Further studies on lead compounds containing the opioid pharmacophore Dmt-Tic

Some reference opioids containing the Dmt-Tic pharmacophore, especially the delta agonists H-Dmt-Tic-Gly-NH-Ph (1) and H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid (4) (UFP-512) were evaluated for the influence of the substitution of Gly with aspartic acid, its chirality, and the importance of the -NH-Ph and N(1)H-Bid hydrogens in the inductions of delta agonism. The results provide the following conclusions: (i) Asp increases delta selectivity by lowering the mu affinity; (ii) -NH-Ph and N(1)H-Bid nitrogens methylation transforms the delta agonists into delta antagonists; (iii) the substitution of Gly with L-Asp/D-Asp in the delta agonist H-Dmt-Tic-Gly-NH-Ph gave delta antagonists; the same substitution in the delta agonist H-Dmt-Tic-NH-CH2-Bid yielded more selective agonists, H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid and H-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid; (iv) L-Asp seems important only in functional bioactivity, not in receptor affinity; (v) H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid(N(1)-Me) (10) evidenced analgesia similar to 4, which was reversed by naltrindole only in the tail flick. 4 and 10 had opposite behaviours in mice; 4 caused agitation, 10 gave sedation and convulsions.     

Steroid secretion following exposure of ovarian follicular cells to three different natural mixtures of persistent organic pollutants (POPs)

This study investigated in vitro endocrine disrupting effects of three mixtures of POPs: 'Marine mix' extracted from Atlantic cod liver, and two mixtures extracted from burbot liver, 'Mj?sa mix' and 'Losna mix'. The POP mixtures were chemically characterized. Co-culture of theca and granulosa cells, were exposed for 48h with different doses of 'Marine mix', 'Mj?sa mix' or 'Losna mix'. As an end point cell viability was determinated by LDH test, steroid analysis by EIA and caspase-3 by colorimetric substrate. Chemical characterization of the mixtures demonstrated that the 'Marine mix' contained high levels of DDTs and PCBs. In the 'Mj?sa mix', the dominant pollutants were BDEs and HBCD. The concentrations of POPs measured in the 'Losna mix' were considerably lower. All mixtures used in the present study had a stimulatory effect on testosterone and estradiol secretion with 'Marine mix'>'Mj?sa mix'>'Losna mix'. These results show that even a mixture containing background concentrations of POPs significantly affected steroid secretion. A higher steroidogenic response in low dose ranges, compared with high dose ranges indicated xenobiotic-conditioning hormesis. This could complicate predictions of effects in risk assessments.     

Method for screening of solid dispersion formulations of low-solubility compounds--miniaturization and automation of solvent casting and dissolution testing

An efficient method has been developed for screening solid dispersion formulations that are intended to enhance the dissolution of poorly soluble compounds. The method is based on miniaturization and automation of sample preparation by solvent casting, and dissolution testing, in a 96-well plate format, using less than 0.1mg of compound per well. To illustrate the method, six polymers and eight surfactants were screened, individually and in combination, for their ability to dissolve a compound with aqueous solubility of < 1 microg/ml in simulated intestinal fluid. Screening was performed at an excipient/compound ratio of 10:1, and a polymer/surfactant ratio of 3:1 for ternary formulations. Sixteen of the 48 ternary formulations dissolved the compound to a level > 100 microg/ml, i.e. at least a 100-fold increase over the aqueous solubility. A number of synergies were observed wherein the performance of a ternary formulation greatly exceeded that of either of the corresponding binary formulations. Thirteen 'hits' from screening were scaled up with melt methods, and approximately 2/3 of these showed comparable dissolution enhancement when tested at larger scale. Five of these were administered to rats, and the absolute oral bioavailability ranged from 10 to 23%, versus less than 1% for the unformulated compound.     

Enantiomeric propanolamines as selective N-methyl-D-aspartate 2B receptor antagonists

Enantiomeric propanolamines have been identified as a new class of NR2B-selective NMDA receptor antagonists. The most effective agents are biaryl structures, synthesized in six steps with overall yields ranging from 11-64%. The compounds are potent and selective inhibitors of NR2B-containing recombinant NMDA receptors with IC 50 values between 30-100 nM. Potency is strongly controlled by substitution on both rings and the centrally located amine nitrogen. SAR analysis suggests that well-balanced polarity and chain-length factors provide the greatest inhibitory potency. Structural comparisons based on 3D shape analysis and electrostatic complementarity support this conclusion. The antagonists are neuroprotective in both in vitro and in vivo models of ischemic cell death. In addition, some compounds exhibit anticonvulsant properties. Unlike earlier generation NMDA receptor antagonists and some NR2B-selective antagonists, the present series of propanolamines does not cause increased locomotion in rodents. Thus, the NR2B-selective antagonists exhibit a range of therapeutically interesting properties.     

