Combination treatment of CC531-lac-Z rat liver metastases by chemoembolization with pemetrexed disodium and gemcitabine

Abstract

Purpose: The aim of this study was to evaluate the combination effect of pemetrexed disodium (MTA; Alimta; LY 231514) and gemcitabine (GEM) administered by hepatic artery and portal vein chemoembolization (HACE and PVCE) in a colorectal cancer rat liver metastasis model.

Materials and methods: Proliferation studies on CC531-lac-Z rat colon cancer cells were performed using the MTT assay to obtain the optimal combination schedule of the two antineoplastic agents. To generate diffuse liver metastasis, 4 x 10(6) tumor cells were implanted into the portal vein of male WAG/Rij rats. MTA (30 mg/kg, 60 mg/kg, and 90 mg/kg) was administered locoregionally by portal vein chemoembolization (PVCE) and compared with repeated systemic intravenous injection. GEM (50 mg/kg) was also given locoregionally by hepatic artery chemoembolization (HACE) as well as systemically. All routes of administration were examined alone as well as in combination. Efficacy of treatment in terms of liver metastases burden was determined at the end of the experiment by measuring the beta-galactosidase activity of CC531-lac-Z cells with a chemoluminescence assay.

Results: Combination experiments in vitro showed a more than additive tumor cell reduction after sequential exposure to MTA preceding GEM (observed/expected ratio [O/E] = 0.73). Experiments with the reverse sequence (GEM-->MTA) resulted only in additive combination effects (O/E ratio = 1.08). Simultaneous drug exposure showed less than additive combination effects (O/E ratios > or = 1.25). In vivo, locoregional administration by HACE with GEM was significantly more effective than systemic intravenous bolus treatment (P = 0.03). Portal vein chemoembolization with MTA performed immediately after tumor cell inoculation was ineffective. Repeated systemic treatment with MTA yielded a slight reduction in tumor cell load that was significant versus control at the medium and high doses (60 mg/kg, P = 0.009; 90 mg/kg, P = 0.046) but not versus intraportal chemoembolization. The combination treatment of systemic (60 and 90 mg/kg) or locoregional (60 mg/kg) MTA with HACE using GEM (50 mg/kg) resulted in more than 80% tumor growth inhibition; this antineoplastic combination effect was maximally additive.

Conclusion: A regimen-dependent synergistic combination effect of both drugs was found in vitro. In animals, hepatic artery chemoembolization with GEM was superior to systemic intravenous bolus treatment. Portal vein chemoembolization with MTA was ineffective. The optimal in vitro regimen of MTA (intravenous or PVCE) preceding GEM (HACE) resulted in a maximally additive tumor growth inhibition. The results indicate that MTA and GEM can successfully be combined and favor further evaluation in patients.

Fig. 1

Operation site: the liver hilus has been prepared for inserting the PVC catheter into the gastroduodenal artery. Before injection of the chemoembolization treatment, the metal clip on the proper hepatic artery will be removed. (1) PVC catheter—tip in the gastroduodenal artery, (2) ligature of the gastroduodenal artery, (3) fixation of the catheter by a silk stitch, (4) straight microforceps, (5) liver, (6) metal clip on the proper hepatic artery, (7) curved microforceps, (8) metal clip on the common hepatic artery, (9) duodenum

Fig. 2

Defined concentrations of tumor cells suspended in 1 g healthy rat liver were measured with the β-galactosidase assay kit to generate a standard curve of chemoluminescence. The data were fitted to obtain a linear trend according to the equation Y=133.66X+5×10⁶ (R²=0.99).

Fig. 3

Single-drug exposure of CC531-lac-Z cells to Alimta (MTA) and gemcitabine (GEM) was tested on days 1–3 (top) and 3–5 (bottom) to determine the optimum range of concentrations for combination schedules. All concentrations were evaluated in 12 wells of a 96-well plate using the MTT assay. Results are shown as mean absorption in percent of untreated control cells with respective standard deviation (vertical bars).

Fig. 4

Results of sequential (a, b) and simultaneous (c, d) combination therapy with MTA and GEM using different schedules. Columns illustrate the absorption in percent control; besides single drug effects, the expected (white columns) and observed (dotted columns) combination effects are given. Bars symbolize standard deviation.

Fig. 5

Summary of treatment effects; columns denote the respective therapeutic efficacy as quotient of treated and control values (T/C%) for (a) tumor cell number determined by β-galactosidase assay and (b) wet liver weight. White columns indicate the expected combination effect. Bars symbolize standard error of the mean.