WO2011156729A2 - Methods of mitigating effects of radiation and reducing the risk of systemic infection - Google Patents
Methods of mitigating effects of radiation and reducing the risk of systemic infection Download PDFInfo
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- WO2011156729A2 WO2011156729A2 PCT/US2011/040006 US2011040006W WO2011156729A2 WO 2011156729 A2 WO2011156729 A2 WO 2011156729A2 US 2011040006 W US2011040006 W US 2011040006W WO 2011156729 A2 WO2011156729 A2 WO 2011156729A2
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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
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/4833—Thrombin (3.4.21.5)
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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
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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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
Definitions
- Amifostin is required to be injected into adjacent tissues prior to fractionated radiotherapy. Therefore, a need exists for new methods for preventing and treating radiation induced injuries resulted from accidental radiation exposure or radiotherapy.
- Applicants have discovered that post-exposure injection of the thrombin peptide derivative TP508 can increase survival time and delay onset of septic bacterial growth in mice that were exposed to a lethal dose of gamma irradiation (Examples 3 and 4).
- either topical treatment or systemic injection of the thrombin peptide derivative TP508 can promote healing of an open dermal wound in mice that were exposed to radiation (Example 5).
- the present invention is directed to a method of reducing the risk of mortality or extending the life expectancy (by, e.g., at least 5%, 10%, 20%, 25%, 50%, 75% or 100%) in a subject exposed to a lethal dose of radiation, or to a dose of radiation that when combined with injury would be lethal, comprising administering to the subject an effective amount of a thrombin peptide derivative comprising Asp-Ala-R, wherein R is a serine esterase conserved sequence.
- the present invention is directed to a method of reducing the risk of developing systemic bacterial, fungal or viral infection in a subject exposed to radiation.
- the method comprises administering to the subject an effective amount of a thrombin peptide derivative comprising Asp-Ala-R, wherein R is a serine esterase conserved sequence.
- the present invention is directed to a method of treating a subject with traumatic injury, dermal injury and/or burn injury who is also exposed to radiation, comprising administering to the subject an effective amount of a thrombin peptide derivative, wherein the thrombin peptide derivative comprises Asp-Ala-R, wherein R is a serine esterase conserved sequence.
- the present invention is also directed to a method of reducing radiation related injury in a subject undergoing radiation therapy, comprising administering to the subject an effective amount of a thrombin peptide derivative, wherein the thrombin peptide derivative comprises Asp-Ala-R, wherein R is a serine esterase conserved sequence.
- the present invention is directed to a method of reducing the risk of developing a radiation induced illness in a subject undergoing radiation therapy, comprising administering to the subject an effective amount of a thrombin peptide derivative, wherein the thrombin peptide derivative comprises Asp-Ala-R, wherein R is a serine esterase conserved sequence.
- the present invention is directed to a method of promoting healing of a wound on a subject that was caused by radiation exposure and or has been exposed to radiation, comprising administering to the wound an effective amount of a thrombin peptide derivative, or comprising administering an effective amount of a thrombin peptide derivative systemically post radiation exposure, wherein the thrombin peptide derivative comprises Asp- Ala-R, wherein R is a serine esterase conserved sequence.
- the present invention is directed to a method of reducing the risk of developing bacterial, fungal or viral infection in the blood of a subject that has not been exposed to radiation and that may be at risk of developing bacterial, fungal or viral infection in the blood. The method comprises administering to the subject an effective amount of a thrombin peptide derivative comprising Asp-Ala-R, wherein R is a serine esterase conserved sequence.
- the present invention is also directed to the use of a thrombin peptide derivative for reducing the risk of mortality in a subject exposed to a lethal dose of radiation, reducing the risk of developing bacterial, fungal or viral infection in a subject exposed to radiation, treating a subject with traumatic injury, dermal injury and/or burn injury who is also exposed to radiation, reducing radiation related injury in a subject undergoing radiation therapy, reducing the risk of developing a radiation induced illness in a subject undergoing radiation therapy; promoting healing of a wound on a subject that was caused by radiation exposure and/or has been exposed to radiation, wherein the thrombin peptide derivative comprises Asp-Ala-R, wherein R is a serine esterase conserved sequence.
- Figure 1 is a graph that shows caspase activity measured in human microvascular endothelial cells (HMVEC) exposed to 0, 5, 10 or 20 Gy of radiation and treated with saline or 30 ug/ml TP508.
- HMVEC human microvascular endothelial cells
- Figure 2 is a graph that shows percent of mice surviving on different days after sustaining 8 Gy radiation exposure and a dermal excision, who have also received treatment with saline placebo or TP508 delivered topically or intravenously.
- Figure 3 is a graph that shows percent of mice surviving at different days after receiving 12 Gy radiation exposure and a single post-exposure bolus dose of either saline or TP508.
- Figure 4 is a bar graph that shows the number of live bacteria (CFU) in the blood of mice 6 and 7 days after exposure to 0 or 12 Gy radiation and either a saline placebo or TP508 injection.
- CFU live bacteria
- Figure 5 is a bar graph that shows the rate of linear wound healing (measured as mm/day) in mice at days 0-5 and 5-16 post-exposure to 0 or 8 Gy irradiation and either saline placebo or TP508 applied topically or by intravenous injection.
- Figure 6 is a panel with two bar graphs.
- the bar graph in panel A shows serum IL-6 levels (ng/ml) measured 11 days post-irradiation in mice irradiated at 0 and 8 Gy and treated with either saline placebo (P) or TP508 administered either topically (TPt) or intravenously (TPiv).
- the bar graph in panel B shows serum IL-6 levels (ng/ml) measured 7 days post- irradiation in mice irradiated at 0 and 12 Gy and treated with either saline placebo (P) or TP508 administered either topically (TPt) or intravenously (TPiv).
- Figure 7 is a bar graph showing fold increase in the sprouting area after 5 days of aortic explant culture.
- the aortas were isolated from mice 24 hours after exposure to 0, 3, 8 or 10 Gy radiation and treatment with either saline placebo or TP508 administered intravenously.
- the present invention is directed to methods of reducing the adverse effects of radiation exposure in a subject comprising administering to the subject an effective amount of a thrombin peptide derivative described therein.
- Radiation exposure can, for example, result from nuclear detonation, nuclear weaponry, accidental radiation exposure (such as due to accidents at nuclear reactors or inadequate protection from radiation source) or radiation therapy.
- Low-dose radiation exposure can be an occupational hazard affecting airline workers and astronauts;
- the present invention is also directed to methods of reducing the risk of developing bacterial, fungal or viral infection in the blood in the subjects who have not been exposed to radiation, comprising administering to the subject an effective amount of a thrombin peptide
- the infection can enter the bloodstream as a complication of
- SIRS 125 response syndrome
- indwelling catheters are a frequent cause of blood infections because they provide a means by which bacteria normally found on the skin can enter the bloodstream.
