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CA 02578212 2007-02-27
WO 2006/026604 PCT/US2005/030798
METHODS FOR PROMOTING WOUND HEALING
1. FIELD OF THE INVENTION
[0001] The present invention relates to methods for using Product R, a peptide-
nucleic acid composition, to promote wound healing and/or treat loss of
appetite or fatigue
in patients suffering from a wound. The methods are applicable for treating a
wide variety
of wounds, including ulcers and burn injuries.
2. BACKGROUND OF THE INVENTION
2.1. Wounds and Wound healin~
[0002] Wound healing is the process through which the repair of damaged
tissue(s)
is accomplished. Wounds can range from minor abrasions to complicated life-
threatening
deep lacerations. Wounds can be classified in general into two main classes:
1) acute
wounds, such as those acquired through trauma, surgical wounds, burn injuries
or the
wounds to donor sites for autologous skin grafts; and 2) chronic wounds, such
as venous
homeostasis ulcers, diabetic ulcers and decubitus ulcers, which are of long
duration and
progressive.
[0003] The evolution of the wound healing process is described in three main
phases: 1) The inflammatory or cleansing stage; 2) the proliferative or
granulation phase;
and 3) the maturation and remodeling or epithelialization phase. The
inflammatory stage
occurs during the first few days after trauma. The wounded area begins its
attempts to
restore its normal state by constricting blood vessels to control bleeding.
Platelets and
thromboplastin are formed in the wound to produce clots. An inflammatory
reaction with
the influx of polymorphonuclear leukocytes to begin cleaning the wound of dead
tissue and
contaminating organisms occurs. The inflammatory process serves mainly to
cleanse the
wound. Too much inflammation interferes with the process of wound healing.
[0004] After the inflammatory phase, the proliferative stage of wound healing
can
last about three weeks or longer. Fibroblasts produce collagen to begin the
granulation
process in the wound. The wound gradually contracts, blood vessels are formed
to nourish
new tissue and a covering epithelial layer is formed.
[0005] The maturation and remodeling stage may last up to two years (even up
to
five years for the healing of bums). The formation of new collagen changes the
shape of
the wound and increases the strength of the scar that forms; however, scar
tissue is at best
80% as strong as the original tissue.
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~Q(~061 1! 1111 ~~I,~yrnprpho,n~G~,~~rf{leukocytes (PMNs) are the first cells
to appear in the
wound within the first 24 hours. Macrophages enter the wound about 48 hours
after injury
with peak numbers achieved about the third day after injury. The macrophages
are derived
from circulating monocytes by a combination of migration and chemotaxis. The
macrophages are longer lived in the wound and linger in the wound until
healing occurs. T-
lymphocytes appear about the fifth day after injury. The presence and
activation of
macrophages and T-lymphocytes are critical to the normal process of wound
healing.
[0007] Cytokines and chemokines play important roles in the process of wound
healing. Burn wounds are also marked by a production of endogenous cytokines
(Stallo et
al., 2003, Burns 29:641-647). Among the cytokines and chemokines important in
wound
healing, interleukin-1- beta (IL-lbeta) is important for chemotaxis during the
inflammation
phase and may also play a role in collagen synthesis (Castagnoli et al., 2002,
Wound Repair
Regen. 10:107-108). Tumor necrosis factor-alpha (TNF-alpha) stimulates
angiogenesis and
is also involved in chemotaxis. Interferon-gamma (IFN-gamma) appears to play a
role
during the proliferative phase. Interleukin-1 beta induces fever,
adrenocorticotropic
hormone (ACTH) release, enhances the.synthesis of TNF-alpha and IFN-gamma and
activates PMNs. Interleukin-2 activates macrophages, T-cells and NK-cells.
Interleukin-6
induces fever and enhances the release of acute phase reactants from the
liver. Interleukin-
8, an important pro-inflammatory chemokine, enhances neutrophil adherence,
chemotaxis
and granule release.
[0008] Immunomodulators have been shown to promote wound healing. For
example, alpha thymosin, a T cell stimulator, has shown positive activity in
wound healing
(Malinda et al., 1998, J. Irnrnunol. 160:1001-1006).
[0009] While minor wounds generally heal completely on their own in a healthy
patient, more severe wound injuries do not heal completely, resulting in scar
tissue and/or
other complications or do not heal at all. Lacerations, however, tend to
become more
inflamed than clean surgical wounds, including wounds resulting from surgical
amputations, and are more difficult to heal. At a simple level this may be due
to the trauma
of the laceration that results in dead tissue and contamination with bacteria,
requiring a
greater inflammatory response to cleanse the wound. Furthermore, vascular
perfusion of
traumatized tissue may be compromised by damage to both large and small blood
vessels
and lymphatic channels.
[0010] The natural healing of wounds is complicated by many factors which can
adversely affect the wound healing process, including the presence of necrotic
debris,
foreign material, infection, medication, and the age, health, and nutritional
status of the
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WO 2006/026604 PCT/US2005/030798
injpr.eq i)xtvic~~}~lu;.,i,In process that impedes peripheral blood
circulation, such
as arteriosclerosis, prolonged pressure, varicose vein disease, and venous
stasis, can
adversely affect the delivery of oxygen, nutrients, chemical signals, and
appropriate cell
types to mediate healing in an injured patient, will impair wound healing.
Certain partial
and full thickness injuries, such as burns, skin grafts, and various types of
ulcers, resist
repair and produce significant pain and discomfort for the afflicted
individual.
[0011] Bacterial wound infection is the most common local cause for prolonged
wound healing. Human skin is typically colonized by a number of
microorganisms,
including Candida albicans, Staphylococcus epidermidis, Staphylococcus aureus,
and some
Streptococcus strains. Thus, any wound which exposes underlying tissues to the
environment becomes infected with at least resident microbial flora. Wounds
which are
well tended and in highly vascularized tissue resist infection, while those in
ischemic tissue
are much more susceptible to infection.
