STUDIES ON THE FOLIC ACID VITAMINS
`
`III. The Duration of the Eflects of the Folic Acid
`Antagonists in Man
`
`PAUL T. CoNn1T, M.}D., PI-LB.“
`
`THE FIRST paper of this series described the
`inhibition by aminopterin of the conver-
`sion of folic acid to compounds with citrovo-
`rum factor (CF) activity in man.“ That the
`duration of this inhibition might have pro-
`found therapeutic significance was suggested
`by the work of Goldin et al.3 on the relative
`effectiveness of different dosage schedules of
`amethopterin in the treatment of mouse leu-
`kemia. They found that an interval of 4 days
`between doses produced the best therapeutic
`results when treatment was begun shortly
`after tumor implantation, before dissemina-
`tion occurred, and that
`this interval was re-
`lated to the recovery of the host from the
`effects of the drug.'’- 9
`The development of an analogous dosage
`regimen of amethopterin in man requires the
`determination of the size of the individual
`doses and the interval between consecutive
`
`doses. The second paper of this series pre-
`sented the results of a study of the acute tox-
`icity of amethopterin in man.” It was found
`that intravenous doses as large as 16 mg. per
`kg. produced little toxicity in patients with
`normal renal and bone marrow function.
`
`The duration of the effects of amethopterin
`in man is the subject of this report. Observa-
`tions on 3 other folic acid antagonists are in-
`
`From the National Cancer Institute, of the National
`Institutes of Health, Public Health Service, Bethesda,
`l\-Id.
`The many microbiological assays were skillfully per-
`formed by Dorothea F. Thomasson. The synthetic fo-
`linic acid used as a standard for the microbiological
`assays and the aminopterin were generously provided
`by Dr. H. P. Broquist and Dr. M. Ruegsegger of the
`Lederle Laboratories Division of the American Cyan-
`amid Company, Pearl River, NJ’. The 3'-chloroame-
`thopterin and 3’.5'-dichloroamethopterin were supplied
`by the Research Division of the American Cyanarnid
`Company. The parenteral solutions of amethopterin
`[50 mg. per 1111.), 3'-chloroarnethoptenn (10 mg. per
`ml.), and 3’,5'-dichloroamethopterin (50 mg. per ml.)
`were prepared as the sodium salts In
`the Pharmaceuti-
`cal Development Service of the
`hartnacy, Clinical
`Center, of the National Institutes of Health, under the
`direction of Dr. John A. Scigliano.
`' Present address: Oklahoma Medical Research Foun-
`dation, Oklahoma City. Okla.
`Received for publication April 28, 1959.
`
`229
`
`cluded. The urinary excretion of citmvorum
`factor derived from folic acid was used as an
`
`index of drug effect. From the results ob-
`tained, a dosage regimen of amethopterin for
`the treatment of human cancer is proposed.
`
`MATERIAIS RND METHODS
`
`Patients. Twelve patients with disseminated
`cancer participated in this study. Eight were
`reported previously in regard to the toxic ef-
`fects produced by single doses of amethopterin.’
`None of these patients had obvious renal in-
`sufficiency.
`Procedure. Amethopterin (4-amino-N“L
`methyl pteroylglutamic acid, Methotrexate)
`was given as a rapid intravenous injection
`to all
`the patients
`receiving this drug
`except patient
`I.L., who was given
`it
`orally. Aminopterin (4—amino pteroylglutamic
`acid) was administered as
`an oral dose.
`’-Chloroamethopterin (4-amino-N10-n1ethyl—3L
`chloropteroylglutamic acid) and 3’,5’-dichlo-
`roamethopterin (4-amino-N1“-methyl-3’,5’-dL
`chloropteroylglutamic acid) were given
`intrarnuscularly.
