KR20200044434A - Composition for treating intractable ulcer comprising substance P - Google Patents

Abstract

The present invention relates to a composition for treating refractory ulcers, containing a substance P, an antioxidant, a surfactant, and a thickener, as active components, and more specifically, relates to a composition which, due to said combination, exhibits excellent effects of reducing the thickness of epithelial tissues and promoting collagen synthesis, and thus can be used in the treatment of various refractory ulcers. The composition containing the substance P of the present invention has the stability of the substance P increased due to a synergistic effect by mixing the substance P and basic characteristics with the antioxidant, the surfactant, and the thickener, and thus exhibits excellent therapeutic effects on refractory ulcers. Accordingly, the composition of the present invention can be beneficially used for treating refractory ulcers, such as diabetic ulcers.

Description

Composition for treating intractable ulcer comprising substance P {Composition for treating intractable ulcer comprising substance P}

The present invention relates to a composition for treating refractory ulcers comprising substance P (substance P), antioxidant, surfactant, and thickener as an active ingredient, and exhibits excellent epithelial tissue thickness reduction and collagen synthesis promoting effect by the combination. It relates to a composition that can be used for the treatment of intractable ulcers.

Intractable ulcers are chronic wounds, and the natural healing mechanism is out of balance, so full healing is impossible. In the case of general wounds, natural healing is possible with a wound healing mechanism of inflammation, proliferation, and remodeling, whereas in case of such intractable ulcers, spontaneous healing is difficult due to problems in one or more of the above three processes, and long-term repeated treatment As it is required, it is a great social burden in terms of productivity reduction and health care burden.

Types of intractable ulcers include diabetic ulcers, pressure ulcers called pressure sores, and venous ulcers. Diabetic ulcer is one of the main complications in diabetic patients. Diabetes patients have about 25% chance of developing diabetes ulcer, which is the most common complication that occurs in 1 out of 4 patients. (Lancet. # 2005; 366: 1719-1724). Diabetic ulcers are caused by significantly slowing the rate of movement of cytokines and growth factors that help wound healing due to blood flow restriction to the wound site and inducing a chronic inflammatory response (Korea Diabetes J, 2009, 33: 83- 90). Diabetic ulcers easily cause infection, leading to body amputation, which is one of the serious causes that accounts for 25-90% of the total number of amputations. Compressed ulcers and venous ulcers are also intractable wounds that have problems with blood flow, like diabetic ulcers. Compressed ulcers are diseases caused by problems with the supply of oxygen and nutrients by restricting blood flow in specific areas, and venous ulcers In the case of calf muscle, it is a representative disease that occurs due to a decrease in the blood flow pump function, which prevents blood flow from circulating and accumulates in the legs.

Treatment of intractable ulcers is basically controlled by blood sugar control, wound area load removal, dead tissue removal, and treatment management for disinfection and infection. Active therapies have been developed that contain growth factors that promote wound healing. Regranex ^® , a topical coating agent containing platelet-derived growth factor (PDGF), is used for diabetic ulcers approved by the US Food and Drug Administration and epidermal growth factor approved by Daewoong Pharm in Korea. factor: EGF) -containing external solution is also used for diabetic ulcers. However, the growth factor contained in such a therapeutic agent is easily degraded in an ulcer environment, so the effect of the drug is not sufficiently delivered to the wound site, and the molecular weight is large, so it is 50 times the amount required for recovery due to the disadvantage that it is not easily absorbed by the skin. There is a problem that the amount should be administered. Therefore, there is an urgent need to develop a therapeutic agent that exerts a sufficient effect in an appropriate amount by improving delivery to a wound site and skin absorption.

Under the above background, the present inventors tried to develop a composition having excellent treatment efficacy for refractory ulcers, and as a composition using substance P, a neuropeptide composed of 11 amino acids, as an antioxidant, surfactant, and thickener in substance P By mixing to confirm that it is possible to maximize the treatment effect of refractory ulcer of substance P and completed the present invention.

An object of the present invention is to provide a pharmaceutical composition for the treatment of refractory ulcers comprising a substance P consisting of the amino acid sequence of SEQ ID NO: 1, an antioxidant, a surfactant, and a thickener.

Another object of the present invention is to provide a quasi-drug composition for the treatment of refractory ulcers comprising the substance P, antioxidant, surfactant and thickener.

