During conventional radiotherapy, the dose that can be delivered to the tumor is limited by the tolerance of the surrounding normal tissue within the treatment volume. Boron Neutron Capture Therapy (BNCT) represents a promising modality for selective tumor irradiation. The key to effective BNCT is selective localization of {sup 10}B in the tumor. We have shown that the synthetic amino acid p-boronophenylalanine (BPA) will selectively deliver boron to melanomas and other tumors such as gliosarcomas and mammary carcinomas. Systemically delivered BPA may have general utility as a boron delivery agent for BNCT. In this paper, BNCT with BPA is used in treatment of experimentally induced gliosarcoma in rats and nonpigmented melanoma in rabbits. The tissue distribution of boron is described, as is response to the BNCT. 6 refs., 4 figs., 1 tab

Coderre, J.A.

Greenberg, D.

Joel, D.D.

Micca, P.L.

Packer, S. (North Shore Univ. Hospital, Manhasset, NY (USA). Div. of Ophthalmology)

Saraf, S. (Brookhaven National Lab., Upton, NY (USA))

UNT Digital Library

,—46148DE91 013808BORON NEUTRON CAPTURE THERAPY OF OCULAR MELANOMA AND INTRACRAN1ALGLIOMA USING p-BORONOPHENYLALANINE.3A. Coderre, S. Packer1, D. Greenberg, P.L. Micca, D.D. Joel and S. SarafMedical Department, Brookhaven National Laboratory, Upton, NY 11973, USA.department of Ophthalmology, North Shore University Hospital, Manhasset, NY 11030,USA.INTRODUCTIONDuring conventional radiation therapy, the dose that can be delivered to the tumor is limited bythe tolerance of the surrounding normal tissue within the treatment volume. Boron neutron capturetherapy (BNCT) represents a promising modality for selective irradiation of tumor tissue. The key toeffective BNCT is the selective localization of 10B in the tumor. The ratio of the 10B concentration in thetumor to that in the normal tissues within the treatment volume will largely determine the therapeutic gainthat can be achieved in any subsequent irradiation.We have shown that intragastric (i.g.) administration of the synthetic amino acid p-boronophenylalanine (BPA) as an aqueous slurry at neutral pH is an effective method of drugadministration (Coderrc, 1988). BPA is non-toxic even at very high doses, is rapidly solubilized in theacidic environment of the stomach and is absorbed in the gastrointestinal tract. The time-course of tumoruptake and clearance of boron following i.g. doses was similar to that observed following i.p. doses cfBPA. BPA is an analogue of the melanin precursor tyrosine and as such was originally intended as anagent for BNCT of melanoma. Subsequent studies with BPA have shown that this amino acid analogueis capable of selectively delivering boron to tumors other than melanoma; a murine mammary carcinomaand a rat gliosarcoma (Coderre, 1990). These results indicate that systemically administered BPA mayhave a general utility as a boron delivery agent for BNCT.Our therapy experiments have progressed from irradiation of s.c. murine thigh tumors (Coderre,1988) to the use of more sophisticated tumor models in order to demonstrate the ability of BNCT toeffectively treat a tumor in the presence of radiosensitive normal tissues. Thermal neutron irradiationshave been carried out at the Brookhaven Medical Research Reactor (BMRR), following i.g.administration of BPA, using the non-pigmented Greene melanoma carried in the anterior chamber ofthe rabbit eye and the GS-9L gliosarcoma implanted in the rat brain.METHODSBPA (as the free araino acid) was administered i.g. by transesophageal intubation with a feedingtube, as an aqueous slurry at neutral pH. Animals received a dose of approximately 750 mg BPA per kgbody weight; rats received either 150 mg of L-BPA or 300 me of D.L-BPA per dose in 3 ml of water,rabbits received up to 2.0 g of D.L-BPA b 15 ml of water. ^B analysis was performed by the promptgamma spectroscopic technique. D l S T RlBUTION OF THIS DOCUMENT IS UNUMITEOThe rabbit ocular melanoma model. The Greene melanoma forms an amclanotic tumor whentransplanted into the anterior or posterior chamber of the rabbit eye. During surgical procedures, rabbitswere anesthetized with intramuscular ketaminc hydrochloride (35mg/kg)/xylazine (5mg/kg). Irismelanomas were initiated in New Zealand White rabbits by implantation of approximately 1 mm3fragments of tumor tissue into the anterior chamber through an incision in the sclera at the iimbus.Tumor size was measured with a hand-held caliper; 2 perpendicular dimensions were recorded. Tumorsgrew to about 200 mg in 2-3 weeks.The rat