Environmental degradation of human metabolites of cyclophosphamide leads to toxic and non-biodegradable transformation products.

The present study assessed the ready biodegradability of the prodrug cyclophosphamide (CPA) and its stable human metabolites in the closed bottle test (CBT). The results of the CBT showed that only the main human metabolite, carboxyphosphamide (CXP), was biodegradable to a certain extent (23 ± 2.4 % ThODNH3). All other metabolites showed neither biodegradation under these conditions nor were any toxic effects on the inoculum observed. Yet, HRMSn results revealed partial primary elimination of all human metabolites and formation of 25 new transformation products. Abiotic degradation via SNi and SN2 reactions was proposed as the main degradation pathway during the CBT. The main degradation products were assigned as 3-(2-chloroethyl)oxazolidin-2-one (COAZ), cytotoxic N-2-chloroethylaziridine (CEZ) and nor­nitrogen mustard (NNM), an analogue of the chemical warfare agent HN2. While the acute ecotoxicity of the detected products is widely unknown, many have already been reported in medical literature to be either mutagenic, genotoxic, cytotoxic or carcinogenic and may therefore cause a greater risk than their precursors. QSAR models predicted that 16 of them are mutagenic and genotoxic, thus classifying the majority of the chemicals as potential environmental hazards. The central intermediates during the degradation process were proposed as CEZ and its corresponding aziridinium ion. However, other degradation products may occur depending on the type and strength of nucleophiles present in the matrices. Overall, the results demonstrated the importance to include human metabolites in the evaluation of the environmental fate of pharmaceuticals and their risk assessment especially when investigating prodrugs. The results underline the importance of identifying possible degradation products of metabolites, as they can be more toxic than related parent compounds and metabolites and can cause a greater risk to the environment and humans.

Analysis of the Fragmentation Pathways for the Collision-Induced Dissociation of Protonated Cyclophosphamide: A Mass Spectrometry and Quantum Mechanical Study.

Cyclophosphamide is a well-known anticancer agent acting by means of DNA alkylation. Associated with its tumor selectivity, it also possesses a wide spectrum of toxicities. As the requirement of metabolic activation before cyclophosphamide exerts either its therapeutic or toxic effects is well recognized, research aiming at elucidating the pathways that lead to the activation of this drug is of key importance. This has created the necessity for developing an effective analytical method for detecting cyclophosphamide and its breakdown products. In this paper, an Acquity TQ tandem quadrupole mass spectrometer equipped with electrospray ionization in positive-ion mode was employed for detecting cyclophosphamide in its protonated form. The full-scan mass spectrum of cyclophosphamide shows two ion clusters displaying the characteristic isotopic pattern of two chlorine atoms and assigned as sodiated cyclophosphamide, [CP + Na]+, and protonated cyclophosphamide, [CP + H]+ or PCP. With the aid of quantum mechanical DFT calculation, free energy differences in the gas phase among PCP protomers were computed with respect to the most stable protomer being protonated on the 2-oxide oxygen of the 1,3,2-oxazaphosphorine-2-oxide ring. In addition, the interconversion mechanisms among the different protomers were also proposed by intercepting the corresponding transition states in the gas phase. Collision-induced dissociation (CID) of PCP generated six characteristic product ions. Fragmentation mechanisms were proposed and supported by computation. The calculated energy barriers for all of the located transition states were found to be accessible under the reported experimental conditions.

Anti-Tumor Effects of Queen Bee Acid (10-Hydroxy-2-Decenoic Acid) Alone and in Combination with Cyclophosphamide and Its Cellular Mechanisms against Ehrlich Solid Tumor in Mice.