Another object of the present invention is to provide a method for treating intractable ulcer comprising administering the pharmaceutical composition to an individual in need thereof.

One aspect of the present invention provides a pharmaceutical composition for the treatment of refractory ulcers comprising the substance P, antioxidant, surfactant and thickener.

The present inventors not only used a conventional commercialized therapeutic agent, but also effectively reduced the wound size of refractory ulcers than using the substance P alone when using the substance P in combination with an antioxidant, a surfactant, and a thickener, It was confirmed that it has an effect of further promoting collagen production.

That is, the present invention uses an antioxidant, a surfactant and a thickener, specifically sodium thiosulfate, polysorbate 80 and hydroxyethylcellulose as an additional component in substance P, thereby improving the treatment effect for refractory ulcers compared to the concentration of substance P. It is based on the idea of being able to enhance, which was first identified in the present invention.

In the present invention, "substance P (substance P)" is SEQ ID NO: 1 consisting of 11 amino acids (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH ₂ ) Means a neuropeptide. It is widely distributed in the central and peripheral nervous systems and is known to perform various functions such as pain induction and vasodilation by secreting rapidly when a specific stimulus reaches the sensory nervous system. However, there is no known composition using the effect of treating the refractory ulcer of the substance P, in particular, the composition P to improve the therapeutic effect compared to the concentration by mixing an antioxidant, a surfactant, and a thickener.

The concentration of the substance P contained in the pharmaceutical composition of the present invention may be 0.1 to 100 μg / ml, and specifically 5 μg / ml, but is not limited thereto. At this time, when the concentration of substance P contained in the pharmaceutical composition of the present invention is 0.1 to 100 μg / ml, the cell proliferation effect of substance P is excellent.

In the present invention, the "antioxidant" acts on free radicals or peroxides generated during oxidation of the active ingredient by oxygen in the air to stop the chain reaction of oxidation, prevent the progress of oxidation, and prevent the deterioration of the active ingredient Refers to the substance added. The antioxidant can be used as an active ingredient of the pharmaceutical composition containing the substance P to prevent the deterioration of the therapeutic effect of refractory ulcers of the pharmaceutical composition.

Antioxidants can be used without limitation conventional antioxidants that can be used in the art, specifically, sodium thiosulfate (sodium thiosulfate), beta-mercaptoethanol (β-mercaptoethanol (β-ME)), glutathione (glutathione, GSH) , Ascorbic acid, vitamin E, beta-carotene, lycopene, coenzyme Q-10, selenium, chromium, magnesium, taurine, hypotaurine, trehalose or these Combination of can be used, but is not limited thereto. Specifically, for the purposes of the present invention, the antioxidant may be sodium thiosulfate.

The content of the antioxidant may be 0.01 to 1% by weight relative to the total weight of the composition of the present invention, specifically 0.1% by weight, but is not limited thereto. The content of the antioxidant is not particularly limited as long as it can prevent deterioration of the therapeutic effect of refractory ulcers in the pharmaceutical composition, and an appropriate dose can be used by those skilled in the art.

In the present invention, "surfactant" means a substance that maintains a uniform liquid composition using a lipophilic oil component, and the surfactant in the present invention is an interface commonly used in the preparation of pharmaceutical compositions in the art. Pharmaceutically acceptable anionic, cationic, nonionic or amphoteric surfactants can be used without limitation.

Specifically, polysorbate 80 (polysorbate 80), polysorbate 60 (polysorbate 60), polysorbate 20 (polysorbate 20), poloxamer, poloxamine, polyvinylpyrrolidone (polyvinyl pyrrolidone) ), Polyvinyl alcohol (polyvinyl alcohol), polyethylene glycol (polyethylene glycol) or a combination of these may be used, but is not limited thereto. Specifically, for the purposes of the present invention, the surfactant may be polysorbate 80.

The content of the surfactant may be 0.001 to 0.02% by weight relative to the total weight of the composition of the present invention, specifically 0.006% by weight. When the content of the surfactant is less than 0.001% by weight, the emulsifying power of the composition of the present invention is lowered, and thus the precipitation stability of the formulation may be lowered. If it is more than 1.0% by weight, the viscosity of the composition of the present invention is too high or phase inversion, etc. Problems may arise.

In the present invention, "thickener" means an additive added to impart viscosity, and is also referred to as a thickener or thickener. In the present invention, the thickener may be used without limitation, a thickener generally used in the preparation of pharmaceutical compositions in the art.

