SPE/RIA vs LC/MS for measurement of low levels of budesonide in plasma.

A radioimmunoassay is described that measures budesonide in plasma after solid-phase extraction (SPE/RIA) of the analyte. The performance of the assay was compared with that of a selective LC/MS method. The limit of quantitation of budesonide determined for the LC/MS and SPE/RIA assay was 50 pg/mL and 120 pg/mL, respectively. Based on quality control samples, a higher variability was observed for the SPE/RIA (CV between 4.5 and 23.0%) than for the LC/MS method (CV between 7.5 and 12.5%). Plasma samples obtained from healthy volunteers after administration of budesonide rectal foam were assayed by both methods. In a subset of samples, these results were compared with those measured by direct RIA to evaluate the selectivity of two assays. About two times higher budesonide levels were measured with the direct RIA (lacking the extraction step), presumably because of cross-reactivity with budesonide metabolites, indicating that the extraction step in SPE/RIA is necessary for selectivity. Both SPE/RIA and LC/MS methods were found to be selective, sensitive and suitable for pharmacokinetic studies. Results obtained from the two methods were compared with a number of statistical methods. Ratios of results obtained for the clinical samples were close to 1 (ratio LC-MS/ SPE/RIA = 0.98 +/- 0.27). Linear regression indicated a slope of 1.17 +/- 0.0378. The concordance correlation (r = 0.91) indicated that the agreement between both methods was fair while the Bland-Altman plot indicated that the agreement was less pronounced at higher concentrations (1-3 ng/mL). In summary, the results confirm that the SPE/RIA is an alternative to HPLC/MS and that among the statistical methods tested the concordance correlation analysis was judged to be the most informative test to assess the comparability of two methods.

Single-dose and steady-state pharmacokinetic and pharmacodynamic evaluation of therapeutically clinically equivalent doses of inhaled fluticasone propionate and budesonide, given as Diskus or Turbohaler dry-powder inhalers to healthy subjects.

Direct comparisons of the pharmacokinetic (PK) and systemic pharmacodynamic (PD) properties of inhaled corticosteroids after single and multiple dosing in the same subjects are scarce. The objective of this study was to compare thePK/PDproperties of clinically equivalent, single, and multiple doses of dry-powder formulations of inhaled fluticasone propionate (FP 200 and 500 microg via Diskus) and budesonide (BUD, 400 and 1,000 microg via Turbohaler). Fourteen healthy subjects completed a double-blind, double-dummy, randomized, placebo-controlled, five-way crossover study consisting of a single dose administered at 8 a.m. on day 1 followed by 4 days of twice-daily dosing at 8 a.m. and 8 p.m. on days 2 to 5. Serum concentrations of FP and BUD were measured using validated liquid chromatography/ mass spectrometry assays. The 24-hour cumulative cortisol suppression (CCS) in serum was monitored as the pharmacodynamic surrogate marker. Peak serum concentrations following single and multiple dosing were observed 10 to 30 minutes after inhalation for BUD and 30 to 90 minutes afterinhalation of FP with no influence of dose ordosingregimen. After a single dose of 1000 microg BUD and 500 microg FP the median estimates of terminal half-life and mean residence time were 3.5 and 3.9 hours for BUD and 10.1 and 12.0 hours for FP, respectively. Using previously reported intravenous data, the mean absorption times (MAT) were calculated to be around 2 hours and 7 hours for BUD and FP respectively. On average, the area under the curve (A UC) at steady state (day 5) was up to 30% higher for BUD compared to that over a 12-hour period following the first dose on day 1, whereas A UC estimates were 50% to 80% higherforFP at steady state, indicating accumulation. However, the steady-state Cmax values were seven to eight times and AUC values three to four times higher for BUD than for FP. Comparison of active treatment data with placebo showed that CCS after a single dose was not pronounced for any of the doses/drugs studied. On day 5, both doses of BUD caused statistically significant suppression (CCS of 19% for the 400 microg dose and 36% for the 1,000 microg dose). For FP only the high dose had a statistically significant effect on serum cortisol (CCS of 14% for the 200 microg dose and 27% for the 500 microg dose). Compared to BUD, FP has slower pulmonary absorption and slower elimination kinetics. However, following inhalation of therapeutically equipotent, multiple twice-daily doses in healthy subjects, the systemic effects of FP delivered via Diskus on AUC24 serum cortisol were relatively low and similar to those of BUD delivered via Turbohaler.

A comparison of structure-activity relationships between spermidine and spermine analogue antineoplastics.

