Voltammetric/UV-Vis study of temozolomide inclusion complexes with cyclodextrin derivatives.

Temozolomide (TMZ) - a chemotherapeutic agent possessing cytotoxic activity is used in single or combined therapies of human glioma. Difficulties in these applications, connected with low solubility and stability of temozolomide lead us to study the inclusion complexes between TMZ, and three cyclodextrins: ß-cyclodextrin (ßCD), monodeoxy-6-monoamino-ß-cyclodextrin hydrochloride (ßCDamine), and ß-cyclodextrin containing galactosamine and triazole ring in the side group (ßCDgal). The voltammetric and spectroscopy studies showed the improvement of the drug solubility and formation of stable complexes. Higuchi and Connors method was used to determine the solubilities of the drug in the presence of the selected cyclodextrins. Phase solubility diagrams showed increase of TMZ solubility and 1:1 stoichiometry of the complexes formed. The stability constant of TMZ- ßCDgal complex was pH - dependent, larger at pH 7.4 (corresponding to the pH of the body fluids), than at pH 5.5, characteristic for the cancer cells environment. ßCDgal ligand was an effective complexing agent for TMZ due to additional strong proton-acceptor π-π interactions between the triazole ring of the cyclodextrin and the ring of TMZ. The increased solubility and sustainability of TMZ complexes with ßCDgal allow to propose this cyclodextrin as a promising TMZ carrier for further studies in the biological cell environment.

Occupational exposure of pharmacy technicians and cleaning staff to cytotoxic drugs in Dutch hospitals.

Many studies into surface contamination of hospital environments have demonstrated that occupational exposure to cytotoxics through the dermal route remains a possible risk. In this study, we assess the actual dermal exposure of the hands of pharmacy technicians and cleaning personnel in a panel of hospitals performing tasks that pose a risk of exposure. We compare the dermal exposure to a tentative limit value for cyclophosphamide. Pharmacy technicians and cleaning personnel were asked for hand rinsing after performance of nine tasks previously identified as posing a risk of occupational exposure. All samples were analyzed for the presence and quantity of eight antineoplastic drugs. By using data on both the frequency of the performance of the tasks and the measured dermal contamination during these tasks, weekly exposure to the marker drug (cyclophosphamide) was calculated. In five Dutch hospitals, 70 hand rinse samples and 8 blanks were collected. These were analyzed and results were used to calculate weekly exposure. The tentative limit value used was 0.74 µg of cyclophosphamide. For cleaning personnel, all results remained below this threshold value. For pharmacy technicians, the compounding itself also remained well below the limit; however, the task involving preparatory work, as well as the checking of compounded drugs, had a 13% chance of exceeding the limit. All of the highest values were found when employees were not wearing gloves on these tasks. Cleaning personnel and pharmacy technicians compounding cytotoxic drugs in our study were sufficiently protected from occupational exposure. In contrast, pharmacy technicians who perform preparatory and finishing tasks (before and after the actual compounding) are not protected enough when they do not wear gloves.

Fastidious chemical decontamination after cyclophosphamide vial breakage in a compounding unit.

An important amount of cytotoxic drug may accumulate in the workplace following the breakage of a vial containing an anticancer drug. Thanks to the monthly monitoring of the surface contamination in our compounding unit, a strong increase of cyclophosphamide contamination was highlighted in the storage area following the breakage of the vial, despite application of the emergency procedure. This study presents an analysis of chemical decontamination in the context of massive contamination. Samples were taken on the floor and on the caster of a storage shelf where the vial broke. The residual contamination was measured with a liquid chromatography-mass spectrometry/mass spectrometry method. An admixture of 10-2 M sodium dodecyl sulfate and 70% isopropanol (SDS/IPA 8:2) was selected as the decontamination solution. High amounts of cyclophosphamide were retrieved. The initial contamination on the floor was over 20 ng/cm2. Three decontaminations with SDS/IPA were carried out at Day 61, Day 68, and Day 71. The amount of cyclophosphamide decreased to 0.45 ng/cm2 at D134. However, high values were still measured on the caster despite successive decontaminations, with a maximal value of 19.78 ng/cm2 observed at Day 106. Continuous monitoring in our unit led us to highlight the inefficiency of our emergency procedure to eliminate high cyclophosphamide contamination. The procedure involving the SDS/IPA admixture was more efficient on the floor compared to the caster, which is a different surface type and porosity. This work highlights the importance of improving the procedures of incident management using contamination monitoring and repeated decontamination procedures adapted to different contaminants and surfaces.

