KR100766820B1 - Transmucosal Delivery System for Proteins or Peptides - Google Patents
Transmucosal Delivery System for Proteins or Peptides Download PDFInfo
- Publication number
- KR100766820B1 KR100766820B1 KR1020060068804A KR20060068804A KR100766820B1 KR 100766820 B1 KR100766820 B1 KR 100766820B1 KR 1020060068804 A KR1020060068804 A KR 1020060068804A KR 20060068804 A KR20060068804 A KR 20060068804A KR 100766820 B1 KR100766820 B1 KR 100766820B1
- Authority
- KR
- South Korea
- Prior art keywords
- chitosan
- protein
- peptide
- insulin
- delivery system
- Prior art date
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/28—Insulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
Abstract
본 발명은 (ⅰ) 키토산; 및 (ⅱ) 상기 키토산에 공유결합된 생체 내 전달 목적의 단백질 또는 펩타이드를 포함하는 단백질의 경점막 운반 시스템(transmucosal delivery system) 및 약제학적 조성물에 관한 것으로서, 본 발명의 단백질 또는 펩타이드-키토산 복합체 및 약제학적 조성물은 생체 점막, 특히, 소화관(특히, 위장관)에 있는 점막에서 흡수율이 우수하고 안정성이 뛰어날 뿐만 아니라, 운반체로 이용된 키토산은 생체적합성이 우수하고 체내에서 분해되기 때문에 경구 투여 시에도 생체이용율이 우수하여 경구 투여를 통한 단백질 의약에 의한 치료를 가능하게 한다.The present invention (i) chitosan; And (ii) a transmucosal delivery system and a pharmaceutical composition of a protein comprising a protein or peptide for in vivo delivery covalently bound to the chitosan, the protein or peptide-chitosan complex of the present invention and The pharmaceutical compositions not only provide good absorption and stability in the mucous membranes of the living body, especially in the gastrointestinal tract (particularly the gastrointestinal tract), but the chitosan used as a carrier has excellent biocompatibility and decomposes in the body. Excellent utilization allows for treatment with protein medications via oral administration.
키토산, 단백질, 펩타이드, 의약, 경점막, 경구 Chitosan, protein, peptide, medicine, transmucosal, oral
Description
도 1은 본 발명의 인슐린-키토산 복합체를 당뇨가 유도된 웅성 래트의 꼬리 정맥에 주사한 후 상대적 혈당치를 측정하여 나타낸 그래프이다.1 is a graph showing the relative blood glucose levels after injecting the insulin-chitosan complex of the present invention into the tail vein of male rats induced with diabetes.
도 2는 본 발명의 인슐린-키토산 복합체 용액을 당뇨가 유도된 웅성 래트에 경구 투여한 후 상대적 혈당치를 측정하여 나타낸 그래프이다.2 is a graph showing the relative blood glucose levels after oral administration of the insulin-chitosan complex solution of the present invention to male rats induced with diabetes.
본 발명은 경점막 운반 시스템(transmucosal delivery system)에 관한 것으로서, 보다 상세하게는 본 발명은 단백질 또는 펩타이드를 위한 경점막 운반 시스템 및 경점막 운반용 약제학적 조성물에 관한 것이다.The present invention relates to a transmucosal delivery system, and more particularly, to a transmucosal delivery system for a protein or peptide and a pharmaceutical composition for transmucosal delivery.
유전자 조작과 생물공정 기술의 발전으로, 현재까지 화학적 합성이 어려웠던 다양한 펩타이드 및 단백질 의약, 즉 바이오 의약을 대량 생산하는 것이 가능해졌다. 그러나 대부분의 단백질은 거대한 분자량과 특이적인 분자구조로 인하여 생 체 점막에서 흡수되지 않는 특성을 보임으로써 경구 유사 제제로 사용되지 못하는 어려움이 있었다. 따라서, 그 투여방법이 주사제로 제한되어 있는 바, 이는 장기 복용시 투약의 어려움 및 대상 환자에게 주사에 대한 두려움과 거부감을 유발한다는 문제가 있다. 따라서, 바이오 의약의 장내 흡수율을 높여 투여에 대한 부담이 없는 경구 제제로 개발할 수 있게 되면, 주사에 대한 두려움과 거부감을 없애고 환자가 복약 지시대로 약물을 복용하기가 용이해지므로 단기 혹은 장기적으로 환자의 삶의 질을 높일 수 있게 될 것이다.Advances in genetic engineering and bioprocessing technology have made it possible to mass produce a variety of peptide and protein medicines, biopharmaceuticals, which until now have been difficult to chemically synthesize. However, most proteins are difficult to be used as oral analogues because of their large molecular weight and specific molecular structure, which prevent them from being absorbed by the living mucosa. Therefore, the method of administration is limited to injections, which causes problems with long-term administration and causes fear and rejection of injections in the target patient. Therefore, if the intestinal absorption rate of biopharmaceuticals can be increased and developed into an oral preparation that does not burden administration, the patient will be able to take the drug as directed by the medication and eliminate the fear and rejection of the injection. You will be able to improve your quality of life.
이러한 이유로 지금까지 치료용 단백질의 생체 내 안정성과 흡수율을 증진시키려는 방법들이 다양하게 시도되었다. 이들 중 가장 잘 알려진 방법이 "PEG화 (PEGylation)"로서, 이는 PEG를 단백질에 화학적으로 부가시키는 방법이다. 이 방법은 처음에는 항원성을 감소시키려는 목적으로 사용되었으나 현재는 목적 단백질의 생체 내 체재 기간을 증가시킴으로써 생체내 안정성과 흡수율을 증진시키려는 목적으로 많이 이용되고 있다.For this reason, various methods have been tried to improve the in vivo stability and absorption rate of therapeutic proteins. The best known of these is "PEGylation", which is a chemical addition of PEG to a protein. This method was initially used for the purpose of reducing antigenicity, but is now widely used for the purpose of enhancing the in vivo stability and absorption rate by increasing the length of stay of the target protein in vivo.
이것 이외에도 바이오 의약과 함께 지방산 혹은 담즙산 등과 같이 장 상피세포막의 침투성(permeability)을 높이는 물질을 사용하는 방법, 장 상피세포막의 수용체와 선택적 결합을 할 수 있는 물질(예: Vitamin B12 및 Fc 수용체)을 이용한 방법, 지질이나 담즙산과 같은 지용성 물질을 인슐린에 직접 화학결합시킨 컨쥬게이트를 만들어 장내 점막에서의 흡수율을 높이는 방법, 생분해성 고분자로 만든 마이크로크기의 입자 또는 나노 크기의 입자에 단백질 의약을 내포시켜 전달하는 방법 등이 연구되고 있다.In addition to biopharmaceuticals, methods using a substance that enhances the permeability of the intestinal epithelial membrane, such as fatty acids or bile acids, and substances capable of selectively binding to receptors of the intestinal epithelial membrane (e.g. Vitamin B12 and Fc receptors) Method to increase the absorption rate in the intestinal mucosa by making conjugate conjugated fat-soluble substances such as lipids and bile acids directly to insulin, and embedding protein medicine in micro-sized or nano-sized particles made of biodegradable polymer Methods of delivery are being studied.
그러나 이러한 방법들은 경구 투여시 여전히 매우 낮은 생체흡수율 및 생체이용율을 보이며, 기존의 경구 제형화 방법들은 장기적으로 복용시 독성을 보일 수 있는 첨가물들을 사용하므로 잠재적인 안전성이 우려되는 단점을 가지고 있다.However, these methods still show very low bioabsorption rate and bioavailability when administered orally, and conventional oral formulation methods use additives that may be toxic when taken in the long term.
