Metabolic Disposition of Lurbinectedin, a Potent Selective Inhibitor of Active Transcription of Protein-Coding Genes, in Nonclinical Species and Patients.

Lurbinectedin is a novel and potent selective inhibitor of active transcription of protein-coding genes, triggering apoptosis of cancerous cells. It has been approved for the treatment of patients with metastatic small-cell lung cancer with disease progression on or after platinum-based chemotherapy. Studies exploring the disposition and metabolism of lurbinectedin were performed in vitro and in vivo (by intravenous administration of lurbinectedin). Low blood cell partitioning for lurbinectedin in rats, nonhuman primates (NHP), and humans was determined as 23.4%, 29.8%, and 9.8%, respectively. Protein binding was very high (>95%) in total plasma (rat, NHP, and human), albumin, and α-1-acid glycoprotein (both human). In vitro, lurbinectedin underwent intense liver microsome-mediated metabolism-in 10 minutes, 80% of the compound is metabolized in human-with CYP3A4 being the isoform involved in that metabolism. Results also showed NHPs being the nonclinical species which, metabolically, most closely resembles humans. Mass balance studies performed in rats (both genders), NHPs (male only), and patients (both genders) demonstrated that the principal route of excretion of 14C-lurbinectedin-related radioactivity was through the feces (88.7% ± 10.1% in patients), with only a minor fraction recovered from the urine (5.6% ± 2.0% in patients). In plasma samples, the majority of lurbinectedin-related radioactivity was attributed to unchanged compound (95% ± 3.1% and 70.2% ± 10.9% in NHPs and humans, respectively). Plasma metabolic profiling demonstrated the major (% compared with unchanged compound) circulating metabolites were N-Desmethyl-lurbinectedin (0.4% ± 0.2% and 10.4% ± 2.2% in NHPs and patients, respectively) and 1',3'-Desmethylene-lurbinectedin (0.9% ± 0.7% and 14.3% ± 10.4% in NHP and patients, respectively). SIGNIFICANCE STATEMENT: Lurbinectedin is a novel and potent selective inhibitor of active transcription of protein-coding genes, triggering apoptosis of cancerous cells, and was recently approved for the treatment of patients with metastatic small-cell lung cancer with disease progression on or after platinum-based chemotherapy. The present study provides a complete set of information on the pharmacokinetics, biotransformation, and elimination of 14C-lurbinectedin and its metabolites, following a single intravenous administration to nonclinical species (rats and nonhuman primates) and patients.

Evaluation of different configurations of hybrid membrane bioreactors for treatment of domestic wastewater.

Four membrane bioreactors (MBRs) with the same dimensions were studied for 180 days: three hybrid growth membrane bioreactors with biofilm attached in different packing media and a conventional MBR (C-MBR). The four MBRs had an identical membrane module of hollow fiber with a nominal porous diameter of 0.4 µm. The MBRs were: (1) a C-MBR; (2) a moving bed membrane bioreactor (MB-MBR), which was packed with 2 L of carrier Kaldnes-K1, presenting an exposed surface area of 678.90 m²/m³; (3) a non-submerged organic fixed bed (OFB-MBR) packed with 6.5 L of organic packing media composed of a mixture of cylindrical pieces of wood, providing an exposed surface area of 178.05 m²/m³; and (4) an inorganic fixed bed non-submerged membrane bioreactor (IFB-MBR) packed with 6 L of spherical volcanic pumice stone with an exposed surface area of 526.80 m²/m³. The four MBRs were fed at low organic loading (0.51 ± 0.19 kgCOD/m³ d). The results were recorded according to the behavior of the total resistance, transmembrane pressure (TMP), permeability, and removal percentages of the nutrients during the experimental time. The results showed that the MB-MBR presented the better performance on membrane filtration, while the higher nutrient removals were detected in the OFB-MBR and IFB-MBR.

Use of air stacking and abdominal compression for cough assistance in people with complete tetraplegia.

