The relationship between a drug’s metabolism and its toxicology has been an issue of debate for a long time. A milestone in this debate was the publication of a paper in 2002 by Baillie et al. [1], which reported the outcome of a multi disciplinary committee sponsored by the pharmaceutical industry. The paper discussed the potential risk to human subjects from expo-sure to drug metabolites that may not be pres-ent in significant amounts in preclinical species. The presence of human specific metabolites may lead to toxic effects in humans but might be undetected in the toxicology species, where there was little or no exposure to the metabolite. In practice the occurrence of truly human-specific metabolites is rare [2], but significant quantita-tive differences between species are common and so the term ‘dis proportionate human metabolite’ to describe the situation where the exposure of a specific metabolite in humans was significantly greater than that in any of the toxicity species quickly became adopted [3]. Regulatory guid-ances followed, the first being published by the US FDA (published in draft form in 2005 and finalized in 2008 [4]). The ICH published their guidance in 2009 [5]. The focus of the guidelines, however, was upon what additional toxicology studies might be required if dispro-portionate human metabolites were discovered and some ambiguity remained on the way a dis-proportionate meta bolite was defined. The FDA guidance states that a metabolite ≥10% of the parent systemic exposure (AUC) at steady state is of concern, whereas the ICH M3 guideline
states that a metabolite ≥10% of the total drug-related exposure is of concern and no mention of steady state is made. As a harmonized guideline, the ICH document technically takes precedence over that from the FDA, and the proportion of metabolites should be expressed relative to total compound-related material. Nevertheless, there is still a good deal of discussion on this point within the industry and the approach adopted varies between companies. This arti-cle, therefore, addresses both the ICH and FDA guidance.
A number of other guidelines have also dis-cussed aspects of metabolites and safety. They emphasize that (major) human metabolites should be covered in safety pharmacology stud-ies (ICH guideline S7a [101]) and carcino genicity protocol submissions [102]. A special situation is the treatment of certain life-threatening indications where specific regulations apply, for example, for cancer indications [103]. In such cases an overall risk–benefit assessment is considered. As the FDA clearly states in their metabolites in safety testing (MIST) guidance, “Finding disproportionate drug metabolites late in development can potentially cause pro-gram and marketing delays.” The lack of reli-able quantitative exposure data of metabolites from early clinical trials can be problematic in a number of ways. For example, decisions have to be made on which metabolites should be monitored during clinical trials, which then makes it necessary to synthesize the appropriate standards. Commitment of the considerable
Meeting the MIST regulations: human metabolism in Phase I using AMS and a tiered bioanalytical approach
The metabolites in safety testing and ICH-M3 guidance documents emphasize the importance of metabolites when considering safety aspects for new drugs. Both guidances state that relevant metabolites should have safety coverage in humans (although the guidelines have different definitions of relevant metabolites). Not having safety coverage for important metabolites in humans may cause significant delay in the overall pharmaceutical development program. This article discusses the regulatory background regarding safety and metabolites, as well as outlines an integrated strategy taken by one pharmaceutical company, Lundbeck A/S. Lundbeck uses metabolite exposure data from first-in-man studies, obtained using an accelerator MS approach followed by a two-tiered bioanalytical investigation. This enables early availability of key data on this aspect and, overall, represents a powerful risk mitigation strategy.
Graham Lappin*1, Mark Seymour1, Gerhard Gross2, Martin Jørgensen2, Morten Kall2 & Lisbet Kværnø3
1Xceleron Inc., Seneca Meadows Parkway, Germantown, MA, USA 2Drug ADME Research, H Lundbeck A/S, 2500 Valby, Copenhagen, Denmark 3Process Research, H Lundbeck A/S, 2500 Valby, Copenhagen, Denmark *Author for correspondence: E-mail: graham.lappin @xceleron.com
resources these decisions demand will only be justified once the human adsorption, metabo-lism and excretion (AME)study, using isotopi-cally labeled compound, is completed. Having said that, many companies may choose which metabolite standards to synthesize much ear-lier, for example, based upon in vitro data (see below): the strategy is very company depen-dent. The problem is compounded by the ICH M3 guideline [5], which requires that metabolite exposures are expressed as a proportion of total compound-related material. The latter is very difficult to measure reliably in the absence of a radioisotope.
As a result, the pharmaceutical industry has been developing a number of strategic risk mitigation approaches to obtain information about metabolites, their systemic exposure and formation across species (with strong emphasis on human metabolism) early in development [6–12]. The strategy adopted by Lundbeck A/S will be discussed in more detail in this article.
