SULFONAMIDE-SPECIFIC HAPTENS DURING SULFONAMIDE THERAPY

Daniel B. Schiedel Graduate Program in Pharmacoiogy and Toxico lo~

Submitted in partial f u I f i e n t of the requirements for the degree of Master of Science

Faculty of Graduate Science The University of Western Ontario

London, Ontario NOV 1998

O Copyright by Daniel Schiedel 1998

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ABSTRACT

The mechanisms underlying idiosyncratic hypersensitivïty reactions to sulfonamide

therapy are not clearly understood. hiblished studies suggest that covalent binding of

s~dfamethoxazole (SMX) to plasma proteins may be involved in the pathogenesis of these

reactions. After raishg a rabbit polyclonal antibody to SMX-keyhole Limpet hemocyanin

(SMX-KLH), we used Western blot techniques to investigate the ability of SMX and its

metabolites to conjugate with semm protein(s) in vitro- In vivo studies were also

conducted, and senim £rom 11 HIV patients undergoing desensihtion with SMX therapy

was assessed for SMX-haptenated proteins. In addition. 15 normal healthy subjects were

ueated with oral SMX (cotrimoxazole) for 10 days and blood was collected on days 0,3,

6,9 and 12 of therapy, and semm was examined for the presence of SMX-haptenated

pro teins. Human serum albumin and a 40 kDa protein identified as aIp ha- 1 acid

glycoprotein ( A M ) were detected in SMX-hydroxlarnine (SMX-HA) treated senun. In

vivo analys is of plasma samples revealed that none of the I 1 HN patients reçeiving

cotrimoxazole therapy nor any of the 15 healthy subjects formed detectable SMX-

haptenated proteins. Total leukocyte and differential counts, cellular toxicity and

glurathione assays as well as liver function tests were dso-performed on 10 of the heaithy

subjects, and there was no indication for an increased risk for development of an adverse

reaction. The lack of SMX-haptenation of s e m proteins suggests that seeking this

marker wiU not predict the onset of hypersensitivity reactions to contemporary

sulfonarnide therapy and may suggest that alternative metabolic or immunoIogical

mechanisms may be k-dved.

Keyworak: Sulfamethoxazole, sulfamethoxazole-hydroxylamine, hapten-prorein conjugate, alpha-2 acid glycoprotein, Western bbt , hypersensitivity reactions

DEDICATION

The thesis is dedicated to my parents, Ken and Doma, and rny two broihers Scott and

Jon, for their unconditional love and invaluabIe suppofi To my fnends, your strength,

compassion and constant encouragement, for that 1 am etemally gratefd.

This research was generously supported by the Medical Research Council of

Canada under the name of Dr. Michad Rieder,

1 wodd like to thank Fust and forernost, Dr. Michael Rieder for the opportunity to

perforrn and successfully cornplete a Masters of Science degree in his laboratory. His

passion and dedication for biomedical research combined with his wit and endless humour

is forever chenshed,

To the members of my advisory cornmittee Dr. David Freernan, Dr. Michael

Clarke and Dr. James Hammond. your insightful thoughts and inteiIectual discussions are

greatly ap preciated and highly treasured.

To Dr. Jane Tucker and John Wijsman whom I deeply value for their integrity.

persistence and cornmitment to excellence. p d e d me through the bumpy and adventurous

road that I passed, 1 am so grateful.

To David Hess and Alice Tschen for their outstanding leadership, wisdom and

friendship. thanks for making a Merence.

TABLE OF CONTENTS

Certificate of Examination

Abstrac t

Dedication

Acknowledgments

List of TabIes

List of Figures

List of Appendices

List of Abbreviations

1-0 Introduction

1 - 1 Overview of sulfonamides

1.2 Hypersensitivity reactions to suIfonamides

1.3 Metabolism of sulîonamides

1.4 Formation of hapten-protein complexes

1 S Adverse reactions in HN patients

2.0 Hypo thesis and Objectives

3.0 Methods

3.1 Generation of polyclonal antibodies to SMX

3.1.1 S ynthesis of SMX-KLH conjugates for immunization

3.1.2 Synthesis of SMX-OVA conjugates for irnmunization

3.1.3 Immunization of rabbits

3.1.4 Confirmation of SMX-antibodies

3.1.5 Purification of polyclonal antibodies

3.1.6 Confirmation of antibodies to SMX by ELISA

3.2 Analysis of human sera for SMX-protein conjugates (in vitro)

3.3 Analysis of serum from HIV patients for SMX-protein conjugate (in vivo)

3.4 Analysis of senun from normal subjects for SMX-protein conjugates (in vivo)

3.5 Predictive tests for reactions to SMX

3-51 Total leukocyte and differential counts

3-52 CeU toxicity using MTT assay

3-53 Glutathione assessrnent

3.5.4 Liver functiond tests

4-1 Production of anti-SMX-KLH antibodies

4.2 Analysis of SMX-haptenated proteins (in vitro)

4.3 Analysis of serum from HN patients

4.4 Assessment of normal subjects receiving SMX therapy

4.4.1 Evaluation of subjects administered SMX therapy

4.4.2 Analysis of SMX-haptenation (in vivo)

4.5 Assessment of predictive tests

4.5.1 Totai leukocyte and differential counts

4.5.2 Cellular toxicity assays

4.5.3 Glu tathione assay

4.5.4 Liver functional ways

5-0 Discussion

5-1 Adverse reac tions to s~arnethoxazole

5.2 In vitro effects of sulfamethoxazole and sulfamethoxazole- hy droxylamine

5-3 In vivo effects of sulfamethoxazole

5.4 Future directions

5.5 Summary and conclusions

References

Vita

LIST OF TABLES

Table 1 Sumrnary of normal individuals participaMg in the couùnoxazole study.

Table 2 The effects oE 25 uM SMX-HA on celIular viability from PB MCs 68 of patients treated with cotrimoxzole therapy.

Table 3 The effects of 50 uM SMX-HA on cellular viability from PBMCs 69 of patients treated with cotrimoxzoIe therapy.

Table 4 The effects of 200 uM SMX-HA on cellular viability from PBMCs 70 of patients treated with cotrimoxzole therapy.

Table 5 Evaluation of glutathione levels in patients over SMX treatment 72 period.

Table 6 Assessrnent of iiver function in patients over the course of SMX 74 treatment.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

The chernical structure of SMX and its metabolites.

The four classes of hypersensitivity reactions-

Pathways of SMX metabolism.

Quantifcation of anti-SMX antibodies using dot blots.

ELISA resdts quantifying the rabbit polyclonal antibody to S m .

Western bIot analysis of SMX, SMX-HA and SMX-NO treated serurn-

Western blot anaiysis of SMX-treated serum with AAG.

Western blot analysis of serum from nomal subjects and HIV patients receiving sulfonamide therapy.

Western blot analysis of semm from normal subjects treâted with sulfonamides using an antibody initiaily conjugated to sepharose beads.

Western blot anaiysis of serum fron normal subjects under reduced and non-reduced conditions.

Western blot analysis of serurn from HN patients and normal subjects using a 7.5% polyacrylamide gel.

Western blot analysis of serum from patients treated with SMX using an altemate source of rabbit ami-SMX-KLH antibody.

Analysis of total leukocytes from patients over SMX treatment period.

Leukocyte differential counts from patients over the SMX treatment period-

LIST OF APPENDICES

Appendix 1

Letter of Idormation

Consent Form

Ethics Approval

LIST OF ABBREVIGTIONS

M G

ADR

AIDS

AP

BSA

CFA

CYP450

DMSO

ECL

Fab

GSH

HA

IHIV

HRP

&A

IgG

IgM

IFA

NADPH

NAC

Alpha- l Acid Glycoprotein

Adverse dmg reaction

Acquired Immune Deficiency Syndrome

Alkaline P hosp hatase

Bovine S e m Albumin

Complete Freund's Adjuvant

Cy-tochrome P450 mixed function oxidase

Dimethyl sulfoxide

Enhanced Cherniluminescence

Fragment antigen binding

Glutathione

Hydroxylamine

Human Immunodeficiency V i m

Horseradis h Peroxidase

Immunoglo bulin A

Immunogiobului E

Immunoglobulin G

Immunoglobulin M

Incomplete Freund's Adjuvant

Nicotinamide adenine dinucleotide phosphate

N-acetylcys teine

NATUNAT2 N-Acety!ûansferase Type l/ Type 2

OVA

PBMC

PBS

PNPP

RPMI

SDS-PAGE

SGPT

SMX

SMX-BSA

S m - H A

Sm-KLH

S m - N O

SMX-OVA

m s

TBS

0valbumi.n

Penpherd Blood Mononuclear Cells

Phosphate Buffered Saline

P-nitrophenyl phosphate

RoseweU Park Memoriai Institue (medium)

Sodium dodecyl sulfate - polyacrylamide gel electrophoresis

Semm glutamic pyruvic transaminase

Sulfarne thoxazole

Sulfamethoxazole - bovine se- albumin conjugate

S ulfamethoxazole - hy droxylarnine

Sulfarnethoxazole - keyhole limpet hemocyanin conjugate

Sulfamethoxazole - nitroso

Sulfamethoxazole - ovalbumin

Tween-tris buffered saline

Tris baered saline

1.1 Ovemew of sulfonamides

Sulfonami& therapy has been used extensively to treat various diseases and

infections since its introduction to the medical comrnunity by Gerhard Domagk in 1935

(Domagk, 1935). This dmg is adminktered primarily to individuals for the treatment of a

wide array of bacterial infections, as well as for complications resulting from

transplantations and immunosuppressive disorciers. In addition. sulfonamides are

commonly prescribed to HIV patients not only for the treatment of the disease but also as

prophylaxis to prevent opportunistic infections such as Pneumqst ï s carinii pneumonia

and toxoplasmic encephalitis. As anti-microbial agents, sulfonamides have been s h o w to

be highly effective in the treamient of these infections and due to the lack of efficacy of

alternative antunicrobials, the use of sulfonamides has been quite commoa The

widespread use of sulfonamides has been associated with a number of adverse reactions-

Approximately 5% of individu& develop a reaction while receiving sulfonamide therapy

(Sullivan, 19û4, Weinshilboum. 1987). These reactions range from nausea, kadaches,

di&ess, to more severe reactions characterized by erythema multiforme, toxic epidemal

necrolysis, high fever, multi-organ toxicity, and in extremely rare cases, anaphylactic

reactions. These types of reactions are commonly classifed as hypersensitivity reactions.

The symptoms that are observed, e.g. skin rash or fever, develop after an interval of

several days to a week after initiation of sulfonamide treatment. Severe reactions such as

Stevens-Johnson syndrome and toxic epidemal necrolysis represent a major cause of

morbidity and rnortality among patierits receiving sulfonamide therapy (Shear et al., 19 86).

The pathogenesis of these hypersensitivity reactions to sulfonamide therapy is

poorly understood 1t bas been postulated that these reactions of a "multïfactorial

nature" and are not attributed to a single etiological factor. Hypersensitivity reactions are

believed to be, in part, initiated by the formation of reactive intemediates or metabolites

resulting from the bioactivation of the parent compound, SMX (Shear et aL, 1985, Rieder

et al., 1988). As a result, these metabolites rnay cause direct cellular toxicity or suppress

components of the adaptive immune response such as lymphocyte proMeration and naturat

killer c d activity (keder et al., 1991)- Altematively, these metabolites may covalently

bind to cellular macromolecules or plasma proteins which c m Iead to the potential

formation of immunogens and induce an undesirable immune response (Pohl et al., 1988,

Park et al,, 1987). The chernical structure of sulfamethoxazole and its metabolites are

illustrated in Figure 1.

As bacteriostatic agents, sulfonamides primady act by competitively inhibiting

with bacterial paminobenzoic acid @ B A ) for the enzyme dihydropteroate synthetase

@S) (Rang and Dale, 199 1). As armatic amines, suIfonamides represent structural

analogues of the substrate pABA which enable them to interact with DS and interfere with

folk acid synthesis. Folate is an absolute requirement by al l m a m a i s for purine

biosynthesis. Mammals cm obtain adequaîe levels of folate through diet. Bactena on the

other hand, Iack a transport system for folic acid and thus are forced to manufacture their

own folate. The inhibition of pABA by suEonanïdes results in the inhibition of bacterial

replication (Rang and Dale, 199 1).

Cotrimoxazole, a combination mixture of sulfamethoxazoIe and trimethoprim, is

often administered to patients for the treamient of pneumonia and other bacterial

infections. The addition of trimethoprim, a dihydrofolate reciuctase inhibitor which

prevents folate from k i n g converted to its active form tetrahydrofolate, aids in the

inhibition of nucleic acid synthesis. The use of cotrimoxazole enables physicians to

prescrïbe lower doses of sulfamethoxazole and expands the speceum of activity. This

approach lowers the risk of potentiai adverse drug reactions to sulfonamide therapy while

stiU maintaining efficacy (Koopans 1995).

Figure 1. The chernical structures of a) sulfamethoxazole b) sulfamethoxazole-

hydroxylamine and c) sulfamethoxazoIe-nitroso.

1 1 Hypersensitivity Reactioos to Sulfonarnides

Reactions to sulfonamids are generally characterized as "drug hypersensitivity

reactions". The term drug hypersensitivity was &st described by Gell and Coombs in 1968

(Figure 2). GelI and Coombs classified dnig hypersensitivity reactions as king four srpes

of reactions commonly refirred to as Type 1 to Type IV (Coombs and Gel, 1968). Each

type of reaction is characterized by the immunological mechanisms of each of the classes

of drug hypersensitivity reactions.

Type 1 hypersensitivity reactions are often referred to as "allergie" reactions. This

type of reaction is identifed by the release of local inflanmatory mediators such as

histamine and various leukotrienes as a result of the interactions between specific antigens

and the immunoglobulin IgE- The antigen binds to the Fab portion of the antibody, already

present on the surface of mast cells from an initial response, &ter subsequent exposure to

a particular antigen. This fanlitaies the cross-linking of IgE antibodies causing release of

contents frorn the mast ce&. including histamine, a potent vasodilator. Histamine release

leads to systemic reactions that include anaphylaxis, bronchospasm and urticaria (Janeway

and Travers, 1994).

