In vivo assessment in tumor-bearing animal models classically complements cell cultures data for comparing cancer treatment efficacies. For this reason, hTNBCSC and hOvCSC xenografts were implanted subcutaneously into immunodeficient mice as described in the Methods section with only 1000 cells given their highly tumorigenic nature. Three days later, the animals began receiving weekly IV bolus administration of either vehicle, docetaxel, or TH1902. Docetaxel was administered at a dose (15 mg/kg/week; for 3 cycles) in accordance with the estimated maximal tolerated dose (MTD) for mice, as well as at 1/4 of the MTD (3.75 mg/kg/week). TH1902 was administered at doses (35 and 8.75 mg/kg/week) which contained quantities of docetaxel equivalent to those in the two administrations of free docetaxel. From the size of the hTNBCSC and hOvCSC tumors, it is apparent that docetaxel had little impact on xenografts growth when administered neither at its MTD, for both hTNBCSC and hOvCSC, nor at 1/4 of this dosage as reflected by the growth curves. TH1902, when administered at a dosage equivalent to docetaxel at its MTD, provided greater tumor growth inhibition than did docetaxel for both xenograft models. Furthermore, higher dosage of administered TH1902 (up to 1.5 equivalent of docetaxel MTD) did not generate significant differences in terms of hOvCSC tumor inhibition without affecting mice body weights suggesting that TH1902, even at higher doses, is better tolerated compared to docetaxel. Then, in order to statistically compare the effects of docetaxel and TH1902 on tumor growth, the tumor sizes measured at the vehicle group endpoint were compared and statistically significant differences between the tumor sizes in vehicle-treated animals found in TH1902-treated animals for hTNBCSC and hOvCSC. Mouse body weight was used as an indicator of the morbidity associated with administration of docetaxel or TH1902. Administration of docetaxel at its MTD provoked a weight loss that approached 10% after the treatments, which is often observed in xenograft models with administration of docetaxel at this level. The body weights of animals treated with an equivalent quantity or a 1.5-fold equivalent of TH1902 were maintained at a roughly constant level throughout the experiment. The animals treated with vehicle recorded a slight weight gain (~5%) over this period while animals treated with the lower dosages of docetaxel or TH1902 were similar to the animals treated with vehicle. The body weight data indicates that TH1902 appears to be better tolerated than the equivalent quantity of free docetaxel, in addition to the fact that TH1902 is more efficacious than docetaxel when administered in vivo in the murine models of CSC tested.

In vivo assessment in tumor-bearing animal models classically complements cell cultures data for comparing cancer treatment efficacies. For this reason, hTNBCSC and hOvCSC xenografts were implanted subcutaneously into immunodeficient mice as described in the Methods section with only 1000 cells given their highly tumorigenic nature. Three days later, the animals began receiving weekly IV bolus administration of either vehicle, docetaxel, or TH1902. Docetaxel was administered at a dose (15 mg/kg/week; for 3 cycles) in accordance with the estimated maximal tolerated dose (MTD) for mice, as well as at 1/4 of the MTD (3.75 mg/kg/week). TH1902 was administered at doses (35 and 8.75 mg/kg/week) which contained quantities of docetaxel equivalent to those in the two administrations of free docetaxel. From the size of the hTNBCSC and hOvCSC tumors, it is apparent that docetaxel had little impact on xenografts growth when administered neither at its MTD, for both hTNBCSC and hOvCSC, nor at 1/4 of this dosage as reflected by the growth curves. TH1902, when administered at a dosage equivalent to docetaxel at its MTD, provided greater tumor growth inhibition than did docetaxel for both xenograft models. Furthermore, higher dosage of administered TH1902 (up to 1.5 equivalent of docetaxel MTD) did not generate significant differences in terms of hOvCSC tumor inhibition without affecting mice body weights suggesting that TH1902, even at higher doses, is better tolerated compared to docetaxel. Then, in order to statistically compare the effects of docetaxel and TH1902 on tumor growth, the tumor sizes measured at the vehicle group endpoint were compared and statistically significant differences between the tumor sizes in vehicle-treated animals found in TH1902-treated animals for hTNBCSC and hOvCSC. Mouse body weight was used as an indicator of the morbidity associated with administration of docetaxel or TH1902. Administration of docetaxel at its MTD provoked a weight loss that approached 10% after the treatments, which is often observed in xenograft models with administration of docetaxel at this level. The body weights of animals treated with an equivalent quantity or a 1.5-fold equivalent of TH1902 were maintained at a roughly constant level throughout the experiment. The animals treated with vehicle recorded a slight weight gain (~5%) over this period while animals treated with the lower dosages of docetaxel or TH1902 were similar to the animals treated with vehicle. The body weight data indicates that TH1902 appears to be better tolerated than the equivalent quantity of free docetaxel, in addition to the fact that TH1902 is more efficacious than docetaxel when administered in vivo in the murine models of CSC tested.

