DNA binding of polyomavirus large T-antigen: kinetics of interactions with different types of binding sites.

Polyomavirus large T-antigen binds to GRGGC sites in double-stranded viral DNA, regulating transcription and replication. Using surface plasmon resonance to record interactions of large T-antigen with different types of binding sites, we found that the configuration of recognition motifs influenced both the association and dissociation rates. Particularly, the complex formed at the origin of DNA replication was labile. A comparison of the interactions between large T-antigen and binding sites with one, two and four GRGGC motifs in tandem showed a strong preference for dimer binding, without detectable co-operativity between dimers. Sodium chloride stabilised the complexes, whereas the dissociation increased rapidly by increasing pH above 7.0.

Preferred DNA-binding-sites of polyomavirus large T-antigen.

Polyomavirus large T-antigen is a multifunctional protein. Its essential function in virus infection is to control the synthesis of viral RNA and DNA. For this activity specific DNA binding is necessary. Large T-antigen can bind to several sites in the regulatory region of viral DNA, consisting of clustered GRGGC nucleotide motifs. Since large T-antigen also regulates the activity of cellular genes and cellular DNA synthesis, it seemed possible that there are alternative, as yet unrecognized, binding sites. To identify sites preferred by large T-antigen, double-stranded polynucleotides with random sequence were used. These polymers had a 31-bp central segment synthesized from a mixture of all four nucleotides and flanking segments of defined sequence. They were subjected to several cycles of binding to large T-antigen with intervening PCR amplification. Individual polynucleotides with affinity for large T-antigen were then isolated by cloning and their nucleotide sequences were determined. The majority of the polynucleotides contained two or three GRGGC motifs separated by between five and eight variable nucleotides. This result suggests that there are not any alternative high-affinity binding sites of large T-antigen. By comparing all the individual binding motifs an extended consensus sequence was observed. The dinucleotide TG was predominant immediately 5' to the binding pentanucleotide. On the 3'-side, at position +2, C residues were very rare. Although the pentanucleotide motif is the same as in polyomavirus DNA, the extended consensus sequence is not observed in viral DNA. In semi-quantitative experiments, binding of purified large T-antigen to a few of the selected DNA molecules was tested. Stable complexes were formed with DNA substrates containing two or three binding motifs in tandem. Binding to DNA with only one complete motif was weaker, but significantly stronger than non-specific association. This result has implications for the number of large T-antigen binding sites in cellular DNA. When mutant bc1081 large T-antigen, that is defective in specific DNA binding, was used in selection of polynucleotides, a different result was obtained. Neither bc1081 nor wild-type large T-antigen bound strongly to these polynucleotides. However, the presence of the motif TTCGGCTT, or part of it, in five of the six isolated polynucleotides suggested that the T-antigen selection was specific.

Characterization of responders and nonresponders to tiaprofenic acid and naproxen in the treatment of patients with osteoarthritis.

OBJECTIVE: To characterize responders/nonresponders to 2 nonsteroidal antiinflammatory drugs by a combination of start variables in the treatment of patients with osteoarthritis (OA) of the hip(s)/knee(s). METHODS: Two hundred eight patients participated in a 3-week randomized double blind parallel group trial. RESULTS: The responder rate to tiaprofenic acid and naproxen was 52 and 59%, respectively. Correctly characterized by use of a combination of 6 baseline variables were 79% of the patients treated with tiaprofenic acid and 81% treated with naproxen. CONCLUSION: Both active drugs were found effective compared with placebo in patients with OA, but with different characteristics.

The acute haemodynamic and renal effects of oral felodipine and ramipril in healthy subjects.

The aim of the present investigation was to compare the acute haemodynamic and renal effects of the calcium antagonist felodipine with the ACE inhibitor ramipril and with placebo. Single oral doses of felodipine 5 and 20 mg, ramipril 2.5 and 10 mg, and placebo were given to ten healthy subjects in a double-blind cross-over study. Blood pressure, heart rate, forearm blood flow (FBF), forearm vascular resistance (FVR), renal blood flow (RBF), renal vascular resistance (RVR), glomerular filtration rate (GFR), filtration fraction (FF), diuresis, and sodium excretion were recorded for 4.75 h after administration. Felodipine 20 mg caused a significant fall in diastolic blood pressure, maximal 12% compared with placebo, while there were no significant effects of felodipine 5 mg or the two doses of ramipril. Heart rate increased significantly after both doses of felodipine, maximal 28% after the 20 mg dose. There was also a small but significant increase in heart rate of 12% after ramipril 2.5 mg. FVR fell significantly after both doses of felodipine, maximal 38% after the 20 mg dose. There were no significant changes in FVR after any of the ramipril doses. Both doses of felodipine and both doses of ramipril caused significant reductions in RVR. Maximal reduction, 33%, was found after felodipine 20 mg. There were no significant changes in GFR or FF with either drug. Felodipine caused a significant increase in natriuresis, maximal 129% while ramipril did not.

