Naproxen Metabolism Research Articles

(133) Preliminary Analysis of the Relationship between Refractory Menstrual Pain and Naproxen Metabolism

Non-steroidal anti-inflammatory drugs are commonly used to treat painful periods, also known as dysmenorrhea. A subset of women, however, do not benefit from this treatment. Since the pharmacological basis of treatment resistance in dysmenorrhea has not been characterized, we assessed the relationship between measures of naproxen metabolism and self-reported menstrual pain relief. While on their menses, dysmenorrheic participants (n=24) and healthy controls (n=11) answered questionnaires assessing their menstrual pain, including a 0-100 visual analog scale (VAS). Participants also graded their discomfort with cramping events in real-time for 10 minutes, using a hand squeeze bulb paradigm. Following baseline assessments, participants received naproxen. Ninety minutes later, participants reported their pain relief and repeated the bulb-squeeze paradigm. We also quantified serum concentrations of naproxen and its metabolite O-desmethylnaproxen from blood collected 90 minutes post ingestion. Serum naproxen and O-desmethylnaproxen concentrations were reliably measured across a range of standards (R2 >0.98). Dysmenorrheic participants had severe menstrual pain (71±4, VAS) and missed 3.5±1 days of work or school over the last three months. Naproxen effectiveness, reported as a percent change in VAS before and after taking naproxen, varied across dysmenorrhea participants: 8 participants reported pain decreased by ≥50%, 6 reported pain decreased between 20-50%, 4 between 0-20%, and 6 reported no change or worsened pain. Reported naproxen effectiveness was negatively correlated to serum naproxen concentration (r= -0.49, p=0.019). Similarly, increased O-desmethylnaproxen concentrations were associated with greater naproxen effectiveness (r= -0.45, p=0.032). No dysmenorrheic participants, however, demonstrated an abnormal metabolism profile. Serum naproxen or O-desmethylnaproxen concentrations did not differ between healthy controls and dysmenorrhea participants (p=0.59 and p=0.92, respectively). Overall, low serum concentrations of naproxen and its metabolite were associated with minimal menstrual pain relief, suggesting naproxen absorption, but not metabolism may be altered in women with NSAID-resistant dysmenorrhea.

Direct Conjugation of Emerging Contaminants in Arabidopsis: Indication for an Overlooked Risk in Plants?

Agricultural use of treated wastewater, biosolids, and animal wastes introduces a multitude of contaminants of emerging concerns (CECs) into the soil-plant system. The potential for food crops to accumulate CECs depends largely on their metabolism in plants, which at present is poorly understood. Here, we evaluated the metabolism of naproxen and ibuprofen, two of the most-used human drugs from the Profen family, in Arabidopsis thaliana cells and the Arabidopsis plant. The complementary use of high-resolution mass spectrometry and 14C labeling allowed the characterization of both free and conjugated metabolites, as well as nonextractable residues. Naproxen and ibuprofen, in their parent form, were conjugated quickly and directly with glutamic acid and glutamine, and further with peptides, in A. thaliana cells. For example, after 120 h, the metabolites of naproxen accounted for >90% of the extractable chemical mass, while the intact parent itself was negligible. The structures of glutamate and glutamine conjugates were confirmed using synthesized standards and further verified in whole plants. Amino acid conjugates may easily deconjugate, releasing the parent molecule. This finding highlights the possibility that the bioactivity of such CECs may be effectively preserved through direct conjugation, a previously overlooked risk. Many other CECs are also carboxylic acids, such as the profens. Therefore, direct conjugation may be a common route for plant metabolism of these CECs, making it imperative to consider conjugates when assessing their risks.

Amiodarone Analog-Dependent Effects on CYP2C9-Mediated Metabolism and Kinetic Profiles

