Journal of Chemotherapy
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Biochemical Rationale for the 5-Fluorouracil Leucovorin Combination and Update of Clinical Experience Y.M. Rustum To cite this article: Y.M. Rustum (1990) Biochemical Rationale for the 5-Fluorouracil Leucovorin Combination and Update of Clinical Experience, Journal of Chemotherapy, 2:sup1, 5-11, DOI: 10.1080/1120009X.1990.11738998 To link to this article: http://dx.doi.org/10.1080/1120009X.1990.11738998
Published online: 15 Jul 2016.
Submit your article to this journal
Article views: 1
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yjoc20 Download by: [University of Tasmania]
Date: 13 March 2017, At: 23:51
Vol. 2 - Supplement n. 1 (5-11) - 1990
Journal of Chemotherapy
Biochemical Rationale for the 5-Fluorouracil Leucovorin Combination
and Update of Clinical Experience Y.M. RUSTUM
Summary - - - - - - - - - - - - - - - -
Although '-fluorouracil has been the drug of choice for the treatment of patients with advanced adenocarcinoma of the colon, the reported response rate does not exceed 20% with a median survival time less than 9 months. Mechanisms of sensitivity to this agent include: 1) inhibition of thymidylate synthase by FdUMP, the active metabolite of j-£luorouracil; 2) incorporation of FUTP into cellular RNA; and J) incorporation of FdUTP into cellular DNA. Recent laboratory preclinical results suggest that the major site of action of '-fluorouracil when combined with '-formyltetrahydrofolate (folinic acid, leucovorin) is thymidylate synthase resulting in pronounced and prolonged inhibition of DNA synthesis. This effect is primarily due to the stabilization of FdUMP binding to thymidylate synthase by the reduced folate cofactor, N', N10methylenetetrahydrofolate. In this paper, the rationale for the modulation of ,fluorouracil by leucovorin, the clinical pharmacology and the clinical experience of this combination will be reviewed.
INTRODUCTION
5-Fluorouracil (FUra) has been utilized extensively as a single agent or in combination with other agents in the clinical management of gastrointestinal malignancies, and carcinomas of the breast, ovary, and squamous carcinomas of the head and neck 1,2. FUra, administered as a single agent, is generally considered the standard therapy for patients with adenocarcinoma of the colon; however, the drug produces a response-rate of less than 20% in this disease 2. Because of the wide spectrum of marginal antitumor activity exhibited by FUra, numerous investigators are trying to enhance the antitumor activity and therapeutic selectivity of FUra by combining this drug with other agents. In this paper, the recent investigations that have attempted to enhance the antitumor. activity of FUra will be reviewed. This includes the use of FUra modulators such as leucovorin (CF). METABOLISM OF FUra
Once transported inside the cell, FUra undergoes extensive metabolism (Figure 1). The antitumor activity and cytotoxicity of this compound have been ascribed to three major mechanisms: (a) metabolism to 5-fluoro-2 -deoxyuridine-5 -monophosphate (FdUMP), which is a potent inhibitor of thymidylate synthase (dTMP-S) 4,5; b) incorporation of the ribonucleoside triphosphate (5-fluorouridine 5 triphosphate), into certain species of RNA 6,7; and c) incorporation of the metabolite 5-fluoro-2 -deoxyuridine 5 -triphosphate into DNA 8-10. In the reaction catalyzed by dTMP-S, the normal substrate 2 -deoxyuridine 5 I
I
I
Key words: 5-Fluorouracil, leucovorin, colon adenocarcinoma.
I
Grace Cancer Drug Center, Roswell Park Memorial Institute, Elm and Carlton Streets, Buffalo, NY 14263 USA. © Edizioni Riviste Scientifiche - Firenze
I
I
I -
ISSN 1120-009X
6
Y.M. RUSTUM
Fdurd - -..
...-----,I - -.....·1
dTMP~ dTDP
Figure 1 - Metabolic pathway of fluoropyrirnidines and folates.
monophosphate (dUMP) is converted to 2'deoxythymidine 5' -monophosphate (dTMP), which is required for DNA synthesis. In this reaction, the reduced folate N,sNIO methylenetetrahydrofolate, (N, SNI0_CH 2 -FH 4 ), serves as the required cofactor, as the methylene group of the folate is added to the 5 position of the heterocyclic ring, and is subsequently reduced to the CH 3 state to form dTMP 11. The FUra metabolite FdUMP inhibits this reaction in a competitive manner with respect to the normal substrate dUMP. In the absence of the reduced folate cofactor, the FdUMP forms a bivalent complex with the enzyme, (Kd = 10 - SM), whereas the presence of N, SN 1 0 -CH 2 - FH 4 results in a covalent ternary complex between the enzyme, the cofactor, and FdUMP, in. which FdUMP is bound 7-8 orders of magnitude more tightly to the complex 10. This observation has led numerous investigators to suggest that the extent of the cytotoxic effects of FUra may be critically dependent on the intracellular levels of the reduced folate cofactor.
BIOCHEMICAL RATIONALE FOR THE LEUCOVORIN/FURA COMBINATION IN CLINICAL INVESTIGATIONS
Waxman et ale 11 performed some of the initial studies to investigate the potential role of reduced folates in potentiating FUra toxicity. These investigators observed that incubating Friend leukemia cells with 10 J.l,M citrovorum factor (CF) increased the sensitivity of cells to
-
dTTP
F-DNA
FUra. They postulated that the intracellular folate cofactor required for FdUMP interaction with dTMP-S may be a limiting factor in FUra cytotoxicity, and it was possible to overcome this metabolic limitation with the addition of exogenous reduced folate. Ullman et ale 12 demonstrated that FdUrd cytotoxicity towards cultured L1210 cells was reduced when intracellular folates were depleted by limiting their source in the medium or by treating the cells with methotrexate to inhibit dihydrofolate reductase. Furthermore, they noted that the intracellular levels of FdUMP associated with dTMP-S were decreased significantly when cells were depleted of folates. Houghton et ale 13-16 have conducted numerous investigations to determine the biochemical bases of responsiveness to FUra in xenografts of human colorectal adenocarcinoma transplanted in immune deprived mice. Their data revealed that FUra sensitive and FUra resistant xenografts could not be distinguished by total drug uptake, incorporation into RNA, maximal concentrations of FdUMP achieved intracellularly, or retention of this metabolite. Likewise, FUra sensitive and resistant xenografts could not be distinguished by the activity of dTMP-S, nor by the ratio of free FdUMP to dTMP-S binding activity. In the FUra sensitive tumors, however, maximal binding was achieved without addition of exogenous cofactor. In contrast, in FUra resistant lines, maximal enzyme inhibition occurred only in the presence of exogenous cofactor. These results led these investigators to conclude that the intracellular availability of the reduced cofactor may
BIOCHEMICAL RATIONALE FOR THE 5-FLUOROURACIL LEUCOVORIN COMBINATION, ETC.
