Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
DEPARTMENT for ENVIRONMENT, FOOD and RURAL AFFAIRS Research and Development CSG 15 Final Project Report (Not to be used for LINK projects) Two hard copies of this form should be returned to: Research Policy and International Division, Final Reports Unit DEFRA, Area 301 Cromwell House, Dean Stanley Street, London, SW1P 3JH. An electronic version should be e-mailed to [email protected] Project title Differential Metabolism of Chiral Compounds DEFRA project code PS2501 Contractor organisation and location Health and Safety Laboratory Broad Lane Sheffield, S3 7HQ Total DEFRA project costs Project start date £ 49,530 01/12/02 Project end date 30/11/03 Executive summary (maximum 2 sides A4) The majority of organic chemicals, including those being developed and used as pesticides, occur as one or more types of isomer. There are several classes of isomer, categorised into structural and stereoisomers. Structural isomers, that share a common molecular formula, generally have vastly different physical, chemical and biological properties, to the extent that they have different synthetic pathways and are readily separated from mixtures. In contrast, stereoisomers contain the same chemical groups and bonds and only differ in their three-dimensional structure so their physical, chemical and biological properties are often similar. Chiral compounds, comprising the mirror-image enantiomers and diastereomers (non-enantiomeric stereoisomers containing multiple chiral centres), belong to this latter group. Enantiomers pose an exceptional challenge to product development and risk assessment since they are physically and chemically identical in a non-chiral environment and are formed in a 50:50 ratio during chemical synthesis. Thus they are often treated as the same compound and, as a result, many of the chiral pesticides in current use are produced as mixtures of isomers. This causes a problem because chiral compounds can be viewed as a mixture composed of the active isomer plus impurities (other chiral isomers), but risk assessments are generally not carried out for the indivudual components of the mixture. While the chiral enantiomers and diastereomers are difficult to separate and distinguish in the laboratory, the body, however, is a chiral environment; biological macromolecules being constructed from single enantiomeric forms of amino acids and sugars. Thus chiral compounds may be distinguished in vivo resulting in stereoselective interactions with protein receptors and enzymes. As a result, one isomer of a chiral compound may have a desirable effect while its enantiomer or diastereomers may be inactive or even have unwanted side effects. In fact, stereoselective behaviour of chiral compounds has been recognised for over a century, but investigation of the mechanisms of these differential effects was not possible until the last few decades. Having initially focussed almost entirely on differences in the intrinsic biological activity of enantiomers, the potential importance of differential toxicokinetics, particularly metabolism, is a more recent consideration. CSG 15 (Rev. 6/02) 1 Project title Differential Metabolism of Chiral Compounds DEFRA project code PS2501 However, despite extensive published literature on chiral compounds, especially pharmaceuticals, differential metabolism of chiral compounds remains poorly characterised. This project has reviewed the scientific literature for chiral compounds in order to assess the potential importance of stereoselective differences in their metabolism. This information is important to guide policy decisions regarding whether toxicological data should be required for each isomer of a chiral compound. Approximately 1/3 of currently registered pesticide active ingredients in the UK contain one or more chiral centres. Only a few of these are available for use as a purified enantiomer. In addition, other non-chiral compounds may form chiral metabolites. Therefore, chirality is likely to be an important consideration in the risk assessment of pesticides. Thus, this review has focussed, wherever possible, on pesticides. However, due to the limited literature for this class of compound, examples of pharmaceuticals and industrial chemicals have also been used to illustrate the possible outcomes of differential metabolism. There are many examples of differential metabolism of chiral compounds in the published literature from the past 40 years. Varying degrees of selectivity (or sometimes complete specificity) have been noted in both metabolism of parent compound (substrate stereoselectivity) and production of metabolites (product stereoselectivity). The majority of chiral compounds exhibit slight differences in metabolism; however, some notable examples are available where major differences in metabolism of enantiomers occur. For example, the chiral pyrethroid insecticide fenvalerate produced a toxic metabolite in experimental animals. Further research indicated that this metabolite was only formed from one isomer, which was not an active insecticide. As a result, the purified active isomer was marketed as esfenvalerate. There are several examples of differential metabolism of chiral pharmaceuticals resulting in adverse side effects and, consequently, pharmaceuticals are now routinely produced as the purified active enantiomer. Conversely, there are other examples where no differences have been found in the metabolism of chiral compounds. Metabolism studies have been conducted on an ad hoc basis and there has been little effort to systematically describe the likelihood of significant differential metabolism arising for a given chiral compound. However, the available data suggests that most chiral compounds will exhibit some degree of differential metabolism. Several factors need to be taken into account when considering any requirement for additional toxicological data for chiral pesticides. Aside from economic factors, these include weight of scientific evidence, availability of suitable analytical technology and availability of test methods. A thorough review of the scientific literature indicates that most compounds exhibit some degree of stereoselective metabolism. Importantly, some compounds exhibit complete stereoselectivity, so different metabolites are produced from the individual enantiomers. It should be noted, however, that orally administered pharmaceuticals may be more sensitive to the effects of first-pass metabolism so differential metabolism might be more prominent for this class of compound than pesticides, where exposures are likely to be low-dose and via other routes than oral. Chiral separation technology has undergone significant developments over the past 30 years, facilitating the routine separation of enantiomers, although some compounds still present considerable analytical challenges. This report highlights several experimental approaches that have been applied to study differential metabolism of chiral compounds. An in vitro approach might be suitable for screening for differential metabolism of chiral compounds. This would help to minimise the cost of these additional tests and would also be in keeping with efforts to develop alternative test methods to reduce, refine or replace the use of laboratory animals. We therefore conclude that, most of the time, currently available technology should enable producers to evaluate the extent of differential metabolism that occurs for chiral compounds if this were to become a regulatory requirement. As noted above, the majority of chiral metabolism research has involved pharmaceuticals, which may behave quite differently from pesticides because of different exposure routes or dose levels. Few studies have been published that describe metabolism of chiral pesticides, so it is difficult to directly demonstrate the relevance of differential metabolism in pesticide toxicology. There is therefore a requirement for further research, focussing specifically on the potential for differential metabolism of chiral pesticides. In particular, there is a need to systematically compare metabolism of a chiral pesticide across a range of experimental animal and other potentially exposed species. We recommend that further work should be conducted to demonstrate a generic approach for evaluating differential metabolism of chiral pesticides and we suggest an in vitro approach using test compounds where metabolism has been previously characterised (e.g. some pyrethroid or organophosphate insecticides or coumarin anticoagulants). This work would demonstrate the importance (or not) of differential metabolism in mediating the toxicity of chiral pesticides. It would form the basis of a standardised assay to screen for differential metabolism that could be part of a tiered approach to gathering CSG 15 (Rev. 6/02) 2 Project title Differential Metabolism of Chiral Compounds DEFRA project code PS2501 toxicological data for chiral pesticides. Data obtained from these in vitro studies would be suitable for subsequent use in various computer-modelling applications either to aid the interpretation of the in vivo situation or as data sets for structure-activity relationship approaches. CSG 15 (Rev. 6/02) 3 Project title Differential Metabolism of Chiral Compounds DEFRA project code PS2501 Scientific report (maximum 20 sides A4) 1 Introduction 1.1 Isomerism and Chirality Many chemicals exist in a number of isomeric forms, which are classified as either structural or stereoisomers (1). Structural isomers share the same molecular formula but have distinct chemical structures that confer different physical, chemical and biological properties. In contrast, stereoisomers contain the same atoms connected by the same sequence of bonds and only differ in their threedimensional structures. Stereoisomers can be further subdivided into geometric and optical isomers. In the case of geometric (cis-/ trans-) isomers, variations in the distance between chemical groups generally confer sufficient differences in physical, chemical and biological properties to allow them to be easily distinguished. This contrasts with optical isomers, which contain one or more chiral (also termed asymmetric or stereogenic) centres and occur as either enantiomers or diastereomers. Optical isomers are generally much more difficult to distinguish and separate from one another. In fact enantiomers have identical physical and chemical properties in a non-chiral environment. Hence, in the non-chiral environment of the laboratory, enantiomers appear, and are often treated as, the same compound. Chirality arises most commonly when an atom such as carbon, silicon, nitrogen, phosphorus or sulphur forms a tetrahedral structure with four different chemical groups attached. Two non-superimposable mirror images called enantiomers are formed that are identical in the connections and spatial separations between groups and only differ in the arrangement of the groups around the chiral centre (figure 1). D D A C C A B B mirror plane Figure 1: Representation of a pair of enantiomers where A, B, C and D are different chemical groups attached to the chiral (asymmetric or stereogenic) centre. Compounds can also exhibit an overall chiral shape. The presence of a rigid feature in the molecule, e.g. a carbon-carbon double bond, may result in enantiomers being formed along a chiral axis. Alternatively, steric effects may arise, particularly in ring structures, where sufficiently high-energy barriers exist to prevent free rotation about a bond allowing enantiomeric atropisomers to exist. At this stage, it is important to note the distinction between enantiomers and diastereomers. Enantiomers are exact mirror images of each other, thus they have identical structures and spatial separation between chemical groups. They are identical in all physical and chemical properties except in the direction of rotation of plane-polarised light and in their reactions with other chiral molecules. Chemical synthesis from non-chiral precursors results in a 50:50 mixture of the enantiomers, known as a racemate. Diastereomers (or diastereoisomers) occur in compounds that contain more than one chiral centre. They are non-enantiomeric optical isomers. The relative distance between chemical groups of diastereomers differs in a similar way as geometric isomers so they are sometimes given cis- and trans- notation. A consequence of this structural difference between diastereomers is that although their physical, chemical and biological properties may be similar they are not identical and diastereomers can be relatively easily distinguished. Chemical synthesis of diastereomeric compounds often produces fixed ratios of diastereomers and this ratio may be stated in the technical product. Inversion of a single chiral centre produces the epimer. All of the chiral centres must be inverted to produce the enantiomer. Thus regardless of the number of chiral centres contained in a compound, any given stereochemical conformation can only exist as one pair of enantiomers. In summary, a molecule with (n) chiral centres exists as a pair of enantiomers, 2 n-2 diastereomers and n epimers. Molecular symmetry within a molecule may reduce the number of possible stereoisomers by formation of meso compounds, where enantiomer pairs are identical. Enantiomers are therefore a unique type of isomer because they are chemically and physically identical (except in their interactions with other chiral molecules) which presents an exceptional challenge to the development and risk assessment of chiral compounds. Stereochemical terms are often used interconvertibly in the literature. Hence ‘stereoisomer’ is often used when ‘enantiomer’ may be more appropriate. In this report, the specific terms ‘enantiomer’ or ‘diastereomer’ will be used wherever possible and ‘stereoisomer’ CSG 15 (Rev. 6/02) 4 Project title Differential Metabolism of Chiral Compounds DEFRA project code PS2501 will be used to describe enantiomers and diastereomers collectively. However, when used in the context of chirality, the term ‘stereoisomer’ generally refers only to optical isomers and excludes other types of stereoisomer, such as geometric isomers. 