Specifically, hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, sodium carboxymethylcellulose, or a combination thereof may be used, but is not limited thereto. Specifically, for the purposes of the present invention, the thickener may be hydroxyethyl cellulose.

The content of the thickener may be 1 to 20% by weight based on the total weight of the composition of the present invention, specifically 15% by weight. If the content of the thickener is less than 1% by weight, the stability of the pharmaceutical composition may occur, and if it exceeds 20% by weight, the viscosity of the pharmaceutical composition may be too high, resulting in an unsuitable composition.

The pharmaceutical composition of the present invention may have different therapeutic effects of refractory ulcers depending on the type of antioxidant, surfactant, and / or thickener, in particular sodium thiosulfate as an antioxidant, polysorbate 80 as a surfactant, and hydration as a thickener When hydroxyethyl cellulose is included, it is possible to maximize the therapeutic effect of refractory ulcers of substance P to concentration.

In one embodiment of the present invention, a pharmaceutical composition comprising sodium thiosulfate as an antioxidant along with substance P, polysorbate 80 as a surfactant, and hydroxyethylcellulose as a thickener was prepared and used in the treatment of refractory ulcers. It was confirmed that the treatment effect of refractory ulcer was remarkably superior to the use of substance P alone and the commercialized treatment.

In the present invention, "intractable ulcer" is a chronic wound or ulcer that does not heal even after a certain period of time has passed through a normal healing process. Specifically, normal wound management and treatment have been performed for a period of 4 weeks to 3 months. It may mean an ulcer whose recovery rate is less than 20%. The normal wound healing mechanism goes through the hemostasis stage after tissue damage and goes through four stages: the inflammatory stage, the proliferation stage, and the maturation stage. In general, the hemostasis stage of several hours to several hours, the inflammatory stage for 2-3 days thereafter, and the proliferative stage for tissue regeneration build up from 2-3 days, and proceeds from about 2 to 6 weeks, and the stabilization process of the wound is then performed. At this time, various growth factors and cells appear at each stage, and the damaged area is restored. The normal healing process is usually healed at the end of the proliferative phase. If the wound does not heal within a certain period of time due to a delay in one stage, it progresses to an intractable ulcer, which is mainly expressed in the form of an ulcer of abnormal depth of the skin and subcutaneous tissue. .

The incurable ulcer of the present invention has a different pathogenesis depending on the nature of the disease, but may ultimately be caused by a decrease or deficiency of a factor that induces a normal wound healing mechanism, which is hypoxia, infection, abnormality of cells, and sensory effects due to neuropathy. The reduction may be caused by repeated trauma, reduced blood circulation due to compression, and ischemic conditions, but is not limited thereto.

Specifically, the refractory ulcer of the present invention may include diabetic ulcer and ischemic ulcer. Ischemic ulcers may include, but are not limited to, compression ulcers, venous ulcers, and arterial ulcers. If a chronic wound does not heal even after a certain period of time has passed, it can be applied to the present invention without being limited to the cause of the onset.

Diabetic ulcer is a disease that occurs in diabetic patients. In diabetic patients, various risk factors are associated with each other to cause ulcers, as well as to develop a chronic skin ulcer because of slow wound healing compared to the general public. Diabetes patients may not be able to supply blood flow to the skin because the arteries are stuck during the progression of large vessel disease, atherosclerosis, and so-called microvascular disease, thereby impairing the control of the microcirculatory. When a wound occurs in a specific area, blood flow becomes faster to promote healing of the wound in normal patients, whereas in patients with microvascular disease and ischemic disorder, this reaction is markedly dull. I can't. Therefore, a substance capable of exhibiting a therapeutic effect even in a hyperglycemic state in which diabetes is induced should be used.

Compressed ulcer is a disease commonly referred to as pressure sore, which is generally defined as a region of repetitive pressure over a defined area over the bulge of the bone, resulting in ischemia, cell death and tissue necrosis. It may be caused by conditions such as age or being tied to a wheelchair, but is not limited thereto.

Venous ulcer may mean chronic lower extremity ulcer leading to destruction of tissue and ulcer due to chronic venous dysfunction, but is not limited thereto.

Arterial ulcers may mean ulcers caused by arterial diseases such as arteriosclerosis, arterial dysfunction, and arterial occlusive disease, but are not limited thereto.

