WO2018190613A1 - Hydrogelized powder composition for wound healing and method for producing same - Google Patents

Hydrogelized powder composition for wound healing and method for producing same Download PDF

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Publication number
WO2018190613A1
WO2018190613A1 PCT/KR2018/004191 KR2018004191W WO2018190613A1 WO 2018190613 A1 WO2018190613 A1 WO 2018190613A1 KR 2018004191 W KR2018004191 W KR 2018004191W WO 2018190613 A1 WO2018190613 A1 WO 2018190613A1
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wound
powder composition
parts
weight
powder
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PCT/KR2018/004191
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French (fr)
Korean (ko)
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유진욱
이주호
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부산대학교 산학협력단
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Priority claimed from KR1020180019353A external-priority patent/KR102086150B1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form

Definitions

  • the present invention relates to a powder composition for treating wounds that is hydrogelized when applied to a wound and a method for preparing the wound.
  • a wound is a state in which the anatomical continuity of a human tissue has lost its original continuity by external action.
  • our skin is composed of epidermis, dermis, and subcutaneous fat, and the loss of continuity such as epidermis, dermis, and subcutaneous fat by trauma such as cut or falling is called wound.
  • wound dressing In general, dressing is used to effectively treat wounds of the skin such as wounds and traumas.
  • Wound dressings have the following characteristics: adequate moisture retention in contact with wounds, ability to control wound discharge, ease of attachment and removal of dressings to wounds, air and vapor permeability between external wounds, external Insulation of wounds, resistance to bacteria invasion, non-toxicity to human body, excellent mechanical properties, etc. are required.
  • GSNO S-nitrosoglutathione
  • a hydrogel-type formulation may be more useful than a film-type formulation.
  • most nitric oxide donors including S-nitrosoglutathione have high moisture content. Since it is unstable and rapidly decomposed in an environment, there is a problem that it is very difficult to ensure the stability of the formulation to be formulated into a film.
  • the powder is excellent in stability, but can not provide a wet environment of the wound site, there is a problem that it is difficult to maintain a concentration that can maintain a therapeutic effect for a long time because the release pattern of NO also rapidly made.
  • S-Nitrosoglutathione S-nitrosoglutathione
  • Another object of the present invention is to provide a method for preparing a powder composition for treating wounds that is hydrogelized rapidly upon application of wounds.
  • the present invention provides a powder composition for wound treatment comprising S-nitrosoglutathione (GSNO), alginate salt, pectin and polyethylene glycol (PEG) as an active ingredient do.
  • GSNO S-nitrosoglutathione
  • alginate salt alginate salt
  • pectin polyethylene glycol
  • PEG polyethylene glycol
  • the present invention comprises the steps of (1) grinding each of the powder form of GSNO, alginate salt, pectin and PEG; (2) mixing all the pulverized products prepared in step (1); And (3) passing the mixture prepared in step (2) through a sieve.
  • the present invention relates to a powder composition for treating wounds and a method for producing the same which is hydrogelized when applied to a wound, and the powder composition of the present invention can be hydrogelized to effectively treat a wound area having a larger area than a film-type preparation.
  • the exudates such as bedsores and burns are more susceptible to infections and have wider potential for wounds than conventional commercialized products.
  • the manufacturing process is simple and easy for mass production, and is also stable at room temperature when stored in a sealed container.
  • Figure 1 shows the moisture absorption and thus the morphological changes of the nitric oxide-containing wound treatment powder composition to be hydrogelized when applying the wound of the present invention.
  • Figure 2 shows the storage stability of the powder composition of the present invention.
  • Pseudomonas Pseudomonas of the powder composition of the present invention aeruginosa shows wound healing test results in an infected animal model.
  • Figure 7 shows the results of the quantification of bacteria in the wound in the in vivo animal model.
  • Figure 8 shows the results of the Wort's gram staining on the wound surface at 14 days after the start of treatment (normal skin (A), untreated group (B, C), powder carrier treated group (D) , Powder composition treatment group (E) of the present invention).
  • Figure 9 shows a comparison of the degree of tissue recovery through histological staining (A, B, C, D is H & E staining, E, F, G, H is a Masson's trichrome staining technique, A , E is a normal tissue, B, F is an untreated group, C, G is a group treated only with a powder carrier, D, H is a group treated with a powder composition of the present invention E: epidermis, F: fiber Fibrous tissue G: granulation tissue, H: hair follicle, I: immune cells, M: muscle).
  • the wound recovery is excellent and the antimicrobial effect can be effectively converted into gel form by effectively absorbing exudates at the wound site, which is why the present invention is more effective than film-type preparations.
  • the present invention has been completed by discovering that powder formulations may be useful for treating large area wounds.
  • the present invention provides a powder composition for wound treatment comprising S-nitrosoglutathione (S-Nitrosoglutathione; GSNO), alginate salt, pectin, and polyethylene glycol (PEG) as an active ingredient.
  • S-Nitrosoglutathione S-Nitrosoglutathione
  • GSNO S-nitrosoglutathione
  • alginate salt alginate salt
  • pectin polyethylene glycol
  • alginate is used as a gelling agent, so that the nitric oxide donor GSNO having a wound healing promoting effect and an antibacterial effect can absorb the exudates at the wound site and quickly change into a gel form, thereby forming hydrogel in in situ. It is possible.
  • the main active ingredient in the powder composition is a nitric oxide donor that is also present in the body, and is involved in cell differentiation, collagen synthesis, and the like by nitric oxide (NO) generated during decomposition, thereby promoting and helping to recover from a wound.
  • NO nitric oxide
  • the oxidative stress caused by the nitric oxide generated has an antimicrobial effect, and this mechanism rarely causes resistance and exhibits an antimicrobial effect without a gram negative / positive distinction and thus is suitable for use in skin wound infection.
  • the powder composition based on 100 parts by weight of the powder composition, 2 parts by weight of GSNO to 10 parts by weight, 10 parts to 40 parts by weight of alginate salt, 5 parts to 20 parts by weight of pectin and 50 parts by weight to PEG
  • 2 parts by weight of GSNO to 10 parts by weight 10 parts to 40 parts by weight of alginate salt, 5 parts to 20 parts by weight of pectin and 50 parts by weight to PEG
  • the numerical range it can be included in the 80 parts by weight, it is preferable because the modification to the uniform and durable gel at the wound site is made quickly, and the wound healing effect is maximized.
  • the GSNO: alginate salt: pectin: PEG may be included in a weight ratio of 4: 21: 11: 64.
  • the powder composition may be modified in a hydrogel form within 1 to 3 minutes at the wound site, the powder composition according to the present invention solves the problems appearing in powder form or film form, all of the advantages of each form Can be represented.
  • the powder composition is used to treat wounds by slowly releasing nitric oxide (NO) at the wound site, or Pseudomonas aeruginosa ) or methicillin-resistant Staphylococcus aureus (MRSA) exhibits antimicrobial activity against any one or more of the bar can be treated wound, in one embodiment of the present invention, the powder composition of the present invention.
  • NO nitric oxide
  • MRSA methicillin-resistant Staphylococcus aureus
  • the sustained release of nitric oxide was improved, and the bactericidal effect of more than 99.99% was observed for two species of Gram-positive bacteria, MRSA and Gram-negative bacteria, P. aeruginosa , which are the most problematic infections in the hospital.
  • the wound may be any one or more selected from abrasions, lacerations, burns, cuts, cuts, crystallizations, penetrating wounds, and left wounds, but is not limited thereto.
  • the present invention comprises the steps of (1) milling GSNO, alginate salt, pectin and PEG in powder form, respectively; (2) mixing all the pulverized products prepared in step (1); And (3) passing the mixture prepared in step (2) through a sieve.
  • the sieve may be a porous structure having an average diameter of 80 ⁇ m to 100 ⁇ m, but is not limited thereto.
  • the prepared wound composition may include GSNO: alginate salt: pectin: PEG in a weight ratio of 4: 21: 11: 64.
  • GSNO, sodium alginate, pectin and PEG were first ground to a standard No. 170 (average diameter of the hole is 90 ⁇ m). Thereafter, GSNO: alginate salt: pectin: PEG was mixed at a weight ratio of 4: 21: 11: 64, and then passed through No. 170 to prepare a powder composition for wound treatment according to the present invention.
  • a gelling powder (Comparative Example 1) prepared by mixing GSNO, sodium alginate, pectin, and PEG (MW: 8000) without grinding in the same weight ratio was used.
  • sodium alginate: pectin: PEG were prepared by mixing in a weight ratio of 2: 1: 6.
  • the in vitro test method was as follows: First, the cellulose membrane was contacted on a simulated wound fluid (SWF) mimicking the wound exudate, and then 40 mg of the powder composition prepared in Example 1 was added to cellulose. Evenly distributed over the membrane. At this time, since the water molecules are diffused over the cellulose membrane and absorbed in the powder composition, the photographs were taken every designated time, and the weight of the absorbed fluid (exudate) was measured.
  • SWF simulated wound fluid
  • Example 1 storage stability was analyzed.
  • the specific experimental method was as follows: The powder composition prepared in Example 1 was placed in a microtube of 10 mg each and then placed in a 4 ° C. refrigerator and 37 ° C. incubator. Thereafter, three microtubes were taken out at regular intervals, dissolved in water, and the absorbance at 335 nm was measured using an ultraviolet visible absorbance spectrometer to quantify and compare the amount of GSNO.
  • the amount of GNSO released according to the degree of moisture absorption was confirmed. Specifically, the following experimental method was carried out: 50 mg of the powder composition prepared in Example 1 was uniformly dispersed on the bottom of a 2 ml microtube having a flat bottom surface, and the exudate was absorbed less (200% absorption). ), The most absorbed state (350%), and the amount of SWF that would have been exposed to more exudates (500%) than the absorbable amount was added to each tube. Thereafter, three microtubes were taken in a 37 ° C. incubator and dissolved in water, and then the remaining amount of GSNO was measured and analyzed using an ultraviolet-visible spectrophotometer.
