Since the early 1900s, allergen-specific immunotherapy

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1 Otolaryngology Head and Neck Surgery (2007) 136, S1-S20 REVIEW Sublingual Immunotherapy: Past, present, paradigm for the future? A review of the literature Bryan D. Leatherman, MD, Sara Owen, MD, Mike Parker, MD, Stephen Chadwick, MD, John A. Fornadley, MD, Doug Colson, MD, and Paul Fass, MD, Gulfport, MS; Dallas, TX; Syracuse, NY; Decatur and Springfield, IL; Camp Hill and Hershey, PA; and Aventura, FL Since the early 1900s, allergen immunotherapy has been recognized as an effective treatment option for patients with inhalant allergies. Subcutaneous injection has traditionally been the main route of antigen delivery for immunotherapy in the United States. Over the past 15 years, sublingual administration of allergen extract has become a widely used method of immunotherapy in other countries, particularly in Europe. Although sublingual immunotherapy (SLIT) has been used by some physicians in the United States, this technique has not found widespread utilization. A growing interest in SLIT use in this country is developing. SLIT offers several potential advantages, including excellent safety and tolerability, increased access to immunotherapy, and improved method of antigen delivery to children. This paper reviews the basic and clinical science data available in the literature concerning the immunology, efficacy, and safety of SLIT. It is written to serve as a springboard for future discussions and clinical investigations regarding the potential expanded use of SLIT in the United States American Academy of Otolaryngology Head and Neck Surgery Foundation. All rights reserved. Since the early 1900s, allergen-specific immunotherapy has been recognized as an effective treatment for inhalant allergies. The concept of allergen immunotherapy began with the passive transfer of antibodies to toxins demonstrated by Pasteur, Von Behring, and Kitasato. 1 The belief that pollen-induced illness was mediated by toxins led to early attempts at immunization against pollens. Some of these early attempts were successful, 2 while others resulted in harm to the patients. The introduction of pollen antigen quantitation by Leonard Noon allowed for the beginning of safe, reproducible antigen dose administration. 1 This paved the road for the ultimate expansion of allergen immunotherapy into a widely used, effective treatment option for patients with allergic diseases refractory to avoidance and other allergy medications. Immunotherapy involves administration of increasing concentrations of antigen-specific extracts to allergic patients with the goal of producing changes in the immune system. The ultimate goal is to provide alleviation or reduction in the symptoms produced during natural exposure to the antigen. Immunotherapy takes ad- From the Department of Otolaryngology Head and Neck Surgery, University of Arkansas for Medical Sciences, Gulfport, MS (Dr Leatherman); Department of Internal Medicine, Division of Allergy and Immunology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX (Dr Owen); Department of Otolaryngology and Communication Sciences, SUNY Upstate Medical University, Syracuse, NY (Drs Parker and Colson); Clinical Research, ENT Allergy, Head and Neck Institute, Decatur Memorial Hospital Systems, Decatur, IL, and Otolaryngology and Family Practice, Southern Illinois University, School of Medicine, Springfield, IL (Dr Chadwick); Associated Otolaryngologists of PA, Camp Hill, PA, and Division of Otolaryngology, Penn State University, Hershey, PA (Dr Fornadley); and private practice, Aventura, FL (Dr Fass). Dr Chadwick is a consultant for Lincoln Diagnostics, Aventis, Pharmacia-Upjohn, Pfizer, Schering-Plough, Merck, ALK-Abello, GSK, Ortho- McNeil, Alcon, Antigen Labs, and Abbott; Dr Leatherman is on the Speaker s Bureau of Pfizer and is a consultant for Alcon; Dr Parker is on the Speaker s Bureau of Aventis, GSK, and Merck; and Dr Fornadley is on the Speaker s Bureau of Schering and is a stockholder of GSK and Johnson & Johnson. Reprint requests: Bryan D. Leatherman, MD, FAAOA, Department of Otolaryngology Head and Neck Surgery, University of Arkansas for Medical Sciences, 1213 Broad Avenue, Suite 4, Gulfport, MS address: leathermanbryand@uams.edu /$ American Academy of Otolaryngology Head and Neck Surgery Foundation. All rights reserved. doi: /j.otohns

2 S2 Otolaryngology Head and Neck Surgery, Vol 136, No 3S, March 2007 vantage of the body s tendency to inhibit the effects of some types of stimulus, if the stimulus is given on a scheduled basis, in appropriate amounts, without abrupt changes in intensity. 1 Immunotherapy utilizes this adaptive ability to modulate the body s immune response to allergen stimulus, allowing the body to tolerate the effects of environmental allergen challenge. Successful immunotherapy results in multiple immune system changes that eventually downregulate the immunological response to the allergen stimulus, allowing better control of the symptoms produced. The most common route of antigen delivery has historically been subcutaneous. Attempts have been made throughout the years to deliver allergens via alternate routes. These alternate routes have carried multiple names including nonparenteral, local, noninjection, and alternative immunotherapy. To avoid confusion, it is generally agreed on that the best collective terms are local and noninjection routes. Early in the 20th century, oral immunotherapy was proposed as a possible route of allergen delivery. 3 Bronchial inhalation and nasal routes of allergen administration were also proposed in the next few decades. 3-6 The British Committee for the Safety of Medicine questioned the risk-benefit ratio of subcutaneous immunotherapy (SCIT) in 1986 after discovering several deaths were caused by SCIT treatment. 