1961
`
`OCTOBER - DECEMBER
`
`VOL. 134
`
`THE JOURNAL OF
`
`P H A R M A C 0 L 0 G Y
`
`AND EXPERIMENTAL THERAPEUTICS
`
`Founded by
`John J. Abel
`1909
`
`Official Publication of the American Society for
`Pharmacology and Experimental Therapeutics, Inc.
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`N. C. Moran
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`INDEX EDI'l'Olt
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`PUBLICATIONS
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`8626
`
`EVALUATION OF BORON COMPOUNDS FOR USE IN NEUTRON CAPTURE
`THERAPY OF BRAIN TUMORS. I. ANIMAL INVESTIGATIONS1
`
`A. H. SOLOWAY, R. L. WRIGHT AND J. R. MESSER
`Neurosurgical Service, Massachusetts General Hospital, and the Department of Surgery, Harvard Medical
`School, Boston, Massachusetts
`
`Received for publication March 10, 1961
`
`The neutron capture irradiation of intra(cid:173)
`cranial neoplasms using nonradioactive boron10
`was first proposed by Sweet and Javid (1952).
`For this form of therapy to be successful, it is
`essential that there be a high differential con(cid:173)
`centration of boron between the tumor and the
`adjacent normal brain. Under local bombard(cid:173)
`ment with thermal neutrons of the area con(cid:173)
`taining residual tumor, the neoplastic tissue
`would be selectively destroyed by the following
`nuclear reaction: boron10 + neutron1-->(boron11)-+
`lithium7 + alpha particle + 2.4 MEV. The
`alpha particle and lithium atom emitted travel a
`maximum of nine microns in tissue, thereby
`releasing this destructive energy only in the
`immediate vicinity of the cell containing the
`original disrupted atom of boron10•
`The feasibility of this form of therapy is based
`upon a difference in permeability between normal
`and neoplastic tissues. Fortunately, there is a
`breakdown of the normal blood-brain barrier
`(BBB) in brain tumors (Moore, 1947) and
`consequently many substances which are re(cid:173)
`stricted in their passage into the brain enter the
`tumor readily (Selverstone et al., 1949; Sweet,
`1951).
`Of 125 boron compounds screened in mice
`(Soloway, 1958; Soloway et al., 1960; Soloway
`and Gordon, 1960; Thiry, 1958), fifteen have
`given higher glioma: brain boron ratios than
`were observed with borate (Locksley and Sweet,
`1954). In the present study, ten of these com(cid:173)
`pounds were compared with each other and with
`boric acid to ascertain which compounds deserved
`additional study on the basis of tumor: brain
`ratios and toxicity in mice. Of the five more
`
`promising compounds, two have been tested in
`cats to determine
`toxic manifestations and
`tissue concentrations. This was done prior to
`their evaluation in terminal patients (Sweet
`et al., unpublished).
`
`METHODS. In other studies (Soloway et al.,
`1960), it has been shown that C3H mice bearing
`subcutaneously transplanted gliomasl provide a
`useful means of assaying the tumor:brain ratios
`of various boron compounds. The present variety
`of glioma, an ependymoma, was used throughout
`the investigations. The methods used for trans(cid:173)
`planting the tumor and determining the boron
`content in tissues have been described by Soloway
`et al. (1960). The animals were injected intra(cid:173)
`venously or intraperitoneally usually under ether
`anesthesia with doses of from 70 to 300 mg of
`boron per kilogram of body weight (mg B/kg).
`They were sacrificed by ether inhalation after
`periods from 15 minutes to 3 hours and various
`tissues were weighed and analyzed for boron
`content.
`In general, the toxicity of the compound which
`proved most favorable was then evaluated by
`intravenous injection of the aqueous solutions into
`the tail veins of white Swiss albino mice. Survivors
`were followed for 72 hours. The solutions were
`prepared in a concentrated form and at a pH range
`of 7.35 to 7.40, whenever possible, to minimize
`volume and pH as factors in the toxicity deter(cid:173)
`minations. However, many compounds were
`soluble in high concentration only in an al(cid:173)
`kaline medium.
