Note: Descriptions are shown in the official language in which they were submitted.
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TREATMENT OF RESISTANT LESIONS
TECHNICAL FIELD
[0001] The inventions relate to the treatment of resistant skin lesions.
The inventions
are useful in various contexts, including to promote healing in subjects with
multiple venous
leg ulcers and multiple diabetic foot ulcers, for example.
RELATED APPLICATION
[0002] This application claims the benefit of U.S. Provisional Patent
Application No.
61/944,566, filed on February 25, 2014, which is expressly incorporated herein
in its entirety.
BACKGROUND
[0003] Normal wound healing moves through phases in a timely and
uncomplicated
fashion (hemo stasis, inflammation, proliferation,
epithelialization, and
remodeling/maturation). Wounds that do not heal normally or at expected rates,
typically
those that have been present from more than one to three or six months,
deviate from the
expected sequence of repair. They are sometimes also referred to as ulcers,
and include
venous leg ulcers and diabetic foot ulcers.
[0004] Diabetic foot ulcers are a common and much feared complication of
diabetes.
Diabetic foot ulcers (DFUs) have major short- and long-term impacts on
patients' quality of
life, morbidity and mortality. Studies suggest that the lifetime risk of
developing a foot ulcer
in diabetic patients may be as high as 25%. Foot ulceration requires long and
intensive
treatment, and is associated with major healthcare costs. According to the
Center for Disease
Control, diabetes is the leading cause of nontraumatic lower-limb amputations
in the United
States. Mortality is high and healed ulcers often recur. Prompers, L. et al.,
Prediction of
outcome in individuals with diabetic foot ulcers: focus on the differences
between individuals
with and without peripheral arterial disease. The EURODIALE Study.
Diabetologia. 2008
May; 51(5): 747-755. Despite good management, DFU healing rates in large
multicentre
trials were reported to be 24% at 12 weeks and 31% at 20 weeks. Margolis DJ,
et al. Healing
of diabetic neuropathic foot ulcers receiving standard treatment: a meta-
analysis. Diab Care
1999; 22: 692-95. Even advanced modalities such as skin substitutes or growth
factors are
said to have demonstrated, at best, a 56% healing rate within 12 weeks.
Shishir Shah, DO,
Clinical and Economic Benefits of Healing Diabetic Foot Ulcers With a Rigid
Total Contact
Cast. Wounds. 2012;24(6):152-159. One study that examined a total of 1,000
consecutive
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DFU patients between December 1997 and April 2004 reported that 40% had
multiple
diabetic foot ulcers (multiple or mDFUs). Wounds on the mDFU patients
reportedly had a
significantly lower probability of healing (p<0.00001), and multivariate
analysis confirmed
this parameter as an independent variable with a significant impact on
healing. Beckert S, et
al. A New Wound-Based Severity Score for Diabetic Foot Ulcers: A prospective
analysis of
1,000 patients. Wounds. 2012;24(6):152-159.
[0005] Approximately 70%-80% of ulcers of the lower limbs are venous leg
ulcers
(VLUs) (Abbade, Venous ulcer: epidemiology, physiopathology, diagnosis and
treatment,
Internat J Dennatol. 2005;44:449-456 (2005); O'Brien, et al., Prevalence and
aetology of leg
ulcers in Ireland, Ir J Med Sci. 2000;169:110-112 (2000). In the United
States, VLUs are
commonly associated with substantial disability, impaired quality of life, and
high economic
costs. Heber, et al., A systematic review on the impact of leg ulceration on
patients' quality
of life, Health and Quality of Life Outcomes 5:44 (2007). The slow rate at
which many VLU
patients heal prolongs these problems. Compression therapy, which is applied
to improve
venous circulation, has remained the standard care for VLUs over several
decades but is often
insufficient to heal VLUs in a timely manner. One recent report shows that
just 61.5% of
patients healed at one year in clinical trials (Rippon, M., et al, The
economic impact of
Chronic Wounds, Wounds UK, 2007, 3, No 2). Nevertheless, compression bandaging
remains the standard of care (SOC) due to a lack of effective alternatives.
VLU-related
treatment costs are directly related to time to achieve complete wound
closure.
[0006] As with DFU patients, some VLU patients have more than one wound
at the
same time (multiple or mVLUs). Like mDFUs, multiple VLUs are considered more
difficult
to heal than single VLUs (sVLU), and increasing VLU number has been associated
with
worse outcome. Margolis et al., Venous leg ulcer: incidence and prevalence in
the elderly,
Wound Rep Reg 2004;12:163-168. The incidence of mVLU appears to be increasing.
Analysis of an Intellicure Chronic Wound Dataset shows that 54% of patients
treated at
wound care centers using the U.S. Wound Registry medical database from 2007 to
2012 had
multiple VLUs vs. 40% as reported by Margolis et al. (2004) from a 1998-2000
dataset. Thus,
reports indicate that the incidence of mVLU has grown significantly, by almost
15%, in just
over a decade.
[0007] Gap junctions are a unique type of intercellular communication
conduit found
in most animal cell types. They form channels that interconnect the cytoplasms
of adjacent
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cells and permit the direct, cell-to-cell exchange of ions, secondary
messengers, water,
electrical impulses and low-molecular-weight metabolites and nutrients,
thereby coordinating
diverse metabolic and electrical functions of cell communities. Gap junctions
cross the
extracellular space between cells by the docking of two hemichannels
(connexons). One
connexon is contributed by each adjacent cell. Each connexon is an oligomer of
six connexin
monomers surrounding a central pore.
[0008] Human connexins are a polygenic family of 21 transmembrane
proteins, and
each is believed to provide permeability and regulatory properties to the
channels they form.
The most prevalent human connexin is connexin 43 (Coutinho et al., Dynamic
changes in
connexin expression correlate with key events in the wound healing process,
Cell Biol Int.
27:525-554 (2003). Connexin 43 is the predominant connexin in human epidermis
(Salomon
et al., Topography of mammalian connexins in human skin. J Invest Dermatol,
1994 103,
240-247) and, after acute cutaneous injury, its expression pattern changes
dynamically at the
edges of acute wounds during the wound-healing process (Coutinho et al.,
2003). Levels of
connexin 43 initially decrease at the wound edge (Goliger & Paul, Wounding
alters epidermal
connexin expression and gap junction mediated intercellular communication, Mol
Biol Cell.
6:1491-1501 (1995); Saitoh et al., Changes in the expression of gap junction
proteins
(connexins) in hamster tongue epithelium during would healing and
carcinogenesis,
Carcinogenesis 18:1319-1328 (1997)) but increase in more distant regions where
cells are
proliferating (Coutinho et al., 2003; Goliger & Paul, 1995). Down-regulation
of connexin 43
has been shown to occur during normal healing of acute wounds. Connexin 43 is
down-
regulated in acute wound edge keratinocytes and dermal fibroblasts as they
become migratory
(Coutinho 2003; Goliger & Paul 1995).
[0009] Connexin 26 is found in cells throughout the body, including the
inner ear and
the skin. Some studies indicate that channels made with connexin 26 help to
maintain the
correct level of potassium ions. Other research suggests that connexin 26 is
required for the
maturation of certain cells in the cochlea. Connexin 26 is also reported to
play a role in the
growth, maturation, and stability of the outermost layer of skin (the
epidermis). Connexin 30
is also found in several different tissues throughout the body, including the
brain, skin, and
inner ear. Some studies indicate that gap junctions made with connexin 30 also
help to
maintain the correct level of potassium ions. Connexin 30 gap junctions are
also said to play
a role in the growth and maturation of the epidermis.
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DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 represents blood vessels in VLUs. Blood vessel staining
(green) in a
representative (a) intact arm skin biopsy and (b) VLU. Chronic wound tissue is
characterized
by an enhanced number of dermal blood vessels. Scale bars - 100 and 500 p.m
respectively.
Cx43 = Red; Blood vessels/alpha smooth muscle actin = Green; Nuclei and
autofluorescent
extracellular matrix = Blue. VLU, venous leg ulcer; Cx, connexin.
[0011] Figure 2 represents Cx43, Cx26 and Cx30 expression in VLUs. (a&c)
Location of quantification sites. (b) Representative VLU. (d-e) Mean of the
individual and
group Cx fold changes compared directly to the reference arm values. (f-y)
Cx43, 26 and 30
expression with associated summary graphs for the WE, 1 mm from the WE and at
the FE.
Scale bar - 10x Montages 1000 pm; 40x Images 100 m. Cx43, 26 and 30 = Green;
Nuclei =
Blue. ** p < 0.01; *** p < 0.001. Error bars - Mean +/- SEM (Epidermis - n =
19 except FE
= 14; Dermis - n = 17 except WE = 15; FE, = 13) VLU, venous leg ulcer; Cx,
connexin; WE,
wound edge; PE, far edge.
[0012] Figure 3 represents Cx43, Cx26 and Cx30 expression in DFUs. (a&c)
Location of Cx43 quantification sites. (b) Representative DFU. (d-e) Mean of
the individual
and group Cx fold changes compared directly to the reference arm values. (f-y)
Cx43, 26 and
30 expression and associated summary graphs for the WE, 1 mm from the WE and
at the FE.
Scale bar - 10x Montages 1000 p.m; 40x Images 100 p.m. Cx43, 26 and 30 =
Green; Nuclei =
Blue. * p < 0.05; ** p < 0.01; *** p < 0.001. Error bars - Mean +/- SEM
(Epidermis - n =
11 except FE = 8; Dermis - n = 6). DFU, diabetic foot ulcer; Cx, connexin; WE,
wound edge;
FE, far edge.
[0013] Figure 4 represents Cx43, Cx26 and Cx30 expression in PRUs. (a&c)
Location of Cx43 quantification sites. (b) Representative PRU. (d-e) Mean of
the individual
and group Cx fold changes compared to directly to the reference arm values. (f-
y) Cx43, 26
and 30 expression and associated summary graphs for the WE, 1 mm from the WE
and at the
FE. Scale bar - 10x Montages 1000 vim; 40x Images 100 p.m. Cx43, 26 and 30 =
Green;
Nuclei = Blue. * p < 0.05; ** p < 0.01; *** p < 0.001. Error bars - Mean +/-
SEM
(Epidermis and dermis - n = 6 except FE = 5). PRU, pressure ulcer; Cx,
connexin; WE,
wound edge; FE, far edge.
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[0014] Figure 5 shows where assessments were taken across a 4 mm biopsy
using an
Olympus FV-1000 inverted confocal microscope to take 40x images of arm skin
and wound.
[0015] Figure 6 shows Cx 43 in dermis normalised to patient baseline
expression.
BRIEF SUMMARY OF THE INVENTION
[0016] The inventions described and claimed herein have many attributes
and
embodiments including, but not limited to, those set forth or described or
referenced in this
Brief Summary. It is not intended to be all-inclusive and the inventions
described and
claimed herein are not limited to or by the features or embodiments identified
in this Brief
Summary, which is included for purposes of illustration only and not
restriction.
[0017] In one aspect this invention relates to pharmaceutical
formulations and
methods for treating resistant lesions with a connexin protein modulating
agent.
[0018] In another aspect the invention relates to pharmaceutical
formulations and
methods for treating lesions on subjects likely to be responsive to treatment
with a connexin
protein modulating agent, based on indicators including those described
herein. Such factors
include, for example, the presence of multiple venous leg ulcers (mVLUs) on a
subject, and
the presence of multiple diabetic foot ulcers (mDFUs). Other factors include
degree of local
or systemic inflammation in or on a subject, including lesion or wound
inflammation, as
described herein. Still other factors include the amount of healing during a
pretreatment or
run-in period with a standard-of-care treatment, and the amount healing during
a pretreatment
or run-in period with standard-of-care treatment together with a hydrogel or
other product
applied to the wound to maintain a moist wound environment, all as measured
by, for
example, percent wound surface area reduction and/or linear wound advance. Yet
other
factors include the size of the lesion or the duration of the lesion or both.
[0019] In one aspect this invention relates to compositions and methods
for treating
an ulcer or lesion on a mVLU or mDFU subject by administering a connexin
protein
modulating agent in amounts effective to promote healing. This invention also
relates to
methods of determining whether subjects are responder subjects likely to
respond to treatment
by a connexin protein modulating agent, based on indicators including the
presence of
mVLUs or mDFUs, for example. It has been found that patients with multiple
lesions, or
multiple resistant lesions, who generally have a poorer prognosis for healing
using standard
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treatments, respond surprisingly well to treatment by, for example, connexin
43 modulating
agents.
[0020] In one aspect, this invention relates to the treatment of
resistant lesions and
responder subjects including subjects who have mVLUs or mDFUs, which are more
resistant
to healing, by administering a therapeutically effective amount of a
composition comprising,
for example, a connexin 43 modulating agent. The compositions and methods
relate in part
to the surprising discovery that patients with resistant lesions, including,
for example, patients
with mVLUs and mDFUs, respond particularly well, and far better than standard-
of-care or
vehicle plus standard-of-care, to treatment with a connexin protein modulating
agent in a dose
dependent manner, in contrast to other subjects, for example, those having a
single VLU or
single DFU, for whom treatment with a connexin protein modulating agent shows
less effect
over treatment with vehicle plus standard-of-care or standard-of-care alone.
Thus, it has been
suprisingly discovered, in the case of VLU, for example, that although
subjects having
multiple lesions generally have a low likelihood of response to treatment with
compression
bandaging, they are very responsive to treatment with a connexin protein
modulating agent,
for example, a connexin 43 modulating agent.
[0021] In some embodiments the connexin protein modulating agent is a
connexin
protein antisense oligonucleotide. In one embodiment the connexin protein
modulating agent
is a connexin protein antisense oligodeoxynucleotide, whether chemically
modified or
unmodified. In some aspects the therapeutically effective amount of the
connexin protein
modulating agent is any amount effective to promote healing of a resistant
lesion in or on a
subject. Connexin 43, connexin 26 and connexin 30 protein modulating agents
are preferred.
Connexin 43 protein modulating agents are particularly preferred.
[0022] Examples of effective doses that may be used for the treatment of
resistant
lesions are described and claimed herein. In some aspects, the therapeutically
effective
amount effective to promote healing of a resistant lesion in or on a subject
is administered,
for example, by applying, coating or filling the lesion with a connexin
protein modulating
agent present at a concentration of about 0.1 mg/mL to about 100 mg/mL, or
more. In other
embodiments, the connexin protein modulating agent is present at a
concentration ranging
from about 0.5 to about 50 mg/mL. In other embodiments, the connexin protein
modulating
agent is present at a concentration ranging from about 0.3 to about 30 mg/mL.
In other
embodiments, the connexin protein modulating agent is present at a
concentration ranging
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from about 0.1 or 1.0 to about 10 mg/mL. In other embodiments, the connexin
protein
modulating agent is present at a concentration ranging from about 0.1 or 1.0
to about 0.3 or
3.0 mg/mL. In other embodiments, the connexin protein modulating agent is
present at a
concentration of about 3.0 mg/mL. In any of these aspects the connexin protein
modulating
agent may be a connexin protein antisense oligonucleotide. When the connexin
protein
modulating agent is a modified connexin protein antisense oligonucleotide,
e.g., a backbone-
modified oligonucleotide, or chemically modified oligonucleotide for increased
half-life, the
above-noted dose concentrations may be the same, or may be decreased or
increased as
appropriate based on potency and specificity, for example. In any of these
aspects, the carrier
(vehicle) may be a pharmaceutically acceptable carrier. Such carries include
poloxamer gel,
for example, poloxamer 407, present in an amount ranging from about 15% to
25%, or 20%
to 30%, for example.
[0023] In some aspects, this invention also relates to methods of
determining whether
subjects are those likely to respond to treatment by a connexin protein
modulating agent,
based on indicators described herein. Such factors include, for example, as
noted, the
presence of multiple ulcers, inflammation, the amount of healing measured
during a
pretreatment or run-in period with standard-of-care treatment and/or a product
to maintain
moisture at the lesion, as well as the size and/or duration of the lesion. In
one aspect, in the
case of VLUs, the indicator is the presence of mVLUs, as noted.
[0024] According to the invention, other indicators that have also
surprisingly been
discovered to increase the likelihood of complete healing in response to
treatment of a VLU
on a patient with more than one lesion using a connexin protein modulating
agent include, for
example, age of the subject and body mass index (BMI). For example, in some
embodiments,
the indicator can be age over 50. In one embodiment the age indicator can
include, for
example, ages over 50-52 years. A body mass index (BMI) of less than 40 or 42,
for example,
is another indicator discovered to affect likelihood of response to treatment
by a connexin
protein modulating agent, for example, a connexin 43 modulating agent. In one
embodiment
the BMI indicator is less than 40. In some embodiments, subjects over 50
having mVLUs
and a BMI of less than 42 exhibit a dose dependent significant response to a
connexin protein
modulating agent such as a connexin protein antisense oligodeoxynucleotide,
and are up to 5
times more likely to heal, or greater, than mVLU subjects over 50 who are
treated with
standard of care.
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[0025] In one aspect the compositions of this invention comprise one or
more anti-
connexin protein, for example, anti-connexin 43, polynucleotides that modulate
connexin
activity. In some aspects the connexin modulating agent may be antisense
oligonucleotides.
Anti-connexin oligonucleotides can inhibit connexin activity by decreasing its
expression. In
one aspect the active ingredient includes a connexin protein, for example,
anti-connexin 43,
modulating agent. In other aspects the connexin protein modulating agents may
be anti-
connexin peptides, peptidomimetics (for example, anti-connexin 43 peptides or
peptidomimetics), gap junction closing compounds, hemichannel closing
compounds, and
connexin carboxy-terminal polypeptides for use in treating subjects with
resistant lesions.
[0026] The connexin modulating agents of this invention may be used alone
or in
combination. In some embodiments, treatment with a connexin protein modulating
agent is
administered in conjunction with standard-of-care, for example, compression
bandaging
and/or off-loading.
[0027] The invention includes a package or kit comprising a
pharmaceutical
composition comprising a pharmaceutically acceptable carrier and a
pharmaceutically
acceptable anti-connexin modulating agent, together with a label and/or
instructions for
administering the composition to subjects with resistant lesions, for example
where the
subject has mVLUs or mDFUs, and the agent is administered in amounts effective
to promote
healing of the lesions in a subject, alone or together with standard-of-care,
for example,
compression bandaging and/or off-loading. In one embodiment, the invention
includes a
package or kit comprising a pharmaceutical composition including a
pharmaceutically
acceptable carrier and a pharmaceutically acceptable anti-connexin protein
modulating agent,
such as an anti-connexin protein oligonucleotide, optionally with a label
and/or instructions
for administering the composition to responder subjects with mVLUs and/or
mDFUs in
amounts effective to promote mVLU and/or mDFU healing in a subject, alone or
under
compression bandaging. Packages and kits include those with a connexin 43
protein
modulating agent, a connexin 26 protein modulating agent and/or a connexin 30
protein
modulating agent.
DETAILED DESCRIPTION
[0028] In one embodiment this invention relates to methods for treating
responder
subjects and subjects with resistant lesions, i.e., one or more resistant
lesions, and
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compositions useful in those methods. The compositions may include
pharmaceutical
formulations or dosage forms, suitable for administration in therapeutically
effective amounts.
[0029] In one embodiment, the compositions and methods are based on the
surprising
discovery that certain subjects including subjects with resistant lesions,
such as, for example,
patients with mVLUs and mDFUs, respond particularly well, and far better than
standard-of-
care or vehicle plus standard-of-care, to treatment with a connexin protein
modulating agent
in a dose dependent manner, in contrast to other subjects, for example, those
having a single
VLU or a single DFU, for whom treatment with a connexin protein modulating
agent shows
less effect over treatment with vehicle plus standard-of-care or standard-of-
care alone. Thus,
it has been suprisingly found, in the case of VLU, for example, that although
subjects having
multiple lesions generally have a low likelihood of response to treatment with
compression
bandaging, they are very responsive to treatment with a connexin 43 modulating
agent. As
described further herein, it has been surprisingly discovered that lesions on
subjects having
resistant lesions, such as mVLUs, are more than three times more likely to
completely heal
following treatment with a connexin 43 modulating agent than a lesion on an
mVLU subject
treated with standard of care or vehicle, even though wounds on patients
having mVLUs
usually have a low likelihood of complete healing as described herein. In some
embodiments,
the connexin modulating agent is a connexin 43, connexin 30 or connexin 26
antisense
oligonucleotide. In one embodiment the connexin 43 modulating agent is a
modified
connexin 43 antisense oligodeoxynucleotide.
[0030] Accordingly, in one embodiment, this invention relates to
compositions and
methods useful in treating subjects with resistant lesions, including, for
example, mVLUs and
mDFUs. Compositions and formulations useful in the invention include a
connexin protein
modulating agent. Particular formulations include connexin 43 modulating
agents, connexin
26 modulating agents, and connexin 30 modulating agents.
[0031] In another embodiment, the invention relates to pharmaceutical
formulations
and methods for treating a wound on a subject having multiple venous leg
ulcers (mVLUs),
i.e, more than one venous leg ulcer at the same time, or other resistant
lesions in responder
subjects likely to respond to treatment by a connexin protein modulating
agent, as described
herein. In another embodiment, the invention relates to pharmaceutical
formulations and
methods for treating a wound on a subject having multiple diabetic foot ulcers
(mDFUs), i.e,
more than one diabetic foot ulcer at the same time, or other resistant lesions
in diabetic
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responder subjects likely to respond to treatment by a connexin 43 modulating
agent, as
described herein. In either case, the one or more ulcers can be on the same
leg, or on
different legs. In either case, the subject may be over about 50 years of age,
and/or have a
BMI of less than about 40.
[0032] It has been found that patients with multiple resistant lesions
such as mVLUs,
who generally have a poorer prognosis for healing using standard treatments
than those with
single lesions such as sVLUs, have a surprising comparatively high likelihood
of responding
to treatment by connexin 43 modulating agents. Accordingly, in some
embodiments, this
invention relates to treating patients with mVLUs by administering an amount
of a
composition comprising a connexin 43 modulating agent in an amount effective
to promote
VLU healing in an mVLU subject. In some embodiments the connexin 43 modulating
agent
is a connexin 43 antisense oligonucleotide. The connexin 43 antisense
oligonucleotide may
be, in some embodiment, an unmodified connexin 43 antisense
oligodeoxynucleotide.
[0033] Thus, in one embodiment, this invention relates to treating
responder subjects
who have resistant lesions such as mVLUs or mDFUs. This invention relates in
one aspect to
formulations and methods for treating resistant lesions in subjects who are
likely to be
responsive to treatment by a connexin 43 modulating agent, based on indicators
including
those described herein, which include the presence of mVLUs or mDFUs, or, in
some
instances, the presence of a biomarker indicative of a resistant lesion, as
discussed above.
[0034] In one aspect, this invention relates to formulations and methods
of treating
responder subjects who have resistant lesions such as mVLUs by administering a
therapeutically effective amount of a composition comprising a connexin 43
modulating
agent to responder subjects.
[0035] According to this invention, indicators in addition to presence of
mVLUs
which have further suprisingly been found to increase the likelihood of
complete healing in
response to treatment for resistant lesions using a connexin 43 modulating
agent include, for
example, age of the subject and body mass index (BMI). For example, in some
embodiments,
the indicator can be age over 50. In one embodiment the age indicator can
include, for
example, age over 52. A body mass index (BMI) of less than 42 is another
indicator found to
affect susceptibility to treatment by a connexin 43 modulating agent. In one
embodiment the
BMI indicator is a BMI less than 40, preferably a BMI less than 35 or 30, and
most preferably
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a BMI less than 25. In some embodiments, subjects aged over 50 (or 52) having
mVLUs and
a BMI of less than 40 (or 42) exhibit a dose dependent significant response to
a connexin 43
modulating agent such as a connexin 43 antisense oligodeoxynucleotide.
[0036] Although the presence of mVLUs and sVLUs in older subjects have
been
associated with more difficulty in healing and a lower likelihood of complete
healing, it has
been surprisingly discovered that the difficult to heal subjects over 50 with
mVLUs are more
likely to respond to treatment with a connexin 43 modulating agent than mVLU
subjects over
50 who are treated with vehicle and/or standard-of-care. For example, mVLU
subjects over
50 show a dose dependent response following treatment with an anti-connexin 43
modulating
agent, such as a connexin 43 antisense oligonucleotide, are up to 5 times or
more likely to
heal than mVLU subjects over 50, for example, those over about 52 years of
age, who are
treated with standard-of-care alone.
[0037] Although the presence of mVLUs and sVLUs in older subjects have
been
associated with more difficulty in healing and a lower likelihood of complete
healing, it has
been surprisingly discovered that the difficult to heal subjects having a BMI
less than about
40 with multiple VLUs are more likely to respond to treatment with a connexin
43
modulating agent than mVLU subjects having a BMI greater than about 40 who are
treated
with standard of care. For example, following treatment with an anti-connexin
43 modulating
agent such as a connexion 43 antisense oligonucleotide, a subject with BMI of
30 is up to 4
times or more likely to heal than one with a BMI of 43.
[0038] This invention also relates to methods of determining whether a
subject is a
responder subject, likely to be responsive to treatment by a connexin 43
modulating agent,
based on indicators described herein, for example. In some embodiments, this
invention
relates to a method of determining whether a patient is a responder to
treatment with a
connexin 43 modulating agent, the method comprising determining one or more
subject
indicators selected from the group of a subject's VLU status (single or
multiple VLUs), age,
and BMI measurement, and, optionally, percent healing during a run-in period
with, e.g.,
compression bandaging alone, and determining whether the indicators predict a
likelihood of
response to treatment with a connexin 43 modulator and treating those subjects
expected or
predicted to respond to treatment. In some embodiments, the method of
determining whether
a subject is a responder can be used in conjunction with any of the methods of
treatment and
uses described herein. As discussed herein, mVLU subjects have been
surprisingly
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discovered to be more likely to heal following treatment with a connexin 43
modulating agent
than patients who do not meet the criteria for responsiveness to treatment,
according to
indicators such as those set forth herein.
[0039] The invention relates in some aspects to pharmaceutical
formulations and
packages and kits including a pharmaceutical formulation comprising a
pharmaceutically
acceptable carrier, and a pharmaceutically acceptable anti-connexin modulating
agent, for
administering to responsive subjects with mVLUs an anti-connexin modulating
agent, in
amounts effective to promote mVLU healing in a subject. The package optionally
comprises
a label and/or instructions for this use.
[0040] In some embodiments, the formulations of this invention for use in
treating
mVLUs, mDFUs, or other resistant lesions may comprise a connexin 43 modulating
agent
and one or more pharmaceutically acceptable vehicles formulated for topical
administration.
In some embodiments the composition is formulated to provide sustained release
of the
connexin 43 modulating agent.
[0041] The terms "modulating agent," "modulator" and "modulation" of
connexin
protein activity, as used herein in its various forms, refers to inhibition in
whole or in part of
the expression, action or activity of a connexin or a connexin hemicharmel or
connexin gap
junction, in whole or in part, and may function as anti-connexin agents,
including as gap
junction modulation agents. In some embodiments the connexin protein
modulating agents of
this invention include anti-connexin 43, 30 or 26 oligonucleotides, anti-
connexin 43, 30 or 26
peptides, anti-connexin 43, 30 or 26 peptidomimetics, or gap junction closing
compounds,
hemichannel closing compounds, and connexin carboxy-terminal polypeptides
useful for
healing wounds on subjects with more than one resistant wound, e.g., more than
one VLU on
one or both legs.
