- Email: [email protected]
[30] Competitive protein binding assay of methotrexate
[30]
CPBA OF METHOTREXATE
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particles or magnetizable charcoal) offers a simple, rapid, accurate, and specific manual method for routine monitoring of serum MTX levels. The automated assay described here provides a rapid means of measuring large numbers of such sera with a high degree of accuracy and precision, and represents the only fully automated assay for MTX described to date. Any automated assay for MTX, using either direct or continuousflow principles, must be capable of handling the wide range of concentrations commonly encountered in patients' sera. In the present system, known high samples are prediluted prior to assay so that concentrations greater than 100 ng/ml are not encountered. Fortunately samples can be readily diluted into the appropriate range if data on time and dosage are available. Carryover, which was a major problem with earlier continuousflow automated systems employing long incubation periods, is not a problem and the sampling rate of 30 samples/hr could be doubled in view of the absence of carryover and the use of high specific activity tracer. The relatively high concentration of BSA in the assay buffer was used to minimize the possible effects of intersample variation in protein content. A key feature of the automated system is the discrete, synchronized addition of both tracer and solid-phase to sample containing segments only. During the wash cycle, these reagents are recirculated and not pumped into the assay stream, which ensures that they are utilized with maximal economy. Acknowledgments We wish to thank Dr. J. Gardner and Dr. G. Forrest for their help and usefuldiscussion and Dr. T. Merrett for help with antiserumpreparation. We would like to thank Dr. G. W. Aherne and Dr. H. Calvert for supplyingpatients' samples and results for correlation with our assays. R. S. Kamelis gratefulfor a grant from the Ministryof Higher Educationand Scientific Research, Iraq.
[30] C o m p e t i t i v e P r o t e i n B i n d i n g A s s a y o f M e t h o t r e x a t e
By CHARLES ERLICHMAN, ROSS C. DONEHOWER, and CHARLES E.
MYERS
Introduction Folic acid analogs with antimetabolic activity have been used in cancer chemotherapy for several decades. The principle antifolate agent in current clinical use is methotrexate (2,4-diamino-N-10-pteroylglutamic METHODS IN ENZYMOLOGY, VOL. 84
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181984-1
448
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acid), which exerts its major effect by potent inhibition of the enzyme dihydrofolate reductase (DHFR) (EC1.5.1.3 tetrahydrofolate dehydrogenase). Inhibition of this enzyme prevents the conversion of dihydrofolate (FH2) to tetrahydrofolate (FH4) (Fig. 1). The cellular pool of reduced folates is necessary for the synthesis of thymidylate from deoxyuridylate, de n o v o synthesis of purine nucleotides, and protein synthesis. Although methotrexate affects the synthesis of nucleic acids and proteins, cells which are most sensitive are those actively synthesizing DNA. 1 Despite a detailed understanding of the cellular mechanism of drug action and the inclusion of methotrexate in many therapeutic regimens, only a rudimentry appreciation of pharmacokinetic correlates with the drug's toxicity was available. The introduction of therapy with high-dose infusions of methotrexate indicated a clear need for rapid, sensitive, and specific methods for the measurement of methotrexate to aid in the identification of patients at high risk for toxicity. In using such high dosages of methotrexate, severe and potentially lethal drug toxicity can be averted only by the subsequent administration of leucovorin (N-5-formyltetrahydrofolate) as a rescue agent. The dose and duration of this rescue should ideally be based on the measurement of circulating levels of methotrexate. Several methods which satisfy the needs of a clinical assay for methotrexate have been described. The assays in common clinical use currently are the radioimmunoassay 2-4 and the competitive protein binding assay 5,6 which will be the subject of this discussion. Other methods for the measurement of methotrexate have been described, including a DHFR inhibition
dUM~dTMP MeFH4 FH2 ~FH4~MT X FIG. 1. Site of biochemical action of MTX. dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; FH~, dihydrofolate; FH4, tetrahydrofolate; MeFH4, methylene tetrahydrofolate; DHFR, dihydrofolate reductase; MTX, methotrexate.
D. G. Johns and J. R. Bertino, in "Cancer Medicine" (J. F. Holland and E. Frei, eds.), p. 739. Lea & Febiger, Philadelphia, Pennsylvania, 1973. 2 G. W. Aherne, E. M. Piail, and V. Marks, Br. J. Cancer 36, 608 (1977). 3 L. J. Loeffler, M. R. Blum, and M. A. Nelsen, Cancer Res. 36, 3306 (1976). 4 V. Raso and R. Schreiber, Cancer Res. 35, 1407 (1975). 5 C. E. Myers, M. E. Lippman, H. M. Eliot, and B. A. Chabner, Proc. Natl. Acad. Sci. U.S.A. 72, 3683 (1975). 6 E. Arons, S. P. Rothenberg, M. DeCosta, C. Fischer, and M. P. Iqbal, Cancer Res. 35, 2033 (1975).
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CPBA OF METHOTREXATE
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assay, 7 an enzyme-linked immunoassay, and high-pressure liquid chromatography techniques. This paper will describe the competitive protein binding assay (CPBA) for methotrexate, discuss its clinical utility, and point out its usefulness in studying the interaction of tight binding competitive inhibitors of D H F R with this enzyme. Materials
and Methods
Competitive protein binding assays using enzymes rather than antibodies as receptor proteins have been developed for several chemotherapeutic agents. The measurement of unknown quantities of an inhibitor (MTX) by making use of its specific binding to its target enzyme (DHFR) is similar in principle to radioimmunoassays. In the case of methotrexate, the advantages that the use of an enzyme receptor protein confers on this method is that the laborious preparation of an appropriate antibody is avoided, the variability of antibody-drug affinity from one lot of antibody to another is eliminated, and, depending on the source of DHFR, meaningful information regarding the action of methotrexate on this enzyme can be derived from in vitro studies. The requisite specificity and sensitivity for successful use of this method and inherent assumptions in the theory of ligand binding assays are similar to those of the radioimmunoassay and have been described previously. 8 The receptor protein in the CPBA of methotrexate described by Myers et al. 5 is D H F R partially purified from a strain of dichloromethotrexateresistant Lactobacillus casei with a specific activity of 4.6/zmol of tetrahydrofolate/min/mg protein at pH 7.5 and 37°. It binds a maximum of 1.9 nmol of methotrexate per mg protein at pH 6.2 and 23 ° as determined by incubating the enzyme with excess radiolabeled methotrexate. Enzyme from this source is commercially available from the New England Enzyme Center, Boston, MA. Prior to assay, the stock DHFR is diluted 3500-fold in 0.5 M potassium phosphate buffer pH 6.2 containing freshly prepared NADPH (Sigma Chemical Co.) resulting in a solution with 5 pmol of methotrexate binding capacity and 2.4 /xmol NADPH per ml. Other sources of D H F R have been utilized such as that derived from L1210 leukemia cells. 9 Unlabeled MTX used in the determination of a standard curve and [3H]MTX are both purified by an ammonium bicarbonate gradient elution from a DEAE-cellulose column similar to that previously described.i° The 7 W. C. Werkheiser, S. F. Zakrewski, and C. A. Nichol, J. Pharmacol. Exp. Ther. 137, 162 (1962). 8 R. P. Ekins, Br. Med. Bull. 30, 3 (1974). 9 S. P. Rothenberg, M. DaCosta, and M. P. Iqbal, Cancer Treat. Rep. 61, 575 (1974).
