A sensitive bioanalytical ultra-high-performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantitation of lactone and carboxylate forms of topotecan in plasma and vitreous.

Topotecan (TPT) is used in the treatment of retinoblastoma, the most common malignant intraocular tumor in children. TPT undergoes pH-dependent hydrolysis of the lactone ring to the ring-opened carboxylate form, with the lactone form showing antitumor activity. A selective, and highly sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed for the determination of both forms of TPT in one mobile phase composition in plasma and vitreous humor matrices. The method showed an excellent linear range of 0.375-120 ng/mL for the lactone. For the carboxylate, the linear range was from 0.75 to 120 ng/mL. The matrix effect and the recovery for the lactone ranged from 98.5% to 106.0% in both matrices, for the carboxylate form, it ranged from 94.9% to 101.2%. The dynamics of the transition between TPT lactone and TPT carboxylate were evaluated at different pH environments. The stability of TPT forms was assessed in plasma and vitreous humor at 8 and 37°C and a very fast conversion of lactone to carboxylate form occurred at 37°C in both matrices. The method developed facilitates the investigation of TPT pharmacodynamics and the release kinetics in the development of the innovative local drug delivery systems.

Self-assembled ordered AuNRs-modified electrodes for simultaneous determination of dopamine and topotecan with improved data reproducibility.

Gold nanomaterials have been widely explored in electrochemical sensors due to their high catalytic property and good stability in multi-medium. In this paper, the reproducibility of the signal among batches of gold nanorods (AuNRs)-modified electrodes was investigated to improve the data stabilization and repeatability. Ordered and random self-assembled AuNRs-modified electrodes were used as electrochemical sensors for the simultaneous determination of dopamine (DA) and topotecan (TPC), with the aim of obtaining an improved signal stability in batches of electrodes and realizing the simultaneous determination of both substances. The morphology and structure of the assemblies were analyzed and characterized by UV-Vis spectra, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray powder diffraction (XRD). Electrochemical studies showed that the ordered AuNRs/ITO electrodes have excellent signal reproducibility among several individuals due to the homogeneous mass transfer in the ordered arrangement of the AuNRs. Under the optimized conditions, the simultaneous detection results of DA and TPC showed good linearity in the ranges 1.75-45 µM and 1.5-40 µM, and the detection limits of DA and TPC were 0.06 µM and 0.17 µM, respectively. The results showed that the prepared ordered AuNR/ITO electrode had high sensitivity, long-term stability, and reproducibility for the simultaneous determination of DA and TPC, and it was expected to be applicable for real sample testing.

Evaluation of a safe infusion device on reducing occupational exposure of nurses to antineoplastic drugs: a comparative prospective study. Contamoins-1.

PURPOSE: Despite the decreasing of environmental contamination throughout the anticancer drug circuit, the administration of chemotherapies remains at risk of occupational exposure for nurses. Many medical devices aim at securing administration, but none have been scientifically evaluated to verify the actual improvement. METHODS: A monocentric comparative before/after study was carried out in an oncology day hospital to evaluate the efficacy of Safe Infusion Devices in reducing drug exposure compared to usual infusion practices. The rate of nurses' gloves contamination was estimated. To avoid false negatives and to ensure sampling reproducibility, each sample of gloves was contaminated with a drop of topotecan. Association between contamination and other variables was investigated using a multivariate logistic regression analysis. RESULTS: The usual practice led to a rate of 58.3% of contaminated samples while Safe Infusion Devices to a rate of 15%: Safe Infusion Devices reduced the risk of gloves contamination by 85% in multivariate analysis (Odds ratio = 0.15; 95% confidence interval = 0.05-0.46; p < 0.001). Topotecan was identified in 100% of the samples. Only one case of cross-contamination has occurred. CONCLUSION: Despite the current practice of using neutral solvent-purged infusers, the occupational exposure remains high for nurses and Safe Infusion Devices significantly reduced this risk of exposure. However, glove contamination is only a surrogate endpoint. The results confirmed that the disconnection of empty bags resulted in occupational exposure. Except a contamination due to the leakage of a bag, no cross-contamination was detected. Safe Infusion Devices were highly effective but did not completely eliminate exposure.

