Synthesis of poly(ethylene glycol)-based saquinavir prodrug conjugates and assessment of release and anti-HIV-1 bioactivity using a novel protease inhibition assay

Simi Gunaseelan, Olivia Debrah, Li Wan, Michael J. Leibowitz, Arnold B. Rabson, Stanley Stein, Patrick J. Sinko

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37 Scopus citations


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 modifying its 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-Tat nonapeptide]-PEG3400, and SQV-cysteine(R.I.CK(stearate)-Tat9)-PEG3400. SQV was linked to cysteine to form a releasable SQV-cysteine ester bond in all of the conjugates. The amino group of the cysteine moiety provided an attachment site for a slower-degrading amide bond with N-hydroxysuccinimide-activated forms of PEG- and PEG-biotin. Bisulfide bonds were used to attach the cationic peptides, R.I.CK-Tat9 and R.I.CK(stearate)-Tat9 to the cysteine moiety in order to provide cell-specific release. An assay was established and validated for measuring the activity of SQV and other protease inhibitors in biological samples. In this assay, cleavage of an internally quenched fluorescent substrate, Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gly-Lys(DABCYL)-Arg by HIV-1 protease was inhibited by SQV in a dose-dependent manner at concentrations of 0.05-0.5 μM. All prodrug conjugates were shown to be inactive in this assay until the ester bond was cleaved and active 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-1 activity (ED50) of the PEG-based SQV prodrug conjugates was evaluated in MT-2 cells using an MTT assay. The activity of conjugated SQV was reduced (ED50 = 900 nM) for the PEG only conjugate, but restored 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 (unconjugated SQV, control, ED 50 = 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 the micromolar range. The difference between the LD50 and ED50 suggests a favorable therapeutic index for the prodrug conjugates. In conclusion, these promising initial results demonstrate that the reconversion of the conjugate prodrugs was complete and that active SQV was released. Since the major delivery advantages of PEG prodrug conjugates can only be observed in vivo, issues of reconversion 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 effective bioanalytical tool that can be used to assess the release and anti-HIV-1 activity of HIV-1 PIs from their prodrug forms.

Original languageEnglish (US)
Pages (from-to)1322-1333
Number of pages12
JournalBioconjugate Chemistry
Issue number6
StatePublished - 2004

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry


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