B16-F10 melanoma syngeneic tumors were generated, with tumor sizes monitored as described in the Methods section. Tumors in xenograft-bearing, vehicle-treated mice grew at an exponential rate. Partial inhibition of tumor growth was observed after IV administration of 15 mg/kg/wk docetaxel, whereas treatment with a docetaxel-equivalent quantity of TH1902 (35 mg/kg/wk) induced tumor regression after two treatments over the period measured. Due to rapid tumor growth, only two administrations of the test articles could be performed on a weekly schedule. B16-F10 melanoma tumors from the mice treated with either vehicle, docetaxel, or TH1902 were then excised, fixed in formalin, and immunohistochemically examined.

In the ES-2 study, mice treated with low and high doses of TH1902 significantly inhibited the growth of tumors by, respectively, 45% and 87%, whereas both docetaxel groups, at equivalent docetaxel content, produced little effect (Figure 6A and Table 1). Low doses of docetaxel and low and high doses of TH1902 were well-tolerated with slight weight gain, whereas three weekly cycles of treatments with high doses of docetaxel (the MTD for this agent in mice) produced a small weight loss when compared to initial mice weights

In the SKOV3 study, mice treated with low and high doses of TH1902 showed significant inhibitions of tumor growth where only high dose of docetaxel produced this effect (Figure 6B). When compared to vehicle endpoint (Day 28), low dose of TH1902 produced similar tumor growth inhibitions when compared to high dose of docetaxel (69% vs. 84%, respectively) while high dose of TH1902 induced stronger inhibition with slight regression of tumors (Table 1). Moreover, mice treated with high dose of TH1902 showed prolonged inhibitory effect (up to Day 46) where docetaxel-treated mice could not sustain this effect, which is marked by tumor regrowth (Figure 6B).

For mice bearing endometrial tumor xenografts, a similar dosage regimen to that for ovarian tumor xenografts was used as described previously for both docetaxel and TH1902. The growth of AN3-CA tumors was inhibited by both dosages of TH1902, whereas only a high dose of docetaxel could produce this effect. When compared to vehicle endpoint, low dose of TH1902 significantly inhibited the growth of AN3-CA tumors by 74%, whereas both high doses of docetaxel and TH1902 induced tumor regressions. Interestingly, a large portion of tumors (5 out of 6) within the high-dose TH1902 group were unmeasurable or remained in regression for a prolonged period (up to Day 54), whereas tumors in the equivalent docetaxel dose were unresponsive and regrew before the end of treatments. The two high doses were diminished by half for the final two treatments due to weight loss in the animals receiving high-dose docetaxel. Rapid weight loss was associated with administration of high dosage docetaxel, but this was reversed when the dosage was halved. In contrast, only mild weight loss was observed in animals administered with high-dosage TH1902.

In the SKOV3 study, mice treated with low and high doses of TH1902 showed significant inhibitions of tumor growth where only high dose of docetaxel produced this effect (Figure 6B). When compared to vehicle endpoint (Day 28), low dose of TH1902 produced similar tumor growth inhibitions when compared to high dose of docetaxel (69% vs. 84%, respectively) while high dose of TH1902 induced stronger inhibition with slight regression of tumors (Table 1). Moreover, mice treated with high dose of TH1902 showed prolonged inhibitory effect (up to Day 46) where docetaxel-treated mice could not sustain this effect, which is marked by tumor regrowth (Figure 6B).

