Maximum Tolerated Dose Of Cyclophosphamide Research Articles

Comparison of the cytotoxic effects of single and divided treatment of 4-hydroxycyclophosphamide at the same total dosage amount in canine lymphoma cell lines

Cyclophosphamide is widely used in combination chemotherapy to treat dogs with lymphoma. The metabolite of cyclophosphamide, acrolein, can irritate the urinary bladder and cause sterile haemorrhagic cystitis. The divided administration of cyclophosphamide across multiple days may reduce the occurrence of the cystitis. However, the therapeutic effect of this modification has not been evaluated and compared to the traditional single maximum-tolerated dose. It is difficult to evaluate the cytotoxic effect by the single chemotherapeutic drug in dogs. In order to verify the effect of the single and divided treatment of cyclophosphamide in canine lymphoma, we used two canine lymphoma cell lines (CLBL-1, B-cell lymphoma and UL-1, T-cell lymphoma) to imitate the clinical conditions. The cell viability in the CLBL-1 and UL-1 cells treated by a single dosage of 4-hydroxycyclophosphamide after 48 h were 70.4% and 61.5%, respectively. The cell viability in the CLBL-1 and UL-1 cells treated by the divided dosage of 4-hydroxycyclophosphamide after 48 h were 109.4% and 50.8%. There were no significant differences between the two administration methods in the T-cell lymphoma cell line (P = 0.215). The single full dosage of 4-hydroxycyclophosphamide exhibited a significant cytotoxic effect rather than the divided dosage in B-cell lymphoma cell line (P = 0.007) did. The maximum-tolerated dose of cyclophosphamide is still recommended to be used in dogs with B-cell lymphoma.

Low-dose metronomic delivery of cyclophosphamide is less detrimental to granulosa cell viability, ovarian function, and fertility than maximum tolerated dose delivery in the mouse.

Chemotherapy can cause early menopause or infertility in women and have a profound negative impact on the quality of life of young female cancer survivors. Various factors are known to influence the risk of chemotherapy-induced ovarian failure, including the drug dose and treatment duration; however, the scheduling of dose administration has not yet been evaluated as an independent risk factor. We hypothesized that low-dose metronomic (LDM) chemotherapy scheduling would be less detrimental to ovarian function than the traditional maximum tolerated dose (MTD) strategy. In vitro, MTD cyclophosphamide exposure resulted in decreased proliferation and increased granulosa cell apoptosis, while cells treated with LDM cyclophosphamide were not different from untreated controls. Treatments of MTD cyclophosphamide induced high levels of follicle atresia and enhanced follicle recruitment in mice. In contrast, LDM delivery of an equivalent dose of cyclophosphamide reduced growing follicle numbers, but was not associated with higher levels of follicle atresia or recruitment. MTD cyclophosphamide induced significant vascular disruption and DNA damage in vivo, while LDM chemotherapy with equal cumulative amounts of cyclophosphamide was not different from controls. MTD chemotherapy also had a negative effect on mouse-fertility outcomes. Our findings suggest that LDM scheduling could potentially minimize the long-term effects of cyclophosphamide on female fertility by preventing follicle depletion from enhanced activation.

Abstract 3773: Identification of ALDH1A3 as a driver of resistance to both low-dose metronomic and conventional cyclophosphamide chemotherapy in prostate cancer

