Paola Porcari

L-DOPA PRELOADING INCREASES THE UPTAKE OF BOROPHENYLALANINE IN C6 GLIOMA RAT MODEL: A NEW STRATEGY TO IMPROVE BNCT EFFICACY

PURPOSE Boron neutron capture therapy (BNCT) is a radiotherapeutic modality based on (10)B(n,alpha)(7)Li reaction, for the treatment of malignant gliomas. One of the main limitations for BNCT effectiveness is the insufficient intake of (10)B nuclei in the tumor cells. This work was aimed at investigating the use of L-DOPA as a putative enhancer for (10)B-drug 4-dihydroxy-borylphenylalanine (BPA) uptake in the C6-glioma model. The investigation was first performed in vitro and then extended to the animal model. METHODS AND MATERIALS BPA accumulation in C6-glioma cells was assessed using radiowave dielectric spectroscopy, with and without L-DOPA preloading. Two L-DOPA incubation times (2 and 4 hours) were investigated, and the corresponding effects on BPA accumulation were quantified. C6-glioma cells were also implanted in the brain of 32 rats, and tumor growth was monitored by magnetic resonance imaging. Rats were assigned to two experimental branches: (1) BPA administration; (2) BPA administration after pretreatment with L-DOPA. All animals were sacrificed, and assessments of BPA concentrations in tumor tissue, normal brain, and blood samples were performed using high-performance liquid chromatography. RESULTS L-DOPA preloading induced a massive increase of BPA concentration in C6-glioma cells only after a 4-hour incubation. In the animal model, L-DOPA pretreatment produced a significantly higher accumulation of BPA in tumor tissue but not in normal brain and blood samples. CONCLUSIONS This study suggests the potential use of L-DOPA as enhancer for BPA accumulation in malignant gliomas eligible for BNCT. L-DOPA preloading effect is discussed in terms of membrane transport mechanisms.

In vivo 19F MRI and 19F MRS of 19F- labelled boronophenylalanine-fructose complex on a C6 rat glioma model to optimize boron neutron capture therapy (BNCT)

Boron neutron capture therapy (BNCT) is a promising binary modality used to treat malignant brain gliomas. To optimize BNCT effectiveness a non-invasive method is needed to monitor the spatial distribution of BNCT carriers in order to estimate the optimal timing for neutron irradiation. In this study, in vivo spatial distribution mapping and pharmacokinetics evaluation of the (19)F-labelled boronophenylalanine (BPA) were performed using (19)F magnetic resonance imaging ((19)F MRI) and (19)F magnetic resonance spectroscopy ((19)F MRS). Characteristic uptake of (19)F-BPA in C6 glioma showed a maximum at 2.5 h after compound infusion as confirmed by both (19)F images and (19)F spectra acquired on blood samples collected at different times after infusion. This study shows the ability of (19)F MRI to selectively map the bio-distribution of (19)F-BPA in a C6 rat glioma model, as well as providing a useful method to perform pharmacokinetics of BNCT carriers.

Boronophenylalanine uptake in C6 glioma model is dramatically increased by L-DOPA preloading.

One of the main limitations for BNCT effectiveness is the insufficient intake of (10)B nuclei within tumour cells. This work was aimed at investigating the use of L-DOPA as enhancer for boronophenylalanine (BPA) uptake in the C6 glioma model. The investigation was first performed in vitro, and then extended in vivo to the animal model. BPA accumulation in C6 glioma cells was assessed, using radiowave dielectric spectroscopy (RDS), with and without L-DOPA preloading. C6 glioma cells were also implanted in the brain of 25 rats, randomly assigned to two experimental branches: (1) intra-carotid BPA infusion; (2) intra-carotid BPA infusion after pre-treatment with L-DOPA, administrated 24 h before BPA infusion. All animals were sacrificed, and assessment of BPA concentrations in tumour tissue, normal brain, and blood samples was performed using high performance liquid chromatography (HPLC). L-DOPA preloading induced a massive increase of BPA concentration either in vitro on C6 glioma cells or in vivo in the animal model tumour. Moreover, no significant difference was found in the normal brain and blood samples between the two animal groups. This study suggests the potential use of L-DOPA as enhancer for BPA accumulation in malignant gliomas eligible for BNCT.

Multi-nuclear MRS and 19F MRI of 19F-labelled and 10B-enriched p-boronophenylalanine-fructose complex to optimize boron neutron capture therapy: phantom studies at high magnetic fields

Reaction yield optimization for the synthesis and the complexation of a boron neutron capture therapy agent (19)F-labelled, (10)B-enriched p-boronophenylalanine-fructose ((19)F-BPA-fr) complex was obtained. (1)H, (19)F, (13)C and (10)B magnetic resonance spectroscopy (MRS) of the (19)F-BPA-fr complex in aqueous and rat blood solution phantoms and its spatial distribution mapping using (19)F magnetic resonance imaging (MRI) results are reported. 7 T and 9.4 T magnetic fields were used to perform MRI and MRS respectively. Our in vitro results suggest that in vivo studies on (19)F-BPA through (19)F NMR will be feasible.

