Novel High-Throughput Strategy for the Aqueous Solubility Assessment of Peptides and Proteins Exhibiting a Propensity for Gelation: Application to the Discovery of Novel Antibacterial Teixobactin Analogues.

A novel high-throughput aqueous solubility assay was developed for peptides and proteins exhibiting a high gelling propensity (in this case, antibacterial teixobactin analogues). By integrating the assessment of gel formation, as indicated by an increase in the solution viscosity, into the peptide equilibrium solubility screening assay, we were able to estimate the "free-flowing solubility", which is defined as the concentration at which the peptide solution not only is fully dissolved but also is a liquid exhibiting ideal flowing characteristics. In this workflow, peptide solutions passing the turbidity assessment were further screened by viscosity measurements based on nanobead-assisted dynamic light scattering analysis in a 96-well plate. The method is able to effectively detect the initiation of peptide gelation and facilitate compound ranking based on their aqueous solubility. The application of such an approach helped confirm that the substitution of Ser3 in teixobactin led to desired physicochemical improvements and provided a focal point for further chemistry structure-activity relationship exploration.

Solubilization of Cyclosporine in Topical Ophthalmic Formulations: Preformulation Risk Assessment on a New Solid Form.

Owing to the discovery of a less soluble crystalline form (form 2) of cyclosporine (CsA), risks in solubility and physical stability of these formulations need to be revisited. This work focused on understanding the solubility behavior of various CsA forms in different media, including water, castor oil, and selected cosolvent micellar systems. In water, form 2 was approximately 8-9 times less soluble than form 1 (aka. tetragonal dihydrate). In neat nonaqueous solvent, for example, castor oil, form 3 (aka. orthorhombic hydrate) was found to have the lowest solubility and therefore the most stable form. In addition, the solubility-temperature relationship of CsA is complex and solvent-dependent. In aqueous vehicles, retrograde temperature dependence of solubility was observed in aqueous vehicles, that is, the solubility of CsA decreased with temperature, which was attributed to the effect of temperature on the strength of hydrogen bonding interactions; conversely, the solubility of CsA increased with temperature in nonaqueous solvents. In addition, the solubility of these CsA forms was very sensitive to temperature. Temperature-dependent form transformation was also observed in the media studied, with faster form conversion occurring at elevated temperatures. These studies provided key information to support the risk assessment for topical ophthalmic formulation development of CsA.

Preparation, characterization and ex vivo-in vivo assessment of candesartan cilexetil nanocrystals via solid dispersion technique using an alkaline esterase activator carrier.

The objective of this study was to improve candesartan cilexetil (CC) efficacy by formulating nanocrystals via solid dispersion (SD) technique using tromethamine (Tris). SD was prepared by solvent evaporation at different drug carrier ratios, evaluated for particle size, vitro dissolution studies, TEM, FTIR, and X-ray powder diffraction. Ex vivo, in vivo pharmacokinetic parameters were conducted on selected formulae compared to drug suspension and marketed product. Size analysis demonstrated formation of particles in the nanorange lower than 300 nm. A burst drug release followed by an improved dissolution was observed indicating instant formation of nanocrystals along with amorphization as confirmed by X-ray diffraction. FTIR studies suggested the absence of chemical interaction between Tris and CC. TEM revealed formation of irregular oval nanoparticles. SD-1:5 has higher apparent permeability coefficient compared to CC suspension. Furthermore, the pharmacokinetic results proved the ability of the formed nanoparticles to enhance the efficacy of CC compared to drug suspension and marketed product. In conclusion, using of Tris as alkaline esterase activator carrier could be a promising tool to bypass the controversial effect of esterase enzymes that may be a source for inter-individual variations affecting ester prodrug candidates' efficacy.

Roadmap to 3D-Printed Oral Pharmaceutical Dosage Forms: Feedstock Filament Properties and Characterization for Fused Deposition Modeling.

Application of additive manufacturing techniques (3D printing) for mass-customized products has boomed in the recent years. In pharmaceutical industry and research, the interest has grown particularly with the future scenario of more personalized medicinal products. Understanding a broad range of material properties and process behavior of the drug-excipient combinations is necessary for successful 3D printing of dosage forms. This commentary reviews recent 3D-printing studies by fused deposition modeling (FDM) technique in pharmaceutical sciences, extending into the fields of polymer processing and rapid prototyping, where more in-depth studies on the feedstock material properties, modeling, and simulation of the FDM process have been performed. A case study of a model oral dosage form from custom-prepared indomethacin-polycaprolactone feedstock filament was used as an example in the pharmaceutical context. The printability was assessed in the different process steps: preparation of customized filaments for FDM, filament feeding, deposition, and solidification. These were linked with the rheological, thermal, and mechanical properties and their characterization, relevant for understanding the printability of drug products by FDM.

