Role of thymidine in biochemical modulation: a review

The role of thymidine in the treatment of cancer has been under clinical investigation for nearly a decade. Clinical trials have demonstrated that it lacks antitumor activity in its own right. In this review, the mechanism of action and rationale for the use of thymidine as a biochemical modulator of standard agents such as 5-fluorouracil, 1-beta-D-arabinofuranosylcytosine, and methotrexate are summarized. With this background, the clinical trials which have been conducted with thymidine, either alone or in combination, are described. We suggest a number of further studies of the role of thymidine in the biochemical modulation of antimetabolites.     

Enhancement of drug sensitivity and a bystander effect in PC-9 cells transfected with a platelet-derived endothelial cell growth factor thymidine phosphorylase cDNA.

5''-Deoxy-5-fluorouridine (5''-DFUR) and 1-(tetrahydro-2-furyl)-5-fluorouracil (tegafur), prodrugs of 5-fluorouracil (5-FU), are anticancer agents activated by thymidine phosphorylase (dThdPase). As it is well known that the levels of dThdPase are higher in tumours than in normal tissue, it should be advantageous to use such pyrimidine antimetabolites for the selective inhibition of tumour growth. However, tumours are not necessarily sensitive to 5''-DFUR and tegafur because their levels of dThdPase vary. In this study, we examined whether transfection of tumour cells with a human platelet-derived endothelial cell growth factor (PD-ECGF) complementary DNA (cDNA) expressing dThdPase would sensitize the cells to the cytotoxic effects of pyrimidine antimetabolites in vitro. A cDNA encoding PD-ECGF was transfected into PC-9 cells (human lung adenocarcinoma). The transfected cells, PC9-DPE2, had a more than 50 times higher activity of dThdPase than the parental PC-9 cells or control PC-9 cells transfected with the pcDNA3 vector alone (PC9-D1). They were more sensitive than parental PC-9 or PC9-D1 cells not only to 5''-DFUR and tegafur but also to 5-FU. In addition, we demonstrated that PC9-DPE2 cells are able to potentiate the cytotoxic effects of 5''-DFUR towards co-cultured parental PC-9 cells. This "bystander effect'' did not require cell-cell contact. These results suggest that transfection of PD-ECGF (dThdPase) genes may be useful as a gene therapy strategy for cancer treatment.     

Interferon effects upon the adenocarcinoma 38 and HL-60 cell lines: antiproliferative responses and synergistic interactions with halogenated pyrimidine antimetabolites

In order to gain insight into the mechanisms affecting combination treatment of tumor cells with interferon and halogenated pyrimidine antimetabolites, in vitro studies of murine colon adenocarcinoma 38 and HL-60 cell lines were undertaken. Interferons exhibited modest antiproliferative effects against these lines with DNA synthesis inhibited greater than RNA greater than protein synthesis, as studied by 3H-precursor incorporation. The adenocarcinoma cell line was considerably more sensitive to recombinant gamma-than to purified alpha/beta-interferon, while HL-60 was slightly more sensitive, in short term studies, to antiproliferative effects of recombinant alpha- than to gamma-interferon. Interferon treatment was further associated with suppression of a hyperdiploid component of the adenocarcinoma cell line, as detected by flow cytometry. Combination treatment of the adenocarcinoma cell line with interferon and halogenated pyrimidines, under 4-day continuous exposure conditions, revealed significant synergy for growth inhibition with gamma- much greater than alpha/beta-interferon and with 5-fluorodeoxyuridine greater than 5-fluorouracil much greater than 5-fluorouridine. Thus, synergy was much greater with the more antiproliferative interferon and with the antimetabolite derivative most likely to lead to thymidylate synthetase inhibition rather than RNA incorporation. Sequential 2-day + 2-day treatment revealed greater synergy when interferon preceded 5-fluorouracil or 5-fluorodeoxyuridine rather than the reverse protocol. The synergy of gamma-interferon and 5-fluorodeoxyuridine could be blocked by thymidine, which bypasses inhibition of thymidylate synthetase. HL-60 also exhibited thymidine antagonized synergistic growth-inhibitory effects of interferon and 5-fluorouracil. Analysis of this interaction by [3H]thymidine incorporation, which can reflect thymidylate synthase inhibition, revealed exaggerated responses of interferon-treated cells to either 5-fluorouracil or 5-fluorodeoxyuridine. These results indicate that interferon-halogenated pyrimidine antimetabolite synergistic interactions may be common to several cell types. Evidence is further presented for a mechanism of synergy entailing enhanced thymidylate synthetase inhibition by the antimetabolite of interferon-treated cells.     