In the ES-2 study, mice treated with low and high doses of TH1902 significantly inhibited the growth of tumors by, respectively, 45% and 87%, whereas both docetaxel groups, at equivalent docetaxel content, produced little effect (Figure 6A and Table 1). Low doses of docetaxel and low and high doses of TH1902 were well-tolerated with slight weight gain, whereas three weekly cycles of treatments with high doses of docetaxel (the MTD for this agent in mice) produced a small weight loss when compared to initial mice weights

For mice bearing endometrial tumor xenografts, a similar dosage regimen to that for ovarian tumor xenografts was used as described previously for both docetaxel and TH1902. The growth of AN3-CA tumors was inhibited by both dosages of TH1902, whereas only a high dose of docetaxel could produce this effect. When compared to vehicle endpoint, low dose of TH1902 significantly inhibited the growth of AN3-CA tumors by 74%, whereas both high doses of docetaxel and TH1902 induced tumor regressions. Interestingly, a large portion of tumors (5 out of 6) within the high-dose TH1902 group were unmeasurable or remained in regression for a prolonged period (up to Day 54), whereas tumors in the equivalent docetaxel dose were unresponsive and regrew before the end of treatments. The two high doses were diminished by half for the final two treatments due to weight loss in the animals receiving high-dose docetaxel. Rapid weight loss was associated with administration of high dosage docetaxel, but this was reversed when the dosage was halved. In contrast, only mild weight loss was observed in animals administered with high-dosage TH1902.

The impact of docetaxel and TH1902 on MDA-MB-231 cellular morphology was evaluated and visualized by light microscopy. A drastic change in cell shape was observed in both docetaxel- and TH1902-treated cells when compared to control cells. As already reported for docetaxel-treated human renal clear cell carcinoma, MDA-MB-231 cells treated with docetaxel appeared more contracted than control cells. Interestingly, TH1902-treated cell morphology appeared even smaller, less flattened, and presented greater inter-cell spacing in comparison to those treated with free docetaxel. This observation confirms the enhanced cytotoxic effect of TH1902 when compared to docetaxel. Docetaxel is further believed to exert its cytotoxic effects through both cell cycle regulation and apoptosis induction. MDA-MB-231 cells were therefore treated for 5 or 24 hours with various concentrations of either docetaxel or TH1902, followed by AnnexinV/PI staining. TH1902 increased MDA-MB-231 cell apoptosis in a dose-dependent manner when compared to docetaxel treatment. Less than 1% apoptotic cells were measured when cells were treated with 50 μM TH19P01 for 24 hours (data not shown). Overall, this suggests that, even within such a short time frame, receptor-mediated events account for the increased effects of TH1902. Finally, to confirm the role of SORT1 in TH1902 internalization and induced apoptosis, pharmacological inhibition of its apoptotic activity was measured after incubating MDA-MB-231 breast cancer cells with TH1902 in the presence or absence of the free unlabeled TH19P01 peptide or of one of the two different SORT1 ligands neurotensin or progranulin. The apoptosis assay showed that excess TH19P01 peptide, neurotensin or progranulin competitively reduced TH1902-induced cell death by 68%, 53% and 87%, respectively. This confirms the involvement of SORT1 in the internalization process of TH1902 leading to cell death in a TNBC cell model.

The impact of docetaxel and TH1902 on MDA-MB-231 cellular morphology was evaluated and visualized by light microscopy. A drastic change in cell shape was observed in both docetaxel- and TH1902-treated cells when compared to control cells. As already reported for docetaxel-treated human renal clear cell carcinoma, MDA-MB-231 cells treated with docetaxel appeared more contracted than control cells. Interestingly, TH1902-treated cell morphology appeared even smaller, less flattened, and presented greater inter-cell spacing in comparison to those treated with free docetaxel. This observation confirms the enhanced cytotoxic effect of TH1902 when compared to docetaxel. Docetaxel is further believed to exert its cytotoxic effects through both cell cycle regulation and apoptosis induction. MDA-MB-231 cells were therefore treated for 5 or 24 hours with various concentrations of either docetaxel or TH1902, followed by AnnexinV/PI staining. TH1902 increased MDA-MB-231 cell apoptosis in a dose-dependent manner when compared to docetaxel treatment. Less than 1% apoptotic cells were measured when cells were treated with 50 μM TH19P01 for 24 hours (data not shown). Overall, this suggests that, even within such a short time frame, receptor-mediated events account for the increased effects of TH1902. Finally, to confirm the role of SORT1 in TH1902 internalization and induced apoptosis, pharmacological inhibition of its apoptotic activity was measured after incubating MDA-MB-231 breast cancer cells with TH1902 in the presence or absence of the free unlabeled TH19P01 peptide or of one of the two different SORT1 ligands neurotensin or progranulin. The apoptosis assay showed that excess TH19P01 peptide, neurotensin or progranulin competitively reduced TH1902-induced cell death by 68%, 53% and 87%, respectively. This confirms the involvement of SORT1 in the internalization process of TH1902 leading to cell death in a TNBC cell model.

