Metabolite radiolabeled compounds

Hyphenation of HPLC with NMR combines the power of sepai ation with a maximum of stiaictural information by NMR. HPLC-NMR has been used in the detection and identification of diaig metabolites in human urine since 1992. The rapid and unambiguous determination of the major metabolites of diaigs without any pretreatment of the investigated fluid represents the main advantage of this approach. Moreover the method is non-destmctive and without the need to use radiolabelled compounds. 

Another refinement, that avoids the necessity of developing suitable fecal extraction and chromatographic methods, is to dose the radiolabeled compound by both the i.v. and p.o. routes in two separate studies. Knowing that, by definition, the whole of the i.v. dose must have been bioavailable, a comparison of the proportion of the dose in the urine after the two different routes allows estimation of the percent absorbed. An analogous approach can be used without the use of a radiolabel, when the urine from the two studies is analyzed either for the parent compound or, more usually, for a major common metabolite. Assuming quantitatively identical clearance after both the i.v. and p.o. doses, the ratio of the amounts of analyte in the two experiments gives the absorption. 

In the early years of analysis data on the bioconcentration of LAS in biota were obtained using radiolabeled compounds and LSC, not distinguishing between parent compounds and metabolites [1,27,30]. These methods were non-specific and the data produced were unreliable due to overestimation. Therefore, it was important to develop accurate and specific analytical techniques in order to isolate and quantify these separately. 

Radiolabeling experiments showed that mirex is not metabolized in humans, rodents, cows, or minipigs the parent compound was the only radiolabeled compound present in the plasma, fat, and feces (Dorough and Ivie 1974 Gibson et al. 1972 Kutz et al. 1974 Mehendale et al. 1972 Morgan et al. 1979). Flowever, a monohydro derivative of mirex was identified in the feces, but not the fat or plasma, of rhesus monkeys given an oral or intravenous dose of mirex (Pittman et al. 1976 Stein et al. 1976 Wener et al. 1976). It is believed that the suspected metabolite may have arisen as a result of bacterial action in the lower gut or in the feces (Stein et al. 1976). 

In the development of most new active substances, it is required to investigate the disposition of the compound and its metabolite(s) and their rates and routes of elimination. This is generally carried out with radiolabelled compound, usually In the United Kingdom, approval of the Administration of Radioactive Substances Advisory Committee (ARSAC) is required for administration of radiolabelled compound to man. The purpose of the submission is to demonstrate that the dose of absorbed radiation is minimised by administration of the lowest dose that is consistent with meeting the objectives of the study. In general, the estimated absorbed radiation dose should be less than 500 xSv, but higher amoimts are permissible if they can be justified. The estimate is based on tissue distribution of radioactivity in animals and the pharmacokinetics in animals and man. 

The radiolabelled compounds were separated by thin layer chromatography on cellulose using 6% acetic acid. of salicylic acid was 0.63 and of the metabolite was 0.76. 

The extraction of two typical agricultural products from environmental matrices were chosen as examples for the operation of this system. Diuron, a phenylmethylurea, was freshly spiked onto Tama soil. This soil was characterized and shown to have 3.1% organic material and 14 % clay fraction. In addition, a phenyl metabolite of NUSTAR, a systemic fungicide, on wheat previously unextractable by SFE was extracted. The wheat sample was not classified for its chemical composition. Both samples were treated with radiolabeled compounds (E. I. du Pont de Nemours and Company, Du Pont Agricultural Products, Wilmington, DE) and extraction results are from liquid scintillation counting of the sample extract. Chromatographic evaluation of the Diuron from soil extracts has previously been published (2). 

High-sensitivity techniques for the quantitation and characterization of circulating metabolites following administration of radiolabeled compounds are of critical importance to understand the safety and efficacy profiles of novel drug candidates. AMS is one of the most sensitive techniques for the detection of radiolabeled components. However, the high cost and slow throughput of AMS analysis preclude the routine use of the techniques for metabolism studies. 

