Hypoallergenicity assessment of an extensively hydrolyzed whey‐protein formula in cow’s milk allergic infants

Abstract Background Extensively hydrolyzed formulas are recommended for the dietary management of infants with cow's milk allergy (CMA). Objectives Hypoallergenicity, growth, and gastrointestinal (GI) tolerability of a new extensively hydrolyzed whey‐protein formula (eHWF) in CMA children were assessed. Methods In this prospective, randomized, international, multi‐center study (Trial NL3889), 34 children with confirmed CMA (74% IgE‐mediated) underwent a double‐blind, placebo‐controlled food challenge (DBPCFC) with an eHWF developed with non‐porcine enzymes, supplemented with prebiotic short‐chain galacto‐ and long‐chain fructo‐oligosaccharides (0.8 g/L, ratio 9:1), arachidonic acid (0.35/100 g), and docosahexaenoic acid (0.35/100 g). If tolerant to the eHWF, children participated in a 7‐day open food challenge with this eHWF. Anthropometrics and GI tolerability were assessed in an optional 16‐weeks follow‐up. Results Of the 34 children who started the DBPCFC with the eHWF, 25 subjects (19 boys, mean age: 61 weeks, 18 with IgE‐mediated CMA) completed the DBPCFC and 7‐day open challenge without major protocol deviations and tested negative at both challenges. One child experienced a late moderate eczematous allergic reaction in the optional follow‐up period, indicating the need for close monitoring of subjects starting new formula. Weight and length gain followed the World Health Organization growth curves. Changes in frequency and consistency of stools upon test formula intake were transient. Conclusions The newly developed eHWF is a suitable option in CMA treatment as all subjects tolerated the product. This result is in line with the international criteria for hypoallergenicity (American Academy of Pediatrics) that state that more than 90% of CMA children must tolerate the formula. Use of the formula is also associated with normal growth curves and GI tolerability. Trial registration Trial NL3889, https://www.trialregister.nl/trial/3889.


| INTRODUC TI ON
Over the last decades, food allergies have become an emerging healthcare issue not only in the western world but also in developing countries. 1 It is estimated that 6-8% of infants under 3 years of age have a food allergy, among which cow's milk allergy (CMA) is the most common. [2][3][4] Clinical symptoms of CMA may start during the first weeks of life, shortly after the introduction of cow's milk proteins (CMP), characterized by cutaneous, gastrointestinal (GI), and/or respiratory symptoms and pain behavior. 2 CMA can be classified as immunoglobulin E (IgE)-mediated, usually characterized by the occurrence of symptoms immediately after antigen exposure, or non-IgE mediated, characterized by delayed allergic symptoms, or a combination of both. Its management in infants and young children requires total avoidance of CMP and, when breastfeeding is not possible, the use of "hypoallergenic" formulas. Hypoallergenic formulas based on extensively hydrolyzed proteins or amino acid mixtures are the only safe option. 2 These infant formula substitutes must be proven to be hypoallergenic and have good tolerability, safety, and nutritional adequacy. The nutritional quality is crucial since studies have shown that the growth of children with CMA may be compromised, 5,6 potentially because the dietary intake of macro-and micronutrients is below the recommendations compared to healthy children. 7 The aim of this study was to investigate the hypoallergenicity and safety (growth and GI tolerability) of an extensively hydrolyzed whey-protein formula (eHWF), according to international guidelines (American Academy of Pediatrics [AAP], European Society for Paediatric Allergology and Clinical Immunology [ESPACI], and the European Society for Paediatric Gastroenterology, Hepatology and Nutrition [ESPGHAN]). [8][9][10] 2 | ME THODS This prospective, randomized, controlled, multi-center study was conducted in the Netherlands, Italy, and Poland. Our primary objective was to assess the hypoallergenicity of a newly developed extensively hydrolyzed whey-protein infant formula in children with CMA according to international guidelines. [8][9][10] We also aimed to assess the effects on growth and tolerability of the formula in a 16-week follow-up. The study was registered in the Dutch Trial Register on 1 July 2013 (registration number NTR4051).

| Study population
We included infants and children with proven CMA from January out under the supervision of a pediatrician. We excluded infants and children with a confirmed history of an anaphylactic reaction to cow's milk, those with intolerance to lactose or any other component of the study formula, those with a previous allergic reaction to any existing eHWF, and those fed any amino acid-based formula (for more than 6 weeks preceding the first part of the DBPCFC) due to potentially severe CMA. Children with major congenital malformations and/or existing illnesses that could interfere with formula acceptance or identification of allergic reaction, children breastfed more than twice daily, participating in another clinical study, or whose parents were judged unable to comply with the protocol requirements were also excluded. The minimum expected consumption of the study formula during the open challenge phase had to be 250 ml per day.

