Treatment of oral mucositis induced by cancer therapy

Abstract

Provided are Streptococcus salivarius and compositions comprising Streptococcus salivarius for the treatment of ENT diseases, in particular cancer therapy-induced oral mucositis and respiratory tract infections associated with cancer therapy.

Description

Technical Field

[0001] This invention relates to *Streptococcus salivarius* for the treatment and prevention of ear, nose, and throat (ENT) diseases. Streptococcus salivarius The invention relates to the use of *Streptococcus salivarius* during or after cancer therapy for maintaining healthy oral mucosa, and to *Streptococcus salivarius* for treating and preventing respiratory infections in immunocompromised patients. Background Technology

[0002] Cancer is a global health problem, causing one in six deaths worldwide. In 2020, an estimated 19.3 million new cancer cases and approximately 10 million cancer deaths occurred globally.

[0003] Cancer treatments can be categorized into conventional (traditional) strategies and advanced, novel, or modern strategies. Currently, over half of all ongoing medical treatment trials focus on cancer. Specific factors, such as cancer type, location, and severity, guide the selection of treatment plans and the treatment process. The most widely used traditional treatments include surgery, chemotherapy, and radiotherapy. Chemotherapy, whether used alone or in combination with radiotherapy, is considered the most effective and widely used method for treating cancer. Modern treatments include hormone therapy, anti-angiogenesis, stem cell therapy (including hematopoietic stem cell transplantation (HSCT)), immunotherapy, and dendritic cell-based immunotherapy (Debela). et al. , SAGE OpenMed. 9 (2021), doi: 10.1177 / 20503121211034366).

[0004] Chemotherapy and radiotherapy are widely used non-surgical cancer treatments that can prolong survival or even completely cure the disease. However, they can cause a variety of toxic side effects, including oral mucosal disorders, which can severely impact quality of life. The most widely observed disorder is oral mucositis. Approximately 40% of patients receiving chemotherapy develop oral mucositis; for patients with head and neck cancer (HNC) receiving combined chemotherapy and radiotherapy, this percentage rises to approximately 90% (Kusiak). et al ., Int JEnviron Res Public Health. 17 (2020), 2464; Pulito et al., J Exp Clin CancerRes 9 (2020), 210).

[0005] Allogeneic HSCT is considered the most effective anti-leukemia therapy after remission in adults with acute lymphoblastic leukemia. However, it is often accompanied by acute oral complications, including mucositis, local and systemic infections, especially in the ear, nose, and throat (ENT), dry mouth, and taste changes (Haverman olfaction). et al. Mediators Inflamm. (2014), 378281). In particular, 85% of patients undergoing HSCT followed by intensive chemotherapy developed oral mucositis; and when patients received total body irradiation (TBI) combined with cyclophosphamide chemotherapy, the incidence of severe oral mucositis (≥ grade 3) was 100%.

[0006] Therefore, oral mucositis (OM) is the most common oral complication in patients undergoing cancer treatment. OM is characterized by inflammation and damage to the oral mucosa. OM can lead to erythema, ulceration, pain, dysphagia, and malnutrition, severely impacting patients' quality of life and potentially disrupting cancer treatment. Severe oral mucositis may lead to unplanned hospitalizations or even treatment adjustments, including pausing radiotherapy or reducing chemotherapy dosages, thereby adversely affecting treatment outcomes.

[0007] To better understand omnia and explore its prevention and treatment, the mechanisms of this disease have been extensively studied. Researchers have found that omnia is far more complex than initially thought. Studies have revealed that the development of mucositis is a dynamic process, typically divided into five stages: initiation, primary damage response, signal amplification, ulceration, and healing. In the initiation stage, cancer treatments such as chemotherapy or radiotherapy cause DNA and non-DNA damage to basal epithelial cells, triggering a cascade of reactive oxygen species (ROS) that directly damage cells, tissues, and blood vessels. In the primary damage response stage, mucositis is driven by three pathways activated by the ROS cascade: the NF-κB pathway, the ceramide pathway, and the matrix metalloproteinase pathway. In the signal amplification stage, a series of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, further damage basal epithelial cells through positive feedback from the three pathways. In the ulceration stage, the disruption of mucosal integrity promotes bacterial colonization; bacterial colonization stimulates macrophages to produce more pro-inflammatory cytokines and exacerbates tissue damage. This stage causes severe pain in patients. Finally, during the healing phase, signals from the submucosal extracellular matrix and mesenchyme promote cell proliferation and differentiation, thereby rebuilding the mucosal barrier (Shu et al. , Oral Oncology 102 (2020), 104559).

[0008] Despite a well-studied mechanism, the management of oral malformation (OM) remains challenging. According to the Clinical Practice Guidelines of the Multinational Association of Supportive Care in Cancer / The International Society of Oral Oncology (MASCC / ISOO), currently recommended interventions include basic oral care, growth factors and cytokines, anti-inflammatory agents, cryotherapy, and low-energy laser therapy. However, currently only palifermin (keratinocyte growth factor-1) is approved by the US Food and Drug Administration and the European Medicines Agency (EMA) for mitigating OM in at-risk populations, and its applicability is very limited (Shu). et al. , Oral Oncology 102 (2020), 104559).

[0009] Therefore, other strategies for treating oral mucositis are of vital importance to the health of patients during cancer treatment and to successful treatment outcomes. Summary of the Invention

[0010] This invention generally relates to methods of using *Streptococcus salivarius* for the treatment or prevention of ear, nose, and throat (ENT) diseases induced or associated with cancer therapy, particularly oral mucositis (OM) induced by cancer therapy or respiratory tract infections (RTi) associated with cancer therapy, wherein the method comprises administering the probiotic microorganism *Streptococcus salivarius* to a subject in need. This invention is based on an unexpected discovery that treatment with the probiotic microorganism *Streptococcus salivarius* K12 before, during, and / or after cancer therapy in cancer patients receiving radiotherapy and concurrent chemoradiotherapy (CCRT) or undergoing HSCT can prevent, alleviate, and improve OM in those patients; and that administration of the probiotic microorganism *Streptococcus salivarius* K12 to patients receiving cancer therapy is safe and even reduces the incidence of respiratory tract infections in this patient population.

[0011] To date, studies have reported on probiotics, such as Lactobacillus brevis (Lactobacillus brevis). Lactobacillus brevis Lactobacillus () Lactobacillus lactis ), and Bifidobacterium longum ( Bifidobacterium longum Lactobacillus and Enterococcus faecalis Enterococcus faeciumA mixture of these compounds has the potential to treat OM induced by cancer therapy, but the results are inconsistent and therefore the reliability is insufficient; see review Shu. et al. (Oral Oncology 102 (2020), 104559). Furthermore, the overall mechanism of protective effects of probiotics against various diseases remains unclear.

[0012] Experiments conducted within the scope of this invention unexpectedly revealed that when administered to patients receiving CCRT as shown in Example 1 or patients undergoing HSCT as shown in Example 2, the probiotic strain Streptococcus salivarius K12 had a significant therapeutic effect on the occurrence of OM and alleviated OM symptoms.

[0013] In particular, such as Figure 1 As shown and described in detail in Example 1, during a 7-week CCRT period, patients receiving the oral probiotic *Streptococcus salivarius* K12 had a reduced incidence of radiation-induced oral mucositis (RIOM) with an RTOG (Radiation Therapy Oncology Group) score ≥2. Furthermore, >60% of patients with RIOM ≥2 experienced a 3-week delay in onset; and compared to patients who did not take the probiotic, those who took the oropharyngeal probiotic were completely protected from severe RIOM classified as RTOG 3 and 4, where an RTOG score of 4 is classified as life-threatening and includes ulceration, bleeding, or necrosis. In contrast, an RTOG score of only 2 was classified as moderate.

[0014] Omniform lesions (OMs) in patients receiving CCRT are classified as RIOMs because, compared to OMs (CIOMs) caused by chemotherapy alone, radiation, in addition to its cytotoxic effects, also produces additional necrosis and inflammation of the oral mucosa.

[0015] like Figure 6 As shown and described in detail in Example 2, during a 130-day study in HSCT patients (HSCT is a therapy used for various malignant and non-malignant diseases that requires prior chemotherapy for pre-transplant myeloablation), the incidence of OM grade ≥ 2 (WHO classification criteria) was reduced in patients receiving the oropharyngeal probiotic *Streptococcus salivarius* K12 compared to the control group that did not receive the probiotic. Notably, no ulcerative OM was observed in patients in the probiotic group during the 100-day oropharyngeal probiotic intervention period. Therefore, thanks to the experiments conducted according to the present invention, a probiotic-based therapy that is effective for treating RIOM and OM induced by HSCT and CIOM induced by prior chemotherapy (CIOM) has been established for the first time in human subjects.

[0016] During the studies described in Examples 1 and 2, the occurrence of adverse events (adverse reactions) was also monitored, and no adverse events were reported in either study. Therefore, *Streptococcus salivarius* was considered safe in patients who had received radiotherapy and those who had undergone HSCT. This is of particular importance because patients receiving anticancer therapy are in an immunosuppressed state, thus requiring additional caution and a comprehensive safety evaluation before administering probiotics. Accordingly, proof-of-concept in clinical trials is essential for the approval of a probiotic therapy for human use, especially in individuals with compromised immune function, such as those undergoing cancer treatment or other immunosuppressive drug therapy.

[0017] Therefore, the human studies conducted according to this invention represent an important step toward the approval of the probiotic strain *Streptococcus salivarius* for the treatment of OM induced by cancer therapy.

[0018] Further mouse studies were conducted to visualize the effects of chemotherapeutic agents and *Streptococcus salivarius* on oral epithelial cells to monitor the efficacy of *Streptococcus salivarius* in treating chemotherapy-induced OM. Anticancer drugs, specifically chemotherapy (busulin combined with cyclophosphamide (Example 3) and 5-fluorouracil (Example 4)), were administered over a five- to six-day timeframe to mimic repeated administration and the onset of OM in human subjects. This administration method is indeed similar to that used in human patients (unlike radiotherapy, which typically requires administration over several weeks, making it difficult to establish suitable mouse models for studying RIOM), because chemotherapy is usually delivered over a shorter period, where mucosal tissue damage tends to be acute. Chemotherapy-induced OM (CIOM) typically appears within 4–7 days of treatment initiation and peaks within 2 weeks. This timeframe is well observed in mice.

[0019] While CIOM and RIOM share similarities in cellular events, their biological pathways differ slightly; as mentioned above, radiation, in addition to its cytotoxic effects, produces additional necrotic and inflammatory effects on the oral mucosa. Specifically, chemotherapy is administered systemically, while radiotherapy targets specific areas of the body. Furthermore, as noted above, the kinetics of treatment differ, thus influencing the clinical course. Chemotherapy can be delivered over a short period, where damage to mucosal tissues tends to be acute. CIOM typically appears within 4–7 days of treatment initiation and peaks within 2 weeks. Radiotherapy has a more gradual clinical course, as it is most commonly administered in small fractions over several weeks. RIOM typically begins at a cumulative dose of approximately 15 Gy (approximately 10 days later) and usually reaches its peak at 30 Gy, lasting for weeks or even months (Raber-Durlacher). et al ., Oral Oncology 46 (2010) 452-456).

[0020] As described in Example 3, and as Figure 10 , 11 As shown in Figure 12, *Streptococcus salivarius* K12 promotes the healing of CIOM in mice. Specifically, chemotherapy treatment in mice resulted in significant mucosal hypoplasia and ulceration of the tongue. Furthermore, the basal layer cells were loosely arranged, and nuclear pyknosis—the irreversible condensation of chromatin within the cell nucleus during necrosis or apoptosis—was observed. Additionally, the tongue tissue had fewer cells in the stratum spinosum and stratum granulosum compared to healthy tissue; see also... Figure 10 A and 10B. Treatment with *Streptococcus salivarius* K12 during chemotherapy restored the integrity of the tongue mucosa and partially restored the basal, spinous, and granular layers; see also Figure 10 C. Further observation revealed that chemotherapy reduced mucosal thickness by nearly 50%, while the reduction was smaller, approximately 25%, when salivarius K12 was administered during chemotherapy; see also Figure 11 A to 11C and Figure 12 The same phenomenon was observed after mice were treated with 5-Fu. (See Example 4 and...) Figure 17 As shown, 5-Fu treatment reduced the oral mucosal area of ​​mice, while treatment with Streptococcus salivarius K12 significantly increased it.

[0021] Based on these experiments, the inventors hypothesized that inactivated *Streptococcus salivarius* K12 might have beneficial effects on the prevention and treatment of oral ulcers (OM). This hypothesis is based on the fact that inactivated probiotics have shown reduced systemic inflammation while preserving the cellular and molecular biological response characteristics of live probiotics (WO 2008 / 106373 A1). Furthermore, a study confirmed that heat-inactivated *Lactococcus salivarius* CECT 5713 prevents *Streptococcus mutans* from adhering to hydroxyapatite, and therefore, using the inactivated form may be a strategy to reduce the saliva concentration of this oral pathogen (Sanudo). et al. Archives of Oral Biology 84 (2017), 58-63). Regarding *Streptococcus salivarius* (… S. salivarius It has also been shown that its cell wall includes antigenic compounds (Montague and Knox, J Gen Microbiol. 54 (1986), 237-246; Weerkamp and Jacobs, Infection and Immunity 38 (1982), 233-242), and thus the inventors have developed the present invention, namely, that the cell wall components are sufficient to elicit an immune response. This completely contradicts the prior art teachings in the field regarding the treatment of oral mucositis. For example, in Wang et alIn Front. Immunol. 12 (2021), 684824, the effect of Streptococcus salivarius on RIOM treatment was attributed to the ability of live Streptococcus salivarius to reconstruct the oral microbiota; and therefore, based on this literature, those skilled in the art would not attempt to use inactivated cells.

[0022] As shown in Example 4, treatment of oral epithelial cells with heat-inactivated Streptococcus salivarius K12 effectively restored their proliferative capacity, which had decreased after 5-FU treatment; see also Figure 16 Furthermore, heat-inactivated Streptococcus salivarius K12 also protects the oral mucosal barrier. For example... Figure 18 As shown, 5-FU treatment reduced the oral mucosal area in mice, while treatment with heat-inactivated Streptococcus salivarius K12 significantly increased it. The same effect was observed with live Streptococcus salivarius K12 (see [link to original text]). Figure 17 This has been demonstrated in clinical studies to be suitable for the treatment of oral mucositis (see, for example, Example 2). Therefore, both live and heat-killed Streptococcus salivarius K12 significantly promote the proliferation of oral mucosal cells, and accordingly both are suitable for the treatment of oral mucositis.

[0023] Accordingly, the present invention also relates to the use of inactivated Streptococcus salivarius K12 in the treatment of oral mucosal diseases, particularly oral mucositis, and most preferably in CIOM.

[0024] Osteoarthritis (OM) is not the only complication that can occur during cancer treatment. Respiratory infections are quite common due to the immunosuppressive effects of anticancer drugs, especially chemotherapy and HSCT. Previous clinical studies in adults and children have demonstrated the efficacy of daily administration of Streptococcus salivarius in reducing respiratory infections and key respiratory pathogens.

[0025] In the study described in Example 2, the effects of *Streptococcus salivarius* K12 on respiratory tract infections in HSCT patients and those with consequently compromised immune systems were analyzed. Results showed that adjuvant therapy with *Streptococcus salivarius* K12 effectively reduced the incidence of RTI (Respiratory Tract Infection) in patients after HSCT, shortened the duration of respiratory symptoms, and reduced the number of days of antibiotic use. This latter observation is particularly important because antibiotic use in HSCT recipients is controversial, and there have been reports of long-term adverse effects of antibiotic use on overall survival after HSCT.

