Immunopotentiation with low-dose cyclophosphamide in the active specific immunotherapy of cancer

This paper reviews the use of low-dose cyclophosphamide (CY) with active specific immunotherapy in patients with advanced melanoma and other metastatic cancers, and outlines the basic scientific research that supports this use. In various animal models, CY augments delayed-type hypersensitivity responses, increases antibody production, abrogates tolerance, and potentiates antitumor immunity. The mechanism of CY immunopotentiation involves inhibition of a suppressor function, as indicated by extensive work in the MOPC-315 plasmacytoma murine model. Human studies of the immunopotentiating effect of CY have yielded both positive and negative results. Toxicity associated with low-dose CY has been mild in these studies. Results of efficacy have been variable for reasons such as small sample sizes, short follow-up periods, and the weaker immunogenicity of human tumor-associated antigens. Although beneficial clinical outcomes have been observed in historically controlled trials, there are few randomized, controlled trials that evaluate outcome in relation to CY immunopotentiation of active specific immunotherapy. Additional randomized, controlled trials should be done to examine the clinical efficacy of CY immunopotentiation of therapeutic cancer vaccines. Received: 3 April 1998 / Accepted: 6 May 1998     

Histological changes in spleen and lymph nodes of mice administered cyclophosphamide and levan

Summary Morphological changes in spleen and lymph nodes of C57B1 mice induced by the cytotoxic agent cyclophosphamide and the polysaccharide levan, separately and in combination were studied. In the spleen, an early decrease (phase 1) and a late increase (phase 2) in weight were found to result from all drug administrations. The hypocellularity of phase 1 was due to a depletion in the white pulp affecting mainly the B-region. Splenic weight decrease was parallel to B-cell depletion and most marked in animals exposed to cyclophosphamide with levan. The splenomegaly observed during phase 2 with all treatments was due to extramedullary hematopoiesis in the red pulp. In the lymph nodes, administration of cyclophosphamide and levan produced opposite effects on the B-cell region: cyclophosphamide eliminated the germinal centers for 8 days, but levan seemed to enhance B-cell activity. In animals given both cyclophosphamide and levan, inhibition of B-cell activity began earlier than with cyclophosphamide alone. Levan does not antagonize the suppressive effect of cyclophosphamide on the B-cell component at the early phase when the drugs are given together.     

Increased radiation toxicity with germline ATM variant of uncertain clinical significance

BackgroundWhile patients with ataxia telangiectasia are known to have increased radiation sensitivity, patients with germline heterozygous ataxia telangiectasia mutated (ATM) mutations can have widely varying functional and clinical effects, which can make management decisions difficult. With an increased prevalence of gene panel-based testing for breast cancer patients, radiation oncologists are increasingly confronted with patients who carry germline ATM variants of uncertain clinical significance. This study describes the clinical courses and outcomes of 5 breast cancer patients with varying germline heterozygous ATM mutations undergoing radiation therapy at our institution in order to provide additional knowledge of the varying clinical effects to aid future decision making.Case SeriesWe identified 5 patients with breast cancer and varying germline heterozygous ATM mutations treated at the University of North Carolina Hospitals between 2015 and 2017. The median age at breast cancer diagnosis for the patient series was 46. Clinical effects of radiation treatment varied amongst the 5 patients. The one patient with a pathogenic ATM mutation had no increased radiation related toxicity. Of the 4 patients with ATM variants of uncertain significance, one patient had increased radiation sensitivity with Grade 3 dermatitis. All patients have remained recurrence free with a median duration of 18 months.ConclusionOur data illustrates that patients with germline heterozygous ATM mutations can have widely varying clinical effects with radiation therapy. Given the possibility of unpredictable deleterious effects, our study highlights the importance of caution and careful consideration when devising the multi-modality management strategy in these patients.     

