Nkx-2.5 Regulates MDR1 Expression via Its Upstream Promoter in Breast Cancer Cells

임정석,정규연,박승윤 대한의학회 2019 Journal of Korean medical science Vol.34 No.12

Background: Increased expression of MDR1 gene is one of the major mechanisms responsible for multidrug resistance in cancer cells. Two alternative promoters, upstream and downstream, are responsible for transcription of MDR1 gene in the human. However, the molecular mechanism regarding the transactivation of MDR1 upstream promoter (USP) has not been determined. Methods: Dual-luciferase reporter gene assays were used to assess the effect of Nkx-2.5 on MDR1 USP activity using reporter plasmids for human MDR1 USP and its mutants. MDR1 mRNA level was examined by quantitative real-time PCR. The direct binding of Nkx-2.5 to the USP of MDR1 was evaluated by promoter enzyme immunoassays and chromatin immunoprecipitation assays. Results: Nkx-2.5 significantly stimulates the transactivation of MDR1 USP and increases MDR1 mRNA expression in MCF7 breast cancer cells. Reporter gene assays with deleted MDR1 USPs showed that the Nkx-2.5-binding site is located between positions -71 and +12. Mutation of the Nkx-2.5-binding site at nucleotide +4 to +10 markedly reduced the Nkx-2.5-mediated activation of MDR1 USP activity. A promoter binding immunoassay and a chromatin immunoprecipitation assay revealed that Nkx-2.5 binds directly to the region +4/+10 of human MDR1 USP. Conclusion: The results in the present study show Nkx-2.5 is a positive regulator for the transactivation of MDR1 USP in MCF7 breast cancer cells. Our findings will help elucidate the regulatory mechanism responsible for the multidrug resistant cancer phenotype.

Nkx-2.5 Regulates MDR1 Expression via Its Upstream Promoter in Breast Cancer Cells

Lim, Jung-Suk,Jung, Gyu Yeon,Park, Seung-Yoon KOREAN ACADEMY OF MEDICAL SCIENCE 2019 JOURNAL OF KOREAN MEDICAL SCIENCE Vol.34 No.12

<P><B>Background</B></P><P>Increased expression of MDR1 gene is one of the major mechanisms responsible for multidrug resistance in cancer cells. Two alternative promoters, upstream and downstream, are responsible for transcription of MDR1 gene in the human. However, the molecular mechanism regarding the transactivation of MDR1 upstream promoter (USP) has not been determined.</P><P><B>Methods</B></P><P>Dual-luciferase reporter gene assays were used to assess the effect of Nkx-2.5 on MDR1 USP activity using reporter plasmids for human MDR1 USP and its mutants. MDR1 mRNA level was examined by quantitative real-time PCR. The direct binding of Nkx-2.5 to the USP of MDR1 was evaluated by promoter enzyme immunoassays and chromatin immunoprecipitation assays.</P><P><B>Results</B></P><P>Nkx-2.5 significantly stimulates the transactivation of MDR1 USP and increases MDR1 mRNA expression in MCF7 breast cancer cells. Reporter gene assays with deleted MDR1 USPs showed that the Nkx-2.5-binding site is located between positions -71 and +12. Mutation of the Nkx-2.5-binding site at nucleotide +4 to +10 markedly reduced the Nkx-2.5-mediated activation of MDR1 USP activity. A promoter binding immunoassay and a chromatin immunoprecipitation assay revealed that Nkx-2.5 binds directly to the region +4/+10 of human MDR1 USP.</P><P><B>Conclusion</B></P><P>The results in the present study show Nkx-2.5 is a positive regulator for the transactivation of MDR1 USP in MCF7 breast cancer cells. Our findings will help elucidate the regulatory mechanism responsible for the multidrug resistant cancer phenotype.</P>

