Late sodium current block for drug-induced long QT syndrome: Results from a prospective clinical trial.

Drug-induced long QT syndrome has resulted in many drugs being withdrawn from the market. At the same time, the current regulatory paradigm for screening new drugs causing long QT syndrome is preventing drugs from reaching the market, sometimes inappropriately. In this study, we report the results of a first-of-a-kind clinical trial studying late sodium (mexiletine and lidocaine) and calcium (diltiazem) current blocking drugs to counteract the effects of hERG potassium channel blocking drugs (dofetilide and moxifloxacin). We demonstrate that both mexiletine and lidocaine substantially reduce heart-rate corrected QT (QTc) prolongation from dofetilide by 20 ms. Furthermore, all QTc shortening occurs in the heart-rate corrected J-Tpeak (J-Tpeak c) interval, the biomarker we identified as a sign of late sodium current block. This clinical trial demonstrates that late sodium blocking drugs can substantially reduce QTc prolongation from hERG potassium channel block and assessment of J-Tpeak c may add value beyond only assessing QTc.

Simultaneous quantification of seven active metabolites of roxifiban in human plasma by LC/MS/MS in the presence of an interfering displacer at millimolar concentrations.

Roxifiban (DMP 754) is a glycoprotein (GP) IIb/IIIa antagonist. Following oral administration to humans, roxifiban is metabolized to its primary active zwitterionic form, XV459, and several minor, active, hydrolyzed and hydroxylated metabolites, namely, M1a (DPC-AD3508), M1b (DPC-AD6128), M2 (SW156), M3 (DPC-AG2185), M8a (DPC-AF5814), and M8b (DPC-AF5818). Quantification of these metabolites in humans was not workable with a previous analytical method due to ion suppression of at least four of the analytes by a competitive displacer, DMP 728. This compound, which is another GP IIb/IIIa antagonist with very high affinity for the platelet receptor, was added to harvested blood samples in millimolar quantity to liberate XV459 from the GP IIb/IIIa receptor. An automated ion exchange solid phase extraction (IX-SPE) procedure was developed to selectively extract the seven metabolites of roxifiban and its deuterated internal standard while specifically excluding DMP 728. Among the six hydroxylation metabolites, there were two pairs of epimeric diastereomers (M1a/M1b and M8a/M8b) and one pair of geometric isomers (M2/M3), corresponding to three critical chromatographic pairs that needed to be base-line resolved because of the lack of specificity of MS/MS detection for these isomers. A new LC/MS/MS assay was developed to simultaneously quantify the seven metabolites in human plasma. The assay method was validated under GLP conditions over the concentration range of 0.5 to 80 nM for each of the analytes and successfully applied to assaying approximately 500 plasma samples from clinical trials.

Delineating novel metabolic pathways of DPC 963, a non-nucleoside reverse transcriptase inhibitor, in rats. Characterization of glutathione conjugates of postulated oxirene and benzoquinone imine intermediates by LC/MS and LC/NMR.

The metabolic activation of (S)-5,6-difluoro-4-cyclopropylethynyl-4-trifluoromethyl-3,4-dihydro-2(1H)-quinazolinone, DPC 963, in rats was investigated by identifying and characterizing the GSH and mercapturic acid conjugates excreted in the bile and urine, respectively. The structures of these adducts, which were unequivocally elucidated by LC/MS/MS and NMR experiments, revealed the existence of at least three distinct metabolic pathways leading to these products. One of the pathways, which has been described previously, involves the activation of the acetylene group after an initial hydroxylation on the methine carbon of the cyclopropyl ring. Metabolite M1 was demonstrated to be formed via this pathway after an enzymatic addition of GSH across the triple bond of the substituted acetylene. The second pathway, also previously described, leads to diastereoisomeric GSH adducts M3 and M4 after the formation of a highly reactive oxirene intermediate. This postulated oxirene subsequently rearranges to an alpha, beta-unsaturated cyclobutenyl ketone intermediate capable of undergoing a 1,4-Michael addition with a nucleophile such as GSH. In addition to these pathways, DPC 963 was found to undergo a metabolic activation previously undescribed for structural analogues of this compound. It is postulated that an oxidative defluorination mediated by cytochrome P450 leads to the formation of a putative benzoquinone imine intermediate which subsequently reacts with GSH to form two aromatic ring-substituted regioisomeric conjugates, M5 and M6. In addition to forming the GSH adducts, the benzoquinone imine was also found to be reduced to its unreactive hydroquinone metabolite, which was excreted as the glucuronide conjugate in rat bile. Studies with induced rat microsomes, cDNA-expressed rat P450 isozymes, and polyclonal antibodies against rat P450 clearly demonstrated that the rat P450s 3A1/3A2 were responsible for the formation of postulated oxirene and benzoquinone intermediates.

