[CANCER RESEARCH 50. 4860-4865. August 15. 1990|
`
`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
`
`R. G. Fairchild,2 S. B. Kahl, B. H. Laster, J. Kalef-Ezra, and E. A. Popenoe
`
`Brookhaven National ¡Mboratory,l'pton, New York 11973 ¡R.G. F., B. H. L., J. K-E., E. A. P.]; University of California, San Francisco. California 94143 fS. B. K.];
`and L'niversity of loannina, ¡oannina,451.10 Greece / J. K-E.J
`
`ABSTRACT
`
`A major problem remaining in the evaluation of boronated compounds
`for neutron capture therapy (NCT)
`is the need to know the intra- or
`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 intra- and extracellular
`location and concentra
`tion of boron. Ilowever, 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.
`
`for NCT is the need to determine intracel
`various compounds
`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 boron-
`free 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
`intra- and 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).
`retention,
`This procedure was used to determine the uptake,
`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 boron-
`carrying 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
`
`INTRODUCTION
`to
`NCT'
`is a binary system in which boron is transported
`tumor, and an external neutron beam is used to deliver thermal
`neutrons
`to produce the 10B(n,«)7Lireaction. 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 microlocaliza-
`tion. 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
`
`revised 4/11/90.
`Received 1/3/90:
`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. hematopor-
`phyrin-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.
`
`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2002 - 1/6
`
`

`
`AN IN VITRO TECHNIQUE FOR NCT
`
`tumors (11-14).
`effective than BSH in the treatment of animal
`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-fun-
`gisone (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 boron-
`ated medium was aspirated from above the cell monolayer.
`Washing Procedures. Cells which were to be irradiated in a boron-
`containing 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 boron-
`free 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 boron-
`free 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 1-
`2 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)
`
`and D0s were obtained from this fit, where S is survival, D is dose, and
`n is extrapolation number.
`Evaluation of Experimental Compounds in 11,'"110, Equivalents. The
`radiation response of cells containing boron compounds with unknown
`distributions was evaluated relative to the response obtained with
`known amounts of 10B from H3'°BO3;it
`is assumed that H3'°BO3
`distribution is uniform and homogeneous
`throughout
`the cells in sus
`pension. For experiments described here, V-79 cells were grown in the
`presence of H3'°BO3inconcentrations of 15 and 30 ^g '°B/gof growth
`medium. Survival curves for H1'°BOiare shown in Fig. 1. If one
`evaluates the slope of the survival curve in the linear portion when
`plotted on semilog paper,
`the inverse of the slope is the dose (D0) in
`terms of time, neutron fluence, or absorbed dose that it takes to reduce
`survival by a factor of "e" (i.e., by 63%). A> is expressed in units of
`time throughout
`this paper, as the latter is the most basic parameter.
`Conversion to absorbed dose, biologically effective dose,
`fluence of
`thermal neutrons, etc. can be carried out directly using data in Table 1.
`DOin rad or cGy is equal to the sum of the mixed field dose components
`in iXiy 'min (as given in Table 1), multiplied by irradiation time in min.
`The slope itself will be a linear function of boron concentration,
`i.e.,
`
`—¿(cid:3)I/Do = a + 2.4C, where a is the contribution
`from the sum of
`components other than 10Bin rad/min (i.e., the "background" radiation)
`and C is the fractional concentration of IOBfrom H3'°BO3(see Table
`1). A plot of I/Do versus 10Bcontent provides a graph with 1 dependent
`variable from which the response of a compound with unknown con
`centration and distribution can be evaluated in terms of an equivalent
`amount of IOBfrom H3'°BO3(Fig. 2) (17, 19). Thus,
`if the negative
`inverse of D0 for any survival experiment
`is evaluated from Fig. 2, the
`boron response in terms of an equivalent concentration of H3'°BO3in
`Mg 10B/g can be obtained (boric acid equivalents)
`free of effects from
`contaminating radiations.
`Boron Analysis by Prompt--/ Neutron Activation Analysis. Unless
`otherwise noted, the growth medium in each experiment was prepared
`with a boron concentration
`calculated to be 30 ¿ig'°B/g.This was
`verified by analyzing 0.5 ml of media from each experiment by the
`prompt-7 technique at the BMRR facility (5). For analysis of IOBin
`cells, ~108 cells (~0.1 g) were grown in boronated growth media and
`then washed, pelletized, and analyzed by prompt-^. This technique is
`capable of measuring 0.5 ppm IOBwith ~15% accuracy in 200 s.
