We obtained IC50 values in the low micromolar range on MCF-7 cells varying between 0.14 and 6.64 μM. In the case of HT-29 colon cancer cells, the determined IC50 values were slightly higher and within a range of 3.31-19.10 μM. With exception of compound 10, no significant difference of the cancer cell growth inhibitory effect of the novel bioconjugates and the control conjugates (K1 and K2) could be detected. However, the replacement of 3Trp by 3d-Tic in connection with the deletion of 2His led to an increased cytostatic effect of 10 on both of the analyzed cell lines. The IC50 value of bioconjugate 10 was more than 15-times lower on ER+ breast cancer MCF-7 cells and 5-times lower on colon cancer cells HT-29 compared to the control compound K2. Based on these promising findings, the growth inhibitory effects of conjugate 10 and the related 2His-3d-Tic (3, 5 and 12) conjugates, as well as the 10Gly-NH-Et (7 and 14) containing compounds, were studied on estrogen receptor negative (ER-) MDA-MB-231 breast cancer cells. The corresponding IC50 values are shown in Table 2. The GnRH-III-Dau conjugate 10 revealed also on this cell line the highest anticancer activity with an IC50 value of 2.49 μM. The comparison of the dose-dependent growth inhibitory effect of 10 and K2 on the three cancer cell lines is shown in Figure 1. Considering the results of all three cell lines, only compound 10 which contains the N-terminal modification 2His-d-Tic-Lys(Bu), displayed clearly a reduced cell viability, while the conjugates bearing other substitutions yielded IC50 values which vary only slightly in comparison to the controls.

The main drug-related toxic side effect of anthracyclines is cardiotoxicity, leading to cardiomyopathy and heart failure. Comparative experiments to determine the cardiotoxicity of peptide-anthracycline conjugates with different linkages might be informative for the other conjugates with different types of drugs as well. For this purpose, human cardiomyocytes (HCM) and human umbilical vein endothelial cells (HUVEC) were used as models. The long-term (0-72 h) cytotoxic effect of sixteen GnRH-based conjugates containing Dox and Dau was determined by real-time impedimetric sensing using the xCELLigence SP system (ACEA Biosciences, San Diego, CA, USA) [75]. The results indicated that the ester-linked GnRH-Dox conjugates, including Zoptarelin Doxorubicin, showed significant toxicity at 100 nM and 1 uM, which was remarkably pronounced on the HCM cells. The cytotoxic effect was comparable to that of the free drug, especially at the highest concentration. In contrast, the conjugates with oxime-linked Dau showed no or only a minor toxicity on both the cell lines (Table 2). These data confirm that the linkage between the payload and homing peptide has a significant influence on early drug release and, consequently, an undesired toxic side effect. We may also conclude that the search for more suitable homing peptides might be more important than the application of cleavable bonds between the drug and the peptide to develop efficient DDSs.

We obtained IC50 values in the low micromolar range on MCF-7 cells varying between 0.14 and 6.64 μM. In the case of HT-29 colon cancer cells, the determined IC50 values were slightly higher and within a range of 3.31-19.10 μM. With exception of compound 10, no significant difference of the cancer cell growth inhibitory effect of the novel bioconjugates and the control conjugates (K1 and K2) could be detected. However, the replacement of 3Trp by 3d-Tic in connection with the deletion of 2His led to an increased cytostatic effect of 10 on both of the analyzed cell lines. The IC50 value of bioconjugate 10 was more than 15-times lower on ER+ breast cancer MCF-7 cells and 5-times lower on colon cancer cells HT-29 compared to the control compound K2. Based on these promising findings, the growth inhibitory effects of conjugate 10 and the related 2His-3d-Tic (3, 5 and 12) conjugates, as well as the 10Gly-NH-Et (7 and 14) containing compounds, were studied on estrogen receptor negative (ER-) MDA-MB-231 breast cancer cells. The corresponding IC50 values are shown in Table 2. The GnRH-III-Dau conjugate 10 revealed also on this cell line the highest anticancer activity with an IC50 value of 2.49 μM. The comparison of the dose-dependent growth inhibitory effect of 10 and K2 on the three cancer cell lines is shown in Figure 1. Considering the results of all three cell lines, only compound 10 which contains the N-terminal modification 2His-d-Tic-Lys(Bu), displayed clearly a reduced cell viability, while the conjugates bearing other substitutions yielded IC50 values which vary only slightly in comparison to the controls.

We obtained IC50 values in the low micromolar range on MCF-7 cells varying between 0.14 and 6.64 μM. In the case of HT-29 colon cancer cells, the determined IC50 values were slightly higher and within a range of 3.31-19.10 μM. With exception of compound 10, no significant difference of the cancer cell growth inhibitory effect of the novel bioconjugates and the control conjugates (K1 and K2) could be detected. However, the replacement of 3Trp by 3d-Tic in connection with the deletion of 2His led to an increased cytostatic effect of 10 on both of the analyzed cell lines. The IC50 value of bioconjugate 10 was more than 15-times lower on ER+ breast cancer MCF-7 cells and 5-times lower on colon cancer cells HT-29 compared to the control compound K2. Based on these promising findings, the growth inhibitory effects of conjugate 10 and the related 2His-3d-Tic (3, 5 and 12) conjugates, as well as the 10Gly-NH-Et (7 and 14) containing compounds, were studied on estrogen receptor negative (ER-) MDA-MB-231 breast cancer cells. The corresponding IC50 values are shown in Table 2. The GnRH-III-Dau conjugate 10 revealed also on this cell line the highest anticancer activity with an IC50 value of 2.49 μM. The comparison of the dose-dependent growth inhibitory effect of 10 and K2 on the three cancer cell lines is shown in Figure 1. Considering the results of all three cell lines, only compound 10 which contains the N-terminal modification 2His-d-Tic-Lys(Bu), displayed clearly a reduced cell viability, while the conjugates bearing other substitutions yielded IC50 values which vary only slightly in comparison to the controls.

