Improved Brain Delivery of Gefitinib via Co-Administration with Dual P-glycoprotein and Breast Cancer Resistance Protein Inhibitors

다국어 초록 (Multilingual Abstract)

Blood-brain barrier (BBB) presents a significant obstacle to drug delivery to the brain. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are the primary efflux transporters at the BBB, hindering drug entry into the brain. As these transporters function cooperatively, dual inhibition of P-gp and BCRP is essential to enhance drug delivery to the brain, rather than mono-inhibition. Gefitinib is a first-line treatment of non-small cell lung cancer (NSCLC), which frequently metastasizes to the brain. However, gefitinib is ineffective against brain metastases due to its inability to cross the BBB. This study aims to evaluate whether the brain penetration of gefitinib increases when co-administered with three dual P-gp/BCRP inhibitors – CDK 4/6 inhibitor Ⅳ, BX795, and CGP60474 – thereby expanding its indications as a treatment for brain tumors. We compared the cytotoxicity of gefitinib alone and in combination with the dual inhibitors in vitro using hCMEC/D3 cells. The results showed a significant increase in cytotoxicity with three dual P-gp/BCRP inhibitors. An analytical method using LC-MS/MS was developed to accurately and reproducibly analyze plasma samples and brain homogenates obtained from in vivo studies. The method was validated in compliance with FDA guidelines. In animal experiments, dual inhibitors were co-administered intravenously with gefitinib. While the plasma concentrations of gefitinib were unaffected, the brain-to-plasma ratio significantly increased in the group treated with gefitinib in combination with the dual inhibitors, especially in CDK 4/6 inhibitor IV and CGP60474. These findings demonstrate that co-administration of gefitinib with dual inhibitors improves drug delivery to the brain without affecting its plasma profile. In conclusion, the dual inhibitors hold promise in overcoming BBB-related challenges, thereby enhancing the efficacy of anticancer drugs in treating brain tumors.

국문 초록 (Abstract)

혈액-뇌 관문(BBB)은 뇌로의 약물 전달에 중요한 장벽으로 작용한다. 이 장벽에서 주된 배출 수송체인 P-당단백질(P-gp)과 유방암 저항성 단백질(BCRP)은 뇌로의 약물 진입을 방해하며, 이 두 수송체는 협력적으로 작용하기 때문에 P-gp와 BCRP의 이중 억제가 뇌로의 약물 전달을 향상하는 데 필수적이다. 게피티닙은 전이성 비소세포폐암(NSCLC)의 1차 치료제로, 이 암은 뇌로 자주 전이된다. 그러나 게피티닙은 BBB를 통과하지 못해 뇌 전이에 대한 효과가 제한적이다. 본 연구는 세 가지 P-gp/BCRP 이중 억제제인 CDK 4/6 억제제 IV, BX795 및 CGP60474와 함께 투여했을 때 게피티닙의 뇌 침투력이 증가하는지 평가하여 뇌종양 치료제로의 적응증을 확장하는 것을 목표로 하였다. hCMEC/D3 세포를 사용하여 in vitro에서 게피티닙 단독 및 이중 억제제 병용 시 세포독성을 비교했으며 그 결과, 세 가지 P-gp/BCRP 이중 억제제를 병용한 경우 세포독성이 유의미하게 증가함을 확인하였다. 동물 실험에서는 이중 억제제를 게피티닙과 정맥으로 병용 투여하였으며, 혈장 내 게피티닙 농도에는 변화가 없었지만, 게피티닙 병용 투여 그룹에서 뇌 대 혈장 비율이 유의미하게 증가함을 확인하였다. 특히, 양성 대조군으로 사용한 elacridar 처리 그룹에서는 모든 시간대에서 통계적인 유의성이 나타났으나, 이중 억제제 처리 그룹에서는 특정 시간대에서 뇌 대 혈장 비율이 유의미하게 증가하였다. 이 결과를 통해 게피티닙과 이중 억제제 병용 투여가 혈장 농도에 영향을 미치지 않으면서도 뇌로의 약물 전달을 개선할 수 있음을 보여주었다. 결론적으로, P-gp/BCRP 이중 억제제는 항암제의 BBB 투과를 증진함으로써 뇌종양에서 항암제의 효능을 향상시킬 수 있을 것으로 기대된다.

목차 (Table of Contents)

• I. Introduction 1
• A. Blood-brain barrier 1
• B. P-glycoprotein and breast cancer resistance protein 4
• C. Gefitinib 5
• D. Dual P-gp/BCRP inhibitor candidates 7
• E. Objectives of thesis 10
• II. Materials and method 11
• A. Materials 11
• B. Instruments 12
• C. In vitro studies 13
• 1. Cell lines and culture conditions 13
• 2. Cytotoxicity assay 13
• D. LC-MS/MS analysis of gefitinib 14
• 1. Liquid chromatographic conditions 14
• 2. Tandem mass spectrometric conditions 14
• 3. Preparation of stock, standard solutions, and QC samples 15
• 4. Sample preparation 15
• 5. Analytical method validation 16
• a. Specificity and linearity 16
• b. Precision and accuracy 16
• c. Stability 17
• d. Extraction recovery 18
• E. In vivo studies 18
• 1. Preparation of rats 18
• 2. Drug formulation 19
• 3. Pharmacokinetic study 19
• 4. Brain distribution study 20
• F. Statical analysis 20
• III. Results 22
• A. In vitro studies 22
• 1. Characterization of cell lines used in cytotoxicity assay with gefitinib 22
• 2. Effect of P-gp/BCRP dual inhibitors on gefitinib cytotoxicity 24
• B. Analytical method validation of LC-MS/MS for gefitinib 26
• 1. Method development 26
• 2. Specificity and linearity 27
• 3. Precision and accuracy 30
• 4. Stability 30
• 5. Extraction recovery 33
• C. In vivo studies 34
• 1. Pharmacokinetic study 34
• 2. Brain distribution study of gefitinib with P-gp/BCRP dual inhibitors 36
• IV. Discussion 42
• References 45
• Abstract (in Korean) 49