Terminalia bellirica extract induces anticancer activity through modulation of apoptosis and autophagy in oral squamous cell carcinoma.

Terminalia bellirica (TB) has been used in traditional Indian medical system, Ayurveda. However, the mechanism underlying the efficacy of the TB extract against oral squamous cell carcinoma (OSCC) is yet to be explored. The present study established a connecting link between the TB extract induced apoptosis and autophagy in relation to reactive oxygen species (ROS). Our study revealed, that gallic acid in the TB extract possess a strong free radical scavenging capacity contributing towards the selective anti-proliferative activity. Furthermore, TB extract markedly enhanced the accumulation of ROS that facilitated mitochondrial apoptosis through DNA damage, indicating ROS as the vital component in regulation of apoptosis. This effect was effectively reversed by the use of a ROS scavenger, N-acetyl cysteine (NAC). Moreover, it was observed to induce autophagy; however, it attenuated the autophagosome-lysosome fusion in Cal33 cells without altering the lysosomal activity. Pharmacological inhibitors of autophagy, namely, 3-methyladenine and chloroquine, were demonstarated to regulate the stage-specific progression of autophagy post treatment with the TB extract, favouring subsequent activation of apoptosis. These findings revealed, presence of gallic acid in TB extract below NOAEL value causes oxidative upset in oral cancer cells and promote programmed cell death which has a potential therapeutic value against oral squamous cell carcinoma.

Evaluating Ternary Metal Oxide (TMO) core-shell nanocomposites for the rapid determination of the anti-neoplastic drug Chlorambucil (Leukeran™) by electrochemical approaches.

A functional ternary nanocomposite (MnO2@NiFe2O4) with hierarchically grown flower-like core-shell assembly has been prepared through a simple two-step hydrothermal methodology. The crystallographic information of the sample was obtained via X-ray diffraction. The morphological details of spherical NiFe2O4 (core) and flower-like MnO2 (shell) were examined over FE-SEM and TEM; finally, the XPS confirms the successful formation of MnO2@NiFe2O4 ternary nanocomposite. The surface area of finely grown hierarchical MnO2 flowers on NiFe2O4 nanoparticles was measured through BET isothermal studies. The ternary nanocomposite was employed for the specific and sensitive detection of anticancer drug Chlorambucil. Under the well-optimized condition, the graphically plotted calibration curve was attained at MnO2@NiFe2O4/GCE towards the detection of anticancer drug Chlorambucil, which possesses the wider linear range of 0.025-574.5 µM, with the minimal detection limit of 4.68 nM. The feasibility of the sensitive layer has also been inspected on the pharmaceutical sample (oral tablets) and the biological matrix (human urine), whereas the appreciable spike recoveries were obtained.

Evaluating containment effectiveness of A2 and B2 biological safety cabinets.

PURPOSE: This study investigates the use of a canopy-connected recirculating class II type A2 biological safety cabinet (BSC) as an alternative to the B2 when preparing volatile, sterile compounded preparations. Selection of the appropriate BSC for processes that use subgram levels of volatile chemicals is difficult due to a lack of quantitative containment evidence by cabinet type. There is a perception that hazardous compounding must be done in a B2 cabinet due to the potential for vapors, and this study seeks to challenge that perception. METHODS: In total, 5 tests, 3 prequalification tests and 2 containment capability tests, were conducted on a single cabinet of each type at sterile compounding pharmacies. Prequalification tests were performed to verify that each BSC was operating properly. Each cabinet was certified to NSF-ANSI 49-2016, particle counted per ISO 14644-1:1999, and subjected to a qualitative video smoke study. Once these tests confirmed the expected working conditions, 2 containment capability tests were conducted. The containment testing included tracer gas testing per ASHRAE 110:2016 section 8.1.1 through 8.1.13, and cyclophosphamide sampling during sterile compounding of the drug material. RESULTS: Both cabinets passed all the prequalification tests. During the ASHRAE tracer gas testing the A2 cabinet was able to contain a tracer gas 92% to 160% as effectively as the B2 cabinet depending on the position of the gas ejection. During sterile compounding the airborne cyclophosphamide sampling captured samples of less than 1.0 ng at all locations for both the A2 and B2 cabinets. CONCLUSION: The data generated from this study demonstrate that use of an A2 for hazardous compounding can provide a comparable level of safety for the environment, users, and product while having less stringent airflow requirements relative to a B2. The simpler requirements for an A2 make them an appealing alternative as they have the potential to reduce the overall operating costs associated with a compounding pharmacy while maintaining safe levels of containment.

