Intravenous Formulation of HET0016 Decreased Human Glioblastoma Growth and Implicated Survival Benefit in Rat Xenograft Models

Abstract

Glioblastoma (GBM) is a hypervascular primary brain tumor with poor prognosis. HET0016 is a selective CYP450 inhibitor, which has been shown to inhibit angiogenesis and tumor growth. Therefore, to explore novel treatments, we have generated an improved intravenous (IV) formulation of HET0016 with HPßCD and tested in animal models of human and syngeneic GBM. Administration of a single IV dose resulted in 7-fold higher levels of HET0016 in plasma and 3.6-fold higher levels in tumor at 60 min than that in IP route. IV treatment with HPßCD-HET0016 decreased tumor growth, and altered vascular kinetics in early and late treatment groups (p < 0.05). Similar growth inhibition was observed in syngeneic GL261 GBM (p < 0.05). Survival studies using patient derived xenografts of GBM811, showed prolonged survival to 26 weeks in animals treated with focal radiation, in combination with HET0016 and TMZ (p < 0.05). We observed reduced expression of markers of cell proliferation (Ki-67), decreased neovascularization (laminin and αSMA), in addition to inflammation and angiogenesis markers in the treatment group (p < 0.05). Our results indicate that HPßCD-HET0016 is effective in inhibiting tumor growth through decreasing proliferation, and neovascularization. Furthermore, HPßCD-HET0016 significantly prolonged survival in PDX GBM811 model.

Figure 1

Preparation of HPßCD-HET0016 complex (a) Structure of HET0016 and HPßCD is shown. HPßCD resembles a shell and it can encase any deliverable drug. To prepare the IV formulation of HET0016, it was first dissolved in DMSO and added to 30% HPßCD prepared in water. The bucket like structure of HPßCD with hydrophilic exterior helps encase HET0016 and delivers to the target area. (b) Mass spectrometry of HET0016 in DMSO (left panel), HET0016 complex in plasma (middle panel). HPßCD-HET0016 complex in tissue lysate (right panel). Plasma and tissue lysate was collected from the rats bearing glioma and injected with single dose of HPßCD-HET0016 for evaluation of pharmacokinetics.

Figure 2. Pharmacokinetics of HPßCD-HET0016 in plasma and tissue after IV and IP dose administration in rat glioblastoma model.

The concentrations of HET0016 in plasma or tissue versus time post dose administration in rat model are shown. Blood samples were obtained for 0–24 hrs after dose administration. Plasma and tissue HET0016 concentrations were determined by LC-MS/MS after liquid phase extraction. Each data point is presented as the mean concentration ±SEM (n = 2–4). Concentration of HET0016 in plasma (a) and tumor tissue (b) after a single HET0016 (10 mg/kg) dose through IV and IP route. Significant values from t Student test and Mann-Whitney’s test are represented by *p < 0.05 in comparison to IV and IP doses in the same time point.

Figure 3. Schematic representation of treatment schedule.

U251 glioma cells were implanted orthotopically in rat’s brain and treated with HPßCD-HET0016 as described in material and methods.

Figure 4. HPßCD-HET0016 reduces tumor growth and vascular parameters in rat glioblastoma model.

(a) Representative post contrast T1-weighted images from in vivo imaging of rat with glioma show tumor size from different groups of animals. Semi-quantitative analysis shows significantly reduced tumor volume in animals that received HET0016 from day 8 and continued for 2 weeks (n = 5 to 8). (b) Relative cerebral blood flow maps created from arterial spin labeling techniques. Semi-quantitative analysis (bar graphs) indicates higher flow in IV HET0016-treated groups (started on day 8) compared to that of vehicle-treated group (n = 5 to 8). (c) Tumor plasma volume maps and semi quantitative analysis are shown. Both IV and IP treatment with HET0016 caused a significant decrease in tumor plasma volume (v_p) compared to vehicle and corresponding IP-treated groups. Each group is represented as the mean of the total measurable MRI sections from animals of each group (n = 5–8 animals but 7–16 sections). Significant values from ANOVA test followed by Fisher’s exact test are represented by *p < 0.05 compared to the corresponding vehicle group. An outlier data point (from a single MRI section) was removed as described in our material and methods.

