Oncogenic Characterization and Pharmacologic Sensitivity of Activating Fibroblast Growth Factor Receptor (FGFR) Genetic Alterations to the Selective FGFR Inhibitor Erdafitinib.

Fibroblast growth factor receptor (FGFR) genetic alterations are frequently observed in cancer, suggesting that FGFR inhibition may be a promising therapy in patients harboring these lesions. Identification of predictive and pharmacodynamic biomarkers to select and monitor patients most likely to respond to FGFR inhibition will be the key to clinical development of this class of agents. Sensitivity to FGFR inhibition and correlation with FGFR pathway activation status were determined in molecularly annotated panels of cancer cell lines and xenograft models. Pathway inhibition in response to FGFR inhibitor treatment was assessed in cell lines (both in vitro and in vivo) and in samples from patients treated with the FGFR inhibitor JNJ-42756493 (erdafitinib). Frequency of FGFR aberrations was assessed in a panel of NSCLC, breast, prostate, ovarian, colorectal, and melanoma human tumor tissue samples. FGFR translocations and gene amplifications present in clinical specimens were shown to display potent transforming activity associated with constitutive pathway activation. Tumor cells expressing these FGFR activating mutants displayed sensitivity to the selective FGFR inhibitor erdafitinib and resulted in suppression of FGFR phosphorylation and downstream signal transduction. Clinically, patients receiving erdafitinib showed decreased Erk phosphorylation in tumor biopsies and elevation of serum phosphate. In a phase I study, a heavily pretreated bladder cancer patient with an FGFR3-TACC3 translocation experienced a partial response when treated with erdafitinib. This preclinical study confirmed pharmacodynamics and identified new predictive biomarkers to FGFR inhibition with erdafitinib and supports further clinical evaluation of this compound in patients with FGFR genetic alterations. Mol Cancer Ther; 16(8); 1717-26. ©2017 AACR.

Phase I Dose-Escalation Study of JNJ-42756493, an Oral Pan-Fibroblast Growth Factor Receptor Inhibitor, in Patients With Advanced Solid Tumors.

PURPOSE: JNJ-42756493 is an orally administered pan-fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor. This first-in-human study evaluates the safety, pharmacokinetics, and pharmacodynamics and defines the recommended phase II dose (RP2D) of JNJ-42756493. PATIENTS AND METHODS: Eligible patients with advanced solid tumors received escalating doses of JNJ-42756493 from 0.5 to 12 mg administered continuously daily or JNJ-42756493 10 or 12 mg administered intermittently (7 days on/7 days off). RESULTS: Sixty-five patients were enrolled. The most common treatment-emergent adverse events included hyperphosphatemia (65%), asthenia (55%), dry mouth (45%), nail toxicity (35%), constipation (34%), decreased appetite (32%), and dysgeusia (31%). Twenty-seven patients (42%) experienced grade ≥ 3 treatment-emergent adverse events, and one dose-limiting toxicity of grade 3 ALT elevation was observed at 12 mg daily. Maximum-tolerated dose was not defined. Nine milligrams daily was considered as the initial RP2D; however, tolerability was improved with intermittent schedules, and 10 mg administered on a 7-days-on/7-days-off schedule was considered the final RP2D. Pharmacokinetics were linear, dose proportional, and predictable, with a half-life of 50 to 60 hours. Dose-dependent elevations in serum phosphate, a manifestation of pharmacodynamic effect, occurred in all patients starting at 4 mg daily. Among 23 response-evaluable patients with tumor FGFR pathway alterations, four confirmed responses and one unconfirmed partial response were observed in patients with glioblastoma and urothelial and endometrial cancer (all with FGFR2 or FGFR3 translocations); 16 patients had stable disease. CONCLUSION: JNJ-42756493 administered at 10 mg on a 7-days-on/7-days-off schedule achieved exposures at which clinical responses were observed, demonstrated pharmacodynamic biomarker activity, and had a manageable safety profile.

Detection, Characterization, and Inhibition of FGFR-TACC Fusions in IDH Wild-type Glioma.