play a critical role in the formation of the ternary complex, and that increasing the intracellular level of the reduced folate cofactor may result in greater ternary complex formation and increase the sensitivity of tumors which may be relatively refractory to fluoropyrimidine treatment 14. Subsequent studies by these investigators have demonstrated that only N, 5NI0_CH 2FH 4 and FH 4 are capable of increasing ternary complex formation or stabilizing the preformed covalent complex in cytosol preparations from human colon tumor xenografts 15. It was also demonstrated that an elevated level of active intracellular folate, (FH 4 or N,5NI0_CH 2-FH 4) was required for the maintenance of a stable ternary complex and furthermore, that a prolonged continuous infusion of leucovorin may enhance the cytotoxicity of FUra by stabilizing the dTMP-S-N,5Nl°-CH 2FH 4-FdUMP ternary complex 16. Lockshin and Danenberg 17 extensively investigated the role of the reduced folate cofactor in enhancing the binding of FdUMP to dTMP-S. The results of these investigations have demonstrated that increasing the concentration of N, 5NI0_CH 2-FH 4 can in fact decrease the K off of FdUMP from the complex; however, the Koff of the reduced folate remains constant regardless of the FdUMP levels. This observation is consistent with a mechanism whereby FdUMP does not dissociate from the _enzyme until the cofgctor has been released. These results are consistent with a general mechanism suggesting that the concentration can markedly influence the effectiveness of FdUMP as an inhibitor of dTMP-S. Evans et ale 18 have evaluated the effect of excess '£olates on the activity and site of action of FUra in the S-180 murine sarcoma highly sensitive to FUra and in the human carcinoma cells Hep-2 in cell culture relatively resistant to FUra. In the absence of exogenous cofactor, S180 cells are 50-fold more sensitive to FUra .than is the human line. Furthermore, in the absence of endogenous folates, dTMP-S inhibition is the growth limiting event in S-180 cells, whereas FUra incorporation into RNA is growth limiting in the Hep-2 cell line. Following a 24-hour incubation of cells with a 20 JlM dL-CF and 3 hour exposure to FUra the toxicity of FUra was potentiated 3-fold in both cell lines, although this potentiation did not appear
7
to be due to greater inhibition of dTMP-S, but rather to the stabilization of the ternary complex of FdUMP-dTMP-S-Ns,Nlo,CH2FH4. These studies also revealed that in the presence of excess CF, the site of action of FUra is shifted from RNA to dTMP-S inhibition, resulting in a greater and more prolonged inhibition of DNA synthesis and increased responsiveness to FUra. PHARMACOKINETICS AND CELLULAR PHARMACOLOGY OF LEUCOVORIN
Leucovorin (CF) is a chemically stable 5formyl derivative of FH 4 which can be administered either orally or parenterally. The calcium salt of CF, which is readily soluble in water, is the preparation which is used in the clinic when leucovorin is prepared by chemical reduction. The final product consists of equal amounts of the d- and diastereoisomers; however, the 1diastereoisomer is the biologically active form 19-21. Straw et ale 20 demonstrated that following an i.v. administration of d,lleucovorin, the natural diastereoisomer was rapidly cleared from the plasma by conversion to 5-CH 3 FH 4 and urinary excretion. In contrast, d-5-CH 3 FH 4 is not metabolized and is slowly excreted in the urine. The plasma half-life of the d isomer and I isomer were 451 ± 24 minutes, and 31.6 ± 1.1 minutes respectively. Furthermore, these investigators determined that the urinary clearance of 1-5-CHO-FH 4 and the 5-CH 3 derivative differed only slightly from the creatinine clearance, whereas urinary clearance of d-5-CHO-FH 4 is only 20% of creatinine clearance indicating that this compound is extensively reabsorbed. Sirotnak et al. 22 have demonstrated that both diastereoisomers of folinic acid as well as 5-CH 3 -FH 4 are transported across the cell membrane by a common reduced folate carrier system, although d-5-CHO-FH 4 competes much less effectively with the natural isomers for transport in several murine lines in vitro. Similar results have been obtained by Straw et ale 20, who noted that absorption of d,l-CF after p.o. administration to man is stereoselective, with the absorption of the natural isomer being approximately 5 times that of the d-isomer. Furthermore, when compared to an i. V. administration of leucovorin, repeated oral adminis-
8
Y.M. RUSTUM
tration resulted in a more favorable plasma ratio of active to inactive isomers. Straw and colleagues observed that the concentration of the inert isomer (d) greatly exceeds the concentration of the biologically active isomer (1) following an i.v. administration of the drug, and they suggest that repeated parenteral administration would result in a selective accumulation of the biologically inert isomer, which may compete with the active isomer for cellular transport and metabolism. Although oral administration of CF results in a preferential absorption of the I-isomer, several investigators have demonstrated that peak plasma levels of I-CF are quite low following an oral dose of the drug 23,24. Hines et at.' 23 examined the pharmacokinetics of d-I-CF doses of 200-1600 mg hourly in four divided oral doses. The peak plasma concentration of the I-reduced folate (l-CF + 5-CH 3 tetrahydrofolate) was achieved 4-6 hours following drug administration and ranged from 3.42 ± 0.65 J.1M to 5.11 ± 1.81 J.1M; however, at the highest dose the peak plasma concentration of l-CF was only 0.34 ± 0.16 J.1M. McGuire et ale 24 determined that absolute bioavailability of .a 200 mg oral dose of CF was 31 % of the same dose administered i. v. The 200 mg oral dose resulted in a < 2.0 J.1M plasma level of I-reduced folates, whereas the same dose administered Lv. achieved a peak plasma concentration approaching 20 J.1M. Trave et ale 21 investigated the pharmacokinetic parameters of high dose dl-CF (500 mg/m 2 ) administered by 2 h continuous i. v. infusion and FUra (600 mg/m 2 ) Lv. infusion and FUra (600 mg/m 2 ) i.v. push at 1 h. The concentrations . of dl~CF, I-CF and 5-CH 3 FH 4 at different time points during and following the 2 h i. v. infusion of dl-CF are shown in Table 1. The dl-CF concentrations peaked at the end of the 2 h infusion, while the 5-CH 3 FH 4 ' peak
TABLE 1 - Plasma half-life (ti) of reduced folates in patients with advanced colorectal cancer.
Concentration
(~M)
tJ2 (H)
Component clCF LCF 5-CH 3 FH4
Lv.
p.o.
i.v.
p.o.
80::1:32 24::1:3.2 17::1:8
5::1:2 12
6+ >12
1000
o • dL-CF
Csse 82f'-M
o • L-CF
c... t.9fLM
•
C.. • t5fLM
• 5- CH J
tr---6 • FUra
tOO
C•• • t.5~
'0
o
0
o
•
•
~
. . ----6 ---
~
-0-------------~~----~~
6.
.O:i
l1JL - _L
4
24
--4'-8-.--71.---91.--~ 6 2
Time (t'loursr
Figure 2.
concentration was observed from one to three hours after the end of treatment. l-CF concentrations above 10 J.1M were reached 1 h after the start of the 2 h infusion and maintained for about 2 h. A plasma concentration of 5CH 3 FH 4 exceeded 10 J.1M for a period of about 6 h. Mean t 1/2 and plasma clearance of I-CF were respectively 10 and 18-fold faster than those of dl-CF. Under these conditions, the percent of I-isomer in the HPLC-detected dl-CF dropped from the initial 50% at the beginning of drug administration to less than 10% 2 h after the termination of the drug infusion. Contribution of I-isomer to dl-CF AUC was less than 4%. Furthermore, the plasma tY2 was similar to those of d-CF. Five-day continuous Lv. infusion of dl-CF (500 mg/m 2/d X 5) yielded a steady state concentration of 82 J.1M, 1.9 J.1M, amd 15 J.1M for dCF, L-CF and 5-CH 3 FH 4 , respectively (Figure 2). Pharmacokinetic studies of oral dL-CF (125 mg/m 2 X 4 h) were carried out in patients with advanced colorectal carcinoma and the results are shown in Table 1. These results indicate the plasma concentrations of I-CF seen in these patients receiving oral dl-CF were below 0.5 J.1M.
9
BIOCHEMICAL RATIONALE FOR THE 5-FLUOROURACIL LEUCOVORIN COMBINATION, ETC.
