Nickel oral hyposensitization in patients with systemic nickel allergy syndrome

Abstract Background: This is the first randomized, double-blind, placebo-controlled trial (EUDRACT No. 2009-013923-43) evaluating nickel oral hyposensitizing treatment (NiOHT) in patients with “systemic nickel allergy syndrome” (SNAS), characterized by Ni-allergic contact dermatitis and systemic reactions after eating Ni-rich food. Methods: Adults with positive Ni-patch test, who reported symptoms suggesting SNAS, which improved after Ni-poor diet, and were positive to Ni-oral challenge were eligible. Patients were randomly assigned to three treatments (1.5 μg, 0.3 μg, or 30 ng Ni/week) or placebo for a year, with progressive reintroduction of Ni-rich foods form the 5th month. Out of 141 patients randomized, 113 completed the trial. Endpoints were efficacy and tolerability of treatment. Results: During Ni-rich food re-introduction, the 1.5 μg Ni/week group had a mean VAS score significantly higher than placebo (p = 0.044), with significant improvement of gastrointestinal symptoms (p = 0.016;) and significantly fewer rescue medications. Cutaneous manifestations also improved but without reaching statistical significance. After the treatment, oral challenge with higher Ni doses than at baseline were needed to cause symptoms to flare-up in significantly more patients given 1.5 μg Ni/week than placebo (p = 0.05). Patients reported no side-effects. Conclusions: NiOHT is effective in SNAS, in particular on gastrointestinal manifestations, with trend toward improvement of cutaneous symptoms.


Introduction
A part of patients suff ering from Ni-allergic contact dermatitis (ACD) -which aff ects 10-20% of the population (1 -3) -experiences skin (urticaria/angioedema, fl ares, itching) and gastrointestinal symptoms (meteorism, colic, diarrhea) aft er eating Ni-rich foods (4,5). Th is condition, that according to a recent report (5) aff ects 20 -30% of Ni-ACD patients, is known as systemic contact dermatitis or systemic nickel allergy syndrome (SNAS), the latter better describing the involvement of organs other than skin and the implied immunologic mechanism that involves Th 2 as well as ACD ' s typical Th 1 cytokines (6 -11).
SNAS patients need to follow a lifelong Ni-poor diet, basically avoiding the majority of vegetables, which poses a potential risk for a defi cit in essential elements. To minimize this, nutritionally balanced diets such as BraMa-Ni are now available (5). On the basis of animal studies demonstrating the induction of nickel tolerance aft er repeated oral doses of metal salts (12,13), the eff ects of nickel oral hyposensitizing treatment (NiOHT) were studied in humans (open, non-randomized trials), with encouraging results (14 -17). Parallel to the clinical improvement, a year of NiOHT signifi cantly reduced in vitro nickel-induced Th 1 and Th 2 cytokines from peripheral blood mononuclear cells compared to baseline. Th ere were no such changes in controls who simply followed a Ni-poor diet for a year (9).
On the basis of these observations, we designed the present trial to study the clinical effi cacy and tolerability of NiOHT in SNAS, evaluating the disappearance or reduction of systemic symptoms during the re-introduction of Ni-rich foods, the use of rescue medications and the appearance of side eff ects.

Patients and Methods
Th is was a multicenter, prospective, phase III, randomized, double-blind, placebo-controlled trial with four parallel groups (three treatment doses and placebo) conducted in eight university or hospital allergology units in Italy (Chieti, Agropoli, Genoa, Latina, Lecce, Messina, Rome Catholic University, Rome IDI). Ethics committees in all centers approved the protocol. Enrolled patients gave their signed informed consent.

Eligibility criteria
Eligible participants were adults who i) had a positive Ni-patch test, ii) reported symptoms suggesting SNAS (5), iii) improved at least 70% from baseline aft er one month on a Ni-poor diet (severity of symptoms rated on a visual analog scale -VAS), and iv) were positive to a Ni-oral challenge (NOC).
Exclusion criteria were pregnancy and lactation, concomitant treatment with steroids and/or antihistamines, inability to give the informed consent, and participation in another study.

