세포침투 인산화 STAT3 항체 개발

함영혜 충북대학교 2017 국내석사

Inhibitory effects of CHI3L1 inhibitor compound K284 through inhibition of lactoferrin on atopic dermatitis

전성희 충북대학교 2021 국내석사

Atopic dermatitis (AD) is a chronic inflammatory skin disease with itching and dry skin as the main symptoms. It begins mainly in infancy and is often accompanied by respiratory atopic diseases such as allergic rhinitis and asthma as it grows. Chitinase 3-like 1 (CHI3L1) protein is a glycoprotein found in diseases characterized by chronic inflammation. It is also known to cause inflammation in the progression of allergic diseases. However, the role of CHI3L1 in AD is not clearly seen. Therefore, in this study, the anti-dermatitis effect of 2-({3-[2-(1-Cyclohexen-1-yl)ethyl]-6,7-dimethoxy-4-oxo-3,4-dihydro-2-quinazolinyl}sulfanyl)-N-(4-ethylphenyl)butanamide (K284-6111; K284) was investigated. I induced AD with phthalic anhydride (5% PA) in C57BL/6J mice. After induction, the detailed correlation between AD and CHI3L1 with the inhibitory effect of K284-6111 (1 and 2 mg/kg) on ​​AD was investigated. Histological analysis showed that treatment with K284-6111 (K284) inhibited the invasion of inflammatory cells after inducing 5% PA. K284 reduced in tissues the release of the inflammatory cytokines IL-1β, IL-6, IL-4, IL-13, and thymic stromal lymphpoietin (TSLP). In 5% PA-treated skin tissue and TNF-α and IFN-γ-treated (20 ng/mL) HaCaT cells, K284 contributed to the inhibition of the expression of NF-κB activity. I confirmed that CHI3L1 and lactoferrin (LTF) are related in protein association network analysis. The expression of LTF was increased in 5% PA-treated animal models and HaCaT cells induced by TNF-α and IFN-γ. However, it was reduced by the treatment of K284 in in vivo and in vitro models. Inhibition of LTF reduced the expression of inflammatory cytokines in TNF-α and IFN-γ-induced HaCaT cells. In addition, it was confirmed that K284 treatment in the human skin model (RHS) reduced AD development with LTF inhibition. In addition, in the further animal model, LTF antibody treatment suppressed 5% PA-induced AD development. As a result of serum analysis, CHI3L1 and LTF showed a significant correlation in patients with AD. These results showed that K284 may be a promising agent for AD therapy by inhibiting the expression of LTF and CHI3L1. 아토피 성 피부염 (AD)은 가려움증과 건성 피부를 주된 증상으로 하는 만성 염증성 피부 질환이다. 주로 유아기에 시작되며 성장함에 따라 알레르기성 비염 및 천식과 같은 호흡기 아토피 질환이 동반되는 경우가 있다. Chitinase 3 -like 1 (CHI3L1) 단백질은 만성 염증을 특징으로 하는 질병에서 발견되는 당 단백질이다. 또한 알레르기 질환의 진행에 염증을 일으키는 것으로 알려져 있다. 그러나 AD에서 CHI3L1의 역할은 명확하지 않다. 따라서 본 연구에서는 Dihydro-2-quinazolinyl} sulfanyl) -N- (4-ethylphenyl) butanamide (K284-6111; K284)의 항 피부염 효과를 연구하였다. C57BL/6J 마우스에서 무수 프탈산 (5 % PA)으로 AD를 유도했다. 유도 후, AD에 대한 K284-6111 (1 및 2 mg/kg)의 억제 효과와 AD와 CHI3L1 사이의 상세한 상관관계를 조사하였다. 조직 학적 분석은 K284-6111 (K284) 처리가 5% PA를 유도한 후 염증 세포의 침입을 억제하는 것으로 나타났다. K284는 조직에서 염증성 사이토카인 IL-1β, IL-6, IL-4, IL-13 및 흉선 기질 림프 포이 에틴 (TSLP)의 방출을 감소시켰으며, 5% PA 처리된 피부 조직과 TNF-α 및 IFN-γ 처리 (20 ng/mL) HaCaT 세포에서 K284는 NF-κB 활성의 발현 억제하였다. CHI3L1과 락토페린 (LTF)이 단백질 연관 네트워크 분석과 관련이 있음을 확인했고, 5% PA 처리된 동물 모델과 TNF-α 및 IFN-γ에 의해 유도된 HaCaT 세포에서 LTF의 발현이 증가함을 확인하였다. 그러나 생체 내 및 시험관 내 모델에서 K284의 처리에 의해 감소되었다. LTF의 억제는 TNF-α 및 IFN-γ- 유도 HaCaT 세포에서 염증성 사이토카인의 발현을 감소시켰으며, 또한 인간피부모델 (RHS)에서 K284 처리가 LTF 억제로 AD 발생을 감소시키는 것으로 확인하였다. 또한, 추가 동물 모델에서 LTF 항체 처리는 5% PA 유발 AD 발생을 억제하였으며, 혈청 분석 결과 CHI3L1과 LTF는 AD 환자에서 유의 한 상관관계를 보였다. 이러한 결과는 K284가 LTF 및 CHI3L1의 발현을 억제함으로써 AD 치료에 대한 유망한 약제가 될 수 있음을 시사한다.