One thing that the guidelines do agree upon, however, is that an assessment should be made of the metabolite profile in the toxicity species and in humans as early as possible, and cer-tainly before the start of Phase III when sig-nificant numbers of patients will be chronically exposed to the drug. The question then arises; how can this be achieved in practice? is the subject of this review.
Conventional approaches to obtaining human metabolism dataObtaining metabolite profiles in preclinical spe-cies is relatively straightforward and, although the use of nonisotopic approaches is becom-ing more common, it is typically achieved using compounds labeled with a radioisotope. However, using radioisotopes in human studies can be problematic because of the radioactive burden, which will be discussed in greater detail below. Tritium is used as an isotopic label; how-ever, the use of this isotope can suffer from tri-tium exchange and significant kinetic isotope effects and, therefore, its use is limited. The use of other isotopes (e.g., 35S) is restricted to com-pounds that contain the relevant atom in their chemical structure. The most favored isotope is 14C, which can be incorporated into the ‘carbon skeleton’ of the molecule where it is metaboli-cally stable and its kinetic isotope effects are minimal. It is, nevertheless, important to give consideration to where the 14C is incorporated into the chemical structure so that it follows
the core of the molecule. In some cases more than one radiolabeled form may be required, for example, where the molecule is cleaved into two or more major fragments. Notwithstanding the above, radiolabeled studies have been used for several decades in both preclinical and clinical regulatory mass balance studies. The presence of the radioisotope provides a means to reliably quantify parent drug or metabolites irrespec-tive of their chemical structure. In addition, because the overall recovery of radioactivity can be monitored there is little risk of miss-ing unusual or unpredicted metabolites [13]. Although both the FDA and ICH guidelines concentrate exclusively on circulating levels of metabolites, it is worth noting that the presence of the isotopic label facilitates the quantifica-tion of metabolites in excreta. Furthermore, because much more sample is available, ana-lysis of excreta is often essential for metabolite identification.
� Obtaining metabolism data without the use of radioisotopesThe historical difficulties of administering radioisotopes to humans have led to the use of nonisotopic methods to study a drug’s metabo-lism in humans in the early clinical phases of development (i.e., prior to the human AME study). A widely used approach to obtain-ing early human metabolism data is the ana-lysis of samples taken from nonradiolabeled Phase I studies, typically first-in-man (FIM) from the single ascending dose (SAD) study. High-resolution MS systems employing tech-niques such as mass defect filtering are often used. Although such approaches may provide valuable insight on the relative abundances of metabolites between species, the methods are not particularly quantitative in the absence of authentic standards. Certain technologies have been developed to reduce these effects, such as corona-charged aerosol detection [14] and nanospray ionization [10].
An alternative to MS is NMR, which is quantitative and can be used hyphenated with HPLC [14]. 1H-NMR is growing in its use and has the advantage that it is independent of the position of any isotopic labeling. NMR can also be particularly useful if the structure of the drug contains fluorine, in which case 19F-NMR can be applied using the 19F as a tracer in its own right [12]. In this case, 19F-NMR is just as reli-ant on the integrity of the fluorine atom on the molecular structure as accelerator MS (AMS)
Key Terms
Steady state: Set of conditions where the drug enters systemic circulation at the same rate that it is cleared.
Accelerator MS: MS method of analyzing isotope ratios by accelerating the isotopic ions to very high energies to facilitate their separation and measurement. Scintillation counting relies on the detection of relatively infrequent atomic decay events whilst accelerator MS measures the actual atoms themselves, making it at least 1 million-times more sensitive.
Dosimetry: The risks of exposing humans to radioactivity from 14C drug is assessed in a series of studies in which the 14C drug is administered to the animals to determine the routes and rates of excretion of radioactivity and the radioactive burden in the organs and tissues (typically conducted in the pigmented rat, using quantitative whole-body autoradiography). An accelerator MS-enabled study is unlikely to require dosimetry as the levels of radioactive exposure are extremely low.
is reliant on the metabolic stability of the 14C-labeling position. Overall, however, NMR is not a particularly sensitive technique, even when used with devices, such as cryoprobes [6] requiring microgram amounts of compound in order to obtain a reasonable spectrum. NMR, therefore, is used primarily where the doses are relatively high leading to sufficient systemic concentrations enabling measurement of metabolites down to 10% of the AUC.
