Springer Science and Business Media LLC

Purpose. To develop and demonstrate a novel particle engineering technology, spray freezing into liquid (SFL), to enhance the dissolution rates of poorly water-soluble active pharmaceutical ingredients (APIs). Methods. Model APIs, danazol or carbamazepine with or without excipients, were dissolved in a tetrahydrofuran/water cosolvent system and atomized through a nozzle beneath the surface of liquid nitrogen to produce small frozen droplets, which were subsequently lyophilized. The physicochemical properties of the SFL powders and controls were characterized by X-ray diffraction, scanning electron microscopy (SEM), particle size distribution, surface area analysis, contact angle measurement, and dissolution. Results. The X-ray diffraction pattern indicated that SFL powders containing either danazol or carbamazepine were amorphous. SEM micrographs indicated that SFL particles were highly porous. The mean particle diameter of SFL carbamazepine/SLS powder was about 7 μm. The surface area of SFL danazol/poloxamer 407 powder was 11.04 m2/g. The dissolution of SFL danazol/poloxamer 407 powder at 10 min was about 99%. The SFL powders were free flowing and had good physical and chemical stability after being stored at 25°C/60%RH for 2 months. Conclusions. The novel SFL technology was demonstrated to produce nanostructured amorphous highly porous particles of poorly water soluble APIs with significantly enhanced wetting and dissolution rates.

Reversible self-association (RSA) has long been a concern in therapeutic monoclonal antibody (mAb) development. Because RSA typically occurs at high mAb concentrations, accurate assessment of the underlying interaction parameters requires explicitly addressing hydrodynamic and thermodynamic nonideality. We previously examined the thermodynamics of RSA for two mAbs, C and E, in phosphate buffered saline (PBS). Here we continue to explore the mechanistic aspects of RSA by examining the thermodynamics of both mAbs under reduced pH and salt conditions. Dynamic light scattering and sedimentation velocity (SV) studies were conducted for both mAbs at multiple protein concentrations and temperatures, with the SV data analyzed via global fitting to determine best-fit models, interaction energetics, and nonideality contributions. We find that mAb C self-associates isodesmically irrespective of temperature, and that association is enthalpically driven but entropically penalized. Conversely, mAb E self-associates cooperatively and via a monomer–dimer-tetramer-hexamer reaction pathway. Moreover, all mAb E reactions are entropically driven and enthalpically modest or minimal. The thermodynamics for mAb C self-association are classically seen as originating from van der Waals interactions and hydrogen bonding. However, relative to the energetics we determined in PBS, self-association must also be linked to proton release and/or ion uptake events. For mAb E, the thermodynamics implicate electrostatic interactions. Furthermore, self-association is instead linked to proton uptake and/or ion release, and primarily by tetramers and hexamers. Finally, although the origins of mAb E cooperativity remain unclear, ring formation remains a possibility whereas linear polymerization reactions can be eliminated.

Purpose. To use a single particle levitation technique to investigate the equilibrium water sorption characteristics in both the evaporation and growth of four respiratory drugs at 37°C: atropine sulfate (AS), isoproterenol hydrochloride (IPHC) and isoproterenol hemisulfate (IPHS) and disodium cromoglycate (DSCG). Methods. The equilibrium water content was measured as a function of relative humidity (RH) by a single particle levitation technique using an electrodynamic balance (EDB). The change of water content was determined by the voltage required to balance the weight of the levitated particle electrostatically. The water activities of bulk samples were also measured. Growth ratios were determined and compared with values in the literature. Results. Crystallization or deliquescence was not observed for AS, IPHC and IPHS. The hysteresis in the water cycle was not observed for any of the drugs. At RH ∼ 0%, AS particles still contain about 5% water but IPHC and IPHS particles do not contain any residual water. The aerodynamic growth ratio from RH 0% to 99.5% is 2.60, 2.86, 2.42 and 1.26 for AS, IPHC, IPHS and DSCG, respectively. Super- saturated droplets of IPHC and IPHS are expected to exist in the am- bient conditions. DSCG is in a solid state in the RH range of 10-90%. Conclusions. It is expected that some aerosolized drugs of low solubility may experience supersaturation before they enter the human body and this could exert a significant influence both on particle loss before inhalation and on the deposition of the drugs in the lungs. The EDB is a convenient and reliable tool for studying the hygroscopic properties of pharmaceutical aerosols, especially for supersaturated solutions.