Uroprotective effect of oleuropein in a rat model of hemorrhagic cystitis

Hemorrhagic cystitis is one of the devastating complications seen after receiving cyclophosphamide chemotherapy. Oleuropein is the most important phenolic compound of olive leaves that mediates most of its beneficial pharmacological properties. Herein, we investigated the possible uroprotective effect of oleuropein against cyclophosphamide induced hemorrhagic cystitis in a rat model. For this purpose, we measured bladder nitric oxide, reduced glutathione, catalase, tumor necrosis factor-alpha and vascular endothelial growth factor levels in addition to the bladder gene expression of intercellular adhesion molecule-1 after induction of hemorrhagic cystitis in the presence or absence of oleuropein. Histopathological examination of bladder tissues was also performed. After cyclophosphamide injection, we demonstrated a significant decrease in bladder reduced glutathione (39%) and catalase (55.4%) levels and a significant increase of nitric oxide (5.6 folds), tumor necrosis factor-alpha (3.3 folds), vascular endothelial growth factor (2 folds) and intercellular adhesion molecule-1 (8 folds) bladder contents when compared to those in normal control rats. Administration of oleuropein induced a marked elevation in bladder reduced glutathione (37.8%), catalase (100.4%) with a prominent reduction of bladder nitric oxide (40%), tumor necrosis factor-alpha (35.9%) and vascular endothelial growth factor (56.2%) levels along with downregulation of intercellular adhesion molecule-1 bladder expression (73.1%) in comparison to cyclophosphamide treated rats levels. Our data demonstrated that oleuropein counteracts the harmful effects of cyclophosphamide on the bladder through its antioxidant and anti-inflammatory activities. Oleuropein exerts a definite uroprotective effect against cyclophosphamide induced hemorrhagic cystitis in rats.     

Simple and selective determination of the cyclophosphamide metabolite phosphoramide mustard in human plasma using high-performance liquid chromatography

A simple and selective assay for the determination of the alkylating cyclophosphamide metabolite phosphoramide mustard (PM) in plasma was developed and validated. PM was determined after derivatisation by high-performance liquid chromatography (HPLC) with ultraviolet detection at 276 nm. Sample pre-treatment consisted of derivatisation of PM with diethyldithiocarbamate (DDTC) at 70°C for 10 min, followed by extraction with acetonitrile in the presence of 0.7 M sodium chloride. Phase separation occurred due to the high salt content of the aqueous phase. The HPLC system consisted of a C₈ column with acetonitrile–0.025 M potassium phosphate buffer, pH 8.0, (32:68, v/v) as the mobile phase. The entire sample handling procedure, from collection at the clinical ward until analysis in the laboratory, was optimised and validated. Calibration curves were linear from 50 to 10 000 ng/ml. The lower limit of quantification and the limit of detection (using a signal-to-noise ratio of 3) were 50 and 40 ng/ml, respectively, using 500 μl of plasma. Within-day and between-day precisions were below 11% over the entire concentration range and the accuracies were between 100 and 106%. PM was found to be stable at −30°C for at least 10 weeks both in plasma and as a DDTC-derivative in a dry sample. A pharmacokinetic pilot study in two patients receiving 1000 mg/m² CP in a 1-h infusion demonstrated the applicability of the assay.     

Uroprotective Effect of Pentoxifylline in Cyclophosphamide‐Induced Hemorrhagic Cystitis in Rats

The role of phosphodiesterase inhibitor, pentoxifylline, in the prevention of cyclophosphamide‐induced hemorrhagic cystitis was evaluated in a rat model. Hemorrhagic cystitis was induced in rats by an intraperitoneal (i.p.) injection of a single dose of cyclophosphamide (150 mg/kg). Pentoxifylline (150 mg/kg/day/ip) was administered for 10 days followed by cyclophosphamide. Hemorrhagic cystitis was well characterized macroscopically, microscopically, and biochemically. Cyclophosphamide induced bladder injury including acute severe inflammation, vascular congestion, severe edema, hemorrhage, inflammatory cell infiltration in the lamina propria, and epithelial denudation; as well as it notably elevated serum inflammatory cytokines (tumor necrosis factor‐α, interleukin‐6, and interleukin‐1β), bladder content of malondialdehyde and total nitrate, accompanied with depletion of bladder antioxidant enzymes activities (glutathione peroxidase, superoxide dismutase, glutathione‐S‐transferase, and catalase). Prior administration of pentoxifylline improved all biochemical and histologic alterations induced by the cytotoxic drug cyclophosphamide. In conclusion, pentoxifylline has proven uroprotective efficacy in the cyclophosphamide‐induced hemorrhagic cystitis model, possibly through modulating the release of inflammatory cytokines and nitric oxide and restoring the oxidant/antioxidant balance. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:343‐350, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21494     

Interleukin-1α synergistic in vivo enhancement of cyclophosphamide- and carboplatin-mediated antitumor activity

