WO2018151823A1 - Composition and methods for preventing radiation injury and promoting tissue regeneration - Google Patents
Composition and methods for preventing radiation injury and promoting tissue regeneration Download PDFInfo
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- WO2018151823A1 WO2018151823A1 PCT/US2018/000035 US2018000035W WO2018151823A1 WO 2018151823 A1 WO2018151823 A1 WO 2018151823A1 US 2018000035 W US2018000035 W US 2018000035W WO 2018151823 A1 WO2018151823 A1 WO 2018151823A1
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/38—Cellulose; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
Definitions
- CRI is defined as dermal injury resulting from direct or indirect exposure to low-penetrating radiation. In fallout events, the deposition of hot particles on the skin can result in high doses of acute localized radiation exposure. Exposure to radiation causes damage to the basal cell layer of the skin and results in inflammation, erythema, desquamation, intense reddening, blistering, debilitating fibrosis, infection, ulceration, and necrosis of the exposed tissue.
- ARS describes one or a combination of clinical syndromes that occur after exposure to ionizing radiation.
- ARS syndromes include hematopoietic syndrome, gastrointestinal syndrome, neurovascular syndrome, cardiac syndrome, and others, and each of these syndromes can occur alone or in combination with others.
- radiation exposure frequently occurs in combination with other traumatic injury, such as burns, blunt trauma, and open wounds, all of which can be complicated by microbial infection.
- Combined radiation injury describes conditions where radiation injuiy is coupled with other insults such as burns, wounds, infection, or blunt trauma.
- an isolated polypeptide comprising a carboxy-terminal amino acid sequence of an alpha Connexin (also referred to herein as an alpha Connexin carboxy-Terminal (ACT) polypeptide), or a conservative variant thereof.
- ACT alpha Connexin carboxy-Terminal
- a method of preventing mitigating, and treating radiation injury in a subject at risk of such injury comprising administering to the subject one or more of the herein provided compositions (e.g., polypeptides, nucleic acids, or vectors).
- provided herein are methods for preventing, mitigating the progression of, and treating cutaneous radiation injury (CRl), acute radiation syndrome (ARS), combined radiation injury, or any combination thereof.
- the present disclosure provides methods for preventing, mitigating the progression of, and treating CRl, ARS, and/or combined radiation injury following exposure to ionizing radiation, for example resulting from radiological dispersal devices, improvised nuclear devices, nuclear weapons, or nuclear power plant explosions.
- provided herein are methods for preventing, mitigating the progression of, and treating radiation dermatitis.
- the composition is administered to the subject at a dose of from about 50 ⁇ , about 100 ⁇ , about 150 ⁇ 5 about 200 ⁇ , about 300 ⁇ , about 400 ⁇ , about 500 ⁇ , about 600 ⁇ , about 700 ⁇ , about 800 ⁇ , about 900 ⁇ , or about 1 ,000 ⁇ .
- the composition is adm inistered in a daily dosing regimen.
- the composition is administered parenterally to the subject.
- the composition is administered to the subject by an intravenous, subcutaneous, intraperitoneal, or intramuscular route.
- the composition is administered to the subject by intravenous injection, subcutaneous injection, intraperitoneal injection, or intramuscular injection.
- the composition is administered parenterally to the subject at a dose of from about 1 mg/kg to about 50 mg/kg. In further embodiments, the composition is administered to the subject at a dose of about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 1 5 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, or about 50 mg/kg. In some embodiments, the composition is adm inistered in a daily dosing regimen. In some embodiments, parenteral administration of the composition treats, prevents, and/or inhibits the progression of internal radiation damage.
- the radiation injury is selected from the group consisting of CRI, ARS, radiation burns, radiation dermatitis, radiation injury to the nervous system or brain, radiation pneumonitis, radiation-induced enteritis, and internal radiation.
- the radiation injury is CRI.
- the radiation injury is ARS.
- the radiation injury is combined radiation injury.
- the radiation injury comprises radiation injuiy in combination with burns, wounds, infection, or blunt trauma.
- the radiation injury results from exposure to a source of radiation selected from the group consisting of weapons of mass destruction (WMD), nuclear explosions, and dirty bombs.
- WMD weapons of mass destruction
- the radiation injury comprises radiation dermatitis, wh ich si a specific type of radiation injury resulting from radiotherapy regimens or other interventional medical procedures.
- the radiation injury results from exposure to the sun, x-ray and other diagnostic machines, or ionizing radiation- emitting sources such as tanning beds.
- the peptides provided herein are provided in a sunscreen, sunblock, or suntan lotion composition.
- the radiation injury occurs in a tissue selected from the group consisting of skin, heart, bone, brain, spinal cord, cornea, retina, hematopoietic system, gastrointestinal system, and peripheral nerve.
- the radiation injury occurs on the skin.
- the average skin score is decreased by at least about 30% in presence of the polypeptide as compared to a control.
- the polypeptide prevents scar formation.
- the polypeptide promotes tissue regeneration.
- the polypeptide prevents tissue damage.
- the alpha connexin polypeptide comprises the carboxy terminal-most 4 to 30 contiguous amino acids of an alpha Connexin. In some embodiments, the polypeptide consists of the carboxy terminal-most 5 to 19 contiguous amino acids of the alpha Connexin. In some embodiments, the alpha Connexin is Connexin 37, Connexin 40, Connexin 43, or Connexin 45. In further embodiments, the polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In some embodiments, the polypeptide further comprises a cellular internalization sequence.
- the cellular internalization sequence comprises an amino acid sequence of a protein selected from a group consisting of Antennapedia, TAT, HTV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II, Transportan, MAP (model amphipathic peptide), -FGF, Ku70, Prion, pVEC, Pep- 1 , SynB 1 , Pep-7, HN- 1 , BGSC (Bis-Guanidinium-Spermidine-Cholesterol) and BGTC (Bis-Guanidinium-Tren- Cholesterol).
- a protein selected from a group consisting of Antennapedia, TAT, HTV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II, Transportan, MAP (model amphipathic peptide), -FGF, Ku70, Prion, pVEC, Pep- 1 , SynB 1 , Pep-7, HN- 1
- the cellular internalization sequence is Antennapedia, and wherein the sequence comprises the amino acid sequence of SEQ ID NO:7.
- the polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l l , and SEQ ID NO: 12.
- the alpha connexin polypeptide comprises a biotin at the N- terminus and/or the C-terminus of the polypeptide.
- the composition comprises SEQ ID NO: 91 .
- the composition is administered topically.
- the composition further comprises hydroxyethylcellulose gel.
- the hydroxyethylcellulose gel is present at a concentration of about 0.25% (w/w), about 0.5% (w/w), about 0.75% (w/w), about 1 .00% (w/w), about 1 .25% (w/w), about 1 .5% (w/w) or about 2.0% (w/w).
- the composition is administered systemically.
- the composition is administered parenterally.
- the composition for systemic administration further comprises one or more pharmaceutically acceptable excipient suitable for systemic administration to a subject.
- the composition may be administered both topically and systemically to a subject in need thereof.
- a subject exposed to radiation injury is administered a composition provided herein both topically and systemically in order to treat, prevent, and/or mitigate the progression of both CRI and ARS.
- a subject is administered a composition comprising a polypeptide provided herein topically, and is administered a composition comprising a polypeptide herein systemically.
- the compositions for topical and systemic administration comprise the same alpha connexin polypeptide.
- a subject exposed to radiation injury is administered a composition provided herein both topically and systemically in order to treat, prevent, and/or mitigate the progression of combined radiation injury involving CRI and/or ARS in combination with burns, wounds, infection, or blunt trauma.
- FIGS 1 A, I B, and 1 C show that topical administration of ACT peptide is effective in the prevention, mitigation, and treatment of cutaneous radiation injury (CRI).
- CRI cutaneous radiation injury
- Yorkshire pigs were anesthetized and exposed to single fractions of low energy electrons (6 MeV). Beginning on Day 1 and every 3 days thereafter, skin sites were scored for erythema and desquamation using a Kumar Scale.
- Topical treatment with Granexin ® 100 ⁇ or 200 ⁇
- vehicle was initiated upon observation of erythema (Kumar score > 1 .0). and was applied once daily to exposure sites.
- Figure 1 A shows average skin score comparisons with Granexin ® gel (200 ⁇ ) treatment and vehicle treatment.
- Figure 2 shows the prophylactic effect of ACT peptide on the prevention and mitigation of CRI post-exposure. Irradiated sites were prophylactically (prior to the presentation of CRI symptoms) treated with vehicle or Granexin ® gel 1 ,000 ⁇ (beginning day 1 ; i.e. the day after radiation exposure) on the same daily treatment regimen. Day 21 photographic comparisons show that prophylactic treatment with Granexin ® gel at 1 ,000 ⁇ prevents injury progression and reduces injury severity as compared to vehicle control.
- Figure 3 illustrates the manufacturing process flow chart of an ACT peptide of the invention.
- Figure 4 shows that ACT1 polypeptide reduces early mortality in a Gl-toxicity model of irradiation.
- compositions and methods provided herein are related to preventing, treating, and/or mitigating the progression of radiation injury.
- the compositions and methods provided herein are related to preventing, treating, and/or mitigating the progression of cutaneous radiation injury (C I).
- the compositions and methods herein are related to preventing, treating, and/or mitigating the progression of acute radiation syndrome (ARS).
- the compositions and methods herein are related to preventing, treating, and/or mitigating the progression of combined radiation injuiy.
- compositions and methods provided herein are capable of prophylactic action in radiation injury.
- the compositions and methods provided herein prevent CRT, ARS, or combined radiation injury, and/or mitigate the progression of injury following radiation exposure but prior to symptom manifestation.
- the provided compositions and methods may be used to prevent, treat, or mitigate the progression of the particular types of radiation injury caused by radiological dispersal devices, improvised nuclear devices, nuclear weapons, nuclear power plant explosions, and the like, in populations of subject that are at high risk of exposure to such devices, weapons, and explosions, and/or in subjects who have recently experienced such an exposure.
- ARS, CRI, and combined radiation injury are particular types of radiation injury that result, for example, from a dirty bomb attack or a nuclear power plant explosion.
- the compositions and method herein are related to preventing, treating, and/or mitigating the progression of internal radiation injury as well as radiation injuiy to the skin and underlying tissues.
- the compositions and methods herein are particularly useful for preventing and/or mitigating the progression of CRI and/or ARS and/or combined radiation injuiy in populations of subjects who are at high risk of experiencing CRI and/or ARS and/or combined radiation injury.
- the compositions provided herein are applied topically to a subject to treat, prevent, and/or mitigate the progression of an injury to the skin and underlying tissues as a result of exposure to radiation.
- the compositions provided herein are administered systemically to a subject to treat, prevent, and/or mitigate the progression of systemic injury, such as injury to internal organs, that results from exposure to radiation.
- the same subject is administered the one or more of the compositions provided herein both topically to treat, prevent, and/or mitigate the progression of injuiy to the skin and underlying tissues and systemically to treat, prevent, and/or mitigate the progression of internal damage (e.g., damage to systemic organs).
- the surprising prophylactic action of the provided methods and compositions is useful for preventing, treating, and mitigating the progression of radiation injury that results from the sun, diagnostic machines, and tanning beds.
- Diagnostic machines that use ionizing radiation include, for example, x ray machines and computed tomography (CT) scanners.
- CT computed tomography
- the methods and compositions may be used to prevent, treat, or mitigate the progression of a radiation injury resulting from the sun, a diagnostic machine, or a tanning bed, in populations of subjects who are at high risk of exposure to levels of ionizing radiation from the sun, diagnostic machine, or tanning bed, or who have recently experienced such exposure.
- compositions and methods herein are applied to a subject prior to the subject's exposure to the sun, diagnostic machine, or tanning bed, and/or are applied to the subject after the subject's exposure to the sun, diagnostic machine, or tanning bed.
- compositions provided herein are in the form of a sunscreen or sunblock, or a product that is available at a facility that provides tanning products and/or tanning bed services (e.g., a suntan lotion, tanning accelerator, or tanning oil).
- tanning products and/or tanning bed services e.g., a suntan lotion, tanning accelerator, or tanning oil.
- the present disclosure provides methods and compositions for administration to a subject prior to the subject's use of a tanning bed or prior to the subject's exposure to the sun, in order to prevent, treat, or mitigate the progression of the radiation injury that may be caused by the use or exposure.
- the present disclosure provides methods and compositions for administration to a subject after the subject's use of a tanning bed or after the subject's exposure to the sun, in order to prevent, treat, or mitigate the progression of the radiation injury caused by the use or exposure.
- the present disclosure provides methods and compositions for administration to a subject prior to exposure to a diagnostic machine or procedure that emits ionizing radiation, such as an x-ray or CT scan, wherein the methods and compositions prevent, treat, or mitigate the progression of the radiation injury that is caused by that exposure.
- the present disclosure provides methods and compositions for administration to a subject after exposure to a diagnostic machine or procedure that emits ionizing radiation, wherein the methods and compositions prevent, treat, or mitigate the progression of the radiation injury that is caused by that exposure.
- the surprising prophylactic action of the provided methods and compositions is useful for preventing, treating, and mitigating the progression of radiation dermatitis.
- Radiation dermatitis results from radiation therapy such as a therapy for treatment of diseases such as cancer.
- the present disclosure provides methods and compositions for administration to a subject after the administration of a radiation therapy, in order to prevent, treat, or mitigate the progression of the radiation injury caused by the radiation therapy.
- the present disclosure provides methods and compositions for administration to a subject after exposure to a radiation therapy, wherein the methods and compositions prevent, treat, or mitigate the progression of the radiation injury that is caused by the radiation therapy.
- the methods and compositions provided herein are administered to the subject throughout the course of treatment with the radiation therapy, wherein the methods and compositions prevent, treat, or mitigate the progression of the radiation injury that is caused by the radiation therapy.
- the methods and compositions provided herein are administered to the subject before, during, and/or after the course of the radiation therapy, wherein the methods and compositions prevent, treat, or mitigate the progression of the radiation injury that is caused by the radiation therapy.
- compositions and methods are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein.
- each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
- any subset or combination of these is also specifically contemplated and disclosed.
- the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
- This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions.
