Note: Descriptions are shown in the official language in which they were submitted.
CA 02683015 2014-10-10
WOUND HEALING COMPOSITIONS CONTAINING KERATIN BIOMATERIALS
Related Applications
This application claims priority to United States Provisional Patent
Application Serial
No. 60/912,265, filed April 17, 2007.
Field of the Invention
The present invention concerns keratin biomaterials and the use thereof in
biomedical
applications.
Background of the Invention
The earliest documented use of keratin in medicine comes from a Chinese
herbalist
named Li Shi-Zhen (Ben Cao Gang Mu. Materia Medica, a dictionary of Chinese
herbs,
written by Li Shi Zhen (1518-1593)). Over a 38-year period, he wrote a
collection of 800
books known as the Ben Cao Gang Mu. These books were published in 1596, three
years
after his death. Among the more than 11,000 prescriptions described in these
volumes, is a
substance known as Xue Yu Tan, also known as Crinis Carbonisatus, that is made
up of
ground ash from pyrolized human hair. The stated indications for Xue Yu Tan
were
accelerated wound healing and blood clotting.
In the early 1800s, when proteins were still being called albuminoids (albumin
was a
well known protein at that time), many different kinds of proteins were being
discovered.
Around 1849, the word "keratin" appears in the literature to describe the
material that made
up hard tissues such as animal horns and hooves (keratin comes from the Greek
"kera"
meaning horn). This new protein intrigued scientists because it did not behave
like other
proteins. For example, the normal methods used for dissolving proteins were
ineffective with
keratin. Although methods such as burning and grinding had been known for some
time,
many scientists and inventors were more interested in dissolving hair and
horns in order to
make better products.
During the years from 1905 to 1935, many methods were developed to extract
keratins using oxidative and reductive chemistries (Breinl F and Baudisch 0, Z
physiol Chem
1907;52:158-69; Neuberg C, U.S Pat. No. 926,999, July 6, 1909; Lissizin T,
Biochem Bull
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1915;4:18-23; Zdenko S, Z physiol Chem 1924;136:160-72; Lissizin T, Z physiol
Chem
1928;173:309-11). By the late 1920s many techniques had been developed for
breaking down
the structures of hair, horns, and hooves, but scientists were confused by the
behavior of
some of these purified proteins. Scientists soon concluded that many different
forms of
keratin were present in these extracts, and that the hair fiber must be a
complex structure, not
simply a . strand of protein. In 1934, a key research paper was published that
described
different types of keratins, distinguished primarily by having different
molecular weights
(Goddard DR and Michaelis L, J Biol Chem 1934;106:605-14). This seminal paper
demonstrated that there were many different keratin homologs, and that each
played a
different role in the structure and function of the hair follicle.
Earlier work at the University of Leeds and the Wool Industries Research
Association
in the United Kingdom had shown that wool and other fibers were made up of an
outer
cuticle and a central cortex. Building on this information, scientists at
CSIRO conducted
many of the most fundamental studies on the structure and composition of wool.
Using X-ray
diffraction and electron microscopy, combined with oxidative and reductive
chemical
methods, CSIRO produced the first complete diagram of a hair fiber (Rivett DE
et al.,
"Keratin and Wool Research," The Lennox Legacy, CSIRO Publishing; Collingwood,
VIC,
Australia; 1996).
In 1965, CSIRO scientist W. Gordon Crewther and his colleagues published the
definitive text on the chemistry of keratins (Crewther WG et al., The
Chemistry of Keratins.
Anfinsen CB Jr et al., editors. Advances in Protein Chemistry 1965. Academic
Press. New
York:191-346). This chapter in Advances in Protein Chemistry contained
references to more
than 640 published studies on keratins. Once scientists knew how to extract
keratins from
hair fibers, purify and characterize them, the number of derivative materials
that could be
produced with keratins grew exponentially. In the decade beginning in 1970,
methods to form
extracted keratins into powders, films, gels, coatings, fibers, and foams were
being developed
and published by several research groups throughout the world (Anker CA, U.S.
Pat. No.
3,642,498, February 15, 1972; Kawano Y and Okamoto S, Kagaku To Seibutsu
1975;13(5):291-223; Okamoto S, Nippon Sholcuhin Kogyo Galdcaishi 1977;24(1):40-
50). All
of these methods made use of the oxidative and reductive chemistries developed
decades
earlier.
In 1982, Japanese scientists published the first study describing the use of a
keratin
coating on vascular grafts as a way to eliminate blood clotting (Noishiki Y et
al., Kobunshi
Ronbunshu 1982;39(4):221-7), as well as experiments on the biocompatibility of
keratins (Ito
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H et al., Kobunshi Ronbunshu 1982;39(4):249-56). Soon thereafter in 1985, two
researchers
from the UK published a review article speculating on the prospect of using
keratin as the
building block for new biomaterials development (Jarman T and Light J, World
Biotech Rep
1985;1:505-12). In 1992, the development and testing of a host of keratin-
based biomaterials
was the subject of a doctoral thesis for French graduate student Isabelle
Valherie (Valherie I
and Gagnieu C. Chemical modifications of keratins: Preparation of biomaterials
and study of
their physical, physiochemical and biological properties. Doctoral thesis.
Inst Natl Sci Appl
Lyon, France 1992). Soon thereafter, Japanese scientists published a
commentary in 1993 on
the prominent position keratins could take at the forefront of biomaterials
development
(Various Authors, Kogyo Zairyo 1993;41(15) Special issue 2:106-9).
Taken together, the aforementioned body of published work is illustrative of
the
unique chemical, physical, and biological properties of keratins. However,
there remains a
great need for optimized keratin preparations for use in biomedical
applications, particularly
for the treatment of wounds.
Summary of the Invention
An aspect of the present invention is a pharmaceutical composition comprising
a
keratin derivative (e.g., keratose, kerateine, or a combination thereof) and
optionally, at least
one additional active ingredient (e.g., analgesics, antimicrobial agents,
additional wound
healing agents, etc.).
Another aspect of the present invention is a method for treating a wound
(e.g., burns,
abrasions, lacerations, incisions, pressure sores, puncture wounds,
penetration wounds,
gunshot wounds, crushing injuries, etc.) in a subject in need thereof
comprising applying a
keratin derivative to the wound in an amount effective to treat the wound. In
some
embodiments, said positively charged composition comprises, consists or
consists essentially
of a keratose, a kerateine, or combinations thereof.
In some embodiments, the keratin derivative comprises, consists of or consists
essentially of alpha keratose, gamma keratose, acidic alpha keratose, basic
alpha keratose,
acidic gamma keratose, basic gamma keratose, alpha kerateine, gamma kerateine,
acidic
alpha kerateine, basic alpha kerateine, acidic gamma kerateine, basic gamma
kerateine, or
combinations thereof.
In some embodiments, the keratin derivative is applied to the wound in an
amount
effective to inhibit wound conversion, promote wound closure, or both. In some
embodiments, the keratin derivative is topically applied. In some embodiments,
the keratin
derivative is applied by injection into the body of the subject.
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A further aspect of the present invention is a method for treating a burn
wound in a
subject in need thereof comprising applying a keratin derivative to the wound
in an amount
effective to treat the bum wound. In some embodiments, said positively charged
composition
comprises, consists or consists essentially of a keratose, a kerateine, or
combinations thereof.
In some embodiments, the keratin derivative comprises, consists of or consists
essentially of alpha keratose, gamma keratose, acidic alpha keratose, basic
alpha keratose,
acidic gamma keratose, basic gamma keratose, alpha kerateine, gamma kerateine,
acidic
alpha kerateine, basic alpha kerateine, acidic gamma kerateine, basic gamma
kerateine, or
combinations thereof.
In some embodiments, the keratin derivative is applied to the wound in an
amount
effective to inhibit wound conversion, promote wound closure, or both. In some
embodiments, the keratin derivative is topically applied. In some embodiments,
the keratin
derivative is applied by injection into the body of the subject.
Another aspect of the present invention is a surgical or paramedic aid,
comprising: a
solid, physiologically acceptable substrate; and a keratin derivative on the
substrate. In some
embodiments the keratin derivative comprises, consists of or consists
essentially of alpha
keratose, gamma keratose, acidic alpha keratose, basic alpha keratose, acidic
gamma
keratose, basic gamma keratose, alpha kerateine, gamma kerateine, acidic alpha
kerateine,
basic alpha kerateine, acidic gamma kerateine, basic gamma kerateine, or
combinations
thereof.
A still further aspect of the present invention is a kit comprising a keratin
derivative
and a container in which said keratin derivative is packaged in sterile form.
In some
embodiments the keratin derivative comprises, consists of or consists
essentially of alpha
keratose, gamma keratose, acidic alpha keratose, basic alpha keratose, acidic
gamma
keratose, basic gamma keratose, alpha kerateine, gamma kerateine, acidic alpha
kerateine,
basic alpha kerateine, acidic gamma kerateine, basic gamma kerateine, or
combinations
thereof.
Another aspect of the present invention is the use of a keratin derivative as
described
herein for the preparation of a composition or medicament for carrying out a
method of
treatment as described herein, or for making an article of manufacture as
described herein.
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According to an aspect, there is provided the use of a hydrogel comprising
keratose,
kerateine, or a combination thereof for the preparation of a composition or
medicament for
use in treatment for a wound,
wherein said keratose comprises at least 80% by weight of basic alpha keratose
or
acidic alpha keratose, said basic or acidic alpha keratose having an average
molecular weight
of from 30 kDa to 200 kDa, and
wherein said kerateine comprises at least 80% by weight of basic alpha
kerateine or
acidic alpha kerateine, said basic or acidic alpha kerateine having an average
molecular
weight of from 30 kDa to 200 kDa.
According to another aspect, there is provided a pharmaceutical composition
comprising:
a) kerateine, wherein said kerateine comprises at least 80% by weight of basic
alpha
kerateine or acidic alpha kerateine, said basic or acidic alpha kerateine
having an average
molecular weight of from 30 kDa to 200 kDa;
b) an aqueous carrier; and
c) optionally, at least one additional active ingredient.
19. The composition of claim 18, wherein said composition further comprises
keratose.
According to another aspect, there is provided a surgical or paramedic aid,
comprising:
a solid, physiologically acceptable substrate; and
a composition comprising a keratin derivative on said substrate
wherein said keratin derivative is at least 80% by weight of acidic alpha
kerateine or
at least 80% by weight of acidic alpha keratose, wherein said keratose or
kerateine has an
average molecular weight of from 30 kDa to 200 kDa.
According to another aspect, there is provided a kit comprising:
a) a keratin derivative; and
b) a container in which said keratin derivative is packaged in said container
in sterile
form, and wherein said keratin derivative is at least 80% by weight of acidic
alpha kerateine
or at least 80% by weight of acidic alpha keratose, wherein said keratose or
kerateine has an
average molecular weight of from 10 kDa to 200 kDa.
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,
,
According to yet another aspect, there is provided a container comprising: a
keratin
derivative, wherein the keratin derivative is packaged in said container in
sterile form, and
wherein said keratin derivative is at least 80% by weight of acidic alpha
kerateine or at least
80% by weight of acidic alpha keratose, wherein said keratose or kerateine has
an average
molecular weight of from 10 kDa to 200 kDa.
Brief Description of the Drawings
Figure 1. Skin component cell proliferation. Keratinocytes (a) and fibroblasts
(b)
treated with soluble keratin biomaterials proliferate more readily than cells
treated with media
alone.
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Figure 2. Changes in chemical burn wound area over time. Mice treated with
phenol
to induce a chemical burn experience a passivation of the wound site such that
the normal
course of wound growth does not occur.
Figure 3. Burn wound area in pigs. Wound areas were determined by digital
image
analysis and normalized to the day zero values. Wounds treated with keratin
gels did not
increase in area appreciably in the first several days and healed more quickly
than controls.
