WO2010061915A1 - 血管新生誘導活性及び抗菌活性を有する新規ポリペプチド並びにその医薬用途 - Google Patents
血管新生誘導活性及び抗菌活性を有する新規ポリペプチド並びにその医薬用途 Download PDFInfo
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- WO2010061915A1 WO2010061915A1 PCT/JP2009/070035 JP2009070035W WO2010061915A1 WO 2010061915 A1 WO2010061915 A1 WO 2010061915A1 JP 2009070035 W JP2009070035 W JP 2009070035W WO 2010061915 A1 WO2010061915 A1 WO 2010061915A1
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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
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
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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
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
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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
- C07K14/475—Growth factors; Growth regulators
- C07K14/515—Angiogenesic factors; Angiogenin
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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
Definitions
- the present invention relates to a novel polypeptide having angiogenesis-inducing activity and antibacterial activity, an angiogenesis-inducing agent containing the same, a skin wound therapeutic agent, and a skin wound infection preventive agent.
- Neovascularization is effective in the treatment of various diseases or injuries such as burns, pressure ulcers, wounds, skin ulcers, lower limb ulcers, diabetic ulcers, occlusive arterial disease and occlusive arteriosclerosis.
- diseases or injuries such as burns, pressure ulcers, wounds, skin ulcers, lower limb ulcers, diabetic ulcers, occlusive arterial disease and occlusive arteriosclerosis.
- an angiogenesis inducing agent having both antibacterial activity and angiogenesis inducing activity is required.
- a wound therapeutic agent having antibacterial activity is also effective for skin wounds accompanied by bacterial infection. Furthermore, it is desirable to use a wound therapeutic agent having antibacterial activity in the sense of preventing future infection even for a wound not accompanied by infection.
- LL-37 is known as a polypeptide having angiogenesis-inducing activity and antibacterial activity. (Non-Patent Documents 1 and 2).
- Nakagami et al. Invented a polypeptide having vascular endothelial growth activity, and thus angiogenesis-inducing activity, and further found that the peptide has a peptide having angiogenesis-inducing activity higher than LL-37.
- a patent application was filed for this (Patent Document 1).
- Nakagami et al. Found a polypeptide AG30-5C consisting of 30 amino acid residues having higher angiogenesis-inducing activity than the peptide according to Patent Document 1, and applied for a patent (Patent Document 2).
- Patent Documents 1 and 2 also suggest that the polypeptide has antibacterial activity.
- a preparation (Fiblast Spray (registered trademark)) containing basic FGF consisting of 154 amino acids (bFGF) as an active ingredient is known.
- bFGF basic FGF consisting of 154 amino acids
- the object of the present invention includes a novel polypeptide having angiogenesis-inducing activity and antibacterial activity that is even better than known polypeptides, and a novel angiogenesis-inducing agent comprising the same as an active ingredient, skin ulcer, and refractory wound It is to provide a therapeutic agent for skin wounds and a therapeutic agent for skin wounds associated with infection.
- the present invention provides the following.
- An angiogenesis inducer comprising the polypeptide according to (1) as an active ingredient.
- An antibacterial agent comprising the polypeptide according to (1) as an active ingredient.
- a method for inducing angiogenesis comprising administering an effective amount of the polypeptide according to (1) to a mammal in need of angiogenesis.
- a polypeptide for inducing angiogenesis which is the polypeptide according to (1).
- a preventive, ameliorating or treating agent for skin wound comprising the polypeptide according to (1) as an active ingredient.
- Skin wound consists of cut wound, surgical wound, erosion, burn, pressure ulcer, refractory wound, skin ulcer, leg ulcer, diabetic ulcer, occlusive arterial disease and occlusive arteriosclerosis A preventive, ameliorating or treating agent for a disease selected from the group.
- a preventive, ameliorating or treating agent for bacterial infection in skin wounds comprising the polypeptide according to (1) as an active ingredient.
- a method for preventing, improving or treating a skin wound or bacterial infection in a skin wound comprising administering an effective amount of the polypeptide according to (1) to a mammal.
- a polypeptide for preventing, ameliorating or treating bacterial infection in a skin wound or skin wound which is the polypeptide according to (1).
- a novel polypeptide having excellent angiogenesis-inducing activity and antibacterial activity has been provided.
- a novel angiogenesis-inducing agent comprising this polypeptide as an active ingredient, a wound, a surgical wound, an erosion, various skin ulcers, a therapeutic agent for skin wounds including intractable wounds, and a therapeutic agent for skin wounds associated with infection sponsored. Since the wound therapeutic agent of the present invention has both an effect of preventing and / or treating infection and an effect of promoting wound tissue regeneration, it is excellent as a therapeutic agent for skin wound therapeutic agents.
- 25 ⁇ g / mL, 250 ⁇ g / mL and 2.5 mg / mL mean doses of 1.0 ⁇ g / disc, 10 ⁇ g / disc and 100 ⁇ g / disc, respectively. It is a figure which shows the measurement result of the granulation tissue formation ability by the paper disk model which impregnated SR-3 which is one of the polypeptides of the present invention.
- 25 ⁇ g / mL, 250 ⁇ g / mL and 2.5 mg / mL mean doses of 1.0 ⁇ g / disc, 10 ⁇ g / disc and 100 ⁇ g / disc, respectively.
- 25 ⁇ g / mL, 250 ⁇ g / mL and 2.5 mg / mL mean doses of 1.0 ⁇ g / disc, 10 ⁇ g / disc and 100 ⁇ g / disc, respectively. It is a figure which shows the measurement result of wound wound tension
- amino acid sequence of the polypeptide of the present invention is represented by each of SEQ ID NOs: 1 to 6. All of these polypeptides are novel substances.
- polypeptide AG30-5C described in Patent Document 2 is shown in SEQ ID NO: 7.
- AG30-5C has antibacterial activity and angiogenesis-inducing activity, as specifically described in Patent Document 2 and the following examples. It became clear that the above-mentioned polypeptide of the present invention has angiogenesis-inducing activity and antibacterial activity. And as specifically described in the following examples, the polypeptides according to the examples of the present invention had higher angiogenesis-inducing activity and antibacterial activity than AG30-5C.
- a sugar chain or a polyethylene glycol (PEG) chain is added to the polypeptide or at least a part of the amino acids constituting the polypeptide in order to increase the stability of the polypeptide in vivo.
- a technique using a D-form amino acid is widely known and used. By adding sugar chains or PEG chains, or by using D-form amino acids as at least part of the amino acids that make up the polypeptide, it is less susceptible to degradation by peptidases in vivo, reducing the polypeptide in vivo by half. The period becomes longer. It is also well known that peptide stability is improved by N-terminal acetylation and / or C-terminal amidation of peptides.
- polypeptide of the present invention has antibacterial activity, it may be those subjected to these known modifications for in vivo stabilization, and the term “polypeptide” in the present specification and claims. Is used in the sense of including those that have been modified for in vivo stabilization, unless otherwise apparent from the context.