- steps in methods of making and using the disclosed compositions are if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
- the herein provided polypeptide can be any polypeptide comprising the carboxy-terminal most amino acids of an alpha Connexin, wherein the polypeptide does not comprise the full-length alpha Connexin protein.
- the provided polypeptide does not comprise the cytoplasmic N-terminal domain of the alpha Connexin.
- the provided polypeptide does not comprise the two extracellular domains of the alpha Connexin.
- the provided polypeptide does not comprise the four transmembrane domains of the alpha Connexin.
- the provided polypeptide does not comprise the cytoplasmic loop domain of the alpha Connexin.
- the provided polypeptide does not comprise that part of the sequence of the cytoplasmic carboxyl terminal domain of the alpha Connexin proximal to the fourth transmembrane domain.
- proline residues consistently positioned some 17 to 30 amino acids from the carboxyl terminal-most amino acid (Table 2).
- proline residue at amino acid 363 is positioned 19 amino acids back from the carboxyl terminal most isoleucine.
- a proline residue at amino acid 362 is positioned 1 8 amino acids back from the carboxyl terminal-most isoleucine.
- a glycine residue at amino acid 377 is positioned 1 9 amino acids back from the carboxyl terminal most isoleucine.
- a proline residue at amino acid 258 is positioned 28 amino acids back from the carboxyl terminal most methionine.
- the provided polypeptide does not comprise amino acids proximal to said conserved proline or glycine residue of the alpha Connexin.
- the provided polypeptide can comprise the c-terminal-most 4 to 30 amino acids of the alpha Connexin, including the c-terminal most 4, 5, 6, 7, 8, 9, 1 0, 1 1 , 1 2, 1 3, 14, 1 5, 1 6, 1 7, 1 8, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acids of the alpha Connexin.
- the carboxy-terminal most amino acids of an alpha Connexin in the provided peptides can be flanked by non-alpha Connexin or non-ACT peptide Connexin amino acids.
- flanking non-alpha Connexin and non-ACT Connexin amino acids are provided herein.
- An example of non-ACT Connexin amino acids are the carboxy-terminal 20 to 120 amino acids of human Cx43 (SEQ ID NO: 72).
- Another example would be the carboxy-terminal 20 to 1 20 am ino acids of chick Cx43 (SEQ ID NO: 73).
- Another example would be the carboxy- terminal 20 to 120 amino acids of human Cx45 (SEQ ID NO: 74).
- Another example would be the carboxy-terminal 20 to 120 amino acids of ch ick Cx45 (SEQ ID NO: 75). Another example would be the carboxy-terminal 20 to 1 20 amino of human Cx37 (SEQ ID NO: 76). Another example would be the carboxy-terminal 20 to 120 amino acids of rat Cx33 (SEQ ID NO: 77).
- Non-alpha Connexin is the 239 amino acid sequence of enhanced green fluorescent protein.
- ACT 1 is shown to be functional when fused to the carboxy terminus of the 239 amino acid sequence of GFP
- ACT peptides are expected to retain function when flanked with non-Connexin polypeptides of up to at least 239 amino acids. Indeed, as long as the ACT sequence is maintained as the free carboxy term inus of a given polypeptide, and the ACT peptide is able to access its targets.
- polypeptides exceeding 239 amino acids in addition to the ACT peptide can function in reducing inflammation, promoting healing, increasing tensi le strength, reducing scarring and promoting tissue regeneration following injury.
- Connexins are the sub-unit protein of the gap junction channel, which is responsible for intercellular communication (Goodenough and Paul, 2003). Based on patterns of conservation of nucleotide sequence, the genes encoding Connexin proteins are divided into two families termed the alpha and beta Connexin genes. The carboxy-terminal-most amino acid sequences of alpha Connexins are characterized by multiple distinctive and conserved features (see Table 2).
- ACT peptides This conservation of organization is consistent with the ability of ACT peptides to form distinctive 3D structures, interact with multiple partnering proteins, mediate interactions with lipids and membranes, interact with nucleic acids including DNA, transit and/or block membrane channels and provide consensus motifs for proteolytic cleavage, protein cross-l inking, ADP-ribosylation, glycosylation and phosphorylation.
- the provided polypeptide interacts with a domain of a protein that normally mediates the binding of said protein to the carboxy- terminus of an alpha Connexin.
- NOV nephroblastoma overexpressed protein
- the provided polypeptide can inhibit the operation of a molecular machine, such as, for example, one involved in regulating the aggregation of Cx43 gap junction channels.
- inhibitor means to decrease an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete loss of activity, response, condition, or disease. This can also include, for example, a 1 0% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 1 0, 20, 30, 40, 50, 60, 70, 80, 90, 1 00%, or any amount of reduction in between as compared to native or control levels.
- the ACT sequence of the provided polypeptide can be from any alpha Connexin.
- the alpha Connexin component of the provided polypeptide can be from a human, murine, bovine, monotrene, marsupial, primate, rodent, cetacean, mammalian, avian, reptilian, amphibian, piscine, chordate, protochordate or other alpha Connexin.
- the provided polypeptide can comprise an ACT of a Connexin selected from the group consisting of mouse Connexin 47, human Connexin 47, Human Connexin 46.6, Cow Connexin 46.6, Mouse Connexin 30.2, Rat Connexin 30.2, Human Connexin 3 1 .9, Dog Connexin 3 1 .9, Sheep Connexin 44, Cow Connexin 44, Rat Connexin 33, Mouse Connexin 33, Human Connexin 36, mouse Connexin 36, rat Connexin 36, dog Connexin 36, chick Connexin 36, zebrafish Connexin 36, morone Connexin 35, morone Connexin 35, Cynops Connexin 35, Tetraodon Connexin 36, human Connexin 37, chimp Connexin 37, dog Connexin 37, Cricetulus Connexin 37, Mouse Connexin 37, Mesocricetus
- the provided polypeptide can comprise the amino acid sequence SEQ ID NO: l , SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:89, SEQ ID NO: 90, or SEQ. ID NO:91 or conservative variants or fragments thereof.
- the 20-30 carboxy-terminal-most amino acid sequence of alpha Connexins are characterized by a distinctive and conserved organization.
- the P and G residues occur in clustered motifs (e.g., Table 2, italicized) proximal to the carboxy-terminal type II PDZ binding motif.
- the S and T phosphor-amino acids of most alpha Connexins also are typically organized in clustered, repeat-like motifs (e.g., Table 2, underlined). This organization is particularly the case for Cx43, where 90% of 20 carboxyl terminal-most amino acids are comprised of the latter seven amino acids.
- ACT peptide organization of Cx43 is highly conserved from humans to fish (e.g., compare Cx43 ACT sequences for humans and zebrafish in Table 2).
- the ACT peptide organization of Cx45 is highly conserved from humans to birds (e.g., compare Cx45 ACT sequences for humans and chick in Table 2).).
- the ACT peptide organization of Cx36 is highly conserved from primates to fish (e.g., compare Cx36 ACT sequences for chimp and zebrafish in Table 2).
- the provided polypeptide comprises one, two, three or al l of the amino acid motifs selected from the group consisting of 1 ) a type II PDZ binding motif, 2) Proline (P) and/or Glycine (G) hinge residues; 3) clusters of phospho-Serine (S) and/or phospho- Threonine (T) residues; and 4) a high frequency of positively charged Arginine (R) and Lysine (K) and negatively charged Aspartic acid (D) and/or Glutamic acid (E) am ino acids).
- the provided polypeptide comprises a type II PDZ binding motif at the carboxy-terminus, Proline (P) and/or Glycine (G) hinge residues proximal to the PDZ binding motif, and positively charged residues ( , R, D, E) proximal to the hinge residues.
- P Proline
- G Glycine
- residues R, D, E
- PDZ domains were originally identified as conserved sequence elements within the postsynaptic density protein PSD95/SAP90, the Drosophila tumor suppressor dlg-A, and the tight junction protein ZO-1 . Although originally referred to as GLGF or DHR motifs, they are now known by an acronym representing these first three PDZ-containing proteins (PSD95/DLG/ZO- 1 ).
- the provided polypeptide can inhibit the binding of an alpha Connexin to a protein comprising a PDZ domain.
- the PDZ domain is a specific type of protein-interaction module that has a structurally well-defined interaction 'pocket' that can be filled by a PDZ-binding motif, referred to herein as a "PDZ motif.
- PDZ motif are consensus sequences that are normally, but not always, located at the extreme intracellular carboxyl terminus.
- the provided polypeptide comprises a type II PDZ binding motif.
- Cx37 represents an exceptional variation on the ACT peptide theme.
- the Cx37 ACT-like sequence is GQKPPSRPSSSASKKQ*YV (SEQ ID NO: 43).
- Cx37 has a neutral Q* at position 2 where a hydrophobic amino acid would be expected.
- Cx37 comprises what might be termed a type II PDZ binding domain-like sequence.
- Cx37 strictly maintains all other aspects of ACT peptide organization including clustered serine residues, frequent R and K residues and a P-rich sequence proximal to the PDZ binding domain-like sequence. Given this overall level of conservation of ACT-like organization in common with the other >70 alpha Connexins listed above, it is understood that the Cx37 ACT-like carboxy terminus functions in the provided capacity.
- Cx26 has no carboxyl terminal type II PDZ binding motif; less than 30% of the carboxyl terminal most amino acids comprise S, T, R, D or E residues; it has no evidence of motifs proximal to a type II PDZ binding motif or PDZ binding like motif containing clusters of P and G hinge residues; and no evidence of clustered, repeat-like motifs of serine and threonine phospho-amino acids.
- Cx26 does have three Lysine (K) residues, clustered one after the other near the carboxy terminus of the sequence.
- the unique functional characteristics of this relatively short stretch of amino acids encompass unexpected roles in reducing inflammation, promoting healing, reducing scarring, increasing tensile strength, and promoting regeneration of complex tissue structure and function following injury in tissues as diverse as skin and brain.
- the amino acids of the provided ACT polypeptide is comprised one or more of Proline (P), Glycine (G), phospho-Serine (S), phospho-Threonine (T), Arginine (R), Lysine ( ), Aspartic acid (D), or Glutamic acid (E) amino acid residues.
- Proline (P), Glycine (G), Arginine (R), Lysine ( ), Aspartic acid (D), and Glutamic acid (E) are necessary determinants of protein structure and function.
- Proline and Glycine residues provide for tight turns in the 3D structure of proteins, enabling the generation of folded conformations of the polypeptide required for function.
- Charged amino acid sequences are often located at the surface of folded proteins and are necessary for chemical interactions mediated by the polypeptide including protein-protein interactions, protein-lipid interactions, enzyme-substrate interactions and protein-nucleic acid interactions.
- Proline (P) and Glycine (G) Lysine (K), Aspartic acid (D), and Glutamic acid (E) rich regions proximal to the type II PDZ binding motif provide for properties necerney to the provided actions of ACT peptides.
- the provided polypeptide comprises Proline (P) and Glycine (G) Lysine ( ), Aspartic acid (D), and/or Glutamic acid (E) rich regions proximal to the type II PDZ binding motif.
- Phosphorylation is the most common post-translational modification of proteins and is crucial tor modulating or modifying protein structure and function. Aspects of protein structure and function modified by phosphorylation include protein conformation, protein-protein interactions, protein-lipid interactions, protein-nucleic acid interactions, channel gating, protein trafficking and protein turnover.
- the phospho-Serine (S) and/or phospho-Threonine (T) rich sequences are necessary for modifying the function of ACT peptides, increasing or decreasing efficacy of the polypeptides in their provided actions.
- the provided polypeptide comprise Serine (S) and/or phospho-Threonine (T) rich sequences or motifs.
- ACT peptide In another example, respecting definition of an ACT peptide, it is highly auspicious, in light of the high degree of tissue/organ regeneration potential in lower animals such as fish, that a methionine occurs near the amino terminus of the ACT sequence of zebrafish Cx43 (Table 2). In addition to encoding methionine, the methionine base pair triplet is an alternate translation start site. If translation initiated from this methionine, the sequence SSRARPDDLDV (SEQ ID NO:89), would be produced. This translation product maintains all the conserved and distinctive features of a canonical ACT peptide.
- this peptide comprises a carboxy terminal type II PDZ binding domain and has a domain enriched in P, R and D residues proximal to the PDZ binding domain.
- the sequence comprises a clustered S motif, with potential to modulate ACT peptide function at its amino terminal. This raises the interesting prospect that animals with high tissue/organ regeneration potential such as fish may translate ACT peptides sequences directly.
- the provided polypeptide can comprise the c-terminal sequence of human Cx43.
- the provided polypeptide can comprise the amino acid sequence SEQ ID NO: l or SEQ ID NO:2.
- the polypeptide can comprise 9 amino acids of the carboxy terminus of human Cx40.
- the polypeptide can comprise the amino acid sequence SEQ ID NO:5.
- variants, derivatives, and fragments are contemplated.
- Protein variants and derivatives are well understood to those of skill in the art and in can involve amino acid sequence modifications.
- amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants.
- Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence.
- variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
- Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known and include, for example, M 13 primer mutagenesis and PCR mutagenesis.
- Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 1 0 amino acid residues. Deletions or insertions preferably are made in adjacent pairs, i .e., a deletion of 2 residues or insertion of 2 residues.
- substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct.
- the mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondaiy mRNA structure unless such a change in secondary structure of the mRNA is desired.
- Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions general ly are made in accordance with the following Table 3 and are referred to as conservative substitutions.
- substitutions include combinations shown in Table 3. Conservatively substituted variations of each explicitly disclosed sequence are included within the polypeptides provided herein.
- conservative substitutions have little to no impact on the biological activity of a resulting polypeptide.
- a conservative substitution is an amino acid substitution in a peptide that does not substantially affect the biological function of the peptide.
- a peptide can include one or more amino acid substitutions, for example 2- 1 0 conservative substitutions, 2-5 conservative substitutions, 4-9 conservative substitutions, such as 2, 5 or 10 conservative substitutions.
- a polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that polypeptide using, for example, standard procedures such as site-directed mutagenesis or PCR.
- a polypeptide can be produced to contain one or more conservative substitutions by using standard peptide synthesis methods.
- An alanine scan can be used to identify which amino acid residues in a protein can tolerate an amino acid substitution.
