In vivo BPA monitoring by means of g y

Magnetic Resonance Spectroscopy and Magnetic Resonance

Imaging

Dr. Silvia Capuani, PhD

Imaging

p

INFM-CNR SOFTCenter for Complex Dynamics in Structured Systems

Nuclear Magnetic Resonance laboratoryPhysics Department, “Sapienza” University of Rome

Piazzale Aldo Moro 5, 00185 Rome, Italy

E-mail: Silvia.Capuani@roma1.infn.it

IntroductionSeveral clinical trials, using BNCT, have reported encouraging

results in patients with malignant gliomas

However, the clinical outcome of BNCT still remains largely

unsatisfactoryunsatisfactory

It is general opinion that one of the major limitations for BNCT g p j

effectiveness is the insufficient incorporation of 10B into the tumor cells

Moreover, the relatively low specificity of 10B-carriers uptake in tumor cells y p y p

as compared to the surrounding normal tissues limits the use of high dose

BNCT protocols.

A second limitation is due to a lack of an effective method to monitor, in

vivo, the pharmacokinetic of boron compounds in order to obtain the optimal p p p

irradiation time

Aims

Our goals were:Our goals were:1) to develop an effective imaging method to monitor, in vivo, the bio-

distribution of BPA 19F-MRI and 19F-MRS were used to obtain in vivo spatial distribution mapping

and pharmacokinetic of BPA

2) to investigate the use of L-DOPA as enhancer for BPA uptake in C6 li m llsC6-glioma cells

The investigation was first performed in vitro using C6-glioma cellsand then extended in vivo to the animal model using the C6-rat glioma modeland then extended in vivo to the animal model using the C6 rat glioma model

19F BPA (98% enriched in 10B)

1) Materials and method19F-BPA (98% enriched in 10B)

Animal model: C6-glioma rat brain

Administration of 19F-BPA-fr complex14 days after tumour implantation rats were infused with ay p19F-BPA-fr-complex solution (300mg/kg (bw)) within carotid artery

Kabalka GW et al. 2000 Org. Prep. Proced. Int. 32 290-293; Reddy NK et al. 1985 J. Lab. Compd. Radiopharm. 33 599-600; Porcari P et al. 2006 Phys. Med. Biol. 51 3141-3154

Animal model

FL CPfrontal cerebellum

lobe

bulbus olfactorius

brainstemRat atlas sagittal view brainstemRat atlas sagittal viewAxial view NMR image

25 Male Wistar rats (300-350g) were anesthetized by intraperitonealinjection of ketamine (60mg/kg) and xylazina before being fixed in ainjection of ketamine (60mg/kg) and xylazina before being fixed in astereotactic frame. A middle scalp incision was made and C6 cell

suspension (106 cells in 10 µl) was slowly injected with a Hamilton syringethrough a burr hole in the right hemisphere, 3 and 4 mm depth from the dura.Then the syringe was slowly removed and the burr hole and the scalp sutured

All procedures related to animal care were strictly conformed in accordance with Decree

Then, the syringe was slowly removed and the burr hole and the scalp sutured.

Survival time of the rats was about 2-3 weeks after tumour implantation.

All procedures related to animal care were strictly conformed in accordance with Decree 116/92 which represents the Italian enforcement of the European Directive 86/609/EEC.

MRI m su m nts t 7T

MethodsMRI measurements at 7T

ParameterProtocol: MSME T2-weighted images

TR/TE 2500/45TR/TESlice thiknessSquare FOVMatrix

2500/45 ms1.5 mm40X40 mm128X128 pixelsMatrix

Resolution128X128 pixels312μmX312 μm

Tumor growth was monitored by Magnetic Resonance Imaging at 7T.

2) Materials and method

BPA-fr to perform HPLC Measurements

And 19F-BPA-fr to perform And F BPA fr to perform Imaging experiments

a large clinical experience has been accumulated in the use of been accumulated in the use of

L-DOPA at different doses

2) In vitro experiments: Methodsand Materialsand Materials

C6 glioma cells were grown in Dulbecco’ s modified Eagle’sMedium supplemented with 10% fetal bovine serum and 50 µg/mlpp g

of gentamicin at 37°C under an atmosphere of 5% CO2 in air.Each sample included about 1.6X108 cells.

