10
A retrospective view of cancer chemotherapy is remarkable, not because of the lack of success in finding a curative anticancer agent, but because agents have been found which so narrowly miss being curative in one or another of the many forms of cancer. Methotrexate is one such agent. Since the introduction of aminoptenin into the medical armamentarium by Farber (13) in 1948, questions about the mode of action of the 4- amino folic acid antagonists have plagued and in tnigued investigators. Not only are these agents, through their currently most effective representa tive—methotrexate-—still to be rated high among antineoplastic drugs, but also they pose a number of problems, some typical, some as yet unique, whose solution could provide guiding principles for future chemotherapeutic attempts. These problems include not only the obvious questions of the specific metabolic insult which is responsible for the effectiveness of the drug and of the differential sensitivity of the host and tumor tissues to this insult, but many ancillary aspects which may easily overshadow these primary ef fects. A partial listing of such auxiliary problems would include side-effects of the drug on the host, chemical modifications of the drug to make more effective agents, special excretory mechanisms, limitation of access of the drug to its site of action due to permeability or active transport restnic tions, intracellular compartmentation of drug, and rate of recovery from the effect of the drug, C The work performed in these laboratories was supported by grants (CA-O@Is00and CA-05@98) from the National Cancer Institute, U.S. Public Health Service. as examples of problems recognized but as yet un solved. In this paper I will omit much important work and will make no attempt to present a comprehen sive review of even the recent literature on the antifolic acid drugs; several such reviews have ap peared recently (10, 12, 24, 30, 46). Rather, I will discuss some of the problems just mentioned in the light of available evidence to see how complete a picture of the action of these drugs can be drawn. At the risk of some oversimplifications or unjusti fled generalizations, in view of the fragmentary nature of some of the evidence available, this treatment may serve to focus attention on a few of the crucial questions which must be answered. Nichol and Welch (33) early demonstrated that the biochemical effect of aminopterin injected into rats or added to liver preparations in vitro was to prevent the conversion of folic acid to reduced cofactor forms (folinic acid). Broquist et a!. (5) fur then demonstrated that tetrahydrofolic acid could prevent the effects of aminoptenin in mice, and therefore it was suspected that the effect of the drug was to prevent the reduction of folic acid, a reaction now known to be catalyzed by the en zyme, folic acid reductase.' The demonstration by Fountain et at. (16) that injected methotrexate persists in certain mouse tissues for as long as 8 months led me to investigate the manner of this retention (48). Although administered metho trexate was localized in the supernatant fraction of rat liver after differential centnifugation, it was 1Also called dihydrofolate reductase and tetrahydrofolate dehydrogenase. 1277 The Biochemical, Cellular, and Pharmacological Action and Effects of the Folic Acid Antagonists* WILLIAM C. WERKHEISER (Department of Experimental Therapeu&s, Roswell Park Memorial lastitute, Buffalo, New York) SUMMARY A brief survey of biochemical and pharmacological information on the action of the 4-amino folic acid antagonists is presented. An attempt is made to organize most of these facts into a coherent description of the mechanism of action of these drugs. It is concluded that, although their biochemical action is clearly a result of inhibition of the enzyme folic reductase, their chemotherapeutic value stems from differences in the permeability of various tissues to the compounds. on March 2, 2020. © 1963 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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Page 1: The Biochemical, Cellular, and Pharmacological Action · the biochemical effect of aminopterin injected into rats or added to liver preparations in vitro was to prevent the conversion

A retrospective view of cancer chemotherapy isremarkable, not because of the lack of success infinding a curative anticancer agent, but becauseagents have been found which so narrowly missbeing curative in one or another of the manyforms of cancer. Methotrexate is one such agent.

Since the introduction of aminoptenin into themedical armamentarium by Farber (13) in 1948,questions about the mode of action of the 4-amino folic acid antagonists have plagued and intnigued investigators. Not only are these agents,through their currently most effective representative—methotrexate-—still to be rated high amongantineoplastic drugs, but also they pose a numberof problems, some typical, some as yet unique,whose solution could provide guiding principlesfor future chemotherapeutic attempts.

