DHEA and response to antidepressant treatment: A Mendelian Randomization analysis.

Treatment response is hard to predict and detailed mechanisms unknown. Lower levels of the dehydroepiandrosterone sulphate (DHEA(S)) - a precursor to testosterone and estrogen - have been associated to depression and to response to antidepressant treatment. Previous studies however may have been ridden by confounding and reverse causation. The aim of this study is to evaluate whether higher levels of DHEA(S) are causally linked to response to antidepressants using mendelian randomization (MR). We performed a Two-sample MR analysis using data the largest publicly available GWAS of DHEA(S) levels (n = 14,846) using eight common genetic variants associated to DHEA(S) (seven single nucleotide polymorphisms and one variant rs2497306) and the largest GWAS of antidepressant response (n = 5218) using various MR methods (IVW, MR Egger, Weighted mean, weighted mode, MR-PRESSO) and single SNP analysis. We further investigated for pleiotropy conducting a look up on PhenoScanner and GWAS Catalog. Results show no evidence for DHEA(S) gene risk score from any of MR methods, however, we found a significant association on individual variant analysis for rs11761538, rs17277546, and rs2497306. There was some evidence for heterogeneity and pleiotropy. This is the first paper to show some evidence for a causal association of genetically-predicted DHEA and improvement of depressive symptoms. The effect is not a simple linear effect, and we were unable to dissect whether the effect was direct effect of DHEA(S), mediated by DHEA(S) or on the pathway is not yet clear. Further studies using more refined instrumental variables will help clarify this association.

An epigenome-wide association study meta-analysis of educational attainment.

The epigenome is associated with biological factors, such as disease status, and environmental factors, such as smoking, alcohol consumption and body mass index. Although there is a widespread perception that environmental influences on the epigenome are pervasive and profound, there has been little evidence to date in humans with respect to environmental factors that are biologically distal. Here we provide evidence on the associations between epigenetic modifications-in our case, CpG methylation-and educational attainment (EA), a biologically distal environmental factor that is arguably among the most important life-shaping experiences for individuals. Specifically, we report the results of an epigenome-wide association study meta-analysis of EA based on data from 27 cohort studies with a total of 10 767 individuals. We find nine CpG probes significantly associated with EA. However, robustness analyses show that all nine probes have previously been found to be associated with smoking. Only two associations remain when we perform a sensitivity analysis in the subset of never-smokers, and these two probes are known to be strongly associated with maternal smoking during pregnancy, and thus their association with EA could be due to correlation between EA and maternal smoking. Moreover, the effect sizes of the associations with EA are far smaller than the known associations with the biologically proximal environmental factors alcohol consumption, body mass index, smoking and maternal smoking during pregnancy. Follow-up analyses that combine the effects of many probes also point to small methylation associations with EA that are highly correlated with the combined effects of smoking. If our findings regarding EA can be generalized to other biologically distal environmental factors, then they cast doubt on the hypothesis that such factors have large effects on the epigenome.

Stereospecific quantitation of 6-prenylnaringenin in commercially available H. lupulus-containing natural health products and dietary supplements.

6-Prenylnaringenin (6PN) is a chiral prenylflavonoid found most prevalently in hops (Humulus lupulus) and present in hops and hop products. It is an isomer of the potent phytoestrogen, 8-prenylnaringenin. An enantiospecific method for quantitation 6PN by LC-ESI-MS has been developed. Baseline enantiomeric resolution of 6PN was attained on a Chiralpak(®) AD-RH column with an isocratic mobile phase consisting of acetonitrile and 10 mM ammonium formate (pH 8.5) (39:61, v/v) and a flow rate of 1.25 mL/min. Quantitative MS data were obtained by selected ion monitoring of the [M-H](-)-ion of both enantiomers of 6PN (m/z 339.10) and the internal standard, 4-acetamidobenzoic acid (m/z 178.05). The method was found to be accurate and precise for enantiospecific quantification of 6PN. The method was successfully applied to the content analysis of 39 commercially available natural health products and dietary supplements reported to contain H. lupulus plant material, extracts and label claims of 6PN. 6PN was present in 25 of 34 products containing plant material or extracts of H. lupulus. Of the five products with claimed amounts of 6PN, all were found to possess <50% of label claims. Results of the content analysis indicated a lack of uniformity in botanical nutraceuticals claiming 6PN content.