PURPOSE: Oncogenic fusions consisting of fibroblast growth factor receptor (FGFR) and TACC are present in a subgroup of glioblastoma (GBM) and other human cancers and have been proposed as new therapeutic targets. We analyzed frequency and molecular features of FGFR-TACC fusions and explored the therapeutic efficacy of inhibiting FGFR kinase in GBM and grade II and III glioma. EXPERIMENTAL DESIGN: Overall, 795 gliomas (584 GBM, 85 grades II and III with wild-type and 126 with IDH1/2 mutation) were screened for FGFR-TACC breakpoints and associated molecular profile. We also analyzed expression of the FGFR3 and TACC3 components of the fusions. The effects of the specific FGFR inhibitor JNJ-42756493 for FGFR3-TACC3-positive glioma were determined in preclinical experiments. Two patients with advanced FGFR3-TACC3-positive GBM received JNJ-42756493 and were assessed for therapeutic response. RESULTS: Three of 85 IDH1/2 wild-type (3.5%) but none of 126 IDH1/2-mutant grade II and III gliomas harbored FGFR3-TACC3 fusions. FGFR-TACC rearrangements were present in 17 of 584 GBM (2.9%). FGFR3-TACC3 fusions were associated with strong and homogeneous FGFR3 immunostaining. They are mutually exclusive with IDH1/2 mutations and EGFR amplification, whereas they co-occur with CDK4 amplification. JNJ-42756493 inhibited growth of glioma cells harboring FGFR3-TACC3 in vitro and in vivo. The two patients with FGFR3-TACC3 rearrangements who received JNJ-42756493 manifested clinical improvement with stable disease and minor response, respectively. CONCLUSIONS: RT-PCR sequencing is a sensitive and specific method to identify FGFR-TACC-positive patients. FGFR3-TACC3 fusions are associated with uniform intratumor expression of the fusion protein. The clinical response observed in the FGFR3-TACC3-positive patients treated with an FGFR inhibitor supports clinical studies of FGFR inhibition in FGFR-TACC-positive patients.

Phase 1 pharmacokinetic and drug-interaction study of dasatinib in patients with advanced solid tumors.

BACKGROUND: The recently developed the Src and Abelson (Abl) kinase inhibitor dasatinib has antitumor effects in epithelial and mesenchymal tumors. Preclinical data have indicated that dasatinib is metabolized primarily through cytochrome P450 3A4 (CYP3A4) and may cause QT prolongation. In light of its improved tolerability, the authors were interested in the safety of a once-daily dasatinib regimen. METHODS: The authors conducted a phase 1 trial of dasatinib in 29 patients with advanced solid tumors. Segment 1 of the trial was short term and sequential and was designed to determine whether the coadministration of the potent CYP3A4 inhibitor ketoconazole had an effect on the pharmacokinetics of dasatinib. Segment 2 was designed to evaluate the safety of dasatinib as dosing was increased. QT intervals were monitored closely in both segments. Efficacy was assessed in Segment 2 using both positron emission tomography and computed tomography. RESULTS: Hematologic toxicities were markedly less than those observed in patients with leukemia, whereas nonhematologic toxicities were similar. The authors determined that the maximum recommended dose was 180 mg once daily based on the incidence of pleural effusion. Coadministration of ketoconazole led to a marked increase in dasatinib exposure, which was correlated with an increase in corrected QT (QTc) values of approximately 6 msec. No adverse cardiac events were observed. CONCLUSIONS: The dose-limiting toxic effect for dasatinib was pleural effusion. The pharmacokinetic and cardiac studies indicated that coadministration of dasatinib with potent CYP3A4 inhibitors or agents that prolong the QTc interval should be avoided if possible. Close monitoring for toxicity and dose reduction should be considered if the coadministration of such agents cannot be avoided.

Phase I study of the effect of gastric acid pH modulators on the bioavailability of oral dasatinib in healthy subjects.