Queen bee acid or 10-hydroxy-2-decenoic acid (10-HDA) is one of the main and unique lipid components (fatty acids) in royal jelly. Previous studies have demonstrated that 10-HDA has various pharmacological and biological activities. The present study aims to evaluate the anti-tumor effects of 10-HDA alone and combined with cyclophosphamide (CP), as an alkylating agent which widely used for the treatment of neoplastic cancers, against the Ehrlich solid tumors (EST) in mice. Methods: A total of 72 female Swiss albino mice were divided into eight groups. EST mice were treated with 10-HDA (2.5 and 5 mg/kg) alone and combined with CP (25 mg/kg) orally once a day for 2 weeks. Tumor growth inhibition, body weight, the serum level of alpha-fetoprotein (AFP) and carcinoembryonic antigen tumor (CAE), liver and kidney enzymes, tumor lipid peroxidation (LPO) and nitric oxide (NO), antioxidant enzymes (e.g. glutathione reductase (GR), glutathione peroxidase (GPx), catalase enzyme (CAT)), tumor necrosis factor alpha level (TNF-α), and the apoptosis-regulatory genes expression were assessed in tested mice. Results: the findings exhibited that treatment of EST-suffering mice with 10-HDA at the doses of 2.5 and 5 mg/kg especially in combination with CP significantly (p < 0.001) decreased the tumor volume and inhibition rate, tumor markers (AFP and CEA), serum level of liver and kidney, LPO and NO, TNF-α level, as well as the expression level of Bcl-2 in comparison with the mice in the C2 group; while 10-HDA at the doses of 2.5 and 5 mg/kg especially in combination with CP significantly (p < 0.001) improved the level of antioxidant enzymes of GPx, CAT, and SOD and the expression level of caspase-3 and Bax genes. Conclusions: According to the results of the present investigations, 10-HDA at the doses of 2.5 and 5 mg/kg especially in combination with CP showed promising antitumor effects against EST in mice and can be recommended as a new or alternative anticancer agent against tumor; nevertheless, further investigations, particularly in clinical setting, are required to confirm these results.

Two birds with one stone: a NIR fluorescent probe for mitochondrial protein imaging and its application in photodynamic therapy.

Mitochondrial proteins, most of which are encoded in the nucleus and the rest of which are regulated by the mitochondrial genome, play pivotal roles in essential cellular functions. However, fluorescent probes that can be used for monitoring mitochondrial proteins have not yet been widely developed, thereby severely limiting the exploration of the functions of proteins in mitochondria. Towards this end, here we propose a near-infrared (NIR) fluorescence probe MPP to effectively illuminate the dynamic changes in mitochondrial proteins in live cells under oxidative stress, with excellent temporal and spatial resolution. Of particular importance, MPP extends the study of the pharmacology involved in apoptosis induced by anti-cancer drugs (hydroxycamptothecin (HCPT), epirubicin (Epi) and cyclophosphamide (CPA)) for the first time. Furthermore, employing a protein-activatable strategy, this probe could serve as an excellent phototherapeutic agent in photodynamic therapy (PDT). Finally, in vivo experiments suggest that this versatile probe can be used to image tumors in HeLa tumor-bearing mice for 24 h, which demonstrates that our probe could play a dual role as a robust phototherapeutic and imaging agent.

Effect of the drug cyclophosphamide on the activity of porcine kidney betaine aldehyde dehydrogenase.

The enzyme betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the synthesis of glycine betaine (GB), an osmolyte and osmoprotectant. Also, it participates in several metabolic pathways in humans. All BADHs known have cysteine in the active site involved in the aldehyde binding, whereas the porcine kidney enzyme (pkBADH) also has a neighborhood cysteine, both sensitive to oxidation. The antineoplastic and immuno-suppressant pre-drug cyclophosphamide (CTX), and its bioactivation products, have two highly oxidating chlorine atoms. This work aimed to analyze the effect of CTX in the activity of porcine kidney betaine aldehyde dehydrogenase. PkBADH was incubated with varying CTX concentration (0 to 2.0 mM) at 25 °C and lost 50 % of its activity with 2.0 mM CTX. The presence of the coenzyme NAD+ (0.5 mM) decreased 95% the activity in 2.0 mM CTX. The substrate betaine aldehyde (0.05 and 0.4 mM, and the products NADH (0.1-0.5 mM) and GB (1 and 10 mM) did not have an effect on the enzyme inactivation by CTX. The reducing agents, dithiothreitol and ß-mercaptoethanol, reverted the pkBADH inactivation, but reduced glutathione (GSH) was unable to restore the enzyme activity. Molecular docking showed that CTX could enter at the enzyme active site, where its chlorine atoms may interact with the catalytic and the neighboring cysteines. The results obtained show that CTX inactivates the pkBADH due to oxidation of the catalytic cysteine or because it oxidizes catalytic and neighborhood cysteine, forming a disulfide bridge with a concomitant decrease in the activity of the enzyme.

