Commentary Themed Issue: Radioisotopic Bioanalysis
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[email protected] Radiolabeled metabolite and disposition studies in support of safety assessment “Radiolabeling allows us to discriminate between test article-related materials and endogenous materials allowing us to make quantitative measurements...” Keywords: distribution • exposure • mass spectrometry • metabolism • radiochromatography • safety assessment
Humans, animals and the environment can become exposed to a range of xenobiotics, either intentionally or following accidental exposure. These xenobiotics can be designed to have a positive effect but are likely to have the potential to have a negative effect on biological systems. The balance between these effects is key to understanding the safety profile of all chemicals that are intended for use as human or veterinary pharmaceuticals, agrochemicals or industrial chemicals. If a biological system is to be exposed to a xenobiotic, we need to develop an understanding of both the distribution and the fate of that molecule within that system as well as the rate/route of excretion/elimination/ dissipation as this can have a direct impact on the safety of the material in that system. Radiolabeling provides an ideal analytical tool to aid with such investigations; indeed, a range of studies using radiolabeled test article are a mandatory part of the regulatory process used to authorize novel agrochemicals and veterinary pharmaceuticals and are also used to support the interpretation of toxicology data during the safety evaluation of new drugs. Radiolabeling allows us to discriminate between test article-related materials and endogenous materials allowing us to make quantitative measurements relatively easily and visualize the distribution of test article-related material within the test system. Challenges associated with radiolabeling The initial challenge when considering performing a study with radiolabeled material
10.4155/BIO.15.18 © 2015 Future Science Ltd
is how to produce the material – which radioligand, which position within the molecule with different molecules bringing their own difficulties, for example, proteins, naturally occurring molecules and molecules with multiple active centers. While 14C is often the label of choice, some limitations do occur with this ligand. In particular, the relatively low specific activity with which 14C materials are produced can often lead to a compromise between overall compound dose and radioactive dose having to be made. In contrast, 3H materials can be produced with much higher specific activity but exchange of tritium with body water is a well-described issue which can confound any data generated. When considering other potential labels, especially those emitting γ radiation, half-life and emission energy are often limiting factors in their use. For example, typical ligands used for PET-scanning studies would not be suitable for xenobiotic disposition studies as their half-lives are often only several minutes, not several weeks or years as would be required. The majority of regulatory metabolism studies are conducted using a 14C-labeled molecule. The position of the radiolabel is critical and will involve input from several stake holders. Medicinal chemists will have an interest in following the fate of particular parts of the molecule; metabolism chemists will contribute on the metabolic stability of preferred labeling positions; and synthetic chemists will offer a view on what is possible and not cost-prohibitive. These discussions will lead to the production of a radiolabeled material that will contribute significantly to
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Ashley Beattie Author for correspondence: Department of Environmental Sciences, Charles River, Edinburgh, EH33 2NE, UK ashley.beattie@ crl.com
Stephen Madden Department of Metabolism & Pharmacokinetics, Preclinical Services, Charles River, Edinburgh, EH33 2NE, UK
Chris Lowrie Department of Environmental Sciences, Charles River, Edinburgh, EH33 2NE, UK
David MacPherson Department of Chemistry, Charles River, Edinburgh, EH33 2NE, UK
