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Xiong Y.,Arena Pharmaceuticals | Ullman B.,Arena Pharmaceuticals | Choi J.-S.K.,Arena Pharmaceuticals | Cherrier M.,Biotage | And 10 more authors.
Bioorganic and Medicinal Chemistry Letters | Year: 2012

A series of fused bicyclic heterocycles was identified as potent and selective 5-HT 2A receptor antagonists. Optimization of the series resulted in compounds that had improved PK properties, favorable CNS partitioning, good pharmacokinetic properties, and significant improvements on deep sleep (delta power) and sleep consolidation. © 2012 Elsevier Ltd. All rights reserved. Source


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All animal procedures were conducted under a protocol (#08–1990) approved by the Genentech Institutional Animal Care and Use Committee in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facility in accordance with the Guide for the Care and Use of Laboratory Animals and applicable laws and regulations. For cloning of antibodies from human B cells, informed written consent was obtained from all donors and was provided in accordance with the Declaration of Helsinki. Approval was obtained from the health research ethics committee of Denmark through the regional committee for the Capital Region of Denmark. All in vivo experiments were done with MRSA-USA300 NRS384 obtained from NARSA (https://www.beiresources.org) unless noted otherwise. The generation of protein-A-deficient strain Δmcr USA300 NRS384, as well as protein-A-deficient USA300 lacking tarM or tarS has been described previously38, 39. The protein-A-deficient strains were used only in some in vitro experiments to determine antibody specificity. The MIC for extracellular bacteria was determined by preparing serial twofold dilutions of the antibiotic in tryptic soy broth. Dilutions of the antibiotic were made in quadruplicate in 96-well culture dishes. MRSA (NRS384 strain of USA300) was taken from an exponentially growing culture and diluted to 1 × 104 c.f.u. ml−1. The bacteria were cultured in the presence of antibiotic for 18–24 h with shaking at 37 °C and bacterial growth was determined by reading the optical density (OD) at 630 nm. The MIC was determined to be the dose of antibiotic that inhibited bacterial growth by >90%. Intracellular MIC was determined on bacteria that were sequestered inside mouse peritoneal macrophages (see later for generation of murine peritoneal macrophages). Macrophages were plated at a density of 4 × 105 cells ml−1 and infected with MRSA at a ratio of 10–20 bacteria per macrophage. Macrophage cultures were maintained in growth media supplemented with 50 μg ml−1 of gentamycin to inhibit the growth of extracellular bacteria and test antibiotics were added to the growth media 1 day after infection. The survival of intracellular bacteria was assessed 24 h after addition of the antibiotics. Macrophages were lysed with Hanks buffered saline solution supplemented with 0.1% bovine serum albumin (BSA) and 0.1% Triton-X, and serial dilutions of the lysate were made in PBS solution containing 0.05% Tween-20. The number of surviving intracellular bacteria was determined by plating on tryptic soy agar plates with 5% defibrinated sheep blood. USA300 stocks were prepared for infection from actively growing cultures in tryptic soy broth. Bacteria were washed three times in PBS and aliquots were frozen at −80 °C in PBS 25% glycerol. Intracellular bacteria infections. Seven-week-old female A/J mice (Stock 000646) were obtained from Jackson Labs and infected by peritoneal injection with 5 × 107 c.f.u. of USA300. Mice were killed 1 day after infection and the peritoneum was flushed with 5 ml of cold PBS. Peritoneal washes were centrifuged for 5 min at 1,000 r.p.m. at 4 °C in a table-top centrifuge. The cell pellet containing peritoneal cells was collected and cells were treated with 50 μg ml−1 of lysostaphin (Cell Sciences, CRL 309C) for 20 min at 37 °C to kill contaminating extracellular bacteria. Peritoneal cells were washed three times in ice-cold PBS to remove the lysostaphin. Peritoneal cells from donor mice were pooled, and recipient mice were injected with cells derived from five donors per each recipient by intravenous injection into the tail vein. To determine the number of live intracellular colony-forming units, a sample of the peritoneal cells were lysed in HB (Hanks balanced salt