2-arylpropionic Acid Derivatives Research Articles

Evaluation of contactless conductivity detection for the determination of UV absorbing and non-UV absorbing species in reversed-phase high-performance liquid chromatography

A capacitively coupled contactless conductivity detector (C 4D) was used for the determination of three groups of ionizable species in reversed-phase HPLC with isocratic elution. These were non-steroidal anti-inflammatory drugs which are arylpropionic acid derivatives and represent anionic analytes, beta-blockers which are amines and therefore cationic, and zwitterionic amino acids. Optimization of the eluents led to detection limits in the order of 1 μM for all species. The precision in peak areas was typically between 0.2 and 2.1% and calibration curves were linear up to 500 μM. The determination of ibuprofen, acetylsalicylic acid and atenolol in real samples was also demonstrated by direct injection of dissolved pharmaceutical formulations into the HPLC-C 4D system.

Antioxidant Activity and Inhibition of Human Neutrophil Oxidative Burst Mediated by Arylpropionic Acid Non-steroidal Anti-inflammatory Drugs

It has been suggested that the anti-inflammatory activity of some non-steroidal anti-inflammatory drugs (NSAIDs) may be partly due to their ability to scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS), as well as to inhibit the respiratory burst of neutrophils triggered by various activating agents. Therefore, the aim of the present work was to evaluate and compare the potential scavenging activity for an array of ROS (O2*-, H2O2, HO*, ROO* and HOCl) and RNS (*NO and ONOO-) using in vitro non-cellular screening systems as well as a cellular screening system (human neutrophil oxidative burst), mediated by the arylpropionic acid derivatives (APAs) NSAIDs ibuprofen, flurbiprofen, fenoprofen, fenbufen, ketoprofen, naproxen and indoprofen. The results obtained in the present work demonstrate that under the present experimental conditions, many of the studied APA NSAIDs showed O2*- scavenging activity (fenbufen approximately equal to flurbiprofen approximately equal to indoprofen approximately equal to ketoprofen), H2O2 (ketoprofen approximately equal to indoprofen approximately equal to fenbufen>flurbiprofen>naproxen), HO* (fenoprofen approximately equal to ibuprofen>fenbufen approximately equal to flurbiprofen>ketoprofen>indoprofen approximately equal to naproxen), *NO (indoprofen>naproxen), ONOO- (indoprofen>naproxen>fenoprofen approximately equal to flurbiprofen approximately equal to ibuprofen), as well as inhibit myeloperoxidase (MPO) activity (indoprofen) and scavenge human neutrophil derived ROS (ketoprofen>indoprofen>fenbufen>flurbiprofen). The observed effects, if confirmed in vivo, may strongly contribute to the anti-inflammatory therapeutical activity observed with these NSAIDs.

Enantioselective Pharmacokinetics of Ibuprofen and Involved Mechanisms

Although dexibuprofen (S-ibuprofen) was marketed in Austria and Switzerland, the racemate at various formulations is still extensively used worldwide, and there are no indications that the racemate will be replaced by the single enantiomer. Thus, elucidation of the characteristics and involved mechanisms of the chiral pharmacokinetics of racemic ibuprofen is of special importance for the understanding of the pharmacological and toxicological consequences, and for prediction of the clinically potential drug interactions and influence of the pathological states. Stereoselective pharmacokinetics and metabolism are common features for chiral nonsteroidal antiinflammatory drugs (NSAIDs) and especially for 2-arylpropionic acid derivatives characterized with a chiral center adjacent to the carboxyl group. Although the enantioselective pharmacokinetic characteristics of different NSAIDs should be treated case by case, they share similar mechanisms underlying the protein binding, metabolism and chiral inversion. Ibuprofen was the most extensively researched drug in terms of chiral characteristics and mechanisms. Therefore, elucidation of the mechanisms derived from research on ibuprofen may provide better understanding and prediction of other chiral drugs. This article attempts to elucidate the chiral pharmacokinetics and involved mechanisms of ibuprofen in comparison with other NSAIDs based on recent developments. Topics on history of ibuprofen, enantioselective analysis method, absorption, protein binding, conventional metabolism, metabolic chiral inversion, gene polymorphism, and biochemical developments were included. It is worth mentioning that some underlying biochemical mechanisms, especially for the metabolic chiral inversion and ethnic differences still remain to be seen. Further research is required to develop human-resourced researching model and to provide more evidence concerning the site of inversion, species variation, CYP450 gene polymorphisms, and biochemical mechanisms.