Structural characterization and effect on leukopenia of fucoidan from Durvillaea antarctica.

Fucoidans from brown seaweed shows various bioactive properties and promising prospects in biomedical field. Here, a novel fucoidan (F-4) was extracted and purified from Durvillaea antarctica. The structure of F-4 was characterized by HPLC, HPGPC, GC-MS, together with IR and NMR spectral analysis. F-4 is a sulfated polysaccharide mainly composed of fucose (Fuc), galactose (Gal), and glucose (Glc) in a molar ratio of 26.4: 7.1: 1.0. The backbone of F-4 is composed of (1→3) and (1→4)-linked-α-L-Fucp residues, which sulfated at C-4 or C-2 positions and branched with α-L-Fuc, ß-D-Gal, and ß-D-Glc residues. Furthermore, F-4 can effectively promote the growth of leukocyte in a mouse model induced by cyclophosphamide, possibly by activating hematopoietic progenitor cells and regulating the hematopoietic microenvironment of bone marrow. Our data provide useful information for further investigation of fucoidan in the treatment of chemotherapy-induced leukopenia.

Extraction of Volatile Anticancer Drugs in Air Using a Solid-Phase Extraction Type Device Followed by Gas Chromatography-Mass Spectrometric Analysis.

Ifosfamide (IF), cyclophosphamide (CP), and bendamustine (BD) are widely used anticancer drugs. These drugs have slight volatility; therefore, medical-staff exposure is of concern in the medical field. However, an accurate and quantitative detection method of these volatile drugs in air has not been reported. In this study, we developed the quantitative extraction and detection method of these volatile anticancer drugs in air. For the extraction of analytes, a solid-phase extraction-type collection device packed with styrene-divinylbenzene polymer particles was used. The extracted analytes were quantitatively eluted with 5 mL of ethanol, and the solution was concentrated to 100 µL with nitrogen purging. The analytes were analyzed using gas chromatography-mass spectrometry (GC-MS). The limit of detection of the proposed method for IF and CP was 0.017 and 0.033 ng L-1, respectively in air at an air sampling volume of 300 L. IF and CP showed slight volatility, whereas BD was not detected in GC-MS due to its lower volatility. The spiked recoveries of IF and CP in the proposed method were within the range of 95.5 to 101%. Finally, the proposed method was applied to determine the exposure of IF and CP during the dispensing of CP within a hospital dispensary room. The investigated volatile anticancer drugs were not detected in real air samples, indicating that the protection measures employed are sufficient.

Novel cyclophosphamide of natural products osalmide and pterostilbene induces cytotoxicity and cell cycle arrest in diffuse large B-cell lymphoma cells.

