Effects of ectoine containing nasal spray and eye drops on symptoms of seasonal allergic rhinoconjunctivitis

Abstract Background Patients are often dissatisfied with the symptom control obtained from available pharmacological treatments for seasonal allergic rhinoconjunctivitis (ARC). Therefore, patients seek for alternative, nonpharmacological options to treat their symptoms. Here, we assessed the efficacy of ectoine nasal spray and ectoine eye drops in comparison to placebo to prevent nasal and ocular symptoms following exposure to pollen in patients with ARC. Methods In this double‐blind, randomized, placebo‐controlled, cross‐over study, 46 patients with ARC applied ectoine eye drops and nasal spray in immediate succession or placebo eye drops and nasal spray for 13 days before ARC symptoms were induced in an environmental exposure chamber. Primary endpoint was the baseline‐adjusted area under the curve (AUC) posttreatment total nasal symptom score (TNSS) and the total ocular symptom score (TOSS) using analysis of covariance. Secondary endpoints were, amongst others, total nonnasal symptoms score (TNNSS) and nasal patency (measured using acoustic rhinometry). Results Treatment with both ectoine and placebo reduced TNSS, TOSS, and TNNSS upon allergen exposure. The analysis of parameters at baseline and after allergen exposure demonstrated that ectoine induced a clinically relevant improvement in ARC symptoms compared to placebo: the least square mean difference for baseline‐adjusted AUC was ‒1.87 for TNSS, ‒1.45 for TOSS and ‒2.20 for TNNSS. The mean change from baseline AUC of TNNSS for ectoine was also significantly greater than for placebo (‒5.49 vs. ‒3.46; p = 0.011). Ectoine significantly improved the singular symptoms “sneezing,” “watery eyes” and “itchy eyes” (p ≤ 0.021) as well as “itchy ear/palate” (p = 0.036) in comparison to placebo. Mean cross sectional areas of the nasal cavity were reduced to a lesser extent after treatment with ectoine (‒0.020 ± 0.022) than with placebo (‒0.047 ± 0.029). The current study also demonstrated a very good safety profile of ectoine treatment. Few AEs with comparable numbers in both treatment groups were reported during the study, which were mild in severity and resolved without medical treatment. Conclusion The study suggests that ectoine is effective in reducing nasal and ocular symptoms associated with ARC. Being a natural, bacteria derived stress protection molecule functioning by a physical mode of action, it therefore represents an alternative nonpharmacological treatment option.


| BACKGROUND
Despite the vast number of drug-based treatments available for seasonal allergic rhinoconjunctivitis (ARC), 1 patient dissatisfaction is still an issue. This is partially due to the available medications which do not control the signs and symptoms adequately enough to match the patients' needs, [2][3][4][5] or because the drugs can induce deleterious side effects. [6][7][8][9] Thus, there is medical need for alternative treatment options.
Most of these alternatives are nonpharmacological treatment options used by the patients as shown in previous literature. [10][11][12][13] The extremolyte ectoine is one of these prospective, alternative, nonpharmacological treatment options. It is an osmolyte of low molecular weight which is generated by extremophilic bacteria and protects biological molecules from external influences such as extreme temperature, pressure, salt concentration, and ultraviolet radiation. Ectoine protects macromolecules (like membranes and proteins) via an entropy-driven, cosmotropic physical mechanism. 14,15 This leads to the stabilization of the native form of proteins, as well as an increase in the fluidity of lipid membranes, 16,17 thus resulting in the increased stability of the membrane barrier to various stressors. 18,19 Ectoine reduces the inflammatory process at the membrane level, as was shown in different preclinical models. [20][21][22] Ectoine is currently used in topical applications for skin diseases such as atopic dermatitis. 20,23 Ectoine nasal spray (ENS) has also been studied in patients with acute rhinosinusitis. 24 To evaluate the general efficacy and safety of ENS for treating allergic rhinitis or rhinoconjunctivitis, several clinical trials have assessed it compared to antihistamines, cromoglycate, and to glucocorticoids. Thus, in a comparative, open-label study, 25 a nasal spray containing ectoine was as effective as cromoglycate for relieving rhinoconjunctivitis symptoms and-because of virtually no side effects-was tolerated significantly better by the patients. Additionally, the ENS used in conjunction with ophthalmic drops containing ectoine were as effective as the corresponding azelastine products in relieving allergic symptoms during the pollen season. 25 In an open randomized study with children, the use of nasal ectoine in combination with oral antihistamines reduced significantly faster the severity of the rhinitis symptoms nasal congestion, nasal discharge, nasal irritation, and sneezing, as well as conjunctivitis symptoms like itchy eyes and conjunctival hyperemia in comparison to oral antihistamines alone. 26 However, the objective measurement of allergy-related symptoms remains a challenge. To overcome this, the present study used a validated environmental exposure chamber (EEC) model which is capable of examining anti-allergic treatments by mimicking a natural, yet controlled airborne allergen exposure. Thus, confounding variables such as unpredictable atmospheric pollen levels due to erratic weather conditions with climate changes seen in traditional field trials can be eliminated.
In this proof-of-concept study, we aimed to assess the extent of relief of rhinoconjunctivitis symptoms through cotreatment with both nasal and ocular applications of ectoine compared to placebo.