[0026] Therefore, on the one hand, the safety of Streptococcus salivarius K12 in immunosuppressed patients was first demonstrated during studies conducted within the scope of this invention, and on the other hand, its therapeutic efficacy against respiratory infections in HSCT patients has been demonstrated. Streptococcus salivarius can be used to treat ENT disease induced by cancer therapy, particularly ENT disease induced by chemotherapy and / or radiotherapy, as well as patients undergoing HSCT, and especially immunosuppressed patients. Attached Figure Description

[0027] Figure 1 Oropharyngeal probiotic *Streptococcus salivarius* (ENT-K12) prevents severe recurrent idiopathic ophthalmopathy (RIOM) in nasopharyngeal carcinoma patients during CCRT. The figure shows the mean RTOG score of patients undergoing 7 weeks of CCRT, where RIOM (RTOG=1) onset began in the CCRT group (control group) and the CCRT-P group (probiotic recipient group) during weeks 2 and 3 of the CCRT course, respectively.

[0028] Figure 2 Kaplan-Meier curve analysis of the first episode of RIOM with RTOG grade I in the CCTR group (control group) and the CCRT-P group (receiving the oropharyngeal probiotic Streptococcus salivarius (ENT-K12)). The horizontal axis begins at the start of the CCRT treatment course. Compared with those in the CCRT-P group, the free rate of RIOM with RTOG grade I was observed to decrease significantly faster in the CCRT group (p<0.05). Y-axis: Patients who did not experience RIOM with RTOG grade I (%).

[0029] Figure 3 Kaplan-Mel curve analysis of the first episode of RIOM with RTOG II in the CCTR group (control group) and the CCRT-P group (receiving the oropharyngeal probiotic Streptococcus salivarius (ENT-K12)). The horizontal axis begins at the start of the CCRT treatment course. Compared with those in the CCRT-P group, the incidence of RIOM with RTOG II decreased significantly faster in the CCRT group (p<0.05). Y-axis: Patients who did not experience RIOM with RTOG II (%).

[0030] Figure 4Kaplan-Mel curve analysis of the first episode of RIOM with RTOG grade III in the CCTR group (control group) and the CCRT-P group (receiving the oropharyngeal probiotic Streptococcus salivarius (ENT-K12)). The horizontal axis begins at the start of the CCRT treatment course. Compared with those in the CCRT-P group, the incidence of RIOM with RTOG III was observed to decrease significantly faster in the CCRT group (p<0.05). Y-axis: Patients who did not experience RIOM with RTOG III (%).

[0031] Figure 5 Kaplan-Mel curve analysis of the first episode of RIOM with RTOG grade IV in the CCTR group (control group) and the CCRT-P group (receiving the oropharyngeal probiotic Streptococcus salivarius (ENT-K12)). The horizontal axis begins at the start of the CCRT treatment course. The incidence of RIOM with RTOG IV did not differ between patients in the CCRT and CCRT-P groups (p>0.05), as only one patient in the CCRT group developed RIOM with RTOG IV, while the other nine patients did not. Y-axis: Patients who did not develop RIOM with RTOG IV (%).

[0032] Figure 6 Oropharyngeal probiotic *Streptococcus salivarius* (ENT-K12) for the prevention of severe ophthalmopathy (OM) in subjects undergoing HSCT. This figure shows the mean event-free rate (OM grade ≥ 2 according to WHO grading criteria) in the probiotic group (patients receiving both HSCT and oropharyngeal probiotic ENT-K12) and the control group (patients receiving HSCT but not ENT-K12). During the 130-day study period, the incidence of OM grade ≥ 2 was reduced in patients receiving the oropharyngeal probiotic.

[0033] Figure 7 The probability of no RTi attacks occurring during the 130-day study period (including a 100-day oropharyngeal probiotic intervention and a 30-day follow-up period). Kaplan-Meil analysis showed that the probability of no RTi attacks was consistently higher in the probiotic group than in the control group (p=0.071).

[0034] Figure 8 The cumulative duration of RTi-like symptoms during the 130-day study period (including a 100-day oropharyngeal probiotic intervention and a 30-day follow-up period) was measured. Kaplan-Mell analysis showed that the cumulative duration of RTi-like symptoms was shorter in the probiotic group (p=0.131).

[0035] Figure 9The cumulative duration of RTi-like symptoms during the 130-day study period (including a 100-day oropharyngeal probiotic intervention and a 30-day follow-up period) was assessed. Kaplan-Mell analysis showed that the cumulative duration of RTi-like symptoms observed in the probiotic group was consistently shorter (p=0.131), resulting in a significantly fewer cumulative days of antibiotic use in the probiotic group throughout the study period (p=0.011).

[0036] Figure 10 Representative images of H&E staining of the tongue dorsum show the integrity of the tongue mucosa (80x). A) Control group (untreated mice); B) Chemotherapy group (mice receiving chemotherapy); C) Chemotherapy + ENT-K12 group (mice receiving chemotherapy and the oropharyngeal probiotic Streptococcus salivarius (ENT-K12)). In the chemotherapy group (B), significant mucosal dysplasia and ulceration were observed in the tongue, as well as nuclear pyknosis (irreversible condensation of nuclear chromatin during cell necrosis or apoptosis), loose arrangement of basal cells, and fewer spinous and granular cells in the tongue tissue than in the control group (A). In the chemotherapy + ENT-K12 group (C), the integrity of the tongue mucosa was restored, and the basal, spinous, and granular layers were partially restored.

[0037] Figure 11 Representative images of H&E staining on the dorsum of the tongue, showing mucosal thickness (80x). A) Control group (untreated mice); B) Chemotherapy group (mice receiving chemotherapy); C) Chemotherapy + ENT-K12 group (mice receiving chemotherapy and the oropharyngeal probiotic Streptococcus salivarius (ENT-K12)). Compared with the control group (A), mucosal thickness was reduced by nearly 50% in the chemotherapy group (B); and by approximately 25% in the chemotherapy + ENT-K12 group (C) compared with the control group (A).

[0038] Figure 12 The chart shows the mucosal thickness in a longitudinal section of the mouse tongue. Compared to untreated mice (healthy mucosa), the mucosal thickness in mice receiving chemotherapy was reduced by nearly 50%; while in mice receiving chemotherapy and the oropharyngeal probiotic Streptococcus salivarius (ENT-K12), the reduction was only about 25%.

[0039] Figure 13 Live cell imaging experiments showed that the growth activity of oral epithelial cells (HOK cells) decreased after 48 hours of 5-Fu treatment.

[0040] Figure 14 The proliferation activity of HOK cells was analyzed in a CCK-8 assay, and mitochondrial metabolic concentrations were measured to reflect cell proliferation activity. Treatment with 5-Fu at concentrations >10 μg / mL for 48 hours induced a significant decrease in the proliferation activity of HOK cells (p<0.05).

[0041] Figure 15The proliferative activity of HOK cells was analyzed in a CCK-8 assay. Heat-killed Streptococcus salivarius ENT-K12 did not show proliferative toxicity to HOK cells at any concentration, and treatment with heat-killed Streptococcus salivarius ENT-K12 significantly improved HOK cell viability at concentrations >1.5 mg / mL.

[0042] Figure 16 The proliferative activity of HOK cells was analyzed in a CCK-8 assay. Treatment with 10 μg / mL 5-Fu significantly decreased HOK cell proliferation; however, treatment with heat-killed Streptococcus salivarius ENT-K12 restored proliferation in a dose-dependent manner.

[0043] Figure 17 Tissue sections (AC) and graphs (D) showing the area of ​​the oral mucosal layer demonstrate that 5-Fu has a significant adverse effect on the integrity and permeability of the oral mucosal barrier in mice. Treatment with 5-Fu reduces the area of ​​the oral mucosal layer in mice, while treatment with live Streptococcus salivarius ENT-K12 significantly increases it.

[0044] Figure 18 Tissue sections (AC) and graphs (D) showing the area of ​​the oral mucosal layer demonstrate that 5-Fu has a significant adverse effect on the integrity and permeability of the oral mucosal barrier in mice. Treatment with 5-Fu reduced the area of ​​the oral mucosal layer in mice, while treatment with heat-killed Streptococcus salivarius ENT-K12 significantly increased it.

[0045] Figure 19 Tissue sections (AC) and graphs (D) showing average fluorescence activity indicate that Ki-67 expression in the 5-Fu group was lower than that in the probiotic group (treated with live Streptococcus salivarius ENT-K12) and the control group. Therefore, treatment with live Streptococcus salivarius ENT-K12 significantly promoted the proliferation of oral mucosal cells.

[0046] Figure 20 Tissue sections (AC) and graphs (D) showing average fluorescence activity indicate that Ki-67 expression in the 5-Fu group was lower than that in the probiotic group (treated with heat-killed Streptococcus salivarius ENT-K12) and the control group. Therefore, heat-killed Streptococcus salivarius ENT-K12 treatment significantly promoted the proliferation of oral mucosal cells.

[0047] Figure 21Cell cycle analysis of HOK cells was performed using flow cytometry. (A) Control group; (B) HOK cells treated with heat-killed Streptococcus salivarius ENT-K12; (C) HOK cells treated with 5-FU; (D) HOK cells treated with both 5-FU and heat-killed Streptococcus salivarius ENT-K12. Compared with the control group, treatment with heat-killed Streptococcus salivarius ENT-K12 for 48 hours did not significantly alter the cell cycle of HOK cells. 5-FU treatment led to S phase and G2 / M phase arrest, which are associated with periods of rapid cell growth and protein synthesis during which cells prepare for mitosis and cytokinesis. Heat-killed Streptococcus salivarius ENT-K12 restored the entire cell cycle after 5-FU treatment.

[0048] Figure 22 The reactive oxygen species (ROS) levels in HOK cells were detected by flow cytometry. ROS levels in HOK cells were induced by 5-FU treatment, and significantly decreased after treatment with heat-killed Streptococcus salivarius ENT-K12. The FITC-A value represents the ROS level in HOK cells, reflecting DNA damage during chemotherapy (the higher the value, the higher the ROS level and the more severe the damage). Detailed Implementation

[0049] This invention relates to the treatment of ear, nose, and throat (ENT) diseases in human subjects, wherein the ENT disease is preferably oral mucositis (OM), and the treatment comprises the administration of *Streptococcus salivarius*. In particular, this invention relates to *Streptococcus salivarius* or compositions comprising *Streptococcus salivarius* for the treatment of ENT diseases in human subjects, wherein the ENT disease is ENT disease induced or associated with cancer therapy, preferably, wherein the ENT disease is OM induced by cancer therapy or respiratory tract infection (RTi) associated with cancer therapy. With regard to respiratory tract infections, this invention particularly relates to its treatment in immunosuppressed patients, such as those undergoing HSCT. This invention also relates to the use of inactivated *Streptococcus salivarius* for the treatment of oral mucosal diseases, particularly oral mucositis. Furthermore, this invention relates to the use of *Streptococcus salivarius* or compositions comprising *Streptococcus salivarius* for maintaining the health of the ear, nose, and throat (ENT), preferably the oral mucosa, in human subjects during cancer therapy. Furthermore, this invention also relates to a tablet, particularly a lozenge comprising *Streptococcus salivarius*, for the above-described uses.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of this invention, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Materials, methods, and examples are illustrative only and not limiting.

[0051] Other features and advantages of the invention will become apparent from the following detailed description and claims.

[0052] To avoid any ambiguity, it is emphasized that the use of phrases such as "in some embodiments," "in certain embodiments," "in some cases," "in certain circumstances," "in other embodiments," and "in one embodiment" implies that when reading any of the embodiments described herein, each feature of those embodiments should be taken into account, and this disclosure must be treated in the same manner as if the combination of features of those embodiments were to be detailed in one embodiment. The same applies to any combination of embodiments and features exemplified in the appended claims and embodiments, which are also intended to be combined with the features of the corresponding embodiments disclosed in the specification, where features of embodiments are subordinate only for the sake of consistency and brevity. In practice, each combination of embodiments and features, which can be interpreted due to (multiple) subordinate relationships, must be considered as already explicitly disclosed, rather than as a choice among different options.

[0053] The terms “human subject” and “patient” are used interchangeably in this document.

[0054] The term "treatment" refers to both "therapeutic treatment" and "preventive treatment," and the treatment of ENT disease and the treatment of OM also refer to patients who have not yet developed ENT disease or OM, but are at risk or susceptible to developing such disease. Patients with ENT disease, and especially OM, are, for example, those receiving cancer therapy or those who are otherwise immunosuppressed.

[0055] The terms “cancer treatment,” “anti-cancer therapy,” “cancer therapy,” and “anti-cancer drug” are used interchangeably in this document. Furthermore, cancer therapy can also be used to treat other diseases besides cancer, such as autoimmune diseases. Therefore, patients receiving anti-cancer therapy do not necessarily have cancer, but may alternatively or incidentally have, for example, an autoimmune disease. Accordingly, when referring to cancer therapy-induced OM, the OM can be induced by cancer therapy used to treat cancer, or by cancer therapy used to treat other diseases (especially autoimmune diseases).

[0056] More specifically, HSCT, or treatment with chemotherapeutic agents or antimetabolites such as methotrexate, is not only a treatment strategy for cancer patients, but also a potentially curative treatment for patients with bone marrow failure syndrome, congenital immunodeficiency, or autoimmune diseases such as multiple sclerosis. Furthermore, low-dose chemotherapy and methotrexate are also potential treatments for autoimmune diseases such as rheumatoid arthritis, as mentioned above regarding cancer patients, which also lead to OM (Haverman dysplasia). et al. , Mediators Inflamm. (2014), 378281; Gobbo et al ., PhotonLasers Med 2 (2013), 71-76; Gobbo et al ., Photon Lasers Med 2 (2013), 71-76).

[0057] When referring to “ENT disease induced by cancer therapy,” “oral mucositis induced by cancer therapy,” “ENT disease induced by chemotherapy therapy,” “OM induced by chemotherapy therapy (CIOM),” “ENT disease induced by radiotherapy therapy,” “OM induced by radiotherapy therapy (RIOM),” “ENT disease induced by HSCT,” “OM induced by HSCT,” or similar expressions, it means that the ENT disease and OM are induced by or related to the mentioned therapy.

[0058] HSCT involves administering healthy hematopoietic stem cells to patients with bone marrow dysfunction or depletion. This helps to enhance bone marrow function and, depending on the disease being treated, can lead to the destruction of malignant tumor cells or the generation of functional cells that can replace dysfunctional cells, as is the case in immunodeficiency syndromes, hemoglobinopathies, and other diseases. Pre-transplant bone marrow ablation is typically performed prior to HSCT via high-dose chemotherapy.

[0059] Streptococcus salivarius K12 has been commercially available as a probiotic for over 15 years. Streptococcus salivarius K12 is deposited at the German Microbiological Collection (DSMZ) of the Leibniz Institute, located at 7B Inhoffenstraße 7B, 38124 Braunschweig, Germany, with accession number DSM 13084; it is also publicly available at the American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108, USA, with accession number BAA-1024. S. salivarius ENT-K12, which is genetically identical to *Streptococcus salivarius* K12, was deposited on February 22, 2023, at the Leibniz Institute DSMZ (German Center for Microbial Collections), located at 7B Inhofenstrasse, Braunschweig, Germany 38124, with the accession number DSM 34540. *Streptococcus salivarius* is also known as *Streptococcus thermophilus* subspecies (…). Streptococcus salivarius subsp. thermophilus )ENT-K12, and therefore, the term "Salinomyces ( )ENT-K12" in this article is used in conjunction with this terminology. Streptococcus salivarius "Salinomyces ( Streptococcus salivarius K12", "Salinomyces streptococcus ( Streptococcus salivarius )ENT-K12", "Salinomyces streptococcus ( S. salivarius "Salinomyces ( S. salivarius K12", "Salinomyces streptococcus ( S. salivarius )ENT-K12", "Streptococcus thermophilus ( Streptococcus thermophilus "Streptococcus thermophilus ( )", "Streptococcus thermophilus ( Streptococcus thermophilus K12", "Thermophilic Streptococcus ( Streptococcus thermophilus )ENT-K12", "Streptococcus thermophilus ( S. thermophilus "Salinomyces ( S. thermophilus K12", "Thermophilic Streptococcus ( S. thermophilus )ENT-K12", "Streptococcus salivarius thermophilus subsp. ( S. salivarius subsp. thermophilus “ENT-K12”, “K12” and “ENT-K12” can be used interchangeably.