COLORECTAL CARCINOMA
TABLE 2 - Protocols of FUra in combination with CF. Reference • FUra CF
600 mg/m2/wk X 6 Lv. push 1 h after the start of CF 500 mg/m 2/wkx6 2 h Lv. infusion 2wks rest, 1 course (High CF dose, HD)
• FUra CF
370 mg/m 2/d X 5 day; Lv. push 200 mg/m 2/d X 5 day; 15 min infusion Q 28 days (Intermediate CF dose)
• FUra
370 mg/m 2/d X 5 day continuous Lv. infusion 500 mg/m 2/d X 5.5 day continuous Lv. infusion Q 28 days (High CF dose)
CF
• FUra CF
600 mg/m 2/wkx 6 Lv. push, 425 mgfm2 d X 5d and 20 mg/m 2 d X 5 25 mg/m 2/wk X 6 15 Lv. infusion (Low CF dose)
Previously untreated patients
27,28
Toxicity. The profile of FUra tOXICIty in combination with CF was found to be dose and schedule dependent of CF administration. The data demonstrated that the dose-limiting toxicity of FUra alone when administered by a daily or weekly schedule was myelotoxicity. In contrast, CF, regardless of the dose and schedule, caused a shift in the profile of toxicity of FUra to gastrointestinal (GI) and stomatitis. In the CF weekly schedule the toxicity was predominantly GI while with the daily schedule, stomatitis was the dose limiting toxicity 28,29,31,32. In the study by Erlichman et all 33, patients receiving FUra + CF had significantly more mucositis and diarrhea from course 1 than those receiving FUra alone (p < 0.005); stomatitis was more severe in the combination group (p < 0.02). In the study of Doroshow et all 29, both stomatitis and severe diarrhea were more frequent in the FUra + CF group than the FUra alone group in the study of Nobile et all 34 and there was more severe GI toxicity in the Petrelli trial 27 • With the more pronounced toxicity, high dose CF was achieved requiring a' dose reduction of FUra. The diarrhea can be severe and in some cases requiring hospitalization. With careful follow-up and dose reduction -of FUra, reversal of toxicity can be achieved, however.
29
30,32
31,33
CLINICAL PROTOCOLS
Although other schedules and doses of CF and FUra have been utilized clinically, those clinical protocols which have been extensively utilized are listed in Table 2. In addition, recently a protocol utilizing oral leucovorin in colon cancer is under investigation. The dose of CF is equivalent to 50-125 mg/m 2fh X 4 p.o. with FUra administration Lv. after the 4th oral dose of CF. Initial clinical results suggest that this form of CF treatment is as active as the i. v. use of CF23,2S.
TABLE 3 - Phase III trials of FUra and CF previously untreated advanced colorectal cancer. Studyl
FUra (mg/m 2 )
Patient (No.)
Response (%)
FUra+CF (mg/m2 )
Patient (No.)
Response (%)
PMH
370 X 5d
61
7
370+200x5d
64
33
RPMI
450x 5d 200xqodx6
19
9
600 + 500/wk X 6
30
40
COH
300x 5d
34
15
370 + 500 X 5.5d*
29
45
GITSG
500x 5d
107
12
600 + 500/wk X 6
109
28
NCCTG
500x5d
39
10
370 +200 X 5d 425 + 20 X 5d
35 37
26 43
NCOG
440 x5d then 555/wk
49
19
400 + 200 X 5d
16
96
Genoa
600/wkx6
44
11
600 + 500/wk X 6
43
31
* Continues Lv. infusion 1 PHM, Princess Margaret Hospital, Canada (29, Erlichman); RPMI, Roswell Park Memorial Inst. (27,28, Petrelli); COH , Ctty of Hope (30, 32 Doroshow); GITSG, Gastrointestinal Tumor Study Group (31, Petrelli); NCCTG, North Central Cancer Treatment Group (32, O'Connel); NCOB, Northern California Oncology Group (35, Valone); Genoa (34, Nobile). •
10
Y.M. RUSTUM
Therapeutic efficacy. The data in Table 3 demonstrate the updated clinical experience (Phase III) with this combination. In all the clinical trials conducted to date, except for the NCOG, the data demonstrated that CF can indeed increase significantly the response rate to FUra in previously untreated patients with advanced colorectal cancer. The controversy, however, remaining to be resolved is whether high dose CF (500 mg/kg) in combination with FUra produces a higher response rate than low (20-25 mg/m 2 ) or intermediate (200 mg/m 2 ) doses of CF. Except for the North Central Cancer Treatment Group (NCCTG) (O'Connel) high dose CF appears to give a higher response rate. In all the Phase III trials, overall ·response rate to FUra was 11.8% (42/365 pts) and to FUra with CF was 28% (154/564 pts). Previously treated patients. Contrary to previously untreated patients with colon cancer, the combination of FUra + CF in previously treated patients is less effective regardless of the CF dose used. The data suggest that this combination should be limited at the present time to previously untreated patients with advanced colorectal cancer. Studies are underway to delineate the underlying nechanisms of action of CF and FUra in this group of patients.
CONCLUSION AND FUTURE PROSPECTS
The CF/5-FUra regimen possesses antitumor activity against colorectal carcinoma (and other tumors such as breast carcinoma and squamous cell head and neck cancers). Recent phase III triaJs demonstrated that the antitumor activity of the combination against colorectal carcinomas is significantly greater than the response obtained with 5-FUra administered as a single agent at the maximally tolerated dose. It is not yet clear, however, whether this combination actually has improved the survival rate of patients with advanced colorectal carcinoma, although initial results are favorable. Future plans with this combination should include: 1) Evaluation of the therapeutic efficacy of this combination against other human solid tumors 2) Studies to determine -whether the thera-
peutic activity of the CF/5-FUra regimen can be enhanced by the addition of other agents (i.e. cisplatin or dipyridamole) to the regimen 3) Identification of the optimal dose and schedule of 5-FUra administered alone and in combination with leucovorin 4) Determination of the therapeutic role of l-CF administration by different routes 5). Biochemical and pharmacologic studies designed to elucidate mechanisms for improvement in the therapeutic selectivity of 5-FU and, thus, provide a rational approach for improving the survival rate of patients treated with the combination of 5-FUra and leucovorin.