STUDY DESIGN: Cross-sectional. OBJECTIVE: To assess cough using air stacking (AS) to assist inspiratory volume with abdominal compression (AC) during expiration in patients with American Spinal Injury Association Impairment Scale (AIS) A. SETTING: Large tertiary hospital in Chile. METHODS: Peak cough flow (PCF) was measured during four different interventions: spontaneous maximal expiratory effort (MEE); MEE while receiving AC (MEE-AC); MEE after AS with a manual resuscitation bag (AS-MEE); and MEE with AS and AC (AS-MEE-AC). RESULTS: Fifteen in-patients with complete tetraplegia (C4-C6) were included. Median age was 33 years (16-56). PCF during the different interventions was PCF for MEE was 183±90 l min(-1); PCF for MEE-AC was 273±119 l min(-1); PCF for AS-MEE was 278±106 l min(-1) and PCF for AS-MEE-AC was 368±129 l min(-1). We observed significant differences in PCF while applying MEE-AC and AS-MEE compared with MEE (P=0.0001). However, the difference in PCF value was greater using the AS-MEE-AC technique (P=0.00001). CONCLUSION: Patients with spinal cord injury (SCI) presented an ineffective cough that constitutes a risk factor for developing respiratory complications. The application of combined techniques (AS-MEE-AC) can reach near normal PCF values. This is a low-cost, simple and easily applied intervention that could be introduced to all patients with tetraplegia.

S-p-bromobenzyl-glutathione cyclopentyl diester (BBGC) as novel therapeutic strategy to enhance trabectedin anti-tumor effect in soft tissue sarcoma preclinical models.

Trabectedin, approved for the treatment of soft tissue sarcoma (STS), interferes with cell division and genetic transcription processes. Due to its strong anti-tumor activity in only certain histotypes, several studies on trabectedin combinations are currently ongoing to improve its efficacy. In this study, we aimed to investigate novel potential therapeutic strategies to enhance the anti-tumor effect of trabectedin using integrated in silico, in vitro, and in vivo approaches. For in silico analysis, we screened two public datasets, GSEA M5190 and TCGA SARC. Fibrosarcoma, leiomyosarcoma, dedifferentiated, and myxoid liposarcoma cell lines were used for in vitro studies. For in vivo experiments, fibrosarcoma orthotopic murine model was developed. In silico analysis identified Glo1 as the only druggable target upregulated after trabectedin treatment and correlated with poor prognosis. The specific Glo1 inhibitor, S-p-bromobenzylglutathione cyclopentyl diester (BBGC), increased trabectedin cytotoxicity in STS cells, and restored drug sensitivity in myxoid liposarcoma cells resistant to trabectedin. Moreover, the combined treatment with BBGC and trabectedin had a synergistic antitumor effect in vivo without any additional toxicity to mice. Based on these results, we believe that BBGC warrants further investigation to evaluate its potential clinical use in combination with trabectedin.

Development of a new method for the quantitation of metabolites in the absence of chemically synthetized authentic standards.

A new method for the quantification of metabolites in the absence of a chemically synthetized authentic standard is described herein. Metabolites to be used as reference standards were obtained biologically from microsomes incubation. The method is a stepwise process in which, only the radiolabeled (14C) and non-radiolabeled parent compound are required. Briefly, the separation and principles of equimolar detection of LC-radioactivity were applied and, a calibration curve of the 14C-parent compound was used to quantify the formation of its 14C-metabolite. In turn, serial dilutions of this 14C-metabolite were the base for the calibration curve that allowed the quantification of the non-radiolabeled metabolite. This method was applied in plasma samples obtained from a dog pharmacokinetic study in which, a PharmaMar compound (lurbinectedin) and its N-desmethylated metabolite were quantified and, the results compared to those obtained by the classical approach (with the chemically synthetized N-desmethylated metabolite). Plasma concentrations obtained with the two methods were very similar, with standard relative errors between -11% to -4%. Similar, main pharmacokinetic parameters were calculated with the concentrations obtained either thru this method or by using a chemically synthetized authentic standard.