- the amino acid sequence of the polypeptide portion is represented by any one of SEQ ID NOs: 1 to 6 as described above (however, the acetylated or amidated amino acid described above) Is considered to be the same amino acid as an amino acid that is not acetylated or amidated), and therefore, the stabilized modified polypeptide is produced in a polypeptide comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 6 PEG-like structure added for stabilization in the body, at least some of its amino acids changed to D-form, and acetylated and / or amidated at the N-terminus of the polypeptide As well as polypeptides modified by combinations of these modifications.
- Glycosylation to polypeptides is well known, for example, Sato M, Furuike T, Sadamoto R, Fujitani N, Nakahara T, Niikura K, Monde K, Kondo H, Nishimura S., "Glycoinsulins: dendritic sialylolulins displaying a prolonged blood-sugar-lowering activity. ", J Am Chem Soc. 2004 Nov 3; 126 (43): 14013-22, Sato M, Sadamoto R, Niikura K, Monde K, Kondo H, Nishimura S," Site- specific introduction of sialic acid into insulin. ", Angew Chem Int Ed Engl. 2004 Mar 12; 43 (12): 1516-20.
- the sugar chain can be bound to the N-terminus, C-terminus, or an amino acid therebetween, but is preferably bound to the N-terminus or C-terminus so as not to inhibit the activity of the polypeptide.
- the number of sugar chains to be added is preferably 1 or 2, and more preferably 1.
- the sugar chain is preferably monosaccharide to tetrasaccharide, more preferably disaccharide or trisaccharide.
- the sugar chain can be bound to a free amino group or carboxyl group of the polypeptide directly or via a spacer structure such as a methylene chain having about 1 to 10 carbon atoms.
- the PEG chain can be attached to the N-terminus, C-terminus, or the amino acid between them, and usually one or two PEG chains are attached to a free amino group or carboxyl group on the polypeptide.
- the molecular weight of the PEG chain is not particularly limited, but is usually 3000 to 7000. Degrees, preferably those of about 5000 is used.
- a method in which at least a part of amino acids constituting a polypeptide is in D form is also known, for example, Brenneman DE, Spong CY, Hauser JM, Abebe D, Pinhasov A, Golian T, Gozes I. orally active and mechanistically nonchiral. ", J Pharmacol Exp Ther. 2004 Jun; 309 (3): 1190-7 and Wilkemeyer MF, Chen SY, Menkari CE, Sulik KK, Charness ME.,” Ethanol antagonist peptides: structural specificity without "J. Pharmacol. Exp. Ther. 2004. Jun; 309 (3): 1183-9. Although some of the amino acids constituting the polypeptide may be D-form, it is preferable that all of the amino acids constituting the polypeptide are D-form amino acids in order not to inhibit the activity of the polypeptide as much as possible.
- the polypeptide which is an active ingredient of the angiogenesis inducer of the present invention can be easily produced by a conventional method such as chemical synthesis using a commercially available peptide synthesizer.
- the stabilization modification can also be easily performed by a known method as described in each of the above documents.
- polypeptide of the present invention Since the polypeptide of the present invention has a high angiogenesis-inducing activity, it can be used as an angiogenesis-inducing agent and also as a skin wound treatment agent.
- angiogenesis-inducing agent refers to a drug capable of inducing and / or increasing angiogenesis at the administration site and its periphery when locally administered to a living body.
- the induction of angiogenesis can be evaluated by a known method such as blood flow measurement at the relevant site.
- induction and / or increase of angiogenesis promotes granulation, regeneration of skin tissue, and promotes healing.
- antibacterial agent has the effect of killing already infected bacteria or suppressing or reducing their growth when administered to a living body, or the effect of suppressing or preventing bacterial infection. Refers to a drug.
- skin wound therapeutic agent refers to a drug that, when locally applied to and / or around a wound site, promotes healing or improvement of the wound or prevents or delays deterioration. Promoting wound healing or improvement refers to the early reduction of the wound site area or the promotion of granulation and / or skin tissue regeneration at the wound site.
- Angiogenesis-inducing agent and skin wound therapeutic agent and prevention, improvement or treatment of skin wound described later, and use as a polypeptide polypeptide blood vessel for prevention, improvement or treatment of bacterial infection in skin wound described later
- a solution, emulsion, suspension, powder, powder, granule, gel, ointment, or transdermal patch particularly preferably as a solution, powder, or transdermal patch.
- a solution dissolved in an aqueous medium such as a buffer solution, particularly preferably a physiological saline buffer can be preferably administered.
- the concentration of the polypeptide in the solution is not particularly limited, but is usually about 0.01 mg / mL to 100 mg / mL, preferably about 0.1 mg / mL to 50 mg / mL, particularly preferably about 1 mg / mL to 10 mg / mL. is there.
- the route of administration is usually local administration such as spraying, application or injection to the site where angiogenesis or wound healing or improvement is needed.
- the dosage is appropriately selected depending on the symptoms and the size of the affected area, but is usually about 0.01 mg to 100 mg, preferably about 0.005 mg to 50 mg, particularly preferably about 0.005 mg to 0.5 mg as the amount of polypeptide. Of course, it is not limited to these ranges. For example, it can be about 0.05 mg per dose.
- the dose can be set according to the area of the site requiring angiogenesis or wound healing or improvement, and can be, for example, 1 to 100 ⁇ g / cm 2 .
- a carrier commonly used in the pharmaceutical field can be used in addition to the above-mentioned aqueous medium.
- external preparations such as ointments, hydrocarbons (hydrophilic petrolatum, white petrolatum, purified lanolin, liquid paraffin, etc.), zinc oxide, higher fatty acids and esters thereof (adipic acid, myristic acid, palmitic acid, stearic acid) Oleic acid, adipic acid ester, myristic acid ester, palmitic acid ester, diethyl sebacate, hexyl laurate, cetyl isooctanoate, waxes (whale wax, beeswax, ceresin, etc.), higher alcohols (cetanol, stearyl alcohol, Cetostearyl alcohol, etc.).
- a solution for example, water, physiological saline, phosphate buffered saline and the like can be mentioned, and in the case of an oral preparation, lactose, starch and the like can be mentioned.
- various pharmaceutical additives such as an emulsifier, a surfactant, a tonicity agent, and a pH adjuster can be blended as necessary.
- diseases and disorders when administered to a living body include burns, pressure ulcers, wounds, skin ulcers, lower limb ulcers, diabetic ulcers, occlusive arterial diseases and occlusive arteriosclerosis. It is not limited.
- the angiogenesis-inducing agent of the present invention can be used as a preventive, ameliorating or therapeutic agent for these diseases or disorders.
- the polypeptide of the present invention has high angiogenesis-inducing activity and also has antibacterial activity.
