Mechanisms in IgE-dependent anaphylaxis to anaesthetic drugs

ANAPHYLACTOID RISK IN ANAESTHESIA

© Masson, Paris. Ann Fr Anesth Roanim, 12: 131-140, 1993

Mechanisms in IgE-dependent anaphylaxis to anaesthetic drugs Mecanismes des reactions anaphylactiques IgE-dependantes aux agents anesthesiques B.A. BALDO *, M.M. FISHER ** * Kolling Institute of Medical Research, ** Intensive Therapy Unit, Royal North Shore Hospital of Sydney, St Leonards NSW 2065, Australia

RI~SUM#: La mise en 6vidence d'anticorps IgE dirig6s contre les myorelaxants, le thiopental et les morphiniques (en particulier la morphine), renforce l'id6e initiale, formul6e ~ partir des tests cutan6s et des observations cliniques, que la plupart des r6actions anaphylactofdes contre ces m6dicaments correspondent •h de v6ritables r6actions d'hypersensibilit6 de type I. Des 6tudes immunochimiques fines ont permis d'dtablir les spdcificit6s structurales des anticorps IgE r6agissant contre ces m6dicaments, et ont permis d'expliquer les m6canismes mol6culaires pour un grand nombre de r6actions crois6es cliniques. De m6me, sont discut6es les particularit6s de I'interpr6tation comparative des tests cutan6s et des dosages immunologiques des IgE sp6cifiques anti-m6dicaments pr6sent6es, ainsi que les m6canismes de lib6ration des m6diateurs, la persistance des IgE sp6cifiques anti-m6dicaments, et la nature du mode de sensibilisation.

Reports of anaphylaxis during anaesthesia were rarely described before the 1970's [10]. Since then the incidence of reactions appears to have increased [22], although this may simply reflect increasing awareness. A p a r t f r o m reactions to the c r e m o p h o r - b a s e d drugs alfathesin and propanidid, reactions were attributed to t h i o p e n t o n e rather than n e u r o m u s c u lar blocking drugs ( N M B D s ) since the i m m u n o l o gical d o g m a of previous exposure was often satisfied with the f o r m e r but not with the latter drugs.

ANAPHYLAXlS TO NEUROMUSCULAR BLOCKING DRUGS (NMBDs)

In addition to the frequently seen lack of documented prior exposure, the well-recognized histamine-releasing properties of N M B D s [16, 48, 52] added to the belief that i m m u n e - m e d i a t e d a n a p h y lactic reactions to these c o m p o u n d s are unlikely, if not impossible. Despite this, skin testing with suitable dilutions of N M B D s were shown to be specific and of diagnostic value [18, 19, 46, 49, 55] and

Presented at the meeting ,, New Trends in Anaphylactoid Risk in Anaesthesia ,,. Nancy (France), 11-12June 1992.

as this form of testing b e c a m e m o r e p o p u l a r [24, 43, 45, 47], the conclusion that reactions were immune mediated b e c a m e increasingly attractive and recognized. This conclusion seemed to be reinforced by early Prausnitz-KListner [42] and leucocyte histamine release studies [1, 55] which suggested the involvement of h o m o c y t o t r o p i c antibodies in the sera of some subjects who experienced a N M B D - i n d u c e d reaction during anaesthesia.

DETECTION OF NMBD-REACTIVE IgE ANTIBODIES

Using sera taken from such patients and accumulated since the early 1970s by FISHER, radioimmunoassay ( R I A ) studies based o n N M B D - s o l i d phases clearly d e m o n s t r a t e d alcuronium-reactive IgE antibodies in the sera of a n u m b e r of the patients [5]. Follow-up investigations with a dtubocurarine-solid phase revealed the presence of lgE antibodies c o m p l e m e n t a r y to the N M B D in the sera of some patients and reactions of some sera with both d-tubocurarine and alcuronium suggested the likelihood of allergenic cross-reactivity between the drugs [6].

TirOs a part : B.A. Baldo.

