A comparison of in vitro and in vivo stability in mice of two morpholino duplexes differing in chain length
A comparison of in vitro and in vivo stability in mice of two morpholino duplexes differing in chain length
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DOI：
10.1021/bc0341019
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发表时间：
2003-09-01
影响因子：
4.7
通讯作者：
Hnatowich, DJ
中科院分区：
文献类型：
--
作者：
He, J;Liu, GZ;Hnatowich, DJ
杂交双链的稳定性是DNA或其类似物如磷二酰吗啉(Morf)放射性药物应用的重要标准。目的：研究两种不同链长的Morf-补体双链(CMORF)在体外和小鼠体内的稳定性，其中15聚体与相同的Morf相比延长至25聚体。方法：用表面等离子体共振(SPR)技术检测15聚体Morf及其互补的15聚体和25聚体与其互补的25聚体Morf的杂交特性。对于~(99m)Tc标记，15和25-聚Morf均通过3‘等效端的10元连接物与NHS-MAG(3)偶联。15和25聚体cMORF通过它们的胺连接到经碳二咪唑处理的聚(甲基乙烯基醚-ALT-马来酸)(PA)上，使得在两种情况下每个聚合物分子上都附着了大约50个cMORF(估计相对分子质量分别约为300和450 kDa)。体外杂交后，用体积排斥法测定PA-cMORF15-TC-99m-MORF15和PA-cMORF25-TC-99m-MORF25同源双链在生理盐水中、37℃C血清孵育后和正常小鼠静脉注射后30min尿液中的含量。在给药后18h内获得了生物分布。结果：SPR测得同源双链的亲和常数均为109M-1，其中25/25的亲和常数仅比15/15高约25%，而15/25和25/15的异源双链的亲和常数相差13倍。经高效液相色谱分析，所有双链在生理盐水中都是稳定的；然而，对含有PAcMORF15-TC-99m-MORF15的血清孵育物和尿液的分析显示，立即出现了一个明显的低分子量峰，经位移分析鉴定为TC-99m-MORF15。在PA-cMORF25-TC-99m-MORF25的情况下，两种液体中的可比峰要明显得多。接受15-聚体结合物的小鼠全身放射性水平也下降得更快(18小时消除65%对30%)，生物分布结果显示15-聚体结合物的肾脏水平更高。结果PA-cMORF25-(99m)TcMORF15异源双链与15聚体的同源双链比25聚体的同源双链更相似。结论：尽管Te-99m-MORF15具有较高的杂交亲和力，但Te-99m-MORF15制备的同源双链和异源双链在血清和体内均不稳定，不能解离为游离的Te-99m-MORF15。与之相比，用~(99)m-MORF25制备的同源双链具有更高的稳定性。杂交稳定性的这些差异可能是放射性药物设计中的重要考虑因素。
The stability of hybridized duplexes is an important criterion for any radiopharmaceutical application of DNAs or their analogues such as phosphorodiamidate morpholinos (MORFs). Objective: The stabilities in vitro and in mice of the duplex between MORF and its complement (cMORF) were investigated for two different chain lengths, a 15-mer MORF compared to the identical MORF but elongated to a 25-mer. Methods: The hybridization characteristics of the 15-mer MORF with its complementary 15-mer and that of the 25-mer with its complementary 25-mer MORF were measured using surface plasmon resonance (SPR) analysis. For radiolabeling with Tc-99m, the 15- and 25-mer MORF, both with a primary amine via a 10-member linker on the 3' equivalent end, were conjugated with NHS-MAG(3). The 15- and 25-mer cMORFs were conjugated via their amines to carbodiimidazole treated poly(methyl vinyl ether-alt-maleic acid) (PA) such that about 50 cMORFs were attached to each polymer molecule in both cases (estimated MWs about 300 and 450 kDa, respectively). After hybridization in vitro, both the PA-cMORF15-Tc-99m-MORF15 and PA-cMORF25-Tc-99m-MORF25 homoduplexes were evaluated by size exclusion HPLC in saline, after incubation in 37 degreesC serum and in urine obtained 30 min post IV administration to normal mice. Biodistributions were obtained up to 18 h post administration. Results: By SPR, the affinity constants for the homoduplexes were both about 109 M-1 with the 25/25 only about 25% higher than the 15/15. However, the affinity constants for the 15/25 and 25/15 heteroduplexes showed a surprisingly 13-fold difference. By HPLC analysis, all duplexes were stable in saline; however, analysis of serum incubates and urine containing PAcMORF15-Tc-99m-MORF15 showed an immediate and pronounced low molecular weight peak that was identified by a shift assay to be Tc-99m-MORF15. The comparable peak in both fluids was much less pronounced in the case of PA-cMORF25-Tc-99m-MORF25. Whole body radioactivity levels also fell much more rapidly in mice receiving the 15-mer conjugate (65 vs 30% eliminated at 18 h) and biodistribution results showed higher kidney levels for the 15-mer conjugate. Results with the PA-cMORF25-(99m)TcMORF15 heteroduplex were more similar to that obtained with the 15-mer homoduplex than the 25-mer homoduplex. Conclusion: Despite what is reported to be high hybridization affinities, both the homoduplex and heteroduplexes prepared with Te-99m-MORF15 were found to be unstable in serum and in vivo toward dissociation to free Tc-99m-MORF15. By contrast, homoduplex prepared with Tc-99m-MORF25 showed higher stability. These differences in hybridization stability may be important considerations in radiopharmaceutical design.