The cellular pharmacology of methotrexate

Pharmac. Ther. Vol.28, pp. 77 to 102, 1985 Printed in Great Britain.All rightsreserved

0163-7258/85 $0.00+0.50 Copyright© 1985PergamonPress Lid

Specialist Subject Editor: I. D. GOLDMAN

THE

CELLULAR

PHARMACOLOGY

OF METHOTREXATE

I. DAVID GOLDMAN and LARRY H. MATHERLY Departments of Medicine and Pharmacology, Medical College of Virginia, Richmond, Virginia 23298, U.S.A.

1. INTRODUCTION Transport of antineoplastic agents into tumor cells is a critical element in drug action since these agents act at intracellular loci and the interactions between drugs and their targets require that the agent first penetrate the cell membrane. Despite the obvious pharmacologic importance of transport little is known about: (i) the mechanisms by which antineoplastic agents traverse cell membranes; (ii) how membrane transport systems determine drug cytotoxicity and selectivity; and (iii) the role that alterations in membrane transport play in drug resistance. Complicated studies designed to specifically address these questions are a variety of observations, described in this paper, that indicate unique elements in the membrane transport of anticancer agents which have not been observed for physiological substrates. In addition, there are other complexities involved in studies designed to elucidate transport of antineoplastic agents that arise from the multifarious interactions between these drugs and intracellular constituents, and their metabolic conversions within host tissues and tumor cells. Nowhere in the pharmacology literature for antineoplastic agents is there more information relating to membrane transport processes than for the 4-aminoantifolates, in particular, methotrexate. Not only is there a comprehensive characterization of the transport of these drugs but there is an evolving understanding of the role of membrane transport as a determinant of the cytotoxicity and selectivity of these agents. Despite what is known, however, the bases for many of the properties of the membrane transport of 4-aminoantifolates remain poorly understood and precise mathematical descriptions of many of the interactions between these drugs and tumor and host cells are not available. In many respects, the history of the development of the understanding of the cellular pharmacology of methotrexate, a prototypical 4-aminoantifolate, is replete with interpretations of data based upon intuition that later have been modified as sophisticated analytical technology and computer modeling analyses have been applied. This paper will first review briefly some controversial issues regarding the membrane transport of methotrexate and then focus on the cellular pharmacology of methotrexate with an emphasis on the correlation between membrane transport properties and the pharmacologic action of the drug within the cell. The section will be developed within the context of a comprehensive review of the current understanding of the mechanism of action of 4-aminoantifolates. Polyglutamylation of methotrexate will be evaluated as a determinant of cytotoxicity, selectivity and drug resistance and the role that membrane transport plays in the regulation of polyglutamylation will be analyzed. Membrane transport as a factor in high-dose methotrexate regimens with 5-formyltetrahydrofolate rescue will be considered and the pharmacologic rationale for high-dose methotrexate regimens will be reviewed. Finally, this section will describe the interesting interactions between methotrexate and 7-hydroxymethotrexate that may occur at the level of the membrane transport carrier as the catabolite accumulates in vivo. 77

78

I.D. GOLDMANand L. H. MATHERLY 2. THE NATURE OF THE INTERACTIONS BETWEEN METHOTREXATE AND MAMMALIAN CELLS THAT ARE CONTROLLED BY THE MEMBRANE TRANSPORT SYSTEM I N V I T R O 2.1. OVERVmW

Membrane transport processes control the rate at which drug penetrates the cell membrane at a particular extracellular drug concentration and the rate at which the drug will exit the cell at a given intracellular drug concentration. It is the net effect of these processes that determines the free intracellular drug level achieved at any given extracellular drug concentration. Transport of methotrexate in most mammalian tumor cell systems sensitive to this agent is mediated by an energy-dependent mechanism. The elements of the transport process that control entry of drug into cells have the characteristics of a transport carrier. Influx is saturable, demonstrates competitive inhibition by structural analogs, is pH- and temperature-dependent, and requires the interaction between the drug and specific receptor sites, such as sulfhydryl groups which are selectively reactive toward reagents such as N-ethylmaleimide and organic mercurials (Goldman et al., 1968). This carrier system has a high-affinity for reduced folate cofactors, its 'usual' substrate, but a very low affinity for folic acid itself (Goldman et al., 1968). The mechanism by which folic acid is transported into cells is not clear. Specific evidence for a carrier mechanism for methotrexate, i.e. a membrane receptor that itself undergoes some physical transposition as it facilitates drug transport, is based upon the demonstration of exchange diffusion phenomena (Goldman, 1971a,b): i.e. methotrexate influx into cells loaded with reduced folates, which utilize the same carrier mechanism, is more rapid than influx into cells not containing these compounds, a phenomenon called trans-stimulation (Goldman, 1971a,b). Of particular interest is the stereospecificity of the transport carrier. While the measured influx Km for the radiolabeled natural and unnatural isomers of 5-methyltetrahydrofolate are comparable (Chello et al., 1982; White et al., 1978; White and Goldman, 1979) the unnatural isomer of 5-formyltetrahydrofolate is a much weaker inhibitor of methotrexate influx than the natural isomer (Sirotnak et al., 1979). Transport of methotrexate has been suggested to be associated with changes in cell cyclic AMP levels (Henderson et al., 1978). However, it was subsequently shown that changes in methotrexate transport that occur with changes in cell cyclic AMP are coincidental since these effects could be temporally dissociated. Hence, increased or decreased cyclic AMP levels could be induced in cells without any changes in methotrexate transport and marked changes in net methotrexate transport could be induced without any change in the level of cyclic AMP (White et al., 1980). 2.2. ANALYSISOF THE RELATIONSHIPBETWEEN INFLUX AND EFFLUX OF METHOTREXATE The mechanism of efflux of methotrexate and its relationship to the influx process is not clearly understood. Under some experimental conditions the exit of methotrexate from cells appears to be mediated at least in part by the same mechanism as entry. For instance, the enhanced or reduced influx Vmaxfor methotrexate induced by positively or negatively charged liposomes, respectively, produces symmetrical changes in the efttux rate constant for methotrexate so that there is no change in net transport (Fry et al., 1979; Fry and Goldman, 1982). This symmetrical change in the bidirectional fluxes of methotrexate was thought to be due to changes in the rate of translocation of the carrier within the cell membrane. Since influx is mediated almost exclusively by the high-affinity tetrahydrofolate-cofactor carrier, this supports the concept that this carrier can mediate the bidirectional fluxes of methotrexate although it does not exclude additional transport routes distinct from this process. However, trans-stimulation of radiolabeled methotrexate influx by intracellular nonlabeled methotrexate is not demonstrable under physiological conditions, although the

Cellular pharmacologyof methotrexate

79

level of methotrexate that accumulates in cells under physiological conditions may be too low to permit the demonstration of this phenomenon (Goldman, 1971a,b). Moreover, efflux of methotrexate cannot be stimulated under usual conditions by the presence of extracellular reduced folates which when present in the intracellular compartment, as indicated above, trans-stimulate methotrexate influx. This has been attributed to the interaction between inorganic ions present at high concentrations in the extracellular compartment under physiological conditions and the tetrahydrofolate-cofactor carrier that results in maximal rates o f carrier exchange in the absence of extracellular folates since in anion-free environments very low levels of either methotrexate or 5-formyltetrahydrofolate trans-stimulate methotrexate efflux (Henderson and Zevely, 1981). Influx of methotrexate or 5-formyltetrahydrofolate can be stimulated in a high potassium environment in cells loaded with methotrexate, indicating that under this condition, at least, the efflux of methotrexate from cells is linked to drug entry into cells by the high affinity tetrahydrofolate-cofactor carrier system (Fry et al., 1980a). It should be pointed out that the use of highly unphysiological buffers that contain high levels of potassium as osmotically active uncharged solutes to study the functional characteristics of cellular transport systems is frought with problems related to the changes in cell size (Fry et al., 1980a), membrane potential (Fry et al., 1980a), energetics of transport (Fry et al., 1980a) and ATP levels (Henderson and Zevely, 1983). All of these changes could in themselves profoundly alter the transport of a charged molecule. Additional support for the concept of distinct entry and efttux routes is the observation that a variety of changes in methotrexate influx can occur without any change in efflux at all. For instance, changes in influx properties contributing to drug resistance are not accompanied by changes in the parameters for methotrexate exit (Hill et al., 1979; Sirotnak et al., 1981a). In L5178Y tumor cells resistant to methotrexate because of a marked reduction in drug influx, efflux is inhibited by metabolic poisons to an extent comparable to the drug sensitive parent line (see Section 2.3), suggesting that drug exit from these cells may be mediated by a route distinct from influx (Hill et al., 1979). Likewise, the disparity in the changes in influx and efflux of methotrexate that occur with variation in the cellular energy state has also been implicated as evidence for separate influx and efflux routes although it is of interest that the decrease in the effiux rate constant and the influx V~x that accompanies energy deprivation are comparable (Dembo et al., 1984). 2.3. ANALYSISOF THE EFFECTSOF ANIONS ON METBOTREXATETRANSPORT Another aspect of the transport of methotrexate and reduced cofactors that requires further clarification relates to the effects of anionic compounds on this process. Shortly after the basic properties of the tetrahydrofolate-cofactor carrier were described, it became clear that this process was profoundly influenced by the ionic composition of the extracellular milieu. While many uphill transport processes have been shown to be dependent upon the transmembrane electrochemical-potential difference for sodium, methotrexate influx was shown to be sodium independent and net drug transport uninfluenced by ouabain (Goldman et al., 1971b). However, unlike its lack of dependence on cations, influx and net transport of methotrexate could be markedly inhibited by the addition of a variety of structurally unrelated organic and inorganic anions to the extracellular medium (Bobzien and Goldman, 1972; Fry et aL, 1980b; Goldman, 1971b, 1977; Jennette and Goldman, 1975). Indeed, even chloride was considered to be inhibitory because transport of methotrexate was stimulated when both sodium and chloride were replaced by either urea or sucrose, manipulations that result in either cell sweUing or shrinkage, respectively (Goldman, 1971b). Recently, there has been renewed interest in the effects of anions on methotrexate transport (Henderson and Zevely, 1980, 1981, 1982a,b). While it is clear that a variety of anions and anionic drugs nonspecifically perturb the transport of methotrexate when added to the extracellular compartment it is unclear as to how the distribution of intracellular and extracellular organic and/or inorganic anions influence transport of methotrexate- and tetrahydrofolate-cofactors under physiological

80

I.D. GOLDMANand L. H. MATHERLY

conditions. The large concentration of organic anions that accumulate within cells represents a potential chemical gradient that could drive methotrexate uphill into cells if these anions could exit the cell, even at a very low rate, by a mechanism shared with the antifolate. This laboratory has suggested how such an interaction between organic phosphates, in general, and adenine nucleotides, in particular, could result in uphill transport into the cell via the tetrahydrofolate-cofactor carrier even under conditions of energy depletion (Goldman, 1969), since the ability of adenine nucleotides to interact with this carrier, at the outer membrane interface, at least, is unrelated to their energy charge (Goldman, 1971b, 1977). This could account for uphill transport into cells in the presence of metabolic poisons which while depleting the high energy pool would not deplete the anion gradient (see Section 2.4 below). Phosphate has also been proposed as an anion that may influence the energetics of methotrexate transport (Henderson and Zevely, 1982a). However, sulfate also interacts with the carrier in the same way as phosphate (Henderson and Zevely, 1982b). Furthermore, there is presently insufficient reliable information on the extent of the phosphate gradient and how changes in this gradient perturb methotrexate transport to permit any conclusions regarding a specific role for these or other inorganic anions as determinants of the energetics of the tetrahydrofolate-cofactor carrier. As indicated above, inherent in experimental designs to evaluate the effects of anions on methotrexate transport are conditions that markedly perturb the cell milieu resulting in changes in cell volume, membrane potential, and energetics. Hence, conclusions regarding the role of changes in anion levels, distinct from other accompanying changes in the physical properties of cells, are difficult to assess. Indeed, much of the data on the effects of anions on methotrexate transport represents phenomena that are fundamentally unexplained. One critical question that remains unanswered is the extent to which anions present in cells influence: (i) the exit of methotrexate from cells; (ii) the unidirectional flux of methotrexate into the cell; and/or (iii) the final gradient for methotrexate that is achieved. To evaluate this will require analysis of the effects of changes in these intracellular species in a cell that is otherwise minimally perturbed. Studies using membrane vesicle systems should be particularly useful in understanding the potential influence of anions on the transport of methotrexate by permitting the assessment of the effects of transvesicular anion gradients in a system uncomplicated by the cellular energy apparatus and drug binding sites (Yang et al., 1979, 1982, 1984). Initial studies, at least, indicate that phosphate and sulfate (but not chloride) trans-stimulate methotrexate or 5-formyltetrahydrofolate fluxes into or out of membrane vesicles but this effect is markedly diminished by the presence of sodium chloride raising the question of the physiological relevance of these interactions (Yang et al., 1984). It will be of interest to learn of the effects of organic anions, in particular, the adenine nucleotides, in this system. 2.4. THE RELATIONSHIP BETWEEN FREE INTRACELLULAR AND EXTRACELLULAR METHOTREXATE; FACTORS THAT INFLUENCE THE FREE INTRACELLULAR DRUG LEVEL; THE EFFECTS OF ENERGY INHIBITORS ON METHOTREXATE TRANSPORT Unlike the entry mechanism which is saturable, the efflux process is first order at most intracellular methotrexate levels that can be achieved (Dembo and Sirotnak, 1976; Fry et al., 1979; Goldman et al., 1968). This assymmetry in the bidirectional fluxes of methotrexate should produce a transmembrane gradient for methotrexate with intracellular drug at a higher concentration that extracellular drug, if undirectional flux measurements made without transport substrate in the trans-compartment are relevant to steady-state conditions (Goldman, 1982). However, since methotrexate is a bivalent anion and mammalian cells carry a negative charge, even when there is active transport into cells the intracellular drug concentration may be less than the extracellular level (Goldman et aL, 1968). Nonetheless, the ratio of the intracellular to extracellular drug concentration may exceed the value predicted from the Nernst equation on the basis of the membrane potential and therefore represent an energy-requiring electrochemical-potential difference for methotrexate across the cell membrane (Goldman, 1982; Goldman et al., 1968). In fact,

