a-video-protocol-randomized-controlled-clinical-trial

A Video Protocol of a Randomized Controlled Clinical Trial - Electrochemotherapy of Cutaneous Metastases with Reduced Dose Bleomycin (BLESS Trial)

Cutaneous metastases in patients with incurable cancer represent a significant problem as they often cause pain, discomfort, and emotional distress that ...

Jožef Stefan Institute

The plasma concentration profile of bleomycin in the distribution phase of patients younger than 65 years is needed to determine the suitable time interval for efficient application of electric pulses during electrochemotherapy. Additionally, bleomycin concentrations in the treated tumors for effective tumor response are not known. In this study, the pharmacokinetic profile of bleomycin in the distribution phase in 12 patients younger than 65 years was determined. In 17 patients, the intratumoral bleomycin concentration was determined before the application of electric pulses. In younger patients, the pharmacokinetics of intravenously injected bleomycin demonstrated a faster plasma clearance rate than that in patients older than 65 years. This outcome might indicate that the lowering of the standard bleomycin dose of 15,000 IU/m with intravenous bleomycin injection for electrochemotherapy is not recommended in younger patients. Based on the plasma concentration data gathered, a time interval for electrochemotherapy of 5-15 min after bleomycin injection was determined. The median bleomycin concentration in tumors 8 min after bleomycin injection, at the time of electroporation, was 170 ng/g. Based on collected data, the reduction of the bleomycin dose is not recommended in younger patients; however, a shortened time interval for application of electric pulses in electrochemotherapy to 5-15 min after intravenous bleomycin injection should be considered.

Bleomycin Concentration in Patients' Plasma and Tumors after Electrochemotherapy. A Study from InspECT Group.

The effectiveness of highly polar agents in cancer treatment is well recognized, but their physicochemical properties make their analytical determination a demanding task. Their analysis requires peculiar sample preparation and chromatographic separation, which heavily impacts the precision of such an analytical method. As a case study, we chose a polar cytotoxic bleomycin, which is a mixture of complexing congeners with relatively high molecular mass, a fact that creates an added challenge in regard to its detection via electrospray mass spectrometry. These issues combined lead to a deprived method performance, so the aim of this study is manifold, i.e., to optimize, validate, and establish quality performance measures for determination of bleomycin in pharmaceutical and biological specimens. Quantification of bleomycin is done at diametrically different concentration levels: at the concentrations relevant for analysis of pharmaceutical dosage forms it is based on a direct reversed-phase HPLC-UV detection, involving minimum sample pretreatment. On the contrary, analysis of bleomycin in biological specimens requires phospholipid removal and protein precipitation followed by HILIC chromatography with MS/MS detection of bleomycin A2 and B2 copper complexes being the predominant species. This study further attempts to solve the traceability issue in the absence of certified reference standards, determines measurement uncertainty, investigates BLM stability and method performance characteristics, and, last but not least, provides an explanatory example of how a method quality assurance procedure should be established in case of an exceedingly complex analytical method.

An effective validation of analytical method for determination of a polar complexing agent: the illustrative case of cytotoxic bleomycin.

Increasing use of pigs as models in translational research, and growing focus on animal welfare are leading to better use of effective analgesics and anaesthetics when painful procedures are performed. However, there is a gap in basic knowledge such as pharmacokinetics of different anaesthetics in these species. The main objective of our study was to determine the pharmacokinetics of levobupivacaine in domestic pigs. Twelve female grower pigs weighing 31.17 ± 4.6 kg were subjected to general anaesthesia and experimental surgery, at the end of which they received 1 mg/kg levobupivacaine via peri-incisional subcutaneous infiltration. Plasma samples were collected before administration of levobupivacaine and at 0.5, 1, 2, 4, 8, 12, 24 and 48 h thereafter. Concentrations of levobupivacaine were determined by liquid chromatography coupled with tandem mass spectrometry. Following single dose of levobupivacaine, all animals had measurable plasma concentrations 0.5 h after drug administration, with most peak concentrations observed at the 1-h time point. In all 12 animals, levobupivacaine was below the limit of quantification 48 h after drug administration. The mean maximum plasma concentration, area under the curve and half-life were determined to be 809.98 μg/l, 6552.46 μg/l h and 6.25 h, respectively. Plasma clearance, volume of distribution and weight-normalized volume of distribution were 4.41 l/h, 35.57 l and 1.23 l/kg, respectively. Peak plasma concentrations in our study were well below concentrations that were found to produce toxicity in pigs.

