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Novel Peripheral CB1R Antagonists Derived from AJ5012 Ameliorate Skin Inflammation in Imiquimod-Induced Psoriatic Mouse Model

AJ5012 유래 신규 말초 CB1 수용체 길항제의 Imiquimod 유도 건선 마우스 모델에서 피부 염증 개선 효과

  • 김현영 (Hyeonyoung Kim, 아주대학교 일반대학원)

초록/요약

Psoriasis is a chronic inflammatory skin disease characterized by the hyperactivation of immune cells. Current therapeutic strategies primarily target cytokines, providing symptomatic relief but failing to correct the underlying immune dysregulation, leading to frequent relapse. Cannabinoid receptor 1 (CB1R) has emerged as a key modulator in chronic inflammatory diseases; however, central nervous system (CNS)-related side effects of existing CB1R antagonists underscore the need for peripherally selective agents. In this study, we developed a novel peripherally selective CB1R antagonist derived from an AJ compound, which exhibits high CB1R affinity and inhibitory activity with markedly reduced blood-brain barrier (BBB) permeability. In vivo evaluations using an imiquimod (IMQ)-induced psoriasis-like dermatitis model demonstrated that this compound significantly ameliorated psoriatic symptoms by reducing epidermal hyperplasia, restoring stratum corneum structure, and suppressing keratinocyte proliferation. In addition, the compound decreased macrophage infiltration into the skin and downregulated the expression of pro-inflammatory cytokines. To assess its therapeutic performance, we compared the efficacy of the compound with that of an anti-IL-17A antibody and methotrexate (MTX). The compound exhibited a comparable level of improvement in psoriatic inflammation. Notably, clodronate-liposome-mediated macrophage depletion markedly attenuated the anti-psoriatic effects of the compound, suggesting a macrophage-dependent mechanism of action. Further in vitro studies using bone marrow-derived macrophages (BMDMs) revealed that CB1R inhibition reduced the expression of psoriasis-associated pro-inflammatory cytokines, supporting the idea that the therapeutic effect is macrophage-associated and may involve broader immunomodulatory actions beyond cytokine neutralization. Collectively, these findings establish peripheral CB1R inhibition as a promising therapeutic strategy for psoriasis and highlight the potential of this novel antagonist as a safe and accessible treatment option for CB1R-mediated inflammatory diseases. Keywords : cannabinoid receptor 1, peripheral, antagonist, psoriasis, macrophage

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초록/요약

건선은 면역세포의 과활성화로 인해 발생하는 만성 염증성 피부질환이다. 현재의 치료법은 주로 사이토카인을 표적하여 증상을 완화하지만, 근본적인 면역 항상성의 이상을 해결하지 못해 치료 중단 시 재발이 빈번하게 발생하는 한계가 있다. 칸나비노이드 수용체 1(CB1R)은 만성 염증성 질환의 주요 조절 인자로 주목받고 있으나, 기존 CB1R 길항제는 중추신경계(CNS) 침투로 인한 부작용이 보고되어 말초 특이적 제제 개발의 필요성이 제기된다. 본 연구에서는 AJ 유도체 기반의 새로운 말초 특이적 CB1R 길항제를 개발하였으며, 이 화합물은 높은 CB1R 결합 친화도와 억제 활성을 보이면서도 혈액-뇌 장벽(BBB) 투과성이 현저히 낮은 특성을 나타냈다. 이미퀴모드(IMQ)-유도 건선 마우스 모델을 이용한 평가에서 본 화합물은 표피 비후 억제, 각질층 구조 회복, 케라티노사이트 증식 억제를 통해 건선 증상을 유의하게 개선하였다. 또한, 피부 내 대식세포 침윤과 염증성 사이토카인 발현을 감소시켰다. 치료 효과를 평가하기 위해 기존 치료제인 메토트렉세이트(MTX) 및 항-IL-17A 항체와 비교한 결과, 본 화합물은 이들과 유사한 수준의 건선 개선 효과를 보였다. 특히, 클로드로네이트 리포좀을 이용한 대식세포 고갈 실험에서 본 화합물의 치료 효과가 유의하게 소실되어, 대식세포 의존적 작용 메커니즘이 확인되었다. 추가적으로, 골수 유래 대식세포(BMDM)를 이용한 실험에서 CB1R 억제가 건선 관련 염증성 사이토카인 발현을 억제함을 확인하였다. 이러한 결과는 말초 CB1R 억제가 건선 치료의 새로운 전략이 될 수 있음을 시사하며, 본 화합물이 CB1R 매개 염증성 질환의 안전하고 접근성 높은 치료제로서의 가능성을 지님을 보여준다.

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목차

Ⅰ. INTRODUCTION 1
Ⅱ. EXPERIMENTAL 4
1. Materials 4
2. Preparation and Characterization of CB1R Antagonist 5
3. Tango™ GPCR Cell-Based β-arrestin Assay 6
4. Mice 7
5. Hypothermia Test 8
6. Catalepsy Test 8
7. Perfusion Assay 9
8. Human Cannabinoid GPCR Cell-Based Antagonist cAMP Assay 10
9. Pharmacokinetic Analysis 11
10. Psoriasis-like Mouse Models 13
11. Histology and Immunohistochemistry staining 14
12. In Vivo Depletion of Macrophages 15
13. Cell Culture 15
14. Flow Cytometric Analysis 16
15. Cell viability test 16
16. Real-Time Quantitative PCR 17
17. Statistical Analysis 17
Ⅲ. RESULTS 19
1. Strategic Design and Binding Affinity analysis of Novel Peripheral CB1R Antagonists. 19
2. Behavioral Assessment of Blood-Brain Barrier Permeability of CB1R Antagonists. 24
3. Brain-to-Plasma (B/P) Ratio of Compounds Using Transcardial Perfusion Assay. 28
4. Novel CB1R Antagonists Pharmacological and Pharmacokinetic Characteristics. 30
5. Efficacy of CB1R Antagonists against Psoriasis-like Skin Dermatitis. 34
6. Dose-Dependent Efficacy of Novel CB1R Antagonists against Psoriasis-like Skin Dermatitis. 37
7. Therapeutic Comparison of novel CB1R Antagonists against Psoriasis-like Skin Dermatitis. 41
8. Macrophage-Dependent Efficacy of Novel CB1R Antagonists against Psoriasis-like Skin Dermatitis. 45
9. Suppression of Pro-inflammatory Gene Expression in BMDMs by Novel CB1R Antagonists. 50
Ⅳ. DISCUSSION 53
Ⅴ. REFFRENCE 55
Ⅵ. ABSTRACT IN KOREAN (국문 초록) 59

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