Philippe Neven

Simple di- and trivanillates exhibit cytostatic properties toward cancer cells resistant to pro-apoptotic stimuli

A series of 33 novel divanillates and trivanillates were synthesized and found to possess promising cytostatic rather than cytotoxic properties. Several compounds under study decreased by >50% the activity of Aurora A, B, and C, and WEE1 kinase activity at concentrations <10% of their IC(50) growth inhibitory ones, accounting, at least partly, for their cytostatic effects in cancer cells and to a lesser extent in normal cells. Compounds 6b and 13c represent interesting starting points for the development of cytostatic agents to combat cancers, which are naturally resistant to pro-apoptotic stimuli, including metastatic malignancies.

Curcumin and resveratrol act by different ways on NADPH oxidase activity and reactive oxygen species produced by equine neutrophils.

In neutrophils (PMNs), superoxide anion (O2*-), the first reactive oxygen species (ROS) produced to kill pathogenic agents, is generated by NADPH oxidase, an enzymatic complex formed by the translocation of cytosolic subunits to the membrane flavocytochrome b558. In horses, excessive activation of PMNs is often associated with deadly pathologies and the modulation of their ROS production by acting on NADPH oxidase is a prime target to manage inflammation. We developed a cell-free assay to measure the activity of equine NADPH oxidase assembled in vitro, in order to test the effects of natural or synthetic compounds on the enzyme activity or assembly. The cell-free assay was validated with diphenyleneiodonium chloride and Gp91ds-tat, two inhibitors largely described for human NADPH oxidase. The anti-oxidant effects of curcumin and resveratrol at final concentration ranging from 10(-4) to 10(-6) M were studied on whole cells by chemiluminescence (CL) and by cell-free assay, in which the molecule was added before or after the enzyme assembly. The CL assay demonstrated that curcumin efficiently inhibited the O2(-) production and easily entered into PMNs or interacted with their membrane. Cell-free assay showed that curcumin acted on the reconstitution of NADPH oxidase even at 10(-5)M, while resveratrol appeared to be an O2*- scavenger rather than an inhibitor of NADPH oxidase activity, since it acted from outside the cell in CL and after the complex assembly in cell-free assay. By acting directly on NADPH oxidase, curcumin should be a good candidate for the treatment of acute or inflammatory diseases involving an excessive ROS production.

Intra- and extracellular antioxidant capacities of the new water-soluble form of curcumin (NDS27) on stimulated neutrophils and HL-60 cells

Phagocytic cells, especially neutrophils (PMNs) are specialized in the production of reactive oxygen species (ROS) to kill pathogenic agents, but an excessive ROS production is associated with tissue damages and inflammatory diseases. Phagocytes are thus prime therapeutic targets to control inflammatory events associated to ROS production. Nowadays, there is a growing interest for the use of polyphenols to modulate the inflammatory response. The aim of this work was to study the antioxidant effect of NDS27, a highly water-soluble form of the polyphenolic molecule curcumin, on in vitro stimulated equine PMNs and human promyelocytic leukemia cells (HL-60). NDS27 was either pre-incubated with cells and eliminated before their activation (intracellular effect) or let in the medium (extracellular effect). Our results indicate that NDS27 significantly and dose-dependently (10(-6) M-10(-4) M) inhibited the ROS production in both cell types without affecting their viability. NDS27 was able to cross and interact with cell membrane, especially for HL-60 cells, while we observed a better intracellular antioxidant effect with PMNs. The activity of myeloperoxidase (MPO) released by PMNs and HL-60 cells, was decreased by NDS27, but more efficiently for PMNs. These results suggested that the greater efficiency of NDS27 in PMNs is due to an inhibitory effect on cells which are more mature for ROS production, probably by targeting the enzymes implied in respiratory burst like MPO. The modulatory effect of NDS27 on the oxidant activity of cells involved in immune and inflammatory responses opens perspectives for a therapeutic control of pathologies with excessive inflammatory reactions.

NDS27 combines the effect of curcumin lysinate and hydroxypropyl-β-cyclodextrin to inhibit equine PKCδ and NADPH oxidase involved in the oxidative burst of neutrophils

Polymorphonuclear neutrophils (PMNs) are involved in host defence against infections by the production of reactive oxygen species (ROS), but excessive PMN stimulation is associated with the development of inflammatory diseases. After appropriate stimuli, protein kinase C (PKC) triggers the assembly of NADPH oxidase (Nox2) which produces superoxide anion (O2 −), from which ROS derive. The therapeutic use of polyphenols is proposed to lower ROS production by limiting Nox2 and PKC activities. The purpose of this study was to compare the antioxidant effect of NDS27 and NDS28, two water‐soluble forms of curcumin lysinate respectively complexed with hydroxypropyl‐β‐cyclodextrin (HPβCD) and γ‐cyclodextrin (γ‐CD), on the activity of Nox2 and PKCδ, involved in the Nox2 activation pathway. Our results, showed that NDS27 is the best inhibitor for Nox2 and PKCδ. This was illustrated by the combined effect of HPβCD and curcumin lysinate: HPβCD, but not γ‐CD, improved the release of curcumin lysinate and its exchange against lipid or cholesterol as demonstrated by the lipid colouration with Oil Red O, the extraction of radical lipophilic probes recorded by ESR and the HPLC measurements of curcumin. HPβCD not only solubilised and transported curcumin, but also indirectly enhanced its action on both PKC and Nox2 activities. The modulatory effect of NDS27 on the Nox2 activation pathway of neutrophils may open therapeutic perspectives for the control of pathologies with excessive inflammatory reactions.