Figure 4. Kaplan-Mayer Survival Graph: Time is presented in minutes on a
logarithmic scale. All animals in the control group died within 60 minutes.
One animal from
the keratin and one from the HemCon hemostatic bandage group was sacrificed
upon
recommendation of the animal care staff. Overall, keratin outperformed the
other groups with
only one death compared with 2 deaths in the QuikClote hemostatic agent and
HemCon
hemostatic bandage groups.
Figure 5. Shed Blood: Blood loss is normalized to body weight and expressed as
percentage of body weight. Keratin and QuikClot hemostatic agent groups lost
significantly
(*) less blood than the control and HemCon hemostatic bandage groups.
Figure 6. Mean Arterial Pressure (MAP): Blood pressure is expressed in
percentage of initial pressure. The negative control and QuikClote hemostatic
agent groups
showed a steep drop in pressure to 40% of initial MAP. Animals treated with
keratin or
HemCon hemostatic bandage were able to stabilize the MAP around 80% of
initial
pressure. These differences were not statistically different compared to the
control group.
Figure 7. Shock Index (SI): The modified shock index was calculated by
dividing
heart rate by MAP. This index is clinically used to assess the severity of a
shock, with low
values being better. The animals in the keratin group showed compensated low
values over
the entire study period, while QuikClote hemostatic agent and HemCon
hemostatic
bandage groups had similar values as the negative control. There was no
statistical
significance between the groups.
Figure 8. Histological Assessment: Representative tissue sections stained with
hematoxylin and eosin, 50x. A) The negative control group shows signs of poor
perfusion
with wide and empty sinusoids. The surface is lacking a functional blood clot.
B) The surface
of the QuikClote hemostatic agent treated samples shows an area of necrosis
(arrow) and
clotting. Only minimal cellular infiltration and tissue regeneration is
visible. The void areas
represent the removed QuikClote hemostatic agent granules. C) Tissue samples
from
HemCon hemostatic bandage treated animals showed patchy areas of adherent
clotted
blood, where there was a low level of cellular infiltration. D) Liver samples
from animals
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treated with keratin show a thick layer of keratin biomaterial attached to the
injured surface.
There are signs of excellent biocompatibility with a high cellular activity
and the formation of
early granular tissue (large arrow) in the spaces between the keratin.
Further, there is a high
level of direct contact of hepatocytes with the keratin biomaterial (small
arrow).
Figure 9. Keratin Treated Group, High Magnification: A) Formation of early
granulation-like tissue within the spaces of the keratin gel, 200x. B)
Interface between keratin
gel and liver tissue showing integration of the biomaterial and tissue, and
early cellular
infiltration, 400x.
Detailed Description of the Preferred Embodiments
The ability of extracted keratin solutions to spontaneously self-assemble at
the micron
scale was published in two papers in 1986 and 1987 (Thomas H et al., Int J
Biol Macromol
1986;8:258-64; van de Locht M, Melliand Textilberichte 1987;10:780-6). This
phenomenon
is not surprising given the highly controlled superstructure whence hair
keratins are obtained.
When processed correctly, this ability to self-assemble can be preserved and
used to create
regular architectures on a size scale conducive to cellular infiltration. When
keratins are
hydrolyzed (e.g., with acids or bases), their molecular weight is reduced and
they lose the
ability to self-assemble. Therefore, processing conditions that minimize
hydrolysis are
preferred.
This ability to self-assemble is a particularly useful characteristic for
tissue
engineering scaffolds for two reasons. First, self-assembly results in a
highly regular structure
with reproducible architectures, dimensionality, and porosity. Second, the
fact that these
architectures form of their own accord under benign conditions allows for the
incorporation
of cells as the matrix is formed. These two features are critically important
to any system that
attempts to mimic the native extracellular matrix (ECM).
Cellular recognition is also an important characteristic of biomaterials that
seek to
mimic the ECM. Such recognition is facilitated by the binding of cell surface
integrins to
specific amino acid motifs presented by the constituent ECM proteins.
Predominant proteins
include collagen and fibronectin, both of which have been extensively studied
with regard to
cell binding. Both proteins contain several regions that support attachment by
a wide variety
of cell types. It has been shown that, in addition to the widely know Arginine-
Glycine-
Aspartic Acid (RGD) motif, the "X"-Aspartic Acid-"Y" motif on fibronectin is
also
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recognized by the integrin a4131, where X equals Glycine, Leucine, or Glutamic
Acid, and Y
equals Serine or Valine.
Keratin biomaterials derived from human hair contain these same binding
motifs. A
search of the NCBI protein database revealed sequences for 71 discrete, unique
human hair
keratin proteins. Of these, 55 are from the high molecular weight, low sulfur,
alpha-helical
family. This group of proteins is often referred to as the alpha-keratins and
is responsible for
imparting toughness to human hair fibers. These alpha-keratins have molecular
weights
greater than 40 kDa and an average cysteine (the main amino acid responsible
for inter- and
intramolecular protein bonding) content of 4.8 mole percent. Moreover,
analysis of the amino
acid sequences of these alpha keratin proteins showed that 78% contain at
least one
fibronectin-like integrin receptor binding motif, and 25% contain at least two
or more. Two
recent papers have highlighted the fact that these binding sites are likely
present on the
surface of keratin biomaterials by demonstrating excellent cell adhesion onto
processed
keratin foams (Tachibana A et al., J Biotech 2002;93:165-70; Tachibana A et
al.,
Biomaterials 2005;26(3):297-302).
Other examples of natural polymers that may be utilized in a similar fashion
to the
disclosed keratin preparations include, but are not limited to, collagen,
gelatin, fibronectin,
vitronectin, laminin, fibrin, mucin, elastin, nidogen (entactin),
proteoglycans, etc. (See, e.g.,
U.S. Pat. No. 5,691,203 to Katsuen et al.).
Theories for the biological activity of human hair extracts include that the
human hair
keratins ('tHHKs"), themselves, are biologically active. Over 70 human hair
keratins are
known and their cDNA-derived sequences published. However, the full compliment
of HHKs
is unknown and estimates of over 100 have been proposed (Gillespie JM, The
structural
proteins of hair: isolation characterization, and regulation of biosynthesis.
Goldsmith LA
(editor), Biochemistry and physiology of the skin (1983), Oxford University
Press. New
York;475-510). Within the complete range of HHKs are a small number that have
been
shown to participate in wound contracture and cell migration (Martin, P,
Science
1997;276:75-81). In particular, keratins K-6 and K-16 are expressed in the
epidermis during
wound healing and are also found in the outer root sheath of the hair follicle
(Bowden PE,
Molecular Aspects of Dermatology (1993), John Wiley & Sons, Inc.,
Chichester:19-54). The
presence of these HIIEKs in extracts of human hair, and their subsequent
dosing directly into a
wound bed, may be responsible for "shortcutting" the otherwise lengthy process
of
differentiation, migration, and proliferation, or for alleviating some
biochemical deficiency,
thereby accelerating the tissue repair and regeneration process.
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It has been known for more than a decade that growth factors such as bone
morphogenetic protein-4 (BMP-4) and other members of the transforming growth
factor-13
(TGF-r3) superfamily are present in developing hair follicles (Jones CM= et
al., Development
1991;111:531-42; Lyons KM et al., Development 1990;109:833-44; Blessings M et
al.,
Genes and Develop 1993;7:204-15). In fact, more than 30 growth factors and
cytolcines are
involved in the growth of a cycling hair follicle (Hardy MH, Trends Genet
1992;8(2):55-61;
Stenn KS et al., J Dermato Sci 1994;7S:S109-24; Rogers GE, Int J Dev Biol
2004;48(2-
3):163-70). Many of these molecules have a pivotal role in the regeneration of
a variety of
tissues. It is highly probable that a number of growth factors become
entrained within human
hair when cytoldnes bind to stem cells residing in the bulge region of the
hair follicle
(Panteleyev AA et al., J Cell Sci 2001;114:3419-31). These growth factors are
predicted to be
extracted along with the keratins from end-cut human hair. This observation is
not without
precedent, as it has previously been shown that many different types of growth
factors are
present in the extracts of various tissues, and that their activity is
maintained even after
chemical extraction. Observations such as these show mounting evidence that a
number of
growth factors may be present in end-cut human hair, and that the keratins may
be acting as a
highly effective delivery matrix of, inter alia, these growth factors.
Keratins are a family of proteins found in the hair, skin, and other tissues
of
vertebrates. Hair is a unique source of human keratins because it is one of
the few human
tissues that is readily available and inexpensive. Although other sources of
keratins are
acceptable feedstocks for the present invention, (e.g. wool, fur, horns,
hooves, beaks,
feathers, scales, and the like), human hair is preferred for use with human
subjects because of
its biocompatibility.
Keratins can be extracted from human hair fibers by oxidation or reduction
using
methods that have been published in the art (See, e.g., Crewther WG et al. The
chemistry of
keratins, in Advances in Protein Chemistry 1965;20:191-346). These methods
typically
employ a two-step process whereby the crosslinked structure of keratins is
broken down by
either oxidation or reduction. In these reactions, the disulfide bonds in
cysteine amino acid
residues are cleaved, rendering the keratins soluble (Scheme 1). The cuticle
is essentially
unaffected by this treatment, so the majority of the keratins remain trapped
within the
cuticle's protective structure. In order to extract these keratins, a second
step using a
denaturing solution must be employed. Alternatively, in the case of reduction
reactions, these
steps can be combined. Denaturing solutions known in the art include urea,
transition metal
hydroxides, surfactant solutions, and combinations thereof. Preferred methods
use aqueous
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solutions of tris in concentrations between 0.1 and 1.0 M, and urea solutions
between 0.1 and
10M, for oxidation and reduction reactions, respectively.
o
H> _______ O¨C terminus
N terminus ---N__
H 'CH 0
S. 2
11,02
N terminus-.-¨ O¨C terminus
or C
a
\
CH3C000H H µCH2
H ICH, ,
N terminus.¨N¨C
H ¨0¨C terminus =
0.
0
0
H ______ C terminus
N termi O-
nus¨N_
H 'CH 0
/ 2
H) __ 0- C terminus
HSCH2COOH N terminus
H 'CH
H /CH, , 2
N terminus ¨"NI =O¨C terminus
Scheme 1. General representations of (a) oxidation and (b) reduction of
disulfide
crosslinks in keratin. These reactions cleave the sulfur-sulfur bond in
cystine residues,
= thereby destroying the superstructure and rendering the keratins soluble
in the reaction
media. The resultant fractions are keratose (a) and kerateine (b).
= If one employs an oxidative treatment, the =resulting keratins are
referred to as
"keratoses." If a reductive treatment is used, the resulting keratins are
referred to as
"kerateines" (See Scheme 1)
Crude extracts of keratins, regardless of redox state, can be further refined
into
"gamma" and "alpha" fractions, e.g., by isoelectric precipitation. High
molecular weight
keratins, or "alpha keratins," (alpha helical), are thought to derive from the
microfibrillar
regions of the hair follicle, and typically range in molecular weight from
about 40-85
kiloDaltons. Low molecular weight keratins, or "gamma keratins," (globular),
are thought to
derive from the extracellular matrix regions of the hair follicle, and
typically range in
molecular weight from about 10-15 kiloDaltons. (See Crewther WG et al. The
chemistry of
keratins, in Advances in Protein Chemistry 1965;20:191-346)
Even though alpha and gamma keratins possess unique properties, the properties
of
subfamilies of both alpha and gamma keratins can only be revealed through more
sophisticated means of purification. For example, keratins may be fractionated
into "acidic"
and "basic" protein fractions. A preferred method of fractionation is ion
exchange
chromatography. These fractions possess unique properties, such as their
differential effects
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on blood cell aggregation (See Table 1 below; See also: U.S. Patent
Application Publication
No. 2006/0051732).
