investigation of toxic metabolites during drug development

www.elsevier.com/locate/ytaap

Toxicology and Applied Pharmac

Review

Investigation of toxic metabolites during drug development

Kevin Park*, Dominic P. Williams, Dean J. Naisbitt, Neil R. Kitteringham, Munir Pirmohamed

Department of Pharmacology and Therapeutics, Drug Safety Research Group, University of Liverpool, Sherrington Buildings,

Liverpool, Merseyside L69 3GE, UK

Received 15 July 2004; revised 1 February 2005; accepted 15 February 2005

Available online 5 July 2005

Abstract

Adverse drug reactions (ADRs) are a significant human health problem. Any organ system can be affected, including the liver, skin and

kidney. Drug-induced liver injury is the most frequent reason for the withdrawal of an approved drug from the market, and it also accounts for

up to 50% of cases of acute liver failure. The clinical picture is often diverse, even for the same drug. Mild, asymptomatic effects occur at a

relatively high frequency with a number of drugs. Idiosyncratic toxicity is rare but potentially life-threatening. Many serious ADRs that occur

in man are unpredictable from routine pathology and clinical chemistry in laboratory animals and are therefore poorly understood. The drug

metabolist can determine the propensity of a novel chemical entity to either accumulate in the hepatocyte or undergo bioactivation in numerous

model systems, from expressed enzymes, genetically engineered cells to whole animals. Bioactivation can be measured using trapping

experiments with model nucleophiles or by measurement of non-specific covalent binding. The chemistry of the process is defined and the

medicinal chemist can address the issue by seeking a metabolically stable pharmacophore to replace the potential toxicophore. However, we

require a more fundamental understanding of the role of drug chemistry and biochemistry in ADRs. This requires knowledge of the ultimate

toxin, signalling in cell defense and the sequence of molecular events, which ultimately lead to cell and tissue damage. It is imperative that such

studies have a clinical level, but then translated into laboratory-based molecular studies. This will provide a deeper understanding of potential

toxicophores for drug design and define candidate genes for pharmacogenomic approaches to individualized medicines.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Drug metabolism; Bioactivation; Bioinactivation; Critical protein antigen

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S425

Drug metabolism and drug toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S426

Chemical detection of bioactivation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S428

Biochemical basis of bioactivation and toxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S428

Molecular relationship between metabolism and toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S429

Relationship between metabolism and T-lymphocyte activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S430

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S433

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S433

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S433

0041-008X/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.taap.2005.02.029

* Corresponding author. Fax: +44 151 794 5540.

E-mail address: [email protected] (K. Park).

Introduction

Adverse drug reactions (ADRs) are a significant health

problem, which contribute to both patient morbidity and

mortality (Pirmohamed et al., 2004). In addition, ADRs

represent a major concern for the pharmaceutical industry

ology 207 (2005) S425 – S434

K. Park et al. / Toxicology and Applied Pharmacology 207 (2005) S425–S434S426

because of drug withdrawal during development (attrition)

and after licensing, both of which represent a considerable

loss of investment in human effort and technical resource.

There are many different types of ADRs, affecting every

organ system in the body, including the liver, skin, and

immune system. Hematological, gastrointestinal and cardi-

ovascular toxicities show good concordance between ani-

mals and man, whereas those ADRs which involve the liver,

skin and immune system show much poorer concordance.

Indeed, drug-induced liver injury is the most frequent reason

for the withdrawal of an approved drug, and it also accounts

for more than 50% of cases of acute liver failure (Lee, 2003).

More than 600 drugs have been associated with hepatotox-

icity. The clinical picture is diverse, even for the same drug

when given to different patients. The manifestations range

from mild, asymptomatic changes in serum transaminases,

which are relatively common, to fulminant hepatic failure,

which although rare, is potentially life threatening and may

necessitate a liver transplant (Park et al., 1998).

All ADRs mimic natural disease, and therefore, lessons

learnt from the study of drug-induced toxicity should not

only enhance drug safety but may also provide new

pharmacological strategies for the treatment of autoimmune

disease and liver disease. The study of drug disposition

(metabolism, pharmacokinetics and toxicokinetics) is essen-

tial for understanding drug action and for drug development.

The definition of the active chemical entities and their

concentrations in plasma, and within cells, is required in the

assessment of benefit and risk (Park et al., 1998).

ADRs may be classified as follows (Park et al., 1998):

& Type A (augmented). ADRs that are predictable from the

known primary or secondary pharmacology of the drug.

