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1.
Curr Drug Metab ; 2(1): 17-36, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11465149

ABSTRACT

The most common drug-drug interactions may be understood in terms of alterations of metabolism, associated primarily with changes in the activity of cytochrome P450 (CYP) enzymes. Kinetic parameters such as Km, Vmax, Ki and Ka, which describe metabolism-based drug interactions, are usually determined by appropriate kinetic models and may be used to predict the pharmacokinetic consequences of exposure to one or multiple drugs. According to classic Michaelis-Menten (M-M) kinetics, one binding site models can be employed to simply interpret inhibition (pure competitive, non-competitive and uncompetitive) or activation of the enzyme. However, some cytochromes P450, in particular CYP3A4, exhibit unusual kinetic characteristics. In this instance, the changes in apparent kinetic constants in the presence of inhibitor or activator or second substrate do not obey the rules of M-M kinetics, and the resulting kinetics are not straightforward and hamper mechanistic interpretation of the interaction in question. These unusual kinetics include substrate activation (autoactivation), substrate inhibition, partial inhibition, activation, differential kinetics and others. To address this problem, several kinetic models can be proposed, based upon the assumption that multiple substrate binding sites exist at the active site of a particular P450, and the resulting kinetic constants are, therefore, solved to adequately describe the observed interaction between multiple drugs. The following is an overview of some cytochrome P450-mediated classic and atypical enzyme kinetics, and the associated kinetic models. Applications of these kinetic models can provide some new insights into the mechanism of P450-mediated drug-drug interactions.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Algorithms , Animals , Cytochrome P-450 Enzyme Inhibitors , Enzyme Activation/physiology , Humans , Kinetics
2.
Bioorg Med Chem Lett ; 11(8): 1059-62, 2001 Apr 23.
Article in English | MEDLINE | ID: mdl-11327589

ABSTRACT

Characterization of the metabolites of the COX-2 inhibitor etoricoxib (MK-0663 and L-791,456) produced in vitro indicate formation of an N-oxide pyridine and hydroxymethyl pyridine that can further be glucuronidated or oxidized to an acid. Significant turnover is observed in human hepatocytes. Several CYPs are involved in the oxidative biotranformations and, from in vitro studies, etoricoxib is not a potent CYP3A4 inducer or inhibitor. Based on an in vitro whole blood assay, none of the metabolites of etoricoxib inhibits COX-1 or contributes significantly to the inhibition of COX-2.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Isoenzymes/antagonists & inhibitors , Mixed Function Oxygenases/metabolism , Pyridines/metabolism , Pyridines/pharmacology , Sulfones/metabolism , Sulfones/pharmacology , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/metabolism , Cyclooxygenase Inhibitors/pharmacology , Cytochrome P-450 CYP3A , Etoricoxib , Hepatocytes/metabolism , Humans , Isoenzymes/blood , Membrane Proteins , Microsomes/metabolism , Oxidation-Reduction , Prostaglandin-Endoperoxide Synthases/blood
3.
Drug Metab Dispos ; 29(5): 638-44, 2001 May.
Article in English | MEDLINE | ID: mdl-11302928

ABSTRACT

The metabolic profile of DFU [5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone], a potent and selective COX-2 inhibitor, was characterized using in vitro microsomal and hepatocyte incubations. A single product, corresponding to p-hydroxylation, p-OH-DFU [(5,5-dimethyl-3-(3-fluoro-4-hydroxyphenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone)], was produced in rat microsomal incubations of DFU. In contrast, three metabolites were produced in incubations using suspensions of freshly isolated rat hepatocytes. Microsomal production of the p-O-glucuronide metabolite of DFU from synthetic p-OH-DFU was shown to have chromatographic and mass spectrometric properties identical to the earliest eluting hepatocyte metabolite (M1). The molecular weights of the other two hepatocyte metabolites were readily obtained using capillary high-performance liquid chromatography continuous-flow liquid secondary ion mass spectrometry (HPLC/CF-LSIMS); however, the elemental composition of these metabolites was not. Unlike typical metabolic products, which produce readily identified increments in molecular weight, metabolites M2 and M3 produced molecular ions in positive- and negative-ion CF-LSIMS that were consistent with oxidation of DFU (+16 Da), followed by addition of glutathione (+306 Da) and subsequent loss of 20 and 18 Da, respectively. Capillary HPLC/high-resolution CF-LSIMS was used to generate accurate mass data for M2 and M3 that provided evidence that the losses of 20 and 18 Da, respectively, corresponded to a rearomatization through loss of HF or H(2)O. Isolation and NMR characterization provided the definitive structural proof for these metabolites. Overall, the metabolism of DFU in rat hepatocytes is proposed to proceed through an epoxide intermediate, which then either rearranges to the p-OH-DFU and is conjugated with glucuronic acid, or is trapped with glutathione, followed by rearomatization with loss of HF (M2) or H(2)O (M3).


