- Email: [email protected]
The effect of dexamethasone on the uptake of p-boronophenylalanine in the rat brain and intracranial 9L gliosarcoma
ARTICLE IN PRESS
Applied Radiation and Isotopes 61 (2004) 917–921
The effect of dexamethasone on the uptake of p-boronophenylalanine in the rat brain and intracranial 9L gliosarcoma G.M. Morrisa,b, P.L. Miccab, J.A. Coderreb,c,* a
Normal Tissue Radiobiological Research Group, Research Institute, Churchill Hospital, Oxford OX3 7LJ, UK b Medical Department, Brookhaven National Laboratory, Upton, NY 11790, USA c Nuclear Engineering Department, Massachusetts Institute of Technology, 150 Albany Street, Cambridge, MA 02139, USA
Abstract The steroid dexamethasone sodium phosphate (DEX) is routinely used to treat edema in brain tumor patients. The objective of the present study was to evaluate the effects of DEX on the uptake of boronophenylalanine (BPA) using the rat 9L gliosarcoma tumor model and surrounding brain tissue. Two steroid dosage protocols were used. The high-dose DEX protocol involved five 3 mg/kg intraperitoneal injections at 47, 35, 23, 11 and 1 h prior to the administration of the BPA for a total dose of 15 mg DEX/kg rat. The low-dose DEX administration protocol involved two doses of 1.5 mg/kg at 17 h and 1 h prior to BPA injection for a total dose of 3 mg DEX/kg rat. The control animals received no pretreatment, prior to the administration of BPA. Seventeen days after tumor implantation, rats were injected i.p. with 0.014 ml/g body weight BPA solution (1200 mg BPA/kg; B59 mg 10B/kg). In all groups, rats were euthanized at 3 h after BPA injection. Administration of the steroid had an effect on tumor weight, which decreased to B78% (p > 0:05) of the control weight in the low-dose DEX group, and B48% (po0:001) of the control weight in the high-dose DEX group. At 3 h after the administration of BPA, the concentration of boron in tumor was comparable (p > 0:1) in the control and high-dose DEX groups. The lowest mean value (73.871.6 mg/g) was obtained in the low-dose DEX group. This was significantly lower (p > 0:02) than the tumor boron contents in the high-dose DEX and control groups, which were 81.171.9 and 79.971.7 mg/g, respectively. Tumor:blood boron partition ratios for the control, low- and high-dose DEX groups were 2.3, 2.3 and 2.5, respectively. Boron concentrations were also measured in the normal brain and in the zone of brain adjacent to the tumor exhibiting edema. Although treatment with DEX had no appreciable effect on boron uptake in the normal brain of the rat, after the administration of BPA, it did impact on the boron levels in the zone of peritumoral edema. After the high-dose DEX administration protocol, boron levels in the zone of edema were reduced by B14% (po0:02). This finding suggests that BPA targeting of tumor cells in the peritumoral zone could be compromised by DEX. These cells appear to play a critical role in tumor recurrence after BNCT or conventional radiotherapy. r 2004 Elsevier Ltd. All rights reserved. Keywords: Dexamethasone; Boronophenylalanine; 9L gliosarcoma; Peritumoral zone; Rat
1. Introduction *Corresponding author. Nuclear Engineering Department, Massachusetts Institute of Technology, 150 Albany Street, Cambridge, MA 02139, USA. Tel.: +1-617-452-3383. E-mail address: [email protected] (J.A. Coderre).
Boron neutron capture therapy (BNCT) is a binary approach that relies upon the preferential accumulation of a boronated compound in the tumor, facilitating a selective irradiation of tumor cells during exposure to
0969-8043/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2004.05.007
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low-energy neutrons. Several reviews on BNCT have appeared (e.g. Slatkin, 1991; Barth et al., 1996; Coderre and Morris, 1999). BNCT is being evaluated clinically in facilities in the United States, Europe, and Japan. The boron carrier being used in the majority of the clinical trials is pboronophenylalanine (BPA). BPA, an analogue of the amino acid tyrosine, crosses the intact blood brain barrier (BBB) during infusion. Most glioblastoma patients are medicated with the corticosteroid dexamethasone (DEX) to control tumor-induced edema. In the Brookhaven clinical BNCT protocol, the patients also received a 15 mg prophylactic bolus dose of DEX on the day of treatment (Chanana et al., 1999). Previous studies involving animal tumor models have demonstrated a decrease in vascular permeability and blood flow after the administration of steroids (Yamada et al., 1983; Reichman et al., 1986). It has been shown that DEX does not significantly affect the uptake of cisplatin in the intracranial 9L gliosarcoma or normal brain of rats bearing this tumor (Straathof et al., 1998). However, in areas of the brain adjacent to the tumor (containing infiltrating tumor cells), cisplatin uptake was reduced by a factor of B2.5 (Straathof et al., 1998). These findings suggest that tumor cell targeting with boron carriers such as BPA could potentially be compromised by DEX. The rat 9L gliosarcoma has been used at BNL throughout the preclinical studies leading up to clinical BNCT trials. Here we report on the use of this tumor model to study the effect of DEX on the uptake of BPA into tumor and surrounding brain tissue.
