Enhancement of the antitumor effects of 5-fluorouracil by folinic acid

Pharmac. Ther. Vol. 47, pp. I-19, 1990 Printed in Great Britain. All rights reserved

0

0163-7258/90 %O.oO+ 0.50 1990 Pergamon Press plc

Specialist Subject Editor: D. S. MARTIN

ENHANCEMENT OF THE ANTITUMOR 5-FLUOROURACIL BY FOLINIC

EFFECTS ACID

OF

ENRICOMINI,* /I FABIOTRAvE,t YOUCEFM. RUSTUM~ and JOSEPHR. BERTIN@ *Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Morgagni 65, 50134 Florence, Italy tFarmitalia Carlo Erba, Via Imbonari 24, 20100 Milan, Italy SRoswell Park Memorial Institute, Grace Cancer Drug Center, 666 Elm Street, Buffalo, NY 14263, U.S.A. &Wemorial Sloan-Keltering Cancer Center, 1275 York Avenue, New York, NY 10021, U.S.A. Abstract-Although S-fluorouracil (FUra) is one of the most effective cytotoxic agents in the treatment of various solid tumors (carcinomas of the gastro-intestinal tract, breast, head and neck), remissions occur in only 20-30% of cases and usually are of short duration. Recently, preclinical studies have shown that the antitumor activity of FUra can be potentiated by modulating the metabolism of this drug by using other substances, in particular 5-formyltetrahydrofolate (folinate, LV). Reduced folates (LV and 5-methyltetrahydrofolate) at concentrations z 1 pM can, by raising the intracellular levels of 5,10methylenetetrahydrofolate, increase and prolong the inhibition of the target enzyme, thymidylate synthase, with formation of a stable ternary complex formed by the enzyme, the folate coenzyme and the fluoropyrimidine inhibitor (5-fluorodeoxyuridylate). After phase II clinical trials reported encouraging results with the combination LV-FUra in the treatment of patients with various solid tumors, randomized controlled studies in patients with colorectal carcinoma have documented an increase in the response rate of the combination compared to treatment with FUra alone. The integration of the LV-FUra combination into multidrug regimens is now under investigation for the treatment of carcinomas of the breast, stomach, and head and neck.

CONTENTS I. Introduction 2. Mechanism of Action of FUra 3. Preclinical Rationale for the Use of FUra and LV 3.1. In vitro experimental models 3.2. In vivo experimental models 4. Clinical Studies 4.1. Pharmacokinetics of LV in man 4.2. Cellular pharmacology in human tumors 4.3. Results of clinical trials 4.3.1. Phase II trials in various solid tumors 4.3.2. Phase III trials in colorectal cancer 4.3.3. Integration of the combination of FUra and LV in multidrug 4.3.4. ToxLity 5. Conclusions References

1, INTRODUCTION An important concept that has emerged in clinical oncology from laboratory studies is that of biochemical modulation. This refers to the pharmacological manipulation of intracellular metabolic pathways of an antineoplastic drug (effector) by a so-called modulating agent to produce either a selective enhancement of its antitumor activity or a selective protection of the host (Martin, 1987). IlAwardee of the Lady Tata Memorial Trust. Supported by Grants CA 08010. CA 18420, CA 21071 and the Brookdale Foundation.

I 2

3

regimens

3 6 7 7 8 9 9 10 11 13 14 14

Thus far, most efforts aimed at potentiating the efficacy of antineoplastic agents have involved the antimetabolites (of which the knowledge of the mechanisms of action is more certain), with the use of a second anticancer drug or frequently a noncytotoxic agent, often a metabohte or a cofactor. Attempts to potentiate the antitumor activity of 54uorouracil (FUra) in the clinic have been reported recently. This fluoropyrimidine, synthesized by Heidelberger and colleagues at the University of Wisconsin about 30 years ago (Heidelberger et al., 1957), is even today among the most effective drugs in the treatment of adenocarcinomas of the gastrointestinal tract (i.e. colon, stomach, pancreas) and

2

E. MINI et al.

breast, squamous cell carcinoma of the head and neck, and in the treatment of some tumors of the female genital tract (i.e. ovary and cervix) (reviewed in Wasserman et al., 1975). In vitro studies have shown marked potentiation of FUra and FdUrd cytotoxicity of 5-formyltetrahydrofolate (i.e. folinic acid, leucovorin, LV, 5-HCOH4PteGlu) against human lymphoblastic leukemia cell lines (Keyomarsi and Moran, 1986; Mini et al., 1987b). Thus, while F U r a or 5-fluorodeoxyuridine (FdUrd) may have little or no single agent activity in leukemias and lymphomas (Costanzi et al., 1979; Hedley, 1987), they may be converted to an active drug in these neoplasms by appropriate modulation. On the basis of preclinical data, several compounds have been used to modulate F U r a in the clinic in attempts to improve its antitumor efficacy (reviewed in Bertino and Mini, 1987; Grem et al., 1987a; Leyland-Jones and O'Dwyer, 1986; Peters et al., 1987; Wooley et al., 1985). For example, thymidine has been used to inhibit the catabolism of F U r a in vivo; methotrexate or 6-methylmercaptopurine riboside have been used to increase formation of its nucleotides; and phosphonacetyloL-aspartic acid or hydroxyurea have been used to selectively diminish, in tumor cells, the levels of uracil metabolites competing with those of FUra. Uridine or allopurinol have been used to augment selectively competing metabolites in normal cells of the host; while folates, mostly LV, have been used to increase the cytotoxicity of a specific F U r a metabolite, fluorodeoxyuridine monophosphate (FdUMP), against tumor cells.

2. M E C H A N I S M OF ACTION O F FUra FUra is known to exert its antiproliferative effects following metabolic activation (Fig. 1). F U r a can be preferentially channeled either in the deoxyribonucleotide pathway (DNA synthesis) or in the ribonucleotide pathway (RNA synthesis) depending on the relative activity of the enzymes involved and the

relative availability of deoxyribose-l-P or ribose-l-P donors. Along the ribonucleotide pathway, FUra is anabolized to 5-fluorouridine triphosphate (FUTP), which is incorporated into R N A (Mandel, 1969; Glazer and Peale, 1979; Glazer and Lloyd, 1982); the consequence of this incorporation is the production of fraudulent R N A which can ultimately cause cell death. Along the deoxyribonucleotide pathway, F U r a is anabolized to FdUMP, which is a potent inhibitor of the enzyme thymidylate synthase (TS) (Hartman and Heidelberger, 1961; Santi et al., 1974; Danenberg and Lockshin, 1981; Houghton et al., 1981), leading to decreased thymidine triphosphate pools and to inhibition of DNA synthesis. Cytotoxicity also may depend on incorporation of fluorodeoxyuridine triphosphate (FdUTP) into DNA, and consequent removal of the altered bases from DNA leading to fragmentation of this macromolecule (Kufe et al., 1983; L6nn and L6nn, 1986). These mechanisms of action cooperate to determine FUra cytotoxicity, but their relative importance to FUra therapeutic effects is different in different tumor cells. A study of Hakala and coworkers (Laskin et al., 1979) showed that eight mouse tumor cell lines were, on the average, seven times more sensitive to FUra than seven human tumor cell lines. In a subsequent investigation (Evans et al., 1980), the same authors assessed the relative significance of TS inhibition and/or FUTP incorporation into R N A to the antiproliferative activity of FUra. Their studies demonstrated that in mouse tumor cells the growth inhibition consequent to administration of 'low' FUra doses can be partially reversed by the use of thymidine, thus indicating that under those experimental conditions (low dose = high efficiency of FUra) TS inhibition was an important determinant of FUra cytotoxicity. On the other hand, the growth inhibition consequent to 'high' FUra doses could not be reversed by thymidine. As far as human tumor cells were concerned, thymidine was not able to act as a rescue drug from F U r a cytotoxicity at any FUra dose, therefore TS inhibition (although present) was