Figure 5. HPßCD-HET0016 influences vascular parameters in rat glioblastoma model.

MRI images from in vivo rat imaging show significant changes in vessel permeability (K^trans) (a), and v_e (interstitial space volume) (C) but no changes in k_ep (backflow rate constant) (b) in early and delayed treatment in IV groups. Significant values from ANOVA test followed by Fisher’s exact test are represented by * and ^#p < 0.05 compared to (*) vehicle 8–21 vs IV HET 8–21 and vehicle 0–21 vs IV HET 0–21, and (#) IP HET 8–21 vs IV HET 8–21 and IP HET 0–21 vs IV HET 0–21. Each group is represented as the mean of the total measurable MRI sections from animals of each group (n = 5–8 animals but 7–16 sections), analyzed by MRI at 0.5 μm thickness in a range up to 7–16 viewed slices of each animal.

Figure 6. HPßCD-HET0016 treatment results in reduced proliferation, migration in rat glioblastoma model.

(a,b) Ki-67 immunohistochemical staining was done as a marker of proliferation in tumor tissues. Rats treated with HET0016 starting on day 8 showed significantly fewer proliferative cells in the tumor in the IV group compared to the IP group. (c,d) Bar graphs represent the proportion of Ki67 positive tumor cells compared to total number of tumor cells in tumor area of each group. Images were taken in 40x magnification. The brown nuclei color shows the positive labelling cells. Statistically significant differences were verified by ANOVA followed by Bonferroni’s test. *p < 0.05 in comparison to the respective vehicle group.

Figure 7. HPßCD-HET0016 treatment results in reduced neovascularization in rat glioblastoma model.

Laminin and αSMA immunohistochemistry staining was done in tumor tissues to determine the neovascularization (a,b,d and e) Representative images from brain tumor tissues are shown at 10x and 20x from the IP (left panel) and IV (right panel) groups. Red arrows indicate blood vessels. Four areas on the tissue section were selected and the number of vessels counted. (c and f) Laminin and αSMA quanfication. Each bar represents an average of four areas and was estimated in multiple samples from each group (n = 2–4). Significant difference is indicated by *p < 0.05 compared to the respective vehicle group.

Figure 8. Treatment with HET0016 and TMZ prolongs survival in PDX models.

(a) Treatment with HET0016 and TMZ inhibit neurospheres growth in vitro: HF2303 was treated with HET0016 and TMZ alone (100 μM), in combination with TMZ and followed up for 14 days. (b) HF2303 and (c) GBM811 show the effect of HET0016 and TMZ on survival rate of the mice in groups 1, 2, 3, 4 and 5 of treatment schedules (as described in Material and Methods) were evaluated from the first day of treatment until death. X-axis represents cumulative survival time in weeks. Table in Fig. 8d summarize the details of duration in weeks and % survival. Athymic nude rats were implanted orthotopic with HF2303 and GBM811. Six to ten weeks after implantation, rats were randomized into five treatment groups receiving PBS, radiation, HET0016, TMZ, HET0016 plus TMZ for another 6 wks, as described in Materials and Methods. ^#The animals were not included due to the technical difficulty. Significant difference is indicated by *p < 0.05 vs irradiation and super-control, ^$p-value was 0.18 vs irradiation control, achieved by Kaplan Meier analysis and Log-rank test.

Figure 9. Summary and hypothetical model.

Tumors consist of an abnormal vasculature, composed mainly of immature vessels with increased permeability. The less densely packed cells allow drugs or complexes to accumulate in tumor tissue. Treatment with HPßCD-HET0016 leads to reduced expression of αSMA and increased expression of angiopoietin-2 and Tie-2, which may lead to reduced tumor vasculature that is inadequate to support tumor growth and may lead to tumor dormancy. Our results suggest that HET0016 reduces cancer cell growth, invasion, and vasculature by reducing the expression of signaling molecules in the MAPK, PI3K/AKT, and inflammation pathways.