Doxycycline and its quaternary ammonium derivative for adjuvant therapies of chondrosarcoma.

PURPOSE: This study was conducted during the development of innovative treatment targeting the microenvironment of chondrosarcoma. In this context, MMP inhibitors were conjugated with a quaternary ammonium (QA) function as a targeting ligand to proteoglycans of chondrosarcoma extracellular matrix. Here we report the proof of concept of this strategy applied to the MMP13 inhibitor, doxycycline (Dox). METHODS: A quaternary ammonium derivative of the MMP13 inhibitor doxycycline (QA-Dox) was synthesized, and its anticancer activity was evaluated in the Swarm rat chondrosarcoma (SRC) model compared with the parent drug doxycycline, in vitro and in vivo. In vivo, dox and QA-Dox efficiency was assessed at equimolar doses according to a q4dx4 schedule by monitoring tumour volume by MRI and PG-targeted scintigraphy. Molecular mechanism (MMP13 expression, proteoglycan level) and histology studies were performed on tumours. RESULTS: The link of QA targeting function to Dox maintained the MMP13 inhibitory activity in vitro. Interestingly, the bacteriostatic activity was lost. SRC cells incubated with both drugs were blocked in S and G2 M phases. Tumour growth inhibition (confirmed by histology) was observed for both Dox and QA-Dox. Undesirable blood effects (leukocyte decrease) were reduced when Dox was targeted to tumour tissue using the QA function. CONCLUSIONS: In the SRC model, the MMP13 inhibitor Dox and its QA derivative are promising as adjuvant therapies for chondrosarcoma management.

Proteoglycans as Target for an Innovative Therapeutic Approach in Chondrosarcoma: Preclinical Proof of Concept.

To date, surgery remains the only option for the treatment of chondrosarcoma, which is radio- and chemoresistant due in part to its large extracellular matrix (ECM) and poor vascularity. In case of unresectable locally advanced or metastatic diseases with a poor prognosis, improving the management of chondrosarcoma still remains a challenge. Our team developed an attractive approach of improvement of the therapeutic index of chemotherapy by targeting proteoglycan (PG)-rich tissues using a quaternary ammonium (QA) function conjugated to melphalan (Mel). First of all, we demonstrated the crucial role of the QA carrier for binding to aggrecan by surface plasmon resonance. In the orthotopic model of Swarm rat chondrosarcoma, an in vivo biodistribution study of Mel and its QA derivative (Mel-QA), radiolabeled with tritium, showed rapid radioactivity accumulation in healthy cartilaginous tissues and tumor after [3H]-Mel-QA injection. The higher T/M ratio of the QA derivative suggests some advantage of QA-active targeting of chondrosarcoma. The antitumoral effects were characterized by tumor volume assessment, in vivo 99mTc-NTP 15-5 scintigraphic imaging of PGs, 1H-HRMAS NMR spectroscopy, and histology. The conjugation of a QA function to Mel did not hamper its in vivo efficiency and strongly improved the tolerability of Mel leading to a significant decrease of side effects (hematologic analyses and body weight monitoring). Thus, QA conjugation leads to a significant improvement of the therapeutic index, which is essential in oncology and enable repeated cycles of chemotherapy in patients with chondrosarcoma. Mol Cancer Ther; 15(11); 2575-85. ©2016 AACR.

Zoledronic acid inhibits pulmonary metastasis dissemination in a preclinical model of Ewing's sarcoma via inhibition of cell migration.

BACKGROUND: Ewing's sarcoma (ES) is the second most frequent primitive malignant bone tumor in adolescents with a very poor prognosis for high risk patients, mainly when lung metastases are detected (overall survival <15% at 5 years). Zoledronic acid (ZA) is a potent inhibitor of bone resorption which induces osteoclast apoptosis. Our previous studies showed a strong therapeutic potential of ZA as it inhibits ES cell growth in vitro and ES primary tumor growth in vivo in a mouse model developed in bone site. However, no data are available on lung metastasis. Therefore, the aim of this study was to determine the effect of ZA on ES cell invasion and metastatic properties. METHODS: Invasion assays were performed in vitro in Boyden's chambers covered with Matrigel. Matrix Metalloproteinase (MMP) activity was analyzed by zymography in ES cell culture supernatant. In vivo, a relevant model of spontaneous lung metastases which disseminate from primary ES tumor was induced by the orthotopic injection of 106 human ES cells in the tibia medullar cavity of nude mice. The effect of ZA (50 µg/kg, 3x/week) was studied over a 4-week period. Lung metastases were observed macroscopically at autopsy and analysed by histology. RESULTS: ZA induced a strong inhibition of ES cell invasion, probably due to down regulation of MMP-2 and -9 activities as analyzed by zymography. In vivo, ZA inhibits the dissemination of spontaneous lung metastases from a primary ES tumor but had no effect on the growth of established lung metastases. CONCLUSION: These results suggest that ZA could be used early in the treatment of ES to inhibit bone tumor growth but also to prevent the early metastatic events to the lungs.

Impact of oncopediatric dosing regimen of zoledronic acid on bone growth: preclinical studies and case report of an osteosarcoma pediatric patient.