Formation of unusual glutamate conjugates of 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423) and its analogs: the role of gamma-glutamyltranspeptidase in the biotransformation of benzylamines.

The role of gamma-glutamyltranspeptidase (GGT) in transferring glutamate from endogenous glutathione (GSH) to the benzylamine moiety of a compound, such as 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423), is described. Studies were performed with structurally related analogs of DPC 423 to demonstrate that this type of reaction was common to compounds possessing a benzylamine group. Synthesizing appropriate standards and confirming by liquid chromatography (LC)/mass spectroscopy and LC/NMR made unambiguous assignments of the structures of glutamate conjugates of DPC 423. The use of stable isotope-labeled GSH for metabolism studies has not been described before. In the present study, we report the novel use of deuterated GSH in conjunction with mass spectral analysis to demonstrate the glutamate transfer to the benzylamines in the presence of GGT. To further demonstrate that the alpha protons on the benzylamines and glutamate (as part of glutathione) were unaffected during the transpeptidation, these protons were replaced with deuterium. Acivicin (AT-125), a potent and selective inhibitor of GGT, was used to abolish the formation of the glutamate conjugates of DPC 423 in vitro and in vivo. This provided further evidence of the role of GGT in forming the glutamate conjugates of benzylamines. This study demonstrated conclusively that GGT was responsible for mediating the transfer of glutamic acid from GSH to the benzylamine moiety of a series of structurally related compounds.

Synthesis and biological activities of potential metabolites of the non-nucleoside reverse transcriptase inhibitor efavirenz.

Studies on the biotransformation of the clinically important non-nucleoside reverse transcriptase inhibitor efavirenz have shown that oxidation and secondary conjugation are important components of the processing of this molecule in vivo. We have synthesized metabolites of efavirenz to confirm their structure and to evaluate their activity as antivirals.

The species-dependent metabolism of efavirenz produces a nephrotoxic glutathione conjugate in rats.

Efavirenz, a potent nonnucleoside reverse transcriptase inhibitor widely prescribed for the treatment of HIV infection, produces renal tubular epithelial cell necrosis in rats but not in cynomolgus monkeys or humans. This species selectivity in nephrotoxicity could result from differences in the production or processing of reactive metabolites, or both. A detailed comparison of the metabolites produced by rats, monkeys, and humans revealed that rats produce a unique glutathione adduct. The mechanism of formation and role of this glutathione adduct in the renal toxicity were investigated using both chemical and biochemical probes. Efavirenz was labeled at the methine position on the cyclopropyl ring with the stable isotope deuterium, effectively reducing the formation of the cyclopropanol metabolite, an obligate precursor to the glutathione adduct. This substitution markedly reduced both the incidence and severity of nephrotoxicity as measured histologically. Further processing of this glutathione adduct was also important in producing the lesion and was demonstrated by inhibiting gamma-glutamyltranspeptidase with acivicin pretreatment (10 mg/kg, IV) prior to dosing with efavirenz. Again, both the incidence and severity of the nephrotoxicity were reduced, such that four of nine rats given acivicin were without detectable lesions. These studies provide compelling evidence that a species-specific formation of glutathione conjugate(s) from efavirenz is involved in producing nephrotoxicity in rats. Mechanisms are proposed for the formation of reactive metabolites that could be responsible for the renal toxicity observed in rats.

Characterization of novel glutathione adducts of a non-nucleoside reverse transcriptase inhibitor, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), in rats. Possible formation of an oxirene metabolic intermediate from a disubstituted alkyne.

