Effects of somatostatin analogs on glucose homeostasis in rats.

Pasireotide (SOM230) is a multireceptor-targeted somatostatin analog with high binding affinity for sstr(1,2,3) and sstr(5). The effects of pasireotide and octreotide on blood glucose, insulin, and glucagon levels in rats were evaluated alone and in combination. Single-dose s.c. pasireotide acutely elevated plasma glucose, whereas single-dose s.c. octreotide had no or a small hypoglycemic effect. Glucose elevation with s.c. pasireotide was transient with tachyphylaxis after repeated or continuous administration. Pasireotide and octreotide caused similar inhibitory effects on insulin secretion, whereas pasireotide had a weaker inhibitory effect on glucagon secretion than octreotide. Continuous infusion of pasireotide or injection of pasireotide long-acting release (LAR) resulted in only small and transient elevations of plasma glucose. Based on these results, and differences in the sstr binding affinity of pasireotide vs octreotide, it was hypothesized that the sstr(5) vs sstr(2) receptor activation ratio is the main driver of hyperglycemia after pasireotide. The results also suggest that stronger activation of sstr(2) may counteract the hyperglycemic effect. Indeed, co-administration of octreotide, which has a high affinity for sstr(2), with a hyperglycemic dose of pasireotide did not cause significant changes in plasma glucose levels. In conclusion, although pasireotide and octreotide inhibited insulin to a similar degree, only pasireotide administration was associated with hyperglycemia. The strong glucagon inhibitory effect exhibited by octreotide but not pasireotide may explain this observation. The lack of hyperglycemia during co-administration of pasireotide and octreotide may be explained by the greater activation of sstr(2) compared with pasireotide alone, causing the insulin-glucagon balance to shift within the normoglycemic range. Extrapolation of these data to humans must account for species differences in islet cell sstr expression.

Discovery of 3-(2,6-dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methyl-urea (NVP-BGJ398), a potent and selective inhibitor of the fibroblast growth factor receptor family of receptor tyrosine kinase.

A novel series of N-aryl-N'-pyrimidin-4-yl ureas has been optimized to afford potent and selective inhibitors of the fibroblast growth factor receptor tyrosine kinases 1, 2, and 3 by rationally designing the substitution pattern of the aryl ring. On the basis of its in vitro profile, compound 1h (NVP-BGJ398) was selected for in vivo evaluation and showed significant antitumor activity in RT112 bladder cancer xenografts models overexpressing wild-type FGFR3. These results support the potential therapeutic use of 1h as a new anticancer agent.

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.

Potent and selective inhibition of polycythemia by the quinoxaline JAK2 inhibitor NVP-BSK805.

The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2(V617F) mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative neoplasms and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2(V617F) and JAK2 wild-type enzymes by a novel substituted quinoxaline, NVP-BSK805, which acts in an ATP-competitive manner. Within the JAK family, NVP-BSK805 displays more than 20-fold selectivity towards JAK2 in vitro, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2(V617F)-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, NVP-BSK805 exhibited good oral bioavailability and a long half-life. The inhibitor was efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2(V617F) cell-driven mouse mechanistic model. Furthermore, NVP-BSK805 potently suppressed recombinant human erythropoietin-induced polycythemia and extramedullary erythropoiesis in mice and rats.

Preclinical antitumor activity of the orally available heat shock protein 90 inhibitor NVP-BEP800.

Heat shock protein 90 (Hsp90) is a ubiquitously expressed molecular chaperone with ATPase activity involved in the conformational maturation and stability of key signaling molecules involved in cell proliferation, survival, and transformation. Through its ability to modulate multiple pathways involved in oncogenesis, Hsp90 has generated considerable interest as a therapeutic target. NVP-BEP800 is a novel, fully synthetic, orally bioavailable inhibitor that binds to the NH(2)-terminal ATP-binding pocket of Hsp90. NVP-BEP800 showed activity against a panel of human tumor cell lines and primary human xenografts in vitro at nanomolar concentrations. In A375 melanoma and BT-474 breast cancer cell lines, NVP-BEP800 induced client protein degradation (including ErbB2, B-Raf(V600E), Raf-1, and Akt) and Hsp70 induction. Oral administration of NVP-BEP800 was well tolerated and induced robust antitumor responses in tumor xenograft models, including regression in the BT-474 breast cancer model. In these tumor models, NVP-BEP800 modulated Hsp90 client proteins and downstream signaling pathways at doses causing antitumor activity. NVP-BEP800 showed in vivo activity in a variety of dosing regimens covering daily to weekly schedules, potentially providing a high degree of flexibility in dose and schedule within the clinical setting. Overall, given the mechanism of action, preclinical activity profile, tolerability, and pharmaceutical properties, NVP-BEP800 is an exciting new oral Hsp90 inhibitor warranting further development. Mol Cancer Ther; 9(4); 906-19. (c)2010 AACR.

Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity.

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin inhibitor (mTOR) pathway is often constitutively activated in human tumor cells, providing unique opportunities for anticancer therapeutic intervention. NVP-BEZ235 is an imidazo[4,5-c]quinoline derivative that inhibits PI3K and mTOR kinase activity by binding to the ATP-binding cleft of these enzymes. In cellular settings using human tumor cell lines, this molecule is able to effectively and specifically block the dysfunctional activation of the PI3K pathway, inducing G(1) arrest. The cellular activity of NVP-BEZ235 translates well in in vivo models of human cancer. Thus, the compound was well tolerated, displayed disease stasis when administered orally, and enhanced the efficacy of other anticancer agents when used in in vivo combination studies. Ex vivo pharmacokinetic/pharmacodynamic analyses of tumor tissues showed a time-dependent correlation between compound concentration and PI3K/Akt pathway inhibition. Collectively, the preclinical data show that NVP-BEZ235 is a potent dual PI3K/mTOR modulator with favorable pharmaceutical properties. NVP-BEZ235 is currently in phase I clinical trials.

NVP-AUY922: a small molecule HSP90 inhibitor with potent antitumor activity in preclinical breast cancer models.

INTRODUCTION: Heat shock protein 90 (HSP90) is a key component of a multichaperone complex involved in the post-translational folding of a large number of client proteins, many of which play essential roles in tumorigenesis. HSP90 has emerged in recent years as a promising new target for anticancer therapies. METHODS: The concentrations of the HSP90 inhibitor NVP-AUY922 required to reduce cell numbers by 50% (GI50 values) were established in a panel of breast cancer cell lines and patient-derived human breast tumors. To investigate the properties of the compound in vivo, the pharmacokinetic profile, antitumor effect, and dose regimen were established in a BT-474 breast cancer xenograft model. The effect on HSP90-p23 complexes, client protein degradation, and heat shock response was investigated in cell culture and breast cancer xenografts by immunohistochemistry, Western blot analysis, and immunoprecipitation. RESULTS: We show that the novel small molecule HSP90 inhibitor NVP-AUY922 potently inhibits the proliferation of human breast cancer cell lines with GI50 values in the range of 3 to 126 nM. NVP-AUY922 induced proliferative inhibition concurrent with HSP70 upregulation and client protein depletion--hallmarks of HSP90 inhibition. Intravenous acute administration of NVP-AUY922 to athymic mice (30 mg/kg) bearing subcutaneous BT-474 breast tumors resulted in drug levels in excess of 1,000 times the cellular GI50 value for about 2 days. Significant growth inhibition and good tolerability were observed when the compound was administered once per week. Therapeutic effects were concordant with changes in pharmacodynamic markers, including HSP90-p23 dissociation, decreases in ERBB2 and P-AKT, and increased HSP70 protein levels. CONCLUSION: NVP-AUY922 is a potent small molecule HSP90 inhibitor showing significant activity against breast cancer cells in cellular and in vivo settings. On the basis of its mechanism of action, preclinical activity profile, tolerability, and pharmaceutical properties, the compound recently has entered clinical phase I breast cancer trials.

Biomarkers for assessment of pharmacologic activity for a vascular endothelial growth factor (VEGF) receptor inhibitor, PTK787/ZK 222584 (PTK/ZK): translation of biological activity in a mouse melanoma metastasis model to phase I studies in patients with advanced colorectal cancer with liver metastases.

