Microtubule rescue control by drugs and MAPs examined with in vitro pedestal assay.

Microtubules are essential cytoskeletal polymers, which exhibit stochastic transitions between assembly and disassembly, known as catastrophes and rescues. Understanding of catastrophes, rescues, and their control by drugs and microtubule associated proteins (MAPs) has been informed by in vitro reconstitutions of microtubule dynamics. In such experiments microtubules are typically observed on a flat surface of the coverslip. In contrast, we have recently proposed a modified setup in which microtubules assemble from stabilized seeds, overhanging from microfabricated pedestals, so that their dynamic extensions are fully isolated from contact with the coverslip. This assay allows to eliminate potential artifacts, which may substantially affect the frequency of microtubule rescues in vitro. Here we use the pedestal assay to study the sensitivity of microtubules to paclitaxel, one of the best-known inhibitors of microtubule dynamics. By comparing observations in the conventional and the pedestal assays, we find that microtubule dynamics are substantially more sensitive to paclitaxel when the polymers can contact the coverslip. We interpret this as a consequence of the coverslip-induced microtubule assembly perturbation, leading to formation of lattice with defects, and thereby enhancing the efficiency of paclitaxel binding to microtubules in the conventional assay. To test this idea, we use vinblastine, another small-molecule inhibitor, which had been previously shown to cause microtubule growth perturbations. We find that in the pedestal assay vinblastine sensitizes microtubules to paclitaxel to the level, observed in the conventional assay. Interestingly, a minimal fragment of MAP called CLASP2, a previously characterized rescue factor, has a strong effect on microtubule rescues, regardless of the type of assay. Overall, our study underscores the role of microtubule damage in promoting rescues and highlights the utility of the in vitro pedestal assay to study microtubule dynamics modulation by tubulin inhibitors and MAPs.

LC-MS-MS Determination of Cytostatic Drugs on Surfaces and in Urine to Assess Occupational Exposure.

The ever-increased usage of cytostatic drugs leads to high risk of exposure among healthcare workers. Moreover, workers are exposed to multiple compounds throughout their lives, leading to cumulative and chronic exposure. Therefore, multianalyte methods are the most suitable for exposure assessment, which minimizes the risks from handling cytostatic drugs and ensures adequate contamination containment. This study describes the development and full validation of two liquid chromatography-tandem mass spectrometry methods for the detection of gemcitabine, dacarbazine, methotrexate, irinotecan, cyclophosphamide, doxorubicinol, doxorubicin, epirubicin, etoposide, vinorelbine, docetaxel and paclitaxel in working surfaces and urine samples. The urine method is the first to measure vinorelbine and doxorubicinol. For surfaces, limits of detection (LOD) and limits of quantification (LOQ) were 5-100 pg/cm2, and linearity was achieved up to 500 pg/cm2. Inaccuracy was between -11.0 and 8.4%. Intra-day, inter-day and total imprecision were <20%, except for etoposide and irinotecan (<22.1%). In urine, LOD and LOQ were 5-250 pg/mL, with a linear range up to 1,000-5,000 pg/mL. Inaccuracy was between -3.8 and 14.9%. Imprecision was <12.4%. Matrix effect was from -58.3 to 1,268.9% and from -66.7 to 1,636% in surface and urine samples, respectively, and extraction efficiency from 10.8 to 75% and 47.1 to 130.4%, respectively. All the analytes showed autosampler (6°C/72 h), freezer (-22°C/2 months) and freeze/thaw (three cycles) stability. The feasibility of the methods was demonstrated by analyzing real working surfaces and patients' urine samples. Contamination with gemcitabine, irinotecan, cyclophosphamide, epirubicin and paclitaxel (5-4,641.9 pg/cm2) was found on biological safety cabinets and outpatients' bathrooms. Analysis of urine from patients under chemotherapy identified the infused drugs at concentrations higher than the upper LOQ. These validated methods will allow a comprehensive evaluation of both environmental and biological contamination in hospital settings and healthcare workers.

Rational Discovery of Microtubule-Stabilizing Peptides.

