In Vitro and In Vivo testing of 3D-Printed Amorphous Lopinavir Printlets by Selective Laser Sinitering: Improved Bioavailability of a Poorly Soluble Drug.

The aim of this paper was to investigate the effects of formulation parameters on the physicochemical and pharmacokinetic (PK) behavior of amorphous printlets of lopinavir (LPV) manufactured by selective laser sintering 3D printing method (SLS). The formulation variables investigated were disintegrants (magnesium aluminum silicate at 5-10%, microcrystalline cellulose at 10-20%) and the polymer (Kollicoat® IR at 42-57%), while keeping printing parameters constant. Differential scanning calorimetry, X-ray powder diffraction, and Fourier-transform infrared analysis confirmed the transformation of the crystalline drug into an amorphous form.

Preparation and Characterization of 3D-Printed Dose-Flexible Printlets of Tenofovir Disoproxil Fumarate.

The aim of this work was to design pediatric-friendly, dose-flexible orally disintegrating drug delivery systems (printlets) of the antiviral drug tenofovir disoproxil fumarate (TDF) by selective laser sintering (SLS) for potential use in hospitals along with other antiviral drugs. In order to obtain a consistent quality of printlets with desired properties, it is important to understand certain critical quality attributes for their main and interactions effect. The printlets were optimized by Box-Behnken's design of the experiment by varying process variables while keeping the composition constant.

Pyrimethamine 3D printlets for pediatric toxoplasmosis: design, pharmacokinetics, and anti-toxoplasma activity.

Objectives: The focus of the present research is to develop printlet formulations of pyrimethamine (PMT).

Methods: Printlets formulation of PMT were developed by screening design by varying laser scanning speed, Kollidon® VA 64, polyvinylpyrrolidone, and disintegrant.

Results: Laser scanning speed, Kollidon® VA, and disintegrant had statistically significant effect on hardness, disintegration time, and/or dissolution (p < 0.

Very-Rapidly Dissolving Printlets of Isoniazid Manufactured by SLS 3D Printing: In Vitro and In Vivo Characterization.

The focus of this research was to understand the effects of formulation and processing variables on the very-rapidly dissolving printlets of isoniazid (INH) manufactured by the selective laser sintering (SLS) three-dimensional (3D) printing method, and to characterize their physicochemical properties, stability, and pharmacokinetics. Fifteen printlet formulations were manufactured by varying the laser scanning speed (400-500 mm/s, ), surface temperature (100-110 °C, ), and croscarmellose sodium (CCS, %, ), and the responses measured were weight (), hardness (), disintegration time (DT, ), and dissolution (). Laser scanning was the most important processing factor affecting the responses.

Activation of CPP32 during apoptosis of neurons and astrocytes.

Members of the interleukin-1 beta-converting enzyme (ICE)/CED-3 protease family have been implicated in apoptosis in both vertebrates and invertebrates. Using primary culture methods, we report that neurons and astrocytes require the activity of the ICE/CED-3 family of proteases to undergo apoptosis induced by staurosporine, ceramide, and serum-free media. We show that specific inhibitors of ICE/CED-3 proteases can inhibit apoptosis and that cytosolic fractions from apoptosing neurons, but not healthy cells, induced apoptosis in a cell-free system.

Bcl-2 expression in neural cells blocks activation of ICE/CED-3 family proteases during apoptosis.

The ICE/CED-3 family of proteases has been implicated in playing a fundamental role in programmed cell death. Bcl-2 protein represses a number of apoptotic death programs, but the biochemical mechanism of its action is not known. We investigated the activation of ICE/CED-3 proteases induced by three apoptotic stimuli (staurosporine, ceramide, and serum withdrawal) in the neuronal cell line GT1-7 and in cells overexpressing Bcl-2.

Shift of the cellular oxidation-reduction potential in neural cells expressing Bcl-2.

Expression of the protooncogene bcl-2 inhibits both apoptotic and in some cases necrotic cell death in many cell types, including neural cells, and in response to a wide variety of inducers. The mechanism by which the Bcl-2 protein acts to prevent cell death remains elusive. One mechanism by which Bcl-2 has been proposed to act is by decreasing the net cellular generation of reactive oxygen species.

