion of claim 1
to the subject's skin such that a cosmetic or therapeutic benefit
is attained.
45. The method of claim 44, wherein the suspension or dispersion is
administered to the subject's skin at an amount effective to
thicken the subject's skin or subcutaneous tissue.
46. The method of claim 44, wherein the step of providing a subject
comprises providing a subject suffering from, susceptible to, or
exhibiting at least one symptom of fine skin lines; and wherein the
suspension or dispersion is administered to the subject's skin at
an amount effective to treat, alleviate, ameliorate, relieve, delay
onset of, inhibit progression of, reduce severity of, or reduce
incidence of the fine skin lines.
47. The method of claim 44, wherein the step of providing a subject
comprises providing a subject suffering from, susceptible to, or
exhibiting at least one symptom of facial wrinkles; and wherein the
suspension or dispersion is administered to the subject's skin at
effective to treat, alleviate, ameliorate, relieve, delay onset of,
inhibit progression of, reduce severity of, or reduce incidence of
facial wrinkles.
48. The method of claim 44, providing a subject; and wherein the
suspension or dispersion is administered to the subject's skin at
effective to improve the appearance of the subject's skin.
49. The method of claim 44, providing a subject; and wherein the
suspension or dispersion is administered to the subject's skin at
an amount effective to maintain the appearance of the subject's
skin.
50. The method of claim 44, wherein the step of providing a subject
comprises providing a subject suffering from a wound; and wherein
the suspension or dispersion is administered to the subject's skin
at an amount effective to treat, alleviate, ameliorate, relieve,
inhibit progression of, or reduce severity of the wound.
51. The method of claim 44, wherein the step of providing a subject
comprises providing a subject suffering from, susceptible to, or
exhibiting at least one symptom of hypertrophic scarring, keloids,
localized sclerosis, systemic sclerosis, or other condition
characterized by excess accumulation of the extracellular matrix;
and wherein the suspension or dispersion is administered to the
subject's skin at an amount effective to treat, alleviate,
ameliorate, relieve, delay onset of, inhibit progression of, reduce
severity of, or reduce incidence of the at least one symptom
associated with hypertrophic scarring, keloids, localized
sclerosis, systemic sclerosis, or other condition characterized by
excess accumulation of the extracellular matrix.
52. The method of any claim 44, wherein the step of administering
is performed by hand, with a gloved finger, with an unprotected
finger, by a device, with an adhesive patch, with a spatula, with a
swab, with a syringe without a needle, or with a device that
permits application of the suspension or dispersion to a target
site on the skin without applying the suspension or dispersion to
non-target sites of the skin.
53-61. (canceled)
62. The method of claim 44, wherein at least 99% of the unmodified
peptide permeates the skin.
63. The method of claim 44, wherein at least 95% of the unmodified
peptide permeates the skin.
64. The method of claim 44, wherein at least 90% of the unmodified
peptide permeates the skin.
65. The method of claim 44, wherein at least 75% of the unmodified
peptide permeates the skin.
66. The method of claim 44, wherein at least 50% of the unmodified
peptide permeates the skin.
67. The method of claim 44, wherein at least 25% of the unmodified
peptide permeates the skin.
68. The method of claim 44, wherein at least 10% of the unmodified
peptide permeates the skin.
69. The method of claim 44, wherein at least 1% of the unmodified
peptide permeates the skin.
70. A method, comprising steps of: providing a premix comprising an
oil, a surfactant, water, and an unmodified peptide; and subjecting
the premix to high shear force or high pressure homogenization for
a period of time and under conditions which achieve the suspension
or dispersion of claim 1.
71. The method of claim 70, wherein the step of subjecting the
premix to high shear force or high pressure homogenization is
achieved by microfluidization.
72. The method of claim 70, wherein the ratio of oil and surfactant
in the premix ranges between approximately 0.5:1 and approximately
2:1 by weight.
73-77. (canceled)
78. A method for identifying unmodified peptides that improve the
appearance of skin, comprising steps of: providing: at least one
sample of skin; and at least one suspension or dispersion, wherein
the suspension or dispersion comprises at least one unmodified
peptide; administering the suspension or dispersion to the sample
of skin; and monitoring the effect of the suspension or dispersion
on the appearance of the sample of skin relative to the effect of a
control substance.
79-80. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. provisional patent application, U.S. Ser. No.
60/872,206, filed Dec. 1, 2006 ("the '206 application"). The entire
contents of the '206 application are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Peptides have been shown to have beneficial cosmetic and
therapeutic effects on the skin. In experimental models, short
peptides (of length up to 30 amino acids) have been shown to
stimulate collagen growth in the extra-cellular matrix of the skin,
which may improve the appearance of skin as well as improve the
healing of damaged skin (Katayama, et al., 1993, J. Biol. Chem.,
268:9941; incorporated herein by reference). Modified peptides have
also been shown to decrease the appearance of wrinkles through the
modulation of enzymes that influence muscular contractions in the
muscles underlying the skin that contribute to wrinkle formation
(Lupo, 2005, Dermatol. Surg., 31:832; incorporated herein by
reference).
[0003] However, a major problem in achieving the potential cosmetic
and therapeutic effects of these peptides in humans has been the
transdermal delivery of the peptides across the outer skin barrier
(stratum corneum) to the site of biological action, e.g., the
extra-cellular matrix or underlying muscle (Robinson, et al., 2005,
International J. Cosmetic Science 27:155; incorporated herein by
reference). To achieve the delivery of transdermal delivery of the
peptides in humans, the peptides have had to be chemically modified
by the addition of chemical moieties such as but not limited to
acetyl and/or palmitoyl groups (Robinson, et al., supra). These
chemical modifications are disadvantageous because they are
expensive and time-consuming, which negatively impact the
commercial manufacture of a product containing these peptides.
Chemical modifications of the peptides can also decrease the
biological activity of the peptide by decreasing its ability to
bind at the cellular receptor site of biological activity (through,
for example, steric interference), thus making it less effective. A
peptide that is less effective biologically would be less effective
for cosmetic or therapeutic purposes. Comparably, a peptide that is
less effective biologically would need to be administered at higher
levels to achieve its desired biological effect (if the effect were
even possible), which would be a cost disadvantage for the
commercial manufacture of a product.
SUMMARY OF THE INVENTION
[0004] The present invention describes nanoparticles that
incorporate unmodified short peptides (2 to 30 amino acids long)
that are biologically active agents in the skin (including
epidermis and dermis), sub-cutaneous tissue (including adipose
tissue), and contiguous muscles.
[0005] Inventive nanoparticles can be applied to the skin of a
subject. In some embodiments, inventive nanoparticles achieve
transdermal delivery of incorporated peptides to the subject.
[0006] Inventive nanoparticles can be applied to the skin as a
simple suspension or dispersion or mixed with one or more
excipients and prepared as a formulation such as, but not limited
to, a skin softener, nutrition lotion, cleansing lotion, cleansing
cream, skin milk, emollient lotion, massage cream, emollient cream,
make-up base, lipstick, facial pack or facial gel, cleaner
formulation (e.g. shampoos, rinses, body cleanser, hair-tonics, and
soaps), and dermatological composition (e.g. lotions, ointments,
gels, creams, patches and sprays).
[0007] Thus, the present invention provides systems and
compositions for the transdermal delivery of unmodified peptides.
Among the many advantages of this invention is the ability to
delivery peptides without injection and further without a
requirement for mechanical or chemical abrasion or alteration of
skin. Additional advantages include an ability to utilize
unmodified peptides, thereby simplifying and reducing the cost of
production of inventive cosmetic and/or pharmaceutical preparations
and, further, preserving biological activity of the peptide.
BRIEF DESCRIPTION OF THE DRAWING
[0008] FIG. 1. Histological Analysis of Mice Treated with Peptide
Nanoparticles. Shown are photomicrographs of skin tissue stained
with Masson's Trichrome stain. The average histologic score was
2.33 out of a possible 4 in the Control Group (nanoparticle
formulation without pentapeptide). The average histologic score was
3.67 out of a possible 4 in the Treatment Group (nanoparticle
formulation with pentapeptide).
DEFINITIONS
[0009] Abrasion: The term "abrasion," as used herein refers to any
means of altering, disrupting, removing, or destroying the top
layer of the skin. In some embodiments, abrasion refers to a
mechanical means of altering, disrupting, removing, or destroying
the top layer of the skin. In some embodiments, abrasion refers to
a chemical means of altering, disrupting, removing, or destroying
the top layer of skin. To give but a few examples, agents such as
exfoliants, fine particles (e.g. magnesium or aluminum particles),
acids (e.g. alpha-hydroxy acids or beta-hydroxy acids), alcohols,
may cause abrasion. In general, permeation enhancers such as those
described, for example, by Donovan (e.g., U.S. Patent Publications
2004/009180 and 2005/175636 and PCT Publication WO 04/06954; all of
which are incorporated herein by reference), and Graham (e.g., U.S.
Pat. No. 6,939,852 and U.S. Patent Publication 2006/093624; both of
which are incorporated herein by reference), etc., are expected to
cause abrasion. Of course, those of ordinary skill in the art will
appreciate that a particular agent may cause abrasion when present
at one concentration, or in association with one or more other
agents, but may not cause abrasion under different circumstances.
Thus, whether or not a particular material is an "abrasive agent"
depends on context. Abrasion can readily be assessed by those of
ordinary skill in the art, for example by observation of redness or
irritation of the skin and/or histologic examination of skin
showing alteration, disruption, removal, or erosion of the stratum
corneum.
[0010] Amino acid: As used herein, term "amino acid," in its
broadest sense, refers to any compound and/or substance that can be
incorporated into a polypeptide chain. In some embodiments, an
amino acid has the general structure H.sub.2NC--(H)--(R)--COOH. In
some embodiments, an amino acid is a naturally-occurring amino
acid. In some embodiments, an amino acid is a synthetic amino acid;
in some embodiments, an amino acid is a D-amino acid; in some
embodiments, an amino acid is an L-amino acid. "Standard amino
acid" refers to any of the twenty standard L-amino acids commonly
found in naturally occurring peptides. "Nonstandard amino acid"
refers to any amino acid, other than the standard amino acids,
regardless of whether it is prepared synthetically or obtained from
a natural source. Amino acids, including carboxy- and/or
amino-terminal amino acids in peptides, can be modified by
methylation, amidation, acetylation, and/or substitution with other
chemical groups that can change the peptide's circulating half-life
without adversely affecting their activity. However, as described
herein, the present invention is specifically directed to
"unmodified peptides", meaning peptides that have not been
chemically modified in order to facilitate or achieve transdermal
delivery. Amino acids may participate in a disulfide bond. The term
"amino acid" is used interchangeably with "amino acid residue," and
may refer to a free amino acid and/or to an amino acid residue of a
peptide. It will be apparent from the context in which the term is
used whether it refers to a free amino acid or a residue of a
peptide.
[0011] Animal: As used herein, the term "animal" refers to any
member of the animal kingdom. In some embodiments, "animal" refers
to humans, at any stage of development. In some embodiments,
"animal" refers to non-human animals, at any stage of development.
In certain embodiments, the non-human animal is a mammal (e.g., a
rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep,
cattle, a primate, and/or a pig). In some embodiments, animals
include, but are not limited to, mammals, birds, reptiles,
amphibians, fish, and/or worms. In some embodiments, an animal may
be a transgenic animal, genetically-engineered animal, and/or a
clone.
[0012] Approximately: As used herein, the term "approximately" or
"about," as applied to one or more values of interest, refers to a
value that is similar to a stated reference value. In certain
embodiments, the term "approximately" or "about" refers to a range
of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,
13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in
either direction (greater than or less than) of the stated
reference value unless otherwise stated or otherwise evident from
the context (except where such number would exceed 100% of a
possible value).
[0013] Biologically active agent: As used herein, the phrase
"biologically active agent" refers to any substance that has
activity in a biological system and/or organism. For instance, a
substance that, when administered to an organism, has a biological
effect on that organism, is considered to be biologically active.
In particular embodiments, where a protein or polypeptide is
biologically active, a portion of that protein or polypeptide that
shares at least one biological activity of the protein or
polypeptide is typically referred to as a "biologically active"
portion.
[0014] Botulinum toxin: The term "botulinum toxin," as used herein,
refers to any neurotoxin produced by Clostridium botulinum. Except
as otherwise indicated, the term encompasses fragments or portions
(e.g., the light chain and/or the heavy chain) of such neurotoxin
that retain appropriate activity (e.g., muscle relaxant activity).
The phrase "botulinum toxin," as used herein, encompasses the
botulinum toxin serotypes A, B, C, D, E, F, and G. Botulinum toxin,
as used herein, also encompasses both a botulinum toxin complex
(i.e., for example, the 300, 600, and 900 kD complexes) as well as
the purified (i.e., for example, isolated) botulinum toxin (i.e.,
for example, about 150 kD). "Purified botulinum toxin" is defined
as a botulinum toxin that is isolated, or substantially isolated,
from other proteins, including protein that for a botulinum toxin
complex. A purified toxin may be greater than 95% pure, and
preferably is greater than 99% pure. Those of ordinary skill in the
art will appreciate that the present invention is not limited to
any particular source of botulinum toxin. For example, botulinum
toxin for use in accordance with the present invention may be
isolated from Clostridium botulinum, may be chemically synthesized,
may be produced recombinantly (i.e., in a host cell or organism
other than Clostridium botulinum), etc.
[0015] Characteristic portion: As used herein, the phrase a
"characteristic portion" of a substance, in the broadest sense, is
one that shares some degree of sequence and/or structural identity
and/or at least one functional characteristic with the relevant
intact substance. For example, a "characteristic portion" of a
protein or polypeptide is one that contains a continuous stretch of
amino acids, or a collection of continuous stretches of amino
acids, that together are characteristic of a protein or
polypeptide. In some embodiments, each such continuous stretch
generally will contain at least 2, 5, 10, 15, 20 or more amino
acids. In general, a characteristic portion is one that, in
addition to the sequence identity specified above, shares at least
one functional characteristic with the relevant intact protein. In
some embodiments, the characteristic portion may be biologically
active.
