Werner Kiefer

Cyclooxygenase inhibitors - current status and future prospects

Prostaglandins are formed from arachidonic acid by the action of cyclooxygenase and subsequent downstream synthetases. Two closely related forms of the cyclooxygenase have been identified which are now known as COX-1 and COX-2. Both isoenzymes transform arachidonic acid to prostaglandins, but differ in their distribution and their physiological roles. Meanwhile, the responsible genes and their regulation have been clarified. COX-1, the pre-dominantly constitutive form of the enzyme, is expressed throughout the body and performs a number of homeostatic functions such as maintaining normal gastric mucosa and influencing renal blood flow and platelet aggregation. In contrast, the inducible form is expressed in response to inflammatory and other physiological stimuli and growth factors, and is involved in the production of the prostaglandins that mediate pain and support the inflammatory process. All the classic NSAIDs inhibit both COX-1 and COX-2 at standard anti-inflammatory doses. The beneficial anti-inflammatory and analgesic effects are based on the inhibition of COX-2, but the gastrointestinal toxicity and the mild bleeding diathesis are a result of the concurrent inhibition of COX-1. Agents that inhibit COX-2 while sparing COX-1 represent a new attractive therapeutic development and could represent a major advance in the treatment of rheumatoid arthritis and osteoarthritis. Apart from its involvement in inflammatory processes, COX-2 seems to play a role in angiogenesis, colon cancer and Alzheimers disease, based on the fact that it is expressed during these diseases. The benefits of specific and selective COX-2 inhibitors are currently under discussion and offer a new perspective for a further use of COX-2 inhibitors.

The pyrrole moiety as a template for COX-1/COX-2 inhibitors

Aroyl- and thiophene-substituted pyrrole derivatives have been synthesized as a new class of COX-1/COX-2 inhibitors. The inhibition of COX-1 was evaluated in a biological system using bovine PMNLs as the enzyme source, whereas LPS-stimulated human monocytes served as the enzyme source for inducible COX-2. The determination of the concentration of arachidonic acid metabolites was performed by HPLC for COX-1 and RIA for COX-2. Variation of the substitution pattern led to a series of active compounds which showed inhibition for COX-1 and COX-2. Structural requirements for the development of COX-1/COX-2 inhibitors are discussed.

Simultaneous determination of olanzapine, clozapine and demethylated metabolites in serum by on-line column-switching high-performance liquid chromatography.

An automated method for simultaneous routine quantification of the antipsychotic drugs clozapine, olanzapine and their demethylated metabolites is described. The method included adsorption on a cyanopropyl (CPS) coated clean-up column (10 microm; 10 x 2.0 mm I.D.), washing off interfering serum constituents to waste, and separation on C18 ODS Hypersil reversed phase material (5 microm; 250 x 4.6 mm I.D.) using acetonitrile-water-tetramethylethylenediamine (37:62.6:0.4, v/v/v) adjusted to pH 6.5 with concentrated acetic acid. UV-detection was performed at 254 nm. The limit of quantification was 10-20 ng/ml. Relative day to day standard variations ranged between 4.5 and 13.5%. The method is suitable for routine monitoring of olanzapine and clozapine including their demethylated metabolites.

Novel Insights and Therapeutical Applications in the Field of Inhibitors of COX-2

The discovery of the two isoenzymes COX-1 and COX-2 and the knowledge of their function, localisation and regulation has initiated the development of COX-2 selective inhibitors (coxibs). Inducible COX-2 at the peripheral site of inflammation has been detected in the early 1990s, the involvement of recently detected spinal COX-2 has led to new insights into mechanisms of pain and may explain analgesic and antipyretic properties of COX-2 selective inhibitors. The coxibs rofecoxib and celecoxib have been introduced into therapy and seem to offer some advantages over the classical non-selective NSAIDs. The search for new COX-2 inhibitors is going on, the development of etoricoxib and lumiracoxib is a step ahead concerning efficacy, tolerability and safety. Until today COX-2 selective inhibitors have found their place in therapy of arthritis, osteoarthritis, dysmenorrhea and acute pain. A new paradigm in pain therapy seems to justify their use in perioperative settings in a preemptive or multimodal therapeutical strategy. In the future COX-2 selective inhibitors as opioid sparing agents could become an important tool in pain therapy. Even a therapeutical benefit of COX-2 selective inhibitors in the treatment of Alzheimers Disease or in the prevention or treatment of colorectal or prostate cancer is presently intensely investigated. Recently some authors reported on COX-3, a splicing variant of COX-1. If COX-3 really represents the target for acetaminophen must be called into question.

Flavonoids as noncompetitive inhibitors of Dengue virus NS2B-NS3 protease: inhibition kinetics and docking studies.

NS2B-NS3 is a serine protease of the Dengue virus considered a key target in the search for new antiviral drugs. In this study flavonoids were found to be inhibitors of NS2B-NS3 proteases of the Dengue virus serotypes 2 and 3 with IC50 values ranging from 15 to 44 μM. Agathisflavone (1) and myricetin (4) turned out to be noncompetitive inhibitors of dengue virus serotype 2 NS2B-NS3 protease with Ki values of 11 and 4.7 μM, respectively. Docking studies propose a binding mode of the flavonoids in a specific allosteric binding site of the enzyme. Analysis of biomolecular interactions of quercetin (5) with NT647-NHS-labeled Dengue virus serotype 3 NS2B-NS3 protease by microscale thermophoresis experiments, yielded a dissociation constant KD of 20 μM. Our results help to understand the mechanism of inhibition of the Dengue virus serine protease by flavonoids, which is essential for the development of improved inhibitors.

