The fibroblast growth factor receptor inhibitor, derazantinib, has strong efficacy in human gastric tumor models and synergizes with paclitaxel in vivo.

Derazantinib (DZB) is an inhibitor of fibroblast growth factor receptors 1-3 (FGFR1-3), with additional activity against colony-stimulating-factor-1 receptor (CSF1R). We have profiled the activity of DZB in gastric cancer (GC) as monotherapy and combined with paclitaxel, and explored means of stratifying patients for treatment. The antiproliferative potency of DZB in vitro was quantified in 90 tumor cell lines and shown to correlate significantly with FGFR expression (<0.01) but not with FGFR DNA copy-number (CN) or FGFR mutations. In four GC cell lines in vitro , little or no synergy was observed with paclitaxel. In athymic nude mice, bearing cell-line derived xenografts (CDX) or patient-derived xenograft (PDX) GC models, DZB efficacy correlated highly significantly with FGFR gene expression ( r2 = 0.58; P = 0.0003; n = 18), but not FGFR mutations or DNA-CN. In FGFR-driven GC models, DZB had comparable efficacy to three other FGFR inhibitors and was more efficacious than paclitaxel. DZB had dose-dependent plasma pharmacokinetics but showed low brain penetration at all doses. GC models (one CDX and six PDX) were tested for sensitivity to the combination of DZB and paclitaxel and characterized by immunohistochemistry. The combination showed synergy (5) or additivity (2), and no antagonism, with synergy significantly associated ( P < 0.05) with higher levels of M2-type macrophages. The association of strong efficacy of the combination in vivo with M2 macrophages, which are known to express CSF1R, and the absence of synergy in vitro is consistent with the tumor microenvironment also being a factor in DZB efficacy and suggests additional means by which DZB could be stratified for cancer treatment in the clinic.

Derazantinib, a fibroblast growth factor receptor inhibitor, inhibits colony-stimulating factor receptor-1 in macrophages and tumor cells and in combination with a murine programmed cell death ligand-1-antibody activates the immune environment of murine syngeneic tumor models.

Derazantinib (DZB) is an inhibitor of the fibroblast growth factor receptors 1-3 (FGFRi) with similar potency against colony-stimulating factor receptor-1 (CSF1R), a protein important in the recruitment and function of tumor-associated macrophages. DZB inhibited pCSF1R in the macrophage cell line RAW264.7, and tumor cells GDM-1 and DEL, and had the same potency in HeLa cells transiently over-expressing FGFR2. DZB exhibited similar potency against pCSF1R expressed by isolated murine macrophages, but as in the cell lines, specific FGFRi were without significant CSF1R activity. DZB inhibited growth of three tumor xenograft models with reported expression or amplification of CSF1R, whereas the specific FGFRi, pemigatinib, had no efficacy. In the FGFR-driven syngeneic breast tumor-model, 4T1, DZB was highly efficacious causing tumor stasis. A murine PD-L1 antibody was without efficacy in this model, but combined with DZB, increased efficacy against the primary tumor and further reduced liver, spine and lung metastases. Immunohistochemistry of primary 4T1 tumors showed that the combination favored an antitumor immune infiltrate by strongly increasing cytotoxic T, natural killer and T-helper cells. Similar modulation of the tumor microenvironment was observed in an FGFR-insensitive syngeneic bladder model, MBT-2. These data confirm CSF1R as an important oncology target for DZB and provide mechanistic insight for the ongoing clinical trials, in which DZB is combined with the PD-L1 antibody, atezolizumab.

Preclinical modeling in glioblastoma patient-derived xenograft (GBM PDX) xenografts to guide clinical development of lisavanbulin-a novel tumor checkpoint controller targeting microtubules.

