Solid Self-Microemulsifying Formulation for Candesartan Cilexetil

Sparingly, water-soluble drugs such as candesartan cilexetil offer challenges in developing a drug product with adequate bioavailability. The objective of the present study was to develop and characterize self-microemulsifying drug delivery system (SMEDDS) of candesartan cilexetil for filling into hard gelatin capsules. Solubility of candesartan cilexetil was evaluated in various nonaqueous careers that included oils, surfactants, and cosurfactants. Pseudoternary phase diagrams were constructed to identify the self-microemulsification region. Four self-microemulsifying formulations were prepared using mixtures of oils, surfactants, and cosurfactants in various proportions. The self-microemulsification properties, droplet size, and zeta potential of these formulations were studied upon dilution with water. The optimized liquid SMEDDS formulation was converted into free flowing powder by adsorbing onto a solid carrier for encapsulation. The dissolution characteristics of solid intermediates of SMEDDS filled into hard gelatin capsules was investigated and compared with liquid formulation and commercial formulation to ascertain the impact on self-emulsifying properties following conversion. The results indicated that solid intermediates showed comparable rate and extent of drug dissolution in a discriminating dissolution medium as liquid SMEDDS indicating that the self-emulsifying properties of SMEDDS were unaffected following conversion. Also, the rate and extent of drug dissolution for solid intermediates was significantly higher than commercial tablet formulation. The results from this study demonstrate the potential use of SMEDDS as a means of improving solubility, dissolution, and concomitantly the bioavailability.     

MiR-886-3p down regulates CXCL12 (SDF1) expression in human marrow stromal cells

Stromal Derived Factor 1 (SDF1 or CXCL12), is a chemokine known to be critical for the migration of cells in several tissue systems including the homing of the hematopoietic stem cell (HSC) to its niche in the bone marrow. A comparative analysis of miRNA expression profiles of two stromal cell lines, distinguishable by function and by CXCL12 expression (CXCL12+ and CXCL12-), revealed that the CXCL12- cells expressed>40 fold more miR-886-3p than the CXCL12+ cells. Screening studies showed that when miR-886-3p was transfected into the CXCL12+ stromal cells, the expression of CXCL12 was down-regulated by as much as 85% when compared to appropriate controls, and results in the loss of CXCL12-directed chemotaxis. Similar reductions in CXCL12 were obtained with the transfection of miR-886-3p into primary stromal cell cultures. Additional studies showed that miR-886-3p specifically targeted the 3' untranslated region (UTR) of CXCL12 mRNA. These data suggest a role for miRNA in modulating the expression of CXCL12, a gene product with a critical role in hematopoietic regulation.     

Detailed comparison of the protein-ligand docking efficiencies of GOLD, a commercial package and ArgusLab, a licensable freeware

Molecular docking and virtual screening based on molecular docking have become an integral part of many modern structure-based drug discovery efforts. Hence, it becomes a useful endeavor to evaluate existing docking programs, which can assist in the choice of the most suitable docking algorithm for any particular study. The objective of the current study was to evaluate the ability of ArgusLab 4.0, a relatively new molecular modeling package in which molecular docking is implemented, to reproduce crystallographic binding orientations and to compare its accuracy with that of a well established commercial package, GOLD. The study also aimed to evaluate the effect of the nature of the binding site and ligand properties on docking accuracy. The three dimensional structures of a carefully chosen set of 75 pharmaceutically relevant protein-ligand complexes were used for the comparative study. The study revealed that the commercial package outperforms the freely available docking engine in almost all the parameters tested. However, the study also revealed that although lagging behind in accuracy, results from ArgusLab are biologically meaningful. This taken together with the fact that ArgusLab has an easy to use graphical user interface, means that it can be employed as an effective teaching tool to demonstrate molecular docking to beginners in this area.     

