WO2024069418A1

WO2024069418A1 - A method for treating asthma

Info

Publication number: WO2024069418A1
Application number: PCT/IB2023/059537
Authority: WO
Inventors: Iain Alexander Douglas BLAIR; John Oakley HUNTER; Rosemary Hope Waring; Fredrik Malte Tobias EDENIUS; Harry KARELIS
Original assignee: Ateria Health Australia Pty Ltd
Priority date: 2022-09-29
Filing date: 2023-09-26
Publication date: 2024-04-04
Also published as: GB202214301D0; GB2622839A

Abstract

The invention relates to a malt extract for use in preventing or treating asthma or at least one of the symptoms of asthma in a mammal or avian. In particular the malt extract comprises one or more active enzymes selected from the group consisting of amylases, maltases, cellulases, fructanases, glucanases, xylanases and deacetylases.

Description

A METHOD FOR TREATING ASTHMA

Asthma is a common chronic inflammatory disease of the lungs which has a high morbidity and mortality. It is prevalent all over the world and the affected population is projected to reach 400 million people by 2025. It is characterised by wheeze, shortness of breath, chest tightness and a cough that varies over time and in intensity. These symptoms are accompanied by variable expiratory airflow limitation and may be triggered by bacterial, viral and fungal infections. The condition is known to be multi-factorial, with input from genetics, co-existing disease, nutrition, the environment, the immune system and also, according to Baehren et al. (Cells, 11, 1287 (2022)), the gut microbiome.

According to Yagi et al. (Int. J. Mol. Sci., 22, 10872 (2021)), the microbiome in lungs differs from that in the gastrointestinal tract in that in healthy lungs there are fewer bacteria (about 10⁴ bacteria/g lung tissue as opposed to about 10¹² bacteria/g gut tissue). Although the gastrointestinal tract and lungs are both lined with mucosa and have the same embryological origin, the lung microbiome is in an aerobic environment where the airflow is bidirectional and there is a greater temperature gradient than that found in the gut. This gives a more dynamic and transient system than that of the gastrointestinal tract.

Liu et al. (Microbiology Spectrum 9 (2) (2021)) has shown that the gut microbiome can modulate inflammatory responses and function in lung tissue. Asthma has been shown by Tunney et al. (Am. J. Respir. Crit. Care Med., 187, 1118 (2013)) to be associated with reduced diversity of the gut microbiome and also with dominance of a single taxon or a small group of taxa. This link between the gut microbiome and lung function appears to be partly determined by post-natal environmental influences that affect the development of the immune system. Consequently, allergies, susceptibility to respiratory infections and autoimmune dysfunction in later life can be linked to early experiences. Correlations between gut microbiome and immune responses are, according to Haahtela et al. (Allergy, 76, 3613, (2021)), thought to be partly modulated by epigenetic factors such as histone modulation and DNA methylation, both driven by environmental input.

Similar effects are seen in adult humans. The possibility of lessening the severity of Covid-19 by modulating the gut microbiome has been reviewed by Li et al. (Viruses, 14, 1774 (2022)), Vestad et al. (J. Internal Med., 291, 801 (2022)), and Kaushal and Noor (Current Microbiol., 79, 184 (2022)) while pulmonary aspergillosis (Cai et al., Frontiers in Immunology, 13, 988708 (2022)), cystic fibrosis (Avalos-Fernandez et al., Respiratory Research, 23, 214 (2022)) and chronic obstructive pulmonary disease (COPD) (Millares and Monso, Int. J. Chron. Obstruct. Pulmon. Dis., 12, 17, 1835 (2022)) have all been suggested as potentially being improved by therapeutic modulation of the gut microbiome.

Short chain fatty acids (SCFAs) such as propionate and butyrate are produced by a wide variety of intestinal bacteria such as Roseburia and Coprococcus through fermentation of dietary fibre. Butyrate acts as an energy source for colonocytes while both compounds enter the peripheral circulation and tissues such as the lungs where they modulate the activity of T-regulatory lymphocytes and cytokines. Their increase, particularly that of butyrate, is associated with reduced allergic airway inflammation and higher levels of butyrate are protective against asthma (Depner et al., Nature Med. 26, 1766 (2020)). According to Dickson (PLoS Pathog., 11, el004923 (2015)), there may be a feed-back loop between the lung and the gut, as viral infections of the lung are known to modulate the gut microbiome. Any subsequent reduction in provision of butyrate and other antiinflammatory molecules would then make asthma attacks more likely. This potential gutlung interaction has been discussed in detail by Depner et al. (Nature Med. 26, 1766 (2020)).

Recent research has highlighted the importance of this gut-lung axis, where gut dysbiosis affects the immune system and impacts on the lung, causing inflammation and altering its microbiome. This appears to be a common pathway in humans, mammals and avians, as molecules derived from the microbiome can have systemic effects and act on distal organs where recognition by the host's immune cells can trigger a range of responses. Since the microbiomes of humans, mammals and avians are similar qualitatively if not necessarily quantitatively, effects in one species are often replicated in another since the bacterial metabolites are also similar (Goncalves et al. (Frontiers in Immunology, 13, 889945 (2022)) and Melo-Gonzalez et al. (Frontiers in Immunology, 13, 877533 (2022))).

In chickens, coli bacil losis is caused by avian pathogenic Escherichia coli and depletion of the gut microbiome by antibiotics results in an inflammatory response in the lung which is partly inhibited by acetate, a short chain fatty acid (SCFA) (Peng et al., Veterinary Microbiol., 261, 109187 (2021)). Similarly, Mycoplasma gallisepticum alters the gut microbiome in chickens and also induces inflammation in the lung which can be partly reversed by treating with baicalin to enrich the gut with Bacteroides fragilis (Wang et al., Food Func., 12, 4092 (2021)). These effects are found in other species as viruses in ducks can cause severe damage to the gut wall and mucosal barrier, allowing colonisation by opportunistic pathogens and decreasing production of SCFAs by beneficial bacteria, leading to immune dysfunction and inflammatory states (Zhu et al. (Veterinary Microbiol., 264, 109286 (2022)) and Luo et al. (Poultry Sci., 100, 101021 (2021))).

Mammals also show associations of gut dysfunction with lung damage. Equine asthma has a relatively high incidence in the horse population (> 14%) and affected animals have an altered metabolome, reflecting the gut microbiome, in bronchoalveolar lavage fluid and exhaled breath condensate (Bazzano et al., BMC Vet. Res., 16, 233 (2020)). This altered gut microbiome, unlike that of healthy horses, does not respond with increases in Fibrobacter when they are fed a hay diet (Leclere and Costa, J. Vet. Intern. Med., 34, 996 (2020)).

Studies in mice have shown that tuberculosis infection (Mycobacterium bovis) in the lung reflects dysbiosis in the gut, probably by limiting the cyclooxygenase-2/endoplasmic reticulum stress pathway (Wang et al., Emerging Microbes & Infections, 11, 1, 1806 (2022)). Infection with Pseudomonas aeruginosa caused lung inflammation in mice with a reduction in the diversity and number of gut microbiota with the lung damage being reversed by restoring the gut flora (Wen et al., Frontiers in Cellular and Infection Microbiol., 12, 856633 (2022)). In a mouse model for asthma, antibiotics were used to deplete the gastrointestinal microbiota. Challenge with an allergen showed that this modulation in the gut was associated with lung dysfunction, including increased allergic airway hyperresponsiveness and reduced lung alveolar volume (Cavalcante et al., J Immunol. Res., 1466011 (2022)). When obese golden hamsters were used as a model, infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) again led to lung inflammation and associated changes in the gut microbiome (Sencio et al., Gut Microbes, 14, 1, e2100200 (2022)).

Pigs are omnivorous and monogastric, with an immune system which has about 80 % similarities to that in humans (Pabst, Cell and Tissue Res., 380, 287 (2020)). Streptococcus suis (S. suis) is an important pathogen which causes simultaneous respiratory and digestive tract infections in pigs. Use of a mouse model showed that this infection caused both gut dysbiosis and lung inflammation via altered immune function (Yang et al., Microbiol. Res., 261, 127047 (2022)). Studies in piglets found that the early life microbiota was a potential determinant for susceptibility to Mycoplasma hyopneumoniae infection, a finding obviously relevant to asthma in children (Surendran et al., Vet. Res., 50, 86 (2019)) where the gut microbiome is clearly involved.