- Other causes of blood infections include dental procedures (occasionally including simple tooth brushing), herpes (including herpetic whitlow), urinary tract infections, peritonitis,
- the peptide of the present invention can be administered in combination with an antibiotic.
- the present invention is directed to a method of reducing the risk of mortality in a subject exposed to a lethal dose of radiation, comprising administering to the subject an effective amount of a thrombin peptide derivative described herein.
- a lethal dose of radiation is a dose that would cause death in half of the tested subjects in 10 days, i.e. LD 50 in 10 days.
- the lethal dose depends on the identity of the subject. For example, a lethal dose for a human is about 3.5 Gy or greater and a lethal dose for a mouse is about 12 Gy or greater.
- the subject who is exposed to a lethal dose of radiation may also have additionally sustained traumatic injury, dermal injury and/or burn injury.
- Burn injury is a type of skin injury caused by heat, electricity, chemicals, light, radiation, friction or heat.
- Burn injury caused by radiation exposure includes, for example, thermal burns from infrared heat radiation, beta burns from shallow ionizing beta radiation, and gamma burns from highly penetrating gamma radiation.
- the burn injury can be first-degree, second-degree or third-degree burn.
- TBSA total body surface area
- TBSA TBSA
- a "traumatic injury” is a physical injury produced by force or shock.
- a traumatic injury is often associated with secondary complications, such as shock, respiratory failure and death.
- a traumatic injury can be caused by force of explosions or force of falling and flying objects.
- a traumatic injury also includes an injury to an internal organ resulting in hemorrhaging from the organ and/or at least partial loss of function.
- an injury to an internal organ can be caused by penetration, such as from a bullet or flying projectile.
- a traumatic injury can also include injuries to musculoskeletal system, such as bones, muscles,
- a traumatic injury is a bone fracture.
- Dermal injury is an injury to the dermis layer of the skin. Severe dermal injury is a dermal injury that results in dermal wound that covers at least 100 mm and/or is full-thickness wound (i.e., wound that penetrates through both the epidermis and dermis layer of the skin).
- the burn injury, traumatic injury or dermal injury sustained by the subject expose the subject to systemic infection.
- Systemic infection is an infection that has entered the blood stream and may affect multiple organs and/or tissues or the body as a whole.
- the thrombin peptide derivatives described herein reduce leucocytopenia and/or neutropenia in the subject being treated by the methods of the present 170 invention.
- the thrombin peptide derivatives described herein reduces the decrease in population of bone marrow progenitor cells from radiation damage in the subject being treated by the methods of the present invention.
- the present invention is a method of reducing the risk of developing systemic bacterial, fungal or viral infection in a subject, comprising administering to the subject 175 an effective amount of a thrombin peptide derivative described herein.
- the subject has been exposed to a bacterial, fungal or viral infection.
- a subject who is at risk of developing systemic bacterial, viral or fungal infection has been exposed to radiation. For example, in the event of nuclear detonation, health workers, hospital patients, rescue workers, sanitarian workers and people who are in an environment or a place that is likely to
- a subject who suffers from burn injury, traumatic injury or dermal injury resulting from radiation exposure, for example, from nuclear detonation is more susceptible to bacterial, fungal or viral infection than a subject who does not suffer such injuries.
- a subject who has an unhealed wound prior to radiation exposure is more likely to develop bacterial, fungal or
- the thrombin peptide derivatives described herein are effective in reducing the risk of developing systemic bacterial, fungal or viral infection in the subjects described above
- the method reduces the risk of developing bacterial, fungal or viral infection in the blood of the subject.
- High doses of radiation exposure can result in acute illness, such as breakdown of intestinal walls, which would render the subject more susceptible to septic infection.
- the present invention is directed to a method of delaying the onset 195 of septic systemic infection in a subject who is exposed to radiation comprising administering to the subject an effective amount of a thrombin peptide derivative described herein.
- the subject may be exposed to a lethal dose of radiation. Alternatively, the subject is exposed to a sub-lethal dose of radiation.
- the subject is at risk of developing bacterial, fungal or viral 200 infection and has not been exposed to radiation.
- These subjects include one or more of the following: a) a subject has sustained traumatic injury, severe dermal injury and/or burn; b) a subject that underwent an invasive medical or dental procedure; c) a subject that underwent insertion of an invasive medical device; d) a subject who has pneumonia or other pulmonary conditions that could lead to acute respiratory distress syndrome or systemic infections; e) a
- subject who is immunocompromised f) a subject who is an infant or is older than 60 years old, or g) subject from one or more of the categories a-d, who is an infant or older than 60 years old.
- An invasive medical procedure that exposes a subject to infection is any procedure that involves either making a surgical cut in the skin or inserting an instrument, such as a needle or a tube, into the body of a subject.
- An invasive medical procedure increases a risk of introducing
- An example of an invasive medical procedure is a surgery for any indication.
- Another example of an invasive medical procedure is a procedure wherein an invasive medical devise is introduced into a subject.
- Examples of invasive medical devises can include an intravenous or an arterial line, a breathing
- feeding tubes can include G-tube/PEG tube, J-tube (jejunostomy tube) and NG-tube (nasogastric tube).
- the present invention is directed to a method of reducing the risk of developing a bacterial, fungal or viral infection in the blood of a subject who has pneumonia.
- 220 Pneumonia is an inflammatory condition of the lung, especially of the alveoli. Infection is the most common cause of pneumonia. Infecting agents can be bacteria, viruses, fungi, or parasites. Chemical burns or physical injury to the lungs can also produce pneumonia. Bacteria are the most common cause of pneumonia, with Streptococcus pneumoniae the most commonly isolated bacteria in the cases of community-acquired pneumonia. Another important Gram-positive cause
- 225 of pneumonia is Staphylococcus aureus, with Streptococcus agalactiae being an important cause of pneumonia in newborn infants.
- Gram-negative bacteria cause pneumonia less frequently than gram-positive bacteria.
- Some of the gram-negative bacteria that cause pneumonia include Haemophilus influenzae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Moraxella catarrhalis. These bacteria often live in the stomach or intestines and may enter the
- "Atypical" bacteria which cause pneumonia include Chlamydophila pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila.
- the present invention is directed to a method of reducing the risk of developing a bacterial, fungal or viral infection in the blood of a subject who is
- An immunocompromised subject is a subject whose immune system is
- immunocompromised subjects can include: a) subjects who have genetic defects that can affect functioning of their immune systems; b) subjects who have diseases such as AIDS or cancers, including leukemia, lymphoma or multiple myeloma; c) subjects who have chronic diseases,
- end-stage renal disease requiring dialysis, diabetes, or cirrhosis such as end-stage renal disease requiring dialysis, diabetes, or cirrhosis; d) subjects who receive treatments that can include steroids, chemotherapy, radiation, imm nosuppressi ve post- transplant medications; and e) subjects who are pregnant.
