CIRCADIAN RHYTHM IN THE BIOAVAILABILITY OF ORALLY ADMINISTERED THEOPHYLLINE

The effect of time (i.e., biologic time structure) of drug administration on the bioavailability of theophylline was investigated in man after both a single dosage as well as after repeated, or chronic, drug administrations. Preliminary laboratory investigations on Balb-C mice showed the toxic

DEVELOPMENT AND APPLICATION OF SHORT-TERM MUTAGENICITY AND CYTOTOXICITY BIOASSAYS FOR INTEGRATED HEALTH ASSESSMENT STUDIES OF COAL FLY ASH EMISSIONS

Two respirable coal fly ash samples ((LESSTHEQ) 3(mu)m), one from a pressurized fluidized-bed combustion miniplant and one from a conventional combustion power plant, were investigated for physical properties, chemical composition and biological activity. Electron microscopy illustrated irregularity in fluidized-bed combustion fly ash and sphericity in conventional combustion fly ash. Elemental analysis of these samples showed differences in trace elements. Both fly ash samples were toxic in rabbit alveolar macrophage and Chinese hamster ovary cell systems in vitro. The macrophages were more sensitive to toxicity of fly ash than the ovary cells. For measuring the cytotoxicity of fly ash, the most sensitive parameters were adenosine triphosphate in the alveolar macrophage system and viability index in the hamster ovary system. Intact fluidized-bed combustion fly-ash particles showed mutagenicity only in strains TA98 and TA1538 without metabolic activation in the Ames Salmonella assay. No mutagenicity was detected in bioassay of conventional combustion fly ash particles. Solvent extraction yielded more mass from fluidized-bed combustion fly ash than from conventional combustion fly ash. The extracts of fluidized-bed combustion fly ash showed higher mutagenic activity than conventional combustion fly ash. These samples contained direct-acting, frameshift mutagens.^ Fly ash samples collected from the same fluidized-bed source by cyclones, a fabric filter, and a electrostatic precipitator at various temperatures were compared for particle size, toxicity, and mutagenicity. Results demonstrated that the biological activity of coal fly ash were affected by the collection site, device, and temperature.^ Coal fly ash vapor-coated with 1-nitropyrene was developed as a model system to study the bioavailability and recovery of nitroaromatic compounds in fly ash. The effects of vapor deposition on toxicity and mutagenicity of fly ash were examined. The nitropyrene coating did not significantly alter the ash's cytotoxicity. Nitropyrene was bioavailable in the biological media, and a significant percentage was not recovered after the coated fly ash was cultured with alveolar macrophages. 1-Nitropyrene loss increased as the number of macrophages was increased, suggesting that the macrophages are capable of metabolizing or binding 1-nitropyrene present in coal fly ash. ^

Novel Use of Dual Anti-Inflammatory Therapy to Overcome Drug Resistance and Improve Functional Recovery Following Spinal Cord Injury

Over 1.2 million Americans are currently living with a traumatic spinal cord injury (SCI). Despite the need for effective therapies, there are currently no proven effective treatments that can improve recovery of function in SCI patients. Many therapeutic compounds have shown promise in preclinical models of SCI, but all of these have fallen short in clinical trials. P-glycoprotein (Pgp) is an active transporter expressed on capillary endothelial cell membranes at the blood-spinal cord barrier (BSCB). Pgp limits passive diffusion of blood-borne drugs into the CNS, by actively extruding drugs from the endothelial cell membrane. Pgp can become pathologically up-regulated, thus greatly impeding therapeutic drug delivery (‘multidrug resistance’). Importantly, many drugs that have been evaluated for the treatment of SCI are Pgp substrates. We hypothesized that Pgp-mediated drug resistance diminishes the delivery and efficacy of neuroprotective drugs following SCI. We observed a progressive, spatial spread of Pgp overexpression within the injured spinal cord. To assess Pgp function, we examined spinal cord uptake of systemically-delivered riluzole, a drug that is currently being evaluated in clinical trials as an SCI intervention. Blood-to-spinal cord riluzole penetration was reduced following SCI in wild-type but not Pgp-null rats, highlighting a critical role for Pgp in mediating spinal cord drug resistance after injury. Others have shown that pro-inflammatory signaling drives Pgp up-regulation in cancer and epilepsy. We have detected inflammation in both acutely- and chronically-injured spinal cord tissue. We therefore evaluated the ability of the dual COX-/5-LOX inhibitor licofelone to attenuate Pgp-mediated drug resistance following SCI. Licofelone treatment both reduced spinal cord Pgp levels and enhanced spinal cord riluzole bioavailability following SCI. Thus, we propose that licofelone may offer a new combinatorial treatment strategy to enhance spinal cord drug delivery following SCI. Additionally, we assessed the ability of licofelone, riluzole, or both to enhance recovery of locomotor function following SCI. We found that licofelone treatment conferred a significant improvement in hindlimb function that was sustained through the end of the study. In contrast, riluzole did not improve functional outcome. We therefore conclude that licofelone holds promise as a potential neuroprotective intervention for SCI.

Targeting TRAF6 for Cancer Therapeutical Development

Tumor necrosis factor (TNF)-Receptor Associated Factors (TRAFs) are a family of signal transducer proteins. TRAF6 is a unique member of this family in that it is involved in not only the TNF superfamily, but the toll-like receptor (TLR)/IL-1R (TIR) superfamily. The formation of the complex consisting of Receptor Activator of Nuclear Factor κ B (RANK), with its ligand (RANKL) results in the recruitment of TRAF6, which activates NF-κB, JNK and MAP kinase pathways. TRAF6 is critical in signaling with leading to release of various growth factors in bone, and promotes osteoclastogenesis. TRAF6 has also been implicated as an oncogene in lung cancer and as a target in multiple myeloma. In the hopes of developing small molecule inhibitors of the TRAF6-RANK interaction, multiple steps were carried out. Computational prediction of hot spot residues on the protein-protein interaction of TRAF6 and RANK were examined. Three methods were used: Robetta, KFC2, and HotPoint, each of which uses a different methodology to determine if a residue is a hot spot. These hot spot predictions were considered the basis for resolving the binding site for in silico high-throughput screening using GOLD and the MyriaScreen database of drug/lead-like compounds. Computationally intensive molecular dynamics simulations highlighted the binding mechanism and TRAF6 structural changes upon hit binding. Compounds identified as hits were verified using a GST-pull down assay, comparing inhibition to a RANK decoy peptide. Since many drugs fail due to lack of efficacy and toxicity, predictive models for the evaluation of the LD50 and bioavailability of our TRAF6 hits, and these models can be used towards other drugs and small molecule therapeutics as well. Datasets of compounds and their corresponding bioavailability and LD50 values were curated based, and QSAR models were built using molecular descriptors of these compounds using the k-nearest neighbor (k-NN) method, and quality of these models were cross-validated.