Drug nanocrystals - Versatile option for formulation of poorly soluble materials.

Poor solubility of drug compounds is a great issue in drug industry today and decreasing particle size is one efficient and simple way to overcome this challenge. Drug nanocrystals are solid nanosized drug particles, which are covered by a stabilizer layer. In nanoscale many physical properties, like compound solubility, are different from the solubility of bulk material, and due to this drug nanocrystals can reach supersaturation as compared to thermodynamic solubility. The most important effect of the smaller particle size is that dissolution rate is highly enhanced mainly due to the increased surface area. In this review the most important properties of nanocrystalline drug compounds are presented, with multiple examples of the development and characterization of nanocrystalline drug formulations.

Assessment and comparison of magnetic nanoparticles as MRI contrast agents in a rodent model of human hepatocellular carcinoma.

The purpose of this study was to synthesize, characterize and tailor the surface properties of magnetic nanoparticles with biocompatible copolymer coatings and to evaluate the efficiency of the resulting nanoconjugates as magnetic resonance imaging (MRI) contrast agents for liver imaging. Magnetic nanoparticles with core diameters of 10 and 30 nm were synthesized by pyrolysis and were subsequently coated with a copolymer containing either carboxyl (SHP) or methoxy groups as termini. All four formulas, and ferumoxides (Feridex I.V.(®)), were individually injected intravenously into separate, normal Balb/C mice (at 2.5, 1.0 and 0.56 mg Fe kg(-1)), and the animals underwent T(2)-weighted MRI at multiple time points post injection (p.i.) to evaluate the hepatic uptake and clearance. Furthermore, we compared the abilities of the new formulas and Feridex to detect tumors in an orthotropic Huh7 tumor model. Transmission electron microscopy (TEM) revealed a narrow size distribution of both the 10 and 30 nm nanoparticles, in contrast to a wide size distribution of Feridex. MTT, apoptosis and cyclin/DNA flow cytometry assays showed that the polymer coated nanoparticles had no adverse effect on cell growth. Among all the tested formulas, including Feridex, SHP-30 showed the highest macrophage uptake at the in vitro level. In vivo MRI studies on normal mice confirmed the superiority of SHP-30 in inducing hypointensities in the liver tissue, especially at clinical dose (0.56 mg Fe kg(-1)) and 3 T field. SHP-30 showed better contrast-to-noise ratio than Feridex on the orthotropic Huh7 tumor model. SHP-30 was found to be an efficient contrast agent for liver MR imaging. The success of this study suggests that, by improving the synthetic approach and by tuning the surface properties of IONPs, one can arrive at better formulas than Feridex for clinical practice.

Arabidopsis plants acclimate to water deficit at low cost through changes of carbon usage: an integrated perspective using growth, metabolite, enzyme, and gene expression analysis.

Growth and carbon (C) fluxes are severely altered in plants exposed to soil water deficit. Correspondingly, it has been suggested that plants under water deficit suffer from C shortage. In this study, we test this hypothesis in Arabidopsis (Arabidopsis thaliana) by providing an overview of the responses of growth, C balance, metabolites, enzymes of the central metabolism, and a set of sugar-responsive genes to a sustained soil water deficit. The results show that under drought, rosette relative expansion rate is decreased more than photosynthesis, leading to a more positive C balance, while root growth is promoted. Several soluble metabolites accumulate in response to soil water deficit, with K(+) and organic acids as the main contributors to osmotic adjustment. Osmotic adjustment costs only a small percentage of the daily photosynthetic C fixation. All C metabolites measured (not only starch and sugars but also organic acids and amino acids) show a diurnal turnover that often increased under water deficit, suggesting that these metabolites are readily available for being metabolized in situ or exported to roots. On the basis of 30 enzyme activities, no in-depth reprogramming of C metabolism was observed. Water deficit induces a shift of the expression level of a set of sugar-responsive genes that is indicative of increased, rather than decreased, C availability. These results converge to show that the differential impact of soil water deficit on photosynthesis and rosette expansion results in an increased availability of C for the roots, an increased turnover of C metabolites, and a low-cost C-based osmotic adjustment, and these responses are performed without major reformatting of the primary metabolism machinery.

An in vitro assessment of a cell-containing collagenous extracellular matrix-like scaffold for bone tissue engineering.

