Laura Guerrero

Benznidazole-Related Adverse Drug Reactions and Their Relationship to Serum Drug Concentrations in Patients with Chronic Chagas Disease

ABSTRACT For treating Chagas disease (CD), a current worldwide health problem, only benznidazole and nifurtimox have been approved to be used. In both cases, unwanted drug-related adverse events (ADRs) are frequent when these drugs are used in adults in the chronic stage. The main objective of this study was to establish benznidazole ADRs and their relationship to serum concentrations in patients with chronic Trypanosoma cruzi infection in order to perform more accurate dosages to minimize ADRs. A total of 54 patients were recruited over 12 months. Of these 54 patients, 53 (98%) experienced at least one ADR during follow-up, and the overall average ADR incidence was 2.4 episodes/patient/month. Benznidazole treatment was discontinued in 11 patients, 7 among them due to severe adverse effects. The mean duration of treatment before withdrawal was 11 days. Benznidazole serum concentrations were recorded on days 15, 30, 45, and 60 of follow-up and evaluated according to clinical and epidemiological variables and ADR severity. No relationship was found between the benznidazole serum concentration and the ADRs. The mean (standard deviation) trough serum benznidazole concentrations (all below 20 mcg/ml) on days 15, 30, 45, and 60 were 6.4 (1.9), 6.1 (1.8), 6.2 (2.2), and 5.7 (1.7) μg/ml, respectively. Benznidazole serum concentrations do not appear to be related to the appearance of serious ADRs. Further, well-controlled studies are necessary to establish the optimal regimen for benznidazole in adults with chronic CD.

Efficacy of linezolid compared to vancomycin in an experimental model of pneumonia induced by methicillin-resistant Staphylococcus aureus in ventilated pigs.

Objective:To assess the efficacy of linezolid compared with vancomycin in an experimental model of pneumonia induced by methicillin-resistant Staphylococcus aureus (MRSA) in ventilated pigs. Methods:Forty pigs (30 kg) were intubated and challenged via bronchoscopy with a suspension of 106 colony forming units of MRSA into every lobe. Afterwards, pigs were ventilated up to 96 hours. Twelve hours after bacterial inoculation, the animals were randomized into 4 groups of treatment: group 1, control; group 2, vancomycin twice daily; group 3, continuous infusion of vancomycin; and group 4, linezolid. Clinical and laboratory parameters were monitored throughout the study. Bacterial cultures of bronchoalveolar lavage fluid and lung tissue samples were performed at the end of the study. Measurements of histopathology derangements of lung samples and studies of intrapulmonary drug penetration were performed. Results:A total of 34 animals completed the study. No differences in clinical and laboratory parameters were observed. The percentage of bronchoalveolar lavage fluid and lung tissue samples with positive cultures for MRSA in controls and groups 2, 3, and 4 was respectively 75%, 11%, 11%, and 0% (p < .01); 52%, 9%, 24%, and 2.5% (p < .01). Histopathology studies demonstrated signs of pneumonia in 95%, 69%, 58%, and 57% and signs of severe pneumonia in 48%, 29%, 22%, and 0% of controls and groups 2, 3, and 4, respectively (p < .01). In addition, pharmacokinetics/pharmacodynamics profile in serum and lung tissue showed better results for linezolid compared with both vancomycin treatments. Conclusions:In this animal model of MRSA pneumonia, linezolid showed a better efficacy than vancomycin showed because of a better pharmacokinetics/pharmacodynamics index.

Linezolid limits burden of methicillin-resistant Staphylococcus aureus in biofilm of tracheal tubes.

