Simulating the influence of plasma protein on measured receptor affinity in biochemical assays reveals the utility of Schild analysis for estimating compound affinity for plasma proteins.

BACKGROUND AND PURPOSE: Plasma protein binding (PPB) influences the free fraction of drug available to bind to its target and is therefore an important consideration in drug discovery. While traditional methods for assessing PPB (e.g. rapid equilibrium dialysis) are suitable for comparing compounds with relatively weak PPB, they are not able to accurately discriminate between highly bound compounds (typically >99.5%). The aim of the present work was to use mathematical modelling to explore the potential utility of receptor binding and cellular functional assays to estimate the affinity of compounds for plasma proteins. Plasma proteins are routinely added to in vitro assays, so a secondary goal was to investigate the effect of plasma proteins on observed ligand-receptor interactions. EXPERIMENTAL APPROACH: Using the principle of conservation of mass and the law of mass action, a cubic equation was derived describing the ligand-receptor complex [LR] in the presence of plasma protein at equilibrium. KEY RESULTS: The model demonstrates the profound influence of PPB on in vitro assays and identifies the utility of Schild analysis, which is usually applied to determine receptor-antagonist affinities, for calculating affinity at plasma proteins (termed KP ). We have also extended this analysis to functional effects using operational modelling and demonstrate that these approaches can also be applied to cell-based assay systems. CONCLUSIONS AND IMPLICATIONS: These mathematical models can potentially be used in conjunction with experimental data to estimate drug-plasma protein affinities in the earliest phases of drug discovery programmes.

Reassessment of the pharmacology of Sphingosine-1-phosphate S1P3 receptor ligands using the DiscoveRx PathHunter™ and Ca2+ release functional assays.

BACKGROUND AND PURPOSE: DiscoverRx's PathHunter™ assay measures GPCR agonist potency, via the recruitment of ß-arrestin, independent of the subtype of G(α) protein activated. This assay is frequently used in drug discovery although little is known about the agonist pharmacology generated. Here we have compared agonist potency, efficacy and affinity values obtained in PathHunter™ assays with those from more established radioligand binding and functional techniques. EXPERIMENTAL APPROACH: Using cells expressing the human sphingosine-1-phosphate S1P(3) receptor at four different densities, we compared pharmacological affinity and efficacy values of four structurally distinct ligands - FTY720-P, VPC24191, CYM5442 and the endogenous agonist S1P - obtained from competition binding, functional Ca(2+) release and PathHunter™ assays. KEY RESULTS: The pK(i) values for S1P were significantly different (9.34 ± 0.10 and 8.92 ± 0.15) in clones expressing different receptor levels using the binding assay. In the PathHunter™ and Ca(2+) assays, S1P and CYM5442 were full agonists, FTY720-P was a partial agonist, while the efficacy of VPC24191 could not be detected in PathHunter™ assays. VPC23019, previously described as a S1P(1/3) receptor antagonist, behaved as an S1P(3) receptor partial agonist in the Ca(2+) release assay. CONCLUSIONS AND IMPLICATIONS: Comparison of data from the PathHunter™ assay with binding and functional Ca(2+) assays suggest that PathHunter™ assays measured a different agonist-bound receptor conformation. While this assay has great utility in drug discovery, care must be taken as high-efficacy, low-affinity agonist compounds would not be detected. Therefore highly amplified, more traditional assays are necessary to identify agonists with low efficacy.

Reduced renal hemodynamic response to atrial natriuretic peptide in elderly volunteers.

Aging is associated with decreased ability to excrete salt and water, thus increasing the susceptibility to volume overload in older individuals. Meanwhile, plasma levels of atrial natriuretic peptide (ANP) increase progressively with age for unknown reasons. We compared the natriuretic and renal hemodynamic responses to low-dose ANP infusion in an elderly group of volunteers (mean age, 74 years) with those of a group of younger subjects (mean age, 29 years). A significant reduction below baseline values in effective renal plasma flow occurred in the young group after the 2-hour peptide infusion (657 +/- 125 v 476 +/- 92 mL/min [mean +/- 1 SD]) when compared with the elderly group (two-way analysis of variance; P < 0.02). A concomitant increase in renal vascular resistance was noted in the young group only during the same period (6,631 +/- 1,384 v 9,136 +/- 2,126 dyn s cm2 x 10(6)). This increase was also significantly higher than that in the elderly group (analysis of variance; P < 0.02). Both groups demonstrated similar natriuretic responses. Absolute sodium excretion had increased significantly above baseline values in both young and elderly subjects at the end of the 2-hour peptide infusion (111 +/- 25 mumol/min to 183 +/- 33 mumol/min v 107 +/- 23 mumol/min to 198 +/- 56 mumol/min) and remained elevated until 1 hour postinfusion. We conclude that the elderly subjects in our study demonstrated a diminished renal hemodynamic response to infusion of ANP while preserving a natriuretic response similar to that found in the younger subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between Korotkoff's sounds and ultrasonics of the brachial artery in healthy and normotensive subjects.

