Plants Fagonia cretica L. and Hedera nepalensis K. Koch contain natural compounds with potent dipeptidyl peptidase-4 (DPP-4) inhibitory activity

Ethnopharmacological relevance
The two plants investigated here (Fagonia cretica L. and Hedera nepalensis K. Koch) have been previously reported as natural folk medicines for the treatment of diabetes but until now no scientific investigation of potential anti-diabetic effects has been reported. 

Materials and methods
In vitro inhibitory effect of the two tested plants and their five isolated compounds on the dipeptidyl peptidase 4 (DPP-4) was studied for the assessment of anti-diabetic activity. 

Results
A crude extract of Fagonia cretica possessed good inhibitory activity (IC₅₀value: 38.1 μg/ml) which was also present in its n-hexane (FCN), ethyl acetate (FCE) or aqueous (FCA) fractions. A crude extract of Hedera nepalensis (HNC) possessed even higher inhibitory activity (IC₅₀value: 17.2 μg/ml) and this activity was largely retained when further fractionated in either ethyl acetate (HNE; IC₅₀: 34.4 μg/ml) or n-hexane (HNN; 34.2 μg/ml). Bioactivity guided isolation led to the identification of four known compounds (isolated for the first time) from Fagonia cretica: quinovic acid (1), quinovic acid-3β-O-β-d-glycopyranoside (2), quinovic acid-3β-O-β-d-glucopyranosyl-(28→1)-β-d-glucopyranosyl ester (3), and stigmasterol (4) all of which inhibited DPP-4 activity (IC₅₀: 30.7, 57.9, 23.5 and >100 μM, respectively). The fifth DPP-4 inhibitor, the triterpenoid lupeol (5) was identified in Hedera nepalensis (IC₅₀: 31.6 μM). 

Conclusion
The experimental study revealed that Fagonia cretica and Hedera nepalensis contain compounds with significant DPP-4 inhibitory activity which should be further investigated for their anti-diabetic potential.

Proteomic identification of plasma proteins as markers of growth promoter abuse in cattle

Growth-promoting agents are continually misused for increasing animal growth and fraudulent gain in the meat industry, yet detection rates from conventional targeted testing for drug residues do not reflect this. This is because testing currently relies on direct detection of drugs or related metabolites and administrators of such compounds can take adaptive measures to avoid detection through the use of endogenous or unknown drugs, and low dose or combined mixtures. New detection methods are needed which focus on the screening of biological responses of an animal to such growth-promoting agents as it has been demonstrated that genomic, proteomic and metabolomics profiles are altered by xenobiotic intake. Therefore, an untargeted proteomics approach using comparative two-dimensional gel electrophoresis (2DE) was carried out to identify putative proteins altered in plasma after treatment with oestradiol, dexamethasone or prednisolone. Twenty-four male cattle were randomly assigned to four groups (n = 6) for experimental treatment over 40 days, namely a control group of non-treated cattle, and three groups administered 17β-oestradiol-3-benzoate (0.01 mg/kg, intramuscular), dexamethasone sodium phosphate (0.7 mg/day, per os) or prednisolone acetate (15 mg/day, per os), respectively. Plasma collected from each animal at day 25 post study initiation was subjected to proteomic analysis by 2DE for comparison of protein expression between treated and untreated animals. Analysis of acquired gel images revealed 22 plasma proteins which differed in expression by more than 50 % (p < 0.05) in treated animals compared to untreated animals. Proteins of interest underwent identification by LC–MS/MS analysis and were found to have associated roles in transport, blood coagulation, immune response and metabolism pathways. In this way, seven proteins are highlighted as novel biomarker candidates including transthyretin which is shown to be significantly increased in all treatment groups compared to control animals and potentially may find use as global markers of suspect anabolic practice.

Nucleoside Azide-Alkyne Cycloaddition Reactions Under Solvothermal Conditions or Using Copper Vials in a Ball Mill

Novel nucleoside analogues containing photoswitchable moieties were prepared using 'click' cycloaddition reactions between 5 '-azido-5 '-deoxythymidine and mono- or bis-N-propargylamide-substituted azobenzenes. In solution, high to quantitative yields were achieved using 5mol% Cu(I) in the presence of a stabilizing ligand. 'Click' reactions using the monopropargylamides were also effected in the absence of added cuprous salts by the application of liquid assisted grinding (LAG) in metallic copper reaction vials. Specifically, high speed vibration ball milling (HSVBM) using a 3/32('') (2.38mm) diameter copper ball (62mg) at 60Hz overnight in the presence of ethyl acetate lead to complete consumption of the 5 '-azido nucleoside with clean conversion to the corresponding 1,3-triazole.

