PG545 enhances anti-cancer activity of chemotherapy in ovarian models and increases surrogate biomarkers such as VEGF in preclinical and clinical plasma samples

Abstract

Background: Despite the utility of antiangiogenic drugs in ovarian cancer, efficacy remains limited due to resistance linked to alternate angiogenic pathways and metastasis. Therefore, we investigated PG545, an anti-angiogenic and anti-metastatic agent which is currently in Phase I clinical trials, using preclinical models of ovarian cancer.

Methods: PG545's anti-cancer activity was investigated in vitro and in vivo as a single agent, and in combination with paclitaxel, cisplatin or carboplatin using various ovarian cancer cell lines and tumour models.

Results: PG545, alone, or in combination with chemotherapeutics, inhibited proliferation of ovarian cancer cells, demonstrating synergy with paclitaxel in A2780 cells. PG545 inhibited growth factor-mediated cell migration and reduced HB-EGF-induced phosphorylation of ERK, AKT and EGFR in vitro and significantly reduced tumour burden which was enhanced when combined with paclitaxel in an A2780 model or carboplatin in a SKOV-3 model. Moreover, in the immunocompetent ID8 model, PG545 also significantly reduced ascites in vivo. In the A2780 maintenance model, PG545 initiated with, and following paclitaxel and cisplatin treatment, significantly improved overall survival. PG545 increased plasma VEGF levels (and other targets) in preclinical models and in a small cohort of advanced cancer patients which might represent a potential biomarker of response.

Conclusion: Our results support clinical testing of PG545, particularly in combination with paclitaxel, as a novel therapeutic strategy for ovarian cancer.

Keywords: HB-EGF; Heparanase; Ovarian cancer; PG545; Tumour microenvironment; VEGF.

Figure 1

Combination of PG545and paclitaxel against A2780 cells was analysed for synergy using the method of Chou-Talalay. A. The fraction affected (Fa) and Combination Index (CI) values were calculated and plotted using Calcusyn software after the combined action of Paclitaxel: PG545 (0.09375:7.125 μM) in A2780 cell for 48h. B. DRI for PG545 and paclitaxel was plotted against Fa.

Figure 2

PG545 significantly inhibits migration and invasion of SKOV3 ovarian cancer cells by targeting growth factors. A and B. PG545 (5μM) significantly inhibited migration of SKOV3 ovarian cancer cells stimulated by each of the following HS binding growth factors: HB-EGF, HGF, FGF-2, VEGF₁₆₅ and SDF-1. C. PG545 was also tested against HB-EGF-dependent SKOV3 cell invasion and the compound was found to be a potent inhibitor of this process. NS: Not significant; *p<0.05, **p<0.01, ***p<0.001. D and E. After the SKOV3 cells (D) or OV202 (E) were cultured in 0.2% serum containing medium for 12hrs, 50ng/ml HB-EGF was added with and without PG545 for the indicated times. Western blot analysis indicated diminished phosphorylation of EGFR, ERK and AKT. Total EGFR, ERK and AKT are shown for equal loading.

Figure 3

Combination treatment using xenograft models. A. PG545 administered either s.c. or i.v. in the A2780 model. Treatment started on day 1 (n = 10/group) using 20mg/kg s.c. (1x/wk × 3), 7.5mg/kg i.v. (2x/wk × 3) or 15mg/kg i.v. (1x/wk × 3) when the tumour mean volumes were 140mm³. Efficacy was determined from tumour growth inhibition (TGI) estimated at 55%, 45%, and 55% for 20mg/kg s.c., 15mg/kg i.v. and 7.5mg/kg i.v., respectively on day 12. All treatment groups were considered significant compared versus vehicle control (p<0.01). B. PG545 treated animals survived ~2.5 times longer compared to untreated controls (**p< 0.01, log-rank test). C. A2780 cells were injected s.c. and treatment started with paclitaxel once mean estimated tumour mass reached 136mg. Two days post-paclitaxel treatment, PG545 was administered and treatment continued for 3 weeks. Tumour Growth Inhibition (TGI) for paclitaxel, PG545 and the paclitaxel/PG545 combination group was 11%, 48% and 79% respectively. D. SKOV3 cells were injected s.c. and treatment started at 37 days when the average tumour volume was approximately 123mm³. Vehicle control and PG545 were administered once weekly, s.c. and carboplatin (40 mg/kg) was administered i.v. once weekly. The dose of PG545 was reduced from 20 to 10mg/kg after the first dose. In the groups receiving combination therapy, PG545 was administered two days after carboplatin. TGI at Day 19 was estimated at 40% for PG545 alone, 43% carboplatin alone and 58% for the combination. One mouse in the combination group was culled due to excess bodyweight on Day12 of the study. Otherwise, the combination was well tolerated (a combination arm using 60 mg/kg carboplatin led to TGI of 73% but also bodyweight loss in two mice which were culled in Day 12, data not shown). *=p<0.05, **=p<0.01 PG545 alone versus vehicle control, ### = p<0.001 PG545 and paclitaxel combination versus paclitaxel alone, and + = p<0.05, +++ = p<0.001 combination groups versus vehicle control.

