Screening procedure for detection of non-steroidal anti-inflammatory drugs and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons by gas chromatography-mass spectrometry after extractive methylation

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used as analgesic and anti-rheumatic drugs, and they are often misused. A gas chromatographic-mass spectrometric (GC-MS) screening procedure was developed for their detection in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons after extractive methylation. The compounds were separated by capillary GC and identified by computerized MS in the full-scan mode. Using mass chromatography with the ions m/z 119, 135, 139, 152, 165, 229, 244, 266, 272, and 326, the possible presence of NSAIDs and their metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra recorded during this study. This method allowed the detection of therapeutic concentrations of acemetacin, acetaminophen (paracetamol), acetylsalicylic acid, diclofenac, diflunisal, etodolac, fenbufen, fenoprofen, flufenamic acid, flurbiprofen, ibuprofen, indometacin, kebuzone, ketoprofen, lonazolac, meclofenamic acid, mefenamic acid, mofebutazone, naproxen, niflumic acid, phenylbutazone, suxibuzone, tiaprofenic acid, tolfenamic acid, and tolmetin in urine samples. The overall recoveries of the different NSAIDs ranged between 50 and 80% with coefficients of variation of less than 15% (n = 5), and the limits of detection of the different NSAIDs were between 10 and 50 ng/mL (S/N = 3) in the full-scan mode. Extractive methylation has proved to be a versatile method for STA of various acidic drugs, poisons, and their metabolites in urine. It has also successfully been used for plasma analysis.     

Inhibitory potential of nonsteroidal anti-inflammatory drugs on UDP-glucuronosyltransferase 2B7 in human liver microsomes

Objective A number of nonsteroidal anti-inflammatory drugs (NSAIDs) are subject to glucuronidation in humans, and UDP-glucuronosyltransferase (UGT) 2B7 is involved in the glucuronidation of many NSAIDs. The objective of this study was to identify a NSAID with potent inhibitory potential against UGT2B7 using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Methods A rapid screening method for detecting the inhibitory potential of various drugs against UGT2B7 was established using a LC-MS/MS system. The effects of nine NSAIDs (acetaminophen, diclofenac, diflunisal, indomethacin, ketoprofen, mefenamic acid, naproxen, niflumic acid, and salicylic acid) against UGT2B7-catalyzed 3′-azido-3′-deoxythymidine glucuronidation (AZTG) were investigated in human liver microsomes (HLM) and recombinant human UGT2B7. Results Mefenamic acid inhibited AZTG most potently, with an IC₅₀ value of 0.3 μM, and its inhibition type was not competitive. The IC₅₀ values for diclofenac, diflunisal, indomethacin, ketoprofen, naproxen, and niflumic acid against AZTG were 6.8, 178, 51, 40, 23, and 83 μM, respectively, while those for acetaminophen and salicylic acid were >100 μM. The IC₅₀ values for NSAIDs against AZTG in recombinant human UGT2B7 were similar to those obtained in HLM. Conclusion The method established in this study is useful for identifying drugs with inhibitory potential against human UGT2B7. Among the nine NSAIDs investigated, mefenamic acid had the strongest inhibitory effect on UGT2B7-catalyzed AZTG in HLM. Thus, caution might be exercised when mefenamic acid is coadministered with drugs possessing UGT2B7 as a main elimination pathway.     

Species differences in inhibition potential of nonsteroidal anti-inflammatory drugs against estradiol 3beta-glucuronidation between rats, dogs, and humans