- Gram-positive bacteria such as Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus), Staphylococcus epidermidis, and Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, and enteric bacteria.
- Gram-positive bacteria such as Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus), Staphylococcus epidermidis, and Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, and enteric bacteria.
- a disease or disorder desired to be concomitantly used such as the above-mentioned diseases or disorders, burns, pressure sores, wounds, skin ulcers, lower limb ulcers, diabetes Ulcers and the like.
- skin wound refers to physical damage to the body surface tissue caused by external or internal factors, and includes cuts, lacerations, stab wounds, bites, Includes wounds resulting from gunshot wounds, wounds, abrasions, surgical wounds, erosions, pressure ulcers, intractable wounds, skin ulcers, leg ulcers, diabetic ulcers, occlusive arterial disease and obstructive arteriosclerosis.
- the polypeptide of the present invention can be used for treatment as a wound treatment agent, antibacterial agent, or infection prevention agent.
- SR-1 SEQ ID NO: 1
- SR-2 SEQ ID NO: 2
- SR-3 SEQ ID NO: 3
- SR-4 SEQ ID NO: 4
- SR-5 SEQ ID NO: 5
- SR-6 sequence
- Any peptide of No. 6 can be used for the treatment, but SR-1 and SR-3 are more preferred, and SR-1 is particularly preferred.
- the polypeptide of the present invention has two effects of healing and prevention of infection and wound healing with one active ingredient.
- the polypeptide of the present invention exhibits a wound healing effect comparable to a drug (fiblast spray (registered trademark), referred to herein as a bFGF preparation) containing bFGF as an active ingredient, which has already been used as a wound treatment agent. sell.
- a drug fiblast spray (registered trademark), referred to herein as a bFGF preparation
- bFGF has no antibacterial action
- the polypeptide of the present invention has an antibacterial action, and therefore can be a more effective therapeutic agent for skin wounds associated with infection.
- the polypeptide of the present invention has an early healing effect in wounds with infection compared to bFGF preparations.
- the examples herein illustrate the effect of SR-1 polypeptides on wounds with S.
- aureus infections but are not limited to the examples and any of the polypeptides of the present invention Has the same effect against Staphylococcus aureus and other bacteria, especially those for which the polypeptides of the present invention exhibit antibacterial properties, such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Neisseria pneumoniae and enteric bacteria. Can be easily understood by those skilled in the art. Further, even when a polypeptide of the present invention is administered to a wound in which no infection is observed, it is also possible to prevent bacterial infection and exhibit an early healing effect, as in the case of this example. It can be easily understood by a contractor.
- the polypeptides of the present invention are incisions, surgical wounds, erosions, burns, pressure sores, refractory wounds, skin ulcers, leg ulcers, diabetic Can be used to treat or ameliorate any skin wound, including skin wounds caused by ulcers, occlusive arterial disease and occlusive arteriosclerosis, without any infection, and also treat any wound with infection Or it can be used for improvement.
- These polypeptides also have the effect of preventing infection against wounds that can cause infection.
- treatment or improvement of a wound includes healing of the wound, reduction of the degree of wound, and suppression of wound deterioration. Since the polypeptide of the present invention can prevent bacterial infection at the wound site, it can suppress the deterioration of the wound.
- blisters such as infectious impetigo and pemphigus, burns, and It is skin damage to the epidermis caused by atopic dermatitis scratching, etc., and refers to a state where the surface formed after blistering is moistened.
- an “ulcer” is known to those skilled in the art and as described in general medical technical books. Generally speaking, it is skin damage that reaches tissues below the dermis. Compared to a deep wound. In many cases, bleeding or exudate is observed. This ulcer may occur following a disease with blood circulation such as collagen disease, diabetes or vasculitis, or a malignant tumor, but also includes pressure ulcers such as burn ulcers and pressure ulcers (bed sores). Scars called scars often remain after healing.
- Intractable wounds include diabetic ulcers, leg ulcers, burn ulcers, pressure ulcers (bed sores), pressure ulcers, necrosis, and the like. These ulcers and necrosis are likely to be accompanied by a bacterial infection, which causes criticism. Therefore, it is desirable that treatment or improvement of these wounds promotes regeneration of skin tissue at the wound site through granulation and prevents and treats bacterial infection.
- the polypeptide of the present invention When used as a preventive, ameliorating or therapeutic agent for bacterial infection (including secondary infection) of wounds, the prevention, amelioration or therapeutic effect of infection and / or angiogenic effect or wound healing of the polypeptide The effect provides early prevention, improvement or treatment effect of the wound.
- Bacterial infections of wounds include various bacterial infections, and in particular, Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus), Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae. It is therapeutically desirable to administer drugs having antibacterial properties to these after infection, but administration before infection for the purpose of preventing infection is also very useful for wound treatment.
- polypeptide of the present invention is more desirable from the cosmetic point of view because scars hardly remain compared to bFGF preparations widely used as wound healing agents.
- the angiogenesis inducer, antibacterial agent, skin wound treatment agent or skin wound infection preventive agent of the present invention can be used alone or in combination of two or more of the polypeptides of the present invention. Further, when further antibacterial properties are desired, it can be used in combination with other antibacterial agents or antibiotics.
- these antibacterial agents or antibiotics include cephem, carbapenem, aminoglycosito, new quinolone, ⁇ -lactam, penicillin and glycopeptide antibiotics, and more specifically , Ceftazidime, meropenem, tobramycin, ciprofloxacin, methicillin, ampicillin, vancomycin and the like, but are not limited thereto.
- This crude peptide was purified using a reverse phase HPLC column (ODS) by elution with a gradient of 0.1% TFA-H 2 O / CH 3 CN. Fractions containing the desired product were collected and lyophilized to obtain the desired peptide.
- the amino acid sequence of the synthesized peptide was confirmed using the amino acid sequencer G1000A (Hewlett Packard), PPSQ-23A (Shimadzu Corporation) or ProciscLC (ABI). The sequence of the peptide is shown below. SR-2, SR-3, SR-4 and SR-6 were amidated at the C-terminus.
- SR-1 (SEQ ID NO: 1) MLKLIFLHRLKRMRKRLKRK SR-2 (SEQ ID NO: 2) ELRFLHRLKRRLRKRLKRKLR-amide SR-3 (SEQ ID NO: 3) ELRFLHRLKRMRKRLKRKLR-amide SR-4 (SEQ ID NO: 4) KLIFLHRLKRMRKRLKRKLR-amide SR-5 (SEQ ID NO: 5) KRMRKRLKRKLRLWHRKRYK SR-6 (SEQ ID NO: 6) MRKRLKRKLRLWHRKRYK-amide AG30-5C (SEQ ID NO: 7) MLKLIFLHRLKRMRKRLKRKLRFWHRKRYK
- MALDI-TOF / MS conditions Laser intensity: 2100 Number of irradiations: 1000
- Results Table 1 shows the theoretical and actual MALDI-TOF / MS values of each polypeptide.