Type II reactions are characterized by the recognition and subsequent binding of

antibodies to sWc antigens located on the cell surface of targeted leukocytes. This

antigen-antibody interaction causes cellular lysis mediated through the activation of n a t d

killer cells, monocytes or neutrophiIs as well as the induction of a host of complement

factors. These reactions are obsemed in patients with hyperacute gr& rejection, blood

transfusions and hemolytic anemia

Type III hypersensitivity reactions are atbibuted to the formation of immune

complexes that associate with specific antigens. In persistent infections, these immune

complexes becorne lodged in various tissues. This antibody deposition promotes the

recruitment of nearby lymphocytes and activates the complement system leading to

cellular and tissue injury- Senun sickness Like reactions (SSLR) represent the classical

Figure 2. GelI and Coombs' classification of the four types of hypersensitiviv reactions

(GeU and Coombs, 1968, ). Adapted from Roit et al. (1985).

Release of

Fc Receptor mediators

n

Antigen

Mast cell

Type 1

Activation of complement or Ieukocytes attracted to tissue

CeU surface antigen

A n t i i y Complement

Activation of natural killer cell or complement induces ce11 lysis

Type II

Antigen

Release of cytokines and macrophage activation

Type IV

Type IIï

manifestations of the Type IlI hypersensitivity reactions in whîch patients develop fever,

skin rash and various foms of arulralgia (Keams et aL, 1994).

Type IV hyperseusitivity reactions generally occur at least 12 hours after an

antigenic insult This typically involves the stimulation of T-lymphocytes followed by the

release of specinc lymphokines and activation of macrophages. These reactions range from

miId skin rashes to severe reactions which may occur more often and rapidly upon

subsequent exposure (Dunagin and Millikan, 1980).

The perplexing nature of these reactions can be observed from some of the

reported skin reactîons of individuals receiving suIfonamide therapy. Responses Vary h m

what appears to be a Type I hypersensitivity response in which urticaria are observed to a

more localized dermatitis reaction that is described b y a Type IV response. Unfortunately ,

reactions to sulfonamides do not correspond directly to any of the classicd categories

described by Coombs and GeU. These classifications only consider the immunological

mechanisms independently when attempting to elucidate the perpetuation of adverse dnig

reactions and do not account for alternative mechanisms of immune modulation-

In the 1 s t decade, an effort has been made by Paterson and colleagues to eIaborate

on the mechanisms of adverse drug reactions by combining and outlining both

immunologicai and non-immunological mechanisms (Paüerson et al., 198 6). niese

mechanisms are classified as either predictable or unpredictable reactions.

Redictable adverse dmg reactions, for the most part, are dose-dependent, and can

be avoided by close monitoring of patients on current drug therapy and understanding the

Limitations of the phamacological actions of the h g in question. For example, reduction

in the dosage of the drug usuaIly alleviates complications resulting from the initiation of

drug therapy (Patterson et al., 1986). Often predictable reactions are associated with drug

overdoses, loiown side effects and dmg interactions including both drug-drug interactions

and drug-disease processes.

Unpredictable adverse drug reactions represent a large proportion of adverse

reactions, many of which appear to be unavoidable (Rieder et al., 1992, Rieder 1993).

These reactions are dose-independent and are not related to the intended pharmacological

actions of the dmg. Discontinuation of drug therapy is requried to alleviate the reactioa In

addition. exaggerated and even disabhg effects. such as extreme nausea and vomitting,

may be observeci with normal dosing, a problern that rem- difficult for physicians to

anticipate. Drug intolerance, idiosyncrasies, allergies aud pseudodergies represent the

geneml domain of unpredictable reactions.

Sulfonamide hypersensitivity reactions are characterized as idiosyncratic reactions.

These reactions are typically immune mediated and are associated with fever followed by a

skin rash, 7 to 10 days after the initiation of sulfonarnide therapy (Rieder et al., 1989).

This rash is commonly erythema multiforme, toxic epidermal necrolysis or morbilliform

eruptions. In some patients, heart, bone rnarrow, b e r and kidney may also be involved in

these reactions (Rieder et al., 1989). Reactions to sulfonamides are unique to each

individual and may be dependent on previous exposure to sulfonarnides or may be

associated with a genetic predisposition. This may UicIude individual deficiencies in

metabolism, enzyrnatic activity or even modification of the normal detoxification pathways

(Eüeder, 1993; Shear and Spielberg. 1985).

1 3 Metabotism of Suifarnethoxazole

The complex nature of adverse reactions to sulfonamides represents a major

hindrance for understanding the pathogenesis of these reactions. It has been proposed that

SMX hypersensitivity reactions are the direct result of the oxidative metabolism of the

parent cornpound SMX to a more reactive intermediate, suifamethoxazole-hydroxylamine

(Sm-HA).

In generai, up to 50 to 80% of an administered oral SMX dose undergoes Phase II

N-acetylation by N-acetyltransferase (NAT) either NAT 1 or NAT 2 (Vree et al., 1994;

van der Ven et al., 1994)- This results in the formation of an acetylated-SMX that is non-

toxic, inactive and readily excreted via the urinary tract However, a small fiaction of the

remaining sulfonarnide component appears to be subject to oxidation via the cytochrome

P450 (CYP) monooxygenase systern, predominantly by the isozymes CYP 2 0 (Cnbb and

Spielberg, 1990; Cnbb et al., 1995; Leeder et al,, 1988). The initial oxidative product

generated is SMX-HA SMX-HA cm undergo M e r metabolism by one of three

potential metabolic pathways. SMX-HA can undergo m e r detoxication which appears

to include acetylation to generate inactive metabolites which, similar to acetylated-SMX,

are readily excreted. Secondly, SMX-HA can undergo spontaneous oxidation under

biological conditions to a nitroso metabolite (SMX-NO) that is inherently unstable and

more reactive than SMX-HA (Rieder et al., 1995)- Altematively, the reactive nature of

SMX-HA suggests that the metabolite may act a s a hapten capable of binding to carrier

ce& or molecules such as plasma proteins. Haptens, on their own, do not provoke an

immune response due in part to their smaller size. Antigen presentuig ceUs fail to process

the hapten, however, if the hapten covalently binds to a plasma protein, this "neo hapten-

protein complex" rnay generate either an antigenic or immunogenic species. An antigenic

species is an entity which can bind to antigen-specific ceiis. An irnmunogenic species has

the ability to elicit an immune response such a s T-celi stimulation or antibody production.

The production of specific antibodies or activated T cells directed against the epitopes of

the hapten-protein complex provide a rnechanism to clear this antigen. This cellular or

humoral response promotes the formation of immune complexes that rnay be associated

with hypersensitivity reactions and dtirnately determine the metabolic fate of the hapten-

protein complex (Figure 3)-

The development of adverse dmg reactions to sulfonamides is believed to be

initiated by the production of the reactive intermediate SMX-HA (S hear et al., 1985,

Rieder et aL, 1988). In vitro studies have demonstrated dose-dependent cellular toxicity of

chemically synthesized SMX-HA when CO-incubated with peripheral blood mononuclear

Figure 3. The proposed mechanism for the metabolism and metabolic fate of SMX. The

production of SMX-HA or SMX-NO metabolites may potentially act as haptens and

conjugate to plasma proteins thereby eliciting an immune response that results in a

hypersensitivity reaction (adapted from Park and Kitteringham, 1990. Cnbb et al., 1994).

NATI, NAT 2 (50-80%)

S m + b Acetylated SMX - = Excreied Deacetylation (minor for S M . )

CYP 2C9

GSH Ascorbic acid

NADPH Clearance

Anergic 4- T Anh'body production

Hypersens itivity Reaction

Cellular response Humoral response + Activated macrophages Hypersensitivity

response

Toxic epidennal necrolysis erythema multiforme multi-organ toxicity

Skin rash, fever, organ toxicity

ce& (PBMCs) acquired h m patients who tolerate and do not tolerate sulfonamide

therapy compared to untreated PBMCs (Rieder et al., 1988, Leeder et al., 1SY8, Cribb et

al., 1990). This evidence suggests that an individuals sensitivity to SMX may be correlawl

with an increase in vulnerability of PBMCs to cellular injury or toxicity in vitro.

Otlier studies in çell culture have also iIlustrated toxic effects of SMX. Not onIy

does chemically synthesized SMX-HA show a dose-dependent toxicity when incuba~d

with PBMCs but also when SMX is introduced into a murine microsomd based system to

mode1 an in vivo metabolic system to generate HA metabolites, a similar dose related

toxicity is observed (Spielberg et al., 1984, Leeder et al., 1988, and Cribb and Spielberg.

1990). The microsomal system serves as a surrogate for rnetabolic activation. The

incorporation of NADW, Glucose -6- phosphate and Glucose -6- phosphate

dehydrogenase, cokt ive ly serve as the CO-factors to drive the metabolic system.

It has aIso been postulated that a metabolic bais for these reactions may be a

direct or indirect result of the metabolic deficiencies or aberrations to the cells that are

unique to each individual. Glutathione, (GSH) an intracellular substrate located in a

number of different ceils including hepatocytes and PBMCs, possess anti-oxidant

properties, is primarily responsible for the prevention and formation of oxidative radicals

and electrophilic compounds which cause cellular destruction (Shear et al., 1985, Rieder

et al., 1988). Reduction in GSH levels may increase the risk for patients developing

reactions to sulfonamides because of higher levels of SMX metabolites. In HiV patients,

as the immune system deteriorates, there appears to be a marked reduction in intraceIlular

Ievels of glutathione (Roederer et al., 199 1, Droge et al., 199 1). As a consequence,

individuals may become increasingly susceptible to a variety of electrophilic compouods

and oxidative insults which can lead to cellular injury and substantial tissue damage.

Moreover, those individuals who have reduced cellular GSH levels are more vulnerable to

the adverse reactions of SMX therapy. It has been proposed that GSH controls the levels

of SMX-nitroso formation because of its ability to neutraiize and prevent the production

of reactive species (Crïbb et aL-, 1991). Thus, reduced leveis of GSH wodd increase the

likelihood of the formation of nitroso groups. Moreover, studies indicate that the

concentration-dependent toxicity of cornpounds can be anenuated by the addition of

exogenous GSH to PBMCs (Shear et al-, 1985, Rieder et al,, 1988, Rieder et al., 1995)-

This provides the cek with an abundant supply of scavengers that c m eliminate

po tentiaüy darnaging oxidative radicalS.

It has also been suggested that pharmacogenetic ciifferences among people may

account for a portion of the adverse reactions exhibited (Spielberg, 1996). Genetic

variations in metabolic systems may pose an increased risk in developing a reaction to

sulfonamide treatment More specincally, acetylator phenotype is believed to be correlated

with the risk of developing a reaction meder er al., 199 1). The proportion of fast

acetylators to slow acetylators in the general population is approximatdy equd (Evans,

1989). These levels of SMX-KA in the circulation may detemine whether a person is at

risk for developing an adverse reaction. In theory, the higher the levels of SMX-HA, the

more likely an ADR. Individuals are categorized as either fast or slow acetylators using

caffeine metabolism tests (Tank et aL, 1991). Subjects who are classifiecf as fast

acetylators, have the inherent capacity to quickly and efficiently acetylate the parent SMX

to its inactive or non-toxic (acetylated-SMX) predominantly by NAT 2 where it wodd be

rapidl y excreted. In contrast, slow acetylators do not metabolize the parent compound as

efficiently as fast acetylators do and therefore require additional tune. As a consequence of

this increased latency period, SMX rnay accumulate in the systemic circulation remaining

more vulnerable to oxidation and subsequent conversion to the SMX-HA form (Carr et

al,, 1994).

Acetylator phenotype among HIV patients is believed to be important in the

initiation of adverse reactions to SMX. In a study by Carr et aL, 15 out of 16 (94%) HIV

patients, who previously experienced a hypersensitivity reaction to cotrimoxazole therapy,

expressed the slow acetylator phenotype. In cornparison with 12 non-hypersensitive

patients, only 5 had the slow acetyIator phenotype (Cam et al., 1994). It was also noted

that the stage of HIV infection rnay contribute to acetylator phenotype. Those patients

with advanceci HIV rnay be more inclined to express the slow acetyiator phenotype. This

observation rnay result h m the propagation of the disease possibly attributed to various

forms of liver disease or disorders, concurrent infections, concomitant an ti-viral therapies

and other drugs which rnay interact with hepatic cytochrome enzymes. Despite these

factors, observations by Lee et al. (1989) suggest that acetylator phenotype does not

subject an individual to an increased risk for developing a hypenensitivity reaction Thus,

the prevalence of the slow acewator phenotype does not constitute an absolute risk for

developing reactions to SMX in HN patients. Moreover, acetylator phenotype rnay play a

role in the onset of reactions but is probably insuffcient as the single culprit responsible or

encompaskg the entire domain of adverse reactions in HIV patients.

SMX-HA may undergo further oxidation to the SMX-nitroso cornpound (SMX-

NO). In this metabolic state, the SMX-NO has increased its electrophilic capacity and thus

a propensity for increasing levels of cytotoxicity. The oxidation of SMX-HA to SMX-

nitroso represents an integral cornponent in the pathogenesis of reactions to suIfonamides.

As a low rnolecular weight thiol with anti-oxidant capacity, GSH may neutralize this effect

by conjugating to this reactive species. Glutathione transferase p (GSTF), an isozyme of

the GST superfamily that is involved with GSH in conjugation reactions, is believed to

play an important role in the regdation of GSH levels. However, it does not appear that

patients with reactions to sulfonamides have deficiencies in the levels of GSTp expression

v e y et al., 199 1).

It has been also suggested that individual levels of expression of critical oxidative

enzymes that are involved in the meiabolism of SMX rnay play a d e in determinhg the

potential for an adverse reaction. Thmretically, if there are higher levels or concentrations

of cytochrome P450 isozymes CYP 2C9 present, then this rnay increase the production

and/or avdability of reactive species gmups Le. SMX-HA. As well, signifcant reductions

in levels of NATl and NAT2 enzymatic activïty may suggest that less of the parent

compound is k ing acetylated and a larger fraction of the sdfonamide component is king

subjected to oxîdative metabolism- The levels of NATl and NATS enzyme activity and

their involvement in hypetsensitivity reactions remain to be determined,

The reactions to sulfonamide treatment may involve the propagation of

immunornodulatory responses. Atypicd Lymphocytes, apparent lack of res ponse to

adrenocorticoid therapy and a high rate of infectious complications suggest the

mechanisms have an immunologicd basis. ClinicaUy, these immunological responses can

be observed from the severity of adverse events such as Stevens-Johnson Syndrome,

erythema multiforme, toxic epidermal necrolysis and multiorgan toxiciity. Snidies in vitro

dernonstrate a dose-dependent reduction in lymphocyte proliferation of PBMCs when

incubated with SMX-KA (Rieder et al., 1992). Varying levels of cellular esterases and

their subsequent levels of activity e.g., cell viability, may be inhibited by

immunomodulatory even ts (Leeder et al., 199 1). The percentage of viable cells represents

an index of susceptibility to the toxic effects of drugs or dmg metabolites.

