[CANCER RESEARCH 49, 5879-5883. November 1, 1989]
`
`Changes in Plasma Methionine and Total Homocysteine Levels in Patients
`Receiving Methotrexate
`Infusions1
`
`Emmett H. Broxson, Jr.,2 Linda C. Stork, Robert H. Allen, Sally P. Stabler, and J. Fred Kolhouse
`
`Department of Pediatrics, Wright-Patterson AFB, Dayton, Ohio 45433 [E. H. B.J; C. Henry Kempe Center, The Children's Hospital, Denver, Colorado 80218
`[L. C. S.J; and Department of Medicine, Division of Hematology, University of Colorado School of Medicine, Denver, Colorado 80262 [R. H. A., S. P. S., J. F. K.]
`
`ABSTRACT
`
`reduces intracellular pools of 5-methyltetrahydrofolate
`Methotrexate
`and could result in reduced conversion of homocysteine to methionine by
`methionine synthetase. This study was designed to investigate the effects
`of moderate dose to very high dose methotrexate on methionine and total
`homocysteine as reflections of methotrexate induced intracellular events.
`Methionine and total homocysteine were measured prior to, during, and
`following twenty-six 24-h i.v. infusions of 33.6 g/m2 methotrexate (very
`high dose methotrexate)
`in 16 children with acute lymphocytic leukemia
`and seven 4-h i.v. infusions of 8 g/m2 methotrexate (high dose metho
`trexate) in 5 children with osteogenic sarcoma. Amino acids were meas
`ured by gas chromatography/mass
`spectrophotometry. Mean methionine
`levels decreased by 70.0 ±3.1% (SE) with very high dose methotrexate
`and 72.6 ±5.9% with high dose methotrexate at 24 and 4.5 h, respec
`tively, after beginning methotrexate infusions. Mean total homocysteine
`levels increased by 61.7 ±3.1% with very high dose methotrexate and
`55.6 ±17.5% with high dose methotrexate at 36 and 24 h, respectively,
`after beginning methotrexate
`infusions. No consistent or significant
`changes were noted in levels of total cysteine,
`leucine,
`isoleucine, or
`valine. Similar changes did not occur in patients receiving prednisone,
`vincristine, daunomycin, and intrathecal methotrexate
`as therapy for
`acute lymphocytic leukemia. These changes in homocysteine and methi
`onine may reflect biological effects of methotrexate that may predict
`cytotoxicity of methotrexate.
`
`INTRODUCTION
`
`that binds to and inhibits
`is a potent antifol
`Methotrexate
`reducÃ(cid:173)ase. Recently, methotrexate,
`especially
`dihydrofolate
`polyglutamates,
`has been shown to inhibit other
`methotrexate
`folate dependent enzymes and in this manner disturbs intracel
`lular folate cycling (1).
`is a key intracellular
`(EC 2.1.1.13)
`Methionine
`synthetase
`cobalamin dependent enzyme that catalyzes the transmethyla-
`tion of homocysteine
`to methionine. The major methyl donor
`for this reaction is 5-methyltetrahydrofolate.
`Although nothing
`is known regarding the effect of methotrexate on the activity of
`this enzyme (1), methotrexate
`could interfere with methionine
`synthetase activity: by direct
`inhibition, which would tend to
`increase cellular 5-methyltetrahydrofolate
`levels; by competi
`tion for cellular uptake with 5-methyltetrahydrofolate
`present
`in serum; or by the known inhibition by methotrexate of méth
`ylènetetrahydrofolate
`reducÃ(cid:173)ase(EC 1.1.1.68), an enzyme re
`sponsible for intracellular
`regeneration of 5-melhyllelrahydro-
`
`revised 4/13/89, 7/19/89; accepted 8/4/89.
`Received 12/19/88;
`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.
`1This work was supported by a gift from the Veterans of Foreign Wars,
`Colorado Chapter (E. H. B., L. C. S.): National Institute of Diabetes and Digestive
`and Kidney Diseases, Department
`of Health and Human Services, Grants
`DK21365-12 (R. H. A.) and DK37165-02 (S. P. S.), NIH Grant DK37165 (S. P.