A reduction in microbiome diversity due to antibiotic therapy was shown to predispose children to develop allergic asthma (Van Engelen et al., Clinical Gastroenterology and Hepatology, 20, 6, 1404 (2022)). Arrieta et al. (J. Allergy Clin. Immunol., 142, 424 (2018)) found that a reduction in Lachnospira, Veillonella, Faecalibacterium and Rothia in the gut correlated with an increased risk of developing childhood asthma. Begley et al. (BMJ Open Respir. Res. 5 e000324 (2018)) reports that faecal samples from children with asthma had a higher proportion of Streptococcus and Bacteroides species and a lower proportion of Ruminococcus and Bifidobacterium, while Arrieta et al. (supra) reported that more acetate was found in faecal samples from asthmatic children. These findings have led to attempts to modulate the gut microbiome to improve therapy. According to Liu et al. (supra), addition of strain-specific probiotics to the asthma treatment regime gave some improvement in diversity and clinical responses in adults. However, randomised clinical trials have not so far shown a reduction in the incidence of asthma in children (Wei et al., J. Asthma, 57, 167 (2020)) and meta-analysis of studies involving administration of probiotics to infants has confirmed this (Theodosiou et al., J. Infection, 82, 6, 247 (2021)). Similar effects are seen in adult humans. The possibility of lessening the severity of Covid-19 by modulating the gut microbiome has been reviewed by Li et al. (Viruses, 14, 1774 (2022)), Vestad et al. (J. Internal Med., 291, 801 (2022)), and Kaushal and Noor (Current Microbiol., 79, 184 (2022)) while pulmonary aspergillosis (Cai et al., Frontiers in Immunology, 13, 988708 (2022)), cystic fibrosis (Avalos-Fernandez et al., Respiratory Research, 23, 214 (2022)) and chronic obstructive pulmonary disease (COPD) (Millares and Monso, Int. J. Chron. Obstruct. Pulmon. Dis., 12, 17, 1835 (2022)) have all been suggested as potentially being improved by therapeutic modulation of the gut microbiome.

In view of the foregoing observations and given that an active, i.e., live, enzyme-rich malt extract has been shown to improve the gut microbiome in man, horses and pigeons, with increased levels of short chain fatty acids (SCFAs), it is proposed that an active enzyme-rich malt extract be used as a therapeutic intervention for the prevention and/or treatment of asthma.

Summary of the invention

In one aspect of the invention, a malt extract for use in preventing or treating asthma or at least one of the symptoms of asthma in a mammal or avian is provided, the malt extract comprising one or more active enzymes selected from the group consisting of amylases, maltases, cellulases, fructanases, glucanases, xylanases and deacetylases.

The malt extract, typically barley malt extract, comprises a plurality of enzymatically active digestive enzymes, in particular alpha- and/or beta-amylase (alpha-amylase breaks starch down yielding maltotriose and maltose from amylose, and maltose, glucose and limit dextrin from amylopectin and beta-amylase breaks down starch into maltose), maltase, cellulase, fructanase (which break down fructans found in grass), glucanase (which break down glucans found in cell walls), xylanase (which break down xylans in plant cell walls), deacetylase (which cleave acetyl groups from xylans and fructans thereby allowing xylanase and fructanase to break down the remainder of the molecular structure). The malt extract also comprises smaller amounts of proteinase and lipase (which break down respectively proteins and fats). The malt extract further comprises maltose, maltotriose and maltose polymers and, depending on the precise parameters used in the process of preparing the malt extract, peptides and/or amino acids. The malt extract does not, however, comprise starch because the starch in the barley seeds is broken down to produce a mixture of the aforementioned maltose, maltotriose and maltose polymers.

The barley malt extract is prepared by soaking seeds, typically barley seeds, in water in order to germinate the seeds. Germination causes the seeds to produce a variety of enzymes that break down, for example, starches into sugars through the production of amylases and other carbohydrases, such as fructanases. The germination process also induces other enzymes such as proteases that break down proteins in the grain. Germination is halted by drying with hot air at a temperature of no higher than about 75, 70, 65, 60, 55, 50, 45 or 40 degrees centigrade thereby to produce a malt. Whilst higher temperatures may be used to dry the germinated seeds, such higher temperatures denature an ever greater proportion of the enzymes present in the malt.

The dried sprouted seeds are then milled and water is added and heated to at least about 40, 45, 50, 55, 60, 65 but below 75 or 70 degrees centigrade in order to form a mash, and stirred for about one hour. The enzymes present are active up to different temperatures. Thus, proteases and beta-amylases are active up to about 50 degrees centigrade. Thereafter up to about 65 degrees centigrade, alpha-amylases still degrade starch to sugars.

The next step is separation of the residual solids ('spent grain') from the liquid ('wort'). The wort is then concentrated by vacuum evaporation to provide an active enzyme rich malt extract, typically comprising about 80 % w/w solids in a solution also rich in sugars.

Brief description of the figures

The invention is described hereinbelow with reference with:

Figure 1 which shows total average daily drink consumption (mL/mouse per day) divided by time that treatments were either receiving ERME (TM) or on control water (data are mean ± SD); Figure 2 which shows total average daily food consumption (g/mouse per day) divided by time that treatments were either receiving ERME (TM) or on control water (data are mean ± SD);

Figure 3 which shows airway resistance (A), tissue damping (B) and tissue elastance (C) at functional residual capacity (data are mean ± SD; bars connect treatments that are significantly different (p < 0.05));

Figure 4 which shows airway resistance (A), tissue damping (B) and tissue elastance (C) at the maximum dose of MCh (data are mean ± SD; bars connect treatments that are significantly different (p < 0.05));

Figure 5 which shows airway resistance (A), tissue damping (B) and tissue elastance (C) presented as the percentage increase from the saline aerosol (data are mean ± SD; bars connect treatments that are significantly different (p < 0.05)); and

Figure 6 which shows evocative concentration 150 for airway resistance (REC150; A), tissue damping (GEC150; B) and tissue elastance (HEC150; C) (for each outcome measurement, there was a significant effect of HDM treatment; data are means + SD);

Figure 7 which shows total number of cells per mL in bronchoalveolar lavage (data are mean ± SD; bars connect treatments that are significantly different (p < 0.05));

Figure 8 which shows the number of macrophages (A), neutrophils (B) lymphocytes (C) and eosinophils (D) per mL in bronchoalveolar lavage (data are mean ± SD; bars connect treatments that are significantly different (p < 0.05));

Figure 9 which shows bronchoalveolar lavage mediators for which there was a significant effect of HDM only (data are mean ± SD; * indicates a treatment is significantly different to WWH; # indicates a significant difference to WWS (p < 0.05); a bar connecting two treatments indicates a significant difference between those treatments (p < 0.05), however this is only shown for "within" ERME (TM) comparisons); Figure 10 which shows bronchoalveolar lavage mediators (data are mean ± SD; * indicates a treatment is significantly different to WWH; # indicates a significant difference to WWS (p < 0.05); a bar connecting two treatments indicates a significant difference between those treatments (p < 0.05), however this is only shown for "within" ERME (TM) comparisons);

Figure 11 which shows bronchoalveolar lavage mediators for which there was no significant effect of HDM or ERME (TM) (data are mean ± SD); and

Figure 12 which shows total IgE (ng/mL) in serum (data are mean ± SD; bars connect treatments that are significantly different (p < 0.05)).

Detailed description of the invention

The at least one of the symptoms of asthma may be selected from the group consisting of shortness of breath, chest tightness or pain, coughing and wheezing.

Preferably the daily dosage of the malt extract is in the range 0.02-2, 0.05-2, 0.1-1, 0.25-0.75 ml per kg body weight mammal or avian.

Preferably, the malt extract additionally comprises:

• one or more proteinases and/or lipases; and/or

• one or more water soluble sugars selected from the group consisting of maltose, maltotriose, and maltose polymers.

The malt extract is typically based on one of the seeds selected from the group consisting of barley, wheat, triticale, sorghum, maize, buckwheat, rice and a mixture thereof, but preferably barley. The diastatic power of the malt extract is preferably above 35 (94 degrees Windisch Kolbach (WK) units), 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 115 degrees Lintner. For comparative purposes, a malt with enough power to self-convert starch to sugars has a diastatic power of about 35 degrees Lintner.

The mammal may be selected from the group consisting of a human being, horse, cattle, and pig, whereas the avian may be selected from the group consisting of Columba livia domestica, Gallus gallus domestica, turkey, duck, and pheasant.

