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.     

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.     

Inhibition by non-steroidal anti-inflammatory agents of the release of rabbit aorta contracting substance and prostaglandins from chopped guinea-pig lungs

The release of prostaglandins (PGs) and rabbit aorta contracting substance (RCS) was investigated using mechanical agitation of chopped lung tissue from unsensitized guinea-pigs. Manual stirring of the lung tissue for 45 s produced maximal release of prostaglandins, a release corresponding to the effect of [20–40 ng ml^?1 of PGE₂ x 45 s at 5 ml min^?1] about 100 ng PGE₂ on the rat stomach strip. Nonsteroidal anti-inflammatory agents inhibited the release of the contracting substances. Indomethacin, the most potent, was active in concentrations of about 20 ng ml^?1, whereas flufenamic acid was twice, phenylbutazone about 60 times, acetylsalicylic acid 100 times and sodium salicylate about 6000 times less active than indomethacin. The method could prove to be a simple test for screening nonsteroidal anti-inflammatory agents for inhibition of prostaglandin synthesis.     

Antagonism by mefenamic and flufenamic acids of the bronchoconstrictor action of kinins in the guinea-pig

In the guinea-pig, N-(2,3-xylyl)anthranilic acid (mefenamic acid) and N-(α,α,α-trifluoro-m-tolyl)anthranilic acid (flufenamic acid), two new anti-inflammatory agents, antagonize bronchoconstriction, but not hypotension, produced by kinins. They do not reduce bronchoconstrictor responses to acetylcholine, histamine or 5-hydroxytryptamine. The antibradykinin potencies of mefenamic and flufenamic acids approximately equal that of acetylsalicylic acid when given intravenously and of phenylbutazone when given into the duodenum. After administration of mefenamic and flufenamic acids, the bronchoconstrictor response can be restored by higher doses of bradykinin. The quantitative relationship between the intravenous dose of sodium mefenamate or flufenamate and the dose of bradykinin needed to surmount either antagonist in bronchial muscle fulfils the requirements for competitive antagonism. Antagonism by calcium acetylsalicylate can also be surmounted with higher doses of bradykinin, but in this instance the relationship of antagonist to agonist fulfils requirements for competitive antagonism only at the lower part of the dose range.     

Inhibition of superoxide anion production in macrophages by anti-inflammatory drugs.

The inhibition by anti-inflammatory drugs of the production of Superoxide anions (O₂^?) by isolated guinea pig macrophages was studied spectrophotometrically using NADH and lactate dehydrogenase. id₅₀ values were: 4 × 10^?7M (diclophenac sodium), 1 × 10^?6M (oxyphenbutazone), 1 × 10^?5M (indomethacin), 4 × 10^?5M (phenylbutazone), 7 × 10^?5M (mefenamic acid), 8 × 10^?5 M (flufenamic acid), 8 × 10^?5M (colchicine), 3 × 10^?4M (aspirin), 3 × 10^?4M (benzydamine), 10^?3M < (dexamethasone) and 10^?3M < (gold sodium thiomalate). They seemed to block the cell membrane-associated mechanism to produce Superoxide anions, since most of them did not abolish the generation of superoxide anions from the xanthine oxidase plus hypoxanthine system. Cytochalasin B, pyrogallol, ascorbate, NEM, l-epinephrine and chlorpromazine also inhibited, the production of Superoxide anion, but many non anti-inflammatory drugs were ineffective. This technique was evaluated as a screening method in vitro for nonsteroidal anti-inflammatory drugs.     