These technologies, however, do not approach the robustness of radioisotopic methods for quantif ication, irrespective of the method of isotopic detection. The pres-ence of a radioisotope allows for the detection and quantification of an analyte, regardless of its chemical structure or physical proper-ties, without reference standards and without matrix effects associated with MS. Moreover, if unusual or unpredicted metabolites are formed in the human, then there is a chance they could be missed using MS methods. For these reasons, the use of 14C-labeled com-pound remains the ‘gold standard’ for drug metabolism studies, regardless of the species being studied. Samples taken from 14C animal metabolism studies can be used to estimate the relative abundance of the metabolites in non-labeled human samples using MS. The signal from the MS system is ‘calibrated’ against the metabolite concentration in the animal sample (which is known from the measurement of the radioisotope). The signal observed from the nonlabeled human samples can then be related to an approximate analyte concentration [12]. Differences in matrix effects between plasma from different species may be minimized by diluting human plasma with control plasma from the animal and vice versa.
The utility of AMS in providing early human metabolism dataThe human AME study with 14C-labeled compound is typically performed in late Phase I or Phase II, after the proof-of-concept studies. Leaving the acquisition of comparative metabo-lism data to this stage, however, can be a risky strategy because if disproportionate human metabolites are discovered, then unplanned delays and additional costs may be incurred. Perhaps more significantly, early metabolism data comparing preclinical species with humans can assist in choosing the most appropriate spe-cies for the pivotal safety toxicity studies. For these reasons, some pharmaceutical companies
considered bringing the human mass balance study forward to earlier in the program but this was unpopular as the expense of the study may end up being unnecessary if the drug failed at proof of concept. In addition, the human mass balance study requires dosimetry data to ensure the radioactive dose does not exceed regulatory or ethical limits.
For the reasons stated above, bringing the regulatory human metabolism AME study forward to early Phase I has been considered problematic. However, the advent of biomedical AMS has provided a solution. AMS was devel-oped in the 1970s for radiocarbon dating and was first applied to pharmaceutical development in the 1990s [8]. It is an ultra-sensitive isotope ratio technique based upon the mass separation of isotopes (12C/13C/14C) followed by their accu-rate and precise measurement. Sensitivity in the attogram (10-18 g) of 14C can be achieved [11]. The use of AMS has enabled a number of human metabolism studies to be performed where very low levels of radioactivity were administered, in the nanocurie (kBq) range [7,15–17]. From a regulatory perspective, such studies can be deemed nonradioactive and, therefore, do not generally require formal approval for the administration of radioactivity (although it is good practice to keep the regulator informed). These types of studies can, therefore, be con-ducted in regular clinics or even with patients [7]. The advent of AMS has, therefore, created the opportunity to include nanocurie amounts of 14C-labeled drug mixed with the nonlabeled test dose in FIM studies, such as the SAD study. Incorporation of 14C into a FIM study adds very little cost (assuming the 14C drug is available, which is discussed further below) and does not adversely affect the clinical objectives of the study. Samples collected from the study are then analyzed by AMS to determine total 14C con-tent (analogous to liquid scintillation measure-ments in a conventional AME study) followed by chromatographic separation, typically using HPLC. Fractions are collected (usually auto-mated in 96-well plates) and are analyzed by AMS to reveal the metabolic profile (Figure 1). Plasma can be collected for ana lysis but excreta should also be considered in order to fully meet the regulatory guidelines. Typically, plasma is pooled proportionally over the different time points to form an ‘AUC pool’ (the principles of which have been previously reported [18]). Pools can be generated across all subjects, groups of subjects or for individual subjects depending
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upon requirements. Excreta are typically pooled across time points (to include a minimum pro-portion of the total amount of radioactivity excreted) and profiled either individually or as a pool across subjects. Initial ana lysis of plasma for total 14C enables a concentration–time plot to be generated (mass equivalents of drug vs time), which can be used to select samples to construct an AUC pool for subsequent profil-ing (Figure 1). The peaks in the profile are pro-portional to the AUC and can be expressed as a percentage of the total drug-related material
(ICH guideline) or as a percentage of the par-ent drug (FDA guideline). By coordinating the chromatographic conditions, the profile from the Phase I study can be compared directly with those from the preclinical studies.
The principle disadvantages of using AMS in meeting the MIST guidance are that the 14C-labeled compound has to be available for administration to human volunteers and, because currently there is no direct interface between LC and AMS, the offline ana lysis takes several days to complete. For many
Figure 1. Metabolite profile obtained using HPLC and accelerator MS. An example from a recent Lundbeck A/S project of accelerator MS plasma data acquired from the inclusion of 500 nCi 14C drug in a Phase I clinical study. The 14C-labeled drug was given to the 4th cohort (six subjects) of a single ascending dose study, who received a dose of 10 mg. (A) The plasma was analyzed for total 14C and the plot was used to select the most appropriate samples for the time-proportional pool (AUC pool). Note: the Y-axis is expressed as disintegrations per min [dpm]/ml. (B) The chromatographic profile resulting from an AUC pool. The plasma pool was extracted (92% extraction efficiency) and analyzed by HPLC followed by accelerator MS of the individual fractions, shown as the data points in on the chromatogram. The data are quantitative, with the Y-axis showing percentage of total radioactivity. In this example, none of the metabolites exceeded the regulatory threshold (either 10% of parent or 10% of total compound-related material).