Abstract Prostate cancer (PCa) is the most common cancer in North American males. Conventional, maximum tolerated dose (MTD) chemotherapy with cyclophosphamide (CPA) has been used in the past for PCa treatment, but has been replaced by docetaxel. However, renewed interest in CPA is emerging given its frequent use in low-dose metronomic (LDM) chemotherapy regimens known for their antiangiogenic properties. While therapeutic resistance limits the clinical utility of both LDM and MTD CPA, such resistance appears to occur through at least partially distinct mechanisms. To investigate the molecular changes contributing to resistance formation, we have generated LDM and MTD CPA resistant PC-3 human prostate cancer cell variants (LCR and MCR, respectively), and corresponding control variants (NS), through in vivo passaging in mice. Using next-generation sequencing and gene expression analysis, we observed 453 differentially regulated genes in LCR vs NS, and 1141 genes in MCR vs NS. An intersecting set of 158 genes found to be differentially expressed in both resistant tumor cell lines was further characterized. The genes most up-regulated (including ALDH1A3, MAGEA2 and ELOVL2) and down-regulated (comprising FN1, IL8 and IGFBP3) were chosen for validation by qRT-PCR studies. We also undertook pathway analysis using the DAVID database and the Reactome FI Cytoscape Plugin. Collectively, our studies revealed that glycolysis/gluconeogenesis, biosynthesis of unsaturated fatty acids and activated drug metabolism may contribute to resistance to both LDM and MTD CPA. In addition, numerous chemokine signaling pathway-related genes were found to be downregulated. Current studies are focusing on the role of ALDH1A3 in LDM and MTD CPA resistance. ALDH1A3 is amongst the most differentially regulated genes found in our analysis, and it has been associated with chemotherapy resistance, ‘stemness’ and aggressive tumor behaviour. Despite different mechanisms of action, there are overlapping mechanisms of resistance to LDM and MTD CPA chemotherapy. If our studies confirm an essential role of ALDH13A in such resistance, this could be rapidly clinically translated by repurposing approved pharmacological ALDH inhibitors as anticancer agents. Citation Format: Van C. Hoang, Annabelle Chow, Amy Wong, Lavarnan Sivanathan, Urban Emmenegger. Identification of ALDH1A3 as a driver of resistance to both low-dose metronomic and conventional cyclophosphamide chemotherapy in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3773. doi:10.1158/1538-7445.AM2014-3773

Tumors That Acquire Resistance to Low-Dose Metronomic Cyclophosphamide Retain Sensitivity to Maximum Tolerated Dose Cyclophosphamide

Low-dose metronomic (LDM) chemotherapy is emerging as an alternative or supplemental dosing strategy to conventional maximum tolerated dose (MTD) chemotherapy. It is characterized primarily, but not exclusively, by antiangiogenic mechanisms of action and the absence of high-grade adverse effects commonly seen with MTD chemotherapy. However, similar to other anticancer therapies, inherent resistance to LDM chemotherapy is common. Moreover, even tumors that initially respond to metronomic regimens eventually develop resistance through mechanisms that are as yet unknown. Thus, we have developed in vivo models of PC-3 human prostate cancer cells resistant to extended LDM cyclophosphamide therapy. Such PC-3 variants show stable resistance to LDM cyclophosphamide in vivo yet retain in vitro sensitivity to 4-hydroperoxy-cyclophosphamide (precursor of the active cyclophosphamide metabolite 4-hydroxy-cyclophosphamide) and other chemotherapeutic agents, namely, docetaxel and doxorubicin. Moreover, LDM cyclophosphamide–resistant PC-3 variants remain sensitive to MTD cyclophosphamide therapy in vivo. Conversely, PC-3 variants made resistant in vivo to MTD cyclophosphamide show varying levels of resistance to metronomic cyclophosphamide when grown in mice. These results and additional studies of variants of the breast cancer cell line MDA-MB-231 suggest that resistance to LDM cyclophosphamide is a distinct phenomenon from resistance to MTD cyclophosphamide and that LDM cyclophosphamide administration does not select for MTD chemotherapy resistance. As such, our findings have various implications for the clinical use of metronomic chemotherapy.

Abstract 3813: Recruitment of macrophages, NK cells, and dendritic cells with metronomic cyclophosphamide-induced regression of gliosarcoma xenografts