[0060] Thanks to experiments conducted within the scope of this invention, the application of *Streptococcus salivarius* has been shown to be effective in treating cancer therapy-induced ophthalmopathy (OM). Specifically, in cancer patients undergoing radiotherapy or HSCT (previously chemotherapy), those treated concurrently with the probiotic *Streptococcus salivarius* showed prevention, reduction, and improvement in the occurrence of OM, respectively. Furthermore, mouse studies have also confirmed the role of *Streptococcus salivarius* in treating chemotherapy-induced OM. Mouse studies have also shown that inactivated, particularly heat-inactivated, *Streptococcus salivarius* can be used to treat OM. In addition, immune checkpoint inhibitor therapy is commonly used to treat cancer or autoimmune diseases, and it often causes oral mucosal immune-related adverse events, which are important factors that can significantly affect the treatment outcomes of cancer / autoimmune diseases.

[0061] Due to their rapid mitotic rate, mucosal cells are natural targets for cancer cytotoxic therapies, where epithelial damage is often the cause of osteogenesis imperfecta (OM). Therefore, in general, any cancer therapy can potentially lead to OM, especially cytotoxic drug-based therapies. Thus, patients with any cancer or any other disease requiring anticancer medication, such as autoimmune diseases, who develop OM due to cancer therapy, can be treated with Streptococcus salivarius. Furthermore, OM is not only associated with cancer therapy but may also be related to a variety of other factors. For example, OM is known to occur in various immunosuppressed patients (Chiappelli, Evid Based Complement Alternat Med. 2 (2005), 489-494).

[0062] Therefore, in one aspect, the present invention relates to the use of *Streptococcus salivarius* in the treatment of oral mucosal diseases, preferably OM (an ear, nose, and throat (ENT) disease), in human subjects. In a preferred embodiment, OM is induced by or associated with cancer therapy. Thus, in a preferred embodiment, the patient to be treated with *Streptococcus salivarius* has OM as well as cancer or an autoimmune disease, and is undergoing, has completed, or is about to begin cancer therapy. Cancer therapy includes, but is not limited to: radiotherapy, chemotherapy, chemoradiotherapy (especially concurrent chemoradiotherapy), hormone therapy, anti-angiogenic stem cell therapy (including hematopoietic stem cell transplantation (HSCT)), immunotherapy, dendritic cell-based immunotherapy, molecularly targeted therapy, proton pump inhibitor therapy, immune checkpoint inhibitor therapy, and hormone therapy.

[0063] In a preferred embodiment, the cancer therapy is a cancer therapy based on cytotoxic drugs. In other words, the preferred treatment according to the invention is OM induced by anticancer drugs, such as OM induced by cytotoxic anticancer drugs.

[0064] In a preferred embodiment of the present invention, the cancer therapy for inducing OM is: chemotherapy, radiotherapy, HSCT, immune checkpoint inhibitor therapy, or chemotherapy combined with radiotherapy, HSCT combined with chemotherapy, HSCT combined with radiotherapy, or a combination of HSCT, chemotherapy, and radiotherapy. Accordingly, the OM treated according to the present invention is CIOM, RIOM, HSCT-induced OM, or any combination thereof.

[0065] Therefore, in one embodiment, the present invention relates to the use of *Streptococcus salivarius* for the treatment of OM in human subjects, wherein OM is induced by chemotherapy, radiotherapy, HSCT, a combination of chemotherapy and radiotherapy, a combination of HSCT and chemotherapy, a combination of HSCT and radiotherapy, or a combination of HSCT, chemotherapy, and radiotherapy. In other words, the patient to be treated with *Streptococcus salivarius* suffers from OM as well as cancer or an autoimmune disease, and is undergoing or has completed cancer treatment, wherein the cancer treatment is: chemotherapy, radiotherapy, HSCT, a combination of chemotherapy and radiotherapy, a combination of HSCT and chemotherapy, a combination of HSCT and radiotherapy, or a combination of HSCT, chemotherapy, and radiotherapy.

[0066] In one embodiment, the present invention relates to the use of Streptococcus salivarius for the treatment of obstructive osmosis (OM), preferably for the treatment of OM in human subjects, wherein the OM is CIOM.

[0067] The present invention also relates to the use of *Streptococcus salivarius* for the treatment of obstructive pulmonary disease (OM), preferably for the treatment of OM in human subjects, wherein the OM is RIOM. As mentioned above, if a patient has received radiotherapy or chemoradiotherapy, their OM is referred to as RIOM.

[0068] The present invention also relates to the use of Streptococcus salivarius for the treatment of OM in human subjects, wherein the OM is HSCT-induced OM.

[0069] Those skilled in the art are familiar with a variety of chemotherapeutic agents, which are listed in numerous public lists, such as “Chemotherapeutic Agents and Their Uses, Dosages, and Toxicities, June 1, 2016, Anne M. McDonnell, PharmD, BCOP” on the Cancer Network homepage. In one embodiment, the chemotherapy is a combination of busulfan and cyclophosphamide. In another embodiment, the chemotherapy is 5-FU chemotherapy.

[0070] As previously mentioned, radiotherapy is typically administered over several weeks, and the appropriate dose can be determined by those skilled in the art. As shown in Example 1, for the treatment of NPC, radiotherapy is administered 5 times per week for 6-7 weeks, at a dose of 2 Gy each time, for a total dose of 60-70 Gy. Therefore, in a preferred embodiment, radiotherapy is administered 1 to 7 times per week for 2 to 10 weeks, preferably 3 to 10 weeks, more preferably 4 to 10 weeks, more preferably 5 to 10 weeks, more preferably 5 to 9 weeks, more preferably 5 to 8 weeks, and most preferably 6 to 7 weeks, preferably 2 to 6 times per week, more preferably 3 to 6 times per week, more preferably 4 to 6 times per week, and most preferably 5 times per week. Preferably, radiotherapy is administered at a dose of 2 Gy per dose, for a total dose of 60-70 Gy; however, the duration of treatment depends on the disease being treated, and the treatment regimen can vary. In a preferred embodiment, radiotherapy is administered concurrently with chemotherapy. This administration modality is used for the treatment of nasopharyngeal carcinoma (NPC).

[0071] As demonstrated by studies conducted within the scope of this invention, *Streptococcus salivarius* is effective and safe in treating OM in patients with NPC and in patients undergoing HSCT, see Examples 1 and 2. NPC belongs to the category of head and neck cancers. Most head and neck cancers originate from the mucosal epithelium of the oral cavity, pharynx, and larynx, and are collectively referred to as head and neck squamous cell carcinoma (HNSCC). However, HNSCC can also occur in other areas of the head and neck, including but not limited to: the oral cavity (lips, buccal mucosa, hard palate, anterior part of the tongue, floor of the mouth, and retromolar triangle), nasopharynx, oropharynx (palatine tonsils, lingual tonsils, base of the tongue, soft palate, uvula, and posterior pharyngeal wall), hypopharynx (the lower part of the pharynx extending from the hyoid bone to the cricoid cartilage), and the mucosal epithelium of the larynx. Human papillomavirus-associated HNSCC primarily originates from the palatine and lingual tonsils of the oropharynx, while tobacco-associated HNSCC primarily occurs in the oral cavity, hypopharynx, and larynx. Therefore, in one embodiment, the patient to be treated according to the invention has head and neck cancer, preferably NPC. Accordingly, in one embodiment, the OM treated according to the present invention is caused by cancer therapy for head and neck cancer (preferably NPC).

[0072] In a preferred embodiment, the present invention relates to the use of *Streptococcus salivarius* for the treatment of RIOM, wherein the cancer to be treated with radiotherapy is a head and neck cancer, preferably an NPC. In another preferred embodiment, radiotherapy is administered 5 times per week for 6 to 7 weeks (2 Gy each time, for a total dose of 60-70 Gy).

[0073] HSCT is used to treat a variety of diseases, including cancer, leukemia, lymphoma, heart failure, neurological disorders, autoimmune diseases, immunodeficiency, metabolic or genetic disorders. HSCT treatment typically begins with high-dose chemotherapy, and therefore, OM (obstructive lesions) in HSCT patients can be classified as CIOM (complicated intraepithelial neoplasia). Therefore, in one embodiment, this invention relates to the use of *Streptococcus salivarius* for the treatment of CIOM, which may occur due to chemotherapy or as a result of chemotherapy followed by HSCT. In a preferred embodiment, this invention relates to the use of *Streptococcus salivarius* for the treatment of CIOM, wherein the cancer to be treated by HSCT and prior chemotherapy is a hematopoietic tumor.

[0074] Consistent and accurate assessment of adverse events (OMs) is crucial for describing their incidence and severity, as well as evaluating the effectiveness of potential interventions. The three most commonly used OM grading standards are: the World Health Organization (WHO) standards, the Radiotherapy oncology Group (RTOG) standards, and the Common Terminology Standard for Adverse Events (CTCAE). et al., Comparisons are provided in Support Care Cancer 29 (2021), 6061-6068. All grading criteria range from grade 0 to grade 4, where grade 0 is defined as no findings and grade 4 represents the most severe form of OM. Therefore, in one embodiment, the OM treated according to the invention can be any grade between 0 and 4. Examples 1 and 2 have demonstrated a reduction in the incidence of RIOM with RTOG ≥2, and patients taking oropharyngeal probiotics were completely free from developing severe RIOM classified as RTOG 3 and 4. Furthermore, administration of oropharyngeal probiotics has been shown to delay the onset of oral mucositis and reduce the risk of severe oral mucositis; to restore decreased proliferation and disordered cell cycle of oral epithelial cells following cancer treatment in a dose-dependent manner; and to prevent reduction in oral mucosal area and restore oral mucosal integrity damaged after chemotherapy.

[0075] Therefore, in one embodiment, the *Streptococcus salivarius* according to the invention is used to prevent patients from developing grade ≥2 oral mucositis (OM). In one embodiment, the *Streptococcus salivarius* according to the invention is used to prevent patients from developing grade 3 or 4 OM. In one embodiment, the *Streptococcus salivarius* according to the invention is used to completely prevent patients from developing OM. In one embodiment, the *Streptococcus salivarius* according to the invention is used to delay the onset of oral mucositis and reduce the risk of severe oral mucositis. In one embodiment, the *Streptococcus salivarius* according to the invention is used to restore, in a dose-dependent manner, the decreased proliferation and disordered cell cycle of oral epithelial cells after chemotherapy. In one embodiment, the *Streptococcus salivarius* according to the invention is used to prevent the reduction of oral mucosal layer area after chemotherapy and to restore the integrity of damaged oral mucosa.

[0076] Furthermore, it is known that immunosuppression can often lead to OM, for example, due to HIV infection (Ehlert). et al ., DtschMed Wochenschr 138 (2013), 1601-1695) or treatment with immunosuppressive drugs. Given that experiments conducted within the scope of this invention have shown that *Streptococcus salivarius* is safe and effective in treating CIOM, RIOM, and HSCT-induced OM, it is reasonable to expect that patients with OM due to their immunosuppressive state will also benefit from treatment with *Streptococcus salivarius*. Therefore, this invention also relates to the use of *Streptococcus salivarius* for the treatment of OM in human subjects who are immunosuppressed. Immunosuppressants are generally known in the art and, for example, those produced by Rathee... et al. A review was conducted in The Pharma Innovation Journal 1 (2013), 90-101.

[0077] As mentioned above, *Streptococcus salivarius* is a probiotic microorganism, and probiotics are generally defined as live microorganisms that, when administered in adequate amounts, provide health benefits to the host. The beneficial effects of probiotics can be mediated through: direct antagonistic effects against specific populations of undesirable organisms, leading to a reduction in their numbers; through effects on the metabolism of these populations; or through an overall stimulatory effect on the immune system of an animal or human host.

[0078] like Figure 4As shown, heat-inactivated *Streptococcus salivarius* has a similar beneficial effect on oral epithelial cells as live *Streptococcus salivarius*, which was previously undiscovered; therefore, the present invention also relates to the use of inactivated *Streptococcus salivarius* for healing oral epithelial cells damaged by cancer therapy, particularly for healing oral epithelial cells damaged by chemotherapy. More particularly, the present invention also relates to the use of inactivated *Streptococcus salivarius* for treating cancer therapy-induced oral malformation (OM), and preferably CIOM. Inactivation can be achieved by various methods, such as by heating, pressurization, or radiation. Preferably, inactivation is achieved by heating, and therefore, the *Streptococcus salivarius* is preferably heat-inactivated (also known as heat-killed) *Streptococcus salivarius*. Heat inactivation is preferably carried out at a temperature range of 60°C to 120°C, more preferably at about 65°C for about 60 min, or at about 120°C for about 20 min.

[0079] As mentioned above, OM is an ENT disease. ENT diseases, also known as ear, nose, and throat conditions, encompass conditions involving the ear, nose and nasopharynx, sinuses, pharynx, oral cavity and oropharynx, larynx, trachea, and neck. For example, the most common ENT conditions are reviewed in the Paradis and Messner, Pediatric Board Study Guide (2015), Mar 28, 469-89, the contents of which are incorporated herein by reference. ENT diseases are typically caused by viral or bacterial infections.

[0080] It is known that some cancer treatments temporarily weaken the immune system because they can cause a decrease in the number of white blood cells produced in the bone marrow. Therefore, cancer treatment often makes patients more susceptible to certain infections, such as the aforementioned ENT disease. A similar situation exists with general immunosuppressive therapies. Experiments conducted within the scope of this invention have shown that administration of *Streptococcus salivarius* is safe for patients who are receiving or have received anticancer drug treatment and are therefore immunosuppressed.

[0081] Furthermore, clinical studies have confirmed the safety and efficacy of daily administration of *Streptococcus salivarius* in reducing respiratory tract infections and key respiratory pathogens. In particular, strain K12 has been shown to be highly suitable as an upper respiratory tract probiotic due to its natural tendency to be present in the human oral cavity and to compete fiercely with a variety of potential pathogens. Additionally, *Streptococcus salivarius* K12 has been shown to induce an anti-inflammatory response. For example, *Streptococcus salivarius* K12 can antagonize *Streptococcus pyogenes* (…). S. pyogenes Streptococcus pyogenes is the most important bacterium causing pharyngeal infections in humans and is also affected by acute otitis media. Prophylactic administration of salivarius streptococcus K12 to children with a history of recurrent oral streptococcal disease significantly reduces the incidence of streptococcal and viral infections; see Di Pierro et al., Drug Health Patient Saf. 6 (2014), 15-20. As detailed in Example 2, this study also confirms that Streptococcus salivarius can be used to treat and prevent respiratory infections in patients who have undergone cancer treatment, particularly chemotherapy and HSCT.

[0082] Therefore, the present invention also relates to *Streptococcus salivarius*, which can be used not only for oral mucosal diseases but also for the treatment of other ENT diseases in human subjects, wherein the ENT diseases are induced by cancer therapy. Accordingly, patients treated with *Streptococcus salivarius* have ENT diseases and cancer, or ENT diseases and autoimmune diseases, and these patients are undergoing or have completed cancer therapy. Furthermore, the present invention also relates to *Streptococcus salivarius* for the treatment of ENT diseases in human subjects, wherein the subjects are in an immunosuppressed state as described above.