pharmaceutical companies, the 14C compound is often already available prior to FIM for pre-clinical studies and this can be used for the AMS MIST study (with certain caveats, see below). Graphitization extracts the carbon from a sam-ple by oxidation to CO
2 and then reduction to
elemental carbon. Although the process does take time, it is nevertheless the ultimate sample preparation method for eliminating matrix effects as all memory of the structure and matrix are lost in the formation of the graphite. This does, of course, mean that AMS cannot provide any structural information and for this MS and/or NMR is required. The techniques together, then AMS for quantification and MS/NMR for structural elucidation, can then be a particularly powerful combination.
� Quality considerations: 14C-labeled active pharmaceutical productThe requirements for the quality of the 14C active pharmaceutical product (API) to be administered to humans vary depending upon country and establishment. From a regulatory perspective, however, the 14C-labeled material may not have to be synthesized according to full GMP standards. Typically, a certificate of ana lysis is required, stating the radiochemical and chemical purities (both ≥95%). A BSE/TSE statement may also be required. The nonla-beled API, of course, must be of the required GMP standard whereas the amount of 14C compound mixed with it is extremely small. An AMS-enabled MIST study typically uses 100–1000 nCi, which for a 14C compound with a MW of 400 and a specific activity of 55 mCi/mmol (2 GBq/mmol) has a mass of between 0.73 and 7.3 µg. If, for example, the drug was administered as a 10 mg oral dose, then 1000 nCi of 14C material would consti-tute just 0.073% of the dose, which is below the threshold for the identification of impuri-ties in the API. As an alternative, to ensure all regulatory requirements are met, the 14C-labeled compound used for the animal metabolism studies (where available before the time of FIM studies) could be repurified according to GMP standards, thus obviating the need for a full GMP-compliant synthesis.
It should be stressed that, as for any clinical study, whatever the quality requirements for the synthesis of the 14C-labeled API, the preparation of the final dosage form (i.e., the manufacture of the IMP) should be conducted according to GMP standards.
� Dose considerations for AMS-enabled studiesAt the time of the FIM study, the clinical dose (or dose range) of the drug may not be known with any confidence. This, therefore, presents a difficulty in choosing the dose in which to include the trace of a 14C-labeled compound. There are a number of ways of dealing with this issue. A mid-range dose could be selected and some flexibility written into the protocol, to allow a choice to be made at the appropri-ate point during the study. The dose chosen is unlikely to be far from the final clinical dose, although there is some risk in this approach if the metabolite profile is sensitive to the dose administered. In one case, 14C-labeled drug was included in all doses of a SAD study from 1 to 10 mg (50–500 nCi) [19]. This approach is logistically challenging; however, it does have the advantage that any dose-dependent metabo-lism would become apparent. Another strategy is to administer the 14C-tracer dose to a separate cohort after the SAD data have been reviewed, perhaps as part of a food-effect investigation. It is a feature of this approach that the inclusion of a few µg of 14C-labeled compound will not compromise the initial objectives of the study to which the tracer is added.
Consideration should also be given to the physical form and formulation of the 14C-labeled dose. As with conventional radiolabeled studies, typically the 14C API is formulated as a simple solution whereas, if the tracer dose is being administered as part of a Phase I study with a primary objective other than metabolism (e.g., assessment of food effects), the nonradiolabeled compound may be given as a capsule or in a specific crystalline form. Even at the very low 14C doses used for AMS-enabled studies, it is unlikely that it will be possible to prepare the 14C API using the same processes as the nonlabeled material. Nevertheless, provided the bioavail-ability from the nonradiolabeled formulation is not extremely low, the tracer will always be handled by the body in the same way as the non-radiolabeled dose and, therefore, the metabolism information obtained will be relevant.
� Use of AMS to provide human metabolism data at steady stateAs discussed above, the FDA guideline [4] refers explicitly to the metabolite profile at steady state, whereas no mention of steady state is made in the ICH guideline [5]. Although the ICH guideline should take precedence, the
Key Terms
Active pharmaceutical product: The active drug substance, irrespective of any formulation.
BSE/TSE statement: A declaration that a compound to be dosed to humans is free from agents known to cause bovine spongiform encephalopathy or transmissible spongiform encephalopathy, required only for clinical studies conducted in the UK.
