U.S. patent application number 09/899372 was filed with the patent office on 2001-11-29 for soluble keratin peptide.
Invention is credited to Blanchard, Cheryl R., Siller-Jackson, Arlene J., Smith, Robert A., Timmons, Scott F., Van Dyke, Mark E..
Application Number | 20010047082 09/899372 |
Document ID | / |
Family ID | 23290261 |
Filed Date | 2001-11-29 |
United States Patent
Application |
20010047082 |
Kind Code |
A1 |
Van Dyke, Mark E. ; et
al. |
November 29, 2001 |
Soluble keratin peptide
Abstract
A peptide derived from keratin, which can be used as a
wound-healing agent. In one method for making the peptide, a
keratin source such as human hair is washed, dried, and treated
with an oxidizing agent such as peracetic acid for a time and
temperature sufficient to swell the keratin and oxidize some of the
disulfide bonds to form sulfonic acid groups. The oxidation is
believed to form a series of water-soluble peptides. The oxidized
hair can be filtered, and the filtrate collected and concentrated
under vacuum distillation to a viscous syrup, which can be
neutralized with base. The concentrate can be mixed with an excess
of a water-miscible organic solvent such as methanol, and the
precipitate collected and dried to form the wound-healing agent.
The wound-healing agent is believed to include peptides having a
molecular weight centered around 850 daltons and having at least
one ionizeable group such as sulfonic acid.
Inventors: |
Van Dyke, Mark E.; (Fair
Oaks Ranch, TX) ; Blanchard, Cheryl R.; (Warsaw,
IN) ; Timmons, Scott F.; (San Antonio, TX) ;
Siller-Jackson, Arlene J.; (Helotes, TX) ; Smith,
Robert A.; (Jackson, MS) |
Correspondence
Address: |
Timothy S. Corder
VINSON & ELKINS LLP
2300 First City Tower
1001 Fannin
Austin
TX
77002-6760
US
|
Family ID: |
23290261 |
Appl. No.: |
09/899372 |
Filed: |
July 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09899372 |
Jul 2, 2001 |
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09330550 |
Jun 11, 1999 |
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6270791 |
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Current U.S.
Class: |
530/357 ;
530/350 |
Current CPC
Class: |
A61P 17/00 20180101;
A61K 8/0208 20130101; A61K 38/17 20130101; A61Q 19/00 20130101;
C07K 14/4741 20130101; A61K 8/66 20130101; A61K 8/64 20130101; A61P
17/02 20180101; A61Q 19/08 20130101; A61K 8/65 20130101; A61K 8/02
20130101 |
Class at
Publication: |
530/357 ;
530/350 |
International
Class: |
C07K 014/435 |
Claims
What is claimed is:
1. A water-soluble peptide made by the process comprising the steps
of: providing keratinous material having disulfide linkages;
oxidizing said keratinous material with an oxidizing agent, such
that some disulfide linkages are cleaved and oxidized to form
sulfonic acid groups; filtering said oxidized keratinous material
and collecting a filtrate; neutralizing the pH of said filtrate;
adding said filtrate to a water-miscible organic solvent, such that
a precipitate is formed; collecting said precipitate; and drying
said precipitate.
2. A peptide as recited in claim 1, further comprising a
concentrating step wherein said filtrate is concentrated before
said neutralizing step.
3. A peptide as recited in claim 1, further comprising a
concentrating step wherein said filtrate is concentrated after said
neutralizing step.
4. A peptide as recited in claim 1, further comprising a
concentrating step wherein said filtrate is concentrated before
being added to said water-miscible organic solvent.
5. A peptide as recited in claim 4, further comprising a
concentrating step wherein said filtrate is concentrated after said
neutralizing step.
