[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.

The novel, orally active, delta opioid RWJ-394674 is biotransformed to the potent mu opioid RWJ-413216.

Although the mu opioid receptor is the primary target of marketed opioid analgesics, several studies suggest the advantageous effect of combinations of mu and delta opioids. The novel compound RWJ-394674 [N,N-diethyl-4-[(8-phenethyl-8-azabicyclo]3.2.1]oct-3-ylidene)-phenylmethyl]-benzamide]; bound with high affinity to the delta opioid receptor (0.2 nM) and with weaker affinity to the mu opioid receptor (72 nM). 5'-O-(3-[(35)S]-thio)triphosphate binding assay demonstrated its delta agonist function. Surprisingly given this pharmacologic profile, RWJ-394674 exhibited potent oral antinociception (ED(50) = 10.5 micromol/kg or 5 mg/kg) in the mouse hot-plate (48 degrees C) test and produced a moderate Straub tail. Antagonist studies in the more stringent 55 degrees C hot-plate test demonstrated the antinociception produced by RWJ-394674 to be sensitive to the nonselective opioid antagonist naloxone as well as to the delta- and mu-selective antagonists, naltrindole and beta-funaltrexamine, respectively. In vitro studies demonstrated that RWJ-394674 was metabolized by hepatic microsomes to its N-desethyl analog, RWJ-413216 [N-ethyl-4-[(8-phenethyl-8-azabicyclo[3.2.1]oct-3-ylidene)-phenylmethyl]-benzamide], which, in contrast to RWJ-394674, had a high affinity for the mu rather than the delta opioid receptor and was an agonist at both. Pharmacokinetic studies in the rat revealed that oral administration of RWJ-394674 rapidly gave rise to detectable plasma levels of RWJ-413216, which reached levels equivalent to those of RWJ-394674 by 1 h. RWJ-413216 itself demonstrated a potent oral antinociceptive effect. Thus, RWJ-394674 is a delta opioid receptor agonist that appears to augment its antinociceptive effect through biotransformation to a novel mu opioid receptor-selective agonist.

Metabolism of two analgesic agents, tramadol-n-oxide and tramadol, in specific pathogen-free and axenic mice.

The in vivo metabolism of both tramadol-N-oxide (TNO) and tramadol was investigated in urine pools obtained from 0-24 h after a single 300 mg kg-1 oral dose administration of each compound to specific pathogen-free and axenic mice. Unchanged TNO (< or =42% of the initial drug sample), tramadol, and 23 metabolites from TNO-treated mice and unchanged tramadol (< or =15% of the sample) plus 20 metabolites from tramadol-treated mice were profiled, quantified and tentatively identified on the basis of atmospheric pressure ionization mass spectrometry (API-MS) and tandem mass spectrometry (MS/MS) data. Of the tramadol metabolites, five (M1-5) have been previously identified in mice. Of the tramadol and TNO metabolites, six (M18-23) are new metabolites. The tramadol and TNO metabolites were formed via the following seven metabolic pathways: N-oxide reduction (TNO), O/N-demethylation, cyclohexyloxidation, oxidative N-dealkylation, dehydration (TNO), N-oxidation (tramadol), and glucuronidation. Pathways 1-3 appear to be predominant steps forming four major O/N-desmethyl and hydroxycyclohexyl metabolites, and in conjunction with pathway 7, formed six minor glucuronides. Both tramadol-N-oxide and tramadol are extensively metabolized in mice, and no significant qualitative or quantitative differences in metabolism were observed between specific pathogen-free and axenic mice with the exception of a greater amount of unchanged TNO in axenic mice than in specific pathogen-free mice, more M2 in specific pathogen-free mice than in axenic mice in the TNO-dosed mice, and visa versa for M2 of tramadol-dosed mice.

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 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.

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 of the antineoplastic and immunosuppressive drug 2-CdA (Leustatin) in animals and humans.

