THE MEASUREMENT OF POLYGLUTAMATE METABOLITES OF THE THYMIDYLATE SYNTHASE INHIBITOR, ICI-D1694, IN MOUSE AND HUMAN CULTURED-CELLS
THE MEASUREMENT OF POLYGLUTAMATE METABOLITES OF THE THYMIDYLATE SYNTHASE INHIBITOR, ICI-D1694, IN MOUSE AND HUMAN CULTURED-CELLS
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DOI：
10.1016/0006-2952(93)90170-2
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发表时间：
1993-02-24
影响因子：
5.8
通讯作者：
JACKMAN, AL
中科院分区：
文献类型：
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作者：
GIBSON, W;BISSET, GMF;JACKMAN, AL
描述了一种测定喹唑啉胸苷合成酶抑制剂N-(5-[N-(3，4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-theonyl)-L-glutamic酸(ICID1694)的多谷氨酸的方法。这包括用[5-H-3]ICI D1694孵育细胞，提取多谷氨酸，并使用离子配对方法用高效液相色谱法进行分析。与ICI D1694及其合成的六聚谷氨酸盐标准品(UV检测)的共层析有助于从高效液相色谱回收的级分中鉴定[H-3]多谷氨酸盐。多聚谷氨酸在提取和分析的各个阶段的回收率都很好(总回收率为77-84%)。聚谷氨酸以三元、四元和五元形式容易积累，偶尔还会发现少量的六谷氨酸。小鼠L1210白血病细胞或人W1L2淋巴母细胞与0.1um[H-3]ICI D1694孵育30分钟后，细胞内浓度效应约为6倍，大多数H-3与多谷氨酸形式有关(L1210和W1L2分别约为75%和96%)。此时甚至可以检测到一些较高链长的四和五谷氨酸盐。培养4小时后，细胞内H-3的总水平上升到2-3微米，其中96%以上与多谷氨酸(主要是四和五谷氨酸)有关。另外四个人类细胞系，两个卵巢细胞系(CH1和41M)，MCF-7乳腺细胞和HT-29结肠细胞系，被检测了它们形成细胞内多谷氨酸的能力。与0.1um[H-3]ICI D1694孵育4小时后，药物以多谷氨酸形式在细胞内大量积累(20-100倍)，仅剩下2-20%作为母体单谷氨酸，具体取决于细胞系。主要的多聚谷氨酸再次依赖于细胞系，从三种形式到五种形式。将孵育时间延长至24小时，可进一步积累药物，更大比例的药物以三至六谷氨酸盐的形式出现。虽然不同的细胞系在形成多谷氨酸的总水平和观察到的链长模式上有所不同，但在所研究的所有细胞类型中，ICID1694都发生了快速和广泛的多谷氨酸。
A method is described for the measurement of the polyglutamates of the quinazoline thymidylate synthase inhibitor, N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-theonyl)-L-glutamic acid (ICI D1694). This involved incubation of cells with [5-H-3]ICI D1694, extraction of the polyglutamates and their analysis by HPLC using an ion-pairing method. Cochromatography with ICI D1694 and its synthetic di-hexaglutamate standards (UV detection) aided identification of the [H-3]polyglutamates in the fractions recovered from the HPLC. Recovery of the polyglutamates at each stage of extraction and analysis was very good (77-84% overall recovery). Polyglutamates readily accumulated as the tri-, tetra and penta forms and occasionally a small amount of hexaglutamate was found. After mouse L1210 leukemia or human W1L2 lymphoblastoid cells were incubated for 30 min with 0.1 muM [H-3]ICI D1694 there was a approximately 6-fold concentration effect intracellularly with most of the H-3 associated with polyglutamate forms (approximately 75% and 96% for the L1210 and W1L2, respectively). Even some of the higher chain length tetra- and pentaglutamates could be detected at this time. After 4 hr incubation the total level of intracellular H-3 had risen to 2-3 muM, greater than 96% of which was associated with polyglutamates (mainly tetra- and pentaglutamates). Four other human cell lines, two ovarian (CH1 and 41M), the MCF-7 breast and the HT-29 colon, were examined for their ability to form intracellular polyglutamates. A 4 hr incubation with 0.1 muM [H-3]ICI D1694 resulted in a substantial intracellular accumulation of the drug (20-100-fold) in its polyglutamate forms with only 2-20% remaining as the parent monoglutamate, depending on the cell line. The major polyglutamate was again cell line dependent, ranging from the tri to the penta form. Prolonging the incubation time to 24 hr allowed a further accumulation of drug with a larger percentage appearing as tri- to hexaglutamates. Although cell lines differed in the total level of polyglutamates formed and the pattern of chain length observed, rapid and extensive polyglutamation of ICI D1694 occurred in all the cell types examined.