BACKGROUND: Glioblastoma (GBM) is an incurable disease with few approved therapeutic interventions. Radiation therapy (RT) and temozolomide (TMZ) remain the standards of care. The efficacy and optimal deployment schedule of the orally bioavailable small-molecule tumor checkpoint controller lisavanbulin alone, and in combination with, standards of care were assessed using a panel of IDH-wildtype GBM patient-derived xenografts. METHODS: Mice bearing intracranial tumors received lisavanbulin +/-RT +/-TMZ and followed for survival. Lisavanbulin concentrations in plasma and brain were determined by liquid chromatography with tandem mass spectrometry, while flow cytometry was used for cell cycle analysis. RESULTS: Lisavanbulin monotherapy showed significant benefit (P < .01) in 9 of 14 PDXs tested (median survival extension 9%-84%) and brain-to-plasma ratios of 1.3 and 1.6 at 2- and 6-hours postdose, respectively, validating previous data suggesting significant exposure in the brain. Prolonged lisavanbulin dosing from RT start until moribund was required for maximal benefit (GBM6: median survival lisavanbulin/RT 90 vs. RT alone 69 days, P = .0001; GBM150: lisavanbulin/RT 143 days vs. RT alone 73 days, P = .06). Similar observations were seen with RT/TMZ combinations (GBM39: RT/TMZ/lisavanbulin 502 days vs. RT/TMZ 249 days, P = .0001; GBM26: RT/TMZ/lisavanbulin 172 days vs. RT/TMZ 121 days, P = .04). Immunohistochemical analyses showed a significant increase in phospho-histone H3 with lisavanbulin treatment (P = .01). CONCLUSIONS: Lisavanbulin demonstrated excellent brain penetration, significant extension of survival alone or in RT or RT/TMZ combinations, and was associated with mitotic arrest. These data provide a strong clinical rationale for testing lisavanbulin in combination with RT or RT/TMZ in GBM patients.

Derazantinib: an investigational drug for the treatment of cholangiocarcinoma.

INTRODUCTION: This review evaluates the clinical role of fibroblast growth factor receptor 2 (FGFR2) inhibition with derazantinib in patients with intrahepatic cholangiocarcinoma (iCCA) harboring actionable oncogenic FGFR2 fusions/rearrangements, mutations and amplifications. FGFR inhibitors such as derazantinib are currently being evaluated to address the unmet medical need of patients with previously treated, locally advanced or metastatic iCCA harboring such genetic aberrations. AREAS COVERED: We summarize the pharmacokinetics, and the emerging safety and efficacy data of the investigational FGFR inhibitor derazantinib. We discuss the future directions of this novel therapeutic agent for iCCA. EXPERT OPINION: Derazantinib is a potent FGFR1‒3 kinase inhibitor which also has activity against colony stimulating factor-1‒receptor (CSF1R) and vascular endothelial growfth factor receptor‒2 (VEGFR2), suggesting a potentially differentiated role in the treatment of patients with iCCA. Derazantinib has shown clinically meaningful efficacy with durable objective responses, supporting the therapeutic potential of derazantinib in previously treated patients with iCCA harboring FGFR2 fusions/rearrangements, mutations and amplifications. The clinical safety profile of derazantinib was well manageable and compared favorably to the FGFR inhibitor class, particularly with a low incidence of drug-related hand-foot syndrome, stomatitis, retinal and nail toxicity. These findings support the need for increased molecular profiling of cholangiocarcinoma patients.

Treating ICB-resistant glioma with anti-CD40 and mitotic spindle checkpoint controller BAL101553 (lisavanbulin).

Glioblastoma is a highly malignant brain tumor with no curative treatment options, and immune checkpoint blockade has not yet shown major impact. We hypothesized that drugs targeting mitosis might affect the tumor microenvironment and sensitize cancer cells to immunotherapy. We used 2 glioblastoma mouse models with different immunogenicity profiles, GL261 and SB28, to test the efficacy of antineoplastic and immunotherapy combinations. The spindle assembly checkpoint activator BAL101553 (lisavanbulin), agonistic anti-CD40 antibody, and double immune checkpoint blockade (anti-programmed cell death 1 and anti-cytotoxic T lymphocyte-associated protein 4; anti-PD-1 and anti-CTLA-4) were evaluated individually or in combination for treating orthotopic GL261 and SB28 tumors. Genomic and immunological analyses were used to predict and interpret therapy responsiveness. BAL101553 monotherapy increased survival in immune checkpoint blockade-resistant SB28 glioblastoma tumors and synergized with anti-CD40 antibody, in a T cell-independent manner. In contrast, the more immunogenic and highly mutated GL261 model responded best to anti-PD-1 and anti-CTLA-4 therapy and more modestly to BAL101553 and anti-CD40 combination. Our results show that BAL101553 is a promising therapeutic agent for glioblastoma and could synergize with innate immune stimulation. Overall, these data strongly support immune profiling of glioblastoma patients and preclinical testing of combination therapies with appropriate models for particular patient groups.