The postulated formation of oxirene-derived metabolites from rats treated with a disubstituted alkyne, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), is described. The reactivity of this postulated oxirene intermediate led to the formation of novel glutathione adducts whose structures were confirmed by LC/MS and by two-dimensional NMR experiments. These metabolites were either excreted in rat bile or degraded to mercapturic acid conjugates and eliminated in urine. To demonstrate the oxidation of the triple bond, an analogue of DPC 961 was synthesized, whereby the two carbons of the alkyne moiety were replaced with (13)C stable isotope labels. Rats were orally administered [(13)C]DPC 961 and glutathione adducts isolated from bile. The presence of an oxygen atom on one of the (13)C labels of the alkyne was demonstrated unequivocally by NMR experiments. Administration of (14)C-labeled DPC 961 showed that biliary elimination was the major route of excretion with the 8-OH glucuronide conjugate (M1) accounting for greater than 90% of the eliminated radioactivity. On the basis of radiochemical profiling, the glutathione-derived metabolites were minor in comparison to the glucuronide conjugate. Studies with cDNA-expressed rat enzymes, polyclonal antibodies, and chemical inhibitors pointed to the involvement of P450 3A1 and P450 1A2 in the formation of the postulated oxirene intermediate. The proposed mechanism shown in Scheme 1 begins with P450-catalyzed formation of an oxirene, rearrangement to a reactive cyclobutenyl ketone, and a 1,4-Michael addition with endogenous glutathione to produce two isomeric adducts, GS-1 and GS-2. The glutathione adducts were subsequently catabolized via the mercapturic acid pathway to cysteinylglycine, cysteine, and N-acetylcysteine adducts. The transient existence of the alpha,beta-unsaturated cyclobutenyl ketone was demonstrated by incubating the glutathione adduct in the presence of N-acetylcysteine and monitoring the formation of N-acetylcysteine adducts by LC/MS. Epimerization of GS-1 to GS-2 was also observed when N-acetylcysteine was omitted from the incubation.

Disposition of glutathione conjugates in rats by a novel glutamic acid pathway: characterization of unique peptide conjugates by liquid chromatography/mass spectrometry and liquid chromatography/NMR.

With the advent of liquid chromatography/mass spectrometry and liquid chromatography/NMR, it has become easier to characterize metabolites that were once difficult to isolate and identify. These techniques have enabled us to uncover the existence of an alternate pathway for the disposition of glutathione adducts of several structurally diverse compounds. Studies were carried out using acetaminophen as a model compound to investigate the role of the glutamic acid pathway in disposition of the glutathione adducts. Although the mercapturic acid pathway was the major route of degradation of the glutathione adducts, it was found that the conjugation of the glutathione, cysteinylglycine, and cysteine adducts of acetaminophen with the gamma-carboxylic acid of the glutamic acid was both interesting and novel. The coupling of the glutathione adduct and the products from the mercapturic acid pathway with the glutamic acid led to unusual peptide conjugates. The natures of these adducts were confirmed unequivocally by comparisons with synthetic standards. This pathway (addition of glutamic acids) led to larger peptides, in contrast to the mercapturic acid pathway, in which the glutathione adducts are broken down to smaller molecules. The enzyme responsible for the addition of glutamic acid to the different elements of the mercapturic acid pathway is currently unknown. It is postulated that the gamma-carboxylic acid is activated (perhaps by ATP) before enzymatic addition to the alpha-amino group of cysteine or glutamate takes place. The discovery of these peptide conjugates of acetaminophen represents a novel disposition of glutathione adducts of compounds. The formation of such conjugates may represent yet another pathway by which drugs could produce covalent binding via their reactive intermediates.

Mass spectrometric and NMR characterization of metabolites of roxifiban, a potent and selective antagonist of the platelet glycoprotein IIb/IIIa receptor.

1. The methyl ester prodrug roxifiban is an orally active, potent and selective antagonist of the platelet glycoprotein GPIIb/IIIa receptor and is being developed for the prevention and treatment of arterial thrombosis. 2. Roxifiban was rapidly hydrolyzed to the zwitterion XV459 in vivo and by liver slices from the rat, mouse and human and by intestinal cores from dog. XV459 was metabolized to only a small extent in vitro and in vivo. 3. Studies with rat and dog given radiolabelled roxifiban showed limited oral absorption with the majority of the radiolabel being excreted in faeces. After i.v. doses of 14C-roxifiban, most of the radioactivity was recovered in the urine of rat whereas the dog excreted significant amounts of radioactivity in bile and urine. 4. XV459 could be metabolized extrahepatically by dog gut flora to produce an isoxazoline ring-opened metabolite. In vitro hepatic metabolism of XV459 was mainly by hydroxylation at the prochiral and chiral centres of the isoxazoline ring. These hydroxylated metabolites were not detected in the urine and plasma of human volunteers administered roxifiban. 5. Initial LC/MS identification of metabolites was achieved by dosing the rat with an equimolar mixture of d0:d4 roxifiban and detecting isotopic clusters of pseudomolecular ions. Unequivocal characterization of these metabolites was achieved by LC/MS, LC/NMR and high-field NMR techniques using synthetic standards of the metabolites. 6. The synthesis of one hydroxylated metabolite enabled the assignment of the correct stereochemistry of the substituted hydroxyl group on the isoxazoline ring.