Enantioselective Pharmacokinetics of Ibuprofen and Involved Mechanisms

Although dexibuprofen (S-ibuprofen) was marketed in Austria and Switzerland, the racemate at various formulations is still extensively used worldwide, and there are no indications that the racemate will be replaced by the single enantiomer. Thus, elucidation of the characteristics and involved mechanisms of the chiral pharmacokinetics of racemic ibuprofen is of special importance for the understanding of the pharmacological and toxicological consequences, and for prediction of the clinically potential drug interactions and influence of the pathological states. Stereoselective pharmacokinetics and metabolism are common features for chiral nonsteroidal antiinflammatory drugs (NSAIDs) and especially for 2-arylpropionic acid derivatives characterized with a chiral center adjacent to the carboxyl group. Although the enantioselective pharmacokinetic characteristics of different NSAIDs should be treated case by case, they share similar mechanisms underlying the protein binding, metabolism and chiral inversion. Ibuprofen was the most extensively researched drug in terms of chiral characteristics and mechanisms. Therefore, elucidation of the mechanisms derived from research on ibuprofen may provide better understanding and prediction of other chiral drugs. This article attempts to elucidate the chiral pharmacokinetics and involved mechanisms of ibuprofen in comparison with other NSAIDs based on recent developments. Topics on history of ibuprofen, enantioselective analysis method, absorption, protein binding, conventional metabolism, metabolic chiral inversion, gene polymorphism, and biochemical developments were included. It is worth mentioning that some underlying biochemical mechanisms, especially for the metabolic chiral inversion and ethnic differences still remain to be seen. Further research is required to develop human-resourced researching model and to provide more evidence concerning the site of inversion, species variation, CYP450 gene polymorphisms, and biochemical mechanisms.

Chiral Inversion of Drugs: Coincidence or Principle?

2-arylpropionic acid derivatives are probably the most frequently cited drugs exhibiting the phenomenon that is best known as chiral inversion. One enantiomer of drug is converted into its antipode either in the presence of a solvent or more often in inner environment of an organism. Mechanistic studies of the metabolic chiral inversion were carried out for several drugs from NSAIDs, and a model of this inversion was suggested and subsequently confirmed. The chiral inversion of NSAIDs has been intensively studied in the context of the pharmacological and toxicological consequences. However, the group of NSAIDs is not the sole group of drugs in which the inversion phenomenon can be observed. There exist several other drugs that also display chiral inversion of one or even both of their enantiomers. These drugs belong to different pharmacotherapeutic groups as monoamine oxidase inhibitors, antiepileptic drugs, drugs used in the treatment of hyperlipoproteinemia or drugs that are effective in the treatment of leprosy. Moreover, some chiral or prochiral drugs are metabolized to give chiral metabolites that undergo chiral inversion too, which can have direct impact on pharmacological properties or toxicity of the drug. As the process of chiral inversion is affected by several factors, so the intensity of chiral inversion of individual substances and at different conditions can differ considerably. Interspecies differences and types of tissue are reported to be the main factors that were recognized to play the key role in the process of chiral inversion. Some of more recent studies have revealed that several other factors, such as the route of administration or interaction with other xenobiotics, can influence the enantiomeric conversion, too. Chiral inversion does not seem to be a phenomenon connected with only several drugs from some unique group of 2-arylpropionic acid derivatives: it is also observed in drugs with rather different chemical structures and is much more frequent than it can be realized.

High performance capillary electrophoresis for determination of the enantiomers of 2-arylpropionic acid derivatives in human serum: Pharmacokinetic studies of ketoprofen enantiomers following administration of standard and sustained release tablets