Clinical trial of sequential N-phosphonacetyl-L-aspartate, thymidine, and 5-fluorouracil in advanced colorectal carcinoma

Preclinical studies have demonstrated enhanced cytotoxic effects of 5-fluorouracil (5-FU) when given in conjunction with N-phosphonacetyl-L-aspartate (PALA) or thymidine in several murine systems. Early clinical studies have demonstrated significant delayed depletion of pyrimidine nucleotides in tumor biopsy specimens following systemic PALA administration and prolonged serum levels of 5-FU after thymidine administration. Each of these biochemical effects would be anticipated to augment the cytotoxic activity of 5-FU. A phase II trial of a timed sequential administration schedule of PALA, thymidine, and 5-FU was conducted in 37 patients with advanced measurable colorectal cancer. Ten of 37 patients (27%) experienced objective tumor responses with a median response duration of 22 weeks, and 18 patients (49%) had stable disease for a median duration of 20 weeks. Six of 13 patients (46%) with anaplastic histology and/or rapidly progressive tumors experienced high-quality tumor responses. Leukopenia and neurologic side effects were the primary toxicities, including one death caused by sepsis. This regimen has demonstrated striking alteration in the 5-FU dose-effect relationship and definite antitumor activity in patients with advanced colorectal cancer. Further trials in patients with anaplastic carcinomas of the colon or other anatomic sites should be considered.     

Antimetabolites

Antimetabolites are cytotoxic agents, which have been developed for more than 50 years. Which cancer patient did not receive or will not receive 5-fluorouracil or methotrexate during the evolution his or her disease? Antimetabolites are defined as interfering with the synthesis of the DNA constituents; they are structural analogues, either of purine and pyrimidine bases (or the corresponding nucleosides), or of folate cofactors, which are involved at several steps of purine and pyrimidine biosynthesis. Their first mechanism of action is, therefore, to induce depletion in nucleotides inducing in turn an inhibition of DNA replication. However, some of them are able to get inserted fraudulently into nucleic acids, inducing structural abnormalities leading to cell death by other mechanisms, including DNA breaks. We present in this paper, for the three classes of antimetabolites, both ancient and recent molecules as well as molecules still in clinical trials, without exhaustivity.     

Methotrexate and dipyridamole combination chemotherapy based upon inhibition of nucleoside salvage in humans

We have carried out a clinical trial in 23 patients to determine whether dipyridamole modulates the clinical effect of methotrexate. This trial was based upon in vitro studies which indicate that dipyridamole potentiates the cytotoxic action of methotrexate through inhibition of thymidine salvage. Methotrexate was given as a bolus injection 24 h after initiation of a high dose dipyridamole infusion. The trial was designed so that methotrexate was escalated in individuals until toxicity occurred and then the methotrexate dose resulting in toxicity was repeated without dipyridamole. During the course of this study the methotrexate dose was escalated from 10 to 130 mg/m2. While individual patient tolerance varied, moderate to severe myelosuppression and/or mucositis occurred frequently in patients receiving the combination with methotrexate doses greater than or equal to 60 mg/m2. Ten of 10 patients who experienced moderate or severe toxicity with the combination had significantly less toxicity when treated with methotrexate alone. Dipyridamole did not increase toxicity by an alteration in methotrexate elimination. The potentiation of methotrexate by dipyridamole in these patients suggests that physiological thymidine levels are sufficient to perturb the clinical effects of methotrexate and that thymidine salvage may represent a mechanism for clinical resistance to methotrexate. These results also suggest that a high dose dipyridamole regimen can be used as a pharmacological approach to test the role of nucleoside membrane flux on the clinical action of other standard chemotherapeutic drugs. Phase II studies testing the clinical efficacy of this combination should use a methotrexate dose of 60 mg/m2 with a provision for methotrexate dose escalation based upon individual patient tolerance.     

