[Development of a Sensitive Enzyme-linked Immunosorbent Assay for Daptomycin].

Daptomycin (DAP) has a completely different mechanism of action compared with conventional drugs for methicillin-resistant Staphylococcus aureus (MRSA) and is widely used as the first-line drug for treatment of dermal soft tissue infection and sepsis caused by MRSA infection in clinical practice. However, DAP has serious side effects, including renal dysfunction and rhabdomyolysis, and thus therapeutic drug monitoring of DAP is recommended. The purpose of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for DAP that is simpler and more sensitive compared with existing assay methods and can be used in pharmacokinetic studies. Anti-DAP antibody was obtained by immunizing mice with an antigen conjugated with mercaptosuccinyl bovine serum albumin using N-(4-maleimidobutyryloxy) succinimide as a heterobifunctional coupling agent. Enzyme labeling of DAP with horseradish peroxidase was performed using pyromellitic dianhydride. The generated antibody and enzyme conjugate were used to develop a highly sensitive and specific ELISA for DAP in human serum. This ELISA shows a linear range of detection from 0.3 to 72.9 ng/mL, and a limit of quantification of approximately 0.3 ng/mL. The developed ELISA should be a valuable tool for pharmacokinetic studies and therapeutic drug monitoring of DAP.

A new validated HPLC-UV method for therapeutic monitoring of daptomycin in comparison with reference mass spectrometry.

Daptomycin, a cyclic lipopeptide antibiotic with a broad spectrum of activity against Gram-positive bacteria, is also active against multi-resistant bacterial strains, as well as methicillin-resistant S. aureus, vancomycin-resistant enterococci or penicillin-resistant S. pneumoniae. For these reasons it is a viable alternative for the treatment of persisting infections. However, the therapeutic drug monitoring of daptomycin is recommended because the known variability in drug disposition and the severe clinical conditions of patients. Therefore, we developed a simple and fast UV-HPLC method according to FDA guidelines to monitor plasma concentrations of the drug. Briefly, after a liquid-liquid extraction, plasma calibration samples, quality controls and patients' samples were injected in a HPLC instrument and peaks of daptomycin and gentamicin (internal standard) were resolved by a C18 250 × 4.6 mm, 5 µm stationary phase and peaks were monitored at UV = 262 nm. Mobile phase (isocratic flow of 1 mL/min) consisted of acetonitrile-buffer (KH2PO4 20 mM pH = 3.2) 46:54, vol/vol. Under these conditions, IS and daptomycin peaked at 4.1 and 5.8 min after injection. Values of limits of detection and quantitation accounted for 1.65 and 5.00 (µg/ml), respectively. Values of method linearity (r2) in range 5-100 mg/L were 0.9975 and 0.9956 plasma samples and solvent standard, respectively. Inter- and intra-day variability coefficients were lower than 15 %. The comparison with a reference, commercially-available LC-MS/MS method on 122 patient plasma samples returned excellent correlation (r2 = 0.9474). In conclusion, the present method demonstrated to be reliable and suitable for daptomycin TDM in clinical routine.

Nonadherence to treatment recommendations is a factor contributing to the clinical failure of daptomycin: Cohort study in Brazil

To evaluate the clinical outcomes of daptomycin therapy and adherence to treatment recommendations, a retrospective cohort study was conducted with patients that received daptomycin during the period of the study. The adherence and nonadherence to clinical guidelines were assessed through organism identification, dose and time of treatment, management of bacteremia, and vancomycin treatment failure. A multiple logistic regression model analyzed the association between independent variables and clinical success (dependent variable), considering 5% of statistical significance. The study presented 52 patients who received daptomycin for the treatment of bacteremia (21.1%) or infections (osteomyelitis [63.5%], synovial fluid [15.4%]). Most patients (86.5%) received daptomycin as the second line of treatment, and 51.9% achieved clinical success. The patients had a better chance of clinical success when they followed the guideline indications (OR = 16.86; 95% CI = 1.45-195.88) and the medication was prescribed by a specialist in infectious diseases (OR = 4.84; 95% CI = 1.11-21.09). The study demonstrated lower clinical success than that described in the literature because of patients who were not eligible according to the clinical guidelines. Adherence to recommendations and appropriate prescription of reserve antibiotics is important in limiting early resistance, and avoiding clinical failure and unnecessary expenditure.