Extracellular matrix (ECM) consists of a complex mixture of macromolecules such as collagens, proteoglycans, glycoproteins, and elastic fibers. ECM is essential to preserving tissue architecture, signaling to cells, and regulating calcification in mineralized tissues. Osteoblasts in culture secrete and assemble an extensive ECM rich in type I collagen, and other noncollagenous proteins that can be mineralized. Three-dimensional matrix models can be used in vitro to most appropriately resemble the geometry and biochemistry of natural ECMs. In the present study, MC3T3-E1 mouse calvarial preosteoblasts were cultured within a dense three-dimensional collagenous ECM-like scaffold produced through the method of plastic compression. Plastic compression rapidly produces scaffolds of collagen density approaching native tissue levels with enhanced biomechanical properties while maintaining the viability of resident cells. The proliferation, morphology, and gene expression of seeded MC3T3s, as well as collagen production and matrix mineralization, were investigated for up to 7 weeks in culture. Soluble collagen secretion ranged in concentration from 5 to 30 microg/mL over a 24-h period, concomitant with a steady rate of collagen mRNA expression. Expression of osteogenic markers such as tissue-nonspecific alkaline phosphatase (Alpl), bone sialoprotein (Bsp), and osteopontin (Opn) examined by biochemical assay and reverse transcription-polymerase chain reaction demonstrated cell differentiation. Pericellular voids of ECM around cells, together with evidence of MMP13 mRNA expression, suggested matrix remodeling. Ultrastructural analyses, X-ray microanalysis, micro-computed tomography, as well as Fourier-transform infrared and imaging all confirmed the formation of a calcium-phosphate mineral phase within the fibrillar collagen matrix. In conclusion, preosteoblastic MC3T3 cells seeded within an ECM-like dense collagen scaffold secrete matrix proteins and induce scaffold mineralization in a manner potentially appropriate for bone tissue engineering uses.

Assessment of the combined approach of N-alkylation and salt formation to enhance aqueous solubility of tertiary amines using bupivacaine as a model drug.

Quaternary prodrug types of poorly water-soluble tertiary amines have been shown to exhibit significantly enhanced solubilities as compared to the parent amine. In the present study the combined effect of N-alkylation and salt formation to enhance aqueous solubility of tertiary amines have been investigated using bupivacaine as a model compound. X-ray structure analyses of selected salts were included to investigate the potential existence of correlations between salt solubility and crystal packing modes. Alkyl groups were methyl, ethyl, propyl, and butyl and the derivatives were isolated as their iodide salts. Chloride, mesylate, formate, acetate, glycolate, and tosylate salts were obtained by anion exchange of the N-methyl-bupivacaine derivative. N-Alkylation and salt formation afforded quaternary ammonium salts possessing pH-independent aqueous solubilities far exceeding that of the parent tertiary amine (up to a factor of 3200 at pH 8). A moderate reduction in solubility with increasing length of the alkyl chain was observed for the iodide salts of the N-alkylated bupivacaine derivatives. In case of the N-methyl-bupivacaine derivative variation of the counterion had a significant impact on the solubility with the iodide salt being 200 times less soluble than the chloride salt. X-ray analysis revealed that both the alkyl substituent and the anionic counterion influenced salt packing modes, however, in an unpredictable manner making establishment of quantitative correlations between crystal packing and solubility difficult even for a series of closely related derivatives.

In vitro-in vivo evaluation of supercritical processed solid dispersions: permeability and viability assessment in Caco-2 cells.

In this study improvement in the bioavailability of carbamazepine (CBZ) prepared as solid dispersions by conventional solvent evaporation and supercritical fluid (SCF) processing methods was assessed, along with the elucidation of the mechanism of improved absorption. Solid dispersions of CBZ in polyethylene glycol (PEG) with either Gelucire 44/14 or vitamin E-TPGS (TPGS) were evaluated by intrinsic dissolution. Directional transport through Caco-2 cell monolayers was determined in the presence and absence of TPGS. Cell viability in presence of various concentrations of amphiphilic carriers was seen. In vivo oral bioavailability was determined in rats. The apparent intrinsic dissolution rates (IDR) of both conventional- and SCF-CBZ/PEG 8000/TPGS solid dispersions were increased by 13- and 10.6-fold, respectively, relative to neat CBZ. CBZ was not a substrate of P-glycoprotein. Higher CBZ permeability was seen in presence of 0.1% TPGS. Cell viability studies showed significant cytotoxicity only at or above 0.1% amphiphilic carrier. Supercritical treated formulation (without amphiphilic carrier) displayed oral bioavailability on par with those conventional solid dispersions augmented with amphiphilic carriers. An in vitro-in vivo correlation was seen between IDR and the AUC of the various CBZ solid dispersions. Bioavailability of CBZ was more a function of dissolution as opposed to membrane effects. Although bioavailability from SCF processed dispersions was better than conventionally processed counterparts (except for one formulation containing Gelucire 44/14), an interaction of processing method and inclusion of an amphiphilic carrier, rather by one factor alone contributed to optimal absorption, thus giving contradictory results for Gelucire 44/14 and TPGS formulations.