In the case of refractory ulcer, due to the hyperglycemic environment, poor blood circulation, difficulty in transporting substances necessary for healing of ulcers to wounds, and accompanying infection, the treatment effect by the same drug is significantly lower and the treatment speed compared to general skin wounds It is also very slow. The composition of the present invention can be used to treat intractable ulcers that are not cured by general wound treatment methods, including diabetic ulcers and the like.

In one embodiment of the present invention, in the case of an ulcer developed in a patient with diabetes, the general wound treatment technique has a slow treatment effect, whereas when the pharmaceutical composition of the present invention is used, an excellent wound treatment effect is exhibited even in individuals with diabetes. Was confirmed. That is, the pharmaceutical composition of the present invention can effectively treat intractable ulcers that cannot be treated with a general wound treatment technique, and in particular, has the advantage of exhibiting a significant wound treatment effect even in a hyperglycemic environment of an individual with diabetes.

In the present invention, "treatment" can mean any action that causes or improves the symptoms of ulcers in individuals with intractable ulcers. Specifically, for the purpose of the present invention, it may mean an act of preventing, healing, reversing, weakening, alleviating, minimizing, suppressing, or stopping the harmful effects of ischemic disorders such as increasing perfusion, but is not limited thereto.

The composition of the present invention may be to promote collagen synthesis. In order to proliferate and regenerate cells at the wound site, a step of decomposing the extracellular matrix is necessary, and this decomposition is performed through matrix metalloproteinase (MMP). The stage of the recovery process is regulated by the balance between different MMPs. Excessive MMP activity is the cause of refractory ulcers, which are characterized by long-term inflammation, decreased collagen synthesis, and high content of MMPs. That is, the treatment effect of intractable ulcer can be obtained by reducing the wound size (length and volume) of the intractable ulcer site, increasing the synthesis and deposition of extracellular matrix such as collagen, and increasing the re-epithelialization of the chronic wound.

In one embodiment of the present invention, as a result of administering the pharmaceutical composition of the present invention to the intractable ulcer site, it was confirmed that the effect of reducing the length and area of the wound tissue. In addition, since the tissue in which the healing of intractable ulcers is completed shows a thin epithelial shape like the epithelial tissue of normal skin, the thickness of the epithelial tissue is a measure of the degree of treatment. When applied to a refractory ulcer, the pharmaceutical composition of the present invention can effectively treat a refractory ulcer through reduction of epithelial tissue thickness and promoting collagen synthesis.

The pharmaceutical composition of the present invention can also be used as a single agent, and can be prepared and used as a combination preparation by further including a drug known to have a recognized wound healing effect, and is formulated by using a pharmaceutically acceptable carrier or excipient It can be produced in a dosage form or incorporated into a multi-dose container.

The "pharmaceutically acceptable carrier" may mean a carrier, excipient, or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the compound to be injected. Specifically, non-naturally (non-naturally occuring carrier). The type of the carrier that can be used in the present invention is not particularly limited, and any carrier that is commonly used in the art and is pharmaceutically acceptable can be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more.

The composition comprising a pharmaceutically acceptable carrier may be various formulations, oral or parenteral. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, etc., which are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the compound, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. It can be prepared by mixing. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents, suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The pharmaceutical composition may be prepared in any formulation according to conventional methods. The composition can be formulated, for example, in oral dosage forms (eg, powders, tablets, capsules, syrups, pills, or granules), or parenteral dosage forms (eg, injections). In addition, the composition may be prepared in a systemic formulation or a topical formulation.

The pharmaceutical composition may be used as an external preparation for skin and applied to the skin. The pharmaceutical composition may be a formulation of a liquid, suspension, emulsion, cream, hydrogel, lotion, ointment, spray, patch, or aerosol, but is not limited thereto. When used as an external preparation for skin, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic Emulsifiers, fillers, metal ion blockers and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or any other ingredients commonly used in external preparations for the skin It may contain adjuvants commonly used in the field of dermatology.

In addition, the ingredients may be introduced in an amount generally used in the field of skin science. The pharmaceutical composition may further include an agent that increases percutaneous absorption, for example, dimethyl sulfoxide, dimethyl acetamide, dimethyl formamide, surfactant, azone, alcohol, acetone, and the like.