Differences in the inhibitory potentials against UDP-glucuronosyltransferase (UGT) between species have been reported only rarely, even though the information would be useful for the precise characterization of drug candidates. In this study, the inhibition potentials of nonsteroidal anti-inflammatory drugs (NSAIDs) against UGT-catalyzed estradiol 3beta-glucuronidation (E3G) in the liver microsomes of rats, dogs, and humans were compared. Rat liver microsomes (RLMs) and human liver microsomes (HLMs) exhibited homotropic activation kinetics with S(50) values of 22 and 12 microM, respectively. However, dog liver microsomes (DLMs), exhibited Michaelis-Menten kinetics with no activation. Among the NSAIDs investigated (diclofenac, diflunisal, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, mefenamic acid, niflumic acid, and sulindac), only niflumic acid and mefenamic acid inhibited E3G potently in all three species. The IC(50) values of NSAIDs against E3G in RLMs and HLMs were within a threefold difference of each other, while those in DLMs was more than three times higher than the other two. In conclusion, RLMs showed an inhibitory pattern similar to that of HLMs, whereas DLMs presented a distinct pattern. These results indicate that a rat animal model would be useful for evaluating the inhibitory potentials of drugs against estradiol glucuronidation, but a dog model would not.     

Metabolism of non-steroidal anti-inflammatory drugs (NSAIDs) by Streptomyces griseolus CYP105A1 and its variants

In the field of drug development, technology for producing human metabolites at a low cost is required. In this study, we explored the possibility of using prokaryotic water-soluble cytochrome P450 (CYP) to produce human metabolites. Streptomyces griseolus CYP105A1 metabolizes various non-steroidal anti-inflammatory drugs (NSAIDs), including diclofenac, mefenamic acid, flufenamic acid, tolfenamic acid, meclofenamic acid, and ibuprofen. CYP105A1 showed 4′-hydroxylation activity towards diclofenac, mefenamic acid, flufenamic acid, tolfenamic acid, and meclofenamic acid. It should be noted that this reaction specificity was similar to that of human CYP2C9. In the case of mefenamic acid, another metabolite, 3′-hydroxymethyl mefenamic acid, was detected as a major metabolite. Substitution of Arg at position 73 with Ala in CYP105A1 dramatically reduced the hydroxylation activity toward diclofenac, flufenamic acid, and ibuprofen, indicating that Arg73 is essential for the hydroxylation of these substrates. In contrast, substitution of Arg84 with Ala remarkably increased the hydroxylation activity towards diclofenac, mefenamic acid, and flufenamic acid. Recombinant Rhodococcus erythrocyte cells expressing the CYP105A1 variant R84A/M239A showed complete conversion of diclofenac into 4′-hydroxydiclofenac. These results suggest the usefulness of recombinant R. erythropolis cells expressing actinomycete CYP, such as CYP105A1, for the production of human drug metabolites.     

In vitro inhibitory effects of non-steroidal anti-inflammatory drugs on 4-methylumbelliferone glucuronidation in recombinant human UDP-glucuronosyltransferase 1A9--potent inhibition by niflumic acid

The inhibitory potencies of non-steroidal anti-inflammatory drugs (NSAIDs) on UDP-glucuronosyltransferase (UGT) 1A9 activity were investigated in recombinant human UGT1A9 using 4-methylumbelliferone (4-MU) as a substrate for glucuronidation. 4-MU glucuronidation (4-MUG) showed Michaelis-Menten kinetics with a Km value of 6.7 microM. The inhibitory effects of the following seven NSAIDs were investigated: acetaminophen, diclofenac, diflunisal, indomethacin, ketoprofen, naproxen and niflumic acid. Niflumic acid had the most potent inhibitory effect on 4-MUG with an IC50 value of 0.0341 microM. The IC50 values of diflunisal, diclofenac and indomethacin were 1.31, 24.2, and 34.1 microM, respectively, while acetaminophen, ketoprofen and naproxen showed less potent inhibition. Niflumic acid, diflunisal, diclofenac and indomethacin inhibited 4-MUG competitively with Ki values of 0.0275, 0.710, 53.3 and 69.9 microM, respectively, being similar to each IC50 value. In conclusion, of the seven NSAIDs investigated, niflumic acid was the most potent inhibitor of recombinant UGT1A9 via 4-MUG in a competitive manner.     