`
`100
`
`10
`
`Ü
`tr
`
`ComponentFast
`neutrons
`
`6
`6
`14N(n,p)14CTotal'°B(n,«)7Li*cGy/min13
`3.122.62.4CCcGy
`6.238.2%
`
`x RBE
`/min"26
`
`of total
`effective
`doserate68
`
`16
`16100
`
`10
`8
`6
`IRRADIATION TIME (min)
`Fig. 1. Survival curves for V-79 Chinese hamster cells incubated for 12 h in
`the presence of 15 and 30 ng '°B/ml of growth medium from 3H'°BO,and
`irradiated in the presence of the same '°Bconcentrations
`at the thermal neutron
`beam of the BMRR.
`4861
`
`°A relative biological effect (RBE) of 2 is assumed for fast neutrons and the
`"N(n,p)MC reaction (21).
`* Dose rate for fVBOs;
`assumed.
`' C is concentration of 10Bin /ig'°B/gcell.
`
`a uniform extra- and intracellular distribution is
`
`0.1
`
`24
`
`12
`
`14
`
`ANACOR EX. 2002 - 2/6
`
`

`
`AN /.V VITRO TECHNIQUE FOR NCT
`
`I
`
`I
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`!
`
`!
`
`I
`
`I
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`!
`
`!
`
`!
`
`I
`
`I
`
`I
`
`I
`
`I
`
`2.0
`
`1.8
`
`1.6
`
`1.4
`
`1.2
`
`1-°
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`I
`
`I
`
`I
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`I
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`8
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`I
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`I
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`I
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`I
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`I
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`I
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`I
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`I
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`I
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`I
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`I
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`I
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`I
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`I
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`I
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`12
`
`16
`
`20
`
`24
`
`28
`
`32
`
`36
`
`40
`
`Fig. 2. Slope of survival curves represented by Fig. 1, plotted versus boron
`content
`in growth medium. Data point at 0 boron content
`is the average of 11
`conlrol cunes.
`
`RESULTS
`
`for each
`and measured parameters
`conditions
`Experimental
`individual experiment
`are summarized in Table 2. Representa
`tive survival curves are shown in Figs. 1, 3, 4, and 5. Values for
`boric acid equivalents were obtained from the average of repli
`cate experiments
`(Table 3).
`BSH. Survival curves for BSH are shown in Fig. 3; data from
`individual experiments
`are given in Table 2. Averaged values
`of/Jo are summarized in Table 3, in units of time of irradiation
`at a reactor power of 1 MW. Actual doses can be obtained from
`Table 1. The biological
`response of BSH can be expressed in
`boric acid equivalents
`as obtained from Fig. 2; these data are
`also summarized in Table 3. It is apparent
`that (a) most of the
`BSH is easily washed out because the radiation
`response
`is
`equivalent
`to only 0.5 ng IOB/g (boric acid equivalents = 0.5 ng
`loB/g) and (¿>)even under ambient conditions
`(i.e.,
`irradiation
`in the presence of 30 ppm IOBfrom BSH) most of the boron is
`excluded from the cell (response in boric acid equivalents = 9.5
`^g 10B/g).
`BSSB. Results with the dimer are shown in Fig. 4. Average
`
`D0s and boric acid equivalents are recorded in Table 3. As with
`BSH,
`it is apparent
`that most of the compound is washed out
`and that most of the boron is excluded from the cell. However,
`it is clear that BSSB has better retention and uptake relative to
`BSH.
`BOPP. Survival curves for BOPP are given in Fig. 5. It is
`obvious that
`the biological efficacy of BOPP is significantly
`greater
`than that of the sulfhydryl boron hydrides.
`In terms of
`boric acid equivalents (Table 3), it is evident that in experiments
`with cells maintained under ambient conditions,
`the effective
`boron concentration
`is greater
`than the equilibrium concentra
`tion (i.e., BOPP is concentrated in cells) and that under washed
`conditions,
`retention
`is ~4 and ~20 times greater
`than that
`obtained with BSSB and BSH, respectively.
`To gain further
`insight
`into the mechanisms of porphyrin
`uptake, washed experiments
`similar
`to those shown in Fig. 5
`were carried out, but
`the time of exposure
`to the drug was
`varied from 1-18.5 h. Biological efficacy in terms of boric acid
`equivalents is summarized in Table 4 and plotted in Fig. 6 after
`normalization
`to 30 ppm IOBin the growth medium (i.e., boric
`acid equivalents were adjusted to reflect
`linearly projected up
`take for cells grown in 30 ppm 10B/g growth medium). Follow
`ing an initial rapid uptake of ~3 ßg'°B/gwithin the first hour,
`accumulation is seen to progress linearly at a rate of ~1 ng 10B/
`g/h. In one experiment boron concentration
`in growth medium
`was deliberately reduced to 2.3 ng 10B/g. Normalization
`to 30
`tig '°B/g showed that
`response was proportional
`to boron
`content
`in media (O, Fig. 6),
`thus
`showing an absence of
`saturation effects at the higher boron concentrations.