These problems include not only the obviousquestions of the specific metabolic insult which isresponsible for the effectiveness of the drug and ofthe differential sensitivity of the host and tumortissues to this insult, but many ancillary aspectswhich may easily overshadow these primary effects. A partial listing of such auxiliary problemswould include side-effects of the drug on the host,chemical modifications of the drug to make moreeffective agents, special excretory mechanisms,limitation of access of the drug to its site of actiondue to permeability or active transport restnictions, intracellular compartmentation of drug,and rate of recovery from the effect of the drug,

C The work performed in these laboratories was supported

by grants (CA-O@Is00and CA-05@98) from the National CancerInstitute, U.S. Public Health Service.

as examples of problems recognized but as yet unsolved.

In this paper I will omit much important workand will make no attempt to present a comprehensive review of even the recent literature on theantifolic acid drugs; several such reviews have appeared recently (10, 12, 24, 30, 46). Rather, I willdiscuss some of the problems just mentioned in thelight of available evidence to see how complete apicture of the action of these drugs can be drawn.At the risk of some oversimplifications or unjustifled generalizations, in view of the fragmentarynature of some of the evidence available, thistreatment may serve to focus attention on a fewof the crucial questions which must be answered.

Nichol and Welch (33) early demonstrated thatthe biochemical effect of aminopterin injected intorats or added to liver preparations in vitro was toprevent the conversion of folic acid to reducedcofactor forms (folinic acid). Broquist et a!. (5) furthen demonstrated that tetrahydrofolic acid couldprevent the effects of aminoptenin in mice, andtherefore it was suspected that the effect of thedrug was to prevent the reduction of folic acid, areaction now known to be catalyzed by the enzyme, folic acid reductase.' The demonstration byFountain et at. (16) that injected methotrexatepersists in certain mouse tissues for as long as 8months led me to investigate the manner of thisretention (48). Although administered methotrexate was localized in the supernatant fractionof rat liver after differential centnifugation, it was

1Also called dihydrofolate reductase and tetrahydrofolatedehydrogenase.

1277

The Biochemical, Cellular, and Pharmacological Action

and Effects of the Folic Acid Antagonists*

WILLIAM C. WERKHEISER

(Department of Experimental Therapeu&s, Roswell Park Memorial lastitute, Buffalo, New York)

SUMMARY

A brief survey of biochemical and pharmacological information on the action of the4-amino folic acid antagonists is presented. An attempt is made to organize most ofthese facts into a coherent description of the mechanism of action of these drugs. It isconcluded that, although their biochemical action is clearly a result of inhibition ofthe enzyme folic reductase, their chemotherapeutic value stems from differences inthe permeability of various tissues to the compounds.

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Cancer Research Vol.923,September19631278

not dialyzable. Further investigation demonstrated that the drug was firmly bound to folicreductase, which was inhibited in consequence.This inhibition had been shown by a number ofinvestigators (17, 34—36, 55, 57). Enzymaticstudies showed that the inhibition of folic reductase by methotrexate (and all other 4-amino folic

acid analogs) was of an unusual nature : thestrength of the association between the drug andenzyme at pH 6 was so great that the drug actedas a titrating agent. A crude estimation suggestedthat the dissociation constant of the complex wasless than S X 10― M, which implies an affinity offolic reductase for methotrexate more than 100,-000 times as great as for folic acid at pH 6 and20,000 times as great as for dihydrofolic acid atpH 7.5. This mode of inhibition, for which thename stoichiometric has been suggested (48), ischaracterized by the fact that the effective dissociation constant of the enzyme inhibitor complex is very small compared with the enzyme concentration and thus, at concentrations of inhibitorinadequate to cause complete inhibition of theenzyme, almost all the inhibitor is enzyme-bound.Goldstein (18) has clearly demonstrated thatstoichiometric inhibition precludes distinction between competitive and noncompetitive mechanisms. So far, it has not been possible to determinewhich mechanism is applicable for methotrexate,although work on this question is in progress inseveral laboratories.

The stoichiometnic behavior of this interactionhas been of considerable value in recent investigations, since it allows a titration of enzyme withdrug, permitting (a) determination of free or totaldrug in unknown samples including tissue extracts(52) and (b) determination of free enzyme inequivalents of drug-binding sites (49), a muchmore consistent measure than that of enzymeactivity which shows appreciable variation in repeated assays.

With the use of this procedure, it was possible toshow that about half of the drug which is retainedin mouse liver 24 hours after a single injection islost extremely slowly, with a half-time of over 90days (49), providing confirmation of the retentionfound by Fountain et a!. (16) and later by Charache et al. (8). Thus, the stoichiometric relationship observed in vitro also exists in vivo.

