A Case for the Use of Aminopterin in Treatment of Patients with Leukemia Based on Metabolic Studies of Blasts in Vitro I

Vol. 2, 69-73, January 1996 Clinical Cancer Research 69

Angela Smith, Martina Hum, Naomi J. Winick, may be useful in children who are identified as poor risks and Barton A. Kamen 2 with respect to MTX uptake. Departments of Pediatrics [A. S., M. H., N. J. W., B. A. K.] and Pharmacology [B. A. K.], University of Texas Southwestern Medical INTRODUCTION Center, Dallas Texas 75235-9063 When Farber et al. (1) ushered in the modern era of chemotherapy for children with ALL 3 using an antifolate, the drug was AMT, not MTX. Subsequent laboratory work showed ABSTRACT that MTX, although less potent than aminopterin, had a better Clinical and laboratory investigations during the past therapeutic index in a murine model (2). These preclinical four decades have resulted in numerous schedules, doses, findings and the unpredictable toxicity of AMT, most likely and routes of delivery for methotrexate (MTX). It remains because it was contaminated with folate and/or because of its as an important drug for the treatment of children with increased potency, resulted in a switch to the use of MTX. acute lymphoblastic leukemia (ALL). Aminopterin (AMT) Today, 45 years after the introduction of antifolates for the was the initial antifolate showing promise as an anticancer treatment of patients with cancer and nonmalignant disorders, drug. It is more potent than MTX and also is known to be MTX remains a mainstay in our armamentarium, especially for accumulated more efficiently than MTX in model systems. the treatment of children with ALL (3). Aminopterin is used Because Whitehead et al. (Blood, 76: 44-49, 1990) have primarily as a folate analogue in the laboratory. shown that MTX accumulation by blasts at diagnosis is of A significant amount of laboratory investigation and clin- prognostic significance in children with ALL, we reasoned ical experience during the past three to four decades (e.g., there that if accumulation of a "stoichiometric inhibitor" of di- are more than 19,000 citations for MTX since 1966) has resulted hydrofolate reductase by leukemic blasts was of prognostic in the testing of numerous schedules, doses, and routes of delivery of MTX in patients with cancer (reviewed in Refs. 3 importance, then whether it was AMT or MTX may be and 4). Biochemical parameters, such as mutations or amplifi- relevant only with respect to the absolute dose. To compare cation of DHFR, decreased transport or metabolism of drug to a MTX and AMT metabolism, we incubated lymphoblasts polygammaglutamyl form, and/or increased catabolism have with 1 pLM radiolabeled drug in vitro. MTX and AMT accu- been associated with resistance to MTX (reviewed in Ref. 4). mulation by ALL cells (n = 24) was 0.7 -- 0.7 and 1.47 -- 0.9 With special regard to the treatment of children with ALL, pmol/106 cells, respectively. Based on the data of Whitehead Whitehead et al. (5) have shown that MTX accumulation (up- et al., this predicts pharmacological success in 59 and 84% of take and metabolism to a polygammaglutamyl form) is of prog- the MTX and AMT groups, respectively. Moreover, 5 of 10 nostic significance. If blasts accumulated >-0.5 pmol MTX patients considered poor risks based on MTX accumulation polyglutamates/106 cells during a 24-h incubation with 1 ~M would be "cures" based on AMT uptake. Even at only 0.1 3H-MTX in vitro, the statistical likelihood of a cure was greater IOLMAMT, a concentration at which there is little accumula- compared with that in children whose blasts accumulated less. tion of MTX, 5 of 11 patients studied would be "pharma- Having a biochemical or pharmacological end point as a cological cures" based on AMT uptake. Accumulation of prognostic indicator, in addition to an immunological or karyo- AMT by blasts from 11 patients with T-cell-lineage ALL and typic marker, is a potentially important marker of why a patient 5 patients with acute myelogenous leukemia was also found will relapse rather than just who will relapse. It may afford the to be twice the uptake of MTX. These data allow the sug- clinician an opportunity to alter therapy based on drug metab- gestion that AMT, despite increased potential for toxicity, olism; i.e., there is a defined pharmacodynamic value required for therapy to succeed. Currently, the Pediatric Oncology Group is studying MTX metabolism in children, as assessed by mon- itoring RBC folate and drugs (6), and a small subset of the group is obtaining data to confirm and extend the initial observations Received 5/31/95; revised 8/11/95; accepted 8/30/95. of Whitehead et al. (5). As part of this study, we have studied This work is supported by American Cancer Society Grant DHP 68 not only 3H-MTX but also the metabolism of the physiological and NIH Grant UO1 CA60431 (to B. A. K., an American Cancer Society Clinical Research Professor). M. H. is supported in part by an (plasma) folate 5-methyl-3H-tetrahydrofolic acid (7) and/or American Cancer Society Clinical Oncology Fellowship. This work was presented in part at the 86th Annual Meeting of the American Associ- ation for Cancer Research, March 18-22, 1995, Toronto, Ontario, Canada. 2 To whom requests for reprints should be addressed, at Department of 3 The abbreviations used are: ALL, acute lymphoblastic leukemia; Pediatrics, University of Texas Southwestern Medical Center, 5323 AMT, aminopterin; MTX, methotrexate (amethopterin); DHFR, dihy- Harry Hines Boulevard, Dallas, TX 75235-9063. Phone: (214) 648- drofolate reductase; AML, acute myelogenous leukemia; HPLC, high- 3896; Fax: (214) 648-3122. performance liquid chromatography.