PTK/ZK is a novel, oral angiogenesis inhibitor that specifically targets all 3 vascular endothelial growth factor (VEGF) receptor tyrosine kinases and is currently in phase III clinical trials. In early clinical trials, PTK/ZK demonstrated a dose-dependent reduction in tumor vascular parameters as measured by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and an acute increase in plasma VEGF levels. The reduction in tumor vascularity was significantly correlated with improved clinical outcome in patients with advanced colorectal cancer and liver metastases. To assess the predictive value of a mouse model of tumor metastases, comparisons were performed for the biological activity of PTK/ZK in the mouse model and in patients with liver metastases in the clinical phase I trials. An orthotopic, syngeneic mouse model was used: C57BL/6 mice injected in the ear with murine B16/BL6 melanoma cells which metastases to the cervical lymph-nodes. The primary tumor and spontaneous metastases express VEGF and VEGF receptors and respond to treatment with VEGFR tyrosine kinase inhibitors. PTK/ZK was administered orally, with assessments by DCE-MRI of the metastases and plasma VEGF taken predose and at 3 days posttreatment and efficacy determined at 7 days posttreatment. Dose-ranging studies in naive mice provided preclinical pharmacokinetic data, while two dose-escalation phase I studies provided clinical pharmacokinetic data. An exposure-response relationship was observed both for mouse metastases (measured as % tumor weight treated/control) and for human liver metastases (measured as % regression). In the B16/BL6 model, the active dose of 50 mg/kg PTK/ZK yielded 62.4 (+/- 16.0) h microM plasma exposure, which is comparable to the plasma area under the concentration time curve (AUC) achieved by the 1000 mg dose of PTK/ZK used in clinical trials. At this exposure level in clinical trials, DCE-MRI showed a reduction in the area under the enhancement curve (IAUC) to 47% of baseline. At a similar exposure in the PTK/ZK-treated mice, a reduction in IAUC to 75% of baseline was observed. Furthermore, at doses of 50 mg/kg PTK/ZK and above, an increase in plasma VEGF level 10 h after drug administration was observed in mice which was consistent with findings from the clinical trials. In conclusion, the preclinical pharmacodynamics of PTK/ZK correlate well with clinical activity in phase I trials over comparable exposures to the drug. Thus, data from this preclinical model proved to be consistent with and thus predictive of the biologic effects of PTK/ZK in phase I/II clinical trials.

Antitumor activity of imatinib mesylate in neuroblastoma xenografts.

Imatinib mesylate has antitumor activity in vitro and in vivo against neuroblastoma cell lines and xenografts characterized by a different expression of receptor tyrosine kinases. In this article, we report that imatinib tumor concentration can be independent of the administered dose and does not correlate with the antitumor effect. In xenografts, high-dose administration does not improve imatinib efficacy. In conclusion, there is no clear-cut correlation between the levels of expression for imatinib-responsive targets and the in vitro and in vivo sensitivity. This further suggests that in neuroblastoma the antitumor activity of imatinib may involve the inhibition of other tyrosine kinases and/or pathways.

PTK787/ZK222584, a tyrosine kinase inhibitor of vascular endothelial growth factor receptor, reduces uptake of the contrast agent GdDOTA by murine orthotopic B16/BL6 melanoma tumours and inhibits their growth in vivo.

Assessment of tumour vascularity may characterize malignancy as well as predict responsiveness to anti-angiogenic therapy. Non-invasive measurement of tumour perfusion and blood vessel permeability assessed as the transfer constant, K(trans), can be provided by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Using the orthotopic murine tumour model B16/BL6 melanoma, the small contrast agent GdDOTA (DOTAREM(R); Guerbet, Paris) was applied to assess the vascular transfer constant, K(trans), and interstitial leakage space, whereas intravascular iron oxide nanoparticles (Endorem(R); Guerbet, Paris) were used to detect relative tumour blood volume (rTBV), and in one experiment blood flow index (BFI). No correlations were observed between these four parameters (r(2) always <0.05). The B16/BL6 primary tumour and lymph-node cervical (neck) metastases produced high levels of the permeability/growth factor, VEGF. To probe the model, the novel VEGF receptor (VEGF-R) tyrosine kinase inhibitor, PTK787/ZK222584 (PTK/ZK) was tested for anti-tumour efficacy and its effects on DCE-MRI measured parameters of tumour vascularity. Data from the non-invasive measure of tumour vascularity were compared with a histological measurement of vasculature using the DNA-staining dye H33342. PTK/ZK inhibited growth of the primary and, particularly, cervical tumour metastases following chronic treatment for 2 weeks (50 or 100 mg/kg daily) of 1-week-old tumours, or with 1 week of treatment against more established (2-week-old) tumours. After chronic treatment with PTK/ZK, DCE-MRI detected significant decreases in K(trans) and interstitial leakage space, but not rTBV of both primary tumours and cervical metastases. Histological data at this time-point showed a significant decrease in blood vessel density of the cervical metastases but not the primary tumours. However, in the cervical metastases, the mean blood vessel width was increased by 38%, suggesting overall no marked change in blood volume. After acute (2-4 day) treatment, DCE-MRI of the cervical metastases demonstrated a significant decrease in K(trans) and interstitial leakage space and also in the initial area under the enhancement curve for GdDOTA (IAUC), but no change in the rTBV or BFI. Thus, significant changes could be detected in the DCE-MRI measurement of tumour uptake of a small contrast agent prior to changes in tumour size, which suggests that DCE-MRI could be applied in the clinic as a rapid and sensitive biomarker for the effects of VEGF-R inhibition on tumour blood vessel permeability and thus may provide an early marker for eventual tumour response.

