Design, synthesis, anticancer evaluation, and molecular modelling studies of novel tolmetin derivatives as potential VEGFR-2 inhibitors and apoptosis inducers.

Novel tolmetin derivatives 5a-f to 8a-c were designed, synthesised, and evaluated for antiproliferative activity by NCI (USA) against a panel of 60 tumour cell lines. The cytotoxic activity of the most active tolmetin derivatives 5b and 5c was examined against HL-60, HCT-15, and UO-31 tumour cell lines. Compound 5b was found to be the most potent derivative against HL-60, HCT-15, and UO-31 cell lines with IC50 values of 10.32 ± 0.55, 6.62 ± 0.35, and 7.69 ± 0.41 µM, respectively. Molecular modelling studies of derivative 5b towards the VEGFR-2 active site were performed. Compound 5b displayed high inhibitory activity against VEGFR-2 (IC50 = 0.20 µM). It extremely reduced the HUVECs migration potential exhibiting deeply reduced wound healing patterns after 72 h. It induced apoptosis in HCT-15 cells (52.72-fold). This evidence was supported by an increase in the level of apoptotic caspases-3, -8, and -9 by 7.808-, 1.867-, and 7.622-fold, respectively. Compound 5b arrested the cell cycle in the G0/G1 phase. Furthermore, the ADME studies showed that compound 5b possessed promising pharmacokinetic properties.

Radiosynthesis of a Bruton's tyrosine kinase inhibitor, [11 C]Tolebrutinib, via palladium-NiXantphos-mediated carbonylation.

Bruton's tyrosine kinase (BTK) is a key component in the B-cell receptor signaling pathway and is consequently a target for in vivo imaging of B-cell malignancies as well as in multiple sclerosis (MS) with positron emission tomography (PET). A recent Phase 2b study with Sanofi's BTK inhibitor, Tolebrutinib (also known as [a.k.a.] SAR442168, PRN2246, or BTK'168) showed significantly reduced disease activity associated with MS. Herein, we report the radiosynthesis of [11 C]Tolebrutinib ([11 C]5) as a potential PET imaging agent for BTK. The N-[11 C]acrylamide moiety of [11 C]5 was labeled by 11 C-carbonylation starting from [11 C]CO, iodoethylene, and the secondary amine precursor via a novel palladium-NiXantphos-mediated carbonylation protocol, and the synthesis was fully automated using a commercial carbon-11 synthesis platform (TracerMaker™, Scansys Laboratorieteknik). [11 C]5 was obtained in a decay-corrected radiochemical yield of 37 ± 2% (n = 5, relative to starting [11 C]CO activity) in >99% radiochemical purity, with an average molar activity of 45 GBq/µmol (1200 mCi/µmol). We envision that this methodology will be generally applicable for the syntheses of labeled N-acrylamides.

The influence of physicochemical properties on the reactivity and stability of acyl glucuronides †.

1. Formation of 1-O-acyl-ß-d-glucuronide conjugates is a significant pathway in the metabolism of drugs containing a carboxylic acid group. The formation of acyl glucuronides results in an increase in both the aqueous solubility and molecular mass of the conjugate in comparison to the parent drug and thus facilitates excretion in both urine and bile. 2. Acyl glucuronides are effectively esters, which undergo first order decomposition by both hydrolysis and the intra-migration of the acyl group around the glucuronide ring to yield 2-, 3- and 4-O-glucuronic acid esters which, unlike the metabolically formed 1-O-acyl-ß-d-glucuronides, are not substrates for ß-glucuronidase. The first order degradation half-life is therefore a composite value of these two reactions and a useful indicator of chemical reactivity and potential toxicity. 3. Intra-molecular migration is expected to be the predominant pathway due to entropic considerations. 4. Such conjugates, together with their isomeric ester derivatives, react with nucleophilic sites on proteins and small endogenous molecules, such as glutathione, which potentially contributes to the observed toxicity and adverse drug reactions associated with some drugs. 5. Examination of the stability of the 1-O-acyl-ß-d-glucuronides of aryl acetic acid, α-carbon substituted aryl acetic acid, aliphatic and aromatic acids, as determined by their first order degradation half-lives, indicates the significance of electronic and steric features that contribute to conjugate stability under physiological conditions. 6. Examination of the of the electronic properties of the carbonyl carbon atom in acyl glucuronides, as measured by the pKa of the parent acid, together with the steric substituents about the acyl carbonyl provides insight into the reactivity of these conjugates. 7. The investigations reported herein on a large number of 1-O-acyl-ß-d-glucuronides has allowed rationalisation of their physicochemical properties in relation to the structure of the parent drug and has the potential to contribute to the design of carboxylic acid containing drug molecules with increased stability of a major metabolite with potential reduction in toxicity and adverse drug reactions.

