Total protein content and in vitro protein penetration rates of sublingual immunotherapy preparations

Sublingual immunotherapy (SLIT) is a safe and efficacious treatment used to desensitize patients to their food allergies, with recent additional evidence of a durable response persisting several months after discontinuation of therapy.1Schworer SA Kim EH. Sublingual immunotherapy for food allergy and its future directions.Immunotherapy. 2020; 12: 921-931Crossref PubMed Scopus (13) Google Scholar,2Kim EH Keet CA Virkud YV Chin S Ye P Penumarti A et al.Open-label study of the efficacy, safety, and durability of peanut sublingual immunotherapy in peanut-allergic children.J Allergy Clin Immunol. 2023; 151: 1558-1565Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar Studies demonstrating the efficacy of food SLIT have relied almost exclusively on the use of glycerinated food extracts used in skin prick testing. Skin test extracts, however, are costly (eg, currently approximately 100 Canadian dollars/5 mL of a single allergen), can be difficult to access, and are not licensed for SLIT by regulatory agencies such as the US Food and Drug Administration or Health Canada. These factors make food SLIT inaccessible to many families. In contrast, food powders are relatively inexpensive and commercially available. We evaluated the in vitro sublingual protein penetration rates of commercially available food powders in comparison to glycerinated extracts (current standard in clinical SLIT research) for 5 common food allergens: peanut, cashew, egg, cow's milk, and sesame. Glycerinated extracts and powdered forms of the food allergens were purchased for testing. Protein content was quantified using the Bradford protein assay (ThermoFisher Scientific, Rockford, USA; No. A55866). Powders were first dissolved in double distilled water at a concentration of 1 mg/mL. Powder solutions and liquid extracts were diluted as necessary to fit the standard curve. Results were corrected for dilution factors. The contents of the proteins were calculated on the basis of the absorbance at 595 nm (Tecan infinite M200 Pro; Tecan, Männedorf, Switzerland). TR146 cells (MilliporeSigma, Oakville, Canada), a human carcinoma cell line, were used as an in vitro model of sublingual mucosa since they can form a stratified nonkeratinized epithelium and have been used as a suitable in vitro model for human buccal drug delivery permeability studies.3Lin GC Leitgeb T Vladetic A Friedl HP Rhodes N Rossi A et al.Optimization of an oral mucosa in vitro model based on cell line TR146.Tissue Barriers. 2020; 81748459Crossref Scopus (25) Google Scholar,4Sander C Nielsen HM Jacobsen J. Buccal delivery of metformin: TR146 cell culture model evaluating the use of bioadhesive chitosan discs for drug permeability enhancement.Int J Pharm. 2013; 458: 254-261Crossref Scopus (46) Google Scholar Approval for the use of human cell lines was obtained from the University of British Columbia (B17-0247-A003). TR146 cells were cultivated in a cell culture dish (100 mm diameter) using Nutrient Mixture F-12 Ham medium containing 10% fetal calf serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin. The culture was kept at an environment of 37°C, 95% relative humidity with 5% carbon dioxide. The cell culture medium was changed every 2 days. The cells were subcultured every 7 days using 0.05% trypsin solution. TR146 cells were seeded in a 24-well Millicell Hanging Cell Culture Insert (Sigma, Darmstadt, Germany) at a density of 5 × 104 cells/cm2 and placed in the 24-well plates. The medium was changed every 2 days for 30 days before the test. The formation of the monolayers was confirmed before and after the experiment by transepithelial electric resistance using the Millicell-ERS-2 system (Millipore Corporation, Bedford, USA). The experiment was conducted from the apical to the basolateral chamber and