[0016] Hydrophilic: As used herein, a "hydrophilic" substance is a
substance that may be soluble in polar solvents. In some
embodiments, a hydrophilic substance can transiently bond with
polar solvents. In some embodiments, a hydrophilic substance
transiently bonds with polar solvents through hydrogen bonding. In
some embodiments, the polar solvent is water. In some embodiments,
a hydrophilic substance may be ionic. In some embodiments, a
hydrophilic substance may be non-ionic. In some embodiments, a
hydrophilic substance may dissolve more readily in water, polar
solvents, or hydrophilic solvents than in oil, non-polar solvents,
or hydrophobic solvents. In some embodiments, a hydrophilic
substance may dissolve less readily in oil, non-polar solvents, or
hydrophobic solvents than in water, polar solvents, or hydrophilic
solvents. In some embodiments, a substance is hydrophilic relative
to another substance because it is more soluble in water, polar
solvents, or hydrophilic solvents than is the other substance. In
some embodiments, a substance is hydrophilic relative to another
substance because it is less soluble in oil, non-polar solvents, or
hydrophobic solvents than is the other substance.
[0017] Hydrophobic: As used herein, a "hydrophobic" substance is a
substance that may be soluble in non-polar solvents. In some
embodiments, a hydrophobic substance is repelled from polar
solvents. In some embodiments, the polar solvent is water. In some
embodiments, hydrophobic substances are non-polar. In some
embodiments, a hydrophobic substance may dissolve more readily in
oil, non-polar solvents, or hydrophobic solvents than in water,
polar solvents, or hydrophilic solvents. In some embodiments, a
hydrophobic substance may dissolve less readily in water, polar
solvents, or hydrophilic solvents than in oil, non-polar solvents,
or hydrophobic solvents. In some embodiments, a substance is
hydrophobic relative to another substance because it is more
soluble in oil, non-polar solvents, or hydrophobic solvents than is
the other substance. In some embodiments, a substance is
hydrophobic relative to another substance because it is less
soluble in water, polar solvents, or hydrophilic solvents than is
the other substance.
[0018] In conjunction with: As used herein, the phrase "delivered
in conjunction with" refers to the co-delivery of two or more
substances or agents. In particular, according to the present
invention, the phrase is used herein in reference to delivery of a
biologically active agent with inventive nanoparticles and/or
nanoparticle compositions. A substance or agent is delivered in
conjunction with nanoparticles when the substance or agent is
combined with nanoparticles and/or nanoparticle compositions; is
encapsulated or completely surrounded by nanoparticles; is embedded
within an nanoparticle micellar membrane; and/or is associated with
the outer surface of an nanoparticle micellar membrane. A substance
or agent to be delivered in conjunction with nanoparticles and/or
nanoparticle compositions may or may not be covalently linked to
the nanoparticles and/or nanoparticle compositions. A substance or
agent to be delivered in conjunction with inventive nanoparticles
and/or nanoparticle compositions may or may not be attached to the
nanoparticles and/or nanoparticle compositions by adsorption
forces.
[0019] Isolated: As used herein, the term "isolated" refers to a
substance and/or entity that has been (1) separated from at least
some of the components with which it was associated when initially
produced (whether in nature and/or in an experimental setting),
and/or (2) produced, prepared, and/or manufactured by the hand of
man. Isolated substances and/or entities may be separated from at
least about 10%, about 20%, about 30%, about 40%, about 50%, about
60%, about 70%, about 80%, about 90%, or more of the other
components with which they were initially associated. In some
embodiments, isolated substances and/or entities are more than 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% pure.
[0020] Microfluidized: As used herein, the term "microfluidized"
means exposed to high shear forces. In some embodiments, such
exposure to high shear forces is accomplished by exposure to high
pressure; in some embodiments such high pressure is within the
range of about 15,000 to about 26,000 psi. In some embodiments,
such exposure to high shear forces is accomplished by cavitation.
In some embodiments, such exposure to high shear forces is
accomplished by passing a sample through an instrument such as, for
example, a Microfluidizer.RTM. (Microfluidics Corporation/MFIC
Corporation) or other like device that may be useful in creating a
uniform nanoparticle composition. In some embodiments of the
present invention, a sample is microfluidized through exposure to
high shear forces for a period of time less than about 10 minutes.
In some embodiments, the period of time is less than about 9, 8, 7,
6, 5, 4, 3, 2, or 1 minute(s). In some embodiments, the period of
time is within the range of about 1-2 minutes. In some embodiments,
the period of time is about 30 seconds. In some embodiments of the
invention, a sample is "microfluidized" through a single exposure
to high shear forces; such embodiments are referred to as "single
pass" microfluidization.
[0021] Nanoparticle: As used herein, the term "nanoparticle" refers
to any particle having a diameter of less than 1000 nanometers
(nm). In some embodiments, a nanoparticle has a diameter of less
than 300 nm, as defined by the National Science Foundation. In some
embodiments, a nanoparticle has a diameter of less than 100 nm as
defined by the National Institutes of Health. In some embodiments,
nanoparticles are micelles in that they comprise an enclosed
compartment, separated from the bulk solution by a micellar
membrane. A "micellar membrane" comprises amphiphilic entities
which have aggregated to surround and enclose a space or
compartment (e.g., to define a lumen).
[0022] Nanoparticle composition: As used herein, the term
"nanoparticle composition" refers to any substance that contains at
least one nanoparticle. In some embodiments, a nanoparticle
composition is a uniform collection of nanoparticles. In some
embodiments, nanoparticle compositions are dispersions or
emulsions. In general, a dispersion or emulsion is formed when at
least two immiscible materials are combined. An "oil-in-water"
dispersion is one in which oily particles (or hydrophobic or
non-polar) are dispersed within an aqueous dispersion medium. A
"water-in-oil" dispersion is one in which aqueous (or hydrophilic
or polar) particles are dispersed within an oily dispersion medium.
Those of ordinary skill in the art will appreciate that a
dispersion can be formed from any two immiscible media and is not
limited strictly to combinations of aqueous and oily media. The
term "dispersion medium" therefore applies broadly to any
dispersion medium notwithstanding that it is common to refer to
"aqueous" and "oily" categories. In some embodiments, nanoparticle
compositions are nanoemulsions. In some embodiments, nanoparticle
compositions comprise micelles. In some particular embodiments, a
nanoparticle composition comprises amphiphilic entity nanoparticles
as described in PCT application serial number PCT/US07/______,
entitled "AMPHIPHILIC ENTITY NANOPARTICLES," filed on Nov. 30, 2007
(incorporated herein by reference). In some embodiments, a
nanoparticle composition is stable. In some embodiments, a
nanoparticle composition includes one or more biologically active
agents to be delivered in conjunction with the nanoparticles.
[0023] Nutraceutical: As used herein, the term "nutraceutical"
refers to any substance thought to provide medical, health, or
biological benefits. In some embodiments, nutraceuticals may
prevent disease. In some embodiments, nutraceuticals may provide
basic nutritional value. In some embodiments, a nutraceutical is a
food or part of a food. In some embodiments, a nutraceutical agent
may be a class of isolated nutrients, dietary supplements,
vitamins, minerals, herbs, fortified foods, healing foods,
genetically engineered foods, and processed foods. Nutraceuticals
may also be known as "phytochemical foods" or "functional
foods."
[0024] Premix: As used herein, the term "premix" refers to any
combination of components that is subsequently used to generate a
nanoparticle composition according to the present invention. For
example, a premix is any collection of ingredients that, when
subjected to high shear forces, generates nanoparticles according
to the present invention. In some embodiments, a premix contains
two or more immiscible solvents. In some embodiments, a premix
contains components that self-assemble into nanoparticles. In some
embodiments, a premix contains components that self-assemble into
micelles. In some embodiments, a premix contains one or more
amphiphilic entities as described in PCT application serial number
PCT/US07/______, entitled "AMPHIPHILIC ENTITY NANOPARTICLES," filed
Nov. 30, 2007 (incorporated herein by reference). In some
embodiments, a premix contains one or more unmodified peptides; in
some embodiments, a premix contains at least one other biologically
active agent. In some embodiments, a premix is agitated, mixed,
and/or stirred; in some embodiments, a premix is agitated, mixed,
and/or stirred prior to being subjected to high shear force. In
some embodiments, a premix comprises at least one solubilized
component (i.e., at least one component that is in solution); in
some such embodiments, the premix is subjected to high shear force
after such solubilization is achieved.
[0025] Pure: As used herein, a substance and/or entity is "pure" if
it is substantially free of other components. For example, a
preparation that contains more than about 90% of a particular
substance and/or entity is typically considered to be a pure
preparation. In some embodiments, a substance and/or entity is at
least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure.
[0026] Shear force: As used herein, the term "shear force" refers
to a force that is parallel to the face of a material, as opposed
to a force that is perpendicular to the face of a material. In some
embodiments, a composition exposed to high shear forces in order to
produce a uniform nanoparticle composition. Any method known in the
art can be used to generate high shear forces. In some embodiments,
cavitation is used to generate high shear forces. In some
embodiments, high pressure homogenization is used to generate high
shear forces. Alternatively or additionally, high shear force may
be administered by exposure to high pressure, for example about
15,000 psi. In some embodiments, such high pressure is within the
range of about 18,000 to about 26,000 psi; in some embodiments, it
is within the range of about 20,000 to about 25,000 psi. In some
embodiments, a Microfluidizer.RTM. Processor (Microfluidics
Corporation/MFIC Corporation) or other like device is used to
generate high shear force. Microfluidizer.RTM. Processors provide
high pressure and a resultant high shear rate by accelerating a
composition through microchannels (typically having dimensions on
the order of 75 microns) at a high velocity (typically in the range
of 50 m/s-300 m/s) for size reduction to the nanoscale range. As
the fluid exits the microchannels it forms jets which collide with
jets from opposing microchannels. In the channels the fluid
experiences high shear (up to 10.sup.7 l/s) which is orders of
magnitude higher than that of conventional technologies. Jet
collisions result in mixing in submicron level. Therefore, in such
devices, high shear and/or impact can achieve particle size
reduction and mixing of multiphase. In some embodiments of the
present invention, a sample is exposed to high shear forces for a
period of time less than about 10 minutes. In some embodiments, the
period of time is less than about 9, about 8, about 7, about 6,
about 5, about 4, about 3, about 2, or about 1 minute(s). In some
embodiments, the period of time is within the range of about 1 to
about 2 minutes or less; in some embodiments, the period of time is
about 30 seconds. In some embodiments of the invention, a sample is
"microfluidized" through a single exposure to high shear forces;
such embodiments are referred to herein as "single pass"
microfluidization.
[0027] Small Molecule: In general, a "small molecule" is understood
in the art to be an organic molecule that is less than about 5
kilodaltons (Kd) in size. In some embodiments, the small molecule
is less than about 3 Kd, about 2 Kd, or about 1 Kd. In some
embodiments, the small molecule is less than about 800 daltons (D),
about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or
about 100 D. In some embodiments, small molecules are
non-polymeric. In some embodiments, small molecules are not
proteins, peptides, or amino acids. In some embodiments, small
molecules are not nucleic acids or nucleotides. In some
embodiments, small molecules are not saccharides or
polysaccharides.
[0028] Subject: As used herein, the term "subject" or "patient"
refers to any organism to which a composition of this invention may
be administered, e.g., for experimental, diagnostic, prophylactic,
and/or therapeutic purposes. Typical subjects include animals
(e.g., mammals such as mice, rats, rabbits, non-human primates, and
humans; insects; worms; etc.).
[0029] Substantially: As used herein, the term "substantially"
refers to the qualitative condition of exhibiting total or
near-total extent or degree of a characteristic or property of
interest. One of ordinary skill in the biological arts will
understand that biological and chemical phenomena rarely, if ever,
go to completion and/or proceed to completeness or achieve or avoid
an absolute result. The term "substantially" is therefore used
herein to capture the potential lack of completeness inherent in
many biological and chemical phenomena.
[0030] Stable: The term "stable," when applied to nanoparticle
compositions herein, means that the compositions maintain one or
more aspects of their physical structure (e.g., size range and/or
distribution of particles) over a period of time. In some
embodiments of the invention, a stable nanoparticle composition is
one for which the average particle size, the maximum particle size,
the range of particle sizes, and/or the distribution of particle
sizes (i.e., the percentage of particles above a designated size
and/or outside a designated range of sizes) is maintained for a
period of time. In some embodiments, the period of time is at least
about one hour; in some embodiments the period of time is about 5
hours, about 10 hours, about one (1) day, about one (1) week, about
two (2) weeks, about one (1) month, about two (2) months, about
three (3) months, about four (4) months, about five (5) months,
about six (6) months, about eight (8) months, about ten (10)
months, about twelve (12) months, about twenty-four (24) months, or
longer. In some embodiments, the period of time is within the range
of about one (1) day to about twenty-four (24) months, about two
(2) weeks to about twelve (12) months, about two (2) months to
about five (5) months, etc. For example, if a nanoparticle
composition is subjected to prolonged storage, temperature changes,
and/or pH changes and a majority of the nanoparticles in the
population maintain a diameter within a stated range (i.e., for
example, between approximately 10 nm-120 nm), the nanoparticle
composition is stable. For some such populations, a majority is
more than about 50%, about 60%, about 70%, about 80%, about 90%,
about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%,
about 99.6%, about 99.7%, about 99.8%, about 99.9%, or more. In
some embodiments of the invention, where a nanoparticle composition
comprises one or more biologically active agents (e.g. unmodified
peptide), the nanoparticle composition is considered stable if the
concentration of biologically active agent is maintained in the
composition over the designated period of time under a designated
set of conditions.
[0031] Substantially free of: An inventive nanoparticle composition
is said to be "substantially free of" particles whose diameter is
outside of a stated range when no more than about 50% of the
particles in that composition have diameters outside of the range.
In some embodiments, no more than 25% of the particles are outside
of the range. In some embodiments, no more than 20%, 19%, 18%, 17%,
16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,
0.5% or less of the particles have diameters outside of the stated
range.
[0032] Suffering from: An individual who is "suffering from" a
disease, disorder, or condition (e.g., facial wrinkles) has been
diagnosed with or exhibits symptoms of the disease, disorder, or
condition.
[0033] Therapeutically effective amount: As used herein, the term
"therapeutically effective amount" means an amount of inventive
nanoparticle composition that is sufficient, when administered to a
patient suffering from or susceptible to a disease, disorder,
and/or condition, to treat the disease, disorder, and/or
condition.
[0034] Therapeutic agent: As used herein, the phrase "therapeutic
agent" refers to any agent that, when administered to a subject,
has a therapeutic effect and/or elicits a desired biological and/or
pharmacological effect.