"Keratin derivative" as used herein refers to any keratin fractionation,
derivative or
mixture thereof, alone or in combination with other keratin derivatives or
other ingredients,
including but not limited to alpha keratose, gamma keratose, alpha kerateine,
gamma
kerateine, meta keratin, keratin intermediate filaments, and combinations
thereof, including
the acidic and basic constituents thereof unless specified otherwise, along
with variations
thereof that will be apparent to persons skilled in the art in view of the
present disclosure. In
some embodiments, the keratin derivative comprises, consists or consists
essentially of a
particular fraction or subfraction of keratin. The derivative may comprise,
consist or consist
essentially of at least 80, 90, 95 or 99 percent by weight of said fraction or
subfraction (or
more).
In some embodiments, the keratin derivative comprises, consists of, or
consists
essentially of acidic alpha keratose.
In some embodiments, the keratin derivative comprises, consists of or consists
essentially of alpha keratose, where the alpha keratose comprises, consists of
or consists
essentially of at least 80, 90, 95 or 99 percent by weight of acidic alpha
keratose (or more),
and where the alpha keratose comprises, consists of or consists essentially of
not more than
20, 10, 5 or 1 percent by weight of basic alpha keratose (or less).
In some embodiments, the keratin derivative comprises, consists of, or
consists
essentially of basic alpha keratose.
In some embodiments, the keratin derivative comprises, consists of or consists
essentially of alpha keratose, where the alpha keratose comprises, consists of
or consists
essentially of at least 80, 90, 95 or 99 percent by weight of basic alpha
keratose (or more),
and where the alpha keratose comprises, consists of or consists essentially of
not more than
20, 10, 5 or 1 percent by weight of acidic alpha keratose (or less).
In some embodiments, the keratin derivative comprises, consists of, or
consists
essentially of acidic alpha kerateine.
In some embodiments, the keratin derivative comprises, consists of or consists
essentially of alpha kerateine, where the alpha kerateine comprises, consists
of or consists
essentially of at least 80, 90, 95 or 99 percent by weight of acidic alpha
kerateine (or more),
and where the alpha kerateine comprises, consists of or consists essentially
of not more than
20, 10, 5 or 1 percent by weight of basic alpha kerateine (or less).
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In some embodiments, the keratin derivative comprises, consists of, or
consists
essentially of basic alpha kerateine.
In some embodiments, the keratin derivative comprises, consists of or consists
essentially of alpha kerateine, where the alpha kerateine comprises, consists
of or consists
essentially of at least 80, 90, 95 or 99 percent by weight of basic alpha
kerateine (or more),
and where the alpha kerateine comprises, consists of or consists essentially
of not more than
20, 10, 5 or 1 percent by weight of acidic alpha kerateine (or less).
In some embodiments, the keratin derivative comprises, consists of or consists
essentially of unfractionated alpha+gamma-kerateines. In some embodiments, the
keratin
derivative comprises, consists of or consists essentially of acidic
alpha+gamma-kerateines. In
some embodiments, the keratin derivative comprises, consists of or consists
essentially of
basic alpha+gamma-kerateines.
In some embodiments, the keratin derivative comprises, consists of or consists
essentially of unfractionated alpha+gamma-keratose. In some embodiments, the
keratin
derivative comprises, consists of or consists essentially of acidic
alpha+gamma-keratose. In
some embodiments, the keratin derivative comprises, consists of or consists
essentially of
basic alpha+gamma-keratose.
In some embodiments, the keratin derivative comprises, consists of or consists
essentially of unfractionated beta-keratose (e.g., derived from cuticle). In
some embodiments,
the keratin derivative comprises, consists of or consists essentially of basic
beta-keratose. In
some embodiments, the keratin derivative comprises, consists of or consists
essentially of
acidic beta-keratose.
The basic alpha keratose is preferably produced by separating basic alpha
keratose
from a mixture comprising acidic and basic alpha keratose, e.g., by ion
exchange
chromatography, and optionally the basic alpha keratose has an average
molecular weight of
from 10 to 100 or 200 kiloDaltons. More preferably, the average molecular
weight is from 30
or 40 to 90 or 100 kiloDaltons. Optionally but preferably the process further
comprises the
steps of re-dissolving said basic alpha-keratose in a denaturing and/or
buffering solution,
optionally in the presence of a chelating agent to complex trace metals, and
then re-
precipitating the basic alpha keratose from the denaturing solution. It will
be appreciated that
the composition preferably contains not more than 5, 2, 1, or 0.1 percent by
weight of acidic
alpha keratose, or less.
The acidic alpha keratose is preferably produced by a reciprocal of the
foregoing
technique: that is, by separating and retaining acidic alpha keratose from a
mixture of acidic
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and basic alpha keratose, e.g., by ion exchange chromatography, and optionally
the acidic
alpha keratose has an average molecular weight of from 10 to 100 or 200
kiloDaltons. More
preferably, the average molecular weight is from 30 or 40 to 90 or 100
kiloDaltons.
Optionally but preferably the process further comprises the steps of re-
dissolving said acidic
alpha-keratose in a denaturing solution and/or buffering solution, optionally
in the presence
of a chelating agent to complex trace metals, and then re-precipitating the
basic alpha
keratose from the denaturing solution. It will be appreciated that the
composition preferably
contains not more than 5, 2, 1, or 0.1 percent by weight of basic alpha
keratose, or less.
Basic and acidic fractions of other keratoses can be prepared in like manner
as
described above for basic and acidic alpha keratose.
The basic alpha kerateine is preferably produced by separating basic alpha
kerateine
from a mixture of acidic and basic alpha kerateine, e.g., by ion exchange
chromatography,
and optionally the basic alpha kerateine has an average molecular weight of
from 10 to 100 or
200 kiloDaltons. Optionally but preferably the process further comprises the
steps of re-
dissolving said basic alpha-kerateine in a denaturing and/or buffering
solution, optionally in
the presence of a chelating agent to complex trace metals, and then re-
precipitating the basic
alpha kerateine from the denaturing solution. It will be appreciated that the
composition
preferably contains not more than 5, 2, 1, or 0.1 percent by weight of acidic
alpha kerateine,
or less.
The acidic alpha kerateine is preferably produced by a reciprocal of the
foregoing
technique: that is, by separating and retaining acidic alpha kerateine from a
mixture of acidic
and basic alpha kerateine, e.g., by ion exchange chromatography, and
optionally the acidic
alpha kerateine has an average molecular weight of from 10 to 100 or 200
kiloDaltons. More
preferably, the average molecular weight is from 30 or 40 to 90 or 100
kiloDaltons.
Optionally but preferably the process further comprises the steps of re-
dissolving said acidic
alpha-kerateine in a denaturing and/or buffering solution), optionally in the
presence of a
chelating agent to complex trace metals, and then re-precipitating the basic
alpha kerateine
from the denaturing solution. It will be appreciated that the composition
preferably contains
not more than 5, 2, 1, or 0.1 percent by weight of basic alpha kerateine, or
less.
Basic and acidic fractions of other kerateines can be prepared in like manner
as
described above for basic and acidic alpha kerateine.
Keratin materials are derived from any suitable source including, but not
limited to,
wool and human hair. In one embodiment keratin is derived from end-cut human
hair,
obtained from barbershops and salons. The material is washed in hot water and
mild
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detergent, dried, and extracted with a nonpolar organic solvent (typically
hexane or ether) to
remove residual oil prior to use.
Keratoses. Keratose fractions are obtained by any suitable technique. In one
embodiment they are obtained using the method of Alexander and coworkers (P.
Alexander
et al., Biochem. J. 46, 27-32 (1950)). Basically, the hair is reacted with an
aqueous solution of
peracetic acid at concentrations of less than ten percent at room temperature
for 24 hours. The
solution is filtered and the alpha-keratose fraction precipitated by addition
of mineral acid to
a pH of approximately 4. The alpha-keratose is separated by filtration, washed
with
additional acid, followed by dehydration with alcohol, and then freeze dried.
Increased purity
can be achieved by re-dissolving the keratose in a denaturing solution such as
7M urea,
aqueous ammonium hydroxide solution, or 20mM tris base buffer solution (e.g.,
Trizma
base), re-precipitating, re-dissolving, dialyzing against deionized water, and
re-precipitating
at pH 4.
A preferred method for the production of keratoses is by oxidation with
hydrogen
peroxide, peracetic acid, or performic acid. A most preferred oxidant is
peracetic acid.
Preferred concentrations range from 1 to 10 weight/volume percent (w/v %), the
most
preferred being approximately 2 w/v %. Those skilled in the art will recognize
that slight
modifications to the concentration can be made to effect varying degrees of
oxidation, with
concomitant alterations in reaction time, temperature, and liquid to solid
ratio. It has also
been discussed by Crewther et al. that performic acid offers the advantage of
minimal peptide
bond cleavage compared to peracetic acid. However, peracetic acid offers the
advantages of
cost and availability. A preferred oxidation temperature is between 0 and 100
degrees Celsius
( C). A most preferred oxidation temperature is 37 C. A preferred oxidation
time is between
0.5 and 24 hours. A most preferred oxidation time is 12 hours. A preferred
liquid to solid
ratio is from 5 to 100:1. A most preferred ratio is 20:1. After oxidation, the
hair is rinsed free
of residual oxidant using a copious amount of distilled water.
The keratoses can be extracted from the oxidized hair using an aqueous
solution of a
denaturing agent. Protein denaturants are well known in the art, but preferred
solutions
include urea, transition metal hydroxides (e.g. sodium and potassium
hydroxide), ammonium
hydroxide, and tris(hydroxymethyl)aminomethane (tris base). A preferred
solution is
Trizma base (a brand of tris base) in the concentration range from 0.01 to
1M. A most
preferred concentration is 0.1M. Those skilled in the art will recognize that
slight
modifications to the concentration can be made to effect varying degrees of
extraction, with
concomitant alterations in reaction time, temperature, and liquid to solid
ratio. A preferred
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extraction temperature is between 0 and 100 degrees Celsius. A most preferred
extraction
temperature is 37 C. A preferred extraction time is between 0.5 and 24 hours.
A most
preferred extraction time is 3 hours. A preferred liquid to solid ratio is
from 5 to 100:1. A
most preferred ratio is 40:1. Additional yield can be achieved with subsequent
extractions
with dilute solutions of tris base or deionized (DI) water. After extraction,
the residual solids
are removed from solution by centrifugation and/or filtration.
The crude extract can be isolated by first neutralizing the solution to a pH
between 7.0
and 7.4. A most preferred pH is 7.4. Residual denaturing agent is removed by
dialysis against
DI water. Concentration of the dialysis retentate is followed by
lyophilization or spray
drying, resulting in a dry powder mixture of both gamma- and alpha-keratose.
Alternately,
alpha-keratose is isolated from the extract solution by dropwise addition of
acid until the pH
of the solution reaches approximately 4.2. Preferred acids include sulfuric,
hydrochloric, and
acetic. A most preferred acid is concentrated hydrochloric acid. Precipitation
of the alpha
fraction begins at around pH 6.0 and continues until approximately 4.2.
Fractional
precipitation can be utilized to isolate different ranges of protein with
different isoelectric
properties. Solid alpha-keratose can be recovered by centrifugation or
filtration.
The alpha keratose can be further purified by re-dissolving the solids in a
denaturing
solution. The same denaturing solutions as those utilized for extraction can
be used, however
a preferred denaturing solution is tris base. Ethylene diamine tetraacetic
acid (EDTA) can be
added to complex and remove trace metals found in the hair. A preferred
denaturing solution
is 20 mM tris base with 20 mM EDTA or DI water with 20 mM EDTA. If the
presence of
trace metals is not detrimental to the intended application, the EDTA can be
omitted. The
alpha-keratose is re-precipitated from this solution by dropwise addition of
hydrochloric acid
to a final pH of approximately 4.2. Isolation of the solid is by
centrifugation or filtration. This
process can be repeated several times to further purify the alpha-keratose.
