U.S. patent application number 16/505910 was filed with the patent office on 2019-11-14 for prefabricated alginate-drug bandages.
The applicant listed for this patent is Beth Israel Deaconess Medical Center, Inc., President and Fellows of Harvard College. Invention is credited to Sidi A. Bencherif, Thomas Michael Braschler, Cathal J. Kearney, David J. Mooney, Uyanga Tsedev, Aristidis Veves.
Application Number | 20190343979 16/505910 |
Document ID | / |
Family ID | 51690047 |
Filed Date | 2019-11-14 |
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United States Patent
Application |
20190343979 |
Kind Code |
A1 |
Kearney; Cathal J. ; et
al. |
November 14, 2019 |
PREFABRICATED ALGINATE-DRUG BANDAGES
Abstract
The invention provides a solution to the drawbacks associated
with conventional alginate dressings. This invention features
improved alginate dressings or bandages as well as a fabrication
process that results in an alginate sheet that is preloaded with
drug, can be stored in a freeze-dried state, and is compliant and
ready to use at the time of administration.
Inventors: |
Kearney; Cathal J.; (Boston,
MA) ; Tsedev; Uyanga; (Cambridge, MA) ;
Mooney; David J.; (Sudbury, MA) ; Veves;
Aristidis; (Quincy, MA) ; Braschler; Thomas
Michael; (Quincy, MA) ; Bencherif; Sidi A.;
(Dorchester, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College
Beth Israel Deaconess Medical Center, Inc. |
Cambridge
Boston |
MA
MA |
US
US |
|
|
Family ID: |
51690047 |
Appl. No.: |
16/505910 |
Filed: |
July 9, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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16003192 |
Jun 8, 2018 |
10391196 |
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16505910 |
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14783559 |
Oct 9, 2015 |
10016524 |
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PCT/US14/33867 |
Apr 11, 2014 |
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16003192 |
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61810854 |
Apr 11, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 15/425 20130101;
A61K 38/046 20130101; A61L 2300/412 20130101; A61L 15/44 20130101;
A61L 15/28 20130101; A61L 15/28 20130101; C08L 5/04 20130101 |
International
Class: |
A61L 15/28 20060101
A61L015/28; A61L 15/44 20060101 A61L015/44; A61K 38/04 20060101
A61K038/04; A61L 15/42 20060101 A61L015/42 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under Grant
No. 1R24DK091210-01A1 awarded by the NIH/NIDDK, and Grant No.
5R01EB014703-02 awarded by the NIH/NBIB. The Government has certain
rights in the invention.
Claims
1. A composition comprising a cryo-organized, crosslinked alginate
structure comprising a therapeutic agent, wherein said composition
comprises a Young's modulus of 10 kiloPascals to 10,000 kiloPascals
at room temperature.
2. The composition of claim 1, wherein said composition comprises a
Young's modulus of 100 kiloPascals to 10,000 kiloPascals at room
temperature.
3. The composition of claim 1, wherein said cryo-organized
structure comprises a network of interconnected crosslinked
pores.
4. The composition of claim 1, wherein said therapeutic agent
comprises substance P.
5. A method of promoting wound healing or tissue repair, comprising
contacting a bodily tissue of a subject with the composition of
claim 1.
6. The method of claim 5, wherein said therapeutic agent comprises
substance P.
7-10. (canceled)
11. The method of claim 5, wherein the wound is a diabetic
wound.
12. The method of claim 11, wherein the diabetic wound comprises an
ischemic wound, optionally wherein the ischemic wound comprises a
neuroischemic wound.
13-23. (canceled)
24. The composition of claim 1, wherein said structure comprises a
Young's modulus of 1,000 kiloPascals to 10,000 kiloPascals at room
temperature.
25. The composition of claim 1, wherein said structure comprises a
Young's modulus of 1,000 kiloPascals to 5,000 kiloPascals at room
temperature.
26. The composition of claim 1, wherein the thickness of said
composition is 0.1 mm-10 mm.
27. The composition of claim 1, wherein said alginate comprises
non-oxidized alginate.
28. The composition of claim 1, wherein said alginate comprises
oxidized alginate.
29. The composition of claim 1, wherein said therapeutic agent is a
wound-healing or tissue repair compound.
30. The composition of claim 29, wherein said therapeutic agent is
selected from the group consisting of substance P, Vascular
Endothelial Growth Factor, Platelet-Derived Growth Factor, Stromal
cell-Derived Factor, Epidermal Growth Factor, Transforming Growth
Factor, Granulocyte Macrophage-Colony Stimulating Factor, and
Fibroblast Growth Factor.
31. The composition of claim 4, wherein said substance P is
selected from the group consisting of a SP peptide, a SP-related
molecule, a SP fragment, and a SP peptide derivative.
32. The composition of claim 31, wherein said substance P
comprises: (i) an amino acid sequence that is at least 50%
identical to the amino acid sequence of SEQ ID NO: 1 or 2; (ii) an
amino acid sequence that is at least residues 1-8 of SEQ ID NO: 1
or 2; (iii) the amino acid sequence of SEQ ID NO: 1; (iv) the amino
acid sequence of SEQ ID NO: 2; or (v) a consensus amino acid
sequence, wherein the consensus amino acid sequence comprises
Xaa.sub.1-Pro-Xaa.sub.2-Pro-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.s-
ub.6 (SEQ ID NO: 12).
33. The composition of claim 3, wherein said pores have a diameter
of 10 .mu.m-800 .mu.m, 10 .mu.m-100 .mu.m, 100 .mu.m-200 .mu.m, 100
.mu.m-400 .mu.m, or 300 .mu.m-800 .mu.m.
34. The composition of claim 33, wherein the alginate is
crosslinked ionically.
35. The composition of claim 1, further comprising a woven
mesh.
36. The composition of claim 35, wherein said woven mesh is a
gauze.
37. The composition of claim 35, wherein said woven mesh is
interspersed within the alginate structure.
38. The composition of claim 1, further comprising a backing
material.
39. The composition of claim 1, further comprising a cell.
40. The composition of claim 1, wherein said structure comprises a
Young's modulus of 10 kiloPascals to 500 kiloPascals at room
temperature.
41. The composition of claim 1, wherein said structure comprises a
Young's modulus of 100 kiloPascals to 500 kiloPascals at room
temperature.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 16/003,192, filed Jun. 8, 2018, which
is a divisional application of U.S. patent application Ser. No.
14/783,559, filed on Oct. 9, 2015, now issued as U.S. Pat. No.
10,016,524, issued Jul. 10, 2018, which is a 35 U.S.C. .sctn. 371
national stage filing of International Application No.
PCT/US2014/033867, filed Apr. 11, 2014, which, in turn, claims the
benefit, and priority to, U.S. Provisional Application No.
61/810,854, filed Apr. 11, 2013. The contents of each of the
aforementioned applications are hereby incorporated by reference in
their entireties.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Nov. 8, 2017, is named 117823-09002_SL.txt and is 4,117 bytes in
size.
FIELD OF THE INVENTION
[0004] The invention relates to wound dressings.
BACKGROUND OF THE INVENTION
[0005] Alginate dressings are wound coverings made from a polymer
derived from seaweed. Such dressings maintain a moist
microenvironment that promotes healing. They can be biodegradable
and have been successfully used for variety of secreting lesions.
The dressing limits accumulation of wound secretions and minimizes
bacterial contamination.
[0006] One drawback of alginate that limits the use of alginate
dressing is its fabrication process. In order to prefabricate the
bandage, it is cross-linked in advance and stored at room
temperature, thus making it incompatible with sensitive or labile
drugs, cells, or proteins that are prone to degradation under room
temperature conditions.