Dasatinib is a tyrosine kinase inhibitor (including BCR-ABL and the SRC family) that is effective in patients with chronic myeloid leukemia. Dasatinib has pH-dependent solubility and is bioavailable as an oral formulation. The effect of gastric pH modifiers on dasatinib pharmacokinetics is evaluated in an open-label, randomized, 3-period, 3-treatment crossover study. Twenty-four healthy subjects receive treatment A (2 doses of dasatinib 50 mg separated by 12 hours), treatment B (famotidine 40 mg given 2 hours after dasatinib 50 mg and 10 hours before another dose of dasatinib 50 mg), and treatment C (30 mL of an antacid containing aluminum/magnesium hydroxides given 2 hours before dasatinib 50 mg and concomitantly with dasatinib 50 mg 12 hours after the previous dasatinib dose); a 7-day washout separates each treatment period. When famotidine is administered 2 hours after dasatinib, dasatinib exposure is similar to dasatinib administered alone. However, dasatinib exposure is reduced by approximately 60% when famotidine is administered 10 hours before dasatinib dosing. In contrast, dasatinib exposure is unchanged when antacid (Maalox) is administered 2 hours before dasatinib; but when the antacid is coadministered with dasatinib, dasatinib exposure is reduced by approximately 55% to 58%. This indicates that H(2)-receptor antagonists should not be coadministered with dasatinib. Dasatinib may be administered with acid-neutralizing antacids if the doses are temporally separated by at least 2 hours.

Identification of candidate predictive and surrogate molecular markers for dasatinib in prostate cancer: rationale for patient selection and efficacy monitoring.

BACKGROUND: Dasatinib is a potent, multi-targeted kinase inhibitor that was recently approved for treatment of chronic myelogenous leukemia resistant to imatinib. To aid the clinical development of dasatinib in prostate cancer, we utilized preclinical models to identify potential molecular markers for patient stratification and efficacy monitoring. RESULTS: Using gene expression profiling, we first identified 174 genes whose expression was highly correlated with in vitro sensitivity of 16 cell lines and, thus, considered as candidate efficacy predictive markers. Among these are important prostatic cell lineage markers, cytokeratin 5, androgen receptor and prostate specific antigen. Our results indicate that 'basal type' cell lines with high expression of cytokeratin 5 and low expression of androgen receptor or prostate specific antigen are sensitive to dasatinib. To identify markers as surrogates for biological activity, we treated cell lines with dasatinib and identified genes whose expression was significantly modulated by the drug. Ten genes, including that encoding urokinase-type plasminogen activator (uPA), were found to not only be potential efficacy markers but also to have reduced expression upon dasatinib treatment. The down-regulation of uPA by dasatinib was drug-specific and correlated with the sensitivity of cell lines to dasatinib. Furthermore, EphA2, a target of dasatinib, was found to be a sensitivity biomarker. CONCLUSION: Using the gene expression profiling approach and preclinical models, we have identified prostatic biomarkers that are associated with sensitivity to dasatinib. This study has provided a basis for clinical evaluation of a potential dasatinib efficacy signature in prostate cancer.

Dasatinib (BMS-354825) pharmacokinetics and pharmacodynamic biomarkers in animal models predict optimal clinical exposure.

PURPOSE: Chronic myeloid leukemia (CML) is caused by reciprocal translocation between chromosomes 9 and 22, forming BCR-ABL, a constitutively activated tyrosine kinase. Imatinib mesylate, a selective inhibitor of BCR-ABL, represents current frontline therapy for CML; however, emerging evidence suggests that drug resistance to imatinib may limit its long-term success. To improve treatment options, dasatinib (BMS-354825) was developed as a novel, oral, multi-targeted kinase inhibitor of BCR-ABL and SRC family kinases. To date, dasatinib has shown promising anti-leukemic activity in preclinical models of CML and in phase I/II clinical studies in patients with imatinib-resistant or imatinib-intolerant disease. EXPERIMENTAL DESIGN: The pharmacokinetic and pharmacodynamic biomarkers of dasatinib were investigated in K562 human CML xenografts grown s.c. in severe combined immunodeficient mice. Tumoral levels of phospho-BCR-ABL/phospho-CrkL were determined by Western blot. RESULTS: Following a single oral administration of dasatinib at a preclinical efficacious dose of 1.25 or 2.5 mg/kg, tumoral phospho-BCR-ABL/phospho-CrkL were maximally inhibited at approximately 3 hours and recovered to basal levels by 24 hours. The time course and extent of the inhibition correlated with the plasma levels of dasatinib in mice. Pharmacokinetic/biomarker modeling predicted that the plasma concentration of dasatinib required to inhibit 90% of phospho-BCR-ABL in vivo was 10.9 ng/mL in mice and 14.6 ng/mL in humans, which is within the range of concentrations achieved in CML patients who responded to dasatinib treatment in the clinic. CONCLUSIONS: Phospho-BCR-ABL/phospho-CrkL are likely to be useful clinical biomarkers for the assessment of BCR-ABL kinase inhibition by dasatinib.

