Hydroxy Diclofenac Research Articles

Identification and Characterization of Efflux Transporters That Modulate the Subtoxic Disposition of Diclofenac and Its Metabolites.

In the present work, in vivo transporter knockout (KO) mouse models were used to characterize the disposition of diclofenac (DCF) and its primary metabolites following a single subtoxic dose in mice lacking breast cancer resistance protein (Bcrp) or multidrug resistance-associated protein (Mrp)3. The results indicate that Bcrp acts as a canalicular efflux mediator for DCF, as wild-type (WT) mice had biliary excretion values that were 2.2- to 2.6-fold greater than Bcrp KO mice, although DCF plasma levels were not affected. The loss of Bcrp resulted in a 1.8- to 3.2-fold increase of diclofenac acyl glucuronide (DCF-AG) plasma concentrations in KO animals compared with WT mice, while the biliary excretion of DCF-AG increased 1.4-fold in WT versus KO mice. Furthermore, Mrp3 was found to mediate the basolateral transport of DCF-AG, but not DCF or 4'-hydroxy diclofenac. WT mice had DCF-AG plasma concentrations 7.0- to 8.6-fold higher than Mrp3 KO animals; however, there were no changes in biliary excretion of DCF-AG. Vesicular transport experiments with human MRP3 demonstrated that MRP3 is able to transport DCF-AG via low- and high-affinity binding sites. The low-affinity MRP3 transport had a V max and K m of 170 pmol/min/mg and 98.2 µM, respectively, while the high-affinity V max and K m parameters were estimated to be 71.9 pmol/min/mg and 1.78 µM, respectively. In summary, we offer evidence that the disposition of DCF-AG can be affected by both Bcrp and Mrp3, and these findings may be applicable to humans.

Hydrolytic stability of selected pharmaceuticals and their transformation products

The fact that pharmaceuticals are present in the environment has been proven in numerous publications. Nevertheless, their transformation products (mainly metabolites) are detected significantly less often, mainly because they are not included in the detecting methods, even though many of them are excreted from organisms at high rates and may be biologically active or have other properties that make them a potential threat to the environment. One of the most common processes that occur in the aqueous environment is hydrolysis, which may be one of the most important factors influencing the persistency of pharmaceuticals. Therefore four pharmaceuticals (carbamazepine, ibuprofen, tramadol and naproxen) as well as ten metabolites (10,11-dihydro-10-hydroxy carbamazepine, 10,11-dihydro-2-hydroxy carbamazepine, carbamazepine epoxide, 2-hydroxy ibuprofen, ibuprofen carboxylic acid, O-desmethyltramadol, hydroxy metronidazole, N-acetylsulfamethoxazole, 4′-hydroxy diclofenac, and O-desmethylnaproxen) were selected for the hydrolytic stability tests in accordance to OECD 111 Guideline. The preliminary test showed that only carbamazepine epoxide at pH 4, hydroxy metronidazole at pH 9 and 4′-hydroxy diclofenac at pH 4 and 9 were unstable and were included in the extended tests, which resulted in calculation of rate constants and half-lives at the temperature of 20, 50 and 70 °C as well as the activation energy at the pH values in which these compounds were unstable. The obtained results show that carbamazepine epoxide is quite unstable and the half-life at 20 °C was a little more than 8 days. Nevertheless, hydroxy-metronidazole and 4′-hydroxy diclofenac did not degrade at 20 °C for 30 days.

Effect of iguratimod on diclofenac metabolism by CYP2C9 in rats and human recombinant CYP2C9 yeast cells