[0042] The polynucleotides of this invention include synthesized
polynucleotides
having a length of less than 80 nucleotides, e.g., from 12-18 to about 50-80
nucleotides,
preferably about 30 nucleotides or less, e.g., from 12 to about 30
nucleotides, and more
preferably from about 15 to about 30 nucleotides. In one example, the
polynucleotide has 30
nucleotides.
[0043] Such formulations include, for example, topical delivery forms and
formulations. Such delivery forms and formulations include those for the
treatment of a
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subject as disclosed herein. In some embodiments the anti-connexin
polynucleotides are anti-
connexin 43 oligonucleotides (ODN). In other embodiments, the connexin protein
modulating compounds are anti-connexin 43, 30 or 26 peptides or
peptidomimetics, e.g.,
anti-connexin 43, 30 or 26 hemichannel blocking peptides or anti-connexin 43
hemichannel
blocking peptidomimetics. In some embodiments the gap junction closing
compounds and
hemichannel closing compounds are connexin 43, 30 or 26 gap junction closing
compounds
and connexin 43, 30 or 26 hemichannel closing compounds. Preferred connexin
carboxy-
terminal polypeptides are connexin 43, 30 or 26 carboxy-terminal polypeptides.
Treatment
of a subject, e.g., for mVLUs, with one or more pharmaceutical compositions of
the invention,
e.g., an anti-connexin ODN and a connexin hemichannel blocking agent, e.g., a
peptide or
peptidomimetic, or a first anti-connexin agent and a second anti-connexin
agent, may
comprise their simultaneous, separate, sequential or sustained administration.
[0044] The
pharmaceutical formulations of this invention may further comprise one
or more pharmaceutically acceptable excipients. In some embodiments the
formulation may
comprise a connexin 43, 30 or 26 antisense oligonucleotides. The connexin 43
antisense
oligonucleotide that are included in the formulation may be, in some
embodiments, an
unmodified connexin 43 antisense oligodeoxynucleotide. In some embodiments the
vehicle
may be or contain a gel, a poloxamer
(liquid or gel), a carboxycellulose (e.g.
carboxymethylcellulose), a collagen (e.g., a Type I collagen), a collagenous
material
comprising tropocollagen, a hyaluronan or derived-hyaluronic acid, and/or an
oil (e.g., Emu
oil). The formulations of this invention do not comprise the connexin 43
modulating agent in
sterile water as the only vehicle.
[0045] In
some embodiments, the pharmaceutically acceptable carrier or vehicle is, or
comprises, a gel. In one aspect the gel can be a reverse-thermosetting gel
which is a liquid at
low temperatures, for example at 2-8 C, and which undergoes a reversible
liquid to gel
transition at temperatures greater than approximately 15 C. Thus, in some
embodiments the
carrier may be a liquid at temperatures below approximately 15 C, but may form
a gel at
temperatures above approximately 15 C, such as room temperature or at body
temperature.
In some instances, the gel is a nonionic polyoxyethylene-polyoxypropylene
copolymer
gel. In some embodiments the gel is a pluronic gel. The pluronic gel may be,
for example,
poloxamer 407, also sometimes referred to as Pluronic F-127 (BASF). In some
embodiments, the formulations of this invention may comprise from about 15 to
about 30 %
(w/v) gel. In some embodiments, the formulations of this invention may
comprise from about
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20 to about 25 % (w/v) gel. In some embodiments, the formulations of this
invention may
comprise about 22.6 % (w/v) poloxamer 407 gel.
[0046] In some embodiments, treatment with a connexin protein modulating
agent is
administered in conjunction with compression bandaging, off-loading, or other
standard-of-
care therapy. Exemplary connexin protein modulating agents include connexin
43, 30 or 26
modulating agents.
[0047] It has also been surprisingly discovered that connexin 43 levels
measured in
the dermis and/or epidermis at the edges of multiple VLUs in humans appear
higher than
connexin 43 levels measured at the edges of single VLUs in humans.
Accordingly, a
determination of high connexin 43 levels in the dermis or epidermis of a
resistant wound may
be used as a method of diagnosing a resistant lesion and/or responder patient
prior to
prescribing treatment with a connexin 43 modulating agent.
[0048] Subjects with mVLUs, mDFUs, or other resistant lesions or
otherwise assessed
to have a likelihood of response to treatment of by a connexin protein
modulating agent,
according to the methods of this invention, are also referred to as
"responder" subjects. By
likelihood of response is meant a likelihood that resistant lesion healing is
promoted with a
connexin protein modulating agent over , for example, it may be promoted
treatment with
standard of care (e.g., compression bandageing or off-loading) and/or vehicle
by at least a
factor of 2 when compared to treatment of lesions on mVLU subjects or subjects
with other
resistant lesions with standard of care, such as treatment by compression
bandaging. The
probability of a resistant wound on a responder subject healing with treatment
with a
connexin protein modulating agent is likely to be at least about 10% to 15%
higher than
treatment with standard of care (e.g., compression bandageing or off-loading)
and/or vehicle.
In other words, the healing delta between responder subjects treated with a
connexin protein
modulating agent will be at least about 10% to 15%. Typically this delta will
be 20% or more,
and can be 25%, 30%, 35%, 40% and 45% or more.
[0049] Resistant lesions or wounds include multiple VLUs, multiple
diabetic foot
ulcers (DFUs), multiple pressure ulcers, wounds whose surface areas change
relatively little
during a screening or pretreatment period with compression bandaging or other
standard-of-
care therapy (e.g., off-loading), and those with relatively few signs of
healing during a
screening period with standard therapy, e.g, compression bandaging therapy in
the case of
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VLUs. In some aspects resistant lesions may also be characterized by less
granulation and
epithelialization during the screening or pretreatment period, or at the time
of treatment with
the connexin 43 modulating agent. The screening or pretreatment period may be
from about
days to about 1-4 weeks, for example, and is typically 2 weeks, 3 weeks or 4
weeks, but
may be longer. A 2-week screening or pretreatment period is common.
[0050] Still other factors include the amount healing during a
pretreatment or run-in
period with standard-of-care treatment, and the amount healing during a
pretreatment or run-
in period with standard-of-care treatment together with a hydrogel or other
agent used to
maintain a moist wound environment, all as measured by, for example, percent
lesion surface
area reduction and/or linear lesion advance. Yet other factors include the
size and/or duration
of the lesion. In one aspect, in the case of VLUs, the indicator is the
presence of mVLUs, as
noted. A resistant lesion is any lesion exhibiting one or more indicators of a
resistant lesion,
as described herein. A resistant lesion may exhibit, for example, one or more
of the
indicators based on % surface area reduction during a
pretreatment/screening/run-in period,
the baseline area of lesion, the % epithelialization of the lesion, and
baseline circumference,
as described herein. In addition, a lesion may be characterized as a resistant
lesion if it is
present on a subject having hemoglobin Alc (HbAlc) levels and/or BMI, as
described herein.
In some instances, the resistant lesion may exhibit two, three, four, five or
all six of these
indicators, including the subject having an HbAlc level and BMI as described
herein.
[0051] In one embodiment, where the amount of healing during a pre-
treatment or
run-in period with standard-of-care treatment is used to characterize a
resistant wound, the
pretreatment or run-in period is generally long enough so that some lesions
can show material
or art-recognized advancement to resolution with standard treatment. In one
embodiment, the
period is generally up to 2-4 weeks, but can be longer. Where a pretreatment
or run-in period
of about 2 weeks is utilized to determine whether the subject has a resistant
lesion, i.e., a
period where the patient is treated with standard-of-care such as compression
bandaging
and/or off-loading, for example, if the treated lesion(s) on the patient does
not increase by
more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 %, or heal by
more than about 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29,
or about 30 % (in percent surface area reduction of the lesion), or by any
range between any
two recited values, or any percentage in between any two recited values. For
example, the
treated lesion may be a persistent lesion if it does not heal by more than
about 25%, more
than about 20-30%, or more than about 25-30%, preferably by less than about
25%, and more
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preferably by less than about 20%, the lesion is a resistant lesion for
purposes of this
invention. In other embodiments, the lesion is identified as a resistant
lesion for treatment
with an anti-connexin 43 modulating agent if the treated lesion (s) on the
patient does not heal
by more than about 10-20%, preferably less than from about 10-15%, for example
less than
about 17.5%. Where a pretreatment or run-in period of about 4 weeks is
utilizied to
determine whether the subject has a persisent lesion, if the treated lesion(s)
on the patient
does not increase by more than about 15% or heal by more than about 30-50% or
30-40%
surface area reduction, preferably less than about 35%, and more preferably
less than about
30%, the lesion is a resistant lesion for purposes of this invention.
[0052] In still other embodiments, the lesion is identified as a
resistant lesion for
treatment with an anti-connexin 43 modulating agent if the wound(s) to be
treated does not
show a surface area reduction of at least about 20% over a 2- to 4-week
pretreatment or run-
in period during with the subject is treated with a hydrogel or other agent to
maintain a moist
wound environment (for example, Curasol Hydrogel, Gentell Hydrogel, poloxamer
gel, or
any other acceptable hydrogel or moistening agent for treating lesions) plus
standard-of-care
(for example, compression and/or off-loading, etc.).
[0053] For example, in some embodiments, the lesion is identified as a
resistant lesion
treatment with an anti-connexin 43 modulating agent if the lesion(s) to be
treated is > about
5.0, 5.1, 5.2, 5.3, 5.4, 5.4, 5.5, 5.6, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2,
6.3, 6.4, 6.5, 6.6, 6.7, 6.8,
6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3,
8.4, 8.5, 8.6, 8.7, 8.8, 8.9,
9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.0 or > about 10.0 cm2 (size).
In some embodiments,
the lesion is identified as a resistant lesion treatment with an anti-connexin
43 modulating
agent if the lesion(s) to be treated is > 5 cm2, preferably >6.0 cm2, more
preferably >7.0 cm2
or >8.0 cm2, or also preferably >8.6 cm2.
[0054] In some embodiments, the lesion is identified as a resistant
lesion treatment
with an anti-connexin 43 modulating agent if the lesion (s) to be treated has
minimal
epilethialization, that is, epilethialization of less than about 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.0, 4.0, 5.0,
5.1, 5.2, 5.3, 5.4, 5.5, 5.6,
5.7, 5.8, 5.8, 5.9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,
7.2, 7.3, 7.4, 7.5, 7.6, 7.7,
7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,
9.3, 9.4, 9.5, 9.6, 9.7, 9.8,
9.9, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0,
22.0, 23.0, 24.0, 25.0,
26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0% of the surface
area of the wound. In
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some embodiments, the lesion is identified as a resistant lesion treatment
with an anti-
connexin 43 modulating agent if the wound(s) to be treated has
epilethialization of less than
about 30%, 20%, 10% or less, preferably 5.0% or less, more preferably 2.5% or
less.
[0055] In some embodiments, the lesion is identified as a resistant
lesion treatment
with an anti-connexin 43 modulating agent if the subject to be treated has a
HbAlc of greater
than about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or about 7.0%, or
greater than any
range between any two recited values, or greater than any value in between any
two recited
values. In some embodiments, the lesion is identified as a resistant lesion
treatment with an
anti-connexin 43 modulating agent if the subject to be treated has a HbAlc of
greater than
about 6.5%. In some embodiments, the lesion is identified as a resistant
lesion treatment with
an anti-connexin 43 modulating agent if the subject to be treated has a HbAlc
of greater than
6.0%, 6.5% or greater than 7.0%.
[0056] In some embodiments, the lesion is identified as a resistant
lesion treatment
with an anti-connexin 43 modulating agent if the lesion(s) to be treated has a
circumference
of less than about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,
5.7, 5.8, 5.8, 5.9, 6.1,
6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,
7.7, 7.8, 7.9, 8.0, 8.1, 8.2,
8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7,
9.8, 9.9, 10.0, 10.1, 10.2,
10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9. 11.0, 11.1, 11.2, 11.3, 11.4, 11.5,
11.6, 11.7, 11.8,
11.9, or < about 12.0 cm, or less than any range between any two recited
values, or less than
any value in between any two recited values. In some embodiments, the
resistant lesion
having a circumference less than the recited length may also have a relatively
convex
circumference. In some embodiments, the lesion is identified as a resistant
lesion treatment
with an anti-connexin 43 modulating agent if the lesion has a circumference of
less than
about 10.0 cm, 9.0 cm, 8.0 cm, 7.0 cm, 6.0 cm or 5.7 cm.
[0057] In other embodiments, where a pretreatment or run-in period of
about 2 weeks
is utilized to help determine whether the subject has a resistant lesion based
on linear lesion
advance (LLA), if the treated lesion (s) on the subject shows a linear lesion
advance of less
than about 0.002, 0.003, 0.004, 0.005, 0.006 or 0.007 cm/day or any range
between any of
those values (.e.g., about 0.002 to about 0.0065 cm/day), or a linear lesion
advance of about
0.03, 0.035, 0.04, 0.042, 0.05 cm/week, or any range between any of those
values (e.g, about
0.035 to about 0.05 cm/week), the lesion is a resistant lesion for purposes of
this invention.
In other embodiments, where a pretreatment or run-in period of more than 2
weeks, for
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example up to about 4 weeks, or any period between about 2 weeks to about 4
weeks, is
utilized to determine whether the subject has a persisent lesion, if the
treated lesion (s) on the
patient shows a linear lesion or wound advance of less than about 0.004,
0.005, 0.006, or
0.0065 cm/day or any range between any of those values (.e.g,, about 0.005 to
about 0.0065
cm/day), or a linear lesion advance of less than about 0.03, 0.035, 0.04,
0.042, or about 0.045
cm/week, or less than about any range between any of those values (e.g, about
0.04 to about
0.045 cm/week)õ the lesion is a resistant lesion for purposes of this
invention. In other
embodiments, where a pretreatment or run-in period of about about 2 weeks or
about 4 weeks
is utilized to determine whether the subject has a LLA-persistent lesion, if
the treated lesion
on the patient shows a linear wound advance of 0.050 cm/week or less, the
lesion is an LLA
resistant lesion for the purposes of this invention
[0058] In some embodiments, the lesion is identified as a resistant
lesion for treatment
with an anti-connexin 43 modulating agent if the lesion(s) to be treated is >
about 8.5 cm2
(size) and a duration of more than 6 months, a minimal degree of
epitheliazation, and/or
hemoglobin Ale (HbAle) of 6.5 % or greater.
[0059] In one embodiment, the lesion is identified as a resistant lesion
for treatment
with an anti-connexin 43 modulating agent if the lesion to be treated is
present on a subject
having a BMI of less than about 40, 39, 38. 37, 36, 35, 34, 33, 32, 31, 30,
29, 28, 27, 26, or
about 25. In some embodiments, the lesion is identified as a resistant lesion
for treatment
with an anti-connexin 43 modulating agent if the lesion to be treated is
present on a subject
having a BMI of less than about 40, preferably less than 35 or 30, and most
preferably less
than 25.
[0060] In yet other embodiments, the wound is identified as a resistant
lesion for
treatment with an anti-connexin protein modulating agent if the lesion (s) to
be treated shows
one or more of the following: (a) low levels of mitotic activity or fewer
cells; (b) high levels
of cytokines and/or proteases or other markers or marker ratios and panels
indicative of
resistant lesions; (c) low levels of growth factors; and/or (d) fibroblast
senescence, in
comparison to healing or acute wounds.
[0061] In one embodiment, the responder patient has one or more of the
following
characteristics: (1) multiple venous leg ulcers on one or both legs; (2) age
equal to 50-52
years of age; (3) BMI less than about 40-42; and, (4) healing by less than
about 30-40%
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during a pretreatment period or a run-in period with standard of care
treatment (e.g.,
compression bandaging). In other embodiments, this invention also relates to
methods of
determining whether subjects are likely to respond to treatment by a connexin
43 modulating
agent based on indicators described herein. Methods of assessing whether a
subject is a likely
responder subject can also be used in conjunction with the methods of
treatment and uses
described herein.
[0062] Other indicators of resistant lesions or wounds include biomarkers
of resistant
lesions as described herein. With respect to markers, resistant lesion fluids
show, for
example, lower ratios of two key cytokines, TNFa and IL-1, and their natural
inhibitors, P55
and IL-1 receptor antagonist. Resistant lesions will show from about 1:1 to
about 5:1 in the
case of P55/TNFa and/or from about 1:1 to about 10:1 in the case of IL-1RATIL-
1. Resistant
wounds will also show high levels of cytokines such as IL-1, IL-6 and TNFa in
fluids
collected from the lesions. In other embodiments, a resistant lesion is
identified by evaluating
the change in levels of cytokines over, for exaample, a 2-4 week pretreatment
period during
which the levels of cytokines are not significantly decreased.
[0063] Other resistant lesions are those that show significantly elevated
levels of
proteases compared to acute wounds. The average level of protease activity in
chronic
wound fluids (87 ag collagenase equivalents/m1) is about 100-fold higher than
in mastectomy
fluids. Also, the range of protease activity in chronic wound fluids is rather
large (from 1 to
584 [tg collagenase equivalents/m1). More importantly, the levels of protease
activity tend to
decrease in chronic venous ulcers 2 weeks after the ulcers begin to heal
(Figure 3). In some
embodimentsthe protease may be, for example, a metalloproteinase.
[0064] In still other embodiments, a resistant wound will contain high
levels of ILL
IL6, and matrix metalloproteinases (MMPs), and an abnormally high MMP to TIMP
ratio.
MMPs are part of the larger family of metalloproteinase enzymes that play an
important role
in wound healing. In normal wound healing, MMPs are produced by activated
cells
(neutrophils and macrophages) and wound cells (epithelial cells, fibroblasts
and vascular
endothelial cells). The MIMPs are inhibited by specific endogenous tissue
inhibitor of
metalloproteinases, which comprise a family of four protease inhibitors: TIMP-
1, TIMP-2,
TIMP-3, and TIIVIP-4. As an example, elevated levels of MMP-2, MMP-8 and/or
MMP-9,
preferably eleveted levels of MMP-9 may characterize a resistant wound.
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[0065] In some embodiments, a resistant wound may also be be
characterized by low
levels of TGFr3 and/or low levels of one or more MMP tissue inhibitors (TIMP),
for example
TIMP-1 or TIMP-2). Resistant wounds may also be characterized by the MMP-
9:TIMP-2
ratio. In some embodiments, the resistant wound may be characterized by
increases in one or
more of IL-1, IL-6, IL-8, MIP-la, TNFa and/or 1L-113 or any combination
thereof. Higher
levels of IL-la, IL-1(3, IFNy, IL-12p40, GM-CSF and IL-1RA may also be present
at elevated
levels in resistant wounds.
[0066] Healing as used herein refers to healing based on one or more
assessments for
wound, or lesion, healing, including healing of a wound on an mVLU or mDFU
subject, such
as complete wound closure, or reduction or percent change in wound surface
area.
[0067] As used herein, "subject" refers to any mammal, including humans,
domestic
and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats,
sheep, pigs,
cows, etc. The preferred mammal herein is a human, including adults, children,
and the
elderly. Preferred sports animals are horses and dogs. Preferred pet animals
are dogs and
cats.
[0068] As used herein, "preventing" means preventing in whole or in part,
or
ameliorating or controlling.
[0069] As used herein, a therapeutically effective amount of the connexin
43
modulating agent is any amount effective to promote healing of a resistant
lesion in a subject.
For example, a therapeutically effective amount of the connexin 43 modulating
agent when
used to treat mVLUs is the amount effective to promote healing of mVLUs.
[0070] The terms "peptidomimetic" and "mimetic" include synthetic or
genetically
engineered chemical compounds that may have substantially the same structural
and
functional characteristics of protein regions which they mimic. In the case of
connexins,
these may mimic, for example, the extracellular loops of opposing connexins
involved in
connexon-connexon docking and cell-cell channel formation, and/or the
extracellular loops of
hemichannel connexins.
[0071] As used herein, the term "peptide analogs" refer to the compounds
with
properties analogous to those of the template peptide and can be non-peptide
drugs.
"Peptidomimetics" (also known as peptide mimetics) which include peptide-based
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compounds, also include such non-peptide based compounds such as peptide
analogs.
Peptidomimetics that are structurally similar to therapeutically useful
peptides can be used to
produce an equivalent or enhanced therapeutic or prophylactic effect.
Generally,
peptidomimetics are structural or functional mimics (e.g., identical or
similar) to a paradigm
polypeptide (i.e., a polypeptide that has a biological or pharmacological
function or activity),
but can also have one or more peptide linkages optionally replaced by a
linkage selected from
the group consisting of, for example, -CH2NH-, -CH2S-, -CH2-CH2-, - CH=CH-
(cis and
trans), -COCH2-, -CH(OH)CH2-, and -CH2S0-. The mimetic can be either entirely
composed of natural amino acids, synthetic chemical compounds, non-natural
analogues of
amino acids, or, is a chimeric molecule of partly natural peptide amino acids
and partly non-
natural analogs of amino acids. The mimetic can also comprise any amount of
natural amino
acid conservative substitutions as long as such substitutions also do not
substantially alter
mimetic activity. In the case of connexins, these can mimic, for example, the
extracellular
loops of opposing connexins involved in connexon-connexon docking and cell-
cell channel
formation. For example, a mimetic composition can be useful as a gap junction
modulating
agent if it is capable of down-regulating biological actions or activities of
connexons, such as,
for example, preventing the docking of connexons to form gap-junction-mediated
cell-cell
communications, or preventing the opening of connexons to expose the cell
cytoplasm to the
extracellular millieu. Peptidomimetics encompass those described herein, as
well as those as
may be known in the art, whether now known or later developed.
[0072] The
term "wound dressing" or "lesion dressing" refers to a dressing for topical
application to a resistant lesion or wound and excludes compositions suitable
for systemic
administration. For example, the one or more anti-connexin 43, anti-connexin
30 or anti-
connexin 26 agents, including gap junction modulation agents, may be dispersed
in or on a
solid sheet of lesion contacting material such as a woven or nonwoven textile
material, or
may be dispersed in a layer of foam such as polyurethane foam, or in a
hydrogel such as a
polyurethane hydrogel, a polyacrylate hydrogel, gelatin, carboxymethyl
cellulose, pectin,
alginate, and/or hyaluronic acid hydrogel, for example in a gel or ointment.
In certain
embodiments the one or more anti-connexin agents, including gap junction
modulation agents
are dispersed in or on a biodegradable sheet material that provides sustained
release of the
active ingredients into the wound, for example a sheet of freeze-dried
collagen, freeze-dried
collagen/alginate mixtures (available under the Registered Trade Mark
1H1BRACOL from
Johnson & Johnson Medical Limited) or freeze-dried collagen/oxidized
regenerated cellulose
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(available under the Registered Trade Mark PROMOGRAN from Johnson & Johnson
Medical Limited).
[0073] As
used herein, "matrix" includes for example, matrices such as collagen,
acellular matrices, crosslinked biological scaffold molecules, tissue-based
matrices (including
pig-based wound healing matrices), cultured epidermal autografts, cultured
epidermal
allografts, tissue-engineered skin, collagen and glycosaminoglycan dermal
matrices
inoculated with autologous fibroblasts and keratinocytes, Alloderm (a
nonliving allogeneic
acellular dermal matrix with intact basement membrane complex), living skin
equivalents
(e.g., Dermagraft (living allogeneic dermal fibroblasts grown on degradable
scaffold),
TransCyte (an extracellular matrix generated by allogeneic human dermal
fibroblasts),
Apligraf (a living allogeneic bilayered construct containing keratinocytes,
fibroblasts and
bovine type I collagen), and OrCel (allogeneic fibroblasts and keratinocytes
seeded in
opposite sides of bilayered matrix of bovine collagen), animal derived
dressings (e.g., Oasis's
porcine small intestinal submucosa acellular collagen matrix; and E-Z Derm's
acellular
xenogeneic collagen matrix), tissue-based bioengineered structural frameworks,
biomanufactured bioprostheses, and other implanted or applied structures such
as for example,
vascular grafts suitable for cell infiltration and proliferation useful in the
promotion of wound
healing.
Additional suitable biomatrix material may include chemically modified
collagenous tissue to reduce antigenicity and immunogenicity. Other suitable
examples
include collagen sheets for wound dressings, antigen-free or antigen reduced
acellular matrix
(Wilson et al., Trans Am Soc Artif Intern 1990; 36:340-343) or other biomatrix
which have
been engineered to reduce the antigenic response to the xenograft material.
Other matrix
useful in promotion of resistant wound healing may include for example,
processed bovine
pericardium proteins comprising insoluble collagen and elastin (Courtman et
al., J Biomed
Mater Res 1994; 28:655-666) and other acellular tissue which may be useful for
providing a
natural microenvironment for host cell migration to accelerate tissue
regeneration (Malone et
al., J Vasc Surg 1984; 1:181-91). In certain embodiments, the matrix material
may be
supplemented with one or more anti-connexin 43 modulating agents, such as anti-
connexin
43 polynucleotides and/or the one or more anti-connexin 43 peptides or
peptidomimetics for
site specific release of such agents.
[0074] As
used herein, "resistant lesion promoting matrix" includes for example,
synthetic or naturally occurring matrices such as collagen, acellular matrix,
crosslinked
biological scaffold molecules, tissue based bioengineered structural
framework, and other
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structures useful in the promotion of resistant wound healing. Additional
suitable biomatrix
material may include chemically modified collagenous tissue to reduce
antigenicity and
immunogenicity. Other suitable examples include collagen sheets for wound
dressings,
antigen-free or antigen reduced acellular matrix (Wilson G J et al. (1990)
Trans Am Soc Artif
Intern 36:340-343) or other biomatrix which have been engineered to reduce the
antigenic
response to the xenograft material. Other matrices useful in promotion of
wound healing may
include for example, proteins comprising insoluble collagen and elastin
(Courtman DW et al.
(1994) J Biomed Mater Res 28:655-666) and other acellular tissue which may be
useful for
providing a natural microenvironment for host cell migration to accelerate
epilethialization
(Malone J M et al. (1984) J Vase Surg 1:181-91). The invention contemplates a
synthetic or
natural matrix comprising one or more anti-connexin protein agents.
[0075] In one embodiment, the formulations of this invention also
include salts of
connexin polynucleotides, including for example sodium salts, potassium salts
or any other
salt suitable for topical administation.
Connexin Protein Anti-Connexin Agents
[0076] Anti-connexin protein agents, or connexin modulating agents, of
the invention
described herein are capable of modulating or affecting the transport of
molecules into and
out of cells (e.g., blocking or inhibiting or downregulating), and modulating
cellular
communication (e.g., cell to cell). The anti-connexin protein agents include,
for example,
anti-connexin 43, anti-connexin 30 or anti-connexin 26 agents. Thus, certain
anti-connexin
protein agents described herein are capable of blocking or inhibiting the
transport of
molecules into and out of cells. Thus certain anti-connexin agents described
herein modulate
cellular communication (e.g. cell to cell). Certain anti-connexin agents
affect transmission of
molecules between the cell cytoplasm and the periplasmic or extracellular
space. Such agents
are generally targeted to hemichannels (also called connexons), which may be
independently
involved in the exchange of small molecules between the cell cytoplasm and an
extracellular
space or tissue. Thus, a compound provided herein may directly or indirectly
reduce coupling
between cells (via gap junctions) or between a cell and an extracellular space
or tissue (via
hemichannels), and the modulation of transport of molecules from a cell into
an extracellular
space is within the scope of certain compounds and embodiments of the
invention.