132

B.A. BALDO, M . M FISHER

Correlations between case histories and the presence of serum NMBD-reactive IgE antibodies were sufficiently impressive to prompt further investigations and RIAs for the detection of serum IgE antibodies reactive with succinylcholine and gallamine [31] and vecuronium/pancuronium [32] were soon developed. Preparation of suitable solid phases f o r the former assays posed a problem since the succinylcholine and gallamine molecules lack suitable functional groups which permit chemical coupling to a carrier. This was overcome by preparing choline and triethylcholine solid phases ; the choline and triethylcholine molecules mimicking the terminal groups on succinylcholine and gallamine respectively. The choline assay, when employed with sera from subjects who reacted to succinylcholine, provided the best correlation with skin tests and case histories of any of the NMBDs [30].

ALLERGENIC CROSS-REACTIVITY AND SPECIFICITY OF IgE ANTIBODIES

The apparent allergenic cross-reactivity suggested by clinical observations and direct binding R I A results, was investigated as part of the study of the specificities of the combining sites of the NMBD-reactive IgE antibodies. Early experiments revealed two striking findings : 1) Binding of most of the sera studied could be inhibited, to greater or lesser extent, by all of the NMBDs examined i.e_ alcuronium, d-tubocurarine, succinylcholine, decamethonium, gallamine and pancuronium [4, 5] (table I) (later studies with

vecuronium [32] arid atracurium [26] showed that these were also inhibitory) but, with an occasional serum, for example serum Wa in our 1983 study [5], only one N M B D (in this case alcuronium) proved inhibitory_ 2) A number of other drugs and chemicals without NMB activity were also inhibitory (table II) [5]. It quickly became apparent that the common structural feature in compounds as apparently structurally and/or pharmacologically diverse as succinylcholine, tetramethylammonium bromide, hexadecyltrimethylammonium bromide, acetylcholine, promethazine HCI, neostigmine bromide, pentolineum tartrate, trimethaphan camphorsulfonate and morphine HC1 (table III) was the presence of a tertiary or quarternary ammonium group and this group formed the dominant, but not necessarily the complete, determinant complementary to the IgE antibody combining sites. Bearing in mind our knowledge of the size and heterogeneity of antibody combining sites [38, 39], one can predict that the combining sites of many of the IgE antibodies that react with NMBDs recognize not only the ammonium group but also neighbouring structures. This was suggested by our early quantitative inhibition results obtained with a series of alkyltrimethylammonium salts [5] which showed that, on a molar basis, the octyltrimethyl salt was over three times as potent as the ethyl and dodecyl derivatives and nearly five times as potent as the hexadecyl derivative (table IV). These findings suggest that as the chain length increases, a higher degree of complementarity is detected but continued addition to the C chain

Table I. - - Cross-reactivity between neuromuscular blocking drugs (NMBDs). Results of RIA inhibition studies with sera containing IgE antibodies to NMBDs.

Serum

Ta Pe Lo Ta Ge Lo Pe Bri Hun Mi

Drug eliciting adversedrug reaction d-tubocurarine Succinylcholine Dccamethonium Succinylcholine d-tubocurarine Succinylcholine Succinylcholine Succinylcholine Decamethonium Alcuronium Alcuronium d-tubocuratine

Drug or analogue on solid phase

Amount (nmol) of NMBD or analogue needed for 50 % inhibition of the uptake of 125I-anti-humanIgE Alcuro- d-tubo- Pancuro- Succinyl Decame- Gallanium curarine nium choline thonium mine

d-tubocurarine d-tubocurarine

39 220

17.1 12.5

130 110

195 67

d-tubocurarine Alcuronium Choline Choline Choline

180 39 88 37 58

10.1 34 1.3 1.8 0.7

42.1 130 17 4.7 7.7

52 100 4.7 6 9.8

14 73 0.5 0.9 1.9

6I 105 t .6 6.8 8.4

12 16 23

16 6.8 20

42 15 23

12 4.3 5

9.4 10 4

4.1 6.1 13

4.0 2 l. 1

8.7 7.8 11

15 20 51

22 25 37

Choline Choline Choline

105 46

Choline Triethylcholine

10(I 85

MECHANISMS IN IgE-DEPENDENT ANAPHYLAXIS

Table II. - - Examples of compounds without muscle relaxant properties that significantly inhibit IgE antibody binding to NMBD-solid phase supports.

133

Table III. - - Cross-reactivity of NMBDs with drugs without NMB activity. Inhibition studies with serum from a subject who experienced anaphylaxis of unknown cause *.

