Application of Stable Isotope-Labeled Compounds ... - ACS Publications

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ReViews Application of Stable Isotope-Labeled Compounds in Metabolism and in Metabolism-Mediated Toxicity Studies Abdul E. Mutlib* Biotransformation Department, Drug Safety and Metabolism, Wyeth Research, Building S3324, CollegeVille, PennsylVania 19426 ReceiVed April 17, 2008

Stable isotope-labeled compounds have been synthesized and utilized by scientists from various areas of biomedical research during the last several decades. Compounds labeled with stable isotopes, such as deuterium and carbon-13, have been used effectively by drug metabolism scientists and toxicologists to gain better understanding of drugs’ disposition and their potential role in target organ toxicities. The combination of stable isotope-labeling techniques with mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, which allows rapid acquisition and interpretation of data, has promoted greater use of these stable isotope-labeled compounds in absorption, distribution, metabolism, and excretion (ADME) studies. Examples of the use of stable isotope-labeled compounds in elucidating structures of metabolites and delineating complex metabolic pathways are presented in this review. The application of labeled compounds in mechanistic toxicity studies will be discussed by providing an example of how strategic placement of a deuterium atom in a drug molecule mitigated specific-specific renal toxicity. Other examples from the literature demonstrating the application of stable isotope-labeled compounds in understanding metabolism-mediated toxicities are presented. Furthermore, an example of how a stable isotope-labeled compound was utilized to better understand some of the gene changes in toxicogenomic studies is discussed. The interpretation of large sets of data produced from toxicogenomics studies can be a challenge. One approach that could be used to simplify interpretation of the data, especially from studies designed to link gene changes with the formation of reactive metabolites thought to be responsible for toxicities, is through the use of stable isotope-labeled compounds. This is a relatively unexplored territory and needs to be further investigated. The employment of analytical techniques, especially mass spectrometry and NMR, used in conjunction with stable isotope-labeled compounds to establish and understand mechanistic link between reactive metabolite formation, genomic, and proteomic changes and onset of toxicity is proposed. The use of stable isotope-labeled compounds in early human ADME studies as a way of identifying and possibly quantifying all drug-related components present in systemic circulation is suggested. Contents 1. Introduction and Background 2. Application of Stable Isotope-Labeled Compounds in Metabolism Studies 2.1. Use of Stable Isotope-Labeled Compounds in Locating Metabolites in Biological Fluids 2.1.1. Metabolism of Benzylamine 2.1.2. Detecting Reactive Metabolites Trapped as GSH Adducts 2.2. Application of Stable Isotope-Labeled Compounds in Elucidating Metabolic Pathways and Structures of Metabolites 2.2.1. Metabolism of a Disubstituted Alkyne Leading to a Reactive Oxirene Intermediate and the Subsequent Formation of GSH Conjugates 2.2.2. Metabolism of a Benzylamine to a Glutamate Conjugate Mediated by γ-Glutamyltranspeptidas

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3. Application of Deuterated Analogues in Mechanistic Metabolism-Mediated Toxicity Studies 4. Application of Stable Isotope-Labeled Compounds in Toxicogenomic Studies 5. Application of Stable Isotope-Labeled Compounds in Clinical ADME Studies 6. Recent Developments and Future Directions

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1. Introduction and Background Stable isotope-labeled compounds have been employed in several areas of biomedical research (1-17). The combination of stable isotope-labeling techniques with mass spectrometry, which allows rapid acquisition and interpretation of data, has promoted greater use of these stable isotope-labeled compounds in a number of fields including absorption, distribu* To whom correspondence should be addressed. Tel: 484-865 7525. Fax 484-865 9408. E-mail: [email protected].

10.1021/tx800139z CCC: $40.75  2008 American Chemical Society Published on Web 08/15/2008

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tion, metabolism, and excretion (ADME)1 studies. The use of stable isotope labeling to study various aspects of the metabolism and pharmacokinetics of drugs and other foreign compounds in animals and humans has been very well-documented (2, 3, 5, 6, 9, 18-66). Compounds labeled with stable isotopes, such as deuterium and carbon-13, have been used effectively in the past by drug metabolism scientists and toxicologists to gain a better understanding of a drug’s disposition and its potential role in target organ toxicities. However, while this approach is extremely valuable in drug discovery and development, a survey of the literature suggests that far fewer studies have been conducted with such labeled compounds as compared to radiolabeled analogues. The radiolabeled analogues have been widely used in pharmaceutical companies, especially to gain qualitative and quantitative assessment of drugs’ distribution and excretion patterns in preclinical species and in humans. This practice originated many decades ago when other detection techniques such as LC/MS were nonexistent or in the early stages of development. With the advent of superior LC/MS technologies, it has become much easier to detect and quantitate drugs and drug-related materials in biological matrices. This evolution of LC/MS technology has shifted the use of radiolabeled compounds to later stages of drug discovery and development as compared to the past when radioactivity was used as the primary means of detecting and quantitating compounds. Inadvertently, this has also created a window of opportunity to make greater use of stable isotope-labeled compounds for early metabolism studies. One of the consequences of the extensive use of LC/ MS for quantitative assays has been an increase in the syntheses of stable isotope-labeled analogues that are often used as internal standards in these studies (67-98). With the availability of these stable isotope-labeled analogues and robust LC/MS instruments, it has become straightforward to quantitate analytes in complex biological mixtures. The focus of this review precludes us from discussing the value of having stable isotope-labeled analytes for quantitation purposes. The readers are directed to a number of publications focused on the application of LC/MS and stable isotope-labeled compounds for quantitative analyses from preclinical and clinical pharmacokinetic studies (77, 80-98). Other quantitative applications of stable isotope-labeled compounds include studies conducted to distinguish in vivo and in vitro disposition of enantiomers where only one of the enantiomers was selectively labeled with stable isotopes (5). Recently, we utilized a stable isotope-labeled glucuronide conjugate of acetaminophen (APAP) to explain in vitro kinetic data (99). Despite the greater availability of stable isotope-labeled compounds, drug metabolism scientists have yet to take full advantage of the potential use of these analogues for mechanistic metabolism and toxicity studies. These stable isotope-labeled compounds can be used more widely to gain a better understanding of a drug’s disposition and in toxicity studies. In this review, we provide examples of the use of stable isotope-labeled compounds in elucidating structures of metabolites and the metabolic pathways leading to these products. Identification of metabolite structures is very important, especially if one is trying to understand metabolism-mediated toxicities. The application of these labeled compounds in metabolism and mechanistic 1 Abbreviations: ADME, absorption, distribution, metabolism, and excretion; APAP, acetaminophen; AUC, area under curve; LC-CRIMS, liquid chromatography-chemical reaction interface mass spectrometry; NAPQI, N-acetyl para-benzoquinone-imine; ESI, electrospray ionization; LC-ESI/ MS, liquid chromatography-electrospray ionization/mass spectrometry; MS/ MS, mass spectrometry/mass spectrometry.