[0083] Since *Streptococcus salivarius* is particularly effective in treating upper respiratory tract infections and otitis media, as demonstrated in Example 2, the ENT disease is preferably an upper respiratory tract infection or otitis media. The upper respiratory tract includes the pharynx, nose, pharynx, larynx, sinuses, and trachea; and bacterial-mediated upper respiratory tract infections include, for example, pharyngitis, tonsillitis, sinusitis, and bronchitis. Severe respiratory infections may lead to unplanned hospitalization or even treatment changes, including suspending radiotherapy or reducing chemotherapy doses, thereby adversely affecting treatment efficacy. Furthermore, *Streptococcus pneumoniae* (… S. pneumoniae Colonization in the upper respiratory tract can become the beginning of colonization in the lower respiratory tract, which can lead to pneumonia and potentially death, especially in immunocompromised patients. Accordingly, in a preferred embodiment, ENT disease is a respiratory infection. Therefore, in a preferred embodiment, the present invention relates to the use of *Streptococcus salivarius* for the prevention and treatment of respiratory infections during cancer therapy.

[0084] Specifically, in one embodiment, administering *Streptococcus salivarius* during cancer treatment prevents upper respiratory tract infections in subjects from developing into lower respiratory tract infections. In one embodiment, administering *Streptococcus salivarius* during cancer treatment shortens the duration of respiratory infection symptoms in subjects compared to a control group that did not receive *Streptococcus salivarius*. In one embodiment, administering *Streptococcus salivarius* during cancer treatment shortens the average duration of respiratory infection episodes in subjects compared to a control group. In one embodiment, administering *Streptococcus salivarius* during cancer treatment reduces the need for antibiotics in subjects compared to a control group.

[0085] Therefore, experiments conducted within the scope of this invention have, for the first time, demonstrated that *Streptococcus salivarius* can be used to treat respiratory infections in patients undergoing cancer therapy and thus in an immunosuppressed state. Prior to this study, administering live bacteria to immunosuppressed patients was generally not recommended due to the high risk of adverse side effects such as sepsis. Accordingly, it has been demonstrated for the first time that *Streptococcus salivarius* can be used to treat oral mucosal disorders, particularly oral ulcers (OM) and respiratory infections, in both patients undergoing cancer therapy and immunosuppressed patients.

[0086] In a preferred embodiment, the cancer therapy includes HSCT, chemotherapy, and / or radiotherapy.

[0087] As detailed above, experiments conducted according to the present invention further confirmed that patients taking oropharyngeal probiotics avoided developing severe OM. Therefore, administering *Streptococcus salivarius* to human subjects during cancer therapy helps prevent OM and maintain the health of the ear, nose, and throat (ENT) tracts, preferably the oral mucosa. Therefore, the present invention also relates to *Streptococcus salivarius* maintaining the health of the ear, nose, and throat (ENT) tracts, preferably the oral mucosa, in human subjects and immunosuppressed patients during cancer therapy.

[0088] The present invention also relates to compositions for use according to the invention, wherein the composition comprises *Streptococcus salivarius* as an active ingredient (in an effective amount) and optionally comprises one or more pharmaceutically acceptable excipients. The present invention also relates to compositions for treating oral mucosal disorders induced by cancer therapy, particularly oral mucositis, wherein the composition comprises inactivated *Streptococcus salivarius* as an active ingredient (in an effective amount) and optionally comprises one or more pharmaceutically acceptable excipients.

[0089] The compositions according to the invention are suitable for administering Streptococcus salivarius to patients in need; particularly those susceptible to ENT disease, more specifically those with OM as defined above, and / or those suffering from ENT disease, especially OM. Typically, the therapeutic compositions consist of Streptococcus salivarius and acceptable excipients.

[0090] Excipients include fillers, diluents, carriers, binders, lubricants, disintegrants, anti-caking agents, preservatives, colorants, or flavorings. Excipients are preferably pharmaceutically acceptable and / or acceptable for human consumption.

[0091] A carrier or filler means a load for delivering probiotic microorganisms to an individual, wherein the load is compatible with cell viability or the activity of an inactivated form of the probiotic (here, Streptococcus salivarius). Acceptable carriers / fillers are well known to those skilled in the art; see, for example, Remington's Pharmaceutical Sciences, 18th ed., Gennaro, ed., 1990, Mack Publishing Co., Easton, Pa., which is incorporated herein by reference. Suitable carriers are generally inert and may be solid or liquid. Acceptable carriers include, but are not limited to, water, buffered salt solutions (e.g., phosphate-buffered saline), pharmaceutically acceptable culture media (e.g., BACa, TSBCaYE agar), or other solutions that maintain bacterial viability and retain the biologically active portion of the bacteria, respectively. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. A variety of pharmaceutically acceptable carriers suitable for oral, topical, nasal, or respiratory administration of live or lyophilized bacteria are well known in the art (see, for example, Remington's mentioned above). Suitable solid carriers known in the art include, for example, magnesium carbonate; magnesium stearate; cellulose; talc; sugars such as fructose, sucrose, mannitol, lactose; starch; flour; oligosaccharides and skim milk, as well as similar edible powders, but not limited thereto. Carriers for applying extracts are similarly known in the art.

[0092] Acceptable carriers / fillers suitable for probiotic active microorganisms in compressed products such as tablets are generally solid carriers known in the art and include, but are not limited to, magnesium carbonate; magnesium stearate; cellulose; talc; sugars such as fructose, sucrose, mannitol, sorbitol, xylitol, lactose; sugar substitutes such as isomaltitol; starch; maltodextrin; flour; (fructose-)oligosaccharides and skim milk, as well as similar edible powders, but are not limited thereto.

[0093] For example, typical diluents are: starch; lactose; mannitol; kaolin; calcium phosphate or calcium sulfate; inorganic salts such as sodium chloride; and powdered sugars and sugar substitutes or cellulose as described above.

[0094] Typical binders include starch; gelatin; sugars such as lactose, fructose, and glucose; and so on. Natural and synthetic gums are also readily available, including gum arabic; alginate; locust bean gum; methylcellulose, for example, hydroxypropyl methylcellulose (HMPC); polyvinylpyrrolidone (PVP); astragalus gum; PVP K-30; PVP K-25; xanthan gum; and so on. Polyethylene glycol (PEG 4000 or PEG 6000); ethyl cellulose; and waxes, as well as Nu-BIND® and CompactCel® DIS, can also be used as binders.

[0095] Lubricants and anti-caking agents used to prevent sticking and clumping during formulation include smooth solids such as talc, silica, magnesium stearate and calcium stearate, polyethylene glycol, stearic acid, hydrogenated vegetable oil, rice extract blends such as Nu-MAG®, CompactCel® LUB, potato starch, gum arabic, CompactCel® FLO, Nu-FLOW®, silica, tricalcium phosphate, and rice husks such as Nu-FLOW® or CompactCel® FLO.

[0096] Disintegrants are substances that swell upon wetting to break down a composition and release Streptococcus salivarius or extracts. Disintegrants include starch; clay; cellulose; alginate and gum; more particularly corn and potato starch; methylcellulose; agar; bentonite; lignocellulose; cation exchange resins; alginate; guar gum; citrus pomace; carboxymethyl cellulose; powdered sponge; silica; and sodium lauryl sulfate.

[0097] Flavoring agents are known to those skilled in the art and can be of any kind that imparts a good (or at least a different) flavor to compressed products. These flavoring agents include, but are not limited to, strawberry, mint, orange, banana, passion fruit, cocoa, menthol, grapefruit, lemon and / or combinations thereof, preferably passion fruit, cocoa and menthol; grapefruit and mint; orange and mint.

[0098] Streptococcus salivarius can be formulated into any of a variety of compositions suitable for oral, nasal, topical, or respiratory administration. In a preferred embodiment, Streptococcus salivarius is formulated for oral administration. The composition may be in liquid, solid, or semi-solid form, but in a preferred embodiment, the composition is in solid form. In another preferred embodiment, the composition is an oil droplet composition, wherein Streptococcus salivarius is mixed with oil, preferably vegetable oil, preferably sunflower oil, and especially sunflower oil (sunflower oil). Helianthus annuus It is formulated with L. and α-tocopherol seed oil.

[0099] Streptococcus salivarius can be administered in the following forms: oral creams, oral films, wound dressings, lotions, gels, ointments, solutions, suspensions, emulsions, powders, granules, drops (especially oil drops), sachet, mouthwash, mouthwash, toothpaste, dental cleaning agents, sprays, mouthwash, capsules, lozenges, syrups, dental floss, films, chewing gum, or chewable tablets, but is not limited to these forms. It can be administered via a nebulizer through the respiratory tract, which may or may not contain a propellant. It is preferably administered in dosage forms with a longer residence time in the oral cavity, such as lozenges, chewable tablets, chewing gum, oil suspensions, powders / sachets, or sprays. In a preferred embodiment, the composition comprising Streptococcus salivarius used according to the invention is an oral solid dosage form selected from tablets, capsules, gels, lozenges, chewable tablets, and powders; preferably, the oral composition is a lozenge or powder, more preferably a lozenge.

[0100] Typically, the amount of saliva streptococcus administered to a patient will be high enough to deliver the desired benefit but low enough to avoid serious side effects.

[0101] In one embodiment, the daily dose of *Streptococcus salivarius* administered according to the present invention is 3 × 10⁻⁶. 6 Up to 4 × 10 10 Between CFUs, 3 × 10 is preferred. 9 Up to 3 × 10 10 Between CFU or 4 × 10 9 Up to 4 × 10 10 Between CFU. The inactivated form of *Streptococcus salivarius* is preferably applied in a similar amount to live *Streptococcus salivarius*, i.e., to effectively provide the following amounts: 3 × 10⁻⁶ 6 Up to 4 × 10 10 Between cell equivalents, 3 × 10⁻⁶ is preferred. 9 Up to 3 × 10 10 Between cell equivalents or 4 × 10 9 Up to 4 × 10 10 Between individual cell equivalents.

[0102] In one embodiment, the composition used according to the invention is preferably in solid dosage form, more preferably in tablet form, and most preferably in tablet form, comprising at least 1 mg of *Streptococcus salivarius*, and may comprise up to 1000 mg of *Streptococcus salivarius*. In a preferred embodiment, the composition used according to the invention is preferably in solid dosage form, more preferably in tablet form, and most preferably in tablet form, comprising 1 to 500 mg of *Streptococcus salivarius*, preferably 5 to 160 mg of *Streptococcus salivarius*, preferably between 10 and 120 mg, more preferably between 10 and 100 mg or between 30 and 120 mg, more preferably 40 mg, 50 mg, 60 mg, 100 mg or 120 mg, and most preferably 50 mg of *Streptococcus salivarius*.

[0103] In one embodiment, the composition used according to the invention is preferably in solid dosage form, more preferably in tablet form, and most preferably in lozenge form, comprising 50 mg of Streptococcus salivarius and 10 mg of Streptococcus salivarius. 6 Up to 10 10 CFU, preferably 1 × 10 9 Up to 10 × 10 9 CFU, preferably 6 × 10 9 Up to 7 × 10 9 CFU of *Streptococcus salivarius*; or an amount of cellular equivalents corresponding to its inactivated form. Preferably, the composition, more preferably the solid dosage form, and most preferably the tablet form, is administered to human subjects multiple times daily, preferably three or four times daily. In one embodiment, for patients who have received HSCT and / or have been treated with chemotherapy, the composition is administered three times daily; for patients who have been treated with radiotherapy and / or whose cancer is NPC, the composition is administered four times daily.

[0104] In one embodiment, the composition used according to the invention, along with *Streptococcus salivarius*, is administered for periods ranging from several days to several weeks; wherein the time period varies depending on the type of cancer / autoimmune disease treatment and the type of cancer therapy administered. For example, as mentioned above, chemotherapy is typically delivered over a short period, where the damage to mucosal tissues tends to be acute. Chemotherapy-induced OM typically appears within 4-7 days after the start of treatment and peaks within 2 weeks. Therefore, administering the composition for approximately 2 weeks may be sufficient to prevent the occurrence of severe OM. In contrast, radiotherapy typically requires several weeks to deliver, as also shown in Example 1, and therefore, in one embodiment, the composition used according to the invention is administered to subjects who have already been treated with radiotherapy and / or whose cancer is NPC for several weeks, preferably approximately 7 weeks. In a preferred embodiment, the composition is even administered as a preventative measure before the start of cancer therapy. For example, in the cases of radiotherapy and CCRT treatment, the composition is administered 2 weeks before the start of treatment, respectively.

[0105] In a preferred embodiment, the composition is applied four times daily. More preferably, it is applied after breakfast, lunch, dinner, and before bedtime after brushing (rinsing or brushing).

[0106] In another embodiment, the composition / salicylic streptococcus used according to the invention is administered to human subjects who have undergone HSCT and whose hematopoietic system has been reconstituted for approximately 100 days, wherein hematopoietic system reconstitution is defined as platelet count >20 after HSCT. 10 9 Cells / L and neutrophils >0.5 10 9 Cells / L, preferably the composition is applied three times daily. It is also preferred to apply it after breakfast, after lunch, and before bedtime after oral hygiene (rinsing or brushing).

[0107] Salivarius can be administered before, during (simultaneously with), and / or after cancer therapy, and can also be administered long-term to immunosuppressed subjects. In a preferred embodiment, for patients with RIOM or CIOM, administration of Salivarius is performed at least during cancer therapy, and preferably also after cancer therapy is discontinued, preferably as long as OM symptoms persist and as long as the patient remains immunosuppressed. For HSCT-induced OM, Salivarius is preferably administered after hematopoietic system reconstitution, for example defined as platelets >20 after HSC. 10 9 Cells / L and neutrophils >0.5 10 9 Cells / L

[0108] In one embodiment, the composition used according to the invention, in addition to *Streptococcus salivarius*, includes isomaltulose and one or more additional excipients, preferably lubricants and / or anti-caking agents, and / or binders, and / or flavoring agents. Preferably, the lubricant is magnesium stearate or a natural substitute, the anti-caking agent is silica, tricalcium phosphate or a natural substitute, the binder is HMPC or a natural substitute, and / or the flavoring agent is a flavoring agent, preferably strawberry flavoring, and optionally, the product further includes one or more active ingredients, preferably vitamins, minerals and / or fructooligosaccharides, preferably vitamin D3.

[0109] Natural lubricants are known in the art and include, for example, blends of soybean flour and rice extract containing crude fat, as described in WO2013 / 165131 A1, such as Nu-MAG® or CompactCel® LUB (CompactCel® Fclear 290.02 LUB, Biogrund GmbH, Huenstetten, Germany), oat fiber blends, such as CompactCel® LUB (CompactCel® F 200.28 LUB, Biogrund GmbH, Huenstetten, Germany), or other products such as potato starch or gum arabic. Natural anti-caking agents are also known in the art and are, for example, powdered cellulose such as JELUCEL®, natural potato starch, inulin, rice fiber, or rice husks, such as Nu-FLOW® or CompactCel® FLO. Natural binders include, for example, CompactCel® DIS, NuBind®, or pregelatinized corn starch.