Isotope labelling by reduction of nitriles: Application to the synthesis of isotopologues of tolmetin and celecoxib.

The aryl methyl group is found in many drug-like compounds, but there are limited ways of preparing compounds with an isotope label in this methyl position. The process of cyanation of an aryl halide followed by complete reduction of the nitrile to a methyl group was investigated as a route for preparing stable and radiolabelled isotopologues of drug-like compounds. Using this methodology, carbon-13, deuterium, carbon-14, and tritium labelled isotopologues of the nonsteroidal anti-inflammatory drug tolmetin were produced, as well as carbon-13, deuterium, and carbon-14 labelled isotopologues of another nonsteroidal anti-inflammatory drug, celecoxib. The radiolabelled compounds were produced at high specific activity and the stable isotope labelled compounds with high incorporation making them suitable for use as internal standards in mass spectrometry assays. This approach provides a common synthetic route to multiple isotopologues of compounds using inexpensive and readily available labelled starting materials.

Toxicity of Carboxylic Acid-Containing Drugs: The Role of Acyl Migration and CoA Conjugation Investigated.

Many carboxylic acid-containing drugs are associated with idiosyncratic drug toxicity (IDT), which may be caused by reactive acyl glucuronide metabolites. The rate of acyl migration has been earlier suggested as a predictor of acyl glucuronide reactivity. Additionally, acyl Coenzyme A (CoA) conjugates are known to be reactive. Here, 13 drugs with a carboxylic acid moiety were incubated with human liver microsomes to produce acyl glucuronide conjugates for the determination of acyl glucuronide half-lives by acyl migration and with HepaRG cells to monitor the formation of acyl CoA conjugates, their further conjugate metabolites, and trans-acylation products with glutathione. Additionally, in vitro cytotoxicity and mitochondrial toxicity experiments were performed with HepaRG cells to compare the predictability of toxicity. Clearly, longer acyl glucuronide half-lives were observed for safe drugs compared to drugs that can cause IDT. Correlation between half-lives and toxicity classification increased when "relative half-lives," taking into account the formation of isomeric AG-forms due to acyl migration and eliminating the effect of hydrolysis, were used instead of plain disappearance of the initial 1-O-ß-AG-form. Correlation was improved further when a daily dose of the drug was taken into account. CoA and related conjugates were detected primarily for the drugs that have the capability to cause IDT, although some exceptions to this were observed. Cytotoxicity and mitochondrial toxicity did not correlate to drug safety. On the basis of the results, the short relative half-life of the acyl glucuronide (high acyl migration rate), high daily dose and detection of acyl CoA conjugates, or further metabolites derived from acyl CoA together seem to indicate that carboxylic acid-containing drugs have a higher probability to cause drug-induced liver injury (DILI).

The influence of API concentration on the roller compaction process: modeling and prediction of the post compacted ribbon, granule and tablet properties using multivariate data analysis.