AEE788: a dual family epidermal growth factor receptor/ErbB2 and vascular endothelial growth factor receptor tyrosine kinase inhibitor with antitumor and antiangiogenic activity.

Aberrant epidermal growth factor receptor (EGFR) and ErbB2 expression are associated with advanced disease and poor patient prognosis in many tumor types (breast, lung, ovarian, prostate, glioma, gastric, and squamous carcinoma of head and neck). In addition, a constitutively active EGFR type III deletion mutant has been identified in non-small cell lung cancer, glioblastomas, and breast tumors. Hence, members of the EGFR family are viewed as promising therapeutic targets in the fight against cancer. In a similar vein, vascular endothelial growth factor (VEGF) receptor kinases are also promising targets in terms of an antiangiogenic treatment strategy. AEE788, obtained by optimization of the 7H-pyrrolo[2,3-d]pyrimidine lead scaffold, is a potent combined inhibitor of both epidermal growth factor (EGF) and VEGF receptor tyrosine kinase family members on the isolated enzyme level and in cellular systems. At the enzyme level, AEE788 inhibited EGFR and VEGF receptor tyrosine kinases in the nm range (IC(50)s: EGFR 2 nm, ErbB2 6 nm, KDR 77 nm, and Flt-1 59 nm). In cells, growth factor-induced EGFR and ErbB2 phosphorylation was also efficiently inhibited (IC(50)s: 11 and 220 nm, respectively). AEE788 demonstrated antiproliferative activity against a range of EGFR and ErbB2-overexpressing cell lines (including EGFRvIII-dependent lines) and inhibited the proliferation of epidermal growth factor- and VEGF-stimulated human umbilical vein endothelial cells. These properties, combined with a favorable pharmacokinetic profile, were associated with a potent antitumor activity in a number of animal models of cancer, including tumors that overexpress EGFR and or ErbB2. Oral administration of AEE788 to tumor-bearing mice resulted in high and persistent compound levels in tumor tissue. Moreover, AEE788 efficiently inhibited growth factor-induced EGFR and ErbB2 phosphorylation in tumors for >72 h, a phenomenon correlating with the antitumor efficacy of intermittent treatment schedules. Strikingly, AEE788 also inhibited VEGF-induced angiogenesis in a murine implant model. Antiangiogenic activity was also apparent by measurement of tumor vascular permeability and interstitial leakage space using dynamic contrast enhanced magnetic resonance imaging methodology. Taken together, these data indicate that AEE788 has potential as an anticancer agent targeting deregulated tumor cell proliferation as well as angiogenic parameters. Consequently, AEE788 is currently in Phase I clinical trials in oncology.

In vivo antitumor activity of NVP-AEW541-A novel, potent, and selective inhibitor of the IGF-IR kinase.

IGF-IR-mediated signaling promotes survival, anchorage-independent growth, and oncogenic transformation, as well as tumor growth and metastasis formation in vivo. NVP-AEW541 is a pyrrolo[2,3-d]pyrimidine derivative small molecular weight kinase inhibitor of the IGF-IR, capable of distinguishing between the IGF-IR (IC50 = 0.086 microM) and the closely related InsR (IC50 = 2.3 microM) in cells. As expected for a specific IGF-IR kinase inhibitor, NVP-AEW541 abrogates IGF-I-mediated survival and colony formation in soft agar at concentrations that are consistent with inhibition of IGF-IR autophosphorylation. In vivo, this orally bioavailable compound inhibits IGF-IR signaling in tumor xenografts and significantly reduces the growth of IGF-IR-driven fibrosarcomas. Thus, NVP-AEW541 represents a class of selective, small molecule IGF-IR kinase inhibitors with proven in vivo antitumor activity and potential therapeutic application.