섬유증 및 종양 미세환경 내에서 활성화되도록 설계된 약물 전달 시스템 개발 연구

학위논문(박사) -- 서울대학교대학원 : 약학대학 약학과, 2023. 2. 이우인.질병 부위 내의 미세환경은 질병의 발달 및 치료에 중요한 역할을 담당한다. 질병 부위 내의 미세환경에서는 세포-세포간, 세포-환경 간의 복잡한 상호작용이 일어난다고 알려져 있다. 따라서 미세환경을 어떻게 조절하느냐에 따라서 치료의 성공 여부에 큰 영향을 끼친다. 약물 전달 시스템을 이용하면 국소적인 질병 부위에 특이적으로 약물을 전달할 수 있다. 본 박사학위 논문에서는 이러한 점에 착안하여 자극에 기반하여 활성화되는 약물 전달시슽템을 개발하고, 이를 통해 질병의 미세환경을 조절하여 질병 치료 효과를 보고자 하였다. 약물 전달 시스템에 이용한 자극 및 미세환경의 활용 방법에 따라서 총 세 가지 주제로 연구가 진행되었다. 첫 번째와 두 번째 주제는 종양 모델에서의 항암 치료 및 미세환경 조절에 관한 연구이며 세 번째 주제는 간섬유증 모델에서의 섬유성 미세환경 특이적인 항섬유화 펩타이드 전달에 관한 연구이다. 첫 번째 연구는 종양 치료 및 종양 미세환경의 면역 활성을 위한 적외선/원적외선 감응형 금 나노클러스터에 대한 연구이다. 두 종류의 빛에 동시에 감응하여 종양미세환경 내에서 광역학치료/광열치료 효과를 동시에 유도할 수 있었다. 또한 면역원성이 있는 CpG DNA 가닥을 금나노클러스터의 주형으로 활용함에 따라 수지상세포를 활성화하였다. 이에 따라 종양 특이적인 T 세포 반응을 유도함과 동시에 원발성 종양 진행을 억제할 수 있었다. 두 번째 연구는 저온 대기압 플라즈마 감응성 하이드로겔을 기반으로 한 면역조절 나노입자 및 종양 항원의 지속적 전달 시스템에 관한 연구이다. 저온 대기압 플라즈마에 의하여 종양 조직 내부에 종양 항원과 면역 조절 나노입자를 포함하는 하이드로겔이 형성된다. 종양 내에 형성된 이러한 네트워크는 수지상세포와 T 세포를 하이드로겔로 유도하고 항암 효과를 나타낼 수 있는 종양 미세면역환경을 형성한다. 세 번째 연구는 간섬유증 미세환경 내에서 특이적으로 높게 발현하는 효소가 있다는 것에 착안하여 개발된, 간섬유증 특이적 항섬유화 펩타이드 전달 시스템에 관한 연구이다. 간섬유화를 일으키는 활성화된 간성상세포를 미세환경의 특징을 이용하여 특이적으로 사멸시킬 수 있었으며, 미세환경 내에서 간성상세포의 비율만 낮출 수 있었다. Bile duct ligation, CCl4-induced fibrosis 그리고 지방간 기반 간섬유증까지 총 세 가지 간섬유화 모델에서 항섬유화 효과를 확인할 수 있었다. 이상의 연구들을 통하여 외부 자극을 통한 면역미세환경 조절 및 미세환경 내 내부 자극을 이용한 미세환경 조절을 약물전달시스템에 적용하였을 때 질병의 치료에 유의한 효과를 유도할 수 있다는 것을 확인할 수 있었다. 하지만 연구 별로 한계점이 존재하였다, 광열치료와 광역학치료에는 이를 유도할 수 있는 치료 물질이 포함되어야 하는데, 이 역시 임상적으로 사용되는 약물을 세심히 고를 필요가 있을 것으로 생각된다. 저온 플라즈마의 항암 치료에의 적용은 신규한 치료 방법이지만 그만큼 임상적으로 확인된 케이스가 적으므로 관련하여 더 많은 데이터를 축적할 필요가 있으며, 조직 내에서 플라즈마 기체가 작용할 수 있는 깊이가 깊지 않을 것이기 때문에 적용하고자 하는 종양의 크기가 치료 효과에 크게 영향을 끼칠 것으로 사료된다. 간섬유증 치료용 펩타이드의 전달은 치료 모델로서 간섬유화 모델을 이용했지만, 섬유아세포가 관여하는 다양한 섬유성 질환에 활용할 수 있을 것으로 사료된다. 다만 미세환경 내의 면역 세포 역시도 섬유증에서 중요한 역할을 담당하고 있다고 알려져있는데, 면역 세포의 변화를 평가하기 위해서는 추가적인 phenotype 연구가 필요하다. 본 연구에서는 흔하게 사용되는 페길화된 리포좀 제형을 사용하였으므로 제형 상 개선의 여지가 있으며, 세포를 표적화 할 수 있는 리간드와 함께 작용한다면 용량 대 약효를 더욱 증진시킬 수 있을 것이다. 질병의 미세환경은 복잡성을 띄고 있으며 질병의 종류 및 질병의 진행 정도에 따라, 그리고 개체 별로 큰 차이가 있을 수 있기 때문에 실제 임상에서는 효과가 어떻게 나타날지는 미지수이다. 또한 종양과 섬유성 질환 이외에도 미세환경 조절을 위한 약물전달 시스템을 개발하기 위해서는 더욱 넓은 범위의 연구가 필요하다. 따라서 자극 기반 약물전달시스템을 질병 미세환경에 적용하기 위해서는 질병 별로 보편성을 가지는 요소를 활용하여 질병에 적절한 자극을 선택하는 것이 중요할 것이다. 특히 저온 대기압 플라즈마를 포함한 외부 자극의 경우 대부분 피부 표면과 가까운 부위로 적용이 제한된다는 한계점이 있으므로 치료용 기기 등을 이용하여 이러한 한계를 극복하려는 노력이 필요하다. 여러 한계점에도 불구하고 미세환경의 조절과 국소적인 자극을 기반으로 한 약물전달 시스템의 시너지 효과는 주목할만한 잠재성을 가지고 있다. 