The growing applications of SuFEx click chemistry.

SuFEx (Sulfur Fluoride Exchange) is a modular, next generation family of click reactions, geared towards the rapid and reliable assembly of functional molecules. This review discusses the growing number of applications of SuFEx, which can be found in nearly all areas of modern chemistry; from drug discovery to materials science.

Quality of web-based information for osteoarthritis: a cross-sectional study.

BACKGROUND: Osteoarthritis (OA) is a chronic condition that affects over 8.75 million people in the UK. Approximately 43% of people in the UK search for health and medical information online. However, health information on the internet is of variable quality. Research into the quality of online OA information is dated and there is a need to evaluate the existing information. OBJECTIVES: To assess the quality of websites which provide educational information for patients with OA. DESIGN: Electronic cross-sectional survey. METHODS: The search term 'osteoarthritis' was entered into the five popular UK-based search engines in order to identify 50 unique websites. These websites were appraised by two assessors using criteria developed from the available literature and recent guidelines of the National Institute of Health and Care Excellence. The appraisal considered the general quality of the website and OA-specific content. RESULTS: Most of the websites evaluated (34/50, 68%) scored more than half of the maximum available quality score (max score 59). The median total score was 41. For general quality of the website, the median score was nine (range three to 16, out of 16), and for OA-specific content, the median score was 31 (range two to 43, out of 43). Websites of higher quality were created more recently, disclosed sources of information, had external seals of approval and directed the reader on to other relevant websites. CONCLUSIONS: The internet is a potentially useful tool for educating and empowering healthcare consumers. The websites evaluated were generally of a 'high' standard; however, there was wide variation in the quality of information.

Jak3 deficiency blocks innate lymphoid cell development.

Loss-of-function mutations in the tyrosine kinase JAK3 cause autosomal recessive severe combined immunodeficiency (SCID). Defects in this form of SCID are restricted to the immune system, which led to the development of immunosuppressive JAK inhibitors. We find that the B6.Cg-Nr1d1tm1Ven/LazJ mouse line purchased from Jackson Laboratories harbors a spontaneous mutation in Jak3, generating a SCID phenotype and an inability to generate antigen-independent professional cytokine-producing innate lymphoid cells (ILCs). Mechanistically, Jak3 deficiency blocks ILC differentiation in the bone marrow at the ILC precursor and the pre-NK cell progenitor. We further demonstrate that the pan-JAK inhibitor tofacitinib and the specific JAK3 inhibitor PF-06651600 impair the ability of human intraepithelial ILC1 (iILC1) to produce IFN-γ, without affecting ILC3 production of IL-22. Both inhibitors impaired the proliferation of iILC1 and ILC3 and differentiation of human ILC in vitro. Tofacitinib is currently approved for the treatment of moderate-to-severely active rheumatoid arthritis. Both tofacitinib and PF-06651600 are currently in clinical trials for several other immune-mediated conditions. Our data suggest that therapeutic inhibition of JAK may also impact ILCs and, to some extent, underlie clinical efficacy.

Concordance between nucleic acid amplification technique and culture for the diagnosis of gonorrhoea.

The objective of the study was to evaluate the concordance between nucleic acid amplification technique (NAAT) and culture for the diagnosis of Neisseria gonorrhoeae among attendees at a genitourinary medicine clinic in East London. All patients testing positive for N. gonorrhoeae on NAAT and/or culture between 1 April 2007 and 31 August 2008 at the Department of Sexual Health at Homerton University Hospital were included. Male patients had a first void urine sample for NAAT and urethral culture; female patients had a self-taken vulval swab or endocervical sample sent for NAAT and an endocervical culture sample. After interim analysis, discrepant results had both NAAT and culture repeated prior to treatment. Of 159 male patients with a positive NAAT, 22 (13%) had a negative culture. Among 135 female patients with a positive NAAT, 36 (27%) had a negative culture. Three men had a positive culture and negative NAAT. Nineteen of the discrepant samples were retested prior to treatment and 12 (63%) had spontaneously revered to negative. In conclusion, there was concordance in 84% of male and 67% of female samples. In two-thirds of the discrepant cases, the previously positive NAAT had become negative prior to treatment. This study highlights the importance of consideration of the clinical picture when assessing results.