Process for the preparation of heterocyclyl chalcogenones via reaction of elemental sulfur, selenium, or tellurium with heterocyclic cationic species.

Heterocyclic chalcogenones were prepd. by reaction of S, Se, or Te with ionic liqs. or salts [I; Ra = (substituted) alkyl, cycloalkyl, aryl, aralkyl, alkylaryl; Q = (unsatd.) (substituted) linker to form a ring of 5-10 members; X- = anion selected from conjugate bases of HX having a pKa value of >2.5]. Thus, 1-butyl-3-methylimidazolium acetate was heated with stoichiometric S at 75° for 48 h to give 1-butyl-3-methylimidazole-2-thione. [on SciFinder(R)]

Which biomarkers are predictive specifically for cardiovascular or for non-cardiovascular mortality in men? Evidence from the Caerphilly Prospective Study (CaPS)

OBJECTIVE: To examine a panel of 28 biomarkers for prediction of cardiovascular disease (CVD) and non-CVD mortality in a population-based cohort of men.

METHODS: Starting in 1979, middle-aged men in Caerphilly underwent detailed medical examination. Subsequently 2171 men were re-examined during 1989-1993, and fasting blood samples obtained from 1911 men (88%). Fibrinogen, viscosity and white cell count (WCC), routine biochemistry tests and lipids were analysed using fresh samples. Stored aliquots were later analysed for novel biomarkers. Statistical analysis of CVD and non-CVD mortality follow-up used competing risk Cox regression models with biomarkers in thirds tested at the 1% significance level after covariate adjustment.

RESULTS: During an average of 15.4years follow-up, troponin (subhazard ratio per third 1.71, 95% CI 1.46-1.99) and B-natriuretic peptide (BNP) (subhazard ratio per third 1.54, 95% CI 1.34-1.78) showed strong trends with CVD death but not with non-CVD death. WCC and fibrinogen showed similar weaker findings. Plasma viscosity, growth differentiation factor 15 (GDF-15) and interleukin-6 (IL-6) were associated positively with both CVD death and non-CVD death while total cholesterol was associated positively with CVD death but negatively with non-CVD death. C-reactive protein (C-RP), alkaline phosphatase, gamma-glutamyltransferase (GGT), retinol binding protein 4 (RBP-4) and vitamin B6 were significantly associated only with non-CVD death, the last two negatively. Troponin, BNP and IL-6 showed evidence of diminishing associations with CVD mortality through follow-up.

CONCLUSION: Biomarkers for cardiac necrosis were strong, specific predictors of CVD mortality while many inflammatory markers were equally predictive of non-CVD mortality.

Comparative Study of Different Methods for the Prediction of Drug− Polymer Solubility

In this study, a comparison of different methods to predict drug−polymer solubility was carried out on binary systems consisting of five model drugs (paracetamol, chloramphenicol, celecoxib, indomethacin, and felodipine) and polyvinylpyrrolidone/vinyl acetate copolymers (PVP/VA) of different monomer weight ratios. The drug−polymer solubility at 25 °C was predicted using the Flory−Huggins model, from data obtained at elevated temperature using thermal analysis methods based on the recrystallization of a supersaturated amorphous solid dispersion and two variations of the melting point depression method. These predictions were compared with the solubility in the low molecular weight liquid analogues of the PVP/VA copolymer (N-vinylpyrrolidone and vinyl acetate). The predicted solubilities at 25 °C varied considerably depending on the method used. However, the three thermal analysis methods ranked the predicted solubilities in the same order, except for the felodipine−PVP system. Furthermore, the magnitude of the predicted solubilities from the recrystallization method and melting point depression method correlated well with the estimates based on the solubility in the liquid analogues, which suggests that this method can be used as an initial screening tool if a liquid analogue is available. The learnings of this important comparative study provided general guidance for the selection of the most suitable method(s) for the screening of drug−polymer solubility.