A stereospecific capillary zone electrophoresis (CZE) method for determination of the enantiomers of some 2-arylpropionic acid derivatives (2-APA, profens) in human serum has been developed. Racemic-ibuprofen (rac-IBP), racemic-flurbiprofen (rac-FBP), racemic-ketoprofen (rac-KTP) and (+)-S-naproxen ((+)-S-NPX—an internal standard) were chosen for these studies. The 2-APA enantiomers were extracted from acidified serum samples using methylene chloride separated in a fused silica capillary. The capillary was filled with a background electrolyte, which consisted of 0.05M heptakis 2,3,6-tri-O-methyl-β-cyclodextrin (TMβCD) (chiral selector) in 0.02M triethanolamine-phosphate buffer of pH 5.0. Separation and resolution of the enantiomer mixture were obtained in one analytical run. The calculated electrophoretic parameters of the analytes were as follows: electrophoretic mobility, μep(−)-R=−0.75×10−4 to −0.30×10−4cm2/Vs; μep(+)-S=−0.83–(−0.38) cm2/Vs and electroosmotic mobility, μEOF=2.35×10−4cm2/Vs, migration times, tmigrR=12.55–16.07min; tmigrS=13.08–16.9min, resolution factors, RS=1.88–3.70 and chiral selectivity, α=1.16–1.34. The method developed for the enantiomers was validated. The calibration curves were linear in the range of 0.5–50.0μg/ml for FBP or KTP and of 1.0–50.0μg/ml for IBP enantiomer concentrations. Recovery of the enantiomers from serum was about 90%. At the limit of quantification (LOQ) precision and accuracy were within 15%. The validated method was successfully applied to pharmacokinetic and bioavailability studies on KTP enantiomers in humans after administration of standard and sustained release tablets of rac-KTP. Significant differences in the pharmacokinetic parameters of both formulations were observed and the studied formulations were not bioequivalent.

Diffusion of arylpropionate non-steroidal anti-inflammatory drugs into the cerebrospinal fluid: a quantitative structure-activity relationship approach.

A quantitative structure-activity relationship (QSAR) analysis of a series of arylpropionic acid non-steroidal anti-inflammatory drugs (NSAIDs) has been performed to determine which physicochemical properties of these compounds are involved in their diffusion into the cerebrospinal fluid (CSF). The penetration of eight arylpropionic acid derivatives into CSF was studied in male Wistar rats. After intraperitoneal administration of each compound (5 mg/kg), blood and CSF samples were collected at different times (0.5, 1, 3 and 6 h). The fraction unbound to plasma protein was determined using ultrafiltration. The areas under the curve of the free plasma (AUCF) and CSF (AUCCSF) concentrations were calculated according to the trapezoidal rule. The overall drug transit into CSF was estimated by the ratio RAUC (AUCCSF : AUCF). The lipophilicity was expressed as the chromatographic capacity factor (log kIAM) determined by high-performance liquid chromatography on an immobilized artificial membrane (IAM) column. A significant parabolic relationship was sought between lipophilicity (log kIAM) and the capacity of diffusion across the blood-brain barrier (log RAUC) (r = 0.928; P < 0.01). The arylpropionic acid NSAIDs exhibiting a lipophilicity value between 1.1 and 1.7 entered the CSF easily (RAUC > 1). The molecular weight (MW) was included in this parabolic relationship by means of a multiple regression analysis. This physicochemical parameter improved the correlation (r = 0.976; P < 0.005). Based on our findings, diffusion of arylpropionic acid NSAIDs into CSF appears to depend primarily on their lipophilicity and MW.

Different in vitro activity of flurbiprofen and its enantiomers on human articular cartilage

The 2-arylpropionic acid derivatives or ‘profens’ are an important group of non-steroidal anti-inflammatory drugs that have been used for the symptomatic treatment of various forms of arthritis. These compounds are chiral and the majority of them are still marketed as racemate although it is known that the ( S)- form is the principal effective in the cyclooxygenase inhibition. However, recent findings suggest that certain pharmacological effect of 2-arylpropionic acids cannot be attributed exclusively to the ( S)-(+) enantiomer. To obtain further insights into the pharmacological effect of profens, the present study investigated the influence of racemic and pure enantiomers of flurbiprofen on the production of nitric oxide and glycosaminoglycans, key molecules involved in cartilage destruction. The culture of human articular cartilage stimulated by interleukin-1β (IL-1β), which plays an important role in the degradation of cartilage, has been established, as a profit experimental model, for reproducing the mechanisms involved in the pathophysiology of arthritic diseases. Our results show that mainly ( S)-(+)-flurbiprofen decreases, at therapeutically concentrations, the IL-1β induced cartilage destruction.