Solution stability of Captisol-stabilized melphalan (Evomela) versus Propylene glycol-based melphalan hydrochloride injection.

PURPOSE: The objective of this study was to compare the stability of recently approved Captisol-stabilized propylene glycol-free melphalan injection (Evomela™) against currently marketed propylene glycol-based melphalan injection. The products were compared as reconstituted solutions in vials as well as admixture solutions prepared from normal saline in infusion bags. METHODS: Evomela and propylene glycol-based melphalan injection were reconstituted in normal saline and organic custom diluent, respectively, according to their package insert instructions. The reconstituted solutions were diluted in normal saline to obtain drug admixture solutions at specific drug concentrations. Stability of the solutions was studied at room temperature by assay of melphalan and determination of melphalan-related impurities. RESULTS: Results show that based on the increase in total impurities in propylene glycol-based melphalan injection at 0.45 mg/mL, Evomela admixture solutions are about 5, 9, 15 and 29 times more stable at concentrations of 0.45, 1.0, 2.0 and 5.0 mg/mL, respectively. Results confirmed that reconstituted Evomela solution can be stored in the vial for up to 1 h at RT or for up to 24 h at refrigerated temperature (2-8 °C) with no significant degradation. After storage in the vial, it remains stable for an additional 3-29 h after preparation of admixture solution in infusion bags at concentrations of 0.25-5.0 mg/mL, respectively. In addition, Evomela solution in saline, at concentration of 5.0 mg/mL melphalan was bacteriostatic through 72 h storage at 2-8 °C. CONCLUSION: Formulation of melphalan with Captisol technology significantly improved stability compared to melphalan hydrochloride reconstituted with propylene-glycol based diluents.

Stability of busulfan solutions in polypropylene syringes and infusion bags as determined with an original assay.

PURPOSE: The stability of busulfan solution in 0.9% sodium chloride and stored in polypropylene syringes or infusion bags was evaluated. METHODS: Busulfan solutions (0.54 mg/mL) were prepared and transferred to 50-mL polypropylene syringes and 100- and 500-mL polypropylene infusion bags and stored at 2-8 and 23-27 °C. Chemical stability was measured using a stability-indicating, ultrahigh performance liquid chromatography coupled to mass spectrometry method. The stability of busulfan was assessed by measuring the percentage of the initial concentration remaining at the end of each time point of analysis. The initial busulfan concentration was defined as 100%. Stability was defined as retention of at least 90% of the initial busulfan concentration. A visual inspection of the samples for particulate matter, clarity, and color without instrumentation of magnification was conducted at each time point of analysis. RESULTS: The visual inspection demonstrated no influence of the storage container when busulfan infusions diluted in 0.9% sodium chloride injection were stored at 23-27 °C. No color change or precipitate was observed at this temperature; however, a rapid decrease of the busulfan content in all containers stored at room temperature was observed. Busulfan in syringes was chemically stable for 12 hours, while busulfan in infusion bags (100 and 500 mL) was stable only for 3 hours at 23-27 °C. CONCLUSION: Busulfan 0.54-mg/mL solution in 0.9% sodium chloride injection was physically and chemically stable for 30 hours when stored in 50-mL polypropylene syringes at 2-8 °C and protected from light.

Human metabolites and transformation products of cyclophosphamide and ifosfamide: analysis, occurrence and formation during abiotic treatments.

This study describes a gas chromatography-mass spectrometry analytical method for the analysis of cytostatic cyclophosphamide (CP), ifosfamide (IF) and their selected metabolites/transformation products (TPs): carboxy-cyclophosphamide (carboxy-CP), keto-cyclophosphamide (keto-CP) and 3-dechloroethyl-ifosfamide/N-dechloroethyl-cyclophosphamide (N-decl-CP) in wastewater (WW). Keto-cyclophosphamide, CP and IF were extracted with Oasis HLB and N-decl-CP and carboxy-CP with Isolute ENV+ cartridges. Analyte derivatization was performed by silylation (metabolites/TPs) and acetylation (CP and IF). The recoveries and LOQs of the developed method were 58, 87 and 103 % and 77.7, 43.7 and 6.7 ng L(-1) for carboxy-CP, keto-CP and N-decl-CP, respectively. After validation, the analytical method was applied to hospital WW and influent and effluent samples of a receiving WW treatment plant. In hospital WW, levels up to 2690, 47.0, 13,200, 2100 and 178 ng L(-1) were detected for CP, IF, carboxy-CP, N-decl-CP and keto-CP, respectively, while in influent and effluent samples concentrations were below LOQs. The formation of TPs during abiotic treatments was also studied. Liquid chromatography-high-resolution mass spectrometry was used to identify CP and IF TPs in ultrapure water, treated with UV and UV/H2O2. UV treatment produced four CP TPs and four IF TPs, while UV/H2O2 resulted in five CPs and four IF TPs. Besides already known TPs, three novel TPs (CP-TP138a, imino-ifosfamide and IF-TP138) have been tentatively identified. In hospital WW treated by UV/O3/H2O2, none of the target metabolites/TPs resulted above LOQs.