Development and validation of a liquid chromatography-tandem mass spectrometry assay for the quantification of lurbinectedin in human plasma and urine.

Lurbinectedin is a novel highly selective inhibitor of RNA polymerase II triggering caspase-dependent apoptosis of cancerous cells. This article describes the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to quantify lurbinectedin in human plasma and urine. Plasma samples were pre-treated with 1 M aqueous ammonia after which they were brought onto supported liquid extraction (SLE) columns. Lurbinectedin was eluted from the columns using tert-butyl methyl ether (TBME). Urine was first diluted in plasma and lurbinectedin was extracted from this matrix by liquid-liquid extraction using TBME. Samples were measured by LC-MS/MS in the positive electron ion spray mode. The method was linear over 0.1-100 ng/mL and 1-1000 ng/mL in plasma and urine, respectively, with accuracies and precisions within ±15% (20% for LLOQ) and below 15% (20% for LLOQ), respectively. The method was developed to support a mass balance study in which patients received a dose of 5 mg lurbinectedin.

Metabolite profiling of the novel anti-cancer agent, plitidepsin, in urine and faeces in cancer patients after administration of 14C-plitidepsin.

PURPOSE: Plitidepsin absorption, distribution, metabolism and excretion characteristics were investigated in a mass balance study, in which six patients received a 3-h intravenous infusion containing 7 mg 14C-plitidepsin with a maximum radioactivity of 100 µCi. METHODS: Blood samples were drawn and excreta were collected until less than 1% of the administered radioactivity was excreted per matrix for two consecutive days. Samples were pooled within-patients and between-patients and samples were screened for metabolites. Afterwards, metabolites were identified and quantified. Analysis was done using Liquid Chromatography linked to an Ion Trap Mass Spectrometer and offline Liquid Scintillation Counting (LC-Ion Trap MS-LSC). RESULTS: On average 4.5 and 62.4% of the administered dose was excreted via urine over the first 24 h and in faeces over 240 h, respectively. Most metabolites were found in faeces. CONCLUSION: Plitidepsin is extensively metabolised and it undergoes dealkylation (demethylation), oxidation, carbonyl reduction, and (internal) hydrolysis. The chemical formula of several metabolites was confirmed using high resolution mass data.

Review: Placental adaptations to the presence of maternal asthma during pregnancy.

Asthma is a highly prevalent chronic medical condition affecting an estimated 12% of pregnant, women each year, with prevalence of asthma greatest (up to 16%) among the socially disadvantaged. Maternal asthma is associated with significant perinatal morbidity and mortality including preterm births, neonatal hospitalisations and low birthweight outcomes each year. We have identified that the placenta adapts to the presence of chronic, maternal asthma during pregnancy in a sex specific manner that may confer sex differences in fetal outcome. The male fetus was at greater risk of a poor outcome than a female fetus in the presence of maternal asthma and an acute inflammatory event such as an asthma exacerbation. This review will examine the role of sex specific differences in placental function on fetal growth and survival.

Liquid chromatography-tandem mass spectrometry assay to quantify plitidepsin in human plasma, whole blood and urine.

Plitidepsin is an anti-cancer drug currently evaluated in phase I/II/III clinical trials. This article describes the development and validation of a bioanalytical assay to quantify plitidepsin in human plasma, urine and whole blood using HPLC-MS/MS. The analyte was extracted from the matrix by liquid-liquid extraction using tert-butyl methyl ether. Final extracts were injected onto a C18 column, gradient elution was applied for chromatographic separation and detection was performed on a triple quadrupole mass spectrometer operating in the positive ion mode. The assay was linear over the range 0.1-100ng/mL, with acceptable accuracy and precision values. This is the first reported bioanalytical assay quantifying plitidepsin using a stable isotopically labelled standard, achieving a lower limit of quantification of 0.1ng/mL in all three matrices, allowing the quantification of trace levels of plitidepsin, and accomplishing this in an analysis time of two minutes only. The presented method was successfully applied in a mass balance study with plitidepsin in patients with advanced cancer.

