NOVEMBEWDECEMBER 1994 VOLUME 7,NUMBER 6 0 Copyright 1994 by the American Chemical Society
Invited Review Structure-Activity Studies on DrugInduced Anaphylactic Reactions Brian A. Baldo* and Nghia H. Pham Molecular Immunology Laboratory, Kolling Institute of Medical Research, Royal North Shore Hospital of Sydney, St. Leonards, NSW 2065, Australia, and Department of Medicine, University of Sydney, Sydney, NSW 2006, Australia Received June 28, 1994
I. Allergy, Anaphylaxis, and Allergens In this review the term allergy will be used to denote an immediate hypersensitivity reaction mediated by IgE antibodies which develops usually within minutes, or even seconds, of contact with the provoking agent. Reactions may range from being mild, as in hay fever, to being violent and dangerous as in anaphylactic shock, a clinical syndrome in which the predominant clinical feature is cardiovascular collapse. Bronchospasm, airway impairment, and pulmonary edema are other lifethreatening manifestations of anaphylaxis (I). Immediate hypersensitivity reactions must be distinguished from delayed or cell-mediated hypersensitivities, which may take hours or even days to appear and which include contact sensitivities to poison ivy, chemicals, and metals such as platinum and reactions to tuberculin, trychophyton, and some drugs (2).Agents that provoke immediate hypersensitivity reactions and interact with IgE antibodies are known as allergens. Allergens occur throughout the biosphere in vertebrates, invertebrates, plants, fungi, bacteria, viruses, chemicals, and drugs and, in structural terms, range from complex macromolecules such as proteins and carbohydrates to simple chemicals and ions. Allergen-inducedreactions may occur in the skin, mucous membranes, airways, gastrointestinal tract, and blood and in organs such as the heart. Allergen sources that
* Address correspondence to this author at the Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, NSW 2065, Australia. Tel (02) 438-7272; Fax: (02) 439-2798.
provoke immediate reactions, for example, house dust mites, pollens, fungi, foods, etc., almost always contain multiple allergens, usually proteins or glycoproteins (3, 41,some of which are more allergenic than others in some subjects. Heterogeneity of allergens is not limited to the different macromolecules in a source but extends to individual allergenic molecules where more than one allergenic (i.e., IgE-binding) determinant usually occurs (5-7). Despite the large number of known and suspected allergenic proteins in many commonly encountered everyday sources, work has barely begun on identifying Band T-cell allergenic determinants (8-11) on different purified and characterized allergens. The study of the molecular basis of allergenicity is easier with “simple” organic chemicals such as drugs where the structures are already known and where it is usually possible to obtain both the compound and a variety of analogs in pure form (see section V).
11. Introduction to Drug Allergy Allergic reactions, sometimes anaphylactic, occur in a few subjects following exposure to some drugs and chemicals, and it seems certain that the frequencies of such reactions will increase. This is because it is now clear that such “small” molecules can provoke allergic responses, and because an ever-growingnumber of drugs are being taken orally, injected, inhaled, and absorbed through the skin and mucous membranes and, in both
0893-228xf94/2707-0703$04.50/00 1994 American Chemical Society
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704 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
metabolite
drug
D
+
+ soluble or cell-bound protein in the body
degradation product d
\
non-allergenic
hapten-protein conjugates P
non-allergenic
potentially allergenic
Figure 1. Diagrammatic representation of possible, and potentially allergenic, hapten-protein complexes that may form in vivo from a drug and/or its metabolite and degradative product(s). contracts airways smooth muscle
i
dilates and increases permeability of blood vessels
allergen cross-linking of IgE molecules degranulation
@ * e
**:
-stimulates secretions of mucous glands attracts eosinophils
-+
mature IgE-secretlng plasma cell
lgE antibodies
mast cell or basophll
“rrn
activates platelets
chemical mediators of hypersensitivity
Figure 2. Diagrammatic representation of allergen-mediated release from mast cell or basophil of pharmacologically-activeagents that cause allergic symptoms.
the home and the workplace, people are being increasingly exposed to a variety of chemical substances. A. Immunogenicity (Allergenicity) of Free and Conjugated Drugs. In addition to the chemical nature of the antigen, molecular size and complexity influence antigenicity. Substances of molecular mass less than 5 kDa, and sometimes up to about 10 kDa, are often poorlyantigenic or even nonantigenic. From the time of some of the earliest immunochemical studies on antigenicity (12)it has been assumed that a nonantigenic substance such as a chemical of “small” molecular mass (500- 1000 Da) can only prime for antibody formation and stimulate synthesis of antibodies after combination with a macromolecular carrier, usually protein. For drugs, the formation of such hapten-protein complexes may occur directly in vivo between the unmodified drug and an endogenous protein or cell membrane (12-14) or after metabolic or degradative changes to the drug (15, 16) (Figure 1). Despite long acceptance of this dogma, it is often not possible to demonstrate protein binding or, from the structure of the chemical, envisage how protein binding could occur even when all possible metabolites or degradation products are considered (16, 17). Covalent interaction with proteins in vivo does occur with the p-lactam antibiotics, penicillins, and cephalosporins (13, 16-21), and this has important clinical implications in the field of drug allergy (see section VII), but in many instances it has not been possible to explain allergic reactions to drugs on the basis of protein reactivity of the parent molecule, its biotransformed products, or a reactive impurity (17). Of course, failure to always find such evidence does not necessarily prove that a conjugate of a particular drug or metabolite does not exist. In allergic subjects, IgE antibodies, as well as being free in serum, are fixed via their Fc portions to the surfaces of mast cells and basophils. Binding of the cellbound IgE antibody combining sites with their complementary allergen produces antigen-antibody complexes and cross-linking of some adjacent IgE molecules. This
v-v a
v-v b
Figure 3. Different ways in which a drug [represented diagrammatically in (a) and (b)], or drug-protein conjugate [shown in (c) and (d)], may cross-link, or bridge, adjacent cellbound IgE molecules, which triggers release of mediators of allergy (see Figure 2). (a)Bridging via an allergenically divalent unconjugated drug molecule. This is the mechanism thought to occur in subjects who experience anaphylaxis following administration of a neuromuscular blocking drug. (b) Bridging via a free, unconjugated drug molecule containing two (or more) different allergenic determinants. ( c ) and (d) Bridging via conjugated drug molecules with cross-linking effected by the same, or different, determinants, respectively. Reproduced from ref 31, with permission.