Objective: To evaluate the effects of systemic treatment with linezolid compared with vancomycin on biofilm formation in mechanically ventilated pigs with severe methicillin-resistant Staphylococcus aureus–induced pneumonia. Design: Prospective randomized animal study. Setting: Departments of Pneumology, Microbiology, and Pharmacy of the Hospital Clínic, Barcelona, and Scientific and Technological Services of the University of Barcelona. Subjects: We prospectively analyzed 70 endotracheal tube samples. Endotracheal tubes were obtained from pigs either untreated (controls, n = 20), or treated with vancomycin (n = 32) or linezolid (n = 18). Interventions: The endotracheal tubes were obtained from a previous randomized study in tracheally intubated pigs with methicillin-resistant Staphylococcus aureus severe pneumonia, and mechanically ventilated for 69 ± 16 hrs. Measurements and Main Results: Distal and medial hemisections of the endotracheal tube were assessed to quantify methicillin-resistant Staphylococcus aureus burden, antibiotic biofilm concentration by high-performance liquid chromatography or bioassay, and biofilm thickness through scanning electron microscopy. We found a trend toward a significant variation in biofilm methicillin-resistant Staphylococcus aureus burden (log colony-forming unit/mL) among groups (p = .057), and the lowest bacterial burden was found in endotracheal tubes treated with linezolid (1.98 ± 1.68) in comparison with untreated endotracheal tubes (3.72 ± 2.20, p = .045) or those treated with vancomycin (2.97 ± 2.43, p = .286). Biofilm linezolid concentration was 19-fold above the linezolid minimum inhibitory concentration, whereas biofilm vancomycin concentration (1.60 ± 0.91 µg/mL) was consistently below or close to the vancomycin minimum inhibitory concentration. Biofilm was thicker in the vancomycin group (p = .077). Conclusions: Systemic treatment with linezolid limits endotracheal tube biofilm development and methicillin-resistant Staphylococcus aureus burden. The potential clinical usefulness of linezolid in decreasing the risk of biofilm-related respiratory infections during prolonged tracheal intubation requires further investigation.

A high-performance liquid chromatographic method for benznidazole quantitation in plasma of patients with Chagas disease

Abstract Background: Chagas disease is endemic in Latin America, affecting 16–18 million people with more than 100 million exposed to risk of infection. Its etiological agent is Trypanosoma cruzi. To date, benznidazole is the only treatment of Chagas disease available in Europe. Methods: A high-performance reversed-phase isocratic liquid chromatographic method for benznidazole analysis in human plasma is described. The mobile phase consists of 60% ultrafiltered water and 40% acetonitrile. Samples were precipitated with trichloroacetic acid (0.3 M) (1/1, v/v). The injection volume was 100 μL. Benzocaine was used as internal standard. Results: The assay was linear over a benznidazole concentration range of 1.6–100 μg/mL. The method showed good agreement of results (n=15): inaccuracy (5.6%), intra- and inter-day variability (1.1% and 3.9%, respectively), recovery (94.9%), limit of detection (0.8 μg/mL), lower limit of quantitation (1.6 μg/mL) and acceptable stability over 24 h in the auto-sampler. Only 25 samples (58%) showed values within the therapeutic range. Three samples were subtherapeutic and 15 were in the toxic range. Conclusions: The method offers a fast and simple approach to determining benznidazole in human plasma which could be of use in pharmacokinetic and safety studies.

An experimental model of pneumonia induced by methicillin-resistant Staphylococcus aureus in ventilated piglets

The objectives of the study were to validate a model of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia in ventilated piglets and to study the time-course of biological markers and histopathological changes. 12 piglets were intubated and inoculated with 15 mL of a suspension of 106 colony forming units of MRSA in every lobe through the bronchoscope channel. The piglets were ventilated for 12 h (n = 6) and 24 h (n = 6). Clinical parameters were assessed every 6 h and pro-inflammatory cytokines were measured in serum and in bronchoalveolar lavage (BAL) at baseline and sacrifice. Histopathology of each lobe and cultures from blood, lungs and BAL were performed. Animals developed histopathological evidence of pneumonia at necropsy. At 12 h, pneumonia was present in all animals and was severe pneumonia at 24 h. Microbiological studies confirmed the presence of MRSA. A significant increase in interleukin (IL)-6, IL-8 and tumour necrosis factor-&agr; values was seen in BAL at 24 h and IL-6 at 12 h. In serum, only IL-6 levels had increased significantly at 24 h. In ventilated piglets, bronchoscopic inoculation of MRSA induces pneumonia at 12 h and severe pneumonia at 24 h. This severity was associated with a corresponding increase in systemic and local inflammatory response.