Discovery of Potent, Highly Selective, and In Vivo Efficacious, Allosteric MALT1 Inhibitors by Iterative Scaffold Morphing.

MALT1 plays a central role in immune cell activation by transducing NF-κB signaling, and its proteolytic activity represents a key node for therapeutic intervention. Two cycles of scaffold morphing of a high-throughput biochemical screening hit resulted in the discovery of MLT-231, which enabled the successful pharmacological validation of MALT1 allosteric inhibition in preclinical models of humoral immune responses and B-cell lymphomas. Herein, we report the structural activity relationships (SARs) and analysis of the physicochemical properties of a pyrazolopyrimidine-derived compound series. In human T-cells and B-cell lymphoma lines, MLT-231 potently and selectively inhibits the proteolytic activity of MALT1 in NF-κB-dependent assays. Both in vitro and in vivo profiling of MLT-231 support further optimization of this in vivo tool compound toward preclinical characterization.

Optimization of the In Vivo Potency of Pyrazolopyrimidine MALT1 Protease Inhibitors by Reducing Metabolism and Increasing Potency in Whole Blood.

The paracaspase MALT1 has gained increasing interest as a target for the treatment of subsets of lymphomas as well as autoimmune diseases, and there is a need for suitable compounds to explore the therapeutic potential of this target. Here, we report the optimization of the in vivo potency of pyrazolopyrimidines, a class of highly selective allosteric MALT1 inhibitors. High doses of the initial lead compound led to tumor stasis in an activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) xenograft model, but this compound suffered from a short in vivo half-life and suboptimal potency in whole blood. Guided by metabolism studies, we identified compounds with reduced metabolic clearance and increased in vivo half-life. In the second optimization step, masking one of the hydrogen-bond donors of the central urea moiety through an intramolecular interaction led to improved potency in whole blood. This was associated with improved in vivo potency in a mechanistic model of B cell activation. The optimized compound led to tumor regression in a CARD11 mutant ABC-DLBCL lymphoma xenograft model.

Probing the Effects of the FGFR-Inhibitor Derazantinib on Vascular Development in Zebrafish Embryos.

Angiogenesis is a fundamental developmental process and a hallmark of cancer progression. Receptor tyrosine kinases (RTK) are targets for cancer therapy which may include their action as anti-angiogenic agents. Derazantinib (DZB) is an inhibitor of the fibroblast growth factor receptors (FGFRs) 1-3 as well as other kinase targets including vascular endothelial growth factor receptor 2 (VEGFR2), colony stimulating factor-1 receptor (CSF1R) and platelet-derived growth factor beta receptor (PDGFRbeta). This study aimed to investigate the effect of DZB on blood vessel morphogenesis and to compare its activity to known specific FGFR and VEGFR inhibitors. For this purpose, we used the developing vasculature in the zebrafish embryo as a model system for angiogenesis in vivo. We show that DZB interferes with multiple angiogenic processes that are linked to FGF and VEGF signalling, revealing a potential dual role for DZB as a potent anti-angiogenic treatment.

Continuous low-dose infusion of patupilone increases the therapeutic index in mouse and rat tumour models.