glioma model. Subcutaneous (s.c.) tumors were initiated in F-344 rats by the injectionof 5 x 106 cultured GS-9L gliosarcoma cells in 0.1 ml of growth medium. Tumors were palpable 4-5 daysafter inoculation and had a volume-doubling time of approximately two days. For initiation of braintumors, 10 cultured cells in 1 /il of medium were injected, at a depth of 3-4 mm, into the left frontal lobe(Joel, 1990). This technique produced a locally expanding tumor with no evidence of blood-bornemetastasis or sesd;ng to ventricular or leptomeningeal surfaces. Rats survived an average of 22 ± 4 (SD)days after implantation.RESULTS AND DISCUSSIONBPA distribution studies in the rabbit. Figure 1 shows the boron concentrations in the anteriorchamber melanoma, blood and lens following a single i.g. dose of BPA. At 20 hrs post-administrationthere was « 2 0 \ig 10B/g tumor with a tumor-to-blood concentration ratio of «4:1. 10B concentrationsin liver and muscle (not shown) were similar to those observed in the blood. Quantitative neutron captureradiography of tumor-bearing eyes following BPA administration indicated that the 10B concentration innormal eye tissues was on the order of 3-5 pg/g with the exception of the lens, which showed higher levelsof 10B (10-15 fig/g) at the periphery. BNCT irradiations were carried out 20 hours after a single i.g. doseof BPA.BPA distribution studies in the rat. The time course of 10B accumulation and washout in thetumor as well as the maximum concentration of '°B attained in the tumor were not significantly differentwhen BPA was given as either the purified L-enantiomer (150 mg) or as the racemic mixture (300 mg).Evidently the D-enantiomer is not absorbed efficiently from the gastrointestinal tract and contributes littleto the levels of 10B in normal tissues.Figure 2 shows the concentration of 10B in tumor and normal tissues following i.g. administrationof BPA (300 mg D,L-BPA per dose). The slow rate of accumulation of boron in the tumor followinga single i.g. dose of BPA (Fig. 2) suggested that a second i.g. dose might produce an additive effect. This£oenCDCP• tumorA lenso blood10 20 30 40Time (hrs)50Figure 1. 10B concentrations in anterior chamber^ Greene melanoma, blood, and lens versus timefollowing one i.g. dose of BPA b the rabbit. Individual data points are shown.was confirmed by comparison of the time courses of boron uptake and clearance from tumor, blood andnormal tissues in intracerebral GS-9L rat gliomas following a single i.g. dose of BPA and following twoi.g. doses of BPA given 3 hours apart (Fig. 2). The second dose in the sequence substantially increasedthe maximum concentration of 10B in the tumor, from 24 to 39 ng 10B/g. Blood l0B was also increased,but a tumor/blood 10B concentration ratio of as 3.3:1 was maintained. BNCT irradiations were carriedout eight hours after the first of two i.g. doses given three hours apart.Dosimetry. The dose from BNCT irradiations is expressed as gray-equivalent (Gy-Eq), calculatedby summation of physical doses (Gy) of the component radiations multiplied by appropriate relativebiological effectiveness (RBE) factors. RBE values of 2.3 and 2.0 have been assumed for the ionizingparticles from the 10B(n,a)7Li and 14N(n,p)14C reactions, respectively, and 2.0 has been assumed for fastneutrons.Tumor-bearing rabbits were irradiated for 6 minutes at 1 megawatt reactor power (6MW-min)using a collimator with a 1.55 cm diameter aperture. The thermal neutron fluence at the tumor positionwas 5.3 x 10 n,|,cm'2. The contributions from the beam components to the total dose within thetreatment volume for the 6 MW-min eye irradiation were as follows: fast neutrons, 1.56 Gy; photons fromthe reactor and the collimator, 0.80 Gy; products of the 14N(n,p)14C reaction, 1.03 Gy; and particles fromthe 10B(n,a)7Li reaction, 0.46 Gy per fig of 10B present per gram wet tissue. In the BPA-BNCT group,the B concentration in tumor and normal tissue at the time of irradiation was 20 and 3 JJg/g,respectively. The total radiation dose was calculated to be 12.62 Gy (29.05 Gy-Eq) to the tumor and 4.77Gy (9.44 Gy-Eq) to the normal eye tissues (cornea, retina). These data imply an average therapeutic gainof approximately 3. The B distribution within the lens was non-uniform, as determined by neutroncapture radiography, a local 10B concentration of 15 fig 10B/g would result in a dose of 10.3 Gy (21.9 Gy-Eq).Tumor-bearing rats were irradiated for 4 minutes at a reactor power of 1.25 megawatts (5 MW-min) using a collimator with a 1.1 cm diameter aperture. The