Linezolid pharmacokinetics in patients with acute renal failure undergoing continuous venovenous hemodiafiltration.

A renal failure (ARF) is relatively common in the intensive care unit (ICU) setting, and it is associated with an increased mortality (23%-52%). Some patients with ARF will need renal replacement therapy. A high percentage of critically ill patients with ARF do not tolerate intermittent hemodialysis because of hemodynamic instability. Continuous renal replacement therapy (CRRT) runs continuously over a 24-hour period (unlike conventional hemodialysis, which generally lasts about 4 hours and is performed every 48 hours) and removes solutes and volume more slowly. Thus, it allows greater hemodynamic stability, is better tolerated, and permits accurate blood volume control, which could be beneficial in some patients. All types of CRRT are based on ultrafiltration (elimination of substances by convection). However, some therapies also incorporate diffusion. A solute is effectively removed by CRRT if: (1) its molecular weight is smaller than the pore size of membranes; (2) it binds to plasma proteins at a low percentage; and (3) it has a low volume of distribution (low distribution to tissues). It is generally accepted that techniques that incorporate diffusion and convection (hemodiafiltration) remove more solutes than techniques that act only by convection (hemofiltration) or diffusion (hemodialysis). Dosages of many drugs need to be adjusted in critical patients treated with CRRT. Many antibiotics are removed in a high percentage by these replacement therapies, thus it is important to administer the correct dose to prevent underdosing. Little information is available for linezolid, which is increasingly used in this special population because of a higher methicillinresistant Staphylococcus aureus incidence. The efficacy of linezolid (oxazolidinone) is related to the time that its plasma concentrations exceed the minimum inhibitory concentration (MIC) of microorganism (T> MIC) and to the area under the plasma concentration time curve to MIC ratio (AUC0-24/MIC). 7 A T> MIC of 40%-45% was required in animal models to produce a bacteriostatic effect. However, in a study in 241 critically ill adults infected by Gram-positive bacteria, linezolid efficacy was maximal with T> MIC of ≥85%. The optimal AUC0-24/MIC ratio should be equal to or greater than 100. With regard to elimination, extrarenal clearance represents approximately 65% of total clearance of linezolid. And because of its pharmacokinetic properties (distribution limited to the extracellular space, low protein binding, and

Development and validation of a high performance liquid chromatography method to determine linezolid concentrations in pig pulmonary tissue.

Abstract Background: Linezolid is the first synthetic compound of a new group of antimicrobials, the oxazolidinones, which inhibit protein synthesis. It shows a broad spectrum of activity against Gram positive organisms. With respect to its pharmacokinetics, linezolid shows a relatively high volume of distribution and good penetration into inflammatory fluids, bone, fat and muscle. Methods: A reversed-phase isocratic high-performance liquid chromatographic method for linezolid analysis in piglet pulmonary tissue is described. Tissue samples and controls were prepared in 1×TBE (1 M Tris, 0.9 M boric acid, 0.01 M EDTA). The mobile phase consisted of 20% ultrafiltered water and 80% of (A) 15 mM potassium monohydrogen phosphate buffer (pH=5) with (B) acetonitrile (80%/20%; v/v). Samples were homogenized and precipitated with HClO4 3% (1/1, v/v). The injection volume was 100 μL. Ofloxacin was used as an internal standard. Results: The assay was linear over a linezolid concentration range: 1.6–100 μg/mL. The method provided good validation data (n=15): inaccuracy (3.6%), intra and inter-day variability (4.2% and 5.2%, respectively), recovery (91.8%), limit of detection (0.8 μg/mL) and quantitation (1.6 μg/mL) and acceptable stability within 24 h in the auto-sampler. Conclusions: The method offers a fast and simple approach to determine linezolid in pulmonary tissue which could be of use in pharmacokinetic studies. Clin Chem Lab Med 2010;48:391–8.