Pharmacokinetics of single dose levobupivacaine after peri-incisional subcutaneous infiltration in anaesthetized domestic pigs.

Endocrine-disrupting chemicals (EDCs), including known and unknown parent compounds, their metabolites, and transformation products, are pervasive in daily life, posing increasing risks to human health and the environment. This study employed a high-resolution mass spectrometry-based nontargeted screening approach, integrating polar (HILIC) and reversed-phase separations to expand the chemical space coverage and, supported by open-science tools and resources, evaluated urinary chemical profiles to assess internal EDC exposure. Among 106 annotated biomarkers of exposure, six exhibited significantly higher normalized intensities in patients with ovarian malignancies compared to healthy controls ( < 0.05). This suggests their greater exposure to phthalates (diethylhexyl phthalate and diethyl phthalate), pesticides (metolachlor metabolite and 4-nitrophenol), a UV filter (benzophenone-1), and an industrial byproduct (4-methyl-2-nitrophenol). These compounds may interfere with hormonal regulation, potentially contributing to cancer development. While these findings highlight potential differences in internal EDC exposure, the study primarily demonstrates the applicability of nontargeted urinary profiling for chemical exposure assessment. By providing new insights into EDCs burden and its pathological implications, this work contributes to advancing next-generation chemical risk assessment within the European Partnership for the Assessment of Risks from Chemicals initiative and supports the development of preventive strategies to mitigate environmental cancer risks.

Nontargeted Urinary Profiling Strategy for Endocrine-Disrupting Chemicals in Women with Ovarian Malignancies.

Bleomycin, a cytotoxic antibiotic, poses substantial challenges for mass spectrometry-based analysis due to its extreme polarity, chelating properties, heterogeneity of fractions, and propensity to form multiple charged species during electrospray ionization. As one of the few effective drugs used in electrochemotherapy, the ability to quantify trace levels of bleomycin is critical for evaluating treatment efficacy, often requiring sensitivity beyond the capabilities of existing analytical methods. Such precise quantification would facilitate the evaluation of electrochemotherapy efficacy, such as comparing the in vitro effects of nanosecond electric pulses with conventional microsecond pulses. To address these challenges, we integrated cell viability assays with a robust chemical analytical approach. This approach employed solid-phase extraction for sample preparation, combined with HILIC-LC-MS/MS, achieving exceptional sensitivity with LLOQ of 0.075 µg/L and overcoming analyte and matrix complexity. Although a significant reduction in cell survival was confirmed when combining nanosecond pulses (25 × 400 ns) with bleomycin, chemical analysis revealed discrepancies, underscoring the complex interaction between electric pulse parameters and drug action. These findings highlight the need for further refinement of treatment protocols and the development of advanced analytical techniques.

Article	Year
Bleomycin Concentration in Patients' Plasma and Tumors after Electrochemotherapy. A Study from InspECT Group. Pharmaceutics\| PubMed ID: 34575400	2021
An effective validation of analytical method for determination of a polar complexing agent: the illustrative case of cytotoxic bleomycin. Analytical and bioanalytical chemistry\| PubMed ID: 37041279	2023
Pharmacokinetics of single dose levobupivacaine after peri-incisional subcutaneous infiltration in anaesthetized domestic pigs. Laboratory animals\| PubMed ID: 39157982	2024
Nontargeted Urinary Profiling Strategy for Endocrine-Disrupting Chemicals in Women with Ovarian Malignancies. Environmental science & technology\| PubMed ID: 40263667	2025
Quantitative analysis of trace-level bleomycin in complex matrices: Application to in vitro electrochemotherapy. Journal of pharmaceutical and biomedical analysis\| PubMed ID: 40347764	2025

Helena Plesnik

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