- the bacterial, fungal or viral infection includes (in the presence or absence of radiation exposure), but is not limited to, infection of staphylococci (e.g.,
- enterococci enterococci
- streptococci e.g., streptococcus pneumoniae
- pseudomonas aeruginosa burkholderia cenocepacia
- mycobacterium avium enterobacter
- bacteroides fragilis streptococcus pyogenes
- enterococcus sp. haemophilus influenzae, legionella sp., chlamydia pneumoniae, escherichia coli, Clostridium sp., staphylococcus sp., enterobacter sp., proteus sp., neiserria meningitidis , listeria monocytogenes, Candida sp.
- the bacterial, fungal or viral infection also includes systemic bacterial, systemic fungal infections and systemic viral infections.
- thrombin peptide derivatives described herein can promote healing of
- the radiation exposure is sub-lethal.
- the radiation exposure is less than 3.5 Gy when the subject is a human.
- the present invention is directed to a method of reducing radiation related injury in a subject who is undergoing a radiation therapy.
- the method comprises 265 administering to the subject an effective amount of a thrombin peptide derivative described herein.
- a "radiation related injury” is an injury due to radiation exposure resulting from nuclear detonation, nuclear weaponry, accidental radiation exposure or a radiation therapy. For example, when a subject is exposed to a high dose of radiation, such as in the event of nuclear detonation,
- the radiation exposure often causes acute illness in the subject, including hematopoietic
- Radiation exposure such as radiation therapy, can also cause various skin injuries, such as intense reddening, blistering and
- the radiation related injury is skin ulceration or late stage breakdown.
- the present invention is directed to a method of promoting healing of a bone in a subject who is exposed and/or has been exposed to radiation exposure, wherein the bone is a fractured bone or has been surgically treated, for example, to remove tumor in the bone.
- the thrombin peptide derivative is administered to normal tissue of the subject that is exposed or is to be exposed to the radiation.
- the thrombin peptide derivative is topically administered to the normal skin that is exposed to the radiation.
- radiation often causes damage to underlying tissues surrounding the
- the thrombin peptide derivative can be directly applied to the underlying tissues, locally (e.g., by injection or implantation of a sustained release device and the like or through a catheter) or systemically, e.g., before, during or after the radiation therapy.
- the thrombin peptide derivative can be applied or delivered 295 locally or systemically during the radiation therapy or after the radiation therapy.
- the present invention is directed to a method of reducing the risk of developing a radiation induced illness in a subject undergoing radiation therapy, comprising administering to the subject an effective amount of a thrombin peptide derivative described herein.
- radiation induced illness refers to a disorder, disease or condition that resulted from cellular damages caused by radiation exposure.
- exposure to radiation can result in various cellular damages, such as damages to hematopeotic cells, decreased availability, viability and function of progenitor cells, delayed angiogensis and revascularization, apoptosis of intestinal microvascular endothelial cells, epithelial cells, crypt cells, neuronal cells in the brain
- radiation induced illnesses include diseases,
- Exemplary radiation induced illness include, but is not limited to, leucocytopenia, neutropenia, infections, systemic inflammatory response syndrome (SIRS), sepsis, multiple organ dysfunction syndrome (MODS), lung damage, lung/airway disease, brain microvascular
- the present invention is directed to a method of promoting healing of a wound on a subject that was caused by radiation exposure and/or has been exposed to radiation.
- the method comprises administering to the wound (e.g., topically or systemically, 315 such as by I.V.) an effective amount of a thrombin peptide derivative described herein.
- a wound is a type of injury in which skin is torn, cut or punctured (an open wound).
- An open wound can include incisions or incised wounds (caused by a clean, sharp-edged object such as a knife, a razor or a glass splinter); lacerations (irregular tear-like wounds caused by blunt trauma); abrasions; puncture wounds; penetration wounds or gunshot wounds (caused by a bullet 320 or similar projectile driving into or through the body).
- thrombin derivative peptides As used herein, the thrombin derivative peptides, the modified thrombin peptide derivatives and the thrombin peptide derivative dimers described below can be collectively referred to as "thrombin peptide derivatives.”
- the thrombin derivative peptides, the modified thrombin peptide derivatives and each polypeptide in the thrombin peptide derivative dimers 325 have 19 to 23 amino acids (i.e., 19-23 amino acids in length).
- Thrombin peptide derivatives are analogs of thrombin that have an amino acid sequence derived at least in part from that of thrombin and are 330 active at the non-proteolytically activated thrombin receptor (NPAR).
- NPAR non-proteolytically activated thrombin receptor
- derivatives can include, for example, peptides that are produced by recombinant DNA methods, peptides produced by enzymatic digestion of thrombin, and peptides produced synthetically, which can comprise amino acid substitutions compared to thrombin and/or modified amino acids, especially at the termini.
- Thrombin peptide derivatives of the present invention include thrombin derivative
- the thrombin peptide derivatives of the present invention is a thrombin peptide derivative or a physiologically functional equivalent, i.e., a polypeptide with no more than about fifty amino acids, preferably no more than about thirty amino acids and having sufficient homology to the
- 340 fragment of human thrombin corresponding to thrombin amino acids 508-530 (Ala-Gly-Tyr-Lys- Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val; SEQ ID NO:6) that the polypeptide activates NPAR.
- the thrombin peptide derivatives of the present invention is a thrombin peptide derivative comprising a moiety represented by Structural Formula (I):
- R is a serine esterase conserved domain.
- Serine esterases e.g., trypsin, thrombin, chymotrypsin and the like, have a region that is highly conserved.
- Serine esterase conserved domain refers to a polypeptide having the amino acid sequence of one of these conserved regions or is sufficiently homologous to one of these conserved regions such that the thrombin peptide derivative retains 350 NPAR activating ability.
- a physiologically functional equivalent of a thrombin derivative encompasses molecules which differ from thrombin derivatives in aspects which do not affect the function of the thrombin receptor binding domain or the serine esterase conserved amino acid sequence. Such aspects may include, but are not limited to, conservative amino acid substitutions (as defined 355 below) and modifications, for example, amidation of the carboxyl terminus, acetylation of the amino terminus, conjugation of the polypeptide to a physiologically inert carrier molecule, or sequence alterations in accordance with the serine esterase conserved sequences.
- a domain having a serine esterase conserved sequence can comprise a polypeptide sequence containing at least 4-12 of the N-terminal amino acids of the dodecapeptide previously 360 shown to be highly conserved among serine proteases (Asp-Xi-Cys-X 2 -Gly-Asp-Ser-Gly-Gly- Pro-X 3 -Val; SEQ ID NO: 13); wherein X 1; is either Ala or Ser; X 2 is either Glu or Gin; and X 3 is Phe, Met, Leu, His, or Val).