R di di l t i t Radiowave dielectric spectroscopy

Changes of electrical conductivity of the intracellular medium σp are proportional to variations in intracellular BPA (Capuani et al Chem Phys Lett 2002)to variations in intracellular BPA (Capuani et al. Chem Phys Lett 2002)

1 lls i b t d ith dditi f 2 l BPA f 2 h s

Experimental conditions1. cells incubated with addition of 2mmol BPA for 2 hours2. cells incubated with addition of 50µg/ml of L-DOPA for 2 hours 3. cells incubated with addition of 50µg/ml of L-DOPA for 4 hours µg

4. cells incubated with addition of 2mmol BPA for 2 hours after a two hour pre-incubation with 50µg/ml of L-DOPA

5 cells incubated with addition of 2mmol BPA for 2 hours after a 4 hour pre5. cells incubated with addition of 2mmol BPA for 2 hours after a 4 hour pre-incubation with 50µg/ml of L-DOPA

2) In vivo experiments: Methods All animals were

N 10N 15

sacrificed 2.5 hours after the end of BPA infusion. Assessment of BPA N=10N=15 Assessment of BPA concentrations in:tumor tissue,normal brain,

Pre-treatment with L-DOPA: 50mg/Kg

Intra-t ll

and blood samples was performed using High-Performance Liquid Chromatography peretoneally Liquid Chromatography (HPLC)

Intra-carotidBP f f

Intra-carotidBP f f BPA-fr infusion:

300mg/Kg BPA-fr infusion:

300mg/Kg

1s 2s Axial 1H MR images of1) In vivo Imaging results

1s 2s Axial H MR images ofrat brain acquired twohours after 19F-BPA-frcomplex infusion

19F axial image of ratbrain acquired after 1HMR scan

2.5h after infusion

3s 4s

Porcari P et al. 2008 Phys. Med. Biol. In press

1) Results: 19F-BPA spatial bio-distribution mappingb f 19F MRIby means of 19F MRI

Superimposition of 19F axial image

(in colour level: low=blue, red=high)

i d 2 5 h acquired 2.5 hours after infusion on the

corresponding p gmorphological 1H

proton reference (in grey levels). gr y s).

P. Porcari, S. Capuani, E. D’Amore et al. 2008 Phys. Med. Biol.

4h after

1) Results: pharmacokinetic of 19F-BPA by means of 19F MRS

infusionblood samplesextracted from theright femoral vein

rat brain

4h

2.5h after infusion2.5h

rat brain

1h

P. Porcari, S. Capuani, E. D’Amore et al. 2008 Phys. Med. Biol.

2) In vitro experiments: resultsMean percentage differences between the electrical conductivity of the intracellular medium σp values measured in test and in reference samples are reported for each of the five experimental conditions.

Condition : 1 2 3 4 5

L-DOPA preloading induces a massive increase of BPA concentration in C6-glioma cells only after 4 hours incubation.

S. Capuani, T. Gili, M.Bozzali et al. Int J Radiat Oncol Biol Phys 2008

2) In vivo L-DOPA results MRI1H 19F

BPA uptake in C6-glioma model is dramatically increased

HPLC

Mean ±SD BPA/tissue

concentration [µg/g]

C t l L-DOPA pre-

by L-DOPA preloading

Control group

Only BPA

N=10

treatment

+BPA

N=15

Tumor 33.5± 7.5 88.3 ±12.1

Normal brain(ipsilateral)

12.0± 5.2 10.5 ±6.2

Normal brain(contralateral)

7.4 ±2.2 6.6±2.8

BPA concentrations assessed by HPLC from each sampled tissue

(contralateral)Blood 5.0±1.8 4.8±2.1

1H T2-w image (a) and 19F image (b) of rat brain pre-treatedwith L-DOPA and then infused with 19F-BPA-fr complex

