Use of Radiolabeled Compounds in Drug ... - ACS Publications

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Use of Radiolabeled Compounds in Drug Metabolism and Pharmacokinetic Studies Emre M. Isin,*,† Charles S. Elmore,‡ Göran N. Nilsson,‡ Richard A. Thompson,§ and Lars Weidolf § †

CVGI iMed DMPK, ADME Section ‡DMPK iMed, Screening & Profiling, Isotope Chemistry, and §DMPK iMed, Centre of Excellence, AstraZeneca R&D, Mölndal, SE 431 83 Sweden ABSTRACT: As part of the drug discovery and development process, it is important to understand the fate of the drug candidate in humans and the relevance of the animal species used for preclinical toxicity and pharmacodynamic studies. Therefore, various in vitro and in vivo studies are conducted during the different stages of the drug development process to elucidate the absorption, distribution, metabolism, and excretion properties of the drug candidate. Although state-of-the-art LC/MS techniques are commonly employed for these studies, radiolabeled molecules are still frequently required for the quantification of metabolites and to assess the retention and excretion of all drug related material without relying on structural information and MS ionization properties. In this perspective, we describe the activities of Isotope Chemistry at AstraZeneca and give a brief overview of different commonly used approaches for the preparation of 14C- and 3H-labeled drug candidates. Also various drug metabolism and pharmacokinetic studies utilizing radiolabeled drug candidates are presented with in-house examples where relevant. Finally, we outline strategic changes to our use of radiolabeled compounds in drug metabolism and pharmacokinetic studies, with an emphasis on delaying of in vivo studies employing radiolabeled drug molecules.

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CONTENTS

1. Introduction 2. Radiolabeling Efforts in Isotope Chemistry 3. Synthetic Strategies for Radiolabeling Commonly Employed at AstraZeneca 4. DMPK Studies Involving Radiolabeled Compounds at AstraZeneca 4.1. Covalent Binding 4.2. Quantitative Whole Body Autoradiography 4.3. ADME/Mass Balance (Preclinical and Clinical) Studies 4.4. Studies Related to Safety Testing of Drug Metabolites 5. Future Perspective and Concluding Thoughts Author Information Corresponding Author Notes Acknowledgments Abbreviations References

characterization of the pharmacokinetic parameters of the drug candidate. However, in addition to the parent compound, a good understanding of metabolites derived from the drug candidate is necessary in evaluating excretion routes, as well as the presence of active and/or reactive metabolites. As with the quantification of the parent compound, LC/MS has become the technique of choice for structural identification of metabolites. Recent advances in LC/MS technology, including increased sensitivity and facile collection of MS/MS data1−7 and automated data analysis approaches,2,3 make it feasible to identify and elucidate the structures of expected metabolites such as simple hydroxylations as well as unexpected metabolites such as ringopening and rearrangements.8,9 Despite all advances in MS technology, there are still some fundamental limitations of this technology, e.g., compounds need to be ionized to be detected by MS, metabolite MS responses are frequently different from that of the parent, and the MS response depends on the MS ion source features. This may not be a problem in the case of the candidate drug, whereas quantification of a metabolite of interest may be challenging since this will require the preparation of the synthetic standard for adequate quantification. Considering these caveats to the application of MS techniques to understand the fate of and to quantify drug candidates and their metabolites,

532 533 533 534 534 536 536 538 540 541 541 541 541 541 541

1. INTRODUCTION In the drug discovery and development process, it is of critical importance to be able to both predict and later on follow the fate of the drug candidate once administered to humans. LC/MS techniques are commonly used to quantify the parent molecule in both in vitro and in vivo samples as part of the © 2012 American Chemical Society

Special Issue: Use of Radioisotope-Labeled Compounds in Drug Metabolism and Safety Studies Received: December 1, 2011 Published: February 28, 2012 532

dx.doi.org/10.1021/tx2005212 | Chem. Res. Toxicol. 2012, 25, 532−542

Chemical Research in Toxicology

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Table 1. Summary of 14C-Labeled Compound Deliveries 2008 2009

total compds delivered

syntheses

repurifications

compds delivered for hADME

final compds outsourced

synthetic steps

144 152

85 71

47 74

5a 7b

12 7

295 325

a

Two compounds were synthesized in a combined total of 12 synthetic steps, and three were repurified from existing batches. bFour compounds were synthesized in a combined total of 20 synthetic steps, and three were repurified from existing batches.

pounds are whole body autoradiography (WBA), initial absorption, distribution, metabolism and excretion (ADME) work, covalent binding, and determination of certain physical properties (e.g., plasma protein binding). Generally, after the project selects a single compound to move toward the clinic, a 14Clabeled analogue is prepared, and the 14C-labeled material is used in the following studies over the course of the drug development process: nonclinical and clinical ADME studies, quantitative whole body autoradiography (QWBA), metabolite identification, cross-species comparison, environmental fate, and reactive metabolite formation. AstraZeneca utilizes a mixture of internal and external sources to deliver these compounds. In 2008 and 2009, we synthesized 75 14C-labeled compounds in an average of 4.0 synthetic steps per synthesis (Table 1). We also delivered 12 compounds over the two year period for use in human ADME studies; 6 of those compounds were synthesized specifically for the particular study (in 5.3 synthetic steps per preparation), and the remainder was prepared by repurifications of existing batches. The 3H deliveries varied much more from year-to-year with an average of 143 deliveries/year and 73 syntheses (Table 2). The average 3H

