[ALK-positive large B-cell lymphoma with EBV infection or cyclin D1 expression: a clinicopathological analysis of 3 cases].

Objective: To investigate the clinicopathological features and misdiagnosis factors of ALK positive large B-cell lymphoma (ALK+LBCL). Methods: The clinicopathological data of 3 patients with ALK+LBCL in the Department of Pathology, the Affiliated Hospital of Xuzhou Medical University from 2010 to 2021 were collected retrospectively. Immunohistochemistry (IHC) was used for immunophenotyping, in-situ hybridization (ISH) for EBV-encoded RNA (EBER) detection, in-situ fluorescence hybridization (FISH, break-apart probes) for ALK, MYC, and CCND1 translocations. Next-generation sequencing (NGS) was used for the detection of gene fusions and mutations. And clinicopathological features and prognosis of patients were analyzed. Results: Among the 3 ALK+LBCL patients, there were 2 males and 1 female, aged 42, 59, and 39 years, respectively, none of which presented with B symptoms. Case 1 showed systemic lymphadenopathy with elevated serum EBV DNA loading, while cases 2 and 3 presented with extranodal lesions in the nasal and hard palate, respectively. Bone marrow biopsies were performed in cases 1 and 3, and neither showed involvement. Case 1 was at clinical stage Ⅲ while both cases 2 and 3 were at stage Ⅰ, and IPI score ranged 0-1 in all cases. The morphology of these cases was similar. The architecture was effaced by sheets of cohesive large cells growing in extensive infiltration and intra-sinus growth pattern. The neoplastic cells showed immunoblastic or plasmablastic morphology, and large anaplastic cells were easily found. The tumor cells expressed ALK protein cytoplasmically in almost all cells, with ALK gene translocations detected by FISH. Common B-cell and T-cell markers, including CD20, PAX5, CD19, CD2, CD3, CD5, CD7, CD43, CD56, and bcl-2, were negative, while plasmacytic differentiation markers, including CD138, CD38, and MUM1, were positive; CD22, BOB1 and OCT2 were variably expressed. CD10 was strongly expressed only in case 3. All cases were negative for bcl-6 but positive for CD4, perforin, CD30 (partial cells), pSTAT3 (diffusely), and MYC (40%-50%). The Ki-67 index was ranged 60%-70%. MYC translocation was not detected in any case by FISH. In case 1, EBER was strongly positive in>90% of tumor cells. Case 3 was diffusely positive for cyclin D1 but negative for SOX11 expression and CCND1 translocation. All cases harbored ALK fusion genes detected by NGS. In case 1, the fusion partner was TFG, which had not been reported in DLBCL, while in the other 2 cases, ALK fused with the CTCL gene, which was commonly seen in ALK+LBCL. Cases 1 and 3 were treated with ECHOP-based chemotherapy for six cycles and were followed up for 70 and 27 months, respectively, and both achieved complete remission. Conclusions: ALK+LBCL cases with diffuse EBER-positivity reported in this study show TGF as a new fusion partner of ALK in DLBCL, together with cyclin D1 expression. These rare cases are easily confused with EBV positive diffuse large B-cell lymphoma, not otherwise specified (EBV+DLBCL, NOS), cyclin D1 positive diffuse large B-cell lymphoma (cyclin D1+DLBCL) and ALK positive anaplastic large cell lymphoma (ALK+ALCL), resulting in misdiagnosis. Being aware of these rare phenotypes is essential for pathologists to diagnose ALK+LBCL and guide appropriate treatment accurately.

Leptin regulated calcium channels of neuropeptide Y and proopiomelanocortin neurons by activation of different signal pathways.