[0012] Medications used to treat disorders can produce impaired wound healing.
Chemotherapy, used to eliminate dividing cells in cancer patients, also
suppresses the
ability of such a patient to heal wounds, which is also dependent upon new
cell growth.
Steroids negatively impact all three phases of wound repair, inhibiting the
initial
inflammatory response, slowing the production of new epithelium and vascular
tissue, and
weakening the collagen matrix in the scar tissue (Bryant, 1987, J.
Enterostomal Therapy,
14: 262-66).
[0013] The physical condition of the patient is also important in wound
healing. As
age increases, the ability to repair injured tissue decreases, as the skin
becomes thinner and
the number of fibroblasts and amount of total skin collagen decrease (Shuster
et al., 1975,
Br. J. Dermatol. 93: 639-43). Disease states such as alcoholism, anemia,
diabetes,
malnutrition, shock, and uremia lead to impaired oxygen and nutrient delivery
to the wound
site, thereby inhibiting the healing process. Also, diseases leading to
monocytopenia can
significantly impair wound healing.
[0014] The severe nature of certain wounds and factors which affect the wound
healing process indicate the need for improved agents for promoting wound
healing. In
particular, there is a need for a non-toxic agent that effects the following:
1) Keep the
wound clean and minimize the development of necrotic tissue; 2) Build a
healthy tissue
base for the wound (granulation tissue), allowing the influx of white blood
cells and the
action of wound healing promoting molecules, including growth factors and
relevant
cytokines and chemokines; 3) Prevent infection of the wound and allow the
wound to heal
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CA 02578212 2007-02-27
WO 2006/026604 PCT/US2005/030798
lp;yjpry intpp-,ti,pp wthq.s~~tiaxeyc~iQsure; 4) Decrease scar formation and
promote natural
epithelialization; and 5) Decrease the time required for wound healing.
2.2. Product R
[0015] Reticulose emerged as an antiviral product in the 1930's. While it was
originally believed to be a product composed of peptone, peptides and nucleic
acids, the
precise composition remains unidentified. A method for preparing Reticulose
is provided
in U.S. Patent No. 5,849,196, herein incorporated by reference in its
entirety. Nevertheless,
Reticulose has demonstrated an ability to inhibit rapidly the course of
several viral
diseases. It is nontoxic, miscible with tissue fluids and blood sera and free
from
anaphylactogenic properties.
[0016] As taught by U.S. Patent No. 5,849,196, the components present in the
conventional composition of Reticulose that are greater than 15 kDa in
molecular weight
are more effective in treating viral diseases such as HIV, influenza virus,
herpes simplex
virus, etc., while the components having a molecular weight in a range of
approximately 1
to 15 KDa function as phagocytosis inhibitors.
[0017] Reticulose suffers from several disadvantages: 1) the method of
preparation
does not ensure that each preparation produces the finished components in the
same ratio,
i.e., the final product is not reproducible; 2) the conventional method of
preparation
produces a wide range of the finished components, which makes the quality
control of the
preparation extremely difficult, if possible, because too many parameters need
to be
determined; 3) the presence of the higher molecular weight components, such as
25 KDa
component, essentially peptides, increases the risk of hypersensitivity or
immune reaction
and renders the product less stable. Therefore, it is desirable to have a
product devoid of the
deficiencies of conventional Reticulose while maintaining its therapeutic
properties.
[0018] U.S. Patent Nos. 6,303,153 and 6,528,098, both of which are herein
incorporated by reference in their entireties, disclose the preparation of
Product R, a
composition derived from the same starting materials as used in preparing
Reticulose , but
distinct from Reticulose . For example, material greater than 14 kDa molecular
weight is
removed when preparing Product R.
[0019] Product R is an immunomodulator with a favorable safety profile
affecting
the production of pro-inflammatory chemokines and cytokines, driving the
immune
response towards a normal state. In particular, Product R modulates the
expression of
chemokines and cytokines. Product R stimulates macrophages to produce pro-
inflammatory cytokines and chemokines, including MCP-1, IL-8 and IL-6
(Lazzarino et al.,
2001, Cytokine, 14:234-239; Lazzarino et al., 2000, Immunol. Lett. 74:189-
195). On the
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CA 02578212 2007-02-27
WO 2006/026604 PCT/US2005/030798
.acxJ.~ted,acrophages, Product R inhibits the synthesis of these pro-
er a~ f'~;. la.
inflammatory ~i .. '~=.1~:==...~?.~.,=.~Y.. =.:,={~ .,.:,
chemokines. Product R may also increase levels of IL-2R. IL-2R can
stimulate populations of both T and B cells.
[0020] Insofar as the applicant knows, Product R has never been used, nor
suggested for promoting wound healing. It is now discovered that Product R can
be used
for the promotion of wound healing.
[0021] Although considerable advances have occurred in the field of wound
healing,
the healing of wounds still presents a formidable challenge to the physician.
This is
particularly true in diabetic patients, those with impaired circulation in the
skin, the elderly,
and those who are prone to infection, such as immunocompromised patients. When
wounds
do heal, they frequently do so with cosmetic disfigurement such as scarring or
discoloration. The increase in cosmetic plastic surgery has also resulted in
increased
incidences of surgical wounds that scar upon healing. Thus, there is a clear
need for
improved methods of wound healing comprising administering compositions such
as
Product R that are non-toxic, well-tolerated and that can be used
concomitantly with other
medications.