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[0077] Any anti-connexin protein agent that is capable of eliciting a
desired inhibition
of the passage (e.g. transport) of molecules through a gap junction or
connexin hemichannel
may be used in embodiments of the invention. Any anti-connexin 43 agents that
modulates
the passage of molecules through a gap junction or connexin hemichannel are
also provided
in particular embodiments (e.g., those that modulate, block or lessen the
passage of molecules
from the cytoplasm of a cell into an extracellular space or adjoining cell
cytoplasm). Such
anti-connexin 43 agents may modulate the passage of molecules through a gap
junction or
connexin hemichannel with or without gap junction uncoupling (blocking the
transport of
molecules through gap junctions). Such compounds include, for example, binding
proteins,
polypeptides, and other organic compounds that can, for example, block the
function or
activity of a gap junction or a hemichannel in whole or in part.
[0078] Certain anti-connexin protein agents, such as anti-connexin 43
agents, provide
downregulation of connexin expression (for example, by downregulation of mRNA
transcription or translation) or otherwise decrease or inhibit the activity of
the connexin
protein, connexin hemichannels or gap junctions. In the case of
downregulation, this will
have the effect of reducing direct cell-cell communication by gap junctions,
or exposure of
cell cytoplasm to the extracellular space by hemichannels, at the site at
which connexin
expression is downregulated.
[0079] As used herein, "anti-connexin protein agent" or "connexin
modulating agent"
may include those agents or compounds that prevent, decrease or modulate, in
whole or in
part, the activity, function, or formation of a hemichannel or a gap junction.
In certain
embodiments, a gap junction modulation agent prevents or decreases, in whole
or in part, the
function of a hemichannel or a gap junction. In certain embodiments, a gap
junction
modulation agent induces closure, in whole or in part, of a hemichannel or a
gap junction. In
other embodiments, a gap junction modulation agent blocks, in whole or in
part, a
hemichannel or a gap junction. In certain embodiments, a gap junction
modulation agent
decreases or prevents, in whole or in part, the opening of a hemichannel or
gap junction. In
certain embodiments, said blocking or closure of a gap junction or hemichannel
by a gap
junction modulation agent can reduce or inhibit extracellular hemichannel
communication by
preventing or decreasing the flow of small molecules through an open channel
to and from an
extracellular or periplamic space. Peptidomimetics, and gap junction
phosphorylation
compounds that block hemichannel and/or gap junction opening are presently
preferred. In
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some embodiments the anti-connexin protein agent may be an anti-connexin 43
agent, an
anti-connexin 30 agent, or an anti-connexin 26 agent.
[0080] In certain embodiments, an anti-connexin agent prevents,
decreases or alters
the activity or function of a hemichannel or a gap junction. As used herein,
modulation of the
gap junction activity or function by the anti-connexin agent may include the
closing of gap
junctions, closing of hemichannels, and/or passage of molecules or ions
through gap junctions
and/or hemichannels.
[0081] Examples of anti-connexin protein agents include agents that
decrease or
inhibit expression or function of connexin protein mRNA and/or protein or that
decrease
activity, expression or formation of connexin protein, connexin hemichannels
gap junctions.
As an examples, an anti-connexin protein agents include anti-connexin 43
agents that
decrease or inhibit expression or function of connexin 43 mRNA and/or protein
or that
decrease activity, expression or formation of connexin 43, connexin
hemichannels gap
junctions. Anti-connexin protein agents include anti-connexin protein
polynucleotides, such
as antisense protein polynucleotides, such as anti-connexin protein
oligonucleotides,
connexin protein oligodeoxynucleotides and other polynucleotides (such as
polynucleotides
having siRNA or ribozyme functionalities), as well as antibodies and binding
fragments
thereof that bind connexin protein, and anti-connexin protein peptides and
polypeptides,
including peptidomimetics and peptide analogs of connexin that modulate
hemichatmel or
gap junction activity or function, and other gap junction blocking agents and
gap junction
protein phosphorylating agents. Anti-connexin protein peptides and
polypeptides may, for
example, bind to connexin protein to inhibit its function, or may inhibit
connexin 43 function
by mimicking regions of connexin protein to inhibit or disrupt its binding to
other gap
junction proteins. The agents may be anti-connexin 43 agents, anti-connexin 30
agents
and/or anti-connexin 26 agents.
Anti-Connexin Protein Polynucleotides
[0082] Anti-connexin polynucleotides include connexin anti s ense
protein
polynucleotides as well as polynucleotides which have functionalities which
enable them to
downregulate connexin protein expression, such as connexin 43 expression.
Other suitable
anti-connexin 43, 30 or 26 polynucleotides include anti-connexin protein
oligonucleotides,
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connexin protein oligodeoxynucleotides, connexin protein RNAi polynucleotides
and
connexin protein siRNA polynucleotides.
[0083] Synthesis of antisense polynucleotides and other anti-connexin 43
polynucleotides such as RNAi, siRNA, and ribozyme polynucleotides as well as
polynucleotides having modified and mixed backbones can be performed by any
suitable
method. See e.g. Stein C.A. and Krieg A.M. (eds), Applied Antisense
Oligonucleotide
Technology, 1998 (Wiley-Liss). Methods of synthesizing antibodies and binding
fragments
as well as peptides and polypeptides, including peptidomimetics and peptide
analogs can also
be performed using suitable methods. See e.g. Lihu Yang et al., Proc. Natl.
Acad. Sci. U.S.A.,
1; 95(18): 10836-10841 (Sept 1 1998); Harlow and Lane (1988) "Antibodies: A
Laboratory
Manuel" Cold Spring Harbor Publications, New York; Harlow and Lane (1999)
"Using
Antibodies" A Laboratory Manuel, Cold Spring Harbor Publications, New York.
[0084] According to one aspect, the downregulation of connexin expression
may be
based generally upon the antisense approach using antisense polynucleotides
(such as DNA
or RNA polynucleotides), and more particularly upon the use of antisense
oligodeoxynucleotides (ODN). These polynucleotides (e.g., ODN) may target the
connexin
43 protein. Typically the polynucleotides are single stranded, but may be
double stranded.
[0085] The antisense polynucleotide may inhibit transcription and/or
translation of a
connexin protein, such as connexin 43, 30 or 26. Preferably the polynucleotide
is a specific
inhibitor of transcription and/or translation from the connexin 43, 30 or 26
gene or mRNA,
and does not inhibit transcription and/or translation from other genes or
mRNAs. Screening
of the polynucleotide sequence in a human genome sequence database for
specificity may
also be performed. The product may bind to the connexin 43, 30 or 26 gene or
mRNA either
(i) 5' to the coding sequence, and/or (ii) to the coding sequence, and/or
(iii) 3' to the coding
sequence.
[0086] The antisense polynucleotide is generally antisense to connexin
protein mRNA,
for example, connexin 43, 30 or 26 mRNA. Such a polynucleotide may be capable
of
hybridizing to connexin protein mRNA and may thus inhibit the expression of
connexin by
interfering with one or more aspects of connexin protein mRNA metabolism
including
transcription, mRNA processing, mRNA transport from the nucleus, translation
or mRNA
degradation. The antisense polynucleotide typically hybridizes to the connexin
protein
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mRNA to form a duplex which can cause direct inhibition of translation and/or
destabilization of the mRNA. Such a duplex may be susceptible to degradation
by nucleases.
[0087] The antisense polynucleotide may hybridize to part of the connexin
protein
mRNA, such as connexin 46, 30 or 26 mRNA. Typically the antisense
polynucleotide
hybridizes to the ribosome binding region or the coding region of the connexin
protein
mRNA. The polynucleotide may be complementary to a region of the connexin
mRNA. For
example, the polynucleotide may be the exact complement of a part of connexin
mRNA.
However, absolute complementarity is not required and polynucleotides which
have
sufficient complementarity to form a duplex having a melting temperature of
greater than
about 20 C, 30 C or 40 C under physiological conditions are particularly
suitable for use in
the present invention.
[0088] Thus the polynucleotide is typically a homologue of a sequence
complementary to the mRNA. The polynucleotide may be a polynucleotide which
hybridizes
to the connexin protein mRNA under conditions of medium to high stringency
such as 0.03M
sodium chloride and 0.03M sodium citrate at from about 50 C to about 60 C.
[0089] For certain aspects, the polynucleotides of this invention include
synthesized
polynucleotides having a length of less than 80 nucleotides, e.g., from 15-18
to about 50-80
nucleotides, preferably about 30 nucleotides or less, e.g., from 15 to about
30 nucleotides, and
more preferably from about 15 to about 20 nucleotides. In one example, the
polynucleotide
has 30 nucleotides.
[0090] Alternatively, the antisense polynucleotides may be part of
compositions
which may comprise polynucleotides to more than one connexin protein.
Preferably, the
connexin protein to which polynucleotides are directed is connexin 43. Other
connexin
proteins to which oligodeoxynucleotides are directed may include, for example,
connexins 26,
30, 30.3, 31.1, 32, 36, 37, 40, 40.1, 45, and 46.6. Suitable exemplary
polynucleotides (and
ODNs) directed to various connexins are set forth in Table 1.
[0091] The polynucleotides for use in the invention may suitably be
unmodified
phosphodiester oligomers. Such oligodeoxynucleotides may vary in length. A 30
mer
polynucleotide has been found to be particularly suitable.
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[0092] Many aspects of the invention are described with reference to
oligodeoxynucleotides. However it is understood that other suitable
polynucleotides (such as
RNA polynucleotides) may be used in these aspects.
[0093] The
antisense polynucleotides may be chemically modified. This may
enhance their resistance to nucleases and may enhance their ability to enter
cells. For
example, phosphorothioate oligonucleotides may be used. Other deoxynucleotide
analogs
include methylphosphonates, phosphoramidates, phosphorodithioates, N3'P5'-
phosphoramidates and oligoribonucleotide phosphorothioates and their 2'-0-
alkyl analogs
and 2'-0-methylribonucleotide methylphosphonates.
Alternatively mixed backbone
oligonucleotides ("MBOs") may be used. MBOs contain segments of phosphothioate
oligodeoxynucleotides and appropriately placed segments of modified oligodeoxy-
or
oligoribonucleotides. MBOs have segments of phosphorothioate linkages and
other segments
of other modified oligonucleotides, such as methylphosphonate, which is non-
ionic, and very
resistant to nucleases or 2' -0-alkyloligoribonucleotides. Methods of
preparing modified
backbone and mixed backbone oligonucleotides are known in the art.
[0094] The
precise sequence of the antisense polynucleotide used in the invention will
depend upon the target connexin protein. In one embodiment, suitable connexin
43 antisense
polynucleotides can include polynucleotides such as oligodeoxynucleotides
selected from
SEQ ID NO:1-3 set forth in Table 1: Suitable polynucleotides for the
preparation of the
combined polynucleotide compositions described herein, for combination with
the connexin
43 modulating agent include polynucleotides for connexins 26, 30, 31.1, 32 and
37 are also
described in Table 1.
TABLE 1
5' GTA ATT GCG GCA AGA AGA ATT GTT TCT (connexin 43) (SEQ.ID.N0:1)
GTC 3'
5' GTA ATT GCG GCA GGA GGA ATT GTT TCT (connexin 43) (SEQ.ID.N0:2)
GTC 3'
5' GGC AAG AGA CAC CAA AGA CAC TAC CAG (connexin 43) (SEQ.ID.N0:3)
CAT 3'
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5' TCC TGA GCA ATA CCT AAC GAA CAA ATA 3' (connexin 26) (SEQ.lD.N0:21)
5' CTC AGA TAG TGG CCA GAA TGC 3'
(connexin 30) (SEQ.lD.N0:22)
5' TTG TCC AGG TGA CTC CAA GG 3'
(connexin 30) (SEQ.ID.N0:23
[0095] Although the precise sequence of the antisense polynucleotide
used in the
invention will depend upon the target connexin protein, for connexin 43,
antisense
polynucleotides having any of SEQ.ID.N0:1-2, SEQ.ID.N0.21 or SEQ.ID.N0.22-23
have
been found to be particularly suitable:
[0096] Polynucleotides, including ODN' s, directed to connexin proteins
can be
selected in terms of their nucleotide sequence by any convenient, and
conventional, approach.
For example, the computer programs MacVector and OligoTech (from Oligos etc.
Eugene,
Oregon, USA) can be used. Once selected, the ODN's can be synthesized using a
DNA
synthesizer.
Polynucleotide Homologues
[0097] Homology and homologues are discussed herein (for example, the
polynucleotide may be a homologue of a complement to a sequence in connexin
mRNA).
Such a polynucleotide typically has at least about 70% homology, preferably at
least about
80%, at least about 90%, at least about 95%, at least about 97% or at least
about 99%
homology with the relevant sequence, for example over a region of at least
about 15, at least
about 20, at least about 40, at least about 100 more contiguous nucleotides
(of the
homologous sequence).
[0098] Homology may be calculated based on any method in the art. For
example the
UWGCG Package provides the BESTFIT program which can be used to calculate
homology
(for example used on its default settings) (Devereux et al (1984) Nucleic
Acids Research 12,
p387-395). The PILEUP and BLAST algorithms can be used to calculate homology
or line
up sequences (typically on their default settings), for example as described
in Altschul S. F.
(1993) J Mol Evol 36: 290-300; Altschul, S, F et al (1990) J Mol Biol 215: 403-
10.
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[0099]
Software for performing BLAST analyses is publicly available through the
National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
This
algorithm involves first identifying high scoring sequence pair (HSPs) by
identifying short
words of length W in the query sequence that either match or satisfy some
positive-valued
threshold score T when aligned with a word of the same length in a database
sequence. T is
referred to as the neighbourhood word score threshold (Altschul et al, supra).
These initial
neighbourhood word hits act as seeds for initiating searches to find HSPs
containing them.
The word hits are extended in both directions along each sequence for as far
as the
cumulative alignment score can be increased. Extensions for the word hits in
each direction
are halted when: the cumulative alignment score falls off by the quantity X
from its
maximum achieved value; the cumulative score goes to zero or below, due to the
accumulation of one or more negative-scoring residue alignments; or the end of
either
sequence is reached.
[0100] The
BLAST algorithm parameters W, T and X determine the sensitivity and
speed of the alignment. The BLAST program uses as defaults a word length (W),
the
BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad.
Sci. USA
89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a
comparison
of both strands.
[0101] The
BLAST algorithm performs a statistical analysis of the similarity between
two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA
90: 5873-
5787. One measure of similarity provided by the BLAST algorithm is the
smallest sum
probability (P(N)), which provides an indication of the probability by which a
match between
two nucleotide or amino acid sequences would occur by chance. For example, a
sequence is
considered similar to another sequence if the smallest sum probability in
comparison of the
first sequence to a second sequence is less than about 1, preferably less than
about 0.1, more
preferably less than about 0.01, and most preferably less than about 0.001.
[0102] The
homologous sequence typically differs from the relevant sequence by at
least about (or by no more than about) 2, 5, 10, 15, 20 more mutations (which
may be
substitutions, deletions or insertions). These mutations may be measured
across any of the
regions mentioned above in relation to calculating homology.
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[0103] The homologous sequence typically hybridizes selectively to the
original
sequence at a level significantly above background. Selective hybridization is
typically
achieved using conditions of medium to high stringency (for example 0.03M
sodium chloride
and 0.03M sodium citrate at from about 50 C to about 60 C). However, such
hybridization
may be carried out under any suitable conditions (see Sambrook et al. (1989),
Molecular
Cloning: A Laboratory Manual). For example, if high stringency is required,
suitable
conditions include 0.2 x SSC at 60 C. If lower stringency is required,
suitable conditions
include 2 x SSC at 60 C.
[0104] In one embodiment, the connexin 43 polynucleotides for use in the
pharmaceutical formulations of this invention are screened against other human
genome
sequences to assess or determine specificity.
[0105] The invention also includes in one aspect pharmaceutical
compositions with
instructions for treating responder subjects having mVLUs. In one embodiment
the anti-
connexin 43 modulating agent is a polynucleotide. In some embodiments the anti-
connexin
43 modulating agent is an oligonucleotide. The oligonucleotide may be an anti-
connexin 43
antisense oligodeoxynucleotide. In one aspect, the polynucleotides of this
invention may be
modified or unmodified.
[0106] The invention also includes a package or kit comprising a
pharmaceutical
composition comprising a pharmaceutically acceptable carrier and a
pharmaceutically
acceptable anti-connexin modulating agent, together with a label and/or
instructions for
administering the composition to one or more wounds on subjects with resistant
lesions such
as mVLUs, where the subject is susceptible to treatment with an anti-connexin
modulating
agent, and the agent is administered in amounts effective to promote healing
of the lesions in
a subject, alone or together with compression bandaging. In one embodiment,
the invention
includes a package or kit comprising a pharmaceutical composition including a
pharmaceutically acceptable carrier and a pharmaceutically acceptable anti-
connexin 43
modulating agent, such as an anti-connexin 43 oligonucleotide, optionally with
a label and/or
instructions for administering the composition to responder subjects with
mVLUs in amounts
effective to promote mVLU healing in a subject, alone or under compression
bandaging.
[0107] In one aspect the pharmaceutical formulations of this invention
comprise an
unmodified oligonucleotide specific to connexin 43 mRNA having the sequence 5'-
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GTAATTGCGGCAAGAAGAATTGTTTCTGTC-3' (SEQ ID NO:1). In one aspect the
olignonucleotide may be a deoxyoligonucleotide. In another aspect the
oligonucleotide is
chemically modified to increase half-life.
[0108] In some aspects the formulations of this invention may be
formulated as a
sterile, non-preserved, buffered gel at physiological pH of between pH 6.0 and
8.0, for
example, pH 7.4, containing a deoxyoligonucletoide having, for example, SEQ ID
NO: 1.
The formulation may also contain other to maintain physiological salt
concentrations, such as
potassium phosphate, sodium phosphate and water-for-injection.
Peptide and Polypeptide Anti-Connexin Agents
[0109] Connexin 43, connexin 30 or connexin 26 binding proteins,
including peptides,
peptidomimetics, antibodies, antibody fragments, and the like, are also
suitable modulators of
gap junctions and hemichannels.
[0110] Anti-connexin protein binding proteins include, for example,
monoclonal
antibodies, polyclonal antibodies, antibody fragments (including, for example,
Fab, F(ab')2
and Fv fragments; single chain antibodies; single chain Fvs; and single chain
binding
molecules such as those comprising, for example, a binding domain, hinge, CH2
and CH3
domains, recombinant antibodies and antibody fragments which are capable of
binding an
antigenic determinant (i.e., that portion of a molecule, generally referred to
as an epitope) that
makes contact with a particular antibody or other binding molecule. These
binding proteins,
including antibodies, antibody fragments, and so on, may be chimeric or
humanized or
otherwise made to be less immunogenic in the subject to whom they are to be
administered,
and may be synthesized, produced recombinantly, or produced in expression
libraries. Any
binding molecule known in the art or later discovered is envisioned, such as
those referenced
herein and/or described in greater detail in the art. For example, binding
proteins include not
only antibodies, and the like, but also ligands, receptors, peptidomimetics,
or other binding
fragments or molecules (for example, produced by phage display) that bind to a
target (e.g.
connexin, hemichannel, or associated molecules).
[0111] Binding molecules will generally have a desired specificity,
including but not
limited to binding specificity, and desired affinity. Affinity, for example,
may be a Ka of
greater than or equal to about 104 M-1, greater than or equal to about 106 M-
1, greater than or
equal to about 107 M-1, greater than or equal to about 108 M-1. Affinities of
even greater
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than about 108 M-1 are suitable, such as affinities equal to or greater than
about 109 M-1,
about 1010 M-1, about 1011 M-1, and about 1012 M-1. Affinities of binding
proteins according
to the present invention can be readily determined using conventional
techniques, for
example those described by Scatchard et al., (1949) Ann. N.Y. Acad. Sci. 51:
660.
[0112] Exemplary gap junction modulation agents may include, without
limitation,
polypeptides (e.g. peptiditomimetics, antibodies, binding fragments thereof,
and synthetic
constructs), and other gap junction blocking agents, and gap junction protein
phosphorylating
agents. Exemplary compounds used for closing gap junctions (e.g.
phosphorylating connexin
43 tyrosine residue) have been reported in U.S. Pat. No. 7,153,822 to Jensen
et al., U.S. Pat.
No. 7,250,397, and assorted patent publications. Exemplary peptides and
peptidomimetics
are reported in Green et al., W02006134494. See also Gourdie et al., see
W02006069181,
and Tudor et al., see W02003032964.
[0113] By using data obtained from hydropathy plots, it has been proposed
that a
connexin contains four-transmembrane-spanning regions and two short extra-
cellular loops.
The positioning of the first and second extracellular regions of connexin was
further
characterized by the reported production of anti-peptide antibodies used for
immunolocalization of the corresponding epitopes on split gap junctions.
Goodenough D.A.
J Cell Biol 107: 1817-1824 (1988); Meyer R.A., J Cell Biol 119: 179-189
(1992).
[0114] The extracellular domains of a hemichannel contributed by two
adjacent cells
"dock" with each other to form complete gap junction channels. Reagents that
interfere with
the interactions of these extracellular domains can impair cell-to-cell
communication.
Peptide inhibitors of gap junctions and hemichannels have been reported. See
for example
Berthoud, V.M. et al., Am J. Physiol. Lung Cell Mol. Physiol. 279: L619 ¨ L622
(2000);
Evans, W.H. and Boitano, S. Biochem. Soc. Trans. 29: 606 ¨ 612, and De Vriese
A.S., et al.
Kidney Int. 61: 177 ¨ 185 (2001). Short peptides corresponding to sequences
within the
extracellular loops of connexins were said to inhibit intercellular
communication. Boitano S.
and Evans W. Am J Physiol Lung Cell Mol Physiol 279: L623-L630 (2000). The use
of
peptides as inhibitors of cell-cell channel formation produced by connexin
(Cx) 32 expressed
in paired Xenopus oocytes has also been reported. Dahl G, et al., Biophys J
67: 1816-1822
(1994). Berthoud, V.M. and Seul, K.H., summarized some of these results. Am
J., Physiol.
Lung Cell Mol. Physiol. 279: L619 ¨ L622 (2000).
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[0115] Anti-connexin agents include peptides comprising an amino acid
sequence
corresponding to a transmembrane region (e.g. 1st to 4th) of a connexin (e.g.
43, 26, 30).
Anti-connexin agents may comprise a peptide comprising an amino acid sequence
corresponding to a portion of a transmembrane region of a connexin 43.
[0116] Anti-connexin agents include peptides having an amino acid
sequence that
comprises about 5 to 20 contiguous amino acids of a connexin protein such as
connexin 43
(SEQ.ID.N0:4), connexin 26 or connexin 30, peptides having an amino acid
sequence that
comprises about 8 to 15 contiguous amino acids of connexin 43 (SEQ.ID.N0:4),
connexin 26
or connexin 30, or peptides having an amino acid sequence that comprises about
11 to 13
contiguous amino acids of connexin 43 (SEQ.ID.N0:4), connexin 26 or connexin
30,. Other
anti-connexin agents include a peptide having an amino acid sequence that
comprises at least
about 5, at least about 6, at least about 7, at least about 8, at least about
9, at least about 10, at
least about 11, at least about 12, at least about 13, at least about 14, at
least about 15, at least
about 20, at least about 25, or at least about 30 contiguous amino acids of
connexin 43
(SEQ.ID.N0:4), connexin 26 or connexin 30,. Other anti-connexin agents
comprise the
extracellular domains of connexin 43. 30 or 26, for example, corresponding to
the amino
acids at positions 37-76 and 178-208 of SEQ.ID.N0:4. Anti-connexin agents
include
peptides described herein, for example, agents having an amino acid sequence
corresponding
to the regions at positions 37-76 and 178-208 of SEQ.ID.N0:4. The peptides
need not have
an amino acid sequence identical to those portions of SEQ.ID.N0:4, and
conservative amino
acid changes may be made such that the peptides retain binding activity or
functional activity.
Alternatively, peptides may target regions of the connexin protein other than
the extracellular
domains (e.g. the portions of SEQ.ID.N0:4 not corresponding to positions 37-76
and 178-
208). In one embodiment, the anti-connexin peptides have an amino acid
sequence that
comprises SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. Still other anti-connexin
agents
include connexin carboxy-terminal polypeptides.
[0117] In functional tests using (i) blockage of dye (Lucifer Yellow)
uptake by cells
in spinal cord slices, and (ii) prevention of oedema in spinal cord segments
(using connexin
43 specific antisense as a positive control), connexin 43 peptides comprising
SEQ ID NO:10,
having sequences SEQ ID NO:8 and SEQ ID NO:9 (synthesised by Sigma-Genosys
(Australia)), were shown to prevent and/or block and/or close the opening of
the
hemichannels by inhibiting dye uptake. In contrast, the level of dye uptake
for slices treated
with the peptides having SEQ ID NOS:5-7 ((FEVAFLLIQWI (SEQ ID NO:5),
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LLIQWYIGFSL(SEQ ID NO:6), SLSAVYTCKRDPCPHQ (SEQ ID NO:7)) and SEQ ID
NOS:11-14 (LGTAVESAWGDEQ (SEQ ID NO:11), QSAFRCNTQQPG (SEQ ID NO:12),
QQPGCENVCYDK (SEQ ID NO:13), and VCYDKSFPISHVR (SEQ ID NO:14)) was
comparable with control slices.
[0118] The connexin 43 peptide having SEQ ID NO:9 (which comprises SEQ ID
NO:10), has also been shown to block swelling of cultured spinal cord segments
compared to
a peptide which does not block dye uptake (e. g., a peptide having SEQ ID
NO:13, which was
used as a negative control). The lowest concentration of peptide (5
micromolar) used in those
studies gave the best result (least oedema) when compared to media alone (p=
0.001). The
middle range 50 micromolar was somewhat less effective than the 5 micromolar
concentration in repeat experiments.
