Hazard assessment of antineoplastic drugs and metabolites using cytotoxicity and genotoxicity assays.

Antineoplastic drugs are among the most toxic pharmaceuticals. Their release into the aquatic ecosystems has been reported, giving rise to concerns about the adverse effects, including cytotoxicity and genotoxicity, that they may have on exposed organisms. In this study, we analyzed the cytotoxicity and genotoxicity of 5-fluorouracil (5-FU) and its metabolite alpha-fluoro-beta-alanine (3-NH2-F); gemcitabine (GEM) and its metabolite 2'-deoxy-2',2'-difluorouridine (2-DOH-DiF); as well as cyclophosphamide (CP) on the HepG2 cell line. Drug concentrations were based on those previously observed in the effluent of a major cancer hospital in Brazil. The study found that GEM, 2-DOH-DiF and 5-FU resulted in reduced cell viability. No reduction in cell viability was observed for CP and 3-NH2-F. Genotoxic assessment revealed damage in the form of nucleoplasmic bridges for CP and 3-NH2-F. The tested concentrations of all compounds resulted in significantly increased MNi and NBUDs. The results showed that these compounds induced cytotoxic and genotoxic effects in HepG2 cells at concentrations found in the environment. To the best of our knowledge, this study is the first to report on the cytogenotoxic impacts of the metabolites 3-NH2-F and 2-DOH-DiF in HepG2 cells. These findings may help in the development of public policies that could minimize potential environmental contamination.

5-Fluorouracil and Capecitabine: Assessment and Treatment of Uncommon Early-Onset Severe Toxicities Associated With Administration.

BACKGROUND: Uncommon early-onset severe toxicities from 5-fluorouracil (5-FU) and capecitabine can be fatal if early warning signs are not recognized and treated promptly. OBJECTIVES: This article delineates the differences between expected side effects and uncommon early-onset severe toxicities from 5-FU and capecitabine. It also provides background for understanding the reasons patients may develop these toxicities and reviews the efficacy of standard supportive care against a novel therapy (uridine triacetate). METHODS: A panel of nurses convened to review the literature about toxicities associated with 5-FU and capecitabine administration and determined methods to educate nurses about toxicities and treatment. FINDINGS: Standard supportive care for 5-FU and capecitabine toxicities is associated with high fatality rates. Uridine triacetate treatment within 96 hours of administration is associated with survival.

Assessment of dose-response relationship of 5-fluorouracil to murine intestinal injury.

5-Fluorouracil (5-FU) is the most frequently prescribed anti-tumor drug, but has been reported to result in intestinal injury. Although some progress has been made in understanding the intestinal toxicity of 5-FU, confusion remains about animal models of 5-FU-induced intestinal injury, especially the dosage of 5-FU. This study aims to assess the dose-response relationship between the severity of intestinal injury and different doses of 5-FU, and to determine a proper dosing for the murine model. We found that mice in the 5-FU groups gradually lost body weight over time. Increasing doses of 5-FU resulted in more severe diarrhea, with a concomitant increase in mortality. Histopathological damage was more severe in mice that received higher doses of 5-FU. In addition, plasma diamine oxidase (DAO) activity decreased in experimental mice with intestinal injury in a dose-dependent way. TUNEL and western blot analysis showed cell apoptosis in the ileum and colon related to 5-FU dosage. However, administration of 200 and 400 mg/kg 5-FU caused extremely high mortality, severe diarrhea and histopathological damage, but 25 mg/kg 5-FU did not result in significant intestinal injury. The severity of intestinal injury induced by 5-FU appeared to be dose-dependent and we concluded that the proper dosage of 5-FU to induce a murine model with intestinal mucositis ranged from 50 mg/kg to 100 mg/kg.

Histopathological and biochemical assessment of kidney damage in albino wistar rats treated with cytotoxic platinum compounds in combination with 5-FU.