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toxicity studies will be discussed by providing examples from the laboratories of the author as well as the laboratories of other investigators. All of the examples presented in detail are from studies previously conducted in the author’s laboratory. References are provided for similar studies conducted by other investigators who employed stable isotope-labeled compounds in their work. Furthermore, an example of how stable isotopelabeled compounds can be utilized to better understand some of the gene changes in toxicogenomic studies will be presented. Toxicogenomics is a rapidly evolving field and is expected to play a very significant role in drug discovery and development in future. However, while significant progress has been made in toxicogenomics techniques, the interpretation of large sets of data produced from these studies can be a challenge. One approach that could be used to simplify interpretation of the data, especially from studies designed to link gene changes with the formation of reactive metabolites thought to be responsible for toxicities, is through the use of stable isotope-labeled compounds. This is a relatively unexplored territory and needs to be further investigated. The employment of analytical techniques, especially mass spectrometry and NMR used in conjunction with stable isotope-labeled compounds to establish and understand the mechanistic link between reactive metabolite formation, genomic and proteomic changes, and the onset of toxicity, appears very logical. This interdisciplinary approach may provide us with potential genomic and/or proteomic biomarkers of target organ toxicities in the future.

2. Application of Stable Isotope-Labeled Compounds in Metabolism Studies Stable isotopes such as deuterium and carbon-13 can be incorporated strategically into drug molecules to facilitate the elucidation of their metabolites’ structures using LC/MS and NMR. Numerous examples exist in the literature whereby investigators used 1:1 w/w mixtures of nonlabeled and isotope-labeled compounds to study in vitro and in vivo metabolic disposition of compounds (19, 22-33, 37, 38, 45-48, 50-53, 55-61). The separation of the molecular weights of the labeled and unlabeled analogues by a couple of mass units, while both retaining almost identical physicochemical properties, has made mass spectrometry the method of choice in simultaneously analyzing these compounds. Analyses of in vitro samples or biological extracts from in vivo studies by GC/MS or LC/MS allow the presence of twin ions to be monitored by the mass spectrometer. Usually, a separation of >2 amu between the two ions is desirable, which leads to the appearance of a twin ion pair pattern for the parent compound and its metabolites in the mass spectra of the biological extracts. This greatly facilitates location of the parent compound as well as its metabolites in complex mixtures such as urine and bile samples. In addition to locating metabolites in biological matrices, this approach can be particularly useful in determining the structures of unusual or unpredictable metabolites formed from compounds (48, 51, 52, 56-58). Furthermore, the incorporation of stable isotopes into compounds also allows us to delineate possible mechanisms or the enzymes responsible for the formation of particular metabolites (48, 50-52). Stable isotope labeling does not necessarily have to be performed on the compound whose metabolism is being studied. At times, moieties (e.g., GSH) that are eventually incorporated into the compound of interest may be labeled with stable isotopes so that the products can be identified as metabolic products. The use of stable isotope-labeled GSH to trap and characterize in vitro-generated reactive metabolites is an example of this approach. The stable isotope-labeled GSH

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allows one to use nonradioactive compounds to study metabolic disposition and potential liabilities of drug candidates, especially in a discovery setting (63, 64). Another example is the use of labeled molecular oxygen (18O2) and water (H218O) in metabolism studies to understand metabolic pathways and structures of metabolites (100-103). The use of deuterium-labeled solvents to assist in the identification of metabolites in discovery stages has also become popular (104-107). This technique has been found to be useful in distinguishing hydroxylations on carbon (e.g., formation of alcohols and phenols) from oxidations on heteroatoms (e.g., formation of sulfoxides and N-oxides) provided that the molecule is capable of undergoing any of these reactions. In the presence of a deuterated solvent (such as D2O), the protons on functional groups such as OH, NH2, or SH will be exchanged with deuterium. This leads to mass shifts corresponding to the number of exchangeable protons in the molecule. Hence, a metabolite formed by hydroxylation at an aliphatic position will show a net addition of 1 amu, while a product resulting from S-oxidation, for example, will not show this change. Unambiguous metabolite identification can be made using this technique of in-source H/D exchange, hence avoiding unnecessary isolation of metabolites for NMR characterization. Of course, LC/MS technology has played a very significant role in making optimal use of these isotope-labeled compounds in each of these metabolism studies. It is equally true that high field NMR can also contribute tremendously in elucidating structures of metabolites of compounds labeled with stable isotopes. Because of the low abundance of 13C in nature, the acquisition of 13C NMR data for metabolites is usually either impossible or takes a long time. By strategically placing the 13 C-label in a molecule, one can acquire the critical 13C NMR data required to elucidate or confirm the structures of metabolites (41, 43, 48, 49, 56-58). Examples of how stable isotope-labeled compounds have been used in combination with mass spectral and NMR techniques in metabolism studies are given below. 2.1. Use of Stable Isotope-Labeled Compounds in Locating Metabolites in Biological Fluids. As mentioned earlier, the presence of twin ions in the mass spectra can greatly assist in the identification of metabolites in biological samples such as urine, bile, and plasma. At times, the isotope pattern resulting from the presence of natural isotopes of atoms such as chlorine and bromine (M + 2) in a molecule can allow detection of metabolites in samples (108). For example, the metabolite profiling of a compound such as efavirenz was greatly accelerated by the presence of a chlorine atom in the molecule. The metabolites were easily identified by observing the characteristic ion pattern in the mass spectra (108). The “twin ions” showed a ratio consistent with the natural abundance of 35Cl and 37Cl isotopes (67 and 33%, respectively). However, in many cases, these elements are not present in a molecule; hence, we have to rely on other means of confirming the presence of metabolites in a complex mixture. Radiolabeling is a very widely used technique to obtain qualitative and quantitative information on the in vitro and in vivo disposition of compounds. However, for a number of reasons, these radiolabeled analogues are not often available early in discovery phase. Often, the syntheses of radiolabeled compounds are complex and conducted in a very regulated environment. However, it must be noted that syntheses of stable labeled drugs can be just as challenging as syntheses of radiolabeled compounds, although a special laboratory is not necessary for the former. The availability of stable isotopelabeled compounds allows one to use a 1:1 w/w mixture of labeled:nonlabeled compound for in vitro and in vivo metabolism studies. This permits the metabolism chemist to distinguish

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Figure 1. Structures of benzylamine and its deuterated analogues that were used to study the metabolism of benzylamine in rats.

the drug-related material from endogenous components in a complex biological extract. Furthermore, one could use this approach to study covalent binding of metabolically generated reactive intermediates to proteins. Upon digestion of proteins, LC/MS analyses could be conducted to locate sites of covalent binding to specific amino acids in peptide fragments as demonstrated recently (109-111). Monitoring twin ion pairs in the mass spectral scans obtained during the LC/MS analyses can lead to detection of metabolites in complex biological samples or identification of sites of covalent binding by reactive intermediates. These twin ions are separated from each other depending on the number of labels incorporated in the labeled analogue. If the mass spectrum of a metabolite shows a pattern different from the substrate, that would suggest a loss of the label. Listed below are some examples of how this technique was used successfully to obtain metabolite profiles and distinguish drug-related compounds from endogenous components. 2.1.1. Metabolism of Benzylamine. The in vivo and in vitro metabolic disposition of benzylamine was studied in rats using mixtures of benzylamines that were labeled with deuterium at different positions (see Figure 1). In vivo studies performed with stable isotope-labeled benzylamine enabled rapid mass spectrometric identification of metabolites present in rat bile and urine (58). These metabolites were very polar and found to elute with the solvent front in preliminary studies. Mass spectral analyses of the early eluting peak suggested the presence of benzylamine-related products; hence, chromatographic conditions were modified to retain the metabolites on column for a length of time. Monitoring the presence of twin ions greatly assisted the development of an HPLC method to separate various components from each other and from endogenous compounds. Hippuric acid, formed by glycine conjugation of benzoic acid, was found as the major metabolite of benzylamine. Benzylamine was metabolized to benzaldehyde via oxidative deamination. The aldehyde was rapidly converted to the carboxylic acid, which was subsequently conjugated with glycine and excreted as hippuric acid. Dosing rats with an equimolar mixture of d0and d7-benzylamine confirmed the oxidative deamination of benzylamine, as two of the deuteriums were lost during the metabolic process. The LC-APCI/MS spectrum showed that the d0- and d7-benzylamine produced protonated ([M + H]+) ions at m/z 180 and 185 of nonlabeled and labeled hippuric acid metabolites, respectively. The use of deuterium-labeled benzylamine also provided evidence that the hippuric acid was indeed a metabolite and was not entirely an endogenous product. Analysis of control bile samples showed that hippuric acid was excreted in the bile and urine of rats as an endogenous product, albeit at a very low level. Hippuric acids are normal constituents in urine of mammals, and its elevated levels in urine of animals are sometimes used as a biomarker of tissue damage (112).