Synovial fluid bupivacaine concentrations following single intra-articular injection in normal and osteoarthritic canine stifles.

Intra-articular bupivacaine helps alleviate pain in animals receiving joint surgery, but its use has become controversial as ex vivo studies have illuminated the potential for chondrotoxicity. Such studies typically involve cell cultures incubated in solutions containing high bupivacaine concentrations for long durations. The aim of this study was to measure the actual synovial fluid bupivacaine concentrations after intra-articular injection. Eight healthy beagles with normal stifles and 22 large and giant-breed dogs with stifle osteoarthritis (OA) were treated with a single intra-articular injection of bupivacaine (1 mg/kg) into a stifle. Joint fluid samples were taken from the treated stifle immediately after injection and 30 min after injection and analyzed for bupivacaine concentrations. Immediately after injection, the median bupivacaine concentrations in normal and OA stifles were 3.6 and 2.5 mg/mL, respectively. Thirty minutes after injection, bupivacaine concentrations in normal and OA stifles were 0.4 and 0.6 mg/mL, respectively. These results provide insight into the pharmacokinetics of bupivacaine after injection into a joint. Given its immediate dilution and rapid drop in synovial fluid concentration, bupivacaine is unlikely to damage chondrocytes when administered as a single intra-articular injection.

Pharmacokinetics of oral rufinamide in dogs.

The objective of this study was to determine the pharmacokinetic properties and short-term adverse effect profile of single-dose oral rufinamide in healthy dogs. Six healthy adult dogs were included in the study. The pharmacokinetics of rufinamide were calculated following administration of a single mean oral dose of 20.0 mg/kg (range 18.6-20.8 mg/kg). Plasma rufinamide concentrations were determined using high-performance liquid chromatography, and pharmacokinetic data were analyzed using commercial software. No adverse effects were observed. The mean terminal half-life was 9.86 ± 4.77 h. The mean maximum plasma concentration was 19.6 ± 5.8 µg/mL, and the mean time to maximum plasma concentration was 9.33 ± 4.68 h. Mean clearance was 1.45 ± 0.70 L/h. The area under the curve (to infinity) was 411 ± 176 µg · h/mL. Results of this study suggest that rufinamide given orally at 20 mg/kg every 12 h in healthy dogs should result in a plasma concentration and half-life sufficient to achieve the therapeutic level extrapolated from humans without short-term adverse effects. Further investigation into the efficacy and long-term safety of rufinamide in the treatment of canine epilepsy is warranted.

Systemic delivery of E6/7 siRNA using novel lipidic particles and its application with cisplatin in cervical cancer mouse models.

Small interfering RNA (siRNA) shows great promise in cancer therapy, but its effectiveness in vivo still remains a crucial issue for its transition into the clinics. Although the successful use of polyethylene glycol (PEG)ylated lipidic delivery systems have already been reported, most of the formulation procedures used are labour intensive and also result in unstable end products. We have previously developed a simple yet efficient hydration-of-freeze-dried-matrix (HFDM) method to entrap siRNA within lipid particles, in which the products exhibited superior stability. Here, we show that these HFDM-formulated particles are stable in the presence of serum and can deliver siRNA efficiently to tumours after intravenous administration. Using these particles, around 50% knockdown of the target gene expression was observed in tumours. With the use of siRNA targeting the E6/7 oncogenes expressed in cervical cancer, we showed a 50% reduction in tumour size. This level of tumour growth suppression was comparable to that achieved from cisplatin at the clinically used dose. Overall, our results demonstrate the feasibility of using HFDM-formulated particles to systematically administer E6/7-targeted siRNA for cervical cancer treatment. The simplicity of preparation procedure along with superior product stability obtained from our method offers an innovative approach for the in vivo delivery of siRNA.

Effect of dehydration on raspberries: polyphenol and anthocyanin retention, antioxidant capacity, and antiadipogenic activity.

