Pholcodine exposure raises serum IgE in patients with previous anaphylaxis to neuromuscular blocking agents

Abstract

Background:  Neuromuscular blocking agents (NMBAs) can cause anaphylaxis through immunoglobulin E (IgE) antibodies that bind quaternary ammonium ion epitopes. These epitopes are present in numerous common chemicals and drugs, exposure to which, theoretically, could be of importance in the development and maintenance of the IgE sensitization promoting allergic reactions. Pholcodine is one such drug, which in a recent pilot study was shown to induce a remarkable increase in serum IgE levels in two IgE-sensitized individuals. The present study explores the effect of pholcodine exposure on IgE in a population with previously diagnosed IgE-mediated anaphylaxis towards NMBAs.

Methods:  Seventeen patients were randomized to 1 week’s exposure with cough syrup containing either pholcodine or guaifenesin. The primary variables serum IgE and IgE antibodies towards pholcodine, morphine and suxamethonium were measured before and 4 and 8 weeks after start of exposure.

Results:  Patients exposed to pholcodine had a sharp rise in levels of IgE antibodies towards pholcodine, morphine and suxamethonium, the median proportional increases 4 weeks after exposure reaching 39.0, 38.6 and 93.0 times that of the base levels respectively. Median proportional increase of IgE was 19.0. No changes were observed in the guaifenesin group.

Conclusion:  Serum levels of IgE antibodies associated with allergy towards NMBAs increase significantly in sensitized patients after exposure to cough syrup containing pholcodine. Availability of pholcodine should be restricted by medical authorities because of the potential risk of future allergic reactions to muscle relaxants.

Anaphylaxis during general anaesthesia is in many countries to a large proportion ascribed to immunoglobulin E (IgE)-mediated allergy towards neuromuscular blocking agents (NMBAs) (1–4). These reactions often occur in patients without any previously known exposure to NMBAs. In 1983 Baldo and Fisher (5) presented evidence that IgE from such patients bind NMBAs through a quaternary ammonium ion epitope (QAI), and that this epitope is shared with numerous common drugs and chemicals. Thus, It has been suspected that primary IgE sensitization, the basic prerequisite for an IgE-mediated anaphylaxis, could occur in contact with substances, other than NMBAs, carrying the QAI structure.

There has been an uneven distribution of reported severe NMBA reactions between countries. In Norway the frequency of anaphylaxis caused by NMBAs has been estimated to approximately 1 : 5200 general anaesthesias encompassing neuromuscular blockage, and NMBAs were found to be the causative agent in 93% of the cases in which IgE-mediated allergy was detected (3). In the neighbouring country, Sweden such reactions are far less common, and reports have indicated the frequency to be six times higher in Norway (6, 7). This difference has been attributed to varying degrees of IgE sensitization to QAI in the two otherwise highly comparable countries (6). In Norway, 0.4% of blood donors and 38.5% of NMBA anaphylactics were sensitized to suxamethonium, as were 5.0% and 66.7% to morphine and 6.0% and 64.6% to pholcodine, respectively, all drugs that carry the QAI epitope. No IgE antibodies to suxamethonium, morphine or pholcodine were detected among blood donors from Stockholm, Sweden. Cough syrup containing pholcodine from 1966 until March 2007, has been available without prescription in Norway, but has not been accessible in Sweden.

A recent pilot study indicated that a low dose of cough syrup containing pholcodine had a strong stimulating effect on the synthesis of IgE and IgE antibodies to pholcodine, morphine and suxamethonium in two IgE-sensitized individuals who had no history of anaphylaxis during anaesthesia (8). To further explore these findings, we conducted a randomized clinical trial on a population with previously diagnosed IgE-mediated anaphylaxis towards NMBAs. The aims were primarily to study whether pholcodine exposure caused changes in serum levels of IgE and IgE antibodies to pholcodine, morphine and suxamethonium in individuals previously IgE-sensitized to NMBAs and, adjunctly, to observe whether the cough syrup exposure had any impact on NMBA skin prick test (SPT) reactivity. As a further measure of changes in IgE, the serum was also analysed for common inhalant and food allergens.

