Metabolism of a dopamine receptor partial agonist in rats, including an unusual N-dearylation reaction

The metabolism of 3,4-dihydro-7-[4-(1-naphthalenyl)-1-piperazinyl]butoxy]-1,8-naphthyridin-2(1H)-one (NPBN) was investigated in rats. Animals were administered 30 mg/kg NPBN that was labeled with both tritium and carbon-14. The mass recovery of drug-related material was 96-98%, with almost all material excreted in feces. Metabolism occurred by oxidation reactions followed by conjugation. The main route of metabolism of NPBN occurred via oxidation of the naphthylene ring, which led to naphthol and dihydrodiol metabolites as well as a relatively novel N-dearylated metabolite in which the naphthylene ring was removed. In vitro investigation in rat liver microsomes also showed a glutathione adduct on the naphthalene and a glutathione adduct of naphthoquinone, which, along with the dihydrodiol metabolite, is consistent with the initial generation of an epoxide. A mechanism is proposed whereby the N-dearylation arises via epoxidation, followed by formation of an exocyclic iminium ion intermediate that is hydrolyzed to yield the N-dearylated metabolite. An additional mechanism involves oxidation of the naphthol metabolite via a radical mechanism, since this metabolite was also shown to undergo N-dearylation.     

Liver and lung microsomal metabolism of the tobacco alkaloid beta-nicotyrine

The in vitro metabolic fate of beta-nicotyrine has been examined in rabbit lung and liver microsomal preparations as part of an effort to characterize the formation of potentially reactive metabolic species that may contribute to the toxic properties of tobacco products. HPLC analysis revealed the formation of an unstable metabolite which displayed HPLC-MS/MS characteristics consistent with the structure 1-methyl-5-(3-pyridyl)-3-pyrrolin-2-one. Attempted synthesis of this pyrrolinone, however, resulted in the isolation of the isomeric 1-methyl-5-(3-pyridyl)-2-pyrrolin-2-one. The HPLC, diode array UV, and mass spectral characteristics of this delta 4,5-isomer proved to be identical with those of the metabolite derived from beta-nicotyrine. Studies in D2O suggest that the 2- and 3-pyrrolinones are in equilibrium in aqueous solution. The metabolite undergoes autoxidation, possibly via radical intermediates, to yield 1-methyl-5-(3-pyridyl)-5-hydroxy-3-pyrrolin-2-one.     

Stereospecific in vivo N-methylation of nicotine in the guinea pig

R-(+)-[3H-N'-CH3]N-methylnicotinium ion has been identified as a urinary metabolite of ip administered R-(+)-[3H-N'-CH3]nicotine in the guinea pig. Under similar conditions, S-(-)-[3H-N'-CH3]nicotine is not converted to the corresponding N-methylated metabolite. R-(+)-N-methylnicotinium salt was isolated from the urine of guinea pigs that had been chronically dosed ip with R-(+)-nicotine. The identification and stereochemical analysis of this metabolite were carried out using analytical and preparative cation exchange high pressure liquid chromatography and chemical reduction followed by GLC-mass spectrometric analysis and 1H NMR spectroscopy. The results show that nicotine is stereospecifically biotransformed into an N-methylated urinary metabolite in the guinea pig.     

Metabolism of BW 1370U87 in rat,dog, and man

BW 1370U87 is a potent, selective inhibitor of rat and human brain MAO-A. The plasma concentrations of BW 1370U87 and its metabolites were determined in rat, dog, and man. After an oral dose, BW 1370U87 undergoes extensive first-pass metabolism in all species studied. The 1-(1,2, dihydroxyethyl) metabolite, BW 1003U88, was a major metabolite in rat, dog, and human plasma. The 1-(1-hydroxyethyl) metabolite, BW 183U88, was a major metabolite in dog plasma, whereas it was present in much lower concentrations in the rat and man. Both BW 1003U88 and BW 183U88 are active MAO-A inhibitors although not as active as the parent compound. The inactive 1-(2-acetic acid) metabolite, BW 1552U88, was a major metabolite in rat plasma but a minor metabolite in the dog. Plasma concentration versus time profiles in both rat and man suggest that the metabolites undergo enterohepatic recycling. Although plasma concentrations of BW 1370U87 were relatively low compared to the metabolites, the concentrations detected in rat and man after a 50 mg/kg or 400 mg oral dose, respectively, exceeded the IC₅₀ value measured in rat and human brain. Furthermore, the time course of MAO-A inhibition appears to follow the plasma concentration versus time profile of BW 1370U87 in the rat. Preliminary experiments in rats indicate that BW 1370U87 and its metabolites distribute into brain and inhibit MAO-A.     