Figure 4

Significant anti-tumour activity of PG545 in the immunocompetent ID8 ovarian cancer model. Twenty mice were injected i.p. with 5×10⁶ ID8 cells. Three days after inoculation, mice were randomized to two different groups of 10 mice/grp: 1. control (i.p. PBS), 2. PG545 20mg/kg/wk i.p. for 10 wks. Abdominal circumference (A), volume of ascites (B), and total excised tumour weight (C) was then calculated for each treatment group and compared to control *=p<0.05. D. Representative images of control and PG545-treated mice in week 2 and week 9 in ID8 tumour-bearing mice using the IVIS luminescence imaging system series 2000. Color bar shows photon intensity.

Figure 5

Combination effect of PG545, cisplatin, paclitaxel in A2780 model. A. Fifty mice were injected i.p. with 1.25 ×10⁶ A2780 cells and randomly assigned to 5 treatment groups. Treatment with drugs commenced on Day 3. Group 1: Vehicle control, Group 2: PG545 20mg/kg twice a week starting at day 3 and maintained till the end of the study, Group 3: cisplatin 6mg/kg and paclitaxel 15mg/kg on days 3, 6 and 9 only. Group 4: Cisplatin and paclitaxel (days 3, 6 and 9) PG545 20mg/kg twice a week starting on day 10 and continued until the end of the experiment. Group 5: Cisplatin and paclitaxel (days 3, 6 and 9) PG545 20mg/kg twice a week starting on day 3 and continued until the end of the experiment. B. Kaplan Meier survival curves reveal the significant enhancement of survival following treatment with PG545 alone, doublet therapy of paclitaxel/cisplatin or triplet therapy of PG545/paclitaxel/cisplatin on days as indicated. *=P<0.05, **=P<0.01. C. Median survival in days and number of mice alive at the end of the study are shown in the various treatment groups.

Figure 6

Increased plasma VEGF is a putative pharmacodynamic marker following treatment via different routes of administration with PG545 in preclinical studies. Preliminary yet robust data were generated from the immunocompetent ID8 model and the immunodeficient A2870 model. A. Plasma VEGF concentration was increased in the PG545- treated group (IP injection) versus control animals with borderline statistical significance (P=0.06). B-D. Time versus concentration profiles for PG545 (black triangles) was determined following administration of either via SC injection (20 mg/kg), or IV injection (7 or 15 mg/kg) led to increases in plasma VEGF concentration (open squares).

Figure 7

Treatment with PG545 increases plasma VEGF levels in patients with solid tumours. Administration with weekly s.c. injections of PG545 leads to increases in plasma VEGF (white squares). PG545 plasma concentrations are indicated by the black circles. Patient Pt003 (thyroid) received 2 doses of 50mg of PG545 and the other three patients (colon, pancreatic, and melanoma) received 7, 3 and 8 doses of 25mg respectively. Dosing regimen: Pt003: days 0, 7; Pt021: days 0, 7, 14, 21, 28, 35, 42; Pt022: days 0, 7, 14; Pt023: days 0, 7, 14, 21, 28, 35, 42, 49.