| Study design
The AAP guidelines for clinical testing of hypoallergenic formulas, which have been supported by the ESPACI and ESPGHAN, suggest that the number of subjects must be sufficient to demonstrate with 95% confidence that 90% of the subjects will not react to the formula. [8][9][10] The number of subjects needed to project with 95% confidence (one-sided interval) that less than 10% of subjects will react to the product is 25 subjects if zero clinical reactions are observed, and 43 subjects if one clinical reaction is observed. These sample size estimates were derived based on binomial distribution techniques using Wilson's method for deriving confidence intervals for single proportions.
Prior to the DBPCFC, a blood sample was drawn from all participating children to determine total IgE and allergen-specific IgE levels. In addition, data on anthropometrics and recent GI symptoms were collected.

Key Message
This study describes the safety and hypoallergenicity of a new extensively hydrolyzed whey-protein formula, derived by non-porcine enzyme hydrolysis, for infants with diagnosed cow's milk allergy (CMA). The use of this formula does not impact growth and is a suitable nutritional option for the dietary management of CMA in infants.
In the challenge, the new formula (manufactured by Nutricia Research, Cuijk, The Netherlands) was tested by a DBPCFC followed by an open food challenge for 1 week in case of a negative DBPCFC.
The DBPCFC consisted of two separate hospital visits (visit 1 and visit 2) within a 1-week time frame; children were randomized to receive either: (1) the test formula on the first test day and the reference formula (amino acid formula) on the second test day or (2) the reference (amino acid formula) on the first test day and the test formula on the second test day. Randomization was computergenerated (using SAS Proc Plan) and stratified by the study site.
Participating clinicians received the treatment order within sealed opaque envelopes.
The reference formula and the eHWF were reconstituted according to the manufacturer's instructions; for blinding, the latter was mixed with the placebo formula at a 1:1 ratio. The prescribed schedule was 1, 5, 10, 25, 50, 80 and 100 ml. The administered formulas were prepared by a staff member who was not involved in the patient's care. The investigator, the nursing staff and the family were therefore not informed of what formula the child was being fed.

| Formulae
The test formula was a nutritionally complete, powdered, newly designed; non-porcine enzymes derived, extensively hydrolyzed wheybased infant formula for infants/children with CMA (see Table 1).
The formula was enriched with long-chain polyunsaturated fatty acids (arachidonic acid and docosahexaenoic acid) and a mixture of prebiotic short-chain galacto-oligosaccharides, and long-chain fructo-oligosaccharides (further characterization of the new extensively hydrolyzed protein will be described in Knipping et al. manuscript in preparation). The reference formula used in this study was a commercially available, amino acid-based, nutritionally complete, powdered, hypoallergenic infant formula for the management of (severe) CMA (Neocate, Nutricia Advanced Medical Nutrition).

| Ethics
The approval of the relevant ethics committees in the participating countries was obtained before the start of the study. The study was conducted according to ICH-GCP principles and in compliance with the principles of the "Declaration of Helsinki" (59th WMA General Assembly, Seoul, October 2008) and with the local laws and regulations of the country where the study was performed. Written informed consent was obtained from all parent(s)/guardian(s) before enrolment in the study.

| Statistical analysis
A challenge was considered positive when objective symptoms appeared and when there were severe, persisting (more than 40 min) subjective symptoms. 11 Based on the results of the DPBCFC and subsequent open challenge, the proportion of children tolerating the test formula was calculated with a one-sided 95% Wilson's score confidence interval (for a single proportion).
If the lower confidence limit of the one-sided 95% confidence interval for the population fraction of children with tolerance to the test formula is larger than or equal to 90%, we can claim with 95% confidence that 90% or more of the subjects will tolerate the formula. For anthropometric data, length-for-age and weight-for-age zscores were calculated according to the World Health Organization (WHO) reference values. 12 Other data were described and/or summarized either by means and standard deviations or medians and interquartile ranges as appropriate for continuous data, or by number and percentages for categorical data.

