STUDIES ON THE FOLIC ACID VITAMINS
`
`VI. The Efiect of Amethopterin on Erythropoiesis in Man
`
`PAUL T. CONDIT, M.D., 1=H.n.,“' NA'rHA_Nu-:L I. BERLIN, M.D., PI-I.D., AND
`
`DAVID C. NATHAN, M.n.-l-
`
`IODO
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`MINUTES AFTER I=e”m.1EcT|oN
`
`see
`
`FIG. 1. Plasma Fe“ disappearance curves. The radio-
`activity in plasma, expressed as counts per minute per
`milliliter is plotted at different times after the injec-
`tion of Fe”. Tm represents the half time for disappear-
`ance of Fe“ from the plasma and was determined
`graphically, The intervals between the administration
`of amethopterin and Fe“ are designated by l, 6, 12, and
`24 hours.
`
`DURING the course of an investigation of
`the effects of large doses of amethopterin
`in man,3 it became apparent
`that. the drug
`temporarily arrested production of the formed
`elements of the peripheral blood. The times
`at which the peripheral blood counts reached
`minimum values (reticulocytes 4.6 days, leuko-
`cytes 6.2 days, and platelets 9.3 days) bore a
`close relationship to the life spans of these
`elements as determined by various methods.‘-
`7-1“ In order to gain more information about
`the effects of amethopterin on erythropoiesis,
`the present study was undertaken.
`
`Mernons
`
`Patients. Included were 1 woman with epi-
`dermoid carcinoma (primary unknown) meta-
`static to cervical lymph nodes.
`1 woman with
`multiple basal cell carcinoma,
`1 man with
`pseudomyxoma peritonei of appendiceal ori-
`gin, and 1 man with hepatic metastases from
`recurrent adenocarcinoma of the kidney. All
`had normal peripheral hematological findings
`at the beginning of the studies, were ambula-
`tory, and in good nutritional status.
`Hematology. Microhematocrits of venous
`blood were done by the method of Strumia
`et al.3 Reticulocyte and platelet counts were
`done by Brecher and co-workers’ n1ethods.1- 3
`Isotope Techniques. Twenty milliliters of
`venous blood were obtained from the patient,
`mixed with 10 ].l.C. of radioactive iron (Fe"°)
`citrate, and incubated for 30 minutes. The
`
`From the General Medicine Branch, National Cancer
`Institute. of the National Institutes of Health, Public
`Health Service. Bethesda. Md.
`We wish to thank Dr. E, j. ‘Van Scott for allowing us
`to study patient S.K. Robert F. Dion assisted with
`counting the many samples and also with the hema-
`tological
`studies. The intravenous preparation of
`amethopterin (Methotrexate) was prepared by the
`Pharmaceutical Development Service of the Pharmacy,
`Clinical Center, of the National Institutes of Health,
`under the direction of Dr. ohn A. Scigliano.
`‘Present
`address: Okla oma Medical Research
`Foundation, Oklahoma City, Okla.
`1'Present address: The Peter Bent Brigham Hospital.
`Boston, Ma.
`Received for publication June 8, 1959.
`
`amount of iron added to the plasma was
`within the iron-binding capacity. A volume of
`blood containing approximately 8 p.c. was re-
`injected. Venous blood samples were obtained
`frequently during the first few hours after in-
`jection to establish the rate of disappearance
`of the isotope from the plasma. Subsequent
`samples were collected at the times that the
`organs were scanned. The count rates over
`the liver, spleen, and sacrum (representing the
`bone marrow) due to Fe” were measured by
`the method of Elmlinger et a1.“ Blood samples
`were measured in a well
`type scintillation
`counter with a single channel spectrometer
`to a maximum counting error of 3.5%. Cor-
`rection was made for physical decay.
`
`245
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`8!! 36!
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`Vol. 13
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`246
`
`RESULTS
`
`2 —
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`Plasma Iron Disappearance. The plasma
`iron disappearance curves for 4 patients are
`presented in Fig. 1. When amethopterin was
`given 1 or 6 hours before the administration
`of Fe”, the rate of disappearance of iron from
`the plasma was either within the range of
`normal or slightly greater than normal. With
`an interval of I2 hours between the adminis-
`
`CANCER March-Aprii 1960
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`tration of Fe“ and amethopterin, the initial
`disappearance curve had a half time of 0.92
`hours. However, a marked change in the slope
`of the curve, greater than that usually ob-
`served in normal individuals,
`took place be-
`tween about 7 and 10 hours after Fe” ad-
`ministration or 19 and 22 hours after the
`
`administration of amethopterin. The fourth
`patient received Fe“ 21 hours after the admin-
`istration of amethopterin and had an abnor-
`mally slow plasma iron disappearance curve.