Cellular pharmacologyof methotrexate

81

uphill transport of methotrexate can often be confirmed only after the membrane potential is taken into consideration. As will be developed further in this paper, it is the free intracellular methotrexate concentration achieved and sustained in cells that is a key element in drug action and it is this same intracellular drug component that is quite susceptible to a variety of cellular perturbations. For instance, virtually any energy poison blocks methotrexate efflux to result in the net augmentation of methotrexate uptake into mammalian cells (Fry et al., 1980b; Goldman, 1969, 1971b). This has been attributed to inhibition of an energydependent efflux mechanism that results in increased net methotrexate transport into the cell that is sustained by some energetic process or gradient which is relatively insensitive to the level of cellular ATP (such as an anion gradient--see above and Goldman, 1971b, 1977). Interestingly, other pharmacologic agents (see Section 6) used clinically also enhance the free intracellular drug level by blocking methotrexate efflux (Fry et al., 1982b; Fyfe and Goldman, 1973; Sirotnak et al., 1981b; Yalowich et al., 1982). Conversely, energy substrates such as glucose reduce the free intracellular methotrexate level--presumably due to the augmentation of an active exit mechanism (Dembo et al., 1984; Fry et al., 1980a,b; Fyfe and Goldman, 1973; Goldman, 1969). According to these considerations, many factors can affect the free drug level within the cell, i.e. the membrane potential, the status of the cellular energy state and the extracellular (and possibly the intracellular) anion composition. Of course, the transmembrane gradient for methotrexate is also determined by the extracellular methotrexate concentration. Since the influx process saturates at a lower concentration than the efflux mechanism, the free level of drug within the cell is also a saturable function of the extracellular drug concentration (Goldman, 1982; Goldman et al., 1968). Hence, as extracellular methotrexate is increased the free drug level within the cell achieved by the cartier mechanism approaches a maximum. For instance, in the L1210 leukemia cells with an influx K,, of 4pM, the maximum free intraceilular drug level that can be achieved by the carrier mechanism is about 6 #M (Goldman et al., 1968). Once the cartier system is saturated, the only way additional free drug can accumulate within the cell is by extra cartier 'leaks' such as equilibration of drug via passive diffusion and/or by transport via some other low affinity process. In practice though, even when the extracellular methotrexate level is increased by orders of magnitude above the influx K~ the free intracellular drug level remains very low (Goldman et al., 1976). This is probably due to the electrical and energetic factors (described above) that restrict drug accumulation and play the major role in controlling the level of free intracellular methotrexate when the uphill transport system is saturated. This relationship between the intracellular and extracellular drug levels becomes even more complex in the in vivo situation where, after a parental pulse of methotrexate, there are changes in the extracellular methotrexate concentration that span several orders of magnitude. The next section considers the interaction between methotrexate and cells in the in vivo setting. 3. THE INTERACTION BETWEEN METHOTREXATE AND MAMMALIAN CELLS I N V I V O Figure 1 is a simulation of the relationship between the free intracellular methotrexate concentration and the extracellular drug level following a large pulse of the drug (200 mg/kg) in vivo. With the initial high extracellular drug levels achieved following drug administration, the free intracellular methotrexate concentration within these cells is far less than the extracellular level. This is attributed to saturation of the cartier mechanism and the constraints on the free intracellular drug concentration imposed by the energetic and electrical factors described above. As the extracellular drug concentration falls in the range of the carrier influx Kin, however, the free intracellular concentration approaches and then exceeds the extracellular drug level, a relationship that is then sustained as the residual drug is eliminated. It should be noted in this simulation that the slope of the decline of J P T 2~, I--~"

82

I . D . GOLDMANand L. H. MATHERLY

i

i0-~ i.v.

10-4

Dose • 2 0 0

rn(j k9 -~

%%

iO-e

iO-e T i

L 2

L 3

i 4

Days

FIG. 1. Computer simulation of plasma and intraccllular levels of methotrexate following injection of a single high dose (200 mg/kg). The solid line represents extracellular drug while the broken line represents the intraeellular level of methotrexate (from White and Goldman, 1981).

free intracellular methotrexate parallels the slope of the decline of the plasma drug concentration at concentrations below the influx Kin. This is due to the fact that the rate of drug clearance from the blood at these concentrations is orders of magnitude slower than the rates of transport of methotrexate across the cell membrane. Under these conditions, free intracellular methotrexate is continuously at a steady state with extracellular drug at a level determined by the extracellular methotrexate concentration and the bidirectional fluxes of the drug. This steady state is exactly the same as the intracellular drug level that would be achieved if any particular extracellular concentration of drug was held constant for any longer period of time. From these considerations it is clear that the rate of decline of the free intracellular methotrexate level in most tumor cell systems in vivo over a broad extracellular range of concentrations is controlled not by the membrane transport system but rather by the plasma pharmacokinetics for drug clearance. Hence, this in vivo system is 'flow limited' and not 'transport limited'. Figure 2 is a simulation which analyzes the intracellular methotrexate levels in four different tumor cell lines with different transport properties for methotrexate (Sirotnak and Donsbach, 1976) at a dose of 12 mg/kg. Here, the rate of decline of the drug in each of the tumor lines is exactly the same and parallels the decline in the plasma level (not shown). What is different amongst the tumor lines is the absolute level of free drug in these cells at any time, a difference that arises from the kinetic constants for influx and efflux that were used in this simulation. While it is possible that at very low extracellular methotrexate concentrations transport could become rate limiting to some intracellular event such as binding to dihydrofolate reductase or folylpolyglutamate synthetase (see Section 5 below), the interaction between methotrexate and intracellular sites over a broad range of drug concentrations achieved in vivo is not limited by the unidirectional flux of drug into the cells. Rather, it is determined by the steady state free drug level in the cell which is determined by the asymmetrical transmembrane drug fluxes. 4. HOW THE MEMBRANE TRANSPORT SYSTEM REGULATES THE INTERACTION BETWEEN METHOTREXATE AND DIHYDROFOLATE REDUCTASE WITHIN CELLS: THE MONOGLUTAMATE OF METHOTREXATE AS A RAPIDLY REVERSIBLE ENZYME INHIBITOR The metabolic significance of dihydrofolate reductase derives from the central role of reduced folate cofactors in the biosynthesis of purines and thymidylate. The formation of

Cellular pharmacology of methotrexate

83

5-

u~ o.~ I

I

I

I

I

I

,4

8

12

16

20

24

I"tO,uf'$

FiG. 2. Computer simulation of the rate of decline o f free methotrexate in 5 tumor lines after a simulated dose o f 12mg/kg (from Fry et al., 1983).

thymidylate from deoxyuridylate, catalyzed by thymidylate synthase, is a step unique in biology since it involves both a one-carbon transfer and the oxidation of the reduced folate cofactor (5,10-methylenetetrahydrofolate), resulting in the generation of dihydrofolate. Normally, the dihydrofolate so formed is reduced by dihydrofolate reductase, in the presence of NADPH, to tetrahydrofolate which equilibrates with the active reduced folate cofactor pool. Because of the critical need for thymidylate in DNA synthesis, mammalian cells generally contain 20 to 30 times more dihydrofolate reductase than thymidylate synthase activity so as to ensure normal tetrahydrofolate cofactor stores for anabolic processes even when the biosynthesis of thymidylate is maximal (Jackson and Harrap, 1973). As a result of this huge excess of dihydrofolate reductase, under normal conditions cellular dihydrofolate concentrations are negligible (Jackson et al., 1977; Moran et al., 1976; White and Goldman, 1976). Because of the extremely tight and rapid binding of methotrexate to dihydrofolate reductase, when cells are exposed to the drug, uptake is unidirectional and negligible free methotrexate accumulates until near molar equivalence with the target enzyme is achieved (Goldman et al., 1968). For most tumors that are sensitive to methotrexate, cell drug levels are in excess of the enzyme binding capacity within minutes or less after exposure to typical concentrations of this agent (Goldman, 1973). The association of the antifolate with dihydrofolate reductase results in the build-up of cellular dihydrofolate behind the block in dihydrofolate reductase activity as tetrahydrofolates are converted to 5,10methylenetetrahydrofolate and subsequently to dihydrofolate in the continued synthesis of thymidylate (Jackson and Harrap, 1973; Jackson et al., 1977; White, 1979; White and Goldman, 1976). Dihydrofolate then interacts with the remaining unoccupied enzyme binding sites to an extent proportional to its increased concentration. Because of the excess cellular dihydrofolate reductase activity, as discussed above, adequate rates of tetrahydrofolate biosynthesis are maintained until approximately 95% of the enzyme is associated with methotrexate (Jackson and Harrap, 1973; Jackson et al., 1977; White, 1979; White and Goldman, 1976). Beyond this point, increasing association of methotrexate with dihydrofolate reductase results in suppression of enzyme activity. However, because of the high cellular dihydrofolate level achieved following exposure to methotrexate, substantial free drug concentrations orders of magnitude above the Ki for dihydrofolate reductase are required to completely abolish enzyme activity (Goldman, 1974; Jackson and Harrap, 1973; Jackson et al., 1977; White and Goldman, 1976; White et al., 1975). Only then does tetrahydrofolate synthesis fall and ultimately cease with a consequent marked suppression of thymidylate and purine biosynthesis.

84

I . D . GOLDMAN and L. H. MATHERLY

Hence, it is the level of the free component of the total cellular methotrexate, determined by the extracellular drug concentration and the properties of the transport system, which is critical to achieving sustained suppression of dihydrofolate reductase activity and tetrahydrofolate-dependent processes in the presence of high concentrations of competing dihydrofolate. The concentrations of intracellular methotrexate that are required to produce this effect will be determined by: (i) the cellular dihydrofolate level which, in turn, is determined by the extent to which cellular folates are converted to 5,10-methylenetetrahydrofolate (White, 1983); (ii) those factors that regulate the rate of thymidylate synthesis from deoxyuridylate (Bowen et al., 1978; Jackson and Harrap, 1973); and (iii) the relative binding affinities of the dihydrofolate reductase for methotrexate and dihydrofolate polyglutamyl derivatives. This competitive interaction between dihydrofolate and methotrexate also explains the rapid reversibility of the pharmacologic activity of the monoglutamate of methotrexate when extracellular methotrexate is eliminated (Goldman, 1974; White, 1979; White and Goldman, 1976; White et al., 1975). When cells have been exposed to sufficient methotrexate to abolish tetrahydrofolate synthesis, dihydrofolate levels are maximal and represent the extent to which intracellular tetrahydrofolate cofactors are converted to 5,10-methylenetetrahydrofolate and, subsequently, dihydrofolate in the synthesis of thymidylate. Upon exposure to drug-free medium in vitro, free intracellular methotrexate declines rapidly, a phenomenon controlled by the properties of the efflux process under these conditions. Because it is present in a polyglutamyl form, dihydrofotate remains within the cell so that the ratio of dihydrofolate to methotrexate initially increases markedly. This results in competition between these compounds for enzyme binding with the net displacement of methotrexate from the critical few dihydrofolate reductase binding sites that are sufficient to maintain normal levels of tetrahydrofolate synthesis. When tetrahydrofolate synthesis resumes, thymidylate synthesis rapidly returns to normal rates and dihydrofolate is consumed so quickly that further net displacement of methotrexate ceases and residual drug remains tightly bound to the major portion of enzyme. Hence, while in excess of 95~, of the dihydrofolate reductase remains associated with the drug when free intracellular MTX becomes negligible, the drug is pharmacologically inert. In vitro, the rate of efflux of free methotrexate from the cell determines the time required for enzyme activity to resume and this process is controlled entirely by the membrane transport system. In vivo, the rate of decline of free intracellular methotrexate is determined by the plasma pharmacokinetics of the drug and the absolute level of methotrexate in the cell at any given extracellular concentration will be controlled by the transport system (see Section 3). The level of intracellular methotrexate could be increased by agents which either inhibit the efflux of drug or stimulate its entry, thereby altering the steady state drug level. Exposure of cells to a negatively charged liposome slows efflux of methotrexate in vitro (Fry and Goldman, 1982). However, in vivo, negatively charged liposomes should not change the steady-state drug levels because they alter the bidirectional drug fluxes symmetrically. In contrast, agents such as vinca alkaloids (Fyfe and Goldman, 1973), epipodophyllotoxins (Yalowich et al., 1982) or probenecid (Sirotnak et al., 1981b) which elevate the level of intracellular free drug by asymmetrically inhibiting drug exit alone (see Section 6) would likely produce a sustained increase in the free intracellular drug level in vivo to the extent to which this effect is achieved at obtainable plasma drug levels. Dihydrofolate, like most intracellular folate cofactors, is present in cells as polyglutamyl derivatives (Moran et al., 1976). These polyglutamyl folate cofactors are, in general, the preferred substrates for folate-dependent reactions and may play an important role in regulating the availability of reduced folate cofactors for the various folate-dependent anabolic pathways (McGuire and Bertino, 1981). Moreover, because they do not readily exit the cell, these derivatives accumulate to appreciable intracellular concentrations assuring their availability for biosynthetic processes. Recently, it has become increasingly evident that methotrexate, as well as certain other 4-aminoantifolates, is also converted to polyglutamyl derivatives in a reaction catalyzed by folylpolyglutamate synthetase. The significance of this metabolism is considered in detail in the next section.