Uptake, metabolism and release of [3H]-adrenaline by human platelets

1. Measurements were made of the uptake, metabolism and release of [(3)H]-adrenaline by human platelets in citrated plasma or in an artificial medium.2. Radioactive adrenaline was not taken up at 0-2 degrees C. At 37 degrees C there was a slow uptake which continued for at least 5 hours.3. About half of the radioactivity in the platelets was intact adrenaline. The other half was an acidic metabolite from which adrenaline was released by acid hydrolysis.4. The immediate uptake of adrenaline was proportional to its concentration in the plasma up to at least 1 x 10(-5)M. Uptake measured after 1 h also increased linearly with concentration up to about 1 x 10(-4)M but less with higher concentrations. The highest concentration ratio was about 12.5. The concentration of metabolite in the platelets increased with the concentration of added adrenaline only up to about 2 x 10(-4)M.6. The immediate uptake of adrenaline was partially inhibited by phentolamine and dihydroergotamine. Measurement of uptake both immediately and after 1 h showed that the inhibition produced was not increased beyond about 50% by these drugs or by (+/-)-propranolol, chlorpromazine or amitriptyline up to 1 x 10(-4)M.7. Formation of the metabolite was inhibited by pyrogallol, 8-hydroxyquinoline, or tropolone. This inhibition was associated with a corresponding increase in the adrenaline accumulated intact. Formation of the metabolite was not inhibited by monoamine oxidase inhibitors.8. Reserpine caused a small decrease in the uptake of adrenaline radioactivity in 1 h and a great increase in the proportion recovered as metabolite.9. Thrombin caused the release from platelets of intact adrenaline but not of the metabolite.10. Platelets of albino patients with spontaneous haemorrhages accumulated adrenaline radioactivity at the normal rate but this radioactivity was wholly accounted for by metabolite and not released by thrombin.11. After taking up adrenaline, platelets resuspended in artificial medium at 37 degrees C slowly released both adrenaline and its metabolite. At the same time, the intracellular adrenaline was slowly metabolized.12. The Result suggest that human platelets take up adrenaline by two processes, one of which is inhibited by both alpha- and beta-adrenoceptor blocking agents ad well as by phenothiazines; and that in the platelets adrenaline is partly stored in organelles from which, like 5-hydroxytryptamine, it can be specifically released.     

Metabolism of a Serotonin-4 Receptor Partial Agonist 4-{4-[4-Tetrahydrofuran-3-yloxy)-Benzo[d]Isoxazol-3-yloxymethyl]-Piperidin-1-ylmethyl}-Tetrahydropyran-4-ol (TBPT): Identification of an Unusual Pharmacologically Active Cyclized Oxazolidine Metabolite in Human

4-{4-[4-Tetrahydrofuran-3-yloxy)-benzo[d]isoxazol-3-yloxymethyl]-piperidin-1-ylmethyl}-tetrahydropyran-4-ol (PF-4995274, TBPT) is a new agent that is a partial agonist of the human serotonin-4 (5-HT4) receptor and is under investigation for neurological disorders. Metabolism of TBPT was examined in vitro in human liver microsomes and human hepatocytes. Metabolites were also identified in the plasma of healthy human subjects in a phase 1 clinical study. Human-derived metabolite profiles were compared with corresponding profiles obtained in laboratory animal species. There were two major routes of metabolism in vitro: N-dealkylation of the methyltetrahydropyran moiety (M1) and hydroxylation at the seven position of the benzisoxazole moiety (M4). These were also observed in human plasma; however, in that matrix, the major metabolite was an unusual cyclized oxazolidine entity (M2). M2 was proposed to be formed via generation of an intermediate 4° iminium ion on the piperidine ring followed by spontaneous cyclization by attack of the β-hydroxyl substituent of the tetrahydropyran ring to form a cyclized oxazolidine product. An authentic standard of the metabolite was generated using a methylene-blue-sensitized photochemical oxidation reaction as well as microbial transformation. Further investigation of this metabolite showed that it also possessed 5-HT4 agonism activity similar to the parent. The metabolite was 150-fold more highly protein bound in human plasma than TBPT, which is consistent with its presence as a major circulating metabolite while being only a minor metabolite in in vitro systems. Overall, this illustrates the importance of understanding the complex dispositional properties of a pharmacologically active metabolite.     

