A selective LC/RIA for dexamethasone and its prodrug dexamethasone-21-isonicotinate in biological fluids.

A combined LC/RIA procedure is described for the selective determination of dexamethasone (DEX) and its prodrug dexamethasone-21-isonicotinate (DIN) in plasma. The low affinity of the employed dexamethasone antiserum for DIN (cross-reactivity less than 0.5%) allowed the direct determination of DEX in plasma extracts. For the determination of DIN, both substances of interest were separated by LC, the DIN containing fraction was collected, hydrolysed and the generated DEX was consequently assayed by radioimmunoassay. The assay detection limits were 0.1 ng ml-1 for DEX and 0.75 ng ml-1 for DIN. For both substances, inter- and intra-day variabilities (RSDs) were 6 and 12%, respectively.     

Differences in the glucocorticoid to progesterone receptor selectivity of inhaled glucocorticoids.

Due to the high affinity of new inhaled corticosteroids (ICS) towards the glucocorticoid receptor (GR), and because of the similarities between the binding domains of the GR and the progesterone receptor (PR), the present study focused on assessing the relative binding affinities (RBA) of glucocorticoids (systemic and ICS) to PR (RBAPR). By comparison with the affinities towards the GR (RBAGR) the binding selectivities were also assessed. In general, the selectivity of the investigated glucocorticoids showed a decreasing trend with increasing lipophilicity. When orally administered, less lipophilic glucocorticoids showed the highest selectivity, with RBAGR/RBAPR ratios of 1,375, 760 and 476 for betamethasone, beclomethasone and dexamethasone, respectively. For ICS, mometasone furoate, the most lipophilic steroid, was the least selective (1.1), followed by beclomethasone monopropionate (9), fluticasone propionate (12), triamcinolone acetonide (18), mometasone (25) and budesonide (44), which shows the highest selectivity among inhaled glucocorticoids. In conclusion, the present study revealed that there are differences in selectivity among commercially available glucocorticoids. Future clinical studies are needed to investigate whether the high affinity of some of the investigated glucocorticoids to the progesterone receptor is of clinical relevance.     

A Biotin–Avidin-Based Enzyme Immunoassay for βh-Endorphin

An avidin–biotin enzyme-linked immunosorbent assay (ELISA) is described for _h-endorphin (_h-EP). Microtiter plates coated with commercially available antibodies were used together with _h-EP tracer derivatives that were biotinylated in positions 24, 28, and 29 via a C₆ spacer arm. Nonspecific binding of biotinylated derivatives to the microtiter plates was blocked with a mixture of 1% casein and 10% ethanolamine in 0.1 M NaHCO₃. A sequential saturation procedure using a high-affinity antiserum in combination with an avidin–alkaline phosphatase complex matched the sensitivity of reported radioimmunoassays (RIAs), with a detection limit of 0.5 fmol/assay. The intra- and interassay coefficients of variation were 5 and 12%, respectively. Results obtained by ELISA and RIA showed good correlations (r = 0.95). The -EP concentration in extracted rat plasma after high-performance liquid chromatographic (HPLC) fractionation was determined by this method to be 1600 fmol/ml.     

Cytogenetic and molecular characterization of simultaneous chronic and acute myelocytic leukemia

We describe a patient initially diagnosed with a chronic myeloproliferative disorder in the accelerated phase. Cytogenetic analysis showed the presence of two independent clones. One clone contained a typical Philadelphia (Ph) chromosome due to t(9;22)(q34;q11), as the sole abnormality which was proven molecularly to result in the b2a2-BCR/ABL fusion. The other clone displayed a complex karyotype with several structural and numerical aberrations including trisomy 11 and 22 but lacking a t(9;22) or any other structural abnormalities involving chromosomes 9 and 22. Fluorescence in situ hybridization demonstrated that the t(9;22) was present only in cells with two copies of chromosomes 11 and 22. In contrast, cells with trisomies 11 and 22 lacked evidence for a BCR/ABL fusion. Based on the genetic findings, simultaneous chronic and acute myelocytic leukemias were diagnosed rather than a blastic phase of a chronic myelocytic leukemia.     

HPLC determination of glucocorticoid alcohols, their phosphates and hydrocortisone in aqueous solutions and biological fluids

High-performance liquid chromatographic (HPLC) assays are described for the determination of dexamethasone phosphate, dexamethasone and hydrocortisone and for the determination of triamcinolone, triamcinolone acetonide, triamcinolone acetonide phosphate and hydrocortisone in aqueous solutions and biological fluids. These assays allow quantification of the glucocorticoids in plasma down to a concentration of 100 ng ml(-1) (dexamethasone phosphate 300 ng ml(-1), hydrocortisone 40 ng ml(-1)). During the development of optimum extraction procedures the pK(a) values of the esters were determined by extractions performed at different pH values. The stability in vitro of the phosphate esters in ampoules, plasma and blood was studied. The esters are stable in their formulated ampoules after long-term storage, whereas the half-life in vitro of dexamethasone phosphate in plasma at 37 degrees C is 5 h and that of triamcinolone acetonide phosphate is 3.5 h. Determination by HPLC of endogenous hydrocortisone in samples from patients who received either dexamethasone phosphate or triamcinolone acetonide phosphate gave results identical with those obtained by radio-immunoassay (RIA).     

A pharmacokinetic/pharmacodynamic approach to predict the cumulative cortisol suppression of inhaled corticosteroids

The suppression of endogenous cortisol release is one of the major systemic side effects of inhaled corticosteroids in the treatment of asthma. The circadian rhythm of the endogenous cortisol release and the resulting plasma concentrations as well as the release suppression during corticosteroid therapy could previously be described with an integrated PK/PD model. Based on this model, a PK/PD approach was developed to quantify and predict the cumulative cortisol suppression (CCS) as a surrogate marker for the systemic activity of inhaled corticosteroid therapy. The presented method was applied to predict CCS after single doses and during short-term multiple dosing of the inhaled corticosteroids flunisolide (FLU), fluticasone propionate (FP), and triamcinolone acetonide (TCA), and after oral methylprednisolone as systemic reference therapy. Drug-specific PK and PD parameters were obtained from previous single-dose studies and extrapolated to the multiple-dose situation. For single dosing, a similar CCS within the range of 16-21% was predicted for FP 250 micrograms, FLU 500 micrograms, and TCA 1000 micrograms. For multiple dosing, a respective CCS of 28-33% was calculated for FLU 500 micrograms bid, FP 250 micrograms, bid, and TCA 1000 micrograms bid. Higher cortisol suppression compared to these single and multiple dosing regimens of the inhaled corticosteroids was predicted after oral doses of only 1 mg and 2 mg methylprednisolone, respectively. The predictive power of the approach was evaluated by comparing the PK/PD-based simulations with data reported previously in clinical studies. The predicted CCS values were in good correlation with the clinically observed results. Hence, the presented PK/PD approach allows valid predictions of CCS for single and short-term multiple dosing of inhaled corticosteroids and facilitates comparisons between different dosing regimens and steroids.