Such reactions usually represent an exaggerated pharma-

cological effect and are dose dependent. They can

therefore usually be avoided by the selection of the

appropriate dose for the patient. Inter-individual variation

in pharmacokinetics and pharmacodynamics may be

confounding factors. Such reactions account for approx-

imately 80% of all ADRs.

& Type B (idiosyncratic). These ADRs are not predictable

from the basic pharmacology of the drug and exhibit

marked inter-individual susceptibility. They do not show

any simple dose–response relationship for the target

patient population, although dose may be important for

the susceptible individual. Such ADRs are feared by

physicians because of their unpredictable nature, and

they may be life-threatening. They are an important cause

of drug attrition. The mechanisms for reactions remain

poorly understood. Drug metabolism, immune respon-

siveness, environment, genetics and the chemical struc-

ture of the drug (metabolite) are all factors which can, in

theory, contribute to inter-individual susceptibility.

Major advances in molecular pharmacology and toxico-

logy over the last decade have provided a conceptual

framework for the mechanism of action of chemical toxins

at the chemical, biochemical, cellular and clinical levels. We

now have the opportunity to define the processes that link

drug metabolism and the formation of toxic metabolites to

changes in cell function and the evolution of a particular

ADR. In this review, we shall consider how the drug

metabolist can contribute to an understanding of drug ADRs

and at what stages such information might be used to assess

the progression through various stages of drug development.

Drug metabolism and drug toxicity

The biotransformation of lipophilic compounds into

water-soluble derivatives that are more readily excreted is

the physiological role of drug metabolism. The primary site

of drug metabolism is the liver. The liver is exposed to

xenobiotics immediately after their absorption from the

gastro-intestinal tract and has a high capacity for both phase

I and phase II biotransformations. Cytochrome P450

enzymes play a primary role in the metabolism of an

incredibly diverse range of foreign compounds, including

therapeutic agents. Such compounds may undergo concen-

tration in the liver by various processes, including active

transport systems, which extract foreign compounds from

the blood and contribute to the first pass elimination of

drugs.

Variability in the rate of drug metabolism can influence

both efficacy and safety. Factors which influence this

process and are of clinical relevance are well documented

(Park and Maggs, 1986; Park and Pirmohamed, 2001; Park

et al., 1992; Pirmohamed and Back, 2001; Pirmohamed and

Park, 2001a, 2001b) and include pharmacogenetics, enzyme

induction, enzyme inhibition, disease and age.

During the past 20 years, a range of test systems have

been developed in academia and industry to explore the

pharmacokinetic basis of human variation in drug response,

with respect to the patient variables, and include: human

liver banks, expression systems, cell lines, in silico

techniques, transgenic animals, volunteers (phenotyped

and genotyped) and patients. These test systems enable

the drug metabolist to relate molecule to man at various

stages in drug development and thus allow the medicinal

chemist to create molecules that the clinician can use safely

and effectively in the target patient population (Williams

and Naisbitt, 2002; Williams and Park, 2003; Williams et

al., 2002). A vast amount of human experience with respect

to drug metabolism by the cytochrome P450 superfamily

has been obtained over the past 20 years, which has been

assimilated in various international databases/web-sites.

Such information has been used retrospectively to improve

the safety/efficacy of established drugs through transfer of

information into clinical practice via the Specific Product

Characteristics for physicians and Patient information leaf-

lets. The same information has been used prospectively in

the design of new chemical entities, which do not possess

K. Park et al. / Toxicology and Applied Pharmacology 207 (2005) S425–S434 S427

the unwanted pharmacokinetic characteristics of past prod-

ucts. Overall, this represents a major contribution of drug

metabolism to human health.

Although the physiological role of drug metabolism is

detoxication and clearance, certain biotransformations can

act as an ‘‘intoxication’’ process. Thus, xenobiotics undergo

biotransformation to toxic metabolites that can interfere with

cellular functions and may have intrinsic chemical reactivity

towards certain types of cellular macromolecules. The

propensity of a molecule to form either toxic and/or

chemically reactive metabolites is simply a function of its

chemistry (Fig. 1). Studies with model chemical toxins, and

also a limited number of therapeutic agents, have provided

the medicinal chemist with a large number of well-defined

structural alerts. A number of drug-metabolizing enzymes,

and in particular the cytochromes P450, can generate toxic

metabolites. The versatility of the superfamily of P450

enzymes, together with the reactivity of the oxygen

intermediate, enables them to functionalize even relatively

inert substrates, but this may also lead to the direct

formation of diverse chemically reactive species. Such

metabolites are short-lived, with half-lives of generally less

than 1 min, and are not usually detectable in plasma. Their

intracellular formation can be inferred from in vitro studies

and animal experiments by either radiometric analysis or

trapping reactions with either endogenous nucleophiles or

chemical reagents in combination with highly sensitive and

specific bioanalytical techniques. However, none of these

experimental procedures is directly applicable to patients,

where the assessment of exposure is almost impossible.