- the biological activity of the protein is not decreased by more than 25%, for example not more than 20%, for example not more than 10%, when an alanine, or other conservative amino acid (such as those listed below), is substituted for one or more native amino acids.
- Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post- translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seiyl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86 [ 1983]), acetylation of the N-terminal amine and, in some instances, amidation of the C-terminal carboxyl.
- amino acid and peptide analogs which can be incorporated into the disclosed compositions.
- D amino acids or amino acids which have a different functional substituent than the amino acids shown in Table 3.
- the opposite stereoisomers of naturally occurring peptides are disclosed, as well as the stereoisomers of peptide analogs.
- These amino acids can readily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog amino acid into a peptide chain in a site specific way (Thorson et al., Methods in Molec. Biol.
- Molecules can be produced that resemble polypeptides, but which are not connected via a natural peptide linkage.
- Amino acid analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, greater ability to cross biological barriers (e.g., gut, blood vessels, blood-brain-barrier), and others.
- enhanced or desirable properties such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, greater ability to cross biological barriers (e.g., gut, blood vessels, blood-brain-barrier), and others.
- D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such.
- Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type e.g., D-lysine in place of L- lysine
- Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations.
- the provided polypeptide can comprise a conservative variant of the c- terminus of an alpha Connexin (ACT). As shown in Table 4, an example of a single conservative substitution within the sequence SEQ ID NO:2 is given in the sequence SEQ ID NO:3. An example of three conservative substitutions within the sequence SEQ ID NO:2 is given in the sequence SEQ ID NO:4.
- the provided polypeptide can comprise the amino acid SEQ ID NQ:3 or SEQ TD NO:4.
- variants of the nucleic acids and polypeptides herein disclosed which have at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 percent sequence identity to the stated or known sequence.
- sequence identity can be calculated after al igning the two sequences so that the sequence identity is at its highest level.
- sequence identity Another way of calculating sequence identity can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local sequence identity algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 ( 1981 ), by the sequence identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 ( 1970), by the search for similarity method of Pearson and Lipman, Proc. Natl . Acad. Sci. U.S.A. 85 :
- nucleic acids can be obtained by, for example, the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 1 83 :281 -306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment.
- the provided polypeptide can comprise an amino acid sequence with at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 percent sequence identity to the c-terminus of an alpha Connexin (ACT).
- ACT alpha Connexin
- the provided polypeptide comprises an amino acid sequence with at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 percent sequence identity to SEQ ID NO: l , SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:3 1 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 , SEQ ID NO:89 or SEQ ID NO:90.
- SEQ ID NO:29 amino acid sequence
- SEQ ID NO:30 amino acid sequence
- the herein provided polypeptides can be added directly to a tissue injury in a subject.
- efficiency of cytoplasmic localization of the provided polypeptide is enhanced by cellular internalization transporter chemically linked in cis or trans with the polypeptide.
- Efficiency of cell internalization transporters are enhanced further by light or co-transduction of cells with Tat-HA peptide.
- the provided polypeptide can comprise a cellular internalization transporter or sequence.
- the cellular internalization sequence can be any internalization sequence known or newly discovered in the art, or conservative variants thereof.
- Non-limiting examples of cellular internalization transporters and sequences include Antennapedia sequences, TAT, HIV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II, Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep- 1 , SynB l , Pep-7, HN-1 , BGSC (Bis- Guanidinium-Spermidine-Cholesterol, and BGTC (Bis-Guanidinium-Tren-Cholesterol) (see Table 5).
- the provided polypeptide can further comprise the amino acid sequence
- SEQ IDNO:7 SEQ ID NO: 14 (Bucci, M. et al.2000. Nat. Med.6, 1362-1367), SEQ ID NO: 15 (Derossi, D., et al. 1994. Biol. Chem.269, 10444-10450), SEQ ID NO: 16 (Fischer, P. M. et al 2000. J. Pept. Res. 55, 1 63- 1 72), SEQ ID NO: 1 7 (Frankel, A. D. & Pabo, C. O. 1 988. Cell 55, 1 1 89- 1 193; Green, M. & Loewenstein, P. M. 1988. Cell 55, 1 1 79- 1 1 88), SEQ ID NO: 1 8 (Park, C.
- SEQ ID NO: 19 (Pooga, M., et al. 1998. FASEB J. 1 2, 67-77), SEQ ID NO:20 (Oehlke, J. et al. 1998. Biochim. Biophys. Acta. 1414, 1 27- 139), SEQ ID NO:21 (Lin, Y. Z., et al. 1 995. J. Biol. Chem. 270, 14255- 14258), SEQ ID NO:22 (Sawada, M, et al. 2003. Nature Cell Biol.
- SEQ ID NO:25 (Morris, M. C., et al. 2001 . Nature Biotechnol. 19, 1 1 73- 1 1 76), SEQ ID NO:26 (Rousselle, C. et al. 2000. Mol. Pharmacol. 57, 679- 686), SEQ ID NO:27 (Gao, C. el al. 2002. Bioorg. Med. Chem. 1 0, 4057-4065), or SEQ ID NO:28 (Hong, F. D. & Clayman, G. L. 2000. Cancer Res. 60, 655 1 -6556).
- the provided polypeptide can further comprise BGSC (Bis-Guanidinium-Spermidine-Cholesterol) or BGTC (Bis-Guanidinium-Tren-Cholesterol) (Vigneron, J. P. et al. 1998. Proc. Natl. Acad. Sci. USA. 93, 9682-9686).
- BGSC Bis-Guanidinium-Spermidine-Cholesterol
- BGTC Bis-Guanidinium-Tren-Cholesterol
- the provided polypeptide can comprise any ACT sequence (e.g, any of the amino acids
- the provided polypeptide can comprise an Antennapedia sequence comprising amino acid sequence
- the provided polypeptide can comprise the amino acid sequence SEQ ID NO:7.
- the provided polypeptide can comprise the amino acid sequence SEQ ID NO:7.
- SEQ ID NO:8 SEQ ID NO:9, SEQ ID NO: 1 0, SEQ ID NO: 1 1 , or SEQ ID NO: 1 2.
- nucleic acids encoding the polypeptides provided herein.
- the disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell, the expressed mRNA will typically be made up of A, C, G, and U.
- isolated nucleic acid also refers to RNA, e.g., an mRNA molecule that is encoded by an isolated DNA molecule, or that is chemically synthesized, or that is separated or substantially free from at least some cellular components, e.g., other types of RNA molecules or polypeptide molecules.
- nucleic acid encoding a polypeptide comprising the amino acid sequence SEQ ID NO: l , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12.
- the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes.
- direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes.
- Appropriate means for transfection, including viral vectors, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA are described by, for example, Wolff, J. A., et al., Science, 247, 1465- 1468, ( 1990); and Wolff, J. A. Nature, 352,
- Transfer vectors can be any nucleotide construction used to deliver genes into cells (e.g., a plasmid), or as part of a general strategy to deliver genes, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).
- plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as SEQ ID NO:6, into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered.
- the promoters are derived from either a virus or a retrovirus.
- Viral vectors are, for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also disclosed are any viral families which share the properties of these viruses which make them suitable for use as vectors.
- Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector.
- Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells.
- Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells.
- Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature. Also disclosed is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens. Vectors of this type can carry coding regions for Interleukin 8 or 10.
- a retrovirus is an animal virus belonging to the virus family of Retroviridae, including any types, subfamilies, genus, or tropisms.
- Retroviral vectors in general, are described by Verma, 1. M., Retroviral vectors for gene transfer. In Microbiology- 1985, American Society for Microbiology, pp. 229-232, Washington, (1985), which is incorporated by reference herein. Examples of methods for using retroviral vectors for gene therapy are described in U.S. Pat. Nos. 4,868, 1 16 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136; and Mulligan, (Science 260:926-932 (1993)); the teachings of which are incorporated herein by reference.
- a retrovirus is essentially a package which has packed into it nucleic acid cargo.
- the nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat.
- a packaging signal In addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging of the replicated virus.
- a retroviral genome contains the gag, pol, and env genes which are involved in the making of the protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell.
- gag, pol, and env genes allow for about 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed, and upon replication be packaged into a new retroviral particle. This amount of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript.
- a packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery, but lacks any packaging signal.
- the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.
- viruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest. 92: 1 580- 1 586 (1993); Kirshenbaum, J. Clin. Invest. 92:381 -387 (1993); Roessler, J. Clin. Invest.
- Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 ( 1973); Svensson and Persson, J. Virology 55:442-449 ( 1985); Seth, et al., J. Virol. 5 1 :650-655 ( 1984); Seth, et al., Mol. Cell. Biol. 4: 1528-1533 ( 1984); Varga et al., J.
- a viral vector can be one based on an adenovirus which has had the E l gene removed, and these virons are generated in a cell line such as the human 293 cell line, in one aspect, both the E l and E3 genes are removed from the adenovirus genome.
- AAV adeno-associated virus
- This defective parvovirus can infect many cell types and is nonpathogenic to humans.
- AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19.
- this vector can be the P4.1 C vector produced by Avigen, San Francisco, Calif, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and/or a marker gene, such as the gene encoding the green fluorescent protein, GFP.
- the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene.
- ITRs inverted terminal repeats
- Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B 19 parvovirus.
- the disclosed vectors thus provide DNA molecules which are capable of integration into a mammalian chromosome without substantial toxicity.
- the inserted genes in viral and retroviral usually contain promoters, and/or enhancers to help control the expression of the desired gene product.
- a promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site.
- a promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.
- compositions can be delivered to the target cells in a variety of ways.
- the compositions can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation.
- the delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro.
- the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.
- delivery of the compositions to cells can be via a variety of mechanisms.
- delivery can be via a liposome, using commercially available liposome preparations such as LlPOFECTiN, LIPOFECTAMJNE (GTBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as well as other liposomes developed according to procedures standard in the art.
- nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Arlington, Ariz.).
- Nucleic acids that are delivered to cells which are to be integrated into the host cell genome typically contain integration sequences. These sequences are often viral related sequences, particularly when viral based systems are used. These viral integration systems can also be incorporated into nucleic acids which are to be delivered using a non-nucleic acid based system of deliver, such as a liposome, so that the nucleic acid contained in the delivery system can become integrated into the host genome.
- Other general techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. These systems typically rely on sequence flanking the nucleic acid to be expressed that has enough homology with a target sequence within the host cell genome that recombination between the vector nucleic acid and the target nucleic acid takes place, causing the delivered nucleic acid to be integrated into the host genome. These systems and the methods necessary to promote homologous recombination are known to those of skill in the art.
- compositions can be delivered to the subject's cells in vivo and/or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like).
- the nucleic acids that are delivered to cells typically contain expression controlling systems.
- the inserted genes in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the desired gene product.
- a promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site.
- a promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.
- Promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus, cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter.
- the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature, 273 : 1 13 ( 1978)).
- the immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment (Greenway, P. J. et al., Gene 1 8: 355-360 ( 1982)).
- promoters from the host cell or related species also are useful herein.
- Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981 )) or 3' (Lusky, M. L., et al., Mol. Cell. Bio. 3 : 1 108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J. L. et al., Cell 33 : 729 ( 1983)) as well as within the coding sequence itself (Osborne, T. F., et al., Mol. Cell. Bio. 4: 1293 (1984)).
- Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, cc-fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression.
- the promotor and/or enhancer may be specifically activated either by light or specific chemical events which trigger their function.
- Systems can be regulated by reagents such as tetracycline and dexamethasone.
- irradiation such as gamma irradiation, or alkylating chemotherapy drugs.
- the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize expression of the region of the transcription unit to be transcribed.
- the promoter and/or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time.
- a promoter of this type is the CMV promoter (650 bases).
- Other such promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTR.
- GFAP glial fibrillary acetic protein
- Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites.
- the transcription unit can also contain a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA.
- the identification and use of polyadenylation signals in expression constructs is well established. Homologous polyadenylation signals can be used in the transgene constructs.
- the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. Transcribed units an contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct.
- the viral vectors can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed.
- Example marker genes are the E. Coli lacZ gene, which encodes ⁇ - galactosidase, and green fluorescent protein.
- the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G41 8, hydromycin, and puromycin.
- DHFR dihydrofolate reductase
- neomycin thymidine kinase
- neomycin analog G41 8 hydromycin
- puromycin puromycin.
- the transformed mammalian host cell can survive if placed under selective pressure.
- selective regimes There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. Two examples are: Chinese hamster ovary (CHO) DHFR-cells and mouse LTK-cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media.
- An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.
- the second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1 :327 (1982)), mycophenolic acid, (Mulligan, R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al., Mol. Cell. Biol. 5: 410-413 (1985)).
- the three examples employ bacterial genes under eukatyotic control to convey resistance to the appropriate drug G41 8 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G41 8 and puramycin.
- a cell comprising one or more of the herein provided vectors.
- “cell”, “cell line”, and “cell culture” may be used interchangeably and all such designations Include progeny.
- the disclosed cell can be any cell used to clone or propagate the vectors provided herein.
- the cell can be from any primary cell culture or established cell line.
- the method may be applied to any cell, including prokaryotic or eukaryotic, such as bacterial, plant, animal, and the like.
- the cell type can be selected by one skilled in the art based on the choice of vector and desired use.
- animals produced by the process of transfecting a cell within the animal with any of the nucleic acid molecules or vectors disclosed herein Disclosed are animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules or vectors disclosed herein, wherein the animal is a mammal. Also disclosed are animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules or vectors disclosed herein, wherein the mammal is mouse, rat, rabbit, cow, sheep, pig, or primate.
- compositions comprising one or more of the herein provided polypeptides, nucleic acids, or vectors in a pharmaceutically acceptable carrier.
- a composition comprising a combination of two or more of any of the herein provided ACT polypeptides in a pharmaceutically acceptable carrier.
- a composition comprising SEQ ID NO: l and SEQ ID NO:5 in a pharmaceutically acceptable carrier.
- pharmaceutically acceptable a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
- the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
- composition can further comprise any known or newly discovered substance that can be administered to a wound, tissue injury, site of inflammation or cancer.
