Risk prediction with a generalized linear mixed effects model for detecting adverse drug event of drug combination

김혜령 Graduate School, Yonsei University 2023 국내박사

Adverse drug events (ADEs) induced by multiple combination drugs are a critical concern when dealing with drug-drug interactions (DDIs). Most studies on multiple drug combination methods mainly focus on pairwise drug reactions, so existing risk prediction approaches for high-dimensional drug interaction are inadequate. To consider ADEs of a high-dimensional situation multiple-combination drugs, we propose a generalized linear mixed-effect model with two levels to predict the multiple- combination drug risk based on a single drug. As the structure of single and multiple combination is a connected structure, it should be considered as a nested structure rather than as a multiple independent structure. In this study, a mixed-effect model was used to estimate the variation between clusters by grouping single drug units. We assume the analyses that do not reflect these nested structures can lead to increase false-positive ADE signal detection. For performance comparison, we use the mixture drug-count response model (MDRM) (Wang, Zhang et al. 2018) for high-dimensional drug interaction. In simulation, we show that our mixed-effect model provides better performance than the conventional MDRM method with power. Then, we apply these methods to the CDM database and compare the predicted risk using the proposed method based on observed risk. 복합약물 복용에 따라 유발된 약물 부작용에 대한 연구는 약물-약물 상호 작용과 관련된 중요한 이슈로 떠오르고 있다. 노인 연령일수록 약물에 대해 복합 약물을 복용하는 경우는 점점 더 높아지고, 최근 시기일수록 3개 이상의 복합 약물을 복용하는 경우도 증가하고 있다. 그러나 기존의 통계방법론에 따라 주로 집중되고 있는 연구는 두개의 약물 조합의 부작용에 집중되어 있기 때문에, 고차원 약물 상호작용의 위험 예측 방법에 대한 연구가 부족한 실정이다. 기존에 복합약물에 대한 혼합 약물 반응 모델이 존재하고 이의 경우에는 여러 개의 복합 약물의 경우를 고려하지만, 이 또한 약물의 개수에 따라 선형적으로 위험을 가정하여 추정할 뿐, 약물 자체의 특성에 대한 고려를 포함하여 모델링 하고 있지 않다. 따라서 우리는 여러 개의 약물 조합에 따른 특성을 반영한 약물 부작용을 고려하기 위해 우리는 단일 약물을 기반으로 여러 개의 약물 조합의 위험을 예측하기 위한 2레벨 일반화 선형 혼합 모형을 제안한다. 이는 약물의 조합에 대한 부분이 하나 존재하고, 또 하나의 조합은 기본 약물을 기반으로 묶인 집단에 의해 약물의 변동성이 존재하는 것을 고려하여, 이에 대한 경우에 약물 부작용을 적절히 예측하기 위한 모형이다. 따라서 본 논문에서는 기본이 되는 약물 단위로 그룹화하여 집단 간의 변동을 추정하고자 했다. 우리는 이러한 특성을 반영하지 않은 모형은 위양성을 증가시키는 것을 제시하고자 했다. 시뮬레이션 과정을 통해 우리의 2레벨 일반화 선형 혼합 모형이 기존 혼합 약물 반응 모형에 비해 성능이 우수한 것을 살펴볼 수 있었고, 실제 공통데이터 모델에서 간독성에 적용하여 임상적으로 유의한 조합을 실제 시그널로 잘 예측하는지를 비교하여 결과를 제시하고자 하였다.

국내 신약 drug lag 와 임상 개발전략의 연관성 분석 (2012~2019 년 국내 허가신약 중심으로)

