Acute metabolic alkalosis enhances response of C3H mouse mammary tumors to the weak base mitoxantrone

Abstract

Uptake of weak acid and weak base chemotherapeutic drugs by tumors is greatly influenced by the tumor extracellular/interstitial pH (pH(e)), the intracellular pH (pH(i)) maintained by the tumor cells, and by the ionization properties of the drug itself. The acid-outside plasmalemmal pH gradient in tumors acts to exclude weak base drugs like the anthracyclines, anthraquinones, and vinca alkaloids from the cells, leading to a substantial degree of "physiological drug resistance" in tumors. We have induced acute metabolic alkalosis in C3H tumor-bearing C3H/hen mice, by gavage and by intraperitoneal (i.p.) administration of NaHCO(3). (31)P magnetic resonance spectroscopic measurements of 3-aminopropylphosphonate show increases of up to 0.6 pH units in tumor pH(e), and 0.2 to 0.3 pH units in hind leg tissue pH(e), within 2 hours of i.p. administration of NaHCO(3). Theoretical calculations of mitoxantrone uptake into tumor and normal (hind leg) tissue at the measured pH(e) and pH(i) values indicate that a gain in therapeutic index of up to 3.3-fold is possible with NaHCO(3) pretreatment. Treatment of C3H tumor-bearing mice with 12 mg/kg mitoxantrone resulted in a tumor growth delay of 9 days, whereas combined NaHCO(3)--mitoxantrone therapy resulted in an enhancement of the TGD to 16 days.

Figure 1

A series of ³¹P MR spectra were obtained from a 975-mm³ C3H tumor before and after i.p. administration of 0.7 mlx1 M NaHCO₃ to the mouse at t=0 minutes. Chemical shifts are calibrated against the α-NTP resonance, which is set to -10.05 ppm. By 1 hour postinjection, the pH_e of the tumor is substantially raised, as indicated by the shift of the 3-APP resonance upfield to lower ppm values, and this increase persists through 4 hours postinjection. Tumor pH_i is increased to a small extent by 4 hours postinjection as well, as indicated by the shift to higher ppm values of the P_i resonance.

Figure 2

A series of ³¹P MR spectra were obtained from the hind leg of a C3H/Hen mouse before and after i.p. administration of 0.7 mlx1 M NaHCO₃ to the mouse at t=0 minutes. Signal intensities of both the 3-APP and P_i peaks were low compared with spectra obtained from tumors. A small shift of the 3-APP peak to lower ppm values is visible by 45 minutes postinjection, indicating alkalinization of tissue pH_e. A small shift of the P_i resonance to higher ppm values is also visible, indicating alkalinization of pH_i. The intensities of the phosphocreatine peak (≈-2.5 ppm) and the NTP peaks ≈-5, -10.05, and -18.5 ppm) did not change significantly during the course of MRS experiment. The inset shows the calculated pH_e and pH_i versus time in hind leg tissue from these spectra. Both pH_e and pH_i are seen to increase slightly following i.p. administration of 0.7 mlx1 M NaHCO₃.

Figure 3

pH_e and pH_i versus time in C3H tumors from four different animals that were administered 0.7 mlx1 M NaHCO₃ (i.p.) at t=0 minutes. The larger tumors in panels B to D can be seen to have more acidic pH_e and pH_i before NaHCO₃ administration than the smaller tumor shown in panel A. The more acidic tumors in B to D show increases of both pH_e and pH_i by 2 hours postinjection of the NaHCO₃, whereas the neutral tumor in A did not alkalinize in response to the NaHCO₃ bolus.

