METABOLISM AND IMMUNE EFFECTS OF SULFA&ETHOXAZOLE AND HYDRO- METABOLITE

Michelle M. Aarts Department of Phoanacology and Toxicology

Submitted m partial Wtithent of the reqyiremnts for the degree of Wster of Science

Faculty of Graduate Studies The University of Westan Ontario

London, Ontuio October, 1996

0 Copyright by Michelle Anrts 1996

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ABSTRACT

Hypersensitivity reactions to sulfonamides are severe, idiosyncratic drug

reactions characterized by late onset fwer, skin ras& and organ toxicity. The

underlying medrmisn b lmlmowabra we pmpose that these reactions m y occur due

to metabolic activation of the drug md production of potential imrmmogenic

complexes. To test this hypothesis, fist and slow acetylrtors, identified by caeine

metabolisn, were given cotrimom1e twice daily for 9 days to ewrPine metabolic

clifkeaces between the 2 groups. Blood and urine samples were taken after 93.6,

9 and 12 days of treatment- Isolated lymphocytes were mitogen stimulated to test

immune cell function. Urine samples were ~~ for caffeine metabolites to

determine acetylrtor pheuotype md also for SMX, SMX-HA and acetyLSMX

content. SMX acecyLtioa was not sigdicantly Mkrent between acetylntion groups

although HA production was s i g d b d y higher for fist acetylators. No dSetences

in lymphocyte proliferation were found over the treatment period for either fast or

slow acetylstors and there was no correlation between urinary SMX-HA levels and

degree of probfhtio~~ Howewr, of serum by Western blot using anti-SMX-

BSA antibody revealed a 42 kDa protein that occured at days 3 and 6 but was

reduced or absent by day 9 in all subjects mdyzed. It is concluded that di&rences

in excretion of HA mt~bolite may be due to Weraces in metabolic Epctors such as

HA generation by cytocbrome P450 enzymes, HA binding to proteins in vivo or

detoxification by ghathione. The appearance of SMX-conjugates suggests that

reactive SMX metabolites are able to covalently bind proteins in vivo and form

potential immune complexes, their disappearance by day 9 may be attriiutable to

antibody production or uptake by phagocytic cells.

iii

DEDICATION

This work is dedicated to my b d y and fiends who put up with me,

suppolted me md loved me throughout my education

ACKNOWLEDGMENTS

This research was supported by a grant Eom The Kidney Foundation of

Canada in the name of Dr. Michael J- Rider.

I would like to thank Dr. David J. Freeman for his help and expertise in

developing and fine tuning the high perfo~natlce lipuid chromatographic assays and

also for his amemus thoughts and iders I would like to thank David Hess and Alice

Tschen for their support and laughter. I would like to acknowledge my supervisor

Dr. Michael J. Rieder for his support, insight and Psdleausl guidance throughout this

project.

TABLE OF CONTENTS

Certificate of Examination

Abstract

Dedication

Acknowledgments

List of Tables

List of Figures

List of Appendices

List of Abbreviations

1-0 Xntroduction

1. I. Sulfonamide Antiiiotics

L.2. Adverse Reictions to Sulfotlatnide Antiotics

L 3 - Metabolism of Sulfonanrides

1.3.1. Metabolism by N-Acetyltransfrrse Enymes

1.3 -2. Generation of Reactive Metabolites of Sulfonamides

by Cytochrome P450 enzymes

1.4. Rot& Conjugation

1.5 Models for Sulfimamide Adverse Drug Reaction

2.0 Hypothesis and Objectives

3.0 Methods

3. L. Subjects

3.2. Materials

3.3. Analysis of Urine for S~llfamethoxazole and Metabolit

3.4. Acetylrtor Status by Analysis of Urine Clffehe Metabolites

3.5. Isolation of Peripheral Blood Mononuclear Cells

3.6. Peripheral Blood Mononuclear Cell RoWeratim Assay

3.7. Western Blot Analysis of Serum Proteins

3.8. Data Analysis

4.0 R e d s

4.1. Subjects

4.2. Cafkbe Metabolism

4.3. S ~ o x u o l e Metabolism

4.4. Peripherai Blood Mononuclear Cell Roliferation

4.5. Westem Blot Analysis of Senan Roteins

5.0 Discussion

5.1. S ~ e t h o x a u , l e Adverse hug Reaction

5.2. S ~ e t h o x a z o k Immune

5.3. In V i o Metabolism of S ~ o x a z o l e

5.4 In Vivo Immune Effkcts of S ~ e t h o x a u , l e Metabolism

5.5. Future Directions

5.6. Conclusions

References

Vita

LIST OF TABLES

Table 1: Clrssiiication of Adverse Drug Reactions

Table 2: Cheracteristics of subjects participating in study.

Table 3: Metabolism ratios for caffeine and ~ o x a z o l e for

subjects treated with SMX

LET OF FIGURES

Egure I:

Figure 2:

Figure 3:

Figure 4:

Figure 5:

Figure 6:

Figure 7:

Figure 8:

Figure 9:

The four clrssificatiotls of hypersasitivity reactions 4

Enymptic patbways invoked m the m e t a b o h of 12

Suhmethoxazole

Measurement o f ~ e t h o x a z o k , ~ o x a u , l e 27

hydroxylrmine and aceryl--0x11u)ie in the urine by

high p e r f b m c e liquid chromatography.

Measurement o f Caffeine metabolites, AFMU, 1U and 29

1 X m urine by high performance liqyid chromatography.

Acetylation of suif'ametho~~~~)le by both frst and slow 39

acetylators

Measurement of slllErmethowzok hydroxybbe 41

excreted in the urine.

Proliferation of peripheral blood mononuclear cells

fiorn controls and treated subjects who are fast or

slow acetylators.

Measurement of labelled protein in the serum by scanning 47

densitometfy

Western blot analysis of serum proteins over the SMX 49

treatment period

LIST OF APPENDICES

Appendix 1

In vivo M e t a b o h of Sdfimethowzole: A Relimvy Study 77

Appendix 2

Sample Size CalCU1Btions 81

Appends 3

Letter of Wormation 83

Consent Form 84

Eth ics Approva l 84a

LIST OF ABBREVIATIONS

acetyl-SMX

ADR

AFMU

AIDS

CPM

cYP450

ECL

FCS

GSH

HA

HEPES

HI[V

HPLC

HRP

[gG

NADPH

NAT INAT2

PBMC

PHA

PMA

RPMI

SDS-PAGE

SMX

Aceryd suhmethoxazole metabolite

Adverse drug reaction

Acmed Imrmmodeficiency Syndrome

Counts per minute

Cytochrom P450 mixed fimctim oxidase

Enhanced cheduminescence

Fetal calf serum

Glutathione

Hydroxylamine

4-(2-hydro*ethyl) 1-pipemhe ethane sulfonic acid

HumPn Imrmmodeficiency V i

High pressure liqyid chromatography

Horseradish peroxidase

Immunoglobulin E

[mrrmnoglobulia G

Nico-de adenine dinucleotide phosphate

N-acetyhransfkrse type l / type 2

Peripheral blood mononuclear cells

Phytohemagghxtanin

Phorbol myristate acetate

Rosewen Park Memod lnstihlte (media)

Sodium dodecyl d t e - potyacrylamide gel electrophoresis

Sdfhethoxazole

SMX-BSA S~e thoxazo l e - bovine serum albumm conjugate

SMX-HA suifkmethoxazoie hydroxyiamine

lTBS Tween - tris bufked soline

IU I-methyhuic acid

IX 1-methylxanthine

1 .O INTRODUCTION

1.1 Sulfonrmide Antibiotics

The sulfonamide class of bacteriostatic drugs has been in use for the treatment

of a variety of infectious diseases for over 50 yean (Meekins et a[., 1994; Mandell and

Sande, 1985). Sulfonamides were primarily used for therapy of bacterial infections but

the indications for sulfonamide therapy decreased as alternate agents appeared. With

the onset of AIDS and increase in transplant therapies, sulfoaamide use has increased as

an effective prophylaxis against opportunistic infections (Koopmans et d. 1995: Lee et

al., 1994; Rieder et al.. 1991). Sulfarnethoxazole (SMX) in particular is commonly

used to prevent and treat P~euntocysns carinii pneumonitis (Koopmans et al.. 1995).

The chemotherapeutic properties of pronmsil, a dye, were frst introduced by

Domagk in the 1930s. who demonstrated that the dye could protect mice from

streptococcal infections (Mandell and Sande, 1985). Prontosil is a pro-drug which

requires metabolism to its active form, sulfanilamide. Sulfanilamide and other

sulfonamides exert their effects by competitively inhibiting bacterial folic acid

synthesis. Sulfonamides are aromatic amines containing the sulfanilamide moiety

which is structurally similar to p-aminobenzoic acid (PABA) (figure 2). Sulfonamides

compete with PABA for the enzyme involved in folate synthesis thereby inhibiting the

metabolism of the bacteria (Mandell and Sande, 1985).

Although sul fonarn ides are effective as bacteriostatic agents, adverse reactions

to this class of drugs have been well documented. Combination therapy, such as

L

002 sulfamethoxazole/trimethoprim mixtures, is now commonly prescribed. This allows

the dose of the sulfonarnide to be lowered while retaining the anti-microbial efficacy of

the therapy (Kooprnans n al.. 19%).

1.2 Adverse Reactions to SulfonPmide Antibiotics

Mild side effects such as nausea, vomiting and gastric upset occur most

frequently and these general toxic effects account for up to a third of sulfonamide

adverse drug reactions (Carrington n al.. 1987). Cutaneous reactions such as urticaria1

skin rashes occur in approximately 5 1 of sulfonarnide reactions (Carrington et al.,

1987; Park and Kitteringharn, 1990). Anaphylactic allergy is not a common side effect

of sulfonamide therapy (Park and Kitteringharn, 1990). A small percentage of

therapies may result in severe reactions such as delayed onset hypersensitivity (Rieder

et al., 1989). These reactions are characterized by high fever, erythema multiforme,

and organ involvement such as kidney and liver damage after LO - 14 days of therapy.

Severe hypersensitivity reactions to sulfonamides are usually classed as "idiosyncratic"

and can result in life threatening illness (Leeder et al., 1991; Kmpmans et al.. 1995;

Gupta et ai., 1992; Rieder et al., 1995).