분자핵의학 기법을 이용한 다약제내성 진단

이재태,안병철 대한핵의학회 2004 핵의학 분자영상 Vol.38 No.2

다약제내성이 발현된 암세포에서 세포내의 항암제를 세포외로 배출시키는 기전을 체내에서 비침습적인 방법으로 영상화 할 수 있는 SPECT와 PET는 악성종양의 진단과 평가에 중요한 역할을 할 것으로 판단되나, 아직까지도 Pgp와 MRP의 운반능력을 적절하게 평가하는 핵의학적 영상방법을 정립하는데는 극복해야할 문제점들이 많다.16) 지금까지의 MDR영상에 관한 연구들은 대부분이 ^(99m)Tc-표지 방사성의약품을 이용한 연구였으나, PET의 임상 응용이 증가함에 따라 보다 특이적이고 쉽게 응용될 수 있는 PET용 방사성 추적자의 개발도 이루어져야 할 것이다. ^(99m)Tc-MIBI의 암 세포내 일방향(unidirectional) 섭취는 음성인 세포막 전하와 세포내 소립체 기질 전하에 의하여 결정되므로, MIBI의 섭취는 다른 지용성 양전하를 띤 막전위 추적자들과 유사하게 작용한다. ^(99m)Tc-표지 방사성의약품은 암조직의 혈류 증가나 소립체 용적이나 활성도가 증가하면 섭취가 증가할 수 있어 보다 특이적인 MDR추적제의 개발이 필요한 것이다. 최근 Lorke 등60)은 약제감수성 및 내성 인체대장암세포인 HT-29par 세포와 HT-29mdr1 세포를 이용한 연구에서, 두세포 모두에서 18F-FDG의 섭취가 있었고, MDR이 발현된 세포와 종양에서 18F-FDG 섭취가 훨씬 낮았고, MIBI는 MDR이 없는 모세포에서도 매우 낮았음을 보고한 바 있다. 이 세포는 전자현미경 검사에서 사립체가 풍부하지 않은 세포였다. 그러므로 이러한 결과로 보아 ^(99m)Tc-MIBI 영상에서 종양이 보이지 않거나 섭취가 미약하다고 해도 MDR이 발현되었다고 단정할 수는 없게 된다. 즉 MDR의 발현유무를 정확하게 감별할 수 있기 위하여는 저항이 없는 세포에 MIBI가 충분하게 섭취되어야 한다는 것이 필수적인 요건이며, 종양세포 종류에 따라서는 FDG가 MDR의 marker가 될 수 있다는 것이다. Pgp 수송체는 ATP의존성 약제배출 펌프이므로 MDR세포는 에너지가 많이 필요하여, MDR세포는 당분해율(rate of glycolysis)이 증가되어 있고 HT-29mdr1 종양세포에서는 포도당 이동과정의 변화로 FDG 섭취가 감소되었다. 또한 Pgp가 점차 증가됨과 함께 plasmam embrane transporter인 GLUT-1 level이 감소된다. 이러한 결과는 다약제내성의 영상화가 지금까지의 예상보다 보다 복합적이고 다양하므로 보다 많은 연구가 필요할 것이라는 점을 시사한다. 최근 시도되고 있는 생체광학 영상을 이용한 다약제내성 유전자 및 Pgp 발현 연구는 아직 시작단계이나, 분자 생물학적 영상법의 발전과 함께 MRI 기술등에도 이용될 수 있으므로 향후 많은 연구가 있을 것으로 기대된다. Although the outcome of cancer patients after cytotoxic chemotherapy is related diverse mechanisms, multidrug resistance (MDR) for chemotherapeutic drugs due to cellular P-glycoprotein (Pgp) or multidrug-resistance associated protein (MRP) is most important factor in the chemotherapy failure to cancer. A large number of pharmacologic compounds, including verapamil, quinidine, tamoxifen, cyclosporin A and quinolone derivatives have been reported to overcome MDR. Single photon emission computed tomography (SPECT) and positron emission tomography (PET) are available for the detection of Pgp and MRP-mediated transporter. ^(99m)Tc-MIBI and other ^(99m)Tc-radiopharmaceuticals are substrates for Pgp and MRP, and have been used in clinical studies for tumor imaging, and to visualize blockade of Pgp-mediated transport after modulation of Pgp pump. Colchicine, verapamil and daunorubicin labeled with ^(11)C have been evaluated for the quantification of Pgp-mediated transport with PET in vivo and reported to be feasible substrates with which to image Pgp function in tumors. Leukotrienes are specific substrates for MRP and N-[11C]acetyl-leukotriene E4 provides an opportunity to study MRP function non-invasively in vivo. SPECT and PET pharmaceuticals have successfully used to evaluate pharmacologic effects of MDR modulators. Imaging of MDR and reversal of MDR with bioluminescence in a living animal is also evaluated for future clinical trial. We have described recent advances in molecular imaging of MDR and reviewed recent publications regarding feasibility of SPECT and PET imaging to study the functionality of MDR transporters in vivo. (Korean J Nucl Med 38(2):180-189, 2004)