Iguratimod (IGU, also known as T-614), a novel disease modifying antirheumatic drug intended to cure patients with rheumatoid arthritis (RA). The purpose of this study is to evaluate the effect of IGU on the pharmacokinetics of CYP2C9 probe drug diclofenac and its metabolite 4′-hydroxy diclofenac in vivo and in vitro. In in vivo experiments, 24 rats were randomly assigned to three groups consisting of the control group (Normal saline), low dose IGU group (10 mg/kg) and high dose IGU group (30 mg/ kg). Blood samples were collected from orbital sinuses vein before 1 hour and serial times of giving diclofenac (15 mg/kg) to all the rats. Plasma concentration of diclofenac and its metabolite 4´-hydroxy diclofenac were assayed by high performance liquid chromatography. Pharmacokinetic parameters were assessed by Winnonlin 6.4 pharmacokinetic software. Moreover, in vitro studies were performed in recombinant human CYP2C9 yeast cell system. IGU at low dose showed no significant differences in the pharmacokinetic parameters of diclofenac and 4-hydroxy diclofenac in vivo when compared with control group (p>0.005). However, at the high dose of IGU, the pharmacokinetic parameters of 4´-hydroxy metabolite of diclofenac increase in half-life (T1/2) and mean area under the curve (AUC0→24), while a decrease in mean clearance (CL, mL/h/kg) and volume of distribution Vz (mL/kg). In addition, in in vitro study, high doses of IGU reduces the metabolism rate of diclofenac. IGU at high dose significantly increase the pharmacokinetics parameters of 4´-hydroxy diclofenac in rats. Additionally, it also showed the potent inhibitory effect on diclofenac metabolism in recombinant human CYP2C9 yeast cells.

Sono-activated persulfate oxidation of diclofenac: Degradation, kinetics, pathway and contribution of the different radicals involved

Degradation of a diclofenac aqueous solution was performed using persulfate anions activated by ultrasound. The objective of this study was to analyze different parameters affecting the diclofenac (DCF) removal reaction by the ultrasonic persulfate (US/PS) process and to evaluate the role played by various intermediate oxidative species such as hydroxyl- and sulfate radicals, superoxide radical anion or singlet oxygen in the removal process as well as to determine a possible reaction pathway. The effects of pH, initial persulfate anion concentration, ultrasonic amplitude and temperature on DCF degradation were examined. Sulfate and hydroxyl radicals were involved in the main reaction pathway of diclofenac. Diclofenac amide and three hydroxy-diclofenac isomers (3´-hydroxy diclofenac, 4´-hydroxy diclofenac and 5-hydroxy diclofenac) were identified as reaction intermediates.

Diclofenac toxicity in human intestine ex vivo is not related to the formation of intestinal metabolites.

The use of diclofenac (DCF), a nonsteroidal anti-inflammatory drug, is associated with a high prevalence of gastrointestinal side effects. In vivo studies in rodents suggested that reactive metabolites of DCF produced by the liver or the intestine might be responsible for this toxicity. In the present study, precision-cut intestinal slices (PCIS) prepared from the jejunum of 18 human donors were used as an ex vivo model to investigate whether DCF intestinal metabolites are responsible for its intestinal toxicity in man. PCIS were incubated with a concentration range of DCF (0-600 µM) up to 24 h. DCF (≥400 µM) caused direct toxicity to the intestine as demonstrated by ATP depletion, morphological damage, caspase 3 activation, and lactate dehydrogenase leakage. Three main metabolites produced by PCIS (4'-hydroxy DCF, 5-hydroxy DCF, and DCF acyl glucuronide) were detected by HPLC. Protein adducts were detected by immunohistochemical staining and showed correlation with the intestinal metabolites. DCF induced similar toxicity to each of the samples regardless of the variation in metabolism among them. Less metabolites were produced by slices incubated with 400 µM DCF than with 100 µM DCF. The addition of the metabolic inhibitors such as ketoconazole, cimetidine, or borneol decreased the metabolite formation but increased the toxicity. The results suggest that DCF can induce intestinal toxicity in human PCIS directly at therapeutically relevant concentrations, independent of the reactive metabolites 4'-OH DCF, 5-OH DCF, or diclofenac acylglucuronide produced by the liver or formed in the intestine.

