Active metabolites characterized

Stages in hazard characterization according to the European Commission s Scientific Steering Committee are (1) establishment of the dose-response relationship for each critical effect (2) identification of the most sensitive species and strain (3) characterization of the mode of action and mechanisms of critical effects (including the possible roles of active metabolites) (4) high to low dose (exposure) extrapolation and interspecies extrapolation and (5) evaluation of factors that can influence severity and duration of adverse health effects. 

Considerable effort is often required to prepare sufficient quantities of key mammalian metabolites of drug candidates for biological activity evaluation or for use as analytical standards. The new regulatory guidance in drug development [5] will certainly lead to more emphasis on key human metabolite characterization. Microbial bioreactors can be used for... 

The reaction of metabolically generated polycyclic aromatic diol epoxides with DNA Ua vivo is believed to be an important and critical event in chemical carcinogenesis Cl,2). In recent years, much attention has been devoted to studies of diol epoxide-nucleic acid interactions in aqueous model systems. The most widely studied reactive intermediate is benzo(a)pyrene-7,8-diol-9,10-epoxide (BaPDE), which is the ultimate biologically active metabolite of the well known and ubiquitous environmental pollutant benzo(a)pyrene. There are four different stereoisomers of BaPDE (Figure 1) which are characterized by differences in biological activities, and reactivities with DNA (2-4). In this review, emphasis is placed on studies of reaction mechanisms of BPDE and related compounds with DNA, and the structures of the adducts formed. 

Reactive Metabolites of PAHs. A wide variety of products have been identified as metabolites of PAHs. These include phenols, quinones, trans-dihydrodiols, epoxides and a variety of conjugates of these compounds. Simple epoxides, especially those of the K-region, were initially favored as being the active metabolites responsible for the covalent binding of PAH to DNA. Little direct experimental support exists for this idea (62.63,64) except in microsomal incubations using preparation in which oxidations at the K-region are favored (65,66). Evidence has been presented that a 9-hydroxyB[a]P 4,5-oxide may account for some of the adducts observed in vivo (67.68) although these products have never been fully characterized. 

Botsch, S., Gautier, J.C., Beaune, P., Eichelbaum, M. and Kroemer, H.K. (1993) Identification and characterization of the cytochrome P450 enzymes involved in N-dealkylation of propafenone molecular base for interaction potential and variable disposition of active metabolites. Molecular Pharmacology, 43 (1), 120-126. 

This lack of information may well relate to bupropion s complex metabolism, whereby biologically active metabolites predominate several-fold over the parent compound these metabolites may ultimately prove to be the basis for therapeutic effects [Golden et al. 1988]. Furthermore, because the full spectrum of action of the metabolites has not been explored, it is difficult to confidently characterize the primary biochemical action of bupropion as noradrenergic. One must therefore be cautious in interpreting any distinct aspects of bupropion s clinical actions as reflecting some noradrenergic mechanisms. And to our knowledge, bupropion is unique in that no other compound available for use in humans has a similar overall profile of preclini-cal and clinical biochemical effects. 

Ramelteon Activates and MT2 receptors in suprachiasmatic nuclei in the CNS Rapid onset of sleep with minimal rebound insomnia or withdrawal symptoms Sleep disorders, especially those characterized by difficulty in falling asleep not a controlled substance Oral activity forms active metabolite via CYP1A2 Toxicity Dizziness fatigue endocrine changes Interactions Fluvoxamine inhibits metabolism... 

Pharmacokinetic—pharmacodynamic characterization of tramadol is difficult because of differences between tramadol concentrations in plasma and at the site of action, and because of pharmacodynamic interactions between the two enantiomers of tramadol and its active metabolites.53... 

Metabolite identification is central to many of the activities in preclinical development. A more complete characterization of pharmacokinetic properties is performed in animals (typically rats and dogs) during this stage. The knowledge of the biotransformation pathways of the lead candidate to its metabolites is used to indicate the magnitude and duration of activity. Metabolite identification is critical to many of these activities and plays an important role in establishing the dose and toxicity levels. 

Johnson MD et al (2004) Pharmacological characterization of 4-hydroxy-N-desmethyl tamoxifen, a novel active metabolite of tamoxifen. Breast Cancer Res Treat 85 151-159... 