Materials and methods

Patients

Patients were recruited from March to May 2006 at the Section for Clinical Allergology, Department of Occupational Medicine, Haukeland University Hospital, Bergen, Norway. This study was approved by the Regional Committee for Medical Research Ethics in Western Norway, the Norwegian Social Science Data Services, the Directorate for Health and Social Affairs and by the Norwegian Medicines Agency. We contacted patients who had previously been diagnosed with NMBA-allergic anaphylaxis at Haukeland University Hospital and who were living in the Bergen area. The diagnostic criteria for inclusion were: selection based on clinical data from the anaphylactic reactions and test results at follow-up examinations, including the presence of IgE antibodies towards suxamethonium and/or positive SPTs and histamine release tests with NMBAs. Exclusion criteria were: pregnancy, serious chronic diseases, acute bacterial or viral infections, age below 18 or above 69 years, use of beta-blocking medication and previous history of anaphylaxis to substances other than NMBAs. The time interval between the anaphylactic reactions and study inclusion varied from 4 months to 8 years (mean 5.8 years). At inclusion, each patient answered a questionnaire on prior use of cough syrup before further involvement.

Cough syrup exposure

The patients were randomized to ingest cough syrup containing either pholcodine (Tuxi^®, Weifa, Oslo, Norway) or guaifenesin (Solvipect^®, Nycomed Pharma, Asker, Norway). Cough syrup, both pholcodine and guaifenesin, was administered at 10 ml once daily [one third of a therapeutic daily dosage – suggested adequate for response by the pilot study (8)] for seven consecutive days. With a limited number of patients available for participation, it seemed feasible to expose the majority of the participants to the probable culprit substance. Participants were therefore randomly drawn at a 2 : 1 ratio without replacement to receive pholcodine or guaifenesin respectively.

Serum IgE analyses

Serum for analyses of IgE, often referred to as ‘total IgE’, and IgE antibodies, often referred to as ‘specific IgE’, was obtained at day 0, before exposure to cough syrup, and then 4 and 8 weeks after the start of exposure. The IgE and IgE antibodies to suxamethonium, morphine, pholcodine and inhalant and food allergen mixes (Phadiatop™ and Fx5™; Pharmacia Diagnostics AB, Uppsala, Sweden) were analysed by ImmunoCAP™ (Phadia, Uppsala, Sweden), with cut-off for a positive IgE antibody test set at 0.2 kU_A/l (samples measured below 0.2 kU_A/l were set to 0.1 kU_A/l for statistical analyses).

Skin prick tests

Skin prick tests were performed at day 0 and repeated 4 weeks thereafter. The tests included positive and negative controls and all NMBAs commercially available in Norway. All tests were performed in duplicates and according to the guidelines of the European Academy of Allergy and Clinical Immunology (9). An SPT result was considered positive when the mean of the duplicate wheal diameters was at least 3 mm larger than that of the negative control. Skin prick test panel: histamine chloride 10 mg/ml and negative control solution (ALK Abelló, Hørsholm, Denmark), cisatracurium 2 mg/ml and mivacurium 2 mg/ml (Nimbex^® and Mivacron^® respectively, GlaxoSmithKline, Brentford, Middlesex, UK), pancuronium 2 mg/ml, rocuronium 10 mg/ml and vecuronium 2 mg/ml (Pavulon^®, Esmeron^® and Norcuron^® respectively, Organon, Oss, The Netherlands) and suxamethonium 10 mg/ml (Curacit^®; Nycomed Pharma). All SPTs were performed by an allergy nurse or physician using a technique that produced a mean histamine wheal diameter of 5.3 mm with a coefficient of variation for the differences between duplicates (n = 14, day 0) of 19.5%.

Statistics

Two-sided Wilcoxon rank sum test was used for comparison of changes in IgE concentrations between the study groups, and Wilcoxon sign rank test for comparison of time-points within groups.

Results

The background characteristics of the patients are summarized in Table 1. The two treatment groups were fairly similar with regard to confounding background factors. However, the relative number of patients, with suxamethonium-specific IgE antibodies and positive SPT with NMBA prior to cough syrup exposure was somewhat higher in the guaifenesin group, while history of atopy was slightly more frequent in the pholcodine group. All patients reported to have ingested cough syrup of some kind in their lives prior to the study, but four of the patients could not remember to have consumed cough syrup containing pholcodine.

Table 1.   Background characteristics of patients with NMBA anaphylaxis

	Pholcodine group, (n = 11)	Guaifenesin group, (n = 6)	Total, (n = 17)
Sux, suxamethonium; SPT, skin prick test; NMBA, neuromuscular blocking agent; HRT, histamine release test *Moderate = not life-threatening; severe = life-threatening †Tryptase increase above the upper reference limit of 24.0 μg/l or at least three times that of the baseline level ‡IgE >0.35 kUA/l.
Age, mean (years)	45.6	40.2
Male : female ratio	2 : 9	1 : 5	3 : 14
Time from anaphylaxis to study inclusion (years)	6.51	4.36	5.75
Anaphylaxis moderate : severe*	3 : 8	1 : 5	4 : 13
Elevated tryptase at the time of reaction†	8	6	14
Mean IgE at the time of the reaction (kU/l)	285 (9/11)	351
Median IgE at the time of the reaction (kU/l)	163 (9/11)	183
Positive IgE–Sux at reaction/primary follow-up‡	6	5	11
Positive SPT–NMBA at the primary follow-up	5	5	10
Positive HRT–NMBA at the primary follow-up	1	2	3
History of atopic disease	6	2	8

Adverse events

All patients completed the study protocol. No obvious adverse events occurred during the study except for one pholcodine-assigned patient that experienced swollen eyelids during the last 3 days of cough syrup exposure.