Fenamates and the potent inhibition of human liver phenol sulphotransferase

1. The inhibition of the human liver phenol sulphotransferase (HL-PST) and catechol sulphotransferase (HL-CST) by five fenamates has been studied and the activities of HL-PST and HL-CST were measured with 4-nitrophenol and dopamine as substrates, respectively. 2. The IC50 for inhibition of HL-PST were 0.02 microM (mefenamic acid); 0.12 microM (tolfenamic acid); 0.28 microM (niflumic acid); 0.87 microM (meclofenamic acid) and 1.50 microM (flufenamic acid). 3. HL-CST was less susceptible than HL-PST to the inhibition by fenamates and the IC50 for HL-CST were 36 microM (tolfenamic acid); 70 microM (flufenamic acid); 76 microM (mefenamic acid); 180 microM (niflumic acid) and 185 microM (meclofenamic acid). 4. The ratios of the IC50 for HL-CST:HL-PST were drug-dependent and ranged from 47 (flufenamic acid) to 3800 (mefenamic acid). Mefenamic acid is a relatively potent and selective inhibitor of HL-PST. 5. The IC50 for HL-PST obtained with mefenamic acid was three orders of magnitude lower than the peak plasma concentration of this drug after an oral dose of 0.5 g. Accordingly, mefenamic acid should impair sulphation in vivo.     

Diclofenac sodium

The pharmacology, pharmacokinetics, clinical efficacy, adverse effects, and dosage of diclofenac sodium are reviewed. Diclofenac, the first nonsteroidal anti-inflammatory agent (NSAID) to be approved that is a phenylacetic acid derivative, competes with arachidonic acid for binding to cyclo-oxygenase, resulting in decreased formation of prostaglandins. The drug has both analgesic and antipyretic activities. Diclofenac is efficiently absorbed from the gastrointestinal tract; peak plasma concentrations occur 1.5 to 2.0 hours after ingestion in fasting subjects. Even though diclofenac has a relatively short elimination half-life in plasma (1.5 hours), it persists in synovial fluid. The drug is metabolized in the liver and is eliminated by urinary and biliary excretion. In clinical trials, diclofenac was as effective as aspirin, diflunisal, indomethacin, sulindac, ibuprofen, ketoprofen, and naproxen in improving function and reducing pain in patients with rheumatoid arthritis. For treatment of osteoarthritis, diclofenac was equivalent in efficacy to aspirin, diflunisal, indomethacin, sulindac, ibuprofen, ketoprofen, naproxen, flurbiprofen, mefenamic acid, and piroxicam. Diclofenac was as effective as indomethacin or sulindac in treating ankylosing spondylitis. The most frequent adverse effects reported for diclofenac were gastrointestinal, but these effects were fewer and less serious than occurred with aspirin or indomethacin; in addition, diclofenac caused fewer central nervous system reactions than indomethacin. Diclofenac is administered in divided doses with meals. The recommended total daily dosage is 100 to 150 mg (osteoarthritis and ankylosing spondylitis) or 150 to 200 mg (rheumatoid arthritis). Diclofenac is effective, but no more so than other NSAIDs. It is structurally distinct and offers another choice in the treatment of rheumatological conditions.     

Multi-residue determination of anti-inflammatory analgesics in sera by liquid chromatography--mass spectrometry