solution supplemented with 10 mM HEPES and 0.1% BSA) with 0.1% Triton-X, and serial dilutions of the lysate were made in PBS with 0.05% Tween-20. Free bacteria infections. A/J mice were infected with various doses of free bacteria using a fresh aliquot of the glycerol stocks used for the peritoneal injections. Actual infection doses were confirmed by c.f.u. plating. For the data shown in Fig. 1a the actual infection dose for intracellular bacteria was 1.8 × 106 c.f.u. per mouse, and the actual infection dose for free bacteria was 2.9 × 106 c.f.u. per mouse. Selected mice were treated with a single dose of 110 mg kg−1 of vancomycin by intravenous injection immediately after infection. Generation of MRSA-infected peritoneal cells. Six-to-eight-week-old female A/J mice (see earlier) were infected with 1 × 108 c.f.u. of the NRS384 strain of USA300 by peritoneal injection. The peritoneal wash was harvested 1 day after infection, and the infected peritoneal cells were treated with 50 μg ml−1 of lysostaphin diluted in HEPES buffer supplemented with 0.1% BSA (HB buffer) for 20 min at 37 °C. Peritoneal cells were then washed twice in ice-cold HB buffer. The peritoneal cells were diluted to 1 × 106 cells ml−1 in RPMI 1640 tissue culture media supplemented with 10 mM HEPES and 10% fetal calf serum, and 5 μg ml−1 vancomycin. Free MRSA from the primary infection was stored overnight at 4 °C in PBS solution as a control for extracellular bacteria that were not subject to neutrophil killing. Infection of osteoblasts, HBMEC and A549 cells. MG63 cell line (CRL-1427) and A549 cells (CCL185) were obtained from ATCC and maintained in RPMI 1640 tissue culture media supplemented with 10 mM HEPES and 10% fetal calf serum (RPMI-10). HBMEC cells (catalogue #1000) and ECM media (catalogue #1001) were obtained from ScienceCell Research Labs. The cells were used without further authentication or testing for mycoplasma contamination. Cells were plated in 24-well tissue culture plates and cultured to obtain a confluent layer. On the day of the experiment, the cells were washed once in RPMI (without supplements). MRSA or infected peritoneal cells were diluted in complete RPMI-10 and vancomycin was added at 5 μg ml−1 immediately before infection. Peritoneal cells were added to the osteoblasts at 1 × 106 peritoneal cells per ml. A sample of the cells was lysed with 0.1% Triton-X to determine the actual concentration of live intracellular bacteria at the time of infection. The actual titre for all infections was determined by plating serial dilutions of the bacteria on tryptic soy agar with 5% defibrinated sheep blood. The human IgG antibodies against anti-β-GlcNAc WTA monoclonal antibody (mAb) and anti-α-GlcNAc WTA mAb were cloned from peripheral B cells from patients after S. aureus infection using a monoclonal antibody discovery technology that conserves the cognate pairing of antibody heavy and light chains40. Antibodies were expressed by transfection of mammalian cells41. Supernatants containing full-length IgG1 antibodies were harvested after 7 days and used to screen for antigen binding by enzyme-linked immunosorbent assay (ELISA). These antibodies were positive for binding to cell wall preparations from USA300. Antibodies were subsequently produced in 200-ml transient transfections and purified with protein A chromatography (MabSelect SuRe, GE Life Sciences) for further testing. Synthesis of the rifalogue linker drug was performed as follows. Protease cleavable linker MC-VC-PAB-OH23 (1.009 g, 1.762 mmol, 1.000, 1,009 mg) was taken up in N,N-dimethylformamide (6 ml, 77 mmol, 44, 5,700 mg). To this was added a solution of thionyl chloride (1.1 equiv., 1.938 mmol, 1.100, 231 mg) in dichloromethane (DCM) (1 ml, 15.44 mmol, 8.765, 1,325 mg) in portions dropwise (half was added over 1 h, stirred for 1 h at room temperature, then the other half was added over another hour). The solution remained a yellow colour. Another 0.6 equiv. of thionyl chloride was added as a solution in 0.5 ml DCM dropwise, carefully. The reaction remained yellow and was stirred sealed overnight at room temperature. The reaction was monitored by liquid chromatography mass spectrometry (LC/MS), indicating 88% conversion to benzyl chloride. Another 0.22 equiv. of thionyl chloride was added dropwise as a solution in 0.3 ml DCM. When the reaction approached 92% benzyl chloride, the reaction was bubbled with