- the detected m / z of each polypeptide agreed with the respective theoretical value, and the sequence of the synthesized polypeptide could be confirmed.
- Angiogenesis-inducing activity of the polypeptide The angiogenesis-inducing activity of SR-1 and SR-2 was measured using AG30-5C as a positive target. Specifically, the ability of the polypeptide to form a lumen was evaluated using an angiogenesis kit (KZ-1000, Kurashiki Boseki Co., Ltd.). A group without the polypeptide (control) was used as a negative control.
- the primary antibody (mouse anti-human CD31 antibody) was diluted 4000 times with a blocking solution (Dulbeccoline buffer solution (PBS ( ⁇ )) containing 1% BSA). 0.5 ⁇ mL of this primary antibody solution was added to each well and incubated at 37 ° C. for 60 minutes. After the incubation, each well was washed 3 times with 1 mL of blocking solution.
- a blocking solution Dulbeccoline buffer solution (PBS ( ⁇ )
- PBS ( ⁇ ) Dulbeccoline buffer solution
- BSA 1% BSA
- a secondary antibody solution (goat anti-mouse IgG alkaline phosphatase complex) diluted 500-fold with blocking solution was added to each well, incubated at 37 ° C. for 60 minutes, and then washed three times with 1 mL of distilled water. .
- two BCIP / NBT tablets were dissolved in distilled water and filtered through a filter having a pore size of 0.2 ⁇ m to prepare a substrate solution.
- 0.5 ⁇ mL of the prepared BCIP / NBT substrate solution was added to each well and incubated at 37 ° C. until the lumen became deep purple (usually 5-10 minutes). After the incubation, each well was washed 3 times with 1 mL of distilled water. After washing, the washing solution was removed by suction and left to stand for natural drying. After drying, photographs of each well were taken under a microscope.
- Each image obtained was quantified using angiogenesis quantification software. Computer analysis was performed with various indices, and the length of the lumen formed in each visual field was measured based on this scale, and the effect of adding the polypeptide to the control was evaluated.
- both the polypeptides SR-1 and SR-2 of the present invention had angiogenesis-inducing activity. Its activity was higher than AG30-5C.
- Antimicrobial activity of polypeptides (1) The antimicrobial activity of the polypeptides was measured using ATP assay.
- the antibacterial activity of the peptide was evaluated from the viability of the bacteria using the PROMEGA BacTiter-Glo Microbial Cell Viability Assay kit. That is, using a microtiter plate or a test tube, the amount of ATP in live bacteria at a peptide concentration of 10 ⁇ g / mL was measured.
- Staphylococcus aureus ATCC29213 (S. aureus ATCC29213) was used as a gram-positive bacterium, or P. aeruginosa ATCC27853 was used as a gram-negative bacterium.
- the bacteria were cultured in the medium for 3 to 4 hours, and then the absorbance at A 600 was measured. According to McFarland # 0.5, the bacterial solution was diluted with Mueller-Hinton broth (MHB). Each strain was added so as to be about 0.5-1 ⁇ 10 5 CFU / mL (final concentration) in terms of E. coli.
- Each peptide was prepared and dispensed to a final concentration of 10 ⁇ g / mL in a microplate or test tube, and a bacterial solution was added.
- the one without peptide was used as a negative control, and tobramycin (TOB) was used as a positive control.
- TOB tobramycin
- the plate was cultured at 37 ° C. for 3 hours, and the amount of ATP in the culture was measured. A relative value was obtained by comparison with the negative control, and was used as the survival rate.
- polypeptides SR-1, SR-2, SR-3, SR-4 and SR-6 of the present invention exhibit superior antibacterial activity against Staphylococcus aureus than AG30-5C. It was. Furthermore, SR-2 and SR-3 showed superior antibacterial activity against Pseudomonas aeruginosa than AG30-5C.
- MIC minimum drug concentration at which fungal growth is inhibited, and is used as an indicator of the efficacy of antibacterial agents, the strength of bacterial sensitivity, etc. If it is more than this, it is the density
- CLSI CLINICAL AND LABORATORY STANDARD INSTITUTE
- the absorbance at A 600 was measured. Diluted with Mueller-Hinton broth (MHB) according to McFarland # 0.5. Each strain was added to a concentration of about 10 5 CFU / ml (final concentration). Each peptide was prepared at an arbitrary concentration, and serial dilution was performed therefrom. The bacterial solution was added to a microplate or test tube into which the polypeptide at each concentration step was dispensed. Those without peptide were used as negative controls, meropenem (MEPM), ciprofloxacin (CPFX), tobramycin (TOB), and oxacillin (OX) as positive controls. The plate was incubated at 37 ° C. for 20 hours, and the minimum concentration at which growth was inhibited was defined as the minimum growth inhibition concentration.
- MPM Mueller-Hinton broth
- CPFX ciprofloxacin
- TOB tobramycin
- OX oxacillin
- the respective strains are: E. coli: ATCC 25922 (Escherichia coli ATCC 25922), Pseudomonas aeruginosa: ATCC 27853 (Pseudomonas aeruginosa ATCC 27853), Staphylococcus aureus: ATCC29213 (Staphylococcus aureus ATCC29213), Pneumoniae bacterium: JCMb166ella le ), Enterobacteria (1): JCM 1232 (Enterobacter cloacae 123 JCM 1232), Enterobacteria (2): JCM 1235 (Enterobacter aerogenes JCM 1235), Staphylococcus epidermidis: JCM 2414 (Staphylococcus epidermidis JCM 2414).
- Tube formation An angiogenesis kit (product number: KZ-1000) of Kurashiki Boseki Co., Ltd. was used to evaluate the tube formation of the polypeptide.
- Vascular endothelial growth factor A (VEGF-A) was used as a positive control, and an unstimulated group without polypeptide was used as a negative control.
- the concentration of each polypeptide was adjusted to 0.5, 2.5, and 10 ⁇ g / mL. It was added angiogenic dedicated medium containing polypeptide in the kit in 24-well to the attached cells were seeded (cell cocultured with optimal concentrations of human vascular endothelial cells and fibroblasts), 37 °C, 5% CO 2 Cultured under conditions. On the 4th, 7th and 9th days of culture, the medium was replaced with the same additive. On the 11th day from the start of culture, staining was performed using a lumen staining kit (for CD31 antibody staining) according to the following procedure.
- the medium was removed, and after washing with Dulbecco's phosphate buffer (PBS (-)), ice-cooled 70% ethanol was added to fix the cells.
- PBS (-) Dulbecco's phosphate buffer
- a well was washed with blocking solution (Dulbecco phosphate buffer (PBS (-)) containing 1% BSA), and CD31-stained primary antibody (mouse anti-human CD31 antibody) diluted 4000 times with blocking solution was added to each well. After addition of 0.5 mL, the mixture was incubated for 60 minutes at 37 ° C. After completion of the incubation, each well was washed 3 times with 1 mL of blocking solution, and then secondary antibody solution (goat anti-mouse IgG alkaline diluted 500 times with blocking solution).