3. SUMMARY OF THE INVENTION
[0022] The present invention provides methods for promoting wound healing in a
patient, comprising administering to said patient an amount of Product R
effective to
promote wound healing. The present invention also provides methods for
treating loss of
appetite and fatigue in a patient, preferably a patient suffering from a wound
or burn injury,
comprising administering to said patient an amount of Product R effective to
treat loss of
appetite and fatigue. In the methods and compositions of the invention,
Product R
comprises nucleotides and peptides have molecular weights not more than 14 KDa
and
substantially not more than 8 KDa, wherein said nucleotides and peptides are
breakdown
products of casein, peptone, RNA and serum albumin, and wherein said
composition has a
light absorption spectrum with typical absorption ratios of 2.0 (~:10%) at 260
nm/280 nm
and 1.4 ( 10%) at 260 nm/230 nm. In a preferred embodiment, the patient is a
human. In
certain embodiments, the amount of Product R effective to promote wound
healing andlor
treat loss of appetite and fatigue is in a range from about 0.5 microliters to
about 100
microliters per kilogram of body weight per day, or from about 2.5 microliters
to about 40
microliters per kilogram of body weight per day, or from about 10 microliters
to about 25
microliters per kilogram of body weight per day. In certain embodiments of the
invention,
Product R is administered parenterally, topically or systemically. In another
embodiment,
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WO 2006/026604 PCT/US2005/030798
11rTduQt P=~i~;.ad~}~k ~api,o Uy to a wound in a dose adequate to encompass
the total
~õ ... ...... .. area of the wound.
[0023] The methods of the invention can be employed to promote wound healing
in
a wide variety of wounds. In certain embodiments, the wound is a result of
decubitus
ulcers, diabetic ulcers, surgical wounds or burn injury.
[0024] The present invention also encompasses methods for promoting wound
healing in a patient, comprising administering to said patient an amount of
Product R
effective to promote wound healing and an effective amount of another
medicament. The
present invention also encompasses methods for treating loss of appetite or
fatigue in a
patient, preferably a patient suffering from a wound or burn injury,
comprising
administering to said patient an amount of Product R effective to promote
wound healing
and an effective amount of another medicament. In certain embodiments, the
other
medicament is an antibiotic, a biological response modifier, e.g., a cytokine,
or a wound
healing factor.
[0025] The present invention also coinprises pharmaceutical compositions and
kits
comprising 1) an amount of Product R effective to promote wound healing; 2) a
biological
response modifier or a wound healing factor agent; and 3) a pharmaceutically
acceptable
carrier.
[0026] Antibiotics useful in the metliods of the invention include, but are
not limited
to, penicillin, cephalosporin, griseofulvin, bacitracin, polymyxin B,
amphotericin B,
erythromycin, neomycin, streptomycin, tetracycline, vancomycin, gentamicin,
and
rifamycin. Biological response modifiers useful in the methods and
compositions of the
invention include, but are not limited to, interferon-a, interferon-y,
interleukin-2,
interleukin-4, interleukin-6, and tumor necrosis factor. Wound healing factors
useful in the
methods and compositions of the invention include, but are not limited to,
interferon (IFN)-
(3, IFN-y, interleukin (IL)-1, IL-2, IL-4, IL-5, IL-15, tumor necrosis factor,
flt-1 ligand,
arginine, connective tissue growth factor, adenosine, cyclic adenosine
monophosphate, the
fibroblast growth factor family, tumor growth factor-a, tumor growth factor-(3
(1 and 2),
vascular endothelial growth factor, the epidermal growth factor family, the
platelet derived
growth factor family, the insulin-like growth factor family, nitric oxide,
macrophage-
stimulating protein, and macrophage-derived growth factor.
4. DETAILED DESCRIPTION OF THE INVENTION
[0027] The inventors have discovered that Product R can promote the wound
healing process, particularly when administered topically, i. e., to the
surface of the wound
site, or systemically. The present invention provides methods and compositions
for
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WO 2006/026604 PCT/US2005/030798
pjQrnQ.ting, pu,p~,}.heali~,g~..uih,,V~c~duct R. Product R so delivered can be
used for all
~ i
wound types, acute or chronic, such that the wound undergoes healing more
rapidly than
similar wounds left to heal naturally or which are treated with currently
available methods.
Without being bound by any theory, Product R is believed to modulate the
immune system
to promote wound healing.
[0028] Product R(AVR118; Advanced Viral Research Corp., Yonkers, NY) is a
composition comprising the breakdown products of casein, peptone, RNA, and
serum
albumin. The manufacturing process, composition, and the chemical and physical
and some
biological properties of Product R are described in U.S. Patent Nos. 6,303,153
and
6,528,098, both of which are herein incorporated by reference in their
entireties.
[0029] Product R has been used as a synonym of Reticulose in some literature.
For the purpose of the present application, Product R and Reticulose represent
two distinct
products.
[0030] Product R is a therapeutic composition for treating viral infections
and
stimulating the immune system comprising nucleotides and peptides have
molecular
weights not more than 14 KDa and substantially not more than 8 KDa, wherein
said
nucleotides and peptides are breakdown products of casein, peptone, RNA and
serum
albumin, and wherein said composition has a light absorption spectrum with
typical
absorption ratios of 2.0 ( 10%) at 260 nm/280 nm and 1.4 ( 10 1 ) at 260
nm/230 nm.
[00311 Generally, Product R is prepared according to the following manner.
[0032] First, the starting materials casein, beef peptone, RNA, bovine serum
albumin (BSA), and sodium hydroxide are suspended in proportions of, by
weight, 35-50%
(casein), 15-40% (beef peptone), 10-25% (RNA), 1-10% (BSA) and 5-25% (sodium
hydroxide) in an appropriate volume of distilled water. All starting materials
are generally
available or otherwise can be readily prepared by a person of ordinary skill
in the art.
While any RNA is suitable for the intended purpose of the present invention,
plant RNA is
preferred and yeast RNA is the most preferred. The ratio of total proteins
versus the
volume of distilled water is generally about 1.5-2.5 to about 100 by weight,
preferably
about 2.2 to about 100 by weight. This means that every 1.5-2.5 grams of the
total proteins
are suspended in about 100 milliliters of distilled water.
