Pharmac. Ther, Vol. 4. pp. 307-327 © Pergamon Press Ltd. 1979. Printed in Great Britain

Specialist Subject E d i t o r s :

0163-725817910201--03071505.0010

ALAN C. SARTORELLI, WILLIAM A. CREASEY and JOSEPH R. BERTINO

BIOCHEMICAL AND KINETIC APPROACHES TO INHIBITION OF MULTIPLE PATHWAYS G. B. GRINDEY and Y. C. CHENG* Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial Institute, Buffalo, New York 14263, USA

IT IS NOW generally accepted that there are thousands of biochemical reactions that occur inside cells and that most, if not all, of these reactions are catalyzed by specific enzymes. The overall characteristics of individual cell types will depend on the makeup of the cellular biochemical network of enzymes, as well as the nutritional environment of the cell. For example, some cells which are deficient in thymidine kinase will depend solely on the de n o v o synthesis of thymine nucleotides for DNA synthesis (Kit et al., 1966), While others may depend exclusively on salvage metabolites for purine nucleotide biosynthesis (Murray, 1971; Scholar and Calabresi, 1973). In this review, we will limit the discussion to the type of components that make up multiple enzyme pathways or networks, the importance of each component in determining drug interactions within that network, and the complications involved in predicting such interactions. Finally, additional biochemical approaches leading to increased drug activity against a specific target cell will be discussed. 1. COMPONENTS OF MULTIPLE-ENZYME SYSTEMS The absolute amount of enzyme in a cell is determined by its overall rate of synthesis and degradation. The rate of synthesis will depend upon the transcription and translation of mRNA, as well as maturation and modification processes, while the rate of degradation will depend upon the stability of the enzyme as well as its availability to proteolytic enzymes. For instance, the increased levels of dihydrofolate reductase which occur in cells treated with methotrexate (Hakala et al., 1961; Hillcoat et al., 1967; Raunio and Hakala, 1967) probably result from increased levels of mRNA for this enzyme (Hangii and Littlefield, 1976; Kellens et al., 1976). In contrast, nucleoside diphosphokinase is stabilized against degradation by trypsin by the presence of its substrates (Agarwal and Parks, 1971). The time required for changes in the level of an enzyme from one steady state to another will depend primarily on its rate of degradation (Schimke, 1973). Kinetically, enzymes can be classified into two broad categories: those that display Michaelis-Menten kinetics and those which exhibit more complex non-Michaelis-Menten kinetics. Complex kinetics are a function of factors such as the interaction between multiple substrates for binding to the enzyme, the effect of metal activators, the presence of a number of different interconverting species of the same enzyme, and the interaction of regulatory ligands. In addition, there are multiple enzyme species which are not spontaneously interconvertible but which catalyze the same reaction within cells. These isozymes may have different kinetic properties. For example, six distinguishable nucleoside diphosphokinases have been isolated from human cells (Cheng et al., 1971), while the thymidine kinase isolated from the cytosol fraction has a different substrate specificity than the enzyme found in the mitochondrial fraction (Leung, et al., 1975; Lee and Cheng, 1976; Cheng et al., 1977). Such isozymes may be coded by different genes, or alternatively, they may represent one enzyme species at different stages of maturation, modification, or *American LeukemiaSocietyScholar. Supported in part by USPHS grants CA-17156. 307

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G. B. GRINDEYand Y. C. CMENG

degradation. Topologically, enzymes in the same pathway may exist in complex form and each individual enzyme may behave differently. The metabolic pools of chemicals inside the cell are controlled by their rates of synthesis and conversion to other metabolic products. However, the total concentration of an intracellular metabolite does not necessarily reflect its concentration at a given site due to the complication of cellular compartmentalization or of binding of the metabolite by macromolecules. An example of this is the proposed compartmentalization of the deoxyribonucleoside triphosphates in cell nuclei (Skoog and Bjursell, 1974). These considerations may complicate predictions of the metabolic activity of some pathways based on the properties and amount of the specific enzymes and the total pool sizes of the substrates involved. Most enzyme networks are regulated to some extent by metabolites that are involved in the network, and, which in many instances are the end-products of that pathway. The effects of these metabolites can be classified into three basic categories: (a) those that regulate the activity of the enzyme by direct interaction, such as feedback inhibitors or activators (Moore and Hurlbert, 1966; Brown and Reichard, 1969); (b) those that regulate the overall amount of enzyme present in the cell, such as inducers, suppressors, stabilizers and destabilizers (Bonney and Maley, 1975); and (c) those that regulate the activity of the enzyme through indirect effects mediated by such functions as the reaction sequence of the enzyme or overall structure of the pathway under consideration (Werkheiser et al., 1973; Dolnick and Cheng, 1978). In the steady-state, the concentration of all components (i.e. enzymes, substrates, intermediates, products and regulators), as well as the velocity of the initial and all other reactions in the network remain constant with time. The origin of a steady-state multi-enzyme network is defined as the reaction whose substrate concentration does not change as a result of physiological or pharmacological perturbations in the system (Webb, 1963; Grindey et al., 1975; Harrap and Jackson, 1975). Such a condition exists for pathways which account for a small amount of the total flux of the initial reactant or for nutrients which are homeostatically controlled (Grindey et al., 1975; Harrap and Jackson, 1975). The network of the multiple enzyme system can be constructed through monolinear chains, branched chains which could be either convergent or divergent, polylinear chains, cyclic systems, distributive systems, regenerative systems, self-regulatory systems, or combinations of these. As discussed by Webb (1963), the dynamics of these systems depend upon their construction. In considering the following examples under steady-state conditions, Vl

A

a

>a

v2

>B

, C

2...~.~B.../

Case I

,D

Case II

for Case I, 1)1 ~--" 1)2, while for Case II, vl = 1)2--I-1)3 = /)4 + 1)5= 1)6"In Case I, a change in v2 would require a change in vl for the system to remain in the steady-state, whereas in Case II, a change in v2 might be compensated for by a change in 1)3 and thus might not have any effect on the overall flux through the pathway (v6). The reversibility of enzyme reactions in the system is also an important factor. For example, in Case I, an increase in B has no effect on v,. However, if this reaction were reversible, then an increase in B would cause some inhibition of v~. The transit time or diffusion rate of an intermediate from one enzyme to the next may also have effects. Transit time is determined by the spatial arrangement of the multiple enzyme system, the diffusion constant of the intermediate, and the microenvironment of the network. The interaction of all of these components must be considered when the effects of multiple inhibitors on such enzyme networks are evaluated. While each component may not play a major role in all drug interactions, each can have important