Diffuse large B-cell lymphoma (DLBCL) is the most common category and disease entity of non-Hodgkin lymphoma. Osalmide and pterostilbene are natural products with anticancer activities via different mechanism. In this study, using a new synthetic strategy for the two natural products, we obtained the compound DCZ0801, which was previously found to have anti-multiple myeloma activity. We performed both in vitro and in vivo assays to investigate its bioactivity and explore its underlying mechanism against DLBCL cells. The results showed that DCZ0801 treatment gave rise to a dose- and time-dependent inhibition of cell viability as determined by CCK-8 assay and flow cytometry assay. Western blot analysis results showed that the expression of caspase-3, caspase-8, caspase-9 and Bax was increased, while BCL-2 and BCL-XL levels were decreased, which suggested that DCZ0801 inhibited cell proliferation and promoted intrinsic apoptosis. In addition, DCZ0801 induced G0/G1 phase arrest by downregulating the protein expression levels of CDK4, CDK6 and cyclin D1. Furthermore, DCZ0801 exerted an anti-tumor effect by down-regulating the expressions of p-PI3K and p-AKT. There also existed a trend that the expression of p-JNK and p-P38 was restrained. Intraperitoneal injection of DCZ0801 suppressed tumor development in xenograft mouse models. The preliminary metabolic study showed that DCZ0801 displayed a rapid metabolism within 30 min. These results demonstrated that DCZ0801 may be a new potential anti-DLBCL agent in DLBCL therapy.

Causes and possibilities to circumvent cyclophosphamide toxicity.

Cyclophosphamide is an inert prodrug converted into 4-hydroxycyclophosphamide (OHCP) by hepatic hydroxylation. OHCP is in equilibrium with its tautomeric aldophosphamide (ALDO). From ALDO, the cytotoxic active metabolites are formed enzymatically by phosphodiesterases; these are the alkylating metabolite phosphoramide mustard (PAM) and the proapoptotic aldehyde 3-hydroxypropanal (HPA). PAM damages the DNA by alkylation; HPA amplifies the thereby induced apoptosis. The generally accepted view that acrolein, which is believed to be formed in the formation of PAM by ß-elimination from ALDO would be mainly responsible for the toxicity of cyclophosphamide, has to be revised because no acrolein is formed in the systemic circulation of patients after cyclophosphamide administration. It is shown that not acrolein, but OHCP itself is the true toxic metabolite of cyclophosphamide. Toxicity tests with OHCP and PAM were carried out, which demonstrated that OHCP unfolds its toxicity, not as a carrier of PAM but is toxic itself by reacting with nucleophilic groups of macromolecules, for example, thiol groups of membrane proteins. Further experiments demonstrate that the toxicity of oxazaphosphorine cytostatics may be drastically reduced if the formation of the pharmacologically active metabolite ALDO bypasses the formation of OHCP. Toxicity experiments in mice with S-ethanol-cyclophosphamide (SECP) that hydrolyzes to OHCP show that SECP is as toxic as OHCP, whereas the thiazolidine of ALDO, which hydrolyzes to ALDO bypassing OHCP is 7-9 times less toxic without loss of antitumor activity.

Simultaneous degradation of the anticancer drugs 5-fluorouracil and cyclophosphamide using a heterogeneous photo-Fenton process based on copper-containing magnetites (Fe3-xCuxO4).

The effect of substitution of iron by copper in the magnetite lattice was investigated in terms of the catalytic activity in the heterogeneous photo-Fenton process. The physicochemical properties of the Fe3-xCuxO4 nanoparticles were characterized by X-ray diffraction (XRD), X-ray fluorescence (WD-XRF), specific surface area measurements, field emission scanning electron microscopy (FEG-SEM), and X-ray photoelectron spectroscopy (XPS). Copper-modified magnetite showed higher catalytic activity for H2O2 conversion to HO• (estimated using 7-hydroxycoumarin), compared to pristine magnetite (Fe3O4). Consequently, improved degradation of the anticancer drugs 5-fluorouracil (5-FU) and cyclophosphamide (CP) was observed, with high efficiencies achieved using Fe2.75Cu0.25O4 (0.125 g L-1) and 15 mmol L-1 H2O2, at pH 6.5, which resulted in complete degradation of 7.7 µmol L-1 5-FU and CP after 150 min. Low leaching of Cu and Fe demonstrated the stability of the catalyst in the Fenton process, with high catalytic activity (>90%) maintained after use in 4 cycles. The addition of radical scavengers such as methanol, tert-butanol and iodide ions indicated that surface-bonded hydroxyl radicals played a major role in the degradation of 5-FU and CP in the Fe3-xCuxO4/H2O2 system. The substitution of octahedral Fe(II) sites of the magnetite lattice by Cu(II) and the partial oxidation of Cu(I) to Cu(II) and Fe(II) to Fe(III) on the catalyst surface after the Fenton reaction were confirmed by analysis of the XPS spectra.

