[Determination of nabumetone and 6-methoxy-2-naphthylacetic acid in plasma using HPLC with UV and MS detection]. / Stanovení nabumetonu a kyseliny 6-methoxy-2-naftyloctové v plazme pomocí HPLC s UV a MS detekcí.

The study aimed to establish and validate an analytical method for the determination of nabumetone and 6-methoxy-2-naphthylacetic acid (6-MNA) in human plasma after a single therapeutic dose of the drug. Two methods based on HPLC with UV and MS detection were compared. Optimal results in sample preparation were achieved using solid phase extraction. The recovery reached approximately 84% and 86-90% for nabumetone and 6-MNA, respectively. A reverse C18 column was used for HPLC separation of the analytes. The limit of UV detection was 50 nM and 0.1 microM for 6-MNA and nabumetone, respectively. The limit of MS detection was 1 microM and 0.5 microM for 6-MNA and nabumetone, respectively. Precision ranged between 4.2-14.4% and 4.6-8.5% using UV and MS detection for nabumetone, respectively. The respective values for 6-MNA were 2.4-12.5% and 2.1-9.4%. Accuracy ranged between 93.4-109.6% in UV detection and 86.2-107.9% using UV and MS detection for nabumetone, respectively. The respective values for 6-MNA were 87.8-107.4% and 86.3-106.4%. The method was subsequently applied to determine the pharmacokinetic parameters of nabumetone and 6-MNA in a group of 24 healthy volunteers.

Simultaneous determination of two anti-inflammatory drugs in serum using isopotential fluorimetry.

The simultaneous determination of 6-methoxy-2-naphthylacetic acid (6MNA) and diflunisal in serum samples using the combination of matrix isopotential synchronous fluorescence (MISF) and first derivative technique is proposed. 6MNA and diflunisal exhibit overlapped spectra and serum produces background fluorescence that precludes the direct determination of these anti-inflammatory drugs by conventional fluorimetry. This method provides good analytical results for determination of compounds in samples with unknown background fluorescence. The method was applied for the simultaneous determination of 6MNA and diflunisal in serum samples at concentrations between 20-200 and 100-1000 ng mL(-1), respectively, by means of absolute values of first derivative of synchronous scan at 247.9/364.0 and 262.6/392.4 nm for 6MNA and diflunisal, respectively. In order to obtain maximum sensitivity and adequate selectivity, factors affecting fluorescence intensity were studied. As a result, the analyses were performed in water at a pH of 7.2, adjusted by using sodium dihydrogen phosphate/hydrogen phosphate (0.1M) as a buffer solution. Serum samples were diluted 200 times. Analytical parameters of the proposed method were calculated according to the error propagation theory. The limit of detection calculated according to Clayton was 15.8 and 63.0 ng mL(-1) for 6MNA and diflunisal, respectively. The sensitivity, repeatability and reproducibility achieved with the proposed method were adequate for the determination of these anti-inflammatory agents in serum samples.

High-throughput LC-MS/MS assay for 6-methoxy-2-naphthylacetic acid, an active metabolite of nabumetone in human plasma and its application to bioequivalence study.

A simple, precise and accurate assay for the determination of 6-methoxy-2-naphthylacetic acid (6-MNA), an active metabolite of nabumetone in human plasma, was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analyte (6-MNA) and propranolol (internal standard, IS) were extracted from 200 microL aliquot of human plasma via solid-phase extraction employing HLB Oasis cartridges and separated on a Discovery HS C18 (50 x 4.6 mm, 5 microm) column. Detection of analyte and IS was done by tandem mass spectrometry with a turbo ion spray interface operating in positive ion and multiple reaction monitoring acquisition mode. The total chromatographic runtime was 3.0 min with retention time for 6-MNA and IS at 1.97 and 1.26 min, respectively. The method was validated over a dynamic linear range of 0.20-60.00 microg/mL for 6-MNA with mean correlation coefficient r > or = 0.9986. The intra-batch and inter-batch precision (%CV) across five validation runs (lower limit of quantiation, low-, medium- and high-quality controls and upper limit of quantitation) was less than 7.5%. The accuracy determined at these levels was within -5.8 to +0.2% in terms of percentage bias. The method was successfully applied for a bioequivalence study of 750 mg nabumetone tablet formulation in 12 healthy Indian male subjects under fasted condition.

Extractionless determination of 6-methoxy-2-naphthylacetic acid, a major metabolite of nabumetone, in human plasma by high-performance liquid chromatography.

Following oral administration of the prodrug nabumetone, the major metabolite 6-methoxy-2-naphthylacetic acid (6-MNA) was determined in human plasma. Minimal sample preparation was followed by reversed-phase liquid chromatography and UV detection, affording high sample throughput. The lower limit of quantification (LLOQ) was 70 ng/ml, at a signal-to-noise ratio of 8:1. The assay method displayed good correlation (r=0.997), and can be readily employed in pharmacokinetic and bioequivalence studies.

WY-50295 tromethamine: a 5-lipoxygenase inhibitor without activity in human whole blood.