Patupilone is a microtubule-targeted cytotoxic agent with clinical efficacy, but causes diarrhoea in more than 80% of patients. The efficacy and tolerability of patupilone delivered continuously by subcutaneous (s.c.) mini-pumps [(mini-pump dose (MPD)] or by intravenous bolus administration [intravenous bolus dose (IVBD)] were compared preclinically to determine whether the therapeutic index could be improved. The antiproliferative potency in vitro of patupilone was determined by measuring total cell protein. Tumours were grown s.c. in rats (A15) or nude mice (KB31, KB8511) or intracranially in nude mice (NCI-H460-Luc). Efficacy was monitored by measuring tumour volumes, bioluminescence or survival. Toxicity was monitored by body weight and/or diarrhoea. Total drug levels in blood, plasma, tissues or dialysates were quantified ex-vivo by liquid chromatography-mass spectroscopy/mass spectroscopy. Patupilone was potent in vitro with GI50s of 0.24-0.28 nmol/l and GI90s of 0.46-1.64 nmol/l. In rats, a single IVBD of patupilone dose dependently inhibited the growth of A15 tumours, but also caused dose-dependent body weight loss and diarrhoea, whereas MPD achieved similar efficacy, but no toxicity. In mice, MPD showed efficacy similar to that of IVBD against KB31 and KB8511 tumours, but with reduced toxicity. In a mouse intracranial tumour model, IVBD was more efficacious than MPD, consistent with patupilone concentrations in the brain. MPD provided constant plasma levels, whereas IVBD had very high C0/Cmin ratios of 70-280 (rat) or 8000 (mouse) over the dosing cycle. Overall, the correlation of plasma and tumour levels with response indicated that a Cave of at least GI90 led to tumour stasis. Continuous low concentrations of patupilone by MPD increased the therapeutic index in s.c. rodent tumour models compared with IVBD by maintaining efficacy, but reducing toxicity.

Continuous low plasma concentrations of everolimus provides equivalent efficacy to oral daily dosing in mouse xenograft models of human cancer.

PURPOSE: Everolimus is a drug used successfully in a number of different oncology indications, but significant on-target toxicities exist. We explored the possibility of improving the therapeutic index (TI) by studying alternative means of administering the drug based upon low continuous dosing. METHODS: All studies were performed using naïve nude mice or nude mice bearing s.c. human renal 786-O tumours or human breast MDA-MB-468 tumours. Everolimus was administered via a standard emulsion, either i.v., p.o., i.p., s.c., or via s.c. osmotic mini-pumps (MP) or via poly-lactic-co-glycolic (PLGA)-microparticles (PLGA-µP) prepared from everolimus powder injected s.c. Total-drug levels in blood, plasma or tissues were quantified ex vivo by LC-MS/MS. Efficacy studies were performed over 2-3 weeks and toxicity assessed by changes in body weight, glucose and white blood cell count. Effects on tumour activity biomarkers were quantified using reverse-phase protein array. RESULTS: Everolimus administration s.c. in an emulsion decreased the absorption rate but increased the C max and bio-availability of everolimus compared to standard approaches of administration p.o. or i.p. Everolimus administration s.c. via MP or PLGA-µP reduced the C max and provided continuous low concentrations of everolimus in the plasma, which inhibited tumour pS6/S6 to a similar degree to oral administration. Toxicities such as changes in body weight or white blood cell count were unaffected. Provided the everolimus concentration was above the free unbound IC50 for proliferation of the tumour cell line, efficacy could be achieved equivalent to that provided by standard oral administration. However, an overall improvement in the TI could not be demonstrated. CONCLUSIONS: Continuous low plasma concentrations of everolimus can provide strong efficacy in preclinical models, which if translatable to the clinic may reduce on-target toxicities and so increase the TI.

A Novel Potent Oral Series of VEGFR2 Inhibitors Abrogate Tumor Growth by Inhibiting Angiogenesis.

This paper describes the identification of 6-(pyrimidin-4-yloxy)-naphthalene-1-carboxamides as a new class of potent and selective human vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitors. In biochemical and cellular assays, the compounds exhibit single-digit nanomolar potency toward VEGFR2. Compounds of this series show good exposure in rodents when dosed orally. They potently inhibit VEGF-driven angiogenesis in a chamber model and rodent tumor models at daily doses of less than 3 mg/kg by targeting the tumor vasculature as demonstrated by ELISA for TIE-2 in lysates or by immunohistochemical analysis. This novel series of compounds shows a potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

Tumour T1 changes in vivo are highly predictive of response to chemotherapy and reflect the number of viable tumour cells--a preclinical MR study in mice.