These findings, taken together, indicate not onlythat methotrexate and related compounds are indeed very potent inhibitors of folic reductase invivo and in vitro, but also that, within 24 hoursafter administration of the drug, no free drug isavailable to inhibit any other enzyme. It therefore seemed reasonable to accept the working hy

pothesis that the action of the antifolic drugs onfolic reductase was the explanation for their toxicmanifestations, and to set aside for the momentsome disquieting observations on the effects offolinic acid that seemed to contradict it.

This hypothesis has been tested in several ways:Greenspan et al. (19, 20) have shown that mice canbe protected from the effect of a lethal injection ofaminopterin by the prior injection of folic acid.

Mice preconditioned in this way could not againbe protected from the toxic effects of the drug byfolic acid injection until at least the 3d day subsequent to the first course of injections. A group ofmice preconditioned in this manner were sacrificedat intervals, and the content of free folic reductaseand of bound aminopterin in liver and intestinalmucosa was determined (49) . The free enzyme content of the intestinal mucosa, a tissue sensitive tothe effect of the drug, was negligible at a timewhen folic acid injection was ineffective and wasrapidly increasing by the time such injections wereeffective. It therefore appeared that an effectivefolic reductase system in this tissue (and probablyothers, such as bone marrow) is necessary for theprotective action of folic acid, and it was inferredthat suppression of this system is the effect mostimmediately responsible for toxicity. These experiments were clearly only suggestive, and moredirect tests of the hypothesis were devised.

Philips, Thiersch, and Ferguson (37) showedthat the acute LD50 of aminopterin in mice is 3mg/kg and that of methotnexate in rats and miceis 15 and 89 mg/kg, respectively. These widelyvarying values suggested the possibility of cornparing the effect of a drug on the intestinal folicreductase of a given animal with the toxicity ofthe same drug in the same animal. Animals weregiven injections of a drug at doses between 0.01and 400 mg/kg, sacrificed 24 hours later, and thefree folic reductase and bound drug in the intestinal mucosa were determined. It was found(50) that the dose necessary to reduce the freeenzyme of the intestinal mucosa to negligiblelevels was in all three cases almost precisely theLD50(Charts 1 and 2).

Similar experiments were performed in ratsbearing, bilaterally, the Walker 256 carcinoma andthe Murphy-Sturm lymphosarcoma. The first ofthese tumors is highly refractory (38), the second,highly sensitive, to methotrexate.2 The results(Chart 2) show that the folic reductase of the sensitive tumor was completely inhibited at nontoxicdoses of methotrexate, whereas the enzyme of theresistant tumor was incompletely inhibited evenby doses above the LDbO (50).

2 F. Rosen and C. A. Nichol, unpublished experiments.

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WERKHEISER—EffeCts of Folic Acid Antagonists 1279

A collaborative study with Law, Roosa, andNichol (Si) has pursued this matter still further.The P-388 leukemia in mice is somewhat sensitiveto treatment with methotrexate, considerable inhibition of tumor growth being observed at theexpense of mild toxicity. This leukemia has beentransferred to cell culture (line R-26C) and three

sub-lines (R-46, R-67, and R-74) successively moreresistant to methotrexate developed from it. Allfour lines have subsequently been transferred backto mice, in both subcutaneous and ascitic forms.The subcutaneous control R-26C remains somewhat sensitive to the drug whereas the other threestrains appear to be resistant. Animals bearingthese tumors were given injections of methotrexate. After 24 hours they were sacrificed, and

the free folic reductase in the tumor was determined. The results are shown in Chart 3. Cornparison of the curves for intestinal mucosa and forthe control (R-26C) shows that the tumor folicreductase is slightly less responsive to the drug

than is that of the intestine. The results are inagreement with the fact that this tumor displaysonly borderline sensitivity to the drug. The curvefor the R-74 shows a great increase in the enzymeinhibitory dose, whereas the curves for the R-46and R-67 tumors are intermediate, as is theirsensitivity in culture.

The correlation between toxicity to animal ortumor and effect on enzyme shown in these experiments provides strong evidence that inhibitionof the folic reductase of sensitive—i.e., rapidly

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reductase was determined by titration with methotrexate (49).

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Cancer Research Vol. 23, September 19631280

dividing—tissues is the metabolic effect responsible for the toxicity of these drugs.

