Susan K. Paulson

Plasma protein binding of celecoxib in mice, rat, rabbit, dog and human

The plasma protein binding of celecoxib was determined for animals and humans using in vitro and ex vivo methods. Eight, healthy, human volunteers (three male, five female, 20–39 years) received celecoxib (600 mg) BID for 7 days, blood samples were collected and concentrations of bound and unbound celecoxib determined. The fraction of bound drug in the volunteers was constant (97.4±0.1%) at total celecoxib plasma concentrations ranging from 0.01 to 4.02 μg/mL. The ex vivo plasma protein binding of celecoxib in the animals was concentration‐independent up to approximately 12, 8 and 10 μg/mL for mouse, rat and dog, respectively. The plasma protein binding of celecoxib after a single oral dose of 10 and 300 mg/kg to mice was 98.3±0.2%, of 1 and 400 mg/kg to rats was 98.3±0.2% and of 1 and 100 mg/kg to dogs was 98.5±0.1%. The percent binding of celecoxib to plasma proteins in vitro was slightly lower than those values determined ex vivo. The in vitro binding of celecoxib to plasma protein was constant over the concentrations of 0.1–10 μg/mL for all species, except rat. Copyright

Tissue Factor Pathway Inhibitor Does Not Influence Inflammatory Pathways during Human Endotoxemia

Activation of coagulation induces a proinflammatory response in in vitro and animal experiments. Inhibition of the tissue factor-dependent pathway of coagulation inhibits cytokine release and prevents death in gram-negative sepsis models in primates. This study investigated the influence of blocking the coagulation system by tissue factor pathway inhibitor (TFPI) on endotoxin-induced inflammatory responses in healthy humans. Eight men were studied in a double-blind, randomized, placebo-controlled cross-over study. They received a bolus intravenous injection of 4 ng/kg of endotoxin, followed by a 6-h continuous infusion of either TFPI (0.2 mg/kg/h after a bolus of 0.05 mg/kg) or placebo. Endotoxin induced-activation of coagulation was prevented completely by TFPI. In contrast, TFPI did not influence leukocyte activation, chemokine release, endothelial cell activation, or the acute phase response. Thus, complete prevention of coagulation activation by TFPI does not influence activation of inflammatory pathways during human endotoxemia.

Characterization of metabolites of Celecoxib in rabbits by liquid chromatography/tandem mass spectrometry

The metabolism of the anti-inflammatory drug Celecoxib in rabbits was characterized using liquid chromatography (LC)/tandem mass spectrometry (MS/MS) with precursor ion and constant neutral loss scans followed by product ion scans. After separation by on-line liquid chromatography, the crude urine samples and plasma and fecal extracts were analyzed with turbo-ionspray ionization in negative ion mode using a precursor ion scan of m/z 69 (CF(3)) and a neutral loss scan of 176 (dehydroglucuronic acid). The subsequent product ion scans of the [M - H] ions of these metabolites yielded the identification of three phase I and four phase II metabolites. The phase I metabolites had hydroxylations at the methyl group or on the phenyl ring of Celecoxib, and the subsequent oxidation product of the hydroxymethyl metabolite formed the carboxylic acid metabolite. The phase II metabolites included four positional isomers of acyl glucuronide conjugates of the carboxylic acid metabolite. These positional isomers were caused by the alkaline pH of the rabbit urine and were not found in rabbit plasma. The chemical structures of the metabolites were characterized by interpretation of their product ion spectra and comparison of their LC retention times and the product ion spectra with those of the authentic synthesized standards.

The influence of reduced liver blood flow on the pharmacokinetics and pharmacodynamics of recombinant tissue factor pathway inhibitor

Recombinant tissue factor pathway inhibitor (rTFPI) has been shown to be an effective treatment in animal models of sepsis and is under investigation for human use. Reduced liver blood flow during septic shock may substantially alter the pharmacokinetics of rTFPI because clearance of rTFPI approaches liver blood flow. The aim of this study was to examine the effect of exercise‐induced reduction in liver blood flow on the pharmacokinetics and pharmacodynamics of rTFPI.

A radioreceptor binding assay for measurement of platelet-activating factor synthesis by human neutrophils

A new convenient method for the measurement of platelet-activating factor produced in vitro has been developed. This method involves incubation of neutrophils with stimuli, lipid extraction, purification of lipid extracts by normal phase high performance liquid chromatography (HPLC) and quantification of platelet-activating factor (PAF) by a competitive radioreceptor binding assay. The recovery of PAF from the extraction and purification procedures is 94.5 +/- 0.9%. The sensitivity of the assay is 10 pg/tube. Human neutrophils stimulated with 0, 0.25, 0.5, 1 and 2 microM A 23187 will produce ND, ND, 720, 840 and 900 pg of PAF, respectively (ND = not detectable). The values obtained for PAF produced by human neutrophils in the present assay are comparable to those obtained with the rabbit platelet aggregation assay.