[0035] Treatment: As used herein, the term "treatment" (also
"treat" or "treating") refers to any administration of a
biologically active agent that partially or completely alleviates,
ameliorates, relives, inhibits, delays onset of, reduces severity
of and/or reduces incidence of one or more symptoms or features of
a particular disease, disorder, and/or condition. Such treatment
may be of a subject who does not exhibit signs of the relevant
disease, disorder and/or condition and/or of a subject who exhibits
only early signs of the disease, disorder, and/or condition.
Alternatively or additionally, such treatment may be of a subject
who exhibits one or more established signs of the relevant disease,
disorder and/or condition.
[0036] Toxic solvent: As used herein, the term "toxic solvent"
refers to any substance that may alter, disrupt, remove, or destroy
an animal's tissue. As would be understood by one of ordinary skill
in the art, an animal's tissue can include living cells, dead
cells, extracellular matrix, cellular junctions, biological
molecules, etc. To give but a few examples, toxic solvents include
dimethyl sulfoxide, dimethyl acetimide, dimethyl foramide,
chloroform, tetramethyl foramide, acetone, acetates, and
alkanes.
[0037] Uniform: The term "uniform," when used herein in reference
to a nanoparticle composition, refers to a nanoparticle composition
in which the individual nanoparticles have a specified range of
particle diameter sizes. For example, in some embodiments, a
uniform nanoparticle composition is one in which the difference
between the minimum diameter and maximum diameter does not exceed
approximately 600, approximately 550, approximately 500,
approximately 450, approximately 400, approximately 350,
approximately 300, approximately 250, approximately 200,
approximately 150, approximately 100, approximately 90,
approximately 80, approximately 70, approximately 60, approximately
50, or fewer nm. In some embodiments, particles (e.g.,
unmodified-peptide-containing particles) within inventive uniform
nanoparticle compositions have diameters that are smaller than
about 600, about 550, about 500, about 450, about 400, about 350,
about 300, about 250, about 200, about 150, about 130, about 120,
about 115, about 110, about 100, about 90, about 80 nm, or less. In
some embodiments, particles (e.g., unmodified-peptide-containing
particles) within inventive uniform nanoparticle compositions have
diameters within the range of about 10 and about 600 nanometers. In
some embodiments, particles (e.g., unmodified-peptide-containing
particles) within inventive uniform nanoparticle compositions have
diameters within the range of about 10 to about 300, about 10 to
about 200, about 10 to about 150, about 10 to about 130, about 10
to about 120, about 10 to about 115, about 10 to about 110, about
10 to about 100, or about 10 to about 90 nm. In some embodiments,
particles (e.g., unmodified-peptide-containing particles) within
inventive botulinum nanoparticle compositions have an average
particle size that is under about 300, about 250, about 200, about
150, about 130, about 120, about 115, about 110, about 100, or
about 90 nm. In some embodiments, the average particle size is
within the range of about 10 to about 300, about 50 to about 250,
about 60 to about 200, about 65 to about 150, about 70 to about 130
nm. In some embodiments, the average particle size is about 80 to
about 110 nm. In some embodiments, the average particle size is
about 90 to about 100 nm. In some embodiments, a majority of the
particles (e.g., unmodified-peptide-containing particles) within
inventive uniform nanoparticle compositions have diameters below a
specified size or within a specified range. In some embodiments,
the majority is more than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more of
the particles in the composition. In some embodiments of the
invention, a uniform nanoparticle composition is achieved by
microfluidization of a sample. In some embodiments of the
invention, a uniform nanoparticle composition is prepared by
exposure to high shear force, e.g., by microfluidization.
[0038] Unmodified peptide: As used herein, the term "unmodified
peptide" refers to a peptide that has not been chemically modified
through the addition of other covalently-bonded functional groups
intended to achieve transdermal delivery of the peptide. In some
embodiments, the peptide has not been chemically modified to add
pendant acetyl and/or palmitoyl groups. In some embodiments, the
peptide has not been chemically modified to add any functional
pendant groups.
DESCRIPTION OF CERTAIN EMBODIMENTS
Nanoparticles
[0039] As discussed herein, the present invention provides
nanoparticle compositions that include one or more unmodified
peptides. In some embodiments, such nanoparticle compositions
further include one or more other biologically active agents in
addition to the unmodified peptides. In some embodiments, the
nanoparticle compositions are formulated with one or more other
components, for example in a pharmaceutical or cosmetic
preparation. In some embodiments, such a pharmaceutical or cosmetic
preparation is formulated to achieve transdermal delivery of the
unmodified peptides (and/or one or more other biologically active
agents).
[0040] In some embodiments, inventive nanoparticle compositions are
stable. In some embodiments, the nanoparticle compositions are
uniform.
[0041] In some embodiments, a uniform nanoparticle composition
comprises a population of particles whose difference between the
minimum and maximum diameters does not exceed approximately 600 nm,
approximately 550 nm, approximately 500 nm, approximately 450 nm,
approximately 400 nm, approximately 350 nm, approximately 300 nm,
approximately 250 nm, approximately 200 nm, approximately 150 nm,
or approximately 100 nm.
[0042] In some embodiments, inventive nanoparticles have diameters
that are smaller than about 1000, about 600, about 550, about 500,
about 450, about 400, about 350, about 300, about 250, about 200,
about 150, about 130, about 120, about 115, about 110, about 100,
about 90, about 80, about 50 nm, or less.
[0043] In some embodiments, inventive nanoparticles have a diameter
of 1 nm to 1000 nm, 1 nm to 600 nm, 1 nm to 500 nm, 1 nm to 400 nm,
1 nm to 300 nm, 1 nm to 200 nm, 1 nm to 150 nm, 1 nm to 120 nm, 1
nm to 100 nm, 1 nm to 75 nm, 1 nm to 50 nm, or 1 nm to 25 nm. In
some embodiments, inventive nanoparticles have a diameter of 1 nm
to 15 nm, 15 nm to 200 nm, 25 nm to 200 nm, 50 nm to 200 nm, or 75
nm to 200 nm.
[0044] In some embodiments, the total particle distribution is
encompassed within the specified range of particle diameter size.
In some embodiments, less than 50%, 25%, 10%, 5%, or 1% of the
total particle distribution is outside of the specified range of
particle diameter sizes. In some embodiments, less than 1% of the
total particle distribution is outside of the specified range of
particle diameter sizes. In certain embodiments, the nanoparticle
composition is substantially free of particles having a diameter
larger than 300 nm, 250 nm, 200 nm, 150 nm, 120 nm, 100 nm, 75 nm,
50 nm, or 25 nm.
[0045] In some embodiments, nanoparticles within inventive
nanoparticle compositions have an average particle size that is
under about 300 nm, about 250 nm, about 200 nm, about 150 nm, about
130 nm, about 120 nm, about 115 nm, about 110 nm, about 100 nm,
about 90 nm, or about 50 nm. In some embodiments, the average
particle size is within the range of about 10 nm to about 300 nm,
50 nm to about 250 nm, 60 nm to about 200 nm, 65 nm to about 150
nm, or 70 nm to about 130 nm. In some embodiments, the average
particle size is about 80 nm to about 110 nm. In some embodiments,
the average particle size is about 90 to about 100 nm.
[0046] In some embodiments, inventive nanoparticle compositions are
substantially free of particles having a diameter in excess of 300
nm. Specifically, in some embodiments, fewer than 50%, of the
nanoparticles in inventive nanoparticle compositions have a
diameter in excess of 300 nm. In some embodiments, fewer than 25%
of the particles have a diameter in excess of 300 nm. In some
embodiments, fewer than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,
12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of the
particles have a diameter in excess of 300 nm. Furthermore, in some
embodiments, the nanoparticles in inventive nanoparticle
compositions have diameters within the range of 10 nm to 300
nm.
[0047] In some embodiments, inventive nanoparticle compositions are
substantially free of particles having a diameter in excess of 200
nm. Specifically, in some embodiments, fewer than 50%, of the
nanoparticles in inventive nanoparticle compositions have a
diameter in excess of 200 nm. In some embodiments, fewer than 25%
of the particles have a diameter in excess of 200 nm. In some
embodiments, fewer than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,
12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of the
particles have a diameter in excess of 200 nm. Furthermore, in some
embodiments, the nanoparticles in inventive nanoparticle
compositions have diameters within the range of 10 nm to 200
nm.
[0048] In some embodiments, inventive nanoparticle compositions are
substantially free of particles having a diameter in excess of 120
nm. Specifically, in some embodiments, fewer than 50%, of the
nanoparticles in inventive nanoparticle compositions have a
diameter in excess of 120 nm. In some embodiments, fewer than 25%
of the particles have a diameter in excess of 120 nm. In some
embodiments, fewer than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,
12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of the
particles have a diameter in excess of 120 nm. Furthermore, in some
embodiments, the nanoparticles in inventive nanoparticle
compositions have diameters within the range of 10 nm to 120
nm.
[0049] In some embodiments, a majority of the nanoparticles within
inventive compositions have diameters below a specified size or
within a specified range. In some embodiments, the majority is more
than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,
99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more of the particles in the
composition.
[0050] Zeta potential is a measurement of the electric potential at
a shear plane. A shear plane is an imaginary surface separating a
thin layer of liquid bound to a solid surface (e.g. the surface of
inventive nanoparticles) and showing elastic behavior from the rest
of liquid (e.g. liquid dispersion medium) showing normal viscous
behavior. In some embodiments, inventive nanoparticles have a zeta
potential ranging between -50 mV to +50 mV. In some embodiments,
inventive nanoparticles have a zeta potential ranging between -25
mV to +25 mV. In some embodiments, inventive nanoparticles have a
zeta potential ranging between -10 mV to +10 mV.
[0051] In some embodiments inventive nanoparticle compositions are
emulsions or dispersions. In general, an emulsion or dispersion is
formed from at least two immiscible materials, one of which will
constitute the dispersion medium (i.e., the liquid medium in which
particles (e.g., nanoparticles, which constituted the "dispersed
medium") are dispersed. An "oil-in-water" dispersion is one in
which oily particles are dispersed within an aqueous dispersion
medium. A "water-in-oil" dispersion is one in which aqueous
particles are dispersed within an oily dispersion medium. Those of
ordinary skill in the art will appreciate that a dispersion can be
formed from any two immiscible media and is not limited strictly to
combinations of aqueous and oily media. The term "dispersion
medium" therefore applies broadly to any dispersion medium
notwithstanding that it is common to refer to "aqueous" and "oily"
categories. For example, emulsions or dispersions can be prepared
from immiscible sets of hydrophobic/hydrophilic materials;
polar/nonpolar materials, etc., regardless of whether such
materials are strictly speaking "aqueous" or "oily."
[0052] In some embodiments, inventive nanoparticle compositions
comprise micellar structures (e.g., the nanoparticles are
micelles). In some embodiments, such micellar structures are
crosslinked. In some embodiments, such micellar structures are not
crosslinked.
[0053] In some embodiments, inventive nanoparticle compositions
self-assemble from a collection of combined components. In some
embodiments, inventive nanoparticle compositions are prepared by
subjecting a combination of components (i.e., a "premix") to high
shear force. In some embodiments, high shear force is applied by
high pressure, by cavitation, by homogenization, and/or by
microfluidization. In some embodiments, combined
nanoparticle-forming components are agitated, stirred, or otherwise
mixed. In some such embodiments, the components are subjected to
high shear force after having been mixed. In some specific
embodiments, mixing may be performed for a period of time such as,
for example, less than one hour or more than 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, or 15 hours. In some embodiments, solubilization is
achieved.
[0054] In some embodiments of the invention, production of
nanoparticle compositions involves dialyzing a collection of
components, for example to remove any organic solvent, and/or
freeze-drying to produce a composition.
[0055] In some embodiments of the present invention that utilize a
premix, it is to be understood that the premix components may
assemble into particles before the application of high shear force.
At least some of such particles may be microparticles or even
nanoparticles. In some embodiments, an inventive nanoparticle
composition is prepared from a premix, wherein the premix is
selected from the group comprising a suspension or a microemulsion.
In some embodiments, however, particle structures do not form in
the premix before application of high shear force.
[0056] In some embodiments of the present invention, all of the
components present in the final nanoparticle composition are
present in the premix and are subjected to high shear force to
produce the nanoparticle composition. In some embodiments of the
present invention, one or more of the components that are present
in the final nanoparticle composition is/are missing from the
premix or is/are present in the premix in a smaller amount than in
the final nanoparticle composition. That is, in some embodiments of
the present invention, one or more materials are added to the
nanoparticle composition after the premix is subjected to high
shear stress.
[0057] In certain embodiments of the invention, the premix is
prepared as a solution prior to application of high shear force. In
particular, for nanoparticle compositions that include at least one
biologically active agent (e.g., an unmodified peptide), it is
often desirable for the biologically active agent to be dissolved
in the premix before the high shear stress is applied. Thus, in
many embodiments, the biologically active agent is soluble in at
least one of the media (or in a combination of media utilized in
the premix). In some embodiments of the invention, such dissolution
requires heating; in other embodiments it does not.
[0058] In some embodiments of the invention, nanoparticle
compositions are prepared from components including one or more
aqueous, polar, or hydrophilic medium(a), one or more oily,
nonpolar, or hydrophobic medium(a), one or more micelle components,
one or more surfactants or emulsifiers, one or more biologically
active agents and/or one or more release retarding agents, etc.
[0059] Those of ordinary skill in the art will be well aware of
suitable aqueous media that can be used as dispersion media or as
media to be dispersed in accordance with the present invention.
Representative such aqueous media include, for example, water,
saline solutions (including phosphate buffered saline), water for
injection, short chain alcohols, 5% dextrose, Ringer's solutions
(lactated Ringer's injection, lactated Ringer's plus 5% dextrose
injection, acylated Ringer's injection), Normosol-M, Isolyte E, and
the like, and combinations thereof.
[0060] Those of ordinary skill in the art will also be well aware
of suitable oily media that can be used as dispersion media or as
media to be dispersed in accordance with the present invention. In
some embodiments, the oil may comprise one or more fatty acid
groups or salts thereof. In some embodiments, the fatty acid group
may comprise digestible, long chain (e.g., C.sub.8-C.sub.50),
substituted or unsubstituted hydrocarbons. In some embodiments, the
fatty acid group may be a C.sub.10-C.sub.20 fatty acid or salt
thereof. In some embodiments, the fatty acid group may be a
C.sub.15-C.sub.20 fatty acid or salt thereof. In some embodiments,
the fatty acid group may be a C.sub.15-C.sub.25 fatty acid or salt
thereof. In some embodiments, the fatty acid group may be
unsaturated. In some embodiments, the fatty acid group may be
monounsaturated. In some embodiments, the fatty acid group may be
polyunsaturated. In some embodiments, a double bond of an
unsaturated fatty acid group may be in the cis conformation. In
some embodiments, a double bond of an unsaturated fatty acid may be
in the trans conformation.
