1960
`
`MAY - AUGUST
`
`VOL. 129
`
`T H E J 0 U R N A L 0 F
`
`PHARMACOLOGY AND
`
`E X P E R I M E N T A L T H E R A P E U T I C S
`
`Founded by
`John J. Abel
`1909
`
`Official Publication of the American Society for
`Pharmacology and Experimental Therapeutics, Inc.
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`EDITOR
`Lawrence Peters
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`ASSISTANT EDITOR
`N. C. Moran
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`H. K. Beecher
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`w. w. Douglas
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`Sidney Ellis
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`R. F. Furchgott
`L. I. Goldberg
`
`EDITORS FOR SPECIFIC FIELDS
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`and Electrolytes
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`Pharmacology
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`Pharmacology
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`P. B. Dews : Behavioral Pharmacology
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`
`H. c.
`Hodge
`P. R. Saunders
`F. w. Schueler
`Jenden
`D. J.
`c. c. Smith
`Eva King Killam
`c. M. Smith
`H. G. Mandel
`H. F. Smyth, Jr.
`T. H. Mar en
`J. L. Strominger
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`E. Leong Way
`Norton Nelson
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`8358
`
`PENETRATION OF BRAIN AND BRAIN TUMOR BY AROMATIC
`COMPOUNDS AS A FUNCTION OF
`MOLECULAR SUBSTITUENTS1
`
`A. H. SOLOWAY, B. WHITMAN AND J. R. MESSER
`NeurOBurgical Service, Ma88achU8etts General H08pital and the Department of Surgery,
`Harvard Medical School, B08ton, Massachusetts
`
`Received for publication November 20, 1959
`
`The possibility of d!'Stroying tumors by neu(cid:173)
`tron capture irradiation was first proposed by
`Kruger (1940). The most favorable tumor for
`such therapy was considered by Sweet et al.
`(1952) and Javid, Brownell and Sweet (1952) to
`be the highly malignant brain tumor, glioblas(cid:173)
`toma multiforme. The rationale was the existence
`of the blood-brain barrier (BBB) which was
`shown
`(Ehrlich, 1885; Lewandowsky, 1900;
`Friedemann, 1942; Tschirgi, 1952; Herlin, 1956;
`Bakay, 1956; Mayer et al., 1959) to restrict the
`passage of various compounds into the central
`nervous system (CNS) relative to other tissues.
`In brain tumors (Moore, 1947) as well as other
`diseases of the brain (Macklin, 1920) there is a
`breakdown in this normal barrier phenomenon
`permitting localization in this lesion of radio(cid:173)
`active isotopes (Selverstone et al., 1949; Sweet,
`1951).
`irradiation of brain
`For neutron capture
`tumors, it is necessary to concentrate in the neo(cid:173)
`plasm an isotope, for example nonradioactive
`B10, with a high cross-section capture for thermal
`neutrons. These nonionizing neutrons would be
`avidly absorbed by the nucleus of the boron10
`atom producing the following nuclear reaction
`\\ith the liberation of 2.5 million electron volts:
`boron10 -> (boron11) -> lithium7 + helium4
`
`Tissues containing sufficient boron would be
`destroyed upon neutron irradiation and thus
`there is a need for compounds which \\ill be ex(cid:173)
`cluded from normal brain and will concentrate
`in the tumor.
`Initial studies by Soloway (1958) showed great
`
`1 This research was supported by a grant from
`the U. S. Atomic Energy Commission under con(cid:173)
`tract No. AT(30-l)-1093, and by the National
`Cancer Institute, U. S. Public Health Service
`Grant No. C-3174(C2) Rad.
`
`differences in penetration of the brain and brain
`tumor by various substituted phenylboronic
`acids. There appeared to be a direct correlation
`between penetration of the brain and a low
`aqueous-benzene partition coefficient. Those com(cid:173)
`pounds which concentrated in the lipid solvent,
`benzene, penetrated brain readily, gave poor
`tumor /brain ratios as measured by boron con(cid:173)
`tent and were considered to have a high lipid sol(cid:173)
`ubility. Substances which were excluded from
`the benzene in this partition were considered to
`have poor lipid solubility. Many of these per(cid:173)
`meated the brain to a limited degree and gave
`high tumor /brain boron localizations.