Cleavage of actin by interleukin 1 beta-converting enzyme to reverse DNase I inhibition.

Three of the predominant features of apoptosis are internucleosomal DNA fragmentation, plasma membrane bleb formation, and retraction of cell processes. We demonstrate that actin is a substrate for the proapoptotic cysteine protease interleukin 1beta-converting enzyme. Actin cleaved by interleukin 1beta-converting enzyme can neither inhibit DNase I nor polymerize to its filamentous form as effectively as intact actin.

Differentiation of BC3H1 and primary skeletal muscle cells and the activity of their endogenous insulin-degrading enzyme are inhibited by the same metalloendoprotease inhibitors.

Upon reduction of serum in their media, mouse BC3H1 muscle cells withdraw from the cell cycle and begin to differentiate. In differentiating cells, the induction of muscle-specific genes is accompanied by a distinct morphological chance. However, differentiated BC3H1 cells do not fuse with each other; they remain mononucleated.

Isolation and sequence of lambda gt11 cDNA clones encoding the 5B4 antigen expressed on sprouting neurons.

Monoclonal antibody (mAb) 5B4 recognizes a developmentally regulated membrane glycoprotein (Mr approximately 185,000-255,000) expressed on sprouting neurons. The amino acid sequence deduced from lambda gt11 cDNA clones encoding the transmembrane and cytoplasmic domains of the 5B4 antigen is co-linear with that of chick NCAM-ld. The significant level of overall sequence identity (75%) demonstrates that the 5B4 antigen is rat brain NCAM.

Metalloendoprotease inhibitors which block the differentiation of L6 myoblasts inhibit insulin degradation by the endogenous insulin-degrading enzyme.

The cultured myoblasts of the rat skeletal muscle cell line L6 proliferate till confluency and then fuse to form myotubes and express a number of muscle-specific proteins. We had shown that this differentiation process is blocked by specific metalloendoprotease inhibitors. We now demonstrate that metabolizing L6 myoblasts and their cell extracts degrade insulin to acid-soluble fragments by a non-lysosomal pathway.

Cloning and overexpression of the colicin E1 immunity gene.

A DNA fragment containing only the putative immunity gene-coding sequence was cloned under the control of the trp and lambda PL promoters, generating pRKA11 and pIPL, respectively. Escherichia coli hosts containing either construction were immune to colicin E1. Cells harboring both pIPL and pNT204, which encodes a temperature-sensitive cI repressor, were sensitive to colicin E1 at 30 degrees C, but became immune after 0.

The 5B4 antigen expressed on sprouting neurons contains alpha-2,8-linked polysialic acid.

Monoclonal antibody 5B4 recognizes a large (approximately 185,000-255,000 Da) developmentally regulated membrane glycoprotein, whose expression on fetal rat neurons is coincident with neuronal sprouting. By the use of two prokaryotic-derived probes specific for recognizing alpha-2,8-linked polysialosyl units, we demonstrate the presence of this unusual carbohydrate moiety (a characteristic of neural cell adhesion molecules, NCAMs) on the fetal form of the 5B4 antigen. The 5B4 antigens expressed in adult rat brain (approximately 140,000 and 180,000 Da) do not contain polysialic acid.

Metalloendoprotease inhibitors that block fusion also prevent biochemical differentiation in L6 myoblasts.

The effect of metalloendoprotease inhibitors on the biochemical differentiation of the rat skeletal muscle line, L6, was investigated. Confluent unfused L6 cells exposed briefly to 1,10-phenanthroline, a chelator of divalent metal cations, or continuously to dipeptide amide metalloendoprotease substrates that are blocked at the NH2-terminals, N-carbobenzyloxyserylleucyl amide and N-carbobenzyloxyglycylleucyl amide, did not fuse or express creatine kinase, myosin heavy chain, or alpha-actin. These effects were reversible and dose-dependent.

Membrane action of colicin E1: detection by the release of carboxyfluorescein and calcein from liposomes.