- the provided composition can further comprise one or more of classes of antibiotics (e.g. Aminoglycosides, Cephalosporins, Chloramphenicol, Clindamycin, Erythromycins, Fluoroquinolones, Macrolides, Azolides, Metronidazole, Penicillin's, Tetracycline's, Trimethoprim-sulfamethoxazole, Vancomycin), steroids (e.g. Andranes (e.g. Testosterone), Cholestanes (e.g. Cholesterol), Cholic acids (e.g.
- antibiotics e.g. Aminoglycosides, Cephalosporins, Chloramphenicol, Clindamycin, Erythromycins, Fluoroquinolones, Macrolides, Azolides, Metronidazole, Penicillin's, Te
- Corticosteroids e.g. Dexamethasone
- Estraenes e.g. Estradiol
- Pregnanes e.g. Progesterone
- narcotic and non-narcotic analgesics e.g. Morphine, Codeine, Heroin, Hydromorphone, Levorphanol, Meperidine, Methadone, Oxydone, Propoxyphene, Fentanyl, Methadone, Naloxone, Buprenorphine, Butorphanol, Nalbuphine, Pentazocine
- chemotherapy e.g.
- anti-cancer drugs such as but not limited to Altretamine, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carrnustine, Chlorambucil, Cisplatin, Cladribine, Cyclophosphamide, Cytarabine, dacarbazine, Diethylstilbesterol, Ethinyl estradiol, Etoposide, Floxuridine, Fludarabine, Fluorouracil, Flutamide, Goserelin, Hydroxyurea, Idarubicin, Ifosfamide, Leuprolide, Levamisole, Lomustine, Mechlorethamine, Medroxyprogesterone, Megestrol, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitoxantrone, Paclitaxel, pentastatin, Pipobroman, Plicamycin, Prednisone, Procarbazine, Streptozocin, Tamoxifen
- Alclofenac Alclometasone Dipropionate; Algestone Acetonide; alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Decanoate; Deflazacort; Delatestryl; Depo- Testosterone; Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Dif
- Prednazate Prifelone; Prodolic Acid; Proquazone; Proxazole; Proxazole Citrate; Rimexolone;
- Tetrydamine Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;
- Triflumidate Triflumidate; Zidometacin; Zomepirac Sodium), or anti-histaminic agents (e.g. Ethanolamines
- Acetaminophen, Pseudoephedrine, Triprolidine Acetaminophen, Pseudoephedrine, Triprolidine.
- compositions may be administered topically, orally, or parenterally.
- the compositions can be administered extracorporeal ly, intracranially, intravaginally, intraanally, subcutaneously, intradermal ly, intracardiac, intragastric, intravenously, intramuscularly, by intraperitoneal injection, transdermal ly, intranasally, or by inhalant.
- intracranial administration means the direct delivery of substances to the brain including, for example, intrathecal, intracisternal, intraventricular or trans-sphenoidal delivery via catheter or needle.
- Parenteral administration of the composition is generally characterized by injection.
- Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
- a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Pat. No. 3,610,795, which is incorporated by reference herein.
- topical intranasal adm inistration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.
- Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
- the compositions provided herein may be applied topically once per day, twice per day, three times per day, four times per day, five times per day, or more. In some embodiments, the compositions provided herein may be applied topically once every two days, once every three days, once every four days, once every four days, once every five days, once every six days, or once per week. In some embodiments, the compositions provided herein may be applied topically on an as-needed basis. For example, in some embodiments, the compositions provided herein may be applied during or after the manifestation or increasing severity of symptoms of the radiation injuiy.
- the compositions provided herein may be applied topically once per day for about 5, about 1 0, about 1 5, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, or more consecutive days.
- the compositions provided herein comprise an ACT peptide provided herein and a gel, and may be topically applied.
- the compositions provided herein comprise SEQ ID NO: 91 .
- the compositions provided herein comprise SEQ ID NO: 91 and hydroxyethylcellulose gel, wherein the compositions are topically applied.
- the compositions provided herein include a topical gel termed "Granexin®".
- Granexin® comprises 1 .25% hydroxyethyl cellulose gel and the ACT1 peptide.
- the chemical structure of the ACT1 peptide in Granexin® is: Biotin-Ahx-Arg-Gln-Pro-Lys-Ile-Trp-Phe-Pro-Asn-Arg-Arg-Lys-Pro-Trp-Lys-Lys-Arg-Pro- Arg-Pro-Asp-Asp-Leu-Glu-Ile-OH (SEQ ID NO: 91 ), wherein Ahx is L-2-aminohexanoic acid (6-aminohexanoic acid).
- Granexin® further comprises one or more preservative, solvent, buffer agent, stabilizer, chelating agent, and/or any additional pharmaceutically acceptable excipient or carrier.
- compositions provided herein are topically and/or systemically administered about 1 , about 2, about 5, about 10, about 15, about 20, about 24, about 48, or more hours prior to exposure to ionizing radiation. In some embodiments, the compositions provided herein are topically and/or systemically administered about 1 , about 2, about 5, about 10, about 1 5, about 20, about 24, about 48, or more hours post exposure to ionizing radiation. In some embodiments, compositions provided herein are topically and/or systemically administered in a window of time of about 1 to about 48 hours post exposure to ionizing radiation, or about 4 to about 24 hours post exposure, or about 10 to about 36 hours post exposure.
- the compositions provided herein are topically applied about 24 hours post exposure to ionizing radiation.
- a thin layer of a composition provided herein e.g., a composition comprising an ACT peptide provided herein and a gel
- the present disclosure provides methods for treatment of exposure to ionizing radiation, comprising topically applying to the area of exposure a layer of a composition comprising SEQ ID NO: 91 and hydroxyethylcellulose gel.
- the compositions provided herein may be administered systemically once per day, twice per day, three times per day, four times per day, five times per day, or more.
- the compositions provided herein may be administered systemically once every two days, once every three days, once every four days, once every four days, once every five days, once every six days, or once per week.
- the compositions provided herein may be administered systemically on an as-needed basis prior to or after exposure to ionizing radiation.
- the compositions provided herein may be administered during or after the manifestation or increasing severity of symptoms of the radiation injury.
- compositions provided herein may be administered systemically once per day for about 5, about 10, about 1 5, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, or more consecutive days.
- the compositions provided herein for systemic delivery to a subject in need thereof comprise an ACT peptide provided herein.
- the compositions provided herein for systemic delivery to a subject in need thereof comprise ACT1 polypeptide (SEQ ID NO: 2).
- the compositions provided herein for systemic delivery to a subject in need thereof comprise one ur inure pharmaceutically acceptable carrier.
- the compositions provided herein are systemically administered about 1 , about 2, about 5, about 10, about 15, about 20, about 24, about 48, or more hours post exposure to ionizing radiation. In particular embodiments, the compositions provided herein are systemically administered about 24 hours post exposure to ionizing radiation. In some embodiments, the compositions provided herein are systemically administered post exposure to ionizing radiation and prior to manifestation of symptoms of ARS. In some embodiments, the compositions provided herein are systemically administered post exposure to ionizing radiation and after manifestation of symptoms of ARS, wherein the compositions treat, inhibit, or mitigate the progression of ARS.
- compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
- the materials may be in solution or suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
- the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451 , (1991 ); Bagshawe, . D., Br. J. Cancer, 60:275-281 , (1989); Bagshawe, et al., Br. J.
- Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
- the following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214- 6220, ( 1989); and Litzinger and Huang, Biochimica et Biophysica Acta, I 104: 179- 187, ( 1992)).
- receptors are involved in pathways of endocytosis, either constitutive or ligand induced.
- receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
- the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991 )).
- Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995.
- an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
- the pharmaceutically- acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
- the pH of the solution can be from about 5 to about 8, from about 7 to about 7.5.
- Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
- compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
- compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
- Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.
- the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. In certain embodiments, administration is by topical administration. In certain embodiments, administration is by systemic administration. Systemic administration includes, for example, enteral or parenteral administration. In some embodiments, systemic administration is by intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or inhalation. In some embodiments, systemic administration is by aerosolized delivery.
- Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions.
- non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
- Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
- Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
- Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
- Formulations for topical administration may include ointments, lotions, creams, gels (e.g., poloxamer gel), drops, suppositories, sprays, liquids and powders.
- Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
- the disclosed compositions can be administered, for example, in a microfiber, polymer (e.g., collagen), nanosphere, aerosol, lotion, cream, fabric, plastic, tissue engineered scaffold, matrix material, tablet, implanted container, powder, oil, resin, wound dressing, bead, microbead, slow release bead, capsule, injectables, intravenous drips, pump device, silicone implants, or any bio-engineered materials.
- the formulation for topical administration is a sunscreen, sunblock, or similar formulation. Such compositions may absorb, filter, reflect, or block UV radiation from the sun or other source of UV radiation. Thus, in some embodiments, the present disclosure provides formulations that function both to reduce UV radiation exposure and to prevent, treat or mitigate the progression of radiation injury from UV radiation.
- the formulation for topical administration is a suntan lotion, tanning accelerator, tanning oil, or similar formulation.
- the sunscreen, sunblock, suntan lotion, tanning accelerator, or tanning oil includes an amount of the polypeptides provided herein that can prevent, treat, or mitigate the progression of a radiation injury caused by UV light (e.g., from the sun, a tanning bed, or other source).
- the sunscreen includes a concentration of a polypeptide provided herein (e.g., from about 0.001 % (w/w) to about 5.0% (w/w), or from about 10 ⁇ to about 2000 ⁇ .
- the provided pharmaceutically acceptable carrier is a poloxamer.
- Poloxamers referred to by the trade name Pluronics®, are nonionic surfactants that form clear thermoreversible gels in water. Poloxamers are polyethylene oxide-polypropylene oxide- polyethylene oxide (PEO-PPO-PEO) tri-block copolymers. The two polyethylene oxide chains are hydrophil ic but the polypropylene chain is hydrophobic. These hydrophobic and hydrophilic characteristics take charge when placed in aqueous solutions. The PEO-PPO-PEO chains take the form of small strands where the hydrophobic centers would come together to form micelles.
- the micelle sequentially, tend to have gelling characteristics because they come together in groups to form solids (gels) where water is just slightly present near the hydrophilic ends. When it is chilled, it becomes liquid, but it hardens when warmed. This characteristic makes it useful in pharmaceutical compounding because it can be drawn into a syringe for accurate dose measurement when it is cold. When it warms to body temperature (when applied to skin) it thickens to a perfect consistency (especially when combined with soy lecithin/isopropyl palmitate) to facilitate proper inunction and adhesion.
- Pluronic® F 127 (F 127) is widely used because it is obtained easily and thus it is used in such pharmaceutical applications.
- F 1 27 has a EO:PO:EO ratio of 100:65 : 100, which by weight has a PEO:PPO ratio of 2: 1 .
- Pluronic gel is an aqueous solution and typically contains 20-30% F- 127. Thus, the provided compositions can be administered in F 127.
- Excipients for use in topical gels are well-known in the art and examples may be found in the Handbook of Pharmaceutical Excipients (Rowe, R. C. et al, APhA Publications; 5 th ed., 2005).
- Exemplary excipients may include waxes, various sugars and types of starch, polymers, gels, emollients, thickening agents, rheology modifiers, humectants, glycerol, organic basic compounds, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and solvents.
- rheology modifiers examples include Carbopol, hydroxypropyl cellulose, C26-28 alkyl dimethicone, C26-28 alkyl methicone, polyphenylsisquioxane, trimethylsiloxysilicate, crosspolymers of cyclopentasiloxane and dimethicone/vinyltrimethylsiloxysilicate, fumed silica (e.g. Cab-O-Sil M5P), and mixtures thereof.
- emollients include glycerine, pentylene glycol, sodium pyrrolidone carboxylic acid, lanolin, saccharide isomerate, stearoxy dimethicone, stearyl dimethicone, and mixtures thereof. Emollients may be useful to prevent stratum corneum dehydration occurring due to the use of anhydrous solvents in the formulation.
- organic bases include 2-amino-2-methyl propanol, niacinamide, methanolamines, triethanolamines, Trisamino, AMP-95, AmP-Ultra PC 2000, triisopropanolam ine, diisopropanoiamine, Neutrol TE, Ethomeen, and mixtures thereof. The organic base may render the pH of the medicament basic or neutral.
- exemplary excipients include water-soluble porogens.
- a water-soluble porogen is an additive that may facilitate water uptake and diffusion into the gel. Any suitable porogen may be used, but in some embodiments, the porogen may include sodium chloride, potassium chloride, sucrose, glucose, lactose, sorbitol, xylitol, polyethylene glycol, polyvinylpyrrollidone, polyvinyl alcohol or mixtures thereof.
- Polymers may also act as excipients in topical gels.
- Exemplary polymers include hydrophilic polyurethanes, hydrophilic polyacrylates, co-polymers of carboxymethylcellulose and acrylic acid, N-vinylpyrrolidone, poly(hydroxy acids), polyanhydrides, polyorthoesters, polyamides, polycarbonates, polyalkylenes (e.g., polyethylene and polypropylene), polyalkylene glycols (e.g., poly(ethylene glycol)), polyalkylene oxides (e.g., polyethylene oxide), polyalkylene terephthalates (e.g., polyethylene terephthalate), polyvinyl alcohols, polyvinyl ethers, polylvinyl esters, polyvinyl halides (e.g., poly(vinyl chloride)), polyvinylpyrrolidone, polysiloxanes, po!y(vinyl acetates), polystyrenes, polyurethane cop
- the polymers may be superabsorbent polymers (SAPs).
- SAPs superabsorbent polymers
- a polymer is considered superabsorbent, as defined per IUPAC, as a polymer that can absorb and retain extremely large amounts of water relative to its own mass.
- SAPs may absorb water up to 500 times their own weight and may swell up to 1000-times their original volume.
- Particular SAPs of interest include sodium polyacrylate, the polyurethane Tecophilic TG-2000, and polymers prepared by the use of polyacrylamide copolymer, ethylene maleic anhydride copolymer, cross-linked carboxy-methyl-cellulose, polyvinyl alcohol copolymers, polyvinylpyrrolindone and cross-linked polyethylene oxide.
- polymers that are relatively hydrophobic may be used. Any suitable hydrophobic polymer may be used.
