Reproducible Quantification of Unbound Fractions of Four Beta-Lactam Antibiotics: Ultrafiltration Versus Microdialysis of Spiked Healthy Donor Plasma.

BACKGROUND: Ultrafiltration (UF) is a conventional method for isolating the protein-unbound plasma fractions of therapeutic drugs. However, the ideal UF conditions for specific compounds remain largely unexplored. By comparing UF-derived unbound concentrations with the corresponding results obtained using a reference method, the authors sought to identify appropriate UF conditions for cefotaxime, cloxacillin, flucloxacillin, and piperacillin. METHODS: In vitro microdialysis (MD) with a no-net-flux approach was used as a reference method for plasma protein separation, for which UF performance was assessed. Four levels of relative centrifugal force (2500-11,290 g ) and 2 levels of temperature (37 vs. 22°C) during 10 minutes of UF centrifugation were evaluated. Ultrafiltrates and reference microdialysates were analyzed using liquid chromatography-tandem mass spectrometry to obtain unbound concentrations. After identifying the appropriate UF conditions in the spiked plasma samples, exploratory analyses of clinical samples (n = 10 per analyte) were performed. RESULTS: Of the evaluated UF alternatives, the best overall agreement with the MD-derived reference concentrations was obtained with 11,290 g UF performed at 22°C. For cloxacillin specifically, 37°C UF yielded better agreement than 22°C UF at 11,290 g. Clinical sample analyses indicated minimal differences between 22°C and 37°C at 11,290 g UF for cefotaxime and piperacillin. However, consistently lower levels of unbound cloxacillin (median: -23%, IQR: -19% to -24%) and flucloxacillin (median: -27%, IQR: -21 to -34%) were observed after UF at 22°C versus 37°C. CONCLUSIONS: For the evaluated UF device, 10 minutes of 11,290 g UF at 22°C is appropriate for flucloxacillin, cefotaxime, and piperacillin, and can arguably be justified for cloxacillin as well for laboratory practice purposes. Maintenance of 37°C during high-centrifugal UF may lead to overestimation, particularly for unbound flucloxacillin.

Pronounced Interindividual But Not Intraindividual Variation in Tamoxifen and Metabolite Levels in Plasma During Adjuvant Treatment of Women With Early Breast Cancer.

BACKGROUND: Tamoxifen is still an important antihormonal treatment option for patients with breast cancer and estrogen receptor-positive tumors. More than 20% of patients relapse despite treatment. The drug is usually dosed 20 mg/d irrespective of interindividual variation in drug clearance. To study interindividual and intraindividual variation in plasma levels we measured tamoxifen and metabolite levels in plasma on 2 occasions, with at least 4 weeks in between, of 39 women (19 premenopausal and 20 postmenopausal women) on adjuvant treatment (20 mg/d) of early breast cancer. METHODS: We used an ultra-performance liquid chromatography with a mass spectrometry detection method for identification and quantification of tamoxifen, N-desmethyltamoxifen, 4-OH-tamoxifen, and endoxifen. Follicle-stimulating hormone, luteinizing hormone, and estradiol levels were also measured. RESULTS: The plasma concentrations of tamoxifen and its metabolites showed a pronounced interindividual variation, whereas intraindividual concentrations were rather stable. Despite the same dosage, interindividual tamoxifen concentrations varied from 51 to 307 ng/mL (124 ± 57, mean ± SD) and endoxifen values showed a range from 3.2 to 19 ng/mL (10.4 ± 5.2, mean ± SD), that is, 6-fold variation for both. CONCLUSIONS: Large interindividual variation of tamoxifen and endoxifen with stable intraindividual levels, and too low levels of endoxifen in a considerable proportion of patients strongly support that therapeutic drug monitoring and individualized dosing could lead to optimal exposure and hopefully better outcome. A randomized outcome study between conventional dosing and therapeutic drug monitoring-guided dosing is needed to show whether this approach works.

Structural studies of the O-antigenic polysaccharide from Plesiomonas shigelloides strain AM36565.

The structure of the repeating unit of the O-antigenic polysaccharide from Plesiomonas shigelloides strain AM36565 has been determined. Component analysis and (1)H and (13)C NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by (1)H,(13)C heteronuclear multiple-bond correlation, (1)H,(1)H-NOESY, and (1)H,(13)C-HSQC-(1)H,(1)H-NOESY experiments. The O-antigen polysaccharide is composed of repeating units with the following structure: →3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→4)[ß-D-GalpNAc-(1→3)]-α-D-GlcpNAc-(1→, in which the monosaccharide side-chain substitutes the backbone in half of the repeating units. A matrix-assisted laser desorption/ionization mass spectrometry experiment suggested that the polysaccharide consists of two regions, one with tetrasaccharide repeating units and one with trisaccharide repeating units.

