Foreword Themed Issue: Radioisotopic Bioanalysis

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Themed Issue on Radioisotopic Bioanalysis “It is safe to assume that radioisotopic bioanalysis will remain an important and valuable tool for researchers for the foreseeable future.” Keywords: Accelerator MS • autoradiography • metabolism • radioisotope • scintillation counting • tracer

Almost since radioactivity itself was discovered, at the end of the 19th century, the idea of utilizing the ‘signature’ of radioactive compounds to track their fate through complex systems in a quantitative manner without the need to develop specific methods each time has been exploited by scientists, engineers and others. In ‘A historical perspective on radioisotopic tracers in metabolism and biochemistry’ [1] , G Lappin tells the story of how modern radioanalysis techniques have developed from those early beginnings. It is clear that the attributes of being fully quantitative, independent of the physicochemical properties of the labeled compound, and providing a ‘tag’ that enables discrimination of the radioactive entity from other components in the system (including the nonradiolabeled analyte) make radioanalysis an extremely powerful analytical tool. As G Lappin points out, the basic design of radiotracer studies has stayed remarkably unchanged since those first studies. However, although the instrumentation involved has (not coincidentally) also remained much the same since the advent of the Tri-Carb in the 1950s, advances in scintillation counting technology that have reduced backgrounds and increased counting efficiency (described by Simon Temple in his paper ‘Liquid scintillation counting: how has it advanced over the years and what does the future hold?’ [2]) and latterly the introduction of the ultrasensitive technique of Accelerator MS (AMS; see G Young and M Seymour, ‘Application of 14C-Accelerator MS in pharmaceutical development’ [3]) have facilitated some

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innovative applications. For example, the use of LC+AMS to measure an intravenous ‘microdose’ of radiolabeled compound administered concomitantly with an extravascular pharmacologically active dose of the nonlabeled version offers a scientifically superior and resource-sparing methodology for determining the fundamental pharmacokinetics of drug candidates as part of the initial series of clinical investigations during pharmaceutical development, and has the potential to transform the development paradigm. The ability to carry out radiotracer studies with relatively small amounts of radioactivity also opens up the possibility of conducting such investigations in vulnerable populations, such as the renally or hepatically impaired, in other words the very people to whom the drugs being developed will be administered and arguably, therefore, those in whom it is most important that the metabolism of the compounds administered should be understood. Interestingly, although the basic design of scintillation counters has not changed since the early days, the author is aware of at least one major pharmaceutical company that, spurred on by the utility of the applications highlighted by the protagonists of AMS, has put significant effort into optimizing their scintillation counting instrumentation and methodologies, thereby significantly reducing the achievable limit of quantification. If other practitioners follow suit, perhaps this will provide fresh impetus to the development groups of the commercial instrument manufacturers.

Bioanalysis (2015) 7(5), 499–501

Mark Seymour Eckert & Ziegler Vitalea Science, Suite B101, 2121 2nd Street, Davis, CA 95618, USA mark.seymour@ ezag.com