6. A peptide made by the process comprising the steps of: providing
vertebrate hair having disulfide linkages; washing said hair in
water; drying said hair in air; oxidizing said hair with about 2
volume percent peracetic acid, in an amount of about 30 grams of
hair to about 500 mL of peracetic acid, at boiling temperature for
about 5 hours, such that some disulfide linkages are cleaved and
oxidized to form sulfonic acid groups; filtering said oxidized hair
and collecting a filtrate; concentrating said filtrate about
ten-fold by vacuum distillation at about 5 to 10 mm Hg using a pot
temperature of about 40 degrees C; neutralizing said concentrated
filtrate pH with about 3 to 4 Normal Ammonium Hydroxide; adding
said neutralized filtrate to methanol, in an amount of about 1
liter of neutralized, concentrated, filtrate to about 6 to 10
liters of methanol, such that a precipitate is formed; collecting
said precipitate; and drying said precipitate using evaporation
without heat application.
7. A peptide as recited in claim 6, wherein said provided
vertebrate hair is human hair.
8. A method for making a water-soluble peptide comprising the steps
of: providing a keratinous material having disulfide linkages;
oxidizing said keratinous material with an oxidizing agent, such
that some disulfide linkages are cleaved and oxidized, such that
water-soluble peptides are formed; and separating said
water-soluble peptides from said keratinous material and collecting
said water-soluble peptides.
9. A method for making a water-soluble peptide as recited in claim
8, further comprising the step of drying said collected
peptides.
10. A method for making a water-soluble peptide as recited in claim
8, wherein said keratinous material includes human hair.
11. A method for making a water-soluble peptide as recited in claim
10, wherein said oxidizing step produces hydrophilic groups from
said cleaved disulfide groups.
12. A method for making a water-soluble peptide comprising the
steps of: providing keratinous material having disulfide linkages;
oxidizing said keratinous material with an oxidizing agent, such
that some disulfide linkages are cleaved and oxidized to form
sulfonic acid groups; filtering said oxidized keratinous material
and collecting a filtrate; neutralizing said filtrate;
precipitating said filtrate by adding said filtrate to a
water-miscible organic solvent, such that a precipitate is formed;
collecting said precipitate; and drying said precipitate.
13. A method for making a water-soluble peptide as recited in claim
12, wherein said oxidizing agent is selected from the group
consisting of peracetic acid, hydrogen peroxide, peroxy carbonates,
ammonium sulfate peroxide, perborates, hypochlorite, chlorine
dioxide, sodium and calcium peroxide.
14. A method for making a water-soluble peptide as recited in claim
12, wherein said oxidizing agent is present in a strength of at
least about 1 volume percent.
15. A method for making a water-soluble peptide as recited in claim
12, wherein said oxidizing step is carried out at sufficient
oxidant concentration and temperature and for sufficient time to
provide at least about 5 weight percent of said keratinous material
as dried precipitated keratin peptide filtrate.
16. A method for making a water-soluble peptide as recited in claim
12, wherein said oxidizing step is carried out at sufficient
oxidant concentration and temperature and for sufficient time to
provide at least about 10 weight percent of said keratinous
material as dried precipitated keratin peptide filtrate.
17. A method for making a water-soluble peptide as recited in claim
12, wherein said oxidizing step is carried out at sufficient
oxidant concentration and temperature and for sufficient time to
provide at least about 15 weight percent of said keratinous
material as dried precipitated keratin peptide filtrate.
18. A method for making a water-soluble peptide as recited in claim
12, wherein said oxidizing step is carried out at sufficient
oxidant concentration and temperature and for sufficient time to
provide about 20 weight percent of said keratinous material as
dried precipitated keratin peptide filtrate.
19. A method for making a water-soluble peptide as recited in claim
12, wherein said oxidizing step is carried out at in at least 1
volume percent of an oxidizing agent selected from the group
consisting of peracetic acid and hydrogen peroxide, wherein said
oxidizing step is carried out at boiling temperature and for at
least about 2 hours.
20. A method for making a water-soluble peptide as recited in claim
19, wherein said oxidizing step is carried out for at least about 4
hours.
21. A method for making a water-soluble peptide as recited in claim
12, wherein said oxidizing step is carried at a temperature of at
least about 90 degrees C and for at least about 5 hours.
22. A method for making a water-soluble peptide as recited in claim
12, wherein sufficient disulfide linkages are cleaved in said
oxidizing step to produce a soluble form of keratin.
