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.

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.

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.

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.

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.

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.

Clarithromycin does not affect phosphorylation of zidovudine in vitro.

Zidovudine (ZDV) and clarithromycin (CLR) are often used simultaneously in the management of patients with AIDS. While pharmacokinetic studies show decreased absorption of ZDV when it is administered with CLR, it is unknown if CLR affects the intracellular metabolism of ZDV. We investigated the effects of CLR on the intracellular metabolism of ZDV in vitro. CEM-T4 cells were coincubated with a microM ZDV ([3 H] ZDV, 3 microCi/ml) either alone or with 1 or 10 microM CLR. Cells were also grown in the presence of CLR for 48 h prior to exposure to ZDV. Samples were analyzed for mono-, di-, and triphosphate metabolites of [3 H] ZDV by high-performance liquid chromatography separation and radiochemical detection. There were no significant differences in levels of intracellular metabolites of ZDV following exposure to ZDV, either alone or with 1 or 10 microM CLR and under both coincubated and preincubated conditions. These results show that treatment with CLR does not alter the formation of phosphorylated metabolites of ZDV in this cell line.

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.

Phase II study of 5-fluoruracil leucovorin and azidothymidine in patients with metastatic colorectal cancer.

The primary objective of this study was to determine the response rate of patients with metastatic colorectal cancer to combined therapy with 5-fluorouracil (5-FU), leucovorin, and intravenous azidothymidine (AZT), a thymidine nucleoside analog. By itself, AZT has limited antineoplastic efficacy. However, experimental studies indicate that 5-FU enhances the antitumor activity of AZT by inhibiting synthesis of normal thymidine nucleotides with which AZT competes for incorporation into nucleic acids. A phase I study defined the maximum tolerated dose of AZT as 7 g/m2 with hypotension during the infusion being the dose-limiting toxicity. A phase II study was performed with oral leucovorin (100 mg p.o. hourly for 4 h prior to 5-FU and 4 h and 8 h after 5-FU), bolus 5-FU (400 mg/m2) followed 1 h later by a 2-h infusion of AZT (7 g/m2). Treatment was given weekly for 4 weeks followed by a 1-week break, which constituted a cycle of therapy. Responses were evaluated after every two cycles. Patients continued on therapy as long as they tolerated treatment and did not have progressive disease. Of 15 evaluable patients who had received no chemotherapy there was 1 complete response and 4 partial responses (a 33% response rate), whereas only 1 of 6 patients who had received prior adjuvant chemotherapy had a partial response (17%). An additional 10 patients had stable disease lasting 2-14 months. Therapy was well tolerated with the only one instance each of grade 3 nausea and vomiting, diarrhea, anemia, and hypotension. Approximately 50% of treatments were accompanied by mild hypotension, which was easily corrected by increasing the rate of normal saline infusion. There was no difficulty administering this regimen in the outpatient setting. While the overall response rate (29%) is comparable to that seen with combinations of 5-FU and leucovorin alone, in most reported series a considerably higher dose of 5-FU was utilized than in this study. Since patients in the present study experienced relatively little 5-FU toxicity, increasing the dose of 5-FU in this regimen would appear to be feasible and might result in a higher response rate.

Quantitative analysis of 3'-azido-3'-deoxythymidine incorporation into DNA in human colon tumor cells.

We have previously reported that 3'-azido-3'-deoxythymidine (AZT) can possess significant antineoplastic activity in vitro and in vivo when combined with agents which inhibit de novo thymidylate synthesis. Under these conditions cytotoxicity is closely associated with the degree to which AZT is incorporated into DNA. We now report a fluorescence postlabeling technique by which AZT incorporation into DNA can be quantitated without employing radiolabeled AZT. Cultured human colon tumor (HCT-8) cells were exposed to various concentrations of AZT alone and in combination with 5-fluorouracil (FUra). Control cells received the same amount of medium. DNA was isolated from harvested cell pellets (2 x 10(7)). Enzymatic digestion of DNA to the mononucleotide level followed by HPLC analysis of the digest showed that the DNA preparation was free of RNA contamination. The DNA digest was conjugated with dansyl chloride in situ via the phosphoramidate derivative with ethylenediamine. HPLC analysis of the postlabeled nucleotides using fluorescence detection detected 105, 245, and 479 fmol of 5'-monophosphate of AZT (AZTMP) per microg of DNA from cells exposed to 20, 50, and 100 microM AZT, respectively. FUra (3 microM) doubled the AZT incorporation per microg of DNA in cells exposed to 50 and 100 microM AZT. These findings generally support our previously reported data which quantitated (3H)AZT incorporation into cellular DNA and are discussed in light of the potential clinical utility of this technique in assessing the relationship between AZT incorporation into DNA and therapeutic action.