Pyridothiadiazinedioxides structurally related to quinazolinones cholecystokinin/gastrin receptor ligands: synthesis and biological evaluation

The synthesis of 3-aralkyl-4-aryl-4H-, 3-aralkylamino-4-aryl-4H- and 3-aralkylsulfanyl-4-aryl-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1, 1-dioxides is described. Moreover, the affinity of the different compounds towards the cholecystokinin CCK-A and CCK-B receptors was evaluated. For selected compounds, affinity on the two receptor subtypes was expressed in the micromolar range. This was comparable to the affinity observed with the naturally occurring CCK receptor antagonist asperlicin.

Synthesis and biological evaluation of new 3-aralkylamino-2-aryl-2H-1,2,4-pyridothiadiazine 1,1-dioxides as potential CCK receptor ligands

A series of 2‐aralkyl‐4H‐pyridothiadiazine 1,1‐dioxides and 3‐aralkylamino‐2‐aryl‐2H‐pyrido[4,3‐e]‐1,2,4‐thiadiazine 1,1‐dioxides structurally related to quinazolinone CCK receptor antagonists were synthesized and evaluated as CCK‐A and CCK‐B receptor ligands.

The soluble curcumin derivative NDS27 inhibits superoxide anion production by neutrophils and acts as substrate and reversible inhibitor of myeloperoxidase

A water-soluble curcumin lysinate incorporated into hydroxypropyl-β-cyclodextrin (NDS27) has been developed and shown anti-inflammatory properties but no comparative study has been made in parallel with its parent molecule, curcumin on polymorphonuclear neutrophils (PMNs) and myeloperoxidase (MPO) involved in inflammation. The effect of NDS27, its excipients (hydroxypropyl-β-cyclodextrin and lysine), curcumin lysinate and curcumin were compared on the release of superoxide anion by PMNs using a chemiluminescence assay and on the enzymatic activity of MPO. It was shown that curcumin and NDS27 exhibit similar inhibition activities on superoxide anion release by stimulated PMNs but also on MPO peroxidase and halogenation activities. The action mechanism of curcumin and NDS27 on the MPO activity was refined by stopped-flow and docking analyses. We demonstrate that both curcumin and NDS27 are reversible inhibitors of MPO by acting as excellent electron donors for redox intermediate Compound I (∼107 M-1 s-1) but not for Compound II (∼103 M-1 s-1) in the peroxidase cycle of the enzyme, thereby trapping the enzyme in the Compound II state. Docking calculations show that curcumin is able to enter the enzymatic pocket of MPO and bind to the heme cavity by π-stacking and formation of hydrogen bonds involving substituents from both aromatic rings. Hydroxypropyl-β-cyclodextrin is too bulky to enter MPO channel leading to the binding site suggesting a full release of curcumin from the cyclodextrin thereby allowing its full access to the active site of MPO. In conclusion, the hydroxypropyl-β-cyclodextrin of NDS27 enhances curcumin solubilization without affecting its antioxidant capacity and inhibitory activity on MPO.

Comparison of the Effects of Nimesulide and Nimesulide‐L‐lysine on PGE2 Production by COX‐1 and COX‐2 and on Chondrocyte Metabolism In‐vitro

Nimesulide, a non-steroidal anti-inflammatory drug and one of a promising class of selective COX-2 inhibitors, has a very interesting therapeutic profile. Unfortunately, it is poorly soluble in water, which leads to important difficulties in the formulation of injectable solutions. This problem can also affect the bioavailability of nimesulide. To increase the aqueous solubility of the drug a nimesulide-L-lysine salt was synthesized in our laboratory; its aqueous solubility was greater than that of nimesulide (solubility in purified water 7.5 mg mL−1, and 0.01 mg mL-1, respectively). The aim of this study was to compare the anti-inflammatory profiles of nimesulide and nimesulide-L-lysine salt in a two-step in-vitro investigation. First, we evaluated the COX-2 selectivity of the drugs by a method using purified COX-1 and COX-2 enzymes. In a second step we evaluated the effects of the drugs on the production of prostaglandin E2 (PGE2) and proteoglycan by chondrocytes from man. The results obtained confirmed the COX-2 selectivity of the two compounds. Nimesulide-L-lysine had the same anti-inflammatory profile as nimesulide on chondrocyte cultures and better water solubility. Nimesulide-L-lysine should, therefore, be used to prepare injectable preparations and should ameliorate bioavailability after oral treatments.

Pharmacomodulation Studies of Torasemide Leading to Original Non‐carboxylic Thromboxane A2 Receptor Antagonists

Because torasemide, a loop diuretic, dose-dependently relaxes canine coronary artery precontracted with thromboxane A2 (TxA2), a series of pyridine derivatives have been investigated with the aim of developing original TxA2 receptor antagonists. A binding test showed the affinity (IC50; the dose reducing binding by 50%) of one sulphonylurea derivative (BM 27) and two sulphonylcyanoguanidine derivatives (BM 114 and BM 115) for the TxA2 receptor of washed platelets from man were 161, 1. 75 and 0.54 μM, respectively. The IC50 of torasemide was only 2.69 μM. Their antagonism was confirmed by the capacity of the compounds to prevent the aggregation of platelets, from man, induced by arachidonic acid, the biological precursor of TxA2 (IC50: BM 114, 29.3 μM; BM 115, 12.0 μM; BM 27, 234 μM; torasemide, 350 μM; sulotroban, 12.3 μM). Similar results were obtained with the best compound BM 115 (IC50 11.5 μM) when U-46619, a stable TxA2 agonist, was used as the aggregating agent. This molecule can be regarded as a template for the design of novel non-carboxylic TxA2 receptor antagonists.