Microtubule (MT) stabilization is an attractive pharmacological strategy to hamper the progress of neurodegenerative diseases. In this regard, seeking peptides with MT-stabilizing properties has awoken great interest. This work reports the rational discovery of two structurally related MT-stabilizing octapeptides using a combination of protein-peptide docking, conventional molecular dynamics, Gaussian accelerated molecular dynamics (GaMD), and tubulin polymerization assays. FASTA sequences for ∼1000 peptides were crafted from single and double mutants of davunetide (NAP) and docked against the Taxol (TX) site on an octameric MT model representing a portion of the MT wall. Docked peptides were rescored after MM minimization and binding free energy refinement through single-point MM/GBSA calculations. The 60 best-ranked peptides were subjected to 50 ns MD simulations on peptide-MT complexes at the terminal TX site in the octameric Tau-MT model resulting in 11 complexes with occupancies greater than 99% and peptide-protein binding free energies less than -40 kcal/mol. Selected peptides were then examined through 300 ns GaMD simulations in complexes containing two identical ligands at the terminal and intermediate TX sites in the Tau-MT model to account for the differential association of MT-binding peptides to different regions of the MT structure. Six candidates showed a favorable MT-binding potential based on the analysis of interaction frequencies and relative mobilities of the complex components, suggesting a pivotal role of Arg278, Gln281, and Arg369 residues for peptides recognition. Four candidates were predicted to preserve an adequate balance of longitudinal and lateral interactions between tubulin dimers in peptide-MT complexes such that MT-stabilizing effects could be expected. MT polymerization experiments confirmed that four peptides (HAPVSIHQ, NYPVSIHQ, NWPVSIWQ, HAPVSIIQ) exhibit MT-stabilizing activity in vitro with NWPVSIWQ (P43) and HAPVSIIQ (P52) being the most active. Tryptophan quenching assays verified that P43 and P52 bind to nonpolymeric tubulin, whereas viability experiments on HEK cells confirmed their safety to pursue future pharmacological studies. The results herein presented are valuable to making progress in the rational design of MT-stabilizing peptides.

Development and application of a liquid chromatography coupled to mass spectrometry method for the simultaneous determination of 23 antineoplastic drugs at trace levels.

The goal of this study was to develop a method for the simultaneous quantification of 23 commonly used antineoplastic drugs in a hospital pharmacy, using ultra-high pressure liquid chromatography separation coupled to tandem mass spectrometry detection (UHPLC-MS/MS). The following drugs were investigated: 5-fluorouracil, cytarabine, ganciclovir, gemcitabine, dacarbazine, methotrexate, pemetrexed, busulfan, topotecan, rentitrexed, ifosfamide, cyclophosphamide, etoposide, irinotecan, doxorubicin/epirubicin, vincristine, docetaxel, paclitaxel, daunorubicin, idarubicin, vinblastine, oxaliplatin and carboplatin. The chromatographic separation was performed on a phenyl-hexyl column (2.1 ×100 mm, 1.7 µm) with a gradient elution of methanol and water containing 10 mM ammonium formate adjusted to pH 4.9. All compounds were analyzed in less than 13 min and detected with a triple quadrupole mass spectrometer operating in MRM mode. Limits of detection (LODs) and limits of quantification (LOQs) were comprised between 0.01 and 5 ng.mL-1, and between 0.5 and 5 ng.mL-1, respectively. Accuracies ranged between 117% and 83% at the LOQ, intermediate and upper LOQ concentrations, with relative standard deviations (RSD) inferior to 8%, for all the antineoplastic drugs. Finally, the UHPLC-MS/MS method was successfully applied to the analysis of surface samples to evaluate the chemical contamination by these highly toxic compounds in a chemotherapy preparation unit in a hospital pharmacy with the purpose of monitoring the exposure of health care professionals.

Paclitaxel use in pregnancy: neonatal follow-up of infants with positive detection of intact paclitaxel and metabolites in meconium at birth.