급성 골수성 백혈병에서 다약제내성(MDR-1) 유전자 발현의 빈도 및 임상적 의의

백진호,박성원,김동환,정진태,곽동석,박소향,손상균,서장수,이건수,이재태,이규보 경북대학교 병원 2001 경북대학교병원의학연구소논문집 Vol.5 No.1

배경:백혈병 치료성적이 나쁜 이유 중 하나는 항암제에 대한 내성이며 특히 다약제내성(MDR-1)유전자의 과발현이 치료실패의 중요한 원인이다.가장 많이 알려진 P-당단백은 MDR-1 유전자에 의해 발현된 transmembrane단백으로 항암제가 암세포내로 유입 축적되는 것을 방해함으로써 내성을 나타낸다.급성 골수성 백혈병에서 MDR-1유전자의 발현빈도와 항암치료에 대한 반응, 환자의 성, 나이, 염색체 이상, 혈청 LDH, 혈청 thymidine kinase, 수행능력, 백혈병의 아형, 혈색소, 말초혈액 및 골수의 골수아세포의 비율, CD34와 CD7 세포면역표지자 등과의 연관성에 대해 알아보았으며, 화학요법을 시행받은 환자를 대상으로 MDR-1유전자 발현과 완전 관해유도, 무병생존기간 및 전체생존기간의 임상경과를 비교 검토하였다. 방법:1994년 3월부터 1999년 6월까지 경북대학교 병원 혈액종양내과에서 처음 진단받은 급성 골수성 백혈병환자 36명을 대상으로 골수 또는 말초혈액을 채취하여 RNA를 추출하여 MDR-1 유전자와 β-actin분절의 시발체를 이용하여 역전이중합효소연쇄반응으로 PCR합성체를 만들어 전기영동하여 MDR-1유전자의 발현유무를 확인하였다. 결과:평균 연령은 43.69세(18∼77세)였으며, 남녀수는 16명과 20명이었다.MDR-1유전자는 19명(52.8%)에서 발현되었으며 MDR-1유전자의 발현율과 CD34와 CD7 세포면역표지자의 연관성을 분석한 결과 통계적으로 유의한 상관관계가 있었다.염색체 검사의 소견에서 예후가 양호한 군이 27명(75.0%), 예후가 나쁜 군은 9명(25.0%)이었고,MDR-1유전자 발현율과 연관성을 분석한 결과 MDR-1유전자 음성군에서 양호한 예후를 나타내는 염색체는 19명 중 11명(57.9%)으로 유의한 연관성을 보였다.관해유도화학요법후 말초혈액에서 골수아세포가 소실될 때까지의 시간은 MDR-1유전자 음성군이 5.4일로 양성군의 12.0일보다 짧았으나 통계적인 의미는 없으며, MDR-1유전자 발현은 환자의 나이, 수행능력, 백혈병 아형, 혈색소, 말초혈액 및 골수의 골수아세포 비율, 혈청LDH, 혈청 thymidine kinase, 비장종대 유무, 알부민 수치 등과 연관성이 없었다.MDR-1유전자 발현과 완전 관해유도와의 연관성을 분석한 결과 MDR-1유전자 음성군에서는 14명 중 13명(92.9%)이, 양성군에서는 12명 중 4명(33.3%)이 완전관해가 유도되어 통계적으로 유의한 차이가 있었으며, 무병생존기간과 전체 생존기간과의 연관성을 분석한 결과 통계적인 차이가 없었다. 결론:급성 골수성 백혈병에서 MDR-1유전자 발현율은 염색체 분류와 더불어 관해유도화학요법의 치료반응 유무를 예견할 수 있는 유용한 지표로 사용될 수 있고, 향후 치료전략을 수립하는데 참고할 자료가 되리라고 본다. Background:The expression of the multidrug resistance-1(MDR-1) gene which encodes p-glycoprotein, is recognized as a biological mechanism possibly contributing to treatment failure in patients with acut myeloid leukemia(AML). Recent studies indicate its association with poor risk factors such as cytogenetic partten and surface phenotype of blasts.We analyzed the role of MDR-1 gene expression in 36 chemo-naive AML patients. Methods: In 36 patients, clinical data were reviewed and compared to MDR-1 gene expression, immunophenocyping results on CD7 & CD34, cytogenetic pattern and other suggestive prognostic factors. Results: Median follow-up period was 250 days. The MDR-1 geng expression was obserned in 19 out of 36 patients(52.8%). Significant correlation between MDR-1 gene and CD7 & CD34 expression was found. SIxteen out of 17(94.1%) MDR-1 negative patients harbored favorable cytogenetic patterns, where as 11 out of 19 (57.9%) MDR-1 positive patients had favorable cytogenetic patterns. MDR-1 gene expression was not correlated to disease ferr survival(DFS), nor overall survival(OS) statistically although it has shown significant correlation to complete remission(CR) rate(p=0.001). Conclusion: We found that lack MDR-1 geng expression was exclusively associated to favorable cytogenetic patterns in our study.In order of MDR-1 geng and clinical outcome or other prognostic features, including cytogenetic pattren,further larger studies would be necessary.(Korean J Hematol 2000;35:117~125)