Structure of compound or cation group with ammonium group highlighted *

Compound

Drugs

NMBDs

Tetraalkylammonium salts

***R4 N IRN + (CH3)3

Alkylt rimethyla mmonium salts

(CH3)3N +CHz CH2OH

Acetylcboline

(CH3)sN +CH2CH2OCOCH3

Q S

NCH2CH - N(CH3)z I CH3

Promethazine

N + (CH3)3

~

Morphine HCI Choline chloride Chlorpromazine HCI Promethazine HCI Neostigmine bromide Pentolineum tartrate Procaine HCI Lignocaine HCI

4.5 83 63 53 23 27 275 > 1 000

* NMBDs not suspected. Reaction occured after use of a local anaesthetic throat spray. ** Morphine-Scpharose used as solid phase in RIA

Neostigmine OOCN(CH))2

O" N - CH~ H

Table IV. - - Inhibition by alkyl quaternary ammonium salts of the binding of NMBD-reactive IgE antibodies* to a NMBD-solid phase** Inhibitory compound

Amount (nmoles) needed for 60 % inhibition of binding

Ethyltrimethylammonium bromide Octyltrimethylammonium bromide Dodecyltrimethylammonium bromide Hexadecyltrimethylammouium bromide Tetramethylammonium bromide Tetrapropylammonium bromide Tetrapentylammonium bromide

8.1 2.5 8.6 12.2 8.6 230 500

Pentolineum CH3 Procaine tt

22 240 30 67

O t h e r drugs :

Choline

Morphine

:

Succinylcholine Alcuronium d-tubocurarine Gallamine

**R3N

Trialkylamirles

Amount of drug (nmoles) needed for 50 % inhibition of IgE binding **

NH2--@COO

CH3 (CH2)2N(CzHsh

* Exact confines of IgE-binding .determinant are not always clear and depend upon IgE antibodies studied - i.e. determinants show heterogeneity. Determinant may be solely ammonium group or extend to attached or surrounding atoms and groupings. ** R = methyl or ethyl ; *** R = methyl, ethyl, propyl, etc. ; t R = methyl, ethyl.., hexadecyl, etc. tt See text below

eventually leads to a decline in inhibitory activity, that is, a poorer ~ fit ,, for the combining sites of the antibody studied. Not surprisinglyl the nature of the alkyl group within the ammonium ion is also important in antibody recognition. This is clearly illustrated by results obtained with a series of three tetraalkylammonium salts (table IV). The tetramethyl salt was approximately 27 and 58 times as active as the tetrapropyl and tetrapentyl derivatives respectively. With an occasional serum, for example serum Wa described in our 1983 study [5], a substituted ammonium group does not seem to constitute the complementary allergenic determinant since reactivity with alcuronium was not inhibited by any of the other NMBDs or other compounds containing tertiary or quarternary ammonium ions.

* Antibodies in serum Wh. ** Alcuronium-Scpharose solid phase.

Thus, IgE antibodies that react with NMBDs appear to fall into three main specificity groups : 1) those that recognize essentially only the ammonium groups of NMBDs and are thus inhibited almost equally well by all NMBDs ; 2) those that recognize not only the ammonium ions but also adjacent and/or adjoining structures. Hence, although these antibodies may recognize the different NMBDs, inhibitory potencies of the NMBDs may vary widely ; 3) antibodies that are not complementary to the ammonium groups but recognize other structures on the NMBDs. Such antibodies are likely to be inhibited only by the N M B D that provoked the patient's reaction.

134

B.A. BALDO, M.M. FISHER

As well as establishing the specificity of NMBDreactive IgE antibodies and before attempting to apply the RIAs to aid the diagnosis of anaphylactold reactions seen during anaesthesia, other essential control investigations were also undertaken. Of these controls, listed in BALDO and HARLE [9], tWO are highlighted here. Firstly, examination of sera from a wide variety of ~ normal ~ and allergic subjects and from subjects with certain pathological conditions, showed that significant uptakes of IgE antibodies by the NMBD supports sometimes occurred with sera containing high total IgE levels, for example, sera from subjects with eczema or allergic aspergillosis. These sera also showed high uptakes with other drugsolid phases. For detailed findings see article by BALDO and FISHE~ in this volume [7]. These findings are similar to R A S T results frequently obtained when sera containing high levels of IgE antibodies, especially sera from subjects with eczema, are used with a battery of inhalant and food allergen solid phases. A second important control that deserves to be highlighted here stems from the practice of obtaining sera often up to 30 days or so after the anaphylactic event. We therefore looked for NMBD-reactive IgE antibodies in the sera of subjects taken 30 days after receiving a N M B D during uneventful surgery. No antibodies were detected in any of the sera. RELATIONSHIP BETWEEN SKIN TEST AND RIA FINDINGS

