Design, synthesis of celecoxib-tolmetin drug hybrids as selective and potent COX-2 inhibitors.

Three novel series of diarylpyrazole 10b-d and triarylpyrazole derivatives 11a-d &12a-d were synthesized through Vilsmier-Haack condition. The structures of prepared compounds were determined through IR, 1H NMR, 13C NMR, Mass spectral and elemental analysis. Docking of the synthesized compounds over COX-2 active site ensure their selectivity. Moreover, the target compounds were evaluated for both in vitro and in vivo inhibitory activity. All compounds were more selective for COX-2 isozyme than COX-1 isozyme and with excellent anti-inflammatory activity. Compounds 11b, 11d and 12b showed the highest anti-inflammatory activity (67.4%, 62.7%, 61.4% respectively), lower ulcerogenic liability (UI = 2.00, 2.75, 3.25 respectively) than indomethacin (UI = 14) and comparable to celecoxib (UI = 1.75) which were confirmed from the histopatholgical study.

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.

Neutrophil depletion protects against zomepirac-induced acute kidney injury in mice.

Acyl glucuronide (AG) metabolites of carboxylic acid-containing drugs have been implicated in drug toxicity. Zomepirac (ZP) is a non-steroidal anti-inflammatory drug that was withdrawn from the market because of anaphylactic reactions and renal injury. We recently established a novel mouse model of ZP-induced kidney injury by increasing zomepirac acyl-glucuronide (ZP-AG) concentration via pretreatment with tri-O-tolyl phosphate, a nonselective esterase inhibitor, and l-buthionine-(S,R)-sulfoximine, a glutathione synthesis inhibitor. Although we have shown that ZP-AG is responsible for ZP-induced kidney injury in mice, the exact pathogenic mechanisms of ZP-induced kidney injury have not been investigated yet. In this study, we aimed to investigate the role of immune cells in the pathogenesis of ZP-induced kidney injury, as a representative of AG toxicity. We found that the counts of neutrophils and inflammatory monocytes increased in the blood of mice with ZP-induced kidney injury. However, clodronate liposome- or GdCl3-induced monocyte and/or macrophage depletion did not affect blood urea nitrogen and plasma creatinine levels in mice with ZP-induced kidney injury. Neutrophil infiltration into the kidneys was observed in mice with ZP-induced kidney injury, whereas anti-lymphocyte antigen 6 complex, locus G (Ly6G) antibody pretreatment prevented the renal neutrophil infiltration and partially protected against ZP-induced kidney injury. The mRNA expression of neutrophil-infiltrating cytokines and chemokines, interleukin-1α and macrophage inflammatory protein-2α, increased in mice with ZP-induced kidney injury, whereas pretreatment with anti-Ly6G antibody resulted in a marked reduction of their expression. These results suggest that ZP-AG might be involved in kidney injury, partly via induction of neutrophil infiltration. Therefore, this study may provide an important understanding on toxicological role of ZP-AG in vivo that helps to understand toxicity of AG metabolites.

Zomepirac Acyl Glucuronide Is Responsible for Zomepirac-Induced Acute Kidney Injury in Mice.

Glucuronidation, an important phase II metabolic route, is generally considered to be a detoxification pathway. However, acyl glucuronides (AGs) have been implicated in the toxicity of carboxylic acid drugs due to their electrophilic reactivity. Zomepirac (ZP) was withdrawn from the market because of adverse effects such as renal toxicity. Although ZP is mainly metabolized to acyl glucuronide (ZP-AG) by UDP-glucuronosyltransferase, the role of ZP-AG in renal toxicity is unknown. In this study, we established a ZP-induced kidney injury mouse model by pretreatment with tri-o-tolyl phosphate (TOTP), a nonselective esterase inhibitor, and l-buthionine-(S,R)-sulfoximine (BSO), a glutathione synthesis inhibitor. The role of ZP-AG in renal toxicity was investigated using this model. The model showed significant increases in blood urea nitrogen (BUN) and creatinine (CRE), but not alanine aminotransferase. The ZP-AG concentrations were elevated by cotreatment with TOTP in the plasma and liver and especially in the kidney. The ZP-AG concentrations in the kidney correlated with values for BUN and CRE. Upon histopathological examination, vacuoles and infiltration of mononuclear cells were observed in the model mouse. In addition to immune-related responses, oxidative stress markers, such as the glutathione/disulfide glutathione ratio and malondialdehyde levels, were different in the mouse model. The suppression of ZP-induced kidney injury by tempol, an antioxidant agent, suggested the involvement of oxidative stress in ZP-induced kidney injury. This is the first study to demonstrate that AG accumulation in the kidney by TOTP and BSO treatment could explain renal toxicity and to show the in vivo toxicological potential of AGs.