Molecular conformations in the pentasaccharide LNF-1 derived from NMR spectroscopy and molecular dynamics simulations.

The conformational dynamics of the human milk oligosaccharide lacto-N-fucopentaose (LNF-1), α-L-Fucp-(1 → 2)-ß-D-Galp-(1 → 3)-ß-D-GlcpNAc-(1 → 3)-ß-D-Galp-(1 → 4)-D-Glcp, has been analyzed using NMR spectroscopy and molecular dynamics (MD) computer simulations. Employing the Hadamard (13)C-excitation technique and the J-HMBC experiment, (1)H,(13)C trans-glycosidic J coupling constants were obtained, and from one- and two-dimensional (1)H,(1)H T-ROESY experiments, proton-proton cross-relaxation rates were determined in isotropic D(2)O solution. In the lyotropic liquid-crystalline medium consisting of ditetradecylphosphatidylcholine, dihexylphosphatidylcholine, N-cetyl-N,N,N-trimethylammonium bromide, and D(2)O, (1)H, (1)H and one-bond (1)H, (13)C residual dipolar couplings (RDCs), as well as relative sign information on homonuclear RDCs, were determined for the pentasaccharide. Molecular dynamics simulations with explicit water were carried out from which the internal isomerization relaxation time constant, τ(N), was calculated for transitions at the ψ torsion angle of the ß-(1 → 3) linkage to the lactosyl group in LNF-1. Compared to the global reorientation time, τ(M), of ∼0.6 ns determined experimentally in D(2)O solution, the time constant for the isomerization relaxation process, τ(N(scaled)), is about one-third as large. The NMR parameters derived from the isotropic solution show very good agreement with those calculated from the MD simulations. The only notable difference occurs at the reducing end, which should be more flexible than observed by the molecular simulation, a conclusion in complete agreement with previous (13)C NMR relaxation data. A hydrogen-bond analysis of the MD simulation revealed that inter-residue hydrogen bonds on the order of ∼30% were present across the glycosidic linkages to sugar ring oxygens. This finding highlights that intramolecular hydrogen bonds might be important in preserving well-defined structures in otherwise flexible molecules. An analysis including generalized order parameters obtained from nuclear spin relaxation experiments was performed and successfully shown to limit the conformational space accessible to the molecule when the number of experimental data are too scarce for a complete conformational analysis.

NMR studies of molecular conformations in alpha-cyclodextrin.

A new approach for analysis of NMR parameters is proposed. The experimental data set includes scalar couplings, NOEs, and residual dipolar couplings. The method, which aims at construction of the conformational distribution function, is applied to alpha-cyclodextrin in isotropic solution and dissolved in a dilute liquid crystal. An attempt to analyze the experimental data using an average molecular conformation resulted in unacceptable errors. Our approach rests on the maximum entropy method (ME), which gives the flattest possible distribution, consistent with the experimental data. Very good agreement between experimental and calculated NMR parameters was observed. In fact, two conformational states were required in order to obtain a satisfactory agreement between calculated and experimental data. In addition, good agreement with Langevin dynamics computer simulations was obtained.

Molecular conformations of a disaccharide investigated using NMR spectroscopy.

The molecular structure of alpha-L-Rhap-(1--> 2)-alpha-L-Rhap-OMe has been investigated using conformation sensitive NMR parameters: cross-relaxation rates, scalar 3J(CH) couplings and residual dipolar couplings obtained in a dilute liquid crystalline phase. The order matrices of the two sugar residues are different, which indicates that the molecule cannot exist in a single conformation. The conformational distribution function, P(phi, psi) related to the two glycosidic linkage torsion angles phi and psi was constructed using the APME method, valid in the low orientational order limit. The APME approach is based on the additive potential (AP) and maximum entropy (ME) models. The analyses of the trajectories generated in molecular dynamics and Langevin dynamics (LD) computer simulations gave support to the distribution functions constructed from the experimental NMR parameters. It is shown that at least two conformational regions are populated on the Ramachandran map and that these regions exhibit very different molecular order.