Robert L. Stolfi

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.

Biochemical Modulation of Tumor Cell Energy in Vivo: II. A Lower Dose of Adriamycin is Required and a Greater Antitumor Activity is Induced when Cellular Energy is Depressed

A quadruple drug combination--consisting of a triple-drug combination of N-(phosphonacetyl)-L-aspartate (PALA) + 6-methylmercaptopurine riboside (MMPR) + 6-amino-nicotinamide (6-AN), designed to primarily deplete cellular energy in tumor cells, + Adriamycin (Adria)--yielded significantly enhanced anticancer activity (i.e., tumor regressions) over that produced by either Adria alone at maximum tolerated dose (MTD) or by the triple-drug combination, against large, spontaneous, autochthonous murine breast tumors. The adenosine triphosphate (ATP)-depleting triple-drug combination administered prior to Adria resulted in a 100% tumor regression rate (12% complete regression; 88% partial regression) of spontaneous tumors. Histological examination of treated tumors demonstrated that the treatment-induced mechanism of cancer cell death was by apoptosis. The augmented therapeutic results (100% tumor regressions) were obtained with approximately one-half the MTD of Adria as a single agent and suggest the potential clinical benefit of longer, more effective, and safer treatment by low doses of Adria when combined with the triple-drug combination. Two likely mechanisms of action are discussed: (1) prevention of DNA repair; (2) complementary disruption of biochemical pathways by both the triple-drug combination and the biochemical cascade of apoptosis that is induced by a DNA-damaging anticancer agents such as Adria.

Perspective: The Chemotherapeutic Relevance of Apoptosis and a Proposed Biochemical Cascade for Chemotherapeutically Induced Apoptosis

Cancer cells that are sufficiently damaged by cancer chemotherapeutic agents (as well as radiotherapy) eventually die in an ordered sequential biochemical process known as apoptosis. Apoptosis is a general physiological mechanism for controlled cell deletion that is an active (i.e., an energy-dependent, at least initially), inherent (gene-directed) program of cell death, and therefore sometimes referred to as cell suicide and programmed cell death (1,2). The apoptotic biochemical events occurring after the anticancer agents interaction with its biochemical target is the actual process of cell death and is a secondary phenomenon following the primary drug-target interaction. Thus, anticancer agents, despite having different primary biochemical targets (e.g., inhibition of thymidylate synthase, microtubule damage, topoisomerase inhibitors, DNA crosslinking agents, etc.), all ultimately kill by inducing the biochemical cascade of apoptosis (3,4). However, there is a “qualitative” and “quantitative” heteroge...

Biochemical modulation of tumor cell energy IV: Evidence for the contribution of adenosine triphosphate (ATP) depletion to chemotherapeutically-induced tumor regression

DNA-damaging agents, e.g. Adriamycin (ADR), are reported to cause tumor regression by induction of apoptosis. A reduction in the intracellular content of ATP is part of the biochemical cascade of events that ultimately results in programmed death of the cell, or apoptosis. A chemotherapeutic three-drug combination (PMA) consisting of N-(phosphonacetyl)-L-aspartate (PALA) + 6-methylmercaptopurine riboside (MMPR) + 6-aminonicotinamide (6AN) significantly lowers levels of ATP in CD8F1 murine breast tumors in vivo and produces tumor regression by apoptosis. Addition of the DNA-damaging antitumor agent ADR to PMA was found to further significantly deplete ATP in CD8F1 murine breast tumors in vivo with a concomitant significant increase in the number of tumor regressions. The correlative biochemical and therapeutic results are consistent with, and support, the hypothesis that ATP depletion is a significant factor and, therefore, is a worthy therapeutic target in the production of apoptosis.