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ISSN 1757-6180

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Foreword  Seymour One area where significant progress in radiodetection may be on the horizon is that of flow detectors. T Deakin (‘Radioisotopic characterisation as an analytical tool – current status, limitations and future challenges’ [4]) gives a very detailed review of the state of scintillation counting-based flow detection and predicts that continuing developments in electronic discrimination techniques will improve signal-to-noise ratios and hence drive down detection limits. However, more of a step change will arrive in the form of a direct LC– AMS interface. A number of groups are working on this and, although commercially available versions are perhaps 2–5 years away, the CO2-accepting ion sources required for their realization are already in use for discreet sample analysis and yielding significant benefits in terms of reduced cost and increased throughput. The availability of a robust and reliable flow-detection system with the sensitivity of AMS could ultimately tip the balance between conventional and microtracer human AME studies in favor of the latter. Since those early days, the use of radioisotopes for the quantitative assessment of metabolism has become a routine element of the human drug development process (notwithstanding the fact that, as Beattie et al. point out in their paper ‘Radiolabelled metabolite and disposition studies in support of safety assessment’ [5] , there is no specific regulatory requirement for this, in contrast to the development of chemicals, agrochemicals and veterinary medicines). Thus, most marketing applications for human pharmaceuticals include data from a battery of preclinical ADME (adsorption, distribution, metabolism and excretion) studies as well as a human mass balance study. Although recently some researchers have advocated that studies with radioisotopes in animal species be largely abandoned [6] , their proposed strategy relies on conducting the pivotal human AME study with an isotopic tracer and earlier in the development life cycle than is often the case presently. A major driver toward this proposal is the rapid evolution of the enabling technique of LC–MS/ MS: since the advent of atmospheric pressure ionization LC–MS interfaces, in the mid 1970s, the ability to directly couple LC systems capable of separating drug molecules and their metabolites in biological samples with the structural elucidation power of MSn (and, latterly, high-resolution MS) has revolutionized the work of drug metabolism and pharmacokinetics (DMPK) departments. Others have gone even further, developing methodologies for the quantification of nonradiolabeled metabolites by LC–MS without the need for authentic reference standards [7,8] and thus potentially removing the need to conduct labeled studies at all. However, the resources required seem more than those for the radiometric methods they seek to replace and

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the data produced perhaps only semiquantitative, suggesting that the conduct of ADME studies using radioisotopes is likely to continue for the foreseeable future. The ability to create images of the biodistribution of radiolabeled compounds, initially relying on the property of ionizing radiation that it will ‘fog’ photographic film and, more recently, using reusable phosphor imaging plates, has been developed into quantitative whole-body autoradiography (QWBA). The development and utility of this and related techniques are well described by A McEwen and C Henson in their paper ‘Quantitative whole body autoradiography: past, present and future’ [9] . As with scintillation counting, the technology is now mature and seemingly not set for transformational change in the near future. Nevertheless, as the authors point out, QWBA remains the ‘gold standard’ for reliable, quantitative distribution data and, because it is nondestructive, lends itself well to combination with other techniques, for example, matrix-assisted laser desorption/ionization imaging or localized identification/quantification of specific analytes in sections using liquid extraction surface analysis and MS/MS. Overall, it is clear that radioanalysis remains an important tool in the drug developers’ box. The paper by Pluim et al. (‘Improved pharmacodynamics assay for dihydropyrimidine dehydrogenase activity in peripheral blood mononuclear cells’ [10]) is a contemporary example of this: the improved assay described achieved outstanding accuracy and precision numbers and solves a material problem in clinical therapy, by overcoming inaccuracy in the existing method for diphenhydramine dehydrogenase (DPD) determination caused by sample hemolysis. Accurate determination of DPD can be instrumental in both fundamental oncology research and personalized medicine, allowing appropriate dose setting for 5-fluorouracil therapy based on phenotypic testing of patients for DPD deficiency. Of course, although it is probably the most widely used in pharmaceutical research, 14C is not the only radioisotope utilized for analysis, others include 3H, 125 35 I, S and 32P. 3H can be and is used in much the same way as 14C. Although its half-life is much shorter than that of 14C (12.3 years compared with 5730 years), that is of course more than long enough to facilitate experiments run over the time courses associated with biological systems and processes and, perhaps with some half-life correction, compatible with programs lasting many months. The shorter half-life also means that higher specific radioactivities can be achieved, thus overcoming the reduced counting efficiencies obtained with this lower-energy radionuclide compared with

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Themed Issue on Radioisotopic Bioanalysis

