An enantio-selective chromatographic stationary phase for S-ibuprofen prepared by stoichiometric molecular imprinting

Molecularly Imprinted Polymers (MIPs) against S-ibuprofen were synthesised using a tailor made functional monomer, 2-acrylamido-4-methylpyridine, following extensive pre-polymerisation studies of template-monomer complexation. An apparent association constant of 340 +/- 22 M-1 was calculated that was subsequently corrected to account for dimerisation of ibuprofen (K-dim = 320 +/- 95 M-1) resulting in an intrinsic association constant of 715 +/- 16 M-1, consistent with previously reported values. Using the synthesised imprinted polymer as a stationary phase, complete resolution of a racemic mixture of ibuprofen was achieved in predominantly aqueous mobile phases. An imprinting factor of 10 was observed, and was found to be in agreement with the difference in the average number of binding sites between MIP and blank polymers, calculated by staircase frontal chromatography. The imprinted polymers exhibited enhanced selectivity for the templated drug over structurally related NSAIDs. When applied as sorbents in solid-phase extraction of ibuprofen from commercial tablets, urine and blood serum samples, recoveries up to 92.2% were achieved. © The Royal Society of Chemistry 2012

Molecularly imprinted polymers for the extraction of imiquimod from biological samples using a template analogue strategy

Molecularly Imprinted Polymers (MIPs) against imiquimod, a highly potent immune response modifier used in the treatment of skin cancer, were synthesised using a template analogue strategy and were compared with imprints of the drug itself. An investigation of the complexation between the functional monomer and the template analogue revealed an association constant of 1,376 ± 122 M-1, significantly higher than previously reported values for similar systems. The binding characteristics of the synthesised imprinted polymers were evaluated and extremely strong binding for imiquimod was observed while imprinting factors as high as 17 were calculated. When applied as sorbents in solid-phase extraction of imiquimod from aqueous, urine and blood serum samples, clean extracts and recoveries up to 95% were achieved, and it is concluded that while imiquimod imprints exhibited higher capacity for the drug, template analogue imprints are more selective. The results obtained suggest potential applications of imiquimod imprints as sorbents in rapid extraction and monitoring of undesirable systemic release of the drug.

Pre-clinical development of a combination microbicide vaginal ring containing dapivirine and darunavir

Objectives: Combination microbicide vaginal rings may be more effective than single microbicide rings at reducing/preventing sexual transmission of HIV. Here, we report the preclinical development and macaque pharmacokinetics of matrix-type silicone elastomer vaginal rings containing dapivirine and darunavir.

Methods: Macaque rings containing 25 mg dapivirine, 300 mg darunavir and 100 mg dapivirine, and 300 mg darunavir were manufactured and characterised by differential scanning calorimetry. In vitro release was assessed into isopropanol/water and simulated vaginal fluid. Macaque vaginal fluid and blood serum concentrations for both antiretrovirals were measured during 28-day ring use. Tissue levels were measured on day 28. Ex vivo challenge studies were performed on vaginal fluid samples and IC50 values calculated.

Results: Darunavir caused a concentration-dependent reduction in the dapivirine melting temperature in both solid drug mixes and in the combination ring. In vitro release from rings was dependent on drug loading, the number of drugs present, and the release medium. In macaques, serum concentrations of both microbicides were maintained between 101–102 pg/mL. Vaginal fluid levels ranged between 103–104 ng/g and 104–105 ng/g for dapivirine and darunavir, respectively. Tissue concentrations ranges for each drug were: vagina (1.8×103–3.8×103 ng/g) > cervix (9.4×101–3.9×102 ng/g) > uterus (0–108 ng/g) > rectum (0–40 ng/g). Measured IC50 values were > 2 ng/mL for both compounds.

Conclusions: Based on these results, and in light of recent clinical progress of the 25mg dapivirine ring, a combination vaginal ring containing dapivirine and darunavir is a viable second-generation HIV microbicide candidate.