The Millers, in their work on chemical carcinogenicity,

developed the concept that small organic molecules can

undergo bioactivation to electrophiles and free radicals and

elicit toxicity by chemical modification of cellular macro-

molecules (Miller and Miller, 1947, 1952). The application

of such concepts to human drug-induced toxicity was

established through the studies of Brodie, Gillette and

Fig. 1. The relationship between drug metabolism and drug toxicity. The figure d

drugs can elicit a toxic response. Examples of some reactive metabolites and tox

Mitchell (Brodie et al., 1971; Gillette et al., 1974) on the

hepatotoxicity of the widely used analgesic paracetamol

(acetaminophen), for which the mouse is an appropriate

animal. Such studies provided a focus on the measurement

of covalent modification of tissues macromolecules as either

a biomarker or actual cause of drug-induced toxicity.

Drug bioactivation does not necessarily lead to drug

toxicity. Indeed, in many cases, bioactivation and bioinacti-

vation operate in concert for the efficient physiological

removal of a drug. The best example of this is the

bioactivation of paracetamol by CYP2E1 to a reactive

metabolite, N-acetyl benzoquinone imine (NAPQI), and

immediate bioinactivation by conjugation to glutathione,

hence the lack of hepatotoxicity with therapeutic doses of

the drug. Important pathways of bioinactivation include

glutathione conjugation, epoxide hydrolysis and quinone

reduction. However, when a reactive metabolite is a poor

substrate for such enzymes, it can escape bioinactivation, or

when it is formed in such quantities that the bioinactivation

process is overwhelmed, there is damage to the cell. Toxic

metabolites have the potential to modify the function of

various critical cellular macromolecules and are therefore

able to cause a diverse range of drug toxicities, including

tissue necrosis, cellular apoptosis, chemical carcinogenesis,

hypersensitivity, reproductive toxicity and idiosyncratic

toxicity.

Clearly, the role of drug metabolism in these processes is

far more complex than that encountered in Type A ADRs.

Nevertheless, the need to develop predictive test systems is

just as great in order to improve the ability of the medicinal

chemist to develop safer drugs. The drug metabolist can

contribute to this process. At present, we are at the stage of

trying to understand and define the role of drug metabolism

in non-pharmacological or chemically based ADRs. Pre-

dictive test systems can only be developed once there is

sufficient conceptual knowledge and information available

in readily accessible databases.

etails the metabolically dependent and independent mechanisms by which

icophores are given.


Chemical detection of bioactivation

A number of bioanalytical techniques have been utilized

to detect chemically reactive metabolites at a very early

stage in drug development, because of the potential hazard

associated with such metabolites. These include the

detection of glutathione adducts, DNA adducts, proteins

and oxidative damage in systems of varying biological

integrity such as microsomes, expressed enzymes, hepato-

cytes, cell lines and animal models including transgenics.

Such an exercise provides a useful iteration between the

drug metabolist and the medicinal chemist, especially when

a novel series of chemical entities is under investigation.

However, such information can also be used to assess

human risk. The availability of radiolabeled compound is

critical for the determination of covalent binding of drug to

tissues and macromolecules. It is essential that such

analyses are interpreted in the context of the overall picture

of drug metabolism. Many drugs, which are perfectly safe in

man, will undergo bioactivation in the presence of micro-

somes and NADPH. This is simply a consequence of

repeated oxidation reactions (removal of electrons) making

an inert molecule electrophilic. This does provide a

chemical marker of potential hazard that can then be

assessed further in progressively more integrated biological

systems. Ultimately, the chemistry of the process must be

related to biological function (Fig. 2).

Fig. 2. Examples of some toxic chemically reactive metabolites. The structure

paracetamol, bromobenzene, carbon tetrachloride and furosemide are given al

pathological outcome occurring in the murine liver 24 h after an acute hepatotox

Biochemical basis of bioactivation and toxicity

The use of transgenic animals has provided definition of

the role of P450 enzymes in chemical-induced toxicity.

Studies on the relationship of xenobiotic-metabolizing

enzymes with the induction of toxicity in whole animals

have been limited and difficult to interpret due to the

multiple forms of cytochrome P450 expressed. The effect of

a single enzyme on chemical toxicity can be precisely

determined by either disrupting expression or by introduc-

ing expression through genetic manipulation of the cyto-

chrome P450 enzymes in mice (Gonzalez, 2002, 2003).