The 5-LO inhibitor, WY-50295 tromethamine (T) prevented leukotriene release (LTB4 production) in calcium ionophore stimulated, purified human and rat neutrophils. However, whereas WY-50295T inhibited both in vitro and ex vivo rat whole blood leukocyte LTB4 formation (IC50= 40 microM and oral ED50 of 18 mg/kg, respectively), it did not inhibit LTB4 production in calcium ionophore stimulated human whole blood at concentrations to 200 microM. To reduce binding of WY-50295T to serum albumin, 250 microM of a naphthalene sulfonic acid (> 99.9% binding to albumin primarily at the carboxylic site) and 250 microM sulfanilamide (binding to nonspecific sites) separately or in combination were preincubated in whole blood prior to addition of WY-50295T; however, WY-50295T still did not inhibit 5-LO and free drug blood levels were unchanged. When purified human neutrophils in the presence of fatty acid saturated albumin (fraction V) was employed, the 5-LO inhibitory activity of WY-50295T was prevented. Zileuton (5 microM) inhibited LTB4 production by 99% in the presence of these albumins. Also, rat albumin presented WY-50295T to purified rat neutrophils more effectively than human albumin (i.e. WY-50295T was more active in the presence of rat albumin). These results suggest that the high affinity binding of WY-50295T to human albumin and possibly the reduction of drug uptake (passive diffusion) using purified human vs rat neutrophils may account for the inactivity of WY-50295T in the human whole blood assay.

[Determination of 6-methoxy-2-naphthylacetic acid, a major metabolite of nabumetone, in human plasma by HPLC].

This paper reports a sensitive and rapid method for determining 6-methoxy-2-naphthylacetic acid (MNA), a major metabolite of nabumetone in human plasma using naproxen as the internal standard. High performance liquid chromatograph model 680 (Waters, USA) with a variable wavelength UV detector and reversed-phase YWG-C18 column (10 microns, 250 x 4.6 mm) was used. After the addition of acetate buffer(pH3.0), the plasma sample was extracted with methylene chloride. The mobile phase of methanol-pH3.0, 0.02 mol/L acetate buffer(74:26) was pumped at 1.0 ml/min through the column. The detector at 0.01AUFS was set at 270 nm. The retention times for MNA and naproxen were 3.98 min and 4.73 min respectively. Standard curve was linear in the concentration range of 0.5 to 64 mg/L. The detection limit in serum was 0.02 mg/L. Extraction recovery was 88%-94%; method recovery 96%-102%; withinday RSD less than 3.5%; inter-day RSD less than 5%.

Heat inactivation of paraoxonase and arylesterase activities in human and rabbit serum.

The heat inactivation of esterases in human and rabbit serum was followed at 50 and 55 degrees C by measuring the decrease of activity with paraoxon, phenylacetate and beta-naphthylacetate as substrates. The rate of inactivation measured with the three substrates was slightly, but significantly different, indicating that the substrates are hydrolysed by different enzymes.

The science--equivalent efficacy and diminished risk.

Nabumetone is an effective anti-inflammatory drug in models of inflammation and in man, comparable in potency to other compounds of this type. It differs from other compounds in several important respects. The parent molecule is non-acidic and virtually inactive but is transformed to an active metabolite (6MNA) by the liver. 6MNA is not excreted in bile and cannot therefore, by reflux from the small intestine, reach the stomach. Many other compounds can reach the stomach in this way even when given by routes other than oral, for example by suppository. Prostaglandin production by the stomach, a protective mechanism, is not markedly affected by 6MNA whereas it is suppressed by other NSAIDs. These studies suggest a favourable therapeutic ratio for nabumetone.

Penetration of the active metabolite of nabumetone into synovial fluid and adherent tissue of patients undergoing knee joint surgery.

The concentration of 6-methoxy-2-naphthylacetic acid (6-MNA) in plasma, synovial fluid, synovial tissue and fibrous capsule tissue was determined in an open study with 20 patients scheduled for knee joint surgery after oral treatment with nabumetone under steady-state conditions. 6-MNA is the principle metabolite of the prodrug nabumetone arising from an extensive first-pass metabolism in the liver. Patients suffering from rheumatoid arthritis (n = 12) or osteoarthritis stage III or IV (n = 8) received a daily dose of nabumetone 1 g in the evening starting 4 days prior to surgery. On day 1 an additional loading dose of nabumetone 1 g was given in the morning. At the time of surgery (day 5), blood, synovial tissue and fibrous capsule tissue were taken simultaneously. The samples were analysed by high performance liquid chromatography. After 4 days of treatment mean 6-MNA concentration in plasma was 40.76 mg/L, in synovial fluid 34.79 mg/L, in synovial tissue 19.33 mg/g and in fibrous capsule tissue 11.43 mg/g. Under steady-state conditions mean synovial fluid levels of 6-MNA were higher than after administration of a single dose and, in common with levels in synovial tissue, persist in a range sufficient for in vitro cyclo-oxygenase inhibition.

A pharmacokinetic study of the active metabolite of nabumetone in young healthy subjects and older arthritis patients.

