Ketoprofen Methyl Ester Research Articles

Formation of ketoprofen methyl ester artifact in GC–MS analysis of basic drugs in horse urine using alkaline liquid-liquid extraction

This study investigates the unexpected formation of ketoprofen methyl ester (KME) during the routine alkaline liquid–liquid extraction (LLE) process for analyzing basic drugs in horse urine samples using GC–MS analysis. An unidentified peak in the GC–MS chromatogram was observed in certain horse urine samples, identified as KME through mass spectral comparison with in situ synthesized KME. Since the KME is not a metabolite of ketoprofen present in urine, it is proposed that its formation occurs during the LLE process due to the reaction between ketoprofen contained in the urine and methanol used as the solvent of the spiked internal standard. Surprisingly, no artifact was detected when negative quality control horse urine samples (absence of ketoprofen) even when spiked with standard ketoprofen and methanol. Further investigation indicated that the presence of lipase enzymes from bacteria in specific urine samples is the key factor in the formation of the KME artifact. This hypothesis was confirmed when negative quality control horse urines were spiked with ketoprofen, methanol, and a lipase enzyme and the KME artifact was detected. Additionally, the formation of a methyl ester artifact was also detected for flunixin, a carboxylic acid NSAID drug, when negative quality control horse urines were spiked with the drug, methanol, and lipase enzyme. These findings will be valuable for scientists analyzing drugs in urine.

Characteristic Evaluation of a 11C-Labeled Leucine Analog, l-α-[5-11C]methylleucine, as a Tracer for Brain Tumor Imaging by Positron Emission Tomography.

Amino acid transporters are upregulated in many cancer cells, and system L amino acid transporters (LAT1-4), in particular, LAT1, which preferentially transports large, neutral, and branched side-chain amino acids, are considered a primary target for cancer positron emission tomography (PET) tracer development. Recently, we developed a 11C-labeled leucine analog, l-α-[5-11C]methylleucine ([5-11C]MeLeu), via a continuous two-step reaction of Pd0-mediated 11C-methylation and microfluidic hydrogenation. In this study, we evaluated the characteristics of [5-11C]MeLeu and also compared the sensitivity to brain tumors and inflammation with l-[11C]methionine ([11C]Met) to determine its potential for brain tumor imaging. Competitive inhibition experiments, protein incorporation, and cytotoxicity experiments of [5-11C]MeLeu were performed in vitro. Further, metabolic analyses of [5-11C]MeLeu were performed using a thin-layer chromatogram. The accumulation of [5-11C]MeLeu in tumor and inflamed regions of the brain was compared with [11C]Met and 11C-labeled (S)-ketoprofen methyl ester by PET imaging, respectively. Transporter assay with various inhibitors revealed that [5-11C]MeLeu is mainly transported via system L amino acid transporters, especially LAT1, into A431 cells. The protein incorporation assay and metabolic assay in vivo demonstrated that [5-11C]MeLeu was neither used for protein synthesis nor metabolized. These results indicate that MeLeu is very stable in vivo. Furthermore, the treatment of A431 cells with various concentrations of MeLeu did not change their viability, even at high concentrations (∼10 mM). In brain tumors, the tumor-to-normal ratio of [5-11C]MeLeu was more elevated than that of [11C]Met. However, the accumulation levels of [5-11C]MeLeu were lower than those of [11C]Met (the standardized uptake value (SUV) of [5-11C]MeLeu and [11C]Met was 0.48 ± 0.08 and 0.63 ± 0.06, respectively). In brain inflammation, no significant accumulation of [5-11C]MeLeu was observed at the inflamed brain area. These data suggested that [5-11C]MeLeu was identified as a stable and safe agent for PET tracers and could help detect brain tumors, which overexpress the LAT1 transporter.

Study of Membrane-Immobilized Oxidoreductases in Wastewater Treatment for Micropollutants Removal.