Dual modulation of blue-fluorescent carbon dots for simultaneous detection of topotecan and pantoprazole.

This study introduces a novel approach for the simultaneous determination of topotecan (TOP) and pantoprazole (PNT), two drugs whose interaction is critical in clinical applications. The significance of this study originates from the need to understand the pharmacokinetic changes of TOP after PNT administration, which can inform necessary dose adjustments of TOP. To achieve this, nitrogen blue emissive carbon dots (B@NCDs) were produced and employed due to their unique fluorescent properties. When TOP is added to B@NCDs, it exhibits strong native fluorescence at 545 nm without influencing the B@NCDs' fluorescence at 447 nm. Conversely, PNT causes quenching of B@NCDs fluorescence, a property that enables the distinct detection of both drugs. The B@NCDs were fully characterized using different techniques, including ultraviolet-visible spectrophotometry, fluorescence analysis, X-ray diffraction (XRD), transmission electron microscopy (TEM), and FTIR spectroscopy. The proposed method demonstrated excellent linearity, selectivity, and sensitivity, with low detection limits (LOD, S/N = 3); 0.0016 µg mL-1 for TOP and 0.36 µg mL-1 for PNT. Applied to spiked rabbit plasma samples, this method offers a new approach for evaluating the pharmacokinetic interaction between TOP and PNT. It enables the determination of all pharmacokinetic parameters of TOP before and after coadministration with PNT, providing essential insights into whether dose adjustments are necessary. This research not only contributes to the field of drug monitoring and interaction studies but also exemplifies the potential of B@NCDs in complex biological matrices, paving the way for further pharmacological and therapeutic applications.

Electrochemical Nanosensor for the Simultaneous Determination of Anticancer Drugs Epirubicin and Topotecan Using UiO-66-NH2/GO Nanocomposite Modified Electrode.

In this work, UiO-66-NH2/GO nanocomposite was prepared using a simple solvothermal technique, and its structure and morphology were characterized using field emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDS), and X-ray diffraction (XRD). An enhanced electrochemical sensor for the detection of epirubicin (EP) was proposed, which utilized a UiO-66-NH2/GO nanocomposite-modified screen-printed graphite electrode (UiO-66-NH2/GO/SPGE). The prepared UiO-66-NH2/GO nanocomposite improved the electrochemical performance of the SPGE towards the redox reaction of EP. Under optimized experimental conditions, this sensor demonstrates a remarkable limit of detection (LOD) of 0.003 µM and a linear dynamic range from 0.008 to 200.0 µM, providing a highly capable platform for sensing EP. Furthermore, the simultaneous electro-catalytic oxidation of EP and topotecan (TP) was investigated at the UiO-66-NH2/GO/SPGE surface utilizing differential pulse voltammetry (DPV). DPV measurements revealed the presence of two distinct oxidation peaks of EP and TP, with a peak potential separation of 200 mV. Finally, the UiO-66-NH2/GO/SPGE sensor was successfully utilized for the quantitative analysis of EP and TP in pharmaceutical injection, yielding highly satisfactory results.

Optimizing the storage of chemotherapeutics for ophthalmic oncology: stability of topotecan solution for intravitreal injection.