The fat-derived hormone leptin regulates food intake and body weight in part by modulating the activity of neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (ARC). To investigate the electrophysiological activity of these neurons and their responses to leptin, we recorded whole-cell calcium currents on NPY and POMC neurons in the ARC of rats, which we identified by morphologic features and immunocytochemical identification at the end of recording. Leptin decreased the peak amplitude of high voltage-activated calcium currents (I(HVA)) in the isolated neurons from ARC, which were subsequently shown to be immunoreactive for NPY. The inhibition was prevented by pretreatment with inhibitors of Janus kinase 2 (JAK2) and mitogen-activated protein kinases (MAPK). In contrast, leptin increased the amplitude of I(HVA) in POMC-containing neurons. The stimulations of I(HVA) were inhibited by blockers of JAK2 and phosphatidylino 3-kinase (PI3-k). Both of these effects were counteracted by the L-type calcium channel antagonist nifedipine, suggesting that L-type calcium channels were involved in the regulation induced by leptin. These data indicated that leptin exerted opposite effects on these two classes of neurons. Leptin directly inhibited I(HVA) in NPY neurons via leptin receptor (LEPR) -JAK2-MAPK pathways, whereas evoked I(HVA) in POMC neurons by LEPR-JAK2-PI3-k pathways. These neural pathways and intracellular signaling mechanisms may play key roles in regulating NPY and POMC neuron activity, anorectic action of leptin and, thereby, feeding.

Hepatic metabolism of two alpha-1A-adrenergic receptor antagonists, phthalimide-phenylpiperazine analogs (RWJ-69205 and RWJ-69471), in the rat, dog and human.

The In vitro metabolism of two alpha-1A-adrenergic antagonists, RWJ-69205 and RWJ-69471 (phthalimide-phenylpiperazine analogs), was assessed after 30 and 60 min incubations with rat, dog and human hepatic S9 fractions in the presence of an NADPH-generating system. Unchanged RWJ-69205 (> or = 72% of the sample in all species) plus 3 metabolites from the RWJ-69205 incubations, and unchanged RWJ-69471 (> or = 60% of the sample in all species) and 7 metabolites from the RWJ-69471 incubations, were profiled, quantified, and tentatively identified on the basis of API-MS and MS/MS data. The formation of RWJ-69205 and RWJ-69471 metabolites are via the following five metabolic pathways: 1. phenylhydroxylation, 2. O-dealkylation, 3. oxidative N-dealkylation, 4. N-dephenylation, and 5. dehydration. Pathway 1 formed 2 major/moderate hydroxy-phenyl metabolites of 2 analogs (4-17%) in all species, and pathway 2 produced 2 O-desisopropyl metabolites of 2 analogs in major/moderate (7-16%) in rat and human, and in trace (< 1%) in dog; in conjunction with pathway 1, yielded a minor diphenolic metabolite (< 1-2%) in RWJ-69471. Pathway 3 formed a minor N-dealkylated metabolite, isopropoxyphenyl piperazine (< 1-6%) in all species of 2 analogs. Pathways 4 and 5 produced 2 minor N-desphenyl metabolite and dehydrated metabolite, respectively, in rat and human S9 (< or = 1-2%) in RWJ-69471. Both RWJ-69205 and RWJ-69471 were less extensively metabolized in the dog. However, rat and human appeared to metabolize RWJ-69471 more extensively than RWJ-69205 in this hepatic system.

Metabolism of the alpha-1A-adrenergic receptor antagonist, pyridine-phenylpiperazine analog (RWJ-69597), in rat, dog and human hepatic S9 fractions -API-MS/MS identification of metabolites.

The In vitro metabolism of the alpha-1A-adrenergic antagonist, RWJ-69597, an analog of pyridine-phenylpiperazines, was conducted after incubation with rat, dog and human hepatic S9 fractions in the presence of an NADPH-generating system. Unchanged RWJ-69597 (> or =43% of the sample in all species) plus 9 metabolites were profiled, quantified, and tentatively identified on the basis of API-MS and MS/MS data. The four metabolic pathways for the formation of RWJ-69597 metabolites are: 1. methyl/phenyl/piperazinylhydroxylation, 2. N/Odealkylation, 3. N-dephenylation, and 4. dehydration. Pathway 1 formed 1 major (8-36%) and 3 minor (<1-3%) hydroxylated metabolites. Pathway 2 produced 2 moderate/minor N/O-dealkylated metabolites (<1- < or =11%), and in conjunction with pathway 1, formed 1 minor diol metabolites (< or =2%). Pathways 3 and 4 generated 2 minor metabolites, N-desphenyl RWJ-69597 (< or =4%) and dehydrated RWJ-69597 (< or =2%), respectively. RWJ-69597 is more extensively metabolized in the rat than the dog or the human in this hepatic system.

Metabolism of the new alpha-1A-adrenergic receptor antagonist, phthalimide-phenylpiperazine analog (RWJ-69442), in rat, dog and human hepatic S9 fractions, and in rats.