OBJECTIVE: While previous research has demonstrated roller compaction operating parameters strongly influence the properties of the final product, a greater emphasis might be placed on the raw material attributes of the formulation. There were two main objectives to this study. First, to assess the effects of different process variables on the properties of the obtained ribbons and downstream granules produced from the rolled compacted ribbons. Second, was to establish if models obtained with formulations of one active pharmaceutical ingredient (API) could predict the properties of similar formulations in terms of the excipients used, but with a different API. MATERIALS AND METHODS: Tolmetin and acetaminophen, chosen for their different compaction properties, were roller compacted on Fitzpatrick roller compactor using the same formulation. Models created using tolmetin and tested using acetaminophen. The physical properties of the blends, ribbon, granule and tablet were characterized. Multivariate analysis using partial least squares was used to analyze all data. RESULTS: Multivariate models showed that the operating parameters and raw material attributes were essential in the prediction of ribbon porosity and post-milled particle size. The post compacted ribbon and granule attributes also significantly contributed to the prediction of the tablet tensile strength. CONCLUSIONS: Models derived using tolmetin could reasonably predict the ribbon porosity of a second API. After further processing, the post-milled ribbon and granules properties, rather than the physical attributes of the formulation were needed to predict downstream tablet properties. An understanding of the percolation threshold of the formulation significantly improved the predictive ability of the models.

The effect of inorganic salt type and concentration on hydrophilic drug loading into microspheres using the emulsion/solvent diffusion method.

AIM: In order to avoid gastric irritation caused by tolmetin sodium (TS), gastro resistant Eudragit® S 100 microsphere formulations were prepared with the emulsion/solvent diffusion method. MATERIALS: Considering the high water solubility of the TS molecule, the effects of the presence of inorganic salt (NaCl, NaBr and KH2PO4; 0.1 M and 1.0 M) in external phase and external phase pH on the encapsulation efficiency were evaluated. RESULTS: Percentage yield value was found to vary between 55.8% and 72.1%. Improvement in encapsulation efficiency was determined by increasing concentrations of NaCl, NaBr and KH2PO4. The microspheres were observed to have a spherical shape and the measured particle size values varied between 52.1 and 81.5 µm. The released amounts of the drug were found to be low as the inorganic salt concentrations increased. CONCLUSION: Conclusively, drug release in stomach pH was significantly prevented by the microspheres prepared using Eudragit® S 100 polymer, and these formulations are considered to be a model for other orally administered drugs with similar problems.

Spectroscopic investigation of tolmetin interaction with human serum albumin.

The interaction of tolmetin (TOL) with human serum albumin (HSA) in physiological buffer solution (pH 7.4) was studied by fluorescence and UV-vis absorption spectroscopy at different temperatures, combined with time-resolved fluorescence measurements. The experimental results showed that there was a strong fluorescence quenching of HSA by tolmetin. Using the continuous variation method, a single class of binding sites for TOL on HSA was put in evidence. The binding constants Ka were calculated at different temperatures, using a nonlinear fit to the experimental data, and the thermodynamic parameters ΔH(0), ΔS(0) and ΔG(0) were given. The obtained thermodynamic signature suggests that at least van der Waals and electrostatic type interactions are present. Quenching efficiency calculations, based on steady state and time-resolved spectroscopy, indicate that both static and dynamic quenching mechanisms are present.

Crystal structures of three classes of non-steroidal anti-inflammatory drugs in complex with aldo-keto reductase 1C3.