Abstract Metronomic chemotherapy displays improved therapeutic activity over conventional schedules for several malignancies in both preclinical and clinical studies. In particular, metronomic cyclophosphamide treatment (140 mg/kg BW every 6 days) induces dramatic, near-complete regression of established xenografts of human U251 and rat 9L gliosarcomas in s.c. scid mouse models, whereas maximum tolerated dose cyclophosphamide effects only moderate tumor growth delays. Furthermore, metronomic cyclophosphamide-induced regression of 9L tumors is accompanied by reduced microvessel density, induction of thrombospondin-1 (TSP1) and impaired drug uptake, all of which are consistent with the prevailing view that anti-angiogenesis is a major factor in the therapeutic effectiveness of metronomic chemotherapy. However, VEGF receptor inhibition induces substantially greater anti-angiogenesis in these tumor models but does not induce tumor regression. Therefore, we sought to investigate other non-anti-angiogenic mechanisms to explain the substantial regression and have found that it is strongly associated with recruitment of the host innate immune system. In regressing 9L and U251 tumors, host (mouse) PEDF (pigment epithelium-derived factor; Serpinf1) and PAI-1 (plasminogen activation inhibitor-1; Serpine1) expression is increased, with the levels of PEDF RNA being strongly correlated (r=0.89) with TSP1 RNA in large sets of 9L and U251 tumors isolated various times after initiation of metronomic cyclophosphamide therapy. Notably, PAI-1 and TSP1 are both expressed by tumor-associated macrophages. Metronomic cyclophosphamide also increased Fas, which stimulates macrophage activation, and the macrophage marker CD68, supporting the involvement of tumor-associated macrophages. Fas is also associated with cytotoxic lymphocytes, such as NK cells, and NK cell markers and cytotoxic effectors were dramatically increased in metronomic cyclophosphamide-treated tumors. Furthermore, dendritic cell markers important for cell-target interactions and antigen presentation were increased by metronomic cyclophosphamide treatment. Cytokines and chemokines were also up regulated in the metronomic cyclophosphamide-treated tumors, suggesting that chemokine and cytokine gradients help mobilize the host innate immune system in regressing 9L and U251 tumors. These findings highlight the ability of metronomic chemotherapy to induce macrophage, natural killer cell, and dendritic cell recruitment, thereby activating innate immunity leading to regression of established gliosarcomas and potentially other tumor types. Supported by NIH grant CA49248 (to DJW). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3813.

Abstract A80: Metronomic cyclophosphamide induces macrophage recruitment in regressing human and rat gliosarcoma xenografts

Abstract Metronomic chemotherapy has shown improved therapeutic activity over conventional schedules, both in preclinical studies and in initial clinical trials for several malignancies. Metronomic cyclophosphamide treatment (140 mg/kg BW every 6 days) induces dramatic, near-complete regression of established xenografts of human U251 and rat 9L gliosarcoma in s.c. scid mouse models, whereas maximum tolerated dose cyclophosphamide effects moderate tumor growth delay without sustained regression. Metronomic cyclophosphamide-induced regression of 9L tumors is accompanied by a reduction in microvessel density, suppression of CD31 expression, induction of the anti-angiogenic factor thrombospondin-1 (TSP1), and impaired drug uptake. These findings are consistent with the high intrinsic sensitivity of proliferating endothelial cells to metronomic chemotherapy shown by others, and with the prevailing view that anti-angiogenesis is a major factor in the therapeutic effectiveness of metronomic chemotherapy. However, VEGF receptor inhibition induces substantially greater anti-angiogenesis in these tumor models, yet does not induce major tumor regression. Presently, we investigate whether non-anti-angiogenic mechanisms may contribute to the observed regression of U251 and 9L gliosarcomas, and demonstrate metronomic cyclophosphamide-induced host macrophage recruitment. This recruitment is associated with induction of the mouse (host) factors PEDF (pigment epithelium-derived factor; Serpinf1) and PAI-1 (plasminogen activation inhibitor-1; Serpine1), with the levels of PEDF RNA being strongly correlated (r=0.89) with TSP1 RNA in large sets of 9L and U251 tumors isolated various times after initiation of metronomic cyclophosphamide therapy. Notably, PAI-1 and TSP1 are both expressed by tumor-associated macrophages. Metronomic cyclophosphamide also induced Fas, which stimulates macrophage recruitment during inflammation, and the macrophage marker CD68, supporting the involvement of tumor-associated macrophages in the response to metronomic chemotherapy. These findings highlight the ability of metronomic chemotherapy to induce macrophage recruitment, thereby activating innate immunity leading to regression of established gliosarcomas and potentially other tumor types. Supported in part by NIH grant CA49248 (to DJW). Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A80.