1.4 Formation of hapten-protein complexes

Although both metabolic and immunomodulatory mechanisms have been

implicated in these reactions to sulfonamides, there has been recent evidence to suggest

that SMX andfor its metabolites may act as haptens and covalently bind with cellular

macrornolecules, in particular, plasma proteins (Shear and Spielberg, 1985). The

production of these hapten-protein complexes rnay potentially cause cellular destruction or

form immunogens that can propagate an immune response.

Immune complexes were fxst described by Eüch in 1942, who suggested that when

patients were treated with SMX it was believed that these complexes formed in their sera

(Rich, 1942). Two years later, Leftwich investigated the effects of injecting senun samples

under the skin of individuals in order to induce an immune response and described the

recipients as "allergie" to sulfonamides (Leftwich, 1944). Reactions to procainamide have

k e n associated with h g induced lupus, whereby the metabolites of procainamide

conjugate to plasma proteins thereb y generating reac tive species (Uetrec t et al., 19 85).

Halothane, a conventional anaesthetic agent, is associated with dnig induced hepatitis

which is relatively rare (1 in 10 000 individu& receiving an anaesthetic) and occurs

between days 2 and 5 after administration, (Marshall and Longnecker, 1990). The

hepatitis-like symptoms appear to arise fiom the oxidative metabolism of halothane to

produce a trifluoroacetyl halide metabolite that has the i n t ~ s i c ability to combine with

plasma proteins (Satoh et al-, 1985). Specific antibodies have been generated in

individuals who sustain these adverse events and the antibodies are directed against

hepatic proteins conjugated to the tntluoroacetyl halide. The antibodies that are formed

are unique to those individuah who have mictions to halothane and are not found in

patients who tolerate halothane therapy nor have they be identifiied in patients with other

forms of disease associated with hepatitis (Uetrecht, 1990).

Other in vino assays M e r support the formation of immune complexes and their

involvement in mediating toxicity. Sulfadiazine, another member of the sulfonamide family

of anti-microbials, when radioactively labelled was shown to covalently bind with

microsomal proteins (Shear and Spielberg, 1985). This conjugation appeared to correlate

with cytotoxicity to mononuclear lymphocytes. These authors also demonstrated that

SKF-525-A, cimetidine, and C% inhibited P450 mediated metabolism as well as

formation of SMX-HA. Conversely, by reducing the amount of anti-oxidant agents GSH

and N-acetylcysteine available to the cdtured lymphocytes, the concentration of SMX-HA

metabolites increased. SMX-HA has also been demonstrated to covalently biod to hepatic

microsomal proteins in vino (Cnbb et al., 199 1).

In 1997, Cribb also investigated the sera of individuals, who received SMX, for the

presence of antibodies that recognized rnicrosomal proteins and SMX-HA microsomal

protein conjugates (Cribb et al., 1997). Patients were divided according to a) those with

no history of adverse reactions to suMonamides, b) those with a history of adverse events

to sulfonamide hypersemitivity reactions and c) patients who had reactions to

sulfonamides. but were not consistent with hypersensitivity reactions e.g. urticaria,

s w e k g , diarrhea, Seventeen of the 21 patients (81%). who had reactions characteristic of

sulfonamide therapy, had specific antibodies directed against SMX-HA microsomal

protek. Fourteen of these patients had antibodies that recognized a 55 kDa microsomal

endoplasmic reticulurn protein identified as disuifide isomerase, while four patients had

antibodies against an 80 kDa protein known as grp 78. Three patients had antibodies

recognizing 96 kDa proteins (Cribb et al., 1997). Only one of the patients, who developed

a reaction, uncharacteristic of the hypersensitivity reactions, had antibodies to a 80 kDa

protein- The control group did not have any microsomal proteins or antibodies present,

This evidence suggests that the onset of hypersensitivity reactions may involve the

recognition of specific protein epitopes by antibodies while antibodies to SMX metaboLites

are of l e s importance. Moreover, SMX-HA that binds to Iiver proteins may m o d e the

protein structurdy and functionaIly thereby generating an immunogenic complex that is

recognized by the immune system.

In vivo studies performed by M e e b and CO-workers demonstrated the

production of specific proteins that become conjugated by SMX or SMX-HA in vivo

(Meekins et al-, 1994). Patients who were treated with oral doses of sulfamethoxazole

(500 mg twice daily) had theù sera subsequently analyzed for haptenated proteins using a

polyclonal antibody to SMX-BSA in a Western blot system. Two individuals had the

presence of a 30 D a protein in their sera This protein band remained detectable 48 hrs

after discontinuhg therapy. Thus, the inability to effectively clear this protein may r e k t

an individuals risk for developing a reaction (Meekins et al., 1994).

The resdts of preliminary studies in our laboratory also suggest that specific-SMX

haptenated proteins are formed during therapy. The s e m of individuals who were treated

with cotrimoxazole therapy was ùivestigated for SMX-haptenated p ro tek using a

Western blot technique and an anti-SMX-BSA polyclonal antibody obtaïned fiom Dr. RS.

Gruchalla, Southwestern Medical Center, Dallas, Texas- It was demonstrated that ail

individu& receiving cotrimoxazole therapy had a 42 kDa protein present in their sera

(Aarts, 1996). This protein band appeared on Days 3 and 6 of therapy but was absent by

Day 9. These resdts suggest that any persons receiving sulfonamide therapy will generate

haptenated proteins. The ability of iodividuals to effectively clear these proteins from the

sptemic circulation rnay account for the variation in tolerance of sulfonamide therapy. As

well, the amount of SMX-HA in the systernic circulation rnay determine an individual's

risk for an adverse reaction.

A study investigating the adverse reactions of twelve HXV patients treated with

sulfonamides also showed the presence of haptenated proteins in the sera (Gmchalla et al.,

1998). Patients were administered coeimoxazole either for prophylaxis or desensitization.

Desensitization studies are designed to switch the type of immune response of patients

who are dependent on a particular drug therapy and the ineffectiveness of alternative

antimicrobial therapy. By incremental administering of coaimoxazole, an attempt is made

to alter the immunological response from IgE to other isotypes such as IgG. Of the four

patients who were king treated prophylactically, the senun of one patient had a single 40

kDa protein present as identified by an ad-SMX-BSA antibody and a Western blot

system. Of the eight patients undergoing desensitization, six showed the presence of the

40 kDa protein in their serum, three of whom had adverse events. Only one of the eight

patients had SMX-IgG antibodies present as measured b y Enzyme-Linked immunosorbent

assay (ELISA) inhibition. These results suggest that although irnmunocompromised

individuals who receive sulfonamide therapy are likely to f o m hapten-protein complexes

in vivo, they are not necessarily associated with an adverse reaction. However, those

patients who present hapten-protein complexes in their sera appear to be at nsk for

developing a reaction. Reactions to sulfonamides observed in HIV patients are more

complex in iden-g a single factor that is responsible for the intolerance to conventionai

sulfonamide therapy- Other ri& factors such as the stage of HIV infkction, levels of CD4

lymphocytes, GSH levels and degree of immunodeficiency when taken into account, may

influence the incidence, degree and severity of reactions.

1.5 Adverse reactions in HIV patients

The frequency of adverse cimg reactions to sulfonamide therapy is greater among

HIV seropositive individuals than the general pop dation. The incidence of these reactions

ranges from 40% to 80% with individuals infected with HIV as compared to 5% of the

normal HIV seronegative population (Gordin et al., 1984, Golden et ai., 1989, Van Der

Ven et al,, 1991, Daftarian et al., 1995). The reactions reported are specific for

hypersensitivity reactions. This marked increase in the occurrence of these reactions has

been attributed to a number of metabolic and Unmunomodulatory factors that may be

directly or indirectly associated with the disease itself, However, the precise mechanism

remains poorly understood. It has k e n suggested that a combination of deficiencies in the

metabolisrn and elimination of the sulfamethoxazole may play a major role in the onset of

these reactions. Impaired clearance mechanisms may also potentiate the risk for

development of reactions. An unequal balance of detofi~cation and bioactivation

mechanisms may account for the propagation of the immune responses.

In some children, unformately, the indication of an adverse reaction to

cotrirnoxazole therapy for a bactend uifection, has led to the suspicion and subsequent

diagnosis of HIV infection (Rieder et al., 1997)- The exact rate and nature of these

reactions however remains elusive in chiidren-

Reduction in the Ievels of SMX-HA excreted in the urine was observed in HIV

patients in contrast to urine from normal individuals during days 3 and 10 of therapy (Lee

et al., 1994). This observation suggests that there may be an apparent increase in the

levels of circulating reactive metabolites and thus subsequent formation of the SMX-HA

in vivo. This rnay also reflect the elimination of SMX-HA It is possible that additional

metaboliies are king generated. Cribb et al., proposed that prostaglandin synthase. an

enzyme located in the kidney responsible for the metabolic fate of prostaglandins, can

form oxïdaiive metabolites Le, SMX-HA, in the urine (Cribb et al,, 1990)- However, the

amount of SMX-HA generated by prostaglandin synthase does not conaibute signincantly

to the overall levels of SMX-HA in the plasma (Mitra et al., 1996).

Augmenteci levels of SMX-HA specific antibody, have also ken correlated with

disease process (O'Neil et al., 1991)- This dmg sensitivîty rnay be dependent on the degree

of immunodeficiency, severity and stage of the disease. Individuais diagnosed with a "les

severe" form of the disease have a sharp rise in the levels of specifk IgG immunoglobulin

levels, In contras, patients with a "more severe" form of the disease have IgE

immunoglobulin levels greater than normal or l e s severe disease. However, the antibody

level and isotype appear to be related to the disease, rather than the sensitivity to the drug.

Clinically, the stage of HIV infection in patients is classified according to levels of

CD4 T-lymphocytes and viral loads. Patients, are generally treated with aggressive

antiviral and prophylaxis treament when CD4 counts fall below 500mm3, while

indWiduals are clinicdy diagnosed with AIDS when levels of CD4 lymphocytes are

d00mm3 (Janeway and Travers 1994). The levels of CD4 lymphocytes may also be

correlated with a propensity for adverse reactions. Some Uivestigators believe that as CD4

lymphocyte levels decrease below a critical level, the incidence of reactions to sulfonamide

therapy also decreases (Carr et al., 1993). Other groups have reported that the probability

of developing skin reaceions to alternative antimicrobials such as amoxiçillin-clavulanate

increased as the number of CD4 lymphocytes were reduced pattegay et al,, 1989). This

contrast in observations suggest that the mechanisms for reactions to cotrimoxazole

therapy may cliffer €rom the mechanisms of alternative anti-microbial therapy.

It is also conceivable that HIV-infected ceils rnay be more susceptible to the

reactive dmg metabolites of SMX. Rieder et al.. (1995) hvestigated the toxic effects of

SMX-HA in W-infected ceus and Human T d lyrnphotrophic virus -V) infected

cells. A concentration-dependent toxicity was observed with the HIV infected cell luies,

while non-infeçted and HTLV cell hes did not exhibit this toxicity, suggesting that the

HTV infection itreif may contribute to the observed toxicity. In addition, glutathione levels

were significantly reduced in the HIV infected cell lines as compared to the non-infected

cells (Rieder, et al., 1995). This marked decrease in htracellular glutathione rnay provide

an environment favouring the fornation of electrophilic and oxidative radicals Le. SMX-

NO, thereby causîng cellular insult

2.0 HYPOTHESIS and OB JECTLVES

We hypothesize that a.ll patients treated with sulfonamides develop unique

suifonamide-haptenated proteins during the course of treatrnent, Those individuals who

develop adverse drug reactions during sulfonamide therapy clear these unique

suifonamide-haptenated proteins through different immunological pathways than do

patients who tolerate sulfonamides without adverse effix ts. In addition, altered clearance

mechanisms, particularly in HIV patients, may contribute to the increased incidence of

reactions to sulfonamide therapy.

Reactive metabolites of SMX are believed to be key elements in the pathogenesis

of adverse reactions among normal individu& and more promiaently in HIV infected

individuals. It is postulated that these reactions are initiated by the formation of a hapten-

protein cornplex, with SMX or metabolites of SMX, thereby acting as haptens and

subsequently conjugating to semm protein(s) to form imrnunogenic complexes. The

production of these immunogenic complexes may be responsible for the induction of an

irnmunological response that is associated with adverse reactions to SMX therapy-

Therefore. the following objectives were generated and investigated accordingly.

1) To iden* and characterize SMX-haptenated semm protein(s) in order to further

understand the precise d e of these proteins and their involvement in hypersensitivity

reac tions,

2) To iden* the type of imrnunological response that individuals mount against the

haptenated proteins and determine how these responses Vary between patients who

toIerate sulfonamide therapy and those patients who have an adverse reaction to

sulfonamide therapy-

3) To investigate the kinetics of hapten formation in sera of HIV infected individuals

administered sulfonamide therapy.

4) To investigate the kinetics of hapten-formation in sera of normal individuais

administered saonamide therapy.

