Nintedanib for the treatment of patients with refractory metastatic colorectal cancer (LUME-Colon 1): a phase III, international, randomized, placebo-controlled study.

Background: Angiogenesis is critical to colorectal cancer (CRC) growth and metastasis. Phase I/II studies have demonstrated the efficacy of nintedanib, a triple angiokinase inhibitor, in patients with metastatic CRC. This global, randomized, phase III study investigated the efficacy and safety of nintedanib in patients with refractory CRC after failure of standard therapies. Patients and methods: Eligible patients (Eastern Cooperative Oncology Group performance status 0-1, with histologically/cytologically confirmed metastatic/locally advanced CRC adenocarcinoma unamenable to surgery and/or radiotherapy) were randomized 1 : 1 to receive nintedanib (200 mg twice daily) or placebo (twice daily), until disease progression or undue toxicity. Patients were stratified by previous regorafenib, time from onset of metastatic disease to randomization, and region. Co-primary end points were overall survival (OS) and progression-free survival (PFS) by central review. Secondary end points included objective tumor response and disease control by central review. Results: From October 2014 to January 2016, 768 patients were randomized; 765 were treated (nintedanib n = 384; placebo n = 381). Median follow-up was 13.4 months (interquartile range 11.1-15.7). OS was not improved [median OS 6.4 months with nintedanib versus 6.0 months with placebo; hazard ratio (HR), 1.01; 95% confidence interval (CI), 0.86-1.19; P = 0.8659]. There was a significant but modest increase in PFS with nintedanib versus placebo (median PFS 1.5 versus 1.4 months, respectively; HR 0.58; 95% CI 0.49-0.69; P < 0.0001). There were no complete or partial responses. Adverse events (AEs) occurred in 97% of 384 nintedanib-treated patients and 93% of 381 placebo-treated patients. The most frequent grade ≥3 AEs were liver-related AEs (nintedanib 16%; placebo 8%) and fatigue (nintedanib 9%; placebo 6%). Conclusions: The study failed to meet both co-primary end points. Nintedanib did not improve OS and was associated with a significant but modest increase in PFS versus placebo. Nintedanib was well tolerated. ClinicalTrials.gov number: NCT02149108 (LUME-Colon 1).

A phase I/II, open-label, randomised study of nintedanib plus mFOLFOX6 versus bevacizumab plus mFOLFOX6 in first-line metastatic colorectal cancer patients.

BACKGROUND: This randomised, open-label, phase I/II study evaluated the efficacy and safety of nintedanib, an oral, triple angiokinase inhibitor, combined with chemotherapy, relative to bevacizumab plus chemotherapy as first-line therapy in patients with metastatic colorectal cancer (mCRC). PATIENTS AND METHODS: Patients with histologically confirmed mCRC (adenocarcinoma), an Eastern Cooperative Oncology Group performance status ≤ 2 and adequate organ function were included. Patients were randomised 2:1 to receive nintedanib 150 mg or 200 mg b.i.d. plus mFOLFOX6 (oxaliplatin 85 mg/m(2), l-leucovorin 200 mg/m(2) or d,l-leucovorin 400 mg/m(2), 5-fluoruracil bolus 400 mg/m(2) followed by 2400 mg/m(2), every 2 weeks) or bevacizumab (5 mg/kg every 2 weeks) plus mFOLFOX6. During phase I, patients underwent a 3 + 3 dose-escalation schema to determine the maximum tolerated dose (MTD) of nintedanib in combination with mFOLFOX6. The primary end point was progression-free survival (PFS) rate at 9 months. Objective response (OR) was a secondary end point. RESULTS: The nintedanib recommended phase II dose was 200 mg b.i.d. plus mFOLFOX6 based on safety data from phase I (n = 12). Of 128 patients randomised in the phase II part, 126 received treatment (nintedanib plus mFOLFOX6, n = 85; bevacizumab plus mFOLFOX6, n = 41). PFS at 9 months was 62.1% with nintedanib and 70.2% with bevacizumab [difference: -8.1% (95% confidence interval -27.8 to 11.5)]. Confirmed ORs were recorded in 63.5% and 56.1% of patients in the nintedanib and bevacizumab groups, respectively. The incidence of adverse events (AEs) considered related to treatment was 98.8% with nintedanib and 97.6% with bevacizumab; the incidence of serious AEs was 37.6% with nintedanib and 53.7% with bevacizumab. The pharmacokinetics of nintedanib and the components of mFOLFOX6 were unaffected by their combination. CONCLUSIONS: Nintedanib in combination with mFOLFOX6 showed efficacy as first-line therapy in patients with mCRC with a manageable safety profile and further studies in this population are warranted.

Matrix metalloproteinases MMP-2, -9 and tissue inhibitors TIMP-1, -2 expression and secretion by primary human osteoblast cells in response to titanium, zirconia, and alumina ceramics.