Fresh and dried raspberries prepared by freeze drying (FD), microwave-vacuum (MIVAC), hot-air drying (HAD), and a combination of hot-air drying and microwave-vacuum (HAD/MIVAC) drying methods were evaluated for polyphenol retention, total polyphenol and anthocyanin contents, total antioxidant capacity, and antiadipogenic activity (the inhibition of fat cell development). Ellagic acid and quercetin were present in the largest concentrations in fresh and dehydrated raspberries. Dehydration led to a loss of polyphenols and anthocyanins and antioxidant capacity. Polyphenols (aglycone form) were retained in the greatest amount: 20% (freeze dried) to 30% (HAD/MIVAC) (fresh = 100%). A total of 30% of polyphenols (glycoside form) were retained in raspberries dried by the HAD/MIVAC methods with 5% of retention observed for raspberries dried by FD, HAD, or MIVAC. FD and MIVAC resulted in higher retention of anthocyanins (aglycone form) than other drying methods. It was also observed that antioxidant activity was reduced by dehydration. Adipogenesis was inhibited by polyphenolic glycosides (30%) and aglycones (30% to 40%) in fresh and HAD/MIVAC raspberries. Extracts from dried raspberries by HAD/MIVAC methods were relatively more effective at inhibiting adipogenesis compared to HAD and FD dried raspberries.

Enhancing absorption of fluorescein and LHRH across hindgut epithelia in a marsupial, the common brushtail possum Trichosurus vulpecula.

We have identified differences in transport properties of intestinal epithelia in the marsupial brushtail possum, compared to eutherian mammals. To determine whether differences in its permeability to hydrophilic compounds also occur, the absorption of sodium fluorescein and luteinizing hormone releasing hormone (LHRH) was assessed in vitro and the ability of chemical enhancers and a metabolic inhibitor to promote their absorption investigated. The apparent permeability of colonic and caecal tissues to fluorescein and LHRH and transepithelial resistance (Rt) in the absence or presence of ethylenediamine tetra-acetic acid (EDTA), sodium deoxycholic acid (SDA), dithiothreitol (DTT), polyacrylic acids (PAA), or the inhibitor bacitracin were determined. The effects of SDA and/or DTT on adherent mucus and the release of lactate dehydrogenase (LDH) were also assessed. In the absence of treatment, both tissues had comparable amounts of adherent mucus, Rt and low permeabilities to fluorescein and LHRH. All chemical enhancers increased fluorescein permeability, but SDA at concentrations >0.5 mM also induced LDH release. DTT alone and in combination with SDA reduced the amount of adherent mucus. Bacitracin inhibited LHRH metabolism and increased LHRH permeability. These data indicate that the possum hindgut epithelium represents a significant barrier to the uptake of hydrophilic compounds, similar to that in eutherians.

W/O microemulsions for ocular delivery: evaluation of ocular irritation and precorneal retention.

Water-in-oil microemulsions (w/o ME) capable of undergoing a phase-transition to lamellar liquid crystals (LC) or bicontinuous ME upon aqueous dilution were formulated using Crodamol EO, Crill 1 and Crillet 4, an alkanol or alkanediol as cosurfactant and water. The hypothesis that phase-transition of ME to LC may be induced by tears and serve to prolong precorneal retention was tested. The ocular irritation potential of components and formulations was assessed using a modified hen's egg chorioallantoic membrane test (HET-CAM) and the preocular retention of selected formulations was investigated in rabbit eye using gamma scintigraphy. Results showed that Crill 1, Crillet 4 and Crodamol EO were non-irritant. However, all other cosurfactants investigated were irritant and their irritation was dependent on their carbon chain length. A w/o ME formulated without cosurfactant showed a protective effect when a strong irritant (0.1 M NaOH) was used as the aqueous phase. Precorneal clearance studies revealed that the retention of colloidal and coarse dispersed systems was significantly greater than an aqueous solution with no significant difference between ME systems (containing 5% and 10% water) as well as o/w emulsion containing 85% water. Conversely, a LC system formulated without cosurfactant displayed a significantly greater retention compared to other formulations.

Preparation of immuno-stimulating complexes (ISCOMs) by ether injection.