특히 종양 및 섬유성 질환처럼 기존 치료법에 한계가 있는 질병에 대해서 본 연구에서 제시된 지극 기반 미세환경 조절용 약물 전달시스템은 유망한 치료 선택지가 될 수 있을 것이다.The microenvironment plays a crucial role in both the progression and treatment of diseases including tumor and hepatic fibrosis. However, current therapeutics are non-targeting or cell-targeting agents which can induce systemic adverse effects. The limited therapeutic efficacy of these agents was mainly associated with the deleterious microenvironment of the tissue. Therefore, strategies for targeted modulation of the microenvironment should be further investigated. Herein, we developed the programmed drug delivery systems that can be activated by exogenous or endogenous stimuli at the tumor and fibrotic microenvironment. This thesis work is composed of two parts, which are the exogenous stimuli-actuated delivery system for tumor immune microenvironment modulation and peptide-delivery nanotherapeutic for regulation of fibrotic microenvironment. In Part I, we developed stimuli-responsive delivery platforms that are programmed to be activated by light or cold atmospheric plasma (CAP). With these delivery systems, our goal was to eradicate primary tumors as well as distant tumors following immune microenvironment activation. In the first chapter, the gold-DNA nanocluster was developed for photo-immunotherapy on a tumor. Previous studies mainly used one light source for a single type of phototherapy, such as photothermal or photodynamic therapy, which has limited therapeutic efficacy as well as insufficient immune reaction. In this context, we designed gold-DNA nanocluster intercalating methylene blue for dual-light activated photo-immunotherapy. By exploiting a CpG sequence as a template for DNA polymer, delivery of nucleic acid to the dendritic cell was achieved to activate the tumor immune microenvironment followed by the prevention of distant tumor growth. Our in vivo study suggest that systemic injection of the nanocluster elicited enhanced tumor eradication and immune activation at the tumor site after the dual-light irradiation. In the second chapter, we developed programmed antigen-releasing hydrogel actuated by CAP irradiation. Previous clinical trials have utilized CAP for residual tumor removal, however, its limited penetration depth has limited its effectiveness in treating primary tumors. Our findings show that the CAP-activated hydrogel system is capable of sustained release of both tumor antigen and TGF-beta inhibitor, leading to increased anti-tumor immune response and