CYP2E1 is well conserved across mammalian species,

metabolizes ethanol and many low molecular weight toxins

and carcinogens. It can be induced by ethanol, acetone and

other small compounds through post-transcriptional mRNA

and protein stabilization. Under treatment with inducers,

CYP2E1 can also be detected in a number of extrahepatic

tissues.

The cytochrome P450 isoforms that have been demon-

strated to be involved in the metabolism of paracetamol to a

toxic reactive quinone imine metabolite are CYP1A2 and

CYP2E1 (Lee et al., 1996; Zaher et al., 1998). These

investigations unequivocally determined the involvement of

these cytochromes in the initiation of paracetamol-induced

liver toxicity in mice. The cyp2e1 gene was isolated, and a

mouse line that lacks expression of CYP2E1 was generated

s of the toxic metabolites that are formed from oxidative metabolism of

ongside the requirement for GSH depletion and covalent binding. The

ic dose of the compound can be seen in context.


by homologous recombination in embryonic stem cells.

When cyp2e1 knockout mice were dosed, they were found

to be considerably less sensitive to its hepatotoxic effects

than wild-type animals, indicating that this P450 is the

principal enzyme responsible for the metabolic conversion

of the drug to its active hepatotoxic metabolite (Lee et al.,

1996). The observation that CYP1A2 and CYP 2E1 double

null mice were much less sensitive than either of the single

knockouts also suggested a major role for CYP1A2 in the

formation of the toxic metabolite of paracetamol. Doses of

1200 mg/kg to double null mice showed infrequent liver

lipidosis and mild kidney lesions, depletion of hepatic

glutathione was retarded, and covalent binding was not

observable (Zaher et al., 1998).

The sensitivity of CYP2E1-null animals to benzene has

also been seen analyzed (Valentine et al., 1996). Benzene

was administered at 200 ppm to null and wild-type mice.

Total urinary radioactivity and all radiolabeled individual

metabolites were reduced in the urine of cyp2e1-null mice

compared to wild-type controls. Also, more urinary radio-

activity could be accounted for as phenylsulfate conjugates

in cyp2e1-null mice compared to wild-type mice, indicating

the importance of CYP2E1 in the oxidation of phenol

following benzene exposure in normal mice. No benzene-

induced cytotoxicity or genotoxicity was observed in

cyp2e1-null mice. By contrast, benzene exposure resulted

in severe genotoxicity and cytotoxicity in wild-type mice.

This study indicates that CYP2E1 is the major determinant

of in vivo benzene metabolism and benzene-induced

myelotoxicity in mice.

Additional studies also conclusively demonstrated that

CYP2E1 is the major enzyme involved in the initiation of

toxicity due to cisplatin, acrylonitrile and chloroform (Con-

stan et al., 1999; Liu et al., 2000; Wang et al., 2002).

Cytochrome P450 1B1 knockout mice have been employed

to determine that CYP1B1 metabolizes 7,12-dimethylben-

z[a]anthracene to a DNA alkylating species, which corre-

lates with the formation of ovarian cancers in mice (Buters

et al., 1999).

Molecular relationship between metabolism and toxicity

Paracetamol is a useful tool with which to explore the

molecular interface between drug metabolism and drug

toxicity. It is also a highly relevant drug with respect to

clinical toxicology. The drug is a major cause of drug-related

morbidity and mortality in humans, producing massive

hepatic necrosis after a single toxic dose. Importantly, the

same pathology and clinical chemistry is observed in rodents.

Toxicity is essentially dose-dependent, although there can be

inter-species, intra-species and inter-individual variability in

susceptibility. For example, mice are generally more suscep-

tible than rats, while in man, alcoholics and patients on

enzyme-inducing drugs can have increased susceptibility. At

therapeutic doses, paracetamol undergoes metabolic clear-

ance by glucuronylation and sulphation to metabolites, which

are rapidly excreted in urine. However, a proportion of the

drug undergoes bioactivation to NAPQI by CYP2E1,

CYP1A2 and CYP3A4 (Raucy et al., 1989; Thummel et

al., 1993) (Figs. 1 and 2).

NAPQI is rapidly quenched by a spontaneous reaction

with hepatic glutathione after a therapeutic dose of para-

cetamol. After a toxic (over) dose, glutathione depletion

occurs, which is an obligatory step for covalent binding and

toxicity (Davis et al., 1974). The standard treatment for

paracetamol intoxication is N-acetylcysteine, which replaces

hepatic glutathione and prevents toxicity, and is most

effective when given within 16 h of the overdose.