This study investigated the application of the solvent dispersion technique, specifically ether injection, which has been successfully used in the preparation of liposomes, as a new, continuous and potentially scaleable method for the preparation of ISCOMs. Phosphatidylcholine (PC) and cholesterol (Chol) were dissolved in ether, which was injected into an aqueous solution, maintained at 55 degrees C, containing Quil A. The influences of the following variables on ISCOM formation were investigated: ratio of PC:Quil A:Chol used, pumping rate, total lipid mass and concentration of buffer salts and Quil A in the aqueous phase. All samples were characterized by negative stain transmission electron microscopy, photon correlation spectroscopy and sucrose ultracentrifugation gradient. It was demonstrated that ISCOMs could be produced by this method but the homogeneity of the preparation was influenced by the conditions used. Homogeneous ISCOM preparations were consistently produced only when the weight ratio of PC:Quil A:Chol was 5:3:2 with a total lipid mass of 20 mg, the Quil A dissolved in a 0.01 M phosphate buffer at a concentration of 6 mg in 4 ml, and the ether solution injected into the warmed buffer solution at a rate of 0.2 ml/min. Changing any of these variables resulted in more heterogeneous preparations in which ISCOMs typically co-existed with other colloidal structures such as worm-like and helical micelles, liposomes, lamellae and lipidic particles.

Gastrointestinal transit in the common brushtail possum measured by gamma scintigraphy.

This paper reports an example of the application of pharmaceutical technology to wildlife management, specifically the design of an oral delivery system for the common brushtail possum in New Zealand. Designing an oral delivery system requires a knowledge of the time taken for particulates to reach target sites within the gastrointestinal tract (GIT). The transit time for fluid and indigestible particles of two different size ranges was determined in the common brushtail possum (Trichosurus vulpecula). Technetium-labelled (99mTc) anion exchange resin particles (75-125 or 500-700 microm diameter) or solution (99mTc-labelled diethylenetriamine pentaacetic acid, 99mTc-DTPA) was administered orally. At predetermined times after dosing (3, 6, 12, 24 or 32 h), the distribution of radioactivity throughout excised gastrointestinal tracts was determined by gamma scintigraphy. The transit profile was similar for the three formulations investigated. Unlike other closely related hindgut fermenting marsupials, there was no evidence to support the presence of a colonic separating mechanism in the common brushtail possum. Gastrointestinal transit was independent of body mass, gender and time of day that the dose is given. To target the hindgut for oral delivery of protein and peptide biocontrol agents, the formulation would need to protect the bioactive for approximately 12 h prior to release.

Using different structure types of microemulsions for the preparation of poly(alkylcyanoacrylate) nanoparticles by interfacial polymerization.

A phase diagram of the pseudoternary system ethyloleate, polyoxyethylene 20 sorbitan mono-oleate/sorbitan monolaurate and water with butanol as a cosurfactant was prepared. Areas containing optically isotropic, low viscosity one-phase systems were identified and systems therein designated as w/o droplet-, bicontinuous- or solution-type microemulsions using conductivity, viscosity, cryo-field emission scanning electron microscopy and self-diffusion NMR. Nanoparticles were prepared by interfacial polymerization of selected w/o droplet, bicontinuous- or solution-type microemulsions with ethyl-2-cyanoacrylate. Morphology of the particles and entrapment of the water-soluble model protein ovalbumin were investigated. Addition of monomer to the different types of microemulsions (w/o droplet, bicontinuous, solution) led to the formation of nanoparticles, which were similar in size ( approximately 250 nm), polydispersity index ( approximately 0.13), zeta-potential ( approximately -17 mV) and morphology. The entrapment of the protein within these particles was up to 95%, depending on the amount of monomer used for polymerization and the type of microemulsion used as a polymerization template. The formation of particles with similar characteristics from templates having different microstructure is surprising, particularly considering that polymerization is expected to occur at the water-oil interface by base-catalysed polymerization. Dynamics within the template (stirring, viscosity) or indeed interfacial phenomena relating to the solid-liquid interface appear to be more important for the determination of nanoparticle morphology and characteristics than the microstructure of the template system.

Fusing subunit antigens to interleukin-2 and encapsulating them in liposomes improves their antigenicity but not their protective efficacy.

