Tesevatinib is a potent oral brain penetrant EGFR inhibitor currently being evaluated for glioblastoma therapy. Tesevatinib distribution was assessed in wild-type (WT) and Mdr1a/b(-/-)Bcrp(-/-) triple knockout (TKO) FVB mice after dosing orally or via osmotic minipump; drug–tissue binding was assessed by rapid equilibrium dialysis. Two hours after tesevatinib dosing, brain concentrations in WT and TKO mice were 0.72 and 10.03 μg/g, respectively. Brain-to-plasma ratios (Kp) were 0.53 and 5.73, respectively. With intraperitoneal infusion, brain concentrations were 1.46 and 30.6 μg/g (Kp 1.16 and 25.10), respectively. The brain-to-plasma unbound drug concentration ratios were substantially lower (WT mice, 0.03–0.08; TKO mice, 0.40–1.75). Unbound drug concentrations in brains of WT mice were 0.78 to 1.59 ng/g. In vitro cytotoxicity and EGFR pathway signaling were evaluated using EGFR-amplified patient-derived glioblastoma xenograft models (GBM12, GBM6). In vivo pharmacodynamics and efficacy were assessed using athymic nude mice bearing either intracranial or flank tumors treated by oral gavage. Tesevatinib potently reduced cell viability [IC50 GBM12 = 11 nmol/L (5.5 ng/mL), GBM6 = 102 nmol/L] and suppressed EGFR signaling in vitro. However, tesevatinib efficacy compared with vehicle in intracranial (GBM12, median survival: 23 vs. 18 days, P = 0.003) and flank models (GBM12, median time to outcome: 41 vs. 33 days, P = 0.007; GBM6, 44 vs. 33 days, P = 0.007) was modest and associated with partial inhibition of EGFR signaling. Overall, tesevatinib efficacy in EGFR-amplified PDX GBM models is robust in vitro but relatively modest in vivo, despite a high brain-to-plasma ratio. This discrepancy may be explained by drug-tissue binding and compensatory signaling.

The therapeutic targeting of epidermal growth factor receptor (EGFR) aberrations in patients with glioblastoma (GBM) has had limited success despite the critical role of this pathway in GBM development. EGFR is a transmembrane receptor tyrosine kinase and overexpression in GBM has been associated with poor prognosis (1). Approximately 57% of GBM have EGFR alterations, the most common of which are focal amplifications of EGFR (2). Constitutive EGFR activation and dimerization leads to phosphorylation of its C-terminal tail and signal activation of several downstream pathways including RAS/RAF/MEK/ERK and PI3K/AKT, which are critical for the regulation of metabolism, proliferation and survival (3). Furthermore, EGFR knockdown induces cell death in EGFR-mutant GBM cell lines (4). Given the importance of this pathway in gliomagenesis, therapies that target EGFR have been extensively examined for GBM; however, this strategy has not yet been successful in clinical trials. First-generation EGFR inhibitors, such as erlotinib (5, 6) and gefitinib (7, 8), do not improve survival despite promising preclinical activity (9, 10). This lack of efficacy has been associated with poor blood–brain barrier (BBB) penetration secondary to multidrug resistant P-glycoprotein (MDR1; ABCB1) and breast cancer resistance protein (BCRP; ABCG2)-mediated efflux transport (9–12). Because robust inhibition of EGFR signaling may require higher drug concentrations in the central nervous system (CNS), EGFR inhibitors with high BBB penetrance may be promising candidates for GBM therapy.

Tesevatinib (KD019; XL647; EXEL-7647) is a potent, orally administered small-molecule inhibitor of EGFR, ERBB2 (HER2/NEU), and the SRC family of nonreceptor tyrosine kinases, and has been reported to have excellent BBB penetration (13, 14). Tesevatinib inhibits wild-type EGFR (IC50 = 0.3 nmol/L; ref. 15) more potently than erlotinib (IC50 = 2 nmol/L; ref. 16). Tesevatinib also inhibits HER2 (IC50 = 16.1 nmol/L), VEGFR2 (IC50 = 1.5 nmol/L), and SRC (IC50 = 10.3 nmol/L; ref. 14). Quantitative whole-body autoradiography in rats demonstrated that a brain/blood radioactivity ratio of approximately 1.0 was achieved 6 to 24 hours after administering a single dose of 14C labeled tesevatinib. BBB penetration was further assessed in mice using LC/MS-MS (liquid chromatography – tandem mass spectrometry), which showed a brain/plasma ratio (Kp) of 2.3 to 4.4 over a 24-hour period after a single oral dose of tesevatinib. Furthermore, tesevatinib therapy has been previously shown to increase median survival by 20% in a GL261 orthotopic syngeneic glioma model (14). This study aimed to extend these early preclinical data by assessing the efflux liability of tesevatinib at the BBB, and by evaluating the impact of tesevatinib in patient-derived xenograft (PDX) GBM models with EGFR amplification. These clinically relevant data would provide additional evidence to support and refine trial development involving tesevatinib therapy in patients with GBM.

Chemicals, reagents, and supplies

The tosylate salt of tesevatinib [N-(3,4-dichloro-2-fluorophenyl)-7-((((3aR,5r,6aS)-2-methyloctahydrocyclopenta[c]pyrrol-5-yl)-methyl)-oxy)-6-(methyloxy)quinazolin-4-amine] was obtained from Kadmon Corporation and used for all experiments (purity >98%). The chemical structure of tesevatinib (XL647) has been previously reported (13). The molecular weights of the tosylate salt and the free base are 663.59 and 491.14 g/mol, respectively. All dosing and concentrations reported here are with respect to tesevatinib free base. Erlotinib was obtained from the NCI (Rockville, MD). For in vitro studies, tesevatinib and erlotinib were dissolved in DMSO and stored at −20°C. For in vivo studies, tesevatinib was suspended in 0.5% METHOCEL E5 premium LV (DOW) and 0.2% Tween 20 (Bio-Rad) in phosphate buffered saline (PBS) and administered by oral gavage. Erlotinib was suspended in 0.5% METHOCEL E5 premium LV in PBS and administered by oral gavage. Analytical-grade reagents (for LC/MS-MS analysis), and a 96-well rapid equilibrium dialysis (RED) base plate and membrane inserts (8 kDa molecular weight cut-off cellulose dialysis membrane) were purchased from Thermo Fisher Scientific.

