Download Differential Metabolism of Chiral Compounds

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.
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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
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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.
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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’
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Differential Metabolism of Chiral Compounds
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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)
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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.
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Differential Metabolism of Chiral Compounds
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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.
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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
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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.
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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 RS 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
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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.
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Differential Metabolism of Chiral Compounds
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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.
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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.
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3.2
Differential Metabolism of Chiral Compounds
DEFRA
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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.
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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)
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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.
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Enantiomer excess (%)
[S]-[R]
Total
Project
title
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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
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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
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Differential Metabolism of Chiral Compounds
DEFRA
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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.
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