Improving chemotherapeutic drug penetration in tumors by vascular targeting and barrier alteration

Abstract

Drug delivery and penetration into neoplastic cells distant from tumor vessels are critical for the effectiveness of solid-tumor chemotherapy. We have found that targeted delivery to tumor vessels of picogram doses of TNF-alpha (TNF), a cytokine able to alter endothelial barrier function and tumor interstitial pressure, enhances the penetration of doxorubicin in tumors in murine models. Vascular targeting was achieved by coupling TNF with CNGRC, a peptide that targets the tumor neovasculature. This treatment enhanced eight- to tenfold the therapeutic efficacy of doxorubicin, with no evidence of increased toxicity. Similarly, vascular targeting enhanced the efficacy of melphalan, a different chemotherapeutic drug. Synergy with chemotherapy was observed with 3-5 ng/kg of targeted TNF (intraperitoneally), about 10(6)-fold lower than the LD(50) and 10(5)-fold lower than the dose required for nontargeted TNF. In addition, we have also found that targeted delivery of low doses of TNF to tumor vessels does not induce the release of soluble TNF receptors into the circulation. The delivery of minute amounts of TNF to tumor vessels represents a new approach for avoiding negative feedback mechanisms and preserving its ability to alter drug-penetration barriers. Vascular targeting could be a novel strategy for increasing the therapeutic index of chemotherapeutic drugs.

Figures

Figure 1

Effect of mTNF and NGR-mTNF on tumor growth and body weight of animals bearing RMA-T lymphomas. Animals bearing RMA-T tumors (five mice per group) were treated intraperitoneally with NGR-mTNF or mTNF at day 12 after tumor implantation (a) or at days 10, 11, and 12 (b), in two separate experiments (Exp. 1 and Exp. 2). Tumor volumes in Exp. 1 (a) and Exp. 2 (b) and animal body weight in Exp. 1 (c) 1–4 days after treatment are shown. The arrowheads in c indicate the time of treatment.

Figure 2

Circulating levels of sTNF-R2 and their role in regulating the activity of NGR-mTNF and NGR-hTNF. (a) Serum levels of sTNF-R1 and sTNF-R2 in B16F1 tumor–bearing mice 1 hour after treatment with various doses of NGR-mTNF or mTNF. Animals (three mice per group) were treated at day 6. (b) Effect of the anti–sTNF-R2 mAb 6G1 on the antitumor activity of NGR-mTNF. The mAb 6G1 (100 μg) was administered to animals bearing B16F1 tumors at day 5 and 8. Each animal was treated 1 hour later with NGR-mTNF at the indicated doses, and 2 hours later with melphalan (90 μg, five mice per group). (c) Effect of NGR-hTNF and hTNF on the growth of RMA-T tumors. Mice were treated with various doses of each cytokine at day 11. NS, not significant (t test).

Figure 3

Effect of melphalan, alone (a) or in combination with NGR-mTNF (c) or mTNF (d), on the tumor growth (a–d) and body weight (e and f) of mice bearing B16F1 melanoma. The animals were treated intraperitoneally with the drugs and the doses indicated in each panel (five animals per group) at days 4, 7, and 9 after tumor implantation (indicated by arrows).

Figure 4

Effect of various doses of doxorubicin, alone (white bars) or in combination with NGR-mTNF (black bars), on the tumor growth (a and b), body weight (c and d), and survival (e) of mice bearing B16F1 melanomas. The drugs were administered to the animals (five mice per group intraperitoneally) 5 days after tumor implantation.

Figure 5

Effect of melphalan or doxorubicin, alone or in combination with NGR-mTNF, on well-established RMA-T and B16F1 tumors. Each animal was treated with the drugs and the doses indicated in each panel (five animals per group) at time points indicated by the arrows. The numbers on each curve indicate the animals that were tumor-free at day 43.

Figure 6

Role of TNF receptors in the synergistic activity of NGR-mTNF and melphalan. (a) Effect of mAb V1q (an anti-mTNF neutralizing antibody) on the antitumor activity of melphalan in combination with NGR-mTNF in the B16F1 model. The drugs were administered at day 5. V1q and NGR-mTNF were premixed and incubated for 1 hour before injection into animals. (b) Effect of melphalan in combination with NGR-hTNF at the indicated doses.

Figure 7

Effect of NGR-mTNF on the penetration of doxorubicin in B16F1 and RMA-T tumors. (a) Bright-field (upper panels) and fluorescence (lower panels) microscopy of B16F1 cells incubated in vitro with 100 μg/ml doxorubicin (30 minutes, 37°C). Inset: Merge of bright-field and fluorescence images. (b) Stability of the B16F1 fluorescence signal after in vitro treatment with doxorubicin. B16F1 cells were incubated with various doses of doxorubicin in culture medium (30 minutes, 37°C), washed with 0.9% sodium chloride, and fixed with 4% formaldehyde. The cells were then incubated for 0 hours or 24 hours in culture medium at 4°C, washed again, and analyzed by FACS. (c and f) Representative FACS analysis of cells recovered from B16F1 (c) or RMA-T (f) tumors 2 hours after in vivo administration of doxorubicin alone (320 μg) or in combination with NGR-mTNF (0.1 ng). Dashed lines indicate the fluorescence interval considered positive. (d and g) Mean ± SE fluorescence of B16F1 (d) or RMA-T (g) cells recovered from tumors. (e and h) Mean ± SE of positive cells recovered from B16F1 (e) or RMA-T (h) tumors. *P < 0.05, statistical analysis by two-tailed t test.

Figure 8

Schematic representation of the hypothetical interactions of low (a), moderate (b), and high (c) doses of NGR-TNF with soluble and membrane receptors in normal vessels (CD13-negative) and in tumor-associated vessels (CD13-positive). Black arrows indicate TNF receptor signaling or extracellular domain shedding.