N . The concentration was increased from 0.3 M to 0.6 M. MC-VC-PAB-Cl (0.9 mmol) was cooled to 0 °C and rifalogue (dimethyl piperazinebenzoxazinorifamycin42 (0.75 g, 0.81 mmol, 0.46, 750 mg)) was added. The mixture was diluted with another 1.5 ml of DMF to reach 0.3 M. Stirred open to air for 30 min. N,N-diisopropylethylamine (3.5 mmol, 3.5 mmol, 2.0, 460 mg) was added and the reaction stirred overnight open to air. Over the course of 4 days, four additions of 0.2 equiv. N,N-diisopropylethylamine base were added while the reaction stirred open to air, until the reaction appeared to stop progressing. The reaction was diluted with DMF and purified on high-performance liquid chromatography (HPLC; 20–60% ACN/FA·H O) in several batches to give MC-VC-PAB-rifalogue (0.38 g, 32% yield) m/z = 1,482.8. The non-cleavable rifalogue linker drug was synthesized using the exact same method, but replacing MC-VC-PAB-OH with MC-V-D-Cit-PAB-OH. Construction and production of the THIOMAB variant of anti-WTA antibody was done as reported previously43. Briefly, a cysteine residue was engineered at the Val 205 position of the anti-WTA light chain to produce its THIOMAB variant. The thio anti-WTA was conjugated to MC-vc-PAB-rifalogue. The antibody was reduced in the presence of 50-fold molar excess dithiothreitol (DTT) overnight. The reducing agent and the cysteine and glutathione blocks were purified away using HiTrap SP-HP column (GE Healthcare). The antibody was re-oxidized in the presence of 15-fold molar excess dehydroascorbic acid (MP Biomedical) for 2.5 h. The formation of interchain disulfide bonds was monitored by LC/MS. A threefold molar excess of the linker drug (MC-VC-PAB-rifalogue) over protein was incubated with the THIOMAB for 1 h. The AAC was purified by filtration through a 0.2 μm SFCA filter (Millipore). Excess-free linker drug was removed by filtration. The conjugate was buffer exchanged into 20 mM histidine acetate pH 5.5/240 mM sucrose by dialysis. The number of conjugated MC-VC-PAB-rifalogue molecules per mAb was quantified by LC/MS analysis. Purity was also assessed by size-exclusion chromatography. LC/MS analysis was performed on a 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS (Agilent Technologies). Samples were chromatographed on a PRLP-S column, 1,000 Å, 8 μm (50 mm × 2.1 mm, Agilent Technologies) heated to 80 °C. A linear gradient from 30–60% B in 4.3 min (solvent A, 0.05% TFA in water; solvent B, 0.04% TFA in acetonitrile) was used and the eluent was directly ionized using the electrospray source. Data were collected and deconvoluted using the Agilent Mass Hunter qualitative analysis software. Before LC/MS analysis, AAC was treated with lysyl endopeptidase (Wako) for 30 min at 1:100 w/w enzyme to antibody ratio, pH 8.0, and 37 °C to produce the Fab and the Fc portion for ease of analysis. The drug-to-antibody ratio (DAR) was calculated using the abundance of Fab and Fab+1 calculated by the MassHunter software. Analysis of bacteria isolated from infected mice. Balb/c mice were infected with 1 × 107 c.f.u. of MRSA (USA300) by intravenous injection and kidneys were harvested on day 3 after infection. Kidneys were homogenized using a GentleMACS dissociator in 5 ml volume per two kidneys using M-Tubes and the program RNA01.01 (Miltenyi Biotec). Homogenization buffer was: PBS plus 0.1% Triton-X-100, 10 μg ml−1 DNAase (bovine pancreas grade II, Roche) and protease inhibitors (complete protease inhibitor cocktail, Roche 11-836-153001). After homogenization, the samples were incubated at room temperature for 10 min and then diluted with ice-cold PBS and filtered through a 40 μm cell strainer. Tissue homogenates were washed twice in ice-cold PBS and then suspended in a volume of 0.5 ml per two kidneys in HB buffer (Hanks balanced salt solution supplemented with 10 mM HEPES and 0.1% BSA). The cell suspension was filtered again and 25 μl of the bacterial suspension was taken for each staining reaction (Fig. 2c). Antibody staining for flow cytometry. Bacteria (1 × 107 of in vitro grown bacteria (Fig. 2d), or 25 μl of tissue homogenate described earlier (Fig. 2c) were suspended in HB buffer and blocked by incubation with 400 μg ml−1 of mouse IgG (Sigma, I5381) for 1 h. Fluorescently labelled antibodies were added directly to the blocking reaction and incubated at room temperature for an additional 10–20 min. Bacteria were washed three times