Enzyme kinetics of cytochrome P450-mediated reactions.

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.

In vitro metabolism considerations, including activity testing of metabolites, in the discovery and selection of the COX-2 inhibitor etoricoxib (MK-0663).

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.

In vitro metabolism of the COX-2 inhibitor DFU, including a novel glutathione adduct rearomatization.

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).

Investigation of the in vitro metabolism profile of a phosphodiesterase-IV inhibitor, CDP-840: leading to structural optimization.

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.

Application of rat hepatocyte culture to predict in vivo metabolic auto-induction: studies with DFP, a cyclooxygenase-2 inhibitor.

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.

Synthesis, characterization, and activity of metabolites derived from the cyclooxygenase-2 inhibitor rofecoxib (MK-0966, Vioxx).

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.

Description of a 96-well plate assay to measure cytochrome P4503A inhibition in human liver microsomes using a selective fluorescent probe.

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.

Mass spectrometric analysis of arachidonyl-containing phospholipids in human U937 cells.

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.

Oxidative bioactivation of the lactol prodrug of a lactone cyclooxygenase-2 inhibitor.

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.

Refinement of an in vitro cell model for cytochrome P450 induction.

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.

Mechanism of selective inhibition of human prostaglandin G/H synthase-1 and -2 in intact cells.

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.

Dioxabicyclooctanyl naphthalenenitriles as nonredox 5-lipoxygenase inhibitors: structure-activity relationship study directed toward the improvement of metabolic stability.

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).

Verlukast (MK-0679) conjugation with glutathione by rat liver and kidney cytosols and excretion in the bile.

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.

Integrated application of capillary HPLC/continuous-flow liquid secondary ion mass spectrometry to discovery stage metabolism studies.

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.

In vitro and in vivo biotransformations of the naphthalenic lignan lactone 5-lipoxygenase inhibitor, L-702,539.

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.

Purification and characterization of recombinant human cyclooxygenase-2.

Recombinant human cyclooxygenase-2 (hCox-2, Prostaglandin G/H synthase-2) has been purified from baculovirus-Sf9 and vaccina virus-Cos-7 cell expression systems. The detergent-solubilized, purified enzyme is heterogeneous in terms of its glycosylation. The vaccinia virus hCox-2 is a mixture of two glycoforms, whereas baculovirus hCox-2 comprises at least four species. The specific cyclooxygenase activities of both enzymes are 43 mumol O2/min/mg with arachidonic acid which is within the range of values reported for ovine Cox-1. The Km values of arachidonic acid for hCox-2 and ovine Cox-1 are 0.9 and 2.7 microM, respectively. Six other C-18 and C-20 fatty acids containing at least one 1,4-cis,cis-pentadiene moiety were also identified as substrates for hCox-2. Linoleic and gamma-linolenic acid were determined by mass spectrometry as being hydroxylated primarily at the C-9 and C-13 positions, whereas linolenic acid was hydroxylated primarily at the C-12 and C-16 positions. hCox-2 binds heme such that maximal activity is observed at a stoichiometry of 1.0 heme per enzyme subunit. The apparent molecular mass of hCox-2, determined by gel filtration chromatography in the presence of 2.0% beta-octylglucoside, is consistent with a dimeric structure. The results of this study indicate that the physical and catalytic properties of recombinant hCox-2 are very similar to that of the extensively studied ovine Cox-1.

Lipoxygenase stimulating effects of hydroxylated docosahexaenoates produced by human platelets.

Human platelet suspensions are capable of lipoxygenating docosahexaenoic acid (22:6n3) to an 11(S)-OH-, 14(S)-OH- or 17(S)-OH-22:6n3. The structure and stereochemical purity of these derivatives were confirmed by GC/MS and chiral phase LC analysis. The purified OH-22:6n3 positional isomers which are formed by human platelets were capable of inducing a concentration-dependent contractile response in the guinea-pig lung parenchymal strip at sub-micromolar concentrations. OH-22:6n3 may act in part through stimulation of leukotriene (LT) production as an increase in peptidyl-LT levels (LTC4, LTD4 and LTE4) occurred during the OH-22:6n3-induced contraction in this preparation. Both specific lipoxygenase inhibitors (caffeic acid, 20 uM and NDGA, 50 uM) and a LT receptor antagonist (FPL55712, 20 uM) significantly inhibited the contractile response. Moreover, the OH-22:6n3 positional isomers induced a concentration-dependent increase in LTB4 and LTC4 production in the guinea-pig chopped lung preparation. Other hydroxylated fatty acids and parent fatty acids which were tested (12-OH-20:4n6, 5-OH-20:4n6, 12-OH-20:5n3, 20:5n3 and 22:6n3) did not significantly contract this airway smooth muscle preparation or alter LT production. The hydroxylated 22:6n3 metabolites may modulate airway smooth muscle function in part through the release of peptidyl-LTs from the guinea-pig lung.

