Introduction: Use of Radioactive Compounds in Drug Discovery and Development

I was introduced to the use of radioactivity as a sophomore in college, in the laboratories I was doing research in (I even used a planchet counter, a piece of equipment that only a few of our readers have probably ever seen). At that time, radiation training was rather empirical. As an undergraduate and in graduate school, the liquid scintillation counters in the departments were very popular instruments and always busy. I was thrilled to finally be able to afford my own and not have to queue up for time on others. Today radioactive methods have lost some of their previous popularity, in the context of the development of some other approaches, e.g. mass spectrometry, and pressures to reduce the generation of radioactive waste. Nevertheless, we still routinely use ³²P in our DNA polymerase work and both ³H and ¹⁴C in many enzyme reactions in our laboratory.

Dr. Abdul Mutlib suggested that we feature a series of articles for a special issue regarding uses of radioactivity, particularly in regard to safety assessment and metabolism issues in the pharmaceutical industry. Radioactivity does have certain distinct advantages, particularly in consideration of some of the quantitative needs associated with the “metabolites in safety testing” (MIST) developments.^1,2 What are some of the issues and questions regarding radioactive methods in drug metabolism and safety assessment?

Several questions arise at the discovery stage, i.e. in this case doing “discovery metabolism” or “discovery toxicology.”³ Are radiochemical studies in order? Is ³H or ¹⁴C (or other, e.g. ¹²⁵I) most appropriate? Some issues include in vitro microsomal covalent protein binding assays to evaluate potential safety risks, in vivo covalent binding assays (for the same goal), and addressing mass balance, routes of excretion, and metabolic routes to understand high clearance problems and accumulation in organs. In these aspects, whole body autoradiography can be very useful. One of the general issues is whether or not radioactivity is necessary in the case of each project. Radioactive chemicals can be useful but do require resources (particularly in cases where the incorporation of ¹⁴C label is not straightforward), which must be committed at a time when a definite lead molecule has not been identified yet. Moreover, some alternate approaches are “label-free,” e.g. NMR spectroscopy and mass spectrometry. Is the added quantitative information worth the allocation of resources (including time) for radiolabeling?

At the stage of the interface between discovery and preclinical development, the availability of radiolabeled material can be useful in pharmacokinetic/pharmacodynamic studies, toxicokinetics, and other issues in toxicology and safety pharmacology. Radiolabeled materials can greatly facilitate studies of routes of metabolism. The quantitative aspect of radioactivity is invaluable in establishing pharmacokinetic patterns when structures of metabolites are unknown (and their UV spectra and relative MS ionization responses are not known). In some cases, accelerator mass spectrometry (AMS) and microdosing have been used at this stage to expedite in vivo studies with humans.

A variety of additional questions arise as a drug proceeds into a more “classic” development phase, and some of these can be facilitated with the use of radioactivity. When should mass balance studies be done in various preclinical species and humans?⁴ What study design should be used? Should mass balance be done in intact animals or those with cannulated bile ducts? What should be the duration of studies? Should whole body autoradiography be done to address distribution? How can (all of) this information be efficiently utilized?

Further, which questions require radioactive material and which might be better addressed with alternate methods? Can MIST issues be addressed without the use of radiolabeled materials? Are high-resolution mass spectrometry (HRMS) methods appropriate to circumvent the need for radioactive material in identifying metabolites in complex mixtures? Can AMS be used to reduce the amount of radioactivity required, and is the information good enough to extrapolate to higher dose situations? Is the information obtained from AMS and non-radioactive approaches going to be acceptable to the U.S. Food and Drug Administration (FDA) and other regulatory agencies?

Further, how critical are preclinical radiolabel studies? Are some proposed only to satisfy anticipated questions from regulatory bodies and do the studies have intrinsic merit in extrapolating to questions of human safety? Are species differences being considered appropriately? Are whole body autoradiography studies useful for the particular project, and what questions are being addressed that will ultimately have bearing on issues of metabolism and safety in humans? What are the regulatory expectations for Investigational New Drug (IND) and New Drug Application (NDA) submissions at the U.S. FDA? Will these be different and additional studies needed for Japan, the European Union, and World Harmonization regulations? How does the nature of the particular drug and disease influence the decisions about strategies? Time is of the essence in pharmaceutical development, and choices are critical regarding which strategies can be used most appropriately in terms of operational timelines and the resources that can be realistically expected to be devoted to a particular project.

This introduction covers a wide variety of issues in pharmaceutical discovery and development. The overall process is complex, difficult, and expensive, so critical decisions must be made as to the approaches to issues of metabolism and safety assessment and how the work will be most effectively integrated into discovery, development, clinical trials, and even postmarket surveillance. We have chosen to focus on the use of radioactive methods and alternatives in this special issue. The papers can be currently summarized (at least by this author) in concluding that (i) some issues require radioactivity (e.g. questions about covalent binding), (ii) some approaches that formerly were relegated to radioactive methods can often be done more appropriately by other methods (e.g., qualitative detection of most metabolites by HRMS mass defect screening,⁵ and (iii) some issues can be addressed using radioactivity or alternate approaches, depending upon the specific situation.

Five papers on aspects of the general subject are included in this issue. All come from industrial groups. The first is a general overview by Penner et al.⁶ and deals with the general issue of quantitation, which as mentioned earlier is of particular interest in the MIST issue. Radiolabeled studies are of use in estimating bioavailability and predicting drug-drug interactions in absorption/distribution/metabolism/excretion (ADME) studies. The second paper, by Isin et al.,⁷ describes the activities of the isotope chemistry group at a major pharmaceutical company, AstraZeneca, both in the context of labeling and application of radiolabeled compounds in development studies. Issues addressed include covalent binding and MIST considerations. Solon,⁸ in the third paper, deals with the subject of autoradiography including whole body autoradiography, quantitative whole body autoradiography, and microautoradiography. The fourth paper, by Zhang et al.,⁹ presents a specific case, a drug candidate at Bristol-Myers Squibb that showed adrenal toxicity. Radiolabeled material was used to help define the metabolites and the extent of covalent binding to protein, which in this case was correlated with toxicity. Of particular interest, one of the “typical” steroid-metabolizing cytochrome P450 enzymes (P450 11A1) was involved in activating the molecule. Finally, in the fifth paper, Mutlib et al.¹⁰ discuss the use of an alternate strategy, that of the use of ¹⁹F NMR spectroscopy for the quantitation of metabolites. Comparisons are made with radioactive measurements.

Collectively these papers address some, but by no means all, of the issues associated with applications of radioactive labeling in drug metabolism and safety assessment. Other aspects include AMS and microdosing, identification of covalently modified proteins, the viewpoints of regulatory agencies, and others. Although we were not able to cover all of these, many of these and other items are dealt with in the primary literature and review articles in Chemical Research in Toxicology, and we encourage you to read it regularly.

ABBREVIATIONS

ADME

absorption/distribution/metabolism/excretion

AMS

accelerator mass spectrometry

FDA

Food and Drug Administration

HRMS

high resolution mass spectrometry

IND

Investigational New Drug

MIST

metabolites in safety testing

NDA

New Drug Application