18-1 Topics Type III hypersensitivity Type IV hypersensitivity.
SULFONAMIDE-SPECIFIC · PDF fileSulfarne thoxazole Sulfamethoxazole - bovine se- albumin...
Transcript of SULFONAMIDE-SPECIFIC · PDF fileSulfarne thoxazole Sulfamethoxazole - bovine se- albumin...
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
National Library m * I ofCanada Bibliothèque nationale du Canada
Acquisitions and Acquisitions et Bibliographic Sewices services bibliographiques
395 Weilington Street 395. rue Wellington Ottawa ON K1A ON4 OttawaON KlAON4 Canada Canada
The author has granted a non- exclusive licence dowing the National Lïbrary of Canada to reproduce, loan, distrïtbute or seli copies of this thesis in microfonn, paper or electronic formats.
The author retains ownership of the copyright in this thesis. Neither the thesis nor substantial extracts f?om it may be printed or otherwise reproduced without the author's permission.
YourNe vouenilénna,
Our fi& NcUre reGkBnte
L'auteur a accordé une licence non exclusive permettant à la Bibliothèque nationale du Canada de reproduire, prêter, distribuer ou vendre des copies de cette thèse sous la forme de microfiche/fÏlm, de reproduction sur papier ou sur format électronique.
L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation.
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.
REFERENCES
Aarts, MM. Metubolism and immune eflects of sulfamethoxuzole [master's thesis]. London, Ontario, The University of Western Ontario; 1 996.
Akerlund, B, Tyne& E, Bratt, G, Bielenstein, M and Lidinan, C (1997) N-acetylcysteine Treatment for the Risk of Toxic ciceactions to Trimethoprim-SUlpbamethoxazoIe in Rimary Pneumqstis carinni Prophy1axk in HIV-infected Patients. J of Infection. 35: 143 - 1 47.
Battegay, M, Opravil, Wuthrich, B and Luthy, R (1989) Rash with a~zloxicillui- chvulanate therapy in HN-mfeced patients (letter). h c e ~ 2: 1 100.
Bradford, MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilking the prhciple of pro tein-dye binding. Anal. Biochem. 72 : 248- 254.
Buhl, R, JafFe, HA, Holroyd, KJ, Wells, FB, Mastrangeli, A, Salt& C, Catin, AM and Crysd, RG (1 989) Systemic gluthathione deficiency in symptom-fkee HIV seropositive indIviduais. Lancet iï, 12941298.
Cam, A Gross, AS Hoskins, -M, Penny, R and Cooper, DA (1994) Acetylation phenotype and cutaneous hypersensitivity to trimethoprim-sulphamethoxazole in HIV- infected patients. HDS. 8: 333-337.
C m , A, Penny, R, and Cooper, DA (1 993) Efficacy and d e t y of rechallenge with low- dose trimethoprim-sulfamethoxazole in previously hypersensitive HIV-infected patients. A I X 7: 65-7 1.
Carr, A, Swanson, C, Penny, R and Cooper, DA (1993) Clinical and laboratory markers of hypersensitivy to triniethoprim-sulf'amethoxazole in patients with Pneumocystis carin ii pneumonia and A I D S . Jinfect Dis. 167: 180-185.
Coombs, RRA and Gell, PGH (1 968) Classification of dergic reactions responsibie for clinical hypersensitivity and disease. Ln: Coombs, RRA-Gell, P.G.H editors. Clinicd aspects of immmology. 2nd ed. New York: FA Davis: 575-596.
Cnib, AE, Leeder, S, J and Spielberg, SP (1 989) Use of a micro plate reader in an assay of glutathione reductase using 5,St Dithiobis (2-nitrobeozoic acid). A n d Biochern. 183: 195- 196.
Cnibb, AE, Miller, M, Leeder, JS, W, J, and Spielberg, SP (1991) Reactions of the nitroso and hydroxylamine metabolites of sulfamethoxazole with reduced glutathione. Drug Metab Dispos. 19: 900-906.