Cell culture and antibodies

EGFR-amplified PDX GBM models were selected from the Mayo Clinic Brain Tumor PDX National Resource (17): GBM12 (EGFR G719A and L62R missense mutations), GBM6 (EGFRvIII mutation), GBM108 (EGFRvIII mutation), GBM76 (EGFRvIII mutation), GBM39 (EGFRvIII mutation), GBM84 (EGFR R222C missense mutation), and GBM08 (EGFR R252C missense mutation). Conventional short-tandem repeat (STR) assessments through the Mayo Genome Facility Genotyping Core were used to ensure the provenance of these PDX models. Mycoplasma testing is regularly performed using the MycoAlert Detection Kit (Lonza; last tested August 10, 2020). Short-term explant cultures were cultured in neural stem cell media (Stem Pro NSC SFM; Invitrogen) supplemented with 1% penicillin/streptomycin (Thermo Fisher Scientific). All in vitro experiments involving PDX models were performed within 1 to 2 passages. GBM12 and GBM6 explant cultures were treated with tesevatinib 0 to 1,000 nmol/L to assess in vitro cell viability, and were analyzed using a CellTiter-Glo 3D Cell Viability Assay (Promega) as per the instruction manual. Notably, the in vitro growth rates of PDX lines differ, so different durations were used for the assay (14 days for GBM12 and 18 days for GBM6). In a separate experiment, GBM108, GBM76, GBM39, GBM84, and GBM08 explant cultures were treated with either tesevatinib or erlotinib (0–10,000 nmol/L) and analyzed using a CellTiter-Glo 3D Cell Viability Assay. Assay durations ranged from 6 to 9 days, depending on in vitro growth rates. For in vitro assessments of the effect of tesevatinib on EGFR signaling, GBM12 and GBM6 explant cultures were treated with tesevatinib 0 to 10,000 nmol/L for 24 hours at 37°C.

Primary neurosphere cultures and subcutaneous flank tumors were harvested and processed for protein extraction and Western blotting as previously described (18). Antibodies used included EGFR (RRID:AB_732106; Abcam), phospho-EGFR (Tyr1173; RRID:AB_331795), phospho-EGFR (Tyr1068; RRID:AB_331701), EGFR (RRID:AB_331707), phospho-AKT (Ser473; RRID:AB_329825), AKT (RRID:AB_329827), phospho-p44/42 ERK1/2 (Thr202/Tyr204; RRID:AB_331646), p44/42 ERK1/2 (RRID:AB_330744), goat anti-rabbit IgG (HRP-linked; RRID:AB_2099233), β-actin (RRID:AB_330288), α-tubulin (RRID:AB_2619646), phospho-SRC (Tyr416; RRID:AB_331697; Cell Signaling Technology), and c-SRC (RRID:AB_2302631; Millipore).

Brain distribution of tesevatinib after a single oral dose

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at either the University of Minnesota and/or Mayo Clinic (as appropriate), and all animal care procedures were performed according to an IACUC approved protocol.

Non–tumor-bearing FVB wild-type (WT) mice and FVB Mdr1a/b(-/-)Bcrp1(-/-) triple knockout (TKO) mice (Taconic Farms), ages 8 to 14 weeks, were used to study brain drug distribution. The sexes of the mice were balanced in each study. WT mice were treated with a single dose of oral tesevatinib (70 mg/kg) and euthanized in a carbon dioxide chamber at eight time points (1, 2, 4, 8, 16, 24, 32, and 48 hours) after drug administration to assess brain and plasma disposition of tesevatinib (n = 4/time point). In a separate study, WT and TKO mice (n = 4/group) were treated with a single oral dose of tesevatinib (70 mg/kg) and euthanized 2 hours after drug administration to assess brain penetrance either with or without efflux transporters (MDR1a/b and BCRP). Plasma and brains were harvested as previously described (19), flash frozen and stored at −80°C until LC/MS-MS analysis to determine tesevatinib concentrations.

Brain distribution of tesevatinib after intraperitoneal infusion at a constant rate

The brain distribution of tesevatinib after intraperitoneal infusion at a constant rate was also investigated in WT and TKO mice (n = 4/group). A drug-loaded osmotic mini pump (ALZET, model 1003D, Durect Corporation) was surgically implanted into the intraperitoneal cavity to administer tesevatinib (10 mg/mL in DMSO) at a constant infusion rate of 1 μL/h as previously described (20). Animals were euthanized 48 hours after intraperitoneal implantation. Plasma and brain were harvested, flash frozen, and stored at −80°C until analysis.

Tissue binding of tesevatinib by RED

The free fraction of tesevatinib in mouse plasma and brain homogenate was determined using a RED device according to the manufacturer's protocol. Brains and plasma were harvested from drug-naïve FVB WT and TKO mice. Brains were mechanically homogenized with 3 volumes (w/v) of PBS buffer (pH 7.4). Tesevatinib was added to plasma and brain homogenates to a final concentration of 5 μmol/L. Samples were loaded into the donor chambers of the RED device in triplicate, with PBS in the corresponding buffer (receiving) chambers. Samples were then sealed with an adhesive lid and agitated for six hours at 300 rpm at 37°C. Postdialysis samples were stored at −80°C until LC/MS-MS analysis. Undiluted unbound fraction in the brain was calculated as previously reported with a dilution factor of 4 (21). Tesevatinib recovery ranged between 84% and 101%.