`The purpose of this investigation was to de(cid:173)
`termine which substituents on phenylboronic
`acid restricted and which aided penetration of
`the CNS. From such a study, information might
`be obtained about the mechanism of action of
`the BBB on various compounds and could serve
`as a guide both to those engaged in therapy based
`on the exclusion of substances from the CNS
`(Market al., 1960) as well as to those involved in
`the synthesis of neurotropic drugs.
`
`METHODS. To determine the aqueous-benzene
`partition coefficient of the various boron com(cid:173)
`pounds, about 10 mg of each substance was dis(cid:173)
`tributed between 50 ml of a phosphate-buffered
`aqueous medium of pH 7.2 and 50 ml of benzene.
`The compounds were partitioned by shaking in a
`separatory funnel and the layers were separated.
`Aliquots of each phase were then analyzed by the
`method of Ellis et al. (1949) for boron content. It
`was essential for maximum accuracy of this colori(cid:173)
`metric method that the samples contain 1 to 5 ,.g
`of boron. In the case of the more lipid soluble
`compounds it was essential to dilute aliquots of
`the partitioned fluids and to analyze an aliquot of
`these diluted liquids. Many of those compounds
`which were largely excluded from the benzene
`
`310
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`1960
`
`PENETRATION OF BRAIN
`
`311
`
`phase required aliquots of 15 ml of this layer to
`obtain significant readings on the Coleman Junior
`Spectrophotometer. The results appear in table 1
`and are listed as ,.g of boron per ml of undiluted
`solution.
`The tumor/brain boron ratios were obtained
`from C3H mice bearing subcutaneous
`trans(cid:173)
`planted gliomas. 2 Fresh tumor tissue was ground
`in normal saline and a cellular suspension was
`injected in the region of the left scapula in 6- to
`8-week-old C3H mice. Within 7 to 10 days the
`tumors were of sufficient size for use. Each com(cid:173)
`pound was dissolved in dilute basjl or water, de(cid:173)
`pending upon the solubility of the compound, and
`was injected intraperitoneally into the glioma
`mice. At fixed time intervals after injection, the
`animals were asphyxiated by ether and five 50-mg
`samples of both tumor and brain were then ana(cid:173)
`lyzed by the above method for boron content. In
`table 2 is recorded the average amount in tissue
`in ,.g of boron per g as well as the tumor/brain
`ratio for each compound. The number of mice
`used for each dose at each time varied from 1 to 3
`und the tissue content listed is an average. Only
`short sacrifice times are recorded since longer
`times may permit metabolic alteration in the
`eompound and would therefore not be a true cri(cid:173)
`terion of the compound's immediate localization
`in tissue. In many cases at times upwards of one
`hour the tumor/brain ratio approached one. It is
`also a possibility that this data might be affected
`by the presence of metabolic breakdo\'\"11 products
`of the various boron compounds. No evidence was
`obtained of the metabolism of these compounds
`in mice since the method of analysis was not for
`the particular compound in question but for boron
`in the form of boric acid.
`
`R.:~uLT:' AND DiscussiON. The aqueous(cid:173)
`benzl'ne partition coefficit'nts in table 1 are of the
`,·arious mono- and disubstituted aromatic boronic
`acids. All compounds with a coefficient ll'SS than
`29 wl'rc toxic at moderate doses producing a de(cid:173)
`pressant action upon the animal's normal activity
`and rl'sponse to stimuli. Each compound in this
`category had a tumor /brain boron ratio of less
`than 1 as shown in table 2. Those with a
`eoeffieil'nt of 2 or less were highly toxic and in
`small doses (18 to 35/~o~g/g of mouse) produced
`death of the animals. Examination of table 1 re-
`2 The authors are greatly indebted to Dr. D. M.