primary as well as distant tumor eradication. These results highlight the potential of the CAP-responsive in situ hydrogel system as a promising therapeutic platform for overcoming the limitations of CAP's depth-related restriction in treating primary tumors. In part II of the thesis, the fibrotic microenvironment was modulated for the treatment of liver fibrosis. We designed an anti-fibrotic peptide delivery system that is programmed to be liberated by activated hepatic stellate cells (aHSCs). Despite the urgent global needs, there is no clinically approved therapeutic for liver fibrosis. In the fibrotic microenvironment, aHSCs play a central role in fibrogenesis and express distinct enzyme proteins such as fibroblast activation protein (FAP). Our goal was selective eradication of aHSCs in the fibrotic liver by exploiting promelittin, which is a FAP-responsive prodrug form of melittin. Treatment of promelittin-conjugated liposome could elicit anti-fibrotic effects in three different hepatic fibrosis models as well as apoptosis of aHSC. Although this peptide-delivery system was tested in liver fibrosis, it could be applied to other fibrotic diseases, such as lung fibrosis and cardiac fibrosis. Taken together, studies on programmed in situ drug delivery systems, which can be activated by stimuli, were achieved in this thesis work and showed promising therapeutic effects following the modulation of the tumor and fibrotic microenvironment. Furthermore, the drug delivery strategies suggested in this thesis address the current limitation of the therapeutics, which can broaden the current therapeutic indication.Abstract 1 Contents 3 List of Tables 5 List of Figures 6 Chapter I. Overview 8 I-1 Disease and the tissue microenvironment 9 I-2. Stimuli-responsive drug delivery systems 12 I-3. Scope of study 14 I-4. References 16 Chapter II. Photosensitizer-trapped gold nanocluster for dual light-responsive phototherapy 20 II-1. Introduction 22 II-2. Materials and methods 24 II-3. Results 28 II-4. Discussion 36 II-5. References 38 Chapter III. Cold plasma-actuated on-site hydrogel for remodeling tumor immune microenvironment 41 III-1. Introduction 42 III-2. Materials and methods 44 III-3. Results 53 III-4. Discussion 69 III-5. References 71 Chapter IV. Liver fibrosis-activated antifibrotic peptide delivery 75 IV-1. Introduction 76 IV-2. Materials and methods 77 IV-3. Results 85 IV-4. Discussion 98 IV-5. References 102 Chapter V. Conclusion 107 국문 초록 109박