bridging of the cell-bound IgE triggers cell degranulation and release of a variety of mediators including histamine, leukotrienes, prostaglandins, etc., that cause the signs and symptoms of allergies including anaphylaxis (Figure 2). A biologically-active allergen must therefore be a t least divalent for effective cross-linking of cell-bound IgE (Figure 3). Hence, in the absence of evidence of the existence of hapten-protein conjugates for many drugs implicated in anaphylaxis, one is left with the problem of explaining the mechanism of monovalent hapteninduced anaphylaxis. For one group of drugs at least, namely, those that block nerve transmission a t the neuromuscular junction, divalency appears to be an inherent part of the molecular structure even in the absence of protein binding. The allergenic or IgEantibody binding determinants on these drugs are wholly,
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Table 1. Constituents of the Hair Preparation Causing Anaphylaxis amodimethicone ceteareth 30 cetyl alcohol fragrance hydroxypropyl guar magnesium chloride magnesium nitrate methylisothiazolinone
methylchloroisothiazolinone methylparaben octyldodecanol stearyl alcohol stearalkonium chlorideu steartrimonium hydroxyethyl hydrolyzed collagenb
a Nfl-Dimethyl-N-octadecylbenzenemethanaminium chloride. bKnown by the trade name Crotein Q. This is a quaternary ammonium derivative of hydrolyzed protein in which approximately 90%of the a-and +amino groups of the protein have been substituted. Used in hair preparations as a conditioner and to improve “body” and gloss.
or in part, the substituted ammonium groups, at least two of which occur in each molecules (see section VI). Hence, even in the absence of conjugation to a carrier protein, the free drug molecules are equipped to initiate mediator release in sensitized subjects (Figure 3). Experimental verification of this appears to have been provided (22,231. B. The Dogma of Sensitization by Prior Exposure. Many subjects who experience a drug-induced anaphylactic reaction have never before received the drug. There is perhaps no better example of this than that provided by subjects who experience allergic reaction that are often life-threatening following the administration of a neuromuscular blocking agent during anesthesia. Approximately 70% of these reactors have never had a previous anesthetic, and since 90% of reactors are women, the possibility of sensitization to environmental chemicals, especially those used in female grooming, suggests itself. Compounds containing substituted ammonium ions, the dominant allergenic structural feature on the drugs, are commonly found in cosmetics, shampoos and other hair products, disinfectants, cleaning materials, fabric softeners, algaecides, throat lozenges, and foods. Hence, sensitization of women by quaternary ammonium compounds as components of hair preparations, cosmetics, and cleaning agents is an attractive, but unproven, explanation. A recent case we studied, however, provides some evidence for this theory of “environmental sensitization”. Following application of a commercial “protein hair treatmentn, a 42 year old woman experienced an anaphylactic reaction which required reversal by injection of epinephrine. Likely sensitization to the hair preparation was demonstrated by skin testing (see section 1111, the subject responding with a pronounced wheal (12 mm diameter) and flare (20 mm) reaction. Direct antibody binding and inhibition studies employing neuromuscular blocking drugs and components of the hair preparation (Table 1) showed that the drugs and two of the hair preparation components, stearalkonium chloride and steartrimonium hydroxyethyl hydrolyzed collagen (Crotein Q), but particularly the latter, reacted with IgE antibodies in the subject’s serum. Screening of sera from patients who experienced a lifethreatening reaction to a neuromuscular blocker revealed two sera which showed an almost identical IgE recognition spectrum to the subject’s serum. With all three sera, Crotein Q was as effective as d-tubocurarine in inhibiting IgE binding to the d-tubocurarine solid phase. It seems likely that antibodies mediated the anaphylactic reaction by reacting with the quaternary ammonium ion determinants present in the hair preparation. Such antibodies may also react with other substituted ammonium groups
that occur in a variety of chemicals and drugs and, if the compounds contain multiple ammonium groups as in Crotein Q, or if the compounds are at least divalent with respect to these groups (as in neuromuscular blocking drugs), then the possibility exists for an allergic reaction in sensitized subjects. At present, it is not known how many sensitized subjects there are in the so-called “normal” population, but such subjects would appear to be at risk should they receive a neuromuscular blocking drug during anesthesia. It seems likely that allergic sensitivities to some other drugs and chemicals may also arise from environmental contacts and consequent immunological reactivity to related chemical structures encountered in the home, workplace, or elsewhere. The identification of allergenic determinants on drugs that provoke anaphylaxis is therefore of clinical as well as of fundamental importance since a knowledge of the specificities of patients’ humoral and cellular responses may lead to identification of previously unsuspected sensitizing compounds and to improved diagnosis and predictive screening.
111. In Vivo and In Vitro Testing for Drug Allergy Skin testing is the simplest, quickest, and most inexpensive procedure for testing for allergic sensitivity. The method is generally sensitive and reproducible and can be performed by almost anybody, and responsiveness may remain detectable for many years. Test material is introduced intradermally by injecting 10-20 yL of solution into the dermal layer or by placing a droplet of a solution of the drug on the skin and pricking or scratching the skin through the droplet with a needle or lancet. The latter procedure, commonly known as the prick test (24),usually employs solutions a t a higher concentration than is used for intradermal testing. Results in the form of a raised central wheal surrounded by a red flare reaction are usually read within 5-15 min. Specific immunoassays for the detection of drugreactive IgE antibodies in patients’ sera are being increasingly used to supplement the patient’s history and skin test results for the diagnosis of drug allergies. In the immunoassay’s simplest form, the drug, or drug hapten-protein conjugate, is covalently coupled to a solid phase (Sepharose beads, paper discs, plastic beads, wells of microtiter tray, cellulose strips, etc.) and the complex is then reacted with the test serum. Specifically bound antibodies that react with the drug are detected with an enzyme-labeled, fluorescent-labeled, or radiolabeled second antibody (Figure 4). Instead of using the assay in its direct form, the procedure may be altered to detect inhibition of binding of the drug to its complementary antibody. In the inhibition format, immunoassay studies provide a powerful and quantitative means of establishing specificity of antibody-binding reactions and identifying the precise structural groupings that constitute the allergenic determinants (see section V).
lV.Preparation of Drug Conjugates for In Vitro Studies For the preparation of drug-carrier complexes to be used in direct binding immunoassay and quantitative hapten inhibition studies, nucleophilic addition reactions have proved to be the most widely and generally useful chemical procedure. In particular, bis-oxirane (1,4butanediol diglycidyl) and divinyl sulfone have been
Baldo and Pham
706 Chem. Res. Toxicol., Vol. 7, No. 6,1994
drug-protein solid support complex
drug-protein conjugate
@E@ + solid support
(or
chemical coupling
*
drug withsuitable functional group)
;I/* antibodies in patient's serum
drug-solid phase complex)
some antibodies react with drug-protein or dfug-solid phase complex
I
W8Sh & add labelled second antibody
-
detect label in tube gives a measure of the amount of drug-specific antibodies in patient's serum
-
*
I-ibC
wash
Figure 4. Diagrammatic representation of the immunoassay procedure for the detection of drug-reactive antibodies in serum.
Scheme 1. Some Strategies for the Preparation of Drug-Carrier Complexes Insolublecarbohydrate or soluble protein carrier
+
Couplingagent
Drug Containing: -Nucleophile (-OH,-NH2,-SH)-+ -or Carboxyl group (-COOH)
+
found to be easy to carry out, versatile, and valuable coupling agents for linking a variety of drugs containing nucleophilic groups to both insoluble carbohydrate polymers and protein carriers (Scheme 1). Divinyl sulfone proved suitable for drugs such as &tubocurarine which are unstable at the high pHs required for some coupling agents such as bis-oxirane. Carbodiimides may be used to form peptide bonds at room temperature by coupling carboxyl groups on drugs to amino groups of proteins. This strategy can be employed to prepare drug-protein conjugates of the p-lactam antibiotics and their metabolic and degradative products and to couple quaternary acid hydrolysis products of neuromuscular blocking drugs (see Scheme 1and section VI).
