ILLUMINATE (Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events), the phase 3 morbidity and mortality trial of torcetrapib, a cholesteryl ester transfer protein (CETP) inhibitor, identified previously undescribed changes in plasma levels of potassium, sodium, bicarbonate, and aldosterone. A key question after this trial is whether the failure of torcetrapib was a result of CETP inhibition or of some other pharmacology of the molecule. The direct effects of torcetrapib and related molecules on adrenal steroid production were assessed in cell culture using the H295R as well as the newly developed HAC15 human adrenal carcinoma cell lines. Torcetrapib induced the synthesis of both aldosterone and cortisol in these two in vitro cell systems. Analysis of steroidogenic gene expression indicated that torcetrapib significantly induced the expression of CYP11B2 and CYP11B1, two enzymes in the last step of aldosterone and cortisol biosynthesis pathway, respectively. Transcription profiling indicated that torcetrapib and angiotensin II share overlapping pathways in regulating adrenal steroid biosynthesis. Hormone-induced steroid production is mainly mediated by two messengers, calcium and cAMP. An increase of intracellular calcium was observed after torcetrapib treatment, whereas cAMP was unchanged. Consistent with intracellular calcium being the key mediator of torcetrapib's effect in adrenal cells, calcium channel blockers completely blocked torcetrapib-induced corticoid release and calcium increase. A series of compounds structurally related to torcetrapib as well as structurally distinct compounds were profiled. The results indicate that the pressor and adrenal effects observed with torcetrapib and related molecules are independent of CETP inhibition.
In this paper, protein-drug interactions were studied by solid-phase microextraction (SPME) using diazepam binding to human serum albumin as a model system. Since drug compounds are normally polar and nonvolatile by nature, direct SPME is used in this work. The SPME extraction is an equilibrium process among the concentrations of the analyte partitioned onto the SPME fiber, free and bound drug in the solution. A calibration curve was first constructed by employing the amount of the analytes partitioned on the fiber versus the free analyte concentration in the solution in the absence of protein. In method I, the extraction was performed in the protein solution with known diazepam concentration. In method II, diazepam was first loaded onto the fiber by extracting in solution with known diazepam concentration. This fiber was subsequently transferred into the protein solution for desorption. The amount of the analyte left on the fiber was analyzed after the system reached equilibrium. The free drug concentration was then obtained from the calibration curve for both methods. The Scatchard plot was finally employed to obtain the number of binding sites and the equilibrium binding constants. Since only a very small amount of the protein solution is required (150 microL for each extraction), method II is very useful for circumstances where the protein amount is very limited. The direct measurement method proposed in this paper does not need a GC response factor, which significantly decreases the experimental error. The only measurement needed is the area count change (ratio) of the fiber injections before and after the protein was introduced into the solution. The difference between the direct measurement method for method I and method II is discussed. The result illustrated that the SPME direct measurement method provided both theoretical accuracy and simplicity in such applications.
A theophylline antiserum was covalently immobilized on the surface of a fused silica fiber, modified with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde, and used as a selective and sensitive extraction medium for the immunoaffinity solid-phase microextraction (SPME) determination of theophylline in serum samples. The specificity of the immunoaffinity SPME fiber was first investigated using a fixed concentration of [3H]theophylline together with various amounts of interference, possessing no cross-reactivity with the theophylline antibody. No significant non-specific binding was observed. The reproducibility of the fiber preparation and the immunoaffinity SPME analysis was also investigated, resulting in a relative standard deviation of 6.1% for five analyses of the same fiber. The antigen-antibody binding isotherm was obtained by analyzing theophylline standards of various concentrations (0.1-5 ng mL(-1)) until saturation values were reached. Initial binding of theophylline was linear with a r2 = 0.968. The cross-reactivity of the theophylline immunoaffinity SPME fiber for the structural analog caffeine was investigated by adding various amounts of caffeine in the presence of theophylline at a saturation concentration and produced a low cross-reactivity value of 0.1%. Finally. spiked serum samples (10 and 50 ng mL(-1)) were successfully analyzed with an excellent correlation with the standard binding isotherm, thus confirming the performance of the immunoaffinity SPME coating for improved bioanalysis.
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