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[0119] Connexin 43 (SEQ ID NO. 4)
Connexin 43 (SEO ID NO. 4)
Met Gly Asp Trp Ser Ala Leu Gly Lys Leu Leu Asp Lys Val Gln Ala
1 5 10 15
Tyr Ser Thr Ala Gly Gly Lys Val Trp Leu Ser Val Leu Phe Re Phe
20 25 30
Arg Ile Leu Leu Leu Gly Thr Ala Val Glu Ser Ala Trp Gly Asp Glu
35 40 45
Gln Ser Ala Phe Arg Cys Asn Thr Gln Gln Pro Gly Cys Glu Asn Val
50 55 60
Cys Tyr Asp Lys Ser Phe Pro Ile Ser His Val Arg Phe Trp Val Leu
65 70 75 80
Gln Ile Ile Phe Val Ser Val Pro Thr Leu Leu Tyr Leu Ala His Val
85 90 95
Phe Tyr Val Met Arg Lys Glu Glu Lys Leu Asn Lys Lys Glu Glu Glu
100 105 110
Leu Lys Val Ala Gln Thr Asp Gly Val Asn Val Asp Met His Leu Lys
115 120 125
Gln lle Glu Ile Lys Lys Phe Lys Tyr Gly Ile Glu Glu His Gly Lys
130 135 140
Val Lys Met Arg Gly Gly Leu Leu Arg Thr Tyr Re Re Ser Ile Leu
145 150 155 160
Phe Lys Ser Ile Phe Glu Val Ala Phe Leu Leu Ile Gln Trp Tyr Ile
165 170 175 -
Tyr Gly Phe Ser Leu Ser Ala Val Tyr Thr Cys Lys Arg Asp Pro Cys
180 185 190
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Pro His Gln Val Asp Cys Phe Leu Ser Arg Pro Thr Glu Lys Thr Ile
195 200 205
Phe lle Ile Phe Met Leu Val Val Ser Leu Val Ser Leu Ala Leu Asn
210 215 220
Ile Ile Glu Leu Phe Tyr Val Phe Phe Lys Gly Val Lys Asp Arg Val
225 230 235 240
Lys Gly Lys Ser Asp Pro Tyr His Ala Thr Ser Gly Ala Leu Ser Pro
245 250 255
Ala Lys Asp Cys Gly Ser Gln Lys Tyr Ala Tyr Phe Asn Gly Cys Ser
260 265 270
Ser Pro Thr Ala Pro Leu Ser Pro Met Ser Pro Pro Gly Tyr Lys Leu
275 280 285
Val Thr Gly Asp Arg Asn Asn Ser Ser Cys Arg Asn Tyr Asn Lys Gln
290 295 300
Ala Ser Glu Gln Asn Trp Ala Asn Tyr Ser Ala Glu Gln Asn Arg Met
305 310 315 320
Gly Gln Ala Gly Ser Thr Ile Ser Asn Ser His Ala Gln Pro Phe Asp
325 330 335
Phe Pro Asp Asp Asn Gln Asn Ser Lys Lys Leu Ala Ala Gly His Glu
340 345 350
Leu Gln Pro Leu Ala Ile Val Asp Gln Arg Pro Ser Ser Arg Ala Ser
355 360 365
Ser Arg Ala Ser Ser Arg Pro Arg Pro Asp Asp Leu Glu Ile
370 375 380
[0120] The anti-cormexin peptides, for example, anti-cormexin 43, 30, or
26 peptides
may comprise sequences corresponding to a portion of the connexin
extracellular domains
with conservative amino acid substitutions such that peptides are functionally
active anti-
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connexin agents. Exemplary conservative amino acid substitutions include for
example the
substitution of a nonpolar amino acid with another nonpolar amino acid, the
substitution of an
aromatic amino acid with another aromatic amino acid, the substitution of an
aliphatic amino
acid with another aliphatic amino acid, the substitution of a polar amino acid
with another
polar amino acid, the substitution of an acidic amino acid with another acidic
amino acid, the
substitution of a basic amino acid with another basic amino acid, and the
substitution of an
ionizable amino acid with another ionizable amino acid.
[0121] Exemplary peptides targeted to connexin 43 are shown below in
Table 2. M1,
2, 3 and 4 refer to the 1st to 4th transmembrane regions of the connexin 43
protein
respectively. El and E2 refer to the first and second extracellular loops
respectively.
Table 2. Peptidic Inhibitors of Intercellular Communication (Cx43)
1-1,VAFLLIQW1 M3 & E2 (SEQ.ID.N0:5)
LLIQWYIGFSL E2 (SEQ.ID .NO: 6)
SLSAVYTCKRDPCPHQ E2 (SEQ.ID .NO:7)
VDCFLSRPTEKT E2 (SEQ.ID .NO: 8)
SRPTEKTIFII E2 & M4 (SEQ.ID.N0:9)
SRPTEKT E2 (SEQ.M.NO: 10)
LGTAVESAWGDEQ M1 & El (SEQ.ID.NO: 11)
QSA1-RCNTQQPG El (SEQ.ID.N0:12)
QQPGCENVCYDK El (SEQ.ID.N0:13)
VCYDKSFPISHVR El (SEQ.ID.N0:14)
KRDPCHQVDCFLSRPTEK E2 (SEQ.ID.NO: 15)
[0122] Table 3 provides the extracellular loops for connexin family
members which
are used to develop peptide inhibitors for use as described herein. The
peptides and provided
in Table 4, and fragments thereof, are used as peptide inhibitors in certain
non-limiting
embodiments. In other non-limiting embodiments, peptides comprising from about
8 to about
15, or from about 11 to about 13 amino contiguous amino acids of the peptides
in this Table 4
are peptide inhibitors. Conservative amino acid changes may be made to the
peptides or
fragments thereof.
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Table 3. Extracellular loops for connexin proteins
El
huCx26 KEVWGDEQADFVCNTLQPGCKNVCYDHYFPISHIR (SEQ.ID.NO: 24)
huCx30 QEVWGDEQEDFVCNTLQPGCKNVCYDHFFPVSHIR (SEQ.ID.NO: 25)
huCx43 ESAWGDEQSAFRCNTQQPGCENVCYDKSFPISHVR (SEQ. ID .NO: 16)
E2 '
huCx26 MYVFYVMYDGFSMQRLVKCNAWPCPNTVDCFVSRPTEKT (SEQ. ID .NO:
26)
huCx30 MYVFYFLYNGYHLPWVLKCGIDPCPNLVDCFISRPTEKT (SEQ.ID.NO:
27)
huCx43 LLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKT (SEQ.ID.NO:
17)
[0123] Table 4 provides the extracellular domain for connexin family
members which
may be used to develop peptide anti-connexin agents. The peptides and provided
in Table 5,
and fragments thereof, may also be used as peptide anti-connexin agents. Such
peptides may
comprise from about 8 to about 15, or from about 11 to about 13 amino
contiguous amino
acids of the peptide sequence in this Table 5. Conservative amino acid changes
may be made
to the peptides or fragments thereof.
Table 4. Extracellular domains
Peptide VDCFLSRPTEKT (SEQ.ID.NO: 8)
Peptide SRPTEKTIFII(SEQ.ID.NO: 9)
Peptide SRP1EKT (SEQ.ID.NO: 10)
huCx43 LLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTIFIE
(SEQ.ID.NO: 18)
huCx26 MYVFYVMYDGFSMQRLVKCNAWPCPNTVDCFVSRPTEKTVFTV
(SEQ.ID.NO: 28)
huCx30 YVFYFLYNGYHLPWVLKCGIDPCPNLVDCFISRPTEKTVFTI
(SEQ.ID.NO: 29)
[0124] In certain embodiments, it is preferred that certain peptide
inhibitors block
hemichannels without disrupting existing gap junctions. While not wishing to
be bound to
any particular theory or mechanism, it is also believed that certain
peptidomimetics (e.g. the
connexin 43 peptide inhibitor, VCYDKSFPISHVR, (SEQ.ID.NO: 14) block
hemichannels
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without causing uncoupling of gap junctions (See Leybeart et al., Cell Commun.
Adhes. 10:
251-257 (2003)), or do so in lower dose amounts.
[0125] A peptide comprising SRPTEKT
(SEQ.ID.NO: 10), for example
VDCFLSRPTEKT(SEQ.ID.NO: 8) or SRPTEKTIFII (SEQ.ID.NO: 9), may also be used,
for
example to block hemichannels without uncoupling of gap junctions. The peptide
SRGGEKNVFIV (SEQ.ID.NO: 19) may be used that as a control sequence (DeVriese
et al.,
Kidney Internat. 61: 177-185 (2002)). The peptides may be 3 or more amino
acids in length.
[0126]
Peptides or variants thereof, can be synthesized in vitro, e.g., by the solid
phase peptide synthetic method or by enzyme-catalyzed peptide synthesis or
with the aid of
recombinant DNA technology. Solid phase peptide synthetic method is an
established and
widely used method, which is described in references such as the following:
Stewart et al.,
(1969) Solid Phase Peptide Synthesis, W. H. Freeman Co., San Francisco;
Merrifield, (1963)
J. Am. Chem. Soc. 85 2149; Meienhofer in "Hormonal Proteins and Peptides,"
ed.; C.H. Li,
Vol.2 (Academic Press, 1973), pp.48-267; and Bavaay and Merrifield, "The
Peptides," eds. E.
Gross and F. Meienhofer, Vol.2 (Academic Press, 1980) pp.3-285. These peptides
can be
further purified by fractionation on immunoaffinity or ion-exchange columns;
ethanol
precipitation; reverse phase HPLC; chromatography on silica or on an anion-
exchange resin
such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel
filtration using, for example, Sephadex G-75; ligand affinity chromatography;
or
crystallization or precipitation from non-polar solvent or nonpolar/polar
solvent mixtures.
Purification by crystallization or precipitation is preferred.
[0127] Table
5A shows the human connexin 43 cDNA sequence. The coding portion
of the sequence is located at nucleotides 251-1399.
Table 5A
Human Connexin 43 from GenBank Accession No. NM_000165 (SEQ.ID.N0:20)
1 gagtcagtgg cttgaaactt ttaaaagctc tgtgctccaa gttacaaaaa agctatacg
61 aggtatcagc acttacttt cattaggggg aaggcgtgag gaaagtacca aacagcagcg
121 gagttttaaa ctttaaatag acaggtctga gtgcctgaac ttgccttttc attttacttc
181 atcctccaag gagttcaatc acttggcgtg acttcactac ttttaagcaa aagagtggtg
241 cccaggcaac atgggtgact ggagcgcctt aggcaaactc cttgacaagg ttcaagccta
301 ctcaactgct ggagggaagg tgtggctgtc agtacttttc attttccgaa tcctgctgct
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361 ggggacagcg gttgagtcag cctggggaga tgagcagtct gcctttcgtt gtaacactca
421 gcaacctggt tgtgaaaatg tctgctatga caagtctttc ccaatctctc atgtgcgctt
48 1 ctgggtcctg cagatcatat ttgtgtctgt acccacactc ttgtacctgg ctcatgtgtt
541 ctatgtgatg cgaaaggaag agaaactgaa caagaaagag gaagaactca aggttgccca
601 aactgatggt gtcaatgtgg acatgcactt gaagcagatt gagataaaga agttcaagta
661 cggtattgaa gagcatggta aggtgaaaat gcgagggggg ttgctgcgaa cctacatcat
721 cagtatcctc ttcaagtcta tctttgaggt ggccttcttg ctgatccagt ggtacatcta
781 tggattcagc ttgagtgctg tttacacttg caaaagagat ccctgcccac atcaggtgga
841 ctgtttcctc tctcgcccca cggagaaaac catcttcatc atcttcatgc tggtggtgtc
901 cttggtgtcc ctggccttga atatcattga actcttctat gttttcttca agggcgttaa
961 ggatcgggtt aagggaaaga gcgaccctta ccatgcgacc agtggtgcgc tgagccctgc
1021 caaagactgt gggtctcaaa aatatgctta tttcaatggc tgctcctcac caaccgctcc
1081 cctctcgcct atgtctcctc ctgggtacaa gctggttact ggcgacagaa acaattcttc
1141 ttgccgcaat tacaacaagc aagcaagtga gcaaaactgg gctaattaca gtgcagaaca
1201 aaatcgaatg gggcaggcgg gaagcaccat ctctaactcc catgcacagc cttttgattt
1261 ccccgatgat aaccagaatt ctaaaaaact agctgctgga catgaattac agccactagc
1321 cattgtggac cagcgacctt caagcagagc cagcagtcgt gccagcagca gacctcggcc
1381 tgatgacctg gagatctaga tacaggcttg aaagcatcaa gattccactc aattgtggag
1441 aagaaaaaag gtgctgtaga aagtgcacca ggtgttaatt ttgatccggt ggaggtggta
1501 ctcaacagcc ttattcatga ggcttagaaa acacaaagac attagaatac ctaggttcac
1561 tgggggtgta tggggtagat gggtggagag ggaggggata agagaggtgc atgttggtat
1621 ttaaagtagt ggattcaaag aacttagatt ataaataaga gttccattag gtgatacata
1681 gataagggct ttttctcccc gcaaacaccc ctaagaatgg ttctgtgtat gtgaatgagc
1741 gggtggtaat tgtggctaaa tatttttgtt ttaccaagaa actgaaataa ttctggccag
1801 gaataaatac ttcctgaaca tcttaggtct tttcaacaag aaaaagacag aggattgtcc
1861 ttaagtccct gctaaaacat tccattgtta aaatttgcac tttgaaggta agctttctag
1921 gcctgaccct ccaggtgtca atggacttgt gctactatat ttttttattc ttggtatcag
1981 tttaaaattc agacaaggcc cacagaataa gattaccat gcatttgcaa atacgtatat
2041 tctttttcca tccacttgca caatatcatt accatcactt tttcatcatt cctcagctac
2101 tactcacatt catttaatgg tttctgtaaa catttttaag acagttggga tgtcacttaa
2161 catttttttt ttgagctaaa gtcagggaat caagccatgc ttaatattta acaatcactt
2221 atatgtgtgt cgaagagttt gttttgtttg tcatgtattg gtacaagcag atacagtata
2281 aactcacaaa cacagatttg aaaataatgc acatatggtg ttcaaatttg aacctttctc
2341 atggattttt gtggtgtggg ccaatatggt gtttacatta tataattcct gctgtggcaa
2401 gtaaagcaca cttttttttt ctcctaaaat gtttttccct gtgtatccta ttatggatac
2461 tggttttgtt aattatgatt ctttattttc tctccttttt ttaggatata gcagtaatgc
2521 tattactgaa atgaatttcc tttttctgaa atgtaatcat tgatgcttga atgatagaat
2581 tttagtactg taaacaggct ttagtcatta atgtgagaga cttagaaaaa atgcttagaQ
2641 tggactatta aatgtgccta aatgaatttt gcagtaactg gtattcttgg gttttcctac
2701 ttaatacaca gtaattcaga acttgtattc tattatgagt ttagcagtct tttggagtga
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2761 ccagcaactt tgatgtttgc actaagattt tatttggaat gcaagagagg ttgaaagagg
2821 attcagtagt acacatacaa ctaatttatt tgaactatat gttgaagaca tctaccagtt
2881 tctccaaatg ccttttttaa aactcatcac agaagattgg tgaaaatgct gagtatgaca
2941 cttttcttct tgcatgcatg tcagctacat aaacagtttt gtacaatgaa aattactaat
3001 ttgtttgaca ttccatgtta aactacggtc atgttcagct tcattgcatg taatgtagac
3061 ctagtccatc agatcatgtg ttctggagag tgttctttat tcaataaagt tttaatttag
3121 tataaacata
[0128] The Cx 26 cDNA coding reference sequence NG_008358.1 (SEQ ID NO.30)
is shown below in Table 5B. An anti-connexin 26 polynucleotide may have the
sequence of
any polynucleotide sequence having 12 to 80 nucleotides of SEQ ID NO:30 (or
any number
of nucleotides between 12 and 80).
Table 5B
1 atggattggg gcacgctgca gacgatcctg gggggtgtga acaaacactc caccagcatt
61 ggaaagatct ggctcaccgt cctcttcatt tttcgcatta tgatcctcgt tgtggctgca
121 aaggaggtgt ggggagatga gcaggccgac tttgtctgca acaccctgca gccaggctgc
181 aagaacgtgt gctacgatca ctacttcccc atctcccaca tccggctatg ggccctgcag
241 ctgatcttcg tgtccacgcc agcgctccta gtggccatgc acgtggccta ccggagacat
301 gagaagaaga ggaagttcat caagggggag ataaagagtg aatttaagga catcgaggag
361 atcaaaaccc agaaggtccg catcgaaggc tccctgtggt ggacctacac aagcagcatc
421 ttcttccggg tcatcttcga agccgccttc atgtacgtct tctatgtcat gtacgacggc
481 ttctccatgc agcggctggt gaagtgcaac gcctggcctt gtcccaacac tgtggactgc
541 tttgtgtccc ggcccacgga gaagactgtc ttcacagtgt tcatgattgc agtgtctgga
601 atttgcatcc tgctgaatgt cactgaattg tgttatttgc taattagata ttgttctggg
661 aagtcaaaaa agccagttta a
[0129] The Cx 30 cDNA coding reference sequence NM_001110219.2
(SEQ.ID.N0:31) is shown below in Table 5C. An anti-connexin 30 polynucleotide
may have
the sequence of any polynucleotide sequence having between 12 to 80
nucleotides (or any
number of nucleotides between 12 and 80) of SEQ ID NO:31.
Table 5C
1 atggattggg ggacgctgca cactttcatc gggggtgtca acaaacactc caccagcatc
61 gggaaggtgt ggatcacagt catctttatt ttccgagtca tgatcctcgt ggtggctgcc
121 caggaagtgt ggggtgacga gcaagaggac ttcgtctgca acacactgca accgggatgc
181 aaaaatgtgt gctatgacca ctttttcccg gtgtcccaca tccggctgtg ggccctccag
241 ctgatcttcg tctccacccc agcgctgctg gtggccatgc atgtggccta ctacaggcac
301 gaaaccactc gcaagttcag gcgaggagag aagaggaatg atttcaaaga catagaggac
361 attaaaaagc agaaggttcg gatagagggg tcgctgtggt ggacgtacac cagcagcatc
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421 tttttccgaa tcatctttga agcagccttt atgtatgtgt tttacttcct ttacaatggg
481 taccacctgc cctgggtgtt gaaatgtggg attgacccct gccccaacct tgttgactgc
541 tttatttcta ggccaacaga gaagaccgtg tttaccattt ttatgatttc tgcgtctgtg
601 atttgcatgc tgcttaacgt ggcagagttg tgctacctgc tgctgaaagt gtgttttagg
661 agatcaaaga gagcacagac gcaaaaaaat caccccaatc atgccctaaa ggagagtaag
721 cagaatgaaa tgaatgagct gatttcagat agtggtcaaa atgcaatcac aggtttccca
781 agctaa
Other Anti-connexin Agents
[0130] As used herein, "gap junction phosphorylating agent" may include
those
agents or compounds capable of inducing phosphorylation on connexin amino acid
residues
in order to induce gap junction or hemichannel closure. Exemplary sites of
phosphorylation
include one or more of a tyrosine, serine or threonine residues on the
connexin protein. In
certain embodiments, modulation of phosphorylation may occur on one or more
residues on
one or more connexin proteins. Exemplary gap junction phosphorylating agents
are well
known in the art and may include, for example, c-Src tyrosine kinase or other
G protein-
coupled receptor agonists. See Giepmans B, J. Biol. Chem., Vol. 276, Issue 11,
8544-8549,
March 16, 2001. In one embodiment, modulation of phosphorylation on one or
more of these
residues impacts hemichannel function, particularly by closing the
hemichannel. In another
embodiment, modulation of phosphorylation on one or more of these residues
impacts gap
junction function, particularly by closing the gap junction. Gap junction
phosphorylating
agents that target the closure of connexin 43 gap junctions and hemichannels
are preferred.
[0131] Still other anti-connexin agents include connexin carboxy-
terminal
polypeptides. See Gourdie et al., W02006/069181.
[0132] In certain another aspect, gap junction modifying agent may
include, for
example, aliphatic alcohols; octanol; heptanol; anesthetics (e.g. halothane),
ethrane, fluothane,
propofol and thiopental; anandamide; arylaminobenzoate (FFA: flufenamic acid
and similar
derivatives that are lipophilic); carbenoxolone; Chalcone: (2',5'-
dihydroxychalcone); CHTs
(Chlorohydroxyfuranones); CMCF (3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-
furanone);
dexamethasone; doxorubicin (and other anthraquinone derivatives); eicosanoid
thromboxane
A(2) (TXA(2)) mimetics; NO (nitric oxide); Fatty acids (e.g. arachidonic acid,
oleic acid and
lipoxygenase metabolites; Fenamates (flufenamic (FFA), niflumic (NFA) and
meclofenamic
acids (MFA)); Genistein; glycyrrhetinic acid (GA):18a-glycyrrhetinic acid and
18-beta -
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glycyrrhetinic acid, and derivatives thereof; lindane; lysophosphatidic acid;
mefloquine;
menadione; 2-Methyl-1,4-naphthoquinone, vitamin K(3); nafenopin; okadaic acid;
oleamide;
oleic acid; PH, gating by intracellular acidification; e.g., acidifying
agents; polyunsaturated
fatty acids; fatty acid GJIC inhibitors (e.g., oleic and arachidonic acids);
quinidine; quinine;
all trans-retinoic acid; and tamoxifen.
Manufacture and Stability
[0133] The polynucleotides of this invention can be manufactured using
solid-phase
chemistries for synthesizing oligonucleotides. In one aspect, the formulations
of this
invention will comprise a salt of the polynucleotides of this invention, such
as the sodium salt
of the polynucleotides of this invention. In one embodiment the formulation
may comprise
the sodium salt of a polynucleotide having SEQ ID NO:1, for example. In some
embodiments, the polynucleotide having SEQ ID NO:1 may be a modified
oligodeoxynucleotide having SEQ ID NO: 1.
[0134] In some embodiments, the formulations of this invention are
substantially pure.
By substantially pure is meant that the formulations comprise less than about
10%, 5%, or 1%,
and preferably less than about 0.1%, of any nucleotide or non-nucleotide
impurity. In some
embodiments the total impurities, including metabolities of the connexin 43
modulating agent,
will be not more than 15%. In some embodiments the total impurities, including
metabolities
of the connexin 43 modulating agent, will be not more than 12%. In some
embodiments the
total impurities, including metabolities of the connexin 43 modulating agent,
will be not more
than 11%. In other embodiments the total impurities, including metabolities of
the connexin
43 modulating agent, will be not more than 10%.
[0135] In some embodiments, the purity of the formulations of this
invention may be
measured using a method selected from anion exchange HPLC (AEX-HPLC) or mass
spectrometry. Mass spectrometry may include LC/MS, or LC/MS/MS. The assay may
in
some embodiments comprise both AEX-HPLC and LC/MS.
[0136] Sterile compositions comprising the connexin 43 modulating agents
of this
invention prepared using aseptic processing by dissolving the anti-connexin
modulating agent
in the formulation vehicle. In one embodiment, the formulation may also be
sterilized by
filtration. Excipients used in the manufacture of of the formulations of this
invention are
widely used in pharmaceutical products and released to pharmacopeial
standards.
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Dosage Forms and Formulations and Administration
[0137] The
connexin protein modulating agents of the invention, for example,
connexin 43, 30 or 26 modulating agents may be administered to a subject in
need of
treatment, having a resistant wound, such as mVLU, or multiple DFU or pressure
ulcers or
other multiple non-healing, slow-healing, or chronic lesions. The anti-
connexin 43
modulating agents may be used in the manufacture of a medicament to treat any
of the
conditions mentioned herein. Thus, in accordance with the invention, there are
provided
formulations by which connexin 43 can be modulated and/or cell-cell
communication can be
downregulated in a transient and site-specific manner.
[0138] The
connexin protein modulating agent, or anti-connexin protein agent, may
be present in a substantially isolated form. It will be understood that the
product may be
mixed with carriers or diluents which will not interfere with the intended
purpose of the
product and still be regarded as substantially isolated. A product of the
invention may also be
in a substantially purified form, in which case it will generally comprise
about 80%, 85%, or
90%, e.g. at least about 88%, at least about 90, 95 or 98%, or at least about
99% of the
polynucleotide (or other anti-connexin 43 agent) or dry mass of the
preparation. In one
embodiment, the anti-connexin agent is an anti-connexin 43, 30 or 26 peptide
or anti-
connexin 43, 30 or 26 peptidomimetic, e.g., an anti-connexin agent that can
block or reduce
hemichannel opening, is administered prior to the administration of an anti-
connexin43
polynucleotide that blocks or reduce connexin expression or the formation of
hemichannels or
gap junctions, e.g., by downregulation of connexin protein expression.
[0139] The
pharmaceutical formulations, or pharmaceutical compositions, combined
preparations and medicaments of the invention may take any suitable form for
topical
administration. For example, the pharmaceutical formulations may take the form
of solutions,
suspensions, instillations, salves, creams, gels, foams, ointments, emulsions,
lotions, paints,
sustained release formulations, or powders, and typically contain about 0.1 %-
95% of active
ingredient(s), preferably about 0.2%-70%. Other suitable formulations include
pluronic gel-
based formulations, carboxymethylcellulose(CMC)-based
formulations, and
hyroxypropylmethylcellulose(HPMC)-based formulations.
[0140] In
some embodiments, the pharmaceutically acceptable carrier or vehicle is, or
comprises, a gel. In one aspect the gel can be a reverse-thermosetting gel
which is a liquid at
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low temperatures, for example at 2-8 C, and which undergoes a reversible
liquid to gel
transition at temperatures greater than approximately 15 C. Thus, in some
embodiments the
carrier may be a liquid at temperatures below approximately 15 C, but may form
a gel at
temperatures above approximately 15 C, such as room temperature or at body
temperature.
In some instances, the gel is a nonionic polyoxyethylene-polyoxypropylene
copolymer gel.
In some embodiments the gel is a pluronic gel. The pluronic gel may be, for
example,
poloxamer 407, also sometimes referred to as Pluronic F-127 (BASF). In some
embodiments,
the formulations of this invention may comprise from about 15 to about 30 %
(w/v) gel. In
some embodiments, the formulations of this invention may comprise from about
20 to about
25 % (w/v) gel. In some embodiments, the formulations of this invention may
comprise
about 22.6 % (w/v) poloxamer 407 gel. In some embodiments, the gel may be a
fluorinated
methacrylamide chitosan hydrogel system. See, Wijekoon et al., Acta Biomater.
2013
Mar:9(3):5653-64.
[0141] Gels or jellies may be produced using a suitable gelling agent
including, but
not limited to, gelatin, tragacanth, or a cellulose derivative and may include
glycerol as a
humectant, emollient, and preservative. Ointments are semi-solid preparations
that consist of
the active ingredient incorporated into a fatty, waxy, or synthetic base.
Examples of suitable
creams include, but are not limited to, water-in-oil and oil-in-water
emulsions. Water-in-oil
creams may be formulated by using a suitable emulsifying agent with properties
similar, but
not limited, to those of the fatty alcohols such as cetyl alcohol or
cetostearyl alcohol and to
emulsifying wax. Oil-in-water creams may be formulated using an emulsifying
agent such as
cetomacrogol emulsifying wax. Suitable properties include the ability to
modify the viscosity
of the emulsion and both physical and chemical stability over a wide range of
pH. The water
soluble or miscible cream base may contain a preservative system and may also
be buffered
to maintain an acceptable physiological pH.
[0142] Foam preparations may be formulated to be delivered from a
pressurized
aerosol canister, via a suitable applicator, using inert propellants. Suitable
excipients for the
formulation of the foam base include, but are not limited to, propylene
glycol, emulsifying
wax, cetyl alcohol, and glyceryl stearate. Potential preservatives include
methylparaben and
propylparaben.
[0143] Preferably the agents of the invention are combined with a
pharmaceutically
acceptable carrier or diluent to produce a pharmaceutical composition.