preparation of radiolabeled analogues of drug candidates becomes a necessity. Once the radiolabeled analogue is available, metabolite profiles of drug candidates in in vitro systems as well as in vivo can be generated unbiased by the ionizability of the metabolites. Furthermore, quantification of metabolites is relatively straightforward provided that the specific activity of the parent drug is known and the label is retained in the metabolite of interest. Also retention of the parent drug and/or metabolites in tissues, and covalent binding to proteins can be assessed without the need of characterizing the bound compound (vide infra). Therefore, despite (and owing to, in the case of accelerated mass spectrometry (AMS)) the recent advances in MS technology, the use of radiolabeled compounds in drug discovery and development is still important. In this perspective, considerations around different labeling options and approaches are discussed, and various applications of radiolabeled analogues to study the drug metabolism and pharmacokinetic (DMPK) properties of drug candidates are presented with relevant in-house examples. Finally, we describe changes to our global strategy with respect to the use of radiolabeled molecules in DMPK studies, which focuses on back-loading of in vivo studies that utilize radiolabeled drug candidates.

Table 2. Summary of 3H-Labeled Compound Deliveries

2. RADIOLABELING EFFORTS IN ISOTOPE CHEMISTRY Isotope Chemistry at AstraZeneca supports a wide range of internal customers with isotopically labeled and unlabeled compounds. In addition to radiolabeled molecules, these include metabolites and stable isotope labeled molecules that will not be discussed in this perspective. The radioactive compounds supplied are primarily 3H- and 14C-labeled. The 14C-labeled compounds are in most cases more difficult to prepare compared to 3 H-labeled compounds due to the fact that the 14C label has to be introduced early on in the synthetic route as part of a basic building block and hence requires the total synthesis of the drug candidate of interest. In approaching a 14C synthesis, the first step is to determine acceptable label locations; the label should be located in a portion of the molecule that will be in the most important or largest fragment of the molecule if cleaved by metabolism. This is based upon initial metabolism data and generally means the label must be in the core of the molecule. Introduction of the 3H label can in many cases be relatively easy. However, depending on the position, there is the risk of losing the 3H label in the form of tritiated water upon oxidative biotransformation of the parent drug.10 Another issue relating to the use of 3H-labeled material is the possibility of inducing metabolic isotope effects (a kinetic hydrogen isotope effect11,12 which could affect the rate of certain biotransformation reactions in vitro13 and in vivo14−16). Typically, early phase projects use 3H-labeled compounds for radioligand binding assays in an attempt to estimate the potency of a drug candidate toward the target receptor. Depending on the needs, mainly from Bioscience organization, some 125I labeled compounds are occasionally prepared by contract research organizations. DMPK related studies (Table 1) in early phases utilizing 3H-labeled com-

2008 2009

total compds delivered

syntheses

repurifications

final compds outsourced

synthetic steps

172 124

96 62

66 56

10 6

129 93

synthesis only required 1.4 synthetic steps to complete. On average, 14C compounds (not for human ADME use) were delivered in 2−4 months after the request was placed by the responsible scientist. This includes the time required to order reagents, conduct purity analyses after the synthesis, and formulate the compound. 3H syntheses typically took a month but were sometimes as short as three days when the need was immediate, provided that the compound was readily accessible and the labeling was carried out on the final compound of interest. Once the radiolabeled molecules are prepared, they are generally stored in a solution in ethanol at −20 or −80 °C to minimize degradation due to radiolysis. The scientist responsible for the assay/study orders and specifies the amount and purity of the labeled compound. The request has to be approved by the designated compound owner prior to being delivered to the assay/study responsible. This approach provides that compound delivery takes place efficiently from a central storage and the right amount and purity is supplied.

3. SYNTHETIC STRATEGIES FOR RADIOLABELING COMMONLY EMPLOYED AT ASTRAZENECA The methods used to incorporate radioactivity into molecules have been reviewed recently.17,18 The primary methods for incorporating a 3H label into a molecule fall into two broad categories: those which rely upon the synthesis of a precursor 533



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Scheme 1. Representative Example of Ir-Catalyzed Hydrogen Isotope Exchange (HIE) Reaction to Incorporate 3H2 Gas Directly into the Molecule

the synthesis to minimize the number of radiochemical steps and to use the label as the limiting reagent. There are a limited number of commercially available reagents for 14C synthesis, and all of these are prepared from Ba14CO3.18 Furthermore, for the past two years a shortage of Ba14CO3 has existed due to problems with the single reactor at which it was produced. It has become difficult to obtain basic starting materials, and the cost of those materials when offered for sale has increased. This shortage is projected to last at least to 2014. Typical 14C starting materials include carbon dioxide, metal cyanides, malonates, acetic acid (and other easily accessible acids), urea, and benzenes (via acetylene), and the more complex the reagent, the more costly it is.18 Many other reagents are available by custom synthesis, but the price rises accordingly, and there may be a significant delay in receiving the compound if it requires novel chemistry to be developed.