1.2 Significance of Chirality in Biological Systems While enantiomers display identical physical and chemical properties in the non-chiral environment of the laboratory, living organisms are chiral, being constructed from the single enantiomeric (L)-form of amino acids and (D)-form of sugars. Hence any interaction in vivo between a chiral compound and a protein receptor or enzyme might be stereoselective. The potential therefore exists for individual enantiomers to exhibit different biological activity, depending on the importance of three-dimensional geometry to the interaction between the chemical and the molecular receptor. Indeed stereo-selective behaviour of chiral compounds has been observed since the experiments of Pasteur in the mid 19 th Century (2, 3). There are numerous examples in the scientific literature where individual enantiomers display quantitatively and qualitatively different pharmacokinetic or pharmacodynamic activities (411). Consequences of this difference in biological activity can range from half of the dose of a racemic mixture being inactive to half of the dose causing unwanted, potentially harmful, side effects. However, there are also examples of chiral compounds where both enantiomers have similar desirable biological activity (12, 13). Therefore, the frequency with which individual enantiomers exhibit different biological effects needs to be better characterised in order to underpin the risk assessment of chiral compounds and give a sound scientific basis for deciding whether further regulatory data should be required for these compounds. 1.3 Development of Single Stereoisomer Products Although the field of stereochemistry originated over 200 years ago, research was limited mainly due to the lack of suitable analytical techniques for resolution of enantiomers. Despite these limitations however, some important experimental work was undertaken, which has been reviewed elsewhere (2, 3), including the development of the nomenclature convention for assignment of configuration at a chiral centre (14). The development of chiral chromatography throughout the 1970s (15-17) facilitated the routine separation (or resolution) of enantiomers for the first time. During the late 1980s the potential importance of chirality began to be recognised by the pharmaceutical industry such that U.S. Food and Drug Administration (FDA) guidelines issued in 1987 included reference to enantiomers for the first time… ‘even in racemates…enantiomers may be considered as impurities’ (18). This was followed in 1992 by a policy statement that was almost entirely concerned with chiral drugs (19). Since the mid 1980s, there has been extensive research of pharmacological differences between enantiomers, principally led by the pharmaceutical industry, where development of single-enantiomer drugs has been favoured for the last decade, driven by technological advances such as large-scale chiral separation procedures and chiral synthesis (20). The proportion of single-enantiomer drugs reaching the market has risen from around 20% 10 years ago to almost 75% today due to de novo development and chiral switches (21). The development of single enantiomer agrochemicals is relatively recent. However, there are now several pesticides on the market that are either enantiopure (e.g. dichlorprop-P, fenoxaprop-P-ethyl, metalaxyl-M, S-methoprene) or contain subsets of stereoisomers (e.g. alpha-cypermethrin, beta-cyfluthrin, bioresmethrin, deltamethrin). Several reviews of chirality in agrochemicals are available (22-27). The issue of chirality in pesticides remains important since the proportion of chiral agrochemicals in use appears to be steadily increasing from 19% in the 1980s (25) to 25% in 1996 (27). A survey of pesticide active ingredients registered by Pesticides Safety Directorate in April 2003 showed that 102/ 339 (30%) contained one or more chiral centres. Moreover, of the 301 ‘organic’ compounds, 34% were chiral. A further14/ 25 non-agricultural active ingredients currently registered by the Health and Safety Executive were chiral. Therefore 32% of currently registered pesticide active ingredients in the UK contain one or more chiral centres. Other compounds contain prochiral centres that may be metabolised to form chiral products. The development of single-enantiomer products has raised some specific problems that need to be evaluated during risk assessment. Firstly, the chiral purity of the product must be determined as small impurities of another stereoisomer could heavily bias toxicology data. For single enantiomer pairs, this may be fairly easily achieved, however the number of stereoisomers increases exponentially with increasing number of chiral centres in a molecule so that a compound containing four chiral centres could have 16 different stereoisomers. This may present significant analytical problems. Secondly, chiral stability of the product must be determined. Again, this may be relatively simple for single enantiomer pairs but more challenging for compounds with multiple chiral centres. In addition to assessing chemical stability of the product, in-vivo metabolic stability must also be determined. Metabolism of chiral compounds will be discussed in detail later, however single enantiomer products may undergo chiral inversion or racemisation in-vivo. The amount of racemisation occurring in-vivo must be quantified, since there would be no point in administering a single enantiomer compound on safety grounds if it was converted to the harmful enantiomer in-vivo. Finally, chiral centres may be introduced into nonchiral compounds by metabolic activity. It may therefore be necessary to evaluate the rate of production and relative toxicity of stereoisomeric metabolites. 1.4 Aims and Scope of the Review Focussing predominantly on mammals and other farm animal species, this review summarises current understanding of the metabolism of chiral compounds, identifies areas for further research and proposes experimental approaches. The review is intended to help guide regulatory policy on whether toxicology data should be requested for all isomers of a chiral pesticide. The objectives of the project were to: CSG 15 (1/00) 5 Project title 1. 2. 3. Differential Metabolism of Chiral Compounds DEFRA project code PS2501 Undertake a comprehensive literature search of metabolism of chiral compounds. Review data on metabolism of chiral pesticides and other classes of chemicals. Produce a strategy for further research. 2 Metabolism of Chiral Compounds 2.1 Introduction Differential actions of chiral compounds may be due to stereoselectivity in both toxicokinetics (metabolism and disposition) and toxicodynamics (toxic or pharmacological effects). Early reports generally concentrated on toxicodynamics and highlighted differences in the intrinsic toxicological activity of enantiomers. However, more recent publications increasingly recognise the potential importance of stereoselective toxicokinetics, particularly metabolism, in mediating the stereoselective effects of chiral compounds (4-6, 8-13, 22, 28). Most chemicals that are absorbed by an organism are metabolised, often by several different pathways to a number of metabolites. Generally, metabolites are formed that are more easily excreted, however, metabolism sometimes results in bioactivation to more toxic intermediates. An enormous array of metabolic enzymes exist, some of which are ubiquitous to all living organisms while others are only found in closely related species. Families of metabolic enzymes (isozymes) exist that vary in their structure and substrate specificity from one species to another, but can be traced back to a common ancestor. Expression levels of some metabolic enzymes vary according to the type of cell or tissue. The activity or expression level of enzymes often varies between individuals within a species because of genetic polymorphisms or enzyme induction due to environmental factors. While most chemical reactions do not distinguish between enantiomers, metabolic reactions are catalysed by enzymes that are themselves constructed from homochiral amino acids. Therefore, enzyme-catalysed reactions occur in a chiral environment so differences in rates of reaction between individual enantiomers might be expected. There is a vast literature detailing studies of the metabolism of chiral compounds in vitro and in vivo dating back to the late 1960s and early 1970s (29-35). Published studies vary in the degree of stereoselectivity reported: from complete enantiospecificity (only one enantiomer is metabolised) to slight enantioselectivity (all enantiomers are metabolised but at different rates). Some studies report no differences between individual enantiomers. Differences in enantioselectivity have been reported between species or even between individuals within a species. These differences are apparently linked to differences in enzyme activity or expression level and will be discussed in detail later (section 2.5). This section will discuss experimental approaches that have been used to study the metabolism of chiral compounds and factors that can affect interindividual and interspecies variation in enantioselectivity. A selection of case studies will be discussed in detail, focussing on pesticides wherever possible, to illustrate the impact of stereoselective metabolism. 2.2 Stereoselectivity of Metabolic Transformation Discrimination between enantiomers during metabolism can occur at both the substrate and the product level (36). Enantioselectivity may be manifest as either a difference in the biotransformation of chiral compounds (substrate enantioselectivity) or in the production of chiral metabolites from non-chiral compounds (product enantioselectivity). The latter phenomenon is particularly important when biologically active metabolites are formed. A further type of transformation is the metabolic inversion of some enantiomers. Thus, metabolic transformation of xenobiotics can be categorised in terms of the following five distinct stereochemical courses (36). (i) (ii) (iii) (iv) (v) Prochiral substrate chiral metabolite Chiral substrate chiral metabolite Chiral substrate diastereomer metabolite Chiral substrate non-chiral metabolite Chiral inversion Metabolic enzymes can (i) introduce a stereogenic centre into a non-chiral molecule to form enantiomeric metabolites, (ii) alter a ligand attached to a stereogenic centre with either retention or inversion of chirality, (iii) introduce an additional stereogenic centre into a chiral molecule to produce diastereomeric metabolites, (iv) convert a pair of enantiomers into a common metabolite by removal of a stereogenic centre or (v) convert one enantiomeric form into the other. An important distinction should be made between chiral inversion, which requires enzyme activity to convert one enantiomeric form into the other, and racemisation, which describes the spontaneous conversion (i.e. not enzyme mediated) of one enantiomer to another. Chiral compounds may be metabolised by more than one pathway so that overall stereoselectivity in clearance of the compound will reflect the net balance of selectivity in different enzymes. Alternatively several isozymes may catalyse the formation of the same product and each may exhibit different steric preferences. In these instances, inter-species and inter-individual variation in enzyme activity could significantly affect the metabolic fate of a chiral compound and impact on its biological activity. CSG 15 (1/00) 6 Project title 2.3 Differential Metabolism of Chiral Compounds DEFRA project code PS2501 Investigation of Differential Metabolism Differential metabolism of chiral compounds has been demonstrated by in vitro and in vivo metabolic studies and by environmental monitoring studies. These three approaches each have their own advantages and disadvantages, which are briefly discussed. The principal advantage of in vitro studies is that they can focus on a specific reaction, or metabolic pathway, and identify the individual enzymes involved. However, it can be difficult to relate findings in vitro to the in vivo situation. Thus while in vitro studies are useful to identify whether stereoselective metabolism is possible, they can not confirm that it definitely occurs in vivo. In vivo studies have the advantage that the impact of stereospecific metabolism can be observed at the whole animal level, taking into account different metabolic pathways, exposure routes and dose level. However, the effects of differential metabolism can not be isolated from other toxicokinetic factors that might affect the enantiomer ratio of metabolites in blood or urine. For example, plasma and tissue protein binding has been widely reported to be stereospecific and active transport processes involved in absorption or excretion of a chemical may also be subject to stereospecific constraints. Furthermore, it is often difficult to determine whether differential toxic effects observed in vivo are due to toxicokinetic factors or intrinsic differences in toxicity of individual enantiomers. Finally, environmental sampling can provide useful information on stereospecific metabolism, particularly for persistent pollutants e.g. some pesticides and industrial chemicals. Enantiomer ratios can be measured at different trophic levels of the food chain and enantiomer enrichment may indicate that selective metabolism and/or storage is occurring. However, this approach suffers from the same limitations as in vivo studies in addition to lacking the tight controls of laboratory-based studies. 