EP3116996A2 - Treatment of resistant lesions - Google Patents
Treatment of resistant lesionsInfo
- Publication number
- EP3116996A2 EP3116996A2 EP15760904.1A EP15760904A EP3116996A2 EP 3116996 A2 EP3116996 A2 EP 3116996A2 EP 15760904 A EP15760904 A EP 15760904A EP 3116996 A2 EP3116996 A2 EP 3116996A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- connexin
- lesion
- subject
- resistant
- modulating agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
Definitions
- 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.
- 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.
- VLUs venous leg ulcers
- VLUs venous leg ulcers
- 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).
- compression bandaging remains the standard of care (SOC) due to a lack of effective alternatives.
- SOC standard of care
- VLU 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.
- sVLU single VLUs
- Margolis et al Venous leg ulcer: incidence and prevalence in the elderly, Wound Rep Reg 2004;12: 163-168. The incidence of mVLU appears to be increasing.
- Analysis of an Intellicure Chronic Wound Dataset shows that 54% of patients treated at wound care centers using the U.S. Wound Registry medical database from 2007 to 2012 had multiple VLUs vs. 40% as reported by Margolis et al. (2004) from a 1998-2000 dataset. Thus, reports indicate that the incidence of mVLU has grown significantly, by almost 15%, in just over a decade.
- 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.
- 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.
- Connexin 26 is found in cells throughout the body, including the inner ear and the skin. Some studies indicate that channels made with connexin 26 help to maintain the correct level of potassium ions. Other research suggests that connexin 26 is required for the maturation of certain cells in the cochlea. Connexin 26 is also reported to play a role in the growth, maturation, and stability of the outermost layer of skin (the epidermis). Connexin 30 is also found in several different tissues throughout the body, including the brain, skin, and inner ear. Some studies indicate that gap junctions made with connexin 30 also help to maintain the correct level of potassium ions. Connexin 30 gap junctions are also said to play a role in the growth and maturation of the epidermis.
- Figure 1 represents blood vessels in VLUs. Blood vessel staining (green) precede 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 ⁇ respectively.
- VLU venous leg ulcer; Cx, connexin.
- Figure 2 represents Cx43, Cx26 and Cx30 expression in VLUs.
- Scale bar - lOx Motages 1000 ⁇ ; 40x Images 100 ⁇ .
- Figure 3 represents Cx43, Cx26 and Cx30 expression in DFUs.
- Figure 4 represents Cx43, Cx26 and Cx30 expression in PRUs.
- Error bars - Mean +/- SEM (Epidermis and dermis - n - 6 except FE 5).
- PRU pressure ulcer; Cx, connexin; WE, wound edge; FE, far edge.
- 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.
- Figure 6 shows Cx43 in dermis normalised to patient baseline expresion.
- this invention relates to pharmaceutical formulations and methods for treating resistant lesions with a connexin protein modulating agent.
- the invention in another aspect 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.
- a connexin protein modulating agent based on indicators including those described herein.
- 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 and/or linear wound advance. Yet other factors include the size of the lesion or the duration of the lesion or both.
- 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.
- 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.
- 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.
- the connexin protein modulating agent is a connexin protein antisense oligonucleotide. In one embodiment the connexin protein modulating agent is a connexin protein antisense oligodeoxynucleotide, whether chemically modified or unmodified. In some aspects the therapeutically effective amount of the connexin protein modulating agent is any amount effective to promote healing of a resistant lesion in or on a subject. Connexin 43, connexin 26 and connexin 30 protein modulating agents are preferred. Connexin 43 protein modulating agents are particularly preferred.
- 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.
- the connexin protein modulating agent is present at a concentration ranging from about 0.5 to about 50 mg/mL.
- the connexin protein modulating agent is present at a concentration ranging from about 0.3 to about 30 mg/mL.
- 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.
- 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.
- the carrier 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.
- 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.
- 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.
- the indicator is the presence of mVLUs, as noted.
- 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).
- the indicator can be age over 50.
- 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.
- the BMI indicator is less than 40.
- 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.
- a connexin protein modulating agent such as a connexin protein antisense oligodeoxynucleotide
- compositions of this invention comprise one or more anti-connexin protein, for example, anti-connexin 43, polynucleotides that modulate connexin activity.
- the connexin modulating agent may be antisense oligonucleotides.
- Anti-connexin oligonucleotides can inhibit connexin activity by decreasing its expression.
- the active ingredient includes a connexin protein, for example, anti-connexin 43, modulating agent.
- 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.
- connexin modulating agents of this invention may be used alone or in combination.
- treatment with a connexin protein modulating agent is administered in conjunction with standard-of-care, for example, compression bandaging and/or off-loading.
- the invention includes a package or kit comprising a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically acceptable anti-connexin modulating agent, together with a label and/or instructions for administering the composition to subjects with resistant lesions, for example where the subject has mVLUs or mDFUs, and the agent is administered in amounts effective to promote healing of the lesions in a subject, alone or together with standard-of-care, for example, compression bandaging and/or off-loading.
- 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.
- 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.
- 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.
- the connexin modulating agent is a connexin 43, connexin 30 or connexin 26 antisense oligonucleotide. In one embodiment the connexin 43 modulating agent is a modified connexin 43 antisense oligodeoxynucleotide.
- 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.
- 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.
- mVLUs multiple venous leg ulcers
- 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.
- the one or more ulcers can be on the same leg, or on different legs.
- the subject may be over about 50 years of age, and/or have a BMI of less than about 40.
- 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.
- 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.
- this invention relates to treating responder subjects who have resistant lesions such as mVLUs or mDFUs.
- This invention relates in one aspect to formulations and methods for treating resistant lesions in subjects who are likely to be responsive to treatment by a connexin 43 modulating agent, based on indicators including those described herein, which include the presence of mVLUs or mDFUs, or, in some instances, the presence of a biomarker indicative of a resistant lesion, as discussed above.
- 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.
- the indicator can be age over 50.
- 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.
- 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.
- mVLUs and sVLUs have been associated with more difficulty in healing and a lower likelihood of complete healing
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the formulations of this invention for use in treating mVLUs, mDFUs, or other resistant lesions may comprise a connexin 43 modulating agent and one or more pharmaceutically acceptable vehicles formulated for topical administration.
- the composition is formulated to provide sustained release of the connexin 43 modulating agent.
- modulating agent 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.
- 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.
- 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.