[0110] In one embodiment, the composition used according to the present invention is (i) A compressed product, i.e., an oral solid dosage form, and in addition to Streptococcus salivarius, includes isomaltulose and additional lubricant / anti-caking agent, preferably wherein the lubricant / anti-caking agent is magnesium stearate or a natural alternative; a flavoring agent, preferably wherein the flavoring agent is a flavoring agent, preferably strawberry flavoring agent; and optionally a natural sweetener and / or one or more active ingredients, preferably vitamins, preferably vitamin D3; (ii) A compressed product, i.e., an oral solid dosage form, which, in addition to *Streptococcus salivarius*, includes isomaltulose and additional lubricant / anti-caking agent, preferably wherein the lubricant / anti-caking agent is magnesium stearate or a natural alternative; a binder, preferably wherein the binder is hydroxypropyl methylcellulose (HMPC) or a natural alternative; a flavoring agent, preferably wherein the flavoring agent is a flavoring agent, preferably strawberry flavoring; and optionally a natural sweetener, and / or one or more active ingredients, preferably vitamins, preferably vitamin D3; (iii) A powder product, which, in addition to Streptococcus salivarius, includes isomaltulose and an additional anti-caking agent, preferably wherein the anti-caking agent is silica, tricalcium phosphate or a natural substitute; a flavoring agent, preferably wherein the flavoring agent is a flavoring agent, preferably strawberry flavoring; fructooligosaccharides; and optionally a natural sweetener and / or one or more active ingredients, preferably vitamins, preferably vitamin D3.

[0111] Natural alternatives to magnesium stearate are preferably blends of rice extract, preferably including rice extract, microcrystalline cellulose, and refined sunflower oil, such as CompactCel® F clear 290.02 LUB; blends of oat fiber, preferably including oat fiber, microcrystalline cellulose, and refined sunflower oil, such as CompactCel® F 200.28 LUB, Nu-MAG®; potato starch, gum arabic, rice husk such as Nu-FLOW® or CompactCel® FLO, but most preferably blends of rice extract. Natural alternatives to HMPC are preferably natural binders such as CompactCel® DIS, NuBind®, or pregelatinized corn starch, most preferably pregelatinized corn starch.

[0112] Therefore, in one embodiment, the composition used according to the invention is a compressed article, i.e., an oral solid dosage form, and includes isomaltulose in addition to Streptococcus salivarius, and further includes rice extract blend (as a lubricant / anti-caking agent), pregelatinized corn starch (as a binder), flavoring agent (as a flavoring agent, preferably strawberry flavoring agent), natural sweetener (preferably stevia) and vitamin D3 (as an additional active ingredient).

[0113] The components included in the exemplary solid dosage forms are shown in Tables 1 to 3.

[0114] Table 1: Ingredients of an exemplary isomaltulose / probiotic tablet.

[0115]

[0116] Table 2: Ingredients of an exemplary isomaltulose / probiotic tablet.

[0117]

[0118] Table 3: Ingredients of an exemplary isomaltulose / probiotic tablet.

[0119]

[0120] Magnesium stearate can be replaced with natural lubricants / anti-caking agents, such as rice extract blends, such as products Nu-MAG® or CompactCel® LUB (CompactCel® F clear 290.02 LUB, Biogrund GmbH, Huenstetten, Germany), oat fiber blends, such as products CompactCel® LUB (CompactCel® F 200.28 LUB, Biogrund GmbH, Huenstetten, Germany), or other products, such as potato starch, or gum arabic, powdered cellulose, such as JELUCEL®, natural potato starch, inulin, rice fiber, or rice husks, such as Nu-FLOW® or CompactCel® FLO, but preferably with rice extract blends, preferably with CompactCel® LUB, and / or HMPC can be replaced with natural binders, such as CompactCel® DIS, NuBind®, or preferably pregelatinized corn starch.

[0121] The compressed product of this invention may also include silica, but may be replaced with a natural anti-caking agent, such as rice husk, for example CompactCel® FLO, or Nu-FLOW®. Preferably, the compressed product does not include magnesium stearate and silica, but includes a blend of rice extract.

[0122] Exemplary powder products include the ingredients shown in Table 4.

[0123] Table 4: Ingredients of an exemplary isomaltulose / probiotic powder.

[0124]

[0125] Silica can be replaced with tricalcium phosphate or a natural anti-caking agent, such as rice husk (Nu-Flow®) or CompactCel® FLO as described above, and can also be omitted entirely.

[0126] The composition is preferably prepared by a method comprising the following steps: (i) mixing *Streptococcus salivarius* with isomaltulose and one or more other excipients and optionally with one or more other active ingredients; and (ii) homogenize the mixture; and (ii) Compressing the mixture into a compressed product, preferably at a pressure of 6 to 10 kN / compressed product, or filling the mixture into small bags, and optionally... (iii) Analyze the CFU / g content and / or water activity; and optionally the disintegration time, average mass, crushability, appearance, friability and / or water activity of the product.

[0127] In a preferred embodiment, step (i) includes the following steps: (a) Mixing isomaltulose with one or more other excipients, preferably with a lubricant / anti-caking agent, and more preferably with magnesium stearate; (b) Spraying the binder solution onto the mixed excipients, preferably wherein the binder solution consists of water and a binder, preferably wherein the binder is HMPC, thereby forming wet particles; (c) Dry the particles, preferably at a temperature of 40 ± 2°C for about 30 minutes; (d) Grinding particles; (e) Add Streptococcus salivarius to the ground particles, and optionally add one or more additional active ingredients.

[0128] For detailed information about the composition and its preparation method, please refer to European patent application EP 23 167 706.3, the contents of which are incorporated herein by reference.

[0129] In another embodiment, the composition used according to the invention, especially in solid dosage forms, includes one or more excipients in addition to Streptococcus salivarius, preferably one or more fillers, lubricants, and optionally anti-caking agents and / or flavorings.

[0130] In one embodiment, the composition used according to the invention, particularly in solid dosage forms, includes fructose and maltodextrin (fillers), magnesium stearate or its natural alternatives (lubricants), flavoring agents, preferably strawberry flavoring agents (flavoring agents), and optionally silica or its natural alternatives (anti-caking agents). The natural alternatives are the same as those described above.

[0131] Exemplary compositions, especially solid dosage forms, are shown in Tables 5 and 6.

[0132] Table 5: Ingredients of Exemplary Probiotic Tablets

[0133] Table 6: Ingredients of Exemplary Probiotic Tablets

[0134] The compositions used according to the present invention (especially solid dosage forms) are preferably prepared by a "briquetting" method, which includes the following steps: (i) The excipient is briquetting, preferably using a pressure of about 7.5 kN / compressed article; (ii) Crush and grind the compressed blocks; (iii) Add the saliva streptococci to the ground briquettes and mix; (iv) Compressing the mixture into the compressed article, preferably using a pressure of about 4.8 kN / compressed article; and optionally... (vii) Analyze the disintegration time, average mass, crushability, appearance, friability and / or water activity of the compressed product.

[0135] In a preferred embodiment, the method further includes the following steps: (a) Before step (i), one or more excipients are dried, weighed and sieved, preferably wherein the drying is carried out in a fluidized bed granulator; (b) Mix and homogenize one or more excipients after step (a) and before step (i); (c) Weighing and sieving the Streptococcus salivarius prior to step (iii), preferably sieving through a 1 mm mesh; and / or (d) Homogenize the mixture after step (iii) and before step (iv).

[0136] For detailed information on the composition and its preparation method, please refer to international application PCT / EP2023 / 059635, the contents of which are incorporated herein by reference.

[0137] In one embodiment, the composition used according to the invention, particularly in solid dosage forms, includes isomaltitol (filler), magnesium stearate or its natural alternatives (lubricant), flavoring agent, preferably strawberry flavoring agent (flavoring agent), and optionally includes silica or its natural alternatives (anti-caking agent). The natural alternatives are the same as those described above.

[0138] Exemplary compositions, particularly solid dosage forms, are shown in Table 7.

[0139] Table 7: Ingredients of Exemplary Probiotic Tablets

[0140] In all tablets, the concentration of vitamin D3 (cholecalciferol) in the premix is ​​100,000 IU / g (1 IU = 0.025 mcg).

[0141] As described above, the oral solid dosage form used according to the present invention is, in a preferred embodiment, a tablet. Therefore, the present invention further relates to tablets comprising *Streptococcus salivarius* for use according to the present invention, and for use of tablets according to the present invention. In a preferred embodiment, the tablet comprises the ingredients mentioned above regarding the oral solid dosage form. Most preferably, the tablet comprises the ingredients listed in Table 6, namely *Streptococcus salivarius*, fructose, maltodextrin, magnesium stearate, and a flavoring agent (preferably strawberry flavoring), and most preferably, the tablet comprises the ingredients in the amounts listed in Table 6.

[0142] In one embodiment, the tablets used according to the present invention are prepared by the above-described "blistering" method.

[0143] In one embodiment, the tablets used according to the invention are sublingually administered by the patient until completely dissolved and must not be chewed or swallowed directly. Preferably, the patient must not drink or swallow anything for at least 2 hours after administration of the tablets. In a preferred embodiment, the disintegration time of the tablets is at least 4 to 5 minutes, which ensures that *Streptococcus salivarius* can colonize the patient's oral cavity. The disintegration time can be determined as described in Chapter 2.9.1 of the 6th edition of the European Pharmacopoeia (ISBN-10: 9287160546; ISBN-13: 978-9287160546), published by the European Commission on 10 May 2008.

[0144] The present invention also relates to *Streptococcus salivarius* and corresponding compositions as defined above, and particularly to lozenges for use according to the invention, wherein *Streptococcus salivarius* or the corresponding composition (especially the lozenges) is packaged in a kit comprising instructions for administering *Streptococcus salivarius* or the corresponding composition (especially the lozenges). In a preferred embodiment, the lozenges are packaged in blister packs.

[0145] In another preferred embodiment, the composition is an oil drop, which is a preferred and convenient form of administration for patients with painful OM. Exemplary oil drop compositions include probiotic Streptococcus salivarius K12, sunflower (sunflower, α-tocopherol) seed oil, and cholecalciferol oil (vitamin D3, α-tocopherol, medium-chain triglycerides).

[0146] The present invention also relates to a method for treating cancer therapy-induced ENT disease, preferably cancer therapy-induced OM, in patients as described above, wherein the method comprises administering *Streptococcus salivarius* as described above, a composition comprising *Streptococcus salivarius*, or a tablet comprising *Streptococcus salivarius*. The present invention also relates to a method for treating ENT disease, preferably OM, in immunosuppressed patients as described above, wherein the method comprises administering *Streptococcus salivarius* as described above, a composition comprising *Streptococcus salivarius*, or a tablet comprising *Streptococcus salivarius*.

[0147] In a preferred embodiment, the *Streptococcus salivarius* used according to the present invention are *Streptococcus salivarius* K12 (American Center for Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108, USA, accession number BAA-1024) and *Streptococcus salivarius* ENT-K12 (DSMZ-German Microbial Collection, Leibniz Institute, Braunschweig 7B, 38124, Germany, accession number DSM 34540).

[0148] This specification incorporates numerous references. All references cited in this application (including references cited in full, granted patents, published patent applications, including background sections and manufacturers' specifications, descriptions, etc.) are hereby incorporated herein by reference; however, it is not acknowledged that any cited reference is prior art to this invention.

[0149] A more complete understanding can be obtained by referring to the following specific embodiments, which are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0150] Example Example 1: Oropharyngeal probiotic ENT-K12 for the prevention of severe radiation-induced oral mucositis in nasopharyngeal carcinoma patients during radiotherapy. Concurrent chemoradiotherapy (CCRT) is the standard treatment strategy for nasopharyngeal carcinoma (NPC). Studies have shown that 59.4% to 100% of head and neck cancer patients undergoing CCRT develop oral mucositis (OM). Radiotherapy-induced oral mucositis (RIOM) is one of the most common complications in head and neck cancer (HNC) patients undergoing CCRT. RIOM is mainly characterized by discomfort or pain in the mouth or throat, and it reduces patient tolerance to radiotherapy, leading to a reduction in the total radiotherapy dose and may even result in radiotherapy discontinuation (Martha). et al ., Radiother Oncol 110 (2014), 9-15; Gobbo et al(LaserSMed Sci 31 (2016), 471-9). Due to the occurrence of RIOM, the incidence and severity of anxiety and depression in NPC patients during radiotherapy are increased. This also reduces quality of life (QoL) and treatment adherence. In addition, RIOM has various sequelae, including pain, sore throat, dysphagia, reduced oral intake, and systemic infections. RIOM often leads to treatment delays or interruptions, which may reduce disease control and worsen patient prognosis. This not only negatively impacts quality of life but also has a significant adverse effect on tumor control and patient survival (Triarico). et al .,Pathogen 11 (2022), 448; Oronsky et al (Transl Oncol 11 (2018), 771-778). Therefore, prevention and treatment of CCRT-induced RIOM have significant clinical implications. Currently, the best treatment for RIOM is preventative measures, and numerous studies and clinical trials on palliative medications have been conducted to attempt to prevent and control RIOM. However, high-level evidence is lacking for single-target therapies for treating RIOM.

[0151] Radiotherapy-induced toxicities include xerostomia and oral mucositis, resulting from damage to the mucosal barrier. This leads to severe oropharyngeal dysbiosis throughout the course of CCRT, often coinciding with the onset of severe oral mucositis. Studies have shown that probiotic supplements altering the gut microbiota reduce the risk of cancer therapy-induced oral mucositis (Shu et al., Oral Oncology 102 (2020), 104559), however, no studies have reported the restoration of the oropharyngeal microbiota during CCRT. Previous research has shown that oropharyngeal probiotics can inhibit the growth of pathogens in periodontal biofilms and help maintain the physiological balance of the oral microbiota (Begi). et al ., Int J Mol Sci 24 (2023), 7249. This probiotic can also enhance the host's immune level, as demonstrated by the anti-inflammatory response induced by this strain and the reduction of pro-inflammatory cytokines (Evivie). et al., Front Microbiol 10 (2019), 782. Previous clinical studies have shown that oropharyngeal probiotics can effectively colonize the oropharynx / nasopharynx and reduce the risk of pharyngeal tonsillitis infection (Wilcox). et al.(Clin Microbiol Infect 25 (2019), 673-680). For these reasons, a preliminary study was designed to investigate the safety of oropharyngeal probiotics and whether they could reduce the incidence of RIOM in NPC patients undergoing CCRT and delay the progression of RIOM.

[0152] The pathogenesis of RIOM is believed to be: radiation-induced damage to rapidly dividing submucosal basal cells, which in turn leads to epithelial cell damage; it is a common complication in nasopharyngeal carcinoma (NPC) patients undergoing concurrent chemoradiotherapy (CCRT).

[0153] Main objectives This study aimed to evaluate the efficacy and safety of the oropharyngeal probiotic ENT-K12 in the incidence, severity, and progression of recurrent obstructive pulmonary disease (RIOM) in NPC patients undergoing CCRT. RIOM was clinically scored five times per week by the same radiation oncologist according to the Radiotherapy oncology Group (RTOG) criteria (see Table 8) to track the dynamic changes of RIOM throughout the treatment course.