Identification and characterization of efavirenz metabolites by liquid chromatography/mass spectrometry and high field NMR: species differences in the metabolism of efavirenz.

Efavirenz (Sustiva, Fig. 1) is a potent and specific inhibitor of HIV-1 reverse transcriptase approved for the treatment of HIV infection. To examine the potential differences in the metabolism among species, liquid chromatography/mass spectrometry profiles of efavirenz metabolites in urine of rats, guinea pigs, hamsters, cynomolgus monkeys, and humans were obtained and compared. The metabolites of efavirenz were isolated, and structures were determined unequivocally by mass spectral and NMR analyses. Efavirenz was metabolized extensively by all the species as evidenced by the excretion of none or trace quantities of parent compound in urine. Significant species differences in the metabolism of efavirenz were observed. The major metabolite excreted in the urine of all species was the O-glucuronide conjugate (M1) of the 8-hydroxylated metabolite. Efavirenz was also metabolized by direct conjugation with glucuronic acid, forming the N-glucuronide (M2) in all five species. The sulfate conjugate of 8-OH efavirenz (M3) was found in the urine of rats and cynomolgus monkeys but not in humans. In addition to the aromatic ring-hydroxylated products, metabolites with a hydroxylated cyclopropane ring (at C14) were also isolated. GSH-related products of efavirenz were identified in rats and guinea pigs. The cysteinylglycine adduct (M10), formed from the GSH adduct (M9), was found in significant quantities in only rat and guinea pig urine and was not detected in other species. In vitro metabolism studies were conducted to show that the GSH adduct was produced from the cyclopropanol intermediate (M11) in the presence of only rat liver and kidney subcellular fractions and was not formed by similar preparations from humans or cynomolgus monkeys. These studies indicated the existence of a specific glutathione-S-transferase in rats capable of metabolizing the cyclopropanol metabolite (M11) to the GSH adduct, M9. The biotransformation pathways of efavirenz in different species were proposed based on some of the in vitro results.

Liquid chromatography/mass spectrometry and high-field nuclear magnetic resonance characterization of novel mixed diconjugates of the non-nucleoside human immunodeficiency virus-1 reverse transcriptase inhibitor, efavirenz.

Efavirenz (Sustiva) is a potent and specific inhibitor of the HIV-1 reverse transcriptase and is approved for the treatment of HIV infection. The metabolism of efavirenz in different species has been described previously. Efavirenz is primarily metabolized in rats to the glucuronide conjugate of 8-OH efavirenz. Electrospray ionization-liquid chromatography/mass spectrometry analyses of bile samples from rats dosed with either efavirenz or with 8-OH efavirenz revealed three polar metabolites, M9, M12, and M13, with pseudomolecular ions [M-H](-) at m/z 733, 602, and 749, respectively. The characteristic mass spectral fragmentation patterns obtained for metabolites M9 and M13 suggested that these were glutathione-sulfate diconjugates, and the presence of a glutathione moiety in metabolite M9 was confirmed by liquid chromatograpy/nuclear magnetic resonance (NMR) analysis of bile extracts. Metabolite M12 was characterized by liquid chromatography/mass spectrometry as a glucuronide-sulfate diconjugate. Unambiguous structures of M9, M12, and M13 were obtained from one-dimensional proton and carbon NMR as well as proton-proton (correlated spectroscopy, two-dimensional shift correlation), proton-carbon heteronuclear multiple quantum correlation, and long-range proton-carbon (heteronuclear multiple bond correlation) correlated two-dimensional NMR analyses of metabolites isolated from rat bile. The mass spectral and NMR analyses of M10, which was isolated from rat urine, suggested a cysteinylglycine-sulfate diconjugate. The isolation of these polar metabolites for further characterization by NMR was aided by mass spectral analyses of HPLC fractions and solid phase extraction extracts during the isolation steps. The complete characterization of these novel diconjugates demonstrates that further phase II metabolism of polar conjugates such as sulfates could take place in vivo.

Application of liquid chromatography/mass spectrometry in accelerating the identification of human liver cytochrome P450 isoforms involved in the metabolism of iloperidone.