- 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.
- the anti-connexin polynucleotides are anti-connexin 43 oligonucleotides (ODN).
- 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.
- 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.
- the pharmaceutical formulations of this invention may further comprise one or more pharmaceutically acceptable excipients.
- 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.
- 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.
- the pharmaceutically acceptable carrier or vehicle is, or comprises, a gel.
- 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.
- 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.
- the gel is a nonionic polyoxyethylene- polyoxypropylene copolymer gel.
- the gel is a pluronic gel.
- the pluronic gel may be, for example, poloxamer 407, also sometimes referred to as Pluronic F-127 (BASF).
- BASF Pluronic F-127
- 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.
- 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.
- connexin 43 levels measured in the dermis and/or epidermis at the edges of multiple VLUs in humans appear higher than connexin 43 levels measured at the edges of single VLUs in humans. Accordingly, a determination of high connexin 43 levels in the dermis or epidermis of a resistant wound may be used as a method of diagnosing a resistant lesion and/or responder patient prior to prescribing treatment with a connexin 43 modulating agent.
- 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 are also referred to as "responder" subjects.
- 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.
- standard of care e.g., compression bandageing or off-loading
- 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.
- the healing delta between responder subjects treated with a connexin protein modulating agent will be at least about 10% to 15%.
- this delta will be 20% or more, and can be 25%, 30%, 35%, 40% and 45% or more.
- 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.
- 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.
- 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.
- the indicator in the case of VLUs, is the presence of mVLUs, as noted.
- a resistant lesion is any lesion exhibiting one or more indicators of a resistant lesion, as described herein.
- a resistant lesion may exhibit, for example, one or more of the indicators based on the baseline area of lesion, the % epithelialization of the lesion, and baseline circumference, as described herein.
- a lesion may be characterized as a resistant lesion if it is present on a subject having hemoglobin Ale (HbAlc) levels and/or BMI, as described herein.
- HbAlc hemoglobin Ale
- 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.
- 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.
- 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.
- standard-of-care such as compression bandaging and/or off-loading
- 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%.
- 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%.
- 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 preferably less than about 30%, the lesion is a resistant lesion for purposes of this invention.
- 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.).
- a hydrogel or other agent to maintain a moist wound environment
- standard-of-care for example, compression and/or off-loading, etc.
- 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 cm 2 (size).
- 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 cm 2 , preferably >6.0
- cm more preferably >7.0 cm " or >8.0 cm , or also preferably >8.6 cm .
- 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
- 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.
- the lesion is identified as a resistant lesion treatment with an anti-connexin 43 modulating agent if the subject to be treated has a HbAlc of greater than about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or about 7.0%, or greater than any range between any two recited values, or greater than any value in between any two recited values.
- 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%.
- the lesion is identified as a resistant lesion treatment with an anti-connexin 43 modulating agent if the subject to be treated has a HbAlc of greater than 6.0%, 6.5% or greater than 7.0%.
- 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, 10.9.9.
- the resistant lesion having a circumference less than the recited length may also have a relatively convex circumference.
- 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.
- 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)
- LLA linear lesion advance
- 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.
- 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.gnati about 0.005 to about 0.0065 cm/day), or a linear lesion advance of less than about 0.03, 0.035, 0.04, 0.042, or about 0.045 cm/week, or less than about any range between any of those values (e.g, about 0.04 to about 0.045 cm/week),
- the lesion is a resistant lesion for purposes of this invention.
- the lesion is an LLA resistant lesion for the purposes of this invention
- 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 cm 2 (size) and a duration of more than 6 months, a minimal degree of epitheliazation, and/or hemoglobin Ale (HbAlc) of 6.5 % or greater.
- HbAlc hemoglobin Ale
- 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.
- 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.
- 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.
- 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).
- this invention also relates to methods of determining whether subjects are likely to respond to treatment by a connexin 43 modulating agent based on indicators described herein. Methods of assessing whether a subject is a likely responder subject can also be used in conjunction with the methods of treatment and uses described herein.
- 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-lRA/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.
- 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.
- protease may be, for example, a metalloproteinase.
- a resistant wound will contain high levels of ILl, IL6, and matrix metalloproteinases (MMPs), and an abnormally high MMP to ⁇ 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: ⁇ -1, TEVIP-2, TEVIP-3, and TIMP-4.
- elevated levels of MMP-2, MMP-8 and/or MMP-9 may characterize a resistant wound.
- a resistant wound may also be be be characterized by low levels of TGF and/or low levels of one or more MMP tissue inhibitors ( ⁇ ), for example TIMP-1 or ⁇ -2). Resistant wounds may also be characterized by the MMP- 9: ⁇ -2 ratio.
- the resistant wound may be characterized by increases in one or more of IL-1, IL-6, IL-8, ⁇ - ⁇ , TNFa and/or IL- ⁇ or any combination thereof. Higher levels of IL-1 a, IL- ⁇ , ⁇ , IL-12p40, GM-CSF and IL- 1RA may also be present at elevated levels in resistant wounds.
- Healing 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.
- 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.
- preventing means preventing in whole or in part, or ameliorating or controlling.
- a therapeutically effective amount of the connexin 43 modulating agent is any amount effective to promote healing of a resistant lesion in a subject.
- a therapeutically effective amount of the connexin 43 modulating agent when used to treat mVLUs is the amount effective to promote healing of mVLUs.
- 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.
- peptide analogs refer to the compounds with properties analogous to those of the template peptide and can be non-peptide drugs.
- “Peptidomimetics” also known as peptide mimetics
- Peptidomimetics 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.
- peptidomimetics are structural or functional mimics (e.g.
- a paradigm polypeptide i.e., a polypeptide that has a biological or pharmacological function or activity
- a paradigm polypeptide i.e., a polypeptide that has a biological or pharmacological function or activity
- 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 -CH2SO-.
- 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.
- connexins these can mimic, for example, the extracellular loops of opposing connexins involved in connexon-connexon docking and cell-cell channel formation.
- 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.
- wound dressing refers to a dressing for topical application to a resistant lesion or wound and excludes compositions suitable for systemic administration.
- 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.
- the one or more anti-connexin agents, including gap junction modulation agents are dispersed in or on a biodegradable sheet material that provides sustained release of the active ingredients into the wound, for example a sheet of freeze-dried collagen, freeze-dried collagen/alginate mixtures (available under the Registered Trade Mark 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).
