Oral zidovudine, continuous-infusion fluorouracil, and oral leucovorin calcium: a phase I study.

A phase I clinical, pharmacologic, and biochemical evaluation of escalating oral zidovudine (AZT) given over 2 days with a fixed dose of continuous-infusion fluorouracil (800 mg/m2 per day X 3 days) and oral leucovorin calcium was performed. Eighteen patients were treated with doses of AZT ranging from 1.0 to 9.0 g/m2 per day. Nausea and vomiting were dose limiting, with a maximally tolerated dose of 7.5 g/m2 per day. Rash and mucositis occurred but were not dose limiting. A dose-related increase in peak plasma levels of AZT was observed, and the alpha half-life of AZT in plasma (75 min) was unaffected by these high doses. At doses above 4.0 g/m2 per day, trough levels significantly increased, perhaps reflecting prolonged absorption from the gut. No responses were observed; however, a significant increase in DNA single-strand breaks was observed in peripheral blood cells after a threshold dose of 4.0 g/m2 per day, confirming a biological effect of AZT in this regimen. Further trials with an intravenous formulation capable of maintaining plasma levels and circumventing dose-limiting toxicity are warranted.

Tissue-specific enhancement of uridine utilization and 5-fluorouracil therapy in mice by benzylacyclouridine.

Benzylacyclouridine (BAU), a potent inhibitor of uridine phosphorylase, delays the disappearance of uridine from plasma, affects the utilization of uridine by selected tissues, and enhances the therapeutic effects of 5-fluorouracil (FUra) in female C57BL/6 mice. A single 30-mg/kg i.v. injection of BAU lengthens the plasma half-life of both a tracer dose of [3H]uridine (3 micrograms/kg) and a pharmacological dose of uridine (250 mg/kg) by 250 and 83%, respectively. This dose of BAU also increases the normal plasma concentration of uridine about 4-fold to 9 microM and sustains these levels for 4 h. Four injections of BAU at 30 mg/kg over 6 h or a single injection at 240 mg/kg increases the plasma concentration of uridine over 10-fold to approximately 50 microM. In addition to affecting the pharmacokinetics of uridine, a 30-mg/kg dose of BAU selectively increases up to 4-fold the ability of normal host tissues to salvage a tracer dose of [3H]uridine for nucleic acid biosynthesis, the uracil nucleotide pool size, and the incorporation of uridine into nucleic acids. However, uridine salvage from plasma by colon tumor 38 is increased only slightly by BAU, while the uracil nucleotide pool size and uridine incorporation into tumor nucleic acids are actually decreased by 15 and 37%. The selective effect of BAU on uridine utilization is reflected in the ability of BAU to modify FUra-induced host toxicity. The dose of FUra required to kill 50% of the treated normal mice (350 mg/kg) is modestly increased by "rescue" regimens consisting of the subsequent administration of repeated injections of either BAU alone (30 mg/kg/injection) or uridine alone (250 mg/kg/injection). However, an increase of 54% is achieved when repeated injections of the combination of BAU and uridine are administered. In C57BL/6 mice bearing advanced transplants of colon tumor 38, the period of tumor growth inhibition resulting from multiple courses of FUra-containing drug regimens can be increased by the delayed administration of BAU alone or BAU combined with uridine.

Tissue uridine pools: evidence in vivo of a concentrative mechanism for uridine uptake.

Pools of free uridine, ranging from 7.3 to 38.0 nmol/g wet weight, have been detected in a variety of freeze-clamped murine tissues. These concentrations average 10-fold greater than that detected in plasma. The kinetics of these pools after an i.v. tracer dose of [3H]uridine suggest that the initial rapid disappearance of [3H]uridine from plasma (t1/2 = 2 min) reflects distribution into tissues as well as catabolism by the liver. Subsequently, the tissue uridine pools turn over with half-lives of 13 to 18 h. Analyses of the activity of the proximal enzymes in uridine metabolism (uridine phosphorylase and uridine kinase) suggest that the phosphorylase correlates with the size of tissue uridine pools. Further evidence for this is seen in the sustained 5- to 15-fold increase in both tissue and plasma uridine concentrations after treatment with benzylacyclouridine, a potent uridine phosphorylase inhibitor. In contrast, a nonphysiological dose of exogenous uridine (250 mg/kg) briefly increases the plasma concentration of uridine to over 1 mM but it returns to below 10 microM within 1 h. Under these conditions as well, tissue concentrations of uridine increase 5- to 10-fold in most tissues, 20-fold in spleen, and 70-fold in kidney. High cellular concentrations of free uridine relative to medium are also observed in dispersed murine splenocytes. Furthermore, splenocytes incubated in 5 microM [3H]uridine achieved a 2-fold higher intracellular concentration of [3H]uridine in less than 1 min independent of phosphorylation. Thymidine was not concentrated in this system nor did nitrobenzylthioinosine inhibit [3H]uridine uptake. These findings suggest that in normal tissues and explanted cells, pools of uridine are sustained by a concentrative transport mechanism and constitute a previously unrecognized reservoir of pyrimidine nucleosides in tissues.