Nude mouse models as predictors of chemotherapy in man: thymidine and pyrimidines.

The National Cancer Institute cancer treatment screening program has been reorganized incorporating, as an important feature, a panel of human tumors growing as xenografts in congenitally athymic mice. The new screening program is a prospective experiment in the search for new and more effective agents for the treatment of clinical neoplasia. The new program is described and questions that are being asked prospectively are presented. Data are summarized on the activity against human tumor xenografts for a number of clinically established antitumor drugs and examples are presented in which there is interest in compounds for the clinic on the basis of activity in the new screen. Studies are outlined in which high dose thymidine inhibited the growth of human melanoma and teratocarcinoma transplanted in athymic mice. Studies are discussed employing murine tumors in which marked augmentation of the in vivo antitumor activity of 5-fluorouracil was obtained by combination therapy with the pyrimidine nucleosides thymidine, uridine and cytidine. The desirability of investigating combination chemotherapy with pyrimidine nucleosides and 5-fluorouracil and other pyrimidine antagonists in the treatment of human tumor xenografts is stressed. There is a broad range of investigations that can be conducted in nude mouse models and it is important to conduct such programs in relation to the clinic.     

Antitumor effects of polyamine antimetabolites with polyamine-free diet

To evaluate the antitumor efficacy of polyamine antimetabolites such as methylglyoxal-bis-guanylhydrazone (MGBG), ethylglyoxal-bis-guanylhydrazone (EGBG), and alpha-difluoromethylornithine (DFMO), the combined therapies of polyamine antimetabolites with a polyamine-free diet were studied. The combination of EGBG plus DFMO or MGBG plus DFMO showed a marked suppression of tumor growth with a polyamine-free diet; and, when compared, EGBG was slightly superior to MGBG. The inhibition of DNA biosynthesis was also parallel to the above-mentioned results. Furthermore, from an analysis for polyamine levels in tumor tissues, it was confirmed that the polyamine depletion can be maintained by polyamine antimetabolites in combination with a polyamine-free diet in vivo. Moreover, as EGBG has lesser side effects than MGBG, EGBG might be more suitable for clinical use.     

Biologic agents as biochemical modulators: pharmacologic basis for the interaction of cytotoxic chemotherapeutic drugs and interferon

Biochemical modulation of cytotoxic cancer chemotherapeutic agents is one means of enhancing the activity and selectivity of antitumor drugs. Traditionally this approach has utilized detailed information regarding a particular enzymatic reaction or biochemical pathway to develop potential modulating agents. In contrast, the reported clinical therapeutic activity of IFN in combination with cytotoxic agents has prompted a reexamination of the biochemical actions of the cytokine. Interferon elicits a number of cellular actions that might contribute to its pharmacologic activity, including both direct antitumor effects and host-mediated actions. The best understood are those related to the cytotoxicity of the fluoropyrimidine antimetabolites and include enzymatic reactions involved in fluoropyrimidine metabolic activation, catabolism, and interaction with its target enzyme. However, even in this instance, a mechanistic association of a specific pharmacologic action with therapeutic activity remains to be determined. These studies demonstrate that cytokines and other biologic agents may exert specific biochemical modulations that augment (or potentially attenuate) the activity of the cytotoxic chemotherapeutic agents.Supported in part by grant CA54422 from the National Cancer Institute and by an educational grant from Schering Corporation. The views expressed in this paper are those of the authors and do not necessarily reflect those of the supporters     

A review of dose-limiting events in phase I trials: antimetabolites show unpredictable relationships between dose and toxicity

