Study of the Stability of Oligodeoxynucleotide–Doxorubicin Conjugate In Vitro and Its Pharmacokinetics In Vivo by RP‐HPLC

Abstract

Oligodeoxynucleotide–doxorubicin conjugate is a novel modified oligodeoxynucleotide (ODN) to inhibit the expression of mdr1 gene. The present study was undertaken to determine the stability of the conjugate in vitro and its pharmacokinetics in vivo using a reverse‐phase HPLC assay. The method employed a short C18 reverse phase column combined with a C8 pre‐column and a linear gradient elution with acetonitrile containing 0.1 M aqueous triethylammonium acetate, pH 7.0. Detection was carried out using a UV‐diode array detector at 254 and 480 nm. Minimum sensitivity of ∼0.15 µg/mL in plasma and 0.1 µg/mL in PBS of the conjugate was achieved. After incubation in 10% activated fetal calf serum for 24 hours, 15.8% of the conjugate was degraded. As a comparison however, 97.2% of the control ODN was degraded within the same incubation time. The pharmacokinetics studies show that the half‐lives of the conjugate is about 8 hours, 4 times longer than ODN as a control. Assay validation studies revealed that the method is accurate, reproducible for determination of the conjugate, and can be used for a pharmacokinetic study of the conjugate.