INTRACELLLJLAR

EXTRACELLULAR /MEMBRANE PRPP FUra ~

FdUrd

FUra ~

PP FUMP

FUDP

~i['~,~

FdUDP

~~

F.RNA ~ F-DNA

.. FdUnl

dUMP~7,,~.~

dTMP-p,,.J~dTrP ~, DNA

S,IO.CHzH4PteGl)u(n HtPleGlu(n)

FIG. 1. Metabolism and sites of action of FUra and FdUrd. Circled numbers refer to the following enzymes (1) orotate phosphoribosyltransferase; (2) uridine phosphorylase; (3) thymidine phosphorylase; (4) thymidine kinase; (5) uridine kinase; (6) thymidylate synthase. Glu,, polyglutamate derivatives of folates.

Antitumor effects of 5-tiuorouracil NUMP

~

dUMP

~, dTMP

5,1~.Clllil4Ptl~lUln )

HtPtIQIg(a)

meth

1Q.HCO-H4PteGlu(,)

• homo 5-CHrH4PImGlu E-r-dUMP

5-HCO-H4PteOIv _

'

"

E+

E - FdUMP - 5,10.CHIH4PIIGlUln )

FdUMP

///

FIG. 2. Biochemical interactions between folate coenzymes and FdUMP with thymidylate synthase. Abbreviations: gly, glycine; ser, serine; homo, homocysteine; meth, methionine. not an important determinant of FUra antiproliferative activity in those experimental systems. These observations led to the idea of increasing the antiproliferative activity by FUra against tumors by enhancing its effects at the TS level. In tumors from patients the inhibition of TS by FdUMP appears in fact to be an important mechanism to achieve a clinically relevant response to FUra treatment (Spears et al., 1984; Peters et al., 1989). The biochemical modulation of FUra activity by LV with the consequent enhancement of TS inhibition can be understood by knowing the molecular mechanisms responsible for the formation of thymidylate (dTMP) in eukaryotic cells (Fig. 2). The conversion of uridylate (dUMP) into dTMP is carried out in two sequential chemical reactions: first TS catalyzes the substitution of the hydrogen in the C-5 position of dUMP with a methylene group; then the enzyme reduces the methylene group to a methyl group. The cofactor that is involved both as the donor of the methylene group and subsequently as the reducing agent is 5,10-methylenetetrahydrofolate (5,10-CH2H4PteGlu) (Friedkin, 1973; Huennekens et al., 1987). Therefore, the conversion of dUMP into dTMP involves the transient formation of a ternary complex constituted by the substrate (dUMP), the enzyme (TS) and the cofactor (5,10-CH~H, PteGIu); at the end of the reaction, the ternary complex dissociates releasing dTMP, the enzyme and dihydrofolate (H2PteGlu), the oxidized cofactor. After administration of FUra, the FdUMP that is generated will compete with the physiological substrate dUMP for TS; however, due to the characteristics of the fluorine-carbon bond, the substitution of the fluorine atom with the methylene group will not occur and the reaction does not proceed further. Activity of TS, size of the cellular pools of dUMP and

3

folate cofactor and amount of FdUMP generated are all relevant to the DNA effects of FUra. In particular, the presence of suitable amounts of the folate cofactor is critical, since a binary complex FdUMP-TS is relatively weak (kd= 10-sM), while the ternary complex FdUMP-TS-5,10-CH2H4PteGIu is more stable (kd-~10-12M) and dissociates much more slowly (Santi et al., 1974; Danenberg et aL, 1974; Danenberg and Danenberg, 1978)~ Increased efficiency of binding of FdUMP to TS is provided by polyglutamate derivatives of 5,10-CH2H4PteGIu (Allegra et al., 1985; Radparvar et al., 1989). The administration of exogenous chemically stable folate precursors of 5,10-CH2H4PteGlu, such as LV and 5-methyltetrahydrofolate (5-CH3H4PteGlu) (Huennekens et al., 1987), can increase the intracelluiar levels of this cofactor and enhance the formation and stability of ternary complexes between FdUMP and TS, leading to increased FUra-mediated DNA effects.

3. PRECLINICAL RATIONALE FOR THE USE OF FUra AND LV 3.1. IN VITRO EXPERIMENTALMODELS

On the basis of the observations concerning the mechanisms of inhibition of TS by FdUMP, Ullman et al. (1978) carried out studies on L1210 leukemia cells cultured in vitro. FdUrd cytotoxicity was enhanced by about 5-fold in the presence of LV at concentrations 10-20 times higher than those required for optimal tumor cell growth (i.e. >0.5-1 #M) (Fig. 3 and Table 1). Ullman et al. (1978) also analyzed the formation of the FdUMP-TS-folate ternary complex following exposure of L1210 leukemia cells to 3H-FdUrd (0.5 #M) in the absence or presence of LV (20/~M). When LV was omitted from the medium only 8% of the ternary complex was formed as when LV was present. It was later demonstrated both in intact cells (Berger and Hakala, 1984; Yin et al., 1982) and in cell extracts (Houghton et al., 1986a,b), that the stability of the ternary complex is dependent on the availability of 5,10-CH2H4PteGlu. Houghton and Houghton (1984) demonstrated that in cell extracts from human

lOO 1,0

5O

-"'~K , \

I

I|

10"7

10-6

!

10-9

10-8

o,5

4~

LV (M)

FIG. 3. Effects of LV on growth of LI210 leukemia cells in culture and on growth inhibition by FdUrd. (Modified from Ullman et al., 1978.) Reprinted with permission of the author.

4

E. MINI et al. TABLE1. Antitumor Activity o f Combined Folate-Fluoropyrimidines in In Vitro Tumor Systems Investigator

Ceil line (species)

Conclusions

UIIman e t al., 1978 Evans e t al., 1981

Ll210 leukemia (mouse) Hep-2 squamous cell carcinoma (human) and S-180 sarcoma (mouse) Waxman and Bruckner, Friend's erythroleukemia et al., 1982 (mouse) Keyomarsi and Moran, 1986 LI210 leukemia (mouse). CCRF-CEM, MOLT-4, CCRF-SB, B-8392 leukemias (human)

LV + FdUrd synergism LV~FUra synergism

Mini et al., 1987b

LV + Fura synergism

CCRF-CEM leukemia (human)

LV + FUra synergism LV + FUra and LV + FdUrd synergism

LV~FdUrd and LV + FdUrd synergism Mini et al.. 1987a

CCRF-CEM leukemia (human)

Park et al.. 1988

Colorectal carcinoma cell lines (human)

