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Chem. Res. Toxicol. 2006, 19, 1570-1579
Metabolites and Safety: What Are the Concerns, and How Should We Address Them? Dennis A. Smith and R. Scott Obach* Pharmacokinetics, Dynamics, and Drug Metabolism, Global Research and DeVelopment, Pfizer, Inc., Groton, Connecticut 06340 ReceiVed August 22, 2006
The issue of the safety of drug metabolites in humans is a complex one. In this commentary, a proposal is made regarding how to deal with drug metabolites observed in humans such that the safety of these molecules can be assured. The human radiolabeled ADME study, in which metabolites are identified and quantified in circulation and excreta, is proposed as the primary source of information on human metabolites from which decisions can be made regarding the need for further risk assessment. Although radiolabel ADME studies yield quantitative metabolite profiles that are commonly reported as a percentage of the total drug related material (for circulating metabolites) and a percentage of total dose (for excretory metabolites), it is essential to convert these values into absolute abundances. The structure of a metabolite, its abundance, the biofluid in which it is observed (circulation or excreta), and the toxicity mechanism of concern serve as the four most important characteristics for determination as to whether further safety consideration is warranted. Metabolites in circulation require consideration for toxicity that can arise by effects on specific receptors and/or enzymes (either target or off-target). Metabolites in excreta require consideration for their potential to indicate a body-burden to chemically reactive intermediary metabolites, which can yield toxicities of nonspecific mechanisms commonly associated with covalent binding (e.g., carcinogenicity, immunoallergic response, etc.). Through an analysis of 24 drugs removed from the market because of human toxicity, it was concluded that further testing of human metabolites would not have yielded any additional information that could have predicted human safety findings because human metabolites would have been present in the animal species routinely used in toxicology testing after the administration of the parent compound. Contents 1. Metabolites in Safety Testing: What Are the Concerns? 2. Metabolite Profile Information Generated by Drug Metabolism Scientists and Used for MIST Decisions 3. Considerations for MIST Decisions Are Highly Interrelated 3.1. Metabolites That Could Reversibly and Specifically Interact with Target Macromolecules: Metabolites in Circulation 3.2. Metabolites That Could Covalently and Nonspecifically React with Macromolecules Leading to Long-Term Effects: Metabolites in Excreta 4. Metabolites in Safety Testing: How Should We Proceed when 1 µM), additional safety evaluation for an appropriate risk assessment may be required, especially if human exposure is greater than that observed in animals. At these elevated concentrations, nonselective reversible binding interactions can occur. This latter group of metabolites may cause effects on off-target receptors/enzymes by virtue of their high concentration coupled with nominal affinity.
3.2. Metabolites That Could Covalently and Nonspecifically React with Macromolecules Leading to Long-Term Effects: Metabolites in Excreta In the human ADME study, metabolites present in the urine and feces are identified, and from the percentage of total drug-
related radioactivity in excreta along with the structural data, a metabolic pathway tree can be constructed. The percentages of the dose that proceed through various branches of the tree can be derived from the data and converted to total amount values. This information is useful in assessing what the maximum total body-burden to any given metabolite would be (Figure 1). The potential concern for these metabolites revolves around whether they are chemically reactive or indicative of the existence of a preceding chemically reactive intermediate. As with circulating metabolites described above, the structure of the metabolite is key in any MIST assessment of excretory metabolites; however, in this case, the focus is on chemically stable structures that are proximal to reactive intermediates or structures that are themselves reactive. The direct observation of reactive metabolites in vivo is rare, although not unprecedented (e.g., quinones of troglitazone and remoxiprde, epoxide of alclofenac (11-13)). Downstream metabolite structures indicative of the presence of a reactive intermediate metabolite include mercapturic acids, dihydrodiols, methyl catechols, and conjugates of dihydroquinones. Also, some stable end product metabolites that would have arisen through oxidative opening of ring systems known to yield reactive intermediate metabolites (e.g., 2-aminothiazoles, furans, cyclopropylamine, etc.) should be included in such a list. Acyl glucuronides provide an example of a class of reactive metabolites in which there is great enough stability so that to varying extents, they can be observed in vivo. The concern regarding these types of metabolites is their potential involvement in toxicities that cannot be ascribed to specific receptor-ligand interactions. When generated, any reactive metabolite can covalently bond to a variety of macromolecular structures, with few of the reactions yielding toxic sequelae. Reaction with DNA can result in gene mutation, which depending on the specific site, can yield no effect, cell