Erythromycin acistrate is a new prodrug of erythromycin. Its bioavailability from hard gelatin capsules containing the drug with or without sodium bicarbonate was studied in healthy volunteers. The plasma levels of erythromycin, anhydroerythromycin, and acetylerythromycin were measured using an HPLC method. Addition of sodium bicarbonate to the capsule markedly enhanced the plasma level of the microbiologically active substance, erythromycin, doubling the C max and AUC values (P < 0.05). At the same time, the lag time in the absorption curve was shortened to one-third. No changes in the plasma levels of the inactive metabolite anhydroerythromycin were noted. It is concluded that adding sodium bicarbonate to an erythromycin acistrate formulation enhances its bioavailability.

Purpose. This work examines the effectiveness of synthetic peptide immunogens derived from immunodominant T-cell epitopes as replacements for their intact parent protein in vaccines. Methods. Fluorescein was conjugated to hen egg lysozyme (FL-HEL, positive control) and three synthetic peptide immunogens: (a) murine B10.A (H-2a) immunodominant T-cell epitope of HEL [FL-(T-cell epitope)]; (b) multiple antigenic peptide (MAP) multimer of this epitope {[FL-(T epitope)]n-MAP, n = 2-4}; and (c) negative control MAP with T-cell epitope residues replaced with glycine [(FL-Gly18)4-MAP]. The dose response of each immunogen was examined over a 300-fold range in B10.A mice. The immune response was monitored using antifluorescein ELISA assays. Results. FL-(T epitope)'s immune response correlated positively with dose, with maximum response comparable to that of [FL-(T epitope)]n-MAP, or FL-HEL. This trend was consistent across 1°, 2°, and 3° responses, although interanimal variability was higher in the latter two because of an all-or-none response in mice immunized with this peptide. [FL-(T epitope)]n-MAP's immune response was consistently high and nearly dose independent, a trend observed across 1°, 2°, and 3° responses. FL-HEL's immune response correlated negatively to dose in the 1° response but was nearly dose independent in the 2° and 3° responses. The magnitude of these latter responses was comparable to that observed for [FL-(T epitope)]n-MAP. (FL-Gly18)4-MAP did not elicit an immune response except at the highest dose. This trend was consistent across 1°, 2°, and 3° responses. Conclusions. The monomeric epitope was 300-fold less potent than its parent carrier protein, but increasing immunogen valency using MAP technology compensated totally for reduced potency. (FL-Gly18)4-MAP's lack of response at all but the highest dose strongly suggests that a specific immunodominant T-cell epitope sequence for HEL is necessary for successful peptide mimicry of HEL. This work also demonstrates the importance of quality assessment of commercial MAP core resins.

Purpose. To investigate differences in the cellular uptake and intracellular distribution of protein-bound doxorubicin in comparison to free doxorubicin and a liposomal formulation (CAELYX®) Methods. LXFL 529 lung carcinoma cells were incubated with an acid-sensitive transferrin and albumin conjugate of doxorubicin, a stable albumin doxorubicin conjugate, and free and liposomal doxorubicin for up to 24 h. The uptake of doxorubicin was detected with confocal laser scanning microscopy (CLSM). To investigate the intracellular localization of the anticancer drug, lysosomes, Golgi apparatus, and mitochondria were also stained by various organelle-specific fluorescent markers. In vitro efficacy of the doxorubicin derivatives was examined with the BrdU incorporation assay. Results. The acid-sensitive albumin and transferrin doxorubicin conjugates showed enhanced cytotoxicity in comparison to liposomal doxorubicin, whereas the stable albumin-doxorubicin conjugate showed only marginal activity. Of all compounds tested, doxorubicin showed the highest cytotoxicity. CLSM studies with specific markers for lysosomes, mitochondria, and the Golgi apparatus demonstrated that protein-bound doxorubicin or liberated doxorubicin was accumulated in the mitochondria and Golgi compartments, but not in the lysosomes after 24 h. Free doxorubicin showed a time-dependent intracellular shift from the nucleus to the mitochondria and Golgi apparatus. Fluorescence resulting from incubation with CAELYX was primarily detected in the nucleus. Conclusions. Our results indicate that other organelles in addition to the cell nucleus are important sites of accumulation and interaction for protein-bound doxorubicin or intracellularly released doxorubicin as well as for free doxorubicin.