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question of steady state can nevertheless still arise. Using non isotopic techniques to analyze samples from Phase I studies involving multiple dosing can provide information on metabolism at steady state. The majority of studies involv-ing radiolabeled drug, however, continue to be single-dose studies. Indeed the examples given in the FDA guideline were all from single-dose AME studies. It is also worth noting that the meaning of the term ‘steady state’ in the FDA guideline is ambi guous. It is generally taken to mean steady state of the parent compound. However, if metabolites were formed with half-lives significantly longer than that of the parent, then they could take a very long time to reach steady state. Over time, their plasma concentra-tions would increase, thus producing a quanti-tatively changing metabolite profile until the point where the parent compound and all of its metabolites were in steady state (Figure 2). The advent of AMS as a bioanalytical technique, however, provides a potential means to address the steady-state question whilst retaining the quantitative advantages of using 14C-labeled compound.
Using AMS, a study could be performed where a pulse 14C dose is given along with the first nonlabeled dose in a repeat dose study, then a second pulse of 14C-labeled drug is given once steady state for the nonlabeled drug has been reached. Reports of these types of steady-state designs, in conjunction with AMS, have looked at changing pharmacokinetics between single and repeat doses [20]. Such a design could be incorporated into the multiple ascending dose study. If enzymes are induced or inhibited following repeat dosing, then this would be reflected by changes in the metabolite profiles obtained after single and multiple nonradiola-beled doses. If the question is one of accumulat-ing metabolites however, due to their half-lives being longer than the parent drug, then a pulse experiment will not suffice.
Because of the inherent sensitivity of AMS, the doses of 14C-labeled compound required to obtain quantitative metabolism data are very small. Consequently, it is possible to administer repeat doses of 14C drug to human subjects over a period long enough to achieve steady state, at least for the parent compound and, therefore, to observe any accumulation of metabolites. For example, some clinical facilities are able to administer up to 1000 nCi of radioactivity without formal approval from the regulatory authorities. The 1000 ni could, for example, be
split into ten 100 nCi doses, each of which is sufficient to allow detection in a biological sam-ple using AMS. Another approach to estimating the relative amounts of metabolite to parent at true steady state (i.e., the situation in Figure 2B) is to estimate the half-lives of the metabolites after a single dose. The steady state concentra-tions are reliant on half-life, dose administered and dosing interval and, therefore, can be cal-culated. By measuring the relative amounts of metabolite from the SAD study over time in the elimination phase, such estimates of steady-state concentrations could be made.
The potential for repeat dosing of 14C-labeled compounds to humans should, however, be balanced against other methodologies, such as the tiered approach proposed by EBF as a consolidated European industry view [21].
Although a metabolite profile at steady state can be achieved in humans as described above, it is worth remembering that the purpose of the MIST guidance is to compare the metabolic profiles between human and preclinical species. ADME studies in laboratory animals are nearly always conducted using single-dose studies and so for an entirely relevant comparison to be made, the animals would also have to be dosed to a steady state analogous to the human. Through a combination of ADME single-dose studies and bioanalytical investigations of metabolite exposure in multiple-dose toxicology studies, however, this can be elegantly resolved. Samples from single- and multiple-doses studies analyzed by quantitative bioanalytical methods offer an alternative approach. By the use of bioana lysis, exposure of relevant metabolites after multiple dosing can be measured in both human and ani-mals early in development to compare systemic exposure. This is only possible, however, due to the introduction of the 14C spike in SAD study to identify relevant human metabolites and sub-sequently synthesize these human metabolites as reference compounds early in development. The combination of an AMS-enabled study and regular MS in this context is particularly powerful.
� Use of early metabolism data to optimize the regulatory human AME studyAn often overlooked advantage in an AMS-enabled MIST study is the ability to obtain information on the rates and routes of excretion in humans. This information can be invaluable in the design of the regulatory mass balance study, conducted at a later date. Usually, the
choice of the duration of a regulatory mass balance study is a play-off between informa-tion on the rates of routes of excretion observed in the preclinical studies and bioanalytical data in humans for the parent drug and per-haps a selected number of metabolites. This information is then coupled with the practi-cal considerations of how long subjects can be kept confined in the clinic. A review in 2007, concluded that a recovery of approximately 80% was the ‘norm’ for human mass balance studies and some fell well short of this number [22]. Although not reported, it is intriguing to postulate how many of the studies with <80% recovery had to be repeated. If the study was left until the late stage of Phase II (which is the developing trend) then having to repeat the study could bring it onto the critical path, potentially causing considerable delays in the drug’s development. Data from an AMS-enabled Phase I study, however, can provide quantitative information on the elimination rates of radioactivity in humans, which is a far better predictor of the outcome of the regu-latory human AME study. Armed with such information, the regulatory mass balance study could be designed more appropriately and post-poning of the AME study would be associated with little risk of delaying the drugs develop-ment, as the risk of having to repeat the study should be negligible.