Toxicokinetics studies are designed to measure the amount and rate of the absorption, distribution, metabolism, and excretion of a xenobiotic. These data are used to construct predictive mathematical models so that the distribution and excretion of other doses can be simulated. Such studies are carried out using radiolabeled compounds to facilitate measurement and total recovery of the administered dose. This can be done entirely in vivo by measuring levels in blood, expired air, feces, and urine these procedures can be done relatively noninvasively and continuously in the same animal. Tissue levels can be measured by sequential killing and analysis of organ levels. It is important to measure not only the compound administered but also its metabolites, because simple radioactivity counting does not differentiate among them. 

The metabolic study, considered separately, consists of treatment of the animal with the radiolabeled compound followed by chemical analysis of all metabolites formed in vivo and excreted via the lungs, kidneys, or bile. Although reactive intermediates are unlikely to be isolated, the chemical structure of the end products may provide vital clues to the nature of the intermediates involved in their formation. The use of tissue homogenates, subcellular fractions, and purified enzymes may serve to clarify events occurring during metabolic sequences leading to the end products. 

The method of test substance synthesis or its source should be made a part of the documentation. This would apply to any test chemical, whether it is a technical material, a formulation, a metabolite, a by-product, or a radiolabeled compound. Any inpurities greater them... 

In vitro studies can only give a limited, mechanistic picture of biotransformation in animals or humans. The quantitative importance of each individual metabolite can only be assessed in vivo. Also, samples collected from in vivo studies give rise to comprehensive metabolite identification work (Watt et al. 2003 Clarke et al. 2001) which is also required from a regulatory point of view (Baillie et al. 2002). Due to the labour-intense nature of these studies and the need of applying radiolabeled compounds in order to get a complete picture of biotransformation these studies are performed at a later stage of development during preclinical and clinical phase. 

The radioactivity represents the sum of the original compound and/or radioactive-labeled metabolites and not to forget possible synthetic side-products which can be present in traces (depending on the purity and content of the synthetic material). Discussing traces of radioactivity, for instance traces crossing the placenta, keep in mind that these traces may be due to synthetic side-products. Thus, whenever possible, try to use radiolabeled compound as clean as possible. 

Reproductive toxicology studies are conducted to reveal the effect of the drug candidate on mammalian reproduction and whether potential reproductive risks may exist for humans. These reproductive studies commonly use pregnant rats and rabbits as the test species. To ensure that the dams and the fetuses are appropriately exposed to the drug candidate and metabolites (11), the dams can be dosed with radiolabeled compound, and the amount of radioactivity that crosses the placenta and into the fetuses at various times after dosing can be... 

C]-1,1-dichloroethane, 7.4% and 29.3% of the dose was metabolized by rats and mice, respectively. The predominant metabolite in both species was [ C]-C02 (Mitoma et al. 1985). It is likely that the ingested radiolabeled 1,1-dichloroethane underwent first-pass extraction by the liver. It has been suggested that high doses such as those used in this study exceed the capacity of the animals to metabolize 1,1 -dichloroethane (Bruckner 1989). The radiolabeled compound that was not excreted unchanged in the expired air was probably largely metabolized in the liver, followed by subsequent redistribution of labeled metabolites to other organs prior to their excretion. 

The first section of this book describes the application of LC/MS to the analysis of agricultural chemicals and their metabolites. Using LC/MS for residue analysis in agricultural chemistry has become routine in many laboratories. Many pesticides, such as the chlorophenoxy acid and sulfonyl urea herbicides or organophosphorus and methyl carbamate insecticides, are too polar or thermally labile for analysis via GC. The use of LC/MS for the identification of polar pesticide metabolites and conjugates, an area traditionally dominated by radiolabeled compounds, stands out as a particularly dramatic demonstration of the power of this technique. 

In the past, studies of the distribution and metabolism of cannabinoids had been hampered by the lack of pure radiolabeled compounds. In 1973, shortly after the radiolabeled cannabinoids became available, Dr. E. G. Lelghty reported the recognition of some unidentified metabolites that were retained In rat liver and spleen up to 15 days after an acute Intravenous or multiple subacute Intraperltoneal Injections of either A -THC or A -THC ). The metabolites could be extracted by methanol and had a greater lipophilicity than the parent THC. These long-retained metabolites were also found In bone marrow ( ) and represented an Increasing percentage of the total cannabinoids found In feces, but not urine (. 

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