Analysis of tesevatinib by LC/MS-MS

Total drug concentrations of tesevatinib in plasma and brain specimens were measured using LC/MS-MS performed on a TSQ Quantum Classic (Thermo Fisher Scientific) coupled with an Agilent 1200 SL series HPLC (Agilent Technologies) using positive electrospray ionization. Tesevatinib was extracted as previously described (19) and reconstituted in the mobile phase (60:40 of 20 mmol/L ammonium formate with 0.1% formic acid:acetonitrile). Five microliters of sample was injected into a liquid chromatography column (Phenomenex Synergi 4 μmol/L Polar-RP 80 A; 75 × 2 mm). Isocratic elution was performed at a flow rate of 0.35 mL/minute for a total run time of 5 minutes per sample. The retention times for tesevatinib and the internal standard (dacomitinib) were 2.30 and 1.77 minutes, respectively, and these were detected with the m/z transition of 419.15 > 138.10 and 470.10 > 385.08, respectively. These methods were linear over a range of 0 to 1,000 ng/mL (weighting factor 1/X). All specimen concentrations were within the range of the calibration curve.

In vivo efficacy and pharmacodynamic studies

In vivo tesevatinib dosing was initially based on recommendations from Kadmon Corporation, in turn based on prior mouse efficacy studies defining the maximum tolerated dose (MTD) as 70 mg/kg orally administered on days 1 to 5 every week (14). However, given that tesevatinib is dosed daily in ongoing clinical trials (22), we later opted to transition to a continuous daily dosing regimen. Preliminary tolerability studies determined that 60 mg/kg is the MTD with daily dosing. Erlotinib dosing (100 mg/kg daily) was selected based on the MTD as previously determined elsewhere (18, 23).

Xenografts were established in female athymic nude mice (Hsd:athymic Nude-Foxn1nu, ages 6–7 weeks; Envigo, Indianapolis, IN, RRID:RGD_5508395) as previously described (24). Mice were randomized into treatment groups: Continuous treatment by oral gavage with vehicle only, tesevatinib (60 mg/kg daily) or erlotinib (100 mg/kg daily). All mice were observed daily by an experienced technician blinded to the treatment received. For orthotopic efficacy studies, mice (n = 10/group) with intracranial xenografts initiated treatment 4 days after tumor implantation and were euthanized when moribund. For heterotopic efficacy studies, mice (n = 8–10/group) with flank tumors initiated treatment once the tumor volume reached approximately 100 to 200 mm3. Flank tumors were measured thrice weekly by an experienced technician blinded to the treatment received, and mice were euthanized when tumor volume exceeded 2,000 mm3. For pharmacodynamic studies, mice (n = 4–5/group) with established heterotopic tumors (>200 mm3) were treated for five days and euthanized two hours after the last dose to harvest tumor. Tumor lysates were analyzed by Western blotting to compare the effect of tesevatinib and erlotinib on EGFR signaling.

Statistical and pharmacokinetic analyses

All in vitro data presented are the mean ± SEM from three or more experiments. Statistical differences were evaluated using either unpaired two-sample t test or Mann–Whitney test, with statistical significance at P < 0.05. IC50 values were calculated by fitting a sigmoidal curve to a log-transformed drug concentration-response curve. Median survival (orthotopic models) and median time to predefined tumor volume endpoint (TTVE; i.e., 1,500 mm3 on two consecutive days or >2,000 mm3 at any time; flank models) were estimated by the Kaplan–Meier method and compared by log rank test. Statistical tests were performed using GraphPad Prism (Version 6; GraphPad Software; RRID:SCR_002798). The sample sizes were estimated to be sufficient to yield approximately 80% power to detect 50% difference between groups based our previous experience.

Pharmacokinetic parameters were calculated from the plasma and brain concentration–time profiles after a single oral dose of tesevatinib. Noncompartmental analysis was performed using Phoenix WinNonlin 6.2 (Pharsight). The AUC from time zero to infinity (AUC0-∞) were calculated for the plasma and brain concentration–time profiles using the log-linear trapezoidal approximation that includes the area extrapolated between the last measured concentration (Clast) to time infinity (∞) using the terminal rate constant capturing the last three or four data points in the concentration-time profiles.

Brain and plasma distribution of tesevatinib in WT mice after a single oral dose

The brain and plasma concentration-time and brain-to-plasma ratio (Kp) profiles after a single 70 mg/kg dose of tesevatinib were evaluated in FVB WT mice. The maximum concentrations (Cmax) in the brain and plasma were 7.57 μg/g and 3.78 μg/mL, respectively, both occurring 16 hours postdose (Kp at Tmax = 2.00; Fig. 1A and B). The AUC0-∞ was 158.0 hour/μg/mL and 83.0 hour/μg/mL in brain and plasma, respectively, with a Kp of 1.90 based on these AUC values (Fig. 1B). These data suggest high brain-penetrance, consistent with previously reported pharmacokinetic evaluations (14, 25). The terminal half-life of tesevatinib in the brain and plasma was 13.3 and 14.0 hours, respectively. The concentration of tesevatinib in the brain exceeded that in the plasma at 4 hours postdose, and remained as such throughout the 48-hour period of observation.

Figure 1.

Pharmacokinetic profile of tesevatinib in non–tumor-bearing wild-type FVB mice after administration of a single oral dose. Plasma and brain samples were obtained over a 48-hour period after administration of a single 70 mg/kg dose of oral tesevatinib. Total brain and plasma concentrations for tesevatinib (A) and brain-to-plasma ratios (B) are shown for 1, 2, 4, 8, 16, 24, 32, and 48 hours postdose.

Figure 1.

Pharmacokinetic profile of tesevatinib in non–tumor-bearing wild-type FVB mice after administration of a single oral dose. Plasma and brain samples were obtained over a 48-hour period after administration of a single 70 mg/kg dose of oral tesevatinib. Total brain and plasma concentrations for tesevatinib (A) and brain-to-plasma ratios (B) are shown for 1, 2, 4, 8, 16, 24, 32, and 48 hours postdose.