Studies of resistance to the 4-amino folic acidanalogs has also provided important informationconcerning the mechanism of action of these drugs.Hakala et at. (2.3) developed strains of culturedSarcoma 180 cells which were 67- and 174-fold resistant to methotrexate. These two lines contained65 and 155 times more folic reductase, respectively, than the parent line, whereas the rate ofmethotrexate uptake was unchanged. The properties of the enzyme were identical in all lines; only

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the amount was changed. Increased levels of folicreductase have also been reported to accompanyresistance to methotrexate in L5178Y leukemiacells in culture (14), in the L1210 leukemia in mice(31), and in acute leukemia in man (3). Althoughthe increased levels of folic reductase appear to bemost commonly associated with selection of agenetically stable resistant cell line, enzyme induction or adaptation also appears to play a partin some instances, both in cell culture (21) and inhuman leukemia (4). This subject will be discussed in another presentation in this Symposium.It now seems clear that, in all these instances, nosistance is a consequence of the increased amountof enzyme which acts as a physiological sequestering agent for the drug. Furthermore, it seems

probable that inhibition of folic reductase is theprimary action responsible for the therapeuticeffect of methotrexate in these sensitive tumors.

However, the differential aspect of the effectsof various 4-amino folic acid compounds on vanous tissues cannot be explained on the basis of folicreductase levels alone. Thus, the dose of aminopterm and methotrexate necessary to inhibit thefolic reductase of intestinal mucosa in mice andrats is widely different (Charts 1 and 2). The levelsof folic reductase in tumors sensitive to methotrexate are not necessarily lower than the levels intumors which are naturally refractory to the drug(Chart 2) (50). Fischer (15) has developed a lineof leukemia L5178Y in which resistance was notaccompanied by increase in folic reductase. Lastly,the R-74 sub-line of the P-388 tumor displays onlya moderate increase in folic reductase associatedwith a profound resistance to the drug as measured both by growth in cell culture and by the expeniment shown in Chart 3. It is clear that somephenomenon other than level of enzyme is responsible for these effects.

One possible explanation of these discrepanciescould be a variation in the rate of entry of thesedrugs into tissues. Only brief forays have yet beenmade into this area of cancer pharmacology, butit may be a fruitful one and undoubtedly deservesmuch further work. Fischer has found (15) thatthe low folic reductase, methotrexate-resistantL5178Y cell line he has developed shows in vitro alower rate of uptake of methotrexate than doesthe parent line. Preliminary experiments in thislaboratory (51) show that,@ hour after administration of a selected dose (0.08 mg/kg) of methotrexate to mice bearing the ascitic forms of the

P-388 tumor, enough drug has entered the cells ofthe sensitive tumor (R-26C) to cause almost cornplete inhibition of the folic reductase present,whereas so little drug has entered the resistant tumor (R-74) that its presence is hard to detect.These results show that between some sensitiveand resistant lines of tumors differences in permeability exist which are great enough to account forthe resistance. In contrast, Hakala (21) has reported that lines of Sarcoma 180 which are resistant in culture and which have high levels offolic reductase have unchanged rates of uptake ofmethotrexate.

Rosen and Nichol have shown that the methotrexate-refractory Walker 2.56 tumor is sensitiveto a mild dietary deficiency of folic acid (38),whereas the methotnexate-sensitive MurphySturm tumor implanted bilaterally in the sameanimals is almost unaffected by the same regimen.2 The folic acid content of these tumors, bi

AMETHOPTERIN Cmg 1kg)

CHART 3.—See legend to Chart 1

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WERKHEISER—EJeCis of Folic Aci4 Antagonists 1281

laterally implanted in the same animals, has beenstudied as a function of dietary folic acid. Whenthe animals were fed commercial chow (containingapproximately 6.2.5 mg folic acid/kg), the MurphySturm tumor contained three times more folicacid than did the Walker tumor. In the absence ofdietary folic acid, the content of the vitamin inboth tumors decreased by a factor of 2—3.Theaddition of 1.25 mg folic acid/kg of the deficient diet resulted in a twofold increase in thefolic acid content of the Murphy-Sturm tumorbut had no effect on that of the Walker tumor.2The tentative conclusion from these results is thatthe Walker tumor is relatively impermeable tofolic acid, as it seems to be to methotrexate, andthat, on the other hand, the Murphy-Sturm tumor has a much higher capacity for the uptake ofboth compounds. Thus, the Murphy-Sturm tumon can compete with the liver for the availablefolic acid and is refractory to the dietary deficiency, but its ability to take up compounds ofthis structure causes it to be sensitive to methotrexate. The Walker tumor behaves in an exactly

opposite fashion.It is tempting at this point to conclude that dif

ferential permeability is responsible for the differential sensitivity of dividing tissues and tumorsin which the folic reductase levels are comparable,but before considering this conclusion some additional experiments must be cited whose explanation is not immediately obvious, but which may infact be the key to the principal problems remaining in this field.