Subject(s)
Cyclooxygenase Inhibitors/metabolism , Furans/metabolism , Glutathione/metabolism , Isoenzymes/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Animals , Cells, Cultured , Chromatography, High Pressure Liquid/methods , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Magnetic Resonance Spectroscopy , Male , Mass Spectrometry/methods , Microsomes/metabolism , Rats , Rats, Sprague-Dawley
4.
Drug Metab Dispos ; 29(3): 232-41, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11181489

ABSTRACT

CDP-840 is a selective and potent phosphodiesterase type IV inhibitor, whose in vitro metabolism profile was first investigated using liver microsomes from different species. At least 10 phase I oxidative metabolites (M1-M10) were detected in the microsomal incubations and characterized by capillary high-performance liquid chromatography continuous-flow liquid secondary ion mass spectrometry (CF-LSIMS). Significant differences in the microsomal metabolism of CDP-840 were found between rat and other species. The major route of metabolism in rat involved para-hydroxylation on the R4 phenyl. This pathway was not observed in human and several other species. The in vitro metabolism profile of CDP-840 was further examined using freshly isolated hepatocytes from rat, rabbit, and human. The hepatocyte incubations indicated more extensive metabolism relative to that in microsomes. In addition to the phase I oxidative metabolites observed in microsomal incubations, several phase II conjugates were identified and characterized by CF-LSIMS. Interspecies differences in phase II metabolism were also found in these hepatocyte incubations. The major metabolite in human hepatocytes was identified as the pyridinium glucuronide, which was not detected in rat hepatocytes. Simple structural modification on R4, such as p-Cl substitution, greatly reduced the species differences in microsomal metabolism. Furthermore, modifications on R3, such as the N-oxide, eliminated the N-glucuronide formation in human. These results not only helped in determining the suitability of animal species used in the preclinical safety studies but also provided valuable directions for the synthetic efforts in finding backup compounds that are more metabolically stable.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Microsomes, Liver/metabolism , Phosphodiesterase Inhibitors/metabolism , Pyridines/metabolism , Animals , Cells, Cultured , Chromatography, High Pressure Liquid , Cyclic Nucleotide Phosphodiesterases, Type 4 , Glucuronides/chemistry , Glucuronides/metabolism , Hepatocytes/metabolism , Humans , Liver/cytology , Liver/metabolism , Magnetic Resonance Spectroscopy , Male , Mass Spectrometry , Molecular Structure , Phosphodiesterase Inhibitors/blood , Phosphodiesterase Inhibitors/chemistry , Phosphodiesterase Inhibitors/pharmacokinetics , Pyridines/blood , Pyridines/chemistry , Pyridines/pharmacokinetics , Rabbits , Rats , Rats, Sprague-Dawley
5.
Drug Metab Dispos ; 29(2): 159-65, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11159806

ABSTRACT

The drug candidate DFP [5,5-dimethyl-3-(2-isopropoxy)-4-(4-methanesulfonylphenyl)-2(5H)-furanone] is a selective cyclooxygenase-2 inhibitor under evaluation for analgesic and anti-inflammatory therapy. The in vitro metabolic pathways (rat microsomes) involve hydroxylation of the isopropyl side chain at either of two positions, the methyl or the methine, thus producing a hydroxylated metabolite (DFHP) or a dealkylated metabolite (DFH). DFH formation was the major pathway. Using hepatic microsomes from rats treated with agents that induce specific CYP isozymes, it was shown that the dexamethasone-inducible rat CYP3A isozyme(s) play a major role in DFH formation. The roles of CYP3A1 and -3A2 were confirmed with genetically engineered rat CYP enzymes. The potential for induction of rat CYP3A by DFP was evaluated by incubating DFP in rat hepatocyte cultures and measuring the CYP3A levels. Both CYP3A immunoreactive protein and enzyme activity were induced in a dose-dependent manner. The induction was confirmed in vivo by dosing rats with DFP at 100 mg/kg for 4 days. Microsomes prepared from the excised livers showed that DFP gave approximately 55% of the induction observed with dexamethasone, as determined by Western blot. In vitro metabolic auto-induction of DFP was assessed by measuring the metabolism of DFP in hepatocytes treated with DFP. DFH formation was significantly enhanced in the DFP-treated cells. In vivo, treating rats with DFP at doses of 10 to 100 mg/(kg.day) for 13 weeks indicated that DFP induced its own metabolism. The C(max) and plasma drug area under the curve values during the thirteenth week were significantly lower than that on the first day, and the effect was dose-dependent.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Benzene Derivatives/pharmacokinetics , Cyclooxygenase Inhibitors/pharmacokinetics , Cytochrome P-450 Enzyme System/biosynthesis , Furans/pharmacokinetics , Hepatocytes/enzymology , Isoenzymes/antagonists & inhibitors , Oxidoreductases, N-Demethylating/biosynthesis , Alkylation , Animals , Benzene Derivatives/metabolism , Biotransformation , Cell Culture Techniques/methods , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/metabolism , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/metabolism , Dexamethasone/pharmacology , Enzyme Induction/drug effects , Furans/metabolism , Hydroxylation , Liver/enzymology , Liver/metabolism , Male , NADP/metabolism , Oxidoreductases, N-Demethylating/metabolism , Predictive Value of Tests , Prostaglandin-Endoperoxide Synthases , Rats , Rats, Sprague-Dawley
6.
Mol Pharmacol ; 59(1): 144-52, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11125035