The development of efficient strategies for wastewater treatment to remove micropollutants is of the highest importance. Hence, in this study, we presented a rapid approach to the production of biocatalytic membranes based on commercially available cellulose membrane and oxidoreductase enzymes including laccase, tyrosinase, and horseradish peroxidase. Effective enzyme deposition was confirmed based on Fourier transform infrared spectra, whereas results of spectrophotometric measurements showed that immobilization yield for all proposed systems exceeded 80% followed by over 80% activity recovery, with the highest values (over 90%) noticed for the membrane-laccase system. Further, storage stability and reusability of the immobilized enzyme were improved, reaching over 75% after, respectively, 20 days of storage, and 10 repeated biocatalytic cycles. The key stage of the study concerned the use of produced membranes for the removal of hematoporphyrin, (2,4-dichlorophenoxy)acetic acid (2,4-D), 17α-ethynylestradiol, tetracycline, tert-amyl alcohol (anesthetic drug), and ketoprofen methyl ester from real wastewater sampling at various places in the wastewater treatment plant. Although produced membranes showed mixed removal rates, all of the analyzed compounds were at least partially removed from the wastewater. Obtained data clearly showed, however, that composition of the wastewater matrix, type of pollutants as well as type of enzyme strongly affect the efficiency of enzymatic treatment of wastewater.

Synthesis and Preclinical Evaluation of 18F-Labeled Ketoprofen Methyl Esters for Cyclooxygenase-1 Imaging in Neuroinflammation.

Cyclooxygenase (COX) is a rate-limiting enzyme in the synthesis of proinflammatory prostanoids from arachidonic acid. In vivo imaging of COX by PET is a potentially powerful tool for assessing the inflammatory response to injury, infection, and disease. We previously reported on a promising PET probe for COX imaging, 11C-labeled ketoprofen methyl ester, which can detect COX-1 activation in models of neuroinflammation and neurodegenerative disorders. In the current study, we aimed to design a fluorine-substituted benzoyl group of ketoprofen (FKTP) and to evaluate its racemate and enantiomers (18F-labeled ketoprofen methyl ester, [18F]FKTP-Me) as PET proradiotracers, potential radiopharmaceuticals for invivo PET study of COX-1. Methods: We performed nucleophilic aromatic 18F-fluorination to obtain the desired racemic radiolabeled probe, (RS)-[18F]FKTP-Me, at a radiochemical yield of 11%-13%. Subsequent high-performance liquid chromatography separation with a chiral column yielded the desired enantiomerically pure (R)- and (S)-[18F]FKTP-Me. We examined the invivo properties of (RS)-, (R)-, and (S)-[18F]FKTP-Me in PET studies using rats in which hemispheric inflammation was induced by intrastriatally injecting a lipopolysaccharide. Results: Racemic (RS)-[18F]FKTP-Me and enantiomeric (R)- or (S)-[18F]FKTP-Me were synthesized with radiochemical and chemical purities of more than 99%. The metabolite analysis revealed that the racemic (RS)-[18F]FKTP-Me crossed the blood-brain barrier and entered the brain, where it was subsequently hydrolyzed to its pharmacologically active acid form. PET images revealed a high accumulation of (R)-, (S)-, and (RS)-[18F]FKTP in the inflamed regions in rat brain. Moreover, the accumulated radioactivity of (S)-[18F]FKTP-Me was higher than that of (RS)-[18F]FKTP-Me and (R)-[18F]FKTP-Me, which was correlated with the stereospecific inhibitory activity of FKTP against COX-1. Conclusion: From the results of this study, we conclude that racemic (RS)-[18F]FKTP-Me and its enantiomers could act as proradiotracers of neuroinflammation in rat brain by the association of their hydrolyzed acid forms with COX-1 in inflamed regions. In particular, (S)-[18F]FKTP-Me demonstrated suitable properties as a COX-1-specific probe in PET imaging of neuroinflammation.