Effects of oxaprozin and of other 2-arylpropionic acid derivatives on nuclear factor κB(NF-κB) activation

The translocation to the nucleus and the binding of NF-κB to DNA is thought to play a fundamental role in the activation of immune and inflammatory cells in order to activate the genes that in turn produce cytokines. We evaluated the ability of some 2-arylpropionic acid derivatives (fenoprofen, flurbiprofen, ketoprofen, S- and R-isomers of ibuprofen, ibuprofen, naproxen, and oxaprozin) to affect NF-κB binding to DNA in human peripheral mononuclear cells. We observed NF-κB inhibition by oxaprozin (IC50 = 50 μM), ibuprofen (185 μM) and S-ibuprofen (51 μM). Since oxaprozin reaches higher concentrations in synovial tissues and fluid (4—5 times higher than in plasma, i.e. around 100 μM) its inhibitory activity is clinically relevant. The inhibitory activity observed with all other tested drugs was outside their clinically relevant concentrations. At the concentrations observed to affect the NF-κB binding, oxaprozin and ibuprofen also inhibited the release of TNF-α and interleukin 1β in cells stimulated by Escherichia coli lipolysaccharide. By administering oxaprozin or ibuprofen at high dosage it should be possible to inhibit NF-κB DNA binding in humans in vivo.

Pharmacology of chiral compounds: 2-arylpropionic acid derivatives.

Molecules exist as three dimensional structures. Therefore they can exist in symmetrical and asymmetrical forms. Molecules with an asymmetric centre are chiral. If the molecule and its mirror image are non-superimposable, the relationship between the two molecules is enantiomeric and the two stereoisomers are enantiomers. Since enantiomers have very similar or identical physicochemical properties, it is very difficult to distinguish between them in an achiral environment. However, once in a chiral environment, as in the body, they exhibit clear differences. In fact, most of the physiological processes in nature are stereospecific. Stereospecificity can occur in pharmacokinetic processes, in particular that utilise a carrier protein, receptor or enzyme. In addition, stereoselectivity occurs in pharmacodynamic processes and the differences between enantiomers can be either qualitative and quantitative. 2-arylpropionic acid derivatives (2APAs - profens) are an important subgroup within the class of NSAIDs. These are chiral compounds marketed as racemic mixtures. Some members of the group in an species-dependent manner undergo a special type of metabolic transformation leading to partial inversion to the optical antipode through a specific conjugation with CoA (coenzyme A) and subsequent epimerization. This metabolic inversion has not only pharmacological consequences (related to clinical effect) but also toxicological consequences such as, formation of hybrid triglycerides and even inhibition of fatty acid beta-oxidation. Differences on inversion rate between compounds and species will be discussed as well as its modification by different patho-physiologic processes such as, inflammation.

Expression of rat liver long-chain acyl-CoA synthetase and characterization of its role in the metabolism of R-ibuprofen and other fatty acid-like xenobiotics

Our investigations of fatty acid metabolism and epimerization of the 2-arylpropionic acid derivative, R-ibuprofen, resulted in the successful purification of an acyl-CoA synthetase from rat liver microsomes that catalyzes the formation of both palmitoyl-CoA and R-ibuprofenoyl-CoA. To investigate whether R-ibuprofenoyl-CoA synthetase and long-chain acyl-CoA synthetase (LACS) are identical enzymes, we cloned the cDNA from LACS into the pQE30 expression vector and transformed the construct into Escherichia coli M15[pREP4]. Induction of the bacterial protein synthesis with 0.2 mM isopropyl-beta- d-galactoside resulted in a strong, time-dependent increase in LACS protein as determined by Western blot analysis using a polyclonal rabbit anti-LACS antibody. Incubations of the recombinantly expressed protein with palmitic acid as physiological LACS substrate or R-ibuprofen in the presence of Mg 2+, ATP, and CoA resulted in a 5-fold increase in the thioesterification of both substrates. Western blot analysis using tissue homogenates of rat liver, heart, kidney, lung, brain, and ileum showed that LACS was found in every tissue investigated, with the greatest expression in the liver. Similar results were obtained with activity measurements using R-ibuprofen and palmitic acid as substrates. Northern blot analysis revealed a hybridization with a 3.8-kb mRNA transcript in rat liver, heart, and kidney, but no signal was observed in lung, brain and ileum, suggesting the expression of different LACS isoform(s) in these organs. In summary, our results further show that R-ibuprofenoyl-CoA synthetase and long-chain acyl-CoA synthetase are identical enzymes that are involved in the metabolism of various xenobiotics.

Uniform-sized molecularly imprinted polymers for 2-arylpropionic acid derivatives selectively modified with hydrophilic external layer and their applications to direct serum injection analysis.