In considering the relationship between results of skin tests with dilutions of NMBDs and RIAs for detection of NMBD-reactive IgE antibodies, it quickly becomes apparent that the RIAs detect more allergenic cross-reactions and these crossreactions are less relevant clinically than reactions detected by skin tests. This is, presumably, a reflection of the difference between recognition by the IgE antibody combining sites of the drug in the test tube and the extra requirement of crosslinking via the antibody combining sites of cellbound IgE molecules in the skin (see below). Hence, although RIA inhibition:studies may provide valuable quantitative data on the potencies and therefore the allergenic similarities of various inhibitory drugs, the relevance of such information to the clinical situation is not always obvious. This point is discussed in more detail in our paper dealing with diagnosis in this volume [7].

accepted that small molecular weight compounds (for example less than about 1 kD) which are themselves non-antigenic, become immunogenic by combining with endogenous macromolecules [41]_ However, apart from a small number of drugs and other chemicals including the penicillins, relatively few chemicals, their impurities or metabolites, have been recognized as having intrinsic protein binding properties [11]. In the absence of data supporting the existence of drug (or chemical) - endogenous protein complexes, the formation and involvement of quarternary (and tertiary) ammonium compound - - cell membrane complexes [12, 13] in the production of IgE antibodies to these chemicals should be investigated. A large proportion of subjects who react to a NMBD do so on first exposure suggesting that allergenic cross-reactivity underlies the sensitization. In other words, the nature of the sensitizing source for many patients is unknown and, presumably, not a NMBD. Results with one female patient we studied recently appears to support this. Serum from the subject who experienced an anaphylactoid attack after using a local anaesthetic throat spray was screened for IgE antibodies to a range of drugs. A strong positive reaction was obtained with the morphine solid phase and weaker, but clear positive, uptakes were also recorded with d-tubocurarine, Choline and, interestingly, the local anaesthetic procaine but not with lignocaine, the local anaesthetic in the throat spray (table III). The latter compounds, although never before tested with NMBD-reactive sera, contain a tertiary ammonium group (table II)_ The weak cross-reactivity of lignocaine detected in both direct binding and inhibition studies, however, failed to implicate this drug in the patient's reaction and leaves open the question of the identity of the drug or other agent that provoked the attack. It would be interesting to know whether or not the patient would react adversely to the administration of a NMBD. Although we have speculated that allergic sensitization to substituted ammonium groups may result from contact with the many compounds in our environment that contain these groups [5], this will be a difficult question to prove one way or the other. Screening large numbers of atopic subjects, especially women, for serum IgE antibodies to some carefully selected tertiary and quaternary ammonium solid phases is one possible, if laborious, initial approach to studying this question.

PERSISTENCE OF ALLERGY TO DRUGS ORIGIN AND SIGNIFICANCE SERUM ANTIBODIES

OF

NMBD-REACTIVE

IgE

From the early years of immunology, and especially the pioneering studies of LANDSTEINER and his contemporaries [40], it has been generally

Apart from a skin test study of this subject in patients with penicillin allergy, little data appears to be available on the length of time patients remain allergic to drugs_ SULLIVAN et al. [53] skin tested 740 subjects with penicillin G, penicilloic

MECHANISMS IN IgE-DEPENDENT ANAPHYLAXIS

acid and penicilloyl poly-L-lysine and found that 93 per cent were skin test-positive 7 to 12 months after reactions and 22 per cent were positive 10 years or more after reactions. These findings do not convincingly accord with the conclusion that skin tests become rapidly negative in subjects with penicillin allergy. DIDIER et al. [14] used skin tests, leucocyte histamine release and RIA to look for the persistence of allergy in 21 patients who had experienced an anaphylacti c reaction to succinylcholine. Skin tests showed that sensitization persisted in 18 of the patients 1 to 4 years later and histamine release experiments and RIAs also remained positive in a majority of the patients. In a recent study we found NMBD-reactive IgE antibodies in the sera and positive intradermal tests in patients 4-29 years after experiencing an anaphylactic reaction during anaesthesia. In the absence of evidence of allergy diminishing with time, we concluded that it would be wise for such patients to avoid the drug(s) responsible for the rest of their lives [21]. There appears to be little or no information available on the persistence of skin test reactivity, serum IgE antibodies or mast cell/basophil sensitivity to other drugs implicated in allergic sensitization and reactions.