Hanks' Balanced Salt Solution with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HBSS-HEPES) was used as media. All solid extracts and liquid extracts were diluted by the media with the same protein content and added to the apical chamber. Samples (50 μL) from the basolateral chamber were obtained at 0.5, 1, 2, and 4 hours with 37°C incubation. The permeated protein from the samples was analyzed by Bradford protein assay. The protein content was calculated as the measured protein content minus any protein detected in the blank control samples, which contained no protein from food extracts or powder solutions. All samples were tested in triplicate and averages (±SD) are presented. Two-way repeated analysis of variance tests was applied to determine differences in penetration rates between food powders and glycerinated extracts at 0.5, 1, 2, and 4 hours after inoculation. If statistically significant differences were detected among means, Tukey tests were applied. A 2-sided significance level of 0.05 was applied. Data were analyzed with Stata BE/18.0 (Stata Corp, College Station, Texas). In comparisons of the measured total protein content with the reported total protein content, more variability was observed for glycerinated extracts (between 64% and 236% of reported total protein content) than in the food powders (between 83% and 114% of the reported total protein content) (Table 12Kim EH Keet CA Virkud YV Chin S Ye P Penumarti A et al.Open-label study of the efficacy, safety, and durability of peanut sublingual immunotherapy in peanut-allergic children.J Allergy Clin Immunol. 2023; 151: 1558-1565Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,5Kim EH Yang L Ye P Guo R Li Q Kulis MD et al.Long-term sublingual immunotherapy for peanut allergy in children: clinical and immunologic evidence of desensitization.J Allergy Clin Immunol. 2019; 144 (1320-1326.e1)Abstract Full Text Full Text PDF Scopus (89) Google Scholar,6Gendo K Orden T Tevrizian A Jacobs J Mozelsio N Gilbert K et al.Food sublingual immunotherapy using consistent, cheaper, and customizable oral immunotherapy solutions.J Asthma Allergy. 2021; 14: 467-470Crossref PubMed Scopus (0) Google Scholar). There was also wide variability in the calculated volume of glycerinated extract required to provide a goal SLIT maintenance dose of 2- to 4-mg dose of protein among the different food extracts (Table 12Kim EH Keet CA Virkud YV Chin S Ye P Penumarti A et al.Open-label study of the efficacy, safety, and durability of peanut sublingual immunotherapy in peanut-allergic children.J Allergy Clin Immunol. 2023; 151: 1558-1565Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,5Kim EH Yang L Ye P Guo R Li Q Kulis MD et al.Long-term sublingual immunotherapy for peanut allergy in children: clinical and immunologic evidence of desensitization.J Allergy Clin Immunol. 2019; 144 (1320-1326.e1)Abstract Full Text Full Text PDF Scopus (89) Google Scholar,6Gendo K Orden T Tevrizian A Jacobs J Mozelsio N Gilbert K et al.Food sublingual immunotherapy using consistent, cheaper, and customizable oral immunotherapy solutions.J Asthma Allergy. 2021; 14: 467-470Crossref PubMed Scopus (0) Google Scholar).Table 1Total Protein and In Vitro Protein Penetration Rates in Food Powders and Glycerinated Extract for 5 AllergensMeasured and reported total protein contentPreparationMeasured total protein content (mean ± SD)Reported total protein contentaReported total protein content was obtained from food manufacturers nutrition facts food labels, published data, and/or by means of communications from the manufacturer, when available. Reported total protein was not available for cashew and sesame extracts.Percent of reported total protein contentCalculated volume of extract needed to deliver 2 mg proteinbValues calculated as goal sublingual immunotherapy dose of 2 mg protein or 4 mg protein (as per previously published