Respiratory tract eicosanoid measurement using microdialysis sampling and GC/MS detection.

Eicosanoids are important mediators in many physiological and pathophysiological conditions. Sampling the eicosanoids remains a challenge, particularly in the respiratory tract. We examined the possibility that microdialysis might offer a means for sampling the large airways which would provide profiles of local eicosanoid levels before and after challenge. Guinea-pigs were anesthetized by ip injection of urethane and a tracheotomy performed. Microdialysis probes were inserted 2 cm into the tracheal opening, and samples were collected for 1 h intervals over 5 h. Injections of arachidonic acid, leukotriene (LT) D4, or saline vehicle were made iv at the beginning of the third hour. Prostaglandins (PG) E2, D2, F2 alpha and 6-keto-F1 alpha and thromboxane (TX) B2 were determined by GC/MS. Significant post-treatment increases in levels were observed following the injection of arachidonic acid for all eicosanoids except 6-keto-PGF1 alpha. The greatest change was an 8.1-fold increase for PGD2 (P < 0.025) while the smallest change was a 3.5-fold increase for TXB2 (P < 0.025). Intravenous administration of LTD4 caused significant increases in levels of PGE2 (P < 0.025), PGF2 alpha (P < 0.05) and 6-keto-PGF1 alpha (P < 0.025) in the first hour after challenge. No increase was observed in control experiments following saline injection. These results indicate that microdialysis provides a useful methodology for sampling local eicosanoid production.

Enhancement of mass spectrometric detection of LTC4, LTD 4, and LTE 4 by derivatization.

Several acylating reagents are synthesized and used to introduce quatemary phosphonium or ammonium or ternary sulfonium functions into a simple model of a peptido leukotriene (PLT). One of these reagents was selected for further study with LTE4, LTD4, and LTC4. We demonstrate that acylation of the free amine function of PLTs to produce the 5-triphenylphosphoniumvaleryl-amide (TPPV) derivatives enhances chemical stabilities and significantly increases responses in fast-atom bombardment and continuous-flow liquid secondary ion mass spectrometry (CF-LSIMS) relative to the native PLTs. With high-performance liquid chromatography inlet to CF-LSIMS, we demonstrate the facile detection in selected ion monitoring of the TPPV derivative of 3 pg of LTD4.

CYP1A1 specificity of Verlukast epoxidation in mice, rats, rhesus monkeys, and humans.

It has previously been shown that Verlukast is converted to Verlukast dihydrodiol in microsomes from beta-naphthoflavone (BNF)-treated, but not uninduced Swiss Webster mice and Sprague-Dawley rats. We have examined the involvement of CYP1A1 in this reaction in more detail. It is concluded that this reaction is catalyzed exclusively by CYP1A1 in rats, mice, and humans based on the following criteria: 1) the epoxidation of Verlukast is negligible in uninduced rats, which express CYP1A2 but not CYP1A1; 2) Verlukast epoxidation is highly inducible by BNF treatment (60- to 200-fold); 3) Verlukast epoxidation in BNF-treated rat microsomes was inhibited by alpha-naphthoflavone (ANF) treatment, indicating that this activity was mediated by the CYP1A subfamily; 4) > 95% of Verlukast epoxidation in BNF-treated rat microsomes was inhibited by antibodies raised against CYP1A1; and 5) Verlukast was epoxidized by human CYP1A1 but not CYP1A2. Thus, Verlukast epoxidation appears to be specific for rat, mouse, and human CYP1A1. Additional studies showed that Verlukast was metabolized to Verlukast dihydrodiol in microsomes from uninduced rhesus monkeys. This reaction was inhibited by nanomolar concentrations of ANF in rhesus monkey microsomes implicating the involvement of the CYP1A subfamily. In addition, the 8-hydroxylation of R-warfarin, a pathway that is selective for rodent and human CYP1A1 activity, was also catalyzed at significant rates by rhesus monkey microsomes. These findings indicate that, unlike rats, mice, and humans, which have very low constitutive levels of hepatic CYP1A1 activity, the uninduced rhesus monkey is able to catalyze reactions specific to CYP1A1 in rodents and humans.(ABSTRACT TRUNCATED AT 250 WORDS)