Exposure-Toxicity Association of Cyclophosphamide and Its Metabolites in Infants and Young Children with Primary Brain Tumors: Implications for Dosing.

PURPOSE: To characterize the population pharmacokinetics of cyclophosphamide, active 4-hydroxy-cyclophosphamide (4OH-CTX), and inactive carboxyethylphosphoramide mustard (CEPM), and their associations with hematologic toxicities in infants and young children with brain tumors. To use this information to provide cyclophosphamide dosing recommendations in this population. PATIENTS AND METHODS: Patients received four cycles of a 1-hour infusion of 1.5 g/m2 cyclophosphamide. Serial samples were collected to measure cyclophosphamide, 4OH-CTX, and CEPM plasma concentrations. Population pharmacokinetic modeling was performed to identify the patient characteristics influencing drug disposition. Associations between drug exposures and metrics reflecting drug-induced neutropenia, erythropenia, and thrombocytopenia were investigated. A Bayesian approach was developed to predict 4OH-CTX exposure using only cyclophosphamide and CEPM plasma concentrations. RESULTS: Data from 171 patients (0.07-4.9 years) were adequately fitted by a two-compartment (cyclophosphamide) and one-compartment model (metabolites). Young infants (<6 months) exhibited higher mean 4OH-CTX exposure than did young children (138.4 vs. 107.2 µmol/L·h, P < 0.0001). No genotypes exhibited clinically significant influence on drug exposures. Worse toxicity metrics were significantly associated with higher 4OH-CTX exposures. Dosing simulations suggested decreased cyclophosphamide dosage to 1.2 g/m2 for young infants versus 1.5 g/m2 for children to attain similar 4OH-CTX exposure. Bayesian-modeled 4OH-CTX exposure predictions were precise (mean absolute prediction error 14.8% ± 4.2%) and had low bias (mean prediction error 4.9% ± 5.1%). CONCLUSIONS: A 4OH-CTX exposure-toxicity association was established, and a decreased cyclophosphamide dosage for young infants was suggested to reduce toxicity in this population. Bayesian modeling to predict 4OH-CTX exposure may reduce clinical processing-related costs and provide insights into further exposure-response associations.

Oxidative degradation of cyclophosphamide using thermal plasma activation and UV/H2O2 treatment in tap water.

There is a growing concern about pharmaceuticals entering the aquatic environment. Many of these compounds cannot be removed completely in sewage treatment plants. To remove these unwanted medicines from water, oxidative degradation techniques may complement the current purification steps. In this paper we studied the effect of advanced oxidation on the cytostatic drug cyclophosphamide (CP) by comparing thermal plasma activation with UV/H2O2 treatment. Plasma activated water (PAW) contains highly reactive oxygen and nitrogen species (RONS) as a result of electric gas discharges in air over water. CP solutions in tap water were oxidized over a period of 120 min and subsequently analyzed by LC-MS/MS to measure the compound degradation. Plasma activation was applied at 50, 100, or 150 W electric power input and UV/H2O2 treatment was carried out by the addition of H2O2 and placing an UV-C source above the test solution for immediate irradiation. The oxidative degradation of CP in PAW resulted in a complete degradation within 80 min at 150 W. CP was also completely degraded within 60 min applying UV/H2O2 oxidation. Both treatment techniques do induce different structural changes, demonstrating that CP is completely degraded in tap water.

Photocatalytic degradation of cyclophosphamide and ifosfamide: Effects of wastewater matrix, transformation products and in silico toxicity prediction.