- exemplary polymers that are relatively hydrophobic include aromatic polyurethanes, silicone rubber, polysiloxanes, polycaprolactone, polycarbonate, polyvinylchloride, polyethylene, poly-L-Iactide, poly-DL-glycolide, polyetheretherketone (PEEK), polyam ide, poiyimide and polyvinyl acetate.
- a hydrophobic gel-base and/or rheology modifier may be used.
- the polymers may act as thickening agents in the medicaments.
- the polymeric portion of the gel may act as a visco- elastic substance and may retain the gel at the site of application, along with the alpha connexin polypeptides dispersed therein.
- a gel that includes a polymer may have spreadabil ity such that it forms a thin film when applied on the skin surface. This film may enable the application of the contained alpha connexin polypeptides over a wide area, and may serve to maintain the alpha connexin polypeptides on the affected area of the skin.
- ionic compounds may include salts such as sodium chloride, potassium chloride; cationic, anionic or zwitterionic surfactants such as sodium dodecyl sulfate (SDS), perfiuorooctanoate (PFOA), perfluorooctanesulfonate (PFOS), ammonium lauryl sulfate (ALS), sodium lauryl ether sulfate (SLES), alkyl benzene sulfonate, cetyl trimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC), benzethonium chloride, dodecyl betaine, cocamidopropyl betaine and cocoampho glycinate.
- SDS sodium dodecyl sulfate
- PFOA perfiuorooctanoate
- PFOS per
- surfactants both ionic and non-ionic may reduce the interfacial surface energy and may facil itate spreading of the topical formulation over a wider area.
- solvent excipients may be used as a carrier vehicle for the alpha connexin polypeptides and other excipients.
- the polymer chains may interact with the solvent and undergo swelling to form a network that may impart visco- elastic properties to the topical formulation.
- the solvent may evaporate upon application, leaving a residual film of the polymer along with the entrapped alpha connexin polypeptides.
- Exemplary solvent excipients that may be useful in hydrophi lic formulations may include dimethyl isosorbide, propylene glycol, glycerol, isopropanol, ethanol, benzyl alcohol, ethylene glycol, polyethylene glycol, ethoxydiglycol or mixtures thereof.
- Exemplary solvent excipients that may be useful in hydrophobic formulations may include capric/caprylic triglycerides, isopropyl myristate, mineral oil, isododecane, isodecyl neopentanoate, butylene glycol, pentylene glycol, hexylene glycol, methoxypolyethyleneglycol, cyclopentasiloxane, cyclotetrasiloxane, dimethicone, caprylyl methicone or mixtures thereof.
- the topical formulation may also include at least one additional therapeutic agent such as antimicrobial agents, anti-acne agents, anti-inflammatory agents, analgesic agents, anesthetic agents, antihistamine agents, antiseptic agents, immunosuppressants, antihemorrhagic agents, vasodilators, wound healing agents, anti-biofilm agents and mixtures thereof.
- additional therapeutic agent such as antimicrobial agents, anti-acne agents, anti-inflammatory agents, analgesic agents, anesthetic agents, antihistamine agents, antiseptic agents, immunosuppressants, antihemorrhagic agents, vasodilators, wound healing agents, anti-biofilm agents and mixtures thereof.
- antimicrobial agents include penicillins and related drugs, carbapenems, cephalosporins and related drugs, erythromycin, aminoglycosides, bacitracin, gramicidin, mupirocin, chloramphenicol, thiamphenicol, fusidate sodium, lincomycin, clindamycin, macrolides, novobiocin, polymyxins, rifamycins, spectinomysin, tetracyclines, vanomycin, teicoplanin, streptogramins, anti-folate agents including sulfonamides, trimethoprim and its combinations and pyrimethamine, synthetic antibacterials including nitrofurans, methenamine mandelate and methenamine hippurate, nitroimidazoles, quinolones, fluoroquinolones, isoniazid, ethambutol, pyrazinamide, para-am inosal icy l
- topical anti-acne agents examples include adapalene, azelaic acid, benzoyl peroxide, clindamycin and clindamycin phosphate, doxycycline, erythromycin, keratolytics such as salicylic acid and retinoic acid ("Retin-A"), norgestimate, organic peroxides, retinoids such as isotretinoin and tretinoin, sulfacetamide sodium, and tazarotene.
- Retin-A salicylic acid and retinoic acid
- Particular anti-acne agents include adapalene, azelaic acid, benzoyl peroxide, clindamycin (e.g., clindamycin phosphate), doxycycline (e.g., doxycycline monohydrate), erythromycin, isotretinoin, norgestimate, sulfacetamide sodium, tazarotene, etretinate and acetretin.
- antihistamine agents include diphenhydramine hydrochloride, diphenhydramine salicylate, diphenhydramine, chlorpheniramine hydrochloride, chlorpheniramine maleate isothipendyl hydrochloride, tripelennamine hydrochloride, promethazine hydrochloride, methdilazine hydrochloride, and the like.
- Examples of local anesthetic agents include dibucaine hydrochloride, dibucaine, lidocaine hydrochloride, lidocaine, benzocaine, p-buthylaminobenzoic acid 2-(die-ethylam ino) ethyl ester hydrochloride, procaine hydrochloride, tetracaine, tetracaine hydrochloride, chloroprocaine hydrochloride, oxyprocaine hydrochloride, mepivacaine, cocaine hydrochloride, piperocaine hydrochloride, dyclonine and dyclonine hydrochloride.
- antiseptic agents include alcohols, quaternary ammonium compounds, boric acid, chlorhexidine and chlorhexidine derivatives, iodine, phenols, terpenes, bactericides, disinfectants including thimerosal, phenol, thymol, benzalkonium chloride, benzethonium chloride, chlorhexidine, povidone iode, cetylpyridinium chloride, eugenol and trimethylammonium bromide.
- anti-inflammatory agents include nonsteroidal antiinflammatory agents (NSAlDs); propionic acid derivatives such as ibuprofen and naproxen; acetic acid derivatives such as indomethacin; enolic acid derivatives such as meloxicam, acetaminophen; methyl salicylate; monoglycol salicylate; aspirin; mefenamic acid; flufenamic acid; indomethacin; diclofenac; alclofenac; diclofenac sodium; ibuprofen; ketoprofen; naproxen; pranoprofen; fenoprofen; sulindac; fenclofenac; clidanac; flurbiprofen; fentiazac; bufexamac; piroxicam; phenylbutazone; oxyphenbutazone; clofezone; pentazocine; mepirizole; tiaramide hydrochloride; steroids
- alclometasone dexamethasone, flumethasone, prednisolone, or methylprednisolone, or may be a higher potency corticosteroid such as clobetasol propionate, betamethasone benzoate, betamethasone dipropionate, diflorasone diacetate, fluocinonide, mometasone furoate, triamcinolone acetonide.
- analgesic agents include alfentanil, benzocaine, buprenorphine, butorphanol, butamben, capsaicin, clonidine, codeine, dibucaine, enkephalin, fentanyl, hydrocodone, hydromorphone, indomethacin, lidocaine, levorphanol, meperidine, methadone, morphine, nicomorphine, opium, oxybuprocaine, oxycodone, oxymorphone, pentazocine, pramoxine, proparacaine, propoxyphene, proxymetacaine, sufentanil, tetracaine and tramadol.
- anesthetic agents include alcohols such as phenol; benzyl benzoate; calamine; chloroxylenol; dyclonine; ketamine; menthol; pramoxine; resorcinol; troclosan; procaine drugs such as benzocaine, bupivacaine, chloroprocaine; cinchocaine; cocaine; dexivacaine; diamocaine; dibucaine; etidocaine; hexylcaine; Ievobupivacaine; lidocaine; mepivacaine; oxethazaine; pri localne; procaine; proparacaine; propoxycaine; pyrrocaine; risocaine; rodocaine; ropivacaine; tetracaine; and derivatives, such as pharmaceutically acceptable salts and esters including bupivacaine HC1, chloroprocaine HCI, diamocaine cyclamate,
- antihemorrhagic agents include thrombin, phytonadione, protamine sulfate, aminocaproic acid, tranexamic acid, carbazochrome, carbaxochrome sodium sulfanate, rutin and hesperidin.
- the instant invention may also contain other active agents such as niacinamide, phytantriol, farnesol, bisabolol and salicylic acid. It is expected that certain additional active agents will act synergistically with the bioactive peptide component, or will enhance the shelf-life of the formulation.
- the gels may also include other compounds that improve the organoleptic properties of the topical formulation.
- Examples of such compounds include perfumes, dyes and colorants; chelating agents including but not limited to edetate disodium (EDTA), EGTA, CP94, citric acid; preservatives including but not limited to quaternary ammonium compounds, such as benzalkonium chloride, benzethonium chloride, cetrimide, dequalinium chloride, and cetylpyridinium chloride; mercurial agents, such as phenylmercuric nitrate, phenylmercuric acetate, and thimerosal; alcoholic agents, for example, chlorobutanol, phenylethyl alcohol, and benzyl alcohol; antibacterial esters, for example, esters of parahydroxybenzoic acid; and other anti-microbial agents such as chlorhexidine, chlorocresol, benzoic acid and polymyxin.
- chelating agents including but not limited to edetate disodium (EDTA), EGTA, CP94, citric acid
- a topical formulation comprising at least one alpha connexin polypeptide and hydroxyethylcellulose gel, wherein the hydroxyethylcellulose gel stabilizes the alpha connexin polypeptide.
- the hydroxyethylcellulose gel stabilizes the alpha connexin polypeptide so that after 3 months of storage at 5 °C, at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 00% of the alpha connexin polypeptide is detectable by analytical methods.
- the alpha connexin polypeptide is present in the formulation at a concentration of about 0.0025% (w/w), of about 0.005% (w/w), of about 0.0075% (w/w), of about 0.010% (w/w), of about 0.01 5% (w/w), of about 0.020% (w/w), of about 0.025% (w/w), of about 0.030% (w/w), of about 0.035% (w/w), of about 0.040% (w/w), of about 0.045% (w/w), of about 0.050% (w/w), of about 0.055% (w/w), of about 0.060% (w/w), of about 0.065% (w/w), of about 0.070% (w/w), of about 0.075% (w/w), of about 0.080% (w/w), of about 0.085% (w/w), of about 0.090% (w/w), of about 0.095% (w/w), of about 0.100% (w/w), of about 0.150% (w/w), of about
- the drug product of the invention is a clear colorless gel which contains 0.0072% (w/w) (20 ⁇ ) of the ACT peptide, 0.018% (w/w) (50 ⁇ ) of the ACT peptide, 0.036% (w/w) ( 100 ⁇ ) of the ACT peptide, 0.072% (w/w) (200 ⁇ ) of the ACT peptide, or 0.36% (w/w) (1000 ⁇ ) of the ACT peptide.
- the ACT peptide may be dissolved in a semisolid dosage form that contains >0% water, >1 0% water, >20% water, >30% water, >40% water, >50% water, >60% water, >70% water, >80% water, or >90% water and 0.25% gelling agent (polymer), 0.55% gelling agent (polymer), 0.75% gelling agent (polymer), 1 .00% gelling agent (polymer), 1 .25% gelling agent (polymer), 1 .50% gelling agent (polymer), 1 .75% gelling agent (polymer), 2.00% gelling agent (polymer), 2.25% gelling agent (polymer), or 2.50% gelling agent (polymer), or 3.00% gelling agent (polymer), or 3.50% gelling agent (polymer), or 4.00%» gelling agent (polymer), or 4.50% gelling agent (polymer), or 5.00% gelling agent (polymer).
- the ACT peptide may be well preserved and adequately buffered to about pH 5.5, about pH 6, about pH 6.5, about pH 7, about pH 7.5 or about pH8.
- the qualitative and quantitative composition is that listed in Table 7.
- ACTl peptide sequence is listed in Table 8 below in which Ahx refers to L-2-aminohexanoic acid, also known as 6-aminohexanoic acid:
- the excipients used in the drug product topical preparation are selected from the group consisting of or are one or more of the following:
- the drug product topical preparation comprises a peptide, D-mannitol, hydroxyethylcellulose, and purified water.
- Said preparation may further comprise one or more of methylparaben, propylparaben, glycerin, sodium phosphate monobasic, sodium phosphate dibasic, propylene glycol, and edetate disodium (EDTA).
- the hydroxyethylcellulose is 250 HHX.
- the peptide is an alpha connexin polypeptide.
- the drug product topical preparation comprises a peptide at a concentration between about 0.001 % (w/w) and about 0.5% (w/w) (for example, at about 0.0072%), 0.01 8%, 0.036%, or 0.072% (w/w)); methylparaben at a concentration between about 0.1 0% (w/w) and about 0.25% (w/w) (for example, about 0.
- the peptide is an alpha connexin polypeptide.
- edetate disodium (EDTA) and Mannitol are incorporated within the drug product to provide stabi lity to the ACTI peptide.
- the Mannitol is present in the formulation at 0.01 % (w/w) to 1 .6% (w/w), 0.01 % (w/w) to 1 .5% (w/w), 0.01 % (w/w) to 1 .4% (w/w), 0.01 % (w/w) to 1 .3% (w/w), 0.01 % (w/w) to 1 .2% (w/w), 0.01 % (w/w) to 1 .1 % (w/w), 0.01 % (w/w) to 1 .0% (w/w), 0.01 % (w/w) to 0.9% (w/w), 0.01 % (w/w) to 0.8% (w/w), 0.01 % (w/w) to 0.7% (w/w), 0.01 % (w/w) to 0.7% (w/
- a buffering agent is included in the topical formulation to maintain the pH in a certain range.
- Suitable buffering agents can include, but are not limited to, acetate buffers, citrate buffers, phosphate buffers, lactic acid buffers, malic acid buffers, succinic acid buffers, borate buffers, sodium hydroxide, potassium hydroxide, and ammonium hydroxide.
- Phosphate salts such as monosodium phosphate (NaFhPCM; also known as monobasic sodium phosphate), disodium hydrogen phosphate (Na 2 HP0 4 ; also known as dibasic sodium phosphate), monopotassium phosphate (KH2P0 4 ), dipotassium phosphate (K2HPO4), and mixtures thereof can also be used.