protocols by Kim et al2,5) divided by the mean measured total protein content (mg/mL), as determined by Bradford assay.Calculated volume of extract needed to deliver 4 mg proteinbValues calculated as goal sublingual immunotherapy dose of 2 mg protein or 4 mg protein (as per previously published protocols by Kim et al2,5) divided by the mean measured total protein content (mg/mL), as determined by Bradford assay.ExtractscGlycerinated extracts (1:20 w/v fully dissolved in 0.2% phenol and 50% glycerinated saline) of peanut (Arachis hypogaea), cashew (Anacardium occidentale), egg (Gallus domesticus), cow's milk (Bos domesticus), and sesame (Sesamum indicum) were obtained from Stallergenes Greer Canada (London, Canada). Peanut7.75 ± 0.001 mg/mLapprox 4-8 mg/mLdReported total protein content obtained from Gendo et al.697-1940.26 mL0.52 mL Cashew10.42 ± 0.002 mg/mLN/AN/A0.19 mL0.38 mL Egg4.71 ± 0.03 mg/mL2-4 mg/mLdReported total protein content obtained from Gendo et al.6118-2360.42 mL0.85 mL Cow's milk2.56 ± 0.01 mg/mL2-4 mg/mLdReported total protein content obtained from Gendo et al.664-1280.78 mL1.56 mL Sesame0.88 ± 0.002 mg/mLN/AN/A2.27 mL4.55 mLPowderseThe following brands of powders were purchased commercially: Sahah Naturals PB&Me Original Powdered Peanut Butter (Montreal, Canada), Nuts.com Cashew Flour (Cranford, USA), Hoosier Hill Farm Whole Egg Granules (Fort Wayne, USA), Carnation Instant Skim Milk Powder (Markham, Canada), and Kevala Organic Sesame Flour (Dallas, USA). Peanut0.33 ± 0.0004 mg/mg0.40 mg/mg83 Cashew0.20 ± 0.002 mg/mg0.18 mg/mg111 Egg0.57 ± 0.004 mg/mg0.50 mg/mg114 Cow's milk0.36 ± 0.003 mg/mg0.36 mg/mg100 Sesame0.44 ± 0.2 mg/mg0.40 mg/mg110Protein penetration rate, mean (SD)Time point, hPeanutCow's MilkCashewEggSesameExtractcGlycerinated extracts (1:20 w/v fully dissolved in 0.2% phenol and 50% glycerinated saline) of peanut (Arachis hypogaea), cashew (Anacardium occidentale), egg (Gallus domesticus), cow's milk (Bos domesticus), and sesame (Sesamum indicum) were obtained from Stallergenes Greer Canada (London, Canada).PowdereThe following brands of powders were purchased commercially: Sahah Naturals PB&Me Original Powdered Peanut Butter (Montreal, Canada), Nuts.com Cashew Flour (Cranford, USA), Hoosier Hill Farm Whole Egg Granules (Fort Wayne, USA), Carnation Instant Skim Milk Powder (Markham, Canada), and Kevala Organic Sesame Flour (Dallas, USA).ExtractcGlycerinated extracts (1:20 w/v fully dissolved in 0.2% phenol and 50% glycerinated saline) of peanut (Arachis hypogaea), cashew (Anacardium occidentale), egg (Gallus domesticus), cow's milk (Bos domesticus), and sesame (Sesamum indicum) were obtained from Stallergenes Greer Canada (London, Canada).PowdereThe following brands of powders were purchased commercially: Sahah Naturals PB&Me Original Powdered Peanut Butter (Montreal, Canada), Nuts.com Cashew Flour (Cranford, USA), Hoosier Hill Farm Whole Egg Granules (Fort Wayne, USA), Carnation Instant Skim Milk Powder (Markham, Canada), and Kevala Organic Sesame Flour (Dallas, USA).ExtractcGlycerinated extracts (1:20 w/v fully dissolved in 0.2% phenol and 50% glycerinated saline) of peanut (Arachis hypogaea), cashew (Anacardium occidentale), egg (Gallus domesticus), cow's milk (Bos domesticus), and sesame (Sesamum indicum) were obtained from Stallergenes Greer Canada (London, Canada).PowdereThe following brands of powders were purchased commercially: Sahah Naturals PB&Me Original Powdered Peanut Butter (Montreal, Canada), Nuts.com Cashew Flour (Cranford, USA), Hoosier Hill Farm Whole Egg Granules (Fort Wayne, USA), Carnation Instant Skim Milk Powder (Markham, Canada), and Kevala Organic Sesame Flour (Dallas, USA).ExtractcGlycerinated extracts (1:20 w/v fully dissolved in 0.2% phenol and 50% glycerinated saline) of peanut (Arachis hypogaea), cashew (Anacardium occidentale), egg (Gallus domesticus), cow's milk (Bos