BACKGROUND:Expression of the colony-stimulating factor 1 (CSF1) gene is elevated in most tenosynovial giant-cell tumors. This observation has led to the discovery and clinical development of therapy targeting the CSF1 receptor (CSF1R).METHODS:Using x-ray co-crystallography to guide our drug-discovery research, we generated a potent, selective CSF1R inhibitor, PLX3397, that traps the kinase in the autoinhibited conformation. We then conducted a multicenter, phase 1 trial in two parts to analyze this compound. In the first part, we evaluated escalations in the dose of PLX3397 that was administered orally in patients with solid tumors (dose-escalation study). In the second part, we evaluated PLX3397 at the chosen phase 2 dose in an extension cohort of patients with tenosynovial giant-cell tumors (extension study). Pharmacokinetic and tumor responses in the enrolled patients were assessed, and CSF1 in situ hybridization was performed to confirm the mechanism of action of PLX3397 and that the pattern of CSF1 expression was consistent with the pathological features of tenosynovial giant-cell tumor.RESULTS:A total of 41 patients were enrolled in the dose-escalation study, and an additional 23 patients were enrolled in the extension study. The chosen phase 2 dose of PLX3397 was 1000 mg per day. In the extension study, 12 patients with tenosynovial giant-cell tumors had a partial response and 7 patients had stable disease. Responses usually occurred within the first 4 months of treatment, and the median duration of response exceeded 8 months. The most common adverse events included fatigue, change in hair color, nausea, dysgeusia, and periorbital edema; adverse events rarely led to discontinuation of treatment.CONCLUSIONS:Treatment of tenosynovial giant-cell tumors with PLX3397 resulted in a prolonged regression in tumor volume in most patients. (Funded by Plexxikon; ClinicalTrials.gov number, NCT01004861.).

In the well-known settlement micro-region of the Małopolska Upland - known for extensive studies into the Neolithic agricultural anthropopressure - the authors have re-examined three selected Holocene soil-sediment sequences derived from soil erosion by water. The macroscopic features of both colluvium and proluvium (deposits of gully erosion) from an area of very strong Neolithic anthropopressure in the Nidzica River Basin have been described, and analyses of the basic characteristics of sediments and fossil soils as well as micro-morphological analyses performed. 40 samples were dated with the SAR OSL method. The results of 14C and OSL dating point to the stabilization of loess slopes in the Holocene until the intensification of agricultural activity in the Funnel Beaker Culture (TRB, Neolith, about 3900 cal BC). Apart from the Neolithic colluvium and proluvium, only colluvia accumulated during the last millennium were found. The presence of Bronze Age colluvium remains a possibility. OSL dating confirmed a strong link between the increase in prehistoric farming activity and the growing intensity of Holocene water erosion. It also revealed the presence of colluvial sequences of erosion gaps, with the geologic record obliterated by later pedogenic processes, and impossible to identify on the basis of classical macroscopic and micromorphological analyses. The study has also shown that the OSL method is an important tool in chronostratigraphy of the Holocene colluvia as long as it is supported by deep paleo-geographical analysis of the entire basin and knowledge of colonization history. This method is not suitable for dating gully erosion sediments - thus the age of the oldest Neolithic gully erosion deposits in Bronocice was determined on the basis of dating of the overlaying colluvia.