Vitamin D3 can be obtained either through the diet or by the conversion of 7-de-hydrocholesterol in the skin by the action of ultraviolet light. Extensive metabolism of vitamin D3 can occur (no less than 30 metabolites of vitamin D have been isolated and chemically characterized). To obtain the most biologically active metabolite, vitamin D3 is first hydroxylated in the liver to form 25-hydroxyvitamin D3, which is then converted to l,25(OH)2D3 by a cytochrome P-450 mitochondrial en-... 

There has been revived interest in a-acetylmethadol as a result of its use in the maintenance of addicts (p. 304) and several pharmacokinetic(19) and pharmacological studies of the ester have been made. The acetate is characterized by a slow onset of action, a feature attributed to its conversion to an active metabolite,(40) and this proposal is supported by the isolation of two metabolic products from rats(41) and opioid addicts(42) that are effective analgesics. The compounds are the secondary and primary amines corresponding to a-acetylmethadol formed by successive N-demethylation and were detected by GCMS after conversion to trichloroacetamide derivatives. Authentic primary amine 15 was initially prepared by oxidizing a-(-)-acetylmethadol... 

Tuk, B., Van Oostenhmggen, M.F., Herhen, V.M.M., Mandema, J.W., Danhof, M. (1999). Characterization of the pharmacodynamic interaction between parent drug and active metabolite in vivo midazolam and alpha-OH-midazolam. J. Pharmacol. Exp. Then 289 1067-74. 

The Streptomyces WEC 85E assay has been used to test fermentation extracts from two thousand actinomycete isolates. Over one himdred strains, representing possibly twenty-seven different species, were foimd to produce metabolites that affect the onset of sporulation. Purification and characterization of the active metabolites present in some of these extracts is currently in progress. 

As for any bioanalytical method, the extent of validation for an immunoassay should be related to the intended application of the assay. Thus, if an immunoassay is intended to support rapid screening in discovery R D, the characterization of specificity and the accuracy and precision specifications may be less stringent than if the assay is used to support pre-clinical and clinical development studies. Indeed, an assay for discovery support may be designed to detect active metabolites as well as parent molecule, so that... 

The metabolic pathways of 5-FU and its prodrugs have been well characterized in the liver as well as in liver tumors and metastases by in vivo 19F MR spectroscopy. The liver and extrahepatic spaces catabolize 5-FU, which is subsequently excreted in the urine [3-5, 7-10, 46, 59], Active metabolites (fluoronucleotides) are created by anabolism in tumors. Even though clinical studies showed significant individual subject variations [3, 5, 7-10, 26,46,47], spectral characteristics such as resonance frequency, linewidth, relaxation time, and amplitude were not related to the therapeutic response. However, dynamic processes, specifically the accumulation and retention of 5-FU in the tumor, were indicative of response. Patients showing tumor half-lives of free 5-FU of 20 minutes or longer observed in patients were characterized by Wolff el al. [47, 53] as trappers. While over 50% of the evaluated population were nontrappers, approximately 60% of the patients who responded to therapy were trappers [3-5, 7-10],... 

When measuring the active metabolites wash-out period and sampling times may need to be adjusted to enable adequate characterization of the pharmacokinetic profile of the metabolite. 

Olefinic bonds in xenobiotic molecules can also be targets of cytochrome P450-catalyzed epoxidation (Figure 32.5b). In contrast to arene oxides, the resulting epoxides are fairly stable and can be isolated and characterized. But like arene oxides, they are substrates of epoxide hydrolase to yield dihydrodiols. This is exempUlied by carbamazepine, whose 10,11-epoxide is a major and pharmacologically active metabolite in humans, and is further metabolized to the inactive dihydrodiol. ... 

As the compound reaches the late discovery and candidate selection stage, the focus is to determine its major metabolic pathways, metabolic difference between species, and to identify potential pharmacologically active or toxic metabolites. Because of the complexity, comprehensive metabolite characterization studies have been typically conducted at this stage with radiolabeled standard. Identification of circulating metabolites is also important at this stage to explain the pharmacokinetic or the pharmacodynamic profile. An NCE may show efficacy that is inconsistent with what is predicted based upon the known concentration of the parent drug. These inconsistencies could be due to the presence of active metabolites. The knowledge of these metabolites will also dictate how the analysis of samples will be conducted in the development and clinical studies. 

Yamazoe, Y., Ishii, K., Kamataki, T., Kato, R., and Sugimura, T., Isolation and characterization of active metabolites of tryptophan-pyrolysate mutagen, TRP-P-2, formed by rat liver microsomes, Chemico-Biol. Interact., 30, 125, 1980. 

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