The number of macro-metastases in peripheral organs, such as spleen, lung, liver and kidneys, was counted, in order to determine the anti-metastatic effect of free Dau and the GnRH-III conjugates on aggressive 4T1 BC orthotopic model (Figure 5A). The number of macro-metastases in spleen was significantly decreased in all treated groups (Dau,1and2) by 64.3, 72.8 and 78.1%. In the lung, the number of macro-metastases was also significantly reduced for all treated groups by 55.4, 55.2 and 64.4%, respectively. The numbers of macro-metastases in the liver and kidneys were decreased under treatments, whereby a significant decrease could be only obtained for conjugate2.

The number of macro-metastases in peripheral organs, such as spleen, lung, liver and kidneys, was counted, in order to determine the anti-metastatic effect of free Dau and the GnRH-III conjugates on aggressive 4T1 BC orthotopic model (Figure 5A). The number of macro-metastases in spleen was significantly decreased in all treated groups (Dau, 1 and 2) by 64.3, 72.8 and 78.1%. In the lung, the number of macro-metastases was also significantly reduced for all treated groups by 55.4, 55.2 and 64.4%, respectively. The numbers of macro-metastases in the liver and kidneys were decreased under treatments, whereby a significant decrease could be only obtained for conjugate 2.

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 anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

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 anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

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.

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 anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The number of macro-metastases in peripheral organs, such as spleen, lung, liver and kidneys, was counted, in order to determine the anti-metastatic effect of free Dau and the GnRH-III conjugates on aggressive 4T1 BC orthotopic model (Figure 5A). The number of macro-metastases in spleen was significantly decreased in all treated groups (Dau,1and2) by 64.3, 72.8 and 78.1%. In the lung, the number of macro-metastases was also significantly reduced for all treated groups by 55.4, 55.2 and 64.4%, respectively. The numbers of macro-metastases in the liver and kidneys were decreased under treatments, whereby a significant decrease could be only obtained for conjugate2.

The number of macro-metastases in peripheral organs, such as spleen, lung, liver and kidneys, was counted, in order to determine the anti-metastatic effect of free Dau and the GnRH-III conjugates on aggressive 4T1 BC orthotopic model (Figure 5A). The number of macro-metastases in spleen was significantly decreased in all treated groups (Dau, 1 and 2) by 64.3, 72.8 and 78.1%. In the lung, the number of macro-metastases was also significantly reduced for all treated groups by 55.4, 55.2 and 64.4%, respectively. The numbers of macro-metastases in the liver and kidneys were decreased under treatments, whereby a significant decrease could be only obtained for conjugate 2.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

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.

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 anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

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 anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

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 anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

The anti-proliferative effect of the GnRH-III conjugates 1 and 2, as well as free Dau, was tested on wide range of cancer cell lines from different origin and also on MRC-5 (human fibroblast) as non-cancerous control cell line. The data showed that both conjugates possess an anti-proliferative effect on all cell types (Table 1). Except for the ovarian cancer cell lines A2780 and OVCAR-8, conjugate 2 displayed higher anti-proliferative activity than conjugate 1, depending on the type of cancer cells. The lowest activity was measured on PANC-1 pancreatic cancer cells, whereby a high IC50 value was also obtained on MRC-5 cells, showing selectivity of the conjugates for cancer cell lines. The obtained IC50 values of the conjugates vary mostly in the low micromolar range and were one to two order of magnitude higher when compared to free Dau that can enter cells non-specifically by passive diffusion. Moreover, the relative potency was calculated as a ratio of conjugates IC50 and free Daus IC50 in order to show the potency of the conjugates independently from the cell line, due to different activity of free Dau. A lower value of relative potency indicates that the conjugates IC50 value is closer to the free Daus IC50 value, which implies that the targeting capacity of the conjugate as well as its anti-tumor effect is stronger on a particular cell line, as compared to a cell line with higher relative potency. The BC cell lines showed good response to the conjugates by IC50 values, as well as by relative potency. Besides, the conjugates showed high anti-proliferative activity on mice CRC cell line C26, while the conjugates showed a moderate anti-proliferative activity on HT-29 human colon adenocarcinoma, but the relative potency was in the same range as for the BC cells.

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.

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.

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

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.

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.

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.

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.

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

Human pituitary and human prostate cancer tissues have been used to evaluate the binding affinities of the new GnRH-III-drug conjugates to GnRH-R. Therefore, increasing compound concentrations were applied and the displacement of radiolabeled [¹²⁵I]-triptorelin from GnRH-Rs was detected. The obtained results were compared with the binding affinities of the oxime bond-linked GnRH-III-Dau conjugates (I, II). All compounds bind to the receptors with high affinities in the low nanomolar range, while GnRH unrelated peptides such as somatostatin or bombesin were not able to displace the radio-labelled triptorelin. However, in comparison to the GnRH-III-homing peptide (I), the self-immolative linker conjugate exhibited a 3- to 10-times reduced affinity to the GnRH receptors. Interestingly, most of the PTX-containing cleavable compounds possessed a slightly higher binding affinity than the corresponding Dau-equivalent, even if the targeting sequence and the cathepsin cleavage site remained the same.

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).

To investigate the anticancer activity of the GnRH-III drug conjugates, cell viability studies have been performed on A2780 ovarian cancer and Panc-1 pancreatic cancer cells. The GnRH-R expression of these cell lines was determined by Western blot studies. In the case of the A2780 cells, a distinct band at 38 kDa could be detected which corresponds to the full-length human GnRH-R. In contrast, the signal intensity of the 38 kDa band was much lower for Panc-1 pancreatic cancer cells being in line with our previous results. Thus, the antiproliferative activity of the GnRH-drug conjugates was studied on high-GnRH-R-expressing A2780 cells and low-GnRH-R-expressing Panc-1 cells. Since the release of free Dau and PTX can be assumed, both drugs were used as controls. The cells were treated for either 24 h (Dau conjugates) or six hours (PTX compounds), followed by additional incubation with fresh growth medium until 72 h after treatment initiation. The obtained results reveal, on the one hand, that the non-cleavable linker-containing conjugates possess a reduced anticancer activity in comparison to the cleavable conjugates and, on the other hand, that the activity of the all GnRH-III-drug conjugates was substantially reduced compared to the free drug. Moreover, all compounds displayed a lower biological activity on Panc-1 cells than on A2780. In the case of the cleavable GnRH-III-Dau conjugates, the IC50 values varied between 2.85-11.18 μM on A2780 cells, whereby the best activity was obtained for compound 13 (2.85 μM) which contained the cathepsin B-cleavage site Val-Ala and the GnRH-III-[2His-3D-Tic-4Lys(Bu)] peptide carrier. Apart from that, the IC50 values of the cleavable PTX conjugates on A2780 cells are in the same sub-micromolar range and vary between 0.51-0.77 μM, while the activity of these conjugates was approximately 10 times lower on Panc-1 cells (5.03-8.15 μM).