Markus Wartmann

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.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 (IC50s: 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 (IC50s: 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.

The biology and medicinal chemistry of epothilones.

Epothilones are naturally occurring 16-membered macrolides with the ability to promote tubulin polymerization in vitro and to stabilize preformed microtubules against Ca(2+)- or cold-induced depolymerization. At the cellular level, interference with microtubule functionality results in potent inhibition of cancer cell proliferation at nM to even sub-nM concentrations. Most significantly, epothilones, unlike paclitaxel (Taxol), are equally active against drug-sensitive and multidrug-resistant cell lines in vitro and epothilone B has also shown potent in vivo antitumor activity in Taxol-resistant human tumor models. Epothilone B is currently undergoing Novartis-sponsored Phase II clinical trials. In addition to naturally occurring epothilones, numerous synthetic and semi-synthetic analogs have been prepared since the absolute stereochemistry of epothilone B was first disclosed in mid-1996 and their in vitro biological activity has been determined. These studies have generated a wealth of SAR data in a remarkably short period of time, given the complexity of the synthetic targets pursued. One of these analogs, BMS-247550, is presently in Phase II clinical trials by Bristol-Myers Squibb. In a first part this review is intended to provide a summary of the basic features of the in vitro biological profile of epothilones A and B, including emerging data on potential cellular epothilone resistance mechanisms. The second and third part will feature a comprehensive discussion of the epothilone SAR as it has emerged from the work of various (industrial and academic) laboratories across the world, including our own, with regard to effects on tubulin polymerization, in vitro antiproliferative activity, and in vivo antitumor activity.

Extended kinase profile and properties of the protein kinase inhibitor nilotinib

As a drug used to treat imatinib-resistant and -intolerant, chronic and advanced phase chronic myelogenous leukaemia, nilotinib is well characterised as a potent inhibitor of the Abl tyrosine kinase activity of wild-type and imatinib-resistant mutant forms of BCR-Abl. Here we review the profile of nilotinib as a protein kinase inhibitor. Although an ATP-competitive inhibitor of Abl, nilotinib binds to a catalytically inactive conformation (DFG-out) of the activation loop. As a consequence of this, nilotinib exhibits time-dependent inhibition of Abl kinase in enzymatic assays, which can be extrapolated to other targets to explain differences between biochemical activity and cellular assays. Although these differences confound assessment of kinase selectivity, as assessed using a combination of protein binding and transphosphorylation assays, together with cellular autophosporylation and proliferation assays, well established kinase targets of nilotinib in rank order of inhibitory potency are DDR-1>DDR-2>BCR-Abl (Abl)>PDGFRalpha/beta>KIT>CSF-1R. In addition nilotinib has now been found to bind to both MAPK11 (p38beta) and MAPK12 (p38alpha), as well as with very high affinity to ZAK kinase. Although neither enzymatic nor cellular data are yet available to substantiate the drug as an inhibitor of ZAK phosphorylation, modeling predicts that it binds in an ATP-competitive fashion.

Structural resemblances and comparisons of the relative pharmacological properties of imatinib and nilotinib

Although orphan drug applications required by the EMEA must include assessments of similarity to pre-existing products, these can be difficult to quantify. Here we illustrate a paradigm in comparing nilotinib to the prototype kinase inhibitor imatinib, and equate the degree of structural similarity to differences in properties. Nilotinib was discovered following re-engineering of imatinib, employing structural biology and medicinal chemistry strategies to optimise cellular potency and selectivity towards BCR-ABL1. Through evolving only to conserve these properties, this resulted in significant structural differences between nilotinib and imatinib, quantified by a Daylight-fingerprint-Tanimoto similarity coefficient of 0.6, with the meaning of this absolute measure being supported by an analysis of similarity distributions of similar drug-like molecules. This dissimilarity is reflected in the drugs having substantially different preclinical pharmacology and a lack of cross-intolerance in CML patients, which translates into nilotinib being an efficacious treatment for CML, with a favourable side-effect profile.

Chemical synthesis and biological properties of pyridine epothilones

BACKGROUND Numerous analogs of the antitumor agents epothilones A and B have been synthesized in search of better pharmacological profiles. Insights into the structure-activity relationships within the epothilone family are still needed and more potent and selective analogs of these compounds are in demand, both as biological tools and as chemotherapeutic agents, especially against drug-resistant tumors. RESULTS A series of pyridine epothilone B analogs were designed, synthesized and screened. The synthesized compounds exhibited varying degrees of tubulin polymerization and cytotoxicity properties against a number of human cancer cell lines depending on the location of the nitrogen atom and the methyl substituent within the pyridine nucleus. CONCLUSIONS The biological screening results in this study established the importance of the nitrogen atom at the ortho position as well as the beneficial effect of a methyl substituent at the 4- or 5-position of the pyridine ring. Two pyridine epothilone B analogs (i.e. compounds 3 and 4) possessing higher potencies against drug-resistant tumor cells than epothilone B, the most powerful of the naturally occurring epothilones, were identified.

The Ret receptor tyrosine kinase pathway functionally interacts with the ERalpha pathway in breast cancer.

A limited number of receptor tyrosine kinases (e.g., ErbB and fibroblast growth factor receptor families) have been genetically linked to breast cancer development. Here, we investigated the contribution of the Ret receptor tyrosine kinase to breast tumor biology. Ret was expressed in primary breast tumors and cell lines. In estrogen receptor (ER)alpha-positive MCF7 and T47D lines, the ligand (glial-derived neurotrophic factor) activated signaling pathways and increased anchorage-independent proliferation in a Ret-dependent manner, showing that Ret signaling is functional in breast tumor cells. Ret expression was induced by estrogens and Ret signaling enhanced estrogen-driven proliferation, highlighting the functional interaction of Ret and ER pathways. Furthermore, Ret was detected in primary cancers, and there were higher Ret levels in ERalpha-positive tumors. In summary, we showed that Ret is a novel proliferative pathway interacting with ER signaling in vitro. Expression of Ret in primary breast tumors suggests that Ret might be a novel therapeutic target in breast cancer.