3H-AMT. We reasoned that if accumulation of a "stoichiomet- 4.0 ric inhibitor" of dihydrofolate reductase by leukemic blasts was of prognostic importance, then whether it was either AMT or 3.5 MTX polyglutamate may be relevant only with respect to the amount of the drug (mg/m 2) needed. Maybe the potency of "~u 30 AMT can be harnessed by applying knowledge obtained during ~ 2,5 the 40 years of empiric and laboratory studies of antifolates. The results of our study of AMT and MTX metabolism by blasts in ,,tff 20 vitro obtained from children with leukemia constitute the sub- -o 1.5 ject of this report. 0 1.0 ! 0.5 ...... MATERIALS AND METHODS ! Patients. In the last 18 months, we had the opportunity to 0.0 study blast cells from 63 children diagnosed with leukemia. Of MTX AMT these, 40 samples provided sufficient numbers of blasts to do a Fig. 1 Accumulation of radiolabeled MTX and AMT by blasts incu- comparative study of AMT and MTX metabolism. Twenty-four bated for 24 h with 1 I,LM drug in vitro. The blasts were freshly isolated patients had B-cell-lineage ALL, 10 had T-cell-lineage ALL, and incubated according to methods presented previously (7) and de- and 6 had primary or secondary AML. tailed in "Materials and Methods." The dotted line marks the value (0.5 Media and Radiolabel. Cells were incubated in RPMI pmol drug/106 cells) that is associated with successful therapy. The solid lines are the median values for MTX (0.63 pmol/106 cells) and AMT 1640 with 25 mM HEPES, 10% FCS (GIBCO 26140-020), and (1.19 pmol/106 cells) in these 24 matched samples. GAT (5 mg glycine, 1 mg adenosine, and 1 mg thymidine/ml). 3H-MTX and 3H-AMT (Moravek MT701 and MT506; 15-30 Ci/mmol) were added to the media with the unlabeled drug to a concentration of 1 ~M (final specific activity, 10 ~Ci/ml). Un- RESULTS labeled polyglutamate standards of MTX-Glu2_ 6 were pur- A comparison of MTX and AMT accumulation by blasts chased from Dr. B. Schircks (Jona, Switzerland). AMT-Glu 2 from newly diagnosed patients with B-cell-lineage ALL incu- was supplied very kindly by Dr. J. Piper (Southern Research bated overnight with 1 p~M radiolabeled drug in vitro is pre- Institute, Birmingham, AL). By convention, the parent drug is sented in Fig. 1. The mean uptakes of MTX and AMT were 0.7 Glu~, i.e., the monoglutamate. The addition of one glutamate _ 0.7 and 1.47 _ 0.9 pmol drug/106 cells, respectively. Thus, residue results in a diglutamate, the addition of a second is a AMT uptake is twice the uptake of MTX. The median values for triglutamate, etc. MTX and AMT uptake were 0.63 and 1.19 pmol/106 cells, Tissue Culture Conditions. Fresh lymphoblasts were respectively. The Wilcoxon two-tailed rank test revealed these isolated from bone marrow or blood through Ficoll-Hypaque populations to be significantly different (P = 0.0006). The (Pharmacia 17-0840-02) by standard methods as detailed by Mann-Whitney statistic confirmed that these two populations Whitehead et al. (5), washed with HBSS (without phenol red; are different (P = 0.007). In only one of the 24 cases was uptake GIBCO 310-4025AJ), counted on a Coulter counter, and incu- of the two drugs equivalent. This was in a sample in which MTX bated in 35-mm dishes (Falcon 3001) at a concentration of 5 × uptake was very large (>3.0 pmol/106 cells). A more important 106/2 ml media containing the drug of interest for 24 h at 37°C determinant of cytotoxicity may be the amount of drug accu- in a humidified atmosphere in 5% CO e. Cells were then col- mulated as a polyglutamate. These species have a long intracel- lected, washed three times in 5 ml HBSS at room temperature, lular half-life and are inhibitors of enzymes in the de novo suspended in 2 ml HBSS, counted again, subjected to viability synthesis of purines, in addition to the inhibition of dihyarofo- determination by trypan blue exclusion, and boiled for 5 min. late reductase (9). The product of the total uptake times the An aliqout was counted on a scintillation counter to determine percentage of metabolism yields a mean MTX-Glu n value of the total radioactive uptake. 0.65 pmol/106 cells and an AMT-Glu n value of 1.43 pmol/106 HPLC. An aliqout of the cell extract containing approx- cells. Although the percentages of drugs metabolized to a poly- imately 4000 cpm was filtered, "spiked" with known polyglu- glutamate were similar, 92 and 97% for MTX and AMT, re- tamate standards, and then injected onto a C 18 column (Waters spectively, the interpatient variability was significantly greater WAT086684) connected to a Flo-OnekBeta (Radiomatic) radio- for MTX (___26.5%) than for AMT (_+5.2%). Whether this active flow detector. The parent drug and polyglutamates were reflects a higher efficiency of uptake and/or substrate preference eluted with a gradient of 5 mM tetrabutylammonium phosphate of folylpolyglutamate synthetase for AMT cannot be ascertained (PicA) (Waters WAT085101) and 5 mM PicA with 40% aceto- completely. In a cell-free system, it has been established that nitrile (Baxter 015-4; Ref. 8). Standards were monitored at a AMT has a lower K m and greater Vma× than MTX (10). Because wavelength of 280 nm. 59% of the patients accumulated >0.5 pmol MTX to a larger Statistical Analysis. A two-tailed Wilcoxon signed rank polyglutamate and thus had drug uptakes consistent with a test and Mann-Whitney tests were done using a commercially favorable outcome (5), we did a separate comparison of the available software package (INSTAT; GraphPad, Inc., San Di- patients who would be considered poor metabolizers of MTX ego, California). and, therefore, at increased risk for relapse. MTX metabolism to

