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.

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.

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.

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.

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.

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.

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.

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.

Retiro de ventilación mecánica prolongada: experiencia de seis años con la aplicación de protocolo especializado / Weaning of prolonged mechanical ventilation: specialized protocol

La Ventilación Mecánica Prolongada (VMP) ha sido definida como aquella mayor de 21 días por al menos 6 horas diarias. Este fenómeno es un creciente problema para las Unidades de Cuidados Intensivos (UCI) en todo el mundo. Los consensos internacionales señalan que los pacientes con VMP deben ser tratados en unidades diferentes a las UCI, con protocolos especializados multiprofesionales dedicados a intentar el retiro del respirador. En nuestro Centro hemos recibido desde el año 2002 a 31 pacientes con VMP y les hemos aplicado un Protocolo de Rehabilitación Respiratoria Integral para ese objetivo. Las patologías predominantes fueron neuromusculares. El protocolo tiene 4 etapas progresivas, las 2 primeras comprenden la evaluación general y el inicio de terapias multiprofesionales. Las 2 siguientes consisten en retiro progresivo del respirador y continuación de terapias. Se midió la presión inspiratoria, presión espiratoria espiratoria máximas (PImáx y PEmáx) y la presión inspiratoria máxima sostenida (PIms), también se evaluó la actividad muscular de cuello, tronco y extremidades, así como el estado general y nutritivo, el nivel de conciencia y la calidad del apoyo familiar. Se entra a etapa 3 cuando los valores de PImáx y PEmáx son 60 y 50 cmH2O, respectivamente. Fueron excluidos de llegar a las últimas 2 etapas 7 pacientes, quienes no lograron este nivel de presiones. Resultados: 19pacientes (61 por ciento) con promedio de edad de 44 años (16-77), fueron retirados del respirador, 5 están aún en etapas 2 y 3; el promedio de VMP fue de 238 días y de 74 días a contar desde el ingreso a etapa 3. El promedio de mejoría de PImax fue de 59 por ciento, la PEmáx 60 por ciento y la PIms 61 por ciento, todos valores con significación estadística (p <0,001). El control cefálico y de tronco mejoró con las terapias. También el daño cognitivo, la depresión y la alteración de la función deglutoria...

Prolonged mechanical ventilation (PMV) is defined as greater than 21 days of mechanical ventilation for at least 6hours per day and is an increasing problem for the Intensive Care Units in all over the world. Many expert consensus groups recommend that long-term facilities may be a useful resource of care to implement integrated and specialized protocols for weaning from PMV. Since 2002 our center admitted 31 patients in PMV who were included on an Integral Respiratory Rehabilitation Protocol that was applied for our primary outcome of weaning success. Neuromuscular diseases were the main cause requiring PMV. The protocol applied consists of 4 successive stages, the first two oriented to evaluation and initiation of multiprofessional therapies, the next two are dedicated to progressive weaning and continuation of therapy. Peak inspiratory and expiratory pressures, and maximal sustained inspiratory pressure (PImax, PEmax and PIms) were measured, additionally muscular activity from neck, trunk and legs, general and nutritional condition, conscience level and family support were evaluated. Entry to stage 3 was defined when the PImax and PEmax were 60 and 50 cmH2O. Seven patients who failed to achieve this pressure levels were excluded. Results: 19 patients (61 percent) with a mean age of 44 years (16-77) had successful weaning and 5 patients are still in stage 2 or 3. Mean duration of PMV was 238 days. Mean Time on Mechanical Ventilation from the beginning of stage3 until the final weaning was 74 days. PImax, PEmax and PIms increased in 59, 60 and 61 percent each, all with statistical significance p <0.001, cephalic and trunk muscle control was increased with integrated therapies. The cognitive impairment, depression and swallowing disorders were professionally treated. Conclusion: A specialized, multiprofessional integrated protocol for PMV in our center permitted a successful weaning rate comparable to the best international series.

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.