Example

Methods

Mice

Young adult (6-week-old) female BALB/c mice (n=96) were purchased from the Animal Resources Centre (Perth, Australia) and housed at the Telethon Kids Institute under Physical Containment level 2 conditions and a 12:12 hour light/dark cycle. Standard mouse chow (Specialty Feeds, Glen Forrest, Western Australia) and water (containing a barley based enzyme rich malt extract (ERME™ available from Ateria Health Limited (England) or not for control) was provided ad libitum. Mice were housed in groups of 2-4 animals in individually ventilated cages (Techniplast "Green Line"). Mice were given approximately 1 week to acclimate to the facility before commencing treatment.

Malt extract treatment

For mice receiving the malt extract, the malt extract was prepared in Hydropacs™ watering systems (Avidity Science, Aylesbury, Hertfordshire, England) fresh daily. This was achieved by weighing each individual Hydropacs™ (already containing non-acidified drinking water) and injecting in a calculated volume of the malt extract using a syringe. This was prepared to a dose equivalent to 2.5 mL malt extract/kg body weight based on a 20 g mouse drinking 3 mL per day. Malt extract containing Hydropacs™ were changed every day to ensure the malt extract solution stayed "fresh" and weighed daily to estimate consumption per box. Control mice received non-acidified drinking water, with Hydropacs™ being weighed and changed every about 4 days. House dust mite sensitisation

An asthma/allergic airways disease phenotype was generated using a house dust mite (HDM) sensitisation method. Briefly, mice were lightly anaesthetised using isoflurane and intranasally inoculated with 25 pg of HDM (Dermatophagoides pteronyssinus) protein dissolved in 50 pL of saline or saline alone (control) by pipetting drops onto the nostrils until aspirated. Mice received this treatment for ten consecutive days. The HDM used contained 29.4 % w/w total protein and 3291 EU endotoxin/mg protein (XPB82D3A25, lot no. 361862; Stallergenes Greer, Lenoir, NC).

Treatment groups

Prior to commencing the study, mice were randomly divided into one of eight treatment groups. The groups were (i) 14 days of prophylactic malt extract (or control) followed by (ii) continued malt extract (or control) for 10 days. During the final 10 days, mice also either received daily HDM or control intranasal inoculations, with all measurements being performed the day after the final inoculation. This achieved a cross-over design whereby it was possible to explore the effects of prophylactic treatment with malt extract only, concurrent treatment of malt extract with HDM only, or prophylactic treatment + concurrent treatment (+ each commensurate control group). This resulted in the groups described in Table 1.

Table 1: Malt extract (ERME™)/HDM study treatment groups.

Observations and clinical scoring Each day that the mice were involved in the study cage-side observations were performed. Each animal was thoroughly assessed for:

• changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, unusual respiratory pattern);

• changes in gait, posture, and response to handling;

• the presence of colonic or tonic movements, stereotypes (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g., self-mutilating, walking backwards); and

• any other deviation from "normal".

Mice were be scored daily on a scale 0 (normal), 1 (some deviation) or 2 (obvious or continuous deviation) for each assessment criteria, and a total clinical score given.

Lung function assessment

Twenty-four hours after their last HDM or saline inoculation exposure, mice were anaesthetized via intraperitoneal injection with ketamine/xylazine (0.4 and 0.02 mg/g body weight respectively; Troy Laboratories, Glendenning, NSW, Australia) prior to lung function and responsiveness to methacholine (MCh) being measured. This was done using a modification of the low-frequency forced oscillation technique (LFOT) and a small-animal ventilator (flexiVent; Scireq, Montreal, QC, Canada).

LFOT is a commonly used technique to assess respiratory mechanics in both humans and animal models, which involves applying an oscillatory pressure signal externally to the respiratory system (in this case to the trachea via the flexiVent piston) and measuring the resultant respiratory output signal (such as pressure or airflow. The standard flexiVent "Primewave" 16 second oscillatory pressure signal which is comprised of 19 mutually prime sinusoidal frequencies ranging between 0.25 and 19.625 Hz was used. When this signal is applied to the airway of a mouse, each frequency applied results in a commensurate measurement of resistance and reactance, both presented in units of cm HzO.s/mL, which forms the impedance spectrum (Z_rs). In brief, following induction of surgical anaesthesia, a polyethylene cannula was inserted into the trachea and secured with silk ligature. Mice were then connected to the ventilator, ventilated at 450 breaths/minute with a tidal volume of 8 mL/kg and positive end expiratory pressure of 2 cmHzO. They were allowed to stabilise for 5 minutes prior to standardisation of lung volume history via three slow deep inflations to a respiratory pressure of 20 cmHzO.

Following this, the flexiVent was used to measure respiratory system impedance (Z_rs). Z_rs is the primary outcome of LFOT and can be defined as the ratio of the amplitude of the input oscillatory pressure signal to the resulting flow, i.e. the relationship between the input and output signals characterised by a transfer function (Bates et al., Comprehensive Physiology, 1, 3, 1233 (2011)). In order to be meaningful in terms of lung function, Z_rs data needs to be physiologically interpreted. This is achieved by fitting a mathematical model to the Z_rs data, with the optimal model currently in use being the Constant Phase Model (Hantos et al., J. Appl. Physio., 72, 1, 168 (1992)). The Constant Phase Model is incorporated into the flexiVent software used to generate these data, and is defined by the equation Zrs — Rn + law + [G - iH]/(na where R_n is Newtonian resistance, l_aw is inertance, G is tissue damping and H is tissue elastance. The key benefits of the Constant Phase Model are that it can be fit to all Z_rs data and that it allows Z_rs data to be partitioned into airway and parenchymal compartments allowing calculation of Newtonian resistance (R_n), inertance (l_aw), tissue damping (G) and elastance (H). In mice, R_n is equivalent to airway resistance (R_aw) because of the high compliance of the chest wall. The calibration procedure in the flexiVent software was used to correct for the resistance of the tracheal cannula and as such, l_aw values were negligible and are not reported.

Once stabilised on the ventilator, LFOT measurements were taken once a minute for five minutes to obtain lung function data at functional residual capacity data. This was followed by a 10-second saline aerosol and increasing doses of methacholine (MCh) delivered by an ultrasonic nebuliser (DeVilbiss UltraNeb, Somerset, PA, USA). After each challenge, LFOT measurements were again taken once a minute for 5 minutes with the peak response used for analyses. Differences in responsiveness were assessed as the maximum responses to 30 mg/mL MCh challenge. Sensitivity to MCh was also assessed by calculating the dose of MCh required for R_aw, G and H to increase by 150 %. Biological sample collection

At the completion of lung function testing, a blood sample via cardiac puncture was taken. This was centrifuged, and serum frozen for subsequent measurement of HDM specific IgE using commercially available kits. This was followed by taking bronchoalveolar lavage (BAL) fluid by washing 0.5mL of chilled saline in and out of the lungs three times. BAL was centrifuged to separate the cell pellet and supernatant. Cells were stained with trypan blue and total cell counts obtained using a haemocytometer. Differential counts were obtained from the cytospin sample, stained with Sirius Red and/or Rapid Diff stains and examined using light microscopy. The supernatant was stored for subsequent analysis of cytokines, chemokines and mediators using commercially available Bioplex kits (Bio-Plex Pro Mouse Cytokine 23-plex Assay).

Statistics

To explore the effects of prophylactic treatment with ERME (TM) only, concurrent treatment of ERME (TM) with HDM only, or prophylactic treatment + concurrent treatment (+ each commensurate control group), 2-way ANOVA was used where possible, with "ERME" and "Allergy" as factors. ERME (TM) treatment was divided into "prophylactic" (EWx), "concurrent" (WEx), "both" (EEx) or "neither" (WWx), and allergy treatment was divided into "HDM" (xxH) or "saline" (xxS). SigmaPlot vl4 and GraphPad Prism v8 were used for statistical analyses. Data were transformed to satisfy the assumptions of normality and equal variance where necessary, p < 0.05 was considered statistically significant. In situations where data were not able to be transformed to satisfy the assumptions of ANOVA, for example mediators in bronchoalveolar lavage fluid, general linear modelling was used.

Results

Observations and clinical scoring

There were no instances of adverse of effects due to ERME (TM) treatment. Every mouse scored "0" (i.e. "normal") for every criterion every day except:

• 1 mouse in the EWS treatment scored a "1" for minor weight loss on one occasion.

This occurred 2 days after it switched from ERME (TM) onto water. The weight of this mouse never dropped below 102.6% of its starting weight, and it recovered this weight within 1 day.