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Commentary Beattie, Madden, Lowrie & MacPherson the understanding of the metabolism of a novel drug molecule. The use of radiolabeling in regulatory studies For human pharmaceuticals, the need to gain an understanding of the absorption, distribution, metabolism and elimination properties of novel compounds is clearly defined in ICH M3 guideline [1] , which states that information should be available prior to the conduct of large-scale clinical trials. However, there is no regulatory mandate on how this information is generated or on the use of radiolabeled studies to assist in the process. Indeed, given the improvements in analytical technology over the last 10 years, and in LC– MS/MS technology in particular, there has been a significant level of debate surrounding the usefulness of conducting such studies with human pharmaceuticals, with some commentators believing that the level of information gathered from radioalabeled metabolism studies should now be attainable from analysis of samples collected in toxicology and human clinical studies using nonradiolabeled test articles [2,3] . There is no doubt that LC–MS/MS technology has developed significantly over the last 20 years and is a very powerful tool in drug development; however, when we are detecting drug metabolites in complex matrices using nonradiolabeled LC–MS approaches, we can never be absolutely certain that all the metabolic products have been detected. To give some confidence we need to quantify the detected metabolites and relate measured quantities to the administered dose to give a material balance. However, quantifying metabolites using LC–MS without the benefit of an analytical standard is difficult because of the variation in ionization between metabolites and because of the confounding influence of matrix effects. Radiolabeling avoids these difficulties and provides robust quantitative data and a material balance. Many individuals (including the authors of this article) and organizations [4] believe that the conduct of radiolabeled metabolism and distribution studies provides the best opportunity to produce the necessary data to generate a complete profile of drug disposition and metabolism and that such studies continue to play an important role in the drug development process. The complex nature of the compounds under investigation will often require the studies to be conducted with multiple radiolabeled forms (different sites of labeling). This ensures that the metabolic fate and disposition are fully understood as the molecule may be subject to cleavage or structural rearrangement that results in incomplete understanding of the transformation products or the molecule may contain
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multiple bioactive moieties that are bridged together using a chemical linker. For all small molecule therapeutic agents the range of studies and information that is gathered from radiolabeled metabolism and distribution studies is broadly similar. Plasma (blood) kinetics, excretion and distribution are normally conducted in, at least, a rodent species. The distribution of radiolabeled material is either conducted by removal and analysis of individual tissues or by quantitative whole body autoradiography (QWBA). For the pharmaceutical industry QWBA has been adopted as the preferred technique for the last 10–15 years and this has proven to be a very powerful analytical tool. However, the technique is only now beginning to be adopted by the other industry sectors. One major drawback of QWBA is that the technique provides information on the distribution of total radiolabeled material and does not allow discrimination between individual chemical entities, in other words, does not differentiate between parent compound and metabolites. MALDI-MS imaging (MALDI-MSI) is an emerging technique that will help address this gap in the data provided by QWBA. Using high-sensitivity MS, MALDI-MSI provides researchers with the ability to scan tissue sections for particular molecular masses of interest. This will allow the distribution of both parent compound and metabolites to be determined. Furthermore, software developments over the last few years have now given us the opportunity to produce quantitative data using this technique. Although in its relative infancy, MALDI-MSI has the potential to play a key role in contributing to our understanding of both the distribution of drugs and their metabolites and how this distribution impacts the toxicological profile seen with these molecules. One of the primary outputs of the radiolabeled metabolism package will be information on the identity of any metabolites formed and the proportion that each of these metabolites contributes to the overall exposure to xenobiotic-related material in both the test system and in the ultimate target test system, for example, humans, animals, plants or the environment. By understanding the range of metabolites formed and the proportion (effective systemic load) of each of these metabolites we can interrogate the data from toxicology studies to ensure that the safety of all metabolites has been suitably assessed prior to significant exposure in the target system [5] . The significance (or not) of metabolites The relative quantities of metabolites are important in establishing the safety of novel pharmaceuticals. At least two toxicology species will be used to assess the toxicity of the compound over a range of dose levels.