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DRUGS
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concentration of the unlabeled drug is determined spectrophotometrically utilizing an extinction coefficient of 7.0 x 10-3 M -1 cm -1 at 370 nm in 0.05 M Tris-HCl pH 7.5.1 Solutions of known MTX concentration are then prepared in 0.15 M potassium phosphate pH 6.2. [3H]Methotrexate (Amersham-Searle) with specific activity of 9.5 Ci/mmol is diluted in 0.1 M potassium phosphate buffer pH 6.2 with 330 units of heparin per ml in a final concentration of 0.033/zCI/ml prior to assay. To separate unbound methotrexate from that bound to DHFR, a charcoal slurry is prepared by combining 10 g of activated charcoal, 2.5 g of bovine serum albumin fraction V, and 0.1 g of high-molecular-weight dextran in 100 ml of distilled water. The pH is then adjusted to 6.2 with 1 N HC1. These reagents are purchased from Sigma Chemical Co. This slurry is highly effective in binding the MTX which is not bound to D H F R in the assay tube. Less than 3% of the unbound [~H]methotrexate remains after addition of 50/zl of the charcoal slurry. Plasma samples containing 100 units of heparin per ml are acidified by the addition of 20/zl of 1 N HCI per ml of plasma to achieve a pH of 6.2. Spinal fluid is similarly adjusted to pH 6.2 by the addition of 10/zl of 1 N HC1 to each ml of fluid. Optimal binding of MTX to L. casei D H F R occurs at pH 6.2. Assay Method. The assay is performed by the rapid sequential addition of (1) 150/~1 of the dilute [~H]methotrexate solution; (2) 200/zl of serially diluted unknown sample or standard solution of unlabeled methotrexate; and (3) 100/zl of the diluted solution of D H F R and NADPH. The assay is immediately agitated and 50/zl of charcoal slurry is added. Assay tubes are mixed again and centrifuged at 700 g for 30 min. A 200-/.d aliquot of the supernatant fluid is removed, being careful not to disturb the charcoal pellet, then added to 10 ml of Aquasol (New England Nuclear Corp.) and counted in a liquid scintillation counter. The supernatant fluid contains the enzyme-bound methotrexate--both labeled and unlabeled. The amount of bound [3H]methotrexate will vary inversely with respect to the amount of unlabeled methotrexate present. Therefore, using known concentrations of methotrexate in the range of 1 x 10-9 to 1 × 10-TM, a sigmoid standard curve may be constructed by plotting percentage of maximum bound, i.e., when no unlabeled methotrexate is present, versus concentration of methotrexate on semilogarithmic paper (Fig. 2). It is possible to increase the linear portion of the curve by plotting the logit transformation on the ordinate (inset, Fig. 2). The logit transformation is calculated as follows:
[ _B/B0 ]
logit = In 1 - B/BoJ 10 V. T. Oliverio, Anal. Chem. 33, 263 (1961).
(1)
[30]
a z D O
C P B A OF METHOTREXATE
451
100
co
80 D x <
60
u
0
%%%%
109 %%
40
MTX
%
1041 CONCENTRATIONIM~
10 7
%'%[~%
20
0
I0-9
10 -8
1(
MTX CONCENTRATION (M)
FIG, 2. Standard curve generated by varying the concentration of methotrexate plotted on x axis and [3H]methotrexate bound as a percentage of maximum bound in the absence of methotrexate on the y axis. Inset is the plot of logit versus methotrexate concentration of the same data points (r2 = 0.990).
where Bo is the amount o f [3H]methotrexate b o u n d to D H F R in the absence of the competing unlabeled m e t h o t r e x a t e and B is the amount o f the [3H]methotrexate bound to D H F R in the p r e s e n c e of the k n o w n or unk n o w n amounts of unlabeled m e t h o t r e x a t e in e a c h assay tube.
Assay R e s u l t s The sensitivity of this a s s a y for m e t h o t r e x a t e is 0.3 pmol per assay tube as originally described. This translates into measurable levels o f m e t h o t r e x a t e in biological fluids as low as 1.5nM. This level of sensitivity is possible b e c a u s e o f the high binding affinity of m e t h o t r e x a t e for D H F R . The association constant which gives a m e a s u r e o f this affinity is 2.1 x 108 M -1 for the m e t h o t r e x a t e - D H F R reaction. F u r t h e r m o r e , the availability o f [3H]methotrexate with high specific activity allows small quantities of [3H]methotrexate to be e m p l o y e d in the a s s a y and still h a v e significant radioactivity to permit statistically acceptable counting of samples. The reproducibility of the assay is good with a coefficient of variation of less than 10% between concentrations of m e t h o t r e x a t e of 1.5 to 50 nM for duplicate samples.
452
DRUGS
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The specificity of [3H]methotrexate for D H F R is high. This is demonstrated by the abolition of binding of [aH]methotrexate in the presence of 2000-fold excess unlabeled methotrexate. Under these conditions, the competition for receptor sites by the ligands is greatly in favor of the unlabeled methotrexate. Assessment of interference which may occur in this assay can be carded out in the presence of increasing concentrations of folate analogs and known metabolites of methotrexate. The degree of interference that any of these substances may cause in the assay can be conveniently expressed as the concentration of the substance which inhibits binding of [3H]methotrexate to 50% of its maximum under the conditions of the assay (I50). The compounds of greatest concern are 7-hydroxymethotrexate, 2,4-diaminoN-10-methylpteroic acid (DAMPA), and N-5-formyltetrahydrofolic acid (leucovorin) (Fig. 3). 7-Hydroxymethotrexate, which is a hydroxylation product of the liver, has an I50 of 40 riM, 8-fold greater than labeled methotrexate. DAMPA, the carboxypeptidase G1 cleavage product of methotrexate, has an I50 of 440 nM, 88-fold greater than the unlabeled methotrexate. Both have been identified in the plasma of patients following high-dose infusion. Leucovorin, used in the "rescue" of patients receiving high-dose methotrexate infusions has an I50 of 0.1 mM or 24,000 times higher than for methotrexate itself. Other folate analogs which have been tested in this assay are 5-methyltetrahydropteroylglutamate, the major circulating folate and N-10-methylfolic acid, a contaminant of the commercially available methotrexate. A summary of the I50 results for all
o
A
.i.~ l.~.- ~..I
,~.C-~ ~
,
,,
~--o.
o
NH 2
B
",N.,,~'-.,1"~-vO.c..
o
NIt 2
c
B2 N
-FT% .-..~ I~L. N
"~.
c.,
o
," ~'--~, "
NH 2
D
.,...........