23. A method for making a water-soluble peptide as recited in claim
12, wherein said keratin material includes human hair.
24. A method for making a water-soluble peptide as recited in claim
12, wherein, at said precipitating step, said filtrate has a
concentration of between about 3 and about 15 grams of soluble
peptides per liter, and between about 5 and about 34 liters of said
water miscible organic solvent is used per gram of soluble peptides
in said liter.
25. A method for making a water-soluble peptide as recited in claim
12, wherein, at said precipitating step, said filtrate has a
concentration of between about 3 and about 15 grams of soluble
peptides per liter, and between about 60 and about 100 liters of
said water miscible organic solvent is used per liter of
filtrate.
26. A method for making a water-soluble peptide as recited in claim
12, wherein, said filtrate is concentrated after said filtration,
and, at said precipitating step, has a concentration of between
about 30 and about 150 grams of soluble peptides per liter, and
between about 6 and about 10 liters of said solvent is used per
liter of concentrated filtrate.
27. A method for making said water-soluble peptide as recited in
claim 12, wherein said filtrate is concentrated after said
neutralizing step.
28. A method for making said water-soluble peptide as recited in
claim 12, wherein said filtrate is concentrated before being added
to said water-miscible organic solvent.
29. A method for making said water-soluble peptide as recited in
claim 12, wherein said solvent is selected from the group
consisting of methanol, ethanol, acetone, and tetrahydrofuran.
30. A water-soluble peptide comprising a peptide chain having
between about 5 and about 15 amino acids and having at least one
ionizable pendant group at physiological pH.
31. A water-soluble peptide as recited in claim 30, wherein said
ionizable pendant group is sulfonic acid.
32. A water-soluble peptide as recited in claim 30, wherein said
peptide is about 10 amino acids long and derived from keratin and
said ionizable pendant group is sulfonic acid.
33. A water-soluble peptide as recited in claim 30, wherein about
90% of said peptides fall between about 300 and about 1300 daltons
in molecular weight and are derived from keratin and said ionizable
pendant group includes sulfonic acid.
34. A water-soluble peptide as recited in claim 30, wherein said
peptide has a mean molecular weight of about 850 daltons.
35. A method for treating a wound comprising the steps of:
providing a water-soluble peptide having between about 5 and about
15 amino acids and having at least one ionizable pendant group at
physiological pH; and applying said peptide to said wound.
36. A method for treating a wound as recited in claim 35, wherein
said providing step provides ionizable pendant groups including
sulfonic acid.
37. A method for treating a wound as recited in claim 35, wherein
said providing step includes said peptide having a mean molecular
weight of about 850 daltons and wherein said providing step
ionizable pendant groups includes sulfonic acid.
38. A method for treating a wound comprising the steps of:
providing a wound healing agent consisting essentially of a
water-soluble peptide having between about 5 and about 15 amino
acids and having at least one ionizable pendant group at
physiological pH; and applying said wound healing agent to said
wound.
39. A method for treating a wound comprising the steps of:
providing a wound-healing agent consisting essentially of a product
made by the process including the steps of providing hair having
disulfide linkages, oxidizing said hair with an oxidizing agent,
such that some disulfide linkages are cleaved and oxidized to form
sulfonic acid groups, filtering said oxidized hair and collecting a
filtrate, neutralizing the pH of said filtrate, adding said
filtrate to a water-miscible organic solvent such that a
precipitate is formed, collecting said precipitate, and drying said
precipitate; and applying said wound healing agent to said
wound.
40. A method for treating a wound as recited in claim 39, wherein
said method does not include applying a substance which
substantially chemically alters said wound healing agent.
41. A method for treating a wound as recited in claim 39, further
comprising combining said dried precipitate with a water-based
formulation.
42. A method for treating a wound as recited in claim 39, further
comprising applying said precipitate to a wound dressing.
43. A method for treating a wound as recited in claim 39, wherein
said wound healing agent applied to said wound is formed of pure
keratin.
44. A method for treating a wound as recited in claim 39, wherein
said wound healing agent is added to a keratin sheet prior to being
applied to said wound.