[0061] In some embodiments, the fatty acid group may be one or more
of butyric, caproic, caprylic, capric, lauric, myristic, palmitic,
stearic, arachidic, behenic, or lignoceric acid. In some
embodiments, the fatty acid group may be one or more of
palmitoleic, oleic, vaccenic, linoleic, alpha-linolenic,
gamma-linoleic, arachidonic, gadoleic, arachidonic,
eicosapentaenoic, docosahexaenoic, or erucic acid.
[0062] In some embodiments, the oil is a liquid triglyceride. In
certain embodiments, the oil is a medium chain (e.g., 6-12 carbons)
triglyceride (e.g., Labrafac WL 1349, coconut oil, palm kernel oil,
camphor tree drupe oil, etc.). In certain embodiments, the oil is a
short chain (e.g., 2-5 carbons) triglyceride. In certain
embodiments, the oil is a long chain (e.g., greater than 12
carbons) triglyceride (e.g., soybean oil, sunflower oil, etc.).
[0063] Suitable oils for use with the present invention include,
but are not limited to, almond, apricot kernel, avocado, babassu,
bergamot, black current seed, borage, cade, camomile, canola,
caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod
liver, coffee, corn, cotton seed, emu, eucalyptus, evening
primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut,
hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender,
lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam
seed, mineral, mink, nutmeg, olive, orange, orange roughy, palm,
palm kernel, peach kernel, peanut, poppy seed, pumpkin seed,
rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana,
savoury, sea buckthorn, sesame, shea butter, silicone, soybean,
sunflower, tea tree, thistle, tsubaki, vetiver, walnut, wheat germ,
and mixtures thereof. Suitable synthetic oils for use with the
present invention include, but are not limited to: caprylic/capric
triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,
isopropyl myristate, octyldodecanol, oleyl alcohol, and
combinations thereof.
[0064] Appropriate micelle components may include, for example, one
or more amphiphilic entities. Useful amphiphilic entities include
natural entities, synthetic entities, and entities that contain
both natural and synthetic components. In some embodiments,
amphiphilic entities may comprise one or more polymers, and/or one
or more compounds with polymeric character.
[0065] In general, an amphiphilic entity is one that has both
hydrophobic and hydrophilic natures. As will be appreciated by
those of ordinary skill in the art, an amphiphilic entity can be
comprised in any number of different ways. In some embodiments, an
amphiphilic entity may comprise one or more individual compounds or
molecules that is itself amphiphilic. To give but a few examples,
such compounds or molecules include polyethylene glycol (PEG),
phospholipids, cholesterols, glycolipids fatty acids, bile acids,
and saponins PEG is generally recognized as safe for use in food,
cosmetics, and medicines by the US Food and Drug Administration.
PEG is water-soluble, non-toxic, odorless, lubricating,
nonvolatile, and nonirritating.
[0066] In some embodiments, an amphiphilic entity may comprise one
or more individual components that is not itself amphiphilic but
that has some hydrophilic or hydrophobic character. In such
embodiments, two or more such non-amphiphilic components will
typically be associated with one another such that the assemblage
of the individual components is amphiphilic. Such association may
or may not involve covalent linkage; such association may involve
non-covalent bonding (e.g., via electrostatic interactions,
affinity interactions, hydrophobic interactions, hydrogen bonding,
Van der Waals interactions, ionic interaction, dipole-dipole
interaction, etc.). In general, such association may involve any
relevant force, bond, or means of adhesion.
[0067] In some embodiments, an amphiphilic entity for use in
accordance with the present invention may be constructed from two
or more individual components having differing degrees of
hydrophilicity or hydrophobicity. In certain embodiments, an
amphiphilic entity may comprise at least one hydrophilic component
and at least one hydrophobic component. In certain embodiments, the
"hydrophilic" and "hydrophobic" components are either hydrophilic
or hydrophobic relative to one another.
[0068] In some embodiments, two or more components of differing
degrees of hydrophilicity or hydrophobicity may be bonded together
by covalent bonds to form a homopolymer or a co-polymer. In some
embodiments, a co-polymer may be a block co-polymer. In some
embodiments, a co-polymer may be a graft co-polymer.
[0069] In some embodiments, an amphiphilic entity may comprise or
consist of an amphiphilic block co-polymer. In some embodiments, an
amphiphilic block co-polymer may be a diblock co-polymer. In
certain embodiments, an amphiphilic diblock co-polymer may comprise
a first polymer block and a second polymer block connected
covalently at the chain ends. In specific embodiments, the first
polymer block may comprise repeating units of a hydrophilic
component, and the second polymer block may comprise repeating
units of a hydrophobic component. In specific embodiments, the
first polymer block may comprise repeating units of a hydrophobic
component, and the second polymer block may comprise repeating
units of a hydrophilic component. In some embodiments, an
amphiphilic block co-polymer may be a multiblock co-polymer. In
certain embodiments, an amphiphilic block co-polymer may comprise
multiple alternating blocks of two or more polymers connected
covalently at the chain ends. In specific embodiments, an
amphiphilic block co-polymer may comprise multiple alternating
hydrophilic blocks and hydrophobic blocks connected covalently at
the chain ends. In specific embodiments, each block of the
alternating blocks may comprise repeating units of either
hydrophilic components or hydrophobic components.
[0070] In some embodiments, an amphiphilic entity may comprise or
consist of an amphiphilic graft co-polymer. In some embodiments, an
amphiphilic graft co-polymer may comprise or consist of blocks of
polymers connected covalently to the side chains of other blocks of
polymers. In specific embodiments, each polymer block may comprise
or consist of repeating units of either hydrophilic or hydrophobic
components. In certain embodiments, an amphiphilic graft co-polymer
may comprise or consist of a first polymer block and a second
polymer block connected covalently to a side chain of the first
polymer block. In certain embodiments, the first polymer block may
comprise or consist of repeating units of a hydrophilic component,
and the second block may comprise repeating units of a hydrophobic
component. In certain embodiments, the first polymer block may
comprise or consist of repeating units of a hydrophobic component,
and the second block may comprise repeating units of a hydrophilic
component.
[0071] In some embodiments, an amphiphilic block or graft
co-polymer may include a hydrophilic polymer block comprising
repeating units of a polysaccharide and a hydrophobic polymer block
comprising repeating units of a polyester or polysaccharide.
Alternatively or additionally, an amphiphilic block or graft
co-polymer may include a hydrophobic polymer block comprising
repeating units of a polysaccharide and a hydrophilic polymer block
comprising repeating units of a polyester or polysaccharide. Such a
hydrophilic polymer block can contain repeating units of any type
of hydrophilic polymer, such as a polysaccharide (e.g. pullulan) or
polyalkene oxide (e.g. polyethylene oxide). The hydrophobic polymer
block can contain repeating units of any type of hydrophobic
polymer, such as a polycaprolactone or polyamide (e.g.
polycaprolactam).
[0072] In some embodiments, the hydrophilic portion of the
amphiphilic entity may be non-ionic. In some embodiments, the
hydrophilic component of an amphiphilic entity comprises one or
more ionic groups. In general, such ionic groups are hydrophilic
and can confer hydrophilic nature on the amphiphilic entity.
[0073] In some embodiments, the ionic group may be cationic. In
some embodiments, the cationic group may be an ammonium
(NH.sub.4.sup.+), nitronium (NO.sub.2.sup.+) nitrosyl (NO.sup.+),
hydronium (H.sub.3O.sup.+), mercurous (Hg.sub.2.sup.2+),
phosphonium (PH.sub.4.sup.+), vanadyl (VO.sup.2+), or salt
thereof.
[0074] In some embodiments, the ionic group may be anionic. In some
embodiments, the anionic group may be a fatty acid, arsenide
(As.sup.3-), azide (N.sub.3.sup.-), bromide (Br.sup.-), chloride
(Cl.sup.-), fluoride (F.sup.-), hydride (H.sup.-), iodide
(I.sup.-), nitride (N.sup.3-), oxide (O.sup.2-), phosphide
(P.sup.3-), selenide (Se.sup.2-), sulfide (S.sup.2-), peroxide
(O.sub.2.sup.2-), arsenate (AsO.sub.4.sup.3-), arsenite
(AsO.sub.3.sup.3-), borate (BO.sub.3.sup.3-), perbromate
(BrO.sub.4.sup.-), bromate (BrO.sub.3.sup.-), bromite
(BrO.sub.2.sup.-), hypobromite (BrO.sup.-), carbonate
(CO.sub.3.sup.2-), hydrogen carbonate (HCO.sub.3.sup.-), chlorate
(ClO.sub.3.sup.-), perchlorate (ClO.sub.4.sup.-), chlorite
(ClO.sub.2.sup.-), hypochlorite (ClO.sup.-), chromate
(CrO.sub.4.sup.2-), dichromate (Cr.sub.2O.sub.7.sup.2-),
perfluorate (BrO.sub.4.sup.-), fluorate (BrO.sub.3.sup.-), fluorite
(BrO.sub.2.sup.-), hypofluorite (BrO.sup.-), periodate
(IO.sub.4.sup.-), iodate (IO.sub.3.sup.-), iodite (IO.sub.2.sup.-),
hypoiodite (IO.sup.-), nitrate (NO.sub.3.sup.-), nitrite
(NO.sub.2.sup.-), phosphate (PO.sub.4.sup.3-), hydrogen phosphate
(HPO.sub.4.sup.2-), dihydrogen phosphate (H.sub.2PO.sub.4.sup.-),
phosphite (PO.sub.3.sup.3-), silicate (SiO.sub.3.sup.2-), sulfate
(SO.sub.4.sup.2-), thiosulfate (S.sub.2O.sub.3.sup.2-), hydrogen
sulfate (HSO.sub.4.sup.-), sulfite (SO.sub.3.sup.2-), hydrogen
sulfite (HSO.sub.3.sup.-), sulfonate (--S(.dbd.O).sub.2--O.sup.-),
acetate (C.sub.2H.sub.3O.sub.2.sup.-), formate (HCO.sub.2.sup.-),
oxalate (C.sub.2O.sub.4.sup.-2), hydrogen oxalate
(HC.sub.2O.sub.4.sup.-), citrate (C.sub.6H.sub.5O.sub.7.sup.3-),
succinate (C.sub.4H.sub.4O.sub.4.sup.2-), fumarate
(C.sub.4H.sub.2O.sub.4.sup.2-), malate
(C.sub.4H.sub.5O.sub.5.sup.2-), hydrogen sulfide (HS.sup.-),
telluride (Te.sup.2-), amide (NH.sub.2.sup.-), cyanate (OCN.sup.-),
thiocyanate (SCN.sup.-), cyanide (CN.sup.-), hydroxide (OH.sup.-),
permanganate (MnO.sub.4.sup.-), or salt thereof.
[0075] In some embodiments, the hydrophilic component of an
amphiphilic entity may comprise or consist of a nucleic acid. For
example, the nucleic acid polymer may include DNA, RNA, or
combinations thereof. In some embodiments, the nucleic acid polymer
may be an oligonucleotide and/or polynucleotide. In some
embodiments, the nucleic acid polymer may be an oligonucleotide
and/or modified oligonucleotide; an antisense oligonucleotide
and/or modified antisense oligonucleotide; a cDNA; a genomic DNA;
viral DNA and/or RNA; DNA and/or RNA chimeras; plasmids; cosmids;
gene fragments; an artificial and/or natural chromosome (e.g. a
yeast artificial chromosome) and/or a part thereof; an RNA (e.g. an
mRNA, a tRNA, an rRNA and/or a ribozyme); a peptide nucleic acid
(PNA); a polynucleotide comprising synthetic analogues of nucleic
acids, which may be modified or unmodified; various structural
forms of DNA including single-stranded DNA, double-stranded DNA,
supercoiled DNA and/or triple-helical DNA; Z-DNA; and/or
combinations thereof.
[0076] In some embodiments, the hydrophilic component of an
amphiphilic entity may comprise or consist of a carbohydrate. In
some embodiments, the carbohydrate may be a polysaccharide composed
of simple sugars (or their derivatives) connected by glycosidic
bonds, as known in the art. Such sugars may include, but are not
limited to, glucose, fructose, galactose, ribose, lactose, sucrose,
maltose, trehalose, cellbiose, mannose, xylose, arabinose,
glucoronic acid, galactoronic acid, mannuronic acid, glucosamine,
galatosamine, and neuramic acid. In some embodiments, the polymer
may be a hydrophilic carbohydrate, including aminated,
carboxylated, and sulfated polysaccharides. In some embodiments,
the hydrophilic carbohydrate may be one or more of pullulan,
cellulose, microcrystalline cellulose, hydroxypropyl
methylcellulose, hydroxycellulose, methylcellulose, dextran,
cyclodextran, glycogen, starch, hydroxyethylstarch, carageenan,
glycon, amylose, chitosan, N,O-carboxylmethylchitosan, algin and
alginic acid, starch, chitin, heparin, konjac, glucommannan,
pustulan, heparin, hyaluronic acid, curdlan, and xanthan. In some
embodiments, hydrophilic polysaccharides can be modified to become
hydrophobic by introducing a large number of side-chain hydrophobic
groups. In some embodiments, a hydrophobic carbohydrate may include
cellulose acetate, pullulan acetate, konjac acetate, amylose
acetate, and dextran acetate.
[0077] In some embodiments, the hydrophilic component of an
amphiphilic entity may comprise or consist of a gum including, but
not limited to, xanthan gum, alginic acid, caraya gum, sodium
alginate, and/or locust bean gum.
[0078] In some embodiments, a component of an amphiphilic entity
may comprise or consist of a protein. In some embodiments, a
protein is a hydrophilic component of an amphiphilic entity. In
other embodiments, a protein is a hydrophobic component of an
amphiphilic entity. Exemplary proteins that may be used in
accordance with the present invention include, but are not limited
to, albumin, collagen, or a poly(amino acid) (e.g. polylysine).