Non-steroidal anti-inflammatories (NSAIDs) are analgesic, antipyretic, and, as their name implies, anti-inflammatory drugs, which are widely used for the treatment of a variety of human and veterinary disease conditions in which control of pain and inflammation is desired. Acetaminophen (ACE) is a common over-the-counter analgesic. Detection of a variety of widely used NSAIDs and ACE in fluid and tissue samples is an important diagnostic tool. A sensitive and selective analytical method has been developed for simultaneous screening of 12 NSAIDs and ACE by liquid chromatography-mass spectrometry with an atmospheric pressure chemical ionization interface set to operate in the negative ion mode of MS. Following sample preparation, all analytes were separated on a C18-reversed-phase column with a gradient elution of acetonitrile and acetic acid. Full-scan mass spectral fragmentation profiles were established for each analyte and individual extracted ion chromatograms were used for quantitation. Linearity of detection was observed over the 0.05-25.0 microg/mL range of standard concentrations. The instrument limits of detection (LOD), based on an individual analyte quantitation ions, fell between 0.05 and 1.0 microg/mL for all compounds. The matrix LODs were determined to be 0.05 microg/mL for phenylbutazone (m/z 307); 0.1 microg/mL for indomethacin (m/z 312), flunixin (m/z 295), and piroxicam (m/z 330); 0.5 microg/mL for ACE (m/z 150), diclofenac (m/z 250), ketoprofen (m/z 209), and mefenamic acid (m/z 240); 1.0 microg/mL for oxyphenbutazone (m/z 323); 5.0 microg/mL for ibuprofen (m/z 205), salicylic acid (m/z 137), and tolmetin (m/z 212); and 10 microg/mL for naproxen (m/z 185).     

Pyrogenic fever and blood plasma glucocorticoids after nonsteroid anti-inflammatory drugs

Rabbits were injected with the lipopolysaccharide from E. coli (LPS) and received orally nonsteroid anti-inflammatory drugs (NSAIDs): acetylsalicylic acid, indomethacin, mefenamic acid, ibuprofen, aminophenazone, metamizole sodium, and phenylbutazone. These NSAIDs exerted antipyretic action without inhibiting the increase in the level of plasma glucocorticoids induced by LPS. This finding indicates the lack of correlation between the pyrogenic action of bacterial pyrogen and pyrogenic increase in the plasma glucocorticoid level. The investigated NSAIDs when given alone to normothermic rabbits differently affected the plasma glucocorticoid level: acetylsalicylic acid, indomethacin and ibuprofen depressed the plasma level of these hormones, mefenamic acid and phenylbutazone elevated it, and aminophenazone and metamizole sodium did not alter it significantly.     

Effects of non-steroidal anti-inflammatory drugs on polymorphonuclear leukocyte functions in vitro: focus on fenamates

Prostanoid-independent anti-rheumatic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are a matter of debate. The aim of the present study was to compare the effects of chemically different NSAIDs (diclofenac, indomethacin, ketoprofen, paracetamol, piroxicam and four fenamates: flufenamic, meclofenamic, mefenamic and tolfenamic acids) on human polymorphonuclear leukocyte (PMN) functions, i.e. calcium ionophore A23187-triggered degranulation, leukotriene 134 (LTB4) release, platelet-activating factor (PAF) production and migration towards LTB4. The four fenamates caused a dose-dependent inhibition of each of the PMN functions tested. Flufenamic, meclofenamic and tolfenamic acids were about equipotent to inhibit PMN degranulation (IC₅₀s 21–32 M) and LTB4 release (IC₅₀s 21–25 M) whereas mefenamic acid achieved similar effects at somewhat higher drug concentrations. Tolfenamic and meclofenamic acids were the most potent fenamates to inhibit PAF synthesis (IC₅₀s 37 and 51 M) as well as migration towards LTB4 (IC₅₀s 61 and 92 M). Out of the other NSAIDs, diclofenac (which is chemically related to fenamates) suppressed degranulation as well as LTB4 and PAF production. Indomethacin inhibited LTB₄ and PAF synthesis whereas ketoprofen reduced degranulation. The inhibitory effects of the non-fenamate NSAIDs occurred only at drug concentrations far higher than those achieved clinically. Paracetamol and piroxicam (up to 300 M) did not influence the PMN functions tested. We conclude that NSAIDs with a fenamate structure differ from other NSAIDs by inhibiting PMN functions induced either by receptor-mediated stimulus (LTB4) or calcium ionophore (A23187) at micromolar drug concentrations. Correspondence to: E. Moilanen at the above address     

In vitro evaluation of the complexation of non-steroidal anti-inflammatory drugs with caffeine

