[CANCER RESEARCH 34, 371-377, February 1974]

Stereochemical Characteristics of the Folate-Antifolate TransportMechanism in L1210 Leukemia Cells1

Francis M. Sirotnak and Ruth C. Donsbach

Memorial Sloan-Kellering Cancer Center, New York. New York 10021

SUMMARY

The rate of influx, extent of concentrative uptake, and therate of efflux (loss) by active transport in L1210 leukemiacells has been compared for the pteridine antifolates, amino-pterin and methotrexate, eight related quinazoline analogs,and two pyrimidine derivatives. The data reveal a differencein the Stereochemical specificity for influx and efflux. Influxis preferential in the order pteridine, quinazoline, and pyrimidine. Influx of aminopterin is more rapid than that ofmethotrexate. L-Glutamylquinazolines were taken up fasterthan L-aspartylquinazolines, but influx of a D-glutamyl-quinazoline was slower than the corresponding D-aspartylderivative. Influx of the quinazolines was faster when therewas a methyl- or chloro- substitution at position 5. Influx ofthe pyrimidines was also faster when a methyl group was atposition 6. Michaelis constants (Km) for influx of the variousanalogs varied from 1.42 x 10~6Mto over 10"4 M. Individ

ual Vmax values were essentially the same (1.87 to 2.22nmoles/min/g dry weight). The relationship between thevalues for initial velocity of influx (v), the Km and Vmaxobtained with each analog are in agreement with that predictedby the Michaelis-Menten equation and is consistent with thenotion that differences in rates of influx are attributable todifferences in the affinity of the carrier for the system. Efflux is preferential in the order pteridine, pyrimidine, andquinazoline. Efflux of aminopterin and methotrexate occursat the same rate. Both aspartyl- and glutamylquinazolinesefflux at about the same rate, but the D-aspartyl and D-glutamyl forms efflux more rapidly than the correspondingL forms. A methyl, and particularly a chloro, substitutionat position 5 of the quinazoline reduces the rate of efflux.The extent of concentrative uptake observed for each analogdirectly reflects the relative magnitude at which the influxand efflux processes operate and may be the physiologicalparameter most relevant to therapeutic efficacy.

INTRODUCTION

Recent findings (24, 25) from this laboratory attribute theselective activity of methotrexate during therapy of theL1210 leukemia to a greater persistence of drug in tumor

' This work supported in part by Grant CA-08748 from the NationalCancer Institute and Grant BC-108 from the American Cancer Society.

Received June 20, 1973; accepted November 7, 1973.

versus normal cells. This apparently occurs because of thelarger potential for concentrative uptake of drug by thetumor cells after the serum concentration has fallen to alow level. Although the significance of these findings to antifolate treatment of human leukemia remains to be determined, the potential for therapeutic exploitation of thisphysiological site seems obvious.

The manner by which antifolates penetrate tumor cellshas been of interest to a number of workers (3, 5, 6-11, 15-17, 20, 21, 23, 27, 28). There is extensive evidence (6-9, 16,17, 20, 21, 23, 27, 28) in vitro indicating that uptake inL1210 cells occurs by active transport. In most other tumorcells, uptake has been shown (3, 5, 17) to at least resemblean active transport process.

The characteristics of uptake in L1210 cells demonstratedin vitro closely approximate that seen in the animal (24, 25).In another aspect of these studies, the Stereochemical requirements of the transport mechanism in L1210 cells wereexamined. Measurements were made of the rate of influxand extent of concentrative uptake, as well as the rate ofefflux (loss) of a variety of folate analogs. The results of preliminary studies comparing aminopterin, methotrexate, andmethasquin have been reported from our laboratory (23).Comparisons between methotrexate and methasquin (20)and methotrexate and a pteroate analog (16) have also beenmade elsewhere. A more extensive kinetic analysis involvinga number of individual Stereochemical differences amonganalogs is presented here.

MATERIALS AND METHODS

The maintenance and transplantation of the ascitic L1210line (V) in vivo has been described (14). Methotrexate andaminopterin were supplied by Lederle Laboratories, PearlRiver, N. Y. The quinazoline and pyrimidine analogs wereprovided by Parke Davis and Co., Detroit, Mich. Aminopterin and methotrexate were purified by chromatography(22). The final purity of all drug samples was evaluatedbioautographically (4). The dihydrofolate reducíasecontent of the LI 210 cells was determined by titration inhibitionwith methotrexate or methasquin (30).