세균백신의 국가표준품 구축 및 품질관리시험 적용 연구

김소영 충북대학교 2021 국내박사

The study was aimed to establish the national standards for diphtheria toxin and purified Vi typhoid vaccine used in potency test. First, diphtheria toxin stocks of the first national standard established in 2007 were exhausted, and in 2019, Green Cross Co., Ltd. freeze-dried the diphtheria toxin stock solution to produce 2 lots of candidate substances for the second national standard, 1,962 vials and 1,942 vials, respectively. The feasibility of manufacturing was evaluated through quality evaluation and long-term and accelerated stability tests for candidate substances. In order to assign the potency of diphtheria toxin secondary national standard candidate materials, the collaborative study was conducted with five institutions including vaccine manufacturers. The potency unit of the candidate substance was L+ unit (minimum amount of toxin that was mixed with 1 unit of standard antitoxin and injected subcutaneously into a guinea pig weighing 250 g and kills the animal on the 4th day), and the result was statistically analyzed. The titer of the candidate MFDS-B-19-002 is 210 L+/vial (95% confidence interval: 208.52~221.58 L+/vial), and the titer of the candidate MFDS-B-19-003 is 210 L+/vial (95% Confidence interval: 208.63~219.61 L+/vial). It was confirmed that there was a significant difference in the basic statistics of institutions through One-way ANOVA, but the degree of difference between institutions was small through the post-hoc test (Tukey's test) and the variability of each institution was very small. And, since the differences between institutions were within the range of the equivalence area, statistical equivalence was confirmed. In addition, the CD50 and LD50 values used in the diphtheria potency test were measured, and when the titer of the commercial vaccine was measured using candidate substances (applicability test), it was confirmed that there was no significant difference compared to the first national standard. In addition, it was confirmed that both types of candidate substances maintained stability for 12 months or longer. In case of typhoid vaccine reference, the 1st national standard established in 2009, was currently exhausted, so Boryung biopharma Co. produced 4,070 vials of 2nd standard candidate in 2018. The manufacture of the standard candidate was guaranteed throughout quality test, accelerated and long-term stability test. Reference standards ensure the accuracy and reliability of quality control tests for vaccines. Thus, collaborative study was conducted to estimate the more reliable potency of the candidate material of 2nd national standard, and performed by using test methods; HPAEC-PAD and rocket immunoeletrophoresis. The results of HPAEDC-PAD were analyzed to be 51.5 μg/vial (95% CI: 50.70~52.29 μg/vial). On the other hands, the results of rocket assay were shown to be 63.4 μg/vial (95% CI: 61.08~65.89 μg/vial). In addition, the precision and proficiency of the institutes was confirmed through the determination of CV and the Robust Z-score, respectively. The candidate substance whose quality has been verified is intended to be registered as the second national standard. After registration of the standard product, it is distributed to manufacturers and vaccine developers, and is used for national shipment approval test of vaccines and for quality control of manufacturers, contributing to securing reliability and consistency of vaccine quality control.

원획분 단계 생략을 통한 정맥주사용 면역글로불린-G 제조 공정의 최적화

이동휘 충북대학교 2021 국내석사

Immunoglobulin-G (IgG) is classified as a blood product obtained from plasma provided by many healthy blood donor and is mainly used in the form of intravenous injection in patients with primary immunodeficiency. The purpose of this study is reduction of the manufacturing cost by improvement of process steps in the manufacturing process. The manufacturing processes of intravenous IgG (IVIG) products manufactured by global pharmaceutical companies were compared and analyzed with the manufacturing process of the products applied in this study in order to discover the improvement of the product. As a result, the improvement plan that is ‘Elimination of one of sterile filtration steps by elimination of intermediate step, purified bulk’ was derived. By applying the improvement plan, the total manufacturing days of the product and the number of quality test items in the process of applied the improvement plan were reduced, and the procedure for approval of intermediate was eliminated. Accordingly, it was possible to increase the flexibility in the manufacturing plan, and it was analyzed that the manufacturing cost could be reduced by 41% in the process of applied the improvement plan. This study is meaningful for reducing the manufacturing cost by improvement of the process of IVIG product, and it will be useful data for the process optimization of pharmaceutical companies aiming at product development or process improvement of similar products.

