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Hydrophobic uncharged drugs such as docetaxel are difficult to encapsulate and retain in liposomal nanoparticles (LNP). In this work we show that a weak base derivative of docetaxel can be actively loaded into LNP using pH gradient loading techniques to achieve stable drug encapsulation and controlled release properties. Docetaxel was derivatized at the hydroxyl group in the C-2' position to form an N-methyl-piperazinyl butanoic acid ester. The free hydroxyl group in this position is essential for anticancer activity and the prodrug has, therefore, to be converted into the parent drug (docetaxel) to restore activity. Cytotoxicity testing against a panel of cancer cell lines (breast, prostate and ovarian cancer) demonstrated that the prodrug is readily converted into active drug; the derivative was found to be as active as the parent drug in vitro. The docetaxel derivative can be efficiently loaded at high drug-to-lipid ratios (up to 0.4 mg/mg) into LNP using pH loading techniques. Pharmacokinetic, tolerability and efficacy studies in mice demonstrate that the LNP-encapsulated prodrug has the long drug circulation half-life required for efficient tumor accumulation (50-100 times higher drug plasma levels compared with free derivative and Taxotere, the commercial docetaxel formulation), is active in a xenograft model of breast cancer (MDA-MB-435/LCC6), and is well tolerated at i.v. doses of 3 times higher than the maximum tolerated dose (MTD) of the parent drug. This is the first demonstration that a therapeutically active, remote-loaded, controlled-release LNP formulation of a taxane can be achieved. The approach reported here has broad applicability to other approved drugs as well as new chemical entities.

Invasive aspergillosis (IA) is a life-threatening systemic mycosis caused primarily by Aspergillus fumigatus. Early diagnosis of IA is based, in part, on an immunoassay for circulating fungal cell wall carbohydrate, galactomannan (GM). However, a wide range of sensitivity and specificity rates have been reported for the GM test across various patient populations. To obtain iron in vivo, A. fumigatus secretes the siderophore, N,N',N"-triacetylfusarinine C (TAFC) and we hypothesize that TAFC may represent a possible biomarker for early detection of IA. We developed an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for TAFC analysis from serum, and measured TAFC in serum samples collected from patients at risk for IA. The method showed lower and upper limits of quantitation (LOQ) of 5 ng/ml and 750 ng/ml, respectively, and complete TAFC recovery from spiked serum. As proof of concept, we evaluated 76 serum samples from 58 patients with suspected IA that were investigated for the presence of GM. Fourteen serum samples obtained from 11 patients diagnosed with probable or proven IA were also analyzed for the presence of TAFC. Control sera (n = 16) were analyzed to establish a TAFC cut-off value (≥6 ng/ml). Of the 36 GM-positive samples (≥0.5 GM index) from suspected IA patients, TAFC was considered positive in 25 (69%). TAFC was also found in 28 additional GM-negative samples. TAFC was detected in 4 of the 14 samples (28%) from patients with proven/probable aspergillosis. Log-transformed TAFC and GM values from patients with proven/probable IA, healthy individuals and SLE patients showed a significant correlation with a Pearson r value of 0.77. In summary, we have developed a method for the detection of TAFC in serum that revealed this fungal product in the sera of patients at risk for invasive aspergillosis. A prospective study is warranted to determine whether this method provides improved early detection of IA.

Translation initiation in eukaryotes is facilitated by the mRNA 5' cap structure (m7GpppX, where X is any nucleotide) that binds the multisubunit initiation factor eIF4F through one of its subunits, eIF4E. eIF4E is a phosphoprotein whose phosphorylation state positively correlates with cell growth. Protein kinase C phosphorylates eIF4E in vitro, and possibly in vivo. Using recombinant eIF4E incubated in vitro with purified protein kinase C and analyzed by solid-phase phosphopeptide sequencing in combination with high performance liquid chromatography coupled to mass spectrometry, we demonstrated that the third amino acid of the peptide SGSTTK (Ser209) is the major site of phosphorylation. This finding is consistent with the newly assigned in vivo phosphorylation site of eIF4E (Joshi, B., Cai, A. L., Keiper, B. D., Minich, W. B., Mendez, R., Beach, C. M., Stepinski, J., Stolarski, R., Darzynkiewicz, E., and Rhoads, R. E. (1995) J. Biol. Chem. 270, 14597-14603). A S209A mutation resulted in dramatically reduced phosphorylation, both in vitro and in vivo. Furthermore, the mutant protein was phosphorylated on threonine (most probably threonine 210) in vivo. Here we show that in the presence of the recently characterized translational repressors 4E-BP1 or 4E-BP2, phosphorylation of eIF4E by protein kinase C is strongly reduced. This suggests a two-step model for the phosphorylation (and activation) of eIF4E by growth factors and hormones: first, dissociation of eIF4E from 4E-BPs, followed by eIF4E phosphorylation.

We describe a simple, fast, sensitive, and nonisotopic bioanalytical technique for the detection of tyrosine-phosphorylated peptides and the determination of sites of protein tyrosine phosphorylation. The technique employs a protein tyrosine phosphatase micro enzyme reactor coupled on-line to either capillary electrophoresis or liquid chromatography and electrospray ionization mass spectrometry instruments. The micro enzyme reactor was constructed by immobilizing genetically engineered, metabolically biotinylated human protein tyrosine phosphatase beta onto the inner surface of a small piece of a 50-microns inner diameter, 360-microns outer diameter fused silica capillary or by immobilization of the phosphatase onto 40-90-microns avidin-activated resins. By coupling these reactors directly to either a capillary electrophoresis column or a liquid chromatography column, we were able to rapidly perform enzymatic dephosphorylation and separation of the reaction products. Detection and identification of the components of the reaction mixture exiting these reactors were done by mass analysis with an on-line electrospray ionization mass spectrometer. Tyrosine-phosphorylated peptides, even if present in a complex peptide mixture, were identified by subtractive analysis of peptide patterns generated with or without phosphatase treatment. Two criteria, namely a phosphatase-induced change in hydropathy and charge, respectively, and a change in molecular mass by 80 Da, were used jointly to identify phosphopeptides. We demonstrate that, with this technique, low picomole amounts of a tyrosine-phosphorylated peptide can be detected in a complex peptide mixture generated by proteolysis of a protein and that even higher sensitivities can be realized if more sensitive detection systems are applied.

