Understanding the degradation pathway of a poorly water-soluble drug formulated in PEG-400.

VX-497 is a poorly water-soluble compound. It is formulated in PEG-400 and encapsulated in softgel capsules. Although the drug product is stable at refrigerated conditions, many degradation peaks have been observed at accelerated storage conditions. An investigation utilizing high performance liquid chromatography-mass spectrometry (HPLC-MS) was conducted to understand the degradation mechanism of the active pharmaceutical ingredient (VX-497) in PEG-400 formulation. Results revealed that the degradation was mainly caused by the reaction between VX-497 with moisture (hydrolysis) and PEG-400 (PEGylation). The numerous degradation peaks observed in the samples stored at accelerated conditions were PEG adducts covalently attached to portions of the VX-497 molecule, which were confirmed by comparison with synthetic markers. Investigation also found that an impurity, which was present in the VX-497 drug substance, reacted with PEG-400 following the same reaction mechanism, and generated additional impurities in the VX-497 drug product. By changing the process for drug substance synthesis, pure batches of VX-497 were obtained. Furthermore, it was found that the reaction between VX-497 and PEG-400 was temperature and time dependent. When the drug product was manufactured at 45 degrees C and the processing time was controlled, the PEG degradants and by-products were reduced to non-detectable levels, resulting in greatly improved drug product quality. This paper presents an integrated effort among analytical, process, and formulation scientists on how to develop a better drug product by understanding the fundamental issues of the drug product, namely the degradation mechanism.

NMR structure of the enzyme GatB of the galactitol-specific phosphoenolpyruvate-dependent phosphotransferase system and its interaction with GatA.

The phosphoenolpyruvate-dependent carbohydrate transport system (PTS) couples uptake with phosphorylation of a variety of carbohydrates in prokaryotes. In this multienzyme complex, the enzyme II (EII), a carbohydrate-specific permease, is constituted of two cytoplasmic domains, IIA and IIB, and a transmembrane channel IIC domain. Among the five families of EIIs identified in Escherichia coli, the galactitol-specific transporter (II(gat)) belongs to the glucitol family and is structurally the least well-characterized. Here, we used nuclear magnetic resonance (NMR) spectroscopy to solve the three-dimensional structure of the IIB subunit (GatB). GatB consists of a central four-stranded parallel beta-sheet flanked by alpha-helices on both sides; the active site cysteine of GatB is located at the beginning of an unstructured loop between beta1 and alpha1 that folds into a P-loop-like structure. This structural arrangement shows similarities with other IIB subunits but also with mammalian low molecular weight protein tyrosine phosphatases (LMW PTPase) and arsenate reductase (ArsC). An NMR titration was performed to identify the GatA-interacting residues.

Alterations in heart looping induced by overexpression of the tight junction protein Claudin-1 are dependent on its C-terminal cytoplasmic tail.

In vertebrates, the positioning of the internal organs relative to the midline is asymmetric and evolutionarily conserved. A number of molecules have been shown to play critical roles in left-right patterning. Using representational difference analysis to identify genes that are differentially expressed on the left and right sides of the chick embryo, we cloned chick Claudin-1, an integral component of epithelial tight junctions. Here, we demonstrate that retroviral overexpression of Claudin-1, but not Claudin-3, on the right side of the chick embryo between HH stages 4 and 7 randomizes the direction of heart looping. This effect was not observed when Claudin-1 was overexpressed on the left side of the embryo. A small, but reproducible, induction of Nodal expression in the perinodal region on the right side of the embryo was noted in embryos that were injected with Claudin-1 retroviral particles on their right sides. However, no changes in Lefty,Pitx2 or cSnR expression were observed. In addition, Flectin expression remained higher in the left dorsal mesocardial folds of embryos with leftwardly looped hearts resulting from Claudin-1 overexpression on the right side of the embryo. We demonstrated that Claudin-1's C-terminal cytoplasmic tail is essential for this effect: mutation of a PKC phosphorylation site in the Claudin-1 C-terminal cytoplasmic domain at threonine-206 eliminates Claudin-1's ability to randomize the direction of heart looping. Taken together, our data provide evidence that appropriate expression of the tight junction protein Claudin-1 is required for normal heart looping and suggest that phosphorylation of its cytoplasmic tail is responsible for mediating this function.

Physicochemical characterization and mechanisms of release of theophylline from melt-extruded dosage forms based on a methacrylic acid copolymer.

The purpose of the current study was to investigate the physicochemical properties of melt-extruded dosage forms based on Acryl-EZE and to determine the influence of gelling agents on the mechanisms and kinetics of drug release from thermally processed matrices. Acryl-EZE is a pre-mixed excipient blend based on a methacrylic acid copolymer that is optimized for film-coating applications. Powder blends containing theophylline, Acryl-EZE, triethyl citrate and an optional gelling agent, Methocel K4M Premium (hydroxypropyl methylcellulose, HPMC, hypromellose 2208) or Carbopol 974P (carbomer), were thermally processed using a Randcastle single-screw extruder. The physical and chemical stability of materials during processing was determined using thermal gravimetric analysis and HPLC. The mechanism of drug release was determined using the Korsmeyer-Peppas model and the hydration and erosion of tablets during the dissolution studies were investigated. The excipient blends were physically and chemically stable during processing, and the resulting dosage forms exhibited pH-dependent dissolution properties. Extrusion of blends containing HPMC or carbomer changed the mechanism and kinetics of drug release from the thermally processed dosage forms. At concentrations of 5% or below, carbomer was more effective than HPMC at extending the duration of theophylline release from matrix tablets. Furthermore, carbomer containing tablets were stable upon storage for 3 months at 40 degrees C/75% RH. Thus, hot-melt extrusion was an effective process for the preparation of controlled release matrix systems based on Acryl-EZE.