Of course, a question that arises if isoto-pic data are generated early is: ‘is a separate regulatory AME study actually necessary?’ Certainly, mass balance data generated using AMS are acceptable to the regulatory authori-ties [7]. If feces are collected as part of a Phase I tracer study, it would be feasible to use the results to provide definitive mass balance data, although in practice it may be advisable to dose a separate group for the purpose rather than run the study as an ‘add on’ to an existing phase of the study. As Roffey et al. emphasized in their review [22], low recovery of radioac-tivity does not necessarily compromise the important information generated from human AME studies.
Strategies to address MIST: an industry perspectiveLundbeck A/S discovered that in one of their projects a human disproportionate metabolite was apparent rather late in overall development. The human disproportionate metabolite was a Phase I metabolite resulting from a complicated
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Figure 2. An illustration of steady state. (A) Steady state, where the parent drug is at steady state but the systemic concentration of a metabolite is still increasing. (B) The AUC metabolite profile will reflect the relative amounts of parent and metabolite for a particular time (in this case 80 h). (C) Steady state, where the parent drug and a metabolite have both reached steady state. (D) The AUC metabolite profile (in this case at 150 h) will reflect the relative amounts of parent and metabolite. The proportion of the metabolite is now higher but should not change with time unless the steady state conditions are perturbed.
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sequence of several metabolic steps and rear-rangements. This finding caused a significant delay in the overall development. Based on that experience and to avoid similar events to take place in another program, a comprehensive risk mitigation strategy has been elaborated, as shown in Figure 3.
A cross-species (x-species) metabolism study in hepatocytes (the metabolically most competent system) is used to provide the first predictions about human metabolism and also to ‘validate’ the species used in toxicology studies. These experiments also enable the chromatographic procedure to separate parent compounds and its metabolites to be developed. Such an in vitro metabolism pattern does not always accurately predict systemic exposure of metabolites, how-ever, as elaborated in numerous publications [23–25]. Therefore, in vivo metabolism studies in animals are performed shortly after the in vitro x-species metabolism investigations to obtain pre-liminary information about the in vitro–in vivo correlation regarding metabolites, in particular, about their systemic exposure. This is followed by further synthesis of additional metabolite standards, if the in vivo results indicate this is appropriate. During the FIM study, low levels of 14C-labeled compound are added to one group of volunteers. Typically a mid-dose level is chosen, as such a dose is not too far away from the final clinical dose. Plasma and urine from the FIM are collected. As a fast and cost-efficient readout is required, these samples are normally pooled. After a work-up procedure they are subjected to HPLC and fractionated. Single fractions (typi-cally collected every 30 s) are then analyzed for their 14C content using AMS. Obtaining the radioactivity count for each fraction collected allows the construction of a complete metabolism profile of the entire sample, including the quan-tification of parent and metabolites. Typically, a plasma pool metabolism profile will provide:
�Conclusive information of systemic parent exposure versus metabolite;
�Metabolite profile: how many major and minor metabolites are present;
�Degree of systemic exposure of single metabolites versus parent;
�Tailored MS investigations of chromatographic fractions that represent metabolites.
As indicated in Figure 3, part of this strategy is also to synthesize metabolite standards very early,
even if it is not entirely clear which metabolites are relevant in terms of MIST and/or ICH M3 guidance at this point of development. Choosing this approach allows delays to be avoided, as any synthesized reference standard significantly facilitates definitive metabolite identification in any subsequent in vivo metabolism studies, such as rat and dog ADME.
The results from the metabolite profiling ana-lysis are followed up by bioanalytical ana lysis to quantify ‘relevant’ metabolites with a ‘fit-for-pur-pose’ assay [26,27]. The final step is a comparison of relative systemic exposure of metabolites in humans (from multiple-dose study) and animals (from toxicokinetic studies). Thus, Lundbeck is able to generate conclusive quantitative metabolism data as early as possible and take appropriate measures, if needed, without causing any delay in overall development. It goes with-out saying that such an approach requires close coordination between several functions within Lundbeck and close management of the overall activities.
ConclusionThe combination of early information from the metabolite profile in a FIM study, followed by a series of bioanalytical approaches in multiple-dose studies in humans and pivotal toxicology studies in animals represents a very powerful tool to obtain key metabolite exposure data very early as part of a risk mitigation strategy.