2.4 Studies of Enantioselective Metabolism Because of their importance in catalysing a wide variety of biotransformations, the cytochrome P450 (CYP) superfamily of metabolic enzymes have been the focus of extensive research. Microsomal fractions can be prepared from tissues that contain CYP as well as some other classes of enzymes. Liver is most commonly used because of its high expression level of most metabolic enzymes. Experimental techniques are routinely used that allow the role of individual CYP isoforms in metabolism of a test compound to be characterised in vitro. Preparation of microsomes from a range of individuals or species also allows interindividual or interspecies comparisons to be made. More recently, recombinant CYP isoforms have been expressed allowing investigation of the role of specific CYP isoforms. Warfarin has received lots of attention in the study of differential metabolism of chiral compounds; principally because of its pharmacological use as an anticoagulant, however, it is also used as a rodenticide. It contains one chiral centre, and is administered as the racemate (figure 2). Warfarin displays substrate enantiospecificity. The pharmacologically more potent (S)-warfarin is metabolised in humans almost exclusively by CYP2C9 to (S)-7-hydroxy-warfarin, and to a much lesser extent to (S)-6- hydroxywarfarin (37, 38). By contrast, (R)-warfarin is also extensively metabolised, but by multiple isoforms of CYP and also by ketoreductase. Recombinant CYP1A2 catalysed formation of (R)-6- and (R)-8-hydroxy-warfarin and CYP3A4 catalysed formation of (R)-10- hydroxy-warfarin (37, 38). All hydroxy-metabolites are inactive, so for the example of warfarin all of these CYP-mediated reactions are detoxification processes. O O CH3 OH CH3 OH H H O O O (S) O (R) Figure 2: Structures of warfarin enantiomers. Arrows show main sites of oxidative attack by human CYP. Another pesticide to display differential metabolism is the organochlorine insecticide methoxychlor, which is not currently registered for use in the UK. In this case, product enantiospecificity occurs; methoxychlor is prochiral but it is demethylated to form a chiral metabolite (figure 3). Recombinant human CYP1A2 and 2A6 enantioselectively formed (R)-hydroxy-methoxychlor, whereas CYP1A1, 2B6, 2C8, 2C9, 2C19 and 2D6 enantioselectively formed (S)-hydroxy-methoxychlor (39). CYP1A2, 2C9 and 2C19 displayed the highest degrees of enantioselectivity (>90% selectivity). In contrast, CYP3A4 and 3A5 were not enantioselective. These two examples of warfarin and methoxychlor highlight the variations in steric preference that can exist between CYP isoforms. Individual isoforms can exhibit enantioselectivity in both affinity for a substrate (e.g. warfarin) and production of metabolite (e.g. methoxychlor). Thus, variations in the activity of individual isozymes between individuals or species can affect enantiomer ratios of either parent compound or metabolites, which will be discussed in more detail later. CSG 15 (1/00) 7 Project title Differential Metabolism of Chiral Compounds H DEFRA project code PS2501 CCl 3 H3C CH3 O O methoxychlor CYP H CCl 3 H CH3 HO O CCl 3 H3C O (R)-hydroxy-methoxychlor OH (S)-hydroxy-methoxychlor Figure 3: Demethylation of methoxychlor. Product enantioselectivity was also observed for O-demethylation of methoxychlor in rat liver microsomes. At low substrate concentrations (<1 μM), (S)-hydroxy-methoxychlor was the major product, comprising 83 – 94% of total metabolites (40). However, at increased substrate concentrations (>2 μM), the relative proportion of the R-enantiomer increased to around 30%. These findings were consistent with multiple CYP isoforms, with different affinities and enantioselectivities, capable of metabolising methoxychlor. Purified CYP isoforms from rat liver microsomes were shown to possess different enantioselectivity as well as different affinity for O-demethylation of methoxychlor (41) and inhibition of specific CYP isoforms altered the enantiomer ratio of metabolite (42). Human liver microsomes from four individual donors and also pooled livers all exhibited similar product enantioselectivity to rat, with an approximate 80:20 ratio of (S)-:(R)-metabolite obtained at the single substrate concentration used in this study (25 μM) (39). As a reflection of the dominance of pharmaceutical compounds in the published literature for metabolism of chiral compounds, the best examples of in vitro studies of enantioselective metabolism have used pharmaceuticals. One such compound, bufuralol (BF), is a β-adrenoreceptor blocking agent that contains a chiral centre yielding two enantiomers. One of the major metabolic pathways of BF in mammals is 1”-hydroxylation which introduces a second chiral centre into the molecule so that the product 1”-hydroxybufuralol (1”OH-BF) can exist as four diastereomers (figure 4) (43). In vitro experimental results indicated that the R enantiomer of BF was preferentially metabolised to 1”-OH-BF and that CYP2D6 was the major enzyme catalysing this biotransformation (44, 45). However, while rat liver microsomes favoured the formation of 1”R-OH-BF over 1”S-OH-BF from both enantiomers of BF, human liver microsomes exhibited the reverse stereoselectivity (45). No data for other animal species are available to further establish interspecies variation in product stereoselectivity. However, interindividual variation in product stereoselectivity has been demonstrated using human liver microsomes prepared from four individual donors (44). Three out of the four livers exhibited product stereoselectivity for formation of 1”S-OH-BF. In contrast, a single liver preparation displayed the opposite stereoselectivity. Further investigation showed that this individual liver possessed low CYP2D6 activity and high CYP2C19 activity. These findings were supported by the observation that recombinant CYP2D6 and CYP2C19 displayed opposite product stereoselectivity. Thus the metabolism of bufuralol provides an example of how variation in activity of individual CYP isoforms can influence the stereochemical profile of chiral metabolites. CH3 H3C H3C H3C CH3 HN CYP O CH3 H3C OH * CH CH3 HN O * * HC HC OH OH 1"-OH-BF BF Figure 4: 1”-hydroxylation of bufuralol. Chiral centres are marked with an asterisk. The pharmaceutical cisapride is a racemate consisting of two enantiomers. Studies of the metabolism of individual enantiomers as well as the racemate were performed in human liver microsomes and recombinant CYP. Both enantiomers were metabolised via Ndealkylation and aromatic hydroxylation to the same products, however, the relative proportions of metabolites varied according to the enantiomer indicating that regioselective and stereoselective metabolism was occurring (46). Experiments using recombinant CYP indicated that CYP3A4 was the major enzyme involved in production of all three metabolites. Interestingly, the rate of racemic cisapride metabolism with both microsomes and recombinant CYP3A4 was slower compared with equimolar concentrations of each enantiomer indicating that the enantiomers inhibited each other’s metabolism. Similar enantiomer-enantiomer interactions have been CSG 15 (1/00) 8 Project title Differential Metabolism of Chiral Compounds DEFRA project code PS2501 observed for metabolism of another pharmaceutical, lansoprazole. Recombinant CYP2C19 metabolised the (+)-enantiomer more rapidly than the (-)-enantiomer, but when the racemate was used, the reverse enantioselectivity was observed (47). As previously discussed, stereoselectivity in metabolism appears to be responsible for much of the pharmacokinetic differences that are often observed between the enantiomers of a chiral compound. However, both pharmacokinetic and pharmacodynamic events contribute to clinically observed stereoselectivity in toxicological studies so it is often difficult to isolate the effects of differential metabolism from in vivo studies. There is a large published literature for in vivo studies of chiral compounds, especially pharmaceuticals. Some of the major classes of pharmaceuticals exhibiting differential chiral metabolism include antiarrhythmics (13, 48-50), ß2-adrenergic agonists (51-53) and 2-arylpropionic acid derivatives (profens) (6, 54, 55). In vivo studies using chiral pesticides are scarce, however, one such study, where toxicity was shown to result from differential metabolism, administered the pyrethroid insecticide fenvalerate to rats and mice. Fenvalerate exists as a mixture of four diastereomers due to the presence of two chiral centers (figure 5). Its main toxic action in mammals is granuloma formation in various tissues. Following administration of the isomers to experimental animals, only one isomer, [2R,α-S], was metabolised to a lipophilic conjugate, cholesteryl [2R]-2-(4-chlorophenyl) isovalerate (56) which has been shown to be the causative agent of granulomatous changes observed in liver, spleen and mesenteric lymph node tissues (57, 58). These findings were repeated in vitro using tissue homogenates from mice, rats, dogs and monkeys and while all of the isomers were hydrolysed to 2-(4-chlorophenyl) isovalerate (CPIA), [2R,α-S] was confirmed to be the only isomer that formed the cholesterol conjugate (59). Interspecies differences were also observed in CPIA-cholesterol ester formation. Generally, mouse tissues showed higher activity than other species and no activity was found in dog kidney and spleen or monkey liver (59). This probably reflects the presence of different carboxylesterases and mirrors interspecies differences in toxicity. As a consequence of these findings, a purified active isomer of fenvalerate, [2S,α-S] was produced and marketed under the name Esfenvalerate which did not produce granulomas. Cl O CH CH3 * N C O * CH3 CH O Figure 5a: Structure of fenvalerate. Note the chiral centers at C2 of the isovaleric acid moiety and the C bonded to the α-cyano group. Figure 5b: 3D structures of esfenvalerate [2S, α-S] on the left and its toxic diastereomer [2R, α-S] on the right. As described earlier, the enantiomers of the chiral anticoagulant warfarin are metabolised by different enzymes (S-warfarin by CYP2C9 and R-warfarin by CYP3A4, 1A2 and ketoreductase (37)). S-warfarin possesses 3 to 5 times greater anticoagulant activity than its enantiomer (60). When racemic warfarin was coadministered with the cytochrome P450 inhibitors cimetidine and sulfinpyrazole, the anticoagulant effect was only increased by the latter (61, 62). Total warfarin clearance was reduced following coadministration of both inhibitors, however, cimetidine only decreased clearance of the R-enantiomer with no effect on the more active S-enantiomer. In contrast, sulfinpyrazole, which inhibits different CYP isoforms, decreased the clearance of S-warfarin therefore increased its anticoagulant effect. CSG 15 (1/00) 9 Project title Differential Metabolism of Chiral Compounds DEFRA project code PS2501 Similar effects on warfarin metabolism are observed due to genetic polymorphisms. CYP2C9, the major CYP responsible for catalysing hydroxylation of S-warfarin in humans, exhibits polymorphism with several allelic variants possibly associated with altered catalytic activities (63). The wild-type allele, 2C9*1, is the most commonly found; its frequency is 80-85% in Caucasians and over 95% in Japanese, Chinese and African-American populations. Variant *2 and *3 alleles are present in approximately 35% of Caucasian individuals; however, these alleles are significantly less prevalent in African-American and Asian populations (63). There is little data for other alleles that have only recently been identified. Clinical studies have shown that patients homozygous for 2C9*1 required the highest doses of warfarin while those individuals homozygous for 2C9*2 or *3 generally required the lowest doses. Heterozygotes, containing a single copy of 2C9*1 and