2. Materials and methods 2.1. Tumor inoculation Twenty-eight male Fischer 344 rats (Taconic Farms, Germantown, NY) weighing 200–225 g were inoculated intracranially with 104 cultured GS9L cells in 1 ml of medium as described previously (Coderre et al., 1994). Rats were housed two per cage with food and water ad libitum and maintained in a 12-h light/dark cycle throughout the experiment. On day 15 after tumor implantation, the rats were sorted into three groups: controls (n ¼ 16), high-dose DEX (n ¼ 18), and lowdose DEX (n ¼ 18). 2.2. Experimental design Dexamethasone sodium phosphate (DEX), 10 mg DEX/ml (Gensia Laboratories, Ltd, Irvine, CA), was diluted with water to 1.5 mg/ml for intraperitoneal injection (i.p.). The high-dose DEX animals received five 3 mg/kg intraperitoneal injections at 47, 35, 23, 11
and 1 h prior to the administration of the BPA for a total dose of 15 mg DEX/kg rat. The low-dose DEX animals received two doses of 1.5 mg/kg at 17 and 1 h prior to BPA injection for a total dose of 3 mg DEX/kg rat. The control animals received no pretreatment, prior to the administration of BPA. p-Boronophenylalanine (BPA; l-enantiomer, > 98% 10 B-enriched; Ryscor, Inc., Raleigh, NC) containing 4.9% boron by weight was used as the boron delivery agent. The BPA was solubilized as the fructose complex at a concentration of 85 mg/ml as previously described (Coderre et al., 1994). Boron analysis was carried out using prompt-gamma spectroscopy (Fairchild et al., 1986) or direct current plasma-atomic emission spectroscopy (Coderre et al., 1994). The two methods of analysis are inter-calibrated using atomic emission calibration standards, 10B-enriched boric acid from the National Institute of Standards and Technology, and standard solutions of BPA (Coderre et al., 1994). 2.3. Statistical analysis Data are presented as the mean7standard error. Treatment groups were evaluated using Student’s t-test, with p values o0.05 being considered as statistically significant.
3. Results The tumor weights were 147716, 115711 and 72710 mg (mean7SE) in the control, low DEX dose and high DEX dose groups, respectively, at 17 days after tumor inoculation. Administration of the steroid clearly had an effect on tumor weight, which decreased to B78% (p > 0:05) of the control weight in the low-dose DEX group, and B48% (po0:001) of the control weight in the high-dose DEX group (Fig. 1A). At 3 h after the administration of BPA, the concentration of boron in tumor was comparable (p > 0:1) in the control and high-dose DEX groups (Fig. 1B). The lowest mean value (73.871.6 mg/g) was obtained in the low-dose DEX group. This was significantly lower (p > 0:02) than the tumor boron contents in the highdose DEX and control groups, which were 81.171.9 and 79.971.7 mg/g, respectively. Levels of boron in the blood, after BPA administration, were slightly higher (po0:01) in the control group, as compared with the high- and low-dose DEX groups (Fig. 1B). Tumor:blood boron partition ratios for the control, low- and highdose DEX groups were 2.3, 2.3 and 2.5, respectively. Boron concentrations were also measured in the normal brain (ipsilateral and contralateral cerebrum) and in the zone of brain adjacent to the tumor exhibiting edema. Levels of boron in the control group were
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Fig. 1. (A) Tumor weights in the control, low-dose and highdose DEX groups. (B) Boron concentrations (mg/g) in the blood and tumor in the control, low-dose and high-dose DEX groups. ’ = blood; = tumor.
did significantly (po0:02) reduce boron levels in this edematous zone (Fig. 2). The concentration of boron in the zone of peritumoral edema was 30.371.1 mg/g in the high-dose DEX group, and 34.271.1 mg/g and 35.370.8 mg/g in the low-dose DEX and control groups, respectively.