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Brain and plasma distribution of tesevatinib in WT and TKO mice after a single oral dose

We have previously demonstrated that the brain penetration of both erlotinib and gefitinib are restricted because of MDR1 and BCRP-mediated efflux transport from brain capillary endothelial cells (11, 12). We assessed the impact of efflux liability on tesevatinib by evaluating the brain/plasma pharmacokinetics in WT and TKO mice. Two hours after tesevatinib dosing, the total plasma concentrations of tesevatinib in TKO and WT mice were not statistically different (2.03 vs. 1.33 μg/mL, P = 0.35; Fig. 2A). However, the total brain concentration of tesevatinib in TKO mice was significantly higher than that in WT mice (10.03 vs. 0.72 μg/mL, P = 0.03). The total tesevatinib Kp in TKO mice was also significantly higher than that of WT mice (5.73 vs. 0.53, P = 0.006). These results suggest that tesevatinib has substantial efflux liability for MDR1a/b and BCRP1 at the BBB.

Figure 2.

Effect of efflux transporters on brain and plasma distribution of tesevatinib. A, Non–tumor-bearing FVB mice [wild-type (WT) and Mdr1a/b(-/-)/Bcrp1(-/-) triple knockouts (TKO)] were dosed once with tesevatinib 70 mg/kg orally (n = 4 per group). Plasma and brain were harvested after 2 hours to assess plasma and brain disposition. The total concentrations and derived unbound concentrations are plotted. B, Non–tumor-bearing FVB mice (WT and TKO) were dosed with tesevatinib using osmotic minipumps implanted intraperitoneally (n = 4/group). Plasma and brain were harvested 48 hours after implantation to assess plasma and brain disposition. The total concentrations and derived unbound concentrations (D) are plotted. Note that the P values for each total and unbound concentration set are the same because the unbound concentrations are derived directly from the total concentrations.

Figure 2.

Effect of efflux transporters on brain and plasma distribution of tesevatinib. A, Non–tumor-bearing FVB mice [wild-type (WT) and Mdr1a/b(-/-)/Bcrp1(-/-) triple knockouts (TKO)] were dosed once with tesevatinib 70 mg/kg orally (n = 4 per group). Plasma and brain were harvested after 2 hours to assess plasma and brain disposition. The total concentrations and derived unbound concentrations are plotted. B, Non–tumor-bearing FVB mice (WT and TKO) were dosed with tesevatinib using osmotic minipumps implanted intraperitoneally (n = 4/group). Plasma and brain were harvested 48 hours after implantation to assess plasma and brain disposition. The total concentrations and derived unbound concentrations (D) are plotted. Note that the P values for each total and unbound concentration set are the same because the unbound concentrations are derived directly from the total concentrations.

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Brain and plasma distribution of tesevatinib in WT and TKO mice after intraperitoneal infusion at a constant rate

We also assessed the brain and plasma distribution of tesevatinib in both WT and TKO mice after intraperitoneal infusion using tesevatinib-loaded osmotic mini pumps. The plasma concentrations of tesevatinib in TKO and WT mice were similar (1.22 vs. 1.73 μg/g, P = 0.48; Fig. 2B). However, the brain concentrations of tesevatinib in TKO and WT mice differed significantly (30.6 vs. 1.46 μg/g, P = 0.003; Fig. 2B). Consistently, the total tesevatinib Kp was also significantly higher in TKO than WT mice (25.10 vs. 1.16, P < 0.001), suggesting enhanced brain penetration in the absence of MDR1a/b and BCRP1.

Nonspecific tissue binding of tesevatinib to plasma and brain homogenate

The significant efflux liability exhibited by tesevatinib despite high concentrations in the brain may be explained by non-specific tissue binding, so this variable was evaluated using rapid equilibrium dialysis. The fraction of unbound tesevatinib in the brain and plasma was 0.0011 and 0.0156, respectively. These unbound fractions were multiplied by relevant total drug concentrations to derive unbound tesevatinib concentrations from prior experiments.

The unbound tesevatinib brain-to-plasma ratio (Kp,uu) based on AUC was 0.13 for the 48 hour pharmacokinetic study. The unbound tesevatinib concentrations in the brain and plasma for the single time-point pharmacokinetic study were 0.78 ng/g and 20.66 ng/mL respectively in WT mice, and 10.86 ng/g and 31.50 ng/mL, respectively, in TKO mice (Fig. 2A). A similar relationship was observed for intraperitoneal infusion related pharmacokinetic data for unbound tesevatinib (WT mice: brain, 1.59 ng/g, plasma, 26.86 ng/mL; TKO mice: brain, 33.11 ng/mL, plasma, 18.96 mL; Fig. 2B). The Kp,uu values calculated from these experiments were substantially less (WT mice, 0.03–0.08; TKO mice 0.40–1.75), respectively, than the values predicted by total concentration ratios (Fig. 2A and B). These results suggest a high degree of non-specific tissue binding which may limit the pharmacologically active drug concentration.

As previously reported, the total concentration of erlotinib in the normal brain (two hours post-single dose of erlotinib 100 mg/kg) and the corresponding Kp are 1,338 ng/g and 0.14, respectively (26); and the unbound fractions of erlotinib in the brain and plasma are 0.096 and 0.045, respectively (27). Therefore, the unbound concentration of erlotinib in the brain and the Kp,uu are 128 ng/g and 0.30, respectively. Given the known inefficacy of erlotinib in orthotopic glioma models, the substantially lower unbound tesevatinib bioavailability in the brain compared to that of erlotinib raises the question of whether in vivo efficacy of tesevatinib may also be limited in glioma models.