Iloperidone, [1-[4-[3-[4-(6-fluoro-1, 2-benzisoxazol-3-yl)-1-piperidinyl]propoxy]-3-methoxyphenyl]eth anone, 1, is currently undergoing clinical trials as a potential antipsychotic agent. The metabolism of iloperidone was studied in human liver microsomes to define the metabolic pathways and to identify the cytochrome P450 (CYP) isoforms responsible for the formation of major iloperidone metabolites. Iloperidone was extensively metabolized in vitro via hydroxylation, reduction and O-demethylation to produce 1-[4-[3-[4-(6-fluoro-1, 2-benzisoxazol-3-yl)-1-piperidinyl]propoxy]-3-methoxyphenyl]-2- hydrox yethanone, 4; 4-[3-[4-(6-fluoro-1, 2-benzisoxazol-3-yl)-1-piperidinyl]propoxy]-3-methoxy-alpha-met hylben zene methanol, 3, and 1-[4-[3-[4-(6-fluoro-1, 2-benzisoxazol3-yl)-1-piperidinyl]propoxy]-3-hydroxyphenyl]etha none, 2, respectively, in decreasing order of abundance. The major in vitro metabolite, 4, present in trace quantities in urine, was postulated to be either eliminated in bile as a conjugate or further metabolized to a phenol, 4-[3-[4-(6-fluoro-1, 2-benzoisoxazol-3-yl)-piperidin1-yl]propoxy]-3-methoxyphenol , 5. The formation of the three major in vitro metabolites 2, 3 and 4 was NADPH dependent. The major circulating and urinary metabolite in humans dosed with 1 was metabolite 3. The mean apparent Km and Vmax for formation of 2 by human liver microsomes was 7.4 +/- 3.0 microM and 0.0343 +/- 0.0134 nmol min-1 mg-1, respectively. The mean apparent Km and Vmax for 3 was 101.2 +/- 34.7 microM and 0.1414 +/- 0.0346 nmol min-1 mg-1, respectively. The mean apparent Km and Vmax for 4 was 39.7 +/- 10.8 microM and 0.1372 +/- 0.056 nmol min-1 mg-1, respectively. The CYP isoenzymes responsible for the formation of metabolites 2, 3 and 4 were determined by using selective chemical inhibitors and by correlation studies. Metabolites 2 and 4 were formed by CYP3A4 and by the polymorphic CYP2D6 respectively. Metabolite 3 is postulated to be produced mainly by a cytosolic enzyme(s), although CYP3A, CYP1A2 and CYP2E1 isozymes were shown to be involved in its formation as well. The power of liquid chromatography/mass spectrometry in greatly accelerating the process of identifying the human liver CYP isoforms involved in the metabolism of iloperidone was demonstrated in this study. Liquid chromatography/mass spectrometry was used in the initial studies to confirm the identities of the metabolites. This was followed by accurate and reliable quantitation of individual metabolites present in biological extracts by operating the mass spectrometer in the selected ion monitoring mode.

Metabolism of an atypical antipsychotic agent, 3-[4-[4-(6-fluorobenzo[b]thien-3-yl)-1-piperazinyl]butyl]-2, 5,5-trimethyl-4-thiazolidinone (HP236).

Metabolism of an atypical antipsychotic agent, 3-[4-[4-(6-fluorobenzo[b]thien-3-yl)-1-piperazinyl]butyl]-2, 5,5-trimethyl-4-thiazolidinone (HP236) by rats is described. HP236 was extensively metabolized both in vitro and in vivo. Metabolites were identified using electrospray interface-LC/MS (ESI-LC/MS) and 1H NMR spectroscopy. Protonated molecular ions, MH+, were observed for all the metabolites of HP236 using ESI-LC/MS. Tandem MS was performed on these quasimolecular ions to provide structural information. Structures of metabolites were confirmed by chromatographic and spectroscopic comparisons to synthetic standards. Metabolic pathways for HP236 both in vivo and in vitro included: a) cleavage of the thiazolidinone ring structure to give N-acetyl-N-[4-[4-(6-fluorobenzo[b] thien-3-yl)-1-piperazinyl]butyl]-2-methyl-propanamide, N-[4-[4-(6-fluorobenzo[b] thien-3-yl)-1-piperazinyl]-butyl]-2-methyl-propanamide, and N-[4-[4-(6-fluorobenzo[b] thien-3-yl)-1-piperazinyl]butyl]-acetamide; b) oxidation of the sulfide to give a mixture of sulfoxide diastereoisomers, 3-[4-[4-(6-fluorobenzo[b]thien-3-yl)-1-piperazinyl]butyl]-1-oxo-2, 5,5-trimethyl-4-thiazolidinone and a sulfone, 1,1-dioxo-3-[4-[4-(6-fluorobenzo[b]thien-3-yl)-1-piperazinyl]butyl ]-2, 5,5-trimethyl-4-thiazolidinone; and c) N-dealkylation at the piperazine ring to produce 3-(4-(1-piperazinyl]butyl]-2, 5,5-trimethyl-4-thiazolidinone and 6-fluoro-3-(1-piperazinyl)benzo[b]thiophene. Metabolites found circulating in the plasma of rats dosed with HP236 were identical to those produced in vitro using rat liver microsomes or S9 fractions; however, LC/MS analysis of rat urine extract showed that the metabolite profile was different than that obtained from plasma or from in vitro extracts. The metabolite resulting from the cleavage of the thiazolidinone ring, N-acetyl-N-[4-[4-(6-fluorobenzo[b] thien-3-yl)-1-piperazinyl]-butyl]-2-methyl-propanamide, was the major circulating metabolite in the plasma of rats dosed with HP236. Results from in vitro studies showed that the metabolite was also produced by incubating the sulfoxides and sulfone analogs of HP236 with rat liver microsomes. A mechanism for the formation of N-acetyl-N-[4-[4-(6-fluorobenzo [b]thien-3-yl)-1-piperazinyl]butyl]-2-methyl-propanamide from HP236 is proposed.