- a biodegradable sheet material that provides sustained release of the active ingredients into the wound, for example a sheet of freeze-dried collagen, freeze-dried collagen/alginate mixtures (available under the Registered Trade Mark 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).
- 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 keratinocyte
- Additional suitable biomatrix material may include chemically modified collagenous tissue to reduce antigenicity and immunogenicity.
- Other suitable examples include collagen sheets for wound dressings, antigen-free or antigen reduced acellular matrix (Wilson et al., Trans Am Soc Artif Intern 1990; 36:340-343) or other biomatrix which have been engineered to reduce the antigenic response to the xenograft material.
- 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 Vase Surg 1984; 1: 181-91).
- 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.
- 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.
- matrices useful in promotion of wound healing may include for example, proteins comprising insoluble collagen and elastin (Courtman DW et al. (1994) J Biomed Mater Res 28:655- 666) and other acellular tissue which may be useful for providing a natural microenvironment for host cell migration to accelerate epilethialization (Malone J M et al. (1984) J Vase Surg 1 :181-91).
- the invention contemplates a synthetic or natural matrix comprising one or more anti-connexin protein agents.
- 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.
- 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.
- certain anti-connexin protein agents described herein are capable of blocking or inhibiting the transport of molecules into and out of cells.
- 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.
- hemichannels also called connexons
- a compound provided herein may directly or indirectly reduce coupling between cells (via gap junctions) or between a cell and an extracellular space or tissue (via hemichannels), and the modulation of transport of molecules from a cell into an extracellular space is within the scope of certain compounds and embodiments of the invention.
- 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.
- 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.
- 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.
- a gap junction modulation agent prevents or decreases, in whole or in part, the function of a hemichannel or a gap junction.
- a gap junction modulation agent induces closure, in whole or in part, of a hemichannel or a gap junction.
- a gap junction modulation agent blocks, in whole or in part, a hemichannel or a gap junction.
- a gap junction modulation agent decreases or prevents, in whole or in part, the opening of a hemichannel or gap junction.
- said blocking or closure of a gap junction or hemichannel by a gap junction modulation agent can reduce or inhibit extracellular hemichannel communication by preventing or decreasing the flow of small molecules through an open channel to and from an extracellular or periplamic space. Peptidomimetics, and gap junction phosphorylation compounds that block hemichannel and/or gap junction opening are presently preferred.
- the anti- connexin protein agent may be an anti-connexin 43 agent, an anti-connexin 30 agent, or an anti-connexin 26 agent.
- an anti-connexin agent prevents, decreases or alters the activity or function of a hemichannel or a gap junction.
- 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.
- 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.
- 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 polynucleotides such as antisense protein polynucleotides, such as anti-connexin protein oligonucleotides, connexin protein oligodeoxynucleotides and other poly
- Anti-connexin protein peptides and polypeptides may, for example, bind to connexin protein to inhibit its function, or may inhibit connexin 43 function by mimicking regions of connexin protein to inhibit or disrupt its binding to other gap junction proteins.
- the agents may be anti- connexin 43 agents, anti-connexin 30 agents and/or anti-connexin 26 agents.
- Anti-connexin 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.
- 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.
- 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).
- antisense polynucleotides such as DNA or RNA polynucleotides
- ODN antisense oligodeoxynucleotides
- these polynucleotides may target the connexin 43 protein.
- the polynucleotides are single stranded, but may be double stranded.
- the antisense polynucleotide may inhibit transcription and/or translation of a connexin protein, such as connexin 43, 30 or 26.
- a connexin protein such as connexin 43, 30 or 26.
- the polynucleotide is a specific inhibitor of transcription and/or translation from the connexin 43, 30 or 26 gene or mRNA, and does not inhibit transcription and/or translation from other genes or mRNAs. Screening of the polynucleotide sequence in a human genome sequence database for specificity may also be performed.
- the product may bind to the connexin 43, 30 or 26 gene or mRNA either (i) 5' to the coding sequence, and/or (ii) to the coding sequence, and/or (iii) 3' to the coding sequence.
- the antisense polynucleotide is generally antisense to connexin protein mRNA, for example, connexin 43, 30 or 26 mRNA.
- 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.
- 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.
- the polynucleotide may be the exact complement of a part of connexin mRNA.
- 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.
- 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.
- 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.
- the polynucleotide has 30 nucleotides.
- the antisense polynucleotides may be part of compositions which may comprise polynucleotides to more than one connexin protein.
- 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.
- 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.
- oligodeoxynucleotides 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.
- the antisense polynucleotides may be chemically modified. This may enhance their resistance to nucleases and may enhance their ability to enter cells.
- 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.
- MBOs mixed backbone oligonucleotides
- 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.
- suitable connexin 43 antisense polynucleotides can include polynucleotides such as oligodeoxynucleotides selected from SEQ ID NO: 1-3 set forth in Table 1: Suitable polynucleotides for the preparation of the combined polynucleotide compositions described herein, for combination with the connexin 43 modulating agent include polynucleotides connexins 26, 30, 31.1, 32 and 37 are also described in Table 1.
- antisense polynucleotide used in the invention will depend upon the target connexin protein, for connexin 43, antisense polynucleotides having any of SEQ.ID.NO:l-2, SEQ.ID.NO.21 or SEQ.ID.NO.22-23 have been found to be particularly suitable:
- Polynucleotides, including ODN's, directed to connexin proteins can be selected in terms of their nucleotide sequence by any convenient, and conventional, approach.
- the computer programs Mac Vector and OligoTech from Oligos etc. Eugene, Oregon, USA
- the ODN's can be synthesized using a DNA synthesizer.
- the polynucleotide may be a homologue of a complement to a sequence in connexin mRNA.
- a polynucleotide typically has at least about 70% homology, preferably at least about 80%, at least about 90%, at least about 95%, at least about 97% or at least about 99% homology with the relevant sequence, for example over a region of at least about 15, at least about 20, at least about 40, at least about 100 more contiguous nucleotides (of the homologous sequence).
- Homology may be calculated based on any method in the art.
- 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.
- 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.
- the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
- the BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787.
- One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
- P(N) the smallest sum probability
- 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.
- 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.
- 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).
- medium to high stringency for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C.
- suitable conditions include 0.2 x SSC at 60°C. If lower stringency is required, suitable conditions include 2 x SSC at 60°C.
- 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.
- the invention also includes in one aspect pharmaceutical compositions with instructions for treating responder subjects having mVLUs.