4. Discussion highest in the zone of peritumoral edema, with intermediate levels in the ipsilateral cerebrum. The boron content was lowest in the contralateral cerebrum (Fig. 2). This relationship of boron concentrations was maintained in the high- and low-dose DEX groups, with the highest concentration of boron in the zone of peritumoral edema and the lowest concentration in the contralateral cerebrum (Fig. 2). Levels of boron in the ipsilateral cerebrum were not affected (p > 0:05) by treatment with low- or high-doses of DEX. In the contralateral cerebrum the concentration of boron was B7% higher (po0:01) in the high-dose DEX group, than in the other two treatment groups. The low-dose DEX treatment protocol did not appreciably alter (p > 0:1) the uptake of boron in the zone of peritumoral edema relative to the controls. However, treatment with the high-dose DEX protocol
Peritumoral edema in brain tumor is routinely treated with steroids. A similar dose of DEX (high-dose group), to that used clinically, was administered in the present study. The dosage protocol was comparable with the one employed by Straathof et al. (1998), in a study involving Fischer 344 rats bearing the intracranial 9L gliosarcoma. This enabled data relating to vascular permeability, detailed by these authors, to be compared with the BPA biodistribution results reported in the present study. Using cisplatin, Straathof et al. (1998) demonstrated that the level of this water-soluble chemotherapeutic compound was a factor of 10 higher in tumor than in the surrounding normal brain. The concentration of cisplatin in the zone of peritumoral edema was also appreciably higher than in normal brain. While pretreatment with DEX had little effect on cisplatin levels
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in the 9L gliosarcoma or normal brain, it reduced the concentration of cisplatin in the zone of peritumoral edema by a factor of 2.6 (Straathof et al., 1998). In another study using the 9L gliosarcoma implanted intracranially in Fischer 344 rats, blood to tissue transport (BTT) of 14C alpha aminoisobutyric acid was estimated using double label autoradiography (Fross et al., 1991). The BTT was found to be highest in tumor and lowest in normal brain. In the zone of peritumoral edema the BTT was about 2.5 times lower than in the tumor, but was a factor of 6–9 times higher than in the normal brain. Viewed overall, the findings of these two studies (Straathof et al., 1998; Fross et al., 1991) indicate that the blood brain barrier (BBB) is disrupted in the 9L tumor and partially disrupted in the zone of peritumoral edema. The administration of DEX appears to reduce blood vessel permeability in the zone of edema. This could explain why levels of boron from BPA, in the present study, were reduced in the zone of peritumoral edema, but were not affected appreciably in tumor or normal brain, subsequent to treatment with DEX (highdose). Additional studies also indicate that the administration of DEX does not significantly affect the uptake of compounds into the normal brain, but it does appear to reduce vascular permeability in the zone of peritumoral edema (Neuwelt et al., 1982; Neuwelt et al., 1990; Braunschweiger and Schiffer, 1986; Nakagawa et al., 1987; Shapiro et al., 1990). DEX has a variable influence on the uptake of a range of compounds in different tumor types (Braunschweiger and Schiffer, 1986; Nakagawa et al., 1987; Shapiro et al., 1990; Molnar et al., 1995; Warnke et al., 1995). These differences are probably related to variations in the vascular architecture and permeability in the various tumor models, and to differences in the compounds used. Studies using the identical compound in different tumor types are limited. 14 C-alpha aminoisobutyric acid (AIB) has been used in conjunction with autoradiography to evaluate the effects of DEX on blood vessel permeability in the rat C6 and RG-2 intracranial gliomas. In the C6 glioma, BTT of 14CAIB was reduced by a factor of 4 relative to controls, and in the RG-2 glioma, BTT was not affected by treatment with DEX (Shapiro et al., 1990; Molnar et al., 1995). It is evident from the present and previous studies e.g. (Coderre et al., 1994; Morris et al., 1997) that BPA can penetrate the BBB and distribute in the normal central nervous system. However, it has been shown in rats that disruption of the BBB using mannitol increases the accumulation of boron from BPA by a factor of 2–3 in both the normal brain and the intracranial F98 glioma (Yang et al., 1996). This indicates that BPA targeting of tumor cells is susceptible to changes in vascular permeability. Further evidence for a vascular effect on BPA targeting is provided by boron microdistribution
studies using ion microscopy. These studies have demonstrated that infiltrating tumor cells from the rat F98 glioma and 9L gliosarcoma, distributed in the zone of peritumoral edema, contain B50% less boron than cells in the main tumor mass (Smith et al., 1996, 1997). Disruption of the BBB with mannitol resulted in a doubling of the boron content of these infiltrating tumor cells (Smith et al., 1996, 1997).
5. Conclusion It can be concluded that, although treatment with DEX had no appreciable effect on boron uptake in the 9L gliosarcoma (main tumor mass) or normal brain of the rat, after the administration of BPA, it did impact on the boron levels in the zone of peritumoral edema. After the high-dose DEX administration protocol, boron levels in the zone of edema were reduced by B14%. This finding suggests that BPA targeting of tumor cells in the peritumoral zone could be compromised by DEX. These cells appear to play a critical role in tumor recurrence after BNCT or conventional radiotherapy. It is essential that they are adequately targeted with boron for effective BNCT. Edema in brain tumor patients is frequently more pronounced than is the case in animal brain tumor models. The limited clinical data available indicates that vascular permeability in the region of peritumoral edema is considerably reduced after DEX administration (Jarden et al., 1985; Yeung et al., 1994). It is possible, therefore, that the negative effects of DEX on boron uptake in tumor cells in the zone of edema may be more pronounced in the clinical situation. This phenomenon could have a critical bearing on the clinical effectiveness BNCT for glioma and merits further investigation.
Acknowledgements D. Greenberg provided technical assistance related to dosimetry. GMM acknowledges funding from the UK Cancer Research Campaign. Additional support was provided by the Office of Biological and Environmental Research, US Department of Energy, under contract number DE-AC02-98CH10886.
References Barth, R.F., Soloway, A.H., Brugger, R.M., 1996. Boron neutron-capture therapy of brain-tumors—past history, current status, and future potential. Cancer Invest. 14, 534–550. Braunschweiger, P.G., Schiffer, L.M., 1986. Effect of dexamethasone on vascular function in RIF-1 tumors. Cancer Res. 46, 3299–3303.