The impact of docetaxel and TH1902 on MDA-MB-231 cellular morphology was evaluated and visualized by light microscopy. A drastic change in cell shape was observed in both docetaxel- and TH1902-treated cells when compared to control cells. As already reported for docetaxel-treated human renal clear cell carcinoma, MDA-MB-231 cells treated with docetaxel appeared more contracted than control cells. Interestingly, TH1902-treated cell morphology appeared even smaller, less flattened, and presented greater inter-cell spacing in comparison to those treated with free docetaxel. This observation confirms the enhanced cytotoxic effect of TH1902 when compared to docetaxel. Docetaxel is further believed to exert its cytotoxic effects through both cell cycle regulation and apoptosis induction. MDA-MB-231 cells were therefore treated for 5 or 24 hours with various concentrations of either docetaxel or TH1902, followed by AnnexinV/PI staining. TH1902 increased MDA-MB-231 cell apoptosis in a dose-dependent manner when compared to docetaxel treatment. Less than 1% apoptotic cells were measured when cells were treated with 50 μM TH19P01 for 24 hours (data not shown). Overall, this suggests that, even within such a short time frame, receptor-mediated events account for the increased effects of TH1902. Finally, to confirm the role of SORT1 in TH1902 internalization and induced apoptosis, pharmacological inhibition of its apoptotic activity was measured after incubating MDA-MB-231 breast cancer cells with TH1902 in the presence or absence of the free unlabeled TH19P01 peptide or of one of the two different SORT1 ligands neurotensin or progranulin. The apoptosis assay showed that excess TH19P01 peptide, neurotensin or progranulin competitively reduced TH1902-induced cell death by 68%, 53% and 87%, respectively. This confirms the involvement of SORT1 in the internalization process of TH1902 leading to cell death in a TNBC cell model.

The impact of docetaxel and TH1902 on MDA-MB-231 cellular morphology was evaluated and visualized by light microscopy. A drastic change in cell shape was observed in both docetaxel- and TH1902-treated cells when compared to control cells. As already reported for docetaxel-treated human renal clear cell carcinoma, MDA-MB-231 cells treated with docetaxel appeared more contracted than control cells. Interestingly, TH1902-treated cell morphology appeared even smaller, less flattened, and presented greater inter-cell spacing in comparison to those treated with free docetaxel. This observation confirms the enhanced cytotoxic effect of TH1902 when compared to docetaxel. Docetaxel is further believed to exert its cytotoxic effects through both cell cycle regulation and apoptosis induction. MDA-MB-231 cells were therefore treated for 5 or 24 hours with various concentrations of either docetaxel or TH1902, followed by AnnexinV/PI staining. TH1902 increased MDA-MB-231 cell apoptosis in a dose-dependent manner when compared to docetaxel treatment. Less than 1% apoptotic cells were measured when cells were treated with 50 μM TH19P01 for 24 hours (data not shown). Overall, this suggests that, even within such a short time frame, receptor-mediated events account for the increased effects of TH1902. Finally, to confirm the role of SORT1 in TH1902 internalization and induced apoptosis, pharmacological inhibition of its apoptotic activity was measured after incubating MDA-MB-231 breast cancer cells with TH1902 in the presence or absence of the free unlabeled TH19P01 peptide or of one of the two different SORT1 ligands neurotensin or progranulin. The apoptosis assay showed that excess TH19P01 peptide, neurotensin or progranulin competitively reduced TH1902-induced cell death by 68%, 53% and 87%, respectively. This confirms the involvement of SORT1 in the internalization process of TH1902 leading to cell death in a TNBC cell model.

The results show that TH1902 induces more efficiently apoptosis in both cell lines when compared to docetaxel especially in SKOV3 cells. Overall, this suggests that receptor-mediated events appear to account for the increased effects of TH1902 within such a short time frame. To confirm the implication of SORT1 in TH1902 internalization and apoptosis induction, SORT1 was transiently silenced in ES-2 and SKOV3 cells.

The results show that TH1902 induces more efficiently apoptosis in both cell lines when compared to docetaxel especially in SKOV3 cells. Overall, this suggests that receptor-mediated events appear to account for the increased effects of TH1902 within such a short time frame. To confirm the implication of SORT1 in TH1902 internalization and apoptosis induction, SORT1 was transiently silenced in ES-2 and SKOV3 cells.

