Oral mucositis represents ulcerative inflammatory changes of oral mucosa due to the toxic effects of chemo- or radiotherapy. It is among the most important challenges in oncology patients’ treatment, especially in head and neck carcinoma [1]. It is initiated by injury of epithelial cells leading to inflammatory changes in the oral mucosa. Depending on the grade, ulcerations can develop and affect the deepest submucosal tissues with microbial infections leading to exacerbation. Among the key aspects of treatment are tissue regeneration, immunomodulation, antimicrobial activity, and pain management [2]. Adequate therapy timing (chronotherapy) could decrease the risk of severe oral mucositis development [3]. Hospital pharmacists and nurses have a crucial role in managing oral mucositis. They implement oral care protocols and treatments and educate patients about self-care at home [4, 5]. Despite the influence of oral mucositis on quality of life and anti-cancer treatment success, prevention and treatment options are scarce [1]. Guidelines combine good oral hygiene, nutrition, and various therapeutics [6]. Palifermin is the only approved drug for the treatment of oral mucositis in hematologic patients or as supportive care for preparative regiments in patients predicted to develop oral mucositis of grade 3 according to the World Health Organization (WHO) Oral Toxicity Scale [7]. It is a recombinant human keratinocyte growth factor 1 which is stimulating the proliferation of epithelial cells [1]. There are many compounded preparations, usually specific for certain clinical sites, that are also used. Benzydamine mouthwashes and oral glutamine [8, 9] together with povidone-iodine, sodium bicarbonate, and natural compounds [10] are often recommended. Photobiomodulation is also a successful and cost-effective approach to the prevention and therapy of oral mucositis [11–13]. However, a standard oral care protocol for oral mucositis is still not available [14].

There are several activities that oral mucositis therapeutics should have. In the first place, anti-inflammatory, antimicrobial, and pro-regenerative activities are beneficial. Also, immunomodulatory effects are needed to overcome exaggerated immune response aggravating oral mucositis conditions. Many therapeutics have pain-reducing effects to prevent decreased food and beverage intake and worsening of patients’ general condition [1]. The natural component of healthy saliva, lysozyme, is among the compounds with several activities beneficial in the treatment of oral mucositis [15, 16]. Eminagić et al. [15] conducted an observational study and demonstrated the advantages of lysozyme-based compounds in the treatment of oral mucositis due to radio- or chemotherapy. Rasic et al. [16] showed that a spray containing lysozyme, cetylpyridinium, and lidocaine is effective and safe in the treatment of oral mucositis. Lysozyme plays a crucial role in innate immunity and has antimicrobial and immunomodulatory effects on the skin and mucous membranes in our body. It kills bacteria mainly by hydrolysis of peptidoglycans or by ionic interactions with the cell wall [17]. Lysozyme also interacts with nucleic acids and possesses antiviral properties [18]. Immunomodulatory effects are partly based on the production of peptidoglycan fragments stimulating immune response [17]. Lysozyme also shows anti-inflammatory effects [19] through interaction with complement and chemotaxis of activated leukocytes [20]. Its anti-nociceptive [19] and analgesic properties are described [21], and studies are showing the pro-regenerative and wound-healing properties of this enzyme [22]. Bianchi showed that lysozyme inhibits the conversion of arachidonic acid into prostaglandins while having no direct effect on the action of prostaglandins themselves [21]. Due to its natural origin and all the mentioned activities, lysozyme could bring added value to oral mucositis treatment protocols. Additional studies are needed to evaluate lysozyme-based medicines in the treatment of oral mucositis.

The aim of this study was to evaluate the effectiveness and safety of lysozyme-based spray compared to mouthwash preparation commonly used in the hospital pharmacy in reducing pain, erythema, and ulceration of the oral cavity and pharynx mucous membranes in patients undergoing head and neck radiotherapy.

A post-marketing, prospective randomized open-label study was conducted at the University Clinical Center of the Republic of Srpska, Banja Luka, Bosnia and Herzegovina from August 2020 until April 2022. The study was registered with the Agency for Medicinal Products and Medical Devices of Bosnia and Herzegovina under the protocol number LCS-OM-01.

The total number of participants was 56, of which 26 were in lysozyme and 30 in the control group. There was no difference between groups regarding age (U = 335, p = 0.370), sex (p = 0.200, Fisher’s Exact Test), and the number of days from the start of radiotherapy to inclusion in the trial with a mean value of 14 days [t (54) = 0.359, p = 0.721]. Also, there were similar characteristics of patients regarding the tumor site and Tumor, Node, Metastasis (TNM) classification (Table 1).