The leaching behavior of cyclophosphamide and ifosfamide from soil in the presence of co-contaminant--Mixture sorption approach.

Anticancer drugs (ACDs) exhibit high biological activity, they are cytotoxic, genotoxic, and are constantly released into the environment as a result of incomplete metabolism. Consequently they pose a serious threat to the environment and human health due to their carcinogenic, mutagenic and/or reproductive toxicity properties. Knowledge of their bioavailability, including their sorption to soils and their impact on the soil-groundwater pathway, is crucial for their risk assessment. Laboratory batch and column leaching tests are important tools for determining the release potential of contaminants from soil or waste material. Batch and column tests were carried out with soils differing in physicochemical properties, each spiked with cyclophosphamide (CK) or ifosfamide (IF). Moreover, due to the fact that environmental pollutants may occur as coexisting compounds in the soil the mobility evaluation for ACDs in the mixture with metoprolol (MET; ß-blocker) as a co-contaminant was performed. In order to assess appropriateness, the batch and column tests were compared. The release depended on the properties of both the soil and the presence of co-contaminants. The faster release was observed for coarse-grained soil with the smallest organic matter content (MS soil: 90% decrease in concentration until liquid-to-solid ratio (L/S) of 0.3 L kg(-1) for all tests' layout) than for loamy sand (LS soil: 90% decrease in concentration until ratio L/S of 0.75 L kg(-1)). ACDs are highly mobile in soil systems. Furthermore, the decrease of mobility of ifosfamide was observed with the presence of a co-contaminant (metoprolol) in both of the soils (in MS soil a decrease of 29%; in LS soil a decrease of 26%). The mobility of cyclophosphamide does not depend on the presence of a contaminant for MS soil, but also exhibits a decrease of 21% in LS soil.

HPLC-MS/MS method to measure trabectedin in tumors: preliminary PK study in a mesothelioma xenograft model.

BACKGROUND: Trabectedin is an anticancer agent registered for the second-line treatment of soft tissue sarcoma and ovarian cancer. No preclinical data are available on its tumor distribution, so a method for quantification in neoplastic tissues is required. METHODS/RESULTS: We validated an LC-MS/MS assay determining the recovery, sensitivity, linearity, precision and accuracy in mouse tumor and liver samples. The limit of quantification was 0.10 ng/ml with a curve range of 0.10-3.00 ng/ml (accuracy 96.1-102.1%). Inter-day precision and accuracy of QCs were 6.0-8.2 and 97.0-102.6% respectively. The method was applied in mesothelioma xenografts treated with therapeutic doses. CONCLUSION: The method was validated for measuring trabectedin in tissues. In a mesothelioma xenograft model, trabectedin distributed preferentially in tumor compared with liver.

Environmental contamination, product contamination and workers exposure using a robotic system for antineoplastic drug preparation.

Environmental contamination, product contamination and technicians exposure were measured following preparation of iv bags with cyclophosphamide using the robotic system CytoCare. Wipe samples were taken inside CytoCare, in the clean room environment, from vials, and prepared iv bags including ports and analysed for contamination with cyclophosphamide. Contamination with cyclophosphamide was also measured in environmental air and on the technicians hands and gloves used for handling the drugs. Exposure of the technicians to cyclophosphamide was measured by analysis of cyclophosphamide in urine. Contamination with cyclophosphamide was mainly observed inside CytoCare, before preparation, after preparation and after daily routine cleaning. Contamination outside CytoCare was incidentally found. All vials with reconstituted cyclophosphamide entering CytoCare were contaminated on the outside but vials with powdered cyclophosphamide were not contaminated on the outside. Contaminated bags entering CytoCare were also contaminated after preparation but non-contaminated bags were not contaminated after preparation. Cyclophosphamide was detected on the ports of all prepared bags. Almost all outer pairs of gloves used for preparation and daily routine cleaning were contaminated with cyclophosphamide. Cyclophosphamide was not found on the inner pairs of gloves and on the hands of the technicians. Cyclophosphamide was not detected in the stationary and personal air samples and in the urine samples of the technicians. CytoCare enables the preparation of cyclophosphamide with low levels of environmental contamination and product contamination and no measurable exposure of the technicians.