V. Strategy for Identifying Allergenic (IgE-Binding)Determinants One of the most fundamental questions in the field of allergy research is what are the structural features that
Drug-Solid phase complex or Drug-Proteln conjugate
make a molecule, be it a protein or a small molecular weight chemical or drug, allergenic? At the level of IgE antibody binding where the recognition spectrum of the antibodies in serum is not known, the easiest and most direct approach is to define the specificities of the antibodies by identifying the precise structural features on the antigenic structures that interact most closely with the antibody combining sites. In practice for drug-allergy studies, this is best achieved by undertaking quantitative inhibition studies (25)with appropriate compounds and as wide a range as possible of carefully selected analogs. Serum containing the antibodies, or free antibodies, is first incubated with the test compounds before addition of the complementary drug-solid phase followed by labeled second antibody (Figure 5). Just how precisely the antibody combining site is defined depends upon how closely the selected compounds match the determinant groups on the allergen in question. Of course, one can rarely be certain that the most inhibitory of the compounds studied is, in fact, the exact complementary
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Invited Review
+# antibodies in patient's Serum
A
incubate RT,lh
v f-drug
some antibodies react with drug
some free antibodies react with drug-protein Q drug-solid phase complex
1
wash & add Iabeiled second antibody
-
34kC
detect label in rube gives a measure of the amount of drug-specific antibodies bound to the solid phase; f------compared with control (no inhibitor) to obtain percent inhibition
Figure 5. Diagrammatic representation of the immunoassay inhibition procedure used in quantitative hapten inhibition studies for the establishment of specificity of antibody binding and for the identification of drug allergenic determinants. Table 2. Classificationof Neuromuscular Blocking Drugs suitable functional other group for functional coupling group
strategy for coupling the drug to a protein carrier or insoluble carbohydrate support
Depolarizing Neuromuscular Blocking Drugs methonium compds succinylcholine (suxamethonium)
none
.employment of structural analog choline coupled via bis-oxirane or betaine coupled via carbodiimide (CDI) decamethonium none none ohydrolysis of ester group(s), then coupling via bis-oxirane, divinyl sulfone (DVS), or CDI Nondepolarizing Neuromuscular Blocking Drugs phenolic ether with quaternary ammonium groups gallamine none none .employment of structural analog triethylcholine coupled via bis-oxirane diallylbisnortoxiferine alcuronium hydroxyl none .coupling via bis-oxirane quaternary benzylisoquinolinium diethers .coupling via DVS d-tubocurarine hydroxyl none metocurine e? use tubocurarine as drug-solid phase none none bisquaternary benzylisoquinolium diesters atracurium none diester .hydrolysis of ester group(s) then coupling via bis-oxirane, mivacurium none diester DVS, or CDI doxacurium none diester steroidal nondepolarizing NMBDs vecuronium none diester ohydrolysis of ester group(s), then coupling via bis-oxirane pancuronium none or DVS diester pipecuronium diester none ORG 9426 (rocuronium) ester hydroxyl
structure especially when the exact confines of the combining site are considered. Ideally, one should aim to employ a range of analogs each with small structural variations to ultimately allow the precise definition of the fine structural features of the determinant. An additional complication arises from the well-known heterogeneity of antibodies (26) at the class, subclass, and combining site levels, and this is seen between individuals and within the same individual reacting subject. Heterogeneity of allergenic determinants on the same molecule probably exists for all allergenic drugs and certainly occurs with all the drug allergies we have studied so far (27) although it appears to be less pronounced with the neuromuscular blocking drugs (27,28) (see section VI). The extent of heterogeneity of drug
diester
allergenic determinants will ultimately only be answered by studies on large numbers of sera from allergic subjects. At present, only those subjects allergic to neuromuscular blocking drugs have been examined in large enough numbers to allow confident conclusions to be drawn on the number and nature of allergenic determinants present. Although considerable progress in understanding drug allergies at the level of IgE antibody recognition has been made in the last decade, much of what we have learned in structure-activity terms has been with the neuromuscular blocking drugs and p-lactam antibiotics. Accordingly, emphasis in this review will be on these two groups of drugs while findings with other drugs will be considered collectively and with an emphasis on the antibody-binding determinants identified so far.
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708 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
Scheme 2. Bisquaternary Benzylisoquinolinium Diester Neuromuscular Blocking Drugs and Strategy for the Preparation of Drug-Carrier Complexes for the Detection of IgE Antibodies to These Drugs
Atracurlum
Mivacurlum
Doxacurium
Quaternary acld ( H O O o ( _ l )
Scheme 3. Steroidal Nondepolarizing Neuromuscular Blocking Drugs and Strategy for the Preparation of Drug-Carrier Complexes for the Detection of IgE Antibodies to These Drugs
i0-C-CHI f
i0-C-CHI f
Vecuronlum (R=H) or Pancuronlum RICH^)
Plpecuronlum
v.t.d
Q
N
ProtebNb
e
\O-
Org 9426 (Rocuronlum)
C ~ C H O H C ~ ~ ( C H ~ ) ~ O C H ~ C H O H C H T ~
Pro8~IwNteCHpCHOHCHpO(CHp)4OCHpCHOHCH&
HO
Quaternary alcohol ( HQ
)
w -1
VI. Neuromuscular Blocking Drugs and Narcotics Neuromuscular blocking drugs, sometimes referred to simply as muscle relaxants, act at the neuromuscular
ProtelwN l t C H z C ~ S O p C ~ C ~
junction where they interrupt transmission of the nerve impulse to skeletal muscle. These drugs are thus distinguishable from centrally-acting muscle relaxants such as the benzodiazepines, meprobamate, mephenesin, baclofen, and so on. On the basis of the mechanism by which
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Invited Review
Scheme 4. Strategy for the Preparation of Drug-Carrier Complexes for the Detection of IgE Antibodies to Succinylcholine and Decamethonium 1. FMPI OYMQT :OF -S
(CH~)~N'CH~CHTOH Choline (Quatomay alcohol: H
(CH3)3N+CHp COOH Betaine
e
)
(amternary acid: nooc-)
Protein-NKm
0
2.