Simultaneous determination of non-steroidal anti-inflammatory drugs in river water samples by liquid chromatography-tandem mass spectrometry using molecularly imprinted polymers as a pretreatment column

A restricted access media-molecularly imprinted polymer (RAM-MIP) for flufenamic acid has been developed for the simultaneous determination of non-steroidal anti-inflammatory drugs (NSAIDs) in river water samples. The RAM-MIP was prepared using 4-vinylpyridine and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multi-step swelling and polymerization method followed by a surface modification technique. The RAM-MIP for flufenamic acid showed excellent molecular recognition abilities for flufenamic acid and mefenamic acid, and moderate molecular recognition abilities for indomethacin, etodolac and ketoprofen. The simultaneous determination of NSAIDs (mefenamic acid, indomethacin, etodolac and ketoprofen) in river water samples was carried out by LC-MS/MS using the RAM-MIP for flufenamic acid as a pretreatment column. The concentrations of mefenamic acid, indomethacin and etodolac in river water samples were determined to be 0.4, 0.7 and 0.3ng/L, respectively, while ketoprofen was below the limit of quantitation.     

Proteinuria-lowering effect of anti-inflammatory drugs

Summary

Indomethacin is known to reduce proteinuria in most cases of the nephrotic syndrome, regardless of the type of the glomerular lesion. This study shows the effect of ibuprofen and flufenamic acid in a total of 25 cases of protein-losing nephropathy. The response was good in 11 out of 14 cases treated with ibuprofen and 6 out of 11 cases treated with flufenamic acid. Ibuprofen appeared to increase the glomerular filtration rate and flufenamic acid to lower it in association with the decrease in protein loss. The authors suggest that this may be a possible parameter for investigating the anti-inflammatory activity of a new compound.     

Contribution of cytochrome P450 and UDT-glucuronosyltransferase to the metabolism of drugs containing carboxylic acid groups: risk assessment of acylglucuronides using human hepatocytes

Abstract

1.?In order to evaluate the inhibition activity of 1-aminobenzotriazole (ABT) and (?)-borneol (borneol) against cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT), the substrates of these metabolic enzymes were incubated with ABT and borneol in human hepatocytes. We found that 3?mM ABT and 300?μM borneol were the most suitable experimental levels to specifically inhibit CYP and UGT.

2.?Montelukast, mefenamic acid, flufenamic acid, diclofenac, tienilic acid, gemfibrozil, ibufenac and repaglinide were markedly metabolized in human hepatocytes, and the metabolism of gemfibrozil, mefenamic acid and flufenamic acid was inhibited by borneol. With regard to repaglinide, montelukast, diclofenac and tienilic acid, metabolism was inhibited by ABT. Ibufenac was partly inhibited by both inhibitors. Zomepirac, tolmetin, ibuprofen, indomethacin and levofloxacin were moderately metabolized by human hepatocytes, and the metabolism of zomepirac, ibuprofen and indomethacin was equally inhibited by both ABT and borneol. The metabolism of tolmetin was strongly inhibited by ABT, and was also inhibited weakly by borneol. Residual drugs, telmisartan, valsartan, furosemide, naproxen and probenecid were scarcely metabolized.

3.?Although we attempted to predict the toxicological risks of drugs containing carboxylic groups from the combination chemical stability and CL_int via UGT, the results indicated that this combination was not sufficient and that clinical daily dose is important.     

Effects of antirheumatic drugs on sponge-induced granulation tissue, rheumatoid synovial tissue, matrix-free tendon cells and fibroblast plasma membranes in vitro.

Slices of sponge-induced, mature granulation tissue were incubated in the presence of antirheumatic drugs (sodium acetylsalicylate, indomethacin and phenylbutazone at concentrations of 10^?7–10^?2M) and labelled precursors of nucleic acids, proteins and acid mucopolysaccharides. Analogous experiments were made with sliced human synovial tissue from rheumatoid patients and with matrix-free embryonic chick tendon cells. The synthetic functions were suppressed by 10^?4–10^?3M indomethacin and phenylbutazone but only by 10^?2m acetylsalicylic acid. At low concentrations (10^?5–10^?6M) there was an increase in the radioactivity in proteins during the early period of the incubation but this was reversed later. Indomethacin and phenylbutazone affected the production of collagen at two points: (i) the synthesis of the peptide chain and. more notably, (ii) the secretion of collagen to the extracellular space. Indomethacin affected the final content of cellular collagen only slightly. Preliminary experiments on the effect of indomethacin on the activities of enzymes in plasma membranes prepared from granulation tissue showed some effect on leucyl-β-naphthylamidase and Na⁺, K⁺ -activated Mg²⁺- adenosine triphosphatase. The use of such systems for the screening of new connective tissue-active drugs is discussed. The secretion of collagen from matrix-free embryonic tendon cells is the most sensitive target for indomethacin in vitro.     