`Repeated test doses of folic acid were given
`before and at intervals of about 4 days after
`the administration of the antagonist. Twenty-
`four—hour urine samples were collected and
`assayed for citrovorum factor activity by the
`method of Sauberlich and Baumann“ as used-
`
`in this laboratory.3 In the illustrations, each
`point represents the urinary excretion of cit-
`rovorum factor during the 24-hour period
`after the subcutaneous administration of 15
`
`mg. of folic acid.
`
`RESULTS
`
`Patient D.W. was given no folic acid an-
`tagonist but was given serial test doses of folic
`acid to determine the variability to be ex-
`pected in the urinary excretion of citrovorum
`factor (Fig. 1). A similar control period for
`patient H.B.
`is presented in Fig. 5. The ob-
`served values ranged from 92 to 116% and
`
`MEDAC Exhibit 2010
`
`ANTARES v. MEDAC
`
`IPR2014-01091
`
`Page 00001
`
`MEDAC Exhibit 2010
`ANTARES v. MEDAC
`IPR2014-01091
`Page 00001
`
`

`
`230
`
`CANCER. March-A pri I 1960
`
`V01» 13
`
`
`
` NO AMETH-
`
`OPTERIN
`
`l
`
`
`lotus,/Kat
`
`
`AMETHOPTERIN
`
`
`
`0
`
`IO
`
`20
`
`DAYS
`
`yo MG/KG.
`AM ETH -
`
`OPTERIN
`
`0
`
`I0
`
`20
`
`DAYS
`
`' gle doses of amethopterin on the urinary excretion of citrovorum factor (CF) derived
`FIG. 1. The effect of
`figures, each point represents the amount
`E citrovorum factor excreted in the 24-
`from Eolic acid. In all
`hour
`iod after the parenteral administration of 15 mg. of folic ac‘
`. In this illustration, and in Figs. 2
`4,
`the v
`'cal arrows indicate the administration of amethopterin. Included are the following patients: D.
`., a
`-il—year—olrl white man with malignant carcinoid: 'R.A., a. 14-31
`-old white boy with fibrosarooma who received
`490 mg. of ameth
`erin; R.B., a 36-year-old white man
`' h
`odgkin's disea who received 690 mg. of ame-
`thopterin; R.S., a
`-
`1'-old white man with seminoma
`0 received 750 mg.
`amethopteri
`' 13.0., a 70-year-
`old white woman w'
`mycosis fungoides who received 470 mg. of amethopterin: L.B., 3 —year-old white
`woman with breast carcinoma who received 1,000 mg. of amethopterin.
`
`Page 00002
`
`Page 00002
`
`

`
`No. 2
`
`STUDIES ON THE Four: ACID VITAMINS. III.
`
`- Candi:
`
`231
`
`AMETHOPTERIN
`
`o I. G M6. / KG.
`
`o
`
`lo
`
`20
`DAYS
`
`so
`
`40
`
`Fin. 2. The elfect of
`repeated doses of amethopterin
`on the urinary excretion of or derived from folic acid
`in patient A.T., a'32-year-old white man with semi-
`notna who received 330 mg. and 325 mg. of ame-
`thopterin. See Fig. 1 for explanation of symbols.
`
`from 82 to 114% of the mean respectively.
`Single Doses of Amethopterin. Patients R.A.,
`R.B., R.S., B.G., and L.B. each received a
`single dose of amethopterin, from 6.5 to 16.
`mg. per kg. (Fig. 1). The initial excretion of
`citrovorum factor derived from folic acid was
`
`5 to 25 pg. per day. After the administration
`of amethopterin, the next dose of folic acid
`produced only 8 to 29% of the pretreatment
`amount. Recovery to initial values or higher
`had occurred by 13 to 24 days in 4 of the 5
`patients. Patient R.S. was not followed for
`longer than 18 days because he received addi-
`tional amethopterin on a different dosage
`regimen at
`that
`time. The higher recovery
`values of citrovorutn factor excretion exhibited
`
`by patients B.G. (159%) and L.B. (195%) sug-
`gest overcompensation during the recovery
`period.