BACKGROUND: . Intravitreal administration of topotecan shows activity against tumor vitreous seeding in the conservative treatment of retinoblastoma, a malignant tumor originated in the retina of small children. Adequate storage of the intravitreal topotecan solution would allow immediate availability for patients at health care institutions. The goal of the work was to address the stability of the intravitreal topotecan formulation upon reconstitution. MATERIALS AND METHODS: . Intravitreal topotecan solutions were reconstituted (at a concentration of 0.2 mg topotecan in 1 mL saline solution vehicle, aliquoted in 1 mL plastic syringes) and stored either frozen or at room temperature for different times. Topotecan content was analyzed at time zero and at different conditions using a high performance liquid chromatography method to quantify topotecan lactone (active) and to detect its pH-dependent hydrolysis product, the open carboxylate. RESULTS: . We found that intravitreal topotecan syringes remained stable at room temperature at least for 24 h, at least for 167 days upon stored frozen at -20°C, and up to 8 h after thawing at day 6. The degradation carboxylate product did not appear in the analyzed thawed samples during the whole study. CONCLUSIONS: . This study confirms the stability of frozen intravitreal topotecan syringes and will help optimize the use of this chemotherapy modality at institutions with low resources. Storage of aliquots will also help reduce personnel exposure to chemotherapy at hospital pharmacies.

Development of an enzyme­linked immunosorbent assay for camptothecin.

The use of camptothecin and its analogues has increased in clinical settings and in agriculture. Therefore, camptothecins and their derivatives, metabolites and degradation products are frequently found in the environment. Therefore, it is important to develop an ELISA for the quantification of camptothecins in human plasma, plants, animal tissues and other matrices. The present study developed a novel competitive indirect ELISA for camptothecin using a monoclonal antibody (MAb). In total, two haptens and various carrier proteins were tested to select the most suitable immunogen for the production of MAbs against camptothecin. Hapten 1 conjugated with keyhole limpet hemocyanin was selected for the preparation of MAb 5A3, and was used to establish a competitive indirect ELISA for camptothecin. A total of three derivatives of camptothecin used in clinical practice were examined. Topotecan showed an IC50 value of 0.68 µg/ml with a detection limit of 0.19 µg/ml, belotecan showed an IC50 value of 0.87 µg/ml with a detection limit of 0.22 µg/ml and irinotecan showed an IC50 value of 2.85 µg/ml with a detection limit of 0.47 µg/ml. The cross­reactivity results suggested that the assay developed in the present study possessed a high sensitivity to camptothecin. Therefore, this immunoassay technique may be suitable for monitoring the levels of camptothecin in compound analysis, clinical applications, and analyses of food and environmental samples.

Concurrent determination of topotecan and model permeability markers (atenolol, antipyrine, propranolol and furosemide) by reversed phase liquid chromatography: utility in Caco-2 intestinal absorption studies.

A simple, sensitive, specific and high-resolution reversed-phase liquid chromatographic method utilizing ultraviolet detection has been developed and validated for simultaneous determination of topotecan and four intestinal permeability markers (atenolol, antipyrine, propranolol and furosemide) as suggested by US-FDA. Chromatography was carried out on C-18 column with mobile phase comprising water (pH 3.0) and acetonitrile gradient pumped at a flow rate of 1 ml min(-1). The validation parameters included specificity, accuracy, precision, sensitivity and stability studies. Topotecan, an anti-cancer drug widely used in metastatic carcinoma, is a P-glycoprotein substrate having oral bioavailability of 30% with large inter-patient variability. The present method was successfully applied for demonstrating P-gp mediated transport of topotecan and its inhibition using verapamil in Caco-2 cell monolayer. The method can be used in identification of novel P-gp inhibitors for topotecan and estimating the contribution of P-gp in affecting oral bioavailability of topotecan. The other applications of method include its use in validation of Caco-2 monolayer assay for getting biowaiver based on Biopharmaceutic Classification System and its extrapolation to in situ and/or in vivo studies.

Fluorescence detection combined with either HPLC or HPTLC for pharmaceutical quality control in a hospital chemotherapy production unit: application to camptothecin derivatives.