Biochemicaland kinetic approaches to inhibitionof multiplepathways

309

consequences for specific drug interactions. In this review, we will discuss the complications that exist in predicting drug effects on metabolic networks by beginning with basic theoretical considerations and then developing more complicated networks. In this way, the role of each component in determining drug interactions may be better understood. 2. COMBINATION OF INHIBITORS OF SINGLE ENZYMES The kinetics of dual inhibition of a single, one substrate or two substrate enzyme have been described in detail previously (Webb, 1963; Segel, 1975). This type of interaction is usually evaluated by comparison of the effects of relative amounts of the two inhibitors with respect to their specific concentration, which is defined as the absolute concentration of drug divided by the K~ for the enzyme that is inhibited by the drug (Webb, 1963; Segel, 1975). For drugs whose primary mechanism of action is not known, drug interactions may be evaluated using fractional concentrations of the two drugs which achieve a constant effect (usually 50 per cent inhibition). For example, if one-half the concentration of the first inhibitor (It) required to achieve a constant effect (i.e. 50 per cent inhibition) added to one-half the concentration of the second inhibitor (12) required to achieve the same effect yields 50 per cent inhibition, then the interaction is additive (Elion et al., 1954; Grindey et al., 1975). In terms of K~, if the degree of inhibition obtained at either It or/2 equal to two K~ values is idcnticai to the degree of inhibition obtained by combination of the two agents at concentrations equal to their respective K~ values, then the interaction is defined as additive (Segel, 1975). Effects greater than those defined as additive are termed synergistic, while those less than expected are termed antagonistic. Kinetic derivations (Webb, 1963; Segel, 1975) clearly indicate that if the binding of the first inhibitor excludes the binding of the second to the enzyme (i.e. mutually exclusive binding), then an additive interaction will occur from a combination of these two agents. The type of inhibition (i.e. competitive, uncompetitive, or non-competitive) of the two inhibitors does not alter this conclusion. If, however, both agents can simultaneously bind to the enzyme, then the interaction will be synergistic. These predicted interactions (Grindey et al., 1975) are summarized in Table I. The above considerations do not necessarily imply the existence of more than one binding site on the enzyme for the inhibitors (Grindey et al., 1975; Segel, 1975); however, certain enzymes may have more than one binding site for inhibition, and these binding sites may or may not act independently. If the binding of one agent to the first site causes a change in the enzyme molecule which facilitates the binding of the second drug, then the degree of synergism between the two agents may be greatly enhanced. Other factors which affect the degree of synergism between two agents that bind simultaneously to the same enzyme include substrate concentrations and the kinetic parameters of the inhibited reaction (Grindey et al., 1975). TABLE !. Predicted Interactions for Inhibition of Either a One-Substrate or Two-Substrate Enzyme °

Type of inhibitof Cli+ cI2 ~Ii+ "12 "11+ "12 ~I1+ cI2 Cll+ "12 "Ii + "12 .... 11+ .... 12 ....

It + c or.12

Bindinginteraction Mutuallyexclusive Mutuallyexclusive Mutuallyexclusive Non-exclusive binding Non-exclusive binding Non-exclusive binding Bindingof I~ partiallyinterferes with bindingof 12 Bindingof 11facilitatesbindingof/'2

Interaction Additive Additive Additive Synergistic Synergistic Synergistic Synergistic Highlysynergistic

°These predictions were taken from G. B. Grindey,R. G. Moran and W. C. Werkheiser (1975). b~l, competitiveinhibitor;"Is non-competitiveor uncompetitiveinhibitor. JPT Vol. 4, No. 2 - - E

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G.B. GRINDEY and Y. C. CHENG

An example of a highly synergistic interaction resulting from dual inhibition of a single enzyme is the dramatic potentiation of the growth-inhibitory activity of adenosine analogs by guanine nucleosides (Grindey et al., 1976). Studies on the isolated enzyme phosphoribosylpyrophosphate (PRPP) amidotransferase, the first committed step in the de n o v o biosynthetic pathway for purine nucleotides, have indicated that it is regulated by the ribonucleotide end-products of that pathway. Kinetic evaluation indicates that there are separate binding sites on the enzyme for the ribonucleotides of 6-hydroxy- and 6-aminopurines, and that both types of nucleotides may act simultaneously (Caskey et al., 1964). Combination of adenine and guanine ribonucleotides has produced more than additive inhibition of the enzyme isolated from various sources (Nierlich and Magasanik, 1965; Hill and Bennet, 1969; Shiio and Ishii, 1969; Tay et al., 1969; Wood and Seegmiller, 1973). In intact cells, Henderson (1963), assaying the inhibition of the formation of 5'-phosphoribosyl-Nformylglycinamide (FGAR) from glycine, demonstrated the strong potentiation of feedback inhibition of the purine nucleotide pathway de n o v o by the combination of adenine and guanine. Concentrations of the two purines i n combination, which produced only weak inhibition when used alone, caused almost complete inhibition of the assay system. Potentiation of the inhibition of FGAR formation by combinations of 2,6-diaminopurine and adenine also were observed. However, since adenine can bypass the inhibition of the de n o v o purine nucleotide pathway by supplying purine nucleotides through utilization by the salvage routes, no growth inhibition was observed with this combination. Certain cells cannot utilize guanine or guanosine as a source of all cellular purines (Schaffer, 1973), thus marked growth inhibition of both leukemia L1210 and Sarcoma 180 cells in culture was produced by a combination of guanine nucleosides with 6-methylmercaptopurine ribonucleoside (MMPR) (Grindey et al., 1976). In L1210 cells, the concentration of MMPR required for 50 per cent inhibition of growth was 0.07 tzM. Addition of either guanosine (10 ~r,t) or deoxyguanosine (20/zM) produced a thirty-five-fold potentiation of MMPR and thus reduced the concentration required for 50 per cent inhibition of growth to 0.002/xrd. The dramatic inhibition of FGAR synthesis by this combination indicated that the site of blockage responsible for this synergism is an early step in the de n o v o purine nucleotide pathway, probably at the level of PRPP amidotransferase. The action of several other purine analogs are also potentiated by guanine or its nucleosides and these include 4-aminopyrazolo(3,4d)pyrimidine (Zimmerman and Mandel, 1964; Schachtschabel and Ferro, 1967), the carboxycyclic analog of adenosine (Hill et al., 1971), N6-(A2-isopentenyl)adenosine, 6-chloropurine ribonucleoside, and several other N6-substituted adenosines (Grindey et al., 1976). Unfortunately, however, no analogs of guanosine have yet been found which dramatically potentiate the activity of these purine antimetabolites. Another interesting example of dual inhibition of a single enzyme involves the potentiation of the activity of certain inhibitors of ribonucleotide reductase by the iron chelator, deferoxamine. As discussed by Brockman (1974), iron is required for the enzymatic reduction of ribonucleotides, and chelation of this cofactor by deferoxamine is capable of inhibiting the isolated enzyme. Against adenocarcinoma 755 cells in culture (Brockman et al., 1972), sub-inhibitory concentrations of ribonucleotide reductase inhibitors (i.e. guanazole, hydroxyurea and pyridine-2-aldehyde thiosemicarbazone) were potentiated by non-inhibitory concentrations of deferoxamine. Enhanced antitumor activity of 5-hydroxy-pyridine-2-carboxaldehyde against leukemia L1210 in combination with deferoxamine also was observed (Brockman, 1974). 3. COMBINATION OF INHIBITORS OF MULTIPLE ENZYMES Potter (1951) proposed the concept of :sequential inhibition' (Table 2) for the combination of agents in cancer chemotherapy. Sequential inhibition was defined as: 'the use of two or more inhibitors, each of which acts on the same metabolic sequence but upon different enzymes within a limited portion of the sequence.' It was proposed

Biochemical and kinetic approaches to inhibition of multiple pathways TABLE 2. Simple Models for Dual Inhibition of Biochemical Pathways Type of interaction

Descriptive model"

1. Sequential inhibition

2. Concurrent inhibition

z,

A

l I" e

A

Jt.a

/2

[[ : C

.¢

oY Ii

I2

z,

;2

3. Sequential (convergent)

o/ 4. Sequential (divergent)

5. Bicentric inhibition o

[I

A

IILe.

6. Bicentric depletion

~C

12

o. I I ~

If 7. Bicentric inhibition and depletion

A. . . .

~----

C

o ,J rl

8. Cyclic inhibition

~

D "~" ~-~C

£z

9. Concerted inhibition

10. Sequential cyclic inhibition

A

It * B

O

"In these simple models, A is considered the origin of the steady-state chain and thus is constant while C is the product of the pathway.