Hypersensitivity reactions are classically characterized into 4 classes using the

scheme outlined by Gell and Coombs in 1%8 (Coombs and Gell, 1%8; Roin et ai.,

1985) (figure 1). Type I hypersensitivities are immediate allergic reactions that result

From the interaction of antigen with IgE and the subsequent release of inflammatory

mediators. T* I reactions are characterized by anaphylaxis, bronchospasm and

003 urticaria (Roitt et ai., 1985). Type II or cytotoxic reactions occur when antibody binds

to antigens on the cell surface leading to lysis by activation of natural killer cells or

complement. Such reactions can lead to red cell lysis in the cases of blood transfusions

or drug induced haernolytic anemia (Roitt et ai., 1985; Rieder, 1992). Immune

complex formation is responsible for inducing Type 111 hypersensitivity. Antigen-

antibody complexes can be formed in large quantities in respow to persistent infection

or environmental antigens. These complexes deposit in tissues, amacting complement

and polymorphic cells resulting in local damage (Roia et ai.. 1985). Serum sickness

reactions are believed to be a result of Type 111 hypersensitivity (Roia et at., 1985;

Kearns et al., 1994). Contact dermatitis is a result of Type IV or delayed type

hypersensitive reaction and is mediated by activated T-cells. Following secondary

contact with an antigen, Tcells release cytokines which induce an inflammatory

response. These reactions can take from 12 hours to 14 days to develop and the

underlying mechanism is not well understood (Roitt et ai., 1985).

The severe hypersensitivity reactions caused by sulfonarnide therapy have

characteristics €rom dl the hypersensitivity groups but do not fit well into any one of

the Gel1 and Coombs classifications (Rieder, 1994). Skin reactions can range from

urticaria1 (IgE mediated) to localized dermatitis (Type IV) (Park and Kineringham,

1990; Koopmans et al., L995; Rieder, 1993) but the mechanisms behind these reactions

do not explain the more severe skin lesions such as Stevens-Johnson syndrome and

toxic epidermal necrol ysis . The delayed time course for sul fonam ide reaction to occur

F i g w 1: The four classifications of hypersensitivity reactions as described by Gel1

and Coombs (1968).

celCsurface antigen

antigen mHd!aa cell

Type II

Complement

cell lysis by activation of complement or NK cells

antigen-

I % I

S body tissues

complement activation or leumcytes attracted to tissue

maaophage activation

Type Ill

006 suggests a Type IV sensitivity but these reactions require previous sensitization and

many severe sulfonamide reactions occur during the fwst exposure (Roitt er ai., 1985:

Rieder et ai.. 1995; Riedet, 1993). The cellular damage found in conditions of

s ul fonamide-induced hepatitis and haemolytic anemia also indicate evolution of a Type

11 reaction (Roitt n al.. 1985; Rieder, 1992; Gupta et ai.. 1992).

Adverse drug reactions can be classified as either predictable or unpredictable

reactions (Rieder, 1992) (table 1). Predictable reactions are those resulting from

known actions of the drug itself. Signs and symptoms related to overdose, side effects

and drug interactions are considered to be predictable reactions as they are

characteristic of the pharmacological actions of the drug in question. Unpredictable

adverse reactions are dependent on the individual rather than on the chemical itself.

Frequently, inter-patient variability in the types of adverse reactions is observed. Such

variability may be due to pharmacogenetic differences in the metabolism of a particular

drug. These reactions include intolerance, allergy and idiosyncratic reactions (Rieder.

1 992; Rieder, 1993). Sulfonamide delayed hypersensitivity reactions have been

described as idiosyncratic reactions, as they are dependent on the individual patient ,

have unknown etiology and are perhaps governed by a genetic predisposition (Rieder,

1993; Shear and Spielberg, 1985).

1.3. Metabolism of Sulfonamides

Until recently little information was available concerning the mechanisms

underlying adverse reactions to sulfonamides . Hypersensitivity reactions to

sulfonamides are believed to be initiated by metabolism of the sulfonamide and

007 mediated by an immune response (Shear and Spielberg, 1985; Shear e? al., 1986:

Rieder et al., 1989). Sulfonamides are acetylated or oxidized to various intermediate

metabolites. Fifty to 80% of a sulfonamide dose is metabolized via arylamine N-

acetyltransferase to N4-acetyl sulfonamide, a stable compound that is excreted in the

urine (figure 2) (Vree ad, 1994; van der Ven et al., 19W; Grant et al., 1992). A

significant portion of the sulfonamide dose also appears to be oxidized via cytochrome

P450 mixed function oxidases to a reactive hydroxylamine metabolite (Cribb and

Spielberg, 1990; Cribb ef ul., 1995; Leeder ef d, 1988). Metabolism of sulfonarnides -

resulting in the production of a hydroxylamine intermediate as well as detoxification of

reactive metabolites are thought to play a key role in the development of

hypersensitivity reactions (Rieder a ale, 1989; Shear e? al., 1986).

1.3.1. Metabolism of Sulfonamides by N-Acetyltraasferase Enzymes

Ar y lamine N- Acetyltransferase catalyses the acety 1 CoAdependant conjugation

of primary amino groups with acetate (Grant n a&.. 1992: Cribb et al.. 1993). N-

Acetyltransferase is a polymorphic enzyme consisting of N-acetyltransferase type 1

(NAT 1) and type 2 (NAT2) enzymes. NATl and NAT;? are separate gene products

encoded by NATl and NA12 genes respectively (Grant et al., 1992; Cribb et al..

1993). The genetic polymorphism associated with acetyltransferase activity, which

results in the fast and slow acetylation phenotypes, comes from the multidlelic gene

locus for NAlT expressed as NAT2A and NAT2B isofom. NATZA is the major gene

product while NAT2B is thought to arise from post-translational processing of 2A as

Table I: Classification of Adverse Drug Reactions (adapted From Rieder, 1992)

I . Predictable adverse reactions

a) Overdose or toxicity

b) S ide-ef fects at normal pharmacological doses

C) Secondary effects that are indirect consequences of therapy

d) Drug Interactions

Unpredictable adverse reactions

a) Intolerance

- a lower threshold to the pharmacological action of the drug in

susceptible individuals

b) Idiosyncratic react ions

- inexplicable adverse reactions that may occur in susceptible individuals

i .e. halothane induced hepatitis

C) Allergic reactions

009 the isoforms are Functionally indistinguishable (Grant et al.. 1992). One allele for

NAT2 encodes the functioning enzymes responsible for the 'fast' phenotype while

multiple alternate alleles code for the 'slow' phenotype (Cribb et ui-. 1993; Grant et

al., 1992; Notarianni et al.. 1996). Slow acetylators have mutant NAR alleles. lack

the functional NAT2 gene product and therefore enzyme function. The presence of

multiple mutant alleles accounts for the 50 to 60% incidence of the slow acetylator

phenotype in Caucasian populations (Grant et d., 1992).

N-acetylation is a common detoxification reaction and toxicity to chemicals

metabolized by NAT has been associated with slow acetylator status (Notarianni et al..

1996). Compounds such as isoniazid, caffeine, procainamide and various sul fonarn ides

are metabotized by NAT2 a d differential acetylation of these compounds can be seen

between individuals with fast and slow enzyme status. These chemicals are designated

'polymorphic substrates' as their acetylation rates correlate with genetically determined

acetylator phenotypes (Grant et ol., 1992). NATl is constitutively expressed and has

functional activity that overlaps that of NAT2. Many compounds, for example, PABA

are NATl specific, do not demonstrate differential acetylation and are therefore said to

be 'monomorphic' substrates. A very few compounds such as 2-aminofluorene are

acetylated efficiently by both NATl and NAT2 enzymes (Grant et al., 1992; Cribb et

al., 1993).

010 1.3.2. Generation of Reactive Metabolite of Sulfomddes by Cytochrome P450

EnZVrnes

In addition to acetylation, part of a sulfonarnide dose undergoes cytochrome

P450 cad ysed N-oxidation to an electrop h il ic hydroxy lamine (HA) intermediate (Crib b

ef al., 1995). Elecuophilic metabolites have been implicated in the toxicity of many

drugs including procainamide (Ueuecht, 1985; Rieder, 1992). If not detoxified,

reactive metabolites such as the HA and nitroso of sulfonamides can bind covalently,

via the N, oxygen species, to cellular macromolecules resulting in cell injury, cell death

or the formation of haptens that can initiate immune responses (Shear and Spielberg,

1985; Rieder et ale, 1988; Cribb and Spielberg, 1990). The suspect isozymes involved

in N,-hydroxylation in humans are CYPlA2 and CYPZC9 (Cribb et d., 1993) (figure

2). The HA metabolite is known to be toxic to peripheral blood mononuclear cells in

vitro (Rieder et al., 1988). Sulfonamide HA can also undergo spontaneous oxidation to

a more reactive nitroso-sulfonamide intemediate that readily binds proteins in vitro and

may be responsible for some of the cellular toxicity and protein binding in vivo (van

der Ven n al., 1995; Carr et al., 1993). Under normal physiologic conditions this

oxidation of HA to the nitroso derivative appears to be prevented by glutathione (GSH)

(Cribb et al., 1991; Cribb et al., 1995).

The HA metabolite of SMX demonstrates a dose-dependent toxicity to

lymphocytes in vitro that is much greater than that of the parent compound (Cribb and

Spielberg, 1990; Leeder et al., 1988; Rieder et al.. 1988). A significant increase in

this in v i m lymphocyte toxicity is observed in the cells of individuals who are sensitive

to sulfonamides. As a result, the level of toxicity found after in v i m exposure of

PBMCs to SMX-HA is currently used as a predictive marker for sulfonamide

hypersensitivity (Rieder et of., 1989). Studies that used liver microsomal enzymes

(i.e., CYP450) and cofactors in the presence of SMX indicated that HA could indeed

be produced by enzymatic bioactivation (Spielberg. 1984: Leeder n al.. 1988: Cribb

and Spielberg, 1990). That the reactive metabolites of sulfonamides are able to

covalently bind ceilular proteins was demonstrated by Shear and Spielberg (Shear and

Spielberg, 1985). They found that sulfadiazine was bound to proteins in cultures using

a microsomal system to generate metabolites. This binding was highest when

phenobarbital induced microsomal enzymes. in the presence of cofactors, were used.

SMX reactive metabolites may also affect the function of exposed cells by binding with

cellular macromolecules. Electrophilic intermediates cause marked decreases in

cellular esterase activity prior to loss of cell viability (Leeder et al., L991; Leeder et

al.. 1991). In PBMC cultures exposed to SMX-HA, dosedependent suppression of

mitogen induced proliferation is also seen (Rieder et al., 1992).

Even though in v i ~ o data implicated HA production as a mechanism of toxicity

for sulfonamides, supporting in vivo evidence is required. The HA metabolite is toxic

but the question remains if it is produced in vivo in sufficient quantities to have

Figure 2: Enzymatic pathways involved in the metabolism of Sulfamethoxazole,

S ul famethoxazole h ydroxy Iamine and Acety 1 sultamethoxazole in the human I iver .

Adapted from A.E.Cribb et ul. (1995).

N-ACETY L TRANSFERME: PrcdominantIy NATl

<

so2 Ducecylation (minor for sulf~~~cthoxuole) AH

- p 7 0 2

NH

9 C H 3 A N

014 toxic effects? Also the question of systemic availability needs to be answered. It is

possible that the HA is covalently bound to cellular macromolecules such as the

CYP450 enzymes in the liver where it is produced. Covalent binding in the liver

would be expected to greatly decrease the systemic availability of HA. If HA or

nitroso metabolites were bound to hepatic proteins, less reactive metabolite would enter

the circulation and therefore would not be available to cause cellular damage outside of

the liver.