경구 투여된 약물은 소화관의 점막을 통하여 생체 내로 흡수되기 때문에, 경구 투여용 약물은 경점막 운반 시스템을 갖추어야 한다. 경점막 운반(transmucosal delivery)은 약물을 투여하는 방법으로서 많은 이점을 가지고 있다. 전신성 및 국소성 약물 효능을 달성할 수 있는 경점막 운반의 능력 때문에, 특정 처방(regimen)에 대한 요구에 맞출 수 있어 경점막 운반은 매력적인 약물운반시스템으로 각광을 받고 있다. 경점막 운반은 신속하게 치료 효능을 발휘할 뿐만 아니라 빠른 약물 청소율(clearance)을 나타내기 때문에, 결국 약물의 생체이용율(bioavailability)을 증가시킨다. 또한, 경점막 운반 시스템은 다른 투여 방식보다 환자 순응도(compliance)가 우수하다.Because orally administered drugs are absorbed in vivo through the mucous membranes of the digestive tract, drugs for oral administration must have a transmucosal delivery system. Transmucosal delivery has many advantages as a method of administering drugs. Because of the ability of transmucosal transport to achieve systemic and topical drug efficacy, transmucosal transport is emerging as an attractive drug delivery system because it can be tailored to the needs of certain prescriptions. Transmucosal delivery not only results in rapid therapeutic efficacy but also results in rapid drug clearance, which in turn increases the bioavailability of the drug. In addition, transmucosal delivery systems have better patient compliance than other modes of administration.
경점막 운반 시스템의 상술한 이점들 때문에, 보다 개선된 경점막 운반 시스템을 개발하려는 많은 시도가 있었다. WO 2005/032554, WO 2005/016321, 미합중국 특허 제6896519호, 제6564092호 및 제6506730호에 경점막 운반 시스템이 개시되어 있다.Because of the aforementioned advantages of transmucosal delivery systems, many attempts have been made to develop more improved transmucosal delivery systems. Transmucosal delivery systems are disclosed in WO 2005/032554, WO 2005/016321, United States Patents 6896519, 6554092 and 6567730.
한편, 단백질 의약을 경점막 운반 방식으로 하여 구강 투여(oral administration)할 수 있는 방법을 개발하려는 시도들은 대부분 성공적인 결과를 얻을 수 없었는데, 특히 단백질 의약의 치료학적 효능이 만족스러울 정도가 아니었다.On the other hand, attempts to develop methods for oral administration of protein drugs by transmucosal delivery have largely failed to achieve successful results, particularly the therapeutic efficacy of protein drugs was not satisfactory.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.
본 발명자들은 종래의 단백질 의약용 운반시스템이 가지고 있는 부작용과 단점들을 극복하면서도, 단백질 의약을 경점막 운반, 특히 구강 투여할 수 있는 운반시스템을 개발하기 위하여 노력하였다. 본 발명자들은 상기의 목적을 달성할 수 있는 운반시스템의 기본물질로서 키토산을 선택하였고, 키토산에 단백질 의약을 공유결합시킨 단백질 또는 펩타이드-키토산 복합체를 구강 투여하는 경우, 생체 내에서 우수한 약리학적 효능을 발휘한다는 놀라운 실험 결과를 얻음으로써, 본 발명을 완성하게 되었다.The present inventors endeavored to develop a delivery system capable of transmucosal delivery of protein medicine, especially oral administration, while overcoming the side effects and disadvantages of conventional protein pharmaceutical delivery systems. The present inventors selected chitosan as a basic substance of a delivery system capable of achieving the above object, and when the oral administration of a protein or peptide-chitosan complex covalently bound to a protein medicine to chitosan, the pharmacological efficacy in vivo is excellent. The present invention has been completed by obtaining surprising experimental results.
따라서, 본 발명의 목적은 단백질 또는 펩타이드의 경점막 운반 시스템(transmucosal delivery system)을 제공하는 데 있다.Accordingly, it is an object of the present invention to provide a transmucosal delivery system of proteins or peptides.
본 발명의 다른 목적은 경점막 운반용 약제학적 조성물을 제공하는 데 있다.Another object of the present invention to provide a pharmaceutical composition for transmucosal delivery.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 (ⅰ) 키토산; 및 (ⅱ) 상기 키토산에 공유결합된 생체 내 전달 목적의 단백질 또는 펩타이드를 포함하는 단백질의 경점막 운반 시스템(transmucosal delivery system)을 제공한다.According to one aspect of the present invention, the present invention is (i) chitosan; And (ii) a transmucosal delivery system of a protein comprising a protein or peptide for in vivo delivery covalently bound to the chitosan.
본 발명의 다른 양태에 따르면, 본 발명은 (a) (ⅰ) 키토산; 및 (ⅱ) 상기 키토산에 공유결합된 생체 내 전달 목적의 단백질 또는 펩타이드를 포함하는 단백질의 경점막 운반 시스템의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 경점막 운반용 약제학적 조성물을 제공한다.According to another aspect of the invention, the invention is (a) (iii) chitosan; And (ii) a pharmaceutically effective amount of a transmucosal delivery system of a protein comprising a protein or peptide for in vivo delivery covalently bound to the chitosan; And (b) provides a pharmaceutical composition for transmucosal delivery comprising a pharmaceutically acceptable carrier.
본 발명자들은 종래의 단백질 의약용 운반 시스템이 가지고 있는 부작용과 단점들을 극복하면서도, 단백질 의약을 경점막 운반, 특히 구강 투여할 수 있는 운반시스템을 개발하기 위하여 노력하였다. 우선, 본 발명자들은 상기의 목적을 달성할 수 있는 운반시스템의 기본물질로서 키토산을 선택하였고, 키토산에 단백질 의약을 공유결합시킨 단백질 또는 펩타이드-키토산 복합체를 구강 투여하는 경우, 생체 내에서 우수한 약리학적 효능을 발휘한다는 놀라운 실험 결과를 얻게 되었다.The present inventors have endeavored to develop a delivery system capable of transmucosal delivery of protein medicine, in particular oral administration, while overcoming the side effects and disadvantages of conventional protein pharmaceutical delivery systems. First, the present inventors selected chitosan as a basic material of a delivery system capable of achieving the above object, and when the oral administration of a protein or peptide-chitosan complex covalently bound to a protein medicine to chitosan, the pharmacological superior in vivo The results of amazing experiments have been shown to be effective.
본 발명의 경점막 운반 시스템은 단백질 또는 펩타이드를 경점막 운반하기 위하여 개발된 것이다. 본 명세서에서 용어 “단백질”은 아미노산이 펩타이드 결합으로 연결된 중합체를 의미한다. 용어 “펩타이드”는 아미노산이 펩타이드 결합으로 연결된 올리고머를 의미한다.The transmucosal delivery system of the present invention has been developed for transmucosal delivery of proteins or peptides. As used herein, the term "protein" refers to a polymer wherein amino acids are linked by peptide bonds. The term “peptide” refers to oligomers in which amino acids are linked by peptide bonds.