Pharmacological evaluation of novel Alzheimer's disease therapeutics: acetylcholinesterase inhibitors related to galanthamine.

Acetylcholinesterase (AChE) inhibitors from several chemical classes have been tested for the symptomatic treatment of Alzheimer's disease; however, the therapeutic success of these compounds has been limited. Recently, another AChE inhibitor, galanthamine hydrobromide (GAL), has shown increased clinical efficacy and safety. Using biochemical, behavioral and pharmacokinetic analyses, this report compares GAL with two of its analogs, 6-O-acetyl-6-O-demethylgalanthamine hydrochloride (P11012) and 6-O-demethyl-6-O[(adamantan-1-yl)-carbonyl]galanthamine hydrochloride (P11149), for their therapeutic potential. P11012 and P11149 were found to be potent, competitive and selective inhibitors of AChE, demonstrating central cholinergic activity, behavioral efficacy and safety. P11012 and P11149, though pharmacokinetic analyses, were shown to act as pro-drugs, yielding significant levels of 6-O-demethylgalanthamine. In vitro, 6-O-demethylgalanthamine was 10- to 20-fold more potent than GAL as an inhibitor of AChE, and it demonstrated greater selectivity for inhibition of AChE vs. butyrylcholinesterase. Like GAL, both P11012 and P11149 showed central cholinergic activity biochemically, by significantly inhibiting rat brain AChE; physiologically, by causing hypothermia; and behaviorally, by attenuating scopolamine-induced deficits in passive avoidance. In addition, GAL, P11012 and P11149 enhanced step-down passive avoidance, another measure of behavioral efficacy. By comparing efficacious doses with primary overt effects, P11012 and P11149 had better oral therapeutic indices than GAL. Oral pharmacokinetic analyses of GAL, P11012 and P11149 revealed differences. Although P11012 and P11149 exhibited similar area under the curve values, 191149 had slower, lower and more sustained concentration maximum levels. P11012 and GAL rapidly reached their concentration maximums, but GAL, in brain had the highest area under the curve and concentration maximum. Because of its composite profile, including duration of action, oral therapeutic index and pharmacokinetics, P11149 is considered the better therapeutic candidate for the treatment of Alzheimer's disease.

Application of hyphenated LC/NMR and LC/MS techniques in rapid identification of in vitro and in vivo metabolites of iloperidone.

Iloperidone, 1(-)[4(-)[3(-)[4-(6-fluro-1,2-benzisoxazol-3-yl)-1- piperidinyl]propoxy]-3-methoxyphenyl]ethanone, is currently undergoing clinical trials as a potential antipsychotic agent. Iloperidone was found to be extensively metabolized to a number of metabolites by rats, dogs, and humans. LC/MS/MS was used to characterize and identify metabolites of iloperidone present in complex biological mixtures obtained from all three species. Identification of some of the unknown metabolites in rat bile was achieved successfully by combination of LC/NMR and LC/MS with a minimum amount of sample cleanup. The utility of coupling a semipreparative HPLC to LC/MS instrument for further characterization of collected metabolites was demonstrated. It was shown that iloperidone was metabolized by O-dealkylation processes to yield 6-fluoro-3(-)[1-(3-hydroxypropyl)-4-piperidinyl]-1,2-benzisoxazole and 1(-)[4(-)[3(-)[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1- piperidinyl]propoxy]-2-hydroxyphenyl]ethanone. Oxidative N-dealkylation led to the formation of 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole and a secondary metabolite, 3(-)[(4-acetyl-2-methoxy)phenoxy]propionic acid. Iloperidone was reduced to produce 4(-)[3(-)[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl]- propoxy]-3-methoxy-alpha-methylbenzenemethanol as the major metabolite in humans and rats. Hydroxylation of iloperidone produced 1(-)[4(-)[3(-)[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1- piperidinyl]propoxy]-2-hydroxy-5-methoxyphenyl]ethanone and 1(-)[4(-)[3(-)[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl]-3 -methoxyphenyl]propoxy]-2-hydroxyethanone, the later of which was found to be the principal metabolite in dogs. The identities of all these metabolites were established by comparing the LC/MS retention times and mass spectral data with synthetic standards.