Paclitaxel is often excluded during pregnancy for women with breast cancer due to limited neonatal follow-up. We confirmed in utero fetal Paclitaxel exposure for 8 newborns. Birth details and follow-up to 36 months of age is reported. Meconium samples from newborns exposed to chemotherapy were screened by liquid chromatography-high resolution mass spectrometry while blinded to maternal treatment during pregnancy. Newborn information at birth and annually was obtained. Mean gestational age (GA) at cancer diagnosis and start of chemotherapy was 8.7 + 6.2 weeks and 17.1 ± 3.5 weeks. Paclitaxel was started at a mean GA of 27.0 ± 5.8 weeks. Paclitaxel followed Doxorubicin/Cyclophosphamide in 6 cases, 5-Fluouracil/Doxorubicin/Cyclophosphamide in 1, and was used alone in 1. Mean number of days between Paclitaxel and birth was 23 ± 15. Identification of Paclitaxel and/or metabolites was made in all meconium from paclitaxel-exposed fetuses. Birthweight was < 10% for GA in 3 infants. Three anomalies occurred: mild hip dysplasia without further treatment and mitral valve stenosis. The third child was diagnosed with Cleidocranial Dysostosis, a familial anomaly. Mean age at pediatric follow-up is 18.7 + 9.3 months. Pediatricians report eczema and recurrent otitis media in 1 child, iron deficiency anemia and upper respiratory infection in 2. One child is < 10% for height and weight at 15 months. All are meeting developmental milestones at median age of 18.7 months, range: 6-36 months. CONCLUSION: Up to 3 years of age, follow-up of neonates exposed to Paclitaxel in utero is reassuring. Continued observation of neonatal development is essential. WHAT IS KNOWN: • Chemotherapy during the second and third trimester of pregnancy does not result in an increase in congenital malformations or developmental delay. • In non-human primate studies by Van Calsteren et al., variable plasma and/or tissue concentrations of taxanes, carboplatin, and trastuzumab were encountered in the fetal compartment. • Pilot data reported by the current investigators proved that paclitaxel crosses the human placenta. WHAT IS NEW: • This current article provides medical and developmental follow up on the newborns from this exposure for 3 years after birth.

Optimization of Taxol Extraction Process Using Response Surface Methodology and Investigation of Temporal and Spatial Distribution of Taxol in Taxus mairei.

Taxus mairei is an important source for industrial extraction of taxol in China. However, the standard and steps of extraction are currently not uniform, which seriously affects the taxol yield. In the present study, the influence of four factors (methanol concentration, solid-liquid ratio, ultrasonic extraction temperature, and ultrasonic extraction time) on the taxol yield was successively explored in T. mairei. A response surface methodology (RSM) was used to optimize the extraction process based on the single-factor experiments above. The optimal conditions were as follows: methanol concentration was 90%, solid-liquid ratio was 1:15 (g/mL), ultrasonic extraction temperature was 40 °C and ultrasonic extraction time was 60 min. Moreover, the twigs and needles from T. mairei with different tree ages were treated by the optimum extraction process, which further revealed temporal and spatial distribution of taxol in the reproducible tissues. Interestingly, the taxol content was relatively higher in needles of T. 'Jinxishan' (a cultivar from T. mairei with yellow aril, FY), but was less in FY twigs. The accumulation of taxol in twigs and leaves of females (with red aril, FR) was significantly higher than that of males (M); however, the content showed a decreasing trend with the increasing tree ages. Therefore, it is suitable to increase the proportion of female trees especially the FY leaves as raw materials for the industrial production of taxol from T. mairei, and the tree ages should be better controlled at 3-7 years.

Occupational exposure to antineoplastic drugs: what about hospital sanitation personnel?