Taurolidine: cytotoxic and mechanistic evaluation of a novel antineoplastic agent.

Bis-(1,1-dioxoperhydro-1,2,4-thiadiazinyl-4)methane (taurolidine) is a synthetic broad-spectrum antibiotic that reacts with bacterial cell membrane components to prevent adhesion to epithelial cell surfaces. Reflecting the key role of adhesion in the growth and development of human solid tumors, studies were initiated to assess the antiproliferative activity of this agent in selected human and murine tumor cell lines. A 3-day exposure to Taurolidine inhibited the growth of all of the cell lines evaluated with IC(50)s ranging from 9.6-34.2 microM. Studies to identify the mechanism responsible for this effect were conducted in NIH-3T3 murine fibroblasts and the PA-1 and SKOV-3 human ovarian tumor cells. These studies revealed that a 48-h exposure to taurolidine had little effect on cell cycle distribution in PA-1 and SKOV-3 cells but significantly increased the appearance of DNA debris in the sub-G(0)/G(1) region, an effect consistent with an induction of apoptosis. In contrast, in NIH-3T3 cells, taurolidine exposure did not increase DNA debris in the sub-G(0)/G(1) region. Additional studies assessed phosphotidylserine externalization after a 24-h exposure to taurolidine using annexin-V binding as a cell surface marker. These studies revealed that taurolidine increased the percentage of annexin-V-positive cells by 4-fold and 3-fold in PA-1 and SKOV-3 cells, respectively. In NIH-3T3 cells, taurolidine exposure slightly increased ( approximately 5%) annexin-V binding. Parallel studies revealed that exposure to taurolidine also resulted in poly(ADP-ribose) polymerase cleavage in both ovarian tumor cell lines but not in NIH-3T3 cells. Finally, murine-based studies were conducted to assess the antineoplastic activity of three consecutive daily i.p. bolus injections of taurolidine at doses ranging from 5-mg injection/mouse to 30-mg injection/mouse. The 20-mg injection dose produced approximately 10% mortality and was identified as the maximally tolerated dose in this model. Administration of this regimen to nude mice bearing i.p. human ovarian tumor xenografts significantly inhibited both tumor formation and growth. These findings are discussed in light of their clinical implications.

Concentrative uridine transport by murine splenocytes: kinetics, substrate specificity, and sodium dependency.

A previous report from this laboratory indicated that the concentration of free uridine (Urd) in many normal murine tissues greatly exceeds that in plasma. We now report that Urd uptake by isolated murine splenocytes is concentrative, and that the rate of uptake from medium containing 10 to 500 microM [3H]Urd conforms to a process that is saturable with a Km of 38.0 +/- 4.1 (SE) microM and Vmax of 2.70 +/- 0.27 pmol/s/microliter cell water. Other ribosyl and deoxyribosyl pyrimidine nucleosides or their analogues were not concentrated by splenocytes; however, ribosyl and deoxyribosyl purine nucleosides and, to a lesser extent, deoxyuridine did inhibit Urd uptake. In this system Urd uptake was not inhibited by 1 microM nitrobenzylthioinosine or 10 microM dipyridamole but was significantly inhibited by 5 mM NaN3 or 250 microM KCN. Transport of Urd involves Na+ cotransport as evidenced by complete inhibition when Na+ is replaced by Li+ in the incubation medium, and it is also inhibited by 3 mM ouabain. Active Urd transport coexists with the nonspecific, carrier mediated, facilitated diffusion of nucleosides as demonstrated by the inhibition of Urd efflux and thymidine influx in splenocytes by nitrobenzylthioinosine and dipyridamole. Under identical conditions, Urd entry into L1210 leukemia cells was nonconcentrative and non-Na+ dependent but inhibited by nitrobenzylthionosine. That nucleosides enter most cultured neoplastic cell lines by facilitated diffusion and not the active transport mechanism for Urd confirms earlier findings and may represent an exploitable target for chemotherapy.