Subunit vaccines commonly lack sufficient immunogenicity to stimulate a comprehensive protective immune response in vivo. We have investigated the potential of specific cytokines (interleukin-2) and particulate delivery systems (liposomes) to enhance antigenicity. Here we report that the IgG1 and IFN-gamma responses to a subunit antigen, consisting of a T and B-cell epitope from Influenza haemagglutinin, can be improved when it is both fused to interelukin-2 and encapsulated in liposomes. However, this vaccine formulation was not able to protect animals against a challenge with live Influenza A/PR/8/34 virus. The addition of more potent immune stimulators may be necessary to improve responses.

A comparison of pseudo-ternary diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by lipid-film hydration and dialysis.

Pseudo-ternary diagrams for Quil A, phospholipid (phosphatidylcholine (PC) or phosphatidylethanolamine (PE)) and cholesterol were established in order to identify combinations that result in the formation of immune-stimulating complex (ISCOM) matrices and other colloidal structures produced by these three components in aqueous systems following lipid-film hydration or dialysis (methods that can be used to produce ISCOMs). In addition, the effect of equilibration time (1 month at 4 degrees C) on the structures formed by the various combinations of the three components was investigated. Depending on the ratio of Quil A, cholesterol and phospholipid, different colloidal particles, including ISCOM matrices, liposomes and ring-like micelles, were found irrespective of the preparation method used. In contrast, worm-like micelles were only observed in systems prepared by lipid-film hydration. For samples prepared by dialysis, ISCOM matrices were predominantly found near the Quil A apex of the pseudo-ternary diagram (> 50% Quil A). On the other hand, for samples prepared by lipid-film hydration, ISCOM matrices were predominantly found near the phospholipid apex of the pseudo-ternary diagram (> 50% phospholipid). The regions in the pseudo-ternary diagrams in which ISCOM matrices were observed increased following an extended equilibration time, particularly for samples prepared by lipid-film hydration. Differences were also observed between pseudoternary diagrams prepared using either PE or PC as phospholipids.

Uptake of antigen encapsulated in polyethylcyanoacrylate nanoparticles by D1-dendritic cells.

Polyethylcyanoacrylate (PECA) nanoparticles were prepared by interfacial polymerization of a water-in-oil microemulsion. Nanoparticles were isolated from the polymerization template by sequential ethanol washing and centrifugation. A nanocapsule preparation yielding the original particle size and distribution following redispersion in an aqueous solution was achieved by freeze-drying the isolated nanoparticles in a solution of 5% w/v sugar. The cytotoxicity and uptake of nanocapsules by dendritic cells was investigated using a murine-derived cell line (D1). PECA nanoparticles were found to adversely effect cell viability at concentrations greater than 10 microg/ml of polymer in the culture medium. In comparison to antigen in solution, cell uptake of antigen encapsulated within nanoparticles was significantly higher at both 4 and 37 degrees C. Following a 24 h incubation period, the percentage of cells taking-up antigen was also increased when antigen was encapsulated in nanoparticles as compared to antigen in solution. The uptake of nanoparticles and the effect of antigen formulation on morphological cell changes indicative of cell maturation were also investigated by scanning electron microscopy (SEM). SEM clearly demonstrated the adherence of nanoparticles to the cell surface. Incubation of D1 dendritic cells with nanoparticles containing antigen also resulted in morphological changes indicative of cell maturation similar to that observed when the cells were incubated with lipopolysaccharide. In contrast, cells incubated with antigen solution did not demonstrate such morphological changes and appeared similar to immature cells that had not been exposed to antigen.

Influence of single versus multiple actuations on the particle size distribution of beclometasone dipropionate metered-dose inhalers.

The particle size distributions of beclometasone dipropionate delivered from Becotide and Respocort inhalers after single and multiple actuations were investigated using the Andersen Mark II Cascade impactor and the drug was quantified using high performance liquid chromatography. The fine particle mass and the mass median aerodynamic diameter were calculated. An apparent increase in mass median aerodynamic diameter was observed when the number of actuations increased. In addition, the fine particle mass decreased as the number of actuations increased. When performing and analysing cascade impaction study data differences between single versus multiple actuations must be considered. Regulatory guidelines should be amended to stipulate the number of actuations to be loaded into devices used to evaluate the particle size distribution of inhaled aerosol products.