OBJECTIVE: Occupational exposure to antineoplastic drugs (ANPs) occurs mainly through dermal contact. Our study was set up to assess the potential exposure of hospital sanitation (HS) personnel, for whom almost no data are available, through contamination of surfaces they regularly touch. METHODS: In the oncology departments of two hospitals around Montreal, surface wipe samples of 120-2000 cm2 were taken at 10 sites cleaned by the HS personnel and five other sites frequently touched by nursing and pharmacy personnel. A few hand wipe samples were collected to explore skin contamination. Wipes were analyzed by ultra-performance liquid chromatography tandem-mass spectrometry for 10 ANPs. RESULTS: Overall, 60.9% of 212 surface samples presented at least one ANP above the limits of detection (LOD). Cyclophosphamide and gemcitabine were most often detected (52% and 31% of samples respectively), followed by 5-fluorouracil and irinotecan (15% each). Highest concentrations of five ANPs were found in outpatient clinics on toilet floors (5-fluorouracil, 49 ng/cm2; irinotecan, 3.6 ng/cm2), a perfusion pump (cyclophosphamide, 19.6 ng/cm2) and on a cytotoxic waste bin cover (gemcitabine, 4.97 ng/cm2). Floors in patient rooms had highest levels of cytarabine (0.12 ng/cm2) and methotrexate (6.38 ng/cm2). Hand wipes were positive for two of 12 samples taken on HS personnel, seven of 18 samples on nurses, and two of 14 samples on pharmacy personnel. CONCLUSIONS: A notable proportion of surfaces showed measurable levels of ANPs, with highest concentrations found on surfaces cleaned by HS personnel, who would benefit from appropriate preventive training. As potential sources of worker exposure, several hospital surfaces need to be regularly monitored to evaluate environmental contamination and efficacy of cleaning.

Evaluation of the efficacy and safety of a new formulation-lipid emulsion-based PTX injection: Pharmacokinetics, tissue distributions and anticancer effect on human gastric cancer cells in vitro.

Paclitaxel (PTX) is one of the most widely used chemotherapeutic agents. The commercial PTX formulation was based on Cremophor EL and ethanol owing to its poor aqueous solubility. However, Cremophor EL has been shown to cause toxic effects such as life-threatening anaphylaxis. In our study, we diluted PTX in a commercially available 20% (w/v) lipid emulsion (Lip-PTX) in order to avoid Cremophor EL. The purpose of this study was to evaluate the pharmacokinetics and tissue distributions between Lip-PTX and PTX injection. We also investigated the effects of Lip-PTX and PTX injection on human gastric cancer cells HGC-27 by MTT assay. The apoptosis was detected by flow cytometry with Annexin V/propidium iodide (PI) double staining. Furthermore, the safety such as acute toxicity was also assessed. The results showed that PTX in Sprague-Dawley rats administered Lip-PTX exhibited extended half-life, increased clearance (P < 0.05) and smaller area under the concentration-time curve compared with PTX injection and there was little significant difference in the distribution of PTX in Sprague-Dawley rats or tumor-bearing mice between Lip-PTX and PTX injection. The cells treated with Lip-PTX had a higher percentage of apoptosis and a higher G2 /M phase ratio, which indicated that the anticancer effect of Lip-PTX was significantly better than that of PTX injection. Moreover, our study highlighted the safety of Lip-PTX. This study demonstrated the feasibility and potential advantages of Lip-PTX for clinical therapy.

Validated HPLC method for paclitaxel determination in PLGA submicron particles conjugated with α-fetoprotein third domain: Sample preparation case study.

OBJECTIVES: The goal of this study was to develop sample preparation method and validate the HPLC method for precise determination of paclitaxel (Ptx) in PLGA submicron particles conjugated with protein vector molecule. METHODS: Ptx loaded PLGA submicron particles were formulated by a single emulsification method. PLGA submicron particles were conjugated with alpha fetoprotein third domain (rAFP3d) via standard carbodiimide technique. The obtained conjugate was analyzed using 1525 binary pump and 2487 UV-VIS detector system (Waters, USA) and Reprosil ODS C-18 analytical column with the dimensions of 150mm×4.6mm ID×5µm (Dr. Maisch GmbH, Germany). Sample preparation method was developed utilizing guard cartridge with С18 stationary phase (Phenomenex, USA). HPLC method was validated according to the international conference on harmonization guidelines. RESULTS: Efficient sample preparation was achieved using 4% of DMSO pre-dissolution, following by 10min of centrifugation at 4500g. Ptx determination was performed using acetonitrile/0.1% phosphoric acid (50:50 v/v) mobile phase at a flow rate of 1.0mL/min, injection volume of 10µL, and at 227nm. The developed method showed linearity, accuracy and precision in the range from 0.03 to 360µg/mL, with LOD and LOQ values of 0.005 and 0.03µg/mL, respectively. The intra- and inter-day precisions presented RSD values of lower than 2%. CONCLUSION: The validated method was successfully applied to calculate Ptx encapsulation efficacy and drug loading in the developed formulation.