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).

Synthesis of Tolmetin Hydrazide-Hydrazones and Discovery of a Potent Apoptosis Inducer in Colon Cancer Cells.

Tolmetin hydrazide and a novel series of tolmetin hydrazide-hydrazones 4a-l were synthesized in this study. The structures of the new compounds were determined by spectral (FT-IR, (1)H NMR) methods. N'-[(2,6-Dichlorophenyl)methylidene]-2-[1-methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]acetohydrazide (4g) was evaluated in vitro using the MTT colorimetric method against the colon cancer cell lines HCT-116 (ATCC, CCL-247) and HT-29 (ATCC, HTB-38) to determine growth inhibition and cell viability at different doses. Compound 4g exhibited anti-cancer activity with an IC50 value of 76 µM against colon cancer line HT-29 (ATCC, HTB-38) and did not display cytotoxicity toward control NIH3T3 mouse embryonic fibroblast cells compared to tolmetin. In addition, this compound was evaluated for caspase-3, caspase-8, caspase-9, and annexin-V activation in the apoptotic pathway, which plays a key role in the treatment of cancer. We demonstrated that the anti-cancer activity of this compound was due to the activation of caspase-8 and caspase-9 involved in the apoptotic pathway. In addition, in this study, we investigated the catalytical effect of COX on the HT-29 cancer line, the apoptotic mechanism, and the moleculer binding of tolmetin and compound 4g on the COX enzyme active site.

GSSG/GSH ratios in cryopreserved rat and human hepatocytes as a biomarker for drug induced oxidative stress.

The formation of reactive oxygen species (ROS) could cause cellular damage and eventually lead to apoptosis and necrosis. The ratio between oxidized glutathione and reduced glutathione (GSSG-to-GSH ratio) has been used as an important in vitro and in vivo biomarker of the redox balance in the cell and consequently of cellular oxidative stress. This paper optimizes a LC-MS/MS method for the simultaneous determination of GSH and GSSG. The proposed method is based on the derivatization of reduced GSH using iodoacetic acid (IAA) in order to prevent its rapid oxidation to GSSG during sample preparation. The optimized analytical method was applied to evaluate the effect of different pharmaceutical agents on GSSG-to-GSH ratio in cryopreserved rat and human hepatocytes in culture. Hepatocyte viabilities were also determined at the same time by using the WST-1 assay as a direct measurement of cell mitochondrial respiration. The results obtained demonstrate that cryopreserved rat and human hepatocytes in culture are reliable in vitro models for the evaluation of cellular oxidative stress. In addition, the GSSG-to-GSH ratio measurements could be a biomarker of hepatotoxicity providing similar results to those of cytotoxicity assay.

Evaluation of intestinal absorption of amtolmetin guacyl in rats: breast cancer resistant protein as a primary barrier of oral bioavailability.

AIMS: The purpose of the present study was to investigate the role of efflux transporters on the intestinal absorption of amtolmetin guacyl (MED-15). MAIN METHODS: The effects of P-glycoprotein (P-gp), multiple resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) inhibitors on intestinal absorption amount of MED-5 (tolmetin-glycine amide derivative), the metabolite formed from MED-15 in the intestinal epithelial cells were studied in the in vitro everted gut sac experiments. Moreover, the in situ single-pass intestine perfusion was adopted to clarify the role of efflux transporters in excreting MED-5 in knockout mice. The plasma concentration of MED-5 and tolmetin, the metabolite formed from MED-5 was determined in Bcrp1 knockout mice and wild-type mice. KEY FINDINGS: BCRP inhibitor Ko143 (50 µM and 100 µM) significantly increased the intestinal absorption amount in jejunum, ileum and colon (p<0.05). However, no effect was observed in the presence of P-gp inhibitor verapamil and MRP2 inhibitor MK571 in each intestinal segment. Furthermore, the plasma concentration MED-5 and tolmetin, metabolites of MED-15, increased 2-fold and 4-fold, respectively, in Bcrp1 knockout mice compared with wild-type mice after the single-pass perfusion of small intestine with MED-15. SIGNIFICANCE: It may be concluded that BCRP plays an important role in the intestinal efflux of MED-5 and limits the bioavailability after oral administration of MED-15.