  • the antimicrobial experiment was performed in vitro with respect to the powder composition prepared in Example 1. Specifically, the following experimental method was carried out: P. aeruginosa (PAO1, purchased from KCTC) and MRSA (USA 300, purchased from ATCC) were incubated for 12 hours, and then the maximum absorption amount was prepared in Example 1 10 8 CFU / ml bacteria were added to the powder composition and incubated for 24 hours, and the CFU was measured and compared. In addition, using the Baclight live / dead kit was stained bacteria exposed to the powder composition prepared in Example 1 for 24 hours, photographed by confocal laser fluorescence microscope.
  • P. aeruginosa P. aeruginosa
  • MRSA USA 300
  • the wound recovery ability was confirmed in the bacterial infection ICR mouse model. Specifically, the following experimental methods were carried out: 6-week-old ICR mice were purchased, acclimated for 1 week, then anesthetized and hair removed. Whole layer wounds were created using 8 mm biopsy punches on the back, and 10 9 CFU of P. aeruginosa was added to each wound, and the wounds were sealed and left for 2 days to make infected whole layer wounds. Thereafter, every two days, the powder composition prepared in Example 1, the powder composition without GSNO (powder carrier) and the untreated group were divided into a total of three groups, and photographed each time, the size was compared and analyzed. It was.
  • the number of bacteria in the wound in the mouse model was quantified.
  • the following experimental methods were performed: 6-week-old ICR mice were purchased, acclimated for 1 week, anesthetized, and hair was removed.
  • Whole layer wounds were created using 8 mm biopsy punches on the back, and 10 9 CFU of P. aeruginosa was added to each wound, and the wounds were sealed and left for 2 days to make infected whole layer wounds.
  • the powder composition prepared in Example 1 and the powder composition without GSNO (powder carrier) were divided into a total of three groups of non-treated groups, and euthanized three mice every fixed day to tear off the wound site. Finely ground and diluted, P. aeruginosa
  • the bacteria were cultured in cetrimide medium where only bacteria were selectively grown, and CFU was measured.
  • Hematoxylin & Eosin staining H & E staining
  • Mason's trichrome staining technique masson's trichrome staining
  • H & E staining hematoxylin and eosin were sequentially stained, and in the case of Mason's trichrome staining, staining was performed according to the staining method sequence provided by ABCAM. Thereafter, tissues were compared, observed and photographed at a magnification of 200 times using an optical microscope.
  • Example 1 The content uniformity of the powder composition prepared in Example 1 and Comparative Example 1 prepared without undergoing grinding was confirmed. That is, each powder was unfolded and randomly sampled at 10 places to measure the content of GSNO. At this time, the weight of the powder of about 10 mg was measured and dissolved in water, and the absorbance at 335 nm by using an ultraviolet visible spectrophotometer to measure and compare the content of GSNO. In addition, the Hausner ratio calculated
  • Example 1 As a result, as shown in FIG. 10, the powder composition of Example 1, which was subjected to the grinding process, was found to have a more uniform distribution of GNSO, thereby preparing a highly uniform formulation. Since the fluidity is lowered in, it was confirmed that when applied to the actual wound it can further reduce the phenomenon of the powder flowing down before it turns into a gel.
  • Powder carrier compositions containing various gelling agents were prepared in the same manner as in Example 1 except that GSNO was not mixed in order to determine whether the gelling ability was different when using other gelling agents instead of alginate. .
  • GSNO hyaluronic acid, carrageenan, poloxamer, gelatin, guar gum, CMC-Na or chitosan are used instead of alginate as a gelling agent, respectively.
  • a powder carrier composition of Comparative Examples 2 to 8. At this time, the content ratio of each composition was the same as the excipient ratio except the GSNO of the powder composition of Example 1, the gelling agent: pectin: PEG 2: 1: 6.
  • the gelling agent except for alginate and hyaluronic acid is 10 minutes until gel formation. Since the above has passed, it can be seen that it is not suitable as a gelling agent of the in situ hydrogel-forming powder. In other words, alginate and hyaluronic acid showed in situ hydrogel formation, but hyaluronic acid showed a slower gelation rate than alginate, and thus alginate was the most optimal gelling agent for rapid gel formation.
  • Sample Average gel formation time S.D Remarks Alginate 0.86 minutes 0.11 NO / GP Hyaluronate 3.27 minutes 0.17 - Carrageenan Not suitable (10 minutes or more) - Not suitable for in situ hydrogel forming powder formulations which must form gels quickly Poloxamer Not suitable (10 minutes or more) - Gelatin Not suitable (10 minutes or more) - Guar gum Not suitable (10 minutes or more) - CMC-Na Not suitable (10 minutes or more) - Chitosan Not suitable (10 minutes or more) -
  • a powder composition is prepared according to the method described in Example 1, but the alginate content is 10% (relative to the total weight), and the effect is verified.
  • a powder containing 2% GSNO and a powder containing 10% GSNO were prepared.
  • the GSNO: alginate salt: pectin: PEG as a content ratio was prepared so that 2: 10: 5: 83 and 10: 10: 5: 75.
  • a powder containing 40% alginate was prepared (GSNO: alginate salt: pectin: PEG 4: 4: 20: 36), and in Example 1 The mouse animal model was treated in the same manner as the prepared powder composition.
  • the specific experimental method was as follows: 6 weeks old ICR mice were purchased and allowed to acclimate for 1 week, followed by anesthesia and hair removal. Whole layer wounds were made using 8 mm biopsy punches on the back, and the drug was treated into the above-mentioned drug and GSNO-free carrier powder composition, and the non-treated group, and the drugs were taken and photographed every two days to compare and analyze the size. .
  • the specific method was as follows: Each powder prepared was evenly spread by 50 mg in a small dish, and then 1 ml of distilled water was evenly added to observe and photograph the shape of the gel formed.

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Abstract

The present invention relates to a powder composition for wound healing comprising S-nitrosoglutathione (GSNO), alginate salt, pectin and polyethylene glycol (PEG) as active ingredients, and a method for producing the same. As the composition according to the present invention uses GSNO in combination with an in situ hydrogel-forming system on the basis of alginate having appropriate drug release control capability, the composition has advantages of both an acid and a film so that the formulation stability is high when stored; can quickly change into a gel to cover a wound or provide a moisture environment when applied to a wound site; effectively removes germs present in exudative wounds; and can promote wound healing through promotion of cell differentiation and collagen synthesis.

Description

하이드로겔화 되는 창상 치료용 파우더 조성물 및 이의 제조방법Hydrogel-ized wound composition and its preparation method
본 발명은 창상 적용 시 하이드로겔화 되는 창상 치료용 파우더 조성물 및 이의 제조방법에 관한 것이다.The present invention relates to a powder composition for treating wounds that is hydrogelized when applied to a wound and a method for preparing the wound.
우리 몸을 밖에서 감싸고 있는 피부는 일상생활에서 여러 가지 위험한 물리적 손상에 노출되어 있다. 따라서 피부의 기계적 손상, 타박상, 화상 등 우리 주위에서 많은 요인들로 인해 상처가 발생한다. 상처는 사람의 조직이 가지는 해부학적인 연속성이 외부의 작용에 의해 그 본래의 연속성을 상실한 상태를 말한다. 예를 들어 우리의 피부는 표피, 진피, 피하지방으로 이루어져 있는데, 베이거나 넘어지는 등의 외상에 의해서 이러한 표피나 진피, 피하지방 등의 연속성을 상실한 것을 상처라고 한다.The skin that surrounds our bodies is exposed to various dangerous physical damages in our daily lives. Therefore, many factors around us, such as mechanical damage to the skin, bruises, burns, wounds occur. A wound is a state in which the anatomical continuity of a human tissue has lost its original continuity by external action. For example, our skin is composed of epidermis, dermis, and subcutaneous fat, and the loss of continuity such as epidermis, dermis, and subcutaneous fat by trauma such as cut or falling is called wound.
일반적으로 창상, 외상 등과 같은 피부의 상처를 효과적으로 치료하기 위하여 드레싱(Wound dressing)을 사용한다. 상처 치료용 드레싱이 가져야 할 특성으로서 상처와의 접촉 면에서 적당한 습기의 유지능력, 상처분비물의 조절 능력, 상처에 대한 드레싱의 부착 및 제거의 용이성, 외부의 상처 부위 간의 공기 및 수증기 관통성, 외부에 대한 상처부위의 단열성, 박테리아의 침입에 대한 저항성, 인체에 대한 무독성, 우수한 기계적 물성 등이 요구된다.In general, dressing is used to effectively treat wounds of the skin such as wounds and traumas. Wound dressings have the following characteristics: adequate moisture retention in contact with wounds, ability to control wound discharge, ease of attachment and removal of dressings to wounds, air and vapor permeability between external wounds, external Insulation of wounds, resistance to bacteria invasion, non-toxicity to human body, excellent mechanical properties, etc. are required.
현재 가장 대중화된 상처의 치유 방법으로는 연고를 바르는 것인데, 가장 많이 사용되는 피부 상처 치유 촉진 제품으로는 마데카솔(동국제약), 센티카(삼진제약) 및 티나덱스(종근당) 등이 있다. Currently, the most popular wound healing methods are ointment. The most popular products for promoting skin wound healing include Madecassol (Dongkuk), Sentica (Samjin Pharm) and Tinadex (Chong Kun Dang).
인체에서 자발적으로 형성되는 산화질소 공여체인 S-나이트로소글루타치온 (S-Nitrosoglutathione; 이하 'GSNO')을 함유한 산화질소 방출성 필름은 인체에 적용할 수 있는 기계적 특성을 가질 뿐 아니라, 서서히 산화질소를 방출하며 상처 감염의 주원인이 되는 병원균을 억제하고 상처를 신속하게 치료할 수 있기 때문에 상기 필름은 상처치료에 유용하게 사용될 수 있다.Nitric oxide releasing films containing S-nitrosoglutathione (GSNO), a nitric oxide donor that spontaneously forms in the human body, not only have mechanical properties that can be applied to the human body, but also gradually oxidize The film can be useful for wound healing because it releases nitrogen and inhibits pathogens that are the main cause of wound infections and can quickly heal wounds.