Cellular pharmacologyof methotrexate

85

5. THE POLYGLUTAMYLATION OF METHOTREXATE AND THE CONTROL OF THIS REACTION BY THE MEMBRANE TRANSPORT SYSTEM: POLYGLUTAMYL DERIVATIVES OF METHOTREXATE AS SLOWLY REVERSIBLE INHIBITORS OF DIHYDROFOLATE REDUCTASE WITHIN CELLS The conversion of methotrexate to its polyglutamyl derivatives, described in a number of normal (Gewirtz et al., 1979; Rosenblatt et al., 1978) and tumor cell lines (Fry et al., 1982a; Galivan, 1980; Jolivet et al., 1982; Whitehead, 1977), is a metabolic event of profound pharmacologic importance. The significance of this metabolism is two-fold: (i) these derivatives have an equal or possibly greater affinity for dihydrofolate reductase than the unaltered drug (Jolivet and Chabner, 1983; Schilsky et al., 1983); and (ii) once formed intracellularly, these derivates exit the cell much more slowly than the unmetabolized drug (Fry et al., 1982a; Gewirtz et al., 1979; Galivan, 1980; Jolivet et al., 1982; Galivan and Nimec, 1983, Rosenblatt et al., 1978). Since the reversibility of the pharmacologic effects of methotrexate derive from the rapid efflux of the monoglutamyl methotrexate from ceils (see Section 4), the polyglutamylation of this agent essentially converts methotrexate from a readily reversible to a much less reversible inhibitor of dihydrofolate reductase activity within cells. The extent of retention of the polyglutamyl derivatives of methotrexate depends on the cell type and glutamyl chain length. In the Ehrlich ascites tumor, for instance, all the polyglutamyl derivatives are retained completely for hours following resuspension into drug-free medium (Fry et al., 1982a). On the other hand, in hepatoma cells (Galivan and Nimec, 1983) and the MCF-7 breast carcinoma cell line (Jolivet et al., 1982), the lower polyglutamyl forms exit the cell, while the long chain derivatives are retained for very long intervals. In some instances, following resuspension of cells in a drug-free medium, the short chain derivatives are further metabolized to long chain polyglutamates (Galivan and Nimec, 1983). In any event, the critical issue is not whether the polyglutamyl derivatives do or do not exit cells in an absolute sense. Rather, polyglutamylation is an important determinant of drug action if eiflux of the derivatives formed is retarded sufficiently to result in a rate of decline of the net cellular antifolate level which is appreciably slower than the rate of decline of the extracellular methotrexate level. The pharmacologic activity of the polyglutamyl derivatives of methotrexate has been clearly established. Hence, continuous exposure to glycine, adenosine and thymidine along with methotrexate protects cells from methotrexate cytoxicity, even after high levels of intracellular methotrexate polyglutamyl derivatives have formed, by circumventing the anabolic requirement for tetrahydrofolate-cofactors (Fabre et al., 1984d; Jolivet et al., 1982; Rosenblatt et al., 1982). If, however, cells that have accumulated methotrexate polyglutamates in the presence of these protective agents are subsequently suspended into a methotrexate-free medium in the absence of glycine, adenosine and thymidine, DNA synthesis and cell growth ceases. Hence, it is the persistence of the polyglutamyl forms of the drug in the absence of the monoglutamate that is responsible for the prolonged pharmacologic activity under these conditions. Furthermore, enhanced activity of methotrexate correlates with enhanced accumulation of polyglutamyl derivatives in tumor cells (Fabre et al., 1984d). As described above, the competitive nature of the interaction between methotrexate and dihydrofolate at the level of dihydrofolate reductase suggested that the primary pharmacologic role of membrane transport in drug action was as a determinant of the level of free monoglutamyl antifolate achieved for direct suppression of dihydrofolate reductase activity. However, with the recognition that methotrexate and related antifolates were metabolized intracellularly to active polyglutamyl derivatives, the role of membrane transport within the context of the overall cellular pharmacology of this agent had to be reassessed. For the Ehrlich ascites tumor, in which this has been studied in detail, polyglutamylation of methotrexate is slow relative to membrane transport and appreciable free intracellular methotrexate levels are necessary for the formation of these derivatives (Fry et al., 1982a). Hence. the formation of polyglutamyl derivatives is clearly limited by the properties of the membrane transport system since the reaction is driven by the

86

I.D. GOLDMANand L. H. MATHERLY 2.5

2.0

~ _.=~ ~E

1.5 ¸

~,

~.o

u~×

0.5

~ i

0

I 24

Km=2

I

~M

I

I

48

72

~

I 96

Hours

FIG. 3. Computersimulationdepicting the role of influx K,, in the accumulationand decline of methotrexat¢polyglutamatesin tumor cells in oivo followinga dose of methotrexateat 12mg/kg. The influx of K,, was varied from 2 to 10/ZM(from Fry et al., 1983). monoglutamyl substrate level within the cell, a component regulated by the transport system. Of particular interest is the relationship between the formation and persistence of methotrexate polyglutamyl derivatives within tumor cells in vivo and drug transport. After a pulse of methotrexate the initial high free cellular drug levels achieved results in an early burst of polyglutamylation, a process that subsequently slows and ultimately ceases as the plasma and intracellular levels of monoglutamyl methotrexate fall. While the short chain derivatives may be converted to longer chain polyglutamates during this later interval of plasma drug decline, the more polyglutamyl derivatives formed during the initial period of active synthesis, the longer will the total antifolate level in excess of the dihydrofolate reductase binding capacity be sustained in cells as the extracellular methotrexate concentration falls. This relationship is illustrated in Fig. 3, a simulation based upon pharmacokinetic parameters obtained in vivo and transport and metabolic parameters obtained from in vitro studies (Fry et aL, 1983). Transport is considered within the context of the influx K=, a measure of the affinity of the carrier for methotrexate which is an important determinant of the free intracellular drug level achieved. It can be seen that by decreasing the influx Kin, i.e. increasing the affinity of the carrier for methotrexate, higher levels of methotrexate polyglutamyl derivatives are formed and persist in the cells over longer intervals. It should be pointed out that in this model the duration over which the polyglutamates are retained intracellularly is determined solely by differences in the peak level of drug accumulated since the rate constant for the decline of these compounds is held constant. This rate constant represents the rate of etflux of the polyglutamates and/or the rates of their hydrolysis by endogenous conjugase with subsequent efflux of the monoglutamate. This parameter may, of course, vary from one cell type to another and might vary amongst different antifolate polyglutamyl derivatives. Nonetheless, it is clear that the membrane transport of methotrexate profoundly influences the formation and the duration of persistence of the active antifolylpolyglutamate derivatives within cells. On the basis of these considerations, the conversion of methotrexate to its polyglutamyl congeners is of such importance to the pharmacologic activity of the drug that underivatized methotrexate might be viewed as a 'prodrug' of the active form(s) of the antifolate. 6. MODULATING THE FREE INTRACELLULAR METHOTREXATE LEVEL AND THE RATE OF POLYGLUTAMYLATION WITH OTHER PHARMACOLOGIC AGENTS Serendipitously, a number of agents were found to block the efflux of methotrexate from tumor cells, thereby increasing the level of free intracellular drug achieved. This elevated

Cellular pharmacology of methotrexate

87

free methotrexate concentration results in enhanced inhibition of cellular dihydrofolate reductase activity. Such an effect has been described for vinca alkaloids (Fyfe and Goldman, 1973) and the epipodophyllotoxins, VM-26 and VP-16 (Yalowich et al., 1982), and low concentrations of probenecid (Sirotnak et al., 1981b). High concentrations of probenecid inhibit methotrexate influx and depress the steady state drug level (Sirotnak et al., 1981b). The mechanism by which low concentrations of probenecid inhibit the etflux of methotrexate from cells has not been established. The vinca alkaloids and the epipodophyllotoxins appear to inhibit the efflux process by their effects on cellular energy metabolism similar to the effects demonstrated with metabolic poisons such as azide or dinitrophenol (Fry et al., 1980b; Goldman, 1969, 1971b). The effects of these compounds can be reversed, at least in part, by energy substrates such as glucose which presumably increase the glycolytic production of ATP (Fry et al., 1980b; Goldman, 1969, 1982). The polyglutamylation of folyl and antifolyl substrates is an ATP-requiring reaction which is abolished by energy inhibitors such as sodium azide. The vinca alkaloids and epipodophyllotoxins appear to affect energy metabolism in a selective fashion which results in inhibition of the energy-dependent efttux of methotrexate without an inhibitory effect on polyglutamylation since intracellular polyglutamylation of methotrexate is enhanced by these agents (Fry et al., 1982b; Yalowich et al., 1982), presumably due to the increased levels of monoglutamyl substrate that accumulate in their presence. The pharmacologic importance of these drug interactions is unclear and the extent to which they can be extrapolated to clinical regimens is uncertain. First, the doses of the drugs required to produce these effects in vitro are relatively high, in particular for the vinca alkaloids, although plasma levels achieved for these drugs are sufficient to perturb transport in at least some human tumor lines (Bender, 1975). In addition, many of these agents are largely protein-bound in the plasma (Gewirtz and Holt, 1984) resulting in still lower free drug levels in vivo. Nonetheless, even in the presence of physiological levels of albumin these interactions occur to some extent/n vitro (Gewirtz and Holt, 1984). Based upon this biochemical interaction between these agents, studies have explored possible synergism between epipodophyllotoxins or vinca alkaloids and methotrexate. Synergy can be demonstrated which depends upon the interval between the administration of the drugs. For vinca alkaloids or epipodophyllotoxins, an optimal interval of administration following methotrexate is in the range of 16-32 hr (Chello et al., 1979; Wampler et al., 1983). Interestingly, for the epipodophyllotoxin-methotrexate interaction, as the interval between administration of the drugs is changed, the optimum dose of each drug is changed as well (Wampler et al., 1983). It is unclear as to whether this synergistic interaction is related to effects on methotrexate transport and/or polyglutamylation or whether it is related to other factors. For instance, synergy could also derive from a perturbation in the cell cycle induced by methotrexate with the release of cells from the antifolate block at a time optimal for the action of vinca alkaloids or epipodophyllotoxins. Compounds which selectively block influx of methotrexate would, of course, reduce the net level of free intracellular drug achieved following drug administration and, consequently, also retard the formation of polyglutamyl derivatives. As described in detail in the following sections, such an effect on methotrexate influx has been reported for the 7-hydroxy catabolite and the natural tetrahydrofolate cofactors. 7. INTERACTIONS BETWEEN 7-HYDROXYMETHOTREXATE AND METHOTREXATE AT THE LEVEL OF THEIR C O M M O N M E M B R A N E TRANSPORT C A R R I E R Another route of metabolism of methotrexate involves its hydroxylation at the 7-position of the pteridine ring by liver aldehyde oxidase to form 7-hydroxymethotrexate. This catabolite has been detected in humans following the administration of moderate to high doses of methotrexate (Breithaupt and Kuenzlen, 1982; Lankelma et al., 1980). Figure 4 illustrates the plasma levels of methotrexate and 7-hydroxymethotrexate in man following the infusion of an intermediate dose of methotrexate (3 g) for 24 hr. The plasma