`The half time of 2.93 hours, 2 to 3
`times
`longer than normal, was nearly identical with
`the late portion of the plasma iron disappear-
`ance curve obtained when the interval be-
`
`tween the administration of
`isotope was 12 hours.
`0ne—Hou-I‘ Interval. The results of giving
`Fe” one hour after amethopterin are depicted
`
`the drug and
`
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`
`25
`
`FIG. 3. Patient H.H.. a 49-year-old white man with
`pseudomyxoma peritonei, who had received 3 mi. of
`Fe“ 6 hours after 1-1 mg. per kg.
`(925 mg.) of ame-
`thopterin. For explanation of
`the abbreviations, see
`Fig. 2.
`
`in Fig. 2. The lower part of the figure shows
`the changes in the Fe" content of the liver,
`spleen, and sacrum corrected for the isotope
`contained in the vascular system. The counts
`over the sacrum increased rapidly to a maxi-
`mum value the first day and thereafter de-
`creased rapidly to nearly 0 by day 18. The
`liver and spleen both showed an initial in-
`crease in activity,
`in each case considerably
`less than the sacrum, followed by a slow dis-
`appearance with time. The appearance of
`radioactivity in erythrocytes is shown in the
`upper part of the figure. The incorporation
`proceeded rapidly, reaching 50% on day 5
`and 80% on day 12 after Fe“ administration-
`The reticulocytes decreased, reaching a mini-
`mum value of 0.1% on day 3, while recovery
`became apparent on day 3. Changes in hema-
`tocrit were erratic but represented some de-
`crease in the initial value of 39.5%. A leuko-
`cyte count of 2.600 cells per cu. mm. on day 8,
`and a platelet count of 168,000 per cu. mm. on
`day 12 suggest a moderate drug effect. Oral
`ulcerations were not seen in this patient.
`
`Page 00002
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`DAYS AFTER Fa“ ADMINISTRATION
`
`FIG. 2. Patient S.K.. a 55-year-old white woman with
`multiple basal cell carcinomas, who had received 8 tie.
`of F125“
`I hour after 10 mg. per kg.
`(800 mg.) of
`amethopterin. The following abbreviations are used:
`plate. platelets per cubic millimeter times 10*’; ‘NBC,
`leukocytes per cubic millimeter times l0“°.
`
`Page 00002
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`

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`N0- 2
`
`Srumes on rm: Fottc Acm VITAMINS. VI.
`
`- Candi: et at.
`
`247
`
`Six-Hour Interval. The results of giving
`Fe” 6 hours after amethopterin are recorded
`in Fig. 3. The changes in isotope content of
`the viscera and its incorporation into periph-
`eral erythrocytes were similar to those ob-
`served after the 1-hour interval. Reticulocytes
`decreased to 0 on day 4 and recovered
`promptly. Changes in hernatocrit were not
`consistent. A leukocyte count of 5,100 cells
`per cu. mm. recorded on day 7 was somewhat
`below the initial count of 3,900 cells per cu.
`mm. but does not indicate serious drug eiiect.
`No reduction in platelet count was observed,
`but the value of 633,000 per cu. mm. recorded
`on day 23 may represent a rebound from the
`effect of amethopterin.
`Twelve-Hour Interval. The results of wait-
`
`ing 12 hours after amethopterin before giving
`Fe” are presented in Fig. 4. Again the changes
`in isotope distribution are similar to those ob-
`served after the 1-hour interval. Reticulocytes
`decreased to 0 on day 4, after which they
`slowly recovered. The hematocrit once again
`showed considerable variation from day to
`day and in general showed some decrease.
`
`Plate
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`Fit'.. 4. Patient ].H., a 53-year-old white man with
`gnu’; :::_"?3":;g":gl.
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`For explanation of the abbreviations, see Fig. 2.
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`ems AFTER Fe“ Anmmisrnimou
`
`Pic. 5. Patient E.R., a 39-year-old white woman with
`metastatic carcinoma, who had received 8 |.|.C. of Fe”
`24- hours after 12 mg. per kg. (750 mg.) of arnethopterin.
`For explanation of the abbreviations see Fig. 2.
`
`The minimum value of 1,500 cells per cu.