`Perese of the Department of Neurosurgery at the
`Roswell Park Memorial Institute in Buffalo, New
`York, for kindly supplying us with the original
`subcutaneously grown ependymoma. This was
`produced by implantation in the brain of a meth(cid:173)
`ylcholanthrene pellet and the intracranial neo(cid:173)
`plasm resulting was transplanted subcutaneously.
`
`TABLE 1
`Aqueous-benzene partition coefficients
`
`~B(OH)s !Aqueous Benzene Partition
`Coefticient
`
`p-Si(CHa)a*t
`2,4,6-tri CHat
`m-CFat
`p-SCHa*
`p-Br
`p-OCtH,
`p-Cl
`o-CHa
`m-CHa
`p-CHa
`p-OCHa§
`p-F
`p-H
`o-NOs
`m-NOs
`m-NHCOOCtH•
`p-CHOt
`3-NOt-4-COOH
`p-COOH
`3-NHt-4-CHa
`p-B(OH)t
`m-NH,
`2-NOt-4-B(OHh
`m-COOH
`p-CH:CHCOO-t
`I
`NHt
`m-OHt
`p-OH*
`2-NOs-4-COOH
`3-NHs-4-COOH
`3-NHCOCHtCHtCOOH
`3-NHCONHt
`
`0.38 12.6
`0.03
`4.0
`9.0
`0.4
`3.4
`7.7
`0.4
`I 5.1
`7.5
`0.7
`I 5.1
`6.4
`0.8
`6.4
`6.3
`1
`6.9
`5.6
`1
`9.8
`6.7
`2
`10.9
`6.8
`2
`8.0
`5.2
`2
`9.4
`3.5
`3
`11.4
`3.5
`3
`14.4
`2.3
`6
`12.6
`1.9
`7
`13.5
`1.1
`12
`10.8
`14
`0.77
`12.4
`0.43
`29
`9.2
`0.18
`51
`11.4
`0.17
`67
`15.4
`0.23
`67
`0.06 >200
`24.5
`12.4
`0.06 >200
`22.1
`0.05 >200
`12.1
`0.04 >200
`10.2
`0.04 >200
`
`17.9
`16.8
`9.8
`12.2
`8.9
`10.9
`
`0.03 >200
`0.02 >200
`>200
`0
`>200
`0
`>200
`0
`>200
`0
`
`* Dr. H. Gilman, Professor of Chemistry at
`Iowa State University, kindly supplied this com(cid:173)
`pound.
`t Dr. H. R. Snyder, Professor of Chemistry at
`the University of Illinois, kindly supplied this
`compound.
`t These compounds existed prior to partition
`as trimeric anhydrides.
`§ The results reported in Science 128: 1572, 1958,
`for this compound were found to be incorrect
`upon successive analyses.
`
`veals a definite order for the halogens substituted
`in the para position on phenylboronic acid. The
`bromo compound concentrated to a greater ex(cid:173)
`tent in the benzene phase than did the chloro
`compound and this in turn had a lower partition
`
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`
`312
`
`SOLOW AT ET AL.
`
`Vol. 1~9
`
`TABLE 2-Continued
`
`Time
`of
`
`Q
`
`i Sacri-
`lice
`- - -
`35 15
`35 30
`70 15
`35 30
`70 30
`140 30
`35 15
`70 15
`35 30
`70 30
`35 15
`70 30
`140 30
`200 30
`140 15
`70 30
`140 30
`140 15
`
`'
`
`..
`..
`!Local-
`iza-
`tiou
`!;
`.9
`Fac-
`~
`tort
`:!
`E-o =
`- - -
`30 33 0.9
`28
`29 1.0
`21
`9 2.3
`18
`8 2.2
`12 3.7
`44
`56 25 2.2
`25 21 1.2
`43 39 1.1
`32 26 1.2
`60 53 1.1
`4 2.5
`10
`7 4.9
`34
`7 6.1
`43
`52
`9 5.8
`57
`10 5.7
`6 7.3
`44
`8 7.9
`63
`34
`4 8.5
`
`TABLE 2
`Tumor/brain boron localization factors*
`
`4-Si(CHah
`
`2,4,6-tri CHa
`
`m-CFa
`
`p-SCHa
`
`p-Br
`
`p-OCsH•
`
`p-Cl
`
`o-CHa
`
`m-CHa
`
`p-CHs
`
`p-OCHa
`
`p-F
`
`p-H
`
`o-NOa
`
`m-NOa
`
`m-NBCOOC,H,
`
`p-CHO
`
`3-NO:r-4-COOH
`
`p-COOH
`
`Time
`
`Local-
`iza-
`
`of -
`.. - tion
`..