Preclinical antitumor activity of BMS-599626, a pan-HER kinase inhibitor that inhibits HER1/HER2 homodimer and heterodimer signaling.

PURPOSE: The studies described here are intended to characterize the ability of BMS-599626, a small-molecule inhibitor of the human epidermal growth factor receptor (HER) kinase family, to modulate signaling and growth of tumor cells that depend on HER1 and/or HER2. EXPERIMENTAL DESIGN: The potency and selectivity of BMS-599626 were assessed in biochemical assays using recombinant protein kinases, as well as in cell proliferation assays using tumor cell lines with varying degrees of dependence on HER1 or HER2 signaling. Modulation of receptor signaling was determined in cell assays by Western blot analyses of receptor autophosphorylation and downstream signaling. The ability of BMS-599626 to inhibit receptor heterodimer signaling in tumor cells was studied by receptor coimmunoprecipitation. Antitumor activity of BMS-599626 was evaluated using a number of different xenograft models that represent a spectrum of human tumors with HER1 or HER2 overexpression. RESULTS: BMS-599626 inhibited HER1 and HER2 with IC50 of 20 and 30 nmol/L, respectively, and was highly selective when tested against a broad panel of diverse protein kinases. Biochemical studies suggested that BMS-599626 inhibited HER1 and HER2 through distinct mechanisms. BMS-599626 abrogated HER1 and HER2 signaling and inhibited the proliferation of tumor cell lines that are dependent on these receptors, with IC50 in the range of 0.24 to 1 micromol/L. BMS-599626 was highly selective for tumor cells that depend on HER1/HER2 and had no effect on the proliferation of cell lines that do not express these receptors. In tumor cells that are capable of forming HER1/HER2 heterodimers, BMS-599626 inhibited heterodimerization and downstream signaling. BMS-599626 had antitumor activity in models that overexpress HER1 (GEO), as well as in models that have HER2 gene amplification (KPL4) or overexpression (Sal2), and there was good correlation between the inhibition of receptor signaling and antitumor activity. CONCLUSIONS: BMS-599626 is a highly selective and potent inhibitor of HER1 and HER2 kinases and inhibits tumor cell proliferation through modulation of receptor signaling. BMS-599626 inhibits HER1/HER2 receptor heterodimerization and provides an additional mechanism of inhibiting tumors in which receptor coexpression and heterodimerization play a major role in driving tumor growth. The preclinical data support the advancement of BMS-599626 into clinical development for the treatment of cancer.

Prediction of active drug plasma concentrations achieved in cancer patients by pharmacodynamic biomarkers identified from the geo human colon carcinoma xenograft model.