Trastorno de déficit de atención e hiperactividad y alergia a alimentos / Attention deficit hyperactivity disorder and food allergy

El trastorno por déficit de atención e hiperactividad (TDAH) es la patología neuropsiquiátrica más común de la infancia. Requiere un manejo multidisciplinario. Su etiología es multifactorial, implica complejos factores ambientales y genéticos, así como diversos factores nutricionales. Se ha relacionado con alergia a algunos alimentos como los aditivos y azúcares refinados. El tratamiento con psicoestimulantes como el methilfenidato ha mostrado disminución de síntomas y mejoría conductual. Otras intervenciones no farmacológicas como las dietas restrictivas y de eliminación, provocan mejoría en este tipo de pacientes; no obstante, los estudios controlados no han llegado a demostrar un mecanismo inmune implicado en la patogenia de este trastorno. No se cuenta con evidencia suficiente para afirmar de manera categórica que existe una relación causa-efecto entre alergia a alimentos y TDAH; sin embargo, ante la comprobada eficacia de las dietas de restricción, es necesaria la búsqueda intencionada de la misma en pacientes hiperactivos. La aplicación de dietas de eliminación es recomendable en aquellos individuos en los que se compruebe su eficacia, pero su aplicación en forma general no es recomendable (AU)

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.

Correlation between in vitro and in vivo activities of GM 237354, a new sordarin derivative, against Candida albicans in an in vitro pharmacokinetic-pharmacodynamic model and influence of protein binding.

The antifungal effect of GM 237354, a sordarin derivative, was studied in an in vitro pharmacokinetic (PK)-pharmacodynamic dynamic system (bioreactor) which reproduces PK profiles observed in a previously described model of drug efficacy against murine systemic candidiasis. Immunocompetent mice infected intravenously with 10(5) CFU of Candida albicans were treated with GM 237354 at 2.5, 10, and 40 mg/kg of body weight every 8 h subcutaneously for 7 days. Free concentrations in serum were calculated by multiplying total concentrations measured in vivo by 0.05, the free fraction determined in vitro by equilibrium dialysis. In the bioreactor the inoculum was approximately 10(6) CFU/ml; and a one-compartment PK model was used to reproduce the PK profiles of free and total GM 237354 in serum obtained in mice, and clearance of C. albicans was measured over 48 h. A good correlation was observed when the in vivo fungal kidney burden and the area under the survival time curve were compared with the in vitro broth "burden," although only when free in vivo levels in serum were reproduced in vitro. GM 237354 displayed a 3-log decrease effect both in vivo and in vitro. The very few reports available on in vitro-in vivo correlations have been obtained with antibiotics. The good in vitro-in vivo correlation obtained with an antifungal agent shows that the in vitro dynamic system could constitute a powerful investigational tool prior to assessment of the efficacy of an anti-infective agent in animals and humans.

Animal pharmacokinetics and interspecies scaling of sordarin derivatives following intravenous administration.

Sordarin derivatives constitute a new group of synthetic antifungal agents that selectively inhibit fungal protein synthesis. They have demonstrated in vitro activity against the most important fungal pathogens, both yeast and filamentous. This new family of compounds has also shown in vivo activity against murine Candida albicans, Histoplasma capsulatum, and Coccidioides immitis experimental infections, as well as against Pneumocystis carinii pneumonia in rats. After intravenous dosing in animals, both the area under the concentration-time curve and the elimination half-life were highest in Cynomolgus monkeys, followed by those in rats, mice, and rabbits. The volume of distribution at steady state for sordarin derivatives was similar in all species tested. The clearance in rats and mice was higher than for other species. GM 237354, a sordarin derivative, was characterized by high serum protein binding in mouse, rat, and monkey serum (unbound fraction, < or =5%). An indirect evaluation of the effect of liver function upon the metabolism of this class of compounds has been made in animals with impaired liver function such as Gunn rats, as well as in allometric studies that showed better correlations of half-life to liver blood flow than to animal body weight. Linearity of the main pharmacokinetic parameters was demonstrated after intravenous dosing of the representative compound GM 193663 at 10 and 20 mg/kg of body weight in rats. Allometry was used to determine whether human pharmacokinetic parameters can be predicted from animal data by regression analysis against body weight and liver blood flow. All these results have demonstrated that the human pharmacokinetics of sordarin derivatives can be forecast from animal data.