Suitable carriers and
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diluents include isotonic saline solutions, for example phosphate-buffered
saline. Suitable
diluents and excipients also include, for example, water, saline, dextrose,
glycerol, or the like,
and combinations thereof. In addition, if desired substances such as wetting
or emulsifying
agents, stabilizing or ph buffering agents may also be present.
[0144] The
term "pharmaceutically acceptable carrier" refers to any pharmaceutical
carrier that does not itself induce the production of antibodies harmful to
the individual
receiving the composition, and which can be administered without undue
toxicity. Suitable
carriers can be large, slowly metabolized macromolecules such as proteins,
polysaccharides,
polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid
copolymers.
[0145]
Pharmaceutically acceptable salts can also be present, e.g., mineral acid
salts
such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and
the salts of
organic acids such as acetates, propionates, malonates, benzoates, and the
like.
[0146]
Suitable carrier materials include any carrier or vehicle commonly used as a
base for creams, lotions, gels, emulsions, lotions or paints for topical
administration.
Examples include emulsifying agents, inert carriers including hydrocarbon
bases, emulsifying
bases, non-toxic solvents or water-soluble bases. Particularly suitable
examples include
pluronics, HPMC, CMC and other cellulose-based ingredients, lanolin, hard
paraffin, liquid
paraffin, soft yellow paraffin or soft white paraffin, white beeswax, yellow
beeswax,
cetostearyl alcohol, cetyl alcohol, dimethicones, emulsifying waxes, isopropyl
myristate,
microcrystalline wax, oleyl alcohol and stearyl alcohol.
[0147] An
auxiliary agent such as casein, gelatin, albumin, glue, sodium alginate,
carboxymethylcellulose, methylcellulose, hydroxyethylcellulose or polyvinyl
alcohol may
also be included in the formulation of the invention.
[0148] Other suitable formulations include pluronic gel-based formulations,
c arboxymethylcellulo se(CMC)-based formulations, and
hyroxypropylmethylcellulose(HPMC)-based formulations. The
composition may be
formulated for any desired form of delivery, including topical, instillation,
parenteral,
intramuscular, subcutaneous, or transdermal administration. Other useful
formulations
include slow or delayed release preparations.
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[0149] Where the anti-connexin agent is a nucleic acid, such as a
polynucleotide,
uptake of nucleic acids by mammalian cells is enhanced by several known
transfection
techniques for example those including the use of transfection agents. Such
techniques may
be used with certain anti-connexin agents, including polynucleotides. The
formulation which
is administered may contain such transfection agents. Examples of these agents
include
cationic agents (for example calcium phosphate and DEAE-dextran) and
lipofectants (for
example lipofectamTM and transfectamTM), and surfactants.
[0150] Where the anti-connexin agent comprises a polynucleotide,
conveniently, the
formulation further includes a surfactant to assist with polynucleotide cell
penetration or the
formulation may contain any suitable loading agent. Any suitable non-toxic
surfactant may
be included, such as DMSO. Alternatively a transdermal penetration agent such
as urea may
be included. In certain non-limiting preferred embodiments, the transdermal
penetration
agent comprises an ethoxylated oil or fatty acid, fatty alcohol, or fatty
amine therein having
about 10 to 19 ethoxylations per molecule. Ethoxylated lipids suitable as a
penetration
enhancer include oils such as an ethoxylated vegetable, nut, synthetic or
animal oil, suitably
ethoxylated emu oil or ethoxylated macadamia nut oil. According to a non-
limiting preferred
aspect, suitable ethoxylated lipids that can be used in the formulations
described herein can
be a vegetable, nut, animal, or synthetic oil or fatty acid, fatty alcohol, or
fatty amine therein
having at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more ethoxylations
per molecule.
Non-limiting preferred ethoxylated oils include macadamia nut oil, meadowfoam
oil
(limnanthes alba) castor oil, jojoba oil, corn oil, sunflower oil, sesame oil
or emu oil.
Optionally, other conventional agents used in pharmaceutical formulations such
as an alcohol
and/or water and/or an aqueous adjuvant can be mixed with the penetration
enhancer to
improve the solubility and/or transport of a particular gap junction
modulation agent.
[0151] The effective dose for a given subject or condition can be
determined by
experimentation or other methods known in the art or later developed. For
example, in order
to formulate a range of dosage values for human subjects, cell culture assays
and animal
studies can be used, and doses providing superior results can be converted to
doses for human
or other mammalian subjects. The dosage of such compounds preferably lies
within the dose
that is therapeutically effective for at least 50% of the population, and that
exhibits little or no
toxicity at this level.
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[0152] The effective dosage of each of the anti-connexin agents employed
in the
methods and compositions of the invention may vary depending on a number of
factors
including the particular anti-connexin agent or agents employed, whether used
alone or in
combination, the combination partner, the mode of administration, the
frequency of
administration, the severity fo the resistant lesion, the route of
administration, the needs of a
patient sub-population to be treated or the needs of the individual patient
which can differ due
to age, sex, body weight, relevant medical condition specific to the patient.
[0153] The dose at which an anti-connexin agent is administered to a
patient will
depend upon a variety of factors such as the age, weight and general condition
of the patient,
the condition that is being treated, and the particular anti-connexin agent
that is being
administered.
[0154] A suitable therapeutically effective dose of an anti-connexin
agent may be at
least about 1.0 mg/mL of the anti-connexin agent. In some embodiments, the
suitable
therapeutically effective dose of the anti-connexin agent may be from about
0.1 mg/mL to
about 100 mg/mL. In some embodiments, the suitable therapeutically effective
dose of an
anti-connexin agent may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,
1.0, 2.0, 3.0, 4.0,
5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0,
19.0, 20.0, 21.0, 22.0,
23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0,
36.0, 37.0, 38.0,
39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 52.5,
55.0, 57.5, 60.0,
62.5, 65.0, 67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 90.0, 92.5,
95.0, 97.5, or about
100.0 mg/mL, or any range or subrange between any two of the recited doses, or
any dose
falling within the range of about 0.1 to about 100 mg/mL. In other
embodiments, the
connexin 43 modulating agent is present at a concentration ranging from about
0.5 to about
50 mg/mL. In other embodiments, the connexin 43 modulating agent is present at
a
concentration ranging from about 0.3 to about 30 mg/mL. In other embodiments,
the
connexin 43 modulating agent is present at a concentration ranging from about
0.1 or 1.0 to
about 10 mg/mL. In other embodiments, the connexin 43 modulating agent is
present at a
concentration ranging from about 0.1 or 1.0 to about 0.3 or 3.0 mg/mL. In
other
embodiments, the connexin protein modulating agent, such as a connexin 43
modulatinge
agent, a connexin 30 modulating agent and/or a connexin 26 modulating agent is
present at a
concentration of about 3.0 mg/mL. In any of these aspects the connexin 43, 30
or 26
modulating agent may be a connexin 43, 30 or 26 antisense oligonucleotide.
When the
connexin 43 modulating agent is a modified connexin 43 antisense
oligonucleotide the above-
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noted dose concentrations may be increased by from about 2- to about 10-fold,
for example.
In any of these aspects, the carrier (vehicle) may be a thermoreversible gel.
For example, the
gel may be a poloxamer gel, for example, poloxamer 407, present in an amount
ranging from
about 15-25 or 30%, for example.
[0155] Alternatively, in the case of anti-connexin oligonucleotides or
anti-connexin
peptidomimetics, the dosage of each of the gap junction modulation agents in
the
compositions may be determined by reference to the composition's concentration
relative to
the size, length, depth, area or volume of the area to which it will be
applied. For example, in
certain topical applications, dosing of the pharmaceutical compositions may be
calculated
based on mass (e.g., grams) of or the concentration in a pharmaceutical
composition (e.g.,
lig/u1) per length, depth, area, or volume of the area of application. Useful
doses of
polynucleotides range from about 3 to about 500 micrograms per square
centimeter of wound
size. Certain doses will be about 2 to about 10 micrograms per square
centimeter of wound
size. Doses may also be from about 3 to about 30 micrograms per square
centimeter of
wound size. Certain doses will be about 3-10, about 10-30, about 30-50, 50-75,
75-100, or
about 30-100 micrograms per square centimeter of wound size. Other useful
doses are
greater than about 20 micrograms per square centimeter of wound size, at least
about 25
micrograms per square centimeter of wound size, about 30 micrograms per square
centimeter
of wound size, at least about 35 micrograms per square centimeter of wound
size, at least
about 40 micrograms per square centimeter of wound size, at least about 50
micrograms per
square centimeter of wound size, and at least about 100 to at least about 150
micrograms per
square centimeter of wound size. Other doses include about 150-200 micrograms
per square
centimeter, about 200-250 micrograms per square centimeter, about 250-300
micrograms per
square centimeter, about 300-350 micrograms per square centimeter, about 350-
400
micrograms per square centimeter, and about 400-500 micrograms per square
centimeter, or
any range or subrange between any two of the recited doses, or any dose
falling within the
range of about 3 to about 500 micrograms per square centimeter of wound size,
or greater.
[0156] Useful doses ranges may also include from about 10 to 500
micrograms per
square centimeter of wound size, including at least about 15 micrograms per
square
centimeter of wound size, at least about 20 micrograms per square centimeter
of wound size,
at least about 25 micrograms per square centimeter of wound size, about 30
micrograms per
square centimeter of wound size, at least about 35 micrograms per square
centimeter of
wound size, at least about 40 micrograms per square centimeter of wound size,
at least about
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50 micrograms per square centimeter of wound size, and at least about 100 to
at least about
150 micrograms per square centimeter of wound size. Othe doses include about
150-200
micrograms per square centimeter, about 200-250 micrograms per square
centimeter, about
250-300 micrograms per square centimeter, about 300-350 micrograms per square
centimeter,
about 350-400 micrograms per square centimeter, and about 400-500 micrograms
per square
centimeter. In other embodiments, the doses will be about 10.0, 11.0, 12.0,
13.0, 14.0, 15.0,
16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0,
29.0, 30.0, 31.0,
32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0,
45.0, 46.0, 47.0,
48.0, 49.0, 50.0, 52.5, 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0,
77.5, 80.0, 82.5,
85.0, 87.5, 90.0, 92.5, 95.0, 97.5, 100.0, 105, 110, 115, 120, 125, 130, 135,
140, 145, 150,
155, 160, 65, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 250, 260, 270,
280, 290, 300,
310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,
460, 470, 480, 490,
or about 500 milligrams per square centimeter, or any range or subrange
between any two of
the recited doses, or any dose falling within the range of about 1.0 to about
500 milligrams
per square centimeter.
[0157] In certain embodiments, the anti-connexin agent composition may be
applied
at about 0.01 micromolar ([A,M) or 0.05 tM to about 200 iM, or up to 300 !AM
or up to 1000
11M or up to 2000 [IM or up to 3200 jtM or more, for example up to about 10
mM, 20 mM, or
30 mM final concentration at the treatment site and/or adjacent to the
treatment site, and any
doses and dose ranges within these dose numbers. In one embodiment, the anti-
connexin
agent composition is applied at greater than about 1000 1A,M. Preferably, the
antisense
polynucleotide composition is applied at about 1000 1AM to about 10 mM final
concentration,
more preferably, the anti-connexin agent composition is applied at about 3 mM
to about 10
mM final concentration, and more preferably, the anti-connexin agent
composition is applied
at about 1-3 mM to about 5-10 mM final concentration.
[0158] Additionally, anti-connexin protein agents, such as anti-connexin
43, 30 or 26
agents, or other resistant wound healing agents may be present at about 8 [iM
to about 20 jiM
final concentration, and alternatively the anti-connexin agent composition is
applied at about
[AM to about 20 tM final concentration, or at about 10 to about 15 !AM final
concentration.
In certain other embodiments, the anti-connexin agent is applied at about 10
1AM final
concentration. In yet another embodiment, the anti-connexin agent composition
is applied at
about 1-15 [iM final concentration. In other embodiments, the anti-connexin
agent is applied
at about a 20 11M, 30 M, 40 11M, 50 pt,M, 60 p,M, 70 tM, 80 p.M, 90 11M, 100
[A,M, 10-200
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[AM, 200-300 M, 300-400 M, 400-500 104, 500-600 [AM, 600-700 !AM, 700-800
1AM, 800-
900 104, 900-1000 or 1000-1500 M , or 1500 [04 ¨ 2000 M, 2000 !AM - 3000
[04, 3000
!AM - 4000 M, 4000 M - 5000 [04, 5000 1AM - 6000 M, 6000 M - 7000 M, 7000
!LAM -
80001AM, 8000 [04 - 9000 [04, 9000 !AM ¨ 10,000 [AM, 10,000 !AM ¨ 11,000 M,
11,000 M
¨ 12,000 M, 12,000 M ¨ 13,000 !AM, 13,000 !AM ¨ 14,000 1t04, 14,000 [04 ¨
15,000 M,
15,000 M ¨ 20,000 M, 20,000 M ¨ 30,000 M, 30,000 [04 ¨ 50,000 1t04, or
greater, or
any range or subrange between any two of the recited doses, or any dose
falling within the
range of from about 20 M to about 50,000 M.
[0159] Still other dosage levels between about 1 nanogram (ng)/kg and
about 1 mg/kg
body weight per day of each of the agents described herein. In certain
embodiments, the
dosage of each of the subject compounds will generally be in the range of
about 1 ng to about
1 microgram per kg body weight, about 1 ng to about 0.1 microgram per kg body
weight,
about 1 ng to about 10 ng per kg body weight, about 10 ng to about 0.1
microgram per kg
body weight, about 0.1 microgram to about 1 microgram per kg body weight,
about 20 ng to
about 100 ng per kg body weight, about 0.001 mg to about 0.01 mg per kg body
weight,
about 0.01 mg to about 0.1 mg per kg body weight, or about 0.1 mg to about 1
mg per kg
body weight. In certain embodiments, the dosage of each of the subject
compounds will
generally be in the range of about 0.001 mg to about 0.01 mg per kg body
weight, about 0.01
mg to about 0.1 mg per kg body weight, about 0.1 mg to about 1 mg per kg body
weight. If
more than one anti-connexin agent is used, the dosage of each anti-connexin
agent need not
be in the same range as the other. For example, the dosage of one anti-
connexin agent may
be between about 0.01 mg to about 10 mg per kg body weight, and the dosage of
another anti-
connexin agent may be between about 0.1 mg to about 1 mg per kg body weight,
0.1 to about
10, 0.1 to about 20, 0.1 to about 30, 0.1 to about 40, or between about 0.1 to
about 50 mg per
kg body weight. The dosage may also be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1.0,
2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0,
16.0, 17.0, 18.0, 19.0,
20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0,
33.0, 34.0, 35.0,
36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0,
49.0, 50.0, 52.5,
55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5, 85.0,
87.5, 90.0, 92.5,
95.0, 97.5, or about 100.0 mg per kg body weight, or any range or subrange
between any two
of the recited doses, or any dose falling within the range of from about 0.1
to about 100 mg
per kg body weight.
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[0160] Conveniently, the anti-connexin agent is administered in a
sufficient amount to
downregulate expression of a connexin protein, or modulate gap junction
formation or
connexon opening for at least about 0.5 to 1 hour, at least about 1-2 hours,
at least about 2-4
hours, at least about 4-6 hours, at least about 6-8 hours, at least about 8-10
hours, at least
about 12 hours, or at least about 24 hours post-administration.
[0161] The dosage of the anti-connexin agents in the compositions and
methods of the
subject invention may also be determined by reference to the concentration of
the
composition relative to the size, length, depth, area or volume of the area to
which it will be
applied. For example, in certain topical and other applications, e.g.,
instillation, dosing of the
pharmaceutical compositions may be calculated based on mass (e.g. micrograms)
of or the
concentration in a pharmaceutical composition (e.g. [igl[t1) per length,
depth, area, or volume
of the area of application. The volume of the wound may also be determined by
imaging.
[0162] The doses of an anticonnexin protein modulating agent may be
administered in
single or divided applications. The doses may be administered once, or
application may be
repeated. Typically, application will be repeated weekly until wound healing
is promoted, or
a repeat application may be made in the event that wound healing slows or is
stalled. Doses
may be applied every 12 hours to 7 days apart, or more. For example, doses may
be applied
12 hours, or 1, 2, 3, 4, 5, 6, or 7 days apart, or at any time interval
falling between any two of
these times, or between 12 hours and 7 days. In the case of a chronic wound,
repeat
applications may be made, for example, weekly, or bi-weekly, or monthly or in
other
frequency for example if and when wound healing slows or is stalled. The anti-
connexin 43
agent, or connexin 43 modulating agent, may be administered for up to four,
six, eight, ten,
twelve, fourteen, sixteen, eighteen, twenty, twenty-two, twenty-four or twenty-
six weeks. For
some indications, such as certain ocular uses, more frequent dosing, up to
hourly may
employed.
[0163] In one aspect of the invention a an anti-connexin 26, 30 or 43
polynucleotide
is administered in one composition and an anti-connexin 26, 30 or 43
polynucleotide is
administered in a second composition. The first and second compositions may be
administered simultaneously, separately or sequentially and in any order. For
example, the
first is administered before the second composition. In one embodiment the
first composition
is administered after the second composition. In one embodiment the first
composition is
administered before and after the second composition. In one embodiment the
second
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composition is administered before and after the first composition. When not
administered as
a fixed combination, preferred methods include the sequential administration
of one or more
anti-connexin polynucleotides or one or more anti-connexin peptides or
peptidomimetics,
either or both of which are provided in amounts or doses that are less that
those used when
the agent or agents are administered alone, i.e., when they are not
administered in
combination, either physically or in the course of treatment of a wound. Such
lesser amounts
of agents administered are typically from about one-twentieth to about one-
tenth the amount
or amounts of the agent when administered alone, and may be about one-eighth
the amount,
about one-sixth the amount, about one-fifth the amount, about one-fourth the
amount, about
one-third the amount, and about one-half the amount when administered alone.
Preferably,
the agents are administered sequentially within at least about one-half hour
of each other.
The agents may also be administered with about one hour of each other, with
about one day
to about one week of each other, or as otherwise deemed appropriate. As noted
herein, the
doses of an anti-connexin polynucleotide, peptide or peptidomimetic
administered in
combination, or other anti-connexin agents administered in combination with
either or both,
can be adjusted down from the doses administered when given alone.
[0164] In one embodiment, the combined use of one or more anti-connexin
polynucleotides or one or more anti-connexin peptides or peptidomimetics
reduces the
effective dose of any such agent compared to the effective dose when said
agent administered
alone. In certain embodiments, the effective dose of the agent when used in
combination is
about 1/15 to about 1/2, about 1/10 to about 1/3, about 1/8 to about 1/6,
about 1/5, about 1/4,
about 1/3 or about 1/2 the dose of the agent when used alone. In another
preferred
embodiment, the combined use of one or more anti-connexin polynucleotides and
one or
more anti-connexin peptides or peptidomimetics, or other anti-connexin agents
in
combination with either or both, reduces the frequency in which said agent is
administered
compared to the frequency when said agent is administered alone. Thus, these
combinations
allow the use of lower and/or fewer doses of each agent than previously
required to achieve
desired therapeutic goals.
[0165] Preferably one or more anti-connexin agents, such as anti-connexin
43
polynucleotides, are delivered by topical administration (peripherally or
directly to a site),
including but not limited to topical administration using solid supports (such
as dressings and
other matrices) and medicinal formulations (such as gels, mixtures,
suspensions and
ointments). In one embodiment, the solid support comprises a biocompatible
membrane or
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insertion into a treatment site. In another embodiment, the solid support
comprises a dressing
or matrix. In one embodiment of the invention, the solid support composition
may be a slow
release solid support composition, in which the one or more anti-connexin
polynucleotides
and one or more anti-connexin peptides or peptidomimetics, or other anti-
connexin agents to
be administered in combination with either or both, is dispersed in a slow
release solid matrix
such as a matrix of alginate, collagen, or a synthetic bioabsorbable polymer.
Preferably, the
solid support composition is sterile or low bio-burden. In one embodiment, a
wash solution
comprising two or more anti-connexin agents can be used.
[0166] The delivery of of a formulation comprising one or more anti-
connexin protein
modulating agents (for example, anti-connexin 43, 30 or 26 modulating agents),
such as
polynucleotides or peptides or peptidomimetics, or other anti-connexin protein
agents to be
administered alone or in combination with either or both, over a period of
time, in some
instances for about 1-2 hours, about 2-4 hours, about 4-6 hours, about 6-8, or
about 24 hours
or longer, may be a particular advantage in more severe injuries or
conditions. In some
instances, cell loss may extend well beyond the site of a procedure to
surrounding cells. Such
loss may occur within 24 hours of the original procedure and is mediated by
gap junction
cell-cell communication, or hemichannel opening. Administration of anti-
connexin agent(s),
e. g., for downregulation of connexin expression, or blockade or inhibition of
connexon
opening or activity, therefore will modulate communication between the cells,
or loss into the
extracellular space in the case of connexon regulation, and minimize
additional cell loss or
injury or consequences of injury.
[0167] While the delivery period will be dependent upon both the site at
which the
downregulation is to be induced and the therapeutic effect which is desired,
continuous or
slow-release delivery for about 0.5-1 hour, about 1-2 hours, about 2-4 hours,
about 4-6 hours,
about 6-8, or about 24 hours or longer is provided. In accordance with the
present invention,
this is achieved by inclusion of one or more anti-connexin polynucleotides
and/or one or
more anti-connexin peptides or peptidomimetics, or other anti-connexin 43
agents or resistant
wound healing agents, alone or in combination with either or both, in a
formulation together
with a pharmaceutically acceptable carrier or vehicle, particularly in the
form of a
formulation for continuous or slow-release administration.
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[0168] The routes of administration and dosages described herein are
intended only as
a guide since a skilled physician will determine the optimum route of
administration and
dosage for any particular patient and condition.
[0169] Any of the methods of treating a subject having a resistant wound
referenced
or described herein may utilize the administration of any of the doses, dosage
forms,
formulations, and/or compositions herein described.
Dressings and Matrices
[0170] In one aspect, one or more anti-connexin polynucleotides and/or
one or more
anti-connexin peptides or peptidomimetics are provided in the form of a
dressing or matrix.
In certain embodiments, the one or more agents of the invention are provided
in the form of a
liquid, semi solid or solid composition for application directly, or the
composition is applied
to the surface of, or incorporated into, a solid contacting layer such as a
dressing gauze or
matrix. The dressing composition may be provided for example, in the form of a
fluid or a
gel. One or more anti-connexin 43 polynucleotides and one or more anti-
connexin 43
peptides or peptidomimetics may be provided in combination with conventional
pharmaceutical excipients for topical application. Suitable carriers include:
Pluronic gels,
Polaxamer gels, Hydrogels containing cellulose derivatives, including
hydroxyethyl cellulose,
hydroxymethyl cellulose, carboxymethyl cellulose, hydroxypropylmethyl
cellulose and
mixtures thereof; and hydrogels containing polyacrylic acid (Carbopols).
Suitable carriers
also include creams/ointments used for topical pharmaceutical preparations, e.
g., creams
based on cetomacrogol emulsifying ointment. The above caniers may include
alginate (as a
thickener or stimulant), preservatives such as benzyl alcohol, buffers to
control pH such as
disodium hydrogen phosphate/sodium dihydrogen phosphate, agents to adjust
osmolarity
such as sodium chloride, and stabilizers such as EDTA.
[0171] In addition to the biological matrices previously mentioned,
suitable dressings
or matrices may include, for example, the following with one or more anti-
connexin
polynucleotides or one or more anti-connexin protein peptides or
peptidomimetics (or other
anti-connexin agents to be administered in combination with either or both):
[0172] 1) Absorptives: suitable absorptives may include, for example,
absorptive
dressings, which can provide, for example, a semi-adherent quality or a non-
adherent layer,
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combined with highly absorptive layers of fibers, such as for example,
cellulose, cotton or
rayon. Alternatively, absorptives may be used as a primary or secondary
dressing.
[0173] 2) Alginates: suitable alginates include, for example, dressings
that are non-
woven, non-adhesive pads and ribbons composed of natural polysaccharide fibers
or xerogel
derived from seaweed. Suitable alginates dressings may, for example, form a
moist gel
through a process of ion exchange upon contact with exudate. In certain
embodiments,
alginate dressings are designed to be soft and conformable, easy to pack, tuck
or apply over
irregular-shaped areas. In certain embodiments, alginate dressings may be used
with a
second dressing.
[0174] 3) Antimicrobial Dressings: suitable antimicrobial dressings may
include, for
example, dressings that can facilitate delivery of bioactive agents, such as,
for example, silver
and polyhexamethylene biguanide (PHMB), to maintain efficacy against
infection, where this
is needed or desirable. In certain embodiments, suitable antimicrobial
dressings may be
available as for example, as sponges, impregnated woven gauzes, film
dressings, absorptive
products, island dressings, nylon fabric, non-adherent barriers, or a
combination of materials.
[0175] 4) Biological & Biosynthetics: suitable biological dressings or
biosynthetic
dressings may include, for example, gels, solutions or semi-permeable sheets
derived from a
natural source, e.g., pigs or cows. In certain embodiments, a gel or solution
is applied to the
treatment site and covered with a dressing for barrier protection. In another
embodiment, a
biological-based (e.g., cultured humans cells, pig intestinal mucosa or
bladder tissue) or
biosynthetic-based sheet is placed in situ which may act as membrane,
remaining in place
after a single application, or the may be biological dressings or biosynthetic
dressings may be
prepared in advance to include one or more, preferably two, anti-connexin
agents.
[0176] 5) Collagens: suitable collagen dressings may include, for
example, gels, pads,
particles, pastes, powders, sheets or solutions derived from for example,
bovine, porcine or
avian sources or other natural sources or donors. In certain embodiments, the
collagen
dressing may interact with treatment site exudate to form a gel. In certain
embodiments,
collagen dressing may be used in combination with a secondary dressing.
[0177] 6) Composites: suitable composite dressings may include, for
example,
dressings that combine physically distinct components into a single product to
provide
multiple functions, such as, for example, a bacterial barrier, absorption and
adhesion. In
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certain embodiment, the composite dressings are comprised of, for example,
multiple layers
and incorporate a semi-or non-adherent pad. In certain embodiment, the
composite may also
include for example, an adhesive border of non-woven fabric tape or
transparent film. In
certain other embodiment, the composite dressing may function as for example,
either a
primary or a secondary dressing and in yet another embodiment, the dressing
may be used in
combination with topical pharmaceutical composition.
[0178] 7) Contact Layers: suitable contact layer dressings may include,
for example,
thin, non-adherent sheets placed on an area to protect tissue from for
example, direct contact
with other agents or dressings applied to the treatment site. In certain
embodiments, contact
layers may be deployed to conform to the shape of the area of the treatment
site and are
porous to allow exudate to pass through for absorption by an overlying,
secondary dressing.
In yet another embodiment, the contact layer dressing may be used in
combination with
topical pharmaceutical composition.
[0179] 8) Elastic Bandages: suitable elastic bandages may include, for
example,
dressings that stretch and conform to the body contours. In certain
embodiment, the fabric
composition may include for example, cotton, polyester, rayon or nylon. In
certain other
embodiments, the elastic bandage may for example, provide absorption as a
second layer or
dressing, to hold a cover in place, to apply pressure or to cushion a
treatment site.