88

I.D. GOLDMANand L. H. MATHERLY

o~ 7-OH-MTX

10-5 o

13 n 10-6 io-r/

MTX/ i

24 t

~

4.8 Hours I

~

72 I

t

96 I

FIG. 4. Plasma concentrationsof methotrexateand 7-hydroxymethotrexatein a patient following a 24 hr infusion of 3 g of methotrexate(courtesyof G. Fabre). levels of 7-hydroxymethotrexate exceed those of methotrexate almost immediately after the cessation of the infusion, attributable to the longer plasma half-life of the catabolite (Breithaupt and Kuenzlen, 1982; Lankelma et al., 1980). Indeed, plasma concentration ratios of 7-hydroxymethotrexate to methotrexate as high as 30 to 1 have been reported after administration of methotrexate. This creates the potential for a number of competitive interactions between these compounds at the level of the membrane transport carrier and intracellular loci as well. In Ehrlich ascites tumor cells, 7-hydroxymethotrexate is transported by the tetrahydrofolate-cofactor carrier (Fabre et al., 1984b; Lankelma et al., 1980). The catabolite binds to the carrier with an affinity (Kin = 9 #M) similar to that for methotrexate (Kin = 5 #M) and competes with both methotrexate and reduced folates for entry into the cell. By virtue of its inhibition of methotrexate influx, the catabolite depresses the free intracellular drug level achieved (Fig. 5, top panel) and slows the rate of association of methotrexate with dihydrofolate reductase (Fig. 5, bottom panel). The catabolite does not, however, directly interfere with the binding of methotrexate to dihydrofolate reductase (Fabre et al., 1984b), consistent with its low affinity for this enzyme (Johns and Loo, 1969). As discussed above, free intracellular methotrexate exceeding drug bound to dihydrofolate reductase is required for maximum suppression of enzyme activity. While 7-hydroxymethotrexate does not bind appreciably to cellular dihydrofolate reductase, the inhibitory effect of the catabolite on the net transport of methotrexate decreases the suppression of dihydrofolate reductase-dependent thymidylate biosynthesis by methotrexate monoglutamate (Fabre et al., 1984b). Further, this depression of the free monoglutamyl substrate level also decreases the net accumulation of methotrexate polyglutamates both in the Ehrlich ascites tumor (Fabre et al., 1984a) and the MOLT 4 human lymphoblastic leukemia (Fabre et al., 1983). Moreover, 7-hydroxymethotrexate is itself a good substrate for the folylpolyglutamate synthetase in the Ehrlich ascites tumor (Fabre et al., 1984a) and the MOLT 4 line (Fabre et al., 1983), as well as in cell-free preparations of this enzyme from rat liver (McGuire et al., 1983). While 7-hydroxymethotrexate forms polyglutamyl derivatives at a rate 2.7-fold greater than that for comparable concentrations of methotrexate (Fabre et al., 1984a), no direct competition between 7-hydroxymethotrexate and/or its polyglutamyl derivatives and methotrexate at the level of the intracellular folylpolyglutamate synthetase has been demonstrated in cells. The time course of formation of 7-hydroxymethotrexate poly-

89

Cellular pharmacology o f methotrexate

4

(o)

3 r

";

2

"10

E ¢o x I-

I

t

I

I io

I 20

I

I

(b)

o

I I I 30 40 50 Time (rain) FIG 5 Effect of 7-hydroxymethotrexate on the transport of methotrexate (a) and binding to dihydrofolat¢ reductase (b) Ehr|ich ascites tumor cells were incubated with ] #M methotrexate with ( O - - O - - O ) and without ( © - - © - - © ) 10/IM 7-hydroxymethotrexate The intracellular level of dihydrofo]ate reductase is 2 nmo]/g dry weight (from Fabre et a l , ]984b)

glutamates in the Ehrlich tumor is illustrated in Fig. 6. As previously reported for methotrexate with this cell line (Fry et al., 1982a), polyglutamylation of 7-hydroxymethotrexate is rapid following drug uptake. Up to three polyglutamyl derivatives can be detected by liquid chromatography. Of particular interest is the reciprocal relationship between the levels of total intracellular 7-hydroxymethotrexate polyglutamates and the monoglutamate; i.e. as the former increases, the latter declines. This relationship was observed also for methotrexate and its polyglutamyl derivatives (Fry et al., 1982a) and was attributed, in part, to the net displacement of monoglutamyl drug from dihydrofolate reductase by the polyglutamates as the intracellular level of the latter increased with the subsequent efflux of the monoglutamate from the cell. However, since 7-hydroxymethotrexate is not bound appreciably to this enzyme another explanation is necessary. It is possible that the presence of polyglutamyl derivatives of 4-aminoantifolates in cells decreases the net transport of the monoglutamate. It is more likely that the rate of polyglutamylation is sufficiently fast, as compared to the rate of entry of the monoI

]

T

'

Totot [ 3H 5

__O~o

"

./"

O ~ • ' ~

--

"

O~O_

•

;o 3 . ~ . . / / . . ~

o

P,

; [

,J

I 60

! 120

I

I

180

240

Minutes FIG. 6, Time course of uptake and metabolism of 7-hydroxymethotrexate [7-OH-MTX] to 7-hydroxymethotrexate polyglutamates [7-OH-MTX-PG] (from Fabre et al., 1984a).

90

I.D. GOLDMANand L. H. MATH~LY

glutamate into the cell, to result in the depression of free intracellular monoglutamate to a level below that expected if there were no metabolic consumption within the cell. As observed for the polyglutamyl derivatives of methotrexate, 7-hydroxymethotrexate polyglutamates are retained within Ehrlich cells much longer than underivatized 7-hydroxymethotrexate (Fabre et al., 1984a). Studies indicate that the polyglutamyl derivatives of 7-hydroxymethotrexate bind to dihydrofolate reductase. Initially, this was thought to be limited to the tetraglutamyl derivative (Fabre et al., 1984a), but more recent studies with a variety of tumor lines indicate binding of the monoglutamate and other polyglutamyl forms as well (Seither and Goldman, unpublished observation). This is currently under further investigation. It is clear that polyglutamyl form(s) of 7-hydroxymethotrexate are cytotoxic since when cells are loaded with these derivatives by incubation with 7-hydroxymethotrexate in the present of glycine, adenosine, and thymidine following which the monoglutamate and the protecting nucleosides and glycine are removed, cytotoxicity due to the retained polyglutamates is expressed "(Fabre et al., 1984c). Hence, while 7-hydroxymethotrexate could compromise the pharmacologic activity of methotrexate by competition for transport, the catabolite could also complement the effectiveness of the drug by preventing transport and utilization of extracellular tetrahydrofolate-cofactors and/or by inhibiting dihydrofolate reductase directly following its polyglutamylation within the cell. 8. INTERACTIONS BETWEEN METHOTREXATE AND 5 - F O R M Y L T E T R A H Y D R O F O L A T E IN THE RESCUE PHENOMENON: THE ROLE OF M E M B R A N E TRANSPORT 8.1. OVERVIEW The provision of a source of reduced folates in conjunction with methotrexate significantly alters the antifolate properties of this agent. For instance, when reduced folates are administered to tumor-bearing animals simultaneously with methotrexate there is a marked decrease in the chemotherapeutic efficacy of the antifolate (Goldin et al., 1952, 1953). Alternatively, if the folate derivative is administered at a suitable interval following the antifolate, the toxic effects of the methotrexate are reduced, however, the tumoricidal response is largely preserved (Goidin et al., 1966; Sirotnak et al., 1978). In fact, sequential scheduling of this type allows the in vivo administration of otherwise lethal doses of methotrexate with enhanced chemotherapeutic efficacy. Within the context of the known interactions between these folates this reversal of the effects of methotrexate could be due to: (i) inhibition of methotrexate transport into cells with reduced direct suppression of dihydrofolate reductase; (ii) decreased polyglutamylation of methotrexate arising from competition at the level of the membrane transport carrier or the folylpolyglutamate synthetase; and/or (iii) the provision of reduced folate cofactors thereby circumventing the block in tetrahydrofolate synthesis within the cells. While the mechanism by which 5-formyltetrahydrofolate achieves selective rescue in animal tumor models is unclear, the success of these regimens in experimental systems has nonetheless led to their clinical implementation (Bertino et al., 1971; Djerassi, 1975; Frei et al., 1975). The original premise of 5-formyltetrahydrofolate rescue was that an added source of active folate cofactors, following exposure of cells to methotrexate, would circumvent the block at the level of dihydrofolate reductase, a phenomenon that should be noncompetitive. However, studies both in vitro and in vivo have shown that increasing doses of methotrexate require higher levels of the natural folate to abolish the antifolate effects of the drug (Borsa and Whitmore, 1969; Greenspan et al., 1951; Moran et al., 1979; Pinedo et al., 1976), a finding inconsistent with this mechanism. Moreover, White (1983) recently pointed out that the reduced folate transport carrier could not deliver 5-formyltetrahydrofolate at a rate sufficient to meet the tetrahydrofolate requirements for cellular replication. This analysis demonstrated that the rate of oxidation of tetrahydrofolates to dihydrofolate during semiconservative DNA synthesis is approximately ten-fold faster than the maximum rate of transport by the carrier; at the 5-formyitetrahydrofolate plasma levels

Cellular pharmacology of methotrexate

91

achieved during low-dose rescue, the actual rates of transport would be even slower. Thus, while 5-formyltetrahydrofolate added after methotrexate equilibrates with and increases the intracellular folate pool this does not appear to be the primary mechanism by which rescue from methotrexate toxicity is achieved. The competitive reversal of the biochemical effects of methotrexate could derive, in part, from an interaction between the folate derivative and methotrexate at the level of their common transport carrier. For instance, as the interval between the dose of methotrexate and 5-formyltetrahydrofolate is lengthened the amount of the folate cofactor required for rescue is decreased (Sirotnak et al., 1978). This may be related to methotrexate inhibition of 5-formyltetrahydrofolate transport into cells, an effect that is diminished with time following methotrexate administration as the drug blood level falls and that can be overcome competitively as the dose of 5-formyltetrahydrofolate is increased. Reduced folates inhibit methotrexate transport with a reduction in free intracellular antifolate monoglutamyl levels (Goldman et al., 1968; White et al., 1978). This diminution of intracellular methotrexate decreases inhibition of dihydrofolate reductase activity by the monoglutamate (White and Goldman, 1979) and also decreases polyglutamylation of methotrexate (Galivan and Nimec, 1983; Rosenblatt et al., 1981). Hence, the unnatural isomer of 5-methyltetrahydrofolate, which is transported by the tetrahydrofolate-cofactor carrier but cannot be used for subsequent folate-dependent carbon transfer reactions in cells, can decrease the inhibitory effects of methotrexate on DNA synthesis by reducing drug transport into cells (White and Goldman, 1979). However, such a membrane-derived effect would not overcome inhibition of dihydrofolate reductase by antifolylpolyglutamyl derivatives already formed. Further, there are instances in which cellular competition between methotrexate and folate cofactors cannot be related at all to interactions at the level of the transport cartier. For example, when the extracellular methotrexate and 5-formyltetrahydrofolate levels are far below the influx Km the degree of carrier saturation is so low that significant interaction at this level is not possible (White, 1983). In addition, rescue interactions have been demonstrated between reduced folates and lipophilic antifolates (see below) which traverse the cell membrane by means other than the tetrahydrofolate-cofactor carrier (Hill et al., 1975; Jackson et al., 1984). 8.2. COMPETITIVEINTERACTIONSAMONG METHOTREXATE, TETRAHYDROFOLATESAND DIHYDROFOLATE REDUCTASEWITHIN CELLS

Recently, there have been a number of important new insights into the mechanism of rescue and the inherent selectivity of this phenomenon. The finding of competition between methotrexate and dihydrofolate at the level of dihydrofolate reductase as described above (Section 4) led to the suggestion that the added 5-formyltetrahydrofolate might increase the total folate pool ultimately leading to higher dihydrofolate levels which competitively reverse the pharmacologic effects of methotrexate (Moran et al., 1979; White, 1983). Further, it has been shown that reduced folates in sufficient concentrations can displace dihydrofolate reductase-bound methotrexate when added to Ehrlich ascites tumor cells (Matherly et al., 1983) or a cell-free preparation (Matherly et al., 1984). Regardless of the actual folate derivative mediating the dissociation of enzyme-bound antifolate within the cell, a primary determinant of this competitive interaction is the concentration of methotrexate in the cellular milieu at the time tetrahydrofolate is provided. In the presence of appreciable free intracellular methotrexate, no significant competition at the level of the dihydrofolate reductase by added folates can be demonstrated. Further, at high extracellular levels of methotrexate, competitive interference at the level of the membrane carrier reduces the amount of rescue agent that enters the cell. 8.3. POLYGLUTAMYLATIONOF METHOTREXATEAS A FACTOR IN THE SELECTIVITY OF LEUCOVORIN RESCUE The presence of methotrexate polyglutamates in cells may also be an important determinant of rescue since reduced folates fail to effectively reverse the pharmacologic