Chang and Bertino, 1989

SQ-I squamous cell carcinoma (human)

colon carcinoma xenografts sensitive (HxHC1) and resistant (HXVRCs) to F U r a growing in athymic mice, the addition of 5,10-CH2H4PteGlu prevented the dissociation of the ternary complex throughout the observation time (60 min). In the sensitive line, however, this occurred with folate concentrations 10-fold lower (10 vs 100 ~M). The synergism of the combination of folates and F U r a has been demonstrated in vitro against various tumor cell lines including those of human origin (Evans et al., 1981; Waxman and Bruckner, 1982; Keyomarsi and Moran, 1986; Mini et al., 1987a,b; Park et al., 1988; Chang and Bertino, 1989) (Table 1). Various concentrations of LV or of 5-CH 3H4PteGlu and both F U r a and F d U r d were used. Also, LV was added to the cultures either before, together with or after F U r a or FdUrd. The simultaneous addition of LV to FUra and FdUrd or sequential exposure (LV~fluoropyrimidine) enhanced growth inhibition in all tumor cell lines. Evans et al. (1981) compared the effects of LV on the cytotoxicity of F U r a in mouse S- 180 sarcoma and human Hep-2 carcinoma cells in vitro. This study demonstrated that 20 ~tM LV in the culture medium was able to potentiate 3-fold the growth inhibition caused by F U r a in Hep-2 cells. Subsequent studies showed that the intracellular concentrations of those folates which are immediate precursors of 5,10-CH2H4PteGlu, i.e. 5-CHaH4PteGlu, tetrahydrofolate (H4PteGlu), 5-HCO-H4PteGIu and 10HCO-H4 PteGlu, were lower in Hep-2 cells than in the F U r a sensitive mouse S-180 cells, thus indicating that the cellular folate cofactor pool might indeed be responsible for the difference in F U r a sensitivity between the two cell lines (Yin et al., 1982). By treating these cells with LV the intracellular folate cofactor pool will be increased and FUra-mediated D N A effects enhanced. A delayed recovery to the pretreatment levels of TS rather than the extent of TS inhibition correlated with the growth inhibition consequent to the combined F U r a - L V treatment (Berger and Hakala, 1984). These findings suggested that the composition and/or the size of the infracellular folate

5-CH3-H4PteGIu + Fura synergism 5-CH3-H4PteGlu~FdUrd and 5-CH1-H4PteGlu + FdUrd synergism LV-FUra and LV-FdUrd synergism in 10~I1 cell lines LV + FUra and LV + FdUrd synergism

pools determine the relevance of TS as the target for growth inhibition by FUra. Waxman and Bruckner (1982) showed that in Friend erythroleukemia cells the inhibitory effects of FUra on cell growth was increased 3-fold when the drug was administered with 10 #~l LV over a period of 72 hr. The inhibitory effects of F U r a on 3Hdeoxyuridine incorporation into D N A were also increased when combined with the same concentration of the folate. Keyomarsi and Moran (1986) studied the growth inhibitory effects and cytotoxicity effects of F U r a and FdUrd in L1210 leukemia cells and in four human lymphoblast cell lines. Two of these human lines were o f T origin (CCRF-CEM and MOLT-4 cells) and two were of B origin (CCRF-SB and B8392 cells). The growth inhibitory potency of both compounds was increased by addition of LV in the medium for all cell lines tested. The total intracellular folate pool increased with every increment of LV added to the medium from 0.032 to 100/.tM, although the cytotoxicity of fluoropyrimidines was not increased at LV concentrations greater than 10/~M. The degree of enhancement of the cytotoxicity of F U r a was similar in mouse and human lines. The effects of FdUrd were enhanced to a greater extent by LV than were the effects of FUra. Further studies on the synergism of FdUrd and LV in L1210 cells led to confirmation of the proposed mechanism of interaction, i.e. expansion of cellular 5,10-CH2H4PteGlu pools with subsequent stabilization of the ternary complex: FdUMP, TS and 5,10-CH2H4PteGlu (Keyomarsi and Moran, 1988). Chang and Bertino (1989) also noted that the human SQ-1 squamous cell carcinoma line potentiation of FUra and FdUrd growth inhibition by LV occurred at a concentration of I/~M. Higher folate concentrations (up to 100/tM) did not further enhance the cytotoxicity of the fluoropyrimidines. Enhancement of F U r a and FdUrd cytotoxicity was observed by Park et al. (1988) in 10 out of the 11 human colorectal carcinoma cell lines tested. LV concentration used in this study was 20/~r~. By

Antitumor effects of 5-fluorouracil

5

I;------

5

'o:"v°:;;o':,7 :I/

:;:.,,o., A.'/

._o

3I 2

0J o o i--

I

o

i

O,

0 L V " ~ FUfQ II I.V ÷ FUro

-I

i 0

I 48

I

, ~FU'*~LV'96 144

I

-I 0

I

48

I

I 96

I

I 144

Time (h)

FIG. 4. Sequence-dependent effects of LV and FUra or FdUrd on CCRF42EM cell growth. Cells were exposed to FUra (100 #M) or FdUrd (0.1 #M) or LV (100/~M). Each drug exposure either alone or in simultaneous or sequential combination was for 4 hr. After 4 hr of exposure to the first drug, cells were washed twice and reexposed to the second drug. After the final wash, cells were resuspended in drug-free medium and their growth was followed. (From Mini et al., 1987b.) comparing the in vitro area under the curve (AUC) of F U r a needed for 50% growth inhibition to the clinically achievable A U C of FUra, the authors noted that 50% inhibition occurred at a clinically achievable AUC in 3 of the 11 lines, while with L V - F U r a one additional line showed 50% growth inhibition at a clinically achievable AUC. In the human T-lymphoblast leukemic cell line C C R F - C E M the sequence of administration of F U r a in relation to folate (4 hr before, F U r a ~ L V ; simultaneous, F U r a + LV; 4 hr after, LV--*FUra) did not influence significantly the degree of synergism; the inhibition of cell growth was marked in all three experimental conditions (Fig. 4) (Mini et al., 1987b). The sequence of administration of F d U r d in relation to the folate was, on the contrary, important in the outcome of the combination: the sequence LV--,FdUrd (4 hr before) and the simultaneous combination (LV + FdUrd) were active, while the inverse sequence F d U r d ~ L V was not (Fig. 4) (Mini et al., 1987b). In both cases pretreatment with LV or at least simultaneous exposure to these drugs is necessary to allow intracellular uptake of the folate to reach equilibrium with the other folate monoglutamate and polyglutamate pools, including 5,10-CH2H4PteGIu, and consequently to maximize the binding of F d U M P to TS. The difference observed in this cell line in the degree and sequence-dependence of synergism might be explained by the different rate of conversion of the two fluoropyrimidines to the common active metabolite, FdUMP. In CCRF-CEM ceils, in fact, F d U r d will presumably reach higher F d U M P levels in a shorter time as compared to FUra. As described by Piper and Fox (1982), these cells lack uridine and thymidine phosphorylases. Thus, F d U r d is entirely activated to F d U M P via thymidine kinase, while F U r a is only partially converted to the fluorodeoxynucleotide via a multi-

