Biochemical Characterizations of Phenylalanine Ammonia-Lyase and its Mutants to Develop an Enzymatic Therapy for Phenylketonuria

Woomi Kim(김우미) 한국생명과학회 2009 생명과학회지 Vol.19 No.9

페닐케톤뇨증은 상염색체 열성으로 유전되며, phenylalanine-4-hydroxylase (PAH, EC 1.14.16.1)의 돌연변이에 의해 효소 불활성화를 초래하는 질환이다. 최근 유전자 재조합된 phenylalanine ammonia-lyse (PAL)에 의한 효소 대체요법이 보고된 바 있다. 이 효소를 경구용 약제로 개발하기 위하여 효소활성을 나타내기 위한 최적 조건들을 알아야 하며, 위장관내 소화효소에 의해 분해되지 않는 구조적 안정성을 유지하여야 한다. 따라서 본 연구에서는 PAL의 생화학적 특성을 규명하고, 이를 바탕으로 위장관내 소화효소로부터 저항할 수 있는 변이형들을 만들고자 하였으며, 이러한 구조적 변화를 통하여 효소의 특이 활성도가 유지될 수 있는지를 보고자 하였다. PAL의 특이 활성도를 측정하였고, 효소 활성을 나타내기 위한 최적 pH, 온도 변화에 따른 효소 활성도, 단백분해효소에 의한 활성도 변화를 측정하였다. PAL의 Vmax는 페닐알라닌과 티로신에 대하여 각각 1.77, 0.47 μ㏖/ ㎎ x protein로 나타났으며, Km은 페닐알라닌에 대하여 4.77×10?⁴ M, 티로신에 대하여 4.37×10?⁴ M로 나타났다. 또한 pH 8.5에서 가장 높은 활성을 나타내었는데, 이는 소장의 평균 pH와 유사하다. PAL의 효소 활성은 -80℃에서 5개월 동안 유지되었으며, 4℃에서 1주일 동안 93.4%의 활성을 유지하였다. PAL은 키모트립신에 의해 쉽게 분해되었으며, 이보다 약한 정도로 트립신, elastase, carboxypeptidase A, B에 의해 분해되었다. 췌장 소화효소에 대한 저항성을 증가시키기 위하여 트립신, 키모트립신 절단부위 아미노산을 변이시켜 유전자 변이형을 만들었고, 효소 활성도를 측정하였다. 6개의 유전자 변이형은 모두 저하된 효소 활성도를 나타내었는데, Y110H는 0.084, Y110A와 Y110L은 0, R123A는 0.11, R123H는 0.074, R123Q는 0.033으로 나타났다. 이러한 결과는 트립신 및 키모트립신 절단부위 아미노산이 PAL의 효소 활성에 필수적인 역할을 하고 있음을 나타낸다. PAL 변이형은 단백분해작용으로부터 보호할 수 있는 전처치 방법이지만, 페닐알라닌을 효과적으로 저하시키기 위해서 효소활성을 유지할 수 있는 다음 단계의 처치가 필요하다. Enzyme substitution with recombinant phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is currently being explored for treatment of phenylketonuria (PKU), an autosomal recessive genetic disorder with mutations of the gene encoding phenylalanine-4-hydroxylase (EC 1.14.16.1). However, oral administration of PAL is limited because of proteolytic digestion in the gastrointestinal tract. The aim of this study was to determine the bi℃hemical properties of PAL and delinate the susceptibility of wild-type PAL to pancreatic proteolysis by exploring several mutants, and to develop therapeutic drugs with PAL for PKU. The specific activity of PAL was assayed and its optimal pH, temperature stability, and intestinal protease susceptibility were investigated. Its Vmax values for phenylalanine and tyrosine were 1.77 and 0.47 μ㏖/ min/㎎ protein, respectively, and its Km values were 4.77×10?⁴ and 4.37×10?⁴ M, respectively. PAL showed an optimal pH at 8.5, corresponding to the average pH range of the small intestine. It showed no loss of activity at -80℃ for 5 months and possessed 93.4% of its activity under 4℃ for 4 wks. PAL was susceptible to chymotrypsin digestion and, to a lesser extent, to trypsin, elastase, carboxypeptidase A, and B. The trypsin and chymotrypsin cleaving sites were mutated to investigate protection from pancreatic digestion and the specific activities of these mutants were evaluated. The six mutants displayed low specific activities compared to the wild- type, suggesting that the primary trypsin and chymotrypsin cleaving sites may be essential for catalytic reaction. The PAL mutants could therefore be applied as a pretreatment modality without susceptibility to proteolytic attack, however, additional modification for enhancing enzymatic activity is needed to reduce the Phe levels effectively.