311

312

G. B.

GRINDEYand

Y. C.

CHENG

that an accumulation of the substrate of the inhibited reaction may partially reverse the activity of an antimetabolite and that inhibition of a prior enzyme in the same sequence would negate this effect. Elion et. al. (1954) evaluated combinations of base analogs and antifolates against bacteria as examples of a group of antimetabolites with at least three sit~s of action arranged sequentially along the same biochemical pathway. In addition to the striking synergism between sulfonamides and diamino, pyrimidines, which has been quoted by Hitchings (1969) as a classic example of sequential inhibition, many combinations of purine and thymine analogs also have been found to be strongly synergistic. The concept of 'concurrent blocking' was proposed to explain the highly synergistic interactions among combinations of i antagonists of nucleic acid synthesis and was defined as 'the simultaneous inhibition of the utilization of two or more precursors of the same product' (Table 2) (Elion et al., 1954). Skipper et al. (1954) described additional examples of these two concepts for cancer chemotherapy. The kinetics of a simplified model of sequential inhibition have been extensively debated in the literature (Black, 1963; Rubin et al., 1964; Webb, 1963; Handschumacher, 1965; Sartorelli, 1969; Grindey and Nichol, 1972; Grindey et al., 1975; Harrap and Jackson, 1975). Black (1963) concluded that synergism was an intrinsic property of dual inhibition of a monolinear pathway; however, as originally pointed out by Rubin et al. (1964), and further discussed by Grindey et al. (1975), the assumptions leading to this conclusion are not applicable to conditions existing in intact cells or isolated systems. Moreover, Jackson (R. C. Jackson, personal communication, 1975) has concluded that mathematical errors exist in Black's derivation. Webb (1963) described the kinetics of a simple monolinear enzyme chain operating at a steady-state (Table 2). He noted that either a competitive or non-competitive inhibitor ( .... 11) acting on El alone (the origin of the steady-state chain) would depress the steady-state rate of formation of C to the same extent that it inhibits the reaction. A non-competitive inhibitor (hi2) acting on the second enzyme (E:), however, would not decrease the formation of C unless this enzyme became rate-limiting and thus removed the system from the steady-state. It should be pointed out that a competitive inhibitor (cI2) acting on E2 would have no effect on the system, since there are no constraints on the concentration of B. Only when the physical state of the system prevents sufficient rise in B to compensate for the inhibition by cI2 (such as diffusion from the compartment) will there be any effect on the system (assuming that the transition time of the system is minimal). In such a system, combination of two inhibitors (. . . . Ii + . . . . /2) would produce no greater inhibition than that observed with the most effective agent alone. If El were considered reversible, there would be some constraints on the accumulation of B. A combination of two inhibitors in such a system would inhibit the overall reaction sequence more strongly than a single agent, but less than that expected for an additive interaction. Thus, Webb (1963) concluded that 'multiple inhibition in simple monolinear chains would seem generally to be incapable of producing an effect much greater than a single inhibitor, and a marked potentiation of inhibition would be out of the question'. Rubin et al. (1964) analyzed the kinetics of sequential inhibition of de n o v o pyrimidine nucleotide biosynthesis in cell-free extracts and in intact cells utilizing 5-azaorotate, an inhibitor of orotidylic pyrophosphorylase, and 6-azauridine, an inhibitor of orotidylate decarboxylase. Kinetic analysis of inhibition of these two sequential enzymes completely supported the theoretical points made by Webb (1963). The addition of 5-azaorotate, an inhibitor of the first enzyme, produced a 50 per cent reduction in the rate of product formation; however, the further addition of 6azauridine, an inhibitor of the second enzyme, in a concentration sufficient to suppress the rate of orotidylate decarboxylase by up to 95 per cent, did not cause suppression of product formation greater, than that produced by the maximally inhibitory agent alone (Rubin et al., 1964). Only when sufficient inhibitor is added to cause the second enzyme to become the rate-limiting reaction, can the effect of this inhibitor be observed (Handschumacher, 1965). Handschumacher (1965) also discussed

Biochemical and kinetic approaches to inhibitionof multiple pathways

313

the kinetics of concurrent inhibition with respect to the simple model shown in Table 2. He concluded, in agreement with Webb (1963), that kinetic consideration of isolated enzymes could not explain potention of growth inhibition by combinations of sequential or concurrent inhibitors, when viewed in terms of simlble monolinear or convergent chains. In intact cells, enzyme networks are more complicated than simple monolinear chains (Webb, 1963, Handschumacher, 1965; Sartorelli, 1969; Hitchings, 1969; Grindey and Nichol, 1972; Nichol et ai., 1972; Grindey et al., 1975; Harrap and Jackson, 1975). Harrap and Jackson (1975) extended these considerations to include more complex examples of dual inhibition of multiple enzymes, as well as effects that occur during the transition time (i.e. time taken to reach a new steady-state). These examples include sequential (monolinear, convergent and divergent), concurrent, bicentric, and cyclic inhibition, bicentric depletion, and bicentric inhibition and depletion (Table 2). On theoretical kinetic considerations, they too concluded that dual inhibition of these relatively simple models of multi-enzyme systems would, for the most part, result in a degree of inhibition similar to that observed with each agent alone and less than that expected for an additive interaction. An analysis of dual inhibition of the cyclic thymidylate synthetase system, using mixtures of purified dihydrofolate reductase and thymidylate synthetase was conducted to verify these kinetic predictions (Harrap and Jackson, 1975). Methotrexate was utilized as an inhibitor of dihydrofolate reductase, while 5-fluorodeoxyuridylic acid was used to inhibit thymidylate synthetase. With thymidylate synthetase made ratelimiting in the cyclic system (dihydrofolate reductase added in twenty-fold excess), the addition of levels of methotrexate which substantially inhibited the total dihydrofolate reductase activity produced levels of inhibition no greater than that observed by 5-fluorodeoxyuridylate alone. These experiments showed that in simple cyclic systems, only inhibition of the rate-limiting enzyme alters the overall flux through the system. Inhibition of the other enzyme has an effect only when the degree of inhibition is sufficient to make it the rate-limiting process. In addition, the transition time from one steady-state to the next was too rapid to detect on a time-scale of minutes. Harrap and Jackson (1975) concluded that: (a) the treatment of enzyme kinetics of multi-enzyme pathways which is most applicable to living organisms is that of an open, steady-state system with constrained inputs; (b) the transition time of such systems probably does not exert a significant effect on drug interactions; (c) the kinetic analysis of simple metabolic networks appears to offer adequate explanations for the effects of two inhibitors on mixtures of isolated enzymes; and (d) the TABLE 3. Predicted Interactions for Sequential Inhibition of a Simple Monolinear Chain under Feedback Regulation ° Type of Feedback Binding interaction inhibitor° inhibitorc between A and Ii Interaction cI! + ci2 ell + cI2 el, + cI2 "I, + Cl 2 Cl~+ "12 "I~ + "I2 "It + "12 "I~ + "12 "I~ + "12 "I~ + "12 "Ii + "I2

cA CA CA cA CA "A nA "A CA CA cA

Mutually exclusive Independent Stimulatory Independent Mutually exclusive Mutually exclusive Independent Stimulatory Mutually exclusive Independent Stimulatory

Additive Synergistic Synergistic Synergistic Antagonistic Antagonistic Additive Synergistic Antagonistic Antagonistic Parameter-dependent

"These predictions were taken from G. B. Grindey, R. G. Moran and W. C. Werkheiser (1975). boil, competitive inhibitor with respect to substrate(S); Cl2, competitive inhibitor with respect to allosteric effector (A), "Ii, non-competitive inhibitor with respect to S; "12, non-competitive inhibitor with respect to A. CCA, competitor inhibitor with respect to S; "A, non-competitive inhibitor with respect to S.

