Jackie Morizet

Poly(ethylene glycol)-Coated Hexadecylcyanoacrylate Nanospheres Display a Combined Effect for Brain Tumor Targeting

The aim of the present study was to evaluate the tumor accumulation of radiolabeled long-circulating poly(ethylene glycol) (PEG)-coated hexadecylcyanoacrylate nanospheres and non-PEG-coated hexadecylcyanoacrylate nanospheres (used as control), after intravenous injection in Fischer rats bearing intracerebrally well established 9L gliosarcoma. Both types of nanospheres showed an accumulation with a retention effect in the 9L tumor. However, long-circulating nanospheres concentrated 3.1 times higher in the gliosarcoma, compared with non-PEG-coated nanospheres. The tumor-to-brain ratio of pegylated nanospheres was found to be 11, which was in accordance with the ratios reported for other carriers tested for brain tumor targeting such as long-circulating liposomes or labels for magnetic resonance imaging. In addition, a 4- to 8-fold higher accumulation of the PEG-coated carriers was observed in normal brain regions, when compared with control nanospheres. Using a simplified pharmacokinetic model, two different mechanisms were proposed to explain this higher concentration of PEG-coated nanospheres in a tumoral brain. 1) in the 9L tumor, the preferential accumulation of pegylated nanospheres was attributable to their slower plasma clearance, relative to control nanospheres. Diffusion/convection was the proposed mechanism for extravasation of the nanospheres in the 9L interstitium, across the altered blood-brain barrier. 2) In addition, PEG-coated nanospheres displayed an affinity with the brain endothelial cells (normal brain region), which may not be considered as the result of a simple diffusion/convection process. The exact underlying mechanism of such affinity deserves further investigation, since it was observed to be as important as specific interactions described for immunoliposomes with the blood-brain barrier.

Therapeutic activity of CPT-11, a DNA-topoisomerase I inhibitor, against peripheral primitive neuroectodermal tumour and neuroblastoma xenografts.

The anti-tumour activity of CPT-11, a topoisomerase I inhibitor, was evaluated in four human neural-crest-derived paediatric tumour xenografts; one peripheral primitive neuroectodermal tumour (pPNET) (SK-N-MC) and three neuroblastomas. Two models, SK-N-MC and IGR-N835, were established in athymic mice from a previously established in vitro cell line. Two new neuroblastoma xenograft models, IGR-NB3 and IGR-NB8, were derived from previously untreated non-metastatic neuroblastomas. They exhibited the classic histological features of immature neuroblastoma along with N-myc amplification, paradiploidy, chromosome 1p deletions and overexpression of the human mdr 1 gene. These tumour markers have been shown to be poor prognostic factors in children treated for neuroblastoma. CPT-11 was tested against advanced stage subcutaneous tumours. CPT-11 was administered i.v. using an intermittent (q4d x 3) and a daily x 5 schedule. The optimal dosage and schedule was 40 mg kg-1 daily for 5 days. At this highest non-toxic dose, CPT-11 induced 100% tumour-free survivors on day 121 in mice bearing the pPNET SK-N-MC xenograft. For the three neuroblastoma xenografts, 38-100% complete tumour regressions were observed with a tumour growth delay from 38 to 42 days, and anti-tumour activity was clearly sustained at a lower dosage (27 mg kg-1 day-1). The efficacy of five anti-cancer drugs commonly used in paediatric oncology or in clinical development was evaluated against SK-N-MC and IGR-N835. The sensitivity of these two xenografts to cyclophosphamide, thiotepa and cisplatin was of the same order of magnitude as that of CPT-11, but they were refractory to etoposide and taxol. In conclusion, CPT-11 demonstrated significant activity against pPNET and neuroblastoma xenografts. Further clinical development of CPT-11 in paediatric oncology is warranted.