Development and validation of a rapid RP-HPLC method for simultaneous quantification of paclitaxel and cetuximab in immunoliposomes.

The development of rational therapies against complex diseases, such as cancer, has increased in the past few years due to the advances of 'omics' technologies. Concomitantly, several efforts have been made to design sophisticated drug delivery systems in order to increase specificity and drug accumulation in tumor sites. The complexity of these drug delivery systems highlights the need for suitable analytical methods to determine encapsulation/conjugation efficiency of drugs and molecules responsible for the targeted delivery. Therefore, this study focuses on the development and validation of a RP-HPLC-DAD methodology for concurrent quantification of paclitaxel (PTX) and cetuximab (CTX) in immunoliposomes. Chromatographic separation was achieved using a wide pore C8 column, and a gradient mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in Milli-Q water/acetonitrile/isopropanol with a flow rate of 1 mL min-1. Drug peaks were fully separated and detected at 280 nm using UV detector. The method was validated according to ICH and FDA guidelines in terms of specificity and forced degradation studies, system suitability, linearity, limit of detection, limit of quantification, repeatability, intermediate precision, accuracy, robustness, and short-term stability. The developed method was linear over the concentration range of 37.5-150 µg mL-1 of PTX and 75-300 µg mL-1 of CTX. All parameters evaluated satisfied the acceptance criteria, according to both FDA and ICH guidelines. The applicability of the analytical method was assessed following the development of PTX-loaded immunoliposomes conjugated with CTX. Thus, the present study shows a novel, simple, stability-indicating and suitable method to quantify simultaneously PTX and CTX in immunoliposomes.

Simultaneous Determination of Docetaxel and Celecoxib in Porous Microparticles and Rat Plasma by Liquid-Liquid Extraction and HPLC with UV Detection: in vitro and in vivo Validation and Application.

PURPOSE: A simple, rapid, sensitive, and reliable HPLC method with UV detection was developed and validated for simultaneous quantitation of docetaxel and celecoxib and paclitaxel for dissolution characterization and pharmacokinetic studies. METHODS: The HPLC assay was performed isocratically on a reversed-phase C18 µ-Bondapack column using a mobile phase of acetonitrile:water (45:55, v/v) at a flow rate of 1.2 mL/min, and the analytes were detected at 230 nm. Paclitaxel was used as an internal standard for analysis of plasma samples following simple liquid-liquid extraction with n-hexane:isoamyl alcohol (97:3). The method was validated for specificity, linearity, sensitivity, precision, accuracy, robustness, and in vitro-in vivo application. RESULTS: The retention times for docetaxel, paclitaxel, and celecoxib were 10.94, 12.4, and 16.81 min, respectively. The standard curves covering 0.1-1 µg/mL and 0.05-4 µg/mL were linear using dissolution medium and rat plasma, respectively. The limit of quantitation of the method was 50 ng/mL using 100 µL of rat plasma sample and injection of 50 µL of the residue. Within- and between-day precision and accuracy did not exceed 16.86% and 12.10%, respectively. This validated method was successfully used to quantify docetaxel and celecoxib simultaneously in the release study of docetaxel- celecoxib -loaded porous microparticles and pharmacokinetics studies. The methods were found to be simple, specific, precise, accurate, and reproducible. In this study, paclitaxel was used as the internal standard while dexamethasone, flutamide, and budesonide proved suitable alternative as an internal standard. CONCLUSION: Since docetaxel and celecoxib could be co-administered for the treatment of a wide range of cancers such as non-small cell lung carcinoma, the developed method is particularly advantageous for routine therapeutic drug monitoring and pharmacokinetic studies of these drugs.

Whole fungal elicitors boost paclitaxel biosynthesis induction in Corylus avellana cell culture.