| Approvals and ethics
The study protocol and informed consent form were approved by

| Study design
This was a single center, double-blind, placebo-controlled, two-way crossover study, which was conducted outside of the ragweed pollen

| Study assessments
Patients underwent a 3-h stay in an EEC with exposure to 3500 ± 500 ragweed pollen grains/m 3 . Rhinoconjunctivitis symptoms were assessed before and after entry to the EEC. In addition, symptoms were also evaluated every 30 min during the EEC sessions.
The scores for the symptoms of runny nose, itchy nose, nasal congestion, and sneezing were summed to obtain the TNSS. The TOSS was the sum of itchy, red, and watery eye symptoms. The total nonnasal symptom score (TNNSS), comprising the ocular symptoms from the TOSS as well as itchy ear/palate was also collected. For grading of all symptoms, a 4-point scale was used with 0 = none, 1 = mild, 2 = definite awareness of sign/symptom that is bothersome but tolerable and 3 = sign/symptom is hard to tolerate.
In addition, acoustic rhinometry (AcR) tests were performed to assess nasal patency before and after each EEC session (pre-EEC and post-EEC, respectively) and the mean cross-sectional nasal areas (MCAs) were determined. For further details on AcR and MCA procedure, please refer to File S1. AcR was carried out by trained operators. Measurements were performed in triplicate, and-where possible-the same equipment and operator were used for the entire study duration for each patient.

| Study objectives and endpoints
The primary objective was the assessment of the efficacy of ENS and  with the clinical study protocol definition of analysis sets, those patients were excluded from the intent-to-treat (ITT) population as they did not participate in any posttreatment efficacy assessment.

| Statistical analysis
No patient was excluded because of nonadherence to the treatment. In total, 42 patients completed all posttreatment assessments and had no major protocol deviations. They were constituted to the per protocol (PP) population, which was identical to the ITT population.
Patients' demography and age distribution are shown in Table 1.

| Effect of ectoine on nasal symptoms (TNSS)
Patients receiving ENS and EOD had a 1.89-fold lower TNSS after 3 h of posttreatment EEC exposures than patients treated with placebo, though the TNSS decreased not only after ectoine, but also after placebo treatment when compared to baseline ( Figure 3A).  Figure 3B). This corresponds to a 7.47% greater reduction at the posttreatment visit by the ectoine treatment than in placebo-treated patients. The mean reduction from baseline AUC of TNSS was 1.61-fold greater in patients treated with ectoine when compared to placebo (LSMD: -4.92 vs. -3.05; Figure 3C, Table 2). This difference showed clinically meaningful improvement in the ectoine treatment group in comparison to placebo, but values did not reach statistical significance (p = 0.065).

| Effect of ectoine on ocular symptoms (TOSS)
Similarly  Figure 4C, Table 2). This difference showed both clinically meaningful and statistically significant improvement in the TOSS after ectoine treatment in comparison to placebo treatment.

| Effect of ectoine on single symptoms
When investigating individual nasal symptoms, treatment with ectoine resulted in significantly greater relief of the symptom "sneezing" (p = 0.020; Table 2), whereas the differences in the changes in the other nasal symptoms were not significant.
For individual ocular symptoms, mean change from baseline AUC values were more reduced after ectoine treatment than with placebo, with a statistically significant greater relief for "watery eyes" (p = 0.020) and "itchy eyes" (p = 0.021): The baseline AUC for "watery eyes" was 5.70 at the EEC screening visit, which decreased  Table 2).
The itchy ear/palate symptom of the TNNSS had a treatment LSMD of 0.62, which was also statistically significant (p = 0.036; Table 2).

