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 APPLICATIONS
[0002] This
Application claims benefit of US Provisional Application No.
61/953,604, filed on March 14, 2014, and of US Provisional Application No.
51/953,608,
filed on March 14, 2014, the contents of each of which are hereby expressly
incorporated
by reference as if set forth in their entirety.
BACKGROUND
[0003] Normal wound healing moves through phases in a timely and
uncomplicated fashion (hemostasis, 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;
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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 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, Intemat 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
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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 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
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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.
DESCRIPTION OF THE DRAWINGS
[0010] Figure 1
represents blood vessels in VLUs. Blood vessel staining (green)
ihn 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
um 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 Cx43 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 and associated 'summary graphs for the WE, 1 mm
from the
WE and at the FE. Scale bar - 10x Motages 1000 um; 40x Images 1001AM. Cx43, 26
and
30 = Green; Nuclei = Blue. "p<0.01; ***p<0.001. Error bars - Mean +/- SEM
(Epidermis - n = 19 except NE = 14; Dermis - n = 17). VLU, venous leg ulcer;
Cx,
connexin; WE, wound edge; FE, 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 NE. Scale bar - 10x Montages 1000 um; 40x Images 100 um. 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 directly to the reference arm
values. (f-y)
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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 pm; 40x Images 100 pm. 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.
[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 Cx43 in dermis normalised to patient baseline
expresion.
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. Another factor which may be
considered in
combination with other factors indicative of resistant lesions includes 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
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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 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
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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 riv/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 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.
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[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.
[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
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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
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
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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 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
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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 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.
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[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 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
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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 hemichannel
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
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.
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[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 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
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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, and those with relatively few signs
of healing
during a screening period with standard therapy, e.g, compression bandaging
therapy in
the case of 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 10 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
another factor includes 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, as measured by, for
example,
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
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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 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 A lc
(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. Still another factor to be considered in
conjunction with
other factors is 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, as measured by, surface area reduction or percent lesion surface
area
reduction.
[0051] In one
embodiment, where the amount of healing during a pre-treatment or
run-in period with standard-of-care treatment is used as one factor, together
with other
factors, in characterizing 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 as one factor in conjunction with other factors 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 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 preferably by less than about 20%. In other embodiments, the
lesion 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 utilized 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
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preferably less than about 30%, the lesion is a resistant lesion for purposes
of this
invention. In still other embodiments, the lesion 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.). These amounts of healing can
be used in
combination with other factors to determine if a wound is a resistant wound.
[0052] 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.
[0053] 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 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.
[0054] 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
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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 HbAl c of greater than 6.0%, 6.5% or greater than
7.0%.
[0055] 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.
[0056] 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 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
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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-resistant 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
[0057] 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 (HbAlc) of 6.5 % or greater.
[0058] 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.
[0059] 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.
[0060] 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%
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
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subject is a likely responder subject can also be used in conjunction with the
methods of
treatment and uses described herein.
[0061] 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-1RA/IL-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.
[0062] 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 g collagenase equivalents/ml) 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 g 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.
[0063] In still
other embodiments, a resistant wound will contain high levels of
1L1, IL6, and matrix metalloproteinases (MMPs), and an abnormally high MMP to
TI1VIP
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 MMPs are inhibited by
specific
endogenous tissue inhibitor of metalloproteinases, which comprise a family of
four
protease inhibitors: TIMP-1, TIMP-2, TIMP-3, and TIMP-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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[0064] In some
embodiments, a resistant wound may also be be characterized by
low levels of TGFI3 and/or low levels of one or more MMP tissue inhibitors
(TIIVIP), for
example TIMP-1 or TEMP-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 IL-1 r3 or
any
combination thereof. Higher levels of IL-la, IL-10, IL-
12p40, GM-CSF and IL-
IRA may also be present at elevated levels in resistant wounds.
[0065] 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.
[0066] 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.
[0067] As used
herein, "preventing" means preventing in whole or in part, or
ameliorating or controlling.
[0068] 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.
[0069] 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.
[0070] 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.