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Radiolabeled metabolite & disposition studies in support of safety assessment
The metabolic pathway will be established and can be compared with the metabolic pathway in humans either following a radiolabeled human volunteer study or following the bioanalytical monitoring of blood/ plasma/urine for key metabolites during clinical trials. If man produces metabolites that are unique (i.e., not present in the toxicology species) or disproportionate (i.e., major in man but relatively minor in the toxicology species), then questions will be asked about the suitability of the toxicology species, confirming the importance of accurate metabolite quantification. Clearly the selection of toxicology species that reflect the metabolic profile in man is important and in vitro studies comparing metabolism between species provided useful data that can facilitate this process. Quantification of metabolites & limits of sensitivity The quantification of unchanged parent compound and transformation products in metabolism studies is generally by chromatography with radiochemical detection. The most common approach is HPLC coupled to a flow scintillation analyzer with a liquid or solid counting cell. This method allows fast realtime acquisition of data and typically allows detection of peaks greater than approximately 1–2 Bq. However, a greater level of sensitivity may be required from the assay. To achieve this, techniques such as a stop-flow system may be applied to the radiochemical detector and HPLC to increase residence time in the count cell. Alternatively, fractions of the HPLC column eluent may be collected and subjected to scintillation counting (liquid or solid scintillators are used). Using these techniques typically allows detection of peaks as low as 0.2–0.3 Bq. However, it takes significantly longer to acquire data (perhaps by as much as a day) and limits are dependent to a greater extent on sample preparation. The use of HPLC is generally preferred as it allows seamless transition to MS for structural elucidation. However, it is possible to drive limits of sensitivity even lower using TLC and phosphor imaging. This technique relies heavily on reducing background interference and can be used to achieve sensitivity around ten-times greater than the most sensitive HPLC radiochemical detection techniques. However, this technique can be very slow (up to 1-week exposure time) in generating data and while not impossible, it is more challenging to align with a mass spectrometer for structural elucidation purposes. Generally speaking, quantification of individual peaks is calculated in relative terms by taking the total area as the denominator and expressing each individual peak as a percentage of that total. The total area may be calculated simply as the sum of all integrated regions
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Commentary
of interest (percentage of ROI method) or by applying background subtraction to the chromatogram and using the net area remaining as the denominator (percentage of total area method). It is important to be clear which method has been used, as the former will result in a remainder, which needs to be considered in any subsequent interpretation of the data. This remainder consists of radioactivity which sits between the limits of detection and quantification and is therefore not integrated as discrete peaks. Whilst it is important to understand sensitivity in terms of the peaks detected, it is equally important to understand what may be missing from the picture. Regardless of industry, significant metabolites require to be quantified and their structures understood. The term ‘significant’ is applied differently depending on factors such as the regulatory body, the chemical nature of the parent compound, toxicology data, etc. As an example, an agrochemical residue study requires characterization data (structural information or characteristic) for metabolites appearing in edible commodities at >10 ppb and requires unequivocal identification (structural assignment) of metabolites at 50 ppb [6,7] . A similar regulatory study of a veterinary pharmaceutical in a food-producing species requires identification of metabolites at 100 ppb in edible commodities [8] . In an environmental context [9] , it is important to understand environmental significance in absolute terms (predicted environmental concentration) and as a percentage of the starting material. As application rates of new active ingredients are driven downward by stricter guidance, the limits of sensitivity of the assays must be adjusted to ensure peaks at levels as low as 0.1% of the applied radioactivity are detectable in the final samples. It is important to set the limits of sensitivity for the assay to ensure peaks are detectable and this is relatively straightforward. Limits of quantification should be set at least ten-times below the regulatory threshold for characterization/identification. A sample injected to HPLC with flow through radiodetection should contain sufficient radioactivity to detect peaks; however, since the assay will not account for radioactivity below 1–2 Bq, low-level peaks may be missing from the integration. This could present a problem if they are of toxicological concern, however, as the peaks are quantified in relative terms (percentage of ROI or total area methods), it is possible that an assignment could be regarded as significant because lots of minor peaks are present but not at a level that results in their inclusion in the maths. This may mean peaks of toxicological or regulatory concern being missed but may also mean there is focus on the ‘wrong peaks.’ Specific activity of the starting radiochemical is an important factor to consider when thinking about assay sensitivity. A
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Commentary Beattie, Madden, Lowrie & MacPherson specific activity which is too low will mean you are unable to detect peaks. A higher specific activity will improve sensitivity; however, depending on the way the radiolabel is incorporated, MS data can become more complex. Generally speaking for small molecules (