`mm. for the leukocytes obtained on day 5 and
`of 66,000 per cu. mm. for the platelet count
`on day 9 indicated a marked drug effect in
`this patient, but no oral ulcerations were
`present.
`Twenty-four-Hour Interval. The results of
`waiting 24 hours before giving Fe” after
`amethopterin are shown in Fig. 5. As shown
`in the lower part of the figure, the counts over
`the sacrum reached a maximum value on the
`
`first day but were sustained for 2 additional
`days before a decrease occurred. The activity
`over the liver rose rapidly to values greater
`than those obtained over the sacrum, were
`maintained for about 3 clays, and then de-
`clined. Counts over the spleen were less than
`those over the sacrum or liver and remained
`
`essentially constant for 8 days, after which
`they decreased steadily to 0 by day 22. As
`shown in the upper part of the figure, incor-
`pm-atjgn of F359 into erythrocytes did not be
`gin until the third day after Fe“ administra-
`tion. Fifty per cent of the incorporation
`
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`248
`
`CANCER March-April 1960
`
`Vol. 15
`
`occurred on day 7, and maximum values of
`about 90% incorporation occurred on day 18.
`Reticulocytes decreased to 0.2% on day 5 and
`then promptly recovered. Hematocrit de-
`creased from 42% to 35% by day 5 and had
`recovered to 42% by day 9. Leukocytes reached
`a minimum value of 3,700 cells per cu. mm-
`on day 7 and were still depressed at the end of
`the period of observation, 23 days after Fe“
`administration. Platelet counts showed little
`
`change during the first 13 days, but a mod-
`erate rise occurred subsequently.
`DISCUSSION
`
`The previous study of the elfects of large
`doses of amethopterin indicated that this drug
`caused a temporary decrease in the number of
`cells in the peripheral blood.” The time that
`elapsed after the administration of amethop-
`terin before the reticulocyte counts reached
`minimum values (4.6 days) corresponded
`closely to the maturation time of transfused
`reticulocytes (5 to 6 days)” and suggested that
`the decrease was due to the normal matura-
`
`tion of reticulocytes at a time when no new
`cells were being liberated from the marrow.
`Such an arrest in the production of erythro-
`cytes should be reflected by alterations in the
`distribution into liver, spleen, and bone mar-
`row of injected Fe59 and its incorporation into
`red cells. When Fe” was given 24 hours after
`the administration of a single dose of ame-
`thopterin, abnormal patterns of isotope dis-
`tribution were obtained. The rate of disap-
`pearance of Fe“ from the plasma was slower
`than normal by a factor of 2 or 3; the uptake
`of Fe” by the liver was greater than that of
`the bone marrow; the activity in the liver and
`sacrum remained approximately constant for
`3 days and in the spleen for 8 days; at day 3
`for the liver and sacrum and day 8 for the
`spleen, the activity of all 3 organs decreased at
`nearly the same rate. Little or no incorpora-
`tion of isotope into peripheral erythrocytes
`occurred until
`the third day after Fe” ad-
`ministration, which was the fourth day after
`the administration of amethopterin. Once he-
`gun, however, the appearance of labeled eryth-
`rocytes proceeded in a normal manner, 50%
`incorporation being achieved at about 4 days
`after production began or 7 days after Fe“
`administration. Thus in patient E.R., Fe“
`was stored in the marrow, represented by the
`sacrum, and liver for about 3 days and the
`spleen for 8 days, and then utilized for eryth-
`ropoiesis.
`I-‘e59 was retained in the liver and
`
`spleen until the bone marrow had begun the
`production of erythrocytes. Even though the
`Fe“ was presumably can‘ied in the plasma be-
`tween the liver and sacrum after erythro-
`poiesis had resumed, it was not possible to
`demonstrate any increase in the concentration
`of Fe“ in the plasma during this period of
`time because of rapid clearance by the mar-
`row and inability to measure extremely low
`concentrations of isotope.
`When shorter intervals of 1 or 6 hours in-
`tervened between the administration of ame-
`
`thopterin and Fe“, no alteration in the dis-
`appearance of Fe” from the plasma or in the
`appearance of labeled erythrocytes could be
`detected. The accumulation in the various or-
`
`gans immediately after the injection of Fe"
`and the subsequent release took place in a
`normal manner.