Osteogenic properties of bone cells are a key parameter governing osseointegration of implant devices. In this context, osteoblasts have a central role via extracellular matrix synthesis and remodeling that they regulate through different protease activity. In this study, we have analyzed the expression of two matrix metalloproteinases (MMPs): MMP-2 (72 kDa) and MMP-9 (92 kDa) and their specific tissue inhibitors TIMP-1 and TIMP-2 in primary human osteoblastic cells. The effect of titanium, zirconia, and alumina ceramics on the synthesis of these proteases was assessed using reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and zymographic analysis. Our results showed that osteoblasts express MMP-2 and -9 mRNA. Furthermore, MMP-2 mRNA expression was decreased by titanium and increased by alumina whereas zirconia did not have any significant effect. Conversely, MMP-9 mRNA expression was stimulated by titanium but decreased with zirconia, whereas alumina induced no significant changes. Zymographic analysis has evidenced pro-MMP-2 gelatinolytic activity in all cell populations with time-dependent increase profile; pro-MMP-9, however, was not detected. Enzyme-linked immunosorbent assay data confirmed the production of MMP-2 and very low levels of MMP-9. In addition, TIMP-1 was secreted in 24-h-cultured cells and increased to maximal level at 48-72 h whereas TIMP-2 levels were very low. The interactions between human osteoblasts and the studied biomaterials altered both MMP-2, -9 and TIMP-1expression indicating that biomaterials may influence osseointegration and bone remodeling.

In vitro reactions of human osteoblasts in culture with zirconia and alumina ceramics.

The biocompatibility of two implantable materials, zirconia and alumina ceramics, was investigated in vitro using human osteoblast cell cultures. The viability of osteoblast cells with the materials was evaluated by the methylthiazole sulfate test that revealed an absence of any cytostatic or cytotoxic effect. Cell proliferation kinetic and total protein synthesis in osteoblasts with zirconia or alumina were similar to that observed in control cells cultured on glass coverslips. Light and scanning electron microscopic examinations showed an intimate contact between osteoblasts and the substrates; well-spread cells were observed on the surfaces of both materials. Adhesion ability and morphological characteristics were preserved in osteoblast cultures with these substrates. Moreover, immunohistochemical staining in osteoblasts with zirconia and alumina showed the capacity of these cells to elaborate the extracellular matrix composed of types I and V collagen, osteocalcin, osteonectin, bone sialoprotein, and cellular fibronectin. Finally, DNA image cytometry and interphase silver-nucleolar organizer regions quantification were applied as complementary biocompatibility tests to detect any changes in DNA synthesis and cell proliferation, respectively. The results showed that neither material altered cell ploidy or cell growth rate in accordance with the absence of any inducing effect on DNA synthesis or proliferation.

DNA image cytometry and Ag-NORs-staining application in biocompatibility studies on human osteoblast cells in vitro.

We report here the study of the biocompatibility of a bone graft material, the Pyrost, using a previously established in vitro model of human osteoblasts. The effect of this material on cell proliferation was evaluated by the MTS assay. Results indicated the absolute absence of cytotoxic or cytostatic effect of Pyrost on cultured osteoblasts. Viability rate was more than 90% in cells cultured with the material compared to the control. Morphological analysis, undertaken by scanning electron microscopy showed a good adhesion and a spreading of osteoblasts in contact with the material that was colonized by cultured cells. In the second part of this work, we have introduced two methods as complementary biocompatibility tests: DNA image cytometry and interphase Ag-NORs quantification. DNA content was measured in cells cultured with or without Pyrost for 3, 9, 15 and 30 days. The determination of DNA indicated that the majority of osteoblasts population was diploid without aneuploidy. The DNA index and cell distribution profile in DNA histograms were similar in all cell populations. The Ag-NORs amount was used as a parameter for cell kinetic evaluation. We have measured the Ag-NORs index like DNA quantification. The proliferation rate, evaluated by Ag-NORs counts in osteoblasts cultured with or without the material, was identical. However, a decrease in Ag-NORs index was observed from day 3 to day 15 of incubation. These results showed a satisfactory biocompatibility of the Pyrost in human osteoblasts culture. The material did not alter cell viability and had no inducing effect either on proliferation rate or on cell ploidy as demonstrated by DNA image cytometry and Ag-NORs proteins staining.

Effects of verapamil and S9788 on MDR-1 mRNA expression studied by in situ hybridization.

The effects of Verapamil and S9788 (triazineaminopiperidine), a new modulator of multidrug resistance, on mdr-1 mRNA expression were determined using in situ hybridization. S9788 appeared to be a better reversal drug by strongly decreasing drug efflux. However, Verapamil only decreased mdr-1 mRNA expression in Human erythroleukemic resistant cells (ADM/k562) and in Human lymphoblastic resistant cells (VLB/CEM). This effect occurred in all Verapamil-treated resistant cells with no difference between cell subpopulations. These results were confirmed by slot-blot experiments. In conclusion, S9788 and verapamil effects are comparable overall although the mechanisms responsible for this resistance modulation are not strictly identical.