`The more promising compounds were tested
`more thoroughly by intravenous and intracarotid
`injection in cats anesthetized with pentobarbital.
`The latter route was included in the event that
`intracarotid administration of boron compounds
`
`1 This research was supported by a grant from
`the U.S. Atomic Energy Commission under Con(cid:173)
`tract No. AT(30-1)-1093 and by the National Can(cid:173)
`cer Institute, U. S. Public Health Service Grant
`No. C-3174 (C2) Rad.
`
`2 Dr. D. M. Perese of the Department of Neuro(cid:173)
`surgery at the Roswell Park Memorial Institute in
`Buffalo, New York, very generously supplied us
`with the original subcutaneously grown ependy(cid:173)
`moma.
`117
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`118
`
`SOLOWAY ET AL.
`
`Vol. 134
`
`becomes the preferable method of injection for
`neutron capture therapy. Continuous electro(cid:173)
`encephalographic and electrocardiographic trac(cid:173)
`ings were made and vital signs followed closely.
`Urinary excretion of several compounds or their
`metabolites was measured by collecting urine
`with indwelling catheters and analyzing aliquots
`for boron content. At intervals of from 15 minutes
`to several days after injection, serial blood
`samples were taken and boron analyses were
`performed on them as well as on other tissues of
`each animal. Vital organs were then examined
`for histologic changes.
`
`RESULTS AND DxscusSION. Mouse studies.
`Tumor:brain boron ratios in mice were approxi(cid:173)
`mately the same whether injected intravenously
`or intraperitoneally. Consequently, in table 1,
`the average boron ratio listed for the ten com(cid:173)
`pounds at the times specified includes mice
`injected by either of these routes of administra(cid:173)
`tion. At each time interval 3 to 10 mice were
`used and from these data, standard deviations
`of each group were calculated to ascertain the
`variation in this study. Though the deviations
`were large, it was apparent that the ratios for all
`of these compounds were appreciably greater
`than was observed in this laboratory with the
`borate ion (Locksley and Sweet, 1954) and
`
`confirmed in this present study. This is especially
`true at times greater than 1 hour after injection,
`since with borate at such times a ratio of only
`one was observed.
`Since all of the compounds described here
`appeared to be quite promising from the stand(cid:173)
`point of ratio, it was essential to compare them
`with regard to toxicity. The LD50 values of each
`of these ten compounds as well as of boric acid
`are listed in table 2. Of those screened the follow(cid:173)
`ing five, based on boron content, were the most
`satisfactory from toxicity considerations: 1) m(cid:173)
`Boronosuccinanilic acid. 2) 3-Amino-4-carboxy(cid:173)
`benzeneboronic acid. 3) 2-Acetamidobenzene-1,
`4-diboronic acid. 4) o-(2-Carboxy-2-acetamido(cid:173)
`ethyl)-benzeneboronic acid. 5) Sodium perhydro(cid:173)
`decaborate. Of these five, m-boronosuccinanilic
`acid, 3-amino-4-carboxybenzeneboronic and more
`recently sodium perhydrodecaborate have been
`investigated more extensively in larger animals
`and finally in terminal glioxna patients (Sweet
`et al., unpublished). In view of its encouragingly
`low toxicity based on boron content, sodium
`perhydrodecaborate would appear to be the
`compound of choice. Doses of 50 mg B/kg have
`been administered to man with no untoward
`effects. Surprisingly enough, it is a boron hydride.