A systematic investigation of the impact of spermidine analogues both in vitro and in vivo is described. The study characterizes the effects of these analogues on L1210 cell growth, polyamine pools, ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, spermidine/spermine N1-acetyltransferase, the maintenance of cellular charge, i.e., cationic equivalence associated with the polyamines and their analogues, and compares their ability to compete with spermidine for transport. The findings clearly demonstrate that the activity of the linear polyamine analogues is highly dependent on the length of the triamines and the size of the N(alpha),N(omega)-substituents. It appears that there is an optimum chain length for various activities and that the larger the N(alpha),N(omega)-alkyls, the less active the compound. Metabolic transformation including N-dealkylation of these compounds is also evaluated. While there is no monotonic relationship between chain length and the ability of the analogue to be metabolized, the dipropyl triamines are clearly more actively catabolized than the corresponding methyl and ethyl systems. A comparison of the triamines with the corresponding tetraamines is made throughout the text regarding both in vitro activity against L1210 cells and in vivo toxicity measurements, suggesting that several triamine analogues may offer therapeutic advantages over the corresponding tetraamines.

Inhibition of pseudomonal ulceration in rabbit corneas by a synthetic matrix metalloproteinase inhibitor.

PURPOSE: To evaluate the effect of the synthetic matrix metalloproteinase inhibitor, Galardin, on proteases produced by Pseudomonas aeruginosa (PA) and on a rabbit model of Pseudomonas keratitis. METHODS: Protease activities of culture broths from Pseudomonas strains PA-28 and W-186 were characterized in vitro by gelatin zymography and by digestion of Azocasein in the presence and absence of Galardin and the serine protease inhibitor, aprotinin. In a noninfectious in vivo experiment, sterile PA culture broth from W-186 was injected intrastromally into rabbit corneas that were treated topically with Galardin or vehicle, then evaluated clinically and histologically. In an infectious in vivo experiment, rabbit corneas were injected with washed PA-28, then treated topically with Galardin or vehicle and clinically scored. RESULTS: Gelatin zymography of culture broth from W-186 and PA-28 detected two proteases that were both inhibited by Galardin. Galardin reduced the digestion of Azocasein by both PA culture broths by 99%, whereas aprotinin did not significantly reduce the protease activity of PA-28 conditioned broth. Intrastromal injection of sterile W-186 culture broth caused rapid corneal destruction that was prevented by topical treatment with Galardin. Intrastromal injection of washed PA-28 bacteria resulted in progressive corneal melting that was significantly (P < 0.005) delayed, but ultimately not prevented, by topical treatment with Galardin. CONCLUSIONS: Pseudomonal protease activity in culture broth consisted predominantly of metalloproteinases and were effectively inhibited by Galardin in vitro. Topical treatment with Galardin prevented destruction of rabbit corneas by bacterial products present in culture broth, and it delayed corneal destruction after injection of PA bacteria. Galardin may be a useful adjuvant when corneal destruction proceeds despite prompt antibiotic treatment.

Metabolism and pharmacokinetics of N1,N11-diethylnorspermine.

The pharmacokinetics and metabolism of N1,N11-diethylnorspermine (DENSPM) is described. When administered to dogs as an intravenous bolus, DENSPM was shown to have a plasma half-life of 72.8 +/- 11.8 min, with an early distribution phase half-life of approximately 4 min and an apparent volume of distribution of 0.216 +/- 0.032 liter/kg. The renal clearance half-life was 59.7 +/- 7.6 min, with 48.8 +/- 12.5% of the drug recovered in the urine between 0-4 hr unchanged. In three other experiments, the drug was administered to dogs by constant rate intravenous infusion over periods ranging from 10 min to 2 hr. Analysis of plasma concentration-time data and urinary excretion data yielded pharmacokinetic parameters in general agreement with the intravenous bolus experiments. DENSPM metabolites were identified in both beagle dog and mouse tissues. Tissues were sampled from a single beagle 24 hr posttreatment, and rodent samples were examined at 12, 24, 48, and 96 hr posttreatment. Both the concentration of DENSPM and the metabolic profile were shown to vary in the lung, liver, spleen, and kidney. Although all the tissues examined contained DENSPM and its metabolites, the liver and kidney had the highest level of metabolites that included N1-ethylnorspermine, N1-ethylnorspermidine, N1-ethyl-1,3-diaminopropane, and norspermidine. These data suggest that DENSPM is metabolized by N-deethylation and step-wise removal of aminopropyl equivalents by spermine/spermidine N1-acetyltransferase/polyamine oxidase, a metabolic pathway unique to the polyamines.