Antineoplastic drugs have been identified in surface water and effluents from wastewater treatment and, once in the environment, may be harmful to aquatic organisms, as these compounds are possibly mutagenic, genotoxic, cytotoxic, carcinogenic and teratogenic. This work investigated the photodegradation of cyclophosphamide (CP) and ifosfamide (IF) using ruthenium doped titanate nanowires (Ru-TNW) in distilled water (DW) and in wastewater (WW) from secondary wastewater treatment, under UV-Vis radiation. The results indicated that Ru-TNW showed photocatalytic activity for the two cytotoxic drugs with the half-life (t1/2) of 15.1 min for CP and 12.9 min for IF in WW. Four CP transformation products (TPs) and six IF TPs from the photodegradation process are here reported. These TPs were elucidated by high-resolution mass spectrometry. For both pollutants, the results showed different time profiles for the TPs when WW and DW were used as matrix. Overall, in the WW there was a higher production of TPs and two of them were detected only in this matrix. In other words, environmental matrices may produce different TPs. Degradation pathways were proposed and both drugs bear similarities. Additionally, in silico toxicity were performed by quantitative structure-activity relationship models. The predictions indicated that the TPs, with the exception of one IF TP, presented high mutagenic potential.

Human constitutive androstane receptor agonist DL5016: A novel sensitizer for cyclophosphamide-based chemotherapies.

The DNA alkylating prodrug cyclophosphamide (CPA), alone or in combination with other agents, is one of the most commonly used anti-cancer agents. As a prodrug, CPA is activated by cytochrome P450 2B6 (CYP2B6), which is transcriptionally regulated by the human constitutive androstane receptor (hCAR). Therefore, hCAR agonists represent novel sensitizers for CPA-based therapies. Among known hCAR agonists, compound 6-(4-chlorophenyl)imidazo-[2,1-b]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) is the most potent and broadly utilized in biological studies. Through structural modification of CITCO, we have developed a novel compound DL5016 (32), which has an EC50 value of 0.66 µM and EMAX value of 4.9 when activating hCAR. DL5016 robustly induced the expression of hCAR target gene CYP2B6, at both the mRNA and protein levels, and caused translocation of hCAR from the cytoplasm to the nucleus in human primary hepatocytes. The effects of DL5016 were highlighted by dramatically enhancing the efficacy of CPA-based cytotoxicity to non-Hodgkin lymphoma cells.

Cyclophosphamide and isophosphamide - DFT conformational studies in the gas phase and solution.

Cyclophosphamide and isophosphamide have been subjected to comprehensive conformational studies in the vacuum and solution using the SMD solvation model. Vacuum calculations were conducted using the B3LYP, M05-2X, M06-2X and ωB97XD functionals. Natural bond orbital (NBO) analysis has been performed for selected geometries. A preference for a chair conformation with the axial P=O bond is shown (1C4). The 5S0 conformation is 1.25-2.31 kcal/mol and 1.72-2.92 kcal/mol higher in energy than the global minimum conformations of cyclophosphamide and isophosphamide, respectively. In the gas phase, the chair conformation with the equatorial P=O bond (4C1) is of comparable stability or less stable than the skew form, depending on the method used, while it is slightly more favored than the 5S0 conformation in solution. The stereoelectronic effects do not differentiate the ring conformer stability. The steric strains between N(EtCl)1-2 and the C4 and C6 carbon atoms mainly influence the stability of cyclophosphamide and isophosphamide conformers.

Protective effects of garlic extract against hematological alterations, immunosuppression, hepatic oxidative stress, and renal damage induced by cyclophosphamide in rats.