The impact of pregnancy on urinary ketorolac metabolites after single intravenous bolus.

Compared to female volunteers or postpartum, ketorolac clearance is higher at delivery. To explore the alterations that explain this higher clearance, urinary ketorolac metabolites collected at delivery (n = 40) were compared to female volunteers (unpaired, n = 8) or postpartum (paired, n = 8) following intravenous administration of 30 mg ketorolac tromethamine. A mean 38 (SD 9) % of the ketorolac dose was retrieved in 8-h urine collections. This was based on mean portions of 56 (20), 10 (14) and 33 (12) % for free ketorolac, ketorolac-glucuronide and p-hydroxy-ketorolac, respectively. The mean ketorolac-glucuronide portion at delivery (5 %) was lower compared to female volunteers (21 %) or postpartum (21 %) (p = 0.003 and p = 0.002, respectively). Similarly, there was a difference in mean portion of free urinary ketorolac at delivery when compared to healthy female volunteers (60-45 %, p = 0.046). Using paired statistics, the mean portion of total urinary ketorolac was lower (62-73 %, p = 0.015) while the portion retrieved as p-hydroxy-ketorolac was significantly higher at delivery compared to postpartum (38-28 %, p = 0.031). The differences in urine metabolites suggest that the increased ketorolac clearance at delivery is in part explained by increased metabolic clearance to p-hydroxy-ketorolac, reflecting increased oxidation activity.

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.

Differential involvement of mitochondrial dysfunction, cytochrome P450 activity, and active transport in the toxicity of structurally related NSAIDs.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of pain and inflammation. However, this group of drugs is associated with serious adverse drug reactions. Previously, we studied the mechanisms underlying toxicity of the NSAID diclofenac using Saccharomycescerevisiae as model system. We identified the involvement of several mitochondrial proteins, a transporter and cytochrome P450 activity in diclofenac toxicity. In this study, we investigated if these processes are also involved in the toxicity of other NSAIDs. We divided the NSAIDs into three classes based on their toxicity mechanisms. Class I consists of diclofenac, indomethacin and ketoprofen. Mitochondrial respiration and reactive oxygen species (ROS) play a major role in the toxicity of this class. Metabolism by cytochrome P450s further increases their toxicity, while ABC-transporters decrease the toxicity. Mitochondria and oxidative metabolism also contribute to toxicity of class II drugs ibuprofen and naproxen, but another cellular target dominates their toxicity. Interestingly, ibuprofen was the only NSAID that was unable to induce upregulation of the multidrug resistance response. The class III NSAIDs sulindac, ketorolac and zomepirac were relatively non-toxic in yeast. In conclusion, we demonstrate the use of yeast to investigate the mechanisms underlying the toxicity of structurally related drugs.

Single dose oral mefenamic acid for acute postoperative pain in adults.

BACKGROUND: Mefenamic acid is a non-steroidal anti-inflammatory drug (NSAID). It is most often used for treating pain of dysmenorrhoea in the short term (seven days or less), as well as mild to moderate pain including headache, dental pain, postoperative and postpartum pain. It is widely available in many countries worldwide. OBJECTIVES: To assess the efficacy of single dose oral mefenamic acid in acute postoperative pain, and any associated adverse events. SEARCH STRATEGY: We searched Cochrane CENTRAL, MEDLINE, EMBASE and the Oxford Pain Relief Database for studies to December 2010. SELECTION CRITERIA: Single oral dose, randomised, double-blind, placebo-controlled trials of mefenamic acid for relief of established moderate to severe postoperative pain in adults. DATA COLLECTION AND ANALYSIS: Studies were assessed for methodological quality and the data extracted by two review authors independently. Summed total pain relief (TOTPAR) or pain intensity difference (SPID) over 4 to 6 hours was used to calculate the number of participants achieving at least 50% pain relief. These derived results were used to calculate, with 95% confidence intervals, the relative benefit compared to placebo, and the number needed to treat (NNT) for one participant to experience at least 50% pain relief over 4 to 6 hours. Numbers of participants using rescue medication over specified time periods, and time to use of rescue medication, were sought as additional measures of efficacy. Information on adverse events and withdrawals was collected. MAIN RESULTS: Four studies with 842 participants met the inclusion criteria; 126 participants were treated with mefenamic acid 500 mg, 67 with mefenamic acid 250 mg, 197 with placebo, and 452 with lignocaine, aspirin, zomepirac or nimesulide. Participants had pain following third molar extraction, episiotomy and orthopaedic surgery. The NNT for at least 50% pain relief over 6 hours with a single dose of mefenamic acid 500 mg compared to placebo was 4.0 (2.7 to 7.1), and the NNT to prevent use of rescue medication over 6 hours was 6.5 (3.6 to 29). There were insufficient data to analyse other doses or active comparators, or numbers of participants experiencing any adverse events. No serious adverse events or adverse event withdrawals were reported in these studies. AUTHORS' CONCLUSIONS: Oral mefenamic acid 500 mg was effective at treating moderate to severe acute postoperative pain, based on limited data. Efficacy of other doses, and safety and tolerability could not be assessed.

