Method of treating chronic ulcers
United States Patent: 7,700,660
Issued: April 20, 2010
Anton-Lewis (Winterville, NC)
Assignee: Encelle, Inc.
Appl. No.: 09/870,414
Filed: May 30, 2001
George Washington University's Healthcare MBA
The invention provides a method of
treating a chronic ulcer, such as a diabetic ulcer, comprising
administering a therapeutic amount of a hydrogel matrix to the ulcer, the
matrix composition comprising gelatin and a long chain carbohydrate. The
matrix may further include polar amino acids, nitric oxide inhibitors and
super oxide inhibitors. Injection is a preferred method of administration.
The matrix may be injected into one or more locations within the ulcer,
underneath the ulcer and/or around the periphery of the ulcer.
Description of the
FIELD OF INVENTION
The invention relates to methods of treating chronic ulcers, such as
BACKGROUND OF THE INVENTION
There are over 15 million diagnosed cases of diabetes in the United States
alone. According to the American Diabetes Association, about 60-70% of
people with diabetes have mild to severe forms of diabetes-related nerve
damage. Diabetic neuropathy is a condition that encompasses a wide range
of dysfunction. Neuropathic ulcers or lesions of the foot resulting from
diabetic neuropathy are a major cause of lower leg amputations. In fact,
progression of diabetic foot ulcers is the leading cause of non-traumatic
lower limb amputations in the United States. The risk of a leg amputation
is 15-40 times greater for a person with diabetes.
Loss of protective sensation and repetitive trauma (e.g. walking) are
major causes of such ulcers. Loss of tone in the small muscles of the feet
cause changes in the architecture of the foot that ultimately result in
increased pressure over the ball of the foot. This increased pressure
causes calluses and eventually ulceration.
These lesions are associated with microcirculatory compromise, resulting
in the breakdown of dermal integrity. The etiology is thought to be
progressive endothelial vessel injury induced by chronic hyperglycemia.
While neuropathy, trauma, and infection secondarily promote foot lesion
extension, the underlying pathology for these conditions and the ulcer
itself is chronic hyperglycemia resulting in compromised vascular flow to
the skin. Once developed, these ulcers become chronic conditions lasting
indefinitely. It is not unusual for ulcers of this type to persist for
many years. Unlike common trauma-induced superficial wounds, chronic
diabetic ulcers penetrate deep into the patient's tissue, often exhibiting
penetration completely through the dermis, leaving the ulcer open and
exposing underlying structures such as tendon, muscle or bone.
Current therapy for diabetic foot ulcers is inadequate, as evidenced by
the high incidence of healing failure (See Ramsey et al., Diabetes Care
22:382-387, 1999). Conventional therapies include debridement of necrotic
tissue, repeated sterile dressings, use of orthotic devices to reduce
pressure, bed rest, and aggressive use of antibiotics to fight infection.
Conventional therapy does not address the underlying pathology of
microangiopathy in the lower extremity, but seeks to provide enough
covering to prevent ulcer extension and possible amputation. Cell-based
coverings are sometimes used to treat ulcers, such coverings including
autologous skin flaps, skin grafts, or cultured skin layers such as
APLIGRAF.TM.. However, providing a covering that may or may not assist in
closure does nothing to treat the underlying pathology, which is
compromised circulation in combination with compromised sensation. As
such, the rate of recurrence of healed ulcers is as high as 80%. There
remains a need in the art for therapeutic methods for treating chronic
ulcers, such as diabetes-related ulcers.
SUMMARY OF THE INVENTION
It has been discovered that the matrix described herein is capable of
successfully treating and healing chronic ulcers, such as ulcers resulting
from diabetes-related vasculoneuropathy. Although ulcers of this type are
often resistant to conventional wound treatments, the method of the
present invention can heal chronic lesions or ulcers in a matter of days
or weeks. The present invention involves the administration of a
therapeutic amount of a hydrogel matrix to the ulcer in a manner that
exposes polar groups of the basement membrane of the patient's tissue to
the components of the matrix (e.g. by injection).
The matrix of the invention preferably comprises a gelatin component, such
as denatured collagen, at a concentration of about 0.01 to about 40 mM.