Interaction between prostaglandin D and chemoattractant receptor-homologous molecule expressed on Th2 cells mediates cytokine production by Th2 lymphocytes in response to activated mast cells.

The mechanisms by which immunologically activated mast cells stimulate the production of proinflammatory cytokines by T helper type 2 (Th2) lymphocytes were investigated in a human cell culture system. Supernatants collected from cord blood-derived mast cells after treatment with immunoglobulin E (IgE)/anti-IgE contained an activity that stimulated the production of interleukin (IL)-4, IL-5 and IL-13 (both mRNA and protein) by Th2 lymphocytes. This activity was not detected in supernatants from unactivated mast cells and its production was inhibited by treatment of activated mast cells with the cyclo-oxygenase inhibitor diclofenac. The concentration of diclofenac used inhibited completely the production of prostaglandin D(2) (PGD(2)) but did not inhibit the release of histamine or leukotriene C(4). The effect of supernatants from activated mast cells was mimicked by exogenous PGD(2) at concentrations similar to those detected in the cultures of activated mast cells, and addition of exogenous PGD(2) to supernatants from diclofenac-treated mast cells restored their ability to stimulate Th2 cytokine production. The ability of the mast cell supernatants to stimulate production of Th2 cytokines was not affected by addition of diclofenac to the Th2 cells directly, indicating that the production, but not the action, of the factor was sensitive to diclofenac treatment. Inhibition of chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) abolished the effect of the mast cell supernatants on Th2 cytokine production. These data indicate that mast cells have the ability to stimulate Th2 cells to elaborate cytokines independently of T cell receptor activation or co-stimulation and this response is mediated by PGD(2) acting upon CRTH2 expressed by Th2 cells.

The results of arthroplasty in osteoarthritis of the shoulder.

We have undertaken a prospective clinical and radiological analysis of 124 shoulder arthroplasties (113 patients) carried out for osteoarthritis. The clinical results showed improvement in the absolute Constant score and the American Shoulder and Elbow Surgeons score of 22 and 43, respectively. Both were statistically significant (p < 0.001). There was no significant difference in the scores after hemiarthroplasty and total arthroplasty in those patients with an intact rotator cuff. When revision was used as the end-point for survival at ten years, survival of 86%, or 90% if glenoid components made of Hylamer sterilised in air were omitted, was obtained in primary osteoarthritis. The most common cause for revision in the hemiarthroplasty group was glenoid pain at a mean of 1.5 years; in the total arthroplasty group it was loosening of the glenoid at a mean of 4.5 years. Analysis of pre-operative factors showed that the risk of gross loosening of the glenoid increased threefold when there was evidence of erosion of the glenoid at operation. Shoulder arthroplasty should not be delayed once symptomatic osteoarthritis has been established and should be undertaken before failure of the cuff or erosion of the glenoid are present.

Liquid chromatographic-tandem mass spectrometric method for the determination of the neuraminidase inhibitor zanamivir (GG167) in human serum.

An LC-MS-MS method for the analysis of the neuraminidase inhibitor, zanamivir, in human serum is described. Zanamivir was extracted from protein precipitated human serum samples using Isolute SCX solid-phase extraction cartridges and analysed using reversed-phase chromatography with TurboIonSpray atmospheric pressure ionisation followed by mass spectrometric detection. The method uses a stable isotope internal standard, is highly specific and sensitive for a compound of this type and has been used for the analysis of human serum and urine samples from clinical studies. The method was extended to the analysis of serum and plasma samples from pre-clinical studies involving the rat, ferret and cell culture media. The method has been shown to be robust and valid over a concentration range of 10-5000 ng/ml using a 0.2-ml sample volume. The main advantages of this method compared to earlier procedures are primarily specificity, sensitivity, ease of sample preparation, small sample volume and short analysis time (ca. 5 min).

An improved method for the determination of fluticasone propionate in human plasma.