13056 Background: The discovery of oncogenic B-Raf mutations in a majority of patients with metastatic melanoma, and in many tumors from patients with colorectal cancer and other cancers, presents the opportunity to develop oncogene-selective inhibitors with a favorable safety profile. Methods: Guided by co-crystallography, a novel chemical scaffold has been developed into a series of potent inhibitors of oncogenic B-Raf with selectivity versus wild-type B-Raf. From this series, a potent inhibitor has been chosen as a candidate for development. Results: Consistent with the structure-guided approach, this compound shows pronounced selectivity versus a wide array of over 70 other kinases covering all branches of the kinome. This selectivity translates to a wide cellular therapeutic index: inhibition of proliferation of a panel of cell lines bearing the V600E oncogenic B-Raf mutation occurs at IC50s ranging from 40–400 nM while inhibition of cell lines lacking oncogenic B-Raf occurs at IC50s greater than 6500 nM. This cell-based selectivity for the oncogenic B-Raf is greater than the selectivity shown in biochemical assays, supporting that tumor cells bearing the oncogenic B-Raf protein are more dependent on the MAP kinase pathway. Combination experiments with a series of cytotoxic and targeted clinical anti-cancer agents reveal multiple examples of synergistic inhibition of proliferation in vitro, and this synergy generally appears selectively in oncogenic B-Raf-bearing cells. The good oral bioavailability (F > 70%) allows for prolonged exposure in both rodents and non-rodents. Robust efficacy is evident in a murine COLO205 tumor xenograft model, with once-daily oral dosing at 20 mg/kg over 14 days resulting in > 80% inhibition of tumor growth with no effect on body weight. Conclusions: Since this compound is highly selective and targets a B-Raf variant that is absent in all non-transformed cells, it may have a broad therapeutic index, alone or in combination, for the treatment of patients with V600E oncogenic B-Raf tumors. [Table: see text]

Chronically hypoxic rats (exposed to 5000 m elevation for 3 weeks) develop pulmonary hypertension (PH) that is reversed upon return to normoxia and is blocked by bradykinin (BK) antagonist B9430 treatment (100 microg/kg s.c. three times per week). Treatment of rats with both the synthetic VEGF receptor-1/2 antagonist 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-indolin-2-one (SU5416) (200 mg/kg, single s.c. injection) and hypoxia (3 weeks) causes irreversible severe PH characterized by marked elevation of pulmonary artery pressure (PAP), right ventricular hypertrophy, and obliteration of pulmonary arteries by proliferating endothelial cells (EC). Between weeks I and 2 of treatment, there is increased apoptotic EC death and caspase-3 activity. The combination of hypoxia with VEGFR-1 and -2 blockade appears to cause death of normal lung EC and proliferation of an apoptosis-resistant proliferating EC phenotype. Cotreatment with BK antagonist B9430 and (or) the broad caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp) (2 mg/kg three times per week) prevented development of severe PH and caused significant reduction of PAP: 39.7 +/- 4.6 mmHg in Z-Asp + SU5416, 37.1 +/- 1.2 mmHg in BK antagonist B9430 + SU5416, 27.2 +/- 0.7 mmHg in Z-Asp alone, and 36.6 +/- 3.0 mmHg in BK antagonist alone versus 48 +/- 1.7 mmHg in SU5416-treated rats and 32.8 +/-1.4 mmHg in vehicle-treated controls. The PAP correlated with the right ventricular mass. Pulmonary arteries of rats treated with Z-Asp and BK antagonist B9430 had a marked reduction of intravascular EC, yet there was still evidence of medial muscular hypertrophy, similar to that observed in chronically hypoxic rats not treated with SU5416. We conclude that EC death induced by VEGFR-2 blockade with SU5416 may trigger an EC selection process that allows for the expansion of apoptosis-resistant EC, possibly driven by mechanisms independent of VEGF and VEGFR-2.

Our understanding of the pathobiology of severe pulmonary hypertension, usually a fatal disease, has been hampered by the lack of information of its natural history. We have demonstrated that, in human severe pulmonary hypertension, the precapillary pulmonary arteries show occlusion by proliferated endothelial cells. Vascular endothelial growth factor (VEGF) and its receptor 2 (VEGFR-2) are involved in proper maintenance, differentiation, and function of endothelial cells. We demonstrate here that VEGFR-2 blockade with SU5416 in combination with chronic hypobaric hypoxia causes severe pulmonary hypertension associated with precapillary arterial occlusion by proliferating endothelial cells. Prior to and concomitant with the development of severe pulmonary hypertension, lungs of chronically hypoxic SU5416-treated rats show significant pulmonary endothelial cell death, as demonstrated by activated caspase 3 immunostaining and TUNEL. The broad caspase inhibitor Z-Asp-CH2-DCB prevents the development of intravascular pulmonary endothelial cell growth and severe pulmonary hypertension caused by the combination of SU5416 and chronic hypoxia.

