JP4096115B2 - Skin wound healing promoter - Google Patents

Description

【００２９】
（２） 皮膚創傷治癒に対する作用
ラット(Wistar)にジエチルエーテルの吸入麻酔下で、クエン酸緩衝液に溶解したストレプトゾトシン（70mg/kg BW）を尾静脈より注射し、糖尿病ラット(DM Rat)を作成した（各群５匹）。また、ラット(Wistar)にクエン酸緩衝液のみを同量静注して非糖尿病ラット(non DM Rat)を作成した（各群５匹）。静注から４週間後にすべてのラットの背部体毛をジエチルエーテル吸入麻酔下でバリカンで剃毛し，脱毛用クリームで除毛した。２４時間後に５ｍｍ径の皮膚生検用トレパンを用いて背部皮膚に表皮・真皮の全層性の創傷を等間隔で５箇所作成し、止血を確認した後に表２および表３の各軟膏を１日１回塗布した。また、１日１回の軟膏塗布の直前に背部の創傷を写真撮影しその面積を測定した。非糖尿病ラット(non DM Rat)についての面積測定結果を表２に、また、糖尿病ラット(DM Rat)についての面積測定結果を表３に示す。なお、各表中の値は５例の平均値を示す。
【００３０】
【表２】

【００３１】
【表３】

【００３２】
【００３３】
【発明の効果】
表１から明らかなように、サブスタンスＰ、ＦＧＬＭおよびＩＧＦ−Ｉをそれぞれ単独使用して組織片を培養しても、皮膚表皮の伸長はコントロール（無添加）使用の場合と同程度であるのに対して、サブスタンスＰ若しくはＦＧＬＭとＩＧＦ−Ｉを共存させて組織片を培養した場合は、これらの薬剤を単独使用した場合の２〜２．５倍もの伸長（相乗的効果）を示す。また、表２は、サブスタンスＰおよびＩＧＦ−Ｉを併用する軟膏がサブスタンスＰ若しくはＩＧＦ−Ｉを単独で使用する場合よりも顕著に皮膚の創傷治癒を促進することを示し、表３は難治性の糖尿病ラット(DM Rat)に対しても優れた効果を発揮することを示すものである。したがって、サブスタンスＰやＦＧＬＭに代表されるサブスタンスＰ類縁体およびその医薬として許容される塩類の少なくとも１つとＩＧＦ−Ｉに代表されるインシュリン様成長因子を併用（混合）して投与すれば、これらの薬剤が相乗的に作用して裂傷、擦過傷、外科的切開、皮膚潰瘍、とりわけ糖尿病患者の難治性皮膚潰瘍、火傷などの皮膚創傷の治癒を効果的に促進できる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a skin wound healing promoter containing substance P analog and at least one of these pharmaceutically acceptable salts and an insulin-like growth factor as active ingredients.
[0002]
[Prior art]
Skin wounds are surface tissue damage such as lacerations, abrasions, surgical incisions, skin ulcers, and burns. In the treatment of such skin wounds, it is common to wait for a natural healing by the living body's own resilience after applying an emergency treatment to the injured site.
[0003]
However, since such natural healing requires a long period of time for recovery and pain continues, it is desirable to actively promote wound healing by administering a wound therapeutic agent to the injured site.
[0004]
In the wound healing process, new epithelial tissues and connective tissues are formed by cell migration and proliferation. Therefore, agents that promote or stimulate the migration, differentiation, and proliferation of cells involved in wound healing can be used as wound healing agents. As such a wound treatment agent, lysozyme chloride, solcoserine and the like are known.
[0005]
However, existing wound treatment agents have a problem in that wound healing is not sufficiently promoted and wounds cannot be completely cured in a short period of time. These wound treatments are thought to be due to their small contribution to re-covering of the epidermis, collagen synthesis, improvement of peripheral circulation, granulation, and angiogenesis, which are important factors in the wound healing process. ing.
[0006]
By the way, substance P analog is a polypeptide composed of 3 to 12 amino acids specifically disclosed in US Pat. No. 3,862,114, and these polypeptides are known to have antihypertensive action. Yes. Substance P, one of the substance P analogs, is a polypeptide released and produced from the nerve terminal and is Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH ₂ It is a polypeptide consisting of 11 amino acids, and is known to exhibit vasodilation, smooth muscle contraction, salivary gland secretion promotion, diuretic action, and the like. In the ophthalmological field, the effect of substance P on the abnormal secretion of conjunctival goblet cells in eye disorders (international patent publication WO95 / 13087) and the dynamics of substance P during inflammation such as keratitis (Journal of the Ophthalmological Society of Japan) 91 , 982-987 (1987), Journal of the Japanese Ophthalmological Society, 92 , 448-452 (1988)). Furthermore, Phe-Gly-Leu-Met-NH ₂ (hereinafter referred to as FGLM), which is a tetrapeptide on the C-terminal side of substance P, has an effect as a corneal disorder therapeutic agent. It is described in.
[0007]
On the other hand, insulin-like growth factor is one of the factors that regulate the growth of normal human cells, such as epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, and transforming growth factor. Are insulin-like growth factor-I (hereinafter referred to as IGF-I) and insulin-like growth factor-II (hereinafter referred to as IGF-II). It stimulates thyroid cell proliferation for IGF-I (J. Biol. Chem., 264 , 18485-18488 (1989)) and regulates muscle growth and differentiation for IGF-II (Hum Mol. Genet., 3 , 1117-1121 (1994)).