1 mouse in the EES treatment scored a "1" for minor weight loss on one occasion. This occurred 16 days into the ERME (TM) treatment. The weight of this mouse never dropped below 102.7% of its starting weight, and it recovered this weight within 1 day.

2 boxes of mice experienced "barbering" (one WES and one WWH). This is a behaviour in which one mouse aggressively grooms other mice, resulting in the removal of fur and/or whiskers. In both instances the barbering was mild and one mouse per box was identified as the "bully" as it was the only one with whiskers. These boxes were provided with wooden chew blocks (made from the same material as their bedding), and the barbering stopped.

From this it can be inferred that ERME (TM) treatment as given in drinking water at a dose equivalent to 2.5mL ERME (TM)/kg body weight (based on a 20g mouse drinking 3 mL per day) did not lead to any adverse health effects in mice.

Food and drink consumption

There were clear trends in drink consumption dependent on treatment. Mice on continuous ERME (TM) (EEH and EES) had the highest drink consumption at 3.03 ± 0.23 mL/mouse/day and 2.92 mL/mouse/day respectively (Table 2). This was roughly 50% greater consumption compared with mice drinking water only (WWH and WWS). Drink consumption was slightly lower in mice that switched to water from ERME (TM) (EWH and EWS), and then similar at around 2 mL/mouse/day for the four remaining treatments.

Table 2: Total average daily drink and food consumption (mL/mouse per day and grams/mouse per day). Data are mean ± SD.

Drink consumption can be broken down further into consumption while mice were receiving ERME (TM) or water, which takes into account the switch experienced by four treatments (Figure 1). These data support those shown in Table 2, with ERME (TM) consumption being higher ( about 2.5 to about 3 mL/mouse/day) in mice that start on ERME (TM) (EEH, EES, EWH and EWS), compared with mice that started on water (WEH, WES, WWH, WWS) which drank between about 2 and 2.25 mL/mouse/day. These data suggest that ERME (TM) in drinking water was palatable to mice.

Food consumption (Table 2 and Figure 2) did not vary as much as drink consumption, with all treatment groups eating between 2.34 and 2.65 grams of chow/mouse/day. Apart from EEH mice, those which started treatment on water (WEH, WES, WWH and WWS) generally consumed slightly more food that those that started on ERME (TM). As food consumption did not vary between treatments, but drink consumption did, it can be inferred that mice receiving ERME (TM) likely had a higher overall caloric intake than mice on water.

Body Weight

There was no significant difference in the weights of mice randomly allocated to the different treatments at the start of the study (i.e. before any treatments started; p > 0.945 in all cases; Table 3). There was also no significant difference in weights between treatments at the conclusion of the study (p > 0.352 in all cases; Table 3). There was, however, a significant effect of ERME (TM) treatment on percentage weight gain, with mice receiving ERME (TM) only concurrently with HDM/saline (WES and WEH) gaining significantly less weight than all other treatments (p = 0.047).

Table 3: Body weight (grams) at the start and end of the experiment, and percentage weight gain. Data are mean ± SD. * indicates significant difference.

Lung function at functional residual capacity

Lung function at functional residual capacity (FRC) is representative of how the lungs of the mice are working when at rest. The technique allows interrogation of the lung to obtain three main outcomes - airway resistance (R_aw) which is a measure of how constricted the main conducting airways/trachea are and reflects the resistance of the central airways/trachea where air is moving by bulk flow, tissue damping (G) which is a measure of peripheral lung constriction/obstruction and reflects the energy dissipation of the lung tissues, and tissue elastance (H) which is a measure of how "stiff" the lungs are and reflects energy conservation of the lung tissues. Typically, increases in any of these are interpreted as worse lung function.

Airway resistance: There was a significant effect of both HDM (p < 0.001) and ERME (TM) (p = 0.041; Figure 3A) on airway resistance at FRC. There was no significant interaction (p = 0.295). Overall, mice treated with HDM had significantly higher R_aw at FRC compared with controls (p < 0.001), however post hoc analyses showed that this was primarily due to R_aw in EWH being significantly higher than in EWS (p = 0.018) and R_aw in WWH being significantly higher than in WWS (p = 0.008). Airway resistance at FRC was not significantly higher in EEH mice compared with EES (p = 0.170) nor was it higher in WEH mice compared with WES (p = 0.709). This is suggestive of treatment with ERME (TM) concurrent with HDM sensitisation partially ameliorating the effect of HDM on this outcome.

Tissue damping: There was a significant of HDM on tissue damping (G) at FRC (p < 0.001), but no overall effect of ERME (TM) on this parameter (p = 0.674). The significant effect of HDM was that each HDM treated group had significantly higher G at FRC compared with its commensurate control (Figure 3B). There was a significant interaction between ERME (TM) and HDM (p = 0.004) which was due to G at FRC for WEH mice being significantly lower than this parameter for EEH mice (p = 0.028). This is suggestive of treatment with ERME (TM) having no effect on peripheral lung resistance.

Tissue elastance: There was a significant effect of HDM on tissue elastance (H) at FRC (p < 0.001), with each HDM treated group having significantly higher H at FRC compared with its commensurate control. There was no effect of ERME (TM) (p = 0.446). There was a significant interaction between the two, however, (p = 0.003) which was due to H at FRC for WEH mice being significantly lower than this parameter for EEH mice (p = 0.002; Figure 3C). This is suggestive of treatment with ERME (TM) having no effect on lung tissue stiffness.

Conclusion

In brief, HDM exposure resulted in significant impacts on all three parameters of lung function measured at FRC. ERME (TM) had limited effects, which were primarily with respect to WEH mice being significantly different to WWH mice (for R_aw) and EEH mice for G and H.

In more detail, HDM exposure resulted in significant impacts on all three parameters of lung function measured at FRC, while ERME (TM) had limited effects, which were primarily with respect to WEH mice. The key effects of ERME (TM) were:

(i) WEH had lower airway resistance at FRC compared with WWH mice indicating giving ERME (TM) during HDM sensitisation reduces resting airway resistance.

(ii) WEH mice did not have significantly higher Raw at FRC compared with their commensurate control (WES) which could also be interpreted as provision of ERME (TM) during allergic sensitisation exerted some protective effects on Raw at FRC. (iii) EEH mice had higher G and H compared with WEH mice, indicating either a slightly positive effect of concurrent ERME (TM) treatment on these parameters, or a negative impact of pre-and concurrent ERME (TM) treatment on these parameters.

Additionally, EEH mice did not have higher R_aw than EES mice, which is supportive of the observation that pre- and concurrent treatment with ERME (TM) may increase airway resistance at FRC.

Typically, studies investigating the effects of HDM on lung-function in mice do not directly present data obtained at FRC (usually only hyperresponsiveness data are published), nor do they present the same parameters presented here. The effects of ERME (TM) were less overt than those due to HDM, and were primarily with respect to WEH and EEH mice (Figure 3). These data could be interpreted as WEH having "better" lung function at FRC than EEH, or EEH being "worse" than WEH. Potentially the most significant outcome from this is that WEH mice did not have higher R_aw at FRC compared with WES (while EWH was different to EWS and WWH was different to WWS). This could be interpreted as ERME (TM) treatment co-concurrent with allergic sensitisation somehow preventing HDM induced increases in R_aw. Similarly, EEH mice were not significantly different to EES in terms of R_aw at FRC, but this appears to be more related to higher Raw for EES mice, rather than reduced R_aw for EEH mice, suggesting that pre- and concurrent treatment with ERME (TM) led to worse lung function for this parameter.

Airway-hyperresponsiveness (AHR) This is a measure of whether the lungs of the mice respond excessively to a bronchoconstrictor called methacholine (MCh). It is similar to lung function assessment used in humans to test whether they have asthma. Mice are exposed to increasing concentrations of methacholine via inhalation, and lung function (partitioned into R_aw, G and H) is assessed after each dose. Dose response curves and sensitivity to MCh can be obtained. As with lung function at FRC, increases in these parameters are interpreted as decreases in lung health. It is important to note that AHR data can be analysed in terms of "absolute" values, or as the % increase from values measured for each individual after their saline aerosol. The former gives actual quantitative data in units of pressure/volume, but it does not take into account differences in lung function at FRC (i.e. a different starting point for the dose response curve). General trends are usually the same between the two analysis methods, which are both described below.