人肝微粒体中尼莫地平及其脱氢代谢物的HPLC测定法及代谢动力学

目的：建立人肝微粒体中尼莫地平及其代谢物的HPLC测定法,并用本法研究尼莫地平在肝微粒体中代谢的动力学。方法：采用HPLC法,色谱柱为Hypersil C₁₈柱,流动相为乙腈—水(62∶38)检测波长UV 238 nm,样品用乙醚—正己烷(1∶1)提取。结果：本法的回收率为94.3%～98.5%,日内和日间RSD为1.45%～9.68%,尼莫地平和其脱氢代谢物的浓度分别在1.31～20.92 μg.mL^-1和122.7～4908.0 ng.mL^-1范围内与峰面积呈良好线性关系。在本实验条件下尼莫地平呈线性消除,而其脱氢代谢物呈线性增加。结论：尼莫地平在人肝微粒体内被迅速代谢,肝微粒体P450酶参与了尼莫地平的脱氢氧化。     

Studies on the biotransformation of 5-vinyl-5-(1-methylbutyl)- and 5-vinyl-5-(1-ethyl-propyl)-barbituric acid (author's transl)]

After taking 5-vinyl-5-(1-methylbutyl)-barbituric acid (vinylbital, Speda) another not yet known metabolite (Sp 1) was isolated from urine and was identified as 5-vinyl-5-(1-methyl-3-carboxy-propyl)-bartiburic acid. Compound Sp 2a is 1-methylhydantoin. Presumably this compound is an artifact from creatinine. We have not yet been able to identify a third mercury(I)-nitrate-positive substance Sp 2b. The supposition that this compound were a metabolite of 5-vinyl-5-(1-ethyl-propyl)-barbituric acid could not be upheld. The three mercury(I)-nitrate-positive substances appearing in the urine after taking 5-vinyl-5-(1-ethyl-propyl)-barbituric acid, behaved in chromatographic analysis completely differently from the unknown compound Sp 2b. The substances were isolated. Two of them are the metabolites 5-vinyl-5-(1-ethyl-2-carboxy-ethyl)-barbituric acid and 5-(1-ethyl-propyl)-barbituric acid. We are not sure whether the third compound identified as 5-hydroxy-5-(1-ethyl-propyl)-barbituric acid, is formed in vivo, as the substance could have developed by oxidation or by the influence of peroxide contained in ether, as well. A product hydroxylized in the side chain, like in vinylbital, was not found in the metabolite urine of 5-vinyl-5-(1-ethyl-propyl)-barbituric acid.     

Metabolism of metyrapone. III. Formation of an alpha-pyridone metabolite by rat hepatic soluble enzymes

Incubation of 2-methyl-1,2-bis(3-pyridyl)-1-propanone (metyrapone) with rat hepatic 140,000 g supernatant fraction and an NADPH-regenerating system leads to the formation of 2-methyl-1,2-bis(3-pyridyl)-1-propanol (metyrapol), and one other metabolite identified as 2-methyl-1-[3-(6-oxopyridyl)]-2-(3-pyridyl)-1-propanone by a combination of various spectrometric techniques (UV, IR, NMR, and mass spectrometry). Incubation of metyrapol under similar conditions did not lead to the formation of any detectable metabolites. Hitherto, no reports have described the in vitro biotransformation of an unquaternized pyridine molecule to an alpha-pyridone metabolite.     

