JP2009179571A - Agent for reinforcing wound-treating effect of g-csf by external application - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new agent for reinforcing the wound-treating effect of G-CSF (globule of colony-forming stimulating factor) by external application. <P>SOLUTION: Provided is an agent for reinforcing the wound-treating effect of the G-CSF by external application, characterized by containing propyleneglycol alginate as an active ingredient. The propyleneglycol alginate does not have such a property for depositing in an acidic region to produce gel, as that of sodium alginate. Thereby, the propyleneglycol alginate has an advantage suitable for preparing a liquid composition in which the G-CSF stable in an acidic region is stably retained. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an agent for enhancing the effect of treating wounds when G-CSF is externally applied.

It is already known that G-CSF (granulocyte colony-stimulating factor) has a wound healing effect in external application. For example, in Patent Document 1, G-CSF is used externally for intractable skin ulcers that are positioned as one of wounds. The therapeutic effect is shown. In Patent Document 1, when alginic acid or a salt thereof is applied to a wound site together with G-CSF, these increase the abundance of G-CSF in a certain period or suppress the attenuation in blood, Moreover, it describes that it contributes to the improvement of the therapeutic effect with respect to an intractable skin ulcer by increasing a patient's blood flow.
International Publication No. 2007/119486 Pamphlet

However, the search for a new substance having an excellent enhancing effect on the wound treatment effect of G-CSF for external use is significant in establishing an excellent treatment method for various wounds including refractory skin ulcers. It is deep.
Then, an object of this invention is to provide the enhancer of the wound treatment effect in the external use of novel G-CSF.

  As a result of intensive studies in view of the above points, the inventor of the present invention is superior in wound healing effect of G-CSF by using alginate propylene glycol ester which is one of polyhydric alcohol esters of alginic acid. It was found to have an enhancing action.

As described in claim 1, the enhancer of the external wound treatment effect of G-CSF of the present invention made on the basis of the above findings is characterized by containing propylene glycol alginate as an active ingredient.
Moreover, the liquid composition for external use for wound treatment of this invention is characterized by including G-CSF and a propylene glycol alginate at least as described in Claim 2.

  ADVANTAGE OF THE INVENTION According to this invention, the enhancer of the wound treatment effect in the external use of novel G-CSF can be provided.

  In the present invention, propylene glycol alginate, which is an active ingredient of an enhancer of wound healing effect for external use of G-CSF, is a known substance in which at least a part of the carboxyl group of alginic acid is ester-bonded with propylene glycol. It is already highly utilized as an additive (gelling agent, thickener, etc.), etc., and has high safety.

  In the present invention, G-CSF, which is a target for enhancing the wound treatment effect by external use with propylene glycol alginate, has an action of differentiating and proliferating granulocytes, and can be recognized as G-CSF by those skilled in the art. It is not limited to human-derived natural products having a known amino acid sequence, but is a product produced by genetic engineering techniques or analogs in which one or more amino acids are substituted, deleted, added or inserted into the amino acid sequence. May be. The main effect of G-CSF in wound healing is mobilization of all cells necessary for tissue repair such as neutrophils from bone marrow, myofibroblasts induced to differentiate into fibroblasts, megakaryocytes induced to differentiate into platelets, etc. Is to help. This effect can be expected to be enhanced by using in combination with other vascular endothelial cell growth factors such as fibroblast growth factor (FGF), and can further amplify granulation ability. Moreover, as an effect by application of G-CSF to a wound site, differentiation promoting action of bone marrow-derived cells having a low degree of differentiation such as stem cells infiltrated in the vicinity of the wound site may be considered.

  For example, when G-CSF is applied to a wound site, the alginate propylene glycol ester can exert its effect on the external wound treatment effect of G-CSF by applying alginic acid propylene glycol ester to the wound site simultaneously. it can. Here, the term “application” means a local administration method to a wound site such as application or spraying. Such an embodiment can be easily realized by using an external liquid composition for wound treatment comprising at least G-CSF and propylene glycol alginate.