BACKGROUND: Effective chemotherapy rapidly reduces the spin-lattice relaxation of water protons (T1) in solid tumours and this change (ΔT1) often precedes and strongly correlates with the eventual change in tumour volume (TVol). To understand the biological nature of ΔT1, we have performed studies in vivo and ex vivo with the allosteric mTOR inhibitor, everolimus. METHODS: Mice bearing RIF-1 tumours were studied by magnetic resonance imaging (MRI) to determine TVol and T1, and MR spectroscopy (MRS) to determine levels of the proliferation marker choline and levels of lipid apoptosis markers, prior to and 5 days (endpoint) after daily treatment with vehicle or everolimus (10 mg/kg). At the endpoint, tumours were ablated and an entire section analysed for cellular and necrotic quantification and staining for the proliferation antigen Ki67 and cleaved-caspase-3 as a measure of apoptosis. The number of blood-vessels (BV) was evaluated by CD31 staining. Mice bearing B16/BL6 melanoma tumours were studied by MRI to determine T1 under similar everolimus treatment. At the endpoint, cell bioluminescence of the tumours was measured ex vivo. RESULTS: Everolimus blocked RIF-1 tumour growth and significantly reduced tumour T1 and total choline (Cho) levels, and increased polyunsaturated fatty-acids which are markers of apoptosis. Immunohistochemistry showed that everolimus reduced the %Ki67+ cells but did not affect caspase-3 apoptosis, necrosis, BV-number or cell density. The change in T1 (ΔT1) correlated strongly with the changes in TVol and Cho and %Ki67+. In B16/BL6 tumours, everolimus also decreased T1 and this correlated with cell bioluminescence; another marker of cell viability. Receiver-operating-characteristic curves (ROC) for everolimus on RIF-1 tumours showed that ΔT1 had very high levels of sensitivity and specificity (ROCAUC = 0.84) and this was confirmed for the cytotoxic patupilone in the same tumour model (ROCAUC = 0.97). CONCLUSION: These studies suggest that ΔT1 is not a measure of cell density but reflects the decreased number of remaining viable and proliferating tumour cells due to perhaps cell and tissue destruction releasing proteins and/or metals that cause T1 relaxation. ΔT1 is a highly sensitive and specific predictor of response. This MRI method provides the opportunity to stratify a patient population during tumour therapy in the clinic.

Microtubule stabilising agents and ionising radiation: multiple exploitable mechanisms for combined treatment.

Combined radiochemotherapy treatment modalities are in use for many indications and therefore of high interest. Even though a combined modality in clinical use is often driven by pragmatic aspects, mechanistic preclinical-based concepts of interaction are of importance in order to translate and implement an optimal combination and scheduling of two modalities into the clinics. The use of microtubule stabilising agents is a promising strategy for anti-cancer therapy as a part of combined treatment modality with ionising radiation. Traditionally, microtubule targeting agents are classified as cytotoxic chemotherapeutics and are mostly used in a maximally tolerated dose regimen. Apart from direct cytotoxicity and similar to mechanisms of molecular targeting agents, microtubule stabilising agents interfere with multiple cellular processes, which can be exploited as part of combined treatment modalities. Recent preclinical investigations on the combination of ionising radiation and microtubule stabilising agents reveal new mechanistic interactions on the cellular and tumour level and elucidate the supra-additive tumour response observed particularly in vivo. The major focus on the mechanism of interaction was primarily based on radiosensitisation due to cell cycle arrest in the most radiosensitive G2/M-phase of the cell cycle. However, other mechanisms of interaction such as reoxygenation and direct as well as indirect endothelial damage have also been identified. In this review we summarise and allocate additive and synergistic effects induced by the combined treatment of clinically relevant microtubule stabilising agents and ionising radiation along a described radiobiological framework encompassing distinct mechanisms relevant for exploiting the combination of drugs and ionising radiation.

Patupilone (epothilone B) for recurrent glioblastoma: clinical outcome and translational analysis of a single-institution phase I/II trial.

