Benzotriazine di-oxide prodrugs for exploiting hypoxia and low extracellular pH in tumours

Abstract

One of the key characteristics of the tumour microenvironment in addition to hypoxia is acidosis. Extracellular acidification in tumours results from the metabolic shift towards glycolysis, reduced clearance of metabolic acids and upregulation of membrane transporters involved in the regulation of intracellular pH (pHi). The resulting regulation of pHi at relatively alkaline values when extracellular pH (pHe) falls establishes an inverse pH gradient across the plasma membrane relative to normal cells. This reversal can be exploited in drug design to potentially superimpose additional tumour selectivity on hypoxia activated prodrugs (HAPs) by targeting acidosis as well as hypoxia. This possibility has been tested by adding acidic sidechains to benzotriazine di-oxide (BTO) prodrugs related to tirapazamine and SN30000. The 1st generation BTO acid prodrugs showed evidence for dual selectivity, but the poor potency and selectivity of these compounds was hypothesised to be due to their very low pKa values resulting in slow kinetics of cellular uptake. The aims of the present study were to measure the trans-plasma membrane pH gradient in relevant cell models, and to test whether an increase in pKa in 2nd generation BTO prodrugs provides an increase in potency. This study illustrated that it is possible to measure hypoxic selectivity using cells that are adapted to 0.2% oxygen for 24 hours, compared to acute assays under anoxia. Overall, the 2nd generation compounds achieved a higher potency under hypoxia and low pHe compared to the 1st generation compounds. However, only one compound with a pKa in the desired range (5-7) has been tested, which exhibited pH selectivity that is consistent with the estimates of pHi at pHe 6.5 and 7.4. But its low potency is not improved relative to the 1st generation compounds. The data investigating pHi using two independent methods- the fluorescent probe BCECF and the radiolabel 14C-dimethadione did not appear to explain pH selectivity of the weak acid chlorambucil based on pH dependent partioning theory for weak acids alone. Preliminary results from this thesis suggest that future studies could explore a longer duration of incubation under 0.2% oxygen, to improve the regulation of pHi in cell culture models.