The matrix also includes a long chain carbohydrate, such as dextran. The
preferably concentration of dextran is about 0.01 to about 10 mM.
Preferred embodiments of the matrix further include an effective amount of
polar amino acids, one or more nitric oxide inhibitors, such as L-cysteine
or L-arginine analogues, and a superoxide inhibitor, such as EDTA or salts
In a preferred embodiment, the administering step comprises injecting the
matrix into one or more superficial locations within the ulcer,
superficial locations around the periphery of the ulcer, and locations
underneath the ulcer. Typically, the total therapeutic amount comprises
about 1 to about 60 ml.
DETAILED DESCRIPTION OF THE INVENTION
The present invention now will be described more fully hereinafter with
reference to the accompanying drawings, in which preferred embodiments of
the invention are shown. This invention may, however, be embodied in many
different forms and should not be construed as limited to the embodiments
set forth herein; rather, these embodiments are provided so that this
disclosure will be thorough and complete, and will fully convey the scope
of the invention to those skilled in the art.
Early in fetal development, a more open form of collagen (compared to
tightly bound mature collagen) is associated with large carbohydrate
molecules, and serves as the predominant tissue scaffolding. It is
believed that attachment of differentiated or incompletely differentiated
cells of mescenchymal origin to this polar, proteoglycan-like, collagen
scaffolding results in a specific host tissue response. This response is
to guide the differentiation of mesenchymal tissue into endothelial cells,
subsequently organizing into blood vessels, and finally differentiating
into primitive blood cells prior to the differentiation of bone marrow.
Although not bound by any particular theory, the present invention is
intended to provide a matrix scaffolding designed to maximize the polar
amino acid hydrogen bonding sites found in alpha chains derived from
collagen. These alpha chains or gelatin are preferably derived from pig
gelatin, and stabilized by 500,000 molecular weight dextran, or other
long-chain carbohydrates, added while the alpha chains are heated. The
positively charged polar groups of the collagen-derived alpha chains are
then able to associate with the negatively charged --OH groups of the
repeating glucose units found in the dextran. The gelatin and the dextran
form a proteoglycan-type structure. FIGS. 1-4 (see Original Patent)
illustrate the interaction between the various components of the preferred
embodiment of the matrix of the invention and interaction between the
matrix and the tissue of a patient.
FIG. 1 (see Original Patent) illustrates the creation of polar alpha
chains 15 from tropocollagen 10 derived from mature collagen. Heating
tropocollagen 10 disrupts the hydrogen bonds that tightly contain the
triple stranded monomers in mature collagen. By breaking these hydrogen
bonds, the polar amine and carboxylic acid groups are now available for
binding to polar groups from other sources or themselves.
FIGS. 2A-2B (see Original Patent) illustrate stabilization of the matrix
monomeric scaffolding by the introduction of a long-chain carbohydrate 20,
such as dextran. As shown in FIG. 2B, without the long-chain carbohydrate
20, the alpha chain 15 will form hydrogen bonds between the amino and
carboxylic acid groups within the linear portion of the monomer and fold
upon itself, thus limiting available sites for cellular attachment. As
depicted in FIG. 2A, the long-chain carbohydrate 20 serves to hold the
alpha chain 15 open by interfering with this folding process.
FIG. 3 (see Original Patent) illustrates the effect of polar amino acids
and/or L-cysteine added to stabilize the monomer/carbohydrate units 25 by
linking the exposed monomer polar sites to, for example, arginine's amine
groups or glutamic acid's carboxylic acid groups. Furthermore, disulfide
linkages can be formed between L-cysteine molecules (thereby forming
cystine), which in turn forms hydrogen bonds to the monomeric alpha chains
15. The hydrogen bonds formed between these additional amino acids and
monomer/dextran units 25 are broken when the matrix is liquefied upon
heating, and the polar groups are freed to attach the monomer/dextran
units to exposed patient tissue surfaces upon injection. In preferred
embodiments, EDTA or a salt thereof is also present to chelate divalent
cations and thereby prevent divalent cations from being preferentially
attracted to the exposed polar groups of the monomer/carbohydrate units 25
to the exclusion of the polar amino acids.