Evaluation of risk of deterministic effects in fluoroscopically guided procedures.

A methodology for the evaluation of skin dose distribution and possible skin injuries on patients undergoing interventional procedures has been developed as part of the European DIMOND programme. Relevant dosimetric data from the procedures are recorded and other specific measurements for skin dose evaluation (slow films for therapy, radiochromic films, thermoluminescence dosemeters and optically stimulated luminescence dosemeters) have been carried out. For non-cardiac and for some cardiac procedures, dose-area product values of 200 and 300 Gy cm2 were proposed, respectively, as a trigger level for further detailed skin dose investigation and possible clinical follow-up. Results from a survey of 191 selected complex procedures are reported. Out of all the patients included in the trial, 16% received skin doses > or =1.5 Gy. No skin injuries were found in any of the patients followed.

The application of image quality measurements for digital angiography.

The image quality (IQ) evaluation of a charge-coupled device (CCD)-based digital angiography system was assessed with respect to modulation transfer function and noise power spectrum. These values were used to calculate the system's frequency-dependent detective quantum efficiency (DQE). The X-ray image detector was an image intensifier (II) lens coupled to a CCD camera. Two measurement setups were used. Setup A is standard IQ assessment, while Setup B more closely represented clinical conditions (polymethyl methacrylate (PMMA) of varying thickness placed between the X-ray tube and II, with test object positioned between PMMA slices 30 cm from the II). Exposure parameters varied according to automatic brightness control settings. Setup B included X-ray radiation scattered by the patient-PMMA. A clinical DQE, describing the transmission of the input signal-to-noise ratio associated with both primary and secondary X-ray spectra, was defined.

Magnetic resonance imaging in the evaluation of inflammatory lesions in muscular and soft tissues: an experimental infection model induced by Candida albicans.

We have developed an experimental model to monitor inflammatory lesions in muscle and soft-tissues during the different stages of the disease by means of Magnetic Resonance Imaging (MRI). MRI of mice legs infected with Candida albicans was performed by standard two-dimensional spin echo and fast spin echo (RARE) using customized coils. The MRI findings were compared with pathologic examinations at the initial acute and established acute inflammatory stages, which provided accurate and detailed information on the evolution of the processes involved. The yeast caused inflammation within the first hours post-inoculation, appearing on T2-weighted images as an inhomogeneous mass with increased signal intensity. The presence of fungal hyphae was observed as hypointense signal areas in both T2 and T1 weighted images, with histologic confirmation. Areas of decreased signal intensity on T2 weighted images were apparent on the last experimental day and were attributed to the granulation tissue located within the capsule surrounding the abscess. The close correlation found between MRI and histopathology suggests that MRI is an ideal radiologic technique for monitoring the clinical and therapeutic follow-up of fungal infections in muscle and soft tissues.

On the correct measurement of relative heavy charged particles to gamma thermoluminescent efficiencies.

In order to better understand the most important experimental aspects for performing correct measurements of relative thermoluminescent (TL) efficiencies, an investigation has been carried out to quantify the effect of using different experimental procedures in the evaluation of 3 MeV proton-to-gamma relative efficiency (etap,gamma) of LiF:Mg,Ti. Variations in batch, presentation, annealing and reader have been studied. When the same protocol is used to measure proton and gamma TL response, efficiency values obtained range from 0.36 to 0.59 for peak 5 and from 0.44 to 0.79 for the total signal. The use of different annealings and different batches leads to 20% and 10% differences in etap,gamma respectively. Large differences (40%) are found between efficiency values measured with TLD-100 chips and those obtained using TLD-100 microcubes. When 'mixed' procedures are used to measure the proton and the gamma response, differences in etap,gamma may increase even more. The main conclusion of this work is to stress the importance of measuring an entire series of experiments in the same laboratory with a carefully defined protocol and using dosemeters from the same batch to obtain heavy charged particle TL response and gamma TL response with identical annealing and readout procedures.