Paclitaxel is an effective natural-source chemotherapeutic agent commonly applied to treat a vast range of cancers. In vitro Corylus avellana culture has been reported as a promising and inexpensive system for paclitaxel production. Fungal elicitors have been made known as the most efficient strategy for the biosynthesis induction of secondary metabolites in plant in vitro culture. In this research, C. avellana cell suspension culture (CSC) was exposed to cell extract (CE) and culture filtrate (CF) derived from Camarosporomyces flavigenus, either individually or combined treatment, in mid and late log phase. There is no report on the use of whole fungal elicitors (the combined treatment of CE and CF) for the elicitation of secondary metabolite biosynthesis in plant in vitro culture. The combined treatment of CE and CF significantly led to more paclitaxel biosynthesis and secretion than the individual use of them. Also, multivariate statistical approaches including stepwise regression (SR), ordinary least squares regression (OLSR), principal component regression (PCR) and partial least squares regression (PLSR) were used to model and predict paclitaxel biosynthesis and secretion. Based on value account for (VAF), root mean square error (RMSE), coefficient of determination (R2), mean absolute percentage error (MAPE) and relative percent difference (RPD) can be concluded that mentioned regression models effectively worked only for modeling and predicting extracellular paclitaxel portion in C. avellana cell culture.

Development and validation of a liquid chromatography tandem mass spectrometry quantification method for 14 cytotoxic drugs in environmental samples.

RATIONALE: Cytotoxic drug preparation in hospital pharmacies is associated with chronic occupational exposure leading to a risk of adverse effects. The objective was to develop and validate a quantification method for the following cytotoxic drugs in environmental wipe samples: cyclophosphamide, ifosfamide, cytarabine, dacarbazine, docetaxel, paclitaxel, doxorubicin, epirubicin, etoposide, 5-fluorouracil, gemcitabine, irinotecan, methotrexate and pemetrexed. METHODS: The quantification method was developed using liquid chromatography coupled to tandem mass spectrometry and a wiping technique using viscose swabs. Linearity, accuracy, precision, limit of quantification, specificity and stability were assessed, from swab desorbed solution, to validate the analytical method, with respect to ICH guidelines. Environmental samples were collected by wiping five work surfaces of 225 cm2 with viscose swabs, during three days. RESULTS: The quantification method was linear over the calibration range with a lower limit of quantification ranging from 0.5 to 5.0 ng mL-1 depending on the cytotoxic drug. The intra-day and inter-day relative biases were below 1.5% and 13.5%, respectively. This method was successfully applied to surface-wipe sampling and environmental contaminations ranged from 0.7 to 1840.0 ng cm-2 for the most contaminated areas. CONCLUSIONS: This quantification method for 14 cytotoxic drugs was successfully applied to environmental contamination monitoring and could therefore be a useful tool for monitoring and toxicological studies.

Development and validation of a HPLC-UV method for the simultaneous detection and quantification of paclitaxel and sulforaphane in lipid based self-microemulsifying formulation.

Paclitaxel (PTX) and sulforaphane (SFN) are known anticancer molecules. Their activity was found to be potentiated when tested concurrently. Only recently, however, a novel SFN enabled PTX self-microemulsifying formulation (SMEDDS) was developed for their simultaneous delivery. This necessitated the development of an analytical method for the simultaneous detection and quantitation of PTX and SFN. In this study, a simple and sensitive isocratic high performance liquid chromatography-ultraviolet (HPLC-UV) analytical method was developed and validated per International Conference on Harmonization guidelines to satisfy this objective. Its application was demonstrated when quantifying the amount of PTX and SFN released from the SMEDDS in various dissolution media. The separation of the analytes was performed with the aid of a reversed phase C18 column at ambient temperature using a 60:40 mixture of acetonitrile and KH2PO4 buffer (pH 5.0) as the mobile phase. PTX and SFN peaks were detected at 202 nm with high resolution without interference from excipients. This method showed linearity within 2.5-100 µg/mL range with r2 > 0.999. The limit of detection and lower limit of quantitation were 0.1638 and 0.4964 µg/mL for PTX and 0.4419 and 1.3389 µg/mL for SFN, respectively. A total of 98-101% of the injected samples was recovered with RSD of 0.06-0.68% indicating the suitability of the method for the simultaneous detection and quantitation of the molecules in dissolution media.

Herb-Drug Interaction Potential of Licorice Extract and Paclitaxel: A Pharmacokinetic Study in Rats.