Pharmacokinetics and hemodynamic and diuretic/natriuretic effects of felodipine administered as an extended-release tablet.

In this study the pharmacokinetics, and the hemodynamic and diuretic/natriuretic effects of three different doses of felodipine ER-10, 20, and 40 mg--were evaluated in healthy subjects. There was a linear correlation between the dose of felodipine, Cmax, and AUC24, showing that the absorption was linearly related to the dose. The diastolic blood pressure was reduced by 15-20% after the two highest doses. The maximal blood-pressure lowering effect was seen 4 hours after drug intake, and a small reduction in diastolic blood pressure was still present after 24 hours. This was, however, not statistically significant but was related to a sustained effective plasma concentration of the drug (6 nmol/l). Systolic blood pressure was not affected. The two highest doses of felodipine ER produced a significant increase in heart rate 2 and 6 hours after the dose, compared with placebo. There was also a significant decrease in forearm vascular resistance after the 20- and 40-mg doses. Both diuresis and natriuresis were significantly increased by about 100% each during the first 4 hours after the 20-mg dose. Following the 40-mg dose, diuresis and natriuresis were lower than after 20 mg and were not significantly different from placebo.

Acute effects of felodipine and nifedipine on hepatic and forearm blood flow in healthy men.

The acute effects of oral administration of felodipine 10 mg and nifedipine 10 mg on heart rate, blood pressure, forearm blood flow and hepatic blood flow were studied in nine healthy men. Both drugs caused an increase in heart rate of 16 and 7 beats.min-1, respectively. Hepatic blood flow was significantly increased by 1.2 and 0.41.min-1 after felodipine and nifedipine. There was also a decrease in diastolic blood pressure, 10 and 5 mm Hg, respectively, after felodipine and nifedipine. The forearm blood flow was increased by about 30 ml.100 ml-1.min-1 after felodipine, but nifedipine had no effect. The haemodynamic effects were most pronounced 50 min after drug administration.

Haemodynamic effects and pharmacokinetics of felodipine at rest and during exercise in hypertensive patients treated with metoprolol or atenolol.

A study has been performed in thirteen patients with essential hypertension, WHO Class I-II, and a diastolic blood pressure greater than or equal to 95 mmHg, on beta-blocker (metoprolol or atenolol) monotherapy, who were also given felodipine 10 mg b.d. for 28 days. The acute and steady state blood pressure response at rest and during exercise, and the pharmacokinetics of felodipine and metoprolol, were examined. Felodipine in combination with the beta-blocker reduced the systolic and diastolic blood pressures acutely and at steady-state. The duration of the effect was longer at steady-state. There was a significant correlation between the plasma concentration of felodipine and the change in blood pressure. The increase in systolic blood pressure during exercise was of the same magnitude before and after felodipine administration. No change in resting supine heart rate was found after the administration of felodipine. There was no significant differences in the pharmacokinetics of felodipine during long-term treatment, except for the trough plasma concentration, which was increased at steady-state, even though cumulation of felodipine and its metabolite did not occur. There was a significant decrease in the maximal plasma concentration and AUC of metoprolol after 28 days of treatment with felodipine, but its elimination half-life was not changed. The adverse reactions reported during this study were those generally seen after dihydropyridines and, except for two patients who were withdrawn after the first study day, the effects were well tolerated.

Dose-dependent effects of felodipine on diuresis and natriuresis in healthy subjects.

We compared the effects of various acute doses of felodipine and placebo on diuresis and natriuresis in healthy men. The subjects were given felodipine, 1 and 3 mg as an intravenous infusion, and 5, 15, and 40 mg as an oral solution on 5 separate days. On each day blood pressure and heart rate were recorded and urine was collected for analysis of volume and sodium for 24 h. Felodipine induced a dose-dependent increase in heart rate and a dose-dependent decrease in diastolic blood pressure. These effects were maximal within 30 min of drug administration. Felodipine induced a maximal increase in diuresis of about 150% compared with placebo and a maximal increase in natriuresis of about 240%. The renal effects were most pronounced during the first 4 h after dose intake. During the 8-24 h interval, diuresis and natriuresis were lower than after placebo, but when the whole 24-h period was considered, no significant differences were found between felodipine and placebo in regard to sodium and water excretion. The most pronounced effects on diuresis and natriuresis were seen after moderate doses (3 mg i.v. and 15 mg orally). The response to the highest dose (40 mg orally) was somewhat less probably due to the excessive drop in diastolic blood pressure.

Pharmacokinetic and pharmacodynamic studies of felodipine in healthy subjects after various single, oral and intravenous doses.