7 Despite clear evidence that the deaths were caused by avoidable human errors and multiple subsequent studies demonstrating the safety of SCIT, 8-10 interest in the development of noninjection routes of immunotherapy has persisted. Improved safety, convenience, and potential cost savings are the main driving forces behind the continued interest in noninjection routes of immunotherapy. SLIT has its origins in the development of an oral route of allergen delivery. Oral allergen administration is an attractive potential route due to the gut-associated lymphoid tissue (GALT), which could potentially serve as a rich portal of antigen exposure to the immune system. 11,12 Several authors have demonstrated that gut absorption of proteins is sufficient to provoke an immune response Specialized membranous epithelial cells (M cells) overlie the ileal GALT and are thought to facilitate macromolecular transport of proteins across the distal ileal mucosal surface for immune presentation. The presence of low-level antibodies to foods in most individuals and the appearance of cow s milk specific IgM in infants as early as 1 week old provide further evidence of immune interaction with ingested proteins. 16 It is also possible for allergen proteins to be absorbed from the intestinal tract and interact with immune cells in a distant location. 14,17-19 During oral immunotherapy, allergen is swallowed in an aqueous solution, capsules, or enteric-coated tablets. Despite the theoretical advantages of oral immunotherapy, subsequent studies demonstrated marginal evidence of efficacy. Only three of nine double-blind placebo-controlled (DBPC) trials showed any statistical improvement in rhinitis symptoms. These studies also demonstrated that gastrointestinal side effects became more frequent as the high doses of antigen necessary to achieve symptom improvement were reached. 3 Sabra et al 19 reported that only 2 percent of ingested protein could be measured in the serum, while another study revealed only 0.1 percent of ingested food proteins could be found antigenically intact in the serum. 20 This is one possible explanation of why significant improvements were only found at high doses. In 1986, SLIT using low doses of dust mite allergen was reported. 21 Since that time, numerous investigators, mostly from European centers, have published data about the clinical efficacy and basic science of SLIT. In 1998, the World Health Organization reported that there were sufficient efficacy and safety data available to conclude that SLIT was an acceptable means of immunotherapy administration. 22 A subsequent publication by the European Academy of Allergology and Immunology, as well as the Allergic Rhinitis and Its Impact on Asthma (ARIA) document, agreed with the utility of SLIT in adults and children. 23,24 Although SLIT has been used for a long time by some physicians in the United States, there has not been widespread experience with or acceptance of the technique in this country. The purpose of this supplement is to investigate the current knowledge of SLIT by reviewing the published data concerning the basic science and clinical experience with SLIT. The supplement reviews the basic science regarding the sublingual absorption of allergen and evidence of immunological changes produced by SLIT, including the potential locations of immune interaction. A review of the clinical efficacy and safety data of SLIT will also be presented. It is hoped that this publication will promote future discussions and clinical investigations regarding the potential expanded use of SLIT in the United States. The literature review for this publication was accomplished by performing an Ovid MEDLINE search to identify publications related to the areas of interest as described above. The search included publications dating back to Some important publications concerning the basic science of SLIT were identified as references in articles read during the review process. Publications were limited to the English language. PREVIOUS SUBLINGUAL IMMUNOTHERAPY REVIEWS Previous reviews of SLIT have been published in the literature. Malling et al 25 published a review in The purpose of this article was to evaluate clinical efficacy of SLIT based on DBPC studies published in peer-reviewed English-language literature between 1990 and June Twenty-three studies were reviewed, encompassing 523 study patients and 492 controls. There was no meta-analysis. Rather, the author set an evaluation standard for efficacy and applied this to each individual study. The paper defined a level of clinically relevant efficacy as a 30 percent improvement of scores evaluating symptom

3 Leatherman et al Sublingual Immunotherapy: Past, present, paradigm for the future?... S3 reduction or reduction in medication use. Less than a 30 percent improvement was considered insufficient to justify therapy. Studies with data showing at least a 30 percent improvement in both symptom and medication scores were graded as efficacious. Studies with improvement in one of the categories were judged as showing possible efficacy. Studies without improvement in either category or those where symptom and medication scores were discordant were graded no efficacy. The results of this analysis showed 6 of the 23 (26%) to be effective, 8 (35%) as possibly effective, and 9 (39%) as not effective. The authors reviewed studies for type of antigen, size of study population, age of population, study parameters, and allergen dose in an effort to identify factors that predicted success or lack of success in the administration of SLIT. There was no specific factor identified to suggest a link to an efficacious or a nonefficacious therapy. It was noted that most of the trials that yielded nonsignificant results were underpowered, that is, they had population sizes too small to detect even relatively large changes (such as 25% 50%) between treatments. The study conclusion was that there was insufficient evidence to recommend SLIT for use in routine clinical practice. While continuing to show promise, further largescale, well-controlled studies are needed to define the role of SLIT in the treatment of allergic diseases. The process used by the authors appears valid. The conclusions drawn are supported by the evaluation within the article. The encouraging finding of at least possible efficacy found in 61 percent of the articles is not overstated. The authors identification of low power present in the studies failing to show efficacy