`
`TABLE 1
`Tumor/brain boron ratios in mice• at various intervals after administration of boron compounds
`
`Compound
`
`1St
`
`30
`
`60
`
`120
`
`180
`
`8.3 ± 2.1
`9.1 ± 2.0
`
`-
`6.9 ± 1.1 6.5 ± 2.1
`6.5 ± 0.9 5.8 ± 2.4 5.2 ± 3.2
`
`6.0 ± 2.2 11.0 :1:: 2.5
`6.4 ± 2.2
`6.8 ± 2.3
`5.7 ± 1.7
`7.8 ± 1.2
`4.6 ± 1.7
`7.0 ± 1.5
`4.2 ± 2.8
`4.9 ± 1.1
`
`7.2 ± 0.6 3.7 ± 2.3 3.5 ± 2.7
`6.4 ± 2.5 4.9 ± 1.2 3.3 ± 1.2
`7.3 ± 1.3 4.4 ± 1.0 5.2 ± 0.6
`7.3 ± 1.7 3.9 ± 0.2 4.0 ± 2.3
`5.4 ± 3.5 4.4 ± 1.0 4.4 ± 2.2
`
`6.1 ± 1.3
`p-Borono-phenylalaninet
`2-Acetamidobenzene-1, 4-diboronic 8.2 ± 2.4
`acid
`m-Ureidobenzeneboronic acid
`m-Boronosuccinanilic acid
`m-Carboxybenzeneboronic acid
`p-Carboxybenzeneboronic acid
`2-Nitrobenzene-1 , 4-diboronic
`acid
`o-(2-Carboxy -2-acetamidoethyl)-
`benzeneboronic acidt
`3-Amino-4-carboxybenzene-
`boronic acid
`Sodium perhydrodecaborate§
`
`4.8 ± 2.4
`
`6.9 ± 1.8
`
`3.9 ± 1.2
`
`7.7 ± 2.6
`
`6.5 ± 2.6 3.4 ± 1.3 2.8 ± 0.6
`
`7.2 ± 1.5
`
`8.5 ± 2.2 6.7 ± 1.6 7.0 ± 0.8
`
`5.4 ± 1.1
`
`7.2 ± 2.2 5.7 ± 1.5 7.3 ± 2.1
`
`• Ratios are recorded with the standard deviations; the mice received doses of 140 to 300 mg B/kg.
`t Time of sacrifice in minutes after injection.
`t Dr. H. R. Snyder, Professor of Chemistry at the University of Illinois, kindly supplied these com(cid:173)
`pounds.
`§Dr. M. F. Hawthorne of the Redstone Arsenal Division of the Rohm and Haas Company kindly
`furnished triethylammonium perhydrodecaborate and the procedure for preparing the sodium salt.
`
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`
`1961.
`
`BORON COMPOUNDS AND BRAIN TUMORS
`
`119'
`
`TABLE 2
`Mouse toxicity study
`
`pH
`
`LDSOt
`
`Toxic Signs of Near Lethal Dose
`
`Compound
`
`Boric acid
`
`3-Amino-4-carboxybenzeneboronic
`acid
`
`p-Boronophenylalanine
`
`m-U reidobenzeneboronic acid
`
`p-Carboxybenzeneboronic acid
`
`m-Carboxybenzeneboronic acid
`
`2-Nitrobenzene-1 ,4-diboronic acid
`
`No.
`Mice•
`- -
`
`12
`
`34
`21
`16
`
`25
`
`If lie mcB/llg
`6.9 2.11
`375
`
`7.4 2.42
`430
`270
`8.8 1.52
`9.4 1.24
`220
`- - - - ---
`8.1 3.29
`200
`
`9.5 2.06
`125
`16
`- - - - - ----
`10.0 1.52
`80
`11
`- -- ----
`100
`10.0 1.02
`- -- ----
`9.4 1.74
`115
`- - - ----
`7.4 2.56
`170
`-- - ----
`9.3 1.68
`175
`
`12
`
`25
`
`20
`
`34
`
`mmol/llg
`34.6 Seizures, respiratory depres-
`sion, ataxia, diarrhea
`
`39.7
`25.0
`20.3
`
`18.5 Seizures, respiratory depres-
`sion
`
`11.5
`
`7.4 Seizures, respiratory depres-
`sion
`
`4.6 Seizures, respiratory depres-
`sion
`
`10.6 Seizures, diarrhea, respiratory
`depression
`
`15.7 None
`
`8.1 Seizures, respiratory depres-
`sion
`
`17.5 None
`
`11.5 Seizures, respiratory depres-
`sion
`
`23.1 None
`
`- - - ----
`190
`7.4 4.09
`- -- ----
`10.6 2.54
`250
`--- ----
`7.5 5.72
`250
`- - - - ---
`6.3 Seizures
`11
`7.2 1.04
`Sodium perhydrodecaborate
`685
`* The numbers of mice refer to those used in the LDOO range and not to the total number of mice
`required for the toxicity study.