본 발명의 경점막 운반 시스템에 의해 운반되는 단백질 또는 펩타이드는 어떠한 천연 또는 합성 단백질 또는 펩타이드도 포함하고, 예를 들어 호르몬, 호르몬 유사체, 효소, 효소저해제, 신호전달단백질 또는 그 일부분, 항체 또는 그 일부분, 단쇄항체, 결합단백질 또는 그 결합도메인, 항원, 부착단백질, 구조단백질, 조절단백질, 독소단백질, 사이토카인, 전사조절 인자 및 혈액 응고 인자를 포함하나 이에 한정되는 것은 아니다. 바람직하게는, 본 발명의 경점막 운반 시스템에 의해 운반되는 단백질 또는 펩타이드는 단백질 의약으로 이용되는 인슐린, IGF-1(insulin-like growth factor 1), 성장호르몬, 인터페론, 에리쓰로포이에틴, G-CSF(granulocyte-colony stimulating factor), GM-CSF(granulocyte/macrophage-colony stimulating factor), IL-2(interleukin-2) 또는 EGF(epidermal grwoth factor)이며, 보다 바람직하게는 인슐린 또는 IGF-1이며, 가장 바람직하게는 인슐린이다.Proteins or peptides carried by the transmucosal delivery system of the present invention include any natural or synthetic protein or peptide and include, for example, hormones, hormone analogs, enzymes, inhibitors, signaling proteins or portions thereof, antibodies or portions thereof. , Short chain antibodies, binding proteins or binding domains, antigens, adhesion proteins, structural proteins, regulatory proteins, toxin proteins, cytokines, transcriptional regulators and blood coagulation factors. Preferably, the protein or peptide carried by the transmucosal delivery system of the present invention is insulin, IGF-1 (insulin-like growth factor 1), growth hormone, interferon, erythropoietin, G used as a protein medicine Granulocyte-colony stimulating factor (CSF), granulocyte / macrophage-colony stimulating factor (GM-CSF), interleukin-2 (IL-2) or epidermal grwoth factor (EGF), more preferably insulin or IGF-1 Most preferably insulin.
상기의 단백질 의약은 우수한 약리학적 효능을 발휘하는 것으로 알려져 있으나, 대부분 정맥내 투여 경로를 이용하기 때문에 주사를 통해 인체에 적용되고 있다. 따라서, 상기 단백질 의약에 대한 환자 순응도가 매우 낮은 문제점이 있다. 본 발명의 경점막 운반 시스템은 단백질 의약의 경점막 운반, 특히 경구 투여가 가능하도록 개발된 것으로서, 단백질 의약의 경구 투여제로서의 새로운 처방(regime) 방식을 개척한 것이다.The protein medicament is known to exert excellent pharmacological effects, but most of them are applied to the human body by injection because it uses the intravenous route of administration. Therefore, there is a problem in that the patient compliance with the protein medicine is very low. The transmucosal delivery system of the present invention was developed to enable transmucosal delivery, particularly oral administration, of protein medicine, and pioneered a new regimen of oral administration of protein medicine.
본 발명의 경점막 운반 시스템에서 단백질 또는 펩타이드는 키토산에 공유결합 되어 있다. 키토산(chitosan)은 자연계에 셀룰로오스 다음으로 가장 많이 존 재하는 천연 유기 고분자이며, 해마다 1,000억 톤 이상 생산되는 키틴으로부터 제조되는데, 게, 새우 등의 갑각류, 메뚜기, 잠자리 등 곤충류, 팽이버섯, 표고버섯 등의 버섯류, 세균의 세포막 등에 분포하고 있는 키틴을 탈아세틸화 시켜 얻는다. 화학적 구조면에서 보면, N-아세틸-D-글루코사민(N-acetyl-D-glucosamine) 단량체가 β-1,4 결합의 직쇄상으로 연결된 키틴에서 아민기에 존재하던 아세틸기가 제거되어 키토산이 형성된다(Errington N, et al., Hydrodynamic characterization of chitosan varing in molecular weight and degree of acetylation. Int J Biol Macromol. 15:1123-7(1993)). 키토산은, 키틴과 비교할 때 아민기에 존재하던 아세틸기가 제거되었으므로 산성 용액에서 다중양이온(polycation)으로 존재하게 된다. 따라서 산성 수용액에서 물에 대한 용해성이 좋아지므로 가공성이 우수하고 건조후의 기계적 강도가 비교적 우수하므로 분말, 섬유, 박막, 겔, 구슬 등의 형태로 성형되어 사용된다(E. Guibal, et al., Ind . Eng. Chem . Res ., 37:1454-1463(1998)). 키토산은 연결되어 있는 단량체의 개수에 따라 단량체 12개 정도 내외의 올리고머와 고분자에 속하는 폴리머로 나뉘고, 폴리머는 분자량 15만 미만의 저분자 키토산, 분자량 70만에서 100만에 이르는 고분자 키토산, 그리고 그 사이의 범위를 가지는 중분자 키토산으로 나뉘어진다.Protein or peptide in the transmucosal delivery system of the present invention is covalently bonded to chitosan. Chitosan is a natural organic polymer that is the second largest in nature after cellulose, and is made from chitin, which is produced over 100 billion tons each year.Insects such as crabs and shrimps, grasshoppers, dragonflies, insects, enoki mushrooms and shiitake mushrooms It is obtained by deacetylation of chitin, which is distributed in mushrooms and bacterial cell membranes. In terms of chemical structure, the N-acetyl-D-glucosamine monomer is removed from the chitin linked to the β-1,4 bond by the acetyl group present in the amine group to form chitosan ( .. Errington N, et al, Biol Hydrodynamic characterization of chitosan varing in molecular weight and degree of acetylation Int J Macromol 15:. 1123-7 (1993)). Chitosan is present as a polycation in acidic solution because the acetyl group that was present in the amine group was removed compared to the chitin. Therefore, since it has good solubility in water in acidic aqueous solution, it has excellent processability and relatively high mechanical strength after drying, so it is used in the form of powder, fiber, thin film, gel, beads, etc. (E. Guibal, et al., Ind .. Eng Chem Res, 37: .. 1454-1463 (1998)). Chitosan is divided into oligomers of about 12 monomers and polymers belonging to the polymer according to the number of monomers connected, and the polymer is composed of low molecular weight chitosan having a molecular weight of less than 150,000, polymer chitosan having a molecular weight of 700,000 to 1 million, and between It is divided into the middle molecule chitosan which has a range.
키토산은 안정성과 환경 친화성, 생분해성 및 생체적합성이 우수하여 여러 산업과 의료용 분야에 많이 활용되고 있다. 또한, 키토산은 안전하고 면역촉진 부작용도 없는 것으로 알려졌다. 생체 내에서는 라이소자임(lysozyme)에 의해 N-아세틸글루코사민으로 분해되고, 이는 당단백질 합성에 사용된 후 이산화탄소의 형 태로 배출된다(Chandy T, Sharma CP. Chitosan as a biomaterial. Biomat Art Cells Art Org. 18:1-24(1990)).Chitosan is widely used in various industrial and medical fields because of its excellent stability, environmental friendliness, biodegradability and biocompatibility. Chitosan is also known to be safe and free of immunostimulating side effects. In vivo lysozyme (lysozyme) and digested with N- acetylglucosamine, which is the discharge state after the type of the carbon dioxide used in the art of protein synthesis by (Chandy T, Sharma CP. Chitosan as a biomaterial. Biomat Art Cells Art Org . 18: 1-24 (1990).
본 발명은 상술한 바와 같이 생체적합성이 우수한 키토산을 경점막 운반 시스템의 운반체(carrier)로 이용한 점에 특징이 있으며, 단백질 또는 펩타이드-키토산 복합체가 경구 투여제로 이용되는 경우에도 단백질 또는 펩타이드 고유의 우수한 약리학적 효능이 인 비보에서 발휘될 수 있음을 최초로 제시하는 것이다.The present invention is characterized in that the use of chitosan excellent in biocompatibility as a carrier of the transmucosal delivery system as described above, even if the protein or peptide-chitosan complex is used as an oral dosage, It is the first suggestion that pharmacological efficacy can be exerted in vivo .