5-Fluorouracil enhances azidothymidine cytotoxicity: in vitro, in vivo, and biochemical studies.

Preliminary in vitro studies suggest that the combination of 5-fluorouracil (FUra) and 3'-azido-3'-deoxythymidine (AZT) is more cytotoxic than either agent alone. Therefore, a biochemical and therapeutic evaluation of this combination was initiated. Quantitation of the cytotoxicity of FUra plus AZT against the growth of HCT-8 cells in vitro revealed that 1 microM FUra (approximately 10% inhibitory concentration) increased the cytotoxicity of AZT and decreased its 50% inhibitory concentration by 60%. Similarly, incubating HCT-8 cells in 5 microM AZT (approximately 10% inhibitory concentration) decreased the 50% inhibitory concentration of FUra by over 50%. Biochemical analysis indicated that AZT did not affect FUra-induced inhibition of thymidylate synthase or [3H]-FUra incorporation into nucleic acids. In contrast, incubation in 5 microM FUra increased the incorporation of [3H]-AZT (5 microM) into the nucleic acid fraction of these cells by 52% (P less than 0.05). Therapeutic evaluation of this combination in athymic (nude) mice bearing HCT-8 xenographs revealed that, while weekly FUra (85 mg/kg) or AZT (600 mg/kg) exerts minimal antineoplastic activity (after 4 courses, treatment/control = 0.81 and 0.70, respectively), their combination, at the same doses, inhibited tumor growth by nearly 70% (P less than 0.01 versus FUra alone). FUra-related host toxicity was not increased by the addition of AZT. Higher doses of FUra alone were not more effective than FUra plus AZT. In vivo, AZT did not affect the incorporation of [3H]-FUra into the nucleic acid fraction of various murine tissues, including HCT-8 xenografts. FUra, however, increased [3H]-AZT incorporation into nucleic acids in a tissue-specific manner. In the presence of FUra, the incorporation of [3H]-AZT in spleen, liver, and gut increased 40, 21, and 4%, respectively, while in HCT-8 xenografts [3H]-AZT incorporation increased more than 2-fold. Analysis of the activities of selected enzymes involved in pyrimidine metabolism suggests that this tissue-specific effect may be related to the pyrimidine salvage capacity of these tissues. These findings are described in light of their potential impact on human colon cancer chemotherapy.

Modulation of p53 expression by human recombinant interferon-alpha2a correlates with abrogation of cisplatin resistance in a human melanoma cell line.

G3361/CP cells, a cisplatin (CDDP)-resistant subclone of the human melanoma cell line G3361, overexpress wild-type p53 protein and demonstrate an increase in the percentage of cells in G0--G1 arrest compared to parental cells. Exposing G3361/CP cells to human recombinant IFN-alpha2a reduces the high basal levels of p53, releases G3361/CP cells from G0-G1 into S phase, and abrogates CDDP resistance. These findings suggest that recombinant IFN-alpha2a disrupts p53-mediated cell cycle regulation to restore CDDP sensitivity in G3361/CP cells.

Enhancement of 5-fluoro-2'-deoxyuridine antineoplastic activity by 5-benzyloxybenzyloxybenzylacyclouridine in a human colon carcinoma cell line.

The pyrimidine acyclonucleoside benzyloxybenzyloxybenzylacyclouridine (BBBAU) showed growth inhibitory activity against the human colon cancer HCT-8 cell line with a 50% inhibitory concentration of 55 microM. Unlike its parent compounds, BBBAU was an extremely weak inhibitor of uridine phosphorylase. This acyclonucleoside analogue is an inhibitor of thymidylate synthase (TS) as determined by inhibition of [6-3H]-2'-deoxyuridine incorporation into DNA, inhibition of 3H release from [5-3H]-2'-deoxyuridine, and decrease in both the free and total TS 5'-fluoro-2'-deoxyuridine 5'-monophosphate binding sites. Kinetic analysis revealed that BBBAUMP, the monophosphate analogue of BBBAU, is a competitive inhibitor of purified human recombinant TS with a Ki of 8.0 microM. Nucleoside transport and uptake studies revealed that BBBAU (30 microM) inhibited the initial rate of transport and the total uptake of thymidine (25 microM). In contrast, while BBBAU (30 microM) inhibited the initial rate of transport of 5-fluoro-2'-deoxyuridine (FdUrd, 25 microM), its intracellular accumulation was increased. BBBAU (10 and 50 microM, respectively) potentiated FdUrd growth inhibition of HCT-8 cells and significantly enhanced the cytotoxic effects of FdUrd (0.3 and 1 microM, respectively) against HCT-8 cells using a clonogenic assay system. This combination resulted in additive inhibitory effects on TS activity resulting in greater depletion of dTTP pools. Moreover, the incorporation of radiolabeled FdUrd into the DNA fraction of HCT-8 cells was enhanced. The potential importance of this novel combination for human colon cancer chemotherapy is discussed.