- Phosphatase complex 0.5 mL was added to each well, incubated for 60 minutes at 37 ° C, then washed 3 times with 1 mL distilled water, during which time two BCIP / NBT tablets were dissolved in distilled water, Prepare a substrate solution by filtering through a 0.2 ⁇ m pore size filter, add 0.5 ⁇ mL of the prepared BCIP / NBT substrate solution to each well, and incubate at 37 ° C. until the lumen becomes deep purple (usually 5-10 minutes). End of incubation After completion, each well was washed three times with 1 mL of distilled water, and after washing, the washing solution was removed by suction and left to stand to dry naturally.After drying, a photograph of each well was taken under a microscope.
- Each image obtained was quantified using angiogenesis quantification software. Measure the area and length of the lumen formed in each field of view based on the scale of Kurashiki Boseki's angiogenesis quantification software (angiogenesis quantification software Ver 1.0). It was evaluated with.
- Table 6-1 luminal area
- Table 6-2 luminal length
- the five polypeptides SR-1-5 were evaluated for serum stability.
- the peptide is dissolved in human serum (purchased from KAC Co., Ltd.) or rat serum obtained by centrifugation at room temperature after blood collection, and allowed to stand at 37 ° C., to a final concentration of 500 ⁇ g / mL. did. After standing for 10 minutes, 30 minutes or 60 minutes, the product was subjected to HPLC chromatogram analysis to detect a degradation product peak, and the ratio of the peptide concentration remaining in the solution to the starting concentration was calculated. In addition, the half-life in serum of each peptide was calculated.
- SR-1 and SR-5 were completely degraded within 1 hour, whereas SR-2, SR-3, and SR-4 were degraded 35-55% at 37 degrees 1 hour. Stayed in. In human serum, over 90% of all peptides were found to be degraded within 1 hour.
- each polypeptide (SR-1 to 5) in PBS was also confirmed.
- the peptide PBS solution with a final concentration of 1 mg / mL was stored at 4 ° C and room temperature.
- the solution was subjected to HPLC analysis under the same conditions as described above except that the injection volume was 5 ⁇ L, and the storage stability in the solution was evaluated by the presence or absence of the degradation product peak. As a result, no degradation product was observed, and it was found that any polypeptide had sufficient storage stability in PBS at both room temperature and 4 ° C.
- SR-1 and SR-3 are 10 ⁇ g / disk (250 ⁇ g / mL x 40 The largest increase in granulation tissue weight was shown at a dose of ⁇ l / disc). From these results, it is considered that SR-1 and SR-3 particularly contribute to granulation tissue formation by promoting angiogenesis even in local administration in vivo.
- a 30-36 mm cut was created with a safety razor on the back of a Crl: CD (SD) rat (male, 7 weeks old, obtained from Charles River Japan), and sutured at three locations at equal intervals. did.
- the day on which this surgical procedure was performed was defined as the first day (day 0).
- a negative subject (Saline) was added with 50 ⁇ L of physiological saline.
- each peptide was dripped once a day until the wound tension measurement day. The yarn was removed on the third day (day ⁇ 3), and the tensile strength was measured on the sixth day (day 6).
- SR-1, SR-3, SR-4 and SR-5 showed the effect of increasing wound tension.
- the bFGF preparation in the individual administered any of the polypeptides of the present invention, particularly SR-1, SR-2, SR-3 and SR-4, the bFGF preparation The scar of the incision clearly disappeared compared to the administered solid, and scar formation was hardly observed by visual observation (data not shown).
- Rat wound wound models include Stenberg BD et al. (J Surg Res. 1991 Jan; 50 (1): 47-50.) And Hayward P et al. (Am J Surg. 1992 Mar; 163 (3): 288-93.) For reference, it was prepared as follows. HWY / slc hairless rats (male, 7 weeks old, obtained from Nippon SLC Co., Ltd.) were used.
- the white blood cell count was measured and cyclophosphamide (100 mg / kg) was administered into the tail vein the day before the full-thickness defect was produced, and the white blood cell count was counted the next day, and only individuals with a white blood cell count of 5000 or less were used.
- wound healing was delayed in the group in which infection was induced in the wound compared to the non-infected group.
- the wound epidermis decreased more quickly and healed faster in the group administered SR-1 to the infected wound than when the bFGF preparation was administered to the infected wound.
- Efficacy equivalent to or better than that of bFGF preparation was observed. From the above, it was found that SR-1 showed a higher healing effect than the bFGF preparation for wounds with infection.
- the polypeptide of the present invention is useful as an angiogenesis-inducing agent, an antibacterial agent, a skin wound prevention, improvement or treatment agent, and a bacterial infection prevention, improvement or treatment agent in skin wounds.
- the C-terminal arginine residue is modified by amidation.
- the C-terminal arginine residue is modified by amidation.
- the C-terminal arginine residue is modified by amidation.
- the C-terminal lysine residue is modified by amidation.