one of either 2C9*2 or *3, required intermediate warfarin doses (64-66). Thus, when polymorphic enzymes are involved in stereoselective metabolism, the likely existence of sensitive sub-populations further complicates the risk assessment of the chemical. Chiral inversion is a phenomenon particularly important for the 2-arylpropionic acid (2APAs) or profen pharmaceuticals (6). Chiral inversion requires enzyme activity, so it is distinct from racemisation, which occurs spontaneously. Chiral inversion may interconvert both enantiomers or, as in the case of the 2APAs, it can be unidirectional; 2APAs undergo RS inversion (6). However, the extent of chiral inversion differs in a compound and species-specific manner (67-71). There have been no reports of chiral inversion of pesticides, however chiral inversion of (S)-mandelic acid, a metabolite of the industrial chemicals styrene and ethylbenzene, to (R)mandelic acid has been reported in rats (72). Not all chiral compounds exhibit stereoselective metabolism. For example methadone, which contains a chiral center and is used as the racemate, is predominantly metabolized via mono-N-demethylation catalysed by the cytochrome P450s (74). An in vitro study of this reaction in human liver microsomes failed to demonstrate any significant enantioselectivity, although V max for (S)-methadone was 15% lower than for (R)-methadone (75). Further investigation using recombinant CYP indicated that CYP3A4 and 2C19 showed similar reaction rates for both enantiomers. However, significant differences in the pharmacokinetics of the two enantiomers have been observed (73) The apparent lack of enantioselectivity in metabolism suggests that other factors, such as protein binding, may be responsible for the observed stereoselective pharmacokinetics. Other compounds where no significant differences in metabolism of individual enantiomers have been observed include cyclophosphamide (76, 77), gacyclidene (78), ofloxacin (79, 80), oxamniquine (81), pantoprazole (82, 83), sotalol (84-86) and terfenadine (87), however these compounds tend to be the exception rather than the rule. 2.5 Differential Metabolism of Chiral Compounds Between Species Research on the metabolic transformation of drugs and other chemicals is mostly based on experimental work in a restricted number of species, usually small animals in laboratory work. However, considerable interspecies variation exists in activity of biotransformation enzymes. It is likely that further variation will be encountered in farm animals, although there are few data available for these species. It is important to identify whether these interspecies differences in enzyme activity, that are a result of minor alterations in amino acid structure of enzymes from one species to another, can alter stereospecificity of enzymes. The examples of bufuralol, fenvalerate and 2-arylpropionates have already been discussed and further examples are given here. Flobufen is a nonstereoidal anti-inflammatory drug that contains one chiral and one prochiral centre. Its principal biotransformation pathway is reduction to 4-dihydroflobufen (DHF), which can exist as four diastereomers (figure 6). This metabolic reaction has been investigated in vitro using hepatocytes and liver homogenates from three ruminant species (88). All four diastereomers were formed from racemic flobufen and individual enantiomers produced two diastereomers each. Interestingly, the ratios of DHF diastereomers obtained from incubations with individual enantiomers of flobufen varied between species to the extent that different species exhibited reverse stereoselectivity. It should also be noted that there was approximately 4-fold variation in rate of total metabolite formation across the three species (88). O CH3 CH OH * OH CH3 CH CH * * O O F OH F F Flobufen F Dihydroflobufen Figure 6: Biotransformation of flobufen to diastereomeric 4-dihydroflobufen. The petrochemical isoprene has large-scale industrial use and undergoes stereospecific metabolism. Its initial metabolism involves cytochrome P450 mediated epoxidation (figure 7), with subsequent metabolism by epoxide hydrolase and glutathione-S-transferase. Slight differences in product enantioselectivity were observed in vitro between rat and mice (89). A more detailed in vitro study of CSG 15 (1/00) 10 Project title Differential Metabolism of Chiral Compounds DEFRA project code PS2501 interspecies variation using liver microsomes showed that rat enantioselectively formed (S)-isopropenyloxirane in an approximate 2:1 ratio, no enantioselectivity was observed for mouse or rabbit, but dog, monkey and human showed slight enantioselectivity for the (R)-enantiomer (90). Problems encountered with the chiral separation of 2-methyl-2-vinyloxirane prevented the determination of enantiomer ratios for this metabolite. A separate study that did not quantify individual enantiomers showed that human CYP2E1 was the principal isoform catalysing this reaction (91). O O S H2C CH3 H2C CH2 CH3 H CYP H2C CH2 R CH3 H 2-methyl-2-vinyloxirane CH3 CH2 H2C H3C H S R CH2 H Isoprene O H H2C CH2 O 2-isopropenyloxirane Figure 7: Isoprene can undergo epoxidation at either double bond, from the front or the back of the molecule, to produce two pairs of monoepoxide enantiomers. Chloroprene, which is structurally related to isoprene possessing a chloride group instead of methyl, also exhibits enantioselective metabolism. Liver microsomes from rats had slight enantioselectivity for formation of the (R)-epoxide while microsomes from mouse and human had the opposite enantioselectivity (92). Although the (R/S) prefix of the respective epoxide metabolites formed from chloroprene and isoprene in rat changes, they have the same absolute stereochemical configuration. Thus, although only slight enantioselectivity was observed, the overall trends in stereochemistry of epoxidation were similar in structurally related chemicals. Albendazole is a prochiral antihelmintic that is sulfoxidated in vivo to its major active metabolite, generating a chiral centre. Opposite product enantioselectivity was observed following oral administration to rats and sheep (93). Rats had slightly higher plasma concentration of the (-) metabolite (SO.ABZ) whereas sheep had much higher plasma concentrations of (+)-SO.ABZ; 36 hours postdosing the enantiomer ratio was 96(+):4(-). A later study comparing albendazole metabolism in sheep, goats and cattle showed that all three species displayed similar product enantioselectivity with predominant formation of the (+)-metabolite, however, small variations were found in the enantiomer ratio between the species (94). The above examples are representative of the scientific literature for interspecies differences in differential metabolism of chiral compounds. Interspecies variation due to enantioselective metabolism is generally fairly low. However, there are occasions where differential metabolism alters the enantiomer ratio significantly and sometimes, different enantiomers are predominantly formed in different species (28, 95-98). In this case, any difference in relative toxicity between enantiomer pairs (especially if toxic effects are associated with just one enantiomer) will be magnified, resulting in significantly different dose-response relationships. There are several examples where one species exhibits differential metabolism of a chiral compound while other species have only slight or no differences (99-101). This can make extrapolations from laboratory toxicology species to man and domestic or agricultural animals difficult. The mechanistic causes of species differences in enantioselective metabolism have been less well studied than interindividual differences. However, some major differences in xenobiotic metabolism have been recognised for many years, in both oxidation and conjugation reactions (102-108). These differences are believed to arise from altered enzyme structures or expression level between species. Differences in enantioselectivity between species may be considered an extension of this variation. Thus differential metabolism of chiral compounds between species could occur due to either (1) altered enzyme structure resulting in different affinity for substrate or formation of different metabolites or (2) altered expression levels (or complete absence) of an enzyme. There are a limited number of studies that have directly compared metabolism across different species, so it is difficult to characterise the likelihood or frequency of differential metabolism. However, differential metabolism does occur for several diverse classes of chemical so a requirement for manufacturers to investigate species differences in enantioselectivity could be justified, especially if enantiomer pairs are known to have different toxic effects. In vitro experiments using suitable tissue extracts from laboratory species and potentially exposed non-target species could offer a relatively cheap initial screen for differential metabolism. If no significant differences were found, then it could be considered safe to proceed with further toxicological testing without the need for enantioselective analytical methods. CSG 15 (1/00) 11 Project title Differential Metabolism of Chiral Compounds DEFRA project code 3 Differential Metabolism of Chiral Pesticides 3.1 Chirality in Pesticides PS2501 Chiral compounds are found among all of the major classes of pesticides currently used in the UK. Among the pesticides that most commonly display chirality are triazole (conazole) fungicides, aryloxyphenoxypropionate herbicides, pyrethroid insecticides and coumarin anticoagulant rodenticides. These classes of pesticide contain common structures, making them particularly likely to possess chiral centres (figure 8). However, many other pesticides that do not belong to the classes noted above are chiral, often because substituent groups contain chiral centres or because they possess chiral axes. About 1/3 of currently registered pesticide active ingredients in the UK are chiral and as synthetic pesticides become increasingly complex, the likelihood that they will contain chiral centres increases. N N OH CH3 OH N C Cl HC F C N N F N Cyproconazole Flutriafol N Cl O Cl HC F C C N N N N N N Fenbuconazole Epoxiconazole Figure 8a: Representative structures of triazole fungicides (chiral centres shown in red). CH3 CH3 CH CH3 O N CH3 O O O CH O O O Cl O Fenoxaprop-P ethyl N O CH3 O F CH O CH3 O F Cl Cl F O Fluazifop-P butyl Diclofop methyl Figure 8b: Representative structures of aryloxyphenoxypropionate herbicides. Note that diclofop methyl is used as the racemate and non-specific stereochemistry is depicted using a wavy bond while single enantiomers of fenoxaprop-P ethyl and fluazifop-P butyl are used and the absolute stereochemical structure is shown. CSG 15 (1/00) 12 Project title Differential Metabolism of Chiral Compounds H3C CH3 HC CH DEFRA project code Cl O Cl O O Permethrin O H3C CH3 HC CH Cl O Cl CH O N Alphacypermethrin H3C CH3 Br C C O Br CH H O H O N Deltamethrin Figure 8c: Representative structures of pyrethroid insecticides. O O CH3 Br CH OH O O O CH Warfarin OH HO CH OH Bromadiolone O CH O Coumatetralyl Figure 8d: Representative structures of coumarin anticoagulant rodenticides. CSG 15 (1/00) 13 PS2501 Project title 3.2 Differential Metabolism of Chiral Compounds DEFRA project code PS2501 Metabolism of Chiral Pesticides Metabolism of most classes of pesticides generally leads to formation of inactive products. However, some pesticides, e.g. the organothiophosphates, are bioactivated to highly potent metabolites. While most pesticides have fairly low mammalian toxicity, for those compounds with appreciable toxicological effect any differential metabolism leading to variation in rates of bioactivation or detoxification could influence the dose-response relationship and may need to be assessed during the risk assessment process. The examples of the rodenticide, warfarin, and the insecticides, methoxychlor and fenvalerate, have already been discussed. The following section reviews the remaining literature for metabolism of chiral pesticides. Differential metabolism of pesticide stereoisomers has been investigated since the mid 1970s, mainly focussing on insecticides that may cause adverse health effects in mammals. In vitro experiments using mouse liver microsomes showed that trans isomers of the pyrethroid resmethrin were hydrolysed very rapidly compared to cis isomers (109, 110). This agreed with in vivo data where rats and mice survived moderate doses of (+)-cis-resmethrin and large doses of (+)-trans-resmethrin. Rabbit liver microsomal metabolism of a chiral organophosphate herbicide, S-2571, produced the same metabolites with both enantiomers, however, the (-) isomer was metabolised faster (111). A series of papers by Lee et al investigated in vitro and in vivo metabolism of the enantiomers of the organophosphate fonofos in mice. (R)-fonofos was metabolised by mouse liver microsomes to fonofos-oxon and subsequently hydrolysed at approximately twice the rate of the (S)-enantiomer (112). Following administration of individual enantiomers of fonofos to mice, the total amount of metabolites excreted over a 96 hour period was