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Table 2. Drugs That Exhibit Toxicity Presumed to Be Caused by the Generation of Reactive Metabolites drug
daily dose (mg)
drug
daily dose (mg)
acetaminophen felbamate isaxonine fenclofenac nitrefazole alclofenac ebrotidine piprofen benoxaprofen chlormezanon clozapine flipexide ibufenac suloctidyl zomepirac bendazac moxisylyte clometacin suprofen tienilic acid troglitazone
4000 2400 1500 1200 1200 1000 800 800 600 600 600 600 600 600 600 500 480 450 400 400 400
tolrestat amineptine fenclozic acid perhexiline tolcapone remoxipride zimeldine nefazodone cyclofenil dilevalol trovafloxicin exifone alpidem bromfenac pemoline nomifensine vesnarinone nialamide mianserin sudoxicam mebanazine
400 300 300 300 300 300 300 300 200 200 200 180 150 150 150 125 120 100 60 50 30
dysfunction, or death due to alteration of the biosynthesis of an essential protein or uncontrolled cell growth (carcinogenesis). Reaction with various proteins can also yield no effect, the accelerated degradation of the protein, disruption of the function of the protein resulting in cellular dysfunction or death, or the generation of an immunogen. The toxicities that arise via covalent alterations of biological macromolecules tend to be long-term effects that require chronic dosing regimens and include cancer via chemical carcinogenesis, teratogenesis and reproductive impairment, and immuno-allergic reactions. The effects can present in the clinic in a variety of ways but most often occur as effects on the skin (ranging in severity from minor rashes to Stevens-Johnson syndrome), liver (ranging from minor alterations in standard liver function tests through to total liver failure), and blood (e.g., aplastic and hemolytic anemias, neutropenia, etc.). Examples of drugs that generate reactive metabolites and have been associated with toxicities are numerous (Table 2). However, the mechanistic link between reactive metabolites and chronic toxicities such as the examples described above is not complete and is proof by association rather than a well-defined biochemical or pharmacological mechanism. There are also many examples of drugs that yield reactive metabolites in vitro and/or show evidence of their formation in vivo that are not associated with these types of toxicities. Furthermore, some of the toxicities, although severe, are observed in humans at very low frequencies and are thus difficult to observe in animal toxicity studies. It is important to note that the vast majority of drugs that have caused toxicities presuming to arise via reactive metabolites are administered at relatively high daily doses (i.e., >100 mg), suggesting that there may be a finite general capacity for total body burden for reactive metabolites. For example, the maximum recommended daily dose of acetaminophen is 4 g, and it has been shown that nearly 10% of a dose of acetaminophen proceeds through the reactive intermediate metabolite N-acetyl p-quinoneimine (14). This corresponds to the body being exposed to a total of ∼400 mg equivalents (∼2500 µmol) of a chemically reactive electrophile and illustrates the body’s capacity for detoxicating a reactive metabolite because acetaminophen is considered a generally safe drug when used as recommended. But it should be noted that in a recent study, Watkins and colleagues (15) observed elevated liver function test parameters in healthy volunteers receiving
multiple doses of acetaminophen at the maximum recommended dose of 4 g per day. Although this report did not question the safety of the drug at this dose in terms of the liver and other organs and referred to other large and detailed safety studies, some degree of liver perturbation may be seen in some individuals at the maximum recommended dose, and a true noobservable-effect dose in terms of any effects on the liver may be lower than 4 g per day. An explanation of adaptation in patients was offered for this finding by the authors. Of course, the capacity of the body to detoxicate reactive metabolites would be expected to differ from drug to drug; however, the acetaminophen example does indicate an example of this capacity and reinforces the conservative proposal that metabolites present at less than 10 mg in excreta should not warrant further toxicicity testing even if present in humans at a greater amount than that in toxicology animal species (3).
