Detection and pharmacokinetics of salmeterol in thoroughbred horses following inhaled administration.

Salmeterol is a man-made beta-2-adrenergic receptor agonist used to relieve bronchospasm associated with inflammatory airway disease in horses. Whilst judicious use is appropriate in horses in training, they cannot race with clinically effective concentrations of medications under the British Horseracing Authority's Rules of Racing. Salmeterol must therefore be withdrawn prior to race day and pharmacokinetic (PK) studies used to establish formal detection time advice. Salmeterol xinafoate (Serevent Evohaler® ) was administered (0.1 mg twice daily for 4.5 days) via inhalation to six horses. Urine and blood samples were taken up to 103 h postadministration. Hydrolysed samples were extracted using solid phase extraction. A sensitive Ultra high performance tandem mass spectrometry (UPLC-MS/MS) method was developed, with a Lower limit of quantification (LLOQ) for salmeterol of 10 pg/mL in both matrices. The majority of salmeterol plasma concentrations, postlast administration, were below the method LLOQ and so unusable for PK analysis. Urine PK analysis suggested a half-life consistent with duration of pharmacological effect. Average estimated urine concentration at steady-state was obtained via PK modelling and used to estimate a urine concentration of 59 ± 34 pg/mL as a marker of effective lung concentration. From this, potential detection times were calculated using a range of safety factors.

Identification of the putative binding pocket of valerenic acid on GABAA receptors using docking studies and site-directed mutagenesis.

BACKGROUND AND PURPOSE: ß2/3-subunit-selective modulation of GABAA receptors by valerenic acid (VA) is determined by the presence of transmembrane residue ß2/3N265. Currently, it is not known whether ß2/3N265 is part of VA's binding pocket or is involved in the transduction pathway of VA's action. The aim of this study was to clarify the localization of VA's binding pocket on GABAA receptors. EXPERIMENTAL APPROACH: Docking and a structure-based three-dimensional pharmacophore were employed to identify candidate amino acid residues that are likely to interact with VA. Selected amino acid residues were mutated, and VA-induced modulation of the resulting GABAA receptors expressed in Xenopus oocytes was analysed. KEY RESULTS: A binding pocket for VA at the ß(+) /α(-) interface encompassing amino acid ß3N265 was predicted. Mutational analysis of suggested amino acid residues revealed a complete loss of VA's activity on ß3M286W channels as well as significantly decreased efficacy and potency of VA on ß3N265S and ß3F289S receptors. In addition, reduced efficacy of VA-induced IGABA enhancement was also observed for α1M235W, ß3R269A and ß3M286A constructs. CONCLUSIONS AND IMPLICATIONS: Our data suggest that amino acid residues ß3N265, ß3F289, ß3M286, ß3R269 in the ß3 subunit, at or near the etomidate/propofol binding site(s), form part of a VA binding pocket. The identification of the binding pocket for VA is essential for elucidating its pharmacological effects and might also help to develop new selective GABAA receptor ligands.

Detection and pharmacokinetics of salbutamol in thoroughbred racehorses following inhaled administration.

Salbutamol sulphate (Ventolin Evohaler) was administrated via the inhalation route to six horses at a dose of 0.5 mg every 4 h during the day for 2 days (total dose 4 mg). Urine and blood samples were taken up to 92 h postadministration. Hydrolyzed plasma and urine were extracted using solid phase extraction (SPE). A sensitive tandem mass spectrometric method was developed in this study, achieving a lower limit of quantification (LLOQ) for salbutamol of 10 pg/mL in plasma and urine. The parent drug was identified using UPLC-MS/MS. Most of the determined salbutamol plasma concentrations, post last administration, lie below the LLOQ of the method and so cannot be used for plasma PK analysis. Urine PK analysis suggests a half-life consistent with the pharmacological effect duration. An estimate of the urine average concentration at steady-state was collected by averaging the concentration measurements in the dosing period from -12 to 0 h relative to the last administered dose. The value was averaged across the six horses and used to estimate an effective urine concentration as a marker of effective lung concentration. The value estimated was 9.6 ng/mL and from this a number of detection times were calculated using a range of safety factors.

An investigation of complete denture teaching in the UK: part 2. The DF1 experience.