그러나 필름 제형의 특성상 삼출물이 나오는 상처, 광범위한 상처 혹은 움직임이 많은 신체부위의 상처 등에는 적용하기에 한계가 있다. 또한, 제조 공정 상의 문제로 -20℃ 외의 냉장, 상온 등의 보관 조건에서는 GSNO의 안정성을 담보할 수 없는 한계점이 있다.However, due to the nature of the film formulation there is a limit to apply to the exudation wounds, a wide range of wounds or wounds of the body parts with a lot of movement. In addition, due to problems in the manufacturing process there is a limit that can not ensure the stability of the GSNO in the storage conditions, such as refrigeration, room temperature other than -20 ℃.
또한, 넓은 면적의 상처 혹은 움직임이 많은 부분의 상처인 경우에는 필름 형태의 제제보다 하이드로겔 형태의 제형이 보다 유용하게 사용될 수 있으나, S-나이트로소글루타치온을 비롯한 대부분의 산화질소 공여체는 수분이 있는 환경에서 불안정하여 빠르게 분해되므로, 필름제로 제형화 하기에는 제제의 안정성을 확보하기가 매우 어렵다는 문제가 있다. 반면, 산제의 경우 안정성은 뛰어나지만 상처 부위의 습윤 환경을 제공할 수 없고 NO의 방출 패턴 또한 급격하게 이루어져 치료 효과를 유지할 수 있는 농도를 장시간 유지하기 어렵다는 문제가 있다.In addition, in the case of a large area wound or a wound with a lot of movement, a hydrogel-type formulation may be more useful than a film-type formulation. However, most nitric oxide donors including S-nitrosoglutathione have high moisture content. Since it is unstable and rapidly decomposed in an environment, there is a problem that it is very difficult to ensure the stability of the formulation to be formulated into a film. On the other hand, the powder is excellent in stability, but can not provide a wet environment of the wound site, there is a problem that it is difficult to maintain a concentration that can maintain a therapeutic effect for a long time because the release pattern of NO also rapidly made.
본 발명의 목적은 상처 회복 촉진 효과와 항균 효과를 지닌 산화질소 공여체 S-나이트로글루타치온(S-Nitrosoglutathione)을 상처 부위의 삼출물을 흡수하여 겔 형태로 신속하게 바뀔 수 있도록 하는, 창상 적용 시 하이드로겔화 되는 창상 치료용 파우더 조성물을 제공하는 것이다.It is an object of the present invention to hydrogelize nitric oxide donor S-nitrosoglutathione (S-Nitrosoglutathione), which has an effect of promoting wound healing and antibacterial effect, to quickly change the gel form by absorbing exudates of the wound site. It is to provide a powder composition for wound treatment.
본 발명의 다른 목적은 창상 적용 시 신속하게 하이드로겔화 되는 창상 치료용 파우더 조성물의 제조방법을 제공하는 것이다.Another object of the present invention is to provide a method for preparing a powder composition for treating wounds that is hydrogelized rapidly upon application of wounds.
상기 목적을 달성하기 위하여, 본 발명은 S-나이트로소글루타치온(S-Nitrosoglutathione; GSNO), 알지네이트염, 펙틴 및 폴리에틸렌글리콜(PEG)을 유효성분으로 포함하는 창상(wound) 치료용 파우더 조성물을 제공한다.In order to achieve the above object, the present invention provides a powder composition for wound treatment comprising S-nitrosoglutathione (GSNO), alginate salt, pectin and polyethylene glycol (PEG) as an active ingredient do.
상기 다른 목적을 달성하기 위하여, 본 발명은 (1) 분말 형태의 GSNO, 알지네이트염, 펙틴 및 PEG를 각각 분쇄하는 단계; (2) 상기 단계 (1)에서 제조한 분쇄물을 모두 혼합하는 단계; 및 (3) 상기 단계 (2)에서 제조한 혼합물을 체에 통과시키는 단계를 포함하는 창상 치료용 파우더 조성물의 제조방법을 제공한다.In order to achieve the above another object, the present invention comprises the steps of (1) grinding each of the powder form of GSNO, alginate salt, pectin and PEG; (2) mixing all the pulverized products prepared in step (1); And (3) passing the mixture prepared in step (2) through a sieve.
본 발명은 창상 적용 시 하이드로겔화 되는 창상 치료용 파우더 조성물 및 이의 제조방법에 관한 것으로, 본 발명의 파우더 조성물은 하이드로겔화 되어 필름 형태의 제제보다 대면적의 창상 부위를 효과적으로 치료할 수 있는 바, 특히, 욕창이나 화상 등과 같은 삼출물이 있고 감염될 여지가 높으며 광범위하게 생길 수 있는 상처에 기존의 상품화 된 제품보다 적합한 특성을 지니고 있고, 사용된 물질들은 모두 생체적합성이 뛰어난 물질들이므로 독성 문제를 해결하였으며, 또한 제조 과정이 단순하여 대량생산에 용이하며, 밀봉용기에 보관 시 상온에서 또한 안정하다.The present invention relates to a powder composition for treating wounds and a method for producing the same which is hydrogelized when applied to a wound, and the powder composition of the present invention can be hydrogelized to effectively treat a wound area having a larger area than a film-type preparation. The exudates such as bedsores and burns are more susceptible to infections and have wider potential for wounds than conventional commercialized products. In addition, the manufacturing process is simple and easy for mass production, and is also stable at room temperature when stored in a sealed container.
도 1은 본 발명의 창상 적용 시 하이드로겔화 되는 산화질소 함유 창상 치료 파우더 조성물의 수분 흡수와 그에 따른 형태 변화를 나타낸 것이다.Figure 1 shows the moisture absorption and thus the morphological changes of the nitric oxide-containing wound treatment powder composition to be hydrogelized when applying the wound of the present invention.
도 2는 본 발명의 파우더 조성물의 저장 안정성을 나타낸 것이다.Figure 2 shows the storage stability of the powder composition of the present invention.
도 3은 본 발명의 파우더 조성물의 수분 흡수 정도에 따른 NO 방출 실험의 결과이다.3 is a result of the NO release experiment according to the moisture absorption of the powder composition of the present invention.
도 4는 본 발명의 파우더 조성물의 in vitro 항균 실험(antibacterial test)의 결과이다.4 is a result of an in vitro antibacterial test of the powder composition of the present invention.
도 5는 본 발명의 파우더 조성물의 공초점 레이저 형광 현미경(Confocal laser fluorescence microscopy)을 이용한 항균 능력 시험의 결과이다.5 is a result of the antimicrobial performance test using a confocal laser fluorescence microscopy of the powder composition of the present invention.
도 6은 본 발명의 파우더 조성물의 녹농균(Pseudomonas aeruginosa) 감염 동물 모델에서의 상처 회복 능력 시험 결과를 나타낸 것이다.6 is Pseudomonas Pseudomonas of the powder composition of the present invention aeruginosa ) shows wound healing test results in an infected animal model.
도 7은 in vivo 동물 모델에서 상처에 있는 균 수 정량 실험의 결과를 나타낸 것이다.Figure 7 shows the results of the quantification of bacteria in the wound in the in vivo animal model.
도 8은 치료 개시 후 14일째 되는 시점에서 상처 표면에 있는 균 확인 시험 (Twort's gram staining) 결과를 나타낸 것이다(정상 피부(A), 비처리군(B, C), 파우더 담체 처리군(D), 본 발명의 파우더 조성물 처리군(E)).Figure 8 shows the results of the Wort's gram staining on the wound surface at 14 days after the start of treatment (normal skin (A), untreated group (B, C), powder carrier treated group (D) , Powder composition treatment group (E) of the present invention).
도 9는 조직학적 염색을 통한 조직 회복 정도를 비교하여 나타낸 것이다(A, B, C, D는 H&E 염색, E, F, G, H는 메이슨 트리크롬(Masson's trichrome) 염색 기법을 사용한 것으로서, A, E는 정상 조직, B, F는 비처리군, C, G는 파우더 담체만을 처리한 그룹, D, H는 본 발명의 파우더 조성물을 처리한 그룹임. E: 표피(epidermis), F: 섬유조직(fibrous tissue) G: 육아조직(granulation tissue), H: 모공(hair follicle), I: 면역세포(immune cells), M: 근육(muscle)).Figure 9 shows a comparison of the degree of tissue recovery through histological staining (A, B, C, D is H & E staining, E, F, G, H is a Masson's trichrome staining technique, A , E is a normal tissue, B, F is an untreated group, C, G is a group treated only with a powder carrier, D, H is a group treated with a powder composition of the present invention E: epidermis, F: fiber Fibrous tissue G: granulation tissue, H: hair follicle, I: immune cells, M: muscle).
도 10은 분쇄 과정을 포함 또한 비포함한 제조방법으로 제조된 조성물 파우더의 GSNO 함량 균일성을 확인한 결과이다.10 is a result of confirming the uniformity of the GSNO content of the composition powder prepared by the manufacturing method including the grinding process and also without.
도 11은 본 발명의 조성물 파우더에서 알지네이트염의 함량에 따른 치료 효과를 비교한 결과이다.11 is a result of comparing the treatment effect according to the content of alginate salt in the composition powder of the present invention.
도 12는 본 발명의 조성물 파우더에서 펙틴 및 PEG의 포함 여부에 따른 겔화 형성 실험 결과를 나타낸 것이다.12 shows the results of gelation formation experiments according to the presence of pectin and PEG in the composition powder of the present invention.
이하, 본 발명을 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명의 발명자들은 GSNO와 알지네이트염을 기반으로 할 경우 상처 회복 촉진 효과가 우수하고 항균 효과로 인하여 상처 부위의 삼출물을 효과적으로 흡수함으로써 겔 형태로 신속하게 변환할 수 있고, 이로 인해 필름형태의 제제보다 파우더 형태의 제제를 사용할 경우 넓은 면적의 상처 치료에 유용할 수 있음을 밝혀내어 본 발명을 완성하였다.When the inventors of the present invention are based on GSNO and alginate salts, the wound recovery is excellent and the antimicrobial effect can be effectively converted into gel form by effectively absorbing exudates at the wound site, which is why the present invention is more effective than film-type preparations. The present invention has been completed by discovering that powder formulations may be useful for treating large area wounds.