The in vitro and in vivo metabolism of RWJ-69442, an alpha-1A-adrenergic receptor antagonist, was investigated after incubation with rat, dog, and human hepatic S9 fractions in the presence of NADPH-generating system, and a single oral/iv dose administration to rats (oral: 100 mg/kg; iv: 10 mg/kg). Unchanged RWJ-69442 (> or =30% of the sample in vitro; < or =47% of the sample in vivo) plus 14 metabolites were profiled, quantified and tentatively identified on the basis of API-MS and MS/MS data. The metabolic pathways for RWJ-69442 are proposed via the 4 steps: 1. phenyl/piperazinylhydroxylation, 2. N/O-dealkylation, 3. N-dephenylation, and 4. dehydration. Pathway 1 formed OH-phenyl-RWJ-69442 (M1, 4-32% in vitro & in vivo), and diOH-RWJ-69442 (M4, <1-4% in vitro & in vivo). Pathway 2 generated O-desisopropyl-RWJ-69442 (M2, <1-21% in vitro & in vivo), N-desmethyl-RWJ-69442 (M3, 2-3% in vitro & in vivo), N-desmethyl-M2 (M6, 1-8% in vitro & in vivo), and N-dealkylated RWJ-69442 (M9, < or =1-17% in vitro & in vivo), and in conjunction with pathway 1 produced 6 minor to major oxidized metabolites, OH-M2 (M5, 1-2% in vitro), OH-M3 (M11, 4-6% in vivo), OH-M9 (M10, <1-34% in vitro & in vivo), O-desisopropyl-M9 (M12, 3-21% in vivo), O-desisopropyl-M10 (M13,2-12% in vivo), and dehydro-M13 (M14, 25% in vivo). Pathways 3 and 4 formed 2 minor metabolites, N-desphenyl-RWJ-69442 (M7, <1-12% in vitro & in vivo) and dehydrated-RWJ-69442 (M8, <1-2% in vitro), respectively. RWJ-69442 is extensively metabolized in vitro in the rat and human (except dog), and in vivo in the rat.

Metabolism and excretion of the antiepileptic/antimigraine drug, Topiramate in animals and humans.

The metabolism and excretion of 2,3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate (TOPAMAX, topiramate, TPM) have been investigated in animals and humans. Radiolabeled [14C] TPM was orally administered to mice, rats, rabbits, dogs and humans. Plasma, urine and fecal samples were collected and analyzed. TPM and a total of 12 metabolites were isolated and identified in these samples. Metabolites were formed by hydroxylation at the 7- or 8-methyl of an isopropylidene of TPM followed by rearrangement, hydroxylation at the 10-methyl of the other isopropylidene, hydrolysis at the 2,3-O-isopropylidene, hydrolysis at the 4,5-O-isopropylidene, cleavage at the sulfamate group, glucuronide conjugation and sulfate conjugation. A large percentage of unchanged TPM was recovered in animal and human urine. The most dominant metabolite of TPM in mice, male rats, rabbits and dogs appeared to be formed by the hydrolysis of the 2,3-O-isopropylidene group.

Recent advances in biotransformation of CNS and cardiovascular agents.

Compound biotransformation is a very important research area for drug discovery and development. In this review, publications from the metabolism studies of ten compounds, seven CNS and three cardiovascular agents, from the Johnson & Johnson Corp. were reviewed. The seven CNS compounds are: three antipsychotic agents, mazapertine (arypiperazine analog), RWJ-46344 (arypiperidine analog) and risperidone (aryisoxazole-piperidine analog), one antidepressant, etoperidone (arypiperazine analog), one anxiolytic agent, fenobam (aryimidazole urea analog), one muscle relaxant, xilobam (pyrrolidinylidene urea analog), and one antiepileptic agent, topiramate (fructopyranose sulfamate analog). The three cardiovascular agents are: two arylalkylamine calcium channel blockers, bepridil and RWJ-26240, and one thioindolaminidine antianginal agent, RWJ-34130. Other antipsychotic and antidepressant agents with similar analogs (ziprasidone, trazodone and nefazodone) as well as other similar analogs of calcium channel blockers (verapamil) are discussed. In this article, excretion and metabolism (in vitro, in vivo) of compounds are reviewed from the CNS agents to the cardiovascular agents, including structures of parent compounds, their metabolites, metabolic pathways, and methods for the isolation, profiling, quantification and structural identification of unchanged compounds and metabolites. Pharmacological activities of parent compounds and their metabolites are also briefly discussed.