92

I . D . GOLDMAN and L. H. MATHERLY

effects of methotrexate in cells which have accumulated appreciable levels of these derivatives (Galivan and Nimec, 1983; Rosenblatt et al., 1982). Moreover, in the presence of added tetrahydrofolates no net loss of enzyme-bound antifolate is observed in cells which have accumulated methotrexate polyglutamates to a concentration exceeding that of dihydrofolate reductase (Matherly et al., 1983). Accordingly, polyglutamylation of methotrexate may be an important factor in the selectivity of rescue in much the same way that this appears to be one key element in the selective activity of the drug. Hence, these derivatives which accumulate rapidly in susceptible tumor cells are formed to a much lesser extent in bone marrow cells (Fabre et al., 1984d) and gastrointestinal cells (Fry et al., 1983), tending to spare host cells from the sustained antifolate effects of the drug as the free monoglutamyl levels in the intracellular and extracellular compartments fall. This is in contrast to drug-sensitive tumor cells in which an antifolate block is induced initially by the monoglutamate and then sustained by the polyglutamyl derivatives. This difference in methotrexate polyglutamate levels will prevent the net displacement of antifolate from tumor enzyme by added folates while the low levels of polyglutamyl derivatives in marrow and intestinal cells should result in the net displacement of the monoglutamate from dihydrofolate reductase with rapid resumption of endogenous tetrahydrofolate biosynthesis in these tissues. Finally, interactions between reduced folates and methotrexate at other key cellular loci could also represent an important element of the competitive nature of rescue by 5-formyltetrahydrofolate from the toxic effects of methotrexate. Potential sites for such competition include thymidylate synthase or the purine biosynthetic enzymes, glycinamide ribotide (GAR) transformylase and aminoimidazolecarboxamide ribotide (AICAR) transformylase. While the binding of monoglutamyl methotrexate to these loci is probably insufficient to be pharmacologically important, the demonstrated enhanced binding of the polyglutamate derivatives of methotrexate to these enzymes (Allegra et al., 1984; Baggott, 1983) may be an element in the failure of the natural folates to effectively reverse the pharmacologic effects of methotrexate in cells which have accumulated appreciable levels of these derivatives (Galivan and Nimec, 1983; Rosenblatt et al., 1982). Again, an important determinant of the selectivity of rescue by 5-formyltetrahydrofolate in vivo may reside in the differential capacity of host and tumor tissues to synthesize methotrexate polyglutamates. 9. MEMBRANE TRANSPORT OF METHOTREXATE AS A FACTOR IN DRUG RESISTANCE 9.1. OVERVIEW The preceding sections describe a number of elements in addition to membrane transport which contribute to the overall cytotoxic response to methotrexate. Nonetheless, e~ficient transport into tumor cells is of paramount importance to chemotherapeutic effectiveness since the intracellular drug level achieved is the critical determinant of such intracellular events as binding to dihydrofolate reductase and conversion to polyglutamates. Indeed, an excellent correlation between transport efficacy and in vivo sensitivity to methotrexate has been documented for a number of murine tumor lines (Kessel et al., 1965; Sirotnak and Donsbach, 1975, 1976). Because of this critical role of transport in drug action, it is not surprising that impaired transport of methotrexate represents a frequent basis of drug resistance in cultured murine (Hill et al., 1979; Jackson et al., 1975; Sirotnak et al., 1968) and human (Niethammer and Jackson, 1975) tumor lines, as well as in murine tumor cells derived in vivo (Sirotnak et al., 1981a). Decreased antifolate transport may arise from a diminished binding affinity of the carrier for methotrexate, a reduction in the number of carrier molecules, and/or a reduced rate of carrier translocation in the cell membrane. Reduced drug influx is generally not accompanied by a corresponding change in the parameters for methotrexate exit (Hill et al., 1979; Sirotnak et al., 1981a), resulting in a net depression of the intracellular methotrexate level achieved at drug levels below saturation of the carrier.

Cellular pharmacologyof methotrexate

93

Impaired transport of methotrexate which renders cells insensitive to conventional doses of drug should be circumvented by increasing the level of extracellular antifolate so as to force the drug into the cell via a carrier with a reduced affinity or by increasing drug entry via passive diffusion or another low affinity transport route. As the extracellular methotrexate level is increased differences in the levels of free intracellular drug in sensitive and resistant cells decrease (Hill et al., 1979). In clinical practice, the use of 'high dose' methotrexate as a means of forcing drug entry requires co-administration of 5-formyltetrahydrofolate after a suitable interval to avoid lethal toxicity. An alternative approach to circumventing transport-related drug resistance involves the use of nonclassical antifolates which enter the cell by routes other than the tetrahydrofolatc-cofactor transport carrier. These approaches are described in the following sections. 9.2. A BASIS FOR THE SELECTIVITYOF HIGH-DOSE METHOTREXATEWITH LEUCOVORIN RESCUE DIRECTED AGAINST RESISTANTTUMORS WITH DEFECTIVE METHOTREXATETRANSPORT Of potential importance is the apparent selectivity of 5-formyltetrahydrofolate rescue in tumors with impaired transport of methotrexate (Hill et al., 1975; Sirotnak et al., 1982). Hence, if in a resistant cell there is reduced transport of methotrcxatc by the tctrahydrofolate-cofactor carrier, increasing the extracellular methotrexate level will overcome this element of resistance either by forcing drug via a carrier with a lower affinity or by increasing drug delivery by passive diffusion. While these high concentrations will enhance mcthotrcxate delivery to susceptible host tissues as well, it is the clement of rescue that provides the inherent selectivity of this approach. Upon addition of low levels of 5-formyltetrahydrofolate, susceptible host tissues, with normal tetrahydrofolate-cofactor carriers, will accumulate the tctrahydrofolatc substrate within the intracellular compartment at an adequate velocity. On the other hand, the tumor cell with a defective or deficient tctrahydrofolate-cofactor transport carrier will not take up the rescue agent at a sufficient rate. This may be one reason why high doses of 5-formyltetrahydrofolate in rescue minimize selectivity (Sirotnak et al., 1978). Hence, a 5-formyltetrahydrofolatc dose is desired that will not penetrate the resistant tumor cell at a rate sufficient to reverse the pharmacologic block induced by methotrexate but will enter susceptible host tissues at an adequate velocity. If in rescue the 5-formyltetrahydrofolate blood level is too high, it will permeate even the methotrexate resistant cells by the same mechanism(s) by which methotrexate, at the high extracellular levels achieved, is delivered to these cells. This concept may account for an element of selectivity inherent in high-dose methotrexate regimens with 5-formyltetrahydrofolate rescue directed against solid tumors in general. Hence, malignant cells within poorly vascularized solid tumors will have decreased access to circulating methotrexate in low dose regimens. With high levels of methotrexate, diffusion of drug into the central core of the tumor should be achieved. On the other hand, in low dose rescue, tetrahydrofolate-cofactors would presumably penetrate the central tumor area to a small extent while the well perfused susceptible host tissues, which receive maximum exposure to the high levels of methotrexate, should nonetheless be rescued by the very low concentrations of 5-formyltetrahydrofolate which will have ready access to this compartment. As described below, these considerations are also important in the implementation of lipophilic antifolates as a means of cirvumventing transport-related methotrexate resistance. 9.3. APPROACHES TO THE MODIFICATIONOF THE 4-AMINO-ANTIFOLATESTRUCTURE TO ENHANCE DELIVERY INTO SENSITIVEAND RESISTANTTUMOR CELL LINES Since antifolate uptake is critical to drug effectiveness, as described above, considerable interest has focused on modifications of the drug structure which promote net intracellular accumulation. The 4-aminopteridine structure is clearly critical to association with the membrane carrier since mcthotrexate is transported much more efficiently than folic acid (Goldman et al., 1968; Lichtcnstein et al., 1969). However, with the reduction of the pteridine ring the 4-amino-group loses its importance as both the 5-methyl and 5-formyl

94

I. D, GOLDMANand L. H. MATI-IERLY

derivaties of tetrahydrofolate are transported very efficiently by this carrier system (Goldman et al., 1968; White et al., 1978). Further, hydroxylation of the 7 position of the pterin of methotrexate has only a negligible effect on transport (Fabre et al., 1984b; see Section 7 above). The Nt°-bridge plays a particularly critical role in carrier binding. The deletion of the N~°-methyl group in aminopterin, alone, decreases the influx Km by a factor or 4 relative to methotrexate (Sirotnak and Donsbach, 1972). In addition, 10-deazaaminopterin has a greater affinity for the tetrahydrofolate cofactor carrier than methotrexate and accumulates intracellularly to a greater extent, resulting in an enhanced activity against murine tumors (Sirotnak et al., 1984a,b). An alternative approach to promoting net antifolate uptake, particularly in cells defective in methotrexate transport, involves eliminating the charge on the molecule to enhance uptake by passive diffusion thus circumventing the requirement for cell entry by the tetrahydrofolate cofactor carrier. Such a modification would, in addition, improve drug penetration of the blood-brain barrier to permit access to tumors within the central nervous system. This modification must not, however, appreciably decrease the high affinity of the drug for intracellular loci such as dihydrofolate reductase. Examples of such antifolates include 2,4-diamino-5-(Y,4'-dichlorophenyl)-6-methyl-pyrimidine (DDMP; metoprine; Hill and Price, 1980), triazinate (Baker's antifol; Skeel et al., 1973, 1976) and recently, trimetrexate [2,4,-diamino-5-methyl-6-(3,4,5-trimethoxyanilino)methyl] quinazoline (Bcrtino et al., 1979; Jackson et al., 1984). These agents circumvent resistance attributable to transport defects (Diddens et al., 1983; Jackson et al., 1984; Kamen et al., 1981; Ohnoshi et al., 1982) in experimental tumor systems. However, since these agents all form tight complexes with intracellular dihydrofolate reductase by virtue of their 2,4-diamino aromatic ring configuration, methotrexate-resistant stains that overproduce dihydrofolate reductase show considerable cross resistance to this class of drugs (Diddens et al., 1983). Significantly, these lipophilic antifolates do not possess the terminal glutamate necessary for binding and polyglutamylation by the folylpolyglutamate synthetase. Hence, these agents are not converted to highly retentive forms, analogous to the polyglutamates of methotrexate, and their pharmacologic effects should be readily reversible in the absence of extracellular drug. This characteristic, of course, would obviate any selective advantage that the polyglutamylation of antifolates might afford (see Section 9 above). For DDMP, uptake to steady-state is rapid in cells resistant to methotrexate due to impaired transport, consistent with a non-facilitated passage through the membrane (Hill et al., 1975). However, these studies are complicated by large and rapid uptake components that may represent adsorption of this lipid soluble agent in the cell membrane. It has been suggested that transport of trimetrexate is carrier-mediated although not via the carrier which facilitates uptake of methotrexate (Besserer et al., 1984; Kamen et al., 1981). In fact, in human lymphoblast cells, resistance to trimetrexate but not methotrexate has been attributed to detective uptake of the lipophilic antifolate (Besserer et al., 1984). An additional approach to the design of alternative antifolate derivatives has been to modify the terminal glutamate of methotrexate so as to abolish the anionic characteristic of the molecule by esterification to enhance passive diffusion into the cell (Rosowsky et al., 1980). Alternatively, methotrexate has been conjugated to carrier molecules such as poly-L-lysine (Galivan et al., 1982; Rysler and Shen, 1978). The rationale for the latter approach is based on the premise that tumor cells have more pinocytotic activity than normal cells and would therefore selectively take up the complex. Once inside the cell, the conjugate is hydrolyzed with the release of methotrexate which can then bind to dihydrofolate reductase and/or undergo conversion to polyglutamyl derivatives (Galivan et al., 1982). Hence these derivatives are 'prodrugs' of methotrexate that facilitate entry by a route other than the tetrahydrofolate-cofactor carrier yet effectively preserve the antifolate properties of the drug. While the clinical applicability of the esters or high molecular weight conjugates of methotrexate has not been explored and might be minimized by the presence of serum esterase activity, these derivatives nonetheless circumvent methotrexate resistance attributable to impaired transport in a variety of cell lines in vitro (Rosowsky et al., 1980; Rysler and Shen, 1978).