in HB buffer and then fixed in PBS 2% paraformaldehyde before FACS analysis. Test antibodies (anti-β-WTA, anti-α-WTA or isotype control-anti CMV-gD) were conjugated with Alexa-488 using amine reactive reagents (Invitrogen, succinimidyl-ester of Alexa Fluor 488, NHS-A488). Antibodies in 50 mM sodium phosphate were reacted with a 5–10-fold molar excess of NHS-A488 in the dark for 2–3 h at room temperature. The labelling mixture was applied to a GE Sepharose S200 column equilibrated in PBS to remove excess reactants from the conjugated antibody. The number of A488 molecules per antibody was determined using the ultraviolet method as described by the manufacturer. For analysis of bacteria in tissue homogenates a non-competing anti-S. aureus antibody (rF1 (ref. 38)) was conjugated to Alexa-647 to distinguish S. aureus from similar sized particles. Test antibodies were examined at a range of doses from 80 ng ml−1 to 50 μg ml−1. Flow cytometry was performed using a Beckton Dickson FACS ARIA (BD Biosciences) and analysis was performed using FlowJo analysis software (Flow Jo LLC). The anti-β-WTA antibody Fab fragment was expressed in Escherichia coli and purified on Protein G Sepharose followed by SP sepharose cation exchange and size-exclusion chromatography. Antibody was concentrated to 30 mg ml−1 in MES buffer (20 mM MES pH 5.5, 150 mM NaCl) and mixed with a 2:1 mol/mol ratio of the WTA analogue (diluted in water) for crystallization trials. Sparse matrix crystallization screening provided initial hits in PEG-8000 based conditions, which were further optimized to provide diffraction quality crystals. Ultimately, data were collected on a crystal grown by the vapour diffusion method in a sitting drop containing 0.5 μl protein and 0.5 μl 0.08 M sodium cacodylate pH 6.5, 0.16 M calcium acetate, 14.4% PEG-8000, and 20% glycerol. Crystals were cryo-protected in mother liquor, flash frozen in liquid nitrogen, and stored for data collection at 100 K. Data were collected to 1.7 Å at beamline 22ID at the Advanced Photon Source (APS) under cryo-cooled conditions (100 K) at a wavelength of 1.0 Å. Data were reduced using HKL2000 and SCALEPACK in the space group P2 2 2 , with unit cell parameters of a = 63.7 Å, b = 111.4Å, c = 158.4 Å (see Extended Data Table 1 for processing statistics). The structure was solved by sequential molecular replacement searches using Fab constant and variable regions (Protein Data Bank accession 4177) as individual search models. Iterative rounds of manual model adjustment with COOT followed by simulated annealing, coordinate, and b-factor refinement with Phenix and BUSTER (Global Phasing) gave a final model with R/R values of 20.6% and 23.7% respectively. Ramachandran statistics calculated by MolProbity indicate that 97.2% of the model residues lie in favoured regions, with 0.5% outliers. Synthesis of dibenzyl phosphorochloridate. A mixture of NCS (3.5 g, 26.6 mmol) was suspended in toluene (80 ml). Then dibenzyl phosphonate (2.0 g, 7.6 mmol) was added. The mixture was stirred at room temperature overnight. The white solid was filtered off and the organic phase was evaporated to give dibenzyl phosphorochloridate (1; 2.1 g, 96%) as light yellow oil. 1H NMR (300 MHz, CDCl , 25 °C) δ 7.36 (s, 10H), 5.20 (m, 4H). Synthesis of 4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-1-O-acetyl-d-ribitol-5-dibenzylphosphate. A mixture of 2 (described in ref. 44) (500 mg, 0.95 mmol) dissolved in pyridine (12 ml) was cooled to −30 °C and 1 (described ref. 44) (595 mg, 2.0 mmol) was added, stirring for 2 h at −30 °C and warmed to room temperature for 4 h. The mixture was added to H O, and concentrated in vacuo. The residue was purified by column chromatography (silica gel: 200 to ~300 mesh; dichloromethane: methanol in a 30:1 as eluent) to give 4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-1-O-acetyl-d-ribitol-5-dibenzylphosphate (3; 190 mg, 24%) as light yellow solid. 