[0180] 9) Foams: suitable foam dressings may include, for example, sheets
and other
shapes of foamed polymer solutions (including polyurethane) with small, open
cells capable
of holding fluids. Exemplary foams may be for example, impregnated or layered
in
combination with other materials. In certain embodiment, the absorption
capability may be
adjusted based on the thickness and composition of the foam. In certain other
embodiments,
the area in contact with the treatment site may be non-adhesive for easy
removal. In yet
another embodiment, the foam may be used in combination with an adhesive
border and/or a
transparent film coating that can serve as an anti-infective barrier.
[0181] 10) Gauzes & Non-Woven dressings: suitable gauze dressings and
woven
dressings may include, for example, dry woven or non-woven sponges and wraps
with
varying degrees of absorbency. Exemplary fabric composition may include, for
example,
cotton, polyester or rayon. In certain embodiment, gauzes and non-woven
dressing may be
available sterile or non-sterile in bulk and with or without an adhesive
border. Exemplary
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gauze dressings and woven dressings may be used for cleansing, packing and
covering a
variety of treatment sites.
[0182] 11) Hydrocolloids: suitable hydrocolloid dressings may include,
for example,
wafers, powders or pastes composed of gelatin, pectin or
carboxymethylcellulose. In certain
embodiment, wafers are self-adhering and available with or without an adhesive
border and in
a wide variety of shapes and sizes. Exemplary hydrocolloids are useful on
areas that require
contouring. In certain embodiments, powders and pastes hydrocolloids may use
used in
combination with a secondary dressing.
[0183] 12) Hydrogels (Amorphous): suitable amorphous hydrogel dressings
may
include, for example, formulations of water, polymers and other ingredients
with no shape,
designed to donate moisture and to maintain a moist healing environments and
or to rehydrate
the treatment site. In certain embodiment, hydrogels may be used in
combination with a
secondary dressing cover.
[0184] 13) Hydrogels: Impregnated Dressings: suitable impregnated
hydrogel
dressings may include, for example, gauzes and non-woven sponges, ropes and
strips
saturated with an amorphous hydrogel. Amorphous hydrogels may include for
example,
formulations of water, polymers and other ingredients with no shape, designed
to donate
moisture to a dry treatment site and to maintain a moist healing environment.
[0185] 14) Hydrogel Sheets: suitable hydrogel sheets may include for
example, three-
dimensional networks of cross-linked hydrophilic polymers that are insoluble
in water and
interact with aqueous solutions by swelling. Exemplary hydrogels are highly
conformable
and permeable and can absorb varying amounts of drainage, depending on their
composition.
In certain embodiment, the hydrogel is non-adhesive against the treatment site
or treated for
easy removal.
[0186] 15) Impregnated Dressings: suitable impregnated dressings may
include, for
example, gauzes and non-woven sponges, ropes and strips saturated with a
solution, an
emulsion, oil, gel or some other pharmaceutically active compound or carrier
agent, including
for example, saline, oil, zinc salts, petrolatum, xeroform and scarlet red as
well as the
compounds described herein.
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[0187] 16) Silicone Gel Sheets: suitable silicone gel sheet dressings
may include, for
example, soft covers composed of cross-linked polymers reinforced with or
bonded to mesh
or fabric.
[0188] 17) Solutions: suitable liquid dressings may include, for
example, mixtures of
multiprotein material and other elements found in the extracellular matrix. In
certain
embodiment, exemplary solutions may be applied to the treatment site after
debridement and
cleansing and then covered with an absorbent dressing or a nonadherent pad.
[0189] 18) Transparent Films: suitable transparent film dressings may
include
polymer membranes of varying thickness coated on one side with an adhesive. In
certain
embodiments, transparent films are impermeable to liquid, water and bacteria
but permeable
to moisture vapor and atmospheric gases. In certain embodiments, the
transparency allows
visualization of the treatment site.
[0190] 19) Fillers: suitable filler dressings may include, for example,
beads, creams,
foams, gels, ointments, pads, pastes, pillows, powders, strands or other
formulations. In
certain embodiment, fillers are non-adherent and may include a time-released
antimicrobial.
Exemplary fillers may be useful to maintain a moist environment, manage
exudate, and for
treatment of for example, partial- and full- thickness wounds, infected
wounds, draining
wounds and deep wounds that require packing.
Kits, Medicaments and Articles of Manufacture
[0191] Optionally, one or more anti-connexin protein polynucleotides
and/or one or
more anti-connexin protein peptides or peptidomimetics and/or other anti-
connexin agents
such as a gap junction or hemichannel phosphorylation agent or connexin
carboxy-terminal
polypeptide, alone or in combinations of any of the anti-connexin protein
modulating agents,
or other resistant wound healing agents, may also be used in the manufacture
of the
medicament, or in a kit. Suitable anti-connexin protein modulating agents,
polynucleotides or
peptides may be anti-connexin 43, 30 or 26 modulating agents, polynucleotides
or peptides.
[0192] In one aspect, the invention provides an article of manufacture
or kit
comprising one or more compositions or formulations described. For example,
the kit may
include a pharmaceutical formulation comprising an effective amount of one or
more anti-
connexin 43 polynucleotides and/or one or more anti-connexin 43 peptides or
peptidomimetics and/or other anti-connexin agents, such as a gap junction or
hemichannel
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phosphorylation agent or connexin carboxy-terminal polypeptide, alone or in
combinations of
any of the anti-connexin 43 modulating agents, or other resistant wound
healing agents,
[0193] Articles of manufacturer are also provided, comprising a vessel
containing a
composition or formulation of the invention as described herein and
instructions for use for
the treatment of a subject. For example, in another aspect, the invention
includes an article of
manufacture comprising a vessel containing a therapeutically effective amount
of one or more
anti-connexin protein polynucleotides and/or one or more anti-connexin protein
peptides or
peptidomimetics and/or other anti-connexin agents, such as a gap junction or
hemichannel
phosphorylation agent or connexin carboxy-terminal polypeptide, alone or in
combinations of
any of the anti-connexin protein modulating agents, or other resistant wound
healing agents,
together with instructions for use, including use for the treatment of a
subject. Suitable anti-
connexin protein modulating agents, polynucleotides or peptides may be anti-
connexin 43, 30
or 26 modulating agents, polynucleotides or peptides.
[0194] In some aspects the article of manufacture may comprise a matrix
that
comprises one or more anti-connexin protein peptides or peptidomimetics or
other anti-
connexin agents, such as a gap junction or hemichannel phosphorylation agent
or connexin
carboxy-terminal polypeptide, alone or in combinations of any of the anti-
connexin 43
modulating agents, or other resistant wound healing agents, Suitable anti-
connexin protein
modulating agents, polynucleotides or peptides may be anti-connexin 43, 30 or
26 modulating
agents, polynucleotides or peptides.
Treatment
[0195] The compositions and formulations of the invention comprising one
or more
connexin protein modulating agents may be used for treating resistant lesions,
such as
mVLUs or mDFUs in responder subjects. The compositions and formulations of the
invention may also be used in conjunction or combination with a second
composition for
promoting and/or improving the healing of resistant lesions.
[0196] As disclosed herein suitable anti-connexin protein
polynucleotides, peptides or
peptidomimetics or modulating agents for use in the methods of treatment of
this invention
may include, for example, anti-connexin 43, 30 or 26 polynucleotides or
peptides or
peptidomimetics.
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[0197] In one
aspect the invention is directed to a method of promoting or improving
resistant lesion healing in a subject, comprising administration a
therapeutically effective
amount of one or more anti-connexin protein modulating agents, which may
include anti-
connexin protein polynucleotides and one or more anti-connexin protein
peptides or
peptidomimetics or, optionally, one or more anti-connexin protein
polynucleotides and/or one
or more anti-connexin 43 peptides or peptidomimetics other anti-connexin
agents, such as a
gap junction or hemichannel phosphorylation agent or connexin carboxy-terminal
polypeptide, or other resistant wound healing agent. In
certain embodiments, the
administration of one or more anti-connexin protein polynucleotides and one or
more anti-
connexin protein peptides or peptidomimetics, or, optionally, one or more anti-
connexin
polynucleotides and/or one or more anti-connexin peptides or peptidomimetics
other anti-
connexin agents, or other resistant wound healing agent, is effective to
improve healing of the
resistant lesion, for example, to facilitate epithelial growth and surface
recovery. In certain
embodiments, the administration of one or more anti-connexin protein
polynucleotides and
one or more anti-connexin protein peptides or peptidomimetics, or, optionally,
one or more
anti-connexin polynucleotides and/or one or more anti-connexin peptides or
peptidomimetics
other anti-connexin agents, or other resistant wound healing agent, is
effective to promote
complete wound closure, or to increase the rate of persitent wound closure.
According to
another aspect of the present invention, re-epithlialization and/or formation
of granulation
tissue is promoted. Methods of promoting re-epithelialization of resistant
skin lesions
comprise administering to a subject having a resistant skin lesion, including,
for example,
mVLUs, in an amount effective to promote re-epithelialization. Analogous
methods can be
used to regulate epithelial basal cell division and growth. In certain
embodiments, the
administration of the anti-connexin protein modulating agent is effective to
promote cell
migration to accelerate closure and healing, to facilitate epithelial growth,
or any combination
thereof. Subjects which may be treated include subjects with mVLU, having one
or more of
the other indicators described herein, for example, age over 50-52 or BMI less
than 40-42.
Suitable anti-connexin protein modulating agents, polynucleotides or peptides
may be anti-
connexin 43, 30 or 26 modulating agents, polynucleotides or peptides.
[0198] In one
aspect the invention is directed to a method of promoting or improving
resistant lesion healing in a subject, comprising administration of one or
more anti-connexin
protein polynucleotides and one or more anti-connexin protein peptides or
peptidomimetics,
or, optionally, one or more anti-connexin protein polynucleotides and/or one
or more anti-
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connexin protein peptides or peptidomimetics other anti-connexin agents, or
resistant lesion
healing agents, in an amount effective to regulate epithelial basal cell
division and growth. In
one embodiment, the anti-connexin agent is a connexin antisense polynucleotide
effective to
regulate epithelial basal cell division and growth. In one embodiment, a
second connexin
antisense polynucleotide is a connexin 26 or connexin 30 antisense
polynucleotide, peptide or
peptidomimetic, a connexin 43 antisense polynucleotide, peptide, or
peptidomimetic or a
mixture thereof. Subjects which may be treated include subjects with mVLU,
having one or
more of the other indicators described herein, for example, age over 50-52 or
BMI less than
40-42.
[0199] In one aspect the invention is directed to a method of promoting
or improving
resistant wound healing, comprising administration of one or more anti-
connexin protein
peptides or peptidomimetics, or, optionally, one or more anti-connexin protein
polynucleotides and/or one or more anti-connexin protein peptides or
peptidomimetics other
anti-connexin agents, or resistant wound healing agents, in an amount
effective to regulate
outer layer keratin secretion. In one embodiment, the anti-connexin agent is a
connexin
antisense polynucleotide effective to regulate outer layer keratin secretion.
In one
embodiment, the connexin antisense polynucleotide is a connexin protein
antisense
polynucleotide, peptide or peptidomimetic, a connexin 43, connexin 26 or
connexin 30
antisense polynucleotide, peptide or peptidomimetic or a mixture thereof.
Subjects which
may be treated include subjects with mVLU, having one or more of the other
indicators
described herein, for example, age over 50-52 or BMI less than 40-42.
[0200] In yet a further aspect, the invention provides a method of
decreasing scar
formation and/or improving scar appearance in a patient who has suffered a
resistant wound.
[0201] In one aspect the invention is directed to sustained
administration of one or
more anti-connexin protein polynucleotides and one or more anti-connexin
protein peptides
or peptidomimetics, or, optionally, one or more anti-connexin protein
polynucleotides and/or
one or more anti-connexin protein peptides or peptidomimetics other anti-
connexin agents, or
resistant wound healing agents. In one embodiment, the anti-connexin agents
are
administered for at least at least about 0.5 hours, about 1- 24 hours, at
least about 2, hours, at
least about 3 hours, at least about 4 hours, at least about 5 hours, at least
about 6 hours, at
least about 7 hours, at least about 8 hours, at least about 9 hours, at least
about 10 hours, at
least about 11 hours, at least about 12 hours or at least about 24 hours. In
one embodiment,
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connexin expression is downregulated over a sustained period of time. In
another
embodiment, connexin hemichannels are blocked or closed, in whole or in part,
over a
preferred period of time. Preferably connexin protein expression is
downregulated and
connexin hemichannel opening is blocked or inhibited, in whole or in part, for
a sustained
period of time. Conveniently, connexin protein expression is downregulated or
hemichannels
blocked or inhibited for at least about 1, 2, 4, 6, 8, 10, 12, or 24 hours.
According to one
embodiment, the wound is a resistant lesion. Suitable subjects include a
diabetic subject.
Other subjects include, for example, those with peripheral edema, vasculitis,
or
cardiovascular disease.
Suitable anti-connexin protein polynucleotides, peptides or
peptidomimetics may be anti-connexin 43, 30 or 26 polynucleotides or peptides
or
peptidomimetics.
[0202] In one
aspect, the present invention provides a method of treating a subject
having a resistant wound which comprises sustained administration of an
effective amount of
one or more anti-connexin protein peptides or peptidomimetics, or, optionally,
one or more
anti-connexin protein polynucleotides and/or one or more anti-connexin protein
peptides or
peptidomimetics other anti-connexin agents, or resistant wound healing agents,
to the wound.
[0203]
According to another further aspect, the present invention provides a method
of promoting or improving resistant wound healing in a subject having a wound
which
comprises sustained administration of one or more anti-connexin protein
peptides or
peptidomimetics, or, optionally, one or more anti-connexin protein
polynucleotides and/or
one or more anti-connexin protein peptides or peptidomimetics other anti-
connexin agents, or
resistant wound healing agents, to a wound area in an amount effective to
increase re-
epithlialization rates in the wound area.
[0204] In one
embodiment, the composition or compositions are administered in a
sustained release formulation. In another embodiment, the composition or
compositions are
administered for a sustained period of time. Conveniently, the composition is
effective to
decrease connexin protein alone, or in combination with reducing connexin 31.1
levels or
activity (e.g., hemichannel or gap junction activity) for at least about 24
hours.
[0205]
Subjects which may be treated include subjects with mVLU, having one or
more of the other indicators described herein, for example, age over 50-52 or
BMI less than
40-42. Subjects which may be treated include diabetic subjects.
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[0206] In one aspect the invention is directed to a method for treatment
or prophylaxis
of a resistant lesion comprising administering to a subject in need thereof an
effective amount
of an anti-connexin agent administered to said resistant wound or a tissue
associated with said
resistant wound in combination with another anti-connexin agent. In another
embodiment,
the resistant wound is a resistant chronic skin lesion and a composition of
the present
invention is administered to the skin or a tissue associated with the skin of
said subject for an
effective period of time. Resistant lesions or wounds include multiple VLUs,
multiple
diabetic foot ulcers (DFUs), multiple pressure ulcers, wounds whose surface
areas change
relatively little during a screening period with compression bandaging or
other standard-of-
care therapy (e.g., off-loading), and those with relatively few signs of
healing during a
screening period with compression bandaging therapy. In some aspects resistant
lesions are
characterized by less granulation and epithelialization during the screening
period, or at the
time of treatment with the connexin 43 modulating agent. The screening period
may be from
about 10 days to about 1-4 weeks, for example, and is typically 2 weeks and
sometimes 4
weeks.
[0207] In some embodiments, the surface of the lesion may be freed of
slough,
exudate and devitalized tissue, preferably without excision of skin edges or
enlargement of
the lesion. In some embodiments, the pharmaceutical formulations of this
invention
comprising one or more connexin protein modulating agens may be applied
topically around
the inside edge of the ulcer to be treated and then applied to the remainder
of the wound bed.
[0208] When not administered as a fixed combination, preferred methods
include the
sequential administration of one or more anti-connexin protein polynucleotides
and one or
more anti-connexin protein peptides or peptidomimetics, or, optionally, one or
more anti-
connexin polynucleotides and/or one or more anti-connexin peptides or
peptidomimetics
other anti-connexin agents, such as a gap junction or hemichannel
phosphorylation agent or
connexin carboxy-terminal polypeptide, or another resistant wound healing
agent. Preferably,
the agents are administered sequentially within at least about one-half hour
of each other.
The agents may also be administered with about one hour of each other, with
about one day
to about one week of each other, or as otherwise deemed appropriate.
Preferably, an anti-
connexin protein peptide or anti-connexin protein peptidomimetic, e.g., an
anti-connexin
agent that can block or reduce hemichannel opening, is administered prior to
the
administration of an anti-connexin agent that blocks or reduce connexin
expression or the
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formation of hemichannels or gap junctions, e.g., by downregulation of
connexin protein
expression.
[0209] In another embodiment for treatment of wounds, including resistant
wounds,
either or both of the one or more anti-connexin protein polynucleotides and
one or more anti-
connexin protein peptides or peptidomimetics, or, optionally, one or more anti-
connexin
polynucleotides and/or one or more anti-connexin peptides or peptidomimetics
other anti-
connexin agents, such as a gap junction or hemichannel phosphorylation agent
or connexin
carboxy-terminal polypeptide, or other resistant wound healinge agents, are
provided in
amounts or doses that are less that those used when the agent or agents are
administered alone,
i.e., when they are not administered in combination, either physically or in
the course of
treatment of a wound. Such lesser amounts of agents administered are typically
from about
one-twentieth to about one-tenth the amount or amounts of the agent when
administered
alone, and may be about one-eighth the amount, about one-sixth the amount,
about one-fifth
the amount, about one-fourth the amount, about one-third the amount, and about
one-half the
amount when administered alone.
[0210] In one embodiment the method for treatment or prophylaxis of a
resistant
wound comprises sustained administration of one or more anti-connexin protein
polynucleotides and one or more anti-connexin protein peptides or
peptidomimetics, or,
optionally, one or more anti-connexin polynucleotides and/or one or more anti-
connexin
peptides or peptidomimetics other anti-connexin agents, such as a gap junction
or
hemichannel phosphorylation agent or connexin carboxy-terminal polypeptide, or
other
resistant wound healing agent. In one embodiment, the composition or
compositions are
administered in a sustained release formulation. In another embodiment, the
composition or
compositions are administered for a sustained period of time. Conveniently,
the composition
is effective to decrease connexin protein levels, or block or reduce connexin
protein
hemichannel opening, for at least about 1-2 hours, about 2-4 hours, about 4-6
hours, about 4-
8 hours, about 12 hours, about 18 hours, or about 24 hours. Subjects which may
be treated
include diabetic subjects, and patients with other ulcers, including venous
ulcers and others
described herein and known in the art.
[0211] The following examples which will be understood to be provided by
way of
illustration only and not to constitute a limitation on the scope of the
invention.
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EXAMPLES
EXAMPLE 1: Lack of Toxicity
[0212] As discussed herein, the unmodified 30-mer anti-connexin
deoxyoligonucleotide having SEQ ID NO:1 ("the Polynucleotide") has been shown
to have
surprising utility in treating responder subjects with mVLUs and other
indicators of
likelihood to respond to treatment with an anti-connexin 43 modulating agent.
[0213]
Moreover, use of the compositions of this invention, comprising synthetic,
unmodified deoxyoligonucleotides with unmodified backbones resulted in low
toxicity with
no systemic exposure, and, importantly with respect to safety, undetectable or
exceedingly
low pK even when very large clinical-multiple doses of an unmodified anti-
connexin
deoxyoligonucleotide having SEQ. ID. NO:1 were repeatedly administered to open
wounds
in the skin of test animals.
[0214] The
low toxicity is due in part to the high specificity of the Polynucleotide.
Human DNA sequence database searches were performed to evaluate the extent to
which a
polynucleotide having SEQ ID NO:1 may have homology with sequences in the
known array
of human genes and to assess whether unwanted inhibitory activity could be
exerted against
expression of human gene products other than the target gene and thereby
induce "off-target"
effects. The human genome database searches for homologies within the genome,
homologies with known and predicted transcripts, and homologies with potential
internal
mismatch sequences, showed that the 30-nucleotide oligonucleotide having SEQ
ID NO:1 is
highly specific for the intended CX43 target with no likely off-target
effects.
[0215] In
addition, in contrast with chemically modified oligonucleotides, which have
been found to cause complement activation and inhibition of the extrinsic
coagulation
pathway, unmodified oligonucleotides of this invention have not shown such
effects.
Furthermore, the Polynucleotide displayed no evidence for genetic toxicity
based on the
results of the complete battery of three genetic toxicity studies (i.e., a
bacterial mutagenicity
assay, an in vitro chromosomal aberrations test and an in vivo micronucleus
study in mice).
Moreover, because the Polynucleotide is a chemically unmodified
polynucleotide, it is
degraded via naturally occurring processes such as depurination followed by
backbone
cleavage.
Furthermore, oligonucleotides with an unmodified backbone like the
Polynucleotide do not bind to plasma proteins (e.g., Brown DA, et al. Effect
of
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phosphorothioate modification of oligodeoxynucleotides on specific protein
binding. J Biol.
Chem. 1994;269:26801-26805), as do the phosphorothioate oligonucleotides, and
hence,
would not be expected to displace other drugs that bind to albumin or
otherwise alter the
balance of free vs. plasma protein-bound drug. Moreover, a single site
modification in the
30-mer Polynucleotide will not dramatically alter the binding energy of the
other 29 base
pairs in the sequence and thus the related impurities will not be expected to
alter the efficacy
or specificity of the drug in a biological system.
[0216] The
Polynucleotide has also been shown to have a short half-life in cells (¨ 20
minutes) and a very short half-life in the circulation
several minutes) due to rapid
metabolism by endogenous nucleases and extremely rapid glomerular filtration.
It has been
shown that systemic exposure to the Polynucleotide is exceedingly low, even
when very large
clinical-multiple doses of a formulation comprising a poloxamer gel and anti-
connexin
deoxyoligonucleotide having SEQ. ID. NO:1 (Polynucleotide Formulation) are
repeatedly
administered to open wounds in the skin of test animals. Pharmacokinetic
studies undertaken
have also shown that the polynucleotide having SEQ ID NO:1 is undetectable in
the plasma
from patients that have been treated topically with the Polynucleotide
Formulation, despite
the use of a highly sensitive hybridization type bioanalytical assay. Hence,
systemic
exposure of topically applied Polynucleotide Formulation across the range of
clinical doses
used is negligible.
[0217] For
example, the Polynucleotide Formulation was well tolerated and revealed
no toxicity when administered weekly to wound sites for 3 months in rats and
rabbits at large
clinical-multiple doses. Toxicokinetic and tissue distribution analyses showed
that while
there was substantial exposure of the wound site tissues to active
oligonucleotide ingredient
having SEQ ID NO:1 over the course of the study, systemic exposure was
negligible.
[0218] In
both rat and rabbit dermal studies, rabbit subjects received weekly topical
application of the Polynucleotide Formulation to excisional wound sites at
doses of 0, 120,
1200 or 9320 ug/dose while rats received doses of 0, 30, 300 or 2330 ug/dose.
The 13-week
dermal toxicity studies in rats and rabbits, from which plasma and tissue
samples were
collected for Polynucleotide bioanalysis, were conducted with weekly topical
application of
the Polynucleotide Formulation to excisional wound sites for 2 weeks or 13
weeks (animals
in the 13 week group were repeatedly wounded every 3 weeks), followed by a 2-
or 4-week
recovery-period. Analysis of plasma concentrations at several post-dosing time
points
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revealed low systemic exposure even at the highest dose levels. Mean
concentrations of the
Polynucleotide were generally less than 100 ng/mL in plasma samples of animals
treated with
the highest dose levels (i.e., 9320 and 2330 tig/dose, for rabbits and rats,
respectively, or
approximately 3 and 4-6 mg/kg, respectively, based average body weights). For
the majority
of the samples collected from the low- and mid-dose rats and rabbits, no
quantifiable levels of
the Polynucleotide were present (levels were below LOQ of 1 ng/mL). Rapid
metabolism and
clearance of the Polynucleotide were observed in both species as evidenced by
the rapid
appearance of metabolites described as shortmers which are primarily N-1 and N-
2
oligonucleotides with subsequent metabolism to shorter oligonucleotide
structures. The
Polynucleotide and shortmers were absent at the 3-hour post-dose time point,
which is
consistent with the expected rapid in vivo metabolism and clearance of an
unmodified
(natural backbone) oligonucleotide. Based on the nature of the bioanalytical
assay employed
(a hybridization assay with electrophorectic resolution), the expected
exonuclease-mediated
metabolism to chain-shortened metabolites was documented.
[0219] The results of the analysis of tissues collected at four time
points in the rabbit
study showed that the levels of the Polynucleotide and metabolites were very
low or not
quantifiable in the two internal organs analyzed (liver and kidney). The
kidney and liver were
chosen to assess systemic absorption because these are the known major organs
of uptake of
oligonucleotides with systemic administration. The absence of the
Polynucleotide or shortmer
metabolites in most of these samples is consistent with the plasma level data
and indicates
minimal systemic absorption of the Polynucleotide following topical
application of the
Polynucleotide Formulation to wound sites. In contrast, there was high and
resistant
exposure of the wound site to the Polynucleotide and metabolites following
topical
application of the Polynucleotide Formulation, with dose related mean levels
of the
Polynucleotide and metabolites present at wound sites, and decreasing amounts
present one to
four weeks after administration of the last dose. Although steady clearance of
the
Polynucleotide from the wound site skin was evident, as well as ongoing
metabolism, it was
found that although the Polynucleotide is unmodified, there was ample exposure
of the
wound site skin to the Polynucleotide throughout the study with the weekly
dosing schedule
that was utilized which was analogous to the clinical dosing schedule for all
human VLU
studies.
[0220] For both species, the levels of intact Polynucleotide or presumed
metabolites
in plasma and systemic tissues were much lower (generally not detectable) when
samples
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were collected on days on which the dose was applied to a wound site that was
largely healed
(i.e., 15-16 days after wounding), as compared to the days when doses were
applied to fresh
wounds. Thus, the extent of systemic exposure to the Polynucleotide and
metabolites was
virtually negligible when the Polynucleotide Formulation was applied to a
largely healed
wound site, indicating that the Polynucleotide has little or no potential to
cross an intact skin
barrier. Overall, the plasma concentration data indicate that systemic
absorption of the
Polynucleotide when applied topically to wounds is very low, particularly when
applied to
partially healed wounds, and that nuclease-mediated metabolism occurs very
quickly.