Kidney cells damage and subsequent renal adverse effects with oxaliplatin are less reported phenomena, whereas cisplatin (CDDP, first generation platinum compound) has therapeutic limitations due to renal toxicity. This experimental study reports oxaliplatin (third generation platinum compound) induced direct damage in rat kidney tissues and alterations in renal biochemical profile. Oxaliplatin was administered in albino wistar rats with 5-FU (5 Fluorouracil) to mimic as model of FOLFOX, the mainstay chemotherapeutic regimen in colorectal cancer (CRC). This study reports changes in renal biochemical profile (serum creatinine and urea) in rats treated in different treatment groups with cisplatin, oxaliplatin, 5-FU, cisplatin+5-FU and oxaliplatin+5-FU which are compared with group of rats treated with normal saline (control group). Subjective renal toxicity in tissues was compared among rats treated with oxaliplatin alone and cisplatin, with and without 5-FU by light microscopy. Cast formation, medial hypertrophy of the vessel wall, vacuolization and necrosis was seen in kidney tissues of oxaliplatin treated rat. Changes in serum creatinine well-above diagnostic risk levels were noted. Apparent tubular degenerative sequence associated with vacuolization and cast formation was observed in 5-FU treated rats. Kidney damage ensued after treatment with 5-FU and oxaliplatin are slightly comparable to massive tubular damages, hemorrhage, casts and vacuolization along with multiple foci of alterations induced by cisplatin.

Acute aquatic toxicity assessment of six anti-cancer drugs and one metabolite using biotest battery - Biological effects and stability under test conditions.

Available ecotoxicological data for anti-cancer drugs and their metabolites are incomplete, and only some studies have been accompanied by chemical analysis. Therefore, the main aim of this study was to evaluate the acute toxicity of the six most commonly used cytostatics, namely cyclophosphamide (CF), ifosfamide (IF), 5-fluorouracil (5-FU), imatinib (IMT), tamoxifen (TAM) and methotrexate (MET) and its metabolite - 7-hydroxymethotrexate (7-OH-MET), towards selected aquatic organisms, namely bacteria Vibrio fischeri, algae Raphidocelis subcapitata, crustaceans Daphnia magna and duckweed Lemna minor. All ecotoxicological tests were accompanied by chemical analysis to determine the differences between nominal and actual concentrations of investigated compounds and their stability under test conditions. For unstable compounds, tests were performed in static and semi-static conditions. It was observed that L. minor was the most sensitive organism. The compounds that were most toxic to aquatic organisms were 5-FU (highly toxic to algae, EC50 = 0.075 mg L-1), MET and TAM (very toxic to highly toxic to duckweed depending on the test conditions; EC50MET 0.08-0.16 mg L-1, EC50TAM 0.18-0.23 mg L-1). It is suspected that MET and 5-FU mainly affected algae and plants most probably because the exposure time was long enough for them to cause a specific effect (they inhibit DNA replication and act predominantly on actively dividing cells). Furthermore, the obtained results also suggest that the toxicity of the metabolites/potentially produced degradation products of MET towards duckweed is lower than that of the parent form, whereas the toxicity of TAM degradation products is in the same range as that of TAM.

Prevention of 5-fluorouracil-induced early severe toxicity by pre-therapeutic dihydropyrimidine dehydrogenase deficiency screening: Assessment of a multiparametric approach.

5-Fluorouracil (5-FU)-based treatments can lead to early-onset severe (4%-5%) even fatal (0.3%) toxicities in patients with dihydropyrimidine dehydrogenase (DPD) deficiency. This multicenter prospective cohort study aimed to assess the clinical benefit of pretherapeutic screening for DPD deficiency using a multiparametric approach. Two parallel cohorts of patients treated with 5-FU-based chemotherapy for colorectal carcinoma were compared in a prospective nonrandomized study. In arm A, patients had DPD deficiency screening before treatment, whereas in arm B no pretherapy screening was performed. Dosing was based on 5-FU administration guidelines of each institution. DPD deficiency screening was performed using a combined multiparametric approach (5-FUODPM Tox). The frequency of early grade 4-5 toxic events potentially induced by 5-FU was compared in the two groups. At total of 1,142 patients (n = 1,116 evaluable) were enrolled. In arm A, out of 718 evaluable patients, nine grade 4 early toxicities potentially related to 5-FU were reported in nine patients (1.2%) with no toxic death despite one complete DPD deficiency and 24 partial deficiencies. The 24 patients with partial deficiency had safe pharmacokinetics (PK)-monitored 5-FU. In arm B, among 398 evaluable patients, 17 grade 4-5 toxic early events potentially related to 5-FU were reported in 12 patients (4.2%). The incidence of early severe toxicity was significantly higher in arm B (P = .0019), confirming the positive impact of pretherapeutic DPD assessment. The percent of patients with a toxicity grade 3 or higher observed in arm A was 10.8% (n = 78) compared to 17.55% (n = 69)  in arm B (P = .0497). The percentage of death was reduced from 2.5/1,000 in arm B to 0 in arm A. The time to occurrence of all grade ≥3 toxicities was determined in both arms and the difference between the two arms was significant (P = .047). Overall, one patient with complete DPD deficiency confirmed retrospectively died within 13 days from grade 5 multivisceral toxicity. Enrollment was prematurely closed after external experts' decision. In conclusion, multiparametric pretherapeutic DPD deficiency screening significantly lowered the risk of early severe toxicity and avoided an early toxic death. This approach should be used for safe administration of 5-FU-based treatments.