A Combination Vaginal Ring Releasing Dapivirine and Darunavir

Background: Combination microbicide vaginal rings, containing two or more antiretrovirals targeting different steps in the HIV replicative process, may be more effective than single microbicide products at preventing sexual transmission of HIV. Here, we report the preclinical development, including in vitro release and macaque pharmacokinetics, of matrix-type silicone elastomer rings containing dapivirine (DPV; an experimental non-nucleoside reverse transcriptase inhibitor) and darunavir (DRV; a marketed protease inhibitor). Methods: Macaque rings containing 25 mg DPV, 300 mg DRV and 100 mg DPV, and 300 mg DRV were manufactured and characterised by differential scanning calorimetry. In vitro release was assessed into isopropanol/water and simulated vaginal fluid. Macaque vaginal fluid and blood serum concentrations for both antiretrovirals were measured during 28-day ring use. Tissue levels were measured on day 28. Ex vivo challenge studies were performed on vaginal fluid samples and IC50 values calculated.
Results: DRV caused a concentration-dependent reduction in the DPV melting temperature in both solid drug mixes and in the combination ring. In vitro release from rings was dependent on drug loading, the number of drugs present, and the release medium. In macaques, serum concentrations of both
microbicides were maintained between 101-102 pg/mL. Vaginal fluid levels
ranged between 103-104 ng/g and 104-105 ng/g for DPV and DRV, respectively. Vaginal tissue concentrations decreased in rank order: vagina
(1.8×103-3.8×103 ng/g) > cervix (9.4×101-3.9×102 ng/g) > uterus (0-108 ng/g) > rectum (0-40 ng/g). Measured IC50 values (HIV-1 BaL) determined from macaque vaginal fluid samples were < 2 ng/mL for both compounds. Conclusions: Based on these results, and in light of the ongoing clinical progress of the 25mg DPV ring, a combination vaginal ring containing DPV and DRV is a viable second-generation HIV microbicide candidate.

Tetrabutylammonium methacrylate as a novel receptor for selective extraction of sulphonylurea drugs from biological fluids using molecular imprinting

Glibenclamide (GLIB), an oral antidiabetic medication of the sulphonylurea drugs family, was stoichiometrically imprinted using tetrabutylammonium methacrylate as the functional monomer, for the first time in molecular imprinting, and utilising the sulphonylurea affinity for carboxylate anions. Solution association between the drug and the novel functional monomer was studied by 1H-NMR titrations, whereby evidence of sulphonylurea deprotonation followed by the formation of “narcissistic” GLIB dimers was found when tested in CDCl3, while an affinity constant in excess of 105 L mol-1 was measured in DMSO-d6. Detailed analysis of GLIB binding on the subsequently prepared imprinted and non-imprinted polymers confirmed deactivation of binding sites by exchange of a proton between GLIB and methacrylate, followed by extraction of the tetrabutylammonium counterion from the polymer matrix, resulting in overall reduced binding capacities and affinities by the imprinted material under equilibrium conditions. An optimised MI-SPE protocol, which included a binding site re-activation step, was developed for the extraction of GLIB from blood serum, whereby recoveries of up to 92.4% were obtained with exceptional sample clean-up.

Leptin concentrations in maternal serum and cord blood in diabetic and nondiabetic pregnancy

OBJECTIVE: The purpose of this study was to examine the relationships between maternal and cord leptin concentrations, maternal and neonatal outcomes, and measures of glycemic control in diabetic and nondiabetic pregnancy.

Serum eosinophilic cationic protein and blood eosinophil counts for the prediction of the presence of airways inflammation in children with wheezing

Background Serum eosinophilic cationic protein (ECP) concentrations may be useful noninvasive markers of airways inflammation in atopic asthma. However, the usefulness of serum ECP measurement for the prediction of airways inflammation in children with a history of wheezing is unknown.

Comparison of serum and whole-blood specimens for the detection of Candida DNA in critically ill, non-neutropenic patients

In contrast to the multitude of studies on fungal PCR assay methods, little work has been reported evaluating Candida PCR performance when using whole blood compared with serum in candidaemic patients. Here, a comparison of the performance of whole-blood and serum specimens using a set of real-time PCR Candida species assays is described. Specimens were collected prospectively from non-neutropenic adults who were recruited to a diagnostic clinical trial, the primary purpose of which was to verify the performance of the assays using serum; in all, 104 participants also had whole-blood specimens submitted for analysis in addition to the serum specimen. Of these participants, 10 had laboratory-confirmed candidaemia and 94 were categorized as being 'unlikely' to have invasive Candida infection. PCR results from the whole-blood specimens are presented here and compared with the results from serum specimens in this subgroup among whom both specimen types were obtained contemporaneously. All participants with candidaemia were PCR-positive from serum samples; however, only seven were PCR-positive from whole blood. All specimens from patients in the 'unlikely' category were PCR-negative in both types of specimen. Moreover, DNA extraction from serum required 1 h; extraction from whole blood required approximately 3 h. These data tentatively suggest that, overall, serum is an appropriate specimen for Candida PCR for detection of candidaemia in non-neutropenic adults.