The gamma keratose fraction remains in solution at pH 4 and is isolated by
addition to
a water-miscible organic solvent such as alcohol, followed by filtration,
dehydrated with
additional alcohol, and freeze dried. Increased purity can be achieved by
redissolving the
keratose in a denaturing solution such as 7M urea, aqueous ammonium hydroxide
solution, or
20mM tris buffer solution, reducing the pH to 4 by addition of a mineral acid,
removing any
solids that form, neutralizing the supernatant, re-precipitating the protein
with alcohol, re-
dissolving, dialyzing against deionized water, and reprecipitating by addition
to alcohol. The
amount of alcohol consumed in these steps can be minimized by first
concentrating the
keratose solution by distillation.
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After removal of the alpha keratose, the concentration of gamma keratose from
a
typical extraction solution is approximately 1-2%. The gamma keratose fraction
can be
isolated by addition to a water-miscible non-solvent. To effect precipitation,
the gamma-
keratose solution can be concentrated by evaporation of excess water. This
solution can be
concentrated to approximately 10-20% by removal of 90% of the water. This can
be done
using vacuum distillation or by falling film= evaporation. After
concentration, the gamma-
keratose solution is added dropwise to an excess of cold non-solvent. Suitable
non-solvents
include ethanol, methanol, acetone, and the like. A most preferred non-solvent
is ethanol. A
most preferred method is to concentrate the gamma keratose solution to
approximately 10
w/v % protein and add it dropwise to an 8-fold excess of cold ethanol. The
precipitated
gamma keratose can be isolated by centrifugation or filtration and dried.
Suitable methods for
drying include freeze drying (lyophilization), air drying, vacuum drying, or
spray drying. A
most preferred method is freeze drying.
Kerateines. Kerateine fractions can be obtained using a combination of the
methods
of Bradbury and Chapman (J. Bradbury et al., Aust. .1. Biol. ScL 17, 960-72
(1964)) and
Goddard and Michaelis (D. Goddard et al., J. Biol. Chem. 106, 605-14 (1934)).
Essentially,
the cuticle of the hair fibers is removed ultrasonically in order to avoid
excessive hydrolysis
and allow efficient reduction of cortical disulfide bonds in a second step.
The hair is placed in
a solution of dichloroacetic acid and subjected to treatment with an
ultrasonic probe. Further
refinements of this method indicate that conditions using 80% dichloroacetic
acid, solid to
liquid of 1:16, and an ultrasonic power of 180 Watts are optimal (H. Ando et
al., Sen7
Gakkaishi 31(3), T81-85 (1975)). Solid fragments are removed from solution by
filtration,
rinsed and air dried, followed by sieving to isolate the hair fibers from
removed cuticle cells.
In some embodiments, following ultrasonic removal of the cuticle, alpha- and
gamma
kerateines are obtained by reaction of the denuded fibers with
mercaptoethanol. Specifically,
a low hydrolysis method is used at acidic pH (E. Thompson et al., Aust. J
Biol. Sci. 15, 757-
68 (1962)). In a typical reaction, hair is extracted for 24 hours with 4M
mercaptoethanol that
has been adjusted to pH 5 by addition of a small amount of potassium hydroxide
in
deoxygenated water containing 0.02M acetate buffer and 0.001M surfactant.
The solution is filtered and the alpha kerateine fraction precipitated by
addition of
mineral acid to a pH of approximately 4. The alpha kerateine is separated by
filtration,
washed with additional acid, followed by dehydration with alcohol, and then
dried under
vacuum. Increased purity is achieved by re-dissolving the kerateine in a
denaturing solution
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such as 7M urea, aqueous ammonium hydroxide solution, or 20mM tris buffer
solution, re-
precipitating, re-dissolving, dialyzing against deionized water, and re-
precipitating at pH 4.
The gamma kerateine fraction remains in solution at pH 4 and is isolated by
addition
to a water-miscible organic solvent such as alcohol, followed by filtration,
dehydrated with
additional alcohol, and dried under vacuum. Increased purity can be achieved
by redissolving
the kerateine in a denaturing solution such as 7M urea, aqueous ammonium
hydroxide
solution, or 20mM tris buffer solution, reducing the pH to 4 by addition of a
mineral acid,
removing any solids that form, neutralizing the supernatant, re-precipitating
the protein with
alcohol, re-dissolving, dialyzing against deionized water, and reprecipitating
by addition to
alcohol. The amount of alcohol consumed in these steps can be minimized by
first
concentrating the keratin solution by distillation.
In an alternate method, the kerateine fractions are obtained by reacting the
hair with
an aqueous solution of sodium thioglycolate.
A preferred method for the production of kerateines is by reduction of the
hair with
thioglycolic acid or beta-mercaptoethanol. A most preferred reductant is
thioglycolic acid
(TGA). Preferred concentrations range from 1 to 10M, the most preferred being
approximately 1.0M. Those skilled in the art will recognize that slight
modifications to the
concentration can be made to effect varying degrees of reduction, with
concomitant
alterations in pH, reaction time, temperature, and liquid to solid ratio. A
preferred pH is
between 9 and 11. A most preferred pH is 10.2. The pH of the reduction
solution is altered by
addition of base. Preferred bases include transition metal hydroxides, sodium
hydroxide, and
ammonium hydroxide. A most preferred base is sodium hydroxide. The pH
adjustment is
effected by dropwise addition of a saturated solution of sodium hydroxide in
water to the
reductant solution. A preferred reduction temperature is between 0 and 100 C.
A most
preferred reduction temperature is 37 C. A preferred reduction time is between
0.5 and 24
hours. A most preferred reduction time is 12 hours. A preferred liquid to
solid ratio is from 5
to 100:1. A most preferred ratio is 20:1. Unlike the previously described
oxidation reaction,
reduction is carried out at basic pH. That being the case, keratins are highly
soluble in the
reduction media and are expected to be extracted. The reduction solution is
therefore
combined with the subsequent extraction solutions and processed accordingly.
Reduced keratins are not as hydrophilic as their oxidized counterparts. As
such,
reduced hair fibers will not swell and split open as will oxidized hair,
resulting in relatively
lower yields. Another factor affecting the kinetics of the
reduction/extraction process is the
relative solubility of kerateines. The relative solubility rankings in water
is gamma-
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keratose>alpha-keratose>gamma-kerateine>alpha-kerateine from most to least
soluble.
Consequently, extraction yields from reduced hair fibers are not as high. This
being the case,
subsequent extractions are conducted with additional reductant plus denaturing
agent
solutions. Preferred solutions for subsequent extractions include TGA plus
urea, TGA plus
tris base, or TGA plus sodium hydroxide. After extraction, crude fractions of
alpha- and
gamma-kerateine can be isolated using the procedures described for keratoses.
However,
precipitates of gamma- and alpha-kerateine re-form their cystine crosslinks
upon exposure to
oxygen. Precipitates must therefore be re-dissolved quickly to avoid
insolubility during the
purification stages, or precipitated in the absence of oxygen.
Residual reductant and denaturing agents can be removed from solution by
dialysis.
Typical dialysis conditions are 1 to 2% solution of kerateines dialyzed
against DI water for
24 to 72 hours. Those skilled in the art will recognize that other methods
exist for the removal
of low molecular weight contaminants in addition to dialysis (e.g.
microfiltration,
chromatography, and the like). The use of tris base is only required for
initial solubilization
of the kerateines. Once dissolved, the kerateines are stable in solution
without the denaturing
agent. Therefore, the denaturing agent can be removed without the resultant
precipitation of
kerateines, so long as the pH remains at or above neutrality. The final
concentration of
kerateines in these purified solutions can be adjusted by the addition/removal
of water.
Regardless of the form of the keratin (i.e. keratoses or kerateines), several
different
approaches to further purification can be employed to keratin solutions. Care
must be taken,
however, to choose techniques that lend themselves to keratin's unique
solubility
characteristics. One of the most simple separation technologies is isoelectric
precipitation. In
this method, proteins of differing isoelectric point can be isolated by
adjusting the pH of the
solution and removing the precipitated material. In the case of keratins, both
gamma- and
alpha- forms are soluble at pH >6Ø As the pH falls below 6, however, alpha-
keratins begin
to precipitate. Keratin fractions can be isolated by stopping the
precipitation at a given pH
and separating the precipitate by centrifugation and/or filtration. At a pH of
approximately
4.2, essentially all of the alpha-keratin will have been precipitated. These
separate fractions
can be re-dissolved in water at neutral pH, dialyzed, concentrated, and
reduced to powders by
lyophilization or spray drying. However, kerateine fractions must be stored in
the absence of
oxygen or in dilute solution to avoid crosslinking.
Another general method for separating keratins is by chromatography. Several
types
of chromatography can be employed to fractionate keratin solutions including
size exclusion
or gel filtration chromatography, affinity chromatography, isoelectric
focusing, gel
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electrophoresis, ion exchange chromatography, and inununoaffinity
chromatography. These
techniques are well known in the art and are capable of separating compounds,
including
proteins, by the characteristics of molecular weight, chemical functionality,
isoelectric point,
charge, or interactions with specific antibodies, and can be used alone or in
any combination
__ to effect high degrees of separation and resulting purity.
A preferred purification method is ion exchange (IEx) chromatography. IEx
chromatography is particularly suited to protein separation owning to the
amphiphilic nature
of proteins in general and keratins in particular. Depending on the starting
pH of the solution,
and the desired fraction slated for retention, either cationic or anionic IEx
(CIEx or AIEx,
__ respectively) techniques can be used. For example, at a pH of 6 and above,
both gamma- and
alpha-keratins are soluble and above their isoelectric points. As such, they
are anionic and
can be bound to an anionic exchange resin. However, it has been discovered
that a sub-
fraction of keratins does not bind to a weakly anionic exchange resin and
instead passes
through a column packed with such resin. A preferred solution for AIEx
chromatography is
__ purified or fractionated keratin, isolated as described previously, in
purified water at a
concentration between 0 and 5 weight/volume %. A preferred concentration is
between 0 and
4 w/v %. A most preferred concentration is approximately 2 w/v %. It is
preferred to keep the
ionic strength of said solution initially quite low to facilitate binding to
the AIEx column.
This is achieved by using a minimal amount of acid to titrate a purified water
solution of the
__ keratin to between pH 6 and 7. A most preferred pH is 6. This solution can
be loaded onto an
AIEx column such as DEAE-Sepharose resin or Q-Sepharose resin columns. A
preferred
column resin is DEAE-Sepharose resin. The solution that passes through the
column can be
collected and further processed as described previously to isolate a fraction
of acidic keratin
powder.
In some embodiments the activity of the keratin matrix is enhanced by using an
AIEx
column to produce the keratin to thereby promote cell adhesion. Without
wishing to be bound
to any particular theory, it is envisioned that the fraction that passes
through an anionic
column, i.e. acidic keratin, promotes cell adhesion.
Another fraction binds readily, and can be washed off the column using salting
__ techniques known in the art. A preferred elution medium is sodium chloride
solution. A
preferred concentration of sodium chloride is between 0.1 and 2M. A most
preferred
concentration is 2M. The pH of the solution is preferred to be between 6 and
12. A most
preferred pH is 12. In order to maintain stable pH during the elution process,
a buffer salt can
be added. A preferred buffer salt is Trizmae base. Those skilled in the art
will recognize that
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slight modifications to the salt concentration and pH can be made to effect
the elution of
keratin fractions with differing properties. It is also possible to use
different salt
concentrations and pH's in sequence, or employ the use of salt and/or pH
gradients to
produce different fractions. Regardless of the approach taken, however, the
column eluent
can be collected and further processed as described previously to isolate
fractions of basic
keratin powders.