FIG. 4 (see Original Patent) shows attachment of the matrix to patient
tissue by hydrogen bonding to exposed tissue amino acids. Exposure of
these amino acids is easily achieved by tearing of the tissue with a
hypodermic needle at the time of injection. The exposed polar groups of
the basement membrane (BM) of the patient's tissue readily bind to the
solid, scaffolding portion of the matrix enhanced by the polar amino
acids. The aqueous portion is believed to be absorbed over a period of
minutes to hours at normal body temperature.
Normally, the tearing of tissue secondary to injection trauma stimulates
production and release of nitric oxide, initiating recruitment of immune
and inflammatory cells that phagocytise or release chemicals to destroy
foreign substances. By providing local and temporal inhibition of nitric
oxide and superoxide release and production, nitric oxide inhibitors, such
as aminoguanidine and cysteine, and superoxide inhibitors, such as EDTA,
allow the collagen derived alpha chain/dextran units 25 to bind and become
integrated on the exposed tissue surface. The alpha chain/dextran units 25
then serve as the scaffolding on which formerly differentiated host cells
de-differentiate into "mesenchymoid" morphology. This de-differentiation
process is followed by integration of these incompletely differentiated
cells into host tissue. These mesenchymoid cells are then able to promote
areas of their genome that leads to differentiation into fibroblasts,
endothelial cells, and primitive blood forms, which results in tissue
healing and regeneration.
By providing a proteoglycan-like scaffolding similar to that found in the
early stages of fetal development, and using structural stabilizers that
serve a secondary purpose in enhancing host response to the scaffolding
upon injection, the matrix serves as a biocompatible device capable of
increasing vascularization and promoting wound healing and local tissue
regeneration, even in the case of diabetic foot ulcers unresponsive to
conventional ulcer treatments. Because the matrix promotes tissue
regeneration, as occurs during embryogenesis and fetogenesis where similar
types of scaffolding are present, it has now been discovered that the
matrix of the invention can be used to successfully treat chronic ulcers
that fail to respond to conventional would therapy, such as ulcers caused
by diabetes, ulcers caused by chronic pressure (decubitus ulcers), venous
stasis ulcers, or trauma-induced ulcers accompanied by surrounding
Components of the Matrix
The matrix comprises a gelatin component. Although denatured collagen is
the preferred gelatin component, other gelatinous components characterized
by a backbone comprised of long chain sequences of amino acids having
polar groups whose intramolecular hydrogen bonds can be broken in order to
expose the polar groups to interaction with other molecules can be used.
For example, boiled agarose, alginate, keratin, aminoglycans,
proteoglycans and the like could be used as the gelatin component. In one
embodiment, the gelatin is porcine gelatin from partially hydrolyzed
collagen derived from skin tissue.
The gelatin is present at a concentration of about 0.01 to about 40 mM,
preferably about 0.05 to about 30 mM, most preferably about 1 to about 5
mM. Advantageously, the gelatin concentration is approximately 1.6 mM. The
above concentrations provide a solid phase at storage temperature (below
about 33.degree. C.) and a liquid phase at treatment temperature (about 35
to about 40.degree. C.). Intact collagen may be added in small amounts to
provide an additional binding network. The final concentration of intact
collagen is from about 0 to about 5 mM, preferably 0 to about 2 mM, most
preferably about 0.05 to about 0.5 mM.
A long chain carbohydrate having a molecular weight of about 20,000 to
about 1,000,000 Daltons is added to the gelatin component. Although
dextran is a preferred carbohydrate, other high molecular weight
carbohydrates may be used, such as amylopectin. The dextran loosely
polymerizes around the gelatin component, thereby facilitating cell
attachment by preventing folding of the gelatin scaffolding. The long
chain carbohydrate is present at a concentration of about 0.01 to about 10
mM, preferably about 0.01 to about 1 mM, most preferably about 0.01 to
about 0.1 mM. In one embodiment, dextran is present at a concentration of
about 0.086 mM.
The gelatin/long chain carbohydrate component of the matrix of the present
invention is mixed with a liquid composition. The liquid composition is
preferably based upon a standard culture medium, such as Medium 199,
supplemented with additives as described below.