ARTICLE IN PRESS G.M. Morris et al. / Applied Radiation and Isotopes 61 (2004) 917–921 Chanana, A.D., Capala, J., Chadha, M., et al., 1999. Boron neutron capture therapy for glioblastoma multiforme: interim results from the phase I/II dose—escalation studies. Neurosurgery 44, 1182–1193. Coderre, J.A., Button, T.M., Micca, P.L., et al., 1994. Neutron capture therapy of the 9L rat gliosarcoma using the pboronophenylalanine–fructose complex. Int. J. Radiat. Oncol. Biol. Phys. 30, 643–652. Coderre, J.A., Morris, G.M., 1999. The radiation biology of boron neutron capture therapy. Radiat. Res. 151, 1–18. Fairchild, R.G., Gabel, D., Laster, B.H., et al., 1986. Microanalytical techniques for boron analysis using the 10 B(n,a)7Li reaction. Med. Phys. 13, 50–56. Fross, R.D., Warnke, P.C., Groothuis, D.R., 1991. Blood flow and blood-to-tissue transport in 9L gliosarcomas: the role of the brain tumor model in drug delivery research. J. Neuro. Oncol. 11, 185–197. Jarden, J.O., Dhawan, V., Poltorak, A., et al., 1985. Positron emission tomographic measurement of blood-to-brain and blood-to-tumor transport of 82Rb: the effect of dexamethasone and whole brain radiation therapy. Ann. Neurol. 18, 636–646. Molnar, P., Lapin, G.D., Groothuis, D.R., 1995. The effects of dexamethasone on experimental brain tumors: I. Transcapillary transport and blood flow in RG-2 rat gliomas. J. Neuro. Oncol. 25, 19–28. Morris, G.M., Coderre, J.A., Micca, P.L., et al., 1997. Central nervous system tolerance to boron neutron capture therapy with p-boronophenylalanine. Brit. J. Cancer 76, 1623–1629. Nakagawa, H., Groothuis, D.R., Owens, E.S., et al., 1987. Dexamethasone effects on [125I] albumin distribution in experimental RG-2 gliomas and adjacent brain. J. Cerebr. Blood Flow Metab. 7, 687–701. Neuwelt, E.A., Barnett, P.A., Bigner, D.D., 1982. Effects of adrenal cortical steroids and osmotic blood brain barrier opening on methotrexate delivery to gliomas in the rodent: the factor of the blood brain barrier. Proc. Natl. Acad. Sci. USA 79, 4420–4423. Neuwelt, E.A., Horaczek, A., Hagel, M.A., 1990. The effect of steroids on gentamicin delivery to brain after blood brain barrier disruption. J. Neurosurg. 72, 123–126.
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Reichman, H.R., Farrell, C.L., Del Maestro, R.F., 1986. Effects of steroids and nonsteroid anti-inflammatory agents on vascular permeability in a rat glioma model. J. Neurosurg. 65, 233–237. Shapiro, W.R., Hiesinger, E.M., Cooney, G.A., et al., 1990. Temporal effects of dexamethasone on blood-to-brain and blood-to-tumor transport of 14C- alpha-aminoisobutyric acid in rat C6 glioma. J. Neuro. Oncol. 8, 197–204. Slatkin, D.N., 1991. A history of boron neutron capture therapy of brain tumors—postulation of a brain radiation dose tolerance limit. Brain 114, 1609–1629. Smith, D.R., Chandra, S., Coderre, J.A., Morrison, G.H., 1996. Ion microscopy imaging of 10B from p-boronophenylalanine in a brain tumor model for boron neutron capture therapy. Cancer Res. 56, 4302–4306. Smith, D.R., Chandra, S., Coderre, J.A., et al., 1997. Quantitative ion microscopy imaging of boron-10 in rat brain tumor models for BNCT. In: Larsson, B., Crawford, J., Weinreich, R. (Eds.), Advances in Neutron Capture Therapy, Chemistry and Biology, Vol. II. Elsevier Science B.V., Lausanne, pp. 308–314. Straathof, C.S.M., van den Bent, M.J., Ma, J., et al., 1998. CSM: the effect of dexamethasone on the uptake of cisplatin in 9L glioma and the area of brain around tumor. J. Neuro. Oncol. 37, 1–8. Warnke, P.C., Molnar, P., Lapin, G.D., et al., 1995. The effect of dexamethasone on transcapillary transport in experimental brain tumors: II. Canine brain tumors. J. Neuro. Oncol. 25, 29–38. Yamada, K., Ushino, Y., Hayakawa, T., et al., 1983. Effects of methylpredisolon on peritumoral brain edema: an autoradiographic study. J. Neurosurg. 59, 612–619. Yang, W., Barth, R.F., Carpenter, D.E., et al., 1996. Enhanced delivery of boronophenylalanine for neutron capture therapy by means of intracarotid injection and blood brain barrier disruption. Neurosurgery 38, 985–992. Yeung, W.T.I., Lee, T.Y., Del Maestro, R.F., et al., 1994. Effects of steroids on iopamidol blood brain transfer constant and plasma volume in brain tumors measured with X-ray computed tomography. J. Neuro. Oncol. 18, 53–60.