BACKGROUND: Patients with glioblastoma (GBM) inevitably develop recurrent or progressive disease after initial multimodal treatment and have a median survival of 6-9 months from time of progression. To date, there is no accepted standard treatment for GBM relapse or progression. Patupilone (EPO906) is a novel natural microtubule-stabilizing cytotoxic agent that crosses the blood-brain barrier and has been found to have preclinical activity in glioma models. METHODS: This is a single-institution, early-phase I/II trial of GBM patients with tumor progression who qualified for second surgery with the goal of evaluating efficacy and safety of the single-agent patupilone (10 mg/m(2), every 3 weeks). Patients received patupilone 1 week prior to second surgery and every 3 weeks thereafter until tumor progression or toxicity. Primary end points were progression-free survival (PFS) and overall survival (OS) at 6 months as well as patupilone concentration in tumor tissue. Secondary end points were toxicity, patupilone concentration in plasma and translational analyses for predictive biomarkers. RESULTS: Nine patients with a mean age of 54.6 ± 8.6 years were recruited between June 2008 and April 2010. Median survival and 1-year OS after second surgery were 11 months (95% CI, 5-17 months) and 45% (95% CI, 14-76), respectively. Median PFS was 1.5 months (95% CI, 1.3-1.7 months) and PFS6 was 22% (95% CI, 0-46), with 2 patients remaining recurrence-free at 9.75 and 22 months. At the time of surgery, the concentration of patupilone in tumor tissue was 30 times higher than in the plasma. Tumor response was not predictable by the tested biomarkers. Treatment was generally well tolerated with no hematological, but cumulative, though reversible sensory neuropathy grade ≤3 was seen in 2 patients (22%) at 8 months and grade 4 diarrhea in the 2nd patient (11%). Non-patupilone-related peri-operative complications occurred in 2 patients resulting in discontinuation of patupilone therapy. There were no neurocognitive changes 3 months after surgery compared to baseline. CONCLUSIONS: In recurrent GBM, patupilone can be given safely pre- and postoperatively. The drug accumulates in the tumor tissue. The treatment results in long-term PFS in some patients. Patupilone represents a valuable novel compound which deserves further evaluation in combination with radiation therapy in patients with GBM.

Adaptation to HIF-1 deficiency by upregulation of the AMP/ATP ratio and phosphofructokinase activation in hepatomas.

BACKGROUND: HIF-1 deficiency has marked effects on tumour glycolysis and growth. We therefore investigated the consequences of HIF-1 deficiency in mice, using the well established Hepa-1 wild-type (WT) and HIF-1ß-deficient (c4) model. These mechanisms could be clinically relevant, since HIF-1 is now a therapeutic target. METHODS: Hepa-1 WT and c4 tumours grown in vivo were analysed by 18FDG-PET and 19FDG Magnetic Resonance Spectroscopy for glucose uptake; by HPLC for adenine nucleotides; by immunohistochemistry for GLUTs; by immunoblotting and by DIGE followed by tandem mass spectrometry for protein expression; and by classical enzymatic methods for enzyme activity. RESULTS: HIF-1ß deficient Hepa-1 c4 tumours grew significantly more slowly than WT tumours, and (as expected) showed significantly lower expression of many glycolytic enzymes. However, HIF-1ß deficiency caused no significant change in the rate of glucose uptake in c4 tumours compared to WT when assessed in vivo by measuring fluoro-deoxyglucose (FDG) uptake. Immunohistochemistry demonstrated less GLUT-1 in c4 tumours, whereas GLUT-2 (liver type) was similar to WT. Factors that might upregulate glucose uptake independently of HIF-1 (phospho-Akt, c-Myc) were shown to have either lower or similar expression in c4 compared to WT tumours. However the AMP/ATP ratio was 4.5 fold higher (p < 0.01) in c4 tumours, and phosphofructokinase-1 (PFK-1) activity, measured at prevailing cellular ATP and AMP concentrations, was up to two-fold higher in homogenates of the deficient c4 cells and tumours compared to WT (p < 0.001), suggesting that allosteric PFK activation could explain their normal level of glycolysis. Phospho AMP-Kinase was also higher in the c4 tumours. CONCLUSIONS: Despite their defective HIF-1 and consequent down-regulation of glycolytic enzyme expression, Hepa-1 c4 tumours maintain glucose uptake and glycolysis because the resulting low [ATP] high [AMP] allosterically activate PFK-1. This mechanism of resistance would keep glycolysis functioning and also result in activation of AMP-Kinase and growth inhibition; it may have major implications for the therapeutic activity of HIF inhibitors in vivo. Interestingly, this control mechanism does not involve transcriptional control or proteomics, but rather the classical activation and inhibition mechanisms of glycolytic enzymes.

Everolimus augments the effects of sorafenib in a syngeneic orthotopic model of hepatocellular carcinoma.