Interventions
As treatment, patients took hard gelatin capsules containing diff erent doses of Ni-as NiSO 4 ⋅ 6H 2 O with microcrystalline cellulose as excipient, or placebo, all identical in appearance and fl avor. Eligible patients used the BraMa-Ni diet (5) for one month, and were then randomly assigned to four groups. Th e fi rst three groups were given nickel at doses increasing in 40 days from 1 ng to the three fi nal maintenance doses (10 ng, 0.1 μ g and 0.5 μ g, corresponding to a dose of NiSO 4 of 0.044 μ g, 0.44 μ g and 2.2 μ g respectively), three times a week for 12 months. To protect the blinding, patients randomized to lower doses received placebo for the fi rst days of the up-dosing phase, according to the scheme in Table 1.
Nickel oral challenge was done by administering weekly increasing doses of nickel, from 1.25 to 6 μ g (with increment of 1.25 μ g), until the appearance of clinical manifestations of SNAS, in particular taking into account of objective data as the appearance of cutaneous (urticaria, angioedema, eczema, erythema) or gastrointestinal (evident meteorism, diarrhea, vomiting, colic). Capsules were made by Lofarma SpA, Milan, Italy.
All patients were allowed rescue medications (desloratadine 5 mg/day and/or prednisone 25 mg/day) if necessary.

Outcomes
Primary endpoints: 1) disappearance or reduction of symptoms during Ni-rich food re-introduction (patients reported their clinical status in a diary and made a VAS at each control visit), and use of rescue medications (reported in the diary); 2) tolerability evaluated on the basis of side eff ects (reported in the diary).
Ancillary analyses: in a substantial number of patients, patch tests became negative aft er the treatment and the NOC doses able to elicit symptoms at the end of the study changed from baseline. Th erefore, since the patch test and NOC were included in the protocol, these fi ndings were analyzed, even though this was not pre-specifi ed.
T1 visit (end of diet): Clinical evaluation, VAS, and NOC in patients whose VAS rating improved at least 70% from baseline. NOC-positive patients were randomized for treatment and received Ni capsules, diary and rescue medications.
T2 visit (3rd month of treatment): clinical and diary evaluation, VAS. Prescription to re-introduce foods with maximum 100 μ g/kg nickel content during the 5th month, then up to 200 μ g/kg during the 6th month of treatment (Table 2).
T3 visit (7th month of treatment): clinical and diary evaluation, VAS. Prescription to re-introduce foods with maximum 500 μ g/kg nickel content for a month, then all other Ni-rich foods ( Table 2).
T4 visit (end of treatment): clinical and diary evaluation, VAS, patch test, NOC, laboratory tests. All eligible patients aft er one month of Ni-pour diet were randomly assigned to one of the four groups of treatment. Nickel dose was progressively increased in 40 days from 1 ng to 3 defi nite maintenance doses (10 ng, 0.1 μ g and 0.5 μ g) administered 3 times a week for a total of 12 months. In order to protect the blinding, patients randomized to lower doses received placebo during the fi rst days of the up-dosing phase. Th e reintroduction of Ni-rich foods started from the 5 th month with foods with maximum 100 μ g/Kg nickel content, until 200 μ g/Kg during the 6 th month, until 500 μ g/Kg during the 7 th month and then all other Ni-rich foods from the 8 th month. List of Ni content in foods has been derived from literature (26 -29).
Unscheduled visits were allowed at patients ' request and clinical data were recorded.

Visual Analogic Scale
Patients were asked to show in a 10 cm Visual Analogic Scale their perceived clinical condition (with 0 the worst and 10 the best) at enrollment and at each control visit.

Sample size/power/level of signifi cance
Published studies report that NiOHT was eff ective (disappearance of symptoms on re-introduction of Ni-rich foods) in 60% of SNAS patients, as opposed to 30% disappearance of symptoms in the placebo or untreated groups. Setting the type I error at 5% ( α ϭ 0.05) in a one-tailed test of signifi cance and a type II error of 20%, which corresponds to a study power of 80%, the calculated sample size indicated 30 patients for each of four treatment arms. Considering a drop-out rate of 30 -40%, a total of 160 patients were enrolled and 40 were randomly assigned per treatment arm. PASS 2005 soft ware (Kaysville, UT) was used.