신한재 성균관대학교 일반대학원 2021 국내석사

Although there have been a few studies on drug lag in Korea, result of studies were limited that factorial analysis on clinical development factors could not be done. It is important to focus on clinical development strategy to analyze the association between clinical development and drug lag from early development stage to shorten drug lag. Using publicly disclosed review report in MFDS website, data was collected for variables including the basic characteristics of drugs such as ‘drug classification (synthetic drugs/biologics)’, ‘orphan drugs designation’, ‘origin of development(US/Europe/Japan)’, ‘therapeutic area (anti-cancer, anti-inflammatory drugs/anti-infective drugs/others)’, ‘clinical development strategies(GCT/FT)’, ‘pivotal clinical trial design’, ‘Korean participation in clinical trials before initial approval worldwide’. Based on the collected database, a multiple regression analysis was conducted to analyze the association between independent variables and dependent variables.「 Korean approval lag 」was significantly reduced if sponsor included Korean sites for clinical trials before the first drug approval worldwide (β=-0.447; p<0.001). In addition, anti-infective drugs (β=-0.399, p=0.003) and anti-cancer drugs and immuno-modulatory drugs (β =-0.287, p=0.020) had significantly shorter「 Korean approval lag 」compared to other drugs. In stepwise analysis, pivotal trials in 'placebo-controlled ' design, which were not significant related to independent variables in the previous analysis with entire variables, significantly increased the 「 Korean approval lag 」 compared to other pivotal trial design(β=-0.447; p<0.001). In Korea, the drug with ‘placebo-control design’ as a pivotal trial had the longest 「 Korean approval lag 」(573.0 months). In addition, in ‘active-control study’, the 「 Korean approval lag 」 for 'superiority' design was 336.0 months, 48 months shorter than the 「 Korean approval lag 」for 'Non-inferiority' design. Additional analysis was performed with the results of the time of Korean clinical participation. The 「 Korean approval lag 」was rather shorter when Korea participated global clinical trial in phase II trials rather than in phase III trials. Factors such as drug characteristics (biologics/synthetic drugs), orphan drug designation and origin countries (US/Europe/Japan) showed no significant association with independent variables. As anti-cancer drugs, AIDS drugs, anti-infective drugs that pose a life-threatening risk are possible to get exemption from the bridge data submission, 「 Korean approval lag 」was shorter as foreign clinical data also can be used for approval. It was confirmed that if Korea participates in global clinical trials before first drug approval worldwide, it significantly expedited its domestic approval. If sponsor considers venturing into the Korean after marketing authorization was obtained from foreign regulatory authorities, the introduction of drugs can be delayed as the decision on conducting bridging trial for generating required bridging data requires additional time & cost. In terms of design of clinical trials, it was also suggested that introduction of drugs in Korea was generally faster if regulatory package was submitted with ‘active-controlled’ pivotal trial rather than ‘placebo-controlled’. Moreover, it is important to provide pharmaceutical companies with specific guidance on bridging data requirement & assessment for sponsors to plan regulatory strategies in early development. 국내 Drug lag 에 관련한 연구가 기존에도 있었지만 임상 개발 요인을 고려한 요인 분석이 이루어지지 못했다는 한계점이 있었다. 임상개발전략에 초점을 맞추어 Drug lag 와 임상개발과의 연관성을 분석하고 약품 개발단계에서 조기에 허가 기간을 단축하기 위한 임상개발전략을 계획하는 것이 중요하다. 식약처 공개 자료원인 의약품 허가, 심사 결과 공개를 통하여 독립변수인 ‘의약품분류(생물의약품/합성의약품)’, ‘희귀의약품 여부(희귀의약품/그 외)’, ‘개발국가(미국/유럽/일본)’, ‘치료영역(항종양제 및 면역조절제/항감염제/그 외)’ 등의 의약품 기본 특성과 함께 ‘임상개발 전략’, ‘Pivotal 임상시험 디자인’, ‘최초 허가 이전 한국 임상시험 실시 여부’로 임상시험 관련 독립변수에 관한 데이터를 수집하고, 종속변수인 「 한국의 허가 lag 」를 산출하기 위한 약물 시판 허가 승인일에 관한 자료를 수집하였다. 수집된 데이터베이스를 바탕으로 독립변수와 종속변수의 연관성을 분석하기 위해 다중회귀분석을 실시한 결과, 세계최초 허가 전 한국 임상참여를 한 약물의 경우 아닌 약물에 비해 유의하게「 한국의 허가 lag 」가 감소하였다. (β=-0.447, p<0.001). 또한, 항감염제. (β=-0.399, p=0.003)와 항종양제 및 면역조절제(Β=-0.287, p=0.020) 의약품도 다른 의약품에 비해 국내에서의 「 한국의 허가 lag 」가 현저하게 짧았다. 전체 변수를 대상으로 한 분석에서 유의하게 도출되지 않았던 위약-대조(위약-대조) 디자인의 임상시험이 stepwise 분석에서는 다른 임상시험 디자인에 비해서 유의하게 「 한국의 허가 lag 」가 증가하였다(β=-0.447, p<0.001).한 국에서는 위약-대조(위약-대조) 디자인을 pivotal 시험으로 가지는 약물은 「 한국의 허가 lag 」가 573.0 개월로 가장 길었다. 또한 활성 대조(활성-대조) 디자인에서는 '우위성(Superiority) 시험 디자인'의 「 한국의 허가 lag 」가 336.0 개월로 '비열등성(Non-inferiority) 시험 디자인'보다 48 개월 짧았다. 또한 다국가 임상시험을 실시한 약물에 대해 한국 2 상에서 임상시험에 참여한 경우와 3 상에서 참여한 경우를 비교하였으며, 2 상 임상시험에 참여하였을 때 대체로 「 한국의 허가 lag 」가 더 짧았다. 이는 초기 임상단계에서 다국가 임상시험에 참여할수록 약물 도입 속도가 개선됨을 시사한다. 의약품특성(생물의약품/합성의약품), 희귀의약품여부(희귀의약품/그 외), 의약품 개발국가(미국/유럽/일본) 등의 요인은 종속변수와 유의한 연관성을 보이지 않았다. 가교자료 제출 면제 요건에 표준요법 또는 이에 준하는 치료법이 없는 항암제와 에이즈 치료제, 생명에 위협을 주는 감염병 치료제가 속하므로 항감염제, 항종양제 및 면역조절제의경우 대체로 국내 허가를 위해 외국 임상자료를 활용 가능하므로 「 한국의 허가 lag 」가 짧은 것을 알 수 있다. 또한 다국가 임상시험에 한국이 FDA, EMA 등의 세계 최초 국가 허가가 이루어 지기 이전부터 2 상, 3 상 단계에서 조기에 참여할 경우 국내 도입이 유의하게 빨라진다는 것을 확인할 수 있다. 임상시험 디자인에서도 유효성을 증명하기 위한 활성 대조군시험(특히, 우위성을 증명하는 경우) 결과로 심사를 요청할 경우 대체로 국내 약물 도입이 더 빨라지는 경향이 있다는 것을 알 수 있다. 이를 위해선 제약사의 노력만이 아닌 조기에 규제기관에서 요청하는 구비요건을 약물 개발에 반영하기 위해 가교자료 요건과 평가기준에 대한 구체적인 가이드라인을 제공하는 것도 중요함을 시사한다.

Multifunctional Scaffolds for Drug-Delivery Therapies

Borges, Thiago Francisco Costa Carpes Case Western Reserve University ProQuest Dissertat 2016 해외박사(DDOD)

Drug delivery systems are responsible for transporting a pharmaceutical agent into a specific part of the body as needed. Besides finding a matrix that releases the drug properly, it is desirable to choose an appropriate process that helps to enhance the therapeutic effect. Electrospinning, a method that draws micro or nanofibers using an electrical charge, provides an opportunity to improve cell attachment, drug loading, and mass transfer properties. Furthermore, the three-dimension of electrospun scaffolds have high porosity similar to the natural extracellular matrix, making them good candidates for tissue engineering and regenerative medicine. Described herein is the work toward combining drug delivery properties with electrospinning to develop multifunctional scaffolds for three different applications.First, electrospun crosslinked poly(acrylic acid) (PAA) fibers were obtained in order to create a hydrogel for chemoembolization procedures. More specifically, electrospun PAA samples were prepared to deliver doxorubicin and irinotecan via ion-exchange. Since both drugs are used in salt form, the gels were first neutralized prior drug loading, which converted the carboxylic form into sodium carboxylate groups. The performance of the gels regarding drug loading and elution profiles of both drugs were compared to two commercial market leaders: Hepasphere™ and DC Bead®. The amount of drugs incorporated was similar to the commercial brands; however, the higher surface area of scaffolds prolonged the release of the drugs. The antiproliferative activity of doxorubicin and irinotecan was successfully assessed in the A375 melanoma and DLD1 adenocarcinoma cell lines. Additionally, induced clotting assays using human plasma showed that the scaffolds also induced thrombus formation, which make them good candidates for chemoembolic device.Secondly, electrospinning were used to develop a drug-eluting biodegradable graft for covered stents. The grafts were prepared using a tri-layered configuration composed of poly(lactic-co-glycolic acid)/polycaprolatone/ poly(lactic-co-glycolic acid) (PLGA/PCL/PLGA). The drug was successfully incorporated in the abluminal PLGA layer and eluted via diffusion. According to the concentration of sirolimus, a range of 95-81% of drug was delivery in an estimated time of 30 days, similar to commercial drug-eluting stents. The PCL layer was melted to provide additional adhesion properties between the graft and the stent platform. Besides acting as an adhesive for the graft, the molten PCL also helped to control the direction of the elution. The elution profile into the abluminal direction showed to be faster than the luminar direction. The probability of a clot formation was tested in ex-vivo experiments using swine blood. The percentage of thrombus formed on the surface of the electrospun graft matched to bare metal platform, demonstrating the safety of the device.Finally, clotrimazole dissolved in oil was successfully incorporated into in-situ crosslinked gelatin scaffolds using emulsion electrospinning. This strategy aims to produce formulation for intra-vaginal device for treatment fungal infections. The final scaffold had a morphology of fibers with oil droplets of drug. The elution profile presented a burst release of approximately 90% of clotrimazole in 20 min, followed by the release of the remained drug in 90 min. The antifungal activity was demonstrated in vitro against Aspergillus fumigatus in samples with dosage of 2.5 and 5.0 mg of clotrimazole per mL of oil. Although no significant difference was observed in the antifungal activity when compared with gelatin gels loaded with the same concentration of clotrimazole, the scaffolds absorbed 4x faster water than gels, which may lead to a faster and stronger mucoadhesion property.