Enzyme Assay for Antifolate. The content of drug in cell-free extracts was determined by titration (30) with a partially purified (29) dihydrofolate reducíasefrom a high-level recombinant strain of Diplococcus pneumoniae (26).The details of the routine tube assay have already been

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Francis M. Sirotnak and Ruth C. Donsbach

described (23). All but 2 of the antifolates (see legend ofTable 1) titrate the microbial enzyme to about 80% inhibition. Both D-deazaaminopterin and D-quinaspar titrate theenzyme only to about 60% inhibition. However, in our experience a good level of reliability in assays for both compounds can still be obtained by using additional samplereplicates. The range for the amount of drug detectable inthis assay varies between 0.05 and 0.5 ng.

Antifolate Uptake by I 1210 Cells. The harvesting of cellshas been described (23, 27). Usually 2 x IO7cells in 1 ml(A6oo = 3.0) of suspending medium (pH 7.5) were incubated with drug. The uptake of drug at 37°corrected fordrug associating with cells at 0°was used as a measure ofuptake by active transport. Uptake at 0°(about 1 to 2% of

the total uptake at 37°)provides a measure of drug rapidly

adsorbed on the cell surface. This is essentially a temperature-independent process (9, 23). In agreement with theknown lipophobic character of the folate analogs, the rateof passive diffusion occurring within the concentration range(2.2 n\i or less) used during these studies would be expected to be negligible (2, 18). This was confirmed (6) formethotrexate by an estimation made at 37°at an external

concentration (100 ßM)well in excess of that necessary tosaturate the carrier mechanism. Similar measurements ofthe diffusion at 37°of methotrexate were also made in the

current study at an external concentration of 500 ¿IM.Therate of diffusion at 0.45 JÕM(the concentration used in therate determinations shown in Table 1) calculated from

Table 1The uptake of folate antagonists by LI210 leukemia cells

Substituents

Compound"AminopterinMethotrexateD-Deaza-aminopterinDeazaaminopterin5-Methyldeaza-aminopterin5-Chlorodeazaami-nopterinD-QuinasparQuinasparMethasquinChlorasquinNSC

110180NSC

110191Basic

ringstructure"2,4-Diami-nopteridine2,4-Diaminop-teridine2.4-Diamino-quinazoline2,4-Diamino-quinazoline2,4-Diamino-quanazoline2,4-Diamino-quinazoline2.4-Diamirio-quinazoline2,4-Diamino-quinazoline2,4-Diamino-quinazoline2,4-Diamino-quinazoline2,4-Diamino-pyrimidine2,4-Diamino-pyrimidineAmino

acid5 6 10moietyL-GlutamylCH,

L-GlutamylD-GlutamylL-GlutamylCH3

L-GlutamylCl

i.-GlutamylD-AspartylL-AspartylCH3

L-AspartylCl

L-Aspartyl*

L-Aspartyl•

CHj L-AspartylRate

ofinflux'

(nmole/min/g drywt)0.7100.2300.0410.2080.2370.3500.1750.0470.0680.0760.00330.0047Km"(X10"M)1.424.9557.206.655.952.777.6538.3027.2023.50>100>100