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"Peptidomimetics" (also known as peptide mimetics) which include peptide-based
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.
[0071] 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
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example a sheet of freeze-dried collagen, freeze-dried collagen/alginate
mixtures
(available under the Registered Trade Mark FIBRACOL from Johnson & Johnson
Medical Limited) or freeze-dried collagen/oxidized regenerated cellulose
(available under
the Registered Trade Mark PROMOGRAN from Johnson & Johnson Medical Limited).
[0072] 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
Ain 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.
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[0073] 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
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 Vasc Surg 1:181-91). The invention contemplates a synthetic or
natural matrix
comprising one or more anti-connexin protein agents.
[0074] 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
[0075] 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
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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.
[0076] 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.
[0077] 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.
[0078] 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
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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 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.
[0079] 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.
[0080] 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 hemichannel 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.
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Anti-Connexin Protein Polynucleotides
[0081] Anti-connexin polynucleotides include connexin antisense 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, connexin protein oligodeoxynucleotides, connexin protein
RNAi
polynucleotides and connexin protein siRNA polynucleotides.
[0082]
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.
[0083]
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.
[0084] 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,
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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.
[0085] 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 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.
[0086] 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.
[0087] 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.
[0088] 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.
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[0089]
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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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
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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'
5' TCC TGA GCA ATA CCT AAC GAA CAA ATA 3' (connexin 26) (SEQ.ID.N0:21)
5' CTC AGA TAG TGG CCA GAA TGC 3' (connexin 30) (SEQ.ID.N0:22)
5' TTG TCC AGG TGA CTC CAA GG 3' (connexin 30) (SEQ.ID.N0:23
[0094] 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:
[0095]
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
[0096] 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%
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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).
[0097] 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.
[0098] 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.
[0099] 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.
[0100] 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
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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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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
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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.
[0106] In one
aspect the pharmaceutical formulations of this invention comprise
an unmodified oligonucleotide specific to connexin 43 mRNA having the sequence
5'-
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.
[0107] 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
[0108] 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.
[0109] 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
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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).
[0110] 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 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.
[0111]
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.
[0112] 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).
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[0113] 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).
[0114] 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.
[0115] 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,
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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.
[0116] 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
((fiEVAFLLIQWI
(SEQ ID NO:5), 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.
[0117] 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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[0118] Connexin 43 (SEQ ID NO. 4)
Connexin 43 (SEQ 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 Ile 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 Ile 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 Ile Ile Ser Ile Leu
145 150 155 160
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te 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
Pro His Gln Val Asp Cys Phe Leu Ser Arg Pro Thr Glu Lys Thr Ile
195 200 205
Phe Ile Ile Phe Met Leu Val Val Ser Leu Val Ser Leu Ala Leu Asn
210 215 220
lle 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
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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
[0119] The anti-
connexin peptides, for example, anti-connexin 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-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.
[0120]
Exemplary peptides targeted to connexin 43 are shown below in Table 2.
Ml, 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)
FEVAFLLIQWI M3 & E2 (SEQ.ID.N0:5)
LLIQWYIGFSL E2 (SEQ.ID.N0:6)
SLSAVYTCKRDPCPHQ E2 (SEQ.ID.N0:7)
VDCFLSRPTEKT E2 (SEQ.ID.N0:8)
SRPTEKTIFII E2 & M4 (SEQ.ID.N0:9)
SRPTEKT E2 (SEQ.ID.NO: 10)
LGTAVESAWGDEQ M1 & El (SEQ.ID.N0:11)
QSAFRCNTQQPG El (SEQ.ID.N0:12)
QQPGCENVCYDK El (SEQ.ID.N0:13)
VCYDKSFPISHVR El (SEQ.ID.N0:14)
KRDPCHQVDCFLSRPTEK E2 (SEQ.ID.NO: 15)
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[0121] 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.