Design and development of nanomaterials for biomolecular detection and cancer therapy

아룬쿠마르 인하대학교 대학원 2017 국내박사

Recent decades researchers are more concern over the utilization of nanomaterial for biological application. The aim of my thesis work is to develop nanomaterials and its composite for Biosensing (Part 1) and Drug Delivery (Part 2) application. The biosensor can detect biological analyte from the different environment. These analytes can be any of the organic molecules or microorganisms. Due to the excellent conductivity and biocompatibility graphene is widely studied for electrochemical biosensors. In this thesis, I am presenting a biosensor platform using graphene/(protein, metal oxide) composite. Graphene oxide is an oxidized form of graphene with a lot of oxygen group on the surface. In my work, I tailored Protamine (peptide) on the surface of the graphene oxide and used it for heparin sensing (chapter 1.1). This approach provides an excellent sensing property with the broad range of heparin detection. Due to the incorporation of a biological molecule (peptide), the stability of this platform is less. In order solve this, metal oxides used as an intermediate in the biosensor. In this thesis, I demonstrated non-enzymatic (nickel oxide as an intermediate) electrochemical sensor for sensing cholesterol (chapter 1.2). This platform provides higher stability and quick response to sensing of cholesterol. In my second part of my thesis, I utilized polymeric nanocarriers as a drug delivery system (Part 2). This system can provide sustained drug release along with the other unique characteristics (bioimaging, ROS). Now a day, researcher giving so much attention to the porous materials such as activated carbons, zeolite, metal-organic frameworks (MOFs). Some of these materials have used in controlled drug delivery system to avoid the ‘burst release.' In this thesis, I utilized Metal-organic frameworks for anticancer drug delivery system (Chapter 2.1). NMOF was coated with PEG to retain the colloidal stability and bioavailability. This as-synthesized NMOF/PEG showed excellent biocompatibility. They also exhibited high drug loading (30 at.% of DOX) due to the presence of NH2 (MOF) and (OH) in PEG. The interaction of NMOF/PEG with the breast cancer cell line was analyzed using BIO-TEM analysis, laser confocal imaging. This NMOF/PEG also utilized to produce ROS in cancer cells upon exposure to the visible light irradiation. This combination of chemo- and ROS - therapy showed excellent efficiency over killing the cancer cells (MCF-7). The hydrothermal stability of this MOF is less due to the presence of the organic group in this structure. To obtain advanced cancer treatments like senescence, we need highly stable nanocarrier. Covalent organic polymers made of covalent bonding with excellent stability, a high electron cloud in the network. I utilized this covalent triazine polymer for increasing senescence activity in cancer cell line (Chapter 2.2). CTP synthesized via the Friedel-Crafts reaction with organized porosity, large surface area. The high electron cloud in this network is used to incorporate less soluble drugs. The NCTP exhibited excellent dispersibility in physiological solution while maintaining its chemical structure and the porosity of the material. NCTP-DOX showed prolonged drug release for over (over 50h), and in acidic pH the drug release was high. The potency of NCTP on cellular senescence confirmed by the expression of senescence-associated marker proteins p53 and p21. These results suggest that NCTP can use as a new platform for imaging and drug delivery, which may find potential applications in diagnosis and therapy. Some of the polymers are unique due to their biomimetic nature. PAMAM is one of those, which capable of binding with the targeted molecules. Poly(amidoamine) (PAMAM) is widely studied for cancer drug delivery system because it is capable of binding to the drugs and proteins through covalent, noncovalent, hydrophobic and hydrophilic interactions (Chapter 2.3). Poly(amidoamine)/5-fluorouracil (PAMAM/5-FU) was prepared and used as a conjugate system for delivering drugs to target E6 and E7 oncoproteins, which are predominant in cervical cancers (Chapter-2). Specifically, molecular docking analysis was used to investigate the interaction between the PAMAM/5-FU and E6/E7 oncoproteins, which showed that the PAMAM/5-FU conjugate had a higher affinity to the oncoprotein than for 5-FU. Different generations of PAMAM dendrimers (0.5G, 1.0G, 1.5G, 2.0G, and 2.5G) were synthesized, characterized and tested as drug carriers for 5-FU. PAMAM/5-FU showed increased toxicity on the cancer cells than the 5-FU. Laser confocal imaging and western blot for tumor suppressor proteins pRb and p53 were used to confirm the interaction of PAMAM/5-FU with E6/E7 oncoproteins. Hematological analysis of PAMAM/5-FU using mice (BALB/c female) with cervical cancer confirmed the less toxic nature of this material. Based on these results, the developed PAMAM/5-FU conjugate is a potential candidate for the treatment of cervical cancer.

Analysis of drug approval status and drug lags in South Korea and Vietnam : application of statin drugs

Kwon, Yongmin Sungkyunkwan university 2020 국내석사

As the Vietnamese population’s average life expectancy is becoming higher so that the population is being exposed to more chronic diseases, uses and prescriptions in statin drugs are also increasing. Considering the exponential growth in Vietnam’s both economy and its pharmaceutical market, analysis in statin drug approval status and drug lags of Vietnam would be crucial for establishment of an ideal export strategy of South Korean statin drugs. Comparison studies on pharmaceutical regulatory systems and drug lags are prevalently established; however, approval status of specific drugs and features in drug approval have not been systematically investigated. In this study, a thorough analysis in Korean and Vietnamese statin drug approval status was fully investigated by using database from Ministry of Food and Drug Safety (MFDS) and the Drug Administration of Vietnam (DAV). Approved statins that were examined in the study were atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, and fluvastatin from 1990 to 2020. Various categories in approval status were analyzed, which included the number of approved statins, most approved statins, annual statin approval status, and others. After approval dates were analyzed, a drug lag analysis among statins was conducted. Types of drug lag analysis included global approval lag, drug lag between Korea and Vietnam, and drug lags of first approved brand name statins in each country. For purpose of this study, only relative drug lag was calculated, by assessing the date difference between the first global approval date and the first approval date of Korea or Vietnam. The number of statin drug approvals was 1.9 times bigger in Korea than Vietnam. In Korea, rosuvastatin (740, 48.7%) occupied the highest proportion whereas in Vietnam, atorvastatin (361, 44.3%) showed the highest proportion. Data indicated that Vietnam has a higher average value of annual approval rate. However, graphs depict that statin approval in Korea are continuously increasing while approval rate stays in range of 55 – 111. Global approval lag for statins was 17.3 months in Korea and 194.8 months in Vietnam. Not only in average value, but also drug approval lag in all statins was found longer in Vietnam than Korea. Data showed Vietnam first approved alternative statins in pravastatin, lovastatin, and fluvastatin, rather than first globally approved statins. 베트남의 평균 수명이 증가함에 따라 베트남 인구는 만성 질환에 위험에 노출되며 스타틴 약물의 사용 및 처방 또한 급증하는 추세이다. 베트남의 경제적 성장과 제약 시장의 기하급수적인 발전 속도를 고려 하였을 때, 국내 스타틴 의약품의 이상적인 수출 전략을 수립하는 것은 국내 제약산업의 성장에 큰 도움이 될것이다. 국내 스타틴 의약품 수출 가능성을 위하여, 베트남과 국내 스타틴 의약품 허가 현황 및 drug lag에 대한 분석을 하였다. 본 연구에서는 두 국가의 규제기관인 한국 식품의약품안전처 (MFDS)와 베트남 Drug Administration of Vietnam (DAV)의 데이터베이스가 참고되었다. 연구에서 조사된 스타틴은 아토르바스타틴, 로수바스타틴, 심바스타틴, 프라바스타틴, 로바스타틴, 플루바스타틴 총 6개의 스타틴이다. 허가 현황과 관련된 분석 방법은 승인된 스타틴의 총 개수, 가장 많이 승인된 스타틴, 연간 스타틴 허가율 등이 있었다. 스타틴 drug lag에 대한 분석 방법은 상대적 durg lag 분석을 기반으로 하였으며, 분석 항목은 global approval lag, 한국과 베트남 사이 drug lag, 그리고 최초 승인 스타틴의 허가 상태가 있었다. 연구 결과에 따르면, 1990년부터 2020년까지 총 허가된 스타틴은 한국이 베트남에 비해 약 1.9배의 양을 차지하였다. 국내에서는 로수바스타틴 (740, 48.7%)이 가장 많이 허가되었고 베트남에서는 아토르바스타틴 (361, 44.3%)의 허가가 가장 잦았다. 총 6개의 스타틴에 대해서 모두 베트남의 drug lag가 국내 drug lag에 비해 더 높다는 결과가 있었으며, global approval lag의 평균값은 국내에서는 17.3개월, 베트남에서는 194.8개월로 분석되었다.