백혈병 세포에서 Multidrug Resistance Gene-1 (mdr1)의 과발현이 ^99m Tc-sestaMIBI 섭취에 미치는 영향

천경아,이재태,이상우,강도영,손상균,이종기,정준기,전수한,이규보 경북대학교 의학연구소 2000 경북대학교병원의학연구소논문집 Vol.4 No.1

Purpose: To determine whether 99mTc-MIBI is recognized by the multidrug resistant P-glycoprotein (Pgp), we have measured quantitatively 99mTc-MIBI uptake in cancer cells. The effects of various Pgp reversing agents on cellular 99mTc-MIBI uptake were also investigated in the presence of multidrug resistance gene-1(mdr1 gene) overexpression. Materisls and Methods: We measured percentage uptake of 99mTc-MIBI at different incubation temperatures both in mdr1 positive and negative cells. The effects of verapamil, cyclosporin, and dipyridamole on cellular uptake of 99mTc-MIBI were also evaluated with or withouts overexpression of mdr1 gene in Cultured murine leukemia L1210 cells. Results: The mdr1 gene expressing cell lines were effectively induced in in vitro with continuous application of low-dose adriamycin or vincristine. Cellular uptake of 99mTc-MIBI was higher in mdr1 negative L 1210 cells than those of mdr1 positive cells, and higher when incubated in 37℃ than 4℃. In the presence of verapamil, cyclosporin or dipyridamole, 99mTc-MIBI uptake was increased upto 604% in mdr1 positive cells. Conclusion: Cellular uptake of 99mTc-MIBI is lower in leukemia cells over-expressing mdr1 gene, and MDR-reversing agents increase cellular uptake. These results suggest that 99mTc-MIBI can be used for characterizing Pgp expression and developing MDR-reversing agents in vitro. (Korean J Nucl Med 1999;33: 152-62)

다약제 내성 유전자를 이용한 항류마티스 약제의 내성 검사 방법 개발

김상경 ( Sang Gyung Kim ),서헌석 ( Hun Suk Suh ),최정윤 ( Jung Yoon Choe ),이종원 ( Jong Won Lee ),서장수 ( Jang Soo Suh ),김신규 ( Think You Kim ) 대한류마티스학회 2003 대한류마티스학회지 Vol.10 No.1