In order to achieve the analytical assessment of the manufactured batches of chemotherapy preparations, post-production quality control has been developed. The common use of camptothecin derivatives (i.e. irinotecan (CPT-11) and topotecan (TPT)) as part of protocols in Institut Gustave Roussy (IGR) has led to develop an efficient analytical method that could assess an increasing number of samples with high throughput, good specificity and practicality. Due to the difference of concentration between batches containing irinotecan or topotecan, HPLC and HPTLC both combined with fluorescence detection were investigated. Those two techniques made identity, purity and quantitation assays possible. The chromatographic conditions that were chosen allowed identification of each drug through their rate of flow (Rf), 0.10 and 0.35, or their retention time (tR), 2 and 7 min for topotecan and irinotecan, respectively. A calibration curve was plotted for each molecule and validated by three quality controls (high, medium and low). Coefficients of variation of repeatability (CVr) and intermediate precision (CVi) were determined for both methods. Considering their values and the concentration ranges (from 100 to 500 mg/L for HPTLC and from 0.1 to 1 mg/L for HPLC), it was decided to perform analysis using HPTLC for irinotecan preparations and HPLC for topotecan preparations. These inferences seemed appropriate regarding the number of preparations to be assayed.

Electrochemical investigation of the interaction between topotecan and DNA at disposable graphite electrodes.

Topotecan (TPT) is a semisynthetic, water soluble analog of the plant alkaloid camptothecin which has been widely used for the treatment of ovarian and cervical cancers. To obtain better understanding on how it can affect DNA structure, electrochemical biosensor platforms for the investigation of TPT-double stranded DNA (dsDNA) interaction were developed for the first time in this study. The electrochemical detection of TPT, and TPT-dsDNA interaction were investigated at the surface of pencil graphite electrodes (PGEs) and single-walled carbon nanotube (SWCNT) modified PGEs by using differential pulse voltammetry (DPV). The changes at the oxidation signals of TPT and guanine were evaluated before/after each modification/immobilization step. An enhanced sensor response was obtained by using SWCNT-PGEs compared to unmodified PGEs with resulting limits of detection (LODs) for TPT as 0.51 µg/mL, 0.45 µg/mL, 0.37 µg/mL (130 pmol, 117 pmol, 96.5 pmol in a 110 µL sample, respectively) by using electrochemically pretreated PGE, unmodified PGE and SWCNT modified PGE. In addition, electrochemical impedance spectroscopy (EIS) measurements were performed for the purpose of modification of PGEs by using SWCNTs and the interaction process at the surface of SWCNT-PGEs by evaluating the changes at the charge transfer resistance (Rct).

Stability and compatibility of topotecan hydrochloride for injection with common infusion solutions and containers.

The stability and compatibility of topotecan hydrochloride with common infusion solutions and containers were studied. During this study, the leaching of diethylhexyl phthalate (DEHP), a major plasticizer of some polyvinyl chloride (PVC) materials was also investigated. A formulation of topotecan hydrochloride was added to 50 ml PVC infusion bags, polyolefin infusion bags and 150 ml glass bottles containing either 5% dextrose injection or 0.9% sodium chloride injection at an initial nominal topotecan concentration of 0.05 mg ml-1. Additionally, the topotecan hydrochloride formulation was added to 50 ml PVC infusion bags containing either 5% dextrose injection or 0.9% sodium chloride injection at an initial nominal topotecan concentration of 0.025 mg ml-1. Containers were maintained at 5 degrees C for 7 days or 23-24 degrees C for 24 h. Samples were analyzed using a stability-indicating HPLC method to determine the concentration of topotecan and the presence of any degradates. The samples were also analyzed by separate HPLC methods to detect the presence of DEHP and the hydrolyzed lactone ring form (SKF 105992) of topotecan hydrochloride. In addition, the pH of each sample was measured initially and at the end of the storage time. There was no significant loss of topotecan observed for any of the conditions studied and no significant increase in degradates was observed. The pH remained unchanged for all samples between the start and end of the study. At the concentrations studied, topotecan hydrochloride was stable for up to 24 h at room temperature and for up to 7 days at 5 degrees C, in PVC and polyolefin infusion bags and glass bottles containing either 5% dextrose injection or 0.9% sodium chloride injection. The presence of topotecan hydrochloride did not contribute to leaching of DEHP in the PVC infusion bags.