In vitro and in vivo genotoxicity assessment of selected pharmaceuticals in relation to Escherichia coli and Cyprinus carpio.

Genotoxicity studies (using SOS chromotest and comet assay) of Escherichia coli and carp (Cyprinus carpio) were performed for three pharmaceutically active compounds, ciprofloxacin, 17α-ethinylestradiol and 5-fluorouracil, used in the treatment of humans. The values of genotoxicity induction coefficient (I) in the SOS chromotest clearly showed genotoxicity for ciprofloxacin, both in the presence and in the absence of S9 fraction; 17α-ethinylestradiol demonstrated slight genotoxicity at the highest tested concentration; and 5-fluorouracil did not induce genotoxic effects in Escherichia coli mutants. Statistical analysis of the results of the comet assay revealed significant differences in cell populations derived from carp placed in a solution of 5-fluorouracil in comparison with the negative control. Statistical analysis also showed a significant increase of "% DNA in tail" of comets in cell populations incubated in solutions of 17α-ethinylestradiol at concentrations of 10000, 2000 and 400 µg/L and in solutions of 5-fluorouracil with S9 fraction at concentrations of 50,000 and 2,000 µg/L in comparison with the negative controls.

Antithymidylate resistance enables transgene selection and cell survival for T cells in the presence of 5-fluorouracil and antifolates.

Antithymidylates (AThy) constitute a class of drugs used in the treatment of cancers such as lung, colon, breast and pancreas. These drugs inhibit DNA synthesis by targeting the enzymes dihydrofolate reductase (DHFR) and/or thymidylate synthase (TYMS). AThys effectively inhibit cancer cells, and also inhibit T cells, preventing anticancer immunity, which might otherwise develop from AThy-induced cancer destruction. We establish that T cells expressing mutant DHFR--DHFR L22F, F31S (DHFR(FS))--and/or mutant TYMS--TYMS T51S, G52S (TYMS(SS))-effectively survive in toxic concentrations of AThys methotrexate, pemetrexed and 5-fluorouracil. Furthermore, we show that DHFR(FS) permitted rapid selection of an inducible suicide transgene in T cells. These findings demonstrate that AThy resistances prevent AThy cytotoxicity to T cells while permitting selection of important transgenes. This technological development could enhance in vitro and in vivo survival and selection of T-cell therapeutics being designed for a broad range of cancers.

Degradation of cyclophosphamide and 5-fluorouracil by UV and simulated sunlight treatments: Assessment of the enhancement of the biodegradability and toxicity.

The presence of pharmaceuticals in the environment has triggered concern among the general population and received considerable attention from the scientific community in recent years. However, only a few publications have focused on anticancer drugs, a class of pharmaceuticals that can exhibit cytotoxic, genotoxic, mutagenic, carcinogenic and teratogenic effects. The present study investigated the photodegradation, biodegradation, bacterial toxicity, mutagenicity and genotoxicity of cyclophosphamide (CP) and 5-fluorouracil (5-FU). The photodegradation experiments were performed at a neutral to slight pH range (7-7.8) using two different lamps (medium-pressure mercury lamp and a xenon lamp). The primary elimination of the parent compounds was monitored by means of liquid chromatography tandem mass spectrometry (LC-IT-MS/MS). NPOC (non-purgeable organic carbon) analyses were carried out in order to assess mineralization rates. The Closed Bottle Test (CBT) was used to assess ready biodegradability. A new method using Vibrio fischeri was adopted to evaluate toxicity. CP was not degraded by any lamp, whereas 5-FU was completely eliminated by irradiation with the mercury lamp but only partially by the Xe lamp. No mineralization was observed for the experiments performed with the Xe lamp, and a NPOC removal of only 18% was registered for 5-FU after 256 min using the UV lamp. Not one of the parent compounds was readily biodegradable in the CBT. Photo transformation products (PTPs) resulting from photolysis were neither better biodegradable nor less toxic than the parent compound 5-FU. In contrast, the results of the tests carried out with the UV lamp indicated that more biodegradable and non-toxic PTPs of 5-FU were generated. Three PTPs were formed during the photodegradation experiments and were identified. The results of the in silico QSAR predictions showed positive mutagenic and genotoxic alerts for 5-FU, whereas only one of the formed PTPs presented positive alerts for the genotoxicity endpoint.