In vitro and in vivo metabolism of the antianxiolytic agent fenobam in the rat.

Fenobam [(Fn); N-(3-chlorophenyl)-N-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea] sulfate is a novel agent with potent anxiolytic activity in rats. [14C]Fn sulfate was administered as an oral solution (250 mg/kg) to male Wistar rats, and 52% of the administered dose was excreted in urine (0-5 days). In vitro metabolism of Fn was studied by incubating [14C]Fn with rat hepatic 9000 x g supernatant preparations. Unchanged Fn and a total of six metabolites were isolated, quantified, and identified from the urine and liver 9000 x g supernatant samples by column chromatography; TLC; UV, IR, and NMR spectroscopy; MS; and comparison with synthetic samples. Four metabolic pathways for Fn are proposed: (1) hydroxylation at the phenyl ring to form 4-hydroxyphenyl-Fn, a major pathway in vivo (12% of the sample radioactivity) but a minor pathway in vitro (4% of the sample radioactivity); (2) hydroxylation at the creatinine ring to form 5-hydroxy-Fn (19%) of the sample radioactivity), a dominant pathway in vitro but not in vivo; (3) oxidative cleavage at the creatinine ring (loss of a ketene unit), a minor pathway for Fn but an important pathway for 4-hydroxyphenyl-Fn in vivo; and (4) N-demethylation, a minor pathway for Fn in vivo.

Synthesis of hydroxylated derivatives of topiramate, a novel antiepileptic drug based on D-fructose: investigation of oxidative metabolites.

To corroborate the structures of two monohydroxylated metabolites of topiramate (1), we synthesized four monosaccharide derivatives from D-fructose: 4,5-O-[(1R)- and 4,5-O-[(1S)-1-hydroxymethylethylidene]-2,3-O-isopropylidene-beta-D -fructopyranose sulfamates (2a and 2b); 2,3-O-[(1R)- and 2,3-O-[(1R)-1-hydroxymethylethylidene]-4,5-O-isopropylidene-beta-D -fructopyranose sulfamates (3a and 3b). The route to 2a and 2b was brief and straightforward, while that to 3a and 3b was more involved. In the latter case, the D-fructose bis-acetal 10 was benzylated and converted to a monoacetal dibenzoate (14) (50% yield), which was then transacetalized to give a mixture of 4,5-dibenzoyl-2,3-O-[(1R)- and 4,5-dibenzoyl-2,3-O-[(1S)-1-benzyloxymethylethylidene]- beta-D-fructopyranose (16a and 16b) (22%). The individual diastereomers were separated and processed via ester saponification, acetonation, sulfamoylation, and hydrogenolysis into 3a (36%) and 3b (27%). Structure 2b was confirmed for one oxidative metabolite, but the other metabolite was found not to correspond with either 2a, 3a, or 3b. On the basis of CI-MS and 1H NMR data, a (2-hydroxy-1,4-dioxano)pyran structure, 4, is proposed for this unidentified metabolite.

Metabolic fate of the hypoglycemic agent pirogliride in laboratory animals and humans.

Metabolism of the hypoglycemic agent, pirogliride, was investigated in the rat, dog monkey and human. Unchanged pirogliride plus six metabolites were isolated and identified using solvent extraction, HPLC and CI and EI-MS from urine and fecal samples. Pirogliride was metabolized in man to a small extent by oxidation of the 4-position of the phenyl ring. The monkey metabolized pirogliride mainly by oxidation of the pyrrolidine rings, while oxidation of the phenyl ring was the minor pathway. In contrast to the monkey, the rat metabolized pirogliride primarily by oxidation of the phenyl ring. The dog showed a balance of oxidation between the phenyl and pyrrolidine rings.

Biotransformation of linogliride, a hypoglycemic agent in laboratory animals and humans.