[0033] The suspension as prepared above is then autoclaved at a pressure of
approximately 5-15 lbs., preferably 8-10 lbs. under an elevated temperature in
a range, for
example, from about 65 -150 C, preferably from about 90 -110 C, over a
period of
approximately 2-10 hours, preferably more than 3 hours. As known to a person
of ordinary
skill in the art, under such conditions RNA may be completely hydrolyzed into
nucleotides.
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CA 02578212 2007-02-27
WO 2006/026604 PCT/US2005/030798
r.=~uto({,''l~vi7~~{'.,~==th~=sc}1 =~ia~,=i. ,cpoled down to room temperature,
and then allowed to
{ . ~fi .J1' a1 sl,:a+
stay at a temperature of 3 0 to 8 C for at least 12 hours to precipitate
insoluble elements.
Alternatively, the cooled solution may be centrifuged at a temperature below 8
C to remove
the precipitates.
[0034] The resulting solution is then filtered through a 2 micron and a 0.45
micron
filters under an inert gas such as nitrogen or argon at a pressure of about 1-
6 psi. In a
similar manner the solution is filtered again through a pyrogen retention
filter, preferably
0.2 micron.
[0035] After the above filtration, the solution may be cooled at 3 to 8 C
again for at
least about 12 hours and filtered again in the same way as described above.
[0036] The resulting filtrate is then assayed for total nitrogen content using
methods
known to a person of ordinary skill in the art such as Kjeldahl method
(Kjeldahl, Z. 1983,
Anal. Chem., Vol. 22:366), and its improvements. Based on the assay, the
filtrate is then
diluted with chilled distilled water to an appropriate volume having a
preferred total
nitrogen content ranging from 165 to 210 mg/ml.
[0037] The pH of the diluted solution is then adjusted with HCl to a
physiologically
acceptable pH, preferably to about 7.3 to 7.6, after which the diluted
solution is filtered
again through a 0.2 micron filter under an inert gas as described above.
[0038] Product R so produced contains essentially nucleotides, nucleosides and
free
nucleic acid bases of low molecular weights from a complete hydrolysis of RNA
and small
peptides from partial hydrolysis of the proteins. It is possible that the base
hydrolysis of the
proteins also produces free amino acids.
[0039] It is understood that the use of a filtration technique is essentially
to remove
bacteria or other particles having similar size to or larger size than
bacteria. Thus, any filter
regardless of its manufacturer or material from which it is made is suitable
for the intended
purpose. All filters used in the present process are widely available to a
person of ordinary
skill in the art.
[0040] The final filtrate is then filled and sealed into appropriate vials,
such as 2 ml
or 10 ml glass vials under an inert gas. The filled vials are autoclaved for
final sterilization,
after which they are ready for use.
[0041] An analysis of the composition of Product R reveals that Product R
contains
two major components, which are exhibited as two bands having molecular
weights of 5.2
kDa and 4.3 kDa on a SDS-polyacrymide gel electrophoresis, namely peptide-A
and
peptide-B, respectively. Peptide-A is a 31 amino acid long peptide of 5.2 KDa
molecular
weight derived from bovine casein. It is a straight chain peptide lacking any
cysteine-
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WO 2006/026604 PCT/US2005/030798
hc,t~~ is remarkable for six proline residues spaced throughout
..
,,.,. , ...,..
the molecule and four basic glutamine residues. Since proline residues can
induce bends in
peptide chains, the structure is likely a highly folded peptide. Peptide-B
comprises a 21
amino acid long linear peptide of 4.3 KDa molecular weight covalently linked
at the
hydroxyl group of a serine residue at position 18 to a diadenosine
dinucleotide unit through
a diphosphodiester linkage at the 3'-position. Peptide-A and peptide-B are
present in the
Product R composition in an approximately equal amount and the total amount of
these two
peptides is about 4.8-5.3 mg/ml, determined by a Lowry protein assay.
[0042] The sequence of peptide A is:
KVLPVPQKAVPYPQRDMPIQAFLLYQEPVLG (SEQ ID NO. 1). The sequence of
peptide B is: GEIPDAGGRIVDYYVGFSDSV (SEQ ID NO. 2). Product R also comprises
nucleosides, nucleoside diphosphates and nucleoside monophosphates. There are
sixteen
identified constituent compounds present in the Product R formulation - 3
nucleosides, two
nucleoside diphosphates and eight nucleoside monophosphates, together with two
peptides
(one of them a peptide-nucleic acid conjugate) and sodium chloride.
[0043] The physical, chemical and biological properties of Product R are
further
described in U.S. Patent Nos. 6,303,153 and 6,528,098, the contents of which
are
incorporated by reference in their entireties.
4.1. Target Wounds and Wound-related Conditions
[0044] The methods of the present invention are applicable for treating any
type of
wound to promote wound healing. The methods of the present invention are also
applicable
for treating conditions, such as loss of appetite and fatigue, in a patient,
preferably a patient
suffering from a wound or burn injury. Certain wounds heal normally without
therapeutic
intervention. In these cases, the methods of the invention can quicken the
healing process.
However, there are numerous disorders in which wound healing plays a role,
such as, for
example, diabetes mellitus, arterial occlusive diseases, psoriasis, Crohn's
disease,
epidermolysis bullosa, age-related skin changes or innervation disorders.
Wound healing
disorders lead to a delayed healing of wounds or to chronic wounds. These
disorders can be
caused by the nature of the wound (e.g. large-area wounds, deep and
mechanically
expanded operation wounds, bums, trauma, decubitus), medicinal treatment of
the patients
(e.g. with corticoids) but also by the nature of the disorder itself. For
example, 25% of the
patients with Type II diabetes thus frequently suffer from chronic ulcers
("diabetic foot"), of
which approximately half necessitate expensive hospitalized treatments and
nevertheless
finally heal poorly. Diabetic foot causes more stays in hospital than any
other complication
associated with diabetes. The number of these cases in diabetes Type I and II
is on the
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WO 2006/026604 PCT/US2005/030798
thospital admissions. Moreover, wounds heal more
..h
poorly with increasing age of the patients. An acceleration of the natural
wound healing
process is often desirable as well in order to decrease, for example, the
danger of bacterial
infections or the rest periods of the patients.