Cribb, AE, Miller, M, Tesoro, A, and Spielberg, SP (1990) Peroxidase-dependent oxidation of sulphonamides by monocytes and neutrophils from humans and dogs. Mol Phannacol- 38: 744-751,
Cribb, AE, Nuss, CE, Alberts, DW, Lamphere, DB, Grant, DM, Grossman, SJ and Spielberg, SP (1 996) Covalent binding of sulfamethoxazole reactive metabolites to human and rat liver subcellular fractions assessed by irnrnunochemical detection. Chem Res, Tuxicol. 9: 500-507,
Cribb, AE, PoN, LR, Spielberg, SP, and Leeder, JS (1997) Patients with delayed-omet sulfonamide hypersensitivi ty reac tions have antibodies recognizing endo plasmic re ticulum luminal proteins. J of Phar Exp Ther. 282: 10644072.
Cribb, AE, and Spielberg, SP (1990) Hepatic microsomal metabolism of sulfamethoxazole to the hydroxylamuie. Drug Metab. Dispos. 18,784787-
Cribb, AE, Spielberg, SP and GrifTin, GP (1995) N4-hydroxylation of sulfamethoxazole by cytochrorne P450 of the cytochrome P4502C s u b f d y and reduction of sulfamethoxazole hyciroxylamine in human and rat hepatic rnicrosornes, Amer Society P h a m Exp 77zerp. 23: 406-4 14.
Daftarian, Filion, LG, Cameron, W, Conway, B, Roy, R, Tropper, F and Diaz-Mitoma, F (1995) Immune Response to S~amethoxazole in Patienîs with AIDS. Clin and Diag Lab Immunol, 2: 199-204.
Domagk G.(1929) Eïn beitrag m g chemotherapie der bahrieuen ùifectionen. Dtsch Med Wochenschr. 61: 250-253.
Droge, W, Eck, HP, Gmunder, H and Mihm, S (199 1) Modulation of lymphocyte functions and immune responses by cysteine and cysteine derivatives. Am J Med. 91: 140s-144s.
Dunagin, WG and Millikan, LE (1980) Dmg eruptions. Med Clin North Am. 64: 983- 1003-
Eck, HP, Gmunder, H, Hartmann, T, Petzoldt, D, Daniel, V and Droge, W (1989) Low concentrations of acid-soluble thiol (cysteine) in the blood plasma of HIV-1 infected patients. Biological Chemistry Hoppe-Seyler. 370: 10 1- 108.
Englehard, D, Stutman, HR, Marks MI. (1984) Interaction of ketoconazole with rifampin and isoniazîd. N Engl Jof Medicine. 311: 1681-1683.
Evans, D AP (19 89) N-acety ltransferase, Pharmacol Ther. 42: 1 57-234.
Fischl, M, Dickenson, GM and Lavoie, L (1988) Safety and efficacy of sulfaniethoxazole and trimethoprim chemoprophylaxis for Pnemcys t i s carinii pneumonia in A I D S . LAMA- 259: 1185-1 189.
Gatti, G, Flaherty, J. Bubp. J, White, J, Borin, M and GarnbertogLio J (1993) Comparative study of bioavailabilities and pharmacokinetics of chdamycin in healthy vohnteers and patients with AIDS- Antimkrob. Agents Chemother. 37: 1 137- 1 243,
GZ, HJ, Sally, JH, Naisbin, DJ, Maggs, JL, Kitteringham, Nit, Pirmohamed, M and Park, KB (1997) The relationship between the disposition and immunogenicity of sulfamethoxazole in the rat J Pharm Exp Ther. 282: 795-80 1.
GoIden, JA (1989) Pulmononary complications of AIDS. In J . A Levy (ed), AIDS pathogenesis and treatment Marcel Dekker, Inc., New York p. 422-425-
Gordin, FM, Simon, GL, Wofsy, CB, and Mills, J (1984) Adverse reactions to trimethoprim-sulphamethoxazole in patients with the aquired irnmunodeficiency syndrome. Ann Intern Med 100: 495-499.
Gruchalla, RS, Pesenko, RD, Thinh, TD, and Skiest, DJ (1998) SuMonamide-induced reactions in desensitized patients with AIDS- The role of covalent protein haptenation by sulfarnethoxazole- J Allergy Clin Immunol. 101: 37 1-378-
Gnichalla, RS and Sullivan, TJ (199 1) Detection of human IgE to sulfamethoxazole by skin testing with sulfamethoxazoy1-poly-L-tyrosine- J Allergy Clin. Immunol. 88: 784- 792.