[0008]
However, neither substance P analogs nor insulin-like growth factors have been reported to have an effect on skin diseases.
[0009]
[Problems to be solved by the invention]
Therefore, it was an interesting subject to conduct a pharmacological test on the skin epidermis by combining substance P analog and insulin-like growth factor to examine the effect of promoting skin epidermal elongation and the effect of promoting skin wound healing.
[0010]
[Means for Solving the Problems]
When the present inventors perform the above pharmacological tests, if substance P analogs typified by substance P and FGLM coexist with insulin-like growth factors typified by IGF-I, these drugs are synergistic. It has been found that it acts on the skin to significantly promote the elongation of the skin epidermis and the healing of skin wounds, and also exhibits an excellent healing effect against refractory diabetic skin ulcers. That is, the skin wound healing promoter of the present invention comprising as an active ingredient at least one substance P analog and pharmaceutically acceptable salts thereof and an insulin-like growth factor is a laceration, abrasion, surgical incision, skin ulcer (diabetic (Including skin ulcers), skin wounds such as burns, and diseases such as gangrene resulting therefrom. In addition, ascorbic acid, ascorbic acid ester, salt of ascorbic acid, pantothenic acid, salt of pantothenic acid and the like that have already been confirmed to have a wound healing effect may be added to the skin wound healing promoter of the present invention. it can.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Substance P as the analogs, for example, Tyr-Arg-Pro-Lys- Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe _{-Gly-Leu-Met-NH 2} ( substance P), Arg-Pro-Lys -Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH 2, Tyr-Pro-Gln-Gln-Phe-Phe _{-Gly-Gln-Met-NH 2} , Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2, Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH 2, Gln- Gln-Phe-Phe-Gly- Leu-Met-NH 2, Asp-Ala-P _{e-Tyr-Gly-Leu-} Met-NH 2, Gln-Phe-Phe-Gly-Leu-Met-NH 2, Ala-Phe-Tyr-Gly-Leu-Met-NH 2, Phe-Phe-Gly-Leu _{-Met-NH 2, Tyr-Phe} -Gly-Leu-Met-NH 2, Gly-Phe-Gly-Leu-Met-NH 2, Phe-Gly-Leu-Met-NH 2 (FGLM), Tyr-Gly- such as Leu-Met-NH ₂ and the like. More preferable examples include substance P and FGLM.
[0012]
As for the amino acid structure of substance P analog, there are L-form, D-form and DL-form, all of which are included in the present invention. A more preferred form in the present invention is a polypeptide in which all three-dimensional structures are L-forms.
[0013]
Substance P analogs, pharmaceutically acceptable salts of substance P and FGLM include, for example, hydrochloride, sulfate, phosphate, lactate, maleate, fumarate, oxalate, methanesulfonic acid Examples thereof include salts and p-toluenesulfonate.
[0014]
The skin wound healing promoter of the present invention can be formulated using a widely used technique. Examples of the dosage form include ointments, jellies, patches, patches, lotions, creams, sprays, aerosols, plasters, suspensions, emulsions, tablets, pills, etc. It can also be used as a liquid agent by selecting an appropriate solvent. In addition, in order to prepare a skin wound healing promoter, filler, excipient, base, disintegrant, extender, binder, film agent, lubricant, colorant, pH adjustment according to the dosage form Agents, solubilizers, suspending agents, buffers, stabilizers, preservatives, preservatives, surfactants, antioxidants, dispersants, emulsifiers, solubilizers, solubilizers and the like can be added.
[0015]
