Over the past decade, increasing data have emphasized both the importance of dihydropyrimidine dehydrogenase (DPD), the initial, rate-limiting enzyme in the catabolism of fluorouracil (5-FU), and its role as a control step in 5-FU metabolism, regulating the availability of 5-FU for anabolism.
ABSTRACT: Over the past decade, increasing data have emphasized both the importance of dihydropyrimidine dehydrogenase (DPD), the initial, rate-limiting enzyme in the catabolism of fluorouracil (5-FU), and its role as a control step in 5-FU metabolism, regulating the availability of 5-FU for anabolism. It is now clear that DPD also accounts for much of the variability observed with therapeutic use of 5-FU, including variabilities in 5-FU levels over a 24-hour infusion, interindividual pharmacokinetics, bioavailability, toxicity, and drug response (resistance). This variability makes effective dosing of 5-FU and related drugs difficult. In order to lessen this variability, and potentially improve 5-FU pharmacology, the pharmaceutical industry has made an effort to develop DPD inhibitors to modulate 5-FU metabolism, which has resulted in the creation of a new subclass of orally administered fluoropyrimidines, known as DPD-inhibiting fluoropyrimidines (DIF). Four drugsuracil and tegafur (UFT) or the combination of UFT and leucovorin, ethynyluracil (eniluracil), S-1, and BOF-A2have recently undergone clinical evaluation in the United States. The biochemical basis for using these drugs is reviewed. [ONCOLOGY 14(Suppl 9):19-23, 2000]
It has now been more than four decades since fluorouracil (5-FU) was first synthesized and introduced as a clinical chemotherapeutic agent. Designed as an analog of the naturally occurring pyrimidine uracil in which a fluorine atom was substituted for a hydrogen atom in the fifth position of the pyrimidine ring, 5-FU has been shown to be a true antimetabolite. As such, it is capable of being taken up into the cell like uracil or thymine and then metabolized by the pyrimidine anabolic and catabolic pathways (see Figure 1).[1,2] The 5-FU nucleotide metabolites that are formed from anabolism, including 5-fluorodeoxyuridine 5´-monophosphate (FdUMP), 5-fluorouridine 5´-triphosphate (FUTP), and 5-formyl-2´-deoxyuridine, 5´-triphosphate (FdUTP) can affect critical sites responsible for blocking cell replication. This includes inhibition of thymidylate synthase or incorporation into RNA or DNA, resulting in cytotoxicity and, in turn, anticancer activity.
While anabolism is clearly important, over the past several years there has been an increasing appreciation for the important role of catabolism in 5-FU pharmacology by regulating the amount of 5-FU available for anabolism. The first enzyme in catabolism, dihydropyrimidine dehydrogenase (also known as DPD, dihydrouracil dehydrogenase, dihydrothymine dehydrogenase, uracil reductase, EC 22.214.171.124) is the critical rate-limiting step in the regulation of 5-FU metabolism. DPD is responsible for converting more than 85% of clinically administered 5-FU to 5-FUH2 (an inactive metabolite) in an essentially irreversible enzymatic step.
The variability in DPD activity, in some cases within individual patients and in others from patient to patient, has been shown to be responsible for much of the variability noted in clinical studies with 5-FU. The affect of variable DPD activity on the clinical pharmacology of 5-FU is summarized in Table 1.
Variation in DPD activity within patients is thought to be responsible for the variable blood levels of 5-FU noted over a 24-hour period. Studies have demonstrated that 5-FU levels can vary with a peak and trough following a cosine wave within patients receiving continuous infusion of 5-FU by automated pumps over a 24-hour period.[4-6] The DPD levels in these patients have been observed to follow a circadian pattern inverse to the circadian pattern observed with 5-FU levels in this cohort. This finding has prompted some chemotherapists to propose the use of time-modified 5-FU infusions to optimize drug delivery during a 24-hour period as a potential benefit in the treatment of certain human cancers.
In studies in different patients who otherwise have similar liver function, demonstrated variation in DPD activity is now thought to be responsible for the large variation in 5-FU pharmacokinetics. Following administration of an intravenous (IV) bolus of 5-FU, the t ½ß was shown to vary almost five-fold among patients studied. DPD enzyme activity in normal tissues (peripheral blood mononuclear cells and liver) has also been shown to vary from individual to individual in a normal distribution pattern, with as much as a six-fold variation from the lowest to the highest values.[9,10] This wide variation in DPD activity is thought to be responsible for the wide variation in the t ½ß observed in patients in population studies.