Propofol is a water-insoluble intravenous anesthetic agent that is actually formulated as a water-in-oil emulsion with known drawbacks such as pain on injection, microorganism growth support and stability. We report on the properties of formulations of propofol in poly (N-vinyl-2-pyrrolidone)-block-poly(d,l-lactide), PVP–PLA, polymeric micelles (Propofol-PM). Microbial growth in these formulations was evaluated with Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 25922) and Candida albicans (ATCC 10231). Sleep-recovery studies in female Sprague–Dawley rats, at a dose of 10mg/kg were performed to compare pharmacodynamic profiles of the new Propofol-PM formulations with those of Diprivan®, a commercially available lipid based propofol formulation. Growth of microorganisms was not supported in the Propofol-PM formulations tested. No significant differences in times to unconsciousness, awakening, recovery of righting reflex and full recovery were observed between Propofol-PM formulations and Diprivan®. Propofol loaded in PVP–PLA micelles (Propofol-PM) is not significantly different in terms of pharmacodynamic but demonstrates no microorganism growth support and improved stability that opens up the door to pain on injection reduction strategy.

Cataloguing endogenous miRNA targets by inhibiting miRNA function is fundamental to understanding the biological importance of each miRNA in gene regulatory pathways. Methods to down-regulate miRNA activity may help treat diseases where over-expression of miRNAs relates to the underlying pathophysiology. This study objectively evaluates the in vitro potency of different anti-miRNA oligonucleotides (AMOs) using various design and modification strategies described in the literature as well as some novel modification strategies. MiR21 and miR16 AMOs, containing chemical modifications such as 2′-O-methyl RNA, locked nucleic acid and 2′-Fluoro bases with or without phosphorothioate linkages, were directly compared by transfection into HeLa cells using a dual-luciferase reporter assay to quantify miRNA inhibition. Potency for the various AMOs ranged from inactive at high dose (50 nM) to strongly inhibitory at both high and low dose (1 nM). Including phosphorothioate linkages improved nuclease stability and generally increased functional potency. Incorporating high binding affinity modifications, such as LNA and 2′F bases, increases AMO potency while maintaining specificity; nevertheless, use of low dose is preferred when using high potency reagents to minimize the potential for cross reactivity. 2′OMe/LNA chimeras with PS modifications were the most potent constructs tested for miRNA inhibition in vitro.

The distribution of chlorpromazine (CPZ) between aqueous buffer solutions and 1-octanol was studied over a wide range of pH, buffer concentration, and temperature. A mathematical model was developed to simulate the distribution profiles. It is assumed that only monomers of CPZ exist in the organic phase, whereas in the aqueous phase, association equilibria were assumed to occur. The model predicted the formation of dimers and no higher aggregates over most of the concentration range covered in this study. Thermodynamic parameters for the partition equilibria were evaluated from the equilibrium partition coefficients measured as a function of temperature. Positive values of ΔH and ΔS were obtained for the transfer of CPZ from the aqueous to the organic phase. The process is entropy controlled indicative of a hydrophobic interaction between CPZ and the aqueous solvent.

Skin and soft tissue infections are increasingly prevalent and often complicated by potentially fatal therapeutic hurdles, such as poor drug perfusion and antibiotic resistance. Delivery vehicles capable of versatile loading may improve local bioavailability and minimize systemic toxicities yet such vehicles are not clinically available. Therefore, we aimed to expand upon the use of glutathione-conjugated poly(ethylene glycol) GSH-PEG hydrogels beyond protein delivery and evaluate the ability to deliver traditional therapeutic molecules. PEG and GSH-PEG hydrogels were prepared using ultraviolet light (UV)-polymerization. Hydrogel loading and release of selected drug candidates was examined using UV-visible spectrometry. Therapeutic molecules and GST-fusion protein loading was examined using UV-visible and fluorescent spectrometry. Efficacy of released meropenem was assessed against meropenem-sensitive and -resistant P. aeruginosa in an agar diffusion bioassay. For all tested agents, GSH-PEG hydrogels demonstrated time-dependent loading whereas PEG hydrogels did not. GSH-PEG hydrogels released meropenem over 24 h. Co-loading of biologic and traditional therapeutics into a single vehicle was successfully demonstrated. Meropenem-loaded GSH-PEG hydrogels inhibited the growth of meropenem-sensitive and resistant P. aeruginosa isolates. GSH ligands within GSH-PEG hydrogels allow loading and effective delivery of charged therapeutic agents, in addition to biologic therapeutics.