4. Metabolites in Safety Testing: How Should We Proceed when Risk Assessment Is Deemed Necessary? On the basis of the discussion above and that by others (13), instances will arise in drug development programs in which a metabolite is observed in the human ADME study and a risk assessment is deemed necessary. Risk assessment is needed for (I) circulating metabolites with a structure similar to that of the parent drug and present at free concentrations greater than 25% of the free drug concentrations of the parent or if structurally distinct from the parent, is present in greater than 1 µM free concentration and (II) when the metabolic pathway reconstructed from human excretory metabolite data indicates the formation of a reactive intermediate and the total amount proceeding through that pathway represents a challenge of greater than 10 mg total to the human. How do we make such a risk assessment for human metabolites using nonclinical approaches? In the first instance, assessing whether the metabolite is also present in the animal species used in safety assessments would be the obvious approach. At the time that human ADME data are available, most if not all of the corresponding data will have been gathered in laboratory animal species using similar approaches (i.e., single dose of radiolabeled drug with quantitative radiometric analysis of plasma and excreta). The metabolite profile data from humans and animals can be directly compared, ensuring that the comparison is made between absolute amounts and not percentages. When done this way, it is not uncommon that the animal exposure will exceed the human exposure, largely due to the fact that body-weight-corrected doses used in animal toxicology studies are considerably greater than doses used in the clinic. If this is the case, it can be concluded that in previous toxicology studies, the animals were exposed to the human metabolite, and the data from these toxicology studies on the parent drug can be used to underwrite the risk assessment for the metabolite. For circulating metabolites, emphasis should be placed on primary and secondary pharmacology screening to mitigate the risk. The combination of pharmacological potency and free concentration allows assessment of any additional risks. If contribution to activity by the metabolite is less than 25% of the parent, it should be deemed as not requiring further evaluation. A shortcoming of simply using the ADME data to make human versus animal comparisons is that these data are from single-dose studies, whereas the more appropriate comparison in almost all cases uses multiple-dose data. If the single dose radiometric ADME data show that animals possess considerably more of a metabolite than humans, an inference that this is also
PerspectiVe
the case with multiple dosing is not unreasonable. However, if the reverse is true, the next step taken would be to conduct a toxicokinetic bridging study. In this study, animal species that were used in previous safety studies are dosed with the parent drug in a manner mimicking the previous studies (dose route, level, and frequency). The number of doses employed should be enough to achieve steady state, at which time, toxicokinetic sampling is done for the appropriate matrix (blood and/or excreta) and the metabolite in question measured using a specific analytical method (e.g., HPLC-MS). The exposure measured in animals is compared to the corresponding human values to determine if previous and future safety studies in animals yield exposure to the metabolite suitable for risk assessment. If this approach fails, the situation begins to become onerous, as alternative approaches to make risk assessments for human metabolites become more complex, time-consuming, and expensive. For in vivo risk assessments, two possible paths exist, each possessing their own shortcomings. A seldom used approach is to seek an alternate laboratory animal species that may do a better job of generating the human metabolite from the parent drug than the more conventional species. (These can include hamsters, guinea pigs, and marmosets, among others.) In vitro systems may be screened to aid in this selection by examining the metabolite profiles from other species compared to those from human in vitro systems. Once identified, the previously used species can be replaced by the new species that yields the metabolite in future toxicology studies. The two greatest problems with this approach are that (a) it is hit-ormiss in that there may be no species that generates the metabolite better than any of the species already used, and (b) using an alternate species in toxicology studies is more difficult because there will be generally less experience with the alternate species and a smaller database of knowledge of background pathology and receptor similarity to those of humans. The more frequently proposed approach is to directly administer the metabolite in question to animal species and conduct toxicology studies similar to those done for the parent drug (Table 1). This requires that bulk material for the metabolite be prepared, which, depending on the structure of the metabolite, may be impractical. For instance, a hydroxylation reaction may introduce a chiral center and a ratio of enantiomers in a metabolite, which may be synthetically unachieveable. It also requires that the metabolite if administered orally, will have acceptable bioavailability and be chemically stable in the GI tract (which for many types of conjugated metabolites will not be the case). The metabolite is assumed to exhibit effects, if any, in the same manner as if it were generated from the parent drug, and it would need to be able to penetrate into the same tissues from which it would have been generated when arising from the parent drug. Furthermore, if the metabolite in question is a trapped metabolite downstream from a reactive metabolite, its direct administration to animals would provide no value at all in risk-assessing the reactive metabolite. To date, we do not have enough experience to know whether the direct administration of human drug metabolites to animals provides any value in making better risk assessments for the parent drug. Furthermore, some practical questions arise as to how far this risk assessment should be taken. Would a single chronic dose toxicology study suffice, or would special studies such as 2-year carcinogenicity or reproductive studies also be necessary? Would this assessment need to be made in all species routinely used in toxicology studies? These questions require answers, with scientifically sound underlying rationales, if direct safety testing of metabolites were to be adopted as the preferred approach