-
CDI, pn5.s ProtelpNHz
OF FS-:
+
(CH3)3N+CH2CHrOH Choline (Quaternary alcohol: H
a
)
1
See above
they decrease the effects of the neurotransmitter acetylcholine, neuromuscular blocking drugs may be classified as depolarizing or non-depolarizing agents (Table 2). k Immunoassays To Detect Antibody Reactivity. IgE antibodies to neuromuscular blocking drugs were first detected by employing solid supports of alcuronium and d-tubocurarine covalently linked to the insoluble carbohydrate polymer Sepharose (Pharmacia, Uppsala, Sweden) (28-30). Detection of antibodies that reacted with succinylcholine and gallamine presented a greater problem since these compounds have no functional groups that can be utilized for direct linkage to either a protein or insoluble carrier. Bearing in mind the importance of terminal groups in the recognition and reaction of antigens with their complementary antibodies, choline and triethylcholine were coupled to Sepharose via their free hydroxyl groups to produce reactive supports suitable for employment in routine immunoassays (Tables 2 and 4;Scheme 4). Another strategy to avoid time-consuming and frequently difficult total synthesis involves the hydrolysis of the neuromuscular blocking drug into compounds that can be employed in inhibition studies or covalently coupled for use in antibody direct binding studies. This strategy was used to develop solid phase test materials for the detection of antibodies to the new generation of neuromuscular blocking drugs atracurium, mivacurium, and doxacurium (Scheme 2). By utilizing the ester linkages of the drugs, the corresponding quaternary acids and alcohols can easilv be generated and these. in turn. can be coupled to either a solid phase or a protein carrier I
for employment in immun0assays.l A similar strategy was applied for detecting antibodies to pipecuronium and ORG 9426 and also for improving the vecuroniud pancuronium immunoassay (Scheme 3). This strategy involving hydrolysis of the ester linkage can also be exploited for the development of solid phase supports directly from succinylcholine. Another alternative for succinylcholine and decamethonium is the employment of the structural analog betaine which can be coupled to a protein carrier via the carboxyl group (Scheme 41.l B. Allergenic Determinants of Neuromuscular Blocking Drugs. Immunochemical investigations in the early 1980s (28-30) demonstrated IgE antibodies that reacted with alcuronium and d-tubocurarine in the sera of subjects who had experienced an anaphylactoid reaction following the injection of these drugs during anesthesia. Quantitative hapten inhibition studies revealed that binding of the drugs to antibody could be inhibited by all neuromuscular blockers examined, viz., alcuronium, d-tubocurarine, decamethonium, suxamethonium, gallamine, and pancuronium, and also by some other drugs and chemicals without neuromuscular blocking activity (Table 3). A common structural feature of all the inhibitory compounds was found to be a tertiary or quaternary ammonium group, and the substituted ammonium ion formed the dominant, but not necessarily the complete, determinant complementary to the IgE antibody combining sites (31). Not surprisingly, the nature of the alkyl group of the ammonium ion is important for recognition; for example, tetramethylam-
-
'Bald0 and Pham, unpublished.
Baldo and Pham
710 Chem. Res. Toxicol., Vol. 7, No. 6, 1994 Table 3. Examples of Compounds without Neuromuscular Blocking Properties That Significantly Inhibit Human IgE Antibody Binding to Neuromuscular Blocking Drugs Compound
1
Structure of compound or cation group with ammonium group highlighteda
J
60Triakylamines Tetraalkylammonium salts
R3N c
+ + RdN, dRN(R')~
40-
+
Choline
(CH3)3NCH2CH20H
Acetylcholine
(CH~)~NCH~CH~OCOCHS
20
+
-
O-?
Promethazine
1
10
100
loo00
Inhibitor concentration (nmol)
Figure 6. Results of quantitative hapten inhibition studies
+
using a morphine-solid phase together with serum from a morphine-allergic subject, morphine, and some of its analogs. IgE antibody binding was detected with a radiolabeled second antibody. Symbols: (0)morphine; (0)codeine; (0)meperidine; (m) nalorphine; (A) naltrexone; (A)methadone; (0)fentanyl; (+) naloxone. Modified from ref 36.
Neostigmine
Morphine
- ......
,
N-CHa
CH,,
I
HO
Pentolineum
Procaine
N
H
z
Morphine
Meperidine
Methadone
Rntanyl
o COO(CHZ)~N(CZ~S)Z
a Exact confines of IgE-binding determinant are not always clear and depend upon the IgE antibodies studied; Le., determinants show heterogeneity. Determinants may be solely the ammonium group or extend to attached or surrounding atoms and groupings. b R = methyl or ethyl. " R = methyl, ethyl, propyl, etc. d R = methyl, ethyl, ..., hexadecyl, etc.; R' = methyl, ethyl, propyl, etc.
monium bromide was a far more potent inhibitor of the binding of alcuronium to IgE than the tetrapropyl and tetrapentyl salts (28). The specificities of IgE antibodies that reacted with neuromuscular blocking drugs were found to fall into three main groups: (1)Those that are complementary to essentially only the ammonium group. These antibodies are inhibited equally well, or almost equally well, by all neuromuscular blocking drugs. (2) Antibodies that recognize not only the ammonium group but also adjacent and/or adjoining structures. Inhibitory potencies of different neuromuscular blockers may therefore vary widely. (3) Antibodies that do not recognize substituted ammonium groups but other structures on the drugs. Such antibodies are rare and are inhibited only by the neuromuscular blocker that provoked the anaphylactic reaction. The locations of the IgE-binding determinants on the most studied neuromuscular blocking drugs are shown in Table 4. C. Morphine and Related Narcotics. These drugs are often potent histamine releasers, and anaphylactoid reactions to them have been attributed to this effect (3234). It has been argued, however, that reactions to these drugs involving bronchospasm are likely to be immune mediated and the direct histamine-releasing effects are unlikely to cause clinical anaphylaxis in normal dosage (35). Analysis of hapten inhibition results (36) (Figure 6; Table 5) showed that the allergenically important struc-
Figure 7. Structures of morphine and some allergenically
cross-reactive compounds detected by inhibition of IgE antibody binding to a morphine-solid phase. Structural sequences thought to be recognized by IgE are outlined. For other inhibitory compounds see Figure 6 and Table 5. Reproduced from ref 36 with permission.
turd features of morphine comprise the cyclohexenyl ring with a hydroxyl at C-6 and, most importantly, a methyl substituent attached to the N atom. Although the inhibition exhibited by meperidine and methadone is surprising if one compares their written structures with morphine (Table 51, examination of space filling models revealed that both compounds contain structural features that are similar to morphine and of interest from an immunological perspective. In particular, all three structures share a sequence of an aromatic ring separated from the N atom by 3 C atoms with a methyl group attached to the N (Figure 7). Even with fentanyl, which showed only 3% inhibition at 200 nmol but 20-36% inhibition in the range 700-1600 nmol (Figure 6), a sequence of atoms similar to part of the morphine molecule can be demonstrated (Figure 7). D. Cross-Reacting Allergenic Determinants on Neuromuscular Blocking Drugs and Morphine. From the time of the earliest studies which demonstrated IgE antibodies to neuromuscular blocking drugs, mor-
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Table 4. Allergenic (IgE Antibody-Binding)Determinants or Fine Structural Features Complementary to Drug-Reactive IgE Antibodies Detected in Sera of Subjects Allergic to Neuromuscular Blocking Drugsa St ructurc
Drug o r analos
Allergenic determinants
tired for tc\ting
Outline of model with determinants shaded
2 quat
Succinylcholinc
Choline (analog)
Space-filling model
I quat J
a
n
+-cnz-cn=cnz
2 quat 2 tert
Alcuronium
U
&Tubocurarine
1 quat 1 tert
Gallamine
3 quat
Triet hylcholine (analog)
I quat
Pancuronium/\.ecuronium
2 quat/ 1 quat I tert
Atrlrcurium
2 q1I;lt
OMe
a Abbreviations: quat, quaternary; tert, tertiary ammonium group. For pancuronium, R = CH3; for vecuronium, R = H. Reproduced from ref 27 with permission.
phine was shown to interact with the antibodies (29). Both direct binding immunoassay and inhibition studies have revealed that morphine cross-reacts with the muscle relaxants, particularly d-tubocurarine. Shape similarities on morphine and d-tubocurarine have been pointed out (37) to explain this close cross-reaction between the two compounds, but in structural terms, allergenic crossreactivity resides in the substituted ammonium groups common to the relaxants and the narcotic.