REFERENCES 1 Chabner BA, Myers CEo Clinical pharmacology of cancer chemotherapy. In: DeVita V, Hellman S, Rosenberg SA. (eds.), Cancer Principles and Practice of Oncology; Philadelphia: ]B Lippincott, 1985: 296. 2 Levin P, Mittelman A, Douglass H, et al. Survival and response to chemotherapy for advanced colorectal adenocarcinoma: an Eastern Cooperative Oncology Group report. Cancer (Phila.), 1980; 46: 1536-1543. 3 Santi DV, McHenry CS, Sommer H. Mechanism of interaction of thymidylate synthetase with 5-£1uorodeoxyuridylate. Biochemistry, 1974; 13: 471-480. 4 Danenberg PV, Langenbach R], Heidelberger C. Structures of reversible and irreversible complexes of thymidylate synthetase and £1uoropyrimidine nucleotides. Biochemistry, 1974; 13: 926-933. S Glazer RI, Pealer AL. The effect of 5-£1uorouracil on the synthesis of nuclear RNA in L1210 cells in vitro. Mol Pharmacol, 1979; 16: 270-277. 6 Glazer RI, Hartman KD. The effect of 5-£1uorouracil on the synthesis and methylation of low molecular weight nuclear RNA in L1210 cells. Mol Pharmacol, 1979; 17: 245-249. 7 Kufe DW, Major PP, Egan EM, Loh E. 5-Fluoro-2'deoxyuridine incorporation into L1210 DNA. ] BioI Chern, 1981; 256: 8885-8888. 8 Danenberg PV, Heidelberger C, Mulkins M, Peterson AR. The incorporation of 5-£1uoro-2' -deoxyuridine into DNA of mammalian tumor cells. Biochem Biophys Res Commun, 1981; 102: 654-658. 9 Schuetz ]D, Wallace H], Diasio RB. 5-fluorouracil incorporation into DNA of CF-l mouse bone marrow cells as a possible mechanism of toxicity. Cancer Res, 1984; 44: 13581363. 10 Santi DV. A biochemical rationale for the use of 5fluorouracil in combination with leucovorin. In: Bruckner HW, Rustum YM. (eds.), The Current Status of 5-Fluorouracil-Leucovorin Calcium Combination. New York: Park Row Publishers, 1984: 1-4. 11 Waxman S, Bruckner H, Wagle A, Schreiber C. Potentiation of 5-fluorouracil (5-FU) antimetabolic effect by leucovorin (LV). Proc Am Assoc Cancer Res, 1978; 19: 149. 12 Ullman B, Lee M, Martin DW]r, Santi DV. Cytotoxicity of 5-fluoro-2' -deoxyuridine: requirement for reduced folate cofactors and antagonism by methotrexate. Proc Nat! Acad Sci USA, 1978; 75: 980-983.
BIOCHEMICAL RATIONALE FOR THE 5-FLUOROURACIL LEUCOVORIN COMBINATION, ETC.
13 Houghton JA, Houghton PJ. On the mechanism of cytotoxicity of fluorinated pyrimidines in four human colon adenocarcinoma xenografts maintained in immune-deprived mice. Cancer (Phila.), 1980; 45: 1159-1167. 14 Houghton JA, Maroda SJ, Phillips JO, Houghton PJ. Biochemical determinants of responsiveness to 5-fluorouracil and its derivatives in xenografts of human colorectal adenocarcinomas in mice. Cancer Res, 1981; 41: 144-149. 15 Houghton JA, Schmidt C, Houghton PJ. The effect of derivatives of folic acid on the fluorodeoxyuridylate-thymidylate synthetase covalent complex in human colon xenografts. Eur J Cancer Clin Oncol, 1982; 18: 347-354. 16 Houghton JA, Houghton PJ. Basis for the interaction of 5-fluorouracil and leucovorin in colon adenocarcinoma. In: Bruckner HW, Rustum YM. (eds.), The Current Status of 5Fluorouracil-Leucovorin Calcium Combination. New York: Park Row Publishers, 1984: 23-32. 17 Lockshin A, Danenberg A. Biochemical factors affecting the tightness of 5-fluorodeoxyuridylate binding to human thymidylate synthetase. Biochem Pharmacol, 1981; 30: 247257. 18 Evans RM, Laskin JD, Hakala MT. Effect of excess folates and deoxyinosine qn the activity and site of action of 5-fluorouracil. Cancer Res, 1981; 41: 3288-3295. 19 Blakley RL. The Biochemistry of Folic Acid and Related Pteridines, New York: American Elsevier Publishing Co., 1969: 82. 20 Straw JA, Szapary D, Wynn WT. Pharmacokinetics of the diastereoisomers of leucovorin after intravenous and oral administration to normal subjects. Cancer Res, 1984; 114: 3114-3119. 21 Trave F, Rustum YM, Petrelli N et al. Plasma and tumor tissue pharmacology of high dose intravenous leucovorin calcium in combination with fluorouracil in patients with advanced colorectal carcinoma. J Clin Oncol, 1988; 6: 1184. 22 Sirotnak FM, Chello RL, Moccio DM, et al. Stereospecificity at carbon 6 of formyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro. Biochem Pharmacol, 1979; 28: 2993-2997. 23 Hines JD, Zakem MH, Adelstein DJ, et al. Bioavailability of high dose oral leucovorin (CF). NCI Monograph: Development of folate and folic acid antagonists for cancer chemotherapy, 1987; 5: 57. 24 McGuire BW, Sia LL, Leese PT, et al. Pharmacokinetics of leucovorin calcium after intravenous, intramuscular and oral administration. Clin Pharmacol, 1988; 6: 52-58. 25 Hines JD, Adelstein DJ, Jefferey L, et al. High dose weekly.oral leucovorin and 5-fluorouracil in previously untreated patients with advanced colorectal carcinoma: A phase
11
I study. In: Rustum and McGuire, eds. Expanding role of Folates and Fluoropyrimidines in Cancer Chemotherapy, Plenum Press, New York, NY, 1989; 44: 213. 26 Madajewicz S, Petrelli N, Rustum YM, et al. Phase I-II trial of high dose calcium leucovorin and 5-fluorouracil in advanced colorectal cancer. Cancer Res, 1984; 44: 4667-4669. 27 Petrelli N, Herrera L, Rustum Y, et al. A prospective randomized trial of 5-fluorouracil versus 5-fluorouracil and high dose leucovorin versus 5-fluorouracil and methotrexate in previously untreated patients with advanced colorectal carcinoma. J Clin Oncol, 1987; 5 (10): 1559-1565. 28 Machover D, Schwarzenberg L, Goldsmith E, et al. Treatment of advanced colorectal and gastric adenocarcinoma with 5-FU combined with high-dose folinic acid: A pilot study. Cancer Treat Rep, 1982; 66: 1803-1807. 29 Doroshow]H, Bertrand M, Newman E, et al. Preliminary analysis of a randomized comparison of 5-fluorouracil and high dose continuous infusion folinic acid in dessiminated colorectal cancer. NCI Monographs, 1987; 5: 171. 30 Petrelli N, Stablein D, Bruckner H, Megibow A, Mayer R, Douglass H. A prospective randomized phase III trial of 5-fluorouracil (5-FU) versus 5-FU and high dose leucovorin (HDCF) versus 5-FU and low dose leucovorin (LDCF) in patients (pts) with metastatic colorectal carcinoma: A report of the Gastrointestinal Tumor Study Group. Proc Am Assoc Cancer Res, 1988; 7: 94 (abst. No. 357). 31 Bertrand M, Doroshow JH, Multhauf P, et al. Highdose continuous infusion folinic acid and bolus 5-fluorouracil in patients with advanced colorectal cancer: a phase II study. J Clin Oncol, 1986; 4: 1058-1061. 32 O'Connel MJ. A controlled clinical trial including folinic acid of two distinct dose levels in combination with 5fluorouracil for the treatment of advanced colorectal cancer. Experience of the Mayo Clinic and North Central Cancer Treatment Group. In: Rusturn and McGuire, eds. The Expanding Role of Folates and Fluoropyrimidines in Cancer Chemotherapy. Advances in Experimental Medicine and Biology, New York: Plenum Press, 1989; 44: 213. 33 Erlichman C, Fine S, Wong A, Elhakin J. A randomized trial of 5-fluorouracil and folinic acid in patients with metastatic colorectal cancer. J Clin Oncol, 1988; 6: 469-475. 34 Nobile MT, Cannobbio L, Sobrero A, et al. A randomized trial of 5-fluorouracil alone versus 5-fluorouracil and high dose leucovorin in untreated and advanced colorectal cancer patients. In: Rustum and McGuire, eds. Expanding Role of Folates and Fluoropyrimidines in Cancer Chemotherapy. Advances in Experimental Medicine and Biology. Symposium held in Buffalo, NY, April 28 and 29, New York, NY: Plenum Press, 1989; 44: 213.