Serum IgE and IgE antibodies

There were no significant differences in the concentrations of ‘total IgE’ or IgE antibodies to pholcodine, morphine or suxamethonium between the study groups prior to cough syrup exposure. During the 4 weeks after start of exposure, there was a large increase in IgE concentrations in the pholcodine group (Table 2 and Fig. 1) and at 8 weeks the IgE concentrations had moderately declined. No changes were seen in the guaifenesin group, neither at 4 nor at 8 weeks. The highest increase relative to baseline occurred with IgE antibodies towards suxamethonium, morphine and pholcodine, whereas the rise in IgE levels was intermediate; IgE antibodies to inhalant and food allergens showed only slight elevation. The differences between the two study groups for all primary variables (IgE and IgE antibodies to pholcodine, morphine and suxamethonium) were highly significant, (P < 0.01), (Table 3).

Table 2.   Median IgE (kU/l) and IgE antibody (kU_A/l) levels before and after cough syrup exposure

	Pholcodine group, (n = 11)			Guaifenesin group, (n = 6)
	Day 0	4 weeks	8 weeks	Day 0	4 weeks	8 weeks*
The 25th and 75th percentiles are given in parentheses. *n = 5
IgE-pholcodine	1.0 (0.4–5.1)	81 (11–232)	31 (10–115)	3.4 (0.9–4.5)	3.2 (1.0–4.2)	2.7 (0.4–5.1)
IgE-morphine	1.1 (0.2–3.1)	57 (12–207)	24 (8.9–93)	2.1 (0.5–3.4)	2.3 (0.5–4.0)	0.8 (0.3–3.1)
IgE-suxamethonium	0.1 (0.1–0.2)	9.3 (3.4–28)	5.5 (1.7–9.5)	0.2 (0.1–1.4)	0.2 (0.1–1.3)	0.1 (0.1–0.3)
IgE	153 (70–289)	5218 (589–8029)	3106 (481–3336)	163 (72–213)	142 (74–210)	143 (51–174)
IgE-inhalants	0.1 (0.1–0.1)	0.45 (0.3–1.2)	0.4 (0.2–0.8)	0.1 (0.1–0.1)	0.1 (0.1–0.1)	0.1 (0.1–0.1)
IgE-foods	0.1 (0.1–0.1)	0.2 (0.1–0.9)	0.1 (0.1–0.6)	0.1 (0.1–0.1)	0.1 (0.1–0.1)	0.1 (0.1–0.1)

Figure 1.  Concentrations of serum IgE and IgE antibodies before and 4 and 8 weeks after cough syrup exposure. Individual values shown as dots and median values shown as bars. Statistical significances of changes within group are tested by Wilcoxon sign rank test. Some comparisons within the guaifenesin group contained insufficient numbers of untied pairs for statistical calculations (<5, marked §).

Table 3.   Increase in concentrations of IgE and IgE antibodies to pholcodine, morphine, suxamethonium, Phadiatop and Fx5 given as ratios between concentrations 4 weeks after and before cough syrup exposure

	IgE-Pho	IgE-Mor	IgE-Sux	IgE	IgE-inhalants	IgE-foods
Pho, pholcodine; Mor, morphine; Sux, suxamethonium. *The hypothesis of no difference between the treatment groups is tested by Wilcoxon rank sum test (two-sided).
Pholcodine group, (n = 11)
Minimum	3.3	1.0	1.0	2.8	1.0	1.0
Median	39.0	38.6	93.0	18.7	3.1	1.8
Maximum	1570	2370	390	370	14.7	15.4
Guaifenesin group, (n = 6)
Minimum	0.81	0.88	0.86	0.84	1.0	1.0
Median	1.0	1.1	1.0	0.95	1.0	1.0
Maximum	1.2	1.3	1.0	1.1	1.2	1.2
P-value*	<0.01	<0.01	<0.01	<0.01	<0.01	<0.05

Skin prick tests

Table 4 summarizes the SPT results from 14 individuals. Three patients in the pholcodine group had taken oral antihistamines on days of testing and were excluded from the SPT section. There was a tendency towards increased numbers of positive SPTs and increased wheal diameters to the NMBA panel in the pholcodine group. A high number of negative results and the relatively low number of participants in both groups did not allow statistical calculations.