Cyclophosphamide is an alkylating agent widely used as anticancer drug, reported to exert cytotoxic effects attributed to oxidative stress. Therefore, this study aimed to explore the protective effect of ethanolic extract of garlic (EEG) against cyclophosphamide (Cyp)-induced hematological disturbance and immunosuppressive and hepatotoxic effects. Forty male Wistar albino rats were randomized into four equal groups: the normal control one, the Cyp-treated group (50 mg/kg BW/IM, once weekly), the EEG-treated group (300 mg/kg BW, orally, daily), and the Cyp & EEG group. All rats received their relevant treatments for four consecutive weeks. This study revealed that Cyp significantly decreased erythrocyte count, hemoglobin (Hb), packed cell volume (PCV), and total leukocyte and lymphocyte counts. However, the counts of neutrophils, eosinophils, and toxic neutrophils were elevated. Additionally, hepatic malondialdehyde (MDA) and levels of liver and renal biomarkers were significantly elevated in the Cyp-treated group. Otherwise, hepatic catalase (CAT), reduced glutathione (GSH), superoxide dismutase (SOD), and serum total antioxidant capacity (TAC) were significantly lower than the control rats. Furthermore, Cyp significantly reduced whole blood respiratory burst activity (NBT), serum lysozyme and bactericidal activities, interlukin-12 (IL-12), and interferon-γ. In contrast, the levels of nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interlukin-1ß (IL-1ß) were elevated. Additionally, Cyp induced hepatic and renal histopathological alterations. Data in the present study demonstrated that EEG has immunomodulatory and antioxidant effects and has the ability to diminish the alterations induced by Cyp.

The Main Metabolites of Fluorouracil + Adriamycin + Cyclophosphamide (FAC) Are Not Major Contributors to FAC Toxicity in H9c2 Cardiac Differentiated Cells.

In the clinical practice, the combination of 5-fluorouracil (5-FU) + Adriamycin (also known as doxorubicin, DOX) + cyclophosphamide (CYA) (known as FAC) is used to treat breast cancer. The FAC therapy, however, carries some serious risks, namely potential cardiotoxic effects, although the mechanisms are still unclear. In the present study, the role of the main metabolites regarding FAC-induced cardiotoxicity was assessed at clinical relevant concentrations. Seven-day differentiated H9c2 cells were exposed for 48 h to the main metabolites of FAC, namely the metabolite of 5-FU, α-fluoro-ß-alanine (FBAL, 50 or 100 µM), of DOX, doxorubicinol (DOXOL, 0.2 or 1 µM), and of CYA, acrolein (ACRO, 1 or 10 µM), as well as to their combination. The parent drugs (5-FU 50 µM, DOX 1 µM, and CYA 50 µM) were also tested isolated or in combination with the metabolites. Putative cytotoxicity was evaluated through phase contrast microscopy, Hoechst staining, membrane mitochondrial potential, and by two cytotoxicity assays: the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and the neutral red (NR) lysosomal incorporation. The metabolite DOXOL was more toxic than FBAL and ACRO in the MTT and NR assays. When in combination, neither FBAL nor ACRO increased DOXOL-induced cytotoxicity. No nuclear condensation was observed for any of the tested combinations; however, a significant mitochondrial potential depolarization after FBAL 100 µM + DOXOL 1 µM + ACRO 10 µM or FBAL 100 µM + DOXOL 1 µM exposure was seen at 48 h. When tested alone DOX 1 µM was more cytotoxic than all the parent drugs and metabolites in both the cytotoxicity assays performed. These results demonstrated that DOXOL was the most toxic of all the metabolites tested; nonetheless, the metabolites do not seem to be the major contributors to FAC-induced cardiotoxicity in this cardiac model.

Transformation kinetics of cyclophosphamide and ifosfamide by ozone and hydroxyl radicals using continuous oxidant addition reactors.