INTRODUCTION: In a sample of NCIC CTG phase I trials we noted that studies of antimetabolites were frequently confounded by the occurrence of dose-limiting toxicities (DLT) at doses well below those ultimately recommended (recommended dose, RD) for further study, necessitating frequent expansion of dose levels and usually a change to more conservative dose escalation. This slowed development, exposed patients to ineffective doses of drugs, and raises concerns about the safety of current trial designs which include a single patient per dose level. Conversely, some patients treated at the RD may be receiving inadequate doses of anticancer drugs. To determine if this was a general phenomenon, we undertook a review of the results of a large group of phase I trials of cytotoxic agents. METHODS: Starting dose (SD), number of dose levels, dose at first DLT (D-DLT), maximum tolerated doses (MTD, dose at which DLT is seen in two or more patients) and RD were extracted from the NCI-Canada phase I trial database, and from a literature survey of phase I studies published between 1985 and 1999. Combination phase I and phase Ib studies were excluded. RESULTS: The review included 33 trials with antimetabolites and 60 with other cytotoxic agents. The median ratio D-DLT/MTD was 0.33 for antimetabolites and 0.75 for other cytotoxic agents (P < 0.01). Similarly, the median ratio D-DLT/RD was 0.43 for antimetabolites and 1 for other cytotoxic agents (P < 0.01). The median number of dose levels tested was nine for antimetabolites and six for other cytotoxic agents. DISCUSSION: Statistically significant differences in the ratios D-DLT/MTD and D-DLT/RD between antimetabolites and other cytotoxic compounds were noted, confirming our initial observations that unpredictable DLT occurs earlier and at lower dose levels in phase I clinical trials of antimetabolites than would be expected as compared to other classes ofcytotoxic agents. Toxicity thus appears to be incompletely predicted by dose alone for antimetabolites. DLT may occur in certain patients at doses well below the RD. Current phase I design may not be ideal for development of these compounds, and should focus on pharmacodynamic endpoints in addition to traditional pharmacokinetic and clinical endpoints.     

Gemcitabine increases systemic 5-fluorouracil exposure in advanced cancer patients

A number of recent clinical trials testing the combination of 5-fluorouracil (5-FU) and gemcitabine in patients with advanced pancreatic adenocarcinoma have shown a significant clinical response rate, but also significant toxicity. As the two antimetabolites may interact at several biochemical levels along their pathways of activation, we investigated whether gemcitabine (GEM) affects 5-FU pharmacokinetics in cancer patients. Thus, we compared 5-FU pharmacokinetics in two groups of patients with various cancers who received the same schedule of 5-FU and folinic acid (FUFA), with or without GEM. There was a significant increase in systemic (5-FU) exposure and toxicity in the FUFA plus GEM group. Our finding may be useful in designing future studies of the combination in order to reduce the occurrence of side-effects and to maximise the antitumour activity.     

Effect of ara-A on differentiation and proliferation of HL-60 cells

The HL-60 human leukemic promyelocytic cell line can be induced to mature into terminally differentiated cells using certain chemotherapeutic agents. We have recently demonstrated that two inhibitors of DNA synthesis, cytosine arabinoside (ara-C) and aphidicolin, can induce HL-60 differentiation with the appearance of monocytic markers. These pyrimidine antimetabolites may have affected DNA methylation patterns and resulted in altered gene expression, or the differentiated phenotype may have occurred by inhibition of DNA replication. Consequently, we have extended these studies by using the purine analog, adenine arabinoside (ara-A), which also acts as an inhibitor of DNA synthesis. The results demonstrate that ara-A also induces HL-60 non-specific esterase activity and enhances expression of myeloid cell surface antigens, MY-4 and MO-1. The induction of a differentiated phenotype by ara-A occurs after partial inhibition of DNA synthesis, a finding similar to that observed with ara-C and aphidicolin. These observations indicate that purine, as well as pyrimidine analog inhibitors of DNA polymerization can induce differentiation of HL-60 cells along a monocytic lineage. These findings may be relevant to recent clinical trials that have employed low doses of ara-C in an attempt to induce differentiation of malignant hematopoietic cells.     