Probing The effects of Experimental Conditions on the Character of Drug-Polymer Phase Diagrams Constructed Using Flory-Huggins Theory

Purpose: Amorphous drug-polymer solid dispersions have been found to result in improved drug dissolution rates when compared to their crystalline counterparts. However, when the drug exists in the amorphous form it will possess a higher Gibb’s free energy than its associated crystalline state and can recrystallize. Drug-polymer phase diagrams constructed through the application of the Flory Huggins (F-H) theory contain a wealth of information regarding thermodynamic and kinetic stability of the amorphous drug-polymer system. This study was aimed to evaluate the effects of various experimental conditions on the solubility and miscibility detections of drug-polymer binary system. Methods: Felodipine (FD)-Polyvinylpyrrolidone (PVP) K15 (PVPK15) and FD-Polyvinylpyrrolidone/vinyl acetate (PVP/VA64) were the selected systems for this research. Physical mixtures with different drug loadings were mixed and ball milled. These samples were then processed using Differential Scanning Calorimetry (DSC) and measurements of melting point (Tend) and glass transition (Tg) were detected using heating rates of 0.5, 1.0 and 5.0°C/min. Results: The melting point depression data was then used to calculate the F-H interaction parameter (χ) and extrapolated to lower temperatures to complete the liquid–solid transition curves. The theoretical binodal and spinodal curves were also constructed which were used to identify regions within the phase diagram. The effects of polymer selection, DSC heating rate, time above parent polymer Tg and polymer molecular weight were investigated by identifying amorphous drug miscibility limits at pharmaceutically relevant temperatures. Conclusion: The potential implications of these findings when applied to a non-ambient processing method such as Hot Melt Extrusion (HME) are also discussed.

The emergence of DNA methylation as a key modulator of aberrant cell death in prostate cancer

It is now well established that cancer cells exhibit a number of genetic defects in the machinery that governs programmed cell death and that sabotage of apoptosis is one of the principal factors aiding in the evolution of the carcinogenic phenotype. A number of studies have implicated aberrant DNA methylation as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many processes including apoptosis. To date, studies on the methylation profile of apoptotic genes have largely focused on cancers of the breast, colon and stomach, with only limited data available on prostate cancer. Here we discuss the major developments in the field of DNA methylation and its role in the regulation of aberrant apoptosis in prostate cancer. The most significant advances have involved the discovery of apoptotic gene targets of methylation, including XAF1, (fragile histidine triad (FHIT ), cellular retinol binding protein 1 (CRBP1), decoy receptor 1(DCR1), decoy receptor 2 (DCR2 ), target of methylation-induced silenceing 1 (TMS1), TNF receptor superfamily, member 6 (FAS), Reprimo (RPRM) and GLI pathogenesis-related 1 (GLIPR1). These genes are reported to be hypermethylated in prostate cancer and some offer potential as diagnostic and prognostic markers. We also introduce the concept of an 'apoptotic methylation signature' for prostate cancer and evaluate its potential in a diagnostic, prognostic and therapeutic setting.

Naturally-occurring TGR5 agonists modulating glucagon-like peptide-1 biosynthesis and secretion

Selective GLP-1 secretagogues represent a novel potential therapy for type 2 diabetes mellitus. This study examined the GLP-1 secretory activity of the ethnomedicinal plant, Fagonia cretica, which is postulated to possess anti-diabetic activity. After extraction and fractionation extracts and purified compounds were tested for GLP-1 and GIP secretory activity in STC-1 pGIP/neo cells. Intracellular levels of incretin hormones and their gene expression were also determined. Crude F. cretica extracts stimulated both GLP-1 and GIP secretion, increased cellular hormone content, and upregulated gene expression of proglucagon, GIP and prohormone convertase. However, ethyl acetate partitioning significantly enriched GLP-1 secretory activity and this fraction underwent bioactivity-guided fractionation. Three isolated compounds were potent and selective GLP-1 secretagogues: quinovic acid (QA) and two QA derivatives, QA-3β-O-β-D-glycopyranoside and QA-3β-O-β-D-glucopyranosyl-(28→1)-β-D-glucopyranosyl ester. All QA compounds activated the TGR5 receptor and increased intracellular incretin levels and gene expression. QA derivatives were more potent GLP-1 secretagogues than QA. This is the first time that QA and its naturally-occurring derivatives have been shown to activate TGR5 and stimulate GLP-1 secretion. These data provide a plausible mechanism for the ethnomedicinal use of F. cretica and may assist in the ongoing development of selective GLP-1 agonists.