[0221] Further evidence of the minimal systemic exposure to the
Polynucleotide was
provided by the toxicokinetic data from the safety pharmacology study in
cynomolgus
monkeys in which large intravenous doses of the Polynucleotide were
administered by bolus
injection. In addition, the potential for large intravenous doses to elicit
class effects that have
been observed with chemically modified oligonucleotides (unlike the
Polynucleotide), i.e.,
complement activation and inhibition of the extrinsic coagulation pathway, was
assessed in
this study. The Polynucleotide was admistered as single escalating doses of 10
and 50 mg/kg
by intravenous bolus injection, and the animals were evaluated for changes in
cardiovascular
parameters by radiotelemetry (blood pressure, heart rate, and
electrocardiographic activity
(both qualitative and quantitative evaluation of ECG intervals), as well as
respiratory function
and neurologic function. Plasma samples for bioanalysis were collected at a
few time points
following intravenous injection. Doses of the Polynucleotide up to 50 mg/kg,
administered
intravenously, were associated with maximal plasma concentrations (at five
minutes after
dosing) of parent and proximal metabolites that ranged from approximately
230,000 to
460,000 ng/mL; however, the plasma concentrations had fallen to just above the
lower limit
of quantification (0.9 ng/mL) at the 2-hour post-dose collection time point.
These data
provide additional evidence for the rapid metabolism and clearance of the
Polynucleotide.
Furthermore, the absence of any adverse effects in this study demonstrated
safety under
conditions when systemic exposure is several orders of magnitude greater than
that detectable
following administration by the intended clinical route, i.e., dermal
application. Thus, the
compositions and formulations of this invention have surprising low toxicity.
[0222] In addition, while chemically modified oligonucleotides have been
found to
cause complement activation and inhibition of the extrinsic coagulation
pathway, these so-
called "class effects" of chemically-modified oligonucleotides were not found
with the
Polynucleotide.
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[0223] Human clinical PK studies also demonstrated that following
administration of
the anti-connexin deoxyoligonucleotide having SEQ ID NO:1, the
deoxyoligonucleotide was
undetectable in the plasma of patients who had been treated topically for VLU
with dose
concentrations up to and including 3.0 mg/mL, using a bioanalytical method
having a lower
limit of quantification (LLOQ) of 1.0 ng/mL, indicating that there was
negligible systemic
exposure. Specifically, clinical use of the Polynucleotide Formulation has
been associated
with no measurable systemic absorption, which is expected for unmodified
oligonucleotides
that are characterized by poor metabolic stability in blood and are very
rapidly eliminated via
glomerular filtration and by nuclease-mediated metabolism. Thus, what little
drug may enter
the systemic circulation is rapidly metabolized to smaller natural-structure
oligonucleotides or
monomers and rapidly cleared by renal filtration. Also, the Polynucleotide has
been shown to
have high specificity to connexin 43 based on human genome database searches.
These
characteristics contribute to the overall favorable safety profile in which
over 200 patients
have been exposed to the Polynucleotide Formulation at 3.0 mg/mL or higher.
[0224] In summary, dermal administration of the Polynucleotide
Formulation results
in drug deposition in skin samples from wound sites. Following dosing of the
wound site, the
Polynucleotide is taken up into the skin and local tissues and persists at
appreciable levels
over a one-week period, which is the intended clinical dosing interval.
However, there is no
appreciable accumulation at the treatment site. Systemic exposure with this
route of
administration is negligible, mainly owing to a DNA structure that undergoes
very rapid
metabolism and elimination when taken up into the systemic circulation such
that there is
virtually no deposition in expected systemic tissues seen with other
oligonucleotides, such as
the kidney and liver.
EXAMPLE 2: Analysis of connexin 43 levels in single and multiple VLUs
[0225] A study was undertaken of Cx43 protein expression in patients who
were
reported to have multiple (n=10; mVLU) or single (n=8; sVLU) venous leg
ulcers. These
patients were being treated for a clinical diagnosis of non-infected VLU of at
least four weeks
duration. They underwent a 4mm punch chronic wound edge biopsy, and a matching
punch
of non-wounded arm skin. Cx43 protein expression was assessed by
immunohistochemistry
at three sites across the wound biopsy. The biopsy measurement sites were: (i)
at the chronic
wound edge side of the biopsy (WE), (ii) lmm away from the chronic WE side,
and (iii) the
opposite side to the chronic WE (far edge). Normal unwounded skin was assessed
in one
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central site in the biopsy. Cx43 expression in the wound was normalized to the
patient's non-
wounded basal Cx43 expression (i.e., reported as the ratio of chronic wound
edge skin Cx43
to the matched patient Cx43 expression in unwounded skin).
[0226] Biopsies: Wound biopsies were taken during an outpatient clinic
visit of a
single treating clinician. A 4mm full thickness skin punch biopsy taken under
local
anaesthetic from the visible wound edge (WE). The side of the biopsy away from
the open
wound-bed was marked with ink to help keep the sample orientated throughout
subsequent
histological processing. Each patient also provided a matched 4mm punch biopsy
of their
normal arm skin, thus providing matched unwounded baseline Cx43 skin
expression levels.
At the time of collection samples were immediately transferred to 4%
paraformaldehyde for
24 hours, and then 20% sucrose in Phosphate-buffered saline ("PBS"). Tissue
blocks were
then embedded in optimal cutting temperature medium (OCT) and stored at -80
C.
[0227] Immunohistochemistry: Tissue was sectioned, stained and imaged by
confocal microscopy using identical parameters per patient to permit
quantification. Standard
approach using primary antibody of Cx43 1:4000 (Sigma - Poole, UK - C6219).
The
Secondary antibody was Alexa Fluor 488 goat anti-rabbit 1:400) (Invitrogen -
Paisley, UK).
Nuclei were stained using HOECHST (Sigma - Poole, UK - B-2883 and B-2261
1:50,000 in
PBS).
[0228] Confocal Microscopy: An Olympus FV-1000 inverted confocal
microscope
was used to take 40x images of the arm skin and wound. The 4mm unwounded arm
biopsies
were assessed at one central site in the biopsy. The 4mm wound biopsies were
examined
across their diameter at three locations: at the wound edge "WE", "1 mm from
the WE", and
at the far edge "FL" of the 4 mm biopsy (i.e., directly opposite the wound
WE). Figure 5
shows where the assessments were taken across the 4mm biopsy.
[0229] Image Quantification and Statistical Analysis: Cx43 quantification
was
carried out using ImageJ. Thresholds were kept constant between all images.
Three different
Cx43 expression measurement locations across the biopsy (i.e., "WE", "lmm",
"Ph") were
independently compared between the multiple and single ulcer groups at those
same three
locations. Data were tested for normality and if necessary underwent a log-
transformation
before proceeding with a simple statistical approach comprising independent t-
tests. Each of
the three wound locations was treated as independent and no multiple
comparison correction
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was applied. The mean raw data and SEM are shown on the graphs. Note: not all
biopsies
were suitable for assessment of Cx43 at all three locations in the biopsy, so
in some cases a
full dataset was not available. Final group sizes were 5-8 wounds from single
wound patients
and 8-10 wounds multiple wound patients.
Table 6: Patient Demographic Summary
I ,-,--ritrw-
,------771, i , 77,,,,,,,f?, , r Tr = e ,,47.
q,',:if,,,_. it. 4.,*/ ,11:_s0450,i,! L '44,' tit )) ,is WrOfil I FrP,1:
;OUT AN'si Iii OA iligali491CA
Li?".4 , r ;1'.1,;Zi= .:f'si -.' '.3,, -,:4: : V ':. 4.; -';..V, - ' ''1'.-
4r,: :-1..'1;7:: 'Liµt. : .V. -'; I ' ' ,. : 'µ.% 6":,''' ,''-:!".
:11'. IL
1-',;1':,t,. , J ilLyaki,1õ,tru , 4.400.VIItal:4. L .:S.eiEiX'.1 Pl'Itik:-
.3:41:LAAthitki1E21tAt' ,Ealak--dig
Multiple 52 9.6 6
5M : 5F
wounds (31 - 70) (2.0 - 36.4) (1 - 36)
Single 59 7.6 6
8 6M : 2F
wound (45 - 79) (2.4- 113.1) (1.5-108)
P value 0.1 0.48 0.36
[0230] As shown in Table 6, the demographics of the two small study
groups were
not significantly different.
[0231] Figure 6 shows Cx43 in dermis normalised to patient baseline
expression
(Ratio Dataset). There was a discernible pattern evident with a higher Cx43
expression ratio
being present in the dermis of the "multiple wounds" compared to the "single
wounds". This
was present across the whole biopsy. The statistical analysis (t-test on
normalised data)
between the groups supported this trend at the WE (p=0.07) and lmm (p=0.07)
sites, and on
the "Far" side of the biopsy it was significant (p=0.046).
[0232] There is usually low Cx43 background expression in the normal
skin dermis
compared to the epidermis. Cx43 upregulation in the dermis maybe due to
several factors
such as increased underlying inflammatory cell invasion, new blood vessel
formation, more
myofibroblast differentiation, and a greater global stimulus on resident cells
to express Cx43
due to the effects of surrounding tissue ischemia and hypoxia. These are all
potential
theoretical sources for the dermal Cx43 upregulation.
[0233] The existence of greater dermal Cx43 upregulation in patients
with multiple
wounds is consistent with "multiple" wounds having more underlying tissue
damage, worse
circulation, and/or more inflammation than "single" wounds. Indeed the
formation of a
"multiple" wound phenotype may even point to a different underlying biology
with more
"field changes" in the surrounding skin of the wounds than in "single
phenotype" VLUs. In
support of this concept, there is a literature on the differences between
multiple or single
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wounds, showing that multiple wounds are associated with slower healing
indices (Margolis
DJ, et al. The accuracy of venous leg ulcer prognostic models in a wound care
system.
Wound Repair and Regeneration. 2004; 12(2): 163-8) and considered a sign of
worse
underlying venous disease (Rutherford RB, et al. Venous severity scoring: An
adjunct to
venous outcome assessment. Journal of Vascular Surgery. 2000; 31(6): 1307-12).
The
increased dermal Cx43 expression in multiple leg ulcers may result from a
greater
inflammatory response and possibly impaired skin perfusion. Thus, the anti-
inflammatory
(Mori R, et al. Acute downregulation of connexin43 at wound sites leads to a
reduced
inflammatory response, enhanced keratinocyte proliferation and wound
fibroblast migration.
Journal of Cell Science. 2006; 119(Pt 24): 5193-203; Cronin M, et al. Blocking
connexin43
expression reduces inflammation and improves functional recovery after spinal
cord injury.
Molecular and Cellular Neurosciences. 2008; 39(2): 152-60; Qiu C, et al.
Targeting
connexin43 expression accelerates the rate of wound repair. Current Biology.
2003; 13(19):
1697-703; Coutinho P, et al. Limiting burn extension by transient inhibition
of Connexin43
expression at the site of injury. British journal of plastic surgery. 2005;
58(5): 658-67;
Gilmartin DJ, et al. Integration of scaffolds into full-thickness skin wounds:
the connexin
response. Advanced Healthcare Materials. 2013; 2(8): 1151-60.), anti-vessel
leak (Cronin M,
et al. Blocking connexin43 expression reduces inflammation and improves
functional
recovery after spinal cord injury. Molecular and Cellular Neurosciences. 2008;
39(2): 152-60)
and vascular regeneration (Ormonde S, et al. Regulation of connexin43 gap
junction protein
triggers vascular recovery and healing in human ocular resistant epithelial
defect wounds. The
Journal of Membrane Biology. 2012; 245(7): 381-8) activities by connexin 43
modulation
(see Example 3 below) i.e., the specific activities not known to be caused
directly by a
vehicle plus compression or compression bandaging alone, are more evident in
this treatment
group. Multiple wounds likely represent the end result of more extensive
underlying tissue
damage and pathology compared to single wounds, and this severity can manifest
as higher
Cx43 expression, the target of connexin modulators such as, for example, a
Cx43 antisense
oligonucleotide. It was surprisingly determined that multiple VLU representing
more severe,
and generally harder to heal lesions, may respond to a wider range of
modulated Cx43
activities, e.g., anti-inflammatory activity, vascular regeneration, and
reduced vascular leak
and edema.
EXAMPLE 3: Efficacy of Connexin 43 Modulating Agent in
Treating Wounds on mVLU Subjects
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[0234] A 10-week randomized, parallel group, dose-ranging, controlled,
multi-center
study was conducted to assess the efficacy and safety of two dose
concentrations of the
Polynucleotide Formulation (1.0 mg/mL and 3.0 mg/mL) plus standard of care
compression
bandaging (SOC) vs. Polynucleotide Formulation Vehicle (poloxamer 407 gel)
plus SOC
("Vehicle") in subjects with a VLU. An additional SOC-alone arm was included
in order to
compare healing with Vehicle-treated subjects.
[0235] The primary objective of the study was to determine whether
Nexagon (1.0
or 3.0 mg/mL) improved healing of VLU. Percent surface area change of the
reference VLU
(RVLU) at 10 weeks was the primary endpoint of the study. Key secondary
endpoints were
incidence of complete RVLU closure and time to complete RVLU closure in the 10-
week
treatment period, both acceptable regulatory endpoints for registration
studies.
[0236] A two-week Screening Period was designed to determine whether
subjects
were eligible to proceed to the treatment period of the study. The
Investigator selected one
RVLU at the first study visit (in patients with multiple VLU, this was the
largest lesion that
met the eligibility criteria for the study). Key eligibility criteria were
patient age >18 years;
confirmed venous insufficiency by venous duplex ultrasonography; non-infected,
full
thickness well-circumscribed VLU located above the malleolus; an ankle
brachial index >
0.80; and a VLU between 2 and 20 cm2 at the end of the screening period.
Centralized
review of the RVLU photos was performed by the Medical Monitor during this
period to
supplement the Investigators' assessments of patient eligibility for
randomization.
[0237] As SOC treatment, all subjects received multi-layer high
compression
bandaging (CobanTM 2; 3M) from the first screening visit until the end of the
Treatment
Period and for up to 2 weeks after the first incidence of RVLU closure was
noted.
[0238] Eligible subjects proceeded to the Treatment Period and all
(except those in
the SOC-alone group) were assigned to double-blind treatment in one of three
dose arms ((l)
3.0 mg/mL Polynucleotide Formulation comprising 3.0 mg/mL Polynucleotide ("3.0
mg/mL
Polynucleotide Formulation" or "3.0 mg/mL"), (2) 1.0 mg/mL Polynucleotide
Formulation,
or (3) Vehicle). Visits were conducted once per week. Subjects progressed to
the Post
Treatment Period for up to 12 weeks of follow-up if the RVLU closed
completely; otherwise
subjects were discharged from the study after the Treatment Period, except SOC-
alone
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subjects who could progress to up 10 weeks of open-label treatment with 3.0
mg/mL
Nexagon .
[0239] 313 subjects met the eligibility criteria and were randomized to
the four
treatment groups with 92, 97, 91 and 33 subjects assigned to the 3.0 mg/mL,
1.0 mg/mL,
Vehicle and SOC-alone treatment groups, respectively. The average age of the
313
randomized subjects was 61.6 years (range 27.0 to 92). The mean BMI was 31.2
m/kg2
(range 16.0 - 45.7).
[0240] All 313 randomized subjects were included in the Intention-To-
Treat (ITT)
population. The Safety Population (SP) population was identical to the ITT
population.
Thirty-one subjects were excluded from the ITT population by the Study
Management
Committee, resulting in a Per Protocol (PP) population of 282 subjects with 87
(94.6%), 83
(85.6%), 85 (93.4%) and 27 (81.8%) in the 3.0 mg/mL, 1.0 mg/mL, Nexagon
Vehicle and
SOC-alone groups, respectively. All analyses presented below were performed on
the entire
ITT population.
[0241] The study included both sVLU and mVLU subjects, enrolled at
random. In
the more severe mVLU population (defined by subjects with more than one VLU),
both raw
and modelled data show a dose response for complete wound healing, and
clinically
significant deltas between the 1.0 mg/mL and 3.0 mg/mL dose concentrations of
SEQ ID
NO:1 and Vehicle. As shown in Table 7 below, the raw values contrast between
3.0 mg/mL
Polynucleotide Formulation and Vehicle is clinically significant at a 25%
delta with mVLU
subjects treated with 3.0 mg/mL showing a greater than 2.4-fold improvement in
healing over
vehicle (a 143% increase in wound healing), and is nearly significant at
p=0.0658. Analysis
with multiple-covariate logistic regression also shows a dose response and
clinically
significant differences between both active doses and Vehicle, and the
difference between 3.0
mg/mL Polynucleotide Formulation and Vehicle was statistically significant at
p=0.0127.
Further analysis with a two-covariate logistic regression model was also
statistically
significant at p=0.042 (showing a 27.5% delta between 3.0 mg/mL and Vehicle,
and a greater
than 5.4-fold improvement in healing over vehicle (a 443% increase in wound
healing)). This
model contained ulcer duration, ratio of baseline wound circumference to area,
wound surface
area reduction during run-in, and baseline wound circumference (with only the
latter two
being statistically significant in the model).
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Table 7: Multiple VLU Subject Data
3.0 mg/mL vs.
3.0 1.0Vehicle
DATA ANALYSIS ing/mL mg/mL Vehicle SOC
Delta P-Value
RAW DATA 42.3% 38.7% 17.4% 25.0%
24.9% 0.0658
LOGISTIC 41.3% 34.4% 7.6% 16.0% 33.7% 0.0127
REGRESSION
[0242] The logical regression in Table 7 was obtained by fitting logistic
model with (a)
treatment, (b) VLU status (multiple vs. single) and (c) all other retained
covariates including
interactions of treatment with BMI and age, to complete closure outcome using
all data).
[0243] The raw value analysis shown above in Table 7 was not adjusted for
covariates.
The logistic regression data was obtained from a model that contains
treatment, multiple VLU
status, treatment by VLU interaction, and retained covariates, including age
and BMI.
[0244] In sum, analysis of the results of the study using statistical
models
recommended by the FDA in its June 2006 Chronic Cutaneous Ulcer Guidance for
Industry
showed that treatment of VLU subjects using the Polynucleotide Formulation
increased the
incidence of complete healing of mVLU subjects and reduced the time to
complete healing of
mVLU when compared to both Vehicle and SOC. This demonstrates that the
pharmaceutical
formulations of this invention, which comprise an anti-connexin 43 modulator
are suprisingly
more effective at treating mVLU.
[0245] There were no safety issues identified in the safety population
(i.e., all subjects
who were randomized into the study whose RVLU was treated with at least one
dose of
investigational product or treatment, according to the randomization schedule;
n = 313), or in
the mVLU population.
[0246] As shown in Table 8, the presence of multiple VLUs is associated
with
multiple covariates that are traditionally known as risk factors for poor
healing. The data
from this study show that in comparison with the single VLU wounds, multiple
VLU lesions
had significantly higher baseline area, circumference, necrotic tissue, HgA lc
and wound
duration; lower degrees of epithelialization and circumference/area ratio at
randomization;
and were found in patients with higher BMI. Other covariates were similar
between single
and multiple VLU subjects. It is noted, in this regard, that the overall
unadjusted incidence of
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complete healing in the combined control groups in this study (i.e., the SOC-
alone and
Nexagon Vehicle groups) was three times less for subjects with multiple
ulcers (20.5%) than
for subjects with only a single VLU (62.4%). This difference is statistically
significant
(p=0.001; chi-squared test), supporting the observation that an ulcer on a
subject with
multiple VLU is harder to heal than a solitary VLU.
TABLE 8: COMPARISON OF COVARIATE VALUES OF SINGLE VS. MULTIPLE
VLU
Covariate mVLU std deviation sVLU std deviation P-value
Baseline area 6.13 4.19 5.04 3.77 0.024
BMI 32.7 6.87 30.7 6.80 0.015
Circumference 11.62 4.53 10.13 4.40 0.006
Necrotic tissue 2.03 6.83 0.71 2.81 0.071
HbAlc 6.13 0.99 5.97 0.80 0.185
Epithelialization 19.71 21.37 26.18 27.03 0.024
Circumference/Area 2.34 0.86 2.59 1.09 0.05
Duration>1 yr 35.42 25.81 0.10
(Fisher Exact)
[0247] The following analyses were performed on the ITT population: (1)
a logistic
model to evaluate the incidence of complete wound closure; (2) a proportional
hazards (Cox)
model to evaluate time to complete healing; and, (3) a linear model to
evaluate wound surface
area reduction. Results for the healing endpoints, showing that the
Polynucleofide
Formulation improved both the incidence of complete wound closure and the time
to
complete healing, are summarized below and in Table 9.
[0248] The 3.0 mg/mL Polynucleotide Formulation was 81.5% more effective
than
Vehicle in incidence of complete RVLU closure (46.1% vs. 25.4%; p = 0.0533),
with the odds
of healing being 151.3% greater in the 3.0 mg/mL group than the Vehicle group
(OR =
2.5132).
[0249] The 3.0 mg/mL Polynucleotide Formulation was also 98.7% more
effective
to Vehicle in time to complete RVLU closure, with the odds of healing first
for unhealed
patients throughout treatment being nearly twice as high in the 3.0 mg/mL
group than the
Vehicle group (HR = 1.9875, p = 0.0712).
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TABLE 9: Key Endpoints for ITT Population in the Study
Incidence of Time to
Dose Complete RVLU Complete RVLIJ
Concentration Closure Closure
(mg/mL) (Probability) (Hazard)
3.0 46.1% 9.06
1.0 29.2% 4.67
0.0 (Vehicle) 25.4% 4.56
SOC-alone 32.3% 2.23
3.0 vs. Vehicle p = 0.053 p = 0.071
Vehicle vs. SOC p = 0.592 p = 0.348
Multiple VLU Treatment ¨ Polynucleotide Formulation Treatment and Other
Indicators of
Responder Subjects
[0250] Indicators important in the logistic regression model validated
the expected
effect of the indicator on overall healing, e.g., baseline wound circumference
(p = 0.0016),
ulcer duration (p = 0.0042), wound surface area change during run-in (p =
0.0150), etc. An
important discovery from the statistical analyses is that Polynucleotide
Formulation treatment
interacted with three prognostic indicators of healing: multiplicity of VLU
(mVLU), patient
age, and BMI. For BMI, the comparison between the 3.0 mg/mL dose concentration
vs.
Vehicle, demonstrated improved odds in favor of 3.0 mg/mL in subjects with BMI
less than
42, including a BMI of less than 40. The interactions of multiplicity of VLU
and age with
treatment indicate that subjects with more severe venous disease and
predisposition to poor
healing are optimal for demonstrating the therapeutic effect of the
Polynucleotide
Formulation over Vehicle. Also of note is that the raw data analysis for
multiple VLU
subjects showed a 24.9% difference between the 3.0 mg/mL dose concentration
and Vehicle
for incidence of complete healing (42.3% vs. 17.4%, respectively; p = 0.0658),
which is a
surprisingly large difference for wound healing treatments. The logistic model-
adjusted
incidence of complete healing data showed a larger, 33.7% difference between
the 3.0 mg/mL
dose concentration and Vehicle (41.3% vs. 7.6%, respectively; p=0.0127).
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[0251] The study demonstrated that the Polynucleotide Formulation was
safe and well
tolerated, and showed clinically meaningful efficacy with the 3.0 mg/mL
Polynucleotide
Formulation. It was surprisingly found that treatment with the Polynucleotide
Formulation
was particularly efficacious in more severely diseased patients with multiple
VLUs. In
summary, the study demonstrated that the 3.0 mg/mL dose concentration was safe
and
resulted in marked clinical activity and clinically meaningful efficacy with
Polynucleotide
Formulation compared to Vehicle or SOC, for example using the 3.0 mg/mL in
more severely
diseased patients with multiple VLU.
EXAMPLE 4: TREATING RESPONDER mVLU SUBJECTS
[0252] Clinical trials are conducted to confirm and demonstrate the
safety, tolerability
and efficacy of a formulation comprising 3.0 mg/mL or 10.0 mg/mL
Polynucleotide in the
treatment of mVLU subjects susceptible to treating with an anti-connexin
modulating agent.
[0253] The harder-to-heal multiple venous ulcer population will be the
focus of this
study. Human test subjects are treated with suitable doses of a suitable anti-
connexin 43
polynucleotide formulation applied to all VLU sites under occlusive
compression bandages.
[0254] In order to treat responder subjects likely to respond to
treatment with the anti-
connexin 43 modulating agent of this invention, subjects who have multiple
(unilateral or
bilateral) VLU are included in the study. These subjects were demonstrated to
be the most
difficult to heal. As discussed above, it was demonstrated by Applicants that
the overall
unadjusted incidence of complete healing in the combined control groups (i.e.,
the SOC-alone
and Vehicle groups) was three times less for subjects with multiple ulcers
(20.5%) than for
subjects with only a single VLU (62.4%).
[0255] The formulation may be any formulation of this invention. In one
aspect the
formulation is the 3.0 mg/mL Polynucleotide Formulation of Example 4. Plasma
will be
obtained for PK measurements pre-dosing and 5, 15, 30, 60, 120 and 240 minutes
post-dosing.
[0256] Additional inclusion and exclusion criteria refinements will be
based on age
over 50 and BMI of less than, for example, 42.
[0257] The primary objective of this study is to confirm that the 3.0
mg/mL
Polynucleotide Formulation plus compression bandaging as SOC can improve the
incidence
of complete wound closure for mVLU subjects compared to Vehicle plus SOC. The
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secondary objectives are to determine whether the 3.0 mg/mL Polynucleotide
Formulation is
safe and tolerable and if the 3.0 mg/mL Polynucleotide Formulation improves
time to
complete wound closure
[0258] Standard-of-care in this study will comprise clinical wound
evaluation by the
Investigator, irrigation of the lesion with warm tap water or normal saline.
Cytotoxic
solutions such as Betadine are prohibited, but brief washing with a mild
antiseptic agent that
is subsequently washed completely off the wound surface is permitted.
Mechanical wound
surface cleaning or debridement will also be used in SOC treatment as
indicated. The
Protocol will require a clean, healthy-appearing wound bed prior to each
application of
Nexagon . The surface of the lesion should be freed of slough, exudate and
devitalized tissue;
however the skin edges should not be excised and therefore, the wound should
not be
enlarged by the procedure. The SOC treatment will also include application of
a primary
dressing to the wound surface (e.g., AllevynTM Non-Adhesive Dressing; Smith &
Nephew)
and wrapping of the mid-foot to the upper calf with a multi-layer compression
secondary
dressing (e.g., CobanTM 2; 3M). The peri-wound skin may be treated with
moisturizing, anti-
fungal or corticosteroid.
[0259] Consented subjects with multiple VLU will enter a two-week
screening period
where baseline assessments and eligibility assessments will be performed,
including: venous
duplex ultrasound to exclude subjects without underlying venous insufficiency,
histopathology to exclude subjects with carcinoma in the VLU, and wound
measurements to
exclude subjects whose VLU is having large changes in size. Centralized review
of the VLU
photographs will be performed by the Medical Monitor to supplement the
Investigator's
judgment of eligibility for randomization, i.e., both the Medical Monitor and
the Investigator
must find that the subject is eligible in order for him/her to be randomized.
[0260] At the first visit, the Investigator will select a VLU that meets
the eligibility
criteria of the protocol to be the reference venous leg ulcer (RVLU). Each
subject will have
only one VLU selected as the RVLU. All other venous ulcers will be
photographed and
identified.