`l Sacri-
`lice A ·~
`Fac-
`tort
`- - - -
`Ill
`- - -
`35 15, <3 20 0.1
`6 26 0.2
`35 20,
`4 31 0.1
`35 30
`35 10, 6 37 0.2
`35 15
`23 40 0.6
`35 30
`29 35 0.8
`35 15,
`7 42 0.2
`18 20, 12 30 0.4
`12 14 0.9
`35 30
`35 9, 4 50 0.1
`18 7, 10 30 0.3
`18 30
`17 0.6
`11
`35 15
`14 57 0.2
`18 15
`9 43 0.2
`12 31 0.4
`18 30
`18 15
`13 23 0.6
`18 30
`11 20 0.6
`35 15
`23 52 0.4
`32 59 0.5
`35 30
`35 15
`23 52 0.4
`32 59 0.5
`35 30
`35 15
`15 47 0.3
`12 25 0.5
`35 30
`35 15
`18 53 0.3
`20 44 0.5
`35 30
`35 15
`44 0.7
`29
`33 40 0.8
`35 30
`35 15
`16 62 0.3
`13 56 0.2
`35 30
`35 15
`34 51 0.7
`34 44 0.8
`35 30
`35 15
`25 41 0.6
`29 41 0.7
`35 30
`35 151[ 16 40 0.4
`35 15
`12 42 0.3
`18 30
`16 19 0.8
`35 15, 18 34 0.5
`18 15
`13 20 0.6
`18 30
`14 19 0.7
`35 15
`9 16 0.6
`13 2.0
`26
`35 30
`70 15
`38 15 2.5
`70 30
`53 12 4.4
`5 3.4
`17
`35 30
`70 15
`8 3.8
`30
`140 15
`57 10 5.7
`70 30
`43
`7 6.1
`140 30
`8 7.9
`63
`
`3-NHa-4-CHa
`
`p-B(OH)t
`
`m-NHa
`
`2-NOa-4-B(OB)s
`
`m-COOH
`
`p-CBaCBCOO-
`I
`NHt
`
`m-OH
`
`p-OH
`
`2-NOz-4-COOH
`
`3-NHs-4-COOH
`
`3-NBCONH2
`
`140 30
`65 10 6.5
`14 1.9
`35 15
`26
`42 38 1.1
`70 15
`33 22 1.5
`35 30
`60 44 1.4
`70 30
`25
`17 1.5
`35 15
`133 72 1.8
`140 15
`30 19 1.6
`35 30
`35 15
`21
`3 7.0
`70 15
`5 8.4
`42
`7 5.6
`70 30
`39
`7 6.4
`200 15
`45
`140 30
`49
`6 8.2
`7 7.2
`200 30
`50
`3-NHCOCHaCHaCOOH 200 15
`9 6.9
`62
`4 7.2
`140 30
`29
`8 6.8
`.200 30
`54
`1140 15
`75 10 7.5
`74
`7 10.5
`!140 30
`I
`* In most eases the tumor and brain concentra(cid:173)
`tions were averages of 2 or 3 mice.
`t Dose in ~&g of boron per g of mouse.
`t Concentrations are in ~&g of boron per g of
`tissue.
`§Localization factor is the tumor/brain boron
`ratio.
`, The toxicity of the compounds resulted in
`death of the animals at these times.
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`
`1960
`
`PENETRATION OF BRAIN
`
`313
`
`coefficient than the fluoro derivative. On this
`basis it would appear that the higher the atomic
`weight of the attached halogen the greater the
`lipid solubility of the compound. As would be
`anticipated, the para ethoxy compound showed a
`greater relative concentration in the benzene
`phase than did the methoxyderivative; its longer
`alkyl chain is knmrn to increase fat solubility. Re(cid:173)
`placement of the oxygen in the methoxy com(cid:173)
`pound with a sulfur atom appreciably shifted the
`partition coefficient to a lower value. In this con(cid:173)
`nection it is interesting to note that Mark et al.