MARKED ENHANCEMENT IN VIVO OF PACLITAXEL'S (TAXOL'S) TUMOR-REGRESSING ACTIVITY BY ATP-DEPLETING MODULATION

Paclitaxel alone Is active against the CD8F1 murine spontaneous mammary cancer, and when administered following an ATP-depleting combination of N-(phosphonacetyl)- L-aspartate (PALA) + 6-methylmercaptopurine riboside (MMPR) + 6-aminonicotinamide (6-AN) (PMA) produced significantly enhanced partial tumor regressions over that produced by either paclitaxel alone at the maximal tolerated dose (MTD), or by the PMA drug combination alone, against advanced, first passage spontaneous murine breast tumors. The anticancer activity of paclitaxel is due to enhancement and stabilization of microtubule polymerization. Pertinently, microtubule disassembly (an ATP-dependent process) is known to sharply decrease in the presence of ATP depletion. Thus, the dramatic therapeutic enhancement observed with paclitaxel in combination with PMA is in agreement with biochemical expectations, since PMA has been shown to deplete ATP in CD8F1 tumor cells. The augmented therapeutic results were obtained with approximately one-third the MTD of paclitaxel as a single agent and suggest the potential clinical benefit of more effective treatment with lesser amounts of drug.

Apoptosis induced in advanced CD8F1-murine mammary tumors by the combination of PALA, MMPR and 6AN precedes tumor regression and is preceded by ATP depletion

Abstract The drug combination N-(phosphonacetyl)-l-aspartic acid (PALA), methylmercaptopurine riboside (MMPR) and 6-aminonicotinamide (6AN), referred to as PMA, induces regressions of advanced CD8F1 murine mammary carcinomas in vivo. We demonstrated that CD8F1 tumor regressions were preceded by the appearance of apoptotic bodies, as observed by microscopic examination of morphology and TUNEL end-labeling, and fragmentation of DNA into nucleosomal “ladder” patterns. These indications of apoptosis were present as early as 6 h after simultaneous administration of MMPR and 6AN and further increased by over fivefold during the next 3 to 6 h, then remained at 7 to 12.8% (0.6 to 2.4% in saline-treated controls) of the cell population for at least 24 h after MMPR + 6AN administration. The 5′-phosphate derivative of MMRP, MMPR-5P, which inhibits de novo purine biosynthesis, was present at a “steady-state” level, and significant (40%) depletion of ATP had occurred by 3 h and both of these events preceded the onset of apoptosis. In addition, MMPR-5P was retained in CD8F1 tumors at a high level over a prolonged period (>96 h) even as tumors were undergoing regression. The prolonged presence of MMPR-5P was important for optimal chemotherapeutic effect, since treatment with iodotubercidin (IodoT), an inhibitor of MMPR/adenosine kinase, 6 h after MMPR + 6AN administration prevented the prolonged accumulation of MMPR-5P and reversed the regression of CD8F1 tumors. In addition, compared to the PMA-treated group, there was a significant restoration of ATP levels after treatment with IodoT. In individual PMA-treated CD8F1 tumors the degree of ATP depletion was found to correlate with the degree of tumor shrinkage at 24 h, after tumors had sufficient time to respond to treatment. These results define the time-course of drug-induced apoptosis in CD8F1 tumors, show that ATP depletion occurs prior to apoptosis and demonstrate that prolonged retention of MMPR-5P is associated with optimal chemotherapy. Collectively, these results suggest that the depletion of ATP by PMA treatment may be a component of the biochemical apoptotic cascade in the CD8F1 tumor.

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.

Enhanced antitumor activity of an adriamycin + 5-fluorouracil combination when preceded by biochemical modulation.

A three-drug combination, PMA, consisting of (phos-phonacetyl)-L-aspartic acid + 6-methylmercaptopurine riboside+5-aminonlcotinamide, preceding either 5-fluorouraell (5-FU) or adriamycin (Adr), produced tumorregressing activity in a murine advanced breast tumor model not attainable with either 5-FU or Adr as single agents, or with any lesser combination of these drugs administered at maximally tolerated doses. Marked tumor-regressing activity was further increased signifi-cantly by using 5-FU and Adr together in conjunction with the modulatory biochemical conditioning (particularly ATP depletion) provided by pretreatment with PMA.