Potentiation of radiation therapy by the oncolytic adenovirus dl1520 (ONYX-015) in human malignant glioma xenografts

In spite of aggressive surgery, irradiation and/or chemotherapy, treatment of malignant gliomas remains a major challenge in adults and children due to high treatment failure. We have demonstrated significant cell lysis and antitumour activity of the E1B-55 kDa-gene-deleted adenovirus ONYX-015 (dl1520, CI-1042; ONYX Pharmaceuticals) in subcutaneous human malignant glioma xenografts deriving from primary tumours. Here, we show the combined efficacy of this oncolytic therapy with radiation therapy. Total body irradiation (5 Gy) of athymic nude mice prior to intratumoral injections of ONYX-015 1 × 108 PFU daily for 5 consecutive days yielded additive tumour growth delays in the p53 mutant xenograft IGRG88. Radiation therapy was potentiated in the p53 functional tumour IGRG121 with a ‘subtherapeutic’ dose of 1 × 107 PFU daily for 5 consecutive days, inducing significant tumour growth delay, 90% tumour regression and 50% tumour-free survivors 4 months after treatment. These potentiating effects were not due to increased adenoviral infectivity or replication. Furthermore, cell lysis and induction of apoptosis, the major mechanisms for adenoviral antitumour activity, did not play a major role in the combined treatment strategy. Interestingly, the oncolytic adenovirus seemed to accelerate radiation-induced tumour fibrosis. Potentiating antitumour activity suggests the development of this combined treatment for these highly malignant tumours.

Oncolytic Activity of p53-Expressing Conditionally Replicative Adenovirus AdΔ24-p53 against Human Malignant Glioma

Prognosis of malignant glioma is poor, and results of treatment remain mediocre. Conditionally replicative adenoviruses hold promise as alternative anticancer agents for the treatment of malignant glioma. Here, we evaluated the conditionally replicative adenovirus AdΔ24 and its recently developed derivative AdΔ24-p53, which expresses functional p53 tumor suppressor protein while replicating in cancer cells, for treatment of malignant glioma. In comparison to its parent AdΔ24, AdΔ24-p53 killed most malignant glioma cell lines and primary glioblastoma multiforme short-term cultures more effectively, irrespective of their p53 status. Moreover, AdΔ24-p53 caused more frequent regression and more delayed growth of IGRG121 xenografts derived from a glioblastoma multiforme in vivo. Five intratumoral injections of 107 pfu AdΔ24 gave 24 days median tumor growth delay (P < 0.01), 30% tumor regressions, and 30% animals surviving >120 days tumor-free or with a minimal tumor residual. The same dose of AdΔ24-p53 caused >113 days of median tumor growth delay (P < 0.001), 70% tumor regressions, and 60% animals surviving >120 days tumor-free or with a minimal tumor residual. Antitumor effects in vivo were associated with extensive conditionally replicative adenovirus replication, apoptosis induction, and tumor morphology changes, including dissociation, inflammatory cell infiltration, and necrosis. We conclude that conditionally replicative adenoviruses expressing p53 are promising new agents for treatment of malignant glioma.

Potent therapeutic activity of irinotecan (CPT-11) and its schedule dependency in medulloblastoma xenografts in nude mice

The anti‐tumor activity of irinotecan (CPT‐11), a DNA‐topoisomerase I inhibitor, was evaluated in 5 advanced stage subcutaneous medulloblastoma xenografts in nude mice, using different schedules of administration. With a 5‐day schedule, the highest i.v. dose tested (40 mg kg−1 day−1) induced complete regressions in all xenografts but 1, and delays in tumor growth always exceeded 30 days. Two xenografts, IGRM11 and IGRM33, were highly sensitive, and animals survived tumor‐free beyond 120 days after treatment. CPT‐11 clearly retained its anti‐tumor activity at a lower dosage (27 mg kg−1 day−1). CPT‐11 was significantly more active than cyclophosphamide, thiotepa and etoposide against the 3 xenografts evaluated. To study the schedule dependency of its anti‐tumor activity, CPT‐11 was given i.v. at the same total doses over the same period (33 days) using either a protracted or a sequential schedule in IGRM34‐bearing mice. With a dose of 10 mg kg−1 day−1 given on days 0–4, days 7–11, days 21–25 and days 28–32 (total dose, 200 mg kg−1), 3 of 6 animals were tumor free on day 378. The same total dose given with a sequential schedule, i.e., 20 mg kg−1 day−1 on days 0–4 and days 28–32, failed to induce complete regression. The plasma pharmacokinetics of CPT‐11 and SN‐38 were studied in IGRM34‐bearing animals after a single i.v. dose of 10 and 40 mg kg−1. The plasma clearance rate of CPT‐11 was dose dependent. The ratio between the SN‐38 and CPT‐11 area under the curve in plasma was 0.4–0.65, i.e., significantly higher than that observed in humans at the maximum tolerated dose (0.01–0.05). Conversely, this ratio was 10‐fold lower in tumor than in plasma. Clinical development of irinotecan is warranted in pediatric malignancies. Int. J. Cancer 73:156–163, 1997.