Effect of Flavonol Derivatives on the Carrageenin-Induced Paw Edema in the Rat and Inhibition of Cyclooxygenase-1 and 5-Lipoxygenase in Vitro

Alkoxyflavonols were synthesized by the Algar‐Flynn‐Oyamada (AFO) cyclization of chalcones. Hydroxyflavonols were prepared by dealkylation of methoxyflavonols by refluxing in hydroiodic acid. The alkoxyflavonols 3‐hydroxy‐2‐(2,3,4‐trimethoxy‐phenyl)‐ 4H‐chromen‐4‐one (6), 2‐(4‐ethoxyphenyl)‐3‐hydroxy‐4H‐ chromen‐4‐one (7), 2‐(4‐butoxyphenyl)‐3‐hydroxy‐4H‐ chromen‐4‐one (10), and 2‐(3‐n‐butoxyphenyl)‐3‐hydroxy‐4H‐ chromen‐4‐one (11) as well as the trihydroxy derivative 3‐hydroxy‐ 2‐(3,4,5‐trihydroxyphenyl)‐4H‐chromen‐4‐one (18) displayed high anti‐inflammatory activity in carrageenin‐induced rat paw edema. Additionally, the inhibition of enzymes of the arachidonic acid cascade by the derivatives was investigated in vitro. In contrast to the natural compound quercetin, the com‐pounds were more potent inhibiting cyclooxygenase‐1 than 5‐lipoxygenase except for 3‐hydroxy‐7‐methoxy‐2‐(4‐methoxyphenyl)‐ 4H‐chromen‐4‐one (5). No correlation between the antiinflammatory activity in the rat paw edema test and the inhibition of 5‐lipoxygenase or cyclooxygenase‐1 could be observed. In conclusion, the present results suggest that other effects than inhibition of these enzymes of the arachidonic acid cascade are important for the anti‐inflammatory activity of the investigated alkoxyflavonols.

Natural products as inhibitors of recombinant cathepsin L of Leishmania mexicana.

Cysteine proteinases (cathepsins) from Leishmania spp. are promising molecular targets against leishmaniasis. Leishmania mexicana cathepsin L is essential in the parasite life cycle and a pivotal in virulence factor in mammals. Natural products that have been shown to display antileishmanial activity were screened as part of our ongoing efforts to design inhibitors against the L. mexicana cathepsin L-like rCPB2.8. Among them, agathisflavone (1), tetrahydrorobustaflavone (2), 3-oxo-urs-12-en-28-oic acid (3), and quercetin (4) showed significant inhibitory activity on rCPB2.8 with IC50 values ranging from 0.43 to 18.03 µM. The mechanisms of inhibition for compounds 1-3, which showed Ki values in the low micromolar range (Ki = 0.14-1.26 µM), were determined. The biflavone 1 and the triterpene 3 are partially noncompetitive inhibitors, whereas biflavanone 2 is an uncompetitive inhibitor. The mechanism of action established for these leishmanicidal natural products provides a new outlook in the search for drugs against Leishmania.

Regioisomeric 3-, 4- and 5-aminomethyl isoxazoles: synthesis and muscarinic activity

Summary A series of 3-, 4- and 5-aminomethyl isoxazoles and isoxazoles with one or two additional methyl groups at the heterocycle were synthesized in order to investigate the structural requirements, ie heterocyclic moiety, regiochemistry and length of an aminoalkyl unit, for muscarinic activity. This was assayed on isolated rabbit vas deferens (M1 receptor subtype) and isolated guinea-pig atrium (M2 receptor subtype) and ileum (M3 receptor subtype). The isoxazoles tested are one to three orders of magnitude less active than furane or oxadiazole derivatives, having similar structural characteristics except for the heterocycle. Thus, the differences in molecular point charges and charge distribution contribute to the muscarinic activity of these compounds more than small differences in molecular shape and conformational energies.

Investigations Concerning the Correlation of COX-1 Inhibitory and Hydroxyl Radical Scavenging Activity

The aim was to study the COX‐1 inhibiting efficacy in context with hydroxyl radical scavenging properties of compounds bearing a carboxylic acid and ester function, respectively. In general, the acids are more potent radical scavengers than the corresponding esters but there is no clear correlation with their COX‐1 inhibiting potencies. A feasible scavenging mechanism of carboxylic acids is discussed.

COX-2 inhibition and pain management: a review summary.

Cyclooxygenase-2 selective inhibitors have long been regarded as potent anti-inflammatory drugs for the treatment of arthritis, osteoarthritis and dysmenorrhea. The reports of cardiovascular risk and the subsequent withdrawal of rofecoxib, and recently valdecoxib, has called the therapeutic potential of coxibs into question. Currently, according to the latest decisions of the US Food and Drug Administration and European Medicines Agency, the approval of valdecoxib has been refused for 1 year due to an increased rate of cardiovascular risks and serious skin reactions. There are restrictions concerning the use of all other coxibs. The short-time use of coxibs, however, in anti-inflammatory treatment and in perioperative settings may become important in the future. Exact insights into the processes of pain modulation explain the benefit of coxibs in reducing peripheral and central nociception, and their use in pre-emptive and multimodal pain management. In the future, coxibs could be established into a personalized therapy in patients with a low cardiovascular risk, but with increased risk of gastrointestinal complications.