is interesting and possibly critical. This review article should be considered as strongly supportive of the safety and strong probability of efficacy from at least some form of SLIT but does not constitute any endorsement of the therapy for clinical treatments. Another review article was recently published by Passalacqua et al. 26 The stated purpose of this article was to evaluate clinical efficacy and safety of SLIT and secondarily to consider mechanisms of action. The studies that made up the evaluation were identified from a MEDLINE search of keywords sublingual immunotherapy, respiratory allergy, asthma, and rhinitis in the English-language peerreviewed literature between 1986 and November The paper has three major sections. The first considers clinical efficacy of sublingual therapy in rhinitis and asthma separately. The second part addresses safety, and the third, mechanisms of action. Finally, there is a short passage devoted to controversies and a conclusion section. In the review of efficacy, the authors draw from 25 DBPC studies considered to have adequate study methods to be included for consideration. The criteria for inclusion, or the articles excluded, were not provided. The results of the articles are accepted at face value without commentary. The authors report that 22 of the studies confirmed the clinical efficacy of SLIT in rhinitis.... No critical analysis of the studies patient selection, methods, statistical analysis, or conclusions is offered. In considering asthma, the authors again relate only the conclusions reached by the authors of the individual studies. Further, the authors draw report data from an open, nonblinded study. The tone of this article appeared to be slanted heavily in favor of SLIT being safe and efficacious. In the review of rhinitis, the only articles subjected to critical review of process and assessment of results were the three studies that did not show support for SLIT. Build-up dosage schedules and mechanisms of action are discussed as if efficacy were already firmly established. The most powerful supporting data presented by the authors would be the reference to Wilson et al, 27 who published a meta-analysis entitled Sublingual Immunotherapy (Cochrane Review), which presumably evaluated the same studies that the present authors identify. Comparative studies with small to moderate numbers of patients do exist, but the literature is hard pressed to yield comparative analyses of such studies, with the exception of Wilson et al s 2005 article Sublingual Immunotherapy for Allergic Rhinitis: Systematic Review and Meta-analysis. 28 This paper, previously printed as a Cochrane Review, deserves attention in deciding the future of SLIT as a modality. This meta-analysis was an excellent effort to pool the data available in multiple small efficacy studies to give a better understanding of the overall efficacy of the SLIT technique. The following comments will attempt to summarize the paper, discuss some of the elements of systematic review and meta-analysis as they pertain to this work, and give an opinion on future directions for developing SLIT. The objectives of Wilson et al s 28 systematic review and meta-analysis were to evaluate the available literature for the efficacy of SLIT compared with placebo or injection immunotherapy for reductions in symptoms and/or medication requirements in allergic rhinitis (not artificially induced) and for alterations in immunological markers in blood and target organs, including in vivo allergen sensitivity. Studies to be selected were all randomized placebocontrolled clinical trials. Potential trials for inclusion were obtained through excellent search engines, including the Cochrane Controlled Clinical Trials Register, EMBASE ( ), MEDLINE ( ), and Scisearch (to 2002). Furthermore, select conferences were canvassed for relevant abstracts, and select allergy specialists were queried for appropriate ongoing trials or unpublished data. Through a collaboration of the authors, 81 abstracts were identified and subsequently reduced to a final 22, studying 979 patients. All studies were DBPC trials of parallel-group design. Reasons for elimination included being review articles, descriptive studies, unsuitable routes of delivery, lack of randomization or control, and duplicate or pending trials. Methods, interventions, and outcomes are listed in their

4 S4 Otolaryngology Head and Neck Surgery, Vol 136, No 3S, March 2007 article. The trials were single-antigen predominant SLIT trials with six for house dust mite, five for Parietaria, five for grass pollen, two for olive pollen, and one each for cat dander, ragweed pollen, and Cupressus. All of the continuous outcome data was fed into RevMan 4.1 for statistical analysis. An analysis method of standardized mean differences (SMD) was used because the trials authors used varied scoring and recording systems for symptoms observed and medications used. SMD produces a difference in means between the immunotherapy and placebo patients in measures of a pooled standard deviation. Random effect models were used for SLIT summary statistics of overall efficacy. These data were presented as SMDs with 95 percent confidence intervals. Significant differences between studies were determined using 2 tests to evaluate heterogeneity with a value of P 0.1. Initial study design subgroup comparisons included seasonal versus perennial allergens, children versus adults, dosage of major allergen delivered, duration of immunotherapy, and sublingual-spit versus sublingual-swallow. World Health Organization guidelines were used to determine the major allergen comparisons: less than 5 g of major allergen versus 5 20 g versus more than 20 g. To evaluate preseasonal, perennial, and prolonged treatment, the duration of immunotherapy intervals were less than 6 months versus 6 12 months versus longer than 12 months. The meta-analysis for the symptoms severity scores indicated that SLIT significantly reduces rhinitic symptoms and the need for medication. There was, however, significant heterogeneity between the trials, thought to be related to the variety in scoring systems used in the trials. Despite this, Wilson