A complimentary procedure is also feasible using CIEx techniques. Namely, the
keratin solution can be added to a cation exchange resin such as SP Sepharose
resin
(strongly cationic) or CM Sepharose resin (weakly cationic), and the basic
fraction
collected with the pass through. The retained acid keratin fraction can be
isolated by salting
as previously described.
Meta Keratins. Meta keratins are synthesized from both the alpha and gamma
fractions of kerateine using substantially the same procedures. Basically, the
kerateine is
dissolved in a denaturing solution such as 7M urea, aqueous ammonium hydroxide
solution,
or 20mM tris buffer solution. Pure oxygen is bubbled through the solution to
initiate
oxidative coupling reactions of cysteine groups. The progress of the reaction
is monitored by
an increase in molecular weight as measured using SDS-PAGE. Oxygen is
continually
bubbled through the reaction solution until a doubling or tripling of
molecular weight is
achieved. The pH of the denaturing solution can be adjusted to neutrality to
avoid hydrolysis
of the proteins by addition of mineral acid.
Keratin Intermediate Filaments. IFs of human hair fibers are obtained using
the
method of Thomas and coworkers (H. Thomas et al., Int. J. Biol. MacromoL 8,
258-64
(1986)). This is essentially a chemical etching method that reacts away the
keratin matrix that
serves to "glue" the IFs in place, thereby leaving the IFs behind. In a
typical extraction
process, swelling of the cuticle and sulfitolysis of matrix proteins is
achieved using 0.2M
Na2S03, 0.1M Na206S4 in 8M urea and 0.1M tris-HC1 buffer at pH 9. The
extraction
proceeds at room temperature for 24 hours. After concentrating, the dissolved
matrix keratins
and IFs are precipitated by addition of zinc acetate solution to a pH of
approximately 6. The
Ws are then separated from the matrix keratins by dialysis against 0.05M
tetraborate solution.
Increased purity is obtained by precipitating the dialyzed solution with zinc
acetate,
redissolving the IFs in sodium citrate, dialyzing against distilled water, and
then freeze drying
the sample.
Further discussion of keratin preparations are found in U.S. Patent
Application
Publication 2006/0051732 (Van Dyke).
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Compositions and formulations. Dry powders may be formed of keratin
derivatives as
described above in accordance with known techniques such as freeze drying
(1yophilization).
In some embodiments, compositions of the invention may be produced by mixing
such a dry
powder composition form with an aqueous solution to produce a composition
comprising an
electrolyte solution having said keratin derivative solubilized therein. The
mixing step can be
carried out at any suitable temperature, typically room temperature, and can
be carried out by
any suitable technique such as stirring, shaking, agitation, etc. The salts
and other constituent
ingredients of the electrolyte solution (e.g., all ingredients except the
keratin derivative and
the water) may be contained entirely in the dry powder, entirely within the
aqueous
composition, or may be distributed between the dry powder and the aqueous
composition.
For example, in some embodiments, at least a portion of the constituents of
the electrolyte
solution are contained in the dry powder.
The formation of a matrix comprising keratin materials such as described above
can
be carried out in accordance with techniques long established in the field or
variations thereof
that will be apparent to those skilled in the art. In some embodiments, the
keratin preparation
is dried and rehydrated prior to use. See, e.g., U.S. Pat. Nos. 2,413,983 to
Lustig et al., U.S.
Pat. No. 2,236,921 to Schollkipf et al., and 3,464,825 to Anker. In preferred
embodiments,
the matrix, or hydrogel, is formed by re-hydration of the lyophilized material
with a suitable
solvent, such as water or phosphate buffered saline '(PBS). The gel can be
sterilized, e.g., by
7-irradiation (800 lcrad) using a Co60 source. Other suitable methods of
forming keratin
matrices include, but are not limited to, those found in U.S. Pat. Nos.
6,270,793 (Van Dyke et
al.), 6,274,155 (Van Dyke et al.), 6,316,598 (Van Dyke et al.), 6,461,628
(Blanchard et al.),
6,544,548 (Siller-Jackson et al.), and 7,01,987 (Van Dyke).
In some composition embodiments, the keratin derivatives (particularly alpha
and/or
gamma kerateine and alpha and/or gamma keratose) have an average molecular
weight of
from about 10 to 70 or 85 or 100 kiloDaltons. Other keratin derivatives,
particularly meta-
keratins, may have higher average molecular weights, e.g., up to 200 or 300
kiloDaltons. In
general, the keratin derivative (this term including combinations of
derivatives) may be
included in the composition in an amount of from about 0.1, 0.5 or 1 percent
by weight up to
3, 4, 5, or 10 percent by weight. The composition when mixed preferably has a
viscosity of
about 1 or 1.5 to 4, 8, 10 or 20 centipoise. Viscosity at any concentration
can be modulated
by changing the ratio of alpha to gamma keratose.
The keratin derivative composition or formulation may optionally contain one
or
more active ingredients such as one or more growth factors, analgesics,
antimicrobials,
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additional coagulants, etc. (e.g., in an amount ranging from 0.0000001 to 1 or
5 percent by
weight of the composition that comprises the keratin derivative(s)), to
facilitate growth or
healing, provide pain relief, inhibit the growth of microbes such as bacteria,
facilitate or
inhibit coagulation, facilitate or inhibit cell or tissue adhesion, etc.
Examples of suitable
growth factors include, but are not limited to, nerve growth factor, vascular
endothelial
growth factor, fibronectin, fibrin, laminin, acidic and basic fibroblast
growth factors,
testosterone, ganglioside GM-1, catalase, insulin-like growth factor-I (IGF-
I), platelet-derived
growth factor (PDGF), neuronal growth factor galectin-1, and combinations
thereof. See, e.g.,
US Patent No. 6,506,727 to Hansson et al. and US Patent No. 6,890,531 to Horie
et al.
As used herein, "growth factors" include molecules that promote the
regeneration,
growth and survival of tissue. Growth factors that are used in some
embodiments of the
present invention may be those naturally found in keratin extracts, or may be
in the form of
an additive, added to the keratin extracts or formed keratin matrices.
Examples of growth
factors include, but are not limited to, nerve growth factor (NGF) and other
neurotrophins,
platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin
(TPO),
myostatin (GDF-8), growth differentiation factor-9 (GDF9), basic fibroblast
growth factor
(bFGF or FGF2), epidermal growth factor (EGF), hepatocyte growth factor (HGF),
granulocyte-colony stimulating factor (G-CSF), and granulocyte-macrophage
colony
stimulating factor (GM-CSF). There are many structurally and evolutionarily
related proteins
that make up large families of growth factors, and there are numerous growth
factor families,
e.g., the neurotrophins (NGF, BDNF, and NT3). The neurotrophins are a family
of molecules
that promote the growth and surv. ival of, inter alia, nervous tissue.
Examples of neurotrophins
include, but are not limited to, nerve growth factor (NGF), brain-derived
neurotrophic factor
(BDNF), neurotrophin 3 (NT-3), and neurotrophin 4 (NT-4). See U.S. Pat. Nos.
5,843,914 to
Johnson, Jr. et al.; 5,488,099 to Persson et al.; 5,438,121 to Barde et al.;
5,235,043 to Collins
et al.; and 6,005,081 to Burton et al.
For example, a growth factor can be added to the keratin matrix composition in
an
amount effective to promote the regeneration, growth and survival of various
tissues. The
growth factor is provided in concentrations ranging from 0.1 ng/mL to 1000
ng/mL. More
preferably, growth factor is provided in concentrations ranging from 1 ng/mL
to 100 ng/mL,
and most preferably 10 ng/mL to 10Ong/mL. See U.S. Pat. No. 6,063,757 to Urso.
The composition is preferably sterile and non-pryogenic. The composition may
be
provided preformed and aseptically packaged in a suitable container, such as a
flexible
polymeric bag or bottle, or a foil container, or may be provided as a kit of
sterile dry powder
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in one container and sterile aqueous solution in a separate container for
mixing just prior to
use. When provided pre-formed and packaged in a sterile container, the
composition
preferably has a shelf life of at least 4 or 6 months (up to 2 or 3 years or
more) at room
temperature, prior to substantial loss of viscosity (e.g., more than 10 or 20
percent) and/or
substantial precipitation of the keratin derivative (e.g., settling detectable
upon visual
inspection). The kit may contain a single unit dose of the active keratin
derivative. A single
unit dose may be 0.1 or 0.5 or 1, to 100 or 200 or 300 grams of the keratin
derivative, or
more, depending upon its intended use.
Other examples of natural polymers that may be utilized in a similar fashion
to the
disclosed keratin preparations include, but are not limited to, collagen,
gelatin, fibronectin,
vitronectin, laminin, fibrin, mucin, elastin, nidogen (entactin),
proteoglycans, etc. (See, e.g.,
U.S. Pat. No. 5,691,203 to Katsuen et al.).
Clotting compositions and methods to control bleeding containing keratin
biomaterials. One aspect of the present invention is a method for treating
bleeding in a
subject afflicted with a bleeding wound comprising: applying a keratin
derivative to= a
bleeding wound in an amount effective to treat the bleeding. In some
embodiments, the
keratin derivative comprises, consists of or consists essentially of
kerateine, alpha kerateine,
gamma kerateine, acidic alpha kerateine, basic alpha kerateine, or
combinations thereof, such
as described above. The bleeding may be that associated with, e.g., severe
trauma producing
rapid, voluminous hemorrhaging, including, but not limited to: surgery;
penetrating trauma
such as stabbing and gunshot wounds; motor vehicle trauma; and head, neck,
chest and
abdominal hemorrhaging; with or without clear access to the site of the
hemorrhaging.
Many different compositions may comprise the hemostatic agent, including, but
not
limited to, keratin derivatives. Other examples of hemostatic agents include,
but are not
limited to, those comprising fibrin or fibrinogen, thrombin, factor XIII,
calcium, chitosan
(deacetylated poly-N-acetyl glucosamine), zeolite (oxides of silicon,
aluminum, sodium,
magnesium, and quartz), chitin (acetylated poly-N-acetyl glucosamine), bovine
clotting
factors, non-zeolite mineral (e.g., hydrophobic polymers and potassium salts),
and molecular
sieve materials from plant sources (e.g., TraumaDEXTm, AristaTm AH, etc.,
Medafor, Inc.,
Minneapolis, MN). It should be noted, however, that not all of these
hemostatic agents are
recommended for all types of bleeding treatments, and those skilled in the art
should select
hemostatic agents for use in the disclosed compositions and methods
accordingly. For
example, zeolite is intended only for external use.
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PCT/US2008/004984
In some embodiments of the invention, gels containing keratin derivatives are
used.
The gels of these embodiments are adherent to the tissue and hydrophilic. In
some
embodiments, when deposited onto a bleeding surface of a wound, the gels are
sufficiently
adhesive to not be washed away, even in the presence of active bleeding. In
some
embodiments the gel= absorbs fluid from the blood and becomes even more
adherent (e.g.,
within minutes of administration). Contact of the gel of some embodiments with
blood can
instigate thrombus formation, probably through platelet activation and/or
concentration of
= clotting factors. Also, without wishing to be bound to any particular
theory, it is thought that
the adherent gel of some embodiments can form a physical seal of the wound
site and provide
= 10 a porous scaffold for cell infiltration and granulation-like tissue
formation, much like clotted
blood.
In some embodiments, the keratin composition is applied directly onto the site
of the
bleeding. In some embodiments, the keratin composition is injected into the
body of a subject
to treat an internal site of bleeding, e.g., where there is no clear access to
the site of
hemorrhaging.