Pharmacologic characterization of the rabbit neutrophil receptor for platelet-activating factor.

Abstract The characteristics of receptors for platelet-activating factor (PAF) on rabbit neutrophils are investigated in this report. The presence of PAF-specific binding to rabbit neutrophils was confirmed using radiolabeled ligand binding assays and a rabbit peritoneal neutrophil membrane preparation. Binding of PAF to the neutrophil membranes was reversible and reached equilibrium within 30 min. Scatchard analysis of PAF-specific binding to the rabbit neutrophil membranes revealed a dissociation constant (Kd ) for PAF of 0.41 ± 0.045 nM and a B max of 0.32 ± 0.11 pmol of PAF receptor/mg of protein. The order of potencies of PAF receptor antagonists to inhibit the binding of 3H-PAF to rabbit peritoneal neutrophil membranes was determined. For the competition assays, 100 μg of neutrophil or platelet membrane protein, 0.18 nM 3H-PAF, and varying amounts of PAF antagonist were incubated at room temperature for 1 hr. PAF receptor antagonists tested were ONO-6240, brotizolam, kadsurenone, WEB-2086, L-652,731, BN-52021, CV-3988, triazolam, alprazolam, and verapamil. The orders of potencies of these PAF receptor antagonists were similar for inhibition of 3H-PAF binding to rabbit peritoneal neutrophil and platelet membranes (correlation coefficient, r = 0.97). PAF had a significantly higher affinity for rabbit neutrophil membranes (Kd = 0.41 ± 0.045 nM), as compared with its affinity for rabbit platelet membranes (Kd = 0.87 ± 0.092 nM). In addition, sodium was found to inhibit 3H-PAF specific binding to rabbit platelet membranes and not to affect 3H-PAF binding to neutrophil membranes. These data indicate that, although PAF receptors on rabbit platelets and neutrophils exhibit similar orders of potencies of PAF receptor antagonists to inhibit the binding of 3H-PAF, the disparity in Kd of PAF for the receptors and the effect of NaCl on the binding of 3H-PAF reveal subtle differences between the cell types.

A nonequilibrium radioimmunoassay for angiotensin II

A method for the measurement of angiotensin II levels in dog plasma is described. The method is similar to previously published assays in that in couples gradient high-performance liquid chromatography (HPLC) with radioimmunoassay (RIA) and requires blood sample collection and processing to plasma in the presence of protease inhibitors. The unique feature of the present method is that it utilized a commercially available angiotensin II RIA run under nonequilibrium conditions. Performing the angiotensin II RIA under nonequilibrium conditions increased RIA sensitivity to allow for a minimal detectable limit of 0.75 pg/mL, a limit of detection not achievable with current commercially available RIAs. This lower limit of detection will now allow for the measurement of circulating levels of angiotensin II. Quality control pools of dog plasma fortified with 4.59-50 pg/mL angiotensin II were assayed and analytical recoveries (ARs) and coefficients of variation (CV) of 72.2%-111% and 3.67%-19.0% were observed for the respective pools.

Role of Preclinical Metabolism and Pharmacokinetics in The Development of Celecoxib

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the principle therapy for the treatment of pain, fever, and inflammation (e.g. the signs and symptoms of arthritic disease). The use of these drugs are associated with significant toxicities in the gastrointestinal (GI) tract, along with renal and platelet side effects (bleeding) (Borda and Koff, 1992). Smith and Willis (1971) and Vane (1971) proposed over 30 years ago that the mechanism of action of NSAIDs was due to the blockade of the production of prostaglandins via inhibition of the enzyme cylooxygenase (COX). The clinical significance of the discovery of two cyclooxygenases (COX-1 and COX-2) in the early 1990s along with their significant role in physiological function and disease has been amply reviewed (Needleman and Isakson, 1997; Hawkey et al., 1999; Silverstein et al., 2000). The subsequent development and approval of inhibitors that target COX-2 relative to COX-1in vivobrought into clinical use new drugs that obviated the toxicological limitations of older NSAIDs. Celecoxib (Celebrex®), the first drug approved based on this COX-2 selectivity, entered clinical use in 1999. In the context of this chapter, COX-2-selective refers to the extent to which a drug inhibits COX-2in vivorelative to COX-1.

Cyclooxygenase-2 (COX-2) Plays a Significant Role in Regulating the Fetal Ductus Asteriosus In Vivo and In Vitro

Cyclooxygenase-2 (COX-2) Plays a Significant Role in Regulating the Fetal Ductus Asteriosus In Vivo and In Vitro