In the P-388 ascites tumors, enough drug entersthe cell within@ hour after injection of methotrexate (0.03 mg/kg) to cause 95 per cent inhibitionof folic reductase and of the conversion of folicacid to folinic acid by the whole cell in tiitro. By24 hours, a large proportion of free enzyme ispresent and the amount of drug per mg. cell protein is considerably lowered (51). This loweringcould, in principle, be due to loss of drug from thecell or to synthesis of new enzyme protein duringthe interval. The first alternative has been excluded by the following experiment. Mice bearingthe R-26C line of the P-388 ascites tumor weregiven injections of amethoptenin (0.03 mg/kg) andsacrificed at intervals thereafter. Total volume ofpacked ascitic cells, free folic reductase, and totalbound drug was determined. The results, expressed in amounts per mouse, are shown inChart 4. It is clear that there is no loss of bounddrug from the cell population but that, as a resultof cell multiplication, the amount of drug per cellprogressively decreases. The synthesis of folicreductase continues to parallel cell growth, unaf

fected by the presence of drug, and the free folicreductase level asymptotically returns to normal.

In this system, then, the only drug which canescape from a cell is that which is in excess of thefolic reductase present; drug, once bound toenzyme, remains bound. This was also implied bythe drug retention studies cited above. In contrast,Fischer has observed that, after exposure ofL5178Y cells to methotrexate, much of the drugthat has entered a cell can again leave upontransfer of the cell to a drug-free medium, and hassuggested that the drug “canbe freed from thefolic acid reductase of these cells―(14). It is possible that the cell culture and whole animalsystems differ in this regard, but before this view

I

is accepted it would be desirable to have directevidence that, during the period of incubation inthe absence of drug, there is a net decrease in theamount of enzyme-bound drug in the total cellpopulation.

Hakala (22) finds, in studying sensitive and nosistant Sarcoma 180 cell culture lines, that at aconcentration of 3 X 1O@ M methotrexate in theexternal medium, no free drug can be found in thecells even after a steady state has been achieved.Yet the presence of 10@ M methotrexate can giverise to appreciable intracellular concentrations offree drug in excess of that bound to folic reductasein the L5178Y cell culture line (15). Free intracellular methotrexate can also be found in theP-388 ascites tumor (strain R-26C) 4 hour afterinjection of 10 mg drug/kg (51) and in theMurphy-Sturm tumor 24 hours after the injectionof SO mg/kg.8 The cell culture lines of Sarcoma 180seem to differ markedly in their manner of druguptake from these other tumors.

DAYS AFTER INJECTION OF AMETHOPTERIN (003mg/kg)

CHART 4.—See text for conditions

3 W. C. Werkheiser, unpublished experiments.

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Cancer Research Vol. 23, September 196312.82

There is a large number of observations on theability of folinic acid (and, by extension, of tetrahydrofolic acid) to prevent methotrexate toxicity.In cell culture and in studies in vitro (1, 23, 54)and in animals (6, 7, 19, 39, 45), this reversal hasbeen shown to be competitive. Numerous investigators (see 12, 23, 25) have drawn the reasonable

conclusion that there must be some enzymic targetof the drug distal to the formation of tetrahydrofolic acid and that it is for this site that folinicacid is a competitor. Yet, no very prominent target has been found, although a number of weakantagonisms have been observed (27, 28, 40, 41).It is true that both Fischer (15) and Hakala (22)have observed inhibition of methotrexate uptakeby folinic acid in cell culture, and this could, inprinciple, provide the explanation for the otherreversals. However, the effect in both systems required much higher concentrations of folinic acidthan those needed to reverse the growth-inhibitoryaction of the drug, and in Hakala's experiments,at least, the effect was definitely not competitive.

