Pharmacokinetics and anti-inflammatory pharmacodynamics of prednisolone in cynomolgus monkey.

The purpose was to investigate whether the pharmacokinetics and pharmacodynamics of prednisolone in the non-human primate was an appropriate surrogate for man. After single intravenous doses of 0.03, 0.3, and 3 mg kg(-1), prednisolone demonstrated a dose-dependent clearance and volume of distribution. When corrected for concentration-dependent protein binding, the free clearance was linear at the tested dose levels. The protein binding-corrected volume of distribution was similar across doses. The serum half-life was estimated as being between 2 and 4 h. Prednisolone exhibits near complete inhibition of the cytokines TNF-alpha, IL-1beta, IL-6 and IL-8 with very similar IC50 estimates from 0.09 to 0.16 microg ml(-1) (from 0.24 to 0.44 microM). The monkey demonstrated a similar pharmacokinetics-pharmacodynamics profile of prednisolone when compared with man (from the literature).

Inhibition of cholesteryl ester transfer protein by substituted dithiobisnicotinic acid dimethyl ester: involvement Of a critical cysteine.

SC-71952, a substituted analog of dithiobisnicotinic acid dimethyl ester, was identified as a potent inhibitor of cholesteryl ester transfer protein (CETP). When tested in an in vitro assay, the concentration of SC-71952 required for half-maximal inhibition was 1 microm. The potency of SC-71952 was enhanced 200-fold by preincubation of the inhibitor with CETP, and was decreased 50-fold by treatment with dithiothreitol. Analogs of SC-71952 that did not contain a disulfide linkage were less potent, did not display time dependency, and were not affected by dithiothreitol treatment. Kinetic and biochemical characterization of the inhibitory process of CETP by SC-71952 suggested that the inhibitor initially binds rapidly and reversibly to a hydrophobic site on CETP. With time, the bound inhibitor irreversibly inactivates CETP, presumably by reacting with one of the free cysteines of CETP. Liquid chromatography/mass spectroscopy (LC/MS) analyses of tryptic digests of untreated or SC-71952-inactivated CETP was used to identify which cysteine(s) were potentially involved in the time-dependent, irreversible component of inactivation by the inhibitor. One disulfide bond, Cys143-Cys184, was unaffected by treatment with the inhibitor. Inactivation of CETP by SC-71952 correlated with a progressive decrease in the abundance of free Cys-13 and Cys-333. Conversion of Cys-13 to alanine had no effect on the rapid reversible component of inactivation by SC-71952. However, it abolished the time-dependent enhancement in potency seen with the inhibitor when using wild-type CETP. These data indicate that Cys-13 is critical for the irreversible inactivation of CETP by SC-71952 and provides support for the structural model that places Cys-13 near the neutral lipid-binding site of CETP.

Development and comparison of two 3T3-L1 adipocyte models of insulin resistance: increased glucose flux vs glucosamine treatment.