T" I T 1 OH
,o H
, ~-~,,
O
C--OH
l
O
,,
C--H II 0
FIG. 3. Structures of antifolates. (A) Methotrexate; (B) 7-hydroxymethotrexate; (C) DAMPA; (D) leucovorin.
[30]
CPBA OF METHOTREXATE
453
these agents is listed in the table. These findings suggest that folate analogs are potential sources of error in the interpretation of the methotrexate levels obtained by this assay. However, the available data suggest that the clinical significance of this interference is likely to be minimal since only in a small number of cases do levels of metabolites accumulate which may interfere with the assay. Results obtained by the CPBA have been compared to those of an enzyme inhibition assay for methotrexate 5 and at least two radioimmunoassays. 11,12The values determined in plasma and CSF by the CPBA and the enzyme inhibition method show a good correlation between the two methods. Comparison of the CPBA to a commercially available radioimmunoassay for methotrexate suggested that plasma samples may give divergent results in the two assays. The divergence was found to be maximal in those samples obtained 48 to 72 hr after the start of the drug infusion and indicated a consistently higher level of methotrexate as measured by the RIA. Further studies revealed that 2,4-diamino-N-10-methylpteroic acid may be a significant metabolite in some patiens which would interfere with the tested radioimmunoassay. The I5o for this metabolite in the radioimmunoassay was only 2.3 times greater than that of unlabeled methotrexate. 11Therefore, small amounts of 2,4-diamino-N- 10methylpteroic acid could contribute significantly to the measured level of methotrexate by this radioimmunoassay. Since antibody specificity may vary from one radioimmunoassay method to another depending on the technique and species in which the antibody is raised, cross-reaction of this nature must be determined in each case. INTERFERENCE OF FOLATE ANALOGS IN THE CPBA AS MEASURED BY I50a Compound Methotrexate 7-Hydroxy-MTX DAMPA N- 10-Methylfolic acid Leucovorin 5-methyltetrahydropteryolglutamate
I5o (riM) 5.0 40 400 100 100,000 1,300,000
I50 is the concentration of a compound required to decrease the binding of [SH]methotrexate to 50% of its maximum. 1~ R. C. Donehower, K. R. Hand¢, J. C. Drake, and B. A. Chabner, Clin. Pharmacol. Ther. 26, 63 (1979). ~2 R. Virtanen, E. Iisalo, M. Pavinen, and E. Nordman, Acta Pharmacol. Toxicol. 44, 296 (1979).
454
DRUGS
[30]
Applications of the Competitive Protein Binding Assay for Methotrexate The competitive protein binding assay has a major role in the clinical monitoring of therapy with high-dose methotrexate. Monitoring multiple infusions given in a standard fashion has allowed the identification of a plasma parameter which will predict with a high degree of probability whether a patient will develop clinical toxicity. After a 6-hr infusion of 50-250 mg/kg, patients having levels of methotrexate greater than 0.9/~M in plasma at 48 hr were identified as having a significant chance of developing methotrexate systemic toxicity. 13 The ability to rapidly determine whether a plasma level exceeding 0.9/zM exists in any given patient on such a regimen allows the physician to increase the dose of leucovorin and thus decrease the likelihood of toxicity. The use of this assay to determine the pharmacokinetic behavior of a small test dose of methotrexate in a given patient in short order has allowed one group of investigators to individualize the patient's high dose of methotrexate using a computer modeling system. 14 Such routine monitoring and potential for tailoring a course of therapy has been greatly simplified by the availability of such a rapid, simple, and inexpensive assay as the competitive protein binding assay. The competitive protein binding technique can also be used to study the interaction of methotrexate and dihydrofolate reductase. As mentioned previously, methotrexate is considered a tight binding inhibitor of DHFR. Difficulties arise in applying steady-state rate equations in determining the dissociation constants of tight binding inhibitors. Certain assumptions and approximations often requiring complex mathematical analyses must be made in order to obtain an estimate of the K i value of methotrexate for DHFR. 15"1eEven after acknowledging these difficulties, application of different techniques may result in large variations in the Ki obtained. ~5 However, using equilibrium binding analysis of methotrexa t e - D H F R interaction, an estimate for methotrexate binding affinity to DHFR can be arrived at simply. Such experiments have been carried out under similar conditions as those for the CPBA. Two series of assay tubes were prepared containing D H F R from L. casei with 7 pmol of methotrexate binding capacity, 0.24 /zmol of NADPH, 0.075 ~mol of potassium phosphate buffer, and varying amounts of [3H]methotrexate from 1 to 20 13 R. G. Stoller, K. R. Hande, S. A. Ja¢obs, S. A. Rosenberg, and B. A. Chabner, New Engl. J. Med. 297, 630 (1977). 14 S. Monjanel, J. P. Rigault, J. P. Cano, Y. Carcassonne, and R. Favre, Cancer Chemother. Pharmacol. 3, 189 (1979). 15 W. P. Greco and M. T. Hakala, J. Biol. Chem. 254, 12104 (1979). 16 S. Cha, Biochem. Pharmacol. 24, 2177 (1975).
[30]
CPBA OF METHOTREXATE
455
pmol. In the second set of tubes, 5 nmol of unlabeled methotrexate was. added to all the reaction mixtures. The final volume was 450/zl. The excess unlabeled methotrexate which was added to the second set of tubes is sufficient to prevent specific binding of [aH]methotrexate to DHFR, allowing assessment of nonspecific binding of the labeled methotrexate to protein. Separation of bound and free [aH]methotrexate was carried out as previously described using the charcoal slurry, and the supernatant containing the bound counts was counted. Plotting the results initially as bound versus total [3H]methotrexate and then according to the method of Scatchard lr lead to results shown in Fig. 4. This Scatchard plot suggests that there is a single homogeneous site on DHFR for methotrexate binding and the slope of this line is equivalent to the -KA or association constant. The inverse of the KA gives a KD (dissociation constant) of 4.76 x 10-9 M. This result is obtained by carrying out the binding studies during a time period when equilibrium exists for formation of the enzyme inhibitor complex and in the absence of dihydrofolate. Thus, the need for steady-
1o 9 8
2.75 2.50 2.25
/
/
4
~_"~
\,
~41.25
\
3
il 1.00
2
0.50 0.00
1 0 0
I
1
5
10
•
li5
• llll I I t I I I I ~1 2 4 6 8 '10 12 14 I[MTXI'I b°und InM) I
20
25
30
35
I 40
M T X , T O T A L (nM)
FIG. 4. Plot of b o u n d [3H]methotrexate v e r s u s total [aH]methotrexate. E x p e r i m e n t a l conditions are described in text. Inset is the s a m e data replotted according to the m e t h o d of Scatchard 17 using an unweighted least-squares fit (r = 0.97) (from Ref. 5).
~7 G. Scatchard, Ann. N . Y . Acad. Sci. 51, 660 (1949).