The pharmaceutical composition of the present invention may contain substances P, antioxidants, surfactants and thickeners in effective amounts. The "effective amount" refers to an amount sufficient to exhibit the effect of treatment when administered to an individual in need of treatment for intractable ulcer. The effective amount can be appropriately selected depending on the cell or individual selected by those skilled in the art. Severity of refractory ulcer disease, patient's age, weight, health, sex, patient's sensitivity to drugs, time of administration, route of administration and rate of discharge, duration of treatment, factors including drugs used in combination or coincidental with the composition used, and others It can be determined by factors well known in the medical field. For the administration, the recommended dosage may be administered once a day, or may be divided into several times.

Another aspect of the present invention provides a quasi-drug composition for the treatment of refractory ulcer comprising the substance P, antioxidant, surfactant and thickener.

The terms "substance P", "antioxidant", "surfactant", "thickener" and "refractory ulcer" as used herein are as described above.

In the present invention, "quasi-drug" is a fiber, rubber product or the like used for the purpose of treating, alleviating, treating or preventing a disease of a person or animal, has a weak effect on the human body or does not directly act on the human body, and is a device Or it means something other than a machine and something similar. In addition, as an article corresponding to one of the agents used for sterilization, pesticide, and similar purposes for the prevention of infection, utensils and machinery among articles used for the purpose of diagnosing, treating, reducing, treating or preventing human or animal diseases Alternatively, it may mean a product that is not a device and is used for the purpose of having a pharmacological effect on the structure and function of a person or animal, except for a device, a machine, or a device. In addition, the quasi-drug may include personal hygiene products. The personal hygiene product may be, but is not limited to, a disinfecting cleaner, shower foam, gagreen, wipes, detergent soap, hand wash, or ointment.

Specifically, in the present invention, the quasi-drug may be an external preparation for skin. The external preparation for skin may be formulated for topical application to the skin by containing a cosmetic or dermatologically acceptable medium or base. These are all formulations suitable for topical application, for example solutions, gels, solids, dough anhydrous products, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) and ratios obtained by dispersing an oil phase in an aqueous phase. It can be provided in the form of an ionic vesicle dispersant, or in the form of a cream, skin, lotion, powder, ointment, spray, or consylstick. It can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. These compositions can be prepared according to conventional methods in the art.

In addition, the external preparation for skin of the present invention is a fatty substance, an organic solvent, a solubilizer, a thickening agent, a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizing agent, a foaming agent, a fragrance, a surfactant, water, an ionic type or a ratio. With ionic emulsifiers, fillers, metal ion blocking agents, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or any other ingredients commonly used in cosmetics It may contain adjuvants commonly used in the same cosmetic or dermatological field. The amount of the adjuvant may be used in a suitable amount generally used in the cosmetic or dermatological field depending on the purpose of use.

When the composition according to the present invention is used as a quasi-drug additive, the composition may be added as it is or used with other quasi-drugs or quasi-drug components, and may be suitably used according to a conventional method.

Another aspect of the present invention provides a method of treating intractable ulcer comprising administering the pharmaceutical composition to an individual in need thereof.

The terms "substance P", "antioxidant", "surfactant", "thickener" and "refractory ulcer" as used herein are as described above.

In the present invention, "individual" may mean any animal, including humans, who have or are likely to develop refractory ulcers. The animal may be, but is not limited to, humans, mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats, which require treatment for similar symptoms.

In the present invention, "administration" means introducing the pharmaceutical composition of the present invention to a patient in any suitable way, and the route of administration of the composition can be administered through any general route as long as it can reach the target tissue. have. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, but are not limited thereto.

 Due to the nature of the present invention, which is effective in the treatment of intractable ulcers, the administration may be applied to the skin, specifically, the surface of the body, that is, the skin, scalp, hair, nails, damaged tissue (eg, skin), Or it may mean applying or spreading the composition of the present invention on the wound surface of a refractory ulcer.

The pharmaceutical composition for treating refractory ulcers containing the substance P, antioxidant, surfactant, and thickener may be administered in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level depends on the individual type and severity, age, sex, drug activity, and drug. Sensitivity to administration, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-drugs, and other factors well known in the medical field.

The composition of the present invention may be administered daily or intermittently, and the number of administrations per day can be administered once or divided into 2-3 times. When the two active ingredients are single agents, the number of administrations may be the same or may be different. In addition, the composition of the present invention can be used alone or in combination with other drug treatments for the treatment of intractable ulcers. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, and can be easily determined by those skilled in the art.