Randomization
We used a computer-generated list of random numbers to allocate participants, with the randomization sequence stratifi ed by center with 1:1:1:1 allocation. Capsules were pre-packed in blister packs and consecutively numbered for each patient according to the randomization schedule. Each patient was assigned an order number and received the corresponding packs. Th e contract research organization that controlled the quality standard throughout the trial prepared the allocation sequence and stored the allocation list. Investigators enrolled patients and assigned them to their specifi c groups.

Statistical analysis
Patients who reached constant dosage were included in the intention-to-treat (ITT) analysis. Categorical variables were summarized as frequency and percentages. Continuous variables were presented as mean and standard deviation (SD). Effi cacy was defi ned as the proportion of patients in each of the four treatment arms whose symptoms were either reduced or eliminated. Th e chi-square test with Yates ' s continuity correction or Fisher ' s exact test was employed to examine diff erences in effi cacy. Intergroup diff erences in continuous variables were analyzed by ANOVA and the t-test. Th e confi dence interval (95% CI) was calculated for the mean diff erence and percentage risk diff erence between groups. Th e equivalent non-parametric tests were used when the data were not normally distributed or the sample was too small. All hypotheses were tested considering a single tail. P values of 0.05 or less were considered statistically signifi cant. SPSS ® Advanced Statistical ™ 13 (2004, Chicago, IL) was used.

Results
Recruitment started in April 2010 and ended in May 2012. Figure 1 illustrates the fl ow diagram of the trial. Out of 141 patients randomized, 129, who reached the constant dosage of NiOHT, were included in the intention-to-treat (ITT) analysis. 113 of them completed the trial.

Symptoms
Th e main demographic characteristics of enrolled patients and their symptom severity at enrolment (rated on the VAS) are summarized in Table 3, where symptoms reported by 129 patients included in the ITT analysis are also reported. As per inclusion criteria, NOC, performed aft er one month of BraMa-Ni diet, elicited the reappearance of SNAS symptoms (cutaneous and gastrointestinal in particular, but not cough and headache).
As expected, considering the inclusion criteria, all clinical parameters of all groups had signifi cantly improved from baseline at T1 (aft er the fi rst month of diet), with no signifi cant diff erences between the groups (non-parametric tests, p Ͻ 0.05) (

Outcomes
Th ere were no signifi cant diff erences in VAS and the various symptoms among the groups at the T2 and T3 visits. At T4, with the re-introduction of the highest Ni-containing foods (500 μ g/ kg and over), group 1 (given the highest Ni dose) showed the best values for all parameters, groups 3 and 4 the worst and group 2 intermediate. Only for group 1 were the changes from baseline signifi cantly better than placebo; the signifi cance was amplifi ed when considering groups 3 and 4 together as placebo. Th ere were no diff erences between groups 3 and 4 in any parameter, and in some cases group 3 patients were worse than placebo. Figure 2 shows the changes in VAS scores from baseline. Th e mean score for group 1 was signifi cantly higher than group 4 (t-test p ϭ 0. Headache and cough did not signifi cantly change during the study, and no signifi cant diff erences were found between groups (data not shown).

Rescue medications
At T4, with the re-introduction of Ni-rich foods, three patients in group 1 took rescue medications compared with 17 in group 2, 12 in group 3 and 11 in group 4 (each group vs. group 1, Fisher ' s exact test, p Ͻ 0.05).