10B-editing 1H-detection and 19F MRI strategies to optimize boron neutron capture therapy

Boron neutron capture therapy (BNCT) is a binary radiation therapy used to treat malignant brain tumours. It is based on the nuclear reaction (10B + n th --> [11B*] --> alpha + 7Li + 2.79 MeV) that occurs when 10B captures a thermal neutron to yield alpha particles and recoiling 7Li nuclei, both responsible of tumour cells destruction by short range and high ionization energy release. The clinical success of the therapy depends on the selective accumulation of the 10B carriers in the tumour and on the high thermal neutron capture cross-section of 10B. Magnetic resonance imaging (MRI) methods provide the possibility of monitoring, through 10B nuclei, the metabolic and physiological processes suitable to optimize the BNCT procedure. In this study, spatial distribution mapping of borocaptate (BSH) and 4-borono-phenylalanine (BPA), the two boron carriers used in clinical trials, has been obtained. The BSH map in excised rat brain and the 19F-BPA image in vivo rat brain, representative of BPA spatial distribution, were reported. The BSH image was obtained by means of double-resonance 10B-editing 1H-detection sequence, named M-Bend, exploiting the J-coupling interaction between 10B and 1H nuclei. Conversely, the BPA map was obtained by 19F-BPA using 19F-MRI. Both images were obtained at 7 T, in C6 glioma-bearing rat brain. Our results demonstrate the powerful of non conventional MRI techniques to optimize the BNCT procedure.

Early detection of human glioma sphere xenografts in mouse brain using diffusion MRI at 14.1 T

Glioma models have provided important insights into human brain cancers. Among the investigative tools, MRI has allowed their characterization and diagnosis.

The effects of ageing on mouse muscle microstructure: A comparative study of time-dependent diffusion MRI and histological assessment.

The investigation of age‐related changes in muscle microstructure between developmental and healthy adult mice may help us to understand the clinical features of early‐onset muscle diseases, such as Duchenne muscular dystrophy. We investigated the evolution of mouse hind‐limb muscle microstructure using diffusion imaging of in vivo and in vitro samples from both actively growing and mature mice. Mean apparent diffusion coefficients (ADCs) of the gastrocnemius and tibialis anterior muscles were determined as a function of diffusion time (Δ), age (7.5, 22 and 44 weeks) and diffusion gradient direction, applied parallel or transverse to the principal axis of the muscle fibres. We investigated a wide range of diffusion times with the goal of probing a range of diffusion lengths characteristic of muscle microstructure. We compared the diffusion time‐dependent ADC of hind‐limb muscles with histology. ADC was found to vary as a function of diffusion time in muscles at all stages of maturation. Muscle water diffusivity was higher in younger (7.5 weeks) than in adult (22 and 44 weeks) mice, whereas no differences were observed between the older ages. In vitro data showed the same diffusivity pattern as in vivo data. The highlighted differences in diffusion properties between young and mature muscles suggested differences in underlying muscle microstructure, which were confirmed by histological assessment. In particular, although diffusion was more restricted in older muscle, muscle fibre size increased significantly from young to adult age. The extracellular space decreased with age by only ~1%. This suggests that the observed diffusivity differences between young and adult muscles may be caused by increased membrane permeability in younger muscle associated with properties of the sarcolemma.

Novel Pharmacological and Magnetic Resonance Strategies to Enhance Boron Neutron Capture Therapy (BNCT) Efficacy in the Clinical Treatment of Malignant Glioma

High-grade glioma, such as anaplastic astrocitomas (AA, WHO grade 3) and glioblastomas multiforme (GBM, WHO grade 4) are extremely aggressive and highly infiltrative brain tumours (Kleihues & Cavenee, 2000; Louis et al., 2007). In most cases they recur locally after applying the standard multimodality treatment based on surgical resection, followed by radiotherapy and/or chemotherapy. Despite advances in medicine, malignant gliomas continue to carry a dismal prognosis, even though a modest increase (by 4.5 months) in median survival and quality of life has been achieved. The main limitation to the effectiveness of surgery and radiotherapy in patients suffering from high-grade glioma is that these techniques, based on the geometric definition of tumour volume, are not suitable to eradicate tumour infiltrating cells within normal brain tissue. Moreover adjuvant chemotherapy has little effect on prolonging survival in patients with GBM (Stupp et al., 2005). As a consequence, novel therapeutic approaches, based on a better understanding of cancer biology, are needed. To this end, experimental therapies such as gene therapy (Mischel et al., 2003), antiangiogenic therapy (Van Meir et al., 2010), monoclonal antibodies (Zhu et al. 2010), cancer immunotherapy (Keunen et al., 2011), vaccines (Hickey et al., 2010), boron neutron capture therapy (BNCT) (Barth et al., 1992, 2005) and radioimmunotherapy (Joensuu, 2000) are under investigation. Among these, BNCT represents a promising adjuvant therapy for malignant glioma, and for other forms of cancer such as head/neck cancer. It is a binary form of radiation therapy based on the selective accumulation of boronated compounds within tumour cells which are then irradiated by low-energy thermal neutrons. The nuclear reaction that occurs between the stable isotope, 10B, and thermal neutrons, yields high-energy alpha particles and recoiling lithium nuclei which release most of their ionizing energy within a few microns (about one cell diameter), therefore limiting radiation damage only to 10B-containing cells. Thus, BNCT can be considered as a biologically targeted form of radiation therapy because of its ability to target tumour cells