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 JAK2V617F 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 JAK2V617F 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 JAK2V617F-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 JAK2V617F cell-driven mouse mechanistic model. Furthermore, NVP-BSK805 potently suppressed recombinant human erythropoietin-induced polycythemia and extramedullary erythropoiesis in mice and rats. Mol Cancer Ther; 9(7); 1945–55. ©2010 AACR.

A drug resistance screen using a selective MET inhibitor reveals a spectrum of mutations that partially overlap with activating mutations found in cancer patients

The emergence of drug resistance is a primary concern in any cancer treatment, including with targeted kinase inhibitors as exemplified by the appearance of Bcr-Abl point mutations in chronic myeloid leukemia (CML) patients treated with imatinib. In vitro approaches to identify resistance mutations in Bcr-Abl have yielded mutation spectra that faithfully recapitulated clinical observations. To predict resistance mutations in the receptor tyrosine kinase MET that could emerge during inhibitor treatment in patients, we conducted a resistance screen in BaF3 TPR-MET cells using the novel selective MET inhibitor NVP-BVU972. The observed spectrum of mutations in resistant cells was dominated by substitutions of tyrosine 1230 but also included other missense mutations and partially overlapped with activating MET mutations that were previously described in cancer patients. Cocrystallization of the MET kinase domain in complex with NVP-BVU972 revealed a key role for Y1230 in binding of NVP-BVU972, as previously reported for multiple other selective MET inhibitors. A second resistance screen in the same format with the MET inhibitor AMG 458 yielded a distinct spectrum of mutations rich in F1200 alterations, which is consistent with a different predicted binding mode. Our findings suggest that amino acid substitutions in the MET kinase domain of cancer patients need to be carefully monitored before and during treatment with MET inhibitors, as resistance may preexist or emerge. Compounds binding in the same manner as NVP-BVU972 might be particularly susceptible to the development of resistance through mutations in Y1230, a condition that may be addressed by MET inhibitors with alternative binding modes.

Patupilone Induced Vascular Disruption in Orthotopic Rodent Tumor Models Detected by Magnetic Resonance Imaging and Interstitial Fluid Pressure

Purpose: Evaluation of vascular disruptive activity in orthotopic models as potential surrogate biomarkers of tumor response to the microtubule-stabilizing agent patupilone. Experimental Design: Mice bearing metastatic B16/BL6 melanoma and rats bearing mammary BN472 tumors received vehicle or efficacious patupilone doses (4 and 0.8-1.5 mg/kg i.v., respectively). Tumor vascularity assessment by dynamic contrast-enhanced or dynamic susceptibility contrast magnetic resonance imaging and interstitial fluid pressure (IFP) occurred at baseline, 2 days (mice and rats), and 6 days (rats) after treatment and were compared with histologic measurements and correlated with tumor response. Results: In B16/BL6 metastases, patupilone (4 mg/kg) induced a 21 ± 5% decrease (P < 0.001) in tumor blood volume and a 32 ± 15% decrease (P = 0.02) in IFP after 2 days and reduced tumor growth and vessel density (>42%) after 2 weeks (P ≤ 0.014). Patupilone dose-dependently inhibited BN472 tumor growth (day 6) and reduced IFP on days 2 and 6 (−21% to −70%), and the percentage change in IFP correlated (P < 0.01) with the change in tumor volume. In both models, histology and vascular casts confirmed decreases in tumor blood volume. One patupilone (0.8 mg/kg) administration decreased (P < 0.01) tumor IFP (54 ± 4%), tumor blood volume (50 ± 6%), and vessel diameter (40 ± 11%) by day 6 but not the apparent diffusion coefficient, whereas histology showed that apoptosis was increased 2.4-fold and necrosis was unchanged. Apoptosis correlated negatively (P < 0.001) with IFP, tumor blood volume, and tumor volume, whereas tumor blood volume and IFP were correlated positively (P = 0.0005). Conclusions: Vascular disruptive effects of patupilone were detected in situ using dynamic contrast-enhanced or dynamic susceptibility contrast magnetic resonance imaging and IFP. Changes in IFP preceded and correlated with tumor response, suggesting that IFP may be a surrogate biomarker for patupilone efficacy.

EPO906 (epothilone B): a promising novel microtubule stabilizer.

EPO906 (epothilone B) is a potent member of a new class of microtubule-stabilizing cytotoxic agents known as epothilones. Although structurally unrelated to the clinically validated taxanes, EPO906 acts similarly to promote the formation and stabilization of microtubules, arresting proliferating cells in mitosis, and eventually causing cell demise by apoptosis. In preclinical studies, EPO906 has shown anticancer activity both in vitro and in vivo against several cancer types, including models that are paclitaxel-resistant. Importantly, in contrast to the taxanes, EPO906 retained activity against cancer cells either overexpressing the P-glycoprotein efflux pump or bearing tubulin mutations. Two phase I studies with EPO906 were conducted to determine the safety and maximal tolerated dose on two different dosing schedules: weekly and every 3 weeks. Diarrhea was the dose-limiting toxicity on both schedules. Tumor responses were seen in colorectal cancer as well as a variety of other tumor types, such as breast, ovarian, lung, and carcinoid in these two phase I trials. Based on the promising results from phase I studies, phase II studies in numerous indications are ongoing.