Aldo-keto reductase 1C3 (AKR1C3) catalyses the NADPH dependent reduction of carbonyl groups in a number of important steroid and prostanoid molecules. The enzyme is also over-expressed in prostate and breast cancer and its expression is correlated with the aggressiveness of the disease. The steroid products of AKR1C3 catalysis are important in proliferative signalling of hormone-responsive cells, while the prostanoid products promote prostaglandin-dependent proliferative pathways. In these ways, AKR1C3 contributes to tumour development and maintenance, and suggest that inhibition of AKR1C3 activity is an attractive target for the development of new anti-cancer therapies. Non-steroidal anti-inflammatory drugs (NSAIDs) are one well-known class of compounds that inhibits AKR1C3, yet crystal structures have only been determined for this enzyme with flufenamic acid, indomethacin, and closely related analogues bound. While the flufenamic acid and indomethacin structures have been used to design novel inhibitors, they provide only limited coverage of the NSAIDs that inhibit AKR1C3 and that may be used for the development of new AKR1C3 targeted drugs. To understand how other NSAIDs bind to AKR1C3, we have determined ten crystal structures of AKR1C3 complexes that cover three different classes of NSAID, N-phenylanthranilic acids (meclofenamic acid, mefenamic acid), arylpropionic acids (flurbiprofen, ibuprofen, naproxen), and indomethacin analogues (indomethacin, sulindac, zomepirac). The N-phenylanthranilic and arylpropionic acids bind to common sites including the enzyme catalytic centre and a constitutive active site pocket, with the arylpropionic acids probing the constitutive pocket more effectively. By contrast, indomethacin and the indomethacin analogues sulindac and zomepirac, display three distinctly different binding modes that explain their relative inhibition of the AKR1C family members. This new data from ten crystal structures greatly broadens the base of structures available for future structure-guided drug discovery efforts.

Lipid-drug interaction: biophysical effects of tolmetin on membrane mimetic systems of different dimensionality.

This work focuses on the application of different biophysical techniques to study the interaction of tolmetin with membrane mimetic models of different dimensionality (liposomes, monolayers, and supported lipid bilayers) composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), used as a representative phospholipid of natural membranes. Several biophysical techniques were employed: Fluorescence steady-state anisotropy to study the effects of NSAIDs on membrane microviscosity and thus to assess the main phase transition of DPPC, surface pressure-area isotherms to evaluate the adsorption and/or penetration of NSAIDs into the membrane, IRRAS to acquire structural information of the lipid membrane upon interaction with the drugs, and AFM to study the dynamic change in surface topography of the lipid bilayers caused by interaction with tolmetin. The experiments were performed taking into account the physiological conditions that tolmetin may find in the course of its in vivo therapeutic activity. Therefore, the studies covered the interactions of tolmetin with lipid membranes in both gel and liquid-crystalline phases at two pH conditions: 7.4 (plasma pH) and 5 (inflamed tissue pH). The applied models and techniques provided detailed information about different aspects of the tolmetin-membrane interaction. The studies have shown that tolmetin-membrane interaction is strongly dependent on the degree of drug ionization and of the lipid phase state, which can be related with the therapeutic action and gastro intestinal toxicity of this drug.

Determination of the scale of segregation of low dose tablets using hyperspectral imaging.

In this work, near infrared (NIR) hyperspectral imaging was used to quantify the spatial distribution of drug in tablets containing tolmetin sodium dihydrate. Hyperspectral data cubes were generated by imaging the same spatial region of a sample while illuminated by a laser at a different wavelength for each image. Images were generated for wavelengths ranging from 1100 to 2200 nm. Ten tablets with concentrations ranging from 0.0 to 10.0% w/w tolmetin were imaged, and the scales of segregation were calculated for the tablets. Lactose anhydrous was used as the diluent, and all mixtures contained 0.5% magnesium stearate as a lubricant. This research has shown hyperspectral imaging to be viable tool for quantifying segregation of low dose drugs in tablets.

Validation and clinical application of an LC-ESI-MS/MS method for simultaneous determination of tolmetin and MED5, the metabolites of amtolmetin guacil in human plasma.

