Terence O'Reilly

Protein kinases as targets for anticancer agents: from inhibitors to useful drugs

Many components of mitogenic signaling pathways in normal and neoplastic cells have been identified, including the large family of protein kinases, which function as components of signal transduction pathways, playing a central role in diverse biological processes, such as control of cell growth, metabolism, differentiation, and apoptosis. The development of selective protein kinase inhibitors that can block or modulate diseases caused by abnormalities in these signaling pathways is widely considered a promising approach for drug development. Because of their deregulation in human cancers, protein kinases, such as Bcr-Abl, those in the epidermal growth factor-receptor (HER) family, the cell cycle regulating kinases such as the cyclin-dependent kinases, as well as the vascular endothelial growth factor-receptor kinases involved in the neo-vascularization of tumors, are among the protein kinases considered as prime targets for the development of selective inhibitors. These drug-discovery efforts have generated inhibitors and low-molecular weight therapeutics directed against the ATP-binding site of various protein kinases that are in various stages of development (up to Phase II/III clinical trials). Three examples of inhibitors of protein kinases are reviewed, including low-molecular weight compounds targeting the cell cycle kinases; a potent and selective inhibitor of the HER1/HER2 receptor tyrosine kinase, the pyrollopyrimidine PKI166; and the 2-phenyl-aminopyrimidine STI571 (Glivec(R), Gleevec) a targeted drug therapy directed toward Bcr-Abl, the key player in chronic leukemia (CML). Some members of the HER family of receptor tyrosine kinases, in particular HER1 and HER2, have been found to be overexpressed in a variety of human tumors, suggesting that inhibition of HER signaling would be a viable antiproliferative strategy. The pyrrolo-pyrimidine PKI166 was developed as an HER1/HER2 inhibitor with potent in vitro antiproliferative and in vivo antitumor activity. Based upon its clear association with disease, the Bcr-Abl tyrosine kinase in CML represents the ideal target to validate the clinical utility of protein kinase inhibitors as therapeutic agents. In a preclinical model, STI571 (Glivec(R), Gleevec) showed potent in vitro and in vivo antitumor activity that was selective for Abl, c-Kit, and the platelet-derived growth factor-receptor. Phase I/II studies demonstrated that STI571 is well tolerated, and that it showed promising hematological and cytogenetic responses in CML and clinical responses in the c-Kit-driven gastrointestinal tumors.

Dual Inhibition of mTOR and Estrogen Receptor Signaling In vitro Induces Cell Death in Models of Breast Cancer

Purpose: RAD001 (everolimus), a mammalian target of rapamycin (mTOR) pathway inhibitor in phase II clinical trials in oncology, exerts potent antiproliferative/antitumor activities. Many breast cancers are dependent for proliferation on estrogens synthesized from androgens (i.e., androstenedione) by aromatase. Letrozole (Femara) is an aromatase inhibitor used for treatment of postmenopausal women with hormone-dependent breast cancers. The role of the mTOR pathway in estrogen-driven proliferation and effects of combining RAD001 and letrozole were examined in vitro in two breast cancer models. Experimental Design: The role of the mTOR pathway in estrogen response was evaluated in aromatase-expressing MCF7/Aro breast cancer cells by immunoblotting. Effects of RAD001 and letrozole (alone and in combination) on the proliferation and survival of MCF7/Aro and T47D/Aro cells were evaluated using proliferation assays, flow cytometry, immunoblotting, and apoptosis analyses. Results: Treatment of MCF7/Aro cells with estradiol or androstenedione caused modulation of the mTOR pathway, a phenomenon reversed by letrozole or RAD001. In MCF7/Aro and T47D/Aro cells, both agents inhibited androstenedione-induced proliferation; however, in combination, this was significantly augmented (P < 0.001, two-way ANOVA, synergy by isobologram analysis). Increased activity of the combination correlated with more profound effects on G1 progression and a significant decrease in cell viability (P < 0.01, two-way ANOVA) defined as apoptosis (P < 0.05, Friedman test). Increased cell death was particularly evident with optimal drug concentrations. Conclusion: mTOR signaling is required for estrogen-induced breast tumor cell proliferation. Moreover, RAD001-letrozole combinations can act in a synergistic manner to inhibit proliferation and trigger apoptotic cell death. This combination holds promise for the treatment of hormone-dependent breast cancers.