" Aminopterin, /V-|p-|((2,4-diamino-6-pteridinyl)methyl]amino|benzoyl|-L-glutamate; methotrexate. A'-(p-|((2,4-diamino-6-pteridinyl)methyl]methylamino|benzoyl|-L-glutamate; D-deazaaminopterin, Ar-|p-|[(2,4-diamino-6-quinazolinyl)methyl]-amino|benzoyl|-i)-glutamate, hemihydrate; deazaaminopterin, N-\p |[(2,4-diamino-6-quinazolinyl)methyl]amino|benzoyl](- L-glutamate: 5-methyldeazaaminopterin, W-|/>-|[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino|benzoyl|-L-glutamate, diso-dium, tetrahydrate; 5-chlorodeazaaminopterin, Ar-(/j-|[(2,4-diamino-5-chloro-6-quinazolinyl)methyl]amino|benzoyl|-L-glutamate,hemihydrate; D-quinaspar, /V-(p-{[(2,4-diamino-6-quinazolinyl)methyl]amino|benzoyl|-D-aspartate, disodium, heptahydrate;quinaspar, /V-|/7-|[(2,4-diamino-6-quinazolinyl)methyl]amino|benzoyl|-L-aspartate; methasquin, /V-(/>-|((2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino|genzoyl|-L-aspartate. disodium, pentahydrate; chlorasquin, /V-|p-|[(2,4-diamino-5-chloro-6-quina-zolinyl)methyl]amino|benzoyl|-L-aspartate, dihydrate; NSC 110180, Ar-(p-|(/)-(2,4-diamino-5-pyrimidinyl)benzoyl]amino|ben-zoyl|-L-aspartate; NSC 110191, /V-jp-|[p-(2,4-diamino-6-methyl-5-pyrimidinyl)benzoyl]amino|benzoyl|-L-aspartate.

"p-Aminobenzoyl moiety attached at position 9 of the pteridinyl ring and benzylaminobenzoyl attached at position 5 of the

pyrimidines.' The initial rate of uptake at 37°corrected for uptake at 0°.Rate = nmoles/min/g dry weight (drug],,«,!,.]= 0.45 M.Values

are an average of 4 to 6 replicate experiments, with a standard deviation of less than 30%. Each compound was always compared tomethotrexate run as an internal control in the same experiment.

" Michaelis constant (molar). Values are an average of 5 to 8 replicate experiments, with a standard deviation of less than 30%.Each compound was always compared to methotrexate run as an internal control in the same experiment. Precautions used in determining rates for true initial velocity were the same as described previously (6, 15).

' Not applicable, since this is a 5-arylpyrimidine (see above).

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Antifolate Transport in L/210 Leukemia Cells

these data was 0.00075 to 0.001 nmole/min/g dry weight.The suspending medium consisted of 107 HIMNaCl, 5.3

mM KC1, 26.2 mM NaHCO3, 1.9 mM CaCl2, 1 mMMgCl2-6H2O, 10 mM glucose, and 10 mM Tris-HCl. Exposure to drug was terminated by the addition of 10 mlof cold 0.02 M potassium phosphate-0.14 M NaCl, pH 7.5.Cells were washed free of drug by resuspension twice in thesame solution to a final volume of 1 ml. The cell numberwas determined by an absorbance measurement using astandard curve relating microscopic cell count to absorbance (27). Drug was removed from the cells by heat extraction, and cellular debris was discarded after centrifugation.

Kinetic Analysis of Antifolate Influx. The Michaelis constant (Km) for the transport system was determined by themethod of Lineweaver and Burk (19). In this system, itrepresents the external drug concentration needed for half-saturation and is used as a relative measure of the affinity ofthe carrier component for drug. The initial rate of influx at37°(minus uptake at 0°)was measured at varying concen

trations of each antifolate. The time of incubation was adjusted to allow for measurement of uptake below the di-hydrofolate reducíasecontent. Since no free drug existsinternally in this range, uptake is essentially unidirectionaland a valid measure of influx. The necessity for this precaution has been stressed in earlier reports (9, 23).

Measurements of Efflux by L1210 Cells. The efflux of thevarious antifolates from LI210 cells was measured (9, 23)by preloading the cells with drug (usually to an intracellularlevel 3 to 4 times the dihydrofolate reducíasecontent) andwashing the cells free of drug with cold 0.02 M potassiumphosphate-0.14 M NaCl, pH 7.5. The cells were then re-suspended in fresh medium and incubated at 37°for vary

ing periods of time.

RESULTS

The Influx of Antifolates by 1.1210 Cells. The folate analogs used during these studies include the pteridines, amino-pterin and methotrexate, 8 quinazolines, and 2 pyrimidinederivatives. In accordance with their gross stereochemicalsimilarity, the 3 groups appear to compete for the sametransport mechanism(s). This conclusion is based on a priordemonstration of inhibition of the rate of influx at 37°ofmethotrexate-3H by methasquin (20, 23) and of inhibition

of the rate of influx of some of these drugs (sometimes bykinetic analysis) by the natural folates folie acid, 5-methyl-tetrahydrofolic acid, and 5-formyltetrahydrofolic acid (7-9,17, 21, 23, 27). A similar measurement of the inhibition ofinflux of the pyrimidine analogs at 37°by normal folates

was also made during the current studies. Like prior results,inhibition of the rate of influx of these analogs was shown tobe competitive or resemble a competitive process and demonstrable only at saturating or near-saturating externalconcentrations. The extent to which the influx of each classof antifolate was inhibited by normal folates was in goodquantitative agreement with the relative rate and kineticsof influx observed for each.