Immobilized Lipase in Resolution of Ketoprofen Enantiomers: Examination of Biocatalysts Properties and Process Characterization

In this study, lipase from Aspergillus niger immobilized by physical immobilization by the adsorption interactions and partially interfacial activation and mixed physical immobilization via interfacial activation and ion exchange was used in the kinetic resolution of the ketoprofen racemic mixture. The FTIR spectra of samples after immobilization of enzyme-characteristic signals can be seen, and an increase in particle size diameters upon immobilization is observed, indicating efficient immobilization. The immobilization yield was on the level of 93% and 86% for immobilization unmodified and modified support, respectively, whereas activity recovery reached around 90% for both systems. The highest activity of immobilized biocatalysts was observed at pH 7 and temperature 40 °C and pH 8 and 20 °C for lipase immobilized by physical immobilization by the adsorption interactions and partially interfacial activation and mixed physical immobilization via interfacial activation and ion exchange, respectively. It was also shown that over a wide range of pH (from 7 to 10) and temperature (from 20 to 60 °C) both immobilized lipases retained over 80% of their relative activity, indicating improvement of enzyme stability. The best solvent during kinetic resolution of enantiomers was found to be phosphate buffer at pH 7, which obtained the highest efficiency of racemic ketoprofen methyl ester resolution at the level of over 51%, followed by enantiomeric excess 99.85% in the presence of biocatalyst obtained by physical immobilization by the adsorption interactions and partially interfacial activation.

Stability of Ketoprofen Methylester in Plasma of Different Species.

Pharmacokinetic and pharmacodynamic assessment of ester-containing drugs can be impacted by hydrolysis of the drugs in plasma samples post blood collection. The impact is different in the plasma of different species. This study evaluated the stability of a prodrug, ketoprofen methylester (KME), in commercially purchased and freshly collected plasma of mouse, rat, dog, cat, pig, sheep, cattle and horse. KME hydrolysis was determined following its incubation in commercially purchased and freshly collected plasma of those species. Different esterase inhibitors were evaluated for prevention of the hydrolysis in rat, dog and pig plasma. KME was rapidly hydrolyzed in both commercially purchased and freshly collected plasma of mouse, rat, and horse. The hydrolysis was initially quick and then limited in cat plasma. KME hydrolysis was minimum in commercially purchased plasma of dog, pig, sheep and cattle but substantial in freshly collected plasma of those species. Different esterase inhibitors showed different effects on the stability of KME in rat, dog and pig plasma. These results indicate that plasma of different species has different hydrolytic activities to estercontaining drugs. The activities in commercially purchased and freshly collected plasma may be different and species-dependent. Esterase inhibitors have different effects on preventing hydrolysis of the ester-containing drugs in the plasma of different species.

Exploratory human PET study of the effectiveness of 11C-ketoprofen methyl ester, a potential biomarker of neuroinflammatory processes in Alzheimer's disease

IntroductionNeuroinflammatory processes play an important role in the pathogenesis of Alzheimer's disease (AD). As a biomarker of neuroinflammatory processes, we designed 11C-labeled ketoprofen methyl ester ([11C]KTP-Me) to increase the blood–brain barrier permeability of ketoprofen (KTP), a selective cyclooxygenase-1 (COX-1) inhibitor. Animal studies indicated that [11C]KTP-Me enters the brain and accumulates in activated microglia of inflammatory lesions. In a first-in-human study, we reported that [11C]KTP-Me is a safe positron emission tomography (PET) tracer and enters the brain; the radioactivity is washed out from normal cerebral tissue. Here we explored the efficacy of [11C]KTP-Me as a diagnostic biomarker of neuroinflammatory processes in AD. Methods[11C]KTP-Me was synthesized by rapid C-[11C]methylation of [11C]CH3I and the corresponding arylacetate precursor. Nine subjects (four healthy subjects, two Pittsburgh compound-B (PiB)-positive patients with mild cognitive impairment (MCI), and three PiB-positive AD patients) underwent a dynamic brain PET scan for 70min after injection. We evaluated differences in cortical retention and washout rate in the brain between healthy subjects and MCI/AD patients. ResultsA brain distribution pattern reflecting blood flow in the early-phase image was seen in both healthy subjects and MCI/AD patients. Cortical activity gradually cleared in all groups. However, we observed no obvious difference in the washout rate between healthy subjects and MCI/AD patients or between MCI and AD patients. Conclusions[11C]KTP-Me cannot be useful as a potential diagnostic biomarker for MCI/AD. Further improvements in binding affinity and specificity, etc., are needed to be a diagnostic biomarker of neuroinflammation in AD. Advances in knowledge and implications for patient care[11C]KTP-Me is a new tracer that targets COX-1. [11C]KTP-Me is expected to be a diagnostic biomarker of neuroinflammation in AD in the future. The effectiveness was limited in a small number of AD patients. Therefore, further studies are needed to clarify the usefulness of [11C]KTP-Me.