Examples of the carrier for the preparation include white petrolatum, liquid paraffin, gelled hydrocarbon, cetyl alcohol, polyethylene glycol, gelatin, corn starch, sodium alginate, methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, plastic base hydrophilic, gelatin, Dextrin, cetyl alcohol, stearyl alcohol, polyethylene glycol, polyvinyl alcohol, methoxyethylene-maleic anhydride copolymer, polyvinyl ether, polymer / copolymer comprising vinylpyrrolidone, sodium stearate, magnesium stearate, chloride Benzalkonium, olive oil, camellia oil, soybean oil and other fats, lactose, water and the like.
[0016]
The amount of substance P analog and pharmaceutically acceptable salt thereof contained in the skin wound healing promoter is 0.001 to 10% by weight, more preferably in the range of 0.005 to 5% by weight. It is. The amount of insulin-like growth factor contained in the skin wound healing promoter is 0.0001 to 5% by weight, more preferably 0.001 to 1% by weight.
[0017]
The skin wound healing promoter of the present invention can be administered in various forms depending on the wound site and the degree of wound. For example, when used as an external preparation, it is desirable to directly apply, spray, or affix this agent to a required site (affected area) such as the skin.
[0018]
The dosage of the skin wound healing promoter of the present invention can be appropriately selected in consideration of symptoms, age, dosage form and the like. The substance P analog and pharmaceutically acceptable salts thereof are usually 0.01 to 5000 mg, preferably 0.1 to 1000 mg per day, and divided into insulin-like growth factor once or several times. It is administered in combination (mixed). The dose of insulin-like growth factor is usually 0.001 to 1000 mg, preferably 0.01 to 500 mg per day. Substance P analogs and pharmaceutically acceptable salts thereof are divided into one or several times. Administered in combination (mixed) with at least one of the above. The skin wound healing promoter of the present invention can be administered in 1 to 5 times a day.
[0019]
Formulation examples and pharmacological test results are shown below, but these examples are for better understanding of the present invention and do not limit the scope of the present invention.
[0020]
【Example】
[Formulation example]
The typical formulation example of this invention is shown below.
[0021]
Example 1 (ointment A-1)
Substance P 100mg in 100g
IGF-I 10mg
Liquid paraffin 10g
White petrolatum appropriate amount [0022]
By changing the amount of substance P added, the concentration was 0.2% (w / w) (ointment A-2), 0.5% (w / w) (ointment A-3), 1.0% (w / W) (ointment A-4) can be prepared.
[0023]
Example 2 (ointment B-1)
FGLM 100mg in 100g
IGF-I 5mg
Liquid paraffin 10g
White petrolatum appropriate amount [0024]
By changing the amount of FGLM added, the concentration was 0.3% (w / w) (ointment B-2), 1% (w / w) (ointment B-3), 2% (w / w) (ointment The ointment of B-4) can be prepared.
[0025]
Example 3 (ointment C-1)
FGLM 50mg in 100g
IGF-I 10mg
Ascorbic acid 3mg
Liquid paraffin 10g
Plastic base hydrophyll appropriate amount [0026]
By changing the amount of FGLM added, the concentration was 0.3% (w / w) (ointment C-2), 1% (w / w) (ointment C-3), 3% (w / w) (ointment C-4) ointments can be prepared.
[0027]
[Pharmacological test]
(1) Action on skin epidermal elongation Rats (Spraque-Dawley) were over-administered with pentobarbital, and then an about 2 × 4 mm ear tissue piece was prepared. 18 ml of each TCM199 medium containing each test drug [Substance P, FGLM, IGF-I, Substance P + IGF-I and FGLM + IGF-I] in Table 1 was placed in a 60 mm dish, and three tissue pieces were added to each dish. (12 cases in each group: 3 sections). After culturing in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 24 hours, the tissue piece was fixed with ethanol-acetic acid (volume ratio 95: 5), and 3 tissue sections per tissue piece were according to a conventional method. Was made. After staining with hematoxylin-eosin, the length of the epidermal cell layer with the cut surface extended was measured. As a control, a culture solution containing no test drug was used and tissue pieces were cultured under the same conditions as described above. These results are shown in Table 1. In addition, the value in a table | surface shows the average value of 12 examples.
[0028]
[Table 1]