1H NMR (300 MHz, Acetone-d , 25 °C) δ 7.29–7.23 (m, 10H), 7.08 (d, 1H), 5.08 (t, 1H), 4.99–4.78 (m, 6H), 4.31–3.97 (m, 8H), 3.82–3.63 (m, 3H), 1.88 (s, 3H), 1.86 (s, 6H), 1.79 (s, 3H), 1.69 (s, 3H). LC/MS (m/z) ES+ 784 [M+H]+. Synthesis of 4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-1-O-acetyl-d-ribitol-5-phosphate. A mixture of 3 (150 mg, 0.19 mmol) dissolved in MeOH (6 ml) was hydrogenated over 10% Pd/C (20 mg) for 2 h at room temperature. Then the mixture was filtered, and the filtrate was evaporated to give 4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-1-O-acetyl-d-ribitol-5-phosphate (4; 100 mg) as light yellow oil. LC/MS (m/z) ES+ 604 [M+H]+. Synthesis of 4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-d-ribitol-5-phosphate (5). A mixture of 4 (80 mg, 0.16 mmol) dissolved in MeOH (10 ml) was cooled to 5 °C and K CO (30 mg, 0.21 mmol) was added and stirred at 5 °C for 3 h. The reaction was then quenched with 1 N HCl, and concentrated in vacuo. The crude product was purified by gel filtration (LH-20, MeOH) to give 4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-d-ribitol-5-phosphate (5; 13.3 mg, 23%) as a white solid 1H NMR (300 MHz, MeOH-d , 25 °C) δ 4.62 (d, 1H), 4.30–4.02 (m, 3H), 3.92–3.32 (m, 9H), 2.03 (s, 3H). LC/MS (m/z) ES+ 436 [M+H]+. S. aureus (USA300) was taken from an overnight stationary phase culture, washed once in PBS and suspended at 1 × 107 c.f.u. ml−1 in PBS with no antibiotic or with 1 × 10−6 M antibiotic in a 10 ml volume in 50 ml polypropylene centrifuge tubes. The bacteria were incubated at 37 °C overnight with shaking. At each time point, three 1 ml samples were removed from each culture and centrifuged to collect the bacteria. Bacteria were washed once with PBS to remove the antibiotic and the total number of surviving bacteria was determined by plating serial dilutions of the bacteria on agar plates. S. aureus (USA300) was taken from an overnight stationary phase culture, washed once in tryptic soy broth (TSB) and then adjusted to a final concentration of 1 × 107 c.f.u. ml−1 in a total volume of 10 ml of either TSB or TSB with ciprofloxicin (0.05 mM). Cultures were incubated with shaking at 37 °C for 6 h and then the second antibiotic, either rifampicin (1 μg ml−1) or the rifalogue (1 μg ml−1) was added. At the indicated times, samples were removed from each culture, washed once with PBS to remove the antibiotic and re-suspended in PBS. The total number of surviving bacteria was determined by plating serial dilutions of the bacteria on agar plates. At the final time point the remainder of each culture was collected and plated. To quantify the amount of active antibiotic released from AACs after treatment with cathepsin B, AACs were diluted to 200 μg ml−1 in cathepsin buffer (20 mM sodium acetate, 1 mM EDTA, 5 mM l-cysteine, pH 5). Cathepsin-B (from bovine spleen, Sigma C7800) was added at 10 μg ml−1 and the samples were incubated for 1 h at 37 °C. As a control, AACs were incubated in buffer alone. The reaction was stopped by addition of 9 volumes of bacterial growth media, TSB pH 7.4. To estimate the total release of active antibiotic, serial dilutions of the reaction mixture were made in quadruplicate in TSB in 96-well plates and MRSA (USA300) was added to each well at a final density of 2 × 103 c.f.u. ml−1. The cultures were incubated overnight at 37 °C with shaking and bacterial growth was measured by reading absorbance at 630 nM using a plate reader. We synthesized and conjugated a maleimide FRET peptide to the anti-β-WTA THIOMAB antibody. We used a FRET pair of tetramethylrhodamine (TAMRA) and fluorescein. The maleimide FRET peptide was synthesized by standard Fmoc solid-phase chemistry using a PS3 peptide synthesizer (Protein Technologies; B.-C.L., M.D. and R.V., manuscript in preparation)27. Briefly, 0.1 mmol of Rink amide resin was used to generate C-terminal carboxamide. We used a Fmoc-Lys(Mtt)-OH at the N- and C-terminal residues in order to remove the Mtt group on the resin and carry out additional side-chain chemistry to attach TAMRA and fluorescein. The sequence of Val-Cit-Leu was added between the FRET pair as a cathepsin-cleavable spacer. The crude maleimide FRET peptide or maleimidocarproyl-K(TAMRA)-G-V-Cit-L-K(fluorescein) cleaved off from the resin was subjected to further purification by reverse-phase HPLC with a Jupiter 5u C4 column (5 μm, 10 mm × 250 mm; Phenomenex). Our FRET probe allows monitoring not only of the intracelluar trafficking of the antibody conjugate, but also the processing of the linker in the phagolysosome. The intact antibody conjugate fluoresces only in red due to the fluorescence resonance energy transfer from the donor. However, upon the substrate cleavage of the FRET peptide in the phagolysosome, the green fluorescence from the donor is expected to appear. Murine peritoneal macrophages were plated on