Sorafenib targets the Raf/mitogen-activated protein kinase, VEGF, and platelet-derived growth factor pathways and prolongs survival patients in advanced hepatocellular carcinoma (HCC). Everolimus inhibits the mammalian target of rapamycin, a kinase overactive in HCC. To investigate whether the antitumor effects of these agents are additive, we compared a combined and sequential treatment regimen of everolimus and sorafenib with monotherapy. After hepatic implantation of Morris Hepatoma (MH) cells, rats were randomly allocated to everolimus (5 mg/kg, 2×/week), sorafenib (7.5 mg/kg/d), combined everolimus and sorafenib, sequential sorafenib (2 weeks) then everolimus (3 weeks), or control groups. MRI quantified tumor volumes. Erk1/2, 4E-BP1, and their phosphorylated forms were quantified by immunoblotting. Angiogenesis was assessed in vitro by aortic ring and tube formation assays, and in vivo with Vegf-a mRNA and vascular casts. After 35 days, tumor volumes were reduced by 60%, 85%, and 55%, relative to controls, in everolimus, the combination, and sequential groups, respectively (P < 0.01). Survival was longest in the combination group (P < 0.001). Phosphorylation of 4E-BP1 and Erk1/2 decreased after everolimus and sorafenib, respectively. Angiogenesis decreased after all treatments (P < 0.05), although sorafenib increased Vegf-a mRNA in liver tumors. Vessel sprouting was abundant in control tumors, lower after sorafenib, and absent after the combination. Intussusceptive angiogenic transluminal pillars failed to coalesce after the combination. Combined treatment with everolimus and sorafenib exerts a stronger antitumoral effect on MH tumors than monotherapy. Everolimus retains antitumoral properties when administered sequentially after sorafenib. This supports the clinical use of everolimus in HCC, both in combination with sorafenib or after sorafenib.

Regulation of VEGF-expression by patupilone and ionizing radiation in lung adenocarcinoma cells.

The use of microtubule stabilizing agents (MSAs) is a promising strategy for anti-cancer therapy alone and as part of combined treatment modalities with ionizing radiation. However MSA-provoked molecular and cellular processes including the regulation of intercellular, paracrine signaling pathways are far from clear. Here we investigated the interference of the novel, clinically relevant MSA patupilone (epothilone B) with the tumor-cell derived vascular endothelial growth factor (VEGF), which is most relevant for tumor angiogenesis. Low-dose, sub-nanomolar concentrations of patupilone specifically reduced hypoxia-driven stabilization of the transcription factor HIF-1α in the patupilone-sensitive lung adenocarcinoma cell line A549, but not in the mutant derivative cell line A549.EpoB40. Patupilone further reduced hypoxia-induced VEGF expression and secretion but only in the A549 wildtype cell line. In the wildtype cell line, ionizing radiation alone induced hypoxia-dependent VEGF-expression but a strong dominant counteracting effect of patupilone was always observed when ionizing radiation was combined with patupilone, on the level of HIF-1α protein stability, VEGF-expression and VEGF-secretion. These results demonstrate that patupilone and ionizing radiation dysregulate hypoxia-induced stress responses, which might contribute to the potency of this promising, combined treatment modality.

Everolimus and PTK/ZK show synergistic growth inhibition in the orthotopic BL16/BL6 murine melanoma model.

PURPOSE: Everolimus (RAD001, Afinitor) is an mTORC1 pathway inhibitor, and vatalanib (PTK/ZK) is a pan VEGF-R tyrosine kinase inhibitor (TKI). These two drugs have been shown to have overlapping but also distinct anti-angiogenic effects. Consequently, we investigated the pharmacokinetics (PK) and pharmacodynamics (PD) of their combination in vivo. METHODS: Murine melanoma B16/BL6 cells were grown orthotopically in BL6/C57 mice by injection into the derma of both ears to create a primary tumour which metastasized rapidly to the cervical lymph nodes. Mice were treated daily p.o. with PTK/ZK (100 mg/kg) or everolimus (1 mg/kg) or their combination, and anti-tumour efficacy (PD) assessed. In the same model, plasma PK of everolimus was measured following single doses of the monotherapy or combination schedules. RESULTS: Two independent experiments showed that combination of everolimus and PTK/ZK caused at least additive increases in anti-tumour activity compared to either monotherapy, without increases in toxicity. Pooling the data to improve the statistical power demonstrated the interactions to be synergistic. PK modelling showed that although PTK/ZK increased everolimus plasma concentrations by about twofold, this PK drug-drug interaction could not account for the increased anti-tumour effect of the combination. Modelling of the PTK/ZK dose-response curve in this model suggested that any effect of everolimus on the PK of PTK/ZK was unlikely to affect efficacy. Measurement of changes in tumour and plasma VEGF levels at the endpoint of therapy confirmed earlier observations of differential effects of these two agents. CONCLUSIONS: The combination of everolimus and PTK/ZK hold promise for the treatment of human cancers.