`S.), and National
`Institute of General and Medical Sciences, Department
`of
`Health and Human Services, Grant GM26486-09
`and March of Dimes Birth
`Defects Foundation Grant 1-805 (J. F. K.).
`The opinions and assertations expressed are those of the authors and are not
`to be construed as official or as if reflecting the policy of the United States Air
`Force.
`Presented in part at the 79th American Society of Clinical Oncology Meeting,
`May 24, 1988.
`2To whom requests for reprints should be addressed.
`
`folale (1), bolh of which would lend lo decrease cellular 5-
`methyllelrahydrofolale
`levels.
`Recent
`tissue culture studies reveal thai incubation of human
`cells with metholrexale
`(10 ^M) results in a dramatic and rapid
`(wilhin hours) fall in inlracellular 5-melhyltelrahydrofolale
`(2,
`3). This acule cellular folate deficiency could result in decreased
`activity of methionine
`synthelase and reduced aclivity of me
`thionine synthetase could result
`in reduced cellular methionine
`(and increased homocysteine). Reductions of intracellular me
`thionine would be predicted lo have major consequences on
`methylalion reaclions involving DNA, RNA, and proteins (Fig.
`1). Individuals who have dielary folale deficiency are known lo
`have increased serum levels of homocysleine
`(4) and normal
`levels of serum melhionine (4).
`1.0 lo 13.6 g of melholrexale
`Refsum et al. (5) adminislered
`i.v. over 2-4 h lo adulls and demonslraled
`a modesl
`increase
`in serum homocysleine that tended to decrease with subsequenl
`Irealmenls. No consislenl changes in plasma melhionine were
`observed. The presenl sludy was designed lo measure the effecls
`of 4-h infusions of 8 g/m2 and a 24-h infusion of 33.6 g/m2 of
`melholrexale
`on plasma methionine
`and lotal homocysleine.
`The resulls demonslrale
`a dramalic
`decline in Ihe plasma
`melhionine
`levels and a less striking increase in plasma tolal
`homocysleine levels. Il is possible thai
`the changes in metabo-
`liles lhal are Ihe subslrale
`and product of Ihe melhionine
`synlhelase reaclion in palienls
`Irealed wilh melholrexate will
`prove lo be important markers of cyloloxicily and therapeutic
`response to methotrexale.
`
`MATERIALS
`
`AND METHODS
`
`Patients. Five patients [age, 14.8 ±1.4 year (SD)] with osteogenic
`sarcoma receiving high dose methotrexate,
`8 g/m2, prior
`to their
`definitive surgery were evaluated. Plasma methionine,
`total cysteine,
`and total homocysteine were measured during seven doses of high dose
`methotrexate.
`Sixteen children [age, 6.6 ±4.4 years (SD)] with acute lymphocytic
`leukemia receiving very high dose methotrexate, 33.6 g/m2, were eval
`uated. Plasma methionine,
`total cysteine, and total homocysteine levels
`were measured during 26 doses of very high dose methotrexate. One
`adult, age 39 years, with Burkitt's lymphoma,
`treated with 3 g/m2 of
`methotrexate over 32 h was also evaluated.
`Protocols. Patients with osteogenic sarcoma were given 8 g/m2 of
`methotrexate i.v. over 4 h followed by leucovorin beginning 24 h from
`starting high dose methotrexate
`infusions. According to Children's
`Cancer Study Group Protocol CCG-782, during high dose methotrex
`ate infusions and until
`the methotrexate
`level was less than 0.1 MM,
`patients were alkalinized to keep the urine pH between 6.5 and 7.5 and
`fluid input was maintained at twice maintenance fluids. No chemother
`apy was administered concomitantly with high dose methotrexate, but
`vincristine was given 24 h after the high dose methotrexate
`infusion.
`The four infusions of high dose methotrexate were given 1 week apart
`for two doses prior to and following a course of bleomycin, cyclophos-
`phamide, and dactinomycin.