 Interleukin-1α (IL-1α) has potent acute antitumor activity in vivo and can enhance the efficacy of chemotherapeutic drug-mediated antitumor responses. Studies were undertaken to examine the ability of IL-1α to enhance the activity of cyclophosphamide (CTX) administered in combination with carboplatin. To determine the in vivo effect of IL-1α, CTX and/or carboplatin, mice bearing 14-day RIF-1 tumors were treated on day 0 with a concurrent i.p. injection of varying doses of CTX (5–150 mg/kg), human IL-1α (125 μg/kg), and carboplatin (50 mg/kg) and examined 24 h later for the surviving fraction by the in vivo excision clonogenic-tumor-cell assay. Even at the lowest doses of CTX, IL-1α significantly enhanced the clonogenic tumor cell kill when compared to treatment with CTX alone. When carboplatin was added to the treatment schema, significantly greater clonogenic cell killing and tumor regrowth delay were observed as compared to any agent alone or a two-drug combination (CTX/IL-1α or CTX/carboplatin). Significant enhancement was observed even at low doses of CTX in combination with carboplatin and IL-1α. The interaction between the three-drug combination was found to be synergistic as determined by the median dose effect with significant dose reduction apparent for IL-1α and CTX when used in this combination. These results demonstrate that IL-1α can synergistically enhance the antitumor efficacy of CTX and the combination of CTX and carboplatin. Received: 11 September 1996 / Accepted: 20 May 1997     

Analysis of cyclophosphamide and five metabolites from human plasma using liquid chromatography–mass spectrometry and gas chromatography–nitrogen–phosphorus detection

An assay method for the quantification of cyclophosphamide (CY) and five metabolites from human plasma is presented. The procedure is adapted to the chemical properties of the compounds of interest: non-polar compounds are extracted into methylene chloride, concentrated and analyzed by GC–NPD after derivatization, and the remaining aqueous fraction is deproteinated with acetonitrile–methanol prior to separation via reversed-phase HPLC and detection using atmospheric pressure ionization (API)-MS. Standard curves are linear over the required range and reproducible over five months. Plasma concentration–time profiles of CY and metabolites from a patient receiving CY by intravenous infusion (60 mg/kg, once a day for two days) are presented.     

The cytotoxic action of activated and non-activated cyclophosphamide in yeast: Comparison of induced DNA damage

The cytotoxic and DNA-damaging effects of cyclophosphamide (CP) and its ‘activated’ derivative 4-OOH-CP were studied using a series of strains of S. cerevisiae which allow a phenotypical classification of genotoxic characteristics as well as direct physicochemical demonstration of key DNA lesions. The concurring results of biological and biochemical experiments indicate that (i) non-activated CP has a weak but detectable monofunctional alkylating potency, leading to DNA strand breaks and (ii) 4-OOH-CP has the ability to induce both DNA strand breaks and interstrand cross-links. The activity of CP is probably due to spontaneous decomposition in aqueous solution.     

The expression of PAC1 increases in the degenerative thymus and low dose PACAP protects female mice from cyclophosphamide induced thymus atrophy

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with cytoprotective ability mediated by its specific receptor PAC1. In this research, firstly the thymus index and the expression of PAC1 in the normal and degenerative thymus with different gender were assayed; secondly PACAP in different dose was used to treat the female mice with cyclophosphamide (CPS) and the changes in thymus index, the expression of PAC1, histopathology, apoptosis, oxidative status and the caspase 3 activity in thymus were determined and compared. It was found that in the mice of age from 1 to 9 weeks in the stage of sex development, the thymus index was significantly higher in female mice than in male mice. And it was found for the first time that the PAC1 expression level in thymus of female mice was significantly higher than that of male mice and the expression of the PAC1 and PACAP increased significantly in the degenerative thymus induced by CPS. After PACAP was co-injected with CPS to the female mice, it was shown that only low dose (1 nmol/kg) of PACAP promoted the thymus index, inhibited the cell apoptosis, ameliorated the oxidative status and decreased the caspase activity significantly, while high dose (10 nmol/kg) of PACAP had no significant protective effects against CPS-induced thymus atrophy. It was concluded that the expression of PAC1 in the thymus changes in reverse ratio with thymus index and in direct ratio with cell apoptosis and only low dose of PACAP had positive effects against the CPS-induced thymus atrophy.