A highly sensitive, rapid assay method has been developed and validated for the simultaneous estimation of tolmetin (TMT) and MED5 in human plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. A simple solid-phase extraction process was used to extract TMT and MED5 along with mycophenolic acid (internal standard, IS) from human plasma. Chromatographic separation was achieved with 0.2% formic acid-acetonitrile (25:75, v/v) at a flow rate of 0.50 mL/min on an X-Terra RP(18) column with a total run time of 2.5 min. The MS/MS ion transitions monitored were 258.1 → 119.0 for TMT, 315.1 → 119.0 for MED5 and 321.2 → 207.0 for IS. Method validation and clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 20 ng/mL and the linearity was observed from 20 to 2000 ng/mL, for both the anlaytes. The intra-day and inter-day precisions were in the range 3.27-4.50 and 5.32-8.18%, respectively for TMT and 4.27-5.68 and 5.32-8.85%, respectively for MED5. This novel method has been applied to a clinical pharmacokinetic study.

Control of encapsulation efficiency in polymeric microparticle system of tolmetin.

Ethylcellulose microparticles containing tolmetin sodium, an anti-inflammatory drug, were prepared by a solvent diffusion method based on the formation of multiple W/O(1)/O(2)-emulsion. The drug used was TOL, which is water-soluble and n-hexane was used as the non-solvent. Important parameters in the evaluation of a microencapsulation technique are actual drug loading, the encapsulation efficiency, the yield, solvent systems, dispersed phase to continuous phase ratio (DP/CP ratio), composition of continuous phase, drug distribution in microparticles and stability of primary emulsion. A small volume of internal aqueous phase and volume of organic solvent were favorable to achieve high drug encapsulation efficiencies. Since drug release during the initial stages depends mostly on the diffusion escape of the drug, major approaches to prevent the initial burst have focused on efficient encapsulation of the drug within the microparticles. For this reason, control of efficiency and the extent of initial burst are based on common formulation parameters. Most parameters affect encapsulation efficiency and initial burst by modifying solidification rate of dispersed phase. In order to prevent many unfavorable events such as pore formation, drug loss, and drug migration that occur while the dispersed phase is in the semi-solid state, it is important to understand and optimize these variables.

A study of the intermolecular interactions of tolmetin/N-acetyl-L-tyrosine ethyl ester complex.

The formation of tolmetin/N-acetyl-l-tyrosine ethyl ester (ATEE) complex has been reported by means of both theoretical and experimental studies, including quantum mechanical calculations as well as UV-vis absorption, fluorescence and time-resolved spectroscopy measurements. It has been found that the fluorescence of ATEE is quenched due to the formation of a non-fluorescent complex between ATEE and tolmetin in the ground state. The geometrical parameters of ATEE/tolmetin complex have been determined with the use of the DFT method applying the B3LYP correlation-exchange functional and 6-31G(d) basis set. The results of experiments indicated the static ATEE quenching by tolmetin. Additionally, the experimental and theoretically predicted Gibbs free energy of complexation has been calculated.

Singlet oxygen scavenging activity of non-steroidal anti-inflammatory drugs.

It has long been known that singlet oxygen ((1)O2) is generated during inflammatory processes. Once formed in substantial amounts, (1)O2 may have an important role in mediating the destruction of infectious agents during host defense. On the other hand, (1)O2 is capable of damaging almost all biological molecules and is particularly genotoxic, which gives a special relevance to the scavenging of this ROS throughout anti-inflammatory treatments. Considering that the use of non-steroidal anti-inflammatory drugs (NSAIDs) constitutes a first approach in the treatment of persistent inflammatory processes (due to their ability to inhibit cyclooxygenase), a putative scavenging activity of NSAIDs for (1)O2 would also represent a significant component of their therapeutic effect. The aim of the present study was to evaluate the scavenging activity for (1)O2 by several chemical families of NSAIDs. The results suggested that the pyrazole derivatives (dipyrone and aminopyrine) are, by far, the most potent scavengers of (1)O2 (much more potent compared to the other tested NSAIDs), displaying IC(50)-values in the low micromolar range. There was a lack of activity for most of the arylpropionic acid derivatives tested, with only naproxen and indoprofen displaying residual activities, as for the oxazole derivative, oxaprozin. On the other hand, the pyrrole derivatives (tolmetin and ketorolac), the indolacetic acid derivatives (indomethacin, and etodolac), as well as sulindac and its metabolites (sulindac sulfide and sulindac sulfone) displayed scavenging activity in the high micromolar range. Thus, the scavenging effect observed for dipyrone and aminopyrine will almost certainly contribute to their healing effect in the treatment of prolonged or chronic inflammation, while that of the other studied NSAIDs may have a lower contribution, though these assumptions still require further in vivo validation.