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Figure 2. Dosing rats with mixtures of d0:d2- or d0:d7-benzylamine greatly facilitated location of the metabolites in bile and urine samples. Metabolites M1-M4 and M12-M13 lost two deuteriums located at the benzyl position, while the other metabolites (M5-M10 and M14-M15) showed retention of all seven deuteriums. The presence of twin ion pairs in the mass spectra, along with the MS/MS data, was used to demonstrate the retention of deuteriums in the aromatic ring or at the benzylic positions of the metabolites (58).

Figure 3. LC/MS spectra of metabolite M3 (cysteinylglycine conjugate) showing the [M + H]+ at m/z 316 and 321 for nonlabeled and d5-labeled analogues, respectively. (A) Loss of two deuteriums from d7-labeled benzylamine during the biotransformation to M3. (B) Loss of two deuteriums was from the benzylic position as no twin ion pair was observed (expected m/z 316 and 318 if deuteriums at benzylic position were retained). It was shown later that the benzamide formation was a prerequisite step prior to the bioactivation of the benzene ring (58).

Following dosing the animals with benzylamine, the levels of hippuric acid in urine increased by greater than 1000-fold. The deuterium-labeled benzylamine demonstrated that the hippuric acid was indeed a metabolite, and its high levels in urine and bile were not due to an injury to a tissue. LC/MS analysis of bile and urine obtained from rats dosed with 1:1 equimolar mixtures of either d0:d7- or d0:d2-benzylamine also showed the existence of several GSH-related adducts in addition to hippuric acid (Figure 2) (58). In addition to locating metabolites in bile/urine samples, mass spectrometry/

mass spectrometry (MS/MS) analyses of the LC/MS peaks that showed characteristic twin ions were used to aid structural assignments. For example metabolite M3, a cysteinylglycine conjugate was found to have lost two deuteriums from the benzylic position after analyzing the same metabolite peak from d0:d7- and d0:d2-benzylamine-dosed rats (Figure 3). Mass spectral analysis of the peak from the d0:d7-benzylamine-dosed rat showed that M3 had lost two deuteriums during the metabolism, while the d0:d2-benzylamine study confirmed that the loss of deuteriums was from the benzylic position. Further-

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Figure 4. Application of stable isotope-labeled GSH and a rapid scanning linear ion trap mass spectrometer to detect and characterize the formation of reactive metabolites from drug candidates (64). The use of a 1:1 mixture of labeled and nonlabeled GSH produces adducts that get detected as twin ions separated by 3 amu in the mass spectra. In most cases, the labeled and nonlabeled GSH adducts have almost identical retention times, hence enabling the appearance of twin ions corresponding to the molecular weights of adducts.

more, one can clearly see in Figure 3 that introduction of deuteriums at the benzylic position had a significant effect on the formation of M3 as evidenced by the much lower abundance of the ion at m/z 321 as compared to the ion at m/z 316 ([M + H]+) of the nonlabeled adduct. The identification of the GSH-related adducts indicated that benzylamine was metabolized to a number of reactive intermediates. Various metabolic pathways, including those independent of P450, were found to produce these intermediates (58). A previously undocumented pathway included the formation of a new carbon-nitrogen bond that led to a potentially reactive intermediate, Ar-CH2-NH(CO)-X, capable of interacting with various nucleophiles. Metabolites that were produced by the reaction of this intermediate, Ar-CH2-NH(CO)-X, with nucleophiles included S-[benzylcarbamoyl] GSH (M5), N-acetylS-[benzylcarbamoyl]cysteine (M8), S-[benzylcarbamoyl] cysteinylglycine (M6), S-[benzylcarbamoyl] cysteinylglutamate (M10), and N-[benzylcarbamoyl] glutamate (M9) (Figure 2). The structures of metabolites clearly show that benzylamine is bioactivated via several mechanisms, including those that are not fully understood. The toxicological significance of such an extensive bioactivation of benzylamine to reactive metabolites is currently unknown and needs to be further investigated. Nonetheless, the studies conducted with benzylamine demonstrated the value of stable isotope-labeled analogues in facilitating the detection and ultimately the characterization of metabolites in the biological fluids (58). 2.1.2. Detecting Reactive Metabolites Trapped as GSH Adducts. An ongoing effort within the pharmaceutical industry is to minimize the attrition of compounds in development due to toxicity. Metabolic bioactivation of potential therapeutic agents, leading to reactive intermediates, has traditionally been

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considered undesirable, and chemistry efforts are now being directed to minimize this potential metabolic liability. Several approaches are currently being used in the pharmaceutical industry to detect and at times obtain quantitative information on the bioactivation of potential drug candidates (113-119). A common approach is to use a nucleophile such as GSH to trap reactive intermediates generated in vitro and apply LC/MS techniques to characterize these metabolites (61). Identification and subsequent characterization of the trapped reactive metabolite often allow one to speculate on the metabolic routes and the possible structures of the reactive intermediates. One approach employed routinely by others and us is the use of stable isotope-labeled GSH for these trapping experiments (63, 64). Not only does it increase the chance of detecting the trapped conjugates, the use of stable isotope-labeled GSH also helps eliminate any potential false positives that may be observed by mass spectral analyses. Figure 4 illustrates the strategy of using stable isotope-labeled GSH for detecting trapped reactive metabolites using a linear ion trap mass spectrometer. The advantage of using a linear ion trap or any fast scanning instrument is the ability to acquire full scan and MSn data during a single analytical run. This allows one to look at the full range of data for every peak in the LC/MS chromatogram and confirm the existence or absence of any potential GSH adduct. For example, the extract from a microsomal incubation conducted according to the procedure outlined in Figure 4 was analyzed, and the presence of the GSH adduct derived from APAP was confirmed at tR ) 7.5 min. The potentially false positive peak at tR ) 6.5 min showing a neutral loss of 129 amu (characteristic fragment of GSH adducts) was shown not to be GSH-related by observing the absence of a twin ion pair at that retention time (Figure 5). An added advantage of using these stable isotope-labeled trapping agents is the ability to gain additional structural information based on how the trapped adducts fragment in the mass spectrometer. The relevance of detecting and quantitating these GSH adducts has been discussed recently (120). 2.2. Application of Stable Isotope-Labeled Compounds in Elucidating Metabolic Pathways and Structures of Metabolites. 2.2.1. Metabolism of a Disubstituted Alkyne Leading to a Reactive Oxirene Intermediate and the Subsequent Formation of GSH Conjugates. The elucidation of the structures of the metabolites derived from a postulated oxirene intermediate produced in rats treated with the disubstituted alkyne, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)3,4-dihydro-2(1H)-quinazolinone (DPC961), was achieved through the use of 13C stable isotope labels. The reactivity of the postulated oxirene would be expected to lead to the formation of novel GSH adducts whose structures were confirmed by LC/ MS and by two-dimensional NMR experiments (48). The postulated metabolic pathway leading to these GSH adducts is shown in Figure 6. To demonstrate the oxidation of the triple bond, an analogue of this compound was synthesized in which the two carbons of the alkyne moiety were replaced with carbon13 (Figure 6). Rats were orally administered with 13C-DPC 961, and the GSH adducts were isolated from the bile. The presence of an oxygen atom on one of the 13C atoms of the alkyne was demonstrated unequivocally by NMR experiments (Figure 7). This was achieved by comparing the 13C resonances of the alkyne carbons of 13C-labeled compound with those of the isolated GSH adduct. The 13C resonances of the labeled carbons had apparently changed due to the change in the hybridization from sp to sp2 and sp3. Figure 7 shows one-dimensional 13C NMR spectra of the labeled compound (top) and the labeled