Effect of dipyridamole on fluorodeoxyuridine cytotoxicity in vitro and in cancer patients

Summary Dipyridamole (DP) has previously been studied both in vitro and in vivo in combination with various antimetabolites, including methotrexate and 5-fluorouracil (5FU). We evaluated in vitro and clinically the effects of adding DP to fluorodeoxyuridine (FUDR) in colorectal cancer. Using a human colony-forming assay, we observed that 0.05 M DP increased the cytotoxicity of FUDR by a median of 33.5-fold vs 1.5-fold for 5FU against human colon-cancer cell lines. The mechanism of the DP-enhanced antitumor activity of FUDR is not completely understood but appears to be related to a profound inhibition by DP of thymidine accumulation in and FUDR efflux from colon-cancer cell lines. On the basis of these in vitro results, 28 patients with metastatic colon cancer were entered in a clinical trial of monthly courses of 0.1 mg/kg FUDR daily for 14 days and 75 mg oral DP 5 times daily for 14 days starting on the 3rd day of continuous i.v. FUDR infusion. The pharmacokinetics of DP was studied in three patients; the results showed that 98% of total serum DP was protein-bound and that free DP levels were significantly lower than the concentrations necessary for the expected in vitro DP/FUDR modulation. Treatment was well tolerated, with only 12 patients developing mild to moderate toxicity. Of 27 evaluable patients, 4 achieved a partial response that lasted 2, 3, 5, and 6+ months. This relatively low response rate (15%), which is similar to that achieved with FUDR alone, may be explained by the low steady-state plasma concentrations of free DP achieved in our patients. Other means of DP administration, such as i.v., i.a., and i.p. injection, may be required to achieve free DP concentrations necessary for successful biochemical modulation of FUDR in patients.Supported in part by grants CA17094, CA23074, and CA39629 from the National Institutes of Health, Bethesda, Md 20205, and a grant from the Arizona Chronic Disease Commission. HSG is a recipient of an American Cancer Society Career Development Award     

Older and new purine nucleoside analogs for patients with acute leukemias

Purine nucleoside analogs (PNAs) compose a class of cytotoxic drugs that have played an important role in the treatment of hematological neoplasms, especially lymphoid and myeloid malignancies. All PNA drugs have a chemical structure similar to adenosine or guanosine, and they have similar mechanisms of action. They have many intracellular targets: they act as antimetabolites, competing with natural nucleosides during DNA or RNA synthesis, and as inhibitors of key cell enzymes. In contrast to other antineoplastic drugs, PNAs act cytotoxically, both in the mitotic and quiescent cell cycle phases. In the last few years, three PNAs have been approved for the treatment of lymphoid malignancies and other hematological disorders: 2-chlorodeoxyadenosine (2-CdA), fludarabine and pentostatin. 2-CdA and fludarabine are also active in the treatment of acute myeloid leukemia (AML). These drugs, in combination with cytarabine and other agents, are commonly used as salvage regimens in relapsed or refractory AML. Moreover, the addition of 2-CdA to the standard induction regimen is associated with an increased rate of complete remission and improved survival of adult patients with AML. More recently three novel PNAs have been synthesized and introduced into clinical trials: clofarabine, nelarabine and forodesine. Clofarabine is the most promising PNA in current clinical trials in pediatric and adult patients with acute leukemias. Nelarabine is more cytotoxic in T-lineage than in B-lineage leukemias. Clofarabine and nelarabine have been approved for the treatment of refractory patients with acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma. Clofarabine is also an active drug in AML treatment when administered either alone or in combination regimens as front-line treatment and in relapsed or refractory patients. Unlike other PNA, forodesine is not incorporated into DNA but displays a highly selective purine nucleoside phosphorylase inhibitory action. Forodesine is undergoing clinical trials for the treatment of T-cell malignancies, including T-cell ALL. This article summarizes recent achievements in the mechanism of action, pharmacological properties and clinical activity and toxicity of PNAs, as well as their emerging role in lymphoid and myeloid acute leukemias.     

