Article Figures & Data
Figures
- Figure 1.
Mechanism of action of IPI-504 on HER2+ breast cancer cell lines. A, effect of IPI-504 on cell proliferation of various HER2+ breast cancer cells examined in cell lines that were sensitive or refractory to trastuzumab. Treatment with IPI-504 was highly effective in inhibiting the proliferation of all HER2+, trastuzumab-sensitive and trastuzumab-resistant lines. B, to determine the kinetics of HER2 turnover, BT-474 (trastuzumab-sensitive) and JIMT-1 (trastuzumab-resistant) cells were probed for HER2 expression at multiple time points after treatment with either 0.1 or 1.0 μmol/L IPI-504. Although no HER2 degradation was observed in BT-474 cells within 8 h of treatment with 0.1 μmol/L IPI-504, marked HER2 degradation was observed in JIMT-1 at the same time point. However, HER2 levels quickly recovered at time points beyond 24 h. Treatment with 1.0 μmol/L IPI-504 resulted in enhanced HER2 suppression beyond 24 h for BT-474 cells. Complete and sustained degradation of HER2 levels beginning at 8 h was achieved at this higher IPI-504 concentration in JIMT-1 cells. C, in BT-474 (HER2+, trastuzumab-sensitive) cells, degradation of HER2, pHER3, pAkt, Akt, and pPRAS40 was detected beginning with 0.05 μmol/L IPI-504. Whereas pHER3 levels are significantly down-regulated, total HER3 expression did not appear to be affected. Dramatic Hsp70 protein induction was observed on treatment with 0.05 to 3.0 μmol/L IPI-504. Similar to IPI-504 effect on BT-474 cells, decreased expression of HER2 and pHER3 was detected in JIMT-1 (HER2+, trastuzumab-resistant) cell lysates. Unlike BT-474, HER3 degradation was evident on treatment with 0.05 to 0.1 μmol/L IPI-504. Interestingly, expression of Akt, a Hsp90 client protein, was unaffected in the BT-474 cell line. In addition, downstream Akt signaling, including pAkt and pPRAS40, were not affected in this HER2+, trastuzumab-resistant cell line.
- Figure 2.
Antitumor activity and mechanism of action of IPI-504 in HER2+, trastuzumab-sensitive human gastric cancer xenograft model. N-87 xenografts were established in athymic nu/nu mice, and on reaching 200 mm3 in size, mice (n = 10 per group) were staged and randomized for the study. A, i.v. administration of 75 or 100 mg/kg IPI-504 on a every-fourth-day schedule (4 doses in total) was compared with 50 or 75 mg/kg IPI-504 on a every-other-day schedule (7 doses in total). Every-other-day schedule was chosen to be the most frequent dose possible via this route of administration given the limited number of tail vein injections that can be done. Treatment with 30 mg/kg trastuzumab i.p. (every fourth day × 4) shows that this is indeed a sensitive HER2+ model. B, p.o. administration of either 50 or 100 mg/kg IPI-504 was examined. Mice were either treated on a daily or every-other-day schedule for a total of 13 and 7 doses, respectively. C, i.v. treatment with 25, 50, or 75 mg/kg IPI-504 was initiated in female athymic nu/nu mice when the BT-474 xenografts reached an average of size of 300 mm3. IPI-504 was administered every other day for a total of 7 doses (every other day × 7) at various IPI-504 doses indicated. Trastuzumab (30 mg/kg) was dosed i.p. every 4 d for a total of 4 doses (every fourth day × 4) and used as a positive control for this line, which is trastuzumab-sensitive. D, p.o. treatment with 25, 50, or 100 mg/kg IPI-504 was administered to mice implanted with BT-474 human breast cancer cells. IPI-504 was administered daily for a total of 13 doses (daily × 13) at doses indicated. Trastuzumab (30 mg/kg), dosed i.p. every 4 d for a total of 4 doses (every fourth day × 4), was used as positive control. E, Western blot analyses were done on tumor lysates collected at the end of the efficacy study described in C, where IPI-504 was administered i.v. in mice bearing BT-474 xenografts. For single-dose effect of IPI-504 on HER2 degradation, the vehicle-treated group during the course of the study was given a single dose of 75 mg/kg IPI-504 and tumors were harvested at 7, 24, and 48 h thereafter. For tumor lysates exposed to cumulative doses of IPI-504, tumors were harvested at 7, 24, and 48 h post-last dose. The same blot was also probed with glyceraldehyde-3-phosphate dehydrogenase as a loading control. F, tumor lysates in D, where IPI-504 was dosed p.o. at various concentrations, were harvested and analyzed for HER2 protein expression by Western blot analyses. Lysates for single-dose effect of IPI-504 on HER2 degradation were isolated from the vehicle-treated group, which was given a single dose of 75 mg/kg IPI-504 at the end of the study and tumors were harvested at 7, 24, and 48 h thereafter. Tumor lysates treated with multiple IPI-504 doses were harvested at 7, 24, and 48 h post-last dose. The same blot was probed with glyceraldehyde-3-phosphate dehydrogenase as a loading control.
- Figure 3.
Antitumor activity and mechanism of action of IPI-504 in JIMT-1, a HER2+, trastuzumab-resistant, human breast cancer xenograft model. A, i.v. treatment with 25, 50, or 75 mg/kg IPI-504 was initiated in female athymic nu/nu mice implanted orthotopically with JIMT-1 cells (n = 9 mice per group). IPI-504 was administered every other day for a total of 10 doses (every other day × 10) at doses indicated. Trastuzumab (30 mg/kg) was dosed i.p. every 4 d for a total of 5 doses (every fourth day × 5) and used to show trastuzumab resistance of this model. B, p.o. treatment with 25, 50, or 100 mg/kg IPI-504 was administered to mice carrying orthotopic JIMT-1 xenografts in the thoracic mouse mammary fat pad. IPI-504 was administered daily for a total of 14 doses (daily × 14) at doses indicated. Trastuzumab (30 mg/kg) was included as a negative control and dosed i.p. every 4 d for a total of 5 doses (every fourth day × 5).
- Figure 4.
Efficacy and tolerability of IPI-504/trastuzumab combination in HER2+, trastuzumab-sensitive and trastuzumab-resistant xenograft model. A and B, athymic nu/nu mice implanted with N-87 (HER2+, trastuzumab-sensitive) cells were staged and randomized when tumors were ∼250 mm3 (n = 9 mice per group). IPI-504 was dosed p.o. on a daily schedule for a total of 14 doses (daily × 14; p.o.) at either 50 or 100 mg/kg. Trastuzumab was dosed every 4 d i.p. for a total of 4 doses (every fourth day × 4; i.p.) at either 10 or 30 mg/kg. To facilitate viewing of multiple arms of this combination study, data from 50 mg/kg IPI-504 combination with trastuzumab are represented in A and results of 100 mg/kg IPI-504 combination with trastuzumab are represented in B. Overall, single-agent IPI-504 treatment showed better efficacy than either 10 or 30 mg/kg trastuzumab as single agent but in combination achieved significant tumor growth delay. Finally, the combined treatment of IPI-504 and trastuzumab was well tolerated with no body weight loss detected. C and D, athymic nu/nu mice implanted with JIMT-1 cells (n = 10 mice per group) were staged and randomized when tumors reached ∼250 mm3 in size. IPI-504 was dosed p.o. on a daily schedule for a total of 14 doses (daily × 14; p.o.) at either 50 or 75 mg/kg. Trastuzumab was dosed every fourth day i.p. for a total of 4 doses (every fourth day × 4; i.p.) at either 10 or 30 mg/kg. To facilitate viewing of multiple arms of this combination study, data from 50 mg/kg IPI-504 combination with trastuzumab are represented in C and results of 100 mg/kg IPI-504 combination with trastuzumab are represented in D. Overall, combined treatment of 75 mg/kg IPI-504 and 10 mg/kg trastuzumab showed enhanced tumor growth delay over single agent and all combination arms were well tolerated with no body weight loss detected.
- Figure 5.
Efficacy and tolerability of IPI-504:lapatinib combination in HER2+, trastuzumab-sensitive breast cancer xenograft model. Athymic nu/nu mice implanted with KPL4, HER2+ human breast cancer cells (n = 10 mice per group) were staged and randomized when tumors were ∼150 mm3. IPI-504 was dosed p.o. on a daily schedule for a total of 14 doses (daily × 14; p.o.) at either 50 or 100 mg/kg. Lapatinib was p.o. dosed daily, twice a day for a total of 28 doses (twice a day × 14; p.o.). Mice would be dosed with lapatinib in the morning and late afternoon and IPI-504 at midday. To facilitate interpretation of multiple combination arms in this study, data from 50 mg/kg lapatinib in combination with 25 and 50 mg/kg IPI-504 are represented in A and results of 100 mg/kg lapatinib in combination with 25 and 50 mg/kg IPI-504 are represented in B. Treatment with 50 mg/kg IPI-504 as a single agent was more effective in TGI than the highest dose of lapatinib tested. The combination of 50 mg/kg IPI-504; 100 mg/kg lapatinib resulted in 20% complete regression and 40% PR in treated mice. Although combination at these concentrations were well tolerated, combination arm of 75 mg/kg IPI-504; 100 mg/kg lapatinib had to be terminated prematurely due to potential drug-drug interaction (data not shown).
- Figure 6.
Effect of IPI-504 drug holiday on IPI-504:trastuzumab in HER2+, trastuzumab-sensitive human tumor model. A and B, athymic nu/nu mice implanted with N-87 cells were staged and randomized when tumors reached between 250 and 270 mm3 in size (n = 10 mice per group). IPI-504 was dosed at 75 mg/kg i.v. to reflect the route of administration in the clinic on either a every-other-day schedule for a total of 8 doses (S3) or a every other day × 4 with a drug holiday incorporated before administration of second cycle of treatment (S1). Trastuzumab was dosed either continuously every fourth day i.p. for a total of 6 doses (S2) to span the dosing period of IPI-504 treatment with drug holiday incorporated or administered only during IPI-504 drug holidays (S4). For better viewing of multiple arms of this combination study, data from single-agent data are represented in A and various combination arms are represented in B. To assess the finer differences between each combination arm, tumors were monitored for regrowth post-last dose (day 33 of treatment). Overall, all combination arms were well tolerated with no body weight loss detected.
Volume 8, Issue 8