따라서 본 발명은 S-나이트로소글루타치온(S-Nitrosoglutathione; GSNO), 알지네이트염, 펙틴 및 폴리에틸렌글리콜(PEG)을 유효성분으로 포함하는 창상(wound) 치료용 파우더 조성물을 제공한다.Accordingly, the present invention provides a powder composition for wound treatment comprising S-nitrosoglutathione (S-Nitrosoglutathione; GSNO), alginate salt, pectin, and polyethylene glycol (PEG) as an active ingredient.
본 발명은 겔화제로서 알지네이트를 사용하는 바, 상처 회복 촉진 효과와 항균 효과를 지닌 산화질소 공여체 GSNO가 상처 부위의 삼출물을 흡수하여 겔 형태로 신속하게 바뀔 수 있도록 하여, in situ에서 하이드로겔 형성이 가능하다.According to the present invention, alginate is used as a gelling agent, so that the nitric oxide donor GSNO having a wound healing promoting effect and an antibacterial effect can absorb the exudates at the wound site and quickly change into a gel form, thereby forming hydrogel in in situ. It is possible.
상기 파우더 조성물에서 주요 활성 성분인 GSNO는 체내에도 존재하는 산화질소 공여체이며, 분해되면서 발생하는 산화질소(NO)에 의해 세포 분화, 콜라겐 합성과정 등에 관여하여 이를 촉진하고 상처의 회복을 돕는다. 또한, 발생한 산화질소에 의한 산화 스트레스는 항균 효과를 갖는데, 이러한 기전은 내성을 거의 일으키지 않으며 그람 음/양성 구분 없이 항균 효과를 나타내므로 피부 상처 감염에 사용하기 적합하다. GSNO, the main active ingredient in the powder composition, is a nitric oxide donor that is also present in the body, and is involved in cell differentiation, collagen synthesis, and the like by nitric oxide (NO) generated during decomposition, thereby promoting and helping to recover from a wound. In addition, the oxidative stress caused by the nitric oxide generated has an antimicrobial effect, and this mechanism rarely causes resistance and exhibits an antimicrobial effect without a gram negative / positive distinction and thus is suitable for use in skin wound infection.
본 발명의 일 실시예에서, 상기 파우더 조성물 100 중량부에 대하여, GSNO 2 중량부 내지 10 중량부, 알지네이트염 10 중량부 내지 40 중량부, 펙틴 5 중량부 내지 20 중량부 및 PEG 50 중량부 내지 80 중량부로 포함될 수 있는 바, 상기 수치 범위로 포함되는 경우, 상처 부위에서 균일하고 튼튼한 겔로의 변성이 신속하게 이루어졌으며, 또한 상처 치유 효과가 극대화되므로 바람직하다.In one embodiment of the present invention, based on 100 parts by weight of the powder composition, 2 parts by weight of GSNO to 10 parts by weight, 10 parts to 40 parts by weight of alginate salt, 5 parts to 20 parts by weight of pectin and 50 parts by weight to PEG When included in the numerical range, it can be included in the 80 parts by weight, it is preferable because the modification to the uniform and durable gel at the wound site is made quickly, and the wound healing effect is maximized.
보다 바람직하게는, 상기 GSNO : 알지네이트염 : 펙틴 : PEG는 4 : 21 : 11: 64의 중량비로 포함될 수 있다.More preferably, the GSNO: alginate salt: pectin: PEG may be included in a weight ratio of 4: 21: 11: 64.
상기 파우더 조성물은 창상 부위에서 1분 내지 3분 내에 하이드로겔 형태로 변성될 수 있는 바, 본 발명에 따른 파우더 조성물은 산제 형태나 필름 형태에서 나타나는 문제점을 해결하고, 각각의 형태의 장점들만을 모두 나타낼 수 있다.The powder composition may be modified in a hydrogel form within 1 to 3 minutes at the wound site, the powder composition according to the present invention solves the problems appearing in powder form or film form, all of the advantages of each form Can be represented.
특히, 상기 파우더 조성물은 창상 부위에서 산화질소(NO)를 서방출함으로써 창상을 치료하거나, 녹농균(Pseudomonas aeruginosa) 또는 메티실린 내성 황색포도알균(methicillin-resistant Staphylococcus aureus; MRSA) 중 어느 하나 이상의 균에 대한 항균 활성을 나타냄으로써 창상을 치료할 수 있는 바, 본 발명의 일 실시예에서, 본 발명의 파우더 조성물은 일산화질소의 서방출성이 향상되었고, 또한, 병원 내 감염균 중 가장 문제가 되는 그람 양성균인 MRSA와 그람 음성균인 P. aeruginosa 의 2종 균에 대해 99.99% 이상의 살균 효과를 나타냄을 검증하였다.In particular, the powder composition is used to treat wounds by slowly releasing nitric oxide (NO) at the wound site, or Pseudomonas aeruginosa ) or methicillin-resistant Staphylococcus aureus (MRSA) exhibits antimicrobial activity against any one or more of the bar can be treated wound, in one embodiment of the present invention, the powder composition of the present invention The sustained release of nitric oxide was improved, and the bactericidal effect of more than 99.99% was observed for two species of Gram-positive bacteria, MRSA and Gram-negative bacteria, P. aeruginosa , which are the most problematic infections in the hospital.
상기 창상은 찰과상, 열상, 화상, 자상, 절상, 결출상, 관통상 및 좌상에서 선택된 어느 하나 이상일 수 있으나, 이에 제한되지는 않는다.The wound may be any one or more selected from abrasions, lacerations, burns, cuts, cuts, crystallizations, penetrating wounds, and left wounds, but is not limited thereto.
더욱이, 본 발명은 (1) 분말 형태의 GSNO, 알지네이트염, 펙틴 및 PEG를 각각 분쇄하는 단계; (2) 상기 단계 (1)에서 제조한 분쇄물을 모두 혼합하는 단계; 및 (3) 상기 단계 (2)에서 제조한 혼합물을 체에 통과시키는 단계를 포함하는 창상 치료용 파우더 조성물의 제조방법을 제공한다.Moreover, the present invention comprises the steps of (1) milling GSNO, alginate salt, pectin and PEG in powder form, respectively; (2) mixing all the pulverized products prepared in step (1); And (3) passing the mixture prepared in step (2) through a sieve.
상기 단계 (1) 내지 (3)에서, 상기 파우더 조성물 100 중량부에 대하여, GSNO 2 중량부 내지 10 중량부, 알지네이트염 10 중량부 내지 40 중량부, 펙틴 5 중량부 내지 20 중량부 및 PEG 50 중량부 내지 80 중량부로 포함될 수 있는 바, 이러한 범위 내로 포함되어 제조될 때 상처 부위에서 균일하고 튼튼한 겔로의 변성이 신속하게 이루어졌으며, 또한 상처 치유 효과가 극대화되는 파우더 조성물이 제조될 수 있다.In the above steps (1) to (3), based on 100 parts by weight of the powder composition, 2 parts by weight of GSNO to 10 parts by weight, 10 parts to 40 parts by weight of alginate salt, 5 parts to 20 parts by weight of pectin and PEG 50 It can be included in the weight part to 80 parts by weight, when it is included in the range prepared when the modification to the uniform and durable gel at the wound site is made quickly, and also a powder composition that maximizes the wound healing effect can be prepared.
상기 단계 (3)에서, 상기 체는 80 ㎛ 내지 100 ㎛의 평균직경의 다공성 구조일 수 있으나, 이에 제한되지는 않는다.In the step (3), the sieve may be a porous structure having an average diameter of 80 ㎛ to 100 ㎛, but is not limited thereto.
보다 상세하게는, 상기 단계 (3)에서, 제조된 창상 치료용 파우더 조성물은 GSNO : 알지네이트염 : 펙틴 : PEG를 4 : 21 : 11 : 64의 중량비로 포함하고 있을 수 있다.More specifically, in the step (3), the prepared wound composition may include GSNO: alginate salt: pectin: PEG in a weight ratio of 4: 21: 11: 64.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이며 본 발명의 내용을 예시하는 것일 뿐이므로 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다.Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are provided to more fully explain the present invention to those skilled in the art, and the scope of the present invention is not limited to the following examples, only to illustrate the contents of the present invention. no.
<< 실시예Example 1> 본 발명에 따른 창상 치료용 파우더 조성물의 제조 1> Preparation of the wound composition powder composition according to the present invention
본 발명에 따른 창상 치료용 파우더 조성물을 제조하기 위하여, 먼저 GSNO, 소듐 알지네이트, 펙틴 및 PEG(MW: 8000)를 170호 표준체(구멍의 평균직경이 90 ㎛임)를 통과할 정도로 분쇄하였다. 그 후, GSNO : 알지네이트염 : 펙틴 : PEG를 4 : 21 : 11 : 64의 중량비로 혼합한 후 170호 체를 통과시킴으로써 본 발명에 따른 창상 치료용 파우더 조성물을 제조하였다. 대조군으로는 분쇄 과정을 거지 않은 GSNO, 소듐 알지네이트, 펙틴 및 PEG(MW: 8000)를 상기와 동일한 중량비로 혼합하여 제조한 겔링 파우더(비교예 1)를 사용하였고, GSNO를 포함하지 않은 담체의 경우, 소듐 알지네이트 : 펙틴 : PEG를 2 : 1 : 6의 중량비로 섞어 제조한 것을 사용하였다.In order to prepare a wound care powder composition according to the present invention, GSNO, sodium alginate, pectin and PEG (MW: 8000) were first ground to a standard No. 170 (average diameter of the hole is 90 μm). Thereafter, GSNO: alginate salt: pectin: PEG was mixed at a weight ratio of 4: 21: 11: 64, and then passed through No. 170 to prepare a powder composition for wound treatment according to the present invention. As a control, a gelling powder (Comparative Example 1) prepared by mixing GSNO, sodium alginate, pectin, and PEG (MW: 8000) without grinding in the same weight ratio was used. And sodium alginate: pectin: PEG were prepared by mixing in a weight ratio of 2: 1: 6.