and those which use the final compound of interest directly. Ir-catalyzed hydrogen isotope exchange (HIE) pioneered by Heys and Hesk19,20 allows properly functionalized molecules to incorporate 3H2 gas directly into the molecule without any modifications (Scheme 1). The iridium complex first associates with the directing group (in this case the carbonyl of an amide) and then oxidatively inserts into the C−H bond of the ortho aromatic proton to give a 5-member or 6-member metallacycle. Ligand isomerization rapidly exchanges the aromatic derived proton with a 3H from 3H2 gas, and reductive elimination forms an aromatic-3H bond. Finally dissociation of the Ir complex from the directing group liberates the tritiated compound. This methodology has been extended to include N-heterocylic carbene containing catalysts which are some of the more powerful and selective catalysts available.21,22 It is common practice at AstraZeneca to assess the feasibility of incorporation using deuterium prior to moving to the synthesis of the radioactive material. The other broad method for incorporation of 3H involves the synthesis of an appropriate precursor which can then be reacted with 3H2 gas or another source of 3H. The most frequently used method employs aromatic halogenation of the final compound (typically with N-iodosuccinimide and trifluoro acetic acid) and subsequent reduction with Pd/C and 3H2 gas. In addition, a compound containing an aryl halide or double bond can also be synthesized to serve as the reactant for the reduction step. Other less prevalent methods include reduction with tritide sources (e.g., NaB3H4), methylation using [3H]-methyl sources (e.g., C3H3I), and acetylation using 3H containing acid chlorides.23 The formation of a C−C bond is more challenging, and the required reagents are more costly than for a 3H synthesis; thus, the synthesis of a 14C-labeled compound involves a larger resource commitment. In designing the synthesis, an assessment of the synthetic route to the unlabeled compound is performed to see if there is a convenient step for incorporation of the radiolabel. It is preferable to incorporate the radiolabel late in

4. DMPK STUDIES INVOLVING RADIOLABELED COMPOUNDS AT ASTRAZENECA As summarized in Figure 1 and briefly mentioned above, there are numerous DMPK studies which have had importance during the different stages of the drug development process. These studies are designed to increase the knowledge around the drug candidate and as the compound progresses through the later stages, to be able to take informed decisions with respect to the safety of the drug candidate in the clinic. The major DMPK studies are described below in detail with internal examples where relevant. 4.1. Covalent Binding. Covalent binding of drugs to biological macromolecules was described more than half a century ago24,25 and subsequently has been proposed to be linked to adverse drug reactions (ADR) in humans with idiosyncratic ADRs (IADR) being the major focus26,27 This proposed link placed a heavy emphasis on covalent binding studies utilizing radiolabeled drug molecules by the pharmaceutical industry in 534



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Figure 1. Summary of DMPK studies at AstraZeneca involving radiolabeled drug candidates with approximate timelines.

an attempt to minimize the ADR risk of the drug.28,29 In recent years, several pharmaceutical companies reported their findings on the potential link between covalent binding and idiosyncratic ADRs and/or hepatotoxicity.30−33 A careful consideration of these publications suggests that the best “correlation” between covalent binding to proteins and ADRs seems to be observed in studies conducted in human hepatocytes, and even then, there are exceptions where drug molecules leading to high covalent binding are safe, and drug molecules with negligible levels of covalent binding have been reported to lead to severe IADRs in patients. As part of the development of a new internal reactive metabolite strategy,34 AstraZeneca undertook an extensive study to assess the utility of covalent binding of drug candidates to human hepatocytes in risk assessment of drug candidates. The details of the risk assessment strategy taking covalent binding, the daily dose, and signals in a panel of in vitro hepatic toxicity assays will be described elsewhere (manuscript in preparation). In summary, our results demonstrated that covalent binding in human hepatocytes when (and only when) taken together with dose and hepatic screening score has high predictive value in filtering high risk drug candidates. For our covalent binding studies, we have adopted a modified version of the semiautomated method developed by Merck.35 As part of our validation work, we have prepared, purchased, or obtained through sharing with other pharmaceutical companies approximately 40 drug molecules with different ADR profiles, and the covalent binding propensities of these compounds were evaluated in cryopreserved human hepatocytes. Both 14C- and 3H-labeled drug molecules were utilized in the study, and good inter and intraday variability was obtained. Currently, as part of the new Reactive Metabolite Strategy, a covalent binding study for all drug candidates approaching a candidate selection decision is mandatory regardless of whether a reactive metabolite alert is present. These results are evaluated together with the predicted daily dose and hepatic screening panel score.34 Following the implementation of this new reactive metabolite strategy, the internal demand for covalent binding studies has increased. In many cases, covalent binding studies are triggered by an indication of reactive metabolite formation during biotransformation studies conducted as part of the lead generation and optimization processes. As a consequence, it is not uncommon to generate covalent binding data early in the lead optimization process and incorporate the outcome into the design−make−test−analyze cycle. (For a detailed description of the design−make−test−analyze

cycle at AstraZeneca, see the reference by Plowright et al.36) Because of the relative ease and speed of preparation, 3H labeling is preferred at this stage. A careful evaluation of biotransformation pathways of the drug candidate is necessary to make sure that the labeling position is not a metabolic soft spot, and hence the label is not lost via metabolism. During the covalent binding study, aliquots from the incubations are taken at the beginning and at the end of the incubation with the primary aim of estimating the turnover of the parent compound so that fraction of metabolites leading to covalent binding can be determined. These aliquots are also analyzed by LC/Radioactivity monitoring (RAM) for elution of 3H2O, which indicates the loss of the label by oxidative metabolism. During our validation work, we have compared the covalent binding levels of 3Hand 14C-labeled ibuprofen and verapamil in cryopreserved hepatocytes (see Figure 2 for labeling positions). For ibuprofen,