Development and validation of the HPLC method for simultaneous estimation of Paclitaxel and topotecan.

A simple, rapid, accurate and precise high performance liquid chromatography (HPLC) method for simultaneous analysis of Paclitaxel and Topotecan was developed. Different analytical parameters, such as linearity, accuracy, precision, specificity with intentional degradation, limit of detection and limit of quantification (LOQ), were determined according to the ICH guidelines. Acetonitrile-water (70:30, 0.1% trifluoroacetic acid) was run on a Phenomenex Luna C-18(2) column in isocratic mode at a flow rate of 1.2 mL/min for simultaneous analysis of the two drugs using a UV detector set at 227 nm. The proposed method showed a retention time (Rt) of 14.56 min for Topotecan and 23.81 min for Paclitaxel with a continuous run up to 30 min. The linearity of the calibration curves for each analyte in the desired concentration range was found to be good (r(2) > 0.9995). The recovery ranged from 97.9 to 101% for each drug with a relative standard deviation (%RSD) of <2%. Peaks corresponding to each of the drugs exhibited  positive values for the minimum peak purity index over the entire range of integrated chromatographic peak indicating high purity of the peaks. Stability analysis revealed that the drugs remained stable for sufficient time. Thus, the developed method was found to be robust and it can be employed to quantify Paclitaxel and Topotecan in commercial sample and rat blood/serum.

Topotecan vitreous and plasma levels and retinal toxicity after transcorneal intravitreal delivery in healthy albino rabbits: alternative retinoblastoma treatment.

AIM: To determine intravitreal and plasma concentrations and retinal toxicity after transcorneal intravitreal injection of 1 µg and 2 µg of topotecan (Hycamtin). METHOD: Twelve healthy albino rabbits were included in this in vivo experiment. Six anesthetized albino rabbits received a single transcorneal intravitreal injection of 1 µg (group A) or 2 µg (group B) of topotecan. Vitreous and blood samples were collected until 168 h. Left eyes were treated with the same volume of saline. Plasma and vitreous levels of topotecan were determined by high-performance liquid chromatography. Area under the plasma concentration time curve (AUC) was calculated using trapezoidal rule. Clinical evidence of toxicity was classified into four grades according to anatomical structures. Electroretinograms (ERGs) were recorded. RESULTS: Time to maximum concentration was observed up to 2 h after drug injection in group A whereas up to 1 h in group B. Low levels of topotecan were detected in plasma in both groups and in the vitreous humor of the contralateral eye in group B. Topotecan levels (mean vitreous AUC in group A 2.55 µg/mL.h and in group B 5.338 µg/mL.h) were detectable up to 6 h in both groups. We observed following structural changes in rabbit eyes: corneal vascularization, cataract, hemophthalmus, choroidal edema and focal retinal atrophy. Abnormal ERGs were obtained. CONCLUSION: Our findings proved that transcorneal intravitreal administration of 1 µg and 2 µg of topotecan results in potentially cytotoxic intraocular concentrations. More studies are needed to establish the safety of topotecan for retinoblastoma in children.

Laser induced fluorescence and photochemical derivatization for trace determination of camptothecin.

Laser-excited fluorescence was used for the selective determination of camptothecin in samples containing anti-cancer camptothecin-analogs (irinotecan and topotecan). The selectivity of the method was based on the UV photochemical derivatization in basic solution which increased the analyte fluorescence (337/450 nm) and eliminated fluorescence from the two campthotecin-analogs. The influence of UV exposure time and sodium hydroxide concentration was studied using an experimental design. Limit of detection was 4 × 10(-10) mol L(-1) with linear fluorescence response up to 1 × 10(-6)mol L(-1). Average recoveries of camptothecin (added to the samples to simulate a contamination) were 92 ± 4 and 94 ± 6% (n=3) respectively in irinotecan and topotecan based pharmaceuticals.