Applied Radiation and Isotopes 61 (2004) 917–921
The effect of dexamethasone on the uptake of p-boronophenylalanine in the rat brain and intracranial 9L gliosarcoma G.M. Morrisa,b, P.L. Miccab, J.A. Coderreb,c,* a
Normal Tissue Radiobiological Research Group, Research Institute, Churchill Hospital, Oxford OX3 7LJ, UK b Medical Department, Brookhaven National Laboratory, Upton, NY 11790, USA c Nuclear Engineering Department, Massachusetts Institute of Technology, 150 Albany Street, Cambridge, MA 02139, USA
Abstract The steroid dexamethasone sodium phosphate (DEX) is routinely used to treat edema in brain tumor patients. The objective of the present study was to evaluate the effects of DEX on the uptake of boronophenylalanine (BPA) using the rat 9L gliosarcoma tumor model and surrounding brain tissue. Two steroid dosage protocols were used. The high-dose DEX protocol involved five 3 mg/kg intraperitoneal injections at 47, 35, 23, 11 and 1 h prior to the administration of the BPA for a total dose of 15 mg DEX/kg rat. The low-dose DEX administration protocol involved two doses of 1.5 mg/kg at 17 h and 1 h prior to BPA injection for a total dose of 3 mg DEX/kg rat. The control animals received no pretreatment, prior to the administration of BPA. Seventeen days after tumor implantation, rats were injected i.p. with 0.014 ml/g body weight BPA solution (1200 mg BPA/kg; B59 mg 10B/kg). In all groups, rats were euthanized at 3 h after BPA injection. Administration of the steroid had an effect on tumor weight, which decreased to B78% (p > 0:05) of the control weight in the low-dose DEX group, and B48% (po0:001) of the control weight in the high-dose DEX group. At 3 h after the administration of BPA, the concentration of boron in tumor was comparable (p > 0:1) in the control and high-dose DEX groups. The lowest mean value (73.871.6 mg/g) was obtained in the low-dose DEX group. This was significantly lower (p > 0:02) than the tumor boron contents in the high-dose DEX and control groups, which were 81.171.9 and 79.971.7 mg/g, respectively. Tumor:blood boron partition ratios for the control, low- and high-dose DEX groups were 2.3, 2.3 and 2.5, respectively. Boron concentrations were also measured in the normal brain and in the zone of brain adjacent to the tumor exhibiting edema. Although treatment with DEX had no appreciable effect on boron uptake in the normal brain of the rat, after the administration of BPA, it did impact on the boron levels in the zone of peritumoral edema. After the high-dose DEX administration protocol, boron levels in the zone of edema were reduced by B14% (po0:02). This finding suggests that BPA targeting of tumor cells in the peritumoral zone could be compromised by DEX. These cells appear to play a critical role in tumor recurrence after BNCT or conventional radiotherapy. r 2004 Elsevier Ltd. All rights reserved. Keywords: Dexamethasone; Boronophenylalanine; 9L gliosarcoma; Peritumoral zone; Rat
1. Introduction *Corresponding author. Nuclear Engineering Department, Massachusetts Institute of Technology, 150 Albany Street, Cambridge, MA 02139, USA. Tel.: +1-617-452-3383. E-mail address: [email protected] (J.A. Coderre).
Boron neutron capture therapy (BNCT) is a binary approach that relies upon the preferential accumulation of a boronated compound in the tumor, facilitating a selective irradiation of tumor cells during exposure to
0969-8043/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2004.05.007
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low-energy neutrons. Several reviews on BNCT have appeared (e.g. Slatkin, 1991; Barth et al., 1996; Coderre and Morris, 1999). BNCT is being evaluated clinically in facilities in the United States, Europe, and Japan. The boron carrier being used in the majority of the clinical trials is pboronophenylalanine (BPA). BPA, an analogue of the amino acid tyrosine, crosses the intact blood brain barrier (BBB) during infusion. Most glioblastoma patients are medicated with the corticosteroid dexamethasone (DEX) to control tumor-induced edema. In the Brookhaven clinical BNCT protocol, the patients also received a 15 mg prophylactic bolus dose of DEX on the day of treatment (Chanana et al., 1999). Previous studies involving animal tumor models have demonstrated a decrease in vascular permeability and blood flow after the administration of steroids (Yamada et al., 1983; Reichman et al., 1986). It has been shown that DEX does not significantly affect the uptake of cisplatin in the intracranial 9L gliosarcoma or normal brain of rats bearing this tumor (Straathof et al., 1998). However, in areas of the brain adjacent to the tumor (containing infiltrating tumor cells), cisplatin uptake was reduced by a factor of B2.5 (Straathof et al., 1998). These findings suggest that tumor cell targeting with boron carriers such as BPA could potentially be compromised by DEX. The rat 9L gliosarcoma has been used at BNL throughout the preclinical studies leading up to clinical BNCT trials. Here we report on the use of this tumor model to study the effect of DEX on the uptake of BPA into tumor and surrounding brain tissue.