Pupillary and visual thresholds in young children as an index of population vitamin A status

A prototype scotopic sensitivity machine was used to evaluate pupillary and visual thresholds for 295 Indonesian children aged 1-5 y, most of whom were initially vitamin A-deficient. Subjects were tested 6 and 9 mo after receiving a high dose of vitamin A. A group of 136 older children was tested at 6 mo after dosing; all subjects underwent testing at 9 mo. After testing at 9 mo, children randomly received either a second high dose of vitamin A or placebo and were tested a final time 2 wk later. Children with abnormal pupillary thresholds had significantly higher relative dose responses (RDRs) (P < 0.01) and significantly lower serum retinol values (P = 0.05) than did normal children. The mean pupillary threshold rose (eg, retinal sensitivity fell) as vitamin A status deteriorated between 6 and 9 mo after initial dosing, and was significantly different from a group of normal American children tested previously (P < 0.001). After placebo-controlled dosing, the decline in pupillary and visual thresholds (rise in retinal sensitivity) was significant for children receiving vitamin A but not for children receiving placebo.

Pupillary threshold as an index of population vitamin A status among children in India

Two hundred seven vitamin A-deficient southern Indian children aged 1-7 y (mean age: 56.9 mo) underwent testing of dark-adapted visual and pupillary thresholds in their village setting according to a previously reported protocol. One hundred thirty (62.8%) of the children also underwent serum retinol testing, and 178 (86.0%) participated in a randomized, placebo-controlled vitamin A dosing trial with pre- and postdose testing of dark-adaptation threshold. Most subjects (184 of 207, 88.9%) were able to complete pupillary testing, an objective sign requiring minimal cooperation, including a high proportion of the youngest children (72.2% of subjects aged 2 y). The proportion of children completing visual threshold testing, which requires greater understanding and cooperation, was significantly smaller than that able to complete pupillary testing (131 of 207, 63.3%; P < 0.0001, chi square). At baseline (predosing), the mean serum retinol concentration declined in linear fashion with a higher pupillary threshold (0.73 mumol/L with a score < or = 4; 0.47 mumol/L with a score > or = 8; P < 0.01). The mean pupillary threshold for these highly vitamin A-deficient Indian children (-0.622 log cd/m2) was significantly higher than that for 136 more moderately deficient Indonesian children (-0.985 log cd/m2; P < 0.001, two-sample t test) and 56 normal American children (-1.335 log cd/m2; P < 0.0001, two-sample t test). The improvement in pupillary dark-adaptation testing was not significant for children receiving vitamin A or placebo, though there was a nonsignificant trend toward greater improvement in children receiving vitamin A (P = 0.2, two-sample t test). Pupillary threshold testing represents a new, noninvasive, practical, and seemingly valid approach to assessing the vitamin A status of a moderately to severely deficient preschool population.

Responsiveness of dark-adaptation threshold to vitamin A and beta-carotene supplementation in pregnant and lactating women in Nepal.

BACKGROUND: Impaired dark adaptation occurs commonly in vitamin A deficiency. OBJECTIVE: We sought to examine the responsiveness of dark-adaptation threshold to vitamin A and beta-carotene supplementation in Nepali women. DESIGN: The dark-adapted pupillary response was tested in 298 pregnant women aged 15-45 y in a placebo-controlled trial of vitamin A and beta-carotene; 131 of these women were also tested at 3 mo postpartum. Results were compared with those for 100 nonpregnant US women of similar age. The amount of light required for pupillary constriction was recorded after bleaching and dark adaptation. RESULTS: Pregnant women receiving vitamin A had better dark-adaptation thresholds (-1.24 log cd/m(2)) than did those receiving placebo (-1.11 log cd/m(2); P: = 0. 03) or beta-carotene (-1.13 log cd/m(2); P: = 0.05) (t tests with Bonferroni correction). Dark-adaptation threshold was associated with serum retinol concentration in pregnant women receiving placebo (P: = 0.001) and in those receiving beta-carotene (P: = 0.003) but not in those receiving vitamin A. Among women receiving placebo, mean dark-adaptation thresholds were better during the first trimester (-1.23 log cd/m(2)) than during the second and third trimesters (-1.03 log cd/m(2); P: = 0.02, t test). The mean threshold of nonpregnant US women (-1.35 log cd/m(2)) was better than that of all 3 Nepali groups (P: < 0.001, t test, for all 3 groups). CONCLUSIONS: During pregnancy, pupillary dark adaptation was strongly associated with serum retinol concentration and improved significantly in response to vitamin A supplementation. This noninvasive testing technique is a valid indicator of population vitamin A status in women of reproductive age.

Physiologic indicators of vitamin A status.