The effect of hydrogen bonding propensity and enantiomeric composition on the dynamics of supercooled ketoprofen - dielectric, rheological and NMR studies.

The aim of this work is to analyze in detail the effect of small hydrogen bonding (HB) structures and enantiomeric composition on the dynamics of glass-forming liquid ketoprofen. For that purpose dielectric relaxation, rheological and NMR studies were performed. Investigated samples are racemic ketoprofen, a single enantiomer of ketoprofen and a racemic ketoprofen methyl ester with no tendency to form HB dimers. The combination of complementary experimental techniques enables us to show that macroscopic viscosity η and α-relaxation time τα have nearly the same temperature dependencies, whereas the relation between the viscosity (or molecular reorientation) and the translational self-diffusion coefficient violates Stokes-Einstein law already at high temperature. Additionally, based on dielectric relaxation studies performed on increased pressure we were able to identify similarities and key differences in the supercooled liquid dynamics of investigated materials affected by their tendency to form intermolecular hydrogen bonds. This includes the effect of pressure on the glass transition temperature Tg, changes in the fragility parameter m and activation volume ΔV, the role of thermal energy and density fluctuations in governing the viscous liquid dynamics (Ev/Ep ratio). Finally, we have also demonstrated that the dynamic behaviour of a single enantiomer and the racemic mixture of the same compound are very much alike. Nevertheless, some slight differences were observed, particularly in the τα(T) dependencies measured in the vicinity of glass transition both at ambient and elevated pressure.

Detection of Cyclooxygenase-1 in Activated Microglia During Amyloid Plaque Progression: PET Studies in Alzheimer's Disease Model Mice.

Cyclooxygenase (COX), a prostanoid-synthesizing enzyme, is considered to be involved in the neuroinflammatory process of neurodegenerative diseases. However, the role of COX in the progression of neurodegeneration is not well understood. We hypothesized that in vivo imaging of COX by PET will contribute to elucidation of the function of COX during the neurodegenerative process in Alzheimer's disease (AD). (11)C-labeled ketoprofen methyl ester (racemic (RS)-(11)C-KTP-Me) developed recently by our group is a useful PET probe for in vivo imaging of COX-1 during neuroinflammation. The (S)-enantiomer of ketoprofen is known to be pharmacologically more active than the (R)-enantiomer. We thus synthesized (11)C-labeled (S)-ketoprofen methyl ester ((S)-(11)C-KTP-Me) as an improved PET probe specific for COX-1 and applied it for investigation of the changes in COX-1 during the progression of AD in a mouse model. The specificity of (S)-(11)C-KTP-Me for COXs was examined in PET studies with rats that had intrastriatal injection of lipopolysaccharide. To determine the details of changes in COX-1 during progression of amyloid-β (Aβ) plaque formation in amyloid precursor protein transgenic (APP-Tg) mice, we performed immunohistochemical studies and ex vivo autoradiography with (S)-(11)C-KTP-Me. PET studies using hemispheric lipopolysaccharide injection into rats revealed that the sensitivity of (S)-(11)C-KTP-Me in neuroinflammation was much higher than that of (RS)-(11)C-KTP-Me and (R)-(11)C-KTP-Me; these results closely corresponded to the inhibitory activities of each enantiomer against COX-1 estimated by an in vitro assay. In APP-Tg mice, (S)-(11)C-KTP-Me administration resulted in progressive and significant increases in accumulation of radioactivity in the brain from 16 to 24 mo old in accordance with the histopathologic appearance of abundant Aβ plaques and activated microglia, whereas few changes in radioactivity accumulation and few Aβ plaques were seen in age-matched wild-type control mice. High-radioactivity accumulation by (S)-(11)C-KTP-Me was markedly observed in the frontal cortex and hippocampus in which COX-1-expressing activated microglia tightly surrounded and enclosed large and more intensely stained Aβ plaques, indicating neuroinflammation that originated with Aβ. (S)-(11)C-KTP-Me is a potent PET probe that is highly selective for COX-1. Studies using APP-Tg mice demonstrated that (S)-(11)C-KTP-Me could detect activated microglia that are associated with amyloid plaque progression, suggesting the involvement of COX-1 in the neuroinflammatory process in AD.