Figure 4

Average pH_e and pH_i values (±SD) from C3H tumors in control mice, and in mice that were administered either NaHCO₃ or NH₄Cl (0.7 mlx1 M) by gavage. Tumors from control mice were separated into three categories based on size. Neither pH_e nor pH_i was significantly affected by tumor size for tumors in the control group (P > .2). Tumor pH_i in NaHCO₃-treated mice was not significantly different from tumor pH_i in control animals (P > .05 against tumors in all three control groups). _#Tumor pH_e was significantly higher in NaHCO₃-treated mice compared to tumors in untreated control mice in the two larger size categories (P < .05). Tumor pH_e in NaHCO₃-treated mice was marginally higher than tumor pH_e in the smallest group of tumors (688±211 mm³) in control mice (P = .06). *Both tumor pH_i and tumor pH_e were significantly lower in NH₄Cl-treated mice than in control mice regardless of tumor size (P < .03 in all cases).

Figure 5

Average pH_e and pH_i values (±SD) from normal hind leg tissue in control mice, and in mice that were administered either NaHCO₃ or NH₄Cl (0.7 mlx1 M) by gavage. Gavage administration of NaHCO₃ did not result in significant changes in either pH_e (P=.5) or pH_i (P=.17). *Gavage administration of NH₄Cl resulted in a considerable drop in pH_e (P=.003) as well as pH_i (P=.04) of hind leg tissue in treated mice compared with control mice.

Figure 6

Theoretically calculated tissue-blood drug partition ratios: (A) from Equation 1, for combined NaHCO₃ (i.p.) and mitoxantrone therapy, and (B) from Equation 2, for combined therapy using NH₄Cl (gavage) and a hypothetical singly charged weak acid drug of pK_a=6.0. For mitoxantrone a pK_a2 of 12 was assumed, because Equation 1 is insensitive to pK_a2 for physiological values of pH_i and pH_e. The following representative values of pH_e and pH_i in control/NaHCO₃-treated/NH₄Cl-treated animals, respectively, were obtained from the data reported in Figures 2–5 — tumor pH_i 7.05/7.3/6.7, tumor pH_e 6.7/7.3/6.16, hind leg pH_i 7.33/7.5/7.15, and hind leg pH_e 7.35/7.6/6.8 — and inserted into Equations 1 and 2 to obtain the drug partition ratios shown here. Pretreatment with NaHCO₃ (i.p.) is predicted to result in a five-fold enhancement in uptake of mitoxantrone into tumor tissue compared to tumor tissue in an untreated host, but only a 1.5-fold increase in uptake of drug into normal tissue in the NaHCO₃-treated animal compared with normal tissue in an untreated animal. Thus, the chemotherapeutic index of mitoxantrone can potentially be increased 3.3-fold by NaHCO₃ pretreatment of the host animal. However, acidosis induced by gavage administration of NH₄Cl to the host animal is not tumor specific, resulting in a two-fold increase in uptake of a weak acid drug of pK_a 6 into normal tissue and only a 1.2-fold increase in uptake into tumor tissue. Thus, the chemotherapeutic index of a weak acid drug is predicted to be reduced by gavage administration of NH₄Cl.

Figure 7

Growth curves for C3H tumors in (A) untreated mice and NaHCO₃ (0.7 mlx1 M, i.p.)-treated mice; (B) mitoxantrone (6 mg/kgx2)-treated mice and mitoxantrone (6 mg/kgx2) plus NaHCO₃ (0.7 mlx1 Mx2, i.p.)-treated mice; (C) mitoxantrone (12 mg/kg)-treated mice and mitoxantrone (12 mg/kg) plus NaHCO₃ (0.7 mlx1 M, i.p.)-treated mice. Each point in panels A to C represents the mean tumor volume from cohorts of four mice per treatment group. Error bars in panels B and C represent standard deviations. NaHCO₃ treatment alone was found to not affect the growth rate of C3H tumors (panel A). Treatment with mitoxantrone alone resulted in a TGD of 9 days, regardless of whether the 12 mg/ kg dose was fractionated into two doses (panel B) or given as a single bolus (panel C). Combined treatment with mitoxantrone and NaHCO₃ resulted in a TGD of 15 to 16 days, regardless of dose fractionation. Pretreatment with NaHCO₃ resulted in an increase in log₁₀ cell kill from 0.9 to 1.56, corresponding to a greater than 4.5-fold increase in cell kill.