Table 3. Extracellular loops for connexin proteins
El
huCx26 KEVWGDEQADFVCNTLQPGCKNVCYDHYFPISHIR (SEQ.ID.NO: 24)
huCx30 QEVWGDEQEDFVCNTLQPGCKNVCYDHH-PVSH1R (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)
[0122] 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 SRP __ IEKT (SEQ.ID.NO: 10)
huCx43 LLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTIFII
(SEQ.ID.NO: 18)
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huCx26
MYVFYVMYDGFSMQRLVKCNAWPCPNTVDCFVSRPTEKTVFTV
(SEQ.ID.NO: 28)
huCx30 YVFYFLYNGYHLPWVLKCGIDPCPNLVDCFISRP _______________ IEKTVFTI
(SEQ.ID.NO: 29)
[0123] 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 without causing uncoupling of gap junctions (See Leybeart et al.,
Cell
Commun. Adhes. 10: 251-257 (2003)), or do so in lower dose amounts.
[0124] A
peptide comprising SRPTEKT (SEQ.ID.NO: 10), for example
VDCFLSRPTEKT(SEQ.ID.NO: 8) or SRP
(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.
[0125] 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.
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[0126] 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 agcttttacg
61 aggtatcagc acttttcttt 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
361 ggggacagcg gttgagtcag cctggggaga tgagcagtct gcctttcgtt gtaacactca
421 gcaacctggt tgtgaaaatg tctgctatga caagtctttc ccaatctctc atgtgcgctt
481 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
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1741 gggtggtaat tgtggctaaa tatttttgtt ttaccaagaa actgaaataa ttctggccag
1801 gaataaatac ttcctgaaca tcttaggtct tttcaacaag aaaaagacag aggattgtcc
1861 ttaagtccct gctaaaacat tccattgtta aaatttgcac tttgaaggta agattctag
1921 gcctgaccct ccaggtgtca atggacttgt gctactatat ttttttattc ttggtatcag
1981 tttaaaattc agacaaggcc cacagaataa gattttccat 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 tat-lava atgtaatcat tgatgcttga atgatagaat
2581 tttagtactg taaacaggct ttagtcatta atgtgagaga cttagaaaaa atgcttagag
2641 tggactatta aatgtgccta aatgaatttt gcagtaactg gtattcttgg gttttcctac
2701 ttaatacaca gtaattcaga acttgtattc tattatgagt ttagcagtct tttggagtga
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
[0127] The Cx 26 cDNA coding reference sequence NG_008358.1 (SEQ ID
NO.30) is shown below in Table 5B. An anti-cormexin 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
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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
[0128] The Cx 30 cDNA coding reference sequence NA/1_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
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
[0129] 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.
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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.
[0130] Still other anti-connexin agents include connexin carboxy-
terminal
polypeptides. See Gourdie et al., W02006/069181.
[0131] 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' -
dihydroxychalc one) ; CHFs (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 -
glycyrrhetinic
acid, and derivatives thereof; lindane; lysophosphatidic acid; mefloquine;
menadione; 2-
Methy1-1,4-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
[0132] 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.
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[0133] 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%.
[0134] 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.
[0135] 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.
Dosage Forms and Formulations and Administration
[0136] 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.
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[0137] 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.
[0138] 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.
[0139] 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
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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.
[0140] 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.
[0141] 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.
[0142] Preferably the agents of the invention are combined with a
pharmaceutically acceptable carrier or diluent to produce a pharmaceutical
composition.
Suitable carriers and 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.
[0143] The term "pharmaceutically acceptable carrier" refers to any
pharmaceutical carrier that does not itself induce the production of
antibodies harmful to
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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.
[0144]
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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
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[0149] 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.
[0150] 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.
[0151] 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.
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[0152] 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.
[0153] 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-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.
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[0154] 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., [tg/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.
[0155] 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 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
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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.
[0156] In
certain embodiments, the anti-connexin agent composition may be
applied at about 0.01 micromolar (IAM) or 0.05 1AM to about 200 [IM, or up to
300 [IM or
up to 1000 1AM or up to 2000 11M or up to 3200 !AM 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
1AM. Preferably, the antisense polynucleotide composition is applied at about
1000 p,M 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.