similar and the relative amounts of each metabolite were virtually identical (113). These data appear to contradict the earlier in vitro data, however the authors note that at the doses used (R)-fonofos caused moderate toxicity while (S)-fonofos did not result in any clinical symptoms. Thus the physiological state of the animals may have affected rates of metabolism and/ or excretion of metabolites. Enantioselective hydrolysis of EPN-oxon was demonstrated in vitro using liver microsomes prepared from rat, mouse and rabbit. While rates of hydrolysis of the racemate displayed interspecies variation, rat having the highest esterase activity and rabbit the lowest, all three species showed similar enantioselectivity, with the (-)-enantiomer being hydrolysed approximately 1.5-times faster than the (+)-enantiomer (114). Similar enantioselectivity has also been reported for another chiral organophosphate, cyanofenphos (115). An example of how stereospecific metabolism may affect the dose-response relationship is found in the oxidative bioactivation of the phosphorothiolate insecticide profenofos (figure 9). In vitro data indicated that (+)-profenofos was the more potent inhibitor of acetylcholinesterase (AChE). However the (-) enantiomer was found to be more toxic to mice in vivo. Incubation with liver microsomes followed by quantification of AChE inhibition indicated that the individual enantiomers reacted in an opposite manner (116). (-)-Profenofos underwent preferential metabolic activation and the metabolite was 34-times more inhibitory to AChE, whereas (+)-profenofos was preferentially detoxified and its capacity to inhibit AChE was reduced 2-fold. This reversal of enantioselectivity in AChE inhibition following metabolism may be due to differences in rates of oxidation, however, the intrinsic potency of metabolite enantiomers to inhibit AChE is not known so this possibility can not be discounted. Figure 9: Structure of profenofos enantiomers. (R)-enantiomer is on the right. Another example of enantioselective metabolism is the product enantioselectivity observed in the in vitro and in vivo demethylation of the non-chiral organophosphate fenitrothion in mice by glutathione-S-transferase (figure 10). This reaction represents a detoxification pathway. Both enantiomers of desmethylfenitrothion were produced in vitro using mouse liver cytosol and in vivo, but at an approximate 2:1 ratio of (R)(+):(S)(-) enantiomers (117). Similar results were obtained in rats in vitro, however the enantioselectivity was much lower than mice (58:42), which may be due to a different enzyme in both species (118). In contrast, a study using five purified glutathione-S-transferase isoforms from rat liver produced mainly the (S)(-)-enantiomer of desmethylfenitrothion indicating that mice and rats display opposite enantioselectivity (119). The discrepancy between these studies remains to be explained. CSG 15 (1/00) 14 Project title Differential Metabolism of Chiral Compounds DEFRA project code PS2501 S O P HO O P O CH3 (R)-desmethylfenitrothion S O CH3 O CH3 N O H3C N O - + O + O S - O CH3 P Fenitrothion O + O N O OH H3C - CH3 (S)-desmethylfenitrothion Figure 10: Enantioselectivity demethylation of fenitrothion. Differential metabolism of the chiral organophosphate isofenfos has been demonstrated in rat liver microsomes (123). (-)-Isofenfos produced approximately four times more ‘oxon’ than its enantiomer. Administration of 1R cis- and IR trans-cyphenothrin to rats resulted in more rapid hydrolysis of the trans-isomer (120). This phenomenon has been previously reported for other pyrethroids (109, 110). Both isomers were metabolised to the same major products. However, in vitro investigation of metabolism by different tissues indicated that enantioselectivity was tissue-dependent. Liver esterase hydrolysed the trans-isomer more rapidly, as did lung and kidney esterase. However, plasma hydrolysed both isomers at equal rates. In addition, when the data using cyphenothrin was compared to similar experiments using phenothrin (no cyano- group on the alcohol moiety), both trans-isomers exhibited similar rates of hydrolysis but cis-cyphenothrin underwent ester hydrolysis at three times the rate of cis-phenothrin (120). Pyrethroids possess two chiral carbon atoms in the chrysanthemic acid portion of the molecule, which contains a cyclopropane ring (figure 11). Additional chiral centres are commonly found in the alcohol portion of the molecule (refer to figure 8c for complete chemical structure showing alcohol and chrysanthemic acid moieties). The two chiral centres in the cyclopropane ring are defined using either their absolute configuration (e.g. 1R, 3S) or using cis- and trans- notation, similar to the system used for geometric isomers. Thus both cis-isomers are enantiomers of each other, whereas the trans-isomers are their diastereomers. Natural chrysanthemic acid occurs as a single enantiomer (1R) and its mirror image (1S) is completely inactive as an insecticide. Differential metabolism of these four isomeric forms of various pyrethroids has been investigated. As outlined above, differences have been observed in rates of hydrolysis between cis- and trans-isomers. However, while no or very small differences in metabolite profile were found between enantiomer pairs of phenothrin (121) and tetramethrin (122) in rats, these data are in contrast to the findings of Ueda et al (109) that there were appreciable differences in oxidation at the isobutenyl group between (+)-cis and (-)-cis-resmethrin. H3C OH C H3C CH3 1 C H H 2 H3C HO C 3 H CH3 O O H H3C CH3 1R, 3S cis CH3 1S, 3R cis O O H OH HO H H3C CH3 H H3C H3C H CH3 H3C 1R, 3R trans CH3 CH3 1S, 3S trans mirror plane Figure 11: Chrysanthemic acid stereoisomers. Numbering of carbon atoms is shown on the top left structure. A study of the carbamate pesticide methiocarb investigated enantioselectivity of different classes of metabolic enzymes in rat liver microsomes. Methiocarb is not chiral, but it is sulfoxidated to a chiral metabolite. Both the cytochrome P450s (CYP) and flavinCSG 15 (1/00) 15 Project title Differential Metabolism of Chiral Compounds DEFRA project code PS2501 containing monooxygenases (FMO) catalysed sulfoxidation to similar extents in microsomes from control rats (124). Absolute stereochemical configuration of the metabolite, methiocarb sulfoxide, were not determined, however, one enantiomer (A) was produced at a higher rate (Vmax) and at higher affinity (K m) than the other enantiomer (B). In control microsomes, (A) was formed at enantiomer excess (e.e.) of 48% (ratio (A)(B)= 2.9:1). Inhibition of CYP by clotrimazole increased e.e. to 88% whereas inhibition of FMO by methimazole or thermal inactivation reduced e.e to 32 and 17% respectively. Thus it appears that FMO are almost completely enantiospecific for formation of A, whereas CYP are only slightly enantioselective for A. 3.3 Potential Importance of Differential Metabolism of Chiral Compounds The potential impact of differential metabolism of chiral compounds can be demonstrated using the following three theoretical examples. Figure 12 shows the effect of product enantioseletivity on production of a chiral metabolite from a prochiral substrate; enantioselective clearance of a chiral substrate (substrate enantioselectivity) would generate the inverse of these plots. In this example, the individual metabolite enantiomers are formed with different K ms and Vmaxs. The (R)-metabolite has Km=1 and Vmax=1 (high affinity, low capacity) while the (S)-metabolite has Km=10 and Vmax=10 (low affinity, high capacity). Production of each enantiomer can be described using a one-compartment pharmacokinetic model, however, these combine to produce a spurious two-compartment model for the racemate. Calculation of kinetic constants for the racemic data gives K m=8.0 and Vmax=10.8, however, at this Km the high affinity production of (R)-metabolite would be almost saturated. In the absence of individual metabolite enantiomer concentrations, use of the racemic data would indicate non-linear reaction kinetics (figure 13). The ratio of enantiomers also varies with substrate concentration (figure 14). In this example, enantiomer excess of the (S)-metabolite increases with substrate concentration from close to zero at low substrate concentrations (i.e. approximately a 50:50 mix of enantiomers) to 80% at high substrate concentrations (i.e. approximately a 9:1 ratio of S:R enantiomer). Rate (V) 15 10 Racemate S-metabolite R-metabolite 5 0 0 25 50 75 100 [Substrate] Figure 12: Theoretical simulation of the effects of product enantioselectivity on reaction kinetics. The (R)-metabolite is formed with high affinity but low capacity; the (S)-metabolite is formed with low affinity but high capacity. 3 V/S 2 1 0 0.0 2.5 5.0 7.5 10.0 12.5 V Figure 13:Eadie-Hofstee plot showing non-linear kinetics. The blue and green lines indicate the individual components for formation of each enantiomer. The dotted red line was plotted using kinetic parameters obtained from the curve fit in figure 12. CSG 15 (1/00) 16 Differential Metabolism of Chiral Compounds Enantiomer excess (%) [S]-[R] Total Project title DEFRA project code PS2501 100 75 50 25 0 0 25 50 75 100 [Substrate] Figure 14: Variation of enantiomer excess over a range of substrate concentrations for enantioselective metabolism where one enantiomer is formed with high affinity but low capacity while the other is formed with low affinity but high capacity. In a second example, a compound exhibiting enantioselective metabolism where chiral metabolites are formed with different K m ((R)=1 and (S)=10) but the same Vmax (1) (differences in affinity) is considered (figure 15). In this case, kinetic analysis of the racemic data produced Km=2.6 and Vmax=1.9. Under this scenario, differential metabolism results in the greatest difference at low substrate concentrations and enantiomer excess decreases with increasing substrate (figure 16). 1.5 2 V 1.0 0.5 Rate 0.0 0 2 4 6 8 10 S 1 Racemate S-metabolite R-metabolite 0 0 25 50 75 100 [Substrate] Enantiomer excess (%) [R]-[S] Total Figure 15: Simulation of the effect of differential metabolism when enantiomers are produced with the same V max but different Km. 100 75 50 25 0 0 25 50 75 100 [Substrate] Figure 16: Variation of enantiomer excess over a range of substrate concentrations for enantioselective metabolism where enantiomers are formed with different affinities. In a final example, if chiral metabolite formation occurred at similar K m but different Vmax (differences in reaction rate) then the high Vmax will mask the role of the low activity component. In this instance, enantiomer excess is determined solely by the difference in Vmax and it remains constant at all substrate concentrations (plots not shown). These examples highlight how the use of racemic data for chiral compounds that undergo differential metabolism may give rise to spurious kinetic data. This is particularly important when the individual enantiomers possess different toxicological effects and the use of racemic data will not produce reliable dose-response relationships. The observed effects of differential metabolism may also be dose-dependent and determined by the relative affinity and capacity for metabolism of the individual enantiomers. Thus in the three CSG 15 (1/00) 17 Project title Differential Metabolism of Chiral Compounds DEFRA project code PS2501 examples outlined here, differential metabolism can be more apparent at either high doses or at low doses or it may be independent of dose. These theoretical examples are supported by literature data where such concentration effects have been noted for metabolism of styrene (125), propafenone (126) and verapamil (127).The effects described here are likely to be more important for in vitro metabolic studies, however they could also arise during in vivo toxicology studies, especially where only single dose levels are administered. 4 Conclusions 1. Chirality is an important consideration in the risk assessment of pesticides. 1/3 of currently registered active ingredients in the UK contain one or more chiral centres and some of the non-chiral ones may produce chiral metabolites. Of these compounds, only a few are currently available for use as pure enantiomers. However, chiral separation, enantioselective analysis and especially asymmetric synthesis technology is rapidly improving, principally driven by the pharmaceutical industry. Therefore it may be reasonable to expect pesticide manufacturers to carry out toxicological testing using the individual pure enantiomers of chiral compounds and to justify whenever chiral products are manufactured as the racemate. 2. There are numerous examples in the scientific literature of individual enantiomers displaying different biological effects. Differential metabolism of chiral compounds has been demonstrated for a wide variety of chemicals; however, differential effects can also result from other factors. The most obvious difference between enantiomers is intrinsic toxicological effect (pharmacodynamics), where one enantiomer has a different mode of action from another or may even be non-toxic. The contribution of pharmacokinetic factors towards differential effects of enantiomers have been recognised more recently and include an important role for metabolism, but also include protein or tissue binding and active transport processes that might occur during absorption or excretion of a chemical. Thus several factors besides metabolism need to be considered when interpreting data from in vivo studies. 