Despite the structural similarity between morphine and its analogs on the one hand and neuromuscular blocking drugs on the other, subjects allergic to the latter drugs invariably tolerate the narcotics and exhibit no allergic signs and symptoms upon receiving them. This is, presumably, because the neuromuscular blockers contain more than one allergenic determinant group and are thus able to cross-link adjacent cell-bound IgE molecules (see Figures 2 and 3a) whereas the narcotics
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712 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
Table 5. Relative Inhibitory Activities of IgE-Morphine Interaction by Morphine and Some Structurally-Related Compounds Compound
Inhibition (%) of IgE antibody binding to morphine-Sepharose with 200 nmol of comuound
Suucture
Compound
Inhibition (W)of IgE antibody binding to morphine-Sepharose with 200 nmol of compound
Suucture
HO
12
Morphine
3
Naltrexone
N-CH,
CI H 2 C H 2 - 0
Codeine
12
Fentanyl
3
N-CHj
0 HO
Nalorphine
Meperidine N
31 02CH2CH3
HO
Naloxone
contain only one N-alkyl substituent. Under current thinking, we assume, therefore, that allergenic reactivity to morphine must be mediated via protein or membrane binding of the drug (Figure 312). A more detailed comparison of findings with subjects allergic to morphine or neuromuscular blocking drugs is contained in the review by Fisher et al. (38).
VII. /3-Lactam Antibiotics Structurally, these antibiotics may be classified into two main groups, monocyclic and bicyclic P-lactams (Figure 8). Members of the former group, known as monobactams, consist solely of a p-lactam ring with an attached amide side chain while fusion of the p-lactam ring with another 5- or 6-membered ring creates the bicyclic compounds. Apart from ring size, these may be further subdivided according to the presence or absence of a ring heterophile atom to give the seven different groups shown in Figure 8. A. Penicillins. Within 20 years of the introduction of penicillins into clinical medicine, extensive immunochemical studies stimulated by a desire to understand penicillin allergy and directed at identifying penicillin antigens were being undertaken by several groups. These studies have contributed significantly to our knowledge of drug allergy and to our understanding of the immune response to low MW chemicals (14, 19-21, 39-43). However, much of the immunological research on penicillins has been directed at the study of penicillin antigens as distinct from allergens, and this is reflected in the many investigations employing laboratory animals rather than humans. Basic to the definition of penicillin antibodies has been the nature of the hapten-protein linkage, with the point of linkage of the carrier t o the drug determining the classification into “major” or “mi-
Methadone
31
nor” antigenic determinants. Structures of the penicilloyl or “major” determinant and three examples of “minor” determinants are shown in Figure 9. Such an (‘entire hapten” view of penicillin determinants places too little emphasis on fine structural features of the molecule and on the nature of the side-chain groups. Our studies with sera from subjects who experienced an immediate allergic reaction to penicillin revealed heterogeneity in the IgE antibody penicillin recognition spectra (44,45). For some subjects, the side-chain group was the dominant structural feature of the IgE-reactive determinant, but with other patients the thiazolidine ring a t the other end of the molecule was the feature that was most complementary to the antibody combining sites. Some recognition of the /3-lactam ring as part of a compound determinant with either the side-chain group or the thiazolidine ring was also found. Examples of these findings are summarized in Tables 6 and 7. Quantitative inhibition immunoassays showed that, for subjects 1and 2, ampicillin, benzylpenicillin, and amoxicillin were the best inhibitors of IgE binding and, most significantly, considerably more potent inhibitors than other penicillins with side-chain structures that are markedly different. Table 6 shows results obtained with six of the best inhibitors. All six contain, to a greater or lesser extent, structural similarities in the side-chain group, and these are shown hatched on the outlined models. By contrast, poor inhibitory activity in the range 1-600 nmol was shown by compounds with marked structural differences in the side chain, for example, piperacillin and flucloxacillin. Consistent with the conclusion that IgE antibody recognition with these 2 patients involved the side-chain structure was the lack of activity shown by 6-aminopenicillanic acid, a compound lacking the side-chain group on the p-lactam ring. For subject 3, a different recogni-
Chem. Res. Toxicol., Vol. 7, No. 6, 1994 713
Invited Review
R
G
O
N
P
g
Z
'COOH Ampklllln
0.g. Pcnklllln0 PonkllllnV
og. knlponom
Amoxlclllln
Phonothlclllh~ Epklllin Proplclllln Bacampklllln Oxrclllb Nafclllln Mlthlclllk,
Tlcrnlllln kbclllk Calb.nlclllln
Cloxsclllin Dkloxrclllln
Yubclllln Plp.nclllln
CLAVAMS
y2;mCw
Flucloxrclllln __ _ _ _ _ _ - - - - - - 0.0. - Chvul8nlc - - - acld ----------
Rl-CONFJR,
COOH
r q . Cophaloxln
Cohcbr CophndlM C . f u n m d O k Crfadroxll Cofonlcld Crphalothln Cotonnldo Cophalorldlno Cofuroxlmo Cophaplrln Cofpmzil Crfuolln
R140Nmb
CARBACEPHEMS
CEPnALOSPORlNSlCEPHEMSl
COOH
Cefolrxlmo kftlzoxlmo
0.Q. LoraCarbOf
Cofpodoxlnto
CoflMXMI.
Coflxlmo Cofluldlme cofoponzw
CEPnAMYClNS
I1-0XA-gUCTAMS (1-ox.cap hrlosporins)
(7-a-methoxycephslosporlna)
i-coNpARzR1-coNFdR2 H s
HC?