The composition of the present invention increases the stability of the substance P by synergistic action by mixing the substance P with an antioxidant, a surfactant, and a thickener, thereby exhibiting an excellent therapeutic effect on intractable ulcers. Therefore, the composition of the present invention can be usefully used as a therapeutic use for intractable ulcers such as diabetic ulcers.

FIG. 1 is a graph quantifying (a) wound size reduction and (b) wound size reduction in skin wound models of normal and diabetic-induced mice.
FIG. 2 shows the substance P (Only SP), the composition containing the substance P (SP gel), Easyy topical solu., And the intrasite gel in the skin wound model of a mouse induced with diabetes. After each treatment, (a) a photograph showing the reduction in wound size after 4, 7 and 14 days, (b) a graph quantifying the extent of wound size reduction after 4, 7 and 14 days, (c) A graph quantifying the extent of wound size reduction after 4 days, (d) a graph quantifying the extent of wound size reduction after 7 days, and (e) a graph quantifying the extent of wound size reduction after 14 days.
FIG. 3 shows the substance P (Only SP), the composition containing the substance P (SP gel), Easyy topical solu., And the intrasite gel in the skin wound model of diabetes-induced mice. After each treatment, (a) histologically showing the wound area (light yellow) after 14 days, (b) a graph quantifying the extent of wound length reduction after 14 days, (c) wound after 14 days This graph quantifies the extent of area reduction.
FIG. 4 shows the substance P (Only SP), the composition containing the substance P (SP gel), Easyy topical solu., And the intrasite gel in the skin wound model of the diabetic-induced mouse. After each treatment, (a) a photograph showing the epithelial cell thickness reduction and collagen synthesis degree after tissue 14 days through tissue staining, (b) a graph quantifying the epithelial cell thickness after 14 days.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

Example 1: Preparation of a composition for treating refractory ulcers containing substance P (hereinafter referred to as SP gel)

A new formulation containing substance P was prepared by including substance P in sodium thiosulfate as an antioxidant, polysorbate 80 as a surfactant, and hydroxyethylcellulose as a thickener in the amounts shown in Table 1 below. Substance P was synthesized through a solid / solution phase using peptide synthesis technology Fmoc-chemistry, and purified by high performance liquid chromatography to finally use a purity of 85% or more.

SP gel Composition of the composition content Substance P 5 μg / ml Sodium thiosulfate 0.1% Polysorbate 80 0.006% Hydroxyethyl cellulose 15%

Example 2: Comparison of wound healing effects of normal mice and mice induced with diabetes

In the case of intractable ulcers such as diabetic ulcers, the natural wound healing mechanism does not work properly, so the degree of wound healing is significantly slower than normal wounds. A screening experiment was conducted to confirm that a diabetic ulcer model with a slow therapeutic effect was correctly established, compared to skin wounds in normal mice without diabetes.

To induce diabetic ulcers, 8-week-old male C57BL / 6 mice (Dooyeol Biotech) were adapted for a week, then dissolved STZ (streptozotocin) 250 mg / kg in physiological saline and administered once intraperitoneally to induce diabetes. Did. The standard of diabetes was set to 350 mg / dl or more without fasting, and mice with diabetes were selected and skin wound models using the same were prepared. At the same time, a skin wound model using normal mice without STZ administration was also prepared.

Specifically, a mixed solution of ketamine and rompun was administered to the abdominal cavity of the mouse, followed by anesthesia, and then the electric shaver and hair removal cream (Veet) were used to remove the hair on the back. The part to remove the epidermis was sterilized using povidin and 70% ethanol, and a full-thickness wound was applied using a 5-mm biopsis punch on both sides of the lower gluteal region avoiding the spine of the mouse. To prevent the wound area from spontaneously shrinking by the epidermal muscles of the mouse, attach a silicone ring with an outer diameter of 15-mm and an inner diameter of 8-mm to the periphery of the wound using a surgical thread, and then attach the wound picture from 0 to 17 The day was taken and the extent of wound size reduction was quantified.

As can be seen in Figure 1, 10 days after causing the wound, the wound size was reduced to 50% compared to the initial level in normal mice, while the wound size was confirmed to remain at about 75% in diabetic-induced mice. Did. Furthermore, on the 17th day after the wound was induced, it was confirmed that the wound size was about 5% and about 60% in normal mice and mice induced with diabetes, respectively.