Determination of daptomycin in human plasma and breast milk by UPLC/MS-MS.

During the lactation, the choice of a proper antibiotic is crucial since the drug can cross into breast milk causing toxicity to the infant. Therefore, an extraction protocol and LC/MS-MS method for the determination of daptomycin in human milk and plasma were developed, validated and applied to a case of a breastfeeding mother affected by a purulent acute soft skin infection treated with daptomycin. Because of daptomycin high protein binding and its high molecular weight, the optimisation of the extraction protocol and analytical conditions were deeply investigated, and several parameters were taken into account: in particular the type of extraction, internal standard, the type of organic modifier, pH of the aqueous solution, and gradient. The use of a protein precipitation protocol coupled to a C8-reverse phase LC-MS/MS allows for a reliable quantification of daptomycin in both plasma (in the range of 19-199 µg/mL) and breast milk (in the range of 0.12-0.32 µg/mL). The determination of milk/plasma (M/P) ratio, which ranged from 0.002 to 0.006, allowed to assess that daptomycin, effective for the mother, was contemporarily safe for the breastfed newborn.

Fourier-Transform Infrared (FTIR) Spectrophotometry: An Ecofriendly Method for the Analysis of Injectable Daptomycin.

Daptomycin (DPT) is an important antimicrobial agent used in clinical practice because it is very active against several types of medicinally challenging Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci. In addition to concerns about the quality of the analytical methods used in the QC of drugs, there is also concern about the impact of these methods on the environment. The trend toward sustainable consumption is increasingly evident and has forced the pharmaceutical industry to reduce the generation of toxic waste. In this context, IR spectrophotometry stands out because it does not use organic solvents and, although it is formally accepted for the identification of individual compounds, also allows the quantification of substances. Therefore, the aim of this work was to develop and validate a green analytical method for the analysis of DPT in a lyophilized powder for injection by FTIR spectrophotometry. The method involved absorbance measurements in the spectral region of 1700-1600 cm-1. The method was properly validated and found to be linear, precise, accurate, selective, and robust for the concentration range between 0.2 and 0.6 mg/150 mg. The validated method was able to quantify DPT powder for injection and can be used as an environmentally friendly alternative for routine analysis in QC.

A fully validated microbiological assay for daptomycin injection and comparison to HPLC method

abstract Daptomycin (DPT) was the first lipopeptide antibiotic available for commercialization. It is active against gram-positive bacteria, including resistant strains. This work aimed to develop and validate a turbidimetric microbiologic assay to determine daptomycin in an injectable form. A 3x3 design was employed, at concentrations of 1, 2 and 4.0 µg/mL. The microorganism test used was Staphylococcus aureus ATCC 6538p, and Antibiotic Medium 3 was used as the culture medium. Method validation demonstrated that the bioassay was linear (r=0.9995), precise (RSD=2.58%), accurate (recovery 100.48± 2.11%), and robust. Degradation kinetics was also performed in an alkaline medium, indicating that daptomycin degradation follows first order kinetics under these conditions. The analyses of degraded solutions showed that daptomycin degradation products do not possess bactericidal activity. The bioassay was compared to HPLC method that was previously developed and no significant difference was found between them (p>0.05). The method proved to be appropriate for daptomycin injection quality control.

resumo A daptomicina (DPT) é o primeiro lipopeptídeo cíclico disponível para comercialização. Possui atividade frente a bactérias gram-positivas, incluindo cepas resistentes. O objetivo deste trabalho foi desenvolver e validar um ensaio microbiológico turbidimétrico para quantificar a daptomicina na forma injetável. Empregou-se delineamento 3x3, nas concentrações de 1,0; 2,0 e 4,0 µg/mL. Como micro-organismo teste foi usado Staphylococcus aureus ATCC 6538p, e Meio para Antibióticos nº 3 foi empregado como meio de cultura. A validação do método demonstrou que o ensaio foi linear (r=0,9995), preciso (RSD=2,55%), exato (recuperação de 100,48 ± 2,11%) e robusto. A cinética de degradação em meio alcalino foi avaliada, indicando que a daptomicina segue cinética de primeira ordem nessa condição. A análise das soluções degradadas mostrou que os produtos de degradação da daptomicina não possuem atividade antimicrobiana. O bioensaio foi comparado com o método por CLAE previamente desenvolvido e não houve diferença significativa entre ambos (p<0,05). O método mostrou-se apropriado para o controle de qualidade da daptomicina injetável.

