Robert C. Sawyer

An overview of thymidine

This review summarizes a body of information suggesting that proper metabolic modulation with certain metabolites can sensitize tumor cells to antimetabolites, and others can de‐sensitize (i .e . protect) normal cells from the toxicity of antimetabolites. This new approach offers the possibility of increasing the selectivity of drug therapy, with the promise of a real advance in cancer chemotherapy. The metabolite thymidine (TdR), long used as a cell synchronizing agent, is known to exert this effect in vitro by metabolic modulation of a number of enzymes in the salvage pathway to DNA synthesis. Against this biochemical background, in vivo effects of TdR employed as an agent for cancer therapy are reviewed as follows: 1) TdR alone, and in combination with, 2) Methotrexate (MTX), or 3) 5‐Fluorouracil (FU), or 4) Cytosine arabinoside (ara‐C). TdR is shown in all instances either to protect against host toxicity (eg. MTX), or to potentiate the anti‐tumor effect (eg. FU and ara‐C). Findings are also presented that a sequential schedule of MTX prior to TdR prior to FU is important for the optimal therapeutic activity of these drugs. The biochemical basis for the MTX → FU augmentation is reportedly due to increased activation of FU by MTX (acting indirectly). On the basis of this biochemical insight, a completely different chemotherapeutic agent methyl‐mercaptopurine rihoside (MMPR) was substituted for MTX, resulting in a dramatic potentiation of anticancer activity. Metabolic modulation with still other metabolites (UR) and a hormone (testosterone) was demonstrated to protect from host toxicity due to certain anti‐cancer agents without offsetting anti‐tumor activity. The ability to prevent leukopenia by these means was particularly impressive. Clinical trials have been initiated with TdR alone, TdR + MTX, and TdR + FU; the available clinical data are summarized.

Potentiation of the anti-tumor activity of 5FU by thymidine and its correlation with the formation of (5FU) RNA

Evidence is presented with two murine tumor systems (CD8F1 mammary carcinoma and CD2F1 colon tumor 26). We question the thesis that the antitumor activity of 5‐fluorouracil (FU) is achieved solely by the inhibition of thymidylate synthetase by the derivative, fluorodeoxymonophosphate (FdUMP). The data described here are more consistent with the formation of FU‐containing RNA (FU‐RNA), an event which can adversely affect a variety of cellular mechanisms requiring RNA processing and function. These are not two mutually exclusive mechanisms. However, if the deleterious effect of FU‐RNA constitutes a significant quantitative component of the anti‐neoplastic activity of FU, the addition of thymidine would be expected to increase the antitumor activity of FU by the following three mechanisms: 1) protection of FU from catabolic degradation by saturation of the relevant enzymes with thymidine; 2) selective arrest by thymidine “feedback” of normal cells compared with malignant cells; 3) feedback repression of the FU anabolic pathways leading to deoxyderivatives, thus encouraging the entry of FU into RNA.

Use of oral uridine as a substitute for parenteral uridine rescue of 5-fluorouracil therapy, with and without the uridine phosphorylase inhibitor 5-benzylacyclouridine

SummaryInitial clinical trials have demonstrated that uridine (Urd) rescue given i.v. over at least 3 days can ameliorate 5-fluorouracil (FUra) toxicity; to avoid Urd-induced phlebitis in the peripheral veins of patients, a central vein is used. The latter necessity, along with the need for 3 days of i.v. administration, makes Urd rescue by parenteral means a cumbersome and complicated clinical procedure. It would appear preferable to use oral Urd; however, the oral Urd dose in the clinic is limited, as high doses cause diarrhea. Therefore, using a tumor-bearing murine model we investigated as to whether low doses of oral Urd coupled with a Urd phosphorylase inhibitor benzylacyclouridine (BAU), would effect safe rescue of FUra toxicity with preservation of antitumor activity. A high-dose FUra-containing drug combination that included parenteral Urd rescue was used as a control; other groups of tumor-bearing mice received the same drug combination, except that p.o. Urd was substituted for i.p. Urd. In the absence of BAU, p.o. Urd could effect rescue while maintaining an antitumor effect comparable to that obtained with i.p. Urd. When given concomitantly with BAU, a 50% reduction in the oral Urd dose (i.e., from 4,000 to 2,000 mg/kg) enabled the achievement of a comparable therapeutic index. Intraperitoneal Urd produces very high (6–8 mM) plasma and tissue Urd levels, which remain above 100 μM for at least 6 h. In contrast, neither oral Urd nor oral BAU alone raised plasma Urd concentrations above about 50 μM. However, the combination of oral Urd plus oral BAU gave a peak plasma Urd level of about 300 μM, and the level was maintained above 100 μM for 6 h. Following oral Urd administration, gut tissue levels of Urd were in the mM range and those of BAU were in the range of 10–20 μg/g tissue, a level sufficient to result in substantial inhibition of Urd phosphorylase. Oral Urd plus oral BAU appears to be a promising clinical alternative to parenteral administration of Urd for selective rescue of FUra toxicity.

