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Bioresponsive Nano-Lipid Cluster for Long-term Injectable Delivery of Peptides

  • 전상원 (Sangwon Jeon, 아주대학교 일반대학원 약학과)

초록/요약

This study designed a novel lipid-based bioresponsive nano-lipid cluster system by utilizing the liquid crystal structure for the drug delivery system and aimed to control the release by encapsulating a hydrophilic material. An in situ forming cluster formulation composed of lipids with biocompatibility and biodegradability such as phospholipids, oleic acid, and propylene glycol laurate to ensure sustained drug release and injection site safety was prepared, and liraglutide, an agonist of glucagon-like peptide-1, was applied. Liraglutide-loaded nano-lipid cluster(pre-BNLC) was prepared by simply mixing phospholipids, oleic acid, propylene glycol, and ethanol. By evaluating the viscosity, rheological characteristics, and injection force, pre-BNLC confirmed that the liraglutide-containing pre-BNLC had a viscosity of 500 mPa.s or less and an injection force 12N or less clinically applicable to injectability. When exposed to an aqueous environment, pre-BNLC formed a semi-solid nano-lipid cluster (BNLC). Through polarized optical microscopy, small-angle X-ray scattering, and atomic force microscopy analysis, the internal structure of the formed BNLC was confirmed to be a typical hexagonal phase. When the gelation characteristics of BNLC were analyzed with a rheometer and a texture analyzer, it was identified that it gels quickly within a few minutes and had a physical strength that is not easily deformed by an external force. In addition, the cryo-FIB-SEM analysis of the structure of the surface and internal section of BNLC revealed that the molecular structural characteristics of the constituent lipids, CPP (critical packing parameter), affect the number and structure of the pores formed. The pre-BNLC containing liraglutide was stable at 4°C for four weeks without changing properties or contents, and the encapsulation efficiency was close to 100%. In the in vitro release test, there was almost no initial burst release, and it showed a typical sustained drug release profile compared to the control formulation, a drug solution. The CPP of the lipids constituting the BNLC system determines the packing level of the internal structure of the BNLC, which affects the physical gel strength, the swelling profile, degradation rate, and the size and number of pores on the surface and inside of the BNLC. We investigated the correlation between the properties of these BNLCs and the release properties of the encapsulated drug from BNLC. The in vivo pharmacokinetics and in vivo local safety studies were evaluated in rats. In vivo pharmacokinetic study in rats was performed using Saxenda® Inj, commercially available as a once-a-day dosage form, as a reference drug, and BNLC continued the drug release action for liraglutide for more than 14 days. Further, at the injection site of rats, BNLC showed biodegradable properties that were slowly degraded during the administration period. No significant pathological abnormalities were observed in the injection site tissue to evaluate local tolerance through autopsy. In this study, the bioresponsive nanoporous lipid cluster showed the potential as a safe, novel long-term injectable delivery of peptides by revealing the correlation between the molecular structural characteristics, gelling properties, degradation profiles of the cluster and release kinetics of encapsulated drug from the cluster.

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초록/요약

본 연구는 약물 전달 시스템 중 액상결정 구조를 활용하여 새로운 지질 기반의 생체반응형 나노액상지질결합체 시스템을 설계하였으며 친수성 물질을 봉입하여 방출 제어하는 것을 목표로 하였다. 지속성 약물 방출 뿐만 아니라 주사 부위 안전성 확보를 위해서 인지질, 올레산 및 프로필렌글리콜 라우레이트와 같은 생체적합성과 생분해성이 확보된 지질들로 구성된 in situ forming cluster 제형을 제조하고, 여기에 글루카곤 유사 펩타이드-1(glucagon-like peptide-1)의 효능제인 리라글루티드(liraglutide)를 적용한 생체반응형 나노액상지질결합체(bioresponsive nanoporous-lipid cluster)를 제조하였다. 리라글루티드를 함유한 나노액상지질결합체(pre-BNLC)는 인지질, 올레산, 프로필렌글리콜 및 에탄올을 단순하게 혼합하는 방식으로 제조되었다. 점도, 레올로지 특성 및 주사압을 평가하여 리라글루티드 함유 pre-BNLC는 점도가 500 mPa.s 이하이고, 주사압은 12N 이하의 임상적으로 적용 가능한 주사능(injectability)이 있음을 확인하였다. 이 Pre-BNLC는 수성 환경에 노출되었을 때, 반고형의 nanoporous-lipid cluster(BNLC)를 형성하였다. 편광현미경(polarized optical microscopy), 소각 X-선 산란(small-angle X-ray scattering) 및 원자힘현미경(atomic force microscopy) 분석을 통해 형성된 BNLC의 내부 구조는 전형적인 hexagonal phase임을 확인하였다. BNLC의 겔화 특성은 레오미터(rheometer) 및 물성측정기(texture analyzer)로 분석하였을 때, 수 분 이내에 빠르게 겔화(gelation) 되고, 외력에 의해 쉽게 변형되지 않는 물리적 강도를 갖음을 규명하였다. 또한 Karl Fischer분석 및 DSC 분석으로 water swelling에 의한 겔화 매커니즘을 확인하였다. 또한 BNLC의 표면과 내부 절단면의 구조를 cryo-FIB-SEM으로 분석한 결과, 구성하는 지질의 분자구조적 특성, CPP(critical packing parameter)이 형성되는 pore 수와 구조에 영향을 주는 것을 규명하였다. 리라글루티드를 함유한 pre-BNLC은 4℃에서 4주동안 성상, 함량 변화없이 안정적이었고, 캡슐화 봉입효율(encapsulation efficiency)은 거의 100%였다. In vitro 방출시험에서 초기 급속 방출(initial burst release)은 거의 없었으며, 대조군인 용액제제에 비하여 전형적인 지속성 약물 방출 패턴을 보여주었다. BNLC 시스템을 구성하는 지질의 CPP는 BNLC 내부 구조의 packing level을 결정하고, 이것은 BNLC의 물리적 겔 강도, swelling profile, 분해특성 및 pore의 크기와 수에 영향을 준다. 이러한 BNLC의 특성과 봉입된 약물의 방출 특성 간의 상관성을 규명하였다. 1일 1회 투여 형태로 시판중인 삭센다(Saxenda) Inj을 대조약으로 이용하여 랫드에서의 체내동태 평가를 수행하였고, BNLC는 리라글루티드에 대하여 14일 이상 약물 방출 작용을 지속하였다. 또한 랫드의 주사부위에서 BNLC는 투여 기간 동안 서서히 분해되는 생분해성 특성을 보여주었고, 부검을 통한 국소 내성 평가에서 주사부위 조직에서는 중대한 병리학적 이상이 관찰되지 않았다. 본 연구를 통해 인지질과 함께 새로운 지질로 구성된 생체반응형 나노액상지질결합체는 산업화 가능성이 높고, 구성되는 지질의 분자구조적 특성과 형성되는 lipid cluster의 겔화 특성, 분해특성 및 구조적 특성 간의 상관성을 밝힘으로써 안전성이 확보된 새로운 펩타이드 약효 지속형 주사 제형으로서 가능성을 보여주었다.