Hughes, W, Leoung, G and Kramer F (1993) Cornparison of atovaquone (55C80) with trirnethopnm-sulfamethoxazole to m a t Pneumocystis carinii pneurnonia in patients with AIDS N Engl J Med 328: 1521-1525.
laneway, CA and Travers, P (1994) Immunobiology: The immune systzm in health and disease. New York and London: Current Biology LtdGarland Publishing Inc. 1 1.1- 1 1.10.
Jansson, M. Wahren, G, Scarlatti, M, Principi, V, Lombardi, S, Livadiotti, L, Elia, A. Plebani, A, Wigzell, H and Rossi, P (1992) Patterns of immunoglobulin G subclass reactivity to HIV-1 envelope peptides in children born to HIV-1 infected mothers. AIDS. 6: 365-37 1.
Jarstrand, C, Akerlund, B and Lindeke, B (1990) hcreased nitroblue tetrazolium (NBT) reduction and Iow cysteine levels in asyrnptomatic HIV positive patients. Lancet. 335: 235.
Jou, Yi-Her, M-erro, PK, Mayers, GL, and B a n k e ~ RB (1983) Methods for the Attachent of Haptens and Proteins to Erythrocytes. Methods in Enzymology 92: 257- 277.
Kearns GL, Wheeler, JG, Childress, SH and Letzig, LG (1994) Serum sickness-like reactions to cefaclor. Role of hepatic metaboikm and individual susceptibility. J of Pediatrics, 125: 799-809,
Kremer, JMH, Wilting, J and Janssen, LHM (1988) Dmg binding to human alpha-1-acid glycoprotein in health and disease. Pharmacol Reviews. 40: 1-47,
Laemmli, UK (1970) Cleavage of strucniral proeins during the assernbly of the head of the bacteriophage T4. Nature. 227: 680-685.
Lambin, P, Gervais, A and Levy, M (1991) Intrathecal synthesis of IgG subclasses in multiple sclerosis and in aquired irnnunodeficiency syndrome (AIDS). Neuroirnmunol. 35: 179-189.
Lee, BL, Delahunty, T, and Safrin, S (1994) The hydroxylamine of sulphamethoxazole and adverse reactions in patients with aquired irnrnunodetlciency syndrome. Clin Pharmacol n e r . 56: 184- 190,
Lee, BL, Medina, 1, Benowitz, NL, et al., (1989) Dapsone, trimethoprim, and sulfamethoxazole blood levels during treatment of pneumocystis pneumonia in patients with aquired immunodeficiency syndrome: evidence of drug interactions. Ann Intern Med. 110: 606-6 1 1,
Lee, BL, Wong, D, Benowitz, NL, and Sullam, PM (1993) Altered patterns of drug metabolism in patients with aquired immunodeficiency syndrome. Clin Pharmacol Ther. 53: 529-535.
Leeder, JS, Cannon, MN, Nakhooda, A, Spielberg, SP (1988) Drug metabolite toxicity assessed in vitro with a purified, reconstituted cytochrorne P-450 system. J of Phamcol Exp mer. 245 : 956-962.
Leeder, JS, Dosch, HM, and Spielberg, SP (1991) Cellular toxicity of sulfamethoxazole reactive metabolites-1. Biochem. Pharmacol. 41: 567-574.
Leeder, JS, Nakhooda, A, Spielberg, SP and Dosch. HM (1991) Cellular toxicity of sdfonamide reactive metabolites. II: Inhibition of natural m e r ceU activity in human peripherd blood mononuclear ceus. Biochem Pharmcol. 41: 575-579.
Leftwich, WB (1944) An intradermal test for the recognition of hypersensitivity to the sulfonamide dmgs. Bull Johns Hopkins Hosp. 74: 26-48.
Marshall, BE, and Longnecker, ED (1990) General anesthetics, in Gilman AG, Rd, TW, Nies, AS, Taylor, P (eds): The Pharmacological Basis of Therapeutics, 8th ed. New York: Pergamon Press, 285-3 10-
Meekins,CV, Sullivan, Tl, and Gruchalla, RS (1994) Immunochemical analysis of sulfonamide drug allergy: IdentiFrcaiion ~Esulf'ethoxazole-substituted human semm proteins. J Allergy Clin Immunol, 94: 10 17- 1024.