Azidothymidine-induced cytotoxicity and incorporation into DNA in the human colon tumor cell line HCT-8 is enhanced by methotrexate in vitro and in vivo.

We have reported that 5-fluorouracil can increase the cytotoxic and antineoplastic activity of 3'-azido-3'-deoxythymidine (AZT). To further evaluate the antineoplastic utility of AZT we now have assessed its effect in combination with methotrexate (MTX) in the human colon tumor model HCT-8. Incubation of these cells for 5 days in AZT and MTX caused a reduction in the 50% inhibitory concentration of AZT and isobologram analysis revealed additive effects which were reversed by the addition of 50 microM thymidine to the incubation media. This enhanced cytotoxicity appeared not to be related to an effect of AZT on MTX activity; in whole-cell assays the ability of MTX to inhibit de novo dTMP synthesis and deplete intracellular pools of dTTP was not affected by AZT. In contrast, although MTX did not alter AZT triphosphate production, it did affect AZT triphosphate utilization in DNA synthesis. Incubation of cells for 24 h in [3H]AZT alone (5 microM, 3 microCi/ml) resulted in 6.6 pmol AZT incorporated into cellular DNA/10(6) cells. Coincubation of these cells in [3H]AZT (5 microM) plus 5 or 15 nM MTX increased AZT incorporation into DNA to 8.0 and 20.5 pmol/10(6) cells, respectively. Biochemically, this effect appeared to correlate with the concentration-dependent ability of 5 or 15 nM MTX to deplete intracellular dTTP pools, which were reduced by 25 and 49%, respectively. Further evidence of the relationship between intracellular dTTP pools and AZT cytotoxicity was that, in the presence of both MTX and 50 microM thymidine, intracellular dTTP pools remained near normal levels and the incorporation of 5 microM AZT into DNA was not enhanced. Therapeutically, studies conducted in athymic (nude) mice bearing HCT-8 xenografts that received six weekly cycles of MTX (87.5 mg/kg) and AZT (300 mg/kg) revealed that the two-drug regimen exerted superior antineoplastic effects compared to either drug alone (treated versus control approximately 0.9 for AZT or MTX and approximately 0.3 for MTX plus AZT). In addition, the combination did not increase toxicity compared to therapy with MTX alone. These findings are discussed in light of their biochemical and clinical implications.

Enhancement of fluorouracil therapy by the manipulation of tissue uridine pools.

Evidence for transport systems that actively concentrate uridine in normal tissues provides a previously unexploited opportunity for manipulation to therapeutic advantage. The ability to expand these pools in a tissue-specific manner by administration of exogenous uridine, inhibition of uridine phosphorylase with BAU or blockade of the facilitated transport of nucleosides with dipyridamole is established. If the apparent defect in the active transport mechanism for uridine in neoplastic cells in culture as well as several model tumors reflect the properties of human neoplasms, a new exploitable therapeutic difference may exist. These approaches may, in the near future, increase the therapeutic effectiveness not only of fluorouracil and the other fluoropyrimidines but also of other agents which disrupt uridine metabolism such as PALA and pyrazofurin.

Phase I clinical and pharmacological studies of benzylacyclouridine, a uridine phosphorylase inhibitor.