In 1992 Cribb and Spielberg demonstrated that SMX-HA was produced in

sufficient quantities to be detected in the urine of patients treated with SMX (Cribb and

Spielberg, 1992). These findings indicated that HA is produced in vivo and distributed

systemically and therefore -available to cause toxicity in organs other than the liver.

Several groups have now investigated the kinetics of SMX in vivo and demonstrated the

presence of SMX-HA in both the plasma and urine (Vree et al., 1994; van der Ven et

al.. 1994; Lee et al., 1994).

1.4. Protein Coqjugation

Another mechanism for hypersensitivity mediated by metatmi ism is the

formation of sulfonarnide conjugated proteins in treated individuals. The formation of

drug-specific haptens has been proposed as a factor in the development of adverse

reactions to many drugs including penicillins, halothane and carbamazapine (Park,

1987; Park and Kitteringham, 1990; Rieder. 1994). Drug-specific haptens may act as

neo-antigens, against which the body would launch an immune response, depending on

the protein and the treated individual (Park and Kineringham, 1990; Rieder. 1994).

015 Rich postulated in 1942 that sulfonamides may form potential immune complexes in the

serum of treated individuals (Rich. 1942). By injecting serum samples underneath the

skin LefoKich subsequently demonstrated that the serum of sulfonamide treated

individuals induced skin reactions in patients allergic to sulfo~rnides (Lefovich, 1944).

Further support for the formation of an immune complex comes from in vim

lymphocyte toxicity studies by Cnib et al. (1995). They showed that SMX

intermediates covalently bound to the microsomal proteins used to generate SMX

metabolites (Cribb ez al., 1995; Shear and Spielberg. 1985). Despite this knowledge

the offending molecule remained elusive until 1994 when Meekins et af. described a

SMX-conjugated protein in the serum (Meekins ef ai.. 1994). This protein was

isolated from the serum of SMX treated individuals and labelled with an anti-SMX-

BSA IgG antibody. The antibody did not label the protein in untreated serum. The

protein was also only found in treated subjects who subsequently developed adverse

reactions to SMX. Meekins et al. (1994) postulated that such a protein-SMX conjugate

could serve as an antigenic compound and that individuals who produced this conjugate

would be at increased risk of SMX reactions.

The SMX-HA is thought to play an important role in the development of

adverse reactions to SMX. The reactive SMX-HA is capable of causing cell injury and

toxicity. It has been postulated that slow acetylators may have an increased incidence

of hypersensitivity reactions as they would have a greater amount of the sulfonamide

dose available for oxidation by CYP450 enzymes (Rieder n of., 1991; Cribb et al.,

016 1993: Grant et al., 1992). Although this theory is supported by a predominance of

slow acetylator status in hypersensitive individuals (Rieder et al.. 1991). polymorphism

in acetyiation has only been demonstrated for a small group of the sulfonamide drugs,

sulfanilarnidopyrimidines and -pyridines (Cribb et al., 1993; Grant et al., 1992; Vree

and Heks ter. 1985). Other sulfonarnides, including the commonly used SMX have

demonstrated only monomorphic metabolism in human populations (Cribb et ai..

1993). Differential formation of the HA metabolite may be accounted for by levels of

CY P4SO isozymes which form the reactive metabolite (Cribb n oi., 1995; Kadlubar,

1994: Page et ol.. 1991). Detoxification of the reactive metabolite may also be

important in determining who is at risk for hypersensitivity reactions. Spontaneous

oxidation of SMX-HA to the nitroso intermediates is prevented by the presence of GSH

and N-acetylcysteine (Cribb et al., 199 1; Rieder et al., 1988). Environmental changes

in sulfonamide metabolism or the balance of HA production and detoxification may

affect susceptibility. Such changes are most evident in patients with AIDS who

demonstrate a marked increase. up to 80% in HtV seropositive individuals. in the

incidence of hypersensitivity to sulfonarnides (Koopmans et al.. 1995; Lee et al.. 1994;

van der Ven et al., 1995).

1.5. Models for Sulfonamide Advecse Drug Reaction

In the past, studies concerning the toxicity of SMX have concentrated on the in

v i t ro exposure of human PBMCs to the drug and its metabolites. Peripheral blood

mononuclear lymphocytes are used as an easily obtainable, immune competent cell

017 model. Lymphocytes contain cell defences such as GSH, are therefore capable of

de toxitication and have a limited capacity to generate toxic metabolites making

1 y mp hocytes useful for studying host defences to challenge by metabolites (Spielberg,

1984). Lymphocytes are treated in v i m with concentrations of a particular drug and its

metabolites. Potential toxicity is then assessed by measuring cell viability and

comparing treated cells with untreated controls. If the metabolites of a drug are not

known, liver microsomal preparations can be used as a source of enzymes to generate

metabolites (Spielberg, 1980; Spielberg, 1984). The use of liver microsomaI

preparations, both human and murine, has demonstrated that SMX metabolites can be

generated enzymatically and that these metabolites are toxic to lymphocytes (Cribb and

Spielberg, 1990; Cribb e? al., 1995; Shear and Spielberg, 1985). In vine cellular

models have also demonstrated that SMX metabolites can have direct effects on the

function of immune cells (Spielberg, 1984; Leeder et aL, 1991; Leeder et al.. 1991).

SMX-HA causes dose-dependent suppression of mitogen induced proliferation (Rieder

et al., 1992). The demonstration of HA binding to cells and proteins in vine provides

a possible mechanism for the generation of SMX-haptens which could activate an

immune response (Cribb et al.. 1991; Shear and Spielberg, 1985). While these studies

have provided valuable insights into the actions of SMX metabolites, supporting in vivo

data is required to account for the actions of the drug in humans.

Several studies have analysed the metabolism of SMX in vivo and characterized

the kinetics of SMX and its metabolites (van der Ven e? ui., 1994; van der Ven et al.,

018 1995; Lee et a/., 1994; Cribb and Spielberg, 1992). These studies have concentrated

on the clearance of SMX and its metabolites in search of differences in metabolism that

may account for the development of hypersensitivity reactions. Thus far no differences

in the acetylation of SMX have been found between fast and slow acetylators (van der

Ven et ul., 1994) or between normals and patients with AIDS who have up to 80 %

incidence of hypersensitivity reactions (van der Ven et a!., 19%; Lee et a/., 1994).

Van der Ven and colleagues did demonstrate a difference in the excretion of SMX-HA

between normals and AIDS patients without differences in acetylation of the drug (van

der Ven et al., 1995). They theorized that the lower levels of HA from AIDS patients

may be due to deficient detoxification, allowing HA to bind in vivo and therefore not

be found in the urine. This study did not, however. go on to demonstrate whether

differences in production, detoxification or binding occurred for these patient groups.

Studies of sulfonamide kinetics in vivo so far have only used one time point to

determine the production of metabolites and have not looked at both the kinetics and

the immune effects of SMX in vivo. Meekins et a' demonstrated for the first time in

1994, a sulfonamide-protein conjugate produced in vivo after five days of SMX

therapy. They hypothesized that this conjugate was a possible immunogenic complex

(Meekins et d , 1994). Although this was a significant finding, it did not attempt to

i d u d e sul fonamide metabolism not account for potential changes over a treatment

period. To date, no studies have been performed which examine the kinetics of

sulfonamide metabolism over a treatment period. Nor have studies compared the in

019 vivo metabolism of sulfonamides with potential immune effects of this class of drugs.

2.0 HYPOTEESIS and OBJECTIVES

In order to examine the metabolism of SMX in vivo and the possible immune

effects of SMX metabolites, the following hypotheses were generated and were

investigated in the following study.

1) Electrophilic metabolites of sulfamethoxazole are generated in vivo and are capable

of binding to serum proteins in SMX treated individuals, forming potential

immunogenic complexes. These SMX-protein complexes may constitute a risk factor

for S MX- hypersensitivity and can be identified in sensitive individuals.

2) Differences in the production of SMX reactive metabolites are observed between

fast and slow acetylators regardless of SMX-acetylation. Although slow acetylators are

at higher risk of sulfonamide hypersensitivity, SMX is known only to be

monomorphically acetylated, implying the involvement of other metabolic factors that

may be associated with slow acetylator status.

3) Since slow acetylator status is a risk factor for hypersensitivity and SMX-haptens

may be markers for hypersensitivity reactions. slow acetylator status will be correlated

with the production of SMX-haptenated proteins in vivo.

4) S MX reactive membol ites produced in vivo have direct toxic effects on lymphocytes

resulting in changes in lymphocyte function in vitro. Based on preliminary data

(appendix 1) and previous in v i m work (Cribb and Spielberg, 1990; Rieder et al.,

L 992; Shear and Spielberg, l98S), decreases in lymphocyte function are correlated with

increased production of S MX reactive metabolites in vivo.

021 In order to test these hypotheses. n o d healthy individuals were given SMX

orally over a treatment period and blood, serum and urine samples were collected for

analysis. To examine hypothesis one, identification of SMX-specific proteins in the

plasma was performed using Western blot analysis. The protocol was based on that of

Meekins et al. (L994) who demonstrated the presence of a SMX-specific protein

conjugate in sulfonamide sensitive subjects treated with SMX.

To examine hypotheses two and three, the acetylator status of each subject was

determined using caffeine metabolism. Differences in the metabolism of SMX were

assessed for fast and slow acetylators. Metabolites of both caffeine and SMX were

detected using HPLC analysis of urine samples.

The lymphocyte suppression work of Rider et al. (1992) was used as a model

to investigate hypothesis four and assess changes in lymphocyte function over time for

S MX-treated subjects. Peripheral blood mononuclear cells were isolated from whole

blood samples and used to examine the effects of in vivo generation of SMX reactive

metabolites.

3.0 METHODS

3.1. Subjects

Sample size dculations were performed prior to begi~ing this study

(Appendix 2). Seventeen normal, healthy volunteers were obtained by advertisement.

7 females and LO males ranging in age fiom 20 to 44 years. Four of the subjects had

taken sulfonamides prior to this study (table 2). Subjects were given cotrimoxazole as

the Bactrimm double strength (800 mg sulfamethoxazole, 160 mg trimethoprim)

formulation twice daily for 9 days. This regime was chosen because it is a standard

therapy for an ambulatory care patient. Subjects were required to refrain fiom the use

of alcohol or other medication during the come of the study. Blood samples used for

lymphocyte isolation (10 mL) and separate plasma (2 mL) samples were obtained by

venipuncture and urine (20 mL) samples were collected on days 0, 3, 6 . 9 and 12 of

treatment fkom each subject. Peripheral blood mononuclear cells were isolated from

whole blood and used in proliferation assays. Plasma and urine samples were stored at

-20°C for later analysis by Western blot and high pressure liquid chromatography

(HPLC) respectively. Samples were also collected fiom five control subjects who were

recruited to participate in this study using the same criteria as for test subjects but did

not receive the cotrimoxazole dose. Each subject gave informed, written consent prior

to beginning the study. Ethics approval for this study was obtained fkom the University

of Western Ontario (p. iii).

