Use of population pharmacokinetic modeling and Monte Carlo simulation to describe the pharmacodynamic profile of cefditoren in plasma and epithelial lining fluid.

Cefditoren is a broad-spectrum, oral cephalosporin that is highly active against clinically relevant respiratory tract pathogens, including multidrug-resistant Streptococcus pneumoniae. This study described its pharmacodynamic profile in plasma and epithelial lining fluid (ELF). Plasma and ELF pharmacokinetic data were obtained from 24 patients under fasting conditions. Cefditoren and urea concentrations were determined in plasma and bronchoalveolar lavage fluid by liquid chromatography-tandem mass spectrometry. Concentration-time profiles in plasma and ELF were modeled using a model with three disposition compartments and first-order absorption, elimination, and transfer. Pharmacokinetic parameters were identified in a population pharmacokinetic analysis (big nonparametric adaptive grid with adaptive gamma). Monte Carlo simulation (9,999 subjects) was performed with the ADAPT II program to estimate the probability of target attainment at which the free-cefditoren plasma concentrations (88%) protein binding and total ELF concentrations exceeded the MIC for 33% of the dosing interval for 400 mg cefditoren given orally every 12 h. After the Bayesian step, the overall fits of the model to the data were good, and plots of predicted versus observed concentrations for plasma and ELF showed slopes and intercepts very close to the ideal values of 1.0 and 0.0, respectively. In the plasma probability of target attainment analysis, the probability of achieving a time for which free, or unbound, plasma concentration exceeds the MIC of the organism for 33% of the dosing interval was <80% for a MIC of >0.06 mg/liter. Similar to plasma, the probability of achieving a time above the MIC of 33% was <80% for MIC of >0.06 mg/liter in ELF. Cefditoren was found to have a low probability of achieving a bacteriostatic effect against MICs of >0.06 mg/liter, which includes most S. pneumoniae isolates with intermediate susceptibility to penicillin, when given in the fasting state in both plasma and ELF.

Promoter cloning and characterisation of the transcriptional regulation of the human thyrostimulin A2 subunit.

The novel heterodimeric glycoprotein hormone thyrostimulin consists of two unique subunits, A2 and B5. To understand its yet unknown transcriptional regulation, we characterised the 3.1-kb immediate 5'-flanking region of the human A2 gene localised on chromosome 11q13. In transient transfection assays this sequence exhibited promoter activity, which could be confined to nucleotides -506 to -347 relative to the ATG start codon. Interestingly, this minimal promoter appeared to be non-tissue-specific. Deletional, mutational and gel shift analyses revealed regulatory elements that are essential for the regulation of the A2 gene expression. Another noteworthy feature of this gene is the presence of silencer elements upstream and downstream of the promoter. To surmise, our results provide an initial step toward a detailed analysis of the underlying molecular mechanisms of the human thyrostimulin gene expression.

The promoter of the human sodium/iodide symporter responds to certain phthalate plasticisers.

The diesters of benzene-1,2-dicarboxylic (phthalic) acid, the phthalates, are used to make plastics flexible and can comprise 40% of the weight of plastic. Human exposure to phthalates can occur via ingestion, inhalation and dermal routes, as well as through parenteral exposure from medical devices containing phthalates. Since earlier morphological studies showed that some phthalates induced thyroid hyperactivity, we thought it important to investigate possible effects of six major phthalates on the transcriptional activity of sodium/iodide symporter (NIS). Di-isodecyl phthalate (DIDP), benzyl butyl phthalate (BBP) and di-octyl phthalate (DOP) increased the activity of the human NIS promoter construct 2.5-, 2.6- and 2.4-fold, respectively. Likewise, these phthalates also enhanced the rat NIS endogenous mRNA expression ca. 2-fold. No effect was observed for bis-(2-ethylhexyl) phthalate (DEHP) and di-isononyl phthalate (DINP), whereas dibutyl phthalate (DBP) appeared to down-regulate hNIS promoter. Although the demonstrated stimulation of NIS gene transcription by DIDP, BBP and DOP is not very strong, this finding is of great importance as humans are routinely exposed for long periods to phthalate plasticisers, the accumulation of which may contribute to thyroid hyperfunction.

Graves' IgG activate upstream enhancer of the sodium/iodide symporter.

Graves' thyroid tissue has been shown to express elevated levels of human sodium/iodide symporter (hNIS) mRNA and protein. In the present work, we demonstrate for the first time that hNIS gene expression in Graves' disease (GD) is up-regulated by Graves' IgG. Here, in transient transfection experiments using FRTL-5 cells, hNIS promoter and enhancer/luciferase construct showed an up to six-fold increase in transcriptional activity after incubation with purified Graves' IgG. Mutation of a CRE site in hNIS enhancer inhibited Graves' IgG response. In addition, mutation of a novel putative regulatory region in hNIS promoter reduced the stimulation three-fold. This discovered putative regulatory sequence might play a role in hNIS up-regulation by Graves' IgG and TSH. The data presented here complement our current knowledge of the pathogenesis of GD and will contribute to a better understanding of mechanisms regulating the thyroid iodide concentrating activity.

Iodination of proteins in TPO transfected thyroid cancer cells is independent of NIS.

This study shows that organification of radioiodide into proteins of thyroid cancer cells exogenously co-expressing the thyroid peroxidase (TPO) and the sodium/iodide symporter (NIS) is independent of NIS function. When administering (125) I to cells constitutively expressing either NIS, or TPO or NIS/TPO, next to iodide accumulation due to NIS activity, organification was exclusively observed in TPO expressing/co-expressing cells. The use of specific inhibitors for TPO and NIS showed that organification is strictly dependent of TPO and not of NIS. An identical pattern of iodoproteins migrating between approximately 75 and 200 kDa in all cell lines tested was observed. Among the five major iodoproteins, two polypeptides appear to be related and three are most probably unrelated, according to their peptide pattern. Our results significantly indicate that co-expression of TPO in NIS transfected cells mediates iodination on the one hand but on the other hand does not contribute to augmentation of a putative NIS-based radioiodide concentrator gene therapy.

Characterization of a thyroid-specific and cyclic adenosine monophosphate-responsive enhancer far upstream from the human sodium iodide symporter gene.

We describe the cloning and characterization of a human sodium iodide (NIS) upstream enhancer (NUE). This putative enhancer was cloned based on its sequence homology (69% identity) to the rat NUE. A 296 base pair (bp) genomic DNA fragment, which is located 9000 bp upstream from the human hNIS gene, was amplified by polymerase chain reaction (PCR) and inserted into a luciferase reporter gene in front of both the homologous NIS promoter and the heterologous SV40 promoter. No enhancer activity could be found after transfection into HeLa cells, but in FRTL-5 cells representing the thyroid model, a threefold stimulation of the NIS promoter was found. This enhancer activity was present in both directions and was stimulated threefold by thyrotropin (TSH) and 14-fold by the cyclic adenosine monophosphate (cAMP) agonist forskolin. A small element (TGACGCA) in this enhancer was found to be of central importance, because its site-directed mutagenesis abolished the enhancer activity. This element bound specifically to proteins in nuclear extracts from FRTL-5 cells and to a lesser extent also from HeLa cells. In summary, we describe a thyroid-specific and cAMP-responsive enhancer far upstream from the human NIS gene, which is located in the intronic region of another gene coding for a ribosomal protein.