5) To provide a predictive modd indicating those individuals who, based on formation of

hapten-protein complexes, may be vulnerable to developing a reaction to sulfonamide

therapy-

3.0 METHODS

3.1 Generation of polyclod adbodies to SMX

3.1.1 Synthesis of SMX-KLH conjugates for immunization

The S M ? conjugates were prepared by coupling the diazonium s d t of SMX to

Keyhole limpet hemocyanin (JCLH) as desclibed previously (Jou et al., 1983). B nefly,

0.75 mm01 of SMX was added to 0.1 M hydrochloric acid and mixed thoroughly. Next, 1

M cold soaium nitrite solution was added dropwise until the immediate appearance of a

purple colour, as indicated by the addition of a droplet to starch-iodide p a p a The

solution was then neuualized by the addition of a few crystals of sulfamic acid. The

reaction was allowed to stabilize for 2 hours at 4% In z separate beaker, 0.50 mmol of

methyl-ghydroxybennmidate (Ki3) was dissolved in 8 ml diçtilled water. HB acts as

molecdar linker between the SMX and K M . The HB solution was then added dropwise

while raising and maintalliing the pH at 8.5 with 1 M sodium hydroxide (NaOH). The pH

was adjusted to 7.0 yielding a solution of 0.24 M SMX-HB. in a separate tube, 10 mg of

KLH was dissolved in water to yield a fmal concentration of Img/ml. Finally, 5 ml of KLH

solution was added to 5 ml of the SMX-HB and placed on a rotator overnight at 4%

Samples were then dialyzed twice over 24 hours with PBS and once with water and then

stored at -200C unid desired,

3.1.2 Synthesis of SMX-OVA conjugates for irnmunization

The procedure used to conjugate SMX to OVA was adopted by Nisonoff (1967)

with sLight modifications. The diazonium salt was fust prepared by dissolving 40 mg of

SMX in 2 5 ml of 1 N hydrochlonc acid. Cold sodium nitrite (14 m g l d ) was added

dropwise to the SMX solution u n d the immediate appearance of a purple colour was

observed, as indicated by the addition of a droplet to starch-iodide paper. The solution

was mixed thoroughly and ailowed to stabilize for 2 hrs. Next, 250 mg of OVA were

dissolved in a separate beaker using 10 ml of 0.13 m o n sodium chloride and 0.16 moVL

of bric acid and adjusted to pH 9. The beaker was placed in an ice bath with a pH probe

and was continuously stirred. The diazonium salt of SMX was then added dropwise to the

OVA solution and the p S was maintained at 9 using sodium hydroxide. The SMX-OVA

solution w s aiXowed to stabilize for 1 hour in the ice bath- The pH was then set to 7.0

using hydrochlonc acid and the SM.-OVA mixture was dialyzed twice with PBS and

once with water over 24 hours and then stored at -200C until required,

3.1.3 Irnmunization of rabbi ts

The SMX-KLH conjugate was sent to Rockland Pharmaceuticals Inc.

(Gihrtsville, Pennsylvania) for immunization of rabbits. The immunization procedure was

perfomed using the same methodology as with SMX-OVA conjugates. In short, two

young male New Zealand white rabbits were prebled and subsequently administered 1.0 ml

(250 uglml) of SMX-OVA prepared in Phosphate Buffer Solution (PBS) and emulsified in

Complete Freund's Adjuvant (CFA) (0.5 ml SMX-OVAPBS: 0.5 ml FCA). The dmg

conjugate was then injected subcutaneously in five to ten sites dong the lower posterior

region of the rabbit. Ten days later, the rabbits were bled and then imrnunized as described

in the primary injection, except the CFA was replaced by Incomplete Freund's Adjuvant

(FA). The same volume and concentrations were used as in the primary injection. The

immunizations (SMX-OVA in ICA) were repeated on days 20 and 30 &ter the initial

injection. On day 30. blood was collected and senun was separated from the whole blood

and analyzed for the presence of polyclonal antibodies to SMX-

3.1.4 Confirmation of SMX-antiboàies

Dot blots were perforrned to confirm the presence of anti-SMX mtibody titres

from rabbits immunized with either SMX-KLH or SMX-OVA. Briefly, one or two drops

of either SMX (0.5 m m ) or SMX conjugated with or without protein carriers @SA,

OVA or EUH) were added to nitrocellulose membranes and allowed to dry for 30

minutes- Pre immune rabbit s e m served as a control, The dots were then blocked for 3

hrs with blocking solution (31 non-fat milk powder containhg 1% BSA in Tris bufFered

saline mS). N e x ~ 10 ml of either pre or post immunization rabbit sem (1: 100) diluted

in 0.1 % Tween containing TBS (TTBS), was added to each swc blot and incubated

overnight at 40C. The blots were washed three tirnes with ï T B S allowing ten minutes for

each wash Ten millilitres of secondary goat-anti-rabbit IgG heavyhight antibody

conjugated with streptavidin horseradish peroxidase (1:3000 in 'TTBS) was then added to

each blot for 1 hr. The membranes were washed thoroughly three àmes. Enhanced

cherniluminescence (ECL) (Amersharn) was added to the membranes and the blots were

detected using autoradiographic film.

3.1.5 Purification of polyclonal antibodies

On Days 37, the rabbits were exsanguinated, blood was collected and serum

separated. Post immunization rabbit serum sarnples (3.0 ml) were then placed over a 20 ml

Econo-PacB Protein A column (BIO-RAD) that was pretreated with 10 ml of binding

buffer (pH 9) (NO-RAD). The Protein A column consists of pufied protein A coupled to

agarose beads through chemicdy stable amide bonds. This kit provides a method for the

purification of ail IgG subclasses h m crude serum. The column was then washed with 20

ml of binding buffer and then eluted in 1 ml volumes of elution buffer (pH 3) (BIORAD)

(Total volume 10 ml). The 1 ml volumes were then run through a desalhg coIumn and

collected in 1 ml fractions using PBS. The absorbance of each of the 1 ml collection

fractions was measured using a spectrophotometer at 280 nrn. Dot blots were repeated

with both SMX-KLH and SMX-OVA anthenim. The antibody collections (2 mglml) were

stored at -20°C und required Pre immune serum samples were also placed over the

Protein A column coiiected as described above.

Semm was collected both before and after subsequent immunizations. Unless

otherwise stated, SMX-KLH anribodies were used as the primary antibody in all the

experiments, Enzyme-linked-immunoassays (ELISA'S) were also completed to further

support the presence of a specific polyclond antibody to SMX.

3.1.6 Confirmation of antibodies to SMX by ELISA

ELISA'S were cornpleted as descnbed by Gruchalla (1998) with some minor

modincations. Briefly, 2 0 ul of OVA, SM'-OVA or SMX-BSA (20 ugfml) was

aliquoted into a 96 Bat bottom weil plate. The samples were allowed to incubate overnight

at 40C. The plate was washed three times with TBS. Next, 200 ul of 1% BSA in TBS was

added for 2 hrs at room temperature to block any non-specific binding. The plate was

washed three times with TBS and then incubated for 1 hr with either pre-immune sera or

pst-immune sera, seridly diluted with 1% BSA and in ï T B S beginning at 1: 100 to

150 MW) and were performed in trïplicate. After the plates were washed, 100 ul of a

secondary goat-anti-rabbit SMX (QG heavy and light chain) antibody conjugated alkalule

phosphatase (1:20 000 in ' I T B S ) was added to each well for 1 hr at room temperature.

The plates were then washed and 100 ul of P-nitrophenyl phosphate (PNPP) subsuate was

added. The optical density (OD) was determined at 405 nm for each sample using an

automated plate reader and Sofunax @ program. The absorbance of the pre-immune sera

compared to the absorbance of the post immune sera was used to ve- rabbit antibody

production to SMX-

3.2 Analysis of human sera for SMX-protein conjugates (in v h )

Nomal hurnan senun samples were assessed over the course of SMX-treatment

from days O to 12. In addition, SMX. SMX-HA and SMX-NO (300uM) were added

separately to day O senun samples and incubated at 370C, 5% CO2 over 24 hours and

also analyzed. Each of the sulfonamide samples was dissolveci in PBS containhg 1 0 8

DMSO. Based on the dye-binding procedure by Bradford (1976). total serum protein was

quaatif5ed using a Bio-Rad protein assay.

S e m samples (40 ug/ul) were diluteà in M sample buffer under reducing

conditions (20 46 v/v Glycerol ,4% sodium dodecyl sulfate (SDS), 0.02 % bromophenol

blue, 0.2 M Dithiothreitol (Dm) or non reciucing sample buffer dong with upper gel

buffer and then boiled for 5 minutes in order to denature the proteins. Using SDS-

polyacrylamide gel electrophoresis (SDS-PAGE) in a Bio-Rad minipro tein II gel

apparatus, proteins were separated according to size. Fiiteen microlitres of sample was

loaded onto a 15 cm 7.546 or 12.5% discontinuous polyacrylamide separating gel with a

5% stacking gel according to the procedure by Laemmli (1970). Gels were run at 130 V

for approximately 1.5 hrs or until the samples reached the bottom of the gel. The gels

were than transferred by tank blotihg to nitroceUulose membranes for 1 hr at 100 V. The

membranes were blocked with 3% nonfat skim milk powder and 1% BSA in TBS at room

temperature for 3 hrs. Immunoblotting was performed at 1: 100 dilution of the anti-SMX

IgG antibody in TBS containing 0.2% non-fat skim milk and 0.1% Tween. Membranes

were incubated ovemight at 4OC. The primary antibody was decanted and the membranes

were thoroughly ~ s e d three times at ten minute intervals with TT'BS. Biotin- labeled goat

anti-rabbit IgG was added as the secondary antibody to the membranes at a dilution of

1:25 000 in TTBS for 1 hour at room temperature. Membranes were subsequently washed

as described above. Streptavidin horseradish-peroxidase was then added to the membranes

at a dilution of 1: 1500 in T ïBS and ailowed to incubate for 20 minutes. The membranes

were washed 3 times in TTBS and the bound IgG was visualized using enhanced

cherniluminescence (ECL) and developed on autoradiographic f i at suitable exposure

times,

3.3 Analysis of senun from HIV patients for SMX-protein conjugates (in vivo)

S e m samples were obtained from the blood of 11 HIV patients undergoing a

desensitization study with cotrimoxazole- Sainples were coliected on days 0-4, 10 and 30

of the therapeutic regimen. The sera were heat inactivated for 30 minutes and diluted with

2 X sample buffer to generate a final concentration of pmtein of 40 uglul, Gel

electrophoresis and detection were perfomed as described in section 3.2 to detect any

SMX-haptenated proteins.

3.4 Analysis of serurn from normal subjeets for SMX-protein conjugates (in vivo)

Fiteen normal, healthy volunteers were recruited from the University of Western

Ontario and John P, Robarts Research Institute, The individuals were either students or

employees of the iiniversity or institute. Each subject was informed of the purpose of the

study and gave informed and written consent pnor to his or her participation in the study.

Ethics approval for the study was obtained from the University of Western Ontario. There

were six fernales and nine males ranging from 19 - 48 years of age. None of the individu&

had pnor exposure to sulfonamide therapy, nor did they have any known adverse reactions

to other antibiotics. Subjects were given NOVO-TEUMEL 8 DS (Cotrimoxazole) which

containeci in each capsule 800 mg suifamethoxazole and 160 mg trimethoprim. Individuals

were required to take two oral tablets daily (one in the moming and one at night at

approximately the same t h e each day) for ten days. The initial loading dose was doubled

for subjects 1 1 through 15 on day O of the study. Each moming on days 0,3,6,9 and 12

of the study, 15 mls of blood was coliected via venipunchire from the subjects and the

semm subsequently isolated. Peripheral blood mononuclear ce& (PBMCs) were also

isolated by layering blood on Ficoli gradients and coliecting the interface after

cenaga t ion . PBMCs were then resuspended in PBS to yield a cell concentration of 106

celis/rnl For each subject (1 through 10) total leukocyte and differential counts, ceU

toxicity and cellular glutathione assays were performed. Serum sainples were analyzed by

Western Blot argdysis and probed with antibodies directed towards SMX to determine the

presence of any SMX-haptenated senun proteins. Liver function tests were &O performed

using serum aquired from the subjects.

3.5 Predictive tests for reactions to SMX

3.5.1 Total leukocyte and differential counts

Total number of viable leukocytes were counted manually under Light microscopy

using a haemocytometer. CeU viability was assessed using trypan blue dye exclusion, a

measure of cell membrane integrity.

Examination of leukocyte subsets were performed for each subject Using Eght

microscopy (40X), a randorn sample of 100 PBMCs were observed, counted and

categorized according to their rnorphological and structural classification (neutrophils,

lymphocytes. monocytes, eosinophils and basophils) using Wright's stain.

3.5.2 Ce11 toxicity using MTT assay

The toxicity of SMX and SMX-HA incubated with PBMCs was measured using an

in viîro toxicity assay as described previously (Shear et al., 1986, Mossman, T. 1983).

PBMCs were counted and distributed in PBS to a 96 weil-microtitre plate (1 -0 x

105/well). SMX or SMX-HA was added to yield concentrations ranging from 25 uM to

2 0 uM for each well. Each concentration was tested in triplkate. The celis were then

incubated for two hours at 37*C/5% C02. The PBMCs were washed, incubated in RPMI

medium containing 10% fetal caK senim, 100 ug/ml strep tornycin, 100 U/ml penicülin and

50 uM 2-beta-mercaptoethanol for 18 hours then washed three times with PBS. Cell

viability was assessed usuig the 3-(4,5-dimethylthiazo1-2-y1)-2$-diphenyl te~azofiurn

bromide @KIT) assay- M T ï is uglized as a measure of cellular viability as only those cells

with functional mitochondrial dehydrogenase can convert the substrate M I T to an

insoluble colored forrnazan produc t that can be measured spectropho torneeically using a

Vmz kinetic microplaie reader. Twenty microlitres of M ï T (5 mg/&) was added to each

welL Following a 3 hr incubation at 370US%C$, the absorbance was measured at

560 nm and the cytotoxicity was measured as a percentage in the reduction in absorbance

relative to controls, which were not incubated with the SMX or SMX-HA,

3.5.3 GIutathione assessrnent

Levels of glutathione were quanbfied in lysates of PBMCs from each subject (1

throughl0) according to the method of Cnbb (1989) with minor modifications. In short,

each sample was prepared in 0.1 mM sodium phosphate buffet (pH 7.5) containhg 1 mM

EDTA Twenty five microlitres of 6 mM Dithio-bis (2-nitrobenzoic acid) (DTNB), 25 ul

of 1.0 rnM NADPH and 25 ul of reductase standard (0.7 uM to 50 uM) or the sample to

be assayed was added to tripkate wells of a 96-well plate. The reaction was initiated by

the addition of 25 ul of 3-25 m M glutathione reductase to each well except those wells

designated as blanks. Reagents were added using a multi-channel pipette. The absorbance

of the product 5-thio-2-nitrobenzoate (TNB) is followed spectrophotometrically at a

wavelength of 405 m. The absorbance was measured every six seconds for a total t h e of

one minute by kinetic plot mode using an automated plate reader and the SofmaxO

program. The rate of the reaction is proportional to the glutathione concentration in each

weU,

3-5.4 Liver function tests

The levels of diagnostic hepatic enzymes in semm were measured to assess liver

damage or inflammation in subjects who received cotrimoxazole therapy. Specifcally.

serum glutamic pyruvic transaminase (SGFT) levels were assesseci using senun fiom

patients which was quantified using a GP Transaminase diagnostic kit (Sigma Diagnostics,

St. Louis, MO, USA)- This hepatic enzyme catalyzes the transfer of a-amino groups from

selective amino acids Le. danine, to a-ketoglutaric acid to generate pyruvic acid The

quantity of keto acids are detennined colorimetricaily from the reaction with a subsh-ate

(2,4-dinitrophenyl-hybzine). The absorbante was read at 505 nm ushg an automated

plate reader and SofmiaxO program and the IeveIs of SGPT in semm of patients were

compared to baseline (day 0) values. This assay was performed according to the

methodology in the diagnostic kit using 200 ul of crude senim. SGIT levels are expressed

in Sigma-Frankel Unittdd (U/mi)- Detection limits are as low as 5 UfmL

4.1 Production of anti-SMX antibodies

To test for the presence of polyclonal antibodies against SMX-KLH, dot blots

were performed. Titres from antisenim were detected against dïfferent drug, hg-carrier

combinations (Figure 4a). The levels of antibody against SMX-KLH were higher

qualitatively in the post immunization serurn of rabbits, as indicated by the colour

development on the nitrocellulose membranes, compared to pre immunized sera fiom the

same rabbits (absence of colour).