Identifying Optimal Biologic Doses of Everolimus (RAD001) in Patients With Cancer Based on the Modeling of Preclinical and Clinical Pharmacokinetic and Pharmacodynamic Data

PURPOSEnTo use preclinical and clinical pharmacokinetic (PK)/pharmacodynamic (PD) modeling to predict optimal clinical regimens of everolimus, a novel oral mammalian target of rapamycin (mTOR) inhibitor, to carry forward to expanded phase I with tumor biopsy studies in cancer patients.nnnPATIENTS AND METHODSnInhibition of S6 kinase 1 (S6K1), a molecular marker of mTOR signaling, was selected for PD analysis in peripheral blood mononuclear cells (PBMCs) in a phase I clinical trial. PK and PD were measured up to 11 days after the fourth weekly dose. A PK/PD model was used to describe the relationship between everolimus concentrations and S6K1 inhibition in PBMCs of cancer patients and in PBMCs and tumors of everolimus-treated CA20948 pancreatic tumor-bearing rats.nnnRESULTSnTime- and dose-dependent S6K1 inhibition was demonstrated in human PBMCs. In the rat model, a relationship was shown between S6K1 inhibition in tumors or PBMCs and antitumor effect. This allowed development of a direct-link PK/PD model that predicted PBMC S6K1 inhibition-time profiles in patients. Comparison of rat and human profiles simulated by the model suggested that a weekly 20- to 30-mg dose of everolimus would be associated with an antitumor effect in an everolimus-sensitive tumor and that daily administration would exert a greater effect than weekly administration at higher doses.nnnCONCLUSIONnA direct-link PK/PD model predicting the time course of S6K1 inhibition during weekly and daily everolimus administration allowed extrapolation from preclinical studies and first clinical results to select optimal doses and regimens of everolimus to explore in future clinical trials.

Comprehensive mapping of p53 pathway alterations reveals an apparent role for both SNP309 and MDM2 amplification in sarcomagenesis

Purpose: Reactivation of p53 tumor suppressor activity in diseases such as soft-tissue sarcoma is considered an attractive means of targeted therapy. By systematically assessing alterations affecting the p53 pathway, we aimed to (a) classify sarcoma subtypes, (b) define a potential role in malignancy, and (c) identify potential patient biomarkers in this heterogeneous disease. Experimental Design: We have mapped mutational events in a panel of 192 benign or malignant bone and soft-tissue sarcomas. Analyses included TP53 and CDKN2A mutational and SNP status, MDM2 and MDM4 amplification and MDM2 SNP309 status. Results: We found an inverse relationship between MDM2 amplification and TP53 mutations, with a predominantly wild-type CDKN2A background. A high rate of point mutations in TP53 was observed uniquely in leiomyosarcoma, osteosarcoma, and MFH. Both MDM2 and MDM4 were also amplified in a subtype-specific manner, which was frequently seen as a coamplification event. We have also analyzed the risk allele frequencies for MDM2 SNP309, and show that the G allele was strongly associated with both liposarcomas and MDM2 amplification. Conclusions: Our data emphasize the critical role of p53 inactivation in sarcomagenesis, whereby different pathway alterations may be related to the heterogeneity of the disease. Moreover, we observed a strong association of malignancy with TP53 mutation, or MDM2 amplification and the presence of a G allele in SNP309, especially in lipoma versus liposarcoma. We propose, therefore, that MDM2 markers along with TP53 sequencing should be considered as patient biomarkers in clinical trials of sarcomas using MDM2 antagonists. Clin Cancer Res; 17(3); 416–26. ©2010 AACR.

