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Evaluation of Epoxyketone-based Immunoproteasome-selective Inhibitors with Enhanced Brain Distribution and Stability In Vitro & In Vivo : 뇌 조직으로의 분포 및 안정성이 개선된 epoxyketone-based 면역프로테아좀 억제제의 평가연구

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dc.contributor.advisor이우인-
dc.contributor.author유지수-
dc.date.accessioned2020-05-07T05:16:11Z-
dc.date.available2020-05-07T05:16:11Z-
dc.date.issued2020-
dc.identifier.other000000160461-
dc.identifier.urihttp://dcollection.snu.ac.kr/common/orgView/000000160461ko_KR
dc.description학위논문(석사)--서울대학교 대학원 :약학대학 약학과,2020. 2. 이우인.-
dc.description.abstractImmunoproteasome (iP) is one of the proteasome subtypes and is induced by immune cytokines. Recent studies have reported that iP inhibitors are effective against neurodegenerative diseases. Among the iP inhibitors used in research, YU102 has an epoxyketone as a pharmacophore and has a peptides backbone. Proteasome inhibitors having epoxyketone and peptides are considered to have a short in vivo half-life. In addition, YU102 is known as the P-glycoprotein (P-gp) substrate, which is the main efflux transporter of the blood-brain barrier, thereby limiting the penetration to the brain. Therefore, in this study it is evaluated the possibility of brain penetration in vivo for several YU102 derivatives. In vivo and in vitro stability was measured for macrocyclic YU102 analogs that are expected to increase metabolic stability. In vitro stability experiments using liver homogenate and whole blood in rats showed that the macrocyclic compounds (DB43, DB49) were generally more stable than the linear form (DB16). In particularly, DB43 showed a significant increase in stability in liver homogenate where metabolism occurs very rapidly. In addition, plasma concentrations were measured by mouse pharmacokinetic test. As a result, macrocyclic compounds (DB43, DB49) showed greater stability & systemic exposure than linear compounds (YU102, DB16). In addition, DB55 showed slightly increased in vivo stability compared to DB49. To determine the potential for penetration into the brain of the compounds, the degree of target (LMP2) inhibition in the brain was measured. Comparing between linear forms (YU102 vs DB16), the target brain was suppressed by about 20 % in the mouse brain injected with DB16. After injection of linear forms (YU102, DB16) or macrocyclic forms (DB43, DB49), the target inhibitions in the brain were reduced by about 8 % in DB43 or about 11 % in DB49 compared to vehicle group. In addition, the blood-to-plasma concentration ratios (B/P ratios) and plasma unbound fraction were measured in mice. As a result, epoxyketone-based inhibitors showed a B/P ratio of greater than 1 and the unbound fractions of the rage of 54-77 % in all compounds except DB43. DB43 has a B/P ratio of about 1 and an unbound fraction of about 98 %, suggesting DB43 had a lower binding to erythrocyte. This study is to evaluate epoxyketone-based immunoproteasome-selective inhibitors with enhanced brain permeability & stability and demonstrated the potential of several compounds. While further research should be conducted, this study may suggest possibilities of epoxyketone-based immunoproteasome inhibitors for brain distribution.-