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Figure 5. Detection and characterization of the GSH adduct of APAP using a linear ion trap instrument. A mixture of labeled and nonlabeled GSH was used to trap the reactive metabolite of APAP formed by incubation with rat liver microsomes. Peaks at tR ) 6.5 and 7.5 produced constant neutral losses of 129 amu characteristic of GSH adducts. However, full scan mass spectral analysis of the peaks at these retention times showed that the peak at tR ) 6.5 min was not a GSH adduct based on the absence of the typical twin ion expected from the use of a 1:1 mixture of labeled and nonlabeled GSH (64).

Figure 6. Proposed mechanism for the formation of the GSH conjugates from 13C-labeled DPC 961, which was postulated to form an oxirene intermediate. The localization of oxygen on one of the initial alkyne carbons during the oxidative metabolism was demonstrated by studying the changes in their carbon-13 NMR chemical shifts.

metabolite (bottom). The spectrum of the unlabeled compound shows acetylene 13C chemical shifts (68 and 92.5 ppm). The spectrum of the GSH conjugate shows a ketone 13C chemical shift (200 ppm) for carbonyl 2 and a methine 13C chemical shift (49.5 ppm) for carbon d (Figure 7). Elucidating the structures of the GSH adducts provided an insight into the possible mechanism leading to reactive intermediates. The formation of

oxirene, as demonstrated through studies with stable isotopelabeled analogues and NMR spectroscopy, was considered as an undesirable characteristic of this compound. Oxirenes are fairly reactive and can potentially interact with biomolecules such as proteins at their site of formation. Further toxicological evaluation of this compound was not conducted, and the

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Figure 7. 13C-labeling of the substrate (DPC 961) enabled us to confirm the oxidation of the triple bond by observing and assigning the C-13 chemical shifts of the original alkyne carbons. Upon oxidation, the carbon retaining the oxygen shows a significant downfield shift to approximately 200 ppm. Because of the low abundance of naturally occurring 13C, the labeling of the molecule enabled rapid acquisition of the carbon-13 NMR data for the carbons of interest as compared to the rest of the carbons in the molecule, as evidenced by the strength of these C-13 signals in the spectra (48).

chemical template was modified due to the potential metabolic liability associated with the oxirene formation. The application of NMR technology along with stable isotopelabeled compounds for understanding metabolic disposition of xenobiotics is still largely ignored. Studies with compounds labeled with either deuterium or 13C or a combination of both could be conducted in conjunction with NMR and mass spectrometry as analytical tools to effectively elucidate metabolic pathways and structures of metabolites. 2.2.2. Metabolism of a Benzylamine to a Glutamate Conjugate Mediated by γ-Glutamyltranspeptidas. Studies conducted in our laboratory showed that benzylamine-containing compounds, such as 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2′(methylsulfonyl)-[1,1′-biphenyl]-4-yl]-3-(trifluoromethyl)-1Hpyrazole-5-carboxamide, were metabolized to a number of unusual products, including a glutamate conjugate (52). It was important to understand the mechanism and metabolic pathway that led to the formation of this glutamate conjugate as it was excreted in urine and bile in abundant quantities. Preliminary studies suggested the possibility of GSH being the donor of the glutamate moiety for this conjugation process. To confirm this hypothesis, stable isotope-labeled GSH was used in conjunction with LC/MS to demonstrate the transfer of glutamate from GSH, a tripeptide, to the benzylamines in the presence of γ-glutamyltranspeptidase. To further demonstrate that the R-protons on the benzylamines and glutamate (part of GSH) were unaffected during the transpeptidation, these protons were replaced with deuterium (see Figure 8). The mass spectrum of the glutamate conjugates formed from d3-GSH and benzylamine and its stable isotope-labeled analogue is shown in Figure 9. The potential mechanism leading to the glutamate conjugate

formation needed to be understood as it involved GSH, which plays an important role in cell homeostasis and defense against toxic insult from oxidants and other reactive products generated from endogenous and exogenous compounds. Depletion of GSH via this metabolic pathway was considered undesirable for this compound. Long-term toxicity studies in rats with this compound demonstrated toxicities in kidney, liver, and other organs. It was postulated that this pathway could have perhaps played a role in these organ toxicities. Hence, the structure of this compound was modified whereby the benzylamine moiety, shown to be a metabolic soft spot in producing undesirable metabolites, was replaced with an alternate functional group that maintained the pharmacological activity. In summary, stable isotope-labeled analogues can play a very important role in identifying metabolites in complex biological samples. Combined with the powerful analytical capabilities of mass spectrometry and NMR, they provide us with a very useful tool in understanding the metabolic pathways and biochemical mechanisms that lead to the formation of metabolites.

3. Application of Deuterated Analogues in Mechanistic Metabolism-Mediated Toxicity Studies Substituting deuterium for hydrogen at a chemically labile site to decrease both reactive intermediate formation and toxicity is well-known (59, 121-125). Carbon-deuterium bonds have a higher activation energy and lower zero-point energy for cleavage than do carbon-hydrogen bonds (59, 126). If a carbon-hydrogen bond cleavage is the rate-determining step of a multistep process, then substitution of deuterium for hydrogen will slow the rate of formation of all metabolites

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Figure 8. Elucidating metabolic pathways and mechanisms for the formation of metabolites through the use of stable isotope-labeled compounds and reagents. In this case, the availability of d3-GSH (labeled on glutamate) enabled us to confirm the source of glutamic acid that was covalently linked to the nitrogen of the benzylamine via an amide bond. The deuteriums on the benzylamine confirmed that no oxidative metabolism occurred at this position prior to the γ-glutamyltranspeptidase-mediated transfer of glutamate from GSH (52).