Stress increases depression-like behaviors in Alzheimer’s disease mice

유승식 충북대학교 2022 국내석사

알츠하이머성 치매 (AD) 는 인지 장애, 기억 상실, 행동 변화 및 기능적 능력 상실을 특징으로 하는 치매의 가장 흔한 질환이다. 또한, 우울증은 AD 환자에게서 흔히 나타나는 동반 질환이다. 최근 AD는 우울증과 병태생리학적으로 공통된 기전을 공유한다는 것이 밝혀졌다. 만성 스트레스는 글루코코르티코이드 (GC)의 분비를 증가시키고 글루코코르티코이드 수용체 (GR)에 결합하여 뇌의 기능을 조절한다. 많은 양의 GC 분비는 아밀로이드 전구체 단백질 (APP)의 잘못된 processing과 아밀로이드 베타 (Aβ) 생성을 유발하여 AD를 유발한다. 또한, 우울증은 스트레스와 관련된 질병으로 잘 알려져 있으며 코르티솔 수치가 우울증의 바이오마커로 사용된다. 나는 우울증과 AD의 관계를 조사하기 위해 Tg2576 마우스와 WT 마우스를 10일 동안 물 회피 스트레스 (WAS)에 노출시켰다. WAS 에 노출시킨 이후 Tg2576 WAS 마우스 를 대상으로 기억 기능 및 우울증 관련 행동실험을 진행하였다. 우울증 관련행동실험에서는 Tg2576 WAS 마우스가 WT WAS 마우스 및 Tg2576 CON 마우스보다 더 우울한 행동을 나타내는 것으로 확인하였다. 또한, Tg2576 CON 마우스는 WT CON 마우스보다 더 우울한 행동을 나타내었다. 나아가,코르티코스테론 수치와 인산화된 글루코코르티코이드 수용체 (p-GR)도 Tg2576 WAS 마우스에서 Tg2576 CON 마우스보다 더 높게 나타났다. SPNS2의 주요 기능은 S1P 를 밖으로 방출하는 역할을 한다. GEO 데이터베이스는 AD 및 우울증 환자에서 SPNS2 유전자 발현을 분석하는 데 사용되었다. 결과는 SPNS2의 유전자 발현이 AD 및 우울증과 상관관계가 있음을 보여 주었다. 다음으로 Tg2576 WAS 마우스에서 SPNS2의 발현정도를 확인하였다. Tg2576 WAS 마우스에서 SPNS2의 발현량은 Tg2576 CON 마우스와 비교하였을 때 크게 증가하였다. 또한 SPNS2 발현이 세포 내 S1P 수준을 감소시키면서 세포외 S1P level이 증가되는지를 확인하기 위해 Tg2576 WAS 마우스 의 뇌와 혈청에서 S1P level을 측정하였다. Tg2576 CON 마우스와 비교하여 뇌의 S1P level은 증가하였고 혈청에서 S1P level은 감소하였다. 또한, stress 는 뇌의 S1P 수치를 더욱 감소시켰다. 그러나 혈청 내 수치는 더욱 증가하였다. 따라서 이러한 결과는 AD와 우울증이 연관성이 있으며, 스트레스 매개 우울증이 SPNS2를 상향 조절함으로써 S1P의 방출을 통해 Tg2576 형질전환 마우스에서 더 많은 영향을 미칠 수 있음을 나타낸다. Alzheimer’s disease (AD) is the most common form of dementia characterized by cognitive impairment, loss of memory, behavioral changes and loss of functional abilities. Depression is a common co-morbidity seen in people with AD. Recent evidence suggests that AD and depression share common mechanisms of pathogenesis. Chronic stress leads to increased secretion of glucocorticoid (GC) and modify brain functions by binding glucocorticoid receptor (GR). High levels of GC secretion, triggers AD by causing the misprocess of amyloid precursor protein (APP) and the production of amyloid-beta (Aβ). Moreover, depression is well-known stress related illness and elevated levels of cortisol are used as a biomarker of depression. To investigate relationship between depression and AD, water avoidance stress (WAS) was induced for 10 days in both Tg2576 mice and wild-type (WT) mice. After WAS, memory function and depression-related test were investigated in Tg2576 WAS mice. On depression-related behavioral tests showed that Tg2576 WAS mice exhibited more depressive behaviors than WT WAS mice and Tg2576 CON mice. Tg2576 CON mice showed more depressive behaviors than WT mice. Moreover, corticosterone levels and phospho-glucocorticoid receptor (p-GR) were also higher in Tg2576 WAS mice than in Tg2576 CON mice. Sphingolipid Transporter 2 (SPNS2) is a Sphingosine-1-Phosphate (S1P) transporter. GEO database was used to analyze SPNS2 gene expression in AD and depression patients. The reults shows that SPNS2 gene expression correlates with AD and depression. Next, expression level of SPNS2 in Tg2576 WAS mice brains was measured. In Tg2576 WAS mice, SPNS2 was significantly increased compared with Tg2576 CON mice. SPNS2 was also higher in Tg2576 CON mice compared with WT CON mice. To study whether increased SPNS2 expression release more level of S1P, the S1P levels in the Tg2576 WAS mice brains and serum were measured. Compared with Tg2576 CON mice, the S1P level in the brains was decreased, but was increased in the serum. Moreover, WAS treatment further decreased S1P level in the brains. However, the level in the serum further increased. Therefore, these results indicate that AD and depression could be associated, and stress mediated depression could affect more in Tg2576 transgenic mice through the release of S1P by up-regulating SPNS2.