- the anti-connexin 43 modulating agent is a polynucleotide.
- the anti- connexin 43 modulating agent is an oligonucleotide.
- the oligonucleotide may be an anti- connexin 43 antisense oligodeoxynucleotide.
- the polynucleotides of this invention may be modified or unmodified.
- the invention also includes a package or kit comprising a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically acceptable anti-connexin modulating agent, together with a label and/or instructions for administering the composition to one or more wounds on subjects with resistant lesions such as mVLUs, where the subject is susceptible to treatment with an anti-connexin modulating agent, and the agent is administered in amounts effective to promote healing of the lesions in a subject, alone or together with compression bandaging.
- a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically acceptable anti-connexin modulating agent, together with a label and/or instructions for administering the composition to one or more wounds on subjects with resistant lesions such as mVLUs, where the subject is susceptible to treatment with an anti-connexin modulating agent, and the agent is administered in amounts effective to promote healing of the lesions in a subject, alone or together with compression bandaging.
- 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.
- 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.
- the pharmaceutical formulations of this invention comprise an unmodified oligonucleotide specific to connexin 43 mRNA having the sequence 5'- GTAATTGCGGCAAGAAGAATTGTTTCTGTC-3' (SEQ ID NO: l).
- the olignonucleotide may be a deoxyoligonucleotide.
- the oligonucleotide is chemically modified to increase half-life.
- 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:l.
- the formulation may also contain other to maintain physiological salt concentrations, such as potassium phosphate, sodium phosphate and water-for-injection.
- Connexin 43, connexin 30 or connexin 26 binding proteins are also suitable modulators of gap junctions and hemichannels.
- 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.
- an antigenic determinant i.e., that portion of a molecule, generally referred to as an epitope
- binding proteins including antibodies, antibody fragments, and so on, may be chimeric or humanized or otherwise made to be less immunogenic in the subject to whom they are to be administered, and may be synthesized, produced recombinantly, or produced in expression libraries. Any binding molecule known in the art or later discovered is envisioned, such as those referenced herein and/or described in greater detail in the art.
- 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).
- 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 10 4 M-l, greater than or equal to about 10 6 M-l, greater than or equal to about 10 7 M-l, greater than or equal to about 10 s M-l.
- Affinities of even greater than about 10 8 M-l are suitable, such as affinities equal to or greater than about 10 9 M-l, about 10 10 M-l, about 10 11 M-l, and about 10 12 M-l.
- 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.
- 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
- Exemplary peptides and peptidomimetics are reported in Green et al, WO2006134494. See also Gourdie et al, see WO2006069181, and Six et al., see WO2003032964.
- 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).
- the extracellular domains of a hemichannel contributed by two adjacent cells "dock" with each other to form complete gap junction channels.
- 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.
- 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.NO: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.NO: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.NO:4), connexin 26 or connexin 30,.
- a connexin protein such as connexin 43 (SEQ.ID.NO: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.NO:4), connexin 26 or connexin 30, or peptides having an amino acid sequence
- 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.NO: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.NO:4.
- Anti-connexin agents include peptides described herein, for example, agents having an amino acid sequence corresponding to the regions at positions 37-76 and 178-208 of SEQ.ID.NO:4.
- the peptides need not have an amino acid sequence identical to those portions of SEQ.ID.NO:4, and conservative amino acid changes may be made such that the peptides retain binding activity or functional activity.
- peptides may target regions of the connexin protein other than the extracellular domains (e.g. the portions of SEQ.ID.NO:4 not corresponding to positions 37-76 and 178-208).
- 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.
- 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.
- 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).
- 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 middle range 50 micromolar was somewhat less effective than the 5 micromolar concentration in repeat experiments.
- Connexin 43 (SEQ ID NO. 4)
- 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.
- 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.
- VDCFLSRPTEKT E2 (SEQ.ID.NO:8)
- SRPTEKT E2 (SEQ.ID.NO: 10)
- LGTAVESAWGDEQ Ml & E1 (SEQ.ID.NO: ll)
- VCYDKSFPISHVR El SEQ.ID.NO: 14
- 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.
- 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 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.
- 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.
- certain peptidomimetics e.g. the connexin 43 peptide inhibitor, VCYDKSFPISHVR, (SEQ.ID.NO: 14
- a peptide comprising SRPTEKT (SEQ.ID.NO: 10), for example VDCFLSRPTEKT(SEQ.ID.NO: 8) or SRPTEKTIFII (SEQ.ID.NO: 9), may also be used, for example to block hemichannels without uncoupling of gap junctions.
- the peptide SRGGEKNVFIV (SEQ.ID.NO: 19) may be used that as a control sequence (DeVriese et al., Kidney Internat. 61 : 177-185 (2002)).
- the peptides may be 3 or more amino acids in length.
- 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.
- the Cx 26 cDNA coding reference sequence NG_008358.1 (SEQ ID NO.30) is shown below in Table 5B.
- An anti-connexin 26 polynucleotide may have the sequence of any polynucleotide sequence having 12 to 80 nucleotides of SEQ ID NO:30 (or any number of nucleotides between 12 and 80).
- the Cx 30 cDNA coding reference sequence NM_001110219.2 (SEQ.ID.NO: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.
- 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.
- 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.
- modulation of phosphorylation on one or more of these residues impacts hemichannel function, particularly by closing the hemichannel.
- 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.
- Still other anti-connexin agents include connexin carboxy-terminal polypeptides. See Gourdie et al, WO2006/069181.
- gap junction modifying agent may include, for example, aliphatic alcohols; octanol; heptanol; anesthetics ⁇ e.g. halothane), ethrane, fluothane, propofol and thiopental; anandamide; arylaminobenzoate (FFA: flufenamic acid and similar derivatives that are lipophilic); carbenoxolone; Chalcone: (2',5'- dihydroxychalcone); 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.
- the polynucleotides of this invention can be manufactured using solid- phase chemistries for synthesizing oligonucleotides.
- 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.
- the formulation may comprise the sodium salt of a polynucleotide having SEQ ID NO:l, for example.
- the polynucleotide having SEQ ID NO: l may be a modified oligodeoxynucleotide having SEQ ID NO: 1.
- the formulations of this invention are substantially pure.
- 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.
- 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%.
- 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.
- 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.
- 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.
- 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.