Picogram determination of iloperidone in human plasma by solid-phase extraction and by high-performance liquid chromatography-selected-ion monitoring electrospray mass spectrometry.

A very sensitive liquid chromatographic-mass spectrometric (LC-MS) method has been developed to quantitate iloperidone, 1, and its principal metabolite, 4-[3-[4-(6-fluoro-1,2- benzisoxazol-3-yl)-1-piperidinyl]propoxy]-3-methoxy-alpha- methylbenzenemethanol, 2, in human plasma. Iloperidone is currently used in clinical trials for the treatment of psychoses. The analytes were extracted from human plasma using mixed-mode Bond-Elut Certify cartridges and quantitated using selected-ion monitoring electrospray ionization mass spectrometery (SIM-ES-MS). No interference was observed from endogenous compounds following the extraction of plasma samples from six different human subjects. The limit of quantitation for 1 and 2 was 250 pg/ml of plasma. The standard curves were linear over a working range of 250 pg to 20 ng/ml. Absolute recoveries from plasma ranged from 82 to 101% and 73 to 97% for 1 and 2, respectively. Overall intra-day precision ranged from 0 to 9% and 0.9 to 12.5% for 1 and 2, respectively. The method was found to be rugged and very reliable due to the high specificity of SIM-ES-MS. The results obtained from this study confirm the application of solid-phase extraction combined with SIM-ES-MS in quantitating basic drugs in small quantities in biological extracts.

Determination of metoclopramide and two of its metabolites using a sensitive and selective gas chromatographic-mass spectrometric assay.

A modified gas chromatographic-mass spectrometric (GC-MS) assay has been developed to quantitate metoclopramide (MCP) and two of its metabolites [monodeethylated-MCP (mdMCP), dideethylated-MCP (ddMCP)] in the plasma, bile and urine of sheep. The heptafluorobutyryl derivatives of the compounds were formed and quantitated using electron-impact ionization in the selected-ion monitoring mode (MCP, m/z 86, 380; mdMCP, m/z 380 and ddMCP, m/z 380). No interference was observed from endogenous compounds following the extraction of various biological fluids obtained from non-pregnant sheep. Sample preparation has been simplified and the method is more selective and sensitive (2 fold) than our previous assay using electron-capture detection. The limit of quantitation for MCP, mdMCP and ddMCP was 1 ng/ml in plasma, urine and bile, requiring 0.5 ml of sample. This represents 2.5 pg of the analytes at the detector. The standard curves were linear over a working range of 1-40 ng/ml. Absolute recoveries in plasma ranged from 76.5-94.7%, 79.2-96.8%, 80.3-102.2% for MCP, mdMCP and ddMCP, respectively. In urine, recoveries ranged from 56.5-87.8%, 61.5-87.5%, 62.6-90.2% for MCP, mdMCP and ddMCP, respectively. Recoveries in bile ranged from 83.5-100.9%, 78.5-90.5%, 66.9-79.2% for MCP, mdMCP and ddMCP, respectively. Overall intra-day precision ranged from 2.9% for MCP in plasma to 12.6% for mdMCP in bile. Overall inter-day precision ranged from 5.9% for MCP in urine to 14.9% for ddMCP in bile. Bias was the greatest at the 1 ng/ml concentration in all biological fluids ranging from a low of 2.4% for mdMCP in plasma to a high of 11.9% for ddMCP in urine. Applicability of the assay for pharmacokinetic studies of MCP, mdMCP and ddMCP in the plasma and urine of a non-pregnant ewe is demonstrated.

Simultaneous analysis of diphenhydramine and a stable isotope analog (2H10)diphenhydramine using capillary gas chromatography with mass selective detection in biological fluids from chronically instrumented pregnant ewes.