3.2. Materials

Hypaque-Ficoll gradient, phorbol myrismte acetate (PMA), phytohemagglutanin

(PHA), ionomycin, 4-(2-hydroxyethy1)-l-piperkine ethane sulphonic acid (HEPES)

buffer and l-methylxanthine (LX) and I-methyluric acid (1U) HPLC standards came

from Sigma Chemical Co. (St. Louis, Missouri). RPMI (Rosewell Park Memorial

Institute) 1640 culture media and FCS was fkom Gibco Life Technologies. The rabbit

anti-SMX-BSA antisera was a kind gift of Dr. Rebecca Gruchdla (Southwest Medical

Center, Dallas, Texas). The 5-acetyiamin~dfomyIamin0-3-methyiuracil (AFMU)

used as a standard for the caffeine HPLC analysis was a kind gift of Dr. B.K. Tang

(University of Toronto, Toronto, Ontario). Sulfamethoxazole-hydroxy lamine HPLC

standard was obtained from Dalton Chemids, Sulfamethoxazole standard was

purchased from Aldrich Chemical Company Incorporated. Protein assay, streptavidin -

H RP , pol yacrylarnide. TBS and Tween-20 were fkom Bio-Rad Laboratories Limited.

Horseradish Peroxidase-conjugated goat anti-rabbit IgG secondary antibody was

purchased from Jackson [mmunoresearch Laboratories. Enhanced chemiluminescence

(ECL) detection system, biotinylated molecular weight markers, ECL-hyperfilm and

scintillation cocktail were from Amersham.

3.3. Analysis of Urine for Sulfamethoxazole and Metabolites

Each subject gave a urine sample on the test days into containers containing

ascor b ic acid. Ascorbic acid stabilizes SMX-HA preventing its auto-oxidation to

nitroso sulfarnethoxazole. Samples were frozen at -20°C until analysed. HPLC

024 analysis of the urine samples followed the method described by Cribb and Spielberg

(1992). To prepare samples for analysis. 100 pL of 15% perchloric acid was added to

900 pL urine and the samples centrifuged for 5 minutes at 13.000 cpm to remove

particulate matter. The supernatant was removed and diluted 1/100 with 1.5%

perchloric acid. Processed samples were transferred to autosampler vials and HPLC

analysis performed using the following conditions:

Chromatograph:

Column:

Mobile phase:

FIow rate:

Injection volume:

Run time:

Temperature:

Waters 501 pump

h-Max model 48 1 detector

254 nm

C8 Spherisorb 5pm

10 cm x 0.32 mm

80:20: 1 :0.05 (vol/vol)

warer/acetoniuile/glacial acetic acidltriethylarnine

0.5 mL/min

20 pL

15 min

40°C

All urinary products were identified by UV absorbance and comparison with standard

soiutions of SMX, SMX-HA and acetyl-SMX. Quantification of SMX and its

me tab01 ites was determined using peak height. Calibration standards were produced by

spiking blank urine from an untreated control subject with known amounts of SMX and

025 SMX-HA. Spiked blank urine was processed in the same manner as the samples and

injected onto the column. The retention times of SMX-HA, SMX and acetyl-SMX

were 7.4. 8 -9 and L 1.0 minutes respectively (figure 3). All SMX metabolites, SMX-

HA and acetyl-SMX were expressed as a percentage of the total drug and metabolites

excreted i.e, (SMX-HA / SMX + SMX-HA + acetyl-SMX) x 100%.

3.4. Acetylator Status by Analysis of Urine Cae ine Metabditos

Urinary caffeine metabolites were analysed by HPLC to determine acetylator

phenotype status of subjects. Caffeine was used as a non-invasive marker of

acetylation (Tang et af-, 1983). Urine samples were centrifuged at 12000g for 2

minutes in an Eppendorf microcentrifuge to remove particulate material. Samples were

then diluted 1:2 with 10 mM sodium acetate (mobile phase A). Processed urine

samples were transferred to autosampler vials and HPLC analysis was carried out using

the following conditions:

Chromatograph: Waters 501 pump

LMax model 481 detector

280 nrn

Prodigy C18 5pm

15 cm x 0.32 mm

A: LO mM sodium acetate

B: 5050 methanol: 10 m M sodium acetate

Column:

Mobile phase:

Flow Rate 0.5 mL/rnin

Injection volume: 20 pL

Run time: 40 min

Temperature: 4iPC

Column effluent was monitored for caffeine metabolites at 280 nrn. The run times for

AFMU. 1-U and 1-X were 6.3, 16.2 and 21.5 minutes respectively (figure 4). Due to

the weak mobile phase needed for resolution of the three caffeine metabolites many

urinary compounds were retained on the column. A second mobile phase (B)

consisting of 5050 methanol: l0mM sodium acetate was used from 26 to 32 minutes to

remove any retained material. The column was allowed to equilibrate using mobile

phase A for 8 minutes before injection of the next sample. All products were

determined by comparison with an internal standard. The ratio of AFMU to AFMU +

1X + LU was calculated for each subject to determine acetylator phenotype. A ratio of

less than 0.3 indicates a slow acetylator (Tang et al., 1983).

Figure 3: Measurement of suifarnethonazole, sulfamethoxazole-hydroxylamine and

acety l-sulfamethoxazdle in the urine by high performance liquid chromatography .

Measurement of blank urine (A), a blank wine sample spiked with SMX and SMX-HA

(B) and a sample urine from a subject treated with SMX (C). Measurements of

metabolite ratios were determined using peak height.

Figure 4: Measurement of caffeine metabolites, 5-acetylaminod-fomylamino-3-

methyluracil (AFMU), 1-methyluracil (1U) and 1-methylxanthine (1X) in urine by high

performance 1 iquid chromatography . Analysis of blank urine (A), blank urine spiked

with AFMU, 1U and 1X (B) and a sample urine from subject taking caffeine (C).

Measurements of metabolite ratios were determined using peak height.

3.5. Peripheral Blood Mononuclear Cell Isolation

All cell culture in this investigation was performed using peripheral blood

mononuclear cells (PBMCs). The cells were isolated from heparinized whole blood

from each of the treated and control subjects on each of the test days. Blood was

layered onto Ficoll gradient and spun in a Beckman bench top centrifuge for 30 minutes

at 1500 rpm. The PBMC layer was removed from the plasma-gradient interface and

washed 3 times by resuspension and resedimentation in HEPES buffer. The cell pellet

was resuspended in 5 mLs RPMI culture media with 10% FCS, 50 pM P-

rnercaptoethanol and 50 mM penicillin-streptomycin. PBMCs were counted and the

concentration adjusted with culture media to 106 cells per mL.

3.6. Peripheral Blood Moaonuclear Cell Proliferation Assay

The PBMC proliferation assay was carried out in 96 well microtitre plates.

Cells were added to control (unstimulated) and test (stimulated) wells at a tinal

concentration of 200,000 cells per well in culture media. Mitogens were added to test

we1 1s at final well concentrations of PHA 5 pgImL, PMA 1 ngImL and ionomyein 10"

M. The cells were stimulated with PHA/PMA or PMAhonomycin combinations. The

plates were then incubated for 72 hours at 37°C and 5% C4, followed by a 6 hour

pulse with tritiated thymidine at 1 pcilwell. Cells were harvested onto filter paper

disks and placed into scintillation vials. Scintillation cocktail was added to the vials

and the radioactivity in counts per minute of each sample determined using a Beckman

LS I701 Scintillation Counter. PBMC proliferation was determined as counts per

minute (CPM) of radioactivity and the Stimulation Index was determined as follows:

Stimulation Index = x 100% Control day CPM(stimulltc~) - CPM(backgmw)

3.7. Western Blot Analysis of Serum Protein

Serum samples were frozen at -20°C until analysis by Western Blot. Toral

serum protein was quantified using the Bio-Rad protein assay [based on dye-binding

procedure by Bradford (1976)l. Proteins were separated on the basis of size using

sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a Bio-Rad

miniprotein 11 gel apparatus. Serum samples or biotinylated molecular weight markers

were diluted in sample buffer containing 10% SDS and 2-mercaptoethanol (as a

reducing agent) and boiled for I0 minutes to denature proteins. Five microlitres of

sample (5 pg total protein) were loaded onto a 12.5% dismntinuous polyacrylamide

gel. Gels were run at 125V for 2 hours so that the 30 kDa molecular weight marker

reached the bottom of the gel. Gel separated proteins were then blotted onto

nitrocellulose membranes by wet transfer for 1 hour at 120V constant current in the

miniprotein 11 appmtlls. Nitrocellulose membranes were blocked overnight in tris

buffered saline with 0.5% Tween (TT'BS) containing 5% non-fat milk powder.

Primary rabbit anti-SMX-BSA antisera was diluted 150 in blocking solution with 0.1%

BSA and incubated overnight at 4OC. Membranes were washed twice with blocking

solution and incubated 1.5 hours in primary antibody solution. After primary antibody

033 labelling, membranes were washed twice for 10 minutes in 10 mL blocking solution

and then incubated for one hour in goat anti-rabbit IgG secondary a n t l i y conjugated .

to HRP, diluted 1:~00,000 in blocking solution. Membranes were then washed 5 times

in blocking solution. The secondary antibody solution also contained strepavidin-HRP

used to label the biotinylated molecular weight marken. ECL developing reagent was

added to the protein side of the nitroceUulose membranes for 1 minute and then the

excess poured off. The protein side of the membrane was then exposed to ECL-

hype- for 30 seconds in a dark room anci the film developed.

The anti-SMX-BSA antisera was obtained from Dr. R. G N C U of the

Southwestern Medical Center, Dallas, Texas and was raised in rabbits using the

following protocol. New Zealand white rabbits were injected intramdarly with 25

pg SMX-BSA emulsified in Hunter's TitreMax and boosted 6 weeks later. Rabbits

were bled one month after boosting and the serum samples analysed for the presence of

SMX-specific IgG antibodies by ELlSA inhibition (Meekins et ul., 1994).

3.8. Data Analysis

Sample size was calculated using the formulas given in Appendix 2. AU results

were log transformed and the geometric means generated to normalize the data.

Comparisons of the acetylation of SMX and the density of chemilluninescent labelled

protein between fast and slow acetylatoa were made using a Student's t-test and were

considered significant if p s 0.05. Comparisons of the levels of SMX-HA excreted

034 were performed using ANOVA followed by a post hoc student's t-test and were

considered significant if p a 0.05. Comparisons of PBMC proliferation were made

using ANOVA @ s 0.05) followed by a post hoc student's t-test and correlations

between PBMC proliferation and SMX-HA excretion were performed using Pearson

product moment coefficient using the SchoolStat program (Apple Computers,

California).