`t The values are accurate to within ± 25 mg B/kg.
`
`m-Boronosuccinanilic acid
`
`2-Acetamidobenzene-1 ,4-diboronic
`acid
`
`o- (2-Carboxy-2-acetamidoethyl)-
`benzeneboronic acid
`
`48
`
`19
`
`14
`
`The literature is replete with information con(cid:173)
`cerning the high toxicity of such materials
`(Hill et al., 1958; Walton et al., 1955; Lowe and
`Freiman, 1957), but the high chemical stability
`of the decaborate ion, B,oH,o- (Hawthorne, un(cid:173)
`published) is possibly responsible for its biological
`inactivity.
`In assessing the toxicity of these compounds,
`it became apparent that the volume of the
`injected solution was not a major factor provided
`it did not exceed such large volumes as 2.5 to 3
`ml per mouse. Interestingly enough, such vol-
`
`umes were well tolerated when injected intra(cid:173)
`venously over a 10- to 20-second period. The pH
`of the material was of greater concern since even
`small volumes (less than 0.4 ml) of alkaline
`solutions produced respiratory depression, sei(cid:173)
`zures, and often death. To determine the effect of
`pH upon the toxicity of these boron compounds,
`we measured the LD50 for boric acid solutions
`at varying hydrogen ion concentrations (table 2).
`As might be anticipated, the lowest toxicity was
`attained at the physiological pH, increasing on
`either side of this value. By raising the pH to
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`
`120
`
`SOLOWAY ET AL.
`
`Vol. 134.
`
`9.4, the toxicity of the boric acid solution ap(cid:173)
`proximately doubled. Similar findings of the
`effect of pH have been reported recently by
`White (1960) with regard to the toxicity of
`nitrogen mustards.
`
`TABLE 3
`Average boron concentrations in cat tissues* in mg
`B/kg
`Boric Acid
`
`Optic chiasm
`Sciatic nerve
`Cerebellum
`Cerebrum-cor-
`tex
`White matter
`Brain stem
`Spinal cord
`Pituitary
`Feces (rectum)
`
`mg B/kg
`
`375-415
`360--400
`320-410
`3()()-390
`
`Kidney
`Heart
`Skull
`Liver
`
`3()()-330 Muscle
`Scalp
`280-340
`230-270
`Fat
`2()()-260
`Blood
`340
`
`mgB/kg
`
`5.'i<Hi20
`475-550
`4()()-520
`450
`
`415-465
`360
`60-80
`143G-500
`
`Cat studies. Preliminary to the injection of
`m-boronosuccinanilie acid and 3-amino-4-car(cid:173)
`boxybenzeneboronic acid in man, their toxic
`effects as well as lethal doses of boric acid were
`determined in cats. A comparison study of the
`toxicity of these aromatic boronic acids to boric
`acid would provide some intimation as to what
`might be an initial safe dose of these compounds
`in man. Much is known regarding the pharma(cid:173)
`cology of boric acid in man (Pfeiffer et al., 1945;
`Goodbloom, 1953; Locksley and Farr, 1955;
`McNally and Rust, 1928; Watson, 1945; Con(cid:173)
`nelly et al., 1958) and this is the reason for its
`use as a reference standard.