ABSTRACT

Gamma-aminobutyric acid (GABA) activates two qualitatively different inhibitory mechanisms through ionotropic GABA(A) multisubunit chloride channel receptors and metabotropic GABA(B) G protein-coupled receptors. Evidence suggests that pharmacologically distinct GABA(B) receptor subtypes mediate presynaptic inhibition of neurotransmitter release by reducing Ca2+ conductance, and postsynaptic inhibition of neuronal excitability by activating inwardly rectifying K+ (Kir) conductance. However, the cloning of GABA(B) gb1 and gb2 receptor genes and identification of the functional GABA(B) gb1-gb2 receptor heterodimer have so far failed to substantiate the existence of pharmacologically distinct receptor subtypes. The anticonvulsant, antihyperalgesic, and anxiolytic agent gabapentin (Neurontin) is a 3-alkylated GABA analog with an unknown mechanism of action. Here we report that gabapentin is an agonist at the GABA(B) gb1a-gb2 heterodimer coupled to Kir 3.1/3.2 inwardly rectifying K+ channels in Xenopus laevis oocytes. Gabapentin was practically inactive at the human gb1b-gb2 heterodimer, a novel human gb1c-gb2 heterodimer and did not block GABA agonism at these heterodimer subtypes. Gabapentin was not an agonist at recombinant GABA(A) receptors as well. In CA1 pyramidal neurons of rat hippocampal slices, gabapentin activated postsynaptic K+ currents, probably via the gb1a-gb2 heterodimer coupled to inward rectifiers, but did not presynaptically depress monosynaptic GABA(A) inhibitory postsynaptic currents. Gabapentin is the first GABA(B) receptor subtype-selective agonist identified providing proof of pharmacologically and physiologically distinct receptor subtypes. This selective agonism of postsynaptic GABA(B) receptor subtypes by gabapentin in hippocampal neurons may be its key therapeutic advantage as an anticonvulsant.


Subject(s)
Acetates/pharmacology , Amines , Anticonvulsants/pharmacology , Cyclohexanecarboxylic Acids , Neurons/drug effects , Potassium Channels, Inwardly Rectifying , Receptors, GABA-B/metabolism , gamma-Aminobutyric Acid , Amino Acid Sequence , Animals , Dimerization , Excitatory Postsynaptic Potentials , G Protein-Coupled Inwardly-Rectifying Potassium Channels , GABA-B Receptor Agonists , Gabapentin , Hippocampus/cytology , Hippocampus/drug effects , In Vitro Techniques , Male , Molecular Sequence Data , Neurons/metabolism , Neurons/physiology , Oocytes , Potassium Channels/biosynthesis , Potassium Channels/genetics , Protein Isoforms , Rats , Rats, Sprague-Dawley , Sequence Homology, Amino Acid , Xenopus laevis
7.
Bioorg Med Chem Lett ; 10(23): 2683-6, 2000 Dec 04.
Article in English | MEDLINE | ID: mdl-11128651

ABSTRACT

Metabolites of the COX-2 inhibitor rofecoxib (MK-0966, Vioxx) were prepared by synthetic or biosynthetic methods. Metabolites include products of oxidation, glucuronidation, reduction and hydrolytic ring opening. Based on an in vitro whole blood assay, none of the known human metabolites of rofecoxib inhibits COX-1 nor contributes significantly to the inhibition of COX-2.