To determine whether docetaxel or TH1902 inhibited TNBC cell proliferation, MTT assays were performed in MDA-MB-231 and HCC-70 cells exposed to various concentrations of TH19P01, docetaxel or TH1902. Inhibition of MDA-MB-231 cell proliferation was effectively triggered by both docetaxel and TH1902 compounds in a concentration-dependent manner whereas TH19P01 had no effect at the concentrations tested. The IC50 value of TH1902 was found to be comparable to that of docetaxel at low nM concentrations in both MDA-MB-231 and HCC-70 cells, which supports the rationale that conjugated docetaxel can indeed be released from TH1902 once internalized and exert its antiproliferative effect inside the targeted cancer cells. The effect of TH1902 on MBA-MB-231 cell-cycle arrest was also tested. The results show that more than 70% of treated cells were arrested in the G2/M phase of the cell cycle in comparison to vehicle-treated control cells where ~14% of the cells remained in the G2/M phase. While TH19P01 appears to be an inert peptide with no in vitro antiproliferative effects, these data confirm both the growth inhibitory effect of TH1902 and, more importantly, that the docetaxel anticancer potency was unaffected by its conjugation to the TH19P01 peptide.

Cells were thus exposed to various concentrations of TH1902 or docetaxel, and the effects on cell proliferation were measured using the MTT detection assay. It is apparent that both reagents exerted a cytotoxic effect with IC50 values of TH1902 comparable to that of docetaxel at low nM concentrations in the four cell lines tested. This supports the rationale that conjugated docetaxel can be released from TH1902 and exert its anti-proliferative effect inside the targeted cancer cells. Parallel experiment showed that cell proliferation was unaffected by the free peptide (TH19P01) at concentrations up to 1000 nM.

SORT1-positive SK-MEL-28 and B16-F10 melanoma cells were selected for testing the anti-proliferative effect of docetaxel and TH1902. When TH1902 biological effects were monitored, the half-maximal inhibitory concentration (IC50) of TH1902 was similar to that of docetaxel, averaging 0.38 vs. 0.39 nM, respectively, in human SK-MEL-28 cells, and 2.57 vs. 1.72 nM in murine B16-F10 cells.

Cells were thus exposed to various concentrations of TH1902 or docetaxel, and the effects on cell proliferation were measured using the MTT detection assay. It is apparent that both reagents exerted a cytotoxic effect with IC50 values of TH1902 comparable to that of docetaxel at low nM concentrations in the four cell lines tested. This supports the rationale that conjugated docetaxel can be released from TH1902 and exert its anti-proliferative effect inside the targeted cancer cells. Parallel experiment showed that cell proliferation was unaffected by the free peptide (TH19P01) at concentrations up to 1000 nM.

To determine whether docetaxel or TH1902 inhibited TNBC cell proliferation, MTT assays were performed in MDA-MB-231 and HCC-70 cells exposed to various concentrations of TH19P01, docetaxel or TH1902. Inhibition of MDA-MB-231 cell proliferation was effectively triggered by both docetaxel and TH1902 compounds in a concentration-dependent manner whereas TH19P01 had no effect at the concentrations tested. The IC50 value of TH1902 was found to be comparable to that of docetaxel at low nM concentrations in both MDA-MB-231 and HCC-70 cells, which supports the rationale that conjugated docetaxel can indeed be released from TH1902 once internalized and exert its antiproliferative effect inside the targeted cancer cells. The effect of TH1902 on MBA-MB-231 cell-cycle arrest was also tested. The results show that more than 70% of treated cells were arrested in the G2/M phase of the cell cycle in comparison to vehicle-treated control cells where ~14% of the cells remained in the G2/M phase. While TH19P01 appears to be an inert peptide with no in vitro antiproliferative effects, these data confirm both the growth inhibitory effect of TH1902 and, more importantly, that the docetaxel anticancer potency was unaffected by its conjugation to the TH19P01 peptide.

SORT1-positive SK-MEL-28 and B16-F10 melanoma cells were selected for testing the anti-proliferative effect of docetaxel and TH1902. When TH1902 biological effects were monitored, the half-maximal inhibitory concentration (IC50) of TH1902 was similar to that of docetaxel, averaging 0.38 vs. 0.39 nM, respectively, in human SK-MEL-28 cells, and 2.57 vs. 1.72 nM in murine B16-F10 cells.