효과적인 암치료를 위한 리소좀 약물전달체 기반 암 질환 선택적 치료제 개발

최우일 전북대학교 일반대학원 2020 국내석사

As the life span of human increases, the biggest concern facing humanity is the pursuit of a healthy life through overcoming incurable diseases such as cancer. Conventional cancer treatments are performed through radiotherapy or chemotherapy. These therapies are non-specific therapies with low therapeutic efficiency and significant side effects. Selective therapeutics is one of spotlighted field in pharmaceutical industry. Target specific drug delivery is main concept of this strategy. This research is also concerned for studying to improve efficiency for drug delivery by target selectivity, suitability and economical production of drug delivery system based on bio-derived materials. The budding yeast, Saccharomyces cerevisiae, is one of the best studied eukaryotic models. S. cerevisiae has prominent vacuole correspond to lysosome. Yeast vacuoles functionally as lysosomes contain acidic hydrolase with acidic lumen in the rage of pH 4-6 and single 7-10 nm phospholipid bilayer membrane. Enzyme system of vacuoles indicate anti cancerous effect. And vacuolar membrane can be manipulated for target specific attachment. In this study, efficacy of vacuoles drug delivery systems to acute myeloid leukemia (AML) cells was evaluated with down-sizing and targeting system. Size control of vacuoles can be constructed through deletion of YPT7 protein that related with inter-vacuolar fusion. In vacuolar fusion pathway, partial deletion of YPT7 cause failure of intermembrane docking, and then SNAREs chaperoning cannot be occurred. After construction of down-sizing recombinant yeast, we confirmed size distribution and anti-cancer effects with encapsulated daunorubicin. For selective therapeutics, targeting peptide to AML was decorated on vacuolar membrane. Novel peptide binding AML cell lines was chosen from reported technical paper, and it was expressed on vacuolar outer membrane. Selectivity of peptide and cellular uptake was confirmed. Finally, enhanced drug delivery of vacuolar carriers was founded. In summary, these studies demonstrate that yeast derived vacuoles with nano-sizing and targeting ability can be used potential bio-based drug delivery system for cancer therapy. Also, this vacuole system provides potential strategies and platform for disease selective treatment through simple manipulation of vacuole. Keywords: Drug delivery system; Vacuole; Lysosome; Selective therapeutics; Cancer therapy; Acute myeloid leukemia

(A) study of goal-directed molecular optimization for AI-based drug design

최종환 Graduate School, Yonsei University 2023 국내박사

Drug design is the process of discovering new drug candidates based on the understanding of diseases and information about target biomolecules associated with the diseases. New drug candidates should be able to bind to target biomolecules, such as proteins, and induce functional changes to treat the diseases. Discovering molecular structures with these chemical properties is the main objective of drug design research. To efficiently uncover such candidates, computer-aided drug design techniques and virtual screening methods are widely used, with deep learning-based goal-directed molecular optimization techniques attracting significant attention. This dissertation covers research on variational autoencoder models and optimization techniques for effective drug design. Two molecular structure generation techniques are proposed. The first is a multi-objective molecular generation model using self-critical sequence training, and the second is a structure-constrained molecular generation model based on reinforcement and metric learning. Through various benchmark experiments, it has been confirmed that the proposed models outperform existing molecular structure generation techniques. Additionally, further experiments have demonstrated the importance of the unique features possessed by each model. Moreover, the proposed molecular structure generation techniques have been shown to successfully discover hit compounds that exhibit the effect of removing aging cells, such as navitoclax, and identify lead molecules overcoming the drug resistance problem of sorafenib, one of the anticancer drugs for hepatocellular carcinoma. Based on these results, it is expected that the techniques proposed in this dissertation will enable effective research in novel drug development and be valuable in advancing research on new drug design and optimization techniques. 약물 설계란 질병에 대한 이해와 질병에 관련된 표적 생체분자의 정보를 바탕으로 새로운 약물 후보물질을 발견하는 과정이다. 신약 후보물질은 인체 내 단백질과 같은 표적 생체분자에 결합하여 질병 치료를 위한 표적의 기능 변화를 일으킬 수 있어야 하며, 이러한 화학적 성질을 갖는 분자 구조를 발견하는 것이 약물 설계 연구의 주된 목표이다. 이러한 후보물질을 효율적으로 발굴하기 위해 컴퓨터를 활용한 약물 설계 기술 및 가상 스크리닝 방법이 널리 활용되고 있으며, 그 중에서도 심층학습을 활용한 목표 지향적 분자 최적화 기법이 큰 주목을 받고 있는 실정이다. 본 학위논문에서는 효과적인 약물 설계를 위한 변분오토인코더 모델 및 최적화 기법에 대한 연구내용을 다룬다. 총 두 가지의 분자구조 생성 기법을 제안하고 있으며, 첫번째는 자기비판적 문자열 훈련 기법을 이용한 다중 성질 제약 분자구조 생성모델이고, 두번째는 강화학습 및 메트릭학습을 활용한 구조 제약 분자 생성모델이다. 여러가지 벤치마크 실험들을 수행하여 제안하는 모델들이 기존의 분자구조 생성 기법들보다 성능적으로 우수함을 확인하였으며, 또한 추가적인 실험을 통해 각 모델이 가진 고유한 기능들의 중요성을 입증하였다. 그리고 제안하는 분자구조 생성 기법들을 이용하여 나비토클락스와 같이 노화세포 제거효과를 나타내는 유효물질 및 간암치료제 중 하나인 소라페닙의 약물저항성을 극복하는 선도물질을 발굴할 수 있음을 보여준다. 이러한 결과들에 의거하여, 본 학위논문에서 제안하는 기법들이 효과적인 신약 개발 연구를 가능케 하고, 나아가 새로운 약물 설계 및 최적화 기술 연구에 유용하게 활용될 수 있을 것으로 기대한다.