Absolute AHR

Airway resistance: There was a significant effect of HDM (p < 0.001) and ERME (TM) (p < 0.001) on Raw at the maximum dose of MCh, but no interaction (p = 0.640; Figure 4A). This means that each HDM treated group was significantly more responsive than their commensurate control (p < 0.001 in all cases; Figure 4A), but there was no difference in responsiveness to MCh for airway resistance between mice treated with saline (p > 0.275 in all cases). Within HDM treated groups, WEH mice were significantly less responsive than EEH (p = 0.014) and WWH mice (p = 0.002), indicating that ERME (TM) given during HDM sensitisation significantly reduces airway constriction compared with mice not given ERME (TM), or mice also given ERME (TM) prior to HDM commencing.

Tissue damping: There was a significant effect of HDM (p < 0.001) and ERME (TM) (p = 0.025) on G at the maximum dose of MCh, and a significant interaction (p < 0.001; Figure 4B). Each HDM treated group was significantly more responsive than their commensurate control (p < 0.001 in all cases). Within HDM treated mice, EEH were significantly more responsive than both EWH and WEH (p < 0.015 in both cases), and WWH was significantly more responsive than WEH (p = 0.018). There was no difference between saline treated mice (p > 0.960 in all cases). As with airway resistance, the significantly lower G in WEH mice indicates that ERME (TM) given during HDM sensitisation significantly reduces tissue damping (peripheral lung resistance) compared with mice not given ERME (TM), or mice also given ERME (TM) prior to HDM commencing.

Tissue elastance: There was a significant effect of HDM (p < 0.001) and ERME (TM) (p = 0.032) on H at the maximum dose of MCh, and a significant interaction (p < 0.001; Figure 4C). Each HDM treated group was significantly more responsive than their commensurate control (p < 0.001 in all cases). Within HDM treated mice, EEH were significantly more responsive than WEH and EWH mice (p < 0.027 in both cases), but EEH mice were not significantly more responsive than WWH mice (p = 0.052). WEH mice were significantly less responsive than all other treatments (p < 0.049 in all cases). There was no significant difference in maximum H between saline treated mice (p > 0.847 in all cases). As with both airway resistance and tissue damping, the significantly lower H in WEH mice indicates that ERME (TM) given during HDM sensitisation significantly reduces tissue elastance (lungs are less "stiff") compared with mice not given ERME (TM), or mice also given ERME (TM) prior to HDM commencing.

Percentage increase AHR

Airway resistance: There was a significant effect of HDM (p < 0.001) and ERME (TM) (p = 0.007) on Raw % increase from saline, but no interaction (p = 0.707; Figure 5A). This means that each HDM treated group was significantly more responsive than their commensurate control (p < 0.001 in all cases), but there was no difference in responsiveness to MCh for airway resistance between mice treated with saline (p > 0.573 in all cases). Within HDM treated groups, WEH and EWH mice were significantly less responsive than WWH mice (p = 0.037 in both cases).

Tissue damping: There was a significant effect of HDM (p < 0.001) and ERME (TM) (p = 0.005) on G % increase from saline, and a significant interaction (p = 0.020; Figure 5B). Each HDM treated group was significantly more responsive than their commensurate control (p < 0.001 in all cases). Within HDM treated mice, WEH mice were significantly less responsive than both WWH and EEH mice (p < 0.023 in both cases), and EWH was significantly less responsive than EEH (p = 0.032). There was no difference between saline treated mice (p > 0.961 in all cases).

Tissue elastance: There was a significant effect of HDM (p < 0.001) and ERME (TM) (p = 0.028) on H % increase from saline, and a significant interaction (p < 0.026; Figure 5C). Each HDM treated group was significantly more responsive than their commensurate control (p < 0.001 in all cases). Within HDM treated mice, EEH were significantly more responsive than WEH mice (p < 0.001). There was a trend towards significance for WEH mice being less responsive than WWH mice (p = 0.063). There was no significant difference in maximum H between saline treated mice (p > 0.906 in all cases). Conclusion

In brief, HDM exposure resulted in significantly higher R_aw, G and H at the maximum dose of MCh (Figures 4 and 5) for all treatments, i.e. all HDM treated groups were significantly more responsive to MCh compared with their commensurate control. The level of increase after HDM exposure did vary between HDM treated groups as a result of ERME (TM). In most cases, WEH mice were the least responsive to MCh of HDM treated groups, followed by EWH. EEH mice were similar to WWH mice for Raw, but significantly more responsive than WWH for G and H.

In detail, HDM exposure resulted in significantly increased responsiveness to MCh for all three parameters of lung function (Figures 4 and 5). ERME (TM) treatment also impacted responsiveness, which (like assessment at FRC) were primarily with respect to WEH mice. There was a slight effect of interpreting the data in terms of absolute changes in lung function (Figure 4) vs percentage increase from FRC, however the majority of the data were similar. The key effects of ERME (TM) were:

(i) For airway resistance (R_aw), WEH mice were significantly less responsive than both EEH and WWH mice (absolute) and significantly less than WWH (% increase). EWH mice were also significantly less responsive than WWH (% increase). EEH was not different to WWH for this parameter. These data indicate that provision of ERME (TM) during HDM sensitisation reduces airway hyper-responsiveness (i.e. a reduction in constriction of the main conducting airways), while treatment pre HDM sensitisation has less of an effect, and pre- combined concurrent treatment has no effect.

(ii) Similar patterns were seen for G. WEH mice were significantly less responsive than WWH and EEH mice for this parameter, while EWH were also less responsive than EEH mice. EEH and WWH were not significantly different to each other. These findings could also be interpreted as provision of ERME (TM) during allergic sensitisation exerting some protective effects on G, i.e. less constriction of the peripheral airways, while pre- combined with concurrent treatment has no effect. (iii) Again, similar patterns were seen for H. In terms of absolute changes in H, WEH mice were less responsive than all other HDM treated groups (they were only less responsive than EEH for % increase in H). EWH mice were also less responsive than EEH mice. EEH were not significantly different to WWH. Like R_aw and G, these findings could be interpreted as provision of ERME (TM) during allergic sensitisation exerting some protective effects on H, i.e. lung tissue is less stiff/more elastic (Bates and Irvin, J. of Appl. Physiology, 94, 4, 1297 (2003)) and/or there has been derecruitment of peripheral lung units (Bates and Irvin, J. of Appl. Physiology, 93, 2, 705 (2002)), while pre- combined with concurrent treatment has no effect.

ERME (TM) also impacted responsiveness to MCh, but only for mice also exposed to HDM, i.e. saline controls were not different to each other regardless of ERME (TM) treatment. There was also a spectrum in terms of the effect ERME (TM) had on responsiveness to MCh, with mice receiving pre- combined with concurrent treatment with ERME (TM) (EEH) mice being as responsive as the comparison treatment receiving no ERME (TM) (WWH) for R_aw, G and H. Pre-treatment with ERME (TM) (EWH) had a minor effect on G and H, however the key impact of ERME (TM) was again for mice provided with ERME (TM) while also undergoing HDM treatment (WEH). Mice treated in this way were less responsive (in terms of absolute response) than non-ERME (TM) treated mice (WWH) and EEH mice for all three parameters, and less responsive than WWH mice for R_aw and G in terms of % increase. Combined, these data indicate that ERME (TM) does impact responsiveness to MCh in this model, and that the timing/duration of the ERME (TM) treatment is important. It is important to note that ERME (TM) treatment did not abolish airway hyperresponsiveness in any treatment as the least responsive ERME (TM)/HDM treated group (WEH) was still significantly more responsive than non-HDM treated mice for all lung function parameters.

Evocative Concentration

Evocative concentration (EC) is a measure of sensitivity to MCh. It reports the dose of methacholine required for a certain level of increase in airway resistance (REC150), tissue damping (GEC150) or tissue elastance (HEC150). The increase is often 150 %. A higher number is indicative of reduced sensitivity to MCh, i.e. the slope of the dose response curve is less. A proportion of animals did not reach a 50 % increase in R_aw, G and H (particularly in saline treated groups), so these data are not normally distributed or have equal variance. As such, general linear modelling was used to analyse these data.

REC 150

There was a significant effect of HDM on REC150 (p < 0.001) with HDM treatment significantly reducing REC150 compared with saline treatment (Figure 6A). Within HDM treated groups, EEH were significantly more sensitive to MCh (lower REC150), compared with EWH and WEH (p < 0.035 in both cases). EEH was not significantly different to WWH (p = 0.499). EWH and WEH were also significantly less sensitive than WWH (p < 0.005 in both cases). There was no difference in REC150 between any of the saline treated groups (p > 0.071 in all cases). These data support other lung function outcomes in that either concurrent (WEH) or prophylactic (EWH) treatment with ERME (TM) reduced sensitivity to MCh.