SUMMARY OF THE INVENTION
[0007] The invention provides a solution to the drawbacks
associated with conventional alginate dressings. This invention
features improved alginate dressings or bandages, e.g., a topical
dressing or bandage, as well as a fabrication process that results
in an alginate sheet that is preloaded with drug, can be stored in
a freeze-dried state, and is compliant and ready to use at the time
of administration. The method is applicable to any ionically
crosslinked hydrogel composition such as alginate, chitosan,
gelatin, and collagen.
[0008] Accordingly, a method of making a compliant bandage
composition includes the steps of providing an alginate solution,
molding said solution into a desired shape, inducing a
cryo-organized structure by freezing and lyophilizing said molded
solution, and contacting said cryo-organized structure with a
crosslinking agent to yield a compliant bandage composition.
[0009] Preferably, the step of contacting the alginate with a
crosslinking agent is carried out after the step of inducing a
cryo-organized structure, e.g., by freezing and then lyophilizing
The crosslinking stabilizes or locks in place the 3-dimensional
pore structure induced by freezing the mixture. The pores are
interconnected. The cryo-organization of alginate resulting from
the freeze-drying process confers flexibility to the composition,
and the crosslinking process stabilizes the structure to maintain
flexibility. Depending on the fabrication conditions, the pores are
generally homogeneous, heterogeneous, and/or aligned. In some
cases, a gauze fabric is added to the alginate solution prior to
inducing the cryo-organized structure and then crosslinking.
[0010] The resulting composition is a compliant bandage or wound
dressing and is suitable for contacting a bodily tissue (human or
non-human animal) immediately. Optionally, the composition is
freeze dried again for short term storage, long term storage, or
for transport to a medical facility at which the product will be
used. Thus, the method optionally further comprises a second
freezing and lyophilizing step. If not subjected to a second
freeze/dry step, the sterile gel composition can be stored for
months at room temperature, at 4.degree. C., or frozen (e.g., at
-20.degree. C. to -80.degree. C.), the length of storage being
dependent on the half-life of the incorporated drug rather than the
alginate composition itself.
[0011] In preferred embodiments, the alginate solution comprises a
therapeutic agent such as a drug. In one example, the agent
includes a wound-healing or tissue repair compound such as
substance P ("SP" or a SP-related molecule, a SP fragment, or a SP
peptide derivative composition), Vascular Endothelial Growth Factor
(VEGF), Platelet-Derived Growth Factor (PDGF), Stromal cell-Derived
Factor (SDF), e.g., SDF-1, Epidermal Growth Factor(EGF),
Transforming Growth Factor (TGF), e.g., TGF-.beta., Granulocyte
Macrophage-Colony Stimulating Factor (GM-CSF), or Fibroblast Growth
Factor (FGF). Alternatively, or in addition, the alginate contains
analgesics such as ibuprofen; opiates (e.g., morphine); or topical
anesthetics (e.g., benzocaine or lidocaine). Alternatively or in
addition, the alginate contains and anti-microbial compound such as
erythromycin, streptomycin, zithromycin, platensimycin, iodophor,
2% mupirocin, triple antibiotic ointment (TAO, bacitracin
zinc+polymyxin B sulfate+neomycin sulfate) and others, as well as
peptide anti-microbial agents. In some examples, the alginate
comprises a hemostatic agent, e.g., microfibrillar collagen,
poly-N-acetyl glucosamine, chitosan, kaolinite, thrombin,
epinephrine, fibrin sealant, gelatin, mineral zeolite, aluminum
chloride, silver nitrate, ferric subsulfate solution, acrylates
(e.g., cyanoacrylates), ostene, bone wax, methylcellulose,
glutaraldehyde, fibrinogen, or polyethylene glycol.
[0012] In some embodiments, the alginate comprises a drug to treat
or alleviate a symptom of a skin/cutaneous lesion (e.g., a chronic
skin lesion) or a mucous membrane lesion. Exemplary skin lesions
include but are not limited to burns, eczema, psoriasis, disease
wounds, acne, actinic keratosis, allergic/contact dermatitis, boils
(infections that develop on hair follicles), bullae (fluid-filled
sacs or lesions that appear when fluid is trapped under a layer of
skin), cellulitis, chemical burns, cherry angiomas, chicken pox,
cold sores, corns, calluses, cysts, dyshidrotic eczema (itchy
blisters on the palms of hands and/or soles of feet), erysipelas
(bacterial infections that affect the skin's upper layers),
frostbite, genital herpes, gout, impetigo, insect sting allergies
(e.g., mosquito bites, wasp stings), keloids (smooth, hard growths
that form when scar tissue grows excessively), keratosis pilaris,
lipomas, molluscum contagiosum (skin infections caused by the
molluscum virus), methicillin-resistant Staphylococcus (Staph)
infections, neurofibromas, nodules (abnormal growths that form
under the skin, often filled with inflamed tissue or fluid),
pemphigoid (a rare autoimmune disorder that more often affects the
elderly), pemphigus vulgarus (an autoimmune disease that leads to
painful blisters on skin and mucous membranes), porphyrias,
scabies, scarlet fever, sebaceous cysts, seborrheic keratosis,
shingles, skin cancer, warts.
[0013] Exemplary drugs that treat or alleviate a symptom (itching,
raised bumps on skin, raw and sensitive skin, thicken/cracked/scaly
skin, red to brownish-gray patches on skin) of eczema include
corticosteroids (e.g., prednisone, methylprednisone, dexamethasone,
prednisolone, or triamcinolone acetonide), antibiotics (e.g.,
fluoroquinolone, cephalosporins, macrocyclics, penicillins,
monobactams, carbapenems, macrolides, lincosamides, streptogramins,
aminioglycosides, quinolones, sulfonamides, tetracyclines,
vancomycin, bacitracin, polymyxin B, or nitrofurantoin), oral
antihistamines (e.g., diphenhydramine, cetirizine, fexofenadine,
hydroxyzine, or loratidine), and immunomodulators (e.g., tacrolimus
or pimecrolimus).
[0014] Exemplary drugs that treat or alleviate a symptom (red
patches of skin covered by scales, dry cracked skin that may bleed,
itching, burning, soreness, swollen or stiff joints,
thickened/pitted/ridged nails) of psoriasis include hydrocortisone,
prednisone, calcipotriene, mometasone, cyclosporine, adalimumab,
and triamcinolone.
[0015] Exemplary drugs that treat or alleviate a symptom (e.g.,
pain, itchiness, infection, inflammation) of burns or disease
wounds include analgesics (e.g., aspirin; non-steroidal
anti-inflammatory drugs (NSAIDS) such as ibuprofen and naproxen;
acetaminophen; COX-2 inhibitors such as rofecoxib, celecoxib, and
etoricoxib; opiods such as morphine, codeine, oxycodone,
hydrocodone, dihydromorphine, buprenorphine, tramadol; or
flupirtine), hydrocortisone, antibiotics (e.g., fluoroquinolone,
cephalosporins, macrocyclics, penicillins, monobactams,
carbapenems, macrolides, lincosamides, streptogramins,
aminioglycosides, quinolones, sulfonamides, tetracyclines,
vancomycin, bacitracin, polymyxin B, or nitrofurantoin), or
antihistamines (e.g., diphenhydramine, cetirizine, fexofenadine,
hydroxyzine, or loratidine).
[0016] Exemplary drugs to treat or alleviate a symptom of frostbite
include corticosteroids (e.g., prednisone, methylprednisone,
dexamethasone, prednisolone, or triamcinolone acetonide),
antibiotics (e.g., fluoroquinolone, cephalosporins, macrocyclics,
penicillins, monobactams, carbapenems, macrolides, lincosamides,
streptogramins, aminioglycosides, quinolones, sulfonamides,
tetracyclines, vancomycin, bacitracin, polymyxin B, or
nitrofurantoin), aloe vera gel/lotion/cream, analgesics (e.g.,
aspirin; non-steroidal anti-inflammatory drugs (NSAIDS) such as
ibuprofen and naproxen; acetaminophen; COX-2 inhibitors such as
rofecoxib, celecoxib, and etoricoxib; opiods such as morphine,
codeine, oxycodone, hydrocodone, dihydromorphine, buprenorphine,
tramadol; or flupirtine).