In vitro efficacy and pharmacodynamic impact of tesevatinib in PDX GBM models

The efficacy of tesevatinib was then investigated in vitro in neurospheres using two EGFR amplified PDX lines: GBM12 (EGFR missense mutation) and GBM6 (EGFRvIII mutation; Fig. 3A). GBM12 was significantly more sensitive to tesevatinib (IC50 11 nmol/L, i.e., 5.5 ng/mL) than GBM6 (102 nmol/L, i.e., 50.1 ng/mL; P = 0.002; Fig. 3B) in an in vitro CellTiter-Glo 3D Cell Viability Assay. GBM12 cell viability was reduced by 99% at 100 nmol/L (49 ng/mL) of tesevatinib, compared with a reduction of only 41% in GBM6 at that concentration (Fig. 3B). Additional in vitro efficacy studies were performed in a separate panel of EGFR amplified PDX GBM models to explore the impact of EGFRvIII mutation on tesevatinib and erlotinib efficacy in EGFR amplified models (Fig. 3C). Both tesevatinib and erlotinib potency was diminished in two of the three EGFRvIII-mutant lines evaluated (GBM108 and GBM76) when compared to the lines with EGFR missense mutations in the extracellular domain (GBM8 and GBM84). This is consistent with prior reports using isogenic cell lines, which demonstrate diminished tesevatinib potency in EGFRvIII (compared with EGFR wild-type and EGFR missense mutation) expressing models (25).

Figure 3.

In vitro efficacy and pharmacodynamic impact of tesevatinib in PDX GBM models. A, Western blot analysis of untreated cell lysates of EGFR-amplified PDX GBM lines shown to demonstrate lines with EGFRvIII mutation. B, PDX GBM12 and GBM6 cells were cultured in stem cell media and treated with tesevatinib 0 to 1,000 nmol/L for 14 days and 18 days, respectively. A CellTiter-Glo 3D Cell Viability Assay was then used to measure cell viability. C, PDX GBM108 (EGFRvIII), GBM76 (EGFRvIII), GBM39 (EGFRvIII), GBM84 (EGFR R222C), and GBM08 (EGFR R252C) cells were cultured in stem cell media and treated with either tesevatinib or erlotinib (0–10,000 nmol/L) for 6 to 9 days. A CellTiter-Glo 3D Cell Viability Assay was then used to measure cell viability. D, Cell lysates of PDX GBM12 and GBM6 cells were cultured in stem cell media and treated with tesevatinib 0 to 10,000 nmol/L for 24 hours to evaluate phosphorylated and total EGFR, SRC, AKT, and ERK. β-Actin was used as a loading control. Western blot images are shown to demonstrate the effect of graded concentrations of tesevatinib on EGFR signaling.

Figure 3.

In vitro efficacy and pharmacodynamic impact of tesevatinib in PDX GBM models. A, Western blot analysis of untreated cell lysates of EGFR-amplified PDX GBM lines shown to demonstrate lines with EGFRvIII mutation. B, PDX GBM12 and GBM6 cells were cultured in stem cell media and treated with tesevatinib 0 to 1,000 nmol/L for 14 days and 18 days, respectively. A CellTiter-Glo 3D Cell Viability Assay was then used to measure cell viability. C, PDX GBM108 (EGFRvIII), GBM76 (EGFRvIII), GBM39 (EGFRvIII), GBM84 (EGFR R222C), and GBM08 (EGFR R252C) cells were cultured in stem cell media and treated with either tesevatinib or erlotinib (0–10,000 nmol/L) for 6 to 9 days. A CellTiter-Glo 3D Cell Viability Assay was then used to measure cell viability. D, Cell lysates of PDX GBM12 and GBM6 cells were cultured in stem cell media and treated with tesevatinib 0 to 10,000 nmol/L for 24 hours to evaluate phosphorylated and total EGFR, SRC, AKT, and ERK. β-Actin was used as a loading control. Western blot images are shown to demonstrate the effect of graded concentrations of tesevatinib on EGFR signaling.

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Tesevatinib is a potent inhibitor of both EGFR and SRC (14, 15). Both AKT and ERK1/2 are downstream signaling components of the EGFR pathway, and we have previously demonstrated that erlotinib suppresses these signaling pathways in several EGFR amplified GBM models including GBM6 and GBM12. (18). Therefore, we opted to examine the pharmacodynamic impact of tesevatinib on EGFR pathway signaling in both of these PDX models. Tesevatinib potently inhibited phosphorylation of EGFR at both the Tyr1173 and Tyr1068 sites at a concentration of 10 nmol/L in GBM12 (Fig. 3D). Tesevatinib also potently inhibited phosphorylation of ERK1/2-Thr202/Tyr204 and partially inhibited phosphorylation of AKT-Ser473 and SRC-Tyr416 in GBM12 at this concentration (Fig. 3B). Robust inhibition of SRC-Tyr416 phosphorylation in GBM12 was observed at higher concentrations (1,000 nmol/L). The pharmacodynamic impact of tesevatinib was less pronounced in GBM6, with inhibition of EGFR and SRC-Tyr416 phosphorylation observed at concentrations over 1,000 nmol/L and inhibition of ERK1/2-Thr202/Tyr204 phosphorylation at 10,000 nmo/L (Fig. 3D).

These cytotoxicity and pharmacodynamics experiments suggest that although the total concentrations of tesevatinib achieved in the brain (1.46 μg/g; 2.97 μmol/L) may be sufficient for cytotoxic efficacy, in vivo efficacy may be hampered by tissue binding if unbound tesevatinib is critical for effect (1.59 ng/g; 3.24 nmol/L). Tesevatinib is more efficacious in GBM12 than GBM6 at nanomolar concentrations with respect to both in vitro cytotoxicity and inhibition of EGFR signaling. Therefore, we opted to study the orthotopic efficacy of tesevatinib in GBM12 PDX models.