G EC 150

Like REC150, there was a significant effect of HDM on GEC150 (p < 0.001) with HDM treatment significantly reducing GEC150 compared with saline treatment (Figure 6B). Within HDM treated groups, WEH were significantly less sensitive to MCh than EEH, WWH and EWH mice (p < 0.017 in all cases). WWH mice were significantly more sensitive to MCh than WEH and EWH (p < 0.005 in both cases). There was no difference in GEC150 between any of the saline treated groups (p > 0.071 in all cases). Again, concurrent treatment with ERME (TM) (WEH) had beneficial effects on sensitivity to MCh with respect to tissue damping.

H EC 150

There was a significant effect of HDM on HEC150 (p < 0.001) with HDM treatment significantly reducing HEC150 compared with saline treatment (Figure 6C). Within HDM treated groups, WEH were significantly less sensitive to MCh than EEH, WWH and EWH mice (p < 0.006 in all cases). There was no difference in GEC150 between any of the saline treated groups (p > 0.128 in all cases). Concurrent treatment with ERME (TM) (WEH) had beneficial effects on sensitivity to MCh with respect to tissue elastance.

Bronchoalveolar lavage cellular inflammation There was a significant effect of HDM treatment on total numbers of cells in bronchoalveolar lavage (p < 0.001 in all cases) with each HDM treated group having significantly more cells than their commensurate control (Figure 7). An increase in total cells in lavage is consistent with HDM exposure in mice. There was no effect of ERME (TM) (p = 0.059 in all cases).

Differential cell count data were generally not normally distributed, and they did not have equal variance, and therefore they were either transformed to satisfy these assumptions (for 2-way ANOVA), or analysed using general linear modelling. Outliers (values > 3 standard deviations from the mean) were excluded before analysis. There were a total of 7 outliers identified, out of a total of 372 measurements (4 cell types x 93 mice: lavage was not obtained from 1 mouse, and 2 cytospins had no identifiable cells). Of potential note, 2 outliers were with respect to numbers of neutrophils for WEH mice, which were 1,153,475 and 186,704 cells/mL respectively (compared to a mean of 4,020 neutrophils/mL for other WEH mice). One other WEH mouse also had an outlier number of lymphocytes (720,439 cells/mL compared with a mean of 5,164 cells/mL for other WEH mice).

There was a significant effect of HDM treatment on the number of macrophages in bronchoalveolar lavage (p < 0.043 in all cases) with each HDM treated group having significantly more cells than their commensurate control (Figure 8). An increase in macrophages in lavage is consistent with HDM exposure in mice. There was a minor effect of ERME (TM) in that EWH mice had significantly more macrophages in their bronchoalveolar lavage (BAL) than WWH mice (p = 0.043).

There was also a significant effect of HDM on the number of neutrophils in bronchoalveolar lavage (BAL) in that each HDM treated group had more neutrophils in their lavage than their commensurate control (p < 0.002 in all cases), except for WEH which were not significantly different to WES once the two outliers were removed (p = 0.368). Similarly, once the two outliers were removed WEH mice had significantly fewer neutrophils in their lavage than EEH mice (p = 0.040). If WEH extreme outliers are retained in the data set, there is still an overall effect of HDM on neutrophils (p < 0.001), but no other significant differences. Additionally, there was a significant effect of HDM on the number of eosinophils in bronchoalveolar lavage (BAL) in that each HDM treated group had more eosinophils in their lavage than their commensurate control (p < 0.001 in all cases). There was also a minor effect of ERME (TM) in that WEH mice had significantly fewer eosinophils in their bronchoalveolar lavage bronchoalveolar lavage (BAL) than EWH mice (p = 0.013). There were no other significant differences (p > 0.103 in all cases). An increase in eosinophils in lavage is consistent with HDM exposure in mice.

Bronchoalveolar lavage mediators

20 out of 23 possible mediators in the Bio-Plex Pro Mouse Cytokine 23-plex Assay were detected in bronchoalveolar lavage samples from this study. Only IL-1 , IL-3 and GM-CSF were not detected above the limit of detection, and therefore data for those mediators are not presented. In most cases, mediator data were not normally distributed, and they did not have equal variance, and therefore they were analysed using general linear modelling. Outliers (values > 3 standard deviations from the mean) were excluded before analysis. For simplicity, graphs indicate whether treatments are significantly different to WWH (designated by *) and/or WWS (designated by #), with other key differences described in the text.

Significant effect of HDM only (Figure 9)

• lnterleukin-5 (IL-5)

There was a significant effect of HDM treatment on IL-5 with HDM treated mice generally having significantly higher levels than saline treated mice. WWH mice had significantly more IL-5 than all saline treated groups (p < 0.035 in all cases). WWS mice had significantly lower levels of IL-5 than all HDM treated mice (p < 0.048 in all cases). Increased IL-5 post HDM treatment was expected due to it being well established as an interleukin associated with allergic disease and asthma (Hamelmann and Gelfand, Immunological Rev, 179, 1, 182 (2001), Gregory et al., Clin. & Exp. Allergy, 39, 10, 1597 (2009), Malaviya et al., Pharmacology Res. & Persp., 9, 3, e00770 (2021), and Mann et al., Am. J. Physiology - Lung Cellular and Mol. Physiology, 322, 5, L683 (2022)). IL-5 is produced by Th2 cells, mast cells and eosinophils in response to an allergic insult (Yasuda et al., Cells, 9, 5, 1178 (2020)). lnterleukin-12(p40) (IL-12(p40))

There was a strongly significant effect of HDM on IL-12(p40), with all HDM treated mice having significantly higher levels of I L-12(p40) than their treatment controls (p < 0.001 in all cases). There was no significant difference between any of the HDM treated groups (p > 0.248 in all cases), or any of the control groups (p > 0.067 in all cases). I L-12(p40) has a variety of functions including being a chemoattractant for macrophages, and it is associated with various inflammatory lung diseases including asthma (Cooper and Khader, Tr. In Immunol., 28, 1, 33 (2007)). Mann et al. (Am. J. Physiology - Lung Cellular and Mol. Physiology, 322, 5, L683 (2022)) previously reported a significant increase in I L-12(p40) post HDM exposure in mice.

• Interleukin-13 (IL-13)

There was a strongly significant effect of HDM on IL-13, with all HDM treated mice having significantly higher levels of IL-13 than their treatment controls (p < 0.009 in all cases). EWH mice also had significantly less IL-13 than EEH mice (p = 0.021). There were no other significant differences (p > 0.078 in all case). Increased IL-13 in HDM treated mice was expected due to critical role IL-13 is known to play in allergic/inflammatory conditions such as asthma (Wynn, Ann. Rev. Immunol., 21, 1, 425 (2003)) including in mouse models of HDM-induced allergic airways disease. Mann et al. (Am. J. Physiology - Lung Cellular and Mol. Physiology, 322, 5, L683 (2022)) previously reported a significant increase in IL-13 post HDM exposure in mice.

• Eotaxin

There was a strongly significant effect of HDM on eotaxin, with all HDM treated mice having significantly higher levels of eotaxin than their treatment controls (p < 0.011 in all cases) except for WEH and EWH, which were not significantly different to WES (p > 0.070 in both cases). Eotaxin is a chemoattractant for eosinophils (hence is key in allergic disease) and is known to be increased after HDM exposure in mice (Gregory et al., Clin. & Exp. Allergy, 39, 10, 1597 (2009)).

Keratinocyte-derived chemokine (KC) There was a strongly significant effect of HDM on KC, with all HDM treated mice having significantly higher levels of KC than their treatment controls (p < 0.021 in all cases). EWS mice also had significantly higher KC compared with EES (p = 0.026), and borderline higher level than WWS (p = 0.057). KC is a chemoattractant for neutrophils, and is known to increase after HDM exposure, especially in older mice (Brandenberger et al., Clin & Exp. Allergy, 44, 10, 1282 (2014)) and in models of viral asthma exacerbations (Clarke et al., Clin. Sci., 126, 8, 567 (2013)). It has previously been shown to be increased in the bronchoalveolar lavage (BAL) of HDM exposed mice (Gregory et al., Clin. & Exp. Allergy, 39, 10, 1597 (2009), Malaviya et al., Pharmacology Res. & Persp., 9, 3, e00770 (2021), and Mann et al., Am. J. Physiology - Lung Cellular and Mol. Physiology, 322, 5, L683 (2022)).