[0154] Table 8: RTOG Standards

[0155] Secondary objectives • Assess the pathogenesis of RIOM (supragingival plaque, saliva, oral microbiome dysbiosis, anaerobic overgrowth, and nitrate reductase gene). napA Increased expression and regulation of the TLR2 / TLR4 signaling pathway in RIOM development) • Assess anti-inflammatory effects on oral mucosa or tissues (CD3+ T cells, CD4+ T cells, and CD8+ T cells). • Nutritional status (monitored weekly based on weight loss, BMI, albumin, prealbumin, and NRS2002 score) • Assess quality of life, such as the reduction and improvement of side effects caused by radiation therapy (sore throat, loss of taste and appetite, dry mouth, skin peeling on the neck, difficulty swallowing, hair loss, ear discharge, nausea or vomiting, fatigue, diarrhea, etc.). • Confirm subject safety through adverse event reporting Recovery rate • Evaluate the efficacy of the oropharyngeal probiotic ENT-K12 in preventing respiratory infection episodes during the study period. • Number of days of antibiotic treatment • Number of days of antiviral drug treatment • Number of days of antipyretic treatment • Number of days since the onset of oral mucositis and tongue ulcers • Number of days since gum bleeding or gum disease occurred Research Summary A randomized controlled clinical trial was conducted to determine the preventive effect of the oropharyngeal probiotic ENT-K12 on radiation-induced oral mucositis (RIOM) in patients with stage II nasopharyngeal carcinoma (NPC). Ten eligible patients, aged 18 to 65 years, were newly diagnosed with at least stage II NPC according to the International Union Against Cancer / American Joint Committee on Cancer TNM staging system (8th edition) and had never previously undergone head and neck radiotherapy. Patients diagnosed with NPC were randomized (1:1) to receive the oropharyngeal probiotic intervention (CCRT-P group) or not receive the probiotic intervention (CCRT group) as a control; all patients received CCRT and basic oral hygiene instruction. Patients in the oropharyngeal probiotic group received additional oropharyngeal probiotic intervention for 2 weeks prior to and during CCRT. In addition, participants underwent a series of assessments, including blood tests, abdominal ultrasound, chest computed tomography, head and neck MRI, and whole-body bone scans. The exclusion criteria for patient inclusion were as follows: presence of immune dysfunction; pre-existing OM or recurrent OM prior to CCRT; inability to undergo OM intervention; history of cancer; and allergy to probiotics. This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Wuhan University of Science and Technology (Approval No.: 2021101). All data were obtained after obtaining informed consent from patients. Researchers used the Radiotherapy for Oncology Group (RTOG) guidelines (Table 8) to evaluate RIOM and classified it into grades I to IV according to the severity of OM.

[0156] Test Plan All patients underwent CCRT according to the National Comprehensive Cancer Network (NCCN) guidelines. Prior to the start of radiotherapy, all patients received a full oral clinical examination and oral hygiene instruction. Patients with caries, pulpitis, or gingivitis underwent surgical procedures, including professional scaling, fillings, root canals, and extractions of unsalvageable teeth. All enrolled patients received radiotherapy at 2 Gy five times a week for a total dose of 60-70 Gy for seven weeks, and concurrent chemoradiotherapy according to the regimens listed in Table 9. Table 9: CCRT Dosage

[0157] Participants were randomly assigned to one of the following two groups: • Oropharyngeal probiotic ENT-K12 (CCRT-P) • Control (Untreated) (CCRT) Participants in the CCRT-P group took a single tablet four times daily, in the form of a slow-dissolving tablet, for two weeks before CCRT initiation and seven weeks during radiotherapy. The tablets were taken after breakfast, lunch, dinner, and bedtime, after brushing their teeth and using mouthwash. Participants were instructed to hold the tablets in their mouths until completely dissolved (approximately 4-5 minutes) and to avoid chewing or swallowing them directly. They were also advised not to drink or swallow any substances for two hours after administration of the oropharyngeal probiotic. Clinical symptom observers, including physicians and nurses with standardized training, recorded patients' recurrent episodes (RIOMs) on a daily basis. The primary objectives were the safety of the oropharyngeal probiotic and the severity and timing of RIOMs during CCRT. During the study, the physicians and nurses also recorded the type and severity of adverse events related to the oropharyngeal probiotic as a precaution. A 18-month follow-up was conducted after CCRT to monitor survival, distant metastasis, and NPC recurrence.

[0158] Selection criteria To be eligible for enrollment in this study, participants must meet the following criteria: • Patients who were first diagnosed with stage II nasopharyngeal carcinoma (NPC) at the time of enrollment • Age 18-65 years old Never experienced radiation therapy • No symptoms of active respiratory infection Symptoms of active asepticemia • Active phase symptoms without organ dysfunction • Able to tolerate and complete this study • Written informed consent has been signed Exclusion criteria Participants who meet the following criteria will be excluded from this study: • Patients diagnosed with stage III or IV nasopharyngeal carcinoma (NPC) at the time of enrollment • Patients who have previously received head and neck radiotherapy • Patients with poor oral hygiene and / or who already have severe periodontal disease • Symptoms of active respiratory infection at the time of recruitment Symptoms of active bacteremia Symptoms of active organ dysfunction • Having a chronic respiratory disease that may affect the results of the observation. • Suffering from chronic oral mucositis and ulcers that may affect the observation results Detailed research process outlined in the clinical trial protocol Screening visits and visits for the 1st to 35th radiotherapy sessions During the screening visit (Visit 1), once the diagnosis and radiotherapy schedule are confirmed, the overall details of the study and the procedures to be carried out will be explained to the study participants. Subsequently, the participants will be asked to read the participant information page and read and sign the informed consent form, receiving a signed copy.

[0159] Specifically, the following information needs to be recorded and the process initiated: • Written informed consent form Subject ID and Visit Date Inclusion and exclusion criteria If the research participants meet the recruitment requirements, the following information must be recorded and the process initiated: • Participant eligibility • Demographic data • Medication history • Oral mucositis grading scale; WHO Randomization Study participants will be randomly assigned to one of two groups: a) oropharyngeal probiotic ENT-K12; and b) control (no treatment). Participants in the probiotic group will be provided with a 7-week supply of the study product and instructed to take a single tablet four times daily as a slow-dissolving tablet during the 7-week period of radiotherapy, after breakfast, lunch, dinner, and before bedtime, after brushing their teeth and using mouthwash. Participants should hold the tablet in their mouth until it is completely dissolved (approximately 4-5 minutes) and ensure that they do not chew or swallow it directly. They are advised not to drink or swallow any substance for at least 2 hours after administering the probiotic. Participants are instructed to return all unused study product at their final visit on the day of their radiotherapy session.

[0160] Throughout the study, participants were instructed to follow their standard medication, care, diet, and exercise regimen.

[0161] At any time during the study, participants were instructed to contact the study physician immediately if they experienced any of the following symptoms: respiratory infection, sore throat, fever above 38°C, headache, muscle aches, difficulty breathing, chest tightness, cough, nasal congestion or runny nose, swollen lymph nodes and / or abscesses (pus) or leukoplakia on the tonsils; and symptoms of oral mucositis such as dry mouth, thick saliva, increased mucus, red and shiny gums, white soft spots or pus on the tongue, oral pain, blood in the mouth, or a mild burning sensation when eating. If there was evidence of a respiratory infection or oral mucositis, participants were required to visit the study site for confirmation of the diagnosis.

[0162] Study participants were required to record the number of days they experienced oral mucositis, tongue ulcers, gingival bleeding, and gingival disease during the 15-week study period.

[0163] Self-assessment of overall health Please inquire about the subject's overall health and record the results using a 5-point scale.

[0164] • Schedule of visits for radiotherapy sessions 1 through 35 The study participants need to schedule 35 visits to return to the study site (5 visits per week for 7 weeks throughout the course of radiotherapy, with each visit taking place concurrently with radiotherapy treatment).

[0165] • Final visit appointment On the day the study subjects complete their full course of radiotherapy, the researchers will schedule their final visit back to the research site.

[0166] Medical visits (diagnostic visits for respiratory infections and oral mucositis) During the study, if any participant develops symptoms such as respiratory infection, oral mucositis, or tongue ulcers, they should be instructed to contact the study physician immediately. These symptoms include: Sore throat, Fever exceeding 38°C ·Headache, Muscle soreness, • Difficulty breathing Chest tightness, ·cough, • Nasal congestion or runny nose • Enlarged lymph nodes and / or abscesses (pus) or white patches on the tonsils.

[0167] Dry mouth, ·Saliva is sticky, Increased mucus production • Red, swollen, and shiny gums • White soft spots or pus on the tongue • Oral pain • Blood in the mouth or a slight burning sensation when eating.

[0168] If there is evidence of a respiratory infection, participants will be required to visit the study site. A throat swab will be collected and a rapid swab test will be performed to detect respiratory pathogens. A physician will provide advice on medication / medical treatment as needed. Throughout the study, participants will continue to take the study product as directed. If antibiotic treatment is required, participants will continue to take the study product during the course of antibiotic treatment.

[0169] The following information must be recorded and procedures must be followed during the "medical visit": Subject ID and Visit Date • Diagnosis (respiratory infection, stomatitis, tongue ulcer) (Please specify the type of respiratory infection (e.g., streptococcal pharyngitis / tonsillitis) and whether the infection has been confirmed by rapid throat swab (RAD) or other microbiological tests) • Prescription medications (specify the dates and duration of antibiotic, antiviral, antipyretic / steroid, and antifungal treatments) • Oral mucositis grading scale; WHO • Number of days of oral mucositis and tongue ulcers • Number of days with gingivitis and gum disease • Upper respiratory tract infection progresses to lower respiratory tract infection (e.g., chest CT shows ground-glass opacity). Hospitalization or ICU treatment is required due to severe symptoms and hypoxemia. • Pulmonary complications (decreased FEV1, decreased oxygen diffusion capacity) • Adverse events (Please inquire whether any adverse events occurred in the study subjects; if adverse events occurred during treatment, the type and severity of the adverse event must be noted in a separate "Adverse Events" document) • Self-assessment of overall health status (Please inquire about the overall health status of the research participants and record the results using a 5-point scale).

[0170] Final visit (on the day of the entire radiotherapy course) The following information must be recorded and procedures performed during the final visit: Subject ID and Visit Date • Medical history over the past 7 weeks (upper respiratory tract infection, progression of URTi to LRTi, hospitalization required, pulmonary complications, incidence and cause of diarrhea) (Please specify: the type and frequency of respiratory tract infection, oral mucositis and tongue ulcers not diagnosed during "one or more medical visits", and whether the infection has been confirmed by rapid throat swab (RAD) or other microbiological tests) • Oral mucositis grading scale; WHO • Number of days in the past 7 weeks experiencing oral mucositis and tongue ulcers • Number of days spent experiencing gingivitis and gum disease in the past 7 weeks Adverse events • Compliance (returning unused research products and assessing compliance) • Self-assessment of overall health status (Please inquire about the subject's overall health status and record the results using a 5-point scale) • Antibiotic treatment over the past 7 cycles • Antiviral drug treatment over the past 7 cycles • Antipyretics / steroid treatment over the past 7 cycles • Antifungal drug treatment over the past 7 cycles • Overall assessment of tolerability and treatment efficacy Treatment ended • Follow-up visit appointment (8 weeks after the final visit) Follow-up visits (8 weeks after the final visit) The following information must be recorded and procedures performed during follow-up visits: Subject ID and Visit Date Medical history over the past 8 weeks (upper respiratory tract infection, progression of URTi to LRTi, hospitalization required, pulmonary complications, incidence and cause of diarrhea) (Please specify: type and frequency of upper respiratory tract infection in the past 8 weeks; and whether the infection has been confirmed by rapid throat swab (RAD) or other microbiological tests) • Antibiotic treatment over the past 8 cycles • Antiviral drug treatment over the past 8 cycles • Antipyretic / steroid treatment over the past 8 cycles • Antifungal drug treatment over the past 8 cycles • Oral mucositis grading scale; WHO • Number of days experiencing oral mucositis and tongue ulcers in the past 8 weeks • Number of days spent experiencing gingivitis and gum disease in the past 8 weeks Adverse events • Self-assessment of overall health status (Please inquire about the subject's overall health status and record the results using a 5-point scale) • Research concluded Combined medication Research participants should be questioned about their medication history. Details of any medications taken should be recorded in their medical records and case report forms.

[0171] Research Products The oropharyngeal probiotic tablets taken by participants in the probiotic group were formulated as oral tablets and included the ingredients described below. Each tablet contained at least 1 billion colony-forming units (CFU) of Streptococcus salivarius ENT-K12 (also known as Streptococcus thermophilus subsp. ENT-K12, DSM 34540) during its shelf life.

[0172] Table 10: Research Products

[0173]

[0174] Adverse event classification and assessment The AE will use a three-tiered rating system and report in detail in the CRF: Mild - Well tolerated, causing only slight discomfort and not affecting daily activities.

[0175] • Moderate - Discomfort level sufficient to interfere with daily activities.

[0176] • Severe - Leading to disability and / or hindering normal daily activities.

[0177] Relevance assessment: Researchers should use the following guidelines to determine the study treatment relevance for each AE: • Irrelevant - The event is clearly related to other factors (such as the patient's clinical condition, the treatment intervention administered to the patient, or concomitant medications) and does not conform to the known response pattern of the investigational product.

[0178] • Possibly related - The event has a reasonable time series in terms of the administration of the study product and / or is consistent with the known response pattern of the study treatment, but may also be caused by other factors such as the patient's clinical status, the treatment intervention administered to the patient, or concomitant medication.

[0179] • Clearly relevant - The event has a reasonable time sequence in terms of the timing of the administration of the study product, is consistent with the known response pattern of the study product, and cannot be reasonably explained by other factors such as the patient's clinical condition or the treatment intervention administered to the patient; or it occurs immediately after the administration of the study product, or improves after the discontinuation of the study product, or recurs upon re-administration, or shows a positive reaction at the application site.

[0180] Statistical analysis For continuous numerical variables that conform to a normal distribution with a chi-square test, a t-test was used to compare the means; for continuous numerical variables that do not conform to a normal distribution, a nonparametric test was used. Continuous variable data are reported as mean ± standard deviation or median and upper and lower quartiles. Fisher's exact test was used to compare ratios between two groups. SPSS 26.0 and GraphPad Prism 9 software were used for data analysis and plotting. All tests were two-tailed, and a p-value < 0.05 was considered statistically significant.

[0181] result This randomized controlled trial enrolled 10 patients. Five patients received four oropharyngeal probiotic tablets daily for nine weeks, including two weeks before CCRT and seven weeks during CCRT; the other five patients did not receive oropharyngeal probiotics and served as the control group. Treatment adherence was 100%, and no patients withdrew from the study. As shown in Table 11, there were no differences in baseline characteristics between the two groups. The CCRT-P group consisted of four men and one woman, with two patients newly diagnosed as stage II, two as stage III, and one as stage IV; the CCRT group consisted of three men and two women, all of whom were newly diagnosed as stage III. The median total target volume for the CCRT group and the CCRT-P group were 48.4 and 51.8, respectively. Baseline characteristic analysis showed no significant differences between the CCRT group and the CCRT-P group in terms of sex, age, total radiation target volume, tumor stage / lymph node stage / metastasis stage (TNM staging system), and clinical stage (P>0.05). In this study, no patients experienced treatment interruption or delay due to RIOM.

[0182] Table 11: Demographics and characteristics of patients meeting inclusion criteria

[0183] Preliminary assessments indicated that oropharyngeal probiotic intervention was safe for NPC patients undergoing radiotherapy; furthermore, as shown in Tables 12 and 13, a reduction in both the incidence and severity of radiation-induced oral mucositis (RIOM) was observed. The incidence of RIOM with RTOG ≥ 2 decreased during the 7-week radiotherapy period. Furthermore, the onset of RIOM with RTOG ≥ 2 was delayed by 3 weeks in >60% of patients (week 3 in the control group and week 6 in the probiotic group); and no patients taking oropharyngeal probiotics developed severe RIOM classified as RTOG 3 or 4.

[0184] Table 12: Severity of RIOM during the 7-week study period (using RTOG score)

[0185] Table 13: Progress of RIOM during the 7-week study period (categorized by RTOG level ≥ 2)

[0186] More detailed evaluation showed that 60% of patients experienced severe RIOM (RTOG ≥ 3), and these patients were all in the CCRT (control) group. The incidence of RIOM of RTOG grades 1, 2, 3, and 4 in the CCRT group and CCRT-P group was 0%, 40%, 40%, and 20%, and 40%, 60%, 0%, and 0%, respectively.