456
DRUGS
[30]
state assumptions of kinetic studies is not applicable and solution of a "stiff" differential equation-defined model is not required. Extension of the equilibrium binding studies allows the determination of the off rate or kon for methotrexate from DHFR. Such kinetic binding studies have been performed for the DHFR derived from L1210 leukemia cells and Escherichia coli MB 1428. ls,la Assuming that the dissociation of methotrexate from the dihydrofolate reductase is a unimolecular event described by E1 ko~) E + I where E is DHFR and I is methotrexate, then a plot of In (bound methotrexate) versus time is linear with a slope ofkoff. The experimental design involves modification of the CPBA for methotrexate and is outlined for the determination of the koff for DHFR purified from L1210 leukemia. DHFR purified from L1210 leukemia cells with specific activity 33 units/mg protein was used in these studies. The reaction volume of 450/~1 consisted of 0.1 units of DHFR, 1 gA/[3H]methotrexate (specific activity 6.4 Ci/mmol), 0.1 mM N A D P H in 0.05 M potassium phosphate pH 7.4, and 0.15 M KCI. After incubation for 20 min at 37°, 50/~1 of the charcoal slurry prepared as previously described was added to the assay tube. Each tube was agitated and then centrifuged at 4° and 700 g for 45 min. The supernatant which contained the enzyme-methotrexate complex is removed and equally divided into a series of tubes containing 100/.tM unlabeled methotrexate and 0.1 mM NADPH. At specific time points, 50/.d of the charcoal slurry is added and centrifuged again. The radioactivity in the supernatant is determined by scintillation counting. The counts represent the remaining DHFR-[3H]methotrexate complex at each time point. Plotting these results yields an off rate of 0.0014 min -1. Similar methodology was applied to determine the koffof methotrexate from D H F R from E. coli. 19 However, separation of bound and free [3H]methotrexate was carried out using a minicolumn technique. The koffdetermined in this fashion was 0.014 min -~. The versatility of the CPBA for methotrexate is demonstrated by the ability to adapt this assay to measure levels of other antifolate agents. Since this assay depends on the competition by [3H]methotrexate and unlabeled methotrexate, other antifolate agents which bind to D H F R can be substituted for the unlabeled methotrexate if these agents bind to the same site as methotrexate. The sensitivity of the assay for any given antiis M. Cohen, R. A. Bender, R. Donehower, C. E. Myers, and B. A. Chabner, Cancer Res. 38, (1978). 19 j. C. White, J. Biol. Chem. 254, 10889 (1979).
[30]
CPBA OF METHOTREXATE
457
folate will depend on the binding constant of the antifolate to be measured. Determination of the I5o for the antifolate to be measured will give an estimate of the range over which the assay will be useful because a linear portion of the sigmoidal curve generated by adding varying amounts of the antifolate to be assayed is centered about the Is0 concentration. The assay range will usually extend above and below the I50 concentration by one log concentration. The application of this general approach has been demonstrated for several antifolate agentsY° Aminopterin has an I50 concentration of 5 x 10-8 M which is 10-fold higher than methotrexate in the CPBA. Therefore, this assay can be easily adapted to measure levels of aminopterin as low as 5 nM. Similarly, methasquin, another antifolate, with an I50 of 5.4 x 10-s M can be assayed to levels of approximately 5 nM also. An attempt to utilize the L. casei DHFR to assay triazinate (another antifolate) revealed that the I50 was 1 x 10-5 M, thus making the sensitivity of this assay only 1/xM. However, this agent has a higher affinity for the DHFR derived from methotrexate-resistant L1210 leukemia cells 21 with an 150 concentration of 1 x 10-7 M. Therefore, by using another source of DHFR, the assay can be adapted to improve the sensitivity of measuring triazinate to levels of 1 × 10-8 M. This principle of changing the source of the DHFR depending on its affinity for the ligand of interest in order to maximize assay sensitivity is possible because of the comparative studies which have been carried out with DHFR from a variety of sourcesY 2 These studies have shown a significant difference in concentration of a given antifol to inhibit DHFR activity from varying sources by 50%. Trimethoprim, an antifolate used in microbial infections, causes 50% inhibition of DHFR activity derived from E. coli, S. aureus, and P. vulgaris in nanomolar concentrations, whereas concentrations of approximately 0.3 mM is required in order to cause similar inhibition of DHFR from human liver or rat liver. Rothenberg et al. 9 have demonstrated that assay of pyrimethamine, an antimalarial antifolate, can be carried out using DHFR from LI210 leukemia cells with a sensitivity of approximately 0.1 nmol per assay tube. Conclusion We have demonstrated that the competitive protein binding assay for methotrexate utilizing principles of equilibrium binding analysis is a remarkably versatile technique for study of DHFR and antifolates. The 2o C. E. Myers, H. M. Eliot, and B. A. Chabner, Cancer Treat. Rep. 60, 615 (1976). 21 A. R. Cashmore, R. T. Skeel, D. R. Makulu, E. J. Gralla, and J. R. Bertino, Cancer Res. 35, 17 (1975). z~ j. j. Burchall, Ann. N . Y . A c a d . Sci. 186, 143 (1971).
458
DRUGS
[31 ]
assay, described herein, is simple and rapidly performed with sensitivity comparable to published radioimmunoassay techniques for methotrexate and excellent specificity in the clinical setting. Its applications in the routine monitoring of patients treated with high-dose methotrexate and in pharmacokinetic modeling are presented. In addition, applications of this technique to the study of binding affinity and kinetics of binding of methotrexate to DHFR are outlined. Finally, data describing the ability of this assay to be modified for measurement of other antifolate agents have been discussed. The broad spectrum of uses to which this assay may be put makes the CPBA for methotrexate extremely useful as a laboratory method.
[31] Ligand-Binding Radioassay of N~-Methyltetrahydrofolate and Its Application to N5-Formyltetrahydrofolate B y M A R I A DA C O S T A a n d S H E L D O N P . R O T H E N B E R G
Introduction NS-Methyltetrahydrofolate (mTHF), the major intracellular and circulating folate cofactor, can now be measured by ligand-binding radioassay using any one of the natural binding proteins isolated from human or animal tissues or biologic fluids. For most of the reported methods, the folate-binding protein present in cow's milk 1 has been used as the binder and radioisotopically labeled folic acid (FA), or some derivative of FA, as the tracer. Most of these naturally occurring folate-binding proteins, however, have greater affinity for FA than for mTHF or other naturally occurring folates. Consequently, if the classical competitive inhibition radioassay is used with the standard mTHF and tracer competing simultaneously for binding sites on the protein, the sensitivity of the dose-response curve will be diminished. There will also be an inherent error in the total folate concentration when a mixture of folates, each with varying affinity for the binding protein, is measured by a competitive inhibition radioassay. This error can be minimized by carrying out the radioassay at pH 9.3 at which the affinity of FA and mTHF for the binder appears to be equal, 2
1 j. Ghitis, Am. J. Clin. Nutr. 20, 1(1967). 2 j. K. Givas and S. Gutcho, Clin. Chem. 21, 427(1975).