Disposition of pentopril, a new orally active angiotensin-converting enzyme inhibitor, and its active metabolite in rats

The disposition characteristics of pentopril (the ethyl ester) and its active carboxylic acid metabolite (CGS 13934) were determined in conscious rats after separate intravenous administrations of both compounds. The relationship between plasma concentration and pharmacological effect was also evaluated. The extent of apparent bioavailability of the active metabolite was determined after oral administration of pentopril. Pharmacokinetic parameters were calculated from the plasma concentration-time data for both the parent drug and its active metabolite after their separate intravenous administrations using a one-compartment model for the drug and a two-compartment model for the metabolite. The elimination half-life for the drug was approximately 1 min. The elimination half-life for the metabolite was 13 min (SD, +/- 3.5, n = 4) after its direct intravenous administration, but increased to an apparent half-life of 20 min (SD +/- 5, n = 5) when formed in vivo as a metabolite. Comparison of the formation rate of the metabolite and the elimination rate of the parent drug indicated that the parent drug was rapidly and completely hydrolyzed to the acid metabolite as soon as it reached the systemic circulation. No parent drug was detected in plasma after its oral administration. The apparent bioavailability of the acid metabolite was 66% after oral drug administration. A close relation between inhibition of pressor response to angiotensin I (AI) and plasma concentration of the active metabolite was observed when plotted against time after drug or metabolite administration. A Michaelis-Menten function correlated (multiple r2:0.995) well between effect and plasma metabolite concentration with mean concentration for 50% of maximum inhibition, IC50, of 3.6 X 10(-7) M (0.11 microgram/mL).     

Identification and synthesis of O-methylcatechol metabolites of phenobarbital and some N-alkyl derivatives

5-Ethyl-5-(4-hydroxy-3-methoxyphenyl) barbituric acid was identified as a new, minor metabolite of phenobarbital in man. The identity of this O-methylcatechol metabolite was confirmed by an unequivocal chemical synthesis, and by GC-MS studies. Mephobarbital and the 1,3-dimethyl, 1-ethyl, and 1,3-diethyl analogues of phenobarbital yielded the corresponding N-alkylated O-methylcatechol metabolites, all of which were confirmed by synthesis. The N-alkyl barbiturates each gave additionally at least one O-methylcatechol metabolite in which N-dealkylation had occurred. These metabolites accounted for approximately 1-5% of the orally administered dose in man.     

Zur Pharmakokinetik von Lipidsenkern, 6. Mitt.: Ist 2-(4-(2,2-Dichlorcyclopropyl)-phenoxy)-propan ein Metabolit des Lipidsenkers Ciprofibrat?

According to earlier investigations 2-(4-(2,2-dichlorocyclopropyl)-phenoxy)-propane (4) ought to be a metabolite of the hypolipidemic agent ciprofibrate (1). However, 4 could not be detected in plasma or in urine after administration of a dose of 2100 mg 1 during the course of a multiple-dose-study. Therefore, the compound described in literature must be an unknown one and the existence of 4 as a metabolite of 1 is excluded.     

A new metabolite of irinotecan in which formation is mediated by human hepatic cytochrome P-450 3A4.

Irinotecan (CPT-11) is an anticancer prodrug. It is converted by carboxylesterase to yield an active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), which acts as a topoisomerase I inhibitor. Several oxidative metabolites of CPT-11 have been identified in humans, including 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin (APC) and 7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecin (NPC), generated by cytochrome P-450 3A4 (CYP3A4). Other minor metabolites in which metabolic pathways and biologic activities have not been identified also exist. To further investigate the metabolism of CPT-11 in human liver, we analyzed metabolites of CPT-11 in human hepatic microsomes using a high-performance liquid chromatography/mass spectrometry (HPLC/MS) system and detected a new metabolite that was the major one produced in the microsomal system. HPLC-tandem mass spectrometry (HPLC/MS/MS) analysis indicated that this compound was an oxidation product formed by the loss of two hydrogen atoms from the terminal piperidine ring. Kinetic analyses indicated that a single enzyme generated the metabolite, and we have identified this enzyme in two in vitro systems. The formation of the new metabolite was significantly inhibited by SKF525A, ketoconazole, and an anti-CYP3A4 antibody and catalyzed specifically by CYP3A4 expressed in insect microsomes. A significant correlation was observed between the generation of this metabolite and the CYP3A4 content in individual human hepatic microsomes. These findings indicate that this newly detected metabolite is a CYP3A4-generated product that may be produced in hepatic microsomes of patients treated with CPT-11.     