3. Differential metabolism of chiral compounds is an established phenomenon and product or substrate enantioselectivity (or sometimes complete specificity) may be observed. On the other hand, metabolic enzymes can sometimes function as ‘racemases’ and interconvert one enantiomer to another. There are few studies that have comprehensively investigated metabolism of chiral pesticides so the majority of the data is for pharmaceuticals. Differential metabolism may be more important for those pharmaceuticals that are administered orally as large bolus doses and are subject to first-pass metabolism. Pesticides are generally not deliberately administered, so doses are likely to be lower and exposure is likely to occur via other routes than oral. While metabolism of some chiral compounds shows major differences between enantiomers, the majority of cases show only slight differences and some chiral compounds do not show any differences in metabolism of the enantiomers. The frequency with which significant differences between enantiomers occur due to metabolism is difficult to establish. Nonetheless, differential metabolism of some chiral compounds (e.g. fenvalerate) has been shown to directly affect toxicity. Therefore, it might be a reasonable requirement for manufacturers to investigate whether differential metabolism of pesticide enantiomers occurs and, if so, to characterise the effect on toxicity of reduced clearance of individual parent compound enantiomers or the metabolites produced from individual enantiomers. 4. The risk assessment of agricultural pesticides is complicated since food producing animals may be exposed and pesticides may be stored or biotransformed before being consumed by humans. Therefore, metabolism of pesticides across a wide range of species, not just standard laboratory toxicology species, needs to be understood. Interspecies variation in metabolic rates and pathways is well characterised, but chiral compounds introduce a further complication since individual enantiomers may exhibit widely different toxic effects. There is substantial evidence showing that different species metabolise some chiral compounds at different rates or even to different products. Therefore it is important that suitable analytical methods, which can distinguish between individual isomers, are used in toxicological studies. Enantiomers could then be treated as separate compounds that vary between species and are covered by relevant safety factors. 5. Interindividual variation is an important consideration for human risk assessment. Several metabolic enzymes exhibit variation due to either genetic polymorphism or induction and distinct variations in enzyme activity between individuals or ethnic populations may be observed. This has significance for chiral compounds where individual enantiomers may be metabolised by different enzymes (e.g. warfarin). The clearest examples are where one or more of the enzymes displays polymorphism and administration of the racemate results in large differences in enantiomer ratios and may even reverse the overall enantioselectivity of the reaction (e.g. bufuralol). 6. Cost is inevitably important when considering the need for additional toxicological data for chiral pesticides. The additional costs involved in preparing enantiomerically pure isomers, conducting additional toxicology tests and developing asymmetric synthetic routes may prevent the development of some pesticides. This is especially significant if individual enantiomers have almost identical toxicological characteristics; marketing a single enantiomer would enhance the development costs without a parallel increase in safety. Any decision to request additional toxicology data for chiral pesticides should therefore be supported by a sound understanding of the factors leading to enantioselectivity in pesticide metabolism and disposition. 7. A tiered approach to screening for differential metabolism might be compatible with improving pesicide safety without imposing potentially prohibitive costs on manufacturers. Relatively inexpensive in vitro methods could be used to assess CSG 15 (1/00) 18 Project title Differential Metabolism of Chiral Compounds DEFRA project code PS2501 whether significant interspecies or interindividual differences occur in enantioselectivity. More costly and time consuming in vivo toxicology testing using enantioselective analytical techniques could be reserved only for those compounds that display significant differential metabolism. 5 Strategy for Future Research 1. Development of in vitro assays to evaluate species differences in differential metabolism. Few studies have investigated differential metabolism of chiral pesticides. No published studies are available that have systematically compared metabolism of chiral pesticides across a range of experimental animal and other potentially exposed species. Therefore, there is a need for research to apply commonly used in vitro techniques to the quantification of differential metabolism in order to demonstrate a generic approach to determine whether differential metabolism occurs for a particular compound. The research should cover several classes of pesticides, although those pesticides with potential to cause adverse human health effects, e.g. pyrethroid and organophosphate insecticides and coumarin anticoagulant rodenticides, might be considered a priority for study. Suitable tissues need to be identified in order to study metabolism in humans, laboratory species and other potentially exposed domestic animals. Depending on the metabolic reaction involved, serum; containing large quantities of some esterases, or liver (microsomes, cytosol or intact hepatocytes); containing a range of xenobiotic metabolising enzymes, are fairly readily available sources of enzyme that can be used to compare metabolism across a range of species. Initial metabolic oxidations (phase I metabolism), such as those catalysed by the cytochrome P450s, are worthy of further investigations since they could determine the stereochemistry of metabolites, which may be maintained through further metabolic reactions. As outlined in the literature review, different activities of cytochrome P450s between species or individuals can influence the toxicokinetic profiles of individual enantiomers. With the exception of some organophosphates, most metabolic processes of interest involve detoxification reactions. Thus, experimental studies could focus on, for example, hydrolysis of pyrethroid enantiomers in the serum of a range of species or hydroxylation of coumarin anticoagulants by cytochrome P450 in liver microsomes. These metabolic reactions have been well characterised in the past, at least for some individual compounds within a given class of pesticide, but chiral separation techniques may not have been used. By selecting compounds whose major metabolic pathways are known, specific reactions can be targeted and studied in vivo. This approach will significantly reduce the amount of effort required; however, for most pesticides, complete metabolic pathways are only available for a limited number of species. Analytical methods that can distinguish between individual enantiomers are now widely available. Typical instrumentation that is currently being used includes high performance liquid chromatography and gas chromatography in conjunction with a suitable chiral separation column. Both of these instruments can use mass spectrometry detection to enable more sensitive quantitation of analytes. Generally, individual enantiomers of pesticides or their metabolites are not commercially available. Therefore, selection of compounds for investigation is likely to depend on whether small, enantiopure, samples can be obtained from manufacturers. Alternatively, they can be custom synthesised, but this could be very expensive. An alternative approach, only suitable for non-chiral compounds that are metabolised to a chiral metabolite might involve chiral separation of individual metabolite enantiomers and quantification using a racemic standard. However this approach would not be able to designate the absolute stereochemistry of the metabolites, so that the extent of any differential metabolism could be quantified, but correlation with enantioselectivity in toxic effects would not be possible. Thus, the unavailability of suitable standard materials is likely to be a limiting factor in the range of pesticides that can be studied. Some compounds are available as purified isomers, e.g. alpha-cypermethrin, but even this is a 50:50 mixture of enantiomers. Development of standardised assays to study in vitro metabolism of chiral compounds would provide an initial screen for differential metabolism that could form part of a tiered approach to gathering toxicological data for chiral pesticides. 2. Incorporation of in vitro biokinetic data into physiologically-based pharmacokinetic (PBPK) models to demonstrate how differential metabolism may affect the dose at target tissues for toxicity. PBPK models provide a tool where in vitro data can be used in conjunction with relevant physiological parameters to describe the fate of a compound in vivo. They involve computer simulation of the uptake, distribution, metabolism and excretion of chemicals. PBPK models separate the body into a number of tissues, or groups of similar tissues, interconnected by the vascular system. The models are based on several physiochemical parameters such as: blood/tissue solubility, metabolic parameters, perfusion rates, organ weight, body fat and protein binding. Some of these parameters are known or can be predicted, whereas others need to be measured for each compound. Where chiral compounds exhibit differential metabolism, PBPK models will greatly aid the interpretation of in vitro enzyme kinetic data. Parameters such as rate of absorption, blood/tissue partition coefficients and excretion are unlikely to be enantioselective, so the effects of differential metabolism on tissue dosimetry can be easily demonstrated. The in vitro metabolic data produced from recommendation 1 would be suitable for incorporation into PBPK models. Therefore, modelling should be considered as a useful inclusion to any experimental work to make the maximum use of the data. CSG 15 (1/00) 19 Project title 3. Differential Metabolism of Chiral Compounds DEFRA project code PS2501 Investigate whether QSAR models can be developed to predict to extent of differential metabolism that might occur for a chiral compound. It would be useful to develop a computational screening tool to predict from a chemical structure whether enantioselective metabolism, and the degree of selectivity, might occur. Molecular modelling approaches involve interactive docking studies of compounds within the putative active sites of various enzymes. Initial studies could use data for metabolism of a panel of compounds that are metabolised by a common enzyme. The model developed could then be used to predict whether enantioselective metabolism would occur for similar compounds. 6 References 1. 