chamber slides (Ibidi, catalogue 80826) in complete media as described for the macrophage intracellular killing assay. USA300 was labelled with Cell Tracker Violet (Invitrogen C10094) at 100 μg ml−1 in PBS 0.1% BSA by incubation for 30 min at 37 °C. The labelled bacteria were opsonized with the anti-β-WTA-FRET probe by incubation for 1 h in HB buffer. Macrophages were washed once immediately before addition of the opsonized bacteria, and bacteria were added to cells at 1 × 107 bacteria per ml. For no-phagocytosis controls, the macrophages were pre-treated with 60 nM Latrunculin A (Calbiochem) for 30 min before and during phagocytosis. The slides were placed on the microscope immediately after addition of bacteria to the cells and movies were acquired with a Leica SP5 confocal microscope equipped with an environmental chamber with CO and temperature controllers from Ludin. The images were captured every minute for a total time of 30 min using a Plan APO CS ×40, N.A: 1.25, oil immersion lens, and the 488 nm and 543 nm laser lines to excite Alexa-488 and TAMRA, respectively. Phase images were also recorded using the 543 nm laser line. Primary murine peritoneal macrophages or RAW 264.7 cells (purchased from ATCC) were infected in 24-well tissue culture dishes as described later for the intracellular killing assay with MRSA opsonized with AAC at 100 μg ml−1 in HB. The RAW 264.7 cells were used without further authentication or testing for mycoplasma contamination. After phagocytosis was complete, the cells were washed and 250 μl of complete media plus gentamycin was added to wells and the cells were incubated for the indicated time points. At each time point, the supernatant and cellular fractions were collected followed by acetonitrile (ACN) addition to 75% final concentration and incubated for 30 min. Cell and supernatant extracts were lyophilized by evaporation under N2 (TurboVap; Biotage) and reconstituted in 100 μl of 50% ACN, filtered using a 0.45 glass fibre filter plate (Phenomenex) and analysed by LC/MS/MS as follows. The rifalogue was separated on an Acquity UPLC (Waters Corporation) under gradient elution using a Phenomenex Kinetex XB-C18 column (100 Å, 50 × 2.1 mm internal diameter, 2.6 μm particle size). The column was maintained at room temperature. The mobile phase was a mixture of 10 mM ammonium acetate in water containing 0.1% formic acid (A) and 90% acetonitrile (B) at a flow rate of 1 ml min−1. The rifalogue was eluted with a gradient of 3–98% B over 1 min, followed by 0.8 min at 98% B, then 0.7 min of 3% B to re-equilibrate the column. The injection volume was 10 μl. The Triple Quad 6500 mass spectrometer (Ab Sciex) was operated in a positive ion multiple reaction-monitoring (MRM) mode. The rifalogue precursor (Q1) ion monitored was 927.6 m/z and the product (Q3) ion monitored was 895.2 m/z with collision energy at 27 eV and declustering potential at 191 V. The MS/MS setting parameters were as follows: ion spray voltage, 5,500 V; curtain gas, 40 psi; nebulizer gas (GS1), 35 psi, (GS2), 50 psi; temperature, 600 °C; and dwell time, 150 ms. Linear calibration curves were obtained for 0.41–100 nM concentration range by spiking rifalogue into cell or supernatant fractions (lacking MRSA or AAC) that were treated similarly to samples. Concentrations of rifalogue were calculated with MultiQuant software (Ab Sciex). Non-phagocytic cell types. MG63 (CRL-1427) and A549 (CCL185) cell lines were obtained from ATCC and maintained in RPMI 1640 tissue culture media supplemented with 10 mM HEPES and 10% fetal calf serum (RPMI-10). HUVEC cells were obtained from Lonza and maintained in EGM endothelial cell complete media (Lonza). HBMEC cells (catalogue #1000) and ECM media (catalogue #1001) were obtained from ScienceCell Research Labs. The cells were used without further authentication or testing for mycoplasma contamination. Murine macrophages. Peritoneal macrophages were isolated from the peritoneum of 6–8-week-old Balb/c mice (Charles River Laboratories). To increase the yield of macrophages, mice were pre-treated by intraperitoneal injection with 1 ml of thioglycolate media (Becton Dickinson). The thioglycolate media was prepared at a concentration of 4% in water, sterilized by autoclaving, and aged for 20 days to 6 months before use. Peritoneal macrophages were harvested 4 days after treatment with thioglycolate by washing the peritoneal cavity