Figure 2. Labeling positions of ibuprofen and verapamil used in the covalent binding study. * denotes 14C; ¤ denotes 3H. Each nuclide was present in one molecular entity only.

the covalent binding levels for both isotopologues were virtually identical at 40 pmol drug eq./mg protein, and there was no indication of loss of the 3H label. However, the results obtained with verapamil was rather unexpected where the 14C-labeled drug (label being on the N-CH3) resulted in covalent binding levels eight times higher than that of the 3H-labeled drug (200 vs 25 pmol drug eq./mg protein). Supernatant analysis did not indicate the loss of 3H label from the 3H-labeled molecule via formation of 3H2O. However, a peak eluting with the solvent front was observed when the supernatants for the 14C-labeled verapamil were analyzed. This early eluting peak is likely to correspond to formaldehyde formed via oxidative N-demethylation of verapamil, which has been previously documented.37−39 In these previous metabolism studies, the major metabolite had been shown to be another N-dealkylation product where the 535



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further work with the compound of interest since the potential risk in humans due to observed retention of the candidate drug was deemed to be too high to be mitigated later on. Recent advances in mass spectrometry imaging (MSI) enabled this technology to be an alternative to QWBA studies in drug discovery.43−45 However, at this time, MSI has not been integrated into the DMPK working routines at AstraZeneca. 4.3. ADME/Mass Balance (Preclinical and Clinical) Studies. ADME/Mass balance (ADME/MB) studies have traditionally been among the key biotransformation studies during the course of the drug development program. Our clinical and preclinical mass balance studies are performed according to the standard designs generally employed in the industry. The radiolabeled test compound is administered to establish rates and routes of excretion of the parent compound and metabolites. As the name implies, the ultimate goal of these studies is to be able to account for the amount of drug administered, and use of radiolabeled material is necessary to achieve this aim. In addition to the QWBA data mentioned above, a rodent mass balance study providing excretion routes and rates of radiolabeled material is a required part of the dosimetry calculations for regulatory approval to perform human studies using the radiolabeled drug in some countries. Although we prefer to use 14C-labeled material for the mass balance studies, there are no regulatory requirements on whether to use 14C or 3H provided that the same isotope used for the preclinical ADME/MB is also used in the human studies. All aspects of the ADME/MB may not be covered in every study. Generally, urine and feces are collected separately and quantitatively at predetermined time intervals following the administration of the dose, e.g., 0−6, 6−12, 12−24, 24−48 h, etc., for urine, while feces are collected in 24 h intervals. More frequent blood sampling is performed to generate pharmacokinetic data for the parent and metabolites. The route of dose administration may be limited to that intended in the clinic, and thus, the absolute bioavailability of a drug intended for oral use will not be established in those studies. Historically, for a drug that is successfully developed through to regulatory submission, preclinical ADME/MB or metabolite profiling studies would have been performed in most animal species employed in the safety assessment of the drug candidate. In general, this would include rat (male and female separately), dog, and mouse to support the general toxicology, carcinogenicity, embryofetal development studies, and studies to assess drug and metabolite excretion into milk of lactating animals. Occasionally, we have included the intravenous route of administration to estimate the bioavailability or fraction absorbed of the radiolabeled material. The radiolabeled clinical ADME/MB study is generally performed in a small group of healthy volunteers (or patients for oncology projects) of less than 10 subjects. If not frontloaded based on issues related to metabolism of the drug candidate (e.g., discovery of active or reactive metabolites), this study is performed later but in time to support applications to start large clinical trials, as directed by regulatory guidance, i.e., phase III in drug development. For preclinical and clinical ADME/MB studies performed for regulatory submission, our standard protocols state that the recovery of radioactivity via excretion should be monitored until a mean of 95% of the dose is recovered or, if this is not achieved within seven days, until less than a mean of 1% of dose is excreted within a 24-h interval. A low recovery or slow elimination rate may indicate irreversible interactions between drug-related material and endogenous macromolecules and will usually trigger further investigation. In addition to establishing the rates and

Figure 3. Elimination of 14C- and 3H-labeled omeprazole in rat blood.