Influence of dietary fiber from coconut kernel (Cocos nucifera) on the 1,2-dimethylhydrazine-induced lipid peroxidation in rats

The influence of dietary fiber from coconut kernel isolated by the neutral detergent fiber method on the antioxidant status in rats treated with the colon specific carcinogen 1,2-dimethylhydrazine (DMH) was studied in rats fed a high-fat diet for 15 weeks. The DMH-treated fiber group showed higher levels of lipid peroxides than the control group treated with DMH at the preneoplastic and neoplastic stages. Free fatty acid levels were found to decrease significantly in the DMH-treated control group, whereas it was near normal in the fiber groups. Superoxide dismutase and catalase activity also were found to be increased in the liver, intestine, proximal colon, and distal colon. Glutathione levels in all the tissues studied showed significant decreases in the fiber group. The results suggest that coconut kernel fiber can protect cells from loss of oxidative capacity with the administration of the procarcinogen DMH.     

Synergism in the Oviposition Behavior of Plutella xylostella: Sinigrin and Wax Compounds

Diamondback moth, Plutella xylostella, host examining and oviposition behaviors were measured in response to sinigrin dosages (10 ^–3 , 10 ^–4 , and 10 ^–5 M) and controls with and without the addition of n-alkanes. Individual females were presented with a treatment and videotaped while an observer documented specific behaviors during 5-min observation periods. Behavior in response to sinigrin alone was not significantly different from that in response to controls. Alkane alone significantly reduced movement rate during treatment contact, but did not significantly affect other behaviors. Sinigrin concentrations combined with alkane significantly slowed the rate of insect movement, increased turning, and led to significantly longer treatment encounter durations. Behavior changes in response to sinigrin + alkane increased insect exposure to the sinigrin concentrations and led to greater oviposition compared to that in response to sinigrin treatments alone. The synergistic effect that mixing sinigrin and alkane has on P. xylostella behavior arises because the additional time females spend in contact with the treatment increases the rate at which they experience the available stimuli. Involvement of the antennae during examining of a treatment, referred to as swabbing, was usually associated with oviposition on alkane-coated sinigrin treatments. The presence of alkane may alter the way sinigrin is perceived. Oviposition in response to the treatment combinations was also tested in overnight bioassays. The pattern of oviposition in response to treatments during bioassays differed from that established during observations. The value of direct observations and the mechanistic interpretations they allow are emphasized.     

Structure and function of CC-chemokine receptor 5 homologues derived from representative primate species and subspecies of the taxonomic suborders Prosimii and Anthropoidea

A chemokine receptor from the seven-transmembrane-domain G-protein-coupled receptor superfamily is an essential coreceptor for the cellular entry of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) strains. To investigate nonhuman primate CC-chemokine receptor 5 (CCR5) homologue structure and function, we amplified CCR5 DNA sequences from peripheral blood cells obtained from 24 representative species and subspecies of the primate suborders Prosimii (family Lemuridae) and Anthropoidea (families Cebidae, Callitrichidae, Cercopithecidae, Hylobatidae, and Pongidae) by PCR with primers flanking the coding region of the gene. Full-length CCR5 was inserted into pCDNA3.1, and multiple clones were sequenced to permit discrimination of both alleles. Compared to the human CCR5 sequence, the CCR5 sequences of the Lemuridae, Cebidae, and Cercopithecidae shared 87, 91 to 92, and 96 to 99% amino acid sequence homology, respectively. Amino acid substitutions tended to cluster in the amino and carboxy termini, the first transmembrane domain, and the second extracellular loop, with a pattern of species-specific changes that characterized CCR5 homologues from primates within a given family. At variance with humans, all primate species examined from the suborder Anthropoidea had amino acid substitutions at positions 13 (N to D) and 129 (V to I); the former change is critical for CD4-independent binding of SIV to CCR5. Within the Cebidae, Cercopithecidae, and Pongidae (including humans), CCR5 nucleotide similarities were 95.2 to 97.4, 98.0 to 99.5, and 98.3 to 99.3%, respectively. Despite this low genetic diversity, the phylogeny of the selected primate CCR5 homologue sequences agrees with present primate systematics, apart from some intermingling of species of the Cebidae and Cercopithecidae. Constructed HOS.CD4 cell lines expressing the entire CCR5 homologue protein from each of the Anthropoidea species and subspecies were tested for their ability to support HIV-1 and SIV entry and membrane fusion. Other than that of Cercopithecus pygerythrus, all CCR5 homologues tested were able to support both SIV and HIV-1 entry. Our results suggest that the shared structure and function of primate CCR5 homologue proteins would not impede the movement of primate immunodeficiency viruses between species.     