본 발명에서 이용되는 키토산으로써, 통상의 어떠한 키토산도 이용 가능하지만, 바람직하게는 분자량 500-20000, 보다 바람직하게는 분자량 500-10000, 보다 더 바람직하게는 분자량 1000-10000, 가장 바람직하게는 분자량 3000-9000의 키토산이다. 본 발명에서 이용되는 키토산의 분자량이 500 미만인 경우에는, 키토산의 전달체(carrier)로서의 기능이 미약한 문제점이 있고, 키토산의 분자량이 20000을 초과하는 경우에는, 수용액 상에서 자기집합체를 형성하는 문제점이 있다. 본 발명에서 이용되는 바람직한 키토산은 올리고머 수준의 키토산이다.As chitosan used in the present invention, any conventional chitosan may be used, but preferably molecular weight 500-20000, more preferably 500-10000, even more preferably molecular weight 1000-10000, most preferably 3000 Chitosan of -9000. When the molecular weight of chitosan used in the present invention is less than 500, there is a problem that the function of the chitosan as a carrier is weak, and when the molecular weight of chitosan exceeds 20000, there is a problem of forming a self-assembly in an aqueous solution. . Preferred chitosans used in the present invention are oligomeric levels of chitosan.
본 발명에서, 단백질 또는 펩타이드가 키토산에 공유결합 되는 방식은, 크게 두 가지로 나눌 수 있다: 직접 결합 및 링커를 통한 간접 결합.In the present invention, the manner in which the protein or peptide is covalently bound to chitosan can be divided into two types: direct binding and indirect binding through a linker.
단백질 또는 펩타이드에 있는 작용기(예컨대, 아미노산의 R-기에 있는 작용기, 예컨대, -SH, -OH, -COOH 및 -NH2)와 키토산에 있는 작용기(예컨대, -OH 및 -NH2)가 직접적으로 반응하여 공유결합을 형성함으로써 단백질 또는 펩타이드-키토산 복합체를 형성할 수 있다. 간접 결합 방식에 따르면, 당업계에서 링커로 통상 적으로 이용되는 화합물이 중개하여 단백질 또는 펩타이드-키토산 복합체를 형성한다.Functional groups in a protein or peptide (eg, functional groups in the R-group of an amino acid, such as -SH, -OH, -COOH, and -NH 2 ) and functional groups in chitosan (eg -OH and -NH 2 ) are directly By reacting to form a covalent bond, a protein or peptide-chitosan complex can be formed. According to the indirect binding mode, a compound commonly used as a linker in the art is mediated to form a protein or peptide-chitosan complex.
본 발명의 바람직한 구현예에 따르면, 단백질 또는 펩타이드와 키토산은 링커를 통하여 공유결합되어 있는 것이다. 본 발명에서 이용되는 링커는 당업계에서 링커로 이용되는 어떠한 화합물도 가능하며, 단백질 또는 펩타이드에 있는 작용기 종류에 따라 적합한 링커를 선택할 수 있다. 예를 들어, 링커는 N-숙시니미딜 요오도아세테이드 (N-succinimidyl iodoacetate), N-히드록시숙시니미딜 브로모아세테이트(N-Hydroxysuccinimidyl Bromoacetate), m-말레이미도벤조일-N-히드록시숙신이미드 에스테르 (m-Maleimidobenzoyl-N-hydroxysuccinimide ester), m-말레이미도벤조일-N-히드록시설포숙신이미드 에스테르 (m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), N-말레이미도부티릴옥시숙신아미드 에스테르 (N-Maleimidobutyryloxysuccinamide ester), (N-Maleimidobutyryloxysulfosuccinamide ester), E-말레이미도카프로산 히드라지드ㆍHCl (E-Maleimidocaproic acid hydrazideㆍHCl), [N-말레이디도카프로일옥시-숙신아미드] ([N-(E-maleimidocaproyloxy)-succinamide]), [N-말레이미도카프로일옥시)-설포숙신아미드] ([N-(E-maleimidocaproyloxy)-sulfosuccinamide]), 말레이미도프로피온산 N-히드록시숙신이미드 에스테르 (Maleimidopropionic Acid N-Hydroxysuccinimide Ester), 말레이미도프로피온산 N-히드록시설포숙신이미드 에스테르 (Maleimidopropionic Acid N-Hydroxysulfosuccinimide Ester), 말레이미도프로피온산 히드라지드ㆍHCl (Maleimidopropionic Acid HydrazideㆍHCl), N-숙신이미딜-3- (2-피리딜디티오)프로피오네이트 (N-Succinimidyl-3-(2-pyridyldithio)propionate), N-숙시니미딜-4-(요오도아세틸)아미노벤조에이트 (N-Succinimidyl-(4-iodoacetyl) aminobenzoate), 숙시니미딜-(N-말레이미도메틸)시클로헥산-1-카르복실레이트 (Succinimidyl-(N-maleimidomethyl)cyclohexane-l-carboxylate), 숙시니미딜-4-(말레이미도페닐)부틸레이트(Succinimidyl-4-(p-maleimidophenyl)butyrate), 설포숙시니미딜-(4-요오도아세틸)아미노벤조에이트 (Sulfosuccinimidyl-(4-iodoacetyl)aminobenzoate), 설포숙시니미딜-4-(N-말레이미도메틸)시클로헥산-1-카르복실레이트 (Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), 설포숙시니미딜-4-(p-말레이미도페닐)부틸레이트 (Sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate), m-말레이미도벤조산 히드라지드ㆍHCl (m-Maleimidobenzoic acid hydrazide ? HCl), 4-(N-말레이미도메틸)시클로헥산-1-카르복실산 히드라지드ㆍHCl (4-(N-Maleimidomethyl)cyclohexane-l-carboxylic acid hydrazideㆍHCl), 4-(4-N-말레이미도페닐)부티르산 히드라지드ㆍHCl (4-(4-N-maleimidophenyl)butyric acid hydrazideㆍHCl), N-숙시니미딜 3-(2-피리딜디티오) 프로피오네이트 (N-succinimidyl 3-(2-pyridyldithio) propionate), 비스(설포숙시니미딜)수베레이트(Bis(sulfosuccinimidyl)suberate), 1,2-디[3’-(2’-피리딜디티오)프로피온아미도] 부탄(1,2-Di[3’-(2’pyridyldithio)propionamido] Butane), 디숙시니디밀 수베레이트(Dissuccinimidyl Suberate), 디숙시니미딜 타르타레이트(Dissuccinimidyl Tartarate), 디설포숙시니미딜 타르타레이트 (Disulfosuccinimdiyl Tartarate), 디 티오-비스-(숙시니미딜 프로피오네이트) (Dithio-bis-(succinimidyl propionate)), 3,3‘-디티오-비스-(설포숙시니미딜-프로피오네이트)(3,3'-Dithio-bis-(sulfosuccinimidyl-propionate)), 에틸렌 글리콜 비스(숙시니미딜숙시네이트 (Ethylene Glycol bis(Succinimidylsuccinate)) 및 에틸렌 글리콜 비스(설포숙시니미딜숙시네이트)(Ethylene Glycol bis(Sulfosuccinimidylsuccinate))를 포함하나, 이에 한정되는 것은 아니다.According to a preferred embodiment of the invention, the protein or peptide and chitosan are covalently linked via a linker. The linker used in the present invention may be any compound used as a linker in the art, and a suitable linker may be selected according to the type of functional group in the protein or peptide. For example, the linker may be N-succinimidyl iodoacetate, N-hydroxysuccinimidyl bromoacetate, m-maleimidobenzoyl-N-hydroxy M-Maleimidobenzoyl-N-hydroxysuccinimide ester, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, N-maleimidobenzoyl-N-hydroxysuccinimide ester N-Maleimidobutyryloxysuccinamide ester, N-Maleimidobutyryloxysulfosuccinamide ester, E-maleimidocaproic acid hydrazide HCl, N-maleimidobutyryloxysuccinamide ester (N-maleimidobutyryloxysuccinamide ester) N- (E-maleimidocaproyloxy) -succinamide]), [N-maleimidocaproyloxy) -sulfosuccinamide] ([N- (E-maleimidocaproyloxy) -sulfosuccinamide]), maleimidopropionic acid N-hydroxysuccinate Mid ester (Maleimidopropionic Acid N-Hydroxys uccinimide Ester), maleimidopropionic acid N-Hydroxysulfosuccinimide Ester, maleimidopropionic acid hydrazide-HCl (Maleimidopropionic Acid HydrazideHCl), N-succinimidyl-3- -Pyridyldithio) propionate (N-Succinimidyl-3- (2-pyridyldithio) propionate), N-succinimidyl-4- (iodoacetyl) aminobenzoate (N-Succinimidyl- (4-iodoacetyl) aminobenzoate), succinimidyl- (N-maleimidomethyl) cyclohexane-1-carboxylate (Succinimidyl- (N-maleimidomethyl) cyclohexane-l-carboxylate), succinimidyl-4- (maleimidophenyl) butylate (Succinimidyl-4- (p-maleimidophenyl) butyrate), Sulfosuccinimidyl- (4-iodoacetyl) aminobenzoate (Sulfosuccinimidyl- (4-iodoacetyl) aminobenzoate), Sulfosuccinimidyl-4- (N Maleimidomethyl) cyclohexane-1-carboxylate (Sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate), Posuk when Niimi dill -4- (p- maleimido phenyl) butyrate (Sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate), m- maleimido benzoic acid hydrazide and HCl (m-Maleimidobenzoic acid hydrazide? HCl), 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid hydrazideHCl (4- (N-Maleimidomethyl) cyclohexane-l-carboxylic acid hydrazideHCl), 4- (4-N- Maleimidophenyl) butyric acid hydrazideHCl (4- (4-N-maleimidophenyl) butyric acid hydrazideHCl), N-succinimidyl 3- (2-pyridyldithio) propionate (N-succinimidyl 3- (2-pyridyldithio) propionate), bis (sulfosuccinimidyl) suberate, 1,2-di [3 '-(2'-pyridyldithio) propionamido] butane ( 1,2-Di [3 '-(2'pyridyldithio) propionamido] Butane), Dissuccinimidyl Suberate, Dissuccinimidyl Tartarate, Disulfosuccinimidyl Tartrate ( Disulfosuccinimdiyl Tartarate), Dithio-bis- (succinimidyl propionate), 3,3'-Dithio-bis- (sulfosuccinimidyl-propionate) ( 3,3'-Dithio-bis- (sulfosuccinimidyl- propionate), ethylene glycol bis (Sthenimidylsuccinate) and ethylene glycol bis (sulfosuccinimidylsuccinate), including but not limited to no.
단백질 또는 펩타이드와 키토산의 공유결합은 다양한 결합 종류에 따라 형성될 수 있으며, 예를 들어, 이황화 결합, 펩타이드 결합, 이민 결합, 에스테르 결합 또는 아미드 결합에 의해 형성될 수 있다.Covalent bonds of proteins or peptides with chitosan can be formed according to various bond types, for example, by disulfide bonds, peptide bonds, imine bonds, ester bonds or amide bonds.
본 발명의 구체적인 일 실시예에 따르면, 단백질 또는 펩타이드(예컨대, 인슐린)와 키토산은 -CO-(CH2)2-S-S-(CH2)2-CO-가 중간에 개입되어 있는 형태를 가지며, 키토산의 -NH2 및 단백질의 -NH2는 각각 상기 링커에 아미드 결합으로 공유결합 되어 있다.According to a specific embodiment of the present invention, the protein or peptide (eg, insulin) and chitosan have a form in which -CO- (CH 2 ) 2 -SS- (CH 2 ) 2 -CO- is intervened, -NH 2 and -NH 2 in the protein of the chitosan is covalently bound to each amide bond to the linker.
본 발명의 바람직한 구현예에 따르면, 단백질 또는 펩타이드와 키토산의 공유결합에는 링커 -CO-(CH2)n-S-S-(CH2)n-CO-가 중간에 개입되어 있으며, 키토산의 -NH2 및 단백질의 -NH2는 각각 상기 링커와 아미드 결합되어 있다. 상기 링커의 화학식에서 n은 1-5의 정수이다.According to a preferred embodiment of the present invention, the linker -CO- (CH 2 ) n -SS- (CH 2 ) n -CO- is intervened in the covalent bond of the protein or peptide and chitosan, and -NH 2 of the chitosan. And -NH 2 of the protein are each amide-bonded with the linker. In the formula of the linker, n is an integer of 1-5.
본 발명의 경점막 운반 시스템은 단백질 또는 펩타이드를 점막(mucous membrane)을 가지는 조직 및 기관으로 운반한다. 본 발명의 바람직한 구현예에 따르면, 본 발명의 경점막 운반 시스템은 구강(buccal cavity), 비강, 직장, 질, 요도, 인후, 소화관(alimentary canal), 복막 또는 눈의 점막을 통하여 단백질 또는 펩타이드를 운반한다. 가장 바람직하게는, 본 발명의 경점막 운반 시스템은 소화관의 점막을 통하여 단백질 또는 펩타이드를 운반하여 단백질 또는 펩타이드의 경구 투여를 가능하게 한다.The transmucosal delivery system of the present invention delivers proteins or peptides to tissues and organs having mucous membranes. According to a preferred embodiment of the present invention, the transmucosal delivery system of the present invention comprises a protein or peptide through the buccal cavity, nasal cavity, rectum, vagina, urethra, throat, alimentary canal, peritoneum or eye mucosa. To carry. Most preferably, the transmucosal delivery system of the present invention delivers proteins or peptides through the mucous membranes of the digestive tract to allow oral administration of proteins or peptides.
본 발명의 약제학적 조성물은 키토산에 결합되는 단백질 또는 펩타이드의 종류에 따라 다양한 치료학적 효능을 발휘한다. 예를 들어, 상기 단백질이 인슐린인 경우에는 당뇨병 치료 효능을 나타내며, IGF-1 또는 성장호르몬인 경우에는 성장촉진 효능을 나타내며, 인터페론인 경우에는 항바이러스 효능을 나타내고, 에리쓰로포이에틴인 경우에는 빈혈 치료 효능을 나타낸다.The pharmaceutical composition of the present invention exhibits various therapeutic effects depending on the type of protein or peptide bound to chitosan. For example, when the protein is insulin, it shows a therapeutic effect on diabetes, and when IGF-1 or growth hormone, it shows growth promoting effect, and when it is interferon, it shows antiviral effect, and when it is erythropoietin Efficacy in treating anemia.
본 발명의 가장 바람직한 구현예에 따르면, 본 발명의 약제학적 조성물은, (a) (ⅰ) 키토산; 및 (ⅱ) 상기 키토산에 공유결합된 인슐린을 포함하는 인슐린의 경점막 운반 시스템의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 인슐린의 경구 투여용 당뇨 치료용도의 약제학적 조성물을 제공한다.According to the most preferred embodiment of the present invention, the pharmaceutical composition of the present invention comprises (a) (iii) chitosan; And (ii) a pharmaceutically effective amount of a transmucosal delivery system of insulin comprising insulin covalently bound to the chitosan; And (b) provides a pharmaceutical composition for the treatment of diabetes for oral administration of insulin comprising a pharmaceutically acceptable carrier.
본 발명의 당뇨 치료용도의 약제학적 조성물은, 인슐린의 경구 투여를 가능하게 한다. 일반적으로 당뇨 환자들은 주사제 인슐린을 투여받으며, 이러한 투여 방식은 환자에게 많은 불편함을 초래한다. 그러나 본 발명은 당뇨 치료용도의 약제학적 조성물은 경구 투여가 가능하기 때문에 당뇨 치료를 획기적으로 개선할 수 있다.The pharmaceutical composition for treating diabetes of the present invention enables oral administration of insulin. In general, diabetic patients receive an injection of insulin, which causes a lot of inconvenience for the patient. However, the present invention can significantly improve the treatment of diabetes because the pharmaceutical composition for the treatment of diabetes can be administered orally.