PURPOSE: Epidermal growth factor receptor (EGFR), a protein tyrosine kinase expressed in many types of human cancers, has been strongly associated with tumor progression. Cetuximab is an IgG(1) anti-EGFR chimeric mouse/human monoclonal antibody that has been approved for the treatment of advanced colon cancer. Using human tumor xenografts grown in nude mice, we have determined the in vivo pharmacodynamic response of cetuximab at efficacious doses. Three pharmacodynamic end points were evaluated: tumoral phospho-EGFR, tumoral mitogen-activated protein kinase (MAPK) phosphorylation, and Ki67 expression. EXPERIMENTAL DESIGN: The pharmacodynamic study was conducted in nude mice bearing Geo tumors following a single i.p. administration of 0.25 and 0.04 mg. The tumors were analyzed by immunohistochemistry. The levels of phospho-EGFR were quantitated by an ELISA assay. RESULTS: At 0.25 mg, phospho-EGFR was maximally inhibited by 91% at 24 hours, whereas the level of inhibition decreased to 72% by 72 hours. At 0.04 mg, the maximum inhibition of phospho-EGFR was 53% at 24 hours, whereas the level of inhibition decreased to 37% by 72 hours. The time course of phospho-EGFR inhibition and recovery seemed to correlate with the pharmacokinetics of cetuximab. Immunohistochemical analysis showed that phospho-MAPK and Ki67 expression were inhibited between 24 and 72 hours at 0.25 and 0.04 mg. A pharmacokinetic/pharmacodynamic model was established and predicted that the plasma concentration of cetuximab required to inhibit 90% of phospho-EGFR was 67.5 mug/mL. CONCLUSIONS: Phospho-EGFR/phospho-MAPK could be useful clinical biomarkers to assess EGFR inhibition by cetuximab.

Membrane transport of camptothecin: facilitation by human P-glycoprotein (ABCB1) and multidrug resistance protein 2 (ABCC2).

BACKGROUND: The purpose of the present study was to continue the investigation of the membrane transport mechanisms of 20-(S)-camptothecin (CPT) in order to understand the possible role of membrane transporters on its oral bioavailability and disposition. METHODS: The intestinal transport kinetics of CPT were characterized using Caco-2 cells, MDCKII wild-type cells and MDCKII cells transfected with human P-glycoprotein (PGP) (ABCB1) or human multidrug resistance protein 2 (MRP2) (ABCC2). The effects of drug concentration, inhibitors and temperature on CPT directional permeability were determined. RESULTS: The absorptive (apical to basolateral) and secretory (basolateral to apical) permeabilities of CPT were found to be saturable. Reduced secretory CPT permeabilities with decreasing temperatures suggests the involvement of an active, transporter-mediated secretory pathway. In the presence of etoposide, the CPT secretory permeability decreased 25.6%. However, inhibition was greater in the presence of PGP and of the breast cancer resistant protein inhibitor, GF120918 (52.5%). The involvement of additional secretory transporters was suggested since the basolateral to apical permeability of CPT was not further reduced in the presence of increasing concentrations of GF120918. To investigate the involvement of specific apically-located secretory membrane transporters, CPT transport studies were conducted using MDCKII/PGP cells and MDCKII/MRP2 cells. CPT carrier-mediated permeability was approximately twofold greater in MDCKII/PGP cells and MDCKII/MRP2 cells than in MDCKII/wild-type cells, while the apparent Km values were comparable in all three cell lines. The efflux ratio of CPT in MDCKII/PGP in the presence of 0.2 microM GF120918 was not completely reversed (3.36 to 1.49). However, the decrease in the efflux ratio of CPT in MDCKII/MRP2 cells (2.31 to 1.03) suggests that CPT efflux was completely inhibited by MK571, a potent inhibitor of the Multidrug Resistance Protein transporter family. CONCLUSIONS: The current results provide evidence that PGP and MRP2 mediate the secretory transport of CPT in vitro. However, the involvement of other transporters cannot be ruled out based on these studies. Since these transporters are expressed in the intestine, liver and kidney variations in their expression levels and/or regulation may be responsible for the erratic oral absorption and biliary excretion of CPT observed in human subjects.

Oxaliplatin biotransformation and pharmacokinetics: a pilot study to determine the possible relationship to neurotoxicity.