[0017] Exemplary drugs to treat or alleviate a symptom of genital
herpes include acyclovir, famciclovir, and valacyclovir.
[0018] Exemplary drugs to treat or alleviate a symptom of acne
include benzoyl peroxide, salicylic acid, topical retinoids (e.g.,
tretinoin, isotretinoin, adapalene, or tazarotene), antibiotics
(e.g., erythromycin, clindamycin, metronidazole, doxycycline,
minocycline, nadifloxacin, or dapsone), oral contraceptives.
[0019] Exemplary drugs to treat or alleviate a symptom of
erysipelas include antibiotics, such as penicillins, clindamycin,
or erythromycin.
[0020] Exemplary drugs to treat or alleviate a symptom of
methicillin-resistant Staphylococcus (Staph) infections include an
antibiotic described above.
[0021] Typically, a therapeutic agent such as a drug is added to
the alginate solution prior to molding and/or freeze-drying.
[0022] The crosslinking agent induces ionic crosslinking. For
example, the crosslinking agent comprises calcium chloride, barium
chloride, magnesium chloride, or potassium chloride.
[0023] Also within the invention is the product of the
above-described fabrication method, e.g., a composition comprising
a cryo-organized, crosslinked alginate structure comprising a
therapeutic agent, wherein the structure comprises a Young's
modulus of 50 kiloPascal to 500 kiloPascal (kPa), e.g., 10 kPa-250
kPa, e.g., 10 kPa-150 kPa, at room temperature. Unlike conventional
alginate structures that have been frozen, these compositions are
characterized by superior strength and flexibility by virtue of the
cryo-organized network that has been cross-linked to stabilize it.
If the composition further comprises a gauze fabric, the entire
composite structure is characterized by a Young's modulus of 100
kPa to 10,000 kPa at room temperature. The function of the gauze is
to confer mechanical integrity to the ionically-crosslinked
composition and typically does not affect drug delivery. The gauze
fabric is typically comprised of cotton of a loose open weave,
e.g., 2-3 mm mesh, but can also be comprised of other polymers such
as nylon or Dacron. In some examples, the composition withstands a
pressure of up to 100 kPa, e.g., up to 100 kPa, 80 kPa, 60 kPa, 40
kPa, 20 kPa, 10 kPa, or 1 kPa, either continuously or
discontinuously, without collapsing or losing the integrity and/or
shape of the cryo-organized, cross-linked structure. In some
examples, if the composition further comprises a gauze fabric, the
entire composite structure is characterized by a tensile strength
of 100 kPa to 10,000 kPa (e.g., 100 kPa to 5,000 kPa, 100 kPa to
1,000 kPa, 100 kPa to 500 kPa, 250 kPa to 10,000 kPa, 250 kPa to
5,000 kPa, 250 kPa to 1,000 kPa, 500 kPa to 10,000 kPa, 500 kPa to
5,000 kPa, 500 kPa to 1,000 kPa, 1,000 kPa to 10,000 kPa, 1,000 kPa
to 5,000 kPa, or 5,000 kPa to 10,000 kPa) at room temperature.
[0024] In some examples, the bandage/device of the invention
maintains the moisture of the bandage/device after topical
administration onto a subject. For example, the bandage/device
loses less than 60%, e.g., less than 60%, 50%, 40%, 30%, 20%, 15%,
10%, 5%, 2%, 1%, or less, of the water content after administration
(e.g., at least 1 hour, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 12, or 24 hours, 1, 2, 3, 4, 5, 6, or 7 days, 1, 2, 3, 4, or 5
weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months, or more after
administration) compared to prior to administration. For example,
the bandage/device loses less than 60%, e.g., less than 60%, 50%,
40%, 30%, 20%, 15%, 10%, 5%, 2%, 1%, or less, of its weight or mass
after administration (e.g., at least 1 hour, e.g., at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 12, or 24 hours, 1, 2, 3, 4, 5, 6, or 7
days, 1, 2, 3, 4, or 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
months, or more after administration) compared to prior to
administration. The dimensions of the bandage or wound dressing are
those typical for topically applied bandages, i.e., the size
corresponds to the wound to be treated. The thickness generally
comprises 0.1 mm -10 mm, e.g., 1 mm-5 mm or 1 mm-2 mm Release of
drug depends on the affinity of the drug for the network structure,
e.g., alginate, as well as diffusion. Thus, a thinner gel, e.g., 1
mm-5 mm in thickness, permits more rapid release of drug to a
tissue than a thicker dressing. The drug release profile associated
with a thickness of 1 mm-2 mm is compatible with the customary
clinical schedule of changing would dressings. For slower release,
the bandage is optionally fabricated at a greater thickness,
thereby retarding drug release due to the greater distance the drug
must travel to exit the dressing.
[0025] The width and length of the bandage or wound dressing vary
depending on the size of the wound or the use. For example, the
width and/or length of the bandage or wound dressing comprises 0.5
cm-12 cm, e.g., 0.5 cm-6 cm, 0.5 cm-3 cm, 0.5 cm-2 cm, 1 cm-12 cm,
2 cm-12 cm, 3 cm-12 cm, 6 cm-12 cm, 8 cm-12 cm, 10 cm-12 cm, 2
cm-10 cm, 2 cm-8 cm, 2 cm-6 cm, or 4 cm-8 cm. For example, the
surface area of the bandage or wound dressing comprises 0.2
cm.sup.2-144 cm.sup.2, e.g., 0.2 cm.sup.2-100 cm.sup.2, 0.2
cm.sup.2-60 cm.sup.2, 0.2 cm.sup.2-30 cm.sup.2, 0.2 cm.sup.2-10
cm.sup.2, 0.2 cm.sup.2-4 cm.sup.2, 1 cm.sup.2-100 cm.sup.2, 1
cm.sup.2-60 cm.sup.2, 1 cm.sup.2-30 cm.sup.2, 1 cm.sup.2-10
cm.sup.2, 5 cm.sup.2-144 cm.sup.2, 5 cm.sup.2-100 cm.sup.2, 5
cm.sup.2-60 cm.sup.2, 5 cm.sup.2-30 cm.sup.2, 25 cm.sup.2-144
cm.sup.2, 25 cm.sup.2-100 cm.sup.2, 100 cm.sup.2-144 cm.sup.2, 25
cm.sup.2-100 cm.sup.2, or 40 cm.sup.2-80 cm.sup.2.
[0026] The cryo-organized structure comprises a network of pores,
which is formed during and after the first freeze/dry/thaw cycle.
The superior physical properties of the alginate composition are
due to the interconnected pores (e.g., network of gaps) that form
between alginate strands as a result of the freezing step. Ice
crystals form during the freezing process, and alginate
concentrates around the ice crystals. The cryo-organized structure
is an alginate network that is left behind after the ice/frozen
water is removed by lyophilization/drying. After removal of the
water by freeze/drying the mixture, the network lacks mechanical
integrity. The ionic crosslinking step (using aqueous or
non-aqueous ionic crosslinking agents) confers structural integrity
upon the structure, while maintaining an interconnected pore
structure.
[0027] As is described above, the composition contains a
therapeutic agent such as an tissue repair/wound healing
compound.
[0028] The device includes one or more therapeutic compositions,
e.g., drugs such as proteins, peptides, small molecules, nucleic
acids, or even whole cells. The compounds are purified
naturally-occurring, synthetically produced, or recombinant
compounds, e.g., polypeptides, nucleic acids, small molecules, or
other agents. The compositions described herein are purified.