Uniform-sized molecularly imprinted polymers (MIPs) for (S)-naproxen and -ibuprofen selectively modified with hydrophilic external layer, restricted access media (RAM)-MIPs, have been prepared. First, the MIP for (S)-naproxen or -ibuprofen was prepared using 4-vinylpyridine and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multistep swelling and thermal polymerization method. Next, a 1:1 mixture of glycerol monomethacrylate and glycerol dimethacrylate was used for hydrophilic surface modification, and it was added directly to the MIP for (S)-naproxen or -ibuprofen 4 h after the start of molecular imprinting. The obtained RAM-MIP material for (S)-naproxen or -ibuprofen was applied for direct serum injection assays of the drug by a column-switching system, consisting of a RAM-MIP material and conventional C18-silica column. However, leakage of the imprint molecule prevented accurate and precise assays of the drug. This problem has been overcome by using the RAM-MIP for (S)-naproxen for the assays of ibuprofen in rat plasma. The optimized column-switching system was applied successfully to the assay of ibuprofen in rat plasma after oral administration.

Photosensitivity to Ketoprofen

The topical use of nonsteroidal anti-inflammatory drugs (NSAIDs), widely used for moderate acute and chronic painful conditions, is one of several strategies used to improve the tolerability profile of NSAIDs, particularly with regard to gastric and renal adverse effects. However, topical NSAIDs can induce photosensitivity. Among the different NSAIDs used topically, ketoprofen has often been implicated in photosensitivity reactions. Photosensitivity includes both phototoxic and photoallergic reactions. Phototoxicity can be studied in the cell system and on biological targets such as cellular membranes or DNA. In hepatocyte cultures, data suggest that radical intermediates play a role in ketoprofen-photosensitised damage by cell membrane lysis. Photosensitised lysis of red blood cells has been employed as an indicator of membrane damage. Ketoprofen irradiation promotes the photolysis of erythrocyte suspensions. The drug is able to induce photoperoxidation of linoleic acid in the photo-induced lipid peroxidation process. The results obtained from the addition of radical scavengers suggest the involvement of free radicals in these processes. Ketoprofen may induce DNA damage in vitro upon irradiation. DNA, in the presence of ketoprofen, undergoes single strand breaks involving hydroxyl radicals as evidenced by the use of scavengers. Simultaneously with single strand breaks, pyrimidine dimers are formed by an energy transfer mechanism. The oxygen-dependence of both processes suggest competition between a radical process leading to DNA cleavage and a poorly efficient energy transfer between ketoprofen and pyrimidines at the origin of the dimerisation process. Photoallergy is due to a cell-mediated hypersensitivity response involving immunological reactions. Therefore, it only occurs in previously sensitised individuals and requires a latency period of sensitisation. Among NSAIDs, ketoprofen is the main drug involved in this photoallergic contact dermatitis. Cross-sensitivity reactions with other arylpropionic acid derivatives, such tiaprofenic acid, fenofibrate or oxybenzone-harbouring benzoyl ketone or benzophenone may also occur. Finally, the higher frequency of such adverse reactions with ketoprofen could be accounted for by its chemical structure and the variety of chemical reactions that give rise to phototoxic effects. The widespread and repeated use of these agents may lead to sensitisation, incurring a greater risk of systemic allergic reactions with oral NSAIDs or other drugs recognised to induce cross-reactions. Physicians and pharmacists should advise patients and inform them of the risks of topical NSAIDs which are often dispensed as over the counter drugs.

Molecularly imprinted uniform-sized polymer-based stationary phase for naproxen: Comparison of molecular recognition ability of the molecularly imprinted polymers prepared by thermal and redox polymerization techniques

A molecularly imprinted uniform-sized polymer-based stationary phase for ( S)-naproxen has been prepared by a multi-step swelling and thermal or redox polymerization method using 4-vinylpyridine and ethylenedimethacrylate as a host functional monomer and cross-linker, respectively. The obtained molecularly imprinted polymers were evaluated using aqueous-rich mobile phases. The molecularly imprinted polymer materials could separate naproxen enantiomers. Further, the materials showed high selectivity for naproxen and moderate selectivity for other 2-arylpropionic acid derivatives. On the other hand, the molecularly imprinted polymer materials showed little selectivity for other acidic compounds, and basic and neutral compounds. With regard to comparison of the molecularly imprinted polymers prepared by thermal and redox polymerization techniques, the latter materials gave higher retentivity and enantioselectivity than the former materials, while the former materials gave higher column efficiency than the latter materials. Thus, naproxen enantiomers were resolved much better on the molecularly imprinted polymer material prepared by a thermal polymerization method.