The Rapid Determination of Gemcitabine by Reversed-phase Ultra-Performance Liquid Chromatography

Dae Jin Park(박대진),Woomi Kim(김우미) 한국생명과학회 2009 생명과학회지 Vol.19 No.12

Gemcitabine은 다양한 고형암 치료에 사용되는 항암제이다. 혈장내에서 cytidine deaminase에 의해 빠르게 대사되며, 친수성 성질로 인해 역상 액체크로마토그라피(reversed-phase liquid chromatography)를 이용한 농도 분석이 어렵다. 본 연구에서는 역상칼럼(reversed-phase column)을 이용한 초고속 액체크로마토그라피(ultra-performance liquid chromatography, UPLC) 방법에 의해 빠르고 정확한 속력(velocity)과 최고효능(peak efficiency)를 갖춘 분석법을 개발하고자 하였다. Gemcitabine과 2’-deoxycytidine의 머무름 시간(retension time)은 293 ㎚에서 각각 3.2분과 2.1분이었다. 검량선의 직선성 검정은 0.1~20 ㎍/㎖ 의 농도범위에서 높은 직선성을 나타내었다(r²>0.999). 일내(intra-day) 변이게수(coefficients of variation)와 일간(inter-day) 변이게수는 모두 10% 이하였다. 정확성(accuracy) 검정을 위한 일내 및 일간 평균농도 측정치가 97.3~113.5% 범위로 나타났다. 이러한 결과를 토대로, gemcitabine 농도를 측정하기 위한 새로운 분석법으로 빠르고 정확한 역상 UPLC 방법을 제안하고자 한다. Gemcitabine is an anticancer drug used to treat a variety of solid tumors. The drug is rapidly inactivated by cytidine deaminase in plasma and its hydrophilicity restricts the extent of quantification that is possible using reversed-phase liquid chromatography. In this paper, we report a rapid and precise method to analyze velocity and peak efficiency using ultra-performance liquid chromatography (UPLC) with a reversed-phase column. The retention periods of gemcitabine and 2’-deoxycytidine at 283 ㎚ were 3.2 and 2.1 min, respectively. The assay provided highly linear results in the range of 0.1~20 ㎍/㎖ (r²>0.999). The coefficients of variation of the intra-day and inter-day assays were less than 10.0%. We observed that the estimated average concentrations of the intra-day and inter-day assays ranged from 97.3 to 113.5% to verify the accuracy. These results suggest that this new reversed-phase UPLC method is a rapid and reliable way of determining gemcitabine levels.

The Feasibility of Cathepsin B Level in Preoperatively Screening Patients with Thyroid Cancer and Nodular Hyperplasia

Young Sik Choi(최영식),Young Ok Kim(김영옥),Woomi Kim(김우미) 한국생명과학회 2009 생명과학회지 Vol.19 No.11

혈장 cathepsin B 활성도 측정이 갑상선암 및 결절성 증식증의 수술 전 진단에 도움이 되는지를 알아보고자 분화갑상선암 32례, 결절성 증식증 7례, 대조군 5례의 혈장 cathepsin B 발현양상을 대조군과 함께 관찰하였다. 수술시 제거된 여포선암의 암조직, 결절성 증식증 조직의 cathepsin B 발현을 정상 조직과 비교하였다. 혈장 Cathepsin B 활성도는 정상 대조군에서 168.94±15.10 (×10?², mU), 결절성 증식증에서 255.45±95.68 (×10?², mU), 악성종양에서 284.87±79.32 (×10?², mU)로써 악성종양군과 결절성 증식증군에서 cathepsin B 발현이 정상 대조군보다 비교적 높게 나타났다(p<0.05). 혈장 cathepsin B의 정량적 비교를 위한 immunoassay결과에서도 결절성 증식증군(17.64±7.49 ng/㎖)과 악성종양군(15.50±7.86 ng/㎖)에서 정상대조군(4.85±0.61 ng/㎖)보다 높은 수치를 나타내었다(p<0.05). 결절성 증식증 및 악성종양군의 조직 내 cathepsin B mRNA발현이 정상 조직에서보다 높게 나타났다. 따라서 혈장 cathepsin B는 갑상선세포의 비정상적인 증식시에 증가됨을 알 수 있으며, 갑상선암 혹은 결절성 증식증을 스크리닝하는데 이용될 수 있을 것으로 사료된다. To evaluate the feasibility of cathepsin-B levels in preoperatively screening patients with thyroid cancer, we assigned these patients to either the thyroid cancer group (n=32) or the nodular hyperplasia group (n=7). Five healthy volunteers served as controls (n=5). We quantified cathepsin-B expressions in cancerous lesions with follicular carcinoma and hyperplastic lesions with nodular hyperplasia, and compared the degrees to those of normal thyroid tissue, which was obtained from matched contralateral lobe. The activity of serum cathepsin B was significantly higher in patients with thyroid carcinoma (284.87±79.32, ×10?² mU) and those with nodular hyperplasia (255.45±95.68, ×10?² mU) than compared to normal control (168.94±15.10, ×10?² mU) (p<0.05). Based on the results of immunoassay, the concentrations of cathepsin B in the thyroid cancer group (15.50±7.86 ng/ml) and the nodular hyperplasia group (17.64±7.49 ng/㎖) were higher than those of the control group (4.85±0.61 ng/㎖). The degree of cathepsin-B mRNA expression was significantly higher in cancerous or hyperplastic lesions than normal thyroid tissues from matched contralateral lobe with follicular carcinoma or non-neoplastic thyroid disease. Our results indicate that the activity of serum cathepsin B is a useful indicator in screening patients with nodular hyperplasia or neoplastic thyroid disease and it may be involved in the abnormal proliferation of cells.