4.0 RESULTS

4.1. Subjects

Sixteen of the 17 subjects completed the trial without developing adverse

reactions (table 2). Two subjects complained of stomach upset and 2 reported urinary

urgency while on the cotrimoxazole dose. One subject with a long history of

sulfonamide use developed a €xed drug eruption on the left hand at day 8 of treatment.

The skin rash was noted upon subsequent SMX exposure in the same subject. One

subject experienced fever at day 7 and a skin rash at day 8. Treatment was withdrawn

from this subject at day 7 and antihistamine and ibuprofen were prescribed for

symptoms. The rash and swelling subsided by day 12. Blood and urine samples were

still taken on days 9 and L2 to complete the trial.

4.2. Caffeine Metabolism

Caffeine metabolites were andysed by HPLC as a marker for acetylator

phenotype. The ratio of caf5eine metabolites, AFMUl AFMU + 1U + 1X, for each

subject is indicated in table 3. A ratio greater than 0.3 indicates a fast acetylator while

a ratio of less than 0.3 indicates a slow acetylator phenotype (Tang et al., 1983). Five

of the subjects were determined to be fast acetylaton and 12 were determined to have

the slow phenotype. This value (70% slow acetylators) is slightly higher than expected

for the population (50 - 60 56).

Subject Sex Age Prior I I I I Exmure 1

2

6 7

Events during treatment period

13 14

oral contrace~tive, occasional as~ir in yes no

F M

F M

occasiod acetaminophen

-

24 24

F F

GI irritation while raking SMX, subsided after dav 10

26 25

slight fever at day 7, urticaria and swelling at day 8, SMX discontinued; antihistamine and ibuprofen given, rash gone at day 12 oral wntrace~tive yes

no

44

30

oral contraceptive Aplastic anemia as child, bone marrow transplant at 15 yrs; history of sulfonamide use. Developed localized rash on left hand at day 9 rash returned upon subsequent sulfonamide treatments Flu-like symptoms at day 6 gone at day 8. urinary frequency during SMX treatment

yes no oral contraceptives

Complained of urinary frequency during treatment oral co ntrace~ tives -- - -

multivitamin

Table 2: Characteristics of subjects participating in the full study of SMX metabolism.

The indications column describes medications the subjects took during SMX treatment

and documents any events that occurred during the course of the study. General side

effects occurred for 3 of the subjects. Subject 9 demonstrated a fixed drug eruption on

his left hand at the end of the treatment period. Subject 5 experienced what appeared

to be a Type I allergy to SMX on day 7 . SMX treatment was withdrawn at this point.

4.3. Sulfamethoxazole Metabolism

The production of SMX-HA relative to the major metabolites of SMX is

indicated in figure 6. SMX, SMX-HA and acetyl SMX were measurable in the urine at

days 3, 6 and 9 of treatment. SMX was also still measurable at day 12 and in most

subjects low levels of the metabolites were also present. The ratio of HA eliminated

overall for treated individuals was 3.5 * 2%. The hydroxylarnine metabolite ratio

(SMX-HA/ SMX + SMX-HA + acetyl-SMX) was significantly higher (p 2 0.05) for

fast acetylators than slow acetylators (figure 6). SMX-HA excretion was not

significantly different day to day within each acetylator group (p r 0.05). Acetylation

of SMX (table 3) (figure 5) was slightly higher for subjects with the fast phenotype, 82

& 5% of the metabolites excreted with a range from 76 to 821, than for slow

acetylators, 73 f 9 % of metabolites with a range from 56 to 89%. Acetylation of

SMX between the 2 phenotypes was not significantly different at the 5% level of

significance.

4.4. Peripheral Blood Mononuclear Cell Rolsteration

There were no significant changes in PBMC proliferation for the treated

subjects over the course of cotrimoxazole therapy (figure 7). The proliferation on

treatment days (days 3, 6. 9 and 12) was not significantly different from that of the 0

day control for subjects (p > 0.1). No significant differences in proliferation were

seen between fast and slow acetylator groups (p > 0.1). PBMC proliferation was also

not significantly (p > 0.05) different from that of control subjects who were not

1 Subject -

SMX- Acety lation

Acety lation Type

fast fast

-

fast

fast - - -

slow

slow - - -

slow slow slow slow slow slow slow slow stow

Table 3: Metabolism ratios for caffeine and sulfamethoxazole determined by HPLC

analysis of metabolites excreted in the urine. Subjects were asked to consume a

caffeinated beverage before urine samples were collected. Acety lator phenotype was

determined using the ratio of caffeine metabolites, AFMU I AFMU + 1U + 1X. fiom

urine samples taken on day 0 of the treatment period. A ratio greater than 0.3 indicates

a fast metaboliser. SMX acetylation is the fraction of SMX metabolites excreted as

acetyl-SMX averaged fiom days 3, 6 =d 9. SMX-HA levels are expressed as the ratio

of urinary SMX metabolites averaged from days 3. 6 and 9 for each subject.

Figure 5: Acetyiation of SMX for subjects participating in the study. The ratio of

acetyl-SMX to SMX metabolites was determined by HPLC analysis of urine samples.

Values are indicated as the average of samples collected on days 3, 6 and 9 of SMX

treatment for each subject. Subjects L to 5 are fast acetylators, as determined by

caffeine metabolism (table 3), while subjects 6 to 17 are slow acetylators.

Figure 6: Measurement of SMX-HA excreted in the urine. Metabolites are measured

as the ratio of SMX-HA compared with SMX-HA + SMX + acetyl-SMX over the

treatment period of 12 days. No significant differences were found within each

acetylator group over the treatment period. Fast acetylators (n=5) demonstrated

significantly higher SMX-HA excretion @ s 0.05) than slow acetylators (n= 12) on all

treatment days as determined by Student's t-test and ANOVA. Data is represented as

the mean for each subject group f standard error of the mean (s.e.m.).

fast acetylators

0 slow acetylators

043 treated with cotrimoxazole (figure 7). There was no correlation between the levels of

SMX-HA found in the urine and proliferation in vim for either fast or slow acetylators

as determined by Pearson product moment coefficient. Also, there were no individual

correlations between SMX-HA excretion and proliferation of lymphocytes as

determined by Pearson product moment coefficient. There was a great deal of

variability for proliferation between sample days in both control and test subjects. The

apparent low proiiferation seen on day 3 for the fast acetylaton (figure 7) can be

accounted for by 2 subjects whose proliferation was lower than baseline on this day and

higher than baseline on day 6. The other 3 fast acetylaton had proliferation that was

slightly higher than baseline.

4.5. Western Blot Analysis of Serum Proteins

Serum samples from each subject were assayed by Western blot analysis and

detected using a rabbit anti-SMX-BSA IgG antibody. Blots were developed usicg

chemiluminescence and representative samples are shown in figure 9. Blots were

scanned and the amount of protein labelled by ECL was quantified (figure 8) using the

Molecular Analyser program from Bio Rad. All subjects treated with couimoxazole

produced a 42 kDa protein that was labelled by the anti-SMX-BSA antisera. This

protein was present only on days 3, 6 and 9 of treatment not on days 0 or 12. In most

subjects the amount of protein declined at day 9. All subjects, including fast

acetylators (figure 9b), slow acetylators (figure 9a) and the individual who had the

reaction (figure 9c) produced the 42 kDa protein. No significant difference was found

044 between fast and slow acetylator groups (p>O.OS). This finding does not agree with

Meekins et al. (1995) who predicted the protein would only be found in subjects

sensitive to sulfonarnides. A second protein approximately 90 kDa in size and

demonstrating the same time panern as the 42 kDa protein was identified in 2 subjects.

This protein was not present on days 0 or 12 but only on days 3, 6 and 9 of treatment.

This may be a second, novel SMX-protein conjugate or possibly a muitisubunit version

of the 42 kDa protein. A third serum protein identified on the blots by the rabbit anti-

SMX-BSA antisera corresponds in size to human serum albumin (HSA). This is most

likely a nonspecific cross reactivity of the antisera due to the fact that it was raised

against bovine serum albumin. HSA is a large component of the serum and therefore

there is a relatively large amount of the protein on the blots. It should be noted that the

protein corresponding in size to HSA is labelled in all serum samples, not just the SMX

treatment days.

Figure 7: Proliferation of peripheral blood mononuclear cells (PBMCs) stimulated

using PMAIPHA from controls (n = 5) and treated subjects who are fast (n = 5 ) or

slow acetylaton (n = 12). The stimulation index (percent of baseline), calculated

using the formula below, was recorded for each subject group over the treatment period

of 12 days. Data is represented as the mean of each subject group f standard error of

the mean (s.e.m.). There were no significant changes in proliferation over the

treatment period for test groups. No significant differences in proliferation were seen

between fast and slow acetylator groups (p > 0.1). PBMC proliferation was also not

significantly (p > 0.05) different from that of control subjects who were not treated

with SMX.

Stimulation Index = v CP- - CPM x 100% Control day CPM(stimulared) - CPM@ackground)

Figure 8: Relative density of a 42 kDa protein isolated from serum by Western blot

analysis and labelled using enhanced chemiluminescence. Serum samples were

quantitated such that 5 pg total protein were analysed on each sample day. Western

blots were scanned and labelled protein analysed by densitomeny using the Molecular

Analyser program fiom Bio Rad. Densities for each treatment day (days 3, 6, 9 and

12) were compared with the day 0 control sample and are represented as the increase in

density relative to day 0. Serum was analysed for both fast and slow acetylators treated

with cotrimoxazole. There was no significant difference in densities between the fast

and siow acetylator groups. The density of labelled protein at day 9 (**) was

significantly lower than that measured on day 6 for both subject groups. Day 9 values

were only significantly different fiom day 3 values in the siow acetylator group. Data

is represented as the average for each subject group f the standard error of the mean.

fast acetylators H slow acetylators

Figure 9: Western blot analysis of serum proteins over the SMX treatment period.

Blots indicate a 42 kDa protein labelled by rabbit antiSMX-BSA antibody in the serum

of a slow acetylator (a), a fast acetylator (b) and a subject who had a reaction to SMX

therapy (c). Blot (b) has a positive (+) control lane which contains the serum of an

individual known to produce the 42 kDa protein (gift of Dr. R.S. Gnrchalla,

Southwestern Medical Center, Dallas, Texas). The negative (-) control lane contains

untreated serum from the same individual. Blots b and c have sample days 3, 6, 9 and

12 represented in duplicate.

+ + 0 3 6 9 control day

5.0 DISCUSSION

5.1. Sulfatnethoxazok Adverse Dnrg Reactions

Hypersensitivity reactions to sulfonamides are complex events and are the most

serious adverse drug reactions accompanying sul fonamide therapy. These idiosyncratic

reactions are primarily characterized by fever, skin rash, erythema multiforme or toxic

epidermal necrolysis that develops I to 2 weeks after beginning therapy. In a few cases

organ involvement such as hepatitis, nephritis or myocarditis is a complication.