Activities of sordarins in experimental models of candidiasis, aspergillosis, and pneumocystosis.

Sordarin derivatives represent a new class of antifungal agents that act as potent inhibitors of fungal protein synthesis and possess a broad spectrum of activity. The in vivo activity of GM193663 and GM237354 was studied in mouse models of disseminated candidiasis and aspergillosis and in a rat model of pneumocystosis. The pharmacokinetic behavior of both sordarin derivatives was studied in mice and rats. In all studies, compounds were administered by the subcutaneous route. After a subcutaneous dose of 50 mg/kg of body weight to mice, the maximum level in serum, area under the concentration-time curve, half-life, and clearance for GM193663 and GM237354 were 51.8 and 23 microg/ml, 79.5 and 46 microg. h/ml, 0.8 and 0.85 h, and 21 and 25 ml/h, respectively. Systemic candidiasis and aspergillosis were established in CD-1 male mice infected with Candida albicans or Aspergillus fumigatus. For systemic candidiasis, compounds were given three times per day for seven consecutive days at 15, 30, 60, or 120 mg/kg/day. GM193663 and GM237354 showed dose-related efficacy against C. albicans, with 50% effective doses, 1 month after infection, of 25.2 and 10.7 mg/kg/dose, respectively. In experimental infections with A. fumigatus, GM237354 was given three times per day at 30, 60, or 120 mg/kg/day for five consecutive days. Animals treated with GM237354 demonstrated irregular responses. The survival of animals treated with GM237354 was 0, 30, and 0% at 30, 60, and 120 mg/kg/day, respectively. The therapeutic efficacy of GM193663 and GM237354 against Pneumocystis carinii was studied in an experimental P. carinii pneumonia (PCP) rat model. After a subcutaneous dose of 10 mg/kg given to rats, the maximum level in serum, area under the concentration-time curve, half-life, and clearance for GM193663 and GM237354 were 6.6 and 7.2 microg/ml, 8.5 and 11.8 microg. h/ml, 0.7 and 0.8 h, and 230 and 133 ml/h, respectively. To induce spontaneous PCP, rats were chronically immunosuppressed with dexamethasone. Infected animals were treated twice daily for 10 days at 0.2, 2, or 10 mg/kg/day. The therapeutic effect was estimated by the reduction in the number of cysts in the lungs of treated versus untreated animals. GM193663 and GM237354 significantly reduced the mean (+/- standard deviation) log number of cysts from 7.6 +/- 0.2 in the untreated group to 4.7 +/- 0.2 and 4.6 +/- 0.1, respectively, when the drugs were administered at a dose of 2 mg/kg/day. The log number of cysts was also reduced in infected animals given lower doses of the compounds (0.2 mg/kg/day). In summary, GM193663 and GM237354 are new sordarin derivatives that may potentially play a major role in the treatment of candidiasis and PCP. Further testing with Aspergillus in other animal models is warranted.

Pharmacokinetics-pharmacodynamics of a sordarin derivative (GM 237354) in a murine model of lethal candidiasis.