Table 1 Accumulationof AMT and MTX in non-B-lineage cells 1.5 MTX AMT Sample n (pmol/106cells) (pmol/106cells) T-cell-lineage ALL 10 0.75 _+ 0.52 1.59 _+ 1.02 AML 6 0.89 _+ 1.33 1.66 _+ 1.19 0 1.0 o

ALL, and antifolates traditionally have not been considered to I"-4 be important in initial therapy. The results are presented in Table .5 ...... 1. Accumulation of AMT by T-cell-lineage leukemia is similar

0 to B-cell-lineage leukemia and is significantly greater than the E uptake of MTX. Four of six AML cell samples accumulated <-0.15 pmol of MTX, and one accumulated more than 3 pmol, whereas in six of six samples, AMT uptake was ->0.5 (range, 0,0 0.7-2.33) pmol/106 cells. MTX AMT To make more definitive comments on differences between Fig. 2 Increasedaccumulation of AMT compared with that of MTX in the metabolism of MTX and AMT (i.e., polyglutamate chain the 10 samples in which the MTX uptake was less than 0.5 pmol/106 length) we would need to obtain a more complete set of AMT cells as shown in Fig. 1 is detailed more completely here. One-half the standards as well as long-term outcome data. Fig. 3, however, samples had sufficient increases in AMT uptake to be considered more likely successes. The value of polyglutamates for MTX was 0.2 _+ 0.11 shows the increased metabolism of AMT compared with MTX pmol and for AMT was 0.61 _+ 0.4 pmol. This 3-fold increase is because in a patient who would be a "MTX failure" but an "AMT the percentage of metabolism in this population was only 85 _+ 17% for success." The total accumulation of MTX was 0.3 pmol/106 MTX and 95.2 +_ 7.9% for AMT. cells, and the total amount of polyglutamates was 0.15 pmol/106 cells (Fig. 3C). Total and polyglutamate uptakes of AMT uptake a polyglutamate in these l0 cases was decreased to 85 _+ 17%, were 0.75 and 0.7 pmol/106 cells, respectively (Fig. 3B). Be- which translates to only 0.20 +_ 0.11 mol MTX polyglutamates/ cause we had only authentic AMT and AMT-Glu 2 (Fig. 3A), we 106 cells, whereas AMT metabolism was still near complete confirmed that the peaks seen in the regions of standard MTX- (95.2 +_ 7.9%), yielding a net accumulation of 0.61 _+ 0.4 pmol Glu3_ 6 were "polyglutamated" inhibitors of DHFR in two AMT polyglutamates/106 cells (Fig. 2). These results allow the ways. First, these peaks all disappeared, and the radiolabel prediction that 5 of 10 of these patients would be considered coeluted with an AMT standard when treated with plasma good risks (i.e., ->0.5 pmol drug/106 cells) if AMT were used. conjugase (y-glutamyl carboxypeptidase), and, second, the tri- This suggests that there may be a therapeutic benefit to using tium was >90% bound by DHFR in the presence of NADPH AMT versus MTX in this select group of patients, whether the using established methodologies for radioligand-binding assays pharmacological reason is either initial transport or subsequent before or after digestion with conjugase (14). metabolism. To address issues of transport and metabolism further, in DISCUSSION addition to the matched incubations done using 1.0 tXM drug, we There are compelling reasons for considering a second look also had enough sample to analyze 3H-AMT uptake in 11 at the clinical usefulness of AMT. Few if any useful new drugs samples incubated in only 0.1 IXM drug. Uptake at only 0.1 IXM have emerged in the past two decades for the treatment of AMT was 0.57 _+ 0.6 pmol/106 cells, and 5 of 11 samples had children with ALL. Looking at antifolates specifically, a li- uptakes greater than 0.5 pmol/106 cells, the amount associated pophilic inhibitor of dihydrofolate reductase, trimetrexate, ap- with successful therapy. Only one sample was not metabolized proved recently for use in the treatment of patients with Pneu- to a polyglutamate completely (i.e., ->Glue). That the uptake of mocystis carinii pneumonia, did not prove useful in the AMT increased only 2.4-fold with a 10-fold increase in the treatment of patients with recurrent leukemia (15, 16). Trime- extracellular drug also suggests that this is a saturable process. trexate is not a substrate for folylpolyglutamate synthetase. Its In contrast, 3H-MTX uptake at 0.1 lXM was generally so small potential superiority to MTX is in its transport characteristics that meaningful data with the number of cells usually acquired (17). Edatrexate, a MTX analogue with good efficacy in pre- cannot be obtained routinely. This finding supports the work of clinical trials (reviewed in Ref. 18) has had mixed results in others and our own studies of human leukemia cells in vitro, i.e., clinical trials in adults with solid tumors, but there are as yet no that AMT is the preferred substrate for transport and/or metab- published reports documenting activity in children with ALL olism compared with MTX (11-13), and that there is a saturable with or without prior exposure to MTX. Other new antifolates process for AMT uptake and metabolism in the 1-5 ~M range, that are inhibitors of either thymidylate synthetase (e.g., Tomu- which is only the range of the K m for MTX transport. dex) or the de novo purine pathway (e.g., Lometrexol) do not yet In addition to the ALL samples, we also had the opportu- have a record in therapy for children with ALL. Because MTX nity to study some T-lineage and AML cells, because these or a metabolite inhibits both thymidine and purine synthesis, it patients often present with markedly increased WBC counts. will be interesting to know whether drugs that are selective for This comparison is also of interest, because these two types of one or the other pathway have activity, especially after patients leukemia generally carry worse prognoses than B-cell-lineage suffer a relapse while receiving MTX.