BACKGROUND: Both oxaliplatin and ormaplatin undergo biotransformation to Pt(dach)Cl2 with studies suggesting a predictive relationship between systemic exposure to Pt(dach)Cl2 and the severity of the delayed sensory neuropathy associated with ormaplatin. Studies characterizing the pharmacokinetic parameters of oxaliplatin and Pt(dach)Cl2 in humans have not been reported. This study was conducted to characterize the pharmacokinetic parameters of oxaliplatin and Pt(dach)Cl2 and to determine the extent to which oxaliplatin undergoes biotransformation to Pt(dach)Cl2 in humans. MATERIALS AND METHODS: Ten adult patients with metastatic colon cancer received oxaliplatin with or without fluorouracil-based chemotherapy. Blood samples were obtained during cycles 1 and 2. Total platinum, oxaliplatin and Pt(dach)Cl2 in the plasma ultrafiltrate were measured using high performance liquid chromatography and atomic absorption spectrometry. All patients underwent a thorough neurological evaluation after each cycle. RESULTS: The median steady-state concentration (C(SS)) (interquartile range, 25% to 75%) for oxaliplatin 85 mg/m2 was 0.33 microg Pt/ml (0.28 to 0.38 microg Pt/ml). The area under the curve (AUC) was 0.79 microg Pt/ml/h (0.62 to 0.88 microg Pt/ml/h) and the elimination half-life was 0.32 h (0.27 to 0.46 h). The median C(SS) for Pt(dach)Cl2 was 0.008 microg Pt/ml (0.004 to 0.014 microg Pt/ml). The C(SS) ratio of oxaliplatin to Pt(dach)Cl2 was 31 (24 to 51). All patients reported acute cold-induced neuropathy following cycles 1 and 2. Only two patients reported delayed sensory neuropathy (grade 1). CONCLUSION: The parent drug oxaliplatin is the major active platinum complex detected in plasma ultrafiltrate for at least the first few hours following oxaliplatin infusion in humans. Therefore, the plasma biotransformation products of oxaliplatin are unlikely to contribute to its efficacy or toxicity. In particular, plasma Pt(dach)Cl2 is unlikely to significantly contribute to the delayed sensory neuropathy associated with oxaliplatin, since only a limited amount (<3%) of oxaliplatin undergoes biotransformation to Pt(dach)Cl2.

Intestinal transport of irinotecan in Caco-2 cells and MDCK II cells overexpressing efflux transporters Pgp, cMOAT, and MRP1.

Irinotecan (CPT-11) is a water-soluble camptothecin (CPT) derivative that has been recently approved in the United States for patients as a first-line therapy in advanced colorectal cancer. Phase I clinical trials using oral CPT-11 have shown poor and variable oral bioavailability. The present study was designed to investigate the intestinal absorption and efflux mechanisms of CPT-11 using in vitro cell culture models, Caco-2 cells, and engineered Madine-Darby canine kidney (MDCK) II cells overexpressing P-glycoprotein (Pgp), canalicular multispecific organic anion transporter (cMOAT), and multidrug resistance-associated protein (MRP1). The intestinal absorptive and secretory transport of CPT-11 was investigated using Caco-2 cell monolayers. Secretory transport was concentration-dependent and saturable. The secretory efflux permeability (P(eff)) of CPT-11 decreased with decreasing temperature, with an estimated activation energy of 19.6 +/- 2.9 kcal/mol suggesting the involvement of active transporters. The involvement of potential secretory transporters was further characterized in MDCK II cells. The secretory efflux carrier permeability (P(c)) was approximately 4- and approximately 2-fold greater in MDCK II/Pgp and MDCK II/cMOAT cells than that in MDCK II/wild-type cells. Furthermore, the secretory efflux P(eff) of CPT-11 was significantly decreased by Pgp inhibitors, elacridar (GF120918) (IC50 = 0.38 +/- 0.06 microM) and verapamil (IC(50) = 234 +/- 48 microM) in MDCK II/Pgp cells and by cMOAT inhibitor 3-([(3-(2-[7-chloro-2-quinolinyl]ethyl)phenyl]-[(3-dimethylamino-3-oxoprphyl)-thio)-methyl]-thio) propanoic acid (MK571) (IC50) = 469 +/- 60 micro;M) in MDCK II/cMOAT cells. Overall, the current study suggests that Pgp and cMOAT are capable of mediating the efflux of CPT-11 in vitro. Since both Pgp and cMOAT are expressed in the intestine, liver, and kidney, it is likely that these efflux transporters play a significant role limiting the oral absorption and disposition of this important anticancer drug.