Cellular pharmacology of methotrexate

95

Of potential significance in the utilization of nonclassical antifolates as a means of circumventing methotrexate resistance arising from impaired transport is the accompanying inability of these resistant tumors to transport and utilize tetrahydrofolatecofactors, either those endogenous in plasma or those supplied during rescue protocols (Sirotnak et al., 1982). Hence, host tissues should accumulate tetrahydrofolates via the cofactor carrier to circumvent the effects of the antifolate while resistant tumors lacking carrier would accumulate fewer folate coenzymes (see Section 9.2 above). In this fashion, the combination of nonclassical antifolates with low dose 5-formyltetrahydrofolate rescue may also provide a selective advantage in the treatment of transport cartier-deficient tumor cells. 10. BASIS FOR THE DOSE-DEPENDENCY OF METHOTREXATE ACTION: ROLE OF M E M B R A N E TRANSPORT AND OTHER FACTORS The introduction of methotrexate therapy into clinical regimens was for the treatment of acute lymphatic leukemia in children, a disease sensitive to relatively low doses of the drug. This experience along with the original demonstration that methotrexate was a stoichiomettic inhibitor of dihydrofolate reductase in cell-free systems resulted in the general application of this agent in relatively low doses. Later studies provided empirical evidence supporting the efficacy of high doses of methotrexate with 5formyltetrahydrofolate rescue in animal tumor systems. On the basis of these animal studies, high dose methotrexate regimens with 5-formyltetrahydrofolate rescue were applied.in the treatment of a variety of human neoplasms. 'High dose' is a genetic term; unfortunately, attitudes towards methotrexate as a dose-dependent drug have been influenced by the concerns about clinical regimens that have utilized as much as 20-50 g of this agent. The important point is not whether doses this high are required to optimize efficacy but whether, in fact, the chemotherapeutic efficacy of methotrexate is indeed dose-dependent and whether different tumors in different stages may require specific doses to achieve a pharmacologic effect--doses that may be in excess of those that might be administered safely without the subsequent administration of 5-formyltetrahydrofolate. Enough is known now about the cellular pharmacology of methotrexate and its mechanism of action to confirm that the effects of this agent are clearly dose-dependent and that a variety of factors and perturbations in cellular processes can markedly increase the levels of methotrexate that are required to produce a pharmacologic effect. These factors include the rate of synthesis of thymidylate from deoxyutidylate (Bowen et al., 1978; Moran et al., 1979; Washtien, 1982; White and Goldman, 1981), the total level of dihydrofolate reductase within cells (Fischer, 1961; Hakala et al., 1961), and the affinity of dihydrofolate reductase for methotrexate (Flintoff and Essani, 1980; Goldie et al., 1980; Jackson and Niethammer, 1977). Other factors, also important but not as well substantiated, include the level of endogenous tetrahydrofolate-cofactors within cells, the ability of cells to interconvert tetrahydrofolates and ultimately generate dihydrofolate, and the affinity of the folylpolyglutamate synthetase for methotrexate. Finally, an element of major importance as a determinant of the dose of methotrexate required is the membrane transport system. Clearly, natural or acquired impaired membrane transport of methotrexate as a basis for resistance will require increased doses of the drug to circumvent this defect. What is less well appreciated is that there are inherent limitations in the transport and accumulation of methotrexate into ceils mediated by the 'normal' tetrahydrofolate-cofactor carrier. These limitations augment any degree of resistance to methotrexate that is a consequence of changes in the other parameters described above. The methotrexate interaction with dihydrofolate reductase is competitive with dihydrofolate, and high levels of free methotrexate are required to achieve suppression of the small percentage of total enzyme activity that is sufficient to sustain tetrahydrofolate-cofactor stores within the cell. Hence, any changes in the enzyme level or dihydrofolate pool that

96

I.D. GOLDMANand L. H. MATHERLY

can be generated will alter the free methotrexate level that is required to produce a pharmacologic effect. For instance, if the level of enzyme activity within cells is increased, the percentage of dihydrofolate reductase sufficient to sustain tetrahydrofolate synthesis is decreased and a higher level of saturation of the enzyme by methotrexate will be required to maximally suppress this process. This will require a higher intracellular methotrexate concentration. Similarly, if the thymidylate synthase activity is decreased so that the fraction of dihydrofolate reductase required to meet cellular demands for tetrahydrofolate is reduced, the extent of enzyme saturation which is necessary to suppress tetrahydrofolate synthesis is increased, requiring higher intracellular methotrexate levels. As the level of tetrahydrofolate-cofactors within cells is elevated, the concentration of dihydrofolate generated when dihydrofolate reductase activity is blocked by methotrexate may be correspondingly increased, resulting in an intensified competitive interaction between dihydrofolate and methotrexate. This, again, will require a higher intracellular methotrexate concentration to achieve enzyme inhibition. Finally, within the context of polyglutamylation of methotrexate as a key factor in drug action, the extent of this conversion will depend upon, among other things, the level of folyipolyglutamates which could compete with methotrexate at the level of the folylpolyglutamate synthetase (Nimec and Galivan, 1983), the absolute amount of folylpolyglutamate synthetase, and the affinity of this enzyme for methotrexate. Any deleterious changes in these parameters would require more methotrexate to maintain a cytotoxic effect. Alterations of any of the factors described above which results in resistance to methotrexate requires higher levels of methotrexate monoglutamyl substrate within the cell to achieve a pharmacologic effect. It is, of course, the membrane transport system that determines the extent to which methotrexate accumulates within the intracellular compartment. As indicated earlier (see Section 2.4), as the extracellular methotrexate level is increased there is not an unlimited proportional increase in the free intracellular methotrexate level achieved. In fact, the carrier system can generate only a finite concentration of free intracellular drug. As the extracellular methotrexate concentration increases above the carrier Km the free intracellular level approaches a maximum. Indeed, even under conditions in which the extracellular methotrexate concentration is increased by orders of magnitude above the influx K~, levels of free intracellular methotrexate achieved may be orders of magnitude lower than the extracellular drug concentration not only because of saturation of the influx mechanism but because of the constraints on the accumulation of free drug due to electrical factors and the energy-driven exit mechanism (see Section 2.4). It is easy to appreciate how relatively modest increases in the cellular dihydrofolate reductase level or changes in any of the above parameters, alone or in combination, might result in a requirement for an intracellular drug concentration that cannot be obtained by the extracellular drug levels achieved clinically.

11. SOME PERSPECTIVES ON FUTURE RESEARCH IN THIS AREA--CHARACTERISTICS OF DRUG ACTION, IN GENERAL, AND DRUG TRANSPORT, IN PARTICULAR, THAT REQUIRE FURTHER CLARIFICATION A number of critical issues in the area of antifolate pharmacology relating in particular to the role of membrane transport in drug action, remain unanswered. If there is one important lesson that has been learned from studies in this area it is that drug transport and drug action in cells cannot be considered separately. While different tools and expertise are required for the study of these different phenomena, a clear understanding of the role of membrane transport in controlling intracellular biochemical processes and as a determinant of drug action will require an experimental and theoretical integration of a variety of diverse perspectives.

Cellular pharmacologyof methotrexate

97

A considerable amount of work remains to be done to further define the mechanism(s) of transport of 4-aminoantifolates. Many unusual properties of the transport of these agents have been established, such as sensitivity to anions and the marked perturbations of drug transport which occur with changes in cellular energy metabolism. However, the basis for these observations and how they relate to the transport of 4-aminoantifolates and their naturally occurring counterparts which utilize the same cartier system under physiological conditions is unclear. We must now move from mere descriptions of phenomena to clear definitions of cellular mechanisms which are relevant to physiological conditions. Likewise, the extent to which transport is mediated by one or more processes must be better defined, in particular the extent to which efltux of these drugs is mediated by a mechanism distinct from entry and the extent to which oxidized and reduced folates use these routes. Moreover, work must be done to relate structural properties of the 4-aminoantifolate and tetrahydrofolate molecule to determinants of transport and how structural determinants of drug transport relate to determinants of polyglutamylation. The recognition of the important role of polyglutamylation in methotrexate cytotoxicity and selectivity and the role of membrane transport in controlling this process has provided a new dimension for 4-aminoantifolate pharmacology that should be a stimulus to the development of new analogs and the reevaluation of other 4-aminoantifolates synthesized decades ago that may be substrates for the folylpolyglutamate synthetase as well as the membrane transport carrier. Differences between 4-aminoantifolate polyglutamation in susceptible tumors vs normal host tissues has allowed new insights into the basis for drug selectively that may yield novel approaches for the administration of these drugs to enhance selectivity and chemotherapeutic efficacy. Enough is now known about membrane transport of 4-aminoantifolates, their interaction with dihydrofolate reductase, the formation of polyglutamyl derivatives and the ramifications of polyglutamyl derivatives in terms of cytotoxicity and selectivity to enable the development of innovative approaches in the clinical application of these agents based upon firm biochemical and pharmacologic observations and principles. For instance, the low level of accumulation of methotrexate polyglutamyl derivatives in susceptible host tissues (i.e. intestinal mucosal cells and bone marrow cells) following a pulse of drug that is accompanied by high levels of polyglutamate formation in sensitive tumors suggests that the most vulnerable period for susceptible host cells is during the relatively brief interval after a pulse of methotrexate when the monoglutamate levels are high. In contrast, tumor cells are exposed to the cytotoxic effects of methotrexate not only during this interval but also over the much longer period during which polyglutamyl derivatives are present within these cells. This has led to the design of an experimental treatment regimen by this laboratory in which thymidine/inosine is administered over a short interval following administration of a pulse of methotrexate to prevent the antipurine and antipyrimidine effects of the antifolate when the monoglutamate level is high without altering polyglutamylation of the drug as would occur with simultaneous infusions of reduced folates. In this way, host cells should be protected during the period when the monoglutamate level is high allowing polyglutamylation to proceed in the tumor. Following cessation of nucleoside protection when free monoglutamate levels in susceptible host and tumor cells are low but polyglutamyl derivatives persists in tumor cells, the tumoricidal effects of the antifolate are selectively expressed. Indeed, initial studies indicate that this approach permits a large increase in the dose of methotrexate without a commensurate increase in toxicity. This results in enhanced chemotherapeutic efficacy to tumor-beating mice in comparison to comparable methotrexate doses administered without protecting nucleosides. This approach is currently under evaluation in humans at this institution.

Acknowledgements--This work was supported by grants CA16906 and CA09340 from the National Cancer

Institute. National Institutes of Health. The excellentsecretarialassistance of Mrs BrendaBrownand Ms Jenifer Goldman is acknowledged. JPT

28 t

G

98

I . D . GOLDMAN and L. H. MATHERLY

REFERENCES ALLEGRA, C. J., DRAKE, J. C., JOLIVET,J. and CHAaNER, B. A. (1984) Inhibition of the folate-dependent enzymes of denovo purine synthesis and folate interconverting enzymes by methotrexate polyglutamates. Proc. Am. Ass. Cancer Res. 25: 6.

BAGGOT, J. E. (1983) Inhibition of purified avian liver aminoimidazolecarboxamide ribotide transformylase by polyglutamates of methotrexate and oxidized folates. Fedn Proc. 42: 667. BI~NDEI~, R. A. (1975) Membrane transport of methotrexate (NSC-740) in human neoplastic cells. Cancer Chemother. Rep. 6: 73-82. BERTINO, J. R., LEVITT, M., M¢CULLOUOH, J. L. and CHABNmt, B. A. (1971) New approaches to chemotherapy with folate antagonists: use of leucovorin 'rescue' and enzymic folate depletion. Ann. N. Y. Acad. Sci. 186: 486-495. BERTINO, J. R., SAWlCKI, W. L., MOROSON, B. A., CASHMORE, A. R. and ELSLAGER, E. F. (1979) 2,4-Diamino-5-methyl-6-(3,4,5-trimethoxy-anilino)methyl quinazoline (TMQ), a potent nonclassical folate antagonist inhibitor: 1. Effect on dihydrofolate reductase and growth of rodent tumors in vitro and in vivo. Biochem. Pharmac. 28: 1983-1987. BESSERER, J. A., FRY, D. W., BORITZKI, T. J. and JACKSON, R. C. (1984) Studies on cellular resistance and membrane transport with the quinazoline antifolate, trimetrexate. Fedn Proc. 43: 1779. BOBZIEN,W. F. and GOLDMAN, I. D. (1972) The mechanism of folate transport in rabbit reticulocytes. J. clin. Invest. 51: 1688-1698. BORSA, J. and WmTMOttE, G. F. (1969) Studies relating to the mode of action of methotrexate. II, Studies on sites of action in L-cells in vitro. Molee. Pharmac. 5: 303-317. BOWEN, D., WHITE, J. C. and GOLDMAN, I. D. (1978) A basis for fluoropyrimidine-induced antagonism to methotrexate in Ehrlich ascites tumor cells in vitro. Cancer Res. 38: 219-222. BREITHAUPT, H. and KUENZLEN, E. (1982) Pharmacokinetics of methotrexate and 7-hydroxymethotrexate following infusions of high-dose methotrexate. Cancer Treat. Rep. 66: 1733-1741. CHELLO, P. L., Smo'rNAK, F. M., DOmCK, D. M. and Mocclo, D. M. (1979) Schedule-dependent synergism of methotrexate and vincristine against LI210 leukemia. Cancer Treat. Rep. 63: 1889-1894. CHELLO, P. L., S1ROTNAK, F. M., WONG, E., KISL1UK, R. L., GAUMONT, Y. and COMBEI'INE,G. (1982) Further studies of stereospecificity at carbon 6 for membrane transport of tetrahydrofolates: diastereoisomers of 5-methyltetrahydrofolate as competitive inhibitors of transport of methotrexate in LI210 cells. Biochem. Pharmac. 31: 1527-1530. DEMBO, M. and SmOTNAK, F. M. (1976) Antifolate transport in L1210 leukemia cells. Kinetic evidence for the non identity of carriers for influx and efltux. Biochim. biophys. Acta. 448: 505-516. DEMBO, M., SIROTNAK, F. i . and Mocclo, D. M. (1984) Effects of metabolic deprivation on methotrexate transport in L1210 leukemia cells: further evidence for separate influx and efflux systems with different energetic requirements. J. Memb. Biol. 78: 9-17. DIDDEN$, H., NIETHAMMER, O. and JACKSON,R. C. (1983) Patterns of cross-resistance to the antifolate drugs trimetrexate, metoprine, homofolate, and CB3717 in human lymphoma and osteosarcoma cells resistant to methotrexate. Cancer Res. 43: 5286-5292. DJERASSI, I. (1975) High dose methotrexate and citrovorum factor rescue: background and rationale. Cancer Chemother. Rep. 6: 3-6. FABRE, G., MATHERLY, L. H., FAVRE, R., CATALIN, J. and CANO, J.-P. (1983) In vitro formation of polygiutamyl derivatives of methotrexate and 7-hydroxymethotrexate in human lymphoblastic leukemia cells. Cancer Res. 43: 4648-4652. FABRE, G., FAaRE, I., MATHERLY, L. H., CANO, J.-P. and GOLDMAN, I. D. (1984a) Synthesis, retention and biological activity of 7-hydroxymethotrexate polyglutamyl derivatives in Ehrlich ascites tumor cells. J. biol. Chem. 259: 5066-5072. FABRE, G., MATHERLY, L. H., FABRE, I., CANO, J.-P. and GOLDMAN, I. O. (1984b) Interactions between 7-hydroxymethotrexate and methotrexate at the cellular level in the Ehrlich tumor in vitro. Cancer Res. 44: 970-975. FABRE, I., FABRE, G. and GOLDMAN, I. D. (1984d) Selectivity of glycine, adenosine and thymidine protection of granulocytic progenitor cells versus tumor cells against methotrexate cytotoxicity/n vitro: correlation with the formation of methotrexate polyglutamate derivatives. Cancer Res. 44: 3190-3195. FtSCHER, G. A. (1961) Increased levels of folic acid reductase to amethopterin in leukemic cells. Biochem. Pharmac. 7: 75-77. FLINTOFF, W. F. and ESSANI, K. (1980) Methotrexate-resistant chinese hamster ovary cells contain a dihydrofolate reductase with an altered affinity for methotrexate. Biochemistry 19: 4321-4327. FREI, E., JAFFE, E., TATTERSALL, i . H. N., PITMAN, S. and PARKER, L. (1975) New approaches to cancer chemotherapy with methotrexate. New Engl. J. Med. 292: 846--851. FRY, D. W. and GOLDMAN, 1. D. (1982) Further studies on the charge related alterations of methotrexate transport in Ehrlich ascites tumor cells by ionic liposomes: correlation with liposome-cell association. J. Memb. Biol. 66: 87-95. FRY, D. W., WHITE, J. C. and GOLDMAN, I. D. (1979) Alteration of carrier-mediated transport of methotrexate in Ehrlich ascites tumor cells by charged liposomes. J. Memb. Biol, 50: 123-140. FRY, D. W., CYRULSKI, R. H. and GOLDMAN, I. O. (1980a) K+-induced alterations of energetics and exchange diffusion in the carrier-mediated transport of the folic acid analog, methotrexate, in Ehrlich ascites tumor cells. Biochim. biophys. Acta 603: 157-170. FRY, D. W., WHITE, J. C. and GOLDMAN, I. D. (1980b) Effects of 2,4--dinitrophenol and other metabolic inhibitors on the bidirectional fluxes, net transport, and intracellular binding of methotrexate in Ehrlich ascites tumor cells. Cancer Res. 40: 3669--3673. FRY, D. W., YALOWICH, J. C. and GOLDMAN, I. D. (1982a) Rapid formation of poly-y-glutamyl derivatives of methotrexate and their association with dihydrofolate reductase as assessed by high pressure liquid chromatography in the Ehrlich ascites tumor cell in vitro. J. biol. Chem. 257: 1890-1896,