`Patients with acute lymphocytic leukemia received 6 g/m2 metho
`trexate i.v. over 1 h followed by 1.2 g/m2/h for the next 23 h, totaling
`33.6 g/m2 methotrexate
`during the 24-h infusion. These patients,
`treated according to Children's Cancer Study Group Protocol CCG-
`
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`
`EFFECTS OF METHOTREXATE ON METHIONINE AND TOTAL HOMOCYSTEINE
`
`DNA
`RNA
`Protein
`
`Ad«nolyl-S-CH,'CH,-C-COOH
`NHi
`
`S-AOENOSYLMETHIONINE
`
`a-KETOBUTYRATE
`
`Fig. 1. Intracellular metabolism of homocysteine and methionine. The role of
`5-methyltetrahydrofolate
`(5-methyl FH4). tetrahydrofolatc
`(FH4), dihydrofolate
`(FH,). 10-formyltetrahydrofolate
`( W-formylFH4) and 5.10-methylenetetrahydro-
`folate (5,10-methylene FH4) are illustrated. FAD, flavin adenine dinueleotide;
`MeCbl, methylcobalamin.
`
`infusion on the second day of a 1-month
`144, received methotrexate
`induction regimen; 1 methotrexate
`infusion on days 1, IS, and 29 of
`consolidation phase; and 1 methotrexate
`infusion on day 1 of six 6-
`month maintenance cycles. The first maintenance cycle was scheduled
`to begin 2-3 weeks following consolidation
`and patients
`received a
`total of 10 infusions of very high dose methotrexate during the 3 years
`of therapy. Alkalinization and hydration were similar to that given for
`patients receiving high dose methotrexate. Leucovorin is begun at hour
`36, 12 h after completion of the very high dose methotrexate infusion.
`Chemotherapy administered concomitant with the first methotrexate
`dose includes prednisone; with the second through fourth dose, pred-
`nisone and vincristine; and with the fifth through tenth dose, predni
`sone, vincristine,
`and 6-mercaptopurine
`(prednisone, 40 mg/kg/day
`P.O.; vincristine, 1.5 mg/m2 i.V.; and 6-mercaptopurine,
`500 mg/m2
`
`for methotrexate (20.0 mg/min/m2 for very high
`infusion rate constant
`dose methotrexate
`and 33.3 mg/min/m2 for high dose methotrexate)
`divided by the steady state serum methotrexate
`concentration as as
`sessed at 24 or 4.5 h after very high dose and high dose methotrexate
`infusions began (8).
`analyses of the data were per
`Statistical Calculations. Statistical
`formed by Student's
`t test,
`linear regression, and Pearson correlation
`using Lotus 1-2-3 and Statistix II software programs. Results are
`reported as mean ±SE unless otherwise indicated.
`Methionine Synthetase Assay. Methionine
`synthetase was purified
`and assayed as described (9). For each assay where methotrexate was
`included, a duplicate containing no methionine synthetase served as a
`control. Methotrexate was obtained from Sigma Chemical Co., St.
`Louis, MO, and methotrexate heptaglutamate was obtained from Dr.
`Charles Baugh.
`
`RESULTS
`
`Effects of High Dose Methotrexate. A decrease in plasma
`methionine occurred in all patients with osteogenic sarcoma
`4.5 h after starting high dose methotrexate. The mean decrease
`of 72.6 ±5.9% at 4.5 h represented a decline from 22.4 ±3.7
`MMat baseline to 5.6 ±1.0 ßM(P< 0.004). The mean plasma
`methionine
`level returned to near baseline at 24 h (Table 1;
`Fig. 2).
`Plasma total homocysteine increased in all patients receiving
`high dose methotrexate
`and the peak occurred at 24 h after the
`
`total homocysteine, and cysteine levels; mean
`Table 1 Mean plasma methionine,
`serum methotrexate levels; and mean homocysteine:methionine
`ratios during and
`following seven 4-h i.v. infusions of S g/m2 of methotrexate in 5 patients with
`osteogenic sarcoma
`infusions begin
`Samples are drawn at the hours specified with methotrexate
`ning at hour 0. Unless otherwise indicated, all levels are expressed as (imol/liter.