NMBD-INDUCED ALLERGIC RELEASE OF MEDIATORS OF ANAPHYLAXlS

In the absence of evidence of the existence and involvement of NMBD-protein complexes in the release of allergic mediators, we suggested [5] that cross-linking of mast cell-bound IgE may occur via ammonium ion determinants on the free drug. Although the term ~ allergen ,, is frequently applied to a molecule that is recognized and bound by serum IgE antibodies, a more stringent definition requires the additional property of crosslinkage of adjacent cell-bound IgE molecules which occurs as a prelude to release. A biologically active allergen must therefore be at least divalent for effective cross-linking. Even in the absence of protein binding, all the NMBDs are, in terms of their allergenic determinants, at least divalent and thus equipped to initiate mediatorrelease as the free drug. Experimental verification of free drug-induced release from human leucocytes appears to have been provided [54]. Binding of a NMBD in vitro demonstrates the presence of NMBD-reactive IgE antibodies in the serum but this gives no indication of the capacity of the lgE antibodies to bind via the IgE Fc piece to mast cells or basophils or of the capacity of a drug to physically bridge the cell-bound antibody molecules. In relation to the latter point, a number of facts and speculations should be mentioned_

135

Skin test results and leucocyte histamine release studies suggest that both the length and the flexibility of the chain bearing the allergenic determinants appear to be important in the induction of mediator release. Examination of a series of diammonium salts of increasing chain length and comparison with succinylcholine and pancuronium in histamine release experiments; revealed that a chain length of greater than 4A was required for release. Optimum length for histamine release appeared to be a chain of 6A or more. It also seems that NMBDs with a rigid structure such as pancuronium, are less active in promoting release of mediators than structures such as succinylcholine which are flexible and thus allow considerable freedom of movement of the terminal ammonium groups [15]. These considerations are relevant to interpretations of in vitro RIA inhibition results and to the problem (referred to above) of relating inhibition findings to skin tests and clinical findings. For example, a finding of even potent inhibition by pancuronium, alcuronium or d-tubocurafine of IgE binding to a choline-solid phase, may not necessarily mean that the inhibitory drug will be active in the skin or promote a reaction in the patient if administered. Of course, binding of cell-bound IgE molecules may not occur at all with compounds such as morphine which, although a good inhibitor in vitro, has only a single IgE-binding determinant group. Binding of such allergenically univalent drugs to a carrier protein (or membrane) would therefore appear to be the only mechanism by which release of mediators could proceed. A possible alternate mechanism, however, has been proposed by SCHNEIDER et al. who examined the question of the efficacy of monovalent drug and chemical elicitors of mediator release in immediate hypersensitivity. SCHNEIDER et al. [50] concluded that chemicals or drugs without conjugation to a carrier may elicit anaphylaxis provided the monovalent compound possesses, in addition to the allergenic determinant or hapten, a so-called auxiliary group. PCA studies in guinea pigs with synthetic peptides containing an N-terminal 2-carboxy-4, 6-dinitrophenyl group as the hapten and phenylalanine and modified phenylalanine as the C-terminus auxiliary group, showed that anaphylactogenicity was high when the haptenic and auxiliary groups were separated by a long peptide chain. Even when the two groups were closely linked, however, anaphylaxis was sometimes observed. These general features and conclusions were also established in a study on penicillin antigens [ 2 5 ] . A number of different possible types of bridging, the trigger for mediator release, were also considered by SCHNEIDER et al. [50]. Four examples which may be relevant here, are summarized in figure 1. It seems likely that bridging may be

136

B.A. BALDO, M.M. FISHER

effccted by cross-linking adjacent lgE molecules via allergenic determinants on an unconjugated allergenical!y bivalent drug. The determinants on the drug may be of the same (fig. la) or of different (fig. lb) structure. Cross-linking of the type depicted in figure la is the mechanism envisaged to occur with the NMBDs. The involvement of the same (fig. lc), and of different determinants (fig. ld), may also be involved with drugs complexed to a carrier molecule. ,, Non-specific ~, bridging of adjacent cell-bound IgE molecules may also proceed via cross-linking of a combining site on one IgE molecule with a site distinct from the combining sites on an adjacent IgE molecule (for example a site on the Fc-piece). Other possibilities are also suggested by SCHNEIDER et al. [50].