et al 28 thought the statistically significant positive treatment effect validated the conclusion. The subgroup analyses did not indicate any outstanding benefit from SLIT based on age of the patient, allergen used, or duration of therapy. However, there was a significant heterogeneity between the trials in almost all subgroup cases, thought to be due to the variety in symptom scoring systems and perhaps the smaller numbers in the subgroup trials included. Therefore, caution is advised in interpreting these results. That being said, trends did emerge. SLIT treatment for the seasonal allergens did show a significant effect, but the numbers were small. SLIT treatment for the perennial allergens trended toward beneficial significance. Concluding a benefit of treatment for seasonal versus perennial allergens in symptoms and medication reduction cannot be proved by this analysis. Beneficial treatment effect in children was not significant, but the numbers were small. There was significant beneficial effect for all ages combined and for adults only. The duration of treatment beyond 12 months trended strongly toward significance but failed, probably due to small numbers. Wilson et al 28 commented that total dose of allergen delivered probably has benefit, but the quantification of major allergen used was not uniform and/or lacking in the trials, making a sound statement of significance difficult. The evaluation of changes in immunological markers was very difficult due to a lack of consensus in measuring antibody levels and the wide variety of in vivo techniques used to evaluate target organ response. Data accepted for meta-analysis, with small numbers being the case, did suggest a trend of increased allergen-specific IgG4 in the SLIT patients. Last, significant side effects were not reported in any of the trials. The incidence of minor oral side effects was high and included tingling, itching, and swelling beneath the tongue. Two of the trials studied, included a comparison of SLIT with injection immunotherapy, indicating similar reduction in symptoms and medication needs between the two modalities. However, based on the data in the literature to date, Wilson et al 28 thought that a direct quantitative comparison between SLIT and injection immunotherapy cannot be made at this time. That being said, given the risk of adverse reactions (even the occasional death) associated with injection immunotherapy, the inconvenience, and the cost of this time-intense management modality, Wilson et al 28 conclude SLIT appears to be the most attractive alternative form of antigen delivery for desensitization. They use the results of their meta-analysis to give direction and call for further investigation of SLIT. Wilson et al 28 are to be complimented for their paper. As noted, SLIT has been poised for a more widespread introduction in this country. Practice standards and management guidelines are continuing to evolve with the aid of evidencebased medicine (EBM) principles. As Bousquet 29 points out, with regard to the efficacy and potential role of SLIT, there has not been a pivotal controlled trial or a metaanalysis published to date, thereby making an evidencebased appraisal impossible. As a first meta-analysis for SLIT trials, Wilson et al 28 had to adhere to rigorous standards of a systematic review and apply them to secondary research, i.e., where the unit of study is other studies or trials. This meta-analysis, having been initially published as a Cochrane Review, 27 had to conform to the newer standards of the time for preparing systematic reviews advocated by the Cochrane Collaboration. 29,30 Having said that, apparently no system or effort in systematic review and meta-analysis (Cochrane included) is free of error, or the suspicion of bias as clinicians and statisticians point out. 31,32 This has to be taken into consideration when looking at Wilson et al s 28 article. Wilson et al 28 well defined the question to be answered about SLIT, in essence, Do randomized, placebo controlled studies support or refute the efficacy of SLIT in the reduction of symptoms and/or medication in naturally occurring allergic rhinitis? From this premise, the objective for the review and the meta-analysis was set along with other secondary objectives. Appropriately, multiple quality sources were used to amass candidate trials for the systematic review. The databases used included the Cochrane Clinical Trials Register, MEDLINE ( ), EMBASE ( ), and Scisearch. Comprehensive catch terms

5 Leatherman et al Sublingual Immunotherapy: Past, present, paradigm for the future?... S5 used included rhin (covers rhinitis, rhinopathy, rhinosinusitis, and rhinoconjunctivitis), hay fever, immunotherapy, desensitization, and sublingual. Reference lists of recent published trials, and reviews were examined. Abstracts from appropriate conferences were studied. Colleagues were polled for unpublished data or ongoing trials. This was a fairly complete effort. Other paramedical databases and gray literature would seem inappropriate for examination. From a bias standpoint, arguments can always be made about the question of positive bias in trials accepted for publication. For example, trials with desired outcome data may be more likely to be submitted for publication compared to trials with disappointing outcome data. Wilson et al 28 commented on these issues. All identified studies were read by one author, and then selection was made after discussion with another author. Additional information was obtained from trial authors when needed in deciding on selection. Methodological quality was then assessed for selection for meta-analysis. An extensive algorithm of paring down 81 studies to 22 finalists was given. All studies were DBPC trials of parallel-group design. Concealment of treatment allocation and binding of study subjects and investigators were addressed. Wilson et al 28 acknowledge some of the difficulty in judging methodological quality due to the less-regulated datedness of some of the studies. The details of weighting the trials are not spelled out in terms of methodological quality, precision, or external validity; reference was given to Cochrane Collaboration guidelines. Relevant details for the extraction sheet were explained. Extracted data were then entered into RevMan 4.1 for statistical analysis. Due to differences between trials in the variety of scores and scales of symptoms evaluation used and in medication usage, the rationale for using the method of SMD in comparing immunotherapy versus placebo patients was explained. Random effects models (as opposed to fixed or combined effects models) were used to evaluate overall efficacy of SLIT given in terms of SMDs with 95 percent confidence limits. Commonly used 2 tests evaluated statistical heterogeneity between studies with significance being P 0.1. This is preferred over P 0.05 used in many studies. 