METHODS IN ENZYMOLOGY, VOL. 84
Copyright © 1982by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-1819fl4-1
CPBA OF METHOTREXATE
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particles or magnetizable charcoal) offers a simple, rapid, accurate, and specific manual method for routine monitoring of serum MTX levels. The automated assay described here provides a rapid means of measuring large numbers of such sera with a high degree of accuracy and precision, and represents the only fully automated assay for MTX described to date. Any automated assay for MTX, using either direct or continuousflow principles, must be capable of handling the wide range of concentrations commonly encountered in patients' sera. In the present system, known high samples are prediluted prior to assay so that concentrations greater than 100 ng/ml are not encountered. Fortunately samples can be readily diluted into the appropriate range if data on time and dosage are available. Carryover, which was a major problem with earlier continuousflow automated systems employing long incubation periods, is not a problem and the sampling rate of 30 samples/hr could be doubled in view of the absence of carryover and the use of high specific activity tracer. The relatively high concentration of BSA in the assay buffer was used to minimize the possible effects of intersample variation in protein content. A key feature of the automated system is the discrete, synchronized addition of both tracer and solid-phase to sample containing segments only. During the wash cycle, these reagents are recirculated and not pumped into the assay stream, which ensures that they are utilized with maximal economy. Acknowledgments We wish to thank Dr. J. Gardner and Dr. G. Forrest for their help and usefuldiscussion and Dr. T. Merrett for help with antiserumpreparation. We would like to thank Dr. G. W. Aherne and Dr. H. Calvert for supplyingpatients' samples and results for correlation with our assays. R. S. Kamelis gratefulfor a grant from the Ministryof Higher Educationand Scientific Research, Iraq.
[30] C o m p e t i t i v e P r o t e i n B i n d i n g A s s a y o f M e t h o t r e x a t e
By CHARLES ERLICHMAN, ROSS C. DONEHOWER, and CHARLES E.
MYERS
Introduction Folic acid analogs with antimetabolic activity have been used in cancer chemotherapy for several decades. The principle antifolate agent in current clinical use is methotrexate (2,4-diamino-N-10-pteroylglutamic METHODS IN ENZYMOLOGY, VOL. 84
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181984-1
448
DRUGS
[30]
acid), which exerts its major effect by potent inhibition of the enzyme dihydrofolate reductase (DHFR) (EC1.5.1.3 tetrahydrofolate dehydrogenase). Inhibition of this enzyme prevents the conversion of dihydrofolate (FH2) to tetrahydrofolate (FH4) (Fig. 1). The cellular pool of reduced folates is necessary for the synthesis of thymidylate from deoxyuridylate, de n o v o synthesis of purine nucleotides, and protein synthesis. Although methotrexate affects the synthesis of nucleic acids and proteins, cells which are most sensitive are those actively synthesizing DNA. 1 Despite a detailed understanding of the cellular mechanism of drug action and the inclusion of methotrexate in many therapeutic regimens, only a rudimentry appreciation of pharmacokinetic correlates with the drug's toxicity was available. The introduction of therapy with high-dose infusions of methotrexate indicated a clear need for rapid, sensitive, and specific methods for the measurement of methotrexate to aid in the identification of patients at high risk for toxicity. In using such high dosages of methotrexate, severe and potentially lethal drug toxicity can be averted only by the subsequent administration of leucovorin (N-5-formyltetrahydrofolate) as a rescue agent. The dose and duration of this rescue should ideally be based on the measurement of circulating levels of methotrexate. Several methods which satisfy the needs of a clinical assay for methotrexate have been described. The assays in common clinical use currently are the radioimmunoassay 2-4 and the competitive protein binding assay 5,6 which will be the subject of this discussion. Other methods for the measurement of methotrexate have been described, including a DHFR inhibition
dUM~dTMP MeFH4 FH2 ~FH4~MT X FIG. 1. Site of biochemical action of MTX. dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; FH~, dihydrofolate; FH4, tetrahydrofolate; MeFH4, methylene tetrahydrofolate; DHFR, dihydrofolate reductase; MTX, methotrexate.
D. G. Johns and J. R. Bertino, in "Cancer Medicine" (J. F. Holland and E. Frei, eds.), p. 739. Lea & Febiger, Philadelphia, Pennsylvania, 1973. 2 G. W. Aherne, E. M. Piail, and V. Marks, Br. J. Cancer 36, 608 (1977). 3 L. J. Loeffler, M. R. Blum, and M. A. Nelsen, Cancer Res. 36, 3306 (1976). 4 V. Raso and R. Schreiber, Cancer Res. 35, 1407 (1975). 5 C. E. Myers, M. E. Lippman, H. M. Eliot, and B. A. Chabner, Proc. Natl. Acad. Sci. U.S.A. 72, 3683 (1975). 6 E. Arons, S. P. Rothenberg, M. DeCosta, C. Fischer, and M. P. Iqbal, Cancer Res. 35, 2033 (1975).
[30]
CPBA OF METHOTREXATE
449
assay, 7 an enzyme-linked immunoassay, and high-pressure liquid chromatography techniques. This paper will describe the competitive protein binding assay (CPBA) for methotrexate, discuss its clinical utility, and point out its usefulness in studying the interaction of tight binding competitive inhibitors of D H F R with this enzyme. Materials
and Methods
Competitive protein binding assays using enzymes rather than antibodies as receptor proteins have been developed for several chemotherapeutic agents. The measurement of unknown quantities of an inhibitor (MTX) by making use of its specific binding to its target enzyme (DHFR) is similar in principle to radioimmunoassays. In the case of methotrexate, the advantages that the use of an enzyme receptor protein confers on this method is that the laborious preparation of an appropriate antibody is avoided, the variability of antibody-drug affinity from one lot of antibody to another is eliminated, and, depending on the source of DHFR, meaningful information regarding the action of methotrexate on this enzyme can be derived from in vitro studies. The requisite specificity and sensitivity for successful use of this method and inherent assumptions in the theory of ligand binding assays are similar to those of the radioimmunoassay and have been described previously. 8 The receptor protein in the CPBA of methotrexate described by Myers et al. 5 is D H F R partially purified from a strain of dichloromethotrexateresistant Lactobacillus casei with a specific activity of 4.6/zmol of tetrahydrofolate/min/mg protein at pH 7.5 and 37°. It binds a maximum of 1.9 nmol of methotrexate per mg protein at pH 6.2 and 23 ° as determined by incubating the enzyme with excess radiolabeled methotrexate. Enzyme from this source is commercially available from the New England Enzyme Center, Boston, MA. Prior to assay, the stock DHFR is diluted 3500-fold in 0.5 M potassium phosphate buffer pH 6.2 containing freshly prepared NADPH (Sigma Chemical Co.) resulting in a solution with 5 pmol of methotrexate binding capacity and 2.4 /xmol NADPH per ml. Other sources of D H F R have been utilized such as that derived from L1210 leukemia cells. 9 Unlabeled MTX used in the determination of a standard curve and [3H]MTX are both purified by an ammonium bicarbonate gradient elution from a DEAE-cellulose column similar to that previously described.i° The 7 W. C. Werkheiser, S. F. Zakrewski, and C. A. Nichol, J. Pharmacol. Exp. Ther. 137, 162 (1962). 8 R. P. Ekins, Br. Med. Bull. 30, 3 (1974). 9 S. P. Rothenberg, M. DaCosta, and M. P. Iqbal, Cancer Treat. Rep. 61, 575 (1974).
