In Vitro Determination of Uptake, Retention, Distribution, Biological Efficacy, and Toxicity of Boronated Compounds for Neutron Capture Therapy: A Comparison of Porphyrins with Sulfhydryl Boron Hydrides1

A major problem remaining in the evaluation of boronated compounds for neutron capture therapy (NCT) is the need to know the intraor extracellular microdistribution of boron. This is a consequence of the short range of the '°B(n,a)7Lireaction products (-10 urn), such that biological efficacy is dependent upon intracellular distribution. In partic ular, if boron location is predominantly extracellular, a significant reduc tion in efficacy would be expected. The in vitro procedure described here was developed mainly to provide information regarding the intraand extracellular location and concentra tion of boron. I lowever, use of the technique also allows the measurement of compound uptake and retention (binding) and the determination of biological efficacy by the evaluation of survival curves obtained following irradiation with thermal neutrons. Comparison is made to results obtained with boric acid ill,'"HO,i and to results calculated for various boron distributions. Concomitanti), an indication of compound toxicity can be obtained from the plating efficiency of unirradiated control cells. Currently, most investigators utilize in vivo systems for testing and evaluating boron uptake from various carrier molecules. Given the large number of boron compounds being synthesized and needing evaluation as to their usefulness for NCT, the in vitro technique described here is simple and advantageous for initial compound screening. In addition to sparing animal lives, it is both time and cost effective and utilizes much smaller quantities of test compound than are required for an in vivo assay. A boronated porphyrin (BOPP) evaluated by the above procedure shows an uptake and retention ~20 times that of sulfhydryl boron hydride monomer (BSH); the latter compound is currently being used clinically for NCT in Japan and is anticipated for use in clinical trials in the United States. If the advantages demonstrated by BOPP in these /// vitro studies are validated in animal experiments, BOPP should be considered for clinical application. INTRODUCTION NCT' is a binary system in which boron is transported to tumor, and an external neutron beam is used to deliver thermal neutrons to produce the 10B(n,«)7Li reaction. The short range of the high linear energy transfer (LET) reaction products (-10 ¡im,or one cell diameter) results in a severe dependency of the biological efficacy (ability to kill cells) on boron microlocalization. For a typical cell, it is calculated that -10 times more boron is needed if it is located extracellularly, as opposed to intracellularly (1, 2). One of the major problems remaining in the evaluation of Received 1/3/90: revised 4/11/90. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Research carried out under the auspices of the United Slates Department of Energy under contract DE-AC02-76CH00016 and contract CA 37961 with NIH. 2To whom requests for reprints should be addressed, at Medical Department. Brookhaven National Laboratory, Associated Universities, Upton. Long Island. NY 11973. 3The abbreviations used are: NCT, neutron capture therapy; BSH. monomer form of Na2B,;H,,SH; BSSB, dimer form of NajB.jHnSH; BOPP. hematoporphyrin-like polyol porphyrin: PBS. phosphate-buffered saline; BMRR. Brookha ven Medical Research Reactor: O0. dose to reduce survival by a factor of ~e~; PE. plating efficiency. various compounds for NCT is the need to determine intracel lular versus extracellular distribution of boron (3, 4). Current analytical procedures, including neutron activation analysis by prompt-7 emission (5), inductively coupled plasma-atomic emission spectroscopy (6), and track-etching techniques (7), do not have resolutions requisite for measurement of intracellular distribution. Thus, this important parameter has been unavail able for compound evaluation. The in vitro procedure described here was developed mainly to provide information regarding boron location within cells. However, use of the procedure also allows the measurement of compound uptake and retention (binding) and the determina tion of biological efficacy by the evaluation of survival curves obtained following irradiation of boronated and nonboronated control cells with thermal neutrons. Concomitantly, an indica tion of compound toxicity can be obtained from the plating efficiency of unirradiated control cells. Briefly, cells are grown for one mitotic cell cycle in the presence of the boron-containing compounds to be tested and then irradiated in the presence of the same compound; similar irradiations are then carried out after a thorough washing of the cells and suspension in boronfree media. Biological response is then compared to that ob tained after irradiation of cells in known amounts of boric