S u l fonam ide hypersensitivity reactions are a significant source of morbidity for SMX

therapy especially upon rechallenge with the drug and can be life threatening (Rieder et

al., 1988; Rieder et al., 1989). Despite nearly 50 years of experience with sulfonamide

therapy the pathogenesis of hypersensitivity reactions remains obscure. Popular theory

to date impiicates metabolism of the sulfonamide as the initiating event generating a

toxic, reactive intermediate (Cribb et a', I991 ; Rieder, 1994; Shear el al., 1986).

Sulfonamides are aromatic amines metabolized primarily by NAT enzymes but a small

amount of the dose can be metabolized by CYP450 enzymes to a reactive

hydroxylarnine intermediate. The importance of reactive drug metabolites in adverse

drug reactions has become evident for many compounds including aromatic

anticonvulsants, halothane and cefaclor (Rieder, 1994; Kearns et aL , 1994).

Metabolites of such compounds, including SMX, can subsequently be detoxified, cause

direct cytotoxicity, or by formation of a hapten, elicit an immune response (Cribb et

052 a , 199 1 ; Cribb et ai., 1995; Meekins et a[-, 1994). Sulfamethoxazole-hydroxylamine

is an electrophilic intermediate generated by oxidation of the drug by cytochrome P4SO

enzymes. SMX-HA can be produced enymaticaily and has been demonstrated to

covalently bind proteins both in v i m and in vivb (Rieder et a[., 1995; Cribb et ai..

1995; Meekins et aL, 1994). The time course for the development of SMX

hypersensitive reactions indicates that a toxic product must accumulate or the body

requires time to develop a response (Rieder. 1992; Rieder, 1994). The build up of

reactive metabolite may also account for the continuation of sulfonarnide adverse

reaction despite the removal of drug therapy. The accumulation of a reactive

metabolite such as SMX-HA could be responsible for the delayed time course of

sulfonamide reaction. The HA metabolite may also be respow ible for the development

of an immune response. Covalent binding of HA to cellular macromolecules could

result in an inflammatory response due to cell death as well as eliciting an immune

response against proteindrug conjugates which could act as neoantigens (Rieder. L 992:

Park and Kineringham, 1990) The present study was carried out to evaluate the

metabolism of SMX in vivo and to determine what effects differences in metabolism

may have on toxicity of SMX and influencing the generation of an immune response.

5.2. SuiTiunethoxazole Immune Eff-

Sulfamethoxazole hydroxylamine either synthetic or generated from SMX using

liver microsomes is toxic to lymphocytes in vitro (Spielberg, 1984; Rieder et al.,

1 98 8). S MX reactive metabolites are capable of a1 tering cell function without changes

033 in cell viability (Leeder et a[-. 199 1; Leeder et ui., 199 1). SMX-HA also demonstrates

dose-dependent suppression of mitogen induced proliferation of lymphocytes in Mtro

(Rider et al., 1992). These toxic effects. caused by exposure of lymphocytes to the

hydroxylarnine of sulfonamides, are exaggerated in patients who experience

hypersensitivity reactions to sulfonamides (Rieder et al., 1989; Shear et aL, 1986).

Based on this dam we predicted that similar changes in lymphocyte function would be

seen in PBMCs taken €tom subjects treated orally with SMX. A preliminary study was

undertaken to test this hypothesis in which five individuals were treated with SMX for

9 days (appendix 1). holiferation assiys were performed on PBMCs taken from the

subjects on days 3, 6 and 9 of treatment. Individual correlations were found between

the levels of SMX-HA excreted in the urine and suppression of proliferation (appendix

1, figure 1). That is there was suppression seen in subjects with higher levels of SMX-

HA excretion. Increased proliferation at day 9 for this subject was probably due to

immune activation during the anaphylactic response. As well, the subject who

experienced a reaction and had to be withdrawn from the study had significantly higher

levels of HA excretion. These findings prompted the design of a more complete study

with a larger sample size, control subjects, control sample days and integration of

acetylator phenotyping to verify the preliminary results.

Having carried out the larger study (n = 17) we found no correlation between

the relative amounts of SMX-HA excreted and the levels of PBMC proliferation in

vitro. Although day to day values varied for some subjects there was no significant

054 change or trend in proliferation over the course of treatment for either slow or fast

acetylatoa. The day to day changes in proliferation were larger for the fast acetylation

group on average but this is most likely due to the smaller sample size and results from

one individual that were extremely low on days 3 and 9 and high on days 6 and 12. It

is difficult to determine the effects of SMX-HA produced in vivo on immune function

using this in viho PBMC proliferation assay. There was a great deal of variability in

PBMC proliferation day to day and this variability was seen for both treatment and

control groups. Perhaps there are too many influences in vivo on lymphocyte function

to determine the specific effects of SMX metabolism. The effects of SMX-HA on

lymphocyte function has been determined in wino but not for cells at more than one

time point (Rieder et ui., f 992; Leeder et al'. , 199 1 ; Leeder et a', 199 1). It would be

interesting to look at PBMC proliferation in response to in vino HA challenge for one

subject at more than one time point. It is likely that there are too many facton

affecting immune function day to day in an individual to be able to draw more global

conclusions from a proliferation test such as this. One function test for PBMC's from

treated patients which may be more specific could be taken from Henl et al. (1995)

who demonstrated SMX metabolites, generated by a microsomal system, induced

proliferation of CD8+ lymphocytes taken from the skin lesion of a patient experiencing

sulfonarnide hypersensitivity. We know from previous in vifro tests that low levels of

SMX-HA can affect the function of PBMCs without killing the cells (Rieder et al.,

1992; Leeder et al.. 199 1: Leeder et al., 1991). Perhaps exposure to SMX-HA in vzno

may induce T-cells from SMX sensitized individuals to proliferate.

5.3. In Vivo MetaboIism of Sulfamethoxazole

As expected from previous studies SMX-HA is produced in vivo at levels that

are easily measured in the urine of treated subjects. The levels of HA eliminated for

this group of individuals (3.5 f 2%) were comparable to that of other SMX

metabolism studies (Cribb and Spielberg, 1992; Lee et al., 1994; van der Ven et a',

1994). Acetylation of SMX was higher for the fast acetylators, although the values

were not significantly different between acetylation groups (p > 0.05). A large

variabii ity in SMX metabolism was observed within the slow acetylator group (33 %)

and a great deal of overlap occurred between the two phenotypes (table 3). Van der

Ven et ul. (1994) also demonstrated slightly higher acetylation of SMX for the fast

phenotype but did not find a significant difference. Sulfamethoxazole, unlike

sulfonamides such as sulfadiazine, is thought to be rnonomorphically acetylated.

Although both NATl and NAT2 metabolize SMX, NATl has a higher affinity for the

drug and acetylation is therefore considered to be monomorphic (Cribb et uL. 1993).

NAT;! activity is thought to provide a secondary pathway which. in fast metabolizen,

can compete with the oxidative metabolism that generates SMX reactive intermediates.

This activity may account for the small differences obsewd in SMX acetylation (Cribb

et al., 1993). There is a predominance of slow acetylaton among sulfonarnide

hypersensitive individuals. The lack of NAT2 activity may result in a greater capacity

056 to produce SMX-HA and increase the risk of adverse reaction (Cribb et al.. 1993).

Surprisingly though, fast acetylators excreted significantly more SMX-HA than slow

acetylators. This apparent difference in HA production may be due to a number of

factors including formation, detoxification or protein binding of SMX-HA,

Sulfamethoxazole can undergo oxidative metabolism by cytochrome P450

enzymes to the hydroxylamine intermediate (Cribb et of-, 1995; Rieder et a', 1988).

CY P 1 A2 functions in the metabolism of a great deal of compounds that pass through

the 1 iver . Kadlubar ( 1994) described 3 phenotypes for 1 A2 activity, slow, intermediate

and fast metabolisers of caffeine. Such a polymorphism could possibly account for

differences in oxidation of SMX. Cytochrome P450 2C9 is the major protein

component of the 2C subfamily (Goldstein and de Morak, 1994). Sulfamethoxazole-

hydroxylamine is produced specifically by the 2C9 enzyme (Cribb et a/., 1995).

Cytochrorne P450 2C9 activity can be induced which may to some extent, account for

differences in HA production. Allelic variants of the CYP2C9 enzyme have been

described (Goldstein and de Morais, 1994) and a polymorphism for the metabolism of

tolbutamide, another 2C9 substrate, also exists (Cribb et al., 1995; Page et d. 1991).

It has yet to be determined how these variants may contribute to differential metabolism

of S MX and perhaps idiosyncratic hypersensitivity .

Detoxification of the electrophil ic hydroxy lamine of SMX may also affect

clearance of the metabolite and be an imponant &tor in the development of

hypersensitivity. Cytochrome P450 isoymes are not only important in the formation

057 of SMX-HA but also in its detoxification. Human CYP3A4 is capable of reducing

SMX-HA back to the parent drug as is NADH-hydroxylamine r e d u c e (Cribb et a/.,

1995). A balance of enzyme function between such enzymes as CYP2C9 and 3A4 may

be important in determining the amount of reactive metabolite that is available to cause

toxicity. Conjugation of the HA intermediate is also an important detoxification

mechanism (Cribb et d, 1991; Shear and Spielberg, 1985). Compounds such as GSH

and other thiols bind elecmphilic metabolites, reducing their toxic effects and targeting

them for elimination (Cribb et aL, 1991: Rider et aL, 1988; Shear and Spielberg,

1985). Differences in the ability to detoxify SMX-HA are thought to be responsible for

the in vino toxicity observed in lymphocytes fiom hypersensitive individuals. Shear

and Spielberg (1985) demonstrated that cells deficient in GSH synthetase were more

susceptible to HA toxicity than control cells. They also showed that covalent binding

of SMX intermediates to the microsomai proteins used in vitro to generate SMX

metabolites and cytotoxicity were reduced in the presence of GSH and N-

acetylcysteine. These results are similar to the metabolism of acetaminophen in which

GS H protects cells fiom and detoxifies reactive acetaminophen metabolites (Cribb et

al., 1991). Unlike acetaminophen though, GSH does not seem to form stable adducts

with SMX-HA. Rather GSH prevents the spontaneous oxidation of HA to the more

reactive nitroso-SMX thereby protecting the cell from covalent binding and toxicity

(Cribb etal., 1991: van der Ven etal., 1995). GSH also functions in returning nitroso-

SMX to the reduced HA (Cribb et a/., 1991). Since GSH does not ultimately detoxify

058 SMX-HA it plays an important cellular protective role rather than affecting elimination

of SMX metabolites. This observation may explain why SMX-HA is available outside

of the liver to cause extra hepatic toxicity. Once outside the liver, SMX-HA would

have the opportunity for further oxidation and be available to bind cellular molecules as

well as extracellular structures such as serum proteins. Van de Ven et d (1995) found

that HIV-seropositive patients excreted relatively less HA than normals but the

metabolism. as measured by acetyl-SMX. SMX-glucuronide, and other SMX

metabolites was not different between the 2 groups. They hypothesized that HIV-

seropositive patients had defective detoxification by GSH, leading to the generation of

more reactive SMX species which would bind cellular molecules, resulting in decreased

urinary recovery. Such defective detoxification might account for the increased

incidence of hypersensitivity reactions in AIDS patients. Similar variations in

detoxification of SMX reactive metabolites may also occur in some hypersensitive

individuals. Glutathione metabolism may act in balance with other enzyme systems in

the body which generate or detoxify SMX reactive metabolites and a change in this

balance may influence susceptibility to hypersensitivities to SMX.