Photocontact dermatitis from ketoprofen and tiaprofenic acid: cross-reactivity study in 12 consecutive patients.

The arylpropionic acid derivatives (APADs) ketoprofen and tiaprofenic acid can provoke photoallergic dermatitis. Possible cross-reactivity between APADs is of importance in patients using nonsteroidal anti-inflammatory drugs. Because of the similarities in chemical structures, we investigated patients with photoallergy to ketoprofen or tiaprofenic acid, in order to study cross-reactivity between APADs and a possible pattern of cross-reactivity between benzophenone-containing molecules, so as to determine the molecular basis of photoallergy to ketoprofen or tiaprofenic acid. 10 patients with photoallergy to topical ketoprofen, 2 with photoallergy to oral tiaprofenic acid, and 15 control subjects with no history of contact dermatitis from APADs, nor from benzophenone-containing molecules, were photopatch tested in triplicate with ketoprofen, tiaprofenic acid, other APADs (alminoprofen, fenoprofen, flurbiprofen, ibuprofen and naproxen), benzophenone-containing molecules (fenofibrate, oxybenzone, sulisobenzone), and unsubstituted benzophenone. 1 set was irradiated with UVA light, 1 with solar-simulated irradiation and 1 dark control. Tests were read at 2, 3 and 4 days. Patients reacted to both ketoprofen and tiaprofenic acid (12/12), fenofibrate (8/12), oxybenzone (3/12) and unsubstituted benzophenone (11/12), but not to other APADs, nor to sulisobenzone. Tests remained negative in control patients. Photoallergy is due to the benzophenone moiety of ketoprofen, or to the very similar thiophene-phenylketone of tiaprofenic acid, but not to their arylpropionic function. This induces cross-reactivity to fenofibrate and oxybenzone but not to APADs without a benzophenone moiety, which may therefore probably be used in such patients. Unsubstituted benzophenone should be added to standard phototesting series.

A mechanistic investigation into the microbial chiral inversion of 2-arylpropionic acids using deuterated derivatives of 2-phenylpropionic acid

Cordyceps militaris has been previously shown to invert the chirality of ( r )-2-phenylpropionic acid to its antipode in the absence of any other biotransformation. To investigate the mechanism of this unusual biotransformation. ( r, s )-[2- 2H 1]-2-phenylpropionic acid, ( r, s )-[2- 2H,3,3,3- 2H 3]-2-phenylpropionic acid, and ( r, s )-[3,3,3- 2H 3]-2-phenylpropionic acid were synthesized and incubated with C. militaris. NMR spectroscopy showed that deuterium exchange of the α-methine proton occurred during the inversion process but there was no exchange of the β-methyl protons. There were no significant differences in the rates of chiral inversion of the three deuterated derivatives and the undeuterated compound, and with all the compounds attaining 84% enantiomeric excess of the ( s )-enantiomer after 48 h incubation. The deuterium exchange is not therefore the rate limiting process in this biotransformation. The recovery profiles demonstrated that there was rapid metabolism of the β-methyl deuterated derivatives which did not occur for the undeuterated 2-phenylpropionic acid or the derivative which was only deuterated at the α-carbon. These studies clearly illustrate that C. militaris is a useful in vitro model of the chiral inversion of 2-arylpropionic acid derivatives in mammalian systems.

Dual effects of anti-inflammatory 2-arylpropionic acid derivatives on a major isoform of human liver 3alpha-hydroxysteroid dehydrogenase.