Development and Validation of the Determination of Sorafenib in Human Plasma using Tandem Mass Spectrometry Coupled with Liquid Chromatography

Daejin Park(박대진),Sunggon Lee(이성곤),Woomi Kim(김우미) 한국생명과학회 2012 생명과학회지 Vol.22 No.11

소라페닙은 멀티카이네즈 억제제로서 신세포암, 전이성 간세포암 환자의 치료에 효과가 입증된 경구용 항암제이다. 이 연구의 목적은 고속액체크로마토그래피 텐덤질량분석기법(LC/MS/MS)을 이용하여 사람 혈장 내 소라페닙의 농도를 측정하는 효율적인 방법을 개발하고 한국식품의약품안전청(KFDA) 기준에 따라 분석법을 검정하는 것이다. 혈장시료(100 μl)에 내부표준물질인 chlorantraniliprole을 첨가한 후 이소프로필알콜과 에틸아세테이트로 구성(1:4, v/v)된 0.1% 포름산 함유 추출용액을 혼합하였다. 원심분리 후 상층액을 취하여 원심감압농축하였다. 잔사를 이동상에 재용해하고 Waters사의 역상 XTerraTM C18 칼럼(입자크기 3.5 μm)을 장착한 고속액체크로마토그래피 장치에 주입하였다. 액체크로마토그래피는 0.1% 포름산과 10 mM 암모늄 포메이트를 함유한 버퍼용액과 메탄올, 아세토나이트릴을 각각 1:6:3으로 혼합한 용액을 이동상으로 사용하였으며 5분 내에 측정을 완료하였다. 분석대상 물질들은 텐덤질량분석기에서 electrospray 양이온 이온화(ES+) 검출방식으로 확인하였으며 소라페닙은 ‘m/z 465.2 → 252.5’, chlorantraniliprole은 ‘m/z 484.4 → 286.2’으로 구성한 multiple reaction monitoring 방법을 사용하였다. 검정 결과, 2-5,000 ng/ml의 농도 구간에서 양호한 직선성(r<SUP>2</SUP>>0.99)과 정확도(90.7-103.9%), 정밀도(10% 이하)를 나타내었다. 새롭게 개발된 LC/MS/MS을 이용한 사람 혈장 내 소라페닙의 농도 측정법은 KFDA 기준을 만족하였으며, 기존의 방법에 비해 민감도가 높은 방법이었다. Sorafenib is a multikinase inhibitor and an oral anticancer drug approved for the treatment of patients with advanced renal cell carcinoma and those with unresectable hepatocellular carcinoma. The purpose of this study was to develop an efficient method of the determination of sorafenib in human plasma using tandem mass spectrometry coupled with liquid chromatography (LC/MS/MS) and validate the method by the guidelines of the Korean Food and Drug Administration (KFDA). Plasma samples (100 μl) were added with chlorantraniliprole as an internal standard and then mixed with the 0.1% formic acid-containing extraction solution composed of isopropyl alcohol and ethyl acetate (1:4, v/v). After centrifugation, the supernatant was concentrated at 45°C under negative pressure and centrifugal force. The residue was reconstituted with a mobile phase and injected into the HPLC instrument using a reverse phase Waters XTerra<SUP>TM</SUP> C18 column (particle size 3.5 μm). Liquid chromatography was carried out within the run time of 5 min using a mobile phase composed of buffer (0.1% formic acid and 10 mM ammonium formate), methanol, and acetonitrile (1:6:3, v/v/v). The analytes were monitored by tandem mass spectrometry in the multiple reaction monitoring method programmed to detect sorafenib at ‘m/z 465.2 → 252.5’ and chlorantraniliprole at ‘m/z 484.4 → 286.2’ with positive electrospray ionization mode (ES+). The result showed the proper linearity (r<SUP>2</SUP>>0.99) over the range of 2,000-5,000 ng/ml with good accuracy (90.7-103.9%) and precision (less than 10%). The newly developed method using LC/MS/MS was validated by the guideline of KFDA and identified as more sensitive compared to the previous methods.