While most individuals have DPD activity within the normal distribution, a small percentage (< 5%) of the population has DPD activity significantly below the normal distribution.[11-13] These individuals are at significant risk if they develop cancer and are subsequently treated with 5-FU. In this setting, the normal degradation of approximately 85% of 5-FU does not occur, resulting in more available 5-FU for anabolism and patients effectively receiving a drug overdose. This is a true pharmacogenetic syndrome, with symptoms not being recognized until affected individuals are exposed to the drug.
Variation in DPD activity from individual to individual has also recently been shown to be responsible for the apparent variable bioavailability of 5-FU. As a result, there has been a recommendation that 5-FU not be administered orally. The erratic bioavailability has not been well understood, particularly since 5-FU is a small molecule with a pKa that should predict excellent absorption and bioavailability. As noted below, the role of DPD in 5-FU bioavailability was not appreciated until pharmacokinetic studies with DPD inhibitors demonstrated that the area under the concentration-time curve (AUC) from oral administration of 5-FU was essentially the same as that produced by IV administration, suggesting almost 100% bioavailability.
Tumors may also express variable levels of DPD activity. This may explain the observed varied tumor response to 5-FU. A recent study of interest demonstrated increased DPD expression in tumors from patients who were resistant to 5-FU, even when thymidylate synthase expression was low.
The above studies demonstrate the variability in DPD levels in both normal and tumor tissues that may explain the observed variability in 5-FU pharmacology. It has become attractive to consider inhibiting DPD in order to eradicate the variability in 5-FU pharmacology. DPD inhibition in 5-FUsusceptible host tissue, such as gastrointestinal mucosa and bone marrow, should decrease dosing variability from patient to patient. This would be an improvement over the current situation with 5-FU and many other cancer chemotherapeutic agents in which dosing decisions are typically based on observed toxicity. Inhibition of DPD in tumor specimens is also attractive, particularly since many tumors may be 5-FU-resistant based on increased DPD activity within the tumor, resulting in increased degradation and thus less anabolism of 5-FU.
Over the years, many attempts have been made to synthesize more effective fluoropyrimidine drugs, of which three generations currently exist (Table 2). 5-FU and its deoxyribonucleoside derivative 5-fluoro-2-deoxyuridine (FdUrd) represent the first generation. While 5-FU continues to be used as an IV bolus, as part of a continuous infusion regimen, or as a protracted ambulatory infusion, FdUrd is used mainly as part of hepatic arterial infusion regimens. The second-generation fluoropyrimidines, including 5´dFUrd and tegafur, were developed with the hope of permitting oral administration of 5-FU. Due to the many undesirable side effects of these agents, they were never approved in the United States; they are approved in other parts of the world. The third-generation agents include several agents that were developed from the second generation, as well as two separate subclasses: the enzymatically activated prodrug capecitabine (Xeloda) and the DPD inhibitory fluoropyrimidines, or DIF drugs.
Four DIF drugs have undergone clinical evaluation, including UFT, eniluracil, S-1, and BOF-A2. These drugs differ both in the type of DPD inhibition and the degree of inhibition produced. The rationale for using DIF drugs is shown in Table 3. Basically, 5-FU, derived either from 5-FU itself or from a prodrug converted to 5-FU, is administered together with another drug that interferes with (or inhibits) the otherwise rapid catabolism of 5-FU. This effect will lead to increased exposure to 5-FU over time.
All four of the new fluoropyrimidine drugs derive a therapeutic advantage from DPD inhibition. This inhibition permits oral delivery of 5-FU (bioavailability > 70%) and results in less variability in the pharmacokinetics of the fluoropyrimidines. In addition, by inhibiting the catabolic pathway, more 5-FU can enter the anabolic pathway and potentially increase the antitumor effect, which is theoretically particularly important for tumors that have become resistant secondary to an increase of intratumoral DPD.
Finally, although not completely understood, it appears that at least several 5-FU toxicities (hand-foot syndrome, some forms of neurotoxicity, and possibly cardiotoxicity) are secondary to the catabolic pathway. Inhibiting the catabolic pathway should decrease their incidence.