BACKGROUND AND OBJECTIVES: Licorice is the dried roots and rhizomes of Glycyrrhiza uralensis Fisch (Leguminosae), which is often used with paclitaxel to alleviate paclitaxel-induced pain in clinics. However, the herb-drug interaction between licorice and paclitaxel is still unknown. Our study evaluates the effects of oral licorice on the paclitaxel in rats via pharmacokinetic studies. METHODS: A simple and rapid ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to determine paclitaxel in rat. SD rats were randomly divided into 3 groups of 6 animals each as follows: two groups of rats that were pretreated with a daily gavage of licorice (3 g/kg) for 1 or 14 successive days; Control group that was administered distilled water. All rats were then intravenously administered with paclitaxel (3 mg/kg). RESULTS: The results showed that 14 days pretreatment of licorice could decrease the area under the curve (AUC0-t) (from 7483.08 ± 528.78 to 6679.12 ± 266.56 mg/L × h) (P < 0.01), and increase the total clearance (CL) (from 0.36 ± 0.02 to 0.39 ± 0.02 L/h/kg) of paclitaxel (P < 0.01). However, a single co-administration of licorice did not significantly alter the pharmacokinetic parameters of paclitaxel, such as AUC0-t (from 7483.08 ± 528.78 to 7201.24 ± 292.76 mg/L × h) (P > 0.05) and CL (from 0.36 ± 0.02 to 0.36 ± 0.01 L/h/kg) (P > 0.05). CONCLUSIONS: The results will contribute to better use of licorice in the adjunctive therapy and provide information to study the interaction between herbs and chemotherapy.

Selection of fluorescent dye for tracking biodistribution of paclitaxel in live imaging.

Fluorescence imaging is widely used to determine biodistribution of drugs in mice. However, the dye distribution may not be able to exactly reflect the true distribution of drug molecules. We synthesized PTX-Cy5.5 and mPEG-PLA-Cy5.5, and then prepared dye-loaded nanoparticles (NPs) (Cy5.5, DiR, PTX-Cy5.5, and mPEG-PLA-Cy5.5), dye and PTX co-loaded NPs, and PTX-loaded NPs, respectively. The particle sizes of resulting NPs were between 42.7 nm and 68.8 nm, and Zeta potential was between -0.86 mV and -8.49 mV. The biodistribution of fluorescent NPs (dye-loaded NPs and dye and PTX co-loaded NPs) on Bel-7402 tumor-bearing mice was studied via in vivo fluorescence imaging assays, results of which suggested that Cy5.5 loaded NPs and Cy5.5 conjugates (PTX-Cy5.5 and mPEG-PLA-Cy5.5) formulated NPs can reflect the tissue distribution of PTX whether it was incorporated or not. However, DiR failed to reflect true tissue distribution of PTX unless it was co-loaded with PTX. Based on these results, a guidance for the selection of dyes in drug distribution investigations and disease-targeted treatment was presented.

Quantitative and qualitative control of antineoplastic preparations: Gravimetry versus HPLC.

This article compares gravimetry vs. high-performance liquid chromatography (HPLC) as quality control (QC) methods for paclitaxel, docetaxel and oxaliplatin preparations. We aimed at assessing the preparation method reliability in our hospital, evaluating compounding accuracy and estimating the influence of personnel training and standardized homogenization on compounding accuracy. Agreement, correlation, concordance, accuracy and precision between methods were evaluated for each drug. Conforming preparation percentages (CPs) at different tolerance limits (TLs) and compounding accuracy were calculated for each method and drug. Compounding accuracy was compared before and after personnel training and standardized homogenization implantation. SPSS v 20.0 and Ene v 2.0 were used. A total of 222 samples (58 docetaxel, 95 paclitaxel and 69 oxaliplatin) were analyzed. Gravimetry and HPLC are comparable methods. Overall CP was 81% for gravimetry at 10% TL and 85% for HPLC at 15% TL. Compounding accuracy is shown to be good for all methods and drugs. Homogenization optimization and personnel training make measurements more accurate for docetaxel and paclitaxel HPLC, but seem to worsen accuracy for docetaxel gravimetry. Gravimetry has shown to be a good alternative to HPLC for routine QC. Coupling with electronic methods should be considered in the future.