The addition to low levels of 14C in a FIM study followed by ana lysis of samples by AMS is providing valuable early quantitative and quali-tative early information on the metabolite pro-file in humans. Analysis of total 14C in plasma enables an informed choice to be made on the choice of samples used to produce the time-proportional AUC pool. The safety-relevant metabolites are then synthesized as bioanalytical standards and samples from multiple-dose stud-ies in humans and pivotal toxicology studies in animals are analyzed for metabolites to elaborate on their particular safety coverage. This way, all the key information is available at a very early stage of development.
Incorporation of a small amount of 14C in a FIM study allows the ana lysis of excreta samples to reveal the metabolic profile but data on the rates and routes of excretion can be equally as important as an aid to the design of the regulatory human mass balance study. In some cases, studies may be also performed with 14C drug at steady state, either using a single-pulse dose following
repeat administration on nonlabeled drug or by repeat administration of the 14C compond.
Future perspectiveAlthough regular MS provides valuable data on the metabolite profile of a drug, reliable quanti-fication in the absence of authentic standards is likely to remain a serious challenge. The use of a radioisotope is going to remain the most reli-able method for quantification and to ensure no metabolites are inadvertently missed. The addi-tion of low levels of 14C in FIM studies is, there-fore, likely to continue to grow. The resulting
data, combined with regular MS will provide a complete picture of the metabolic profile in humans.
Financial & competing interests disclosureG Lappin and M Seymour are employees of Xceleron Ltd, a company offering accelerator MS-enabled metabolites in safety testing studies. The authors have 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.
Executive summary
� Accelerator MS enables quantitative 14C-metabolism data to be acquired in humans during first-in-man studies.
� Very low levels of 14C are employed, below those that would normally require regulatory approval for the administration of radioactivity to humans.
� In addition to data showing the systemic metabolite burden, preliminary information such as routes and rates of excretion can be obtained and can be used to design the regulatory human adsorption, metabolism and excretion study.
� The combination of accelerator MS and other bioanalytical approaches in multiple-dose studies in human and animals represents a very powerful tool to obtain key metabolite exposure data very early as part of a risk mitigation strategy.
In vitro metabolismFirst information on cross-species metaboliteprofiles
In vivo metabolismStudy in animals with 14C-labeled compound. First in vitro–in vivo comparison
FIM study with14C-tracerInformation on metaboliteprofile and systemic exposurein human after single dose obtained using accelerator MS
Validatedbioanalytical methodComparison of systemic exposurein human after multidoseversus animal species at steady state
Synthesize major in vitrometabolites
Synthesize major systemic in vivo metabolites
Use metabolite standards to identify and quantify keymetabolites; potentialsynthesis of additionalmetabolites
Use metabolite standards for‘fit-for-purpose’ bioanalyticalmethod
Use metabolite standards tocompare systemic exposurecross-species
Figure 3. Lundbeck A/H metabolites in safety testing strategy.
Human metabolism in Phase I using AMS & a tiered bioanalytical approach | Review
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ReferencesPapers of special note have been highlighted as:� of interest�� of considerable interest
1 Baillie TA, Cayen MN, Fouda H et al. Drug metabolites in safety testing. Toxicol. Appl. Pharmacol. 182(3), 188–196 (2002).
� Milestone paper where the issue of metabolite safety was discussed, leading to the issue of regulatory guidelines.
2 Anderson S, Luffer-Atlas D, Knadler MP. Predicting circulating human metabolites: how good are we? Chem. Res. Toxicol. 22(2), 243–256 (2009).
3 Gross G, Wilson I. Issues in the safety testing of metabolites. Future Med. Chem. 1(8), 1381–1390 (2009).
4 US FDA, US Department of Health and Human Services. Guidance for Industry Safety Testing of Drug Metabolites (2008).
5 ICH Topic M3 Note for Guidance on nonclinical safety pharmacology studies for human pharmaceuticals CPMP/ICH/286/95 (2009).
6 Caceres-Cortes J, Reily MD. NMR spectroscopy as a tool to close the gap on metabolite characterization under MIST. Bioanalysis 2(7), 1263–1276 (2010).
7 Comezoglu SN, Ly VT, Zhang D et al. Biotransformation profiling of [14C]ixabepilone in human plasma, urine and feces samples using accelerator mass spectrometry (AMS). Drug Metab. Pharmacokinet. 24(6), 511–522 (2009).
8 Garner RC. Accelerator mass spectrometry in pharmaceutical research and development – a new ultrasensitive analytical method for isotope measurement. Curr. Drug Metab. 1(2), 205–213 (2000).
� Early overview on the use of accelerator MS in drug development.
9 Ghauri FY, Blackledge CA, Wilson ID, Nicholson JK. Studies on the metabolism of fluorinated xenobiotics in the rat using 19F-NMR and 1H-NMR spectroscopy. J. Pharm. Biomed. Anal. 8(8–12), 939–944 (1990).