with cold PBS. Macrophages were plated in DMEM supplemented with 10% fetal calf serum, and 10 mM HEPES, without antibiotics, at a density of 4 × 105 cells well−1 in 24-well culture dishes. Macrophages were cultured overnight to permit adherence to the plate. Human M2 macrophages. CD14+ monocytes were purified from normal human blood using a Monocyte Isolation Kit II (Miltenyi, catalogue 130-091-153) and plated at 1.5 × 105 cells cm−2 on tissue culture dishes pre-coated with fetal calf serum (FCS) and cultured in RPMI 1640 media with 20% FCS plus 100 ng ml−1 rhM-CSF. Media was refreshed on day 1 and on day 7, the media was changed to 5% serum plus 20 ng ml−1 IL-4. Macrophages were used 18 h later. Assay protocol. In all experiments bacteria were cultured in TSB. To assess intracellular killing with AACs, USA300 was taken from an exponentially growing culture and washed in HB. AACs or antibodies were diluted in HB (Hanks balanced salt solution supplemented with 10 mM HEPES and 0.1% BSA) and incubated with the bacteria for 1 h to permit antibody binding to the bacteria (opsonization), and the opsonized bacteria were used to infect macrophages at a ratio of 10–20 bacteria per macrophage (4 × 106 bacteria in 250 μl of HB per well). Macrophages were pre-washed with serum-free DMEM media immediately before infection, and infected by incubation at 37 °C in a humidified tissue culture incubator with 5% CO to permit phagocytosis of the bacteria. After 2 h, the infection mix was removed and replaced with normal growth media (DMEM supplemented with 10% FCS, 10 mM HEPES) and gentamycin was added at 50 μg ml−1 to prevent growth of extracellular bacteria45. At the end of the incubation period, the macrophages were washed with serum-free media, and the cells were lysed in HB supplemented with 0.1% Triton-X (lyses the macrophages without damaging the intracellular bacteria). Serial dilutions of the lysate were made in PBS solution supplemented with 0.05% Tween-20 (to disrupt aggregates of bacteria) and the total number of surviving intracellular bacteria was determined by plating on tryptic soy agar with 5% defibrinated sheep blood. Cell wall preparations (CWPs) were generated from protein-A-deficient S. aureus by incubating 40 mg of pelleted bacteria per ml of 10 mM Tris-HCl (pH 7.4) supplemented with 30% raffinose, 100 μg ml−1 of lysostaphin (Cell Sciences), and EDTA-free protease inhibitor cocktail (Roche), for 30 min at 37 °C. The lysates were centrifuged at 11,600g for 5 min, and the supernatants containing cell wall components were collected. ELISA experiments were performed using standard protocols. Briefly, plates were pre-coated with CWP and then incubated with human IgG preparations: purified human IGIV Immune Globulin (ASD Healthcare), pooled serum from healthy donors or from MRSA patients. The concentrations of anti-staphylococcal IgG present in the serum or purified IgG were calculated by using a calibration curve that was generated with known concentrations of anti-peptidoglycan mAb (4479) against peptidoglycan. Seven-week-old female mice, Balb/c, were obtained from Jackson West, or SCID mice were obtained from Charles River Laboratories. Infections were carried out by intravenous injection into the tail vein. SCID-huIgG model: CB17.SCID mice were reconstituted with IGIV Immune Globulin (ASD Healthcare) using a dosing regimen optimized to achieve constant serum levels of >10 mg ml−1 of human IgG. IGIV was administered with an initial intravenous dose of 30 mg per mouse followed by a second dose of 15 mg per mouse by intraperitoneal injection after 6 h, and subsequent daily dosings of 15 mg per mouse by intraperitoneal injection for 3 consecutive days. Mice were infected 4 h after the first dose of IGIV with 2 × 107 c.f.u. of MRSA diluted in PBS by intravenous injection. The wild-type USA300, protein-A-sufficient strain was used for all in vivo experiments. Mice that received vancomycin were treated with twice daily intraperitoneal injections of 110 mg kg−1 of vancomycin starting between 6 and 24 h after infection for the duration of the study. Experimental therapeutics (AAC, anti-MRSA antibodies or free rifalogue antibiotic) were diluted in PBS and administered with a single intravenous injection 30 min to 24 h after infection. All mice were killed on day 4 after infection, and kidneys were harvested in 5 ml of PBS. The tissue samples were