[0157]
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
04 to about
20 !AM final concentration, and alternatively the anti-connexin agent
composition is
applied at about 1011M to about 201AM final concentration, or at about 10 to
about 15 1AM
final concentration. In certain other embodiments, the anti-connexin agent is
applied at
about 10 !AM final concentration. In yet another embodiment, the anti-connexin
agent
composition is applied at about 1-15 1tM final concentration. In other
embodiments, the
anti-connexin agent is applied at about a 20 1tM, 30 [tM, 40 p,M, 50 1tM, 60
1tM, 70 1tM,
80 !AM, 90 1tM, 100 1tM, or from about 10-200 1AM, 200-300 !AM, 300-400 11M,
400-500
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!AM, 500-600 M, 600-700 1AM, 700-800 M, 800-900 [AM, 900-1000 or 1000-1500
M ,
or 1500 [AM ¨ 2000 jiM, 2000 [AM - 3000 [AM, 3000 [AM - 4000 [AM, 4000 [AM -
5000 [AM,
5000 jtM - 6000 [AM, 6000 1AM - 7000 jtM, 7000 jiM - 8000 jtM, 8000 [AM - 9000
[AM,
9000 M ¨ 10,000 [AM, 10,000 jAM ¨ 11,000 !AM, 11,000 jtM ¨ 12,000 [AM, 12,000
!AM ¨
13,000 [AM, 13,000 [AM ¨ 14,000 M, 14,000 [AM ¨ 15,000 !AM, 15,0001AM ¨
20,000 jtM,
20,000 jtM ¨ 30,000 M, 30,000 [AM ¨ 50,000 [AM, 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.
[0158] 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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[0159]
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.
[0160] 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. 1,ig/11,1) per
length, depth,
area, or volume of the area of application. The volume of the wound may also
be
determined by imaging.
[0161] 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.
[0162] 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
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embodiment the first composition is administered before and after the second
composition. In one embodiment the second 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.
[0163] 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.
[0164]
Preferably one or more anti-connexin agents, such as anti-connexin 43
polynucleotides, are delivered by topical administration (peripherally or
directly to a site),
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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 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.
[0165] 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.
[0166] 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
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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.
[0167] 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.
[0168] 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
[0169] 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 pepticlomimetics 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 carriers 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.
[0170] In
addition to the biological matrices previously mentioned, suitable
dressings or matrices may include, for example, the following with one or more
anti-
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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):
[01711 1)
Absorptives: suitable absorptives may include, for example, absorptive
dressings, which can provide, for example, a semi-adherent quality or a non-
adherent
layer, 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.
[0172] 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.
[0173] 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.
[0174] 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.
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[0175] 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.
[0176] 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
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.
[0177] 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.
[0178] 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.
[0179] 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
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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.
[0180] 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 gauze dressings and woven dressings may be used for cleansing,
packing and
covering a variety of treatment sites.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] 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
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their composition. In certain embodiment, the hydrogel is non-adhesive against
the
treatment site or treated for easy removal.
[0185] 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.
[0186] 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.
[0187] 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.
[0188] 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.
[0189] 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
[0190]
Optionally, one or more anti-connexin protein polynucleotides and/or one
or more anti-connexin protein peptides or peptidomimetics and/or other anti-
connexin
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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.
[0191] 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
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,
[0192] 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.
[0193] 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.
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Treatment
[0194] 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.
[0195] 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.
[0196] 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
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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.
[0197] 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-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.
[0198] 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
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or more of the other indicators described herein, for example, age over 50-52
or BMI less
than 40-42.
[0199] 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.
[0200] 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, 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.
[0201] 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.
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[0202]
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.
[0203] 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.
[0204] 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.
[0205] 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 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.
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[0206] 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.
[0207] 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 formation of
hemichannels or gap
junctions, e.g., by downregulation of connexin protein expression.
[0208] 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-
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fourth the amount, about one-third the amount, and about one-half the amount
when
administered alone.
[0209] 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.
[0210] 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.
EXAMPLES
EXAMPLE 1: Lack of Toxicity
[0211] 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.