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.     

Toxicity assessments of nonsteroidal anti-inflammatory drugs in isolated mitochondria,rat hepatocytes,and zebrafish show good concordance across chemical classes

To reduce costly late-stage compound attrition, there has been an increased focus on assessing compounds in in vitro assays that predict attributes of human safety liabilities, before preclinical in vivo studies are done. Relevant questions when choosing a panel of assays for predicting toxicity are (a) whether there is general concordance in the data among the assays, and (b) whether, in a retrospective analysis, the rank order of toxicity of compounds in the assays correlates with the known safety profile of the drugs in humans. The aim of our study was to answer these questions using nonsteroidal anti-inflammatory drugs (NSAIDs) as a test set since NSAIDs are generally associated with gastrointestinal injury, hepatotoxicity, and/or cardiovascular risk, with mitochondrial impairment and endoplasmic reticulum stress being possible contributing factors. Eleven NSAIDs, flufenamic acid, tolfenamic acid, mefenamic acid, diclofenac, meloxicam, sudoxicam, piroxicam, diflunisal, acetylsalicylic acid, nimesulide, and sulindac (and its two metabolites, sulindac sulfide and sulindac sulfone), were tested for their effects on (a) the respiration of rat liver mitochondria, (b) a panel of mechanistic endpoints in rat hepatocytes, and (c) the viability and organ morphology of zebrafish. We show good concordance for distinguishing among/between NSAID chemical classes in the observations among the three approaches. Furthermore, the assays were complementary and able to correctly identify “toxic” and “non-toxic” drugs in accordance with their human safety profile, with emphasis on hepatic and gastrointestinal safety. We recommend implementing our multi-assay approach in the drug discovery process to reduce compound attrition.     

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 HLPST and HL-CST were measured with 4-nitrophenol and dopamine as substrates, respectively. 2. The IC₅₀ for inhibition of HL-PST were 0.02 μM (mefenamic acid); 0.12 μM (tolfenamic acid); 0.28 μM (niflumic acid); 0.87 μM (meclofenamic acid) and 1.50 μM (flufenamic acid). 3. HL-CST was less susceptible than HL-PST to the inhibition by fenamates and the IC₅₀ for HL-CST were 36 μM (tolfenamic acid); 70 μM (flufenamic acid); 76 μM (mefenamic acid); 180 μM (niflumic acid) and 185 μM (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.     

Section 2 The comparison of four non-steroidal antirheumatic agents in the treatment of rheumatic diseases

Summary

A comparative open study was made of the effectiveness of four antirheumatic agents in the treatment of 69 hospitalised patients with rheumatic diseases who had failed to respond satisfactorily to previous therapies. Simple subjective and objective assessments showed that approximately half the patients treated with ibuprofen, oxyphen-butazone or mefenamic acid showed improvement, but that fewer did so with flufenamic acid. Full therapeutic effect of the drugs was evident in approximately 3 weeks.     

Flow-injection spectrofluorimetric determination of flufenamic and mefenamic acid in pharmaceuticals

Two sensitive and rapid flow-injection (FI) spectrofluorimetric methods are proposed for the determination of flufenamic acid (FF) and mefenamic acid (MF), based on the formation of complexes of these compounds with Al(III) in an ethanolic medium. The calibration graphs resulting from the measurements of the fluorescence at λ_exc = 351 nm and λ_em = 440 nm, and λ_exc = 355 nm and λ_em = 454 nm for the complexes with FF and MF, respectively, are linear over the range 0.030–1.20 μg ml⁻¹ for FF and 0.30–16.1 μg ml⁻¹ for MF. The methods have been applied to the determination of these drugs in pharmaceutical preparations.     