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Biochemical Rationale for the 5-Fluorouracil Leucovorin Combination and Update of Clinical Experience Y.M. Rustum To cite this article: Y.M. Rustum (1990) Biochemical Rationale for the 5-Fluorouracil Leucovorin Combination and Update of Clinical Experience, Journal of Chemotherapy, 2:sup1, 5-11, DOI: 10.1080/1120009X.1990.11738998 To link to this article: http://dx.doi.org/10.1080/1120009X.1990.11738998
Published online: 15 Jul 2016.
Submit your article to this journal
Article views: 1
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yjoc20 Download by: [University of Tasmania]
Date: 13 March 2017, At: 23:51
Vol. 2 - Supplement n. 1 (5-11) - 1990
Journal of Chemotherapy
Biochemical Rationale for the 5-Fluorouracil Leucovorin Combination
and Update of Clinical Experience Y.M. RUSTUM
Summary - - - - - - - - - - - - - - - -
Although '-fluorouracil has been the drug of choice for the treatment of patients with advanced adenocarcinoma of the colon, the reported response rate does not exceed 20% with a median survival time less than 9 months. Mechanisms of sensitivity to this agent include: 1) inhibition of thymidylate synthase by FdUMP, the active metabolite of j-£luorouracil; 2) incorporation of FUTP into cellular RNA; and J) incorporation of FdUTP into cellular DNA. Recent laboratory preclinical results suggest that the major site of action of '-fluorouracil when combined with '-formyltetrahydrofolate (folinic acid, leucovorin) is thymidylate synthase resulting in pronounced and prolonged inhibition of DNA synthesis. This effect is primarily due to the stabilization of FdUMP binding to thymidylate synthase by the reduced folate cofactor, N', N10methylenetetrahydrofolate. In this paper, the rationale for the modulation of ,fluorouracil by leucovorin, the clinical pharmacology and the clinical experience of this combination will be reviewed.
INTRODUCTION
5-Fluorouracil (FUra) has been utilized extensively as a single agent or in combination with other agents in the clinical management of gastrointestinal malignancies, and carcinomas of the breast, ovary, and squamous carcinomas of the head and neck 1,2. FUra, administered as a single agent, is generally considered the standard therapy for patients with adenocarcinoma of the colon; however, the drug produces a response-rate of less than 20% in this disease 2. Because of the wide spectrum of marginal antitumor activity exhibited by FUra, numerous investigators are trying to enhance the antitumor activity and therapeutic selectivity of FUra by combining this drug with other agents. In this paper, the recent investigations that have attempted to enhance the antitumor. activity of FUra will be reviewed. This includes the use of FUra modulators such as leucovorin (CF). METABOLISM OF FUra
Once transported inside the cell, FUra undergoes extensive metabolism (Figure 1). The antitumor activity and cytotoxicity of this compound have been ascribed to three major mechanisms: (a) metabolism to 5-fluoro-2 -deoxyuridine-5 -monophosphate (FdUMP), which is a potent inhibitor of thymidylate synthase (dTMP-S) 4,5; b) incorporation of the ribonucleoside triphosphate (5-fluorouridine 5 triphosphate), into certain species of RNA 6,7; and c) incorporation of the metabolite 5-fluoro-2 -deoxyuridine 5 -triphosphate into DNA 8-10. In the reaction catalyzed by dTMP-S, the normal substrate 2 -deoxyuridine 5 I
I
I
Key words: 5-Fluorouracil, leucovorin, colon adenocarcinoma.
I
Grace Cancer Drug Center, Roswell Park Memorial Institute, Elm and Carlton Streets, Buffalo, NY 14263 USA. © Edizioni Riviste Scientifiche - Firenze
I
I
I -
ISSN 1120-009X
6
Y.M. RUSTUM
Fdurd - -..
...-----,I - -.....·1
dTMP~ dTDP
Figure 1 - Metabolic pathway of fluoropyrirnidines and folates.
monophosphate (dUMP) is converted to 2'deoxythymidine 5' -monophosphate (dTMP), which is required for DNA synthesis. In this reaction, the reduced folate N,sNIO methylenetetrahydrofolate, (N, SNI0_CH 2 -FH 4 ), serves as the required cofactor, as the methylene group of the folate is added to the 5 position of the heterocyclic ring, and is subsequently reduced to the CH 3 state to form dTMP 11. The FUra metabolite FdUMP inhibits this reaction in a competitive manner with respect to the normal substrate dUMP. In the absence of the reduced folate cofactor, the FdUMP forms a bivalent complex with the enzyme, (Kd = 10 - SM), whereas the presence of N, SN 1 0 -CH 2 - FH 4 results in a covalent ternary complex between the enzyme, the cofactor, and FdUMP, in. which FdUMP is bound 7-8 orders of magnitude more tightly to the complex 10. This observation has led numerous investigators to suggest that the extent of the cytotoxic effects of FUra may be critically dependent on the intracellular levels of the reduced folate cofactor.
BIOCHEMICAL RATIONALE FOR THE LEUCOVORIN/FURA COMBINATION IN CLINICAL INVESTIGATIONS
Waxman et ale 11 performed some of the initial studies to investigate the potential role of reduced folates in potentiating FUra toxicity. These investigators observed that incubating Friend leukemia cells with 10 J.l,M citrovorum factor (CF) increased the sensitivity of cells to
-
dTTP
F-DNA
FUra. They postulated that the intracellular folate cofactor required for FdUMP interaction with dTMP-S may be a limiting factor in FUra cytotoxicity, and it was possible to overcome this metabolic limitation with the addition of exogenous reduced folate. Ullman et ale 12 demonstrated that FdUrd cytotoxicity towards cultured L1210 cells was reduced when intracellular folates were depleted by limiting their source in the medium or by treating the cells with methotrexate to inhibit dihydrofolate reductase. Furthermore, they noted that the intracellular levels of FdUMP associated with dTMP-S were decreased significantly when cells were depleted of folates. Houghton et ale 13-16 have conducted numerous investigations to determine the biochemical bases of responsiveness to FUra in xenografts of human colorectal adenocarcinoma transplanted in immune deprived mice. Their data revealed that FUra sensitive and FUra resistant xenografts could not be distinguished by total drug uptake, incorporation into RNA, maximal concentrations of FdUMP achieved intracellularly, or retention of this metabolite. Likewise, FUra sensitive and resistant xenografts could not be distinguished by the activity of dTMP-S, nor by the ratio of free FdUMP to dTMP-S binding activity. In the FUra sensitive tumors, however, maximal binding was achieved without addition of exogenous cofactor. In contrast, in FUra resistant lines, maximal enzyme inhibition occurred only in the presence of exogenous cofactor. These results led these investigators to conclude that the intracellular availability of the reduced cofactor may
BIOCHEMICAL RATIONALE FOR THE 5-FLUOROURACIL LEUCOVORIN COMBINATION, ETC.