NMBDs. Although the true incidence of reactions to the drug is hard to estimate, figures of around 1 in 20,000 have been discussed [8]. Despite similarities between thiopentone and N M B D s in the severity and clinical features of the anaphylactic episodes, some differences are noted. In particular, whereas a large proportion of the reactors to a N M B D experienced no previous exposure, patients who react to thiopentone invariably have received the drug before, that is, in contrast to NMBDs, exposure is usually a predisposing factor. Another major difference between reactions to thiopentone and N M B D s is the occasional finding of a delayed reaction with the former but not the latter drugs.

SERUM IgE ANTIBODIES THAT REACT WITH THIOPENTONE

a

c

b

d

Fig. 1 (a, b, c, d). - - Types of cross-linking, or bridging, of adjacent cell-bound IgE antibody molecules predicted to trigger release of mediators of allergy, a : Bridging via an allergenically divalent unconjugated drug molecule, that is, the mechanism envisaged for NMBD-induced mediator release in allergic subjects, b: Bridging via an unconjugated drug containing two different allergenic determinant groups, e and d : Bridging via conjugated drug molccules with cross-linkingeffected by the same or different determinants respectively. The clear allergenic cross-reactivity detected by lgE-antibody binding of morphine with the NMBDs, particularly d-tubocurarine, deserves closer study. Consistent with the serological findings, molecular models of morphine and d-tubocurarine revealed clear structural similarity between conformations of the trialkylammonium groups of each molecule [33]. Bearing in mind the pronounced histamine releasing properties of both morphine and d-tubocurarine, we speculated that the common structure identified may be complementary to a mast cell receptor site important in the initiation of histamine liberation by these drugs. ANAPHYLAXIS TO THIOPENTONE

Anaphylactic reactions to thtopentone are rare and occur far less frequently than reactions to

Although an R I A for the detection of thiopentone-reactive IgE antibodies has been developed [36] and the method has proved to be a valuable supplement to skin testing for the detection of thiopentone-allergic sensitivity, the method has some deficiencies, both in performance, and in the interpretation of results. Thiopentone is labile at high p H and in the production of the drug-solid phase where coupling is carried out under alkaline conditions, the possibility exists for some degradation of the drug. This seems to be reflected in the high backgrounds generally obtained in the R I A and in the relatively poor inhibition achieved with the free drug when used with sera from thiopentone-allergic patients. Despite this, two allergenic determinant regions have so far been identified on the molecule - - the secondary pentyl and ethyl groups on one side of the molecule and the thio region on the other (table V) [9, 35]. In applying the R I A to the study of sera from subjects who experienced an anaphylactic reaction during anaesthesia, it quickly became apparent that peculiar ~
MECHANISMS IN [gE-DEPENDENT ANAPHYLAXlS

137

Table V. - - IgE-binding structures identified so far in thiopentone.

Dominant structure in IgE-binding determinant* (in bold face)

Structure

0 CH~CH_, ~/~-- N)H=S CH~CH2CH2 ICH O/~-NH

CH3CH2~-

CH3CH_,CH2CH (~-- NH I

O

CH~

CH3 Thiopentone

N ~ S**

Serological findings***

Clinical relevance

Free drug (thiopentone) inhibits binding of patient's serum to ,~ thiopentone . solid phase t

Presence of IgE of this specificity indicates allergy to thiopentone

As above

As above

**CH3CH20~>~- N ~ S CH3CHzCHzCH ~ - - NH I

O

CH3 2 - Mercaptopyrimidine H, Reactive IgE in sera unlike thiopentone, from some inhibits binding of IgE NMBD-allergic t o , thiopentone ,, subjects. Subjects not solid phase allergic to thiopentone

N / ~ " SH* 2 - Mercapt0pyrimidine

* Exact confines of determinant not always clearly defined with all sera. ** Dominant feature of determinant shown Other structures, in particular the pyrimidine ring, probably has an auxiliary function. *** Findings when used with sera from subjects allergic to thiopentonc or a NMBD. * Thiopentone used to prepare drug-solid phase but coupling conditions probably leads to some decomposition of the thiopcntone. Exact structure of attached species therefore uncertain. 11 2 - Merc"a ptopyrimidine also inhibits binding of IgE antibodies reactive with the thio region of thiopentone [8, 34].