Advanced glycation end products induce blood-retinal barrier dysfunction in normoglycemic rats

Advanced glycation end products (AGEs) have been implicated in the progressive vascular dysfunction which occurs during diabetic retinopathy. In the current study we have examined the role of these adducts in blood-retinal barrier (BRB) breakdown and investigated expression of the vasopermeabilizing agent vascular endothelial growth factor (VEGF) in the retina. When normoglycemic rats were injected with AGE-modified albumin daily for up to 10 days there was widespread leakage of FITC-dextran and serum albumin from the retinal vasculature when compared to control animals treated with nonmodified albumin. Ultrastructural examination of the vasculature revealed areas of attenuation of the retinal vascular endothelium and increased vesicular organelles only in the AGE-exposed rats. Quantitative RT-PCR and in situ hybridization demonstrated a significant increase in retinal VEGF mRNA expression (P <0.05). These results suggest that AGEs can initiate BRB dysfunction in nondiabetic rats and a concomitant increase in retinal VEGF expression. These findings may have implications for the role of AGEs in the pathogenesis of diabetic retinopathy.

Nature or nurture BMI and blood pressure at 90. Findings from the Belfast Elderly longitudinal free-living aging study Belfast

Hypertension is a key risk factor for stroke, cardiovascular disease and dementia. Although the link between weight, sodium and hypertension is established in younger people, little is known about their inter-relationship in people beyond 80 years of age. Associations between blood pressure, anthropometric indices and sodium were investigated in 495 apparently healthy, community-living participants (age 90, SD 4.8; range 80–106), from the cross-sectional Belfast Elderly Longitudinal Free-living Aging STudy (BELFAST) study. In age-sex-adjusted logistic regression models, blood pressure =140/90 mmHg significantly associated with body mass index (BMI) [odds ratio (OR)?=?1.28/ kg/m2], with weight (OR?=?1.22/kg) approaching significance (P?=?0.07). In further age-sex-adjusted models, blood pressure above the 120/80 mmHg normotensive reference value significantly associated with BMI (OR?=?1.44/kg/m2), weight (OR?=?1.36/kg), skin-fold-thickness (OR?=?1.33/mm) and serum sodium (OR?=?1.37 mmol/l). In BELFAST participants over 80 years old, blood pressure =140/90 mmHg is associated with BMI, in apparently similar ways to younger groups.

Cord blood erythropoietin, pH, PaO2 and haematocrit following caesarean section before labour

We have investigated the relationship between erythropoietin (Epo) and pH, PaO2 and haematocrit in 100 cord blood samples obtained at Caesarean section prior to labour. Of 82 term (> 37 weeks) infants, 64 were appropriately grown (10th-90th centiles), and their mean cord serum Epo and cord blood Epo was 23 +/- 8 mU/ml (mean +/- SD). Strong inverse correlations were found between cord serum Epo and cord blood pH (r = -0.74; p <0.0001), and between cord serum Epo and cord blood PaO2 (r = -0.55; p <0.0001), but not between cord serum Epo and cord haematocrit (r = 0.02; p <0.9). For the 18 preterm babies (gestation 32.4 +/- 4.1 weeks, birth weight 1,820 +/- 476 g), the Epo level was 36 +/- 8 mU/ml, which was not significantly greater than for the term babies. Strong inverse correlations were again found between Epo and pH (r = -0.87; p <0.0001) and Epo and PaO2 (r = -0.69; p <0.002). Babies from complicated pregnancies (intra-uterine growth retardation, pre-eclampsia, antepartum haemorrhage, diabetes mellitus and fetal distress) tended to have higher Epo levels. Thirteen babies had Epo levels > 40 mU/ml, and 11 (85%) of these required neonatal intensive care. Cord serum Epo correlates better with oxygen tension and pH at birth than with fetal growth and haematocrit, which are measures of chronic stress to the fetus.