- the serine esterase conserved sequence comprises the amino acid sequence of SEQ ID NO: 14 (Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val) or a C-terminal
- amino acids in the serine esterase conserved sequence which differ from the corresponding amino acid in SEQ ID NO: 14 are conservative substitutions as defined below, and are more preferably
- C-terminal truncated fragment refers to a fragment
- said fragment having at least six and more preferably at least nine amino acids.
- the serine esterase conserved sequence comprises the amino acid sequence of SEQ ID NO: 15 (Cys-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val; Xi is Glu or Gin and X 2 375 is Phe, Met, Leu, His or Val) or a C-terminal truncated fragment thereof having at least six
- the thrombin peptide derivative comprises a serine esterase conserved sequence and a polypeptide having a more specific thrombin amino acid sequence Arg-Gly- Asp-Ala (SEQ ID NO: 16).
- SEQ ID NO: 16 One example of a thrombin peptide derivative of this type
- 380 comprises Arg-Gly-Asp-Ala-Cys-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val (SEQ ID NO: 1).
- ⁇ and X 2 are as defined above.
- the thrombin peptide derivative can comprise the amino acid sequence of SEQ ID NO:6 (Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp- Ser-Gly-Gly-Pro-Phe-Val) or an N-terminal truncated fragment thereof, provided that zero, one, two or three amino acids at positions 1-9 in the thrombin peptide derivative differ from the amino acid sequence of SEQ ID NO:6 (Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp- Ser-Gly-Gly-Pro-Phe-Val) or an N-terminal truncated fragment thereof, provided that zero, one, two or three amino acids at positions 1-9 in the thrombin peptide derivative differ
- N-terminal truncated fragment refers to a fragment remaining after removing an amino acid or block of amino acids from the N-terminus, preferably a block of
- the thrombin peptide derivatives described herein can be amidated at the C- terminus and/or acylated at the N-terminus.
- the thrombin peptide derivatives comprise a C-terminal amide and optionally comprise an acylated N-terminus, wherein said C-terminal amide is represented by -C(0)NR a R b , wherein R a and R b are
- R 395 independently hydrogen, a Ci-Cio substituted or unsubstituted aliphatic group, or R a and R b , taken together with the nitrogen to which they are bonded, form a CI -CIO non-aromatic heterocyclic group, and said N-terminal acyl group is represented by R c C(0)-, wherein RL. is hydrogen, a Ci-Cio substituted or unsubstituted aromatic group, or a Ci-Cio substituted or unsubstituted aromatic group.
- the N-terminus of the thrombin 400 peptide derivative is free (i.e., unsubstituted) and the C-terminus is free (i.e., unsubstituted) or amidated, preferably as a carboxamide (i.e., -C(0)NH 2 ).
- the thrombin peptide derivative comprises the following amino acid sequence: Ala-Gly-Tyr-Lys-Pro-Asp-Glu- Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 6).
- the thrombin peptide derivative comprises the amino sequence of
- the thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO: 18: Asp-Asn- Met-Phe-Cys-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser- Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe.
- the thrombin peptide derivates comprising the amino acids of SEQ ID NO: 6, 17, or 18 can optionally be amidated at the C-terminus and/or
- the N-terminus is free (i.e., unsubstituted) and the C- terminus is free (i.e., unsubstituted) or amidated, preferably a carboxamide (i.e., -C(0)NH 2 ).
- a carboxamide i.e., -C(0)NH 2
- 415 thrombin peptide derivative can differ from the corresponding amino acid in SEQ ID NO: 18.
- amino acids in the thrombin peptide derivative which differ from the amino acids in the thrombin peptide derivative which differ from the amino acids in the thrombin peptide derivative
- amino acid in SEQ ID NO: 6 or SEQ ID NO: 18 are conservative substitutions as defined below, and are more preferably highly conservative substitutions.
- eighteen amino acids can be used in the methods of the present invention.
- C-terminal truncated fragment refers to a fragment remaining after removing an amino acid or block of amino acids from the C-terminus.
- N-terminal truncated fragment refers to a fragment remaining after removing an amino acid or block of amino acids from the N- 425 terminus. It is to be understood that the terms “C-terminal truncated fragment” and “N-terminal truncated fragment” encompass acylation at the N-terminus and/or amidation at the C-terminus, as described above.
- a preferred thrombin peptide derivative for use in the disclosed method comprises the amino acid sequence SEQ ID NO:2: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-GIy-Lys-Arg-Gly-Asp-Ala-
- Another preferred thrombin peptide derivative for use in the disclosed method comprises the amino acid sequence of SEQ ID NO: 19: Asp-Asn-Met- Phe-Cys-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Xi-Gly-Asp-Ser-Gly- Gly-Pro-X 2 -Val-Met-Lys-Ser-Pro-Phe.
- Xi is Glu or Gin;
- X 2 is Phe, Met, Leu, His or Val.
- the thrombin peptide derivatives of SEQ ID NO:2 and SEQ ID NO: 19 can optionally comprise a C-
- N-terminus is free (i.e., unsubstituted) and the C-terminus is free (i.e., unsubstituted) or amidated, preferably as a carboxamide (i.e., -C(0)NH 2 ).
- a carboxamide i.e., -C(0)NH 2
- TP508 is an example of a thrombin peptide derivative and is 23 amino acid residues long, wherein the N-terminal amino acid residue Ala is unsubstituted and the COOH of the C-terminal amino acid Val is modified to an amide represented by -C(0)NH 2 (SEQ ID NO:3).
- thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO:6, wherein both N- and C-termini are unsubstituted (“deamide ⁇ 508") ⁇
- Other examples of thrombin peptide derivatives which can be used in the disclosed method include N-terminal truncated fragments of TP508 (or deamide TP508), the N-terminal truncated fragments having at least fourteen amino acids, or C-terminal truncated fragments of TP508 (or deamide TP508), the 450 C-terminal truncated fragments having at least eighteen amino acids.
- a "conservative substitution" in a polypeptide is the replacement of an amino acid with another amino acid that has the same net electronic charge and approximately the same size and shape.
- Amino acids with aliphatic or substituted aliphatic amino acid side chains have approximately the same size when the total number of carbon and heteroatoms in 455 their side chains differs by no more than about four. They have approximately the same shape when the number of branches in their side chains differs by no more than one.
- Amino acids with phenyl or substituted phenyl groups in their side chains are considered to have about the same size and shape. Listed below are five groups of amino acids. Replacing an amino acid in a polypeptide with another amino acid from the same group results in a conservative substitution:
- Group I glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, and non- naturally occurring amino acids with C1-C4 aliphatic or C1-C4 hydroxyl substituted aliphatic side chains (straight chained or monobranched).
- Group II glutamic acid, aspartic acid and non-naturally occurring amino acids with carboxylic acid substituted C1-C4 aliphatic side chains (unbranched or one branch point).