Metabolism of doxophylline by rat liver microsomes

The metabolic transformation of the bronchospasmolytic agent doxophylline (2-(7'-theophyllinemethyl)-1,3-dioxolane) was studied in vitro with phenobarbital-induced rat liver microsomal fraction containing the NADPH-generating system. Doxophylline was poorly metabolized as 95% of the recovered material was parent compound. The major metabolite resulted: 2'-hydroxyethyl ester of theophylline acetic acid. This was an unusual metabolite which possibly arose from dioxolane C2 enzymatic oxydation and subsequent ring opening. Theophylline was a minor metabolite. The per cent ratio of the two metabolites was 4:1; as we did not detected the presence of any compounds formed from N-demethylation, we concluded that doxophylline underwent a regiospecific metabolism on the N7 side chain.     

Sphingosine-1-phosphate: an emerging therapeutic target

Sphingosine-1-phosphate (SPP) is a polar sphingolipid metabolite that has received increasing attention as both an extracellular mediator and an intracellular second messenger. SPP is the ligand of a family of specific cell surface G-protein coupled receptors (GPCR), known as the endothelial differentiation gene-1 (EDG-1) family. These receptors, which include EDG-1, -3, -5, -6 and -8, regulate diverse processes including cell migration, angiogenesis, vascular maturation, heart development, neurite retraction and soma rounding. In addition, abundant evidence indicates that SPP also acts as an intracellular lipid messenger, regulating calcium mobilisation, cell growth and survival. The relative intracellular level of SPP and ceramide, another sphingolipid metabolite associated with cell death and cell growth arrest, is an important factor in determining cell fate. Changes in SPP and ceramide have been implicated in a number of pathological conditions in which apoptosis plays an important role, including cancer and neurodegenerative disorders, as well as in atherosclerosis and allergic responses. This review will examine the biosynthesis, metabolism and potential functions of SPP in diverse diseases in order to illuminate targets for the pharmaceutical and therapeutic manipulation of SPP levels.     

Cross-reactivities of active metabolites in the radioreceptor assay of an antiallergic agent, 1-(2-ethoxyethyl)-2-(hexahydro-4-methyl-1H-1,4-diazepin-1-yl)- 1H-benzimidazole difumarate (KG-2413)

A new antiallergic agent, 1-(2-ethoxyethyl)-2-(hexahydro-4-methyl-1H-1,4-diazepin-1-yl)-1H- benzimidazole difumarate (KG-2413), was orally administered to guinea pigs at a dose of 2 mg/kg. The drug levels in the plasma measured by the radioreceptor assay (RRA) method were significantly higher by 7% than those by the gas chromatographic (GC) method which was selective for the intact drug. Among the metabolites of KG-2413, 1-(2-ethoxyethyl)-2-(hexahydro-1H-1,4-diazepin-1-yl)- 1H-benzimidazole (desmethyl metabolite) and 1-(2-ethoxyethyl)-5-hydroxy-2-(hexahydro-4-methyl-1H-1,4-diazepin- 1-yl)-1H- benzimidazole (5-hydroxy metabolite) had relatively high cross-reactivities of 29% and 21%, respectively, on the RRA method. However, the 5-hydroxy metabolite was not extracted with benzene under strongly basic conditions on the extraction procedure carried out prior to the receptor assay. Therefore, it was suggested that the desmethyl metabolite might affect the RRA method. In order to determine the amount of the desmethyl metabolite in the plasma, a high-performance liquid chromatographic method was established to determine the dansyl derivative of the desmethyl metabolite. By using this method, it was found that the desmethyl metabolite corresponding to 21-32% of the intact drug was present in the plasma after oral administration of KG-2413 at a dose of 2 mg/kg. Taking account of the cross-reactivity (29%) and extraction ratio (83%) of the desmethyl metabolite, this active metabolite affects the RRA method to give 5.1-7.7% overestimation of the intact drug concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural characterization of an unknown metabolite of ciprofloxacin]