- phosphate buffer provides superior stability compared to citrate buffer. In certain aspects, a buffer capacity at 25 mM was found to be adequate for the drug product. Buffer is needed to control pH of the gel system and to maintain stability of the peptide drug.
- the pH range of the topical formulation may be pH 2 to pH 12, pH 4 to pH 10, or pH 6 to pH 8. In some embodiments, an optimal pH range is about pH 5.0 to about pH 7.0. In other embodiments, the pH of the topical formulation of the present invention is 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. In other aspects, propylene glycol in the amount of 3% provides better solubilization of parabens in the aqueous system.
- hydroxyethylcellulose e.g., HEC 250 HHX
- HEC 250 HHX hydroxyethylcellulose
- Pluronic gel may require a cold bath to bring its viscosity into a desired range and require more energy than the manufacturing process with hydroxyethylcellulose.
- the final formulation in Pluronic gel may be very thin at a storage condition of 5 °C.
- hydroxyethylcellulose is a suitable gelling agent and acceptable carrier of the drug product of the present invention.
- the gelling agent is Hydroxyethylcellulose (HEC), 250 HHX.
- the percent (w/w) of HEC is in the range of 1 -5%. In other embodiments, the percent (w/w) of HEC is 1 .25%.
- a purified cellulose is reacted with sodium hydroxide to produce a swollen alkali cellulose.
- the alkali-treated cellulose is more chemically reactive than cellulose.
- HEC Hydroxyl Ethyl Cellulose
- HEC Hydroxyethyi Cellulose 100H (celocell l OOh); TYLOSE MH-
- HEC-Unicel HEC (Hydroxyethyi cellulose); Cellosize; HEC-A1 5000; Fuji HEC-AL 15; HEC-Unicel QP 09L; Cellulose, ethers, 2- hydroxyethyl ether; Unicel QP 52000H; HEC-QP 4400; SP 250 (cellulose); Hetastarch;
- Daicel SP 750 Sanhec
- BL 15 cellulose derivative
- Unicel QP 300H Tylomer H 200; J 164;
- HEMC Hydroxyethyi Methyl Cellulose
- HEC HYDROXYETHYL CELLULOSE
- HEC Hydroxyethyi Starch
- Ethyl Cellulose Hydroxy Ethyl Cellulose
- “Natrosol” [Aqualon] Hydroxy Ethyl Cellulose
- HEC 2-HYDROXYETHYL CELLULOSE
- NATROSOL 1 50L TYLOSE MHB-YP
- the drug product of the present invention is packaged in
- the present invention includes a method of reating or preventing radiation injury in a subject at risk of such injury, comprising administering to the subject a topical formulation comprising at least one alpha connexin polypeptide and hydroxyethylcellulose gel, wherein the hydroxyethylcellulose gel stabilizes the alpha connexin polypeptide.
- the drug product of the present invention may be used to mitigate excessive scar formation associated with radiation injury.
- the drug product is manufactured with the following steps:
- Step 1 In a suitable size of beaker, add propylene glycol, glycerin, methylparaben and propylparaben. Mix with a propeller until the parabens are completely dissolved.
- Step 2 In a manufacturing vessel, add purified water (part I), EDTA, monobasic sodium phosphate, dibasic sodium phosphate and D-mannitol. Mix with a propeller until a clear solution is obtained.
- Step 3 Add the solution from step 1 to the manufacturing vessel. Rinse the beaker with purified water (part II, divided into approximately 3 equal portions) and add the rinse back to the vessel. Continue with propeller mixing until the solution is visually homogeneous.
- Step 6 With continuous propeller mixing, add the drug solution from step 5 to the manufacturing vessel. Rinse the beaker with purified water (part IV, divided into approximately 3 equal portions) and add the rinse back to the vessel. Mix until the gel is homogeneous.
- compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
- compositions may potentially be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycoiic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
- inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
- organic acids such as formic acid, acetic acid, propionic
- Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
- the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms disorder are effected.
- the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
- the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
- the dosage can be adjusted by the individual doctor in the event of any counterindications.
- Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
- Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. The range of dosage largely depends on the application of the compositions herein, seventy of condition, and its route of administration.
- the ACT peptide compositions can be used in doses as low as 0.01 % w/v.
- the dosage can be as low as 0.0002% w/v and possibly as high as 20% w/v in topical skin wound treatments.
- Significantly higher concentrations of the compositions by themselves or in combination with other compounds may be used in applications like cancer/tumor therapy or as an early concentrated bolus immediately following an acute tissue injury, such as CRI.
- Recommended upper limits of dosage for parenteral routes of administration for example intramuscular, intracerebral, intracardicardiac and intraspinal could be up to 1 % w/v or v/v depending on the severity of the injury. This upper dosage limit may vary by formulation, depending for example on how the polypeptide(s) is combined with other agents promoting its action or acting in concert with the polypeptide(s).
- upper limits of 0.01 g/ g body weight over time courses determined by the doctor based on improvement in the condition can be used.
- upper limits of concentration of the provided nucleic acids delivered topically, for example, in skin wounds would be 5- 1 0 ⁇ g/cm 2 of wound depending for example on how the nucleic acid is combined with other agents promoting its action or acting in concert with the nucleic acids. This would be repeated at a frequency determined by the Doctor based on improvement.
- upper limits of concentration of the provided nucleic acids delivered internally for example, intramuscular, intracerebral, intracardiac and intraspinal would be 50- 100 ⁇ g/ml of solution. Again, the frequency would be determined by the Doctor based on improvement.
- the concentration of the polypeptides can be 10-200 ⁇ mixed in with 10-30% pluronic gel or any such carrier that enables penetration of the peptide(s) within the site of interest for a period of at least 3-6 hours prior to surgery.
- This pre-procedural conditioning can improve the subsequent healing response to surgery, including reduced inflammatory response.
- the treatment of a subject at risk of exposure to radiation with the provided polypeptides prevents subsequent radiation injury, such as cutaneous radiation injury.
- the provided polypeptides are administered at a concentration of about 10 ⁇ to about 1 000 ⁇ .
- the provided polypeptides are administered at a concentration of about ⁇ ⁇ , about 5 ⁇ , about 10 ⁇ , about 1 5 ⁇ , about 20 ⁇ , about 25 ⁇ , about 30 ⁇ , about 35 ⁇ , about 40 ⁇ , about 45 ⁇ , about 50 ⁇ , about 55 ⁇ , about 60 ⁇ , about 65 ⁇ , about 70 ⁇ , about 75 ⁇ , about 80 ⁇ , about 85 ⁇ , about 90 ⁇ , about 95 ⁇ , about ⁇ ⁇ , about 1 10 ⁇ , about 120 ⁇ , about 130 ⁇ , about 140 ⁇ , about 150 ⁇ , about 160 ⁇ , about 170 ⁇ , about 1 80 ⁇ , about 190 ⁇ , about 200 ⁇ , about 225 ⁇ , about 250 ⁇ , about 275 ⁇ , or about 300 ⁇ , or about 400 ⁇ , or about 500 ⁇ , or about 600 ⁇ , or about 700 ⁇ , or about 800 ⁇ , or about 900 ⁇ or about 1000 ⁇ , or about 1200 ⁇ , or about 1500 ⁇ , or about 2000 ⁇
- the provided polypeptides are administered at a concentration of at least about 100 ⁇ . In other embodiments, the provided polypeptides are administered at a concentration of at least about 200 ⁇ . In still other embodiments, the provided polypeptides are administered at a concentration of at least about 1000 ⁇ .
- the composition is administered systemically to the subject.
- the composition is administered by inhalation.
- the composition is administered parenterally to the subject.
- the composition is administered to the subject by intravenous injection, subcutaneous injection, intraperitoneal injection, or intramuscular injection.
- the composition is administered parenterally to the subject at a dose of from about 0.01 mg/kg to about 100 mg/kg, or about 0.1 mg/kg to about 100 mg/kg, or about 0.5 mg/kg to about 50 mg/kg, or about 1 mg/kg to about 50 mg/kg, or about 2 mg/kg to about 25 mg/kg, or about 5 mg/kg to about 25 mg/kg.
- the composition is administered to the subject at a dose of about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 1 0 mg/kg, about 1 5 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, or about 1 00 mg/kg.
- the provided polypeptides are administered to the subject in a daily dosing regimen. In other aspects, the provided polypeptides are administered to the subject in a weekly dosing regimen. In other aspects, the provided polypeptides are administered to the subject in a monthly dosing regimen.
- the provided polypeptides are administered to the subject at least about 2 years, at least about 1 year, at least about 6 months, at least about 60 days, at least about 30 days, at least about 20 days, at least about 1 0 days, at least about 7 days, at least about 3 days, at least about 1 day, at least about 23 hours, at least about 22 hours, at least about 21 hours, at least about 20 hours, at least about 1 9 hours, at least about 1 8 hours, at least about 1 7 hours, at least about 1 6 hours, at least about 1 5 hours, at least about 14 hours, at least about 13 hours, at least about 1 2 hours, at least about 1 1 hours, at least about 1 0 hours, at least about 9 hours, at least about 8 hours, at least about 7 hours, at least about 6 hours, at least about 5 hours, at least about 4 hours, at least about 3 hours, at least about 2 hours, or at least about 1 hour prior to exposure to radiation.
- the provided polypeptides are administered to the subject at least about 1 hour, 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 1 0 hours, at least about 1 1 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 1 5 hours, at least about 1 6 hours, at least about 17 hours, at least about 1 8 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 1 day, at least about 3 days, at least about 7 days, at least about 1 0 days, at least about 20 days, at least about 30 days, at least about 60 days, at least about 6 months, least about 1 year, or least about 2 years after exposure to radiation.
- the provided polypeptides are administered from at least about 1 day to at least about 30 days after exposure to radiation. In specific embodiments, the provided polypeptides are administered at least about 1 day after exposure to radiation.
- the tissue exposed to radiation includes, without limitation, skin, heart, bone, brain, spinal cord, cornea, retina, and peripheral nerve. In certain aspects, the tissue exposed to radiation is the skin. In certain aspects, the prevention of radiation injury by the provided polypeptides to a subject at risk of exposure to such injury is assessed by measuring the average skin score.
- the average skin score is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% in the presence of the provided polypeptides as compared to a control. In specific embodiments, the average skin score is reduced by at least about 30% in the presence of the provided polypeptides as compared to a control.
- the term “skin score” refers to any means for assessing the extent of damage to the skin and underlying tissues.
- the skin score is obtained using a scale known as the Kumar scale, an example of which is provided herein at Table 1 1 .
- the term “average skin score,” as used herein, is the average of the scores obtained using any method for assessing and scoring injury to the skin and underlying tissues.
- the radiation injury resulting from exposure to the radiation includes, without limitation, cutaneous radiation injury (CRI; i.e., injury to the skin and underlying tissues from acute exposure to radiation), acute radiation syndrome (ARS; serious illness that occurs following an acute high dose of penetrating whole body irradiation), radiation burns, radiation dermatitis, radiation injury to the nervous system or brain, radiation pneumonitis, and radiation-induced enteritis.
- CRI cutaneous radiation injury
- ARS acute radiation syndrome
- radiation burns radiation dermatitis
- radiation injury to the nervous system or brain radiation pneumonitis
- radiation-induced enteritis the radiation injury is cutaneous radiation injuiy.
- the radiation injury is radiation burns.
- the radiation injury is radiation dermatitis.
- ARS includes any one or more of hematopoietic syndrome or bone marrow syndrome; gastrointestinal syndrome; neurovascular syndrome; cardiovascular syndrome, or central nervous system syndrome.
- hematopoietic targeting ARS "bone marrow targeting ARS”
- GI gastrointestinal
- a subject exposed to ionizing radiation may have any one or any combination of these syndromes.
- the syndromes are referred to as, for example,
- compositions and methods provided herein are useful for treating a subject having combined radiation injury.
- a subject having combined radiation injury has dermal trauma, radiation burns, CRI, ARS, radiation dermatitis, and/or any other injury or syndrome related to exposure to ionizing radiation.
- the present disclosure provides methods and compositions for treatment of various complications of exposure to ionizing radiation, and may include topical as well as systemic application of the provided compositions.
- the radiation injury results from a dirty bomb attack.
- a dirty bomb is an explosive device that disperses harmful radiological substances. Exposure to high levels of radiation from a dirty bomb could result in symptoms of acute radiation syndrome or radiation burns. Exposure to radiation causes damage to the basal cell layer of the skin and results in inflammation, erythema, desquamation, intense reddening, blistering, and ulceration of the irradiated site.
- radionuclide-contaminated wounds and burns promote systemic uptake of radionucl ides that greatly complicate topical decontamination and triage.
- radionuclide contamination also interferes with wound healing. Therefore, in certain aspects, the provided polypeptides prevent systemic uptake of radionuclides from a site of radiation injury. In other aspects, the provided polypeptides prevent or treat radiation injury by reducing hemorrhage, severity of thermal burn injury, and formation of scar tissue at the site of radiation injury. In yet other aspects, the provided polypeptides promote tissue regeneration at the site of radiation injury.
- compositions comprising the herein provided compositions (e.g., polypeptides, nucleic acids, or vectors).
- materials used to treat wounds and/or radiation injury wherein the materials are coated with an ACT polypeptide.
- materials used to treat wounds include bandages, steri-strip, sutures, staples, Or grafts (e.g., skin grafts).
- the material e.g., bandage, steri-strip, suture, staple, graft
- the material can be soaked in the provided polypeptide at a concentration ranging from 1 0-200 ⁇ .
- the material can then be dried and sealed in a sterile container.
- the material can also be immersed in liquid 1 0- 30% pluronic gel at 4 °C. containing polypeptide at 10-200 ⁇ concentration.
- the material can then be brought to approximate room temperature so that the gel polymerizes, leaving a coat of polypeptide-impregnated gel surrounding the material, which can be sealed in a sterile container.
- the polypeptide can also be incorporated into a cross-linkable hydrogel system, such as the poly(lactic-co-glycolic acid) (PLGA) or polyurethane, which can then be fashioned into materials for treating wounds (e.g., bandage, steri-strip, suture, staple, graft).
- PLGA poly(lactic-co-glycolic acid)
- polyurethane polyurethane
- a common problem in such implant surgeries is the formation of a contraction capsule around the implant from scar tissue formation that leads to undue hardening, contraction and ultimately misshaping of the tissue of interest.