[0261] Subjects who continue to meet all of the inclusion criteria and
have none of the
exclusion criteria after completing the Screening Period will be randomized in
a blinded
fashion in a 1:1 ratio into either the Polynucleotide Formulation or Vehicle
group. Either the
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Polynucleotide Formulation or Vehicle, will be applied weekly to the RVLU
during the
Treatment Period. Other VLU will not receive IP but will receive SOC treatment
as
prescribed by the protocol, including wrapping the entire lower study leg and
proximal foot
compression dressing provided for the study. Polynucleotide Formulation is
applied topically
around the inside edge of the ulcer to be treated and then applied to the
remainder of the
wound bed. This provides approximately 0.3 mg of product exposure per cm2 of
wound
surface area. Due to the safety profile of the Polynucleotide Formulation, and
the lack of any
substantive safety issues as revealed by any nonclinical or clinical study,
there are no special
precautions or recommendations.
[0262] Treating only one reference ulcer with randomized investigational
product
accomplishes important objectives:
[0263] It enables use of well-defined endpoints comparable to those
employed in
other registration studies (incidence and time to complete wound closure of
the reference
ulcer).
[0264] Use of 'a single reference ulcer provides an objective endpoint
that avoids the
complication of grading multiple wounds. By contrast, including the criterion
that all wounds
heal may result in a patient being categorized as a failure even though, for
an example, 3 of 4
wounds completely close at the end of the 12-week study period.
[0265] The presence of SOC-treated non-reference wounds could enable the
in-
patient comparison of vehicle vs. SOC treatment in patients who are randomized
to receive
vehicle. The power of this observation will be high since same-patient
analysis removes any
confounding effects of covariates such as, e.g., diabetes, age, concomitant
medications and
patient compliance.
[0266] For each subject, the Treatment Period will end:
[0267] At the first instance where 100% re-epithelialization of the RVLU
is noted. In
this instance the subject will immediately move to the Post-Treatment Period,
or,
[0268] If the RVLU has not achieved 100% re-epithelialization after the
completion
of the T10 visit. These subjects will be contacted in 30 days to assess for
any serious adverse
events.
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[0269] The Post-Treatment Period is designed to confirm RVLU complete
closure,
determine durability of closure and to continue to monitor for any serious
adverse events.
Complete wound closure is defined as 100% re-epithelialization without
drainage confirmed
at two visits, 14 (+ 1) days apart. If the RVLU opens in the Post-Treatment
Period the subject
will exit the study.
[0270] Following completion of the study, the study is unblinded and the
results
analyzed. The results confirm that treatment of mVLU patients with 3.0 mg/mL
Polynucleotide Formulation results in surprisingly high levels of complete
closure of mVLU
in this difficult to heal VLU population.
Example 5: Increased Expression of Connexin 26 and 30 in Chronic Wounds
[0271] The expression of connexins 26 and 30, in addition to connexin 43,
was
examined in patients with a variety of chronic wounds, including venous leg,
diabetic foot or
pressure ulcers. Wound edge punch biopsies were taken from a cohort of
patients with
venous leg, diabetic foot or pressure ulcers. Wound connexin expression in
each patient was
compared to that in a matched, non-wounded arm punch. Tissue was sectioned,
stained and
imaged by confocal microscopy using identical parameters per patient to permit
quantification. Epidermal Cx43, 26 and 30 and dermal Cx43 were discovered to
be strikingly
up-regulated in every ulcer from all three wound types, indicating that
connexin up-regulation
is a common feature between different types of chronic wounds. This result
supports the
therapeutic targeting of Cx26 and Cx30, alone or in combination with Cx43, to
promote cell
migration and wound healing in chronic ulcers.
[0272] Connexins show dynamic changes in expression following acute
wounding.
In animal studies, Cx43 was shown to be naturally down-regulated in wound edge
(WE)
keratinocytes and fibroblasts as they become migratory, whilst Cx26 and Cx30
were up-
regulated in the epidermal leading edge. (Goliger & Paul (1995), Wounding
alters epidermal
connexin expression and gap junction-mediated intercellular communication, Mol
Biol Cell
6: 1491-501; Coutinho, et al (2003), Dynamic changes in connexin expression
correlate with
key events in the wound healing process, Cell Biol Int 27: 525-41; Mendoza-
Naranjo et al.
(2012a), Targeting Cx43 and N-cadherin, which are abnormally upregulated in
venous leg
ulcers, influences migration, adhesion and activation of Rho GTPases. PloS One
7: e37374;
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Mendoza-Naranjo, et al. (2012b), Overexpression of the gap junction protein
Cx43 as found
in diabetic foot ulcers can retard fibroblast migration, Cell Biol Int 36: 661-
7. In biopsies
from patients with mixed ulcers and DFUs, Cx43, 26 and 30 were detected at
epidermal
wound margins as well as in cells at some distance from the epidermal wound
edge (WE)
(Brandner, et al (2004), Connexins 26, 30, and 43: differences among
spontaneous, chronic,
and accelerated human wound healing, J Invest Dermatol 122: 1310-20), but the
involvement
of Cx regulation within the epidermis in chronic wound persistence has not
been thoroughly
investigated. The Cx status of the cells of the dermis may also be very
important. Recently it
has been reported that Cx43 expression in fibroblasts changes their cell-to-
cell adhesion and
cytoskeletal response during wound healing, with Cx43 up-regulation retarding
their rate of
migration (Mendoza-Naranjo et al., 2012b). Determining the levels of Cx
expression in a
variety of chronic wounds is an important step in our understanding of the
link between Cx
expression and impaired healing.
Biopsy Acquisition, Preservation and Cryosectioning
[0273] Patients were eligible for study inclusion if they were over
18yrs years and had
an uninfected chronic wound present for at least 4 weeks, irrespective of
current or previous
treatments. Wound etiology was taken from the clinician's notes. Table 10
shows the
clinical characteristics of the patients in this study.
Table 10: Clinical Characteristics
VLU (n = 19)1 DFU (n = 11) PRU (n = 6) 2
59 59 62
Age
[31-79] [48-82] [34-88]
Male (%) 63% 64% 83%
Wound Location
Gaiter/lower leg (%) 79 % NA NA
Ankle (%) 16 % NA NA
Foot (%) 5% NA NA
Dorsal foot (%) NA 46% NA
Plantar foot (%) NA 36 % NA
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Toe (%) NA 9% NA
Ankle (%) NA 9 % NA
Sacral (%) NA NA 67 %
Malleolus (%) NA NA 17 %
Heel (%) NA NA 17 %
Median Wound Age 6 (17) 3 4 21
(Months) [1.5-108] [1-26] [4-48]
3 months (%) 35% 46% 0%
> 3 - 6 months (%) 24% 27% 17%
> 6 - 12 months (%) 12% 18% 17%
> 12 months (%) 29 % 9 % 66 %
Median Wound Size 9.9 6.6 7.0
(cm2) [2-113] [0.56-22.2] [3.4-40.5]
Diabetes Present (%) 58 % (17) 100 % 4 60 % (5)
IDD 11% 55% 33%
10.4 % (4) 7.3 % (5)
HbAlc (%) Unknown
[6.5-13.5] [6.4-9.9]
46(12) 34 27
BMI
[28-70] [24-44] [25-29]
[0274] All values in Table 10: Median [range]; (n) = number of subjects
with data
available when lower than the full cohort. Abbreviations used in Table 10
include: VLU,
venous leg ulcer; DFU, diabetic foot ulcer; PRU, pressure ulcer; lDD, insulin
dependent
diabetes; HbAlc, hemoglobin Alc; BMI, body mass index; NA, not applicable.
[0275] Wound edge biopsies of chronic wound tissue (VLU: n=19 patients;
DFU:
n=11; PRU: n=6) were obtained during the Outpatients Clinic visit by a single
operator (TES)
via a 4 mm full thickness punch biopsy taken from the visible WE. The biopsy
side away
from the open wound was marked with ink to keep the sample orientated
throughout
processing. Each patient also supplied a matched 4 mm punch biopsy of arm
skin, providing
unwounded baseline Cx expression levels.
[0276] All biopsies were immediately immersed in 4 % paraformaldehyde
for 24
hours and then transferred into 20 % sucrose in phosphate buffered saline
(PBS). Tissue
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blocks were embedded in optimal cutting temperature medium (OCT) (BDH - Poole,
UK)
and stored at -80 C. Frozen sections, 14 gm thick, were obtained using a
Leica CM1900 UV
cryostat and positioned on gelatine-coated slides.
Immunohistochemistry
[0277] Frozen sections were defrosted and immersed in PBS to dissolve
excess OCT.
The tissue was permeabilized for 5 minutes in acetone and non-specific binding
was blocked
using PBS-lysine (0.1 M) over a 30 minute period. Primary antibodies were
prepared in
PBS-lysine (Cx43 1:4000 (Sigma - Poole, UK - C6219), Gap28H (Cx26) 1:200 (Diez
et al.
(1999), Assembly of heteromeric connexons in guinea-pig liver en route to the
Golgi
apparatus, plasma membrane and gap junctions, Eur J Biochem 262: 142-8), Cx30
1:200
(Invitrogen - Paisley, UK - 71-2200), Smooth Muscle Actin 1:200 (Sigma -
Poole, UK -
A2547)). The tissue was incubated in a humid staining chamber with the primary
antibody
for 1 hour at room temperature. The tissue was washed with PBS-lysine for 3x5
minutes
followed by application of the secondary antibody (Invitrogen - Paisley, UK -
Alexa Fluor
488 goat anti-rabbit 1:400 or Alexa Fluor 568 goat anti-mouse 1:400) in
conditions
identical to those used with the primary antibody for 1 hour. Nuclei were
stained using
HOECHST (Sigma - Poole, UK - B-2883 and B-2261 1:50,000 in PBS) for a 5 minute
period
followed by 2x10 minute PBS washes. Coverslips were mounted using Citiflour
(Glycerol/PBS solution, Citiflour Ltd, London, UK).
Confocal Microscopy
[0278] For the 4 mm biopsy samples an Olympus FV-1000 inverted confocal
microscope was used to obtain 10x and 20x qualitative montage images of whole
tissue
sections and 40x quantitative images (epidermis and dermis) of the arm and
wound. The 4
mm biopsies were examined (epidermis and dermis) across their diameter at
three locations:
at the WE, 1 mm from the WE, and at the far edge (14h). Hoescht was excited by
a 405 nm,
Alexa Fluor 488 by a 488 nm and Alexa Fluor 568 by a 565 nm wavelength
laser.
Image Quantification and Statistical Analysis
[0279] Cx quantification was carried out using ImageJ. Epidermal and
dermal
thresholds were kept constant between all images being set at 80 and 100-255
respectively
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with a recognized pixel threshold size of 2-infinity utilized for all images
(Wang et al. (2007),
Abnormal connexin expression underlies delayed wound healing in diabetic skin,
Diabetes 56:
2809-17). In the epidermis Cx expression was related to the cell number as
pixels/cell and in
the dermis as pixe1s/ium2.
[0280] The data from the connexin measurements is presented in the
results section as
the "absolute Cx expression level" which was used for the statistical analysis
(below) and is
presented in the graphs (Fig 2-4). The corresponding fold change data is in
the tables as i)
"fold difference of the group means", this being the fold difference between
the forearm
biopsy group mean and the various wound location group means (WE, 1 mm, FE));
and ii)
the "mean of the individual fold changes." This was based on calculating each
individual's
unique fold difference by first normalizing their wound biopsy Cx expression
to their
matched forearm Cx level. Then a mean individual fold difference was
calculated for each
study group (i.e., this was the mean of the individually normalized Cx fold
changes). This
dataset gives an indication of how much the individual Cx fold differences
could vary
between patients.
Statistical Analysis
[0281] The Cx expression data were analyzed using a two-way ANOVA, the
two
factors/variables being location (i.e., arm, WE, 1 mm from the WE, and 1-1,)
and patient. The
residuals were tested for normality using the Kolmogorov-Smirnoff test; with a
parametric
distribution being assumed in all cases where the p-value ?. 0.05. Normality
was not reached
in three groups: VLU Cx30, DFU Cx30 and DFU Cx43 epidermal values. These
specific
data sets were independently transformed using the natural log before
analysis.. A Dunnett's
post-hoc test compared all three wound measurements back to the reference
group, i.e., arm
values. Significance was taken at values p 0.05.
Features of Chronic Wound Biopsies
[0282] The histology of chronic wound biopsies varied but consistent
features were
identified that distinguished them from healthy tissue as seen in Fig. 1, a
representative VLU.
These include increased depth to the epidermal rete pegs, a greater number of
blood vessels,
and a large abundance of neutrophils both within dead tissue at the WE and
throughout the
dermis.
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[0283] In acute wounds the early hallmark of active healing is the
formation of a thin
keratinocyte tongue at the WE, indicating the start of re-epithelialization.
These cells have a
migratory phenotype and crawl forward across the wound bed. None of the DFU
biopsies
presented with a thinning of the epidermal WE. However, a thinning tongue of
WE
keratinocytes was identified in some VLUs (6/19 biopsies), which may represent
the
beginning of healing or attempts to heal in some wounds. In the pressure ulcer
(PRU) cohort,
two out of the six wounds examined had this feature.
Biopsies from Venous Leg Ulcers
[0284] Biopsies from VLUs revealed several consistent features (Fig. 2).
The
epidermis of the 4 mm biopsies were typically hyper-thickened, increasing in
depth with
distance from the WE. However, in some samples, the epidermis consistently
thinned
towards the WE and had the appearance consistent with a migratory phenotype,
as noted
above. The epidermal expression of Cx43 and Cx30 were increasingly elevated
along the
length of the biopsy as the epidermis became increasingly thickened upon
moving away from
the WE, whereas Cx26 was uniformly elevated in the epidermis along the biopsy.
The levels
of Cx43 at the epidermal WE of these biopsies, whilst having a 4-fold higher
absolute group
mean than that seen in the normal unwounded arm tissue were not significantly
different.
However, 1 mm from the WE the absolute group mean elevation in Cx43 was 8-fold
higher
than the reference arm tissue and highly statistically significant (p< 0.01),
whilst on the far
edge (14E,) of the biopsy the increase was on average 14-fold and very highly
statistically
significant (p< 0.001). Cx26 and Cx30 are normally expressed at relatively low
levels in the
intact skin in comparison to Cx43 but were reported to be increased in hyper-
proliferative
human keratinocytes (Rivas et al. (1997), Identification of aberrantly
regulated genes in
diseased skin using the cDNA differential display technique, J Invest Dermatol
108: 188-94;
Labarthe et al. (1998), Upregulation of connexin 26 between keratinocytes of
psoriatic
lesions, J Invest Dermatol 111: 72-6). These two proteins had a many-fold
greater elevation
than that observed for Cx43 in the chronic wound tissues examined. For
example, epidermal
WE Cx30 was significantly elevated by an average of 213-fold rising to 226-
fold at the
thicker, FE location (p < 0.01 and p < 0.001). Cx26 was also significantly
elevated, 73-fold
at the WE epidermis, rising to 123-fold at the FE, of the biopsy when compared
to the
matched, intact reference tissue (p < 0.001).
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[0285] A common feature within the dermis of VLUs was an increased
number of
blood vessels (Fig. 1) along with a loss of the auto-fluorescent extracellular
matrix in the
upper third of the dermis (Fig. 2c). Dermal fibroblasts do not express Cx26 or
Cx30 but do
express Cx43 and this was significantly elevated across the dermis, increasing
by 20-fold at
the WE and 32-fold at the PE when compared to matched, unwounded tissue (p <
0.01 and p
< 0.001). The corresponding mean of the individual normalized fold changes for
each Cx are
found in Fig. 2d.
Biopsies from Diabetic Foot Ulcers
[0286] Biopsies from DFUs also had common features (Fig. 3). The
epidermis was
hyper-thickened but, unlike VLUs, this was more uniform in DFU samples. None
of the
biopsies showed any signs of thinning towards the WE and had no appearance of
healing (Fig.
3c). Like the VLU, the DFU also had elevated levels of Cx expression but this
was fairly
consistent across the length of the biopsy. The Cx43 absolute group mean was
elevated by 9-
fold at the WE and 7-fold at the FE when compared to the unwounded forearm
(both p <
0.001). Cx26 and Cx30 were also significantly increased, by 62-fold (p < 0.05)
and 201-fold
(p < 0.001) respectively, at the WE and 64-fold (p < 0.05) and 115-fold (p <
0.001) at the FE
side of the biopsy.
[0287] The dermis of the DFUs was distinctly different from that of the
VLUs, as in
many cases it lacked any signs of auto-fluorescent signal from the fibers of
the dermal
extracellular matrix or, if auto-fluorescence remained, the organizational
pattern was absent.
This suggested that a large proportion of the native collagen and elastin had
either been
degraded or was no longer being arranged into mature fibrils. The DFU dermis
featured
significantly increased levels of Cx43, by an average of 20-fold at the WE and
18-fold on the
PE of the biopsy (p < 0.05 and p < 0.01). These data and the means of the
individual
normalized fold changes for each Cx are found in Fig. 3d.
Biopsies from Pressure Ulcers (PRUs)
[0288] Biopsies from PRUs were variable in their appearance (Fig. 4).
The epidermis
was typically thickened along the length of the biopsy with the formation of
deep rete pegs.
The degree of healing was variable, manifesting in some instances as a
thinning tongue of
WE epidermis and reminiscent of the VLUs. Again, Cx expression was elevated in
the
epidermis but unevenly so at the WE. The Cx43 absolute group mean was
significantly
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increased at 1 mm by 10-fold (p < 0.01), whilst Cx26 was elevated 90-fold and
Cx30 by 471-
fold when compared to the low baseline levels found in intact arm skin (p <
0.01 and p<
0.001).
[0289] The dermis of the PRUs could be distinguished from the VLU and DFU by
the
consistent presence of an auto-fluorescent signal from the extracellular
matrix. Cx43
expression was significantly increased on average 58-fold at the WE and 37-
fold on the 1-E
side of the wound (p < 0.05). These data and the means of the individual
normalized fold
changes for each Cx are found in Fig. 4d.
Distribution of Epidermal Connexin (Cx) Overexpression
[0290] The distribution of connexins within the epidermis varied along
the length of
the biopsies and with depth corresponding to the varying layers of the
epidermis. The deep
rete pegs were characterized by a dominance of Cx26 and 30. In some regions, a
large
proportion of the cell membrane appeared to be taken up by connexins, giving
the staining a
"fish scale" appearance.
[0291] As discussed above, it has therefore been demonstrated that a
statistically
significant, substantial up-regulation of three connexin gap junction proteins
in VLUs, DFUs
and PRUs, i.e., epidermal connexin 26, connexin 30 and connexin 43 and dermal
connexin 43.
Precise spatial and temporal control of connexin proteins has been shown to be
integral to the
regular wound reparatory process, where down-regulation of the Cx43 at the
wound edge is
correlated to keratinocyte and fibroblast migration. The Cx misregulation we
have identified
here may serve to slow healing and/or prolong ulceration (Wang et al. 2007).
Epidermal Over-Expression of Connexin26 (Cx26) and Connexin30 (Cx30)
[0292] To date most research on Cx dynamics throughout wound repair has
focused
on elucidating the role of Cx43. Cx26 and Cx30 are usually only detected at
very low levels
within the intact interfollicular epidermis but are significantly up-regulated
post-wounding
within the migratory epidermal leading edge (Coutinho et al., 2003).
Examination of these
proteins within chronic wound tissue found them both to be significantly over-
expressed
across the entirety of the epidermis, which correlates with a variety of skin
proliferative
conditions. For example, up-regulation of Cx26 and/or Cx30 has previously been
reported in
psoriasis (Lemaitre, et al. (2006), Connexin 30, a new marker of
hyperproliferative epidermis,
Br J Dermatol 155: 844-6; Lucke et al. (1999), Upregulation of connexin 26 is
a feature of
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keratinocyte differentiation in hyperproliferative epidermis, vaginal
epithelium, and buccal
epithelium, J Invest Dermatol 112: 354-61), warts (Lucke et al., 1999) and a
variety of
genetically inherited conditions that lead to skin abnormalities, such as
Porokeratosis of
Mibelli (Hivnor et al. (2004), Gene expression profiling of porokeratosis
demonstrates
similarities with psoriasis, J Cutan Pathol 31: 657-64), and Clouston
syndrome(Lemaitre et al,
2006). Based on the data from the experiments as descried herein, it has been
recognized that
the common phenotypic factor between these syndromes and chronic wounds is
keratinocyte
hyper-proliferation.
[0293] Keratinocyte proliferation and differentiation is misregulated in
DFUs and
VLUs (Stojadinovic et al, 2008; Usui et al, 2008). In VLUs there is a loss of
cell cycle
control, along with the mis-expression of activation and differentiation
pathways
(Stojadinovic et al. (2008), Deregulation of keratinocyte differentiation and
activation: a
hallmark of venous ulcers, J Cell Mol Med. 12: 2675-90). In DFUs,
keratinocytes at the WE
are hyper-proliferative, independent of ulcer edge thickness. Interestingly
this extends into
the non-ulcerated region, with tissue of a histologically "normal" phenotype
staining strongly
for the cell proliferation marker, Ki67 (Usui et al. (2008), Keratinocyte
migration,
proliferation, and differentiation in chronic ulcers from patients with
diabetes and normal
wounds, J Histochem Cytochem 56: 687-96). The over-expression of Cx26 and
Cx30, as
detected along the entire length of the 4 mm punch biopsy independent of ulcer
type, could
reflect their direct involvement in the epidermal thickening.
[0294] Studies of acute incisional wound healing in transgenic mice,
where Cx26 is
ectopically expressed within keratinocytes, showed delayed wound healing and a
hyper-
proliferative epidermal state (Djalilian et al. (2006,. Connexin 26 regulates
epidermal barrier
and wound remodeling and promotes psoriasiform response, J Clin Invest 116:
1243-53.
After 21 days post-wounding only 42% of the heterozygous mice had healed
whilst full
epidermal barrier restoration was seen in all wild-type animals by day 14
(Djalilian et al,
2006). Alternatively, Cx26 and 30 may be markers of hyper-proliferation with
their
expression predominantly influencing cellular differentiation.
Epidermal and Dermal Connexin43 (Cx43) Over-Expression
[0295] In this Example, human chronic wounds of the three major
etiologies were
found to have abnormally high connexin expression at the WE. By way of
example, Cx43
WE expression was 9 times greater in the DFU cohort than basal, unwounded skin
levels.
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When this is considered in the context of the substantial preclinical data
that links delayed
healing with elevated Cx43 expression, it strongly indicates that the up-
regulation of this
protein is likely a common feature of chronic wound pathology.
[0296] In summary, it has been demonstrated that the over-expression of
Cx26, Cx30,
and Cx43 in the epidermis and that of Cx43 in the dermis of ulcer biopsies is
a signature
feature of chronic wounds, identified in all patients irrespective of ulcer
type, i.e., VLU, DFU
or PRU.
[0297] The present invention is not limited by the aforementioned
embodiments. It
will occur to those ordinarily skilled in the art that various modifications
may be made to the
disclosed embodiments with-out diverting from the concept of the invention.
All such
modifications arc intended to be within the scope of the present invention.
[0298] All patents, publications, scientific articles, web sites, and
other documents
and materials referenced or mentioned herein are indicative of the levels of
skill of those skilled
in the art to which the invention pertains, and each such referenced document
and material is
hereby incorporated by reference to the same extent as if it had been
incorporated by reference in
its entirety individually or set forth herein in its entirety. Applicants
reserve the right to
physically incorporate into this specification any and all materials and
information from any such
patents, publications, scientific articles, web sites, electronically
available information, and other
referenced materials or documents.
[0299] The written description portion of this patent includes all
claims. Furthermore,
all claims, including all original claims as well as all claims from any and
all priority documents,
are hereby incorporated by reference in their entirety into the written
description portion of the
specification, and Applicants reserve the right to physically incorporate into
the written
description or any other portion of the application, any and all such claims.
Thus, for example,
under no circumstances may the patent be interpreted as allegedly not
providing a written
description for a claim on the assertion that the precise wording of the claim
is not set forth in
haec verba in written description portion of the patent.
[0300] All of the features disclosed in this specification may be
combined in any
combination. Thus, unless expressly stated otherwise, each feature disclosed
is only an example
of a generic series of equivalent or similar features.
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[0301] It is to be understood that while the invention has been
described in
conjunction with the detailed description thereof, the foregoing description
is intended to
illustrate and not limit the scope of the invention, which is defined by the
scope of the appended
claims. Thus, from the foregoing, it will be appreciated that, although
specific embodiments of
the invention have been described herein for the purpose of illustration,
various modifications
may be made without deviating from the spirit and scope of the invention.
Other aspects,
advantages, and modifications are within the scope of the following claims and
the present
invention is not limited except as by the appended claims.
[0302] The specific methods and compositions described herein are
representative of
preferred embodiments and are exemplary and not intended as limitations on the
scope of the
invention. Other objects, aspects, and embodiments will occur to those skilled
in the art upon
consideration of this specification, and are encompassed within the spirit of
the invention as
defined by the scope of the claims. It will be readily apparent to one skilled
in the art that
varying substitutions and modifications may be made to the invention disclosed
herein without
departing from the scope and spirit of the invention. The invention
illustratively described herein
suitably may be practiced in the absence of any element or elements, or
limitation or limitations,
which is not specifically disclosed herein as essential. Thus, for example, in
each instance herein,
in embodiments or examples of the present invention, the terms "comprising",
"including",
"containing", etc. are to be read expansively and without limitation. The
methods and processes
illustratively described herein suitably may be practiced in differing orders
of steps, and that they
are not necessarily restricted to the orders of steps indicated herein or in
the claims.
[0303] The terms and expressions that have been employed are used as
terms of
description and not of limitation, and there is no intent in the use of such
terms and expressions to
exclude any equivalent of the features shown and described or portions
thereof, but it is
recognized that various modifications are possible within the scope of the
invention as claimed.
Thus, it will be understood that although the present invention has been
specifically disclosed by
various embodiments and/or preferred embodiments and optional features, any
and all
modifications and variations of the concepts herein disclosed that may be
resorted to by those
skilled in the art are considered to be within the scope of this invention as
defined by the
appended claims.
[0304] The invention has been described broadly and generically herein.
Each of the
narrower species and subgeneric groupings falling within the generic
disclosure also form part of
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the invention. This includes the generic description of the invention with a
proviso or negative
limitation removing any subject matter from the genus, regardless of whether
or not the excised
material is specifically recited herein.
[0305] It is also to be understood that as used herein and in the
appended claims, the
singular forms "a," "an," and "the" include plural reference unless the
context clearly dictates
otherwise, the term "X and/or Y" means "X" or "Y" or both "X" and "Y", and the
letter "s"
following a noun designates both the plural and singular forms of that noun.
In addition, where
features or aspects of the invention are described in terms of Markush groups,
it is intended, and
those skilled in the art will recognize, that the invention embraces and is
also thereby described in
terms of any individual member and any subgroup of members of the Markush
group, and
applicants reserve the right to revise the application or claims to refer
specifically to any
individual member or any subgroup of members of the Markush group.
[0306] Other embodiments are within the following claims. The patent may
not be
interpreted to be limited to the specific examples or embodiments or methods
specifically and/or
expressly disclosed herein. Under no circumstances may the patent be
interpreted to be limited
by any statement made by any Examiner or any other official or employee of a
Patent Office
unless such statement is specifically and without qualification or reservation
expressly adopted in
a responsive writing by Applicants.
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