- Group III lysine, ornithine, arginine and non-naturally occurring amino acids with amine or guanidino substituted C1-C4 aliphatic side chains (unbranched or one branch point).
- Group IV glutamine, asparagine and non-naturally occurring amino acids with amide substituted CI -C4 aliphatic side chains (unbranched or one branch point).
- Group V phenylalanine, phenylglycine, tyrosine and tryptophan.
- a "highly conservative substitution" in a polypeptide is the replacement of an amino acid with another amino acid that has the same functional group in the side chain and nearly the same size and shape.
- Amino acids with aliphatic or substituted aliphatic amino acid side chains have nearly the same size when the total number of carbon and heteroatoms in their side chains differs by no more than two. They have nearly the same shape when they have
- substitutions include valine for leucine, threonine for serine, aspartic acid for glutamic acid and phenylglycine for phenylalanine. Examples of substitutions which are not highly conservative include alanine for valine, alanine for serine and aspartic acid for serine.
- the thrombin peptide derivatives are modified relative to the thrombin peptide derivatives described above, wherein cysteine residues of aforementioned thrombin peptide derivatives are replaced with amino acids having similar size and charge properties to minimize dimerization of the peptides.
- suitable amino 485 acids include alanine, glycine, serine, or an S' -protected cysteine.
- cysteine is replaced with alanine.
- the modified thrombin peptide derivatives have about the same biological activity as the unmodified thrombin peptide derivatives. See Publication No. US 2005/0158301 Al, which is hereby incorporated by reference.
- modified thrombin peptide derivatives disclosed herein can 490 optionally comprise C-terminal amides and/or N-terminal acyl groups, as described above.
- the N-terminus of a thrombin peptide derivative is free (i.e., unsubstituted) and the C- terminus is free (i.e., unsubstituted) or amidated, preferably as a carboxamide (i.e., -C(0)NH 2 ).
- the modified thrombin peptide derivative comprises a polypeptide having the amino acid sequence of SEQ ID NO:4: Arg-Gly-Asp-Ala-Xaa-Xi-Gly-
- the thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO:20: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa- Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:20.
- the thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO:20: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa- Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:20.
- the thrombin peptide derivative comprises the amino acids 10-18 of SEQ ID NO:20.
- SEQ ID NO: 5 Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa- Xi-Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val, or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:5.
- Xaa is alanine, glycine, serine or an S-protected cysteine.
- Xi is Glu or Gin and X 2 is Phe, Met, Leu, His or Val.
- Xi is Glu
- X 2 is Phe
- Xaa is alanine.
- a thrombin peptide derivative of this type is a polypeptide having the amino acid
- thrombin peptide derivative of this type is the polypeptide H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Ala-Glu- Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH 2 (SEQ ID NO:22).
- Another example of a thrombin peptide derivative of this type is the polypeptide H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-
- the thrombin peptide derivative comprises a polypeptide
- the thrombin peptide derivative comprises a polypeptide having the amino acid sequence SEQ ID NO:24: Asp-Asn-Met-Phe-Xbb-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-Xi-Gly-
- Xaa and Xbb are independently alanine, glycine, serine or an S-protected cysteine.
- Xi is Glu or Gin and X 2 is Phe, Met, Leu, His or Val.
- X 1 is Glu
- X 2 is Phe
- Xaa and Xbb are alanine.
- thrombin peptide derivative of this type is a polypeptide comprising the amino acid sequence Asp-Asn-Met-Phe-Ala-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-
- a further example of a thrombin peptide derivative of this type is the polypeptide H- Asp-Asn-Met-Phe-Ala-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Ala-Glu-Gly- Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe-NH 2 (SEQ ID NO:26).
- Zero, one, two or three amino acids in the thrombin peptide derivative can differ from the amino acid at the
- Xaa and Xbb are independently alanine, glycine, serine or an S-protected cysteine.
- the difference is conservative as in conservative substitutions of the thrombin peptide derivatives.
- S-protected cysteine is a cysteine residue in which the reactivity of the thiol moiety, -SH, is blocked with a protecting group. Suitable protecting groups are known in the art and arc 535 disclosed, for example, in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic
- Suitable protecting groups should be nontoxic, stable in pharmaceutical formulations and have minimum additional functionality to maintain the activity of the thrombin peptide derivative.
- a free thiol can be protected as a
- thioether a thioester, or can be oxidized to an unsymmetrical disulfide.
- the thiol is protected as a thioether.
- Suitable thioethers include, but are not limited to, S-alkyl thioethers (e.g., C1-C5 alkyl), and S-benzyl thioethers (e.g, cysteine-S-S-i-Bu).
- the protective group is an alkyl thioether. More preferably, the S-protected cysteine is an S-methyl cysteine.
- the protecting group can be: 1 ) a cysteine or a cysteine-containing peptide
- protecting peptide attached to the cysteine thiol group of the thrombin peptide derivative by a disulfide bond; or 2) an amino acid or peptide (“protecting peptide") attached by a thioamide bond between the cysteine thiol group of the thrombin peptide derivative and a carboxylic acid in the protecting peptide (e.g., at the C-terminus or side chain of aspartic acid or glutamic acid).
- the protecting peptide can be physiologically inert (e.g., a polyglycine or polyalanine of no more
- the thrombin peptide derivatives of the methods are thrombin peptide derivative dimers. See Publication No. US 2005/0153893, which is hereby 555 incorporated by reference.
- the dimers essentially do not revert to monomers and still have about the same biological activity as the thrombin peptide derivatives monomer described above.
- a "thrombin peptide derivative dimer" is a molecule comprising two thrombin peptide derivatives linked by a covalent bond, preferably a disulfide bond between cysteine residues.
- Thrombin peptide derivative dimers are typically essentially free of the corresponding monomer, e.g., 560 greater than 95% free by weight and preferably greater than 99% free by weight.
- the polypeptides are the same and covalently linked through a disulfide bond.
- the thrombin peptide derivative dimers of the present invention comprises the thrombin peptide derivatives described above. Specifically, thrombin peptide derivatives have less than about fifty amino acids, preferably less than about thirty-three amino acids. Thrombin peptide 565 derivatives also have sufficient homology to the fragment of human thrombin corresponding to thrombin amino acid residues 508-530: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp- Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO:6) so that the polypeptide activates NPAR.
- each thrombin peptide derivative comprising a dimer
- 570 comprises a polypeptide having the amino acid sequence SEQ ID NO:l: Arg-Gly-Asp-Ala-Cys- Xi-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val, or a C-terminal truncated fragment thereof comprising at least six amino acids.
- each thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO: 6: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys- Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val, or a fragment thereof comprising amino acids 10-18 of 575 SEQ ID NO. 5.
- the thrombin peptide derivative comprises the amino acid sequence SEQ ID NO:2: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys- Xi-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val, or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:2.
- Xi is Glu or Gin and X 2 is Phe, Met, Leu, His or Val.