- formulations by which connexin 43 can be modulated and/or cell-cell communication can be downregulated in a transient and site-specific manner.
- 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.
- 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.
- the pharmaceutical formulations, or pharmaceutical compositions, combined preparations and medicaments of the invention may take any suitable form for topical administration.
- 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.
- the pharmaceutically acceptable carrier or vehicle is, or comprises, a gel.
- 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.
- 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.
- the gel is a nonionic polyoxyethylene- polyoxypropylene copolymer gel.
- the gel is a pluronic gel.
- the pluronic gel may be, for example, poloxamer 407, also sometimes referred to as Pluronic F-127 (BASF).
- the formulations of this invention may comprise from about 15 to about 30 % (w/v) gel.
- the formulations of this invention may comprise from about 20 to about 25 % (w/v) gel.
- the formulations of this invention may comprise about 22.6 % (w/v) poloxamer 407 gel.
- the gel may be a fluorinated methacrylamide chitosan hydrogel system. See, Wijekoon et al, Acta Biomater. 2013 Mar:9(3):5653-64.
- 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.
- 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.
- 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.
- 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.
- substances such as wetting or emulsifying agents, stabilizing or ph buffering agents may also be present.
- pharmaceutically acceptable carrier refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity.
- Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers.
- 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.
- 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.
- 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.
- compositions include pluronic gel-based formulations, carboxymethylcellulose(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.
- 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.
- transfection agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectamTM and transfectamTM), and surfactants.
- the formulation further includes a surfactant to assist with polynucleotide cell penetration or the formulation may contain any suitable loading agent.
- a surfactant to assist with polynucleotide cell penetration
- the formulation may contain any suitable loading agent. Any suitable non-toxic surfactant may be included, such as DMSO.
- a transdermal penetration agent such as urea may be included.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 90.0, 92.5
- 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.
- 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.
- the connexin 43, 30 or 26 modulating agent may be a connexin 43, 30 or 26 antisense oligonucleotide.
- the carrier (vehicle) may be a thermoreversible gel.
- the gel may be a poloxamer gel, for example, poloxamer 407, present in an amount ranging from about 15- 25 or 30%, for example.
- 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.
- dosing of the pharmaceutical compositions may be calculated based on mass (e.g., grams) of or the concentration in a pharmaceutical composition (e.g., ⁇ g/ul) 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.
- 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.
- 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.
- 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,
- the anti-connexin agent composition may be applied at about 0.01 micromolar ( ⁇ ) or 0.05 ⁇ to about 200 ⁇ , or up to 300 ⁇ or up to 1000 ⁇ or up to 2000 ⁇ or up to 3200 ⁇ 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.
- the anti-connexin agent composition is applied at greater than about 1000 ⁇ .
- the antisense polynucleotide composition is applied at about 1000 ⁇ 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.
- 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 ⁇ to about 20 ⁇ final concentration, and alternatively the anti-connexin agent composition is applied at about 10 ⁇ to about 20 ⁇ final concentration, or at about 10 to about 15 ⁇ final concentration. In certain other embodiments, the anti-connexin agent is applied at about 10 ⁇ final concentration. In yet another embodiment, the anti-connexin agent composition is applied at about 1-15 ⁇ final concentration.
- the anti-connexin agent is applied at about a 20 ⁇ , 30 ⁇ , 40 ⁇ , 50 ⁇ , 60 ⁇ , 70 ⁇ , 80 ⁇ , 90 ⁇ , 100 ⁇ , or from about 10-200 ⁇ , 200-300 ⁇ , 300-400 ⁇ , 400-500 ⁇ , 500-600 ⁇ , 600-700 ⁇ , 700-800 ⁇ , 800-900 ⁇ , 900-1000 or 1000-1500 ⁇ , or 1500 ⁇ - 2000 ⁇ , 2000 ⁇ - 3000 ⁇ , 3000 ⁇ - 4000 ⁇ , 4000 ⁇ - 5000 ⁇ , 5000 ⁇ - 6000 ⁇ , 6000 ⁇ - 7000 ⁇ , 7000 ⁇ - 8000 ⁇ , 8000 ⁇ - 9000 ⁇ , 9000 ⁇ - 10,000 ⁇ , 10,000 ⁇ - 11,000 ⁇ , 11,000 ⁇ - 12,000 ⁇ , 12,000 ⁇ - 13,000 ⁇ , 13,000 ⁇ - 14,000 ⁇ ,
- 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.
- 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, 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,
- 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.
- 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.
- concentration of the composition relative to the size, length, depth, area or volume of the area to which it will be applied.
- dosing of the pharmaceutical compositions may be calculated based on mass (e.g. micrograms) of or the concentration in a pharmaceutical composition (e.g. ⁇ g/ l) per length, depth, area, or volume of the area of application.
- the volume of the wound may also be determined by imaging.
- 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.
- 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.
- 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.
- repeat applications may be made, for example, weekly, or biweekly, 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.
- 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.
- the first is administered before the second composition.
- the first composition is administered after the second composition.
- the first composition is administered before and after the second composition.
- the second composition is administered before and after the first composition.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- these combinations allow the use of lower and/or fewer doses of each agent than previously required to achieve desired therapeutic goals.
- one or more anti-connexin agents are delivered by topical administration (peripherally or directly to a site), including but not limited to topical administration using solid supports (such as dressings and other matrices) and medicinal formulations (such as gels, mixtures, suspensions and ointments).
- the solid support comprises a biocompatible membrane or insertion into a treatment site.
- the solid support comprises a dressing or matrix.
- 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.
- a wash solution comprising two or more anti-connexin agents can be used.
- anti-connexin protein modulating agents for example, anti-connexin 43, 30 or 26 modulating agents
- 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.
- 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.
- 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.
- 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.
- this is achieved by inclusion of one or more anti-connexin polynucleotides and/or one or more anti-connexin peptides or peptidomimetics, or other anti-connexin 43 agents or resistant wound healing agents, alone or in combination with either or both, in a formulation together with a pharmaceutically acceptable carrier or vehicle, particularly in the form of a formulation for continuous or slow-release administration.
- 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.
- 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.
- the one or more agents of the invention are provided in the form of a liquid, semi solid or solid composition for application directly, or the composition is applied to the surface of, or incorporated into, a solid contacting layer such as a dressing gauze or matrix.
- the dressing composition may be provided for example, in the form of a fluid or a gel.