Objective: A number of disease-modifying anti-rheumatic drugs (DMARDs) have been shown to be more effective than placebo in the management of rheumatoid arthritis (RA). However, most course of DMARDs, except methotrexate, are discontinued after 2 or 3 years, because of toxicity, lack of efficacy or escape from control. The multi-drug resistance (MDR) is a phenomenon in which cells develop cross-resistance to many agents such as anthracyclin, vinca alkaloids and colchicine. In our hypothesis, MDR phenomenon could be implicated in acquired resistance to DMARDs in RA. We have established a mdr1 cell line and tested whether DMARDs are substrate for P-glycoprotein (P-gp). Methods: The mdr1-cDNA was cloned into retroviral vector, and the recombinant retroviral vector was transfected into PA317 cells. The target cells, NIH3T3, were infected with recombinant retroviruses. A colony most resistant to vinblastin was selected for the following experiments; expression of mdr1 gene in NIH3T3 cells was confirmed by RT-PCR, and biological function of mdr1 gene product, P-gp, was tested using Rhodamine-123 (Rh123) efflux assay. Resistance of the target cells expression P-gp which can survive against hydroxychloroquine (HCQ) and methotrxate (MTX) were measured by MTT assay. Results: RT-PCR for mdr1 gene showed successful transfer of the gene into the NIH3T3 cells. Rh123 assay revealed expression of P-gp on the selected cells as follows; Rh123 efflux activity of uninfected NIH3T3 cells was 6%, that of PLXSN was 0.2%, and that of selected cells was 44%. The 50% proliferation inhibitory capacity of the selected cells were twice for HCQ but there was no difference of that for MTX. Conclusion: We established a mdr1 cell line and using the cell line, HCQ was a substrate of MDR, but MTX was not related to MDR.

난소암 세포주와 cisplatin 내성주에서의 MDR 관련 유전자의 발현에 관한 연구

문영진(Young Jin Moon),황윤영(Youn Yeung Hwang),이재규(Jai Kyu Lee),나영정(Young Jeong Na),이영미(Young Mi Lee),장성렬(Sung Yeoul Chang),조삼현(Sam Hyun Cho),김경태(Kyung Tai Kim) 대한산부인과학회 2001 Obstetrics & Gynecology Science Vol.44 No.9

N/A Objective : Expressions of P-glycoprotein, the multidrug resistance-associated protein (MRP) and the lung resistance protein (LRP) with the MDR phenotype widely divergent in human cancer cell lines. This study focused on the altered gene expression related drug transport. Methods : To examine correlations between MDR-associated genes and PKC isozyme with cisplatin resistance on the level of the mRNA expression, we analyzed MDR-associated gene (LRP, MDR1/P-gp and MRP) expression and PKC isozyme, topoisomerase II alpha and beta in cisplatin-sensitive ovarian cancer cell line A2780 and cisplatin - resistant cell line A2780cp using cDNA-PCR approach. Results : LRP mRNA levels were significantly increased in A2780cp compared to the drug sensitive variant. In contrast, MRP mRNA levels were not significantly correlated with drug sensitivity. A modest increase in PKCη and MDR1/P-gp mRNA expression activity was also observed in ovarian cancer A2780cp cell lines that were resistant to CDDP. The level of topoisomerase II alpha and beta were not affected. Conclusion : These results showed that MDR1/P-gp expression may be an important determinant of the MDR phenotype in cell lines intrinsically resistant to cancer chemotherapeutic agents and a multifactorial emergence of MDR phenotype of tumor with a possible involvement of the PKC isozymes may be associated with CDDP resistant ovarian cancer cell line.

Multi-Drug Resistance (MDR1) Gene Expression in de novo Acute Leukemia Cells : Correlations with CD Surface Markers and Treatment Outcome

Jiang, En Zi,Chang, Yoon Jong,Lee, Joong Won,Lee, Won Kil,Kim, Jay Sik,Sohn, Sang Kyun,Lee, Kyu Bo,Suh, Jang Soo 경북대학교 병원 2003 경북대학교병원의학연구소논문집 Vol.7 No.1