NMR structural study of TcUBP1, a single RRM domain protein from Trypanosoma cruzi: contribution of a beta hairpin to RNA binding.

TcUBP1 is a trypanosome cytoplasmic RNA-binding protein containing a single, conserved RNA-recognition motif (RRM) domain involved in selective destabilization of U-rich mRNAs such as the Trypanosoma cruzi small mucin gene family mRNA, TcSMUG. TcUBP1 binds specific transcripts in vivo and co-localizes in the perinuclear part of the cell with components of the mRNA-stability determinant pathway such as poly(A)-binding protein 1 (PABP1) and TcUBP2, a closely related RRM-containing protein. In TcUBP proteins, the RRM domain is flanked by N-terminal Gln-rich and C-terminal Gly-Gln-rich extensions, which are involved in protein-protein interactions. In this work, we determined the solution structure of the TcUBP1 RRM domain by nuclear magnetic resonance (NMR) spectroscopy. The domain has a characteristic betaalphabetabetaalphabeta fold, consisting of a beta sheet composed of four antiparallel betastrands and two alpha helices packed against one face of the beta sheet. A unique aspect of TcUBP1 is the participation of a beta hairpin (beta4-beta5) in the beta sheet, resulting in an enlarged RNA-binding surface. Detailed analysis of the TcUBP1 interaction with a short single-stranded RNA derived from the 3' UTR of TcSMUG was carried out by titration experiments using both NMR spectroscopy and isothermal titration calorimetry. This analysis revealed that amino acids located within the beta hairpin (beta4-beta5) contribute to complex formation. This enlarged protein-RNA interface could compensate for the lack of additional RNA-binding domains in TcUBP1, as observed in many other RRM-containing proteins. The structure of TcUBP1 reveals new aspects of single RRM-RNA interactions and insight into how N- and C-terminal extensions can contribute to RNA binding.

Compression of controlled-release pellets produced by a hot-melt extrusion and spheronization process.

The purpose of this study was to investigate the physicomechanical and dissolution properties of tablets containing controlled-release pellets prepared by a hot-melt extrusion and spheronization process. A powder blend of anhydrous theophylline, Eudragit Preparation 4135 F, and functional excipients was melt-extruded, pelletized, and then spheronized. The pellets were compressed into tablets using forces of 5, 10, 15, and 20 kN. Tablet diluents included microcrystalline cellulose, a mixture of spray-dried lactose and microcrystalline cellulose, modified food starch, and soy polysaccharides. The effective porosity of the compressed pellets was measured using mercury porosimetry and helium pycnometry, while the surface area was determined using Brunauer, Emmett, and Teller (BET) analysis. The disintegration time, hardness, and friability of compacts were determined. Drug release studies were performed according to USP 27 Apparatus 3 guidelines in 250 mL of medium (pH 1.0, 3.0, 5.0, 6.8, and 7.4) 37 degrees C and 20 dpm. Samples were analyzed by high pressure-liquid chromatography (HPLC). Effective porosity and surface area determinations of the melt-extruded pellets were not influenced by compression. The percent of theophylline released from rapidly disintegrating tablets was not affected by compression force or excipient selection, but tablets with prolonged disintegration times exhibited delayed drug release in acidic media. However, dissolution profiles of uncompressed pellets and all compacts were identical after transition from 0.1 N HCl to media increasing in pH from 3.0 to 7.4. Furthermore, pellet to filler excipient ratio and filler excipient selection did not influence the rate of drug release from compacts.

A novel powder coating process for attaining taste masking and moisture protective films applied to tablets.

A novel powder coating process was developed for the application of taste masking and moisture protective films on tablets while avoiding the use of solvents or water. The coalescence of particles to form a polymeric film was investigated through studies of dry powder layering of micronized acrylic polymer (E PO) to produce free films. Theophylline containing tablets were coated with the same acrylic polymer in a laboratory scale spheronizer using a powder coating technique. The dry powder layer delayed the onset of drug release in pH 6.8 medium, depending on the coating level, while no delay was observed in pH 1.0 medium. The presence of hydrophilic polymers in the acrylic coating layer decreased the lag time for drug release in pH 6.8 medium, while only the presence of HPMC in the film slowed the drug release rate in acidic medium. The dry coating process was demonstrated to be a reliable alternative to solvent or aqueous film coating technologies for applying taste masking and moisture protective film coats onto compressed tablets. A controlled drug release profile was achieved in pH 6.8 media.

Production of spherical pellets by a hot-melt extrusion and spheronization process.

Controlled-release theophylline containing spherical pellets were successfully produced by a hot-melt extrusion (HME) and spheronization process. A powder blend of anhydrous theophylline, Eudragit Preparation 4135 F, microcrystalline cellulose and polyethylene glycol 8000 powder was sieved, blended and then melt-extruded in a Randcastle Microtruder. The hot-melt extruded pellets were prepared by first cutting a thin, extruded composite rod into symmetrical pellets. The pellets were then spheronized in a traditional spheronizer at an elevated temperature. Thermal properties of the pellet formulation components and the hot-melt extrudate were studied to determine suitability of the formulation for HME. Pellets were examined using scanning electron microscopy to determine the effect of spheronization time on surface morphology. The rate of release of theophylline from the hot-melt extruded spherical pellets was characterized using USP 24 Apparatus 2 dissolution testing after initial pellet production and after 1 year storage in sealed HDPE containers at 25 degrees C/60% RH.