Enhancement of intratumoral cyclophosphamide pharmacokinetics and antitumor activity in a P450 2B11-based cancer gene therapy model

Abstract

The therapeutic utility of cytochrome P450-based enzyme prodrug therapy is well established by preclinical studies and in initial clinical trials. The underlying premise of this gene therapy is that intratumoral P450 expression leads to in situ activation of anticancer P450 prodrugs, such as cyclophosphamide (CPA), with intratumoral accumulation of its activated 4-OH metabolite. In mice bearing 9L gliosarcomas expressing the CPA 4-hydroxylase P450 2B6, enhanced tumor apoptosis was observed 48 h after CPA treatment; however, intratumoral 4-OH-CPA levels were indistinguishable from those of P450-deficient tumors, indicating that the bulk of activated CPA is derived from hepatic metabolism. In contrast, in 9L tumors expressing P450 2B11, a low K(m) CPA 4-hydroxylase, intratumoral 4-OH-CPA levels were higher than in blood, liver and P450-deficient tumors. Intratumoral 4-OH-CPA increased dose-dependently, without saturation at 140 mg kg(-1) CPA, suggesting restricted tumor cell permeation of the parent drug. To circumvent this problem, CPA was administered by direct intratumoral injection, which increased the maximum concentration and area under the curve of drug concentration x time (AUC) of intratumoral 4-OH-CPA by 1.8- and 2.7-fold, respectively. An overall 3.9-fold increase in intratumoral 4-OH-CPA AUC, and in antitumor activity, was obtained when CPA release to systemic circulation was delayed using the slow-release polymer poloxamer 407 as vehicle for intratumoral CPA delivery. These findings highlight the advantage of gene therapy strategies that combine low K(m) P450 prodrug activation enzymes with slow, localized release of P450 prodrug substrates.

Figure 1. Caspase 3 activity in CPA-treated tumors

Scid mice implanted with 9L or 9L/2B6 tumors were treated with a single i.p. injection of CPA (140 mg/kg BW) or were untreated (UT). Tumors were collected 24 or 48 h later. Caspase 3 activity was determined in the presence and absence of the caspase 3-selective inhibitor Casputin as described in Materials and Methods. Data shown are mean ± SE values (n = 4 for CPA-treated tumors; n = 2 untreated tumors) relative to untreated (UT) tumors. Data were analyzed using one way ANOVA and nonparametric Bonferroni’s Multiple Comparison test: * and **, p< 0.05 and p<0.01, respectively, for CPA treated vs. untreated; +, p<0.05 for 9L/2B6 vs. 9L tumors at the same CPA treatment time.

Figure 2. Tissue levels of 4-OH-CPA and 4-OH-IFA in mice bearing 9L/2B6, 9L/2B1 and 9L tumors

Panel A, Scid mice bearing 9L, 9L/2B6 or 9L/2B1 tumors were treated with CPA or IFA, following which tissues were collected and assayed for 4-OH-CPA or 4-OH-IFA by HPLC. Mice bearing 9L or 9L/2B6 tumors were killed 15 min after CPA treatment at 140 mg/kg. Values shown are mean ± range for blood and liver (n=2) and mean ± SE for tumors (n=4). Data were analyzed using one way ANOVA and nonparametric Bonferroni’s Multiple Comparison test: **, p< p<0.01 for liver and tumor vs. blood; +, p<0.05 for tumor vs. liver. Panel B, mice bearing 9L/2B1 tumors were killed 15 min after CPA treatment at 140 mg/kg or IFA treatment at 300 mg/kg. Values are shown for blood and liver (n=1) and for tumors (mean ± range, n=2).

Figure 3. 4-OH-CPA levels in mice bearing 9L/2B11 tumors

Scid mice bearing 9L or 9L/2B11 tumors were killed 15 min after i.p. injection of CPA at 20, 50 or 140 mg/kg BW. (A) 4-OH-CPA levels in blood, liver and tumors of mice treated with CPA at 140 mg/kg. Data shown are mean ± range for blood and liver (n=2) and mean ± SE for tumors (n=4). (B) 4-OH-CPA in 9L and 9L/2B11 tumors from mice treated at the indicated CPA doses. Data shown are mean ± SE (n=4) values. * and ** indicate significance at p< 0.05 and p< 0.01 for 9L/2B11 vs. 9L, respectively. (C) Increase in tumor-associated 4-OH-CPA in 9L/2B11 tumors compared to 9L tumors as a function of CPA dose. Data shown are mean values ± SE (n=4), calculated by subtracting the mean 4-OH-CPA level in 9L tumors from the mean 4-OH-CPA level in 9L/2B11 tumors at each CPA dose.

Figure 4. Effect of intratumoral CPA delivery on pharmacokinetics of 4-OH-CPA in mice bearing 9L/2B11 tumors

Scid mice bearing 9L (panel A) or 9L/2B11 tumors (panels B–D) were killed 6 to 240 min after treatment with CPA by i.p. (A and B) or i.t. (C and D) injection at 50 mg/kg BW. CPA was dissolved in 0.2% NaCl (panels A–C) or in 23% F127/NaCl (panel D). Data shown are 4-OH-CPA levels (mean ± SE; n=3 for blood and liver, n=6 for tumors). Tissue collection and 4-OH-CPA analysis were as described in Materials and Methods.

Figure 5. Effect of intratumoral CPA delivery on growth of 9L/2B11 tumors

9L/2B11 tumors were implanted s.c. and grown in male scid mice. Tumor volumes (panel A) and body weight (panel B) were measured twice a week. X axis, days after first CPA injection. Pair of arrows, MTD CPA administration schedule: two CPA injections, each at 150 mg/kg and spaced 24 hr apart. Solid square, untreated group; solid triangle, CPA injection i.p. without polymer; inverted solid triangle, intratumoral CPA delivery with 23% F127. Data shown are mean ± SE values (n=10). Tumor doubling time was 6.4 ± 0.7 days for untreated tumors vs. >36 days for the CPA-treated tumors. Tumors treated with CPA/23% F127 regressed more rapidly, and more completely than following i.p. CPA treatment.