Radhakrishnan Venkatasamy

Fucosylated chondroitin sulfates from the body wall of the sea cucumber Holothuria forskali. Conformation, selectin binding and biological activity

Background: An acidic polysaccharide, fCS, from the sea cucumber Holothuria forskali has a range of biological activities. Results: The conformation of fCS was determined, and resulting oligosaccharides were shown to retain desirable biological properties. Conclusion: The conformation of the fCS repeating unit underpins binding to L- and P-selectins. Significance: Exploitation of the fCS-selectin interaction may open new avenues for therapeutic intervention using fCS fragments or their mimetics. Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: →3)GalNAcβ4,6S(1→4) [FucαX(1→3)]GlcAβ(1→, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Lex blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L- and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu2+-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention.

In vivo evaluation of piperine and synthetic analogues as potential treatments for vitiligo using a sparsely pigmented mouse model

Background  Piperine and its analogues have been reported to stimulate melanocyte replication in vitro and may be useful in treating the depigmenting disease, vitiligo.

Amides from Piper nigrum L. with dissimilar effects on melanocyte proliferation in‐vitro

Melanocyte proliferation stimulants are of interest as potential treatments for the depigmentary skin disorder, vitiligo. Piper nigrum L. (Piperaceae) fruit (black pepper) water extract and its main alkaloid, piperine (1), promote melanocyte proliferation in‐vitro. A crude chloroform extract of P. nigrum containing piperine was more stimulatory than an equivalent concentration of the pure compound, suggesting the presence of other active components. Piperine (1), guineensine (2), pipericide (3), N‐feruloyltyramine (4) and N‐isobutyl‐2E, 4E‐dodecadienamide (5) were isolated from the chloroform extract. Their activity was compared with piperine and with commercial piperlongumine (6) and safrole (7), and synthetically prepared piperettine (8), piperlonguminine (9) and 1‐(3, 4‐methylenedioxyphenyl)‐decane (10). Compounds 6–10 either occur in P. nigrum or are structurally related. Compounds 1, 2, 3, 8 and 9 stimulated melanocyte proliferation, whereas 4, 5, 6, 7 and 10 did not. Comparison of structures suggests that the methylenedioxyphenyl function is essential for melanocyte stimulatory activity. Only those compounds also possessing an amide group were active, although the amino component of the amide group and chain linking it to the methylenedioxyphenyl group can vary. P. nigrum, therefore, contains several amides with the ability to stimulate melanocyte proliferation. This finding supports the traditional use of P. nigrum extracts in vitiligo and provides new lead compounds for drug development for this disease.

The role of heparanase in pulmonary cell recruitment in response to an allergic but not non-allergic stimulus

Heparanase is an endo-β-glucuronidase that specifically cleaves heparan sulfate proteoglycans in the extracellular matrix. Expression of this enzyme is increased in several pathological conditions including inflammation. We have investigated the role of heparanase in pulmonary inflammation in the context of allergic and non-allergic pulmonary cell recruitment using heparanase knockout (Hpa-/-) mice as a model. Following local delivery of LPS or zymosan, no significant difference was found in the recruitment of neutrophils to the lung between Hpa-/- and wild type (WT) control. Similarly neutrophil recruitment was not inhibited in WT mice treated with a heparanase inhibitor. However, in allergic inflammatory models, Hpa-/- mice displayed a significantly reduced eosinophil (but not neutrophil) recruitment to the airways and this was also associated with a reduction in allergen-induced bronchial hyperresponsiveness, indicating that heparanase expression is associated with allergic reactions. This was further demonstrated by pharmacological treatment with a heparanase inhibitor in the WT allergic mice. Examination of lung specimens from patients with different severity of chronic obstructive pulmonary disease (COPD) found increased heparanase expression. Thus, it is established that heparanase contributes to allergen-induced eosinophil recruitment to the lung and could provide a novel therapeutic target for the development of anti-inflammatory drugs for the treatment of asthma and other allergic diseases.

UV irradiation affects melanocyte stimulatory activity and protein binding of piperine.