45. A method for treating a wound as recited in claim 39, wherein
said wound healing agent is added to a keratin hydrogel prior to
being applied to said wound.
46. A method for treating a wound as recited in claim 39, wherein
said hair includes human hair.
47. A composition for topical application to skin made by the
process comprising the steps of: providing hair having disulfide
linkages; oxidizing said hair with an oxidizing agent, such that
some disulfide linkages are cleaved and oxidized to form sulfonic
acid groups; filtering said oxidized hair and collecting a
filtrate; neutralizing the pH of said filtrate; adding said
filtrate to a water-miscible organic solvent such that a
precipitate is formed; collecting said precipitate; drying said
precipitate; and mixing said precipitate with a carrier selected
from the group consisting of lotions, creams, and gels.
48. A composition as recited in claim 47, wherein said hair
includes human hair.
49. A tissue engineered implant comprising a keratin scaffold
including therein a keratin peptide made by the process comprising
the steps of: providing hair with disulfide linkages, oxidizing
said hair with an oxidizing agent, such that some disulfide
linkages are cleaved and oxidized to form sulfonic acid groups,
filtering said oxidized hair and collecting a filtrate,
neutralizing the pH of said filtrate, adding said filtrate to a
water-miscible organic solvent such that a precipitate is formed,
collecting said precipitate, and drying said precipitate, wherein
said peptide is disposed within said keratin scaffold to act as a
cell stimulant.
50. A tissue engineered implant as recited in claim 49, wherein
said hair includes human hair.
51. A method for treating keratinous tissue comprising the steps
of: providing a wound-healing agent consisting essentially of a
product made by the process including the steps of providing hair
with disulfide linkages, oxidizing said hair with an oxidizing
agent, such that some disulfide linkages are cleaved and oxidized
to form sulfonic acid groups, filtering said oxidized hair and
collecting a filtrate, neutralizing the pH of said filtrate, adding
said filtrate to a water-miscible organic solvent such that a
precipitate is formed, collecting said precipitate, and drying said
precipitate; and applying said tissue healing agent to said
keratinous tissue.
52. A method for treating keratinous tissue as recited in claim 51,
wherein said tissue forms part of a gastrointestinal tract and said
applying step includes ingesting said tissue healing agent.
53. A. method for treating keratinous tissue as recited in claim
51, wherein said hair includes human hair.
54. A method for treating keratinous tissue as recited in claim 51,
wherein said tissue forms a part of a gastrointestinal tract and
said applying step includes intravenously injecting said tissue
healing agent.
Description
RELATED APPLICATIONS
[0001] The present application is related to U.S. Pat. No.
5,358,935 entitled NONANTIGENIC KERATINOUS PROTEIN MATERIAL; U.S.
patent application Ser. No. 09/057,161, filed Apr. 8, 1998,
entitled KERATINOUS PROTEIN MATERIAL FOR WOUND HEALING APPLICATIONS
AND METHOD; U.S. patent application Ser. No. 08/979,456, filed Nov.
26, 1997, entitled KERATIN-BASED HYDROGEL FOR BIOMEDICAL
APPLICATIONS AND METHOD OF PRODUCTION; U.S. patent application Ser.
No. 08/979,526, filed Nov. 26, 1997, entitled KERATIN-BASED SHEET
MATERIAL FOR BIOMEDICAL APPLICATIONS AND METHOD OF PRODUCTION; and
U.S. patent application Ser. No. 09/198,998, filed Nov. 24, 1998,
entitled METHOD OF CROSSLINING KERATIN-BASED FILMS, SHEETS AND BULK
MATERIALS, entitled WATER ABSORBENT KERATIN AND GEL FORMED
THEREFROM, all of which are herein incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention is related generally to water-soluble
peptides. More specifically, the present invention is related to
water-soluble peptides derived from keratin. In particular, the
present invention is related to water-soluble peptides formed from
chemically processed hair, which can be used as wound-healing
agents
BACKGROUND OF THE INVENTION
[0003] Chronic wounds can be caused by a variety of events,
including surgery, prolonged bedrest and traumatic injuries.