Subject(s)
Cyclooxygenase Inhibitors/chemical synthesis , Cyclooxygenase Inhibitors/pharmacology , Lactones/chemical synthesis , Lactones/pharmacology , Animals , Cyclooxygenase 1 , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/chemistry , Drug Evaluation, Preclinical , Humans , Isoenzymes/blood , Lactones/chemistry , Membrane Proteins , Prostaglandin-Endoperoxide Synthases/blood , Rats , Sulfones
8.
Anal Biochem ; 276(2): 215-26, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10603245

ABSTRACT

The standard method to evaluate CYP3A inhibition is to study the conversion of the specific CYP3A probe testosterone to its 6 beta-hydroxy metabolite in human liver microsomes, in the absence and presence of potential inhibitors. Quantification of the 6 beta-hydroxy metabolite is achieved by HPLC resulting in a tedious and time-consuming assay. In order to increase the P450 inhibition throughput, efforts were made to find a CYP3A probe that would produce a fluorescent metabolite. This paper reports the discovery of DFB as a potential CYP3A fluorescent probe. DFB was significantly metabolized in human microsomes (approximately 1-2 nmol/(min. mg protein)) to give the fluorescent compound DFH. The involvement of CYP3A in the metabolism of DFB was determined using multiple approaches. First, incubations conducted with microsomes made from cell lines expressing single CYPs (Gentest Supersomes) indicated that CYP3A played a major role in the metabolism of DFB. Secondly, immunoinhibition studies conducted with CYP3A antibody resulted in >95% inhibition of DFB metabolism in HLM. Thirdly, inhibition studies with specific CYP1A1, 1A2, 2C8/9, 2C19, 2D6, and 2E1 chemical inhibitors did not suppress DFB activity in HLM. However, ketoconazole, miconazole, nicardipine, and nifedipine, all known CYP3A inhibitors, completely abolished the formation of DFH in HLM. The potency of several inhibitors determined using DFB and testosterone as CYP3A probes was consistent (R = 0.98). Finally, a good agreement was obtained for the formation of DFH and production of 6 beta-hydroxytestosterone when DFB and testosterone were incubated separately with various human liver microsome preparations (R = 0.94, N = 11). In order to use DFH as a fluorescent CYP3A marker in a 96-well plate format, it was important to remove the excess of NADPH at the end of the incubation because the fluorescence of NADPH interferes with DFH detection. This was achieved by adding oxidized glutathione and glutathione reductase to convert NADPH to NADP(+) which is not fluorescent. The liquid-handling steps were fully automated in a 96-well plate format and a template was designed to generate IC(50) curves and to address potential fluorescent interferences from the test compounds. The assay was found to be reproducible (intraday variability <10% and interday variability indicated less than a 2-fold variation in the IC(50) values) and is now routinely used in our laboratory to evaluate CYP3A inhibition of NCEs.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Chemistry Techniques, Analytical/methods , Cytochrome P-450 Enzyme Inhibitors , Fluorescent Dyes , Fluorobenzenes , Furans , Microsomes, Liver/enzymology , Oxidoreductases, N-Demethylating/antagonists & inhibitors , Cell Line , Chemistry Techniques, Analytical/instrumentation , Chromatography, High Pressure Liquid , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/metabolism , Fluorescent Dyes/metabolism , Fluorobenzenes/metabolism , Fluorometry , Furans/metabolism , Humans , In Vitro Techniques , Oxidoreductases, N-Demethylating/metabolism
9.
J Mass Spectrom ; 34(5): 521-36, 1999 May.
Article in English | MEDLINE | ID: mdl-10390857

ABSTRACT

The human histiocytic lymphoma U937 cell line contains a rich source of the 85 kDa cytosolic phospholipase A2 (cPLA2). DMSO-differentiated U937 cells were used as a model to investigate the free arachidonic acid release, the arachidonate distribution and the phospholipid source of arachidonate upon Ca2+ ionophore stimulation. A combination of several chromatographic and mass spectrometric techniques was employed in this study. The amount of free arachidonic acid (AA) released upon stimulation, the arachidonate content in total lipids and in each of the phospholipid classes were determined by gas chromatography/mass spectrometry (GC/MS). Glycerophosphoethanolamine (GPE) was found to be the major pool of arachidonate in differentiated human U937 cells (55%) and glycerophosphocholine (GPC) and glycerophosphoinositol (GPI) contributed 22 and 8%, respectively. Upon Ca2+ ionophore stimulation, GPE class lost the largest amount of arachidonate, followed by GPC class. GPI class, however, gained a substantial amount of arachidonate. Most of the arachidonate depleted from GPE and GPC was recovered as free AA, some of which was rapidly esterified into GPI species. GC/MS with electron capture negative chemical ionization provided excellent sensitivity for the measurement of arachidonic acid which was derivatized to its pentafluorobenzyl ester. Intact phospholipid molecular species including the arachidonyl-containing phospholipid species were identified using capillary high-performance liquid chromatography/continuous-flow liquid secondary ion mass spectrometry (CF-LSIMS). No specificity was found for releasing free AA among the arachidonyl-containing GPE and GPC species upon Ca2+ ionophore stimulation. CF-LSIMS provided a sensitive and effective means of detecting intact phospholipid species.


Subject(s)
Arachidonic Acid/metabolism , Gas Chromatography-Mass Spectrometry , Phospholipids/metabolism , Spectrometry, Mass, Secondary Ion , U937 Cells/metabolism , Gas Chromatography-Mass Spectrometry/methods , Humans , Spectrometry, Mass, Secondary Ion/methods
10.
Drug Metab Dispos ; 27(3): 403-9, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10064573

ABSTRACT

The lactol derivative of a lactone cyclooxygenase-2 inhibitor (DFU) was evaluated in vivo and in vitro for its potential suitability as a prodrug. DFU-lactol was found to be 10 to 20 times more soluble than DFU in a variety of aqueous vehicles. After administration of DFU-lactol at 20 mg kg-1 p.o. in rats, a Cmax of 7.5 microM DFU was reached in the plasma. After oral administration, the ED50s of DFU-lactol in the carrageenan-induced paw edema and lipopolysaccharide-induced pyresis assays in rats are comparable with the ED50s observed when dosing with DFU. Incubations of DFU-lactol with rat and human hepatocytes demonstrated that the oxidation of DFU-lactol can be mediated by liver enzymes and that a competing pathway is direct glucuronidation of the DFU-lactol hydroxyl group. Assays with subcellular fractions from rat liver indicated that most of the oxidation of DFU-lactol occurs in the cytosolic fraction and requires NAD(P)+. Human liver cytosol can also support the oxidation of DFU-lactol to DFU when NAD(P)+ is added to the incubations. Fractionation of human liver cytosolic proteins showed that at least three enzymes are capable of efficiently effecting the oxidation of DFU-lactol to DFU. Incubations with commercially available dehydrogenases suggest that alcohol and hydroxysteroid dehydrogenases are involved in this oxidative process. These data together suggest that lactols may represent useful prodrugs for lactone-containing drugs.


Subject(s)
Cyclooxygenase Inhibitors/pharmacokinetics , Furans/pharmacokinetics , Isoenzymes/drug effects , Lactones/pharmacokinetics , Prodrugs/pharmacokinetics , Prostaglandin-Endoperoxide Synthases/drug effects , Animals , Biotransformation , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Humans , Isoenzymes/metabolism , Male , Membrane Proteins , Oxidation-Reduction , Oxidoreductases/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Rats , Rats, Sprague-Dawley
11.
Drug Metab Dispos ; 26(5): 490-6, 1998 May.
Article in English | MEDLINE | ID: mdl-9571231

ABSTRACT

Induction of cytochromes P450 (P450s) by drugs can lead to drug-drug interactions. Primary hepatocytes have been reported to retain inducible P450s. To optimize the use of primary hepatocytes for predicting induction of P450 (CYP 3A and 2B) expression in vivo, both culture conditions and expression of induction potentials were investigated. In rat hepatocytes, basal CYP 3A1/2 expression was better maintained in cells cultured on Matrigel compared with collagen when low concentrations of dexamethasone were used. However, CYP 3A1/2 induction was not affected by either matrix. In contrast, induction of CYP 2B1/2 by phenobarbital was markedly stronger in hepatocytes cultured on Matrigel. To further validate the in vitro model, Sprague-Dawley rats and isolated hepatocytes cultured on Matrigel were exposed to a series of compounds. In an attempt to minimize large variability between experiments, a novel approach for calculating induction potential was applied. In vitro results for CYP 3A1/2 and 2B1/2 induction correlated well with those observed in vivo. In contrast with rat hepatocytes, basal CYP 3A4 expression in human hepatocytes decreased rapidly in cells cultured on either Matrigel or collagen. However, CYP 3A4 inducibility was retained in cells cultured on either matrix. Interestingly, induction of CYP 3A4 in human hepatocytes by several model compounds did not correlate with the induction of CYP 3A1/2 in rat hepatocytes. This in vitro assay should facilitate the demand for a fast and reproducible method for addressing P450 induction by numerous compounds at the drug discovery stage.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/biosynthesis , Liver/enzymology , Animals , Cell Culture Techniques/methods , Cytochrome P-450 CYP2B1/biosynthesis , Cytochrome P-450 CYP3A , Enzyme Induction , Male , Mixed Function Oxygenases/biosynthesis , Rats , Rats, Sprague-Dawley , Steroid Hydroxylases/biosynthesis
12.
J Lipid Mediat Cell Signal ; 17(1): 15-9, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9302651

ABSTRACT

Protein expression of microsomal GST-II and LTC4 synthase was analyzed by Western blot. Correlation between a 17 kDa band and LTC4 formation was observed for both enzymes. The expression of microsomal GST-II was several fold more efficient than the expression of LTC4 synthase. In addition to catalyzing the biosynthesis of LTC4, microsomal GST-II also produces another product, which has been subjected to mass spectrometric analysis. This analysis demonstrates that the novel product is an isomer of LTC4.


Subject(s)
Glutathione Transferase/chemistry , Leukotriene C4/chemistry , Microsomes/enzymology , Animals , Blotting, Western , Catalysis , Chromatography, High Pressure Liquid , Glutathione Transferase/metabolism , Isomerism , Mass Spectrometry , Spodoptera
14.
Biochem Pharmacol ; 52(7): 1113-25, 1996 Oct 11.
Article in English | MEDLINE | ID: mdl-8831731

ABSTRACT

Selective inhibitors of prostaglandin synthase-2 (PGHS-2) possess potent anti-inflammatory, antipyretic, and analgesic properties but demonstrate reduced side-effects (e.g. gastrotoxicity) when compared with nonselective inhibitors of PGHS-1 and -2. We investigated the mechanism of the differential inhibition of human PGHS-1 (hPGHS-1) and -2 (hPGHS-2) in intact cells by nonsteroidal anti-inflammatory drugs (NSAIDs) and examined factors that contribute to the increased potency of PGHS inhibitors observed in intact cells versus cell-free systems. In intact Chinese hamster ovary (CHO) cell lines stably expressing the hPGHS isozymes, both PGHS isoforms exhibited the same affinity for arachidonic acid. Exogenous and endogenous arachidonic acid were used as substrates by both CHO [hPGHS-1] and CHO [hPGHS-2] cell lines. However, differences were observed in the ability of the hPGHS isoforms to utilize endogenous arachidonic acid released intracellularly following calcium ionophore stimulation or released by human cytosolic phospholipase A2 transiently expressed in the cells. Cell-based screening of PGHS inhibitors demonstrated that the selectivities and potencies of PGHS inhibitors determined using intact cells are affected by substrate concentration and differ from that determined in cell-free microsomal or purified enzyme preparations of PGHS isozymes. The mechanism of inhibition of PGHS isozymes by NSAIDs in intact cells involved difference in their time-dependent inhibition. Indomethacin displayed time-dependent inhibition of cellular hPGHS-1 and -2. In contrast, the selective PGHS-2 inhibitor NS-398 exhibited time-independent inhibition of hPGHS-1 but time-dependent inhibition of hPGHS-2 in intact cells. Reversible inhibition of cellular CHO [hPGHS-1] and CHO [hPGHS-2] was observed with the nonselective NSAIDs ibuprofen and indomethacin, whereas inhibition by the selective PGHS-2 inhibitor DuP-697 was reversible against hPGHS-1 but irreversible against hPGHS-2.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Prostaglandin-Endoperoxide Synthases/drug effects , Prostaglandins/metabolism , Animals , Arachidonic Acid/pharmacology , CHO Cells , Cricetinae , Dose-Response Relationship, Drug , Humans , Ibuprofen/pharmacology , Indomethacin/pharmacology
15.
J Med Chem ; 39(20): 3951-70, 1996 Sep 27.
Article in English | MEDLINE | ID: mdl-8831761

ABSTRACT

Naphthalenic lignan lactone 3a (L-702,539), a potent and selective 5-lipoxygenase (5-LO) inhibitor, is extensively metabolized at two different sites: the tetrahydropyran and the lactone rings. Early knowledge of the metabolic pathways triggered and directed a structure-activity relationship study aimed toward the improvement of metabolic stability in this series. The best modifications discovered, i.e., replacement of the lactone ring by a nitrile group, replacement of the tetrahydropyran ring by a 6,8-dioxabicyclo[3.2.1]octanyl moiety, and replacement of the pendant phenyl ring by a 3-furyl ring, were incorporated in a single molecule to produce inhibitor 9ac (L-708,780). Compound 9ac inhibits the oxidation of arachidonic acid to 5-hydroperoxy-eicosatetraenoic acid by 5-LO (IC50 = 190 nM) and the formation of leukotriene B4 in human polymorphonuclear leukocytes (IC50 = 3 nM) as well as in human whole blood (IC50 = 150 nM). The good inhibitory profile shown by naphthalenenitrile 9ac is accompanied by an improved resistance to oxidative metabolism. In addition, 9ac is orally active in the functional model of antigen-induced bronchoconstriction in allergic squirrel monkeys (95% inhibition at 0.1 mg/kg).


Subject(s)
Benzofurans/chemistry , Lipoxygenase Inhibitors , Lipoxygenase Inhibitors/chemistry , Naphthalenes/chemistry , Nitriles/chemistry , Animals , Arachidonate 5-Lipoxygenase/metabolism , Arachidonic Acid/metabolism , Bronchoconstriction/drug effects , Drug Stability , Humans , Leukotriene B4/biosynthesis , Leukotriene B4/blood , Leukotrienes/metabolism , Lipoxygenase Inhibitors/pharmacology , Male , Microsomes, Liver/enzymology , Molecular Structure , Naphthalenes/pharmacology , Neutrophils/metabolism , Nitriles/pharmacology , Oxidation-Reduction , Rats , Rats, Sprague-Dawley , Saimiri , Structure-Activity Relationship
16.
Drug Metab Dispos ; 23(12): 1325-34, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8689939

ABSTRACT

Hepatic microsomes from different species were used to study the oxidative metabolism of L-746,530 and L-739,010, two potent and specific 5-lipoxygenase inhibitors. HPLC analysis of the incubates obtained from the microsomal incubations of L-739,010 and L-746,530 showed only traces of metabolites. However, recovery of the starting material in the supernatant was less than quantitative in all of the species studied (approximately 90% in rat, approximately 70% in the dexamethasone-induced rat, approximately 70-90% in humans, and approximately 60% in the rhesus monkey for both compounds). The recovery of the starting material was found to be time- and NADPH-dependent, suggesting that metabolite(s) were formed and reacting with the microsomal proteins. Evidence that the cytochrome P4503A (CYP3A) contributed to the formation of the reactive metabolite(s) was shown by the low recovery of material that was observed upon incubation with microsomes obtained from dexamethasone-treated rats (a CYP3A inducer), compared with microsomes obtained from untreated rats. Also, the recovery of material was improved when troleandomycin, a CYP3A inhibitor, was added to rhesus monkey microsomal incubations (25% more parent compound detected in the supernatant with 100 microM of troleandomycin). Using radiolabeled L-746,530 and gel electrophoresis analysis, it was confirmed that radiolabeled material was covalently bound to the microsomal protein. Incubations of L-739,010 and L-746,530 in the presence of semicarbazide resulted, in both cases, in the formation of two adducts. Using a combination of NMR, liquid secondary-ion MS, and UV techniques, these adducts were identified as isomers of an oxidized metabolite that had been trapped by semicarbazide. The site of oxidation was determined to be on the dioxabicyclo moiety. The importance of this moiety in the formation of reactive metabolite(s) was verified by incubating analogs of the 5-lipoxygenase inhibitors that contained blocking methyl groups at the proposed site of oxidation on the bicyclo moiety. Incubations of these gemdimethyl analogs of L-746,530 and L-739,010 with microsomes from different species resulted in significantly improved recovery of the starting material (approximately 94% in the rat, 85% in the dexamethasone-induced rat, 95% in humans, and 85% in the rhesus monkey for both compounds) and significantly less radioactive binding to the microsomal protein.


Subject(s)
Bridged Bicyclo Compounds/metabolism , Lipoxygenase Inhibitors/metabolism , Microsomes, Liver/metabolism , Quinolines/metabolism , Animals , Blotting, Western , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Humans , In Vitro Techniques , Macaca mulatta , Magnetic Resonance Spectroscopy , Mass Spectrometry , Oxidation-Reduction , Protein Binding , Rats , Rats, Sprague-Dawley , Spectrophotometry, Ultraviolet
17.
Drug Metab Dispos ; 23(10): 1085-93, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8654196

ABSTRACT

Verlukast (MK-0679) is a potent leukotriene D4 antagonist that was under development for the treatment of bronchial asthma. A previously uncharacterized metabolite of verlukast was formed in incubations using rat liver cytosol fortified with glutathione (GSH). The metabolite was detected by HPLC and characterized by UV spectroscopy (photodiode array detection after HPLC) and capillary HPLC continuous flow-liquid secondary-ion mass spectrometry. After a large-scale incubation and isolation, it was further characterized by 500 MHz proton NMR. The metabolite is a 1,4-Michael addition product in which GSH has added to position 12 of the styryl quinoline double bond of verlukast. There is no apparent stereoselectivity because a mixture of the two possible isomers, in equal amounts, was observed by NMR. Although there was spontaneous chemical addition of GSH to verlukast (0.18 nmol/min), the reaction was shown to be enzyme-catalyzed in studies using three different preparations of rat liver cytosol at pH 7.4. Using Lineweaver-Burk analysis of experiments in which the effect of verlukast concentration on the rate of conjugation was studied, the apparent KM and Vmax were determined to be 107 +/- 22 microM (SD, N=3) and 0.66 +/- 0.21 nmol/min/mg protein, respectively. In similar studies with GSH as the variable substrate, the apparent KM and Vmax were 2.32 +/- 0.68 mM and 0.69 +/- 0.14 nmol/min/mg protein, respectively. Incubations with kidney cytosol produced the GSH, cysteinylglycine, and cysteine conjugates of verlukast. In bile collected from rats dosed intravenously with 50 mg/kg of verlukast, approximately 80% of the dose was recovered up to 4 hr postdose. The GSH conjugate accounted for 16.5% of the dose. The cysteinylglycine, cysteine, and N-acetylcysteine conjugates were observed and together accounted for 7.5%. Verlukast accounted for 14.5%, and the remainder of the metabolites (40.5%) were oxidation or acyl glucuronide metabolites.


Subject(s)
Bile/metabolism , Bronchodilator Agents/metabolism , Glutathione/metabolism , Kidney/metabolism , Liver/metabolism , Propionates/metabolism , Quinolines/metabolism , Animals , Chromatography, High Pressure Liquid , Cytosol/metabolism , Male , Rats , Rats, Sprague-Dawley
18.
Anal Chem ; 67(17): 2931-6, 1995 Sep 01.
Article in English | MEDLINE | ID: mdl-8779418

ABSTRACT

The application of capillary HPLC/continuous-flow liquid secondary ion mass spectrometry (CF-LSIMS) as part of an integrated approach for characterizing discovery stage in vitro metabolites, using a specific inhibitor for 5-lipoxygenase as a model compound, was investigated. CF-LSIMS demonstrated excellent sensitivity in detecting the metabolites in both the positive and the negative ion modes, with a good full-scan mass spectrum obtained when 5 pmol of metabolite was injected onto the capillary column. Strong pseudomolecular ions and key fragment ions were observed in the primary spectra of the parent drug and its three oxidative in vitro metabolites, allowing the site of metabolism to be pinpointed to particular substructures. This technique demonstrated versatility and offered a very rapid screening procedure for metabolite identification.


Subject(s)
Chromatography, High Pressure Liquid/methods , Spectrometry, Mass, Secondary Ion/methods , Animals , Benzofurans/pharmacokinetics , Biotransformation , In Vitro Techniques , Lipoxygenase Inhibitors/pharmacokinetics , Macaca mulatta , Microsomes, Liver/metabolism
19.
Drug Metab Dispos ; 23(1): 65-71, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7720527

ABSTRACT

It has been reported previously that the tetrahydropyranyl naphthtalenic lignan lactone L-702,539 is a potent nonredox, 5-lipoxygenase inhibitor that has the advantage that it can be dosed either as the lactone or as the corresponding nonactive hydroxy acid L-702,618 (opened lactone). Studies with hepatic microsomes from the rat, rhesus monkey, and human were undertaken in a phosphate buffer and suggested that the closure of the hydroxy acid L-702,618 to the lactone L-702,539 was an enzymatic process. The incubation of L-702,539 under oxidative conditions with these specific hepatic microsomes resulted in the formation of three significant metabolites (> 0.4 nmol/mg protein/hr) as determined by HPLC with UV detection. These metabolites were isolated from large microsomal incubations and were characterized by MS and NMR spectroscopy. Data showed that the lactone and tetrahydropyran portions of the molecule were both susceptible to hydroxylation, and the hydroxylated tetrahydropyran was further oxidized to the hydroxy acid. Analysis of plasma samples obtained from rat and rhesus monkeys following L-702,618 administration indicated that the in vivo metabolic pathway was similar to the one observed in vitro using hepatic microsomes. Studies conducted with microsomes from genetically engineered human cell lines expressing individual cytochrome P450s indicated that the isozyme responsible for the metabolism at the tetrahydropyran ring, was P4503A4. These findings were supported by studies conducted in human microsomes using an inhibitory P4503A4 antibody and troleandomycin, which is a potent P4503A inhibitor.


Subject(s)
Benzofurans/pharmacokinetics , Lipoxygenase Inhibitors/pharmacokinetics , Animals , Biotransformation , Blotting, Western , Cell Line , Cytochrome P-450 Enzyme System/metabolism , Humans , In Vitro Techniques , Macaca mulatta , Male , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Oxidation-Reduction , Rats , Rats, Sprague-Dawley , Species Specificity
20.
Agents Actions Suppl ; 46: 159-68, 1995.
Article in English | MEDLINE | ID: mdl-7610986

ABSTRACT

The main target of non-steroidal anti-inflammatory drugs (NSAIDs) is prostaglandin G/H synthase (PGHS), also known as cyclooxygenase (COX), which exists as two isoforms. In order to evaluate the contributions of PGHS isoforms to physiological and pathological conditions and their sensitivity to inhibition by non-steroidal anti-inflammatory drugs, we have established high level expression systems of recombinant human PGHS isoforms. The inducible form of PGHS, termed PGHS-2, has been purified and characterized with respect to substrate specificity, product formation, enzymatic activity, glycosylation, heme content, quaternary structure, and modification by aspirin. Pharmacological profiles of the recombinant PGHS isoforms indicate that conventional NSAIDs show little selectivity for either enzyme, however, the recently described NSAID, NS-398, exhibits a high degree of specificity for PGHS-2 through a time dependent mechanism.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Baculoviridae/genetics , Cyclooxygenase Inhibitors/pharmacology , Isoenzymes/antagonists & inhibitors , Prostaglandin-Endoperoxide Synthases/genetics , Vaccinia virus/genetics , Animals , Cells, Cultured , Prostaglandin-Endoperoxide Synthases/biosynthesis , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/biosynthesis
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