- One or more anti-connexin 43 polynucleotides and one or more anti-connexin 43 peptides or peptidomimetics may be provided in combination with conventional pharmaceutical excipients for topical application.
- Suitable carriers include: Pluronic gels, Polaxamer gels, Hydrogels containing cellulose derivatives, including hydroxyethyl cellulose, hydroxymethyl cellulose, carboxymethyl cellulose, hydroxypropylmethyl cellulose and mixtures thereof; and hydrogels containing polyacrylic acid (Carbopols).
- Suitable carriers also include creams/ointments used for topical pharmaceutical preparations, e.g., creams based on cetomacrogol emulsifying ointment.
- the above 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.
- 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
- stabilizers such as EDTA.
- suitable dressings or matrices may include, for example, the following with one or more anti- connexin polynucleotides or one or more anti-connexin protein peptides or peptidomimetics (or other anti-connexin agents to be administered in combination with either or both):
- 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.
- absorptives may be used as a primary or secondary dressing.
- 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.
- alginate dressings are designed to be soft and conformable, easy to pack, tuck or apply over irregular-shaped areas.
- alginate dressings may be used with a second dressing.
- 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.
- bioactive agents such as, for example, silver and polyhexamethylene biguanide (PHMB)
- 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.
- 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.
- a gel or solution is applied to the treatment site and covered with a dressing for barrier protection.
- a biological -based e.g.
- 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.
- the collagen dressing may interact with treatment site exudate to form a gel.
- collagen dressing may be used in combination with a secondary dressing.
- 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.
- the composite dressings are comprised of, for example, multiple layers and incorporate a semi-or non-adherent pad.
- the composite may also include for example, an adhesive border of non-woven fabric tape or transparent film.
- 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.
- 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.
- 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.
- the contact layer dressing may be used in combination with topical pharmaceutical composition.
- suitable elastic bandages may include, for example, dressings that stretch and conform to the body contours.
- the fabric composition may include for example, cotton, polyester, rayon or nylon.
- 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.
- 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.
- the absorption capability may be adjusted based on the thickness and composition of the foam.
- the area in contact with the treatment site may be non-adhesive for easy removal.
- 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.
- 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.
- 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.
- suitable hydrocolloid dressings may include, for example, wafers, powders or pastes composed of gelatin, pectin or carboxymethylcellulose.
- wafers are self-adhering and available with or without an adhesive border and in a wide variety of shapes and sizes.
- Exemplary hydrocoUoids are useful on areas that require contouring.
- powders and pastes hydrocoUoids may use used in combination with a secondary dressing.
- 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.
- hydrogels may be used in combination with a secondary dressing cover.
- 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.
- suitable hydrogel sheets may include for example, three-dimensional networks of cross-linked hydrophilic polymers that are insoluble in water and interact with aqueous solutions by swelling.
- Exemplary hydrogels are highly conformable and permeable and can absorb varying amounts of drainage, depending on their composition.
- the hydrogel is non-adhesive against the treatment site or treated for easy removal.
- 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.
- suitable silicone gel sheet dressings may include, for example, soft covers composed of cross-linked polymers reinforced with or bonded to mesh or fabric.
- suitable liquid dressings may include, for example, mixtures of multiprotein material and other elements found in the extracellular matrix.
- exemplary solutions may be applied to the treatment site after debridement and cleansing and then covered with an absorbent dressing or a nonadherent pad.
- suitable transparent film dressings may include polymer membranes of varying thickness coated on one side with an adhesive.
- transparent films are impermeable to liquid, water and bacteria but permeable to moisture vapor and atmospheric gases.
- the transparency allows visualization of the treatment site.
- suitable filler dressings may include, for example, beads, creams, foams, gels, ointments, pads, pastes, pillows, powders, strands or other formulations.
- 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.
- one or more anti-connexin protein polynucleotides and/or one or more anti-connexin protein peptides or peptidomimetics and/or other anti-connexin agents such as a gap junction or hemichannel phosphorylation agent or connexin carboxy- terminal polypeptide, alone or in combinations of any of the anti-connexin protein modulating agents, or other resistant wound healing agents, may also be used in the manufacture of the medicament, or in a kit.
- Suitable anti-connexin protein modulating agents, polynucleotides or peptides may be anti-connexin 43, 30 or 26 modulating agents, polynucleotides or peptides.
- the invention provides an article of manufacture or kit comprising one or more compositions or formulations described.
- 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,
- Articles of manufacturer comprising a vessel containing a composition or formulation of the invention as described herein and instructions for use for the treatment of a subject.
- 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.
- the article of manufacture may comprise a matrix that comprises one or more anti-connexin protein peptides or peptidomimetics or other anti- connexin agents, such as a gap junction or hemichannel phosphorylation agent or connexin carboxy-terminal polypeptide, alone or in combinations of any of the anti- connexin 43 modulating agents, or other resistant wound healing agents, Suitable anti- connexin protein modulating agents, polynucleotides or peptides may be anti-connexin 43, 30 or 26 modulating agents, polynucleotides or peptides. Treatment
- 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.
- 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.
- 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.
- 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
- 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.
- 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.
- re-epithlialization and/or formation of granulation tissue is promoted.
- Methods of promoting re-epithelialization of resistant skin lesions comprise administering to a subject having a resistant skin lesion, including, for example, mVLUs, in an amount effective to promote re-epithelialization.
- Analogous methods can be used to regulate epithelial basal cell division and growth.
- 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.
- 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.
- the anti-connexin agent is a connexin antisense polynucleotide effective to regulate epithelial basal cell division and growth.
- 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.
- 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.
- the anti- connexin agent is a connexin antisense polynucleotide effective to regulate outer layer keratin secretion.
- the connexin antisense polynucleotide is a connexin protein antisense polynucleotide, peptide or peptidomimetic, a connexin 43, connexin 26 or connexin 30 antisense polynucleotide, peptide or peptidomimetic or a mixture thereof.
- Subjects which may be treated include subjects with mVLU, having one or more of the other indicators described herein, for example, age over 50-52 or BMI less than 40-42.
- the invention provides a method of decreasing scar formation and/or improving scar appearance in a patient who has suffered a resistant wound.
- 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.
- 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.
- connexin expression is downregulated over a sustained period of time.
- connexin hemichannels are blocked or closed, in whole or in part, over a preferred period of time.
- connexin protein expression is downregulated and connexin hemichannel opening is blocked or inhibited, in whole or in part, for a sustained period of time.