Comparative Impact of Trastuzumab and Cyclophosphamide on HER-2–Positive Human Breast Cancer Xenografts

Metronomic chemotherapy is a minimally toxic and frequently effective new treatment strategy that is beginning to show promising phase II clinical trial results, particularly for metastatic breast cancer when combined with various molecularly targeted antitumor agents. Here, we assessed a treatment strategy that uses trastuzumab plus daily oral metronomic cyclophosphamide on metastatic Her-2-positive human breast cancer models. Treatments were initiated on orthotopic transplanted primary tumors as well as established visceral metastatic disease of two independent Her-2-positive breast cancer models, both independently derived from the human MDA-MB-231 breast cancer cell line. Outcome was assessed by noninvasive measurements of tumor cell-secreted human choriogonadotropin in the urine as a surrogate marker of relative tumor burden, or by whole body bioluminescent imaging, in addition to prolongation of survival. Orthotopic primary tumors responded to trastuzumab monotherapy with significant growth delays, whereas minimal antitumor effect was observed when mice with metastatic disease were treated. Nevertheless, trastuzumab showed a benefit in this latter setting when combined with metronomic low-dose cyclophosphamide as assessed by prolongation of survival. This benefit was similar to trastuzumab plus maximum tolerated dose cyclophosphamide, but was associated with lesser toxicity. Trastuzumab combined with metronomic cyclophosphamide may be an effective long-term maintenance strategy for the treatment of Her-2-positive metastatic breast cancer.

In Vivo Bioluminescence Imaging Monitoring of Hypoxia-Inducible Factor 1α, a Promoter That Protects Cells, in Response to Chemotherapy

Bioluminescence imaging is a powerful technique that has shown that hypoxia-inducible factor 1 (HIF-1), a transcription factor that protects tumor cells from hypoxia, is up-regulated in tumors after radiation therapy. We tested the hypothesis that bioluminescence imaging would successfully and noninvasively depict an increase in HIF-1 in the novel therapeutic environment of chemotherapy and that, as in radiation therapy, the underlying mechanism involves inducible nitric oxide synthase originating in macrophages. Active HIF-1 consists of alpha and beta subunits that bind to promoter sequences in many genes, including those that protect endothelial cells, promote angiogenesis, and alter metastasis and tumor cell metabolism. We grew 4T1 murine breast carcinoma cells with an HIF-1alpha luciferase reporter construct to 7 mm in the right rear flanks of 18 Balb-C mice. The mice were evenly randomized to receive one of the following single intraperitoneal doses: maximum tolerated dose cyclophosphamide (231.5 mg/kg), maximum tolerated dose paclitaxel (10 mg/kg), or control saline solution. Immunohistochemical analysis of tumor sections from the cyclophosphamide and control groups was performed 10 days after treatment to assess the intensity and distribution of HIF-1alpha expression, hypoxia, macrophage infiltration, and expression of macrophage-derived inducible nitric oxide synthase in tumor tissues treated with maximum tolerated dose cyclophosphamide compared with control tumors. Cyclophosphamide, but not paclitaxel, significantly inhibited tumor growth and caused a significant increase in HIF-1alpha protein levels, which peaked at a 10-fold increase from baseline on day 10 after administration. In contrast, paclitaxel did not have an antitumor effect in this model and did not cause a significant increase in HIF-1alpha. Immunohistochemical analysis showed increased and more evenly dispersed levels of HIF-1alpha protein, macrophage infiltration, and expression of inducible nitric oxide synthase originating in macrophages after cyclophosphamide treatment. We successfully monitored increased expression of a tumor protective protein in a noninvasive manner. Such monitoring may be a means of detection of resistance to therapy, and it may be possible to use the monitoring findings to alter treatment strategies in real time. The tumor microenvironment seen at immunohistochemical analysis supports the hypothesized mechanism that the cytotoxic effects of radiation therapy that attract macrophages, causing the release of macrophage-derived inducible nitric oxide synthase and production of HIF-1alpha under aerobic conditions, also underlie chemotherapy. Such noninvasive imaging may be a means to development of therapeutic strategies that prevent HIF-1 up-regulation after chemotherapy treatments.

A comparative analysis of low-dose metronomic cyclophosphamide reveals absent or low-grade toxicity on tissues highly sensitive to the toxic effects of maximum tolerated dose regimens.