Physiologic indicators reflect the functional consequences of vitamin A deficiency and may be particularly useful for detecting early perturbations in vitamin A status. In conjunctival impression cytology (CIC), epithelial morphology and the presence or absence of mucin spots and goblet cells allow samples, obtained by applying filter paper to the temporal conjunctiva, to be characterized as normal or typical of vitamin A-deficient keratinizing metaplasia. The validity of CIC has been established with reference to other indicators of vitamin A status, and a prevalence of > or =20% abnormal results has been suggested as indicative of a public health problem. However, interpretation of specimens requires considerable training, and nonresponsiveness to supplementation is a frequent problem, which limits the utility of CIC as a method for evaluating the impact of intervention programs. Several simplified field protocols for dark adaptation have been developed, including one in which dark adaptation is assessed by the responsiveness of the pupil to light. Night blind subjects have consistently shown abnormal results on this test, and a significant response to placebo-controlled dosing has been demonstrated for children and pregnant women. Scores have correlated significantly with serum retinol and relative dose response. Pupillary dark adaptation testing is acceptable to most children as young as 2 y old. Limitations of this technique include a time course for recovery after dosing as long as 4-6 wk, a testing time of 20 min, and the need for 1-3 d of training. Given its low cost, noninvasive nature, and lack of the need to transport samples, pupillary dark adaptation offers advantages over other techniques for assessing a population's vitamin A status.

Validation of night blindness reports among children and women in a vitamin A deficient population in rural Tanzania.

OBJECTIVE: This study validates different definitions of reported night blindness (XN) in a vitamin A deficient African population with no local term for XN. DESIGN: Case-control study with follow-up after treatment. SETTING: Eight primary schools and health centres in rural Tanzania. SUBJECTS: A total of 1214 participants were screened for reported XN and other eye signs of xerophthalmia: 461 children aged 24-71 months, 562 primary school-age children and 191 pregnant or breast-feeding women. All 152 cases of reported XN were selected for the validation study and group matched with 321 controls who did not complain of XN. XN reports were validated against serum retinol concentrations and pupillary dark adaptation measurements in cases and controls. INTERVENTION: All children and women who reported XN or had other signs of active xerophthalmia were treated with vitamin A and followed up 3-4 weeks later. Half of the untreated control group who had their serum retinol examined in the baseline examination were also followed up. RESULTS: The overall prevalence of reported XN was 12.5%. At baseline, mean pupillary threshold (-1.52 vs -1.55 log cd/m(2), P=0.501) and median serum retinol concentrations (0.95 vs 0.93 micromol/l, P=0.734) were not significantly different in cases and controls either overall or in each population group. More restricted case definitions reduced the prevalence of reported XN to 5.5% (P<0.001), but there was still no significant difference between cases and controls although the results were in the expected direction. After treatment, the median serum retinol concentration improved significantly only in the most deficient group, the young children. Dark adaptation improved in all the subgroups but the difference was only significant for young children and primary school-age children when the restricted case definitions were used. CONCLUSIONS: XN reports are a poor indicator of vitamin A deficiency in this population. SPONSORSHIP: Task Force Sight and Life, Basel, Switzerland.

Sustained release of the candidate antiretroviral peptides T-1249 and JNJ54310516-AFP from a rod insert vaginal ring

Administration of biomacromolecular drugs in effective quantities from conventional vaginal rings is hampered by poor drug permeability in the polymers from which rings are commonly constructed. Here, we report the formulation development and testing of rod insert rings for sustained release of the candidate antiretroviral peptides T-1249 and JNJ54310516-AFP (JNJ peptide), both of which have potential as HIV microbicides. Rod inserts were prepared comprising antiviral peptides T-1249 or JNJ peptide in combination with a hydrophilic excipient (sodium chloride, sodium glutamate, lactose or zinc acetate) dispersed at different loadings within a medical grade silicone elastomer. The inserts were tested for weight change and swelling when immersed in simulated vaginal fluid (SVF). Dye migration into the inserts was also assessed visually over 28 days. In vitro release of T-1249 and JNJ peptide from rings containing various insert types was tested. Weight change and degree of swelling of rods immersed in SVF was dependent on the type and concentration of excipient present. The rods displayed the following rank order in terms of weight change: sodium glutamate > zinc acetate ≈ sodium chloride > lactose. The weight change and degree of swelling of the inserts did not correlate with the level of dye uptake observed. In vitro release of T-1249 was improved through addition of lactose, sodium chloride and sodium glutamate, while release of JNJ peptide was improved through addition of sodium chloride or sodium glutamate. Sustained release of hydrophobic peptides can be achieved using a rod insert ring design formulated to include a hydrophilic excipient. Release rates were dependent upon the type of excipient used. The degree of release improvement with different inserts partially reflects their ability to imbibe surrounding fluid and swell in aqueous environments.