Occurrence of diclofenac and selected metabolites in sewage effluents

Many pharmaceuticals along with their metabolites have been detected in environmental water samples in the recent decades. The analgesic diclofenac is widely used and thus enters the aquatic environment. Already at realistic environmental concentration levels harmful effects to different organisms have been demonstrated. As this could also be expected for its metabolites, their fate was examined. Six wastewater treatment plant effluents collected throughout Germany were analyzed for the drug and two of its hydroxylated metabolites, 4′-hydroxy diclofenac (4′-OHD) and 5-hydroxy diclofenac (5-OHD), together with the lactam of 4′-OHD, 4′-hydroxy diclofenac dehydrate (4′-OHDD). A quantitative analytical method has been developed using solid-phase extraction followed by LC-ESI-MS/MS. The limits of quantitation (LOQ) in sewage effluent were 0.06 μg/l for diclofenac and its hydroxyl metabolites and 0.07 μg/l for 4′-OHDD. Recoveries ranged from 62 to 81%. The metabolites were detected in the samples in median concentration ranges of < LOQ to 0.71 μg/l, < LOQ to 0.45 μg/l, and < LOQ to 0.42 μg/l for 4′-OHD, 5-OHD, and 4′-OHDD, respectively, while median diclofenac concentrations ranged from 1.3 to 3.3 μg/l. The wide occurrence of its metabolites is highly relevant on account of their structural similarity and the toxicological properties of diclofenac and needs further examination of both toxicity and environmental concentrations of the metabolites.

Isolation and characterization of a new human urinary metabolite of diclofenac applying LC–NMR–MS and high-resolution mass analyses

The nonsteroidal anti-inflammatory drug diclofenac is widely used. Diclofenac is extensively metabolized to several hydroxylated derivatives and their conjugates. The lactam-dehydrate of 4′-hydroxy diclofenac (4′-OHDD) has now been detected as a new urinary metabolite of diclofenac. Isolation was successfully performed using preparative HPLC in three different steps using water, methanol, and acetonitrile, respectively. The structural characterization of 4′-OHDD was achieved by LC–NMR–MS. In addition, specific mass fragmentation pattern could be obtained using LC–high-resolution MS with both positive and negative electrospray ionization.

The disposition of diclofenac in camels after intravenous administration

The pharmacokinetics of diclofenac was studied in camels ( Camelus dromedarus) ( n=6) following intravenous (i.v.) administration of a dose of 2.5 mg kg −1 body weight. The metabolism and urinary detection time were also studied. The results obtained (median and range) were as follows: the terminal elimination half-life ( t 1/2 β ) was 2.35 (1.90–2.73) h, total body clearance (Cl T) was 0.17 (0.16–0.21) l h kg −1. The volume of distribution at steady state ( V SS) was 0.31 (0.21–0.39) l −1 kg −1, the volume of the central compartment of the two compartment pharmacokinetic model ( V C) was 0.15 (0.11–0.17) l kg −1. Five metabolites of diclofenac were tentatively identified in urine and were excreted mainly in conjugate form. The main metabolite was identified as hydroxy diclofenac. Both diclofenac and hydroxy diclofenac, appear to be the main elimination route for diclofenac when administered i.v. in camels. Diclofenac could be identified up to 4 days following i.v. administration in camels using a sensitive gas chromatography/mass spectrometry (GC/MS) method.

Residues Glutamate 216 and Aspartate 301 Are Key Determinants of Substrate Specificity and Product Regioselectivity in Cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) metabolizes a wide range of therapeutic drugs. CYP2D6 substrates typically contain a basic nitrogen atom, and the active-site residue Asp-301 has been implicated in substrate recognition through electrostatic interactions. Our recent computational models point to a predominantly structural role for Asp-301 in loop positioning (Kirton, S. B., Kemp, C. A., Tomkinson, N. P., St.-Gallay, S., and Sutcliffe, M. J. (2002) Proteins 49, 216-231) and suggest a second acidic residue, Glu-216, as a key determinant in the binding of basic substrates. We have evaluated the role of Glu-216 in substrate recognition, along with Asp-301, by site-directed mutagenesis. Reversal of the Glu-216 charge to Lys or substitution with neutral residues (Gln, Phe, or Leu) greatly decreased the affinity (K(m) values increased 10-100-fold) for the classical basic nitrogen-containing substrates bufuralol and dextromethorphan. Altered binding was also manifested in significant differences in regiospecificity with respect to dextromethorphan, producing enzymes with no preference for N-demethylation versus O-demethylation (E216K and E216F). Neutralization of Asp-301 to Gln and Asn had similarly profound effects on substrate binding and regioselectivity. Intriguingly, removal of the negative charge from either 216 or 301 produced enzymes (E216A, E216K, and D301Q) with elevated levels (50-75-fold) of catalytic activity toward diclofenac, a carboxylate-containing CYP2C9 substrate that lacks a basic nitrogen atom. Activity was increased still further (>1000-fold) upon neutralization of both residues (E216Q/D301Q). The kinetic parameters for diclofenac (K(m) 108 microm, k(cat) 5 min(-1)) along with nifedipine (K(m) 28 microm, k(cat) 2 min(-1)) and tolbutamide (K(m) 315 microm, k(cat) 1 min(-1)), which are not normally substrates for CYP2D6, were within an order of magnitude of those observed with CYP3A4 or CYP2C9. Neutralizing both Glu-216 and Asp-301 thus effectively alters substrate recognition illustrating the central role of the negative charges provided by both residues in defining the specificity of CYP2D6 toward substrates containing a basic nitrogen.