HSCP
H 0
COOH
COOH
0.0. Cobxltln Cohnoluok
0.g. Moralactam
C.fol.1M
Figure 8. Classification of ,&lactam drugs. R-co~H-c~c/s~c(c~,
I 1NH-CH-COOH I 0-C
R-CONH-CbC~s~C(CH& ocltct..-c-,,
I NH
I 1
I I
NH
I
Proleh
Protein
Penlcllloyl 'Major'
Penlclllanyl 'Mlnor' ,S-Proteh
N----C=CH
I
0
IS C(Cy),
I l l 1N H - C H -IC O O H R--C\o,%o Penlcillenate 'Mfnor'
Proteh -S,
R-CONH-CH-CH
C(CH&
I NIIH - C H -IC O O H HOOC
Penameldate 'Mlnor'
Figure 9. Structures of the so-called "major" and three of the "minor" penicillin antigenic- allergenic determinants showing different points of attachment of the drug to the carrier.
tion specificity pattern emerged (Table 6). In this case, the activity of all penicillins tested and the weak, but definite, inhibition caused by 6-aminopenicillanic acid (Figure 10) suggested that IgE antibody recognition was primarily directed toward the other end of the penicillin molecule, that is, the thiazolidine ring. The recognition pattern with serum 3, however, is more complicated since benzylpenicllin was a slightly more potent inhibitor than any of the other compounds tested. This finding suggests
7
10
'/ 100
Inhibitor concentration (mol)
Figure 10. Results of quantitative hapten inhibition studies using an ampicillin-solid phase together with serum from a penicillin-allergic subject and a number of different #?-lactams as inhibitors. IgE antibody binding was detected with a radiolabeled second antibody. Symbols: (0) benzylpenicillin; ( 0 ) amoxicillin; (0) phenoxymethylpenicillin; (W) flucloxacillin; (A) piperacillin; (A)mezlocillin; (0)cephalothin; (+) B-aminopenicillanic acid. Modified from ref 44.
714 Chem. Res. Toxicol.,Vol. 7,No. 6, 1994
Baldo and Pham
Table 6. Allergenic or IgE Antibody-Binding Regions Identified on Penicillins in Immunoassays Employing Allergic Patients’ Sera and an Ampicillin-Solid Phasea Drug
Structure
Outline of model with allergenic determinant hatched (sera 1 & 2) or dotted (serum 3)
Space-filling model
Amount (nmol) of compound needed for 50% inhlbition of the uptake of W-anti-human IgE with serum.: (1)
(2)
100
150
230
200
75
260
125
320
380
380
580
230
R-
Benzylpenicillin (Pen G)
Phenoxymethylpenicillin (Pen V)
Ampicillin
Amoxicillln
I
1
Epicillin
Piperacillin O
F
HNH-
OIS Y ” azcHr
6-Aminopenicillank acid
Allergenic determinant identified:
0:; C h -
a Sera 1, 2, and 3 used at a dilution of 1:2. Hatched areas: side-chain regions involved in antibody binding; dotted areas: other end of molecules (thiazolidine ring) involved in antibody binding.
an extra population of antibodies present in serum 3 that recognizes the benzyl side chain (44). Further evidence of side-chain recognition was found in studies on the sera of three subjects who each experienced an anaphylactic reaction after receiving flucloxacillin. Quantitative hapten inhibition experiments with a wide range of penicillins clearly implicated the 5-methyl-3-phenyl-4-isoxazolyl side chain (as in oxacillin, see Table 7) as a structure recognized by the IgE antibodies in all three sera. In addition, the halogen
atoms C1 and F on flucloxacillin and dicloxacillin, shown in Table 7 as heavily dotted and lightly dotted regions, respectively, were important regions of antibody recognition for sera 4 and 5. For IgE in these sera, the 3-(2chloro-6-fluorophenyl)-5-methyl-4-isoxazolyl group comprises the complementary allergenic determinant. The close similarity of the shapes of the fluoro and dichloro derivatives are apparent in Table 7. Serum 6 appears to contain at least two populations of penicillin-reactive IgE antibodies-one that recognizes the side chain as in
Invited Review
Chem. Res. Toxicol., Vol. 7, No. 6, 1994 715
Table 7. Allergenic or IgE Antibody Binding-RegionsIdentified on Isoxazolyl Penicillins in Immunoassays Employing Allergic Patients’ Sera and a Flucloxacillin-Solid Phasea Outline of model with
Drug
Structure
Space-filling model
Amount (nmol) of compound needed
shaded
(4)
(5)
(6)
0.19
3.7
95
2.6
78
15
88
215
50
R-CONH 0
H ‘COOH
R-
Flucloxacillin
&CH
Xcloxacillin
&CH
Cloxacillin
dn ‘0
CHI
Oxacillin 2.4 W
C
H3
Benzylpenicillin (Pen G) 1589 O
C
H
,2000
302
Y
X
/d
rl
Allergenic determinant identified: W
C
H
a Sera 4,5,and 6 used at dilutions of 1:3,1:16, and 1:4,respectively. Hatched and dotted areas: side-chainregions involved in antibody binding; heavily dotted areas: chlorine atom; lightly dotted areas: fluorine atom.
oxacillin, and as in its halogenated derivatives cloxacillin, dicloxacillin, and flucloxacillin, and the other that recognizes a structural common to most, if not all, penicillins. Inhibitory activity by benzylpenicillin and other penicillins suggests the latter conclusion and also suggests that the commonly recognized structure is the p-lactam andor thiazolidine rings or part thereof (45). B. Cephalosporins. Cephalosporins are p-lactam antibiotics structurally and pharmacologically related to penicillins. In the cephalosporins, the P-lactam ring is fused with a 6-membered dihydrothiazine ring instead of the 5-membered thiazolidine ring found in the penicillins. As with the penicillins, addition of a variety of different groups at the so-called side chain (R1 at position 7) or at position 3 (R2) on the dihydrothiazine ring (Table 8) produces compounds with differences in activity related to resistance to hydrolysis by P-lactamases, absorption, and so on.
Development of a radioimmunoassay employing cephalothin linked to a solid phase permitted the detection of IgE antibodies in the sera of some subjects who experienced anaphylaxis following the ingestion of a cephalosporin (46). As with many penicillin sensitivities, specificity investigations based on hapten inhibition experiments revealed that antibody binding occurred with side-chain groups on the cephalosporins. In the example summarized in Table 8, clear inhibitory activity was obtained with cephalothin, cephaloridine, cefoxitin, benzylpenicillin, and 2-thiopheneacetic acid, suggesting that the allergenic determinant encompasses the 2thiophene group with particularly strong recognition of the methylene substituent. Some recognition of the p-lactam ring was also likely since the p-lactam antibiotics were better inhibitors than 2-thiopheneacetic acid, but as in all such hapten inhibition studies outlined here, the exact confines of the determinants complementary
Baldo and Pham
716 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
Table 8. Allergenic or IgE Antibody-BindingRegions Identified on Some Cephalosporins, Benzylpenicillin,and 2-Thiopheneacetic Acid in Immunoassays Employing a Cephalothin-Solid Phasea Structure
Space-filling model
Inhibition (%) of IgE Outline of model with allergenic determinant binding to CePhalOthi'' Seph with 400 nmol of hatched inhibitor in serum ( 7 ) s :
cddn
57
58
33
9
44
12
2-Thiopheneacetic a c M 31
1
Allergenic determinant identified:
a Serum 7 used at a dilution of 1:2. Cefoxitin is a cephamycin with a methoxy group a t position 7 of the cephalosporin nucleus. Hatched areas: side-chain regions involved in antibody binding.
to the antibody combining sites are often difficult to define. C. Other P-LactamAntibiotics. Although allergies to monobactams and other bicyclic P-lactams are known (47),little or no structure-activity information has been reported. In a serological and immunochemical study involving a range of p-lactam antibiotics, allergenic crossreactivities based on IgE recognition of side-chain groups and the 6-membered rings were demonstrated between
a carbocephem and some penicillins and cephalosporins.2
VIII. Other Drug Allergens Apart from the neuromuscular blocking drugs and B-laCtam antibiotics, infOI"IIlati0nOn allergenic StrUCtUreS is available for only a few of the many drugs used in 2Baldo and Pham, unpublished.