코어/쇌 구조를 가지는 나노입자의 형성과 약물전달 체계로의 응용에 관한 연구

오근상 한남대학교 일반대학원 2010 국내박사

This thesis is related to formation of core/shell nanoparticles and their application as a drug delivery system. In the first parts, there is described literature review of characteristics and trends of research of core/shell nanoparticles compared with the liposomal formulations. In addition, Recent trends of research of multi-functional delivery system with diagnosis and therapy was shown for extensive in vivo application of nanoparticles. The second parts respectively contained on the 1) core/shell nanoparticles as a hydrophobic drug delivery system for cancer therapy, 2) core/shell nanoparticles as a protein drug delivery system for myocardial infarction therapy and 3) temperature-responsible core/shell nanoparticles/hydrogel composites for the effective regeneration of ischemic heart as a study on the core/shell nanoparticles with lecithin lipid cores and their application as a drug delivery system . The third parts respectively contained on the 1) preparation and characterizations of core/shell nanoparticles for Cancer therapy; temperature-induced phase transition and 2) a study on the surface modification of paclitaxel-loaded core/shell nanoparticles for effective tumor targeting as a study on the self-assembled core/shell nanoparticles in solvent-free Process. The third part respectively contained on the in vivo characteristics and a formation of gold-deposited super-paramagnetic iron oxide nanoparticles (SPION) for tumor imaging In the second part, a novel preparation method for core/shell nanoparticles with a drug-loaded lipid core developed. The lipid core is composed of lecithin and a drug, and the polymeric shell is composed of Pluronics (poly(ethylene oxide)-poly (propylene oxide)-poly(ethylene oxide) triblock copolymer, F-127). For the formation of stabilized core/shell nanoparticles, freeze-drying was performed in the presence of trehalose used as a cryoprotectant. Cryogenic transmittance electron microscopy (cryo-TEM), and a particle size analyzer were used to observe the formation of the stabilized core/shell nanoparticles. For the formation of polymeric shell composed of F-127, the freeze-drying was performed. The spherical supramolecular structure was disintegrated during the freeze-drying process. This phenomenon is usually found in the freeze-drying of a protein solution. With freeze-drying, the protein activity is significantly decreased due to the disintegration of the supramolecular structure. However, adding 5 wt % trehalose to the F-127 aqueous solution, the freeze-drying was performed to induce the formation of polymeric shell. The nanoparticles with diameters of 267.4 nm were observed, indicating the minimal disintegration of the spherical supramolecular structure. For the application of the core/shell nanoparticles as a drug carrier, paclitaxel, a potent anticancer drug, was loaded into the core/shell nanoparticles, and the drug loading amount and the drug release pattern were observed. Without the polymeric shell (0 wt % of F-127 aqueous solution), a significant burst effect was observed that resulted in the precipitation of paclitaxel into the release medium. With the formation of a polymeric shell, the paclitaxel released from the lipid phase was diffused through the polymeric shell (F-127 phase), resulting in a significant decrease in the burst effect and an almost zero-order release pattern was observed with nanoparticles formed from the 10 wt % F-127 aqueous solution. Also, cy5.5/PTX-loaded core/shell nanoparticles in tumor bearing mice (10 mg/kg) showed a strong NIR fluorescence signal in the tumor area within 1 h of injection, indicating rapid nanoparticles circulation. I could distinguish tumors from surrounding background tissue at 1 h post-injection, with a maximum NIR signal at 9 hours post-injection and continued fluorescence for up to 48 hours. A further study has been performed to evaluate the core/shell nanoparticles as a protein delivery system. VEGF (vascular endothelial growth factor) was used as a model drug. The lecithin forms an anionic nanolipid, and it can form an ionic complex with positively charged molecules. the complex formation between lecithin and protein drugs can be induced by choosing a proper protein with a high isoelectric point (>8). In the freeze-drying process, trehalose used as a cryoprotectant plays two roles. The first is to preserve the core/shell structure of nanoparticles, and the other is to preserve the activity of loaded protein drugs. In the case of VEGF-loaded lecithin nanolipids without F-127, the average diameter of the nanolipids was 96.8 ± 3.1 nm, and the surface charge from zeta potential measurements was -40.4 ± 2.1 mV. With the formation of core/shell nanoparticles, an anionic character of lecithin nanolipids used as core was diminished due to the presence of the polymeric shell. In the case of 0 of the lecithin/F-127 (w/w) ratio, the freeze-dried mixture of VEGF and F-127, which was resuspended in release medium for the release experiment, was used to observe the interaction between VEGF and F-127. A significant burst was observed, releasing about 85% of the initial loading amount during a 9-day period. These results indicate that the sustained release pattern was accomplished with the formation of the core/shell nanoparticles and the electrostatic interactions between lecithin cores, and the loaded proteins play an important role in determining the release pattern. Histological analysis revealed that the core/shell nanoparticles could regenerate heart muscle from fibrous tissue formation in the infarcted area. In the MI group, the wall of the infarcted area was thinner, and this area was stained blue, indicating the formation of fibrous tissue. In the VEGF-loaded NPs-injected group, the wall thickness of the infarcted area was similar to the non-infarcted area. The Quantitative analysis of the infarct sizes of the MI and VEGF-loaded NPs-injected groups were 41±11.5 and 13±6%, resp 본 논문의 코어/쇌 나노입자의 형성과 약물전달 체계로의 응용에 관한 내용이다. 논문은 총 4부로 나누어져 있으며, 1부에서는 리포좀 제형과 비교하여 코어/쇌 나노입자의 연구동향과 특성에 관한 전반적인 개요를 서술하였으며, 2부에서는 약물전달체로서 레시틴 지질 코어를 가지는 코어/쇌 나노입자에 관한 연구와 그 응용에 관한 내용으로 1) 항암치료를 위한 소수성 약물전달체로서의 코어/쇌 나노입자, 2) 허지혈성 심질환 치료를 위한 단백질 약물전달체로서의 코어/쇌 나노입자와 3) 허지혈성 심질환의 효과적인 재생을 위한 온도 감응형 코어/쇌 나노입자/하이드로젤 복합체에 관한 내용을 각각 서술하였다. 3부에서는 용매를 사용하지 않는 공정 하에 제조된 자기집합형 코어/쇌 나노입자에 관한 내용으로 1) 온도 유도형 상전이 현상을 이용하여 항암치료를 위한 코어/쇌 나노입자의 제조 및 특성과 2) 효과적인 종양 표적화를 위한 파클리탁셀이 함유된 코어/쇌 나노입자의 표면 개질화에 관한 내용을 각각 서술하였다. 4부에서는 종양 영상을 위한 금이 개질화된 초상자성 산화철 나노입자의 형성과 in vivo 특성에 관한 내용을 서술하였다. 2부에서는 약물이 함유된 지질을 갖는 코어/쇌 나노입자의 제조방법을 고안하였고, 특성을 알아보았다. 지질코어는 약물과 레시틴 (lecithin)으로 구성되어 있으며, 고분자 쇌은 PEO와 PPO 삼중공중합체로 구성된 pluronic으로 구성되었다. 안정한 코어/쇌 나노입자의 형성을 위해, 동결건조는 동결건조보호제로서 트레할로스 존재 하에 수행하였다. Cryo-TEM과 입자 사이즈 분석으로 안정화된 코어/쇌 나노 입자를 관찰하였다. F-127로 구성된 고분자 쇌을 형성하기 위해 동결건조를 수행하였는데, 구형의 초분자 구조 (supermolecular structure)가 동결건조 과정에서 붕괴되는 것을 관찰하였다. 이 현상은 일반적으로 단백질 용액의 동결건조에서 나타난다. 동결건조의 수행으로 단백질의 활성도는 초분자 구조의 붕괴로 인해 감소하게 된다. 이것은 천연적으로 발생하는 osmolyte인 트레할로스가 동결-건조 상태에서 단백질의 효과적인 안정제로, 용액 속에 있는 효소의 활성 유지에 도움을 준다는 보고가 있다. F-127 용액에 5wt% 트레할로스를 첨가하여 고분자 쇌의 형성을 유도하였다. 구형의 superamolecular structure의 붕괴를 최소화하여 267.4nm의 입자가 관찰하였다. 항암제로 사용되는 Paclitaxel을 약물 전달체로 나노 입자에 적용시키기 위해 나노 입자 안에 봉입하였다. 약물 방출 속도를 polymeric shell의 두께 변화에 따라 관찰하였다. polymeric shell이 없을 경우(0wt% F-127수용액) 예상대로 burst effect로 인하여 release medium에서 paclitaxel이 침전되는 결과로 관찰할 수 있었다. polymeric shell이 형성되면 방출 속도는 감소하였고, zero-order 방출 패턴은 10wt% F-127 수용액으로 형성된 나노 입자로 관찰할 수 있었다. 또한 종양유발 마우스들에 형광 물질인 Cy 5.5/PTX가 함유된 코어/쇌 나노입자를 정맥주사 했을 경우 투여 후 1시간 이내에 종양 부위에 형광신호가 관찰되었다. 투여 후 9시가쐴서 최대 종양 주위에 축적되었으며, 48시간 이상 형광이 지속되는 것을 관찰 할 수 있었다. 다음 연구로 단백질 약물 전달체로서 코어/쇌 나노입자를 평가해 보았다. 모델 단백질 약물로 VEGF (vascular endothelial growth factor, isoelectric point 8.4)를 사용하였다. 레시틴은 음이온성 나노지질을 형성하고, 이것은 양이온성 물질과 이온성 복합체를 형성할 수 있다. 레시틴과 단백질 약물간의 복합체 형성은 높은 등전점 (isoelectric point)을 갖는 단백질을 선택함으로서 유도할 수 있다. F-127 없을 경우 VEGF가 함유된 레시틴 지질의 경우 사이즈는 96.8 ± 3.1 nm이고, 표면전하는 -40.4 ± 2.1 mV으로 관찰되었다. 코어/쇌 나노입자를 형성함에 따라 레시틴 나노지질의 음이온성 특성은 고분자 쇌의 존재에 의해 감소됨을 확인하였다. 동결-건조 과정에서 동결보호제로 사용된 트레할로스는 두 가지 역할을 하게된다. 하나는 나노입자의 코어/쇌 구조를 유지하는 것이며, 다른 하나는 함유된 단백질 약물의 활성도을 유지시키는 것이다. VEGF와 F-127간의 결합을 관찰하기 위해 동결-건조된 VEGF/F-127 화합물을 방출 용액에 분산하였다. 약 85%의 초기 과대 방출이 관찰되었다. 이것은 지속형 방출을 위해서는 레시틴 코어와 함유된 단백질 약물간의 정전기적 인력과 코어/쇌 나노입자를 형성함으로서 달성될 수 있다는 중요한 역할임을 알 수 있었다. 조직학적 분석을 통해 코어/쇌 나노입자가 허지혈성 부위에서 섬유화 조직형성을 통해 심장 복원을 확인하였다. 허지혈성 그룹 (MI group)에서 허지혈성 부위의 벽은 더 얇아졌으며 섬유화된 조직 형성을 통해 알 수 있었다. VEGF가 함유된 나노입자를 투여한 그룹에서 허지혈성 부위의 벽 두께는 정상그룹과 유사했다. 허지혈성과 VEGF가 함유된 나노입자를 투여한 그룹들의 허지혈된 크기의 정량분석에서 각각 41±11.5 와 13±6%를 나타내었다. VEGF가 함유된 나노입자를 투여한 그룹의 허지혈된 크기는 51.8% 감소하였고, 허지혈된 부위의 벽 두께는 허지혈성 그룹보다 VEGF가 함유된 나노입자를 투여한 그룹이 30