Quantification of topotecan by liquid chromatography-mass spectrometry (LC-MS). Application to intestinal transport using rat everted gut sacs.

Topetecan is an important anti-cancer drug that has recently become available as an oral formulation. In order to study its intestinal absorption in vitro and a potential drug-drug interaction with the anti-emetic ondansetron, a sensitive and accurate method for the analysis of topotecan in biological media was required. We developed a liquid-liquid extraction method at pH 7.0-7.5 with a recovery of 85% and which took into account the complex chemical behaviour of topotecan related to the lactone opening and the keto-enol tautomerism. This enabled us to validate a new specific and sensitive LC-MS method of analysis, with satisfactory inter- and intra-day repeatability and accuracy. The method was applied to a study of topotecan uptake in rat everted gut sacs that showed that, despite being a P-glycoprotein substrate like topotecan, ondansetron did not interfere with topotecan uptake.

Quantitation of camptothecin and related compounds.

Camptothecin and congeners represent a clinically very useful class of anticancer agents. Proper identification and quantitation of the original compounds and their metabolites in biological fluids is fundamental to assess drug metabolism and distribution in animals and in man. In this paper we will review the recent literature available on the methods used for separation and quantitative determination of the camptothecin family of drugs. Complications arise from the fact that they are chemically labile, and the pharmacologically active lactone structure can undergo ring opening at physiological conditions. In addition, a number of metabolic changes usually occur, producing a variety of active or inactive metabolites. Hence, the conditions of extraction, pre-treatment and quantitative analysis are to be carefully calibrated in order to provide meaningful results.

Determination of camptothecin analogs in biological matrices by high-performance liquid chromatography.

Several analogs of the topoisomerase I inhibitor camptothecin (CPT) have been introduced in clinical practice in the last decade. All CPT analogs are sensitive to a pH-dependent reversible conversion between a pharmacologically active lactone form and its inactive, lactone ring-opened, carboxylate form. The reversible conversion is also dependent on the, sometimes species-dependent, protein binding properties of the two forms, resulting in different lactone to carboxylate plasma ratios for the various analogs. Pharmacokinetic analysis of the CPT analogs is helpful in understanding the pharmacodynamic outcome of drug treatment, in clinical as well preclinical studies. Measurement of these analogs is habitually complicated by the chemical instability of the lactone moiety and necessitates a rapid centrifugation of the blood sample, preferably at the bedside of the patient, to collect the plasma supernatant. Since the lactone forms of these drugs are able to diffuse across cell membranes, including those of the red blood cells, rapid collection and processing is even necessary in the case where only the total concentrations of the CPT analogs are to be measured. Sample pretreatment procedures of the CPT analogs topotecan, irinotecan, 9-aminocamptothecin and lurtotecan are summarized and discussed in this review.

Determination of topotecan by ELISA.

A highly sensitive ELISA for the determination of (s)-9-dimethylaminomethyl-10-hydroxy-camptothecin (topotecan) capable of measuring as low as 80 pg/ml was developed. Anti-topotecan antibody was obtained by immunizing rabbits with topotecan conjugated with bovine serum albumin using diazotized m-aminobenzoic acid as a cross-linker. Enzyme labeling of topotecan with beta-D-galactosidase was performed by utilizing another cross-linker, N-(4-diazophenyl)maleimide. The specificity of this ELISA appears to be primarily toward the lactone moiety of topotecan, showing a very slight cross-reactivity with the lactone opened-ring of topotecan. The values for the topotecan concentrations detected using this assay were comparable with those detected using HPLC. There was a good correlation between the values determined by the two methods. These findings suggest that this ELISA can detect the natural amounts of the lactone form. Using this assay, drug levels were easily determined in the blood and urine of rats for 5 h after i.v. administration of topotecan at a single dose of 1 mg/kg. The sensitivity and specificity of the ELISA should provide a useful tool for developing pharmacokinetic and pharmacodynamic studies of topotecan.