Abstract Piperine, the major alkaloid of black pepper (Piper nigrum L.; Piperaceae), stimulates melanocyte proliferation and dendrite formation in vitro. This property renders it a potential treatment for the skin depigmentation disorder vitiligo. However, piperine does not stimulate melanin synthesis in vitro, and treatments based on this compound may therefore be more effective with concomitant exposure of the skin to ultraviolet (UV) radiation or sunlight. The present study investigated the effect of UVA and simulated solar radiation (SSR) on the chemical stability of piperine, its melanocyte stimulatory effects and its ability to bind protein and DNA. Chromatographic and spectroscopic analysis confirmed the anticipated photoisomerization of irradiated piperine and showed the absence of any hydrolysis to piperinic acid. Isomerization resulted in the loss of ability to stimulate proliferation of a mouse melanocyte cell line, and to bind to human serum albumin. There was no evidence of DNA binding by piperine either before or after irradiation, showing the absence of photoadduct formation by either piperine or its geometric isomers. This is unlike the situation with psoralens, which form DNA adducts when administered with UVA in treating skin diseases. The present study suggests that exposure to bright sunlight should be avoided both during active application of piperine to the skin and in the storage of piperine products. If UVA radiation is used with piperine in the treatment of vitiligo, application of the compound and irradiation should be staggered to minimize photoisomerization. This approach is shown to effectively induce pigmentation in a sparsely pigmented mouse strain.

Use of within-group designs to test anti-tussive drugs in conscious guinea-pigs

INTRODUCTION Cough is a common medical problem for which there are few effective drug treatments. A limited understanding of the mechanisms of induction and maintenance of cough and a paucity of suitable animal models frustrate drug discovery efforts to find novel anti-tussives. As in humans, guinea-pigs evoke a cough reflex upon exposure to tussive agents such as citric acid and capsaicin; both of which have been widely used to assess novel anti-tussive drugs. However, the potential for using within-group designs in drug development has received little attention and such designs may offer a way of assisting the drug discovery effort in the area of cough as well as other areas. METHODS Cough can be monitored in conscious guinea-pigs by placing animals in a Perspex chamber, in which air is continually exchanged by use of negative pressure and drug delivery of aerosols to the chamber can be accurately timed. Cough in response to a tussive agent (e.g. 0.2-0.4M citric acid; 10-30 microM capsaicin) is detected by the simultaneous microphonic recording of audible signals characteristic of the cough response as well as by positive pressure changes in the chamber generated by a cough dependent rapid expiration of air from the lungs. Both the sound and pressure signals are recorded using an online analyzer, whilst the number of coughs can be analyzed off-line. The number of coughs over a 15 min period is used to quantitate tussive events. RESULTS Reproducible cough can be detected in animals using cross-over designs that lend themselves to drug studies. Both the time and concentration dependence of anti-tussive drug action can be evaluated in the same animal. Furthermore, the effect of different anti-tussive drugs can be evaluated thereby reducing between group error and thereby improving the sensitivity of the test. DISCUSSION Repeated measures design improves the precision with which to evaluate anti-tussive drugs in preclinical models and could be used to make the drug discovery process more efficient.

The Effect of Phytocannabinoids on Airway Hyper-Responsiveness, Airway Inflammation, and Cough