As weil, rabbits irnrnunized with SMX-OVA, ais0 had higher levels of polyclonal

antibodies to SMX-OVA in post immunization senun cornpared to pre immunized sera

(Fig 4b).

In order to establish sufficient production of specific antibodies to SMX-KLH

direct ELISA'S were perfomed. Antisera was diluted from 1: 100 to 150 000 and added

to 96 weU plates coated with either OVA, SMX-OVA or SMX-BSA, The rabbits

developed high antibody titres against SMX as compared with the titres of the rabbit pre-

sera as determined by ELJSA (Figure 5). Both Rabbit # I l and Rabbit #12 had similar

levels of antibody production to SMX and antiser~ fiom Rabbit 4612 was used in al l the

experiments unless otheINvise stated. There were low antibody titres to OVA which may

be attributed to the non-specific binding of the secondary goat-anti-rabbit antibody IgG

conjugated alkaline phosphatase or cross reactivity with OVA.

4.2 Analysis of SMX-haptenated proteins (in vitro)

S e m samples of five individuals not receiving concurrent sulfonarnide therapy

were each treated with either 300 uM SMX or 300 uM SMX-HA. Each sample was

rnaintahed at 370C, 5% CO;? for either 0,0.25,2,4,6,12, or 24 hrs and frozen until

analysis via gel electrophoresis and detection by ECL. Samples treated with SMX only,

Figure 4% Confirmation of the presence of anti-SMX-KLH antibodies using dot blots.

Sarnples of A Rabbit s e m B. OVA C. SMX-OVA and D. KLH were applied to

nitrocellulose membranes and immunoblotting was performed using either pre or post

rabbit semm as described in the methods section 3.2.2. Row 1. Pre immune sera Rabbit

#Il 2. Post immune sera Rabbit #11 3. Pre immune sera Rabbit #12 4. Post immune

sera Rabbit # 12 5. Secondary antibody alone 6. Secondq antibody with Goat serum.

4b. ConCrnation of the presence of anti-SMX-OVA antibodies using dot blots. Sarnples

of A. BSA B. SMX-BSA C. OVA D. SMX-OVA E. KLH and F. Rabbit sera were

applied to nitrocellulose membranes and imrnunoblonuig was peiformed using either pre

or post rabbit semm as described in the Methods section, Row 1. Pre immune sera Rabbit

#11 2. Post immune sera Rabbit #Il 3. Pre immune sera Rabbit A 4. Post immune sera

Rabbit A 5. Pre immune sera Rabbit B 6. Post immune sera Rabbit B 7. Secondary

antibody done.

b) A B C D E F -.-

A

. . a 1 - O

Figure 5. The quantification of specific rabbit anti-$MX-KLH antibodies by ELISA The

weUs were coated with OVA, SMX-OVA or SMX-BSA and diluted rabbit senun (either

pre immunization or p s t Minunkation) was subsequently added- The absorbance at 405

nrn was measured using an automated plate reader and SofunaxO program. Samples were

performed in triplkate. Each data point represents the mean + standard error absorbance

for each dilution.

- - PRE OVA POST OVA - - PRE SMX-OVA

-POST SMX-OVA - - * - - PRE SMX-BSA -POST SMX-BSA

O ! I 1 1 1 r l 1 1 4 100 500 1000 5000 1 0000 50000

Dilution

did not reveal the presence of any specific SMX-haptenated proteins over the course of 24

hours (Egure 6a). Samples treated with SMX-HA however, showed S M - H A haptenated

bands after incubation for 15 minutes and more prominently &ter 24 hrs. The number of

haptenated proteins detected by the anti-SMX antibody ranged from 3 to 6 proteins, with

the haptenated proteins at molecular masses of 66 m a , 40 kDa and 30 kDa

predominating. The 66 kDa protein corresponded with the molecula. weight of human

serum albumin. Samples of SMX-HA were then assessed over the course of 24 hours as

perfomed with serum samples treated with SMX (Figure 6b). SMX-HA haptenated

proteins were detected at 66 kDa and 40 kDa after 15 min of incubation with the drug

370C/5% C02. Also, SMX-NO was incubated for 24 hours with semm and SMX-

haptenated proteins were detected at approximately 66 kDa and 40 kDa (Figure 6 ~ ) .

Published reports suggest that a 42 kDa protein may be involved in reactions to

SMX therapy (Gruchdla et aL, 1998). Therefore, it was important to identify and

characterize the 40 kDa protein band that was observed in SMX-HA treated serum. To

investigate the unlaiown identity of the 40 kDa protein band, a monoclonal rabbit anti-

alpha-1 acid glycoprotein (MG) antibody was conjugated to sepharose beads and was

incubated with SMX-EL4 treated serurn or untreated serum to immunoprecipitate the

specifïc haptenated protein.

In short, 200 ul of Protein A-agarose beads (2.6 mg/ml) were mked in a 1.5 ml

Eppendorf tube at 40C with 200 ul PBS and 7 ul(10 uglul) of a monoclonal anti-rabbit

AAG antibody on a rotary mixer for 2 hrs. After 2 hrs, the tube was centrifuged at 12 000

g for 10 minutes. The supematant was removed and the pellet was washed 10 times with

PBS. The pellet was resuspended and aliquoted equally into two tubes containhg either

10 ul of untreated crude semm or SMX-HA treated crude s e m . The samples were mixed

with 300 ul of PBS and placed on a rotary mixer at 40C for 2 hrs. After the 2 hrs the tubes

were centrifuged at 12 000 g for 10 minutes. The supematant was removed and the pellet

was washed twice with PBS. The pellet was resuspended and 30 ul of sample buffer was

Figure 6a. Western blot analysis of serum samples that were treated in vitro with SMX

(300 uM) or SMX-HA (300 uM) and incubated over the course of 24 hours at 37OC/5%

CO2 (n=5). Haptenated protek correspondmg to 66 kDa, 40 kDa and 30 kDa were

detected ushg the ad-SMX-KLH antiidy after 15 minutes (0.25 hrs) of incubation with

SMX-HA and were more intensely stained d e r 24 hom.

6b. Western blo t a i s of serum samples treated in vitro with SMX-HA (300 uM) and

incubated over the course of 24 hours at 370C/5% CO;? (~5). SMX-HA haptenation

appears to be present d e r 15 minutes (0.25 hrs) and is quite prevaient after 1 hour of

incubation with the SMX-HA as indicated by the 66 kDa and 40 kDa protein bands. The

66 kDa protein appears to correspond with human serum albumin (HSA).

6c. Western blot anaiysis of serum treated in vitro with SMX-NO (300 uM) and incubated

for 24 hours 37OC/5% CO2 (n=3). A 40 kDa protein in the SMX-NO treated s e m was

detected and corresponded to approhteiy the same rnolecular weight as the 40 kDa

protein detected m sem treated with SMX-HA. L represents the molecular weia

Iadder.

SMX SMX-HA ! O 3 6' 9 12 24!-

added. The sample was then boiled for 5 minutes and the upper portion of the sample

buffer was removed for subsequent analysis via elecuophoresis.

Samples were analyzed and detected using the anti-SMX-KLH antibody and

Western blot analysis (Figure 7). In lane B (SMX-HA treated) an SMX-haptenated

protein appears at approximately 40 kDa Moreover, the serum sample that was treated

with SMX-HA and immunoprecipitated with anti-AAG antibody (lane D), showed the

presence of a protein at 66 kDa and more prominently a protein band at 40 kDa The

untreated samples (Lanes A, C) did not show any specific SMX-HA haptenated proteins.

4.3 AnaIysis of s e m from HIV patients

S e m samples from 11 HIV patients receiving cotrimoxazole therapy as part of

the desensitization protocol were examined. Blood was collected on days 0,4, 10 and 30

of the drug regimen and serum samples were isolated. S e m from 11 HIV patients in

total were examined on Days 0,4,10 and 30 ushg the Western blot technique. None of

the HIV patients had any specific SMX- haptenated proteins present (Figure 8a). The non-

specific banding pattern most iikely attributed from the secondary antibody on day O

serum did not differ from that serum on days 4,10 or 30 in any of the patients. SMX-HA

ueated sera (day O) of an HIV patient was used as a positive controI.

4.4 Assesment of normal subjects receiving SMX therapy

4.4.1 Evaiuation of subjects administered SMX therapy

F'iteen subjects were given cotrimoxazoIe for 10 days. Seven of the 15 subjects

had adverse reactions over the course of therapy. An outline of the complications from

subjects receiving cotrimoxazole therapy is presented in Table 1. Two of the subjects (#6

and #15) had severe maculopapular rashes covering their entire body and were treated

with anti-histamines and both subjats discontinued therapy on day 8. Blood samples were

st3.I coltected on day 9 and 12 to complete the shidy. Three subjects (#7, #11 and #14)

Figure 7. Western blot analysis of semm of subject #6- S e m was treated for 24 hours

with SMX-HA and immunoprecipitated with a rabbit monoclonal alpha 1 acid

glycoprotein antibody (AAG). Samples were than run on gel elecirophoresis and detected

with anti-SMX-KLH antibody using the Western blot technique as previousiy described.

Lane A, Untreated senun, day O B. SMX-HA treated serum (24 hour incubation)

C. Untreated serurn immunoprecipitated with monoclonal AAG antibody on day 0. D.

SMX-HA treated s e m immunoprecipitated with monoclonal M G antibody. Lane L

represents the molecular weight ladder. A 40 kDa protein was detected in Iane D

correspondhg to the 40 kDa protein detected in Lane B.

Figure Sa Western blot andysis of semm from HIV patients #8 and #11 undergoing a

desensitization study using cotrimoxazole therapy. Semm samples were taken on days O,

4,10 and 30 of treatment SMX-HA was bcubated for 24 hours with s e m from day O of

HIV patient #11 and used as a positive conirol. L represents the molecular weight ladder-

Sb. Western blot analysis of sera from subjects #9 and #10 on days 0.3 .6 and 9 of

cotirnoxazoIe therapy. SMX-HA was incubated for 24 hours with serum from day O of

subject #9 and used as a positive control. L represents the molecular weight ladder.

Subject # I O Su bject #9

Table 1. O v e ~ e w of the characteristics of the normal subjects who participated in the

cotrimoxazole snidy. Clinical and laboratory findings and complications were documented

for each patient during the course of the snidy. Subjects 1 through 10 were administered

NOVO-TRIMEL @ DS (800 mg sulfarnethoxazole/ 160 mg trimethoprim) twice dafiy for

10 days. Subjects 11 through 15 were also administered cotrimoxazole twice d d y excepr

for the Iùst day when the initial loading dose was doubled.

Gender

M

Complications reported

Day 10, developed mild rash on ankles, pniritis

Day 4, developed mild &ver, discontinued therapy

Day 8, developed rash on legs discontinued therapy; Day 9, developed severe maculopapuiar rash

Day 10, developed mild rash on arms, pruritis

Day 4, developed mild rash on m s , pmritis, discontinued therapy

Day 10, mild rash on arms, pniritis

Day 9, developed severe maculopapular rash, discontinued therapy

had mild maculopapular rashes and pruritis localized to the arm. Subject #l had a mild

ankle rash and pniritis, while subject #4 had a fever that was believed to be attributed to

the dmg therapy and discontinued therapy &ter day 4- Subject #4 had bIood collected on

day 6 but not day 9 or 12.

4.4.2 Analysis of SMX-haptenation (in vivo)

Blood samples (15 ml) were c o k t e d by venipuncture from each subject on days

0,3,6,9 and 12 of the 10 day SMX dosuig schedule. A fraction of the blood obtained (5

ml) was added to non-heparinized tubes for isolation of serum. The remaining 10 mls of

blood was added to heparinized tubes for isolation of PBMCs. S e m sampies from each

of the subjects were analyzed using the anti-SMX-KLH antibody and Western blot

technique as described with the in virru samples- In the 15 subjects whose sera was

investigated, there were approxirnately three to four protein bands evident. However,

these bands tha.t were detected were attributed to non-specikïc cross reactivity and binding

of the secondary labeled biotin goat-anti-rabbit IgG an tibody and streptavidin-horseradish

peroxidase agents. Moreover, the bands present on day 3,6 ,9 and 12 were not

significantly different than that from each patient (subject #9 and #IO) on day O (Figure

8B). Serum from individuals who tolerated covirnoxazole therapy did not show any

ciifference in banding pattern from those subjects who developed a reaction- SMX-HA

treated sera (day O) in vimu was used as a positive control to r e c o n f i that the Western

blot system was functioning pmperly-

In an atternpt to further investigate the formation of any SMX-haptenated proteins

in vivo, a number of modifications to the original Western blot protocol were made, First,

rabbit anti-Sm-KLH antibody was added to sepharose beads as described with the

irnmunoprecipitation with the monoclonal PLAG (Results 4.2). The antisera conjugated to

the beads was treated with either sera (day O) from subject % or #8 incubated with SMX-

HA or sera collected from days O and 6 and Western blot analysis was performed. No

SMX-haptenated protein bands were present on days O or 6 while a 40 kDa S M - H A

haptenated protein was present (positive control) in both subjects' sera treated with SMX-

HA (Figure 9)-

Next, the reducing agent DïT was excluded £rom the standard conditions as

previous reports suggest that reducing agents may disrupt the covalent bonds between

haptens and selective proteins (Cnbb er al., 1996)- Therefore, Western blots were run with

and without D m in the sample buffer (Figure 10). The serum proteins that were run

under non-reduced conditions migrated a slightiy shorter distance than those proteins

under reducing conditions, as observed by the 40 kDa SMX-HA treated semm sample-

More irnportantly, there was no detection of any SMX-haptenated protein bands in any of

the sera from subjects receiving coaimoxazole therapy.