Osteosarcoma and Ewing sarcoma represent the two most frequent primary bone tumors that arise in the pediatric population. Despite recent improvement in their therapeutic management, no improvement in survival rate has been achieved since early 1980 s. Among new therapeutic approaches, bisphosphonates are promising candidates as potent inhibitors of bone resorption. However, their effects on bone growth must be studied at dosing regimen corresponding to pediatric protocols. To this aim, several protocols using zoledronic acid (ZOL) were developed in growing mice (50 µg/kg every 2 days × 10). Parameters of bone remodeling and bone growth were investigated by radiography, micro-computed tomography, histology, and biologic analyses. Extramedullar hematopoiesis was searched for in spleen tissue. A transient inhibitory effect of ZOL was observed on bone length, with a bone-growth arrest during treatment owing to an impressive increase in bone formation at the growth plate level (8- to 10-fold increase in BV/TV). This sclerotic band then shifted into the diaphysis as soon as endochondral bone formation started again after the end of ZOL treatment, revealing that osteoclasts and osteoblasts are still active at the growth plate. In conclusion, endochondral bone growth is transiently disturbed by high doses of ZOL corresponding to the pediatric treatment of primary bone tumors. These preclinical observations were confirmed by a case report in a pediatric patient treated in the French OS2006 protocol over 10 months who showed a growth arrest during the ZOL treatment period with normal gain in size after the end of treatment.

Preclinical evidence that use of TRAIL in Ewing's sarcoma and osteosarcoma therapy inhibits tumor growth, prevents osteolysis, and increases animal survival.

PURPOSE: Osteosarcoma and Ewing's sarcoma are high-grade neoplasms typically arising in the bones of children and adolescents. Despite improvement in therapy, the five-year survival rate is only 20% for patients not responding to treatment or presenting with metastases. Among new therapeutic strategies, the efficacy of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily with strong antitumoral activity and minimal toxicity to most normal cells and tissues, was investigated by complementary approaches both in vitro and in preclinical models. EXPERIMENTAL DESIGN: The sensitivity of osteosarcoma and Ewing's sarcoma cell lines to TRAIL was investigated in vitro by determining TRAIL receptor expression together with TRAIL effects on cell viability and apoptosis. Complementary preclinical studies were carried out in respective tumor models by inoculation of osteosarcoma or Ewing's sarcoma tumor cells in paraosseous location. In addition, a model of lung nodule dissemination was developed by i.v. injection of osteosarcoma cells. RESULTS: In vitro, both osteosarcoma and Ewing's sarcoma cells that express the TRAIL death receptors were highly sensitive to TRAIL-induced caspase-8-mediated apoptosis. TRAIL administered in vivo by nonviral gene therapy inhibited primary bone tumor incidence and growth by 87% and prevented tumor-induced osteolysis, leading to a significant 2-fold increase in animal survival 40 days after tumor induction. Furthermore, TRAIL inhibited tumor nodule dissemination in lungs and increased survival in an osteosarcoma model. CONCLUSION: These findings suggest that TRAIL is a promising candidate for the development of new therapeutic strategies in the most frequent malignant primary bone tumors.

First preclinical imaging of primary cartilage neoplasm and its local recurrence using 99mTc-NTP 15-5 radiotracer.

UNLABELLED: This study on a rat model of grade II chondrosarcoma aimed to determine whether the radiotracer N-(triethylammonium)-3-propyl-[15]ane-N5 radiolabeled with (99m)Tc ((99m)Tc-NTP 15-5), which binds to cartilage proteoglycans, has pathophysiologic validity for in vivo imaging of cartilage tumoral tissue. METHODS: We used 2 experimental approaches with the Swarm chondrosarcoma rat model: that is, a primary paratibial location and local recurrence after intralesional curettage. (99m)Tc-NTP 15-5 scintigraphy and (99m)Tc-hydroxymethylenediphosphonate ((99m)Tc-HMDP) scanning were performed at regular intervals during 50 d after tumor implantation in a paratibial location (primary model; n = 12 animals) and after intralesional curettage in a femoral condyle location (recurrence model; n = 9 animals). For each animal, positive scans were analyzed at each time point using the target-to-background ratio (TBR), with the target region of interest delineated over the tumor and the background region of interest over muscle. In each model, the TBR time course was followed against primary tumoral growth or recurrence. Tumor volume was monitored for 2 mo by measuring the 2 perpendicular diameters. At study end, animals were sacrificed for histopathologic analysis. RESULTS: For both models, (99m)Tc-NTP 15-5 scans showed tracer accumulation at the site of implantation or curettage. For the primary tumor model, the mean TBR was 1.6 +/- 0.14 by day 4 after implantation and increased over time as the disease progressed, with a mean TBR of 4.25 +/- 0.25 on day 45. For the recurrence model, mean TBR was 3.27 +/- 0.24 by day 4 after curettage and increased with recurrence, with a mean value of 5.25 +/- 0.49 on day 50. (99m)Tc-HMDP bone scans were negative for both models throughout the study; at a later stage of the study, an area of (99m)Tc-HMDP accumulation was seen in the diaphysis of the bone adjacent to the tumor and was attributed to remodeling. CONCLUSION: These experimental results in 2 preclinical models of grade II chondrosarcoma bring forward data in favor of (99m)Tc-NTP 15-5 radiotracer for imaging primary growth of chondrosarcoma and its local recurrence after surgery.