Wound healing compositions containing keratin biomaterials and methods to
promote
wound healing. An aspect of the present invention is a method of treating a
wound (e.g.,
= burns, abrasions, lacerations, incisions, pressure sores, puncture
wounds, penetration wounds,
gunshot wounds, crushing injuries, etc.) in a subject in need thereof,
comprising: topically
applying a keratin derivative to the wound in an amount effective to treat the
wound. In some
embodiments the keratin derivative comprises, consists of or consists
essentially of keratose,
alpha keratose, gamma keratose, acidic alpha keratose, acidic gamma keratose,
basic alpha
keratose, basic gamma keratose, kerateine, alpha kerateine, gamma kerateine,
acidic alpha
kerateine, acidic gamma kerateine, basic alpha kerateine, basic gamma
kerateine, etc., or
combinations thereof, such as described above.
The keratin derivative can be topically applied as a dry powder formulation
or, in
some embodiments, applied in an aqueous carrier (e.g., in the form of a gel).
In some
embodiments, the keratin derivative is provided in an ointment (a water-in-oil
preparation in
which the amount of oil exceeds the amount of water in the emulsion), or in a
cream (an oil-
in water preparation in which the amount of water is equal to or exceeds the
amount of oil in
the emulsion). In some embodiments, the keratin derivative is injected under
the skin to reach
an internal site of injury.
"Subjects" (or "patients") to be treated with the methods and compositions
described
herein include both human subjects and animal subjects (particularly other
mammalian
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subjects such as dogs, cats, horses, monkeys, etc.) for veterinary purposes.
Human subjects
are particularly preferred. The subjects may be male or female and may be any
age, including
neonate, infant, juvenile, adolescent, adult, and geriatric subjects.
Examples of wounds that can be treated with the present invention include burn
wounds. Burn wounds are tissue injuries that can result from heat, chemicals,
sunlight,
electricity, radiation, etc. Burns caused by heat, or thermal burns, are the
most common.
Chemical burns resemble thermal burns. Though burn wounds tend to occur most
often on
the skin, other body structures may be affected. For example, a severe burn
may penetrate
down to the fat, muscle or bone.
Wounds are often characterized by the depth of injury. For example, the degree
of a
burn is characterized as first, second or third depending on the depth of the
tissues injured. In
a first-degree burn, only the top layer of skin (the epidermis) is damaged. In
second-degree
burns, the middle layer of skin (the dermis) is damaged. Finally, in a third-
degree burn, the
most severe type, the damage is deep enough to affect the inner (fat) layer of
the skin.
Similarly, pressure sores of the skin are characterized as stage I (red,
unbroken skin,
erythema does not fade with release of pressure), stage II (disrupted
epidermis, often with
invasion into the dermis), stage III (injury of the dermis), and stage IV
(subcutaneous tissue is
exposed).
In pressure sore wounds, pressure-induced constriction of local capillaries
results in
ischemia in the affected skin. Similarly, a burn wound is ischemic due to
associated capillary
thrombosis. A diabetic ulcer is another example of a poorly perfused wound.
For these types
of wounds, where blood is not readily available to aid in the normal course of
wound healing,
in some embodiments compositions containing keratin derivatives are useful not
only for
providing a physical seal of the wound site, but also for providing a porous
scaffold for cell
infiltration and granulation-like tissue formation, much like clotted blood.
Wounds can be evolving injuries in that the area of tissue damage grows after
the
initial insult. For example, a burn wound is typically an evolving injury
characterized by
three zones: the zone of necrosis, the zone of injury, and the zone of
hyperemia. The zone of
necrosis is the area that directly received the external insult (e.g., heat or
chemical insult), and
contains irreversibly damaged tissue. The zone of injury is peripheral to and
below the zone
of necrosis, and the tissue is initially viable but fragile. In a typical
burn, the area of the zone
of necrosis increases as the tissue in the zone of injury becomes damaged. The
term "wound
conversion" describes the process by which the zone of injury progresses to
the zone of
necrosis, increasing the overall area of the wound. See, e.g., U.S. Patent No.
5,583,126 to
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Daynes et al. The zone of hyperemia is peripheral to and below the zone of
injury. It has
minimal cell injury, but is vasodilated.
Without wishing to be bound to any particular theory, it is thought that the
keratin
derivatives disclosed herein both suppress wound growth due to wound
conversion (e.g.,
measured by area of the wound over time, determined by observations of the
severity of
tissue damage with and/or without treatment, etc.) and promote and/or
accelerate the healing
of wounds (e.g., the measured wound area decreases at a faster rate over time
with treatment,
the wound is observed to be healing at a faster rate upon treatment, etc.).
The normal course
of healing for superficial and partial-thickness wounds is by regeneration of
epithelium from
existing basal cells at the surface of the wound, with or without mild
contraction. Deep
wounds heal by a combination of regeneration of epithelium and contraction.
Contraction
serves to decrease the area of the wound, and the epithelium regenerates from
the margins of
the wound.
In some embodiments, the keratin biomaterials are useful in promoting wound
healing
by enhancing the proliferation of fibroblasts (e.g., dermal fibroblasts)
and/or keratinocytes
(e.g., epidermal keratinocytes). See, e.g., U.S. Patent No. 6,673,603 to
Baetge et al. and U.S.
Patent No. 5,840,309 to Herstein et al. Without wishing to be bound to any
particular theory,
it is thought that the enhancement of fibroblast and/or keratinocyte
proliferation is promoted
by growth factors naturally found within the keratin preparations.
In some types of wounds, treatment includes the control of bleeding. For
instance,
abrasions, lacerations, incisions, penetration wounds, and crushing injuries,
etc. often involve
bleeding. Crushing injuries, for example, may involve an open wound (i.e.,
where the skin is
torn and tissues are exposed to the environment), or a closed wound (i.e.,
where the skin is
intact, but the underlying tissue is damaged).
Abrasions are superficial wounds in which the epidermis of the skin is scraped
off.
Abrasions can be caused by, e.g., falling upon a rough surface. Lacerations
are typically
irregular wounds caused by, e.g., a blunt impact to soft tissue (e.g., skin)
that lies on top of
hard tissue (e.g., bone), or involves the tearing of soft tissues (e.g.,
lacerations associated with
childbirth). Lacerations typically show bridging, where connective tissue
and/or blood vessels
flatten against the surface of the underlying hard tissue. Sometimes injury
caused by sharp
objects is also termed a laceration. In the case of injury caused by a sharp
object, there is
normally no bridging because the connective tissue and blood vessels are
severed.
Incisions or incised wounds are normally caused by clean, sharp-edged objects.
Superficial incisions (involving only the epidermis) are typically referred to
as "cuts."
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Incisions may be =caused by a knife, razor, glass splinter, etc., or may be
caused by a scalpel
during surgery or other medical procedure. Penetration wounds are caused by
objects
entering the body (e.g., a knife). Puncture wounds are caused by objects
penetrating the skin,
such as a needle or nail. Gunshot wounds are caused by bullets entering,
driving through, and
sometimes exiting, the body.
Wound injuries carry a risk of infection. In some embodiments, the wound
healing
composition includes an antimicrobial agent. Examples of antimicrobial agents
that can be
topically applied include, but are not limited to, bacitracin (e.g., 400-500
U/g of ointment),
polymyxin B sulfate (e.g., 5,000 or 10,000 U/g of ointment), neomycin (e.g.,
3.5 mg/g of
ointment), Polysporine antibiotic (a blend of polymyxin B sulfate and
bacitracin in an
ointment base), Neosporine antibiotic (a blend of neomycin, bacitracin and
polymyxin B
sulfate in an ointment base), povidone-iodine, silver sulfadiazine (e.g., 1%
cream), mafenide
acetate (a methylated topical sulfonamide compound, e.g., as a 0.5% cream),
nystatin (a
fungicide), nitrofurazone (e.g., 0.2%) and gentamicin (e.g., 0.1% cream).
Antimicrobial
solutions include, but are not limited to, acetic acid (e.g., 0.5% or 0.25%),
sodium
hypochlorite (Dalcin's solution, e.g., 0.5% or 0.25% Na0C1), silver nitrate
(e.g., 0.5%), and
chlorhexidine gluconate (e.g., 0.5%). In some embodiments, the wound healing
composition
includes additional wound healing components. Some antimicrobials are thought
to also
promote wound healing by mechanisms apart from their effects as a bactericide
or fungicide,
etc. For example, there is evidence that silver sulfadizine, the most commonly
used topical
agent for burn care in the United States, has this dual action (Ward RS and
Saffle JR,
Physical Therapy 1995;75(6) 526-38).
In some embodiments, the wound healing composition includes analgesics or
anesthetics for pain relief, surfactants, anti-inflammatory agents, etc. See,
e.g., U.S. Patent
No. 6,562,326 to Miller.
The keratin compositions disclosed herein are useful in both controlling the
bleeding
and in promoting the healing of wounds. The compositions are useful for both
open wounds
and closed wounds. In the case of closed wounds, the keratin may be applied
into the wound
site by, for example, injection with a syringe or from a pressurized canister.
In the case of
blunt trauma, the keratin compositions can be, for example, injected into the
abdomen
through the skin into the site of internal bleeding. However, as those skilled
in the art will
appreciate, tissue swelling must be taken into account so as to avoid over-
expansion and
possible tissue and/or organ damage. Methods of attenuating swelling, such as
treatment with
cold (e.g., cool water, ice, etc.) and elevation of the affected area, may
also be used.
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The dose of the keratin material applied to the wound will depend upon the
particular
wound suffered, the age and overall condition of the subject, the route of
administration, etc.,
and can be optimized in accordance with known techniques. In some embodiments,
the
dosage is 0.1 or 0.5 or 1, to 100 or 200 or 300 grams of the keratin
derivative, or more (e.g.,
in powder or aqueous carrier), depending upon its intended use. In some
embodiments, the
keratin is provided at a concentration from 0.001 to 10 mg/mL, or from 0.01 to
5 mg/mL. In
some embodiments, the keratin is provided at a concentration of from 0.1% to
80% (w/v), or
from 1% to 50% (w/v), or from 5% to 30% (w/v).
In some embodiments, the wound is treated by application of a keratin
preparation in
the form of a gel, cream or ointment. The would may also be treated by a "wet-
to-moist"
dressing, where the keratin (and optionally other additives) is generally
added in its powder
form to an aqueous carrier (e.g., distilled water or saline), and a dressing
(e.g., gauze) is
soaked with the aqueous preparation and placed onto the wound. The aqueous
preparation
should be reapplied as necessary to prevent the dressing from drying.
Alternatively, the
keratin preparation can be formed as a sheet wound dressing as described in
U.S. Patent No.
6,274,163 to Blanchard et al. In further embodiments, the keratin preparation
is formulated
for use as a spray, e.g., solutions such as aqueous preparations that can be
sprayed upon the
wound with an aerosol pump.
In some embodiments, the wound is treated by reapplication of the disclosed
compositions, e.g., several times a day, or as needed. Cleansing to remove
bacteria and
debridement to remove necrotic debris may also be warranted during the course
of treatment.
Application of a moisturizing cream or ointment may be used to soften wound
eschar in order
to assist in debridement.
Surgical or paramedic aids. Another aspect of the invention is a surgical or
paramedic
aid that includes a solid, physiologically acceptable substrate and a keratin
derivative on said
substrate. "Substrate" includes sponges, pacicings, wound dressings (such as
gauze or
bandages), sutures, fabrics, and prosthetic devices.
Kits comprising keratin derivatives. Another aspect of the invention is a kit
comprising, consisting of, or consisting essentially of a keratin derivative
in a container. The
keratin derivative is preferably packaged in the container in sterile form.
The kit may include
a physiologically acceptable substrate, such as sponges, packings, wound
dressings (such as
gauze or bandages), sutures, fabrics, and prosthetic devices.