The complexation of five non-steroidal anti-inflammatory drugs (NSAIDs) with caffeine was evaluated. Mixtures were prepared by powder mixing, trituration in a mortar with a pestle and recrystallisation from a common solvent. The properties of the mixtures and the recrystallised materials, evaluated by differential scanning calorimetry (DSC) and dissolution rate measurements, differed significantly (P<0.05). Disappearance of peaks in DSC thermograms, or lowering of melting points of the NSAIDs in NSAID-caffeine co-precipitates were ascribed to the complexation of caffeine with the NSAIDs. The dissolution rate of ketoprofen was not affected by the addition of caffeine but the dissolution rates of ibuprofen, mefenamic acid, naproxen and indomethacin were significantly enhanced (P0.05) after being co-precipitated, or mixed in a mortar or V-blender, with caffeine. Higher dissolution rates, because of the complexation of caffeine and the drugs in solution, could translate into changed bioavailability and different pharmacokinetic parameters. Less gastrointestinal irritation and quicker onset of required blood levels might improve the efficacy of NSAIDs, a factor that, if confirmed, should be considered when prescribing these drugs to patients.     

Inhibition of human liver phenol sulfotransferase by nonsteroidal anti-inflammatory drugs

Objective: The aim of this investigation was to study the inhibition of 11 nonsteroidal anti-inflammatory drugs (NSAIDs) on the human liver phenol sulfotransferases (HL-PST) and catechol sulfotransferase (HL-CST). Methods: The activities of HL-PST and HL-CST were measured with 4 μM 4-nitrophenol and 60 μM dopamine (the sulfate acceptors) and 0.4 μM 3′-phosphoadenosine-5′-phosphosulfate [³⁵S] (the sulfate donor). Samples of liver were obtained from five patients, aged 55–79 years, undergoing clinically indicated hepatectomy. The inhibition curves were constructed with at least five concentrations of the inhibitor. Results: With the exception of piroxicam, NSAIDs inhibited HL-PST, and the estimates of the inhibitory concentration for 50% of responses (IC₅₀; μM) were: 0.02 (mefenamic acid), 3.7 (diflunisal), 5.4 (nimesulide), 9.5 (diclofenac), 30 (salicylic acid), 41 (ketoprofen), 74 (indomethacin), 159 (ibuprofen), 245 (ketoralac) and 473 (naproxen). With 4-nitrophenol as the variable substrate, the inhibition of salicylic acid on HL-PST was non-competitive and the K_i and K_ies were 18 μM and 21 μM (n = 5; P = 0.548), respectively. HL-CST was less susceptible than HL-PST to inhibition by NSAIDs, with only five drugs inhibiting this enzyme. The IC₅₀ estimates for these drugs (μM) were 76 (mefenamic acid), 79 (diflunisal), 103 (indomethacin), 609 (salicylic acid) and 753 (diclofenac). Conclusion: The comparison of the IC₅₀ estimates of HL-PST with the therapeutic plasma concentrations of NSAIDs corrected for the plasma unbound fraction was consistent with the view that mefenamic acid and salicylic acid, when administered at therapeutic doses, should impair the hepatic sulfation of those compounds that are substrates of phenol sulfotransferase. Received: 7 June 1999 / Accepted in revised form: 13 January 2000     

Mycophenolic acid glucuronidation and its inhibition by non-steroidal anti-inflammatory drugs in human liver and kidney