Benzylacyclouridine (BAU, IND 039655) is a potent and specific inhibitor of uridine phosphorylase (UrdPase; EC 2.4.2.3). This enzyme plays a major role in regulating uridine homeostasis and also catalyzes the conversion of fluoropyrimidine nucleosides to their respective bases. Inhibition of UrdPase enzyme activity 18-24 h after 5-fluorouracil (5-FU) administration increased plasma levels of uridine and enhanced the therapeutic index of 5-FU by rescuing normal tissues. Moreover, in vitro preclinical studies have also shown that inhibiting UrdPase enzyme activity by BAU prior to administration of 5-FU increased cytotoxicity in a number of human cancer cell lines. A series of preclinical studies was performed in dogs and pigs to evaluate the pharmacological and pharmacodynamic properties of BAU. These data showed a sustained elevation in plasma uridine concentration in both animal models. The rapid degradation of a tracer dose of uridine into uracil was virtually arrested by BAU administered both p.o. or i.v. The t1/2 of BAU was 1.8-3.6 h in dogs, with bioavailability levels of 85% (30 mg/kg) and 42.5% (120 mg/kg). In pigs, the half-life varied from 1.6 to 2.3 h, with a bioavailability of 40% at 120 mg/kg. The drug was distributed into most tissues with a tissue: plasma ratio of approximately 0.7. On the basis of these preclinical studies, we performed a Phase I clinical trial of BAU in patients with advanced cancer. Patients received 200, 400, 800, and 1600 mg/m2 BAU as a single oral dose. Toxicities included grade 2 anemia, grade 1 fever, grade 1 fatigue, grade 1 constipation, and grade 1 elevation in alkaline phosphatase; none of these toxicities were observed to be dose dependent. The maximum tolerated dose and dose-limiting toxicity were not reached at the doses given. BAU plasma concentrations and area under the curve correlated linearly with the oral dose level. The pharmacokinetics of BAU were consistent with a first-order clearance, with average peak concentrations ranging from 19 microM (200 mg/m2) to 99 microM (1600 mg/m2) and tbeta1/2 ranging from 3.0 to 3.9 h at the four dose levels. Compared with baseline plasma uridine, treatment of patients with 200, 400, 800, and 1600 mg/m2 BAU increased peak uridine concentrations by 120, 150, 250, and 175%, respectively. On the basis of this clinical study, the suggested Phase II starting dose of BAU in combination with 5-FU is 800 mg/m2. Studies combining BAU with 5-FU and incorporating appropriate molecular and biochemical end points to assess the effects of this drug combination on tumor and/or surrogate tumor tissue are under way.

Recombinant interferon alpha2a synergistically enhances ganciclovir-mediated tumor cell killing in the herpes simplex virus thymidine kinase system.

The herpes simplex virus thymidine kinase (HSV-TK) gene is being developed in the treatment of many different types of tumors. The HSV-TK gene sensitizes tumor cells to the antiviral drug ganciclovir (GCV) and mediates the bystander effect in which unmodified tumor cells are killed as well. Although this approach has shown a significant antitumor effect, the need to potentiate this therapy exists. The results of this study indicate that recombinant interferon alpha2a (1FNalpha2a) acts synergistically with GCV to kill HSV-TK-expressing PA1 human ovarian tumor cells. Furthermore, it enhances the bystander killing of nearby unmodified tumor cells that do not express the HSV-TK gene. Previous studies have suggested that in vitro and in vivo bystander effects may be mediated by different mechanisms. However, IFNalpha2a enhanced bystander killing in both systems, with the survival of mice bearing preexisting tumors being significantly prolonged when they were treated with IFNalpha2a and HSV-TK/GCV compared with either treatment alone. Mechanism studies have shown that treatment with IFNalpha2a and GCV caused an increase in cells in S phase 24 hours after therapy in the HSV-TK-expressing cells, but the mechanism of action of IFNalpha2a does not seem to be related to an increase in DNA damage, because GCV incorporation was not increased after treatment with IFNalpha2a. These findings suggest that IFNalpha2a may be a useful adjunctive therapy for the HSV-TK/GCV system.

3'-Azido-3'-deoxythymidine cytotoxicity and metabolism in the human colon tumor cell line HCT-8.