right side of the molecule possessing our 3H label is oxidatively cleaved (which is consistent with our hepatocyte work). One possible explanation for the higher covalent binding observed for 14C-labeled verapamil, is the contribution of the left side of the molecule to reactive metabolite formation to a larger extent (e.g., imine formation via α-carbon oxidation pathway) than the right side of the molecule, although covalent binding of 14 C formaldehyde, formed via N-demethylation, to proteins cannot be excluded. Regardless of the reasons leading to this difference, the verapamil example demonstrates the importance of understanding the biotransformation pathways prior to choosing the labeling position on the molecule in particular for molecules likely to undergo cleavage reactions. 4.2. Quantitative Whole Body Autoradiography. QWBA is a technique for imaging the distribution and time course of elimination of a radiolabeled drug and its metabolites, commonly performed in rodents.40−42 The obvious advantage of the QWBA studies is to assess whether all the drug related material can be completely eliminated once the drug is administered without the need for characterization of metabolites derived from the drug candidate. At AstraZeneca, these studies are performed typically in the following way: Rodents are sacrificed at predetermined time points after administration of the radiolabeled drug, frozen, and tissue slices produced using a microtome tissue slicer. The slices are then exposed to photographic emulsions or phosphoimaging devices for localization and quantification of radiolabeled material in selected tissues. These studies can be performed to evaluate tissue retention in a limited set of tissues, e.g., skin and the eye, to support phototoxicity assessment, or on a large selection of tissues to assess whether some tissues retain drug and metabolites stronger than others. In the case of strong retention, we have developed a procedure of exhaustive solvent washing of tissues slices. Radioactive material not removed via such solvent washing may indicate covalent binding to macromolecules and may require further investigations in, e.g., human hepatocytes to provide data on human safety hazard and risk assessment. Data from QWBA studies are utilized in AstraZeneca to assess the potential for the accumulation of drug and metabolites, but also in support of regulatory approval to perform human studies employing the radiolabeled compound. On the basis of the tissue elimination rate, dosimetry calculations are performed to set the maximum radiolabeled dose to humans. As part of our drug development programs, we have in a few cases taken advantage of QWBA studies following candidate drug selection to support high level decisions on project progression. Recently, the strong retention of a drug candidate resulted in the termination of 536



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Table 3. Comparison of Information Obtained from in Vivo ADME Studies Using Either the 12C or 12C/14C Drug Candidatea metabolite profiles analytical technique

excretion route (% dose)

excretion rate (% dose/hour)

plasma

urine

feces

bile

metabolite quantification

isotope clusters

metabolite identification

LC/MS [12C] LC/MS [12C/14C]

low high

low high

int. high

int. high

low high

int. high

int. high

NA high

high high

a

Quality of information obtained is indicated as low, intermediate (int.), or high. LC/MS: liquid chromatography hyphenated with high resolution accurate mass spectrometry. LC/RAM-MS: liquid chromatography hyphenated with radioactivity monitoring and high resolution accurate mass spectrometry.

the point, we include here the example of an AstraZeneca drug candidate H 259/31. A partial metabolism scheme and the positions of radiolabeled isotopologs of H 259/31, a compound from our proton pump development program, are shown in Scheme 2. On the

routes of excretion, samples generated in an ADME/MB study are used for metabolite profiling and identification. Metabolite profiling utilizes the full benefit of the radiolabel in that optimization of chromatographic conditions for separation and quantification of metabolites can be achieved without the need for authentic metabolite standard. Metabolite profiling based on radioactivity monitoring of the LC eluent will provide the number of metabolites excreted via each route as well as the quantity (% of dose) or concentration (e.g., μmolEq/L). The amount of radioactivity in the analyzed sample will guide us to use either flow-through RAM for high levels, e.g., preclinical urine or bile samples, or fraction collection into 96-well plates and static plate reader liquid scintillation counting analysis (LSC) for low levels, e.g., plasma. Metabolite structural characterization is initially done using mass spectrometry followed by NMR if required. The doses administered in these studies are generally composed of a mixture of the labeled and unlabeled species. If the dose levels allow, the compounds are mixed with the radiolabeled compound constituting at least 20% and preferably closer to 50% of the total dose given. This allows for facile metabolite scouting by mass spectrometry in the very complex matrix of samples generated in vivo because the isotope clusters of the unlabeled and 14C-labeled protonated molecules (e.g., MH+/ [M+2]H+) can be distinguished and the relationship to the administered drug candidate established. Also, the LC/RAM/MS analysis will indicate whether one or more metabolites are not detectable by MS due to low MS response, while for those that are detected, the protonated molecule cluster will aid in metabolite scouting. In order to obtain further information on the ADME properties of a drug candidate and its metabolites that are not accessible in clinical studies, we frequently perform additional preclinical studies as in the case of bile duct cannulated animals. Collection and analysis of bile can be used for comparison of metabolite profiles in bile and feces to establish whether the anaerobic environment and microflora in the intestine have an impact on, for example, reductive metabolism or hydrolysis of conjugated metabolites. Also, by analyzing urine, bile, and feces we assess whether the drug clearance is governed by metabolism or excretion of the unchanged parent compound. The differences in quality and amount of information generated between unlabeled and radiolabeled ADME/MB studies are summarized in Table 3. It is obvious that a radiolabeled study will provide more information but at a higher cost and time consumption. The selection of the position of a radiolabel requires careful consideration to obtain the maximum benefit and information from these rather costly studies. In the event that a major biotransformation reaction results in the cleavage of a certain moiety of a drug candidate, depending on the position of the radiolabel, it may not be possible to follow an important biotransformation pathway. This may trigger the synthesis of several labeled versions of the drug molecule and administration of each entity in separate studies or as a mixture in the same study. To demonstrate

Scheme 2. Partial Biotransformation Pathways of AstraZeneca Drug Candidate H 259/31