Colicin E1 induces the efflux of carboxyfluorescein and calcein from liposomes whose phospholipid composition is similar to that of Escherichia coli. This colicin action takes place at protein-to-liposome ratios and within pH ranges that are physiologically meaningful. Colicin-induced permeability of carboxyfluorescein is not limited to the initial phase of colicin membrane interaction but is sustained thereafter.

Determination of the molecularity of the colicin E1 channel by stopped-flow ion flux kinetics.

A fluorescence technique that measures fast ion fluxes across liposome membranes was used to determine the molecularity of the colicin E1 channel. The rate of flux of Tl+ (used as a K+ analogue) into large unilamellar vesicles was measured by its ability to quench the fluorescence of a membrane-impermeable fluorophore entrapped in the vesicles. The dependence of Tl+ flux rate on the concentration of ionophore in the vesicle suspension reveals the molecularity of the ionophore.

Correlation between fusion and the developmental regulation of membrane glycoproteins in L6 myoblasts.

Expression of membrane glycoproteins in L6 myoblasts during the course of myogenesis was investigated. The effects of several inhibitors of myoblast fusion and differentiation were also studied. The predominant change in plasma membrane proteins concomitant with fusion was the reduction in the expression of a major 105-kDa glycoprotein and the appearance of a 90-kDa glycoprotein.

Identification of the plasmid-encoded immunity protein for colicin E1 in the inner membrane of Escherichia coli.

A set of plasmids containing portions of the Col E1 plasmid were transformed into recA- cells. These cells, after UV irradiation, only incorporate labelled amino acids into plasmid-encoded proteins. UV-irradiated cells label a 14.

Genetic study of the functional organization of the colicin E1 molecule.

Colicin E1 fragments obtained by genetic manipulations of the ColE1 plasmid were tested for bactericidal activity, binding to bacterial cells, and reactions with a series of anticolicin monoclonal antibodies. Two of the fragments were also tested for ability to form channels in liposomal vesicles. The results are in agreement with studies from chemically and enzymatically derived colicin fragments, assigning the receptor binding activity to the central part of the molecule and the killing activity to a region near the carboxyl terminus.

Comparison of two liposome fusion assays monitoring the intermixing of aqueous contents and of membrane components.

Divalent cation-induced fusion of large unilamellar vesicles (approx. 0.1 micron diameter) made of phosphatidylserine (PS) or phosphatidylglycerol (PG) has been studied.

An alternating site sequence for oxidative phosphorylation suggested by measurement of substrate binding patterns and exchange reaction inhibitions.

Catalysis by beef heart submitochondrial particles of the medium Pi in equilibrium HOH, Pi in equilibrium ATP, and the ATP in equilibrium HOH exchanges is strongly inhibited while the ATPase and intermediate Pi in equilibrium HOH exchange are accelerated when medium ADP is removed by pyruvate kinase action. Arsenate readily blocks completely the Pi in equilibrium ATP and medium Pi in equilibrium HOH exchange reactions, but not the ATP in equilibrium HOH exchange reaction. The residual ATP in equilibrium HOH exchange in presence of arsenate is inhibited by 2,4-dinitrophenol.

Evidence for energy-dependent change in phosphate binding for mitochondrial oxidative phosphorylation based on measurements of medium and intermediate phosphate-water exchanges.

Characteristics of the exchange reactions catalyzed by beef heart submitochondrial particles give new insight into energy transducing steps of oxidative phosphorylation. The uncoupler-insensitive portion of the total Pi in equilibrium HOH exchange in presence of ATP, ADP, and Pi is the intermediate Pi in equilibrium HOH exchange, that is the exchange occurring with Pi formed by hydrolysis of ATP prior to release of Pi from the catalytic site. The exchange of medium Pi with HOH is as sensitive to uncouplers as the Pi in equilibrium ATP exchange and net oxidative phosphorylation, demonstrating a requirement of an uncoupler-sensitive energized state, probably a transmembrane potential or proton gradient, for bringing medium Pi to the reactive state.