acid (H,'"BO.,), in which a homogeneous intraand extracellular distribution is assumed. A comparison of results indicates whether the test compound is taken into the cell, whether it is retained (binds) despite washings, and how much is present in terms of known amounts of H,IOBO3 (boric acid equivalents). Gross (average) amounts of 10B retained in washed cells can then be determined by the prompt--/ method (5). The evaluation of biological efficacy in terms of boric acid equivalents in addition to the quantification of the average cellular boron content can then, in principle, indicate the intracellular (or extracellular) distribution. The analysis utilizes Monte-Carlo calculations of nuclear dose as a function of boron location as described by Gabel et at. (1). This procedure was used to determine the uptake, retention, distribution, biological efficacy, and toxicity of a boronated natural porphyrin synthesized by one of us (S. B. K.). Also, information bearing on the mechanism of porphyrin incorpo ration was obtained by measuring uptake as a function of duration of exposure to the drug. Porphyrins are expected to be a particularly effective vehicle for boron transport; they have been suggested for NCT since they are known to be taken up avidly by all tumors investigated to date, to have long-term retention in tumors, and to have a singularly large boroncarrying capacity (8, 9). In addition, BSH and BSSB were evaluated. BSH has been used in Japan to treat >100 patients with brain tumors with NCT and has been proposed for further clinical trials by a number of groups in the United States and Europe (3, 4). BSSB has been shown to have better uptake and retention in tumors than BSH (10, 11) and, in addition, has been shown to be more 4860 on July 24, 2017. © 1990 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from AN IN VITRO TECHNIQUE FOR NCT effective than BSH in the treatment of animal tumors (11-14). Our comparison of BSH and BSSB with the porphyrin, using this system, indicates that BSSB is taken up and bound more effectively than BSH and that the porphyrin is at least 4 times more effective than BSSB. MATERIALS AND METHODS Boronated Porphyrins. We have synthesized and characterized an unusual tetracarborane carboxylic acid ester of BOPP, with low toxicity, in which high aqueous solubility was achieved by two propionic acid side chains (15, 16). The 95% '0B-enriched potassium salt of the compound has a molecular weight of 1363, so that the molecule is 29.3% boron by weight. Na2B|2HnSH and Na4B24H22S2. These compounds 95% enriched in IOBwere purchased from the Gallery Chemical Co., Gallery, PA. Preirradiation Cell Procedures. V-79 Chinese hamster cells in loga rithmic growth were propagated in Dulbecco's modified Eagle's media (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 1% penicillin-streptomycin-fungisone (Gibco), and 2.0 mivi L-glutamine (Gibco). BSH, BSSB, boric acid (H3BO3), or BOPP were added to the growth medium at a 10B concentration of ~30 ppm for each experiment. After 12 h, the boronated medium was aspirated from above the cell monolayer. Washing Procedures. Cells which were to be irradiated in a boroncontaining medium ("ambient" experiments) were processed with re agents (PBS, trypsin) containing 30 ppm 10Bfrom the experimental compound. In the experiments using BOPP, the cells did not survive trypsinization when the trypsin contained 30 ppm 10Bof BOPP (cause unknown); therefore, BOPP was excluded from the trypsinization pro cedure. Cells were suspended in boron-containing medium (30 ppm 10B)in preparation for irradiation. In the "washed" experiments, every effort was made to remove unbound boron prior to irradiation in boronfree growth medium. Cells which were to be irradiated in a boron-free environment were washed 3 times with PBS, trypsinized, and harvested with boron-free reagents prior to suspension and irradiated in boronfree medium. Cell Irradiations. Cells were irradiated in suspension in growth me dium at a population density of 3.0 x IO5 cells/ml. Irradiations were carried out at the thermal neutron beam port of the BMRR within 12 h following suspension in growth medium. The thermal neutron fluence rate at the center of the sample (1.5-ml Eppendorf microfuge tubes) was 2.8 x 10" n cm"2 min"'. Beam parameters are summarized in Table 1. The irradiation apparatus is described in Ref. 17. Survival Assay. Cells were plated for undisturbed colony growth for 5-6 days. Colonies were then washed with PBS, fixed with buffered formalin, and stained with Giemsa, prior to optoelectronic counting with an Artec colony counter. Survival Curve Analysis. Data were analyzed on the basis of D0s obtained from the linear portion of the survival curves. Curves were fitted with the mult ihil formula (18): 5=1 -[1 exp(-ö/D„)r, Table 1 Dose rates for cell irradiations at the BMRR Power level = 1 MW, thermal neutron flux density = 2.8 x 10" n/(cm2 min)