Systemic Neutralization of Interleukin-8 Markedly Reduces Neutrophilic Pleocytosis during Experimental Lipopolysaccharide-Induced Meningitis in Rabbits

ABSTRACT Interleukin-8 (IL-8) is elevated in the cerebrospinal fluid (CSF) of patients with meningitis and is proposed to participate in subarachnoid-space pleocytosis. However, intracisternal injection of IL-8 into rabbits failed to induce indices typical of meningitis (leukocyte, tumor necrosis factor, or protein accumulation in the CSF or histopathological changes), indicating that merely increasing the CSF level of this chemokine is insufficient to induce inflammation in this anatomical site. IL-8 treatment did not affect inflammatory responses to subsequently intracisternally administered lipopolysaccharide (LPS). IL-8 was chemotactic for rabbit neutrophils in vitro, and subcutaneous injection of IL-8 (diluted in buffer or CSF) proved the in vivo activity of this peptide and suggested the absence of an IL-8 inhibitor in normal rabbit CSF. LPS-dependent pleocytosis was only slightly diminished by intracisternally administered murine anti-rabbit IL-8 monoclonal antibody (MAb) WS-4 but was dramatically reduced by intravenously administered MAb. Therefore, elevated CSF IL-8 levels may contribute to, but cannot solely account for, neutrophil influx into the subarachnoid space during meningitis. However, inhibition of IL-8 activity of the bloodstream side of the blood-brain barrier effectively reduces pleocytosis, indicating a central role of IL-8 in neutrophil influx into CSF during bacterial meningitis. Thus, inhibition of IL-8 is a possible therapeutic target for adjunct treatment of meningitis.

Research and innovation in the development of everolimus for oncology.

Introduction: The critical role of increased activity of mammalian target of rapamycin (mTOR) in the pathophysiology of multiple diseases is well established. Inhibition of the mTOR pathway may block disease progression and improve patient outcomes. Everolimus, an mTOR inhibitor, began in clinical development as part of a regimen (Certican, Zortress) for prevention of organ transplant rejection and is now an approved oncology agent. Areas covered: The objective of this review is to discuss the history of key findings and innovative cancer research undertaken to successfully develop everolimus as an oncology therapy (Afinitor) now approved for patients with advanced renal cell carcinoma (RCC) and for subependymal giant cell astrocytomas (SEGAs) associated with tuberous sclerosis. In addition, data for the use of everolimus in the treatment of other cancers and rare diseases are also discussed. A PubMed search of English articles without time restrictions was conducted using the search terms ‘everolimus or rapamycin’ and ‘cancer’. Bibliographies of retrieved articles were manually searched for additional relevant articles. Major cancer congresses were also searched. Expert opinion: The clinical efficacy of everolimus alone and in combination with other agents has been observed in recently completed Phase II–III studies in a wide spectrum of tumors, including RCC, neuroendocrine tumors, tuberous sclerosis complex, SEGAs and angiomyolipomas, lymphoma and gastric, breast and hepatocellular cancers. These findings emphasize the importance of mTOR in diverse cancers and rare diseases and underscore the potential role for everolimus as an effective agent in multiple indications.

Influence of the blood bacterial load on the meningeal inflammatory response in Streptococcus pneumoniae meningitis.

BackgroundDespite bacteraemia is present in the majority of patients with pneumococcal, little is known about the influence of the systemic infection on the meningeal inflammatory response.MethodsTo explore the role of systemic infection on the meningeal inflammation, experimental meningitis was induced by intracisternal injection of ~1 × 106 CFU Streptococcus pneumoniae, type 3, and the 26 rabbits were either provided with ~1 × 106 CFU S. pneumoniae intravenously at 0 hour (bacteraemic rabbits, n = 9), immunized with paraformaldehyde-killed S. pneumoniae for 5 weeks prior to the experiment (immunized rabbits, n = 8), or not treated further (control rabbits, n = 9). WBC and bacterial concentrations were determined in CSF and blood every second hour during a 16 hours study period together with CSF IL-8 and protein levels. We also studied CSF and blood WBC levels in 153 pneumococcal meningitis patients with and without presence of bacteraemia.ResultsAs designed, blood bacterial concentrations were significantly different among three experimental groups during the 16 hours study period (Kruskal Wallis test, P < 0.05), whereas no differences in CSF bacterial levels were observed (P > 0.05). Blood WBC decreased in bacteraemic rabbits between ~10–16 hours after the bacterial inoculation in contrast to an increase for both the immunized rabbits and controls (P < 0.05). The CSF pleocytosis was attenuated in bacteraemic rabbits as compared to the two other groups between 12–16 hours from time of infection (P < 0.017), despite accelerated CSF IL-8 levels in bacteraemic rabbits.In patients with pneumococcal meningitis, no significant difference in CSF WBC was observed between patients with or without bacteraemia at admission (n = 103, 1740 cells/μL (123–4032) vs. n = 50, 1961 cells/μL (673–5182), respectively, P = 0.18), but there was a significant correlation between CSF and blood WBC (n = 127, Spearman rho = 0.234, P = 0.008).ConclusionOur results suggest that a decrease in peripheral WBC induced by enhanced bacteraemia in pneumococcal meningitis results in an attenuated CSF pleocytosis.