심혈관질환치료제 중 항혈소판제인 cilostazol과 sarpogrelate의 in vitro와 in vivo에서의 약물상호작용 평가 : Evaluation of in vitro and in vivo drug-drug interactions of various cardiovascular disease drugs focused on antiplatelet agents, cilostazol and sarpogrelate : Asses

배수현 가톨릭대학교 일반대학원 2014 국내박사

Cardiovascular disease is one of the leading causes of death worldwide. About one third of deaths in the world (~17.3 million) are caused by cardiovascular dysfunctions each year. By 2020, cardiac disease and stroke will become the leading cause of both death and disability worldwide, with the number of fatalities projected to increase to over 20 million a year and by 2030 to over 24 million a year. Eighty percent of all global cardiovascular disease related deaths occurs in a low and middle income countries. Furthermore, there are many types of cardiovascular diseases which are inter-linked with each other. For example, high cholesterol causes atherosclerosis, hypertension linked with stroke, myocardial infarction, heart failure and peripheral arterial disease. In addition, non-cardiovascular diseases are also strongly related with the cardiac complications. Obesity, diabetic mellitus, periodontal infections and depression promotes the development of cardiovascular risk factors. In result, cardiovascular drugs alone are not sufficient to cure the cardiac patients. Thereby multiple disease states create the demand for multiple drug therapy. In this case combination drugs are used in the USA and have been permitted for use in parts of Europe, Asia, and Latin America. Thus, multiple drug therapy is widely used for the treatment of problems including hypertension, ischemic heart disease and heart failure together. Consequently, multiple drug therapy creates the concept of ‘poly pharmacy’. However, poly pharmacy is not always efficacious and safe owing to drug-drug interactions, reduced patient compliance, and medication errors. The aim of the present study is to conduct in vitro/in vivo drug-drug interactions for safe use of cardiovascular drugs after analyzing the prescription pattern and poly pharmacy in a comprehensive manner using electronic medical record (EMR) data system. For this, we conducted a retrospective observational study using EMR data was introduced on 1995 of Ajou University Medical Center. At first, we found out two drugs, cilostazol and sarpogrelate, that the potential of clinical drug-drug interactions have not been evaluated. Both cilostazol (Pletal®; 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxyl]-3,4-dihydro-2(1H)-quinoline), a specific inhibitor of cyclic AMP phosphodiesterase III in platelets and smooth muscle, and sarpogrelate ((R,S)-1-{2-[2-(3-methoxyphenyl)ethyl]phenoxy}-3-(dimethylamino)-2-propyl hydrogen succinate), a highly specific 5-HT2A receptor antagonist, are widely used for chronic peripheral arterial occlusive disease in Japan, China, and South Korea. Cilostazol is metabolized by CYP3A4/5 and CYP2C19 and sarpogrelate is formed M-1 by hydrolysis of the succinate ester portion from sarpogrelate. However, there had been little information reported for the metabolism-mediated inhibitory effects of cilostazol and sarpogrelate. Here, at first, we investigated in vitro drug interaction studies of both cilostazol and sarpogrelate as predators. The reversible inhibitory effects and time-dependent inactivation effects of cilostazol and its main metabolites, 3,4-dehydrocilostazol (OPC-13015) and 4’-trans-hydroxycilostazol (OPC-13213) on CYP isoforms were conducted using human liver microsomes. Evaluations of inductive effect using human hepatocytes were also conducted. In the same manner, sarpogrelate and M-1 were determined their inhibitory effects on CYP isoforms using human liver microsomes. Secondly, based on in vitro data and many known physiochemical properties of cilostazol and sarpogrelate, possible drug-drug interactions in vivo were predicted using Simcyp® program (version 12 release 2). Cilostazol-known CYP3A4/5 substrates and sarpogrelate-known CYP2D6 substrate, metoprolol, interactions were predicted and evaluated the possibilities of drug-drug interactions. Finally, conducting drug-drug interaction studies of sarpogrelate-metoprolol in vivo, we confirmed the susceptibility of simulation data that are made up using in vitro data and discussed the possibilities of drug interactions with concomitant drugs of sarpogrelate. The concentration profiles, pharmacokinetic properties, and pharmacodynamic effect (blood pressure) of metoprolol (once at 7 day) were evaluated with or without sarpogrelate (single dose or multiple doses). Cilostazol and OPC-13015 exerted time-dependent inhibition towards CYP3A4/5 with KI of 12.1 uM and 1.31 uM and kinact of 0.0365 min-1 and 0.0255 min-1, respectively. 