Non-proliferating plasma cells detected in the salivary glands and bone marrow of autoimmune NOD.B10.H2b mice,a model for primary Sjögren’s syndrome

Autoantibody secreting plasma cells (PCs) are essential contributors in the development of autoimmune conditions such as primary Sjögren’s syndrome (pSS). Particularly, the long-lived PC subset residing in the bone marrow has shown to continuously produce autoantibodies, whilst remaining unaffected by immunosuppressive treatment. We have previously shown accumulation of potentially long-lived PCs in chronically inflamed salivary glands of pSS patients. In this study, we aimed to characterise the PC compartment in the salivary glands (the target organ for pSS) and bone marrow before the onset of the murine pSS like disease versus advanced diseases progression. Bromodeoxyuridine (BrdU) was incorporated to distinguish the long-lived PCs. Double immunohistochemical staining and immunofluorescence were then conducted on submandibular gland and bone marrow sections from 8- and 40-week-old mice to identify BrdU and CD138. BrdU⁺ cells were detected in the submandibular glands of 8-week-old mice, and observed within all focal infiltrates by 40 weeks of age. Most CD138⁺ PCs were however BrdU^? and located predominantly on the periphery of these infiltrates. This observation was verified through immunofluorescence. A comparable staining pattern was observed in the bone marrow of 8- and 40-week-old NOD.B10.H2b mice, where some of the CD138⁺ cells also expressed BrdU. Interestingly, megakaryocytes in the bone marrow of NOD.B10.H2b mice were detected in close proximity to CD138⁺ cells, illustrating a possible presence of PC survival niches. Our results demonstrate the presence and accumulation of potentially long-lived PCs in NOD.B10.H2b mice as the disease advances.     

Evaluation of the irritancy and hypersensitivity potential following topical application of didecyldimethylammonium chloride

Didecyldimethylammonium chloride (DDAC) is a dialkyl-quaternary ammonium compound that is used in numerous products for its bactericidal, virucidal and fungicidal properties. There have been clinical reports of immediate and delayed hypersensitivity reactions in exposed individuals; however, the sensitization potential of DDAC has not been thoroughly investigated. The purpose of these studies was to evaluate the irritancy and sensitization potential of DDAC following dermal exposure in a murine model. DDAC induced significant irritancy (0.5 and 1%), evaluated by ear swelling in female Balb/c mice. Initial evaluation of the sensitization potential was conducted using the local lymph node assay (LLNA) at concentrations ranging from 0.0625–1%. A concentration-dependent increase in lymphocyte proliferation was observed with a calculated EC3 value of 0.17%. Dermal exposure to DDAC did not induce increased production of IgE as evaluated by phenotypic analysis of draining lymph node B-cells (IgE?⁺?B220⁺) and measurement of total serum IgE levels. Additional phenotypic analyses revealed significant and dose-responsive increases in the absolute number of B-cells, CD4?⁺?T-cells, CD8?⁺?T-cells and dendritic cells in the draining lymph nodes, along with significant increases in the percentage of B-cells (0.25% and 1% DDAC) at Day 10 following 4 days of dermal exposure. There was also a significant and dose-responsive increase in the number of activated CD44?⁺?CD4?⁺?and CD8?⁺?T-cells and CD86?⁺?B-cells and dendritic cells following exposure to all concentrations of DDAC. These results demonstrate the potential for development of irritation and hypersensitivity responses to DDAC following dermal exposure and raise concerns about the use of this chemical and other quaternary ammonium compounds that may elicit similar effects.     

Interaction of Proteasomes and Complement C3, Assay of Antisecretory Factor in Blood

Antisecretory factor (AF) is a protein complex which inhibits inflammation and regulates fluid transport. In this article, two new immunoassays (ELISA) are developed. The first ELISA establishes a 26S proteasome concentration of 0.41±0.03 μg/mL in normal plasma; the second ELISA discloses the binding of proteasomes to complement factor C3. The latter test values increased about tenfold following intake of processed cereals, paralleling with the old AF ELISA. The proteasome/C3 complex is purified and shown to expose hidden antisecretory peptide sequence and contain the inactive C3c protein. These findings might explain the antisecretory and anti-inflammatory effect during AF complex formation.     

Bone morphogenetic protein 6—a possible new player in pathophysiology of heart failure

Derangement of bone morphogenetic protein (BMP) signalling was observed in cardiovascular disorders. The present study assesses the diagnostic and prognostic value of BMP6 plasma concentration in chronic heart failure (CHF). 130 CHF patients and 32 controls participated in the study. BMP6 plasma level was measured at baseline. During 12‐month follow‐up death and hospitalisation with CHF exacerbation were recorded. BMP6 was significantly increased in CHF patients with highest concentration in most advanced disease. Individuals with pulmonary congestion or peripheral oedema had higher levels of BMP6 than isovolemic patients. BMP6 was not a predictor of all‐cause mortality or CHF hospitalisation. BMP6 may be involved in pathophysiology of systolic CHF. BMP6 plasma level is related to the disease severity and signs of exacerbation.