The data in figure 6 seems to imply an increase in SMX-HA excretion on day

12 of the treatment period. It should be kept in mind when interpreting this data that

the subjects were no longer taking the cotrimoxazole dose and that there is a large

decrease in the amounts of both SMX and SMX-metabolites excreted. The actuai

amount of HA excreted is lower on day 12 than the other treatment days but its ratio

compared to SMX and acetyl-SMX increases. There may be several reasons for this

059 apparent increase in ratio. SMX and its metabolites demonstrate separate elimination

kinetics. In the kidneys. SMX undergoes net passive resorption whereas acetyl-SMX

has a net active excretion. SMX-HA elimination is not affected by urine pH and

undergoes limited passive resorption from the renal ~bules (van der Ven et a[. , MM).

There may be a limited capacity for SMX reabsorption in the kidneys and subtle

differences in SMX elimination may not be appreciated at the high doses of drug taken

during the treatment period. Resorbed SMX would undergo funher metabolism and be

excreted as metabolites. While the ratio of HA produced in vivo may not change at day

12. SMX resorption appear to increase the relative amounts of metabolites excreted in

the urine. A second explanation for the apparent increase in HA metabolite could be

more effective detoxification by GSH at the lower in vivo levels of HA. More effective

detoxification would result in decrease oxidation of HA to nitroso-SMX. This would

result in decreased protein binding by the reactive nitroso and a higher urinary recovery

of SMX-HA (Van der Ven et al., 1995). SMX-HA and nitroso-SMX conjugated with

serum and cell proteins may also be released as the proteins are degraded. Since serum

proteins undergo constant turnover metabolite release would occur throughout the SMX

treatment but only become observable in the urine after the SMX dose is halted and the

levels of SMX and acetyl-SMX decline.

5.4. In Vivo Immune Effects of Sulfamethorvazole Metabolism

Conjugation of reactive metabolites with serum and cellular proteins is a

potential mechanism for development of adverse reactions to sulfonarnides. S MX-

060 haptenated proteins could act as immunogenic complexes leading to activation of both

cellular and humoral responses. Many drugs ate metabolized to potentially reactive

intermediates capable of binding ro cellular macromolecules and immune involvement

is indicated in the adverse reactions to many types of drugs (Park and Kitteringham,

1990; Rieder. 1992; Leeder et a', 1988). Conjugation of the drug to proteins in vivo

could provoke an immune response against the hapten bound to an endogenous

molecule such as a serum or cellular protein. Such a response could be expected to

propagate an adverse drug reaction despite discontinuation of the drug as is the case

with delayed onset hypersensitivities (Rieder, 1992). The reanion would continue in

this case due to immunization against the self-molecule. The type of reaction

manifested during hypersensitivity depends on the immune response that is generated.

Production of IgE antibodies to a drug hapten can activate mast cells to release immune

med iaton resulting in urticaria, ang ioedema and bronchospasm (Park and

Kitteringham. 1990; Rieder. 1992). An IgG response mounted against a drug can

result in organ and tissue-specific immune responses such as haemolysis or hepatitis

(Rieder, 1992; Rieder, 1994). Binding of IgG to a cell which has SMX-HA conjugated

to it would attract immune cells that could cause lysis of the IgG bound cell.

Activation of a cell mediated response can also occur. manifesting as fuced drug

eruptions and skin cash (Park and Kitteringham, 1990). Many of the ideas on how

S MX metabolism could be involved in the development of an adverse immune reaction

have come from the study of adverse reactions to other drugs. The peniciiloyl

determinant of penicillin is a reactive molecule that is known to form haptens which are

061 capable of eliciting the common allergies associated with therapy (Park and

Kitteringham, 1990; Christie et of-, 1988). Anaphylactic (IgE mediated) reactions

make up only a f k t i o n of the reactions to penicillin (Park and Kitteringham, 1990;

Eflmeyer, 198 1). High dose therapy is also associated with hemolytic anemia and cell

mediated immunity in the form of drug eruptions (Park and Kitteringham, 1990).

Halothane-induced hepatitis is a rare event that occurs associated with the metabolism

of' the anaesthetic (Rieder, 1992). Sera from patients who develop hepatitis contain

antibodies against hepatic proteins bound to halotham derived determinants. These

antibodies are not produced in non-sensitive halothane treated patients (Kenna et ai-,

1988). Procainamide therapy is associated with the development of drug induced lupus

(Rieder, LW; Uetrecht, l988). Like sulfonamides, procainamide is metabolized by

cytochrome P450 enzymes to a hydroxylamine intermediate which can undergo

spontaneous oxidation to a nitroso derivative, capable of forming covalent interactions

with proteins (Uetrecht, 1985; Uetrecht, 1988). Formation of drug haptens by the

reactive metabolites of sulfonamides is thought to be an important factor in the

development of immune reactions to this class of drugs (Lee et a/., 1994; Rieder.

1992). SMX-specific antibodies have been identified in the sera of treated individuals

indicating that an immunogenic SMX complex is formed (Carrington et al., 1987;

Harle et ui., 1988). Lymphocytes taken from the skin at the site of a futed drug

eruption can be activated in response to sulfonarnide exposure (Hertl et ai., 1995; Hertl

and Merk, 1995). Covalent binding of sulfonamide metabolites to proteins and cells

062 has been demonstrated in vim (Rieder et ai., 1988; Shear and Spielberg, 1985) but

until recently SMX-haptens produced in vivo remained elusive. Using anti-SMX-BSA

IgG. Meekins et al. identified a protein in the sera of SMX treated individuals and

postulated that this was a potential hapten that could elicit an immune response in

sensitive individuals (Meekins et af., 19%). Using the same antibody we analysed the

serum of the individuals who participated in this study for SMX-protein conjugates.

All subjects treated with SMX had a 42 kDa protein in theu sera that was not found on

untreated days or in untreated controls. Contrary to the findings of Meekins. this

protein was produced in both sensitive (figure 9) and insensitive subjects and was not

identified as a predisposing factor to hypersensitivity to SMX. No significant

difference in the production of SMX occurred between fast and slow acecylaton. The

protein was apparent in the serum on days 3 and 6 of treatment but was greatly reduced

or not present by day 9 even though the subjects were still on the SMX dose at this

point (figure 8). The protein was not found in any subjects on day 12 after treatment

was stopped. Since the subjects were still taking the wtrimoxazole dose on day 9, they

would be expected to still produce the protein at this sample day. The apparent

decrease in the amount of protein observed on day 9 may be due to breakdown of the

protein in the blood although turnover of plasma proteins would occur throughout

treatment and should not affect the production of haptens. The protein could be taken

out of circulation by an antibody evolved against it. We used denaturing SDS-PAGE

to separate serum proteins. This procedure should remove the 42 kDa protein from a

063 bound antibody. SDS-PAGE denatures proteins so that only proteins or protein

subunits associated by covalent bonds remain intact. A protein bound to an antibody

should still be identifiable by Western blot analysis of the serum. More likely. the

protein may be removed from the circulation by uptake by phagocytic cells such as

macrophages. Whether or not this SMX-protein conjugate acts as an antigen capable of

eliciting an immune response was not determined in this study. One of the subjects

developed urticaria after 7 days of treatment. It is likely that her serum would contain

SMX-specific IgE antibodies as her reaction was anaphylactic (Type 1) rather than a

delayed onset hypersensitivity. If this 42 kDa protein is indeed a potential antigen the

type of reaction elicited would depend on the type of antibody generated, whether or

not there was activation of a cellular immune response, the amount of protein produced

and the origin of the protein. Uptake of the protein by phagocytic cells may result in

its presentation to lymphocytes which could elicit an immune response i.e. activated T-

cells in a delayed hypersensitivity reaction. The nature of the 42 kDa protein which is

covalently bound to the SMX metabolite has yet to be identified. A protein of hepatic

origin may account for the liver toxicity which is seen in severe hypersensitivity

reactions (Rider, 1992). Since HA is available systemically and this 42 kDa protein is

readily detectable in the serum, it is possible that it is a small serum protein. We

incubated an untreated serum sample with increasing concentrations of SMX-HA and

found several bands labelled with the anti-SMX-BSA antibody including one that was

about 42 kDa in size. This pattern of binding was expected due to the reactive nature

of SMX-HA and its nitroso derivative which would form in v i m (Cribb et 01.. 1991;

064 Shear and Spielberg, 1985). Such binding is also in keeping with the results of studies

which examined in vie0 exposure of cells to SMX and metabolites (Cribb et al.. 199 1 ;

Shear and Spielberg, 1983. More that one drug-protein conjugate may also be formed

in vivo. In the serum samples €torn two subjects a protein approximately 90 kDa in

size was labelled that was present on treatment days but not in controls. One of the

subjects who produced this larger protein was also the individual who had an

anaphylactic reaction to SMX (subject 5). This 90 kDa protein may be a separate

protein or possibly a muitisubunit version of the 42 kDa protein. It is also possible that

other drug haptens are formed in individuals and that these haptens have not yet been

identified. Without a proper detoxification system to prevent nitroso formation, SMX

metabolites may bind to serum proteins once leaving the liver.

5.5. Future directions

Even though this study has demonstrated the generation of SMX haptens in vivo

and differences in the formation of a reactive SMX metabolite between acetylator

groups, many questions need to be answered before the mechanism behind the

incidence of sulfonamide adverse drug reactions can be elucidated. Acetylation of

SMX does not appear to account for differences in the production of HA metabolite.

Aithough the slow acetylator phenotype is considered to be a risk factor in the

development of sulfonamide hypersensitivities, it may not directly affect the production

of reactive metabolites such as SMX-HA. Other enzymes involved in the production

and detoxification of SMX-HA were not considered in this study. Analysis of enzymes

065 such as the cytochrome P450 isozymes is important to determine whether other

polymorphisms or variations exist for the metabolism of sulfonamides. Recently

studies have been undertaken to investigate the effects of inhibiting the formation of

SMX-HA during oreatment by co-administration of drugs which compete for the P450

enzymes involved in sulfonamide oxidation (Mitra et ai., 1996). The importance of

detoxification of sul fonamide reactive metabolites has been demonstrated in vitro and

may be a key factor in hypersensitivity reactions. Characterization of the role of GSH

in vivo is necessary to firrther the understanding of sulfonamide toxicity. Perhaps most

importantly studies need to focus not on a single variation in metabolism but on the

balance of enzymes that ultimately influence the reactive metabolites produced.