Comprehensive Assessment of Host Responses to 5-Fluorouracil-Induced Oral Mucositis through Transcriptomic Analysis.

BACKGROUND: Chemotherapy plays an important role in current cancer therapy; however, several problems remain unsolved on the issue of host-therapeutics interaction. The purpose of this study was to investigate the host responses after 5-flurouracil (5-FU) administration and to find the target genes and their relationship with other cytokines in the 5-FU-induced oral mucositis (OM) mouse model through transcriptomic analysis. MATERIALS AND METHODS: Thirty-six 6 to 8 week-old male BALB/c mice were randomly divided into the control group and 5-FU-treated group. In the 5-FU group, mice received 5-FU (100 mg/kg, intraperitoneally) on day 1, day 8, day 15, day 22, and day 29, respectively. We evaluated the oral mucosal change under macroanalysis and histological examination at indicated periods, and then applied transcriptomic analysis of gene expression profile and Immunohistochemical stain to identify the target molecules related to 5-FU-induced OM. RESULTS: The most prominent histological change in this model was observed in the fifth week. The gene expression of Bone gamma-carboxyglutamate protein, related sequence 1 (Bglap-rs1) (-12.69-fold) and Chitinase 3-like 4 (Chi3l4) (-6.35-fold) were significantly down-regulated in this phase. The quantitative real-time PCR results also revealed the expression levels were 0.62-fold in Bglap-rs1 and 0.13-fold in Chi3l4 compared with the control group. Immunohistochemical stain showed significant expression of cluster of differentiation 11b (p<0.01), interleukin-1ß (p<0.001) and tumor necrosis factor-α (p<0.05), and down-regulation of Bglap-rs1 (p<0.01) compared with the control group. By Kyoto Encyclopedia of Genes and Genomes pathway analysis, there were twenty-three pathways significantly participated in this study (p<0.05). CONCLUSIONS: Through comprehensively transcriptomic analysis and IHC stain, we discovered several valuable pathways, verified the main pro-inflammatory cytokines, and revealed two significantly down-regulated genes in the 5-FU-induced OM model. These findings highlighted the way of seeking effective therapeutic agents for chemotherapy-induced OM in future.

Assessment of toxicity and genotoxicity of low doses of 5-fluorouracil in zebrafish (Danio rerio) two-generation study.

Residues of anti-neoplastic drugs represent new and emerging pollutants in aquatic environments. Many of these drugs are genotoxic, and it has been postulated that they can cause adverse effects in aquatic ecosystems. 5-Fluorouracil (5-FU) is one of the most extensively used anti-neoplastic drugs in cancer therapy, and this article describes the results of the first investigation using a two-generation toxicity study design with zebrafish (Danio rerio). Exposure of zebrafish to 5-FU (0.01, 1.0 and 100 µg/L) was initiated with adult zebrafish (F0 generation) and continued through the hatchings and adults of the F1 generation, and the hatchings of the F2 generation, to day 33 post-fertilisation. The exposure did not affect survival, growth and reproduction of the zebrafish; however, histopathological changes were observed in the liver and kidney, along with genotoxic effects, at all 5-FU concentrations. Increases in DNA damage determined using the comet assay were significant in the liver and blood cells, but not in the gills and gonads. In erythrocytes, a significant, dose-dependent increase in frequency of micronuclei was observed at all 5-FU concentrations. Whole genome transcriptomic analysis of liver samples of F1 generation zebrafish exposed to 0.01 µg/L and 1 µg/L 5-FU revealed dose-dependent increases in the number of differentially expressed genes, including up-regulation of several DNA-damage-responsive genes and oncogenes (i.e., jun, myca). Although this chronic exposure to environmentally relevant concentrations of 5-FU did not affect the reproduction of the exposed zebrafish, it cannot be excluded that 5-FU can lead to degenerative changes, including cancers, which over long-term exposure of several generations might affect fish populations. The data from this study contribute to a better understanding of the potential consequences of chronic exposure of fish to low concentrations of anti-neoplastic drugs, and they demonstrate that further studies into multi-generation toxicity are needed.