domesticus), and sesame (Sesamum indicum) were obtained from Stallergenes Greer Canada (London, Canada).PowdereThe following brands of powders were purchased commercially: Sahah Naturals PB&Me Original Powdered Peanut Butter (Montreal, Canada), Nuts.com Cashew Flour (Cranford, USA), Hoosier Hill Farm Whole Egg Granules (Fort Wayne, USA), Carnation Instant Skim Milk Powder (Markham, Canada), and Kevala Organic Sesame Flour (Dallas, USA).ExtractcGlycerinated extracts (1:20 w/v fully dissolved in 0.2% phenol and 50% glycerinated saline) of peanut (Arachis hypogaea), cashew (Anacardium occidentale), egg (Gallus domesticus), cow's milk (Bos domesticus), and sesame (Sesamum indicum) were obtained from Stallergenes Greer Canada (London, Canada).PowdereThe following brands of powders were purchased commercially: Sahah Naturals PB&Me Original Powdered Peanut Butter (Montreal, Canada), Nuts.com Cashew Flour (Cranford, USA), Hoosier Hill Farm Whole Egg Granules (Fort Wayne, USA), Carnation Instant Skim Milk Powder (Markham, Canada), and Kevala Organic Sesame Flour (Dallas, USA).0.536.7 ± 1.2%30.3 ± 1.5%42.1 ± 0.5%40.7 ± 0.9%28.0 ± 0.7%32.2 ± 0.6%40.4 ± 1.0%40.7 ± 0.9%38.5 ± 2.9%24.2 ± 1.1%182.2 ± 2.0%72.1 ± 1.5%89.5 ± 1.9%84.3 ± 1.5%74.0 ± 2.3%76.3 ± 3.2%86.0 ± 1.0%fValues in each column are statistically different (P < .05).84.3 ± 1.5%fValues in each column are statistically different (P < .05).82.4 ± 1.5%71.4 ± 1.8%289.8 ± 1.5%80.0 ± 2.0%92.8 ± 4.9%92.6 ± 2.5%91.0 ± 1.4%81.2 ± 1.5%92.6 ± 2.5%fValues in each column are statistically different (P < .05).91.7 ± 1.4%fValues in each column are statistically different (P < .05).85.5 ± 1.4%78.3 ± 2.1%495.5 ± 1.5%96.1 ± 1.5%94.9 ± 3.2%93.8 ± 0.7%94.9 ± 1.0%91.1 ± 1.5%96.5 ± 1.7%fValues in each column are statistically different (P < .05).93.8 ± 0.7%fValues in each column are statistically different (P < .05).92.1 ± 1.1%93.8 ± 1.1%Abbreviations: approx, approximately; N/A, not applicable; w/v, weight by volume.a Reported total protein content was obtained from food manufacturers nutrition facts food labels, published data, and/or by means of communications from the manufacturer, when available. Reported total protein was not available for cashew and sesame extracts.b Values calculated as goal sublingual immunotherapy dose of 2 mg protein or 4 mg protein (as per previously published protocols by Kim et al2Kim EH Keet CA Virkud YV Chin S Ye P Penumarti A et al.Open-label study of the efficacy, safety, and durability of peanut sublingual immunotherapy in peanut-allergic children.J Allergy Clin Immunol. 2023; 151: 1558-1565Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,5Kim EH Yang L Ye P Guo R Li Q Kulis MD et al.Long-term sublingual immunotherapy for peanut allergy in children: clinical and immunologic evidence of desensitization.J Allergy Clin Immunol. 2019; 144 (1320-1326.e1)Abstract Full Text Full Text PDF Scopus (89) Google Scholar) divided by the mean measured total protein content (mg/mL), as determined by Bradford assay.c Glycerinated extracts (1:20 w/v fully dissolved in 0.2% phenol and 50% glycerinated saline) of peanut (Arachis hypogaea), cashew (Anacardium occidentale), egg (Gallus domesticus), cow's milk (Bos domesticus), and sesame (Sesamum indicum) were obtained from Stallergenes Greer Canada (London, Canada).d Reported total protein content obtained from Gendo et al.6Gendo K Orden T Tevrizian A Jacobs J Mozelsio N Gilbert K et al.Food sublingual immunotherapy using consistent, cheaper, and customizable oral immunotherapy solutions.J Asthma Allergy. 