Mitra, AK, Thummel, KE, Kalhorn, TF, Kharasch, ED, Unadkat, JD, and Slattery, JT (1996) Inhibition of sulfamethoxazole hydroxylamine formation by fluconazole in human liver microsornes and healthy volunteers. Clin P h a m c o l Ther. 59: 332-340.
Mossman, T (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assay- J Immun01 Methools. 65: 55-63.
Nisonoff, A (1957) Coupling of diazonium compounds to proteins. ln: Williams C A Chase MW eds- Methods in immunology and immunochemistry, vol 1. New York: Academic Press, 120- 126.
O'Neil, CE, Reed, MA, Lopez, M. Hyslop, NT Gutierrez, E, and Salvaggio, J (199 1) Evaluation of immune parameters in HIV+ subjects reporthg adverse reactions to sulfamethoxazole. Int Arch AlZergy Appl Immunol. 94: 246-247-
Park, BK, Coleman. JW, and Kitteringham. NR (1987) Drug disposition and dmg hypersensitivity. Biochem Pharmacol. 36: 58 1-590.
Patîerson, R, DeSwarte, RD, Greenberger, PA, and Grammer, LC (1986) Drug allergy and protocols for management of drug allergies. N Engl Rev Allergy Proc. 35: 1 13 1-47.
PohI, LR, Satoh, H, Christ, DD, and K e ~ a , JG (1988) The immunologie and metabolic bais of drug hypersensitivities- Annu. Rev. P h a m c o l . Toxicol. 28: 367-387.
Rang, HP and Dale, MM (1991) Pharmacology. 2nd edition 807-810.
Rich, AR (1942) The ro1e of hypersensitivity in periarteritis nodosa-as indicated by seven cases developing during serum sichess and saonamide therapy. Bull Johns Hopkins Hosp, 71: 123-140.
Rieder, MJ (1993) Immunophamacology and Adverse Drug Reactions. J Clin Pharmacol, 33: 3 16-323.
Rieder, MI, King, SM, and Read, S (1997) Adverse reactions to trimethoprim- sulfamethoxamle among children with human imrnunodeFiciency v h s infection. The Ped Inf Dis J . 16: 1028- 103 1-
Rieder, UI, Krause, R and Bird, IA (1995) Tune-course of toxicîty of reactive sulfonamide metabolites. Toxicology. 95: 14 1 - 146.
Rieder, UT, Krause, R Bird, IA, and Dekaban, GA (1995) Toxicity of SuIfonamide- Reactive Metabolites in W-Wected, HTLV-Infected, and Noninfected Celis. J of Aquire immun DefSc Syndro and Human Retro. 8: 134- 140,
Rieder, MJ, Shear, NH, Kanee, A, Tang, BK, and Spielberg, SP (199 1) Prominence of slow acetylator phenotype among patients with sulfonamide hypersensitivity reactions. Clin Pharmacol n e r . 49: 13-17,
Rieder, UT, Sisson, E, Bird, IA, and AImawi, WY (1992) Proliferation of T-lymphocyte proliferation by sulphonamide hydroxylamine. Int J lmmunopharacol. 14: 1 175- 1 180.
Rieder, MJ, Uetrecht, J, Shear, NH. Cannon, M. Milier, M and Spielberg, SP (1989) Diagnosis of sulfonamide hypersensitivity reactions by in-vitro "rechallenge" with hydroxylamine metabolites. Annak of Int Med. 110: 286-289.
Rieder, UT, Uetrecht, J, Shear, NH and Spielberg, SP (1988) Synthesis and in vitro toxicity of hydroxylamine metabolites of suIfonamides. J of Phannacol Exp Ther. 244: 724-728,
Riley, RJ, Cribb, AE, and Spielberg, SP (199 1) Glutathione tramferase-CL deficiency is not a marker to predispostions to sulphonamide toxicity. Biochem Pharmacol. 42,696-699.
Roederer, M, Staal, FJ, Osada, H and Herzenberg. LA (199 1) CD4 and CD8 cells with high intracellular glutathione leveis are selectively lost as the HIV infection progresses. Int Immunol. 3: 933-937-
Roitt, IM, Brostoff, J and Male. DK (1985) Immunology, 19.1-22.10, London, England, Gower Medical Publishhg Limited.