33 When methodological or clinical heterogeneity is apparent, the question always is whether it is statistically significant. Dealing with statistical heterogeneity can be a difficult issue in systematic reviews and is a subject of discussion among statisticians. The 2 test has been criticized as demonstrating poor power when too few trials are involved and too much power in identifying clinically unimportant heterogeneity when the trial numbers are large in the meta-analysis. 34,35 Other measures of the impact of heterogeneity on the meta-analysis have been offered, including using mathematical criteria independent of the number of trials or treatment effect measure. 34 Details of the subgroup comparisons were given, from which, after looking at the characteristics of the included trials, one might have expected some degree of the heterogeneity subsequently statistically shown, which should give rise to caution in drawing conclusions. Cautions are acceptable, as long as they are so identified. They force the clinician to put the result in perspective (positive or negative) when making clinical decisions or deciding about future study. The meta-analysis of scores representing reduction in symptoms and decrease in medication usage confirms the said efficacy of SLIT. Wilson et al 28 give caution to the results because of the heterogeneity between the studies but give an acceptable rationale for viewing these as valid conclusions. Even with further studies, the subgroup analyses did not seem to confer disproportionate advantage of SLIT in any select patient group or antigen class. Heterogeneity was common. Variability in scoring systems and patient number issues were thought to probably be causative and noted as such. Seasonal pollen patients were in sufficient numbers to show significance for SLIT in symptoms reduction and medication usage, but the fewer numbered perennials only trended positive for SLIT. Children as a group did not seem to benefit from SLIT as did the adults, but the numbers of children were small, and caution was given to assuming the negative result. Extending the treatment duration to longer than 12 months positively trended to efficacy but was short of significance due to a small number of trials. Total antigen dose may have a positive impact, but the analysis fell short of such a conclusion due to the lack of reporting of antigen quantity and doses in the trials. Selective immunological markers were not well evaluated due to lack of uniform reporting in the trials. However, increases in IgG4 trended in the immunotherapy patients. No significant side effects were reported in any of the studies; minor oral tingling, itching, and swelling under the tongue were common. Wilson et al 28 have done well with this systematic review and meta-analysis. No review is without potential criticism, especially when the controversies of statistical theory and construct are applied. 36 A great deal of thought and effort were applied to the project and the exceptions and shortcomings were identified and commented on, and the appropriate cautions given. This analysis gives a vote of efficacy for SLIT in the reduction of symptoms scores and medication needs in allergic rhinitis. As Bousquet 29 points out, this analysis gives further credence to the Allergic Rhinitis and Its Impact on Asthma (ARIA) guidelines on SLIT. The ARIA guidelines recognize SLIT as a treatment option for allergic rhinitis in adults. 37 It gives some direction for further areas of study and sets the stage for a large, perhaps pivotal study in efficacy. While a meta-analysis is a valuable means of pooling small studies to gain a larger patient population from which to draw conclusions, the findings in individual studies are still valuable. It is important to review the efficacy literature, including some studies that have a lower evidence grade than a randomized, DBPC study. The findings of lower evidence grade articles may be valuable to stimulate thought and demonstrate needs for potential future studies

6 S6 Otolaryngology Head and Neck Surgery, Vol 136, No 3S, March 2007 to confirm or refute findings. This review also puts more emphasis on exploring the basic science data related to SLIT than previous review articles, particularly the evidence of sublingual absorption and immunological changes observed with SLIT. BASIC SCIENCE Sublingual Absorption Motivation to develop an alternative or noninjection route for the administration of immunotherapy has been based on the desire to deliver that therapy with enhanced safety compared to conventional SLIT. The ideal alternative route would allow for rapid antigen absorption without significant degradation of antigen, such as that which might occur in the gastrointestinal tract. The sublingual site was suggested because of the widely recognized ability of this site to absorb small nonprotein molecules, such as ethanol and topical nitrate preparations. Clinical studies documenting the efficacy of noninjection immunotherapy have led to the study of the absorption and distribution kinetics of antigen in noninjection techniques, although very little data are available in the literature on this topic. Several studies contribute to the understanding of the pharmacokinetics of antigen administered by noninjection routes. The most studied routes of noninjection immunotherapy include oral, sublingual-swallow, and sublingualspit techniques. The oral route involves antigen