• RANTES

There was a strongly significant effect of HDM on RANTES (regulated on activation, normal T cell expressed and secreted), with all HDM treated mice having significantly higher levels of RANTES than their treatment controls (p < 0.014 in all cases). RANTES is a pro-inflammatory chemotactic mediator that attracts a wide range of cells (including T cells, eosinophils, basophils and monocytes) to sites of inflammation, particularly infection (Appay and Rowland-Jones, Trends in Immunol., 22, 2, 83 (2001)). It is increased in the airways of asthmatic people (Li et al., Science, 24, 10, 103163 (2021)), in mouse lung homogenates post HDM exposure (Malaviya et al., Pharmacology Res. & Persp., 9, 3, e00770 (2021)) and in mouse lavage post HDM exposure (Mann et al., Am. J. Physiology - Lung Cellular and Mol. Physiology, 322, 5, L683 (2022)).

Other significant differences (Figure 10)

• Interleukin 2 (IL-2)

There was a significant effect of treatment on IL-2, with WEH mice having significantly higher levels of this mediator compared with all treatments (p < 0.045 in all cases) except WWH and WES (p > 0.142 in both cases). IL-2 has a wide range of functions, particularly with respect to immunity, whereby it promotes differentiation of T cells into effector and memory T-cells post antigen exposure (Liao, Lin et al. 2011). In a recent study using a similar HDM exposure model, exogenous delivery of a complex of IL-2 and an anti-IL-2 antibody (1C6) significantly reduced key features of the asthmatic phenotype, including responsiveness to MCh, bronchoalveolar lavage (BAL) Th2 cytokines and IgE (Klein et al., Allergy, 77, 3, 933 (2022)). Mann et al. (Am. J. Physiology - Lung Cellular and Mol. Physiology, 322, 5, L683 (2022)) have previously shown that HDM exposure alone does not significantly alter levels of bronchoalveolar lavage (BAL) IL-2.

• Interleukin 4 (IL-4)

There was a significant effect of HDM treatment on IL-4 with HDM treated mice generally having significantly higher levels than saline treated mice. WWH mice had significantly more IL-4 than all saline treated groups (p < 0.003 in all cases), and also more than EWH mice (p = 0.050). WWS mice had significantly lower levels of IL-4 than all HDM treated mice (p < 0.004 in all cases). There was an effect of ERME (TM) in that WEH mice did not have significantly higher IL4 than WES mice (p = 0.058). IL-4 has previously been shown to increase in bronchoalveolar lavage (BAL) and lung homogenates of mice exposed to HDM (Gregory et al., Clin. & Exp. Allergy, 39, 10, 1597 (2009), Malaviya et al., Pharmacology Res. & Persp., 9, 3, e00770 (2021), and Mann et al., Am. J. Physiology - Lung Cellular and Mol. Physiology, 322, 5, L683 (2022)).

• Interleukin 10 (IL-10)

WWH mice had significantly more IL-10 than EES (p = 0.014), and borderline more than WWS (p = 0.051). WEH mice also had significantly more IL-10 than EES (p = 0.034). There were no other significant differences (p > 0.095 in all cases). IL-10 has many functions, typically with respect to immunoregulation and inflammation whereby it has been shown to inhibit synthesis of a range of pro-inflammatory cytokines (Ouyang et al., Ann. Rev. Immunol., 29, 1, 71 (2011)). It has previously been shown to have anti-inflammatory effects when produced by mast cells in a mouse model of allergic skin disease (Grimbaldeston et al., Nature Immunol., 8, 10, 1095 (2007)). Mann et al. (Am. J. Physiology - Lung Cellular and Mol. Physiology, 322, 5, L683 (2022)) previously reported a significant increase in IL-10 post HDM exposure in mice.

• lnterleukin-12(p70) (IL-12(p70))

There were no clear ERME (TM) or HDM induced differences in I L-12(p70), however there were significant differences between treatments. WEH mice had significantly higher levels of I L-12(p70) compared with EWH, EES and EEH mice (p < 0.022 in all cases). WEH was not significantly different to WES (p = 0.566). WES mice also had significantly higher levels of IL- 12(p70) than EWH and EES mice (p < 0.037 in both cases). No treatments were significantly different to WWH or WWS (p > 0.084 in all cases). IL-12(p70) is a potent Thl inducing cytokine with a key role in inducing the synthesis of interferon gamma (IFN-y) in response to infections (Sam and Stevenson, Clin. Exp. Ummunol., 117, 2, 343 (1999)). Mann et al. (Am. J. Physiology - Lung Cellular and Mol. Physiology, 322, 5, L683 (2022)) previously reported that HDM exposure had no impact on levels of I L-2(p70).

• Granulocyte colony-stimulating factor (G-CSF)

There were no distinct effects of HDM or ERME (TM) on G-CSF. EES mice had significantly lower levels of G-CSF compared with WEH (p = 0.049), and there was a trend towards lower levels of G-CSF in EES mice compared with other HDM treated groups (p < 0.092 in all cases). G-CSF is important in mouse models of neutrophilic asthma (Kim et al., Europ. Respiratory J., 55, 2, 1900827 (2020)), so it is not surprising there are few effects in this eosinophilic dominated model.

• Interferon gamma (IFNy)

There was a weak effect of ERME (TM) treatment on IFNy, with WES mice having significantly higher levels than EES and EEH mice (p < 0.048 in both cases). There was a trend towards significance for WEH mice also having higher levels of IFNy compared with EEH and EES (p < 0.062 in both cases). IFNy plays critical roles in innate and adaptive immunity against pathogenic infections (viruses, bacteria, etc). It has been shown to be associated with aryl hydrocarbon receptor (AHR) in humans, and to also have beneficial effects on mucin secretion (Naumov et al., Europ. Respiratory J., 54, Suppl. 63, PA4378 (2019)). In two previous mouse models of HDM-induced allergic airways disease, IFNy was not detected in bronchoalveolar lavage (BAL) (Gregory et al. (Clin. & Exp. Allergy, 39, 10, 1597 (2009)), and Malaviya et al. (Pharmacology Res. & Persp., 9, 3, e00770 (2021)), however it was elevated post-HDM exposure when measured in lung homogenates (Malaviya et al. (2021) (supra). Mann et al. (Am. J. Physiology - Lung Cellular and Mol. Physiology, 322, 5, L683 (2022)) previously reported that HDM exposure had no impact on levels of IFNy. Monocyte chemoattractant protein 1 (MCP-1)

There were complex effects of HDM and ERME (TM) treatment on MCP-1. EEH mice had significantly more MCP-1 than all other treatments (p < 0.004 in all cases). WWH mice had significantly higher levels of MCP-1 than all other treatments except EWH (p < 0.006 in all case) and EEH. Other HDM treated groups had significantly higher amounts of MCP-1 than their treatment controls (p < 0.001 in both cases), except for EWH vs EWS (p = 0.073) and WEH vs WES (p = 0.161). WEH mice did not have significantly higher MCP-1 than WWS mice (p = 0.054). MCP-1 is a potent macrophage/monocyte chemoattractant (Deshmane et al., J. Interferon Cytokine Res., 29, 6, 313 (2009)) that has been shown to be increased in mouse BAL after HDM exposure (Malaviya et al. (Pharmacology Res. & Persp., 9, 3, e00770 (2021)), however in another study using a very similar model, no increase in MCP-1 post HDM exposure was observed (Mann et al., Am. J. Physiology - Lung Cellular and Mol. Physiology, 322, 5, L683 (2022)).

• Macrophage inflammatory protein la (MIP-la)

There was a strong effect of HDM on MIP-la, and also an effect of ERME (TM). HDM treated groups had significantly higher levels of MIP-la than saline treated groups (p < 0.044 in all cases) except for EEH mice which only had significantly more MIP-la than EES mice (p = 0.039). MIP-la is produced by a range of cells (particularly macrophages) after they are stimulated with an insult, and it is important in the inflammatory host response via its role in recruiting pro-inflammatory cells (Maurer and von Stebut, Int. J. Biochem. & Cell Biol., 36, 10, 1882 (2004)). It has been previously shown to be significantly increased post HDM exposure in mice (Ulrich et al. (Pulmonary Pharmacol & Thera., 21, 4, 637 (2008)) and Mann et al. (Am. J. Physiology - Lung Cellular and Mol. Physiology, 322, 5, L683 (2022))).