`Multiple Doses of Amethopterin. Patients
`A.T., R.]., and C.M. were all given more than
`1 dose of amethopterin, from 6 to 15 mg. per
`kg. at intervals of 18 to 23 days (Figs. 2 to 4).
`The initial urinary excretion of 19 pg. of
`citrovorutn factor during the control 24 hours
`by patient A.T. was never equalled after
`either of the 2 doses of atnethopterin the pa-
`
`R. J.
`
`tient received (Fig. 2). The highest value of
`6 pg. per day 16 days after the first dose and
`19 days after the second dose was only 32%
`of the initial one. The minimum values of 7
`
`and 6% respectively occurred with the first
`test dose of folic acid given after each dose of
`atnethopterin.
`Patient 11.]. was given atnethopterin in 3
`doses of 6, 8, and 10 mg. per kg. respectively
`(Fig. 3). The initial excretion of citrovorutn
`factor was 6 pg. per day, which was regained
`20 days after the first and 11 days after the
`second dose of atnethopterin. The minima
`that occurred after these doses were 48 and
`
`51% respectively. After the ‘second dose, a
`maximum of 12.8 pg. per day (2l3% of the
`initial value) was obtained on the twenty-
`second day. No consistent change was ob-
`served after the third dose of arnethopterin.
`The initial citrovorum factor excretion by
`patient C.M. of 3 pg. per day was lower than
`that of the other patients (Fig. 4). This value
`was further reduced after the first dose of 10
`
`mg. per kg. of amethopterin to 1.4 pg. per day.
`Recovery to 11.5 pg. per day had occurred by
`16 days, but a prompt reduction to 0.3 pg. per
`day was produced by a second dose of 15 mg.
`per kg. of amethopterin. Recovery to 7.6 pg.
`per day was evident 14 days after amethopterin,
`1 day before the patient died of melanoma.
`Aminopterin. Patient H.B. was given 1 oral
`dose of 0.1 mg. per kg. of aminopterin (Fig. 5).
`Pretreatment values for the urinary excretion
`of citrovorum factor varied from 5 to 7 pg.
`per day. Five days after aruinopterin, it had
`fallen to 2 pg. per day and recovery did not
`occur until 26 days after administration of
`the antagonist.
`Daily Doses of Amethopterin. Patient I.L.
`was given amethopterin as a daily oral dose of
`5 mg. (Fig. 6). Thirteen closes (a total dosage
`of 65 mg. or 1.3 mg. per kg.) of amethopterin
`
`AMETHOPTERIN
`
`3," s, A no MG./KG.
`
`o
`
`to
`
`20
`
`40
`
`so
`DAYS
`
`5,;
`
`.0
`
`-m
`
`FIG. 3. The effect of repeated
`doses of amethopterin on the
`urina
`excretion of CI? derived
`from olic acid in patient R._].,
`a 54-year-old white man with
`bronchogenic carcinoma who re-
`ceived 270 mg., 360 mg.. and
`450 mg. of amethopterin. See
`Fig.
`I
`for explanation of
`the
`symbols.
`
`Page 00003
`
`Page 00003
`
`

`
`232
`
`CANCER. March-April 1960
`
`Vol. 13
`
`AMETHOPTERIN
`to 3. I5
`
`MG./KG.
`
`o
`
`IO
`
`20
`
`so
`
`DAYS
`
`Fla. 4. The effect of repeated doses of amethopterin
`on the urinary excretion of CF derived from folic acid
`in patient C.M.. a 33-year-old white man with mela-
`noma who received 750 mg. and 950 mg. of amethop-
`teriu. See Fig.
`1 for explanation of the symbols.
`
`reduced the urinary excretion of citrovorum
`factor from 16.5 to 3.3 pg. per day. Recovery
`was slow, and pretreatment values had not
`been regained 65 days after the last dose of
`amethopterin.
`re-
`3’-Chloraamethopterin. Patient M.C.
`ceived 0.05 mg. per kg.
`(3 mg.) of 3’-dtloro-
`amethopterin by intramuscular injection once
`daily for 14 doses (a total dosage of 42 mg. or
`0.7 mg. per kg.) (Fig. 7). This treatment re-
`duoed the urinary excretion of citrovorum fac-
`tor from 24.9 to 8.09 pg. per day. Seven days
`after completion of the first series of doses, at
`second series of 0.1 mg. per kg.
`(6 mg.) per
`day was begun and continued for 8 closes (a
`total dosage of 48 mg. or 0.8 mg. per kg.). The
`citrovorum factor excretion after this second
`
`series was 3.35 pg. per day, and it gradually
`returned to essentially pretreatment amounts
`22 days after the last dose of the drug.
`3',5’-Dichioroamethopterin. After recovery
`from 3’-chloroamethopterin was complete, pa-
`tient M.C. was given 2 mg. per kg. (120 mg.)
`per day of 3’,5’-(lichloroamethopterin by in-
`tramuscular injection. Four doses (given on
`days 0, 1, 2, and 4; a total dosage of 480 mg.
`or 8 mg. per kg.) temporarily reduced the uri-
`nary excretion of citrovorum factor from 22.9
`to 8.53 pg. per day. Recovery to nearly control
`levels had occurred I 1 days after the last dose,
`and the drug was resumed 3 days later (on day
`20). Except for the omission of days 25 and 26,
`treatment was continued through day 45 (24
`doses; a total dosage of 2,880 mg. or 48 mg.
`per kg.). Citrovorum factor excretion, which
`was only 1.59 pg- per day 4 days after the last
`
`dose, gradually recovered but had not reached
`pretreatment values 20 days after the drug
`was discontinued.
`
`DISCUSSION
`
`The available evidence indicates that the
`
`primary action of the folic acid antagonists is
`to prevent
`the reduction of
`folic acid to
`tetrahydrofolic acid. Further metabolism of
`the latter gives rise to compounds that sup-
`port
`the growth of Leuconostoc citrouorum
`and hence possess citrovorum factor activ-
`ity.*- “» 13 The observation in man that ami-
`nopterin reduced the urinary excretion of
`citrovorum factor derived from administered
`folic acid3 was consistent with this evidence.
`
`At the time this earlier study was done,-3 some
`of the patients exhibited the inhibitory effects
`of this drug on the excretion of citrovorum
`factor as long as 8 days after cessation of
`aminopterin administration. It was suggested
`at that time that aminopterin might combine
`irreversibly with the enzyme system responsible
`for the utilization of folic acid and that recov-
`
`ery from its inhibitory effects might require re-
`generation of enzyme protein.3
`The present investigation was designed to
`ascertain in man the duration of the inhibi-
`
`tory effects of the folic acid antagonists upon
`the urinary excretion of citrovorum [actor de-
`rived from folic acid. The validity of the use
`of serial
`test doses of folic acid to measure
`
`these changes was demonstrated by the rela-
`
`is H.B.
`
`cF
`
`10
`
`9P [day
`
`5
`
`AMINOPTERIN
`
`0.1 MG./KG.
`
`
`
`0
`
`O
`
`20
`
`DAYS
`Fin. 5. The effect of a single dose of aminopterin on
`the urinary excretion of citrovorum factor (CF) derived
`from folic acid in patient H.B.. a 45-year-old white
`woman with Hodgkin's disease. The patient received
`5.5 mg. of aminopterin (indicated by the vertical ar-
`row). See Fig. 1 for explanation of symbols.
`
`Page 00004
`
`Page 00004
`
`

`
`No. 2
`
`STUDIES ON THE Foue Acn) VITAMINS. III.
`
`- Comiit
`
`233
`
`AME TH OPTERIN
`
`5 MG./DAY
`
`O
`
`20
`
`40
`
`60
`
`80
`
`. DAYS
`
`daily
`Fla. 6. The effect of
`doses of amethopterirt on pa-
`tient LL.,
`3. 65-year-old white
`woman with malignant carci-
`noid. who received 65 mg. of
`amethopterin in 13 days. In this
`illustration and in Figs. 7 and
`8. the boxes indicate the periods
`of drug administration. See Fig.
`l for explanation of the other
`symbols.
`
`the time required for recovery from
`more,
`this inhibition tends to support the concept
`of irreversible combination or alteration of
`
`the enzyme system responsible for the utiliza-
`tion of folic acid.3 Recovery to the pretreat-
`ment state during the period of observation
`did not occur in 4 patients, suggesting that the
`elfects of
`these drugs are not always com-
`pletely reversible.
`The importance of the duration of the ef-
`fects of the folie acid antagonists in the treat-
`ment of mouse leukemia has been demon-
`
`strated by Goldin et al.3 They found that the
`best therapeutic results were obtained in the
`treatment of early lymphoid leukemia Ll2l0
`(i .e., shortly after implantation) when doses of
`amethopterin were given at
`intervals of 4
`days. On the other hand, when treatment was
`delayed until dissemination of the tumor oc-
`curred, daily treatments with atnethopterin
`were superior. They further showed by serial
`measurements of the median lethal dose (LD50)
`of aminopterin7 and of the protection af-
`forded by the administration of folic acid 1
`hour before a lethal dose of aminopterin9
`M.C.
`
`tively constant urinary excretion of citro-
`vorum factor over a period of several weeks by
`patients who had received no prior folic acid
`antagonist.
`The reduction in citrovorum factor excre-
`
`tion that occurred in every case after the ad-
`ministration of single doses of amethopterin
`was prompt and prolonged. The time re-
`quired for recovery to pretreatment values
`varied from a minimum of about 2 weeks to
`
`nearly 4 weeks and appeared to be roughly
`proportional to the size of the dose. A second
`dose of the antagonist given 21/2 to 3 weeks
`after the first dose usually caused similar
`changes. A single dose of aminopterin caused
`alterations identical with those produced by
`arnethopterin but at a dose some 60 times
`smaller.
`
`The administration of amethopterin by a
`more conventional dosage regimen, consisting
`of consecutive daily doses, also greatly de-
`pressed the urinary excretion of citrovorum
`factor derived from folie acid. The recovery
`from this treatment was somewhat slower than
`
`in that
`that observed after the single doses,
`pretreatment values of citrovorum factor ex-
`cretion had not been regained 2 months after
`cessation of amethopterin administration. The
`total amount of drug administered was 1.3
`mg. per kg, which in its elfects appeared to
`be at least equal to, and possibly more toxic
`than, the largest single dose employed of 16 mg.
`per kg. The other folic acid antagonists stud-
`ied, 3’-chloroamethopterin and 3’,5’-dichloro-
`amethopterin, were administered in consecu-
`tive daily doses and produced elfects similar
`to arnethopterin but at dilferent dosages.
`Thus, inhibition of the urinary excretion of
`citrovorum factor derived from administered
`
`folic acid in man appears to be a general
`property of the folie acid antagonists. Further-
`
`
`
`o
`
`20
`
`40
`
`so
`
`m<\\\\V
`3'-catoaome TH 091: am
`3 as H atom
`
`so
`
`OF
`
`“it
`‘"1’
`
`2°
`
`no
`
`0
`
`DRY 5
`
`Fin. 7. The effect of daily doses of 3'-clIloroan'Iel.hop-
`terin on patient M.C., a 45-year-old white woman with
`renal carcinoma, who received the drug under the fol-
`lowing regimen: 42 mg. in 14 days and 48 mg. in 8
`days. See Figs. 1 and 6 for explanation of the symbols.
`
`Page 00005
`
`Page 00005
`
`

`
`234
`
`CANCER March-April 1960
`
`Vol. 13
`
`cs
`
`30
`
`20
`
`M,
`day
`
`0
`
`.
`effect of daily
`Pic. 8. The
`doses of 3',5'-dichloroametl1op-
`terin on the same patient as in
`Fig. 7, who received the drug
`under
`the following regimen:
`4-80 mg.
`in -1- doses
`(over
`a
`eriod of 5 days) and 2.830 mg.
`P
`.
`ll’! 24 doses {over a period of 26
`days). See Figs. 1 and 6 for ex-
`planation of the symbols.
`
`M.C.
`
`as sssasxss
`
`\‘
`ll
`3',5"'DlCHLOROAMETHOPTERlN
`
`IZOM 6./DAY
`
`0
`
`20
`
`40
`
`60
`
`the interval of 4 days corresponded
`that
`closely to the recovery of the host from the
`effects of a nonlethal dose of the drug. The
`enhanced therapeutic effectiveness of this in-
`termittent dosage regimen in early leukemia
`further suggested a temporal dissociation in
`the recovery of the host and of the tumor
`from the effects of amethopterin. By the time
`the next dose of amethopterin was given, 4
`days later,
`the host had recovered from the
`eliects of the first dose but the tumor had not
`
`and therefore was more severely damaged
`than the host. Another way of stating this re-
`sult is that proper spacing of doses (4 days in
`the mouse) minimizes the toxicity for the host
`so that larger doses can be given.
`Ideally,
`the selection of a dosage regimen
`of amethopterin for use in man, which corre-
`sponds to the one used in the mouse, should be
`based upon a comparison of the recovery of
`both the host and the tumor from the elfects
`
`of the drug. The data presented show that for
`the host the size of the individual dose is di-
`
`DAYS
`
`and the preceding one,” a dosage regimen of
`amethopterin consisting of doses of approxi-
`mately fi mg. per kg. every 2 weeks is proposed
`for the treatment of patients with cancer. Pa-
`tients with bone marrow and renal insufficiency
`of even mild degree probably should not receive
`doses of this magnitude. In order to test the
`therapeutic elfectiveness of this regimen,
`it
`should be tried in diseases that are known to
`
`respond to the folic acid antagonists, specifi-
`cally
`acute
`leukemialfi
`and choriocarci-
`norna.1" Trials in patients with other tumors
`would be interesting because little response
`has been obtained by treatment with daily
`dosage regimens of
`the folic acid antago-
`nist5.1, 12, 14
`
`CoNcLusIoNs
`
`l. The inhibitory effect of amethopterin on
`the urinary excretion of citrovorum factor de-
`rived from folic acid in man persists for sev-
`eral weeks after exposure to the drug.
`2. This inhibitory effect appeals to be a
`general property of the folic acid antagonists,
`being exhibited also by aminopterin, 3’-chlo-
`roamethopterin, and 3’,5’dichloroamethopte-
`rm.
`
`rectly related to the duration of its elfects. Re-
`covery in 2 weeks did not usually occur after
`doses larger than 6 mg. per lcg., while smaller
`doses produced iew toxic effects? In the ab-
`sence of comparable information about hu-
`man tumors, further modification of the dos-
`age regimen becomes arbitrary. It is suggested
`that the dosage regimen consist of individual
`4. Based on these observations, an inter-
`doses of amethopterin of approximately 6 mg.
`mittent dosage regimen of amethopterin for
`per kg. repeated at intervals of about 2 weeks.
`Based on the data presented in this paper
`the treatment of human cancer is proposed.
`REFERENCES
`
`3. The duration of the effects is roughly
`proportional to the size of the dose adminis-
`tered.
`
`I. Buncni-:mu., ]. H.; Ksnnorsxv, D. A.; K1Ncst.Ev-
`PILLERS, E. M.; Sotmum, C. M.; Mums, W. P. L.:
`Estzm-:n, G. C.; Clutvnt, L. F.;
`l'J.mt:EoN, H. W., and
`
`Ruoans, C. P.: Effects of folic acid antagonists and 2.