AIM: This paper, the second in a series of two (see 2013; 215: 177-181), investigates the opinions of a cohort of dental foundation year 1 (DF1) practitioners regarding their skills and competence in relation to their educational background in complete dentures. MATERIALS AND METHODS: With the permission of the Dean of the London Deanery a questionnaire was emailed to the ten London Foundation year 1 training schemes for distribution to approximately 100 DF1s. Five schemes responded with total of 56 completed questionnaires (56%). RESULTS: The average number of complete dentures made as undergraduates was three. Forty-six percent had no experience in making copy dentures. An average of 2 (median 2.05) immediate replacement dentures were made; only 10% made 8-15 dentures. None had experience in implant-supported dentures. Thirty-five percent enjoyed their undergraduate training whereas 45% did not. Thirty-seven percent felt that their training had given them experience and confidence in complete dentures but 32% were of the opposite opinion. Sixty-three percent felt complete dentures were an important or very important aspect of dentistry. Six percent completely disagreed. The majority were confident in making impressions while 39% lacked confidence in registering jaw relations. Thirty-five percent were confident with chairside adjustments at the intermediary treatment stages but 28% were not. Sixty-three percent were confident in the fitting of new dentures and 64% with the after-care. There was no significant gender difference in the responses. There was a significant difference between the London and non-London trained DF1s. The London trained respondents made significantly fewer dentures than the non-London trained cohort. The latter also rated complete denture treatment as being more important. The comments section revealed that 43% felt that they had a lack of experience; only 5% were confident, 16% thought that complete denture treatment would become obsolete and only 5% recognised the continuing importance of complete denture treatment. CONCLUSION: There is a disparity between the comments which indicate a lack of confidence in complete denture treatment and the response to the questionnaire. Other authors have commented on the lack of experience that has resulted in new graduates entering vocational training with little confidence in complete denture techniques. This report has highlighted these difficulties with respect to a current cohort of DF1s.

An investigation of complete denture teaching in the UK: part 1. A survey of undergraduate teaching.

AIM: This paper is the first in a series of two that investigate the undergraduate teaching of complete dentures in the UK. MATERIALS AND METHODS: A questionnaire was sent by email to 13 UK dental schools enquiring about the number of hours spent in the lecture theatre, laboratory and clinic. The schools were also asked to give their views about the preparedness of the new graduates for dealing with complete dentures in general practice. RESULTS: There was great disparity between schools in the overall number of lectures delivered. Two schools claimed 28 hours and the remainder varied between 6-15 hours. Four schools devoted more time to laboratory work whereas three schools spent more time in the clinic. One school reported no dedicated clinical time for complete dentures. There appears to be a regional variation with northern UK dental schools spending more time on the teaching of complete dentures. The quota for complete dentures varied from three in most schools to eight in one school and none in another. Comments from the dental schools varied from a reported lack of suitable complete denture cases, to concerns with insufficient curricular time devoted to the subject and a subsequent lack of clinical competence on graduation. Some schools have integrated complete denture teaching into the general prosthodontic course as it is considered inappropriate to teach different aspects of prosthetics in isolation. CONCLUSION: Competence in complete dentures falls short of what is expected. With a single exception all the schools seem to have low expectations for their undergraduate students to be practically trained and experienced in the production of complete dentures. Despite the advent of implants and the introduction of clinical dental technicians, there is and will continue to be a need for the competent treatment of the edentulous population by general practitioners.

Identification of etamiphylline and metabolites in equine plasma and urine by accurate mass and liquid chromatography/tandem mass spectrometry.

Etamiphylline camsylate (Millophylline V) was administered intravenously to two horses at a dose of 2.8 mg/kg. Urine and blood samples were taken up to 32 h post administration. Unhydrolyzed plasma and urine was extracted using solid phase extraction (SPE). The identity of the parent drug and metabolites was confirmed using a linear ion trap mass spectrometer and accurate mass analysis on an orbitrap mass spectrometer. Desethyletamiphylline (molecular weight 251) was the main metabolite observed in the urine and plasma samples and resulted from the N-deethylation of etamiphylline. The second metabolite detected in urine and plasma resulted from the demethylation of etamiphylline (molecular weight 265). The third minor metabolite detected in urine was proposed to have resulted from a simultaneous N-deethylation and demethylation of etamiphylline (molecular weight 238).

Regulation of oxytocin receptor concentration in rat uterine explants by estrogen and progesterone.

Incubation of uterine explants from immature rats with 0.01-100 ng of 17 beta-estradiol/mL resulted in approximately a fivefold increase in the number of oxytocin receptors per milligram of protein in 48 h. This increase was maintained for at least an additional 48 h in the presence of estrogen. When the explants were incubated with 1 microgram progesterone/mL from the outset, the concentration of oxytocin receptors was the same as initial (0 time) levels. The estrogen-induced increase in oxytocin receptor concentration was blocked by incubation with cycloheximide, an inhibitor of protein synthesis. Once increased, however, the concentration of oxytocin receptors exhibited no turnover for at least a 48-h period in the presence of estrogen. The addition of progesterone and estrogen to explants with elevated receptor levels resulted in almost a 60% reduction in oxytocin receptor concentration by 24 h, with no change in affinity of the receptor for oxytocin. The reduction in receptor concentration by progesterone was not prevented by cycloheximide. The progesterone effect may involve inactivation or degradation of oxytocin receptors or activation of substances that are inhibitory to oxytocin binding. The effects of estradiol and progesterone on oxytocin receptor concentration in uterine explants are similar to those seen when the steroids are administered in vivo. The explant system, therefore, should prove useful in clarifying factors and processes that are involved in regulation of oxytocin receptor concentration in the uterus and in the initiation of parturition in the rat.