UFT was the first of the DIF drugs to be synthesized and is the one for which we have the most experience. This drug is actually a combination of the naturally occurring pyrimidine uracil with the fluoropyrimidine tegafur (ftorafur) in a 4:1 molar ratio. The presence of uracil in excess of 5-FU results in competition at the level of DPD such that 5-FU formed from tegafur will not be degraded rapidly and will remain present for a longer time. While not true inhibition of DPD, the competition between 5-FU and uracil for DPD produces an effect similar to that which is achieved with a true DPD inhibitor.
In contrast to the true DPD inhibitors and inactivators (see below), the affect on DPD by UFT is more rapidly reversible. The rapidly reversible inhibition may avoid some of the problems observed with the earlier DPD inhibitors and may account for a more favorable toxicity profile compared to some of the earlier DPD inhibitors  as well as some of the newer DIF drugs.
Extensive data from Japan, as well as from Europe, South America, and the United States, demonstrate that orally administered UFT has antitumor activity in several tumor types (particularly breast and colon cancer) either as a single agent or combined with oral leucovorin (a combination being developed under the trade name Orzel).[21-23] Studies conducted thus far have shown that it is at least as effective as intravenously infused 5-FU. Furthermore, the toxicity profile has proven quite tolerable with the typical fluoropyrimidine toxicities (eg, diarrhea and nausea) seen at the maximum tolerated dose.
Notably, other toxicities , particularly hand-foot syndrome and neurologic and cardiologic toxicities, are absent. Although not well understood, these toxicities may be secondary to 5-FU catabolites. 5-FU catabolites are less likely to form from UFT, therefore, these toxicities are not typically observed. Within this issue, several articles provide evidence of UFT efficacy and tolerable toxicity.
Recently, a new DPD inhibitor, ethynyluracil (eniluracil or GW776), has been synthesized and demonstrated to be a potent inactivator of DPD. This compound is a pyrimidine possessing a structure similar to both uracil and 5-FU.
Initial phase I and II clinical studies with ethynyluracil demonstrated that DPD was rapidly and completely inactivated, with inhibition maintained for more than 1 day at clinically used doses.[26,27] Phase III studies are close to accrual and await investigators survival data to evaluate the effectiveness of coadministering low-dose 5-FU and ethynyluracil in a number of different malignancies, particularly colorectal cancer and breast cancer.
In Japan, several attempts have been made to further develop this concept. S-1 is a triple-drug combination, consisting of the prodrug, tegafur, together with a DPD inhibitor 5-chloro-2,4-dihydroxypyridine (CDHP) and potassium oxalate in a molar ratio of 1:0.4:1, respectively. This combination not only provides the sustained 5-FU release from the use of the prodrug and DPD inhibitor, but also utilizes potassium oxalate to theoretically lessen the chance of bothersome GI toxicity, particularly diarrhea. Potassium oxalate has been shown in preclinical studies to selectively inhibit 5-FU phosphorylation by the enzyme orotate phosphoribosyltransferase, especially in the GI tract but not in the tumor. Preclinical studies have been encouraging, demonstrating excellent antitumor activity. Clinical studies, thus far, have demonstrated S-1 to be quite tolerable.[31,32]
Based on its clinical efficacy in gastric cancer, S-1 has been approved in Japan for treatment of this condition, where the agent has been observed to be associated with less diarrhea. Unfortunately, early clinical studies in Western Europe and the United States have been notable because diarrhea continues to be the dose-limiting toxicity. The basis for this is unclear, but may be secondary to genetic differences in drug metabolism in the different populations.
BOF-A2 represents another attempt to develop an improved fluoropyrimidine drug. In this two-drug combination, the prodrug 1-ethoxymethyl 5-fluorouracil (EM-FU) is combined with the DPD inhibitor 3-cyano-2,6-dihydroxypyridine (CNDP) in a 1:1 molar ratio.[33,34] EM-FU is relatively resistant to degradation and is metabolized to 5-FU by the liver microsomes. Preclinical studies demonstrated antitumor activity in several animal models, with sustained 5-FU levels resulting from the release of 5-FU from EM-FU.
Clinical studies have been undertaken in both Japan and, more recently, in the United States. While some clinical responses were noted in early phase I and II studies, further research has been placed on hold in these countries due to severe fluoropyrimidine-type toxicities that occurred in several patients.