Purified compounds are at least 60% by weight (dry weight) the
compound of interest. Preferably, the preparation is at least 75%,
more preferably at least 90%, and most preferably at least 99%, by
weight the compound of interest. Purity is measured by any
appropriate standard method, for example, by column chromatography,
polyacrylamide gel electrophoresis, or HPLC analysis. Cells are
also purified or enriched for a particular cell type or phenotype.
For example, a sample of cells of a particular tissue type or
phenotype is "substantially pure" when it is at least 60% of the
cell population. Preferably, the preparation is at least 75%, more
preferably at least 90%, and most preferably at least 99% or 100%,
of the cell population. Cells are purified or enriched using a
variety of known methods, e.g., selection by cell surface markers
using marker-specific antibodies or other ligands. Purity is
measured by any appropriate standard method, for example, by
fluorescence-activated cell sorting (FACS).
[0029] The invention also features a method of promoting wound
healing or tissue repair. The method includes contacting a bodily
tissue such as an injured or diseased tissue of a subject with a
composition, e.g., bandage/device, described herein. For example,
the composition, e.g., bandage/device comprises a therapeutic
agent. For example, the composition, e.g., bandage/device mediates
controlled release, e.g., sustained or delayed release, of the
therapeutic agent to a tissue/wound of the subject.
[0030] Exemplary therapeutic agents are described above. For
example, the therapeutic agent comprises substance P.
[0031] In some embodiments, the size of a wound of the subject is
reduced compared to the size of the wound prior to contacting the
subject with a composition, e.g., bandage/device, described herein.
For example, the size of the wound is reduced by at least 1.5-fold,
e.g., at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold,
15-fold, 20-fold, 30-fold, 40-fold, 50-fold. In some examples, the
size of a wound is a perimeter or area of the wound. In some cases,
administration of the composition, e.g., bandage/device, results in
a complete closure of a wound in the subject. For example, complete
closure of the wound occurs within 6 months, e.g., within 6 months,
5 months, 4 months, 3 months, 2 months, 1 month, 5 weeks, 4 weeks,
3 weeks, 2 weeks, 1 week, 7 days, 6 days, 5 days, 4 days, 3 days, 2
days, 1 day, or less, after administration of the
device/bandage.
[0032] In some embodiments, the devices/bandages of the invention
are effective in treating or alleviating a symptom of a skin
lesion, e.g., a chronic skin lesion. Exemplary skin lesions are
described above. For example, the devices/bandages of the invention
are effective in treating or alleviating a symptom of eczema,
psoriasis, frostbite, bacterial infections, or burns.
[0033] In some embodiments, the device/bandage directly contacts an
injured, damaged, or diseased tissue, e.g., at the site of a
cutaneous/mucous membrane injury, damage, or disease (such as a
skin/mucous membrane lesion or disease described above). In some
cases, the device/bandage covers the site of a cutaneous/mucous
membrane injury and extends to and/or overlaps onto healthy
unaffected tissue, e.g., healthy unaffected skin or mucous
membrane.
[0034] In some embodiments, the devices/bandages of the invention
are effective in reducing pain in a subject. In some examples, the
devices/bandages of the invention are effective in reducing pain in
a subject by at least 5%, e.g., at least 5%, 10%, 15%, 20%, 25%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater, compared to the
level of pain prior to administration of the device/bandage. In
some cases, pain, e.g., at the site of administration, is
eliminated after administration of the device/bandage.
[0035] In some embodiments, the subject comprises diabetes. For
example, the subject comprises a diabetic wound, e.g., an ischemic
wound. In some examples, the ischemic wound comprises a
neuroischemic wound.
[0036] In some embodiments, the composition, e.g., bandage/device,
of the invention is not suitable for injection into a subject.
[0037] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims. All references cited
herein are incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 is a photograph of an ionically cross-linked alginate
sheet following freeze-drying, which breaks easily during
handling.
[0039] FIG. 2 is a flow diagram and set of photographs showing a
fabrication process that uses an intermediary freeze-drying step to
create pores in the alginate that introduce flexibility into the
sheet prior to crosslinking. The starting material contains
alginate+drug (optionally+gauze). Photographs of an alginate-only
dressing and alginate+gauze dressing are shown in the right
panel.
[0040] FIG. 3 are a panel of photographs. The left panel is a
photograph showing freeze-dried non cross-linked cryo-organized
alginate, and the right panel is a scanning electron microscope SEM
image depicting a typical macroporous network structure. The
network structure (comprises of pores) is generally homogenous.
[0041] FIG. 4 is a flow diagram showing the preparation of calcium
crosslinked cryo-organized alginate (COA) gels with an
interconnected macrostructure. Alginate was first dissolved in
water, frozen at -20.degree. C. (1), and then freeze-dried to
generate a cryo-organized macroporous architecture (2). COA was
subsequently ionically calcium cross-linked (3) and re-equilibrated
in water (4). COA was freeze-dried a second time for long-term
storage (5).
[0042] FIG. 5 is a photograph and illustration showing an alginate
bandage application to a wound.
[0043] FIG. 6 is a graph and a table showing percent of Substance P
(remaining after processing loss) released from the bandage
material over time. The sheets incorporate either 32 .mu.g or 64
.mu.g of Substance P with a given processing loss (Sample size n=3
per time point).
[0044] FIG. 7 is a graph and a table showing percent of Trypan
Blue, Mitoxantrone, and bovine serum albumin (BSA) (remaining after
processing loss) released from the bandaging material over time.
(Sample size of study n=3 per time point).
[0045] FIG. 8 is a graph and a table showing tensile properties of
the bandaging material strengths with and without gauze at 2% and
4% alginate. (Sample size n=3). Mean Failure Stress is a measure of
strength of the composition, and Young's Modulus is a measure of
flexibility. The x-axis in the graph (Engineering Strain)
represents a relative change in length of the composition when
subjected to applied stress/original length of the composition.
[0046] FIG. 9 is a graph of mechanical fatigue test data and a set
of before and after images of the alginate bandage. The bandage
dried out in the test, but stays hydrated in situ on a subject, as
the environment is sealed.
[0047] FIG. 10A is a graph showing the ability of devices/bandages
to absorb and retain fluid over several days. FIG. 10B is a graph
showing the ability of devices/bandages to resist degradation in a
fluid environment. Testing was performed in Ca.sup.2+ free
phosphate buffered saline (PBS). As there are no natural alginate
lyases in humans and as Ca.sup.2+ is present in wound exudate,
these testing conditions are good models for wound environments.
Based on these results, the bandages likely perform at least this
well in a wound environment.
[0048] FIG. 11 shows the mechanical properties of the bandages as a
function of time. The loss of calcium when the device/bandage is
placed in calcium-free phosphate buffered saline (PBS) resulted in
a reduction in mechanical properties, e.g., engineering stress and
Young's modulus. However, calcium ions in the wound exudate prevent
such a reduction in mechanical properties when the bandage is
applied to a wound of a subject.
[0049] FIG. 12 is a set of photographs of a rabbit neuroischemic
diabetic wound healing model showing the alginate bandage in place.
The ear was subsequently covered with a standard off-the-shelf
adhesive covering that is routinely used in wound care. These
bandages remained in place and were easily exchanged at three
days.
DETAILED DESCRIPTION
[0050] Alginate has been used for scaffolds and/or cell and drug
carriers. It has also been used as a non-drug delivering bandage in
topical wound healing situations. The drawback of alginate for this
application is its fabrication process. In order to prefabricate
the bandage, it is cross-linked in advance and stored at room
temperature. Such storage conditions make it incompatible with many
drugs, cells, or proteins that degrade in room temperature. One way
of overcoming this problem is to prepare the alginate delivery
device as an injectable solution at the time of administration;
however, this approach is time consuming and impractical for a
surgical setting.