Ionic liquid-based dynamic liquid-phase microextraction: application to the determination of anti-inflammatory drugs in urine samples.

Dynamic liquid-phase microextraction (dLPME) using an ionic liquid as acceptor phase is proposed for the determination of six non-steroidal anti-inflammatory drugs (NSAIDs) in human urine samples for the first time. The extraction is carried out in a simple and automatic flow configuration. The chemical affinity between the extractant (1-butyl-3-methylimidazolium hexafluorophosphate) and the analytes permits a selective isolation of the drugs from the sample matrix allowing also their preconcentration. The whole analytical method has been optimized taking into account all the chemical, physical and hydrodynamic variables. The proposed method is a valuable alternative for the analysis of these drugs in urine within the concentration range 0.1-10 microg mL(-1), allowing their determination at therapeutic and toxic levels. Limits of detection were in the range from 38 ng mL(-1) (indomethacin) to 70 ng mL(-1) (naproxen). The repeatability of the proposed method expressed as RSD (n=5) varied between 2.1% (flurbiprofen) and 3.8% (tolmetin).

Determination of degradation pathways and kinetics of acyl glucuronides by NMR spectroscopy.

Acyl glucuronides have been implicated in the toxicity of many xenobiotics and marketed drugs. These toxicities are hypothesized to be a consequence of covalent binding of the reactive forms of the acyl glucuronide to proteins. Reactive intermediates of the acyl glucuronide arise from the migration of the aglycone leading to other positional and stereoisomers under physiological conditions. In order to screen for the potential liabilities of these metabolites during the early phase of pharmaceutical development, an NMR method based on the disappearance of the anomeric resonance of the O-1-acyl glucuronide was used to monitor the degradation kinetics of 11 structurally diverse acyl glucuronides, including those produced from the known nonsteroidal anti-inflammatory drugs (NSAIDs). The acyl glucuronides were either chemically synthesized or were isolated from biological matrices (bile, urine, and liver microsomal extracts). The half-lives attained utilizing this method were found to be comparable to those reported in the literature. NMR analysis also enabled the delineation of the two possible pathways of degradation: acyl migration and hydrolytic cleavage. The previously characterized 1H resonances of acyl migrated products are quite distinguishable from those that arise from hydrolysis. The NMR method described here could be used to rank order acyl glucuronide forming discovery compounds based on the potential reactivity of the conjugates and their routes of decomposition under physiological conditions. Furthermore, we have shown that in vitro systems such as liver microsomal preparations can be used to generate sufficient quantities of acyl glucuronides from early discovery compounds for NMR characterization. This is particularly important, as we often have limited supply of early discovery compounds to conduct in vivo studies to generate sufficient quantities of acyl glucuronides for further characterization.

Studies on the metabolism of tolmetin to the chemically reactive acyl-coenzyme A thioester intermediate in rats.