본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제 시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다.Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and agents are Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 한편, 본 발명의 약제학적 조성물의 경구 투여량은 바람직하게는 1일 당 0.001-100 mg/kg(체중)이다.Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. On the other hand, the oral dosage of the pharmaceutical composition of the present invention is preferably 0.001-100 mg / kg (body weight) per day.
본 발명의 약제학적 조성물은 바람직하게는 경구 투여된다.The pharmaceutical composition of the present invention is preferably administered orally.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.
본 명세서에서 용어 “약제학적 유효량”은 단백질 또는 펩타이드-키토산 복 합체의 단백질 또는 펩타이드가 가지는 고유의 치료학적 효능을 달성하는 데 충분한 양을 의미한다.As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the inherent therapeutic efficacy of the protein or peptide of the protein or peptide-chitosan complex.
본 발명의 단백질 또는 펩타이드-키토산 복합체 및 약제학적 항암 조성물은, 경점막 투여, 특히 구강 투여하는 경우에도 우수한 약리학적 효능을 발휘한다. 하기의 실험예 4에는 본 발명의 인슐린-키토산 복합체의 인 비보 혈당 강하 효능이 예시되어 있으며, 여기에서, 키토산이 결합되지 않은 프리(free) 인슐린과 비교하여 매우 월등한 혈당 강하 효능을 발휘하고 있음을 확인할 수 있다. 또한, 동물을 대상으로 하여 실험한 하기의 실험예 4의 데이터는 본 발명의 단백질 또는 펩타이드-키토산 복합체가 점막(특히, 위장관 점막)에 대하여 흡수율이 우수하다는 것을 보여준다.The protein or peptide-chitosan complex and the pharmaceutical anticancer composition of the present invention exhibit excellent pharmacological efficacy even in transmucosal administration, especially when administered orally. In Experimental Example 4 below, the in vivo blood glucose lowering effect of the insulin-chitosan complex of the present invention is exemplified, wherein the blood glucose lowering effect is superior to that of free insulin to which chitosan is not bound. can confirm. In addition, the data of Experimental Example 4, which was conducted in animals, shows that the protein or peptide-chitosan complex of the present invention has an excellent absorption rate against mucosa (particularly, gastrointestinal mucosa).
본 발명의 단백질 또는 펩타이드-키토산 복합체 및 약제학적 조성물의 기술적 달성(accomplishment) 및 장점을 요약하면 다음과 같다:The technical achievements and advantages of the protein or peptide-chitosan complexes and pharmaceutical compositions of the present invention are summarized as follows:
(ⅰ) 본 발명의 단백질 또는 펩타이드-키토산 복합체는 생체 점막, 특히, 소화관(특히, 위장관)에 있는 점막에서 흡수율이 우수하다.(Iii) The protein or peptide-chitosan complex of the present invention has excellent absorption in the mucosa of the living body mucosa, especially in the digestive tract (especially the gastrointestinal tract).
(ⅱ) 본 발명의 단백질 또는 펩타이드-키토산 복합체는 생체 내, 특히 위장관 내에서 우수한 안정성을 나타낸다.(Ii) The protein or peptide-chitosan complexes of the invention exhibit good stability in vivo, particularly in the gastrointestinal tract.
(ⅲ) 운반체로 이용된 키토산은 생체적합성이 우수하고 체내에서 분해되기 때문에 본 발명의 단백질 또는 펩타이드-키토산 복합체는 인체에 안전하며 장기적인 복용 시에도 우수한 안전성을 나타낸다.(Iii) Since the chitosan used as a carrier has excellent biocompatibility and is degraded in the body, the protein or peptide-chitosan complex of the present invention is safe for human body and shows excellent safety even in long-term administration.
(ⅳ) 따라서 본 발명의 단백질 또는 펩타이드-키토산 복합체 및 약제학적 조 성물은 경구 투여 시에도 생체이용율이 우수하여 경구 투여를 통한 단백질 의약의 투여를 가능하게 한다.(Iii) Therefore, the protein or peptide-chitosan complex and pharmaceutical composition of the present invention have excellent bioavailability even when administered orally, thereby allowing the administration of protein medicine through oral administration.
(ⅴ) 본 발명의 약제학적 조성물을 경구 투여하는 경우, 종래의 주사제와 비교하여 환자 순응도가 크게 개선된다.(Iv) In the case of oral administration of the pharmaceutical composition of the present invention, patient compliance is greatly improved as compared with conventional injections.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
실시예Example
실시예Example 1: 인슐린과 링커가 1: insulin and linker 결합된Combined 인슐린 중간체의 제조 Preparation of Insulin Intermediates
인슐린 0.1 g (17.22× 10-6 mol)(Serologicals Corp.)을 10 ml의 염산 용액에 용해시키고, SPDP[N-succinimidyl 3-(2-pyridyldithio) propionate, Pierce] 0.008 g (25.83× 10-6mol)을 0.2× 10-3 ml의 DMF(Sigma)에 용해시켜서 상기 인슐린 용액에 첨가하였다. 인슐린 B 사슬의 29번 라이신에 위치-선택적으로 SPDP를 컨쥬게이션 하기 위해서 NaOH 용액으로 pH를 9-10으로 맞추어 실온에서 30분 동안 교반하였다. 상기 교반 용액을 역상 HPLC(Shimadzu)를 이용하여 분리하고, 동결건 조 하여 인슐린 중간체를 제조하였다(반응식 1).0.1 g (17.22 × 10 −6 mol) (Serologicals Corp.) of insulin was dissolved in 10 ml of hydrochloric acid solution and 0.008 g (25.83 × 10 −6 ) of SPDP [N-succinimidyl 3- (2-pyridyldithio) propionate, Pierce] mol) was dissolved in 0.2 x 10 -3 ml of DMF (Sigma) and added to the insulin solution. The pH was adjusted to 9-10 with NaOH solution for site-selective conjugation of SPDP to lysine 29 of the insulin B chain and stirred at room temperature for 30 minutes. The stirred solution was separated using reverse phase HPLC (Shimadzu) and lyophilized to prepare an insulin intermediate (Scheme 1).
실시예Example 2: 키토산과 링커가 2: chitosan and linker 결합된Combined 키토산 중간체의 제조 Preparation of Chitosan Intermediates
분자량 3000, 6000 및 9000의 키토산 각 0.1 g(16.67× 10-6 mol, 단량체 몰수 = 0.67× 10-3 mol)(KITTOLIFE, Korea)을 인산 완충용액 2 ml에 용해시키고, SPDP 0.016 g (50.01× 10-6 mol)을 0.2× 10-3 ml의 DMF에 용해시켜 상기 키토산 용액에 첨가한 후, 실온에서 2시간 동안 교반하였다. 상기 교반 용액에 아세톤을 가하여 펠렛을 침전시킨 후, 이를 증류수에 녹이고 동결건조시켜 키토산 중간체를 제조하였다(반응식 1).0.1 g (16.67 × 10 −6 mol, number of monomer moles = 0.67 × 10 −3 mol) (KITTOLIFE, Korea) each of chitosan with molecular weights 3000, 6000 and 9000 was dissolved in 2 ml of phosphate buffer solution and 0.016 g of SPDP (50.01 ×). 10 -6 mol) was dissolved in 0.2 x 10 -3 ml of DMF and added to the chitosan solution and then stirred at room temperature for 2 hours. Acetone was added to the stirred solution to precipitate the pellet, which was dissolved in distilled water and lyophilized to prepare a chitosan intermediate (Scheme 1).