Pulmonary emphysema, a significant global health problem, is characterized by a loss of alveolar structures. Because VEGF is a trophic factor required for the survival of endothelial cells and is abundantly expressed in the lung, we hypothesized that chronic blockade of VEGF receptors could induce alveolar cell apoptosis and emphysema. Chronic treatment of rats with the VEGF receptor blocker SU5416 led to enlargement of the air spaces, indicative of emphysema. The VEGF receptor inhibitor SU5416 induced alveolar septal cell apoptosis but did not inhibit lung cell proliferation. Viewed by angiography, SU5416-treated rat lungs showed a pruning of the pulmonary arterial tree, although we observed no lung infiltration by inflammatory cells or fibrosis. SU5416 treatment led to a decrease in lung expression of VEGF receptor 2 (VEGFR-2), phosphorylated VEGFR-2, and Akt-1 in the complex with VEGFR-2. Treatment with the caspase inhibitor Z-Asp-CH2-DCB prevented SU5416-induced septal cell apoptosis and emphysema development. These findings suggest that VEGF receptor signaling is required for maintenance of the alveolar structures and, further, that alveolar septal cell apoptosis contributes to the pathogenesis of emphysema.

To determine whether angiogenesis is necessary for normal alveolarization, we studied the effects of two antiangiogenic agents, thalidomide and fumagillin, on alveolarization during a critical period of lung growth in infant rats. Newborn rats were treated with daily injections of fumagillin, thalidomide, or vehicle during the first 2 wk of life. Compared with control treatment, fumagillin and thalidomide treatment reduced lung weight-to-body weight ratio and pulmonary arterial density by 20 and 36%, respectively, and reduced alveolarization by 22%. Because these drugs potentially have nonspecific effects on lung growth, we also studied the effects of Su-5416, an inhibitor of the vascular endothelial growth factor receptor known as kinase insert domain-containing receptor/fetal liver kinase (KDR/flk)-1. As observed with the other antiangiogenic agents, Su-5416 treatment decreased alveolarization and arterial density. We conclude that treatment with three different antiangiogenic agents attenuated lung vascular growth and reduced alveolarization in the infant rat. We speculate that angiogenesis is necessary for alveolarization during normal lung development and that injury to the developing pulmonary circulation during a critical period of lung growth can contribute to lung hypoplasia.

Vascular endothelial growth factor (VEGF) plays a fundamental role in mediating tumor angiogenesis and tumor growth. Here we investigate the direct effect of a novel small molecule inhibitor of the Flk-1-mediated signal transduction pathway of VEGF, SU5416, on tumor angiogenesis and microhemodynamics of an experimental glioblastoma by using intravital multifluorescence videomicroscopy. SU5416 treatment significantly suppressed tumor growth. In parallel, SU5416 demonstrated a potent antiangiogenic activity, resulting in a significant reduction of both the total and functional vascular density of the tumor microvasculature, which indicates an impaired vascularization as well as significant perfusion failure in treated tumors. This malperfusion was not compensated for by changes in vessel diameter or recruitment of nonperfused vessels. Analyses of the tumor microcirculation revealed significant microhemodynamic changes after angiogenesis blockage such as a higher red blood cell velocity and blood flow in remnant tumor vessels when compared with controls. Our results demonstrate that the novel antiangiogenic concept of targeting the tyrosine kinase of Flk-1/KDR by means of a small molecule inhibitor represents an efficient strategy to control growth and progression of angiogenesis-dependent tumors. This study provides insight into microvascular consequences of Flk-1/KDR targeting in vivo and may have important implications for the future treatment of angiogenesis-dependent neoplasms.