Prevention of toxicity by dihydrofolic acid,which has also been reported (9), is somewhatdifficult to reconcile with present ideas, since thiscompound is presumably not in itself an active cofactor. It is to be hoped that this question will beresolved shortly.

So we are faced with a dilemma. The folinicacid reversal is surely significant, yet it seems unlikely that short-lived inhibitions caused by theantagonisms so far observed can be responsible forthe toxicity of drugs in animals, since this toxicityis so notably cumulative (37).

In an attempt to resolve this problem, I willpresent a counter hypothesis and cite such published data as can be marshaled in its favor atpresent. These data are not conclusive, but avariety of approaches designed to make a crucialtest are in progress.

Burchenal and Babcock (7) observed that folinicacid could not reverse methotrexate toxicity whengiven more than 4 hours after the drug. This canbe interpreted as indicating that the affected cellscan withstand a certain period of folic reductaseinhibition, but that if this period is too long, inreversible damage ensues. Now let us consider thecourse of events following administration of asingle dose of methotrexate. The drug enters thecell rather rapidly, perhaps in amounts in excessof that needed to abolish folic reductase activity.This results in a profound inhibition of thyminesynthesis, a lesser, but still very strong, inhibitionof purine synthesis, and a much more moderateinhibition of protein synthesis (43, 47, 53). Continuation of protein synthesis during a period of

obvious methotrexate toxicity has also been doduced from microscopic observation of increasedcytoplasmic volume (42) and enlarged epithelialtonofibrils (44). We can conclude that the presenceof methotrexate in the cell causes an abruptcessation of cell multiplication but permits amoderate synthesis of proteins, including folicreductase. That this enzyme can be synthesized ata significant rate is shown by the fact that theinhibitory level of methotrexate for resistant 8-180cells in culture is determined by the balance between rate of drug entry and rate of enzymesynthesis (21).

The free drug in the cell now has two pathsopen to it. Part of it will pass out of the cell at arelatively slow rate (15) and part will combinewith newly formed enzyme. After a variableperiod of time, which will be a function of drugdose, there will be no free drug, and henceforthnew enzyme will remain free. When enough freeenzyme is present, normal division processes willresume, provided that the period of their paralysis

did not result in irreversible damage. There will, ofcourse, be a particular dose of drug which will behigh enough to permit sufficient drug to remain inthe cell for sufficient time to inactivate all theenzyme that can be formed during the criticalperiod. Such cells will suffer irreversible damage.From the data presented in Charts 1—3,this dosecould be related to the dose which causes the folicreductase activity to be abolished 24 hours afterinjection and which is so closely correlated withtoxicity and tumor sensitivity (SO, 51).

In view of the stoichiometnic association between folic reductase both in t,itro (48) and in vivo(49) and of the observations shown in Chart 4,I assume that the only drug which can escapefrom a cell is that which is in excess of the folicreductase present. Further apparent decreases indrug content per cell are then due to dilution resuiting from increase in total cytoplasm or celldivision.

A slow rate of entry of methotrexate into atissue would then be reflected by (a) low peakintracellular concentrations following injection ofthe drug, (b) a short period of time before free folicreductase would emerge, and (c) refractoriness tothe drug. A fast rate of entry would have theopposite effect. Hence, the primary parameter fordefining the sensitivity of a tissue to methotrexatewould be the rate of entry of drug into that tissue,as has been proposed (15, 50). Tissues in whichresistance has been developed accompanied by increase in folic reductase would be a special casewhere the balance of the various factors discussedabove has been shifted and folic reductase level is

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WEmuiEIsER—Effects of Folic Acid Antagonists 1283

the controlling factor at drug leveLs to which theparent tissues are responsive.

Liver also appears to be a special case, forseveral reasons. The folinic acid stores in liver areenormously larger than those of other tissues, andfolic reductase is probably not critical to theeconomy of the hepatic cell. Liver is not a dividingtissue, and its demand for thymine—the metabolite whose formation is most sensitive to methotrexate inhibition—is probably minimal. The synthesis of folic reductase by the normal liver mustbe extremely slow, since no free enzyme could bedemonstrated for at least a week following a singleinjection of aminopterin, despite the absence offree aminoptenin in the cell (49). Finally, hepaticdamage is not associated with the toxic effects of

the antifolic acid drugs. It can be concluded thatstudies on the action of these drugs solely on livenare unlikely to provide an explanation for theirchemotherapeutic effects.