10 Ramanathan R, Zhong R, Blumenkrantz N, Chowdhury SK, Alton KB. Response normalized liquid chromatography nanospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 18(10), 1891–1899 (2007).
11 Salehpour M, Possnert G, Bryhni H. Subattomole sensitivity in biological accelerator mass spectrometry. Anal. Chem. 80(10), 3515–3521 (2008).
12 Yi P, Luffer-Atlas D. A radiocalibration method with pseudo internal standard to estimate circulating metabolite concentrations. Bioanalysis 2(7), 1195–1210 (2010).
13 Dalvie D. Recent advances in the applications of radioisotopes in drug metabolism, toxicology and pharmacokinetics. Curr. Pharm. Des. 6(10), 1009–1028 (2000).
14 Ramanathan R, Josephs JL, Jemal M, Arnold M, Humphreys WG. Novel MS solutions inspired by MIST. Bioanalysis 2(7), 1291–1313 (2010).
15 Garner RC, Goris I, Laenen AA et al. Evaluation of accelerator mass spectrometry in a human mass balance and pharmacokinetic study-experience with 14C-labeled (R)-6-[amino(4- chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1- methyl-2(1H)-quinolinone (R115777), a farnesyl transferase inhibitor. Drug Metab. Dispos. 30(7), 823–830 (2002).
16 Young G, Ellis W, Ayrton J, Hussey E, Adamkiewicz B. Accelerator mass spectrometry (AMS): recent experience of its use in a clinical study and the potential future of the technique. Xenobiotica 31(8–9), 619–632 (2001).
� Account from the pharmaceutical industry on the use of accelerator MS in drug development.
17 Young G, Ellis WJ. AMS in drug development at GSK. Nuc. Instrum. Methods Phys. Res. B 259, 752–757 (2007).
18 Lappin G, Seymour M. Addressing metabolite safety during first-in-man studies using 14C-labeled drug and accelerator mass spectrometry. Bioanalysis 2(7), 1315–1324 (2010).
19 Waldmeier F, Garner RC. Application of AMS for human ADME. Drug Metab. Rev. 33, 121 (2001).
20 Sarapa N, Hsyu PH, Lappin G, Garner RC. The application of accelerator mass spectrometry to absolute bioavailability studies in humans: simultaneous administration of an intravenous microdose of 14C-nelfinavir mesylate solution and oral nelfinavir to healthy volunteers. J. Clin. Pharmacol. 45(10), 1198–1205 (2005).
21 Timmerman P, Anders Kall M, Gordon B et al. Best practices in a tiered approach to metabolite quantification: views and recommendations of the European Bioanalysis Forum. Bioanalysis 2(7), 1185–1194 (2010).
22 Roffey SJ, Obach RS, Gedge JI, Smith DA. What is the objective of the mass balance study? A retrospective analysis of data in animal and human excretion studies employing radiolabeled drugs. Drug Metab. Rev. 39(1), 17–43 (2007).
23 Dalvie D, Obach RS, Kang P et al. Assessment of three human in vitro systems in the generation of major human excretory and circulating metabolites. Chem. Res. Toxicol. 22(2), 357–368 (2009).
24 Leclercq L, Cuyckens F, Mannens GS et al. Which human metabolites have we MIST? Retrospective analysis, practical aspects and perspectives for metabolite identification and quantification in pharmaceutical development. Chem. Res. Toxicol. 22(2), 280–293 (2009).
25 Yu H, Bischoff D, Tweedie D. Challenges and solutions to metabolites in safety testing: impact of the International Conference on Harmonization M3(R2) guidance. Expert Opin Drug Metab. Toxicol. 6(12), 1539–1549 (2010).
26 Gao H, Obach RS. Addressing MIST (metabolites in safety testing): bioanalytical approaches to address metabolite exposures in humans and animals. Curr. Drug Metab. 12(6), 578–586 (2011).
27 Nedderman AN, Wright P. Looking back through the MIST: a perspective of evolving strategies and key focus areas for metabolite safety analysis. Bioanalysis 2(7), 1235–1248 (2010).
�� General overview of stratergies addressing metabolite safety.
� Websites101 ICH S7A safety pharmacology studies for
huma pharmaceuticals. www.fda.gov/cder/guidance/4461fnl.pdf
102 Guidance for industry carcinogenicity study protocol submissions. www.fda.gov/cder/guidance/4804fnl.pdf
103 ICH S9 nonclinical evaluation for anticancer pharmaceuticals, draft guidance. www.fda.gov/cder/guidance/8681dft.pdf
