Comparison of the antithrombotic effect of PEG-hirudin and heparin in a human ex vivo model of arterial thrombosis. (57/6090)

Polyethylene glycol (PEG)-hirudin is a derivative of hirudin with a long plasma half-life. We have compared the efficacy of PEG-hirudin with unfractionated heparin (UH) in preventing arterial thrombosis. Arterial thrombus formation was induced ex vivo in 12 healthy human volunteers by exposing a tissue factor-coated coverslip positioned in a parallel-plate perfusion chamber to flowing nonanticoagulated human blood drawn directly from an antecubital vein at an arterial wall shear rate of 2600 s-1 for 3.5 minutes. PEG-hirudin, UH, or saline (as control) were administered ex vivo through a heparin-coated mixing device positioned proximal to the perfusion chamber. Platelet and fibrin deposition was quantified by immunoenzymatic measure of the P-selectin and D-dimer content of dissolved plasmin-digested thrombi, respectively. UH was administered to a plasma concentration of 0.35 IU/mL. This concentration prolonged the activated partial thromboplastin time from 32+/-1 seconds to 79+/-4 seconds (P<0.01). UH did not significantly prevent platelet deposition. However, fibrin deposition was reduced by 39% (P<0.05). PEG-hirudin in plasma concentrations of 0.5, 2.5, and 5 microg/mL prolonged the activated partial thromboplastin time to 48+/-2, 87+/-4, and 118+/-4 seconds, respectively. In contrast to UH, PEG-hirudin prevented both platelet and fibrin deposition in a dose-dependent manner with a >80% reduction at 5 microg/mL (P<0.01). Furthermore, the plasma level of PEG-hirudin required to significantly prevent fibrin deposition (0.5 microg/mL) corresponded to a much shorter prolongation of activated partial thromboplastin time (48+/-2 seconds) than that needed for UH (79+/-4 seconds). Thus, our results are compatible with the view that thrombin is greatly involved in recruitment of platelets in evolving thrombi, and that PEG-hirudin is an effective agent for preventing arterial thrombosis in a human ex vivo experimental model.  (+info)

Biological characterization of recombinant human follicle stimulating hormone isoforms. (58/6090)

It has been established that follicle stimulating hormone (FSH) circulates in the bloodstream as a heterogeneous population of molecules. Individual FSH isoforms, while displaying identical amino acid sequences, differ in their extent of post-translational modification. As a result of these variations, the FSH isoforms exhibit differences in overall charge, degree of sialic acid or sulphate incorporation, receptor binding affinity and plasma half-life. Taking advantage of the fact that these forms can be separated from each other on the basis of their charge, we have evaluated in rats the metabolic clearance rates of the acidic [with an isoelectric point (pI) 4.8) isoforms of recombinant human FSH (rhFSH) obtained after chromatofocusing. The less acidic isoform group was found to have a faster clearance from the circulation in rats as compared with the acidic isoform group. This finding is in agreement with the lower bioactivity in vivo (as determined by the Steelman-Pohley assay) of the less acidic isoform group, compared with the acidic one. The mass spectra of the two groups of isoforms showed a difference in the sialic acid content thus highlighting the importance of these residues on the in-vivo activity of FSH. Conversely, when the two groups of isoforms were tested in vitro by using the Y1 human FSH receptor (Y1 hFSHR) assay and a reporter gene assay, no significant differences in the biological activities between these preparations were detected when test concentrations were based on mass.  (+info)

Systemic availability and pharmacokinetics of nebulised budesonide in preschool children. (59/6090)

AIM: To evaluate the systemic availability and basic pharmacokinetic parameters of budesonide after nebulisation and intravenous administration in preschool children with chronic asthma. METHODS: Plasma concentrations of budesonide were measured for three hours after an intravenous infusion of 125 micrograms budesonide. The children then inhaled a nominal dose of 1 mg budesonide through the mouthpiece of a Pari LC Jet Plus nebuliser connected to a Pari Master compressor, and the plasma concentrations of budesonide were measured for another six hours. The amount of budesonide inhaled by the patient ("dose to subject") was determined by subtracting from the amount of budesonide put into the nebuliser, the amount remaining in the nebuliser after nebulisation, the amount emitted to the ambient air (filter), and the amount found in the mouth rinsing water. RESULTS: Ten patients aged 3 to 6 years completed both the intravenous and the inhaled treatment. The mean dose to subject was 23% of the nominal dose. The systemic availability of budesonide was estimated to be 6.1% of the nominal dose (95% confidence intervals (CI), 4.6% to 8.1%) or 26.3% of the dose to subject (95% CI, 20.3% to 34.1%). Budesonide clearance was 0.54 l/min (95% CI, 0.46 to 0.62), steady state volume of distribution 55 litres (95% CI, 45 to 68), and the terminal half life was 2.3 hours (95% CI, 2.0 to 2.6). CONCLUSIONS: Approximately 6% of the nominal dose (26% of the dose to subject) reached the systemic circulation of young children after inhalation of nebulised budesonide. This is about half the systemic availability found in healthy adults using the same nebuliser.  (+info)

Pharmacokinetics and metabolism of rectally administered paracetamol in preterm neonates. (60/6090)