MORPHINE AND RELATED OPIOIDS

Although opioid analgesics are among the most commonly prescribed drugs in hospitals [17], anaphylactoid reactions to these drugs are rare and nowhere near as common as reactions to NMBDs and even thiopentone [23, 43]. The histamine releasing properties of many narcotics are thought, by some, to be responsible for anaphylactoid reactions (references in 23) but FISHER [20] has argued that if significant bronchospasm occurs in such reactions the reactions are more likely to be immune-mediated. In fact, we believe that the direct histamine-releasing effects of drugs in normal dosage are unlikely to cause clinical anaphylaxis in normal patients.

Allergenic determinants identified so far on morphine encompass the N-methyl group and the cyclohexenyl ring with a hydroxyl at C-6. Not surprisingly, quantitative hapten inhibition experiments revealed that codeine and nalorphine, that is, analogues with identical or closely related structures to the morphine determinants, also strongly bound the IgE antibodies (fig. 2). Pethidine and methadone were found to cross-react weakly with the morphine-reactive IgE antibodies [9, 29].

Morphine

IgE ANTIBODIES REACTIVE WITH MORPHINE AND CODEINE

IgE antibodies appear to have been detected to morphine [29] and pethidine (meperidine) [44] only. No details of methods or specificity were given in the latter study. Apart from morphine then, immunochemical information on allergenic determinants of opium narcotics and related drugs is limited and confined to a recent study [9, 23, 29]. Even with morphine, too few allergic subjects have been investigated to assess the extent of heterogeneity of IgE-binding determinants.

CH30~~ N__CH 3 HO Codeine

H(~~~ N__CH2CH= CH2 HO Nalorphine

Fig. 2. - - Structure of morphine with the allergenic determinant groups namely, the N - methyl and the cyclohexenyl ring with a hydroxyl at C-6, highlighted. The close structural similarities of codeine and nalorphine with morphine reveal the molecular basis of the observed allergenic (IgE-binding) cross-reactivity and why cross-reaction with codeine is greater than with nalorphine.

138

CROSS-REACTING DETERMINANTS ON MORPHINE AND NMBDS

As discussed earlier in this review, both direct binding RIA and inhibition studies have revealed that morphine shows clear cut cross-reactivity with NMBDs, particularly d-tubocurarine. Despite this, subjects allergic to NMBDs invariably tolerate morphine and show no allergic signs and symptoms. An obvious explanation for this, but one that still lacks definitive experimental proof, is that the NMBDS contain at least two substituted ammonium groups while morphine contains only one N-methyl substituent. Thus, cross-linking of adjacent IgE molecules on the mast cell can be effected by the divalent drugs but not by the univalent drug. This leads to the further assumption that allergenic reactivity of morphine must be mediated via protein or membrane binding of the drug, a mechanism for which evidence is usually lacking (see earlier this article). A more detailed comparison of findings with sera from subjects allergic to NMBDs or morphine are set out in the review by FISHER et al. [23].

DRUGS AND CHEMICALS AS ALLERGENS

Despite the frequent absence of evidence of both prior contact with the drug and conjugation of the drug to a carrier macromolecule, both of these requirements have, since the time of LANDSTEINER [40, 41], become part of immunological dogma [2, 3]. Although we have speculated that NMBDs may act as allergens in an unconjugated or free form, and there appears to be some evidence for this, the induction agent thiopentone and narcotics such as morphine and codeine and probably a vast range of other drugs, are allergenically univalent suggesting that conjugation to a carrier is a necessary prelude for allergic mediator release with these compounds. As mentioned above, no clear-cut evidence exists for proteinbinding of most of the drugs responsible for allergic sensitivities but the question of whether or not drug allergens are in a conjugated or free form is important to resolve for our ultimate understanding of mechanisms involved in allergic sensitization and drug-induced allergic release of mediators. Coupled with this is the belief that the employment of drug-protein conjugates is necessary for the demonstration of skin test sensitivity to drugs. It is clear, however, that many drugs in free form produce positive skin reactions that allows one to distinguish allergic from non-allergic subjects. Apart from NMBDs which are now routinely used for skin testing, we have found that a range of free drugs, including trimethoprim, sulfamethoxazole, narcotics, atropine, thiopentone, neostig-