<< 실험예Experimental Example 1> 수분 흡수와 그에 따른 형태 변화 분석 1> Absorption of moisture and its shape change
상기 실시예 1에서 제조한 파우더 조성물을 대상으로, 수분 흡수 실험을 통해 시간에 따른 형태 변화를 확인하였다. 구체적으로, in vitro 실험 방법은 다음과 같았다: 먼저 셀룰로오스 멤브레인을 상처 삼출물을 흉내 낸 모조의 상처 유체(simulated wound fluid ; SWF) 위에 접촉시킨 후, 상기 실시예 1에서 제조한 파우더 조성물 40 mg을 셀룰로오스 멤브레인 위에 고르게 분포시켰다. 이때, 물 분자가 셀룰로오스 멤브레인 너머로 확산되어 파우더 조성물에 흡수되므로 지정된 시간마다 사진을 촬영하고 그 무게를 측정하여 흡수한 유체(삼출물)의 양을 측정하였다.For the powder composition prepared in Example 1, the change in form over time was confirmed through a water absorption experiment. Specifically, the in vitro test method was as follows: First, the cellulose membrane was contacted on a simulated wound fluid (SWF) mimicking the wound exudate, and then 40 mg of the powder composition prepared in Example 1 was added to cellulose. Evenly distributed over the membrane. At this time, since the water molecules are diffused over the cellulose membrane and absorbed in the powder composition, the photographs were taken every designated time, and the weight of the absorbed fluid (exudate) was measured.
또한, in vivo 실험 방법은 다음과 같았다: 7주령 ICR 마우스를 마취한 후 등 쪽 부분의 털을 제거하고 8 mm 생검 펀치(biopsy punch)로 전층 상처를 생성하였다. 그 위에 상기 실시예 1에서 제조한 파우더 조성물 40 mg을 고르게 뿌린 후 지정된 시간에 따라 사진을 촬영하였다.In vivo experimental methods were also as follows: 7 weeks old ICR mice were anesthetized and the dorsal hairs were removed and full layer wounds were generated with an 8 mm biopsy punch. After spraying evenly 40 mg of the powder composition prepared in Example 1 on it was taken pictures according to the specified time.
그 결과, 도 1에 나타난 바와 같이, 실시예 1에서 제조한 파우더 조성물은 350 % 까지의 수분을 흡수할 수 있는 능력이 있으며 겔 형태로 신속하게 변화됨을 확인하였다(도 1(A): in vitro 상 수분 흡수 실험에서 시간에 따른 파우더의 상태 변화를 나타낸 것, 도 1(B): in vivo 마우스 모델에서의 풀 두께 절제 상처(full thickness excision wound)에 파우더를 처리하였을 때 파우더의 상태 변화를 나타낸 것, 도 1(C): 파우더의 수분 흡수 능력을 측정한 것).As a result, as shown in Figure 1, it was confirmed that the powder composition prepared in Example 1 has the ability to absorb up to 350% moisture and quickly change to a gel form (Fig. 1 (A): in vitro Figure 1 (B): Changes in the state of the powder when the powder is treated in a full thickness excision wound in an in vivo mouse model. 1 (C): measuring the water absorption capacity of the powder).
<< 실험예Experimental Example 2> 저장 안정성 분석 2> storage stability analysis
상기 실시예 1에서 제조한 파우더 조성물을 대상으로, 저장 안정성을 분석하였다. 구체적인 실험 방법은 다음과 같았다: 상기 실시예 1에서 제조한 파우더 조성물을 10 mg 씩 마이크로튜브에 넣은 후 4℃ 냉장고 및 37℃ 인큐베이터에 넣었다. 그 후, 일정 시간마다 3개의 마이크로튜브를 꺼내어 물에 녹인 후 자외가시부 흡광광도계로 335 nm에서의 흡광도를 측정하여 GSNO의 양을 정량, 비교하였다.For the powder composition prepared in Example 1, storage stability was analyzed. The specific experimental method was as follows: The powder composition prepared in Example 1 was placed in a microtube of 10 mg each and then placed in a 4 ° C. refrigerator and 37 ° C. incubator. Thereafter, three microtubes were taken out at regular intervals, dissolved in water, and the absorbance at 335 nm was measured using an ultraviolet visible absorbance spectrometer to quantify and compare the amount of GSNO.
그 결과, 도 2에 나타난 바와 같이, 140일 동안, 4℃와 37℃ 보관 조건에서도 파우더 조성물의 의미 있는 GSNO의 분해는 관찰되지 않은 것을 확인할 수 있었고, 이러한 점을 볼 때, 상기의 저장 조건에서는 약물의 분해 우려 없이 보관이 가능하다는 것을 알 수 있었다.As a result, as shown in Figure 2, for 140 days, even in the storage conditions of 4 ℃ and 37 ℃ can be confirmed that no significant degradation of the GSNO of the powder composition was observed, in view of these points, It can be seen that the storage can be carried out without fear of decomposition of the drug.
<< 실험예Experimental Example 3>  3> GNSO의GNSO 수분 흡수 정도에 따른 NO 방출 실험 분석 Analysis of NO emission test according to moisture absorption
상기 실시예 1에서 제조한 파우더 조성물을 대상으로, 수분 흡수 정도에 따른 GNSO 방출량을 확인하였다. 구체적으로, 다음과 같은 실험 방법을 수행하였다: 50 mg의 상기 실시예 1에서 제조한 파우더 조성물을 바닥면이 평평한 2 ml 마이크로 튜브 바닥에 고루 분산시킨 후, 삼출물을 적게 흡수한 상태(200 % 흡수), 가장 많이 흡수한 상태(350 %), 흡수할 수 있는 양보다 더 많은 양의 삼출물에 노출되었을 상태(500 %)에 해당되는 양의 SWF를 각각의 튜브에 넣어주었다. 그 후 37℃ 인큐베이터에 넣고 일정 시간마다 3개씩의 마이크로 튜브를 취하여 물에 녹인 후 자외가시부 분광광도계를 이용하여 남아있는 GSNO의 양을 측정, 비교 분석하였다.For the powder composition prepared in Example 1, the amount of GNSO released according to the degree of moisture absorption was confirmed. Specifically, the following experimental method was carried out: 50 mg of the powder composition prepared in Example 1 was uniformly dispersed on the bottom of a 2 ml microtube having a flat bottom surface, and the exudate was absorbed less (200% absorption). ), The most absorbed state (350%), and the amount of SWF that would have been exposed to more exudates (500%) than the absorbable amount was added to each tube. Thereafter, three microtubes were taken in a 37 ° C. incubator and dissolved in water, and then the remaining amount of GSNO was measured and analyzed using an ultraviolet-visible spectrophotometer.
그 결과, 도 3에 나타난 바와 같이, 파우더가 적은 양의 수분에 노출되었을 때, 많은 양의 수분에 노출되었을 때, 최대 흡수점 이상의 수분 환경에 있을 때의 모든 조건에서 24시간 이상의 NO 방출 패턴을 나타내었다.As a result, as shown in Figure 3, when the powder is exposed to a small amount of moisture, when exposed to a large amount of moisture, the NO emission pattern of more than 24 hours under all conditions when in a moisture environment above the maximum absorption point Indicated.
<< 실험예Experimental Example 4> in vitro 항균 실험(antibacterial test) 4> in vitro antibacterial test
상기 실시예 1에서 제조한 파우더 조성물을 대상으로, in vitro에서 항균 실험을 수행하였다. 구체적으로, 다음과 같은 실험 방법을 수행하였다: P. aeruginosa (PAO1, KCTC에서 구입) 및 MRSA (USA 300, ATCC에서 구입) 균을 12시간 배양한 후, 최대 흡수량만큼의 실시예 1에서 제조한 파우더 조성물에 108 CFU/ml 정도의 균을 가하여 24시간 배양하였고, CFU를 측정함으로써 비교, 분석하였다. 또한, Baclight live/dead kit를 사용하여 상기 실시예 1에서 제조한 파우더 조성물에 24시간 노출시킨 균을 염색하였고, 공초점 레이저 형광 현미경으로 촬영하였다.The antimicrobial experiment was performed in vitro with respect to the powder composition prepared in Example 1. Specifically, the following experimental method was carried out: P. aeruginosa (PAO1, purchased from KCTC) and MRSA (USA 300, purchased from ATCC) were incubated for 12 hours, and then the maximum absorption amount was prepared in Example 1 10 8 CFU / ml bacteria were added to the powder composition and incubated for 24 hours, and the CFU was measured and compared. In addition, using the Baclight live / dead kit was stained bacteria exposed to the powder composition prepared in Example 1 for 24 hours, photographed by confocal laser fluorescence microscope.
그 결과, 도 4에 나타난 바와 같이, in vitro에서 P. aeruginosa와 MRSA 균에 처리 시 모두 99.99% 이상의 균이 감소함을 확인할 수 있었다.As a result, as shown in Figure 4, it was confirmed that more than 99.99% of the bacteria in the treatment of both P. aeruginosa and MRSA bacteria in vitro reduced.
또한, 도 5에 나타난 바와 같이, 조성물을 처리한 그룹에서는 거의 모든 점들이 빨간색으로 보였는데, 이는 대부분의 균이 사멸했다는 것을 나타낸다(초록색 형광은 살아있는 균을, 빨간색 형광은 죽어있는 균을 나타냄).In addition, as shown in FIG. 5, almost all of the dots appeared red in the group treated with the composition, indicating that most of the bacteria were killed (green fluorescence indicates living bacteria and red fluorescence indicates dead bacteria). .