Invited Review
Chem. Res. Toxicol., Vol. 7, No. 6,1994 717
Table 9. IgE-Binding Structures Identified on the Thiopentone Molecule Dominant smcture
Sniological findings
Clinical relevance
Table 10. Inhibition by Sulfonamides of IgE Antibody Binding to Sulfamethoxazole-Sepharoae in Sera from a Sulfamethoxazole-AllergicPatient
in IgE-binding detcrminan?
CH3~H2CH2C~ cH1cH2$
I
N&sb NH
Compound Free drug (thiopentone) inhibits binding of patient's serum to"thiopcntone"*lid phasec
Inhibition (W)of binding to sulfamethoxazoleScphmse by 1 pmol of compound
Sh'UCNrC
Presence of IgE ~
~
to thiopentone
~
t
Sulfamethoxamh
~
~
H2No S 0 2 N H
~
CH3
$
66
~
CHI 62
Sulfamerazine As above
Sulfamethirole
39
CH3
2-Mcrcaptopyrimidined C > s H a to "thiopentone"-solidphase
Reactive IgE in sera from some subjects allergic to muscle relaxants. Subjects not allergic thiopentone
a Exact confines of determinant not always clearly defined with all sera. Dominant features of determinant shown. Other structures, in particular, the pyrimidine ring, probably have an auxiliary function. Thiopentone was used to prepare drug-solid phase, but coupling conditions probably lead t o some decomposition of the thiopentone. Exact structure of attached species was therefore uncertain. 2-Mercaptopyrimidinealso inhibits binding of IgE antibodies reactive to the thio region of thiopentone (50).
everyday medicine. Detailed studies on three intensively used compounds, namely, the anesthetic induction agent thiopentone and the antibacterials sulfamethoxazole and trimethoprim, have been undertaken, and the findings are summarized here. A. Thiopentone. Application of a radioimmunoassay for the detection of thiopentone-reactive antibodies (48) resulted in the identification of two allergenic determinants-the secondary pentyl and ethyl groups on one side of the molecule and the thio region on the other (Table 9) (49, 50). Lability of thiopentone at the high pH used for preparation of the drug-solid phase support appears to be a basis of a thiopentone-IgE interaction unrelated to allergic sensitivity to the drug. This clinically-irrelevant binding appears to be due to reaction of IgE antibodies with ring Ns of the pyrimidine nucleus of thiopentone (Table 9) (51) detected by the thiopentonesolid phases prepared at high pHs. Specificity of the binding was demonstrated by inhibition of IgE binding with 2-mercaptopyrimidine but not by thiopentone (511. B. Sulfamethoxazole. Pronounced inhibitory activity shown by sulfamerazine, sulfamethizole, sulfamethazine, sulfisoxazole, and sulfamoxole of the binding of IgE antibodies to sulfamethoxazole-Sepharose complex (Table 10) indicated that a methyl group attached to the heterocyclic ring nucleus (Figure 11) was a dominant allergenic feature on the sulfonamides. Collectively, the inhibition studies suggested that the 5-methyl-3-isoxazolyl group is an important allergenic determinant of sulfamethoxazole (52). The failure of sulfanilamide to significantly inhibit the binding of IgE antibodies in any of the allergic sera studied appears to show that the free amino end of the molecule is not directly complementary to the antibodies involved in allergic reactions to sulfamethoxazole. Apparent lack of allergenic reactivity of the p-aminobenzenesulfonamido group, however, may merely be a reflection of the point of attachment of the drug to the solid phase, attachment via the free amino group making that end of the molecule inaccessible to the antibody combining sites.
Sulfamethazine
52 'CHI
Sulfisoxazole
Sulfamoxole
Sulfanilamide
14
C. Trimethoprim. This antibacterial agent is used extensively in formulations with sulfamethoxazole, the combination being known as co-trimoxazole. Adverse reactions to this drug combination occur more frequently than to almost any other drug or group of drugs except the p-lactam antibiotics. Identification of the 3,4dimethoxybenzyl group as an allergenic determinant of trimethoprim was achieved primarily on the basis of the inhibitory activities of diaveridine and (3,4-dimethoxyphenyl)ethylamine, and this conclusion was supported by the marked inhibition exhibited by 3,4-dimethoxybenzoic acid, 3,4,5-trimethoxycinnamicacid, and 3-(3',4',5'trimethoxypheny1)propionic acid (Table 11). Taken together, these results suggested that the IgE antibodies were recognizing the aromatic ring with two methoxy substituents. Reinforcement of this conclusion and evidence that the opposite end of the trimethoprim molecule was not being recognized were obtained from results with some pyrimidine derivatives, each of which showed little or no activity (Table 11). With sera from some trimethoprim-allergic patients, however, inhibition studies clearly showed that only trimethoprim and its chloro and hydroxy derivatives were active, suggesting that the entire molecule was involved in binding to the IgE antibody combining sites (53). The trimethoprim determinant structures identified so far are shown in Table 12.
M.Conclusion and Future Directions A. Applied Investigations. At the applied level, research on drug allergies should continue to be directed at developing specific diagnostic tests €or the detection of individual drug allergies of the immediate type. In the first instance, and in the light of present knowledge, these tests will be designed to detect drug-reactive IgE antibodies, but as our knowledge of mechanisms underly-
~
Baldo and Pham
718 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
Table 11. Inhibition of Trimethoprim-IgE Antibody Interaction by Trimethoprim and Some Structurally Related Compounds. Patterns of Reactivity Implicating the 3.4-Dimethowbenzyl Determinant Compound Sulfamethoxazole
J
StIIICNII?