The availability of the DPD-inhibiting fluoropyrimidines, or DIF drugs, provides a new strategy in which 5-FU may be administered orally at reduced doses. This schedule produces an effect somewhat similar to continuous infusion of 5-FU with potentially less intrapatient or interpatient variability in 5-FU pharmacokinetics. Thus far, clinical studies with several of these drugs demonstrate tolerable toxicities. Clinical trials with at least one of these drugs, single-agent UFT or combination UFT and leucovorin (described elsewhere in this issue), demonstrate that 5-FU can be administered orally and achieve similar therapeutic efficacy as that obtained with the more standardized IV regimen of 5-FU.
1. Daher GC, Harris BE, Diasio RB: Metabolism of pyrimidine analogues and their nucleosides, in: Metabolism and Reactions of Anticancer Drugs, Vol. 1, in: The International Encyclopedia of Pharmacology and Therapeutics, chapter 2. Oxford, Pergamon Press, 1994.
2. Grem J: 5-Fluoropyrimidines, in Chabner BA, Longo DL, (eds): Cancer Chemotherapy and Biotherapy, pp 180-224. Philadelphia, Lippincott-Raven,1996.
3. Lu Z-H, Zhang R, Diasio RB: Purification and characterization of dihydropyrimidine dehydrogenase from human liver. J Biol Chem 267: 17102-17109, 1992.
4. Harris BE, Song R, He Y, et al: Circadian rhythm of rat liver dihydropyrimidine dehydrogenase: Possible relevance to fluoropyrimidine chemotherapy. Biochem Pharm 37: 4759-4763, 1988.
5. Harris BE, Song R, Soong S-J, et al: Circadian variation of 5-fluorouracil catabolism in isolated perfused rat liver. Cancer Res 49: 6610-6614, 1989.
6. Harris BE, Song R, Soong SJ, et al: Relationship of dihydropyrimidine dehydrogenase activity and plasma 5-fluorouracil levels: Evidence for circadian variation of 5-fluorouracil levels in cancer patients receiving protracted continuous infusion. Cancer Res 50: 197-201, 1990.
7. Levi F: Cancer chronotherapy. J Pharm Pharmacol 51:891-898, 1999.
8. Heggie GD, Sommadossi JP, Cross DS, et al: Clinical pharmacokinetics of 5-fluorouracil and its metabolites in plasma, urine, and bile. Cancer Res 47: 2203-2206, 1987.
9. Lu Z, Zhang R, Diasio RB: Dihydropyrimidine dehydrogenase activity in human peripheral blood mononuclear cells and liver: Population characteristics, newly identified patients, and clinical implication in 5-fluorouracil chemotherapy. Cancer Res 53: 5433-5438, 1993.
10. Lu Z, Zhang R, Diasio RB: Dihydropyrimidine dehydrogenase activity in human liver: Population characteristics and clinical implication in 5-FU chemotherapy. Clin Pharmacol Ther 58: 512-522, 1995.
11. Diasio RB, Beavers TL, Carpenter JT: Familial deficiency of dihydropyrimidine dehydrogenase: Biochemical basis for familial pyrimidinemia and severe 5-fluorouracil-induced toxicity. J Clin Invest 81: 47-51, 1998.
12. Harris BE, Carpenter JT, Diasio RB: Severe 5-fluorouracil toxicity secondary to dihydropyrimidine dehydrogenase deficiency: A potentially more common pharmacogenetic syndrome. Cancer 68: 499-501, 1991.
13. Takimoto CH, Lu Z-H, Zhang R, et al: Severe neurotoxicity following 5-fluorouracil-based chemotherapy in a patient with dihydropyrimidine dehydrogenase deficiency. Clin Cancer Res 2: 477-481, 1996.
14. Lu Z, Diasio RB: Polymorphic drug metabolizing enzymes, in Schilsky RL, Milano GA, Ratain MJ, (eds): Principles of Antineoplastic Drug Development and Pharmacology , pp 281-305. New York, Marcel Dekker, Inc., 1996.
15. Baccanari DP, Davis ST, Knick VC, et al: 5-Ethynyluracil: Effects on the pharmacokinetics and antitumor activity of 5-fluorouracil. Proc Natl Acad Sci 90: 11064-11068, 1993.
16. Jiang W, Lu Z, He Y, Diasio RB: Dihydropyrimidine dehydrogenase activity in hepatocellular carcinoma: Implication for 5-fluorouracil-based chemotherapy. Clin Cancer Res 3: 395-399, 1997.
