Metabolic profiling radiolabeled compounds

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. 

Generation of metabolic profiles especially by use of radiolabeled test compounds in preclinical development makes sure that the animal model allows a qualitative and/or quantitative prediction to human. This is crucial for proof of validity of pharmacological and toxicological data obtained in animal models for humans. 

The two most common drug metabolism studies are mass balance and tissue distribution. Mass balance studies are usually conducted in both the rodent and the nonrodent species used for toxicology evaluations, whereas tissue distribution is performed only in the rodent. For mass balance, a radio-labeled compound is administered to the test species and urine, feces, and, if necessary, expired air are collected at intervals and counted for total radioactivity. Commonly used intervals are 0-4, 4-8, 8-12, 12-24, and then daily, up to 168 hours or until more than 95% of the administered dose has been excreted. Depending on the pharmacokinetic profile of the candidate, other collection intervals can be selected to give a better picture of the excretion profile. For tissue distribution, a radiolabeled compound is administered to the test species, and after predefined times, usually 2, 4, 8, 24, and 48 hours, the test species... 

If little or no previous data on the metabolism of the compound in the target species is available, it is prudent to consider conducting a probe residue depletion study in three animals sacrificed at three widely spaced intervals following the last dose. For example, one animal might be sacrificed at 12 hours (zero withdrawal) and another each at 72 and 120 hours post final dose (the exact times selected will depend on the tissue clearance of the drug under development). The information obtained will allow one to select more accurately sacrifice intervals for the definitive study which encompass proposed safe concentrations of total drug-related residue. Additionally, the probe study residue data will allow for adjustments in the specific activity of the radiolabeled dose to ensure tissue concentrations of total radioactivity at later sacrifice intervals which are sufficient for metabolite profiling and isolation. 

In contrast to these data, a reasonable literature base exists on the disposition of topically applied fenthion, a systemic organophosphate pesticide. Topical treatment of dairy cows with fenthion at clinically effective doses resulted in detectable but nonviolative residues in milk (24) or body fat (25). In this latter study, 90% of the residues detectable in the fat of treated cattle was parent compound. When the metabolic profile of fenthion was compared after dermal and intramuscular administration, very few differences were noted except for the time course of radiolabel clearance from the animals (26). It was not possible in these studies to study the mechanism of dermal fenthion metabolism more precisely. 

Mass Balance Studies. Pharmacokinetic mass balance studies apply unlabeled, stable isotopes or radiolabeled compounds to study the extent of absorption and first-pass metabolism, distribution, and excretion of a given compound. In the microdosing approach, a C-labeled compound is administered to human volunteers at doses from as low as one microgram blood, urine, and fecal samples are collected over time and analyzed for C content by accelerator mass spectroscopy to determine half-life, plasma AUC, and maximal concentration (Cmax)- However, these methods are not very popular even when very low doses of radioactivity are involved. Highly sensitive, and more readily available, tech-niques for separation and analysis (e.g., LC-MS, LC-MS/MS) are frequently used alternatives that enable pharmacokinetic investigations and metabolite profiling of nonradiolabeled compounds. 

Compound A produces a predominant fragment at miz 264, which corresponds to a neutral loss of 175, which in this case corresponds to the loss of 4-trifluo-romethylbenzylamine [37]. This neutral loss can be used to monitor the presence of other species sharing this common feature. Radioactively labelled compound A was incubated in rat liver microsomes, analysed by LC-MS-MS and the TIC for the neutral loss of a mass of 175 (Figure 6.20a) was obtained. When compared with the radioactivity profile in Figure 6.20b, two extra components were detected in the TIC. This was due to the loss of the radiolabel during metabolism. LC-MS-MS is very useful as a complementary detection method where the radiolabel is lost during metabolism or in situations where a radiolabel is not available. 

All the techniques described in this chapter contribute to improved metabolite characterization. Notably, these techniques will enable the generation of extended time profiles for plasma radioactivity, the assessment of free metabolite concentrations following plasma dialysis, and the characterization of metabolites at very low levels of exposure, for example, following inhaled or dermal routes of administration. Furthermore, the use of high-sensitivity techniques could impact the design of radiolabel studies, for example, by enabling a lower radiolabel dose to be administered to human subjects without compromising the ability to characterize the metabolic fate of the compound of interest. 

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. 

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