A Comparison of in Vitro and in Vivo Stability in Mice of Two Morpholino Duplexes Differing in Chain Length

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• 作者：Jiang He ; Guozheng Liu ; Surong Zhang ; Jean-Luc Vanderheyden ; Ning Liu ; Changbin Liu ; Yumin Zhang ; Suresh Gupta ; Mary Rusckowski ; and Donald J. Hnatowich
• 刊名：Bioconjugate Chemistry
• 出版年：2003
• 出版时间：September 2003
• 年：2003
• 卷：14
• 期：5
• 页码：1018 - 1023
• 全文大小：70K
• 年卷期：v.14,no.5(September 2003)
• ISSN：1520-4812

文摘

The stability of hybridized duplexes is an important criterion for any radiopharmaceutical applicationof DNAs or their analogues such as phosphorodiamidate morpholinos (MORFs). Objective: Thestabilities in vitro and in mice of the duplex between MORF and its complement (cMORF) wereinvestigated for two different chain lengths, a 15-mer MORF compared to the identical MORF butelongated to a 25-mer. Methods: The hybridization characteristics of the 15-mer MORF with itscomplementary 15-mer and that of the 25-mer with its complementary 25-mer MORF were measuredusing surface plasmon resonance (SPR) analysis. For radiolabeling with ^99mTc, the 15- and 25-merMORF, both with a primary amine via a 10-member linker on the 3' equivalent end, were conjugatedwith NHS-MAG₃. The 15- and 25-mer cMORFs were conjugated via their amines to carbodiimidazoletreated poly(methyl vinyl ether-alt-maleic acid) (PA) such that about 50 cMORFs were attached toeach polymer molecule in both cases (estimated MWs about 300 and 450 kDa, respectively). Afterhybridization in vitro, both the PA-cMORF15-^99mTc-MORF15 and PA-cMORF25-^99mTc-MORF25homoduplexes were evaluated by size exclusion HPLC in saline, after incubation in 37 C serum andin urine obtained 30 min post IV administration to normal mice. Biodistributions were obtained upto 18 h post administration. Results: By SPR, the affinity constants for the homoduplexes were bothabout 10⁹ M^-1 with the 25/25 only about 25% higher than the 15/15. However, the affinity constantsfor 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 PA-cMORF15-^99mTc-MORF15 showed an immediate and pronounced low molecular weight peak that wasidentified by a shift assay to be ^99mTc-MORF15. The comparable peak in both fluids was much lesspronounced in the case of PA-cMORF25-^99mTc-MORF25. Whole body radioactivity levels also fell muchmore rapidly in mice receiving the 15-mer conjugate (65 vs 30% eliminated at 18 h) and biodistributionresults showed higher kidney levels for the 15-mer conjugate. Results with the PA-cMORF25-^99mTc-MORF15 heteroduplex were more similar to that obtained with the 15-mer homoduplex than the25-mer homoduplex. Conclusion: Despite what is reported to be high hybridization affinities, boththe homoduplex and heteroduplexes prepared with ^99mTc-MORF15 were found to be unstable in serumand in vivo toward dissociation to free ^99mTc-MORF15. By contrast, homoduplex prepared with^99mTc-MORF25 showed higher stability. These differences in hybridization stability may be importantconsiderations in radiopharmaceutical design.