314

G.B. GmNDEVand Y. C. CnF3~6

synergistic interaction between certain combinations of drugs cannot be explained by kinetic consideration of these simple models. Grindey et al. (1975) have evaluated the effect of feedback regulation on dual inhibition of a simple monolinear chain (Concerted Inhibition, Table 2). The interactions predicted for combinations of different inhibitors in this simple model are summarized in Table 3. Whereas for the previous simple models only antagonistic interactions (i.e. inhibitions not significantly greater than those produced by the more active agent) were predicted, several synergistic interactions were predicted in this system. With respect to sequential inhibition of a simple monolinear chain constrained by feedback regulation, the authors concluded that: (a) two inhibitors of sequential enzymes can result in any pattern of interaction (i.e. synergistic, additive, or antagonistic); (b) there is no simple correlation between the type of inhibition of each enzyme and the interaction predicted; (c) in the majority of cases the affinity of substrate, inhibitor, or feedback effector; the maximal velocities of the inhibited enzymes; or the substrate concentrations did not affect the type of interaction predicted (i.e. most cases were parameter independent); and (d) the type of interaction depended on the mechanism of inhibition by each agent and the characteristics of feedback regulation. 4. DUAL INHIBITION OF FOLATE METABOLISM One of the most interesting and useful combinations to date involves the inhibition of the synthesis of folate cofactors in bacteria by sulfonamides and the simultaneous inhibition of dihydrofolate reductase by trimethoprim (Bushby and Hitchings, 1968). In addition to the high degree of synergism between the two agents, the spectrum of activity is altered and the development of resistance is retarded (Bushby, 1969). The combination of these two drugs is effective against organisms that are insensitive, or only marginally sensitive, to either agent alone and, thus, best exemplifies metabolic synergism. Hitchings (1969) has discussed the biochemical aspects of these two types of inhibitor. The diaminopyrimidines block the reduction of dihydrofolate cofactors to tetrahydrofolate by inhibition of dihydrofolate reductase, while the sulfonamides interfere with the synthesis of these cofactors by competing with the utilization of p-aminobenzoic acid. Thus, this dual inhibition of the pathway of folate cofactor synthesis constitutes a form of sequential blockade (Hitchings, 1969). As discussed in the previous sections, theoretical considerations of dual inhibition of a simple monolinear chain indicated that sequential inhibition may result in an antagonistic interaction. However, Harrap and Jackson (1975)considered this combination to be an example of a sequential convergent system (Table 2), while Grindey et al. (1975) proposed that the system consisted of a linear sequence feeding into a cycle (Table 2). Consideration of this sequential-cyclic system, led to the conclusion that the combination should be highly synergistic. These different theoretical predictions for the same combination, utilizing similar kinetic approaches, highlight the role that the basic structure of a metabolic pathway plays in determining drug interactions within the network. While the simple model described by Grindey and co-workers (1975) is adequate to predict the synergistic interaction for this combination, more detailed models are required for a better understanding of the complexities of the system and for the utilization of these models for further predictions. Such models of folate metabolism have been described for mammalian cells by Jackson and Harrap (1973) and for E. coli by Harvey and Dev (1975). The simpler models, however, are useful as tools to explore the role of individual factors in certain drug interactions. Consider the antagonistic interaction between methotrexate and 5-fluorodeoxyuridine (Grindey et al., 1975) or 5-fluorouracil (Harrap and Jackson, 1975; Tattersall et al., 1973). Methotrexate has been shown to be a competitive, albeit extremely tight binding, inhibitor of dihydrofolate reductase (Werkheiser, 1961), while 5-fluorodeoxyuridine and 5-fluorouracil, after conversion to 5-fluorodeoxyuridylic acid, inhibit thymidylate synthetase competitively with dUMP

Biochemicaland kineticapproachesto inhibitionof multiplepathways

315

(Reyes and Heidelberger, 1965). The kinetics of inhibition by these two agents have been described by Harrap and Jackson (1975), and Grindey et al. (1975) as dual inhibition of a cyclic system of the type discussed in detail by Webb (1963). As pointed out by Harrap and Jackson (1975), the behavior 'of this system closely resembles that of a sequential monolinear system and thus the predicted interaction for the combination of methotrexate and 5-fluorouracil or 5-fluorodeoxyuridine should be antagonistic rather than additive or synergistic. As discussed earlier (Section 3), Harrap and Jackson (1975) verified this prediction by evaluating the effect of these two inhibitors on this cyclic system, utilizing mixtures of the two purified enzymes. While such treatment outlined the role of the basic structure of the pathway in determining the antagonistic interaction, clearly other factors are also important. Thus, Tattersall et al. (1975)found that the addition of methotrexate to L5178Y cells in culture elevated the pool of dUMP eight-fold. Utilizing the characteristics of the isolated enzyme and the cellular pools of the inhibitor, these authors calculated that this increase in dUMP would substantially reduce the inhibition of thymidylate synthetase by 5-fluorodeoxyuridylic acid, even though the ratio of the K~Km is greater than 1000. The toxicity of methotrexate to mammalian cells results from depletion of the tetrahydrofolate polyglutamate cofactors (Moran et al., 1975, 1976), due to inhibition of dihydrofolate reductase (Werkheiser, 1961). Since thymidylate synthetase is the only reaction in the cell which converts these cofactors to dihydrofolate polyglutamates, the rate of this enzymatic reaction is a major factor in determining the toxicity of methotrexate. In the presence of thymidine, 5-fluorouracil partially reverses the inhibition of growth of LS178Y cells in culture caused by methotrexate (Tattersall et al., 1973). In L1210 cells in culture, the addition of 5-fluorodeoxyuridine (2x 10-6M) plus thymidine increases the concentration of methotrexate required for 50 per cent inhibition of growth fifty-fold (R. G. Moran, personal communication). The addition of thymidine in these experiments is required to reverse the toxicity of 5-fluorouracil or 5-fluorodeoxyuridine. These results indicate that inhibition of thymidylate synthetase by 5-fluorodeoxyuridylic acid can have profound effects on methotrexate toxicity and also contributes to the observed antagonistic interaction (Grindey et al., 1975; Tattersall et al., 1973). The reaction mechanism for thymidylate synthetase is also important. As shown by Reyes and Heidelberger (1965) with thymidylate synthetase isolated from Ehrlich ascites carcinoma cells, the mechanism is ordered-sequential such that 5,10-methylene-tetrahydrofolate must bind to the enzyme prior to dUMP or 5-fluorodeoxyuridylic acid. In contrast, Dolnick and Cheng (1977a) have reported that thymidylate synthetase obtained from human acute myelocytic leukemia cells is also orderedsequential, with dUMP binding to the enzyme prior to the folate cofactor. Santi et al. (1977) have proposed that decreased levels of tetrahydrofolate cofactors produced by methotrexate would reduce the binding of 5-fluorodeoxyuridylic acid to mouse thymidylate synthetase and decrease the effectiveness of this agent. Indeed, the toxicity of 5-fluorodeoxyuridine was decreased in L1210 cells grown in culture on low levels of folic acid and, under these conditions, the (efficacy) of this agent was increased five-fold by the addition of folinic acid to the culture medium. These results imply that this mechanism can also decrease the toxicity of the combination. On the other hand, Bertino et al. (1977) have proposed that administration of methotrexate prior to 5-fluorouracil might enhance the binding of the anabolic metabolite 5-fluorodeoxyuridylic acid to thymidylate synthetase. While methotrexate is a weak inhibitor of the mouse enzyme, the drug will enhance the binding of 5-fluorodeoxyuridylic acid by replacing the tetrahydrofolate cofactor (Santi et al., 1977). Enhanced survival of mice bearing Sarcoma 180 was observed with this combination when the methotrexate was administered 2 hr prior to 5-fluorouracil, as compared to simultaneous administration (Bertino et at., 1977). A similar schedule dependency for this combination was observed by Martin et al. (1976) in a spontaneous murine mammary cancer. In contrast, Dolnick and Cheng (1977b) have demonstrated that methotrexate antagonizes the effect of 5-fluorodeoxyuridylic acid on

