Raffaele Longo and Giampietro Gasparini
Division of Medical Oncology, "S. Filippo Neri" Hospital, Rome, Italy
Chemotherapy has been the mainstay of medical approaches to the treatment of solid neoplasia. It is usually given in the form of bolus infusion at maximum tolerated doses (MTDs) with the goal of complete tumor kill. With the exception of a few tumors of the adult, such as lymphoid, germ cell, and some pediatric cancers, however, eradication of advanced cancer has been elusive, even with high doses and autologous bone-marrow rescue . It was suggested that the rest periods between each cycle of therapy provide the tumor endothelium an opportunity to repair the chemotherapy-induced damages. The harsh side effects and the ultimate failures of conventional chemotherapy fueled broad investigation of therapeutic alternatives, including drugs targeting not only tumor cells, but also genetically stable cells of tumor stroma, such as endothelial cells (ECs). The emerging paradigm is that patient survival is not incompatible with tumor persistence. A therapy aimed at making the cancer a chronic disease, with tumor burden held at the lowest achievable volume, may prove to be a more appropriate therapeutic strategy for human solid tumors. (Figure 1)
Based on the results of experimental studies [2, 3], Hanahan et al.  proposed the term metronomic chemotherapy for schedules of cytotoxic agents given regularly at subcytotoxic doses and with suppression of the "activated" endothelium as the principal target (i.e., the antiangiogene-sis chemotherapy paradigm).
Angiogenesis is necessary to sustain the growth of primary tumor and metastases. Metronomic schedules are more effective in targeting tumor "activated" ECs than large single high-dose bolus doses followed by long rest periods , because intratumor ECs, in contrast to quiescent mature ECs of normal adult tissues, proliferate rapidly and are more vulnerable to cytotoxic agents . However, the rest period between cycles of conventional chemotherapy permits the survival and regrowth of a fraction of ECs, allowing tumor angiogenesis to persist . Indeed, other functions such as EC motility, invasion, and vessel remodeling may be blocked or altered by metronomic chemotherapy . Suppression of mobilization of bone marrow-derived EC progenitors to sites of angiogenesis is another possibility [2, 3]. Tumor-cell heterogeneity may allow the coexistence of cells having different sensitivities to the same therapeutic agent, and the ability of those cells to shift among the different sensitivity compartments over time, with a "resensitization" effect that may best be exploited using metronomic chemotherapy.
A further theoretical advantage of metronomic chemotherapy is that it minimizes the toxic effects, allowing combinations of potentially synergistic selective inhibitors of angiogenesis [3, 5].
However, a phenomenon that may limit the advantages of low-dose metronomic or continuous-dose delivery is a threshold effect for drug activity. Specifically, there may exist a minimum concentration of drug below which no tumor inhibition takes place .
Recently, Miller et al. defined the criteria for antiangio-genic activity of cytotoxic agents: (a) camptothecin analogs, vinca alkaloids, and taxanes are active against ECs at doses lower than those required for tumor-cell cytotoxicity; (b)
cisplatin, cyclophosphamide, methotrexate, and doxorubicin interfere with EC function without cytotoxic effects; and (c) purine analogues, cisplatin, and anthracyclines block specific steps of the angiogenic cascade .
Recent studies have demonstrated that the antitumor activity of metronomic schedules of chemotherapy is mediated through an antiangiogenic effect .
Browder et al. compared cyclophosphamide given by a metronomic schedule with conventional single, high-dose chemotherapy . The metronomic schedule was more effective in eradicating Lewis lung carcinoma and L1210 leukemia. When the drug was given at the MTD, which requires long periods of rest to allow bone marrow recovery, apoptosis of ECs in the tumor microvasculature was observed, but this damage was largely repaired during the rest periods between the cycles .
However, if the same drug was administered chronically on a once a week schedule, without long breaks, at a lower dose, the repair process was compromised and the anti-angiogenic effects of the drug were enhanced. It was also observed that tumors resistant to conventional dosage of cyclophosphamide responded to the metronomic schedule. Reversal of acquired resistance was attributed to the shifting of the target from the cancer cell to the genetically stable and sensitive ECs.
Similarly, in human orthotopic breast or ectopic colon cancer xenografts in nude or SCID mice, cyclophosphamide given PO at low doses through drinking water showed a relevant antitumor effect, particularly when used in combination with an anti-VEGF-R2 antibody .
In an in vivo mouse corneal model, O'Leary et al. showed a significant antiangiogenic activity of camp-
Figure 1 Metronomic chemotherapy interferes with tumor microvasculature and the stromal compartment, in contrast to standard chemotherapy that presents a direct cytotoxic effect. (see color insert)
tothecin analogs at a dose of 359nmol/kg (210mg/kg) delivered over 6 days .