Preliminary reports identified SMX-haptenated proteins from sera of HIV patients

undergoing desensitization with coaimoxazole therapy using 7.5% gels in Western blots

(Gruchalia et al., 1998). Thus, the percentage acrylarnide that was currently being used

was reduced from 12.5% to 7.5% in an attempt to detect SMX-haptenated proteins. The

serum samples were serially diluted to 1:4 in PBS and were run on a 7.5 % gel under non-

reducing conditions. The serum of subject #6 was treated with SMX-HA and &O

assessed. A non-selective band was present corresponding to a molecdar weight of

approximately 55 kDa However, no SMX-haptenated protein bands were detected in the

HIV serum while a 40 kDa SMX-HA haptenated protein was detected in SMX-HA in

vitro treated semm from subject #6 (Figure 1 1).

Fially, we investigated serum samples with a primary antibody also generated in

rabbits against SMX-KLH, donated by Dr. Alastair Cnbb (University of Prince Edward

Island). Western blots were performed at 7.5% acrylamide and under non-reduced

conditions. Upon analysis, no SMX-haptenated proteins were detected. A non-selective

SMX-haptenated protein approximately 55 kDa was present in both HIV patients and

normal subjects (Figure 22).

Figure 9. Western blot analysis of serum samples of normal subjects treated with

cotrimoxazole therapy. Anti-SMX-KLH antibody was fïrst conjugated to sepharose beads

and then incubated with either SMX-HA treated senun (day O), day O senun (untreated)

or day 6 serum of subject #6 and #8 . A 40 kDa protein was identified in both SMX-HA

treated s e m from the subjects. R is pre immune rabbit serum. L represents the molecular

weight ladder.

Subject #8 Subject #6

Figure 10. Western blot analysis of serum from subjects #6 and #8 during cotrimoxazole

therapy with (a) sample buffer containhg a reducing agent e-g. DTï or (b) sample buffer

with no reducing agent. A 40 kDa protein was detected with SMX-HA treated serum bot .

under reduced or non reduced conditions-

Su bject #6 Subject #8

Su bjectt #6 Subject #8 -

Figure 11. Western Blot analysiç of senun h m HIV patient B and semm (day O) from

subject #6 pretreated with SMX-HA. S e m samples were diluted up to 1:4 in PBS. The

gels were run using 7.5% polyacrylamide under non-reduced conditions. A non specific

band approximatley 55 kDa was present in HTV patient B. A 40 kDa protein was present

in the SMX-HA treated serum. H[V+ represents senun fkom an HIV patient undergoing

desensitization with cotrimoxazole. L represents the rnolecular weight ladder.

H IV+(B) Su bject #6 1:4 1:2 1:li L ;1:1 1:2 1:4 HIV+

66 kDa -b

55 kDa 4

40 kDa -+

Figure 12. Western Blo t analysis of serum from subject #15 over the course of

cotrimoxazole therapy. Rabbit anti-SMX-KLH antibody from Dr- Cribb was used as the

primary antibody (MW) to detect any SMX-haptenated proteins. The secondary

detecting system was perfomed as in the previous Western Blots. 7.5% gels were run

under non-reduced conditions- A non-specific protein band was present in all serum

samples at approximately 55 D a . Serum from HIV patients A and B were aiso

investigated for SMX-haptenated protehs.

H IV+ Subject #15

A B L O 3 6 9 12

4.5 Assesment of predictive tests

4.5.1 Total leukocyte and differential counts

On each day of blood collection the total number of leukocytes were estimated

using a haemocytometer under Light microscopy (Figure 13) for each subject (1 through

10). Subject # 4, who discontinued SMX therapy on day 4 &ter developing a fever, had an

increase in total leukocyîe counts on day 3 (18 million) h m day O (1 1.2 million). In

general, the total number of leukocytes over the course of therapy was quite variable for

each individual and did not change substantially from each day of drug therapy-

In addition, 100 leukocytes were counted, recorded and classified according to

their particular leukocyte subtype (e-g. neutrophils, lymphocytes, monocytes, eosinophils

or basophils) based on morphological examination. There did not appea. to be detectable

changes in the different leukocyte populations in subjects over the course of the 10 day

cotrirnoxazole therapy period (Figure 14). Of note, subject #3 had an increase in

eosinophil levels on day 6 which peaked by day 9, while subject #6 had elevated levels of

monocytes on day 9.

4.5.2 Ce11 toxicity assays

Investigation of the sensitivity of PBMCs to SMX-HA was assessed over the

course of cotnmoxazole therapy using a M ï T assay as a measure of cellular viability. A

concentration-dependent increase in toxicity by day 9 of PBMCs of subjects incubated

with SMX-HA (25 uM, 50 uM) was observed in comparison with baseline values (day O +

SMX-HA)(Table 2, Table 3). This reduction in cellular viability was most evident at

concentrations of 200 uM of SMX-K4 by day 9 of therapy compared to day O (SMX-HA)

(Table 4). Cellular viabitity of PBMCs incubated with SMX over the tirne course did not

change.

Figure 13. Analysis of total leukocyte counts of patients over the course of 10 day

cotrimoxazole therapy. Subject # 4 had increased levels of total white blood cells on day 3

with respect to baseline. There was no strong correlation between the levels of leukocytes

over the therapy penod.

Figure 14% Differential leukocyte counis from subjecrs on day O of cotrimoxazole

therapy and (b) day 3 of cotrimoxazole therapy. Each leukocyte was delegated to one of

five categories: neutrophils (neut), lymphocytes (lymph), monocytes (mono), eosinophils

(eosin) or basophils (baso) based on morphological examination using Wright's stain. A

total of 100 cells were counted,

Figure 14c. Differential leukocyte counts from subjects on day 6 and (d) day 9 of

CO trirnoxazole therapy .

Figure 14e. Dinerential leukocyte counts from subjects on day 12 of cotrimoxazole

therap y.

Percent Ce11 Viability

SMX-HA (25 uM) Cells SMX

Subject DayO DayO DayO Day3 Day 6 Day 9 Day 12

Table 2. The effects of 25 uM SMX-HA on cellular viability using an MTT reduction

assay. Each value was performed in triplicate and the data represents the mean percentage

of cell viability + standard error of the mean. The results are expressed as the percentage

of ceU viability compared to baseluie (day O). ND represents not detennined

Percent Cell Viability

SMX-HA (50 UM) Cells SMX

Subject Day O Day O DayO Day3 Day 6 Day 9 Day 12

- . - -

Table 3. The effects of 50 uM SMX-HA on cellular viability using an M I T reduction

assay. Each value was performed in triplicate and the data represents the mean percentage

of cell viability + standard error of the mean. The results are expressed as the percentage

of cell viability compared to baseline (day O). ND represents not deterrnined.

Percent Ceil Viability

SMX-HA (200 UM)

Ceils SMX Subject DayO DayO DayO Day3 Day 6 Day 9 Day 12

Table 4. The effects of 200 uM SMX-HA on cellular viability using an M?T reduction

assay. Each value was performed in triplicate and the data represents the mean percentage

of cell viability t standard error of the mean. The results are expressed as the percentage

of cell viability cornpared to baseline (day O). ND represena not determined-

4.5.3 Glutathione assay

Levels of GSH, were assessed by the GSH kinetic plot assay using cell Lysates of

PBMCs frorn each subject In general. there was no ciifference in GSH Levels over the

course of dmg therapy with respect to baseluie (day O) values (Table 5). Protein Levels

were nomalized according to the method descnbed by Bradford (1976). A standard curve

using known glutathione concentrations was completed before determining the

concentrations of the unknown GSH samples.

4.5.4 Liver function tests

SGPT Ievels in sera frorn subjects were examined over the course of cotrimoxazole

therapy in each of the subjects (# 1- #IO) (Table 6)- There was no apparent change

between the levels of SGPT over the period of dmg therapy with respect to b a d i n e

values. AU SGPT Ievels under 21 U/ml are normal values while values greater than 28

U/ml may indicate liver damage. A standard cuve of known pyruvic acid concentrations

was prepared prior to analysis of se- sarnples.

Glutathione concentration [UM]

Subject Day O Day 3 Day 6 Day 9 Day 12

TabIe 5. LeveIs of inuacellular glutathione of PBMCs were assessed using the kinetic plot

mode. Each sample was performed in triplicate. Each value represents the mean GSH

concentration standard e m r of the mean. The leveis of glutathione on days 3 ,6 ,9 and

12 of therapy were cornpared against baseiine glutathione concentrations (day O). ND

represents not determined-

Table 6. SGPT levels were quantined from serum of patients over the course of therapy

accorduig to the procedure outlined in the diagnostic kit. The absorbance was converted

into U/ml based on the SGPT standard calibration. Levels of SGPT below 21 U/ml

indicate normal transaminase levels. Water was used as a control (blank). ND represents

not determined.

Leveis of SGPT (Ufmi)

Subject Day O Day 3 Day 6 Day 9 Day 12

Water

5.0 DISCUSSION

5.1 Adverse reactions to sulfonamides

Sulfonamide therapy is used for the treatment of bacterial infections and treatment

and prophylaxis of PCP in patients ùifected with HIV. Unforturiately, this widespread use

is associated with a number of adverse reactions, most noticeably hypersensitivity

reactions. The onset of these reactions typically occur 7 to 14 days &ter initiation of dnig

therapy. Skin rash, fever, GI upset, multi-organ toxicity, urticaria, erythema mukifonne

and toxic epidermal necrolysis comprise a s p e c m of symptoms associated with these

idiosyncratic dmg reactions. The most serious adverse reactions to sulfonamides are

potentiaLly lilè threatening (Rieder, 1989). Therefore, it is increasingly important to

understand and elucidate the pathogenesis of adverse reactions to sulfonamide therapy.

The complex nature of these reactions remains a major obstacle to the

identification of factors that are direcdy or indirectly responsible for the development of

these unwanted events. Reports published over the 1 s t decade, although not conclusive,

suggest that production of a reactive intermediate or metabolite of sulfamethoxazole is

partidy responsible for the initiation of these reactions (Sbear et al., 1986, Cnbb et al-,

199 1, Rieder et al., 1994). Although a large proportion of the sulfamethoxazole dosage is

metabolized via N-acetylation to a non toxic intemediate that is readily excreted, a smaü

fraction of the parent compound remains vulnerable to oxidation via the cytochrome P450

monooxygenase sys tem. Of particuiar interest is the metabolite SMX-HA- This compound

whether generated in vitro metabolically by a murine rnicrosomal based system or

chemically synthesized, has demonstrated dose-dependent toxicity to PBMCs (Shear et

al-, 1986, Rieder et aL, 1989, Rieder et al., 1995). In addition, SMX-HA incubated with

microsoma1 fractions from iiver has k e n s h o w to bind covalently to proteins (Cribb et

al.. 1995, Cribb et aL, 1997). These fmdings suggest that not only is SMX-HA directly

cytotoxic but that it may dso act as a hapten and conjugate to plasma proteins thereby

acting as an immunogen and eliciting an immune response. Therefore, it is conceivable that

SMX-HA is largely responsible for the onset of these hypersensitivity reactions. Further

spontaneous oxidation of SMX-HA to a nitroso compund (SMX-NO) rnay also be

associated with augmented levels of cytotoxicity (Rieder et al., 1992).

Other metabolic and immunological mechanisms rnay also be associated with the

initiation of reactions. Inter-individual differences in metabolism of SMX rnay predispose

patients with an increased risk of development of adverse events. An individual possessuig

the "slow" acetylator phenotype rnay provide a metabolic environment encouraging the

production of reactive intermediates of SMX and thus increase the vulnerability of these

individuals to suIfonamide therapy (Rieder et al., 1991). Reduction in levels of protective

metabolic xavengers, such as glutathione, rnay also promote increased formation of

electrophilic compounds like SMX-HA which c m undergo fuaher spon taneous oxidation

to a more reactive nitroso group (SMX-NO). These products of rnetaboiic degradation

can lead to cellular toxicity and tissue destruction. Potential irnmunogenic SMX-protein

conjugates rnay not be effectively cleared or metabolized from the systemic circulation.

Thus, an irnmunological response rnay be generated in order to eliminate the undesirable

antigen,

Antibodies were generated to SMX-KLH in order to provide an accurate rnethod

of detecting the presence of SMX or SMX-HA haptenated proteins both in vitro and in

vivo. Utilization of this system would potentially provide valuable insight into the type,

size and nature of the protein k i n g haptenated. The ident5cation of a SMX-haptenated

protein rnay be clinicaIly beneficial in that this would serve or provide a predictive model

to determine the susceptibility of individuals towards adverse reactions to sulfonamide

therapy. Thus, patients at nsk for developing adverse reactions to sulfonamide therapy

could avoid the side effects by selecting alternative antimicrobial agents the9 cm tolerate.

Furthemore, a greater understanding of the rnechansims of adverse reactions to

sulfonamide therapy would be anticipated.

5.2 In Vibo Effects of Sulfamethoxazole and Sulfamethoxazole-Hydroxy lamine

Previous studies suggest that SMX or metabolites of SMX i.e. SMX-HA or SMX-

NO, may act as haptens and thus covalentiy bind to cellular macromolecules which may

facilitate an immune response (Meekins et al., 1994, Gruchalla et aL, 1998). The kinetics

of SMX and SMX-HA hapten-protein conjugation were investigated to determine the t h e

course of the potential binding of the drug to a plasma protein(sf in vitro. Human sera

acquired from patients not adrninistered suifonamide therapy and incubated with SMX and

analyzed using Western blots. reveded no conjugation of SMX to any selective plasma

protein . These findings indicate that in vitro, SMX does not covdently bind to plasma

proteins over the course of 24 hrs. Perhaps SMX is not inherently reactive with proteins

because of the Nq-amino group. However, when chemically synthesized SMX-HA was

incubated over the course of 24 hours with senun, Western Blot analysis revealed the

presence of three to six SMX-haptenated proteins. This indicates that although there is an

abundance of protein, there appears to be some degree of selectivity of binding of the

polyclonal antibody-SMX-KLH to SMX-haptenated proteins.

Most notable of these proteins were a 66 kDa and a 40 kDa protein. These

proteins generaily appeared after 15 minutes of incubation with SMX-HA and increased in

intensity over the course of the 24 hour period. The 66 kDa protein observed

corresponded to human serum albumin (HSA). Thus. a large fraction of the apparent

SMX-HA may bind non-selectively to HSA This is most iikely due to the relative

abundance of HSA in plasma (4 g/10 ml). HSA has a invinsic capacity to bind to neutral,

acidic and basic drugs. The other prominent haptenated protein, at 40 Da, was unknown.