13.0% of cilostazol and 12.7% OPC-13015 were bound to human liver microsomes and no inductive effects were measured on CYP activity. Sarpogrelate potently and selectively inhibited CYP2D6-mediated dextromethorphan O-demethylation with an IC50 (Ki) value of 3.05 uM (1.24 uM), in a competitive manner. M-1 also markedly inhibited CYP2D6 activity; its inhibitory effect with an IC50 (Ki) value of 0.179 uM (0.120 uM) was more potent than that of sarpogrelate, and was similarly potent as quinidine (Ki, 0.129 uM), a well-known typical CYP2D6 inhibitor. In addition, sarpogrelate and M-1 strongly inhibited both CYP2D6-catalyzed bufuralol 1’-hydroxylation and metoprolol α-hydroxylation activities. However, upon 30-min preincubation of human liver microsomes with sarpogrelate or M-1 in the presence of NADPH, no obvious shift in IC50 was observed in terms of inhibition of the nine CYP activities, suggesting that sarpogrelate and M-1 are not time-dependent inactivators. Based on in vitro data, we simulated and prediected clinical drug-drug interactions of cilostazol and sarpogrelate using Simcyp® version 12.0. The Cmaxs and AUCs of alprazolam, midazolam, nifedipine, sildenafil, simvastatin, and triazolam which were all well-known CYP3A substrates were increased with cilostazol compared to those without cilostazol. However, in the case of sarpogrelate that was a potent CYP2D6 inhibitor in vitro, the AUC ratios of metoprolol were 1.23 (1.07–1.49) for single sarpogrelate 심혈관계 질환은 전세계 사망률의 원인 중 중요한 부분을 차지하며, 사망자의 약 3분의 1정도가 매년 심혈관계 질환으로 사망하고 있다. 심혈관계 질환은 복합질환으로 다양한 질환이 복합적으로 발생하는 경우가 많다. 예를 들어, 높은 콜레스테롤 은 죽상동맥경화증과 고지혈증을, 고혈압은 심근경색, 뇌졸증, 심부전, 그리고 말초동맥질환을 일으킬 수 있다. 또한, 당뇨나 비만과 같은 비심혈관계 질환에 의해서도 심혈관계 질환이 합병증으로 나타날 수 있다. 이러한 이유들 때문에, 심혈관계 질병 치료에는 한 가지 약물 사용 보다 복합적인 약물 사용이 필요할 수 있으며, 미국이나 유럽, 아시아, 라틴 아메리카 등에서 많이 사용되고 있다. 이러한 복합적인 약물 사용은 고혈압, 허혈성 심질환, 심부전등에 많이 쓰이고 있으며, 이러한 복합적인 약물 사용을 ‘poly pharmacy’ 라고 한다. ‘poly pharmacy’는 복합적 약물 사용으로 인한 약물상호 작용, 환자의 약물 순응도를 고려해야 한다. 심혈관계 약물의 in vitro와 in vivo에서의 약물상호작용의 안전성을 평가하기 위해 우리는 아주대학교 병원의 최근 15년간의 전자의무기록 중 환자의 처방전 목록을 분석하여 심혈관 질환에 많이 사용되는 임상약물의 치료 현황을 살펴보았다. 그 중, 항혈소판제는 다른약물과의 병용 처방이 많았으며, 특히 스텐팅 시술환자와 관상동맥 및 심근경색의 치료와 예방에 많이 사용되었다. 우리는 항혈소판제 중에서 병용투여로 인한 약물상호작용 가능성은 높지만, 안전한 약물사용을 위한 연구가 많이 이루어지지 않은 cilostazol 과 sarpogrelate에 대한 약물상호작용을 평가하기로 하였다. Cilostazol은 혈소판의 phosphodiesterase III를 저해하여 항혈소판 응집효과를 나타내며, 체내에서 CYP3A4/5와 CYP2C19을 통해 대사되어 활성대사체인 OPC-13015와 OPC-13213을 생성한다. Sarpogrelate는 혈소판과 혈관 평활근에 많이 존재하는 5-HT2A 수용체의 높은 선택적 길항제로 작용하여 항혈소판 응집효과를 나타내며, 체내에서 esterase와 CYP 동효소들에 의해 활성 대사체인 M-1을 생성한다고 알려져있다. 현재까지 cilostazol과 sarpogrelate에 의한 약물상호작용이 잘 알려져 있지 않기 때문에, 우선 in vitro에서 두 약물의 저해제로서의 약물상호작용을 평가하였다. 9가지 CYP 동효소에 대한 가역적 저해능과 비가역적 저해능을 살펴보았으며, cilostazol과 sarpogrelate, 그리고 두 약물의 활성대사체들인 OPC-13015, OPC-13213, M-1에 대해서도 약물상호작용 평가 실험을 수행하였다. 그 결과, cilostazol과 그 활성 대사체인 OPC-13015는 CYP3A4/5에 대해 강력한 비가역적 저해능을 보였으며, 그때의 KI 와 kinact 값은 cilostazol은 각각 12.1 uM 과 0.0365 min-1 이었으며, OPC-13015는 1.31 uM 과 0.0255 min-1 이었다. Sarpogrelate와 활성 대사체인 M-1은 CYP2D6에 대해 강력하고 선택적인 가역적 (경쟁적) 저해능을 보였으며, 이때의 IC50 (Ki) 값은 sarpogrelate는 3.05 uM (1.24 uM), M-1은 0.179 M (0.120 uM)로 M-1이 sarpogrelate 보다 더 강력한 CYP2D6 저해능을 보였다. 또한, cilostazol의 대사효소 유도효과를 평가하기 위한 실험에서 cilostazol은 CYP3A에 대한 유도능을 보이지 않았다. 이러한 in vitro 데이터를 바탕으로 cilostazol과 sarpogrelate의 임상에서의 약물상호 작용을 Simcyp®를 이용하여 예측하였다. Cilostazol과 CYP3A의 기질로 잘 알려진 alprazolam, midazolam, nifedipine, sildenafil, simvastatin, 그리고 triazolam과의 약물상호 작용 가능성을 평가한 결과, cilostazol에 의해 모든 기질의 Cmax ratio와 AUC ratio가 정도는 다르지만, 95% 신뢰구간에서 equivalence 범위인 0.8-1.25를 벗어나 임상에서의 약물상호작용 가능성이 높을 것으로 평가되었다. 하지만, sarpogrelate와 CYP2D6의 잘 알려진 기질인 metoprolol과의 약물상호작용을 예측한 결과, sarpogrelate에 의해 metorpolol의 AUC ratio 가 sarpogrelate 단회 복용하였을 때에는 1.23 (1.07-1.49), 반복 복용하였을 때에는 1.27 (1.07-1.60)로, sarpogrelate의 metoprolol에 대한 임상에서의 약물상호 작용은 ‘약’할 것으로 예측되었다. 실제 9명의 건강한 성인들을 대상으로 임상시험을 통한 sarpogrelate의 metoprolol에 대한 약물상호작용을 평가한 결과, metorpolol의 AUC ratio 가 sarpogrelate 1일 복용하였을 때에는 1.53 (1.09-2.32), 4일 복용하였을 때에는 1.51 (1.04-2.30)로 예측결과와 비슷하였다. 하지만, pharmacodynamic 실험 결과를 살펴보면, sarpogrelate에 의해 metorpolol의 증가된 약물 농도가 심박동, 수축기 혈압, 이완기 혈압을 변화시키지 않았다. 본 연구 결과를 종합해보면, cilostazol과 그 활성 대사체인 OPC-13015는 in vitro에서 CYP3A4/5를 강력하게 비가역적으로 억제하며, simcyp®를 이용하여 예측한 결과, 임상에서 CYP3A4/5의 기질과 병용할 경우, 약물 상호작용을 일으킬 가능성이 있다. 그리고 sarpogrelate와 그 활성 대사체인 M-1은 in vitro에서 CYP2D6에 대해 선택적이고 강력한 경쟁적 저해능을 보이지만, simcyp®를 이용한 임상에서의 약물사호작용을 예측한 결과처럼 실제 임상에서 metoprolol과의 약물상호작용은 크지 않았다. In vitro 결과를 통해 임상에서의 약물상호작용을 예측할 때, simcyp®를 통한 예측은 좋은 도구가 될 수 있으며, 임상에서의 cilostazol과 sarpogrelate를 다른 약