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Abstract
Description
(1) アミノ酸配列が配列番号1ないし6のいずれかで表されるポリペプチド。
(2) (1)に記載のポリペプチドを有効成分として含有する血管新生誘導剤。
(3) 前記ポリペプチドのアミノ酸配列が、配列番号1、2又は3で表わされる(2)記載の血管新生誘導剤。
(4) (1)に記載のポリペプチドを有効成分として含有する抗菌剤。
(5) 有効量の(1)に記載のポリペプチドを、血管新生が必要な哺乳動物に投与することを含む、血管新生誘導方法。
(6) (1)に記載のポリペプチドの、血管新生誘導剤の製造のための使用。
(7) (1)に記載のポリペプチドである、血管新生誘導用ポリペプチド。
(8) (1)に記載のポリペプチドを有効成分として含有する、皮膚創傷の予防、改善又は治療剤。
(9) 皮膚創傷が、切創、手術創、びらん、熱傷、褥瘡、難治性創傷、皮膚潰瘍、下肢潰瘍、糖尿病性潰瘍、閉塞性動脈疾患及び閉塞性動脈硬化症に起因する皮膚創傷から成る群から選択される疾患の予防、改善又は治療剤。
(10) 皮膚創傷が、難治性創傷である、(9)記載の予防、改善又は治療剤。
(11) 皮膚創傷が、感染を伴う創傷である、(9)記載の予防、改善又は治療剤。
(12) (1)に記載のポリペプチドを有効成分として含有する、皮膚創傷における細菌感染の予防、改善又は治療剤。
(13) 有効量の(1)に記載のポリペプチドを哺乳動物に投与することを含む、皮膚創傷または皮膚創傷における細菌感染の予防、改善又は治療方法。
(14) (1)に記載のポリペプチドの皮膚創傷または皮膚創傷における細菌感染の予防、改善又は治療剤の製造のための使用。
(15) (1)に記載のポリペプチドである、皮膚創傷または皮膚創傷における細菌感染の予防、改善又は治療用ポリペプチド。
さらに、感染が認められない創傷に対して本発明のポリペプチドの投与後、細菌と接触させた場合でも、該実施例と同様、細菌感染を予防し、早期治癒効果を示すこともまた、当業者であれば容易に理解できる。
文献Solid Phase Peptide Synthesis, Pierce (1984)、Fmoc solid synthesis:a practical approach, Oxford University Press(2000)および第5版 実験化学講座16 有機化合物の合成IV等に記載の方法に従い、全自動固相合成機を用いて、保護ペプチド樹脂をFmoc法で合成した。得られた保護ペプチド樹脂にトリフルオロ酢酸(TFA)とスカベンジャー(チオアニオール、エタンジチオール、フェノール、トリイソプロピルシラン、水などの混合物)を加えて、樹脂から切り出すとともに脱保護して、粗ペプチドを得た。この粗ペプチドを、逆相HPLCカラム(ODS)を用いて、0.1%TFA-H2O/CH3CNの系でグラジエント溶出し、精製を行った。目的物を含む分画を集め凍結乾燥して、目的のペプチドを得た。合成したペプチドのアミノ酸配列は、アミノ酸シーケンサーG1000A(Hewlett Packard)、PPSQ-23A(島津製作所)又はProciscLC(ABI社)を用いて確認した。ペプチドの配列を以下に示す。SR-2、SR-3、SR-4およびSR-6はC末端をアミド化した。
MLKLIFLHRLKRMRKRLKRK
SR-2(配列番号2)
ELRFLHRLKRRLRKRLKRKLR-amide
SR-3(配列番号3)
ELRFLHRLKRMRKRLKRKLR-amide
SR-4(配列番号4)
KLIFLHRLKRMRKRLKRKLR-amide
SR-5(配列番号5)
KRMRKRLKRKLRLWHRKRYK
SR-6(配列番号6)
MRKRLKRKLRLWHRKRYK-amide
AG30-5C(配列番号7)
MLKLIFLHRLKRMRKRLKRKLRFWHRKRYK
合成したポリペプチドの配列をMALDI-TOF/MSを用いた分析結果により確認した。最終濃度100μg/mLのポリペプチド0.1% TFA/50% アセトニトリル溶液 1μLにマトリクス溶液(α-シアノ4-ヒドロキシ桂皮酸(α-Cyano 4-Hydroxy Cinnamic Acid))1μLを加え、MALDI用測定試料とした。MALDI用測定試料(0.4μL)をMALDIターゲットプレートに塗布し、乾燥させた。MALDI-TOF/MSを用いて測定した。
レーザー強度: 2100
照射回数: 1000
各ポリペプチドのMALDI-TOF/MSの理論値及び実測値を表1に示した。各ポリペプチドの検出されたm/zは、それぞれの理論値と一致し、合成したポリペプチドの配列を確認することができた。
AG30-5Cを陽性対象として、SR-1およびSR-2の血管新生誘導活性を測定した。具体的には、血管新生キット(Angiogenesis Kit, KZ-1000, 倉敷紡績株式会社)を用いてポリペプチドの管腔形成能を評価した。陰性対照としてポリペプチドなしの群(コントロール)を用いた。
ATPアッセイ法を用いてポリペプチドの抗菌活性を測定した。
さらに、上記ポリペプチドの抗菌活性を明らかにするために、上記黄色ブドウ球菌、緑膿菌に加えて大腸菌、腸内細菌2種(それぞれ腸内細菌(1)及び腸内細菌(2))、肺炎桿菌、および表皮ブドウ球菌に対する抗菌活性も測定した。
次に、これらポリペプチドのヒト皮膚繊維芽細胞(NHDF)の増殖に対する影響を検討した。同仁化学のCell Counting Kit(WST-1)を用いてポリペプチドの細胞増殖活性を調べた。陰性対照(Control)として、ポリペプチドなしの群を用いた。細胞(正常ヒト新生児包皮皮膚線維芽細胞:NHDF(NB))を96ウェルプレートに播種する(0.5×104 cells/well/100 μL、血清1%)。細胞を播種してから約3時間後、ポリペプチド(1,3,10,30,または100μg/ml)、陽性対照としてFGF(100ng/ml)を100μL添加した。無刺激の群には培地のみを100 μL添加した。CO2インキュベータ内に約48時間静置した後、WST-1試薬を各ウェルに20μL添加し、CO2インキュベータ内で約 2時間静置した。Wallac 1420 ARVOsx(プログラム:WST-1)にて波長450 nm,620 nmの吸光度を測定した。各測定値のO.D.450 - O.D.620を求めた。測定ウェルのO.D.450 - O.D.620の値から細胞を含まない空ウェルのO.D.450 - O.D.620の平均値を引いたものをNet O.D.450 とした。細胞増殖活性は無刺激のNet O.D.450に対するポリペプチド添加時の割合で評価した。
倉敷紡績株式会社の血管新生キット(製品番号:KZ-1000)を用いてポリペプチドの管腔形成を評価した。陽性対照として血管内皮増殖因子A(VEGF-A)、陰性対照としてポリペプチドなしの無刺激の群を用いた。
続いて、SR-1~5の5つのポリペプチドについて、血清中の安定性を評価した。
ペプチドを最終濃度500μg/mLとなるように、ヒト血清(株式会社ケー・エー・シーより購入)またはラットより採血後室温放置後遠心分離により得られたラット血清に溶解させ、37℃で静置した. 10分もしくは30分または60分の静置後、HPLCクロマトグラム分析に供し分解物ピークを検出し、溶液中に残存するペプチド濃度の開始濃度に対する割合を算出した。また、各ペプチドの血清中の半減期を算出した。
カラム:CAPCELL PAK C18 MGII (S-3μm,4.6×150PE,資生堂)
ガードカラム:GUARD CARTRIDGE CAPCELL C18 MG (S-3μm,4.0×10PE,資生堂)
カラム温度:50℃
移動相A:0.025% トリフルオロ酢酸溶液
移動相B:0.025% トリフルオロ酢酸 アセトニトリル
流量:1.0 mL/min
検出器:紫外吸光光度計(測定波長:220 nm)
注入量:100 μL
グラジェント条件:
続いて、SR-1~5の5つのポリペプチドのin vivoにおける肉芽組織形成能について検討した。Crl:CD(SD)系ラット(雄、9週齢、日本チャールス・リバー株式会社より入手)に、25、250、2500μg/mLの濃度の被験物質である各ペプチド、2.5、25、250μg/mLの濃度のbFGF製剤(陽性対照)または生食(陰性対照)を40μlずつディスク(disc)に添加し、最終的には、各ペプチドは1, 10及び100μg/disc、bFGF製剤は0.1, 1及び10μg/discの用量とし、添加したペーパーディスク(FILTER PAPER φ8 mm , ADVANTEC)をラット背部皮下に埋め込んだ。8日後、ペーパーディスクを摘出し、ペーパーディスク周辺部の肉芽組織を採取し、その湿重量を測定した。
μl/disc)の用量で最大の肉芽組織重量の増加を示した。この結果から、特に、SR-1およびSR-3は、in vivoの局所投与においても血管新生を促進し、肉芽組織形成に寄与するものと考えられる。
次に、ラット切創モデルを用いて、SR-1~5の5つのポリペプチドのin vivoにおける切創治癒効果を評価した。ラット切創モデルの作製および創耐張力の測定は、山浦哲明ら(応用薬理22,565~579(1981))に記載のとおり行った。