2. North, M. (1998) Principles and Applications of Stereochemistry, Stanley Thornes Ltd., Cheltenham De Camp, W. H. (1993) Chiral drugs: the FDA perspective on manufacturing and control. J Pharm Biomed Anal 11, 11671172 Bentley, R. (1995) From optical activity in quartz to chiral drugs: molecular handedness in biology and medicine. Perspect Biol Med 38, 188-229 Triggle, D. J. (1997) Stereoselectivity of drug action. Drug Discovery Today 2, 138-147 Landoni, M. F., Soraci, A. L., Delatour, P., and Lees, P. (1997) Enantioselective behaviour of drugs used in domestic animals: a review. J Vet Pharmacol Ther 20, 1-16 Landoni, M. F., and Soraci, A. (2001) Pharmacology of chiral compounds: 2-arylpropionic acid derivatives. Curr Drug Metab 2, 37-51 Strong, M. (1999) FDA policy and regulation of stereoisomers: paradigm shift and the future of safer, more effective drugs. Food Drug Law J 54, 463-487 Shah, R. R., Midgley, J. M., and Branch, S. K. (1998) Stereochemical origin of some clinically significant drug safety concerns: lessons for future drug development. Adverse Drug React Toxicol Rev 17, 145-190 Mehvar, R., and Jamali, F. (1997) Bioequivalence of chiral drugs. Stereospecific versus non-stereospecific methods. Clin Pharmacokinet 33, 122-141 Seiler, J. P. (1995) Chirality--from molecules to organisms. Archives of Toxicology Supplement 17, 491-498 Tucker, G. T., and Lennard, M. S. (1990) Enantiomer specific pharmacokinetics. Pharmacology & Therapeutics 45, 309-329 Eichelbaum, M. (1995) Side effects and toxic reactions of chiral drugs: a clinical perspective. Archives of Toxicology Supplement 17, 514-521 Mehvar, R., Brocks, D. R., and Vakily, M. (2002) Impact of stereoselectivity on the pharmacokinetics and pharmacodynamics of antiarrhythmic drugs. Clin Pharmacokinet 41, 533-558 IUPAC (1976) Commission on Nomenclature of Organic Chemistry, Section E: Stereochemistry (Recommendations 1974). Pure and Applied Chemistry 45, 11-30 Pirkle, W. H., House, D. W., and Finn, J. M. (1980) Broad spectrum resolution of optical isomers using chiral highperformance liquid chromatographic bonded phases. Journal of Chromatography 192, 143-158 Gil-Av, E., Feibush, B., and Charles-Sigler, R. (1966) Separation of enantiomers by gas liquid chromatography with an optically active stationary phase. Tetrahedron Letters 7, 1009-1015 Baczuk, R. J., Landram, G. K., Dubois, R. J., and Dehm, H. C. (1971) Liquid chromatographic resolution of racemic [beta]3,4-dihydroxyphenylalanine. Journal of Chromatography A 60, 351-361 De Camp, W. H. (1989) The FDA perspective on the development of stereoisomers. Chirality 1, 2-6 FDA (1992) FDA's policy statement for the development of new stereoisomeric drugs. Chirality 4, 338-340 Williams, R. C., Riley, C. M., Sigvardson, K. W., Fortunak, J., Ma, P., Nicolas, E. C., Unger, S. E., Krahn, D. F., and Bremner, S. L. (1998) Pharmaceutical development and specification of stereoisomers. Journal of Pharmaceutical and Biomedical Analysis 17, 917-924 Agranat, I., Caner, H., and Caldwell, J. (2002) Putting chirality to work: the strategy of chiral switches. Nat Rev Drug Discov 1, 753-768 Kurihara, N., Miyamoto, J., Paulson, G. D., Zeeh, B., Skidmore, M. W., Hollingworth, R. M., and Kuiper, H. A. (1997) Chirality in synthetic agrochemicals: bioactivity and safety considerations. Pure and Applied Chemistry 69, 2007-2025 Magrans, J. O., Alonso-Prados, J. L., and Garcia-Baudin, J. M. (2002) Importance of considering pesticide stereoisomerism-proposal of a scheme to apply Directive 91/414/EEC framework to pesticide active substances manufactured as isomeric mixtures. Chemosphere 49, 461-469 Kurihara, N., and Miyamoto, J., eds (1998) Chirality in agrochemicals, John Wiley and Sons Ariens, E. J., van Rensen, J. J. S., and Welling, W., eds (1988) Stereoselectivity of Pesticides Vol. 1, Elsevier, Amsterdam Williams, A. (2000) The role of chirality in the agrochemical industry. Phytoparasitica 28, 1-4 Williams, A. (1996) Opportunities for chiral agrochemicals. Pesticide Science 46, 3-9 Wsol, V., Szotakova, B., Kvasnickova, E., and Fell, A. F. (1998) High-performance liquid chromatography study of stereospecific microsomal enzymes catalysing the reduction of a potential cytostatic drug, oracin. Interspecies comparison. J Chromatogr A 797, 197-201 Beckett, A. H. (1969) The importance of steric, stereochemical and physico-organic features in drug metabolism and drug action. Pure Appl Chem 19, 231-248 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. CSG 15 (1/00) 20 Project title 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. Differential Metabolism of Chiral Compounds DEFRA project code PS2501 Hanson, K. R. (1975) Reactions at prochiral centers. Interdependence in the estimation of enzyme stereospecificity toward prochiral centers and the configurational purity of labeled substrates. J Biol Chem 250, 8309-8314 Hewick, D. S., and Fouts, J. R. (1970) The metabolism in vitro and hepatic microsomal interactions of some enantiomeric drug substrates. Biochem J 117, 833-841 Testa, B. (1973) Some chemical and stereochemical aspects of diethylpropion metabolism in man. Acta Pharm Suec 10, 441454 Triebwasser, K. C., Swan, P. B., Henderson, L. M., and Budny, J. A. (1976) Metabolism of D- and L-tryptophan in dogs. J Nutr 106, 642-652 Warren, R. J., and Fotherby, K. (1975) Metabolism of D- and L-norgestrel in humans. Arzneimittelforschung 25, 964-965 Wright, J., Cho, A. K., and Gal, J. (1977) The metabolism of amphetamine in vitro by rabbit liver preparations: a comparison of R(-) and S(+) enantiomers. Xenobiotica 7, 257-266 Caldwell, J. (1995) Stereochemical determinants of the nature and consequences of drug metabolism. J Chromatogr A 694, 39-48 Rettie, A. E., Korzekwa, K. R., Kunze, K. L., Lawrence, R. F., Eddy, A. C., Aoyama, T., Gelboin, H. V., Gonzalez, F. J., and Trager, W. F. (1992) Hydroxylation of warfarin by human cDNA-expressed cytochrome P-450: a role for P-4502C9 in the etiology of (S)-warfarin-drug interactions. Chem Res Toxicol 5, 54-59 Kaminsky, L. S., and Zhang, Z. Y. (1997) Human P450 metabolism of warfarin. Pharmacol Ther 73, 67-74 Hu, Y., and Kupfer, D. (2002) Enantioselective metabolism of the endocrine disruptor pesticide methoxychlor by human cytochromes p450 (p450s): major differences in selective enantiomer formation by various p450 isoforms. Drug Metab Dispos 30, 1329-1336 Kurihara, N., and Oku, A. (1991) Effects of added protein (bovine serum albumin) on the rate and enantiotopic selectivity of oxidative O-demethylation of methoxychlor in rat liver microsomes. Pesticide Biochemistry and Physiology 40, 227-235 Kishimoto, D., Oku, A., and Kurihara, N. (1995) Enantiotopic Selectivity of Cytochrome P450-Catalyzed Oxidative Demethylation of Methoxychlor: Alteration of Selectivity Depending on Isozymes and Substrate Concentrations. Pesticide Biochemistry and Physiology 51, 12-19 Kishimoto, D., and Kurihara, N. (1996) Effects of Cytochrome P450 Antibodies on the Oxidative Demethylation of Methoxychlor Catalyzed by Rat Liver Microsomal Cytochrome P450 Isozymes: Isozyme Specificity and Alteration of Enantiotopic Selectivity. Pesticide Biochemistry and Physiology 56, 44-52 Narimatsu, S., Takemi, C., Kuramoto, S., Tsuzuki, D., Hichiya, H., Tamagake, K., and Yamamoto, S. (2003) Stereoselectivity in the oxidation of bufuralol, a chiral substrate, by human cytochrome P450s. Chirality 15, 333-339 Narimatsu, S., Takemi, C., Tsuzuki, D., Kataoka, H., Yamamoto, S., Shimada, N., Suzuki, S., Satoh, T., Meyer, U. A., and Gonzalez, F. J. (2002) Stereoselective metabolism of bufuralol racemate and enantiomers in human liver microsomes. J Pharmacol Exp Ther 303, 172-178 Weerawarna, S. A., Geisshusler, S. M., Murthy, S. S., and Nelson, W. L. (1991) Enantioselective and diastereoselective hydroxylation of bufuralol. Absolute configuration of the 7-(1-hydroxyethyl)-2-[1-hydroxy-2-(tertbutylamino)ethyl]benzofurans, the benzylic hydroxylation metabolites. J Med Chem 34, 3091-3097 Desta, Z., Soukhova, N., Morocho, A. M., and Flockhart, D. A. (2001) Stereoselective metabolism of cisapride and enantiomer-enantiomer interaction in human cytochrome P450 enzymes: major role of CYP3A. J Pharmacol Exp Ther 298, 508-520 Katsuki, H., Hamada, A., Nakamura, C., Arimori, K., and Nakano, M. (2001) Role of CYP3A4 and CYP2C19 in the stereoselective metabolism of lansoprazole by human liver microsomes. Eur J Clin Pharmacol 57, 709-715 Vickers, S., Duncan, C. A., Kari, P. H., Homnick, C. F., Elliott, J. M., Pitzenberger, S. M., Hichens, M., and Vyas, K. P. (1993) In vivo and in vitro metabolism studies on a class III antiarrhythmic agent. Drug Metab Dispos 21, 467-473 Niwa, T., Shiraga, T., Mitani, Y., Terakawa, M., Tokuma, Y., and Kagayama, A. (2000) Stereoselective metabolism of cibenzoline, an antiarrhythmic drug, by human and rat liver microsomes: possible involvement of CYP2D and CYP3A. Drug Metab Dispos 28, 1128-1134 Volz, M., Mitrovic, V., and Schlepper, M. (1994) Steady-state plasma concentrations of propafenone--chirality and metabolism. Int J Clin Pharmacol Ther 32, 370-375 Vakily, M., Mehvar, R., and Brocks, D. (2002) Stereoselective pharmacokinetics and pharmacodynamics of anti-asthma agents. Ann Pharmacother 36, 693-701 Mehvar, R., and Brocks, D. R. (2001) Stereospecific pharmacokinetics and pharmacodynamics of beta-adrenergic blockers in humans. J Pharm Pharm Sci 4, 185-200 Egginger, G., Lindner, W., Vandenbosch, C., and Massart, D. L. (1993) Enantioselective bioanalysis of beta-blocking agents: focus on atenolol, betaxolol, carvedilol, metoprolol, pindolol, propranolol and sotalol. Biomed Chromatogr 7, 277-295 Tan, S. C., Patel, B. K., Jackson, S. H., Swift, C. G., and Hutt, A. J. (2002) Stereoselectivity of ibuprofen metabolism and pharmacokinetics following the administration of the racemate to healthy volunteers. Xenobiotica 32, 683-697 Caldwell, J., Hutt, A. J., and Fournel-Gigleux, S. (1988) The metabolic chiral inversion and dispositional enantioselectivity of the 2-arylpropionic acids and their biological consequences. Biochem Pharmacol 37, 105-114 Kaneko, H., Matsuo, M., and Miyamoto, J. (1986) Differential metabolism of fenvalerate and granuloma formation. I. Identification of a cholesterol ester derived from a specific chiral isomer of fenvalerate. Toxicol Appl Pharmacol 83, 148-156 Okuno, Y., Seki, T., Ito, S., Kaneko, H., Watanabe, T., Yamada, T., and Miyamoto, J. (1986) Differential metabolism of fenvalerate and granuloma formation. II. Toxicological significance of a lipophilic conjugate from fenvalerate. Toxicol Appl Pharmacol 83, 157-169 CSG 15 (1/00) 21 Project title 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. Differential Metabolism of Chiral Compounds DEFRA project code PS2501 Kaneko, H., Takamatsu, Y., Okuno, Y., Abiko, J., Yoshitake, A., and Miyamoto, J. (1988) Substrate specificity for formation of cholesterol ester conjugates from fenvalerate analogues and for granuloma formation. Xenobiotica 18, 11-19 Miyamoto, J., Kaneko, H., and Takamatsu, Y. (1986) Stereoselective formation of a cholesterol ester conjugate from fenvalerate by mouse microsomal carboxyesterase(s). J Biochem Toxicol 1, 79-93 O'Reilly, R. A. (1974) Studies on the optical enantiomorphs of warfarin in man. Clin Pharmacol Ther 16, 348-354 Toon, S., Low, L. K., Gibaldi, M., Trager, W. F., O'Reilly, R. A., Motley, C. H., and Goulart, D. A. (1986) The warfarinsulfinpyrazone interaction: stereochemical considerations. Clin Pharmacol Ther 39, 15-24 Toon, S., Hopkins, K. J., Garstang, F. M., and Rowland, M. (1987) Comparative effects of ranitidine and cimetidine on the pharmacokinetics and pharmacodynamics of warfarin in man. Eur J Clin Pharmacol 32, 165-172 Lee, C. R., Goldstein, J. A., and Pieper, J. A. (2002) Cytochrome P450 2C9 polymorphisms: a comprehensive review of the in-vitro and human data. Pharmacogenetics 12, 251-263 Furuya, H., Fernandez-Salguero, P., Gregory, W., Taber, H., Steward, A., Gonzalez, F. J., and Idle, J. R. (1995) Genetic polymorphism of CYP2C9 and its effect on warfarin maintenance dose requirement in patients undergoing anticoagulation therapy. Pharmacogenetics 5, 389-392 Aithal, G. P., Day, C. P., Kesteven, P. J., and Daly, A. K. (1999) Association of polymorphisms in the cytochrome P450 CYP2C9 with warfarin dose requirement and risk of bleeding complications. Lancet 353, 717-719 Takahashi, H., Kashima, T., Nomizo, Y., Muramoto, N., Shimizu, T., Nasu, K., Kubota, T., Kimura, S., and Echizen, H. (1998) Metabolism of warfarin enantiomers in Japanese patients with heart disease having different CYP2C9 and CYP2C19 genotypes. Clin Pharmacol Ther 63, 519-528 Brune, K., Geisslinger, G., and Menzel-Soglowek, S. (1992) Pure enantiomers of 2-arylpropionic acids: tools in pain research and improved drugs in rheumatology. J Clin Pharmacol 32, 944-952 Iwakawa, S., Spahn, H., Benet, L. Z., and Lin, E. T. (1991) Stereoselective disposition of carprofen, flunoxaprofen, and naproxen in rats. Drug Metab Dispos 19, 853-857 Soraci, A., Jaussaud, P., Benoit, E., and Delatour, P. (1996) Chiral inversion of fenoprofen in horses and dogs: an in vivo-in vitro study. Vet Res 27, 13-22 Soraci, A., Benoit, E., and Delatour, P. (1995) Comparative metabolism of R(-)-fenoprofen in rats and sheep. J Vet Pharmacol Ther 18, 167-171 Castro, E., Soraci, A., Fogel, F., and Tapia, O. (2000) Chiral inversion of R(-) fenoprofen and ketoprofen enantiomers in cats. J Vet Pharmacol Ther 23, 265-271 Drummond, L., Caldwell, J., and Wilson, H. K. (1990) The stereoselectivity of 1,2-phenylethanediol and mandelic acid metabolism and disposition in the rat. Xenobiotica 20, 159-168 Kristensen, K., Blemmer, T., Angelo, H. R., Christrup, L. L., Drenck, N. E., Rasmussen, S. N., and Sjogren, P. (1996) Stereoselective pharmacokinetics of methadone in chronic pain patients. Ther Drug Monit 18, 221-227 Iribarne, C., Berthou, F., Baird, S., Dreano, Y., Picart, D., Bail, J. P., Beaune, P., and Menez, J. F. (1996) Involvement of cytochrome P450 3A4 enzyme in the N-demethylation of methadone in human liver microsomes. Chem Res Toxicol 9, 365373 Foster, D. J., Somogyi, A. A., and Bochner, F. (1999) Methadone N-demethylation in human liver microsomes: lack of stereoselectivity and involvement of CYP3A4. British Journal of Clinical Pharmacology 47, 403-412 Holm, K. A., Kindberg, C. G., Stobaugh, J. F., Slavik, M., and Riley, C. M. (1990) Stereoselective pharmacokinetics and metabolism of the enantiomers of cyclophosphamide. Preliminary results in humans and rabbits. Biochem Pharmacol 39, 1375-1384 Corlett, S. A., and Chrystyn, H. (1996) High-performance liquid chromatographic determination of the enantiomers of cyclophosphamide in serum. J Chromatogr B Biomed Appl 682, 337-342 Hoizey, G., Kaltenbach, M. L., Dukic, S., Lamiable, D., Millart, H., D'Arbigny, P., and Vistelle, R. (2001) Pharmacokinetics of gacyclidine enantiomers in plasma and spinal cord after single enantiomer administration in rats. Int J Pharm 229, 147153 Verho, M., Malerczyk, V., Damm, D., and