thermal neutron fluence at the tumorcenter was 2.0 x 10 nthcm"2. The contributions from dostmetricaUy significant beam components to thetotal dose within the treatment volume for a 5 MW-min brain irradiation were as follows- fast neutrons,1.35 Gy; photons from the reactor and the collimator, 0.55 Gy; products of the 14N(n,p)14C reaction, 0.38Gy; and particles from the 10B(n,a)7Li reaction, 0.17 Gy per fig of 10B present per gram wet tissue. Inthe BPA-BNCT group, boron concentrations in the tumor, normal brain, and the blood at the time ofirradiation were estimated to be 39, 12 and 10 p% 10B/g, respectively. The total radiation dose wasE605040302010I r— j -r Ione dose• tumorv bloodi ' ' I 'two doses* tumorD blood• livermusclebrain•I :12 15Time (hrs)Figure 2. 10B concentration in intracranial GS-9L gliosarcomas and normal tissues versus timefollowing either one i.g. dose of BPA at t=0 hrs or two i.g. doses of BPA at t=0 and t=3hours. The experiment was; carried out 18 days after tumor initiation. Each point represents4 or 5 rats, mean 1 1 SD.calculated to be 8.9 Gy (19.3 Gy-Eq) to the tumor, 4.3 Gy (8.7 Gy-Eq) to the blood, 4.0 Gy (7.9 Gy-Eq)to the brain, and 4.1 Gy (8.2 Gy-Eq) to the capillary endothelium. The dose to the capillary endothelialcell was calculated as 1/3 of the dose to the blood plus 2/3 of the dose to the normal brain. These dataimply average therapeutic gains of 2.2 - 2.4.BNCTof ocular melanoma. A total of 31 rabbits with anterior chamber melanomas were dividedinto three groups. Figure 3 shows the tumor growth versus time in the groups that received no treatment(Fig.3A), reactor irradiation only (Fig.3B), or BPA-based BNCT (Fig.3C). Three of 13 BNCT-treatedtumors grew (Fig.3C); the remaining tumors were monitored for various periods of time. Rabbits withcontrolled tumors were euthanized at increasing time intervals and the eyes examined histologically. Atapproximately 3 to 6 months post-irradiation cataract formation was observed in treated eyes as well asa mild to moderate keratitis; the vasculature in the treated eyes appeared normal. All 9 tumors in thereactor-irradiation-only group (Fig.3B) as well as all 9 control tumors (Fig.3A) grew at roughly the samerate.CslCDOoE0 20 40 60Time (days)100Figure 3. Growth of the Greene melanoma in the anterior chamber of the rabbit eye (tumor areaversus time) following (A) no treatment (n=9); (B) reactor irradiation only (n=9) and (C)BPA-based BNCT (n=13).BNCT of iniracranial glioma. A total of 44 rats bearing intracerebral tumors were divided intothree treatment groups; 1) no treatment (n=13); 2) reactor irradiation alone (n=15); and 3) reactorirradiation after two i.g. doses of BPA (n = 16). Reactor irradiations alone caused a transient weight loss.There was no evidence of radiation-induced nasopharyngitis. No post-irradiation supportive therapy wasgiven. Figure 4 shows survival versus time following tumor initiation. The median survival of untreatedrats with implanted gliosarcomas was 22 ± 4 (SD) days. Reactor irradiation was performed on day 14after tumor initiation. Rats receiving reactor irradiations alone had a median survival of 25 days. Therewere no survivors in group 1 or group 2. The median survival of rats in group 3 was 80 days. Seven ofthe 16 animals in group 3 are long-term survivors; 4 are alive 13 months post-irradiation and 3 are alive10 months post-irradiation. All of the seven long-term surviving rats developed cataracts in the left eye3 to 6 months after BNCT; 4 developed cataracts in the right eye 6-9 months after irradiation.Joel et al. (1990) have recently reported the results of successful BNCT trials in the GS-9L ratgliosarcoma usmg the dimer form of the sulfhydryl borane (Na4B24H22S2). There are differences in thedistributions of B in blood and normal brain between BPA and dimer which may have radiobiologicalimplications in vivo. Table 1 shows, for BPA and the two dimer experiments reported by Joel et al.(1990), radiation doses to critical tissues based on comparable tumor 10B concentrations and thecorresponding fractions of long-term survivors. These data suggest a dose-response relationship with a50% tumor control dose of roughJy 23 Gy-Eq. Barker et al. (1979) reported a long-term survival of only1 of 20 rats bearing the intracerebral GS-9L gliosarcoma following 20 Gy of x-rays to the whole head.Joel et al. (1990) also reported long-term survival fractions of 1/10 and 4/16 following x-ray doses of 15.0and 22.5 Gy, respectively. X-ray doses in the 20 Gy range may be effective in controlling tumor growthin a small percentage of animals but have also been reported to cause delayed brain damage up to twoyears later (van der Kogel, 1983).