In Vivo Expression of Cyclooxygenase-1 in Activated Microglia and Macrophages During Neuroinflammation Visualized by PET with 11C-Ketoprofen Methyl Ester

Cyclooxygenase (COX)-1 and -2 are prostanoid-synthesizing enzymes that play important roles in the regulation of neuroinflammation and in the development of neurodegenerative disorders. However, the specific functions of these isoforms are still unclear. We recently developed (11)C-labeled ketoprofen methyl ester as a PET probe that targets the COXs for imaging neuroinflammation, though its responsible isoform is yet to be determined. In the present study, we performed ex vivo and in vivo imaging studies with (11)C-ketoprofen methyl ester and determined the contributions of the COX isoforms during the neuroinflammatory process. To identify the COX isoform responsible for (11)C-ketoprofen methyl ester in the brain, we examined the ex vivo autoradiography of (11)C-ketoprofen methyl ester using COX-deficient mice. Time-dependent changes in accumulation of (11)C-ketoprofen methyl ester during the neuroinflammatory process were evaluated by PET in rats with hemispheric neuroinflammation induced by intrastriatal injection of lipopolysaccharide or quinolinic acid. In both rat models, cell-type specificity of COX isoform expression during neuroinflammation was identified immunohistochemically. Ex vivo autoradiographic analysis of COX-deficient mice revealed a significant reduction of (11)C-ketoprofen methyl ester accumulation only in COX-1-deficient mice, not COX-2-deficient mice. PET of rats after intrastriatal injection of lipopolysaccharide showed a significant increase in accumulation of (11)C-ketoprofen methyl ester in the inflamed area. This increase was evident at the early phase of 6 h, peaked at day 1, and then returned to basal levels by day 7. In addition, immunohistochemical analysis revealed that the population of activated microglia and macrophages was elevated at the early phase with COX-1 expression but not COX-2. A significant increase in (11)C-ketoprofen methyl ester accumulation was also observed at day 1 after intrastriatal injection of quinolinic acid, with increased COX-1-expressing activated microglia and macrophages. We have identified (11)C-ketoprofen methyl ester as a COX-1-selective PET probe, and using this, we have also demonstrated a time-dependent expression of COX-1 in activated microglia and macrophages during the neuroinflammatory process in the living brain. Thus, COX-1 may play a crucial role in the pathology of neuroinflammation and might be a critical target for the diagnosis and therapy of neurodegenerative disorders.

General Method for the 11C‐Labeling of 2‐Arylpropionic Acids and Their Esters: Construction of a PET Tracer Library for a Study of Biological Events Involved in COXs Expression