Investigation of Genetic Variation in Scavenger Receptor Class B, Member 1 (SCARB1) and Association with Serum Carotenoids

Objective: To investigate association of scavenger receptor class B, member 1 (SCARB1) genetic variants with serum carotenoid levels of lutein (L) and zeaxanthin (Z) and macular pigment optical density (MPOD).
Design: A cross-sectional study of healthy adults aged 20 to 70.
Participants: We recruited 302 participants after local advertisement.
Methods: We measured MPOD by customized heterochromatic flicker photometry. Fasting blood samples were taken for serum L and Z measurement by high-performance liquid chromatography and lipoprotein analysis by spectrophotometric assay. Forty-seven single nucleotide polymorphisms (SNPs) across SCARB1 were genotyped using Sequenom technology. Association analyses were performed using PLINK to compare allele and haplotype means, with adjustment for potential confounding and correction for multiple comparisons by permutation testing. Replication analysis was performed in the TwinsUK and Carotenoids in Age-Related Eye Disease Study (CAREDS) cohorts.
Main Outcome Measures: Odds ratios for MPOD area, serum L and Z concentrations associated with genetic variations in SCARB1 and interactions between SCARB1 and gender.
Results: After multiple regression analysis with adjustment for age, body mass index, gender, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, smoking, and dietary L and Z levels, 5 SNPs were significantly associated with serum L concentration and 1 SNP with MPOD (P<0.01). Only the association between rs11057841 and serum L withstood correction for multiple comparisons by permutation testing (P<0.01) and replicated in the TwinsUK cohort (P = 0.014). Independent replication was also observed in the CAREDS cohort with rs10846744 (P = 2×10-4), an SNP in high linkage disequilibrium with rs11057841 (r2 = 0.93). No interactions by gender were found. Haplotype analysis revealed no stronger association than obtained with single SNP analyses.
Conclusions: Our study has identified association between rs11057841 and serum L concentration (24% increase per T allele) in healthy subjects, independent of potential confounding factors. Our data supports further evaluation of the role for SCARB1 in the transport of macular pigment and the possible modulation of age-related macular degeneration risk through combating the effects of oxidative stress within the retina.
Financial Disclosure(s): Proprietary or commercial disclosures may be found after the references. Ophthalmology 2013;120:1632–1640 © 2013 by the American Academy of Ophthalmology.

Serum lipoproteins in the diabetes control and complications trial/epidemiology of diabetes intervention and complications cohort:associations with gender and glycemia

To relate the nuclear magnetic resonance (NMR)-determined lipoprotein profile, conventional lipid and apolipoprotein measures, and in vitro oxidizibility of LDL with gender and glycemia in type 1 diabetes.

Serum apolipoprotein AI and B are stronger biomarkers of diabetic retinopathy than traditional lipids

OBJECTIVE - To describe and compare the associations of serum lipoproteins and apolipoproteins with diabetic retinopathy. RESEARCH DESIGN AND METHODS - This was a cross-sectional study of 224 diabetic patients (85 type 1 and 139 type 2) froma diabetes clinic. Diabetic retinopathy was graded from fundus photographs according to the Airlie House Classification system and categorized into mild, moderate, and vision-threatening diabetic retinopathy (VTDR). Serum traditional lipids (total, LDL, non-HDL, and HDL cholesterol and triglycerides) and apolipoprotein AI (apoAI), apolipoprotein B (apoB), and the apoB-to-apoAI ratio were assessed. RESULTS - Diabetic retinopathy was present in 133 (59.4%) individuals. After adjustment for age, sex, diabetes duration, A1C, systolic blood pressure, and diabetes medications, the HDL cholesterol level was inversely associated with diabetic retinopathy (odds ratio 0.39 [95% CI 0.16-0.94], highest versus lowest quartile; P = 0.017). The ApoAI level was inversely associated with diabetic retinopathy (per SD increase, 0.76 [95% CI 0.59-0.98]), whereas apoB (per SD increase, 1.31 [1.02-1.68]) and the apoB-to-apoAI ratio (per SD increase, 1.48 [1.13-1.95]) were positively associated with diabetic retinopathy. Results were similar for mild to moderate diabetic retinopathy and VTDR. Traditional lipid levels improved the area under the receiver operating curve by 1.8%, whereas apolipoproteins improved the area by 8.2%. CONCLUSIONS - ApoAI and apoB and the apoB-to-apoAI ratio were significantly and independently associated with diabetic retinopathy and diabetic retinopathy severity and improved the ability to discriminate diabetic retinopathy by 8%. Serum apolipoprotein levels may therefore be stronger biomarkers of diabetic retinopathy than traditional lipid measures. © 2011 by the American Diabetes Association.