Ancillary analyses
At the end of the study, a signifi cantly larger number of group 1 patients than at baseline needed a NOC with higher Ni doses to induce a fl are-up of symptoms compared to placebo (Fisher ' s exact test p ϭ 0.05; risk diff erence 27.6; 95% CI, 0. 8 to 49.3) or placebo plus group 3 (Fisher ' s exact test p ϭ 0.002; risk diff erence 38.1; 95% CI 14.5 to 55.7) (Figure 3). Signifi cantly more patients had a negative patch test at the end of the study in group 1 than group 4 (Fisher ' s exact test p ϭ 0.008; risk diff erence 32.9; 95% CI 9.0 to 54.8) and group 3 ϩ 4 (Fisher ' s exact test p ϭ 0.011; risk diff erence 27.7; 95% CI 0.8 to 49.3) (Figure 4).

Side eff ects
Only one patient in group 1 reported gastrointestinal symptoms aft er the 10th dose of 0.5 μ g Ni. Th e patient took desloratadine and symptoms disappeared within 3 hours. Th e patient concluded the study as scheduled.

Discussion
Th is is the fi rst double-blind, randomized, placebo-controlled trial evaluating the effi cacy of NiOHT in patients with SNAS. Th e treatment was eff ective. During the re-introduction of Ni-rich foods, symptoms improved signifi cantly in patients given the highest Ni dose, compared to placebo, with a VAS score similar to that of patients in Ni-pour diet. Th e eff ect of NiOHT seemed dose-dependent, as 1.5 μ g Ni/week gave the best results, 30 ng Ni/ week and placebo the worst and 0.3 μ g Ni/week was intermediate. Gastrointestinal symptoms signifi cantly improved, parallel with VAS scores, compared to placebo (Table 4 and Figure 2), and were more sensitive to NiOHT than cutaneous manifestations, which decreased in frequency, but without reaching statistical signifi cance (p Ͼ 0.05) compared to group 3 and placebo. Th is is not altogether surprising, as the skin contact with nickel, which can never be completely avoided, might have induced symptoms linked to ACD, confounding the results. Cough and headache, reported by patients as linked to the ingestion of Ni-rich foods, did not signifi cantly change during the study, nor were induced by NOC. A recent publication (5) confi rmed that they seemed not to be part of the syndrome as they were never induced by NOC, and did not reappear with the re-introduction of Ni-rich foods aft er amelioration induced by a nickel low diet. Th e eff ectiveness of NiOHT with 1.5 μ g Ni/week is corroborated by the observation that during the re-introduction of Ni-rich foods only three patients (10.3%) took rescue medications, compared to signifi cantly more in other groups (group 1 vs. each group, p Ͻ 0.05). Th e subjective data, symptoms and VAS ratings, which show post-NiOHT tolerance to nickel, are supported by objective tests such as NOC and patch test. At the end of treatment, signifi cantly more patients in group 1 than in group 3 and the placebo group needed a higher nickel dose at the oral challenge to elicit symptoms than before treatment. Similar signifi cant diff erences were found between group 1 and groups 4 or 4 ϩ 3 for patients with a negative patch test at the end of the study.
No comparison is possible with other trials, as all previous studies on the eff ects of NiOHT were unregistered and open (14 -17), only one randomized trial comparing patients treated with active medications and controls receiving only a Ni-poor diet (9). Results were positive, as only the treated patients were able to re-introduce the majority of Ni-rich foods. Other trials in patients with ACD but no systemic symptoms gave contrasting results. Open studies (18 -20) were positive, while the double-blind one indicated that NiOHT reduced the T-cell in vitro response to nickel but failed to improve the clinical expression of ACD (21). In addition, the various trials on SNAS are hard to compare because of the wide range of nickel doses (from 0.1 ng to 0.5 μ g), frequency of dosing (daily to weekly) and diagnostic methods (essentially based on history, though a few on specifi c NOC). One open randomized trial found that, parallel with clinical effi cacy, lymphocytes from NiOHT-treated patients released signifi cantly less nickel-induced IFN-γ , IL-13 and IL-5 in vitro than at baseline, whereas there were no signifi cant changes in Ni-poor diet controls (9).   placebo controlled NOC, anyway being the study blinded and randomized the possible biases were distributed among groups and do not aff ect the overall result of the trial.
In conclusion, the results of the study have substantial clinical implications, considering the large numbers of patients with SNAS (in a recent study rated as 20 -30% of all Ni-ACD patients), whose quality of life is diminished on account of their lifelong dietary restrictions. NiOHT allows patients to re-introduce Ni-rich foods in the absence of substantial side eff ects.