Oxytocin receptors in rat involuting mammary gland.

Oxytocin-receptor concentrations in the rat mammary gland were determined by Scatchard analyses with [3H]oxytocin. There was about a 100-fold increase in the number of receptors per mammary gland between the 1st day of pregnancy and late lactation. The number of receptors then fell markedly during postweaning mammary regression, but rose again during a second pregnancy and lactation cycle. The changes in oxytocin-receptor number corresponded to changes in alkaline phosphatase activity per mammary gland. These results strongly support data suggesting that alkaline phosphatase, like oxytocin receptors, is a specific marker for mammary myoepithelial cells. Despite the fall in oxytocin-receptor number per mammary gland during postweaning regression, the concentration of receptors, expressed per milligram of protein, increased 10-fold over lactating levels on the 6th day of regression. Thereafter, receptor concentrations declined, but were still elevated about fivefold over lactating levels on the 15th day of regression. It is likely that the increased concentration of receptors was due to a decrease in the relative amount of nontarget secretory cells. The factors that regulate the concentration of oxytocin receptors on mammary myoepithelial cells are presently unknown; however, the involuting mammary system may be practical for obtaining enriched populations of oxytocin-sensitive myoepithelial cells.

Retinal vessel photocoagulation: a quantitative comparison of argon and krypton laser.

Retinal arteries of owl monkeys were photocoagulated with single exposures to argon laser green light (514 nm), krypton laser green light (531 nm), and krypton laser yellow light (568 nm). The distribution of damage along the vessel in different retinal layers was characterized geometrically after serial sectioning of the histopathologic material. Krypton laser radiation (green and, to an even greater extent, yellow) produced measurably greater effects on retinal vessels and adjacent structures than on deeper retinal levels; argon laser radiation (green) produced greater effects on the pigment epithelium. Krypton yellow produced the greatest total effect. The location of damage to the retinal arteries and pigment epithelium differed, with the peak of the damage around the arteries being farther from the optic disc than was the peak of the pigment epithelial damage.

Purification and characterization of mammary myoepithelial and secretory cells from the lactating rat.

Myoepithelial and secretory cells from the mammary gland of the lactating rat have been isolated, purified, and characterized. Mammary tissue was dissociated with collagenase into basket-like networks of myoepithelial cells and single secretory cells. Because of their larger size, the myoepithelial cell networks could be separated from other mammary and blood cells by differential centrifugation. Isolated secretory cells were purified by isopycnic centrifugation in 25% bovine serum albumin. The purified myoepithelial and secretory cells were viable, as shown by the incorporation of 32P into distinct macromolecules that were separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both myoepithelial and secretory cells retained their characteristic morphology after isolation and purification, as shown by light, transmission, and scanning electron microscopies. The isolated myoepithelial cells were unique and, thus, distinguishable from other mammary cells in a number of respects; they 1) contracted in response to the addition of oxytocin, 2) bound [3H]oxytocin specifically, 3) accounted for the content of alkaline phosphatase and [Na+ + K+]ATPase in mammary tissue, and 4) reacted specifically with antiserum prepared against purified myoepithelial cells. The purified secretory cells were unique in possessing glucose-6-phosphate dehydrogenase activity. The different cell markers not only gave independent estimates of the purity of the cell fractions, but they also may be helpful in identifying mammary cells in stages of differentiation and neoplastic transformation.

[On the photography of larger areas of the optic fundus-trials with the wide angle camera Clinitex CA-2 (author's transl)]. / Zur Fotografie grosser Flächen des Augenhintergrundes-Versuche mit der Weitwinkelkamera Clinitex CA-2.

The wide angle fundus camera Clinitex CA-2 was tried out on patients and normal volunteers on evaluating the numerous photographs we found the three optic heads to deliver good results. The quality of the 60 degrees- and 100 degrees-pictures improved with the increasing dilatation of the pupil. Even retinal detachments and other changes with a different level were recoreded very well. In black-and-white- photography, uneven illumination of the fundus was successfully compensated by using a two-step developer with increased utilization of film speed. This procedure allowed good wide angle documentation of the fundus at comparatively low costs.