Validation of a fast and low-cost alkaline lysis method for gDNA extraction in a pharmacogenetic context.

PURPOSE: Translation of pharmacogenetic findings from the research laboratory to the clinical practice demands simple and efficient procedures. In this sense, we evaluated the suitability of a modified protocol for genomic DNA extraction based on alkaline lysis of cells. METHODS: Dried blood samples were obtained from 48 patients diagnosed with colorectal cancer. A total of 11 mutations in the dihydropyrimidine dehydrogenase gene and related to 5-fluorouracil toxicity were searched by amplicon sequencing and real-time PCR with fluorescent probes. RESULTS: Genomic DNA extracted with the alkaline lysis method, both from dried blood samples and buccal swabs, fulfilled the quality requirements of the two genotyping methods assayed, which yielded 100 % concordant results for 11 genetic variants with relevance to cancer chemotherapy. CONCLUSIONS: The assessed protocol has shown to be a very fast and economical approach to perform genetic analyses in the clinical laboratory for pharmacological purposes.

Assessment of 5-fluorouracil and 4-nitroquinoline-1-oxide in vivo genotoxicity with Pig-a mutation and micronucleus endpoints.

Genotoxicity assessments were conducted on male Sprague Dawley rats treated with 5-fluorouracil (5-FU) and 4-nitroquinoline-1-oxide (4NQO) as part of an international validation trial of the Pig-a mutant phenotype assay. Rats were orally exposed to 0, 11.5, 23, or 46 mg/kg/day 5-FU for three consecutive days (Days 1-3); blood was sampled on Days -1, 4, 15, 29, and 45. Pig-a mutant phenotype reticulocyte (RET(CD59-)) and mutant phenotype erythrocyte (RBC(CD59-)) frequencies were determined on Days -1, 15, 29, and 45, and percent micronucleated reticulocytes (%MN-RET) were measured on Day 4. Rats were treated with 4NQO for 28 consecutive days by oral gavage, at doses of 1.5, 3, or 6 mg/kg/day. RBC(CD59-) and RET(CD59-) frequencies were determined on Days -1, 15, and 29, and MN-RET were quantified on Day 29. Whereas 5-FU was found to increase %MN-RET, no significant increases were observed for RBC(CD59-) or RET(CD59-) at any of the time points studied. The high dose of 4NQO (6 mg/kg/day) was observed to markedly increase RBC(CD59-) and RET(CD59-) frequencies, and this same dose level caused a weak but significantly elevated increase in MN-RET (approximately twofold). Collectively, the results provide additional support for the combination of Pig-a mutation and MN-RET into acute and 28-day repeat-dose studies.

Assessment of genotoxic effects in nurses handling cytostatic drugs.

Several antineoplastic drugs have been classified as carcinogens by the International Agency for Research on Cancer (IARC) on the basis of epidemiological findings, animal carcinogenicity data, and outcomes of in vitro genotoxicity studies. 5-Fluorouracil (5-FU), which is easily absorbed through the skin, is the most frequently used antineoplastic agent in Portuguese hospitals and therefore may be used as an indicator of surface contamination. The aims of the present investigation were to (1) examine surface contamination by 5-FU and (2) assess the genotoxic risk using cytokinesis-block micronucleus assay in nurses from two Portuguese hospitals. The study consisted of 2 groups: 27 nurses occupationally exposed to cytostatic agents (cases) and 111 unexposed individuals (controls). Peripheral blood lymphocytes (PBL) were collected in order to measure micronuclei (MN) in both groups. Hospital B showed a higher numerical level of contamination but not significantly different from Hospital A. However; Hospital A presented the highest value of contamination and also a higher proportion of contaminated samples. The mean frequency of MN was significantly higher in exposed workers compared with controls. No significant differences were found among MN levels between the two hospitals. The analysis of confounding factors showed that age is a significant variable in MN frequency occurrence. Data suggest that there is a potential genotoxic damage related to occupational exposure to cytostatic drugs in oncology nurses.