The initial rate of temperature-dependent influx of thevarious analogs, at a concentration of 0.45 ¿tM,is shown inTable 1. The relative rate of influx among the group variedmore than 200-fold. Influx of aminopterin was the mostrapid, while influx of the pyrimidine analogs, NSC 110180and NSC 110191 was the least rapid. The rate of influx ofmethotrexate, and the related L-glutamylquinazoline, de-azaaminopterin, was only one-third to one-fourth as rapidas that of aminopterin. The L-aspartylquinazoline analogswere transported at one-fifth the rate of the correspondingL-glutamylquinazolines. 5-Chlorodeazaaminopterin and the5-chloro-L-aspartylquinazoline (chlorasquin) were transported at a rate almost twice that of the corresponding un-substituted derivatives. A somewhat smaller increase in therate of influx occurred with 5-methyl-deazaaminopterin andmethasquin. The 6-methyl 2,4-diaminopyrimidine analog(NSC 110191) had a higher rate of influx when comparedto the unsubstituted 2,4-diaminopyrimidine (NSC 110180).The influx of the D-glutamylquinazoline, D-deazaamino-pterin, occurred at only one-fifth the rate obtained withL-deazaaminopterin. In contrast, the rate of influx of theD-aspartylquinazoline (o-quinaspar) was 4-fold greaterthan that of the corresponding i.-aspartyl form (quinaspar).

Kinetic Analysis of Antifolate Influx. Saturation (Mi-chaelis-Menten) kinetics for temperature-dependent uptakewas demonstrated for all of the analogs except the 2 pyrimidine analogs (NSC 110180 and NSC 110191). The apparentMichaelis constant (Km) derived for each analog is shown inTable 1. As anticipated from the data on the rate of influx,the transport mechanism appears to exhibit the greatestaffinity for aminopterin (Km = 1.42 x 10"6 M)and somewhat less for methotrexate (Km = 4.95 x 10"6 M) and theL-glutamylquinazolines (Km = 2.77 to 6.65 x 10~8M). The

mechanism seems to have even less affinity for the D-aspartylquinazoline (Km = 7.6 x 10~6 M) and the L-aspartylquinazolines (Km = 23.5 to 38.3 x 10"6 M) andleast for the D-glutamylquinazoline (Km = 57.2 x 10~6M).

No Km value could be obtained with the 2 pyrimidine analogs because of the extremely high concentrations apparently necessary to saturate the system. The maximum rateof influx obtainable (Vmax)with the pteridine and quanazo-line analogs was calculated graphically or from the basicMichaelis-Menten equation, v = (Vmax-S)/(K.m + S), wherev is initial velocity of influx and S is [drug]externai-Valuesfor each analog are approximately the same, varying from1.87 to 2.22 nmoles/min/g dry weight.

The Intracellular Concentration of Antifolate at SteadyState. The kinetics and extent of concentrative uptake foreach folate analog were measured by incubating L1210 cellsat 37°with drug (0.45 or 2.2 fiM)until the free intracellular

drug concentration was at equilibrium (steady state). Thisusually requires a period of 40 to 60 min. The data shownin Table 2 and Charts 1 and 2 were corrected for uptake at0°.This value is usually very small and has been shown (9,

23) to represent mainly adsorption on the cell surface. Thekinetics of concentrative uptake of the various analogsdiffered considerably. The accumulation of the pteridines(Chart 1) is similar. Uptake occurs at essentially a constantrate (only influx occurs) until the dihydrofolate reducíase

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Francis M. Sirotnak and Ruth C. Donsbach

Table 2The concentrative uptake of folate antagonists by Li 210 leukemia cells

IntracellulardrugCompound"AminopterinMethotrexateD-DeazaaminopterinDeazaaminopterin5-Methyldeazaaminop-terin5-Chlorodeazaaminop-terinD-QuinasparQuinasparMethasquinChlorasquinNSC