Cyclooxygenase (COX) is a critical enzyme in prostaglandin biosynthesis that modulates a wide range of biological functions, such as pain, fever, and so on. To perform in vivo COX imaging by positron emission tomography (PET), we developed a method to incorporate (11)C radionuclide into various 2-arylpropionic acids that have a common methylated structure, particularly among nonsteroidal anti-inflammatory drugs (NSAIDs). Thus, we developed a novel (11)C-radiolabeling methodology based on rapid C-[(11)C]methylation by the reaction of [(11)C]CH(3)I with enolate intermediates generated from the corresponding esters under basic conditions. One-pot hydrolysis of the above [(11)C]methylation products also allows the synthesis of desired (11)C-incorporated acids. We demonstrated the utility of this method in the syntheses of six PET tracers, [(11)C]Ibuprofen, [(11)C]Naproxen, [(11)C]Flurbiprofen, [(11)C]Fenoprofen, [(11)C]Ketoprofen, and [(11)C]Loxoprofen. Notably, we found that their methyl esters were particularly useful as proradiotracers for a study of neuroinflammation. The microPET studies of rats with lipopolysaccharide (LPS)-induced brain inflammation clearly showed that the radioactivity of PET tracers accumulated in the inflamed region. Among these PET tracers, the specificity of [(11)C]Ketoprofen methyl ester was demonstrated by a blocking study. Metabolite analysis in the rat brain revealed that the methyl esters were initially taken up in the brain and then underwent hydrolysis to form pharmacologically active forms of the corresponding acids. Thus, we succeeded in general (11)C-labeling of 2-arylpropionic acids and their methyl esters as PET tracers of NSAIDs to construct a potentially useful PET tracer library for in vivo imaging of inflammation involved in COXs expression.

An extended model for biochemical kinetic resolution of enantiomers

An extended model, which relates the parameters of the conversion ratio of the substrate ( c), the optical purity expressed as enantiomeric excess (ee), the enantiomeric ratio ( E) and the equilibrium constant ( K) in biochemical kinetic resolution of enantiomers, has been developed. The model predications have been verified experimentally via the enantioselective hydrolysis of ketoprofen methyl ester and the enantioselective esterification of ketoprofen in organic solvent.

Preparation of S-(+)-ketoprofen by coupling the enantioselective hydrolysis of ketoprofen methyl ester with the photo-oxidation of methanol

A novel method for preparation of S-(+)-ketoprofen is presented involving coupling enantioselective hydrolysis of ketoprofen methyl ester catalyzed by a surfactant-coated-lipase with the photo-oxidation of methanol in a water-saturated organic solvent. The effect of photocatalytic conversion of methanol into water and carbon dioxide on the hydrolysis of ketoprofen methyl ester and the stability of the enzyme was investigated. The photo-oxidation of methanol shifted the equilibrium of the hydrolysis toward the formation of ketoprofen, increasing the equilibrium conversion ratio and improving the enantioselectivity. Because the surfactant-coated lipase and ketoprofen methyl ester dissolved in the organic solvent and ketoprofen was absorbed on the TiO2 photocatalyst particles, the separation procedures could be simplified and the stability of the enzyme was increased.

Optimization for two-step biochemical kinetic resolution of enantiomers

A method is presented to optimize the two-step biochemical kinetic resolution of enantiomers. The optimization results were verified experimentally via the enantioselective hydrolysis of ketoprofen methyl ester and the enantioselective esterification of ketoprofen.

Design and realization of a tailor-made enzyme to modify the molecular recognition of 2-arylpropionic esters by Candida rugosa lipase

Within a research project aimed at probing the substrate specificity and the enantioselectivity of Candida rugosa lipase (CRL), computer modeling studies of the interactions between CRL and methyl (±)-2-(3-benzoylphenyl)propionate (Ketoprofen methyl ester) have been carried out in order to identify which amino acids are essential to the enzyme/substrate interaction. Different binding models of the substrate enantiomers to the active site of CRL were investigated by applying a computational protocol based on molecular docking, conformational analysis, and energy minimization procedures. The structural models of the computer generated complexes between CRL and the substrates enabled us to propose that Phe344 and Phe345, in addition to the residues constituting the catalytic triad and the oxyanion hole, are the amino acids mainly involved in the enzyme–ligand interactions. To test the importance of these residues for the enzymatic activity, site-directed mutagenesis of the selected amino acids has been performed, and the mutated enzymes have been evaluated for their conversion and selectivity capabilities toward different substrates. The experimental results obtained in these biotransformation reactions indicate that Phe344 and especially Phe345 influence CRL activity, supporting the findings of our theoretical simulations.