Benzotriazine Di-Oxide Prodrugs for Exploiting Hypoxia and Low Extracellular pH in Tumors

Abstract

Extracellular acidification is an important feature of tumor microenvironments but has yet to be successfully exploited in cancer therapy. The reversal of the pH gradient across the plasma membrane in cells that regulate intracellular pH (pHi) has potential to drive the selective uptake of weak acids at low extracellular pH (pHe). Here, we investigate the dual targeting of low pHe and hypoxia, another key feature of tumor microenvironments. We prepared eight bioreductive prodrugs based on the benzotriazine di-oxide (BTO) nucleus by appending alkanoic or aminoalkanoic acid sidechains. The BTO acids showed modest selectivity for both low pHe (pH 6.5 versus 7.4, ratios 2 to 5-fold) and anoxia (ratios 2 to 8-fold) in SiHa and FaDu cell cultures. Related neutral BTOs were not selective for acidosis, but had greater cytotoxic potency and hypoxic selectivity than the BTO acids. Investigation of the uptake and metabolism of representative BTO acids confirmed enhanced uptake at low pHe, but lower intracellular concentrations than expected for passive diffusion. Further, the modulation of intracellular reductase activity and competition by the cell-excluded electron acceptor WST-1 suggests that the majority of metabolic reductions of BTO acids occur at the cell surface, compromising the engagement of the resulting free radicals with intracellular targets. Thus, the present study provides support for designing bioreductive prodrugs that exploit pH-dependent partitioning, suggesting, however, that that the approach should be applied to prodrugs with obligate intracellular activation.

Mimicking tumor hypoxia and tumor-immune interactions employing three-dimensional in vitro models

“…While most of the cancer drugs are designed to target rapidly proliferating cells, tumor cells in a hypoxic niche evade the therapeutic effect of these drugs by demonstrating low proliferation [105]. Low oxygen can result in an acidic environment, relative to the pH heterogeneity inside the TME [106], which inhibits drug uptake rates in cells due to the impediment of molecule diffusion as a result of unnecessary cell membrane charging [107]. Most cancer drugs are weakly basic drugs that become ionized in the acidic hypoxia-driven TME and hence fail to exert their therapeutic effects [108].…”

Zwitterionic Block Copolymer Prodrug Micelles for pH Responsive Drug Delivery and Hypoxia-Specific Chemotherapy

“…22,29 Recently, some TPZ derivatives with increased hydrophobicity and functional groups, such as alkyl carboxylic acid and alkyl amine groups, have been reported with similar bioactivity to TPZ, providing a new approach for the development of TPZD-nanocarrier conjugates through facile chemical reactions. 30,31 Amphiphilic block copolymers (BCPs) have been intensively studied as promising nanomedicines due to their excellent selfassembly ability and biocompatibility. 32 Recently, some exciting strategies in constructing versatile BCPs have been developed for stimuli-triggered drug delivery systems, significantly improving the therapeutic effect of low-molecular drugs or prodrugs in anticancer therapy.…”

“…Over the last few decades, a limited number of drug delivery systems including inorganic − and polymer-based nanoparticles ,, have been developed for the physical encapsulation of the TPZ prodrug, showing encouraging outcomes for hypoxic tumor therapy. Nonetheless, due to the hydrophilic nature of TPZ (2.4 mg/mL in saline), these physically TPZ-encapsulated nanoparticles could not provide efficient and controlled entrapment of TPZ, impeding its full therapeutic potential. , Recently, some TPZ derivatives with increased hydrophobicity and functional groups, such as alkyl carboxylic acid and alkyl amine groups, have been reported with similar bioactivity to TPZ, providing a new approach for the development of TPZD-nanocarrier conjugates through facile chemical reactions. , …”