실시예Example 3: 인슐린-키토산 3: insulin-chitosan 컨쥬게이트Conjugate 제조 Produce
키토산 중간체를 환원시키기 위해서, 실시예 2의 키토산 중간체 0.008 g (1.24× 10-6 mol)와 DTT (24.9× 10-6 mol)(Pierce)를 0.3 ml의 인산 완충용액에 용해시키고 실온에서 4시간 교반하였다. 실시예 1의 인슐린 중간체 0.005 g (0.83× 10-6 mol)를 구연산 완충용액(500 μl)에 용해시킨 후, 상기 환원된 키토산 중간체 용액(100 μl)를 첨가하여 실온에서 12-24시간 동안 교반시켰다. 이것을 역상 HPLC를 이용하여 분리하고, 동결건조시켜 인슐린 및 키토산이 연결된 컨쥬게이트 분자, 인슐린-키토산 컨쥬게이트를 제조하였다(반응식 2).To reduce the chitosan intermediate, 0.008 g (1.24 × 10 −6 mol) and DTT (24.9 × 10 −6 mol) (Pierce) of the chitosan intermediate of Example 2 were dissolved in 0.3 ml of phosphate buffer and 4 hours at room temperature. Stirred. 0.005 g (0.83 × 10 −6 mol) of the insulin intermediate of Example 1 was dissolved in citric acid buffer (500 μl), and then the reduced chitosan intermediate solution (100 μl) was added and stirred at room temperature for 12-24 hours. I was. This was separated using reverse phase HPLC and lyophilized to prepare the conjugated insulin and chitosan conjugated molecules, the insulin-chitosan conjugate (Scheme 2).
실험예Experimental Example 1: 키토산 중간체에서 링커의 치환도 측정 1: Determination of Linker Substitution in Chitosan Intermediates
본 발명의 키토산 중간체에 SPDP가 치환된 정도를 알아보기 위하여 1H NMR을 이용하여 이를 측정하였다. 링커의 치환도는 D2O 용매에서 적분비로 계산하였다. 측정된 치환 분자수는 표 1에 나타내었다.In order to determine the degree of substitution of the SPDP in the chitosan intermediate of the present invention, it was measured using 1 H NMR. The degree of substitution of the linker was calculated by the integral ratio in the D 2 O solvent. The measured number of substituted molecules is shown in Table 1.
표 1에 나타난 바와 같이, 분자량이 증가함에 따라 링커의 치환도가 비례적으로 감소하므로, 저분자량의 키토산에 보다 용이하게 치환됨을 알 수 있다.As shown in Table 1, since the substitution degree of the linker decreases proportionally as the molecular weight increases, it can be seen that it is more easily substituted with low molecular weight chitosan.
실험예Experimental Example 2 : 인슐린-키토산 2: insulin-chitosan 컨쥬게이트에To the conjugate 존재하는 인슐린 양 측정 Measure the amount of insulin present
본 발명의 인슐린-키토산 컨쥬게이트(분자량 6000의 키토산을 이용한 컨쥬게이트)에 포함된 인슐린의 양을 측정하기 위해 1 mg 인슐린-키토산 컨쥬게이트를 1 ml의 구연산 완충용액에 용해시킨 후, UV 275 nm에서 흡광도를 측정하였다. 기준 곡선으로는 인슐린(0.1, 0.5, 1 및 2 mg)을 구연산 완충용액 1 ml에 용해한 후, 흡광도를 측정하여 도시하였고, 이것을 이용하여 인슐린-키토산 컨쥬게이트에 포함된 인슐린 양을 계산하였다. 측정 결과 인슐린-키토산 컨쥬게이트가 함유한 인슐린 양은 44%로 나타났다.In order to measure the amount of insulin contained in the insulin-chitosan conjugate (conjugate using chitosan of molecular weight 6000) of the present invention, 1 mg insulin-chitosan conjugate was dissolved in 1 ml of citric acid buffer, and then UV 275 nm. Absorbance was measured at. As a reference curve, insulin (0.1, 0.5, 1, and 2 mg) was dissolved in 1 ml of citric acid buffer, and then absorbance was measured and shown, and the amount of insulin contained in the insulin-chitosan conjugate was calculated using this. As a result, the insulin-chitosan conjugate contained 44% of the amount of insulin.
실험예Experimental Example 3: 인슐린-키토산 3: insulin-chitosan 컨쥬게이트를The conjugate 이용한 생체 내( In vivo ( inin vivovivo ) 인슐린 활성 측정 실험 Insulin activity measurement experiment
본 발명의 인슐린-키토산 컨쥬게이트(분자량 6000의 키토산을 이용한 컨쥬게이트)를 구연산 완충용액에 용해한 후, 생리 식염수로 희석하여 인슐린 농도 1 U/ml의 인슐린-키토산 컨쥬게이트 용액을 제조하였다. 인슐린 투여 전 당뇨가 유도된 휘스터계 웅성 래트(Wistar rats, 6-7주령)들을 6시간 동안 절식시킨 후 꼬리 정맥에서 혈액을 채취하여 혈당치를 측정하였고 이 값을 초기 값으로 사용하였다. 측정 직후, 0.5 IU/kg 인슐린 또는 1 IU/kg 인슐린 -키토산 컨쥬게이트(Insulin-6K LMWC)를 꼬리 정맥으로 주사하였다. 0.5 IU는 17.4 ㎍에 해당되는 것이다. 또한, 0.5 IU/kg 인슐린을 s.c.(subcutaneous) 주입하였다. 그 결과, 도 1에 나타난 바와 같이, 본 발명의 인슐린-키토산 컨쥬게이트 용액(-▽-)에 포함된 인슐린의 생리 활성은 대조군의 인슐린 용액에 대하여 약 40%를 나타내었으므로 정상적인 생리활성을 가짐을 알 수 있었다.The insulin-chitosan conjugate (conjugate using chitosan of molecular weight 6000) of the present invention was dissolved in citric acid buffer solution and diluted with physiological saline to prepare an insulin-chitosan conjugate solution with an insulin concentration of 1 U / ml. Diabetic-induced diabetic male rats (Wistar rats, 6-7 weeks old) before insulin administration were fasted for 6 hours, blood was collected from the tail vein, and blood glucose levels were measured and used as initial values. Immediately after the measurement, 0.5 IU / kg insulin or 1 IU / kg insulin-chitosan conjugate (Insulin-6K LMWC) was injected into the tail vein. 0.5 IU corresponds to 17.4 μg. In addition, 0.5 IU / kg insulin was injected subcutaneous. As a result, as shown in Figure 1, the physiological activity of the insulin contained in the insulin-chitosan conjugate solution (-▽-) of the present invention showed about 40% of the insulin solution of the control group has a normal physiological activity And it was found.