- Xi is Glu
- X 2 is Phe.
- thrombin peptide derivative of this type is a polypeptide comprising 580 the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu- Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 6).
- a further example of a thrombin peptide derivative of this type is a polypeptide having the amino acid sequence H-Ala-Gly-Tyr-Lys-Pro- Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH 2 (SEQ ID NO:3).
- Z Zambin peptide derivative of this type is a polypeptide having the amino acid sequence H-Ala-Gly-Tyr-Lys-Pro- Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH 2 (SEQ ID NO:3).
- Zero, one, two or three amino acids in the thrombin peptide derivative differ from the amino acid sequence H-Ala-Gly-Tyr-L
- the difference is conservative as for conservative substitutions of the thrombin peptide derivatives.
- thrombin peptide derivative dimer of the present invention is represented by Formula (IV):
- each thrombin peptide derivative comprising a dimer comprises a polypeptide comprising the amino acid sequence SEQ ID NO:27: Ala-Gly-Tyr-Lys- Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys- Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr, or a C-terminal truncated fragment thereof having at least twenty-three amino acids. More preferably, each thrombin peptide derivative comprises the amino acid sequence SEQ ID NO:27: Ala-Gly-Tyr-Lys- Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met
- amino acid sequence SEQ ID NO:28 Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-
- Xi is Glu or Gin and X 2 is Phe, Met, Leu, His or Val.
- Xi is Glu
- X 2 is Phe.
- a thrombin peptide derivative of this type is a polypeptide comprising the amino acid sequence
- a further example of a thrombin peptide derivative of this type is a polypeptide comprising the amino acid sequence H- Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro- Phe-Val-Met-Lys-Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr-NH 2 (SEQ ID NO:29).
- the difference is conservative as defined for conservative substitutions of the thrombin peptide derivatives.
- a "subject” is preferably a human, but can also be an animal in need of treatment with a thrombin peptide derivative disclosed herein, e.g., companion animals (e.g., dogs, cats, and the 610 like), farm animals (e.g., cows, pigs, horses and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like).
- companion animals e.g., dogs, cats, and the 610 like
- farm animals e.g., cows, pigs, horses and the like
- laboratory animals e.g., rats, mice, guinea pigs and the like.
- an "effective amount” is the quantity of the thrombin peptide derivative described herein that results in an improved clinical outcome of the condition being treated with the thrombin peptide derivative compared with the absence of treatment.
- 615 derivative administered will depend on the degree, severity, and type of the disease or condition, the amount of therapy desired, and the release characteristics of the pharmaceutical formulation. It will also depend on the subject's health, size, weight, age, sex and tolerance to drugs.
- the thrombin peptide derivative is administered for a sufficient period of time to achieve the desired therapeutic effect. Typically, from about 1 ⁇ g per day to about 1 mg per day
- thrombin peptide derivatives preferably from about 5 ⁇ g per day to about 100 ⁇ g per day
- the thrombin peptide derivatives can also be administered at a dose of from about 0.1 mg/kg/day to about 15 mg/kg/day, with from about 0.2 mg/kg/day to about 3 mg/kg/day being preferred, especially for systemic means of administration.
- Typical dosages for the thrombin peptide 625 derivative of the invention are also 5-500 mg/day, preferably 25-250 mg/day, especially for systemic means of administration.
- the thrombin peptide derivative or composition can be administered before, during or after the radiation exposure.
- the peptide of the present invention can be administered in combination with an angiogenic growth
- angiogenic growth factor is a polypeptide which stimulates the development of blood vessels, e.g., promotes angiogenesis, endothelial cell growth, stability of blood vessels, and/or vasculogenesis.
- angiogenic factors include, but are not limited to, e.g., VEGF-A and members of the VEGF family, PIGF, PDGF family, fibroblast growth factor family (FGFs), TIE ligands (Angiopoietins), ephrins, ANGPTL3, ANGPTL4, etc.
- polypeptides such as growth hormone, insulin-like growth factor-I (IGF-I), VIGF, epidermal growth factor (EGF), CTGF and members of its family, and TGF-a and TGF- ⁇ .
- Treating means that following a period of administering the thrombin peptide derivative or composition comprising a thrombin peptide derivative, a beneficial therapeutic and/or prophylactic result is achieved, which can include a decrease in the severity of symptoms 640 or delay in or inhibition of the onset of symptoms, increased longevity and/or more rapid or more complete resolution of the disease or condition, or other improved clinical outcome as measured according to the site that is being observed or the parameters measured for a particular disease or disorder.
- Reducing the risk refers to decreasing the probability of developing a disease, disorder 645 or medical condition, in a subject, wherein the subject is, for example, a subject who is at risk for developing the disease, disorder or condition.
- Reducing radiation related injury refers to a decrease in the severity of injuries induced by radiation exposure.
- the disclosed thrombin peptide derivative can be administered by any suitable route, 650 locally (e.g., topically) or systemically, including, for example, by parenteral administration.
- Parenteral administration can include, for example, intramuscular, intravenous, subcutaneous, or intraperitoneal injection or vascular administration, and can also include transdermal patch and implanted slow-release devices such as pumps.
- Topical administration can include, for example, creams, gels, ointments or aerosols.
- Respiratory administration can include, for example, 655 inhalation or intranasal drops.
- the thrombin peptide derivative can be advantageously administered in a sustained release formulation.
- the thrombin peptide derivative can be administered chronically, wherein the peptide derivative is administered over a long period of time (at least 60 days, but more typically, for at least one year), at intervals or by a 660 continuous delivery method, to treat a chronic or recurring disease or condition.
- the thrombin peptide derivative can be administered to the subject in conjunction with an acceptable pharmaceutical carrier as part of a pharmaceutical composition.
- the formulation of the pharmaceutical composition will vary according to the route of administration selected.
- Suitable pharmaceutical carriers may contain inert ingredients which do not interact with the 665 compound.
- the earners should be biocompatible, i.e., non-toxic, non-inflammatory, non- immunogenic and devoid of other undesired reactions at the administration site.
- pharmaceutically acceptable carriers include, for example, saline, aerosols, commercially available inert gels, or liquids supplemented with albumin, methyl cellulose or a collagen matrix. Standard pharmaceutical formulation techniques can be employed, such as those described in 670 Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
- Other suitable pharmaceutical carriers include those described in U.S. Patent No. 7,294,596, the entire teaching of which is incorporated herein by reference.
- compositions used in the methods of the present invention can additionally comprise a pharmaceutical carrier in which the thrombin peptide derivative is dissolved or suspended.
- Examples of pharmaceutically acceptable carriers include, for example, saline, aerosols,
- Gels are comprised of a base selected from an oleaginous base, water, or an emulsion-suspension base, as previously described. To the base is added a gelling agent that forms a matrix in the base, increasing its viscosity to a
- gelling agents are hydroxypropyl cellulose, acrylic acid polymers, and the like.