The matrix preferably includes an effective amount of polar amino acids,
such as arginine, lysine, histidine, glutamic acid, and aspartic acid,
which further enhance the bioadhesiveness of the matrix. An effective
amount is the amount necessary to increase the rigidity of the matrix and
allow direct injection of the matrix into the patient. In one embodiment,
the concentration of polar amino acids is about 3 to about 150 mM,
preferably about 10 to about 65 mM, and more preferably about 15 to about
Advantageously, the added polar amino acids comprise L-glutamic acid,
L-lysine, L-arginine, or mixtures thereof. The final concentration of L-glutamic
acid is about 2 to about 60 mM, preferably about 5 to about 40 mM, most
preferably about 10 to about 20 mM. In one embodiment, the concentration
of L-glutamic acid is about 15 mM. The final concentration of L-lysine is
about 0.5 to about 30 mM, preferably about 1 to about 15 mM, most
preferably about 1 to about 10 mM. In one embodiment, the concentration of
L-lysine is about 5 mM. The final concentration of L-arginine is about 1
to about 40 mM, preferably about 1 to about 30, most preferably about 5 to
about 15 mM. In one embodiment, the final concentration of L-arginine is
about 10 mM.
Additionally, the matrix preferably contains one or more nitric oxide
inhibitors. Nitric oxide inhibitor is defined as any composition or agent
that inhibits the production of nitric oxide or scavenges or removes
existing nitric oxide. Nitric oxide, a pleiotropic mediator of
inflammation, is a soluble gas produced by endothelial cells, macrophages,
and specific neurons in the brain, and is active in inducing an
inflammatory response. Nitric oxide and its metabolites are known to cause
cellular death from nuclear destruction and related injuries. Preferred
nitric oxide inhibitors include L-cysteine, L-arginine analogues (such as
aminoguanidine, N-monomethyl-L-arginine, N-nitro-L-arginine, D-arginine
and the like), cystine, heparin, and mixtures thereof.
In one embodiment, the matrix contains L-cysteine. L-cysteine acts as a
nitric oxide scavenger and provides disulfide linkages, which increase the
matrix's rigidity and resistance to force. The final concentration of L-cysteine
is about 5 to about 500 .mu.M, preferably about 10 to about 100 .mu.M,
most preferably about 15 to about 25 .mu.M. In one embodiment, the final
concentration is about 20 .mu.M.
Advantageously, aminoguanidine is also added to the matrix of the present
invention. As indicated above, aminoguanidine is an L-arginine analogue
and acts as a nitric oxide inhibitor. The final concentration of
aminoguanidine is about 5 to about 500 .mu.M, preferably about 10 to about
100 .mu.M, most preferably about 15 to about 25 .mu.M. In one embodiment,
the final concentration is about 20 .mu.M.
Additionally, the matrix of the present invention may include a superoxide
inhibitor. A preferred superoxide inhibitor is ethylenediaminetetraacetic
acid (EDTA) or a salt thereof. Superoxide is a highly toxic reactive
oxygen species, whose formation is catalyzed by divalent transition
metals, such as iron, manganese, cobalt, and sometimes calcium. Highly
reactive oxygen species such as superoxide (O.sub.2.sup.31) can be further
converted to the highly toxic hydroxyl radical (OH.sup.31) in the presence
of iron. By chelating these metal catalysts, EDTA serves as an
antioxidant. EDTA is also a divalent cation chelator, which increases the
rigidity of the matrix by removing inhibition of --NH.sub.2 to --COOH
hydrogen bonding. The concentration range for the superoxide inhibitor is
about 0.01 to about 10 mM, preferably 1 to about 8 mM, most preferably
about 2 to about 6 mM. In a preferred embodiment, the superoxide inhibitor
is present at a concentration of about 4 mM.
Table 1 (see Original Patent) lists particularly preferred key components
of the matrix of the present invention along with suitable concentrations
as well as preferred concentrations for each component.
