Sabrina Pesnel

Tumor burden influences exposure and response to rituximab: pharmacokinetic-pharmacodynamic modeling using a syngeneic bioluminescent murine model expressing human CD20

Clinical studies have shown a large interindividual variability in rituximab exposure and its significant influence on clinical response in patients receiving similar doses of antibody. The aim of this study was to evaluate the influence of tumor burden on dose-concentration-response relationships of rituximab. Murine lymphoma cells (EL4, 8 x 10(3)), transduced with human CD20 cDNA and transfected with luciferase plasmid (EL4-huCD20-Luc), were intravenously injected into C57BL/6J mice. Tumor burden detection, dissemination, and progression were evaluated quantitatively by in vivo bioluminescence imaging. Different doses of rituximab (6, 12, 20, or 40 mg/kg) were infused 13 days after lymphoma cell inoculation, and rituximab serum concentrations were measured by enzyme-linked immunosorbent assay. Without rituximab, all mice developed disseminated lymphoma and died within 30 days, whereas a significant dose-response relationship was observed in mice receiving rituximab. The 20-mg/kg dose was adequate to study interindividual variability in response because 23% of mice were cured, 59% had partial response, and 18% had disease progression. Rituximab concentrations were inversely correlated with tumor burden; mice with low tumor burden had high rituximab concentrations. Furthermore, rituximab exposure influenced response and survival. Finally, using a pharmacokinetic-pharmacodynamic model, we demonstrated that tumor burden significantly influenced rituximab efficacy.

Preclinical activity of F14512, designed to target tumors expressing an active polyamine transport system

SummaryWe have exploited the polyamine transport system (PTS) to deliver selectively a spermine-drug conjugate, F14512 to cancer cells. This study was aimed to define F14512 anticancer efficacy against tumor models and to investigate whether fluorophor-labeled polyamine probes could be used to identify tumors expressing a highly active PTS and that might be sensitive to F14512 treatments. Eighteen tumor models were used to assess F14512 antitumor activity. Cellular uptake of spermine-based fluorescent probes was measured by flow cytometry in cells sampled from tumor xenografts by needle biopsy. The accumulation of the fluorescent probe within B16 tumors in vivo was assessed using infrared fluorescence imaging. This study has provided evidence of a major antitumor activity for F14512. Significant responses were obtained in 67% of the tumor models evaluated, with a high level of activity recorded in 33% of the responsive models. Complete tumor regressions were observed after i.v., i.p. or oral administrations of F14512 and its antitumor activity was demonstrated over a range of 2–5 dose levels, providing evidence of its good tolerance. The level of cellular fluorescence emitted by the fluorescent probes was higher in cells sampled from tumors sensitive to F14512 treatments than from F14512-refractory tumors. We suggest that these probes could be used to identify tumors expressing a highly active PTS and guide the selection of patients that might be treated with F14512. These results emphasize the preclinical interest of this novel molecule and support its further clinical development.

Monitoring of tumour progression using bioluminescence imaging and computed tomography scanning in a nude mouse orthotopic model of human small cell lung cancer

Human small cell lung carcinoma (SCLC) is the most aggressive type of lung cancer but no clinically relevant animal model has been developed to date. Such a model would be valuable to study the molecular aspects of tumour progression and to test the effectiveness of new treatment agents. We generated a reproducible and reliable nude mouse orthotopic model of human SCLC with NCI-H209 tumour cells genetically modified to express firefly luciferase. Cells were analysed for long-term stability of bioluminescence and a clone was passaged twice subcutaneously to enhance tumorigenicity. Cells resuspended in Matrigel and/or EDTA RPMI medium containing a (99m)Tc-labelled tin colloid used as tracer were implanted intrabronchially with a catheter inserted into the trachea and positioned in the main bronchus using X-ray-guided imaging. Deposition of cells into the lung was then assessed by scintigraphy. The growth of the primary tumour was sensitively and non-invasively followed by bioluminescence imaging that allowed real-time monitoring of tumour progression in the same animals over a 2-12-week period. Additional 3D bioluminescence imaging and computed tomography scanning were used to document tumour location and measurements that were confirmed by histological analyses. In conclusion, this original nude mouse orthotopic model resembles various stages of human small cell lung cancer, and therefore could be used to evaluate new treatment strategies.

