Synthesis of Poly(ethylene glycol)-Based Saquinavir Prodrug Conjugates and Assessment of Release and Anti-HIV-1 Bioactivity Using a Novel Protease Inhibition Assay

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• 作者：Simi Gunaseelan ; Olivia Debrah ; Li Wan ; Michael J. Leibowitz ; Arnold B. Rabson ; Stanley Stein ; and Patrick J. Sinko
• 刊名：Bioconjugate Chemistry
• 出版年：2004
• 出版时间：November 2004
• 年：2004
• 卷：15
• 期：6
• 页码：1322 - 1333
• 全文大小：257K
• 年卷期：v.15,no.6(November 2004)
• ISSN：1520-4812

文摘

Various poly(ethylene glycol)(PEG)-based prodrug conjugates of the HIV-1 protease inhibitor (PI)saquinavir (SQV) were prepared using several types of chemical groups potentially capable of modifyingits pharmacokinetic properties. These prodrug conjugates included SQV-cysteine-PEG3400, SQV-cysteine-PEG3400-biotin, SQV-cysteine(R.I.CK-Tat9) [a cationic retro-inverso-cysteine-lysine-Tatnonapeptide]-PEG3400, and SQV-cysteine(R.I.CK(stearate)-Tat9)-PEG3400. SQV was linked tocysteine to form a releasable SQV-cysteine ester bond in all of the conjugates. The amino group ofthe cysteine moiety provided an attachment site for a slower-degrading amide bond with N-hydroxysuccinimide-activated forms of PEG- and PEG-biotin. Disulfide bonds were used to attachthe cationic peptides, R.I.CK-Tat9 and R.I.CK(stearate)-Tat9 to the cysteine moiety in order to providecell-specific release. An assay was established and validated for measuring the activity of SQV andother protease inhibitors in biological samples. In this assay, cleavage of an internally quenchedfluorescent substrate, Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gly-Lys(DABCYL)-Arg by HIV-1protease was inhibited by SQV in a dose-dependent manner at concentrations of 0.05-0.5 M. Allprodrug conjugates were shown to be inactive in this assay until the ester bond was cleaved andactive SQV was released. The prodrug reconversion half-lives in 0.1 N HCl, phosphate-buffered saline(PBS) at pH 7.4 and in spiked plasma at 37 C were 9, 14, and 0.9 h, respectively. The anti-HIV-1activity (ED₅₀) of the PEG-based SQV prodrug conjugates was evaluated in MT-2 cells using an MTTassay. The activity of conjugated SQV was reduced (ED₅₀ = 900 nM) for the PEG only conjugate, butrestored with the addition of biotin (ED₅₀ = 125 nM), R.I.CK-Tat9 (ED₅₀ = 15 nM), and R.I.CK(stearate)-Tat9 (ED₅₀ = 62 nM) as compared to maximum achievable anti-HIV-1 activity (unconjugatedSQV, control, ED₅₀ = 15 nM), suggesting enhanced cellular uptake of conjugates. Cytotoxicity (LD₅₀)was assessed for all prodrug conjugates using non-HIV-1 infected cells and was found to be in themicromolar range. The difference between the LD₅₀ and ED₅₀ suggests a favorable therapeutic indexfor the prodrug conjugates. In conclusion, these promising initial results demonstrate that thereconversion of the conjugate prodrugs was complete and that active SQV was released. Since themajor delivery advantages of PEG prodrug conjugates can only be observed in vivo, issues ofreconversion and elimination half-lives in plasma will have to be further studied in an in vivo model.The current results also demonstrate that the protease inhibition assay is a simple yet effectivebioanalytical tool that can be used to assess the release and anti-HIV-1 activity of HIV-1 PIs fromtheir prodrug forms.