- the active ingredients are added to the formulation at the desired concentration at a point preceding addition of the gelling agent or can be mixed after the gelation process.
- injectable delivery formulations may be administered intravenously or directly at the site 685 in need of treatment.
- the injectable carrier may be a viscous solution or gel.
- Delivery formulations include physiological saline, bacteriostatic saline (saline containing about 0.9% mg/mL benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer' s-lactate, or liquids supplemented with albumin, methyl cellulose, or hyaluronic acid.
- Injectable matrices include polymers of poly(ethylene oxide) and copolymers of ethylene and 690 propylene oxide (see Cao et al, J. Biomater. Sci 9:475 (1998) and Sims et al, Plast Reconstr.
- compositions such as in a coating of hard gelatin or cyclodextran
- Methods for encapsulating compositions are known in the art (Baker, et al, "Controlled Release of Biological Active Agents", John Wiley and Sons, 1986).
- Ointments are typically prepared using an oleaginous base, e.g., containing fixed oils or hydrocarbons, such as white petrolatum or mineral oil, or an absorbent base, e.g., consisting of an absorbent anhydrous substance or substances, for example anhydrous lanolin. Following formation of the base, the active ingredients are added in the desired concentration.
- an oleaginous base e.g., containing fixed oils or hydrocarbons, such as white petrolatum or mineral oil
- an absorbent base e.g., consisting of an absorbent anhydrous substance or substances, for example anhydrous lanolin.
- Creams generally comprise an oil phase (internal phase) containing typically fixed oils, 700 hydrocarbons, and the like, such as waxes, petrolatum, mineral oil, and the like, and an aqueous phase (continuous phase), comprising water and any water-soluble substances, such as added salts.
- the two phases are stabilized by use of an emulsifying agent, for example, a surface active agent, such as sodium lauryi sulfate; hydrophilic colloids, such as acacia colloidal clays, beegum, and the like.
- an emulsifying agent for example, a surface active agent, such as sodium lauryi sulfate; hydrophilic colloids, such as acacia colloidal clays, beegum, and the like.
- Gels contain a base selected from an oleaginous base, water, or an emulsion-suspension base, as previously described.
- a gelling agent which forms a matrix in the base, increasing its viscosity to a semisolid consistency.
- gelling agents are hydroxypropyl cellulose, acrylic acid polymers, and the like.
- the active ingredients are added to 710 the formulation at the desired concentration at a point preceding addition of the gelling agent.
- thrombin peptide derivatives Diseases and conditions that are treatable with the disclosed thrombin peptide derivatives are often accompanied by symptoms and infirmities such as pain and infection. In certain instances it may be advantageous to co-administer one or more additional pharmacologically active agents along with a thrombin peptide derivative to address such issues. For example, 715 managing pain and inflammation may require co-administration with analgesic or an antiinflammatory agents. Managing infection may require co-administration with antimicrobial, antibiotic or disinfectant agents.
- a thrombin peptide derivative can be administered to a subject alone or in combination with one or more other therapeutics, for example, a cholesterol-lowering agent, an anti- 720 hypertensive agent, a beta-blocker, an anti-coagulant, a thrombolytic agent, an analgesic, an antiinflammatory agent, an anti-plaque agent, insulin, a nitric oxide generating agent, an antiviral agent or an antibiotic.
- a thrombin peptide derivative can be administered to a subject in combination with arginine, for example, with arginine as an oral nutritional supplement.
- Thrombin peptide derivatives and modified thrombin peptide derivatives can be
- Thrombin peptide derivative dimers can be prepared by oxidation of the monomer.
- Thrombin peptide derivative dimers can be prepared by reacting the thrombin peptide derivative with an excess of oxidizing agent.
- oxidizing agent is iodine.
- non-aromatic heterocyclic group is a non-aromatic carbocyclic ring system that has 3 to 10 atoms and includes at least one heteroatom, such as nitrogen, oxygen, or 740 sulfur.
- heteroatom such as nitrogen, oxygen, or 740 sulfur.
- examples of non-aromatic heterocyclic groups include piperazinyl, piperidinyl,
- alkyl is a straight chain or branched saturated hydrocarbon radical. Typically, an alkyl group has from 1 to about 10 carbon atoms, preferably from 1 to about 4 carbon atoms. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n- 745 butyl, sec -butyl, tert-butyl, pentyl, cyclopentyl, hexyl, cyclohexyl, octyl and cyclooctyl.
- Example 1 Effects of TP508 on apoptosis of human micro vasular endothelial cell
- HMVEC Human dermal microvascular endothelial cells
- mice were irradiated ( 137 Cs Gamma Irradiator Mark 30, Shephard and 760 Associates, San Fernando, CA) with exposures of 8 Gy or 3 Gy. After 4 hours or 24 hours, mice were anesthesized and prepared for surgery. A single 1.5 cm square full dermal excision was created and treated topically with saline (25 ⁇ ) or saline plus TP508 (0.25 ⁇ g) and covered with Opsite® occlusive dressing. There was a significant decrease in survival in mice that have sustained radiation combined injuries (Table 1 below). Significant increase in survival was 765 observed when mice were injected with TP508 24 hours after radiation exposure (see Table 1).
- Example 3 Effect of TP508 on mouse survival following radiation exposure to the lethal dose of 12 Gy.
- TP508 mice were exposed to a lethal dose of 137 Cs gamma irradiation (12 Gy). Injection of a single bolus dose of TP508 (500 ⁇ g) within 2 hours after exposure delayed the mortality of the first mouse in the treated group by about 3 days and increased the group mean survival time by about 15%. (See Figure 3). TP508 has a short half-life and may thus only be present in blood at an effective concentration for the first two to three hours. This may explain why it only extends survival for a few days.
- Example 4 Effect of TP508 on bacterial growth in blood of animals post irradiation
- mice had an average of 1.6 x 10 6 CFU/ml while those injected with TP508 were just beginning to show infection, with an average of just over 100 CFU/ml.
- Example 5 Effect of TP508 on healing of open dermal wounds
- mice were irradiated ( 137 Cs Gamma Irradiator Mark 30, Shephard and Associates, San Fernando, CA) with exposures of 8 Gy or 3 Gy. After 4 hours or 24 hours, mice were anesthesized and prepared for surgery. A single 1.5 cm square full dermal excision was created and treated topically with saline (25 ul) or saline plus TP508 (0.3 ug) and covered with Opsite® occlusive dressing. At 8 Gy, radiation delayed wound healing in mice receiving dermal wounds 4 hours after irradiation, but a single topical treatment with TP508 accelerated healing.
- TP508 appears to restore normal rates of healing to irradiated mice. This was confirmed by calculating the linear rate of healing in these wounds (See Figure 5). Interestingly, the linear rates of healing for all groups was similar during the first 5 days after wounding, perhaps due to contraction that was not affected by radiation. From 5 to 16 days, however, radiation significantly impairs healing, but TP508 treatment overcomes this impairment.