Figure 9. Mass spectral data are very critical in confirming the proposed metabolic pathways based on the use of stable isotope-labeled compounds. The top panel (A) shows the [M + H]+ at m/z 663 obtained for the glutamate conjugate formed between d3-GSH and nonlabeled benzylamine (see Figure 7). The bottom pane shows an addition of 3 amu to the [M + H]+, giving m/z at 669 when a stable isotope-labeled (13C/deuterium) analogue was used in the incubation (52).

downstream of this step. One can take advantage of this primary kinetic isotope effect and use it as a tool to investigate metabolism-mediated target organ toxicities. Primary kinetic isotope effects on toxicity have been described previously for a number of compounds. The deuterium isotope effect on the metabolism and toxicity of 1,2-dibromomethane (DBE) and its deuterated analogue, tetradeutero-1,2-dibromoethane (d4-DBE), was studied in mice and has been described previously (121). In vitro studies that measured the release of bromide ion from the substrate as a measure of the rate of metabolism showed

that the liver microsomal metabolism of DBE was reduced by deuterium substitution, whereas the formation of a GSH conjugate mediated by GSH transferase was not affected. Three hours after intraperitoneal administration of DBE or d4-DBE (50 mg/kg), there was 42% less bromide in the plasma of d4DBE-treated mice than in plasma of DBE-treated mice. This difference demonstrated a significant deuterium isotope effect on the in vivo metabolism of DBE. Although the metabolism of d4-DBE was less than that of DBE 3 h after dosing, the DNA damage caused by both analogues was similar. At later time

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Figure 10. Structures of efavirenz (Sustiva, DMP266) and its monodeuterated analogue (d1-efavirenz) used to demonstrate the significance of metabolism at the cyclopropyl position in causing species-specific renal lesions in rats.

points (8, 24, and 72 h), d4-DBE caused greater DNA damage than DBE, suggesting that deuterium substitution led to a decrease in microsomal oxidation and perhaps higher levels of d4-DBE at these late time points. The mechanism of toxicity was attributed to the formation of a GSH adduct, which occurred independent of the microsomal oxidation process. Deuterochloroform (CDCl3) was reported to be 5-70% less toxic in rodents than CHCl3 (122). In this study, chloroform (CHCl3) was postulated to cause nephrotoxicity via its metabolism to toxic phosgene (COCl2) in the DBA/2J male mice. The investigators showed that kidney homogenates from mice metabolized CHCl3 in the presence of GSH to 2-oxothiazolidine-4-carboxylic acid (OTZ). This catabolite appeared to have been formed via renal processing of the GSH conjugate formed from phosgene. Introduction of deuterium led to a decrease in the formation of phosgene and subsequently a reduction in renal toxicity as less of the GSH adduct was being formed. In another study carried out with deuterated N-methylformamide (NMF) (deuteration on the formyl group), hepatotoxicity was reduced substantially (124). Metabolism of NMF to methylamine, N-acetyl-S-(N-methylcarbamoyl)cysteine, and S(N-methylcarbamoyl) GSH was demonstrated by mass spectrometry. The formation of these metabolites was found to be subject to large intermolecular primary kinetic isotope effects when hydrogen was substituted with deuterium in the formyl group (kH/kD ≈ 5-7). These numbers suggested the possible existence of a common metabolic precursor, methyl isocyanate (MIC), for each of these metabolites. It was suggested that N-methyl formamide was perhaps oxidized to MIC, which was trapped as the S-(N-methylcarbamoyl)GSH conjugate. Further processing of this conjugate led to the formation of N-acetylS-(N-methylcarbamoyl)cysteine, which was excreted primarily in urine. The presence of deuterium in the formyl moiety of NMF dramatically reduced the degree of hepatotoxicity in mice as shown by measurements of the biomarker hepatic enzymes in plasma. In a study conducted in our laboratory, the species-specific formation of a nephrotoxic GSH conjugate in rats was demonstrated through the use of a stable isotope-labeled compound (125). High doses of efavirenz produced nephrotoxicty only in rats, while similar exposures to the compound in other species did not elicit such a toxic response. Figure 10 shows the structures of efavirenz and its monodeuterated analogue, which was used to understand the mechanism of renal toxicity in rats. A cysteinylglycine diconjugate (see Figure 11), shown to be formed through a series of metabolic processes involving both phase 1 and phase II enzymes, displayed a unique presence in rat urine. This observation prompted further investigation into the possible role of metabolism in causing this renal toxicity.

Figure 11. By incorporating a single deuterium at the cyclopropyl methine position (marked with an asterisk), the formation of metabolite M10A downstream in the metabolic/catabolic pathway was significantly reduced (see Table 1). The isotope effect leading to the initial reduction in the formation of cyclopropanol precursor, M11A, decreased the incidence and severity of renal lesions in rats (125).

Table 1. Concentrations of Metabolites M1A (Glucuronide) and M10A (Cysteinylglycine) in the Urine of Rats Administered Efavirenz or d1-Efavirenz (125) efavirenz (700 mg/kg) d1-efavirenz (700 mg/kg)

M1A (mg/mL)

M10A (µg/mL)

3.4 ( 0.8 3.2 ( 0.7

28.1 ( 13.0 4.0 ( 1.1

Table 2. Incidence and Severity of Renal Cortical Epithelial Cell Necrosis in Rats Given Efavirenz (700 mg/kg po) and d1-Efavirenz (700 mg/kg po) no. of rats with renal cortical epithelial cell necrosis histologic severity 0 1 2 3 4 a

(unaffected) (minimal) (mild) (moderate) (severe)

efavirenz (700 mg/kg PO)a 0 2 4 2 2

d1-efavirenz (700 mg/kg PO)

a

2 4 3 1 0

N ) 10.

Hence, after elucidating the structures and confirming the metabolic pathways in various species, mechanistic studies involving the use of deuterium-labeled compound were initiated. Deuterium replacement of the methine hydrogen in efavirenz at the site of hydroxylation on the cyclopropyl ring markedly reduced the amounts of GSH and subsequently the cysteinylglycine conjugates produced in rats. Moreover, the incidence and severity of nephrotoxicity in rats were also reduced (Table 2 and Figure 12). The mechanism by which these changes occur is via a primary kinetic isotope effect (127, 128) that reduces the cytochrome P450-mediated hydroxylation of efavirenz to the cyclopropanol intermediate, which then undergoes enzymecatalyzed conjugation to form the GSH adduct (Figure 11). The site-specific effect of deuterium on efavirenz metabolism was demonstrated by quantitative assessment of metabolites pro-

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Figure 12. Incidence and severity of lesions in rats given equivalent doses (700 mg/kg, po) of efavirenz or d1-efavirenz (125). In a blinded study, tissue sections were graded for the severity of necrosis using the following scale: 0, no lesions; 1, minimal lesions (single to few necrotic cells); 2, mild lesions (10-25% necrotic cells); 3, moderate lesions (25-40% necrotic cells); and 4, severe (>40% necrotic cells).