Therapeutic monitoring of the potent, highly lipophilic glucocorticoid, fluticasone propionate (FP), was initially performed by a radioimmunoassay method. However an improved method with a lower limit of quantitation (LLOQ) of at least 25 pg per ml (pg/ml(-1)) was needed to measure the low levels of FP present in human plasma following inhalation administration of doses in the range 50-250 microg twice daily. A sensitive and specific liquid chromatographic, tandem mass spectrometric method (LC-MS/MS) with automated solid phase extraction (SPE) was developed and validated. Fluticasone propionate was extracted from plasma using Bond Elut C18 cartridges and analysed using reverse-phase chromatography with atmospheric pressure chemical ionisation followed by selective reaction monitoring. The method used a 13C-labelled internal standard and was validated over a concentration range of 25-500 pg/ml(-1). The method was shown to be specific, sensitive and reliable in the analysis of clinical samples. The main advantages of this method over the radioimmunoassay method previously used were improved sensitivity, specificity, ease of sample preparation and shortened analysis time.

Molecular characteristics of very virulent European MDV isolates.

Marek's disease virus (MDV) strains with increasing virulence have been reported from many parts of the world. Many of these recent MDV isolates produce an acute early cytolytic disease with high mortality and severe atrophy of the lymphoid organs, thymus and the bursa of Fabricius. Although the degree of the atrophic changes and the virulence of the virus are correlated, the molecular basis of the increased virulence is not known. We examined the characteristics of the disease induced by 3 such MDV isolates, C12/130, MR36 and MR48, isolated from Europe. All the three viruses produce high early mortality and atrophy of the lymphoid organs. As a first step in understanding the determinants of the increased virulence of these isolates, we have compared the sequences of MEQ and the ICP4 genes of these three viruses with that of the published sequences. Some of the amino acid changes seen within the Meq and ICP4 proteins were conserved in all the three isolates and could account for the increased virulence characteristics.

Validation of a liquid chromatographic tandem mass spectrometric method for the determination of sumatriptan in human biological fluids.

A liquid chromatographic tandem mass spectrometric method for the quantitative determination of sumatriptan base in human plasma and urine has been developed and validated over the concentration range 0.2-20 ng base ml-1. Sumatriptan is a 5-HT1 receptor agonist which has found widespread use in the treatment of migraine. Sumatriptan and its internal standard (D3-sumatriptan) were extracted from human matrices using C2 solid phase cartridges. The extracts were chromatographed on a C18 column, ionised using a heated nebuliser assisted atmospheric pressure ionisation (API) interface and detected by MS/MS in the multiple reaction monitoring mode. The completed validation demonstrated the method to be robust, accurate, precise and specific for the direct quantification of sumatriptan in human fluids. The method was used on a routine basis to determine the levels of sumatriptan in human volunteers following the oral administration of a 25 mg dose of sumatriptan succinate.

off absorption, pharmacodynamics, metabolism and excretion of 14C-sumatriptan following intranasal administration to the beagle dog.

The pharmacodynamics, pharmacokinetics, metabolism, and excretion of 14C-sumatriptan have been studied in the beagle dog following administration by the intranasal and other routes. The pharmacological response which was monitored, an increase in carotid arterial vascular resistance, correlated with the plasma levels of unchanged sumatriptan following intranasal, intravenous, or intraduodenal administration to the anaesthetised dog. The pharmacokinetics and metabolism of sumatriptan were then confirmed in conscious male and female dogs. Intranasal administration of 14C-sumatriptan resulted in rapid absorption of part of the dose. The overall bioavailability of sumatriptan was 40-50%. Sumatriptan was eliminated from plasma with a half-life of 1.5 or 1.9 h after intravenous or intranasal dosage respectively. Radioactivity was largely excreted in urine (up to 75% of the dose) with small amounts in the bile and faeces after intravenous and intranasal dosing, as sumatriptan and a major metabolite. The results from these studies suggest that intranasal administration provides a viable method for delivering sumatriptan to the systemic circulation.

Phylogenetic relationships among eight Eimeria species infecting domestic fowl inferred using complete small subunit ribosomal DNA sequences.