- mice with 3 Gy exposures underwent dermal wound 805 surgery 24 hours after irradiation. These wounds also demonstrated delayed healing relative to non-irradiated control mice.
- TP508 topical treatment accelerated healing to overcome the effect of radiation.
- Example 6 Effect of TP508 on apoptosis and proliferation and migration of intestinal 815 crypt progenitor cells.
- mice exposed to 12 Gy whole body irradiation contain a large number of apoptotic cells within the intestinal cryps, as determined by tunnel staining. Mice injected with TP508 appear to have fewer apoptotic cells. This effect of TP508 was confirmed by measuring EdU incorporation (DNA synthesis) at 5 days post-exposure.
- Example 7 Effect of TP508 on radiation and RCI-induced up-regulation of IL-6.
- mice 830 ICR white male mice were exposed to 0 Gy (control) or 8 Gy of gamma radiation and wounded 24 hours later. Wounds were treated topically with saline placebo (P) or TP508 in saline (TPt). A separate group of mice were injected IV with 500 micrograms of TP508 2 hours post 8 Gy exposure. After eleven (11) days, serum from mice was analyzed for amount of IL-6A using enzyme-linked immunosorbent assay (ELISA) ( Figure 6, Panel A).
- ELISA enzyme-linked immunosorbent assay
- mice 835 ICR white male mice were exposed to 0 Gy, or 12 Gy nuclear irradiaton without wounds and were injected IV with placebo or TP508 post-exposure. Serum was isolated from mice seven (7) days later and the amount of IL-6 was determined by enzyme-linked immunosorbent assay (ELISA) ( Figure 6, Panel B).
- ELISA enzyme-linked immunosorbent assay
- TP508 The combination of 8Gy radiation exposure and wounding increases IL-6 levels above 840 wounding alone.
- Topical TP508 treatment of wounds reduces IL-6 levels by -75%.
- Systemic IV injection of TP508 reduces IL-6 levels by more than 90%.
- 12Gy exposure alone without wounds also increases IL-6 levels.
- TP508 injection reduces IL-6 production measured at day 7 by approximately 50%.
- Example 8 Effect of TP508 on endothelial function as demonstrated by aortic explant endothelial cell sprouting assays.
- an established 850 angiogenesis assay was used. In these experiments mice were either non-irradiated or given exposures of 3 Gy, 8 Gy or 10 Gy. Approximately 2 hours post-exposure mice were injected IV with saline or saline plus TP508 (15 mg/kg). Mice were sacrificed 24 hours after exposure, aortas removed and aortic segments were placed on matrigel® and cultured in endothelial growth medium with growth supplement containing VEGF and FGF2 for 5 days.
- TP508 more than doubled the amount of endothelial sprouting from the aortic segments during 5 day incubations as determined by measuring area occupied by sprouts or longest sprout projections.
- Aortic segment explants from 3 Gy exposed mice had some sprouting in the saline injected group, but again this sprouting was more than doubled in mice injected with TP508.
- In the 8 Gy and 10 Gy groups there was virtually no sprouting from 860 aortic segments isolated from placebo mice, while visible sprouting continued to be observed at the edges of explants from T508-treated mice.
- Example 9 Effect of TP508 on hematopoietic recovery and increases proliferation of bone marrow progenitor cells.
- the bone marrow was isolated from non-exposed and mice exposed to 8 Gy with or 865 without TP508 post-exposure injection.
- the samples were subjected to the complete blood count (CBC) analysis, which demonstrated earlier recovery of leukocyte, erythrocyte and thrombocyte numbers in mice treated with TP508. This result suggests that TP508 stimulates hematopoiesis or protects bone marrow cells (BMCs).
- CBC complete blood count
- Histology of bone marrow 8 days after exposure to 8 Gy shows depletion of BMCs in 870 marrow of 8 Gy exposed mice relative to 0 Gy mice.
- TP508 treatment of these mice increases number and density of BMCS.
Abstract
Description
Claims
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AU2011265228A AU2011265228A1 (en) | 2010-06-11 | 2011-06-10 | Methods of mitigating effects of radiation and reducing the risk of systemic infection |
EP11728735.9A EP2579891A2 (en) | 2010-06-11 | 2011-06-10 | Methods of mitigating effects of radiation and reducing the risk of systemic infection |
JP2013514392A JP2013529600A (en) | 2010-06-11 | 2011-06-10 | How to reduce the effects of radiation and reduce the risk of systemic infection |
CA2802176A CA2802176A1 (en) | 2010-06-11 | 2011-06-10 | Methods of mitigating effects of radiation and reducing the risk of systemic infection |
US13/711,291 US20130101574A1 (en) | 2010-06-11 | 2012-12-11 | Methods of using thrombin peptide derivatives |
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US5352664A (en) | 1986-10-31 | 1994-10-04 | Board Of Regents, The University Of Texas System | Thrombin derived polypeptides; compositions and methods for use |
US20050153893A1 (en) | 2002-07-02 | 2005-07-14 | The Board Of Regents, The University Of Texas System | Thrombin peptide derivative dimers |
US20050158301A1 (en) | 2002-07-02 | 2005-07-21 | The Board Of Regents, The University Of Texas System | Thrombin peptide derivatives |
US7294596B2 (en) | 2005-03-14 | 2007-11-13 | Diamorph Ceramic Ab | Sintered ceramic material with improved properties and method for its manufacturing |
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US8334259B2 (en) * | 2006-09-22 | 2012-12-18 | The Board Of Regents, The University Of Texas System | Method of treating endothelial dysfunction comprising administration of a thrombin peptide derivative |
US20100330028A1 (en) * | 2007-04-10 | 2010-12-30 | The Board Of Regents The Universityof Texas System | Combination therapy for chronic dermal ulcers |
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2011
- 2011-06-10 WO PCT/US2011/040006 patent/WO2011156729A2/en active Application Filing
- 2011-06-10 JP JP2013514392A patent/JP2013529600A/en not_active Withdrawn
- 2011-06-10 EP EP11728735.9A patent/EP2579891A2/en not_active Withdrawn
- 2011-06-10 CA CA2802176A patent/CA2802176A1/en not_active Abandoned
- 2011-06-10 AU AU2011265228A patent/AU2011265228A1/en not_active Abandoned
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4132948A4 (en) * | 2020-04-07 | 2024-01-03 | Chrysalis Biotherapeutics | Tp508 acute therapy for patients with respiratory virus infection |
Also Published As
Publication number | Publication date |
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AU2011265228A1 (en) | 2013-05-23 |
EP2579891A2 (en) | 2013-04-17 |
CA2802176A1 (en) | 2011-12-15 |
JP2013529600A (en) | 2013-07-22 |
WO2011156729A3 (en) | 2012-05-10 |
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