[0187] Figure 1 The mean RTOG score of patients during the 7-week CCRT is shown. In the CCRT group and CCRT-P group, the onset of RIOM (RTOG=1) occurred during weeks 2 and 3 of the CCRT course, respectively. At week 7 of CCRT, the mean maximum severity of RIOM decreased by 42.9% in the CCRT-P group; the mean RTOG was observed in the CCRT group and CCRT-P group at 2.8±0.84 and 1.6±0.55, respectively. The mean interval from the start of CCRT to the attainment of maximum RIOM severity was 5 weeks and 4 weeks in the CCRT group and CCRT-P group, respectively. At week 5 of the CCRT course, a significant difference in RIOM distribution was observed between the two groups (p=0.0309), with mean RTOGs of 2.6±0.55 and 1.0±0.71 observed in the CCRT group and CCRT-P group, respectively. Table 14 shows the detailed average RTOG scores of patients at different clinical stages during the 7-week CCRT course. It can be seen that there is no significant correlation between the average change in RIOM severity and clinical stage, but the severity of RIOM gradually increases over time as the CCRT course progresses. Overall, a trend of approximately half the RIOM severity and delayed onset was observed in the CCRT-P group. During the 7-week CCRT course, administration of oropharyngeal probiotics significantly protected patients from severe RIOM (RTOG ≥ 3) onset. Table 15 shows the average interval between the start of the CCRT course and the onset of different grades of RIOM during the 7-week CCRT course. In the CCRT group, the average time to RIOM onset of RTOG I to IV was 17 days, 20 days, 27 days, and 41 days, respectively; while in the CCRT-P group, the average time to RIOM onset of RTOG I to II was 26 days and 34 days, respectively. Furthermore, no patients in the CCRT-P group experienced severe RIOM with RTOG ≥ 3. Compared with the CCRT group, the median delay in RIOM for RTOG I was observed in the CCRT-P group by 9 days, and the median delay in RIOM for RTOG II was observed by 14 days. Figure 2-5 The Kaplan-Mel curves in the figure show a comparison of the time to first RTOG I-IV RIOM in each patient in the two groups. The time from the start of the CCRT course to the first RTOG I RIOM was 14 to 23 days in the CCRT group, with a median of 17 days; while in the CCRT-P group experiencing mild pain, it was 19 to 37 days, with a median of 26 days. Figure 2 Similarly, the time from the start of CCRT treatment to the first RTOG II RIOM attack ranged from 18 to 25 days in the CCRT group, with a median of 20 days; while in the CCRT-P group, 3 out of 5 patients experienced 28 to 39 days, with a median of 34 days, and the other 2 patients in the CCRT-P group did not experience moderate pain. Figure 3 The time from the start of CCRT treatment to the first RTOG grade III RIOM was 24 to 32 days in 3 out of 5 patients in the CCRT group, with a median of 27 days; while no patients in the CCRT-P group experienced severe RIOM. Figure 4 Only one patient in the CCRT group experienced bleeding and necrosis, while no patients in the CCRT-P group experienced this type of pain. Figure 5 ).

[0188] Table 14: Mean RTOG score of patients during 7 weeks of CCRT

[0189] Table 15: Average interval between the start of CCRT and the onset of different grades of RIOM during a 7-week CCRT course.

[0190] No adverse events related to oropharyngeal probiotics were reported throughout the study. All 10 patients in this study survived for more than 18 months after CCRT, and all patients were observed to have stable disease during the 18-month follow-up period, with no NPC recurrence or distant metastasis. This indicates that the oropharyngeal probiotic DSM 34540 is safe for NPC patients during CCRT and has no cariogenic effect.

[0191] RIOM (Recurrent Occurrence of Occurrence) negatively impacts patients' quality of life, physical function, emotional function, and role function, and carries a risk of malnutrition—all factors that influence treatment outcomes. Therefore, advanced clinical practice is needed to help healthcare professionals prioritize effective interventions to reduce the burden of cancer therapy and improve their patients' clinical outcomes. However, treating RIOM remains challenging. Although various preventative drugs and natural preparations have undergone clinical trials, cost-effective or uncontroversial data are scarce. For example, drugs that stimulate epithelial cell proliferation may simultaneously promote cancer cell growth, requiring careful consideration in their application; they can significantly increase healthcare costs for cancer patients and create additional health consultation needs and financial burdens. This study demonstrates that supplementing with oropharyngeal probiotics two weeks before and during CCRT significantly benefits NPC patients undergoing CCRT: delaying the onset of RIOM and substantially reducing the incidence of severe RIOM. Safety was confirmed by the absence of any adverse events related to oropharyngeal probiotics during the 7-week study period, and no NPC recurrence or distant metastasis was observed during the 18-month follow-up.

[0192] Example 2: Prevention of oral mucositis in subjects who underwent hematopoietic stem cell transplantation by the oropharyngeal probiotic ENT-K12 Autologous hematopoietic stem cell transplantation (autologous HSCT) is a standard treatment option for patients with multiple myeloma who meet the criteria for high-dose therapy, lymphoma patients who have undergone second-line treatment, and patients with acute myeloid leukemia. Pre-HSCT immune system damage and chemotherapy significantly increase the risk of infection within 100 days post-transplantation, especially early-onset respiratory infections (Scarlatania). et al ., Eur Respir J. 49 (2017), 1601902. Therefore, respiratory viral infections are more common in patients who have already experienced (HSCT), and these infections in this patient population often present as upper respiratory tract infections, but can rapidly progress to lower respiratory tract infections and are associated with mortality.

[0193] Approximately 10% of patients with autologous HSCT develop signs or symptoms of common respiratory viral infections after hospitalization. Common respiratory viral pathogens include influenza virus, parainfluenza virus, adenovirus, human rhinovirus, and human coronavirus. Of these respiratory viral infections, approximately 45% develop lower respiratory tract infections, and 7% require transfer to the intensive care unit (Morley). et al., Infect Dis (Lond). 53 (2021), 274-280; Marinelli et al., BiolBlood Marrow Transplant. 26 (2020), 782-788. In hematopoietic stem cell transplant recipients and patients with hematologic malignancies, the progression of respiratory viral infections, along with high mortality, has increasingly been recognized as a significant cause of morbidity and mortality. (Fontana and Strasfeld, Infect Dis Clin North Am. 33(2019), 523-544.) Prevention of respiratory infections in HSCT patients is of great importance. It has been reported that specific drugs can be successfully used after molecular diagnostic testing and multiplex assays; however, nasal swabs are not routinely performed during hospitalization of transplant recipients, and clinicians often prescribe antibiotics based on clinical experience. Regarding the reconstitution of gut microbiota dysbiosis and its immunomodulatory effects after HSCT, numerous studies have shown that the administration of gut probiotics and prebiotics is a safe and well-tolerated treatment in adult and pediatric autologous and allogeneic HSCT patients, and may help reduce the incidence of gastrointestinal toxicity or graft-versus-host disease (GVHD); see Mizutani, for example. et al ., Intern Med. 62 (2023), 2949-2958; Andermann et al ., TransplantCell Ther. 27 (2021), 932.e1-932.e11; Ladas et al ., Bone Marrow Transplant. 51(2016), 262-266; Gorshein et al ., Clin Transplant. 31 (2017), 10.1111 / ctr.12947; Yazdandoust et al ., Transpl Immunol.78 (2023), 101836. To explore other methods to reduce the risk of RTI and the progression of upper respiratory tract infection to lower respiratory tract infection in patients with autologous HSCT, this exploratory randomized clinical trial aimed to investigate whether oropharyngeal probiotics are associated with the prevention or reduction of respiratory tract infection incidence and severity, thereby reducing antibiotic use after autologous HSCT.

[0194] Furthermore, oral mucositis is a common disabling dose-limiting toxicity in HSCT. The oral microbiota undergoes significant changes after HSCT and takes up to three months to recover to its original composition, making it one of the microbial-driven risk factors for oral mucositis. Specifically, changes in microbial diversity and similarity are more pronounced and rapid in patients developing ulcerative oral mucositis. Studies have reported that *Streptococcus salivarius* species can be detected before HSCT, but this genus gradually decreases and is replaced by coagulase-negative staphylococci (Salivarius species). Staphylococci ) bacteria replacement, Candida spp. after HSCT ( Candida ) species increase and Enterococcus spp. ( Enterococcus Species identification was significantly associated with ulcerative oral mucositis after HSCT; while patients who did not develop ulcerative oral mucositis had a more stable microbial ecosystem.

[0195] This study's product contains the oropharyngeal probiotic strain *Streptococcus thermophilus* subsp. *salivarius* ENT-K12 (DSM 34540), a probiotic strain targeting the oral cavity and upper respiratory tract, associated with ear, nose, and throat health. Previous clinical studies have demonstrated the safety and efficacy of daily administration of *Streptococcus thermophilus* subsp. *salivarius* ENT-K12 in reducing respiratory infections and key respiratory pathogens. The rationale for this study is to verify the safety and efficacy of the oropharyngeal probiotic ENT-K12 in patients undergoing hematopoietic stem cell transplantation (HSCT). The primary objective of this study is to evaluate the effectiveness of the oropharyngeal probiotic ENT-K12 in reducing respiratory infections and oral mucositis in patients undergoing HSCT.

[0196] Table 16: Oral Mucositis Grading Scale Developed by WHO (World Health Organization)

[0197] Main objectives To conduct a multicenter, open-label, randomized controlled exploratory trial to evaluate the efficacy of the oropharyngeal probiotic ENT-K12 in the prevention of respiratory tract infection and oral mucositis within 100 days after hematopoietic stem cell transplantation (HSCT).

[0198] Secondary objectives • Evaluate the efficacy of the oropharyngeal probiotic ENT-K12 in reducing the incidence of viral respiratory infections during 100 days post-HSCT. • Evaluate the efficacy of the oropharyngeal probiotic ENT-K12 in reducing the incidence of bacterial respiratory infections. • Evaluate the efficacy of the oropharyngeal probiotic ENT-K12 in reducing the incidence of fungal respiratory infections. • Evaluate the efficacy of the oropharyngeal probiotic ENT-K12 in reducing the severity of oral mucositis and xerostomia (based on the Oral Mucositis Grading Scale; WHO). • Evaluate the effect of oropharyngeal probiotic ENT-K12 on the recovery of T lymphocyte subsets • Evaluate the effects of oropharyngeal probiotic ENT-K12 on immune regulation via salivary cytokines • Number of days of antibiotic treatment • Number of days of antiviral drug treatment (ribavirin / pallvizumab) • Number of days of antipyretic / steroid treatment • Number of days of antifungal medication treatment ·Number of days experiencing oral mucositis ·Number of days experiencing gingivitis • Progression of URTi to LRTi (chest CT shows ground-glass opacity) • Hospitalization (ICU) required due to severe symptoms and hypoxemia. • Pulmonary complications (decreased FEV1, decreased oxygen diffusion capacity) • Incidence and causes of diarrhea • Incidence and grading of acute graft-versus-host disease (GvHD) ·mortality rate • Confirm subject safety through adverse event reporting Research Summary This study aimed to determine the preventive effect of the oropharyngeal probiotic ENT-K12 on respiratory tract infections and oral mucositis in patients who have undergone hematopoietic stem cell transplantation (HSCT).

[0199] According to the 2016 WHO guidelines, 60 patients were identified as eligible for HSCT (Hospital-Heart Transplantation) recipients; aged 18 to 65 years; with an Eastern Cooperative Oncology Group (ECOG) performance status between 0 and 2; good hematopoietic reconstitution after HSCT; and no active symptoms of lower respiratory tract infection, chronic respiratory disease, or organ dysfunction. Ultimately, 16 patients meeting these criteria participated in the study. Participants were randomly assigned to one of the following two treatment groups: Oropharyngeal probiotics ENT-K12 • Control group (untreated) Subjects in the probiotic group showed good reconstitution of their hematopoietic system after HSCT (defined as platelet count >20). 10 9 Cells / L, and neutrophils >0.5 10 9 For 100 days after administration of the oral probiotic tablets (cells / L), participants took the tablets three times daily in slow-dissolving tablet form, after breakfast, lunch, and before bedtime, after brushing their teeth and rinsing with mouthwash. Participants were instructed to hold the tablets in their mouths until completely dissolved (approximately 4-5 minutes) and ensure that they did not chew or swallow the tablets directly; they were also advised not to drink or swallow any substances for 2 hours after administration of the oral probiotics.

[0200] From the start of the pretreatment protocol to day 130, including a 100-day intervention and a 30-day follow-up period, participants' diagnoses and pretreatment protocols were extracted from their medical records. Each episode of respiratory infection was recorded weekly; if a participant developed respiratory infection symptoms during the study, such as sore throat, fever greater than 38°C, headache, muscle aches, dyspnea, chest tightness, cough, nasal congestion or runny nose, swollen lymph nodes, tonsillar abscess, or white patches, the participant was instructed to contact the study physician. Any adverse events occurring at each visit were recorded. At the end of the study, participants were required to return any unused probiotics; adherence was assessed by counting the remaining tablets at the end of each visit, with adherence considered satisfactory if ≥90% of the dispensed tablets had been used.

[0201] Study population Patients diagnosed as HSCT recipients according to the 2016 WHO criteria, aged 18 to 65 years, with an Eastern Cooperative Oncology Group (ECOG) performance status between 0 and 2, good hematopoietic reconstitution after HSCT, and no active symptoms of lower respiratory tract infection, chronic respiratory disease, or organ dysfunction, were eligible for this study. Accordingly, 16 patients meeting these criteria were recruited and randomly assigned to either the probiotic group or the control group.

[0202] Selection criteria To be eligible for enrollment in this study, participants must meet the following criteria: • Patients diagnosed as HSCT recipients according to the 2016 WHO guidelines • Age 18-65 years old • Eastern Cooperative Oncology Group (ECOG) performance status 0-2 • No symptoms of active respiratory infection Symptoms of active asepticemia • Active phase symptoms without organ dysfunction • Able to tolerate and complete this study • Written informed consent has been signed Exclusion criteria Participants who meet the following criteria will be excluded from this study: • Eastern Cooperative Oncology Group (ECOG) performance status ≥3 • Failure of hematopoietic reconstitution, defined as platelet count <20 after HSCT. 10 9 Cells / L and neutrophils <0.5 10 9 Cells / L

[0203] • Symptoms of active respiratory infection at the time of recruitment Symptoms of active bacteremia Symptoms of active organ dysfunction • Having a chronic respiratory disease that may affect the results of the observation. Research Process During screening visits, once a platelet count ≥ 20 is achieved after HSCT... 10 9 Cells / L and neutrophils ≥ 0.5 10 9 Upon confirmation of hematopoietic reconstitution with a cell count / L, the study participants will be informed of the overall details of the study and the procedures to be performed. Subsequently, participants will be asked to read the participant information page and sign the informed consent form, receiving a signed copy.

[0204] Specifically, the following information needs to be recorded and the process initiated: • Written informed consent form Subject ID and Visit Date Inclusion and exclusion criteria If the research participants meet the recruitment requirements, the following information must be recorded and the process initiated: • Participant eligibility • Demographic data • Medication history • Oral mucositis grading scale; WHO Randomization Study participants will be enrolled in the study by randomization and will be assigned to one of the following two treatment groups: a) oropharyngeal probiotic ENT-K12; b) control (no treatment).

[0205] Study participants will receive a 100-day supply of the research product and will be instructed to take three single tablets daily after breakfast, lunch, and before bedtime, after brushing their teeth and using mouthwash. Participants should leave the tablets sublingually until completely dissolved (approximately 4-5 minutes) and ensure they do not chew or swallow them directly. They are advised not to drink or swallow any substances for at least 2 hours after administering the probiotics. Participants are instructed to return any unused research product at their final visit.