Single dose oral fenoprofen for acute postoperative pain in adults.

BACKGROUND: Fenoprofen is a non-steroidal anti-inflammatory drug (NSAID), available in several different countries, but not widely used. OBJECTIVES: To assess the efficacy of single dose oral fenoprofen in acute postoperative pain, and associated adverse events. SEARCH STRATEGY: We searched Cochrane CENTRAL, MEDLINE, EMBASE and the Oxford Pain Relief Database for studies to December 2010. SELECTION CRITERIA: Single oral dose, randomised, double-blind, placebo-controlled trials of fenoprofen for relief of established moderate to severe postoperative pain in adults. DATA COLLECTION AND ANALYSIS: Studies were assessed for methodological quality and data extracted by two review authors independently. Summed total pain relief (TOTPAR) or pain intensity difference (SPID) over 4 to 6 hours was used to calculate the number of participants achieving at least 50% pain relief. These derived results were used to calculate, with 95% confidence intervals, the relative benefit compared to placebo, and the number needed to treat (NNT) for one participant to experience at least 50% pain relief over 4 to 6 hours. Numbers of participants using rescue medication over specified time periods, and time to use of rescue medication, were sought as additional measures of efficacy. Information on adverse events and withdrawals was collected. MAIN RESULTS: Five studies (696 participants) met the inclusion criteria; 24 participants were treated with fenoprofen 12.5 mg, 23 with fenoprofen 25 mg, 79 with fenoprofen 50 mg, 78 with fenoprofen 100 mg, 146 with fenoprofen 200 mg, 55 with fenoprofen 300 mg, 43 with zomepirac 100 mg, 30 with morphine 8 mg, 77 with codeine 60 mg, and 141 with placebo. Participants had pain following third molar extraction, laparoscopy, minor day surgery and episiotomy. The NNT for at least 50% pain relief over 4 to 6 hours with a single dose of fenoprofen 200 mg compared to placebo was 2.3 (1.9 to 3.0). There were insufficient data to analyse other doses or active comparators, time to use of rescue medication, or numbers of participants needing rescue medication. There was no difference in numbers of participants experiencing any adverse events between fenoprofen 200 mg and placebo. No serious adverse events or adverse event withdrawals were reported in these studies. AUTHORS' CONCLUSIONS: Oral fenoprofen 200 mg is effective at treating moderate to severe acute postoperative pain, based on limited data for at least 50% pain relief over 4 to 6 hours. Efficacy of other doses, other efficacy outcomes, and safety and tolerability could not be assessed.

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.

Glycerolysis of acyl glucuronides as an artifact of in vitro drug metabolism incubations.

During an investigation of the in vitro glucuronidation of benoxaprofen by human liver S-9 fraction, an unusual drug-related entity possessing a protonated molecular ion that was 74 mass units greater than the parent drug was observed. It was identified as the glycerol ester of benoxaprofen. Formation of this entity required inclusion of uridine diphosphoglucuronic acid (UDPGA) in the incubation, suggesting the formation of benoxaprofen acyl glucuronide followed by transesterification with the glycerol present in the incubation due to its presence as a stabilizer for liver subcellular fractions. Formation occurred during the sample work-up procedure while the samples were subjected to evaporation in vacuo, which does not remove glycerol. Conversion of purified benoxaprofen acyl glucuronide to the glycerol ester was demonstrated in glycerol at 37 degrees C. Other drugs that are converted to acyl glucuronides in vitro (diclofenac, mefenamic acid, tolmetin, and naproxen) were also shown to form corresponding glycerol esters when incubated with human liver S-9 fraction and UDPGA. The potential formation of glycerol esters of carboxylic acid drugs undergoing acyl glucuronidation in vitro represents an experimental artifact to which drug metabolism scientists should be aware.