Pharmacokinetics and pharmacodynamics of high-dose zidovudine administered as a continuous infusion in patients with cancer.

STUDY OBJECTIVE: To investigate the pharmacokinetics and pharmacodynamics of high-dose intravenous zidovudine (ZDV). DESIGN: Phase 1, dose-escalating, unblinded study in patients with cancer. SETTING: A university-affiliated cancer treatment center. PATIENTS: Fourteen patients (6 women) with solid tumors that were unresponsive to standard therapy received 31 courses of therapy. INTERVENTIONS: Intravenous ZDV was administered in doses of 2, 3, 4, 5.5, 7, 8.5, 10, 12, 15, or 20 g/m2/day as a continuous infusion over 48 hours. Patients also received fluorouracil plus leucovorin for 24 hours before the start of and during the ZDV infusion. If no dose-limiting toxicities were encountered, subsequent doses were escalated. Blood samples were collected at 24 and 48 hours after the start of the infusion, and hourly for 4 hours after stopping the infusion. Urine was collected in five patients during the infusion and for 24 hours after stopping it. Blood for measuring peripheral white blood cells was collected before and at the end of the infusion in seven patients to measure DNA chain breaks due to incorporation of ZDV. MEASUREMENTS AND MAIN RESULTS: Zidovudine was measured in plasma by high-performance liquid chromatography and in urine fluorescence polarization immunoassay. Its incorporation into DNA was measured by determining DNA strand breakage in peripheral white blood cells using fluorescence analysis. Pharmacokinetic models were fit to plasma ZDV concentrations using extended least squares regression. Short-term high-dose ZDV was generally well tolerated, with adverse effects related to large amounts of free water administered during the infusion. The mean (SD) ZDV pharmacokinetic values were total clearance 1.44 (1.09) L/hr/kg, volume of distribution 2.72 (2.97) L/kg, and half-life 1.2 (0.6) hours. There was considerable interpatient variability in total drug clearance. Although ZDV exposure increased proportionately with increasing dose, two of three patients receiving the highest dose (20 g/m2/day) had markedly low total drug clearances. The relation between the percentage of abnormal DNA in peripheral white blood cells and zidovudine area under the plasma ZDV versus time curve was described by the Emax pharmacodynamic model. CONCLUSIONS: The pharmacokinetics of high-dose ZDV administered by continuous infusion to patients with cancer are similar to those reported with lower doses in patients with infection due to the human immunodeficiency virus. Further study of potential nonlinear pharmacokinetic behavior at doses above 20 g/m2/day is necessary. The high between-patient variability in ZDV clearance results in variable levels of exposure in vivo, and indicates the need for concentration- or effect-controlled study designs in the further evaluation of the agent's antineoplastic effects.

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.

Hydroxyurea enhances 3'-azido-3'-deoxythymidine (AZT) cytotoxicity in human chronic myeloid leukemia models.

In this report we have evaluated the cytotoxic activity of 3'-azido-3'-deoxythymidine (AZT) used in combination with hydroxyurea (HU), an agent which disrupts de novo thymidylate synthesis. In 2 chronic myeloid leukemia (CML) cell lines, K562 and RWLeu4, the IC50 of AZT was 8 mumol/l and 28 mumol/l respectively, after a 5-day exposure, and the IC50 of HU was 80 mumol/l and 70 mumol/l respectively. In the presence of various concentrations of HU (1 mumol/l-100 mumol/l) the IC50 of AZT in both cell lines was significantly reduced and subsequent isobologram analysis revealed synergistic activity. Similarly, analysis of [3H]AZT incorporation into the DNA fraction of these cells indicated that exposure to AZT+HU resulted in an increased incorporation of AZT into DNA when compared to incubation in AZT alone. Biochemically, this effect appeared to be related to a decrease in dTTP pools caused by HU. The combination AZT+HU has also been demonstrated to exert a synergistic effect in inhibiting colony growth of bone marrow granulocyte-macrophage progenitors (CFU-GM) from patients affected by Ph1+ CML in chronic phase. These results are promising in view of a possible in vivo utilization of this drug combination.

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.

High-dose intravenous zidovudine with 5-fluorouracil and leucovorin. A phase I trial.