Extrapolation of diclofenac clearance from in vitro microsomal metabolism data: role of acyl glucuronidation and sequential oxidative metabolism of the acyl glucuronide.

Diclofenac is eliminated predominantly (approximately 50%) as its 4'-hydroxylated metabolite in humans, whereas the acyl glucuronide (AG) pathway appears more important in rats (approximately 50%) and dogs (>80-90%). However, previous studies of diclofenac oxidative metabolism in human liver microsomes (HLMs) have yielded pronounced underprediction of human in vivo clearance. We determined the relative quantitative importance of 4'-hydroxy and AG pathways of diclofenac metabolism in rat, dog, and human liver microsomes. Microsomal intrinsic clearance values (CL(int) = V(max)/K(m)) were determined and used to extrapolate the in vivo blood clearance of diclofenac in these species. Clearance of diclofenac was accurately predicted from microsomal data only when both the AG and the 4'-hydroxy pathways were considered. However, the fact that the AG pathway in HLMs accounted for ~75% of the estimated hepatic CL(int) of diclofenac is apparently inconsistent with the 4'-hydroxy diclofenac excretion data in humans. Interestingly, upon incubation with HLMs, significant oxidative metabolism of diclofenac AG, directly to 4'-hydroxy diclofenac AG, was observed. The estimated hepatic CL(int) of this pathway suggested that a significant fraction of the intrahepatically formed diclofenac AG may be converted to its 4'-hydroxy derivative in vivo. Further experiments indicated that this novel oxidative reaction was catalyzed by CYP2C8, as opposed to CYP2C9-catalyzed 4'-hydroxylation of diclofenac. These findings may have general implications in the use of total (free + conjugated) oxidative metabolite excretion for determining primary routes of drug clearance and may question the utility of diclofenac as a probe for phenotyping human CYP2C9 activity in vivo via measurement of its pharmacokinetics and total 4'-hydroxy diclofenac urinary excretion.

Aceclofenac blocks prostaglandin E 2 production following its intracellular conversion into cyclooxygenase inhibitors

Aceclofenac, 2-[(2,6-dichlorophenyl) amino] phenylacetoxyacetic acid, is a novel non-steroidal anti-inflammatory drug. We investigated the effects of aceclofenac on prostaglandin E 2 production by several kinds of human cells. Aceclofenac inhibited interleukin-1β-induced prostaglandin E 2 production by human rheumatoid synovial cells, but had no inhibitory effect on cyclooxygenase-1 or cyclooxygenase-2 activities by itself. We also observed that part of the aceclofenac was converted into diclofenac, the cyclooxygenase-1 and cyclooxygenase-2 inhibitor, when aceclofenac was incubated with human rheumatoid synovial cells. Aceclofenac was also converted into diclofenac and 4′-hydroxy diclofenac by human polymorphonuclear leukocytes and monocytes. 4′-Hydroxy diclofenac suppressed prostaglandin E 2 production specifically by blocking cyclooxygenase-2 activity. These findings suggested that aceclofenac can be metabolized to cyclooxygenase inhibitors (diclofenac and/or 4′-hydroxy diclofenac) by these inflammatory cells. Although detailed examinations in non-inflammatory cells remain to be studied, we concluded that aceclofenac is shown to be a new type of non-steroidal anti-inflammatory drug which is intracellulary converted into active metabolites that inhibit the prostaglandin E 2 production.