450
DRUGS
[30]
concentration of the unlabeled drug is determined spectrophotometrically utilizing an extinction coefficient of 7.0 x 10-3 M -1 cm -1 at 370 nm in 0.05 M Tris-HCl pH 7.5.1 Solutions of known MTX concentration are then prepared in 0.15 M potassium phosphate pH 6.2. [3H]Methotrexate (Amersham-Searle) with specific activity of 9.5 Ci/mmol is diluted in 0.1 M potassium phosphate buffer pH 6.2 with 330 units of heparin per ml in a final concentration of 0.033/zCI/ml prior to assay. To separate unbound methotrexate from that bound to DHFR, a charcoal slurry is prepared by combining 10 g of activated charcoal, 2.5 g of bovine serum albumin fraction V, and 0.1 g of high-molecular-weight dextran in 100 ml of distilled water. The pH is then adjusted to 6.2 with 1 N HC1. These reagents are purchased from Sigma Chemical Co. This slurry is highly effective in binding the MTX which is not bound to D H F R in the assay tube. Less than 3% of the unbound [~H]methotrexate remains after addition of 50/zl of the charcoal slurry. Plasma samples containing 100 units of heparin per ml are acidified by the addition of 20/zl of 1 N HCI per ml of plasma to achieve a pH of 6.2. Spinal fluid is similarly adjusted to pH 6.2 by the addition of 10/zl of 1 N HC1 to each ml of fluid. Optimal binding of MTX to L. casei D H F R occurs at pH 6.2. Assay Method. The assay is performed by the rapid sequential addition of (1) 150/~1 of the dilute [~H]methotrexate solution; (2) 200/zl of serially diluted unknown sample or standard solution of unlabeled methotrexate; and (3) 100/zl of the diluted solution of D H F R and NADPH. The assay is immediately agitated and 50/zl of charcoal slurry is added. Assay tubes are mixed again and centrifuged at 700 g for 30 min. A 200-/.d aliquot of the supernatant fluid is removed, being careful not to disturb the charcoal pellet, then added to 10 ml of Aquasol (New England Nuclear Corp.) and counted in a liquid scintillation counter. The supernatant fluid contains the enzyme-bound methotrexate--both labeled and unlabeled. The amount of bound [3H]methotrexate will vary inversely with respect to the amount of unlabeled methotrexate present. Therefore, using known concentrations of methotrexate in the range of 1 x 10-9 to 1 × 10-TM, a sigmoid standard curve may be constructed by plotting percentage of maximum bound, i.e., when no unlabeled methotrexate is present, versus concentration of methotrexate on semilogarithmic paper (Fig. 2). It is possible to increase the linear portion of the curve by plotting the logit transformation on the ordinate (inset, Fig. 2). The logit transformation is calculated as follows:
[ _B/B0 ]
logit = In 1 - B/BoJ 10 V. T. Oliverio, Anal. Chem. 33, 263 (1961).
(1)
[30]
a z D O
C P B A OF METHOTREXATE
451
100
co
80 D x <
60
u
0
%%%%
109 %%
40
MTX
%
1041 CONCENTRATIONIM~
10 7
%'%[~%
20
0
I0-9
10 -8
1(
MTX CONCENTRATION (M)
FIG, 2. Standard curve generated by varying the concentration of methotrexate plotted on x axis and [3H]methotrexate bound as a percentage of maximum bound in the absence of methotrexate on the y axis. Inset is the plot of logit versus methotrexate concentration of the same data points (r2 = 0.990).
where Bo is the amount o f [3H]methotrexate b o u n d to D H F R in the absence of the competing unlabeled m e t h o t r e x a t e and B is the amount o f the [3H]methotrexate bound to D H F R in the p r e s e n c e of the k n o w n or unk n o w n amounts of unlabeled m e t h o t r e x a t e in e a c h assay tube.
Assay R e s u l t s The sensitivity of this a s s a y for m e t h o t r e x a t e is 0.3 pmol per assay tube as originally described. This translates into measurable levels o f m e t h o t r e x a t e in biological fluids as low as 1.5nM. This level of sensitivity is possible b e c a u s e o f the high binding affinity of m e t h o t r e x a t e for D H F R . The association constant which gives a m e a s u r e o f this affinity is 2.1 x 108 M -1 for the m e t h o t r e x a t e - D H F R reaction. F u r t h e r m o r e , the availability o f [3H]methotrexate with high specific activity allows small quantities of [3H]methotrexate to be e m p l o y e d in the a s s a y and still h a v e significant radioactivity to permit statistically acceptable counting of samples. The reproducibility of the assay is good with a coefficient of variation of less than 10% between concentrations of m e t h o t r e x a t e of 1.5 to 50 nM for duplicate samples.
452
DRUGS
[30]
The specificity of [3H]methotrexate for D H F R is high. This is demonstrated by the abolition of binding of [aH]methotrexate in the presence of 2000-fold excess unlabeled methotrexate. Under these conditions, the competition for receptor sites by the ligands is greatly in favor of the unlabeled methotrexate. Assessment of interference which may occur in this assay can be carded out in the presence of increasing concentrations of folate analogs and known metabolites of methotrexate. The degree of interference that any of these substances may cause in the assay can be conveniently expressed as the concentration of the substance which inhibits binding of [3H]methotrexate to 50% of its maximum under the conditions of the assay (I50). The compounds of greatest concern are 7-hydroxymethotrexate, 2,4-diaminoN-10-methylpteroic acid (DAMPA), and N-5-formyltetrahydrofolic acid (leucovorin) (Fig. 3). 7-Hydroxymethotrexate, which is a hydroxylation product of the liver, has an I50 of 40 riM, 8-fold greater than labeled methotrexate. DAMPA, the carboxypeptidase G1 cleavage product of methotrexate, has an I50 of 440 nM, 88-fold greater than the unlabeled methotrexate. Both have been identified in the plasma of patients following high-dose infusion. Leucovorin, used in the "rescue" of patients receiving high-dose methotrexate infusions has an I50 of 0.1 mM or 24,000 times higher than for methotrexate itself. Other folate analogs which have been tested in this assay are 5-methyltetrahydropteroylglutamate, the major circulating folate and N-10-methylfolic acid, a contaminant of the commercially available methotrexate. A summary of the I50 results for all
o
A
.i.~ l.~.- ~..I
,~.C-~ ~
,
,,
~--o.
o
NH 2
B
",N.,,~'-.,1"~-vO.c..
o
NIt 2
c
B2 N
-FT% .-..~ I~L. N
"~.
c.,
o
," ~'--~, "
NH 2
D
.,...........