[0051] Described herein is a novel fabrication process that results
in an alginate sheet that is preloaded with drug, can be stored in
a freeze-dried state, and is compliant and ready to use at the time
of administration. The device can be manufactured in a variety of
sizes (e.g., ready to use sizes) and can also be easily cut to size
at the time of use. The alginate sheet has a defined physical form
that matches the mold in which it is manufactured. The compositions
and methods are useful for a variety of payloads, i.e., compounds
to be delivered to injured or diseased tissues. For example,
alginate bandages have been fabricated to contain Substance P, a
peptide drug. Delivery of Substance P from the bandage has efficacy
in wound healing in diabetics. The alginate dressings/bandages have
applicability over a large range of small molecule drugs, peptides,
proteins, and cells. The alginate sheets/devices of the invention
are stored, e.g., at room temperature, for long periods of time
without loss of mechanical integrity, drug delivery function, or
tissue protective function, e.g., at least 1 day, e.g., at least 1,
2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5 weeks, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12 months, 1, 2, 3, 4, 5 years, or longer. Also, the
stored alginate devices are compliant immediately upon hydration.
Some previously available devices or gels containing drug(s) that
are sensitive to degradation at room temperature have a short shelf
life due to the instability of the drug(s). In contrast, the
alginate devices/bandages of the invention permit storage of
degradation-sensitive drug(s) within the devices for longer periods
of time, e.g., at least 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5
weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1, 2, 3, 4, 5
years, or longer, e.g., at room temperature, without degradation of
the drug(s). As such, the devices are highly portable and are
useful for applications such as field combat, hiking/expeditions,
sailing, and use in third world countries.
[0052] In addition, the alginate dressings/bandages provide
protection of wounds, e.g., open wounds and/or surgical wounds from
physical force. The dressings/bandages also provide controlled
(e.g., delayed or sustained) delivery of therapeutic agents (e.g.,
topically) without the need for any invasive procedures, e.g.,
without the need for implantation or injection. The hydrogel nature
of the device (e.g., high water content) helps maintain moisture in
the wound site, which is favorable for wound healing.
[0053] In addition, the devices/bandages can be modified to
customize them for the intended use. For example, the
devices/bandages can be customized for use as a wound covering that
is easily removed, e.g., for cleaning or replacement. Alginate does
not have any natural ligands for cells to adhere to; thus by using
alginate hydrogels, the device does not become infiltrated by cells
and tissue does not form in the device. In this way, the design of
the bandage is such that the bandage does not become integrated
with any newly formed tissue at the wound site. This property makes
the device removable from the wound site, e.g., without re-damaging
the wound site when the device is removed. This is unlike gauze
alone, which when left on a wound too long can become enveloped by
newly formed tissue--the wound site can then be damaged when the
gauze is removed.
[0054] On the other hand, if the intended use is to build
cells/tissue in and/or around the device, e.g., in tissue
engineering, the alginate is chemically modified by attaching cell
adhesion ligands to the alginate. In some examples, the alginate is
chemically modified to undergo degradation (e.g., oxidation). The
cell adhesion ligands, in combination with the macroporous
structure of the device, then permit cell infiltration. For
example, the alginate bandage is incorporated into/around the
healing tissue (e.g., around or in a wound).
Alginate
[0055] Alginate is a linear polysaccharide consisting of
(1,4)-linked b-D-mannuronate (M) and its C-5 epimer
.alpha.-L-guluronate (G). The monomers can appear in homopolymeric
blocks of consecutive G-residues (G-blocks), consecutive M-residues
(M-blocks), alternating M and G-residues (MG-blocks) or randomly
organized blocks. Chemical composition, primary structure and
average block lengths are conveniently determined by NMR
spectroscopy. Commercial alginates are generally extracted from
brown algae, and the relative amount of each block type varies with
the origin of the alginate. Physical-chemical and biological
properties of alginate vary widely with chemical composition.
G-blocks form stable cross-linked junctions with divalent cations
(e.g. Ca2+, Ba2+, Sr2+, among others) leading to a
three-dimensional gel network. Alginate can also form gels under
acidic conditions without cross-linking agents.
Fabrication Technique
[0056] Freeze-drying is commonly used in preparing sensitive
biological agents for long-term storage as it helps reduce
hydrolytic effects on them. Ionically cross-linked alginate
materials, however, become extremely brittle and are difficult to
handle after undergoing freeze-drying due to a propensity to break
or shatter (FIG. 1). This makes it difficult for the end user to
apply the bandage, while also complicating the devices storage and
transport. Manufacture of the cryo-organized pore structure confers
flexibility on the composition, and crosslinking after
cryo-organization preserves the flexible physical property, which
in part, results in compliance to pressure
[0057] An alternative approach to fabricate the device (drug
delivery bandage or dressing) is described herein (as described in
FIGS. 2 and 4). Initially, the alginate and drug is molded into the
proposed end shape of the device. For example, the device is
fabricated in two forms: alginate alone or alginate in combination
with gauze. The addition of gauze to the bandage increases the
mechanical stiffness and strength of the device and improves
handling. Afterwards, the alginate alone device or alginate+gauze
device is passed through a freeze-drying process to form
cryo-organized structures. A therapeutic agent is added to the
alginate solution before the induction of cryo-organization
(freezing) or after the lyophilization step. The terms freeze-dry
and lyophilization are synonymous and used interchangeably herein.
The process imparts a microstructure in the uncross-linked alginate
prior to it being cross-linked. The alginate solution is chilled at
the desired temperature to assure a completely frozen
state.(typically at least 8 hours, e.g., overnight). Lyophilization
is carried out under standard conditions, e.g., at approximately
150 millibars of pressure over a period of 1-3 days to dry the
composition following freezing. For example, alginate alone
(without gauze) is suitable for directly contacting a wound. In
other examples, alginate contains a woven mesh (e.g., gauze)
interspersed within the layer of alginate. In this case, in order
to fix the alginate portion of the bandage to a wound, the alginate
layer (with or without a woven mesh) is placed on the wound site
(where the alginate is shaped to the size of the wound or greater).
A standard wound cover (e.g., TEGADERM.TM.) is overlaid on the
alginate bandage, extending in all directions past the wound margin
and attaching to the skin.
[0058] In other examples, a bandage is constructed by contacting an
alginate sheet with a backing material (e.g., woven or non-woven
backing material, e.g., plastic, latex, gauze, cloth, or film).
Exemplary backing materials include MEPITEL.TM., JELONET.TM.,
OPSITE.TM., TEGADERM.TM., CARBOFLEX.TM., LYOFOAM C.TM., silicone
(e.g., silicone tape), or cotton). The backing material can be
porous or non-porous. For example, the backing material comprises a
tape or adhesive, e.g., an adhesive plastic strip or an adhesive
medical tape. For example, the contacting step is performed by
gluing, fusing, spraying (e.g., spraying foam onto), or otherwise
attaching the alginate sheet with the backing material. In some
examples, a bandage comprises a backing material and alginate
containing a woven mesh interspersed within a layer of
alginate.
[0059] The presence of a backing material and/or woven mesh on or
within the alginate in the bandage provides increased durability,
guard against moisture loss, and protection against abrasion
compared to alginate alone.