Cells were thus exposed to various concentrations of TH1902 or docetaxel, and the effects on cell proliferation were measured using the MTT detection assay. It is apparent that both reagents exerted a cytotoxic effect with IC50 values of TH1902 comparable to that of docetaxel at low nM concentrations in the four cell lines tested. This supports the rationale that conjugated docetaxel can be released from TH1902 and exert its anti-proliferative effect inside the targeted cancer cells. Parallel experiment showed that cell proliferation was unaffected by the free peptide (TH19P01) at concentrations up to 1000 nM.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The in vivo efficacy of TH1902 and docetaxel against a TNBC xenograft model was next investigated in vivo. Thus, nude mice were implanted in the right flank with MDA-MB-231-luc cancer cells, and luminescence was measured to monitor tumor growth. Mice were treated with intraperitoneal injections of either docetaxel at the MTD of 15 mg/kg/wk, or with TH1902 at the maximal injectable dose of 50 mg/kg/wk, both for five cycles of treatment. Unlike the vehicle-treated control group, where the average tumor luminescence increased over time, a significant decline in luminescence intensity was observed, starting at day 5 in the TH1902-treated group. The level of luminescence intensity was also significantly lower in the free docetaxel-treated group, when compared to that in the vehicle-treated control group, but remained higher than that for the TH1902-treated group. Interestingly, tumor relapse was observed in the docetaxel-treated group beginning at day 46, whereas a sustained decrease of tumor volume was maintained in the TH1902-treated group until day 74, at which time point a complete disappearance of the tumor was achieved. Representative images of luminescence are shown for vehicle-, docetaxel-, or TH1902-treated mice at day 14 when the control group reached the tumor volume endpoint and at day 74 post-treatment at the end of the experiment. Quantification of the residual tumor burden at days 14 and 74 after docetaxel or TH1902 treatment was performed, and the analysis confirmed a much better in vivo TH1902 efficacy profile than was seen with unconjugated docetaxel. Given the lack of apoptotic or antiproliferative effects of TH19P01, no rationale supports its further assessment in vivo. As TH1902 is considered a new chemical entity, its best control condition therefore is the unconjugated docetaxel molecule itself. Body weight of mice on intraperitoneal administration of either docetaxel or TH1902 remained within endpoint limits (-20%, data not shown).

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The in vivo efficacy of TH1902 and docetaxel against a TNBC xenograft model was next investigated in vivo. Thus, nude mice were implanted in the right flank with MDA-MB-231-luc cancer cells, and luminescence was measured to monitor tumor growth. Mice were treated with intraperitoneal injections of either docetaxel at the MTD of 15 mg/kg/wk, or with TH1902 at the maximal injectable dose of 50 mg/kg/wk, both for five cycles of treatment. Unlike the vehicle-treated control group, where the average tumor luminescence increased over time, a significant decline in luminescence intensity was observed, starting at day 5 in the TH1902-treated group. The level of luminescence intensity was also significantly lower in the free docetaxel-treated group, when compared to that in the vehicle-treated control group, but remained higher than that for the TH1902-treated group. Interestingly, tumor relapse was observed in the docetaxel-treated group beginning at day 46, whereas a sustained decrease of tumor volume was maintained in the TH1902-treated group until day 74, at which time point a complete disappearance of the tumor was achieved. Representative images of luminescence are shown for vehicle-, docetaxel-, or TH1902-treated mice at day 14 when the control group reached the tumor volume endpoint and at day 74 post-treatment at the end of the experiment. Quantification of the residual tumor burden at days 14 and 74 after docetaxel or TH1902 treatment was performed, and the analysis confirmed a much better in vivo TH1902 efficacy profile than was seen with unconjugated docetaxel. Given the lack of apoptotic or antiproliferative effects of TH19P01, no rationale supports its further assessment in vivo. As TH1902 is considered a new chemical entity, its best control condition therefore is the unconjugated docetaxel molecule itself. Body weight of mice on intraperitoneal administration of either docetaxel or TH1902 remained within endpoint limits (-20%, data not shown).