110180NSC110191(drugJe.uMT.ai6(MM)2.22.22.20.452.22.20.452.22.22.22.22.211.011.0Total(nmolesdry

wt)21.915.912.931.244.841.651.380.135.522.935.340.32.21.6Freec(UM)11.127.465.6216.9025.2022.7029.1946.8019.4511.7519.3322.39Ratio,(drug)liu.rn.,/(drug)»tern»5.063.392.5537.5911.4010.6366.5021.258.855.358.8010.18

°See Table 1 for structural details.' External drug concentration.' Based on a volume of 0.005 ml for the water content of the intracellular free space in 2 x IO7

cells (20). The amount of drug bound to dihydrofolate reducíase(3.75 nmoles/g dry weight) wassubstracted from the total amount accumulated intracellularly. Values are based on 4 to 6 replicateexperiments with a standard deviation of less than 30%.

60r

40 [drug]ext = 2.2 u M

dea/a .iminopterin

aminopterín

methotrexate

60

Chart I. The rate of concentrative uptake at 37°of pteridine and py-

rimidine antifolates in LI210 leukemia cells. The uptake of each analogwas always related in 4 to 6 replicate experiments to the uptake of methotrexate. Total uptake corrected for uptake at 0°.ext, external.

level is reached. The rate then gradually diminishes as thesteady-state level is approached. With the exception ofD-deazaaminopterin, quinaspar, and possibly D-quinaspar,

60

Chart 2. The rate of concentrative uptake at 37°of quinazoline anti

folates in L1210 leukemia cells. The uptake of each analog was always related in 4 to 6 replicate experiments to the uptake of methotrexate. Totaluptake corrected for uptake at 0°.ext, external.

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Antifolate Transport in L/2/0 Leukemia Cells

the kinetics of uptake for the quinazoline analogs appears(Chart 2) to be quite different. Uptake of drug continues ata constant rate to internal concentrations far greater thanthe drug equivalence of the dihydrofolate reducíasecontent. In these cases linear kinetics of uptake continue againsta sizable concentration gradient (see below). This is dramatically apparent in the case of 5-chlorodeazaaminopterinwhere drug is accumulated to nearly 14-fold the dihydrofolate reducíaselevel. At an exlernal concenlralion of 2.2¿tM,the total accumulation of the pyrimidine analogs(Chart 1) did not reach the dihydrofolate reducíaselevel.

The extenl of concenlralive uplake observed for each fo-lale analog varied over a wide range. The inlernal concen-Iralions of drug, al Ine sleady-slale levels shown in Charts1and 2, are given in Table 2. At an exlernal concentration of2.2 /iM, uptake of ihe pleridine (Charl 1), aminoplerin,and melholrexale was at the least concentrative (3- to 5-fold higher internal concentralion). Uplake of all of thequinazoline analogs (Chart 2), excepl D-deazaaminoplerinand quinaspar, was considerably more concenlralive (alleasl 8.8-fold higher inlernal concenlralion). 5-Chloro-deazaaminoplerin was concenlrated to Ihe grealesl exlent.Al an exlernal concenlralion of 2.2 ¡J.M,ihe inlernal levelal equilibrium was over 21 limes grealer. Al an externalconcenlralion of 0.45 ¿tM,ihe difference was 70 timesgrealer. Al ihe same exlernal concenlration (0.45 jtM),deazaaminoplerin was concenlraled almosl 40-fold. Con-cenlrative uptake of the pyrimidine analogs, NSC 110180and NSC 110191, could not be demonstraled al ihese concentrations.

The Efflux of Folate Analogs by L1210 Cells. Previous

20

•¿�aminopterino methotrexateA NSC 110. 180A NSC 110. 191

25 r

t (min)

Chart 3. The efflux of pteridine and pyrimidine antifolates by L1210leukemia cells. The data shown are based on the average of 3 to 5 replicateexperiments in which the efflux of methotrexate was measured as an internal control.