`(1958) found that the rate of penetration of the
`CNS by thiobarbituric acid is greater than that
`of the oxygen analogue and in agreement with its
`higher lipid solubility. In general it appears that
`in a single group of nonmetallic elements in the
`periodic table, the higher the atomic weight of an
`element attached to the same organic component,
`the more lipid soluble the compound becomes.
`m-Tolylboronic acid is quite lipid soluble but
`replacement of the hydrogens on the methyl
`group with fluorine atoms shifts the partition
`coefficient to an even lower value. The m-tri(cid:173)
`fluoromethylphenylboronic acid corresponds, with
`regard to aqueous-benzene partition, with the
`2,4,6-trimethyl derivative. It is expected that
`the trimethyl derivative would be more lipid
`soluble than the monomethyl compound, for
`increasing the number of alkyl groups increases
`fat solubility. Evidence of the effect of a methyl
`substituent on lipid solubility can be seen by a
`comparison of 3-aminophenylboronic acid with
`the 3-amino-4-methyl compound and phenyl(cid:173)
`boronic acid with o-, m- and p-tolylboronic acids.
`In each case the addition of the methyl group re(cid:173)
`sults in a lower aqueous-benzene partition co(cid:173)
`efficient.
`For the purpose of neutron capture therapy, it
`is highly desirable to have compounds which con(cid:173)
`tain a high percentage of boron. If other factors
`such as toxicity, stability and solubility were the
`same, those substances with a larger percentage
`would be the compounds of choice. They would
`permit large doses of boron to be given and con(cid:173)
`sequently higher levels of B10 could be attained
`in the tumor. Such consideration resulted in a
`comparison of phenylboronic acid \\ith benzene-
`1.4-diboronic acid. Introduction of a second
`boronic acid moiety markedly decreased lipid
`solubility as shown in table 1. From table 2 it is
`apparent that such an alteration appreciably im(cid:173)
`proved the tumor/brain boron ratio. It was con-
`
`sidered that decreased lipid solubility might be
`the result of the formation of a diboronic acid
`anion under physiological conditions, since it is
`probable that the un-ionized form is the lipid
`soluble one. Both anions and cations in general
`show a decreased rate of penetration from plasma
`into the CNS (Tschirgi, 1952). For the purpose
`of determining whether the observed difference
`between phenylboronic acid and benzene-1,4-di(cid:173)
`boronic acid was due to ionization of the latter
`under physiological conditions, the pK. values of
`both compounds in 50% ethanol was measured.
`Phenylboronic acid had a pKa of 10.7 whereas
`the diboronic acid had one of 10.2. Thus the de(cid:173)
`creased lipid solubility of the latter compound is
`not attributable to its ionization.
`Three other groups attached to phenylboronic
`acid were effective in decreasing lipid solubility
`and at the same time increasing appreciably the
`tumor /brain boron ratio. They are the aliphatic
`and aromatic carboxyl groups and the ureido
`function. Introduction of the carboxyl group in
`the 4-position of m-nitrophenylboronic acid
`shifted the partition function to higher values,
`decreased CNS toxicity and gave a larger tumor/
`brain ratio. Anion formation might also be a fac(cid:173)
`tor in the low lipid solubility of the carboxylic
`acid derivatives and their failure to penetrate
`the BBB readily. The pKa of p-carboxyphenyl(cid:173)
`boronic acid in 50% ethanol was 5.2 (for the car(cid:173)
`boxylic acid function) compared to 10.7 for
`phenylboronic acid. This reasoning of ion for(cid:173)
`mation, however, could not be applied to the
`ureido group.