BACKGROUND: The inhibition of pyrimidine metabolism by 5-fluorouracil (5-FU) enhances the anti-cancer effects of zidovudine (formerly called AZT) in in vitro and in vivo model systems without additive toxicity. Zidovudine-induced DNA damage correlates with cytotoxicity. METHODS: A Phase I trial of high-dose continuous-infusion intravenous zidovudine therapy in combination with 5-FU and leucovorin therapy was performed. Eighteen patients with advanced malignant tumors were treated with 43 courses of oral leucovorin (50 mg every 4 hours); continuous-infusion 5-FU (800 mg/M2/day) for 72 hours (3 days); and zidovudine, begun 24 hours after the start of 5-FU and leucovorin, for 48 hours, and terminating with the end of the 5-FU infusion. Zidovudine plasma levels and zidovudine-induced DNA damage were assessed. RESULTS: Zidovudine administered in doses of 2-20 g/M2/day, added no obvious toxicity to the basic chemotherapeutic treatment with 5-FU and leucovorin but resulted in a dose-dependent biologic effect manifested by an increase in DNA strand breaks in peripheral blood cells. At doses greater than 15 g/M2/day, altered plasma kinetics of zidovudine were observed; plasma zidovudine levels increased dramatically in relation to the dose of zidovudine. Limitations in drug administration restricted administration of higher intravenous doses without achieving a maximally tolerated dose. No responses were seen in this heavily pretreated population. CONCLUSIONS: Based on the results of preclinical studies, plasma zidovudine levels greater than those achieved at the maximal dose (133 microns) are required for increased anti-cancer activity with 5-FU. Additional studies using a bolus or rapid infusion as a method of achieving higher peak levels are indicated.

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 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.

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.

Benzylacyclouridine reverses azidothymidine-induced marrow suppression without impairment of anti-human immunodeficiency virus activity.

Increased extracellular concentrations of uridine (Urd) have been reported to reduce, in vitro, azidothymidine (AZT)-induced inhibition of human granulocyte-macrophage progenitor cells without impairment of its antihuman immunodeficiency virus (HIV) activity. Because of the clinical toxicities associated with chronic Urd administration, the ability of benzylacyclouridine (BAU) to effect, in vivo, AZT-induced anemia and leukopenia was assessed. This agent inhibits Urd catabolism and, in vivo, increases the plasma concentration of Urd in a dose-dependent manner, without Urd-related toxicity. In mice rendered anemic and leukopenic by the administration of AZT for 28 days in drinking water (1.5 mg/mL), the continued administration of AZT plus daily BAU (300 mg/kg, orally) partially reversed AZT-induced anemia and leukopenia (P less than .05), increased peripheral reticulocytes (to 4.9%, P less than .01), increased cellularity in the marrow, and improved megaloblastosis. When coadministered with AZT from the onset of drug administration, BAU reduced AZT-induced marrow toxicity. In vitro, at a concentration of 100 mumol/L, BAU possesses minimal anti-HIV activity and has no effect on the ability of AZT to reverse the HIV-induced cytopathic effect in MT4 cells. The clinical and biochemical implications of these findings are discussed.

Different effect of benzylacyclouridine on the toxic and therapeutic effects of azidothymidine in mice.

It has been reported that in vitro uridine (Urd) can reverse azidothymidine (AZT) cytotoxicity without decreasing anti-human immunodeficiency virus (HIV) activity. Our studies in mice have shown that daily oral doses of benzylacyclouridine (BAU), an inhibitor of Urd breakdown, also reduces AZT hematologic toxicity, presumably by elevating the plasma concentration of Urd. We now extend these murine studies and report the effect of various doses of exogenous Urd, various doses of BAU, or the combination of BAU and Urd, administered daily, on AZT-induced toxicity. In mice receiving concomitant AZT, daily doses of Urd of 1,000 to 2,000 mg/kg increase peripheral reticulocytes and slightly reduce AZT-induced hematologic toxicity. However, the range of effective doses is narrow, and higher doses of Urd (greater than 3,000 mg/kg/d) significantly enhance hematologic toxicity. At its most effective dose, (2,000 mg/kg/d), Urd produces 28% mortality. In contrast, BAU doses up to 300 mg/kg/d reduced AZT-related hematologic toxicity in a dose-dependent manner without mortality. Higher daily doses of BAU and the combination of BAU with low doses of Urd were not more effective. Studies conducted in mice infected with the Rauscher murine leukemia virus (RLV) indicate that BAU does not impair the antiretroviral effect of AZT when administered at doses that reduce AZT-induced anemia and leukopenia. These findings may be significant for the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex.