A method for rapid screening of interactions of pharmacologically active compounds with albumin.

We determine the association constants for ligand-protein complex formation using the flow injection method. We carry out the measurements at high flow rates (F=1 mL min(-1)) of a carrier phase. Therefore, determination of the association constant takes only a few minutes. Injection of 1 nM of the ligand (10 µL of 1 µM concentration of the ligand solution) is sufficient for a single measurement. This method is tested and verified for a number of complexes of selected drugs (cefaclor, etodolac, sulindac) with albumin (BSA). We obtain K=4.45×10(3) M(-1) for cefaclor, K=1.00×10(5) M(-1) for etodolac and K=1.03×10(5) M(-1) for sulindac in agreement with the literature data. We also determine the association constants of 20 newly synthesized 3ß- and 3α-aminotropane derivatives with potential antipsychotic activity--ligands of 5-HT1A, 5-HT2A and D2 receptors with the albumin. Results of the studies reported here indicate that potential antipsychotic drugs bind weakly to the transporter protein (BSA) with K≈10(2)-10(3) M(-1). Our method allows measuring K in a wide range of values (10(2)-10(9) M(-1)). This range depends only on the solubility of the ligand and sensitivity of the detector.

The R-enantiomer of the nonsteroidal antiinflammatory drug etodolac binds retinoid X receptor and induces tumor-selective apoptosis.

Prostate cancer is often slowly progressive, and it can be difficult to treat with conventional cytotoxic drugs. Nonsteroidal antiinflammatory drugs inhibit the development of prostate cancer, but the mechanism of chemoprevention is unknown. Here, we show that the R-enantiomer of the nonsteroidal antiinflammatory drug etodolac inhibited tumor development and metastasis in the transgenic mouse adenocarcinoma of the prostate (TRAMP) model, by selective induction of apoptosis in the tumor cells. This proapoptotic effect was associated with loss of the retinoid X receptor (RXRalpha) protein in the adenocarcinoma cells, but not in normal prostatic epithelium. R-etodolac specifically bound recombinant RXRalpha, inhibited RXRalpha transcriptional activity, and induced its degradation by a ubiquitin and proteasome-dependent pathway. The apoptotic effect of R-etodolac could be controlled by manipulating cellular RXRalpha levels. These results document that pharmacologic antagonism of RXRalpha transactivation is achievable and can have profound inhibitory effects in cancer development.

Carboxyl nonsteroidal anti-inflammatory drugs are efficiently glucuronidated by microsomes of the human gastrointestinal tract.

Limited studies have been carried out on the biotransformation of carboxyl nonsteroidal anti-inflammatory drugs (NSAIDs) in the liver. However, the role of the intestine in NSAID metabolism has not been investigated. In this report, the contribution of UDP-glucuronosyltransferases (UGTs) in the human gastrointestinal (GI) tract from five donors to the glucuronidation of the NSAIDs, RS-ketoprofen, S-naproxen, RS- and S-etodolac, was investigated. UGT activity and, for some donors, mRNA levels were evaluated. All NSAIDs were glucuronidated throughout the GI tract; however, glucuronidation was low in stomach and duodenum as compared to the remainder of the intestine. RT-PCR analysis demonstrated that the UGT1A isoforms, UGT1A3, 1A8, and 1A10, and UGT2B7 were expressed in the GI tract. Human recombinant UGT1A3, 1A9, 1A10 and 2B7 were actively involved in the glucuronidation of all NSAIDs while UGT1A7 and the intestine-specific UGT1A8 had no glucuronidating activity towards those compounds. Despite interindividual variations in both the levels of mRNA and the distribution of activity through the intestine, UGTs in the GI tract may contribute significantly to the first pass metabolism of orally administered NSAIDs.

Stereoselective glucuronidation and hydroxylation of etodolac by UGT1A9 and CYP2C9 in man.

1. In vitro metabolic studies with etodolac were performed. S- and R-etodolac were converted to the acylglucuronide and hydroxylated metabolites by UDP-glucuronosyltransferase (UGT) and cytochrome P450 in microsomes. However, the stereoselectivities of UGT and P450 for the isomers were opposite. S-etodolac was glucuronidated preferentially than R-etodolac by UGT. In contrast, R-etodolac was hydroxylated preferentially than S-etodolac by P450. 2. Of several human P450 enzymes, CYP2C9 had the greatest activity for hydroxylation of R-etodolac. Sulfaphenazole, an inhibitor of CYP2C9, and anti-CYP2C9 antibody inhibited the hydroxylation of R-etodolac in human liver microsomes. CYP2C9 therefore contributes to the stereoselective hydroxylation of R-etodolac. 3. Of several human UGT enzymes, UGT1A9 had the greatest activity for glucuronidation of S-etodolac. Propofol and thyroxine, inhibitors of UGT1A9, inhibited the glucuronidation of S-etodolac in human liver microsomes. Therefore, UGT1A9 is mainly responsible for the stereoselective glucuronidation of S-etodolac. 4. Because S-etodolac was metabolized more rapidly than R-etodolac in human cryopreserved hepatocytes, the stereoselectivities of UGT1A9 for etodolac substantially influenced the overall metabolism of S- and R-etodolac in man.

Isolation of an unknown metabolite of the non-steroidal anti-inflammatory drug etodolac and its identification as 5-hydroxy etodolac.

The non-steroidal anti-inflammatory drug etodolac is extensively metabolized in the liver. Renal elimination of etodolac mainly as glucuronide and its other phase I and phase II metabolites is the primary route of excretion. High-performance liquid chromatography assays of human urine after application of etodolac indicated the existence of a further monohydroxylated metabolite (metabolite X) that was identified as 5-hydroxy etodolac. For the identification, electrospray ionization mass spectrometry (ESI-MS) as well as 1H-nuclear magnetic resonance (1H-NMR) and 13C-NMR spectroscopy have been used.

Separation and identification of etodolac and its urinary phase I metabolites using capillary electrochromatography and on-line capillary electrochromatography-electrospray ionisation mass spectrometry coupling.

Capillary high-performance liquid chromatography (capillary HPLC), pressure-assisted capillary electrochromatography (pCEC) and capillary electrochromatography (CEC) were performed in the same capillary packed with 5 microm octadecylsilica (C18) as stationary phase. These three separation modes were compared from the viewpoint of peak efficiency and separation selectivity in order to critically evaluate the advantages which CEC may offer compared to capillary HPLC for the solution of practical biomedical problems. The separation of the non-steroidal anti-inflammatory drug etodolac (ET, 1) and its phase I metabolites, 6-hydroxy etodolac (6-OH-ET, 2), 7-hydroxy etodolac (7-OH-ET, 3) and 8-(1'-hydroxyethyl) etodolac (8-OH-ET, 4) was selected as an example. Baseline separation of all compounds was achieved in different modes and conditions. The effect of pure electrophoretic separation mechanism on the overall separation selectivity observed in CEC has been shown. A high electroosmotic flow (EOF) was observed in C18 packed capillary even at pH 2.5 in various buffers. Furthermore, these separations were coupled on-line with electrospray ionisation mass spectrometry (ESI-MS) and the parent drug and its metabolites were identified in urine. For the coupling of CEC with ESI-MS a laboratory-made electrophoretic device was used in order to overcome some technical disadvantages of commercial instrumentation.

Pharmacokinetic analysis of enterohepatic circulation of etodolac and effect of hepatic and renal injury on the pharmacokinetics.

This study was designed to evaluate the enterohepatic circulation of racemic etodolac in rats. Additionally, the effect of hepatic and renal failure on the pharmacokinetics was estimated. The biliary excretion and the reabsorption of the drug excreted in bile were examined in order to clarify the effect of enterohepatic circulation on the disposition, and a pharmacokinetics model was applied to describe the enterohepatic circulation. The relatively rapid elimination of etodolac was seen in the bile-exteriorized rats (BE rat) compared with that in control rats. Total biliary excretion of etodolac, mainly as glucuronides, after intravenous administration was about 45% of the dose, indicating extensive enterohepatic circulation of the drug. The plasma concentrations of the drug in bile duct-linked rats approximately agreed with the simulation curve by the model, with the peak concentration 6-7 h after dosing. The elimination of the drug was markedly retarded in rats with hepatic (CCI4-induced) and renal (uranyl acetate-induced) failure, and high plasma levels were maintained over the longer times, due to greatly decreased distribution volume. The biliary excretion of etodolac enantiomers was not significantly different between the control and CCI4-groups, suggesting that hepatic glucuronyl transferase activity was preserved in rats impaired by CCI4.

Simultaneous determination of the phase II metabolites of the non steroidal anti-inflammatory drug etodolac in human urine.

Etodolac, an antirheumatic and analgetic drug, is metabolized in humans by hydroxylation and acyl glucuronidation to yield the corresponding 1-O-glucuronides. The acylglucuronides of etodolac and one of its hydroxylated metabolites were determined by HPLC using a RP18 column. In order to increase the lipophilicity of these metabolites the hydroxy groups were acetylated and the carboxylic functions were methylated. In a study with five human volunteers a stereoselective excretion of the acylglucuronides could be observed. The S-etodolac-glucuronide is mainly excreted during the first 6 hours after administration of 400 mg of etodolac whereas the elimination of the R-glucuronide predominates at longer periods of time. The S-glucuronide of 7-hydroxy-etodolac was preferenetively excreted during the period of time observed. An unknown metabolite of etodolac could be characterized by chemical and enzymatically hydrolysis and by MS and NMR.

Stereoselective binding of etodolac to human serum albumin.

The protein binding of etodolac enantiomers was studied in vitro by equilibrium dialysis in human serum albumin (HSA) of various concentrations varying from 1 to 40 g/liter, by addition of each enantiomer at increasing concentrations. In the 1 g/liter solution, at the lowest drug levels, the (R)-form is more bound than its antipode, the contrary being observed at the highest drug levels. For higher albumin concentrations, S was bound in a larger extent than R. Using the displacement of specific markers of HSA sites I and II, studied by spectrofluorimetry, it was suggested that R and S are both bound to site I, while only S is strongly bound to site II.