We have reported that 3'-azido-3'-deoxythymidine (AZT) possesses significant cytotoxicity in human tumor models when combined with agents that inhibit de novo thymidylate (dTMP) synthesis, such as 5-fluorouracil (FUra) and methotrexate (MTX). To aid in the further development of these and related cancer chemotherapeutic regimens, this study was undertaken to identify the biochemical processes relevant to the induction of AZT cytotoxicity in the model human colon tumor cell line HCT-8. The IC50 of AZT in this cell line after a 5-day exposure was 55 microM. In cells incubated for 5 days with various concentrations of [3H]AZT alone, both [3H]AZT nucleotide pools and [3H]AZT incorporation into DNA increased as the concentration of AZT in the medium increased. In addition, a 5-day exposure to AZT, at medium concentrations < or = 100 microM, resulted in a reduction in dTMP synthase (EC 2.1.1.45; methylene tetrahydrofolate:deoxyuridine-5'-monophosphate C methyltransferase) and dTHd kinase (EC 2.7.1.27; ATP: thymidine phosphotransferase) activities, compared with cells incubated without drug. The IC50 of AZT was unchanged when the medium concentration of dThd was increased from 0.1 to 50 microM. Increasing the concentration of dThd to 50 microM also did not affect intracellular pools of [3H]AZTDP and [3H]AZTTP or the degree to which [3H]AZT was incorporated into cellular DNA, but did reduce intracellular [3H]AZTMP by approximately 75%. The degree to which 3'-amino-3'-deoxythymidine (AMT) was generated from AZT and incorporated into DNA also was not affected by varying the medium concentration of dThd. However, the amount of [3H]-AMT detected in DNA, < or = 3 pmol/10(6) cells at medium concentrations of [3H]AZT < or = 100 microM, was below that associated with significant cytotoxicity in these cells. These data support the notion that, in this model, AZT cytotoxicity is determined by the relative size of AZTTP pools and its utilization in DNA synthesis. Studies to verify this relationship assessed the effect of alterations in the concentration of dTTP and [3H]AZTTP on [3H]AZT incorporation into newly synthesized DNA in vitro, using DNA polymerases isolated from HCT-8 cells. The results of these studies confirmed that alterations in the concentration of either dTTP or AZTTP to reduce the dTTP/AZTTP ratio resulted in an increase in AZT incorporation into DNA. These findings are discussed in light of their biochemical implications and relevance to ongoing clinical trials.

Benzylacyclouridine. Pharmacokinetics, metabolism and biochemical effects in mice.

The pharmacokinetics, tissue distribution and urinary excretion of the uridine (Urd) phosphorylase (EC 2.4.2.3) inhibitor 5-benzylacyclouridine (BAU) were studied in C57BL/6 female mice by reverse-phase HPLC. The plasma clearance of BAU after i.v. administration followed first-order kinetics with a half-life of approximately 36 min. Other pharmacokinetic parameters such as volume of distribution (17 ml), clearance rate (0.3 ml/min) and the elimination rate constant (0.019 hr-1) were relatively constant over a dose range of 5 to 240 mg/kg when based on a first-order clearance model. Following oral administration, BAU was rapidly absorbed from the gut; peak plasma concentrations occurred within 30 min and were approximately 60% of equivalent i.v. doses. The distribution of BAU between plasma and most major organs was rapid and efficient, the exceptions being spleen and brain, which maintained only 40% and 10%, respectively, of the plasma BAU concentration. Approximately 41% of the injected dose of BAU was recovered intact in urine within 24 hr. Another 27% appeared as a more polar metabolite which, at a concentration of 50 microM, did not inhibit murine Urd phosphorylase. A near linear relationship was observed between the injected dose of BAU and its ability to increase the plasma concentration of Urd; i.v. injections of 30, 120 and 240 mg/kg increased plasma Urd 3-, 7- and 15-fold respectively. The utility of these data in the design of combination chemotherapy regimens containing BAU and related compounds is discussed.

Human recombinant interferon alpha-2a plus 3'-azido-3'-deoxythymidine. Synergistic growth inhibition with evidence of impaired DNA repair in human colon adenocarcinoma cells.