Partial thickness wounds can include second degree bums, abrasions,
and skin graft donor sites. Healing of these wounds can be
problematic, especially in cases of diabetes mellitus or chronic
immune disorders. Full thickness wounds have no skin remaining, and
can be the result of trauma, diabetes (e.g., leg ulcers) and venous
stasis disease, which can cause full thickness ulcers of the lower
extremities. Full thickness wounds tend to heal very slowly or not
at all. Proper wound care technique including the use of wound
dressings is extremely important to successful chronic wound
management. Chronic wounds affect an estimated four million people
a year, resulting in health care costs in the billions of dollars.
T. Phillips, O. Kehinde, and H. Green, "Treatment of Skin Ulcers
with Cultivated Epidermal Allografts," J. Am. Acad. Dermatol, V.
21, pp. 191-199 (1989).
[0004] The wound-healing process involves a complex series of
biological interactions at the cellular level which can be grouped
into three phases: homeostasis and inflammation; granulation tissue
formation and reepithelization; and remodeling. R. A. F. Clark,
"Cutaneous Tissue Repair: Basic Biological Considerations," J. Am.
Acad. Dermatol, Vol. 13, pp. 701-725 (1985). Keratinocytes
(epidermal cells that manufacture and contain keratin) migrate from
wound edges to cover the wound. Growth factors such as transforming
growth factor-.beta. (TGF-.beta.) play a critical role in
stimulating the migration process. The migration occurs optimally
under the cover of a moist layer. Keratins have also been found to
be necessary for reepithelization. Specifically, keratin types K5
and K14 have been found in the lower, generating, epidermal cells,
and types K1 and K10 have been found in the upper, differentiated
cells. I. K. Cohen, R. F. Diegleman, and W. J. Lindblad, eds.,
Wound Healing: Biochemical and Clinical Aspects, W. W. Saunders
Company, 1992. Keratin types K6 and K10 are believed to be present
in healing wounds, but not in normal skin. Keratins are major
structural proteins of all epithelial cell types and appear to play
a major role in wound healing.
[0005] An optimum wound dressing would protect the injured tissue,
maintain a moist environment, be water permeable, maintain
microbial control, deliver healing agents to the wound site, be
easy to apply, not require frequent changes and be non-toxic and
non-antigenic. Although not ideal for chronic wounds, several wound
dressings are currently on the market, including occlusive
dressings, non-adherent dressings, absorbent dressings, and
dressings in the form of sheets, foams, powders and gels. S.
Thomas, Wound Management and Dressing, The Pharmaceutical Press,
London, 1990.
[0006] Attempts have been made to provide improved dressings that
would assist in the wound-healing process using biological
materials such as growth factors. These biologicals have proven
very costly and, due to the lack of an appropriate delivery
vehicle, have shown minimal clinical relevance in accelerating the
chronic wound-healing process relative to their cost. In cases of
severe full thickness wounds, autografts (skin grafts from the
patient's body) are often used. Although the graft is
non-antigenic, it must be harvested from a donor site on the
patient's body, creating an additional wound. In addition,
availability of autologous tissue may not be adequate. Allografts
(skin grafts from donors other than the patient) are also used when
donor sites are not an option. Allografts essentially provide a
"wound dressing" that provides a moist, water-permeable layer, but
are rejected by the patient, usually within two weeks, and do not
become part of the new epidermis.
[0007] What would be advantageous is a non-toxic, non-antigenic,
inexpensive wound-healing agent having the ability to accelerate
the rate of wound healing and allow non-healing wounds to heal.
SUMMARY OF THE INVENTION
[0008] A water-soluble peptide derived from a keratinous source
such as hair and methods for making same are provided. One method
includes providing a keratinous material having disulfide linkages
and oxidizing the keratinous material with an oxidizing agent, such
that some disulfide linkages are cleaved and oxidized, forming
water-soluble peptides. The water-soluble peptides can be
separated, collected, dried, and used as a wound-healing agent. A
preferred source of keratinous material is hair, such as human
hair.