Antagonistic interplay between antimitotic and G1-S arresting agents observed in experimental combination therapy.

Paclitaxel is a naturally occurring antimitotic agent that has been shown to stabilize microtubules, induce mitotic arrest, and ultimately induce apoptotic cell death. The favorable clinical activity of paclitaxel has prompted considerable interest in combining paclitaxel with numerous other antineoplastic agents. Our previous studies have suggested 5-fluorouracil (5-FU), an antineoplastic agent that usually arrests tumor cells at the G1-S phase of the cell cycle, in combination with paclitaxel significantly represses paclitaxel-induced mitotic arrest and apoptosis. In the present study, we have extended this investigation to include several other antimitotic agents (vinblastine, colchicine, and nocodazole) in various combination schedules with the G1-S arresting agents 5-FU and hydroxyurea (HU). We found 5-FU, as well as HU, could significantly interfere with the overall cytotoxicity as compared with treatment with antimitotic agents alone. It appeared that 5-FU or HU severely limited the antimitotic agents' cytotoxic effects on both mitotic arrest and apoptosis. No combination of a G1-S arresting agent with an antimitotic agent in any schedule produced an antitumor effect greater than that of the antimitotic agent alone. In addition, biochemical examination revealed that 5-FU and HU blocked the antimitotic agent-induced increase of p21WAF1/CIP1 protein levels, as well as prevented the hyperphosphorylation of the bcl-2 and c-raf-1 proteins. These findings suggest that careful considerations may be necessary when combining antineoplastic agents that exert their cytotoxic action at different phases of the cell cycle.     

Fluorodeoxyuridine-induced radiosensitization and inhibition of DNA double strand break repair in human colon cancer cells

The halogenated pyrimidine, fluorodeoxyuridine (FdUrd), has been used in combination with radiation for the treatment of human neoplasms. In an attempt to improve the clinical use of this combination, FdUrd-radiation interactions were studied in vitro using human HT29 colon cancer cells. It was found that FdUrd produced radiosensitization at clinically achievable (1-100 nM) concentrations. Sensitization depended critically on the timing of exposure. When cells were irradiated after a 12-hr exposure to 100 nM FdUrd, marked sensitization was produced (mean inactivation dose (MID) = 2.01 +/- 0.01, compared to control of 4.35 +/- 0.16, p less than .01). No radiosensitization occurred when cells were irradiated 4 hr prior to incubation (MID = 3.95 +/- 0.05, p greater than 0.4). Radiosensitization appeared to result from an inhibition of thymidylate synthase since concentrations of FdUrd which produced radiosensitization depleted intracellular TTP pools and blocked the incorporation of deoxyuridine into DNA. Furthermore, radiosensitization was completely inhibited by co-incubation with thymidine. FdUrd also decreased the repair, but not the formation, of radiation-induced DNA double strand breaks (DSB's). These data are consistent with the hypothesis that FdUrd produces radiosensitization by depleting thymidine pools which leads to a decreased rate of DNA DSB repair. Furthermore, they suggest that in clinical trials FdUrd should be infused at least 8 hr before irradiation.     

Therapeutic potential of the dual-targeted TAS-102 formulation in the treatment of gastrointestinal malignancies