`
`An interval of 12 hours between amethop-
`terin and Fe” administration also produced
`a normal pattern of storage and release of
`Fe“ by the various organs, as well as the ap-
`pearance of labeled erythrocytes in the periph-
`eral blood. However,
`the disappeatance of
`Fe“ from the plasma suggested that a change
`took place 7 to 10 hours after the administra-
`tion of Fe”. The half time of Fe" disappear-
`ance during the first 6 hours after injection
`was slightly less than 1 hour, or within the
`range of normal. An abrupt change in slope
`occurred after this time, representing a much
`slower rate of elimination of Fe“ from the
`
`plasma. This change in the rate of disappear-
`ance occurred 19 to 22 hours after amethop-
`terin administration, which is consistent with
`the data obtained for patient E.R.—-that 24
`hours after amethopterin there was dimin-
`ished uptake of Fe” by the marrow.
`If it is assumed that amethopterin reaches
`the bone marrow in physiologically active con-
`centration rapidly (i.e., minutes) after admin-
`istration,
`then the results suggest
`that
`the
`inhibition of erythrocyte production by ame-
`thopterin takes place before iron is incorpo-
`rated into the developing cell. Since Fe“ has
`been shown to enter pronormoblasts9 and to
`be utilized for hemoglobin synthesis in these
`young cells,“ amethopterin may act at
`the
`earliest stage in erythrocyte production. It is
`also apparent
`that an interval of 20 to 24
`hours is required for the cell to mature from
`the site of action of amethopterin to the stage
`at which Fe” is incorporated. At
`the doses
`employed in this study,
`this inhibition ap-
`pears to be reversible.
`
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`No. 2
`
`Sruoms ON 1111: Fouc Acio VITAMINS. VI.
`
`- Candi! et at‘.
`
`249
`
`CONCLUSIONS
`
`terin must be given at least 20 hours before
`the administration of Fe”.
`
`1. Amethopterin temporarily inhibits incor-
`poration of radioactive iron in erythrocytes.
`2. In order to achieve this effect, amethop-
`
`3. The method employed in this study can
`be utilized to determine the effect of drugs
`upon erythropoiesis.
`
`REFERENCES
`
`1. Beacons, (3.: New methylene blue as reticulocyte
`stain. Am. J. Clin. Path.
`I9: 895-896. 1949.
`2. BRECHEII, G.; SCIINEIDERMAN, M., and CRONKITE.
`E. P.: Reproducibility and constancy of platelet count.
`Am. J. Clio. Path. 23: I5-26, 1953.
`3. CONDIT, P‘. T.: Studies on folic acid vitamins; ll.
`acute toxicity of amethopterin in man. Cancer I3: 222-
`228, 1960.
`'R.;
`l’_.; FLIEDNER, T. M.; Ruoml, J.
`4. C|toN|ttTE, E.
`Bonn, V. F., and HUGHFS. W. L.: Dynamics of prolifer-
`ating cell systems of man studied with tritiated thy-
`midine. [Abstr.] J’. Ciin. Invest. 37: 887, 1958.
`5. ELMLINCI-ZR. P. j.; HUFF, R. L.; Tosuts, C. A... and
`LAWRENCE, J. H.: Iron turnover abnormalities in pa-
`tienls having anemia; serial blood and in oivo tissue
`studies with Fe“. Acta haemat. 9: 73-96. 1953.
`6. Kant‘-s.
`l... M.. and Mo|uuso.V. 1). B.: In nitro in-
`oorporation of iron-59 into hemoglobin S visualized by
`
`autoradiography. Pros. Soc. Exper. Biol. er Med. 39:
`598-502, 1955.
`7. L1-:E.|tslm\. C. H. W., and COHEN, J. A.: Determina-
`tion of life of human blood platelets using labelled
`cliisopropylfluorophosphonate. Nature, London
`175:
`552-553, I955.
`8. S1‘IttI.VuA. M. M.: SAMPLE, A. B., and HART, E. D.:
`Improved micro hematocrit method. Am. J’. Clio. Path.
`24: 1016-1024, 1954-; [corr. 25: 298. 1955].
`9. SUIT, H. D.; LAJTHA, L. G.: OLIVER, R., and ELLIS,
`F.: Studies on “Fe 1:
`take by normoblasts and failure
`of X-irradiation to a
`t uptake. Brit. J. Haematot. 3:
`165-174, I957.
`10. Younc, L. E., and LAWRENCE, J. 5.: Maturation
`and destruction of
`transfused human reticulocytes;
`evaluation of reticulocyte experiments for measure-
`ment of hemoglobin metabolism.
`J’. Clin. Invest. 24:
`554-563, 1945.
`
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