1. The in vivo metabolism of the antineoplastic and immunosuppressive drug 2-CdA (Leustatin) was investigated in mice, monkeys and humans after a single subcutaneous dose of cladribine 60 mg kg(-1) to eight male and eight female mice and 10 mg kg(-1) to one male and one female monkey, and an intravenous infusion dose of cladribine 22-45 mg(-1) per subject to 12 male patients. 2. Plasma (1 h), red blood cells (1 h) and faecal samples (0-24 h) were obtained from mice and monkeys, and urine samples (0-24 h) were obtained from these species and humans. 3. Unchanged cladribine (urine: 47% of the sample in human; 60% of the sample in mouse; 73% of the sample in monkey) and 10 metabolites, consisting of four phase I metabolites (M1-3, M7) and six phase II metabolites -- five glucuronides (M4, M6, M8-10) and one sulfate (M5) -- were profiled, characterized and tentatively identified in plasma, red blood cells, and faecal and urine samples on the basis of API ionspray-mass spectrometry (MS) and MS/MS data. 4. Metabolites were formed via the following three metabolic pathways: oxidative cleavage at the adenosine and deoxyribose linkage (A); oxidation at adenosine/deoxyribose (B); and conjugation (C). 5. Pathways A and B appear to be major steps, forming four oxidative/cleavage metabolites (M1-3, M7) (each 3-20% of the sample). 6. Pathway C along or in conjunction with pathways A and B produced cladribine glucuronide, cladribine sulfate and four glucuronides of oxidative/cleavage metabolites in minor/trace quantities (each < or = 5% of the sample). 7. In addition, the in vitro metabolism of cladribine was conducted using rat and human liver microsomal fractions in the presence of an beta-nicotinamide adenine dinucleotide phosphate-generating system. Unchanged cladribine (> or = 90% of the sample) plus three minor metabolites, M1-3 (each < 8% of the sample), were profiled and tentatively identified by thin-layer chromatography and MS data. 8. Cladribine is not extensively metabolized in vitro and in vivo in all species. However, humans appear to metabolize cladribine to a greater extent than other animals.

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.

In vitro identification of metabolic pathways and cytochrome P450 enzymes involved in the metabolism of etoperidone.

1. In vitro studies have been carried out to investigate the metabolic pathways and identify the hepatic cytochrome P450 (CYP) enzymes involved in etoperidone (Et) metabolism. 2. Ten in vitro metabolites were profiled, quantified and tentatively identified after incubation with human hepatic S9 fractions. Et was metabolized via three metabolic pathways: (A) alkyl hydroxylation to form OH-ethyl-Et (M1); (B) phenyl hydroxylation to form OH-phenyl-Et (M2); and (C) N-dealkylation to form 1-m-chlorophenylpiperazine (mCPP, M8) and triazole propyl aldehyde (M6). Six additional metabolites were formed by further metabolism of M1, M2, M6 and M8. 3. Kinetic studies revealed that all metabolic pathways were monophasic, and the pathway leading to the formation of OH-ethyl-Et was the most efficient at eliminating the drug. On incubation with microsomes expressing individual recombinant CYPs, formation rates of M1-3 and M8 were 10-100-fold greater for CYP3A4 than that for other CYP forms. The formation of these metabolites was markedly inhibited by the CYP3A4-specific inhibitor ketoconazole, whereas other CYP-specific inhibitors did not show significant effects. In addition, the production of M1-3 and M8 was strongly correlated with CYP3A4-mediated testosterone 6beta-hydroxylase activities in 13 different human liver microsome samples. 4. Dealkylation of the major metabolite M1 to form mCPP (M8) was also investigated using microsomes containing recombinant CYP enzymes. The rate of conversion of M1 to mCPP by CYP3A4 was 503.0 +/- 3.1 pmole nmole(-1) min(-1). Metabolism of M1 to M8 by other CYP enzymes was insignificant. In addition, this metabolism in human liver microsomes was extensively inhibited by the CYP3A4 inhibitor ketoconazole, but not by other CYP-specific inhibitors. In addition, conversion of M1 to M8 was highly correlated with CYP3A4-mediated testosterone 6beta-hydroxylase activity. 5. The results strongly suggest that CYP3A4 is the predominant enzyme-metabolizing Et in humans.

In vitro metabolism of the analgesic agent, tramadol-N-oxide, in mouse, rat, and human.

Tramadol-N-oxide (TNO, RWJ-38705) is a new analgesic agent, which is believed to produce its analgesic effect following metabolic conversion to tramadol. In the present study, API ionspray-MS and MS/MS techniques were used to profile the in vitro metabolism of TNO in mouse, rat, and human hepatic S9 fractions in the presence of an NADPH generating system. Unchanged TNO represented 60, 24, and 26% of the sample in mouse, rat, and human, respectively. Tramadol, and seven other metabolites were profiled and tentatively identified on the basis of MS analysis and by comparison to synthetic reference samples. TNO metabolites were formed via four Phase I reactions: (1) N-oxide reduction, (2) O-demethylation, (3) N-demethylation, and (4) cyclohexylhydroxylation. TNO was found to be substantially metabolized in hepatic S9 from all three species. The metabolism of TNO to tramadol via N-oxide reduction was greater in rat and human than in mouse.

Metabolism of the analgesic drug ULTRAM (tramadol hydrochloride) in humans: API-MS and MS/MS characterization of metabolites.