Presta et al. reported that 6-methylmercaptopurine riboside, a purine analog, inhibits angiogenesis induced by FGF-2 in in vitro and in vivo rabbit cornea and chorioallantoic membrane models at topical doses of 100nmol twice a day for 10 days, or at the single dose of 25 |mmol onto the implants, respectively .
Low doses of vinblastine (0.1-1pmol/L in vitro, and 0.5-1 pmol/L in vivo) had reversible antiangiogenic activity, without cytotoxicity . The highest antiangiogenic dose was 1 pmol/L, which is equivalent to a dose of 16 |mg in a 70-kg adult, a much lower dose than currently used in the clinic.
More recently, Klement et al. demonstrated that vinblastine at one tenth to one twentieth of the MTD caused a significant inhibition of angiogenesis with partial tumor regression in subcutaneous implanted tumors. This effect was significantly enhanced by combination with an anti-VEGF R2 (flk-1) antibody .
In Swiss male nude mice implanted intracranially with human glioblastoma cells, Bello et al. showed that low and semicontinuous chemotherapy in combination with the recombinant human PEX (a fragment of matrix metallopro-teinase-2) was associated with longer survival, marked decreases in tumor volume, vascularity, and proliferative index, and increased apoptosis, without major side effects .
Paclitaxel selectively inhibits the proliferation of human ECs at noncytotoxic concentrations (0.1 to 100pM) by blocking the formation of sprouts and tubes in the three-dimensional fibrin matrix. This activity does not affect the cellular microtubule structure, and the treated cells do not show G2/M cell cycle arrest and apoptosis, suggesing a novel, but as yet unknown, mechanism of action .
Available data obtained from studies in tumor-bearing animals specifically aimed to investigate the antiangiogenic effect of cytotoxic agents have found two patterns of anti-angiogenic effect . The first "type" is defined by an angiogenesis inhibition due to a direct action on ECs regardless of the tumor cell line used and observed at dose levels lower than that required for the cytotoxic effect (e.g., bleomycin, vinca alkaloids, paclitaxel). The type-2 antiangiogenic effect is not consistently present within a variety of tumor cell lines resulting from an antiproliferative effect on tumor cells or non-ECs of the tumor host. In the alginate tumor angiogenesis model, the type-2 reaction pattern has been shown for doxorubicin, epirubicin, etoposide, and 5-fluorouracil. However, these effects may be dependent on the scheduling of drug administration .
All these preclinical studies present certain important problems: (i) the experimental model used: The studies with subcutaneous transplantation of rapidly proliferating tumors do not reflect the slow growth of spontaneous tumors and the characteristics of the stromal component of the organ of origin ; (ii) the lack of systematic comparative studies with conventional schedules; and (iii) the possible mecha nisms of acquired resistance have not been adequately investigated .
In addition, the best results were obtained using combinations with selective antiangiogenic inhibitors, leading to additive and/or synergistic effects.
In conclusion, before proceeding to clinical trials, more compelling and appropriate experimental studies on metronomic chemotherapy are needed, and, although several cytotoxic agents affect ECs in vitro, only a few have been shown to produce substantial antiangiogenic activity in vivo, namely, cyclophosphamide, vinblastine, and paclitaxel [2, 3, 5].
There is currently no published clinical study in which metronomic chemotherapy is prospectively compared with conventional schedules, or that describes appropriate in vitro or in vivo methods of monitoring the antiangiogenic activity .
In a Phase I study, Retain et al. explored the continuous infusion of low doses of vinblastine up to 36 weeks and found that 0.7mg/mq daily was the most effective dose, associated with a good tolerability and some clinical benefit .
Colleoni et al. demonstrated the activity of the combination of low oral doses of cyclophosphamide and methotrex-ate in patients with metastatic breast cancer, without relevant side effects. Serum VEGF levels measured at 2 months were lower than at baseline, with statistically significant reduction only in the subgroup of responding patients . The major weakness of the study are the dosages administered, being in the range of cytotoxic effects; the low response rate in previously untreated patients; and, finally, the fact that the method of determination of VEGF is not standardized.
Other Phase I-II studies are investigating low, continuous oral doses of cytotoxic agents (uracil/tegafur and leucovorin, etoposide, 5-fluorouracil) or continuous intravenous infusion (5-fluorouracil, idarubicin, methotrexate, irinotecan) resembling an antiangiogenic schedule . Forty percent of the patients with nonsmall-cell lung cancer not responsive to standard doses of etoposide responded to the same drug given orally at a much lower single dose, with only a 1-week break every month .
Promising results have been reported with weekly taxane treatment in breast and ovarian cancer, even in patients with progressive disease after the same drug given at the MTD every 3 weeks [11,12]. Thus, reversal of an apparent state of clinical drug resistance could be achieved by altering the dosing and frequency of the drug. However, at present, it is unknown if this effect is really related to an antiangiogenic activity.