`MTX, methotrexate; HCYS, homocysteine; METH, methionine; CYS, cysteine.
`Time
`<hr)0
`
`ratio0.49
`±0.1
`±
`±3.7
`±
`5.6 ±1.0°
`2.50 ±0.4*
`4.5
`1.32.1C
`215.7±
`11.9±
`722.9 ±52.8
`
`220.0 ±15.2
`15.2 ±
`26.4 ±7.6
`3.9 ±1.1
`24
`0.82 ±0.2
`248.0 ±15.412.7
`
`
`12.3±1.12.0METH22.417.1 ±2.0CYS244.7
`0.5 ±0.2HCYS9.9
`48MTX0
`0.2"
`
`13.9HCYS/METH0.79 ±
`P < 0.004.
`'/>< 0.002.
`
`50
`
`Homocysteine
`
`12
`Time (hrs)
`
`i.V.).
`their legal guardian gave written informed con
`All patients and/or
`sent prior
`to participation
`in this study and in the chemotherapy
`protocols
`in accordance with the policies of the Medical Research
`Committee of The Children's Hospital and University Hospital
`in
`Denver.
`Sample Collection and Analysis. One to 2 ml of blood were collected
`into EDTA sample tubes when methotrexate
`levels were drawn. The
`plasma was separated from the RBC after centrifugaron
`and frozen at
`—¿(cid:3)20"Cuntil amino acids were measured. Methotrexate
`levels were
`drawn at 0, 4.5, 24, and 48 h after beginning high dose methotrexate
`and at 0, 6, 24, 36, 48, and 72 h after beginning the very high dose
`methotrexate
`infusions. To be évaluablea baseline sample and all or
`all but one of the remaining samples had to be available. Plasma
`methionine,
`total cysteine, and total homocysteine were measured as
`described by Stabler et al. (6). Normal
`ranges (mean ±2 SD after log
`normalization)
`for these three amino acids were: methionine,
`13.5-
`36.8 JIM;total homocysteine, 5.4 -16.2 MM;and cysteine, 186-335 V.M.
`Plasma levels of leucine, isoleucine, and valine were also measured (7)
`during seven methotrexate
`infusions. Serum methotrexate
`levels were
`measured using an enzyme immunoassay (Emit assay; Syva Co., Palo
`Alto, CA).
`Pharmacokinetic Analysis. Systemic clearance of methotrexate
`min/m2) was calculated after each infusion from the zero-order
`
`(ml/
`i.v.
`
`total homocysteine, and
`Fig. 2. Percentage of change of plasma methionine,
`cysteine from baseline levels during and following a 4-h i.v. infusion of 8 g/m2
`methotrexate
`in a patient with osteogenic sarcoma.
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`
`EFFECTS OF METHOTREXATE ON METHIONINE AND TOTAL HOMOCYSTEINE
`
`f-Methotrexate -\
`
`24
`
`48
`
`Time (hrs)
`total homocysteine. and
`Fig. Ì.Percentage of change of plasma methionine,
`cysteine from baseline levels during and following a 24-h i.v. infusion of 33.6 g/
`nr' methotrexate in a child with acute lymphocytic leukemia.
`
`72
`
`infusions began in 5 of 7 doses administered. This
`methotrexate
`elevation of mean plasma total homocysteine
`from 9.9 ±1.1
`MMat baseline to 15.2 ±2.1 MMat 24 h (P < 0.05) represented
`a 55.6 ±17.5% increase. This elevation was concomitant with
`the decline in methotrexate
`levels and the administration
`of
`leucovorin. The mean total homocysteine
`decreased to near
`mean baseline levels at 48 h after
`the methotrexate
`infusion
`began.
`ratio was signifi
`The mean total homocysteine:methionine
`cantly elevated above the mean baseline level at 4.5 h (P <
`0.002)
`representing
`a 462.4 ±95.5% increase. This ratio ap
`proached the baseline value at 48 h.