Uridine allows dose escalation of 5-fluorouracil when given with N-phosphonacetyl-L-Aspartate, methotrexate, and leucovorin

Background. In a previous trial in which methotrexate and N‐phosphonacetyl‐L‐aspartate (PALA) were used to modulate 5‐fluorouracil (5‐FU), four of six patients could not tolerate treatment at the 600 mg/m2 5‐FU dose level because of mucositis, diarrhea, and a decrease in performance status. The current study examines the ability of uridine rescue to prevent such toxic effects in the same regimen and, thereby, allow additional dose escalation of 5‐FU.

A phase II trial of biochemical modulation using N‐ phosphonacetyl‐L‐aspartate, high‐dose methotrexate, high‐dose 5‐fluorouracil, and leucovorin in patients with adenocarcinoma of unknown primary site

Background. 5‐Fluorouracil (5‐FU) has modest activity as a single agent in a number of human adenocarcinomas. The technique of biochemical modulation has been used preclinically to increase the activity of 5‐FU.

Decreased host toxicity in vivo during chronic treatment with 5-flourouracil.

SummaryChronic weekly administration of FUra to CD8F1 female mice bearing spontaneous mammary tumors produced body weight loss during the first 2 weeks of treatment, which became less severe during subsequent weeks of therapy. To our knowledge, the development of such a decrease in FUra toxicity in vivo during chronic treatment with the drug has not been described previously, and a study of this phenomenon was therefore underfaken in tumor-free CD8F1 female mice. Weekly administration of FUra at 85 mg/kg resulted in toxicity expressed in body weight loss and in depressed peripheral WBC levels; however, the magnitude of these toxic effects decreased significantly by the 5th week of treatment. Pretreatment of normal mice with FUra for 7 weeks resulted in a dose-related shift in the LD50 of FUra administered as a subsequent challenge. Compared with an LD50 of 240 mg/kg for FUra in normal mice, the LD50 in mice pretreated with FUra at 50 or 85 mg/kg per week was found to be significantly elevated to 370 and 460 mg/kg, respectively. Pretreatment with FUra at 85 mg/kg for 7 weeks did not alter the activity of the enzymes responsible for the activation of FUra, namely uridine kinase or orotate phosphoribosyltransferase, in the intestinal epithelium or bone marrow, but it did decrease the 24-h urinary excretion of intact [3H]FUra by almost 40% (P<0.01). In addition, the FUra pretreatment schedule resulted in a 31% (P=0.14) increase in the activity of dihydrouracil dehydrogenase in the liver. These results suggest that increased degradation of FUra can be induced by chronic treatment with the drug. Finally, knowledge of the development of increased drug catabolism was used to increase the therapeutic effectiveness, of FUra by its incorporation into an increasing-dose regimen. Mice bearing 24-h transplants of the murine breast tumor were treated with a constant dose of FUra for 12 weeks or with a dose that was increased, after 7 weeks, to a dose normally causing a high degree of drug-related mortality. The group receiving the incremented FUra dose had a significantly slower tumor growth rate without an increase in drug-related toxicity. These results are discussed in light of their obvious clinical implications.

Effect of uridine diphosphoglucose on levels of 5-phosphoribosyl pyrophosphate and uridine triphosphate in murine tissues.

The purpose of the present investigation was to determine whether a single bolus intravenous injection (2000 mg/kg) of uridine diphosphoglucose (UDPG) could affect levels of PRPP in a transplanted mammary adenocarcinoma and in liver of CD8FI mice. Six hours following a single intravenous injection of UDPG, 2000 mg/kg, tumor PRPP was lowered to 80 pmol/mg protein, a 53% decrease compared to saline control tumors. Liver was more sensitive than tumor to the 5-phosphoribosyl pyrophosphate (PRPP)-depleting effects of a single bolus intravenous injection of UDPG, since significantly lower levels of PRPP were found in liver, but not in tumor, at doses of 500–1000 mg/kg of UDPG. Maximal depression (30% of saline control) or PRPP occurred in liver 6 hr after intravenous UDPG at 1000–2000 mg/kg. Enhanced levels of UDPG in plasma (half-life less than 10 min) and tumor was detected at 30 min after intravenous UDPG at 2000 mg/kg. There was no detectable increase in endogenous levels of UDPG in liver at this time, probably as a result of rapid metabolism of UDPG by liver. At this same time, a twofold increase in uridine triphosphate (UTP) was measured in liver after intravenously administered UDPG. In contrast, the level of UTP was not increased significantly above control values in tumor. These data suggest the potential use of UDPG to elevate UTP pools in normal tissues in the delayed rescue of cancer chemotherapeutic drugs such as 5-fluorouracil which function as a uridine analogue in these tissues.