2. Materials and methods 2.1. Tumor inoculation Twenty-eight male Fischer 344 rats (Taconic Farms, Germantown, NY) weighing 200–225 g were inoculated intracranially with 104 cultured GS9L cells in 1 ml of medium as described previously (Coderre et al., 1994). Rats were housed two per cage with food and water ad libitum and maintained in a 12-h light/dark cycle throughout the experiment. On day 15 after tumor implantation, the rats were sorted into three groups: controls (n ¼ 16), high-dose DEX (n ¼ 18), and lowdose DEX (n ¼ 18). 2.2. Experimental design Dexamethasone sodium phosphate (DEX), 10 mg DEX/ml (Gensia Laboratories, Ltd, Irvine, CA), was diluted with water to 1.5 mg/ml for intraperitoneal injection (i.p.). The high-dose DEX animals received five 3 mg/kg intraperitoneal injections at 47, 35, 23, 11
and 1 h prior to the administration of the BPA for a total dose of 15 mg DEX/kg rat. The low-dose DEX animals received two doses of 1.5 mg/kg at 17 and 1 h prior to BPA injection for a total dose of 3 mg DEX/kg rat. The control animals received no pretreatment, prior to the administration of BPA. p-Boronophenylalanine (BPA; l-enantiomer, > 98% 10 B-enriched; Ryscor, Inc., Raleigh, NC) containing 4.9% boron by weight was used as the boron delivery agent. The BPA was solubilized as the fructose complex at a concentration of 85 mg/ml as previously described (Coderre et al., 1994). Boron analysis was carried out using prompt-gamma spectroscopy (Fairchild et al., 1986) or direct current plasma-atomic emission spectroscopy (Coderre et al., 1994). The two methods of analysis are inter-calibrated using atomic emission calibration standards, 10B-enriched boric acid from the National Institute of Standards and Technology, and standard solutions of BPA (Coderre et al., 1994). 2.3. Statistical analysis Data are presented as the mean7standard error. Treatment groups were evaluated using Student’s t-test, with p values o0.05 being considered as statistically significant.
3. Results The tumor weights were 147716, 115711 and 72710 mg (mean7SE) in the control, low DEX dose and high DEX dose groups, respectively, at 17 days after tumor inoculation. Administration of the steroid clearly had an effect on tumor weight, which decreased to B78% (p > 0:05) of the control weight in the low-dose DEX group, and B48% (po0:001) of the control weight in the high-dose DEX group (Fig. 1A). At 3 h after the administration of BPA, the concentration of boron in tumor was comparable (p > 0:1) in the control and high-dose DEX groups (Fig. 1B). The lowest mean value (73.871.6 mg/g) was obtained in the low-dose DEX group. This was significantly lower (p > 0:02) than the tumor boron contents in the highdose DEX and control groups, which were 81.171.9 and 79.971.7 mg/g, respectively. Levels of boron in the blood, after BPA administration, were slightly higher (po0:01) in the control group, as compared with the high- and low-dose DEX groups (Fig. 1B). Tumor:blood boron partition ratios for the control, low- and highdose DEX groups were 2.3, 2.3 and 2.5, respectively. Boron concentrations were also measured in the normal brain (ipsilateral and contralateral cerebrum) and in the zone of brain adjacent to the tumor exhibiting edema. Levels of boron in the control group were
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Fig. 2. Boron concentrations in the zone of peritumoral edema and normal brain in the control, low-dose and high-dose DEX groups. ’ = zone of peritumoral edema; = ipsilateral cortex; = contralateral cortex.
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Fig. 1. (A) Tumor weights in the control, low-dose and highdose DEX groups. (B) Boron concentrations (mg/g) in the blood and tumor in the control, low-dose and high-dose DEX groups. ’ = blood; = tumor.
did significantly (po0:02) reduce boron levels in this edematous zone (Fig. 2). The concentration of boron in the zone of peritumoral edema was 30.371.1 mg/g in the high-dose DEX group, and 34.271.1 mg/g and 35.370.8 mg/g in the low-dose DEX and control groups, respectively.