In vivo assessment in tumor-bearing animal models classically complements cell cultures data for comparing cancer treatment efficacies. For this reason, hTNBCSC and hOvCSC xenografts were implanted subcutaneously into immunodeficient mice as described in the Methods section with only 1000 cells given their highly tumorigenic nature. Three days later, the animals began receiving weekly IV bolus administration of either vehicle, docetaxel, or TH1902. Docetaxel was administered at a dose (15 mg/kg/week; for 3 cycles) in accordance with the estimated maximal tolerated dose (MTD) for mice, as well as at 1/4 of the MTD (3.75 mg/kg/week). TH1902 was administered at doses (35 and 8.75 mg/kg/week) which contained quantities of docetaxel equivalent to those in the two administrations of free docetaxel. From the size of the hTNBCSC and hOvCSC tumors, it is apparent that docetaxel had little impact on xenografts growth when administered neither at its MTD, for both hTNBCSC and hOvCSC, nor at 1/4 of this dosage as reflected by the growth curves. TH1902, when administered at a dosage equivalent to docetaxel at its MTD, provided greater tumor growth inhibition than did docetaxel for both xenograft models. Furthermore, higher dosage of administered TH1902 (up to 1.5 equivalent of docetaxel MTD) did not generate significant differences in terms of hOvCSC tumor inhibition without affecting mice body weights suggesting that TH1902, even at higher doses, is better tolerated compared to docetaxel. Then, in order to statistically compare the effects of docetaxel and TH1902 on tumor growth, the tumor sizes measured at the vehicle group endpoint were compared and statistically significant differences between the tumor sizes in vehicle-treated animals found in TH1902-treated animals for hTNBCSC and hOvCSC. Mouse body weight was used as an indicator of the morbidity associated with administration of docetaxel or TH1902. Administration of docetaxel at its MTD provoked a weight loss that approached 10% after the treatments, which is often observed in xenograft models with administration of docetaxel at this level. The body weights of animals treated with an equivalent quantity or a 1.5-fold equivalent of TH1902 were maintained at a roughly constant level throughout the experiment. The animals treated with vehicle recorded a slight weight gain (~5%) over this period while animals treated with the lower dosages of docetaxel or TH1902 were similar to the animals treated with vehicle. The body weight data indicates that TH1902 appears to be better tolerated than the equivalent quantity of free docetaxel, in addition to the fact that TH1902 is more efficacious than docetaxel when administered in vivo in the murine models of CSC tested.

In vivo assessment in tumor-bearing animal models classically complements cell cultures data for comparing cancer treatment efficacies. For this reason, hTNBCSC and hOvCSC xenografts were implanted subcutaneously into immunodeficient mice as described in the Methods section with only 1000 cells given their highly tumorigenic nature. Three days later, the animals began receiving weekly IV bolus administration of either vehicle, docetaxel, or TH1902. Docetaxel was administered at a dose (15 mg/kg/week; for 3 cycles) in accordance with the estimated maximal tolerated dose (MTD) for mice, as well as at 1/4 of the MTD (3.75 mg/kg/week). TH1902 was administered at doses (35 and 8.75 mg/kg/week) which contained quantities of docetaxel equivalent to those in the two administrations of free docetaxel. From the size of the hTNBCSC and hOvCSC tumors, it is apparent that docetaxel had little impact on xenografts growth when administered neither at its MTD, for both hTNBCSC and hOvCSC, nor at 1/4 of this dosage as reflected by the growth curves. TH1902, when administered at a dosage equivalent to docetaxel at its MTD, provided greater tumor growth inhibition than did docetaxel for both xenograft models. Furthermore, higher dosage of administered TH1902 (up to 1.5 equivalent of docetaxel MTD) did not generate significant differences in terms of hOvCSC tumor inhibition without affecting mice body weights suggesting that TH1902, even at higher doses, is better tolerated compared to docetaxel. Then, in order to statistically compare the effects of docetaxel and TH1902 on tumor growth, the tumor sizes measured at the vehicle group endpoint were compared and statistically significant differences between the tumor sizes in vehicle-treated animals found in TH1902-treated animals for hTNBCSC and hOvCSC. Mouse body weight was used as an indicator of the morbidity associated with administration of docetaxel or TH1902. Administration of docetaxel at its MTD provoked a weight loss that approached 10% after the treatments, which is often observed in xenograft models with administration of docetaxel at this level. The body weights of animals treated with an equivalent quantity or a 1.5-fold equivalent of TH1902 were maintained at a roughly constant level throughout the experiment. The animals treated with vehicle recorded a slight weight gain (~5%) over this period while animals treated with the lower dosages of docetaxel or TH1902 were similar to the animals treated with vehicle. The body weight data indicates that TH1902 appears to be better tolerated than the equivalent quantity of free docetaxel, in addition to the fact that TH1902 is more efficacious than docetaxel when administered in vivo in the murine models of CSC tested.