T" I T 1 OH
,o H
, ~-~,,
O
C--OH
l
O
,,
C--H II 0
FIG. 3. Structures of antifolates. (A) Methotrexate; (B) 7-hydroxymethotrexate; (C) DAMPA; (D) leucovorin.
[30]
CPBA OF METHOTREXATE
453
these agents is listed in the table. These findings suggest that folate analogs are potential sources of error in the interpretation of the methotrexate levels obtained by this assay. However, the available data suggest that the clinical significance of this interference is likely to be minimal since only in a small number of cases do levels of metabolites accumulate which may interfere with the assay. Results obtained by the CPBA have been compared to those of an enzyme inhibition assay for methotrexate 5 and at least two radioimmunoassays. 11,12The values determined in plasma and CSF by the CPBA and the enzyme inhibition method show a good correlation between the two methods. Comparison of the CPBA to a commercially available radioimmunoassay for methotrexate suggested that plasma samples may give divergent results in the two assays. The divergence was found to be maximal in those samples obtained 48 to 72 hr after the start of the drug infusion and indicated a consistently higher level of methotrexate as measured by the RIA. Further studies revealed that 2,4-diamino-N-10-methylpteroic acid may be a significant metabolite in some patiens which would interfere with the tested radioimmunoassay. The I5o for this metabolite in the radioimmunoassay was only 2.3 times greater than that of unlabeled methotrexate. 11Therefore, small amounts of 2,4-diamino-N- 10methylpteroic acid could contribute significantly to the measured level of methotrexate by this radioimmunoassay. Since antibody specificity may vary from one radioimmunoassay method to another depending on the technique and species in which the antibody is raised, cross-reaction of this nature must be determined in each case. INTERFERENCE OF FOLATE ANALOGS IN THE CPBA AS MEASURED BY I50a Compound Methotrexate 7-Hydroxy-MTX DAMPA N- 10-Methylfolic acid Leucovorin 5-methyltetrahydropteryolglutamate
I5o (riM) 5.0 40 400 100 100,000 1,300,000
I50 is the concentration of a compound required to decrease the binding of [SH]methotrexate to 50% of its maximum. 1~ R. C. Donehower, K. R. Hand¢, J. C. Drake, and B. A. Chabner, Clin. Pharmacol. Ther. 26, 63 (1979). ~2 R. Virtanen, E. Iisalo, M. Pavinen, and E. Nordman, Acta Pharmacol. Toxicol. 44, 296 (1979).
454
DRUGS
[30]
Applications of the Competitive Protein Binding Assay for Methotrexate The competitive protein binding assay has a major role in the clinical monitoring of therapy with high-dose methotrexate. Monitoring multiple infusions given in a standard fashion has allowed the identification of a plasma parameter which will predict with a high degree of probability whether a patient will develop clinical toxicity. After a 6-hr infusion of 50-250 mg/kg, patients having levels of methotrexate greater than 0.9/~M in plasma at 48 hr were identified as having a significant chance of developing methotrexate systemic toxicity. 13 The ability to rapidly determine whether a plasma level exceeding 0.9/zM exists in any given patient on such a regimen allows the physician to increase the dose of leucovorin and thus decrease the likelihood of toxicity. The use of this assay to determine the pharmacokinetic behavior of a small test dose of methotrexate in a given patient in short order has allowed one group of investigators to individualize the patient's high dose of methotrexate using a computer modeling system. 14 Such routine monitoring and potential for tailoring a course of therapy has been greatly simplified by the availability of such a rapid, simple, and inexpensive assay as the competitive protein binding assay. The competitive protein binding technique can also be used to study the interaction of methotrexate and dihydrofolate reductase. As mentioned previously, methotrexate is considered a tight binding inhibitor of DHFR. Difficulties arise in applying steady-state rate equations in determining the dissociation constants of tight binding inhibitors. Certain assumptions and approximations often requiring complex mathematical analyses must be made in order to obtain an estimate of the K i value of methotrexate for DHFR. 15"1eEven after acknowledging these difficulties, application of different techniques may result in large variations in the Ki obtained. ~5 However, using equilibrium binding analysis of methotrexa t e - D H F R interaction, an estimate for methotrexate binding affinity to DHFR can be arrived at simply. Such experiments have been carried out under similar conditions as those for the CPBA. Two series of assay tubes were prepared containing D H F R from L. casei with 7 pmol of methotrexate binding capacity, 0.24 /zmol of NADPH, 0.075 ~mol of potassium phosphate buffer, and varying amounts of [3H]methotrexate from 1 to 20 13 R. G. Stoller, K. R. Hande, S. A. Ja¢obs, S. A. Rosenberg, and B. A. Chabner, New Engl. J. Med. 297, 630 (1977). 14 S. Monjanel, J. P. Rigault, J. P. Cano, Y. Carcassonne, and R. Favre, Cancer Chemother. Pharmacol. 3, 189 (1979). 15 W. P. Greco and M. T. Hakala, J. Biol. Chem. 254, 12104 (1979). 16 S. Cha, Biochem. Pharmacol. 24, 2177 (1975).
[30]
CPBA OF METHOTREXATE
455
pmol. In the second set of tubes, 5 nmol of unlabeled methotrexate was. added to all the reaction mixtures. The final volume was 450/zl. The excess unlabeled methotrexate which was added to the second set of tubes is sufficient to prevent specific binding of [aH]methotrexate to DHFR, allowing assessment of nonspecific binding of the labeled methotrexate to protein. Separation of bound and free [aH]methotrexate was carried out as previously described using the charcoal slurry, and the supernatant containing the bound counts was counted. Plotting the results initially as bound versus total [3H]methotrexate and then according to the method of Scatchard lr lead to results shown in Fig. 4. This Scatchard plot suggests that there is a single homogeneous site on DHFR for methotrexate binding and the slope of this line is equivalent to the -KA or association constant. The inverse of the KA gives a KD (dissociation constant) of 4.76 x 10-9 M. This result is obtained by carrying out the binding studies during a time period when equilibrium exists for formation of the enzyme inhibitor complex and in the absence of dihydrofolate. Thus, the need for steady-
1o 9 8
2.75 2.50 2.25
/
/
4
~_"~
\,
~41.25
\
3
il 1.00
2
0.50 0.00
1 0 0
I
1
5
10
•
li5
• llll I I t I I I I ~1 2 4 6 8 '10 12 14 I[MTXI'I b°und InM) I
20
25
30
35
I 40
M T X , T O T A L (nM)
FIG. 4. Plot of b o u n d [3H]methotrexate v e r s u s total [aH]methotrexate. E x p e r i m e n t a l conditions are described in text. Inset is the s a m e data replotted according to the m e t h o d of Scatchard 17 using an unweighted least-squares fit (r = 0.97) (from Ref. 5).
~7 G. Scatchard, Ann. N . Y . Acad. Sci. 51, 660 (1949).