* Denotes 14C; ¤ Denotes 3H. Each nuclide was present in one molecular entity only.

basis of earlier findings, the cleavage pathway induced by glutathione attack on the carbon-2 of the benzimidazole moiety was expected.46 In order to quantify this pathway and to monitor the subsequent metabolic fate of the two cleavage products, we synthesized two radiolabeled compounds, one with 3H label in the pyridine and one with 14C label in the benzimidazole moieties. These two radiolabeled isotopologues of H 259/31 were mixed and administered to rats and dogs. Urine samples analyzed using LC/RAM allowed for acquisition of the individual chromatograms using energy windows focused on each nuclide. In the urine samples, the cleavage pathway amounted to 10 to 15% of the dose given, with the N-acetylcysteine conjugate of the benzimidazole moiety being the major product of the right-hand side of the molecule. The pyridinylsulfenic acid metabolite was further converted enzymatically to several products, sequentially formed via reduction of the sulfenic acid to thiol followed by S-methylation and oxidation to the sulfoxide and sulfone. We also synthesized the cyclopropylmethyl 14C-labeled isotopomer to investigate the mechanism behind findings in 537



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Scheme 3. Modification of Rat Hemoglobin β-Chain (Cys-125) by Omeprazolea

a

* denotes 14C; ¤ denotes 3H. Each nuclide was present in one molecular entity only.

shown in Scheme 3 would then be analogous to that of the triazine herbicides. The prolonged elimination of 14C-labeled omeprazole was not observed in the human ADME/MB study, which is explained by the lack of adequately exposed free thiols in human hemoglobin.50 4.4. Studies Related to Safety Testing of Drug Metabolites. A basis for safety testing in the pharmaceutical industry is that the preclinical toxicity species are adequate surrogates for humans. An important part of this is an understanding of the exposure of not only the parent compound but also its metabolites. The studies performed to gain this understanding are usually referred to as Metabolites In Safety Testing (MIST) studies, which has been the topic of a previous Special Issue of this journal.51 It is fair to say that the publication of the Food and Drug Administration (FDA)’s Guidance for Industry: Safety Testing of Drug Metabolites,52 increased our attention and those of our colleagues in the pharmaceutical industry on drug metabolism studies that most likely would not have occurred otherwise. Not that safety testing of metabolites had been neglected up until then, but the harmonization that followed on how we look at and what we look for in metabolites has significantly boosted the interest and scientific efforts in the field of drug metabolism. The FDA Guidance is colloquially referred to as the MIST Guidance, and it was followed a year later by guidance from the International Conference on Harmonization (ICH M3 (R2))53 providing essentially guidance similar to that of the FDA but on a global agreement level. The top level guidance from these documents tells us that the first evaluation of metabolite exposures starts with metabolite analysis in plasma samples from the first clinical studies, preferably after repeat rather than single dose administration. These studies are almost exclusively performed using the unlabeled drug candidate. From these studies, data on metabolite exposures in humans will guide us to define which metabolites are of concern and need further evaluation of exposure in the animal species used in the safety assessment of the compound. Prior to or in parallel to this stage, however, biotransformation teams perform preclinical metabolism studies using the radiolabeled drug candidate, preferably incorporating 14C. The in vitro metabolism cross-species comparison study is an early study performed after the compound has been selected for development to clinical testing. These in vitro studies are conducted in suspensions of primary hepatocytes from humans and the rodent and nonrodent animal species to be included in the first pivotal toxicity studies. The purpose of these studies is to see whether metabolites observed in human in vitro systems are also observed at comparable levels in animals so that the safety

dog toxicity studies and if they were related to metabolites of the drug candidate. These findings consisted of accumulation of fat in the liver and also depletion of carnitine in muscle tissue and did not present themselves in the rat. Previous literature reports47,48 led us to postulate that these findings might be caused by the formation of a carnitine conjugate of cyclopropyl carboxylic acid (CPCA) generated via pyridine O-dealkylation and subsequent oxidation. The 14C-labeled compound was administered to rats, dogs, and humans, and urinary metabolite profiles were recorded. On the basis of these studies, we were able to conclude that all species formed significant amounts of CPCA and that the rat favored conjugation with glycine and the dog with carnitine and that humans formed about equal amounts of each. These very polar and low molecular weight metabolites were difficult to retain and separate from endogenous interferences using LC/MS, but the quantification was facile using LC/RAM. The link between CPCA-carnitine and the toxicity findings in dog was corroborated in a dog study in which the dog chow was supplemented with carnitine. In this study, no toxicity findings related to CPCA were detected. Thus, the toxicity findings were explained by carnitine depletion via CPCA conjugation, possibly leading to the inhibition of fatty acid transport into mitochondria via palmitoylcarnitine transferase (unpublished studies conducted at Gastrointestinal DMPK, AstraZeneca R&D, Mölndal, Sweden). Another example to demonstrate the power of radiolabeling different parts of the molecule is given by early metabolism studies in rats using omeprazole, our original proton pump inhibitor, where we noticed very slow elimination of 14C from the blood when the compound was labeled with 14C in the benzimidazole moiety (unpublished results). The elimination appeared to follow zero order kinetics with a time course similar to that of the turnover of red blood cells. In an attempt to explain this finding, we repeated the study, dosing the rats orally (100 μmol/kg) with a mixture of unlabeled omeprazole, omeprazole with a 3H label in the pyridine moiety, and omeprazole with the benzimidazole moiety labeled with 14C and followed the elimination from blood. The data again indicated the very long half-life of 14C, while the 3H-labeled material declined much more rapidly and with a more typical pharmacokinetic profile (Figure 3). On the basis of studies in the public domain, we explained the different elimination kinetics between 3H and 14C with a mechanism similar to what has been shown for triazine herbicides in the rat.49 The Cys-125 thiol residue of a β-chain of rat hemoglobin is exposed to the outer surface of the protein and acts as an efficient trapper of electrophilic substrates. The proposed mechanism for omeprazole 14C retention 538