`Boric acid in intravenous doses of 600 mg
`B/kg produced generalized seizures, severe
`diarrhea, and ataxia in 2 cats. Occurrence of
`diarrhea after parenteral administration sug(cid:173)
`gested the possibility that the intestinal wall was
`actively excreting the substance. Fecal specimens
`were taken from the large intestine and showed
`high concentrations of boron, corroborating
`
`BEFORE
`
`INJECTION DURING
`
`INJECTION
`
`5 MINUTES AFTER
`
`CAT A
`
`R-PO
`
`R-FP ~ ~ ~
`~ ~ ~
`
`I 50 mv
`
`,__.. sec.
`
`CAT 8
`
`R-FP ~ ------
`
`~
`
`""''""'
`
`R-PO ~ ~ ~
`,__.. sec.
`I 50 mv
`
`FIG. 1. Electroencephalographic tracings.
`Leads: R-FP = fronto-parietal; R-PO = right parieto-occipital.
`Cat A received 3-amino-4-carboxybenzeneboronic acid, 40 mg Bjkg, by injection into the right com(cid:173)
`mon carotid artery after ligation of the facial portion of the external carotid artery. The pH of the solu(cid:173)
`tion was 8.1 and it contained 3 mg B/ml. A generalized seizure and respiratory arrest ensued.
`Cat B received m-boronosuccinanilic acid, 40 mg B/kg. by the same route. This solution contained
`5 mg B/ml and the pH was 7.4. It was well tolerated. The animal was sacrificed 110 hours later, and
`there was no sign of toxicity.
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`
`1961
`
`BORON COMPOUNDS AND BRAIN TUMORS
`
`121
`
`this hypothesis. Thus boric acid is excreted both
`by the kidney and the intestine, the latter route
`becoming more apparent at higher dose levels.
`In table 3 are listed the boron concentrations in
`other body tissues of these 2 cats. As might be
`expected, the kidney has the highest concentra(cid:173)
`tion, and at this lethal dose, large amounts
`appear also in brain, heart, liver, and muscle.
`Fat, on the other hand, has one of the lower
`values.
`3-Amino-4-carboxybenzeneboronic acid in a
`dose of 40 mg B/kg was injected either intra(cid:173)
`venously or via the carotid artery in 3 cats.
`Carotid injection of the solution at pH 8.1 was
`poorly tolerated. Electroencephalographic trac(cid:173)
`ings were made during these injections and
`showed spiking patterns on the EEG (fig. 1),
`generalized seizures, and respiratory depression
`in the animals.a Intravenous injections, however,
`were well tolerated. In table 4 are shown the
`tissue levels of boron at varying intervals after
`injection. Kidney and scalp had high boron
`concentrations whereas normal brain was in(cid:173)
`variably low.
`m-Boronosuccinanilic acid, the next compound
`considered, goes readily into a solution containing
`10 mg B/ml at pH 7.4. This is in contrast to the
`foregoing compound, 3-amino-4-carboxybenzene(cid:173)
`boronic acid, which is soluble to the extent of
`only 3 mg B/ml even at pH 8.1. Injection of
`m-boronosuccinanilic acid in a dose of 86 mg
`B /kg via the carotid artery produced a marked
`alteration in the EEG together with respiratory
`arrest which failed to respond to assisted respira(cid:173)
`tion. Death occurred shortly after the injection.
`Doses of 40 mg B/kg, however, at a concentration
`of 5 to 10 mg B/ml were well tolerated by both
`intravenous and intracarotid routes.
`Tissue concentrations are listed for 3-amino-4-
`carboxybenzeneboronic acid and for m-borono(cid:173)
`succinanilic acid in table 4. One cat was utilized
`at each
`time for 3-amino-4-carboxybenzene(cid:173)
`boronic acid and at least 2 cats for each interval
`with m-boronosuccinanilic acid. Of
`special
`interest is the fact that normal brain shows a
`very low content of either agent independent of
`the route of administration. The latter compound
`in fact gave comparable results whether by
`intravenous or
`intracarotid
`administration
`a Wright (1960) has observed that injection of
`alkaline solutions via the carotid artery produced
`seizures, damage to the blood-brain barrier and
`respiratory depression.