Evaluation of the mTOR inhibitor, everolimus, in combination with cytotoxic antitumor agents using human tumor models in vitro and in vivo.

The aim was to determine the potential of the allosteric mammalian target of rapamycin inhibitor, everolimus, to act in combination with cytotoxic anticancer compounds in vitro and in vivo. A concomitant combination in vitro showed no evidence of antagonism, but enhanced the antiproliferative effects (additive to synergistic) with cisplatin, doxorubicin, 5-fluorouracil, gemcitabine, paclitaxel, and patupilone. Everolimus (1-5 mg/kg/d orally) was evaluated for antitumor activity in vivo alone or in combination with suboptimal cytotoxic doses using athymic nude mice bearing subcutaneous human H-596 lung, KB-31 cervical, or HCT-116 colon tumor xenografts. Everolimus monotherapy was very well tolerated and caused inhibition of tumor growth, rather than regression, and this was associated with a dose-dependent decline in tumor pS6 levels, a key downstream protein of mammalian target of rapamycin. At the doses used, the cytotoxics inhibited tumor growth and caused tolerable body-weight loss. Concomitant combinations of cisplatin, doxorubicin, paclitaxel, or patupilone with everolimus produced cooperative antitumor effects, in some cases producing regressions without clinically significant increases in toxicity. In contrast, combinations with gemcitabine and 5-fluorouracil were less well tolerated. Alternative administration schedules were tested for cisplatin, gemcitabine, or paclitaxel combined with everolimus: these did not dramatically affect cisplatin or gemcitabine activity or tolerability but were antagonistic for paclitaxel. Everolimus showed promising maintenance activity after treatment with doxorubicin or paclitaxel ceased. Overall, the results confirm that everolimus is an effective, well-tolerated suppressor of experimental human tumor growth, and although it did not show strong potentiation of efficacy, antitumor activity in vivo was increased without marked increases in toxicity, supporting clinical use of everolimus as a partner for conventional cytotoxics.

Anti-Angiogenic/Vascular Effects of the mTOR Inhibitor Everolimus Are Not Detectable by FDG/FLT-PET.

Noninvasive functional imaging of tumors can provide valuable early-response biomarkers, in particular, for targeted chemotherapy. Using various experimental tumor models, we have investigated the ability of positron emission tomography (PET) measurements of 2-deoxy-2-[(18)F]fluoro-glucose (FDG) and 3'-deoxy-3'-[(18)F]fluorothymidine (FLT) to detect response to the allosteric mammalian target of rapamycin (mTOR) inhibitor everolimus. Tumor models were declared sensitive (murine melanoma B16/BL6 and human lung H596) or relatively insensitive (human colon HCT116 and cervical KB31), according to the IC(50) values (concentration inhibiting cell growth by 50%) for inhibition of proliferation in vitro (<10 nM and >1 microM, respectively). Everolimus strongly inhibited growth of the sensitive models in vivo but also significantly inhibited growth of the insensitive models, an effect attributable to its known anti-angiogenic/vascular properties. However, although tumor FDG and FLT uptake was significantly reduced in the sensitive models, it was not affected in the insensitive models, suggesting that endothelial-directed effects could not be detected by these PET tracers. Consistent with this hypothesis, in a well-vascularized orthotopic rat mammary tumor model, other antiangiogenic agents also failed to affect FDG uptake, despite inhibiting tumor growth. In contrast, the cytotoxic patupilone, a microtubule stabilizer, blocked tumor growth, and markedly reduced FDG uptake. These results suggest that FDG/FLT-PET may not be a suitable method for early markers of response to antiangiogenic agents and mTOR inhibitors in which anti-angiogenic/vascular effects predominate because the method could provide false-negative responses. These conclusions warrant clinical testing.