<< 실험예Experimental Example 5> 상처 회복 능력 시험 분석 5> wound recovery test analysis
상기 실시예 1에서 제조한 파우더 조성물을 대상으로, 세균 감염 ICR 마우스 모델에서 상처 회복 능력을 확인하였다. 구체적으로, 다음과 같은 실험 방법을 수행하였다: 6주령의 ICR 마우스를 구입하여 1주 동안 적응시킨 후 마취하고 털을 제거하였다. 등 부분에 8 mm의 생검 펀치를 이용하여 전층 상처를 생성하였고 상처마다 109 CFU의 P. aeruginosa 균을 가한 후 상처를 밀봉, 2일간 방치하여 감염된 전층 상처를 만들었다. 그 후, 2일마다 상기 실시예 1에서 제조한 파우더 조성물 및 GSNO가 없는 파우더 조성물(파우더 담체), 비처리 그룹의 총 3 그룹으로 나누어 처리를 하고 그때마다 사진을 촬영하여 그 크기를 비교, 분석하였다.With respect to the powder composition prepared in Example 1, the wound recovery ability was confirmed in the bacterial infection ICR mouse model. Specifically, the following experimental methods were carried out: 6-week-old ICR mice were purchased, acclimated for 1 week, then anesthetized and hair removed. Whole layer wounds were created using 8 mm biopsy punches on the back, and 10 9 CFU of P. aeruginosa was added to each wound, and the wounds were sealed and left for 2 days to make infected whole layer wounds. Thereafter, every two days, the powder composition prepared in Example 1, the powder composition without GSNO (powder carrier) and the untreated group were divided into a total of three groups, and photographed each time, the size was compared and analyzed. It was.
그 결과, 도 6에 나타난 바와 같이, 시간에 따라 상처의 크기가 현저하게 감소하는 것을 확인할 수 있었고, 파우더를 처리한 그룹이 비처리군이나 파우더 담체(carrier)만 넣은 그룹에 비해 현저하게 뛰어난 상처 크기 감소 효과를 나타내었다.As a result, as shown in Figure 6, it can be seen that the size of the wound is significantly reduced with time, the powder-treated group is significantly superior to the non-treated group or the group containing only the powder carrier (carrier) Size reduction effect was shown.
<< 실험예Experimental Example 6> in  6> in vivovivo 균  Fungus 수 정량Water quantification 및  And Twort의Twort 그람 염색법 Gram Staining
상기 실시예 1에서 제조한 파우더 조성물을 대상으로, 마우스 모델에서 상처에 있는 균 수를 정량하였다. 이때 다음과 같은 실험 방법을 수행하였다: 6주령의 ICR 마우스를 구입하여 1주 동안 적응시킨 후 마취하고 털을 제거하였다. 등 부분에 8 mm의 생검 펀치를 이용하여 전층 상처를 생성하였고 상처마다 109 CFU의 P. aeruginosa 균을 가한 후 상처를 밀봉, 2일간 방치하여 감염된 전층 상처를 만들었다. 그 후 상기 실시예 1에서 제조한 파우더 조성물 및 GSNO가 없는 파우더 조성물(파우더 담체), 비처리 그룹의 총 3 그룹으로 나누어 처리를 하고 정해진 날짜마다 3마리의 마우스를 안락사시켜 상처 부위를 뜯어낸 후 잘게 분쇄하고 희석하여, P. aeruginosa 균만 선택적으로 성장 가능한 세트리미드(cetrimide) 배지에서 배양하였고, CFU를 측정하였다.For the powder composition prepared in Example 1, the number of bacteria in the wound in the mouse model was quantified. At this time, the following experimental methods were performed: 6-week-old ICR mice were purchased, acclimated for 1 week, anesthetized, and hair was removed. Whole layer wounds were created using 8 mm biopsy punches on the back, and 10 9 CFU of P. aeruginosa was added to each wound, and the wounds were sealed and left for 2 days to make infected whole layer wounds. Thereafter, the powder composition prepared in Example 1 and the powder composition without GSNO (powder carrier) were divided into a total of three groups of non-treated groups, and euthanized three mice every fixed day to tear off the wound site. Finely ground and diluted, P. aeruginosa The bacteria were cultured in cetrimide medium where only bacteria were selectively grown, and CFU was measured.
또한, Twort의 그람 염색법을 다음과 같이 수행하였다: 실험 마지막 날인 14일째에 동물을 안락사시킨 후 상처 부위를 채취하여 파라핀 블록(paraffin block)으로 만들어 보존하였다. 상기 블록을 5 um 두께로 절단하여 슬라이드 글라스 위에 붙이고 12시간 37℃ 오븐에서 건조시켰다. 자일렌을 이용하여 파라핀을 제거하고 100 % 에탄올, 95 % 에탄올을 각각 가하고 증류수로 세척하였다. 크리스탈 바이올렛으로 1차 염색, 내추럴 레드(Natural red) 및 패스트 그린(fast green) 용액으로 2차 염색한 후, 자일렌으로 투명 과정을 거쳐서 마운팅 미디어를 넣고 커버글라스를 덮었다. 마지막으로 광학 현미경을 이용하여 1000배 확대한 배율에서 1 um정도 길이의 빨간색을 띄는 P. aeruginosa 균을 관찰, 촬영하였다.In addition, Gram staining of Twort was performed as follows: On the 14th day of the last day of the experiment, animals were euthanized and wounds were harvested to make paraffin blocks and preserved. The block was cut to a thickness of 5 um, pasted onto a slide glass and dried in an oven at 37 ° C. for 12 hours. Paraffin was removed using xylene, and 100% ethanol and 95% ethanol were added thereto, and washed with distilled water. After primary dyeing with crystal violet, secondary dyeing with natural red and fast green solution, the mounting medium was covered with xylene and then covered with a cover glass. Finally, P. aeruginosa, which is about 1 um long red at 1000x magnification using an optical microscope. The bacteria were observed and photographed.
그 결과, 도 7에 나타난 바와 같이, P. aeruginosa 감염 ICR 마우스 모델에서 상처에 존재하는 균의 양을 측정하였을 때, 상기 파우더 조성물 처리군에서 세균의 양이 현저히 감소하였는 바, 본 발명에 따른 파우더 조성물이 상처 부위의 균을 효과적으로 제거할 수 있다는 것을 증명하였다.As a result, as shown in Figure 7, P. aeruginosa When the amount of bacteria in the wound was measured in the infected ICR mouse model, the amount of bacteria in the powder composition treatment group was significantly reduced, indicating that the powder composition according to the present invention can effectively remove the bacteria in the wound area. Proved.
또한, 치료 개시 후 14일째 되는 시점에서 상처 표면에 있는 균 확인 시험 (Twort's gram staining)의 결과를 나타내는 도 8의 내용과 같이, 치료 개시 후 14 일째 되던 날의 상처 부분을 조직학적으로 관찰하여 거기에 P. aeruginosa 균이 있는지 시각화하는 조직 염색 기법을 통해, 비처리군에서는 매우 많은 수의 균을 시각적으로 관찰할 수 있었으나, 상기 파우더 조성물을 처리한 그룹에서는 탐지가 되지 않을 정도로 적은 균만 존재한다는 것을 볼 수 있었다(정상 피부(A), 비처리군(B, C), 파우더 담체 처리군(D), 본 발명의 파우더 조성물(E) 처리군).In addition, as shown in FIG. 8 showing the results of Wort's gram staining on the wound surface at the point of 14 days after the start of treatment, the wound was observed histologically on the day 14 after the start of treatment. P. aeruginosa Tissue staining techniques to visualize the presence of bacteria showed that a large number of bacteria could be visually observed in the untreated group, but in the group treated with the powder composition, only a small number of bacteria were present that could not be detected. Normal skin (A), untreated group (B, C), powder carrier treated group (D), powder composition (E) treated group of the present invention).
<< 실험예Experimental Example 7> 조직 회복 정도 분석 7> Analysis of tissue recovery
상기 실시예 1에서 제조한 파우더 조성물을 대상으로, 조직 확인을 위한 헤마톡실린(Hematoxylin) & 에오신(Eosin) 염색(H&E 염색)과 콜라겐 양 확인을 위한 메이슨의 트리크롬 염색 기법(masson’s trichrome staining)을 수행하였다. 구체적으로, 다음과 같은 방법으로 수행하였다: 실험 마지막 날인 14일째에 동물을 안락사시킨 후 상처 부위를 채취하여 파라핀 블록으로 만들어 보존하였다. 그 후 5 um 두께로 절단하여 슬라이드 글라스 위에 붙이고 12시간동안 37℃ 오븐에서 건조시켰다. 자일렌을 이용하여 파라핀을 제거하고 100 % 에탄올, 95 % 에탄올을 각각 가하였고, 증류수로 세척하였다. H&E 염색법의 경우, 헤마톡실린과 에오신으로 차례대로 염색하였고, 메이슨의 트리크롬 염색의 경우, 판매자(ABCAM)가 제공한 염색 방법 순서에 따라 염색하였다. 그 후 광학 현미경을 이용하여 200배의 배율에서 조직을 비교, 관찰하여 촬영하였다.Hematoxylin & Eosin staining (H & E staining) and Mason's trichrome staining technique (masson's trichrome staining) to confirm the amount of collagen for the powder composition prepared in Example 1 Was performed. Specifically, the method was carried out as follows: On the 14th day of the last day of the experiment, the animals were euthanized and the wounds were collected and preserved by making paraffin blocks. It was then cut to 5 um thickness, pasted onto a slide glass and dried in an oven at 37 ° C. for 12 hours. Paraffin was removed using xylene, and 100% ethanol and 95% ethanol were added, respectively, and washed with distilled water. In the case of H & E staining, hematoxylin and eosin were sequentially stained, and in the case of Mason's trichrome staining, staining was performed according to the staining method sequence provided by ABCAM. Thereafter, tissues were compared, observed and photographed at a magnification of 200 times using an optical microscope.
그 결과, 도 9A 내지 D를 참조하면, 파우더 조성물을 처리하였을 때, 비처리군이나 파우더 담체만을 처리한 그룹에 비해 보다 정상 조직에 가까운 정도로 회복이 잘 된 것을 볼 수 있었다. 또한, 도 9E 내지 9H를 참조하면, 상기 파우더 조성물을 처리한 그룹에서 다른 두 그룹에 비해 많은 콜라겐 양을 관찰할 수 있었다.As a result, referring to Figures 9A to D, when the powder composition is treated, it can be seen that the recovery to a degree closer to normal tissue than the group treated with only the untreated group or the powder carrier. 9E to 9H, the amount of collagen was observed in the group treated with the powder composition compared to the other two groups.