현탁제 제조공정 중에 발생하는 기포로 인한 주성분 함량분포의 변화

강신국 충북대학교 2022 국내박사

This study aimed to investigate the influence of bubbles generated in the pharmaceutical suspension manufacturing process on finished products' active pharmaceutical ingredient (API) contents. In particular, we aimed to evaluate the possibility of having a higher API content in pharmaceutical suspensions containing a large amount of bubbles. We also investigated content analysis results based on prior studies related to the attachment of solid particles to bubbles. Each test tube was agitated at 1,200 rpm for 60 min to generate bubbles. To measure the API content, samples were collected from each layer (lower, middle, upper, foam). The API content analysis results were further analyzed by general properties (pH and density), surface hydrophobicity (contact angle), and morphological properties (particle size and shape). The API content in five out of seven pharmaceutical suspensions increased toward the upper layer, and the API concentration of the foam layer was the highest. The other two suspensions showed an opposite trend. The contact angle of API particles of products with an increased toward the upper layer was greater than 62º, the particle size exceeded 4μm, and the particles were needle-shaped and had angular fragments. Our findings indicate that API particles are highly concentrated in the foam generated during the manufacturing process depending on various properties, including surface hydrophobicity, particle size, and shape. Therefore, improvements in formulations and the manufacturing process of pharmaceutical suspensions such as bubble suppression or breakage rather than foam removal from the intermediate products are required.

Effect of Reducing Intraocular Pressure of Brinzolamide Combination Therapy in Patients with Primary Open-angle Glaucoma and Ocular Hypertension

Kim, Jin A 충북대학교 2022 국내석사

Anti-inflammatory effect of Colchicine in skin inflammation

Woo MinSik 충북대학교 2022 국내석사

Chitinase-3-like-1 inhibitor K284­6111 ameliorates acute liver injury through down regulation of CXCL3