[0009] In one method, hair is oxidized with a sufficient
concentration of oxidizing agent for a sufficient time and
temperature so as to cleave a significant portion of the hair
disulfide bonds, such that some disulfide bonds are oxidized to
form hydrophilic groups such as sulfonic acid and such that
water-soluble peptides are produced. Examples of oxidizing agents
include, but are not limited to, hydrogen peroxide, peracetic acid,
percarbonates, persulfates, chlorine dioxide, sodium and calcium
peroxides, perborates, and hypochlorite. The oxidized hair can be
filtered, the filtrate collected, and neutralized with base. Water
soluble peptides from the neutralized filtrate can be precipitated
from solution by mixing the filtrate with a water-miscible organic
solvent such as methanol. The precipitate can be collected using
centrifugation and the collected filtrate dried. In one method,
about 20 percent of the original hair mass is collected as peptide
material after drying. The dried precipitate can be ground into a
fine powder.
[0010] Peptides produced according to the present invention are
largely water soluble and have an average molecular weight of about
850 daltons and an average chain length of about 10 amino acids.
The peptide chains are believed to have attached a hydrophilic
group, for example, an ionizable group such as sulfonic acid. In
particular, most peptides are believed to have at least one
sulfonic acid group formed from the oxidization of disulfide
linkages of hair. One product made according to the present
invention is a powder that is whitish to yellow in color and
readily soluble in water.
[0011] In use, the peptide powder can be placed over a wound as a
powder. The peptide powder can also be formulated into any
water-based solution, cream, gel, or other vehicle for convenient
application to a wound. In addition, a peptide solution could be
incorporated into or cast onto a polymer wound dressing or a
keratin wound dressing sheet for application to a wound. In in
vitro trials, the peptide wound-healing agent was shown to enhance
proliferation of human skin keratinocytes, human dermal
fibroblasts, and microvascular endothelial cells. Applicants
believe that the peptide fraction isolated according to the present
invention is a highly active form of a wound-healing agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a chromatogram showing the molecular weight
distribution of one sample of the soluble peptide made according to
the present invention; and
[0013] FIG. 2 is a table of experimental results illustrating
proliferation of cells critical to the wound-healing process after
application of a keratin peptide wound-healing agent made according
to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention provides a wound-healing agent which
can include a peptide having an ionizable pendant group such as
sulfonic acid which can be derived from an oxidized protein
disulfide linkage. A preferred source of protein is keratin. One
preferred source of keratin is hair. While hair is a preferred
source of keratinous material, other keratinous materials such as
animal hair, skin, beaks, hooves, feathers, and nails are also
suitable for use in the present invention. The patient or a human
donor are some preferred sources of hair, as hair from these
sources is most likely to result in a non-antigenic wound-healing
product, although animal hair may be acceptable for many
individuals. In one method according to the present invention, hair
is provided, preferably clean and unbleached. In another method,
the hair is washed with Versa-Clean TM (Fisher Scientific,
Pittsburgh, Pa.), rinsed with deionized water, and dried.
[0015] The hair can be oxidized in peracetic acid or another
suitable reagent such as H.sub.2O.sub.2. One method utilizes
between about 1% to 32% peracetic acid, at a temperature between
about 0 degrees C and 100 degrees C for between 0.5 and 24 hours.
One method treats 30 grams of hair with 500 mL of 4 volume percent
peracetic acid at 4 degrees C for 24 hours. Another method treats
the hair at room temperature for 24 hours. Yet another method
treats the hair at about 90 degrees C for about 10 hours. In a
preferred method, the hair is treated by boiling the hair in the
oxidizing agent for at least about 2 hours, and preferably for
about 5 hours. The higher oxidizing temperature is believed to
produce higher yields relative to the lower oxidizing temperature.
In various embodiments, at least 5, 10, and 15 weight percent of
the original hair weight is eventually recovered as peptides by
application of sufficient oxidant, heat, and time. In one
embodiment, about 20 weight percent of the original hair can be
eventually recovered as peptides, useful for the present invention.