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purified human thymidylate synthetase. Thus, observations on purified enzyme obtained from one species may not always be applicable to the characteristics of the enzyme in another species, a situation which stresses the difficulty in directly extrapolating observations in mice to the clinical situation. These results also point out the importance of scheduling as a determinant of the nature of drug interactions. Combinations which are antagonistic when given concurrently may well be synergistic on a different schedule of administration. Finally, Tattersall et al. (1973) have proposed that perturbations in other metabolic pools within the network by methotrexate may decrease the conversion of 5-fluorouracil to the active metabolite, 5-fluorodeoxyuridylic acid, a factor which also contributes to the observed antagonistic interaction. Two additional considerations are the transport characteristics of methotrexate and the overall regulatory properties of the network of DNA synthesis. As described by Zager et al. (1973), a large number of drugs affect the transport characteristics of methotrexate into the cell. While hydrocortisone, L-asparaginase, cephalothin, hydroxyurea, bleomycin, and methylprednisolone all inhibit the uptake of methotrexate, vincristine and vinblastine increases the rate of uptake and the total amount of drug accumulated inside the cell. 5-Fluorouracil did not affect the uptake of methotrexate, and 5-fluorodeoxyuridine was not evaluated (Zager et al., 1973). Werkheiser et al. (1973) examined the effects of the feedback control of DNA synthesis on the type of interaction between combinations of five specific inhibitors of the system: methotrexate, l-formylisoquinoline thiosemicarbazone, arabinosylcytosine, arabinosyladenine and 5-fluorodeoxyuridine. A simplified, open, steady-state model of the system was constructed, and the predicted drug interactions were compared with those determined utilizing L1210 cells in culture (Grindey and Nichol, 1972). The authors (Werkheiser et al., 1973; Grindey and Nichol, 1972) concluded that the feedback regulation involved in the system was a primary determinant of the type of interaction between pairs of inhibitors of DNA synthesis. These studies, (Tattersall et al., 1973; Zager et al., 1973; Santi et al., 1974; Grindey et al., 1975; Harrap and Jackson, 1975; Bertino et al., 1977;) indicate that at least eight factors may play a role in determining the interaction between methotrexate and fluoropyrimidines. They include: (a) the basic structure of the system; (b) the regulatory properties of the system; (c) the reaction mechanism of the target enzyme; (d) the formation of active metabolites; (e) transport phenomena; (f) perturbations in metabolite pools; (g) perturbations in cofactor pools; and (h) the schedule of administration, The prediction of drug interactions within metabolic networks requires consideration of these factors, as well as the development of sophisticated models of the network under consideration. Such models are currently under development (Grindey et al., 1975, 1979; Jackson and Harrap, 1973), and form the subject of a recent review by Jackson and Harrap (1979). 5. INDUCED ALTERATIONS IN ENZYME LEVELS The levels of enzymes inside cells are subject to specific control mechanisms which alter their rates of synthesis and degradation. As a result of this regulation, the turnover rate of each enzyme or protein is different in various mammalian cells and tissues (Schimke, 1973), and the level of each enzyme may respond uniquely to exogenous factors. The basic molecular mechanisms responsible for controlling the rates of enzyme synthesis and degradation will not be discussed in this section. The discussion will be restricted to several known factors which can induce specific changes in the level of critical enzymes. It is well documented that variation in the diet can cause changes in enzyme levels in various organs. A direct relationship exists between caloric intake and the level of amino acids that act on catabolic enzymes such as those involved in the urea cycle (Schimke, 1962). In addition, the thymidine content of serum has been shown to alter the amount of thymidine kinase (Cramer and Sartorelli, 1969). The serum folate

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concentration can also induce changes in thymidylate synthetase in regenerating liver (Labow et al., 1969). Whether a given organ or cell will induce changes in enzyme levels in response to hormone stimulation depends on the presence of specific cellular receptors for that hormone. For example, steroid hormones do not exert any effect on cells if specific binding proteins are absent (Jensen et al., 1968; Gehring et al., 1971). The effects of hormones on different levels of enzymes have been examined extensively by various investigators in different systems. It is generally agreed that each hormone may have different effects on various organs and in the same organ, different hormones may have different effects. Thus, for example, glucagon can induce tyrosine aminotransferase but not tryptophan pyrrolase in rat liver (Greengard and Dewey, 1968); in contrast, both enzymes are induced by glucocorticoids (Kenney, 1970). The phenomenon whereby pre-exposure to one drug interferes with the action of the second has been recognized for years. Thus, phenobarbital can induce the mixed function oxygenase system in the endoplasmic reticulum of liver; this induction results in an increased capacity of liver to metabolize or activate certain drugs such as cyclophosphamide (Sladek, 1972). Methotrexate causes a progressive increase in the level of dihydrofolate reductase in mammalian cells, which returns to the basal level when the drug is removed (Hakala et al., 1961). In addition, methotrexate can also increase the level of thymidylate synthetase in regenerating liver, but not in normal liver. In the human lymphoblastic leukemia cell line, CCRF-CEM, the addition of methotrexate to the culture medium elevates thymidylate Synthetase activity, while the addition of arabinosylcytosine lowers the activity of this enzyme (Roberts and Loehr, 1971). The simultaneous addition of both drugs results in levels of enzyme activity intermediate between those observed for each drug alone, the activity depending upon the ratio of the two drugs. In some instances, induced changes in the level of enzyme activity may be permanent; thus, as described by Raunio and Hakala (1967), continuous exposure of Sarcoma 180 cells in culture to methotrexate resulted in a methotrexate resistant cell line with the activity of dihydrofolate reductase increased 1400-times over that of the original culture. Kit et al. (1966) obtained a cell line resistant to bromodeoxyuridine which lacked cytoplasmic thymidine kinase by growing the cells in the presence of increasing amounts of bromodeoxyuridine. A cell line resistant to one agent sometimes demonstrates a greater sensitivity to a second drug; this concept of 'collateral sensitivity' was first described by Szybalski and Brysan (1952). It was subsequently shown by Law (1953) that 6-mercaptopurine resistant L1210 lymphoma cells had greater sensitivity to antifolates than the parent LI210 cells. Wallerstein et al. (1972) demonstrated that the elevated levels of dihydrofolate reductase observed in methotrexate resistant cells were reduced following treatment with 6-mercaptopurine and folic acid, while the decreased levels of hypoxanthine-guanine phosphoribosyltransferase occurring in 6-mercaptopurine resistant cells increased following treatment with methotrexate and hypoxanthine. This return of antimetabolic sensitivity in these resistant cell lines was termed 'adaptive selection' b y the authors. The levels of certain enzymes fluctuate dramatically through the cell cycle. Ribonucleotide reductase activity, which is responsible for the de n o v o formation of deoxyribonucleotides, is not detectable in the GI phase of the cell cycle and increases sharply in S phase (Cheng et al., 1977). The trigger responsible for this alteration in the levels of ribonucleotide reductase is at present unknown. Variation in the inducibility of an enzyme from one individual to another based on genetic makeup has been described. The variation in induced levels of aryl hydrocarbon hydroxylase in humans, an enzyme which metabolizes steroids, drugs, insecticides and carcinogens, has been attributed to genetic as well as environmental determinants (Kellerman et al., 1976; Paigen et al., 1977). BIoch (1974) has given the term 'metabolic conditioning' to the sensitive manipulation of the metabolism of tumor and host tissues by exogenously supplied metabolites designed to achieve a more selective response to the action of antimetabolites. After