[0079] In some embodiments, the hydrophobic component of an
amphiphilic entity may comprise or consist of one or more fatty
acid groups or salts thereof. In general, such groups are typically
hydrophobic and can confer hydrophobic nature onto the amphiphilic
entity. In some embodiments, the fatty acid group may comprise
digestible, long chain (e.g., C.sub.8-C.sub.50), substituted or
unsubstituted hydrocarbons. In some embodiments, the fatty acid
group may be a C.sub.10-C.sub.20 fatty acid or salt thereof. In
some embodiments, the fatty acid group may be a C.sub.15-C.sub.20
fatty acid or salt thereof. In some embodiments, the fatty acid
group may be a C.sub.15-C.sub.25 fatty acid or salt thereof. In
some embodiments, the fatty acid group may be unsaturated. In some
embodiments, the fatty acid group may be monounsaturated. In some
embodiments, the fatty acid group may be polyunsaturated. In some
embodiments, a double bond of an unsaturated fatty acid group may
be in the cis conformation. In some embodiments, a double bond of
an unsaturated fatty acid may be in the trans conformation.
[0080] In some embodiments, the fatty acid group may be one or more
of butyric, caproic, caprylic, capric, lauric, myristic, palmitic,
stearic, arachidic, behenic, or lignoceric acid. In some
embodiments, the fatty acid group may be one or more of
palmitoleic, oleic, vaccenic, linoleic, alpha-linoleic,
gamma-linoleic, arachidonic, gadoleic, arachidonic,
eicosapentaenoic, docosahexaenoic, or erucic acid.
[0081] In some embodiments, the hydrophobic component of an
amphiphilic entity may comprise or consist of one or more
biocompatible and/or biodegradable synthetic polymers, including,
for example, polycarbonates (e.g. poly(1,3-dioxan-2one)),
polyanhydrides (e.g. poly(sebacic anhydride)), polyhydroxyacids
(e.g. poly(.beta.-hydroxyalkanoate)), polypropylfumarates,
polycaprolactones, polyamides (e.g. polycaprolactam), polyacetals,
polyethers, polyesters (e.g. polylactide and polyglycolide),
biodegradable polycyanoacrylates, polyvinyl alcohols, and
biodegradable polyurethanes. For example, the amphiphilic entity
may comprise one or more of the following biodegradable polymers:
poly(lactic acid), poly(glycolic acid), poly(caprolactone),
poly(lactide-co-glycolide), poly(lactide-co-caprolactone),
poly(glycolide-co-caprolactone), and
poly(DL-lactide-co-glycolide).
[0082] In some embodiments, the hydrophobic component of an
amphiphilic entity may comprise or consist of one or more acrylic
polymers. In certain embodiments, acrylic polymers include, for
example, acrylic acid and methacrylic acid copolymers, methyl
methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl
methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic
acid), poly(methacrylic acid), methacrylic acid alkylamide
copolymer, poly(methyl methacrylate), poly(methacrylic acid
anhydride), methyl methacrylate, polymethacrylate, poly(methyl
methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate
copolymer, glycidyl methacrylate copolymers, and combinations
comprising one or more of the foregoing polymers. The acrylic
polymer may comprise fully-polymerized copolymers of acrylic and
methacrylic acid esters with a low content of quaternary ammonium
groups.
[0083] In some embodiments, the hydrophobic component of an
amphiphilic entity may comprise or consist of a polyester.
Exemplary such polyesters include, for example, polyalkylene
glycols, poly(glycolide-co-lactide), PEGylated
poly(lactic-co-glycolic acid), poly(lactic acid), PEGylated
poly(lactic acid), poly(glycolic acid), PEGylated poly(glycolic
acid), co-polymers of polylactic and polyglycolic acid, and
derivatives thereof. In some embodiments, polyesters include, for
example, polyanhydrides, poly(ortho ester) PEGylated poly(ortho
ester), poly(caprolactone), PEGylated poly(caprolactone),
polylysine, PEGylated polylysine, poly(ethylene imine), PEGylated
poly(ethylene imine), and derivatives thereof. In some embodiments,
polyesters may include, for example, polycaprolactone,
poly(L-lactide-co-L-lysine), poly(serine ester),
poly(4-hydroxy-L-proline ester),
poly[.alpha.-(4-aminobutyl)-L-glycolic acid], and derivatives
thereof.
[0084] Suitable surfactants or emulsifying agents include, but are
not limited to, phosphoglycerides; phosphatidylcholines;
dipalmitoyl phosphatidylcholine (DPPC); dioleylphosphatidyl
ethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA);
dioleoylphosphatidylcholine; cholesterol; cholesterol ester;
diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol
(DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol
(PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid,
such as palmitic acid or oleic acid; fatty acids; fatty acid
amides; sorbitan trioleate (Span 85) glycocholate; sorbitan
monolaurate (Span 20); polysorbate 20 (Tween-20); polysorbate 60
(Tween-60); polysorbate 65 (Tween-65); polysorbate 80 (Tween-80);
polysorbate 85 (Tween-85); polyoxyethylene monostearate; surfactin;
a poloxomer; a sorbitan fatty acid ester such as sorbitan
trioleate; lecithin; lysolecithin; phosphatidylserine;
phosphatidylinositol; sphingomyelin; phosphatidylethanolamine
(cephalin); cardiolipin; phosphatidic acid; cerebrosides;
dicetylphosphate; dipalmitoylphosphatidylglycerol; stearylamine;
dodecylamine; hexadecyl-amine; acetyl palmitate; glycerol
ricinoleate; hexadecyl sterate; tyloxapol; poly(ethylene
glycol)5000-phosphatidylethanolamine; poly(ethylene
glycol)-400-monostearate; and phospholipids. The surfactant
component may be a mixture of different surfactants. These
surfactants may be extracted and purified from a natural source or
may be prepared synthetically in a laboratory. In a preferred
embodiment, the surfactants are commercially available.
[0085] In certain embodiments of the invention, relative amounts of
components utilized to prepare inventive nanoparticle compositions
are selected or adjusted to generate nanoparticles having desired
characteristics. In some embodiments, the oil and surfactant are
utilized at a ratio ranging between 0.25-10. In some embodiments,
the ratio of oil to surfactant is approximately 0.25:1,
approximately 0.5:1, approximately 1:1, approximately 2:1,
approximately 3:1, approximately 4:1, approximately 5:1,
approximately 6:1, approximately 7:1, approximately 8:1,
approximately 9:1, or approximately 10:1. In some embodiments, the
ratio of surfactant to oil is approximately 0.5:1, approximately
1:1, approximately 2:1, approximately 3:1, approximately 4:1,
approximately 5:1, approximately 6:1, approximately 7:1,
approximately 8:1, approximately 9:1, or approximately 10:1. In
some embodiments, the oil and surfactant are utilized at a ratio
ranging between 0.25-2. In some embodiments, the ratio of oil to
surfactant is approximately 0.25:1, approximately 0.5:1,
approximately 1:1, or approximately 2:1. In some embodiments, the
ratio of surfactant to oil is approximately 0.5:1, approximately
1:1, or approximately 2:1. In certain embodiments, the ratio of oil
to surfactant is approximately 1:1.
[0086] In some embodiments, the percent of oil in the composition
from which nanoparticles are prepared (e.g., in the premix) ranges
between 0% to 30%. In some embodiments the percent of oil in the
composition from which nanoparticles are prepared (e.g., in the
premix) is approximately 1%, approximately 2%, approximately 3%,
approximately 4%, approximately 5%, approximately 6%, approximately
7%, approximately 9%, approximately 10%, approximately 11%,
approximately 12%, approximately 13%, approximately 14%,
approximately 15%, approximately 16%, approximately 17%,
approximately 18%, approximately 19%, approximately 20%,
approximately 21%, approximately 22%, approximately 23%,
approximately 24%, approximately 25%, approximately 26%,
approximately 27%, approximately 28%, approximately 29%, or
approximately 30%. In some embodiments the percent of oil is
approximately 8%. In some embodiments the percent of oil is
approximately 5%.
[0087] In some embodiments, where one or more amphiphilic entities
is/are utilized, the percent of amphiphilic entity in the
composition from which nanoparticles are prepared (e.g., in the
premix) can range from 40% to 99%, from 50% to 99%, from 60% to
99%, from 70% to 99%, from 80% to 99%, from 80% to 90%, or from 90%
to 99%. In some embodiments the percent of amphiphilic entity in
the composition from which nanoparticles are prepared (e.g., in the
premix) is approximately 75%, approximately 76%, approximately 77%,
approximately 78%, approximately 79%, approximately 80%,
approximately 81%, approximately 82%, approximately 83%,
approximately 84%, approximately 85%, approximately 86%,
approximately 87%, approximately 88%, approximately 89%,
approximately 90%, approximately 91%, approximately 92%,
approximately 93%, approximately 94%, approximately 95%,
approximately 96%, approximately 97%, approximately 98%, or
approximately 99%.
[0088] The percent of substances with surfactant activity in the
premix can range from 0% to 99%, from 10% to 99%, from 25% to 99%,
from 50% to 99%, or from 75% to 99%. In some embodiments, the
percent of substances with surfactant activity in the premix can
range from 0% to 75%, from 0% to 50%, from 0% to 25%, or from 0% to
10%. In some embodiments, the percent of surfactant in the
composition from which nanoparticles are prepared (e.g., in the
premix) ranges between 0%-30%. In some embodiments the percent of
surfactant in is approximately 1%, approximately 2%, approximately
3%, approximately 4%, approximately 5%, approximately 6%,
approximately 7%, approximately 9%, approximately 10%,
approximately 11%, approximately 12%, approximately 13%,
approximately 14%, approximately 15%, approximately 16%,
approximately 17%, approximately 18%, approximately 19%,
approximately 20%, approximately 21%, approximately 22%,
approximately 23%, approximately 24%, approximately 25%,
approximately 26%, approximately 27%, approximately 28%,
approximately 29%, or approximately 30%. In some embodiments the
percent of surfactant is approximately 8%. In some embodiments the
percent of surfactant is approximately 5%.
[0089] In some embodiments, the nanoparticle composition does not
contain more than one oil. In some embodiments, the nanoparticle
composition may comprise two or more oils. In some embodiments, the
nanoparticle composition does not contain more than one surfactant.
In some embodiments, the nanoparticle composition may comprise two
or more surfactants. In some embodiments, the nanoparticle
composition is completely free or substantially free of toxic
components.
[0090] In some embodiments, the nanoparticle composition consists
essentially of water, an oil, a surfactant, and at least one
biologically active agent (e.g., and unmodified peptide). In some
embodiments, the nanoparticle composition consists essentially of
water, an oil, a surfactant, at least one biologically active
agent, and at least one substance used to produce and/or preserve
the nanoparticle composition.
[0091] In some embodiments, the nanoparticle composition consists
of water, an oil, a surfactant, and an unmodified peptide. In some
embodiments, the nanoparticle composition consists of water, an
oil, a surfactant, an unmodified peptide, and at least one
substance used to produce and/or preserve the nanoparticle.
Unmodified Peptides
[0092] Any of a variety of peptides may be incorporated in
nanoparticle compositions according to the present invention. In
most embodiments, it a peptide is less than about 100 amino acids
in length; in some embodiments, a peptide is less than about 90,
about 80, about 70, about 65, about 60, about 55, about 50, about
45, about 40, about 35, about 30, about 25, about 20, about 15,
about 13, about 12, about 10, about 9, about 8, about 7, about 6,
or about 5 amino acids in length. In some specific embodiments, the
peptide is a penta peptide. In some embodiments, a peptide to be
incorporated in a nanoparticle compositions is comprised solely of
naturally occurring amino acids. In some embodiments, a peptide
comprises one or more non-naturally occurring amino acid.
[0093] Unmodified short peptides for use in accordance with the
present invention, generally, are ones that have biological
activity in the skin (including epidermis and dermis),
sub-cutaneous tissue (including adipose tissue) and/or contiguous
muscles. Such peptides include, but are not limited to, peptides to
promote extra-cellular matrix production (e.g., KTTKS, SEQ ID NO.:
1; EYKTTKSSRL, SEQ ID NO.: 2; VIEYKTTK, SEQ ID NO.: 3; KTTK, SEQ ID
NO.: 4; GKTVIEYKTTKS, SEQ ID NO.: 5; GKTVIEYKTTKSSRL, SEQ ID NO.:
6; WGKTVIEYKTTKSSRLPIID, SEQ ID NO.: 7; CTSHTGAWGKTVIEYKTTKS, SEQ
ID NO.: 8; TTKS, SEQ ID NO.: 9), peptides that may decrease
wrinkles (e.g., EEMQRR, SEQ ID NO.: 10), peptides to improve wound
healing (e.g., gastrin-releasing peptide, VGVAPG, SEQ ID NO.: 11;
YYRADA, SEQ ID NO.: 12; GHK, SEQ ID NO.: 13, interferon, interferon
inducer), and peptides (e.g., P144; TSLDASIIWAMMQN, SEQ ID NO.: 14)
to treat excessive accumulation of extra-cellular matrix that are
result in conditions such as hypertrophic scarring, keloids, and
localized or systemic sclerosis (scleroderma) (Katayama, et al.;
supra, Lupo, supra; Robinson et al., supra; Bhartiya et al., 1992,
J. Cell. Physiol., 150:312; and Santiago et al., 2005, J.
Investigative Dermatology, 125:450; all of which are incorporated
herein by reference). See Table 1 below for definitions of peptide
abbreviations.