Clinical Experience and Outcomes of Adult Pleomorphic Xanthoastrocytoma

Introduction: PXA is known for clinically indolent tumor, however clinical behavior of PXA is not uniform. The predicting factors of aggressive behaviors of PXA have not been fully elucidated. The purpose of this report is to demonstrate the experiences and clinical outcomes of PXA in our institution, to find prognostic factors of PXA. Method: Our institutional database was searched for patients aged 18 years or older who underwent surgery and were diagnosed with PXA between 2002 and 2016. Total of 25 patients were identified and analyzed. Result: There were 8 (32%) male and 17 (68%) female patients. Mean age was 29.9. Most common presenting symptoms was seizure. Most common location of PXA was temporal lobe (11, 44%). Mean tumor size was 33.6 mm (range: 10 to 70 mm). 21 (84%) patients were diagnosed as PXA, WHO grade II and 4 (16%) patients were diagnosed anaplastic PXA, WHO grade III. The perilesional edema was seen on 13 patients (52%). Twenty-one (84%) patients underwent GTR, 4 (16%) of patients underwent STR. Seven (28%) patients received adjuvant radiation therapy. No patient received adjuvant chemotherapy. Recurrence occurred in 11 (44%) patients. High grade transformation was observed in 4 patients (36.4%). Six (24%) patients were died during follow up period. The OS rate of PXA in 1,2,3,5,7 and 10 years were 100%, 89.5%, 89.5%, 89.5%, 61.4% and 40.9% and OS rate of anaplastic PXA in 1,2,3,5,7,10 years were 100%, 100%,100%, 0%, 0% and 0%. The PFS rate of PXA in 6 months, 1,2,3,5 and 7 years were 90.5%, 81%, 70.5%, 65.1%, 65.1% and 52% and PFS rate of anaplastic PXA in 6 months, 1,2,3,5 and 7 years were 100%, 75%, 50%, 50%, 0% and 0%. The differences of OS and PFS between PXA and anaplastic PXA were not statistically significant. (p value= 0.379 and 0.213, respectively. We did comparison the advanced MR imaging (DWI, PWI) and PET finding between PXA and anaplastic PXA. This result failed to show statistical significance. We analyzed the impact of advanced MR findings and PET findings to recurrence rate of PXA grade II. In recurrence group, There were no differences of DWI, PWI and PET findings between recurrence and non-recurrence group. Tumor size larger than 40mm, solid with mixed cystic and hemorrhagic tumor, presence of perilesional edema were poor prognostic factors for PFS (HR=4.394, 11.846, 15.239, p value=0.036, 0.013, 0.01 respectively) were poor prognostic factors in univariate analysis. In multivariate analysis, only perilesional edema was significant poor prognostic factor. (HR=20.523, p value=0.009) We did comparison characteristics between post-treatment silent PXA grade II group and recurrent PXA (grade II) group. In recurrent PXA grade II group, the rate of presence of peritumoral edema was 87.5%, while in silent PXA grade II group, the rate of presence of peritumoral edema was 23.1%. This difference showed statistically significance. (p value= 0.008). A PXA grade II grading system was constructed using the 3 variable, size, peritumoral edema and tumor type. Score 1 to 2 were classified low risk and score 3 to 4 were high risk. Tumor progression and recurrence was predicted according to PXA grade II group, 5-year progression-free survival was 80.2% in low risk group and 20% in high risk group. (p value= 0.025) Conclusion:. To our limited knowledge, tumor size, solid plus cyst and hemorrhage tumor type and peritumoral edema of PXA is associated with poorer progression free survival in univariate analysis. DWI, PWI and PET findings of PXA can’t expect clinical behavior and grade of the PXA. PXA show high recurrence rate, thus close follow up is needed. In the future, multicenter larger size prospective study should be neededMaste

Clinical Outcomes of Large (> 10 mm) Unruptured Posterior Circulation Aneurysms and Their Predictors

Objective : The treatment of large aneurysms of the posterior circulation is complicated and remains challenging. We here analyzed our institutional clinical outcomes of large unruptured aneurysms of the posterior circulation. Methods : This study included 56 patients who presented with a large (>10 mm) unruptured aneurysm of the posterior circulation between 2002 and 2018. Results : There were 18 (32.1%) male and 38 (67.9%) female patients, with a mean age of 53.4 years. The most common location was the vertebral artery, followed by the basilar tip and posterior cerebral artery. The median follow-up duration was 29 months. Eighteen patients (32.1%) were treated by transcranial surgery and 38 (67.9%) were treated by endovascular treatment (EVT). Post-treatment complications occurred in 16 patients (28.6%), with there being no significant difference between the transcranial surgery and EVT groups. Complete obliteration was achieved in 30 patients (53.6%), with there being no statistically significant difference between the transcranial surgery and EVT groups. Recurrence occurred in 17 patients (30.4%), and the rate of recurrence was higher in the EVT group than in the transcranial surgery group (39.5% vs. 11.1%, p=0.03). Forty-four (84%) of 56 patients showed a favorable functional outcome. In saccular aneurysm, EVT was negative predictor of worsening of functional status. Conclusion : Treatment of these aneurysms harbors an inherent high risk of morbidity. No superiority was found between transcranial surgery and EVT in terms of complications and complete obliteration, but transcranial surgery showed a higher treatment durability than EVT