B.A. BALDO, M.M. FISHER

mine, penicillins, cephalosporins and metoclopramide, produce specific positive skin reactions in allergic patients. Although, in relative terms, few drugs and few sera from drug-allergic patients have been studie so far, the range of allergenic determinant structures already identified on a small number of drugs already indicates both an interesting diversity of allergenic structures and the importance of determinant groups containing substituted Ns. In relation to the diversity of structures, it has been acknowledged since the pioneering immunochemical studies of LANDSTEINER[40] that antibodies may be formed to a wide range of low MW chemicals or haptens. Even so, the findings of IgE antibodies complementary to quaternary and tertiary ammonium groups and to secondary pentyl and ethyl groups on thiopentone were surprising. Other B-cell (IgE) determinants identified, for example, the 3,4-dimethoxybenzyl group in trimethoprim [51], the 5-methyl-3-isoxazolyl substituent of sulfamethoxazole [37] and various side chain groupings on penicillins [27] and cephalospotins [28[, suggest that accessibility of groups on the molecule, rather than the particular chemical structure, might be an important factor in the induction of B-cell responses.

THE SENSITIZING AGENT(S)

We sometimes find in the serum of drug-allergic patients, IgE antibodies to drugs other than the drug(s) given (see case reports and discussion in BALDO and FISHER, this volume [7]). This raises the following q u e s t i o n s 1) What was the sensitizing source of the antibodies and the drug allergic sensitivity ? 2) Was the subject previously exposed to that drug(s) ? 3) Is the sensitizing source a drug at all ? The answers to these questions are not always readily apparent and more detailed investigations other than simple direct IgE binding studies are usually needed to gain greater insights. Employment of quantitative hapten inhibition methodology with the suspected drug(s) and with well chosen analogues (if available) is usually the most direct and informative experimental approach to obtain information on the structural features of the haptenic groups complementary to the IgE antibodies detected [5, 9]. This information can, in turn, throw light on the nature and origin of the sensitizing compound and it is often then possible to decide between suspected drugs in cases where more than one was given. The dogma of prior exposure, however, should not obscure consideration of the possibility that allergic sensitization may have occurred via a non-

MECHANISMS IN IgE-DEPENDENTANAPHYLAXIS

related pharmacological agent, or for example, via a chemical encountered in the subject's environfllCllt-

CONCLUSION As more and more immediate type 1 drug allergies are studied at the level of IgE antibodies, it is difficult to escape the conclusion that cross-reactivity to drugs and other chemicals occurs just as it does with ~ normal >~ protein and carbohydrate allergens. Immunochemical studies designed to identify allergenic determinants on drugs and chemicals will increasingly provide us with insights into the chemical nature and the. diversity of structures of , small ~, molecules with allergenic properties and the size of the allergenic repertoire of ~ small ~ allergens. It is already apparent, however, that these ,~ small ~ molecules in the form of drugs and other chemicals contain structures of previously unsuspected immunogenicity and this provides a new dimension to the immunologist's and allergist's studies on the relationship between disease in man and antigenicity and allergenicity. Acknowledgements: This work was funded by the National Health and Medical Research Council of Australia. We thank Gail Knowland and Nghia Pham for technical help.

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ABSTRACT: The discovery of lgE antibodies to neuromuscular blocking drugs, to thiopentone and narcotics, particularly morphine, reinforced earlier beliefs formed from skin test and other clinical findings that many ,, anaphylactoid ~ reactions to drugs were true type 1 immediate hypersensitivity reactions. Immunochemical studies established the fine structural specificities of the drug-reactive IgE antibodies and provided an explanation in molecular terms for a number of observed clinical cross-reactions. Subtleties in interpreting relationships between skin tests and IgE radioimmunoassays are pointed out and mechanisms of drug-induced mediator release, persistence of IgE antibodies and the nature of the sensitizing sources are discussed.