[0212]
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. TD. NO:1 were repeatedly
administered to
open wounds in the skin of test animals.
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[0213] 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.
[0214] 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 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.
[0215] 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 (5. 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
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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.
[0216] 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.
[0217] 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 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 pig/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)
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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.
[0218] 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.
[0219] For both
species, the levels of intact Polynucleotide or presumed
metabolites in plasma and systemic tissues were much lower (generally not
detectable)
when samples 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.
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[0220] 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.
[0221] 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.
[0222] 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,
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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.
[0223] 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
[0224] 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 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).
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[0225]
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.
[0226]
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).
[0227] 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 "PE" of the 4 mm biopsy (i.e.,
directly opposite
the wound WE). Figure 5 shows where the assessments were taken across the 4mm
biopsy.
[0228] Image
Quantification and Statistical Analysis: Cx43 quantification was
carried out using IrnageJ. Thresholds were kept constant between all images.
Three
different Cx43 expression measurement locations across the biopsy (i.e., "WE",
"lmm",
"FE") 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 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
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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
uzi ___________________________ ] ____________
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
[0229] As shown
in Table 6, the demographics of the two small study groups
were not significantly different.
[0230] 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).
[0231] 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.
[0232] 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 wounds, showing that multiple wounds are associated
with
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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.
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EXAMPLE 3: Efficacy of Connexin 43 Modulating Agent in
Treating Wounds on mVLU Subjects
[0233] 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.
[0234] The
primary objective of the study was to determine whether the
Polynucleotide Formulation (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.
[0235] 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.
[0236] 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.
[0237] 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 ((I) 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
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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 subjects who could progress to up 10 weeks
of
open-label treatment with 3.0 mg/mL Polynucleotide Formulation.
[0238] 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).
[0239] 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, Vehicle and
SOC-
alone groups, respectively. All analyses presented below were performed on the
entire
ITT population.
[0240] 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
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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).
Table 7: Multiple VLU Subject Data
3.0 mg/mL vs.
3.0 1.0 Vehicle
i SOC
DATA ANALYSIS mg/mL mg/mL Vehicle
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
[0241] 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).
[0242] 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.
[0243] 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.
[0244] 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.
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[0245] 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, HgAlc 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 complete healing in the combined control
groups in this
study (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%). 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 sVLU std P-value
deviation deviation
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)
[0246] 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 Polynueleotide Formulation improved both the incidence of complete wound
closure
and the time to complete healing, are summarized below and in Table 9.
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[0247] 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).
[0248] 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).
TABLE 9: Key Endpoints for ITT Population in the Study
Incidence of Time to
Dose Complete RVLU Complete RVLU
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
[0249]
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
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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).
[0250] 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
[0251] 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.
[0252] 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.
[0253] 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
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(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%).
[0254] 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.
[0255]
Additional inclusion and exclusion criteria refinements will be based on
age over 50 and BMI of less than, for example, 42.
[0256] 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 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
[0257] 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 Polynucleotide Formulation. 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.
[0258]
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
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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.
[0259] 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.
[0260] 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 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.
[0261] Treating
only one reference ulcer with randomized investigational product
accomplishes important objectives:
[0262] 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).
[0263] 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.
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[0264] 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.
[0265] For each subject, the Treatment Period will end:
[0266] 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,
[0267] 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.
[0268] 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.
[0269] 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
[0270] 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
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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.
[0271]
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; 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
[0272] 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.
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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
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]
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[0273] 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; IDD, insulin
dependent
diabetes; HbAlc, hemoglobin Alc; BMI, body mass index; NA, not applicable.
[0274] 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
(1ES) 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.
[0275] All biopsies were immediately immersed in 4 % paraformaldehyde
for 24
hours and then transferred into 20 % sucrose in phosphate buffered saline
(PBS). Tissue
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
[0276] 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).
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Confocal Microscopy
[0277] 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 (FE). 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
[0278] 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 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 pixels/ m2.
[0279] 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
[0280] 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 Ph) and
patient.
The residuals were tested for normality using the Kolmogorov-Smirnoff test;
with a
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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
[0281] 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.