Determinación de linezolid en fluidos biológicos mediante cromatografía líquida de alta eficacia

OBJECTIVE Evaluation of an analytic method for determining linezolid concentrations in biological fluids including plasma, vitreous humour and cerebrospinal fluid using high-efficiency liquid chromatography and subsequent ultraviolet detection. METHOD The method was validated by studying the following parameters: accuracy, precision, sensitivity, linearity and recovery. The drug was extracted from the biological matrix by means of a protein precipitation with perchloric acid. Chromatographic separation was performed by eluting linezolid with a mobile phase consisting of 80% K2HPO4 buffer solution (15 mM; pH=5) and 20% acetonitrile, and a stationary phase, NOVAPAK C18 150x3.9 mm with precolumn. The wavelength reading was 254 nm and the working flow rate was 1 ml/min. RESULTS We obtained values with accuracies between 94.4 and 106.1%, and precisions between 0.88-6% and 3.7-5.6% for intra-and inter-day variability, respectively. Recovery obtained after analysing the plasma samples was at 93%. The method showed itself to be linear for the concentration levels under study. DISCUSSION The methods behaviour can be described as linear, precise and accurate. Furthermore, the method is fast, sensitive, and inexpensive. It is useful for determining linezolid concentrations in multiple biological matrices. It can also be used as a basis for further clinical pharmacokinetic studies.

Physicochemical compatibility of high concentration drugs usually Y-site administered in intensive care units

Objective To study the physical compatibility of 63 binary mixtures of drugs usually Y-administered in intensive care units, and to evaluate the chemical stability of the most relevant binary mixtures. Methods Binary mixtures were aseptically prepared in a 1:1 ratio. End-points were measured in triplicate immediately after and 4 and 24 h after preparation. Chromatographic techniques were all validated under the study conditions, following international guidelines. All methods were proven to be stability-indicating. Main outcome measures Incompatibility was defined as the presence of haziness, colour change or precipitation compared with the parent drug solution. Chemical instability was considered present when >5% variation in the concentration of any of the components in the mixture was observed and/or pH values were outside the stability range for any of the drugs in the mixture. Results This study demonstrated the physicochemical Y-site compatibility of: (i) dopamine or dobutamine at 8 mg/mL with methadone 0.2 mg/mL in both 5% dextrose (5%D) and normal saline (NS); (ii) dopamine or dobutamine with midazolam 4 mg/mL in 5%D but not in NS; (iii) methadone with midazolam in 5%D and NS, and methadone with esomeprazole or pantoprazole 0.32 mg/mL in NS. Results demonstrated the incompatibility of: (i) dobutamine with furosemide in NS; and (ii) midazolam 4 mg/mL with esomeprazole in NS. Conclusions This study provides information about the physicochemical compatibility of ordinary mixtures, at high concentrations, commonly used in intensive care units. These results might help to improve drug safety management in the critically ill patient.

DEVELOPMENT AND VALIDATION OF A HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD TO DETERMINE VANCOMYCIN CONCENTRATIONS IN PLASMA AND PIG PULMONARY TISSUE

Simple and specific analytical methods were developed and validated to quantify vancomycin in plasma and lung tissue, which were obtained from a model of pneumonia in mechanically ventilated piglets. Tissue and plasma samples were precipitated and centrifuged; 100 µL of the supernatant were injected into the chromatographic system. Ceftazidime was used as the internal standard. The stationary phase was a silica based column Symmetry300 C18 (150 × 4.6 mm) with pre-column. The mobile phase consisted of 20% ultrafiltered water and 80% of (A) 75 mM sodium acetate buffer (pH = 3) with (B) acetonitrile (92%/8%;v/v). Isocratic flow rate was set at 0.8 mL/min and 0.7 mL/min for plasma and tissue samples, respectively. UV absorbance detection was set at 230 nm. Standard curves showed good linearity for plasma and pulmonary tissue (r2 > 0.99). The lower limits of quantitation were 1.56 µg/mL and 3.13 µg/mL for plasma and tissue, respectively. The intra- and inter-day precisions and accuracy all satisfied the acceptance criteria. Both HPLC assays to quantify vancomycin in plasma and pulmonary tissue are rapid, simple, and inexpensive. These methods could be helpful to develop further pharmacokinetic studies of vancomycin penetration in pulmonary tissue.