  The liquid composition for external use for wound treatment comprising at least G-CSF and propylene glycol alginate is, for example, G-CSF and propylene glycol alginate, purified water, physiological saline, phosphate buffer, hydrochloric acid solution, etc. It can be prepared by dissolving in a solvent. If necessary, a surfactant such as polysorbate or a stabilizer such as mannitol may be added as an additive. The concentration of G-CSF in the liquid composition is preferably 1 μg / mL to 2 mg / mL, more preferably 5 μg / mL to 1 mg / mL, and more preferably 7.5 μg / mL, where the wound treatment effect is dependent on concentration. mL to 250 μg / mL. If the concentration is too low, the wound healing effect of G-CSF will not be exhibited, but if the concentration is too high, the effect will not improve so much. The concentration of propylene glycol alginate contained in the liquid composition is preferably 0.001% to 5%, more preferably 0.01% to 3%, and even more preferably 0.05% to 1% (weight% concentration). ). If the concentration is too high, the viscosity of the liquid composition becomes high and spraying becomes difficult. On the other hand, if the concentration is too low, sufficient enhancement of the wound treatment effect of G-CSF is obtained. And the problem that the viscosity of the liquid composition is low and G-CSF cannot be sufficiently retained on the wound surface (therefore, the viscosity of the liquid composition is 1.5 mPa · s to 20 mPa · s). Desirably 5 mPa · s to 10 mPa · s is more desirable). The pH of the liquid composition is preferably 2.5 to 5.0, more preferably 3.0 to 4.5 in order to keep G-CSF stable in the composition. More preferably, it is 5 to 4.3. The pH of the liquid composition can be adjusted as necessary using an inorganic acid (such as hydrochloric acid) or an organic acid (such as citric acid or acetic acid) as a pH adjuster. When sodium alginate is dissolved in a solvent, the pH of the solution is about 7. Therefore, when preparing a liquid composition containing G-CSF and sodium alginate, in order to keep G-CSF stable in the composition, it is necessary to shift the pH to an acidic region using a pH adjuster. Sodium alginate has the property of precipitating and gelling in the acidic region (especially when the pH is 4.3 or lower). Therefore, when preparing a liquid composition in which G-CSF is stably maintained, sodium alginate has a problem that it is difficult to handle. In contrast, when the propylene glycol alginate is dissolved in a solvent, the pH of the solution is about 4. Alginic acid propylene glycol ester does not have the property of being precipitated and gelled in an acidic region like sodium alginate. Moreover, the propylene glycol alginate has less influence on the stability of G-CSF than sodium alginate. Therefore, propylene glycol alginate has an advantage that it is convenient for preparing a liquid composition in which G-CSF is stably maintained.

The liquid composition for external use for wound treatment comprising at least G-CSF and propylene glycol alginate can be formulated into a liquid, a lotion, a spray (spray), or the like according to a commonly performed method. In formulation, various components such as stabilizers, thickeners, solubilizers, preservatives, extenders, tonicity agents, bactericides, preservatives, gelling agents, etc., known per se may be added. Needless to say. The topical liquid composition, for example, once a day to several times or 1 week to 1 month at once / day 1-7 days, preferably a ^{G-CSF 0.1μg / cm 2 ~500μg} / cm 2 at a rate of, more preferably in a ratio of ^{0.5μg / cm 2 ~100μg / cm 2} , more preferably by or sprayed or applied to the wound site at a rate of ^{1μg / cm 2 ~10μg / cm 2} , Excellent wound treatment effect can be expected. In addition, this liquid composition for external use is infiltrated into a base sheet such as a collagen sheet (collagen spun into a cotton fiber) or an alginate sheet (a non-woven fabric made from alginate fiber). You may use it by formulating into an agent. By using in this way, synergistic or additive effects with the moisture retention and wound healing promoting effects of the base sheet can be expected (the dose of G-CSF to the wound site is the same as above). .

  In the present invention, the term “wound” means damage to the body surface such as skin and mucous membrane, and specifically, trauma and burns, pressure ulcers (bed sores), diabetic skin ulcers, ischemic skin ulcers Examples include skin ulcers such as intractable skin ulcers.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is limited to the following description and is not interpreted.

Experimental Example 1: Evaluation of potentiating effect on external wound treatment effect of G-CSF by propylene glycol alginate (Part 1)
(A) About the liquid composition used as a sample Soba Co., Ltd. genetically modified human G-CSF formulation (trade name: Gran Injection 150), sodium alginate (Kimika), propylene glycol alginate (Kimika) The following three liquid compositions were prepared using 10 mM acetate buffer as physiological saline and a pH adjuster. Table 1 shows the pH and viscosity of each liquid composition.
(1) G-CSF 150 μg / 0.6 mL (trade name: Gran Injection 150)
(2) 1.75 mL of G-CSF 150 μg / 0.5% sodium alginate solution
(3) 1.75 mL of G-CSF 150 μg / 0.5% alginate propylene glycol ester solution

(B) About preparation of wound model and evaluation of wound treatment effect of sample using this model Rat (SPF), Crl: CD (SD) strain (purchased from Japan Charles River Co., Ltd.), skin as wound model A cut model was made. Specifically, after anesthetizing the rat with sodium pentobarbital (Dainippon Sumitomo Pharma Co., Ltd.) (40 mg / 0.8 mL / kg, ip), the back was shaved with an electric clipper. The shaved skin was disinfected with alcohol cotton, and then a 3.4 cm cut was made on the midline with a scalpel. A total of three places, 1 cm each from the central part of the incision part to the head side and the tail side from the central part, were sutured with Miher (Michel suture rod: large 3.0 × 14 mm, Natsume Seisakusho Co., Ltd.). Each cut was applied to the cut site so that 50 μg of G-CSF was administered per cut site on the first day, with the cut creation date as the first day. On the 4th day, the michel was removed under diethyl ether anesthesia. On the fifth day, the rats were weighed and killed by diethyl ether overanesthesia, and then the skin around the wound site was peeled off to form a strip-shaped skin piece (short piece about 2 cm × long piece about 3 cm). ), One of the short pieces is fixed at a position of about 0.5 cm from the end, and the wound tension (load required for the cut to be cut) is measured with a tensile tester for measuring wound healing (TK-251, finite). The ratio of increase from the average wound strength of the control (non-treatment) of the average wound strength of each sample was calculated (n = 10). The results are shown in Table 2.