TABLE-US-00001 TABLE 1 Peptide Abbreviations Trivial name.sup.a
Symbols.sup.b Systematic Name.sup.c Formula Alanine Ala A
2-Aminopropanoic acid CH.sub.3--CH(NH.sub.2)--COOH Arginine Arg R
2-Amino-5-
H.sub.2N--C(.dbd.NH)--NH--[CH.sub.2].sub.3--CH(NH.sub.2)--
guanidinopentanoic COOH acid Asparagine Asn.sup.d N.sup.d
2-Amino-3- H.sub.2N--CO--CH.sub.2--CH(NH.sub.2)--COOH
carbamoylpropanoic acid Aspartic acid Asp.sup.d D.sup.d
2-Aminobutanedioic HOOC--CH.sub.2--CH(NH.sub.2)--COOH acid Cysteine
Cys C 2-Amino-3- HS--CH.sub.2--CH(NH.sub.2)--COOH mercaptopropanoic
acid Glutamine Gln.sup.d Q.sup.d 2-Amino-4-
H.sub.2N--CO--[CH.sub.2].sub.2--CH(NH.sub.2)--COOH
carbamoylbutanoic acid Glutamic acid Glu.sup.d E.sup.d
2-Aminopentanedioic acid HOOC--[CH.sub.2].sub.2--CH(NH.sub.2)--COOH
Glycine Gly G Aminoethanoic acid CH.sub.2(NH.sub.2)--COOH Histidine
His H 2-Amino-3-(1H-imidazol-4- yl)- propanoic acid ##STR00001##
Isoleucine Ile I 2-Amino-3-methylpentanoic
C.sub.2H.sub.5--CH(CH.sub.3)--CH(NH.sub.2)--COOH acid.sup.c Leucine
Leu L 2-Amino-4-methylpentanoic
(CH.sub.3).sub.2CH--CH.sub.2--CH(NH.sub.2)--COOH acid Lysine Lys K
2,6-Diaminohexanoic acid
H.sub.2N--[CH.sub.2].sub.4--CH(NH.sub.2)--COOH Methionine Met M
2-Amino-4- CH.sub.3--S--[CH.sub.2].sub.2--CH(NH.sub.2)--COOH
(methylthio)butanoic acid Phenylalanine Phe F
2-Amino-3-phenylpropanoic
C.sub.6H.sub.5--CH.sub.2--CH(NH.sub.2)--COOH acid Proline Pro P
Pyrrolidine-2-carboxylic acid ##STR00002## Serine Ser S 2-Amino-3-
HO--CH.sub.2--CH(NH.sub.2)--COOH hydroxypropanoic acid Threonine
Thr T 2-Amino-3-hydroxybutanoic
CH.sub.3--CH(OH)--CH(NH.sub.2)--COOH acid.sup.c Tryptophan Trp W
2-Amino-3-(1H-indol-3-yl)- propanoic acid ##STR00003## Tyrosine Tyr
Y 2-Amino-3-(4- hydroxyphenyl)- propanoic acid ##STR00004## Valine
Val V 2-Amino-3-methylbutanoic
(CH.sub.3).sub.2CH--CH(NH.sub.2)--COOH
Other Components
[0094] As indicated herein, inventive nanoparticle compositions may
contain or be combined with one or more other components. Certain
exemplary such other components are discussed here.
Biologically-Active Agents
[0095] Any biologically active agents, including, for example,
therapeutic, diagnostic, prophylactic, nutritional, cosmetic,
and/or dermatological agents, may be delivered according to the
present invention. Such biologically active agents may be small
molecules, organometallic compounds, nucleic acids, proteins
(including multimeric proteins, protein complexes, etc.), peptides,
lipids, carbohydrates, herbs, hormones, metals, radioactive
elements and compounds, drugs, vaccines, immunological agents,
etc., and/or combinations thereof. Such biologically agents may be
encapsulated within, adsorbed to the surface of, present at the
interface of and/or present within a micellar membrane of inventive
nanoparticles.
[0096] In some embodiments, the percent of biologically active
agent in the composition used to prepare inventive nanoparticles
(e.g., in the premix) and/or in the nanoparticles ranges from
0.1%-25%. In some embodiments, the percentage of biologically
active agent ranges from 0.1%-20%, from 0.1%-15%, from 0.1%-10%,
from 0.1%-5%, or from 0.1%-1%. In some embodiments, the percentage
of biologically active agent ranges from 1%-20%, from 5%-20%, from
10%-20%, from 15%-20%, or from 15%-25%. In some embodiments, the
percentage of biologically active agent is less than 0.1%. In some
embodiments, the percentage of biologically active agent is greater
than 25%. In some embodiments, the percentage of biologically
active agent is approximately 0.1%, approximately 0.5%,
approximately 1%, approximately 2%, approximately 3%, approximately
4%, approximately 5%, approximately 6%, approximately 7%,
approximately 8%, approximately 9%, approximately 10%,
approximately 11%, approximately 12%, approximately 13%,
approximately 14%, approximately 15%, approximately 16%,
approximately 17%, approximately 18%, approximately 19%,
approximately 20%, approximately 21%, approximately 22%,
approximately 23%, approximately 24%, approximately 25%, or
greater.
[0097] Relevant biologically active agents can be produced or
obtained according to any available method or approach.
Biologically active agents may contain, or be modified to contain,
one or more moieties intended to facilitate their use or delivery
in conjunction with inventive nanoparticles. Such modification
should not interfere with the biological activity of the agent. In
some embodiments, the modification can optionally be removed in
vivo. For example, biologically active agents may be detectably
labeled and/or may be provided in a "pro" form that is converted or
modified after delivery into an active form.
[0098] In some embodiments, the biologically active agent is a
small molecule and/or organic compound with pharmaceutical
activity. In some embodiments, the biologically active agent is a
clinically-used drug. In some embodiments, the drug is an
antibiotic, anti-viral agent, anesthetic, anticoagulant,
anti-cancer agent, inhibitor of an enzyme, steroidal agent,
anti-inflammatory agent, anti-neoplastic agent, antigen, vaccine,
antibody, decongestant, antihypertensive, sedative, birth control
agent, progestational agent, anti-cholinergic, analgesic,
anti-depressant, anti-psychotic, .beta.-adrenergic blocking agent,
diuretic, cardiovascular active agent, vasoactive agent,
non-steroidal anti-inflammatory agent, etc. Of particular interest
are biologically active agents suitable for transdermal
administration.
[0099] The biologically active agents delivered may be a mixture of
pharmaceutically active agents. For example, a local anesthetic may
be delivered in combination with an anti-inflammatory agent such as
a steroid. Local anesthetics may also be administered with
vasoactive agents such as epinephrine. To give but another example,
an antibiotic may be combined with an inhibitor of the enzyme
commonly produced by bacteria to inactivate the antibiotic (e.g.,
penicillin and clavulanic acid).
[0100] In some embodiments, the biologically active agent is a
diagnostic agent. In some embodiments, diagnostic agents include
gases; commercially available imaging agents used in positron
emissions tomography (PET), computer assisted tomography (CAT),
single photon emission computerized tomography, x-ray, fluoroscopy,
and magnetic resonance imaging (MRI); and contrast agents. Examples
of suitable materials for use as contrast agents in MRI include
gadolinium chelates, as well as iron, magnesium, manganese, copper,
and chromium. Examples of materials useful for CAT and x-ray
imaging include iodine-based materials.
[0101] In some embodiments, the biologically active agent is a
prophylactic agent. In some embodiments, prophylactic agents
include vaccines. Vaccines may comprise isolated proteins or
peptides, inactivated organisms and viruses, dead organisms and
virus, genetically altered organisms or viruses, and cell extracts.
Prophylactic agents may be combined with interleukins, interferon,
cytokines, and adjuvants such as cholera toxin, alum, Freund's
adjuvant, etc. Prophylactic agents may include antigens of such
bacterial organisms as Streptococccus pnuemoniae, Haemophilus
influenzae, Staphylococcus aureus, Streptococcus pyrogenes,
Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus
anthracis, Clostridium tetani, Clostridium botulinum, Clostridium
perfringens, Neisseria meningitidis, Neisseria gonorrhoeae,
Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi,
Haemophilus parainfluenzae, Bordetella pertussis, Francisella
tularensis, Yersinia pestis, Vibrio cholerae, Legionella
pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae,
Treponema pallidum, Leptospirosis interrogans, Borrelia
burgdorferi, Camphylobacter jejuni, and the like; antigens of such
viruses as smallpox, influenza A and B, respiratory syncytial
virus, parainfluenza, measles, HIV, varicella-zoster, herpes
simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus,
rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies,
rubella, coxsackieviruses, equine encephalitis, Japanese
encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C,
D, and E virus, and the like; antigens of fungal, protozoan, and
parasitic organisms such as Cryptococcus neoformans, Histoplasma
capsulatum, Candida albicans, Candida tropicalis, Nocardia
asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma
pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium
falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma
gondii, Trichomonas vaginalis, Schistosoma mansoni, and the like.
These antigens may be in the form of whole killed organisms,
peptides, proteins, glycoproteins, carbohydrates, or combinations
thereof.
[0102] In some embodiments, the biologically active agent may be a
protein. As used herein, the terms "protein" and "peptide" can be
used interchangeably. In certain embodiments, peptides range from
about 5 to about 40, about 10 to about 35, about 15 to about 30, or
about 20 to about 25 amino acids in size. Peptides from panels of
peptides comprising random sequences and/or sequences which have
been varied consistently to provide a maximally diverse panel of
peptides may be used.
[0103] In some embodiments, the biologically active agent may be an
antibody. In some embodiments, antibodies may include, but are not
limited to, polyclonal, monoclonal, chimeric (i.e. "humanized"),
single chain (recombinant) antibodies. In some embodiments,
antibodies may have reduced effector functions and/or bispecific
molecules. In some embodiments, antibodies may include Fab
fragments and/or fragments produced by a Fab expression
library.
[0104] In some embodiments, the biologically active agent may be a
nucleic acid. In some embodiments, the oligonucleotides comprise
DNA, RNA, chimeric mixtures, derivatives, characteristic portions,
and/or modified versions thereof. The oligonucleotides of the
present invention may be single-stranded and/or double-stranded.
The oligonucleotide may be modified at the base moiety, sugar
moiety, and/or phosphate backbone, for example, to improve
stability of the molecule, hybridization, etc.
[0105] In specific embodiments, a nucleic acid comprises an
antisense molecule that binds to a translational start site,
transcriptional start site, and/or splice junctions. Antisense
oligonucleotides will bind to a target mRNA and/or prevent
translation. Alternatively or additionally, the antisense
oligonucleotide may bind to DNA of a target gene, such as, for
example, a regulatory element.
[0106] In some embodiments, a nucleic acid comprises a ribozyme
designed to catalytically cleave target mRNA transcripts may be
used to prevent translation of a target mRNA and/or expression of a
target (see, e.g., PCT publication WO 90/11364; and Sarver et al.,
1990, Science 247:1222; both of which are incorporated herein by
reference).
[0107] Alternatively or additionally, endogenous target gene
expression may be reduced by targeting deoxyribonucleotide
sequences complementary to the regulatory region of the target gene
(i.e., the target gene's promoter and/or enhancers) to form triple
helical structures that prevent transcription of the target gene in
target muscle cells in the body (see generally, Helene, 1991,
Anticancer Drug Des. 6:569; Helene et al., 1992, Ann, N.Y. Acad.
Sci. 660:27; and Maher, 1992, Bioassays 14:807; all of which are
incorporated herein by reference).
[0108] In some embodiments, the biologically active agent is a
nutraceutical agent. In some embodiments, the nutraceutical agent
provides basic nutritional value. In some embodiments, the
nutraceutical agent provides health or medical benefits. In some
embodiments, the nutraceutical agent is a dietary supplement.
[0109] In some embodiments, the nutraceutical agent is a vitamin.
In some embodiments, the vitamin is one or more of vitamin A
(retinoids), vitamin B1 (thiamine), vitamin B2 (riboflavin),
vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6
(pyroxidone), vitamin B7 (biotin), vitamin B9 (folic acid), vitamin
B12 (cyanocobalamin), vitamin C (ascorbic acid), vitamin D, vitamin
E, or vitamin K.
[0110] In some embodiments, the nutraceutical agent is a mineral.
In some embodiments, the mineral is one or more of bismuth, boron,
calcium, chlorine, chromium, cobalt, copper, fluorine, iodine,
iron, magnesium, manganese, molybdenum, nickel, phosphorus,
potassium, rubidium, selenium, silicon, sodium, strontium, sulfur,
tellurium, titanium, tungsten, vanadium, or zinc.
[0111] In some embodiments, the nutraceutical agent is an essential
amino acid. In some embodiments, the amino acid is one or more of
arginine, glutamine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, threonine, tryptophan, or valine.
[0112] In some embodiments, nutraceutical agents may include fatty
acids and/or omega-3 fatty acids (e.g. DHA or ARA), fruit and
vegetable extracts, lutein, phosphatidylserine, lipoid acid,
melatonin, glucosamine, chondroitin, aloe vera, guggul, green tea,
lycopene, whole foods, food additives, herbs, phytonutrients,
antioxidants, flavonoid constituents of fruits, evening primrose
oil, flaxseeds, fish and marine animal oils (e.g. cod liver oil),
and probiotics. In some embodiments, nutraceutical agents may
include bio-engineered foods genetically-engineered to have a
desired property (also known as "pharmafoods").
[0113] Exemplary nutraceutical agents and dietary supplements are
disclosed, for example, in Roberts et al., (Nutriceuticals: The
Complete Encyclopedia of Supplements, Herbs, Vitamins, and Healing
Foods, American Nutriceutical Association, 2001; incorporated
herein by reference). Nutraceutical agents and dietary supplements
are also disclosed in Physicians' Desk Reference for Nutritional
Supplements, 1st Ed., 2001, and Physicians' Desk Reference for
Herbal Medicines, 1st Ed., 2001 (both of which are incorporated
herein by reference).
[0114] In some embodiments, inventive nanoparticles loaded with
nutraceutical agents can be incorporated into food substances. For
example, the nutraceutical-loaded nanoparticles can be dissolved
into liquids, such as beverages.
[0115] In some embodiments, the biologically active agent is a
cosmetic and/or dermatological agent. In some embodiments, the
cosmetic and/or dermatological agent may include vitamins and their
derivatives (e.g. vitamin E and its esters, vitamin C and its
esters, vitamins B, vitamin A alcohol or retinol and its esters),
provitamins (e.g. panthenol, niacinamide or ergocalciferol),
antioxidants, phenolic compounds (e.g. benzoyl peroxide), essential
oils, humectants, sunscreen agents, moisturizing agents, proteins,
ceramides, and pseudoceramides.
[0116] In some embodiments, the biologically active agent may be
one or more botulinum toxin peptides or protein complexes. In some
embodiments, the botulinum toxin may be one or more of botulinum
toxin serotypes A, B, C.sub.1, C.sub.2, D, E, F, or G. In some
embodiments, the botulinum toxin may be an isolated and/or purified
botulinum toxin. In some embodiments, the botulinum toxin may be a
partially-isolated and/or partially-purified botulinum toxin. In
some embodiments, the botulinum toxin may be a native botulinum
complex. In some embodiments, the botulinum toxin may be associated
with non-toxin proteins. In some embodiments, the botulinum toxin
may be a recombinantly-made botulinum toxin.
[0117] Those skilled in the art will recognize that this is an
exemplary, not comprehensive, list of biologically active agents.
Any biologically active agent may be encapsulated within or bound
to the surface of nanoparticles.