Although the explosion of knowledge and techniques in A °1 in / molecular biology and genetics has given us tremendous in- sights into the biology of leukemia and who will most likely relapse, exploitable reasons for why a relapse occurs have not emerged. Clinical trials continue to show the importance of antimetabolites and especially the advantage of prolonged pa- AI tient exposure (e.g., Ref. 19). Recent studies of 6-mercaptopurine and MTX metabolism both in vitro and in vivo have begun 45 G O O to shed light on potential reasons for the empiric success and the O O failure of these two agents in the treatment of children with ALL 3 (20-23; reviewed in Ref. 24). The studies of AMT and MTX ¢,oO metabolism reported here, coupled with the likely significance of antifolate accumulation by blasts as a prognostic indicator (5), support the idea that AMT may be a useful drug in those patients deemed to be at increased risk for relapse. B ,-81 I! 2 Because there has been little progress made by way of introducing new classes of drugs into the armamentarium, finding a safe dose and schedule for a more potent analogue suc- cessfully based on pharmacological end points should be a worthwhile endeavor. Currently, we are developing plans for studying AMT in patients. Increased and new knowledge of folate and antifolate metabolism and homeostasis (e.g., Refs. 7 and 25-28), as well the development of assays that allow an 4 5 assessment of pharmacodynamic parameters, should allow a safe and constructive reassessment of AMT in the clinical arena. The early preclinical and clinical literature suggested that AMT is 10-20 times more potent than MTX (2, 29-32). A basis for the difference in potency has been explored in an animal model 0 m (33, 34). There are little, if any, significant head-to-head com- C 8 parisons in clinical trials. A clinical trial of "high dose" AMT, which included some pharmacokinetic data in adults, suggests that AMT has a longer t,/2-f3 (35). This information will be incorporated into a planned Phase 1 trial of AMT in children with cancer. Three other points regarding the use of AMT are important. First, AMT free of significant amounts of folate can be prepared. Second, we have a better understanding of acute O O neurological and renal toxicity, the latter being noted especially O4 for AMT by Glode et al. (35), which may be mediated by adenosine and ameliorated by aminophylline (36). Third, as ¢DO noted above, radioligand-binding assays and HPLC detection will allow detailed pharmacodynamics and kinetics to be done on biopsies, plasma, RBC, etc., in a timely fashion. It may be m "1" that the algorithm for the treatment of patients with ALL will C3 ~ 0 I~ 0 U3 0 include not only age, WBC, DNA index, and karyotype consid- TIME (min) erations, but also a biochemical phenotype with respect to drug Fig. 3 A, HPLC analysis of MTX and AMT standards according to metabolism. methodology detailed elsewhere (8). Approximately 1-3 nmol each standard were applied to obtain observed UV peaks. 1-6, MTX parent, ACKNOWLEDGMENTS diglutamate, triglutamate, tetraglutamate, pentaglutamate, and hexaglu- tamate, respectively. A1, AMT; A2, AMT diglutamate. B, chromato- We acknowledge the hard work of our nurses in procuring the graphic distribution of tritium from blasts incubated with 3H-AMT. samples in a timely fashion, and others in the laboratory, in particular, Based on the shorter retention times of A1 and A2 compared with MTX Dr. Richard Lark and Margaret Marling Cason, for processing some of and MTX diglutamate, the numbers of the peaks refer to the likely the samples. numbers of glutamate residues. As noted in the text, peaks 2-5 cochro- matograph with A1 (AMT) after treatment with conjugase and bind to DHFR before or after treatment. Total AMT uptake was 0.75 pmol/106 REFERENCES cells, 94% of which was a polyglutamate. C, chromatographic distribu- 1. Farber, S., Diamond. L. K., Mercer, R. D., Sylvester, R. F., and tion of tritium from an aliquot of deproteinized extract of same ALL Wolff, J. A. Temporary remissions in acute leukemia in children pro- blasts as in B, but incubated with 1 ~M 3H-MTX, not AMT. Total MTX duced by folic acid antagonist 4-aminopteroylglutamic acid (aminopter- uptake was 0.3 pmol/106 cells, and only 46% was a polyglutamate. in). N. Engl. J. Med., 238: 787-793, 1948.

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A Smith, M Hum, N J Winick, et al.

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