Through these results, a mouse model inducing diabetic ulcer was established as a representative model for intractable ulcer, and it was confirmed that it has distinctly different characteristics from simple wounds, not intractable ulcer.

Example 3: Wound treatment effect of SP gel in diabetic ulcer model

Example 3-1. SP gel administration in diabetic ulcer model

To compare the healing effect of P and SP gel on refractory ulcers, a diabetic skin wound mouse model was prepared. As a positive control group, EZF external solution (Daewoong Pharmaceutical) and intrasite gel (Smith & Nephew) were used as a wound treatment commercialized for the treatment of diabetic ulcer.

After 8 weeks old male C57BL / 6 mice (Dooyeol Biotech) were adapted for a week, STZ 250 mg / kg was dissolved in physiological saline and administered once intraperitoneally to induce diabetes. The standard of diabetes was set to 350 mg / dl or more without fasting, and only the mice in which diabetes was induced were selected to conduct the experiment.

A mixture of ketamine and rompun was administered to the abdominal cavity and anesthetized, followed by removal of the hair on the back using an electric shaver and hair removal cream (Veet). The part to remove the epidermis was sterilized using povidin and 70% ethanol, and a full-thickness wound was applied using a 5-mm biopsis punch on both sides of the lower gluteal region avoiding the spine of the mouse. In order to prevent the wound area from spontaneously shrinking by the epidermal muscles of the mouse, a silicone ring with an outer diameter of 15-mm and an inner diameter of 8-mm was attached around the wound using a surgical thread.

Subsequently, a phosphate buffer solution (posphate buffered saline: PBS) was administered as a negative control to the wound site, and EZF external solution and intrasite gel were administered as a positive control, and substances P and 5 at a concentration of 5 g / ml were used as the experimental group. SP gel at a concentration of g / ml was administered. The positive control, substance P and SP gel, were administered to the entire wound once every 3 to 4 days at 14 days.

Example 3-2. SP gel's wound size reduction effect in diabetic ulcer model

After administering the substance P and SP gel to the diabetic ulcer model as in Example 3-1, the effect of reducing the wound size was compared.

Specifically, the wounds were photographed and observed on the 0th, 4th, 7th and 14th days after administration of the substance P and SP gel. In addition, the size of the wound area was quantified using Photoshop to evaluate the degree of wound reduction.

As a result of visually confirming the size of the wound, as shown in FIGS. 2 (a) and 2 (b), it was confirmed that the effect of reducing the wound size of the SP gel was superior to that of the single administration of the substance P. Specifically, at the time of 7 days after administration of SP gel, the wound size was significantly reduced compared to the treatment with substance P alone, and it was confirmed that the wound reduction effect was superior to that of the conventional commercial product, EZF external solution and intrasite gel. .

As a result of quantifying the wound size over time, as shown in Figs. 2 (c) and (d), after 7 days of administration of the substance, the wound size of the SP gel administration group was only about 40%, whereas the substance P alone treatment group In the case of about 80% of the wounds remained, it was confirmed that the EZF external solution treatment group was about 70% and the intrasite gel treatment group was about 80%.

In addition, as can be seen from FIG. 2 (e), on the 14th day of administration, the wound size of the SP gel administration group was 20% or less, while the substance P alone treatment group was about 40%, the easy F external solution was about 30%, intra Sight gel was about 70%, and it was confirmed that there were still a lot of wounds compared to SP gel.

Through these results, it was confirmed that the SP gel has a roughly twice the effect of reducing the size of the wound compared to the treatment of the substance P alone, and furthermore, the SP gel is a conventional diabetic wound treatment agent compared to the external solution and intrasite gel commercially available. It was also found that there is a better effect.

Example 3-3. Effect of SP gel on wound tissue length and area reduction in diabetic ulcer model

After the SP gel was administered to the diabetic skin wound mouse model as in Example 3-2, the tissue was biopsied 14 days later to confirm the effect of reducing the wound tissue length and wound area. Specifically, the skin tissue of the wound was biopsied and fixed in 10% formalin, and then hematoxylin and eosin (H & E) staining was performed. After staining, the extent of tissue length and area reduction in the remaining wound site was quantified.