1. Metabolism of the analgesic agent tramadol hydrochloride has been investigated after a single oral administration of tramadol to three male volunteers (100 mg/subject), and a urine pool (4-12h) was obtained. 2. Unchanged tramadol and a total of 23 metabolites, consisting of 11 Phase I metabolites (M1-11) and 12 conjugates (seven glucuronides, five sulphates), were profiled, characterized and tentatively identified in urine on the basis of API ionspray-MS and MS/MS data. 3. Of the metabolites, five (M1-5) had been previously identified. 4. The metabolites were formed via the following six metabolic pathways: (1) O-demethylation, (2) N-demethylation, (3) cyclohexyl oxidation, (4) oxidative N-dealkylation, (5) dehydration and (6) conjugation. 5. Pathways 1-3 appear to be major routes, forming seven O-desmethyl/N-desmethyl and hydroxycyclohexyl metabolites. 6. Pathways 1-3 in conjunction with pathway 6 produced seven glucuronides along with five sulphates. 7. In addition, the in vitro metabolism of tramadol was conducted using a human liver microsomal fraction in the presence of an NADPH-generating system. Unchanged tramadol (82% of the sample) plus eight metabolites (M1, M2, M4-6, tramadol-N-oxide (M31), OH-cyclohexyl-M1 (M32) and dehydrated tramadol-N-oxide), were profiled and tentatively identified on the basis of MS and MS/MS data.

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.

Evaluation of the absorption, excretion, and metabolism of the antihypertensive agent RWJ-26899 in male and female CR Wistar rats and Beagle dogs.

The absorption, excretion and metabolism of N-(2, 6-dichlorophenyl)-beta-[[(1-methylcyclohexyl)methoxylmethyl]-N-(phenylmethyl)-1-pyrrolidineethanamine (RWJ-26899; McN-6497) has been investigated in male and female CR Wistar rats and beagle dogs. Radiolabeled [14C] RWJ-26899 was administered to rats as a single 24 mg/kg suspension dose while the dogs received 15 mg/kg capsules. Plasma (0-36 h; rat and 0-48 h; dog), urine (0-192 h; rat and dog) and fecal (0-192 h; rat and dog) samples were collected and analyzed. There were no significant gender differences observed in the data. The terminal half-life of the total radioactivity for rats from plasma was estimated to be 7.7 +/- 0.6 h while for dogs it was 22.9 +/- 4.4 h. Recoveries of total radioactivity in urine and feces for rats were 8.7 +/- 2.9% and 88.3 +/- 10.4% of the dose, respectively. Recoveries of total radioactivity in urine and feces for dogs were 4.1 +/- 1.4% and 90.0 +/- 4.7% of the dose, respectively. RWJ-26899 and a total of nine metabolites were isolated and tentatively identified in rat urine, and fecal extracts. Unchanged RWJ-26899 accounted for approximately 1% of the dose in rat urine and 8% in rat feces. RWJ-26899 and a total of four metabolites were isolated and identified in dog urine, and fecal extracts. Unchanged RWJ-26899 accounted for approximately 1% of the dose in urine and 63% in feces in dog. Five proposed pathways were used to describe the metabolites found in rats: N-oxidation, oxidative N-debenzylation, pyrrolidinyl ring hydroxylation, phenyl hydroxylation and methyl or cyclohexyl hydroxylation. Two biotransformation pathways in dogs are proposed: N-oxidation and methyl or cyclohexyl ring hydroxylation.

Metabolism of the analgesic drug, tramadol hydrochloride, in rat and dog.

1. Metabolism of the analgesic agent, tramadol hydrochloride, was investigated after a single oral administration of 14C-tramadol to four rats (50)mgkg(-1) and two dogs (20)mg kg(-1). 2. Recovery of total radioactivity in rat and dog urine samples over 24 h was 73 and 65% of the radioactive dose, respectively. 3. Unchanged tramadol and a total of 24 metabolites, consisting of 16 Phase I metabolites and eight conjugates (seven glucuromides, one sulphate), were isolated and tentatively identified, which accounted for > 52% of the dose in urine of both species. 4. Of the metabolites, five (M1-5) were previously identified. 5. The metabolites were formed via the following six metabolic pathways: O-demethylation, N-demethylation, cyclohexyl oxidation, oxidative N-dealkylation, dehydration and conjugation. 6. Pathways 1-3 appear to be major steps, forming seven O-desmethyl/N-desmethyl and hydroxy-cyclohexyl metabolites in major quantities. 7. Pathways 1-3 in conjunction with pathway 6 produced four glucuronides along with four minor conjugates. 8. In addition, the in vitro metabolism of tramadol was conducted using rat hepatic S9 fraction in the presence of an NADPH-generating system. Unchanged tramadol (30% of the sample) plus nine metabolites, M1-7, tramadol-N-oxide (M31) and OH-cyclohexyl-M1 (M32), were profiled and tentatively identified based on MS and MS/MS data.