Table 4.   Skin prick test results to the NMBA panel before and after cough syrup exposure in 14* patients

	Pholcodine-group, (n = 8)		Guaifenesin-group, (n = 6)
	Day 0	4 weeks	Day 0	4 weeks
NMBA, neuromuscular blocking agent; SPT, skin prick test. *Number reduced from 17 to 14 because three of the participants (in the pholcodine group) used antihistamines in the study period. †Total number of positive SPTs to NMBAs in the study groups.
SPT positive patients	3	5	4	4
No. positive SPTs†	5	10	5	5
Mean SPT diameter	4.8	5.7	4.8	4.3

Discussion

This study shows that individuals, who have suffered allergic anaphylaxis during general anaesthesia and are IgE-sensitized to an NMBA, respond with a remarkable and statistically highly significant increase in IgE production when exposed to small doses of cough syrup containing pholcodine. The effects of pholcodine on the serum levels of IgE and IgE antibodies to pholcodine, morphine and suxamethonium in sensitized individuals observed in a recent pilot study (5) are thereby confirmed.

In the study population, there was a clear tendency of IgE antibody levels to pholcodine and morphine to be higher than those to suxamethonium. Only one of the 17 patients had higher concentrations of IgE antibodies to suxamethonium than to pholcodine and morphine. A similar relation was previously observed in a study on IgE antibodies in Norwegian and Swedish blood donors and atopic patients (6). If most individuals with an IgE sensitization to pholcodine and suxamethonium have IgE antibodies only to QAIs, the observed concentrations could be explained by antibodies with the same specificity displaying a higher affinity towards QAI on pholcodine and morphine than on suxamethonium. However, an additional allergenic epitope, not cross-reacting with the QAI was found on pholcodine and morphine (6). Higher concentrations of IgE antibodies to this epitope, and also more IgE-sensitized individuals, to this epitope than to QAI were found, e.g. among blood donors 6.0% were positive to pholcodine, 5.0% to morphine and only 0.4% to suxamethonium. As a consequence, it appears that morphine cannot be used to screen for IgE-sensitization to QAI, as suggested (10).

We did not include nonallergic negative controls in the study for ethical reasons. In Norway, IgE antibodies towards pholcodine and suxamethonium have prevalences that are approximately 300 and 20 times higher respectively than the frequency of NMBA-allergic anaphylaxis would suggest (3, 6). Studies on assumed negative controls with no history of anaphylaxis may reveal subjects that are QAI antibody positive at inclusion, or that during the exposure test boost their QAI antibodies to clinically significant levels or even become primary sensitized. These individuals would have to be considered at greater risk of NMBA-induced anaphylaxis than the general population. However, induction of general anaesthesia without the use of an NMBA is under certain conditions associated with increased risk of other complications (11–13). Information on presence of QAI antibodies in patients with no history of anaphylaxis is therefore difficult to apply in practical clinical decision-making. It could even make future anaesthesia more difficult to be applied. Healthy volunteers should not be offered this uncertainty as a result of participation in a clinical trial.

To test whether pholcodine is the actual primary sensitizer, a study would need to include a larger population of nonexposed, nonsensitized controls. It is questionable whether this hypothesis could be tested in Norway representing a highly pholcodine consuming population that since 1966 has been subjected to the over-the-counter availability of pholcodine from the age of 5 years. The inclusion of truly pholcodine-naive individuals would, therefore, seem a practical challenge.

The IgE reaction to pholcodine exposure seems to include both allergen- and nonallergen-specific polyclonal responses. The dominant response was the increase of IgE antibodies to pholcodine, morphine and suxamethonium, which was higher than that of IgE. As all participants presumably entered the study IgE-sensitized to the QAI and the additional epitope shared by morphine and pholcodine, the IgE antibody increases were presumably secondary immune responses – the boosting of established sensitizations to the two allergenic epitopes. By not including nonallergic controls in the trial, we have not studied the possibility that pholcodine may stimulate IgE by a nonimmunological mechanism. It is, however, theoretically difficult to explain how a nonspecific mechanism could result in this predominantly allergen-specific response. However, the study also demonstrated allergen-specific responses to common airway and food allergens, represented by Phadiatop and Fx5, but much weaker than those of IgE antibodies towards the pholcodine related epitopes. The maximum serum concentrations of IgE were much higher than what could be accounted for by the sum of the identified IgE antibodies alone, thus demonstrating an additional, probably nonallergen-specific polyclonal IgE stimulatory, property of pholcodine. The pholcodine exposed patients had a median increase in IgE of >5000 kU/l. Assuming that detectable IgE antibodies to different allergens constitute <10% of IgE in these patients, the median nonallergen-specific IgE production would be >4500 kU/l. Why pholcodine has such a strong mitogenic effect needs to be further studied. Pholcodine is structurally quite similar to morphine, the only difference being a morpholine side chain. Compared with other opioids, it also has a long metabolic half life of 35–50 h. The immunological properties of the substance may be linked to these two factors.