The treatment with oxidizing agent is believed to open the cuticle
structure of the hair and to swell the keratin. Treatment with
oxidizing agent (e.g., H.sub.2O.sub.2 and peracetic acid) is
believed to at least partially oxidize the naturally occurring
disulfide linkages to produce a protein with cysteic acid side
groups (--CH.sub.2SO.sub.3H), which are also referred to as
sulfonic acid groups in the present application. The treatment with
oxidizing agent is not believed to break a substantial portion of
peptide backbone bonds. The result is believed to include
short-chain peptides that have at least one ionizable pendant group
such as sulfonic acid, where the peptides are soluble in water at
physiological pH. In general, the present invention includes the
production of water-soluble peptides from a keratinous material
including peptides having hydrophilic groups such as sulfonic acid
groups.
[0016] The oxidized hair can be recovered, for example with
filtration through a coarse fritted glass filter, and the filtrate
collected. Filtration thus separates the water-soluble peptides
from the remaining keratin source such as hair, allowing collection
of the water-soluble peptides in the filtrate. While filtration is
a preferred separation method, other suitable separation methods
such as decantation or dialysis are also within the scope of the
invention. The hair can be rinsed numerous times with deionized
water to increase the amount of soluble protein that is washed off
the hair. The hair can be discarded or kept for other uses. The
filtrate in one embodiment is concentrated about ten-fold using
vacuum distillation, leaving a viscous syrup. In one method, the
filtrate is concentrated until the concentrated filtrate contains
about 120 grams of peptide per liter. One vacuum distillation
method uses between about 5 and 10 mm Hg pressure at a pot
temperature of about 40 degrees C. In some methods, concentration
is performed at a later stage of the process.
[0017] The filtrate can be neutralized using a base. The filtrate
is neutralized to facilitate the precipitation of the peptides. In
one method, 3 to 4 normal ammonium hydroxide is used as a base in
the amount of about 0.1 mL to 1 mL of the viscous syrup formed from
concentrating the filtrate. In another method, about 1 mL of base
is added to 100 mL of unconcentrated filtrate. The base can be
added until the pH is about 7.
[0018] Neutralized filtrate can then be mixed with a water-miscible
organic solvent such as methanol, ethanol, acetone, or
tetrahydrofuran. In one embodiment, about 6 to 10 mL of methanol is
added to 1 mL of the viscous syrup containing the concentrated
filtrate. In another embodiment, about 60 to 100 mL of methanol is
added to 1 mL of the unconcentrated filtrate. The water-soluble
peptide has lower solubility in the organic solvent/water mixture
and cleanly precipitates out.
[0019] The precipitate can then be collected using well known
methods such as centrifugation, filtration, or decanting. The
precipitate can then be dried using evaporation, preferably without
the application of heat. In one method, the precipitate is dried at
room temperature. In another method, the precipitate is dried under
vacuum, again without the application of heat. No grinding is
required, as the precipitate obtained, when dry, is already a
powder. In some embodiments, the dried precipitate is further
processed by grinding into a fine powder using a mortar and pestle
or equivalent grinding instrument.
[0020] The powder obtained from one method is whitish to yellow in
color and is completely soluble in water. Analysis of these samples
have shown them to be Gaussian distributions of low molecular
weight peptides, as shown in the chromatogram of FIG. 1. Elemental
analysis has shown the carbon content to be between 38.39 and 41.59
weight percent; the hydrogen content to be between 5.74 and 6.16
weight percent; the nitrogen content to be between 15.19 and 15.89
weight percent; the oxygen content to be between 23.67 and 26.97
weight percent; and the sulfur content to be between 3.80 and 4.78
weight percent. Analysis of mass spectra shows a distribution of
molecular weight species, centered at approximately 850
daltons.