>in100755025 -irradiation + BPA1 (n=M6)control (n = 13)•irradiation only (n=15)50 100 150 200 250Days post-implantationFigure 4. Survival of control and BNCT-treated rats bearing intracerebral GS-9L gliosarcomas versustime. Control rats (n=13) received no treatment. All reactor irradiations were performed14 days following tumor initiation. Fifteen rats' received reactor irradiation only. Sixteentumor-bearing rats received 2 i.g. doses of BPA (300 mg D.L-BPA each) given 3 hoursapart; the rats were irradiated 5 hours after the second dose.Table 1. BNCT of the Intracerebral GS-9L Gliosarcoma: Comparison of BPA with the SulfhydrylBorane Dimer.BNCT Treatment ConditionsDimer BPA DimerSMW-min1 5MW-min2 7.5 MW-min3Tumor Dose (Gy-Eq) 14.2 19.3 25.6Endothelial Cell Dose (Gy-Eq) 8.8 8.2 15.2Long-Term Survivors 2/12 7/16 6/101 At the time of irradiation, 10B concentrations in tumor, blood and brain were 26, 35, and 1.410B/gram, respectively (Joel, 1990).2 At the time of irradiation, 10B concentrations in tumor, biood and brain were 39, 12, and 1010B/gram, respectively.3 At the time of irradiation, 10B concentrations in tumor, blood and brain were 35, 45, and 1.410B/gram, respectively (Joel, 1990).The radiation dose to the vascular endothelium in the brain is more significant, in terms ofdelayed damage, than similar doses to brain parenchyma (Hopewell, 1989). The lower 10B concentrationsin blood following BPA administration compared to the concentrations in blood following dimer infusionand the resulting lower radiation doses to the vascular endothelium may be advantageous in theprevention of delayed damage to the vasculature.ACKNOWLEDGEMENTSThe research described in the report involved animals maintained in animal care facilities fullyaccredited by the American Association for Accreditation of Laboratory Animal Care. This research wassupported b part by the US Department of Energy under contract DE-AC02-76CH00016, and by GrantNo. CA42446 (JAC) from the NIH.REFERENCESBarker, M., D.F. Deen, and D.G. Baker, BCNU and x-ray therapy of intracerebral 9L rat tumors. Int. J.Rad. Oncol. Biol. Phys. 5:1581-1583 (1979).Coderre, J A., JA. Kalef-Ezra, R.G. Fairchild, P.L. Micca, L.E. Reinstein, and J.D. Glass, Boron neutroncapture therapy of a murine melanoma. Cancer Res. 48:6313-6316 (1988).Coderre, JA., J.D. Glass, R.G. Fairchild, P.L. Micca, I. Fand, and D.D. Joel, Affinity of the melaninprecursor analog p-boronophenylalanine for tumors other than melanoma. Cancer Res. 50:138-141 (1990).Hopewell, J.W., W. Calvo, D. Campling, H.S. Reinhold, M. Rezvani, and T.K. Yeung, Effects of Radiationon the microvasculature: Implications for Normal-Tissue Damage. In Radiation Tolerance of NormalTissues. Frontiers of Radiation Therapy and Oncology Vol. 23:85-95 (1989).Joel, D.D., R.G. Fairchild, JA. Laissue, S.K. Saraf, JA. Kalef-Ezra, and D.N. Slatkin, Boron NeutronCapture Therapy of Intracerebral Rat Gliosarcomas. Proc. Natl. Acad. Sci, USA (1990) in press.van der Kogel, AJ., The cellular basis of radiation-induced damage in the central nervous system.In: Cytotoxic Insult to Tissue; Effects on Cell Lineages (C.S. Potten, and J.H. Hendry, eds) pp. 329-352,Churchill Livingstone, New York, 1983.DISCLAIMERThis report was prepared as an account of work sponsored by an agency of theUnited States Government. Neither the United States Government nor any agencythereof, nor any of their employees, makes any warranty, express or implied, orassumes any legal liability or responsibility for the accuracy, completeness, or use-fulness of any information, apparatus, product, or process disclosed, or representsthat its use would not infringe privately owned rights. Reference herein to any spe-cific commercial product, process, or service by trade name, trademark, manufac-turer, or otherwise does not necessarily constitute or imply its endorsement, recom-mendation, or favoring by the United States Government or any agency thereof.The views and opinions of authors expressed herein do not necessarily state orreflect those of the United States Government or any agency thereof.

Boron neutron capture therapy of ocular melanoma and intracranial glioma using p-boronophenylalanine

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Boron neutron capture therapy of ocular melanoma and intracranial glioma using p-boronophenylalanine

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