Delivery of peptide-mimetic drugs to tumors utilizing oligopeptide transporters

利用寡肽转运蛋白将肽模拟药物递送至肿瘤

• 批准号: 12557204
• 负责人: TSUJI Akira
• 金额: $ 6.21万
• 依托单位: KANAZAWA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12557204/
• 关键词: transporter; oligopeptide; anti-cancer reagent; bestatine; quantitative PCR; tumor; nude mouse; drug delivery; アデノウィルス; ベスタチン; グリシルサルコシン; ヌードマウス; トラックデリバリー

In the present study, we studied feasibility of drug delivery using oligopeptide transporter which expressed in some tumor cell lines. During this study period, we obtained following findings.1. A stable cell line which over expresses human oligopeptide transporter PEPT1 was established by transfecting hPEPTl cDNA into HeLa cells. Its transport actiyity was confirmed by measuring glycylsarcosine and termed the cell line HeLa-hPEPT1.2. Nude mice (Balb/c nu/nu mice) inoculated with HeLa-hPEPT1 bared tumor within several weeks. In these mice, I.v. injected an anti-tumor drug bestatine, and dipeptide carnosine were accumulated in the hPEPT1-expressing tumors.3. HeLa-hPEPT1 was more sensitive to bestatine than parent HeLa cell line by in vitro MTT assay. Tiimor growth of HeLa-hPEPT1 in nude mice was strongly suppressed by oral dose of bestatine to the mice, while that of HeLa inoculated mice was not affected.4. Messenger RNA expression levels of known oligopeptide transporters that is PEPT1 and PEPT2 were quantitatively analyzed by the real time PCR in iridivisual 25 tumbf lines. Among them ML-1, Nakajima and Caco-2 cells expressed PEPT1in high level on the other hand, PEPT2 expression was observed majority of cell lines examined. Comparing mRNA expression and transport activity profiles, we found little correlation between them. Therefore, existence ofnovel transporter that represents the uptake of peptides has been suggested.5. Molecular cloning of CDNA of organic cation transporter OCTNs and OATPs were performed and some of which were expressed in tumor cell lines.6. We have proved that expression of amino acid transporter LAT1 and LAT2, which play roles in tumor growth, in the blood-brain barrier.In conclusion, detailed characterization of many kinds of transporters which expressed in tumors will provide us valuable information to establish method for tumor specific delivery of drugs.

在本研究中，我们研究了使用在某些肿瘤细胞系中表达的寡肽转运蛋白进行药物递送的可行性。在本次研究期间，我们得到了以下研究结果： 1．通过将hPEPT1 cDNA转染入HeLa细胞来建立过表达人寡肽转运蛋白PEPT1的稳定细胞系。通过测量甘氨酰肌氨酸证实了其转运活性，并将其命名为细胞系HeLa-hPEPT1.2。接种 HeLa-hPEPT1 的裸鼠（Balb/c nu/nu 小鼠）在几周内就会露出肿瘤。在这些小鼠中，I.v.注射抗肿瘤药物bestatin，二肽肌肽在表达hPEPT1的肿瘤中积累。 3．体外MTT测定显示，HeLa-hPEPT1对贝他汀比亲本HeLa细胞系更敏感。口服贝他汀可强烈抑制裸鼠HeLa-hPEPT1的生长，而接种HeLa的小鼠则不受影响。 4．通过实时 PCR 在 iridivisual 25 个 tumbf 细胞系中定量分析了已知寡肽转运蛋白 PEPT1 和 PEPT2 的信使 RNA 表达水平。其中ML-1、Nakajima和Caco-2细胞高水平表达PEPT1，另一方面，在大多数检测的细胞系中观察到PEPT2表达。比较 mRNA 表达和转运活性谱，我们发现它们之间几乎没有相关性。因此，有人提出了代表肽摄取的新型转运蛋白的存在。 5．对有机阳离子转运蛋白OCTNs和OATPs的cDNA进行了分子克隆，并在肿瘤细胞系中得到表达。 6．我们已经证明，在肿瘤生长中发挥作用的氨基酸转运蛋白LAT1和LAT2在血脑屏障中表达。 总之，对肿瘤中表达的多种转运蛋白的详细表征将为我们建立肿瘤特异性药物递送方法提供有价值的信息。

项目成果

Tamai, I.: "Transporter-mediated permeation of drugs across the blood-brain barrier"J. Pharm. Sci.. 89. 1371-1388 (2000)

Tamai，I.：“转运蛋白介导的药物跨血脑屏障渗透”J.

辻 彰: "消化管吸収過程における薬物間相互作用"月刊薬事. 42. 287-293 (2000)

Akira Tsuji：“胃肠道吸收过程中的药物相互作用”月刊药事42。287-293（2000）。

Mayatepek, E.: "Two novel missense mutations of the OCTN2 gene (W283R and V446F) in a patient with primary systemic carnitine deficiency"Hum. Mutat.. 15. 118 (2000)

Mayatepek, E.：“原发性全身性肉碱缺乏症患者中 OCTN2 基因的两个新错义突变（W283R 和 V446F）”Hum。

Naruhashi K: "Active intestinal secretion of new quinolone antimicrobials and the partial contribution of P-glycoprotein"J.Pharm. Pharmacol.. 53・5. 699-709 (2001)

Naruhashi K：“新型喹诺酮类抗菌剂的活性肠道分泌和 P-糖蛋白的部分贡献”J.Pharmacol.. 53・5 (2001)。

玉井郁巳: "OCTNトランスポーターファミリーの組織分布・輸送の多様性"薬物動態. 15. 182-188 (2000)

Ikumi Tamai：“OCTN 转运蛋白家族的组织分布和转运多样性”药代动力学 15. 182-188 (2000)。