[0282] 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
[0283] 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 (FE) of the biopsy the increase was on
average 14-fold
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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).
[0284] 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 FE 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
[0285] 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 Ph side of the biopsy.
[0286] 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
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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 FE 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)
[0287] 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 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).
[0288] 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
FE 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
[0289] The
distribution of connexins within the epidermis varied along the length
of the biopsies and with depth con-esponding 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.
[0290] 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
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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)
[0291] 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 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.
[0292]
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
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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.
[0293] 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
[0294] 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. 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.
[0295] 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.
Figure Legends
[0296] Figure 1. 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
pm respectively. Cx43 = Red; Blood vessels/alpha smooth muscle actin = Green;
Nuclei
and autofluorescent extracellular matrix = Blue. VLU, venous leg ulcer; Cx,
connexin.
[0297] Figure 2. 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.
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Scale bar - 10x Montages 1000 gm; 40x Images 100 gm. 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; FE, far edge.
[0298] Figure
3. 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 gm; 40x Images 100 gm. Cx43, 26 and 30 =
Green;
Nuclei = Blue. * p < 0.05; ** p < 0.01; *** p < 0.001. Error bars - Mean +/-
SEM
(Epidermis - n = 11 except 1HE = 8; Dermis - n = 6). DFU, diabetic foot ulcer;
Cx,
connexin; WE, wound edge; Eh, far edge.
[0299] Figure
4. 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 14E. Scale bar - 10x Montages 1000 gm; 40x Images 100 gm. 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.
EXAMPLE 6: Efficacy of Connexin 43 Modulating Agent in
Treating Wounds on mDFU Subjects
[0300] A Phase
2, 12-week, randomized, four-arm, double-blind, vehicle-
controlled, dose-ranging, multi-center study was conducted to assess the
efficacy and
safety of three dose concentrations of the Polynucleotide Formulation (3.0
mg/mL, 10.0
mg/mL, and 30 mg/mL) compared to Vehicle when applied to diabetic foot ulcers.
Throughout the entire duration of the study, from the start of Screening until
the end of
each subject's participation, each subject received best practice standard of
care (SOC)
for diabetic foot ulcers. The SOC consisted of debridement/cleaning and
dressing the
wound, then off-loading with a removable cast walker (RCW). During the
Treatment
Period of the study, the assigned treatment was applied to the reference
diabetic foot ulcer
(RDFU) twice weekly in addition to the SOC.
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[0301] The
primary objective of the study was to evaluate use of the
Polynucleotide Formulation (3.0 mg/mL, 10.0 mg/mL, and 30 mg/mL) to improve
healing of DFU. Endpoints included the Incidence of Complete RDFU Closure
(Primary
Endpoint) and Time to Complete RDFU Closure within the 12-week treatment
period,
both acceptable regulatory endpoints for registration studies. A key secondary
endpoint
was Percent Surface Area Change of the RDFU within the 12 weeks.
[0302] A two-
week Screening Period was designed to determine whether subjects
were eligible to proceed to the treatment period of the study. For eligible
patients, the
Investigator selected one RDFU at the start of the Screening Period (in
patients with
multiple DFU, this was the largest DFU that met the eligibility criteria for
the study). The
Screening Period was designed to exclude diabetic foot ulcers that had large
changes in
size and to exclude subjects who were non-compliant with the standard-of-care
regime.
Centralized review of RDFU photographs was performed during this period to
assess the
wound appearance and confirm eligibility.
[0303] In the
parallel group phase of the trial, eligible subjects proceeded to the
Treatment Period and all were assigned to double-blind treatment in one of
four treatment
arms [(1) 3.0 mg/mL Polynucleotide Formulation comprising 3.0 mg/mL
Polynucleotide
("3.0 mg/mL Polynucleotide Formulation" or "3.0 mg/mL"), (2) 10.0 mg/mL
Polynucleotide Formulation, (3) 30.0 mg/mL Polynucleotide Formulation, or (4)
Vehicle].