Embodiments of the invention are further described in the following non-
limiting
examples.
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Example 1: Keratin Derivatives/Fractions
Keratose fractions were obtained using a method based on that of Alexander and
= coworkers. However the method was substantially modified to minimize
hydrolysis of
peptide bonds. Briefly, 50 grams of clean, dry hair that was collected from a
local barber
shop was reacted with 1000 mL of an aqueous solution of 2 w/v c/o peracetic
acid (PAA) at
room temperature for 12 hr. The oxidized hair was recovered using a 500 micron
sieve,
rinsed with copious amounts of DI water, and the excess water removed.
Keratoses were
extracted from the oxidized hair fibers with 1000 mL of 100mM Trizma base.
After 3
hours, the hair was separated by sieve and the liquid neutralized by dropwise
addition of
hydrochloric acid (HC1). Additional keratoses were extracted from the
remaining hair with
two subsequent extractions using 1000 mL of 0.1M Trizma base and 1000 mL of
DI water,
respectively. Each time the hair was separated by sieve and the liquid
neutralized with HC1.
All three extracts were combined, centrifuged, and any residual solid material
removed by
filtration. The combined extract was purified by tangential flow dialysis
against DI water
with a 1 KDa nominal low molecular weight cutoff membrane. The solution was
concentrated and lyophilized to produce a crude keratose powder.
Kerateine fractions were obtained using a modification of the method described
by
Goddard and Michaelis (J Biol Chem 1934;106:605-14). Briefly, the hair was
reacted with an
aqueous solution of 1M TGA at 37 C for 24 hours. The pH of the TGA solution
had been
adjusted to pH 10.2 by dropwise addition of saturated NaOH solution. The
extract solution
was filtered to remove the reduced hair fibers and retained. Additional
keratin was extracted
from the fibers by sequential extractions with 1000 mL of 100mM TGA at pH 10.2
for 24
hours, 1000 mL of 10mM TGA at pH 10.2 for 24 hours, and DI water at pH 10.2
for 24
hours. After each extraction, the solution was centrifuged, filtered, and
added to the dialysis
system. Eventually, all of the extracts were combined and dialyzed against DI
water with a 1
KDa nominal low molecular weight cutoff membrane. The solution was
concentrated, titrated
to pH 7, and stored at approximately 5% total protein concentration at 4 C.
Alternately, the
concentrated solution could be lyophilized and stored frozen and under
nitrogen.
Just prior to fractionation, keratose samples were re-dissolved in ultrapure
water and
titrated to pH 6 by addition of dilute HC1 solution. Kerateine samples were
titrated to pH 6 by
careful addition of dilute HC1 solution as well. The samples were loaded onto
a 200mL flash
chromatography column containing either DEAE-Sepharose (weakly anionic) or Q-
Sepharose (strongly anionic) exchange resin (50-100 mesh; Sigma-Aldrich,
Milwaukee,
WI) with gentle pressure and the flow through collected (acidic keratin). A
small volume of
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mM Trizma base (approximately 200 mL) at pH 6 was used to completely wash
through
the sample. Basic keratin was eluted from the column with 100mM tris base plus
2M NaC1 at
pH 12. Each sample was separately neutralized and dialyzed against DI water
using
tangential flow dialysis with a LMWCO of 1K.Da, concentrated by rotary
evaporation, and
5 freeze dried.
As previously described, a sample of alpha-keratose was produced, separated on
a
DEAE-Sepharose IEx column into acidic and basic fractions, dissolved in PBS,
and the pH
adjusted to 7.4. These solutions were prepared at 5 weight percent
concentration and their red
blood cell (RBC) aggregation characteristics grossly evaluated with fresh
whole human blood
10 by mixing at a 1:1 ratio. Samples were taken after 20 minutes and
evaluated by light
microscopy. The ion exchange chromatography was highly effective at separating
the
aggregation phenomenon (data not shown). Basic alpha-keratose was essentially
free from
interactions with blood cells, while the acidic alpha-keratose caused
excessive aggregation.
Samples of acidic and basic alpha-keratose, unfractionated alpha+gamma-
kerateines,
unfractionated alpha+gamma-keratose, and beta-keratose (derived from cuticle)
were
prepared at approximately 4 w/v % and pH 7.4 in phosphate buffered saline
(PBS). Samples
were tested for viscosity and RBC aggregation. These results are shown in
Table 1:
Table 1. Results of viscosity and RBC aggregation tests on keratin solutions.
Fluid formulations
were prepared at approximately 4 w/v % in PBS at pH 7.4 and tested with human
whole blood at a
ratio of 1:1.
Sample Description Viscosity (centipoise) RBC
Aggregation*
acidic alpha-keratose (1X AtEx) 5.65 3
acidic alpha-keratose (2X AlEx) 19.7 5
basic alpha-keratose 1.57 2
alpha+gamma-keratose (hydrolyzed) 1.12 1
alpha+gamma-kerateine (unfractionated) 1.59 2
* Degree of aggregation: 1 = none, 5 = high
Example 2: Cell Proliferation and Wound Healing in Animal Models
Several in vitro and in vivo studies were conducted to demonstrate the
biological
activity of keratin biomaterials. They involved the use of keratin proteins
derived from
human hair using oxidation and reduction reactions to break down the tertiary
structure of the
cortex and extract soluble proteins according to the following methods.
Keratin biomaterials derived from human hair mediated the growth behavior of
skin
component cells. In cell culture experiments, certain types of keratins were
mitogenic toward
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fibroblasts and keratinocytes. Keratin-based hydrogels were shown to be
capable of
passivating chemical and thermal burns in a mouse and pig model, respectively.
Keratose: Clean, dry hair was cut into small fibers and oxidized with
peracetic acid.
Free proteins were extracted using a denaturing solution, neutralized,
purified by dialysis,
concentrated, and isolated by lyophilization. A hydrogel was formed by re-
hydration with
phosphate buffered saline (PBS).
Kerateine: Clean, dry hair was cut into small fibers and reduced with
thioglycolic
acid. Free proteins were extracted using a denaturing solution, dialyzed,
neutralized, and
concentrated. Upon concentration, a viscous hydrogel formed upon exposure to
air.
Cell proliferation: Keratose powder (unfractionated keratose, alpha + gamma)
was
dissolved in culture media with and without serum at several concentrations
and used to
culture human dermal fibroblasts and keratinocytes. The cells had been grown
to
approximately 50% confluency in serum-containing media and serum starved for
24 hours
prior to exposure to the keratin-containing solutions. After 24 hours of
culture with the
keratin-containing media, cell proliferation was evaluated using a
mitochondria metabolic
assay (MTT assay). Cell proliferation assays using keratinocytes and
fibroblasts showed
statistically significant increases in the keratose treated groups (Figure 1).
Wound healing: Immune competent mice were de-haired and a chemical burn
induced between the shoulders using phenol. The wounds were treated after 20
minutes with
a keratin hydrogel and occlusive bandage. The keratin used was unfractionated
(alpha +
gamma) keratose. Dressings were changed every three days for up to 10 days.
Digital photos
of the wounds were taken and animals sacrificed at various time points so that
the wound area
could be excised for histological examination. Wound healing studies in mice
demonstrated
an interesting passivation of the chemical burn. Figure 2 shows the normal
course of wound
progression in a wound treated only with an occlusive dressing as initially
increasing in
wound area. This is due to destruction of vascular support of the peripheral
tissue followed
thereafter by necrosis at the wound margins. The result is a characteristic
growth of the
wound area. In the keratin treated groups, however, the trend was toward
stabilization of the
wound area at the onset of injury. This is thought to be due to a protective
mechanism that
limits morbidity, and/or rapid induction of angiogenesis that counteracts the
initial loss of
vascular support.
In a subsequent large animal study, pigs were cleaned and shaved, and a series
of
deep partial thickness burns were produced along the dorsal rnidline using a
heated brass
block. Wounds were treated every three days for up to 27 days. Keratins used
were
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unfractionated (alpha + gamma) keratose and unfractionated (alpha + gamma)
kerateine.
Digital photos of the wounds were taken and animals sacrificed at various time
points so that
the wound area could be excised for histological examination. In this large
animal study, the
previous findings from the mouse study detailed above were confirmed, with the
keratin
treatments suppressing wound growth and accelerating healing compared to
control groups
(Figure 3).
Example 3: Control of Bleedin2 in an Animal Model
The hemostatic potential of keratin gel was evaluated in a modestly
challenging
animal model. The keratin gel comprised unfractionated kerateine
(alpha+gamma). Liver
injuries are notoriously problematic as both the size of the liver and of the
wound increase.
This rabbit model can produce both profuse and lethal hemorrhage. Controlled
liver
transection was used as a means to establish a consistent set of conditions
that would result in
exsanguination in the absence of treatment (negative control), yet provide for
the recovery of
test animals when a conventional hemostat was applied (positive control). It
should be noted
that the hemostats used as positive controls in this study are indicated for
topical wounds and
require concomitant pressure; they were applied without compression in this
study. This was
done to avoid the confounding contribution compression would add as it was not
used with
the keratin gel.
A total of 16 New Zealand rabbits (3.7 kg average) were used in this study.
The
animals received a standardized liver injury that consisted of transection of
approximately
one third of the left central lobe and were then randomized into one of four
groups. Four
animals served as negative controls and received no treatment, four animals
received
treatment with QuikClot hemostatic agent, four animals were treated with
HemCon
hemostatic bandage, and four animals were treated with keratin gel. No
resuscitation fluids
were given and all animals were closely monitored during surgery. After one
hour the
surgical wound was closed and the animals transferred to the housing facility.
All surviving
animals were sacrificed after 72 hours. At the time of sacrifice, liver tissue
was retrieved for
histological analysis.
Surgeries and Postoperative Treatment. All procedures were performed in
accordance
with Wake Forest University's Animal Care and Use Committee guidelines, which
encompass regulatory and accreditation agencies' guidelines. The animals were
weighed
immediately before surgery. All animals were sedated using a combination of
Ketamine 10
mg/kg and Xylazine 4 mg/kg through an intramuscular injection, intubated and
maintained on
2-3% Isoflurane for the remainder of the procedure. The animals were then
placed in a supine
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position, shaved and connected to the monitoring devices. All animals were
connected to
ECG leads, pulse oximeter cuff on the tail, and an intra-esophageal probe for
temperature
monitoring. After sterile prepping and draping, the abdominal incision was
performed and the
liver exposed. Prior to the liver injury, the abdominal aorta of the animals
was exposed and
canulated using a 23 gauge needle connected to a pressure transducer (Lab-
stat,
ADInstruments Pty. Ltd. Castle Hill, Australia) which in turn was connected to
a PowerLab
(ADInstruments) system for data acquisition. The mean arterial pressure (MAP)
was recorded
continuously throughout the procedure. All animals were monitored for several
minutes and
assured to be in a stable state prior to liver injury. The median lobe of the
liver was used for
the injury due to its ample size and easy accessibility.
Preliminary data during model development showed that a consistent liver
injury
cross sectional area could be created that resulted in death when left
untreated, but that when
treated with a control material could rescue the animal. A 2.0 cm2 surface
area ring was used
to inflict a consistent sized injury to the liver by pulling the left central
lobe through the ring
and cutting inunediately adjacent to the ring with a surgical blade. The MAP,
temperature,
heart rate, 02 saturation, and shed blood were recorded throughout the
procedure at 30
seconds, 5, 15, 30, 45 and 60 minutes. Shed blood was measured at each time
point using pre-
weighed sterile surgical gauze that was placed under the liver injury. In
addition, blood
samples were taken for CBC through an ear vein.