김민지 충북대학교 2023 국내석사

키티나제 3 like 1 단백질 (CHI3L1)은 다양한 유형의 감염 질환, 특히 패혈증에 관여한다. CHI3L1의 비정상적인 발현은 염증과 면역 반응에 중요한 역할을 한다고 알려져 있다. 이전 보고에서, 화합물 K284­6111(K284)이 CHI3L1에 가장 높은 결합 활성을 갖는다는 것을 발견했다. 본 연구에서는 지방다당류 (LPS) 유도 C57BL/6 마우스 간 패혈증 모델을 이용하여 K284가 간 손상에 미치는 영향을 조사하였다. 이번 연구에서 LPS+K284 주입 생쥐에서 생존율이 65%인 반면 LPS 처리 생쥐에서는 0%라는 것을 발견했다. 간 손상 관련 효소 (ALT, AST, LDH) 의 혈청 수준은 K284 주입 마우스에서 감소하였으며, 이는 K284에 의해 패혈성 간 손상이 완화되었음을 나타낸다. 또한 K284 주사 마우스의 간 조직에서 M2 마크로파지 마커의 발현 수준이 유의하게 감소하였다. LPS 처리된 THLE­2 세포에서 CHI3L1의 고갈은 M2 마크로파지 마커 단백질의 발현을 유의하게 감소시켰다. 다음으로 지금까지 관찰된 효과와 관련된 주요 경로를 조사했고 CXCL3 발현이 CHI3L1의 발현과 상관관계가 있다는 것을 GeneMania 프로그램을 통해 발견했다. 그리고 LPS+K284 처리 생쥐의 간 조직에서 CXCL3의 발현이 LPS 처리 생쥐에 비해 낮다는 것을 발견했다. 또한, M2 마크로파지 관련 사이토카인 수치는 LPS와 재조합 CXCL3의 조합에 의해 유의하게 증가하였으나, K284에 의해 유의하게 감소하였다. M2 마크로파지 관련 사이토카인과 CHI3L1 발현 수준은 CXCL3 siRNA에 의해 유의하게 감소하였으며, CXCL3 siRNA와 K284의 결합 처리는 더욱 감소하였다. 따라서, CXCL3 발현은 CHI3L1 억제 화합물인 K284에 의한 LPS 유도 급성 간 손상 회복과 관련된 중요한 인자일 수 있음을 발견했다. 이번 연구 결과는 K284가 간 손상과 같은 패혈증에 적합한 치료제가 될 수 있음을 시사한다. CHI3L1 is involved in various types of infectious disease, especially sepsis. Aberrant expression of CHI3L1 could play a immune responses and critical role in inflammation. Previous studies have shown that compound K284­6111 (K284) has the highest binding activity to CHI3L1. In this study, the effect of K284 on sepsis liver damage was investigated using a lipopolysaccharide (LPS) induced C57BL/6 mouse model. Survival was 65% in LPS+K284 injection mice, while 0% in LPS-treated mice. The serum levels of hepatic injury­related enzymes were decreased in K284 injected mice, indicating that hepatic liver damage was alleviated by K284. In addition, depletion of CHI3L1 in LPS­treated THLE­2 cells decreased the expression of M2 polarization­related marker proteins (MRC1, ARG1, IL­4, and IL­10). It was discovered by examining the main pathways associated with the observed effects that CXCL3 expression correlated with the expression of CHI3L1 through the GeneMania program. The expression of CXCL3 was lower in liver tissue in LPS+K284 treated mice than in LPS treated mice. Moreover, M2 polarization­related cytokine levels were increased by the combination of LPS and recombinant CXCL3, but decreased by CXCL3 siRNA and K284. M2 polarization­related cytokine levels, and expression of CHI3L1 and CXCL3 were decreased by CXCL3 siRNA, and combination treatment of CXCL3 siRNA and K284 was further decreased. Therefore, CXCL3 expression might be a significant factor involved in the reduction of LPS­induced sepsis by the CHI3L1­inhibiting compound, K284. In other words, these data indicate that K284 could be a suitable treatment for hepatic sepsis.

헤모필루스 인플루엔자 b형 백신에 범용 적용 가능한 다당류와 접합단백질 동시확인법 개선

이인영 충북대학교 2023 국내석사

Haemophilus influenzae type b (Hib) vaccine is a type of polysaccharide vaccine, which is used by introducing conjugated proteins to enable children to obtain immunity. In Korea, only Hib vaccine which uses tetanus toxin as a conjugated protein is approved and supplied. Both PRP, the Hib antigen of the Hib vaccine, and TT, the conjugated protein, must be completely linked to each other to obtain as much immunogenicity as expected when vaccinated into the human body. As part of vaccine quality control, National Lot Release system is in operation. Identification test is the basic item of the national lot release test item. Currently, the identification test of the Hib vaccine is carried out by applying different methods for each vaccine manufacturer, making it difficult to manage consistent quality control. To overcome this problem, the purpose of this study is to establish a test method that can be applied to all Hib vaccines approved and supplied in Korea and can be determined to be positive only in a structure in which PRP, a Hib antigen, and TT, a conjugated protein, are completely bound through linker molecule. The existing test method was reviewed and applied to the improved test method using sandwich ELISA, a method suitable for the purpose of this study. Candidates were selected for commercial antibodies that can be purchased and compared and tested. Antibody combinations showing a high Signal to Noise ratio (S/N ratio) for Hib vaccines approved and distributed in Korea were selected as coating antibodies, primary and secondary antibodies, respectively. The protocol of the test method was established by comparing and experimenting with dilution factor and reaction time. The specificity was verified by conducting test method validation on the improved test method protocol, and it was proved to be a better test method than the existing test method through comparison and applicability evaluation with the existing test method. The establishment of improved test method that can be applied to all approved and supplied Hib vaccines in Korea set up for the purpose of this study and can confirm both PRP and TT has been achieved. As the demand for combined vaccines containing Hib vaccines is increasing, I hope that the improved national lot release identification test method of Hib vaccines through this study will contribute to vaccine quality control.