99mTc-HYNIC-spermine for imaging polyamine transport system-positive tumours: preclinical evaluation

PurposeF14512 exploiting the polyamine transport system (PTS) for tumour cell delivery has been described as a potent antitumour agent. The optimal use of this compound will require a probe to identify tumour cells expressing a highly active PTS that might be more sensitive to the treatment. The aim of this study was to design and characterize a scintigraphic probe to evaluate its uptake in cancer cells expressing the PTS.MethodsThree polyamines coupled to a hydrazinonicotinamide (HYNIC) moiety were synthesized and labelled with 99mTc. Their radiochemical purity was determined by HPLC. The plasma stability of the 99mTc-HYNIC-spermine probe and its capacity to accumulate into PTS-active cells were also evaluated. In vitro internalization was tested using murine melanoma B16/F10 cells and human lung carcinoma A549 cells. Biodistribution was determined in healthy mice and tumour uptake was studied in B16/F10 tumour-bearing mice. A HL-60-Luc human leukaemia model was used to confront single photon emission computed tomography (SPECT) images obtained with the 99mTc-labelled probe with those obtained by bioluminescence.ResultsThe 99mTc-HYNIC-spermine probe was selected for its capacity to accumulate into PTS-active cells and its stability in plasma. In vitro studies demonstrated that the probe was internalized in the cells via the PTS. In vivo measurements indicated a tumour to muscle scintigraphic ratio of 7.9±2.8. The combined bioluminescence and scintigraphic analyses with the leukaemia model demonstrated that the spermine conjugate accumulates into the tumour cells.ConclusionThe 99mTc-HYNIC-spermine scintigraphic probe is potentially useful to characterize the PTS activity of tumours. Additional work is needed to determine if this novel conjugate may be useful to analyse the PTS status of patients with solid tumours.

Comparison of Image Restoration Methods for Bioluminescence Imaging

Bioluminescence imaging is a recent modality to visualize biological effects, especially for small animals. However, the acquired images are degraded by diffusion and absorption phenomena from the tissue and by the acquisition system itself. In this paper, we use restoration methods to enhance the quality of bioluminescence images. We propose a model for image formation and an experimental determination of the PSF (Point Spread Function). Several restoration methods are compared on test images generated according to the model and on real data. This comparison is insured by using MSE (Mean Square Error) and two other quantitative criteria. Results showed that the statistical methods give more accurate restoration and are well adapted for Bioluminescence Imaging.

Optical Imaging of Disseminated Leukemia Models in Mice with Near-Infrared Probe Conjugated to a Monoclonal Antibody

Background The assessment of anticancer agents to treat leukemia needs to have animal models closer to the human pathology such as implantation in immunodeficient mice of leukemic cells from patient samples. A sensitive and early detection of tumor cells in these orthotopic models is a prerequisite for monitoring engraftment of leukemic cells and their dissemination in mice. Therefore, we developed a fluorescent antibody based strategy to detect leukemic foci in mice bearing patient-derived leukemic cells using fluorescence reflectance imaging (FRI) to determine when to start treatments with novel antitumor agents. Methods Two mAbs against the CD44 human myeloid marker or the CD45 human leukocyte marker were labeled with Alexa Fluor 750 and administered to leukemia-bearing mice after having verified the immunoreactivity in vitro. Bioluminescent leukemic cells (HL60-Luc) were used to compare the colocalization of the fluorescent mAb with these cells. The impact of the labeled antibodies on disease progression was further determined. Finally, the fluorescent hCD45 mAb was tested in mice engrafted with human leukemic cells. Results The probe labeling did not modify the immunoreactivity of the mAbs. There was a satisfactory correlation between bioluminescence imaging (BLI) and FRI and low doses of mAb were sufficient to detect leukemic foci. However, anti-hCD44 mAb had a strong impact on the tumor proliferation contrary to anti-hCD45 mAb. The use of anti-hCD45 mAb allowed the detection of leukemic patient cells engrafted onto NOD/SCID mice. Conclusions A mAb labeled with a near infrared fluorochrome is useful to detect leukemic foci in disseminated models provided that its potential impact on tumor proliferation has been thoroughly documented.

Abstract B186: Preclinical antileukemic activity of F14512, a novel targeted cytotoxic agent

Despite advances in the therapy of acute myeloid leukemia (AML), the majority of patients die from their disease. Therefore, the lack of effective therapy mandates the development of novel compounds to improve the outcome of patients with relapsed and refractory leukemias. F14512 is a potent spermine‐epipodophyllotoxin conjugate exploiting the polyamine transport system for tumor cell delivery. In this study, we report the in vivo antitumor activity of F14512 against experimental models of AML cell lines and of patient AML samples. F14512 markedly reduced the growth of HL‐60 and U937 cell lines in an in vivo xenotransplantation model, resulting in a highly significant increase of survival of leukemia‐bearing mice. Etoposide evaluated concurrently demonstrated only moderate in vivo activity against these models. F14512 induced in vivo apoptosis of HL‐60 cells, as shown by caspase‐3 activation and PARP cleavage. In an effort to mimic the human disease, we injected approximately 106 AML cells collected from a patient into NOD/SCID mice and allowed them to establish as xenografts for 8 weeks. Subsequent treatment with F14512 was carried out for 2 or 3 weeks followed by the analysis at the end of treatment and 1 week after the end of treatment. Two human AML samples were analyzed. Multiple i.v. administrations of F14512 at 0.32 mg/kg, induced an extensive reduction of the number of leukemic cells in mouse bone marrow and blood (97–99%), assessed by flow cytometry analysis, quantitative RT‐PCR and histology. To identify leukemic cells expressing an active polyamine transport system, we developed a functional method based on the measurement of the cellular uptake of a nitrobenzoxadiazole fluorescent probe (F96982) combining the same spermine moiety as F14512. The level of fluorescence emitted by the probe F96982 was high in HL‐60 cells as well in the 2 patient AML samples that proved to be sensitive to F14512 in vivo. Collectively, these results demonstrated that F14512 exhibits a marked in vivo antileukemic activity, supporting its clinical development. Phase I clinical trials in onco‐hematology are now initiated with this novel promising drug candidate. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B186.

Monitoring of tumor progression using bioluminescence imaging in a nude mice orthotopic model of human small cell lung cancer

Introduction Lung cancer is the main cause of cancer death throughout the world and a clinically relevant animal model of human small cell lung carcinoma (SCLC) should be useful to study the molecular aspects of the tumor progression and test the efficiency of new therapeutic agents. In this study, we generated a reproductible and reliable nude mice orthotopic model of human SCLC based on NCI-H209 tumor cells genetically modified to express firefly luciferase. Methods NCI-H209 cells were transfected with pCMV-luc plasmid and clones highly expressing luciferase were isolated and amplified. Cells were analyzed for long-term bioluminescent stability and a clone was subcutaneously passaged twice in vivo to enhance tumorigenicity. Cells resuspended in Matrigel ® and/or EDTA RPMI medium containing a Tc 99M colloid were implanted intrabronchially using a catheter inserted into the trachea and positioned into the right main bronchus using interventional imaging. Punctual deposition of cells was then assessed by scintigraphy. Tumor progression was then followed using bioluminescence imaging. Results Only tumor nodules were observed into lung and trachea when cells were implanted with EDTA Lung tumor invading parenchyma were present in 40% of the mice with Matrigel ® and improved to 75% with EDTA and Matrigel ® . The growth of the primary tumor was sensitively and non-invasively followed and quantified by bioluminescence imaging using a CCD-camera. This tool allows a real-time monitoring of tumor progression on the same animals over a 2-12 week period. Combination of 3D bioluminescence imaging and computed tomography scanning was used to further document tumor location and measurement. Subsequently, the histological analysis of tissue sections confirmed the presence of a lung tumor. Conclusion Our nude mice orthotopic model resembles various stages human small cell lung carcinoma, and then could be useful for evaluating new therapeutic strategies.