`Survival curves were also obtained following 12 h growth in
`BOPP and subsequent
`irradiation with l37Cs -y-rays at 50 rad/
`h. No radiation sensitization (dose enhancement) was observed
`for BOPP with the 661 keV photons (data not shown).
`
`DISCUSSION
`
`If actual boron concentra
`Evaluation of Boron Distribution.
`tion within or on the cell
`is measured
`for an "unknown"
`compound,
`an evaluation of its location can be made if calcu
`lations are available giving biological efficacy as a function of
`cellular
`location. This procedure
`has been followed for
`the
`"washed" experiments.
`It is possible to grow large amounts of
`cells (~100 mg, or ~108 cells), and following removal of sur
`rounding media and thorough washing,
`to measure IOBretained
`in cells by prompt--y analysis.
`Monte-Carlo calculations have shown that for a hamster V-
`70 cell (with a spherical volume of ~1150 /¿m1and a centrally
`
`Table 2 Experimental conditions and measured parameters for individual experiments used in preparing Fig. 2 and Table 3
`Compounds evaluated
`
`H3'°BO,
`
`BSH
`
`BSSB
`
`BOPP
`
`Experimental parameters and
`status during irradiation
`Boron concentration
`in
`growth media (/ig 10B/g)
`Ambient"AmbientWashed*Plating
`
`
`
`efficiency(%)AmbientAmbientWashedDO
`
`(min)AmbientAmbientWashed13.829.551.055.00.950.6014.830.065.065.01.10.6028.140.864.081.01.353.427.924.688.082.01.453.628.027.560.069.01.22.227.528.776.099.01.12.430.228.265.081.00.551.230.725.777.072.00.401.126.583.01.4
`
`indicate that cells were irradiated in growth medium containing the same concentration of boron compound as they were grown in.
`irradiations
`" Ambient
`* Washed conditions indicate that cells were washed and irradiated in boron-free medium.
`
`4862
`
`ANACOR EX. 2002 - 3/6
`
`

`
`AN IN VITRO TECHNIQUE FOR NCT
`
`!
`
`!
`
`BSH
`30ng10B/ml
`
`—¿(cid:3)
`
`I
`:
`
`100
`
`•¿(cid:3)CONTROL CELLS -
`o WASHED CELLS
`AMBIENT CELLS
`
`•¿(cid:3)CONTROL CELLS
`o WASHED CELLS
`A AMBIENT CELLS
`
`>r
`
`rz
`
`>
`CO
`
`Ü
`CE
`LU
`CL
`
`2
`
`8
`6
`4
`IRRADIATION TIME (min)
`Fig. 3. Survival curves for V-79 Chinese hamster cells incubated for 12 h in
`the presence of ~30 jig '°B/mlof growth medium from BSH. Cells were then
`irradiated at the thermal neutron beam of the BMRR either
`in the presence of
`BSH (ambient cells) or in boron-free growth medium (washed cells).
`
`10
`
`8
`6
`4
`IRRADIATION TIME (min)
`Fig. 4. Survival curves for V-79 Chinese hamster cells incubated for 12 h in
`the presence of ~30 ^g '"B/ml of growth medium from BSSB. Cells were then
`irradiated at the thermal neutron beam of the BMRR cither
`in the presence of
`BSSB (ambient cells) or in boron-free growth medium (washed cells).
`
`10
`
`12
`
`Table 3 Response of V-79 cells lo thermal neutron irradiation following growth
`for 12 h (I cell cycle time) in the presence of 30 ng '°B/ggrowth medium from
`various compounds
`
`grown and irradiated in the presence of 30 ng '°B/gof growth
`medium,
`the biological efficacy was equivalent
`to only 9.5 and
`12 Mg"'B/g of uniformly distributed boron for BSH and BSSB,
`respectively. These results are in agreement with the finding
`that BSH is bound to albumin in vivo and that
`the albumin
`molecule, because of its size and charge,
`remains extracellular
`(20). Perhaps most striking was the finding that, of the small
`amounts of sulfhydryl boron hydrides which were bound, BSSB
`retention was significantly higher than BSH. The higher uptake
`of BSSB in vitro correlates well with the observations
`that
`BSSB uptake and retention is greater
`than BSH in vivo and
`located nuclear volume of 230 ^m3) in suspension in Hj^BOj
`that BSSB is more efficient
`than BSH in the treatment of small
`(ambient experiments) ~10% of the dose would be expected to
`animal
`tumors
`(11-14). The results
`showing that 6.2 ¿¿g
`come from external
`IOB,while ~45% of the dose would come
`loBSS'°B/gcell produced a response equivalent
`to 3.0 ng IOBof
`H,'°BO, is consistent with the hypothesis
`that BSSB distribu
`from IOBin the cytoplasm and 45% from the nucleus (details
`given in Ref.