We have designed a simple system to allow visualization of electroosmotic flow across a capillary junction. Two capillaries are placed in close proximity by careful micropositioning, and the distance between the capillaries is controlled with submicron resolution. The junction thus produced is placed at the focal point of a stereomicroscope, where the image collected by the microscope is imaged onto a CCD camera. Illumination of the junction (immersed in a buffer solution) allows visualization of the electroosmosis of a dye across the liquid junction

An analytical procedure involving trypsin immobilized on the inner surface of a 50-μm-I.d. fused-silica capillary has been developed for on-line digestion of minute amounts of protein. Trypsin was immobilized onto the surface of an aminoalkyl-silane-treated fused-silica capillary via blotin-avidin-biotin coupling. The enzyme-modified capillary was used to digest β-casein simply by flowing a solution of the denatured protein through the capillary at a rate of 40 nL/min and collecting the effluent

The utility of fluorescence detection in capillary electrophoresis is demonstrated using several methodologies and applications. Techniques such as direct and indirect fluorescence detection, pre-column and post-column derivatizations and a comparison of lamps and laser-based sources are reviewed.

Porous membrane phase separators are used to study the adsorption of the cation tetrahexylammonium (Q+), of the anion bromothymol blue (HB−) and of the ion-pair formed between them (QHB) at the liquid–liquid interface in a rapidly stirred mixture of chloroform and aqueous buffer. Adsorption isotherms in all three cases follow the Langmuir equation. The anion HB− is much more strongly adsorbed than the ion-pair QHB. The porous membrane technique readily permits measurement of simultaneous adsorption of the two species HB− and QHB, and thereby allows a study of their competitive adsorption. When QHB is adsorbed in the presence of an excess of HB− both the saturated (monolayer) interfacial concentration of QHB and the logarithm of the adsorption equilibrium constant for QHB decrease linearly with an increase in interfacial concentration of HB−. This shows quantitatively that coadsorption of QHB and HB− involves a direct competition for space at the interface and also that the presence of adsorbed HB− changes the adsorbent properties of the interface. Analytical implications for solvent extraction are discussed. Key words: interfacial adsorption, ion-pairs, liquid–liquid interface.

Two membrane phase separators, made of porous Teflon and of paper, are used to measure isotherms for adsorption of tetrahexylammonium picrate (QP) at the chloroform-water interface in a rapidly stirred liquid-liquid dispersion

The oligomer distribution of several poly(oxyalkylene)diamine(Jeffamine) polymers, ranging in molecular weight from 600 to 2000, has been characterized by precolumn derivatization and capillary zone electrophoresis with fluorescence detection. The separation mechanism is solely based on the differences between the electrophoretic mobilities of the oligomer units. The effect of the precolumn derivatization reaction on oligomer separation is also presented

The investigation of cell differentiation, development, and signal transduction pathways are examples of current research projects which have in common the focus on complex, highly regulated systems consisting of numerous interacting elements. A complete understanding of such processes can only be achieved if the problem is approached globally, considering the temporal and spatial interactions of all the elements involved. This task is supported by large amounts of data stored and annotated in databases such as nucleic acid and amino acid sequence databases and two dimensional (2D)†† protein databases.

We have used a previously described rapid stir cell with remote sample injection, porous Teflon phase separator, and low hold-up volume to study the rate of the homogeneous oxidation of I − by Fe 3+ in the aqueous phase, by monitoring the rate of appearance of I 2 in the chloroform phase. One purpose of this study is to discover the upper limit of reaction rate that can be measured with this improved rapid-stir apparatus

Description d'un appareillage destine a permettre les etudes de la vitesse d'extraction dans des conditions de turbulence. Etude de l'extraction d'o-nitroaniline en solution aqueuse par du chloroforme et d'o-nitrophenol dans le chloroforme par de l'eau

A new method, based on the ion-translocating properties of the ionophores nigericin and A23187, is described for loading large unilamellar vesicles (LUVs) with the drugs vincristine and ciprofloxacin. LUVs composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) (55:45 mol/mol) or sphingomyelin (SPM)/Chol (55:45 mol/mol) exhibiting a transmembrane salt gradient (for example, internal solution 300 mM MnSO4 or K2SO4; external solution 300 mM sucrose) are incubated in the presence of drug and, for experiments involving divalent cations, the chelator EDTA. The addition of ionophore couples the outward movement of the entrapped cation to the inward movement of protons, thus acidifying the vesicle interior. External drugs that are weak bases can be taken up in response to this induced transmembrane pH gradient. It is shown that both nigericin and A23187 facilitate the rapid uptake of vincristine and ciprofloxacin, with entrapment levels approaching 100% and excellent retention in vitro. Following drug loading, the ionophores can be removed by gel exclusion chromatography, dialysis, or treatment with biobeads. In vitro leakage assays (addition of 50% mouse serum) and in vivo pharmacokinetic studies (in mice) reveal that the A23187/Mn 2÷ system exhibits superior drug retention over the nigericin/K + system, and compares favorably with vesicles loaded by the standard ApH or amine methods. The unique features of this methodology and possible benefits are discussed. © 1998 Elsevier Science B.V. All rights reserved.