Complete 18S ribosomal RNA gene sequences were determined for 8 Eimeria species of chickens and for Eimeria bovis of cattle. Sequences were aligned with each other and with sequences from 2 Sarcocystis spp., Toxoplasma gondii, Neospora caninum, and 4 Cryptosporidium spp. Aligned sequences were analyzed by maximum parsimony to infer evolutionary relationships among the avian Eimeria species. Eimecia bovis was found to be the sister taxon to the 8 Eimeria species infecting chickens. Within the avian Eimeria species, E. necatrix and E. tenella were sister taxa: this clade attached basally to the other chicken coccidia. The remaining Eimeria spp. formed 3 clades that correlated with similarities based on oocyst size and shape. Eimeria mitis and Eimeria mivati (small, near spherical oocysts) formed the next most basal clade followed by a clade comprising Eimeria praecox. Eimeria maxima, and Eimeria brumetti (large, oval oocysts), which was the sister group to Eimeria acervulina (small, oval oocysts). The 4 clades of avian Eimeria species were strongly supported in a bootstrap analysis. Basal rooting of E. necatrix and E. tenella between E. bovis and the remaining Eimeria species and the apparent absence of coccidia that infect the ceca of jungle fowl all suggest that E. necatrix and E. tenella may have arisen from a host switch, perhaps from the North American turkey, Meleagris gallopavo.

Absorption, pharmacokinetics and metabolism of 14C-sumatriptan following intranasal administration to the rat.

1. Studies have been carried out to investigate the absorption of sumatriptan after intranasal administration to rats. The pharmacokinetics, metabolism and excretion of 14C-sumatriptan were compared following intranasal and intravenous dosing to male and female albino rats using an aqueous buffered formulation at pH 5.5. 2. Following intravenous administration sumatriptan was eliminated from plasma with a half-life of about 1.1 h. After intranasal administration there was rapid absorption of part of the dose and two peak plasma concentrations were observed, initially at 0.5 and then at 1.5-2 h. The elimination half-life after the second peak was estimated as being about 4 h. 3. Radioactivity was largely excreted in urine (up to 89% of dose in 168 h) after both intravenous and intranasal administration, with a faster rate of excretion after intravenous dosage (73% males, 64% females within 6 h) than after intranasal dosage (37% males, 40% females within 6 h). 4. 14C-sumatriptan was the major component in urine and in extracts of faeces after both intravenous and intranasal administration. The major metabolite excreted in urine and faeces was GR49336, the indole acetic acid analogue. 5. The results of this in vivo rat study suggest that absorption of the dose via the nasal mucosa is incomplete after intranasal administration and that there is a secondary absorption phase probably reflecting oral absorption of part of the dose. The bioavailability is estimated as about 30%, for the period 0-6 h.

Kinetics and disposition of picumeterol in animals.

1. The pharmacokinetics and disposition of picumeterol, a novel beta 2 receptor agonist agent, have been studied in the rat and dog following administration by inhalation, intravenous and oral routes at various dose levels. 2. Picumeterol was found to be transferred across the lung of the rat and dog following inhalation dosage. After i.v. dosage picumeterol was eliminated from plasma with a half-life of about 1 h in the rat and about 2 h in the dog. Plasma clearance in the rat was about twice liver blood flow and the plasma levels of picumeterol were low after oral administration. 3. Following instillation of 14C-picumeterol to the trachea of isolated respiring rat lung preparations radioactivity was transferred from the airways to perfusion media as unchanged drug within 2 min. After 2 h perfusion, no metabolites were detected in the recirculation perfusate or lung. 4. Picumeterol was extensively metabolized in vivo in the rat (about 95%) and dog (about 90%) and in vitro in microsomal preparations of rat, dog and human liver. O-dealkylation and beta-oxidation are important as routes of metabolism. 5. Radioactivity was largely excreted in the urine of the rat and dog (> 50% of dose), as metabolites, following i.v. administration. There was some excretion of radioactivity in dog bile. Extensive first-pass metabolism was found after oral administration in the rat.

Cloning, expression and characterization of human alpha adrenergic receptors alpha 1a, alpha 1b and alpha 1c.

The three human alpha adrenergic receptor subtypes, alpha 1a, alpha 1b, and alpha 1c, have been cloned, expressed in COS-7 cells, and characterized pharmacologically. Competition binding studies of several adrenergic ligands with the three human receptor subtypes reveals a distinct pharmacological profile for each receptor subtype. RNase protection analysis demonstrates that the three receptor subtypes have different patterns of distribution in human tissue. The availability of the three human alpha 1 receptor subtypes will facilitate the development of subtype-selective alpha 1 antagonists for a variety of therapeutic indications.

Disposition of salmeterol xinafoate in laboratory animals and humans.