17. Salonga D, Danenberg K, Johnson M, et al: Colorectal tumors responding to 5-fluorouracil have low gene expression levels of dihydropyrimidine dehydrogenase, thymidylate synthase, and thymidine phosphorylase. Clin Cancer Res 6: 1322-1327, 2000.
18. Diasio RB: Oral administration of fluorouracil: A new approach utilizing modulators of dihydropyrimidine dehydrogenase activity. Cancer Therapeutics 2:97-106, 1999.
19. Majima H: Phase I and preliminary phase II study of co-administration of uracil and FT-207 (UFT therapy). Gan To Kagaku Ryoho 7:1383-1387, 1980.
20. Naguib FNM, el Kouni MH, Cha S: Structure-activity relationship of ligands of dihydrouracil dehydrogenase from mouse liver. Biochem Pharm 38:1471-1480, 1989
21. Takino T: Clinical studies on the chemotherapy of advanced cancer with UFT (uracil plus futraful preparation). Gan To Kagaku Ryoho 7:1804-1812, 1980.
22. Gonzalez Baron M, Colmenarejo A, Feliu J, et al: Preliminary results of phase I clinical trial: UFT modulated by folinic acid (PO) in the treatment of advanced colorectal cancer. Therapeutic Res 13:451-458, 1992.
23. Pazdur R, Lassere Y, Diaz-Canton E, et al: Phase I trials of uracil-tegafur (UFT) using 5- and 28-day administration schedules: Demonstration of schedule-dependent toxicities. Anticancer Drugs 7:728-733, 1996.
24. Pazdur R, Lassere Y, Diaz-Canton E, et al. Phase I trial of uracil-tegafur (UFT) plus oral leucovorin: 14-day schedule. Invest New Drugs 15:123-128, 1997.
25. Spector T, Porter DJT, Nelson DJ, et al: 5-Ethynyluracil (776C85), a modulator of the therapeutic activity of 5-fluorouracil. Drugs of the Future 19: 566-571, 1994.
26. Baker SD, Khor SP, Adjei AA, et al: Pharmacokinetics, oral bioavilability, and safety study of fluorouracil in patients treated with 776C85, an inactivator of dihydropyrimidine dehydrogenase. J Clin Oncol 14: 3085-3096, 1996.
27. Schilsky RL, Burris H, Ratain M, et al: Phase I clinical and pharmacologic study of 776C85 plus 5-fluorouracil in patients with advanced cancer. J Clin Oncol 16: 1450-1457, 1998.
28. Shirasaka T, Shimamato Y, Ohsimo H, et al: Development of a novel form of oral 5-fluorouracil derivative (S-1) directed to the potentiation of the tumor selective cytotoxicity of 5-fluorouracil by two biochemical modulators. Anticancer Drugs 7:548-557, 1996.
29. Shirasaka T, Shimamato Y, Fukushima M: Inhibition by oxonic acid of gastrointestinal toxicity of 5-fluorouracil without loss of its antitumor activity in rats. Cancer Res 53:4004-4009, 1993.
30. Shirasaka T, Nakano K, Takechi, T, et al: Antitumor activity of 1M Tegafur, 0.4M 5-chloro-2,4-dihydroxypyridine and 1M potassium oxonate (S-1) against human colon carcinoma orthotopically implanted into nude rats. Cancer Res 56:2602-2606, 1996.
31. Koizumi W, Kurihara M, Nakano S, et al: Phase II study of S-1, a novel oral derivative of 5-fluorouracil, in advanced gastric cancer. For the S-1 Cooperative Gastric Cancer Study Group. Oncology 58:191-197, 2000.
32. Sugimachi K, Maehara Y, Horikoshi N, et al: An early phase II study of oral S-1, a newly developed 5-fluorouracil derivative for advanced and recurrent gastrointestinal cancers. The S-1 Gastrointestinal Cancer Study Group. Oncology 57:202-210, 1999.
33. Shirasaka T, Fujita F, Fujita M, et al: Antitumor activity and metabolism of BOF-A2, a new 5-fluorouracil-derivative with human cancers xenografted in nude mice. Gan To Kagaku Ryoho 17:1871-1876, 1997.
34. Sasaki T. New anti-cancer drugs for gastrointestinal cancers. Gan To Kagaku Ryoho.24:1925-1931, 1997.