CYP2C9 substrates can exhibit both hyperbolic and atypical kinetic profiles, and their metabolism can be activated or inhibited depending on the effector studied. CYP2C9 genetic variants can also affect both substrate turnover and kinetic profile. The present study assessed whether analogs of the effector amiodarone differentially altered the atypical kinetic profile of the substrate naproxen and whether this effect was genotype-dependent. Amiodarone, desethylamiodarone, benzbromarone, and its dimethyl analog (benz(meth)arone) were incubated with naproxen and either CYP2C9.1 or CYP2C9.3. Amiodarone activated naproxen demethylation at lower concentrations, regardless of the CYP2C9 allele, and inhibited metabolism at higher concentrations without altering the kinetic profile. Desethylamiodarone was a potent inhibitor of naproxen demethylation, irrespective of the CYP2C9 allele. Benzbromarone altered naproxen demethylation kinetics from a biphasic profile to that of a hyperbolic form in CYP2C9.1 and CYP2C9.3, resulting in inhibition and activation, respectively. In contrast, benz(meth)arone activated naproxen demethylation in both CYP2C9.1 and CYP2C9.3. In addition, the kinetic profile of naproxen demethylation became more hyperbolic at lower concentrations of benz(meth)arone and then reverted back to biphasic as the benz(meth)arone was increased further. Equilibrium binding and multiple-ligand docking studies were used to propose how such similar compounds exerted very different effects on naproxen metabolism. In summary, effectors of CYP2C9 metabolism can alter not only the degree of substrate turnover (activation or inhibition) but also the kinetic profile of metabolism of CYP2C9 substrates through effects on substrate binding and orientation. In addition, these kinetics effects are concentration- and genotype-dependent.

Polymorphic variants (CYP2C9*3 and CYP2C9*5) and the F114L active site mutation of CYP2C9: effect on atypical kinetic metabolism profiles.

CYP2C9 wild-type protein has been shown to exhibit atypical kinetic profiles of metabolism that may affect in vitro-in vivo predictions made during the drug development process. Previous work suggests a substrate-dependent effect of polymorphic variants of CYP2C9 on the rate of metabolism; however, it is hypothesized that these active site amino acid changes will affect the kinetic profile of a drug's metabolism as well. To this end, the kinetic profiles of three model CYP2C9 substrates (flurbiprofen, naproxen, and piroxicam) were studied using purified CYP2C9*1 (wild-type) and variants involving active site amino acid changes, including the naturally occurring variants CYP2C9*3 (Leu359) and CYP2C9*5 (Glu360) and the man-made mutant CYP2C9 F114L. CYP2C9*1 (wild-type) metabolized each of the three compounds with a distinctive profile reflective of typical hyperbolic (flurbiprofen), biphasic (naproxen), and substrate inhibition (piroxicam) kinetics. CYP2C9*3 metabolism was again hyperbolic for flurbiprofen, of a linear form for naproxen (no saturation noted), and exhibited substrate inhibition with piroxicam. CYP2C9*5-mediated metabolism was hyperbolic for flurbiprofen and piroxicam but linear with respect to naproxen turnover. The F114L mutant exhibited a hyperbolic kinetic profile for flurbiprofen metabolism, a linear profile for naproxen metabolism, and a substrate inhibition kinetic profile for piroxicam metabolism. In all cases except F114L-mediated piroxicam metabolism, turnover decreased and the K(m) generally increased for each allelic variant compared with wild-type enzyme. It seems that the kinetic profile of CYP2C9-mediated metabolism is dependent on both substrate and the CYP2C9 allelic variant, thus having potential ramifications on drug disposition predictions made during the development process.

Directly coupled HPLC-NMR and HPLC-MS approaches for the rapid characterisation of drug metabolites in urine: application to the human metabolism of naproxen

High resolution nuclear magnetic resonance (NMR) spectroscopy is a very powerful tool for the structural identification of xenobiotic metabolites in complex biological matrices such as plasma, urine and bile. However, these fluids are dominated by thousands of signals resulting from endogenous metabolites and it is advantageous when investigating drug metabolites in such matrices to simplify the spectra by including a separation step in the experiment by directly-coupling HPLC and NMR. Naproxen (6-methoxy-alpha-methyl-2-naphthyl acetic acid) is administered as the S-enantiomer and is metabolised in vivo to form its demethylated metabolite which is subsequently conjugated with beta-D-glucuronic acid as well as with sulfate. Naproxen is also metabolised by phase II metabolism directly to form a glycine conjugate as well as a glucuronic acid conjugate at the carboxyl group. In the present investigation, the metabolism of naproxen was investigated in urine samples with a very simple sample preparation using a combination of directly-coupled HPLC-1H NMR spectroscopy and HPLC-mass spectrometry (MS). A buffer system was developed which allows the same chromatographic method to be used for the HPLC-NMR as well as the HPLC-MS analysis. The combination of these methods is complementary in information content since the NMR spectra provide evidence to distinguish isomers such as the type of glucuronides formed, and the HPLC-MS data allow identification of molecules containing NMR-silent fragments such as occur in the sulfate ester.