The intensity of this band increased as a function of t he . This rnay result from saturation

of HSA with SMX-HA. As weLl, SMX-HA rnay undergo extensive degradation over tirne

and thus contribute to the non-selective binding of free groups of SMX-HA to proteins.

Sunilarly, various penicillin compounds have shown when incubated with serum

derived from CD1 Swiss white mice to haptenate certain serum proteins in vitro

(Warbrick et al., 1995)- Using analysis with Western blots, benzylpenicillin, cephdothin

and phenoxymethylpenicillin potassium were demonstrated, using a polyclonal antibody to

benzyIpenicillin, to conjugate to a serum protein that intensely stained at approximately

66 kDa, presumably mouse albumin. A nurnber of lower molecular weight bands were also

present but were Les intensely stained The nature and role of these haptenated-proteins in

mediating an immunolo@cal response was not examined The exact identity and functional

significance of the 1ess predominant conjugated proteins requires further investigation.

Nonetheless, it appears that binding of these penicillin compounds confers some degree of

selectivity, albeit rninor, to certain plasma proteins.

Incubation of exopnous SMX-NO with patient serum for 24 hrs, produced a 40

kDa haptenated protein when anaiyzed by Western blots. This observation reconGrms that

the rabbit antibodies generated to SMX-KLH recognize the same epitope from SMX-NO

as SMX-HA. As well, the N4 terminus of the SMX-NO appears to bind to a protein of

interest comparable in size to that of which SMX-HA conjugates. The chernical structure

of SMX-NO is slightly different than SMX-HA in that the hydroxyl group is replaced by a

nitroso species. This chernical modification may potentiate the binding of the nitroso

group to plasma proteins. SMX-NO is fomed via spontaneous non-enzymatic oxidation

of SMX-HA and may covalently bind to cellular macromolecdes inducing a grea t r

cytotoxicity than SMX-HA (Ccibb et at., 1991, Rieder, et al., 1995). Under physiological

conditions, cofactors such as GSH, NADPH and ascorbic acid may convert the SMX-NO

back the SMX-HA form where it can undergo further metabolism and detoxification.

The covalent binding of either SMX-HA or S M - N O to a plasma protein rnay

result in the formation of an antigenic or immunogenic complex. The formation of this

cornplex may provoke an Unmune response because of the production of thïs

"neoantigentf. Previous in vino studies involving covalent binding of radiolabelled

sulfadiazine to liver microsomal proteins have also demonstrated cellular toxicity (Shear

and Spielberg. 1986). This toxicity was ~ i ~ c a n t l y attenuated after supplementhg

human lymphocytes with the exogenous anti-oxidant GSH.

After observing this 40 kDa SMX-HA haptenated protein NI vitro, we attempted

to i d e n e and characterize this protein. Examination of a number of plasma proteins

ranging from 30 to 55 kDa range was performed. A thorough Literature search of plasma

proteins reveaied an abundance of a 40 kDa protein in the plasma commody kmwn as

alpha- 1 acid glycoprotein (AAG). Therefore, we investigated the possibility îhat this

protein rnay bind to SMX-HA, After treatment of semm with SMX-HA and

imrnunoprecipitation with a monoclonal antibody to AAG . we analyzed the semm using

Western blot analysis. Upon m e r observation. a 40 kDa protein was identified that

corresponded to the 40 kDa protein that was present SMX-HA treated serum. Thus, it

appeared that the protein haptenated by SMX-HA was most likely the plasma protein

AAG.

The identifcation of this AAG and its conjugation to SMX-HA in retrospect, can

be anticipated from the biological role of this pro tein. AAG, like HSA. is an important

binding protein in plasma, in particular to basic and neutral compounds. Levels of AAG

range between 50 and 100 mgllûû ml of plasma (Kremer et al., 1988). These levels of

AAG can Vary considerably depending on the pathological and physiological conditions of

individuals. Significant increases in the levels of AAG have been reported under various

circumstances of stress, burns, myocardial itifarctions, inflammation and infections in

cornparison with normal, healthy individuals (Kremer, et al., 1988, Voulgari et al., 1982).

Thus, it is plausible that patients receiving cotrimoxazole therapy for the treatment of

bactenal infections or PCP may have higher levels of the AAG plasma protein. This wouid

provide an excess or abundance of protein to which SMX-HA or SMX-NO could

potentially conjugate, leading to the formation of immunogenic complexes. Theoretically,

high levels of AAG in an individual combined with a slow acetylator phenotype may

potentially augment levels of SMX-HA in the semm and thus an increased tendency for

hapten-protein conjugation. However, considering all the participants in the study were

relatively healthy it would seem unlikely that individuais had higher levels of AAG before

initiation of SMX ueatment. Levels of AAG of subjects were not examined in this study,

Therefore it remains to be detennined, at least in vivo, if AAG is a major contributor in the

onset of adverse reactions-

HIV patients may also have increased levek of AAG, most likely attributed to the

disease state itself, degree of immunodeficiency, or opportunistic infections such as

Pneumocystis carinii pneunonia This may also account for the higher frequency of

adverse reactions to sulfonamide therapy. Previous snidies of Hnr patients treated with

clindamycin, an alternative antimicro bial to suifonamides, suggest that these patients have

altered pharmacokinetics of cLindarnycin relative to healthy individuals. These include

greater bioavailability, reduced plasma dmg clearance and lower steady-state volume of

disnibutions (Gatti, et aL, 1993). Under chronic conditions, individuals with HV may

have increased binding of basic drugs such as SMX or SMX-HA to semm proteins.

Consequently, the pharmacokinetics and pharmacodynamies of the antimicrobial agent

may be si@~cantly altered and therapeutic and desirable drug levels may not be achieved

or obtained. The mechanisrns of adverse reactions with clindamycin have not been clearly

understood. Therefore, it remains to be determined whether levels of AAG have a

signifîcant role in mediating immune responses to SM.-HA in HIV patients.

Based on the current findings in vitro, it is possible that AAG may conjugate to

SMX-HA or SMX-NO. Whether or not AAG binds to SMX-HA in vivo requires further

investigation. Indeed, the AAG has a propensity for binding to SMX-HA and SMX-NO,

but ihis conjugation may represent an indiscriminate or non speczc event that is observed,

and may not actually be a causative agent for the hypersensitivity reactions.

5.3 In vivo effects of Suïfamethoxazoie

After extensive analysis of the in vitro s e m samples treated with SMX -HA, it

became more important clinicdy to investigate the eEects of suIfonamide therapy in vivo.

Previous studies have demonstrated that patients receiving oral sulfonamide therapy rnay

generate SMX-haptenated proteins in their sera (Meekins et aL, 1994). These haptenated

proteins rnay be involved or responsible for the pathogenesis of adverse reactions

observed in patients treated with sulfonarnides. Therefore, we examined the sera from

HIV patients undergohg desensitization with cotrimoxazole prophylaciically to determine

the presence of any specific SMX-haptenated proteins. We also assessed the sera of

normal subjects receiving sulfonamide therapy for the potentid formation of hapten-

protein conjugates.

In the examination of the s e m from 11 HIV patients receiving oral cotrimoxazole

therapy as part of desensitization study, none of the patients had the presence of a SMX-

haptenated protein. However, the in vitro SMX-HA treated serum sample of a patient on

day 0, showed the presence of a 66 kDa protein and a 40 kDa protein as observed in the

normal patients.

Similady, 15 n o m d healthy subjec ts who received CO trimoxazole therapy twice

daily over the course of a 10 day penod, eight of the subjects tolerated suifonamide

therapy while seven of the subjects had mitd to severe drug reactions. Serum samples of

each individual were assessed via Western blots for the presence of the SMX-haptenated

proteins. None of the 15 subjects evaluated had any SMX-haptenated proteins present in

their sera. In each subject, a s e m sample from day O was treated with SMX-HA (300

uM) and incubated for 24 hrs, and m on each Western blot and used as a positive

indicator to ensure that our detection system was functioning properly. Each in vitro

treated SMX-HA semm sample revealed the presence of both a 66 kDa protein and a 40

D a protein. Thus, it appeared that our detection system using a polyclonal rabbit

antibody to SMX-KLH to characterize haptenated proteins was recognizing the SMX-HA

epitope in vitro.

These observations, in both sera of the 1 1 HIV patients and 15 normal subjects

suggest that SMX or metabolites of SMX, do not appear to conjugate with semm

proteins, at least not within the detectable limits of our Western Blot system. Our positive

control (SMX-HA treated se=) remains the only semm wherein the 40 kDa protein is

detected. Based on the in vitro data of SMX treated serum the absence of protein

conjugation in the sera may be the result of low Ievels of SMX-HA or SMX-NO in the

plasma If this is the case, there may indeed be hapten conjugation occurring with the

metabolites of S M . and the serum proteins, However, the system we employed may not

be sensitive enough to effectively measure or detect this SMXconjugated protein- The

arnount of SMX or its metabolites that is covalently bound is most likely dependent on the

quantity of protein available, the amount of covaiently bound SMX to the prirnary

antibody and epitope density on the target protein (Cnbb et aL, 1996). In addition. it is

possible rhat the hapten may dissociate from a conjugated protein during preparation of

the serurn sarnples or at other critical steps in the analysis such as denaturing, boiling and

reduction. It has been postulated that addition of a reducing agents such as DTT or

mercaptoethano1 in the sample buffer may encourage dissociation of the d m g from the

protein of interest (Cribb et aL, 1996). Although this does not seem iikely in the case of

covaiently bonded dnigs such as SMX-HA or SMX-NO, Western b1ot.s were performed

under both reduchg and non-reducing conditions. No SMX-haptenated proteins were

detected under either condition.

Revious reports suggested the presence of SMX-haptenated proteins in Westem

blots under conditions of 7.5 1 polyacrylamide (Gruchalla et al.. 1998). Therefore, we

investigated semm of subjects receiving cotrirnoxazole therapy using Western blots under

the same conditions (7.5% polyacrylamide). However, no SMX-haptenated proteins were

detected using this method. An attempt was made to use another source of rabbit-anti-

SMX-KLH antibody (from Dr. Cribb) for detection of haptenated proteins but none were

observed,

Results from the dot blots and ELISA suggest that the polyclonal antibody to

SMX-KLH does indeed recognize SMX epitopes. Alternatively, subjects may be clearing

the hapten-protein complex metabolically before the W u n e system recognizes the

potentiai antigen. Conceivably, there may a certain level of SMX or SMX-HA in the

systemic circulation required for the immune system to elicit a response. This potentid

"threshold" in plasma Ievels of SMX-HA. may potentially determine whether an individual

is more likely to develop ao adverse reaction. Levels of SMX-HA in the plasma or urine

were not investigated in this study.

Previous studies investigating the role of SMX haptenated proteins, indicate that a

SMX-haptenated protein may indeed exist (Meekîns et al., 1994). It was demonstrated

that two of three patients receiving sulfamethoxazole therapy had the presence of a 30

kDa protein in their sera after five days of treatrnent This haptenated protein was also

detectable 48 hrs &ter therapy had discontinuecl. These Fmdings represent the fxst

example of haptenation of human serum proteins by sulfonamides in vivo. Thus, based on

these observations, protein-haptenation appears to be selective for a particular protein

rather than indiscriminate (Meekuis et aL, 1994). However. the production of this hapten-

protein complex does not necessitate an absolute immune response as none of the three

patients reported any side effec ts or complications from sulfonamide therap y. Therefore,

this serum protein rnay become bound to SMX-HA M vivo, but whether or not this

increases the likelihood of an individual to develop an adverse reaction remains to be

detennined.

Studies investigating adverse dmg reactions in ElIV patients treated with

sulfonamide therapy also demonstrate SMX-protein haptenation (GruchaUa et aL, 1998).

Of four individuals treated prophylactically with SMX, one had the presence of a 40 kDa

protein in the senun. Six of eight patients, undergoing desensitization with coaimoxazole,

also had SMX-haptenated proteins present in the serum samples. Only three of the six

patients who showed evidence of a 40 kDa protein. had a skin rash. One of these patients

also had antibodies to SMX. Therefore, these fmdings indicate or suggest that although

SMX and or its metabolites may conjugate to a selective 40 kDa protein. dus does not

provoke an immune response in everyone. Three of the six (50%) HIV patients who had

protein-conjugation with sulfonarnides developed a noticeable rash, indicatuig hapten

formation may predispose an individual to a greater risk for development of an adverse

dmg reaction This does not account for the other three (50%) HN individuals who

developed reactions. Therefore, it appears that additional mechanisms, either rnetabolic or

irnrnunological are likely involved in the onset of these reactions.

The adverse reactions of the seven normal patients in the study who were

administered conimoxazole therapy are unexplaineci. The incidence is very high for the

number of healthy volunteers. The absence of any SMX-haptenated protein in these

patients suggests alternative mechanisms are involved in these reactions,

Adverse reactions to cotrimoxazole therapy rnay have occurred because of an

inability of the individuals to tolerate large doses of cotrimoxazole. Thus, a concentration-

dependent mechanisrn rnay be responsible for the induction of the response. The

prescribed dosing of cotrimoxazole therapy rnay Vary according to the extent and seventy

of infection. The patients in the present study were administered sulfonamides in doses

that represented the upper limïts for bacterial infections. Patients with severe foms of

PCP have k e n reported to have received 1600 mg sulfamethoxazole and 320 mg

trimethoprim, three times daily in order to successfully treat the infection (Hughes et al.,

1993). The patients in the current study received cotrimoxazole (sulfamethoxazole 800 mg

and nimethoprirn 160 mg) twice d d y , for 10 days. The patients who participated in this

study were considered hedthy and did not have any recurrhg infections. This dosage may

be sufficient to induce a reaction as evident by the 47% of individu& in the present study

reporthg adverse events. Similarly, Fischl and colleagues reported 15 of 30 (50%) HBr

patients receiving the same cotrimoxazole drug regimen and tirne course for the

prophylactic treatment of PCP, had skin rashes atuibuted to the antimicrobial therapy

(F i ih l et al., 1988). Therefore. it would seem logical that the oral dosage administered

should be reduced, as long as the dmg eff~cacy for the particular infection was sustained.