Development and validation of a rapid and sensitive UPLC-MS/MS assay for simultaneous quantification of paclitaxel and cyclopamine in mouse whole blood and tissue samples.

The prominent stromal compartment surrounds pancreatic ductal adenocarcinoma and protects the tumor cells from chemo- or radiotherapy. We hypothesized that our nano formulation carrying cyclopamine (CPA, stroma modulator) and paclitaxel (PTX, antitumor agent) could increase the permeation of PTX through the stromal compartment and improve the intratumoral delivery of PTX. In the present study a sensitive, reliable UPLC-MS/MS method was developed and validated to quantify PTX and CPA simultaneously in mouse whole blood, pancreas, liver and spleen samples. Docetaxel was used as the internal standard. The method demonstrated a linear range of 0.5-2000 ng/mL for whole blood and tissue homogenates for both PTX and CPA. The accuracy and precision of the assay were all within ±15%. Matrix effects for both analytes were within 15%. Recoveries from whole blood, liver, spleen and pancreas homogenates were 92.7-105.2% for PTX and 72.8-99.7% for CPA. The stability was within ±15% in all test biomatrices. The validated method met the acceptance criteria according to US Food and Drug Administration regulatory guidelines. The method was successfully applied to support a pharmacokinetic and biodistribution study for PTX and CPA in mice biomatrices.

Past-in-the-Future. Peak detection improves targeted mass spectrometry imaging.

Mass spectrometry imaging is a valuable tool for visualizing the localization of drugs in tissues, a critical issue especially in cancer pharmacology where treatment failure may depend on poor drug distribution within the tumours. Proper preprocessing procedures are mandatory to obtain quantitative data of drug distribution in tumours, even at low intensity, through reliable ion peak identification and integration. We propose a simple preprocessing and quantification pipeline. This pipeline was designed starting from classical peak integration methods, developed when "microcomputers" became available for chromatography, now applied to MSI. This pre-processing approach is based on a novel method using the fixed mass difference between the analyte and its 5 d derivatives to set up a mass range gate. We demonstrate the use of this pipeline for the evaluating the distribution of the anticancer drug paclitaxel in tumour sections. The procedure takes advantage of a simple peak analysis and allows to quantify the drug concentration in each pixel with a limit of detection below 0.1 pmol mm-2 or 10 µg g-1. Quantitative images of paclitaxel distribution in different tumour models were obtained and average paclitaxel concentrations were compared with HPLC measures in the same specimens, showing <20% difference. The scripts are developed in Python and available through GitHub, at github.com/FrancescaFalcetta/Imaging_of_drugs_distribution_and_quantifications.git.

Novel resources of Taxol from endophytic and entomopathogenic fungi: Isolation, characterization and LC-Triple mass spectrometric quantification.

This work presents a second-to-none method for Taxol isolation from the Endophytic fungus Cladosporium sphaerospermum (AUMC 6896) and the Entomopathogenic fungus Metarizium anisopliae (AUMC 5130). The extracts were analyzed by high performance liquid chromatography (HPLC) and Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. This is rapid, consistent, reproducible, accurate, and sensitive for quantifying Taxol across multiple samples. The yield of crude Taxol product obtained from Potato Dextrose broth (PDB) medium inoculated with Cladosporium sphaerospermu and Metarizium anisopliae was found to be 3.732, and 0.0023 µg L-1 respectively. The yield can be improved by adding ammonium acetate or salicylic acid to the culture broth. Addition of ammonium acetate (AA) (20 mg L-1) to culture media resulted in an increase of Taxol yield to 30.365 and 27.289 µg L-1 respectively. Production of Taxol was 29.844 and 67.254 µg L-1 for the two fungus species when ammonium acetate was substituted by 90 mg L-1 salicylic acid (SA). Adding both AA (20 mg L-1) and SA (90 mg L-1) to the culture media resulted in an increase of the Taxol yield to 4.054 and 116.373 µg L-1 respectively. Our proposed analytical method offers very fast (3 min) quantitation of Taxol in comparison with other published methods. These findings represent a new bioprospecting of the endophytic fungi that may serve as a potential material for the production of Taxol for anticancer treatment.