Nonsteroidal anti-inflammatory drugs have been shown to be potent inhibitors of mammalian 3alpha-hydroxysteroid dehydrogenase. Here, we report that the drugs of the 2-arylpropionic acid class act as both activators and inhibitors for a predominant isoform of the human liver enzyme which is involved in the metabolism of steroid hormones, bile acids, drug ketones and xenobiotic aromatic hydrocarbons. Flurbiprofen, fenoprofen, ibuprofen, naproxen, ketoprofen and suprofen stimulated the activity of the human enzyme (1.5-2.4-fold) at low concentrations of less than 20-100 microM, whereas at higher concentrations they inhibited the activity. Comparison of the effects of the structurally related compounds with the drugs revealed that the essential structure required as the activator molecule is 2-phenylpropionic acid with a hydrophobic substituent on the aromatic ring. In addition, an R-enantiomer of ibuprofen showed higher activation (3-fold) than its S-enantiomer. Kinetic analysis with respect to NADP+ concentration indicated that R- and S-ibuprofens are nonessential activators showing binding constants of 23 and 34 microM, respectively. Neither enantiomers activated, but rather inhibited the enzyme, with Met replacing Arg-276 which has been shown to interact with the 2'-phosphate of NADP+. The inhibitions of the mutant enzyme by R- and S-ibuprofens were competitive with respect to the substrate, giving Ki values of 95 and 18 microM, respectively. The results suggest that the human liver 3alpha-hydroxysteroid dehydrogenase isoform possesses the two distinct sites, activator and inhibitor sites, to which anti-inflammatory 2-arylpropionates stereoselectively bind.

Molecularly Imprinted Uniform-Sized Polymer-Based Stationary Phase for Naproxen

Abstract A molecularly imprinted uniform-sized polymer-based stationary phase for (S)-naproxen has been prepared using 4-vinylpyridine and ethylenedimethacrylate as a host functional monomer and cross-linker, respectively. The imprinted polymer material could separate naproxen enantiomers with separation factor of 1.74. Further, the materials showed high selectivity for naproxen and moderate selectivity for other 2-arylpropionic acid derivatives. On the other hand, the imprinted polymer material showed little selectivity for other acidic, basic and neutral compounds.

1-(5-Dimethylamino-1-naphthalenesulphonyl)-(S)-3-aminopyrrolidine (DNS-Apy) as a fluorescence chiral labelling reagent for carboxylic acid enantiomers.

1-(5-Dimethylamino-1-naphthalenesulphonyl)-(S)-3-aminopyrrolidi ne (DNS-Apy) has been synthesized for the separation of carboxylic acid enantiomers by high-performance liquid chromatography (HPLC) and sensitive detection. The reagent reacts with carboxylic acids at room temperature in the presence of activation agents 2,2'-dipyridyl disulphide (DPDS) and triphenylphosphine (TPP). The maximum emission of the diastereomeric amide derived from (S)-phenylpropionic acid and ketoprofen derivatives of DNS-Apy was at 530 nm with excitation at 340 nm. The emission wavelength shifted towards the blue and the fluorescence intensities increased with increasing acetonitrile concentration in the medium. The diastereomers derived from anti-inflammatory drugs were efficiently resolved with a reverse-phase column using water:acetonitrile mixture as mobile phase. All of the racemate of arylpropionic acid derivatives gave equal fluorescence intensity of the two enantiomers with the exception of ketoprofen derivatives where the intensity of the first eluting enantiomer was half that of the second.

Enantioselectivity of bovine serum albumin-bonded columns produced with isolated protein fragments: II. Characterization of protein fragments and chiral binding sites

Enantioselectivity of bovine serum albumin (BSA)-bonded columns produced with isolated protein fragments has been investigated. The BSA fragment, BSA-FG75, was isolated by size exclusion chromatography following peptic digest of BSA. The isolated BSA-FG75 was further fractionated to two fractions, BSA-F1 and BSA-F2, by anion-exchange chromatography. BSA-F1 and BSA-F2 had molecular mass of about 35 000 daltons, estimated by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. BSA-F1 has amino acid residues 1–307 estimated by electrospray ionization (ESI) mass spectrometry, while BSA-F2 is an N-terminal half BSA fragment. The BSA, BSA-FG75, BSA-F1 and BSA-F2 proteins were bound to aminopropyl-silica gels activated by N, N′-disuccinimidyl carbonate. The bound amounts of the BSA fragments were 2.2–2.7 times more than that of the intact BSA. Chiral recognition of 2-arylpropionic acid derivatives, benzodiazepines, warfarin and benzoin was obtained with the BSA fragment-bonded columns. The non-enantioselective interactions of benzoin and benzodiazepines except for clorazepate with BSA fragments were increased with protein surface coverages, while those of 2-arylpropionic acid, clorazepate and warfarin were decreased. The BSA fragment columns gave higher enantioselectivity for lorazepam and benzoin, and lower enantioselectivity for other compounds tested, compared with the BSA column. These results might be due to changes in the globular structure of the BSA fragment and/or changes in the local environment around the binding sites.