Reactive Oxygen Species Scavenger in Acute Intracerebral Hemorrhage Patients A Multicenter, Randomized Controlled Trial

Background and Purpose: Patients with intracerebral hemorrhage (ICH) have oxidative stress. Oxidative stress contributes to the development and progression of perihematomal edema (PHE) in brain hemorrhage patients. We hypothesized that reactive oxygen species (ROS) scavengers might have a neuroprotective role in the acute period of patients with ICH. Methods: This prospective, multicenter, single-blind, randomized study was conducted between June 2017 and October 2019. Intracranial bleeding, including spontaneous ICH, secondary ICH due to vascular anomalies, venous thrombosis, neoplasms, or hemorrhagic infarction, were included in our study. These ROS scavengers were given for 14 days with a dose of N-acetylcysteine 2000 mg/d and selenium 1600 mu g/d intravenously. Other patients received a placebo. The primary outcome was hemorrhage and PHE volume changes in 2-week follow-up computed tomography between ROS scavenger versus placebo groups. Results: In total, 448 patients were enrolled with 123 patients remaining after applying the inclusion and exclusion criteria. There were no significant differences in baseline characteristics between the ROS scavenger (n=57) and placebo (n=66) groups. No significant differences in baseline hematoma and PHE volumes were observed but 2 weeks follow-up computed tomography showed significant differences in PHE volume (21.90 +/- 17.63 versus 30.66 +/- 32.35, P<0.01) and PHE ratio (1.19 +/- 0.73 versus 2.05 +/- 1.27, P<0.01). Among clinical factors, time to reach target Richmond Agitation Sedation Scale (5.98 hours [95% CI, 4.82-7.241 versus 8.42 hours], [95% CI, 6.57-10.77], P<0.01) and the length of intensive care unit stays (6.46 days [95% CI, 2.38-10.55 versus 12.66 days], [95% CI, 8.47-16.85], P<0.01) were significantly shortened among patients who received ROS scavengers than among patients who did not receive ROS scavenger. Conclusions: ROS scavenger showed a significantly reduced PHE volume, time to reach target Richmond Agitation Sedation Scale, and shortened length of intensive care unit stay in patients with acute ICH. Early and high doses of ROS scavengers in a combination regimen may have played a key role in obtaining a favorable outcome in our study. Registration: URL: ; Unique identifier: KCT0004628

Clinical Interrogation of Mandatory Insertion of Central Venous Catheter for Clipping Surgery of Unruptured Intracranial Aneurysm: A Propensity Score Matched Study

Background and Objective: Microsurgical clipping, along with endovascular treatment, has evolved in the treatment of unruptured intracranial aneurysms (UIA), and these developments have resulted in a reduction of the complication rate. We discuss the need for a central venous catheter (CVC) insertion as an anesthetic preparation for microsurgical clipping. Methods: Between January 2019 and September 2019, 722 patients with UIA were treated at our institution. We excluded patients with a history of endovascular treatment or bypass surgery, recurrent aneurysms after coil embolization, brain tumors, or subarachnoid hemorrhages. A total of 272 patients were enrolled. Eighty-four patients underwent CVC insertion, and 188 patients underwent clipping surgery without CVC insertion. Outcome-related factors were compared between the 2 groups. We performed propensity score matching of the 2 groups to increase comparability. Results: There were no significant differences in outcome, sex, aneurysm location, aneurysm multiplicity, aneurysm size, or comorbid disease between the 2 groups. The mean age at the time of surgery was higher in the non-CVC insertion group than in the CVC insertion group. There were no meaningful differences in primary outcomes, including premature rupture and intraoperative motor evoked potential/somatosensory evoked potential change, and secondary outcomes, including estimated blood loss, duration of intensive care unit stay, duration of hospitalization, and Glasgow Outcome Scale score at discharge. Conclusions: CVC insertion for clipping surgery for UIA is not mandatory. Considering the possible complications associated with CVCs, we cautiously suggest aneurysm surgery with CVC insertion in patients with serious medical comorbidities, aneurysm sizes >10 mm, and difficult proximal parent artery control