Current treatment modalities for cancer combine cytotoxic drugs against DNA and novel targeted drugs affecting signal transduction pathways, which are required for growth progression and metastasizing tumors. Classical chemotherapeutic regimens for gastro-intestinal tumors include antimetabolites based on 5-fluorouracil (5FU), the platinum analog oxaliplatin and the topoisomerase inhibitor irinotecan. The thymidine analog trifluorothymidine (TFT) has been shown to bypass resistance pathways for 5FU derivatives (S-1, UFT, Xeloda) in model systems, while concurrent application with a thymidine phosphorylase inhibitor (TPI) increases the bioavailability of TFT, thereby potentiating the in vivo efficacy of TFT. The formulation TAS-102 is given orally in a 1.0:0.5 molar ratio (TFT:TPI). The formulation is dual-targeted due to the cytotoxic effect of TFT, which is enhanced by TPI, while TPI also exerts antiangiogenic effects by inhibiting thymidine phosphorylase (TP), also known as platelet-derived endothelial cell growth factor. Evidence is accumulating from in vitro and in vivo preclinical studies that these properties favor further combinations with other cytotoxic agents currently being used in the treatment of gastro-intestinal tumors. Also treatment with targeted agents will synergistically down-regulate signal transduction pathways responsible for growth and progression of tumors. In this review, we summarize the available information on (clinical) pharmacology, mechanisms of action, pharmacodynamic and pharmacokinetic properties, early clinical trials and future directions of the new potent combination drug TAS-102. ( Cancer Sci 2007; 98: 779–789)     

Immune enhancement of nitroreductase-induced cytotoxicity: studies using a bicistronic adenovirus vector

The nitroreductase (NR)/CB1954 enzyme prodrug system has given promising results in preclinical studies and is currently being assessed in phase I clinical trials. It is well established that there is an immune component to the bystander effect observed with other systems such as thymidine kinase and cytosine deaminase; however, such an effect has not previously been described using NR. We have preliminary data suggesting an immune bystander effect with NR to further examine these effects and their potential enhancement by cytokines, an adenoviral vector containing CMV-NR, an internal ribosome entry site (IRES) and the gene for murine GM-CSF (mGM-CSF) was constructed. The NR-GM-CSF virus was validated in 2 experimental models and demonstrated increased therapeutic efficacy in the MC26 murine colorectal tumour model. These data illustrate that the combination of suicide gene therapy using NR and CB1954 with immune stimulation via GM-CSF gives an improved response compared to either modality alone and suggests that the immune component of this response may be beneficial in combating unresectable, metastatic disease and preventing tumour recurrence.     

Potentiation of antimetabolite antitumor activity in vivo by dipyridamole and amphotericin B

Summary Previous studies have shown that dipyridamole (DP), a potent nucleoside transport inhibitor blocking the rescue effect of exogenous nucleosides, markedly potentiates the cytotoxicity of antimetabolites. However, no enhancement of the chemotherapeutic effect of antimetabolites by DP in vivo has yet been reported. This study provided evidence that the combination of DP and amphotericin B (AmB) significantly potentiated the inhibitory effect of 5-fluorouracil (FU) or methotrexate (MTX) against a panel of transplantable tumors including sarcoma 180, cervical carcinoma U14, and Lewis lung carcinoma in mice. No significant increase in toxicity was induced by this combination in treated mice. Our results indicate that the combination of DP and AmB with antimetabolites is potentially useful in cancer chemotherapy.     

Preliminary results of a phase II trial for the treatment of metastatic breast cancer with 5-fluorouracil and leucovorin

The active metabolite of FUra, 5-fluorodeoxyuridine monophosphate (5-FdUMP), requires the presence of reduced folates to form a covalent ternary complex with the target enzyme thymidylate synthase (TS). In vitro and in vivo studies have demonstrated a potentiation of the cytotoxic effects of FUra when combined with the reduced folate, leucovorin. We have applied this concept to the treatment of metastatic breast cancer in a phase II trial, as recent clinical studies on patients with colorectal carcinoma have suggested an enhanced efficacy for the combination of FUra plus leucovorin. Patients entered on the present study are undergoing treatment with a 5-day daily regimen of leucovorin (500 mg/m2, iv) followed by FUra (375 mg/m2, iv). Toxicity and response data are currently being collected on patients who have failed "standard" combination regimens that included FUra. In patients with accessible tumor, serial biopsies are being obtained during treatment with the combination of FUra and leucovorin and during therapy with FUra alone to assess the degree of 5-FdUMP binding to the target enzyme, TS, in the presence and absence of exogenously administered leucovorin. Preliminary results from the biochemical studies suggest an enhanced saturation of TS by the fluorinated pyrimidine when administered with leucovorin.