EGFR tyrosine kinase inhibition radiosensitizes and induces apoptosis in malignant glioma and childhood ependymoma xenografts

Malignant gliomas and childhood ependymomas have a high rate of treatment failure. Epidermal growth factor receptor (EGFR) activation has been implicated in the tumorigenesis and radioresistance of many cancers, including brain tumors. Therefore, combining EGFR targeting with irradiation is a potentially attractive therapeutic option. We evaluated the tyrosine kinase inhibitor gefitinib for its antitumor activity and potential to radio‐sensitize in vivo in two xenograft models: an EGFR amplified glioma and an EGFR expressing ependymoma, both derived from primary tumors. When administered at 100 mg/kg for 5 consecutive days, gefitinib‐induced partial tumor regression in all treated EGFR amplified IGRG88 glioma xenografts. The addition of 1 Gy of irradiation prior to gefitinib administration resulted in 5 complete and 4 partial regressions for the 9 treated tumors as well as a significant tumor growth delay of 33 days for the combined treatment compared to 19 days for each therapy alone, suggesting additive antitumor activity. Tumor regression was associated with inhibition of AKT and MAPK pathways by gefitinib. In contrast, the ependymoma IGREP83 was sensitive to irradiation, but remained resistant to gefitinib. Combined treatment was associated with inhibition of radiation‐induced MAPK phosphorylation and significant induction of apoptotic cell death though radiation‐induced AKT phosphorylation was maintained. Depending on the scheduling of both therapies, a trend towards superior antitumor activity was observed with combined treatment. Thus, EGFR targeting through tyrosine kinase inhibition appears to be a promising new approach in the treatment of EGFR‐driven glioma, particularly in combination with radiation therapy.

Expression of p53, or targeting towards EGFR, enhances the oncolytic potency of conditionally replicative adenovirus against neuroblastoma

Advanced stage and relapsing neuroblastoma (NB) has a poor prognosis with frequent treatment failures, warranting new treatment options and enhanced local tumor control. Treatment with conditionally replicative adenoviruses (CRAds) has shown effectiveness in various preclinical cancer models, but has not yet been evaluated for local control of NB. Here, we tested the efficacy of the CRAd AdΔ24 and of two AdΔ24 derivatives against NB. Derivative AdΔ24‐425S11 infects cells deficient in coxsackie/adenovirus receptor (CAR) via the epidermal growth factor receptor (EGFR). Derivative AdΔ24‐p53 expresses the tumor suppressor protein p53 to promote oncolysis.

In Vivo Treatment with CPT-11 Leads to Differentiation of Neuroblastoma Xenografts and Topoisomerase I Alterations

Topoisomerase I inhibitors, such as CPT-11, are potent anticancer drugs against neuroblastoma (NB). Differentiating agents, such as retinoids, improve the survival of children with metastatic NB. To characterize the biological effects associated with exposure to CPT-11 in vivo, athymic mice bearing a human NB xenograft, named IGR-NB8 and characterized as an immature NB with poor prognostic markers, were treated with CPT-11. Prolonged stable disease was observed, resulting in an overall tumor growth delay of 115 days. During treatment, tumors differentiated into ganglioneuroblastomas (GGNB), which reverted into an immature phenotype when treatment was discontinued. In contrast, 13-cis retinoic acid failed to induce differentiation of IGR-NB8 in vivo. Tumor differentiation was associated with decreased N-myc expression, induction of p73 expression in the perinuclear area and cytoplasm, and a dramatic 35-fold decrease in topoisomerase I (topo I) catalytic activity. The full-length Mr 100,000 topo I protein was present in both pre and post-treatment immature NB xenografts. In contrast, differentiated GGNBs did not contain the Mr 100,000 protein but an intense Mr 48,000 topo I fragment. Furthermore, redistribution of the Mr 48,000 and 68,000 forms to the cytoplasm was observed in differentiated tumors. The same pattern of topo I expression and catalytic activity was observed in NBs and GGNBs obtained from pediatric patients. Our data suggest that prolonged in vivo exposure to CPT-11 induces differentiation of NB xenografts, which is associated with truncation of the topo I enzyme, relocation of the degraded forms to the cytoplasm, and decreased catalytic activity.