enzymatic pathway including orotate phosphoribosyltransferase and ribonucleotide reductase, which presumably will require longer time. When CCRF-CEM ceils were treated with varying concentrations of LV (0.1-100 #M) for 4 hr and with F U r a (250 pM) for the last 2 hr of exposure, observed synergism was dependent on LV concentrations with a maximum at the higher concentration (100 pM), but with a significant degree at the intermediate concentration (10/~M) (Mini et al., 1987b) (Fig. 5). In the clinic it is possible to reach levels between 10 and 100 #M of the physiological diastereoisomer (l) and of its metabolite 5-CH3 H4PteGlu in the plasma using (dl) LV doses of 200-500 mg/m 2 (Machover et al., 1986; Madajewicz et al., 1984; Newman et al., 1985). A certain degree of synergism was observed also at a lower concentration (1 #M) (Fig. 5). This level can be reached in the clinic with LV doses > 50 mg (Straw et al., 1984). Synergism did not occur at 0.1/~M LV in this experimental system (Mini et al., 1987b), nor had it been previously observed in others (Keyomarsi and Moran, 1986; Ullman et al., 1978). Similar results have been obtained using 5CH3H4PteGIu at equimolar concentrations (Evans et al., 1981; Mini et al., 1987a). These in vitro data appear particularly important from the clinical point of view since 5-CH3H4PteGlu represents the main metabolite o f LV, which is formed rapidly in the plasma following oral or parenteral administration, and reaches within 2 hr the level of the physiological diastereoisomer (l) (Mehta et al., 1978; Nixon and Bertino, 1972; Straw et al., 1984). Radparvar et al. (1989) recently evaluated the role of polyglutamylation of (I)-5,10-CH:H4PteGIu in the formation and stability of 6-3H-FdUMP TS-folate coenzyme ternary complexes using enzyme purified from a human colon carcinoma xenograft (HxVRCs). Longer chain polyglutamates species of (I)-5,10-CH2H4PteGlu (Glu3_6) at concentrations of

6

E. MINi et al.

o Control A LV, I~M G FUra, 100pM x kVl FUra

/

//

4

---

/

o FUrl, 100/llll / x LVIFUra

G

O

2

o

I

I 48

I

I 98

I 0

I 48

I

I 96

0

48

96

Time (h)

FIG. 5. Inhibitory effects of sequential LV-FUra treatments on growth of CCRF-CEM cells. Cells were exposed to LV (Panel A, 1 #M; Panel B, 10#M; Panel C, 100#M) for 4hr and to FUra (100/~M) alone (17) or in combination (X) for the last 2 hr of folate exposure. After drug exposure, cells were washed twice, and their growth followed in drug free medium. (Modified from Mini et al., 1987b.)

0.9 to 1.6#M were >200-fold more effective than (l)-5,10-CH2 H4 PteGlu (335/1 M)at stabilizing ternary complexes for a t~/2 for dissociation of 100 min. Polyglutamates of 5,10-CH2H4PteGIu also increased the affinity of 6-3H-FdUMP for TS as demonstrated by a 7- to 10-fold decrease in kd determined by Statchard analysis at a folate concentration of 10/~M. These differences were in the range of those reported previously for TS purified from human MCF-7 breast cancer cells (Allegra et al., 1985). For the enzyme of that source, a 2.4-fold increase in kon and a 3-fold decrease in ko~ were responsible for the difference in kd reported for the polyglutamate species. However, Radparvar et al. (1989) reported that the increasing binding affinity for (I)-5,10-CH2H4PteGIu appeared to be solely due to a decrease in the rates of dissociation (ko~). The Vmax for the initial velocity of 6-3H-FdUMP binding was achieved at 10/t M folate. Consequently, at concentrations of 5,10-CH2H4PteGlu polyglutamates present in tumors (about 0.2-2 #M) (Houghton et aL, 1988), inhibition of TS by F d U M P in vivo would be expected to be transient based upon the concentration of (I)-5,10-CH2H4PteGlu required for maximal formation and stability of the covalent ternary complex. The relevance of polyglutamylation in the potentiation of F d U r d cytotoxicity by LV has been also investigated by Romanini et al. (1989) utilizing CCRF-CEM/P cells resistant to methotrexate due to impaired polyglutamylation (Pizzorno et al., 1988). Using short-term exposure (4hr) to LV concentrations up to 100/~M, enhancement of F d U r d cytotoxicity did not occur in C C R F - C E M / P mutants which showed decreased retention of 5,10CH2H4PteGIu polyglutamate pools and a more rapid recovery of dTMP synthesis following LV exposure, as compared with methotrexate-sensitive parent C C R F - C E M cells. Recently Davis and Berger (1989) have shown that mechanisms additional to the capacity of

accumulate 5,10-CH 2H4PteGlu derivatives may contribute to the variations associated with response to F d U r d - L V combinations. The response of two human colorectal carcinoma cell lines, RCA and C, increased with increasing LV concentrations, but the effect was more pronounced in cell line RCA. RCA was 4-fold less sensitive than cell line C to FdUrd at low LV concentrations, whereas both cell lines exhibited similar sensitivity at high LV concentrations. RCA accumulated lower 5,10CH2H4PteGlu after LV than did C. The difference in response was also associated with a reduction in the affinity of the RCA TS for 5,10-CH2H4PteGIu relative to enzyme C. Folic acid has also been used with success in modulating the cytotoxicity of FUra in murine and human tumor cell lines; in L1210 leukemia and in Hep-2 carcinoma, the concentration of folic acid necessary to potentiate the activity of fluoropyrimidine (respectively F U r a and FdUrd) was, however, significantly higher when compared to that of LV (25 vs 1/tM, and 300 vs 20 pM, respectively (Evans et al., 1981; Ullman et al., 1978)). 3.2. IN V w o EXPERIMENTALMODELS The results of in vivo studies of combined folates and F U r a in murine transplantable tumors are summarized in Table 2. The first study of Klubes et al. (1981) in mouse L1210 leukemia did not demonstrate enhancement of F U r a efficacy by LV. Single treatments with F U r a alone or with FUra plus LV infusion of mice bearing the i.p. or the s.c. tumor (one day explants), resulted in equivalent increase in median life span and similar host toxicity as measured by either changes in body weight or by mortality. The potentiation of the antitumor activity of FUra has been shown, however, in two different tumor models sensitive or resistant to this fluoropyrimidine (murine colon tumors 26 and 38, respectively) (Nadal

Antitumor effects of 5-fluorouracil TABLE2. Antitumor Activity of the Combination Folates-FUra in Murine Transplantable Tumors End point Tumor

Investigator

system

Klubes et al., 1981

Leukemia LI210 IP

Klubes et al. 1981

Leukemia LI210 SC

Parchure et al., 1984

Leukemia P388 IP

Nadal et al., 1987

Colon carcinoma 26 SC Colon carcinoma 38 SC CD8FI mammary carcinoma SC

Treatment schedule LV i.v. infusion 11.5-115 mg/kg/day, days 1~; after 0.5 hr: FUra i.p. 100-200 mg/kg/day LV i.v. infusion 11.5-115mg/kg/day, days l ~ ; after 0.5 hr: FUra i.p. 100-200 mg/kg/day FA i.p. 187.5mg/kg/day, days I, 5, 9; after O, 12, 24 hr: FUra 135 mg/kg/day days I (2), 5 (6), 9 (10)

Martin e t al., 1988

LV i.p., 50, 100-200 mg/kg/wk x 4; FUra i.p. 109mg/kg/wk

Interval* (hr)

FUra

Folate-FUra

+0.5

28-65t

28-59t

Lack of synergism

+0.5

56-94¢

56-94t

Lack of synergism

0 + 12

72¢ 78t

+24

86~"

0/+1

1795 102++

LV i.p, 500mg/kg/wk at - 1 , +4, +21, +28 hr prior to FUra i.p. 100 mg/kg/wk x 3

+ I

88++

67t 109t (p < 0.001) l14t (p < 0.001) 311++ (p < 0,002) 245++ (p < 0.01) 94++

Conclusions

Synergism at + 12 hr, and 24 hr

Synergism

Occasional synergism, increased host toxicity

*Interval of drug administration is defined as the time between the beginning of the administration of the first drug and the beginning of the administration of the second drug. tlncrease in life span (% of controls). *lnhibition of tumor growth (% of control tumor weight or doubling time).