homogenized using a GentleMACS dissociator (Miltenyi Biotec). The total number of bacteria recovered per mouse (two kidneys) was determined by plating serial dilutions of the tissue homogenate in PBS 0.05% Tween on tryptic soy agar with 5% defibrinated sheep blood. All experiments were performed on biological replicates. Sample size for each experimental group per condition is reported in appropriate figure legends and Methods. For cell culture experiments, sample size was not predetermined, and all samples were included in the analysis. In animal experiments no statistical methods were used to predetermine sample size (n = number of mice per group), and all animals were used for analysis unless the mice died or had to be euthanized when found moribund. These cases are annotated in the figures. The mice were not randomized after infection, and the investigators were not blinded to outcome assessment. When appropriate, statistically significant differences between control and experimental groups were determined using Mann–Whitney tests.


Wooding K.M.,Aurora University | Wooding K.M.,University of Colorado at Denver | Hankin J.A.,University of Colorado at Denver | Johnson C.A.,University of Colorado at Denver | And 5 more authors.
Steroids | Year: 2015

Background A high-throughput, sensitive, specific, mass spectrometry-based method for quantitating estrone (E1), estradiol (E2), and testosterone (T) in postmenopausal human serum has been developed for clinical research. The method consumes 100 μl human serum for each measurement (triplicates consume 300 μl) and does not require derivatization. We adapted a commercially available 96-well plate for sample preparation, extraction, and introduction into the mass spectrometer on a single platform. Methods Steroid extraction from serum samples and mass spectrometer operational parameters were optimized for analysis of estradiol and subsequently applied to other analytes. In addition to determining the limit of detection (LOD) and limit of quantitation (LOQ) from standard curves, a serum LOQ (sLOQ) was determined by addition of known steroid quantities to serum samples. Mass spectrometric method quantitative data were compared to results using a state-of-the-art ELISA (enzyme-linked immunosorbent assay) using stored serum samples from menopausal women. Results The LOD, LOQ, sLOQ was (0.1 pg, 0.3 pg, 1 pg/ml) for estrone, (0.3 pg, 1 pg, 3 pg/ml) for estradiol, and (0.3 pg, 1 pg, 30 pg/ml) for testosterone, respectively. Mass spectrometry accurately determined concentrations of E2 that could not be quantified by immunochemical methods. E1 concentrations measured by mass spectrometry were in all cases significantly lower than the ELISA measurements, suggesting immunoreactive contaminants in serum may interfere with ELISA. The testosterone measurements broadly agreed with each other in that both techniques could differentiate between low, medium and high serum levels. Conclusions We have developed and validated a scalable, sensitive assay for trace quantitation of E1, E2 and T in human serum samples in a single assay using sample preparation method and stable isotope dilution mass spectrometry. © 2015 Elsevier Inc. All rights reserved. Source


Marino-Repizo L.,Institute Quimica Of San Luis Inquisalconicet | Kero F.,Biotage | Vandell V.,Biotage | Senior A.,Biotage | And 3 more authors.
Food Chemistry | Year: 2014

A novel and advanced technology on solid phase extraction column prior to liquid chromatography coupled to tandem mass spectrometry has been used for the determination of ochratoxin A in red wine samples. Due to the need of a reliable and rugged method according to current regulations and with the aim of minimize heuristic efforts associated with analytical method development, the statistical design of experiment was employed. On other hand, the method validation according to European Commission 2002/657/EC was achieved. The values obtained for decision limit (CCα), detection capability (CCβ), limits of detection and quantification were 0.07 μg L-1, 0.14 μg L-1, 0.13 μg L-1 and 0.41 μg L-1, respectively. The recoveries values were ranged from 95.7% to 107.2%. These values were compatible with the 2.0 μg L-1 maximum allowable concentration limit established by different international regulations. © 2014 Elsevier Ltd. All rights reserved. Source

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