Animal models as predictors of the safety and efficacy of antibiotics

As opposed to the testing of safety, the testing of the efficacy of antibiotics in animals is not specified in any directives or guidelines and not explicitly required by regulatory authorities. There exists, however, no doubt that in the evaluation of new compounds testing of both safety and efficacy forms an essential link between in vitro tests and clinical trials. It is inconceivable that clinicians would be prepared to conduct a trial in patients without evidence of the efficacy of the antibiotic in question in an appropriate animal model of infection. Both the models for testing safety and those for testing efficacy suffer from a number of shortcomings. If investigators are aware of these deficiencies and take them into account when interpreting the results, the predictive value of the models can be significantly enhanced.

The natural products epothilones A and B as lead structures for anticancer drug discovery: Chemistry, biology, and SAR studies

Publisher Summary This chapter outlines some of the structural variables that have been investigated as a part of research in laboratories. The chapter tries to provide some insight into what kind of structural changes of the epothilone structural framework may or may not be tolerated without affecting biological activity and to what extent. Epothilone research has produced at least four clinical development compounds, including epothilone B, epothilone B lactam, C21-amino epothilone B, and deoxyepothilone B. Although none of these compounds has yet successfully completed clinical development, they, nevertheless, attest to the great potential of epothilone-based agents to become clinically useful anticancer drugs.

Systemic inflammation alters the inflammatory response in experimental lipopolysaccharide-induced meningitis

Experiments to evaluate the effect of the level and duration of endotoxaemia on the meningeal inflammatory response were performed in order to determine if systemic inflammation alters meningitis. Rabbits received either saline or Escherichia coli O111:B4 lipopolysacharide (LPS) intravenously at various doses (1, 3 or 10u2003µg) and times (−8, −2 or 0u2003h) before an intracisternal injection of 20u2003ng LPS. An intracisternal LPS injection together with saline intravenously produced a peak cerebrospinal fluid (CSF) tumour necrosis factor (TNF) level (95u2003±u200326u2003ng/ml) at 2u2003h and peak leucocyte level (5413u2003±u2003764 cells/µl) at 4u2003h post‐injection. Blood leucocytes were slightly elevated (12u2003000u2003±u2003500/µl at 0u2003h; 16u2003900u2003±u2003280/µl at 8u2003h) but plasma TNF was always undetectable (<u20030·05u2003ng/ml). Conversely, intravenous injection of 3 or 10u2003µg LPS 2u2003h prior to intracisternal LPS injection impaired pleocytosis (peaku2003<u2003220 cells/µl) and delayed (∼4u2003h) and reduced peak CSF TNF levels (3u2003µg LPS 5·0u2003±u20031·2u2003ng/ml; 10u2003µg LPS 6·9u2003±u20031·9; Pu2003<u20030·05). Intravenous administration of 1u2003µg LPS was less inhibitory to CSF inflammation, but delayed onset (peak 1100u2003±u200360 leucocytes/µl CSF at 8u2003h; 6·3u2003±u20030·3u2003ng TNF/ml CSF at 4u2003h; both Pu2003<u20030·05). Neutropenia nadirs were dependent on LPS dose (1u2003µg, 4500u2003±u20031700; 3u2003µg, 1900u2003±u200360; 10u2003µg, 1100u2003±u2003100 all at 4u2003h post‐intravenous dose). Peak plasma TNF levels were not dose‐dependent (>u20038u2003ng/ml), but plasma TNF was always detectable (>u20030·2u2003ng/ml at 10u2003h post‐intravenous dose). Intravenous LPS administration at 0u2003h also blocked pleocytosis, but the inhibitory effect was lost when administration at −8u2003h. In conclusion, the degree and duration of endotoxaemia affect the meningeal inflammatory response to LPS in experimental meningitis.