| Demographics and baseline characteristics
The children in the PP population were recruited in Italy (72%) and the Netherlands (28%). A 76% of the recruited children were males and their age at baseline ranged from 15.1 to 142.7 weeks of age.
Their gestational age, weight for age z-score, and length for age zscore at screening were all within the normal range.
In the PP population, 80% of the children reported skin symptoms and 68% GI symptoms when consuming CMP. At study entry, the majority of the children were fed an eHWF (n = 14), others reported consumption of rice hydrolysate (n = 3), soy (n = 6), and amino acid-based formulae consumption outside exclusion criterium of severe allergy (n = 2; Table 2). Ten children were mixed-fed with a combination of formula feeding and breastfeeding at the time of enrolment in the study. Data are depicted in Table 2.
The demographics and baseline characteristics for AST can be found in Table S1.

| Allergic reactions
None of the 25 children in the PP population showed allergic reactions to the test formula during the DBPCFC, nor in the open challenge (see Table 3). No reaction was reported among the 9 children that were excluded from the PP analysis. In accordance with the international guidelines, [8][9][10]

| Anthropometrics
Because of the wide variation in baseline age of the children individual length-for-age and weight-for-age growth data per gender were evaluated (Table S2). In the follow-up phase, weight and length gain followed the normal WHO growth curves, within the WHO ranges (The WHO Multicentre Growth Reference Study (MGRS)).  c One subject consumed 2 types of formula.

TA B L E 2 Demographic data and subject characteristics at baseline for PP population
d The extensively hydrolyzed formula could be either whey or casein-based. Thirteen severe AEs were reported in 4 subjects. These were GI disorders, infections, investigations, nervous system disorders, and skin and subcutaneous tissue disorders. None of these events were indicated by the investigator to be related to the test formula intake.

| Safety outcomes
Most of them had a short duration and did not require medication.
No serious adverse events were reported in this study.

| GI tolerability
By collecting parent-reported, non-allergy-related GI symptoms from the first day of the DBPCFC (hospital visit 2) up to and including the 16-week follow-up ( Figure S1), the GI tolerability of the test formula was assessed. At baseline, the percentage of children with GI symptoms was low (<20%). At baseline, the majority of the children had stool consistency reported as "soft" or "formed." The use of an infant formula or changing to another infant formula can cause transient changes in stool patterns as also seen in other studies, 14,15 reflected in Figure 1 showing that the first couple of weeks of 16- week follow-up (weeks 3-8) the consistency slightly differed compared to baseline (predominantly softened). This observation seems to be transient as the consistency was back to baseline level by the end of the 16-week follow-up. Other GI symptoms were hardly reported during the first weeks of the follow-up period. Overall, almost all parent-reported symptoms were mild. Stool frequency was very consistent throughout the study (Table S4).  (Table S5).

| DISCUSS ION
This study was designed according to international guidelines (AAP,  among infants with more complex forms of the disease. 18,19 Infants, who react to eHWFs, develop delayed non-IgE-mediated GI reactions to cow's milk and severe eczema more frequently than those who do not react to eHWFs. 20 The one late allergic reaction in the follow-up phase is in line with this information from the literature. The IgE sensitization patterns of the subjects as determined by the ISAC method (see Table S5) could roughly be divided into It has long been known that there is a broad range of attributes among hydrolyzed formulas with significant differences, especially in their taste. 24 In this study only one parent reported aversion to the taste of the test product. This is in line with the observation of Maslin et al., who described that whey-based lactose-containing eHWFs are generally considered to be more palatable and accepted in comparison to other hydrolysates (non-lactose, casein-based). 25,26 The main strength of the present study was that it has been designed according to the AAP guidelines for testing hypoallergenicity of a hydrolyzed formula, 8 which have also been adopted by the A limitation of the study is the exclusion of infants that were on the amino acid formula for scientific and safety reasons. So, this study does not completely represent the total, global population of cow's milk allergic infants and children.
In conclusion, the study formula was tolerated by more than 90% of infants with a diagnosed CMA, with a 95% confidence interval, and is therefore in line with the criteria of hypoallergenicity and safety of the international guidelines (i.e., AAP) for eHWF. The formula also supported proper growth and GI tolerability in those infants. The palatability of the formula tested makes it a suitable option in the treatment of CMA in terms of efficacy, nutritional adequacy, acceptance, and tolerance.

CO N FLI C T O F I NTE R E S T
Johan Garssen is parttime employee of Danone Nutricia Research as well as Utrecht University.

PEER R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1111/pai.13814.