The survival benefits of traditional maximum tolerated dose (MTD) cytotoxic therapy have been modest for the treatment of most types of metastatic malignancy and, moreover, often come with increased acute and chronic toxicity. Recent studies have demonstrated that the frequent administration of comparatively low doses of cytotoxic agents, with no extended breaks [low-dose metronomic (LDM) chemotherapy], may not only be at least as efficient as MTD therapy but also less toxic. This coincides with an apparent selectivity for "activated" endothelial cells of the tumor vasculature. However, the impact of LDM chemotherapy on the most sensitive target cell populations normally affected by MTD therapy (i.e., bone marrow progenitors, gut mucosa, and hair follicle cells) has not been analyzed in experimental detail. Therefore, we compared effects of LDM and MTD cyclophosphamide (CTX) on bone marrow and gut mucosa. Furthermore, we studied the potential impact of LDM CTX on angiogenesis in the context of wound healing and evidence of organ toxicity. We show absent or moderate hematologic and intestinal toxicity of LDM as opposed to MTD CTX. Of note was the finding of sustained lymphopenia, which is not unexpected given the use of CTX as immunosuppressive drug. There was no negative impact on wound healing or evidence of organ toxicity. LDM offers clear safety advantages over conventional MTD chemotherapy and therefore would appear to be ideal for long-term combination therapy with targeted antiangiogenic drugs.

Phase I trial of dose escalation with growth factor support in patients with previously untreated diffuse aggressive lymphomas: determination of the maximum-tolerated dose of ProMACE-CytaBOM.

The aim of this study was to determine the maximum-tolerated dose (MTD) of cyclophosphamide, doxorubicin, etoposide, prednisone, bleomycin, cytarabine, methotrexate, and leucovorin (ProMACE-CytaBOM) when the myelotoxic drugs cyclophosphamide, doxorubicin, etoposide, and cytarabine are escalated. Thirty-eight eligible patients with diffuse aggressive non-Hodgkin's lymphoma were treated on a phase I trial of dose escalation using the ProMACE-CytaBOM regimen and granulocyte-macrophage colony-stimulating factor (GM-CSF; Schering, Kenilworth, NJ). Patients were treated with recombinant (r)GM-CSF 10 microg/kg/d subcutaneously from day 9 to 19. Twenty-seven patients had stage IV disease, four had stage III, and seven had bulky stage II. Half of the patients had bone marrow involvement. The median age was 45 years. We found that the MTD was 200% for the escalated drugs in this regimen (although we never escalated above the MTD or defined by dose-limiting toxicity) and that the normalized dose-intensity (NDI; defined as the ratio of the received dose-intensity to the 100% dose-intensity of ProcMACE-CytaBOM) decreased with each cycle and was lower for the day-8 drug (cytarabine) than for the day-1 drugs. The complete response (CR) rate was 66%, and 92% of patients who achieved CR are alive without disease with a median follow-up time for survival of 3.6 years. The MTD of cyclophosphamide, doxorubicin, etoposide, and cytarabine in the ProMACE-CytaBOM regimen given with growth factor support is 200%, and this dose should be tested in larger phase II trials.

Hydroxyurea potentiation of the antineoplastic activity of cyclophosphamide and 4'-(9-acridinylamino)methanesulfon-M-anisidide (AMSA) in the brown Norway rat myelocytic leukemia model.

The activities of hydroxyurea (HU), 4'-(9-acridinylamino)methanesulfon-M-anisidide (AMSA) and cyclophosphamide (CY) were examined in the brown Norway rat myelocytic leukemia model in experiments designed to determine the synergy, optimal drug sequencing, and therapeutic index of combinations of these agents. A single dose of CY or four consecutive daily doses of AMSA produced increased survival in leukemic rats, with a positive-slope dose-response curve up to the maximum tolerated dose (MTD). HU at 1/2 MTD produced a minimal antileukemic effect but significantly potentiated the antineoplastic activity of 1/2 MTD of CY or AMSA with no significant toxic death rate. Drug-sequence experiments demonstrated that maximal synergy was achieved when HU was given immediately after CY but immediately before or during AMSA administration. No significant cure rate was seen with any CY/HU or HU/AMSA sequence. The three drugs given in the sequence of CY followed 3 days later by HU and AMSA simultaneously, however, was curative in the majority of rats with advanced leukemia, whereas other sequences were more toxic or less effective. Each of the drugs in these experiments was given at 1/2 of its single-agent MTD. HU significantly potentiates the antineoplastic effect of CY and AMSA in a drug-sequence-dependent manner in this model, apparently with an improved therapeutic index.