duced in vivo in rats. The formation of the cysteinylglycine (M10A) and the glucuronide conjugates (M1A) was assessed in the urine of animals administered either efavirenz or its deuterated analogue. Table 1 shows a marked decrease in the levels of M10A, attributed to the downstream effect of deuterium substitution at the metabolic soft spot on the molecule. In contrast, deuterium substitution did not affect the formation of other metabolites such as the glucuronide conjugate (M1A), which is formed at a distal site away from the point of modification on the molecule. It is important to demonstrate in such mechanistic studies conducted with deuterium-labeled analogues that metabolic pathways other than the one of particular interest are not affected, hence complicating interpretation of the data. The results from a short-term study conducted in rats demonstrated quite clearly that the deuterated analogue was far less toxic than the unlabeled efavirenz. The unique metabolism of efavirenz in rats combined with results from mechanistic toxicity studies conducted with deuteriumlabeled analogue facilitated the approval of this compound to treat HIV-infected patients. As demonstrated in these examples, this approach of using stable isotope-labeled compounds (especially deuterium-labeled analogues) is a powerful tool to study chemical mechanisms involving the formation of reactive intermediates. Studies such as the one described above, if planned and conducted carefully, can be used to effectively demonstrate species differences in metabolism and toxicity of a compound, especially if a potential toxic metabolite is formed in a speciesspecific manner. Establishing a link between the formation of metabolite and the organ toxicity is always a challenge. However, as demonstrated in this example, through the use of stable isotope-labeled analogues, one can get a better understanding of species-specific metabolism-mediated toxicities. The resulting data can be presented to the scientific community and regulatory agencies to demonstrate metabolism-mediated toxicity in a preclinical species, and a valid argument can be presented showing that such a metabolic pathway and its potential liability

are not applicable to the human population. Mechanistic studies such as those employing stable isotope-labeled compounds can be conducted so that potentially useful therapeutic agents meant to treat human diseases can be progressed into development and not terminated unnecessarily due to toxicity findings in a preclinical species. The pay-off from such studies can be tremendous; however, it is always a challenge to obtain sufficient resources and support from diverse functional groups within a drug discovery establishment to fully understand mechanisms of toxicity.

4. Application of Stable Isotope-Labeled Compounds in Toxicogenomic Studies Efforts are currently directed within the pharmaceutical industry to address potential toxicity issues earlier in the drug discovery and development process (129-131). Traditionally, toxicity studies have been focused on evaluating the effects of exogenous chemicals, one chemical at a time, through a battery of tests in vitro or in animals. Toxicology studies rely heavily on the use of animals, an expensive endeavor in both time and money with debatable relevance to human safety. The problem lies mostly with our inability to elucidate mechanisms of toxicity for the majority of compounds tested in animals, hence limiting our power to predict potential toxicities of new chemical entities, especially in humans. In several instances, the toxicities have been directly attributed to the parent compound; however, in several cases, metabolites have been identified as the causative agents (114, 122, 123, 132-137). Nonetheless, our mechanistic understanding of many of these toxicities is still lacking. Hence, a significant effort is being made within the pharmaceutical industry to find effective screening assays that would assist in identification of chemical templates with safety concerns, as well as provide an insight into the mechanisms of potential toxicities. Reactive metabolites formed via metabolic bioactivation of compounds are of concern, as these are often circumstantially linked to toxicities. Establishing a link between

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Figure 13. Structures of NMF and its deuterated analogues, d1-NMF and d3-NMF, used to study gene changes in mice livers.

reactive metabolite formation and observed target organ toxicity is often a challenge and requires considerable expenditure of resources. Hence, quite frequently, the possible association between metabolic bioactivation and toxicity is either ignored or not fully studied. Therefore, alternate, less resource-consuming strategies are needed to predict toxicities. Recently, toxicogenomics, which has gained popularity as a predictive tool in toxicology (138-144), offers some hope as both a screening tool and for providing insights into the possible mechanisms of toxicity. Many drugs that elicit an adverse drug reaction form reactive metabolites capable of interacting with macromolecules such as proteins. Unfortunately, there are no simple rules to predict the target macromolecules for particular reactive metabolites or the biological consequences of a particular modification. To better understand the potential role of reactive metabolites in causing toxicities, a number of studies involving toxicogenomics approaches have recently been undertaken (145-151). The ultimate goal has been to identify potential biomarker genes that show significant alterations in expression as a result of reactive metabolite formation that is capable of producing a toxic insult. Of course, one must realize that the gene changes could take place as a consequence of adaptive response to tissue injury as well. Furthermore, the interaction of the compound with several receptors in the body can also elicit gene changes, complicating the assessment of gene changes. Accordingly, one of the major challenges in these toxicogenomic studies has been the inability to pinpoint gene expression changes attributable to the formation of reactive metabolites. There is a need for further studies that will enable differentiation in gene changes due to the reactive metabolites as compared to off-target effects. The ability to modulate reactive metabolite formation by using stable isotope-labeled compounds offers the opportunity to focus the search for predictive biomarker genes of target organ toxicities. It has been well-documented that a stable isotope-labeled compound and its nonlabeled counterpart are similar in terms of target interaction. However, because of the reduced metabolism of the deuterated analogues, the onset and severity of target organ toxicities attributed to reactive metabolite formation can be modulated effectively (123). As stated before, deuterium analogues of compounds have been used to effectively demonstrate the involvement of reactive metabolites in causing organ toxicities (121-125). The decreased toxicities demonstrated by deuterium-labeled analogues should also show concurrent reductions in gene changes as compared to those from treatment with nonlabeled compounds. These differences in toxicity produced by stable isotope and nonlabeled analogues can be used to better understand specific gene changes elicited by reactive intermediates. In a recent study, we utilized a known hepatotoxic compound NMF and its two analogues labeled with deuterium at different positions to block metabolic oxidation at the formyl (d1) and methyl (d3) moieties (Figure 13) (152). NMF was chosen because of its small size and the limited number of metabolites that could be formed from this compound. The metabolism of NMF has been previously described in the literature (124, 153, 154), whereby it was shown to be metabolized primarily via two metabolic pathways: (i) hydroxy-