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conversion to the triphosphate level, 3-deazauridine inhibits the activity of CTP synthetase, which results in a depletion of cytidine nucleotides in the cell. When 3-deazauridine was injected into female mice bearing the L1210 leukemia, toxicity was observed in intestinal epithelium; this toxicity was not seen in male mice. When testosterone was injected together with 3-deazauridine into female mice, the toxicity was alleviated without loss of antitumor activity against leukemia L1210 (Bloch et al., 1974). Although the mechanism of this protection of intestinal epithelium against 3-deazauridine by testosterone is not clear, it is conceivable that testosterone causes changes in the levels of specific proteins which are involved in the expression of drug action. 6. ALTERATIONS IN T H E C E L L U L A R U P T A K E AND METABOLISM OF AGENTS In the preceding sections, the effect of one agent on the metabolism of a second compound was indicated as one of the primary factors to be considered in determining drug interactions. These effects are often proposed as the primary mechanisms leading to synergistic drug interactions. As discussed previously (Section 4), the transport of methotrexate into cells can be inhibited by a variety of antibiotics and other chemotherapeutic agents, while the vinca alkaloids increase the intracellular accumulation of this antimetabolite (Zager et al., 1973). Since resistance to several anticancer agents is related to decreased permeability of cells to the agent, several groups have attempted to increase permeability with membrane-active agents. Yamada et al. (1963) indicated that the uptake and toxicity of nitrogen mustard in rat hepatoma cells could be increased by pretreatment with Tween 80, while Riehm and Biedler (1972) evaluated the effects of a variety of agents on the uptake and toxicity of actinomycin D in Chinese hamster cells sensitive and resistant to this agent. Of twelve agents tested, only Tween 80 markedly enhanced uptake (about thirteen-fold) and toxicity of this agent in the resistant cells. Other studies (Roth and Kochen, 1971) indicated that acridine orange also enhanced the uptake of actinomycin D two- to three-fold in human lymphocytes. More recently, Medoff et al. (1973) demonstrated a dramatic potentiation of the toxicity of N,N'-bis(2-chloroethyl)-N-nitrosourea(BCNU) to mouse L-cells in culture using amphotericin B, and synergism by this combination in the treatment of advanced murine leukemia in AKR mice (Medoff et al., 1974). However, the immunological enhancement observed with amphotericin B complicates the interpretation of the mechanism responsible for the in vivo effect. One of the most interesting approaches to increased drug uptake across semi-permeable cellular membranes is the phenomenon of 'ion trapping' as discussed by Ross (1961). In order for a compound to pass into a cell by simple diffusion, the agent must be able to dissolve in a lipid-rich membrane and exist in the unionized form. Under conditions in which there is no pH difference across the cell membrane, the concentration of total drug inside and outside of the cell will become equal, with the rate of diffusion dependent upon the concentration of the unionized form. Under conditions where a pH difference exists, however, increased uptake of the unionized form of the drug can be achieved. In certain transplanted tumors, the acidic environment of the cancer cell can be increased by pretreatment with glucose (Rosenoer, 1966). This approach was effective with certain aromatic nitrogen mustards (Ross, 1961) and with mannitolmyleran whose activity was potentiated ten-fold by glucose pretreatment (Rosenoer, 1966). Once inside the cell, many anticancer agents, especially nucleoside analogs, have to be activated by specific enzymes. Under certain circumstances, an increase in the activity of these enzymes through drug-induced effects can potentiate toxicity. Paterson and Moriwaki (1969), using lymphoma L5178Y cells in culture, demonstrated that the synergistic interaction between 6-mercaptopurine and 6-methylmercaptopurine ribonucleoside previously observed in animals (Wang et al., 1967) resulted from an interaction at the level of the target cell rather than the host. In these and

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other studies, Paterson and co-workers (1969, 1970a, 1970b) proposed that the mechanism of this synergistic interaction involved increased activation of 6-mercaptopurine to 6-thioinosinic acid by hypoxanthine-guanine phosphoribosyltransferase, an enzyme which requires 5-phosphoribosyl- l-pyrophosphate. 6-Methylmercaptopurine ribonucleoside is a potent inhibitor of 5-phosphoribosyl-l-pyrophosphate amidotransferase, the first committed step in the de n o v o synthetic pathway for purine nucleotides. It was proposed that inhibition of this enzyme by 6-methylmercaptopurine ribonucleoside resulted in increased levels of 5-phosphoribosyl-1pyrophosphate in the cell which then stimulated the activation of 6-mercaptopurine. Additional studies by Scholar et al. (1972) confirmed the synergistic interaction between these two agents in Sarcoma 180 cells and lent additional support to the proposed mechanism. As discussed by Paterson and Wang (1970b), this combination exhibited some of the characteristics of the synergistic interactions previously observed with combinations of thiopurines and the glutamine antagonists, azaserine and 6-diazo-5-oxo-norleucine (Tarnowski and Stock, 1957; Sartorelli, 1965; Sartorelli et al., 1958). As demonstrated by Nelson and Parks (1972), these latter synergistic interactions apparently also result from increased levels of 5-phosphoribosyl-l-pyrophosphate due to inhibition of the purine nucleotide synthetic pathway de n o v o by the glutamine analogs. The incorporation of iododeoxyuridine into DNA is increased by the administration of either methotrexate (Hampton and Eidinoff, 1961; Cooper et al., 1972) or 5fluorodeoxyuridine (Kriss et al., 1962). The mechanism of this stimulation in incorporation of iododeoxyuridine into DNA involves the inhibition of the de n o v o synthesis of thymidylate by either methotrexate or 5-fluorodeoxyuridine. Since dTTP is an effective inhibitor of thymidine kinase and dTMP and dTDP will compete with IdUMP and IdUDP, respectively, for phosphorylation, a decrease in thymidylate pools would be expected to increase the incorporation of IUDR into DNA. After conversion to an active metabolite, modification in the biological half-life of the active species can also alter drug activity. Such a mechanism has been proposed by Nelson and Parks (1972) as the basis for the synergistic interaction against Sarcoma 180 cells of 6°thioguanine and 6-methylmercaptopurine ribonucleoside. In these cells, the addition of 6-methylmercaptopurine ribonucleoside substantially increased the biological half-life of 6-thioguanylate from 7 to 10 hr. The effect was even more dramatic in Sarcoma 180 cells resistant to thioguanine, with an increase in half-life from a low of 3 hr up to 7 hr (Nelson and Parks, 1972). Since an increase in a particulate-bound alkaline phosphatase seems to account for resistance to the 6thiopurines in these cells, and perhaps for the clinical resistance of acute lymphocytic leukemia as well, recent studies by Lee et al. (1975) have attempted to identify more effective inhibitors of this enzyme. While alterations in the systemic catabolism of anticancer agents may alter dramatically the relative potency of a given compound, improvement in the overall therapeutic index or tissue specificity should not usually be expected from such drug interactions (Grindey et al., 1975).. On the other hand, when a change in catabolic activity resides at the level of the target cell, increased drug effectiveness may be expected to result from an inhibition of this activity (LePage, 1970). Thus, many antimetabolites of adenosine are degraded to inactive products by adenosine deaminase, an enzyme present in high concentrations in many tumor cells (LePage, 1970). Using mouse L-cells in culture, Plunkett and Cohen (1975) demonstrated that nontoxic concentrations of erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase, greatly potentiated the biological activity of both arabinosyladenine and cordycepin. Cass and Au-Yeung (1976) evaluated the effects of another potent inhibitor of adenosine deaminase, 2'-deoxycoformycin, on growth inhibition and cytotoxicity of L1210 cells in culture produced by arabinosyladenine. As a single agent, 2'-deoxycoformycin was not growth inhibitory or cytotoxic to these cells; however, its combination with arabinosyladenine resulted in potentiation of the activity of the adenine analog through inhibition of intracellular adenosine deaminase.