`
`TABLE 4
`Average boron concentrations in cat tissues in
`mgB/kg
`
`3-Amino-4-carboxybenzeneboronic acid
`
`Scalp
`Muscle
`Skull
`Brain
`Pituitary
`Optic nerve
`Intestine
`Liver
`Kidney
`
`I
`
`I
`I
`I
`
`30min
`
`160
`30
`50
`5
`100
`40
`60
`70
`180
`
`60min
`
`120 min
`
`190
`20
`20
`4
`-
`30
`50
`110
`360
`
`70
`20
`20
`7
`20
`10
`30
`30
`130
`
`m-Boronosuccinanilic acid
`
`IS min
`
`60min
`30min
`90min
`540min
`- - - - - - - - - ---
`90-100 130-150 55-60 55-65 Trace
`7-13 Trace
`9-11
`15-25 15-25
`20-25 15-20 15-25 1Q-20
`10
`75-105 40-60 3o-40 30-35 Trace
`Trace Trace Trace Trace Trace
`2Q-40
`25-35 25-35
`20
`10
`-
`Trace
`0
`0
`0
`-
`-
`-
`20-25
`20-25
`27Q-330 25o-280 25o-320 60-65
`5
`4o-50 40-60 25-35 15-30 Trace
`25--40 20-30 Trace
`50-55 60--70
`4Q-45 5o-60 15-25 1Q-15
`0
`50--65 60-65 25-35 20-35
`2
`
`4o-55 40-60 15-25 15-20
`25-40 3Q-35 15-20 1Q-20
`-
`-
`5-15 1Q-15
`-
`7-10
`1Q-15
`5-7
`25-35 4Q-45 1Q-20 1Q-15
`5o-60 55-60 20-45 1Q-20
`
`-
`-
`
`3
`2
`
`3
`0
`
`Scalp
`Muscle
`Skull
`Dura
`Brain
`Pituitary
`Lens
`Retina
`Kidney
`Liver
`Intestine
`Adrenal
`Lung
`Lymph
`node
`Spleen
`Fat
`Feces
`Heart
`Thyroid
`
`(Wright and Soloway, unpublished) and con(cid:173)
`sequently, values in table 4 represent values for
`2 cats or range of values for 3 cats at each time
`interval following injection. Kidney concentra(cid:173)
`tions remained at a very high level even several
`hours after injection but blood samples taken 8
`or 9 hours after administration of these doses
`showed only trace amounts of boron. As occurs
`with the 3 compounds, boric acid (Locksley and
`Sweet, 1954), p-carboxybenzeneboronic acid and
`triisopropanolamine borate (Soloway and Gor(cid:173)
`don, 1960), these agents, 3-amino-4-carboxy-
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`
`SOLOWAY ET AL.
`
`Vol. 134
`
`benzeneboronic acid and m-boronosuccinanilic
`acid, are rapidly excreted by the kidney. They
`all behave in this respect in a similar manner,
`namely as "low-threshold substances" in a class
`with urea and sulfate.
`
`SUMMARY
`
`Ten organic boron compounds and boric acid
`have been evaluated in mice for toxicity. Tumor:
`brain boron ratios have been studied in animals
`with subcutaneous transplanted gliomas.
`Two of the more promising compounds, 3-
`amino-4-carboxybenzeneboronic acid and m(cid:173)
`boronosuccinanilic acid have been studied in
`cats by both
`intravenous and
`intracarotid
`injection. Tissue concentrations have been
`determined.
`
`to
`AcKNOWLEDGMENTS. The authors wish
`thank Dr. William H. Sweet, Associate Pro(cid:173)
`fessor of Surgery at the Harvard Medical School
`and Chief of the Neurosurgical Service of the
`Massachusetts General Hospital, for his great
`interest and encouragement during this entire
`investigation. The technical assistance of Miss
`Beverly Whitman, Miss Winnie Crane, Mrs.
`
`Cynthia Provost, and Mrs. Mary Lee Bossert is
`gratefully acknowledged.
`
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