[0045] Clinically, wounds are divided into three categories (Clinical Guide to
Wound Care, Hess, Ed., Lippincott Williams & Wilkins; 4th edition (2002)).
These
categories and representative examples are provided below.
[0046] Wounds
[0047] Acute wound: a wound caused by trauma or surgery; usually requiring
limited local care.
[0048] Chronic wound: a wound which takes longer than usual to heal because of
underlying conditions, such as pressure, diabetes mellitus, poor circulation,
poor nutritional
state, immunodeficiencies, or infection.
[0049] Full-thickness wound: tissue destruction extending through the second
layer
of skin (dermis) to involve subcutaneous tissue and possibly muscle or bone.
[0050] Laceration: a torn or jagged wound.
[0051] Partial-thickness wound: tissue destruction through the first layer of
skin
(epidermis), extending into, but not through, the dermis.
[0052] Ulcers
[0053] Arterial ulcer: an ulcer caused by poor blood supply; related to the
presence
of arterial occlusive disease; symptoms include pain and tissue loss.
[0054] Diabetic ulcer: an ulcer caused by trauma or pressure secondary to
neuropathy or vascular disease related to diabetes mellitus.
[0055] Pressure ulcer (decubitus ulcer): an ulcer caused by poor blood supply
from
pressure, also called a bedsore or pressure sore.
[0056] Venous ulcer: local losses of epidermis and various levels of dermis
and
subcutaneous tissue, occurring over or near the malleoli of the distal lower
extremities;
caused by edema and other sequellae of impaired venous return.
[0057] Burns
[0058] Superficial (first-degree bum): damage limited to the epidermis
characterized by erythema, hyperemia, tenderness, and pain.
[0059] Partial-thickness (second-degree burn): superficial to deep partial-
thickness
wound characterized by large blisters, edema, pain, and wet, weeping, and
shiny surface.
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1~,P060J,, .,, 1111 ,1.,-r ~,~,~1' thicl~~ss;,(t~r~-degree burn): full-
thickness wound characterized by
!!., a : t
deep-red, black, or white appearance; edema; painless nerve ending damage; and
exposed
subcutaneous fat layer.
4.2. Dosage and Administration
[0061] The individual, patient or subject in whom promotion of wound healing,
or
treatment of loss of appetite or fatigue, is desired is an animal, preferably
a mammal, a non-
human animal or primate, and most preferably human. The term "animal" as used
herein
includes, but is not limited to, companion animals, such as cats and dogs; zoo
animals; wild
animals, including deers, foxes and racoons; farm animals, livestock and fowl,
including
horses, cattle, sheep, pigs, turkeys, ducks, and chickens, as well as any
rodents.
[0062] The methods of the invention comprise administering an amount of
Product
R effective to promote wound healing, or treat loss of appetite or fatigue. An
amount of
Product R effective to promote wound healing, or treat loss of appetite or
fatigue, within the
meaning of the present invention can be determined by a patient's attending
physician or
veterinarian. Such amounts are readily ascertained by one of ordinary skill in
the art and
can enable accelerated wound healing when administered in accordance with the
present
invention. Factors which influence what an amount of Product R effective to
promote
wound healing, or treat loss of appetite or fatigue, will be include, the
specific activity of
the therapeutic agent being used, the wound type (mechanical or thermal, full
or partial
thickness, etc.), the size of the wound, the wound's depth (if full
thickness), the absence or
presence of infection, time elapsed since the injury's infliction, and the
age, physical
condition, existence of other disease states, and nutritional status of the
patient.
Additionally, other medication the patient may be receiving will effect the
determination of
the amount of Product R to administer. In certain embodiments, the amount of
Product R
effective to promote wound healing, or treat loss of appetite or fatigue, is
in a range from
about 0.5 microliters to about 100 microliters per kilogram of body weight per
day, or from
about 2.5 microliters to about 40 microliters per kilogram of body weight per
day, or from
about 10 microliters to about 25 microliters per kilogram of body weight per
day. As used
herein, "about" entails normal experimental variation. Alternatively, Product
R may be
administered to the patient according to the conventional doses or any dosages
that are
apparent to a person of ordinary skill in the art.
[0063] The desired dose may be administered as two, three or more sub-doses at
appropriate intervals, generally equally spread in time, throughout the day.
The dosage
used for topical administration to the wound will vary depending on the size
and location of
the wound, and will preferably encompass the entire wound area.
11
CA 02578212 2007-02-27
WO 2006/026604 PCT/US2005/030798
~64~ ypical,.xppe;,~~ ~}ministration for Product R may include, without
T
![Q. ,
i'
limitation, oral, topical, parenteral, sublingual, rectal, vaginal, ocular,
and intranasal.
Parenteral administration includes subcutaneous injections, intravenous,
intramuscular,
intraperitoneal, intrapleural, intrasternal injection or infusion techniques.
In a preferred
embodiment of the invention, Product R is administered topically. In addition
to direct
topical application to the wound, Product R can be used systemically, by the
subcutaneous
route, to stabilize a wounded patient. It will be appreciated that the
preferred route may
vary with, for example, the condition and age of the recipient.
[0065] In certain embodiments, Product R is administered by both systemic and
topical routes to promote the wound healing, or treat loss of appetite or
fatigue,. First,
systemic and topical administration of Product R can be used acutely to
stabilize the
wounded patient. Product R then can be used topically to promote wound
healing, or treat
loss of appetite or fatigue, as the patient is further stabilized, has
undergone any necessary
surgery to debride the wound, and has entered the recuperative phase.