Kinetics of metabolism and degradation of mometasone furoate in rat biological fluids and tissues.

Mometasone furoate (MF) is a potent glucocorticoid developed for the treatment of glucocorticoid-responsive inflammatory disorders. The in-vitro and ex-vivo kinetics of the degradation and metabolism of MF were studied in selected biological fluids of rat and subcellular fractions of different rat tissues. In-vitro, MF was found to degrade slowly into four products in serum and urine, and metabolized rapidly and extensively in rat liver, minimally in extrahepatic tissues, including intestine, stomach, lung and kidney. Further investigation found that the microsomal fraction was the major intracellular site of MF 6 beta-hydroxylation in rat liver. Using chemical inhibitors, CYP3A was found to be the major enzyme involved in the in-vitro MF 6 beta-hydroxylation in rat liver microsomes. Enzyme kinetic studies in rat liver microsomes showed that the overall metabolic process of MF followed biphasic Michaelis-Menten kinetics, while 6 beta-hydroxylation obeyed monophasic Michaelis-Menten kinetics. The kinetic parameters derived from the kinetic models along with the enzyme inhibition studies suggest that MF is mainly metabolized via 6 beta-hydroxylation mediated by CYP3A primarily, and also biotransformed via other pathway(s) catalysed by other enzymes in rat liver in-vitro.

Inhibition of proteolysis in luminal extracts from the intestine of the brushtail possum.

The proteolytic activity of luminal extracts from five regions (duodenum, jejunum, ileum, caecum and colon) of the brushtail possum intestine towards bovine serum albumin (BSA) and human luteinizing hormone releasing hormone (LHRH) was investigated. There were no significant differences in degradation rates between fresh and previously frozen extracts from any region of the possum intestine. The inhibition of degradation of BSA by luminal extracts from two regions (jejunum and ileum) and of LHRH from four regions (jejunum, ileum, caecum and colon) was evaluated. Soybean trypsin-chymotrypsin inhibitor (SBTI), sodium deoxycholate, Carbopol 934P, bacitracin and bestatin significantly inhibited the degradation of both LHRH and BSA (P < 0.05). SBTI almost totally inhibited the proteolysis of BSA and the peptidolysis of LHRH in extracts from the small intestine. This finding suggests that serine proteases such as chymotrypsin are responsible for the protein and peptide degradation in luminal extracts. It is concluded that including serine protease inhibitors in a formulation may enhance oral delivery of bioactive peptides and proteins to possums.

Enzymatic degradation of luteinizing hormone releasing hormone (LHRH) by mucosal homogenates from the intestine of the common brushtail possum (Trichosurus vulpecula).

The peptidolytic activity of fresh and frozen mucosal homogenates from five regions (duodenum, jejunum, ileum, caecum and colon) of possum intestine from Trichosurus vulpecula towards human Luteinizing Hormone Releasing Hormone (LHRH) was investigated. The rank of order of specific peptidolytic activity of the mucosal homogenates was jejunum > ileum > caecum> duodenum = colon, with a 3 to 4 fold difference between the least and the most active segment in both frozen and fresh samples. The formation of peptides LHRH (1-3), LHRH (1-4) and LHRH (1-5) suggest endopepetidase-24.18, endopeptidase-24.15 and angiotensin converting enzyme (ACE) might be responsible for the peptide degradation in mucosal homogenates. The inhibition of LHRH degradation by mucosal homogenates was evaluated in four regions (jejunum, ileum, caecum and colon) of possum intestine. Ethylenediaminetetraacetic acid (EDTA, 5 mM), sodium deoxycholate (SDA, 10 mM) and bacitracin (3.5 or 9 mM) inhibited the degradation of LHRH in mucosal homogenates from small intestine and hindgut. However, the serine protease inhibitor, soybean trypsin-chymotrypsin inhibitor (SBTI), did not prevent degradation of LHRH. It is concluded that combining peptides with inhibitors may enhance oral delivery of bioactive peptides or proteins to possums.