| DISCUSSION
This study aimed to investigate whether ectoine could alter the natural development of rhinoconjunctivitis signs and symptoms in patients exposed to airborne allergens. We were able to show that  Note: Shown are the data of all randomized patients.
Abbreviations: %, percentage based on N; ENS/EOD, treatment with ectoine nasal spray and eye drops; N, number of subjects randomized; n, number of subjects with data available; PNS/POD, treatment with placebo nasal spray and eye drops; SD, standard deviation. a Subjects who checked two or more races were classified as mixed.

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guidelines for an optimal approach. 1,29,30 According to these guidelines, oral antihistamines or leukotriene antagonists should be administered, as well as topical antihistamines or nasal glucocorticoids to treat allergy symptoms. However, there seems to be still room for improvement in the management of ARC patients because patients still report dissatisfaction with or do not find relief with commonly prescribed antihistamines, or glucocorticoids. This is partially due to the fact that available medications do not adequately reduce the signs and symptoms of rhinoconjunctivitis 2-4 or because they cause unwanted side effects. 6,8,9,31 Generally, patients will not adhere if the efficacy of the therapy is not satisfactory. On the other hand, if reduction in symptoms is not sufficient, this may-at least partly-be due to nonadherence of patients to the treatment. This is also the case for unwanted side effects as their occurrence can also result in nonadherence to the treatment. This is a double-edged issue: either the preparation does not work because it is not taken, or it does not work sufficiently, which is why it is not taken.
Because   The reduction in symptom scores following ectoine treatment is comparable with pharmacological therapies. This was shown in two noninterventional studies comparing the effectiveness of ENS and eye drops to azelastine eye drops and cromoglycic acid nasal spray. 25 In the aforementioned study, the TNSS (as assessed by the physician) was reduced by 58.85% in the ectoine group, thus being similar to the 57.11% in the cromoglycic acid group. TOSS decreased by 45.96% in ectoine treated patients and by 44.98% in patients treated with cromoglycic acid nasal spray. 25 This was also shown in a metaanalysis comparing ectoine with different pharmacological treatments. 35 These studies were all designed to treat currently present symptoms in contrast to the study presented here, applying a prophylactic approach. However, so far only a few noninterventional studies analyzed the effect of ectoine treatment. Therefore, further studies (with better evidence) are needed to substantiate the efficacy of ectoine.
Recently, Patel et al. 36 conducted a study similar to the study presented here to test the effect of a combined olopatadinemometasone nasal spray (GSP301) and other treatments (olopatadine) in an EEC by analyzing the LSMDs of the different treatments.
They showed that treatment with olopatadine improved instantaneous nasal symptoms in comparison to placebo with an LSMD of -0.81, which is much less than the LSMD for ectoine presented here.
However, the other treatments tested in the study had all higher Salapatek et al. 37 also conducted a study with mometasone furoate nasal spray as pre-treatment before EEC exposure. The reduction of TNSS was similar to the reduction in the current study (data not shown), demonstrating that ectoine exerts equivalent effects in comparison to pharmacological treatment. The approach of treating eyes and nose simultaneously is based on the fact that over half of the allergy sufferers experience conjunctivitis symptoms in addition to their rhinitis symptoms when exposed to allergens. 38,39 This is due to two mechanisms: allergen introduced in the eyes drains with the tears on a direct way into the nose via the nasolacrimal duct and vice versa via indirect reflex connections from the nose to the eyes. 40 clearing of allergens with saline solutions from the nose and eyes have been shown to provide temporary relief of symptoms. 43 Importantly, the magnitude of the placebo effect seen in this study was at the lower end of the range compared to that seen in other studies of pharmaceuticals, which have reported placebo effects up to 40%. 44 The EEC is an appropriate system to study allergic patients, since it provides a much more controlled environment than a real-life situation. For example, it is difficult to assess the efficacy during a season with a poor pollen flight. However, harsher environmental situations/changes, like simultaneous exposure do different allergens, cannot be done in an EEC. Therefore, conducting studies in a real-life setting would be an interesting approach to further analyze the efficacy of ectoine.