et al., 1987). By measuring the inhibition of tumor

growth following 4 weekly i.p. injections as well as weight loss and hematological toxicity, the authors demonstrated that a significantly longer tumor growth delay occurs when LV administration preceeded or was simultaneous to FUra, as compared to the fluoropyrimidine alone (Table 2), with a substantially similar degree of toxicity. In contrast, Rustum did not observe any significant modification of therapeutic efficacy of FUra by LV against these tumors (Rustum, unpublished results). Similar results have been obtained in P388 leukemia bearing mice with the use of folic acid instead of LV when administered 12 or 24 hr prior but not simultaneously, to FUra (Parchure et al., 1984) (Table 2). Treatments were repeated three times starting on 1, 5 and 9 days after i.p. transplantation of the tumor. Antitumor activity and host toxicity were evaluated by measuring the survival time and deaths in control animals up to 7 days after the last drug injection, respectively. In a study of Martin et al. (1988) the combination of LV and FUra was tested in mice bearing advanced CD8F1 breast tumors at the maximally tolerated dose (MTD) of FUra with and without LV in 3 weekly courses. The antitumor effects (inhibition of tumor growth) and the host toxicity (body weight and mortality) were recorded 5 days after the 3rd weekly course. Overall, therapy with FUra at the MTD was not improved by LV. Further, although the activity of FUra doses lower than the MTD could be increased by LV, the therapeutic results were comparable to those of single agent FUra at MTD. Recently it has been shown that high-dose LV (180mg/kg/day, day 1-8) can expand 5,10CH2H4PteGIu pools in EMT6 mammary adenocarcinoma tissues of mice, but not in bone marrow (Wright et al., 1989). LV given 2 hr prior to FUra (30 mg/kg/day, days 1-5) resulted in a modest but significant augmentation of tumor growth delay compared with FUra alone. The benefit afforded by the use of LV in this regimen was obtained without an increase in host toxicity.

4. CLINICAL STUDIES 4.1.

P H A R M A C O K I N E T I C S GF L V IN M A N

The commercial formulation of LV available for clinical use is a soluble calcium salt consisting of a 50:50 mixture of two diastereoisomers: (l)-LV and (d)-LV. Of the two isomers, only (I)-LV is believed to be biologically active (Blakley, 1969). The dose of LV that has been indicated by in vitro studies as optimal to increase FUra antiproliferative activity, 20/~M, refers to (dl)-LV, i.e. 10/.2M(I)-LV. Therefore, LV dose and schedule of administration for clinical trials should be adequate to produce systemic concentrations of (/)-LV above the 10/~M threshold for a certain period of time. To complicate the issue, (d)-LV and (I)-LV have shown to behave quite differently as far as their pharmacokinetics is concerned (Fig. 6 and Table 3). Following intravenous injection of relatively low doses of LV (Straw et al., 1984; Straw et al., 1987), (I)-LV is cleared from the systemic circulation much faster than (d)-LV (tl/2 of about 0.5 and 8.0hr, respectively). This fact is, in part, due to (/)-LV metabolic conversion to 5-CH3H4PteGlu, since plasma clearance of (I)-LV greatly exceeded its renal clearance. 5-CH3H4PteGIu reached a plasma peak 2 4 hr from LV injection and then disappeared with a tl/2 that was several times slower than t~/2 of the parental compound (I)-LV. Plasma clearance exceeded renal clearance also for 5-CH 3H4PteGlu, indicating the presence of an extrarenal (metabolic) clearance. In contrast, plasma and renal clearances of (d)-LV were equal, confirming that this 'unnatural' stereoisomer did not undergo metabolism. Higher push doses of LV (200mg/m 2) yielded similar pharmacokinetic results as far as the elimination tl/2 of (d)-LV, (l)-LV and 5-CH3H4PteGlu is concerned (Machover et al., 1986). The pharmacokinetics of LV given by continuous i.v. infusion, either short term infusion (Arbuck et al., 1987; Trave et al., 1988; Rustum et al., 1987) or long term infusion (Straw et al., 1987; Trave et al., 1988; Rustum et al., 1987), confirmed the different disposition of the two stereoisomers. With respect to the

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FIG. 6. Plasma pharmacokinetics of (dl)-LV (O 0), (I)-LV ( 0 - - - 0 ) and 5-CH3H4PteGIu (O O) in patients with advanced colorectal carcinoma. Patients received 2 hr infusion of 500 mg/m2 (dl)-LV and at various times, blood samples were obtained, plasma separated and extracted and analyzed by high pressure liquid chromatography. biologically active (I)-LV, (d)-LV achieved higher steady-state plasma levels and its plasma clearance was slower. Since its volume of distribution resulted quite small (2 1), impaired renal excretion rather than accumulation in tissues is likely to be responsible for the high steady-state plasma levels of the 'unnatural' isomer. As for the i.v. push studies, there was no evidence of a (d)-LV metabolism (renal clearance almost matched plasma clearance) (Straw et al., 1987). Under conditions of continuous i.v. infusion, the metabolite 5-CHaH4PteGlu accumulated in the systemic circulation at much higher concentrations than (I)-LV (Straw et al., 1987; Trave et aL, 1988; Rustum et aL, 1987). Its relatively large volume of distribution, although roughly estimated, might reflect the intracellular accumulation of this metabolite, probably in the form of 5-CH3H4PteGlu polyglutamates (Straw et al., 1987). As far as the l0 pM (I)-LV threshold is concerned, 200mg/m 2 LV by i.v. push (Machover et al., 1986) and 500mg/m 2 LV by short term i.v. infusion (Arbuck et al., 1987; Trave et al., 1988; Rustum et al., 1987) yielded plasma concentrations of the natural isomer above the 10 pM level for about I and 2 hr, respectively. If all the natural reduced folates are taken into account ((I)-LV plus 5-CHjH4PteGlu, assuming that all 5-CH 3H 4PteGlu arise from (I)-LV), plasma levels of l0/~M were detected for more than 4 hr in the systemic circulation of patients treated with 500 mg/m 2 by 2 hr i.v. infusion (Arbuck et al., TABLE 3. Peak and Steady State Plasma Concentration of Folates following a 2hr i.v. Infusion of 500mg/m 2 (dl)-LV, 500mg/me/day x 5 i.v. Infusion and 125mg/m2/Q l hr x 4 in the Oral Route

(d/)-LV

(I).LV 5-CH3H4PteGlu

2 hr infusion (peak plasma) ~)

5 day infusion (steady-state) O~M)

Q I hr x 4 p.o. (peak plasma) (~M)