The chemical structure of an unknown metabolite of ciprofloxacin (CAS 85721-33-1) is characterized by means of reversed phase ion pair liquid chromatography, absorption and fluorescence spectroscopy, partition coefficients as well as chemical and enzymatic hydrolytic degradation. A chemical structure of the unknown metabolite is proposed: N-formyl-desethylen-ciprofloxacin. It can be formed as an intermediate in the oxidative degradation of ciprofloxacin via oxociprofloxacin to desethylen-ciprofloxacin, or it may be formed by conjugation of desethylen-ciprofloxacin with formic acid. The amounts found in plasma and urine of patients were in the range of desethylen-ciprofloxacin, i.e. about 1% of the parent compound.     

Isolation and identification of metabolites of 5-ethyl-2'-deoxyuridine from rat urine

The metabolites of 5-ethyl-2'-deoxyuridine (Aedurid) have been isolated and purified from rat urine by preparative thin-layer and column chromatography. With the aid of 1H-NMR and mass spectroscopy and by comparison with an authentic sample, the major metabolite could be identified as 5-(1-hydroxyethyl)uracil. The minor metabolite was identified as 5-ethyluracil.     

Pharmacokinetics of etretin and etretinate during long-term treatment of psoriasis patients

The aromatic retinoic acid derivative etretin has recently been introduced in the treatment of severe psoriasis and other dyskeratoses. Hitherto, the use of the carboxylic acid ester analogue, etretinate, has been hampered by an extremely long elimination half-life of up to 120 days for this drug. Seven patients of either sex from whom we recently reported single-dose pharmacokinetics have been studied after 1 and 3 months multiple dose administration of the drugs. Four were given etretin and three etretinate. Etretin, both as drug and as metabolite, was absorbed faster than etretinate as judged from t-lag, tm and t 1/2 ka. Etretin as drug was eliminated faster than the metabolite etretin, t 1/2 beta 2.39 +/- 1.16 days compared to 6.51 +/- 2.06 days. In patients receiving etretinate the terminal disposition or elimination half-lives for cisetretin (t 1/2 lambda 3 15.9 +/- 9.9 days) were longer than for the metabolite etretin and exhibit a pronounced interindividual variation from 4.25 to 22.8 days. Similarly, cis-etretin accumulated very marked in comparison to the metabolite etretin of the drug etretinate. Assuming 40% systemic availability for both drugs, the central compartment of distribution constituted about 12-32% in case of etretin and about 0.8-3.6% in case of etretinate of the calculated apparent total volume of distribution at steady state, which showed mean values of 3.5 and 39.6 1.kg.-1, respectively, presumably reflecting the higher lipophilic nature of the latter compound.     

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 3. Approaches to eliminate opioid agonist metabolites by using substituted phenylpiperazine side chains

Dihydropyrimidinones, such as 1, represent a novel class of alpha(1a) adrenoceptor antagonists with potential for the treatment of benign prostatic hyperplasia (BPH) (see part 1 of this series). Analysis of the metabolites of 1 revealed that 4-methoxycarbonyl-4-phenylpiperidine is formed as the major metabolite and is an agonist at the mu-opioid receptor. To circumvent any potential liability resulting from the metabolite, we decided to identify alternate templates devoid of agonist activity at the mu-opioid receptor to replace the 4-methoxycarbonyl-4-phenylpiperidine moiety. The present study describes the synthesis and SAR of dihydropyrimidinones linked to substituted 4-phenylpiperazine containing side chains. Compound (+)-38 was identified as a lead compound with a binding and functional profile comparable to that of 1. The putative metabolite 2-carboxamidophenylpiperazine has negligible affinity for the mu-opioid receptor.