•¿�D -dea/a-aminopterin

o deaza-aminopterin

A 5-methyl, deaza- aminopterin

A 5-chloro. deaza aminopterin

•¿�D-quinaspar

O quinaspar

X methasquin-+•chlorasquin

60

t (min)

Chart 4. The efflux of quinazoline antifolates by L12IO leukemia cells.The data shown are based on the average of 3 to 5 replicate experiments inwhich the efflux of methotrexate was measured as an internal control.

studies (8, 9, 17, 23) have shown thai the loss of folate analogs from L1210 cells is temperalure dependent. This is inagreemenl wilh ihe idea lhal the same carrier is ulilized forbolh influx and efflux. A similar lemperature dependencefor efflux of all of the analogs has been observed during thecurrent study. The efflux of the various folate analogs fromL1210 cells in drug-free medium at 37°is shown in Charts

3 and 4. Efflux of both the pteridine and pyrimidine analogs(Chan 3) occurred very rapidly. The inlracellular drug levelwas al ihe dihydrofolale reducíaselevel within 10 to 15 minafter incubation was initiated. As reported earlier (8, 9, 23),further loss of drug with lime occurred al an almosl imper-ceplible rale. Efflux of all of Ihe quinazoline analogs occurred (Charl 4) al a much slower rate. The internal level ofonly D-deazaaminopterin, deazaaminopterin, and D-quinaspar reached enzyme level during ihe 60-min efflux period.

Since Ihe decrease in concenlration of free drug in L1210cells approximates a 1si-order process, il was possible moreaccuralely lo quanlilale Ihe relalive rale of efflux for eachanalog. A /i/2 value (lime required for a 50% decrease inconcenlralion) was first calculaled from a linear semilog-arilhmic plol of ihe internal concentration of free drug atvarious time inlervals. Values derived for each analog aregiven in Table 3. The rale conslanl for efflux for each is ob-lained from ihe expression k = In 2//i/2. These values arealso given in Table 3. A relalive difference in efflux rale ofaboul 20-fold was demonslraled for Ihe various analogs.

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Francis M. Sirotnak and Ruth C. Donsbach

Table 3The rale of efflux affatale antagonists by LI 210 leukemia cells

Compound"AminopterinMethotrexateD-DeazaaminopterinDeazaaminopterin5-

Methyldeazaaminopterin5-ChlorodeazaaminopterinD-QuinasparQuinasparMethasquinChlorasquinNSC

110180NSC110191l¿

(min)2.93.414.520.526.263.813.136.240.739.84.54.4Rate

constant'(min-1)0.2390.2020.0470.0340.0270.0110.0530.0190.0170.0180.1470.158

°Structural details are given in the legend of Table 1."Time required for internal concentration of free drug to decrease by

one-half. Values are averages of 3 to 5 replicate experiments with a standard deviation of less than 30%.

' k = In 2/t*.

Aminopterin had the highest rate of efflux (k = 0.239),followed by methotrexate and the 2 pyrimidine analogs,NSC 110180 and NSC 110191. Efflux of D-deazaamino-pterin and D-quinaspar was slower, but at nearly the samerate. The rate of efflux of the L-aspartylquinazoline analogswas less than one-tenth the rate observed for aminopterin.Two of the L-glutamylquinazolines, deazaaminopterin and5-methyldeazaaminopterin effluxed somewhat faster thanthe corresponding L-aspartyl forms. The rate of efflux forthe 5-chlorodeazaaminopterin was the lowest of the entiregroup (k = 0.011).

DISCUSSION

The function of the antifolate transport mechanism inL1210 leukemia cells exhibits a considerable degree of diversity with respect to stereochemical specificity. Moreover,the data reveal a difference in the stereochemical basis ofthe influx and efflux processes. Based on the respectiveapparent Michaelis constants for influx, the mechanismseems to exhibit the greatest affinity for the 2,4-diaminoanalog (aminopterin) of folie acid. The affinity was somewhat less in the case of the 2,4-amino, /V'-methyl analog

(methotrexate). The poor affinity of the mechanism for folieacid itself has already been demonstrated (8, 9, 17, 23, 29).On the other hand, 5-formyltetrahydrofolate (citrovorumfactor) and 5-methyltetrahydrofolate are probably transported by this mechanism to about the same extent asmethotrexate (8, 9, 17, 21, 23, 27). The mechanism has reduced affinity for quinazoline derivatives and a severelyreduced affinity for the corresponding pyrimidine analogs.Interaction with the L-aspartyl analogs is poor in comparison to the L-glutamyl analogs. The opposite seems to betrue with regard to analogs bearing D-glutamyl or D-aspartyl moieties. The affinity of the mechanism is greaterfor quinazoline analogs bearing a methyl or especially achloro group at position 5. The system has greater affinityfor a pyrimidine analog, if a methyl group is substituted atposition 6.