`The mean plasma total cysteine level appeared to decrease
`during the infusions of high dose methotrexate, but the maximal
`decrease of 11.4 ±3.4% at 4.5 h was not statistically different
`from the mean baseline value (P = 0.17).
`Effects of Very High Dose Methotrexate. Plasma methionine
`decreased in all patients receiving very high dose methotrexate.
`The nadir
`for each patient occurred during the methotrexate
`infusions at either 6 or 24 h (14 at 6 h and 12 at 24 h) and then
`returned to baseline by 48 h. The mean methionine
`level de
`creased 70.03 ±0.1% from a baseline of 22.1 ±1.9-5.8 ±0.5
`MMat 24 h (P < 0.0001)
`(Table 2; Fig. 3).
`levels occurred in
`An increase in plasma total homocysteine
`92% of the patients with the maximal change occurring between
`24 and 72 h (41.8 ±14.0 h) (SD). The mean plasma total
`homocysteine
`level increased significantly between 0 and 36 h
`(P < 0.003) and remained significantly elevated at 48 h (P <
`0.02) before returning
`to near baseline values by 72 h. This
`change at 36 h represented a 61.7 ±12.8% mean increase over
`baseline
`levels. Plasma
`total homocysteine
`levels tended to
`return to baseline values concurrent with the administration
`of
`leucovorin and the initial decline in serum methotrexate
`levels.
`The mean total homocysteine:methionine
`ratio was signifi
`cantly elevated above the mean baseline ratio at 6, 24, and 36
`h (P < 0.0001, P < 0.0004, and P < 0.0004,
`respectively) with
`the largest
`increase of 550.4 ±133.0% occurring at 24 h. This
`ratio tended to return to the baseline value by 48-72 h. A small
`(less than 15%) but statistically insignificant apparent decrease
`in the mean total cysteine level was observed at 6, 24, and 36 h
`(P = 0.0575, P = 0.0919, P = 0.2152, respectively).
`Serum Methotrexate
`Levels. Comparable
`levels of serum
`methotrexate were attained in patients receiving high dose and
`very high dose methotrexate
`(Tables 1 and 2). As assessed by
`the Pearson correlation
`coefficient,
`the percentage of change
`from baseline levels in plasma methionine,
`total homocysteine,
`and the total homocysteine:methionine
`ratio did not appear to
`
`levels at each point of as
`correlate with serum methotrexate
`sessment
`for either high dose or very high dose methotrexate.
`Similarly,
`the peak serum methotrexate
`levels did not appear
`to correlate with the maximum changes in plasma methionine
`or total homocysteine
`for high dose and very high dose meth
`otrexate.
`Methotrexate Clearance. The peak homocysteine:methionine
`ratio did not appear
`to be related to the peak methotrexate
`level, but the peak homocysteine:methionine
`ratio was 2.50 for
`the high dose methotrexate
`patients whose clearance was 59.5
`±15.6 mg/min/m2 while the peak homocysteine:methionine
`ratio was 1.33 for the very high dose methotrexate
`patients
`whose clearance was 105.0 ±25.6 mg/min/m2. The systemic
`clearance of methotrexate may have prognostic significance in
`children with acute lymphocytic leukemia (10, 11) and in these
`studies a higher homocysteine:methionine
`ratio was obtained
`in patients with the slower clearance rates.
`Effects in Patients not Receiving High Dose or Very High
`Dose Methotrexate. None of the changes in plasma methionine
`or total homocysteine noted with very high dose and high dose
`methotrexate were seen in 3 patients receiving intrathecal meth
`otrexate, daunorubicin,
`prednisone,
`and vincristine during in
`duction for acute lymphocytic
`leukemia. Mean plasma total
`homocysteine
`at baseline, 7.2 ±1.7 MM,was similar
`to the
`mean at time of maximum change, 7.0 ±0.4 MM.The mean
`plasma level of methionine
`increased from 16.6 ±3.9 MMat
`baseline to 29.0 ±2.7 MMat the points of maximum change.
`Mean plasma cysteine decreased from 176.3 ±23.7 MMat
`baseline to a mean of 153.7 ±9.6 MMat the points of maximum
`ratio0.27
`change.