Thus, the chemical and biochemical basis of paracetamol

toxicity is well established. More recently, the molecular

toxicology of the process has been explored by a number of

groups (Goldring et al., 2004; Kitteringham et al., 1999,

2000; Laskin and Pilaro, 1986; Laskin et al., 1986; Reilly et

al., 2001; Williams et al., 2004). We have been particularly

interested in the role of drug metabolism in cell defense and

cell destruction and have adopted an integrated approach in

which we have simultaneously explored:

& The chemistry of drug metabolism

& The biochemistry of drug metabolism

& Activation of transcription factors

& Gene expression (Williams et al., 2004)

& Analysis of the hepatoproteome and plasma proteome

& Proteomic analysis of modified proteins

& Conventional pathology and clinical chemistry

The massive chemical stress mediated by a single over-

dose leads to an immediate adaptive defense response in the

hepatocyte. This involves various mechanisms, including the

nuclear translocation of redox-sensitive transcription factors

such as Nrf-2, which ‘‘sense’’ chemical danger and orches-

trate cell defense. The Nrf-2 genes of immediate relevance

are those involved in glutathione synthesis such as g-

glutamylcysteine synthetase (g-GCS), glutathione-S-trans-

ferases (GSTs), glucuronyltransferases and heme oxygenase

(Goldring et al., 2004). Thus, the gene expression seems

designed, in a chemical and biochemical sense, to respond to

the toxic insult of the metabolite. Importantly, in terms of the

development of potential biomarkers that might be used in

drug development, it was shown that nuclear translocation of

Nrf-2 occurs at non-toxic doses of paracetamol and at time-

points before overt toxicity is observed. We have also

observed nuclear translocation of this redox sensitive tran-

scription with other compounds that cause hepatotoxicity

after bioactivation to a toxic metabolite, such as carbon

tetrachloride, furosemide and bromobenzene. We are cur-

rently exploring the usefulness of this transcription factor

and related downstream genes as markers of those forms of

chemicals that can lead to idiosyncratic drug-induced

hepatotoxicity in man (Fig. 2). It will also be important to

determine inter-individual variability in the responsiveness

of this system and its relevance to idiosyncratic drug toxicity.


Over the past few years, especially since the elucidation

of the human and murine genomes, numerous technologies

have been developed that are transforming pharmaceutical

research (Pennie et al., 2001; Waring et al., 2001). In

particular, the application of DNA micro-arrays and Gene

Chips to the quantitative comparison of the expression

levels of thousands of individual genes after exposure to

potential therapeutic candidates is termed toxicogenomics.

The comparison of expression profiles from known tox-

icants to those of novel compounds in a searchable data set

could elucidate underlying mechanisms and identify and

predict potential toxicities without the need to conduct

extensive, long-term animal or human clinical studies

(Bulera et al., 2001; Williams et al., 2004). Toxicogenomic

applications can be divided into mechanistic research and

predictive toxicology (Pennie et al., 2001). In mechanistic

research, the experimental model chosen should follow the

biological/toxicological process as closely as possible, with

similar clearly defined end-points.

Since the initial discovery that covalent binding of

paracetamol to hepatic proteins was associated with

hepatotoxicity, a number of techniques have been used to

identify the protein targets. Thus, radiolabeled drug and

Western blotting enable the detection and quantification of

protein adducts. This provides a non-specific biomarker of

drug bioactivation but does not provide any insight into

altered biological function and thus cannot be used in risk

assessment. More recently, proteomics has allowed the

simultaneous identification of several adducted proteins,

and also the determination of the amino acids modified

within the target protein. The question always asked with

respect to drug development is—Is there a particular protein

that will serve as biomarker of a particular form of drug

toxicity?

Before attempting to answer this question, we need to

define which protein modifications are important for cell

function, for both cell defense and cell destruction, and

separate these protein modifications that are of no functional

consequence and thus can be regarded as ‘‘white noise’’. At

least 17 liver enzymes that show a loss of activity ex vivo

after the administration of a toxic dose of paracetamol to a

rodent species have now been investigated. In addition,

another 14 liver enzymes are known to be adducted by

paracetamol in vivo and in vitro, but have yet to be shown to

be inhibited.