약물-유전체 bipartite network 기반 gene set enrichment analysis를 통한 Drug-Target Score와 Drug-Sensitivity Score의 characterization

이채원 차의과학대학교 일반대학원 2020 국내석사

The hypothesis of this study is “The relationship between target score and sensitivity score of genes for drugs would be independent and exclusive.” To prove this, we conducted drug-target network analysis based on the organicity of live cell. We used FDA-approved 382 drugs which have both protein binding information and microarray experiment data in DSigDB database. Based on drug-gene bipartite network, we analyzed relationship between drug target genes and drug sensitive genes. From analyses, It was turned out that the target score and sensitivity score of genes were in reciprocally independent relationships in their composition, cellular locations, molecular functions, and in biological processes they involved. In addition, similar to yin and yang, we found a symmetric balance between target score and sensitivity score of genes. We think this balance is due to the homeostasis of cells. As we hypothesised, although all microarray experiment data from DSigDB was based on human genes, the composition and characteristics of the target gene and sensitive gene showed mutual exclusiveness. These results indicate that the expression of target gene was barely affected by drug treatments. It could be interpreted that pharmaceutical effect of drug is due to the drug sensitive gene whose expression level is significantly changed by drug treatment rather than the direct action of the target gene. The exclusive relationship, we found on this research, of drug target gene and drug sensitive gene would suggest a new way of drug-gene study design. 본 연구는 약물에 대해 유전자가 갖는 target score와 sensitivity score는 그 정도와 경향이 상이하고, 상호 배타적이며 독립적인 관계를 가질 것이라는 가설을 기반으로 수행되었다. 위와 같은 가설의 입증을 위해 DSigDB 데이터베이스를 사용, 2014년도까지 FDA 승인 된 모든 약물 중 protein binding 정보과 microarray 실험 데이터가 모두 존재하는 382개의 approved drugs를 대상으로 유기성을 갖는 생물체의 drug-gene network 기반 약물의 타겟유전자와 drug sensitive gene의 관계분석 연구를 수행하였다. 그 결과, 약물에 대한 유전자의 target score와 sensitivity score는 유전자의 구성과 세포 내 위치, 분자생물학적 기능 및 관여하는 생물학적 process 모두에서 서로 독립적인 reciprocal distribution을 나타내는 것이 밝혀졌다. 또한, 음과 양 이론과 유사하게 유전자의 target score와 sensitivity score 간에 대칭적 균형을 갖는 것이 확인되었으며, 이러한 균형은 항상성을 유지하고자 하는 세포의 특징으로부터 기인 한 것이라는 결론을 내릴 수 있었다. DSigDB에서 보유하고 있는 microarray 실험은 사람의 거의 모든 유전자를 대상으로 하였으나, 분석에서 보여진 약물의 타겟유전자와 drug sensitive gene의 구성은 상호 배타적인 특징을 나타내었다. 이러한 결과는 타겟 유전자의 발현량은 약물 투여에 의해 크게 영향을 받지 않는다는 것을 의미하며, 이것은 곧 약물이 작용하는 약리학적 효과는 약물이 직접적으로 결합하는 타겟단백질 자체에 의한 것이기 보다, 약물 투여에 의해 그 발현량이 현저히 변화하는 drug sensitive gene에 의한 것이라는 해석으로 이어질 수 있다. 본 연구에서 발견 된 약물 타겟유전자와 dug sensitive gene의 상호 배타적인 관계를 이용한다면 약물-유전자 연구에 있어서 새로운 연구방법론을 제시할 수 있을 것이다.