play a critical role in the formation of the ternary complex, and that increasing the intracellular level of the reduced folate cofactor may result in greater ternary complex formation and increase the sensitivity of tumors which may be relatively refractory to fluoropyrimidine treatment 14. Subsequent studies by these investigators have demonstrated that only N, 5NI0_CH 2FH 4 and FH 4 are capable of increasing ternary complex formation or stabilizing the preformed covalent complex in cytosol preparations from human colon tumor xenografts 15. It was also demonstrated that an elevated level of active intracellular folate, (FH 4 or N,5NI0_CH 2-FH 4) was required for the maintenance of a stable ternary complex and furthermore, that a prolonged continuous infusion of leucovorin may enhance the cytotoxicity of FUra by stabilizing the dTMP-S-N,5Nl°-CH 2FH 4-FdUMP ternary complex 16. Lockshin and Danenberg 17 extensively investigated the role of the reduced folate cofactor in enhancing the binding of FdUMP to dTMP-S. The results of these investigations have demonstrated that increasing the concentration of N, 5NI0_CH 2-FH 4 can in fact decrease the K off of FdUMP from the complex; however, the Koff of the reduced folate remains constant regardless of the FdUMP levels. This observation is consistent with a mechanism whereby FdUMP does not dissociate from the _enzyme until the cofgctor has been released. These results are consistent with a general mechanism suggesting that the concentration can markedly influence the effectiveness of FdUMP as an inhibitor of dTMP-S. Evans et ale 18 have evaluated the effect of excess '£olates on the activity and site of action of FUra in the S-180 murine sarcoma highly sensitive to FUra and in the human carcinoma cells Hep-2 in cell culture relatively resistant to FUra. In the absence of exogenous cofactor, S180 cells are 50-fold more sensitive to FUra .than is the human line. Furthermore, in the absence of endogenous folates, dTMP-S inhibition is the growth limiting event in S-180 cells, whereas FUra incorporation into RNA is growth limiting in the Hep-2 cell line. Following a 24-hour incubation of cells with a 20 JlM dL-CF and 3 hour exposure to FUra the toxicity of FUra was potentiated 3-fold in both cell lines, although this potentiation did not appear
7
to be due to greater inhibition of dTMP-S, but rather to the stabilization of the ternary complex of FdUMP-dTMP-S-Ns,Nlo,CH2FH4. These studies also revealed that in the presence of excess CF, the site of action of FUra is shifted from RNA to dTMP-S inhibition, resulting in a greater and more prolonged inhibition of DNA synthesis and increased responsiveness to FUra. PHARMACOKINETICS AND CELLULAR PHARMACOLOGY OF LEUCOVORIN
Leucovorin (CF) is a chemically stable 5formyl derivative of FH 4 which can be administered either orally or parenterally. The calcium salt of CF, which is readily soluble in water, is the preparation which is used in the clinic when leucovorin is prepared by chemical reduction. The final product consists of equal amounts of the d- and diastereoisomers; however, the 1diastereoisomer is the biologically active form 19-21. Straw et ale 20 demonstrated that following an i.v. administration of d,lleucovorin, the natural diastereoisomer was rapidly cleared from the plasma by conversion to 5-CH 3 FH 4 and urinary excretion. In contrast, d-5-CH 3 FH 4 is not metabolized and is slowly excreted in the urine. The plasma half-life of the d isomer and I isomer were 451 ± 24 minutes, and 31.6 ± 1.1 minutes respectively. Furthermore, these investigators determined that the urinary clearance of 1-5-CHO-FH 4 and the 5-CH 3 derivative differed only slightly from the creatinine clearance, whereas urinary clearance of d-5-CHO-FH 4 is only 20% of creatinine clearance indicating that this compound is extensively reabsorbed. Sirotnak et al. 22 have demonstrated that both diastereoisomers of folinic acid as well as 5-CH 3 -FH 4 are transported across the cell membrane by a common reduced folate carrier system, although d-5-CHO-FH 4 competes much less effectively with the natural isomers for transport in several murine lines in vitro. Similar results have been obtained by Straw et ale 20, who noted that absorption of d,l-CF after p.o. administration to man is stereoselective, with the absorption of the natural isomer being approximately 5 times that of the d-isomer. Furthermore, when compared to an i. V. administration of leucovorin, repeated oral adminis-
8
Y.M. RUSTUM
tration resulted in a more favorable plasma ratio of active to inactive isomers. Straw and colleagues observed that the concentration of the inert isomer (d) greatly exceeds the concentration of the biologically active isomer (1) following an i.v. administration of the drug, and they suggest that repeated parenteral administration would result in a selective accumulation of the biologically inert isomer, which may compete with the active isomer for cellular transport and metabolism. Although oral administration of CF results in a preferential absorption of the I-isomer, several investigators have demonstrated that peak plasma levels of I-CF are quite low following an oral dose of the drug 23,24. Hines et at.' 23 examined the pharmacokinetics of d-I-CF doses of 200-1600 mg hourly in four divided oral doses. The peak plasma concentration of the I-reduced folate (l-CF + 5-CH 3 tetrahydrofolate) was achieved 4-6 hours following drug administration and ranged from 3.42 ± 0.65 J.1M to 5.11 ± 1.81 J.1M; however, at the highest dose the peak plasma concentration of l-CF was only 0.34 ± 0.16 J.1M. McGuire et ale 24 determined that absolute bioavailability of .a 200 mg oral dose of CF was 31 % of the same dose administered i. v. The 200 mg oral dose resulted in a < 2.0 J.1M plasma level of I-reduced folates, whereas the same dose administered Lv. achieved a peak plasma concentration approaching 20 J.1M. Trave et ale 21 investigated the pharmacokinetic parameters of high dose dl-CF (500 mg/m 2 ) administered by 2 h continuous i. v. infusion and FUra (600 mg/m 2 ) Lv. infusion and FUra (600 mg/m 2 ) i.v. push at 1 h. The concentrations . of dl~CF, I-CF and 5-CH 3 FH 4 at different time points during and following the 2 h i. v. infusion of dl-CF are shown in Table 1. The dl-CF concentrations peaked at the end of the 2 h infusion, while the 5-CH 3 FH 4 ' peak
TABLE 1 - Plasma half-life (ti) of reduced folates in patients with advanced colorectal cancer.
Concentration
(~M)
tJ2 (H)
Component clCF LCF 5-CH 3 FH4
Lv.
p.o.
i.v.
p.o.
80::1:32 24::1:3.2 17::1:8
5::1:2 12
6+ >12
1000
o • dL-CF
Csse 82f'-M
o • L-CF
c... t.9fLM
•
C.. • t5fLM
• 5- CH J
tr---6 • FUra
tOO
C•• • t.5~
'0
o
0
o
•
•
~
. . ----6 ---
~
-0-------------~~----~~
6.
.O:i
l1JL - _L
4
24
--4'-8-.--71.---91.--~ 6 2
Time (t'loursr
Figure 2.
concentration was observed from one to three hours after the end of treatment. l-CF concentrations above 10 J.1M were reached 1 h after the start of the 2 h infusion and maintained for about 2 h. A plasma concentration of 5CH 3 FH 4 exceeded 10 J.1M for a period of about 6 h. Mean t 1/2 and plasma clearance of I-CF were respectively 10 and 18-fold faster than those of dl-CF. Under these conditions, the percent of I-isomer in the HPLC-detected dl-CF dropped from the initial 50% at the beginning of drug administration to less than 10% 2 h after the termination of the drug infusion. Contribution of I-isomer to dl-CF AUC was less than 4%. Furthermore, the plasma tY2 was similar to those of d-CF. Five-day continuous Lv. infusion of dl-CF (500 mg/m 2/d X 5) yielded a steady state concentration of 82 J.1M, 1.9 J.1M, amd 15 J.1M for dCF, L-CF and 5-CH 3 FH 4 , respectively (Figure 2). Pharmacokinetic studies of oral dL-CF (125 mg/m 2 X 4 h) were carried out in patients with advanced colorectal carcinoma and the results are shown in Table 1. These results indicate the plasma concentrations of I-CF seen in these patients receiving oral dl-CF were below 0.5 J.1M.
9
BIOCHEMICAL RATIONALE FOR THE 5-FLUOROURACIL LEUCOVORIN COMBINATION, ETC.