`0.4±0.4±0.7±0.7*±0.4'±0.3METH22.17.55.813.124.1±±¿±±28.3±9.1158.0 ±0.030.931.330.690.380.234-±•±•£±0.14°0.20°O.IO*0.060.03
`
`
`
`in an Adult. Fig. 4
`Effect of Moderate Dose Methotrexate
`±6.6159.2164.9185.8175.0±
`shows the results of treatment of an adult patient with a 3-g/
`7.8±7.2±6.2±7.3HCYS/METH
`m2 dose of methotrexate. As observed with children receiving
`higher doses of methotrexate,
`this adult patient with Burkitt's
`lymphoma also showed the characteristic
`decrease in methio
`nine and rise in total homocysteine
`although the decrease in
`methionine was less pronounced at this lower dose of metho
`trexate.
`5881
`
`total homocysteine, and cysteine levels; mean
`Table 2 Mean plasma methionine,
`serum methotrexate levels; and mean homocysteine.-methionine ratios during and
`following twenty-six 24-h i.v. infusions of 33.6 g/m2 of methotrexate in 16
`children with acute lymphocytic leukemia
`Samples were drawn at hours specified with methotrexate infusions beginning
`at hour 0. Unless otherwise indicated, all levels are expressed as ^mol/liter. MTX,
`methotrexate; HCYS, homocysteine; METH, methionine; CYS, cysteine.
`Time
`(hr)MTX0
`06
`±24
`±36
`±48
`±72
`±"
`P<0.0001.*
`P <0.003.c
`P <0.0002."
`P < 0.0004.56.142.25.41.10.1HCYS5.25.36.67.86.95.3±
`
`±1.91.0°0.5°1.0e2.62.5CYS180.0
`
`877.8
`790.0
`28.7
`4.7
`0.8
`
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`
`EFFECTS OF METHOTREXATE ON METHIONINE AND TOTAL HOMOCYSTEINE
`
`Methotrexate
`
`5-Formyl FH4
`
`70
`
`Fig. 4. Percentage of change of plasma methionine,
`cysteine following i.v. infusion of 3 g/m2 methotrexate
`lymphoma.
`
`total homocysteine, and
`in an adult with Burkitt's
`
`Table 3 Effect of methotrexate (10 IÃ(cid:141)M)and methotrexate heptaglutamate (10
`\IM) on partially purified and homogeneous human placenta methionine
`synthetase activity
`
`AdditionNone
`
`±0.3"
`±0.3
`9.9 ±0.3
`9.8 ±0.1
`Methotrexate
`9.9 ±0.4Homogeneous10.0
`9.9 ±0.3
`Methotrexate heptaglutamateCrude10.1
`" The /' value for all values shown in this table is 0.20 or greater.
`
`toxicity of methotrexate may result from reduced biosynthesis
`of methionine which leads to a deficiency of certain vital lipo-
`tropes formed via one-carbon metabolism. Many malignant cell
`lines require exogenous methionine for growth in vitro ( 15). By
`decreasing protein synthesis the profound protracted hypome
`thioninemia induced by methotrexate
`in this study may contrib
`ute to the cytotoxicity of methotrexate. However, until patients
`who are treated with low dose methotrexate where clinical
`toxicity can be severe in the absence of rescue with leucovorin
`are studied,
`the role of hypomethioninemia
`in the toxicity of
`methotrexate
`cannot be determined. Furthermore, whether or
`not such patients develop hypomethioninemia
`associated with
`low doses of methotrexate
`is presently unknown.
`Total homocysteine
`levels increased during the infusions of
`methotrexate
`and continued to rise after
`the infusions were
`completed. The decline toward baseline occurred concomitantly
`with the administration
`of leucovorin and decreasing metho
`trexate levels. Reduced 5-methyltetrahydrofolate
`pools would
`be expected to cause a build up of intracellular homocysteine
`(Fig. 1). When normal and malignant mouse fibroblasts are
`exposed to methotrexate
`in culture there is a marked efflux of
`homocysteine with only a small
`increase in intracellular hom
`ocysteine
`(18). This efflux of homocysteine
`is much more
`pronounced in malignant
`than in normal mouse fibroblasts and
`is almost completely prevented by the addition of leucovorin to
`the culture media (18).