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lation of the N-methyl group leading to the formation of a fairly stable carbinolamide and (ii) formation of S-linked conjugates via a reactive MIC intermediate (Figure 14). The pathway leading to MIC has been studied extensively and has been implicated in NMF-induced liver toxicity (152-154). MIC is categorized as being a reactive soft electrophile capable of reacting with various cellular components. The reactivity of the carbinolamide, while not clearly established, could lead to the formation of the harder electrophiles formamide and formaldehyde, capable of affecting DNA bases. Hence, we hypothesized that these two metabolic pathways would differentially affect various genes and proteins. It has been demonstrated that introduction of deuterium at the formyl position (N-methylform-d1-amide or d1-NMF) leads to a significant kinetic deuterium isotope effect (124) in reducing the formation of MIC (trapped as N-acetylcysteine or GSH adducts). Not surprisingly, studies conducted in mice showed that administration of stable isotope-labeled d1-NMF led to a reduction in the degree of hepatotoxicity. Studies conducted previously by other investigators have shown that the centrilobular hepatic toxicity caused by NMF is most likely due to the formation of MIC (124, 153). Hence, modulating the formation of MIC by introducing a deuterium atom in the molecule gave us an opportunity to decrease hepatotoxicity as well as to study the gene changes brought about by the altered drug metabolism. It is postulated that a deuterium at the formyl position decreased MIC formation and/or shifted metabolism of NMF to hydroxylation of the N-methyl group. Hence, a comparison of gene changes in livers of mice dosed with d0and d1-NMF aided us in identifying potential genomic biomarkers responsive to a reactive metabolite such as MIC. To further assist us in this process, the d3-NMF analogue was also included in this study. The d3-analogue (Figure 14) was postulated to lead to a decrease in the formation of N-(hydroxymethyl)formamide as a result of a deuterium isotope effect. It was shown that d3-NMF produced greater quantities of reactive MIC as compared to the nonlabeled NMF since a greater fraction of the dose was shunted through oxidation to MIC. A comparison of the gene expression changes observed in livers of mice dosed with stable isotope-labeled and nonlabeled NMF analogues revealed distinct expression patterns that could be attributed to differences in metabolism of these compounds (Table 3). Data (in vitro and in vivo) suggested that d3-NMF led to a greater production of MIC as compared to d0-NMF. If a particular gene showed changes in signal intensity in the order d3-NMF > d0-NMF > d1-NMF > saline control (up-regulated) or d3-NMF < d0-NMF < d1-NMF < saline control (down-regulated), it was more likely to be associated with pathway b (MIC formation). However, if the order was d1-NMF > d0-NMF > d3-NMF > saline control (up-regulated) or d1-NMF < d0-NMF < d3-NMF < saline control (downregulated), that gene’s regulation was more likely to be associated with metabolic pathway a (Figure 14). Some of the specific gene changes attributed to pathway b were confirmed by real-time polymerase chain reaction (RT-PCR) (Figure 15). Furthermore, mice were dosed with a synthetic standard of MIC, and the gene changes attributable to this reactive intermediate were confirmed. In addition to deciphering the liver genomic changes produced by administering NMF and its stable isotope-labeled analogues, the changes in serum enzymes indicative of liver injury (i.e., ALT and AST) were also monitored. Finally, a time-matched histopathologic evaluation of the liver samples was conducted. The intention was to demonstrate that the gene expression

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Figure 14. Metabolic pathways of NMF leading to the formation of N-(hydroxymethyl)formamide (pathway a) and MIC (pathway b). The deuterium on the formyl position is lost during the P450-catalyzed oxidation to MIC. The placement of deuterium(s) at either the formyl or the methyl positions can lead to metabolic switching via either of these two pathways. For example, deuteriums on the methyl group can lead to greater levels of MIC formation through pathway b. This was demonstrated by showing greater levels of the N-acetylcysteine conjugate in urine of mice dosed with d3-NMF (152).

Table 3. Genes Showing the Following Order of Fold-Change in Signal Intensities as Compared to Saline Control: d3-NMF > d0-NMF > d1-NMF (Negative Numbers, Down-Regulated Genes; Positive Numbers, Up-Regulated Genes)a gene symbol

d1-NMF

d0-NMF

d3-NMF

down-regulated genes thyroid hormone responsive SPOT14 homologue nuclear distribution gene E homologue 1 sterol regulatory element binding factor 1 lipase, endothelial annexin A6 epidermal growth factor receptor adenosine monophosphate deaminase 2 (isoform L) βGlcNAc β-1,3-galactosyltransferase, polypeptide 3 serum deprivation response ankyrin repeat domain 10 fibronectin 1 cyclin D1

Thrsp Nde1 Srebf1 Lipg Anxa6 Egfr Ampd2 B3galt3 Sdpr Ankrd10 Fn1 Ccnd1

-3.9 -3.7 -3.6 -3.6 -3.3 -2.8 -2.6 -2.6 -2.4 -2.4 -2.3 -2.2

-8.7 -3.9 -5.6 -6.2 -4.7 -3.5 -8.1 -5.5 -3.1 -4.7 -4.2 -3.8

-13.3 -4.0 -5.8 -10.4 -7.4 -5.4 -10.9 -7.1 -4.6 -7.0 -6.1 -5.1

up-regulated genes fibroblast growth factor inducible 15 UDP-glucose pyrophosphorylase 2 GrpE-like 2, mitochondrial angiopoietin-like 4 nuclear receptor subfamily 1, group D, member 2 serum/glucocorticoid regulated kinase 2 dual specificity phosphatase 6 DNA segment, Chr 17, ERATO Doi 808, expressed chemokine (C-C motif) ligand 3 serum glucocoticoid regulated kinase

Fin15 Ugp2 Grpel2 Angptl4 Nr1d2 Sgk2 Dusp6 D17Ertd8 CCl3 Sgk

2.1 2.2 2.3 2.4 2.8 3.1 4.6 5.1 3.4** 1.5**

2.6 2.6 3.3 3.9 3.1 3.6 6.6 9.7 9.7** 9.3

3.2 4.5 3.9 4.4 3.4 3.7 9.0 10.2 12.1** 13.8

a Pathway b (MIC formation) for the metabolism of NMF most likely produced these gene changes (see Figure 14). P values for each comparison were less than 0.05 unless otherwise stated; **p > 0.1.

changes were being monitored before the onset of full-blown hepatotoxicity. Because some of the gene changes were directly attributed to the formation of reactive intermediates, a deuterium isotope effect leading to significant metabolic switching had to be demonstrated. For example, it was postulated that having three deuteriums on the methyl group of NMF would slow the formation of the carbinolamide metabolite, favoring the alternate metabolic pathway of MIC formation (Figure 14). Hence, some of the gene changes in livers of d3-NMF-dosed animals as compared to d0-NMF-dosed animals could be attributed directly to MIC formation. Lastly, we demonstrated corroboration between gene changes, serum biomarker enzyme levels, and histopathology data. At the 6 h time point, there were minimal

histopathologic findings, while significant alterations in the expression of potential biomarker genes were evident. One of the important observations from this study was that despite greater formation of MIC (reactive metabolite) from d3-NMF, the degree of liver toxicity was less than that observed with d0-NMF. These results suggest that while reactive MIC definitely plays a role in liver toxicity, other factors such as oxidative stress may be important as well. The number of studies conducted using stable isotope-labeled compounds to study gene changes is very limited. There are abundant opportunities to conduct toxicogenomic studies with isotope-labeled compounds to get a better understanding of the relationship between the metabolic processes and the gene

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Figure 15. Example of how gene changes can be studied using stable isotope-labeled analogues. These gene changes were attributed to the formation of MIC produced via pathway b shown in Figure 14. The d3-NMF is expected to produce more MIC (due to metabolic shunting) than either d0- or d1-NMF, while d1-NMF is expected to produce the least amount of MIC. RT-PCR of these genes believed to be associated with MIC formation was conducted (152).

changes. This is an area of research that needs to be actively pursued, especially by those interested in establishing a mechanistic link between the formation of reactive metabolites, gene changes, and target organ toxicities. However, one of the major challenges in toxicogenomics studies is to be able to distinguish gene changes as a result of direct toxic insult by a reactive intermediate from those derived from adaptive response as a consequence of tissue injury. The approach that we took in our laboratory was to study gene changes at time points whereby the histological evaluation of tissues did not show any evidence of tissue damage (152).