96+38 24 ± 6 17 ± 8

51 ± 17 1.2 2=0.5 12 ± 5

5±2 < 0.5 4± 2

1987; Trave et aL, 1988; Rustum et al., 1987). Highdose LV (500mg/m2/day) given by prolonged i.v. infusion (5.0-5.5 days) yielded steady-state plasma concentrations of (I)-LV in the 1.0-3.0/~M range (Straw et al., 1987; Trave et al., 1988; Rustum et aL, 1987). Under these conditions, plasma steady-state concentrations of the metabolite 5-CH3H4PteGIu were detected in the range of 3.5-6.0/~M (Lactobacillus casei bioassay on HPLC-collected fractions) (Straw et al., 1987) or in the range of 7-18/~M (HPLC assay) (Rustum et al., 1987). 4.2. CELLULARPHARMACOLOGYIN HUMAN TUMORS At the cellular level (tumor tissue from colorectal cancer patients), the relationship between i.v. administration of high-dose LV in combination with conventional F U r a doses, increase of the folate cofactor pool and inhibition and recovery of TS has been investigated (Trave et al., 1988; Rustum et al., 1987). The results of this study can be summarized as follows: under conditions of continuous i.v. infusion of 500 mg/m2/day of LV and 400-500 mg/m2/day of FUra, 5,10-CH2H4PteGlu pool was increased to a lesser extent and TS was inhibited at a lesser extent than following a short-term (2hr) infusion of 500 mg/m 2 LV and 600 mg/m 2 i.v. push of FUra. It is not clear, however, whether the observed differences in the folate cofactor cellular pool and degree of TS inhibition achieved in patients treated by the two different i.v. schedules (i.e. different pharmacokinetics and pharmacodynamics) to modulate the metabolism of folates and their effectiveness in combination with FUra. Studies are in progress to evaluate the role of dose and schedule in cellular modulation of folate metabolism and F U r a effects. The role of ( d l ) - L V dose and route of administration in the therapeutic modulation of FUra activity against rats bearing colon carcinoma were evaluated. The data in Table 4 summarize these results.

Antitumor effects of 5-fluorouracil

9

TABLE 4. Pharmacokinetic

Parameters of (dl)-LV (400mg/kg i.v.s at lOOmg/kg/l hr x 4 p.o.) and Antitumor Activity in Combination with FUra (100mg/kg) against Rats bearing Colon Carcinoma. Tumors were Inoculated s.c. on day '0' and Treatment was Initiated 7-10 days later when Tumor Sizes were 0.5-0. Z Drug Effects were Evaluated on day 15 after Treatment (,urn)

5-CH3 H4PteGlu (uM)

Inhibition of tumor growth* (% control)

734 _+97 253 4- 36

i20 + 96 115 4- 22

27 5

10 + 3

5 4- I

2

(dl)-LV Schedule Route 2 hr infusion, i.v. 48 hr infusion QI x 4 h r Oral

*On day 15 rats treated with FUra alone at the maximally tolerated doses of 100 mg/kg produced 70% inhibition of tumor growth (30% control).

These results demonstrate that this rat tumor which is sensitive to F U r a becomes more sensitive to the combination of F U r a and (dl)-LV, especially when (dl)-LV was administered by continuous infusion and/or by the oral route. In contrast, (dl)-LV did not modify significantly the therapeutic efficacy of F U r a against mouse colon 26 and 38 (Rustum, unpublished results). These data suggest that modulation of F U r a by (dl)-LV varies with the tumor of choice. The basis for this heterogeneity in response is under investigation in Dr Rustum's laboratory. 4,3.

R E S U L T S OF C L I N I C A L T R I A L S

The preclinical observation that an excess of intracellular reduced folates may be necessary for maximum inhibition of TS and cytotoxicity by F U r a has led to several phase I/II clinical studies of the combination of LV and FUra. In these studies, a variety of dose schedules of both LV and F U r a were used. Bolus administration, as well as short-term or longterm infusions, was used for the two drugs, while time intervals between LV and F U r a and dose levels of the two drugs also varied markedly. Recently LV has also been given by the oral route (Brenckman et al., 1988; Hines et al., 1989). Two treatment regimens have been most frequently employed. Machover et al. (1982) administered LV by i.v. bolus and F U r a by i.v. short-term infusion (over 15 min) daily for 5 consecutive days. In the original protocol patients received 200 mg/m 2 of LV and 370 mg/m 2 of F U r a with adjustment of dosage in subsequent courses of therapy, depending on tolerance. Courses were repeated at 28 day intervals. In further studies utilizing this treatment regimen the two drugs were both administered by i.v. bolus or i.v. infusion lasting up to 2 hr. Time intervals between LV and F U r a varied between 0 and 2 hr. The dose of LV used varied in the different studies among 'low' and 'high' levels (i.e. 60-500 mg/m2/day), which are able to produce plasma concentration of reduced folates of 1/~ M

or higher. Dose levels of F U r a also varied between 340 and 770 mg/m2/day in the different studies. The second treatment schedule proposed by Madajewicz et aL (1984) utilizes a high dose of LV (500 mg/m2/day) administered as a 2 hr infusion with escalating bolus doses of F U r a given midway through the infusion. This schedule was administered weekly x 6 followed by a two-week rest period. Although the maximum tolerated dose was 750 mg/m 2, Madajewicz et al. (1984) recommended the dose of 600 mg/m 2 which yielded nearly the same response rate as the 750 mg/m 2 dose with less toxicity. Further studies conducted according to this schedule always included high-dose LV but varied administration modalities (i.v. bolus or infusion up to 2 hr). Doses of F U r a also varied between 300 and 850 mg/m 2, always given by i.v. bolus. The interval between LV and F U r a administration was between - 30 min (FUra given before) and + 1 hr (LV given before). 4.3.1. Phase H Trials in Various Solid Tumors The results obtained in the most relevant phase II clinical studies performed up to the present are summarized in Table 5, as a function of tumor type and pretreatment characteristics. Out of 693 patients with colorectal carcinoma treated in 21 studies (Barone et al., 1987; Bertrand et al., 1984, 1986; Brenckman et al., 1988; Bruckner et al., 1982, 1983; Byrne et al., 1984; Cunningham et al., 1984; Greene et al., 1986; Hines et al., 1988, 1989; Kaplan and Rivkin, 1984; Laufman et al., 1987; Machover et al., 1986; Madajewicz et al., 1984; Mortimer and Higano, 1988; Schmoll and LeBlanc, 1985; Valone et al,, 1986; van Groeningen et al., 1988; Wilke et al., 1988b) 157 (23%) responded to therapy with combined LV-FUra. Patients who had been treated previously with FUra-containing chemotherapy are included in these results. In FUra-untreated patients the response rate was higher (28°/'0). Tumor

TABLE 5. Summary of Phase H Clinical Studies of the Combination L V-FUra Response Tumor type Colon and rectum Stomach Breast Prostate Liver Head and neck

No. of studies

No. of FUra pretreated pts

No. of FUra untreated pts

Total no. of pts

21 4 6 I l 1

32/250 (13%) 2/13 05%) 36/161 (22%) 0/0 0/0 NR

125/443(28%) 15/54 (28%) 18/41 (44%) 0/17 0/14 NR

157/693(23%) 17/67 (25%) 54/202 (27%) 0/17 0/14 3/16 (19%)

Abbreviations: NR, not reported.

10

E. MINI et al.

remission was also observed, although in a limited percentage of cases (13%), in patients resistant to FUra single-agent therapy. These data indicate that with current FUra-LV regimens, the number of patients who will benefit from combination therapy after tumor progression with single-agent FUra is small, and such patients are candidate to other investigational approaches, including addition of other modulating agents to FUra. An equivalent if not superior percentage of objective response was obtained in patients with cancer of the stomach (Arbuck et al., 1987; Becher et al., 1988; Machover et al., 1986; Marini et al., 1987b) and breast (Becher et al., 1988; Doroshow et al., 1989; Fine et al., 1988; Loprinzi et al., 1988; Marini et al., 1987a; Swain et aL, 1989) (25 and 27%, respectively). In both these tumors, the response rates were higher in previously untreated patients (28 and 44%, respectively) than in previously FUra-treated cases (15 and 22%, respectively).