The clinical measurement of blood pressure is generally obtained by auscultation for Korotkoff's sounds over the brachial artery using a sphygmomanometer and cuff. Real-time two dimensional ultrasound and Doppler techniques were used to study the brachial artery movements and blood flow patterns during these pressure measurements. Simultaneous recordings of Korotkoff's sounds were obtained. A strong correlation in time was demonstrated between Korotkoff's five phases, brachial artery wall movements and specific blood flow patterns. The evidence largely supports what to date has been speculation as to the origin of Korotkoff's sounds in that Korotkoff's phases 1 and 3 appear to be produced by vigorous arterial wall motion and phase 5 by cessation of this movement. Phase 2 is produced by blood flow disturbance. Furthermore, the lack of correlation between phase 4 and any specific vascular event may go some way to explaining why in clinical practice phase 5 readings are now preferred for diastolic pressure.

Lumbar sagittal mobility and low back symptoms in patients treated with manipulation.

The relationship between lumbar sagittal mobility and symptoms was explored in a 1-year prospective study of 55 patients undergoing manipulative treatment. A significant increase in mean mobility was found to occur in the 1st month, together with a concomitant significant decrease in mean pain values; symptoms continued to decrease thereafter, but mobility remained unchanged. More detailed analysis of the data revealed that symptomatic improvement was as common in patients with unaltered or reduced mobility as it was in those who showed an increase; changes in mobility at 1 month had no predictive value for symptomatic status at 1 month or 1 year. It is concluded that if any benefits actually result from manipulative therapy they are not a direct function of increased overall lumbar sagittal mobility. It remains possible that manipulation may influence other mobility parameters, such as coupled motions, so future studies should be directed toward investigation of three-dimensional movement patterns.

Measurement of Pseudomonas aeruginosa phenazine pigments in sputum and assessment of their contribution to sputum sol toxicity for respiratory epithelium.

The phenazine pigments pyocyanin and 1-hydroxyphenazine were resolved by high-pressure liquid chromatography from the sputum sol phase from 9 of 13 patients with cystic fibrosis or bronchiectasis colonized by Pseudomonas aeruginosa. The concentrations measured were each sufficient to inhibit ciliary beating in vitro and contributed a significant proportion of sol phase toxicity for respiratory epithelium.

Deleterious effects of purulent sputum sol on human ciliary function in vitro: at least two factors identified.

Patients with chronic bronchial sepsis have impaired mucociliary clearance. A study was carried out on the effect of sputum sol (obtained by rapid centrifugation of purulent sputum) from 20 patients with chronic bronchial sepsis on the beating of human nasal cilia in vitro by a photometric technique. Thirteen sols caused significant (p less than 0.001) ciliary slowing. Two patterns of slowing were observed: firstly, a gradual onset associated with epithelial disruption (inhibited by alpha 1 antiprotease) and, secondly, an immediate onset associated with ciliary dyskinesia and ciliostasis (inhibited by chloroform extraction). The ciliary slowing activity of sputum sols was associated with the isolation of Pseudomonas aeruginosa (p less than 0.01). It is concluded that purulent sputum contains at least two factors that impair ciliary beating--one a serine protease, which is probably a product released by the host's phagocytic defences, and the other, which is chloroform extractable and probably a bacterial product.

Relative importance of antibiotic and improved clearance in topical treatment of chronic mucopurulent rhinosinusitis. A controlled study.

50 patients with chronic mucopurulent rhinosinusitis were randomly allocated to treatment with nasal sprays of dexamethasone, tramazoline, and neomycin, dexamethasone and tramazoline with no antibiotic, or matched placebo (propellant alone) four times daily to both nostrils for 2 weeks. The patients were assessed in a double-blind manner for symptomatic response and improvement in nasal mucociliary clearance, nasal airway resistance, sinus radiographs, and intranasal bacteriology and appearance. Both active preparations (with antibiotic 14 of 20 patients responded; without antibiotic 12 of 20 patients responded) were more effective than the placebo (2 of 10 patients responded). There was no significant difference in response between the active preparations with and without antibiotic. Thus, in treatment of chronic mucopurulent rhinosinusitis, reduction of the inflammatory response and decongestion make topical antibiotic unnecessary, probably by allowing host clearance mechanisms to recover.

Beat frequency of cilia from sites of purulent infection.

Mucociliary clearance depends on the interaction between cilia and mucus; it is delayed in the presence of purulent secretions. Nasal mucociliary clearance was examined by the saccharin method and nasal ciliary beat frequency by a photometric technique. Four groups were studied: normal controls, patients with bronchiectasis without nasal symptoms, patients with chronic mucopurulent sinusitis alone, and patients with chronic mucopurulent sinusitis and bronchiectasis. Nasal mucociliary clearance was prolonged in infected patients. Cilia obtained from the site of purulent secretions were found to beat more slowly in vitro (mucopurulent sinusitis 12.1 Hz, mucopurulent sinusitis and bronchiectasis 11.6 Hz), than those obtained from normal controls (14.3 Hz) and from patients with bronchiectasis alone (13.6 Hz). The cause of the ciliary slowing seemed most likely to be the release of host factors during the inflammatory response, rather than the particular organism isolated. Ciliary slowing may contribute to the observed delay of mucociliary clearance in conditions in which purulent secretions are present.