5. Application of Stable Isotope-Labeled Compounds in Clinical ADME Studies The majority of clinical ADME studies conducted within the pharmaceutical industry entail the administration of C-14 radiolabeled compounds to healthy human volunteers. Subsequently, the metabolism and mass balance information are obtained from these single-dose studies. Traditionally, these clinical ADME studies have been performed fairly late in the development; however, with the recent release of the FDA guidance on metabolites for safety testing, it is quite possible that these ADME studies will be conducted much earlier to ensure the coverage of human metabolites in toxicity species (155). The recent demands from the regulatory agencies could potentially lead to human drug metabolism studies being conducted much earlier with the intent of characterizing and quantitating major [>10% of parent area under curve (AUC)] circulating metabolites. One possibility is the use of stable isotope-labeled analogues of potential therapeutic agents in obtaining preliminary qualitative and semiquantitative information on the circulating metabolites in humans. Obtaining quantitative information on the levels of circulating metabolites in the absence of synthetic standards of metabolites is a challenge, especially in the absence of radiolabeled compounds. Administration of a 1:1 mixture of labeled:nonlabeled compound could be performed so that relevant metabolites could be identified in plasma from first in human (FIH) studies. Subsequently, the presence and coverage of these metabolites in preclinical toxicity species could be evaluated. The mass spectrometry field has advanced to a stage where the administration of 1:1 mixture may not be entirely necessary for qualitative metabolite identification purposes; however, such an approach could be useful if additional quantitative information could be derived from these studies, especially if synthetic standards of metabolites are not available. One approach that

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has received limited publicity is the application of chemical reaction interface mass spectrometry (CRIMS) in combination with stable isotope isotope-labeled compounds (156-160). With CRIMS, LC or GC eluents are directly combusted inside a microwave-induced plasma chamber followed by further reaction with an oxidizing gas such as sulfur dioxide (160). CRIMS signal response for a given element is a direct function of the label on analyte, and thus, this technique has direct application for detecting and quantitating isotopically labeled compounds. Compounds labeled with carbon-13 are detected at m/z 45 (13CO2) while 15N-labeled compounds are detected at m/z 31 as 15NO. This method eliminates the variation in ionization efficiencies that are often encountered during mass spectral analyses of different analytes, such as metabolites of compounds. The reason for uniform response is that all drug-related compounds maintaining the stable isotope labels get converted to the same neutral elements prior to mass spectral analyses. The hyphenation of liquid chromatography with CRIMS presents an opportunity to perform quantitative measurement of metabolites in the absence of authentic standards and radiolabeled compounds (156-158). Nonetheless, there are certain drawbacks and limitations with this technology. It is not widely available commercially and suffers from low sensitivity. Furthermore, it cannot be used in lieu of radiolabeled compounds to conduct mass balance studies. Nonetheless, it has the potential to provide us with quantitative information on the levels of metabolites present in plasma from humans and preclinical species. However, despite its huge potential, this technology has not been fully developed and explored for routine metabolism studies. The availability of a robust commercial instrument could lead to greater use of this technology in combination with stable isotope-labeled compounds in early human ADME studies in the future.

6. Recent Developments and Future Directions The greater availability of stable isotope-labeled analogues, especially synthesized to be used as internal standards for quantitative studies, has made it possible to use these compounds to conduct routine and mechanistic metabolism studies. Often, a 1:1 mixture of labeled:nonlabeled compound is used to create recognizable mass spectral ion patterns showing the presence of drug-related materials in complex biological mixtures. Technology has advanced to a point where a combination of mass spectrometry and stable isotope-labeled compounds can be used to provide a wealth of information on the metabolic disposition and identities of metabolites in the absence of radiolabeled compounds or authentic metabolite standards. Furthermore, we can anticipate other analytical techniques such as NMR to be more widely used in conjunction with stable isotope-labeled compounds and mass spectrometry to better understand metabolic disposition and in elucidating structures of metabolites. Recent advancements in NMR technology that have allowed significant gain in sensitivity will make it more amenable in elucidating structures of metabolites of compounds labeled with stable isotopes such as carbon-13. Strategic placement of stable isotope label(s) in a compound can also allow us to better understand some of the gene changes attributed to reactive metabolite formation or to a particular metabolic pathway. Target organ toxicities can be modulated by selective introduction of stable isotopes, such as deuterium, in a molecule. Studying and comparing gene changes produced by labeled and nonlabeled compounds can provide an idea of critical genes that may be involved in the onset of toxicities. This is an area of further research in the future as we make

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attempts to obtain signature genes that could be used as potential biomarkers for specific target organ toxicities. Furthermore, one can use stable isotope-labeled compounds to delineate potential metabolism-mediated toxicities. If one suspects that a particular metabolic pathway or a metabolite is involved in causing toxicity, stable isotope labels can be placed in such a manner as to modulate the formation of the specific metabolite, hence potentially mitigating the toxicity. Obviously, one can conduct in vitro studies with labeled and nonlabeled compounds to understand the effect of labeling (such as deuterium isotope effect) on the formation of a metabolite before an extensive toxicity study is conducted. Stable isotope-labeled compounds, although used widely in pharmaceutical industry, are perhaps being underutilized in metabolism and toxicity studies. Significant resources are being spent on making labeled analogues usually for the purpose of using these as internal standards for routine quantitative studies. The greater availability of these labeled analogues should see more routine and mechanistic metabolism studies being conducted with such analogues in the future. The use of stable isotope-labeled compounds in understanding the interaction between reactive metabolites and proteins needs to be explored. Having stable labels on reactive intermediates can greatly assist the identification of sites on proteins modified through covalent binding. Studies can be designed to investigate if particular proteins are targeted by reactive intermediates using stable isotope-labeled compounds. Studies encompassing the simultaneous use of radio- and stable isotope-labeled compounds to study proteomic and genomic changes as a consequence of reactive metabolite-mediated toxicity should potentially lead to a better understanding of some target organ toxicities and perhaps may lead to the identification of potential genomic or proteomic biomarkers. The recent FDA guidance on the safety testing of metabolites will probably lead some investigators to revisit the application of stable isotope-labeled compounds in ADME studies. Our ability to demonstrate human-specific metabolite coverage in preclinical species as early as possible has become a challenge with the issuance of this guidance. Hence, identification of major human metabolites (considered to be greater than 10% of parent AUC values) during early drug development has become very important. The administration of a 1:1 mixture of labeled and nonlabeled analogues is one approach that will enable us to rapidly identify all drug-related components in the plasma of humans during early stages of drug development. Even though major progress has been made in the field of mass spectrometry in detecting and identifying metabolites, one can still possibly miss unexpected or unusual metabolites using the existing LC/ MS technology. The appearance of twin ion pairs in the mass spectra of plasma extracts can be used to scan for all possible metabolites in circulation in the absence of synthetic metabolite standards or radiolabeled compounds. This approach will furnish only qualitative and semiquantitative information on metabolites found in human circulation. However, it remains to be seen whether pharmaceutical companies will commit resources to conduct such early metabolism studies in humans administered with mixtures of stable isotope-labeled and nonlabeled compounds. The application of LC-CRIMS in combination with stable isotope-labeled compounds to obtain both qualitative and quantitative information on metabolites of potential therapeutic agents administered in early human studies also appears logical and appealing. LC-CRIMS could potentially play a more significant role in the future as we make attempts to demonstrate quantitative coverage of human-specific metabolites in toxicol-


ogy studies. However, further refinement of CRIMS technology is needed before it gets widely used in such studies. Note Added after ASAP Publication. There was a production error in which the author list for reference 103 was published as the author list for the perspective in the version published ASAP August 15, 2008; the corrected version was published ASAP August 16, 2008.

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