Following oral administration of linogliride, a hypoglycemic agent, to rat (50 mg kg-1), dog (30 mg kg-1), and man (100 mg per subject), plasma, urine, and fecal extract sample pools were obtained. Nine metabolites plus unchanged linogliride were isolated and identified. The number of metabolites identified were: rat (5), dog (9), and man (1). In each species, more than 78% of the administered dose was recovered in the urine pools. Identified metabolites were estimated to account for > 82% of the total amounts of drug-related sample in urine pools and > 50% in plasma and fecal extract pools. Formation of linogliride metabolites in the three species can be described by four proposed pathways: pyrrolidine hydroxylation, aromatic hydroxylation, morpholine hydroxylation, and imino-bond cleavage. Comparison of the proposed metabolic pathways among species reveals a similarity between rat and dog. In these two species, pyrrolidine hydroxylation was quantitatively the most important pathway, with 5-hydroxylinogliride and dominant hypoglycemic active metabolite in all sample pools. Further oxidation of 5-hydroxylinogliride resulted in the formation of five minor metabolites. The other three pathways appeared to be quantitatively unimportant. Metabolism of linogliride in man occurred to a very limited extent. More than 90% of the total linogliride-related material in plasma was the unchanged drug. Greater than 76% of the administered dose was excreted unchanged in the urine. Only 5-hydroxylinogliride was identified in minor amounts in human samples.

Characterization and identification of the metabolites of fenoctimine using in vitro drug metabolizing systems.

Fenoctimine sulphate (4-(diphenylmethyl)-1-[(octylimino)methyl]piperidine sulphate) and one of its metabolites, 1-formyl-4-(diphenylmethyl) piperidine (RWJ-34321), were incubated with a rat liver post-mitochondrial supernatant preparation and an NADPH generating system. The metabolites, 7-hydroxyoctyl fenoctimine and 7-oxoocytl fenoctimine were identified as in vitro oxidative metabolites of fenoctimine on the basis of mass spectrometry and thin layer chromatography in comparison to authentic samples. RWJ-34321, a third metabolite, was confirmed as a hydrolyzed product of fenoctimine on the same basis. In separate incubations with RWJ-34321, one metabolite (4-(diphenylmethyl)piperidine), was identified as an in vitro metabolite of RWJ-34321 by mass spectrometry and thin layer chromatography. Thus, the in vitro metabolism of fenoctimine by rat liver homogenates resulted in the oxidation of the aliphatic chain at the seven carbon, initially to an alcohol and then to a ketone. The metabolism of RWJ-34321 resulted in decarbonylation of the formyl carbon.

Evaluation of the excretion and metabolism of the new analgesic agent RWJ-22757 in male and female CR Wistar rats.

The excretion and metabolism of (+/-)-trans-3-(2-bromophenyl)octahydroindolizine hydrochloride (RWJ-22757) have been investigated in male and female CR Wistar rats. Radiolabeled [14C] RWJ-22757 was administered orally to each of the rats as a single 60 mg/kg suspension dose. Plasma (0-48 h), urine (0-168 h) and fecal (0-168 h) samples were collected and analyzed. There were no significant gender differences observed in the data. The estimated elimination half-life of the total radioactivity from plasma was 19 h while the estimated elimination half-life of RWJ-22757 was 15 h. Recoveries of total radioactivity in urine and feces were 58.4+/-5.8 and 42.4+/-6.3%, respectively. RWJ-22757 and a total of 11 metabolites were isolated in rat plasma, urine, and fecal extracts. The structures of four of these metabolites were tentatively identified. Unchanged RWJ-22757 accounted for < 4% of the dose in plasma and urine and 28% in feces; thus, indicating the drug was extensively metabolized and either not absorbed well or biliary excreted. Identified metabolites accounted for > 80% of the total radioactivity contained in the samples. The following pathways were used to describe the formation of the metabolites identified in rats: octahydroindolizine ring oxidation, phenyl hydroxylation, octahydroindolizine ring oxidation followed by ring opening to a carboxylic acid function and octahydroindolizine ring oxidation followed by ring opening and N-methylation.

In vitro biotransformation of the new antipsychotic agent, RWJ-46344 in rat hepatic S9 fraction: API-MS/MS/MS identification of metabolites.