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목차

1. Introduction 1
1.1. Limitations in administration and formulation of biopharmaceuticals 1
1.2. Long-acting drug delivery system and long-acting injection technology 2
1.3. Liquid crystal-based long-acting drug delivery technology 7
1.4. The aims and objective of the study 13

2. Materials and Methods 15
2.1. Materials 15
2.2. Preparation and fabrication of pre-BNLC 16
2.3. Ternary phase diagram 17
2.4. Characterization of pre-BNLC and BNLC 18
2.4.1. Bioresponsive nano-lipid cluster (pre-BNLC) 18
2.4.1.1. Appearance and pH 18
2.4.1.2. Viscosity of pre-BNLC 18
2.4.1.3. Rheological properties of pre-BNLC 19
2.4.1.4. Injectability 19
2.4.2. Bioresponsive nano-lipid cluster (BNLC) 19
2.4.2.1. Polarized optical microscopy (POM) 19
2.4.2.2. In vitro cluster formation and differential scanning calorimeter (DSC) 20
2.4.2.3. Gelation time and gel strength 20
2.4.2.4. Swelling and in vitro degradation 21
2.4.2.5. Morphology and internal structure of BNLC 22
2.5. Design of LGT-loaded formulations 23
2.5.1. Solubilization of liraglutide 23
2.5.2. Formulation of LGT-loaded BNLC 24
2.6. Evaluation of LGT-loaded BNLC 24
2.6.1. Encapsulation efficiency 24
2.6.2. In vitro release study 25
2.7. Stability of liraglutide 26
2.7.1. Stability of liraglutide in LGT-loaded pre-BNLC 26
2.7.2. HPLC analysis 26
2.8. Pharmacokinetic studies and statistical analysis 26
2.9. Local Tolerance study 28
2.9.1. Palpation of the injection site 28
2.9.2. Histology 28
2.9.3. In vivo degradation 29

3. Results and Discussion 30
3.1. Selection of components for BNLC system 30
3.1.1. Selection of novel nano-lipid cluster forming adjuvant 30
3.1.1.1. Properties as pre-BNLC 30
3.1.1.2. Properties as BNLC 35
3.2. Ternary phase diagram 37
3.2.1. Ternary phase diagram of Phospholipid/PGL/Water 37
3.2.2. Ternary phase diagram of Phospholipid/OA/Water 37
3.2.3. Design of experiments (DoE) of Phospholipid/PGL/OA 43
3.3. Characterization of pre-BNLC and BNLC 51
3.3.1. Pre-BNLC 51
3.3.2. Formation of BNLC 59
3.3.2.1. In vitro formation of BNLC 59
3.3.2.2. Gelation time and gel strength 62
3.3.2.3. Swelling and in vitro degradation 72
3.3.3. Assessing the internal structure of BNLC 78
3.3.3.1. Cluster formation and phase behavior 78
3.3.3.2. Morphology of BNLC 81
3.3.4. In vitro release profile of hydrophilic material 86
3.3.4.1. In vitro release profile of methylene blue from BNLC 86
3.3.4.2. In vitro degradation of BNLC 87
3.3.5. In vivo safety test 93
3.3.5.1. In vivo local tolerance of BNLC 93
3.4. Preparation of liraglutide (LGT)-loaded formulations 96
3.4.1. Solubility of liraglutide in pH buffer 96
3.4.2. Degradation profile of liraglutide in forced conditions 96
3.4.3. Formulation of LGT-loaded BNLC 101
3.5. Evaluation of LGT-loaded BNLC 105
3.5.1. Encapsulation efficiency 105
3.5.2. In vitro characterization of LGT-loaded BNLC 107
3.5.3. In vitro release 114
3.6. Pharmacokinetics studies in rats 117
3.7. Local tolerance study 120

4. Conclusions 125

5. References 126






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