[0060] Pore size is controlled by the temperature at which the
alginate solution is frozen and the rate of temperature change. Ice
crystal size is dependent on the rate of freezing. The solution is
frozen, e.g., by placement in a constant temperature device, at
-20.degree. C. (standard freezer), -80.degree. C. (deep freezer),
or using liquid nitrogen, e.g., -160.degree. C., -180.degree. C.,
-200.degree. C., or any temperature in between to customize pore
size (average diameter). For example, pores with 300-800 .mu.m
diameter, e.g., 500 .mu.m diameter, are formed at -20.degree. C.;
pores with 100-400 .mu.m diameter, e.g., 100-200 .mu.m diameter,
are formed at about -70 to -88.degree. C.; and pores with 10-99/100
.mu.m diameter, e.g., 50 .mu.m diameter, are formed using liquid
nitrogen, e.g., at about -180.degree. C. The pores are
interconnected and generally homogeneous throughout the alginate
composition. To promote formation of homogeneous pores, the mold
into which the alginate solution is poured is optionally
pre-chilled/frozen prior to pouring the solution into the mold. To
form heterogeneous pores, e.g., pores that are oriented or form
channels, cryo-organization is induced by creating a temperature
gradient. For example, a temperature gradient is created by placing
the alginate solution-containing mold between 2 plates, one plate
having a warmer temperature than the other plate. In this manner,
ice crystal formation occurs in a single direction, e.g., from the
cold surface toward the warmer surface.
[0061] During the rapid cross-linking phase a minimum volume of
calcium chloride solution (100 mM) is used to crosslink the device.
By rapid crosslinking phase is generally meant one hour or less,
e.g., 5, 10, 15, 30, 45 minutes. Typically, crosslinking time is
about 15 min. The cryo-organized alginate structure is dry
(freeze-dried) prior to being contacted with the ionic crosslinking
solution. Thus, the volume of crosslinking solution added to the
dry composition is at least the volume of the composition and
typically 1-10 times, e.g., 1-2 times, the volume of the
composition (mold volume).
[0062] The rapid crosslinking in a small volume ensures that the
microstructure of freeze-drying is maintained and a minimum amount
of drug is lost. FIG. 2 shows the fabrication process that uses an
intermediary freeze-drying step to create micropores in the
alginate that introduce flexibility into the sheet, and FIG. 3
shows a freeze-dried non cross-linked cryo-organized alginate
(left) and a SEM image (right) depicting a typical macroporous
network structure.
[0063] Two strategies have been used to ionically crosslink the
cryo-organized alginate. First, a minimum volume of an aqueous
calcium chloride solution (100 mM), e.g., calcium chloride
dissolved in a physiologically acceptable buffer such as phosphate
buffered saline (PBS) or HEPES buffer, was used to crosslink the
device. The rapid crosslinking in a small volume ensures that the
microstructure of freeze-drying is maintained and a minimum amount
of drug is lost. A second method has been used to crosslink
alginate in a non-aqueous solvent such as ethanol. An advantage of
non-aqueous crosslinking solution is that it minimizes swelling of
the alginate. As shown in FIG. 3, a macroporous cryo-organized
structure can be formed after freeze-drying an aqueous solution of
alginate. The lyophilized cryo-organized alginate (FIG. 4) was
cross-linked in a solution of ethanol containing calcium nitrate
(0.2M). Alginate being insoluble in organic solvents, especially in
alcohols, the cryo-organized interconnected defined polymer
structure does not get disrupted due to the lack of water, which
usually leads to polymer dispersion and subsequent dissolution.
Ethanol crosslinking provides a better retention and definition of
the initial cryo-organized microstructure when compared to the
aqueous crosslinking. Therefore, the macroporous structure is
preserved during the calcium cross-linking process while entrapping
molecules of drugs within the polymer walls.
[0064] There are two options for the final step with regard to the
aqueously crosslinked alginate structure. In the first scenario,
the device is cross-linked and applied immediately to the wound. In
this case, the sterile freeze-dried alginate sheet+drugs and the
sterile calcium chloride cross-linking solution are both supplied
to the end user in a `smart packaging` design that separates the
alginate from the crosslinking solution until the user activates a
push-and-pop mechanism (smartpack) that brings them together to
permit easy cross-linking. This configuration is particularly
applicable to patients with diabetic foot ulcers as they are
familiar with such a system. Diabetic patients with skin/tissue
ulcers such as foot ulcers already pre-wet bandages in saline
before applying them, and this approach only differs in that the
pre-wetting solution corresponds to the aqueous crosslinking
solution, e.g., calcium chloride in water or aqueous buffer. The
freeze-dried alginate bandage packaged dry, and the user contacts
the dry bandage with the crosslinking solution immediately prior to
applying the bandage to the skin or other tissue to be treated.
[0065] Alternatively, since the cross-linking process maintains the
microstructure, it is cross-linked by the manufacturer and once
again freeze-dried for long-term storage. Testing data in which the
bandages underwent the dual freeze-drying process indicated that
that the device maintains its desirable flexibility and mechanical
properties after the second freeze-drying step. Likewise, for the
ethanol cross-linking route, freeze-dried cryo-organized
alginate-based drug-loaded bandages are readily provided for
long-term storage or embedded in a saline solution for immediate or
short-term use. The alginate bandage is applied to the skin using
standard coverings, e.g., TEGADERM.TM.. FIG. 5 shows application of
alginate bandaging to a wound.
[0066] In some embodiments, the devices/bandages of the invention
comprise a SP peptide, SP-related molecule, SP fragment, or SP
peptide derivative composition having a particular consensus amino
acid sequence. For example, the consensus amino acid sequence
comprises
Xaa.sub.1-Pro-Xaa.sub.2-Pro-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6(SEQ
ID NO: 12). For example, Xaa.sub.1 and Xaa.sub.2 are positively
charged amino acids, Xaa.sub.3 and Xaa.sub.4 are any amino acids
other than Pro, and Xaa.sub.5 and Xaa.sub.6 are hydrophobic amino
acids. Xaa.sub.5 and Xaa.sub.6 are preferably aromatic amino acids.
For example, Xaa.sub.5 and Xaa.sub.6 are Phe or Trp.
[0067] In some cases, the amino acid sequence of the peptide
contains at least residues 1-8 of
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met (SEQ ID No: 1). In
other cases, the amino acid sequence of the peptide contains at
least residues 1-8 of
Arg-D-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-Leu-Met (SEQ ID No:
2).
[0068] Other exemplary SP peptide, SP-related molecule, SP
fragment, or SP peptide derivative compositions include bradykinin,
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (SEQ ID NO: 3); neurotensin,
Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu (SEQ ID NO: 4)
or Xaa-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu (SEQ ID NO:
13; where Xaa is Pyr or Tyr); indolicidin,
Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH2 (SEQ ID NO:
5), Lys-Pro-Arg-Pro-Gly-Gln-Phe-Phe-Gly-Leu-Met (SEQ ID NO: 6),
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met (SEQ ID NO: 7),
Arg-Pro-Arg-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met (SEQ ID NO: 8),
Lys-Pro-Arg-Pro-Gln-Gln-Phe-Ile-Gly-Leu-Met (SEQ ID NO: 9),
Lys-Pro-Arg-Pro-His-Gln-Phe-Phe-Gly-Leu-Met (SEQ ID NO: 10), or
Ala-Lys-His-Asp-Lys-Phe-Tyr-Gly-Leu-Met (SEQ ID NO: 11).
[0069] In some examples, the peptide contains levorotatory (L)
and/or dextrorotatory (D) forms of an amino acid. For example, the
peptide has at least one D amino acid.
[0070] For example, a SP peptide, SP-related molecule, SP fragment,
or SP peptide derivative composition has antimicrobial activity and
contains an amino acid sequence that is at least 50% identical to
the amino acid sequence of SEQ ID NO: 1. In some cases, the
peptides are at least 75% identical, 85%, 95%, and 99% identical to
the sequences of SEQ ID NO: 1 or 2. Nucleotide/amino acid sequence
comparisons can be carried out using the Clustal W method or
Clustal V method. (Higgins et al., 1989, CABIOS 5(2):151-153).