[Cerebrovascular accident in young adults. A study of 52 cases]. / Accidente cerebrovascular en adultos jóvenes. Estudio de 52 casos.

OBJECTIVE: A retrospective study of ischaemic and haemorrhagic cerebral vascular accidents in patients under 45 years of age, admitted to hospital Severo Ochoa (Area sanitaria 9 de la Comunidad de Madrid) during a 6 year period. METHOD: 23 CVAs in women and 29 in men were studied. The aetiology of each episode was determined following preestablished criteria. RESULTS: Aetiology: cardiac 13 (25%), atherosclerosis: 8 (15%), haematological: 6 (11%), non-atherosclerotic: 8 (15%), migraine: 1 (2%), hypertensive: 2 (3%), unknown: 15 (29%). There was a predominance of men in the patients with atherosclerosis. These also had more cardiovascular risk factors and were older than the other patients. All patients with CVA due to antiphospholipid antibodies had multiple CVAs. Examinations used to establish the aetiology were: echocardiogram, angiography, carotid doppler, hypercoagulability studies and the clinical history. CONCLUSIONS: In spite of extensive study, 30% of the CVAs in young people still have no aetiological diagnosis. In general in ischaemic CVAs, there is good recovery of the neurological defects and a low mortality. The detection and oral anticoagulation of patients with a source of cardiac emboli, and the elimination of cardiovascular risk factors would probably considerably reduce the incidence of ischaemic stroke in young adults.

[Survival of patients with AIDS treated with protease inhibitors]. / Supervivencia de pacientes con SIDA tratados con inhibidores de la proteasa.

OBJECTIVE: Make a volarization of the effect on survival of the protease inhibitors used on patients with established-AIDS. METHODS: Retrospective study on patients diagnosed of AIDS between January 1989 and March 1998. The main objective is the time between diagnosis and dead. It is compared the survival curve of the patients on treatment with protease inhibitors (PI) with the ones without them. We use the methods of Kaplan-Meier and log-rank. RESULTS: We analyzed 99 patients diagnosed of AIDS. Fifteen were treated with PI in combination and eighty-four with regimens without them. The number of CD4 and the age at the beginning of the study, the type of transmission and the gender were similar at the two groups. The median survival of the treated with PI was 47 (4 months (CI 95%: 39-55), and the one of the no treated as 26(3 months (CI 95%: 20-32) (p = 0.0027) CONCLUSIONS: The treatment with Protease inhibitors plus other antiviral medications is associated with a survival prolongation in patients with AIDS.

[Carcinomatous lymphangitis. Clinical study of 10 cases]. / Linfangitis carcinomatosa. Estudio clínico de 10 casos.

We analyze the clinical characteristics, the diagnostic criteria and the radiological aspects of 10 cases of Carcinomatous Lymphangitis. The quickly progressing exercise dyspnea and the constitutional syndrome were the most frequent symptoms. In nine patients, the tumor was previously unknown. The image methods (adbominal CAT and Echography) and the fibrobronchoscopic exam allowed a correct diagnosis. The most frequent neoplasia had a pulmonary origin and the most frequent histology was adenocarcinoma. The histological tests (bronchial and transbronchial biopsies) were more effective for the diagnosis than the cytological test (bronchoalveolar lavage, bronchoaspiration and bronchial brushing), although these were complemented. The radiology showed a reticulo-linear pattern in all the cases. The finding of macroscopic disorders in the fibrobronchoscopy, the presence of a local infiltration or a node/mass image in the thoracic radiography were highly suggestive of primary pulmonary neoplasia.

[Impact of HIV infection in hospital environment]. / Impacto de la infección por VIH en el medio hospitalario.