2021; 14: 467-470Crossref PubMed Scopus (0) Google Scholare The following brands of powders were purchased commercially: Sahah Naturals PB&Me Original Powdered Peanut Butter (Montreal, Canada), Nuts.com Cashew Flour (Cranford, USA), Hoosier Hill Farm Whole Egg Granules (Fort Wayne, USA), Carnation Instant Skim Milk Powder (Markham, Canada), and Kevala Organic Sesame Flour (Dallas, USA).f Values in each column are statistically different (P < .05). Open table in a new tab Abbreviations: approx, approximately; N/A, not applicable; w/v, weight by volume. Sublingual protein penetration rates were significantly different by time point (0.5, 1, 2, and 4 hours) for all foods tested (P < .05) (Table 12Kim EH Keet CA Virkud YV Chin S Ye P Penumarti A et al.Open-label study of the efficacy, safety, and durability of peanut sublingual immunotherapy in peanut-allergic children.J Allergy Clin Immunol. 2023; 151: 1558-1565Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,5Kim EH Yang L Ye P Guo R Li Q Kulis MD et al.Long-term sublingual immunotherapy for peanut allergy in children: clinical and immunologic evidence of desensitization.J Allergy Clin Immunol. 2019; 144 (1320-1326.e1)Abstract Full Text Full Text PDF Scopus (89) Google Scholar,6Gendo K Orden T Tevrizian A Jacobs J Mozelsio N Gilbert K et al.Food sublingual immunotherapy using consistent, cheaper, and customizable oral immunotherapy solutions.J Asthma Allergy. 2021; 14: 467-470Crossref PubMed Scopus (0) Google Scholar). No significant differences were observed in penetration rates by food form (extract vs powder) for peanut, cow's milk, cashew, or sesame (P > .05) (Table 12Kim EH Keet CA Virkud YV Chin S Ye P Penumarti A et al.Open-label study of the efficacy, safety, and durability of peanut sublingual immunotherapy in peanut-allergic children.J Allergy Clin Immunol. 2023; 151: 1558-1565Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,5Kim EH Yang L Ye P Guo R Li Q Kulis MD et al.Long-term sublingual immunotherapy for peanut allergy in children: clinical and immunologic evidence of desensitization.J Allergy Clin Immunol. 2019; 144 (1320-1326.e1)Abstract Full Text Full Text PDF Scopus (89) Google Scholar,6Gendo K Orden T Tevrizian A Jacobs J Mozelsio N Gilbert K et al.Food sublingual immunotherapy using consistent, cheaper, and customizable oral immunotherapy solutions.J Asthma Allergy. 2021; 14: 467-470Crossref PubMed Scopus (0) Google Scholar). For egg, there was a significant difference in penetration rates by food form (P = .0168), with penetration rates of the glycerinated egg extract significantly higher than the powder at 1 hour (86.0 ± 1.0% vs 84.3 ± 1.5%), 2 hours (92.6 ± 2.5% vs 91.7 ± 1.4%), and 4 hours of incubation (96.5 ± 1.7% vs 93.8 ± 0.7%) (P < .05), but not at 0.5 hours (40.4 ± 1.0% vs 40.7 ± 0.9%) (P > .05) (Table 12Kim EH Keet CA Virkud YV Chin S Ye P Penumarti A et al.Open-label study of the efficacy, safety, and durability of peanut sublingual immunotherapy in peanut-allergic children.J Allergy Clin Immunol. 2023; 151: 1558-1565Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,5Kim EH Yang L Ye P Guo R Li Q Kulis MD et al.Long-term sublingual immunotherapy for peanut allergy in children: clinical and immunologic evidence of desensitization.J Allergy Clin Immunol. 2019; 144 (1320-1326.e1)Abstract Full Text Full Text PDF Scopus (89) Google Scholar,6Gendo K Orden T Tevrizian A Jacobs J Mozelsio N Gilbert K et al.Food sublingual immunotherapy using consistent, cheaper, and customizable oral immunotherapy solutions.J Asthma Allergy. 2021; 14: 467-470Crossref PubMed Scopus (0) Google Scholar). In this study, we observed less variability in the measured protein content of whole food powders than in glycerinated extracts, and statistically significant differences in penetration rates were only observed for egg, with higher rates for glycerinated extract at 1, 2, and 4 hours. However, the percent differences in penetration rates for egg were minimal and would likely have limited practical significance. Since SLIT is believed to promote a tolerogenic environment in the oral cavity through the uptake of allergens by myeloid dendritic cells, several factors, including the influence of pH, saliva, tongue movement, protein weight, allergen solubility, bioavailability, and absorption in the oral cavity1Schworer SA Kim EH. Sublingual immunotherapy for food allergy and its future directions.Immunotherapy. 