Modification of proteins can occur in most intracellular

compartments of the hepatocyte, including endoplasmic

reticulum, cytosol, mitochondria and plasma membrane, an

indication of the intracellular stability and mobility of the

reactive metabolite NAPQI. The loss of hepatocyte viability

is likely to be a consequence of the disruption of certain

critical proteins. Thus, the inhibition of g-GCS, glyceralde-

hyde 3-phosphate dehydrogenase (GAPDH) and Ca2+/Mg2+

ATPase will severely impair hepatocyte function by

uncoupling mitochondria, depleting glutathione and ATP,

and disturbing Ca2+ homeostasis which could lead to the

expression of TNFa and Fas receptors on cell membranes.

g-Glutamylcysteine synthetase catalyzes the rate-limiting

step in glutathione synthesis, the primary biochemical

defense of the hepatocyte against NAPQI. GAPDH, which,

as a component of the glycolytic pathway, contributes to

ATP production, is more than 80% inhibited at 2 h after a

toxic dose of paracetamol. On the basis of a reaction with

NAPQI in vitro, inhibition is thought to be due to the

modification of a critical cysteine (cys-149) residue within

the active site of the enzyme.

The rapid disruption of several proteins suggests that

cellular failure is a consequence of multiple parallel events

rather than a single protein target. It is well established that

one of the main events in isolated hepatocytes is overall

energy failure (Andersson et al., 1990; Burcham and

Harman, 1991), which is accompanied by the generation

of megamitochondria that are apparently ATP-depleted and

non-functional (Karbowski et al., 1999). The final destruc-

tion of hepatocytes involves interplay between hepatocyte

damage mediated by chemical stress and the activation of

non-parenchymal cells and the subsequent release of various

mediators. The role of Kupffer cells has been demonstrated

by the fact that mice treated with clodronate (dichlorome-

tyhylene bisphosphonate, DMDP), which depletes 99% of

macrophages from the liver, were protected against para-

cetamol (Goldin et al., 1996). Furthermore, infiltrating

macrophages are observed (Laskin and Pilaro, 1986).

Neutralization of Fas ligand (Zhang et al., 2000) and TNFa

(Blazka et al., 1996) affords a degree of protection against

the early apoptotic processes and the final overwhelming

necrosis, which is the overriding feature of paracetamol’s

hepatotoxicity. Thus, measurement of extracellular signal-

ling pathways may provide biomarkers of drug-induced

hepatotoxicity. This is extremely important in a clinical

context. Any observation in the plasma compartment that

reflects drug-induced organ toxicity, impending or actual,

would be of immense value in phase I and phase II studies.

From a physiological point of view, it is essential that

sensors in the cell defense system recognize the same

toxicophore in the metabolite that is responsible for damage

of critical macromolecules. The lack of an appropriate

response to chemical ‘‘danger’’, for chemical, biochemical

or genetic reasons, could lead to idiosyncratic drug toxicity.

Relationship between metabolism and T-lymphocyte

activation

Many idiosyncratic drug reactions have all the clinical

hallmarks of an immunological reaction. However, the

mechanism(s) of T-cell activation are not fully understood.

The majority of drugs associated with severe hypersensi-

tivity reactions also form chemically reactive metabolites.

Is this coincidence or consequence? There is good

circumstantial experimental evidence to relate the formation

of a reactive drug metabolite to immune activation.


Halothane, a drug associated with immune-mediated hep-

atitis, is possibly the best example where there is direct

experimental data to relate metabolic activation and the

development of clinical symptoms of hypersensitivity.

Approximately 20–50% of halothane is converted by

CYP 2E1 to a reactive trifluoroacetyl chloride intermediate,

which binds covalently to protein. The structural modifica-

tion of halothane, which reduces the extent of metabolic

activation by over 95%, dramatically reduces the incidence

of severe hepatotoxicity (Park and Kitteringham, 1994).

Chemically reactive metabolites can stimulate T-cells

via two pathways. Pathway 1 is a classical hapten

mechanism (Fig. 3). The covalently modified protein

conjugate is taken up by antigen presenting cells,

processed into peptide fragments, which translocate to

the cell surface in the context of the major histocompat-

ibility complex (MHC) for presentation to T-cells (Naisbitt

et al., 2000). As yet, it is not clear whether T-cell receptor

activation requires the presence of a haptenated drug

bound to the peptide embedded in the MHC. Drug

metabolites also bind directly to MHC molecules

expressed on the surface of antigen presenting cells. This

pathway avoids the requirement of an antigen presenting

cell’s processing machinery (Schnyder et al., 2000). A

recent study has shown that acid elution of pre-existing

peptides on the MHC binding groove does not prevent

drug presentation to T-cells (Burkhart et al., 2002); thus,

drugs might ignore the peptide embedded in MHC binding

groove and bind directly to MHC itself.