In vivo efficacy of tesevatinib in an orthotopic and flank PDX GBM models

The efficacy of tesevatinib was first evaluated in mice with established orthotopic GBM12 tumors. Tesevatinib treatment (60 mg/kg orally daily) was associated with a minor but statistically significant increase in median survival compared with vehicle (23 vs. 18 days, P = 0.003; Fig. 4A). However, the median survival with tesevatinib therapy was significantly less than that with erlotinib (37 days, P < 0.001; Fig. 4A).

Figure 4.

In vivo efficacy of tesevatinib in PDX GBM models. A, Athymic nude mice bearing intracranial PDX GBM12 tumors were treated with either vehicle, tesevatinib 60 mg/kg orally daily or erlotinib 100 mg/kg orally daily, until progression (n = 10/group). Kaplan–Meier plots show days to endpoint between treatment groups (i.e., the time between treatment onset and moribund state). The arrow indicates treatment initiation. B and C, Athymic nude mice bearing flank PDX GBM12 (B) and GBM6 (C) tumors were treated with either vehicle, tesevatinib 60 mg/kg orally daily, or erlotinib 100 mg/kg orally daily, until progression (n = 8–10/group). Kaplan–Meier plots show days to endpoint between treatment groups (i.e., the time between treatment onset and tumor volume >1,500 mm3 on two consecutive days or >2,000 mm3 at any time). The arrow indicates treatment initiation.

Figure 4.

In vivo efficacy of tesevatinib in PDX GBM models. A, Athymic nude mice bearing intracranial PDX GBM12 tumors were treated with either vehicle, tesevatinib 60 mg/kg orally daily or erlotinib 100 mg/kg orally daily, until progression (n = 10/group). Kaplan–Meier plots show days to endpoint between treatment groups (i.e., the time between treatment onset and moribund state). The arrow indicates treatment initiation. B and C, Athymic nude mice bearing flank PDX GBM12 (B) and GBM6 (C) tumors were treated with either vehicle, tesevatinib 60 mg/kg orally daily, or erlotinib 100 mg/kg orally daily, until progression (n = 8–10/group). Kaplan–Meier plots show days to endpoint between treatment groups (i.e., the time between treatment onset and tumor volume >1,500 mm3 on two consecutive days or >2,000 mm3 at any time). The arrow indicates treatment initiation.

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The efficacy of tesevatinib was subsequently evaluated in flank GBM12 tumor models to reduce the potential confounding influence of variable drug delivery across the BBB. Although tesevatinib (60 mg/kg orally daily) significantly increased the median time to predefined tumor volume endpoint (TTVE) compared with vehicle, the degree of benefit remained modest (41 vs. 33 days, P = 0.007, Fig. 4B). In contrast to the orthotopic studies, there was no difference between tesevatinib and erlotinib efficacy (median TTVE 39 days) in this GBM12 flank model (P = 0.51; Fig. 4B). As part of this experiment, the efficacy of an alternative tesevatinib regimen (70 mg/kg orally on days 1–5 every week) was also evaluated however efficacy was no different from the daily dose tesevatinib regimen (Supplementary Fig. S1).

A similar flank efficacy study was also performed in the less sensitive GBM6 model. Interestingly, similar improvements in median TTVE were demonstrated for each of the treatments compared to vehicle (vehicle: 33 days; tesevatinib 60 mg/kg daily: 44 days, P < 0.001; erlotinib 100 mg/kg daily: 47 days, P < 0.001; Fig. 4C). However, there was no significant difference in efficacy between tesevatinib and erlotinib (P = 0.28).

In summary, tesevatinib efficacy was modest and inferior to that of erlotinib in an orthotopic PDX GBM12 model, despite significantly higher brain penetrance based on total drug levels. However, both drugs displayed similar degrees of modest efficacy in flank models.

Pharmacodynamic analysis of tesevatinib in a flank PDX GBM model using Western blotting

The pharmacodynamic impact of tesevatinib on EGFR signaling was compared with that of erlotinib in xenografts of GBM12 and GBM6 using Western blots generated from tumor lysates. Both tesevatinib and erlotinib therapy markedly inhibited phosphorylation of EGFR-Tyr1173 and SRC-Tyr416 in GBM12 flank tumors (Fig. 5A). However, EGFR phosphorylation at Tyr1068 was more robustly suppressed by erlotinib than tesevatinib. Furthermore, erlotinib suppressed the phosphorylation of ERK1/2-Thr202/Tyr204 robustly and that of AKT-Ser473 partially while tesevatinib had no significant effect at either of these phosphorylation sites. Interestingly, the degree of inhibition of phosphorylation observed is similar to the in vitro pharmacodynamic effect observed with 1 to 10 nmol/L of tesevatinib in GBM12.

Figure 5.

Effect of tesevatinib on EGFR signaling in flank GBM xenografts. Athymic nude mice bearing flank xenografts of either GBM6 (A) or GBM12 (B) were treated orally with either vehicle, erlotinib (100 mg/kg), or tesevatinib (60 mg/kg) daily for five consecutive days (n = 4–5/group). Mice were euthanized two hours after dosing on day 5 to harvest flank tumors. Tumor lysates were analyzed to evaluate phosphorylated and total EGFR, SRC, AKT, and ERK. Western blot images are shown to compare the effect of tesevatinib and erlotinib on EGFR signaling. α-Tubulin was used as a loading control.

Figure 5.

Effect of tesevatinib on EGFR signaling in flank GBM xenografts. Athymic nude mice bearing flank xenografts of either GBM6 (A) or GBM12 (B) were treated orally with either vehicle, erlotinib (100 mg/kg), or tesevatinib (60 mg/kg) daily for five consecutive days (n = 4–5/group). Mice were euthanized two hours after dosing on day 5 to harvest flank tumors. Tumor lysates were analyzed to evaluate phosphorylated and total EGFR, SRC, AKT, and ERK. Western blot images are shown to compare the effect of tesevatinib and erlotinib on EGFR signaling. α-Tubulin was used as a loading control.