- the use of the present polypeptides in or on the implant can reduce or prevent this misshaping.
- Non-limiting examples of medical implants include: l imb prostheses, breast implants, penile implants, testicular implants, artificial eyes, facial implants, artificial joints, heart valve prostheses, vascular prostheses, dental prostheses, facial prosthesis, tilted disc valve, caged bal l valve, ear prosthesis, nose prosthesis, pacemakers, cochlear implants, and skin substitutes (e.g., porcine heterograft/pigskin, BIOBRANE, cultured keratinocytes).
- skin substitutes e.g., porcine heterograft/pigskin, BIOBRANE, cultured keratinocytes.
- a method of promoting wound healing following tissue injury in a subject comprising administering to the subject one or more of the herein provided compositions (e.g., polypeptides, nucleic acids, or vectors) in a pharmaceutically acceptable carrier.
- the wound is a result of a cutaneous radiation injury.
- a method of treating a subject with tissue injury comprising administering to the subject one or more of the herein provided compositions (e.g., polypeptides, nucleic acids, or vectors) in a pharmaceutically acceptable carrier.
- Promoter refers to an increase in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the initiation of the activity, response, condition, or disease. This may also include, for example, a 1 0% increase in the activity, response, condition, or disease as compared to the native or control level. Thus, the increase can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of increase in between as compared to native or control levels.
- treat or “treatment” is meant a method of reducing the effects of a disease or condition.
- Treatment can also refer to a method of reducing the underlying cause of the disease or condition itself rather than just the symptoms.
- the treatment can be any reduction from native levels and can be but is not limited to the complete ablation of the disease, condition, or the symptoms of the disease or condition.
- a disclosed method for promoting wound healing is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject with the disease when compared to native levels in the same subject or control subjects.
- the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
- the present disclosure provides compositions and methods for preventing ARS, CRI, combined radiation injury, and/or radiation dermatitis; or mitigating the progression of injury as a result of ARS, CRI, combined radiation injury, and/or radiation dermatitis, in a particular patient population, wherein the patient population comprises subjects who are at risk of such an injury.
- subject includes, but is not limited to, animals, plants, bacteria, viruses, parasites and any other organism or entity that has nucleic acid.
- the subject may be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian.
- the subject can be an invertebrate, more specifically an arthropod (e.g., insects and crustaceans). The term does not denote a particular age or sex.
- a patient refers to a subject afflicted with a disease or disorder.
- a patient population refers to a particular, defined set of subjects having a disease or disorder or at risk of developing a particular disease or disorder.
- the present disclosure provides methods for treating, preventing, or mitigating the severity of radiation injury in a patient population comprising soldiers and/or civilians present in an area subject to war and/or terrorism.
- the present disclosure provides methods for treating, preventing, or mitigating the severity of radiation injury in a patient population comprising cancer subjects receiving radiation therapy.
- patient includes human and veterinary subjects.
- the provided method can reduce scar tissue formation in a subject following tissue injury.
- scar tissue is meant the fibrous (fibrotic) connective tissue that forms at the site of injury or disease in any tissue of the body, caused by the overproduction of disorganized collagen and other connective tissue proteins, which acts to patch the break in the tissue.
- Scar tissue may replace injured skin and underlying muscle, damaged heart muscle, or diseased areas of internal organs such as the liver. Dense and thick, it is usually paler than the surrounding tissue because it is poorly supplied with blood, and although it structurally replaces destroyed tissue, it cannot perform the functions of the missing tissue, it is composed of collagenous fibers, which will often restrict normal elasticity in the tissue involved.
- Scar tissue may therefore limit the range of muscle movement or prevent proper circulation of fluids when affecting the lymphatic or circulatory system.
- Glial scar tissue following injury to the brain or spinal cord is one of the main obstacles to restoration of neural function following damage to the central nervous system.
- a reduction in scar tissue can be assessed by the population of cell types within the injured site. For example, a reduction in glial scar tissue can be estimated by an increased ratio of neuronal to astrocytic cells.
- a reduction in scar tissue formation can be measured by a simple measurement of scar width or area of scar tissue (Wilgus et al., 2003).
- histological assessments can be made about the restoration of structural complexity within healed tissue in comparison to normal tissue.
- psoriasis a doctor would assess the subject's skin to determine whether there has been a reduction in patches of raised red skin covered by flaky white buildup. Certain kinds of psoriasis, are characterized by a pimple-ish (pustular psoriasis) or burned (erythrodermic) appearance. In such cases, the doctor would determine whether treatment has resulted in the reduction of these symptoms.
- tissue fragments of bioposies would be prepared and tissue histological structure would be assessed by a clinical pathologist and/or trained histopathologist to determine if reduction in fibrosis and restoration of normal tissue structure and function has occurred.
- the area of fibrosis to normal tissue could also be quantitatively assessed on such histological preparations.
- the provided method can restore normal tissue mechanical properties such as tensile strength following tissue injury in a subject.
- Tensile strength refers to the amount of stress or strain required to break the tissue or wound.
- the tensile strength of treated wounds can be 60, 65, 70, 75, 80, 85, 90, 95, 1 00% that of uninjured tissue within 3 months after treatment.
- a method of restoring tissue mechanical properties including increasing tensile strength of a healed injury to approach or reach that of normal uninjured tissue, in a subject comprising administering to the subject one or more of the herein provided compositions (e.g., polypeptides, nucleic acids, or vectors) in a pharmaceutically acceptable carrier.
- the type of wounds that would be important with respect to tensile strength/extensibility would include injuries to musculoskeletal structures/tissues, and the skin covering these structures.
- the provided methods can improve tensile strength of articulating joints, bone, cartilage, tendons, or ligaments.
- the provided methods can also improve tensile strength of skin under higher degrees of stress/strain, such as the skin covering the elbow, knee, or foot.
- the most common problems associated with healing of joint injuries is that excessive scarring in these areas leads to contraction, and non-extensibility of the healed joint area. This has serious cosmetic and psychological consequences.
- the properties of the peptides will help modulate and lessen the formation of such scar tissue leading to greater mobility of the joint.
- tissue regeneration can improve tissue regeneration following tissue injury in a subject.
- tissue regeneration is meant the renewal, re-growth, or restoration of a body or a bodily part, tissue, or substance after injury or as a normal bodily process.
- tissue regeneration involves the restoration of the tissue to its original structural, functional, and physiological condition. This is also referred to herein as tissue "complexity".
- the restoration can be partial or complete, meaning 1 0, 20, 30, 40, 50, 60, 70, 80, 90, 1 00% restoration, or any amount of restoration in between as compared to native or control levels.
- tissue regeneration can involve the restoration of hair follicles, glandular structures, blood vessels, muscle, or fat.
- tissue regeneration can involve maintenance or restoration of neurons.
- an improvement in tissue regeneration can be assessed by measurements of the volume of fibrous scar tissue to normal regenerated skin as a ratio.
- counts can be made of discrete regenerating structures such as regenerating skin glands normal ized to the volume of the wound area.
- tissue regeneration involves the recruitment and differentiation of stem cells to replace the damaged cells.
- a "stem cell” is an undifferentiated cell found among differentiated cells in a tissue or organ, or introduced from an external source for e.g., Embryonic stem cells, Adult Bone Marrow stem cells, that can renew itself and differentiate to yield the major specialized cell types of the tissue or organ.
- the primary roles of stem cel ls in a living organism are to maintain and repair the tissue in which they are found.
- stem cell differentiation is meant the process whereby an unspecialized cell (e.g., stem cell) acquires the features of a specialized cell such as a skin, neural, heart, liver, or muscle cell.
- tissue regeneration can involve the differentiation of stem cells present in the epithelium into hair follicles (Alonso and Fuchs, 2003).
- tissue regeneration can involve the differentiation of stem cells into neurons.
- the provided method can enhance stem cell differentiation following tissue injury in a subject.
- Enhanced stem cel l differentiation can be measured by providing a clinically acceptable genetic or other means of marking endogenous or engrafted stem cells and determining the frequency of differentiation and incorporation of marked stem cells into normal tissue structures.
- certain structures such as hair follicles are known to be regenerated from endogenous Stem cells following tissue injury.
- the provided method can reduce inflammation in a subject.
- inflammation By “inflammation”, “inflammatory response” or “immune response” is meant the reaction of living tissues to injury, infection or irritation characterized by redness, warmth, swelling, pain, and loss of function, produced as the result of increased blood flow and an influx of immune cells and secretions. Inflammation is the body's reaction to invading infectious microorganisms and results in an increase in blood flow to the affected area, the release of chemicals that draw white blood cells, an increased flow of plasma, and the arrival of monocytes (or astrocytes in the case of the brain) to clean up the debris.
- the provided compositions and methods can also be used to treat disorders associated with pathological increases in levels of inflammatory cells, including, for example, asthma, eczema, sinusitis, atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, cutaneous and systemic mastocytosis, psoriasis, and multiple sclerosis.
- Treatment with the provided polypeptide can also reduce itching, for example of healing wounds. Generally, itching results from histamine release by mast cells.
- the provided polypeptide can reduce mast cell de- granulation and histamine release.
- the provided polypeptide can be used to treat conditions involving histamine release, including, but not limited to, itching, scratching, sinus irritation, allergic cough, red eyes, asthma, and eczema.
- a reduction in inflammation can be measured by a reduction in the density of inflammatory cell types such as, for example, monocytes or astrocytes.
- a reduction in inflammation can be measured by a reduction in the density of inflammatory cell types such as, for example, neutrophils, mast cel ls, basophils, and monocytes.
- a reduction in inflammation can be calculated by an in vivo measurement of neutrophil activity (Jones et al., 1 994).
- factors like frequency of mast cell degranulation or measurement of histamine levels or levels of reactive oxygen species can be used as measurements of reduction in inflammation.
- the level of inflammation can also be indirectly measured by checking for transcription levels of certain genes by qRT-PCR for e.g.
- ACT peptide action may be by inhibition of inflammatory cell migration and/or inhibition of pro-inflammatory chemicals (histamine, reactive oxygen species) and pro-inflammatory cytokines such as Interleukin (IL)- l , IL-6, IL-8 and tumor necrosis factor (TNF).
- pro-inflammatory chemicals histamine, reactive oxygen species
- pro-inflammatory cytokines such as Interleukin (IL)- l , IL-6, IL-8 and tumor necrosis factor (TNF).
- the provided method can inhibit proliferation of a transformed cell in a subject.
- transformed cell is meant a neoplasm, cancer, or tumor cell that divides and reproduces abnormally with uncontrolled growth.
- inhibition of proliferation (i.e., hyperplasia) of said transformed cell results in a reduction in the growth and thus malignancy of the cancer.
- a representative but non-limiting list of cancers that the disclosed compositions and methods can be used to treat is the following: glioma, lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma, gl ioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers, testicular cancer, colon and
- histopathologists are skilled in assessing biopsy tissue sections using simple qualitative and quantitative indices of mitosis to determine proliferation in transformed cell populations
- Various mouse models have been developed for cancer research. There are specific mouse models for specific types of cancers. For example, Bladder cancer, Cervical cancer, Endometrial cancer, Gastrointestinal cancer, Genitourinary cancer, Head and Neck cancer, Hematopoietic cancer, Kidney cancer, Lung cancer, Mammary Gland cancer, Melanoma, Myeloma, Nervous System cancer, Oral cancer, Ovarian cancer, Pancreatic cancer, Prostate cancer, Sarcoma, Skin cancer. These models are well described and used.
- the favorable effects of the polypeptides, nucleic acids or vectors provided herein can be studied in any of these models.
- the skin cancer mouse model can be easily used for demonstration. Cancers can be cultivated applying the xenograft model of growing human cancerous tissues using the specific pathogen free, homo inbred mouse (a nude mouse) (Yoo, 2004).
- the polypeptides, nucleic acids or vectors provided herein can be locally administered for e.g. bioengineered materials such as a hollow fiber membranes (Orlandini and Margaria.
- microfibers, slow release beads, hypodermic needles, indwelling catheters which can be inserted locally into the cancerous growth, or systemically administered to reach its target for e.g. intravenous infusions, intramuscularly, intraperitoneal injection.
- This treatment can be administered by itself or in combination with other therapeutic compounds for e.g. Chemotherapeutic agents.
- the provided method can inhibit metastasis of a transformed cell in a subject.
- metastasis is meant the transmission of cancer cells from an original site to one or more sites elsewhere in the body, usually by way of the blood vessels or lymphatics. Metastasis can be broken down into a series of events. First, cancer cell migration begins the process by which tumor cells leave the primaiy site of growth, often penetrating the basement membrane and moving towards the local vasculature. Intravasation describes the process of cancer cell entry into the vasculature, and distribution to distant sites. Extravasation refers to the process of cancer cell egression from the vasculature.
- compositions and methods can inhibit metastasis of a transformed cell in a subject by inhibiting migration (i.e., metastatic migration) of said cell.
- Tumourigenesis is the result of cell cycle disorganization, leading to an uncontrolled cellular proliferation.
- Specific cellular processes-mechanisms that control cell cycle progression and checkpoint traversation through the intermitotic phases are deregulated. Normally, these events are highly conserved due to the existence of conservatory mechanisms and molecules such as cell cycle genes and their products.
- An inhibition in metastatic migration can be measured by the levels of such cell cycle genes and products for e.g. cyclins, cyclin dependent kinases (Cdks), Cdk inhibitors (CKI) and extra cellular factors (i.e. growth factors).
- Cdks cyclin dependent kinases
- CKI Cdk inhibitors
- extra cellular factors i.e. growth factors.
- tissue injury can result from, for example, a scrape, cut, laceration wound, crush wound, compression wound, stretch injury, bite wound, graze, bullet wound, explosion injury, body piercing, stab wound, burn wound, wind burn, sun burn, chemical burn, surgical wound, surgical intervention, medical intervention, host rejection following cell, tissue or organ grafting, pharmaceutical effect, pharmaceutical side-effect, bed sore, radiation injury, cosmetic skin wound, internal organ injury, disease process (e.g., asthma, cancer), infection, infectious agent, developmental process, maturational process (e.g., acne), genetic abnormality, developmental abnormality, environmental toxin, allergen, scalp injury, facial injury, jaw injury, foot injury, toe injury, finger injury, bone injury, sex organ injury, joint injury, excretoiy organ injury, eye injury, corneal injuiy, muscle injury, adipose tissue injury, lung injury, airway injury, hernia, anus injury, piles, ear injury, retinal injury, skin
- the tissue injury results from radiation injury.