- connexin protein expression is downregulated or hemichannels blocked or inhibited for at least about 1, 2, 4, 6, 8, 10, 12, or 24 hours.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- an anti-connexin protein peptide or anti-connexin protein peptidomimetic e.g., an anti-connexin agent that can block or reduce hemichannel opening
- 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.
- 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.
- 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.
- the composition or compositions are administered in a sustained release formulation.
- the composition or compositions are administered for a sustained period of time.
- 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.
- the unmodified 30-mer anti-connexin deoxyoligonucleotide having SEQ ID NO: l 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.
- compositions of this invention comprising synthetic, unmodified deoxyoligonucleotides with unmodified backbones resulted in low toxicity with no systemic exposure, and, importantly with respect to safety, undetectable or exceedingly low pK even when very large clinical-multiple doses of an unmodified anti- connexin deoxyoligonucleotide having SEQ. ID. NO: 1 were repeatedly administered to open wounds in the skin of test animals.
- 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: l 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 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. 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 proteins e.g., Brown DA, et al. Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding. Biol. Chem. 1994;269:26801-26805
- the Polynucleotide has also been shown to have a short half -life in cells ( ⁇ 20 minutes) and a very short half-life in the circulation ( ⁇ several minutes) due to rapid metabolism by endogenous nucleases and extremely rapid glomerular filtration. It has been shown that systemic exposure to the Polynucleotide is exceedingly low, even when very large clinical-multiple doses of a formulation comprising a poloxamer gel and anti- connexin deoxyoligonucleotide having SEQ. ID. NO:l (Polynucleotide Formulation) are repeatedly administered to open wounds in the skin of test animals.
- 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:l over the course of the study, systemic exposure was negligible.
- 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 ⁇ g/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).
- 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.
- 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.
- 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.
- 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) 1mm 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.
- 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.
- FIG. 6 shows Cx43 in dermis normalised to patient baseline expression (Ratio Dataset).
- Cx43 background expression 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.
- Blocking connexin43 expression reduces inflammation and improves functional recovery after spinal cord injury. Molecular and Cellular ⁇ euro sciences. 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.
- connexin43 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.
- 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.
- 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 cm 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.
- 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 ((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) 1.0 mg/mL Polynucleotide Formulation, or (3) Vehicle). Visits were conducted once per week.
- Subjects progressed to the Post Treatment Period for up to 12 weeks of follow-up if the RVLU closed completely; otherwise subjects were discharged from the study after the Treatment Period, except SOC-alone subjects who could progress to up 10 weeks of open-label treatment with 3.0 mg/mL Polynucleotide Formulation.
- 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 kg 2 (range 16.0 - 45.7).
- ITT Intention-To-Treat
- SP Safety Population
- PP Per Protocol
- 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: l and Vehicle.
- mVLU multiplicity of VLU
- 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.
- 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.
- the formulation may be any formulation of this invention.
- 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.
- Additional inclusion and exclusion criteria refinements will be based on age over 50 and BMI of less than, for example, 42.
- 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
- 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).
- a primary dressing e.g., AllevynTM Non- Adhesive Dressing; Smith & Nephew
- a multi-layer compression secondary dressing e.g., CobanTM 2; 3M.
- the peri- wound skin may be treated with moisturizing, anti-fungal or corticosteroid.
- Consented subjects with multiple VLU will enter a two- week screening period where baseline assessments and eligibility assessments will be performed, including: venous duplex ultrasound to exclude subjects without underlying venous insufficiency, histopathology to exclude subjects with carcinoma in the VLU, and wound measurements to exclude subjects whose VLU is having large changes in size.
- Centralized review of the VLU photographs will be performed by the Medical Monitor to supplement the Investigator's judgment of eligibility for randomization, i.e., both the Medical Monitor and the Investigator must find that the subject is eligible in order for him/her to be randomized.
- VLU reference venous leg ulcer
- 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.
- Polynucleotide Formulation results in surprisingly high levels of complete closure of mVLU in this difficult to heal VLU population.
- connexins 26 and 30, in addition to connexin 43 was examined in patients with a variety of chronic wounds, including venous leg, diabetic foot or pressure ulcers.
- Wound edge punch biopsies were taken from a cohort of patients with venous leg, diabetic foot or pressure ulcers. Wound connexin expression in each patient was compared to that in a matched, non-wounded arm punch. Tissue was sectioned, stained and imaged by confocal microscopy using identical parameters per patient to permit quantification.
- Epidermal Cx43, 26 and 30 and dermal Cx43 were discovered to be strikingly up-regulated in every ulcer from all three wound types, indicating that connexin up-regulation is a common feature between different types of chronic wounds. This result supports the therapeutic targeting of Cx26 and Cx30, alone or in combination with Cx43, to promote cell migration and wound healing in chronic ulcers.
- Connexins show dynamic changes in expression following acute wounding.
- 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.
- WE wound edge
- Cx26 and Cx30 were up- regulated in the epidermal leading edge.
- 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, 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.
- 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.
- Wound edge biopsies of chronic wound tissue were obtained during the Outpatients Clinic visit by a single operator (TES) via a 4 mm full thickness punch biopsy taken from the visible WE. The biopsy side away from the open wound was marked with ink to keep the sample orientated throughout processing. Each patient also supplied a matched 4 mm punch biopsy of arm skin, providing unwounded baseline Cx expression levels.
- 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/ ⁇ .
- 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.
- 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 FE) and patient.
- the residuals were tested for normality using the Kolmogorov-Smirnoff test; with a parametric distribution being assumed in all cases where the p-value > 0.05. Normality was not reached in three groups: VLU Cx30, DFU Cx30 and DFU Cx43 epidermal values. These specific data sets were independently transformed using the natural log before analysis..
- a Dunnett's post-hoc test compared all three wound measurements back to the reference group, i.e., arm values. Significance was taken at values p ⁇ 0.05.
- Fig. 1 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.
- 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 DFUs also had common features (Fig. 3). The epidermis was hyper-thickened but, unlike VLUs, this was more uniform in DFU samples. None of the biopsies showed any signs of thinning towards the WE and had no appearance of healing (Fig. 3c). Like the VLU, the DFU also had elevated levels of Cx expression but this was fairly consistent across the length of the biopsy. The Cx43 absolute group mean was elevated by 9-fold at the WE and 7-fold at the FE when compared to the unwounded forearm (both p ⁇ 0.001).