A green approach for the quantification of daptomycin in pharmaceutical formulation by UV spectrophotometry

abstract Daptomycin is the first approved drug from a new class of antimicrobials, the cyclic lipopeptides, and is a very important antimicrobial agent in current clinical practice. Currently, there are no "green" analytical methods described in the literature to analyze the typical pharmaceutical dosage form of daptomycin. Thus, the aim of this work was to validate an environment-friendly spectrophotometric method in the UV region, for the analysis of daptomycin as a lyophilized powder. Water was used as diluent and the analyses were carried out on a spectrophotometer at 221 nm. The method met all validation requirements of the ICH guidelines, over a concentration range of 6-21 µg mL-1. A Student's t-test demonstrated that the proposed method was comparable to an HPLC method previously validated. Thus, the validated spectrophotometric method could quantify daptomycin in a powder form for injectable solutions, while being an economical, rapid, and "green" alternative for routine analysis in quality control.

resumo A daptomicina é o primeiro membro aprovado de uma nova classe de antimicrobianos, os lipopeptídeos cíclicos, e é muito importante para a prática clínica atualmente. Não existem métodos analíticos "verdes" descritos na literatura para a análise da daptomicina na forma farmacêutica. Desta forma, o objetivo deste trabalho foi a validação de método espectrofotométrico na região do UV ambientalmente favorável para análise da daptomicina em pó liofilizado. A água foi escolhida como diluente e as análises foram realizadas em 221 nm. O método atendeu a todas as exigências de validação dos guias do ICH, na faixa de 6-21 µg mL-1. Teste t de Student mostrou que o método proposto é intercambiável com método de HPLC previamente validado. Assim, o método espectrofotométrico validado é capaz de quantificar a daptomicina em pó para solução injetável e é uma opção econômica, rápida e "verde" para análises de rotina do controle de qualidade deste fármaco.

Development, Optimization, and Validation of a Green and Stability-Indicating HPLC Method for Determination of Daptomycin in Lyophilized Powder.

Daptomycin is an antimicrobial that plays an important role in clinical practice today because it is considered a promising drug to combat resistant strains, such as methicilin and vancomycin-resistant Gram-positive bacteria. Considering the analysis of daptomycin in a pharmaceutical dosage form, the only method found in literature uses potentially toxic organic solvents. Therefore, the objective of this work was to develop a green and stability-indicating HPLC method for determination of daptomycin in lyophilized powder. The mobile phase was ethanol-water (55+45, v/v) at pH 4.5 pumped at a flow rate of 0.6 mL/min. A C18 column was used, and UV detection was performed at 221 nm. Stress degradation studies were conducted in order to demonstrate the specificity and stability-indicating capability of the method. The method was validated according to International Conference on Harmonization guidelines, proving to be linear (r=0.9996), precise, accurate, robust (demonstrated by the Plackett-Burman model), and specific within the range 20-70 µg/mL. The retention time of daptomycin was 5.8 min. It can be concluded that the validated method can be a fast, safe, and environmentally friendly alternative for the analysis of daptomycin.

Stability of daptomycin 5 mg/mL and heparin sodium 100 units/mL combined in lactated Ringer's injection and stored in polypropylene syringes at 4 and -20°C.