[0021] The peptide provided by the present invention can be used in
several applications. The skin healing properties of the peptide
can be used to promote healing, repair, and cell growth in
keratinous tissue generally. The peptide can be used to treat
damaged skin and skin wounds including, for example, rashes,
including diaper rash, bums including sunburn, cuts, abrasions,
punctures, sores including bed sores, ulcers including diabetic
ulcers and other skin injuries or irritations. The peptide can also
be used to treat aging, weakened or damaged skin, including, for
example, wrinkled skin. In one use, the keratinous tissue is
damaged tissue located either externally or internally. In one
example of use, an external wound can be treated by applying the
peptide to the wound. In one method, the peptide is admixed with a
cream, lotion, or gel before application to the skin. In another
method, the peptide is added to a keratin hydrogel prior to
application to the skin. A keratin hydrogel can be made according
to, for example, U.S. patent application Ser. No. 08/979,456, filed
Nov. 26, 1997, entitled KERATIN-BASED HYDROGEL FOR BIOMEDICAL
APPLICATIONS AND METHOD OF PRODUCTION. In another method, the
peptide can be added to a wound dressing prior to application. For
example, the peptide can be added to a keratin sheet as described
in U.S. patent application Ser. No. 08/979,526, filed Nov. 26,
1997, ENTITLED KERATIN-BASED SHEET MATERIAL FOR BIOMEDICAL
APPLICATIONS AND METHOD OF PRODUCTION. In another use of the
invention, the peptide can be applied internally to damaged
keratinous tissue lining the GI tract by orally administering the
peptide. Examples of such damage can result from ulcers, colitis,
or Crohn's disease.
[0022] The peptide can also be added as a cell growth stimulant to
a tissue engineering scaffold such as the sheet described in U.S.
patent application Ser. No. 09/198,998, filed Nov. 24, 1998,
entitled METHOD OF CROSS-LINKING KERATIN-BASED FILMS, SHEETS, AND
BULK MATERIALS. The peptide is believed suitable to speed repair of
sun or weather damaged skin. The peptide can be mixed with a
carrier lotion such as lanolin and applied to the skin. The peptide
can also be added to cosmetics to impart a skin healing property to
the cosmetic. Cosmetic bases are believed suitable for inclusion of
peptides made according to the present invention.
Experimental Results
[0023] Referring now to the table in FIG. 2, cell studies were
performed on human skin keratinocytes, human dermal fibroblasts,
and microvascular endothelial cells, as indicated, to determine the
effect of the keratin peptide on proliferation of cells critical to
the wound-healing process. Known growth factors for each cell line
were used as positive controls. The following concentrations of
keratin peptide were used: 0 (control, media alone); 0.005; 0.01;
0.05; 0.1; 0.5; 1; 5; and 10 micrograms per milliliter. At day 5 of
the study, the cells were analyzed using a technique that counts
the number of cells. The purpose of this study was to assess cell
proliferation as a result of their exposure to the keratin peptide,
relative to no exposure (media alone) and to known stimulants for
each cell type, the "positive control." Exposure to media alone was
considered the baseline of the study, so the average number of
cells in the baseline cultures was subtracted out of the average
number of cells in the cultures containing the soluble peptides and
the positive control for each cell line. This process
mathematically reduces the baseline to zero and everything else
becomes relative to zero, as seen in the first column of data.
These subtracted numbers are divided through by the average
baseline value and become a percent above or below baseline. The
numbers in the table represent the percent above baseline.
[0024] As can be seen from inspection of FIG. 2, the
keratin-derived peptide stimulated growth of the selected cell
lines and compares favorably with the known growth factors for each
cell line. In particular, at 0.5 micrograms per milliliter of
peptide, skin keratinocytes grew over 28% percent more than with
media alone, and at 5 micrograms peptide per milliliter, dermal
fibroblasts grew over 12 percent more relative to the baseline. At
0.05 micrograms of peptide per milliliter of media, microvascular
endothelial cells grew more than 9 percent more than the media only
baseline. Application of the keratin-derived peptide is thus
believed to be useful as a wound-healing agent.
[0025] Numerous advantages of the invention covered by this
document have been set forth in the foregoing description. It will
be understood, however, that this disclosure is, in many respects,
only illustrative. Changes may be made in details, particularly in
matters of reagents, concentrations, and step order, without
exceeding the scope of the invention. The inventions's scope is, of
course, defined in the language in which the appended claims are
expressed.
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