prepared as aqueous solutions, tablets, or gastroresistant capsules that are swallowed immediately. With sublingual-swallow immunotherapy, the antigen is held under the tongue for a period of time, usually 1 to 2 minutes and then swallowed. The sublingual spit technique requires the antigen to be spit out after remaining under the tongue for 1 to 2 minutes. The pharmacokinetics of noninjection routes were investigated in humans using an experimental model of purified allergen (ParJ 1) labeled with radioactive 123 I. 38 In this study, nonallergic individuals were administered radiolabeled allergen by oral, sublingual, or intranasal routes. Sequential scintiscans, plasma radioactivity, and plasma chromatography were used to follow the radiolabeled antigen at intervals after antigen presentation to the various sites. Based on the profile of plasma radioactivity over time, no direct evidence of systemic absorption of the allergen appeared to occur through the oral mucosa. Up to 2 percent of radioactivity (labeled antigen) was retained in the mucosa of the sublingual area for up to 40 hours after exposure. When free 123 I was presented to the sublingual space, it disappeared from the space rapidly. Antigen that was subsequently swallowed lent itself to gastrointestinal absorption, but only peptide fragments could be identified in the bloodstream. This suggests that deiodination and degradation of the antigen occurred during transit in the gastrointestinal tract and that only free 123 I and small peptide fragments were absorbed. These data imply that a critical aspect of SLIT is the contact of antigen with the oral mucosa, where persistent presence may play a role in the mechanism of SLIT s clinical success, with immune processing of the locally retained allergen occurring through a local immune response. Sublingual pharmacokinetics did not differ between allergic and healthy subjects 39 or between modified allergens (monomeric allergoid) and native allergens, but the modified allergen did appear in slightly increased amounts in the plasma. 40 There was no significant difference in local pharmacokinetics between the sublingual-swallow route and sublingual-spit route, suggesting there was rapid binding of the allergen to oral mucosa following sublingual administration. After allowing for mucosal exposure, spitting out the remainder of the dose had little effect on biodistribution of antigen. Only 30 percent of the antigen was recovered in the spit saliva. 41 Pharmacokinetic and absorption data on SLIT support the notion that it is not rapid absorption of antigen from the sublingual space that contributes to the success of this method but rather a local event (perhaps immunological) occurring in the mucosa of the sublingual space, where antigen is retained for an extended period of time. Greater understanding of events occurring in the mucosal after antigen exposure will contribute to a better appreciation of the immunological events that occur after sublingual antigen administration. Immunology of SLIT A substantial body of research has been devoted to understanding the immunological mechanisms by which subcutaneous, allergen-specific immunotherapy improves allergy symptoms. As the interest in SLIT has increased, a greater number of investigators are also reporting data on the immunological changes produced by this method of immunotherapy administration. Because the majority of research has involved SCIT, this section begins with a brief review of the published data involving immunological changes and the proposed mechanisms of action with SCIT. This will be followed by a review of the more recent information regarding immunological changes with SLIT. One of the most common parameters to measure when investigating immunological changes is serum antibody levels. An increase in allergen-specific IgG levels is usually the first change measurable with SCIT. It has been hypothesized that the IgG antibodies function as blocking antibodies by preventing allergen binding to IgE located on the surfaces of mast cells and basophils, thus thwarting transmembrane signal transduction and the subsequent degranulation of these cells There is experimental evidence that allergen-specific IgG can provide passive protection against anaphylaxis. 45,47-49 Analysis of IgG subsets reveals that typically IgG1 predominately increases in the early months of immunotherapy but then tends to slowly decrease in the later phases of treatment. In contrast, IgG4 levels begin to rise later in the course of treatment. The levels tend to remain elevated several years after discontinuation of successful immunotherapy before eventually declining toward

7 Leatherman et al Sublingual Immunotherapy: Past, present, paradigm for the future?... S7 pretreatment levels The rise in IgG4 strongly correlates with clinical improvement in symptom-medication scores, more so than any other immunological parameter IgG antibodies are also thought to inhibit the process of serumfacilitated antigen presentation, a process that allows IgEmediated presentation of allergens to allergen-presenting cells at very low serum concentrations. As a result, a higher concentration of allergen is necessary to induce T-lymphocyte proliferation and proallergic cytokine production. 58 Another immunoglobulin to rise in the first few months of SCIT is allergen-specific IgE, usually increasing by more than 2-fold. The levels gradually decrease over the next 2 years, eventually falling below pretreatment levels. 51,59-62 It has been proposed that the initial increase is secondary to an early stimulation of the T-helper 2 cell (T H 2) response and increased interleukin (IL)-4 synthesis The gradual decrease may be a result of the stimulation and generation of allergen-specific suppressor T cells. 66,67 Surprisingly, there is no correlation between the decline in allergen-specific IgE and clinical improvement. 46 The decline in IgE does correlate with the rise in IgG antibodies. This relationship has been called the scissors pattern of antibody response to immunotherapy. 68 Patients on successful immunotherapy also have a blunting of the rise in allergen-specific IgE that typically occurs in allergic and nonallergic subjects after natural exposure during the allergen s season. 