The objective of this single-dose study was to evaluate the pharmacokinetics and haemodynamic changes in healthy male subjects following the administration of three oral (5, 15, and 40 mg) and two intravenous (1 and 3 mg) doses of felodipine, a new calcium antagonist with a selective effect on the peripheral resistance vessels. Felodipine was rapidly absorbed within 1 h when administered as an oral solution, but underwent extensive presystemic elimination. The systemic availability varied between 10 and 23 per cent. The disposition was adequately described by a two-compartment model: the disposition was essentially dose-independent up to 40 mg orally and 3 mg intravenously. Felodipine produced significant dose-dependent reduction of diastolic blood pressure and a significant reflexogenic increase in heart rate, without having any major effect on systolic blood pressure. These changes indicate that felodipine acts predominantly as an arteriodilator. The decrease in diastolic blood pressure and increase in heart rate were closely correlated with the plasma concentrations of unchanged felodipine, being maximal at 0.5 h and lasting for at least 4 h after the highest dose.

Felodipine kinetics in healthy men.

In a randomized, crossover study, the absorption, distribution, and elimination of intravenous and oral felodipine were investigated in eight healthy men 22 to 31 years old. Felodipine was given as a 2.5 mg iv infusion over 30 minutes and as a 27.5 mg oral solution. Both doses were labeled with 25 microCi 14C-felodipine. Given as an oral solution, felodipine is rapidly (mean time to peak concentration 64 minutes; range 30 to 90 minutes) and completely absorbed. Presystemic elimination reduced the availability to 16% (range 10% to 25%). Felodipine kinetics can be described by a multicompartmental model with three distinct phases. The t1/2 for the initial phase was 6.4 minutes (range 1.7 to 10.4 minutes) and felodipine was distributed to a volume of 0.6 L/kg (range 0.4 to 0.9 L/kg), which approximately corresponds to the total body water. The second distribution phase reached pseudoequilibrium with a t1/2 of 1.6 hours (range 1.3 to 2.2 hours). The volume of distribution at the end of this phase was 9.7 L/kg (range 6.0 to 18.2 L/kg). The terminal phase had t1/2 of 10.2 hours (range 6.7 to 20.7 hours). The contribution of the three phases to the AUC was 15%, 40%, and 45% in the order of increased t1/2. Total body clearance of felodipine was 1.2 L/min (range 0.9 to 1.6 L/min). Within 72 hours after drug dosing, 62% to 81% of the felodipine doses were excreted in the urine and feces as metabolites. The rate of excretion by the kidneys had a biphasic pattern, with t1/2 values of 4 and 18 hours. Approximately 10% of the doses was excreted in the feces.

Acute haemodynamic effects of felodipine and verapamil in man, singly and with metoprolol.

In a single-blind randomised study in 9 healthy men we compared the acute haemodynamic effects of the calcium antagonists felodipine and verapamil, singly and in combination with metoprolol. Three different cumulative intravenous doses of 0.25, 0.75 and 1.5 mg felodipine and of 2.0, 4.0 and 8.0 mg verapamil or placebo were given as constant infusions over 5 minutes on 3 occasions and were followed by intravenous metoprolol (15 mg). Felodipine caused a significant and dose-dependent decrease in the total peripheral resistance, and an increase in the forearm blood flow by 8, 48 and 163% with progressively increasing doses showing that the drug is a potent arteriolar vasodilator. A significant and dose-dependent increase in heart rate and a decrease in the pre-ejection period/left ventricular ejection time (PEP/LVET) ratio of up to 15% was also recorded, mainly reflecting a reflexogenic increase in the sympathetic tone. Total peripheral resistance, forearm blood flow, heart rate and the systolic time intervals were mainly unchanged after verapamil, whereas the PQ interval was prolonged. Metoprolol given after the 2 calcium antagonists caused a decrease in heart rate and blood flow and an increase in the total peripheral resistance and PEP/LVET ratio. The tolerability was good to all infusions.

Acute diuretic/natriuretic properties of felodipine in man.

The natriuretic/diuretic effect of felodipine was investigated in 2 studies. The first was performed as an open study using intravenous and oral felodipine in healthy male subjects. The second was a double-blind study where a high and a low dose of oral felodipine were given to hypertensive patients on long term treatment with beta-blockers; the different doses of felodipine were chosen to decrease and to have no effect on the blood pressure, respectively. In both studies an oral placebo solution was used as a reference. Felodipine caused a significant increase in natriuresis. Compared with placebo and corrected for total 24-hour excretion, the sodium output during the first 4 hours after drug administration was increased by 219 +/- 53% (mean +/- SEM) after intravenous administration in healthy subjects (p less than 0.01) and by 80 +/- 43% in the first 3 hours after the high dose in hypertensive patients (p less than 0.05). For the same period, the urine excretion was increased by 114 +/- 38% (p less than 0.05) in the healthy subjects and by 36 +/- 22% in the hypertensive patients (not significant). However, the 24-hour excretion of urine, Na+ and K+ was not significantly changed from placebo. A significantly lower blood pressure was recorded after the higher dose (0.10 mg/kg) when given to hypertensive patients, but no such effect was seen after the lower dose (0.01 mg/kg) or in healthy subjects. The changes in diastolic blood pressure seem to be negatively correlated with the diuretic but not with the natriuretic effect.