PURPOSE: The stability of an admixture containing reconstituted daptomycin and heparin in lactated Ringer's injection was evaluated. METHODS: Two samples of the admixture of daptomycin 5 mg/mL and heparin sodium 100 USP units/mL diluted in lactated Ringer's injection were prepared and divided into 5-mL portions for storage in syringes at 4 and -20 °C for 14 days. The percentage of the initial concentration of the drugs remaining in the syringes was assessed using a high-performance liquid chromatographic (HPLC) method with diode-array detection previously validated as stability indicating for both drugs. Forced degradation studies were performed independently with each drug diluted in lactated Ringer's injection. One sample from each stored syringe was analyzed in triplicate on days 0, 1, 2, 3, 4, 7, and 14; quality-control samples of each concentration tested were used throughout the analysis. The admixture samples were visually inspected for color, clarity, and the formation of particulate matter. RESULTS: The HPLC analysis indicated no significant reduction (loss of ≤5%) in the concentration of daptomycin and heparin diluted in lactated Ringer's injection stored in syringes refrigerated at 4 °C and frozen at -20 °C. None of the chromatographic peaks observed in samples subjected to forced degradation were detected in any sample during the 14-day study. All of the syringe-stored samples remained clear and colorless on visual inspection for the duration of the study. CONCLUSION: The admixture of daptomycin 5 mg/mL and heparin sodium 100 USP units/mL was stable when stored in polypropylene syringes for up to 14 days at 4 and -20 °C.

Correlation of daptomycin resistance in a clinical Staphylococcus aureus strain with increased cell wall teichoic acid production and D-alanylation.

Cell wall thickening is a common feature among daptomycin-resistant Staphylococcus aureus strains. However, the mechanism(s) leading to this phenotype is unknown. We examined a number of cell wall synthesis pathway parameters in an isogenic strain set of S. aureus bloodstream isolates obtained from a patient with recalcitrant endocarditis who failed daptomycin therapy, including the initial daptomycin-susceptible parental strain (strain 616) and two daptomycin-resistant strains (strains 701 and 703) isolated during daptomycin therapy. Transmission electron microscopy demonstrated significantly thicker cell walls in the daptomycin-resistant strains than in the daptomycin-susceptible strain, a finding which was compatible with significant differences in dry cell weight of strain 616 versus strains 701 to 703 (P < 0.05). Results of detailed analysis of cell wall muropeptide composition, the degree of peptide side chain cross-linkage, and the amount of the peptidoglycan precursor, UDP-MurNAc-pentapeptide, were similar in the daptomycin-susceptible and daptomycin-resistant isolates. In contrast, the daptomycin-resistant strains contained less O-acetylated peptidoglycan. Importantly, both daptomycin-resistant strains synthesized significantly more wall teichoic acid (WTA) than the parental strain (P < 0.001). Moreover, the proportion of D-alanylated WTA species was substantially higher in the daptomycin-resistant strains than in the daptomycin-susceptible parental strain (P < 0.05 in comparing strain 616 versus strain 701). The latter phenotypic findings correlated with (i) enhanced tagA and dltA gene expression, respectively, and (ii) an increase in surface positive charge observed in the daptomycin-resistant versus daptomycin-susceptible isolates. Collectively, these data suggest that increases in WTA synthesis and the degree of its D-alanylation may play a major role in the daptomycin-resistant phenotype in some S. aureus strains.

Polypyrrole solid phase microextraction: A new approach to rapid sample preparation for the monitoring of antibiotic drugs.

Simple or even rapid bioanalytical methods are rare, since they generally involve complicated, time-consuming sample preparation from the biological matrices like LLE or SPE. SPME provides a promising approach to overcome these limitations. The full potential of this innovative technique for medical diagnostics, pharmacotherapy or biochemistry has not been tapped yet. In-house manufactured SPME probes with polypyrrole (PPy) coating were evaluated using three antibiotics of high clinical relevance - linezolid, daptomycin, and moxifloxacin - from PBS, plasma, and whole blood. The PPy coating was characterised by scanning electron microscopy. Influences of pH, inorganic salt, and blood anticoagulants were studied for optimum performance. Extraction yields were determined from stagnant media as well as re-circulating human blood using the heart-and-lung machine model system. The PPy-SPME fibres showed high extraction yields, particularly regarding linezolid. The reproducibility of the method was optimised to achieve RSDs of 9% or 17% and 7% for SPME from stagnant or re-circulating blood using fresh and re-used fibres, respectively. The PPy-SPME approach was demonstrated to meet the requirements of therapeutic monitoring of the drugs tested, even from re-circulating blood at physiological flow rates. SPME represents a rapid and simple dual-step procedure with potency to significantly reduce the effort and expenditure of complicated sample preparations in biomedical analysis.