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Figure 4. In vitro metabolite profiles of esomeprazole in hepatocytes from a female (A) rat, (B) dog, and (C) human. Indicated metabolites have been identified to form via demethylation and hydroxylation (M1), demethylation (M2), hydroxylation (M3 and M4), demethylation and reduction (M5), and sulfoxidation (M6) of esomeprazole.

testing of metabolites can be done adequately before moving into humans. Typically, a standard protocol utilizes 10 μmol/L of the [14C]-labeled substrate, and cells (one or two million cells/mL) are incubated 2 to 5 h. The samples generated are analyzed by LC/RAM and high resolution accurate MS, to provide metabolite profiles (number and relative amounts of metabolites) and preliminary structural assignment. Comparison of findings across species will provide knowledge on metabolic turnover of the parent compound and whether biotransformation pathways are similar. An example of a typical in vitro hepatocyte metabolism cross-species comparison study is given in Figure 4 where the biotransformation pathways of esomeprazole (the (S)-enantiomer of omeprazole) are compared between different species. In this particular case, all metabolites formed in human hepatocytes are also observed in the metabolite profiles from at least one of the other species. In the instance that we see major quantitative differences in metabolite formation between species, with human hepatocytes being more efficient than, e.g., the rat, we frequently perform a limited in vivo metabolism/excretion study in rats to generate plasma, urine, and bile samples that will provide an in vitro in vivo comparison from rat hepatocytes to the in vivo situation. With the intact rat being a highly effective metabolite factory, we most often find that metabolites that were considered minor in rat hepatocytes are formed at significant levels in vivo. This will give us confidence in using the rat as the rodent tox species and to continue the development program toward First Time in Man (FTIM) studies. If there are no issues requiring targeted problem solving studies, the study to follow the in vitro cross-species study utilizing the radiolabeled compound will be the regulatory rodent ADME study that will provide us with knowledge on the rates and routes of excretion of total drug related material based on measurement of radioactivity by LSC.

Our strategy for biotransformation work along the MIST evaluation process recommends that, if not performed previously, the regulatory radiolabeled rat ADME study is performed before or in parallel with the FTIM single ascending dose study at the latest. This is motivated by the amount of useful knowledge generated from analysis of the rat samples prior to addressing those from the unlabeled clinical studies. Comparison of the chromatograms generated from MS or RAM detection provides a first indication of whether there are significant differences in MS response for metabolites in relation to the absolute quantities given by RAM. Such data has been used to generate response factors to be applied to backcalculate actual concentrations of metabolites based on MS peak areas in those studies where the unlabeled compound was administered.54,55 Recently, other approaches to estimating metabolite coverage in preclinical vs clinical studies have been proposed,56−59 which we are currently evaluating for inclusion in our MIST strategy. In such analyses, the actual concentrations of metabolites are not determined, but the comparisons are rather based on peak areas alone. This can be rationalized by preparation of the samples to cancel out differences in sample matrix, investigation of the linearity of the MS response of the metabolites in the relevant concentration range, and time proportional pooling of plasma samples60 to cover the same time range after dose administration in the investigated animal species and humans. It is acknowledged that there frequently are quantitative and qualitative differences in metabolites formed in the rat compared to those in humans, but knowledge generated in the rat ADME study is nonetheless of great value in preparation for metabolite analysis in the unlabeled FTIM and preclinical toxicology studies. We generally perform the in vivo MIST metabolite exposure evaluation based on analysis of clinical samples from the 539