- Cx26 and Cx30 were also significantly increased, by 62-fold (p ⁇ 0.05) and 201-fold (p ⁇ 0.001) respectively, at the WE and 64-fold (p ⁇ 0.05) and 115- fold (p ⁇ 0.001) at the FE side of the biopsy.
- the dermis of the DFUs was distinctly different from that of the VLUs, as in many cases it lacked any signs of auto-fluorescent signal from the fibers of the dermal extracellular matrix or, if auto-fluorescence remained, the organizational pattern was absent. This suggested that a large proportion of the native collagen and elastin had either been degraded or was no longer being arranged into mature fibrils.
- the DFU dermis featured significantly increased levels of Cx43, by an average of 20-fold at the WE and 18-fold on the FE of the biopsy (p ⁇ 0.05 and p ⁇ 0.01).
- 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 pronounced of the VLUs.
- 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).
- 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.
- 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.
- Keratinocyte proliferation and differentiation is misregulated in DFUs and VLUs (Stojadinovic et al, 2008; Usui et al, 2008).
- 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, Cell Mol Med. 12: 2675-90).
- DFUs keratinocytes at the WE are hyper-proliferative, independent of ulcer edge thickness.
- Cx26 and 30 may be markers of hyper- proliferation with their expression predominantly influencing cellular differentiation.
- FIG. 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 ⁇ respectively.
- VLU venous leg ulcer; Cx, connexin.
- FIG. 1 Figure 2. Cx43, Cx26 and Cx30 expression in VLUs.
- FIG. 1 Cx43, Cx26 and Cx30 expression in DFUs.
- SOC standard of care
- the assigned treatment was applied to the reference diabetic foot ulcer (RDFU) twice weekly in addition to the SOC.
- 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.
- 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.
- 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.
- 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.
- sDFU single DFU
- mDFU multiple DFU
- both raw results and data from a simple statistical model comparing results for the 30 mg/mL dose compared to other groups combined 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.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Epidemiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Veterinary Medicine (AREA)
- Plant Pathology (AREA)
- Public Health (AREA)
- Microbiology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201461953608P | 2014-03-14 | 2014-03-14 | |
US201461953604P | 2014-03-14 | 2014-03-14 | |
PCT/US2015/020786 WO2015139047A2 (en) | 2014-03-14 | 2015-03-16 | Treatment of resistant lesions |
Publications (2)
Publication Number | Publication Date |
---|---|
EP3116996A2 true EP3116996A2 (en) | 2017-01-18 |
EP3116996A4 EP3116996A4 (en) | 2018-01-10 |
Family
ID=54072608
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP15760904.1A Withdrawn EP3116996A4 (en) | 2014-03-14 | 2015-03-16 | Treatment of resistant lesions |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP3116996A4 (en) |
CA (1) | CA2941140A1 (en) |
WO (1) | WO2015139047A2 (en) |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5431949B2 (en) * | 2006-12-11 | 2014-03-05 | コーダ セラピューティクス, インコーポレイテッド | Anti-connexin polynucleotides as impaired wound healing compositions |
EP2245061A2 (en) * | 2007-12-11 | 2010-11-03 | Coda Therapeutics, Inc. | Impaired wound healing compositions and treatments |
EP2237796A2 (en) * | 2007-12-21 | 2010-10-13 | Coda Therapeutics, Inc. | Use of anti-connexin peptides, alone or in combination with anti-connexin polynucleotides, for the treatment of surgical adhesions |
WO2013058778A1 (en) * | 2010-11-01 | 2013-04-25 | Nanoderm Sciences, Inc. | Polymers and hydrogels |
US9156896B2 (en) * | 2013-03-15 | 2015-10-13 | Coda Therapeutics, Inc. | Wound healing compositions and treatments |
-
2015
- 2015-03-16 CA CA2941140A patent/CA2941140A1/en not_active Abandoned
- 2015-03-16 EP EP15760904.1A patent/EP3116996A4/en not_active Withdrawn
- 2015-03-16 WO PCT/US2015/020786 patent/WO2015139047A2/en active Application Filing
Also Published As
Publication number | Publication date |
---|---|
CA2941140A1 (en) | 2015-09-17 |
WO2015139047A2 (en) | 2015-09-17 |
EP3116996A4 (en) | 2018-01-10 |
WO2015139047A3 (en) | 2015-12-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20100279921A1 (en) | Impaired wound healing compositions and treatments | |
RU2521329C2 (en) | Compositions and methods of treating persistent wounds | |
US20110300130A1 (en) | Impaired wound healing compositions and treatments | |
US20110038920A1 (en) | Wound healing compositions and treatments | |
JP2015057430A (en) | Use of anti-connexin polynucleotide and anti-connexin peptide for therapy of abnormal scar or excess scar | |
US20170056468A1 (en) | Treatment of resistant lesions | |
EP2831233A1 (en) | Compositions and treatments based on cadherin modulation | |
US20180028468A1 (en) | Sustained release drug delivery devices | |
WO2015130840A2 (en) | Treatment of resistant lesions | |
US20160244759A1 (en) | Compositions and treatments based on cadherin modulation | |
EP3116996A2 (en) | Treatment of resistant lesions | |
AU2017202281A1 (en) | Impaired wound healing compositions and treatments |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
17P | Request for examination filed |
Effective date: 20161006 |
|
AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
AX | Request for extension of the european patent |
Extension state: BA ME |
|
DAV | Request for validation of the european patent (deleted) | ||
DAX | Request for extension of the european patent (deleted) | ||
RIC1 | Information provided on ipc code assigned before grant |
Ipc: A61K 38/00 20060101ALI20170901BHEP Ipc: A61K 31/7088 20060101ALI20170901BHEP Ipc: C07H 21/04 20060101ALI20170901BHEP Ipc: C12N 15/11 20060101AFI20170901BHEP |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: OCUNEXUS THERAPEUTICS, INC. |
|
A4 | Supplementary search report drawn up and despatched |
Effective date: 20171208 |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: OCUNEXUS THERAPEUTICS, INC. |
|
RIC1 | Information provided on ipc code assigned before grant |
Ipc: A61K 38/00 20060101ALI20171204BHEP Ipc: A61K 31/7088 20060101ALI20171204BHEP Ipc: C07H 21/04 20060101ALI20171204BHEP Ipc: C12N 15/11 20060101AFI20171204BHEP |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20180714 |