[0071] A conservative substitution of one amino acid for another is
a replacement by an amino acid having a similar chemical functional
side group, e.g., replacement of a positively charged amino acid by
another positively charged amino acid, or replacement of a
hydrophobic amino acid by another hydrophobic amino acid. The
charge and hydrophobicity of amino acids is well known in the
art.
[0072] In some cases, antimicrobial synthetic peptides having at
least 50% identity to SP are produced by commonly known methods,
such as the Merrifield solid-phase chemical synthesis method or by
recombinant techniques involving the expression in cultured cells
of recombinant DNA molecules encoding a gene for a desired portion
of a natural or analog SP molecule. See, e.g., U.S. Pat. No.
7,723,467, the contents of which are incorporated herein by
reference in its entirety.
[0073] The invention also includes synthetic SP peptide derivative
compounds, which can comprise amino acid analogs such as D-amino
acids, or which can be non-peptide compositions or peptide
mimetics. The SP peptide derivative compounds and peptide mimetics
have functional antimicrobial activity comparable to that of known
SP peptides. The antimicrobial activity is for example, from about
half of the activity of SP peptide, to about 2-fold, about 4-fold,
or about 10-fold greater than that of SP Peptide. For example, a SP
derivative is a small molecule with a molecular weight of about 100
to about 1000 Da. In other examples, a SP derivative includes
analogs in which at least 1 peptide bond is replaced with an
alternative type of covalent bond (a "peptide mimetic") that is
resistant to cleavage by peptidases. In some examples, an L-amino
acid is replaced by a D-amino acid residue; this replacement
reduces the sensitivity of the compound to enzymatic destruction.
In some embodiments, the SP derivative includes an amino acid
analog, e.g., norleucine, norvaline, homocysteine, homoserine, or
ethionine. In some cases, the SP derivative is derivatized with an
amino-terminal blocking group such as a t-butyloxycarbonyl, acetyl,
methyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl,
dansyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl,
methoxyaselayl, methoxyadipyl, methoxysuberyl, and a
2,3-dinitrophenyl group. For example, blocking the charged amino-
and carboxy-termini of the peptide derived compound enhances the
solubility of the compound in the hydrophobic environment of the
cell membrane of the target microorganism. Such mimetics and
methods of incorporating them into peptides, are well known in the
art. See, e.g., U.S. Pat. No. 7,723,467, the contents of which are
incorporated herein by reference in its entirety.
[0074] In some embodiments, the devices/bandages of the invention
are effective in reducing pain in a subject. Measurements and
scales of pain intensity are known in the art (see, e.g.,
Minimising pain at wound dressing-related procedures. A consensus
document. London: MEP Ltd, 2004). For example, pain is quantified
by the Wong-Baker FACES scale from 0-5 (where 0 indicates no pain
and 5 indicates that it hurts worst); the visual analogue scale
from 0-10 (where 0 indicates no pain and 10 indicates the worst
pain), in which the patient is asked to pick a point on the
continuum that best reflects how he/she is feeling; the numerical
rating scale from 0-10 (where 0=no pain and 10=worst possible
pain), in which the patient is asked to choose an integer that best
places his/her current pain level; or the verbal rating scale in
which the patient is asked which word best describes his/her
current pain level (e.g., no pain, mild pain, moderate pain, or
severe pain).
[0075] In some examples, the devices/bandages of the invention are
effective in reducing pain in a subject by at least 5%, e.g., at
least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, or greater, compared to the level of pain prior to
administration of the device/bandage or is reduced by one or more
units on the scale of 0-10 or 0-5 as described above. In some
cases, pain, e.g., at the site of administration, is eliminated
after administration of the device/bandage.
EXAMPLE 1
Evaluation of Exemplary Drug Eluting Bandages
[0076] Alginate is a preferred ionically crosslinked substance from
which to fabricate bandages or wound dressings due to its ability
maintain moisture at the tissue site and its generally non-adhesive
properties (i.e., does not stick to wounds). To demonstrate this
device, a high molecular weight (HMW) medical grade alginate
(ProNova Biomedical (Norway), HMW MVG alginate) was used at 2%
(w/v), 4% (w/v) solution. In this example, the alginate was medium
viscosity (>200 mPas) sodium alginate where minimum 60% of the
monomer units are guluronate (G/M ratio.gtoreq.1.5), with molecular
weight of >200 kDa. Alginate for fabrication of biomaterials is
well known in the art, e.g., Augst et al., 2006, Macromol Biosci 6,
623-633, see e.g., FIG. 1a; contents of publication hereby
incorporated by reference. Unoxidized alginate is preferred for
fabrication. For drug delivery, non-derivitized alginate, i.e.,
without L-arginine, glycine, and L-aspartic acid. (RGD)
modification, is preferred so that the alginate does not stick to
wounded or diseased tissue. For cell delivery, the alginate is
optionally derivitized with RGD.
[0077] Alginate structures were made with and without an additional
standard cotton gauze mesh embedded. This type of cotton gauze is
used routinely in wound dressing; however, other types of gauze can
be used as well. Further manipulation and enhancement of the
bandage mechanical properties is accomplished through the choice of
the incorporated mesh. In this example, Dynarex Conforming Stretch
Sterile Gauze for testing. To embed the gauze mesh, it was placed
in a mold prior to addition of the alginate. The compositions are
fabricated in any desired size and shape Unless otherwise noted in
this example, the bandage dimensions were 3 cm.times.5 cm and 2 mm
thick and they were cross-linked for 15 mins in 3 ml (for 2%) or 6
ml (for 4%) of 100 mM CaCl.sub.2 in a commercially available HEPES
buffer.
Drug Release
[0078] The release characteristics of the test agent, Substance P
(Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met; RPKPQQFFGLM; SEQ ID
NO: 1)), from the 2% (w/v) and 4% (w/v) bandaging sheets
demonstrated its ability to retain the substance and control a
sustained release of the peptide from the bandage for at least 3
days (typical duration prior to bandage change in the clinic).
[0079] The release was performed by placing the bandage sample of 8
mm diameter and 2 mm thickness in 2 mls of PBS and moving it to a
new solution at defined time intervals. The release was examined
using a commercially available ELISA assay. FIG. 6 shows the
percent of Substance P (remaining after processing loss) released
from the bandage material over time. The sheets incorporate either
32 .mu.g or 64 .mu.g of Substance P with a given processing loss
(Sample size n=3 per time point). Small molecule compounds (less
than 1000 daltons) are typically added to the composition in a
volume of 250 .mu.l for a dressing that is about 12 mm in diameter
and 2 mm thick. Thus, substance P was added at 128 .mu.g/ml at the
low concentration and 256 .mu.g/ml at the higher concentration.
Actual clinical doses are the same or similar Since small molecules
may diffuse out of the dressing quickly, it is advantageous for
small molecules to have an affinity for the alginate, e.g., since
alginate is negatively charged, positively charged small molecule
drugs elute at a clinically acceptable and beneficial rate (and
consistent with standard practice for changing of wound dressings).
For example, substance P is slightly positively charged and is
therefore attracted to the alginate. Charge is less or not relevant
for larger molecules, e.g., BSA. Generally, the dose is determined
based on the therapeutic dose of the drug. For example, VEGF is
administered to the dressing at about 50 .mu.g/ml (3 .mu.g in 60
.mu.l of liquid).
[0080] The data also demonstrates the ability of the bandage
constituents to be adjusted to further control the release
characteristics; increased drug release from the alginate material
is achieved through lower sheet fiber density and/or higher drug
incorporation.
Exemplary Agents
[0081] The applicability of the alginate material to the release of
other types of drugs and factors was also examined Three additional
model drugs, representing classes of drugs, were tested: [0082] 1)
Trypan blue dye: a small molecule that does not interact with the
scaffold [0083] 2) Mitoxantrone: a cationic small molecule that
interacts with the anionically charged alginate chains [0084] 3)
Bovine Serum Albumin: a protein that is not subject to steric
hindrance in a cross-linked alginate network, which controls its
diffusion through the gel.
