Novel Cyclic Peptide-Drug Conjugate P6-SN38 Toward Targeted Treatment of EGFR Overexpressed Non-Small Cell Lung Cancer

Abstract

Background/Objectives: Here, we report on the synthesis and biological evaluation of a novel peptide-drug conjugate, P6-SN38, which consists of the EGFR-specific short cyclic peptide, P6, and the Topo I inhibitor SN38, which is a bioactive metabolite of the anticancer drug irinotecan. Methods: SN38 is attached to the peptide at position 20 of the E ring's tertiary hydroxyl group via a mono-succinate linker. Results: The developed peptide-drug conjugate (PDC) exhibited sub-micromolar anticancer activity on EGFR-positive (EGFR+) cell lines but no effect on EGFR-negative (EGFR-) cells. In vivo studies have shown that this PDC specifically accumulates in EGFR+ non-small cell lung cancer (NSCLC) xenografts and presents superior anticancer activity compared to the EGFR-specific antibody cetuximab (Erbitux^TM) and free SN38. The 10 mg/kg dose of P6-SN38 in a side-by-side EGFR+/EGFR- xenograft shows eradication of the EGFR+ tumor with good tolerance, but no inhibition of tumor growth of the EGFR- counterpart. Conclusions: The PDC examined in this study was proven to be highly efficient for NSCLC, broadening its utilization for targeted cancer therapy in EGFR overexpressed cancers.

Keywords: EGFR; NSCLC; SN38; peptide–drug conjugate; targeted cancer therapy; topo I inhibitor.

Figure 1

Structure of P6 and SN38. To present the locations of the functional groups more clearly, the key positions of the SN38 structure were numbered.

Scheme 1

Synthesis of TBDMS-SN38-suc intermediate.

Scheme 2

Solid phase synthesis of P6-SN38 peptide conjugate. The N and C terminus of peptide are highlighted with bold.

Figure 2

Cell viability after 6 h of pre-incubation of EGFR+ H1299 (A) and EGFR− HEK293 (B) cell lines with P6-SN38 and SN38. H1299 and HEK293 cells were cultured in a 96-well plate and pre-incubated with six different concentrations (0.5, 2.5, 5, 10, 15, and 25 μM) of P6-SN38 or SN38 for 6 h, washed out, and incubated additionally for 24 h, 48 h, and 72 h. Cell viability was assessed using an XTT assay, with the viability of untreated control cells set at 100%. The viability of treated cells was expressed as a percentage relative to the control. Data are presented as the mean ± standard deviation (SD).

Figure 3

Apoptosis of H1299 cells estimated through cell survival percentage by binding with annexin V-FITC. H1299 cells were pre-incubated with P6-SN38 and SN38 as described in Section 2.6. Cells were stained with annexin and PI and analyzed by flow cytometry; * p < 0.05, *** p < 0.001, **** p < 0.0005. “ns” means non-significant.

Figure 4

Cell cycle arrest analysis of treatment with SN38 and P6-SN38. Cell cycle distribution of H1299 cells detected by flow cytometry after 24 h (A) and 48 h (B) treatment with SN38 and P6-SN38 at 2.5 µM. A total of 1 × 10⁶ cells were pre-incubated with P6-SN38 or SN38 for 6 h, followed by additional incubation for 24 h and 48 h. The cells were then stained with propidium iodide (PI) and analyzed by flow cytometry (C). The data in A and B are presented as the mean ± SD (n = 3). “ns” means non-significant. * p < 0.05, *** p < 0.001, **** p < 0.0005. “ns” means non-significant.

Figure 4

Cell cycle arrest analysis of treatment with SN38 and P6-SN38. Cell cycle distribution of H1299 cells detected by flow cytometry after 24 h (A) and 48 h (B) treatment with SN38 and P6-SN38 at 2.5 µM. A total of 1 × 10⁶ cells were pre-incubated with P6-SN38 or SN38 for 6 h, followed by additional incubation for 24 h and 48 h. The cells were then stained with propidium iodide (PI) and analyzed by flow cytometry (C). The data in A and B are presented as the mean ± SD (n = 3). “ns” means non-significant. * p < 0.05, *** p < 0.001, **** p < 0.0005. “ns” means non-significant.

Figure 5

Representative side-by-side xenograft bearing K562/NSCLC tumors before injection (A), white light image of resected tumors and organs at 24 h post-injection (B), fluorescence image of resected tumors and organs at 24 h post-injection (C).

Figure 6

In vivo effect of P6-SN38 conjugate in NSCLC model (10 mg/kg, once a week). Effect of P6-SN38, free SN38, and Erbitux on tumor volume compared to non-treated control (A) and mice weight (B). The resected tumors are shown in (C). N = 4 in each group. * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001.

Figure 6

In vivo effect of P6-SN38 conjugate in NSCLC model (10 mg/kg, once a week). Effect of P6-SN38, free SN38, and Erbitux on tumor volume compared to non-treated control (A) and mice weight (B). The resected tumors are shown in (C). N = 4 in each group. * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001.