456
DRUGS
[30]
state assumptions of kinetic studies is not applicable and solution of a "stiff" differential equation-defined model is not required. Extension of the equilibrium binding studies allows the determination of the off rate or kon for methotrexate from DHFR. Such kinetic binding studies have been performed for the DHFR derived from L1210 leukemia cells and Escherichia coli MB 1428. ls,la Assuming that the dissociation of methotrexate from the dihydrofolate reductase is a unimolecular event described by E1 ko~) E + I where E is DHFR and I is methotrexate, then a plot of In (bound methotrexate) versus time is linear with a slope ofkoff. The experimental design involves modification of the CPBA for methotrexate and is outlined for the determination of the koff for DHFR purified from L1210 leukemia. DHFR purified from L1210 leukemia cells with specific activity 33 units/mg protein was used in these studies. The reaction volume of 450/~1 consisted of 0.1 units of DHFR, 1 gA/[3H]methotrexate (specific activity 6.4 Ci/mmol), 0.1 mM N A D P H in 0.05 M potassium phosphate pH 7.4, and 0.15 M KCI. After incubation for 20 min at 37°, 50/~1 of the charcoal slurry prepared as previously described was added to the assay tube. Each tube was agitated and then centrifuged at 4° and 700 g for 45 min. The supernatant which contained the enzyme-methotrexate complex is removed and equally divided into a series of tubes containing 100/.tM unlabeled methotrexate and 0.1 mM NADPH. At specific time points, 50/.d of the charcoal slurry is added and centrifuged again. The radioactivity in the supernatant is determined by scintillation counting. The counts represent the remaining DHFR-[3H]methotrexate complex at each time point. Plotting these results yields an off rate of 0.0014 min -1. Similar methodology was applied to determine the koffof methotrexate from D H F R from E. coli. 19 However, separation of bound and free [3H]methotrexate was carried out using a minicolumn technique. The koffdetermined in this fashion was 0.014 min -~. The versatility of the CPBA for methotrexate is demonstrated by the ability to adapt this assay to measure levels of other antifolate agents. Since this assay depends on the competition by [3H]methotrexate and unlabeled methotrexate, other antifolate agents which bind to D H F R can be substituted for the unlabeled methotrexate if these agents bind to the same site as methotrexate. The sensitivity of the assay for any given antiis M. Cohen, R. A. Bender, R. Donehower, C. E. Myers, and B. A. Chabner, Cancer Res. 38, (1978). 19 j. C. White, J. Biol. Chem. 254, 10889 (1979).
[30]
CPBA OF METHOTREXATE
457
folate will depend on the binding constant of the antifolate to be measured. Determination of the I5o for the antifolate to be measured will give an estimate of the range over which the assay will be useful because a linear portion of the sigmoidal curve generated by adding varying amounts of the antifolate to be assayed is centered about the Is0 concentration. The assay range will usually extend above and below the I50 concentration by one log concentration. The application of this general approach has been demonstrated for several antifolate agentsY° Aminopterin has an I50 concentration of 5 x 10-8 M which is 10-fold higher than methotrexate in the CPBA. Therefore, this assay can be easily adapted to measure levels of aminopterin as low as 5 nM. Similarly, methasquin, another antifolate, with an I50 of 5.4 x 10-s M can be assayed to levels of approximately 5 nM also. An attempt to utilize the L. casei DHFR to assay triazinate (another antifolate) revealed that the I50 was 1 x 10-5 M, thus making the sensitivity of this assay only 1/xM. However, this agent has a higher affinity for the DHFR derived from methotrexate-resistant L1210 leukemia cells 21 with an 150 concentration of 1 x 10-7 M. Therefore, by using another source of DHFR, the assay can be adapted to improve the sensitivity of measuring triazinate to levels of 1 × 10-8 M. This principle of changing the source of the DHFR depending on its affinity for the ligand of interest in order to maximize assay sensitivity is possible because of the comparative studies which have been carried out with DHFR from a variety of sourcesY 2 These studies have shown a significant difference in concentration of a given antifol to inhibit DHFR activity from varying sources by 50%. Trimethoprim, an antifolate used in microbial infections, causes 50% inhibition of DHFR activity derived from E. coli, S. aureus, and P. vulgaris in nanomolar concentrations, whereas concentrations of approximately 0.3 mM is required in order to cause similar inhibition of DHFR from human liver or rat liver. Rothenberg et al. 9 have demonstrated that assay of pyrimethamine, an antimalarial antifolate, can be carried out using DHFR from LI210 leukemia cells with a sensitivity of approximately 0.1 nmol per assay tube. Conclusion We have demonstrated that the competitive protein binding assay for methotrexate utilizing principles of equilibrium binding analysis is a remarkably versatile technique for study of DHFR and antifolates. The 2o C. E. Myers, H. M. Eliot, and B. A. Chabner, Cancer Treat. Rep. 60, 615 (1976). 21 A. R. Cashmore, R. T. Skeel, D. R. Makulu, E. J. Gralla, and J. R. Bertino, Cancer Res. 35, 17 (1975). z~ j. j. Burchall, Ann. N . Y . A c a d . Sci. 186, 143 (1971).
458
DRUGS
[31 ]
assay, described herein, is simple and rapidly performed with sensitivity comparable to published radioimmunoassay techniques for methotrexate and excellent specificity in the clinical setting. Its applications in the routine monitoring of patients treated with high-dose methotrexate and in pharmacokinetic modeling are presented. In addition, applications of this technique to the study of binding affinity and kinetics of binding of methotrexate to DHFR are outlined. Finally, data describing the ability of this assay to be modified for measurement of other antifolate agents have been discussed. The broad spectrum of uses to which this assay may be put makes the CPBA for methotrexate extremely useful as a laboratory method.
[31] Ligand-Binding Radioassay of N~-Methyltetrahydrofolate and Its Application to N5-Formyltetrahydrofolate B y M A R I A DA C O S T A a n d S H E L D O N P . R O T H E N B E R G
Introduction NS-Methyltetrahydrofolate (mTHF), the major intracellular and circulating folate cofactor, can now be measured by ligand-binding radioassay using any one of the natural binding proteins isolated from human or animal tissues or biologic fluids. For most of the reported methods, the folate-binding protein present in cow's milk 1 has been used as the binder and radioisotopically labeled folic acid (FA), or some derivative of FA, as the tracer. Most of these naturally occurring folate-binding proteins, however, have greater affinity for FA than for mTHF or other naturally occurring folates. Consequently, if the classical competitive inhibition radioassay is used with the standard mTHF and tracer competing simultaneously for binding sites on the protein, the sensitivity of the dose-response curve will be diminished. There will also be an inherent error in the total folate concentration when a mixture of folates, each with varying affinity for the binding protein, is measured by a competitive inhibition radioassay. This error can be minimized by carrying out the radioassay at pH 9.3 at which the affinity of FA and mTHF for the binder appears to be equal, 2
1 j. Ghitis, Am. J. Clin. Nutr. 20, 1(1967). 2 j. K. Givas and S. Gutcho, Clin. Chem. 21, 427(1975).
METHODS IN ENZYMOLOGY, VOL. 84
Copyright © 1982by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-1819fl4-1