Nuclear DNA content and chromatin texture in multidrug-resistant human leukemic cell lines.

Nuclear morphological alterations associated with multidrug resistance (MDR) were evaluated by image cytometry in various human leukemic cell sub-lines: 3 cell lines with P-gp-mediated resistance (CEM-VLB, HL60/Vinc, K562-Dox), the non-Pgp-mediated MDR HL60/AR leukemic cell line with over-expression of MRP, and the at-MDR CEM-VMI leukemic cell line with alteration of topoisomerase II. All these MDR cell sub-lines were obtained by drug selection and were compared with their sensitive counterparts and with the hamster LR73-R cell line obtained by transfection of mouse mdrl cDNA. All MDR cell sub-lines obtained by drug selection displayed decreased DNA Feulgen stainability as compared with their respective sensitive parental cell line, a phenomenon not observed in the transfected LR73-R cells. Nuclear texture analysis on G0/G1-selected cell nuclei revealed 2 types of textural phenotype. The first phenotype was characterized by chromatin decondensation with small but compact chromatin clumps, and was observed in drug-selected P-gp-mediated MDR cells (CEM-VLB, HL60-Vinc, K562-Dox) and in the non-P-gp-mediated MDR HL60/AR cell line. The second phenotype was characterized by a condensed and homogeneous chromatin pattern, and was observed in the at-MDR CEM-VMI cell line. LR73-R cells transfected with mdrl cDNA did not display any significant changes in textural phenotype as compared with sensitive LR73 cells, suggesting that P-gp over-expression alone cannot account for the cytological modifications observed in MDR cells. These data suggest that multidrug resistance could be associated with specific nuclear morphological changes which appeared to be a consequence of alterations occurring during selection by cytotoxic drugs rather than of P-gp over-expression.

[Quantitative cytological study of the activity of a new resistance modulator, S 9788, on human leukemic cells using multiparametric image analysis]. / Etude cytologique quantitative de l'activité d'un nouvel agent modulateur de la résistance, le S 9788, sur des cellules leucémiques humaines par analyse d'image multiparamétrique.

The triazinoaminopiperidine derivative S 9788 is a new multidrug resistance modulator. The modulating activity of S 9788, comparatively to those of verapamil and the combination of S 9788 and verapamil, was demonstrated on the human leukemic T cell line CCRF-CEM resistant (about 6000 fold) to vinblastine using Microculture Tetrazolium Assay. S 9788 at 5 microM, strongly potentialized the cytotoxic activity of vinblastine but the reversion of resistance remained partial. Verapamil and the combination S 9788-verapamil, tested at equimolar concentrations, were respectively 1000 and two times less active than S 9788 alone. The impact of S 9788, verapamil and their combination on the cytological modifications bound to vinblastine resistance of CEM cells was evaluated by multiparametric quantitative cytological analysis (21 nuclear parameters measured) using a SAMBA 2005 cell image processor. Treatments with the different modulators, in absence or in presence of vinblastine, had no significant effects on the morphology of sensitive CEM cells. On vinblastine resistant CEM cells, S 9788 and the combination S 9788-verapamil induced significant cytological modifications. These modifications were characterized by a partial reversion of some parameters (more specifically nuclear texture parameters) to values close to those observed in parental sensitive cells and permitted an automatic classification of these treated resistant cells in cells of "sensitive" type with a percentage superior to 50%. In conclusion, the reversion of resistance induced by S 9788 on CEM cells resistant to vinblastine does not fit only with a biological phenomenon like the efflux of cytotoxic agents but is associated with a set of cellular alterations involved in multidrug resistance.

[Study of erythroid differentiation of K 562 cells resistant to adriamycin]. / Etude de la différenciation érythroïde des cellules K562 résistantes à l'adriamycine.

The induction of differentiation is at present one of the most promising pathway used in the field of cancer chemotherapy. As part of this research, the study of the human K562 erythroleukemic cells differentiation has shown that these cells are potent to synthesize hemoglobin when treated by 40 nmoles/l of adriamycin, 30 nmoles/l of aclacinomycin or 3.10(4) nmoles/l of hemin. This functional differentiation is accompanied by some changes in three membrane antigens expression: GPA, CD15 antigen and TfR. To complete these result, we have studied the differentiation of resistant K562 cells to adriamycin by these three inducers. Our results suggest that this cell population is able to synthesize hemoglobin after its exposition to 2.10(3) nmoles/l of adriamycin, 100 nmoles/l of aclacinomycin or 7, 5.10(4) nmoles/l of hemin. In contrary to aclacinomycin and adriamycin, hemin did not affect the three markers expression. On the other hand, only the induction by adriamycin causes membrane phenotypical changes which are erythroid differentiation specific.