3 (b) and (c), it was confirmed that the length and area of the wound site tissue were reduced in most of the administration groups including the substance P compared to the negative control group. Specifically, on the 14th day after administration, the wound length and area of the negative control group were 1.5 mm and 45 mm ² , respectively, whereas in the substance P alone administration group, they were reduced to 1.2 mm and 40 mm ² , respectively. It decreased to 0.9 mm and 30 mm ² , respectively. Intrasite gel showed a lower wound healing effect than the negative control.

Among them, SP gel administration was the best, specifically, when SP gel administration, wound tissue length and area decreased to 0.6 mm and 20 mm ² . That is, it showed a wound healing effect that is 2 times superior to the material P alone treatment and about 1.5 times superior to the easy F external solution treatment.

Example 3-4. Effect of SP gel to reduce epithelial thickness and promote collagen synthesis in diabetic ulcer model

After SP gel was administered to the diabetic skin wound mouse model as in Example 3-3, tissue was biopsied 14 days later to confirm the effect of reducing epithelial tissue thickness and promoting collagen synthesis. In general, the tissue that has been cured has a thin epithelial shape like the epithelial tissue of normal skin, whereas the wounded tissue has a thicker epithelial shape than the normal epithelial tissue (Experimental & Molecular Medicine (2018) 50:68).

Specifically, after fixing the skin tissue of the wound by biopsy, hematoxylin-eosin staining was performed to confirm the thickness of the epithelial tissue, and further, the biopsy skin tissue was stained with Masson's Trichrome to synthesize collagen. The degree of palpation was confirmed.

As a result, as shown in Figure 4 (b), the negative control group showed an epithelial tissue condition of about 300% thicker than that of normal skin tissue, and in the case of the intrasite gel-treated group, the initial wound was not formed properly in the epithelial tissue itself. Showed the steps. In addition, when the EGF external solution and the substance P alone were treated, they showed epithelial tissue states of about 220% and 150%, respectively.

On the other hand, SP gel showed a thickness of about 100% of epithelial tissue similar to that of normal skin tissue, showing the most excellent wound healing effect among treatment groups. In addition, as shown in Fig. 4 (a), through SP gel administration, the degree of staining was stronger than that of the substance P alone treatment group and the easy F external solution treatment group, thereby confirming that the collagen increase effect was much better.

Therefore, SP gel is to demonstrate a significantly superior wound healing effect, compared to the material P alone and commercial products, EZF external solution and intrasite gel.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without changing its technical spirit or essential characteristics. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the following claims rather than the detailed description and equivalent concepts thereof.

Claims (11)

A pharmaceutical composition for treating refractory ulcers comprising Substance P consisting of the amino acid sequence of SEQ ID NO: 1, antioxidant, surfactant and thickener.
According to claim 1, The antioxidant is sodium thiosulfate (sodium thiosulfate), pharmaceutical composition for the treatment of refractory ulcers.
According to claim 1, The surfactant is polysorbate 80 (polysorbate 80), a pharmaceutical composition for the treatment of refractory ulcers.
According to claim 1, wherein the thickener is hydroxyethyl cellulose (hydroxylethyl-cellulose), pharmaceutical composition for the treatment of refractory ulcers.
According to claim 1, The intractable ulcer is diabetic ulcer, pressure ulcer, venous ulcer, arterial ulcer will include, pharmaceutical composition for the treatment of intractable ulcer.
According to claim 1, The composition is to promote collagen synthesis, a pharmaceutical composition for the treatment of intractable ulcer.
According to claim 1, wherein the composition is a liquid, suspension, emulsion, cream, hydrogel, lotion, ointment, spray, patch or aerosol formulation, pharmaceutical composition for treating intractable ulcers.
A quasi-drug composition for the treatment of refractory ulcer, comprising a substance P consisting of the amino acid sequence of SEQ ID NO: 1, an antioxidant, a surfactant, and a thickener.
The composition of claim 8, wherein the composition comprises the substance P consisting of the amino acid sequence of SEQ ID NO: 1, sodium thiosulfate, polysorbate 80, and hydroxyethylcellulose.
The composition of claim 8, wherein the intractable ulcer comprises diabetic ulcer, pressure ulcer, venous ulcer, and arterial ulcer.
A method of treating intractable ulcer comprising the step of administering the pharmaceutical composition of any one of claims 1 to 7 to an individual other than a human.

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