• Macrophage inflammatory protein 16 (MIP-16)

There were statistically significant effects of both HDM and ERME (TM) on levels of MIP-ip. All HDM treated groups had significantly higher levels of MIP-ip than their treatment controls (p < 0.049 in all cases). EEH mice had significantly higher MIP-ip than all other treatments (p < 0.011 in all cases), except for WWH (p = 0.071). EES mice had significantly lower MIP-ip than all treatments (p < 0.032 in all cases), except for EWS and WWS (p > 0.057 in both cases). MIP-1 has similar functions to MIP-la (Maurer and von Stebut, Int. J. Biochem. & Cell Biol., 36, 10, 1882 (2004)), and is known to be increased in the bronchoalveolar lavage (BAL) of HDM treated mice (Mann et al., Am. J. Physiology - Lung Cellular and Mol. Physiology, 322, 5, L683 (2022)).

• Tumour necrosis factor a (TN Fa)

There was a treatment effect on TNFa, in that WEH mice had significantly higher levels of TNFa than all other treatments (p < 0.042 in all cases), except for WWH and WES (which were both borderline significant at p = 0.074 and p = 0.054 respectively). There was no defined effect of HDM treatment. TNFa is a cytokine and adipokine with a wide range of functions produced by a range of cells including macrophages and mast cells. It is important in innate immunity (Berry et al., Curr. Opin. Pharmacol., 7, 3, 279 (2007)) and it is a potent chemoattractant for neutrophils and eosinophils (Lukacs et al., J. Immunol., 154, 10, 5411 (1995)). It was previously shown that there is no effect of HDM exposure on TNFa (Mann et al., Am. J. Physiology - Lung Cellular and Mol. Physiology, 322, 5, L683 (2022)), however it has been measured in mouse lung homogenates post HDM exposure (Malaviya et al. (Pharmacology Res. & Persp., 9, 3, e00770 (2021)).

No effect of ERME (TM) or HDM (Figure 11)

There were no significant differences between treatments for four of the measured mediators: interleukin-la (IL-la; p > 0.098 in all cases), interleukin-6 (IL-6; p > 0.132 in all cases), interleukin-9 (IL-9; p > 0.103 in all cases) and interleukin-17A (IL-17A; p > 0.191 in all cases). In a previous study using a very similar model, HDM exposure did not result in significant changes in IL-la, IL-9 or IL-17A, but IL-6 was increased (Mann et al., Am. J. Physiology - Lung Cellular and Mol. Physiology, 322, 5, L683 (2022)). In other previous studies of HDM-induced allergic airways disease using similar models, IL-la and IL-17A were increased in lung homogenates, but not in bronchoalveolar lavage (Malaviya et al. (Pharmacology Res. & Persp., 9, 3, e00770 (2021)). IL-6 and IL-9 are implicated in allergic asthma (Kearley et al. (Am . J. Resp. and Crit. Care Med., 183, 7, 865 (2011)), and Gubernatorova et al. (Frontiers in Immunol., 9, Article 2718 (2018))), so it is surprising that an effect was not observed.

Total serum IgE Total IgE was measured (BioLegend ELISA MAX™ Deluxe Set Mouse IgE). These data were box-cox transformed to satisfy the assumptions of ANOVA. There was a significant effect of HDM exposure on total IgE (p < 0.001), with HDM treated mice having significantly higher levels of serum IgE (Figure 12). There was no overall effect of ERME (TM) (p = 0.100) and no significant interaction (p = 0.588). Post-hoc analyses indicated that serum IgE was not significantly different between WEH and WES mice (p = 0.021), but all other HDM treated groups had significantly more IgE than their commensurate controls (EEH vs EES p = 0.01, WWH vs WWS p < 0.001, EWH vs EWS p = 0.021), indicating that ERME (TM) treatment during the HDM sensitisation period reduces the IgE allergic response. Of note, serum IgE in WEH treated mice was not significantly lower than that of WWH treated mice (p = 0.217).

This study aimed to assess the efficacy of oral ERME (TM) administration on ameliorating disease induced in an allergic airways disease (asthma) model in mice. Mice were provided with ERME (TM) in their drinking water before and/or during house dust mite sensitisation, and a range of respiratory outcomes assessed. Key conclusions include:

1. ERME (TM) at a dose of about 2.5mg/kg was well tolerated by mice with no adverse clinical outcomes observed. ERME (TM) also appeared to be attractive to the mice in that they consumed slightly higher volumes of water containing ERME (TM) compared to water with no ERME (TM) added.

2. The house dust mite induced allergic airways disease model functioned as expected, with HDM exposed mice exhibiting altered lung function at functional residual capacity (FRC), increased airway-hyperresponsiveness, predominantly eosinophilic airways inflammation, increased serum IgE and increased levels of several relevant "allergic" mediators such as IL-5, IL-13, eotaxin and others.

3. The cross-over study design proved to be beneficial in elucidating the non-dose dependent effects of ERME (TM) on HDM-induced disease. ERME (TM) given during HDM sensitisation (WEH) had the largest effect on modulating measures of disease severity, followed by pre-treatment (EWH). Pre-treatment combined with concurrent treatment (EEH) appeared to elicit no positive effect on reducing disease, and in some cases, it exacerbated HDM-induced disease (e.g. EEH mice were the most responsive to MCh for tissue damping and tissue elastance).

4. The beneficial effects of concurrent ERME (TM) (WEH) were consistently seen across a range of lung function parameters, including airway resistance at functional residual capacity (FRC) (Figure 3A), absolute airway resistance, tissue damping and tissue elastance at the maximum dose of MCh (Figure 4A-C), percent increase in airway resistance and tissue damping (Figure 5A and B) and sensitivity to MCh (Figure 6).

5. The beneficial effects of WEH were also associated with reduced neutrophil numbers in bronchoalveolar lavage (Figure 8B), but not with other cell populations.

6. There were also some synergies between improved respiratory function in WEH mice and levels of mediators in bronchoalveolar lavage (BAL) (particularly IL-2, IL-4, MCP-1 and TNFa (Figure 10), indicating some immunomodulatory effects of WEH.

7. Concurrent ERME (TM)/HDM exposure (WEH) was also the only ERME (TM)/HDM treatment not associated with increased total serum IgE (Figure 12). Although WEH mice did not have significantly less serum IgE than WWH controls, this may be indicative of reduced sensitisation to HDM in WEH treated mice.

Combined, these data indicate that the WEH treatment elicited a range of effects in this model consistent with modified immune and inflammatory responses to concurrent HDM exposure. These changes were associated with altered/improved physiological function.

Claims

1. A malt extract for use in preventing or treating asthma or at least one of the symptoms of asthma in a mammal or avian, the malt extract comprising one or more active enzymes selected from the group consisting of amylases, maltases, cellulases, fructanases, glucanases, xylanases and deacetylases.

2. A malt extract for use in preventing or treating asthma or at least one of the symptoms of asthma in a mammal or avian according to claim 1, wherein the at least one of the symptoms of asthma is selected from the group consisting of shortness of breath, chest tightness or pain, coughing and wheezing.

3. A malt extract for use in preventing or treating asthma or at least one of the symptoms of asthma in a mammal or avian according to claim 1 or claim 2, wherein daily dosage is in the range 0.02-2, 0.05-2, 0.1-1, 0.25-0.75 ml per kg body weight mammal or avian.

4. A malt extract for use in preventing or treating asthma or at least one of the symptoms of asthma in a mammal or avian according to any one of the preceding claims, wherein the malt extract additionally comprises one or more proteinase and/or lipase.

5. A malt extract for use in preventing or treating asthma or at least one of the symptoms of asthma in a mammal or avian according to any one of the preceding claims, wherein the malt extract additionally comprises one or more water soluble sugars selected from the group consisting of maltose, maltotriose, and maltose polymers.

6. A malt extract for use in preventing or treating asthma or at least one of the symptoms of asthma in a mammal or avian according to any one of the preceding claims, wherein the malt extract is based on one of the seeds selected from the group consisting of barley, wheat, triticale, sorghum, maize, buckwheat, rice and a mixture thereof. A malt extract for use in preventing or treating asthma or at least one of the symptoms of asthma in a mammal or avian according to any one of the preceding claims, wherein the diastatic power of the malt extract is above 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 115 degrees Lintner. A malt extract for use in preventing or treating asthma or at least one of the symptoms of asthma in a mammal or avian according to any one of the preceding claims, wherein the mammal is selected from the group consisting of a human being, horse, cattle, and pig. A malt extract for use in preventing or treating asthma or at least one of the symptoms of asthma in a mammal or avian according to any one of the preceding claims, wherein the avian is selected from the group consisting of Columba livia domestica, Gallus ga II us domestica, turkey, duck, and pheasant.