Daptomycin determination by liquid chromatography-mass spectrometry in peritoneal fluid, blood plasma, and urine of clinical patients receiving peritoneal dialysis treatment.

A 13-min LC-MS method was developed for the determination of daptomycin, a new potent antibiotic, in peritoneal fluid, blood plasma, and urine of patients receiving renal replacement therapy. Chromatography was performed on a C(18) column and detection was performed by a single-quadrupole mass spectrometer coupled to LC via an electrospray interface (ESI). The column effluent was also monitored at 370 nm using a photodiode-array detector. The developed method provided a linear dynamic range for concentrations from 0.5 microg mL(-1) to 100 microg mL(-1). Method precision and accuracy were found to be satisfactory for clinical application, thus the method was successfully used for the analysis of daptomycin in pharmacokinetic studies. The drug was preventively administered against Gram-positive infections to 19 clinical patients undergoing peritoneal dialysis. Peritoneal fluid, blood plasma, and urine samples were collected at 13 time points over a period of 48 h. Clinical samples were analysed following simple sample-preparation procedures and daptomycin was unambiguously detected and quantified.

Effect of divalent cations on the structure of the antibiotic daptomycin.

Daptomycin, a cyclic anionic lipopeptide antibiotic, whose three-dimensional structure was recently solved using solution state NMR (Ball et al. 2004; Jung et al. 2004; Rotondi and Gierasch 2005), requires calcium for function. To date, the exact nature of the interaction between divalent cations, such as Ca(2+) or Mg(2+), has not been fully characterized. It has, however, been suggested that addition of Ca(2+) to daptomycin in a 1:1 molar ratio induces aggregation. Moreover, it has been suggested that certain residues, e.g. Asp3 and Asp7, which are essential for activity (Grunewald et al. 2004; Kopp et al. 2006), may also be important for Ca(2+) binding (Jung et al. 2004). In this work, we have tried: (1) to further pinpoint how Ca(2+) affects daptomycin structure/oligomerization using analytical ultracentrifugation; and (2) to determine whether a specific calcium binding site exists, based on one-dimensional (13)C NMR spectra and molecular dynamics (MD) simulations. The centrifugation results indicated that daptomycin formed micelles of between 14 and 16 monomers in the presence of a 1:1 molar ratio of Ca(2+) and daptomycin. The (13)C NMR data indicated that addition of calcium had a significant effect on the Trp1 and Kyn13 residues, indicating that either calcium binds in this region or that these residues may be important for oligomerization. Finally, the molecular dynamics simulation results indicated that the conformational change of daptomycin upon calcium binding might not be as significant as originally proposed. Similar studies on the divalent cation Mg(2+) are also presented. The implication of these results for the biological function of daptomycin is discussed.

Studies on the reactions between daptomycin and glyceraldehyde.

The objectives of this project were to determine the reaction pathways of daptomycin in the presence of glyceraldehyde in acidic solutions, and to quantitate the kinetics of the major pathways. In the presence of glyceraldehyde (pH range 1-7 at 25 to 60 degrees C), daptomycin formed two major products separable by RP-HPLC. The products were identified using UV spectroscopy, fluorimetry, mass spectrometry, and 2D-1H NMR. The reaction scheme involved the reversible formation of imine and anilide derivatives. Carbinolamine was believed to be a common intermediate in formation pathways of both products. The carbinolamine intermediate underwent either acid catalyzed dehydration resulting in imine formation or intramolecular hydrogen bonding and bond cleavage giving rise to anilide formation. In mild acid conditions, both products reversed to daptomycin. The reaction between daptomycin and glyceraldehyde was first-order with respect to both reactants. In a pH range of 1-7, the imine formation rate was pH dependent with a maximum rate at approximate pH values of 3-4. The observed pH dependence was consistent with the pH dependence of typical amine-aldehyde reactions.