• Bioreductive activation of tumor targeted drug delivery systems containing melphalan

      Weerapreeyakul, Natthida; Chikhale, Prashant J. (2001)
      Solid tumors provide an environment conducive to bioreduction due to hypoxia and overexpression of bioreductive enzymes. Therefore, TDDS were designed with various substituents to modulate bioreductive activation. TDDS contain a quinone-based carrier coupled to the model drug, melphalan methyl ester (MME). Controlled bioreductive activation of TDDS can lead to specific drug release only at the tumor sites and hence reduce toxicity during systemic distribution. The rate and extent of bioreductive activation of TDDS was determined in presence of DT-diaphorase, xanthine oxidase, human breast tumor cells (MCF-7) and colon tumor cells (Caco-2). Stability of TDDS under aqueous buffer conditions and in the presence of glutathione was also evaluated. Anticancer activity of TDDS was determined based on alkylating activity, cytotoxicity and apoptotic induction in both the tumor cell lines. Results show that all TDDS especially CH3-TDDS improved stability of melphalan under pH 7.4. H-TDDS possessed significantly high bioreductive ability in both tumor cells, which was predominantly catalyzed by the reductive enzymes, DT-diaphorase. TDDS with the electron-withdrawing substituent (Br-TDDS) underwent rapid glutathione-catalyzed degradation in tumor cell homogenates. TDDS with electron-donating substituents exhibited either relatively low (CH3-TDDS) or no (C4H4-TDDS or CH3NH-TDDS) bioreduction in tumor cell homogenates. Thus, bioreductive activation of TDDS was influenced by the electronic properties of the substituents. Bioreduction of TDDS (in the presence of purified reductase enzymes and in the MCF-7 and Caco-2 cells) was consistent with their reductive ability (reduction potentials and sodiumborohydride reduction). Both H-TDDS and CH3-TDDS exhibited alkylating activity similar to MME and melphalan. While H-TDDS demonstrated relatively high cytotoxicity and apoptosis induction in the MCF-7 cells, CH 3-TDDS demonstrated relatively high cytotoxicity and apoptosis induction in the Caco-2 cells. MME and melphalan exhibited higher cytotoxicity and apoptosis induction than TDDS in the tumor cells. Based on stability under physiological pH and susceptibility to DT-diaphorase in human tumor cells, H-TDDS and CH3-TDDS appear to be most promising candidates for in vivo antitumor efficacy studies. Such bioreductive TDDS could be useful in targeting a variety of anticancer agents to solid tumors.
    • Modulation of cerebrovascular LAT1 for brain delivery of amino acid-containing anticancer agents

      Killian, Dennis Matthew; Chikhale, Prashant J. (2001)
      The blood-brain barrier (BBB) restricts brain transport for many therapeutic agents. Strategies to improve brain uptake of potential central nervous system therapeutics are needed. This research demonstrates that drug transport via the cerebrovascular large, neutral amino acid transporter (LAT) can be modulated to potentially improve brain drug delivery. We have created two novel drug delivery systems for amino acid-containing agents, BTDS and TDDS. BTDS possesses high recognition for cerebrovascular LAT while TDDS is not recognized by LAT, thereby demonstrating modulation of BBB transport. An in situ rat brain perfusion was utilized to examine cerebrovascular interactions as well as to determine brain uptake parameters. Radiotracer [14C]-L-Leu, an endogenous LAT ligand, was used to probe for cerebrovascular LAT expression. TDDS did not significantly inhibit [14C]-L-Leu brain uptake, suggesting that these agents are not transported into brain via LAT. Quantitative brain uptake of TDDS by UV-HPLC demonstrated rapid brain uptake. Additionally, evidence of TDDS brain uptake was provided using ESI-based mass spectrometry. In contrast, BTDS significantly inhibited [14C]-L-Leu PA product (down to 8--36% of control), to show high recognition for cerebrovascular LAT. Cerebrovascular LAT Ki determination for IBM-BTDS yielded 11.3 +/- 2.8 muM in parietal cortex of rat brain, which is similar to the high affinity values observed for endogenous ligands, L-Leu and L-Phe. Additionally, the LAT1 isoform was shown to be functionally predominantly at the cerebrovasculature. This work demonstrates that chemical alterations on amino acid-based compounds to form drug delivery systems modulate cerebrovascular LAT-mediated BBB transport. This research can potentially lead to therapeutically and pharmaceutically usefully brain drug delivery systems for the effective treatment of various CNS disorders and diseases.