[0085] Drug release was recorded for 4% (w/v) alginate pieces 8 mm
in diameter and 2 mm in thickness that were cross-linked for 15
minutes. FIG. 7 shows percent of Trypan Blue, Mitoxantrone &
BSA (remaining after processing loss) released from the bandaging
material over time. (Sample size of study n=3 per time point).
[0086] The group of drugs differ in their affinity to the
negatively charged nodes of the alginate fibers: mitoxatrone
(alginate binding), trypan blue dye (negatively charged small
particles easily escape the alginate structure), bovine serum
albumin (negatively charged at pH 7 and hence is repelled from the
alginate fibers). Their release profiles from the alginate dressing
material reflect the drug-alginate interaction accordingly: the
trypan is lost very rapidly (24% loss during wetting step and the
majority within one day); mitoxantrone undergoes a sustained
release from the scaffold, which is still ongoing at the
termination of the experiment; the BSA (m.w. approximately 66.5
kDa) is repelled from the alginate scaffold, however, given its
size and the density of alginate it undergoes steric hindrance and
retarded diffusion from the scaffold. Taken collectively, these
data demonstrate the ability of alginate to exhibit controlled
release of a range of drugs from the bandage.
Mechanical Properties
[0087] The ability of the wetted bandages to handle the wear and
tear of use is characterized by various tensile loading tests done
on 2 mm thick pieces of the alginate sheets that were 12.5 mm wide
and 25 mm between the grips. Engineering strength and elasticity
(Young's modulus) are determined using standard methods, e.g.,
those described in engineering textbooks such as Ashby M F and
Jones, D R H, 2011, Engineering Materials I, Fourth edition,
Elsevier. FIG. 8 shows the tensile properties of the bandaging
material strengths with and without gauze at 2% and 4% alginate.
(Sample size n=3). These tensile properties demonstrate excellent
handling potential of the dressing in clinical use, allowing for
intact application and removal even at highly stressed wound areas.
Based on a survey of existing bandages conducted by the inventors,
150 kPa is approximately the average failure stress of those
dressings; our dressing when combine with gauze exceeds this by
approximately 4-fold.
Fatigue Testing
[0088] To ensure that the loading from application of the bandage
under a foot was well sustained by the bandage, the device was
subject to cyclic compression testing. The compression cycle was
constructed to imitate the loading on a foot during walking and
accounted for the average daily steps taken by a diabetic subject
(4000/day) for three days, with the force adjusted to available
diabetic foot pressure values (1 kPa; see FIG. 9). The device
survived the loading regimen, demonstrating that it was able to
endure a greater number of step simulations or greater force
loading with steps.
Mechanical Properties as a Function of Time
[0089] To evaluate the ability of the device to absorb and maintain
moisture, tests were conducted on a bandage of 2 mm thickness (FIG.
10). A stability test (measured by weight change) was also
conducted to determine if the bandaging experienced significant
degradation and loss of mechanical robustness (FIG. 10). (Note: all
testing sample size n=3). At 37.degree. C., the device was able to
absorb liquid (phosphate buffered saline containing physiologic
levels of calcium and magnesium) up to 20 times the weight of the
alginate material itself.
[0090] The device also retained most of this moisture with loss of
less than 20% of the contained liquid with each passing day (at
37.degree. C. in non-sealed containers). In vivo, any losses may in
fact be remediated by exudate absorption as well as the sealing
provided by a secondary covering over the alginate dressing.
[0091] When the device was placed in a calcium free environment,
there was an initial mass loss (attributed to calcium removed from
the device), but afterwards the weight remained constant. The
diffusion of Ca.sup.2+ reduced the mechanical strength and Young's
Modulus of the material (FIG. 11). However in vivo, calcium in the
serum/exudate in the wound environment and minimizes these effects
on mechanical properties of the device.
EXAMPLE 2
Animal Studies
[0092] To test the device handling in a clinical setting, a rabbit
study was performed with two rabbits. Diabetes was induced in the
rabbits using alloxan and their blood glucose monitored and
controlled. The central auricular artery and nerve were isolated
and cut (neuroischemia) or left intact (sham). Four full thickness
skin wounds were then created using a biopsy punch, and when
bleeding had ceased, the wounds were filled with the prefabricated
alginate bandages as shown in FIG. 12.
[0093] The reports back from the surgeons and animal care staff
regarding the ability of the bandage to be applied, to stay in
place, and to be easily removed were positive.
EXAMPLE 3
Rabbit Ear Wound Healing Model
[0094] Experiments are performed to test the efficacy of the
bandage in a neuroischemic and a neuroischemic+diabetes rabbit ear
wound healing model using known methods, e.g., those described in
Pradhan et al., J. Vasc. Surg. 2013, 58(3): 766-775, incorporated
herein by reference. The bandages are tested alone; with
Substance-P alone (32 .mu.g); with VEGF alone (3 .mu.g); or with a
combination of VEGF and Substance-P. Bandages are exchanged every 3
days (a typical timeframe for cleaning) and the wound area examined
at 10 days for % percent healing and histological analysis.
[0095] The described alginate sheet material hence has numerous
applications as a wound dressing that maintains moist and
non-traumatic healing environment and deliver therapeutic elements
in a controlled manner A highly absorptive alginate bandage with
good handling properties that can control release of select drugs
was developed utilizing a medical grade alginate, e.g., alginate
that has FDA approval in other applications, and a standard gauze
material. The fabrication process described herein yields a strong,
flexible, crosslinked alginate product that is superior to
conventional alginate bandages or dressings.
OTHER EMBODIMENTS
[0096] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. All United States patents and published or unpublished
United States patent applications cited herein are incorporated by
reference. All published foreign patents and patent applications
cited herein are hereby incorporated by reference. All other
published references, documents, manuscripts and scientific
literature cited herein are hereby incorporated by reference.
[0097] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
13111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met1 5
10211PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(2)..(2)D-amino acidMOD_RES(7)..(7)D-amino
acidMOD_RES(9)..(9)D-amino acid 2Arg Pro Lys Pro Gln Gln Trp Phe
Trp Leu Met1 5 1039PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 3Arg Pro Pro Gly Phe Ser Pro Phe Arg1
5413PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Glu Leu Tyr Glu Asn Lys Pro Arg Arg Pro Tyr Ile
Leu1 5 10513PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideC-term NH2 5Ile Leu Pro Trp Lys Trp Pro
Trp Trp Pro Trp Arg Arg1 5 10611PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 6Lys Pro Arg Pro Gly Gln
Phe Phe Gly Leu Met1 5 10711PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 7Arg Pro Lys Pro Gln Gln Phe
Phe Gly Leu Met1 5 10811PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 8Arg Pro Arg Pro Gln Gln Phe
Phe Gly Leu Met1 5 10911PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 9Lys Pro Arg Pro Gln Gln Phe
Ile Gly Leu Met1 5 101011PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 10Lys Pro Arg Pro His Gln Phe
Phe Gly Leu Met1 5 101110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 11Ala Lys His Asp Lys Phe Tyr
Gly Leu Met1 5 10128PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(1)..(1)Any positively charged
amino acidMOD_RES(3)..(3)Any positively charged amino
acidMOD_RES(5)..(6)Any amino acid other than ProMOD_RES(7)..(8)Any
hydrophobic amino acid 12Xaa Pro Xaa Pro Xaa Xaa Xaa Xaa1
51313PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)Pyr or Tyr 13Xaa Leu Tyr Glu Asn
Lys Pro Arg Arg Pro Tyr Ile Leu1 5 10
* * * * *