At our center, we are evaluating the combination of weekly paclitaxel, at 80mg/mq, and celecoxib, at 400 mg bid, in patients with nonsmall-cell lung cancer, as second-
line chemotherapy. The preliminary data regarding both the tolerability and efficacy are encouraging and in accordance with the results of other similar ongoing Phase II studies.
In a Phase I dose-finding study, we evaluated the combination of rofecoxib with intravenous weekly irinotecan on days 1, 8, 15, 22 and infusional 5-fluorouracil at the fixed dosage of 200 mg/mq/day, as second-line therapy of metastatic colorectal cancer. The dose levels of irinotecan explored were from 75 to 125mg/mp. Seven of 15 patients assessable for response obtained a partial response (46%) with a median duration of 5 months and another six had a stable disease with a median duration of 5 months. The acute and subacute hematological toxicity was moderate, and mucosal side effects were less than those expected with the same regimen without rofecoxib (submitted). A Phase II study testing the activity of such a schedule is ongoing.
There are several theoretical advantages and opportunities for metronomic chemotherapy (Table I). However, there are also potential problems and challenges in terms of appropriate experimental study design and clinical testing .
First, combined metronomic chemotherapy should be tested by adequate experimental models, such as orthotopic and metastatic tumors. The EC heterogeneity, which also extends to morphology, function, and response to antiangio-genic molecules, suggests that agents that affect angiogene-sis in one organ may not be effective in other sites. Tumor cells implanted into mice usually produce rapidly growing lesions, which can double in size every few days and contain a high proportion of dividing ECs.
Second, human solid tumors are heterogeneous, with different molecular abnormalities, even in the same tumor histotype . Gene expression profiling and cDNA microarrays may categorize tumors into biologically homogeneous subgroups and may be of help to design individually tailored treatments .
Third, the identification of specific surrogate biomarkers can allow the selection of patients as well as the possibility of monitoring tumor response. "Biological" response crite-
Table I Potential Advantages of Metronomic over Conventional Schedules of Chemotherapy.
• The targets are genetically stable ECs
• Activity against both the parenchymal and stromal tumor components
• Enhanced antiangiogenic and proapoptotic activity
• Reduced likelihood of emergence of acquired resistance
• Fewer systemic side effects
• Feasibility of long-term administration
• Possibility of combination with other cytostatic, molecular-targeted treatments ria should replace the currently used the clinical end point based on the objective response.
Fourth, experimental results have emphasized the critical need for combining metronomic regimens with selective antiangiogenic agents. The intrinsic elevated sensitivity of activated ECs to metronomic chemotherapy, compared with that of other cells, may not be related to the presence of high concentrations of EC-specific survival factors, such as VEGF . Such combinations may be particularly effective in inducing higher levels of apoptosis in activated ECs coupled with the inhibition of cell proliferation. It is possible that the inhibition of ECs proliferation or induction of apoptosis may not be a direct effect of the drug, but rather an induced secondary event: e.g., a change in expression of genes or proteins that mediate the antiangiogenic effects.
In conclusion, certain cytotoxic drugs with antiangio-genic properties retain a potent antitumor activity when used in a protracted manner at very low concentrations, being able to affect both the tumoral parenchyma and vascular compartments. In contrast to the concept of "more is better," it appears that survival depends more on a cytostatic effect of chemotherapy than on rapid tumor shrinkage .
It is unlikely that metronomic chemotherapy will lead to significant clinical benefit if given alone. We suggest that it should be considered a novel approach making feasible for long periods the administration of cytotoxic agents in combination with selective molecular-targeting compounds directed against specific growth factors or blocking angiogenesis.
Even though the initial development of these novel combined treatments is in the context of advanced disease, the major therapeutic benefits are expected in the adjuvant setting or as maintenance therapy.
Angiogenesis: Intratumor formation of new blood vessels from a preexisting vascular network.
Anticancer therapy: Conventional cytotoxic chemotherapy. Metronomic chemotherapy: Chemotherapy given regularly at subcy-totoxic soles with the "activated" endothelium as principal target.
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Raffaele Longo is medical doctor and physician researcher at the Division of Medical Oncology, "S. Filippo Neri" Hospital, Rone (Italy), since March 2002. Ongoing research topics are tumor angiogenesis, Cox-2 inhibitors in solid cancers, and clinical development of new targeted molecular drugs.
Professor Gasparini has been Director of the Medical Oncology Unit at the San Filippo Neri Hospital in Rome since 2000. His scientific interests primarily focus on translational research on angiogenesis and molecular-targeted anticancer treatments. He is author of 250 publications and a member of the editorial boards of 15 international oncological journals.
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