<< 실험예Experimental Example 8> 실험 방법에 따른 파우더 입자 특성의 비교 8> Comparison of Powder Particle Characteristics According to Experimental Method
상기 실시예 1에서 제조한 파우더 조성물과 분쇄 과정을 거치지 않고 제조된 비교예 1을 대상으로 함량 균일성을 확인하였다. 즉, 각 파우더를 펼쳐 놓고 무작위로 10 곳에서 샘플링하여 GSNO의 함량을 측정하였다. 이때, 10 mg 정도의 파우더의 무게를 측정하고 물에 녹인 후 자외가시부 분광광도계를 이용하여 335 nm에서 흡광도를 측정하여 GSNO의 함량을 측정, 비교하였다. 또한, 하우스너 비율은 탭 밀도와 벌크 밀도를 구하여 이들의 비로 계산하였다.The content uniformity of the powder composition prepared in Example 1 and Comparative Example 1 prepared without undergoing grinding was confirmed. That is, each powder was unfolded and randomly sampled at 10 places to measure the content of GSNO. At this time, the weight of the powder of about 10 mg was measured and dissolved in water, and the absorbance at 335 nm by using an ultraviolet visible spectrophotometer to measure and compare the content of GSNO. In addition, the Hausner ratio calculated | required tap density and bulk density, and calculated it by these ratios.
그 결과, 도 10에 개시된 바와 같이, 분쇄 과정을 거친 실시예 1의 파우더 조성물은 GNSO가 더 고르게 분포되어 균일성이 높은 제제를 제조할 수 있음을 확인하였고, 또한 하우스너 비율이 증가하여 분체 상태에서 유동성이 저하되므로, 실제 상처에 적용 시 파우더가 겔로 변하기 전에 흘러내리는 현상을 더 감소시킬 수 있음을 확인하였다.As a result, as shown in FIG. 10, the powder composition of Example 1, which was subjected to the grinding process, was found to have a more uniform distribution of GNSO, thereby preparing a highly uniform formulation. Since the fluidity is lowered in, it was confirmed that when applied to the actual wound it can further reduce the phenomenon of the powder flowing down before it turns into a gel.
<< 실험예Experimental Example 9> 다른  9> other 겔화제와의With gelling agent 겔 형성 시간 비교 Gel Formation Time Comparison
알지네이트 대신 다른 겔화제를 사용할 경우 겔 형성 능력에 차이가 생기는지 여부를 확인하기 위하여, GSNO를 혼합하지 않은 것을 제외하고는 상기 실시예 1과 동일한 방법으로 다양한 겔화제를 함유하는 파우더 담체 조성물을 제조하였다. 구체적으로, 비교예로서 겔화제인 알지네이트 대신 각각 히알루론산(Hyaluronate), 카라기난(Carrageenan), 폴록사머(Poloxamer), 젤라틴(Gelatin), 구아검(Guar gum), CMC-Na 또는 키토산(Chitosan)을 사용하여 비교예 2 내지 8 파우더 담체 조성물을 제조하였다. 이때, 각 조성물의 함량 비율은 실시예 1의 파우더 조성물의 GSNO를 제외한 부형제 비율과 동일하게, 겔화제 : 펙틴 : PEG = 2 : 1 : 6 이었다.Powder carrier compositions containing various gelling agents were prepared in the same manner as in Example 1 except that GSNO was not mixed in order to determine whether the gelling ability was different when using other gelling agents instead of alginate. . Specifically, hyaluronic acid, carrageenan, poloxamer, gelatin, guar gum, CMC-Na or chitosan are used instead of alginate as a gelling agent, respectively. To prepare a powder carrier composition of Comparative Examples 2 to 8. At this time, the content ratio of each composition was the same as the excipient ratio except the GSNO of the powder composition of Example 1, the gelling agent: pectin: PEG = 2: 1: 6.
이후, 10 mg의 각 조성물을 바닥이 평평한 2 ml 튜브에 넣고 100 ㎕의 PBS를 떨어뜨린 다음 겔이 되는 시점을 측정하였다. 이때, 겔이 되는 시점으로서 튜브를 뒤집어서 가볍게 두드렸을 때 생성된 겔이 5초 이상 떨어지지 않고 튜브에 붙어 있는 시점을 기준으로 하였다.Thereafter, 10 mg of each composition was placed in a flat bottom 2 ml tube, and 100 μl of PBS was dropped, and the time point of the gel was measured. At this time, as a time point to become a gel, the gel generated when the tube was turned upside down and lightly knocked was based on the time point when the gel was attached to the tube without dropping for 5 seconds or more.
그 결과, in situ 하이드로겔 형성 파우더로 사용하기 위해서는 3분 이내의 신속한 겔 형성 능력이 필수적인 바, 하기 <표 1>을 참조하면, 알지네이트와 히알루론산을 제외한 나머지 겔화제의 경우 겔 형성까지 10분 이상 경과하였으므로 in situ 하이드로겔 형성 파우더의 겔화제로서는 부적합함을 알 수 있다. 즉, 알지네이트 및 히알루론산이 in situ 하이드로겔 형성 현상을 보였으나, 히알루론산은 알지네이트에 비해 느린 겔화 속도를 보였으므로, 신속한 겔 형성을 위해서는 알지네이트가 가장 최적의 겔화제임을 증명하였다.As a result, in order to use as an in situ hydrogel-forming powder, a rapid gel formation ability within 3 minutes is essential. Referring to Table 1 below, the gelling agent except for alginate and hyaluronic acid is 10 minutes until gel formation. Since the above has passed, it can be seen that it is not suitable as a gelling agent of the in situ hydrogel-forming powder. In other words, alginate and hyaluronic acid showed in situ hydrogel formation, but hyaluronic acid showed a slower gelation rate than alginate, and thus alginate was the most optimal gelling agent for rapid gel formation.
SampleSample 평균 겔 형성 시간Average gel formation time S.DS.D 비고Remarks
AlginateAlginate 0.86 분0.86 minutes 0.110.11 NO/GPNO / GP
HyaluronateHyaluronate 3.27 분3.27 minutes 0.170.17 --
CarrageenanCarrageenan 부적합(10분 이상)Not suitable (10 minutes or more) -- 빠른 시간에 겔을 형성하여야 하는 in situ 하이드로겔 형성 파우더 제제에 적합하지 않음Not suitable for in situ hydrogel forming powder formulations which must form gels quickly
PoloxamerPoloxamer 부적합(10분 이상)Not suitable (10 minutes or more) --
GelatinGelatin 부적합(10분 이상)Not suitable (10 minutes or more) --
Guar gumGuar gum 부적합(10분 이상)Not suitable (10 minutes or more) --
CMC-NaCMC-Na 부적합(10분 이상)Not suitable (10 minutes or more) --
ChitosanChitosan 부적합(10분 이상)Not suitable (10 minutes or more) --
<< 실험예Experimental Example 10>  10> 알지네이트Alginate 함량에 따른 치료 효과 비교 Comparison of treatment effects by content
낮은 농도의 알지네이트를 포함할 경우에도 치료 효과가 있는지 확인하기 위해, 상기 실시예 1에서 개시한 제조방법대로 파우더 조성물을 제조하되, 알지네이트 함량을 10%(전체 중량에 대하여)로 하여, 효과가 검증된 GSNO의 중량비인 2%의 GSNO를 함유하는 파우더 및 10%의 GSNO를 함유하는 파우더를 제조하였다. 이때 함량 비율로서 GSNO : 알지네이트염 : 펙틴 : PEG가 2 : 10 : 5 : 83 및 10 : 10 : 5 : 75 가 되도록 제조하였다. 그리고 높은 농도의 알지네이트가 실제 상처에 미치는 영향을 알아보기 위해, 40 %의 알지네이트를 함유하는 파우더를 제조(GSNO : 알지네이트염 : 펙틴 : PEG는 4 : 40 : 20 : 36)하였고, 실시예 1에서 제조한 파우더 조성물과 동일하게 마우스 동물 모델에 처리하였다.In order to check whether there is a therapeutic effect even when a low concentration of alginate is included, a powder composition is prepared according to the method described in Example 1, but the alginate content is 10% (relative to the total weight), and the effect is verified. A powder containing 2% GSNO and a powder containing 10% GSNO were prepared. At this time, the GSNO: alginate salt: pectin: PEG as a content ratio was prepared so that 2: 10: 5: 83 and 10: 10: 5: 75. And to determine the effect of the high concentration of alginate on the actual wound, a powder containing 40% alginate was prepared (GSNO: alginate salt: pectin: PEG 4: 4: 20: 36), and in Example 1 The mouse animal model was treated in the same manner as the prepared powder composition.
이때, 구체적인 실험 방법은 다음과 같았다: 6주령의 ICR 마우스를 구입하여 1주동안 적응시킨 후 마취, 털을 제거하였다. 등 부분에 8 mm의 생검 펀치를 이용하여 전층 상처를 생성하고 위의 약물 및 GSNO가 없는 담체 파우더 조성물, 비처리그룹으로 나누어 약물을 처리하고 2일마다 사진을 촬영하여 그 크기를 비교, 분석하였다.At this time, the specific experimental method was as follows: 6 weeks old ICR mice were purchased and allowed to acclimate for 1 week, followed by anesthesia and hair removal. Whole layer wounds were made using 8 mm biopsy punches on the back, and the drug was treated into the above-mentioned drug and GSNO-free carrier powder composition, and the non-treated group, and the drugs were taken and photographed every two days to compare and analyze the size. .