The in vivo efficacy of TH1902 and docetaxel against a TNBC xenograft model was next investigated in vivo. Thus, nude mice were implanted in the right flank with MDA-MB-231-luc cancer cells, and luminescence was measured to monitor tumor growth. Mice were treated with intraperitoneal injections of either docetaxel at the MTD of 15 mg/kg/wk, or with TH1902 at the maximal injectable dose of 50 mg/kg/wk, both for five cycles of treatment. Unlike the vehicle-treated control group, where the average tumor luminescence increased over time, a significant decline in luminescence intensity was observed, starting at day 5 in the TH1902-treated group. The level of luminescence intensity was also significantly lower in the free docetaxel-treated group, when compared to that in the vehicle-treated control group, but remained higher than that for the TH1902-treated group. Interestingly, tumor relapse was observed in the docetaxel-treated group beginning at day 46, whereas a sustained decrease of tumor volume was maintained in the TH1902-treated group until day 74, at which time point a complete disappearance of the tumor was achieved. Representative images of luminescence are shown for vehicle-, docetaxel-, or TH1902-treated mice at day 14 when the control group reached the tumor volume endpoint and at day 74 post-treatment at the end of the experiment. Quantification of the residual tumor burden at days 14 and 74 after docetaxel or TH1902 treatment was performed, and the analysis confirmed a much better in vivo TH1902 efficacy profile than was seen with unconjugated docetaxel. Given the lack of apoptotic or antiproliferative effects of TH19P01, no rationale supports its further assessment in vivo. As TH1902 is considered a new chemical entity, its best control condition therefore is the unconjugated docetaxel molecule itself. Body weight of mice on intraperitoneal administration of either docetaxel or TH1902 remained within endpoint limits (-20%, data not shown).

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

In vivo assessment in tumor-bearing animal models classically complements cell cultures data for comparing cancer treatment efficacies. For this reason, hTNBCSC and hOvCSC xenografts were implanted subcutaneously into immunodeficient mice as described in the Methods section with only 1000 cells given their highly tumorigenic nature. Three days later, the animals began receiving weekly IV bolus administration of either vehicle, docetaxel, or TH1902. Docetaxel was administered at a dose (15 mg/kg/week; for 3 cycles) in accordance with the estimated maximal tolerated dose (MTD) for mice, as well as at 1/4 of the MTD (3.75 mg/kg/week). TH1902 was administered at doses (35 and 8.75 mg/kg/week) which contained quantities of docetaxel equivalent to those in the two administrations of free docetaxel. From the size of the hTNBCSC and hOvCSC tumors, it is apparent that docetaxel had little impact on xenografts growth when administered neither at its MTD, for both hTNBCSC and hOvCSC, nor at 1/4 of this dosage as reflected by the growth curves. TH1902, when administered at a dosage equivalent to docetaxel at its MTD, provided greater tumor growth inhibition than did docetaxel for both xenograft models. Furthermore, higher dosage of administered TH1902 (up to 1.5 equivalent of docetaxel MTD) did not generate significant differences in terms of hOvCSC tumor inhibition without affecting mice body weights suggesting that TH1902, even at higher doses, is better tolerated compared to docetaxel. Then, in order to statistically compare the effects of docetaxel and TH1902 on tumor growth, the tumor sizes measured at the vehicle group endpoint were compared and statistically significant differences between the tumor sizes in vehicle-treated animals found in TH1902-treated animals for hTNBCSC and hOvCSC. Mouse body weight was used as an indicator of the morbidity associated with administration of docetaxel or TH1902. Administration of docetaxel at its MTD provoked a weight loss that approached 10% after the treatments, which is often observed in xenograft models with administration of docetaxel at this level. The body weights of animals treated with an equivalent quantity or a 1.5-fold equivalent of TH1902 were maintained at a roughly constant level throughout the experiment. The animals treated with vehicle recorded a slight weight gain (~5%) over this period while animals treated with the lower dosages of docetaxel or TH1902 were similar to the animals treated with vehicle. The body weight data indicates that TH1902 appears to be better tolerated than the equivalent quantity of free docetaxel, in addition to the fact that TH1902 is more efficacious than docetaxel when administered in vivo in the murine models of CSC tested.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.