Molecular imaging coupled to pattern recognition distinguishes response to temozolomide in preclinical glioblastoma.

Non-invasive monitoring of response to treatment of glioblastoma (GB) is nowadays carried out using MRI. MRS and MR spectroscopic imaging (MRSI) constitute promising tools for this undertaking. A temozolomide (TMZ) protocol was optimized for GL261 GB. Sixty-three mice were studied by MRI/MRS/MRSI. The spectroscopic information was used for the classification of control brain and untreated and responding GB, and validated against post-mortem immunostainings in selected animals. A classification system was developed, based on the MRSI-sampled metabolome of normal brain parenchyma, untreated and responding GB, with a 93% accuracy. Classification of an independent test set yielded a balanced error rate of 6% or less. Classifications correlated well both with tumor volume changes detected by MRI after two TMZ cycles and with the histopathological data: a significant decrease (p < 0.05) in the proliferation and mitotic rates and a 4.6-fold increase in the apoptotic rate. A surrogate response biomarker based on the linear combination of 12 spectral features has been found in the MRS/MRSI pattern of treated tumors, allowing the non-invasive classification of growing and responding GL261 GB. The methodology described can be applied to preclinical treatment efficacy studies to test new antitumoral drugs, and begets translational potential for early response detection in clinical studies.

Syringe plunger contamination by hazardous drugs: a comparative study.

AIM: The purpose of this study was to perform a comparative cyclophosphamide contamination level test with Becton Dickinson® syringe plungers with Phaseal® Closed System Transfer Devices and Equashield® syringe plungers under routine oncological compounding conditions. METHOD: The ChemoGlo™ sampling kit and analysis services were used to test for cyclophosphamide contamination levels on the syringe plungers of Becton Dickinson® Phaseal and Equashield® syringes that underwent cycles of drug transfer in a Forma Class II, 2A Biological Safety Cabinet. Prior to testing, the syringes were divided into three equal groups for the Equashield® and Becton Dickinson® syringes. A 50 mL aliquot of cyclophosphamide was drawn into each syringe and then injected back into the cyclophosphamide vial. This drug transfer procedure was immediately repeated twice for the syringes in group 1, four times for group 2, and eight times for group 3. After the completion of the drug transfers with the Equashield® and Becton Dickinson® Phaseal syringes, the plungers were retracted back to the nominal syringe marking, and a wipe test of the exposed plunger was done using the ChemoGlo™ sampling kit. RESULTS: Significant contamination levels of 2000 ng and greater were detected on most Becton Dickinson® syringe plungers with Phaseal® Closed System Transfer Devices, whereas all Equashield® syringes remained uncontaminated at undetectable levels.

Leukemia from dermal exposure to cyclophosphamide among nurses in The Netherlands: quantitative assessment of the risk.

Several studies showed that oncology nurses are exposed to antineoplastic drugs via the skin during daily activities. Several antineoplastic drugs (including cyclophosphamide) have been classified as carcinogenic to humans. This study aims to assess the leukemia risk of occupational exposure to cyclophosphamide. Average task frequencies from the population of oncology nurses in the Netherlands and task-based dermal exposure intensities were used to calculate oncology nurses' dermal exposure levels. A dermal absorption model in combination with a physiologically based pharmacokinetic model was used to assess the delivered dose of cyclophosphamide and its active metabolites in the bone marrow. This delivered dose was subsequently related to pharmacodynamic and epidemiological information from a longitudinal study with cyclophosphamide-treated patients to estimate the excess lifetime leukemia risk at age 80 for Dutch oncology nurses after 40 years of exposure to cyclophosphamide. The excess lifetime leukemia risk at age 80 of an exposed oncology nurse after 40 years of dermal exposure to cyclophosphamide was estimated to be 1.04 per million oncology nurses. This risk could potentially increase to a maximum of 154 per million if a nurse performs all cyclophosphamide-related tasks with the maximum frequency (as observed in this population) and is exposed to maximum exposure intensities for each task without using protective gloves for 40 years. This study indicates that the risk of an oncology nurse in a Dutch hospital with an average dermal exposure to cyclophosphamide is well below the maximum tolerable risk of one extra death from cancer per 250 deaths after 40 years of occupational exposure, and that this level is not exceeded in a worst-case scenario.