`Though lipid solubility is an important cri(cid:173)
`terion, it is probably not the only mechanism in(cid:173)
`volved in brain permeability. The problem of ex(cid:173)
`plaining active transport of inorganic ions and
`glucose into the CNS would appear to eliminate
`solubility in lipid as the sole process for CNS
`permeability. In this series of boron compounds
`we have found that those which concentrate
`well in a lipid solvent show invariably a facile
`penetration of the brain. However, the converse
`proposition does not always follow; that is, those
`compounds which concentrate in the aqueous
`layer do not necessarily give high tumor /brain
`ratios and, in certain instances, are quite toxic to
`the CNS. Two types of compounds are in this
`category, the amines with the exception of those
`containing a carboxylic acid function and the
`phenols. These compounds penetrate the brain
`nearly as well as the tumor and are moderately
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`
`SOLOWAY ET AL.
`
`Vol. 129
`
`toxic. It may be that benzene is not the solvent
`of choice in testing the lipid solubility of these
`compounds. If one assumes, however, that the
`unpredictably high concentration
`in normal
`brain is a result of a rapid metabolism of the
`compound to a more lipid soluble substance there
`is no evidence in the literature of such a trans(cid:173)
`formation. Aromatic compounds in general are
`altered by oxidative hydroxylation (Brodie et
`al., 1958; Mitoma et al., 1956). The introduction
`of such hydrophilic groups would decrease rather
`than increase lipid solubility and this would not
`account for the high brain content. Another
`possible pathway in the metabolism of the aro(cid:173)
`matic boronic acids is oxidative deboronation to
`boric acid and phenols. This reaction occurs
`readily under in vitro conditions (Ainley et al.,
`1930; Johnson et al., 1938) but Soloway et al. (un(cid:173)
`published) have found that p-carboxyphenyl(cid:173)
`boronic acid was isolated unchanged in the urine
`of a terminal glioma patient who was given this
`compound intravenously. However, even were
`this reaction to occur in vivo, producing boric
`acid, this would not account for the results.
`Tumor/brain boron ratios of the borate ion have
`been shown in mice by Locksley and Sweet (1954)
`to be consistently higher than was obtained with
`the phenolic and amino boronic acids. Brodie et
`al. (1950) and Mayer et al. (1959) have alluded
`to the possibility of affinity for cellular com(cid:173)
`ponents as a contributing factor in CNS penetra(cid:173)
`tion. Such binding to brain constituents may
`permit ready entrance of such hydrophilic com(cid:173)
`pounds.
`In conclusion, an important criterion in de(cid:173)
`termining the degree of penetration of the brain
`by some compounds is lipid solubility, a concept
`reintroduced by Krogh (1946). Compounds which
`concentrate in
`lipid solvents relative
`to an
`aqueous phase are presented with no BBB; in
`fact, the nervous tissue behaves as a sponge to
`the compound in question. However, low lipid
`solubility does not preclude the rapid entrance of
`a drug into the CNS but the mode of penetration
`of these hydrophilic compounds remains un(cid:173)
`known.
`
`8UMM!.RY
`A correlation is made between aqueous-ben(cid:173)
`Zl'JJe partition coefficients of a series of substi(cid:173)
`tuted phenylboronic acids and
`tumor /brain
`ratios of these compounds in mice having sub(cid:173)
`eutant>ously transplanted gliomas. Those sub(cid:173)
`stances which coneentrate preferentially in the
`
`benzene phase invariably penetrated normal
`brain more readily than tumor and were quite
`toxic to the CNS. The converse proposition, how(cid:173)
`ever, is not always true but compounds which
`did give good tumor /brain ratios wen• only found
`in the hydrophilic group. In general it would ap(cid:173)
`pear that lipid solubility of a compound is an im(cid:173)
`portant factor but certainly not the only one in
`determining the penetration of the brain.
`ACKNOWLEDGMENTS. The authors are greatly
`indebted to Dr. William H. Sweet, Associate
`Professor in Surgery at the Harvard :.\ledical
`School, for his kind interest and sincere en(cid:173)
`couragement of this entire investigation. Tech(cid:173)
`nical assistance of :\liss Winnie Crane, Mrs.
`Cynthia Provost, Paul Szabady, and Mrs. Joan
`Crowley is gratefully acknowledged.
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