OBJECTIVE: The aims of this investigation were to study the glucuronidation of mycophenolic acid (MPA) in human liver and kidney and to search for a compound that inhibits MPA glucuronidation among the non-steroidal anti-inflammatory drugs (NSAIDs). METHODS: A sensitive and reproducible radiometric assay was developed to measure the rate of MPA glucuronidation in human liver and kidney microsomes. The assay employed uridine 5'-diphosphate-[U-14C]-glucuronic acid (UDPGA) and MPA-glucuronide was isolated by TLC. The final concentrations of UDPGA and MPA necessary were 1 mM (liver), and MPA concentration was 0.5 mM (kidney). The inhibition of MPA glucuronidation was studied with 18 NSAIDs and tacrolimus. RESULTS: Glucuronosyl transferase activity followed Michaelis-Menten kinetics and the Km (mean +/- SD; mM) was 0.31+/-0.06 (liver; n = 5) and 0.28+/-0.07 (kidney; n = 5; P = 0.555); the Vmax (mean SD; nmol/mg per minute) was 5.2+/-1.4 (liver; n = 5) and 10.5+/-1.2 (kidney; n = 5; P = 0.0005). The MPA glucuronidation rates (mean +/- SD; nmol/min/mg) were 3.3+/-0.9 (liver; n = 10) and 7.8+/-1.5 (kidney; n = 10; P = 0.0002). The rate of MPA glucuronidation ranged between 2.0 and 5.1 nmol/ mg per minute with a 2.5-fold variation (liver) and between 5.7 and 9.8 nmol/mg per minute with a 1.7-fold variation (kidney). The inhibition study was performed in liver and revealed that the percentage of control ranged from 8%+/-3% (niflumic acid) to 119%+/-16% (Ketoralac). The inhibition curves for MPA glucuronidation rate were determined with the four most effective inhibitors: niflumic acid, flufenamic acid, mefenamic acid and diflunisal. Their IC50 estimates (microM) were 8+/-1, 19+/-9, 63+/-8 and 109+/-15, respectively (liver), and 8+/-2, 13+/-2, 49+/-4 and 122+/-18, respectively (kidney). The IC50 estimate for niflumic acid was eightfold lower than the peak plasma levels after a single oral dose of 250 mg of this drug. CONCLUSION: The human liver and kidney are important sites of MPA glucuronidation. MPA glucuronidation was inhibited to various extents by different NSAIDs and the four most effective inhibitors were niflumic acid, flufenamic acid, mefenamic acid and diflunisal. These drugs have similar molecular structures consisting of two aromatic rings bearing a carboxylic group.     

Glucuronidation of fenamates: kinetic studies using human kidney cortical microsomes and recombinant UDP-glucuronosyltransferase (UGT) 1A9 and 2B7

Mefenamic acid, a non-steroidal anti-inflammatory drug (NSAID), is used commonly to treat menorrhagia. This study investigated the glucuronidation kinetics of flufenamic, mefenamic and niflumic acid using human kidney cortical microsomes (HKCM) and recombinant UGT1A9 and UGT2B7. Using HKCM Michaelis-Menten (MM) kinetics were observed for mefenamic (K(m)(app) 23 microM) and niflumic acid (K(m)(app) 123 microM) glucuronidation, while flufenamic acid exhibited non-hyperbolic (atypical) glucuronidation kinetics. Notably, the intrinsic renal clearance of mefenamic acid (CL(int) 17+/-5.5 microL/minmg protein) was fifteen fold higher than that of niflumic acid (CL(int) 1.1+/-0.8 microL/minmg protein). These data suggest that renal glucuronidation of mefenamic acid may result in high intrarenal exposure to mefenamic acyl-glucuronide and subsequent binding to renal proteins. Diverse kinetics were observed for fenamate glucuronidation by UGT2B7 and UGT1A9. Using UGT2B7 MM kinetics were observed for flufenamic (K(m)(app) 48 microM) and niflumic acid (K(m)(app) 135 microM) glucuronidation and atypical kinetics with mefenamic acid. Similarity in K(m)(app) between HKCM and UGT2B7 suggests that UGT2B7 may be the predominant renal UGT isoform catalysing niflumic acid glucuronidation. In contrast, UGT1A9 glucuronidation kinetics were characterised by negative cooperativity with mefenamic (S(50) 449 microM, h 0.4) and niflumic acid (S(50) 7344 microM, h 0.4) while atypical kinetics were observed with flufenamic acid. Additionally, potent inhibition of the renal glucuronidation of the UGT substrate 'probe' 4-methylumbelliferone by flufenamic, mefenamic and niflumic acid was observed. These data suggest that inhibitory metabolic interactions may occur between fenamates and other substrates metabolised by UGT2B7 and UGT1A9 in human kidney.     