실험예Experimental Example 4: 인슐린-키토산 4: insulin-chitosan 컨쥬게이트를The conjugate 이용한 생체 내 경구 투여 실험 In vivo oral administration experiment
인슐린-키토산 컨쥬게이트(분자량 3000, 6000 또는 9000의 키토산을 이용한 컨쥬게이트)를 구연산염 완충용액에 용해한 후, 생리 식염수로 희석하여 인슐린 농도 100 U/ml의 인슐린-키토산 컨쥬게이트 용액을 제조하였다. 당뇨가 유도된 랫트를 6시간 동안 절식시킨 후 꼬리 정맥에서 혈액을 채취하여 혈당치를 측정하였고 상기 값을 약물 투여 전 초기값으로 사용하였다. 상기 실험동물에 위 존데(gastric sonde)를 사용하여 실시예에서 제조된 인슐린-키토산 컨쥬게이트 용액을 인슐린 농도가 50 IU/㎏이 되도록 위 존데로 경구 투여하였다(50 IU는 1.77 mg에 해당됨). 대조군으로 50 IU/㎏ 인슐린 및 분자량 9000의 키토산을 동일한 방법으로 경구투여 하였다. 약물 투여 후 1, 2, 3 및 4시간째에 꼬리 정맥에서 혈액을 채취하여 혈당치를 측정하였다. 투여 전 초기값을 100%로 하여 각 시간대의 혈당치를 환산하였다. 그 결과, 도 2에 나타난 바와 같이, 본 발명의 인슐린-키토산 컨쥬게이트 용액으로 인슐린이 50 IU/㎏ 농도로 투여된 래트 실험군에서는 초기 혈당치에 비하여 2시간 경과 후, 40% 이상 혈당치가 감소된 반면, 인슐린을 함유하지 않은 염수, 인슐린 자체 및 키토산 자체는 경구 투여한 경우에는 혈당치가 저하되지 않음을 확인하였다.Insulin-chitosan conjugates (conjugates using chitosan of molecular weight 3000, 6000 or 9000) were dissolved in citrate buffer and diluted with physiological saline to prepare an insulin-chitosan conjugate solution with an insulin concentration of 100 U / ml. Diabetic-induced rats were fasted for 6 hours, blood was collected from the tail vein, and blood glucose levels were measured. These values were used as initial values before drug administration. In the experimental animal, the insulin-chitosan conjugate solution prepared in Example using gastric sonde was orally administered to gastric sonde so that the insulin concentration was 50 IU / kg (50 IU corresponds to 1.77 mg). As a control group, 50 IU / kg insulin and chitosan having a molecular weight of 9000 were orally administered in the same manner. Blood glucose levels were measured by taking blood from the tail vein at 1, 2, 3 and 4 hours after drug administration. The blood glucose level of each time slot was converted to the initial value before administration as 100%. As a result, as shown in Figure 2, in the rat experimental group administered insulin at a concentration of 50 IU / kg of the insulin-chitosan conjugate solution of the present invention, after 2 hours compared to the initial blood glucose level, the blood glucose level was reduced by more than 40% It was confirmed that the blood glucose level was not lowered by oral administration of insulin, saline containing no insulin, insulin itself and chitosan itself.
실험예Experimental Example 5: 인슐린-키토산 5: insulin-chitosan 컨쥬게이트의Conjugate 생체이용률 분석 Bioavailability Analysis
분자량 3000, 6000 또는 9000의 키토산을 이용한 컨쥬게이트인 본 발명의 인슐린-키토산 컨쥬게이트, 인슐린-3K LMWC, 인슐린-6K LMWC 및 인슐린-9K LMWC를 마우스에 정맥 또는 피하 주입하고 생체이용률을 분석하였다. 또한, 비교군으로서, 인슐린, 단백질분해효소-키토산 컨쥬케이트 및 글루타티온(환원제)를 포함하는 티올화(thiolated) 키토산-인슐린 타블렛(Krauland AH, et al., J. Control Release, 24;95(3):547-555(2004))을 마우스에 주입하여 생체이용률을 분석하였다. 분석 결과는 다음 표 1과 같다.Insulin-chitosan conjugates of the present invention, insulin-3K LMWC, insulin-6K LMWC and insulin-9K LMWC, which are conjugates using chitosan with molecular weight 3000, 6000 or 9000, were injected intravenously or subcutaneously and analyzed for bioavailability. In addition, as a comparative group, thiolated chitosan-insulin tablets including insulin, protease-chitosan conjugates and glutathione (reducing agents) (Krauland AH, et al., J. Control Release, 24; 95 (3) ): 547-555 (2004)) was injected into mice to analyze bioavailability. The analysis results are shown in Table 1 below.
*티올화(thiolated) 키토산-인슐린 타블렛을 투여한 군 * Group administered with thiolated chitosan-insulin tablet
상기 표 1에서 확인할 수 있듯이, 본 발명의 인슐린-키토산 컨쥬게이트는 생체이용률도 매우 우수하다는 것을 알 수 있다.As can be seen in Table 1, it can be seen that the insulin-chitosan conjugate of the present invention is very excellent in bioavailability.
위에서 상세히 설명한 바와 같이, 본 발명은 단백질 또는 펩타이드를 위한 경점막 운반 시스템(transmucosal delivery system)을 제공한다. 또한, 본 발명은 경점막 운반용 약제학적 조성물을 제공한다. 본 발명의 단백질 또는 펩타이드-키토산 복합체 및 약제학적 조성물은 생체 점막, 특히, 소화관(특히, 위장관)에 있는 점막에서 흡수율이 우수하고 안정성이 뛰어날 뿐만 아니라, 운반체로 이용된 키토산은 생체적합성이 우수하고 체내에서 분해되기 때문에 경구 투여 시에도 생체이용율이 우수하여 경구 투여를 통한 단백질 의약에 의한 치료를 가능하게 한다.As detailed above, the present invention provides a transmucosal delivery system for proteins or peptides. The present invention also provides a pharmaceutical composition for transmucosal delivery. The protein or peptide-chitosan complexes and pharmaceutical compositions of the present invention not only have good absorption and stability in the mucous membranes of the living body, especially the gastrointestinal tract (particularly the gastrointestinal tract), but also the chitosan used as a carrier has excellent biocompatibility. Since it is decomposed in the body, the bioavailability is excellent even during oral administration, thereby enabling the treatment by protein medicine through oral administration.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당 업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이 며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the present invention, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
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| EP07701046A EP1973952A4 (en) | 2006-01-23 | 2007-01-23 | Conjugate comprising pharmaceutical active compound covalently bound to mucoadhesive polymer and transmucosal delivery method of pharmaceutical active compound using the same |
| PCT/KR2007/000403 WO2007083984A1 (en) | 2006-01-23 | 2007-01-23 | Conjugate comprising pharmaceutical active compound covalently bound to mucoadhesive polymer and transmucosal delivery method of pharmaceutical active compound using the same |
| JP2008531031A JP2009508852A (en) | 2006-01-23 | 2007-01-23 | Conjugate in which pharmacologically active substance and mucoadhesive polymer are covalently bonded, and method for transmucosal delivery of pharmacologically active substance using the same |
| US11/847,237 US20070292387A1 (en) | 2006-01-23 | 2007-08-29 | Transmucosal delivery of pharmaceutical active substances |
| JP2011268031A JP5491485B2 (en) | 2006-01-23 | 2011-12-07 | Transmucosal agent for use in transmucosal administration of a conjugate in which a pharmacologically active substance and a mucoadhesive polymer are covalently bonded |
| US14/286,969 US20170252453A9 (en) | 2006-01-23 | 2014-05-23 | Transmucosal delivery of pharmaceutical active substances |
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| CN104072765B (en) * | 2014-07-09 | 2017-07-28 | 中国科学院长春应用化学研究所 | Modified polyethyleneimine and preparation method thereof, drug gene carrier system and preparation method thereof |
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| US6482413B1 (en) | 2001-02-26 | 2002-11-19 | Council Of Scientific And Industrial Research | Vitamin B12 —biodegradable micro particulate conjugate carrier systems for peroral delivery of drugs, therapeutic peptides/proteins and vaccines |
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| WO2005077346A1 (en) | 2004-02-10 | 2005-08-25 | Innovative Drug Delivery Systems, Inc. | Controlled release formulations |
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| US6835393B2 (en) | 1998-01-05 | 2004-12-28 | University Of Washington | Enhanced transport using membrane disruptive agents |
| US6482413B1 (en) | 2001-02-26 | 2002-11-19 | Council Of Scientific And Industrial Research | Vitamin B12 —biodegradable micro particulate conjugate carrier systems for peroral delivery of drugs, therapeutic peptides/proteins and vaccines |
| WO2005054301A1 (en) | 2003-11-14 | 2005-06-16 | Chugai Seiyaku Kabushiki Kaisha | Crosslinked polysaccharide microparticles and process for producing the same |
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