We reported that 3'-azidothymidine-3'-deoxythymidine (AZT) plus 5-fluorouracil or methotrexate produces additive cytotoxicity in HCT-8 cells: a reflection of increased AZT metabolism when de novo thymidylate (dTMP) synthesis was inhibited. We now report that AZT plus human recombinant interferon alpha-2a (rIFN-alpha 2a) produces synergistic growth inhibition in these cells. Evaluation of the effect of rIFN-alpha 2a on dTMP metabolism revealed that exposure to rIFN-alpha 2a (+/-AZT) did not affect dTMP synthase activity significantly but increased thymidine (dThd) kinase activity significantly. Consequently, AZT nucleotide production and incorporation into DNA were increased by coexposure to rIFN-alpha 2a. This alone, however, cannot explain the observed synergism. Therefore, the effect of these agents on DNA excision/repair processes was assessed. Isotope clearance studies demonstrated that rIFN-alpha 2a did not alter the rate of [3H]AZT excision from DNA. In contrast, filter-elution studies revealed that rIFN-alpha 2a (+/-AZT) produced more DNA damage and delayed repair compared with the effects produced by AZT alone. Since DNA polymerases alpha and beta are directly involved in gap-filling repair synthesis, experiments next assessed the effect of rIFN-alpha 2a and/or 3'- azido-3'-deoxythymidine-5'-triphosphate (AZTTP) on their activities. Polymerase alpha was inhibited slightly by AZTTP but not by rIFN-alpha 2a. Polymerase beta activity, however, was inhibited dramatically by rIFN-alpha 2a + AZTTP. Finally, western analysis revealed that a 24-hr exposure to 5000 IU/mL rIFN-alpha 2a (+/-20 microM AZT) significantly reduced wild-type p53 expression compared with AZT-exposed cells. We conclude that rIFN-alpha 2a enhances AZT-induced tumor cell growth inhibition by (i) increasing AZT metabolism, and (ii) inhibiting DNA repair and p53-mediated cell cycle control processes.

Tissue-specific expansion of uridine pools in mice. Effects of benzylacyclouridine, dipyridamole and exogenous uridine.

The concentration of uridine (Urd) in murine tissues appears to be controlled by Urd catabolism, concentrative Urd transport, and the non-concentrative, facilitated diffusion of Urd. Previous reports document the tissue-specific disruption of these processes, and subsequently intracellular pools of free Urd in mice, by the administration of exogenous Urd (250 mg/kg) or the Urd phosphorylase (EC 2.4.2.3; uracil:ribose-1-phosphate phosphotransferase) inhibitor 5-benzylacyclouridine (BAU) (240 mg/kg). We now report the effect of combinations of BAU (120 mg/kg, p.o.), the nucleoside transport inhibitor dipyridamole (DP) (25 mg/kg, i.p.), and exogenous Urd (250 mg/kg, i.v.) on Urd pools in mice. This dose of BAU increased Urd pools 2- to 6-fold, in a tissue-specific manner, for up to 5 hr. DP increased Urd pools 3-fold in spleen, over a 4-hr period, but did not affect other tissues. Administration of BAU 1 hr prior to exogenous Urd resulted in a 50- to 100-fold expansion of tissue normal after 6 hr. Administration of DP 1 hr prior to exogenous Urd caused a tissue-specific 40- to 100-fold increase in Urd pools which, except in spleen, returned to normal within 2 hr. The marked additive effects of these combinations were in contrast to those obtained following the administration of BAU 1 hr prior to DP. This regimen increased Urd pools from 4- to 9-fold, in a tissue-specific manner. In addition, Urd pools remained elevated for up to 9 hr, except in spleen where the Urd concentration was elevated for up to 15 hr. Analysis of enzyme activities indicated that DP does not enhance the inhibitory effect of BAU against murine liver Urd phosphorylase. However, DP did inhibit plasma clearance of BAU, and this effect may partially explain the apparent synergistic effect of this combination. In spite of the prolonged and dramatic expansion of tissue Urd pools produced by BAU + DP, the total Ura nucleotide content in spleen, gut and colon tumor 38 (CT38) increased by less than 70% over a 12-hr period following administration of this combination. These findings are discussed in light of their biochemical and therapeutic implications.

Circadian rhythm of hepatic uridine phosphorylase activity and plasma concentration of uridine in mice.

The activity of hepatic uridine phosphorylase (EC 2.4.2.3.) in male mice (24-29 g) maintained in standardized conditions of 12 hr light (0600-1800 hr) alternating with 12 hr darkness (1800-0600 hr), food and water ad lib., exhibited a circadian rhythm (P less than 0.0001, Cosinor analysis). The peak of enzyme activity (559 +/- 25 pmol/min/mg protein) occurred at 15 hr after light onset (HALO) with the nadir (139 +/- 25 pmol/min/mg protein) at 3 HALO when samples were taken every 4 hr. Female mice showed essentially the same pattern. A circadian rhythm (P less than 0.0001, Cosinor analysis) was also observed when the light-dark cycle was shifted (reverse cycle) so that the lights went on at 2200 hr and off at 1000 hr. Under the reverse cycle condition, there was a corresponding shift in the enzyme activity with a lag period of 3.5 hr in the time of maximum and minimum enzyme activities (i.e. the peak at 11 HALO and the nadir at 23 HALO) after a 2-week adaptation period. The lag period was reduced to 1 hr after 4 weeks of adaptation, and no further change was observed after 6 weeks of adaptation. The plasma concentration of uridine also exhibited a circadian rhythm (P less than 0.0001, Cosinor analysis) with peak concentration (10 microM) occurring at 2 HALO and a nadir (5 microM) at 14 HALO. The circadian rhythm observed in the plasma concentration of uridine is the inverse of that for uridine phosphorylase activity. These results demonstrate that hepatic uridine phosphorylase plays an important role in the regulation of the uridine level in the blood which, in turn, may be involved in the humoral control of sleep by uridine. This may also be of clinical significance in enhancing the antitumor efficacy of the 5-fluorinated pyrimidines by modulating the time of their administration.