Comparison of inhibition potentials of drugs against zidovudine glucuronidation in rat hepatocytes and liver microsomes.

Hepatocytes and liver microsomes are considered to be useful for investigating drug metabolism catalyzed mainly via glucuronidation. However, there have been few reports comparing the glucuronidation inhibition potentials of drug in hepatocytes to those in liver microsomes. 3'-Azido-3'-deoxythymidine (AZT, zidovudine) glucuronidation (AZTG) is the major metabolic pathway for AZT. In this study, the inhibition potentials of drugs against UDP-glucuronosyltransferase (UGT)-catalyzed AZTG in the hepatocytes and liver microsomes of rats are compared. The AZTG inhibition potentials of diclofenac, diflunisal, fluconazole, indomethacin, ketoprofen, mefenamic acid, naproxen, niflumic acid, and valproic acid in liver microsomes and hepatocytes were investigated using liquid chromatography with tandem mass spectrometry. Diflunisal (inhibition type: noncompetitive) inhibited AZTG most potently in rat liver microsomes (RLMs) with an IC(50) value of 34 microM. The IC(50) values of diclofenac, fluconazole, indomethacin, ketoprofen, mefenamic acid, naproxen, niflumic acid, and valproic acid against AZTG in RLMs ranged from 34 to 1791 microM. Diclofenac, diflunisal, indomethacin, ketoprofen, naproxen, and valproic acid inhibited AZTG in hepatocytes with IC(50) values of 58, 37, 88, 361, 486, and 281 microM, respectively. These values were similar to those obtained in RLMs. In conclusion, the AZT glucuronidation inhibition potentials of drugs in the hepatocytes and liver microsomes of rats were found to be similar, and liver microsomes can be useful for evaluating UGT isozyme inhibition potentials.     

Determination of diclofenac sodium, flufenamic acid, indomethacin and ketoprofen by LC-APCI-MS

A sensitive, selective and accurate high-performance liquid chromatography-mass spectrometry (LC-MS) assay for the determination of selected non-steroidal anti-inflammatory drugs (NSAIDs), namely diclofenac sodium (DIC), flufenamic acid (FLU), indomethacin (IND) and ketoprofen (KET), either individually or in mixtures, was developed. The examined drugs were injected onto Shim-pack GLC-CN column and were eluted with a mobile phase consisting of acetonitrile and 20 mM ammonium acetate solution (5:1 v/v)/pH 7.4 at a flow rate l ml min(-1). The mass spectrometer, operated in the single ion monitoring mode, was programmed to admit the negative ions [M-H] at m/z 295.9 (DIC), 280.1 (FLU), 355.8 (IND) and 252.9 (KET), respectively. The calibration curves were linear (r > or = 0.9993) over the concentration range 50-300 ng ml(-1) (FLU, DIC) and 100-500 ng ml(-1) (KET, IND) with detection limits of 0.5-4.0 ng. The mean predicted concentrations for the analytes were in the range -5.9 and 5.2% of the nominal concentrations. Within-day and between-day precision were in the range of 0.8-9.1% of the R.S.D. Mean recovery percentages of the individual compounds from laboratory-made mixtures and pharmaceutical formulations were (99.5-101.5%) and (100.6-102.2%), respectively.     

Synthesis and Biological Evaluation of Ω-(N,N,N-Trialkylammonium)alkyl Esters and Thioesters of Carboxylic Acid Nonsteroidal Antiinflammatory Agents