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EGFR signaling inhibition was even less pronounced in tesevatinib-treated GBM6 flank models. EGFR-Tyr1173 and SRC-Tyr416 phosphorylation were only partially inhibited in GBM6, rather than the more pronounced inhibition observed in GBM12 (Fig. 5B). Erlotinib treated flank GBM6 models demonstrated more pronounced inhibition of EGFR-Tyr1173 phosphorylation and relatively less inhibition of SRC-Tyr416 phosphorylation when compared to tesevatinib treated flank GBM6 models. Otherwise and similar to GBM12, tesevatinib induced partial inhibition of EGFR-Tyr1068 and there was no significant inhibition of AKT-Ser473 and ERK1/2-Thr202/Tyr204; while erlotinib more robustly inhibited EGFR-Tyr1068, AKT-Ser473 and ERK1/2-Thr202/Tyr204.

These results suggest that tesevatinib inhibits phosphorylation of EGFR-Tyr1173 more robustly than EGFR-Tyr1068, while erlotinib inhibits phosphorylation at both sites. Furthermore, EGFR-Tyr1068 phosphorylation seems to be associated with phosphorylation of both AKT-Ser473 and ERK1/2-Thr202/Tyr204 in these models.

The development of BBB penetrant EGFR inhibitors has great potential for GBM therapy but has yet to translate into clinical benefit. First- and second-generation EGFR inhibitors such as erlotinib, gefitinib, and afatinib are ineffective in patients with primary brain tumors, possibly because of limited brain delivery of these drugs across an intact BBB (27). Newer generation EGFR inhibitors such as osimertinib and AZD3759 are CNS-penetrant but were developed to target the EGFR mutations in the tyrosine kinase domain that are more commonly observed in non–small cell lung cancer (NSCLC; refs. 28, 29). Because EGFR aberrations in GBM are typically in the ectodomain (e.g., EGFRvIII mutation) or involve EGFR wild-type amplification, these drugs are unlikely to be effective in most patients with GBM (27). Tesevatinib is a small molecule that was previously reported to potently inhibit EGFR, while demonstrating excellent BBB penetration and efficacy in an orthotopic syngeneic EGFR-amplified GBM model (14). However, although we confirmed high concentrations of tesevatinib in the brain despite an intact BBB, the efficacy of tesevatinib was modest in an orthotopic PDX EGFR-amplified GBM model, and relatively inferior to that of erlotinib.

The current clinical trajectory of tesevatinib as an efficacious CNS penetrant EGFR inhibitor sharply contrasts with these preclinical data. A phase II trial of advanced stage, treatment naïve EGFR mutant NSCLC demonstrated that tesevatinib yields a response rate of 57% and a progression-free survival of 9.3 months, similar to the outcomes achieved with erlotinib and gefitinib while maintaining a comparable adverse effect profile to these drugs (15, 22, 30). Given clinical efficacy in EGFR mutant NSCLC and the high CNS concentrations of tesevatinib, another phase II trial is currently evaluating the activity of tesevatinib in EGFR mutant NSCLC with CNS metastases (NCT02616393; ref. 31). Preliminary data from this trial have revealed radiographic responses in CNS metastases and symptomatic improvement in patients with leptomeningeal disease (32). These data, along with preliminary preclinical evidence of efficacy in glioma models, have led to the ongoing clinical development and evaluation of tesevatinib in recurrent GBM (NCT02844439).

However, efficacy in brain metastases cannot be extrapolated to malignant gliomas because brain metastases have higher degrees of BBB permeability (33). Erlotinib and gefitinib maintain modest clinical activity against brain metastases from NSCLC despite no effect in GBM (5–8, 34). One potential reason for this may be that BBB disruption in malignant gliomas is more heterogeneous than that of brain metastases, and the partially intact BBB throughout the tumor may limit drug delivery and efficacy (35). Furthermore, GBM and associated capillary endothelial cells have significantly higher MDR1 expression than brain metastases, resulting in a more critical role for drug–efflux transporter liability (31, 36). The efflux liability of EGFR inhibitors such as erlotinib and gefitinib for MDR1 and BCRP has been previously described as the main mechanism behind subtherapeutic drug levels in the CNS and consequent poor intracranial efficacy of these drugs in GBM (11, 12, 37). The current generation of brain penetrant EGFR inhibitors seem to be successful in controlling CNS disease either by inhibiting MDR1/BCRP function (osimertinib; ref. 38) or by having no efflux liability to these transporters (AZD3759; ref. 27). Given these findings, the dramatic efflux liability of tesevatinib despite the high concentrations in the brain was surprising and raised concerns for drug-tissue binding.

The need to specifically consider unbound drug concentrations in the brain to assess the potential for glioma therapeutics efficacy is gaining traction (39). Several kinase inhibitors have been designed to achieve high concentrations of free drug in the brain relative to plasma. The ratio of unbound brain drug concentration to unbound plasma drug concentration ratios (i.e., Kp,uu) for AZD3759, GDC-0084, and osimertinib are 1.3, 0.4, and 0.3, respectively (27, 39, 40). This contrasts with the significantly lower Kp,uu for tesevatinib (0.08), given that only 0.11% of tesevatinib is unbound in brain tissue. Extrapolating from the in vivo pharmacokinetic studies, this reveals that the bioavailable concentration of tesevatinib in the brain was only 1.56 ng/g (3.18 nmol/L) while the IC50 for in vitro cell viability in GBM12 was 11 nmol/L. This subtherapeutic concentration of unbound tesevatinib probably contributed toward preventing efficacy in the orthotopic GBM12 model. Furthermore, the significantly higher unbound concentration of erlotinib in brain tissue (128 ng/g) and the corresponding Kp,uu (0.30) may partially explain the superior efficacy of erlotinib in this model compared with tesevatinib despite the apparent difference in BBB penetrance based on total drug concentrations. Notably, although the dose of erlotinib used for these in vivo experiments (100 mg/kg) was based on the MTD as detailed earlier, this dose is actually much higher than the clinically relevant dose (41). Nevertheless, the overall necessity for low tissue binding and a high Kp,uu for novel therapeutics remains uncertain. Osimertinib is more efficacious against CNS metastases compared to erlotinib, despite a similar Kp,uu. Furthermore, patients with GBM treated with the CDK4/6 inhibitor abemaciclib have demonstrated durable stable disease in trials (42) despite the high tissue binding observed with this molecule (>99.6% in brain, Kp,uu = 0.03; ref. 39). Therefore, a high Kp,uu cannot be used as a solitary predictor to optimize glioma-directed therapeutics.