The disposition of [14C]salmeterol xinafoate, a new inhaled beta 2-adrenoceptor agonist with both bronchodilator and antiinflammatory activity, has been studied in laboratory animals and humans following intravenous and oral administration. [14C]Salmeterol was rapidly absorbed in animal species and humans with Cmax observed within 2 hr. Cmax was similar for normalized oral dose level in mice, rats, and rabbits. In dogs, Cmax was higher and reflected the greater oral bioavailability in this species. The plasma t1/2, after intravenous administration, was 5 hr in rats and 2 hr in dogs. The volume of distribution of salmeterol was significantly greater than total body water in both rats (40 liters/kg) and dogs (6 liters/kg) and indicated high tissue uptake of the compound. Plasma clearance was high in rats (95 ml/min/kg) and dogs (30 ml/min/kg). Radioactive drug-related material was widely distributed throughout body tissues in rats. The highest concentrations were present in kidney, liver, gastrointestinal tract, pituitary, lung, heart, and bone marrow. Transfer of radioactive drug-related material across the placental barrier or into milk, studied in rats, was low. In all species the majority of an oral or intravenous dose (55-75%) was excreted in feces. Biliary excretion in rats and dogs accounted for 53% (0-27 hr) and 40% (0-8 hr) of an oral dose, respectively, indicating good absorption across the gastrointestinal tract. Enterohepatic circulation was significant in rats. Salmeterol was cleared predominantly by metabolism in animals and humans.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the disposition of a 5-nitroimidazole in laboratory animals.

The disposition and metabolism of a 5-nitroimidazole compound (SC 28538) was investigated in the rat, beagle dog and rhesus monkey. The absorption of [14C]-SC 28538 was rapid and essentially complete after oral dosage in male animals, and also after intravaginal dosage in the female rat. Peak plasma levels of radioactivity occurred within 2 h of dosage. In the rat and dog the radioactivity was excreted predominantly in the faeces (greater than 60%) but in the monkey more than 60% was excreted in the urine. In both the male and pregnant female rat radioactivity was concentrated in the gastro-intestinal tract, liver and harderian gland and the concentrations of radioactivity in other tissues was generally lower than in plasma. Radioactivity was cleared more rapidly from plasma than from the majority of tissues. SC 28538 was extensively metabolised to form glucuronide and amino acid conjugates. The half-life of SC 28538 was of the order of 1 h in the dog and 3.7 h in the monkey.

Disopyramide elimination in the isolated perfused rat liver.

The elimination kinetics of disopyramide, [14C]disopyramide and [2H]disopyramide have been studied in the isolated perfused rat liver. Disappearance of disopyramide from perfusate was dose- and time-dependent over the dose range 0.3-7.5 mg. Although the mechanism underlying these observations is unclear, the data are consistent with the presence of enzyme saturation and product inhibition. Biliary secretion of conjugated metabolites appeared to be the rate-limiting step in the perfusate clearance of total radioactivity. At doses of 0.3 and 7.5 mg the kinetics of [2H]disopyramide showed a small isotope effect probably of negligible importance.

Haloperidol pharmacokinetics: a preliminary study in rhesus monkeys using a new radioimmunoassay procedure.

The development, validation, and application of a new radioimmunoassay for haloperidol in biological fluids is described. The antiserum, raised against N-amino-butyl chlorophenyl piperidine bovine albumin conjugate, could not distinguish between haloperidol and its reduced metabolite, but it could discriminate against chlorophenyl piperidine (cross-reaction 2.6%). The fluorophenyl metabolites of haloperidol were not recognized by the antiserum. Haloperidol determinations were made on less than 100 microliter aliquots of human and rhesus monkey plasma or diluted urine without prior extraction of the sample. The radioimmunoassay was applied to the study of the pharmacokinetics of intravenous haloperidol administration to two male rhesus monkeys. Salient features of the results are as follows. As with man, the plasma concentration versus time curve could be resolved into three compartments, but there were differences in the distribution of haloperidol between the compartments. The apparent volume of distribution for the two monkeys examined was 5.87 L kg-1 and 7.37 L kg-1, considerably smaller than in man, a difference almost entirely due to a much smaller tissue compartment. The biological half-life of 15.97 hr and 7.56 hr was similar to man. The mean hepatic extraction ratio was calculated to be 0.032 and 0.056, and the data suggested that hepatic metabolism of haloperidol may be of lesser importance in rhesus monkey than in man. An insignificant proportion (0.01%) of the administered dose was excreted as haloperidol in the urine.