Lehr, K. H. (1996) Pharmacokinetics of levofloxacin in comparison to the racemic mixture of ofloxacin in man. Drug Metabol Drug Interact 13, 57-67 Lehr, K. H., and Damm, P. (1988) Quantification of the enantiomers of ofloxacin in biological fluids by high-performance liquid chromatography. J Chromatogr 425, 153-161 Noctor, T. A., Fell, A. F., and Kaye, B. (1990) High-performance liquid chromatographic resolution of oxamniquine enantiomers: application to in vitro metabolism studies. Chirality 2, 269-274 Masubuchi, N., Yamazaki, H., and Tanaka, M. (1998) Stereoselective chiral inversion of pantoprazole enantiomers after separate doses to rats. Chirality 10, 747-753 Tanaka, M., and Yamazaki, H. (1996) Direct determination of pantoprazole enantiomers in human serum by reversed-phase high-performance liquid chromatography using a cellulose-based chiral stationary phase and column-switching system as a sample cleanup procedure. Anal Chem 68, 1513-1516 Sallustio, B. C., Morris, R. G., and Horowitz, J. D. (1992) High-performance liquid chromatographic determination of sotalol in plasma. I. Application to the disposition of sotalol enantiomers in humans. J Chromatogr 576, 321-327 Carr, R. A., Pasutto, F. M., and Foster, R. T. (1994) Stereospecific evaluation of sotalol pharmacokinetics in a rat model: evidence suggesting an enantiomeric interaction. Biopharm Drug Dispos 15, 109-120 Carr, R. A., Foster, R. T., Lewanczuk, R. Z., and Hamilton, P. G. (1992) Pharmacokinetics of sotalol enantiomers in humans. J Clin Pharmacol 32, 1105-1109 CSG 15 (1/00) 22 Project title 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. Differential Metabolism of Chiral Compounds DEFRA project code PS2501 Chan, K. Y., George, R. C., Chen, T. M., and Okerholm, R. A. (1991) Direct enantiomeric separation of terfenadine and its major acid metabolite by high-performance liquid chromatography, and the lack of stereoselective terfenadine enantiomer biotransformation in man. J Chromatogr 571, 291-297 Skalova, L., Szotakova, B., Lamka, J., Kral, R., Vankova, I., Baliharova, V., and Wsol, V. (2001) Biotransformation of flobufen enantiomers in ruminant hepatocytes and subcellular fractions. Chirality 13, 760-764 Wistuba, D., Weigand, K., and Peter, H. (1994) Stereoselectivity of in vitro isoprene metabolism. Chem Res Toxicol 7, 336343 Small, R. D., Golding, B. T., and Watson, W. P. (1997) Species differences in the stereochemistry of the metabolism of isoprene in vitro. Xenobiotica 27, 1155-1164 Bogaards, J. J., Venekamp, J. C., and van Bladeren, P. J. (1996) The biotransformation of isoprene and the two isoprene monoepoxides by human cytochrome P450 enzymes, compared to mouse and rat liver microsomes. Chem Biol Interact 102, 169-182 Cottrell, L., Golding, B. T., Munter, T., and Watson, W. P. (2001) In vitro metabolism of chloroprene: species differences, epoxide stereochemistry and a de-chlorination pathway. Chem Res Toxicol 14, 1552-1562 Delatour, P., Benoit, E., Caude, M., and Tambute, A. (1990) Species differences in the generation of the chiral sulfoxide metabolite of albendazole in sheep and rats. Chirality 2, 156-160 Delatour, P., Garnier, F., Benoit, E., and Caude, I. (1991) Chiral behaviour of the metabolite albendazole sulphoxide in sheep, goats and cattle. Res Vet Sci 50, 134-138 Eriksson, U. G., Lundahl, J., Baarnhielm, C., and Regardh, C. G. (1991) Stereoselective metabolism of felodipine in liver microsomes from rat, dog, and human. Drug Metab Dispos 19, 889-894 Paris, S., Blaschke, G., Locher, M., Borbe, H. O., and Engel, J. (1997) Investigation of the stereoselective in vitro metabolism of the chiral antiasthmatic/antiallergic drug flezelastine by high-performance liquid chromatography and capillary zone electrophoresis. J Chromatogr B Biomed Sci Appl 691, 463-471 Yasumori, T., Chen, L., Nagata, K., Yamazoe, Y., and Kato, R. (1993) Species differences in stereoselective metabolism of mephenytoin by cytochrome P450 (CYP2C and CYP3A). J Pharmacol Exp Ther 264, 89-94 Kern, R., Brode, E., and Schobel, D. (1992) Internally standardized simultaneous assay of verapamil and N-demethylverapamil enantiomers in human plasma by means of high performance liquid chromatography. Methods Find Exp Clin Pharmacol 14, 637-644 Caccia, S., Ballabio, M., Guiso, G., Rocchetti, M., and Garattini, S. (1982) Species differences in the kinetics and metabolism of fenfluramine isomers. Arch Int Pharmacodyn Ther 258, 15-28 Weil, A., Caldwell, J., Guichard, J. P., and Picot, G. (1989) Species differences in the chirality of the carbonyl reduction of [14C] fenofibrate in laboratory animals and humans. Chirality 1, 197-201 Wang, C. P., Howell, S. R., Scatina, J., and Sisenwine, S. F. (1992) The disposition of venlafaxine enantiomers in dogs, rats, and humans receiving venlafaxine. Chirality 4, 84-90 Lin, J. H. (1995) Species similarities and differences in pharmacokinetics. Drug Metab Dispos 23, 1008-1021 Nebbia, C. (2001) Biotransformation enzymes as determinants of xenobiotic toxicity in domestic animals. Vet J 161, 238252 Caldwell, J. (1992) Problems and opportunities in toxicity testing arising from species differences in xenobiotic metabolism. Toxicol Lett 64-65 Spec No, 651-659 Guengerich, F. P. (1997) Comparisons of catalytic selectivity of cytochrome P450 subfamily enzymes from different species. Chem Biol Interact 106, 161-182 Lang, M., and Pelkonen, O. (1999) Metabolism of xenobiotics and chemical carcinogenesis. IARC Sci Publ, 13-22 Lewis, D. F., Ioannides, C., and Parke, D. V. (1998) Cytochromes P450 and species differences in xenobiotic metabolism and activation of carcinogen. Environ Health Perspect 106, 633-641 Shimada, T., Mimura, M., Inoue, K., Nakamura, S., Oda, H., Ohmori, S., and Yamazaki, H. (1997) Cytochrome P450dependent drug oxidation activities in liver microsomes of various animal species including rats, guinea pigs, dogs, monkeys, and humans. Arch Toxicol 71, 401-408 Ueda, K., Gaughan, L. C., and Casida, J. E. (1975) Metabolism of four resmethrin isomers by liver microsomes. Pesticide Biochemistry and Physiology 5, 280-294 Soderlund, D. M., and Casida, J. E. (1977) Effects of pyrethroid structure on rates of hydrolysis and oxidation by mouse liver microsomal enzymes. Pesticide Biochemistry and Physiology 7, 391-401 Ohkawa, H., Mikami, N., and Miyamoto, J. (1976) Stereospecific Metabolism of O-Ethyl O-2-Nitro-5-methylphenyl NIsopropyl Phosphoramidothioate (S-2571) by Liver Microsomal Mixed Function Oxidase. Agricultural and Biological Chemistry 40, 2125-2127 Lee, P. W., Allahyari, R., and Fukuto, T. R. (1978) Studies on the chiral isomers of fonofos and fonofos oxon part 2: In vitro metabolism. Pesticide Biochemistry and Physiology 8, 158-169 Lee, P. W., Allahyari, R., and Fukuto, T. R. (1978) Studies on the chiral isomers of fonofos and fonofos oxon part 3: In vivo metabolism. Pesticide Biochemistry and Physiology 9, 23-32 Nomeir, A. A., and Dauterman, W. C. (1979) Stereospecific hydrolysis of the optimal isomers of O-ethyl O-p-nitrophenyl phenylphosphonate by liver microsomes. Biochem Pharmacol 28, 2407-2408 Ohkawa, H., Mikami, N., and Miyamoto, J. (1977) Stereoselectivity in metabolism of the optical isomers of cyanofenphos (o-p-cyanophenyl o-ethyl phenylphosphonothioate) in rats and liver microsomes. Agric. Biol. Chem. 41, 369-376 CSG 15 (1/00) 23 Project title 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. Differential Metabolism of Chiral Compounds DEFRA project code PS2501 Wing, K. D., Glickman, A. H., and Casida, J. E. (1983) Oxidative bioactivation of S-alkyl phosphorothiolate pesticides: stereospecificity of profenofos insecticide activation. Science 219, 63-65 Miyazaki, A., Kawaradani, M., Marumo, S., and Tomizawa, C. (1983) Enantiotopic demethylation of fenitrothion into partially racemised (R)p-(+)-desmethylfenitrothion by mouse liver homogenate and mice. Journal of Pesticide Science 8, 115-120 Miyazaki, A., Kawaradani, M., and Marumo, S. (1993) Analysis of an enantiomeric composition of desmethylfenitrothion, a major metabolite of achiral fenitrothion, by chiral HPLC. Journal of Pesticide Science 18, 131-133 Wakabayashi, K., Hizue, M., Nozaki, H., Nakayama, M., and Kurihara, N. (1994) Enantiotopic selectivity in monodemethylation of fenitrothion by rat liver glutathione S-transferase isoenzymes and mouse liver cytosol. Journal of Pesticide Science 19, 277-284 Kaneko, H., Matsuo, M., and Miyamoto, J. (1984) Comparative metabolism of stereoisomers of cyphenothrin and phenothrin isomers in rats (Pyrethroid insecticide). Journal of Pesticide Science 9, 237-247 Izumi, T., Kaneko, H., Matsuo, M., and Miyamoto, J. (1984) Comparative metabolism of the six stereoisomers of phenothrin in rats and mice (Pyrethroid insecticide, toxicology). Journal of Pesticide Science 9, 259-267 Kaneko, H., Izumi, T., Ueda, Y., Matsuo, M., and Miyamoto, J. (1984) Metabolism and placental transfer of stereoisomers of tetramethrin isomers in pregnant rats (Pyrethroid insecticides). Journal of Pesticide Science 9, 249-258 Ueji, M., and Tomizawa, C. (1987) Metabolism of chiral isomers of isofenphos in the rat liver microsomal system. Journal of Pesticide Science 12, 269-272 Buronfosse, T., Moroni, P., Benoit, E., and Riviere, J. L. (1995) Stereoselective sulfoxidation of the pesticide methiocarb by flavin-containing monooxygenase and cytochrome P450-dependent monooxygenases of rat liver microsomes. Anticholinesterase activity of the two sulfoxide enantiomers. J Biochem Toxicol 10, 179-189 Wenker, M. A., Kezic, S., Monster, A. C., and De Wolff, F. A. (2001) Metabolism of styrene in the human liver in vitro: interindividual variation and enantioselectivity. Xenobiotica 31, 61-72 Zhou, Q., Yao, T. W., and Zeng, S. (2001) Chiral metabolism of propafenone in rat hepatic microsomes treated with two inducers. World J Gastroenterol 7, 830-835 Mehvar, R., and Reynolds, J. (1995) Input rate-dependent stereoselective pharmacokinetics. Experimental evidence in verapamil-infused isolated rat livers. Drug Metab Dispos 23, 637-641 CSG 15 (1/00) 24 Project title Differential Metabolism of Chiral Compounds DEFRA project code PS2501 Glossary There is a wide assortment of terminology used in stereochemistry. Many terms have been superseded and there is inevitably some misuse of definitions. This glossary gives a brief definition of important terminology used in this report. More detailed lists can be found in many text books such as Morris (2001) and IUPAC recommendations are published on their internet site (Moss, 1996). Chirality: The geometric property of a rigid object (or spatial arrangement of points or atoms) being non-superimposable on its mirror image. Most commonly arises when a molecule possesses one or more asymmetrically substituted atoms (stereogenic centre (chiral or asymmetric centre): usually, but not always tetrahedral carbon with four different chemical groups attached). Some molecules have an overall chiral shape due to steric effects. Diastereomer (Diastereoisomer): Non-enantiomeric optical isomer arising when more than one chiral centre is present in a molecule. Exist as pairs of enantiomers (or meso compound). Enantiomer: One of a pair of non-superimposable mirror images that only differ in the spatial arrangement of groups around the stereogenic centre(s). Enantiomerically pure: A sample containing a single enantiomer of a chiral compound. Enantiomer excess (e.e.)/ enantiomer ratio: A measure of the enantiomeric make up of a sample. Enantiomer excess is defined as Conc R - Conc S Conc Total and is often expressed as a percentage e.g. a sample that consists of 90% R and 10% S has an e.e. of 80% and an enantiomer ratio of 9:1. Epimer: A diastereomer obtained by inversion of a single stereogenic centre. Isomer: Molecule with the same number and types of atoms but with a distinct structure. Meso-compound: An achiral molecule that has stereogenic centres but also contains a plane of symmetry so that both enantiomeric forms of a diastereomer are identical. Optical activity: The ability to rotate the plane of plane-polarised light. Optical isomer: A type of stereoisomer containing one or more stereogenic centres. Better described as either an enantiomer or a diastereomer. Racemate (Racemic mixture): An equimolar mixture of enantiomers, often given the prefix rac- or (±)-. The use of ‘racemic mixture’ is discouraged since it is also a synonym for racemic conglomerate, which describes a mixture of crystals each one of which contains a single enantiomer. Resolution: The separation of a racemate into its component enantiomers. Scalemic: A sample that is partially racemised or enantiomerically enriched. One enantiomer is present in excess. Stereoisomer: A type of isomer made up of the same atoms connected by the same sequence of bonds that differs only in its three dimensional structure. Stereoselectivity: The preferential formation of one stereoisomer over another. Also known as enantioselectivity or diastereoselectivity when the stereoisomers are enantiomers or diastereomers respectively. Morris, D.G., 2001. Stereochemistry. The Royal Society of Chemistry, Cambridge, UK. Moss, G.P., 1996. Basic terminology of stereochemistry, International Union of Pure and Applied Chemistry. CSG 15 (1/00) 25