61,66,69,70 There are other proposed mechanisms by which immunoglobulin changes could result in symptom improvement. Immunotherapy stimulates allergen-specific B lymphocytes to increase IgA and IgM production, which could boost the barrier function against antigen penetration at the mucosal surfaces. This has been demonstrated clinically with an observed increase in allergen-specific IgA in nasal secretions during the course of immunotherapy. 71,72 It is well established that T-lymphocytes play a major role in the allergic inflammatory response. 73 Studies have shown that immunotherapy alters the function of T cells in many ways. It has been proposed that there are two subsets of T cells defined by the types of cytokines they produce. T H 1 cells produce cytokines, such as interferon (INF)-, IL-2, and tumor necrosis factor (TNF)- that induce phagocytic and T-cell mediated microbial defense reactions. T H 2 cells produce cytokines, including IL-4, IL-5, IL-6, and IL-13, that promote IgE-mediated mast-cell degranulation and eosinophil activity associated with type I hypersensitivity. 74 The cytokines produced by the two subsets also have an inhibitory effect on each other. Immunotherapy results in a switch from a T H 2 cytokine profile to a T H 1 response. 75 There are conflicting data about the absolute values of change in specific cytokines, but the increased ratio of T H 1toT H 2 is a consistent finding in the literature Studies have also demonstrated that there is a decreased recruitment of CD4 T cells to an area of provocation after treatment with immunotherapy. The proportion of T H 1 cells in the area of provocation also has been found to increase. 75 An increase in the number of allergenspecific suppressor T cells has been demonstrated with immunotherapy. The cells result in an inhibition of allergenspecific IgE or a decrease in the production of cytokines associated with the allergic response. 67,82-84 It has also been suggested that immunotherapy may exert its effects by altering the expression of the certain T-cell receptor subtypes, producing an increase in receptor types that are more anergic or suppressive with regard to the particular antigen. 85 More recently, the importance of T-cell IL-10 production as a possible mediator and marker of successful immunotherapy has been described. 86 Whatever the mechanisms involved, there is good evidence that T-cell alterations play a central role in the process by which immunotherapy exerts its positive effects on allergy symptoms. Besides immunoglobulin and T-cell changes, other immune alterations have been observed with SCIT. Changes in histamine release have been observed. Studies have shown a downregulation of basophil reactivity and sensitivity. The threshold for histamine release is increased and the total histamine levels are decreased with SCIT Reductions in histamine release can be demonstrated before alterations in IgE and IgG levels are produced at the onset of immunotherapy. Immunotherapy also has pronounced effects on eosinophils. There is suppression of factors that induce eosinophil adhesion, thus inhibiting recruitment in inflammation. 90 A decreased adhesion of eosinophils to intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 has been reported. 91 Immunotherapy results in a blunting of the seasonal increase in bronchoalveolar eosinophils often seen in allergic asthmatics. 92 Nasal secretions contain lower levels of eosinophilic cationic protein (ECP) and tryptase after immunotherapy, both of which are markers of eosinophil activity. 93 After SCIT, there is a decrease in allergen-induced migration of eosinophils and mast cells to nasal mucosa and skin Multiple other potential processes by which immunotherapy decreases clinical allergy symptoms have been proposed. There is much ongoing research into the immunological alterations produced by SCIT, and it is certainly reasonable to assume that many more potential mechanisms of SCIT symptom improvement will be discovered. During the past 15 years, numerous papers have been published describing the immunological changes that result from SLIT. Table 1 lists many of the immunological changes that have been observed with SLIT. Similar to SCIT, T-cell alterations have been reported with SLIT. Early in vitro experiments demonstrated a reduction in the proliferative response of T cells in allergic subjects. 98 A recent study has demonstrated an increase in IL-10 production in allergic patients treated with SLIT compared with control subjects. IL-10 has been linked to the development of tolerance to allergens. 99 This study also showed a reduction in T-cell proliferation in the SLIT group. Other studies have shown that SLIT reduces ICAM-1 expression on epithelial cells. This reduction parallels reduced numbers of eosinophils and neutrophils observed in the nose and con-

8 S8 Otolaryngology Head and Neck Surgery, Vol 136, No 3S, March 2007 Table 1 Immunologic changes observed with SLIT treatment Decreased antigen-specific IgE Reduced postseasonal rise in IgE Early rise in IgG1, with a decline after 2yof treatment Slow initial rise in IgG4, rapid increase between 18 and 24 mo Increased IL-10 production Reduction in T-cell proliferation Reduced eosinophils Reduced neutrophils Decreased serum eosinophilic cationic protein (ECP) Decreased serum IL-13 Decreased serum prolactin Decreased nasal tryptase and specific IgE Decreased nasal tryptase during allergen challenge Decreased urinary leukotriene levels junctiva after allergen challenge and a decreased bronchial reactivity to methacholine. 