  As is clear from Table 2, the wound healing effect of G-CSF applied externally was enhanced by the coexistence of sodium alginate or propylene glycol alginate, but the degree of enhancement was greater for propylene glycol alginate than for sodium alginate. Was far superior.

Experimental Example 2: Evaluation of enhancing action on external wound treatment effect of G-CSF with propylene glycol alginate (Part 2)
In the same manner as in Experimental Example 1, four types of liquid compositions (G-CSF containing 0.5) were administered by applying 0.2 mL per wound site by applying 1.5 μg, 5 μg, 15 μg, and 50 μg of G-CSF. % Alginic acid propylene glycol ester solution) was prepared, and in the same manner as in Example 1, the increase ratio from the average wound strength of the average wound strength control (no treatment) of each sample was calculated (n = 10). The results are shown in Table 3.

  As is apparent from Table 3, it was found that the coexistence of propylene glycol alginate ester enhances the wound treatment effect of G-CSF externally in a dose-dependent manner in a dose range of at least 1.5 μg to 50 μg.

Experimental Example 3: Evaluation of the effect of propylene glycol alginate on the stability of G-CSF In the same manner as in Experimental Example 1, a 0.5% alginate propylene glycol ester solution having a G-CSF concentration of 86 μg / mL, A 0.5% sodium alginate solution having a CSF concentration of 86 μg / mL was prepared, and the stability of G-CSF after 3 weeks storage at 4 ° C. and after 4 weeks storage was evaluated by SDS electrophoresis (SDS-PAGE). did. The results are shown in FIG. 1 (the details of the lane are as follows).
1: Control (G-CSF before starting storage test)
2: G-CSF in propylene glycol ester alginate solution after storage for 4 weeks
3: G-CSF in propylene glycol alginate solution after storage for 3 weeks
4: G-CSF in sodium alginate solution after 4 weeks storage
5: G-CSF in sodium alginate solution after 3 weeks storage
As is clear from FIG. 1, it was found that the G-CSF in the propylene glycol alginate solution was less likely to deteriorate than the G-CSF in the sodium alginate solution.

Formulation Example 1: Lotion (Part 1)
(G / 100mL)
Glycerin 10
Ethanol 10
1,3-butylene glycol 5
Hydroxyethyl cellulose 1
Cetanol 1
G-CSF 0.02
Atelocollagen 0.3
Alginic acid propylene glycol ester 0.25
Citric acid monohydrate 0.66
Trisodium citrate dihydrate 0.27
Methyl paraoxybenzoate 0.1
Sodium dihydrogen phosphate 3
Purified water 68.4
A lotion preparation for treating skin ulcer having the above component composition was produced by a method for producing a lotion preparation known per se.

Formulation Example 2: Lotion (Part 2)
(G / 100mL)
Glycerin 10
Ethanol 10
1,3-butylene glycol 5
Hydroxyethyl cellulose 1
Cetanol 1
G-CSF 0.02
Alginic acid propylene glycol ester 0.25
Citric acid monohydrate 0.66
Trisodium citrate dihydrate 0.27
Methyl paraoxybenzoate 0.1
Sodium dihydrogen phosphate 3
Purified water 68.7
A lotion preparation for treating skin ulcer having the above component composition was produced by a method for producing a lotion preparation known per se.

Formulation Example 3: Sheet (Part 1)
(G / 100mL)
Glycerin 7
1,3-butylene glycol 3
Pentylene glycol 5
Phenoxyethanol 0.5
Methylparaben 0.1
G-CSF 0.02
Alginic acid propylene glycol ester 0.5
Sodium dihydrogen phosphate 3
Purified water 80.88
By infiltrating a medicinal stock solution having the above component composition into a low antigenic collagen sheet (Nippon Organ Pharmaceutical Co., Ltd.) obtained by spinning atelocollagen (collagen from which an antigenic expression site has been removed by a proteolytic enzyme) into a cotton fiber shape. A skin ulcer treatment sheet was produced.

Formulation Example 4: Sheet (Part 2)
Formulation Example 3 except that instead of the collagen sheet in Formulation Example 3, an alginate sheet (Combatec's trade name: Caltostat in which a mixed salt of calcium and sodium alginate was made into a fibrous form to form a nonwoven fabric) was used. Similarly, a sheet for treating skin ulcer was produced.

  INDUSTRIAL APPLICABILITY The present invention has industrial applicability in that it can provide a novel agent for enhancing wound healing effect when G-CSF is applied externally.

It is a result of SDS-PAGE which shows the influence which it has on the stability of G-CSF of the propylene glycol alginate in Experimental example 3.

Claims (2)

  An agent for enhancing the effect of treating wounds by external use of G-CSF containing propylene glycol alginate as an active ingredient. An external liquid composition for wound treatment comprising at least G-CSF and propylene glycol alginate.