Prostatic and hepatocellular carcinoma apparently do not benefit from therapy with LV-FUra combination (Becher et al., 1988; Zaniboni et al., 1988a). Data from Bruckner et al. (1982, 1988a) on a limited number of patients (16 and 8, respectively) with squamous cell carcinoma of the head and neck and with pancreatic cancer, indicate that this combination may be active in these diseases. 4.3.2. Phase III Trials & Colorectal Cancer

Based on phase II data suggesting activity of FUra combined with LV in metastatic colorectal carcinoma, various phase III studies have been initiated in this disease to compare the efficacy of this combination with standard therapy with FUra alone (Table 6) (Di Costanzo et al., 1989; Doroshow et al., 1987; Erlichman et al., 1988; Nobile et al., 1988; O'Connell, 1989; Petrelli et al., 1987, 1988; Valone et al., 1988). The results of the majority of these trials

TABLE6. Summary o f Randomized L V - F U r a Trials in Colorectal Carcinoma Dosage and schedule Investigator Erlicfiman, 1988

LV 200 mg/m2/day x 5 (i.v. bolus)

_

Valone et al., 1988

Petrelli et al., 1987

200 mg/m2/day × 5 (i.v, bolus)

Nobile et al., 1988

Dorosbow et aL, 1987

O'Connell, 1989

vs

vs

500 mg/m2/wk x 6 (i.v. 2 hr infusion)

_

500 mg/m"/wk (i.v. 2 hr infusion) __ 500 mg/m2/day × 6 (i.v. 144hr infusion) __

vs

Petrelli et al., 1988

200 mg/m2/day x 5 (i,v. bolus) --

__

Budd et al.. 1987

200 mg/m2/day × 5 (i.v. bolus) 200 mg/m2/day x 4 (i.v. bolus)

Median survival (months)

0

64

33 (p = 0.0005)

12.6 (p = 0.05)

--

61

7

9.6

400 mg/m2/day x 5 (i.v. bolus) every 4 weeks loading course, 12 mg/kg/day × 5 (i.v. bolus); maintenance. 15 mg/kg/wk

0

49

16

11.0

96

18

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600 mg/m2/wk x 6 (i.v, bolus); rest for 2 weeks, then repeated 450mg/m2/day x 5; then 200 mg/m2/wk every other day x 6 doses (i.v. bolus): repeated every 6 weeks

1

25

48 (p = 0.0009)

10.9

19

11

9.6

370 mg/m2/day × 5 (i.v. bolus) increased to 440 mg/m2/day × 5 as tolerated; every 4 weeks 370 mg/m2/day x 5 (i.v, bolus) increased to 440 mg/m2/day x 5 as tolerated; every 4 weeks

I

43

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--

39

24

29

45

vs

370 mg/m2/day bolus) every 370mg/m:/day bolus) every

--

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370 mg/m2/day x 5 (i.~. bolus) × 2 times every 4 weeks; then every 5 weeks 425 mg/m2/day x 5 (i.v. bolus) x 2 times every 4 weeks; then every 5 weeks 500mg/m2/day x 5 (i.v. bolus) every 5 weeks

0

35

26 (p = 0.04)

NR (p = 0.03)

0

37

43 (p = 0.001)

NR (p = 0.03)

400 mg/m2/day × 5 (i.v. 15 min) every 4 weeks 13.5 mg/kg/day × 5 (i.v. bolus) every 4 weeks

vs

vs

500 mg/m2/wk x 6 (i.v. 2 hr infusion) 25 mg/m2/wk x 6 (i.v. 10min infusion)

Response (%)

600 mg/m2/wk (i.v. bolus)

vs

Di Costanzo et al., 1989

No. patients

vs

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LV-FUra interval (hr)

FUra

vs

vs

x 4 x 4

5 (i.v, weeks 5 (i.v. weeks

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vs

1000 mg/m2/24 hr x 4 (i.v. 96 hr infusion)

Abbreviations: NS, no statistically significant difference; NR, not reported.

16 (p = 0.05) 5

N R (NS) NR 14.6 (p = 0 . 1 6 ) 13.2

39

10

NR

0

58

15

NR

--

6I

16

NR

1

109

I

112

19

NR

--

107

12

NR

0

63

22

10.3

62

21

11.0

28 (p = 0.01)

NR

Antitumor effects of 5-fluorouracil (6/8) demonstrate a significant advantage of the combination of LV and F U r a over single agent chemotherapy with F U r a in terms of response rates (Erlichman et aL, 1988; Nobile et al., 1988; Petreili et al., 1987, 1988; Doroshow et al., 1987; O'Connell, 1989). Only two studies show, however, a significant effect on patient survival (O'Connell, 1989; Erlichmann et al., 1988). In these studies LV was used at low and high doses (between 20 and 500mg/mZ/day). Other treatment variables were length of LV administration, interval of administration of folate and fluoropyrimidine, and F U r a dosage. The optimal dose levels of LV in regard to maximal enhancement of clinical efficacy of F U r a have not yet been established. O'Connell's study (1989) demonstrated that the low-dose schedule (20 mg/m2/day) was superior to the high-dose one (500 mg/m2/day) with regard to both response and survival. Petrelli et al. (1988) showed the contrary, that is, highdose LV (500 mg/m2/day) had a greater enhancing effect on F U r a efficacy compared to low-dose folate (25 mg/m2/day). It should be noted, however, that the dose of F U r a used in the O'Connell study with low-dose LV was higher than with high-dose LV arm. No significant differences were observed in response rate and survival as a function of length of administration of F U r a (i.e.i.v. bolus or continuous infusion over 96 hr) (Budd et al., 1987, Table 6). All arms received the same LV dose; this study did not include, however, a treatment arm with FUra alone. The planned dose intensities for single agent F U r a ranged from 463 to 760 mg/m2/week and the actual delivered dose intensities, established in two of the trials (Erlichman et al., 1988; Nobile et al., 1988) were 531 and 533 mg/m2/week. The planned dose intensity for the F U r a - L V combination ranged from 463 to 600 mg/m2/week compared with the delivered dose intensity of 443 and 463 mg/mZ/week in the same two studies. When used with LV, F U r a could not be administered at the single-agent MTD because of unacceptable toxicity. Nevertheless, a lower dose of FUra, when combined with LV, had higher response rates than F U r a alone. 4.3.3. Integration o f the Combination o f FUra and L V in Multidrug Regimens More recently, combined L V - F U r a has been added with some success in multidrug regimens for the treatment of tumors of the head and neck, stomach and breast. Table 7 summarizes the results of phase II clinical studies of combined L V - F U r a and cisplatin in the treatment of head and neck cancer (Wendt et al., 1989; Loeffler et al., 1988; Vokes et al., 1988). The efficacy of this combination is very marked with response rates on a total number of patients varying from 56 to 100% of cases, obtained with nearly equivalent treatment modalities. In the study reporting the highest activity (Wendt et al., 1989) patients also received radiation therapy and had a 4 years overall survival rate of 48% with marked but