EGFR-activated signal transduction pathways are extremely complex and compensatory signaling in GBM may also prevent brain-penetrant EGFR inhibitor efficacy. Our experiments using subcutaneous models demonstrated modest tesevatinib efficacy in two PDX GBMs despite inhibition of EGFR-Tyr1173 and SRC-Tyr416 phosphorylation in a corresponding pharmacodynamics study. Notably, EGFR-Tyr1068 phosphorylation was less robustly inhibited than EGFR-Tyr1173 and downstream targets (ERK1/2, AKT) remained unaffected. EGFR-Tyr1068 phosphorylation activates both the PI3K/AKT and RAS/RAF/MEK/ERK pathways by directly binding to GRB2 (growth factor receptor binding protein 2; ref. 43). One consideration is that the partial inhibition of EGFR-Tyr1068 with tesevatinib may have been insufficient to suppress these pathways. However, erlotinib was associated with more robust inhibition of EGFR-Tyr1068, AKT-Ser473, and ERK1/2-Thr202/Tyr204. Despite more robust suppression of these signaling pathways, erlotinib was not associated with increased efficacy when compared with tesevatinib in either of these subcutaneous PDX GBM models. Alternatively, previous studies involving EGFR-amplified GBM tumors collected from patients being treated with gefitinib have demonstrated significant inhibition of EGFR phosphorylation without any effect on phospho-ERK (44). This suggests that compensatory signaling may also be playing a role in preventing efficacy in EGFR inhibitors alongside inadequate BBB penetration. For example, EGFR inhibition of EGFR amplified GBM triggers a rapid adaptive response driven by TNF which leads to activation of a TNF–JNK–AXL–ERK signaling axis, thus leading to this phenomenon of ERK activation despite robust EGFR activation (45). Similarly, activation of the Met–HGF signaling axis in tumors treated with EGFR inhibitors may preserve PI3K/MTOR signaling (46, 47). Finally, tumor heterogeneity with respect to EGFR expression may have also been a barrier preventing efficacy (48). Thus, there is a clear need to account for multiple variables when developing EGFR inhibitors for glioma therapy.

Ultimately, additional data is necessary through further clinical development of “brain penetrant” small molecules to determine the true relative value of these variables for GBM therapeutics. Specific to brain-penetrant EGFR inhibitors, the phase II trial evaluating tesevatinib in recurrent GBM (NCT02844439) is in progress. In addition, a first-in-human trial involving WSD0922 is evaluating the safety, tolerability and antitumor activity of this highly brain penetrant EGFR inhibitor (Kp,uu∼1.0) in patients with either EGFR-activated high grade gliomas or NSCLC with CNS metastases (NCT04197934; ref. 49). Finally, JCN068 is another highly BBB penetrant EGFR inhibitor with promising preclinical activity, which is currently progressing toward clinical development for GBM (50). Comparisons of the clinical efficacy of these drugs with respect to these pharmacokinetic properties will shed light on relevant variables that need special attention for the successful design of glioma-directed therapeutics.

S.H. Kizilbash reports grants from NIH during the conduct of the study; nonfinancial support from Kadmon Corporation, grants and nonfinancial support from Orbus Therapeutics, Inc. and Apollomics, Inc., grants from Celgene, grants and nonfinancial support from Wayshine Biopharma, nonfinancial support from Calithera Biosciences, and grants and nonfinancial support from Loxo Oncology outside the submitted work. G. Gampa reports grants from NCI during the conduct of the study. J.N. Sarkaria reports grants from Basilea, Glaxo Smith Kline, Bristol-Myers Squibb, Curtana, Forma, AbbVie, Boehringer Ingelheim, Bayer, Celgene, Cible, Wayshine, Boston Scientific, AstraZeneca, Black Diamond, and Karyopharm outside the submitted work. No disclosures were reported by the other authors.

S.H. Kizilbash: Conceptualization, data curation, formal analysis, investigation, methodology, writing–original draft, writing–review and editing. S.K. Gupta: Investigation, methodology, writing–review and editing. K.E. Parrish: Writing–review and editing. J.K. Laramy: Investigation, methodology, writing–review and editing. M. Kim: Writing-review and editing. G. Gampa: Writing–review and editing. B.L. Carlson: Investigation, methodology, writing–review and editing. K.K. Bakken: Investigation, methodology, writing–review and editing. A.C. Mladek: Investigation, methodology, writing–review and editing. M.A. Schroeder: Investigation, methodology, writing–review and editing. P.A. Decker: Formal analysis, writing–review and editing. W.F. Elmquist: Resources, supervision, writing–review and editing. J.N. Sarkaria: Resources, supervision, writing–review and editing.

This study was supported by funding from the NCI [U54 CA 210180 (J.N. Sarkaria, W.F. Elmquist, S.H. Kizilbash); R01 CA138437 (W.F. Elmquist); K12 CA90628 (S.H. Kizilbash)]. We appreciate the help of Clinical Pharmacology Analytical Services (CPAS), College of Pharmacy, University of Minnesota, for assistance with LC/MS-MS.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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