The largest puzzle in understanding sulfonamide hypersensitivity is the immune

mechanisms involved in these reactions. Although these reactions possess many

characteristics of the various hypersensitivities described by Gell and Coombs (1%8),

they defy classification into any single category thereby creating problems for clinical

diagnosis and treatment. The discovery of a SMX-specific haptea by Meekins et ai. in

1994 was a significant finding providing support for many theories that implicate

' neoantigens ' in the development of sulfonamide hypersensitivities. The

characterization of this hapten, whether it is a protein of cellular or plasma origin may

help to explain the type of immune responses generated against it. The presence of

sulfonamide-specific IgE, IgG and IgM antibodies was not examined in this study but

has been demonstrated in both sensitive and insensitive individuals treated with

066 sulfonarnides (Daftarian et 41.. 1995)- The specificity of these anti i ies and their

involvent in the pathogenesis of adverse reactions has not been determined. Immune

responses to reactive metabolite formation do not necessarily depend on the formation

of an antibody to a hapten. Whether or not SMX-HA or nitroso-SMX cause direct

cellular damage in viva has not been determined nor has the histological evidence for

such toxicity ken demonstrated. The generation of a SMX-hapten may also result in

an activated T-cell response (Rieder, 1992; Mami-HelIweg et a'. 1995). In v i m

testing of lymphocyte function in response to sulfoddes, such as assays developed

by Henl and Merk (1995), for patients suffering fkom hypersensitivity reactions may

help to determine the role of an activated cell response in the generation of these

reactions (Mauri-Hellweg et a%, 1995; Hertl axid Merk, 1995).

5.6. Conclusions

In this study we have shown the differential excretion of a SMX reactive

metabolite (SMX-HA) from subjects treated with SMX. We have also demonstrated

that differential excretion is possible without significant differences in the acetylation of

SMX. Although the slow acetylation phenotype is a risk factor for delayed onset

hypersensitivity, the generation of adverse reactions to sulfamethoxazole is too diverse

and complex to be explained by acetylation phenotype alone. What part acetylation

plays in affecting differential metabolism of donamides has not been established.

These differences may be due to other enzymes involved in the production of SMX-HA

067 such as cytochrome P450 2C9 or more likely due to differences in the detoxification of

reactive metabolites.

We demonstrated in this study that SMX-specific haptens are indeed formed in

vivo during SMX therapy and that these haptens disappear from the circulation with

time. No differences in the formation of SMX-protein conjugates between fast and

slow acetylamrs were obsewed in this study. Whether or not an immune response was

raised against SMX, such as the generation of SMX-specific antibodies, either IgE, IgG

or IgM was not determined. This is the fust demonstration to our knowledge of the

formation and disappearance of a drug hapten over a treatment period. The removal of

this protein from the circulation may indicate its recognition by the immune system and

its potential for eliciting an immune response in susceptible individuals. The deciding

factor for an individual to have an anaphylactic, IgG mediated, immune cell activation

or a combination of reactions to SMX is unknown.

At though S MX metabolites are capable of causing dose-dependent suppression

of lymphocytes when exposed in vitm, this effect was not observed for lymphocytes

taken from subjects treated with SMX in vivo. It is likely that there are too many

factors affecting immune cell function in vivo to be able to extrapolate the findings of a

general lymphocyte function test. Perhaps a SMX-specific test, as described by Hen1

et al. (1995) would be more useful for fume in vivo studies

This work supports the hypothesis that differential metabolism occurs for

sulfonamides and may be a factor in the development of hypersensitivity adverse drug

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APPENDIX 1

In Vivo Metabolirm of Sutfrmethosamk; A Preliminary Study

A preliminary study was performed in order to examine the metabolism of SMX over the course of a treatment period and the possible immune effmts of differences in metabolism. By carrying out this preliminary work we hoped to identify correlations between the metabolism of sulfonamides, in particular sulfmethoxazole, and effect. on immune cell function. We hypothesized that increases in the production of SMX-HA would have suppressive effects on the lymphocytes as was seen in previous SMX studies (Rieder et a', 1992)

Methods

Five normal healthy volunteers, 4 males and 1 female, ranging in age from 24 to 35 participated in the preliminary study. These subjects were given cotrimoxazole (800 mg sulfarnethoxamle, 160 mg mmethoprim) twice daily for 9 days. Blood and urine samples were collected after 3, 6 and 9 days of study. PBMC's were isolated from whole blood and used in proliferation assays. Urine samples were frozen at -20°C until analysis by HPLC for SMX and metabolites. Informed consent was obtained from all subjects before beginning the study. HPLC analysis of the urine samples for SMX, SMX-HA and acetyLSMX content were carried out using the methods descnied in Cnbb et al. (1992). Excretion of SMX-HA detemined as the ratio of urinary SMX metabolites. Lymphocyte proliferation assays were carried out using the methods descn'bed by Rieder et UL (1992). Rolifiion was measwed as counts per minute radioactivity incorporated into growing cells.

Results

Four of the 5 subject completed the trial and one was withdrawn at day 7 due to high fever and skin rash. The skin rash was unicarial in nature. A blood, but not a urine sample, was obtained from this patient on day 9. Individual correlations were found between the levels of HA excreted in the urine and changes in proliferation during treatment. Two subjects showed no change in proliferation over the study period and excreted lower levels of SMX-HA throughout the treatment period. Two subjects excreted increasing levels of SMX-HA from day 3 to day 9 and had a decrease in PBMC proliferation over this period (figure 1). The final subject, who was withdrawn at day 7, excreted significantly higher levels of SMX-HA by day 6 and had large increases in proliferation by days 6 and 9. The activation of PBMC proliferation in this case may be due to immune activation during the development of the sulfonamide reaction.

Conclusions

It was concluded from this preliminary study that increases in the production of SMX-HA in vivo are carrelated with the suppression of mitogen induced proliferation of lymphocytes in vim. These findings also support theories that increased production of sulfonamide reactive metabolites may be responsible for hypersensitivities to sulfonarnides.

Figure 1: Proliferation of PBMCs and urinary SMX-HA levels for 5 subjects participating in a preliminary study. Plots indicate proliferation of PBMC's (bars) as counts per minute (cpm) radiactivity in response to PMA/PHA and urinary SMX-HA (symbols) measured by HPLC over the 9 day treatment period. Plot (a) represents subjects (n = 2) who had decreases in p r o ~ o n and hcreases in urinrry SMX-HA over the treatment period. Two subjects had no chmge m p r o ~ o n and excreted low levels of SMX-HA m the urine 0). One suject (c) experienced an allergic reaction while on the cotrimoxamle dose and had very high leveis of urhury HA The luge increase in proliferation seen on day 9 for this subject is probably due to immme cell activation during the donatm'de reaction.

DAY

APPENDIX 2

Sample Size Calculation

Sample size calculation for the study ' In Vivo Metabolism and Immune E f f i s of Suifhethoxazole and EQdroxylamine Metabolite"was pertonaed using the following equation taken fiom Dcmu, A (1984) Approaches to srmpk sip calculation in the design of clinical nirls - a review. StHcs in Medicine 3: 199-217. Alpha and beta error were determined using the sample size calculation table fkom this same reference.

where; n = sample size = a aror (probability of Wly decking treatment difkence)

q, = f3 aror (probability of-iy decking no treatment difkence) s = standard deviation (estimated percentage based on pilot study) D = difkence between control and test groups that is clinically important to

detect

A difference between control and test groups of 15% was anticipated as clinically important and a standard deviation of 12% was estimated for this study. The aim was to achieve the estimated difference between control and test groups at 95% power with a two-tailed test at the 5% level of signZcance. Thus 4, = 1.96, g = 1.64, s = 12 and D = 15. Substituting these values into the above form&,

Therefore L7 subjects were chosen to participate in the above mentioned study.

A post hoc power calcuIation was performed following completion of the study to determine the validity ofthe study.

let z, =a , d

using this value for 4 in the sample size calculation,

The value of 5.3 17 indicates a weak statistical power. Therefore the ability to make a conclusion from this study is weak

APPENDIX 3

Lcttrcr of Information

Re: In vivo Metabolism and Immune E m s of Sdfbethoxazole and Hydroqhmine Metabolites

Investigators: J k Michael J. Rieder Michelle M Aarts

Place: Robarts Research b t h t e LOO Perth Dr., Loadon 663-5777 e x 4209

You are being asked to participate m a study that will examine the metabolism and imnnme effkcts of Eulfirmeth0~~~)1e. Suhnetho~zo1e is and antimicrobid drug currently used to combat a variety of ~ O I ~ S I Some individuals may experience side eBbs such as gastric upset, loss of appetite, fCVg and skin rash. A smrll percentage of the population is anergic to donamide dnrgs and my side effects should be reported to the researchers as soon as possible. Any adverse reactions will be assessed and we may ask you to withdraw fiom the study.

In the course of the study you win be asked to take the medication Bactrim (cotrimoxazole) (thethoprim 160 mg and submethowole 800 mg) twice daily for 9 days. A one ounce blood sample will be drawn between 8 am and 10 am the morning before you begin taking the drug. One ounce blood samples win Plso be taken between 8 am and 10 am on days 3,6 ,9 and 12 a f k beginning treatment. Each visit should take approximately 15 minutes. Some volunteers may experience bruising due to drawing blood White blood cells win be isolated fiom the blood samples and used m cefl cuhure.

Urine samples will be collected the same morning as the blood srmples (5 samples in total). Urine samples will be used to determine sulfunerhoxamle, sdhnethoxazole hydroxyIamiae and d i e levels. CafEeine Levels win be used to determine your metabolism type. You will be asked to drink one cup of coffee, 2 cups oftea or 2 350 mL cans of a caffkhated soft drink before the &st urine sample is taken.

Codidenti.lity of the participants will be protected and in my publications the participants wiU not be identified by name.

You will be compensated $50.00 for your time and inconvenience. This amount will be pro-rated if you withdraw eom the study. Participation m the study is voluntary. You may refuse to participate or withdraw Born the study at any t h e without any effed on your UWO employmeat or academic standing.

Consent Form

In Vivo M e t a b o h md Innnme EiExts ofSulfmwthoxazo1e pad Hydroxylamine Metabolite

I have read the accotupmy~g letter of infodcm, hnve had the name ofthe study explained to me, and I agree to participate.

All questions have been answered to my satisfkction.

- * - - - - L I I I I I I I o I W I o o - - I - - I H N - m H -

Witness' Signature

~ ~ ~ ~ ~ ~ o ~ - m o o ~ o ~ ~ ~ ~ ~ ~ m ~ ~ ~ ~ ~ ~ ~ ~ ~ o - n - -

Date