[0118] Release Retarding Agents
[0119] In some embodiments of the invention, particularly those
containing one or more biologically active agents (e.g., unmodified
peptides), inventive nanoparticle compositions further include or
are formulated with one or more release-retarding ingredients to
allow for controlled release of the agent. Any release-retarding
ingredient known in the art is suitable for use in making the
inventive nanoparticles. In some embodiments, release-retarding
ingredients are hydrophilic and/or hydrophobic polymers.
Release-retarding ingredients include, for example celluloses or
derivatives thereof, acrylic polymers, ester polymers,
vinyl-pyrrolidone-based polymers, gums, other natural polymers,
and/or combinations of these.
[0120] In some embodiments, the release-retarding ingredient is
cellulose or a derivative thereof. In certain embodiments, the
cellulose or derivative thereof comprises one or more of
hydroxypropyl methylcellulose, methylcellulose,
carboxymethylcellulose, sodium carboxymethylcellulose,
hydroxypropyl ethylcellulose, hydroxyethylcellulose, and
hydroxypropyl cellulose. In certain embodiments, the cellulose or
derivative thereof is methylcellulose or a derivative thereof. In
certain embodiments, the cellulose or derivative thereof is
hydroxypropyl methylcellulose (HPMC). Those skilled in the art will
appreciate that other cellulosic polymers, including other alkyl
cellulosic polymers, can be utilized.
[0121] In some embodiments, the release-retarding ingredient is an
acrylic polymer. In certain embodiments, acrylic polymers include,
for example, acrylic acid and methacrylic acid copolymers, methyl
methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl
methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic
acid), poly(methacrylic acid), methacrylic acid alkylamide
copolymer, poly(methyl methacrylate), poly(methacrylic acid
anhydride), methyl methacrylate, polymethacrylate, poly(methyl
methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate
copolymer, glycidyl methacrylate copolymers, and combinations
comprising one or more of the foregoing polymers. The acrylic
polymer may comprise fully polymerized copolymers of acrylic and
methacrylic acid esters with a low content of quaternary ammonium
groups.
[0122] In some embodiments, the release-retarding ingredient is a
polyester. In some embodiments, polyesters include polyalkylene
glycols, poly(glycolide-co-lactide), PEGylated
poly(lactic-co-glycolic acid), poly(lactic acid), PEGylated
poly(lactic acid), poly(glycolic acid), PEGylated poly(glycolic
acid), co-polymers of polylactic and polyglycolic acid, and
derivatives thereof. In some embodiments, polyesters include, for
example, polyanhydrides, poly(ortho ester) PEGylated poly(ortho
ester), poly(caprolactone), PEGylated poly(caprolactone),
polylysine, PEGylated polylysine, poly(ethylene imine), PEGylated
poly(ethylene imine), and derivatives thereof. In some embodiments,
polyesters include, for example, polycaprolactone,
poly(L-lactide-co-L-lysine), poly(serine ester),
poly(4-hydroxy-L-proline ester),
poly[.alpha.-(4-aminobutyl)-L-glycolic acid], and derivatives
thereof.
[0123] In some embodiments, the release-retarding ingredient is a
cross-linked polymer of poly(vinyl-pyrrolidone). In some
embodiments, the polymer is crosspovidone. In some embodiments, the
polymer is un-cross-linked poly(vinyl-pyrrolidone). In some
embodiments, the polymer is povidone.
[0124] In some embodiments, the release-retarding ingredient may be
a natural polymer. In some embodiments, the natural polymer is a
gum, including, for example, xanthan gum, alginic acid, caraya gum,
sodium alginate, and/or locust bean gum. In some embodiments, the
natural polymer may be a protein (e.g. albumin), lipid, nucleic
acid, or carbohydrateiments, the release-retarding ingredient is a
polyester. In some embodiments, polyesters include polyalkylene
glycols, poly(glycolide-co-lactide), PEGylated
poly(lactic-co-glycolic acid), poly(lactic acid), PEGylated
poly(lactic acid), poly(glycolic acid), PEGylated poly(glycolic
acid), co-polymers of polylactic and polyglycolic acid, and
derivatives thereof. In some embodiments, polyesters include, for
example, polyanhydrides, poly(ortho ester) PEGylated poly(ortho
ester), poly(caprolactone), PEGylated poly(caprolactone),
polylysine, PEGylated polylysine, poly(ethylene imine), PEGylated
poly(ethylene imine), and derivatives thereof. In some embodiments,
polyesters include, for example, polycaprolactone,
poly(L-lactide-co-L-lysine), poly(serine ester),
poly(4-hydroxy-L-proline ester),
poly[.alpha.-(4-aminobutyl)-L-glycolic acid], and derivatives
thereof.
[0125] In some embodiments, the release-retarding ingredient is a
cross-linked polymer of poly(vinyl-pyrrolidone). In some
embodiments, the polymer is crosspovidone. In some embodiments, the
polymer is un-cross-linked poly(vinyl-pyrrolidone). In some
embodiments, the polymer is povidone. In some embodiments, the
release-retarding ingredient may be a natural polymer. In some
embodiments, the natural polymer is a gum, including, for example,
xanthan gum, alginic acid, caraya gum, sodium alginate, and/or
locust bean gum. In some embodiments, the natural polymer may be a
protein (e.g. albumin), lipid, nucleic acid, or carbohydrate
[0126] Formulating Agents
[0127] Inventive nanoparticle compositions may be formulated for
administration to a subject. In certain embodiments, inventive
nanoparticle compositions are formulated for application to the
skin, to achieve transdermal delivery to the subject. For example,
inventive nanoparticle compositions may be formulated in cosmetic
or other preparations intended to be topically applies.
[0128] Human skin comprises the dermis and the epidermis. The
epidermis has several layers of tissue, namely, stratum corneum,
stratum lucidum, stratum granulosum, stratum spinosum, and stratum
basale (identified in order from the outer surface of the skin
inward). The stratum corneum presents the most significant hurdle
in transdermal delivery of medications generally, and presumably of
unmodified peptides in particular. The stratum corneum is typically
about 10-15 m thick, and it consists of flattened, keratised cells
(corneocytes) arranged in several layers. The intercellular space
between the corneocytes is filled with lipidic structures, and may
play an important role in the permeation of substances through skin
(Bauerova et al., 2001, European Journal of Drug Metabolism and
Pharmacokinetics, 26:85; incorporated herein by reference).
[0129] The rest of the epidermis below the stratum corneum is
approximately 150 .mu.m thick. The dermis is about 1 mm-2 mm thick
and is located below the epidermis. The dermis is innervated by
various capillaries as well as neuronal processes.
[0130] Traditionally, attempts at transdermal administration of
medication have been focused in increasing the permeability of the
stratum corneum. Some attempts have included using chemical
enhancing agents that increase the permeability of molecules
through the skin. Some attempts have included using mechanical
apparatus to bypass or ablate portions of the stratum corneum. In
addition, attempts have included use of ultrasound or iontophoresis
to facilitate the permeation of pharmaceuticals through the skin.
In most cases, the goal has been to a pharmaceutical agent,
typically a small molecule, through the skin, typically so that an
agent may pass to the capillary bed in the dermis where the agent
may be systemically incorporated into the subject to achieve a
therapeutic effect.
[0131] The present invention provides, among other things, methods
of administering unmodified peptides transdermally that do not
require use of abrasive or other disrupting agents (whether
chemical, mechanical, electrical, magnetic, etc.). Rather, the
present inventors have surprisingly found that botulinum toxin
incorporated into inventive nanoparticle compositions is
effectively delivered transdermally without further steps to
permeabilize or disrupt the stratum corneum. Use of such agents or
steps with inventive botulinum nanoparticle compositions is not
necessarily precluded in all embodiments of the present invention,
but also is not required.
[0132] The present invention therefore provides methods of
administering unmodified peptides through the topical application
of an inventive nanoparticle composition. In some embodiments, the
inventive nanoparticle composition is applied directly to the skin
and for absorption through the epidermal layers. In some
embodiments, the nanoparticle composition can penetrate the top
layer of the skin, including the stratum corneum, dermal pores,
and/or dermal glands, without the use of chemical or mechanical
skin permeation enhancers or other agents that cause abrasion.
[0133] It will be appreciated by those of ordinary skill in the art
that inventive compositions for topical administration may be
prepared as a cosmetic formulation such as skin softener, nutrition
lotion type emulsion, cleansing lotion, cleansing cream, skin milk,
emollient lotion, massage cream, emollient cream, make-up base,
lipstick, facial pack or facial gel, cleaner formulation such as
shampoos, rinses, body cleanser, hair-tonics, or soaps, or
dermatological composition such as lotions, ointments, gels,
creams, patches or sprays.
[0134] Such formulation of inventive nanoparticle compositions
typically includes combination with one or more excipients such as,
for example, fillers, sequestering agents, softeners, coloring
materials (e.g. pigments and dyes), and fragrances.
[0135] In some embodiments, inventive nanoparticle compositions are
formulated as a cream. The term "cream" refers to a spreadable
composition, typically formulated for application to the skin.
Creams typically contain an oil and/or fatty acid based-matrix.
Creams formulated according to the present invention may contain
nanoparticles and may be capable of substantially complete
penetration (e.g., of such nanoparticles) through the skin upon
topical administration. Such a cream could also act as a carrier
for incorporated materials (e.g., for example, for a botulinum
toxin).
[0136] Those of ordinary skill in the art will appreciate that
inventive nanoparticle compositions may be incorporated into a
device such as, for example, a patch.
[0137] A variety of transdermal patch structures are known in the
art; those of ordinary skill will appreciate that inventive
nanoparticle compositions may readily be incorporated into any of a
variety of such structures. In some embodiments, a transdermal
patch may further comprise a plurality of needles extending from
one side of the patch that is applied to the skin, wherein the
needles extend from the patch to project through the stratum
corneum of the skin. In some embodiments, the needles do not
rupture a blood vessel.
[0138] In some embodiments of the present invention, a nanoparticle
composition can be provided in a depot in a patch so that pressure
applied to the patch causes unmodified peptide to be directed out
of the patch (optionally through needles) and through the stratum
corneum.
[0139] In some embodiments of the present invention, a transdermal
patch includes an adhesive. Some examples of adhesive patches are
well known (for example, see U.S. Pat. Des. 296,006; U.S. Pat. Nos.
6,010,715; 5,591,767; 5,008,110; 5,683,712; 5,948,433; and
5,965,154; all of which are incorporated herein by reference).
Adhesive patches are generally characterized as having an adhesive
layer, which will be applied to a person's skin, a depot or
reservoir for holding a pharmaceutical agent, and an exterior
surface that prevents leakage of the pharmaceutical from the depot.
The exterior surface of a patch is typically non-adhesive.
[0140] Those of ordinary skill in the art will appreciate that a
transdermal patch is but one example of a device with which
inventive nanoparticle compositions may be administered. To give
but a few other examples, a device may be employed that allows the
composition to be applied without first applying the composition to
one's fingers, which may lead to undesirable paralysis of the
fingers. Suitable devices include spatulas, swabs, syringes without
needles, and adhesive patches. Use of spatulas or swabs, or the
like may require the device to be inserted into a container
containing the composition. Using syringes may be accomplished by
filling the syringe with the composition. The composition may then
be topically spread by the spatulas or swabs, or may be expelled
from the syringes onto the person's skin.
[0141] In many embodiments of the invention, it may be desirable to
limit delivery of unmodified peptides to only an intended delivery
area. In some embodiments, such limited delivery may be
accomplished by utilizing an inventive nanoparticle composition in
an application device that permits application of the composition
to a target site on the skin without applying the composition to
non-target site areas of the skin. Clearly, a transdermal patch may
be utilized to this end. Alternatively or additionally, if modified
peptides are to be applied topically to only a selected area, other
areas may be covered or pre-treated or otherwise protected from
exposure.
EXEMPLIFICATION
[0142] The following examples are only intended to provide
illustrations of specific embodiments contemplated by the present
invention. The examples are not intended in any way to be
limiting.
Example 1
Pentapeptide Nanoparticle Formulation
[0143] This example presents peptide nanoparticle compositions
comprising a nanoemulsion containing a pentapeptide, KTTKS (SEQ ID
NO.: 1), that is known to have biological activity on the skin
structures (Katayama et al., supra).
[0144] A pentapeptide nanoemulsion preparation was prepared as
follows: [0145] 800 mg of soybean oil and 800 mg of Tween 80 were
stirred in a sterile vial for 5 minutes; [0146] 8.4 ml water with
0.0001 g of the peptide KTTTS (SEQ ID NO.: 1) was added and stirred
for 20 minutes; [0147] The sample was homogenized for 1 minute; The
sample was stirred for 20 minutes; and [0148] The sample was
microfluidized once at 23,000 psi.
[0149] The resulting pentapeptide nanoemulsion was evaluated for
particle size using the Malvern Nano S particle sizer capable of
sizing particles between about 0.6 nm 6000 nm. The pentapeptide
nanoemulsion preparation had two particle size peaks having an
average particle size of 106 nm (Table 2).
TABLE-US-00002 TABLE 2 Particle Size Distribution of a Pentapeptide
Nanoparticle Size Range Percent of Particles 10-20 nm 1.3% 21-100
nm 30.2% 101-120 nm 10.4% 121-150 nm 22.4% 151-200 nm 19.3% 201-300
nm 14.7% 301-400 nm 1.7% Total 100.0%
Example 2
Pentapeptide Nanoparticle Formulation and Transdermal Penetration
with Biological Effect
[0150] This example presents peptide nanoparticles comprising a
nanoemulsion containing a pentapeptide, KTTKS (SEQ ID NO.: 1), that
is known to have biological activity on the skin structures
(Katayama et al., supra; incorporated herein by reference). This
example demonstrates the biological efficacy on the skin of
transdermally applying a peptide nanoparticle, in this case KTTKS
(SEQ ID NO.: 1).
Materials and Methods
[0151] A pentapeptide nanoemulsion preparation was prepared as
follows: [0152] 5.6 g of Labrafac WL 1349 oil and 5.6 g of Tween 80
were stirred in a sterile beaker for 5 minutes; [0153] 58.8 g
Reagent Grade water was placed in a separate beaker; 0.010 g of the
peptide KTTTS (SEQ ID NO.: 1) was added into the water and stirred
for 20 minutes; [0154] The contents of the first beaker were added
to the contents of the beaker (i.e., the water and peptide) and
then stirred for 20 minutes; and [0155] The entire sample was
microfluidized once at 23,000 psi.