Evaluation of atypical cytochrome P450 kinetics with two-substrate models: evidence that multiple substrates can simultaneously bind to cytochrome P450 active sites.

Some cytochrome P450 catalyzed reactions show atypical kinetics, and these kinetic processes can be grouped into five categories: activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. A two-site model in which the enzyme can bind two substrate molecules simultaneously is presented which can be used to describe all of these observed kinetic properties. Sigmoidal kinetic characteristics were observed for carbamazepine metabolism by CYP3A4 and naphthalene metabolism by CYPs 2B6, 2C8, 2C9, and 3A5 as well as dapsone metabolism by CYP2C9. Naphthalene metabolism by CYP3A4 and naproxen metabolism by CYP2C9 demonstrated nonhyperbolic enzyme kinetics suggestive of a low Km, low Vmax component for the first substrate molecule and a high Km, high Vmax component for the second substrate molecule. 7, 8-Benzoflavone activation of phenanthrene metabolism by CYP3A4 and dapsone activation of flurbiprofen and naproxen metabolism by CYP2C9 were also observed. Furthermore, partial inhibition of 7, 8-benzoflavone metabolism by phenanthrene was observed. These results demonstrate that various P450 isoforms may exhibit atypical enzyme kinetics depending on the substrate(s) employed and that these results may be explained by a model which includes simultaneous binding of two substrate molecules in the active site.

Oxidative stress in isolated rat hepatocytes during naproxen metabolism

Naproxen, a non-steroidal anti-inflammatory drug, induced lipid peroxidation in isolated hepatocytes of rats. The viability of the hepatocytes decreased upon lipid peroxidation, and this effect was accompanied by the formation of high molecular weight protein aggregates in the hepatocytes. Protein aggregation occurred slowly compared with the formation of thiobarbituric acid reactive substances (TBARS). The increase of TBARS was strongly correlated with the decrease of intracellular glutathione. Chemiluminescence was produced from the hepatocyte suspension during naproxen metabolism, and was correlated with the formation of TBARS. These results indicate that lipid peroxidation in the hepatocytes was provoked by reactive oxygens produced in the process of naproxen metabolism.

Lipid Peroxidation and Cherniluminescence during Naproxen Metabolism in Rat Liver Microsomes

1. Rat liver microsomal suspension containing NADPH and MgCl2 was incubated at 37 degrees C with naproxen, a non-steroidal anti-inflammatory drug. Thiobarbituric acid reactive substances (TBA-RS), high molecular weight protein aggregates and fluorescent substances were formed in the microsomal suspension. 2. Chemiluminescence was produced from the microsomal suspension. This chemiluminescence production was well correlated to the TBA-RS formation, indicating that the chemiluminescence production was closely associated with the lipid peroxidation. 3. The addition of SKF-525A to the microsomal suspension inhibited the production of TBA-RS, chemiluminescence and 6-demethylnaproxen (6-DMN), the oxidative product of naproxen. Further, the antioxidant, alpha-tocopherol and singlet oxygen quenchers like histidine, dimethylfuran and 1,4-diazabicyclo[2,2,2]octane strikingly inhibited the productions of chemiluminescence and TBA-RS. 4. Neither naproxen nor 6-DMN caused lipid peroxidation in the absence of NADPH. Thus, lipid peroxidation and chemiluminescence during the oxidation of naproxen in liver microsomes was suggested to be provoked by reactive oxygen species and an origin of chemiluminescence was shown to be singlet oxygen.

Lipid peroxidation in rat liver microsomes during naproxen metabolism

Naproxen, a non-steroidal anti-inflammatory drug, is known to be highly effective and relatively safe, but some side-effects in the liver have been reported. In the present study, the effect of naproxen metabolism on rat liver microsomes was studied by determining lipid peroxidation in terms of thiobarbituric acid reactive substances (TBA-RS), high molecular weight protein aggregates and fluorescent substances formed in the microsomal suspension containing naproxen, NADPH and MgCl 2. Lipid peroxidation was found to occur at 10 mM naproxen. Production of chemiluminescence from the microsomal suspension was observed during naproxen metabolism. The time course of 6-demethylnaproxen formation by O-demethylation of naproxen appeared to be comparable to that of the chemiluminescence production in their initial periods of production. These results suggest that the lipid peroxidation was provoked through the reactive oxygen species generated during the oxidative metabolism of naproxen.