Cannabis has been demonstrated to have bronchodilator, anti-inflammatory, and antitussive activity in the airways, but information on the active cannabinoids, their receptors, and the mechanisms for these effects is limited. We compared the effects of Δ9-tetrahydrocannabinol, cannabidiol, cannabigerol, cannabichromene, cannabidiolic acid, and tetrahydrocannabivarin on contractions of the guinea pig–isolated trachea and bronchoconstriction induced by nerve stimulation or methacholine in anesthetized guinea pigs following exposure to saline or the proinflammatory cytokine, tumor necrosis factor α (TNF-α). CP55940 (2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl) cyclohexyl]-5-(2-methyloctan-2-yl)phenol), a synthetic cannabinoid agonist, was also investigated in vitro. The cannabinoids were also evaluated on TNF-α– and lipopolysaccharide-induced leukocyte infiltration into the lungs and citric acid–induced cough responses in guinea pigs. TNF-α, but not saline, augmented tracheal contractility and bronchoconstriction induced by nerve stimulation, but not methacholine. Δ9-Tetrahydrocannabinol and CP55940 reduced TNF-α−enhanced nerve-evoked contractions in vitro to the magnitude of saline-incubated trachea. This effect was antagonized by the cannabinoid 1 (CB1) and CB2 receptor antagonists AM251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-caroxamide] and JTE907 [N-(1,3-benzodioxol-5-ylmethyl)-1,2-dihydro-7-methoxy-2-oxo-8-(pentyloxy)-3-quinolinecarboxamide], respectively. Tetrahydrocannabivarin partially inhibited the TNF-α−enhanced nerve-evoked contractions, whereas the other cannabinoids were without effect. The effect of cannabidiol and Δ9-tetrahydrocannabinol together did not differ from that of the latter alone. Only Δ9-tetrahydrocannabinol inhibited TNF-α−enhanced vagal-induced bronchoconstriction, neutrophil recruitment to the airways, and citric acid−induced cough responses. TNF-α potentiated contractions of airway smooth muscle in response to nerve stimulation by enhancing postganglionic acetylcholine release. Δ9-Tetrahydrocannabinol and CP55940 inhibited the TNF-α−enhanced acetylcholine release, and hence contraction and bronchoconstriction, through activation of presynaptic CB1 and CB2 receptors. The other cannabinoids did not influence cholinergic transmission, and only Δ9-THC demonstrated effects on airway hyper-responsiveness, anti-inflammatory activity, and antitussive activity in the airways.

Protease inhibitors in respiratory disease: focus on asthma and chronic obstructive pulmonary disease

Respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD), are a major health burden on society and current treatment modalities for these diseases have not significantly changed over the past 40 years. The only major pharmacological advancement for the treatment of these diseases has been to increase the duration of action of bronchodilators (asthma: salmeterol; COPD: tiotropium bromide) and glucocorticosteroids (asthma: fluticasone propionate) and, increasingly, to formulate these agents in the same delivery device. Despite our increasing understanding of the cell and molecular biology of these diseases, the development of novel treatments remains beyond the reach of the scientific community. Proteases are a family of proteins with diverse biological activity, which are found in abundance within the airways of asthma and COPD, and have been implicated in the pathogenesis of these diseases. The targeting of proteases, including mast cell tryptase, neutrophil elastase and matrix metalloprotease with low-molecular-weight inhibitors, has highlighted the potential role of these enzymes in mediating certain aspects of the disease process in preclinical studies. Several challenges remain regarding the development of protease inhibitors, including the synthesis of highly potent and specific inhibitors, and target validation in man.

Steroid sparing effects of doxofylline

Glucocorticosteroids are widely used in the treatment of asthma and chronic obstructive pulmonary disease (COPD). However, there are growing concerns about the side effect profile of this class of drug, particularly an increased risk of pneumonia. Over the last two decades there have been many attempts to find drugs to allow a reduction of glucocorticosteroids, including xanthines such as theophylline. Use of xanthines has been shown to lead to a reduction in the requirement for glucocorticosteroids, although xanthines also have a narrow therapeutic window limiting their wider use. Doxofylline is another xanthine that has been shown to be of clinical benefit in patients with asthma or COPD, but to have a wider therapeutic window than theophylline. In the present study we have demonstrated that doxofylline produces a clear steroid sparing effect in both an allergic and a non-allergic model of lung inflammation. Thus, we have shown that concomitant treatment with a low dose of doxofylline and a low dose of the glucocorticosteroid dexamethasone (that alone had no effect) significantly reduced both allergen-induced eosinophil infiltration into the lungs of allergic mice, and lipopolysaccharide (LPS)-induced neutrophil infiltration into the lung, equivalent to a higher dose of each drug. Our results suggest that doxofylline demonstrates significant anti-inflammatory activity in the lung which can result in significant steroid sparing activity.

Novel relaxant effects of RPL554 on guinea pig tracheal smooth muscle contractility.

We investigated the effectiveness of RPL554, a dual PDE3 and 4 enzyme inhibitor, on airway smooth muscle relaxation and compared it with that induced by salbutamol, ipratropium bromide, glycopyrrolate or their combination on bronchomotor tone induced by different spasmogenic agents.