[0066] The present invention also encompasses methods for promoting wound
healing in a patient, comprising administering to said patient an amount of
Product R
effective to promote wound healing and an effective amount of another
medicament. The
present invention also encompasses methods for treating loss of appetite or
fatigue in a
patient, preferably a patient suffering from a wound or burn injury,
comprising
administering to said patient an amount of Product R effective to promote
wound healing,
or treat loss of appetite or fatigue, and an effective amount of another
medicament. In
certain embodiments, the other medicament is an antibiotic, a biological
response modifier,
e.g., a cytokine, or a wound healing factor. Antibiotics useful in the methods
of the
invention include, but are not limited to, penicillin, cephalosporin,
griseofulvin, bacitracin,
polymyxin B, amphotericin B, erythromycin, neomycin, streptomycin,
tetracycline,
vancomycin, gentamicin, and rifamycin. Biological response modifiers useful in
the
methods of the invention include, but are not limited to, interferon-a,
interferon-y,
interleukin-2, interleukin-4, interleukin-6, and tumor necrosis factor. Wound
healing
factors useful in the methods of the invention include, but are not limited
to, interferon
(IFN)-(3, IFN-y, interleukin (IL)-1, IL-2, IL-4, IL-5, IL- 15, tumor necrosis
factor, flt-1
ligand, arginine, connective tissue growth factor, adenosine, cyclic adenosine
monophosphate, the fibroblast growth factor family, tumor growth factor-a,
tumor growth
factor-(3 (1 and 2), vascular endothelial growth factor, the epidermal growth
factor family,
the platelet derived growth factor family, the insulin-like growth factor
family, nitric oxide,
macrophage-stimulating protein, and macrophage-derived growth factor.
12
CA 02578212 2007-02-27
WO 2006/026604 PCT/US2005/030798
As usedk,:,~p,,'t~Tterm "in combination" refers to the use of more than one
prophylactic and/or therapeutic agents. The use of the term "in combination"
does not
restrict the order in which prophylactic and/or therapeutic agents are
administered to a
subject with a disorder. A first prophylactic or therapeutic agent can be
administered prior
to (e.g., up to 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1
hour, 2 hours, 4
hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2
weeks, 3 weeks,
4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with,
or
subsequent to (e.g., up to 1 minute, 5 minutes, 15 minutes, 30 minutes, 45
minutes, 1 hour,
2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1
week, 2
weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the
administration
of a second prophylactic or therapeutic agent to a subject which had, has, or
is susceptible
to a wound. The prophylactic or therapeutic agents are administered to a
subject in a
sequence and within a time interval such that the agent of the invention can
act together
with the other agent to provide an increased benefit than if they were
administered
otherwise. Any additional prophylactic or therapeutic agent can be
administered in any
order with the other additional prophylactic or therapeutic agents.
4.3. Monitoring of Effects During Therapy
[0068] The effects/efficacy of treatment of a wound according to the present
invention can be detected, for example, on the level of the molecular and
cellular agents
involved in the immune response (e.g., macrophages, B cells, or T cells) or on
the level of
an affected tissue including, but not limited to, stimulation of macrophages
to secrete
growth factors, synthesis and cross-linking of collagen, and decrease in wound
size, using
standard methods well known to one of ordinary skill in the art.
4.4. Pharmaceutical Formulations
[0069] While it is possible for Product R to be administered as part of a
pharmaceutical formulation, it is preferable to present it alone, although it
may be
administered at about the same time as one or more other pharmaceuticals are
independently administered. If Product R is administered as part of a
pharmaceutical
formulation, the formulations of the present invention comprise at least one
administered
ingredient, i.e., Product R, as above defined, together with one or more
acceptable carriers
thereof and optionally one or more additional medicaments. Suitable additional
medicaments are provided in Section 4.2. The carrier(s) must be
"pharmaceutically
acceptable" in the sense of being compatible with the other ingredients of the
formulation
13
CA 02578212 2007-02-27
WO 2006/026604 PCT/US2005/030798
t.,..~p"I;leGi~~F ",t thereof (e.g., suitable for in vivo use, in the
patient).
Preferably, Product R in a pharmaceutical formulation is sterile.
[0070] The formulations may conveniently be presented in unit-dose or multi-
dose
containers, e.g., sealed ampules and vials. Preferred unit dosage formulations
are those
containing a daily dose or unit, daily sub-dose, or an appropriate fraction of
the
administered ingredient.
[0071] The compositions of the invention can be in the form of a solid, liquid
or gas
(aerosol). Pharmaceutical compositions of the invention can be formulated so
as to allow a
compound of the invention to be bioavailable upon administration of the
composition to a
subject. Compositions can take the form of one or more dosage units, where for
example, a
tablet can be a single dosage unit, and a container of a compound of the
invention in aerosol
fonn can hold a plurality of dosage units. A syringe containing a unit dose of
Product R is
also provided.
[0072] For oral administration, the pharmaceutical preparation can be in
liquid
form, for example, solutions, syrups or suspensions, or can be presented as a
drug product
for reconstitution with water or other suitable vehicle before use. Such
liqLtid preparations
can be prepared by conventional means with pharmaceutically acceptable
additives such as
suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated
edible fats);
emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g.,
almond oil, oily
esters, or fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-
hydroxybenzoates or sorbic acid). The pharmaceutical compositions can take the
form of,
for example, tablets or capsules prepared by conventional means with
pharmaceutically
acceptable excipients such as binding agents (e.g., pregelatinized maize
starch, polyvinyl
pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline
cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium
stearate, talc or
silica); disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g.,
sodium lauryl sulphate). The tablets can be coated by methods well-known in
the art.
[0073] Preparations for oral administration can be suitably formulated to give
controlled release of the active compound.