`l). Similarly,
`for washed experiments,
`boron
`tion is primarily intracytoplasmic.
`attached to the cell membrane will be only -10% as effective
`The evaluation of BOPP in terms of boric acid equivalents
`the same quantity of IOBuniformly distributed intracellularly.
`shows clearly that BOPP is ~4 times more effective than BSH
`or BSSB. The actual boron content of 28 pg 10B/g of washed
`Uptake, Retention, Distribution and Biological Efficacy. A
`comparison of measured parameters
`from individual and aver
`cells which produced
`damage
`equivalent
`to 12.0 ng/g of
`H3loBOj is also indicative of a cytoplasmic
`location; however,
`age experiments
`in Tables 2 and 3, respectively, demonstrates
`that
`the experimental
`error observed in replicate experiments
`the greater
`intracellular
`concentration
`and retention of BOPP
`is much less than the differences obtained when experimental
`accounts for its increased biological efficacy.
`The linear accumulation
`of BOPP during the 18-h period
`conditions
`(i.e.. compound,
`status
`during
`irradiation)
`are
`changed.
`investigated is significantly different
`from the rapid diffusion
`in Table 3 indicates
`An evaluation of boric acid equivalents
`processes characteristic
`of H3BOj, BSH, and BSSB (Table 3
`that BSH and BSSB are to a significant extent excluded from
`and Refs. 10, 11, 20, and 22). The linear accumulation
`and
`retention over extended periods (i.e., 1-18.5 h) suggest an active
`the cell. This is evident from the fact that, while the cells were
`4863
`
`(min)Washed"3.5
`CompoundBSH
`
`acid
`
`equivalents<Mg/g)Washed0.5
`
`B Content
`for washed
`cells(Mg/g)6.228.0
`
`12.0
`BSSB
`3.0
`2.3
`1.20.5Boric
`41.0Measured
`12.0Ambient9.5
`1.2Ambient1.4
`BOPPDo
`" Conditions for individual experiments are given in Table 2.
`
`ANACOR EX. 2002 - 4/6
`
`

`
`AN IN IÃ(cid:143)TROTECHNIQUE FOR NCT
`
`T
`
`T
`
`T
`
`T
`
`T
`
`T
`
`T
`
`BOPP RESPONSE vs INCUBATION TIME
`(30 /ig
`IOB/ml)
`
`20
`
`18
`
`16
`
`14
`
`12
`
`IO
`
`8
`
`Io
`
`o5
`
`uÂ
`
`m
`
`§ 6
`
`PORPHYRIN-BOPP
`30ng1°B/ml
`
`•¿(cid:3)CONTROL CELLS
`o WASHED CELLS
`A AMBIENT CELLS
`
`I
`m
`2
`
`z
`
`iti
`
`100
`
`30
`
`10
`
`OC
`
`CO
`
`LU
`Ü
`OC111
`CL
`1.0
`
`0.3
`
`0.1
`
`l
`4
`
`l
`6
`
`l
`8
`
`l
`10
`
`l
`14
`
`l
`16
`
`12
`
`18
`
`HOURS EXPOSURE TO DRUGS
`Fig. 6. The response of cells incubated for various times in —¿(cid:3)30/ig loB/g/ml
`of growth medium from BOPP and then washed prior to irradiation in boron-
`free medium at the thermal neutron beam of the BMRR. Responses expressed in
`boric acid equivalents
`(i.e.,
`/ig IOBfrom H310BO3which would give the same
`response). Data point marked with O was obtained with a boron concentration of
`2.3 ppm and normalized to 30 ppm.
`
`here are attributed
`
`to the
`
`2
`
`4
`
`6
`
`8
`
`10
`
`12
`
`IRRADIATION TIME (min)
`Fig. 5. Survival curves for V-79 Chinese hamster cells incubated for 12 h in
`the presence of -30 /ig ">B/ml of growth medium from BOPP. Cells were then
`irradiated at the thermal neutron beam of the BMRR cither
`in the presence of
`BOPP (ambient cells) or in boron-free growth medium (washed cells).