Cellular pharmacology of methotrexate

99

FRY, D. W., YALOWICH, J. C. and GOLDMAN, I. D. (1982b) Augmentation of the intracellular levels of polyglutamyl derivatives of methotrexate by vincristine and probenecid in Ehrlich ascites tumor cells. Cancer Res. 42: 2532-2536. FRY, D. W., ANDERSON, L. A., BORST, M. and GOLDMAN, I. D. (1983) Analysis of the role of membrane transport and polyglutamylation of methotrexate in gut and the Erlich tumor in vivo as factors in drug sensitivity and selectivity. Cancer Res. 43: 1087-1092. FYFE, M. J. and GOLDMAN,I. D. (1973) Characteristics of the vincristine-induced augmentation of methotrexate uptake in Ehrlich ascites tumor cells. J. biol. Chem. 248: 5067-5073. GALIVAN, J. (1980) Evidence for the cytoxic activity of polyglutamate derivatives of methotrexate. Molec. Pharmac. 17: 105-110. GALIVAN, J., BALINSKA, M. and WmTELEY, J. M. (1982) Interaction of methotrexate poly(1-1yine) with transformed hepatic cells in culture. Archs Biochem. Biophys. 216: 544-550. GALIVAN, J. and NIMEC, Z. (1983) Effects of folinic acid on hepatoma cells containing methotrexate polygiutamates. Cancer Res. 43: 551-555. GEWIRTZ, D. A. and HOLT, S. (1984) Protein binding modifies the effects of probenecid on the cellular pharmacokinetics of methotrexate in the Ehrlich ascites tumor cell. Proc. Am. Ass. Cancer Res. 25: 308. GEWlRTZ, D. A., WroTE, J. C., RANDOLPH, J. K. and GOLDMAN, I. D. (1979) Formation of methotrexate polyglutamates in rat hepatocytes. Cancer Res. 39: 2914-2918. GOLDIE, J. H., KRYSTAL, G., HARTLEY, D., GUDAUSKAS,G. and DEDrtAR, S. 0980) A methotrexate insensitive variant of folate reductase present in two lines of methotrexate-resistant L5178Y cells. Eur. J. Cancer 16:1539-1546. GOLDIN. A., GREENSPAN, E. M., VENDI'I'I'I, J. and SCHOENBACH,E. B. (1952) Studies on the biological interrelationships of folic acid, citrovorum factor, and the antimetabolite aminopterin. J. num. Cancer Inst. 12: 987-1002. GOLDIN, A., MANTEL, N., GREENHOUSE, S. W., VENDI'rTI, J. and HUMPHREYS, S. R. (1953) Estimation of the antileukemic potency of the antimetabolite aminopterin, administered alone and in combination with citrovorum factor or folic acid. Cancer Res. 13: 833-850. GOLDIN, A., VENDITTL J. M., KLINE, I. and MANTEL, N. (1966) Eradication of leukemic cells (L1210) by methotrexate and methotrexate plus citrovorum factor. Nature 212: 1548-1550. GOLDMAN, I. D. (1969) Transport energetics of the folic acid analogue, methotrexate, in LI210 cells: enhanced accumulation by metabolic inhibitors. J. biol. Chem. 244: 3779-3785. GOLDMAN, I. n . (1971a) A model system for the study of heteroexchange diffusion: methotrexate-folate interactions in L1210 leukemia and Ehrlich aseiteg tumor cells. Biochim. biophys. Acta 233: 624-633. GOLDMAN, I. D. (1971b) The characteristics of the membrane transport of amethopterin and the naturally occurring folates. Ann. N. Y. Acad. Sci. 186: 400-422. GOLDMAN, I. D. (1973) Uptake of drugs and resistance. In: Drug Resistance and Selectivity: Biochemical and Cellular Basis, Vol. 1, pp. 299-358, MIHICH, E. (ed.) Academic Press, New York. GOLDMAN,I. n . (1974) The mechanism of action of methotrexate. I. Interaction with a low affanity intracellular site required for maximum inhibition of deoxyribonucleic acid synthesis in L-cell mouse fibroblasts. Molec. Pharmac. 10: 257-274. GOLDMAN, I. D. (1977) Membrane transport of antifolates as a critical determinant of drug cytotoxocity. In: Proceedings of the Ninth Rochestor International Conference on Environmental Toxicity--Membrane Transport, pp. 85-113, Plenum Publishing, New York. GOLDMAN, I. D. (1982) Pharmacokinetics of antineoptastic agents at the cellular level: membrane transport, metabolism, association and dissociation from the intracellular target. In: Clinical Pharmacology of Antitumor Drugs, pp. 15--44, CrlABNER, B. (ed.) W. B. Saunders, Philadelphia, PA. GOLDMAN, I. D., LICrn'ENSTEIN, N. S. and OLIVEmO, V. T. (1968) Carrier-mediated transport of the folic acid analogue, methotrexate, in the L1210 leukemia ceil. J. biol. Chem. 243: 5007-5017. GOLDMAN, I. D.. GUPTA V., WroTE, J. C. and LoIrrV'IELD, S. (1976) Exchangeable intracellular methotrexate levels in the presence and absence of vincristine at extracellular drug concentrations relevant to those achieved in high-dose methotrexate-folinic acid "rescue" protocols. Cancer Res. 36: 276-279. GREENSPAN, E. M., GOLDIN, A. and SCNOENBACrt, E. B. (1951) The relationships of folic acid and citrovorum factor to the toxicity of aminopterin. Cancer Res. 11: 252-253. HAKALA, M. T., ZAKRZEWSK1, S. F. and NlCrtOL, C. A. (1961) Relation of folic acid reductase to amethopterin resistance in cultured mammalian cells. J. biol. Chem. 236: 952-958. HENDERSON. G. B. and ZEVELY, E. M. (1980) Transport of methotrexate in L1210 cells: effect of ions on the rate and extent of uptake. Archs Biochem. Biophys. 200: 149-155. HENDERSON, G. B. and ZEVELY, E. M. (1981) Anion exchange mechanism for transport of methotrexate in LI210 cells. Biochem. biophys. Res. Commun. 99: 163-168. HENDERSON, G. B. and ZEVELY, E. M. (1982a) Intracellular phosphate and its possible role as an exchange anion for active transport of methotrexate in L1210 ceils. Biochem. biophys. Res. Commun. 104: 474482. HENDERSON, G. B. and ZEVELY, E. M. (1982b) Functional correlations between the methotrexate and general anion transport systems of L1210 cells. Biochem. Int. 4: 493-502. HENDERSON, G. B. and ZEVELY, E. M. (1983) Use of non-physiological buffer systems in the analysis of methotrexate transport in LI210 cells. Biochem. Int. 6: 507-515. HENDERSON, G. B., ZEVELY, E. M. and HUENr,mKENS, F. M. (1978) Cyclic adenosine 3',5'-monophosphate and methotrexate transport in L1210 cells. Cancer Res. 38, 859-861. HILL, B. T. and PRICE. L. A. (1980) DDMP [2,4-diamino-5-(3',4'-dichlorophenyl)methylpyrimidine]. Cancer Treat. Rev. 7: 95-112. HILL. B. T., BAILEY. B. D.. WHtTE, J. C. and GOLDMAN, I. D. (1979) Characteristics of transport of 4-aminoantifolates and folate compounds by two lines of L-5178Y lymphoblasts, one with impaired transport of methotrexate. Cancer Res. 39: 2440-2446.