This report describes both the synthesis of a stable isotope analog of the H1 receptor antagonist diphenhydramine (DPHM), and the simultaneous quantitation of DPHM and a deuterated stable isotope analog of DPHM, viz. (2H10)DPHM in biological fluids from the chronically instrumented pregnant ewe. (2H10)DPHM was synthesized and purified, and both its structure and purity were verified. Biological samples were prepared for analysis using liquid-liquid extraction prior to capillary gas chromatography/mass spectrometry. The method employed electron impact ionization with selective ion monitoring of ions with m/z 165 for DPHM and m/z 173 for (2H10)DPHM. The minimal quantifiable concentration of DPHM and (2H10)DPHM from a 1.0 ml sample was 2.0 ng ml-1 in fetal and maternal plasma, fetal tracheal fluid and amniotic fluid. The method was validated from 2.0 ng ml-1 to 200.0 ng ml-1 for both DPHM and (2H10)DPHM in plasma, fetal tracheal fluid and amniotic fluid. Differences in the disposition between DPHM and (2H10)DPHM were not apparent during a control experiment in which both labeled and unlabeled DPHM were administered to a chronically instrumented fetal lamb. This method provides the required sensitivity and selectivity for the simultaneous quantitation of unlabeled and labeled DPHM during pharmacokinetic experiments conducted in near-term pregnant sheep.

Characterization of metabolites of xylazine produced in vivo and in vitro by LC/MS/MS and by GC/MS.

The metabolic fate of xylazine, 2-(2,6-dimethylphenylamino)-5,6-dihydro-4H-1,3-thiazine, in horses is described. The major metabolites identified in the hydrolyzed horse urine were 2-(4'-hydroxy-2',6'-dimethylphenylamino)-5,6-dihydro-4H-1,3-thiazi ne, 2-(3'-hydroxy-2',6'-dimethylphenylamino)-5,6-dihydro-4H-1,3-thiazi ne, N-(2,6-dimethylphenyl)thiourea, and 2-(2',6'-dimethylphenylamino)-4-oxo-5,6-dihydro-1,3-thiazine. These metabolites were also produced by incubating xylazine with rat liver microsomes. The major metabolite produced in vitro by rat liver preparations was found to be the ring opened N-(2,6-dimethylphenyl)thiourea. The identities of these metabolites were confirmed by spectroscopic comparisons with synthetic standards. Phenolic metabolic standards were synthesized efficiently by the use of Fenton's reagent. This reagent was used to monohydroxylate multiply substituted aromatic ring systems. LC/MS/MS, with an atmospheric pressure chemical ionization source, was found to be particularly useful in confirming the presence of phenolic metabolites in hydrolyzed equine urine and microsomal extracts. These phenolic metabolites could not be analyzed by GC/MS even after derivatization with silylating agents. The advantage of LC/MS/MS was that no or little sample preparation of urine or microsomal extract was necessary prior to the analysis. A mechanism is also proposed for the formation of the major metabolite, N-(2,6-dimethylphenyl)thiourea, from xylazine.

Isolation and characterization of carbinolamide and phenolic glucuronide conjugates of (+-)-N-methyl-N-(1-methyl-3,3-diphenylpropyl) formamide and N-formylmethamphetamine by FAB/MS, LC/MS/MS, and NMR.

The metabolic disposition of (+-)-N-methyl-N-(1-methyl-3,3- diphenyl-propyl)formamide, especially with regard to the formation of water soluble glucuronides, is described. The glucuronide conjugates, (+-)-N-hydroxymethyl-N-(1-methyl-3,3-diphenylpropyl)formamide glucuronide, (+-)-N-methyl-N-[1-methyl-3-(4'-hydroxyphenyl)-3-phenylpropyl]formamide glucuronide, and (+-)-N-methyl-N-[1-methyl-3-(4'-hydroxy-3'-methoxyphenyl)-3- phenylpropyl]formamide glucuronide were isolated from the bile of rats dosed with the parent compound. These conjugates were characterized spectroscopically by 1H-NMR, FAB/MS, and LC/MS/MS. Because it is becoming more common to isolate the intact glucuronide conjugates of xenobiotics, we investigated some common mass spectral fragmentation patterns of these conjugates, especially by LC/MS/MS. The fragmentation patterns for each of the conjugates were obtained under MS/MS conditions and compared. Specifically, the fragmentation patterns of phenolic glucuronide and an aliphatic O-glucuronide, in particular a carbinolamide glucuronide, were investigated. The data obtained from these studies was used to predict the nature of glucuronide conjugates obtained from rats dosed with the formamide analog, N-formylmethamphetamine. This is the first spectroscopic characterization of an intact carbinolamide glucuronide conjugate isolated from the bile of rats.