- radiation injury includes, without l imitation, cutaneous radiation injury (i.e., injury to the skin and underlying tissues from acute exposure to radiation), acute radiation syndrome (i.e., serious illness that occurs following an acute high dose of penetrating whole body irradiation), radiation burns, radiation dermatitis, radiation injury to the nervous system or brain, radiation pneumonitis, and radiation-induced enteritis.
- the radiation injury is cutaneous radiation injury.
- the radiation injury is radiation burns.
- the radiation injury is radiation dermatitis.
- the cutaneous radiation injury results from a dirty bomb attack.
- the radiation injury occurs in a tissue including, without limitation, skin, heart, bone, brain, spinal cord, cornea, retina, and peripheral nerve.
- the radiation injury occurs on the skin.
- radiation-induced tissue injury causes damage to the basal cell layer of the skin.
- radiation-induced damage to the basal cell layer of the skin results in symptoms including, but not limited to, inflammation, erythema, desquamation, intense reddening, blistering, and ulceration of the irradiated site.
- radionucl ide-contaminated wounds and burns promote systemic uptake of radionuclides that greatly complicate topical decontamination and triage.
- radionuclide contamination also interferes with wound healing. Therefore, in certain aspects, the provided polypeptides prevent or treat radiation injury by preventing systemic uptake of radionuclides from a wound or burn at the site of radiation injury. In other aspects, the provided polypeptides prevent or treat radiation injury by reducing hemorrhage, severity of thermal burn injury, and scar tissue formation at the site of radiation injury. In yet other aspects, the provided polypeptides promote tissue regeneration at the site of radiation injury.
- the peptides and/or other formulations embodying the invention will modulate cell migration and proliferation, thereby reducing inflammation, accelerating wound healing, reduce scarring and ultimately promote repair, regeneration and restoration of structure and function in all tissues.
- Healing of wounds, post-peptide application will involve significantly reduced fibrosis, consequently reduced scarring in skin wounds and fibrous patches in internal tissue injuries, promoting tissue regeneration and restoring tissue and organ structure and function.
- Said peptides and/or other formulations can be used to treat internal injury caused by an external wound.
- the external wound is caused by radiation injury.
- internal injury may be caused by radionucieotides that cause internal systemic contamination via contaminated burns and wounds.
- the methods and compositions provided herein provide treatment, prevention, and/or inhibition of the progression of internal radiation damage.
- the internal radiation damage in some embodiments, results from systemic contamination with radiation. Systemic contamination with radiation may result from direct administration of radionucieotides or entiy of radionucieotides from contaminated burns and wounds.
- radiation injury includes, without limitation, cutaneous radiation injury (i.e., injury to the skin and underlying tissues from acute exposure to radiation), acute radiation syndrome (i.e., serious illness that occurs following an acute high dose of penetrating whole body irradiation), radiation burns, radiation dermatitis, radiation injury to the nervous system or brain, radiation pneumonitis, and radiation-induced enteritis.
- the radiation injury is cutaneous radiation injury.
- the radiation injury is radiation burns.
- the radiation injury is radiation dermatitis.
- the cutaneous radiation injury is caused by a dirty bomb attack.
- Additional embodiments of the invention comprises the use of said peptides and/or other formulations for tissue and organ regeneration, when administered in association with stem cells and/or drugs and/or other endogenous and/or clinical regimens promoting stem cell mobilization and/or tissue regeneration.
- Stem cells will help in tissue regeneration and our treatment will promote differentiation directly and/or indirectly by processes that include, but are not limited to reduced fibrotic/astrocytic scar formation, thereby restoring normal tissue structure and function.
- compositions and methods promote the generation of a permissive environment in vivo for regeneration and restoration of structure and function of tissues and organs.
- Regenerative processes aided by our peptide include, but are not limited to internal and external injury, regeneration of tissues, organs, or other body parts, healing and restoration of function fol lowing vascular occlusion and ischemia, brain stroke, myocardial infarction, spinal cord damage, brain damage, peripheral nerve damage, retinal damage, bone damage, exposure to radiation, and other insults to tissues causing destruction, damage or otherwise resulting from, but not limited to, injury, surgery, cancer, congenital and developmental malformation, and diseases causing progressive loss of tissue structure and function, including but not limited to diabetes, bacterial, viral and prion-associated diseases, Alzheimer's disease, Parkinson's disease, AlDs and other genetically determined, environmentally determined or idiopathic disease processes causing loss of tissue/organ/body part structure and function.
- the provided peptides can be administered with drugs or other compounds promoting tissue and cellular regeneration including, but not limited to, trophic factors in processes including, but not limited to, brain, retina, spinal cord and peripheral nervous system regeneration (e.g., NGFs, FGFs, Neurotrophins, Neuregulins, Endothelins, GDNFs, BDNF, BMPs, TGFs, Wnts).
- drugs or other compounds promoting tissue and cellular regeneration including, but not limited to, trophic factors in processes including, but not limited to, brain, retina, spinal cord and peripheral nervous system regeneration (e.g., NGFs, FGFs, Neurotrophins, Neuregulins, Endothelins, GDNFs, BDNF, BMPs, TGFs, Wnts).
- trophic factors in processes including, but not limited to, brain, retina, spinal cord and peripheral nervous system regeneration (e.g., NGFs, FGFs, Neurotrophins, Neuregulins
- Additional embodiments of the invention comprise the delivery of said peptides and/or other formulations using techniques such as, but not limited to, all Antennapedia sequences, and related cell internalization vectors (e.g., TAT protein transduction domain, all TAT peptides, all TAT fusion proteins), viral gene delivery vectors, DNA expression vectors, and any other delivery method that can help get our peptide to the tissue and/or cellular site of action b itself or in association with other agents including but not limited to co-factors assisting this delivery (e.g., including but limited to TAT-HA2) and/or stem cells, drugs and other formulations which help in repair, regeneration and restoration of organ and tissue structure and function.
- co-factors assisting this delivery e.g., including but limited to TAT-HA2
- stem cells drugs and other formulations which help in repair, regeneration and restoration of organ and tissue structure and function.
- compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted.
- the provided nucleic acids can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratoiy Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System l Plus DNA synthesizer (for example, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, Mass. or ABI Model 380B).
- One method of producing the disclosed polypeptides is to link two or more peptides or polypeptides together by protein chemistry techniques.
- peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert- butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, Calif.).
- Fmoc 9-fluorenylmethyloxycarbonyl
- Boc tert- butyloxycarbonoyl
- a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a peptide or protein can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment.
- peptide condensation reactions these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form a protein, or fragment thereof.
- peptide or polypeptide is independently synthesized in vivo as described herein. Once isolated, these independent peptides or polypeptides may be linked to form a peptide or fragment thereof via similar peptide condensation reactions.
- enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al., Biochemistry, 30:41 51 (1991 )).
- native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 ( 1994)).
- the first step is the chemoselective reaction of an unprotected synthetic peptide—thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site (Baggiolini M et al. (1992) FEBS Lett. 307:97- 101 ; Clark-Lewis I et al., J. Biol. Chem., 269: 16075 ( 1994); Clark-Lewis I et al., Biochemistry, 30:3 128 (1991 ); Rajarathnam K et al., Biochemistry 33 :6623-30 ( 1994)).
- unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al. Science, 256:221 ( 1992)).
- This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton R C et al., Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257-267 ( 1992)).
- nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid encoding a polypeptide disclosed herein and a sequence controlling the expression of the nucleic acid.
- cells produced by the process of transforming the cell with any of the herein disclosed nucleic acids.
- animals produced by the process of transfecting a cell within the animal with any of the nucleic acid molecules disclosed herein Disclosed are animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules disclosed herein, wherein the animal is a mammal. Also disclosed are animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules disclosed herein, wherein the mammal is mouse, rat, rabbit, cow, sheep, pig, or primate. Also disclose are animals produced by the process of adding to the animal any of the cells disclosed herein.
- kits for promoting wound healing comprising one or more of the polypeptides, nucleic acids or vectors provided herein in a pharmaceutically acceptable carrier.
- kits for promoting wound healing can also include gels, bandages, Millipore tapes, Medicated Q-tips, Sprays, props, Syrups, Liquids, Disposable tubes or pouches.
- the kits also can contain instructions for proper use and safety information of the product or formulation.
- the kits may contain dosage information based on the application and method of administration as determined by a doctor.
- the disclosed methods and compositions are applicable to numerous areas including, but not limited to, laboratory research tools. These formulations play regulatory roles in several cellular processes for e.g. Cell Proliferation, Cell Migration. These formulations can be used in the laboratory in both in vitro and in vivo model systems for studying various cellular processes, cell cycle regulations, cell behavior, responses of cells, organs or tissues to test compounds etc.
- the formulations can be supplied by themselves or in combination with other compounds or as part ot a kit, such as a kit for cell proliferation assay.
- the kit may contain the formulations mentioned herein by themselves or in combination with other compounds. Such a kit would include instructions designed to facilitate the experiment. Other uses are disclosed, apparent from the disclosure, and/or will be understood by those in the art.
- Example 1 Efficacy of topical administration of Granexin ® in the treatment of, and the prevention of a cutaneous radiation injury - delayed application study
- Tissue equivalent bolus material (8 mm) was placed on the skin surface to ensure that greater than 90% of the prescribed dose was limited to a maximum depth of 2 cm.
- MU Dose/((Total Output Factor) X (Normalization Factor)) where: MU is the machine monitor unit setting; Dose is the nominal delivered dose, in cGy; Total Output Factor is the output factor which includes the appropriate cone and lead cutout at 100 cm source to surface distance; and Normalization factor is the output measured on the day of irradiation.
- the objective of this study was to evaluate the efficacy of topical administration of Granexin ® in the treatment of, and the prevention of a cutaneous radiation injury model using the Yorkshire pig. More specifically, the study was conducted to assess whether Granexin® gel could slow the progression and/or mitigate the severity of symptoms associated with CRI.
- Granexin® is a topical gel comprising 1 .25% hydroxyethyl cellulose gel and the ACT1 peptide.
- the chemical structure of the ACT1 peptide in Granexin® is: Biotin-Ahx- Arg-Gln-Pro-Lys-Ile-Trp-Phe-Pro-Asn-Arg-Arg-Lys-Pro-Trp-Lys-Lys-Arg-Pro-Arg-Pro-Asp- Asp-Leu-Glu-Ile-OH (SEQ ID NO: 91 ), wherein Ahx is L-2-aminohexanoic acid (6- aminohexanoic acid).
- Granexin® used in the present example also included the following inactive ingredients:
- Chelating agent edetate disodium (0.05% w/w)
- Stabilizer D-mannitol (0.05% w/w)
- Granexin® gel was packaged as single-patient-use-containers, containing 5g of gel per tube.
- Granexin® is a clear, odorless, hypoallergenic, and easy-to-apply gel.
- Results of treatment are shown in Table 10 below and illustrated in Figure 1 A.
- the Kumar Scale is shown in Table 1 1 .
- Granexin® (100 ⁇ or 200 ⁇ ) showed a decrease in erythema and development of desquamation, as evaluated by the Kumar scale as compared to vehicle treated sites.
- the composition was effective in prevention of injury progression and severity, a decrease in erythema and development of desquamation. ⁇
- Example 2 Treatment with ACT polypeptide composition to prevent and mitigate the progression of CRI following exposure to ionizing radiation - early application study (treatment initiation 24 hours post irradiation)
- Granexin® To evaluate the potential of Granexin ® to prevent cutaneous CRI progression, severity, and ulceration, i r r a d i a t e d sites were prophylactically treated (prior to the presentation of CRI symptoms) with vehicle or Granexin ® 1 ,000 ⁇ .
- Granexin® was applied starting at 24 hours post exposure and continued once daily for 30 days. Briefly, on Day 0, each animal was anesthetized and exposed to single fractions of low energy electrons (6 MeV) using a Varian CLINAC 21 EX Linear Accelerator at a target dose of 45 Gy.
- Granexin® or vehicle gel was applied once a day starting at 24 hours post radiation exposure.
- Irradiated sites treated with Granexin® gel showed delayed injury progression and ulceration and reduced injury severity of CRI as compared to sites treated with vehicle gel.
- Day 21 photographic comparisons show that prophylactic treatment with Granexin ® at 1 ,000 ⁇ prevents cutaneous radiation injury development as compared to control ( Figure 2).
- Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each ot the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise.
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KR20210104319A (en) | 2020-02-17 | 2021-08-25 | 노태성 | Compositions for Treating Diabetic Ulcers |
CN114522165A (en) * | 2022-01-21 | 2022-05-24 | 贵州省中国科学院天然产物化学重点实验室(贵州医科大学天然产物化学重点实验室) | Application of sanguinarine chloride in preparation of medicine or health product for treating liver injury caused by arsenic |
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Also Published As
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EP3582754A4 (en) | 2020-12-30 |
SG11201907196YA (en) | 2019-09-27 |
BR112019017087A2 (en) | 2020-04-14 |
US20230147795A1 (en) | 2023-05-11 |
KR20190135470A (en) | 2019-12-06 |
RU2019128841A3 (en) | 2021-06-01 |
IL268661A (en) | 2019-10-31 |
JP7145163B2 (en) | 2022-09-30 |
RU2019128841A (en) | 2021-03-16 |
AU2018221221B2 (en) | 2022-03-24 |
ES2970509T3 (en) | 2024-05-29 |
AU2018221221A1 (en) | 2019-08-22 |
JP2020507600A (en) | 2020-03-12 |
CN110520108A (en) | 2019-11-29 |
EP3582754A1 (en) | 2019-12-25 |
SG10202107725RA (en) | 2021-09-29 |
CA3053096A1 (en) | 2018-08-23 |
EP3582754B1 (en) | 2024-01-10 |
MX2019009753A (en) | 2019-12-18 |
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