2020; 12: 921-931Crossref PubMed Scopus (13) Google Scholar,7Guo Y Pratap Singh A Emerging strategies for enhancing buccal and sublingual administration of nutraceuticals and pharamaceuticals.J Drug Deliv Sci Technol. 2019; 52: 440-451Crossref Scopus (38) Google Scholar, 8Yu J Ahmedna M Goktepe I. Peanut protein concentrate: production and functional properties as affected by processing.Food Chem. 2007; 103: 121-129Crossref Scopus (309) Google Scholar, 9Koppelman SJ Smits M Tomassen M De Jong GAH Baumert J Taylor SL et al.Release of major peanut allergens from their matrix under various pH and simulated saliva conditions—ara h2 and ara h6 are readily bio-accessible.Nutrients. 2018; 10: 1281Crossref Scopus (13) Google Scholar, 10Kopper RA Odum NJ Sen M Helm RM Stanley JS Burks AW. Peanut protein allergens: the effect of roasting on solubility and allergenicity.Int Arch Allergy Immunol. 2005; 136: 16-22Crossref PubMed Scopus (0) Google Scholar should be considered when attempting to translate these in vitro findings to in vivo absorption. Furthermore, TR146 cells use endocytosis and passive and active transport mechanisms, similar to in vivo sublingual mucosa, although to varying degrees because of differences in their biologic environments.3Lin GC Leitgeb T Vladetic A Friedl HP Rhodes N Rossi A et al.Optimization of an oral mucosa in vitro model based on cell line TR146.Tissue Barriers. 2020; 81748459Crossref Scopus (25) Google Scholar,4Sander C Nielsen HM Jacobsen J. Buccal delivery of metformin: TR146 cell culture model evaluating the use of bioadhesive chitosan discs for drug permeability enhancement.Int J Pharm. 2013; 458: 254-261Crossref Scopus (46) Google Scholar,7Guo Y Pratap Singh A Emerging strategies for enhancing buccal and sublingual administration of nutraceuticals and pharamaceuticals.J Drug Deliv Sci Technol. 2019; 52: 440-451Crossref Scopus (38) Google Scholar In this study, samples were collected for up to 4 hours, since in vitro absorption by means of TR146 cells is slower than in vivo sublingual absorption (which can occur within minutes because of a high degree of vascularization and a different physiological environment). Further research is needed to understand how the penetration rate in vitro translates to the in vivo sublingual environment since SLIT doses are held under the tongue for only a matter of minutes. Detailed in vitro protein studies for more food allergens, and powder and extract products from a variety of manufacturers are also warranted. Overall, the comparable penetration rates of powders and extracts in vitro support that food SLIT using powders could have clinical usefulness, although further research is needed. Dr Chan has received research support from DBV Technologies; has been a member of advisory boards for Pfizer, Miravo, Medexus, Leo Pharma, Kaleo, DBV, AllerGenis, Sanofi Genzyme, Bausch Health, Avir Pharma, AstraZeneca, and ALK; and was co-lead of the Canadian Society of Allergy and Clinical Immunology oral immunotherapy guidelines. Dr Pratap-Singh holds the BC Ministry of Agriculture Endowed Professorship in Food and Beverage Innovation. Drs Williams, Guo and Soller have no conflicts to disclosure. The authors thank Ally Baaske and Bryant Hartono from the University of British Columbia for their assistance with statistical and laboratory analysis, respectively. This research was funded by the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition Foundation Council for Pediatric Nutrition Professionals Research Grant; Natural Science and Engineering Research Council of Canada's Discovery Research Grant (No. RGPIN-2018-04735); and Canada Foundation for Innovation John Evans Leaders Fund Award (No. 37498).