COLORECTAL CARCINOMA
TABLE 2 - Protocols of FUra in combination with CF. Reference • FUra CF
600 mg/m2/wk X 6 Lv. push 1 h after the start of CF 500 mg/m 2/wkx6 2 h Lv. infusion 2wks rest, 1 course (High CF dose, HD)
• FUra CF
370 mg/m 2/d X 5 day; Lv. push 200 mg/m 2/d X 5 day; 15 min infusion Q 28 days (Intermediate CF dose)
• FUra
370 mg/m 2/d X 5 day continuous Lv. infusion 500 mg/m 2/d X 5.5 day continuous Lv. infusion Q 28 days (High CF dose)
CF
• FUra CF
600 mg/m 2/wkx 6 Lv. push, 425 mgfm2 d X 5d and 20 mg/m 2 d X 5 25 mg/m 2/wk X 6 15 Lv. infusion (Low CF dose)
Previously untreated patients
27,28
Toxicity. The profile of FUra tOXICIty in combination with CF was found to be dose and schedule dependent of CF administration. The data demonstrated that the dose-limiting toxicity of FUra alone when administered by a daily or weekly schedule was myelotoxicity. In contrast, CF, regardless of the dose and schedule, caused a shift in the profile of toxicity of FUra to gastrointestinal (GI) and stomatitis. In the CF weekly schedule the toxicity was predominantly GI while with the daily schedule, stomatitis was the dose limiting toxicity 28,29,31,32. In the study by Erlichman et all 33, patients receiving FUra + CF had significantly more mucositis and diarrhea from course 1 than those receiving FUra alone (p < 0.005); stomatitis was more severe in the combination group (p < 0.02). In the study of Doroshow et all 29, both stomatitis and severe diarrhea were more frequent in the FUra + CF group than the FUra alone group in the study of Nobile et all 34 and there was more severe GI toxicity in the Petrelli trial 27 • With the more pronounced toxicity, high dose CF was achieved requiring a' dose reduction of FUra. The diarrhea can be severe and in some cases requiring hospitalization. With careful follow-up and dose reduction -of FUra, reversal of toxicity can be achieved, however.
29
30,32
31,33
CLINICAL PROTOCOLS
Although other schedules and doses of CF and FUra have been utilized clinically, those clinical protocols which have been extensively utilized are listed in Table 2. In addition, recently a protocol utilizing oral leucovorin in colon cancer is under investigation. The dose of CF is equivalent to 50-125 mg/m 2fh X 4 p.o. with FUra administration Lv. after the 4th oral dose of CF. Initial clinical results suggest that this form of CF treatment is as active as the i. v. use of CF23,2S.
TABLE 3 - Phase III trials of FUra and CF previously untreated advanced colorectal cancer. Studyl
FUra (mg/m 2 )
Patient (No.)
Response (%)
FUra+CF (mg/m2 )
Patient (No.)
Response (%)
PMH
370 X 5d
61
7
370+200x5d
64
33
RPMI
450x 5d 200xqodx6
19
9
600 + 500/wk X 6
30
40
COH
300x 5d
34
15
370 + 500 X 5.5d*
29
45
GITSG
500x 5d
107
12
600 + 500/wk X 6
109
28
NCCTG
500x5d
39
10
370 +200 X 5d 425 + 20 X 5d
35 37
26 43
NCOG
440 x5d then 555/wk
49
19
400 + 200 X 5d
16
96
Genoa
600/wkx6
44
11
600 + 500/wk X 6
43
31
* Continues Lv. infusion 1 PHM, Princess Margaret Hospital, Canada (29, Erlichman); RPMI, Roswell Park Memorial Inst. (27,28, Petrelli); COH , Ctty of Hope (30, 32 Doroshow); GITSG, Gastrointestinal Tumor Study Group (31, Petrelli); NCCTG, North Central Cancer Treatment Group (32, O'Connel); NCOB, Northern California Oncology Group (35, Valone); Genoa (34, Nobile). •
10
Y.M. RUSTUM
Therapeutic efficacy. The data in Table 3 demonstrate the updated clinical experience (Phase III) with this combination. In all the clinical trials conducted to date, except for the NCOG, the data demonstrated that CF can indeed increase significantly the response rate to FUra in previously untreated patients with advanced colorectal cancer. The controversy, however, remaining to be resolved is whether high dose CF (500 mg/kg) in combination with FUra produces a higher response rate than low (20-25 mg/m 2 ) or intermediate (200 mg/m 2 ) doses of CF. Except for the North Central Cancer Treatment Group (NCCTG) (O'Connel) high dose CF appears to give a higher response rate. In all the Phase III trials, overall ·response rate to FUra was 11.8% (42/365 pts) and to FUra with CF was 28% (154/564 pts). Previously treated patients. Contrary to previously untreated patients with colon cancer, the combination of FUra + CF in previously treated patients is less effective regardless of the CF dose used. The data suggest that this combination should be limited at the present time to previously untreated patients with advanced colorectal cancer. Studies are underway to delineate the underlying nechanisms of action of CF and FUra in this group of patients.
CONCLUSION AND FUTURE PROSPECTS
The CF/5-FUra regimen possesses antitumor activity against colorectal carcinoma (and other tumors such as breast carcinoma and squamous cell head and neck cancers). Recent phase III triaJs demonstrated that the antitumor activity of the combination against colorectal carcinomas is significantly greater than the response obtained with 5-FUra administered as a single agent at the maximally tolerated dose. It is not yet clear, however, whether this combination actually has improved the survival rate of patients with advanced colorectal carcinoma, although initial results are favorable. Future plans with this combination should include: 1) Evaluation of the therapeutic efficacy of this combination against other human solid tumors 2) Studies to determine -whether the thera-
peutic activity of the CF/5-FUra regimen can be enhanced by the addition of other agents (i.e. cisplatin or dipyridamole) to the regimen 3) Identification of the optimal dose and schedule of 5-FUra administered alone and in combination with leucovorin 4) Determination of the therapeutic role of l-CF administration by different routes 5). Biochemical and pharmacologic studies designed to elucidate mechanisms for improvement in the therapeutic selectivity of 5-FU and, thus, provide a rational approach for improving the survival rate of patients treated with the combination of 5-FUra and leucovorin.