Recently, Pichler has proposed an alternative pathway of

T-cell receptor activation by drugs. Based on findings from

in vitro experiments with T-cell clones generated from the

peripheral blood of hypersensitive patients, the authors

proposed that drugs might bind directly in the absence of

drug metabolism, covalent binding and antigen processing

Fig. 3. Hapten hypothesis of drug hypersensitivity. The scheme illustrates how d

antigen formation. Antigen formation is thought to initiate hypersensitive reactions

presenting cells in conjunction with a Fdanger_ signal.

to MHC molecules (Fig. 3). The resultant ‘‘pharmacolog-

ical’’ bridging interaction between MHC and the T-cell

receptor, although relatively weak in a chemical sense and

readily reversible, is sufficiently stable to stimulate all the

activation events of the T-cell receptor (Schnyder et al.,

1997).

To study the chemical mechanisms of drug hyper-

sensitivity reactions, we have used sulfamethoxazole as a

paradigm. Sulfamethoxazole is a sulphonamide-containing

compound that was first developed in the 1960’s as an

antibacterial agent and is still used today as a cost-effective

alternative to the new generation expanded spectrum

antibacterial agents to decrease or delay the development

of resistance. The drug has been associated with a range of

severe immunological reactions that include anaphylaxis

and Stevens–Johnson syndrome. The experimental evi-

dence for a role for metabolism in the toxicity associated

with this drug is as follows:

& Sulphamethoxazole undergoes bioactivation by both

CYP2C9 and myeloperoxidase to form protein-reactive

metabolites.

& Sulphamethoxazole undergoes metabolism to a hydrox-

ylamine and protein adduct in keratinocytes in vitro and

in animal models.

& The nitroso metabolite of sulphamethoxazole is an

extremely potent immunogen in rat, mouse and rabbit.

& The nitroso metabolite can stimulate IL-5 production in

an animal model.

& T-cells have been detected in hypersensitive patients that

recognize sulphamethoxazole and covalently bound

nitroso metabolite.

& The nitroso metabolite of sulpamethoxazole can stim-

ulate naı̈ve T-cells from healthy volunteers (not exposed

to drug).

rugs and/or their metabolites can interact with cellular proteins, leading to

via interaction with major histocompatibility complex (MHC) II on antigen


To generate a drug antigen, sulfamethoxazole is metab-

olized in the liver, blood cells and keratinocytes to a

hydroxylamine metabolite (Cribb and Spielberg, 1990;

Reilly et al., 2000). These reactions are catalyzed by

CYP2C9 and/or myeloperoxidase. Sulfamethoxazole

hydroxylamine is not chemically reactive, is sufficiently

stable to circulate in the periphery, and is excreted

unchanged in urine (Gill et al., 1997). Further auto-

oxidation of sulfamethoxazole hydroxylamine generates

nitroso sulfamethoxazole, which is chemically reactive and

has been shown to haptenate cellular protein (Naisbitt et al.,

1999). In further studies, we have used LC-MS and NMR

technology to investigate the stability of nitroso sulfame-

thoxazole (SMX). Here, we demonstrated that nitroso

sulfamethoxazole is rapidly degraded in solution; degrada-

tion yielded products of oxidation (nitro SMX), reduction

(SMX, SMX hydroxylamine) and dimerization (azo and

azoxy adducts) (Naisbitt et al., 1996, 2002). Despite this, we

have shown by flow cytometry, using a specific anti-

sulfamethoxazole antibody, that nitroso sulfamethoxazole

circulates in the periphery in rats and binds covalently to

epidermal keratinocytes (Naisbitt et al., 2001). These data

are somewhat paradoxical; however, it is our view that they

provide experimental evidence to support the hypothesis

that a futile redox cycle between hydroxylamine, nitroso and

nitro metabolites of sulfamethoxazole is established in vivo

in patients following the conversion of sulfamethoxazole to

sulfamethoxazole hydroxylamine.

To investigate the role of drug metabolism and covalent

binding in the immunogenicity of sulfamethoxazole, we

have developed an in vivo rat model. Splenocytes from rats

administered the nitroso metabolite, but not the hydroxyl-

amine or parent drug proliferated following in vitro

stimulation with nitroso sulfamethoxazaole. No proliferation

was seen following in vitro stimulation with sulfamethox-

azole. Antigen-specific T-cells proliferated in the presence

of nitroso sulfamethoxazole bound covalently to cellular,

but not serum protein, in an MHC restricted fashion

(Naisbitt et al., 2001). The antigenic threshold of nitroso

sulfamethoxazole for T-cell activation was estimated to be

between 0.5 and 1 AM, which is less than the concentration

of sulfamethoxazole metabolites found in human plasma

after the administration of a therapeutic dose of sulfame-

thoxazole (Gill et al., 1996). Interestingly, we have recently

observed similar results in mice and rabbits (Farrell et al.,

2003). The essential aspect of these animal models is that

there is a T-cell response to nitroso sulfamethoxazole but not

the parent drug.