`6-diaminopurine on neoplastic disease; with a
`‘:11
`reference to acute leukemia. Cancer 4-: 549-569,
`I 51.
`
`Page00006
`
`Page 00006
`
`

`
`No. 2
`
`STUDIES ON THE Fouc Acm VITAMINS. III.
`
`- Candi!
`
`235
`
`2. Comm, P. T.: Studies on folic acid vitamins; II,
`acute toxicity of amethopterin in man. Cancer l3: 222-
`228, 1950.
`3. Connrr. P. 'I‘., and (3303. D.: Studies on Eolic acid
`vitamins;
`I, observations on metabolism of folic acid
`in man and on effect of arninopterin. Cancer I1: 525-
`536, 1958.
`4. Docrolz, V. M.: In vitro studies on conversion of
`folic acid to citrovorum factor. J. Biol. Chem. 222:
`959-968, 1956.
`5. FARBER, 5.: Dmmonn, L. K.; Mmczn, R. D.: SYL-
`vrsree, I1.
`I-‘., _]a., and Wotrr, J. A.: Tempcmu-y re-
`missions in acute leukemia in children produced by
`Eolic
`acid antagonist.
`-Laminoptcroyl-glutamic acid
`(aigiflnoptefin). New England J’. Med. 238: 793-793.
`1
`.
`6. Furnnumm, S., and SILVERMAN, M.: "Inactivation"
`of folic acid by liver. J’. Biol. Chem. 221: 31-40, I957.
`7. Gotnm, A.: Employment of methods of inhibition
`analysis in normal and tumor—bearing mammalian or-
`ganism. Advances Cancer Res. 4: 113-143, 1956.
`8. GoLoIN, A.; Vennlrn, ]. M.: HUMPHREYS, S. R.,
`and MANTEL, N.: Modification of treatment schedules
`in management of advanced mouse leukemia with
`
`arnethopterin. ]. Nat. Cancer Inst. I7: 203-212. 1956.
`9. GRI-IENSPAN, E. M.: GOLIJIN, A., and SCHOENB.-tcfl.
`E. 11.: Studies on mechanism of action of chemothera-
`peutic agents in cancer;
`ll, requirements for preven-
`tion of aminopterin toxicity by folic acid in mice.
`Cancer‘ 3: 856-863, 1950.
`10. L], M. C.: Hliltrc. 11.. and SPENCER, D. B.: Effect
`of methotrexate therapy upon choriocarcinoma and
`chorioadenoma. Proc. Soc. Exper. Biol. er Med. 93:
`36l-366, I956.
`ll. SAUIIERLICH, H. I-2., and Bnummn. C. 2\.: Factor
`required for growth of Leuconosioc citrovorum. J. Biol.
`Chem. 176:
`I65-I73, 1948.
`12. Scnoenmtcn, E. 11.: Cotsxv, j., and CREENSPAN.
`E. M.: Observations on eifects of Eolic acid antagonists,
`amino terin and amethopterin,
`in patients with ad-
`vance
`neoplasms. Cancer 5: 1201-1220, 1952.
`13. SEVEKMAN, M.: EBAUGI-I, F. G., ]R., and GARDINER,
`‘R. (1.: Nature of labile citrovorum [actor in human
`urine. J. Biol. Chem. 223: 259-270, 1956.
`I4. WRIGHT, J. C.; Plucor. A.: WRIGHT, B. P.]; Wam-
`1-mum, 8.. and WRIGHT. I... T.: Evaluation 0 tolic acid
`antagonists in adults with neoplastic diseases: study of
`93 patients with incurable neoplasms. J. Nat. M. A. 43:
`2ll-2-10, 1135!.
`
`Page 00007
`
`Page 00007