The in vitro biotransformation of the antipsychotic agent, RWJ-46344 was studied after incubation with rat hepatic S9 fraction in the presence of an NADPH-generating system. Unchanged RWJ-46344 (approximately 37% of the sample) plus 12 metabolites were profiled, quantified, and tentatively identified on the basis of API (ionspray)-MS/MS/MS data. The proposed metabolic pathways for RWJ-46344 are proposed, and the six metabolic pathways are 1, O-dealkylation; 2, piperidinyl oxidation; 3, N-debenzylation; 4, phenyl hydroxylation; 5, dehydration; and 6, reduction. Pathways 1 to 3 formed O-desisopropyl RWJ-46344 (M3, approximately 13% of the sample) and its hydroxy-metabolite (M5, approximately 8%), hydroxypiperidinyl RWJ-46344 (M1, approximately 5%) and a phenylpiperidinyl metabolite (M8, approximately 24%) as major and moderate metabolites. Eight minor metabolites (each < 2%) were formed via a combination of six steps. RWJ-46344 is metabolized substantially by this rat hepatic system.

In vitro metabolic products of RWJ-34130, an antiarrythmic agent, in rat liver preparations.

The in vitro metabolism of RWJ-34130, an antiarrhythmic agent, was conducted using rat hepatic 9000 x g supernatant (S9) and microsomes in an NADPH-generating system, and the rat liver perfusion. The 100 and 20 microg ml(-1) concentrations of RWJ-34130 aqueous solution were used for microsomal incubation and liver perfusion, respectively. Unchanged RWJ-34130 (approximately 77-78% of the sample in both S9 and microsomes) plus a major metabolite, RWJ-34130 sulfoxide (20% of the sample in both S9 and microsomes) were profiled, isolated and identified from both hepatic S9 and microsomal incubates (60 min) using HPLC and mass spectrometry (MS), and by comparison to a synthetic RWJ-34130 sulfoxide, which was synthesized by reacting RWJ-34130 with MCPBA (meta-chloroperoxy benzoic acid). No unchanged RWJ-34130 was detected in the 3 h liver perfusate, however, 1-phenyl-2-oxo-pyrrolidine was profiled, isolated and identified as a major hydrolyzed metabolite of liver perfusate. RWJ-34130 is not extensively metabolized in vitro in rat hepatic S9 and microsomes. All HPLC metabolic profiles of hepatic S9 and microsomal samples (30 min, 60 min) were qualitatively and nearly quantitatively identical.

Human hepatic metabolism of the anxiolytic agent, RWJ-51521--API-MS/MS identification of metabolites.

The In vitro metabolism of the anxiolytic agent, RWJ-51521 was conducted after incubation with human hepatic S9 fraction in the presence of an NADPH-generating system. Unchanged RWJ-51521 (30% of the sample) and a total of 11 metabolites were profiled, quantified, and tentatively identified on the basis of API (ionspray)-MS/MS data. The 4 proposed metabolic pathways for RWJ-51521 are: (1) N/O-dealkylation, (2) phenylhydroxylation, (3) pyrido-oxidation, and (4) dehydration. Pathway 1 formed 2 major and 3 minor N/O-desalkyl metabolites (M1 & M3, 50%) and in conjunction with pathway 4, formed 2 moderate dehydrated metabolites (M4 & M5, 14%). Pathways 2 and 3 alone, and in conjunction with pathway 4, produced 4 minor metabolites (each < or =2%). RWJ-51521 is extensively metabolized in human hepatic S9 fraction.

Metabolism of the new nonbenzodiazepine anxiolytic agent, RWJ-51204, in mouse, rat, dog, monkey and human hepatic S9 fractions, and in rats, dogs and humans.

The in vitro and in vivo metabolism of the nonbenzodiazepine anxiolytic agent, RWJ-51204 was investigated after incubation with mice, rat, dog, monkey, and human hepatic S9 fractions in the presence of NADPH-generating system, and a single oral dose administration to rats (100 mg/kg), dogs (5 mg/kg), and humans (2.5 mg/subject). Plasma and red blood cells (2 h, rat) and urine samples (0-24 h, rat, dog and human) were obtained postdose. Unchanged RWJ-51204 (39-93% of the sample in vitro; < or =5% of the sample in vivo) plus 14 metabolites were profiled, quantified and tentatively identified on the basis of API-MS and MS/MS data, and by comparison of synthetic samples. The in vitro and in vivo metabolic pathways for RWJ-51204 are proposed, and the metabolite formations are via the following five pathways: 1. phenyl oxidation, 2. pyrido-oxidation, 3. N-deethoxymethylation, 4. dehydration, and 5. glucuronidation. Pathway 1 formed 4-hydroxy-2-fluoro-phenyl-RWJ-51204 (M1, 7-24% in vitro; 5-60% in vivo) in major amounts, OH-benzimidazole-RWJ-51204 (M2, 5-8% in vitro and in vivo) and diOH-phenyl-RWJ-51204 (< or =5-16% in vitro and in vivo); in conjunction with pathway 5 produced M1 glucuronide (60% in rat & dog; 17% in human), M2 glucuronide (16% in human). Pathways 2-4 formed minor/trace oxidized, and dehydrated metabolites. RWJ-51204 is extensively metabolized in vitro (except dog) and in vivo in rats, dogs and humans.