Inhibition (%) of IgE antibody binding to uimethoprim-Sepharose with 100 nmol of compound
Trimethoprim
77
Diavendine
81
3,4,5-Trimethoxy cinnamic acid
64
3- (3’,4‘.5‘-Trimethoxyphenyl) propionic acid
69
3.4-Dimethoxyphenylethylamine
81
3.4-Dimethoxybenzoic acid
66
4-Methoxyphcnylacetic acid
13
Pyrimethine
16
2-Amino-4-hydroxy-6methylpyrimidine
18
4-Amino-5-aminomethyl-2-methylpyrimidine
15
Sulfamoxole
Figure 11. Structures and space-filling models of (a) sulfamethoxazole, (b) sulfamerazine, (c) sulfamethizole, and (d) sulfamoxole. The arrows identify the methyl group on each molecule which is the most important structural feature involved in binding to drug-specific IgE antibodies. Reproduced from ref 27 with permission.
ing immediate hypersensitivity reactions increases, particularly T-cell involvement in allergy (see section E - C below), other investigative approaches for adverse drug reactions in general are likely to evolve. In the last decade, it has become apparent that IgE antibodies occur to some drugs that were previously considered to be unlikely candidates as allergens, and from now on, every drug should be thought of as a t least having the potential to promote an allergic reaction. It has also become increasingly clear that, for some drugs, or groups of drugs, allergenic cross-reactivity occurs but, even so, specific assays will often be required for precise diagnosis of allergies to structurally related, but different compounds. This situation is seen most clearly with the p-lactam antibiotics and to a lesser extent with the neuromuscular blocking drugs and narcotics. B. Fundamental Studies. Since Landsteiner’s pioneering studies on hapten immunochemistry (12) in which he coupled “low” MW chemicals to protein carriers, it has been known that antibodies may be formed in laboratory animals to a wide range of chemicals that are not themselves antigenic such as steroids, sugars, purines, pyrimidines, nucleosides, compounds with aromatic rings such as benzene and phenol, etc. It is now apparent that humans also show a wide spectrum of antibody reactivity to a variety of “small” chemical structures, and at the level of IgE a t least, antibodies form following “natural” exposure rather than direct immunization. In the last decade, more drug allergenic determinants have been identified than in all prior years, and structures as diverse as a substituted ammonium ion and halogenated
derivatives of a (methylpheny1)isoxazolylgroup are now known to be recognized by, and to bind to, IgE antibodies in the sera of some drug-allergic subjects. It is also becoming clear that more than one IgEbinding determinant occurs on some drugs (27),and as more drug allergies and more allergic individuals are studied, the extent of this allergenic heterogeneity and the diversity of structures recognized will emerge. This information will be necessary if we are to ultimately understand the structural basis of drug allergenicity and decide whether there is a wide range of structurally diverse determinants or a relatively few determinants common to many drugs. In view of the large number of drugs in use and the wide variety of structures represented, it might seem more likely that the number of drug allergenic determinants is large. However, even with the relatively small number of drugs studied so far, one structure, the substituted ammonium group in acyclic or cyclic form, occurs widely in many different drugs and binds IgE antibodies in the sera of subjects with different drug allergies (see section VI). C. Drugs and T-cell Responses. It is now well established that CD4+ T-lymphocytes are critical for the induction of IgE-mediated allergic reactions (10,54,55). In many cases it appears that activation of CD4+ T-cells results from interactions between T-cell receptor, peptide fragments of processed antigen, and major histocompatibility complex (MHC) class I1 gene products (56-59). Significant progress has been made in the molecular cloning of peptide allergens (9)and in the use of purified
Invited Review
Chem. Res. Toxicol., Vol. 7, No. 6, 1994 719 Table 12. Trimethoprim Allergenic Determinants Structure
Allergenic determinants identified
house dust mite allergens to stimulate CD4+ T-cells in vitro (60,61).Such studies allow characterization of the peptide determinants recognized by T-cells in association with HLA molecules and suggest that the peptide can also be employed t o inactivate T-cells in vitro. The ability of T-cells to recognize small (haptenic) molecules raises questions of importance both to our understanding of fundamental immunology and to our ability to treat patients suffering from allergic responses to drugs and other haptens. For drug allergens that are not peptide in nature, the molecular conformation of the epitopes recognized by T-cells remains unclear, although there is convincing evidence that T-cells are capable of recognizing many small molecules, ranging from metal ions such as Ni2+, drugs such as penicillamine, and experimental haptens such as trinitrophenol and oxazolone (62-64). In some cases, conjugation of drug to carrier proteins, followed by conventional processing and presentation by MHC class I1 molecules, may be implicated (63, 64) whereas in others there is no convincing evidence yet that carrier proteins are involved (63). For D-peniCillamine, mice were shown to develop MHC class I1 D-penicillaminespecific T-cells which responded to drug-haptenated stimulator cells but not to untreated control cells or to free drug (64). Processing was not required since chemically fixed antigen presenting cells could present the drug effectively to T-cells. In humans, D-penicillamine sensitivity appears to be under control of the HLA complex, reinforcing the argument that T-cells are involved in these reactions. Recently, gold-specific T-lymphocytes were isolated from a subject treated with sodium aurothiomalate. The T-cell clones required histocompatible antigen presenting cells, and gold recognition did not require antigen processing (65). In investigating drug determinants recognized by T-cells, a number of alternative mechanisms involving presentation of the drug seem possible. For example, the drug may be coupled to a carrier protein(s) which is processed into haptenated peptide fragments via the known class 11-dependent pathway. Recognition may be specific to hapten in association with only one peptide or may encompass several peptides. As this pathway is critically dependent upon processing, it can be interrupted by agents that prevent antigen processing. Instead of binding to a free carrier protein, drug may couple directly to a peptide already in the MHC groove at the time of hapten exposure. Once again, recognition may
Spacefilling model
W m of model with determinant shaded
be specific for only one hapten-peptide complex or may allow several possible peptides to be seen. This pathway depends on the presence of particular peptides in the MHC groove which may be displaced experimentally using high affinity peptides. In another possible alternative, drug may couple directly to class I1 MHC itself, presumably on either of the exposed helices which are thought to bind to the T-cell receptor. Recognition may be restricted to MHC-hapten containing only a limited subset of all possible peptides or may be “promiscu~us~~, that is, independent of peptide. In this case, the constraints on binding would be similar to those that are present during recognition of allogeneic MHC. In this scenario it should be possible to demonstrate the physical link between MHC and hapten.
X. Summary As a result of recent research and development, an expanding range of in vitro assays for the detection of drug-reactive IgE antibodies is now available for the diagnosis of individual drug allergies. Continuing immunochemical studies on drugs implicated in allergic reactions via further development of specific immunoassays and production of drug-protein conjugates will allow us to build up a picture of the repertoire of drug and drug-derived B-cell allergenic determinant structures and to identify and predict cross-reactivities. Although we have made good progress in identifying some drug B-cell determinants, we remain ignorant of drug T-cell determinants and, indeed, of the nature of T-cell recognition of all nonpeptide molecules. Demonstration that drugs specifically recognize T-cells from drug-allergic patients may reveal associations with HLA phenotypes, the nature and location of interaction between drug and MHC molecules, and the nature and identity of drug or drug-derived T-cell antigens. A knowledge of the molecular nature of T-cell determinants inducing allergic responses is fundamental to therapeutic attempts to modulate these deleterious reactions. The possibility now exists for the allergenic screening of drugs as part of their toxicological evaluation, and identification of allergenic structures on drugs also has implications for the testing of the allergenic activity and sensitizing potential of other chemicals in our environment. Obvious and important areas for the application of our methods and findings are the pharmaceutical and cosmetic industries.
720 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
Acknowledgment. We thank Gail Knowland for help in preparing figures and tables and the National Health and Medical Research Council for support.
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