[0074] For buccal administration, the compositions can take the form of
tablets or
lozenges formulated in conventional manner.
[0075] For administration by inhalation, the compounds for use according to
the
present invention are conveniently delivered in the form of an aerosol spray
presentation
from pressurized packs or a nebulizer, with the use of a suitable propellant,
e.g.,
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane,
carbon dioxide
14
CA 02578212 2007-02-27
WO 2006/026604 PCT/US2005/030798
otl~er,s~~t~~la~gas.: In ~l~e ~ase; { ~,f a pressurized aerosol the dosage
unit can be determined
::;t
by providing a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin
for use in an inhaler or insufflator can be formulated containing a powder mix
of the
compound and a suitable powder base such as lactose or starch.
[0076] The compounds can be formulated for parenteral administration by
injection,
e.g., by bolus injection or continuous infusion. Formulations for injection
can be presented
in unit dosage form, e.g., in ampoules or in multi-dose containers, with an
added
preservative. The compositions can take such forms as suspensions, solutions
or emulsions
in oily or aqueous vehicles, and can contain formulatory agents such as
suspending,
stabilizing and/or dispersing agents. Alternatively, the active ingredient can
be in powder
form for constitution with a suitable vehicle, e.g., sterile pyrogen-free
water, before use.
[0077] The compounds can be formulated in compositions such as creams,
lotions,
gels, opthalmic drops, ointments, solutions, suspensions, shampoos, or other
forms known
to one of skill in the art and described in, for example, Remington's
Pharmaceutical
Sciences, 16th and 18th eds., Mack Publishing, Easton Pa. (1980 & 1990), and
Introduction
to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia (1985).
Actual
methods for preparing pharmaceutical compositions are known or apparent to
those skilled
in the art and are described in detail in, for example, Remington's
Pharmaceutical Sciences,
16th and 18th eds., Mack Publishing, Easton Pa. (1980 & 1990).
[0078] The coinpounds can also be formulated in rectal compositions such as
suppositories or retention enemas, e.g., containing conventional suppository
bases such as
cocoa butter or other glycerides.
[0079] In addition to the formulations described previously, the compounds can
also
be formulated as a depot preparation. Such long acting formulations can be
administered by
implantation (e.g., subcutaneously or intramuscularly) or by intramuscular
injection. Thus,
for example, the compounds can be formulated with suitable polymeric or
hydrophobic
materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins,
or as sparingly
soluble derivatives, for example, as a sparingly soluble salt. Liposomes and
emulsions are
well known examples of delivery vehicles or carriers for hydrophilic drugs.
4.5. Kits
[0080] The invention also provides kits for carrying out the methods and/or
therapeutic regimens of the invention. In one embodiment, such kits comprise
in one or
more containers, Product R. In another embodiment, such kits comprise in one
or more
containers an amount of Product R effective to promote wound healing, or treat
loss of
appetite or fatigue, in pharmaceutically acceptable form.
CA 02578212 2007-02-27
WO 2006/026604 PCT/US2005/030798
~4n~ainer of a kit of the invention may be in the form of a
pharmaceutically acceptable solution, e.g., in combination with sterile
saline, dextrose
solution, or buffered solution, or other pharmaceutically acceptable sterile
fluid.
Alternatively, Product R may be lyophilized or desiccated; in this instance,
the kit
optionally further comprises in a container a pharmaceutically acceptable
solution (e.g.,
saline, dextrose solution, etc.), preferably sterile, to reconstitute Product
R to form a
solution for injection purposes.
[0082] In another embodiment, a kit of the invention further comprises a
needle or
syringe, preferably packaged in sterile form, for injecting Product R, and/or
a packaged
alcohol pad. Instructions are optionally included for administration of
Product R by a
clinician or by the patient.
[0083] Kits are also provided for carrying out the combination therapies of
the
present invention. In one embodiment, a kit comprises a first container
containing Product
R and a second container containing an additional medicament. Suitable
additional
medicaments are provided in Section 4.2.
[0084] The kit may for example comprise metal or plastic foil, such as a
blister
pack. The kit may be accompanied by one or more reusable or disposable
device(s) for
administration (e.g, syringes, needles, dispensing pens) and/or instructions
for
administration.
2 0 4.6. Animal models
[0085] Animal models can optionally be used to demonstrate use of particular
formulations and/or dosages of Product R in wound healing. Suitable animal
models for
wound healing are known to one of ordinary skill in the art.
[0086] The following examples only serve to further illustrate, but not to
limit the
scope of the present invention.
5. EXAMPLES
5.1. Example 1: Method for Preparing Product R
[0087] About 35.0 g of casein, about 17.1 g of beef peptone, about 22.0 g of
nucleic acid (RNA), about 3.25 g bovine serum albumin were suspended in about
2.5 liters
of water for injection USP at about 3 to 7 C in a suitable container and
gently stirred until
all the ingredients have been properly wet. About 16.5 g of sodium hydroxide
(reagent
grade ACS) is added with stirring. Stirring is continued until the sodium
hydroxide is
completely dissolved. The reaction is autoclaved at about 9 lbs pressure and
200-230 F for
16
CA 02578212 2007-02-27
WO 2006/026604 PCT/US2005/030798
a;p,Fri,qd of~,t~me,~ ~~letely digested, for example, about 4 hours. At the
end
it t,.,, ,,..,8 .,= .,,... ,.,.t at ,,,
of the period, the autoclave was stopped and the reaction flask and contents
were permitted
to slowly cool to ambient temperature. The reaction was then cooled for at
least six hours
at about 3-8 C. The resulting solution was filtered through 2 micron and 0.45
micron
filters using an inert gas such as nitrogen or argon at low pressure (1-6
psi). In a similar
manner, the solution was filtered again through 0.2 micron pyrogen retention
filters. The
resulting filtrate was sampled and assayed for total nitrogen. A calculation
was then
performed to determine the quantity of cooled water for injection to be added
to the filtrate
to yield a diluted filtrate with a nitrogen content between about 165-210
mg/100 ml, the
final volume is approximately 5 liters. The pH was then adjusted with either
concentrated
HCl (reagent grade ACS) or 1.0 normal NaOH to about 7.3-7.6. The diluted
solution was
then filtered again through 0.2 micron filters with inert gas at low pressure.