In our experiments, GnRH-III was applied as a targeting peptide. The conjugate [8Lys(Dau=Aoa-GFLG)]-GnRH-III, in which Dau was connected to the side chain of Lys in position 8 through an aminooxyacetylated cathepsin B-labile GFLG spacer, showed significant tumor growth inhibition in s.c.-developed fast-growing C26 murine colon cancer-bearing Balb/c mice. The effect highly depended on the treatment schedule. In the first attempt, the conjugate was administered i.p. at a dose of 8.86 umol (5 mg Dau-content/kg body weight) five times on days 7, 9, 11, 14 and 16 after tumor transplantation (Treatment schedule A). The tumor volume inhibition was only 15.7% on day 26 when the experiment finished. A slight enhancement in inhibition (22.3%) was detected when a dose of conjugate corresponding to 15 mg Dau content/kg body weight (26.6 μmol) was injected only once on day 7 (Treatment schedule B). An additional treatment on day 10 (Treatment schedule C) did not result in any further improvements (21.9% on day 29). However, when the treatment schedule was changed to two treatments with the same dose on days 4 and 7, 46.3% inhibition was observed on day 29 (Treatment schedule D). In contrast, the treatments with free Dau at a dose of 2 mg/kg body weight (3.55 umol) on days 7, 9, 11, 14 and 16 showed only 22.6% inhibition on day 26. The median survival rates of the treated animals in comparison to the control group were 1 (A), 1.23 (B), 1.21 (C) and 1.38 (D), respectively, and 0.81 for free Dau. These results indicate the lower toxicity and the higher tumor volume inhibition effect of the conjugates in comparison with those of free Dau, as well as the importance of the treatment schedule. In another experiment, HT-29 human colon cancer was developed s.c. in immunodeficient SCID mice. Dau and two conjugates (with or without a GFLG spacer between the GnRH-III homing peptide and the payload) were used for the treatment. The first i.p. administrations were performed on day 13 after tumor inoculation. All the mice treated once with 2.5 mg/kg body weight (4.43 umol) free Dau died within 10 days. In contrast, the conjugates at a dose of 15 mg Dau content/kg body weight (26.6 umol) that refers to 52 mg/kg [8Lys(Dau=Aoa)]-GnRH-III and 62.5 mg/kg [8Lys(Dau=Aoa-GFLG)]-GnRH-III conjugates, respectively, did not show significant toxicity. The treatment was repeated on days 23 and 30. Because of the significant weight loss in several mice in the control group, the experiment was terminated on day 35. The tumor growth inhibition could be calculated as reductions in the tumor volume by 44.3% and 57.6% and the tumor weight by 41% and 50%, respectively. Interestingly, the conjugates were poorly effective on orthotopically developed tumors. In the case of the C26 colon tumor-bearing female Balb/c mice, only a 7% reduction in tumor weight was detected on day 13 after the two treatments on days 4 and 7 with [8Lys(Dau=Aoa)]-GnRH-III, at a 26.6 umol/kg (15 mg Dau content) dose. The effect of free Dau (2 mg/kg on days 4 and 7) showed a better inhibitory effect (24.4%). Interestingly, our novel developed GnRH-III derivative, in which Ser in position 4 was replaced by Lys(Ac), was much more potent, with 49.3% inhibition. It is worth mentioning that the rate of cellular uptake of the [4Lys(Ac), 8Lys(Dau=Aoa)]-GnRH-III conjugate by the tumor cells was significantly higher than that of the conjugate with the native GnRH-III sequence.

The main drug-related toxic side effect of anthracyclines is cardiotoxicity, leading to cardiomyopathy and heart failure. Comparative experiments to determine the cardiotoxicity of peptide-anthracycline conjugates with different linkages might be informative for the other conjugates with different types of drugs as well. For this purpose, human cardiomyocytes (HCM) and human umbilical vein endothelial cells (HUVEC) were used as models. The long-term (0-72 h) cytotoxic effect of sixteen GnRH-based conjugates containing Dox and Dau was determined by real-time impedimetric sensing using the xCELLigence SP system (ACEA Biosciences, San Diego, CA, USA) [75]. The results indicated that the ester-linked GnRH-Dox conjugates, including Zoptarelin Doxorubicin, showed significant toxicity at 100 nM and 1 uM, which was remarkably pronounced on the HCM cells. The cytotoxic effect was comparable to that of the free drug, especially at the highest concentration. In contrast, the conjugates with oxime-linked Dau showed no or only a minor toxicity on both the cell lines (Table 2). These data confirm that the linkage between the payload and homing peptide has a significant influence on early drug release and, consequently, an undesired toxic side effect. We may also conclude that the search for more suitable homing peptides might be more important than the application of cleavable bonds between the drug and the peptide to develop efficient DDSs.