Enhancement of hyperthermia effect in vivo by amiloride and DIDS

Intracellular pH is regulated mainly by Na+/H+ antiport and Cl-/HCO3- exchange through the cell membrane. Amiloride (3,5-diamino-6-chloro-N-(diaminomethylene)pyrazine carboxamide) is a diuretic drug that blocks Na+/H+ antiport and DIDS (4,4-diisothiocyanatostilbene-2,2'-disulfonic acid) is an inhibitor of Cl-/HCO3- exchange. We investigated the potency of these drugs to lower pHi and increase the thermosensitivity of tumors in vivo.

The cytocidal effect of heat in combination with drug effect in vivo was studied using the in vivo-in vitro clonogenic assay method and the tumor growth delay method with SCK tumors, a mammary adenocarcinoma, on the hind limbs of A/J mice. The effects of amiloride and DIDS on tumor pHi and high energy phosphate levels were investigated using 31P-NMR.

We observed that amiloride or DIDS alone increased the effect of hyperthermia at 42.5 degrees C or 43.5 degrees C to suppress tumor growth. The thermosensitization was greater when the two drugs were combined. For example, hyperthermia at 43.5 degrees C alone resulted in a tumor growth delay of about 4 days. When 10 mg/kg amiloride or 25 mg/kg DIDS was injected prior to heating, the growth delay increased to about 6 days. When both drugs were injected prior to heating, a total growth delay of 8 days was obtained. In vivo-in vitro excision assays for cell survival demonstrated that these drugs enhanced the heat-induced tumor cell death. An i.p. injection of 10 mg/kg amiloride plus 25 mg/kg DIDS did not lower the tumor pHi over a 120 min interval. Heating the tumors at 42.5 degrees C for 1 hr significantly lowered the pHi and when the tumor-bearing mice were injected i.p with amiloride and DIDS, and the tumors were heated 1 hr later, the drop in pHi was greater relative to that by heating alone. Heating alone significantly lowered the tumor energy levels as indicated by PCr/Pi and beta-ATP/Pi ratios and an i.p. injection of 25 mg/kg amiloride prior to heating further reduced the energy status in the tumors.

Amiloride or its analogs and DIDS may be useful in increasing the therapeutic efficacy of hyperthermia treatments by enhancing the reduction in tumor pHi.