One important mechanism of drug resistance in acute leukemia is the over-expression of the multi-drug resistance(MDR1) gene that encodes a 170-kDa membrane protein called P-glycoprotein. To estimate the incidence and role of MDR1 gene expression in patients with acute leukemia, we investigated the expression of MDR1 by using the RT-PCR method in blast cells from 40 cases of de novo acute leukemia. We found a high frequency of MDR1 gene expression: 10 out of 20 with de novo acute myeloid leukemia(ALM), 8 OUT OF 17 with de novo acute lymphoblastic leukermia(ALL), and none of the 3 with de novo acute mixed leukemia, were MDR1 mRNA-positive. No correlation between cluster designation(CD) surface markers(CD19, CD7, CD13, CD33, CD34, CD14, HLA-DR) and MDR1 gene expression in AML was found. The complete remission rate was correlated with MDR1 gene expression. Among 40 evaluable patients examined, 17%(3 of 18) with MDR1 mRNA-postitive reached complete remission versus 77%(17 of 22) with MDR1 mRNA-negative(p=0.044). These results suggest that MDR1 gene expression can be used as a prognostic factor and may be helpful in determining chernotherapeutic protocol for patients with acute leukemia.

세무대리인 보고의무 강화방안에 관한 연구

김병일(Kim, Byung-Il),두철(Doo, Cheol) 한국조세법학회 2020 조세논총 Vol.5 No.3

본고는 일반적 조세회피방지규정(GAAR)과 상호보완책으로서 일부 국가에서 도입되고 있는 조세회피거래의 의무보고제도(MDR)에 관한 연구로 우리의 경우 동 제도가 아직 존재하지 않는다. 이는 조세회피를 억제하고 조세회피행위에 신속하게 대처하기 위해 조세회피상품의 개발･판매 등에 종사하는 자 소위 조장자 및 그 상품을 구입한 투자자인 납세자로 하여금 과세당국에 보고의무를 지우는 것이다. 본고에서는 이미 MDR을 도입하고 있는 주요국의 사례 등을 참고로 하여 구체적인 도입방안을 제시하고자 한다. 이를 요약하면 다음과 같다. 첫째, MDR 입법과정에서 헌법상 법치국가원리의 하나인 명확성 원칙에 위배되지 않도록 하여야 할 것이다. 둘째, MDR과 GAAR은 납세순응 측면에서 상호보완적이다. 따라서 MDR 도입시 어떤 거래가 조세회피행위에 해당하는지의 여부를 명확히 나타내주는 GAAR을 정비하는 것이 바람직하다. 다만, GAAR을 MDR과 연계시키거나 연계시키지 않는 어느 방식도 가능하다. 셋째, 보고대상거래의 범위와 관련하여 동 거래의 기준 내지 징표를 마련하여야 할 것이다. 조세회피거래 MDR을 도입한 국가들의 경우 역외거래에 한정하고 있지 않으므로 모든 거래에 적용하는 것이 타당할 것이나, 납세협력비용 등을 고려하여 역외거래와 연관된 국내거래나 국내거래 중 표준화된 금융상품으로 한정하는 안을 제안한다. 넷째, 보고대상세목은 소득세, 법인세, 부가가치세, 상속･증여세 등 상대적으로 조세구조가 복잡한 국세를 대상으로 하되, 향후 지방세로 확대해 나가야 할 것이다. 다섯째, 보고의무자는 원칙적으로 일정 매출액 이상의 대형법인이나 일정한 수입 이상을 거두는 조장자로 하고, 점진적으로 확대하는 것이 바람직할 것이다. 다만, 예외적으로 납세자가 자체개발하거나 조장자가 해외에 있는 경우 등에는 납세자에게 의무를 지운다. 여섯째, 보고의무를 이행했다고 해서 해당 거래가 합법으로 인정받거나 불법으로 처벌되는 것은 아니므로 과세당국은 이러한 사실을 보고의무자에게 알려주어야 할 것이다. This manuscript is a study on the reporting system of tax avoidance transactions that is introduced by some countries as trade-off with general anti-avoidance rule(GAAR) on tax avoidance product. This scheme’ purpose restrains tax avoidance and to promptly cope with tax evasion activity, and burdens duty to report to taxation authorities on promotor who sells and develops tax avoidance product and taxpayer that purchases it. As this manuscript refers to example of main countries introducing mandatory discloure rule(MDR), it presents specific plan after reviewing challenge. Thus, improvement plan for this is set out as follows. First, in the legislative process of MDR, the concept definition should suitable for the principle of clarity which is one of the constitutional principles. Second, the MDR of tax avoidance transactions and GAAR are trade-off in term of tax compliance perspective. Thus, in case of introducing this system, it is important to judge whether a certain trade is GAAR’s tax avoidance transactions and this method does not need to be definite. Third, in relation to range of reportable transactions, there needs to arrange a sign or a standard of reportable transactions. As countries intro- ducing GAAR’s tax avoidance transactions don’t limit off-shore transactions, this range propers to be applied to all of transaction, but its rage needs to apply to be restricted as off-shore transactions being related local trade and local trade standardized financial product. Fourth, there needs to first apply to national tax and then local tax on reportable transactions. Fifth, a person liable for reporting basically set-ups large companies of more than certain sales or promotor of certain import, and then needs to gradually magnify subject of application. Sixth, since performing this duty is not that its transactions is legally valid or illegally punished, tax authorities should notify a person liable for reporting its fact.