4. Discussion highest in the zone of peritumoral edema, with intermediate levels in the ipsilateral cerebrum. The boron content was lowest in the contralateral cerebrum (Fig. 2). This relationship of boron concentrations was maintained in the high- and low-dose DEX groups, with the highest concentration of boron in the zone of peritumoral edema and the lowest concentration in the contralateral cerebrum (Fig. 2). Levels of boron in the ipsilateral cerebrum were not affected (p > 0:05) by treatment with low- or high-doses of DEX. In the contralateral cerebrum the concentration of boron was B7% higher (po0:01) in the high-dose DEX group, than in the other two treatment groups. The low-dose DEX treatment protocol did not appreciably alter (p > 0:1) the uptake of boron in the zone of peritumoral edema relative to the controls. However, treatment with the high-dose DEX protocol
Peritumoral edema in brain tumor is routinely treated with steroids. A similar dose of DEX (high-dose group), to that used clinically, was administered in the present study. The dosage protocol was comparable with the one employed by Straathof et al. (1998), in a study involving Fischer 344 rats bearing the intracranial 9L gliosarcoma. This enabled data relating to vascular permeability, detailed by these authors, to be compared with the BPA biodistribution results reported in the present study. Using cisplatin, Straathof et al. (1998) demonstrated that the level of this water-soluble chemotherapeutic compound was a factor of 10 higher in tumor than in the surrounding normal brain. The concentration of cisplatin in the zone of peritumoral edema was also appreciably higher than in normal brain. While pretreatment with DEX had little effect on cisplatin levels
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in the 9L gliosarcoma or normal brain, it reduced the concentration of cisplatin in the zone of peritumoral edema by a factor of 2.6 (Straathof et al., 1998). In another study using the 9L gliosarcoma implanted intracranially in Fischer 344 rats, blood to tissue transport (BTT) of 14C alpha aminoisobutyric acid was estimated using double label autoradiography (Fross et al., 1991). The BTT was found to be highest in tumor and lowest in normal brain. In the zone of peritumoral edema the BTT was about 2.5 times lower than in the tumor, but was a factor of 6–9 times higher than in the normal brain. Viewed overall, the findings of these two studies (Straathof et al., 1998; Fross et al., 1991) indicate that the blood brain barrier (BBB) is disrupted in the 9L tumor and partially disrupted in the zone of peritumoral edema. The administration of DEX appears to reduce blood vessel permeability in the zone of edema. This could explain why levels of boron from BPA, in the present study, were reduced in the zone of peritumoral edema, but were not affected appreciably in tumor or normal brain, subsequent to treatment with DEX (highdose). Additional studies also indicate that the administration of DEX does not significantly affect the uptake of compounds into the normal brain, but it does appear to reduce vascular permeability in the zone of peritumoral edema (Neuwelt et al., 1982; Neuwelt et al., 1990; Braunschweiger and Schiffer, 1986; Nakagawa et al., 1987; Shapiro et al., 1990). DEX has a variable influence on the uptake of a range of compounds in different tumor types (Braunschweiger and Schiffer, 1986; Nakagawa et al., 1987; Shapiro et al., 1990; Molnar et al., 1995; Warnke et al., 1995). These differences are probably related to variations in the vascular architecture and permeability in the various tumor models, and to differences in the compounds used. Studies using the identical compound in different tumor types are limited. 14 C-alpha aminoisobutyric acid (AIB) has been used in conjunction with autoradiography to evaluate the effects of DEX on blood vessel permeability in the rat C6 and RG-2 intracranial gliomas. In the C6 glioma, BTT of 14CAIB was reduced by a factor of 4 relative to controls, and in the RG-2 glioma, BTT was not affected by treatment with DEX (Shapiro et al., 1990; Molnar et al., 1995). It is evident from the present and previous studies e.g. (Coderre et al., 1994; Morris et al., 1997) that BPA can penetrate the BBB and distribute in the normal central nervous system. However, it has been shown in rats that disruption of the BBB using mannitol increases the accumulation of boron from BPA by a factor of 2–3 in both the normal brain and the intracranial F98 glioma (Yang et al., 1996). This indicates that BPA targeting of tumor cells is susceptible to changes in vascular permeability. Further evidence for a vascular effect on BPA targeting is provided by boron microdistribution
studies using ion microscopy. These studies have demonstrated that infiltrating tumor cells from the rat F98 glioma and 9L gliosarcoma, distributed in the zone of peritumoral edema, contain B50% less boron than cells in the main tumor mass (Smith et al., 1996, 1997). Disruption of the BBB with mannitol resulted in a doubling of the boron content of these infiltrating tumor cells (Smith et al., 1996, 1997).
5. Conclusion It can be concluded that, although treatment with DEX had no appreciable effect on boron uptake in the 9L gliosarcoma (main tumor mass) or normal brain of the rat, after the administration of BPA, it did impact on the boron levels in the zone of peritumoral edema. After the high-dose DEX administration protocol, boron levels in the zone of edema were reduced by B14%. This finding suggests that BPA targeting of tumor cells in the peritumoral zone could be compromised by DEX. These cells appear to play a critical role in tumor recurrence after BNCT or conventional radiotherapy. It is essential that they are adequately targeted with boron for effective BNCT. Edema in brain tumor patients is frequently more pronounced than is the case in animal brain tumor models. The limited clinical data available indicates that vascular permeability in the region of peritumoral edema is considerably reduced after DEX administration (Jarden et al., 1985; Yeung et al., 1994). It is possible, therefore, that the negative effects of DEX on boron uptake in tumor cells in the zone of edema may be more pronounced in the clinical situation. This phenomenon could have a critical bearing on the clinical effectiveness BNCT for glioma and merits further investigation.
Acknowledgements D. Greenberg provided technical assistance related to dosimetry. GMM acknowledges funding from the UK Cancer Research Campaign. Additional support was provided by the Office of Biological and Environmental Research, US Department of Energy, under contract number DE-AC02-98CH10886.
References Barth, R.F., Soloway, A.H., Brugger, R.M., 1996. Boron neutron-capture therapy of brain-tumors—past history, current status, and future potential. Cancer Invest. 14, 534–550. Braunschweiger, P.G., Schiffer, L.M., 1986. Effect of dexamethasone on vascular function in RIF-1 tumors. Cancer Res. 46, 3299–3303.