BPA accumulation in tumor samples wassignificantly higher in treated group

compared to control group (p<0.0001)

p1H T2-w image (c) and 19F image (d) of rat brain not pre-loadedwith L-DOPA (control) and then infused with 19F-BPA-fr complex

19F-BPA tumour signal was observed only in L-DOPA pre-treated rat but not in the other case confirming an increased

19F BPA tumour uptake after L DOPA administration19F-BPA tumour uptake after L-DOPA administrationS. Capuani, T. Gili, M.Bozzali et al. Int J Radiat Oncol Biol Phys 2008 P. Porcari S. Capuani, E. D’Amore et al.

Appl. Rad. Isot. 2009

Discussion and Conclusion

The 19F-MRI in combination with 1H-MRI selectively maps thespatial-distribution of 19F-BPA in C6 tumour-bearing rats

19F MRI is a useful method to investigate and evaluate theF MRI is a useful method to investigate and evaluate thepharmacokinetics of the fluorinated-containing drugsCorrelation between 19F MRI and 19F MRS results highlights an improvedunderstanding of 19F-BPA uptake in tumour and systemic circulation

The results obtained in vitro and in vivo demonstrate that L-DOPA promotes the cellular uptake of BPA

g f F p m y mshowing the optimal irradiation time

promotes the cellular uptake of BPAIt has been proposed (Wittig et al. 2000) that both BPA and L-DOPA penetrate

through the cell membrane using two main mechanisms: by slow diffusion process driven by concentration gradients and by fast process due to active carriers driven by concentration gradients, and by fast process due to active carriers

Previous experiments suggested the presence of specific membrane anti-port carriers with high affinity for L-substrates. The activation of these carriers is

Our results suggest that 4 hours are needed to reach in C6-glioma cells, by slow diffusion, a critical intracellular concentration of L-

g ysupposed to be driven by a chemical gradient of L-molecules across cell membranes

yDOPA to trigger the faster L-antiport system

Discussion and Conclusion

The most striking findings of our work are the convergent results obtained with C6 glioma cells in vitro and using the results obtained with C6 glioma cells in vitro and using the

equivalent animal model in vivo.

L DOPA d i ist ti d d i th C6 li t L-DOPA pre-administration produced in the C6 glioma rat model an enhancement of tumor BPA accumulation which was

2.7 times higher than in the control condition

Our study demonstrates that L-DOPA preloading induces a remarkable increase of BPA uptake in tumor but not in normal

brain tissues

BNCT Study Group in Romey p

F.S. Pastore Department of Neuroscience, Institute of Neurosurgery, University “Tor Vergata”

S. Capuani, P P i Ph i D “ i ” U i i f R P. Porcari Physics Department “Sapienza”, University of Rome, B. Maraviglia

E. D’Amore Service for Biotechnology and Animal Welfare, Istituto Superiore di Sanità, Rome, Italy

R. Marini Bettolo Department of Chemistry, Sapienza University of Rome

M. Bozzali Neuroimaging Laboratory, IRCCS Santa Lucia Foundation;

Research activities in BNCT field: Research activities in BNCT field: MR Imaging and Spectroscopy applied to studies of 19F-BPA and BSH pharmacokinetics, with the aim to

optimize the timing of neutronic irradiation according to the spatial distribution of Boron agents.

In vivo and in vitro investigations on L-DOPA as an agent able to selectively increase the BPA uptake In vivo and in vitro investigations on L DOPA as an agent able to selectively increase the BPA uptake in glioma cells. Quantitative techniques employed are: NMR, HPLC, Dielelectric Spectroscopy.

P. Porcari et al. Phys Med Biol 2006;51:3141. S. Capuani et al. Magn Reson Imaging 2008;26:987Recent publications:

y p g g gS. Capuani et al. Int J Radiat Oncol Biol Phys 2008; 72:562. P. Porcari et al. Phys Med Biol 2008;53:6979S. Capuani et al. Appl. Rad. Isot. 2009;67:S34. P. Porcari et al. Appl. Rad. Isot. 2009;67:S365.