Absorption, distribution and elimination of felodipine in man.

The objectives of these investigations were to study the absorption and disposition characteristics of felodipine in young healthy male volunteers following acute administration of different intravenous and oral doses, and to study urinary metabolites of [14C]felodipine following oral administration. Felodipine is rapidly and extensively absorbed from the gastrointestinal tract but owing to presystemic elimination, probably primarily in the liver, only 15% on average is systemically available. The systemic availability is independent of the oral dose in the 5 to 40 mg dose interval. The major fraction of the felodipine dose is localised extravascularly with a volume of distribution of about 10 L/kg. Less than 1% is confined to the blood. Felodipine is extensively bound to plasma proteins (greater than 99%). The mean elimination half-life of felodipine is greater than 10 hours. The urinary metabolic pattern of felodipine, using high pressure liquid chromatography, reveals 3 major metabolites (carboxylic acids of oxidised felodipine) in human urine.

Pharmacokinetics of prenalterol in healthy subjects and patients with congestive heart failure.

1. Prenalterol is rapidly and completely absorbed after oral administration with peak concentrations reached after 30 minutes. 2. Prenalterol is rapidly distributed to extravascular tissues after intravenous administration. 3. The extent of bioavailability after administration of prenalterol as a solution is about 25% of an intravenous dose. After administration of 20 mg prenalterol in a controlled release preparation the bioavailability is increased to about 45%. 4. The half-life of the elimination phase is close to 2 hours. 5. 60% of an intravenous dose and 15% of an oral dose is eliminated unchanged by renal excretion. The rest is mainly excreted as the sulphate ester of prenalterol.

Compartment model of prenalterol.

In previous pharmacokinetic studies in healthy subjects the time course of plasma concentration of prenalterol was described by a short distribution phase (alpha-phase) with a mean half-life of about 8 minutes and an elimination phase (beta-phase) with an average half- life of about two hours [1, 2]. The aim of this joint study was to check the pharmacokinetic data obtained after intravenous single dose administration with the computer program TOPFIT [3] using different compartment models and to test the predictive power of the chosen kinetic model for plasma concentration data after repetitive intravenous prenalterol dosing.

Relative absorption of disopyramide as determined by liquid chromatography following acute administration of standard capsules and controlled-release tablets.

The absorption of disopyramide has been studied in healthy volunteers following an acute dose of 300 mg in standard capsules (C) and three controlled-release (CR) tablets with different in vitro release rates. Plasma concentrations and the urinary excretion of unchanged compound and its main metabolite, N-deisopropyldisopyramide, were determined simultaneously by using a rapid and sensitive method based on liquid-solid chromatography. The rate of absorption was rapid following C, and maximal concentrations were reached within 1.5-2 h. A CR formulation produced a slower rate of absorption and the rate correlated with the drug in vitro release rate. The maximal concentration of disopyramide was reduced following CR tablets. The extent of absorption was the same following C and two of the CR formulations, whereas the CR composition with the slowest in vitro release rate presented a somewhat reduced extent of absorption. About 70% of the given dose was recovered in urine as disopyramide (50%) and metabolite (20%). The elimination half-life of disopyramide and N-deisopropyldisopyramide was close to 4 and 7 h, respectively.

Pharmacokinetics of metoprolol in patients with hepatic cirrhosis.

The selective beta 1-adrenoceptor antagonist metoprolol is eliminated primarily by hepatic metabolism and usually less than 5% of an oral dose is excreted unchanged in the urine. The effects of impaired liver function on the pharmacokinetics of metoprolol were studied in 10 patients with hepatic cirrhosis. All subjects were given single doses of 20mg intravenously and 50mg orally on separate days. The mean fraction of the drug available systematically was 84 +/- 10% in patients and 50 +/- 11% in a control group of 6 healthy subjects (p less than 0.05). The total body clearance of metoprolol in the cirrhotics was 0.61 +/- 0.13L/min and in the controls 0.80 +/- 0.11L/min. These values correspond to elimination half-lives of 7.2 +/- 1.2 and 4.2 +/- 1.1 hours, respectively. The differences were not statistically significant. Impaired liver function had no effect on the volume of distribution of metoprolol. Total clearance was weakly but linearly related to galactose clearance (r2 = 0.52; p less than 0.05), and the half-life was related to serum bilirubin (r2 = 0.74; p less than 0.01).