`
`Table 4 Biological efficacy of BOPP following exposure limes of1-20 h
`
`Boronconcentrationsin
`
`results presented
`shown). Thus,
`'"B(n,«)7Lireaction.
`in vitro analysis is ca
`that
`The above discussion illustrates
`acidequivalents(normalizedto
`and relative biological
`pable of revealing uptake mechanisms
`growthmedium(«
`ofexposure
`acidequivalents(/ig
`efficacies for boronated biomolecules. This in turn makes it
`toBOPP
`10B/g)26.025.026.928.02.324.3Duration
`10B/g)3.06.68.512.01.015.6Boric
`(h)159121218.5A,(min)2.41.71.51.22.91.0Boric
`30„¿(cid:3)g/g)3.457.99.412.813.019.2
`possible to predict and interpret data obtained in vivo. Clearly,
`however,
`in vivo studies will still be necessary in order to verify
`absolute and differential uptake in tumor and biological efficacy
`in the more complex and realistic animal
`tumor models.
`Toxicity to Cells in Vitro. An indication of the toxicity of
`boron compounds
`can be obtained by comparing
`the PE of
`unboronated cells with the PE of unirradiated
`controls which
`were incubated for 12 h in the presence of the compound.
`metabolic process and perhaps can be ascribed to a receptor-
`Concentrations
`of boron used here (i.e., 30 tig '°B/gof growth
`mediated mechanism (23, 24). The proportionality
`of uptake at
`medium) are typical of concentrations
`used in vivo (19, 21).
`both high (24-28 ppm) and low (2.3 ppm) boron concentrations
`The PE for nonboronated controls in these experiments was 72
`indicates that, at the levels investigated here, saturation of the
`±12 (SD) for a total of 11 curves. Boric acid showed a slight
`process has not occurred.
`toxicity with an average PE of 59. The remaining compounds,
`to visible light, and
`Porphyrins
`are known to be sensitizers
`BSH, BSSB, and BOPP, showed no evidence of toxicity for the
`this mechanism is the basis for photodynamic
`therapy (25).
`times and concentrations
`used, as indicated in Table 2. This
`Similar sensitization has been noted for boronated porphyrins,
`result
`is not surprising, because these compounds
`have been
`so that
`these experiments
`have been protected from exposure
`investigated in part because of their lack of debilitating toxicity.
`to ambient
`light (shielded and manipulated under low intensity
`It is understood that
`in vivo toxicity is too complicated to be
`yellow light so that no reduction
`in plating efficiency was
`uniquely established by an in vitro test and that, ultimately,
`observed). As noted above, exposure of cells with BOPP to
`toxicity must be evaluated in animals. However,
`it should be
`'•"Cs7-rays
`showed no dose enhancement
`from BOPP;
`in
`noted that
`the majority of compounds
`synthesized for possible
`addition, cells grown with 30 ppm BOPP with both natural and
`use in NCT cannot be successfully administered
`because of
`95% IOBenrichment
`showed a response proportional
`to IOB
`overt toxicity;
`this behavior is readily evidenced by a significant
`reduction of PE in cell culture.
`concentration,
`and
`not
`chemical
`concentration
`(data
`not
`4864
`
`ANACOR EX. 2002 - 5/6
`
`

`
`AN IN VITRO TECHNIQUE FOR NCT
`
`in
`Conclusion. The in vitro evaluation of boron compounds
`terms of boric acid equivalents provides data with regard to
`uptake, retention, and intra- or extracellular distribution which
`is otherwise unavailable.
`In addition,
`this simple and reproduc
`ible test system using Chinese hamster cells provides an assay
`of biological efficacy and in vitro cell toxicity which facilitates
`initial evaluation and intercomparison
`of compounds.
`Currently, most
`investigators utilize in vivo systems for test
`ing 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 BNCT,
`the in
`vitro system and technique
`herein described is simple and
`advantageous
`for the initial screening of compounds.
`In addi
`tion 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 here showed an
`uptake and retention ~20 times that of BSH;
`the latter com
`pound is currently being used clinically for NCT in Japan and
`is anticipated for use in clinical
`trials in the United States. Our
`data indicate that, providing the advantage demonstrated
`by
`BOPP in these in vitro studies are validated in animal experi
`ments, BOPP should be considered for clinical application.
`
`ACKNOWLEDGMENTS
`
`The authors would like to acknowledge Chris Gordon for his out
`standing technical assistance and Bernice Armstrong and Amalia Rug-
`giero for their patience in preparing this manuscript.
`
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