[0029]
(2) Effect on skin wound healing Rats (Wistar) are injected with streptozotocin (70 mg / kg BW) dissolved in citrate buffer through the tail vein under diethyl ether inhalation anesthesia to create diabetic rats (DM Rat) (5 in each group). In addition, non-diabetic rats (non DM Rats) were prepared by intravenously injecting the same amount of citrate buffer alone to rats (Wistar) (5 rats per group). Four weeks after the intravenous injection, the back body hairs of all rats were shaved with a clipper under diethyl ether inhalation anesthesia and removed with a hair removal cream. After 24 hours, 5 mm diameter skin biopsy trepans were used to create 5 layers of full-thickness epidermis and dermis on the back skin at regular intervals, and after confirming hemostasis, each ointment in Tables 2 and 3 was 1 It was applied once a day. Moreover, the wound of the back was photographed immediately before ointment application once a day, and the area was measured. The area measurement results for non-diabetic rats (non DM Rat) are shown in Table 2, and the area measurement results for diabetic rats (DM Rat) are shown in Table 3. In addition, the value in each table | surface shows the average value of 5 examples.
[0030]
[Table 2]

[0031]
[Table 3]

[0032]
[0033]
【The invention's effect】
As is apparent from Table 1, even when the tissue pieces were cultured using each of substance P, FGLM and IGF-I alone, the elongation of the skin epidermis was almost the same as in the case of using the control (no addition). On the other hand, when a tissue piece is cultured in the presence of substance P or FGLM and IGF-I, the elongation (synergistic effect) is 2 to 2.5 times that when these drugs are used alone. Table 2 also shows that an ointment that combines substance P and IGF-I promotes skin wound healing significantly more than when substance P or IGF-I is used alone, and Table 3 shows that refractory It also shows that it exhibits excellent effects on diabetic rats (DM Rat). Therefore, if a substance P analog represented by substance P or FGLM and at least one of its pharmaceutically acceptable salts and insulin-like growth factor represented by IGF-I are administered in combination (mixed), The drugs can act synergistically to effectively promote healing of lacerations, abrasions, surgical incisions, skin ulcers, especially skin wounds such as refractory skin ulcers and burns in diabetic patients.

Claims (8)