Carboxylic acids may be metabolized to acyl glucuronides and acyl-coenzyme A thioesters (acyl-CoAs), which are reactive metabolites capable of reacting with proteins in vivo. In this study, the metabolic activation of tolmetin (Tol) to reactive metabolites and the subsequent formation of Tol-protein adducts in the liver were studied in rats. Two hours after dose administration (100 mg/kg i.p.), tolmetin acyl-CoA (Tol-CoA) was identified by liquid chromatography-tandem mass spectrometry in liver homogenates. Similarly, the acyl-CoA-dependent metabolites tolmetin-taurine conjugate (Tol-Tau) and tolmetin-acyl carnitine ester (Tol-Car) were identified in rat livers. In a rat bile study (100 mg/kg i.p.), the S-acyl glutathione thioester conjugate was identified, providing further evidence of the formation of reactive metabolites such as Tol-CoA or Tol-acyl glucuronide (Tol-O-G), capable of acylating nucleophilic functional groups. Three rats were treated with clofibric acid (150 mg/kg/day i.p. for 7 days) before dose administration of Tol. This resulted in an increase in covalent binding to liver proteins from 0.9 nmol/g liver in control rats to 4.2 nmol/g liver in clofibric acid-treated rats. Similarly, levels of Tol-CoA increased from 0.6 nmol/g to 4.4 nmol/g liver after pretreatment with clofibric acid, whereas the formation of Tol-O-G and Tol-Tau was unaffected by clofibric acid treatment. However, Tol-Car levels increased from 0.08 to 0.64 nmol/g after clofibric acid treatment. Collectively, these results confirm that Tol-CoA is formed in vivo in the rat and that this metabolite can have important consequences in terms of covalent binding to liver proteins.

A simple in vitro model to study the stability of acylglucuronides.

INTRODUCTION: Compounds containing the carboxylic functional group (e.g. non-steroidal anti-inflammatory drugs) can be metabolized to form acylglucuronides. Acylglucuronides are intrinsically reactive metabolites capable of undergoing hydrolysis, intra-molecular rearrangement, and formation of covalent adducts with proteins, which may generate potential toxicity. The purpose of this study is to develop an in vitro screening model to assess degradation kinetics of acylglucuronides. METHOD: Zomepirac, ibuprofen, gemfibrozil, and compounds A, B, C, and D were incubated in the presence of rat microsomal protein and uridine 5'-diphosphoglucuronic acid (UDPGA), followed by addition of human plasma to evaluate degradation kinetics of the acylglucuronides. As a comparison, authentic acylglucuronide standards of zomepirac, ibuprofen, gemfibrozil, and compounds A, B, C, and D were chemically synthesized and were evaluated for degradation kinetics. RESULTS: The results demonstrate that degradation half-life values of acylglucuronides of zomepirac, ibuprofen, gemfibrozil, and compounds A, B, C, and D determined by the in vitro formation/degradation model were in the same rank-order with those of the authentic acylglucuronide standards. DISCUSSION: For the seven compounds tested, the model placed the stability of the acylglucuronides formed in vitro in a rank-order consistent with authentic acylglucuronide standards. The method allows for a rapid assessment of the stability of acylglucuronides.

Kinetic studies on the intramolecular acyl migration of beta-1-O-acyl glucuronides: application to the glucuronides of (R)- and (S)-ketoprofen, (R)- and (S)-hydroxy-ketoprofen metabolites, and tolmetin by 1H-NMR spectroscopy.

Conjugation of carboxylate drugs with D-glucuronic acid is of considerable interest because of the inherent reactivity of the resulting beta-1-O-acyl glucuronides. These conjugates can degrade by spontaneous hydrolysis and internal acyl migration. beta-1-O-acyl glucuronides and their acyl migration products can also react covalently with macromolecules with potential toxicological consequences. The spontaneous degradation of the diastereoisomeric beta-1-O-acyl glucuronide metabolites of the racemic drug ketoprofen, two of its ring-hydroxylated metabolites and of tolmetin beta-1-O-acyl glucuronide was investigated by (1)H-NMR spectroscopy in buffer solutions, at pH 7.4 and 37 degrees C. A plot of the logarithm of the peak integrals against time revealed first-order kinetics. Degradation rates and half-lives were calculated for each glucuronide using first-order reaction equations. Tolmetin glucuronide had the fastest degradation rate, whilst all of the ketoprofen-related glucuronides had similar degradation rates. The degradation of the diastereoisomeric glucuronides was stereoselective, with the rate for the (S)-isomer always slower compared with the (R)-isomer by approximately a factor of 2.