[0156] The resulting pentapeptide nanoemulsion was evaluated for
particle size using the Malvern Nano S particle sizer capable of
sizing particles between about 0.6 nm about 6000 nm. The
pentapeptide nanoemulsion preparation had an average particle size
of 114.4 nm. Approximately 95% of the particles were below 130 nm
in size.
[0157] The pentapeptide nanoemulsion was then mixed with an equal
volume of a skin cream, (Base PCCA Vanishing Cream Light) and then
vortexed into a uniform cream to yield the "Treatment Cream."
[0158] A "Control Cream" was prepared by the same method as the
Treatment Cream, except that no peptide was added in the
process.
[0159] Ten Swiss Webster mice were purchased that were each
approximately 20 grams of weight. Upon arrival, all animals were
acclimated to their cages for one week (group housed 5 mice per
cage per group as defined below) and provided with standard cage
bedding and Purina 5001 chow. After one week, the following
treatment paradigms were applied:
[0160] Treatment Paradigms
[0161] Group 1 (Control): Each day for eight weeks, 5 mice each had
75 .mu.l of the Control Cream applied to their backs with a gloved
finger until no cream was visible. The mice had their backs shaved
with an electric shaver two days prior to the first treatment and,
thereafter, in one week intervals.
[0162] Group 2 (Treatment): Each day for eight weeks, 5 mice each
had 75 .mu.l of the Treatment Cream applied to their backs with a
gloved finger until no cream was visible. The mice had their backs
shaved with an electric shaver two days prior to the first
treatment and, thereafter in one week intervals.
[0163] Assessment
[0164] The skin from the each mouse's back that was treated with
either the Control or Treatment Cream was preserved and then
processed with Masson's Trichrome histologic stain. The intensity
of the staining was evaluated at a magnification of 400.times.
using on a histologic scale of 1 to 4 for staining intensity:
1=almost no staining, collagen fibrils were very thin, 2=minimal
staining and minimal collagen fibril width, 3=moderate staining and
moderate fibril width, and 4=intense staining and wide fibrils.
Results
[0165] Histological Assessment
[0166] The average histologic score of the skin tissue stained with
Masson's Trichrome stain was 2.33 out of a possible 4 in the
Control Group. By comparison, the average histologic score of the
stained skin of the mice in the Treatment Group was 3.67 out of a
possible 4. This represents a 57% increase in collagen-staining
intensity of the Treatment group over the Control Group. See FIG. 1
for examples of photomicrographs of skin tissue specimens from each
of the Control and Treatment Groups.
[0167] Assessment of Skin Thickness Effects
[0168] The thickness of skin thickness is measured using a Skin
Layer Thickness Test to determine the depth (in mm) of each skin
layer following examination of a histologic cross-section of the
mouse back skin that was microtomed and placed on a glass
slide.
[0169] Assessment of Extracellular Matrix Production
[0170] Collagen is a major component of the content of the
ExtraCellular Matrix. Collagen content is assessed using two
separate histologic stains (Picro Sirius Red and Pterocarpus Osun)
for collagen in two separate tests of mouse back skin that had
microtomed and placed on a glass slide.
[0171] Collagen content is assessed by using the Western Blot
technique to detect the hydroxyproline content of a homogenized
preparation of the mouse's back skin. Hydroxyproline content is
representative of collagen content.
Conclusion
[0172] The result show that, on average, the Treatment Group had
statistically more collagen than the Control Group by histologic
assessment. These controlled data show that the topical
pentapeptide nanoemulsion preparation had a measurable biological
effect on the skin when compared to a control cream without such a
pentapeptide. Prior studies have shown that the peptide cannot
penetrate the intact skin without chemical modification (Katayama
et al., supra). Therefore, these data show that the inventive
nanoemulsion formulation enabled penetration of unmodified peptide,
resulting in effects to the skin consistent with the known
biological action of the peptide in increasing collagen production
in the skin and resultant increase in skin thickness.
[0173] The results are expected to show that, on average, the
Treatment Group does have statistically thicker skin than the
Control Group. The results are expected to show that, on average,
the Treatment Group does have statistically more collagen than the
Control Group and as measured by the two histologic stains and
Western Blot measurement of hydroxyproline.
Example 3
Skin Thickening and Extra-Cellular-Matrix Stimulator Effects on
Mice Through Transdermal Application of a Peptide Nanoparticle:
Effect of Varying Concentration of Peptide in the Nanoparticle
[0174] This example demonstrates the impact of varying the
concentration of peptide in the nanoparticle on the biological
efficacy on the skin of transdermally applying a peptide
nanoparticle.
Materials and Methods
[0175] The experiment described Example 3 is repeated, except that
the concentration of peptide in the Treatment cream is decreased by
a factor of ten or increased by a factor of ten.
Results and Conclusion
[0176] The results are expected to show that, on average, those
mice treated with the peptide concentration increased by a factor
of ten have statistically thicker skin those mice treated with the
increased peptide concentration. The results are expected to show
that, on average, those mice treated with the peptide concentration
increased by a factor of ten have statistically more collagen than
those with decreased peptide concentration as measured by the two
histologic stains and Western Blot measurement of hydroxyproline.
In sum, these controlled data are expected to suggest that
biological effect on the skin of the peptide nanoemulsion varies
depending on the concentration of peptide incorporated.
Example 4
Administration of Pentapeptide Nanoparticle to Human Subjects to
Reduce Skin Lines
[0177] This example demonstrates the biological efficacy on the
human skin of transdermally applying a peptide nanoparticle.
Materials and Methods
[0178] A pentapeptide nanoemulsion prepared in accordance with
Example 1 or 2 is prepared and mixed with an equal volume of a skin
cream, (Base PCCA Vanishing Cream Light) and then vortexed into a
uniform cream to yield the "Treatment Cream."
[0179] A "Non-Nano Treatment Cream" is prepared by creating mixing
the same amount of pentapeptide into the same amount of water as
Example 1 and then vortexing with the same amount of skin cream as
was used to prepare the Treatment Cream.
[0180] A "Control Cream" is prepared by vortexing the same amount
of water as Example 1 or 2 and with the same amount of skin cream
as was used to prepare the Treatment Cream.
[0181] Thirty healthy human subjects with prominent facial lines
(such as observed in people with photo-damaged skin) are enrolled
in a double-blind, placebo-controlled, split-faced study with
left-right randomization. All subjects are graded with a five point
scale by an observer blinded to treatment status. Score 0 of the
scale is normal skin with a score of 5 being severe facial lines
and wrinkles (primarily in the periocular or "crow's feet" area).
Cheek skin texture is also assessed in terms of pore size (small to
large) and smoothness (smooth to rough/pebbly). Subjects are only
enrolled if they have a score on initial examination of 2.5 or
greater. The face of the subjects will be photographed using
standardized views and distances and lighting conditions.
[0182] Treatment Paradigms
[0183] The patient agrees not to use any facial skin care products
for 3 weeks except for a Control Cream they can use twice daily at
12 hour intervals. After this initial "wash-out" period, each
patient is given two tubes of cream marked "Right" and "Left" with
a unique numerical code for each tube. They are instructed to use
the Right Tube on the right side of the face and the Left Tube on
the left side of the face twice daily at 12 hour intervals. They
are instructed to apply a "pea-sized" amount of cream
(approximately 0.4 g) to each side of the face. They are also
instructed not to use other facial skin care products. For 10 of
the subjects (the Control Group), the Right Tube contains the
Control Cream and the Left Tube contains the Control Cream. For 10
of the subjects (the Non-Nano Treatment Group), the Right Tube
contains the Control Cream and the Left Tube contains the Non-Nano
Treatment Cream. For 10 of the subjects (the Nano Treatment Group),
the Right Tube contains the Control Cream and the Left Tube
contains the Nano Treatment Cream.
[0184] Assessment
[0185] The subjects are observed and photographed at 4, 8, and 12
weeks after beginning the treatment protocols following the
wash-out period. In addition, an observer blinded to treatment
status of the subject as well as the subject herself scores skin
texture with the aforementioned scale for each of the right and
left sides of the face.
Results and Conclusion
[0186] The results are expected to show that, on average, the Nano
Treatment Group has statistically greater differences between the
Right and Left facial texture scores (showing skin appearance
improvement) than the differences observed between Right and Left
scores for the Control Group and that the Non-Nano Treatment Group.
In sum, these controlled data are expected to show that the topical
pentapeptide nanoemulsion preparation has a measurable cosmetic
effect on the skin when compared to a control cream without such a
pentapeptide and a simple cream (Non-Nano Cream) with the same
pentapeptide that was not in a nanoparticle formulation.
EQUIVALENTS AND SCOPE
[0187] The foregoing has been a description of certain non-limiting
preferred embodiments of the invention. Those skilled in the art
will recognize, or be able to ascertain using no more than routine
experimentation, many equivalents to the specific embodiments of
the invention described herein. Those of ordinary skill in the art
will appreciate that various changes and modifications to this
description may be made without departing from the spirit or scope
of the present invention, as defined in the following claims.
[0188] In the claims articles such as "a," "an," and "the" may mean
one or more than one unless indicated to the contrary or otherwise
evident from the context. Claims or descriptions that include "or"
between one or more members of a group are considered satisfied if
one, more than one, or all of the group members are present in,
employed in, or otherwise relevant to a given product or process
unless indicated to the contrary or otherwise evident from the
context. The invention includes embodiments in which exactly one
member of the group is present in, employed in, or otherwise
relevant to a given product or process. The invention also includes
embodiments in which more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process. Furthermore, it is to be understood that the invention
encompasses all variations, combinations, and permutations in which
one or more limitations, elements, clauses, descriptive terms,
etc., from one or more of the claims or from relevant portions of
the description is introduced into another claim. For example, any
claim that is dependent on another claim can be modified to include
one or more limitations found in any other claim that is dependent
on the same base claim. Furthermore, where the claims recite a
composition, it is to be understood that methods of using the
composition for any of the purposes disclosed herein are included,
and methods of making the composition according to any of the
methods of making disclosed herein or other methods known in the
art are included, unless otherwise indicated or unless it would be
evident to one of ordinary skill in the art that a contradiction or
inconsistency would arise. For example, it is to be understood that
any of the compositions of the invention can be used for inhibiting
the formation, progression, and/or recurrence of adhesions at any
of the locations, and/or due to any of the causes discussed herein
or known in the art. It is also to be understood that any of the
compositions made according to the methods for preparing
compositions disclosed herein can be used for inhibiting the
formation, progression, and/or recurrence of adhesions at any of
the locations, and/or due to any of the causes discussed herein or
known in the art. In addition, the invention encompasses
compositions made according to any of the methods for preparing
compositions disclosed herein.
[0189] Where elements are presented as lists, e.g., in Markush
group format, it is to be understood that each subgroup of the
elements is also disclosed, and any element(s) can be removed from
the group. It is also noted that the term "comprising" is intended
to be open and permits the inclusion of additional elements or
steps. It should be understood that, in general, where the
invention, or aspects of the invention, is/are referred to as
comprising particular elements, features, steps, etc., certain
embodiments of the invention or aspects of the invention consist,
or consist essentially of, such elements, features, steps, etc. For
purposes of simplicity those embodiments have not been specifically
set forth in haec verba herein. Thus for each embodiment of the
invention that comprises one or more elements, features, steps,
etc., the invention also provides embodiments that consist or
consist essentially of those elements, features, steps, etc.
[0190] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and/or the understanding of one of
ordinary skill in the art, values that are expressed as ranges can
assume any specific value within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates otherwise.
It is also to be understood that unless otherwise indicated or
otherwise evident from the context and/or the understanding of one
of ordinary skill in the art, values expressed as ranges can assume
any subrange within the given range, wherein the endpoints of the
subrange are expressed to the same degree of accuracy as the tenth
of the unit of the lower limit of the range.
[0191] In addition, it is to be understood that any particular
embodiment of the present invention may be explicitly excluded from
any one or more of the claims. Any embodiment, element, feature,
application, or aspect of the compositions and/or methods of the
invention (e.g., any peptide, any peptide modification, any
nanoparticle, any nanoemulsion, any surfactant, any oil, any premix
component, any method of preparing nanoemulsions, any method of
treatment, etc.), can be excluded from any one or more claims. For
purposes of brevity, all of the embodiments in which one or more
elements, features, purposes, or aspects is excluded are not set
forth explicitly herein.
Sequence CWU 1
1
1415PRTArtificial sequencepeptide that may promote extra-cellular
matrix production 1Lys Thr Thr Lys Ser1 5210PRTArtificial
sequencepeptide that may promote extra-cellular matrix production
2Glu Tyr Lys Thr Thr Lys Ser Ser Arg Leu1 5 1038PRTArtificial
sequencepeptide that may promote extra-cellular matrix production
3Val Ile Glu Tyr Lys Thr Thr Lys1 544PRTArtificial sequencepeptide
that may promote extra-cellular matrix production 4Lys Thr Thr
Lys1512PRTArtificial sequencepeptide that may promote
extra-cellular matrix production 5Gly Lys Thr Val Ile Glu Tyr Lys
Thr Thr Lys Ser1 5 10615PRTArtificial sequencepeptide that may
promote extra-cellular matrix production 6Gly Lys Thr Val Ile Glu
Tyr Lys Thr Thr Lys Ser Ser Arg Leu1 5 10 15720PRTArtificial
sequencepeptide that may promote extra-cellular matrix production
7Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys Ser Ser Arg Leu1 5
10 15Pro Ile Ile Asp 20820PRTArtificial sequencepeptide that may
promote extra-cellular matrix production 8Cys Thr Ser His Thr Gly
Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys1 5 10 15Thr Thr Lys Ser
2094PRTArtificial sequencepeptide that may promote extra-cellular
matrix production 9Thr Thr Lys Ser1106PRTArtificial sequencepeptide
that may decrease wrinkles 10Glu Glu Met Gln Arg Arg1
5116PRTArtificial sequencepeptide that may improve wound healing
11Val Gly Val Ala Pro Gly1 5126PRTArtificial sequencepeptide that
may improve wound healing 12Tyr Tyr Arg Ala Asp Ala1
5133PRTArtificial sequencepeptide that may improve wound healing
13Gly His Lys11414PRTArtificial sequencepeptide that may treat
accumulation of excess extracellular matrix 14Thr Ser Leu Asp Ala
Ser Ile Ile Trp Ala Met Met Gln Asn1 5 10
* * * * *