Studies on NMBA-allergic populations generally report a female predominance, but conflicting results have been obtained with respect to the association between atopy and NMBA allergy (1, 3, 14, 15). In the present study, there were both a clear female predominance and indications of a high frequency of atopy. These tendencies may reflect susceptibility factors for QAI IgE sensitization, a possibility further supported by the finding that among Norwegian ‘allergic’ patients 3.7% had IgE antibodies to suxamethonium and 10% had IgE antibodies to morphine, whereas the figures among Norwegian blood donors were 0.4% and 5% respectively (6).

The IgE sensitization to QAI seems to be related to pholcodine exposure, but it is not known to what extent the increased concentrations of IgE antibodies to the QAI allergenic epitopes immediately after pholcodine exposure would temporarily increase the risk of NMBA anaphylaxis. In a follow-up study from Norway (3) it was noted (unpublished data) that only nine out of 55 NMBA-allergic patients had serum levels of IgE to suxamethonium above class 2, i.e. above 3.5 kU_A/l, at the time of reaction. It can further be inferred from Norwegian estimates on anaphylaxis frequency and the prevalence of IgE antibodies to suxamethonium that only about 5% of patients sensitized to suxamethonium, i.e. with an IgE antibody level >0.35 kU_A/l, will develop anaphylaxis when exposed intravenously to NMBAs. Consequently, the serum concentration of NMBA-specific IgE antibodies per se does not appear to be a reliable predictor of IgE-mediated NMBA anaphylaxis. When sorting the samples, in the present study, according to increasing ratios of IgE antibodies to suxamethonium over IgE, a tendency for high ratios to be linked to positive SPTs to suxamethonium was noticed. Of the seven highest ratios six were SPT positive. Interestingly, in this context, basophile allergen threshold sensitivity (CD-sens), has been shown to correlate much better with the relative rather than the absolute serum IgE antibody concentration (16). Evidently, the clinical relevance of the ratio between NMBA-specific IgE antibodies and IgE deserves further attention.

Several studies have indicated uneven geographical distributions of NMBA-induced allergic anaphylaxis (1, 7, 17). A recent study related the difference in frequency reports of anaphylaxis between Norway and Sweden to varying degrees of exposure and sensitization to pholcodine (6) that contains the QAI epitope. The indicated associations, as underlined by the results of this study appear to advocate a more cautious access to the antitussive agent pholcodine. Based on these findings and those of the pilot study (8) the Norwegian producer decided not to renew the marketing license for the drug, which thereby was taken off the Norwegian market in March 2007. An international multi-centre study on the associations between pholcodine consumption, IgE sensitization and number of reported NMBA-allergic anaphylaxis is at present ongoing.

Acknowledgments

The authors are most grateful to Anne Berit Guttormsen PhD (Associate Professor, Department of Anaesthesia and Intensive Care, Haukeland University Hospital, Bergen, Norway) who contributed with organizational assistance to the research project and collection of historical data on the study population. Göran Nilsson (Statistician, Measurement Quality Co, Uppsala, Sweden) and Ågot Irgens (Statistician, Department of Occupational Medicine, Haukeland University Hospital, Bergen, Norway) were most helpful by performing statistical analyses. We also thank Sigrid Løken (Nurse, Section of Clinical Allergology, Department of Occupational Medicine, Haukeland University Hospital, Bergen, Norway) and Agnete Hvidsten (RNA, Section of Clinical Allergology, Department of Occupational Medicine, Haukeland University Hospital, Bergen, Norway) for their skilful performance of SPTs and project logistics and Ingegerd Ågren-Andersson (BMA, MIAB, Uppsala, Sweden) for performing the IgE analyses. Paul Husby PhD (Professor, Department of Anaesthesia and Intensive Care, Haukeland University Hospital, Bergen, Norway) provided useful advice for the presentation of results. This project was funded by the Western Norway Regional Health Authority (Helse Vest RHF), Stavanger, Norway.