그 결과, 도 11a를 참조하면, 15% 미만의 낮은 함량의 알지네이트(2% 및 10%)를 이용한 조성물 파우더의 경우, 삼출물을 적절히 흡수하지 못해 겔 형태를 유지하지 못했고, GSNO가 쉽게 제거되었으며, 특히 마우스에 처리한 경우 유의미한 치료 효과가 나타나지 않았다. 반면, 도 11b를 참조하면, 15 내지 30%의 알지네이트를 포함하는 파우더 조성물은 삼출물을 흡수하여 겔 형태를 유지하면서 지속적으로 NO를 방출하였고, 특히 마우스 모델에서 유의미한 치료 효과를 나타내었다. 또한, 도 11c를 참조하면, 알지네이트가 너무 높은 비율(30% 초과)로 포함된 경우, 흡수력이 너무 높아 창상 부위가 건조해지고 드레싱이 상처에 강하게 엉겨 붙는 현상이 발생하여, 일반적인 창상에 사용하기에는 어려울 것으로 판단하였다.As a result, referring to Figure 11a, in the case of the composition powder using a low content of alginate (2% and 10%) of less than 15%, the exudates did not properly absorb the gel form, GSNO was easily removed, In particular, mice did not show a significant therapeutic effect. On the other hand, referring to Figure 11b, the powder composition comprising 15 to 30% of the alginate absorbed the exudate and continuously released NO while maintaining the gel form, especially in the mouse model showed a significant therapeutic effect. In addition, referring to FIG. 11C, when the alginate is included in an excessively high ratio (greater than 30%), absorption is so high that the wound is dried and the dressing is strongly entangled in the wound, which makes it difficult to use in general wounds. It was judged that.
<< 실험예Experimental Example 11> 펙틴 및 PEG의 역할 확인 실험 11> Confirmation of the role of pectin and PEG
펙틴 및 PEG의 역할을 확인하기 위해 알지네이트를 22 중량부로 고정하고 펙틴과 PEG의 양을 변경하여 실험하였다. 즉, 상기 실시예 1에서 개시한 제조방법대로 파우더 조성물을 제조하되, 알지네이트 22 중량부와 펙틴 77 중량부(알지네이트:펙틴 = 2:7 중량비)만 포함된 경우(A), 알지네이트 22 중량부와 PEG 77 중량부 (알지네이트:PEG = 2:7 중량비)만 포함된 경우(B), 및 알지네이트 22 중량부와 펙틴 11 중량부 및 PEG 66 중량부(알지네이트:펙틴:PEG = 2:1:6 중량비)가 포함된 경우(C)의 파우더를 각각 제조한 뒤, 겔 형성 실험을 수행하였다. 구체적인 방법은 다음과 같았다: 제조한 각각의 파우더를 작은 디쉬에 50 mg씩 고르게 퍼트린 다음, 1 ml의 증류수를 고르게 가하여 형성되는 겔의 형상을 관찰, 촬영하였다.In order to confirm the role of pectin and PEG, alginate was fixed at 22 parts by weight and experimented by changing the amount of pectin and PEG. That is, when preparing a powder composition according to the production method disclosed in Example 1, but containing only 22 parts by weight of alginate and 77 parts by weight of pectin (alginate: pectin = 2: 7 weight ratio) (A), 22 parts by weight of alginate and Only 77 parts by weight of PEG (alginate: PEG = 2: 7 weight ratio) and 22 parts by weight of alginate and 11 parts by weight of pectin and 66 parts by weight of PEG (alginate: peptine: PEG = 2: 1: 6 weight ratio ) Was prepared in each case of the powder (C), and then gel formation experiments were performed. The specific method was as follows: Each powder prepared was evenly spread by 50 mg in a small dish, and then 1 ml of distilled water was evenly added to observe and photograph the shape of the gel formed.
그 결과, 도 12에 나타난 바와 같이, PEG가 없는 경우(A), 서로 파우더 입자들끼리 엉기는 현상이 발생하여 파우더가 뿌려진 부분에만 점도 높은 겔이 형성되었고, 펙틴이 없는 경우(B), 균일한 겔이 형성되지만 겔의 강도가 약했고, 겔이 완전히 흐르지 않을 때까지의 시간이 펙틴이 있는 경우보다 오래 걸리는 것을 확인할 수 있었다. 본 발명의 실시예 1과 같이, 알지네이트, 펙틴, PEG가 모두 포함되어 있는 경우(C), 신속하면서도 균일하게 겔이 형성되는 것을 확인하였다.As a result, as shown in Figure 12, when there is no PEG (A), the powder particles are entangled with each other to form a gel having a high viscosity only in the portion where the powder is sprayed, there is no pectin (B), uniform One gel was formed, but the strength of the gel was weak, and it was confirmed that the time until the gel did not flow completely took longer than that with pectin. As in Example 1 of the present invention, when all of the alginate, pectin, PEG is contained (C), it was confirmed that the gel is formed quickly and uniformly.
이러한 실험 결과로부터, 과량의 펙틴 또는 PEG는 균일하고 튼튼한 겔의 형성에 좋지 않고, 펙틴은 5 내지 20 중량부, PEG는 50 내지 80 중량부로 포함된 제제가 in situ 하이드로겔 형성 파우더로 적합함을 알 수 있었다.From these experimental results, it was found that excess pectin or PEG was not good for the formation of a uniform and durable gel, and a formulation containing 5 to 20 parts by weight of pectin and 50 to 80 parts by weight of PEG was suitable as an in situ hydrogel forming powder. Could know.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 즉, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다.Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. Do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (10)

  1. S-나이트로소글루타치온(S-Nitrosoglutathione; GSNO), 알지네이트염, 펙틴 및 폴리에틸렌글리콜(PEG)을 유효성분으로 포함하는 창상(wound) 치료용 파우더 조성물.Powder composition for wound treatment comprising S-nitrosoglutathione (GSNO), alginate salt, pectin and polyethylene glycol (PEG) as an active ingredient.
  2. 제 1 항에 있어서, 상기 파우더 조성물 100 중량부에 대하여, GSNO 2 중량부 내지 10 중량부, 알지네이트염 10 중량부 내지 40 중량부, 펙틴 5 중량부 내지 20 중량부 및 PEG 50 중량부 내지 80 중량부로 포함되는 것을 특징으로 하는 창상 치료용 파우더 조성물.According to claim 1, To the 100 parts by weight of the powder composition, 2 to 10 parts by weight of GSNO, 10 to 40 parts by weight of alginate salt, 5 to 20 parts by weight of pectin and 50 to 80 parts by weight PEG Powder composition for wound treatment, characterized in that it is included as a part.
  3. 제 1 항에 있어서, 상기 GSNO : 알지네이트염 : 펙틴 : PEG는 4 : 21 : 11 : 64의 중량비로 포함되는 것을 특징으로 하는 창상 치료용 파우더 조성물.The powder composition of claim 1, wherein the GSNO: alginate salt: pectin: PEG is included in a weight ratio of 4: 21: 11: 64.
  4. 제 1 항에 있어서, 상기 파우더 조성물은 창상 부위에서 1분 내지 3분 내에 하이드로겔 형태로 변성되는 것을 특징으로 하는 창상 치료용 파우더 조성물.The method of claim 1, wherein the powder composition is a wound composition for the wound composition, characterized in that denatured in hydrogel form within 1 to 3 minutes at the wound site.
  5. 제 1 항에 있어서, 상기 파우더 조성물은 창상 부위에서 산화질소(NO)를 서방출함으로써 창상을 치료하는 것을 특징으로 하는 창상 치료용 파우더 조성물.The method of claim 1, wherein the powder composition is wound treatment powder composition, characterized in that for treating the wound by releasing nitric oxide (NO) at the wound site.
  6. 제 1 항에 있어서, 상기 파우더 조성물은 녹농균(Pseudomonas aeruginosa) 또는 메티실린 내성 황색포도알균(methicillin-resistant Staphylococcus aureus) 중 어느 하나 이상의 균에 대한 항균 활성을 나타냄으로써 창상을 치료하는 것을 특징으로 하는 창상 치료용 파우더 조성물.The method of claim 1, wherein the powder composition is Pseudomonas Aeruginosa ) or methicillin-resistant Staphylococcus aureus (W) shows a antimicrobial activity against any one or more bacteria, wound powder composition for the treatment of wounds, characterized in that.
  7. 제 1 항에 있어서, 상기 창상은 찰과상, 열상, 화상, 자상, 절상, 결출상, 관통상 및 좌상에서 선택된 어느 하나 이상인 것을 특징으로 하는 창상 치료용 파우더 조성물.The method of claim 1, wherein the wound is a wound, a wound, a burn, a cut, a cut, a cut out, a penetrating wound and a wound wound, characterized in that any one or more selected from the powder composition.
  8. (1) 분말 형태의 GSNO, 알지네이트염, 펙틴 및 PEG를 각각 분쇄하는 단계;(1) grinding GSNO, alginate salt, pectin and PEG in powder form, respectively;
    (2) 상기 단계 (1)에서 제조한 분쇄물을 모두 혼합하는 단계; 및(2) mixing all the pulverized products prepared in step (1); And
    (3) 상기 단계 (2)에서 제조한 혼합물을 체에 통과시키는 단계를 포함하는 창상 치료용 파우더 조성물의 제조방법.(3) A method of producing a powder composition for wound treatment comprising passing the mixture prepared in step (2) through a sieve.
  9. 제 8 항에 있어서, 상기 단계 (1) 내지 (3)에서, 상기 파우더 조성물 100 중량부에 대하여, GSNO 2 중량부 내지 10 중량부, 알지네이트염 10 중량부 내지 40 중량부, 펙틴 5 중량부 내지 20 중량부 및 PEG 50 중량부 내지 80 중량부로 포함되는 것을 특징으로 하는 창상 치료용 파우더 조성물의 제조방법.The method according to claim 8, wherein in the steps (1) to (3), based on 100 parts by weight of the powder composition, 2 parts to 10 parts by weight of GSNO, 10 parts to 40 parts by weight of alginate salt, 5 parts by weight to pectin 20 parts by weight and 50 parts by weight of PEG to 80 parts by weight of the method for producing a powder composition for treating wounds.
  10. 제 8 항에 있어서, 상기 단계 (3)에서, 제조된 창상 치료용 파우더 조성물은 GSNO : 알지네이트염 : 펙틴 : PEG를 4 : 21 : 11 : 64의 중량비로 포함하는 것을 특징으로 하는 창상 치료용 파우더 조성물.The wound treatment powder of claim 8, wherein the wound composition prepared in step (3) comprises GSNO: alginate salt: pectin: PEG in a weight ratio of 4: 21: 11: 64. Composition.
PCT/KR2018/004191 2017-04-10 2018-04-10 Hydrogelized powder composition for wound healing and method for producing same WO2018190613A1 (en)

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