A series of novel -(N,N,N-trialkylammonium)alkyl ester and thioester derivatives [RCOM(CH₂) _n NR ₃ ⁺ X ^–, M = O or S, n = 2–6, X = I or Cl] of 11 nonsteroidal antiinflammatory carboxylic acid agents (naproxen, ketorolac, indomethacin, ibuprofen, sulindac, ketoprofen, flufenamic acid, mefenamic acid, zomepirac, etodolac, and tifurac) was prepared and evaluated for their antiinflammatory, analgesic, and gastrointestinal erosive properties. In general, each prodrug retained the antiinflammatory activity characteristic of the corresponding parent drug but exhibited moderately to greatly reduced gastrointestinal erosive properties and significantly reduced analgetic potencies. This profile is likely due to a combination of factors including the rate of hydrolysis of the esters in the stomach, gut, and plasma, changes in the locus of absorption of the prodrug or nonsteroidal antiinflammatory drug (NSAID), and altered metabolic disposition patterns resulting from these changes. The results obtained from the compounds of this series indicate that esters of this general class may offer a means to modulate both the aqueous/lipid solubility and the hydrolytic/enzymatic cleavage indices of NSAID prodrugs which potentially possess a more favorable therapeutic ratio of antiinflammatory to gastrointestinal erosive activities.     

Superoxide anion mediates the L-selectin down-regulation induced by non-steroidal anti-inflammatory drugs in human neutrophils

Non-steroidal anti-inflammatory drugs (NSAIDs) induce the shedding of L-selectin in human neutrophils through a mechanism still not well understood. In this work we studied both the functional effect of NSAIDs on the neutrophils/endothelial cells dynamic interaction, and the potential involvement of reactive oxygen species (ROS) in the NSAIDs-mediated down-regulation of L-selectin. When human neutrophils were incubated with diclofenac, a significant reduction in the number of cells that rolled on activated endothelial cells was observed. Different NSAIDs (flufenamic acid, meclofenamic acid, diclofenac, indomethacin, nimesulide, flurbiprofen, meloxicam, phenylbutazone, piroxicam, ketoprofen and aspirin) caused variable increase in neutrophil intracellular ROS concentration, which was inversely proportional to the change produced in L-selectin surface expression. Pre-incubation of neutrophils with superoxide dismutase, but not with catalase, showed both a significant protective effect on the L-selectin down-regulation induced by several NSAIDs and a diminished effect of diclofenac on neutrophil rolling. Interestingly, diclofenac and flufenamic acid but not piroxicam significantly increased the extracellular superoxide anion production by neutrophils, and inhibition of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity with diphenyleneiodonium prevented the down-regulation of L-selectin by diclofenac. In accordance with these results, neutrophils from patients with chronic granulomatous disease, a hereditary disease in which neutrophils show a reduced capacity to form superoxide radicals, exhibited a lower down-regulation of L-selectin (IC50: 15.3 μg/ml) compared to normal controls (IC50: 5.6 μg/ml) in response to diclofenac.ConclusionA group of NSAIDs is capable of interfering with the ability of neutrophils to interact with endothelial cells by triggering L-selectin-shedding through the NADPH-oxidase-dependent generation of superoxide anion at the plasma membrane.     

Influence of non-steroid anti-inflammatory and immunosuppressive drugs on hepatic tryptophan pyrrolase activity in rats

High oral doses of the anti-inflammatory drugs indomethacin, acetylsalicyclic acid, sodium salicylate, salicylamide, mefenamic acid, flufenamic acid, amidopyrine, phenylbutazone and benzydamine administered repeatedly, did not influence tryptophan pyrrolase activity in livers of intact rats. The nonresponsiveness of tryptophan pyrrolase was in contrast to a stimulation of tyrosine aminotransferase caused by flufenamic acid.Induction of tryptophan pyrrolase due to hydrocortisone was not inhibited generally by non-steroid drugs; exceptions were flufenamic acid and benzydamine which depressed hormonal induction. The authors' recent statement is confirmed that inhibition of protein synthesis due to induction is no essential property of non-steroid antiinflammatory drugs.The immunosuppressive drugs cyclophosphamide, triaziquone and azathioprine increased tryptophan pyrrolase activity, probably due to an inhibition of enzyme degradation; chloroquine, 6-mercaptopurin, 6-azauridin and amethopterin did not influence enzyme activity. Induction of tryptophan pyrrolase due to hydrocortisone was depressed by azathioprine, 6-mercaptopurin, 6-azauridin and amethopterin.