100,101 Immunoglobulin profile changes have been observed after SLIT. Tari et al 102 showed SLIT resulted in a significant decrease in antigen-specific IgE. The placebo group had a typical peak-season rise in specific IgE, but this rise was absent in the treated group. IgG changes were identified in the treatment group only. There was an early rise in IgG1 antibodies, with a decline after 2 years of treatment. The IgG4 levels had a slow initial rise, followed by a rapid increase between 18 and 24 months. Most of the data concerning immunoglobulin changes are found in efficacy studies. Multiple studies have reported changes in IgE and/or IgG levels similar to those found with SCIT In contrast, a few studies either showed no changes in IgE or IgG levels, or only one of the two immunoglobulin levels changed It is unclear why there is a discrepancy in immunoglobulin levels between studies, although it may have to do with differences in SLIT techniques and durations. Other inflammatory parameter changes have been reported by various authors. Ippoliti et al studied serum markers of inflammation before and after SLIT. They found a significant decrease in serum ECP, IL-13, and prolactin levels. It is thought that prolactin, produced by activated T cells, may play a role in the immune response by acting as a growth factor for previously stimulated immunocompetent cells to undergo clonal expansion. It also helps induce IL-2 production and alterations in T-cell receptors IL-13 isat H 2 cytokine that induces B cells to switch toward IgE and IgG4 production and is important in the formation of memory cells. 116,117 A recent study measured several parameters in active SLIT patients versus placebo controls. After 1 year of treatment, the basal levels of nasal tryptase and specific IgE were significantly increased in the placebo group but not in the active group. Nasal allergen challenge produced a significant nasal tryptase increase in the placebo group that was not seen in the active group. Neither group had a change in nasal or sputum ECP levels. 118 High urinary leukotriene levels have been reported in allergic rhinitis and asthma patients. 119,120 In 1999, Yuksel et al 121 looked for changes in urinary leukotriene levels after SLIT. They found a significant decrease in urinary leukotriene levels in allergic rhinitis patients undergoing SLIT compared with placebo control subjects. This decrease did not reach significance in the asthma group. The data on the immunological effects of SLIT are growing, adding to the ever-increasing understanding of the immunological changes that accompany the improved symptoms obtained with both subcutaneous and sublingual allergen immunotherapy. CLINICAL SCIENCE Efficacy of SLIT In 1986, the first randomized SLIT trial was published. 21 Since that time, multiple additional efficacy studies have been published evaluating the effectiveness of SLIT in treating allergic manifestations. Multiple antigen preparations have been used, including oral drops, dissolving tablets, or a combination of the two. Dosing and escalation techniques have varied somewhat, but generally they are quite similar. A diverse range of treatment durations have been used. SLIT has been administered preseasonal only, preseasonal continuing through the season, and continuous without regard to season. Total treatment time has ranged from 2 months to 5 years in the published efficacy studies. Success with treatment has been demonstrated with all these various treatment techniques. A MEDLINE review was performed to identify clinical studies concerning the efficacy of SLIT. Only studies that compared SLIT treatment with placebo or controls were included in the review. Using these criteria, 36 studies were identified and reviewed. An additional six studies were identified that included a comparison of results between SLIT and SCIT. It is generally agreed on that efficacy with allergen immunotherapy is best determined by evaluation of medication use scores and subjective symptom scores before and after treatment. There is no consensus to what degree of improvement in these scores, either individually or in combination, is required to declare clinical efficacy. In most SLIT clinical efficacy studies, the authors declared clinical efficacy if there was a statistically significant reduction in either medication or symptom scores. In order to avoid creating arbitrary efficacy guidelines that have not been validated, the conclusion of the authors was used to determine if the SLIT treatment was effective for each publication reviewed. Of the 36 publications comparing active SLIT treatment with placebo or controls, 30 made the determination that SLIT was effective for rhinitis, conjuncitivitis, and/or asthma (Table 2). All of these studies included subjects with symptomatic sensitization to only one allergen. The allergens studied included pollens from trees, grasses and weeds,

9 Leatherman et al Sublingual Immunotherapy: Past, present, paradigm for the future?... S9 Table 2 Summary of randomized placebo-controlled SLIT studies Author and year Study design Age (y) Study size* Allergen treated Time Disease treated outcome measures (P value) Efficacy Evidence grade level Tari et al, RDBPC /28 Mites 18 mo R SS (P 0.001) A SS (P 0.001) Nasal provocation (P 0.01) Nelson et al, RDBPC /21 Cat 3. 5mo R SS (P NS), nasal blockage index (P 0.001) Casanovas et RDBPC /6 O. al, europaea 2mo R SS (P 0.05), MS (P NS) A SS (P 0.05), MS (P NS) Sabbah et al, RDBPC /29 Grasses 17 wk R SS (P 0.01, weeks 6, 7), MS (P 0.01) C(eye redness) SS (P 0.01, week 17), C (tears) SS (P 0.03, week 15) C MS (P NS) Feliziani et al, RDBPC /16 Grasses 3. 5mo R C SS (P 0.010), MS (P 0.002) A SS (P 0.026), MS (P 0.049) R C A SS (P 0.008), MS (P 0.002) Troise et al, RDBPC /16 Parietaria 10 mo R SS (P NS), MS (P 0.05) Hirsch et al, RDBPC /15 Mites 1y R SS (NS), MS (NS) A SS (P 0.05), MS (NS) Passalacqua et RDBPC /9 Mites 2y R A C SS (P al, ), MS not given Vourdas et al, RDBPC /31 Olive 2y R SS (P NS), MS (P NS) A SS (P 0.03), MS (P NS) C SS (P 0.05), MS (P NS) Clavel et al, RDBPC /28 Grasses 6mo R SS (P NS) C SS (P NS) A A(P 0.01), MS (P 0.01) Horak et al, RDBPC /16 Birch 4mo R challenge chamber (P 0.028) C conjunctival provocation (P 0.008) Hordijk et al, RDBPC /27 Grasses 6 mo R/C SS (P 0.03), MS (NS) No R No R Yes C C A C for MS No A C No R Yes C Yes C /C (Continued on S10) A, asthma; C, conjunctivitis; D, double-blind; MS, medication score; NS, not significant; OC, open-controlled; P, placebocontrolled; PR, prospective; R, randomized or rhinitis (under column, Disease treated... ); SS, symptom score. *Number of participants. SLIT/placebo. Treatment duration.