Pharmacokinetics and Metabolism of Naproxen in Children

The metabolism and pharmacokinetics of orally administered naproxen were studied in 10 children aged 6-13 years. 3 patients received the drug for antipyresis and 7 for postoperative pain. The mean elimination rate constant was greater in the febrile children than the postoperative patients, 0.064 h-1 vs. 0.051 h-1. 71% of total drug recovered in urine was naproxen, and 29% was excreted as the desmethyl metabolite. 60 and 63% of naproxen and desmethyl naproxen, respectively, were excreted as conjugates. Area under the curve and fraction of dose recovered in the urine were reduced in the postoperative patients, suggesting reduced gastrointestinal absorption of the drug compared to the febrile patients.

Naproxen-probenecid interaction.

Probenecid induced major alterations in the half‐life. renal clearance, and metabolism of naproxen. In 6 healthy subjects who received 500‐mg single oral doses of naproxen alone and (following two days of probenecid loading) with 500 mg of probenecid 4 times a day, there was an increase in naproxen plasma half‐life from the normal 14 to 37 hr. Twenty‐four hour naproxen plasma levels were doubled by probenecid. but the rate and extent of absorption were not affected. In an 8‐day chronic administration experiment (250 mg twice daily). naproxen steady‐state plasma levels measured before the morning dose were increased 50% by coadministration of 500 mg probenecid twice daily. The major probenecid effects on naproxen metabolism were a 66% reduction of the naproxen conjugates excreted in the urine and an increase in urinary 6‐0‐desmethyl naproxen. The renal clearance of unchanged naproxen was markedly depressed. We conclude that probenecid retards naproxen plasma clearance by inhibiting naproxen glucuronide formation and renal clearance of naproxen. The renal clearance effect is the result of the blocking of tubular secretion of organic anions by probenecid. Inhibition of conjugate formation by probenecid may be the result of successful competition for conjugative processes since a large percentage of both drugs are cleared from the plasma by conjugation. Because of this interaction, patients who receive naproxen and probenecid in combination will have higher steady‐state plasma levels of naproxen and form more of the 6‐0‐desmethyl metabolite than when probenecid is not used.

Studies on the fate of naproxen. II. Metabolic fate in various animals and man.

The species differences in the metabolism of naproxen [(+)-6-methoxy-α-methyl-2-naphthaleneacetic acid] were investigated in 6 species of animals and man. 3H-naproxen was orally administered to mice, rats, rabbits, guinea pigs, dogs and miniature pigs (OHMINI-875) and cold naproxen to human subjects, and their urinary and biliary (rats only) metabolites were separated and characterized by thin-layer chromatography, gas chromatography, high-performance liquid chromatography and gas chromatographymass spectrometry. The excretion rates as % of dose of naproxen and its metabolites in the 24 hr urine were, in a decreasing order, miniature pig 82.5, mouse 80.4, rat 74.7, rabbit 70.3, guinea pig 48.5 and dog 23.0%. Naproxen and its metabolites, i.e., 6-hydroxy-α-methyl-2-naphthaleneacetic acid (6-DMN), naproxen glucuronide, 6-DMN glucuronide and 6-DMN sulfate, were found in the urines of all species. Urinary naproxen in the rat was only 0.2% and those in the other species of animals were 4.8-13.7% of the urinary radioactivity, while in man it was 1.3% of dose. The main urinary metabolite in rat, guinea pig, dog, mouse and rabbit was 6-DMN sulfate (87.3, 46.7, 42.9, 39.9 and 36.2% of the urinary radioactivity, respectively), while those in man and miniature pig were naproxen glucuronide (25.3% of dose) and 6-DMN (78.6% of the urinary radioactivity), respectively. The animals whose urinary metabolic profiles were the nearest to that of man were the guinea pig and mouse, while the animals that differed most from man in this regard were the rat and miniature pig.

Naproxen metabolism in man.

In summary, naproxen is an acidic, highly albumin-bound drug. After oral administration, it is promptly and fully absorbed. The mean half-life of the drug in man is 13 hours, close to ideal for twice-daily administration. The only metabolite detected in man is the 6-desmethyl compound. Both it and naproxen itself are excreted in the urine, primarily as conjugates. The kinetics of naproxen binding to serum albumin tend to limit attainable plasma levels. They increase little if the dose is increased beyond 500 mg twice daily, since greater concentrations are rapidly cleared. Albumin binding and competitive displacement are also responsible for potential interactions of naproxen with drugs such as warfarin, sulfonylureas, and aspirin. Experience thus far does not indicate that any of the potential interactions are clinically meaningful.