Traditionalists believe that the immune system has

evolved to differentiate between self and non-self; the

presence of non-self stimulates an immune response.

Matzinger (1994) has recently proposed an alternative

‘‘danger’’ hypothesis. Matzinger’s theory states that the

primary signal controlling whether the presence of an

antigenic signal results in tolerance (ignorance) or immune

activation is the presence of danger, not non-self (Matzinger,

1994). Danger signals derive from damaged cells that

release intracellular molecules such as heat shock proteins

into extracellular matrix (Gallucci and Matzinger, 2001; Shi

et al., 2000). In terms of drug hypersensitivity, we have

recently shown that non-toxic and toxic concentrations of

nitroso sulfamethoxazole up- and down-regulate, respec-

tively, the expression of CD40 (cluster of differentiation 40),

a co-stimulatory receptor on antigen presenting cells. In on-

going experiments, we are studying the complex relation-

ship between covalent binding cell death and regulation of

stimulatory receptors expressed on antigen presenting cells.

Although certain aspects of Matzinger’s danger theory

remain contentious (Vance, 2000), the hypothesis might

explain why drug hypersensitivity reactions occur much

more frequently with concomitant infection.

It is important to note that results from the studies

described thus far derive from models of drug immunoge-

nicity and not hypersensitivity (i.e., there was no sign of

tissue damage). Thus, we have recently changed the focus of

our research to concentrate on the only currently available

model of drug hypersensitivity—lymphocytes isolated from

the peripheral blood of hypersensitive patients. Lympho-

cytes from patients hypersensitive to sulfamethoxazole

proliferated in the presence of nitroso sulfamethoxazole

(Burkhart et al., 2001; Farrell et al., 2003; Schnyder et al.,

2000). These data show that patients are exposed to nitroso

sulfamethoxazole at the time of the adverse drug reaction;

however, and in contrast to our animal models of

sulfamethoxazole immunogenicity, lymphocytes from

hypersensitive patients also proliferated in the presence of

sulfamethoxazole. It is possible that there may be ongoing

metabolism in human cells from hypersensitive patients,

which is below the limit of detection using available

analytical techniques. Since the number of antigen mole-

cules required to stimulate T-cells is incredibly low–Irvine

et al. (2002) estimated that T-cells can be stimulated when as

little as 10 antigenic ligands are present–it is not surprising

that the immune system is more sensitive than analytical

methodology (Irvine et al., 2002). It is also possible that

human T-cells may be hyper-responsive in a chemical sense

whereby non-covalently bound parent drug may bind in a

‘‘pharmacological-like’’ interaction with the MHC-restricted

T-cell receptor.

These recent advances in our understanding of how drugs

stimulate T-cells has provided a framework to develop in

vitro cell culture assays that might be useful for patient

diagnosis and drug evaluation. In this respect, we are

validating an in vitro induction protocol using lymphocytes

from drug naive volunteers to study the ability of

sulfamethoxazole and sulfamethoxazole metabolite to

induce a primary T-cell response using cells from drug

naive healthy volunteers. Lymphocytes from the vast

majority of individuals reacted in response to the reactive

metabolite nitroso sulfamethoxazole (Engler et al., 2004),

which suggests that T-cell recognition of nitroso sulfame-

thoxazole may not be MHC allele restricted. Following


future development, we hope that this in vitro induction

protocol might be used to predict the potential of a new

chemical entity to cause T-cell-mediated reactions in man

(Engler et al., 2004).

Conclusion

Toxic metabolites can be formed from various functional

groups present in drugs by normal physiological biotrans-

formations. Constitutive and inducible systems exist for the

efficient bioinactivation of such reactive intermediates. Any

failure in cell defense to toxic metabolites can lead to

chemical and/or functional modification of critical cellular

macromolecules. The modification of critical macromole-

cules provides a credible explanation for certain types of

ADRs. This is now a major concern in drug development

and one that must be approached at various stages in the

process. Preclinical systems are available to detect chemical

hazard from metabolites and assess comparative (chemical)

risk. The prediction of individual (idiosyncratic) toxicity

induced by such metabolites is not yet possible, and will

require novel systems that incorporate individual suscepti-

bility factors.

Acknowledgments

The authors wish to acknowledge the support of The

Wellcome Trust and Pfizer PLC.

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investigation of toxic metabolites during drug development

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