Trial Registration
Th e trial was registered in EUDRACT with No. 2009 -013923-43 Declaration of interest: Th e authors declare the following confl ict of interest: their institutions received a grant by Lofarma for the present work. Mario Di Gioacchino and Luisa Ricciardi received payment from Lofarma for lessons on allergy to Lofarma ' s employees.
Funding source: Th is study was funded by Lofarma SpA (Milan, Italy).
Th e involvement of Th 2 cytokines in the pathogenesis of SNAS has been reported in various studies (10,11), showing a signifi cant dose-dependent increase of serum IL-5 in SNAS, but not in ACD patients. Signifi cant decreases of CD3 ϩ CD45RO ϩ CLA ϩ and CD8 ϩ CD45RO ϩ CLA ϩ blood lymphocytes have also been seen, with massive infi ltration of CD4 ϩ cells in the duodenal lamina propria and epithelium (to our knowledge the only pathology with CD4 ϩ cell infi ltration in the epithelium) (7). A study on the immunomodulatory properties of NiOHT also showed an increase of IL-10 (17), a regulatory cytokine involved in the action of vaccines for inhalant and hymenoptera venom allergy (22). Th e cytokine changes, fi rst of all of regulatory cytokines, led to the hypothesis that nickel tolerance aft er NiOHT might be a consequence of the diff erentiation and proliferation of nickel-specifi c T regulatory lymphocytes, which can maintain immune tolerance to Ni in healthy subjects (23). Th is also can explain the eff ect of the low Ni doses administered. In fact, high doses of antigen favor an anergy-driven pathway to tolerance while low doses of antigen promote a suppressive pathway via regulatory T cells producing IL-10 and TGF-β (24).
Clinical aspects of SNAS was described by Braga et al (5). Th e most frequent manifestation of SNAS was the fl are-up of previous ACD eczematous lesions reported by all patients, followed by a fl are-up of a previously positive nickel patch test. Such symptoms were variably associated with eczema in regions not in contact with the metal and/or with urticaria and angioedema. In almost all cases patients reported meteorism and dyspepsia combined with colic, gastric acidity, vomit, diarrhea or throat acidity; less than 10% of patients experienced gut symptoms without skin manifestations. Lactose intolerance was found in a high percentage of SNAS patients.
We think that SNAS is closely related to the systemic contact dermatitis (SCD), were only cutaneous symptoms are considered, but in our experience almost all patients previously diagnosed as SCD to nickel have gastrointestinal symptoms. Many other metals are considered to induce SCD, such as cobalt, chromium, and zinc that are ubiquitous in our environment, with cutaneous manifestations like urticaria/angioedema, lichen planus, palmoplantar pustulosis, and maculopapular rash. Also in these cases, the diagnosis of sensitivity to metal is established by epicutaneous patch testing and oral metal challenge (25). In these cases, contrary to Nickel sensitivity, we never found gastrointestinal symptoms.
Limitations of the present study are related to the determination of nickel doses, sample size, duration of the treatment. Nickel doses during the trial were established in relation to those used in the majority of studies, but the lowest gave exactly the same results as placebo, and we do not know whether doses higher than the largest we used would have given better results or might have caused side eff ects. Th e diffi culty in determining Ni doses for treatment is also underlined by the fact that Ni given with NiOHT is immediately bioavailable, on the contrary little is known about Ni speciation and bioavailability of Ni introduced with foods. Furthermore, the lack of published data made it hard to select the best parameters for adequate sample size. Th e number of patients enrolled seems to have been too low, and in fact, on combining group 3 (patients receiving the lowest nickel dose) with the placebo group, the statistical signifi cance appeared clearer. In a larger sample, we might have been able to gain a better picture of NiOHT ' s eff ects on skin symptoms. Similarly, we do not know if one year is long enough to obtain stable results, so this needs to be verifi ed by an adequate period of follow-up. Furthermore, we did not perform