Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs

Abstract

Poor penetration of anticancer drugs into tumors can be an important factor limiting their efficacy. We studied mouse tumor models to show that a previously characterized tumor-penetrating peptide, iRGD, increased vascular and tissue permeability in a tumor-specific and neuropilin-1-dependent manner, allowing coadministered drugs to penetrate into extravascular tumor tissue. Importantly, this effect did not require the drugs to be chemically conjugated to the peptide. Systemic injection with iRGD improved the therapeutic index of drugs of various compositions, including a small molecule (doxorubicin), nanoparticles (nab-paclitaxel and doxorubicin liposomes), and a monoclonal antibody (trastuzumab). Thus, coadministration of iRGD may be a valuable way to enhance the efficacy of anticancer drugs while reducing their side effects, a primary goal of cancer therapy research.

Fig. 1

Comparison of the drug delivery efficiency of the iRGD combination regimen and conjugated drug delivery. (A and C) Nab-paclitaxel (Abraxane; ABX) quantification in orthotopic human breast tumor (BT474)(A) and human prostate tumor (22Rv1)(C) xenograft models. ABX with free iRGD (combination), ABX coated with iRGD (conjugate), or ABX alone, was intravenously injected into tumor-bearing mice. Three hours later, ABX was captured from tumor extracts with an anti-taxol antibody, followed by detection with a human albumin antibody. n = 3 per group. (B and D) Long-term treatment of tumor mice with ABX. Mice bearing orthotopic BT474 (B) or 22Rv1 (D) tumors were intravenously injected with the indicated ABX formulations every other day at 3 mg paclitaxel/kg/injection, or with phosphate-buffered saline (PBS) only. The treatment was continued for 24 days in (B) and 16 days in (D). n = 8 per group in (B) and n = 9 in (D). Statistical analyses were performed with Student's t-test in (A) and (C) and ANOVA in (B) and (D). Error bars, mean ± SEM; n.s., not significant; *p < 0.05; ***p < 0.001.

Fig. 2

Enhanced anti-tumor effect of free DOX co-injected with iRGD. (A and B) Mice bearing orthotopic 22Rv1 human prostate tumors were intravenously injected with a mixture of DOX (10 mg/kg), and 4 μmol/kg of iRGD or PBS. Tumors and tissues were collected 1 hour later. n = 3 per group. In (A), the tumors were sectioned and stained for blood vessels with an anti-CD31 and the native fluorescence was used to detect DOX. Scale bars = 100 μm. In (B), DOX in the tissues was quantified. (C) Mice bearing orthotopic 22Rv1 tumors implanted 2 weeks earlier received intravenous injections every other day of DOX (1 or 3 mg/kg) or PBS, combined with 4 μmol/kg of iRGD or PBS. The tumors were harvested and weighed after 24 days of treatment. n = 10 per group. (D) TUNEL staining was performed on tumors and hearts from the treatment study, and quantified for positive staining. Statistical analyses were performed with Student's t-test in (B), and ANOVA in (C) and (D); Error bars, mean ± SEM; n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 3

Enhanced anti-tumor effect of DOX-liposomes co-injected with iRGD. (A) Nude mice bearing orthotopic 22Rv1 human prostate tumors implanted 2 weeks earlier received daily intravenous injections of DOX-liposomes (1 or 3 mg/kg) or PBS, combined with 2 μmol/kg of iRGD or cyclo(-RGDfK-), or PBS. The tumors were harvested and weighed after 17 days of treatment. n = 5 per group in the left panel, and n = 8 per group in the right panel. (B) Mice bearing orthotopic 22Rv1 tumors were intravenously injected with DOX-liposomes (5 mg/kg) followed by 4 μmol/kg of iRGD or PBS. The tumors and tissues were collected 3 hours later, and DOX in the tissues was quantified. n = 3 per group. (C) TUNEL staining was performed on tumors and hearts from the treatment study in (A), and quantified for positive staining. Statistical analyses were performed with ANOVA in (A, left panel) and (C), and Student's t-test in (A, right panel) and (B). Error bars, mean ± SEM; n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 4

Enhanced anti-tumor effects of trastuzumab co-injected with iRGD. (A and B) Mice bearing orthotopic BT474 human breast tumors were intravenously injected with trastuzumab (3 mg/kg) followed by 4 μmol/kg of iRGD, or PBS. Tissues were collected 3 hours later. n = 3 per group. In (A), tumor sections were stained for trastuzumab with an anti-human IgG antibody, and quantified for positive staining. Scale bars = 200 μm. In (B), trastuzumab in the tissues was quantified with a competitive ELISA. n = 3 per group. (C) Tumor treatment study with coadministration of trastuzumab and iRGD. BT474 tumor mice were intravenously injected every 4 days with trastuzumab at 3 or 9 mg/kg on the first day of treatment (day 0) and 1.5 or 4.5 mg/kg in subsequent injections, or PBS. The treatment was combined with daily injections of 4 μmol/kg iRGD or PBS on the days of trastuzumab injection, and 2 μmol/kg iRGD or PBS on the other days. n =10 per group. One of 5 experiments that gave similar results is shown. Statistical analyses were performed with Student's t-test in (A) and (B), and ANOVA in (C). Error bars, mean ± SEM; n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001.