In an earlier dose escalation phase eligible subjects (n=43) were assigned to
one of two
treatment arms until the study opened up to the Parallel Group Phase. The dose-
rising
safety assessment phase began with the 3.0 mg/mL Polynucleotide Formulation,
proceeded to the 10.0 mg/mL Polynucleotide Formulation, and concluded with the
30.0
mg/mL Polynucleotide Formulation, during which no safety issues or concerns
were
observed.
[0304] A total
of 168 subjects met the eligibility criteria and were randomized to
the four treatment groups with 42, 43, 41 and 42 subjects assigned to the 3.0
mg/mL, 10.0
mg/mL, 30 mg/mL, and Vehicle, respectively. The assigned treatment or Vehicle
was
applied twice a week by clinical staff up to and including Day 84 of the 12-
week
Treatment Period. If an initial assessment of 100% re-epithelialization was
made at a
study visit prior to Day 84, the subject entered the Post-Treatment Period and
was
followed up for a further 14 days to confirm complete closure of the RDFU.
[0305] Only
subjects whose RDFU was 100% re-epithelialized during the
Treatment Period progressed to the Post-treatment Period. The duration of this
period for
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each subject depended on the status of the RDFU at each assessment visit. For
those
subjects whose RDFU remain healed beyond the first 14 days of the Post-
treatment
Period, this follow-up extended up to a maximum of 12 weeks, to determine
durability of
RDFU complete closure and continued assessment of safety.
[0306] The study included both single DFU (sDFU) and multiple DFU (mDFU)
subjects, enrolled at random. In the more severe mDFU population (defined by
subjects
with more than one DFU), both raw results and data from a simple statistical
model
comparing results for the 30 mg/mL dose compared to other groups combined
(Vehicle
with the lower 3.0 and 10 mg/mL doses) showed a positive treatment response
for
complete wound healing, and clinically significant increases in healing using
the 30.0
mg/mL dose concentration of the Polynucleotide Formulation compared with other
groups.
[0307] Multiple DFU subjects treated with the 30.0 mg/mL Polynucleotide
Formulation showed a complete closure rate of 58.3%, while mDFU subjects
treated with
Vehicle had a 29% healing rate, i.e., a 100% improvement in healing was
observed in the
30mg/mL group. Expected 12-week DFU complete closure rates with standard-of-
care
treatment in clinical practice are understood to average about 30-35%. The raw
values
contrast between mDFU subjects treated with 30.0 mg/mL Polynucleotide
Formulation
and mDFU subjects in the other groups combined showed a 145% improvement in
healing over combined (Vehicle + lower doses); with 30.0 mg/mL (showing 58%
RDFU
complete closure compared to 23.8% RDFU complete closure, p=0.0473).
[0308] In addition, further analysis using three simple statistical
models taking
into account only mDFU status, treatment, and mDFU status x treatment
interaction
showed that subjects treated with the 30.0 mg/mL Polynucleotide Formulation in
comparison to mDFU subjects in the other groups combined had a clinically
meaningful
increase in complete wound closure (linear regression model; p = 0.0554), a
clinically
meaningful faster time to complete wound healing (proportional hazards model;
p =
0.0459), and a clinically meaningful percent reduction in wound surface area
(generalized
linear model; p = 0.036).
[0309] Additionally, the trial data show that wounds on mDFU patients
are harder
to heal than sDFU wounds (Odds Ratio = 2.0638 for the complete healing in
favor of
sDFU; p = 0.0133 for the differences in surface area reduction, also in favor
of sDFU
wounds). The study demonstrated that the Polynucleotide Formulation was safe
and well
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tolerated, and showed clinically meaningful efficacy with the 30.0 mg/mL
Polynucleotide
Formulation and that treatment with the Polynucleotide Formulation was
particularly
efficacious in resistant wounds on patients with multiple DFUs.
* * *
[0310] 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.
[0311] 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.
[0312] 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.
[0313] 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.
[0314] 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
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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.
[0315] 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.
[0316] 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.
[0317] 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 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.
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[0318] 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.
[0319] 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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