100

I . D . GOLDMAN and L. H. MATHERLY

HILL, B. T.. PRICE, L. A., GOLDIE, J. H. and HARRISON S. I. (1975) Methotrexate resistance and uptake of DDMP by L5178Y cells. Eur. J. Cancer 11: 545-553. JACKSON, R. C. and HARRAP, K. R. (1973) Studies with a mathematical model of folate metabolism. Archs Biochem. Biophys. 158: 827-841, JACKSON, R. C. and NIETHAMMER, D. (1977) Acquired methotrexate resistance in lymphoblasts resulting from altered kinetic properties of dihydrofolate reductase. Eur. J. Cancer 13: 567-575. JACKSON, R. C., NIETHAMMER, D. and HUENNEKENS, F. M. (1975) Enzymic and transport mechanisms of amethopterin resistance in LI210 mouse leukemia cells. Cancer Biochem. Biophys. 1: 151-155. JACKSON, R. C., NIETHAMMER, O. and HART, L. I. (1977) Reactivation of dihydrofolate reductase inhibited by methotrexate or aminopterin. Archs Biochem. Biophys. 182: 646-656. JACKSON, R. C., FRY, D. W., BORITZKI, T. J., BESSERER,J. A., LEOPOLD, W. R., SLOAN, B. J. and ELSLAGER, E. F. (1984) Biochemical pharmacology of the lipophilic antifolate, trimetrexate. Adv. Enzyme Reg. 22: 187-206. JENNETTE, J. C. and GOLDMAN, [. D. (1975) Inhibition of membrane transport of folates by anions retained in uremia. J. Lab. clin. 29[ed. 86: 834-843. JOHNS, D. G. and Loo, T, L. (1969) Metabolite of the 4-amino-4-deoxy-N-10-methylpteroyiglutamic acid (methotrexate). J. Pharm. Sci. 56: 356-359. JOL1VET,J. and CHABNER,B. A. (1983) Intracellular pharmacokinetics of methotrexate polyglutamates in human breast cancer ceils. Selective retention and less dissociable binding of 4-NH~_-10-CH3-pteroytgtutamate 4 and 4-NH2-10-CH3-pteroylglutamate5 to dihydrofolate reductase. J. clin. Invest. 72: 773-778. JOLIVET, J., SCHILSKY, R. L., BAILEY, B. n., DRAKE, J. C. and CHABNER, B. A. (1982) Synthesis, retention, and biological activity of methotrexate polyglutamates in cultured human breast cancer cells. J. clin. Invest. 70: 351-360. KAMEN, B. A., EIBL, B., CASHMORE,A. R., WHYTE, W. L., MOROSON, B. A. and BERTINO, J. R. (1981) Efficacy and transport of a new soluble antifol, 2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl] quinazoline (TMQ; JB-II) in methotrexate resistant cells. Proc. Am. Ass. Cancer Res. 22: 26. KESSEL, n., HALL, T. C. and ROBERTS, D. W. (1965) Uptake as a determinant of methotrexate response in mouse leukemia. Science 150: 752-754. LANKELMA, J., VAN DER KLEIN, E. and RAMAEKERS, F. (1980) The role of 7-hydroxymethotrexate during methotrexate anticancer chemotherapy. Cancer Lett. 9: 133-142. LICHTENSTEIN, N. S., OLIVERIO, V. T. and GOLDMAN, I. D. (1969) Characteristics of folic acid transport in the LI210 leukemia cell. Biochim. biophys. Acta. 193: 456-467. MATHERLY, L. H., FRY, D. W. and GOLDMAN, I. n . (1983) Role of methotrexate polyglutamylation and cellular energy metabolism in inhibition of methotrexate binding to dihydrofolate reductase by 5-formyltetrahydrofolate in Ehrlich ascites tumor cells in vitro. Cancer Res. 43: 2694-2699. MATFIERLY, L. H., ANDERSON, L. A. and GOLDMAN, I. D. (1984) Role of the cellular oxidation-reduction state in methotrexate binding to dihydrofolate reductase and dissociation induced by reduced folates. Cancer Res. 44: 2325-2330. McGUIRE, J. J. and BERTINO, J. R. (1981) Enzymatic synthesis and function of folylpolyglutamates. Molec. cell Biochem: 38: 19-48. McGUIRE, J. J., MXNI, E., HSIEH, P. and BERTINO, J. R. (1983) Folylpolyglutamate synthetase: relation to methotrexate action and as a target for new drug development. In: Development of Target-Oriented Anticancer Drugs, CHENG, Y.-C., Goz, B. and MINKOFF, M. (eds) Raven Press, New York. MORAN, R. G., WERKHEISER, W. C. and ZAKRZEWSKI, S. F. (1976) Folate metabolism in mammalian cells in culture: 1. Partial characterization of the folate derivatives present in L1210 mouse leukemia cells. J. biol. Chem. 251: 3569-3575. MORAN, R. G., MULKINS, M. and HEIDELBERGER, C. (1979) Role of thymidylate synthetase activity in development of methotrexate cytotoxicity. Proc. natn. Acad. Sci. U.S.A. 76: 5924-5928. NIETHAMMER, D. and JACKSON,R. C. (1975) Changes of molecular properties associated with the development of resistance against methotrexate in human lymphoblastoid cells. Eur. J. Cancer 11: 846-854. NIMEC, Z. and GALIVAN, J. (1983) Regulatory aspects of the glutamylation of methotrexate in cultured hepatoma cells. Archs Biochem. Biophys. 226: 671-680. OHNOSHL T., OHNUMA, T., TAKAHASHI, I., SCANLON, K.. KAMEN, B. A. and HOLLAND, J. T. (1982) Establishment of methotrexate-resistant human acute lymphoblastic leukemia cells in culture and effects of folate antagonists. Cancer Res. 42: 1655-1660. PINEDO, H. M., ZAHARKO, D. S., BULL, J, M. and CHABNER, B. A. (1976) The reversal of methotrexate cytotoxicity to mouse bone marrow cells by leucovorin and nucleosides. Cancer Res. 36: 4418-4424. ROSENaLA'rr, D. S., WHrrEHEAD, V. M., VERA, N., POT'rIER. A., DuPONT, M. and VUCHICH, M.-J. (1978) Prolonged inhibition of DNA synthesis associated with the accumulation of methotrexate polyglutamates in cultured human cells. Molec. Pharmac. 14: 1143-1147. ROSENBLATT, D. S., WHITEHEAD V, M., VUCHICH, M.-J., POTTIER, A., VERA, N. and BEAULIEU, D. (1981) Inhibition of methotrexate polyglutamate accumulation in culture of human cells. Molec. Pharmac. 19: 87-97. ROSENBLATT, n. S., WHITEHEAD, V. M., MATIASZAK, N. V., POTTIER, A., VUCHICH, M.-J. and BEAULIEU, D. (1982) Differential effects of folinic acid and glycine, adenosine, and thymidine as rescue agents in methotrexate-treated human cells in relation to the accumulation of methotrexate polyglutamates. Molec. Pharmac. 21: 718-722. RosowsKY, A., LAZARUS, H., YUAN, G. C., BELTZ, W. R., MANGINI, L., ABELSON, H. T., MODEST, E, J. and FREI, E. (1980) Effects of methotrexate esters and other lipophilic antifolates on methotrexate-resistant human leukemic lymphoblasts, Biochem. Pharmac. 29: 648-652. RYSLER, H. J.-P. and SHEN, W.-C. (1978) Conjugation of methotrexate to poly(L-lysine) increases drug transport and overcomes drug resistance in cultured cells. Proc. natn. Acad. Sci. U.S.A. 75: 3867-3870.

Cellular pharmacology of methotrexate

101

SCHILSKY, R. L.. JOLIVET, J., BAILEY, B. D. and CHABNER, B. A. (1983) Synthesis, binding and intracellular retention of methotrexate polyglutamates by cultured human breast cancer cells. In: Folyl and Ant!folyl Polyglutamates, pp. 247-258, GOLDMAN, I. D., CHABNER, B. A. and BERTINO, J. R. (eds) Plenum Press, New York. SIROTNAK, F. M. and DONSBACH, R. C. (1972) Comparative studies on the transport of aminopterin, methotrexate, and methasquin by the L1210 leukemia cell. Cancer Res. 32: 2120-2126. SIROTNAK, F. M. and DONSBACH, R. C. (1975) Further evidence for a basis of selective activity and relative responsiveness during antifolate therapy of murine tumors. Cancer Res. 35: 1735-1744. SIROTNAK, F. M. and DONSBACH, R. C. (1976) Kinetic correlates of methotrexate transport and therapeutic responsiveness in murine tumors. Cancer Res. 36: l151-1158. SIROTNAK, F. M., KURITA, S. and HUTCHINSON, D. J. (1968) On the nature of a transport alteration determining resistance to amethopterin in the LI210 leukemia. Cancer Res. 28: 75-80. SIROTNAK, F. M., MOCOO, D. M. and DORICK, D. M. (1978) Optimization of high-dose methotrexate with leucovorin rescue therapy in the LI210 leukemia and sarcoma 180 murine tumor models. Cancer Res. 38: 345-353. SIROTNAK, F. M., CHELLO, P. L., MOCCIO, D. M., KISLIUK, R. L., COMBEPINE, G., GAUMONT, Y. and MONTGOMERY, J. A. (1979) Stereospecificity at carbon 6 at formyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro. Biochem. Pharmac. 28: 2993-2997. S1ROTNAK, F. M., MOCCIO, D. M., KELLEHER, L. E. and GOUTAS, L. J. (1981a) Relative frequency and kinetic properties of transport-defective phenotypes among methotrexate-resistant LI210 clonal cell lines derived in vivo. Cancer Res. 41: 4447-4452. SIROTNAK, F. M.. MoccIo, D. M. and YOUNG, C. W. (1981b) Increased accumulation of methotrexate by murine tumor cells in vitro in the presence of probenecid which is mediated by a preferential inhibition of efflux. Cancer Res. 41: 966-970. SIROTNAK. F. M., MOCCIO, D. M., GOUTAS, L. J., KELLEHER,L. E. and MONTGOMERY,J. A. (1982) Biochemical correlates of responsiveness and collateral sensitivity of some methotrexate-resistant murine tumors to the lipophilic antifolate, metoprine. Cancer Res. 42: 924-928. SmOTNAK, F. M., DEGRAW, J. I., MOCCIO, D. M., SAMUELS, L. L. and GOUTAS, L. J. (1984a) New folate analogs of the 10-deaza-aminopterin series. Basis for structural design and biochemical and pharmacologic properties. Cancer Chemother. Pharmae. 12: 18-25. SIROTNAK, F. M., D~GRAW, J. I., SCHMID, F. A., GOUTAS, L. J. and MOCCIO, D. M. (1984b) New folate analogs of the 10-deaza-aminopterin series. Further evidence for markedly increased antitumor efficacy compared with methotrexate in ascitic and solid murine tumor models. Cancer Chemother. Pharmac. 12: 26-30. SKEEL. R. T., SAWICKI,W. L., CASHMORE,A. R. and BERaaNO, J. R. (1973) The basis for the disparate sensitivity of L1210 leukemia and Walker 256 carcinoma to a new triazine folate antagonist. Cancer Res. 33: 2972-2976. SKEEL. R. T., CASHMOgE, A. R. and BER~NO, J. R. (1976) Clinical and pharmacologic evaluation of triazinate in humans. Cancer Res. 36: 48-54. WAMPLER, G. L., CARTER, W. H., CAMBELL, E. D. and GOLDMAN, I. D. (1983) Exploration of methods for demonstrating therapeutic synergism utilizing a time-interval between the interacting drugs methotrexate and teniposide. Proc. Am. Ass. Cancer Res. 24: 1060. WASHTIEN, W. L. (1982) Thymidylate synthetase levels as a factor in 5-fluorodeoxyuridine and methotrexate cytotoxicity in gastrointestinal cells. Molec. Pharmac. 21: 723-728. WHITE, J. C. (1979) Reversal of methotrexate binding to dihydrofolate reductase by dihydrofolate. Studies with purified enzyme and computer modelling using network thermodynamics. J. biol. Chem. 254: 10889-10895. WHITE, J. C. (1983) Predictions of a network thermodynamics computer model relating to the mechanism of methotrexate rescue by 5-formyltetrahydrofolate and to the importance of inhibition of thymidylate synthase by methotrexate polyglutamates. In: Folyl and Antifolvl Polyglutamates, pp. 305-327, GOLDMAN, I. D., CHABNER, B. A. and BER~NO, J. R. (eds) Plenum Press, New York. WHITE. J. C. and GOLDMAN, I. D. (1976) Mechanism of action of methotrexate. IV. Free intracellular methotrexate required to suppress dihydrofolate reduction to tetrahydrofolate by Ehrlich ascites tumor cell in vitro. Molec. Pharmac. 12: 711-719. WHITE, J. C. and GOLDMAN, I. D. (1979) Lack of stereospecificity at carbon 6 of methyltetrahydrofolate transport: possible relevance to rescue regimens with methotrexate and leucovorin. In: Chemistry and Biology o f Pteridines, pp. 625-627, KISLIUK, R. L. and BROWN G. M. (eds) Elsevier, New York. WHITE, J. C. and GOLDMAN, I. D. (1981) Methotrexate resistance in an L1210 cell line resulting from increased dihydrofolate reductase, decreased thymidylate synthase, and normal membrane transport: computer simulations based on network thermodynamics. J. biol. Chem. 256: 5722-5727. WHITE, J. C., LOF~IELD, S. and GOLDMAN, I. D. (1975) The mechanism of action of methotrexate. III. Free intracellular methotrexate is required for maximum suppression of ~4C-formate incorporation into nucleic acids and proteins. Molec. Pharmac. 11: 287-297. WHITE, J. C., BAILEY, B. D. and GOLDMAN, I. D. (1978) Lack of stereospecificity at carbon 6 of methyltetrahydrofolate transport in Ehrlich ascites tumor cells: carrier-mediated transport of both stereoisomers. J. biol. Chem. 253: 242-245. WHITE, J. C., CARCHMAN, R. A., FRY. D. W. and GOLDMAN, I. D. (1980) Relationship between membrane transport of methotrexate and endogenous cyclic adenosine 3',5'-monophosphate in the Ehrlich ascites tumor. Cancer Res. 40: 2400-2404. WHITEHEAD. V. M. (1977) Synthesis of methotrexate polyglutamates in L1210 murine leukemia cells. Cancer Res, 37: 408-412. YALOWICH. J. C., FRY. D. W. and GOLDMAN. I. D. (1982) VM-26 and VP-16-213 induced augmentation Of methotrexate uptake and intracellular levels of polyglutamyl derivatives in Ehrlich ascites tumor cells /n ritro. Cancer Res. 42: 3648-3653.

102

I . D . GOLDMAN and L. H. MATHERLY

X/'ANG, C.-H., PETERSON, R. H. F., SlrtOTNAK, F. M. and CHELLO. P. L. (1979) Folate analog transport by plasma membrane vesicles isolated from L1210 leukemia cells, d. biol. Chem. 25: 1402-1407. YANG, C.-H., DEMBO, M. and SIROTNAK, F. M. (1982) Two-compartment behavior during transport of folate compounds in LI210 cell plasma membrane vesicles. J. Memb. Biol. 68: 19-28. YANG, C.-H., SIROTNAK, F. M. and DEMBO, M. (1984) Interaction between anions and the methotrexate transport system in LI210 cell plasma membrane vesicles. Fedn Proc. 43: 1779.