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Independently, LePage et al. (1976) and Schabel et al. (1976) evaluated the activity of this combination against leukemia L1210, a tumor which is not responsive to arabinosyldenine, and several other tumors in vivo. Both groups concluded (LePage et al., 1976; Schabel et al., 1976) that 2-deoxycoformycin increased the activity of arabinosyladenine against the responsive tumors and converted the non-responsive L1210 leukemia into a responsive one. Similar effects have been proposed for the combination of tetrahydrouridine, an inhibitor of cytidine deaminase, with arabinosylcytosine (Chou et al., 1977). More importantly, this combination substantially improves the oral activity of arabinosylcytosine in experimental systems (Neil et al., 1970). 7. INTERACTION OF METABOLITES AND ANTIMETABOLITES Interest in the utilization of combinations of metabolites and antimetabolites was stimulated by the early observation of Goldin and co-workers (1957, 1966) that the host toxicity of methotrexate could be reduced by administration of leucovorin (5-formyltetrahydrofolic acid) without significant reduction in antitumor activity. Under conditions of co-administration of the metabolite-antimetabolite combination to mice bearing leukemia L1210, both the antitumor activity and toxicity of methotrexate were prevented. However, when administration of leucovorin was delayed from 12 to 24hr after the methotrexate, significant reduction in host toxicity was observed without the abolishment of antitumor activity (Goldin and Mantel, 1957; Goldin et al., 1966). Similar approaches utilizing purine analogs and metabolites were less successful (Goldin, 1954). The molecular mechanism responsible for this increased therapeutic selectivity is not clear. Capizzi and co-workers (1970) have proposed differences in cell-cycle times between tumor and normal cells as a plausible mechanism and have indicated that tumor cells may become lethally intoxicated at a faster rate then normal cells. One explanation for this phenomenon may be that a significant proportion of marrow cells are in a non-cycling stage and thus would be protected from the cycle-specific cytotoxic effects of methotrexate (Capizzi et al., 1970). To date, this combination (i.e. methotrexate and leucovorin) has shown the most clinical promise of those discussed in this review, with good activity observed in the treatment of head and neck cancer (Capizzi et al., 1970) as well as in several other solid tumors (Djerassi et al., 1976). Another approach aimed at decreasing the toxicity of methotrexate involves the delayed administration of carboxypeptidase G1, an enzyme which inactivates this agent (Chabner et al., 1972). The administration of this enzyme 24-48 hr after methotrexate prevented lethal toxicity without a corresponding reduction in antitumor activity. As originally described by Hakala (1957) and Hakala and Taylor (1959) using Sarcoma 180 cells in culture, the cytotoxic effects of methotrexate can also be completely reversed by the end-products of folate metabolism (i.e. thymidine and a source of preformed purine, such as hypoxanthine). More recently, Borsa and Whitmore (1969) and Tattersall et al. (1974) observed, that in certain cell lines, the inhibition of growth by methotrexate could be partially reversed by the addition of thymidine alone while other tumor cell lines required both thymidine and a preformed purine for any substantial reversal of toxicity (Grindey et al., 1975). Thus, in some cell lines, toxicity was related primarily to an inhibition of thymidylate synthesis, while in others, blockage of both thymidylate and purine nucleotide synthesis were involved. In vivo, Grindey et ai. (1975) found that co-administration of allopurinol blocked the antitumor, but not the toxic effects of methotrexate against the Ll210 leukemia, an effect attributed to the increased availability of hypoxanthine induced by aUopurinol. This finding indicated that the antitumor activity of methotrexate against the L1210 leukemia was primarily related to an inhibition of purine nucleotide biosynthesis, and was consistent with the observation by Hryniuk (1972) that L5178Y cells in culture were killed by the antifolate through purine nucleotide depletion. When Tattersall and co-workers (1975) combined methotrexate with thy-

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midine, which prevents the antithymidylate effect of the folate antagonist, a reduction in the toxicity of methotrexate was observed without any interferance with antitumor activity. Semon and Grindey (1976), Using a system for continuous infusion of unrestrained mice developed by Paul and Dave (1975), reduced the toxicity of methotrexate eight-fold with a marked increase in the survival time of tumor-bearing mice by the co-infusion of thymidine. In similar experiments, the infusion of thymidine and a preformed purine completely blocked both toxicity and antitumor activity. Thus, in mice bearing the L1210 leukemia, the depletion of intracellular folate cofactors produced by methotrexate affects purine nucleotide biosynthesis more seriously than thymidylate synthesis in tumor cells, while the toxicity of methotrexate seems to be more closely related to reduced formation of thymidylate. Based on these observations (Tattersall et al., 1975; Frei et al., 1975; Semon and Grindey, 1976), the clinical evaluation of this combination was initiated and substantial protection of patients against the toxicity of high doses of methotrexate were observed (Ensminger and Frei, 1977). Further evaluation is necessary, however, to define the full therapeutic potential of this combination. A complete review of approaches to high dose methotrexate therapy will be published shortly (Ensminger et al., 1978). Other approaches to the utilization of metabolites have involved attempts to increase the activation of antimetabolites. Saslaw et al. (1968) and Grindey et al. (1968) evaluated the effects of the coadministration of uridine on the antineoplastic effects of arabinosylcytosine in mice bearing leukemia L1210. While uridine potentiated the antitumor activity of this agent, it also enhanced toxicity. The mechanism of this potentiation involved increased phosphorylation of arabinosylcytosine, through a stimulation of deoxycytidine kinase by UTP (Grindey et al., 1968; Kessel, 1968). Using Ehrlich ascites cells, Gotto et al. (1969) enhanced the incorporation of 5fluorouracil into cellular nucleic acids ten to fifty-fold by the addition of inosine to the culture medium. The addition of deoxyinosine to the medium increased the uptake of 5-bromouracil into cellular nucleic acids by about fifteen-fold. The authors concluded that the availability of ribose-l-phosphate and deoxyribose-l-phosphate may be rate-limiting for the incorporation of these base anaiogs into RNA and DNA, respectively (Gotto et al., 1969). Cooper et al. (1972) evaluated the ability of deoxyguanosine to stimulate incorporation of 5-iodouracil into DNA of mice bearing the Dunning leukemia. While a five-fold increase in the incorporation of iodouracil into tumor DNA was observed, a comparable stimulation of incorporation of iodouracil into the DNA of spleen and small intestine also occurred. The addition of diazouracil, an inhibitor of the catabolism of iodouracil, to the combination further stimulated the utilization of this agent for DNA synthesis. While a potentiation of growth-inhibitory activity also occurred with the combination of diazouracil and 5-fluorouracil, an overall increase in therapeutic index was not achieved (Cooper et al., 1972). Early studies by Burchenal and co-workers (1960) demonstrated a dramatic enhancement of the activity of 5-fluorodeoxyuridine by thymidine against several mouse leukemias. Uracil, thymine, and azathymine were also able to potentiate the antineoplastic activity of 5-fluorodeoxyuridine and 5-fluorouracil, but not that of 5fluorouridine against these tumors. However, none of the combinations significantly improved the antitumor activity observed at the maximum tolerated doses of 5fluorouracil or 5-fluorodeoxyuridine given alone (Burchenal et al., 1960). This potentiation was attributed to inhibition of the catabolism of 5-fluorouracil by these metabolites. More recently, Jackson and Weber (1976) reported that low concentrations of thymidine could protect certain hepatoma cells in culture from the toxic effects of 5-fluorodeoxyuridine, a phenomenon related to differential catabolic activity towards thymidine by these cells. Such differences might lead to improved therapeutic selectivity in certain types of cancer (Jackson and Weber, 1976). In B a c i l l u s cereus, Hahn and Mandel (1974) stimulated the incorporation of 5-fluorouracil into RNA about eight-fold by the addition of thymidine. Martin and Stolfi (1977) have recently reported that this combination has good activity in the treatment of spontaneous mammary tumors of mice.