Sordarins are a new class of antifungal agents which selectively inhibit fungal protein synthesis (FPS) by impairing the function of elongation factor 2. The present study investigates possible correlations between sordarin pharmacokinetic (PK) properties and therapeutic efficacy, based on a murine model of invasive systemic candidiasis, and provides a rationale for dose selection in the first study of efficacy in humans. A significant correlation between PK parameters and the in vivo activity of GM 237354, taken as a representative FPS inhibitor, was demonstrated in a murine model of lethal systemic candidiasis. Area under the concentration-time curve (AUC) and maximum concentration of drug in serum (C(max)) over 24 h were determined after a single GM 237354 subcutaneous (s.c.) dose (50 mg/kg of body weight) in healthy animals (no significant PK changes with infection were observed for other sordarin derivatives). These results have been used to simulate PK profiles obtained after several doses and/or schedules in animal therapy. A PK-pharmacodynamic (PD) parameter such as the time that serum drug concentrations remain above the MIC (t > MIC) was also determined. Treatment efficacies were evaluated in terms of the area under the survival time curve (AUSTC), using Kaplan-Meier survival analysis and in terms of kidney fungal burden (log CFU/gram) after s.c. doses of 2.5, 5, 10, 20, and 40 mg/kg every 4, 8, or 12 h (corresponding to total daily doses of 5 to 240 mg/kg). The results show all treatments to significantly prolong survival versus that of infected and nontreated controls (P < 0.05). Relationships between simulated PK and PK-PD parameters and efficacy were explored. A good correlation independent of the dosing interval was observed with AUC (but not C(max) or t > MIC) and both AUSTC and kidney burden. Following repeated dosing every 8 h, AUC(50) (AUC at which 50% of the maximum therapeutic efficacy is obtained) was estimated as 21.7 and 37.1 microg. h/ml (total concentrations) for AUSTC and kidney burden using a sigmoid E(max) and an inhibitory sigmoid E(max) PK-PD model, respectively. For an efficacy target of 90% survival, AUC was predicted as 67 microg. h/ml. We conclude that the PK-PD approach is useful for evaluating relationships between PK parameters and efficacy in antifungal research. Moreover, the results obtained with this approach could be successfully applied to clinical studies.

In vitro pharmacodynamic parameters of sordarin derivatives in comparison with those of marketed compounds against Pneumocystis carinii isolated from rats.

Pneumocystis carinii pneumonia remains one of the most serious complications of immunosuppressed patients. In this study, the in vitro pharmacodynamic parameters of four sordarin derivatives (GM 191519, GM 237354, GM 193663, and GM 219771) have been evaluated by a new quantitative approach and compared with the commercially available drugs pentamidine, atovaquone, and trimethoprim-sulfamethoxazole (TMP-SMX). In vitro activities and in vivo therapeutic efficacies of sordarin derivatives against P. carinii were also evaluated. In vitro activity was determined by the broth microdilution technique, comparing the total number of microorganisms in treated and drug-free cultures by using Giemsa staining. The in vitro maximum effect (E(max)), the drug concentrations to reach 50% of E(max) (EC(50)), and the slope of the dose-response curve were then estimated by the Hill equation (E(max) sigmoid model). Sordarin derivatives were the most potent agents against P. carinii, with EC(50)s of 0.00025, 0.0007, 0.0043, and 0. 025 microg/ml for GM 191519, GM 237354, GM 193663, and GM 219771, respectively. The EC(50)s of pentamidine, atovaquone, and TMP-SMX were 0.025, 0.16, and 26.7/133.5 microg/ml, respectively. The results obtained with this approach showed GM 237354 and GM 191519 to be approximately 35- and 100-fold more active in vitro than pentamidine, the most active marketed compound. All sordarin derivatives tested were at least 5,000-fold more active in vitro than TMP-SMX. The three sordarin derivatives tested in vivo-GM 191519, GM 237354, and GM 219771-showed a marked therapeutic efficacy, defined as reduction of cyst forms per gram of lung. GM 191519 was the most potent (daily dose reducing 50% of the P. carinii burden in the lungs [ED(50)], 0.05 mg/kg/day) followed by GM 237354 and GM 219771 (ED(50)s, 0.30 and 0.49 mg/kg/day, respectively). Good agreement between in vitro parameters and in vivo outcome was obtained when P. carinii pneumonia in rats was treated with sordarin derivatives.