Place 835 ml of Medium 199 into a stirred beaker. While stirring, heat the
solution to 50.degree. C. Pipette 63.28 .mu.l of cysteine, 1 ml of
L-glutamine and 200 .mu.l of aminoguanidine into the stirred beaker. Add
the following gamma-irradiated dry raw materials: 120 grams of denatured
collagen, 50 grams of dextran, and 0.1 grams of intact collagen. Use a
glass stirring rod to aid mixing of the dry materials into solution.
Pipette 8 ml of EDTA into the solution. Pipette 5 ml of L-glutamic acid, 5
ml of L-lysine acetate, and 5 ml of arginine HCl into the stirred beaker.
Note that the solution will turn yellow. Use 10% NaOH to adjust the pH of
the matrix solution to a final pH of 7.40.+-.0.1. Osmolality is preferably
adjusted with sodium chloride and/or sterile water as need to a final
osmolality of about 200 to about 400 mOsm.
Preferably, a therapeutic amount of the matrix of the invention is
administered to a patient suffering from a ulcer, such as a chronic foot
ulcer caused by diabetes-related vasculoneuropathy. The patient can be any
animal, including mammals such as dogs, cats and humans. The term
"therapeutic amount" refers to the amount required to promote ulcer
healing via tissue regeneration as evidenced by, for example, reduction in
the size of the ulcer. The therapeutic amount will be primarily determined
by the size of the chronic lesion. Typically, the volume of matrix applied
to the ulcer is about 1 to about 60 mL. In other terms, the therapeutic
amount is approximately 0.1 to about 5 ml/2.5 cm of the "injection track,"
which is the total linear distance that will be traversed during matrix
administration. Preferably, the therapeutic amount is sufficient to
provide a uniform scaffolding for cellular attachment and differentiation
in the subdermal/subcutaneous interface beneath the ulcer crater and under
the ulcer periphery. In the case of a diabetic foot ulcer, wherein the
compromised microvasculature extends to contiguous tissue underneath the
ulcer, the physician must use clinical judgment to inject subdermal or
subcutaneous tissues beneath the ulcer that he/she feels would benefit
from regeneration of healthy tissue. The matrix is warmed to a temperature
of about 35 to about 40.degree. C. prior to administration in order to
liquefy the matrix.
The method of application of the matrix should result in contact between
the matrix and exposed polar groups of the basement membrane of the
patient's tissue. A preferred method of administering the matrix is by
injection, wherein the needle itself provides the necessary tearing of
tissue that exposes cellular attachment sites capable of integration with
the injected matrix.
In one embodiment, the matrix is injected intradermally or subdermally and
circumferentially around the perimeter of the lesion (skin side) as well
as intralesionally across the lesion width in parallel tracks separated by
about 1 cm. Thus, a typical dose consists of multiple superficial
injections at spaced locations around and/or within the lesion. The
preferred target location of the superficial injections is the area of the
dermal/subdermal tissue junction, which is typically about 0.5 to about
2.0 mm beneath the surface. The skin will be pierced superficially as if
intending to give an intradermal injection, applying pressure on the
plunger. The matrix will not flow at this juncture, so the needle should
slowly be moved deeper at a wider angle until the matrix flows with gentle
plunger pressure. At that point, the needle should be in the desired
subdermal space. The plunger should be pulled back to ensure that the
needle is not directly entering a vein or an artery. If the ulcer is a
full thickness ulcer, that is, with no dermal tissue exposed in the wound
center, then the intralesional matrix injections should be injected at the
most superficial angle possible to allow the matrix to contact the surface
tissue. In this approach, the needle will be visible as it is advanced
just beneath the exposed ulcer surface and the surface will be visible
expanded as the matrix volume is injected. As noted above, in addition to
the superficial injections around and within the ulcer, it may be
advisable to inject an additional volume of matrix underneath the ulcer.
Claim 1 of 54 Claims
1. A method of treating an ulcer,
comprising administering a therapeutic amount of a hydrogel matrix in
liquid form to the ulcer, the matrix composition comprising gelatin and a
long chain carbohydrate, wherein said administering step comprises
injecting the hydrogel matrix into one or more locations in the area of
the dermal/subdermal tissue junction beneath the ulcer or at the periphery
of the ulcer.
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