Identification of metabolites of MDR-1339, an inhibitor of β-amyloid protein aggregation, and kinetic characterization of the major metabolites in rats

Son, Jun-Hyeng,Jeong, Yoo-Seong,Lee, Jong-Hwa,Kim, Min-Soo,Lee, Kyeong-Ryoon,Shim, Chang-Koo,Kim, Young Ho,Chung, Suk-Jae Elsevier 2018 Journal of pharmaceutical and biomedical analysis Vol.151 No.-

<P><B>Abstract</B></P> <P>We previously reported that MDR-1339, an inhibitor of β-amyloid protein aggregation, was likely to be eliminated by biotransformation in rats. The objective of this study was to determine the chemical identity of metabolites derived from this aggregate inhibitor and to characterize the kinetics of formation of these metabolites in rats. Using high performance liquid chromatography coupled with mass spectrometry with a hybrid triple quadrupole-linear ion trap, 7 metabolites and 1 potential metabolic intermediate were identified in RLM incubations containing MDR-1339. In addition to these, 3 glucuronide metabolites were detected in urine samples from rats receiving a 10 mg/kg oral dose of MDR-1339. When the kinetics of the formation of two major metabolites, M1 and M2, were analyzed assuming simple Michaelis-Menten kinetics, the V<SUB>max</SUB> and K<SUB>m</SUB> values were found to be 0.459 ± 0.0196 nmol/min/mg protein and 28.3 ± 3.07 μM for M1, and 0.101 ± 0.00537 nmol/min/mg protein and 14.7 ± 2.37 μM for M2, respectively. When chemically synthesized M1 and M2 were individually administered to rats intravenously at the dose of 5 mg/kg respectively, the volume of distribution and elimination clearance were determined to be 4590 ± 709 mL/kg and 68.4 ± 5.60 mL/min/kg for M1 and 15300 ± 8110 mL/kg and 98.0 ± 19.5 mL/min/kg for M2, respectively. When MDR-1339 was intravenously administered to rats at a dose of 5 mg/kg, the parent drug and M1 were readily detected for periods of up to 6 h after the administration, but M2 was observed only from 2 to 4 h. A standard moment analysis indicates that the formation clearance of M1 is 6.01 mL/min/kg, suggesting that 19.7% of the MDR-1339 dose was eliminated in rats. These observations indicate that the hepatic biotransformation of MDR-1339 results in the formation of at least 10 metabolites and that M1 is the major metabolite derived from this aggregation inhibitor in rats.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Identification of ten metabolites (i.e., 7 Phase 1 + 3 Phase 2) of MDR-1339 in rats. </LI> <LI> Proposal for the major metabolic pathways to be CYP3A4, 2B6 and 2C9. </LI> <LI> Simultaneous quantification of MDR-1339 and its major metabolite M1 and M2. </LI> <LI> Formation of M1 accounted for ∼19.7% of the total MDR-1339 elimination in rats. </LI> </UL> </P>