Comparative assessment of the therapeutic effects of the topical and systemic forms of Hypericum perforatum extract on induced oral mucositis in golden hamsters.

Oral mucositis is a common and irritating complication of chemotherapy and radiotherapy for malignancies. Current treatments have failed to achieve complete remission of this complication. The St. John's wort plant (Hypericum perforatum) has long been known for its anti-inflammatory and antibacterial effects. The current study was designed to investigate the therapeutic efficacy of the topical and systemic administration of H. perforatum extract on oral mucositis. Oral mucositis was induced in 72 male golden hamsters by administration of 5-fluorouracil (60mg/kg), on days 0, 5, and 10 of the study. The cheek pouch was scratched with a sterile needle on days 1 and 2. On days 12-17, H. perforatum extract topical gel 10%, oral H. perforatum extract (300mg/kg), and gel base groups were treated and then compared with a control group. Weights and blood samples were evaluated, biopsies from buccal lesions were examined histopathologically, and tissue malondialdehyde (MDA) was measured. Both of the H. perforatum extract treatment groups saw a significant relief in oral mucositis compared to the control and base gel groups; the systemic form was superior to the topical form. H. perforatum extract, administered orally or topically, expedited the healing of chemotherapy-induced oral mucositis in hamsters.

Novel microbially triggered colon specific delivery system of 5-Fluorouracil: statistical optimization, in vitro, in vivo, cytotoxic and stability assessment.

The present study aimed to statistically optimize a colon specific formulation of 5-Fluorouracil for the treatment of colon cancer. A 3(2) full factorial design was used for optimization. The independent variables employed were amount of pectin and amount of starch paste, each at three levels. The evaluated responses were hardness, percent cumulative drug release (% CDR) at 5th h and t(90%) (time required for 90% of drug release). Drug release studies were carried out using change over media [pH 1.2, 7.4 and 6.5 in presence of 4% (w/v) rat caecal contents]. The optimized formulation was subjected to in vivo roentgenographic studies in New Zealand white rabbits to analyze the in vivo behaviour of the developed tablets. This formulation was also evaluated for cytotoxic potential using HT-29 human colon cancer cell lines. Pharmacokinetic studies in New Zealand white rabbits were conducted to determine the extent of systemic exposure provided by the developed formulation in comparison to an immediate release tablet. The optimized formulation consisting of pectin (66.67%, w/w) and starch paste (15%, w/w) released negligible amount of drug at pH 1.2 and pH 7.4 whereas significant (p < 0.05) drug release was observed at pH 6.5 in presence of 4% (w/v) rat caecal contents. Roentgenographic studies corroborated the in vitro observations, thus providing the "proof of concept". Pharmacokinetic studies revealed significant reduction in systemic exposure and cytotoxicity studies demonstrated enhanced cellular uptake of drug by the developed formulation. Shelf life of the formulation was found to be 2.83 years. The results of the study established pectin-based coated matrix tablet to be a promising system for the colon specific delivery of 5-FU so as to treat colon carcinoma.

Ecotoxicity and genotoxicity assessment of cytotoxic antineoplastic drugs and their metabolites.