`in total
`(5) noted an increase
`Previously, Refsum et al.
`homocysteine which peaked at 24 h after 2-4-h infusions of 1-
`13.6 g of methotrexate
`in 7 adults. Hypomethioninemia was
`not noted except
`in one patient
`receiving 13.6 g of methotrex
`ate. This lack of hypomethioninemia may be due to several
`factors:
`(a) samples were not obtained during methotrexate
`infusions in all patients;
`(/;) the duration of administration
`of
`methotrexate was shorter
`than that
`in our study; and (c) the
`dose for 5 of 7 patients was only l g of methotrexate.
`The changes in total homocysteine and methionine noted in
`our study appear
`to be specific for methotrexate,
`since these
`changes were seen with high dose methotrexate when it was
`used without concomitant
`chemotherapy.
`In addition,
`the ad
`ministration
`of intrathecal methotrexate
`and systemic vincris-
`tine, daunomycin,
`and prednisone to patients with acute lym-
`phocytic leukemia did not produce similar alterations
`in plasma
`methionine and total homocysteine levels.
`Hypomethioninemia
`(35% of normal) has been found to
`occur following i.v. cisplatin which was more pronounced when
`etoposide was given concomitantly with cisplatin intraperito-
`neally in humans
`(19). Cisplatin complexes with methionine
`(20) which may explain the hypomethioninemia
`observed. Such
`a complex of methionine with methotrexate
`has not been de
`scribed.
`In addition to the increase in plasma total homocysteine with
`methotrexate
`infusions, Refsum et al. (5) noted reduced plasma
`levels of total homocysteine
`and decreasing magnitude of the
`changes in total homocysteine with subsequent doses of meth
`otrexate. Only one patient entered on the present
`study had
`four sequential doses of methotrexate
`evaluated from diagnosis
`of acute lymphocytic leukemia. Her baseline total homocysteine
`level increased by the second dose and remained stable for the
`next two doses. However,
`the maximum percentage of change
`in plasma total homocysteine
`did decrease from 165.4% to
`26.9% over four doses. Her baseline plasma methionine
`de
`creased from 33.1 *<Mto I3.9 ¿IMwith a concurrent decrease in
`the percentage
`of change of methionine
`from -81.0% to
`-47.5%.
`This decrease
`in percentage
`of change of plasma
`5882
`
`Determination of Direct Inhibition of Methionine Synthetase
`by Methotrexate. To determine
`if direct
`inhibition of human
`methionine
`synthetase occurs with methotrexate
`(monogluta-
`mate) or methotrexate
`polyglutamate,
`both homogeneous
`and
`partially purified human placenta! methionine synthetases were
`incubated with 10 /¿Mmethotrexate
`(monoglutamate)
`and 10
`Ã(cid:141)¿Mmethotrexate
`heptaglutamate
`(Table 3). Less than 10%
`inhibition of the activity of human methionine
`synthetase was
`observed in triplicate experiments. No difference in inhibition
`was noted between partially purified or homogeneous methio
`nine synthetase. Thus, we conclude that methotrexate or meth
`otrexate polyglutamates do not directly inhibit methionine syn
`thetase in vitro. The concentration
`of methotrexate
`of 10 ¿JM
`results in complete
`inhibition of dihydrofolate
`reductase (12)
`and greater than 90% inhibition of DNA synthesis in fibroblasts
`(13).
