보고서 정보
주관연구기관 |
이화여자대학교 Ewha Womans University |
연구책임자 |
이경림
|
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2017-01 |
주관부처 |
(범부처사업) NTIS |
등록번호 |
TRKO202000003029 |
DB 구축일자 |
2020-04-25
|
키워드 |
인슐린.막 투과 도메인.비강투여.비강 인슐린제제.당뇨병.insulin.protein transduction domain.TCTP-PTD.nasal administration.nasal insulin formulation.diabetes.
|
초록
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○ 연구 목표: 기존 침습적인 인슐린 주사제의 단점을 극복하기 위하여 점막 투과를 촉진하는 막투과 도메인(PTD)인 TCTP-PTD를 이용하여 비강투여 인슐린제제를 개발하고자 함.
○ 연구 내용
- Pre-formulation
• PTD/인슐린 복합체의 열역학 기반 물리 화학적 안정성 평가를 통해, 총 4종의 PTD 중 1종의 PTD 선발
• 동물실험을 통해 최적의 제형 조건 탐색(PK/PD 평가)
- 최종 formulation
• 최종 선정된 비강 인슐린제제의 PK 평가
• 제1형 당뇨
○ 연구 목표: 기존 침습적인 인슐린 주사제의 단점을 극복하기 위하여 점막 투과를 촉진하는 막투과 도메인(PTD)인 TCTP-PTD를 이용하여 비강투여 인슐린제제를 개발하고자 함.
○ 연구 내용
- Pre-formulation
• PTD/인슐린 복합체의 열역학 기반 물리 화학적 안정성 평가를 통해, 총 4종의 PTD 중 1종의 PTD 선발
• 동물실험을 통해 최적의 제형 조건 탐색(PK/PD 평가)
- 최종 formulation
• 최종 선정된 비강 인슐린제제의 PK 평가
• 제1형 당뇨쥐에서 비강 인슐린제제의 PD 평가
• 제1형 당뇨쥐에서 비강 인슐린제제의 용량에 따른 PD 평가
- 최종 formulation의 저장성 평가
• 동물실험을 통해 비강 인슐린제제의 보관수명 평가
- 최종 formulation의 독성 평가
• 최종 formulation 1회 투여 후 비강 점막 독성 평가
• 최종 formulation 1일 1회, 10일간 반복투여 후 독성 평가(비강 점막 독성 평가, 장기 조작학적 평가 및 혈액 생화학검사
○ 결론: 마일스톤1에서 비강 인슐린 전달체로써 우수한 4종의 PTD를 도출하였고, 마일스톤2에서는 PTD/인슐린 복합체의 물리 화학적 안정성 평가를 통해 4종의 PTD 중 1종의 PTD 선정함.
PTD를 기반으로 한 인슐린제제의 제형 최적화 연구를 통해 비강 인슐린제제를 개발함.
(출처 : 요약서 3p)
Abstract
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Intranasal administration is a noninvasive route for drug delivery, which has long been recognized as a convenient route of administration for drugs. The absorption area of nasal mucosa is approximately 150 cm2. It has a dense vascular network of subepithelial capillaries, providing an ex
Intranasal administration is a noninvasive route for drug delivery, which has long been recognized as a convenient route of administration for drugs. The absorption area of nasal mucosa is approximately 150 cm2. It has a dense vascular network of subepithelial capillaries, providing an excellent absorptive site. In addition, intranasally administered drugs can avoid first-pass hepatic metabolism. Nevertheless, the bioavailability of intranasal administered peptides and proteins, including insulin, is low because of their high molecular weight (>1kDa) and hydrophilicity. Therefore, numerous strategies have been developed to enhance nasal absorption of large peptide drugs.
However, increased absorption of poorly absorbable dugs using absorption enhancers such as surfactants, bile salts and fatty acids often correlated with nasal mucosa damage. Therefore, a new strategy has been required to avoid unwanted local side effects.
Protein transduction domains (PTDs), also known as cell-penetrating peptides(CPPs) are short peptides that facilitate delivery of various biologically active molecules including plasmid DNA, siRNA, peptides and proteins into a variety of cell lines and in vivo. The PTDs has been recently presented as a novel tool that is able to enhancenasal absorption of hydrophilic peptide drugs without causing detectable damage in mucosal membrane. Therefore, the further study of PTD-based nasal drug delivery is required to reduce unwanted side effects.
