The in vitro metabolism of irinotecan (CPT-11) by carboxylesterase and beta-glucuronidase in human colorectal tumours

Abstract

Aims: Irinotecan (CPT-11) is a prodrug that is used to treat metastatic colorectal cancer. It is activated to the topoisomerase poison SN-38 by carboxylesterases. SN-38 is metabolized to its inactive glucuronide, SN-38 glucuronide. The aim of this study was to determine, the reactivation of SN-38 from SN-38 glucuronide by beta-glucuronidase may represent a significant pathway of SN-38 formation.

Methods: The production of SN-38 from irinotecan and SN-38 glucuronide (2.4, 9.6 and 19.2 microm) was measured in homogenates of human colorectal tumour, and matched normal colon mucosa from 21 patients).

Results: The rate of conversion of irinotecan (9.6 microm) was lower in tumour tissue than matched normal colon mucosa samples (0.30+/-0.14 pmol min-1 mg-1 protein and 0.77+/-0.59 pmol min-1 mg-1 protein, respectively; P<0.005). In contrast, no significant difference was observed in beta-glucuronidase activity between tumour and matched normal colon samples (4.56+/-6.9 pmol min-1 mg-1 protein and 3.62+/-2.95 pmol min-1 mg-1 protein, respectively, using 9.6 microm SN-38 glucuronide; P>0.05). beta-Glucuronidase activity in tumour correlated to that observed in matched normal tissue (r2>0.23, P<0.05), whereas this was not the case for carboxylesterase activity. At equal concentrations of irinotecan and SN-38 glucuronide, the rate of beta-glucuronidase-mediated SN-38 production was higher than that formed from irinotecan in both tumour and normal tissue (P<0.05). However, at concentrations that reflect the relative plasma concentrations observed in patients, the rate of SN-38 production via these two pathways was comparable.

Conclusions: Tumour beta-glucuronidase may play a significant role in the exposure of tumours to SN-38 in vivo.

Figure 1

Production of SN-38 from irinotecan and SN-38 glucuronide (SN-38G) in one of the human colorectal tumours studied. Each point and bar represents the mean ± SD value for each substrate concentration

Figure 2

The relationship between incubation time and SN-38 production following incubation of 200 µg colorectal tumour with (1) 9.6 µm irinotecan or (2) 9.6 µm SN-38 glucuronide. Each point and bar represents the mean ± SD value for each time point

Figure 3

Carboxylesterase and β-glucuronidase activity in normal colon and colorectal tumour tissue as a function of substrate concentration. Homogenates were incubated with (1) irinotecan or (2) SN-38 glucuronide (SN-38G) for 10 min at 37 °C. Data are presented as mean ± SD (n = 21). (Note that the SD bars are too small to be visible.) Normal (♦), tumour (▪)

Figure 4

Relationship between normal and tumour tissue β-glucuronidase activity, using SN-38 glucuronide (final concentration 19.2 µM) as the substrate. Normal and tumour β-glucuronidase activity was significantly correlated (r² = 0.40, P = 0.003), which was also observed at substrate concentrations of 2.4 and 9.6 µM

Figure 5

Inhibition of β-glucuronidase activity by saccharolactone, using SN-38 (left panel) and p-NP (right panel) as substrates. Normal colon mucosa and colorectal tumour (from patient 12) and plasma (from patients 12 and 15) were incubated in the presence and absence of 100 µM saccharolactone. Note that the bar for tumour in the presence of saccharolactone is too small to be visible (0.11 pmol min⁻¹ mg⁻¹ protein). Control (▪), saccharolactone

Figure 6

Relationship between assays of β-glucuronidase activities using p-NPG and SN-38 glucuronide as substrates. Plasma from 12 cancer patients was incubated in duplicate with either p-NPG or SN-38 glucuronide for 2 h