上記で切創治癒促進効果が認められたが、さらに、黄色ブドウ球菌に感染した創傷に対する治癒効果を評価した。ラット感染創モデルは、Stenberg BD他(J Surg Res. 1991 Jan;50(1):47-50.)およびHayward P 他(Am J Surg. 1992 Mar;163(3):288-93.)を参考に下記のとおり作製した。HWY/slc系ヘアレスラット(雄、7週齢、日本エスエルシー株式会社より入手)を用いた。全層欠損作製前日に白血球数の測定及びサイクロフォスファマイド(100 mg/kg)の尾静脈内投与を行い、その翌日に白血球数をカウントし、白血球数5000以下の個体のみを使用した。ラットの背部に1.73 x 1.73 cm(約3cm2)の正方形の全層欠損を作製し、欠損部に黄色ブドウ球菌(約105CFU/25μL)及び被験物質としてSR-1ペプチド 2 mg/mLを25μL滴下(投与量として50μg)した。なお、bFGF製剤(陽性対照)群は3μg/25μLを、陰性対象群は生理食塩水を25μL滴下した。被験物質投与後、創部を被覆剤(パーミロール, 日東メディカル)で被覆した(day 0)。その後、黄色ブドウ球菌及び被験物質を1日1回連続4日間(day 0, 1, 2, 3)滴下した。全層欠損作製日の6日目(day 6)に、まだ治癒していない創傷面積を測定し、創傷を与えた面積に対する割合を算出し、6日目創傷面積率とした。
[配列番号2] SR-2。C末端のアルギニン残基がアミド化により修飾されている。
[配列番号3] SR-3。C末端のアルギニン残基がアミド化により修飾されている。
[配列番号4] SR-4。C末端のアルギニン残基がアミド化により修飾されている。
[配列番号5] SR-5
[配列番号6] SR-6。C末端のリジン残基がアミド化により修飾されている。
Claims (15)
- アミノ酸配列が配列番号1ないし6のいずれかで表されるポリペプチド。
- 請求項1に記載のポリペプチドを有効成分として含有する血管新生誘導剤。
- 前記ポリペプチドのアミノ酸配列が、配列番号1、2又は3で表わされる請求項2記載の血管新生誘導剤。
- 請求項1に記載のポリペプチドを有効成分として含有する抗菌剤。
- 有効量の請求項1に記載のポリペプチドを、血管新生が必要な哺乳動物に投与することを含む、血管新生誘導方法。
- 請求項1に記載のポリペプチドの、血管新生誘導剤の製造のための使用。
- 請求項1に記載のポリペプチドである、血管新生誘導用ポリペプチド。
- 請求項1に記載のポリペプチドを有効成分として含有する、皮膚創傷の予防、改善又は治療剤。
- 皮膚創傷が、切創、手術創、びらん、熱傷、褥瘡、難治性創傷、皮膚潰瘍、下肢潰瘍、糖尿病性潰瘍、閉塞性動脈疾患及び閉塞性動脈硬化症に起因する皮膚創傷から成る群から選択される請求項8記載の皮膚創傷の予防、改善又は治療剤。
- 皮膚創傷が、難治性創傷である、請求項9記載の予防、改善又は治療剤。
- 皮膚創傷が、感染を伴う創傷である、請求項9記載の予防、改善又は治療剤。
- 請求項1に記載のポリペプチドを有効成分として含有する、皮膚創傷における細菌感染の予防、改善又は治療剤。
- 有効量の請求項1に記載のポリペプチドを哺乳動物に投与することを含む、皮膚創傷または皮膚創傷における細菌感染の予防、改善又は治療方法。
- 請求項1に記載のポリペプチドの皮膚創傷または皮膚創傷における細菌感染の予防、改善又は治療剤の製造のための使用。
- 請求項1に記載のポリペプチドである、皮膚創傷または皮膚創傷における細菌感染の予防、改善又は治療用ポリペプチド。
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Cited By (5)
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WO2010137594A1 (ja) * | 2009-05-25 | 2010-12-02 | アンジェスMg株式会社 | 血管新生誘導活性及び抗菌活性を有するポリペプチドおよびそれを含有する創傷治療剤 |
JP2013014583A (ja) * | 2011-06-10 | 2013-01-24 | Anges Mg Inc | メチオニン・スルホキシドを含む新規ポリペプチド |
US8470765B2 (en) | 2009-03-06 | 2013-06-25 | Anges Mg, Inc. | Polypeptides and antibacterial or antiseptic use of same |
WO2014157485A1 (ja) | 2013-03-29 | 2014-10-02 | 国立大学法人大阪大学 | 抗老化作用を有するペプチドおよびその利用 |
WO2016047763A1 (ja) * | 2014-09-26 | 2016-03-31 | 国立大学法人大阪大学 | 新規ペプチドおよびその用途 |
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005090564A1 (ja) | 2004-03-19 | 2005-09-29 | Genomidea Inc. | 血管内皮増殖促進遺伝子 |
WO2006054947A1 (en) * | 2004-11-17 | 2006-05-26 | Dermagen Ab | Novel antimicrobial peptides |
WO2008096816A1 (ja) | 2007-02-09 | 2008-08-14 | Genomidea Inc. | 血管新生誘導剤及びそれに用いられるポリペプチド |
WO2008096814A1 (ja) * | 2007-02-09 | 2008-08-14 | Genomidea Inc. | 新規ポリペプチド及びそれを有効成分として含有する抗菌剤 |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4939224A (en) * | 1987-02-26 | 1990-07-03 | The Salk Institute Biotechnology/Industrial Associates, Inc. | Vasoactive intestinal peptide analogs |
WO2001012668A1 (en) | 1999-08-18 | 2001-02-22 | University Of Iowa Research Foundation | Cathelicidin-derived peptides with broad spectrum antimicrobial activity |
US7674771B2 (en) | 2003-10-29 | 2010-03-09 | Toagosei Co., Ltd | Antimicrobial peptides and utilization of the same |
US20050214321A1 (en) | 2003-12-01 | 2005-09-29 | Dow Global Technologies Inc. | Recombinant icosahedral virus like particle production in pseudomonads |
US7452856B2 (en) | 2004-07-06 | 2008-11-18 | Seikagaku Corporation | Antibacterial peptide |
EP1837034A4 (en) | 2004-11-30 | 2009-08-05 | Anges Mg Inc | MEDICATION FOR ALZHEIMER'S DISEASE |
JP4730584B2 (ja) | 2004-12-06 | 2011-07-20 | 東亞合成株式会社 | 抗菌ペプチド及びその利用 |
EP1892293A4 (en) | 2005-06-06 | 2008-12-10 | Anges Mg Inc | TRANSCRIPTION FACTOR DECOY |
JP5134964B2 (ja) | 2005-10-05 | 2013-01-30 | ジェノミディア株式会社 | 単離されたヒト細胞、その取得方法及び用途 |
EP1978096A1 (en) | 2005-12-22 | 2008-10-08 | Genomidea Inc | Novel oligonucleotide and nf-kappa b decoy comprising the same |
EP2404932B1 (en) | 2009-03-06 | 2014-07-23 | AnGes MG, Inc. | Polypeptides and antibacterial or antiseptic use of same |
JP5750766B2 (ja) | 2009-05-25 | 2015-07-22 | 株式会社ファンペップ | 血管新生誘導活性及び抗菌活性を有するポリペプチドおよびそれを含有する創傷治療剤 |