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In addition to altering host toxicity, circulating metabolites may also decrease antitumor activity in certain instances. Recently, potent inhibitors of nucleoside transport such as nitrobenzylthioinosine have. been developed (Warnick et al., 1972). This agent, although inactive alone, was synergistic with arabinosylcytosine against leukemia L1210; in addition, nitrobenzylthioinosine slightly increased the plasma halflife of arabinosylcytosine (Cass et al., 1975). Since nitrobenzylthioinosine did not potentiate the effects of arabinosylcytosine against L1210 cells in culture, it was assumed that the synergistic interactions were host-mediated (Cass et al., 1975). While the results suggested that the synergistic interaction involved reduced availability to the tumor cells of exogenous deoxycytidine, a slow conversion of nitrobenzylthioinosine to 6-mercaptopurine could not be ruled out as responsible for the observed effects. Another purine analog, N6-phenyladenosine, has been described by Hakala and Kenny (1972) and Divekar and Hakala (1971) as a potent inhibitor of both nucleoside transport and adenosine kinase. This agent effectively blocked the utilization of adenosine by Sarcoma 180 cells in culture although it was not cytotoxic by itself (Grindey et al., 1973). Although N6-phenyladenosine had only slight antitumor activity in vivo, it potentiated the effects of 6-mercaptopurine, but not those of 6-thioguanosine, against leukemia L1210. In addition, N6-phenyladenosine prevented the acute 'shock-like' toxicity caused by single large doses of either 6-mercaptopurine or 6-thioguanine (Grindey et al., 1973). Although the authors concluded that inhibitors of the uptake and phosphorylation of adenosine may have some value in cancer chemotherapy, further evaluation is necessary to determine the overall utility of this approach for the development of combinations that might be clinically useful. The concept of 'metabolic activation' was introduced by Bloch (1974), who described the utilization of an antimetabolite to interfere with the metabolism of an exogenously supplied normal metabolite, causing it to accumulate in the cell. This accumulation resulted in cellular cytotoxicity via inhibition of metabolic pathways by regulatory mechanisms. An example of this concept is the effect of 6-methylmercaptopurine ribonucleoside on guanosine metabolism in S. f a e c i u m (Bloch 1974). At low concentrations, guanosine completely reversed the growth inhibitory effects of this agent, while higher concentrations of guanosine resulted in a second phase of growth inhibition which was reversed by pyrimidines; guanosine alone was not inhibitory in this system. The mechanism of this interaction involved increased intracellular concentrations of guanosine which interfered with de novo pyrimidine nucleotide biosynthesis in the presence of 6-methylmercaptopurine ribonucleoside (Bloch, 1974). More recently, Jackson et al. (1976) have proposed the concept of enzyme patterndirected chemotherapy, whereby the specialized enzymatic pattern characteristic of a tumor might be exploited to enhance drug selectivity. The enhanced growth inhibitory activity of 3-deazauridine by D-galactosamine was utilized as an example of this concept. The D-galactosamine produced a depletion of the intracellular pools of UTP primarily in both hepatic tissue and well-differentiated hepatoma lines. Since 3deazauridine, after conversion to the triphosphate, is a competitive inhibitor, with respect to UTP, of CTP synthetase (McPartland et al., 1974), the depletion of the UTP pools by D-galactosamine should potentiate selectively the activity of this agent against certain hepatomas (Jackson et al., 1976). The combination was strongly synergistic against a Morris hepatoma and several other hepatoma cell lines in culture, but only additive interactions were observed in the non-differentiated Novikoff hepatoma and in nonhepatic cell lines. Keppler (1977) has recently reported the potentiation of the anticancer effects of 6-azauridine, an inhibitor of de n o v o pyrimidine nucleotide biosynthesis, by D-galactosamine in rat ascites hepatoma cells. Thus, the normal metabolite, D-galactosamine, may be utilized to sensitize hepatomas selectively to inhibitors of pyrimidine nucleotide biosynthesis. 8. CONCLUDING REMARKS As described previously, mammalian cells contain thousands of biochemical reactions catalyzed by enzymes, most of which are interrelated either directly or in-

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directly. In such interactive metabolic networks, inhibition of a single enzyme by a specific inhibitor may result in perturbations in the concentration of many metabolites in the network, rather than simply in the end-products of the inhibited reaction. Moreover, the intracellular concentrations of the end-products of the inhibited reaction need not necessarily decrease. Rational prediction of the interaction between combinations of drugs in such systems requires knowledge of the properties and content of each c o m p o n e n t of the metabolic network, as well as an understanding of the interrelationships and spatial arrangements of these components. It is encouraging that some drug interactions derived experimentally are consistent with predictions made on the basis of general concepts or simple mathematical models which rely on our limited knowledge of the characteristics of the system under study; these observations however, do not necessarily prove the validity of the model involved. In most cases, the experimental results are not consistent with predictions. Such limitations should not discourage the continuous development of new approaches to combination c h e m o t h e r a p y based upon general concepts or mathematical models, as long as the limitations of these approaches are understood. The approaches involved in combination chemotherapy can be classified in two general categories: (a) mixtures of drugs designed to interact within the same target cells; and (b) mixtures of drugs acting on different cell populations. This manuscript dealt primarily with the first category. When multiple drugs are used in v i v o , as in the type of combination chemotherapy currently used in the clinic, the mechanisms responsible for their interaction should be proposed with caution. In most cases, these drugs affect more than one population of cells, so that the outcome of the combination is sometimes unpredictable. It is the hope of the authors that eventually, as more knowledge of the molecular biology and biochemistry of normal and tumor cells is accumulated, a predictable effect of combinations of drugs on target cells can be achieved. REFERENCES AGARWAL,R.P. and PARKS,R.E., JR. (1971) Erythrocytic nucleoside diphosphokiuase. J. biol. Chem. 246: 2258-2264. BERTINO, J.R., SAWICKI,W.L., LINDQUIS'r,C.A. and GUPTA, V.S. (1977) Schedule-dependent antitumor effects of methotrexate and 5-fluorouracil. Cancer Res. 37: 327-328. BLACK, M.L. (1963) Sequential blockage as a theoretical basis for drug synergism. J. Med. Chem. 6: 145-153. BLOCH, A. (1974) Metabolic conditioning and metabolic actuation: Experimental approaches to cancer chemotherapy involving combinations of metabolites and antimetabolites. Cancer Chemother. Rep. 58: 471-477. BEECH, A., DUTSCHMAN,G., GI~NDEV,G. and SIMPSON,C.L. (1974) Prevention by testosterone of the intestinal toxicity caused by the antitumor agent 3-deazauridine. Cancer Res. 34: 1299-1303. BONNEV, R. J. and MALEV, F. (1975) Effect of methotrexate on thymidylate synthetase in cultured parenchymal cells isolated from regenerating rat liver. Cancer Res. 35: 1950-1956. BORSA,J. and WnrrMoRE,G.F. (1969) Studies relating to the mode of action of methotrexate. II. Studies on sites of action in L-cells in vitro. Molec. Pharmac. $: 303-317. BROCr,~IAN,R.W. (1974) Biochemical aspects of drug combinations. Cancer Chemother. Rep. 4:115-129. BROCK~N, R.W., SHADDIX,S., STalNOER,V. and AOAMSON,D. (1972) Enhancement by deferoxamine of inhibition of DNA synthesis by ribonucleotide reductase inhibitors. Proc. Am. Ass. Cancer Res. 13: 88. BRowN, N.C. and REICH--, P. (1969) Role of effector binding in allosteric control of ribonucleoside diphosphate reductase. J. molec. Biol. 46: 39-55. BURCHENAL,J.H., OL'rrGEN, H.F., REPI'r~T, J.A. and COLEV, V. (1960) Studies on the synergism of fluorinated pyrimidines and certain pyrimidine and purine derivatives against transplanted mouse leukemia. Cancer Chemother. Rep. 6: 1-5. BUSHaV, S.R.M. (1969) Combined antibacterial action in vitro of trimethoprim and sulfouamides: the in vitro nature of synergy. Post-graduate reed. Z 45: 10-18. BUSHBY,S.R.M. and HITCmNOS,G.H. 0968) Trimethoprim, a sulpl~onamide potentiator. Br. J. Pharmac. 33: 72-90. CAPlZZL R.L., DECONTLR.C., MARSH,J.C. and B~TINO, J.R. (1970) Methotrexate therapy of head and neck cancer: improvement in therapeutic index by the use of leucovorin 'rescue'. Cancer Res. 30: 1782-1788. CASKEV, C.T., Asn'roN, P.N. and WVNO~E~, J.B. (1964) The enzymology of feedback inhibition of glutamine phosphoribosylpyrophosphate amidotransferase by purine ribonucleotides. Z biol. Chem. 239: 2570-2579. CAss, C.E. and Au-YLroNG,T.H. (1976) Enhancement of 9-B-D-arabinofuranosyladeniue cytotoxicity to mouse leukemia L1210 in vitro by 2'-deoxycoformycin.Cancer Res. 36: 1486-1491. CAss, C.E., Muzlr, H. and PATERSON,A.R.P. (1975) Combination therapy of mouse leukemia L1210 by

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