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not limiting toxicity. It should be noted that the efficacy was also relevant in previously FUra-treated patients (67-77%) (Vokes et al., 1988; Loeffler et al., 1988). In the treatment of patients with advanced gastric cancer, the combination L V - F U r a was employed together with mitomycin C (Becouarn et al., 1988) or doxorubicin + mitomycin C (Arbuck et al., 1988) or with methotrexate + cisplatin (Bruckner et al., 1988b) or with etoposide (Wilke et al., 1988b). Preliminary results of all these studies demonstrate a satisfactory response rate of 50% or higher (Table 8). Zaniboni et al. (1988b) in a phase II trial, used the combination of cyclophosphamide, epirubicin, L V - F U r a for first-line treatment of advanced breast cancer: 23 out of 30 patients (73%) obtained an objective response (Table 9). Since FUra is a cycleactive drug and might be expected to be more effective against rapidly proliferating populations, the combination L V - F U r a has potential value against breast cancer cells synchronized by hormonal treatment. Marini et al. (1988) utilized premarin to synchronize breast cancer cells with the addition of prednimustine at the start of the cycle and L V - F U r a for 5 days at usual dosages to treat heavily chemo and hormonally pretreated patients. Despite its considerable activity as a salvage regimen, the results obtained (30% response rate) do not seem better than those achieved with L V - F U r a alone (Marini et al., 1987a). The results obtained in the above phase II multidrug studies await confirmation by randomized trials before the use of such multidrug regimens including L V - F U r a can be recommended for clinical practice. Preliminary results of a study using a combination of L V - F U r a and cisplatin in nonsmall cell lung cancer did not indicate a substantial benefit for this protocol over other current combination regimens (20% response rate in 5 patients) (Essesse et al., 1987) (Table 9). A low response rate (23%) was observed in 17 previously untreated patients with squamous cell carcinoma of the esophagus by Zaniboni et al. (1987) when L V - F U r a was added to cisplatin (Table 9). This is similar to response rate observed with other cisplatin-based regimens (Kelsen, 1984; Dinwoodie et al., 1984). The combination of L V - F U r a has also been used in the treatment of colorectal cancer together with cisplatin or etoposide, dipyridamol, cytarabine, semustine or methotrexate (Allen et al., 1987; Chiarion Sileni et al., 1988; De Marco et al., 1988; Hiddemann et al., 1988; Scheithauer et al., 1988; Schmoll et al., 1988; Smith et al., 1984; Weeks et al., 1988) (Table 10). In the majority of these studies (with the exception of those of Schmoll et al., 1988 and of Hiddemann et al., 1988) limited numbers of patients (8-18 cases) have been treated. In 35 patients treated with dipyridamol-LV-FUra the first study demonstrated a response rate of 36%, not substantially higher than that observed for the association of L V - F U r a (Schmoll et al., 1988). In the second study (Hiddemann et al., 1988) the addition of etoposide to the L V - F U r a combination did not appear to cause any significant variation in the response rate (29% vs 26%).

14

E. MINI et al.

4.3.4. T o x i c i t y The combination of LV and FUra is usually well tolerated if attention is paid to early signs of gastrointestinal toxicity. Substantial or severe toxicity (grade 3 and 4 of the W.H.O. scale) do not or rarely occur when the FUra dose does not exceed 370 mg/mZ/day in the treatment schedule that utilized a 5-day administration, or 500 mg/m 2 in those based on single F U r a administration at weekly intervals. Thus, in the presence of LV the dose of FUra has to be decreased to approximately 20% of that used when this fluoropyrimidine is used alone. The analysis of type and degree of toxic effects observed in evaluable clinical studies demonstrated however that, at higher dosages, the toxicity of the combination may reach grade 3 and 4, and is mainly stomatitis, diarrhea and other toxic effects on the gastrointestinal tract. In some studies deaths have been reported (reviewed in Greta et al., 1987b).

5. CONCLUSIONS The use of LV combined with FUra to enhance the therapeutic index of the fluoropyrimidine is based on a biochemical rationale defined by numerous laboratory studies. The enhancement of antitumor potency of FUra by exogenous folates is most likely due to an increase of the 5,10-CH2H4PteGlu pool and consequent increased formation and stabilization of the ternary complex, TS-folate coenzyme and FdUMP. The results of phase II clinical studies in gastric, breast and colorectal cancer have been thus far encouraging with response rates higher than those expected with the use of F U r a alone (summarized in Table 5). Results of phase IlI studies in advanced colon adenocarcinoma appear to show an advantage of the combination as compared to single F U r a treatment in terms of response rate, but definitive data on survival are not yet available (summarized in Table 6). On the basis of the positive therapeutic results and good tolerance of the combination, adjuvant chemotherapy studies using this combination have been initiated by cooperative groups in patients with colorectal cancer, Dukes stages B and C (i.e. NSABP, Wolmark, 1988; NCI of Canada, Erlichman, 1988; NCCTG; an intergroup study of NCCTG, ECOG, CALGB, and SWOG; IST of Genoa; GIVIO of Milan; COG of Florence, Italy). The results of these studies are of great importance, since the effectiveness of this combination may be greater and have more impact on survival in the adjuvant situation. The integration of LV FUra treatment with other drugs is also possible and potentially worthwhile, since the toxicity of this combination is mostly gastrointestinal (reviewed in Grem et al., 1987b). The activity of L V - F U r a in combination with other agents that modulate fluoropyrimidine cyotoxicity, in particular those that enhance inhibition of TS, also deserves further study, e.g. in head and neck squamous cell carcinomas (Loeffler et al., 1988; Vokes et al., 1988: Wendt et al., 1989).

Further clinical studies in other potentially FUraresponsive solid tumors and in leukemia-lymphomas are warranted, based upon experimental tumor results (Keyomarsi and Moran, 1986; Mini et al., 1987b). Further experimental studies using human tumor cells of different origin and normal tissues are needed for complete elucidation of these complex drug interactions and future clinical applications. The evaluation of the administration of FUra and LV by alternative routes (i.e. oral or regional administration) warrants further investigation in view of preclinical rationale and of recently published preliminary clinical data (Brenckman et al., 1988; Hines et al., 1989; Smith et al., 1989; Arbuck et al., 1986; Budd et al., 1986; Valeri et al., 1987). Since studies of orally administered LV indicate that absorption of the (l)-isomer is approximately 5 times greater than that of the (d)-isomer due to stereoselectivity (Straw et al., 1984), repeated oral administration of LV might be useful in achieving persistently higher levels of the (l)-isomer as compared to the (d)-isomer. This event would avoid possible competition between the (d) and the (l) forms for cellular uptake and for formation of stable ternary complexes, which may occur at high (d)-LV concentrations following i.v. administration. Preliminary results of Bertrand and Jolivet (1989) in an in vitro human tumor model (CCRF-CEM) suggest however that interference on (/)-LV-FUra synergism would not occur even at high (d)-LV concentrations (1 mM). A multicenter double-blind randomized trial comparing FUra with oral LV or placebo is currently ongoing (Laufman et al., 1989b). The use of regional administration of a fluoropyrimidine (FUra or FdUrd) and LV by the intraperitoneal and hepatic artery routes, although still investigational, might provide a means of enhancing drug efficacy and decreasing systemic toxicity (Smith et al., 1989; Arbuck et at., 1986; Budd et al., 1986; Valeri et al., 1987; Kemeny et al., 1988). Clinical studies with combinations of other folates (i.e. 5-CH3H4PteGIu or folic acid) and FUra have also been performed, but a limited number of cases do not allow at present a final evaluation of therapeutic results (Nobile et al., 1985; Asbury et al., 1987; Valeri et al., 1987). Since preclinical synergism observed with L V - F d U r d has been usually greater than that observed with L V - F U r a (Keyomarsi and Moran, 1986; Mini et al., 1987b), preliminary clinical studies of this combination have been performed (Marsh et al., 1988; Laufman et al., 1989b) and warrant further testing in the future.

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