Amifostine cytotoxicity and induction of apoptosis in a human myelodysplastic cell line.

Amifostine (AMF), a phosphorylated aminothiol, has been used to treat myelodysplastic syndrome (MDS), where it produces a stimulatory effect on hematopoiesis in bone marrow. To determine if AMF also produced a direct effect on human MDS cells, we planned a study to evaluate the effect of a continuous exposure to AMF on a human MDS cell line. AMF was shown to have a growth-inhibitory effect on MDS cells, with an IC(50) of 14 microM after a 5 day exposure. Cell cycle analysis revealed that a 5 day exposure to 20 microM AMF increased the percentage of cells in G0/G1 and this was accompanied by a decrease in the percentage of cells in S phase. Cytoflorometric and agarose-gel electrophoretic analysis revealed that this effect correlated with cell membrane alterations and DNA fragmentation consistent with an induction of apoptosis without affecting the expression of p53 protein or inducing any lymphoid or myeloid differentiation in the MDS cell line. We conclude that the continuous exposure of a human MDS cell line to AMF is cytotoxic and associated with an induction of apoptosis independent of alterations in p53 expression.

Taurolidine: preclinical evaluation of a novel, highly selective, agent for bone marrow purging.

Taurolidine has been shown to have remarkable cytotoxic activity against selected human tumor cells at concentrations that spare normal cells. In this study we have extended this observation and assessed the ability of Taurolidine to purge tumor cells from chimeric mixtures of bone marrow (BM) and neoplastic cells. Normal murine BM and human leukemic (HL-60) or ovarian (PA-1) tumor cell lines were used as models. Exposure of tumor cells to 2.5 mM Taurolidine for 1 h resulted in the complete elimination of viable cells. In contrast, exposure of BM to 5 mMTaurolidine for 1 h reduced CFU-GM, BFU-E and CFU-GEEM colony formation by only 23.0%, 19.6% and 25.2%, respectively. Inhibition of long-term BM culture (LTBMC) growth following a 1 h exposure to 5 mM Taurolidine also was approximately 20% compared to untreated LTBMC. Finally, chimeric cultures were generated from BM and HL-60GR or PA-1GR cells (tumor cells transfected with the geneticin resistance gene). Exposure of these chimeric cultures to 5 mM Taurolidine for 1 h totally eliminated viable cancer cells while minimally reducing viable BM cells. This finding was confirmed by subsequent positive selection for surviving tumor cells with geneticin. These findings reveal that Taurolidine holds promise for use in BM purging.

Isolated pelvic perfusion for unresectable cancer using a balloon occlusion technique.

Previously irradiated recurrent pelvic malignancy is refractory to most treatment modalities. Ten patients with local recurrences (six with rectal cancer; three, anal cancer; and one, anorectal melanoma) were treated with a total of 17 courses of isolated pelvic perfusion chemotherapy (12 with multiple agents) using standard hemodialysis technology. Aortic and inferior vena caval occlusion was maintained via transfemoral balloon catheters, with a single intraoperative balloon disruption. Mean pelvic-systemic drug exposure ratios were 9.8:1 for fluorouracil, 4.8:1 for cisplatin, and 4.4:1 for mitomycin C. Results were three partial responses (two patients subsequently underwent resection) and three minor responses, all in patients with a visible tumor. Pelvic pain was relieved in six of eight symptomatic patients (mean duration, 4 months). Using limited access, this procedure produces high pelvic-systemic concentration gradients, prolonged palliation for recurrent pelvic cancers, and increased resectability in selected patients.