C. The relatively low cost of producing 3H-labeled compounds is attractive, as is the fact that nonlabeled compounds can be postlabeled (as opposed to requiring synthesis from a 14C-labeled precursor). Care must be taken that 3H exchange with other components in the biological system does not compromise the experimental results obtained. Nevertheless, for molecules metabolized by cleavage of the parent to produce more than one substantial (and potentially pharmacologically active) fragment, labeling one half of the molecule with 14C and the other with 3H and using the energy discrimination capabilities of modern scintillation counters to quantify both radioisotopes independently in the same sample is an elegant solution. Tritium-labeled compounds are also suitable for use in micro autoradiography analysis [9] , due to the relatively short path length of the β-particles it emits. As an aside, it is technically possible to quantify 3H using AMS, with comparable sensitivity to that obtained for 14C [11] ; however, most of the AMS instruments currently in use for routine bioanalysis are not capable of such measurements. Regrettably, the use of radioisotopes in PET is not covered in any detail in this issue. The ability to obtain quantitative information using this 3D imaging technique, which utilizes positron-emitting radionuclides with very short half-lives (mostly 18F and 11C; t½ ≈ 2 h and 20 min, respectively), makes it an invaluable tool for understanding what is going on in inaccessible tissues such as the human brain. However, the need

to conduct studies in facilities in close proximity to a cyclotron and the short experimental durations possible are restrictive. Many other radioisotopes are also utilized in bioanalysis, including 32P (e.g., for studying the formation of DNA adducts), and 125I and 89Zn (e.g., used in biodistribution studies for large molecules, such as proteins). Each of the techniques mentioned above makes use of the facts that the amount of radioactivity in a sample is proportional to the mass of the labeled compound(s) present, based on its specific radioactivity, and that radioactivity is detectable by a range of ‘universal’ techniques that are not dependent on the physicochemical properties of the labeled chemical. These attributes make radiodetection a powerful and broadly applicable tool and one that is complementary to a great many other analytical techniques. It is safe to assume that radioisotopic bioanalysis will remain an important and valuable tool for researchers for the foreseeable future.

References

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Yi P, Luffer-Atlas D. A radiocalibration method with pseudo internal standard to estimate circulating metabolite concentrations. Bioanalysis 2(7), 1195–1210 (2010).

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Ma S, Chowdhury S. Analytical strategies for assessment of human metabolites in preclinical safety testing. Anal. Chem. 83(13), 5028–5036 (2011).

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McEwen A, Henson C. Quantitative whole body autoradiography: past, present and future. Bioanalysis 7(5), 557–568 (2015).

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Pluim D, Jacobs BA, Deenen MJ et al. Improved pharmacodynamics assay for dihydropyrimidine dehydrogenase activity in peripheral blood mononuclear cells. Bioanalysis 7(5), 519–529 (2015).

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Dingley KH, Roberts ML, Velsko CA, Turteltaub, KW. Attomole detection of 3H in biological samples using Accelerator mass spectrometry: application in low-dose, dualisotope tracer studies in conjunction with 14C Accelerator mass spectrometry. Chem. Res. Toxicol. 11, 1217–1222 (1998).

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Lappin G. A historical perspective on radioisotopic tracers in metabolism and biochemistry. Bioanalysis 7(5), 531–540 (2015). Temple S. Liquid scintillation counting: how has it advanced over the years and what does the future hold? Bioanalysis 7(5), 503–505 (2015). Young GC, Seymour M. Application of 14C-Accelerator MS in pharmaceutical development. Bioanalysis 7(5), 513–517 (2015).

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Deakin T. Radioisotopic characterisation as an analytical tool – current status, limitations and future challenges. Bioanalysis 7(5), 541–555 (2015).

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Beattie A, Madden S, Lowrie C, MacPherson D. Radiolabelled metabolite and disposition studies in support of safety assessment. Bioanalysis 7(5), 507–511 (2015).

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Obach RS, Nedderman AN, Smith DA. Radiolabelled mass-balance excretion and metabolism studies in laboratory animals: are they still necessary? Xenobiotica 42, 46 (2012).

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Foreword

Financial & competing interests disclosure The author is employed by a CRO providing biomedical AMS services. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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