In spite of growing scientific concern about pharmaceuticals in the environment, there is still a lack of information especially with regard to their metabolites. The present study investigated ecotoxicity and genotoxicity of three widely used cytostatic agents 5-fluorouracil (5-FU), cytarabine (CYT) and gemcitabine (GemC) and their major human metabolites, i.e. alpha-fluoro-beta-alanine (FBAL), uracil-1-beta-D-arabinofuranoside (AraU) and 2',2'-difluorodeoxyuridine (dFdU), respectively. Effects were studied in acute immobilization and reproduction assays with crustacean Daphnia magna and growth inhibition tests with alga Desmodesmus subspicatus and bacteria Pseudomonas putida. Genotoxicity was tested with umu-test employing Salmonella choleraesius subsp. chol. Toxicity was relatively high at parent compounds with EC(50) values ranging from 44 microg L(-1) (5-fluorouracil in the P. putida test) to 200 mg L(-1) (cytarabine in D. magna acute test). In general, the most toxic compound was 5-FU. Studied metabolites showed low or no toxicity; only FBAL (metabolite of 5-FU) showed low toxicity to D. subspicatus and P. putida with EC(50) values 80 and 140 mg L(-1), respectively. All parent cytostatics showed genotoxicity with minimum genotoxic concentrations (MGC) ranging from 40 to 330 mg L(-1). From metabolites, only FBAL was genotoxic in high concentrations. To our knowledge, the present study provides some of the first ecotoxicity data for both cytostatics and their metabolites, which might further serve for serious evaluation of ecological risks. The observed EC(50) values within the microg L(-1) range were fairly close to concentrations reported in hospital sewage water, which indicates further research needs, especially studies of chronic toxicity.

Combined environmental risk assessment for 5-fluorouracil and capecitabine in Europe.

An environmental risk assessment (ERA) was made for the old cytostatic active pharmaceutical ingredient 5-fluorouracil (5-FU) and for capecitabine (CAP), which is a prodrug of 5-FU. This ERA is based on published and company internal data as well as new test results for physicochemical, human metabolism, biodegradability, environmental partitioning and fate, and acute and chronic ecotoxicity properties of the active substance 5-FU as well as on use sales data for 5-FU and CAP in Europe. Predicted environmental concentrations (PECs) were extrapolated following the EMEA 2006 Guideline on ERA for human pharmaceuticals and the European Union 2003 Technical Guidance Document (TGD) for risk assessment as well as the TGD-based application EUSES v2.0. Actual amounts sold were taken from IMS Health Databases, in order to refine the default use and EMEA penetration factor as well as the PECs. Moreover, available measured environmental concentrations (MECs) were used to supplement PECs. A predicted no-effect concentration (PNEC) for 5-FU was derived from chronic ecotoxicity data. Except for the simplistic EMEA Phase I default PEC, the risk characterization by PEC:PNEC and MEC:PNEC ratios for various environmental compartments resulted in no significant risk. As the EMEA Phase I PEC does not integrate documented human metabolism and environmental degradation, in contrast to refined PEC derivations, it is inferred that the current use of CAP and 5-FU does not present any evident risk to the environment. An additional evaluation of persistence, bioaccumulation, and toxicity (PBT) properties supports the conclusion of no significant environmental risk for 5-FU and CAP.

Development and assessment of novel all-in-one parenteral formulations with integrated anticoagulant properties for the concomitant delivery of 5-fluorouracil and calcium folinate.

5-Fluorouracil in combination with its biomodulator folinic acid maintains a pivotal position in current anticancer treatment regimens. However, limitations in clinical management persist with the administration of these drugs. These limitations are associated with the use of a high pH to maintain 5-fluorouracil in solution, resulting in high rates of phlebitis and catheter blockages. Herein, we describe and compare initial studies on novel all-in-one formulations of 5-fluorouracil and folinic acid incorporating either sulfated or hydroxypropyl beta-cyclodextrins at physiological pH that potentially address these issues. All formulations markedly improved the stability of supersaturated solutions of 5-fluorouracil in the presence of folinic acid. In-vitro evaluation of the PC-3, HCT-116, MDA-MB-231, PC-14, and COLO-201 human carcinoma cell lines showed that all formulations exhibited equivalent or better cytotoxicity compared with cells exposed to 5-fluorouracil and folinic acid. Thus, these cyclodextrins do not compromise the cytotoxicity of 5-fluorouracil. Preliminary in-vivo dose tolerance profiles of the formulations were also equivalent to 5-fluorouracil and folinic acid administered separately. Furthermore, given the association between thrombosis and cancer, the potentially beneficial anticoagulant activity of the sulfated cyclodextrin-based formulations was also confirmed in vitro. Extended activated partial thromboplastin times and prothrombin times were observed for the sulfated cyclodextrins in human plasma both as individual compounds and as components of the formulations. In conclusion, these novel all-in-one formulations maintain the in-vitro potency while overcoming the accepted incompatibility of 5-fluorouracil and folinic acid, and represent improved injectable forms of 5-fluorouracil that may reduce phlebitis, catheter blockages, and thromboembolic events.