Insulin resistance can be induced in vivo by intravenous infusion of glucosamine or in cells by incubation with glucosamine. However, a publication (Hresko, R. C., et al. (1998) J. Biol. Chem. 273, 20658-20668) suggests a trivial explanation of glucosamine-induced insulin resistance whereby intracellular ATP pools are depleted presumably due to the phosphorylation of glucosamine to glucosamine 6-phosphate, a hexosamine pathway intermediate. The reduced ATP level impaired insulin receptor (IR) autophosphorylation and tyrosine kinase activity toward substrates. The present work describes the development and comparison of two methods for inducing insulin resistance, by treating 3T3-L1 adipocytes overnight using either 25 mM glucose/5 nM insulin or 2 mM glucosamine. Under these conditions basal glucose transport rates were comparable with controls. Insulin-stimulated 2-deoxyglucose uptake, however, was reduced by approximately 45% in response to both high glucose/insulin and glucosamine treatment, relative to control cells. The total relative amounts of the insulin-responsive glucose transporter, Glut4, remained constant under both treatment conditions. The relative phosphotyrosine (Tyr(P)) contents of the insulin receptor and its substrate 1 (IRS-1) were assessed in whole cell homogenates. With both methods to induce insulin resistance, IR/IRS-1 Tyr(P) levels were virtually indistinguishable from those in control cells. Insulin-stimulated phosphorylation of Akt on Ser(473) was not impaired in insulin-resistant cells. Furthermore, the relative Tyr(P) content of the PDGF receptor was comparable in high glucose/insulin- or glucosamine-treated 3T3-L1 adipocytes upon subsequent challenge with PDGF. Finally, the relative amounts of glutamine:fructose-6-phosphate amidotransferase and O-linked N-acetylglucosamine transferase, two important hexosamine pathway enzymes, were similar in both treatments when compared with controls. Thus, 3T3-L1 adipocytes can be used as a model system for studying insulin resistance induced by increased influx of glucose. Under appropriate experimental conditions, glucosamine treatment can mimic the effects of increased glucose flux without impairment of tyrosine phosphorylation-based signaling.

Large-scale purification of myeloperoxidase from HL60 promyelocytic cells: characterization and comparison to human neutrophil myeloperoxidase.

A large-scale purification procedure was developed for the isolation of myeloperoxidase from HL60 promyelocytic cells in culture. Initial studies showed the bulk of peroxidase-positive myeloperoxidase activity to be located in the cetyltrimethylammonium bromide solubilized particulate fraction of cell homogenates. The myeloperoxidase was then chromatographically purified using concanavalin A followed by gel filtration. SDS-PAGE analysis of the final preparation showed the presence of only two proteins with molecular masses of approximately 55 and 15 kDa, corresponding to the large and small subunits of myeloperoxidase. These data, along with Reinheit Zahl (RZ) values (A(430)/A(280)) of greater than or equal to 0.72, indicate that the myeloperoxidase prepared by this method is apparently homogeneous. Preparations routinely yielded 12-20 mg of pure myeloperoxidase per 10 ml of cell pellet. The HL60 myeloperoxidase was shown to be indistinguishable from purified human neutrophil myeloperoxidase by size exclusion chromatography, analytical ultracentrifugation, SDS-PAGE, Western blot, and NH(2)-terminal sequence analysis. The activities of the two myeloperoxidase samples, as measured using either the tetramethylbenzidine or the taurine chloramine assay, were indistinguishable. Finally, both enzymes responded identically to dapsone and aminobenzoic acid hydrazide, known inhibitors of myeloperoxidase. A protocol is presented here for the rapid, large-scale purification of myeloperoxidase from cultured HL60 cells, as well as evidence for the interchangeability of this myeloperoxidase and that purified from human neutrophils.

Phosphoinositides and phosphoinositide-utilizing enzymes in detergent-insoluble lipid domains.

Recent evidence has implicated caveolae/DIGs in various aspects of signal transduction, a process in which polyphosphoinositides play a central role. We therefore undertook a study to determine the distribution of phosphoinositides and the enzymes that utilize them in these detergent-insoluble domains. We report here that the polyphosphoinositide phosphatase, but not several other phosphoinositide-utilizing enzymes, is highly enriched in a low density, Triton-insoluble membrane fraction that contains caveolin. This fraction is also enriched in polyphosphoinositides, containing approximately one-fifth of the total cellular phosphatidylinositol (4,5)P2. Treatment of cells with the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), did not alter the distribution of polyphosphoinositides or the polyphosphoinositide phosphatase. However, PMA treatment did lead to a decrease in the mitogen-activated protein kinase and actin present in these domains. PMA also induced the recruitment of protein kinase C alpha to the caveolae/DIGs fraction. These findings suggest that polyphosphoinositides, the polyphosphoinositide phosphatase and protein kinase C play an important role in the structure or function of detergent-insoluble membrane domains.