REFERENCES 1 Chabner BA, Myers CEo Clinical pharmacology of cancer chemotherapy. In: DeVita V, Hellman S, Rosenberg SA. (eds.), Cancer Principles and Practice of Oncology; Philadelphia: ]B Lippincott, 1985: 296. 2 Levin P, Mittelman A, Douglass H, et al. Survival and response to chemotherapy for advanced colorectal adenocarcinoma: an Eastern Cooperative Oncology Group report. Cancer (Phila.), 1980; 46: 1536-1543. 3 Santi DV, McHenry CS, Sommer H. Mechanism of interaction of thymidylate synthetase with 5-£1uorodeoxyuridylate. Biochemistry, 1974; 13: 471-480. 4 Danenberg PV, Langenbach R], Heidelberger C. Structures of reversible and irreversible complexes of thymidylate synthetase and £1uoropyrimidine nucleotides. Biochemistry, 1974; 13: 926-933. S Glazer RI, Pealer AL. The effect of 5-£1uorouracil on the synthesis of nuclear RNA in L1210 cells in vitro. Mol Pharmacol, 1979; 16: 270-277. 6 Glazer RI, Hartman KD. The effect of 5-£1uorouracil on the synthesis and methylation of low molecular weight nuclear RNA in L1210 cells. Mol Pharmacol, 1979; 17: 245-249. 7 Kufe DW, Major PP, Egan EM, Loh E. 5-Fluoro-2'deoxyuridine incorporation into L1210 DNA. ] BioI Chern, 1981; 256: 8885-8888. 8 Danenberg PV, Heidelberger C, Mulkins M, Peterson AR. The incorporation of 5-£1uoro-2' -deoxyuridine into DNA of mammalian tumor cells. Biochem Biophys Res Commun, 1981; 102: 654-658. 9 Schuetz ]D, Wallace H], Diasio RB. 5-fluorouracil incorporation into DNA of CF-l mouse bone marrow cells as a possible mechanism of toxicity. Cancer Res, 1984; 44: 13581363. 10 Santi DV. A biochemical rationale for the use of 5fluorouracil in combination with leucovorin. In: Bruckner HW, Rustum YM. (eds.), The Current Status of 5-Fluorouracil-Leucovorin Calcium Combination. New York: Park Row Publishers, 1984: 1-4. 11 Waxman S, Bruckner H, Wagle A, Schreiber C. Potentiation of 5-fluorouracil (5-FU) antimetabolic effect by leucovorin (LV). Proc Am Assoc Cancer Res, 1978; 19: 149. 12 Ullman B, Lee M, Martin DW]r, Santi DV. Cytotoxicity of 5-fluoro-2' -deoxyuridine: requirement for reduced folate cofactors and antagonism by methotrexate. Proc Nat! Acad Sci USA, 1978; 75: 980-983.
BIOCHEMICAL RATIONALE FOR THE 5-FLUOROURACIL LEUCOVORIN COMBINATION, ETC.
13 Houghton JA, Houghton PJ. On the mechanism of cytotoxicity of fluorinated pyrimidines in four human colon adenocarcinoma xenografts maintained in immune-deprived mice. Cancer (Phila.), 1980; 45: 1159-1167. 14 Houghton JA, Maroda SJ, Phillips JO, Houghton PJ. Biochemical determinants of responsiveness to 5-fluorouracil and its derivatives in xenografts of human colorectal adenocarcinomas in mice. Cancer Res, 1981; 41: 144-149. 15 Houghton JA, Schmidt C, Houghton PJ. The effect of derivatives of folic acid on the fluorodeoxyuridylate-thymidylate synthetase covalent complex in human colon xenografts. Eur J Cancer Clin Oncol, 1982; 18: 347-354. 16 Houghton JA, Houghton PJ. Basis for the interaction of 5-fluorouracil and leucovorin in colon adenocarcinoma. In: Bruckner HW, Rustum YM. (eds.), The Current Status of 5Fluorouracil-Leucovorin Calcium Combination. New York: Park Row Publishers, 1984: 23-32. 17 Lockshin A, Danenberg A. Biochemical factors affecting the tightness of 5-fluorodeoxyuridylate binding to human thymidylate synthetase. Biochem Pharmacol, 1981; 30: 247257. 18 Evans RM, Laskin JD, Hakala MT. Effect of excess folates and deoxyinosine qn the activity and site of action of 5-fluorouracil. Cancer Res, 1981; 41: 3288-3295. 19 Blakley RL. The Biochemistry of Folic Acid and Related Pteridines, New York: American Elsevier Publishing Co., 1969: 82. 20 Straw JA, Szapary D, Wynn WT. Pharmacokinetics of the diastereoisomers of leucovorin after intravenous and oral administration to normal subjects. Cancer Res, 1984; 114: 3114-3119. 21 Trave F, Rustum YM, Petrelli N et al. Plasma and tumor tissue pharmacology of high dose intravenous leucovorin calcium in combination with fluorouracil in patients with advanced colorectal carcinoma. J Clin Oncol, 1988; 6: 1184. 22 Sirotnak FM, Chello RL, Moccio DM, et al. Stereospecificity at carbon 6 of formyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro. Biochem Pharmacol, 1979; 28: 2993-2997. 23 Hines JD, Zakem MH, Adelstein DJ, et al. Bioavailability of high dose oral leucovorin (CF). NCI Monograph: Development of folate and folic acid antagonists for cancer chemotherapy, 1987; 5: 57. 24 McGuire BW, Sia LL, Leese PT, et al. Pharmacokinetics of leucovorin calcium after intravenous, intramuscular and oral administration. Clin Pharmacol, 1988; 6: 52-58. 25 Hines JD, Adelstein DJ, Jefferey L, et al. High dose weekly.oral leucovorin and 5-fluorouracil in previously untreated patients with advanced colorectal carcinoma: A phase
11
I study. In: Rustum and McGuire, eds. Expanding role of Folates and Fluoropyrimidines in Cancer Chemotherapy, Plenum Press, New York, NY, 1989; 44: 213. 26 Madajewicz S, Petrelli N, Rustum YM, et al. Phase I-II trial of high dose calcium leucovorin and 5-fluorouracil in advanced colorectal cancer. Cancer Res, 1984; 44: 4667-4669. 27 Petrelli N, Herrera L, Rustum Y, et al. A prospective randomized trial of 5-fluorouracil versus 5-fluorouracil and high dose leucovorin versus 5-fluorouracil and methotrexate in previously untreated patients with advanced colorectal carcinoma. J Clin Oncol, 1987; 5 (10): 1559-1565. 28 Machover D, Schwarzenberg L, Goldsmith E, et al. Treatment of advanced colorectal and gastric adenocarcinoma with 5-FU combined with high-dose folinic acid: A pilot study. Cancer Treat Rep, 1982; 66: 1803-1807. 29 Doroshow]H, Bertrand M, Newman E, et al. Preliminary analysis of a randomized comparison of 5-fluorouracil and high dose continuous infusion folinic acid in dessiminated colorectal cancer. NCI Monographs, 1987; 5: 171. 30 Petrelli N, Stablein D, Bruckner H, Megibow A, Mayer R, Douglass H. A prospective randomized phase III trial of 5-fluorouracil (5-FU) versus 5-FU and high dose leucovorin (HDCF) versus 5-FU and low dose leucovorin (LDCF) in patients (pts) with metastatic colorectal carcinoma: A report of the Gastrointestinal Tumor Study Group. Proc Am Assoc Cancer Res, 1988; 7: 94 (abst. No. 357). 31 Bertrand M, Doroshow JH, Multhauf P, et al. Highdose continuous infusion folinic acid and bolus 5-fluorouracil in patients with advanced colorectal cancer: a phase II study. J Clin Oncol, 1986; 4: 1058-1061. 32 O'Connel MJ. A controlled clinical trial including folinic acid of two distinct dose levels in combination with 5fluorouracil for the treatment of advanced colorectal cancer. Experience of the Mayo Clinic and North Central Cancer Treatment Group. In: Rusturn and McGuire, eds. The Expanding Role of Folates and Fluoropyrimidines in Cancer Chemotherapy. Advances in Experimental Medicine and Biology, New York: Plenum Press, 1989; 44: 213. 33 Erlichman C, Fine S, Wong A, Elhakin J. A randomized trial of 5-fluorouracil and folinic acid in patients with metastatic colorectal cancer. J Clin Oncol, 1988; 6: 469-475. 34 Nobile MT, Cannobbio L, Sobrero A, et al. A randomized trial of 5-fluorouracil alone versus 5-fluorouracil and high dose leucovorin in untreated and advanced colorectal cancer patients. In: Rustum and McGuire, eds. Expanding Role of Folates and Fluoropyrimidines in Cancer Chemotherapy. Advances in Experimental Medicine and Biology. Symposium held in Buffalo, NY, April 28 and 29, New York, NY: Plenum Press, 1989; 44: 213.