건강한 한국인 자원자에서 메트포민의 Targeted Metabolite Profiling

임호섭,차재민,서정주,박정현,이주미,이혜원,배균섭,김우미,윤영란,Lihm, Ho-Seob,Cha, Jaemin,Seo, Jeong Ju,Park, Jeonghyeon,Lee, Joomi,Lee, Hae Won,Bae, Kyun Seop,Kim, Woomi,Yoon, Young-Ran 대한임상약리학회 2012 臨床藥理學會誌 Vol.20 No.2

Background: Metformin is an effective oral antihyperglycaemic agent for type 2 diabetes mellitus, with a variety of metabolic effects. In addition to controlling blood glucose level, it has been appeared to decrease the long-period complications of diabetes, including macrovascular disease. Few reports have addressed the metabolite profiling of metformin. The study was to evaluate if targeted metabolic profiling approach is sensitive enough to predict the therapeutic effects of metformin after a single oral dose. Methods: A randomized, open-label, single-dose study was conducted in twenty eight healthy Korean male volunteers. To determine the concentrations of endogenous metabolites in their pre-dose and post-dose plasma samples, blood samples were collected before and at 2 and 6 h after a single oral dose of 500 mg metformin. Both Modular P/Modular D analyzer and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)-based metabolic profiling was performed. Results: We quantified pre-dose and post-dose creatinine, blood urea nitrogen (BUN), lactic acid, 7 amino acids (lysine, glutamic acid, alanine, valine, leucine, phenylalanine, tryptophan), and 5 lysophosphatidylcholines (14:0, 16:0, 17:0, 18:0, and 18:1) using autoanalyser and UPLC-MS/MS. The postdose levels of alanine, lactic acid, glutamic acid, lysine, valine, leucine, phenylalanine, tryptophan, and lysoPC (18:1) were slightly decreased with statistical significance, but there is no clinical significance. Conclusion: In order to explore the potential endogenous metabolites associated with the therapeutic effects of metformin, further study including non-targeted (global) metabolite profiling is needed.

Enzyme therapy for phenylketonuria : future directions

Kim, Woomi KOSIN UNIVERSITY COLLEGE OF MEDICINE 2006 高神大學校 醫學部 論文集 Vol.21 No.1

The dietary therapy for phenylketonuria (PKU) is unpalatable and ineffective in controlling systemic phenylalanine (Phe) levels during pregnancy. Alternative therapies are currently being investigated, particularly ones that break down Phe. This review underscores the progress made in enzyme replacement therapy for PKU. Two modalities are discussed, the enzymes phenylalanine hydroxylase (PAH) and phenylalanine ammonia-lyase (PAL). Developing stable and functional forms of both enzymes have proven difficult, but recent success in producing PEG-modified form of active and stable PAH shows promise. In addition, microencapsulation (ENC) could partially protect proteolysis and gastric acidity. If the immunologic problems can be overcome by PEGylation, and the activity of PEGylated enzyme can be protected by additional encapsulation, it may provide a new prospect for both the oral and parenteral enzyme therapies in PKU.

Tetracycline계 항균제에 의한 사람 호중구 Elastase의 효소 활성도 억제 및 그 작용 기전

김우미,강구일 고신대학교 의학부 1993 高神大學校 醫學部 論文集 Vol.9 No.1

Human neutrophil elastase, which is known to be a possible mediator in the pathophysiology of connective tissue breakdown, was effectively inhibited by tetracycline, oxytetracycline and demeclocycline. Among them, oxytetracycline showed the most potent inhibitory effect on elastase activity. Tetracycline inhibited human neutrophil elastase non-competitively, however oxytetracycline inhibited human neutrophil elastase competitively. Ki values of tetracycline and oxytetracycline were 4.9mM and 0.39mM, respectively. De-dimethylaminotetracycline, which showed no antibiotic activity since the active dimethylamino radical was removed from the position #4 of the tetracycline, inhibited the activity of human neutrophil elastase by tetracyclines not depend on the dimethylamino radical which is a critical active site for antibiotic effect, rather it depend on the hydoxyl radical of tetracyclines. The property of inhibiting elastase may be an additional molecular biochemical mechanism of action of these drugs as an inhibitor of tissue destruction at the inflammatory sites.