ABSTRACT
The tendency for mycobacteria to aggregate poses a challenge for their use in microplate based assays. Good dispersions have been difficult to achieve in high-throughput screening (HTS) assays used in the search for novel antibacterial drugs to treat tuberculosis and other related diseases. Here we describe a method using filtration to overcome the problem of variability resulting from aggregation of mycobacteria. This method consistently yielded higher reproducibility and lower variability than conventional methods, such as settling under gravity and vortexing.
Subject(s)
Antitubercular Agents/pharmacology , Filtration/methods , Mycobacterium smegmatis/drug effects , Mycobacterium tuberculosis/drug effects , Drug Evaluation, Preclinical/methods , Filtration/instrumentation , High-Throughput Screening Assays/methods , Micropore Filters , Mycobacterium smegmatis/physiology , Mycobacterium tuberculosis/physiology , Reproducibility of ResultsABSTRACT
A novel series of potent substituted anilinoquinolines were discovered as c-fms inhibitors. The potency could be manipulated upon modification of the C4 aniline and C7 aryl functionality. Pharmacokinetic analysis identified a metabolically stable analog suitable for further investigative work.
Subject(s)
Aniline Compounds/chemistry , Quinolines/chemical synthesis , Quinolines/pharmacology , Receptor, Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Animals , Binding Sites , Cell Line , Drug Evaluation, Preclinical , Drug Stability , Inhibitory Concentration 50 , Models, Animal , Models, Molecular , Molecular Structure , Quinolines/chemistry , Rats , Structure-Activity RelationshipABSTRACT
OBJECTIVES: To assess the use of the Biojector B2000 needle-free gas-powered injection system for subcutaneous administration of enfuvirtide in HIV-infected patients and to compare this system with standard needles and syringes with respect to ease of use, severity of injection site reactions (ISR), and enfuvirtide plasma levels. DESIGN: An observational study among 32 treatment-experienced HIV clinic patients receiving enfuvirtide. METHODS: Adult patients were assessed before and after switching from standard needles to the Biojector for enfuvirtide administration. Patients used the Biojector for up to 24 weeks and rated ease of use from 0 (easy) to 3 (difficult). ISR were graded from 0 to 31 for signs and symptoms (erythema, induration, pruritus, nodules/cysts, ecchymosis), duration of individual lesions, and number of lesions. Plasma was collected pre-dose and 1 h post-dose for enfuvirtide measurement. The high-pressure liquid chromatography with tandem mass spectrometry method used was specific for enfuvirtide over its known plasma metabolite. Wilcoxon rank sum tests were used to compare needle-based and Biojector outcomes. RESULTS: The Biojector was rated as being significantly easier to use (P < 0.001) and reduced the occurrence of ISR compared with standard needles (P < 0.001). Enfuvirtide plasma levels were not statistically different between the two administration methods at either pre-dose trough (P = 0.41) or 1 h post-dose (P = 0.74). CONCLUSIONS: The Biojector needle-free injection system was easy to use for enfuvirtide administration and was associated with a decreased severity of ISR. Plasma enfuvirtide levels pre-dose and 1 h post-dose were comparable when injecting with standard needles or the Biojector.
Subject(s)
HIV Envelope Protein gp41/administration & dosage , HIV Fusion Inhibitors/administration & dosage , HIV Infections/drug therapy , HIV-1 , Peptide Fragments/administration & dosage , Adult , Enfuvirtide , Equipment Design , Follow-Up Studies , HIV Envelope Protein gp41/therapeutic use , HIV Fusion Inhibitors/pharmacokinetics , HIV Fusion Inhibitors/therapeutic use , HIV Infections/metabolism , Humans , Injections, Jet/instrumentation , Injections, Jet/methods , Needles , Patient Satisfaction , Peptide Fragments/pharmacokinetics , Peptide Fragments/therapeutic use , Skin/drug effects , Statistics, Nonparametric , SyringesABSTRACT
Colony-stimulating-factor-1 (CSF-1) signaling through cFMS receptor kinase is increased in several diseases. To help investigate the role of cFMS kinase in disease, we identified GW2580, an orally bioavailable inhibitor of cFMS kinase. GW2580 completely inhibited human cFMS kinase in vitro at 0.06 microM and was inactive against 26 other kinases. GW2580 at 1 microM completely inhibited CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibited bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone. In contrast, GW2580 did not affect the growth of mouse NS0 lymphoblastoid cells, human endothelial cells, human fibroblasts, or five human tumor cell lines. GW2580 also did not affect lipopolysaccharide (LPS)-induced TNF, IL-6, and prostaglandin E2 production in freshly isolated human monocytes and mouse macrophages. After oral administration, GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice, inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity, and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly, GW2580 inhibited LPS-induced TNF production in mice, in contrast to effects on monocytes and macrophages in vitro. In conclusion, GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cells in vivo makes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes.