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when confronted with human metabolites that were not covered in animal toxicology studies. A different set of considerations may exist when considering human metabolites in in vitro toxicology studies. (These include broad ligand screens used in safety pharmacology as well as in vitro genotoxicity and phototoxicity tests.) Toxicology studies in vitro have the intent of identification of the hazard as an end point rather than the conventional risk assessment, that is, direct comparisons of active concentrations between in vitro tests and those in human are not made. For broad ligand screens and in vitro phototoxicity, the follow up needed for positive results are generally straightforward and short term. (It should also be stated here that the conduct of additional in vitro phototoxicity tests on metabolites should be unnecessary in cases where the parent drug was already shown to be negative in in vitro phototoxicity tests and for which no modification has occurred on the chromophore when the parent is converted to the metabolite.) However, in the case of in vitro genotoxicity tests, because the ultimate in vivo end point that the tests are attempting to predict is carcinogenicity, which is an effect that is not readily observed in clinical studies, a great deal of consideration is warranted for metabolites. Genotoxicity tests in vitro typically utilize a rat liver subcellular fraction (S-9) as the activation system that has the ability to generate metabolites. If the human metabolite is generated in this system when testing the parent drug, the question arises as to whether that represents a suitable test of the genotoxic potential of the metabolite as well. Consensus has not been achieved regarding this question, although it has been recognized as an important topic (Ku, et al., in press).
5. Drugs Withdrawn from the Market for Safety Concerns: Would a MIST Strategy Have Prospectively Identified a Problem? Table 3 lists drugs that were approved for use in the markets in the USA and/or Europe but subsequently displayed safety concerns of such seriousness that they were withdrawn or relegated to second-line therapeutic options (16). These represent the worst case scenarios for those engaged in the development of new drugs and for government officials charged with the responsibility of regulating new drug approvals because they undermine the faith of patients and physicians in the research done to underwrite the safety of new drugs. For a consideration of MIST, these drugs can be used retrospectively to ask the question as to whether a riskassessment of human metabolites would have provided clues to the toxicity ultimately ascribed to these drugs. This analysis is listed in Table 3. In the vast majority of cases, there would have been no readily recognized rationale for requiring further testing of human metabolites because it would have been a reasonable expectation from available drug metabolism data that human metabolites would have been present in the animal species routinely used in toxicology testing after the administration of the parent compound. Although it is rare among this set of drugs that actual toxicokinetic data are available on metabolites, comparisons of the metabolite profiles generated in radiolabel ADME studies leads to the conclusion that animals frequently do an adequate job of generating the same metabolites as humans. Doses used in toxicology studies are high enough compared to the levels of clinically relevant doses that even metabolites considered minor in animals and major in humans (based on percentage measurements) are present in animals in greater quantities than those in humans. Also, drugs that give rise to chemically reactive metabolites in humans appear to
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Table 3. Drugs Withdrawn from the Market and an Analysis of Whether the Risk-Assessment of Metabolites Would Have Aided in Their Safety Evaluation
drug
presumed toxicity mechanism
presumed to be metabolites involved?
would further risk assessment of a human circulating or excretory metabolite have led to the identification of human safety risk?
comment
alosetron
target pharmacology
no
no
The parent drug is responsible for toxicity (primary pharmacology) (23), and human metabolites are present in excess in animals (24).
astemazole
off-target pharmacology
yes
no
The parent drug and desmethyl metabolite are responsible for toxicity (HERG blockers), (25,26) but exposure to the metabolite in dogs should cover its effect (27).
benoxaprofen chemical reactivity
yes
no
The major metabolite is an acyl glucuronide in humans and dogs (28).
bromfenac
chemical reactivity
yes
possibly
The major metabolite in human is an acyl glucuronide (29). Comprehensive animal metabolism data is unavailable in the literature, preventing the determination of whether animals would have been exposed to metabolic pathways representing bioactivation, although one report states that acyl glucuronidation is minor in rats(30), suggesting that the acyl glucuronide may not have been present in abundance in rat toxicology studies with bromfenac.
cerivastatin
target pharmacology
no
no
Primary pharmacology and high systemic levels of drug (cf. other statins) lead to toxicity. Circulating metabolites are active but contribute