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as support to carcinogenicity studies and possibly rabbits as support to reproductive toxicology. Often the rat ADME/MB studies could be front-loaded to take place before the first dosing to humans. Obach et al. recently proposed a strategic direction where the only ADME/MB study routinely performed would be the human study.62 The authors give three key arguments for this: (1) the improved clarity from the MIST guidance (assessment of circulating metabolites), (2) improved analytical technologies that facilitate this assessment even with unlabeled compounds, and (3) the need to focus on whether human metabolites are present in the relevant preclinical toxicity species and not on obtaining a complete understanding of the metabolic profiles of drug candidate in animals. In many ways, we agree with the authors that we need to rethink this routine use of ADME/MB studies; however, one area where we have concern is the emphasis on assessing only circulating metabolites. We are of the opinion that a full understanding of the biotransformation pathways of a drug candidate in at least one other species besides humans is important. The rat ADME/MB study will provide information on potentially active or reactive metabolites that are not found at high levels in circulation. For example, a significant fraction of the dose may be excreted into bile as conjugated metabolites while being more or less undetected in blood. The value of being able to track and quantify such metabolites using the radiolabeled drug candidate should not be underestimated. Also, it is important to note that what is under discussion is the routine use of these studies, and there are of course a number of circumstances under which ADME/MB studies would still be critical. We have recently launched a new strategy to address reactive metabolite risks based on the combined read out from a set of in vitro toxicity assays and covalent binding in human hepatocytes.34 Our expectations are that this shift to a stronger emphasis on covalent binding will increase the demand on radiolabeled drug candidates rather early in the drug development process resulting in a net increase in radiolabeling efforts. From our in-house experience, we are of the opinion that with careful evaluation of biotransformation pathways, 3H labeling can be readily accessible during the early stages of the drug discovery process with significantly less cost and faster synthesis times than 14C labeling while providing the same quality of data. In addition to covalent binding data, the availability of the 3H-labeled drug candidate early is likely to facilitate the generation of quantitative biotransformation data which in turn will minimize the possibility of last minute surprises with respect to previously unidentified biotransformation pathways. In conclusion, we see a shift in the use of radiolabeled compounds in DMPK studies moving forward. Our view is that we as an industry are moving away from the routine use of a large numbers of ADME/MB studies. From our perspective, we can see a program based on a strong understanding of the metabolism of the compound at candidate selection complemented with covalent binding studies in human hepatocytes and an in vitro cross-species comparison of metabolite profiles in hepatocytes. Following candidate selection, the understanding of the relevance of our preclinical toxicity species to safety testing in humans would shift to being primarily based on analysis of circulating metabolites using unlabeled compound. However, we would still see the combined use of the rat QWBA and ADME/MB as important to understanding the full metabolism picture of the drug candidate. Also, while we see a

multiple ascending dose study in comparison with animal exposures in repeat dose toxicity studies. Using state of the art technology, i.e., high resolution accurate MS and software tools for metabolite evaluation operated by biotransformation scientists, we generally rely on the exposure comparisons generated from preclinical and clinical studies where the unlabeled drug candidate was administered. However, further on in the development of the drug candidate in vivo ADME studies utilizing the radiolabeled compound are performed in other animal species used in the safety assessment of the drug candidate as well as in humans. Data from the radiolabeled ADME study in humans will confirm whether all metabolites of concern were evaluated in the unlabeled studies during the early development phase, bearing in mind that the MIST evaluations are based on repeat dose studies, while ADME studies are single dose only.

5. FUTURE PERSPECTIVE AND CONCLUDING THOUGHTS Despite the recent advances in LC/MS techniques, radiolabeled compounds still have an important place in the drug discovery and development process. This article to a large extent describes how and when we have worked historically with radiolabeled compounds. There is, however, internally within AstraZeneca, as well as in other pharmaceutical companies, an ongoing discussion around the future direction of the use of a number of the radiolabeled studies discussed in this article, the two key points being: (1) assessment of circulating metabolites in humans vs preclinical toxicity species and (2) timing of ADME/MB studies.61 The focus of this debate is on the value and not least the timing of some of these studies in relation to the benefit they bring to safety assessment in particular. The main drivers are the desire within the pharmaceutical industry to reduce or replace animal use when possible and to optimize the use of resources in projects, i.e., what is really decision making. When it comes to the assessment of circulating metabolites in humans versus preclinical toxicity species, alternate approaches have been proposed to follow the fate of drug candidates in preclinical studies in relation to MIST without the need of radiolabeled molecules.56−59 These approaches involve direct comparison of metabolite MS peak areas rather than actual concentrations and may decrease the need for radiolabeled molecules for quantification of metabolites in preclinical species. The clear advantages with such methods are that the relationship between concentration and MS response does not have to be assessed for each metabolite, neither via the determination of response factors, e.g., via radioactive versus MS response nor via access to authentic metabolite standards. It is also not necessary to quantify metabolites in order to determine their fraction of total drug related material and whether they fall above a threshold of concern, as the comparison between systemic exposure levels in animals and humans can be done on any metabolite as long as it is detected by MS. AstraZeneca biotransformation scientists are currently developing this concept for inclusion in our MIST evaluation strategy with the expected outcome that MIST assessment will be simpler, faster, and less resource demanding. As we described previously, a routine ADME/MB package could include studies performed in humans, rats, and nonrodents (usually dogs) and then complemented with mice 540



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decrease in a wide scale routine use of, e.g., ADME/MB studies, they will still retain value as issue driven studies.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Ryan Bragg, Johan Sandell, John Harding, and David Wilkinson for collecting the historic data included in this perspective.

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ABBREVIATIONS AMS, accelerated mass spectrometry; DMPK, drug metabolism and pharmacokinetics; WBA, whole body autoradiography; ADME, absorption, distribution, metabolism, and excretion; QWBA, quantitative whole body autoradiography; HIE, hydrogen isotope exchange; ADR, adverse drug reactions; IADR, idiosyncratic adverse drug reactions; RAM, radioactivity monitoring; MSI, mass spectrometry imaging; ADME/MB, absorption, distribution, metabolism, and excretion/mass balance; LSC, liquid scintillation counting; CPCA, cyclopropyl carboxylic acid; MIST, metabolites in safety testing; FDA, food and drug administration; FTIM, first time in man

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