The efficacy parameters evaluated by medical doctors were similar between the groups. Both treatments decreased the number of patients with local findings after 21 days compared to the baseline (Table 2). Local findings at the palate were the most common, followed by local findings at cheeks and tongue. Local findings at lips were present in a small number of patients (Table 2). A Friedman test was performed to determine whether there were differences in local findings for the lips, cheeks, tongue, and palate over time for each group individually. No significant difference and no deterioration of symptoms within both groups was found.

After 21 days, pain was present mainly when eating solid food, while a small number of patients experienced pain when eating soft food or speaking. A significantly lower pain intensity when eating solid food after 21 days was observed in the lysozyme group compared to the control group. Also, the median intensity of pain when eating soft food or speaking after 21 days was lower in lysozyme compared to the control group, but these differences were not statistically significant (Figure 1, Table 3).

Most of the subjects had palatal sensitivity recorded throughout the entire test period. In the control group, 21 days after the start of therapy, ulcerations developed in 2 subjects, while in the lysozyme group, one subject had a normal palate with no changes (Table 3). During the study, no adverse reactions to applied treatments were observed.

Both groups (lysozyme and control) achieved proper oral mucositis management with no significant deterioration of oral mucosa condition during 21 days of radiotherapy. The efficacy of both therapies was evident as a slight decrease in the oral mucositis grade at the palate, cheeks, tongue, or lips. The results were not statistically significant indicating mild effects towards healing but also activity against further development of oral mucositis. Also, the presence and intensity of pain did not significantly change during the follow-up. It is important to point out that in the lysozyme group, the percentage of patients with pain when eating solid food increased but the pain intensity decreased during the 21 days of follow-up, while the opposite results were seen in the control group. In the lysozyme group, the percentage of patients with pain during soft food eating decreased but the pain intensity increased from two to three, while in the control group, the percentage of patients with pain remained the same, but the pain intensity increased from three to six. While differences were not statistically significant, clinical significance should be considered. However, measurement of the minimum clinically important difference (MCID) is very challenging when different types and intensities of pain are analyzed [27]. The only statistically significant difference between groups was determined for decreased pain intensity when eating solid food after 21 days of follow-up in lysozyme compared to the control group. Although the control group received a higher dose of lidocaine compared to the lysozyme group, a better reduction of pain in the lysozyme group could be due to the direct effects of lysozyme. Hen egg white lysozyme effects on pain were investigated in mice where the alleviation of static mechanical pain was observed. Lysozyme caused epigenetic changes and influenced the NRF1-Parkin-TACAN signaling axis in sensory neurons [28]. Also, antimicrobial [29] and immu-nomodulatory [30] effects of lysozyme could lead to decreased pain due to inflammation. Additional studies are needed to confirm and explain in more detail the obtained results.

The form of a spray in the lysozyme group could have an advantage compared to mouthwash applied in the control group. When the oropharynx is the therapy target, spray has advantages over oral rinse. Patients need to be educated on appropriate rinsing techniques to achieve the maximum therapeutic potential of mouthwashes [31]. Very often, this education of patients is performed by nurses. The form of spray offers advantages due to easier application and less time needed for the education of patients [31].

This study had several limitations. Different forms of applied therapies (the lysozyme group received therapy in the form of mouth spray while the control group received therapy in the form of mouthwash) could lead to different therapy compliance. However, therapy compliance was not monitored. The small number of participants was based on the sample size calculation. Only one study could be used as a reference, and based on that study, the effect size was large [16]. However, the difference between groups in this study was not observed potentially due to a small sample size. The study was open-label which could lead to bias. Also, the study was conducted at only one clinical center. Another randomized, blinded, multicentric study on a larger number of patients should be performed with the same spray containing lysozyme or without lysozyme to confirm the role of lysozyme in oral mucositis treatment.

In conclusion, the effectiveness and safety of lysozyme-based spray for the treatment and prevention of deterioration of radiotherapy-induced oral mucositis was shown. The availability of new treatment options based on lysozyme, a natural enzybiotic present in the saliva of healthy subjects, could bring added value in treating oral mucositis and preventing its complications. However, a larger randomized, blinded study is needed to confirm our results.