-
2009
- 2009-11-27 US US13/131,796 patent/US9376470B2/en active Active
- 2009-11-27 JP JP2010540523A patent/JP5691049B2/ja active Active
- 2009-11-27 ES ES09829160.2T patent/ES2467691T3/es active Active
- 2009-11-27 WO PCT/JP2009/070035 patent/WO2010061915A1/ja active Application Filing
- 2009-11-27 EP EP09829160.2A patent/EP2371840B1/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005090564A1 (ja) | 2004-03-19 | 2005-09-29 | Genomidea Inc. | 血管内皮増殖促進遺伝子 |
WO2006054947A1 (en) * | 2004-11-17 | 2006-05-26 | Dermagen Ab | Novel antimicrobial peptides |
WO2008096816A1 (ja) | 2007-02-09 | 2008-08-14 | Genomidea Inc. | 血管新生誘導剤及びそれに用いられるポリペプチド |
WO2008096814A1 (ja) * | 2007-02-09 | 2008-08-14 | Genomidea Inc. | 新規ポリペプチド及びそれを有効成分として含有する抗菌剤 |
Non-Patent Citations (16)
Title |
---|
"Fmoc solid synthesis: a practical approach", 2000, OXFORD UNIVERSITY PRESS, article "Fmoc solid synthesis: a practical approach" |
"Solid Phase Peptide Synthesis", 1984, PIERCE |
BRENNEMAN DE, SPONG CY, HAUSER JM, ABEBE D, PINHASOV A, GOLIAN T, GOZES I.: "Protective peptides that are orally active and mechanistically nonchiral.", J PHARMACOL EXP THER., vol. 309, no. 3, June 2004 (2004-06-01), pages 1190 - 7 |
DHARAP SS, WANG Y, CHANDNA P, KHANDARE JJ, QIU B, GUNASEELAN S, SINKO PJ, STEIN S, FARMANFARMAIAN A, MINKO T.: "Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide.", PROC NATL ACAD SCI USA., vol. 102, no. 36, 6 September 2005 (2005-09-06), pages 12962 - 7 |
HAYWARD P ET AL., AM J SURG., vol. 163, no. 3, March 1992 (1992-03-01), pages 288 - 93 |
HIRONORI NAKAGAMI ET AL.: "Kokin Peptide to Kekkan Shinsei", THE JOURNAL OF JAPANESE COLLEGE OF ANGIOLOGY, vol. 48, no. 5, March 2009 (2009-03-01), pages 437 - 440, XP008152918 * |
RAAKE P, PFOSSER A, BOEKSTEGERS P, WELSCH U, HIEMSTRA PS, VOGELMEIER C, GALLO RL, CLAUSS M, BALS R.: "An angiogenic role for the human peptide antibiotic LL-37/hCAP-18.", J CLIN INVEST., vol. 111, no. 11, June 2003 (2003-06-01), pages 1665 - 72 |
SATO M, FURUIKE T, SADAMOTO R, FUJITANI N, NAKAHARA T, NIIKURA K, MONDE K, KONDO H, NISHIMURA S.: "Glycoinsulins: dendritic sialyloligosaccharide- displaying insulins showing a prolonged blood-sugar-lowering activity.", J AM CHEM SOC., vol. 126, no. 43, 3 November 2004 (2004-11-03), pages 14013 - 22 |
SATO M, SADAMOTO R, NIIKURA K, MONDE K, KONDO H, NISHIMURA S: "Site-specific introduction of sialic acid into insulin.", ANGEW CHEM INT ED ENGL.., vol. 43, no. 12, 12 March 2004 (2004-03-12), pages 1516 - 20 |
See also references of EP2371840A4 * |
STENBERG BD ET AL., J SURG RES., vol. 50, no. 1, January 1991 (1991-01-01), pages 47 - 50 |
TETSUAKI YAMAURA ET AL., OYO YAKURI, vol. 22, 1981, pages 565 - 579 |
THE FIFTH SERIES OF EXPERIMENTAL CHEMISTRY, VOL.16, SYNTHESIS OF ORGANIC COMPOUNDS IV, vol. 16 |
ULBRICHT K, BUCHA E, POSCHEL KA, STEIN G, WOLF G, NOWAK G.: "The use of PEG-Hirudin in chronic hemodialysis monitored by the Ecarin Clotting Time: influence on clotting of the extracorporeal system and hemostatic parameters.", CLIN NEPHROL., vol. 65, no. 3, March 2006 (2006-03-01), pages 180 - 90 |
WILKEMEYER MF, CHEN SY, MENKARI CE, SULIK KK, CHARNESS ME.: "Ethanol antagonist peptides: structural specificity without stereospecificity", J PHARMACOL EXP THER., vol. 309, no. 3, June 2004 (2004-06-01), pages 1183 - 9 |
ZANETTI M.: "Cathelicidins, multifunctional peptides of the innate immunity.", J LEUKOC BIOL., vol. 75, no. 1, 22 July 2003 (2003-07-22), pages 39 - 48 |
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Also Published As
Publication number | Publication date |
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EP2371840B1 (en) | 2014-04-16 |
EP2371840A4 (en) | 2012-07-04 |
US9376470B2 (en) | 2016-06-28 |
EP2371840A1 (en) | 2011-10-05 |
JPWO2010061915A1 (ja) | 2012-04-26 |
US20120122766A1 (en) | 2012-05-17 |
JP5691049B2 (ja) | 2015-04-01 |
EP2371840A9 (en) | 2012-02-22 |
ES2467691T3 (es) | 2014-06-12 |
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