All animals were randomized into the previously mentioned four experimental
groups. The negative control group did not receive any treatment and the time
of death was
recorded in minutes after infliction of the injury. As for the other
experimental groups, the
treatment was administered at the 5 minute time point unless the MAP fell to
half of the
starting value. For standardization, the hemostatic materials applied were
measured or
weighed. The keratin gel does not require compression so no compression was
used in any of
the other treatment groups so as not to confound the results. In the HemCon
hemostatic
bandage treatment group, a 4.5 x 2.5 cm piece of bandage that was placed on
the bleeding
surface of the liver throughout the procedure and was removed prior to
closure. In the
QuikClot hemostatic agent treated group, 2.5 grams of autoclave sterilized
material per
animal was used. The material was spread on the bleeding surface and was left
after closure
in the surviving animals. In the case of the keratin treatment group, 2 ml of
the gel was used
per animal. Sterile keratin gel was applied to the bleeding surface through a
1 ml syringe.
The keratin was also left in place after closure of the animals. These
parameters were
determined during initial model development based on complete coverage of the
wound site.
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For the surviving animals, the monitoring continued for 60 minutes, after
which the animal
was considered to have survived the initial trauma and the bleeding stopped.
The animals that
were treated with HemCon hemostatic bandage had to undergo removal of the
material
since it could not be left intraabdominally as indicated by the manufacturer.
The aortic
cannula was removed and hemostasis established at the insertion site. No
aortic bleeding was
observed in any animal at necropsy. The fascia and skin of the abdomen was
closed in two
layers. After complete closure of the abdomen, the animals were allowed to
recover and
transported to the housing facility where they were monitored every 15 minutes
until
complete recovery from anesthesia, then three times per day thereafter for the
following three
days. Blood samples were taken from all surviving animals every day for CBC
analysis.
Upon sacrifice at the 72 hour time point, the liver of each animal was
harvested for
histological evaluation.
All presented data is expressed as averages and the corresponding standard
deviations.
For statistical analysis, SPSS v.11 (SPSS Inc, Chicago, IL) was used. Outliers
were defined
as having a z-score larger then +3.0 or smaller then -3.0 using a modified z-
score (median of
the absolute deviation). Data at all time points were analyzed by one-way
analysis of variance
(ANOVA). If significant F values were found, the groups were further analyzed
by Fischer's
Least Significant Difference Test (LSD). An alpha of p<0.05 was considered
significant. The
probability of a Type I error was minimized by limiting comparisons; only
negative control
versus the 3 treatment groups were performed. In order to compensate for bias
generated by
early drop out of dead animals (i.e. animals that exsanguinated before the
=end of the 60
minute operative period), polynomial regression to known pathologic endpoints
was used to
estimate values during the first 60 minutes. For the percent blood loss
graphical data
(Figure 5) where statistical relevance was reached with some groups, values
are expressed as
means with their corresponding standard error.
Negative control animals (i.e. no treatment), as expected, exsanguinated
within the 60
minute operative period (31 19 minutes). Two animals in the QuikClot
hemostatic agent
group and one in the HemCon hemostatic bandage group did not survive beyond
the initial
60 minute operative period. Also in the HemCon hemostatic bandage group, one
animal
was euthanized 24 hours post-op on the advice of the veterinary staff. This
animal was not
ambulatory and could not eat or drink. One animal in the keratin group was
also sacrificed at
48 hours. Although the animal was moving freely in its cage, it was not eating
or drinking. At
necropsy, these animals showed no evidence of additional bleeding after the
operative period.
All other surviving animals recovered without incident, were freely moving in
their cages
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within 24 hours, and had normal CBC by 72 hours (data not shown). A summary of
the
survival data is shown in Figure 4.
Mean Arterial Pressure. The mean arterial pressure (MAP) was recorded using a
23
gauge needle placed into the lower part of the abdominal aorta. The needle was
connected to
a PE 50 tube, which in turn was connected to a pressure transducer (Lab-stat)
that was
connected to a PowerLab system for pressure recording. The MAP was
continuously
monitored during the entire course of the procedure or until the death of the
animal. To
further evaluate the significance of a change in MAP and heart rate, shock
index was used.
Shock index is a well established clinical scoring system for fast assessment
of trauma
patients. The modified shock index was calculated by dividing heart rate by
MAP (mmHg).
The mean arterial pressure in the abdominal aorta was recorded for 60 minutes.
Animals in the keratin and HemCon hemostatic bandage group were able to
achieve stable
MAPs after 5 minutes at 75% of the starting value. The QuikClot hemostatic
agent and
control groups failed to stabilize MAP and dropped to 45% of the starting
value after 60
minutes (Figure 6). However, these data did not reach statistical significance
between
groups.
The shock index (SI), a predictive score grading system for the severity of
blood loss,
showed a beneficial outcome for the keratin group with low values throughout
the first 60
minutes (Figure 7). The high values of QuikClot hemostatic agent matched with
two early
deaths during the first 20 minutes of observation supporting the predictive
nature of this
measure. Although a trend was noted, these data did not reach statistical
significance in the
present study.
Temperature, ECG and Heart Rate. The central temperature was recorded with an
esophageal probe connected to the surgery room monitor. The temperature of the
animal was
continuously monitored throughout the procedure and recorded at the previously
mentioned
time points. The ECG and heart rate were monitored using a three lead system
connected to
the surgery room monitor and was maintained throughout the entire procedure.
Flat line or
irregular electrical activity with electrical mechanical dissociation was used
to define the time
of death.
The liver damage model employed in this study represented severe trauma with
significant, rapid blood loss. The liver transection produced a lethal injury,
typically
involving one or two large vessels of approximately 1 nun diameter and several
in the 0.5 to
1.0 mm diameter ranges. The severity of the injury was such that untreated
rabbits all
exsanguinated within the 60 minute operative period. None of the animals were
able to
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compensate for loss of blood volume with an increase in heart rate. All
animals showed a
comparable decrease from 263 bpm to 188 bpm after 30 minutes and 154 bpm after
one hour.
There were no statistically significance differences between the groups.
However, the keratin
group showed a trend toward compensation and recovery with an increase in
heart rate in the
second half of the surgical period from 30 min to 60 min. The temperature of
all animals
dropped in a similar fashion with a step drop of 0.8 C in the first 5 minutes
and a total of
2.7 C over 60 minutes. There was no statistically significant difference
between the
experimental groups.
Shed Blood Shed blood was measured by weight after subtracting the weight of
the
pre-weighed gauze. Weights were recorded at each time point and fresh gauze
placed under
the liver injury. The shed blood was represented as a percent of the original
body weight for
each animal. CBC was determined from samples taken from an ear vein on a
HEMAVet
multi-species hematology system (Model 950FS, Drew Scientific, Dallas, TX).
Blood loss was measured by weighing the surgical gauze placed below the
injured
liver lobe. The blood loss was expressed as percentage of starting body
weight. As expected
in uncontrolled hemorrhage studies, all animals showed an initial phase of
profuse bleeding
followed by a linear phase with a lower bleeding rate, as MAP falls (Figure
5). A comparison
of the keratin and QuikClot hemostatic agent groups to the negative controls
shows a
significantly decreased amount of blood loss at the 30, 45, and 60 minute time
points (p
values for keratin vs. negative control were 0.018, 0.011 and 0.007; p values
for QuikClot
hemostatic agent vs. negative control were 0.009, 0.005 and 0.004,
respectively).
As one would expect, the survivability of the animals appeared to be dependent
on the
vascular anatomy at the injury site, which was not consistent from animal to
animal even
though the total surface area transected was controlled. When a single very
large bleeder (> 1
mm), or multiple large bleeders (> 2 to 3 in the 1 m size range) were
encountered within the
injury area, the animal's chance of survival was negligible in the QuikClot
hemostatic agent
and HemCon hemostatic bandage groups. In the QuikClot hemostatic agent group
in
particular, a single very large bleeder or an excess of 2 to 3 large bleeders
would ensure
lethality. It should be noted however, that when used according to
manufacturer's instructions
with concomitant pressure, other studies have shown better survival rates
using QuikClot
hemostatic agent and HemCone hemostatic bandage. In all cases of treatment
with keratin
gel, which was also used without any compression, the animals survived for at
least 24 hours,
regardless of the size of the severed vessels. Although a small number of
animals were used
in all test groups (n=4), these outcomes are encouraging.
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The keratin hemostatic gel consistently performed well by each outcome
measure,
particularly shed blood volume, MAP, and (importantly) survival. One
particularly
distinguishing outcome was shock index. In most cases of hemorrhage, cardiac
output is
increased to compensate for the drop in blood pressure. Once this mechanism
takes over, the
value of shock index increases rapidly and survivability becomes doubtful.
Remarkably, the
shock index in the keratin treatment group remained the lowest of all the
materials tested,
consistent with early effective hemostasis.
Histology. A tissue sample including the damaged liver surface was removed
from
each animal within one hour of euthanasia. Each sample was placed in Tissue-
Tek O.C.T.
Compound 4583 (SakuraZ) and frozen in liquid nitrogen. The frozen blocks were
sectioned
into 8[1m slices using a cryostat (Model CM 1850, Leica Microsystems,
Bannockburn, IL) to
include the transected portion of the liver and mounted onto microscope
slides. The slides
were fixed and stained with Hematoxylin and Eosin (H&E). Technical
difficulties in
sectioning arose with both the QuikClot hemostatic agent and the HemCon
hemostatic
bandage sections. The brittle QuikClote hemostatic agent made level sectioning
difficult and
created voids in the sections. The HemCon hemostatic bandage was removed
before
abdominal closure and therefore the clotted blood was only partially visible.
Digital images
were taken (Zeiss Axio Imager M1 Microscope, Carl Zeiss, Thornwood, NY) at
varying
magnifications to observe the interactions between the hemostat and the
damaged area of the
liver. A magnification of 100X showed the overall response of the tissue,
while
magnifications of 200X and 400X were used to visualize the cellular response.
The transected liver surfaces were examined by light microscopy of H&E stained
sections. The negative control group showed a clean cut with no tissue
response or necrosis
(Figure 8A). Moreover, no functional clotting was observed with little
thrombus adhered to
the surface. The QuikClote hemostatic agent samples were difficult to process
due to the
presence of this hard, granular zeolite in the clot. Histology revealed
necrotic tissue mixed
with blood clots (Figure 8B). The transparent areas represent QuikClot
hemostatic agent
particles removed during processing. The HemCon hemostatic bandage group
showed some
areas with clotted blood and adjacent cellular infiltration (Figure 8C). Since
the HemCon
hemostatic bandage was removed after 60 minutes, most of the liver surfaces
had only a thin
layer of blood clots. The keratin group showed a thick layer of biomaterial
attached to the
damaged liver surface (Figure 8D). Granulation-like tissue with cellular
infiltration had
formed in the pores of the keratin biomaterial gel (Figure 9).
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The keratin hemostatic gel was adherent to the tissue and hydrophilic. When
deposited onto the bleeding surface of the liver it was sufficiently adhesive
to not be washed
away, even in the presence of profuse bleeding. The gel absorbed fluid from
the blood and
became even more adherent within a few minutes of administration. Clotting and
adherence
was almost instantaneous with contact. Interestingly, the keratin gel formed a
thick seal of
granulation-like tissue over the wound site by 72 hours. Upon inspection of
histological
sections, 3 days after injury host cells could be seen infiltrating the gel.
It is believed that the
keratin gel used in these experiments serves two purposes. First, contact of
the gel with whole
blood instigates thrombus formation, probably through platelet activation or
concentration of
clotting factors. Second, the adherent gel forms a physical seal of the wound
site and provides
a porous scaffold for cell infiltration and granulation-like tissue formation,
much like clotted
blood.
The foregoing is illustrative of the present invention, and is not to be
construed as
limiting thereof. The invention is defined by the following claims, with
equivalents of the
claims to be included therein.
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