In vitro effects of nonsteroidal anti-inflammatory drugs on oxidative phosphorylation in rats liver mitochondria.

Representative potent anti-inflammatory drugs have been studied for their ability to affect energy metabolism in vitro in rat liver mitochondria. Like 2,4-dinitrophenol, flufenamic acid activated ATPase, inhibited the incorporation of ³²Pi into the ATP fraction, stimulated glutamate oxidation and coupled substrate-level phosphorylation, and promoted state-4 respiration. Aspirin and ibuprofen caused similar but lesser effects on these parameters. These three drugs are considered to be uncouplers of oxidative phosphorylation. Indomethacin and phenylbutazone inhibited both oxidative and substrate-level phosphorylations. Based on their abilities to inhibit glutamate oxidation rather directly and to block the electron transport chain, it was concluded that indomethacin and phenylbutazone could inhibit the generation of energy-rich phosphate compounds by suppressing respiration at multiple sites. Thus, these anti-inflammatory drugs could prevent mitochondrial energy metabolism in different manners.     

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     

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     

Effect of suspending agents on the characteristics of some anti-inflammatory suspensions

The effect of suspending agents on the physical stability and in vitro availability of mefenamic acid, flufenamic acid, glafenine, ibuprofen and azapropazone suspensions was studied. The ulcerogenic effect of these formulated suspensions on the stomach of rats was also investigated. The results revealed that 2% veegum and 2% sorbitol gave the best formulated suspension for glafenine as compared to other formulations. On the other hand, 2% veegum, 2% sobitol and 1% avicel was found to improve the physical stability of mefenamic acid and flufenamic acid suspensions. Also, the combination of 1% veegum, 1% sorbitol and 1% algin produced excellent suspension for ibuprofen and azapropazone as compared to other combinations. The results of in vitro release data proved an optimal availability of the above mentioned formulations. In addition, significant reduction in the gastric erosions in stomach of rats was observed in all mentioned suspensions except glafenine suspension.     

Cytoprotective effect of nonsteroidal antiinflammatory drugs in rat brain slices subjected to reoxygenation after oxygen-glucose deprivation

The aim of this study was to assess the possible neuroprotective effect of the main nonsteroidal antiinflammatory drugs (NSAIDs) in an experimental model of hypoxia-reoxygenation in rat brain slices. After reoxygenation the increase in lactate dehydrogenase (LDH) efflux was inhibited by nimesulide, celecoxib and meloxicam with an IC(50) in the 10(-6)M range, by flurbiprofen, ibuprofen and diclofenac in the 10(-5)M range, and by salicylic acid, indomethacin, acetylsalicylic acid and mefenamic acid the 10(-4)M range. The effect of other NSAIDs was seen with an IC(50) greater than 10(-3)M. A statistically significant linear correlation between the values of LDH efflux and prostaglandin E(2) was found for NSAIDs whose IC(50) of cytoprotection (LDH efflux) was below 10(-4)M. The concentration of interleukin 10 was increased with nimesulide, celecoxib, meloxicam, flurbiprofen, ibuprofen and diclofenac. Flurbiprofen and diclofenac significantly inhibited the production of lipid peroxides. The increase in brain nitrite levels was significantly reduced with celecoxib, flurbiprofen, diclofenac and salicylic acid. Concentrations of 3-nitrotyrosine were significantly reduced with celecoxib, flurbiprofen, ibuprofen, salicylic acid and ketorolac. In conclusion, NSAIDs with the greatest cytoprotective effect (nimesulide, celecoxib and meloxicam) may exert their effect mainly through the blockade of cyclooxygenase-2 (COX-2) activity. Other compounds with neuroprotective activity may complement their lower anti-COX-2 effect with a slight increase in interleukin 10 and reduced oxidative and nitrosative stress in our model of hypoxia-reoxygenation in rat brain slices.