dc.description.abstract면역프로테아좀 (Immunoproteasome, iP)은 프로테아좀 subtypes 중 하나로 면역 사이토카인에 의해 유도된다. iP는 특정 질병 상태에서 발현되는 것으로 알려져 있으며, 최근 연구에서는 iP 억제제가 신경퇴행성 질병에 대해 효과를 나타낸다고 보고되었다. 연구용으로 사용되는 iP 억제제 중 YU102는 epoxyketone을 pharmacophore와 peptides 골격을 가진다. 이 같은 공통구조를 가지는 억제제의 경우 짧은 in vivo 반감기를 가지는 것으로 생각된다. 또한, YU102의 경우 혈관-뇌 장벽 (blood-brain barrier)의 주요 efflux transporter인 P-glycoprotein (P-gp) 기질로 알려져 있어 뇌로의 이행이 제한된다. 따라서, 본 연구에서는 in vitro 실험에서 P-gp를 회피하는 것으로 보이는 몇 개의 YU102 유도체들에 대해 in vivo에서 뇌 투과 가능성을 평가하였다. 또, 대사 안정성이 증가할 것으로 기대되는 macrocyclic 형태의 YU102 유도체들에 대해 in vivo & in vitro 안정성을 비교하였다. 이를 통해 뇌로의 이행 및 안정성이 증가된 iP 억제제들의 가능성을 평가하고자 하였다. Rat의 liver homogenate와 whole blood를 이용한 in vitro 안정성 실험에서 macrocyclic compounds인 DB43, DB49가 linear forms인 DB16에 비해 전반적으로 안정성이 증가하였다. 특히, 대사가 매우 빠르게 일어나는 liver homogenate에서 DB43의 경우 큰 안정성 증가를 보였다. 또, in vivo stability를 비교하기 위해 진행된 mouse plasma pharmacokinetic test 결과 macrocyclic compounds (DB43, DB49)가 linear compounds (YU102, DB16)에 비해 큰 안정성과 systemic exposure을 나타냈다. DB55의 경우 DB49에 비해 안정성이 소폭 증가하였다. YU102에 비해 P-gp와 덜 interaction한 것으로 예상되는 compounds (DB16, DB43, DB49)의 뇌 투과 잠재력을 알아보기 위해 약물 주입 후 뇌에서의 target (LMP2) inhibition 정도를 측정하였다. 같은 linear form (YU102, DB16)을 비교했을 때 DB16을 주입한 mouse brain에서 약 20% 정도 target이 억제되었다. 반면에 YU102의 경우 brain target을 거의 inhibition하지 못하였다. YU102, DB16, DB43, DB49를 주입한 mouse brain에서는 vehicle group에 비해 DB43의 경우 약 8%, DB49의 경우 약 11% 감소되었다. mouse에서 이 compounds의 blood-to-plasma ratio (B/P ratio)와 plasma unbound fraction을 측정한 결과, DB43을 제외한 모든 compounds에서 1 이상의 B/P ratios, 54-77%의 unbound fraction을 나타내어 erythrocyte에 binding이 큰 것으로 생각된다. 본 연구에는 P-gp 기질성을 회피하는 성질과 구조적 변화(macrocyclization)를 통한 안정성 증가가 epoxyketone-based iP 억제제의 뇌로의 이행에 유리할 것이라는 가정 하에 진행되었으며, 그 잠재력을 생체 외 및 생체 내 실험을 통해 입증하였다. 추가 연구를 통한 근거가 더 필요하지만, 본 연구를 통해 epoxyketone-based inhibitor의 뇌 투과 가능성이 제시될 수 있다.-
dc.description.tableofcontents1. Introduction 1
2. Materials and Methods 5
2.1. YU102 derivative compounds 5
2.2. In vitro metabolic stability in SD rat liver homogenate and whole blood 5
2.3. Plasma pharmacokinetic (PK) study in ICR mice 7
2.4. LMP2 activity measurement in tissue homogenates from drug treated mice 8
2.5. Blood-to-Plasma concentration (B/P) ratio and Plasma protein binding assessment 9
2.6. Statistical analysis 11
3. Results 12
3.1. In vitro metabolic stability comparison in SD rats 12
3.2. In vivo plasma pharmacokinetics in ICR mice 12
3.3. In vivo LMP2 immunoproteasomal inhibition in the brain and other tissues 13
3.4. Blood-to-Plasma concentration (B/P) ratios amd Plasma protein binding in mice 15
4. Discussion 17
5. References 21
국문초록 34
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dc.language.isoeng-
dc.publisher서울대학교 대학원-
dc.subject.ddc615-
dc.titleEvaluation of Epoxyketone-based Immunoproteasome-selective Inhibitors with Enhanced Brain Distribution and Stability In Vitro & In Vivo-
dc.title.alternative뇌 조직으로의 분포 및 안정성이 개선된 epoxyketone-based 면역프로테아좀 억제제의 평가연구-
dc.typeThesis-
dc.typeDissertation-
dc.contributor.AlternativeAuthorYoo, Jisu-
dc.contributor.department약학대학 약학과-
dc.description.degreeMaster-
dc.date.awarded2020-02-
dc.identifier.uciI804:11032-000000160461-
dc.identifier.holdings000000000042▲000000000044▲000000160461▲-
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