Experiments showing a rapid loss of aminopterin andmethotrexate suggest an interaction with the efflux mechanism which again is most efficient in the case of the pteridineanalogs. However, in contrast to that seen during influx, themethyl group at A"°has little effect on efflux. Moreover,

both of the pyrimidine analogs efflux almost as rapidly asthe pteridines, suggesting a nearly equivalent affinity for thesame system. The quinazoline analogs as a group appear tohave the lowest affinity for the efflux mechanism. These arelost from the L1210 cells slowly, with little difference observed between the L-aspartyl and L-glutamyl derivatives.Both D-aspartyl and D-glutamyl analogs efflux more efficiently than the corresponding L forms. Whereas a methylor chloro substitution at position 5 of the quinazoline ringwas found to potentiate influx, the opposite is true forefflux.

It is vividly apparent that both influx and efflux play asignificant role in determining the internal level of free drugachievable at a specific external concentration. Moreover,the kinetics and extent of concentrative uptake actually observed for each analog quantitatively reflects the magnitudeat which each can operate. For example, when both influxand efflux are relatively rapid (aminopterin) or slow (meth-asquin), fairly high intracellular levels are achievable atrelatively low extracellular concentrations. When influx isslow and efflux is rapid, as in the case of the pyrimidineanalogs (NSC 110180 and NSC 110191), only low intracellular levels are possible. On the other hand, when influxis rapid but efflux is slow, phenomenally high intracellularlevels are possible, as with 5-chlorodeazaaminopterin.

Knowledge as to general therapeutic relevance of achieving high steady-state levels of free drug in tumor cells willrequire further study of these drugs at a pharmacologicallevel. As suggested (8, 23), the rate of influx alone is probably an inadequate parameter for evaluating possible therapeutic efficacy. This, in fact, has proved to be the case withmethasquin, which was relatively inert during experimentsmeasuring influx but is a fairly effective antileukemic agent(12).

Although we have not directly demonstrated competitionamong various folate analogs for the efflux mechanism, it isassumed that each analog utilizes the same carrier for thispurpose. This is probably true, if both influx and efflux occur by a single carrier, since some evidence has been provided (Refs. 7 to 9, 17, 20, 21, 23, and 27, and in the presentstudy) which indicates that all of the analogs compete forthe same influx mechanism. Additional evidence for the involvement of a single carrier for influx and efflux comesfrom a demonstration of both countertransport (9, 21) and atranstimulation effect (7) in the L1210 system. The latterwas based on data showing that methotrexate influx is morerapid in cells preloaded with 5-formyltetrahydrofolate. Wehave confirmed this finding and have also obtained the sameresult in connection with the influx of the quinazolines andpyrimidines (F. M. Sirotnak and R. C. Donsbach, unpublished results).

The large spectrum of interaction between folate analogsfor the transport carrier is quite different from that seenwith the target enzyme, dihydrofolate reducíase.All of the

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Antifolate Transport in L1210 Leukemia Cells

analogs examined here are excellent inhibitors of the enzyme in L1210 cells (Ref. 13; F. M. Sirotnak and R. C.Donsbach, unpublished results) with dissociation constants(K,) in the vicinity of 10~" M. The greater stereochemical

flexibility of the interaction with enzyme undoubtedly relates to the multisite nature (1) of the contact demonstratedwith this class of inhibitor. It would appear that relatedstudies (24, 25) in tumor versus normal cells, at the level ofmembrane transport, might offer more opportunity forelucidating exploitable stereochemical differences.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the technical assistance of MarthaA. Ward and the interest and support of Dr. Dorris J. Hutchison duringthese studies.

REFERENCES

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FEBRUARY 1974 377

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1974;34:371-377. Cancer Res   Francis M. Sirotnak and Ruth C. Donsbach  Transport Mechanism in L1210 Leukemia CellsStereochemical Characteristics of the Folate-Antifolate

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