The detection of pharmaceuticals in water and wastewater has triggered human and ecological health concerns. As highly toxic compounds, chemotherapy agents (CAs), such as the cyclophosphamide (CYP) and ifosfamide (IFO) structural isomers, represent a unique threat. This research elucidated the fate of CYP and IFO during ozonation and advanced oxidation by hydroxyl radicals (HO•). Novel semi-batch reactors were used to determine the second-order rate constants for CYP and IFO with O3 and HO•. These reactors provided independent control of the oxidant exposure through continuous and constant aqueous ozone and peroxone (O3-H2O2) addition. The rate constants for transformation of CYP and IFO by ozone were 2.58 ± 0.40 M-1s-1 and 6.95 ± 0.21 M-1s-1, respectively, indicating that ozone alone is not suitable for treating CAs. Transformation of CYP and IFO by hydroxyl radicals was fast, with rate constants of 2.69(±0.17)×109 M-1s-1 and 2.73(±0.16)×109 M-1s-1, respectively. The major transformation products formed by O3 and HO attack consisted of the 4-hydroxy-, 4-keto-, dechloroethyl-, and imino- derivatives of CYP and IFO. Low yields of the active metabolites of the CAs, namely phosphoramide mustard and isophosphoramide mustard, were detected. These findings suggest that treated water may retain the ability to alkylate DNA and confer toxicity.

In Vitro and In Vivo Detection of Drug-induced Apoptosis Using Annexin V-conjugated Ultrasmall Superparamagnetic Iron Oxide (USPIO): A Pilot Study.

PURPOSE: To investigate the binding potential of newly developed Annexin V-conjugated ultrasmall superparamagnetic iron oxide (V-USPIO) for detection of drug-induced apoptosis in vitro and in vivo. METHODS: Apoptotic cells induced by camptothecin were incubated with or without Annexin V-USPIO at a concentration of 0.089 mmol Fe/L in vitro. T2 values of the two cell suspensions were measured by 0.47T nuclear magnetic resonance (NMR) spectrometer. Tumor-bearing mice were subjected to 1.5T MR scanner at 2 h after intraperitoneal injection of etoposide and cyclophosphamide. Following the pre-contrast T1- and T2-weighted imaging (0 h), the post-contrast scan was performed at 2, 4, 6 and 24 h after intravenous injection of Annexin V-USPIO (100 µmol Fe/kg). As a control, MRI was also obtained at 4 h after injection of USPIO without Annexin V. The ratio of tumor signal intensity (SI) on post-MRI for that on pre-MRI (Post/Pre-SI ratio) was calculated. After scanning, tumors were resected for pathological analysis to evaluate the distribution of iron and apoptotic cells. RESULTS: The suspension of apoptotic cells incubated with Annexin V-USPIO showed shorter T2 value than that without it. On T1-weighted imaging post/pre-SI ratio at 4 h after injection of Annexin V-USPIO showed 1.46, while after injection of USPIO without Annexin V was 1.17. The similar distribution of iron and apoptotic cells was observed in concordance with high signal intensity area on post-T1-weighted imaging. CONCLUSION: A newly developed Annexin V-USPIO could have the potential for detection of drug-induced apoptosis.

Crystallization of Cyclophosphamide Monohydrate During Lyophilization.

The purpose of this study was to investigate the phase behavior of cyclophosphamide (CPA) during various stages of lyophilization, with special emphasis on obtaining crystalline CPA monohydrate (CPA-MH) in the lyophilized product. Subambient differential scanning calorimetry and low-temperature X-ray diffractometry (LTXRD) were used to study the phase behavior of CPA solution (3.7% w/v). In situ lyophilization in LTXRD chamber was used to monitor the phase transitions occurring during the drying stages. Finally, the implications of these findings were confirmed by freeze-drying the aqueous solution in a laboratory-scale freeze-dryer. The results suggested that CPA remains amorphous during freeze concentration, with a Tg' of -50°C. However, its crystallization as CPA-MH can be induced by annealing the frozen solution between -5°C and -10°C. In situ lyophilization in LTXRD showed that the CPA-MH crystallized during annealing, rapidly dehydrated during primary drying, thereby causing structural collapse. The dehydration of CPA-MH can be prevented by lowering the escaping tendency of water molecules from the crystal lattice of CPA-MH by maintaining the chamber pressure to 300, 400, or 500 mTorr. This study highlights the relationship of process parameters used during lyophilization with the solid form of lyophilized CPA.