Tumorigenicity of cerebellar primitive neuro-ectodermal tumors in athymic mice correlates with poor prognosis in children

The histogenesis of medulloblastoma, also described as a cerebellar primitive neuro‐ectodermal tumor (PNET), remains controversial and unresolved. In addition, genetic markers which characterize cerebellar PNETs with poor prognosis in children have not been identified. Since xenografts can be valuable tools for better understanding the genetic events involved in cerebellar PNETs, small fragments of tumor samples from 17 children with newly diagnosed cerebellar PNETs were transplanted s.c. into female athymic Swiss mice. Eleven were non‐metastatic and 6 were metastatic PNETs. Eight tumors (47%) were tumorigenic. Histological analysis showed 6 typical medulloblastomas, 1 PNET with melanin pigment and 1 PNET with a rhabdoid phenotype. Wide heterogeneity was observed in tumor growth, with a doubling time ranging from 8 to 81 days after the first passage. Tumorigenicity was correlated with the metastatic phenotype of the tumor (p < 0.001). All the patients but one with a tumorigenic tumor relapsed and died. The survival of patients with a non‐tumorigenic PNET (67%) was significantly higher than that of patients with a tumorigenic PNET (13%) (p < 0.02). None of the xenografts or tumors from patients exhibited N‐myc‐gene alteration. Only one xenograft showed c‐myc amplification, with an abnormal 15‐kilobase fragment. None of the 17 tumors from patients showed amplification or c‐myc‐gene rearrangement. In conclusion, tumorigenicity of cerebellar PNETs strongly correlates both with the metastatic phenotype of the tumors and with the disease‐free survival of the patients. In addition, genetic events other than c‐myc‐gene amplification might be involved in cerebellar PNETs with poor prognosis.

Busulphan is active against neuroblastoma and medulloblastoma xenografts in athymic mice at clinically achievable plasma drug concentrations

SummaryHigh-dose busulphan-containing chemotherapy regimens have shown high response rates in children with relapsed or refractory neuroblastoma, Ewing’s sarcoma and medulloblastoma. However, the anti-tumour activity of busulfan as a single agent remains to be defined, and this was evaluated in athymic mice bearing advanced stage subcutaneous paediatric solid tumour xenografts. Because busulphan is highly insoluble in water, the use of several vehicles for enteral and parenteral administration was first investigated in terms of pharmacokinetics and toxicity. The highest bioavailability was obtained with busulphan in DMSO administered i.p. When busulphan was suspended in carboxymethylcellulose and given orally or i.p., the bioavailability was poor. Then, in the therapeutic experiments, busulphan in DMSO was administered i.p. on days 0 and 4. At the maximum tolerated total dose (50 mg kg–1), busulphan induced a significant tumour growth delay, ranging from 12 to 34 days in the three neuroblastomas evaluated and in one out of three medulloblastomas. At a dose level above the maximum tolerated dose, busulphan induced complete and partial tumour regressions. Busulphan was inactive in a peripheral primitive neuroectodermal tumour (PNET) xenograft. When busulphan pharmacokinetics in mice and humans were considered, the estimated systemic exposure at the therapeutically active dose in mice (113 μg h ml–1) was close to the mean total systemic exposure in children receiving high-dose busulphan (102.4 μg h ml–1). In conclusion, busulphan displayed a significant anti-tumour activity in neuroblastoma and medulloblastoma xenografts at plasma drug concentrations which can be achieved clinically in children receiving high-dose busulphan-containing regimens.