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.

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.

Evidence for the bioactivation of zomepirac and tolmetin by an oxidative pathway: identification of glutathione adducts in vitro in human liver microsomes and in vivo in rats.

Although zomepirac (ZP) and tolmetin (TM) induce anaphylactic reactions and form reactive acyl glucuronides, a direct link between the two events remains obscure. We report herein that, in addition to acyl glucuronidation, both drugs are subject to oxidative bioactivation. Following incubations of ZP with human liver microsomes fortified with NADPH and glutathione (GSH), a metabolite with an MH+ ion at m/z 597 was detected by LC/MS/MS. On the basis of collision-induced dissociation and NMR evidence, the structure of this metabolite was determined to be 5-[4'-chlorobenzoyl]-1,4-dimethyl-3-glutathionylpyrrole-2-acetic acid (ZP-SG), suggesting that the pyrrole moiety of ZP had undergone oxidation to an epoxide intermediate, followed by addition of GSH and loss of the elements of H2O to yield the observed conjugate. The oxidative bioactivation of ZP most likely is catalyzed by cytochrome P450 (P450) 3A4, since the formation of ZP-SG was reduced to approximately 10% of control values following pretreatment of human liver microsomes with ketoconazole or with an inhibitory anti-P450 3A4 IgG. A similar GSH adduct, namely 5-[4'-methylbenzoyl]-1-methyl-3-glutathionylpyrrole-2-acetic acid (TM-SG), was identified when TM was incubated with human liver microsomal preparations. The relevance of these in vitro findings to the in vivo situation was established through the detection of the same thiol adducts in rats treated with ZP and TM, respectively. Taken together, these data suggest that, in addition to the formation of acyl glucuronides, oxidative metabolism of ZP and TM affords reactive species that may haptenize proteins and thereby contribute to the drug-mediated anaphylactic reactions.

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.

In vitro and in vivo studies on acyl-coenzyme A-dependent bioactivation of zomepirac in rats.

Zomepirac [ZP, 5-(chlorobenzoyl)-1,4-dimethylpyrrole-2-acetic acid] was withdrawn from the market because of unpredictable allergic reactions that may have been caused by ZP-protein adducts formed by reaction of the reactive acyl glucuronide of ZP (ZP-O-G) with endogenous proteins. To test the hypothesis that the reactive ZP acyl coenzyme A thioester (ZP-CoA) was formed and potentially could contribute to formation of ZP-protein adducts, we investigated the acyl CoA-dependent metabolism of ZP in freshly isolated rat hepatocytes (1 mM) and in vivo (100 mg ZP/kg, ip) in rat livers (2 h after dose administration), rat bile (0-4 h), and rat urine (0-24 h). ZP-CoA was detected in freshly isolated hepatocytes and in vivo in rat livers by LC/MS/MS. In addition, the ZP glycine conjugate (ZP-Gly) and ZP taurine conjugates (ZP-Tau) were identified by LC/MS/MS in rat hepatocytes and in vivo in rat livers, rat urine, and rat bile. The identities of ZP-CoA, ZP-Gly, and ZP-Tau were confirmed by comparison of retention times and MS/MS spectra with those of authentic standards. Moreover, the ZP acyl carnitine ester was detected in rat urine and rat bile based upon (i) the chlorine isotope pattern, (ii) MS/MS spectra showing significant ions characteristic for carnitine (m/z 60, 144 and loss of m/z 59) and ZP (m/z 139), and (iii) accurate mass measurements with a mass accuracy of 0.2 ppm. ZP-CoA serves as an obligatory intermediate in the formation of ZP-Gly, ZP-Tau, and ZP carnitine ester, and it is therefore of mechanistic significance that these conjugates were identified. Finally, time-dependent concentration profiles obtained in experiments with rat hepatocytes and in vivo from quantitative analysis of rat livers indicate that ZP-CoA, in addition to ZP-O-G, may contribute to formation of the potentially toxic covalent ZP-protein adducts.