AIM: To investigate the pharmacokinetics, metabolism, and dose-response relation of a single rectal dose of paracetamol in preterm infants in two different age groups. METHODS: Preterm infants stratified by gestational age groups 28-32 weeks (group 1) and 32-36 weeks (group 2) undergoing painful procedures were included in this study. Pain was assessed using a modified facies pain score. RESULTS: Twenty one infants in group 1 and seven in group 2 were given a single rectal dose of 20 mg/kg body weight. Therapeutic concentrations were reached in 16/21 and 1/7 infants in groups 1 and 2, respectively. Peak serum concentrations were significantly higher in group 1. Median time to reach peak concentrations was similar in the two groups. As serum concentration was still in the therapeutic range for some infants in group 1, elimination half life (T1/2) could not be determined in all infants: T1/2 was 11.0 +/- 5.7 in 11 infants in group 1 and 4.8 +/- 1.2 hours in group 2. Urinary excretion was mainly as paracetamol sulphate. The glucuronide:sulphate ratio was 0.12 +/- 0.09 (group 1) and 0.28 +/- 0.35 (group 2). The pain score did not correlate with therapeutic concentrations. CONCLUSIONS: A 20 mg/kg single dose of paracetamol can be safely given to preterm infants in whom sulphation is the major pathway of excretion. Multiple doses in 28-32 week old neonates would require an interval of more than 8 hours to prevent progressively increasing serum concentrations.  (+info)

Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells. (61/6090)

Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism.  (+info)

Both transcriptional and posttranscriptional mechanisms regulate human telomerase template RNA levels. (62/6090)

The human telomerase RNA component (hTR) is present in normal somatic cells at lower levels than in cancer-derived cell lines. To understand the mechanisms regulating hTR levels in different cell types, we have compared the steady-state hTR levels in three groups of cells: (i) normal telomerase-negative human diploid cells; (ii) normal cells transfected with the human telomerase catalytic subunit, hTERT; and (iii) cells immortalized in vitro and cancer cells expressing their own endogenous hTERT. To account for the differences in steady-state hTR levels observed in these cell types, we compared the transcription rate and half-life of hTR in a subset of these cells. The half-life of hTR in telomerase-negative cells is about 5 days and is increased 1.6-fold in the presence of hTERT. The transcription rate of hTR is essentially unchanged in cells expressing exogenous hTERT, and the increased steady-state hTR level appears to be due to the increased half-life. However, the transcription rate of hTR is greatly increased in cells expressing endogenous hTERT, suggesting some overlap in transcriptional regulatory control. We conclude that the higher hTR level in cells expressing an endogenous telomerase can be a result of both increased transcription and a longer half-life and that the longer half-life might be partially a result of protection or stabilization by the telomerase catalytic subunit. The 4-week half-life of hTR in H1299 tumor cells is the longest half-life yet reported for any RNA.  (+info)

HuD, a neuronal-specific RNA-binding protein, is a putative regulator of N-myc pre-mRNA processing/stability in malignant human neuroblasts. (63/6090)

N-myc gene copy numbers and transcription rates are similar in N (neuroblastic, tumorigenic) and S (non-neuronal, non-tumorigenic) neuroblastoma cells with chromosomally integrated amplified N-myc genes. However, N cells show significantly higher N-myc mRNA levels than S cells. Therefore, post-transcriptional control of N-myc gene expression must differ between these cell types. Since no differences in N-myc mRNA half-life were found between N and S cells from two cell lines, steady-state levels of N-myc pre-mRNA processing intermediates were analysed. Results suggest that the differences in N-myc expression arise primarily at the nuclear post-transcriptional level. The neuronal-specific RNA-binding Hu proteins are present in cytoplasmic and nuclear fractions of N cells and one of them, HuD, binds specifically to both exonic and intronic N-myc RNA sequences. In sense and antisense HuD-transfected N cells, there are coordinate changes in HuD and N-myc expression levels. Thus, we propose that HuD plays a role in the nuclear processing/stability of N-myc pre-mRNA in N-type neuroblastoma cells.  (+info)

Pharmacokinetic changes of a new carbapenem, DA-1131, after intravenous administration to spontaneously hypertensive rats and deoxycorticosterone acetate-salt-induced hypertensive rats. (64/6090)

The pharmacokinetics of a new carbapenem, DA-1131, were compared after i.v. administration of the drug, 50 mg/kg, to spontaneously hypertensive rats (SHRs) at 16 weeks of age (an animal model for human primary hypertension) and at 6 weeks of age (corresponding to the early phase of the development of hypertension, at which time blood pressure remains within the normotensive range) and their respective age-matched control normotensive Kyoto-Wistar rats (KW rats), and deoxycorticosterone acetate-salt-induced hypertensive rats at 16 weeks of age (an animal model for human secondary hypertension) and their age-matched control Sprague-Dawley rats. The total area under the plasma concentration-time curve from time zero to time infinity (AUC) (4720 versus 7070 microg x min/ml) was significantly smaller, and the nonrenal clearance (CLNR) (5.37 versus 3.57 ml/min/kg) was significantly faster in 16-week-old SHRs than those in their control KW rats. Similar results were also obtained from 6-week-old SHRs in AUC (3800 versus 4680 microg x min/ml) and CLNR (7.73 versus 3.31 ml/min/kg). However, the values were reversed in 16-week-old deoxycorticosterone acetate-salt rats in AUC (5310 versus 3870 microg.min/ml) and CLNR (2.57 versus 4.90 ml/min/kg). The significantly faster CLNR of DA-1131 in both 6- and 16-week-old SHRs could be supported at least partly by the results of the in vitro metabolism with kidney homogenate and considerably greater total renal dehydropeptidase-I activity. The data above indicated that the significantly faster CLNR of DA-1131 in 16-week-old SHRs than that in their age-matched control KW rats was due to any hereditary characteristics of SHRs and was not due to the hypertensive state itself.  (+info)