`
`DISCUSSION
`
`study
`evaluated in this
`The three doses of methotrexate
`during infusion. The
`produced profound hypomethioninemia
`degree of hypomethioninemia
`decreased as the serum metho
`trexate level decreased and methionine
`rebounded above base
`line levels at 48-72 h after methotrexate
`infusions began. Baram
`et al. (2, 3) have shown that
`the intracellular concentration of
`5-methyltetrahydrofolate
`is reduced within hours in cells ex
`posed to methotrexate. Thus decreased intracellular conversion
`of homocysteine
`to methionine
`(Fig. 1) would be expected to
`occur and cause an intracellular depletion of methionine
`as is
`seen in genetic defects of methionine synthetase (14). Metabolic
`functions of methionine
`include (a) utilization for protein syn
`thesis and (/>)conversion to .S-adenosylmethionine which serves
`as the predominant methyl group donor, a precursor
`in polyam-
`ine synthesis, and as an intermediate
`in the transsulfuration
`pathway (15, 16). Borsi et al. (17) have suggested that hepato-
`
`Lilly Ex. 2122
`Sandoz v. Lilly IPR2016-00318
`
`

`
`EFFECTS OF METHOTREXATE ON METHIONINE AND TOTAL HOMOCYSTEINE
`
`levels may reflect decreas
`and total homocysteine
`methionine
`ing tumor burden since methotrexate
`appears
`to cause more
`pronounced efflux of homocysteine in malignant
`than in normal
`cells in vitro (18). However,
`this observation could also reflect
`the development of methotrexate
`resistance following repetitive
`treatment. Studies in patients who have developed methotrexate
`resistance would help distinguish whether
`the changes in hom
`ocysteine and methionine reflect a generalized metabolic effect
`of methotrexate or if these changes reflect changes in the tumor
`burden.
`on methi
`the effect of methotrexate
`It is unknown whether
`onine and homocysteine levels is related to organ specific effects
`such as on the liver or changes
`in peripheral utilization and
`metabolism of methionine
`and homocysteine. Lower extracel
`lular methotrexate
`concentrations
`(0.1-10
`/Ã(cid:141)M)appeared
`to
`inhibit methionine uptake by LI 210 mouse leukemia cells (21).
`Although the peak levels of methotrexate
`reached in the present
`studies were much higher, we would have expected serum levels
`of methionine to increase, not decrease,
`if inhibition of cellular
`uptake of methionine by cells in general was the sole effect of
`methotrexate
`on methionine metabolism. Others
`(22) have
`shown that
`the antimetabolic
`effect of methotrexate measured
`in human bone marrow cells and leukemic cells by the deox-
`yuridine suppression test was aggravated by methionine supple
`mentation
`and improved by homocysteine
`supplementation
`(22), an effect opposite of the effect
`in hepatocytes
`suggesting
`that organ specificity is significant. Studies of isolated perfused
`liver and injection of radiolabeled forms of homocysteine
`and
`methionine in animals will be required to sort out the effects of
`methotrexate on intracellular
`folate levels and effects on specific
`organs as well as peripheral
`tissues. Furthermore,
`the levels of
`methotrexate
`required to produce depletion of intracellular 5-
`methyltetrahydrofolate
`were considerably less (1-10 ^M) than
`the serum levels of methotrexate
`reached in the present study.
`The higher extracellular methotrexate
`levels of the present
`study could have additional or different mechanisms that result
`in the observed changes in methionine and homocysteine levels.
`Regardless of the mechanisms
`of hypomethioninemia
`and
`homocystinetnia,
`administration
`of moderate dose to very high
`dose methotrexate
`in this study was associated with decreases
`in plasma methionine
`levels and late increases in plasma total
`homocysteine levels. Further studies are indicated to determine
`if the effects on plasma methionine
`and total homocysteine
`occur with even lower doses of methotrexate
`and with various
`methods of administering methotrexate,
`as well as to determine
`if methionine and total homocysteine
`levels or changes in their
`levels can be used to predict clinical
`toxicity or therapeutic
`responses to methotrexate. These measurements might be used
`to reduce toxicity,
`to monitor
`for the development of resistance,
`and to enhance the tumor cytotoxicity of methotrexate.
`
`ACKNOWLEDGMENTS
`
`The assistance of the following people was invaluable: the oncology
`unit staff at Denver Children's Hospital for help with sample collection;
`Barbara Fenton and Lynn Barczuk for coordination
`and help with
`sample handling; Beverly Raab for assistance in assaying amino acid
`levels; and Susan A. Veach for the preparation of the manuscript.
`
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