In vitro metabolism of the analgesic agent, RWJ-37874, in rat and human.

RWJ-37874, an analogue of aroyl(aminoacyl)pyrrole, is a new analgesic agent. The in vitro metabolism of RWJ-37874 was conducted using rat and human hepatic S9 in the presence of an NADPH generating system, and API-ionspray-MS and MS/MS techniques for metabolite profiling and identification. Unchanged RWJ-37874 (66 & 86% of the sample in rat & human, respectively) plus four metabolites were profiled and tentatively identified on the basis of MS data. RWJ-37874 metabolites were formed via the following two metabolic pathways: 1. oxidative N-deethylation, and 2. pyrrole-oxidation. Pathway 1 produced a mayor and a minor metabolites, N-desethyl-RWJ-37874 (M1; 34% in rat; 13% in human) and N,N-didesethyl-RWJ-37874 (M3; <0.5% in both species), respectively. Pathway 2 formed hydroxypyrrole-RWJ-37874 (M2; <0.5% in all species), and in conjunction with step 1, formed hydroxy-M1 (M4; <0.5% in rat). RWJ-37874 is substantially metabolized in rat and human hepatic S9 fractions. However, rat appears to metabolize RWJ-37874 more extensively than human via N-dealkylation forming N-desethyl-RWJ-37874 as a major metabolite.

In vitro biotransformation of the analgesic agent, RWJ-51784, in rat, dog and human.

RWJ-51784, an analogue of phenyl isoindoles, is a new analgesic agent. The in vitro metabolism of RWJ-51784 was conducted using rat, dog and human hepatic S9 in the presence of an NADPH generating system, and API-ionspray-MS and MS/MS techniques for the metabolite profiling and identification. Unchanged RWJ-51784 (82, 80 & 86% of the sample in rat, dog & human, respectively) plus 6 metabolites were profiled and tentatively identified on the basis of MS data. RWJ-51784 metabolites were formed via the following 3 metabolic pathways: 1. N-demethylation, 2. phenylhydroxylation, and 3. isoindole-oxidation. Pathway 1 produced a moderate or minor metabolite, N-desmethyl-RWJ-51784 (M1; 6% in rat; 5% in dog, 2% in human). Pathway 2 formed 4-hydroxyphenyl-RWJ-51784 (M2; 3-6% in all species). Step 3 formed 2 isoindole-oxidized metaboliotes, OH-indole (M3; 7-8% in all species) and oxo-indole (M4; <1% in all species)-RWJ-51784, and in conjunction with pathway 2 produced 2 trace metabolites, OH-phenyl-OH-isoindole (M5) and OH-phenyl-oxo-isoindole (M6) metabolites. RWJ-51784 is not extensively metabolized in rat, dog and human hepatic S9 fractions.

Hepatic metabolism of the new antipsychotic agent, mazapertine, in rat--API-MS/MS identification of metabolites.

The In vitro biotransformation of the antipsychotic agent, mazapertine was studied after incubation with rat hepatic S9 fraction in the presence of an NADPH-generating system. Unchanged mazapertine (42% of the sample) plus 12 metabolites were profiled, quantified, and tentatively identified on the basis of API (ionspray)-MS/MS data. The proposed metabolic pathways for mazapertine are proposed, and the 6 metabolic pathways are: (1) phenylhydroxylation, (2) piperidyl oxidation, (3) O-dealkylation, (4) N-dephenylation, (5) oxidative N-debenzylation, and (6) dehydration. Pathways 1 to 3 formed 4-OH-phenyl (M1, 10%) and 4-OH-piperidyl (M2, 9%)-mazapertine, O-desisopropyl mazapertine (M3, 17%), and N-desbenzoylpiperidine-mazapertine (M8, 14%) as 4 major metabolites. Mazapertine is extensively metabolized in rat hepatic S9 fraction.