Satoh, H, Gilette, JR, Davies, HW, Schulick, RD, and Pohl, LR (1985) Immunochemical evidence of uifuloracetylated cytochrome P-450 in the liver of halothane-treated rats. Mol Phamcol . 28: 468-474.
Schneider, MME, Hoepelman, A M , Eeftinck-Schattenkerk, JKM, Nielsen, TL. van der Graaf. Y, Frissen, JPHJ et al. (1992) A controlled trial of aerosolized pentamidine or trimethoprim-sulphamethoxazole as primary prop hy laxis against Pneunw cystis carinii pneumonia in patients with human immunodeficiency virus infection New England Journal of Medicine. 327: 1836-1841.
Shear, MI, and Spielberg, SP (1985) In vitro evaluation of a toxic metabolite of sulfadiazine. Can J Physio Pharmacol. 63: 1370- 1372.
Shear, NH, Spielberg, SP, Grant, DM, Tang, BK, and Kalow, W (1986) Differences in metabolism of sulfonamides prediposing to idiosyncratic toxicity. Ann Intern Med. 105: 179- 184.
Spielberg, SP (1984) In vitro assessrnent of pharmacogenetic suscepâbility to toxic dmg metabolites in humans. P roc Fed A m Soc. Exp. BioL 43: 2308-23 13,
Spielberg, SP (1996) N-acetyltransferaîes: Pharmacogenetics and Clinical Consequences of Polyrnorphic Dmg Metabolism. J of Phannacok and Biopharm. 24: 509-5 19.
Staal, F, Ela, S, Roederer, M, Anderson, M, and Herzenberg, L (1992) Glutathione deficiency and hurnan immunodeficiency virus infection. Lancet. 339: 909-9 12-
Sullivan, TJ (1984) Allergic reactions to antirnicrobial agents: a review of reactions to drugs not in the beta lactarn class. J of Allergy Clin Irnmunol74: 594-599.
Tank, BK, Qian, L, Iriah, J, Yip, K. and Kalow, W (1991) Caffeine as a metabolic probe: validation of its use f ~ r acetylator phenotyping. Clin P h a m c o l mer. 49: 648-657.
Uetrecht, J (1985) Reactivity and possible significance of hydroxylarnine and nitroso metabolites of procainamide. J of Phannacol Exp Ther- 232: 420-425.
Uetrecht, J (1990) Dnig metabolism by Ieukocym and its role in dmg-induced lupus and other idiosyncratic drug reactions. Cris Rev Toxicol. 20: 2 13-235.
Van Der Ven, AJAM, Koopmans, PP, Vree, TB and Van Der Meer, JWM (1991) Adverse reac bons to cotrimoxazole in HIV infection. Lancet. 338: 43 1 -433.
Van der Ven, NAM, Mantel, MA, Vree, TB, Koopmans, PP, Van der Meer, JWM (1 994) Formation and elimination of sulphamethoxazole h ydroxylamine after oral administration of sulphamethoxazole. Bris Journal of Clin Pharmacol- 38: 147- 150.
Voulgari, F. Cummins, P, Gardecki. T, Beeching, NJ, Stone, PCW and Stuart, J (1982) Senun levels of acute phase and cardiac proteins after myocardial infarction, surgery and infection- Br. Hean J- 48: 353-356-
Vree, TE!, van der Ven, NAM, Venvey-van Wissen, CPWGM, van Ewijk-Beneken Kolmer, EWJ, Swolfs, AEM, van Galen, PM et al. (1994) Isolation, identification and determination of sulfamethoxazole and its known metabolites in hurnan plasma and urine by high-performance liquid chromatopphy. Jof Chromatography. 658: 327-340.
Warbrick, EV, Thomas, AL, Stejskal, V and Coleman, JW (1995) An andysis of 8-lactam derived antigens on spleen ceii and serum proteins by ELISA and Western blotting. Allergy- 50: 9 10-9 17.
Weinshilboum, RM (1987) The therapeuctic revolution. Clin P h a m mer . 42: 48 1-484.
Winston, DJ, Lau WK and Young, LS (1980) Trimethoprim-sulfamethoxazde for the marnent of Pnemocystis carntii pneurnonia Ann Intent Med. 92: 762-769.
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