Synthesis of retro acyl carrier protein (74-65) fragment on a new glycerol based polystyrene support

Retro-ACP (74-65) fragment was synthesized on a novel 4% tri-(propylene glycol) glycerolate diacrylate cross-linked polystyrene (PS-TRPGGDA) support. The peptide is grown from the functional site present in the cross-linker, which makes it unique and cost-effective among other styrene based polymer supports. A comparative study with Merrifield resin indicates high yield and purity of the peptide synthesized on the novel support.     

Protein kinase C-associated kinase can activate NFkappaB in both a kinase-dependent and a kinase-independent manner

Protein kinase C-associated kinase (PKK, also known as RIP4/DIK) activates NFkappaB when overexpressed in cell lines and is required for keratinocyte differentiation in vivo. However, very little is understood about the factors upstream of PKK or how PKK activates NFkappaB. Here we show that certain catalytically inactive mutants of PKK can activate NFkappaB, although to a lesser degree than wild type PKK. The deletion of specific domains of wild type PKK diminishes the ability of this enzyme to activate NFkappaB; the same deletions made on a catalytically inactive PKK background completely ablate NFkappaB activation. PKK may be phosphorylated by two specific mitogen-activated protein kinase kinase kinases, MEKK2 and MEKK3, and this interaction may in part be mediated through a critical activation loop residue, Thr184. Catalytically inactive PKK mutants that block phorbol ester-induced NFkappaB activation do not interfere with, but unexpectedly enhance, the activation of NFkappaB by these two mitogen-activated protein kinase kinase kinases. Taken together, these data indicate that PKK may function in both a kinase-dependent as well as a kinase-independent manner to activate NFkappaB.     

Biosynthesis of triacylglycerol molecular species in an oleaginous fungus,Mortierella ramanniana var. angulispora

The incorporation of radiolabeled lipid precursors into triacylglycerol (TG) molecular species in Mortierella ramanniana var. angulispora, an oleaginous fungus, was studied to determine the biosynthetic pathways for TG molecular species. Radiolabeled TG molecular species were separated and quantified by reverse-phase high performance liquid chromatography with a radioisotope detector. The major TG molecular species labeled by [1-(14)C]oleic acid at 30 degrees C were OOP, OOO, and OPP (TG molecular species designations represent three constituent acyl groups. G, gamma-linolenic acid; L, linoleic acid; O, oleic acid; S, stearic acid; P, palmitic acid), which were abundant TG molecular species in this fungus. The incorporation of [1-(14)C]oleic acid at 15 degrees C into these molecular species was the same, while that into most other species was decreased, suggesting that biosynthesis of major molecular species such as OOP, OOO, and OPP differs from that of other TG molecular species. [1-(14)C]Linoleic acid incorporation indicated that the major labeled molecular species were LOP and LOO, which may be due to acylation of oleoyl, palmitoyl-glycerol, or dioleoyl-glycerol by exogenous linoleic acid. This is basically the same mechanism as for OOP and OOO biosynthesis from exogenous oleic acid. [(14)C(U)]Glycerol incorporation suggested that TG molecular species containing palmitic acid such as OPP were more readily synthesized through the de novo pathway. Further experiments involving inhibitors such as sodium azide and cerulenin suggested that OOO biosynthesis included a mechanism differing from that in the cases of OOP and OPP. Trifluoperazine, which inhibits the conversion from phosphatidic acid to TG, decreased [1-(14)C]oleic acid incorporation into all molecular species, suggesting that the incorporation into all molecular species included the de novo TG biosynthetic pathway via phosphatidic acid. These results revealed that the biosynthetic pathways for TG molecular species can be classified into several groups, which exhibit different sensitivities to low temperature and inhibitors of lipid metabolism. This implies that the composition of TG molecular species is regulated through different biosynthetic pathways responsible for specific TG molecular species, providing a new insight into the biosynthesis of TG molecular species.