The final
filtrate was then filled and sealed into 2 ml glass ampoules or 2mL vials
while in an inert
gas atmosphere. The ampoules or vials were collected and autoclaved for final
sterilization
at 240 F and 20 to 30 psi pressure for about 30 minutes. Following the
sterilization cycle,
the ampules with Product R were cooled and washed.
[0088] All quantities are subject to plus or minus 2.5% variation for pH,
volume,
and analytical adjustments.
5.2. Example 2
[0089] The patient was an 85 year old active male suffering from a chronic
skin
ulcer on the right leg due to venous insufficiency. The patient generally
enjoyed good
health. He has a history of chronic iron deficiency anemia on the basis of
small amounts of
bleeding from the bowel due to small arterio-venous malformations.
[0090] The patient previously had bilateral vein strippings of the lower
extremities
for varicose veins. Prior to Product R treatment, the patient developed a
superficial skin
ulcer under the lateral malleolus of the right tibia. He applied ointments and
creams such as
antibiotic ointments and zinc oxide cream. The lesion would heal with
formation of an
eschar but then the ulcer would re-open.
[0091] Product R was applied topically to the open lesion, which measured 3/8
inch,
twice a day by dropping liquid Product R on the lesion from an insulin
syringe. Within
seven days, the chronic ulcer epithelialized and healed.
[0092] The lesion did not open and remained closed after three weeks of follow-
up.
This was the first time that this lesion had totally epithelialized. Although
there is a
depression in the skin at the site of the lesion, the color of the new skin
over the area of the
lesion is normal.
17
CA 02578212 2007-02-27
WO 2006/026604 PCT/US2005/030798
5' 'k.,lF 14..,.,,.t.. .,~ ,.,ii
[0093] The patient was an 89 year old woman who was in her usual state of good
health until she began to suffer from mild mental disorders, including a
vacant look for
variable periods, falling asleep at odd times and decreased ability to perform
daily acts of
living. Her appetite remained intact and she continued to interact socially.
[0094] When her physical condition began to deteriorate and she lost her
appetite,
physical therapy was begun. Subsequently, she developed a movement disorder
requiring
therapy with a sedative.
[0095] Prior to Product R therapy, a bulge was noted over her left hip; X-ray
showed only a bone deformity. A month later, the overlying skin began to open
and a foul
smelling wound developed. Initially the wound was long and narrow. The wound
was
diagnosed by her physician as a decubitus ulcer. Treatment with antibiotics
and Varihesive
(ConvaTec, Skillman, NJ) did not affect the wound.
[0096] Treatment with Varihesive Hydrogel was also witllout effect. Her
physical
condition continued to deteriorate and she developed signs of cachexia with
weight loss,
reaching a weight of 35 kilograms. Over the next month, the wound enlarged to
3 times the
initial size both in length and depth; the maximum diameter of the wound was
now 10 cm
in length and 4 cm in width. The underlying bones and tendons showed through
the wound
but the patient was without pain. The wound extended under the margins of the
skin
allowing a hand to be placed underneath the skin of the wound. A month prior
to Product R
treatment, eschars began to form on the patient's back but they did not
ulcerate. At the time
when Product R therapy was initiated, two smaller decubitus ulcers appeared in
the sacral
area.
[0097] Topical therapy was initially initiated with Product R. 1 ml of Product
R was
applied to the wound twice a day; the patient continued with antibiotic
therapy. Two weeks
after the initiation of Product R therapy the wound showed improvement and was
beginning
to close.
[0098] Due to the continued lack of appetite, and the resulting cachexia,
systemic
therapy with Product R by subcutaneous injection of lml once a day was
initiated two
months later. Within two weeks of initiation of systemic AVR1 18 therapy, the
patient
showed marked improvement in appetite and her general physical condition. She
began to
gain weight, was mentally more alert and began to interact with her
environment. She grew
new hair whereas she had been losing hair before Product R therapy was
initiated.
[0099] Both topical and systemic therapy (lmi subcutaneously 3 times per week)
with Product R was continued. The patient continued to show improvement and
the large
18
CA 02578212 2007-02-27
WO 2006/026604 PCT/US2005/030798
Oo~bltus }tip~~ ,rsi~a~~b~3~s;d=eased in size. Especially noteworthy, the
decubitus
ulcer has filled in with healthy looking granulation tissue. The margins of
the wound are no
longer detached from the underlying tissue so that a hand no longer can be
placed under the
skin of the wound. From the beginning time point when Product R therapy was
initiated, to
eight months subsequently, the length of the wound decreased from 10 cm to
approximately
4 1/2 cm. The two smaller decubitus ulcers have closed and healed. The patient
has shown
marked increase in her mental awareness and her general physical condition,
including her
appetite, greatly improved. The patient suffered no side effects from either
topical or
systemic therapy with Product R. The patient is maintaining treatment with
Product R
topically to the wound and by subcutaneous injection.
[00100] The present invention is not to be limited in scope by the specific
embodiments described which are intended as single illustrations of individual
aspects of
the invention, and functionally equivalent methods and components are within
the scope of
the invention. Indeed, various modifications of the invention, in addition to
those shown
and described herein will become apparent to those skilled in the art froni
the foregoing
description and accompanying drawings. Such modifications are intended to fall
within the
scope of the appended claims.
[00101] Various references are cited herein, including scientific
publications, patent
applications, and patents, the disclosures of which are incorporated by
reference in their
entireties.
19
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