We have performed a detailed pharmacokinetic study of the plasma concentrations of the major active metabolite of nabumetone, 6-methoxy-2-naphthylacetic acid (6 MNA), attained after a single dose and during chronic administration comparing the results of a group of young healthy volunteers with those of a group of elderly arthritic patients. The latter had higher peak plasma concentrations of 6 MNA and slower rates of elimination but there is no tendency for the drug to accumulate unpredictably in the old. Disease activity also influences plasma concentration, those with more active disease, and lower serum albumin concentrations had lower AUC values.

Metabolism of isobutylnaphthyl acetic acid in rats: determination of the chemical structures of metabolites.

After oral and intravenous administration of radiolabelled isobutylnaphthyl acetic acid (INAA) to rats two metabolites were isolated from urine and plasma by HPLC. Field desorption, high resolution electron impact mass spectrometry as well as GC-MS after derivatization were used for structure elucidation and identification of the metabolites. The main biotransformation product in rat urine was found to be 5-(2'-hydroxy-2'-methyl-propyl)-1-naphthyl acetic acid (M1). The main metabolite in plasma was derived and was found to be 5-(2'-carboxypropyl)-1-naphthyl acetic acid (M2).

Liver insufficiency as a factor modifying the pharmacokinetic characteristic of the preparation nabumetone.

The effectiveness of the nonsteroidal anti-inflammatory agent nabumetone is related to the formation of an active metabolite: 6-methoxy-2-naphthylacetic acid. The plasma concentrations of nabumetone and its active metabolite, after administration of 1,000 mg single dose p.o., are followed at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 30 and 48 h in 6 patients with laboratory evidence for functional liver insufficiency caused by liver cirrhosis, confirmed by biopsy. In the patients with liver insufficiency the mean values of Cmax and AUC0-24 h for 6-methoxy-2-naphthylacetic acid were 26.75 +/- 8.45 mg/l and 623.64 +/- 161.8 mg X h/l respectively. They did not differ significantly from the values observed in healthy volunteers [v. Schrader et al. 1983]. The Tmax-value was prolonged to 8 h, compared to 4 h Tmax-value of the volunteers. There is a tendency towards a reduced bioavailability of 6-methoxy-2-naphthylacetic acid after nabumetone administration in patients with a more severely expressed pathologic impairment, compared to patients with slight morphologic changes of the liver parenchyma.

Nabumetone--a novel anti-inflammatory drug: bioavailability after different dosage regimens.

The bioavailability of nabumetone after different multiple dosing regimens was investigated in healthy male volunteers by determining the main plasma metabolite 6-methoxy-2-naphthylacetic acid. The mean steady state morning plasma level was higher when 1000 mg nabumetone were administered, once daily, at night than after the application of 500 mg twice daily (in the morning and in the evening). There was only a small further increase of the mean steady state morning level when the dose was increased to 1000 mg b.d. In all application regimens the steady state was reached on day 3. The dosage of 1000 mg, once daily, at night with and without a loading dose of 1000 mg in the morning of the first day were compared. When a loading dose was administered the plasma levels rose very quickly to higher values but reached the same mean on day 3 as when given without the loading dose. The half-lives of the terminal beta-phase with mean values of 22.77 h and 22.0 h are of the same order of magnitude as those found in single dose studies. It can be concluded from these results that a dose regimen of 1000 mg once daily, at night would be preferable.

Naproxen plasma levels in volunteers after single-dose administration by oral and rectal routes.

The bioavailability (plasma concentrations, AUC) of a rectal formulation (suppository) of naproxen was investigated in six healthy volunteers by comparison with an oral preparation (tablets). Plasma half-lives after both formulations were identical 10 hr 15 min+/-25 min (S.D.). Determined by the AUC the bioavailability of naproxen in the suppositories was 94.8%+/-6.3% of the bioavailability of naproxen in the tablets. This paper describes also a new gas-liquid chromatographic method for determining unchanged naproxen in human plasma which is quick, sensitive, and specific.

[Clinical-pharmacological and pharmacokinetic studies with naproxen]. / Klinisch-pharmakologische und pharmakokinetische Studien mit Naproxen

d-2-(6'-Methoxy-2'-naphthyl)-propionic acid (naproxen) is bound in blood to a high degree to plasma protein. After oral application naproxen is absorbed rapidly and completely. The mean biological half-life in man is fourteen hours. This gives us the possibility of controlling the symptoms of disease with two daily doses. In man, 99% of naproxen is excreted in the urine, either unchanged or glucuronized and as its 6-des-methyl metabolite. There is a linear increase of plasma levels with daily doses of up to 500 mg. Higher naproxen doses do not produce higher blood levels but lead to a faster excretion. This probably depends in part on a saturation of the binding sites in plasma. Differences in affinity of some compounds for binding sites in plasma are responsible for a potential interaction between naproxen and other agents, such as warfarin, acetyl salicylic acid (ASA) and sulfonylureas. These interactions are discussed. In a double-blind trial the influence of naproxen and ASA on the gastric mucosa of healthy volunteers has been compared. The results of gastroscopic findings as well as laboratory tests are reported.