_{Tyr-Arg-Pro-Lys-} Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH _{2, Arg-Pro-Lys-} Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH 2, Tyr-Pro-Gln-Gln-Phe-Phe-Gly-Gln-Met-NH 2, Pro- _{Gln-Gln-Phe-Phe-} Gly-Leu-Met-NH 2, Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH 2, Gln-Gln-Phe-Phe-Gly-Leu-Met- _{NH 2, Asp-Ala-Phe} -Tyr-Gly-Leu-Met-NH 2, Gln- _{Phe-Phe-Gly-Leu-} Met-NH 2, Ala-Phe-Tyr-Gly-Leu-Met-NH 2, Phe-Phe-Gly-Leu-Met-NH 2, Tyr-Phe-Gly-Leu-Met A substance P analog selected from the group consisting of —NH ₂ , Gly-Phe-Gly-Leu-Met-NH ₂ , Phe-Gly-Leu-Met-NH ₂ and Tyr-Gly-Leu-Met-NH ₂ or its A skin wound healing promoter containing pharmaceutically acceptable salts and insulin-like growth factor as active ingredients. _{Tyr-Arg-Pro-Lys-} Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH _{2, Arg-Pro-Lys-} Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH 2, Tyr-Pro-Gln-Gln-Phe-Phe-Gly-Gln-Met-NH 2, Pro- _{Gln-Gln-Phe-Phe-} Gly-Leu-Met-NH 2, Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH 2, Gln-Gln-Phe-Phe-Gly-Leu-Met- _{NH 2, Asp-Ala-Phe} -Tyr-Gly-Leu-Met-NH 2, Gln- _{Phe-Phe-Gly-Leu-} Met-NH 2, Ala-Phe-Tyr-Gly-Leu-Met-NH 2, Phe-Phe-Gly-Leu-Met-NH 2, Tyr-Phe-Gly-Leu-Met A substance P analog selected from the group consisting of —NH ₂ , Gly-Phe-Gly-Leu-Met-NH ₂ , Phe-Gly-Leu-Met-NH ₂ and Tyr-Gly-Leu-Met-NH ₂ or its A skin wound healing promoter comprising as an active ingredient 0.001 to 10% by weight of a pharmaceutically acceptable salt and 0.0001 to 5% by weight of an insulin-like growth factor. Arg-Pro-Lys-Pro- Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2, Phe-Gly-Leu-Met-NH 2 and at least one insulin-like salts which are acceptable their pharmaceutically A skin wound healing promoter containing growth factor-I as an active ingredient. Is acceptable 0.001-10 wt% of Arg-Pro-Lys-Pro- Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2, Phe-Gly-Leu-Met-NH 2 and their pharmaceutically A skin wound healing promoter comprising as an active ingredient at least one of the following salts and 0.0001 to 5% by weight of insulin-like growth factor-I.   The skin wound healing promoter according to any one of claims 1 to 4, wherein the skin wound is a laceration, abrasion, surgical incision, skin ulcer or burn.   The skin wound healing promoter whose skin ulcer of Claim 5 is a diabetic skin ulcer. _{Tyr-Arg-Pro-Lys-} Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH _{2, Arg-Pro-Lys-} Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH 2, Tyr-Pro-Gln-Gln-Phe-Phe-Gly-Gln-Met-NH 2, Pro- _{Gln-Gln-Phe-Phe-} Gly-Leu-Met-NH 2, Pro-Gln -Gln-Phe-Tyr-Gly-Leu-Met-NH 2, Gln-Gln-Phe-Phe-Gly-Leu-Met- _{NH 2, Asp-Ala-Phe} -Tyr-Gly-Leu-Met-NH 2, Gln _{Phe-Phe-Gly-Leu-} Met-NH 2, Ala-Phe-Tyr-Gly-Leu-Met-NH 2, Phe-Phe-Gly-Leu-Met-NH 2, Tyr-Phe-Gly-Leu-Met A substance P analog selected from the group consisting of —NH ₂ , Gly-Phe-Gly-Leu-Met-NH ₂ , Phe-Gly-Leu-Met-NH ₂ and Tyr-Gly-Leu-Met-NH ₂ or its A skin epidermal elongation promoter comprising pharmaceutically acceptable salts and insulin-like growth factor as active ingredients. _{Tyr-Arg-Pro-Lys-} Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH _{2, Arg-Pro-Lys-} Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH 2, Tyr-Pro-Gln-Gln-Phe-Phe-Gly-Gln-Met-NH 2, Pro- _{Gln-Gln-Phe-Phe-} Gly-Leu-Met-NH 2, Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH 2, Gln-Gln-Phe-Phe-Gly-Leu-Met- _{NH 2, Asp-Ala-Phe} -Tyr-Gly-Leu-Met-NH 2, Gln- _{Phe-Phe-Gly-Leu-} Met-NH 2, Ala-Phe-Tyr-Gly-Leu-Met-NH 2, Phe-Phe-Gly-Leu-Met-NH 2, Tyr-Phe-Gly-Leu-Met A substance P analog selected from the group consisting of —NH ₂ , Gly-Phe-Gly-Leu-Met-NH ₂ , Phe-Gly-Leu-Met-NH ₂ and Tyr-Gly-Leu-Met-NH ₂ or its A skin epidermal elongation promoter containing as an active ingredient 0.001 to 10% by weight of a pharmaceutically acceptable salt and 0.0001 to 5% by weight of an insulin-like growth factor.