It is now appropriate to reconsider the possibleaction of folinic acid. Since the folinic acid contentof small intestine and bone marrow (32) and ofseveral rat2 and mouse tumors3 is relatively low,it is probable that this substance cannot be stonedin these tissues. Consequently, it might be expected that, following an injection of folinic acid,this material, having entered a cell, wouldgradually leave it in much the same way as doesexcess methotrexate. As long as the intracellularconcentration of folinic acid remains above somecritical level, the cell is completely protected frommethotrexate toxicity by the mere availability ofproducts of the inhibited reaction. At any particulan dose of methotrexate, there will then be a doseof folinic acid which is just sufficient to keep thefolinic acid level above the critical value until allthe free methotrexate has escaped from the cell.Folic reductase synthesis will then resume itsrole as cell protector. Although the mathematicsof this cannot be worked out without knowledgeof the kinetic equations for cellular loss of methotrexate and of folinic acid, intuition suggests that

a constant ratio between dose of methotnexategiven and dose of folinic acid necessary for protection might well prevail over a wide concentrationrange. This phenomenon, then, could explain thecompetitive prevention of methotrexate toxicityby folinic acid that has so often been observed.

If confirmation of this hypothesis can be obtamed, I believe that a reasonably complete doscniption of the mode of action of the 4-amino folicacid antagonists will be at hand. Certain remaining discrepancies (see 12, 25) largely concern differential metabolic effects within a particular

tissue. It is easily possible that mere reduction oftetrahydrofolic acid cofactor concentration dueto folic reductase inhibition can cause such effects

as a consequence of differences in Km valuesamong the various enzymes. Further work isneeded to clarify this matter.

In conclusion, I would like to discuss briefly theimplications for the future in cancer chemotherapyresearch which seem to me to reside in the recenthistory of methotrexate. Many excellent metabolicand enzymatic studies were performed in order toelucidate the biochemical mechanism of action ofthis drug. A further consequence of this work wasthe explanation of the mechanism of the drugresistance which was displayed by some strains oftumors after prolonged exposure to methotrexate.

However, I think it has become evident thattumors in general make use of the same metabolicmachinery in very much the same way as do anumber of normal animal tissues. Explanations ofthe selective antineoplastic activity which has beenobserved in one system or another for a surprisingnumber of compounds can hardly be drawn fromthis sort of biochemical evidence alone. This is notto minimize the importance of identifying theintracellular target(s) of a drug nor to neglect theguidance this information can provide, but to suggest that the biochemical studies with isotopes andenzymes, which by now have become almost traditional, be regularly supplemented with measurements of pharmacodynamic properties whichseem more likely to reflect whatever selectivity thedrug in question displays. Variation in rates of ingress and egress between sensitive and refractorytumors and in rates of enzyme synthesis, as suggested by the work presented here, are, I am sure,but a few of the possible avenues which can condition selectivity. Others, too, will be found as thereal mechanism of the selective action of presentlyknown chemotherapeutic agents is revealed. Inparticular, it seems probable. that, as with methotrexate, variation in penetrability may be a

powerful factor conditioning the effective action ofmany drugs, although the time scale will probablybe much shorter, since drug retention in the manner of methotrexate is not a frequent occurrence.

Furthermore, recognition of the significance ofcellular pharmacodynamics in relation to selectivity immediately suggests attempts to alter thedegree of selective action shown by known agentseither by pretreatments intended to change theproperties of the cells on by structural alterationsof the agents themselves. In this context, as well asin the context of the search for completely newantifolic agents, the di- and tetrahydrofolic acid

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Cancer Research Vol. 23, September 19631284

analogs (28, 29, 56), dideazatetrahydroaminopterin (11) and folic acid (10), and the open-chainpyrimidine structures resembling folic acid beingdeveloped by Baker (2) all have great potentialimportance.

ACKNOWLEDGMENTS

The author wishes to express his appreciation to Dr. C. A.Nicholand Dr. M. T. Hakala for many stimulating discussions,salutary criticisms, and cogent ideas, and to thank Mrs.Magda Horvath and Mrs. Barbara Domin for their excellenttechnical assistance.

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1963;23:1277-1285. Cancer Res   William C. Werkheiser  Effects of the Folic Acid AntagonistsThe Biochemical, Cellular, and Pharmacological Action and

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