We previously reported that a ten amino acid at the N-terminus of human translationally controlled tumor protein (TCTP) contains PTDs (TCTP-PTD), MIIYRDLISH (residues 1-10), allowing for the delivery of macromolecules into the cells through the lipid bilayer. We also found the amino acid residues critical for cellular uptake by a method of systematic alanine substitution in TCTP-PTD. The alanine scanning results has enabled us to design PTD variants with improved solubility and cellular uptake through sequential modification of TCTP-PTD. During the optimization process, we delineated the advantageous composition of sequences for cellular uptake. Especially, TCTP-PTD 13 showed to have improved cell penetrating activity when compared to that of TCTP-PTD wild-type, with negligible cytotoxicity. We previously reported that the TCTP-PTD 13 enhanced the nasal absorption of insulin. We also reported that threeTCTP-PTD 13 analogs improved the nasal insulin absorption when compared with that of TCTP-PTD 13.
The objective of the present study was to design and optimize TCTP-PTD-based formulations for nasal delivery of insulin. We optimized The TCTP-PTD-based formulation of components during formulation development.
Pharmacokinetic analysis
To determine plasma insulin concentrations, insulin with or without PTD-based formulation was administered intranasally to the nostril of rats. Doses of insulin were adjusted to 1 IU/kg for nasal administration and 0.25 IU/kg for subcutaneous injection. Blood samples were taken from the rat tail at 5, 10, 20, 30, 60, 90, 120 and 180 min after nasal administration. Plasma was separated and then the plasma insulin concentration was determined using a human insulin ELISA kit. As shown in Fig. 1, two PTD-based formulations (3-3 and 3-5 formulations) enhanced the insulin nasal absorption compared with free insulin+PTD. The relative bioavailability (BA) of free insulin, free insulin+PTD, 3-3 formulation and 3-5 formulation were 3.3%, 38.7%, 60.7% and 45.9%, respectively.
Hypoglycemic activity in STZ-induced mice
Type 1 diabetes was induced in rats by intraperitoneal injections of alloxan. After 7 days, animals with blood glucose levels in the range of 230 and 300 mg/dL were selected for animal studies. As shown in Fig. 2, the pharmacodynamic effect of PTD-based formulations (dose of insulin: 2 IU/kg) were compared to subcutaneous injection (dose of insulin: 1 IU/kg). The relative pharmacological bioavailability (F%) of free insulin solution was 4%. On the other hand, the F% of 3-3 formulation and 3-5 formulation were 49.3% and 37.5%, respectively.
Toxicity analysis
To assess the potential toxicity of PTD-based formulation for nasal delivery of insulin, A 10-day repeated-dose study was conducted with two PTD-based formulation (3-3 and3-5 formulations). As shown in Fig. 3, nasal toxicity study using excised nasal mucosa showed comparatively no damage to epithelium when compared with control mice.
Blood samples for serum biochemical analysis were also taken at day 10 from mice nasally administrated one times a day for 10 days with PTD-based formulations.
Aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to evaluate hepatocyte damage, and blood urea nitrogen (BUN) and creatinine (CRE) were measured to evaluate kidney damage. As shown in Fig. 4, no significant difference wasfound between the control and the PTD-based formulation. Therefore, the PTD-based formulations were considered safe for nasal administration.
Our unique technologies and findings will facilitate the development of absorption enhancers in nasal insulin delivery for the treatment for diabetes. TCTP-PTDs canpotentially be used to increase the nasal absorption efficiency of a great variety of macromolecules such as vaccines, peptide and protein drugs.
(출처 : SUMMARY 10p)
목차 Contents
- 표지 ... 1
- 제 출 문 ... 2
- 보고서 요약서 ... 3
- 요 약 문 ... 4
- SUMMARY ... 10
- CONTENTS ... 14
- 목차 ... 15
- 제1장 연구개발과제의 개요 ... 16
- 제1절. 연구개발의 필요성 ... 16
- 제2절. 연구개발의 목적 ... 20
- 제3절 연구개발의 범위 ... 20
- 제2장 국내외 기술개발 현황 ... 22
- 제1절. 국외 기술개발 현황 ... 22
- 제2절. 국내 기술개발 현황 ... 24
- 제3장 연구개발수행 내용 및 결과 ... 25
- 제1절. Pre-formulation ... 25
- 제2절. 최종 formulation 평가 ... 35
- 제4장 목표달성도 및 관련분야에의 기여도 ... 47
- 제1절. 목표달성도 ... 47
- 제2절. 관련분야에의 기여도 ... 49
- 제5장 연구개발결과의 활용계획 ... 50
- 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 51
- 제7장 연구시설ㆍ장비 현황 ... 53
- 제8장 참고문헌 ... 54
- 끝페이지 ... 56
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