In contrasf other studies have strong arguments that lower doses (480 mg daily) of

cotrimoxazole may have a greater association with side effects than higher doses (960 mg

daily), with die onset of reactions occurrïng at a later time (Schneider et al., 1992). It

remains mcuIt to assess and compare normal healthy individuais with those patients who

are HIV sero positive with respect to mechanisms of reactions as the disease state itself

rnay contribute to altered drug tolerance levels. Nonetheless, it appears that alteration of

dosing intervals and concentrations of suifamethoxazole therapy need to be thoroughly

addressed In addition. l e s toxic sulfonamides rnay be warranted provided they are still

effective,

A cellular viability assay using M T ï was used to investigate the sensitivity of an

individuais PBMCs after treatment in viro with increasing concentrations of SMX-HA

over the course of cotrimoxazole therapy. At high doses of SMX-HA (200uA4, cellular

viability was markedly reduced in PBMCs on day 9 with respect to baseline (day O). By

day 12, the cellular viability , in general, retumed to levels comparable to baseLine. This

evidence suggests that at high concentrations of SMX-HA, that PBMCs rnay be more

vulnerable to dnig induced toxicity after a period of suifonamide therapy. However, this

did not provide an indication of whether an individual rnay or rnay not develop a reachon

as the toxicity Ievels varied in each subject.

Alterations or modifications in normal metabolic scavengers rnay also contribute to

the reactions observed in the patients receiving cotrimoxazole. Antioxidant substrates such

as GSH and N-acetylcysteine, under normal conditions, have a major role in eliminating

oxidative stress by acting as endogenous substrates during xenobiotic metabolism.

However, in HIV patients, reductions in GSH levels have been reported in patients

PBMCs, plasma and broncho-alveolar lavage (Buhl et al., 1989, Eck et al, 1989, van der

Ven 1995)- This reduction in the cellular defense mechanism rnay allow for the increased

formation of intracelluiar oxidants and reactive intemediates such as SMX-HA or

SMX-NO that rnay potentially bind to cellular macromokcuies faciLitating tissue and

cellular destruction- Thus, a s i m c a n t change in thiol capacity rnay contribute to the

increased incidence of these adverse reactions- Perhaps the subjects who demonstrared

adverse events to cotrimoxazole therapy had altered Ievels of GSH or Limited capabilities

of generating sufficient production of GSH cofactors. Therefore, GSH levels were

examined in subjects 1 through 10- However, levels of GSH were not different over the

course of the 10 day therapy as compared with baseline values. This observation suggests

that GSH levels are not altered in normal individuals receiving cotrimoxazoie therapy and

that additional factors rnay be contributing to the reactions observed.

Reduction in the levels of GSH rnay be associated with the increase in incidence of

reactions to sulfonamide therapy in HN patients. Concentrations of GSH, either free or

protein bound, rnay also indicate physiologicd States of oxidative stress. GSH that remains

free in circulation fùnctions in the preservation of thiol redox status. GSH that is bound to

proteins, in particuiar, CD4 lymphocytes of HIV patients, suggest the presence of

oxidative stress (van der Ven, 1995). In contrast, the addition of exogenous GSH to

PBMCs has reduced cytotoxicity as weil as inhibiting the conversion of the SMX to the

more reactive SMX-HA (Shear and Spielberg, 1985, Rieder et al., 1988, Cribb et al.,

199 1). Thus, it appears that anti-oxidant properties of GSH have a more important role in

inhibiting toxicity than those pathways involving glutathione dependency. IndividuaIs who

have had reactions to sulfonamide therapy do not have deficiencies in glutathione

tramfersase activity (Riley et al., 1991). Additionally, the rote of SMX-NO and how it

mediates sulfonamide toxicity requires hrther investigation in order to fully characterize

these reactions.

To determine if individuals were experiencing liver dyshinction or in£Iammation,

measurement of SGPT levels were examined Increases in SGPT levels over 28 U/ml may

indicate hepatic damage. LeveIs of SGPT were not altered in serum from subjects who

either tolerated or were sensitive to cotrimoxazole therapy over the ten day period. This

Einding suggests that a rnetabolic basis rnay not be involved or associated with the

O bserved reactions.

Total leukocyte and differential counts were d s o examined to determine whether

alterations would predict a patient at risk for developing a hypersensitivity reaction Total

white blood c e h counts remauied relative normal over the course of cotrïmoxazole

therapy. Leukocyte differential counts for each subjectwere also recorded However, there

was no substantial change over the course of therapy in leukoc yte populations. Subject #3

exhibited higher levels of eosinophils on day 6 and 9 but did not have any side effects.

Subject #6. who expenenced a severe maculopapular rash on day 8. had elevated levels of

monocytes on day 9 suggesting inflammation occurring* However, other individuals who

experienced reactions to cotrîmoxazole therapy did not have differences in the leukocyte

levels compared to baseline. Therefore, monitoring of leukocyte levels did not pravide an

accurate indication of whether certain s u b j e c ~ are at greater risk for a reaction.

The primary route of sulfonamide metabolism is mediated via Kacetylation of the

aromatic amine of sulfamethoxazole. The NAT enzyme represents a genetic polyrnorphic

enzyme in which individuals express the inherited phenotype of either a fast acetylator or

slow acetylator based on the caffeine standard measurements. Patients who possess the

slow acetylator phenotype are more likely to have the parent compound, SMX. subjected

to oxidative rnetabolisrn and subsequent formation of reactive intermediates such as SMX-

HA and SMX-NO. Previous studies by Rieder et al., reported 19 of 21 (90%) healthy

patients who previously experienced hypersensitivity reactions to sulfonarnides. were slow

acetylators (Rieder et al., 199 1). None of the patients in the current study was assessed

for acetylator p h e n o ~ . The risk of developing h ypersensitivity reactions appears to be

much greater in HIV sero positive patients than those individuds who are MV sero

negative. The degree of imrnunodeficiency, dosage of sulfonamicie therapy, concomitant

dnig therapy. coexistence of additional viral infections (e-g. cytomegalovim, Epstein-Ban

virus) and reductions in GSH levels may act hdependently or in combination, m e r

conaibuhg to the increased incidence of reactions in EIIV patients (Carr et al., 1993).

As well, in some HIV patients the levels of CD4 lymphocytes rnay provide an

indication of whether a person is at risk for an adverse reaction. Historically. as the disease

progresses in patients, levek of CD4 Lymphocytes slowly decline. This reduction in cells

may occur in a penod ranging from two to hfteen years and when CD4 levels drop below

200 x 106 cells L, the patients are diagnosed with AIDS (Janeway and Travers, 1994). In

the current study, a nurnber of critical dinical feahires regarding the HIV patients who

participated in the study have been lacking. These characteristics include CD4 lymphocyte

counts, viral loads. severity of infection and concomitant therapy. Previous snidies have

reported patients rechdenged with coVimoxazole therapy, who had CD4 counts less than

200 x 106 celldL, had a higher frequency of tolerating this dmg regimen as opposed to

those patients who had higher CD4 counts (Englehard et al., 1984). Ln another study. H W

patients with lower than 25 x 106 c e W L were l e s likeiy to sustain or develop a reaction

(Cm et al., 1993). In contrast, HIV patients with lower than 200 x 106 c e l l a were

reported to have a higher incidence of arnoxicilh-induced rashes (Battegay et al., 1989).

Nevertheless, it appears that correlating levels of CD4 lymphocytes and frequency of

reactions may not be an accurate estimate or predictive method of determinhg an

individu& potentid for a reaction.

5.4 Future directions

The underlying mechanisms of adverse reactions to sulfonamide therapy remain

unclear and poorly understood, An immunological and rnetabolic basis have been

proposed as possible contributors to these reactions. The incidence of these reactions

remains quite prevalent in HIV patients as compared with patients who have normal

hinctioning immune systems or patients with other irnrnunocompromised disorders

(Winston, et al.. 1980). A nurnber of po tential markers of hypersensitivity reactions need

to be further explored. Each individual's reaction to sulfonamide therapy is unique which

makes it increasingly difficult to characterize and predict an adverse event, Therefore, a

number of alternative measures need to be examined,

An important and novel approach would be to further investigate the

immunological role and its involvement in these reactions. Analysis and identification of

not o d y the cellular responses to sulfonamides but also the humoral responses. These

include examinhg the selective Unmunoglobulins and isotypes present in those patients

receiving sulfonamides. CorrelaMg antibody isotypes with reactions may indicate the type

of hypersensitivity reactions present. Gmchda and colleagues illustrated through the use

of skui prick tests that IgE may play a role in the onset of these reactions (Gruchalla et al.,

1991). As well, previous studies have demorrstrated that AXDS patients have higher Ievels

of immunoglobulins IgM and IgGI. and IgG3 that are specific for SMX (Jansson et al.,

1992, Lambin et al., 199 1. Daftarian et al.. 1995). It has been suggested that IgGl and

IgG3 bind to Fc receptors Iocated on both neutrophils and monocytes resuiting in the

formation of immune complexes whic h could potentially release inflammatory mediators,

The precise role of these hmunoglobulins remains uncertain but they may îndeed be

associated with the pathogenesis of the hypersensi tivity reactions in A I ' S patients.

Another strategy to employ would be to develop a practical and efficient animal

model to investigate hypersensitivity reactions associated with SMX. This would provide a

more reliable model to evaluate the metabolic and immunological effects observed in man.

This would allow for a more accurate assessrnent of the quantity of SMX and its

metabolites in the Iiver and the systemic circulation. Moreover, the role of hapten-

conjugation could be thoroughly examined and determined using an in vivo system. Levek

of GSH, N-acetylcysteine and other anti-oxidants could be measured to elaboraie on the

metabolic role of these idios yncratic reactions. Definencies in de toxification and

bioactivation of SMX and its metabolites could be assayed to determine if these factors

are directly or hdirectly involved. A study incorporating the use of a rat as a mode1 for

SMX-induced hypersensitivïty reactions has been demibed previously ( G a et al., 1997).

These fmdings suggeswt that the production of SMX-NO is a likely candidate for

induction of the observed hypersensitivity reactions and that an inability to effectively

detoxify these highly reac tive metabolites may prevent individu& frorn pro tectïng

themselves from the toxic effects of the metabolites, This approach would be potentially

beneficial to normal individuals who remain vulnerable to hypersensitivity reactions. In the

case of HIV-patients, an appropriate immunosuppressed animal mode1 wodd be needed

in order to explore the effects of SMX induced reactions.

Administration and treatment of exogenous antioxidants such as GSH and or N-

acetylcysteine to patients may aid in the reduction of hypersensitivity reactions,

particularly in HIV patients. These patients have a ~ i g ~ c a n t decrease in the levels of

GSH in the plasma as well as intracellulady (Staal et al., 1992). Also, plasma levels of the

GSH precursor, cysteine have been attenuated in HIV patients (Jarstrand et al,, 1990).

Therefore, it would seem logical to restore or replenish these metabolic scavengers

through the treatment of oral cysteine therapy. However, a study involving the treatment

of KXV patients with oral N-acetylcysteine did not increase Ievels of either GSH or

cysteine in the plasma of patients who were also treated with SMX-TMP therapy

(Akerlund et al., 1997). The dosage administered and the duration reported may have

been suboptirnal and further require higher doses of cysteine to sustain efficacy.

5.5 S u m m q and Conclusions

The intent of this study was to further illustrate the role of SMX-haptenated

pro teins and their association with adverse reactions to sulfonamide therap y, It was

dernonstrated in vitro that metabolites of SMX, Le. SMX-HA and SMX-NO when

incubated with s e m selectively bind to proteins that inciude human senun albumin and a

40 kDa protein. This 40 kDa protein was Iater idenùfed as AAG. It was dso shown that

the parent compound SMX does not bind to semm proteins.

Based on the in vitra results, investigation of SMX-haptenated proteins in vivo

was of paramount importance. S e m samples from subjects receiving coaimoxazole

therapy over the course of a ten day period a s well as semm from H l V patients who were

administered conimoxazole therapy during a 30 day period were evaluated using a SMX-

KLH antibody for the presence of any SMX-haptenated proteins. No SMX-haptenated

proteins were detected in either of the 15 normal subjects or 1 1 HIV patients receiving

cotrimoxazole therapy. Seven of the normal subjects exhibited adverse events to

coaimoxazole therapy. Modifications of die original Western blot protocol were

performed using 10 wer percentage gels, non-reduced sam ple buffers and alternative SMX-

p h a r y antibodies but there was no detection of any SMX-haptenated protein.

Additional analysis of patient total Ieukocyte and differen tial counts, cellular

toxicity assays, glutathone assays and liver function tests were completed in order to

predict those individuals who may be susceptible to sulfonamide therapy. Unfortunately, in

retrospect, none of these parameters that were assessed had predicitive value for which

individuals may be sensitive to SMX therapy.

It appears that these reactions are much more complex than originally anticipated.

The absence of any detectable SMX-haptenated proteins in the serum from patients

ueated with sulfonamides suggest that alternative mechanisms are involved in the onset of

hypersensitivity reactions. The nature of these mechanisms rernains elusive but both

imrnunologic and metabolic roles require further investigation to account for the

pathogenesis of these adverse reactions in patients receiving sulfonamide therapy.

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Letter of Information

Re: In vivo metabolism and immune effects of sulfamethoxazole and hydroxylamine metabolite

I nvestigators: Dr. Michael Rieder Dan Schiedel

Room 2.1 1 John P. Robarts Research lnstitute 100 Perth Drive, London, ON Phone: 663-5777 ext.4209

You are being asked to participate in a study that w'll examine the metabolism and immune effects of suIfamethoxazole. Sulfamethoxazole is an antimicrobial agent currentiy used to treat a wide array of bacterial infections. During treatment some individuals may experienœ side effects such as gastnc upset. loss of appetite, %ver and skin rashes. A small percentage of the general population (5%) is allergic to sulfonamide dnigs and any side efkcts should be reported to the researchers as soon as possible. Any adverse reaction, as minor as it is, will be assessed and we rnay ask you to withdraw from the study.

In the course of the study, you will be asked to take the medication Novo-Trimel (cotrimoxazole) which contains sulfamethoxazole (800 mg) and trirnethoprim (160 mg)/ per capsule, twice daily for 10 days. A 15 mL sample of blood will be collected on Days 0,3,6,9, and 12. Each visit should take approxirnately 10 minutes. Some volunteers rnay experience some bruising from the blood collection. The serum obtained will be subsequently analyzed for the presence of sulfonamide-haptenated proteins.

Confidentiality of the participants will be protected and in any publications the participants will not be identified by name.

You will be compensated $50.00 for your tirne. effort and inconvenience. This amount will be pro-rated if you have to wïthdrawal from the study at any time. Participation in the study is strictly volunteer. You may refuse to participate or withdrawal from the study at any time without any effect on yaur W O employment or academic standing.

Consent Form

In vivo metabolisrn and immune effects of sulfamethoxazole and hydroxylamine metabolite-

I have read the accompanying letter of information and have had the nature of the study explained to me and I agree to participate.

Ail questions have been answered to my satisfaction.

Date