OBJECTIVE: Retrospective study to review the admissions at the Hospital Marina Alta due to infection for HIV or its complications and look for risk factors. METHODS: Clinical charts of patients admitted at the hospital from 1989 to 1996 were analyzed. RESULTS: From 11,932 admissions, 199 (1.7%) were due to patients with infection from HIV, resulting in the 2.4% of the total stay. The medium stays were higher (8.6 +/- 7.4 vs 6 +/- 4.5) more re-admissions (42.7% vs 25.5%) and higher mortality (11% vs 7.8%). The parasitic infestations of the nervous central system and cardiovascular were the most numerous number of admissions and also the longer stays. Throughout the years we saw a increase in the patients at the outpatient clinic with HIV infection and a paradogic decrease in the inpatient admissions, and also a decrease in the media stay and total stays. CONCLUSIONS: There is a decrease in the admissions at the inpatient level in contrast with a increment of the prevalence in the outpatients with HIV infection. The improved treatments, the experience of the physicians, the use of the Day Hospital and the use of the service of Home Care Hospitalization allows to keep more patients with less admissions and more outpatient visits.

Comparison of pharmacokinetic profiles of PM02734 loaded lipid nanoparticles and cyclodextrins: in vitro and in vivo characterization.

PM02734 is a chemically synthesized depsipeptide derived from the marine kahalalides family with a broad spectrum of activity against solid tumors in vitro and in vivo, but presenting low bioavailability. In this work, solid lipid nanoparticles made of Precirol ATO 5 have been developed using a hot homogenization method followed by high shear homogenization and ultrasonication. These solid lipid nanoparticles show suitable size (around 150 nm) and encapsulation efficiency (nearly 70%) for the oral administration of the compound PM02734. A physical-chemical stability study was performed after 6 months of storage at different thermical conditions, concluding that solid lipid nanoparticles stored at 4 degrees C were more stable than solid lipid nanoparticles stored at 25 degrees C. The pharmacokinetic profile of drug-loaded solid lipid nanoparticles was also evaluated in Beagle dogs and compared with that of a cyclodextrin-based delivery system by means of AUC, C(max) and T(max) parameter estimation. Solid lipid nanoparticle based formulation provided a sustained release of the drug for a longer period of time than the cyclodextrins.

PM01183, a new DNA minor groove covalent binder with potent in vitro and in vivo anti-tumour activity.

BACKGROUND AND PURPOSE: PM01183 is a new synthetic tetrahydroisoquinoline alkaloid that is currently in phase I clinical development for the treatment of solid tumours. In this study we have characterized the interactions of PM01183 with selected DNA molecules of defined sequence and its in vitro and in vivo cytotoxicity. EXPERIMENTAL APPROACH: DNA binding characteristics of PM01183 were studied using electrophoretic mobility shift assays, fluorescence-based melting kinetic experiments and computational modelling methods. Its mechanism of action was investigated using flow cytometry, Western blot analysis and fluorescent microscopy. In vitro anti-tumour activity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the in vivo activity utilized several human cancer models. KEY RESULTS: Electrophoretic mobility shift assays demonstrated that PM01183 bound to DNA. Fluorescence-based thermal denaturation experiments showed that the most favourable DNA triplets providing a central guanine for covalent adduct formation are AGC, CGG, AGG and TGG. These binding preferences could be rationalized using molecular modelling. PM01183-DNA adducts in living cells give rise to double-strand breaks, triggering S-phase accumulation and apoptosis. The potent cytotoxic activity of PM01183 was ascertained in a 23-cell line panel with a mean GI(50) value of 2.7 nM. In four murine xenograft models of human cancer, PM01183 inhibited tumour growth significantly with no weight loss of treated animals. CONCLUSIONS AND IMPLICATIONS: PM01183 is shown to bind to selected DNA sequences and promoted apoptosis by inducing double-strand breaks at nanomolar concentrations. The potent anti-tumour activity of PM01183 in several murine models of human cancer supports its development as a novel anti-neoplastic agent.