ARTICLE IN PRESS G.M. Morris et al. / Applied Radiation and Isotopes 61 (2004) 917–921 Chanana, A.D., Capala, J., Chadha, M., et al., 1999. Boron neutron capture therapy for glioblastoma multiforme: interim results from the phase I/II dose—escalation studies. Neurosurgery 44, 1182–1193. Coderre, J.A., Button, T.M., Micca, P.L., et al., 1994. Neutron capture therapy of the 9L rat gliosarcoma using the pboronophenylalanine–fructose complex. Int. J. Radiat. Oncol. Biol. Phys. 30, 643–652. Coderre, J.A., Morris, G.M., 1999. The radiation biology of boron neutron capture therapy. Radiat. Res. 151, 1–18. Fairchild, R.G., Gabel, D., Laster, B.H., et al., 1986. Microanalytical techniques for boron analysis using the 10 B(n,a)7Li reaction. Med. Phys. 13, 50–56. Fross, R.D., Warnke, P.C., Groothuis, D.R., 1991. Blood flow and blood-to-tissue transport in 9L gliosarcomas: the role of the brain tumor model in drug delivery research. J. Neuro. Oncol. 11, 185–197. Jarden, J.O., Dhawan, V., Poltorak, A., et al., 1985. Positron emission tomographic measurement of blood-to-brain and blood-to-tumor transport of 82Rb: the effect of dexamethasone and whole brain radiation therapy. Ann. Neurol. 18, 636–646. Molnar, P., Lapin, G.D., Groothuis, D.R., 1995. The effects of dexamethasone on experimental brain tumors: I. Transcapillary transport and blood flow in RG-2 rat gliomas. J. Neuro. Oncol. 25, 19–28. Morris, G.M., Coderre, J.A., Micca, P.L., et al., 1997. Central nervous system tolerance to boron neutron capture therapy with p-boronophenylalanine. Brit. J. Cancer 76, 1623–1629. Nakagawa, H., Groothuis, D.R., Owens, E.S., et al., 1987. Dexamethasone effects on [125I] albumin distribution in experimental RG-2 gliomas and adjacent brain. J. Cerebr. Blood Flow Metab. 7, 687–701. Neuwelt, E.A., Barnett, P.A., Bigner, D.D., 1982. Effects of adrenal cortical steroids and osmotic blood brain barrier opening on methotrexate delivery to gliomas in the rodent: the factor of the blood brain barrier. Proc. Natl. Acad. Sci. USA 79, 4420–4423. Neuwelt, E.A., Horaczek, A., Hagel, M.A., 1990. The effect of steroids on gentamicin delivery to brain after blood brain barrier disruption. J. Neurosurg. 72, 123–126.
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Reichman, H.R., Farrell, C.L., Del Maestro, R.F., 1986. Effects of steroids and nonsteroid anti-inflammatory agents on vascular permeability in a rat glioma model. J. Neurosurg. 65, 233–237. Shapiro, W.R., Hiesinger, E.M., Cooney, G.A., et al., 1990. Temporal effects of dexamethasone on blood-to-brain and blood-to-tumor transport of 14C- alpha-aminoisobutyric acid in rat C6 glioma. J. Neuro. Oncol. 8, 197–204. Slatkin, D.N., 1991. A history of boron neutron capture therapy of brain tumors—postulation of a brain radiation dose tolerance limit. Brain 114, 1609–1629. Smith, D.R., Chandra, S., Coderre, J.A., Morrison, G.H., 1996. Ion microscopy imaging of 10B from p-boronophenylalanine in a brain tumor model for boron neutron capture therapy. Cancer Res. 56, 4302–4306. Smith, D.R., Chandra, S., Coderre, J.A., et al., 1997. Quantitative ion microscopy imaging of boron-10 in rat brain tumor models for BNCT. In: Larsson, B., Crawford, J., Weinreich, R. (Eds.), Advances in Neutron Capture Therapy, Chemistry and Biology, Vol. II. Elsevier Science B.V., Lausanne, pp. 308–314. Straathof, C.S.M., van den Bent, M.J., Ma, J., et al., 1998. CSM: the effect of dexamethasone on the uptake of cisplatin in 9L glioma and the area of brain around tumor. J. Neuro. Oncol. 37, 1–8. Warnke, P.C., Molnar, P., Lapin, G.D., et al., 1995. The effect of dexamethasone on transcapillary transport in experimental brain tumors: II. Canine brain tumors. J. Neuro. Oncol. 25, 29–38. Yamada, K., Ushino, Y., Hayakawa, T., et al., 1983. Effects of methylpredisolon on peritumoral brain edema: an autoradiographic study. J. Neurosurg. 59, 612–619. Yang, W., Barth, R.F., Carpenter, D.E., et al., 1996. Enhanced delivery of boronophenylalanine for neutron capture therapy by means of intracarotid injection and blood brain barrier disruption. Neurosurgery 38, 985–992. Yeung, W.T.I., Lee, T.Y., Del Maestro, R.F., et al., 1994. Effects of steroids on iopamidol blood brain transfer constant and plasma volume in brain tumors measured with X-ray computed tomography. J. Neuro. Oncol. 18, 53–60.