[0206] Throughout the study, participants were instructed to follow their standard medication, care, diet, and exercise regimen. At any time during the study, participants were instructed to immediately contact the study physician if they developed respiratory infection-related symptoms, such as sore throat, fever above 38°C, headache, muscle aches, difficulty breathing, chest tightness, cough, nasal congestion or runny nose, swollen lymph nodes and / or tonsillar abscesses (pus) or leukoplakia; and symptoms of oral mucositis such as dry mouth, thick saliva, increased mucus, red and shiny gums, white soft spots or pus on the tongue, oral pain, blood in the mouth, or a mild burning sensation when eating. If there was evidence of a respiratory infection or oral mucositis, participants were required to visit the study site for confirmation of the diagnosis.

[0207] • Collect blood samples to detect T lymphocyte subsets (according to standard medical procedures). • Saliva samples were collected to detect immune regulation (0, 1, 3, 7, 100, 130). • Collect pharyngeal and oral mucosal swab samples to detect major respiratory pathogens (including Candida albicans). C. albicans ()) and the reconstruction of the oral microbiota) • Self-assessment of overall health status (Please inquire about the subject's overall health status and record the results using a 5-point scale) • Final visit appointment Study participants will need to schedule their final visit back to the study site (on day 100). Participants will be asked to record the number of days they experienced symptoms of respiratory infection, oral mucositis, and gingivitis over the past 100 days (while healthcare professionals or medical students will conduct weekly telephone follow-ups to monitor their health).

[0208] Medical visits (diagnostic visits for respiratory infections and oral mucositis) During the study, if any participant develops symptoms of respiratory infection or oral mucositis, they should be instructed to contact the study physician immediately. These symptoms may include: Sore throat, Fever exceeding 38°C ·Headache, Muscle soreness, • Difficulty breathing Chest tightness, ·cough, · Nasal congestion or runny nose • Swollen lymph nodes and / or abscesses or white patches on the tonsils.

[0209] Dry mouth, ·Saliva is sticky, Increased mucus production • Red, swollen, and shiny gums • White soft spots or pus on the tongue • Oral pain • Blood in the mouth or a slight burning sensation when eating If there is evidence of a respiratory infection, participants will be required to visit the study site. A throat swab will be collected and a rapid swab test will be performed to detect respiratory pathogens. A physician will provide advice on medication / medical treatment as needed. Throughout the study, participants will continue to take the study product as directed. If antibiotic treatment is required, participants will continue to take the study product during the course of antibiotic treatment.

[0210] The following information must be recorded and procedures must be followed during the "medical visit": Subject ID and Visit Date • Diagnosis (respiratory infection, stomatitis, diarrhea, acute GvHD) (Please specify the type of respiratory infection (e.g., streptococcal pharyngitis / tonsillitis, respiratory syncytial virus infection), and whether the infection has been confirmed by rapid throat swab (RAD) or other microbiological tests) Prescription medications (specifying antibiotics, antiviral drugs, antipyretics / steroids, and antifungal drugs) • Oral mucositis grading scale; WHO ·Number of days of oral mucositis ·Number of days of gingivitis • Upper respiratory tract infection progresses to lower respiratory tract infection (e.g., chest CT shows ground-glass opacity). Hospitalization or ICU treatment is required due to severe symptoms and hypoxemia. • Pulmonary complications (decreased FEV1, decreased oxygen diffusion capacity) • Adverse events (Please inquire whether any adverse events occurred in the study subjects; if adverse events occurred during treatment, the type and severity of the adverse event must be noted in a separate "Adverse Events" document) • Self-assessment of overall health status (Please inquire about the overall health status of the research participants and record the results using a 5-point scale).

[0211] Follow-up visit (30 days after the final visit) The following information must be recorded and procedures performed during follow-up visits: Subject ID and Visit Date • Medical history in the past 30 days (upper respiratory tract infection, progression of URTi to LRTi, need for hospitalization, pulmonary complications, incidence and cause of diarrhea, incidence and grading of acute GvHD) • Oral mucositis grading scale; WHO • Number of days with oral mucositis in the past 30 days • Number of days you have experienced gingivitis in the past 30 days (please specify the type and frequency of any respiratory infections and oral mucositis experienced in the past 30 days, and whether the infection has been confirmed by rapid throat swab (RAD) or other microbiological tests) Adverse events • Self-assessment of overall health status (Please inquire about the subject's overall health status and record the results using a 5-point scale) Antibiotic treatment within the past 30 days • Antiviral drug treatment (ribavirin / palizumab) within the past 30 days • Antipyretics / steroid treatment within the past 30 days Antifungal medication treatment within the past 30 days • Research concluded Subjects withdraw from study Participants will be withdrawn from the study under the following circumstances: • Participants may choose to withdraw from this study on their own initiative; • If any research participant violates the original criteria; • If a research subject is deemed unfit to continue the study at any point in time, the researcher has the authority to make that decision.

[0212] Combined medication Research participants should be questioned about their medication history. Details of any medications taken should be recorded in their medical records and case report forms.

[0213] Research Products Participants in the probiotic group will take oropharyngeal probiotic tablets, formulated as oral tablets containing the ingredients mentioned below. Each tablet contains at least 1 billion colony-forming units (CFU) of Streptococcus salivarius ENT-K12 (also known as Streptococcus thermophilus subsp. ENT-K12) during its shelf life.

[0214] Table 17: Research Products

[0215]

[0216] Adverse event classification and assessment The AE will use a three-tiered rating system and report in detail in the CRF: Mild - Well tolerated, causing only slight discomfort and not affecting daily activities.

[0217] • Moderate - Discomfort level sufficient to interfere with daily activities.

[0218] • Severe - Leading to disability and / or hindering normal daily activities.

[0219] Relevance assessment: Researchers should use the following guidelines to determine the study treatment relevance for each AE: • Irrelevant - The event is clearly related to other factors (such as the patient's clinical condition, the treatment intervention administered to the patient, or concomitant medications) and does not conform to the known response pattern of the investigational product.

[0220] • Possibly related - The event has a reasonable time series in terms of the administration of the study product and / or is consistent with the known response pattern of the study treatment, but may also be caused by other factors such as the patient's clinical status, the treatment intervention administered to the patient, or concomitant medication.

[0221] • Clearly relevant - The event has a reasonable time sequence in terms of the timing of the administration of the study product, is consistent with the known response pattern of the study product, and cannot be reasonably explained by other factors such as the patient's clinical condition or the treatment intervention administered to the patient; or it occurs immediately after the administration of the study product, or improves after the discontinuation of the study product, or recurs upon re-administration, or shows a positive reaction at the application site.

[0222] Statistical analysis For continuous numerical variables that follow a normal distribution with homoscedasticity, t-tests were used to compare means; for continuous numerical variables that do not follow a normal distribution, nonparametric tests were used. Data for continuous variables are expressed as mean ± standard deviation or median and upper and lower quartiles. Chi-square tests were used to compare rates between two groups. Data were analyzed and plotted using SPSS 26.0 and GraphPad Prism 9 software. All tests were two-tailed tests; a p-value < 0.05 was considered statistically significant.

[0223] result Sixteen patients (six women and ten men, mean age 39.8 years (range 28–53 years)) with hematopoietic malignancies who had undergone autologous HSCT were enrolled in this study. Seven patients were randomly assigned to receive oropharyngeal probiotics, while the remaining nine served as a control group and did not receive probiotics. Treatment adherence was good in this trial, with no patients withdrawing from the study. Patient characteristics under randomization are summarized in Table 18.

[0224] Table 18: Demographic characteristics of autologous HSCT recipients at enrollment

[0225] The most common underlying disease was non-Hodgkin's lymphoma (100% in the probiotic group, 88.9% in the control group), with the remainder being Hodgkin's lymphoma. In the probiotic group, 2, 4, and 1 patients, respectively, received BEAM, BuCyE, and BUCY conditioning regimens before autologous HSCT; while in the control group, 5, 1, and 3 patients, respectively, received BEAM, BuCyE, and BUCY conditioning regimens before autologous HSCT. The two groups were similar in the following aspects: age, sex distribution, BMI, disease status at the time of autologous HSCT, and CD34 infusion. + The median number of cells / kg, serum albumin level, length of hospital stay, weight loss during hospitalization, duration of thrombocytopenia, duration of fever, and number of days of prophylactic antibiotic use before HSCT were also compared between the two groups. The incidence of febrile neutropenia, positive sore throat, and xerostomia was also similar between the two groups. Patients in the control group had higher mean C-reactive protein levels and longer duration of neutropenia at enrollment, but the differences were not statistically significant. One patient in the probiotic group had bloodstream infection (BSI) recorded at enrollment, but the incidence of BSI was not significantly different between the two groups.

[0226] Results regarding the effects of the oropharyngeal probiotic Streptococcus salivae ENT-K12 on oral mucositis During the 100-day period following hematopoietic reconstitution, ulcerative oral mucositis was monitored three times monthly, with a follow-up period of 30 days. Preliminary assessment indicated that oropharyngeal probiotic intervention was safe (no adverse events observed) in HSCT recipients whose hematopoietic system was well reconstituted after HSCT, with a reduced incidence of ulcerative oral mucositis observed throughout the 130-day study period. Specifically, according to WHO classification criteria, the incidence of ≥2 grade oral mucositis was reduced in patients receiving oropharyngeal probiotics, i.e., a higher event-free rate compared to the control group. No ulcerative oral mucositis was observed in patients in the probiotic group during the 100-day oropharyngeal probiotic intervention period. See [link to relevant documentation]. Figure 6 .

[0227] Given that HSCT patients are in an immunosuppressed state, extra caution and a comprehensive safety evaluation are required before administering probiotics; in this study, patients only began probiotic administration after successful hematopoietic reconstitution. Sepsis is one of the most serious consequences of cancer therapy-induced mucositis, which can lead to treatment interruption and even death. In this preliminary study, no patients developed sepsis from probiotic administration, and no probiotic-related side effects were reported throughout the study period. Furthermore, patients taking oropharyngeal probiotics were completely protected from developing ulcerative oral mucositis.

[0228] Results regarding the effects of the oropharyngeal probiotic *Streptococcus salivarius* ENT-K12 on respiratory tract infections. In addition to evaluating the clinical trial results related to oral mucositis, the efficacy and safety of oropharyngeal probiotics in treating respiratory infection-like symptoms in lymphoma patients after autologous hematopoietic stem cell transplantation were also analyzed.

[0229] During the 100-day period following HSCT, a trend of more than half reduction in the incidence of respiratory tract infections (RTi) was observed in patients administering oropharyngeal probiotics (29% vs. 67%; p=0.131). The incidence of upper respiratory tract infection (URTi) progressing to lower respiratory tract infection (LRTi) was 0% in the probiotic group and 16.7% in the control group. Similarly, during the 100-day period following HSCT, a trend of approximately two-thirds reduction in the incidence of RTi was observed in the probiotic group (0.43% vs. 1.00%; p=0.094). Regarding RTi-like symptoms, the duration of cough, runny nose, and sore throat was shorter in the probiotic group than in the control group at 100 days post-HSCT (3 days, 1 day, 0 days vs. 17 days, 15 days, 4 days; p=0.090, 0.059, 0.102). Furthermore, during the 100-day period following HSCT, the number of medication days in the probiotic group was observed to be more than 1 / 10 shorter than that in the control group, particularly with a significant statistical difference in the number of days of antibiotic use (0 days of antiviral agents, 1 day of antifungal agents, and 0 days of antibiotics vs. 1 day of antiviral agents, 0 days of antifungal agents, and 15 days of antibiotics; p=0.378, 0.257, 0.024). However, during the 30-day follow-up period, the mean number of medication days was similar between the two groups (0 days of antibiotic use vs. 0.7 days; p=0.170). During the 100-day period following HSCT, the mean duration of each RTI flare-up in the probiotic group was shortened by half compared to the control group. However, with the aforementioned reduced antibiotic use, the mean duration of each RTI flare-up was very similar between the two groups (1.4 days vs. 0.7 days; p=0.816). Statistically significant differences were observed in the average number of days of antibiotic use throughout the 130-day study period: the number of days of antibiotic use per person in the probiotic group was significantly lower than that in the control group (0 days vs. 15 days; p=0.011), as shown in Table 21.

[0230] Table 19. Clinical outcomes associated with respiratory tract infections during 100 days of oropharyngeal probiotic administration.

[0231] Mann-Whitney U test Chi-square test Table 20: Clinical outcomes related to respiratory infection during the 30-day follow-up period.

[0232] Mann-Whitney U test Chi-square test Table 21: Clinical outcomes related to respiratory infection during the entire 130-day study period

[0233] Mann-Whitney U test Chi-square test The patient in the probiotic group who had a positive blood culture and bacteremia at enrollment did not develop sepsis throughout the study period. In the control group, one of the nine patients developed sepsis complicated by pneumonia after HSCT.

[0234] Kaplan-Meyer analysis showed that the probability of not experiencing RTi attacks was consistently higher in the probiotic group than in the control group (p=0.071). Figure 7 Meanwhile, the cumulative duration of RTi-like symptoms was consistently shorter in the probiotic group (p=0.131). Figure 8 This resulted in the probiotic group having a significantly fewer cumulative days of antibiotic use throughout the study period (p=0.011). Figure 9 As can be seen, the cumulative number of days of antibiotic use in the control group increased significantly with the cumulative duration of their RTI-like symptoms; meanwhile, because the RTI-like symptoms were milder in the probiotic group, clinicians did not prescribe antibiotics. Therefore, during this study, patients in the probiotic group did not require prescriptions for antibiotic treatment or prophylactic antibiotics.

[0235] No adverse events related to oropharyngeal probiotics were reported throughout the study period; all 7 patients in the probiotic group survived and did not show signs of BSI, distant metastasis or lymphoma recurrence during the 24 months following the completion of the study, indicating that oropharyngeal probiotic administration is safe for patients with well-reconstructed hematopoietic systems after HSCT.

[0236] Prior to allogeneic or autologous HSCT, recipients must undergo chemotherapy or radiation therapy pretreatment to eliminate their own hematopoietic stem cells and, as far as possible, residual leukemia cells, thereby promoting successful engraftment during HSCT; however, this chemotherapy or radiation therapy can lead to immune system suppression, microbiome dysbiosis, bacterial translocation, and pro-inflammatory cytokine responses. Common sources of inflammation in HSCT patients include bacterial, viral, and fungal infections, as well as chronic inflammatory diseases, autoimmune diseases, and neurological disorders. Antibiotic prophylaxis has been used to protect HSCT recipients from infection and has prolonged their survival, but its long-term consequences have remained controversial for decades (Sepkowitz). 29 ( ., Bone Marrow Transplant.2002), 367-371; Tabarraee et al., Iran J Pharm Res. 15(Suppl) (2016), 159-163; César-Arce et al ., Transplant Proc. 49 (2017), 1444-1448; Wang et al ., J Microbiol Immunol Infect. 51 (2018), 123-131; Kimura et al., J Infect. 69 (2014), 13-25. There are also reports that antibiotic exposure is not associated with immediate post-transplant infectious complications, but it can adversely affect long-term overall survival after HSCT (Gromowsky). et al., Blood 140 (Supplement 1)(2022), 7716-7717; Mokhtar et al., Nutrients. 14 (2022), 112. Similarly, the rational and prudent use of appropriate antifungal treatment remains a key component of the treatment regimen.

[0237] During this study, it was demonstrated that adjuvant therapy with oropharyngeal probiotics effectively reduced the incidence of RTI in patients after HSCT, shortened the duration of respiratory symptoms, and reduced the number of days of antibiotic use.

[0238] Example 3: Salinomyces ENT-K12 promotes the healing of chemotherapy-induced oral mucositis in mice.

[0239] Having successfully demonstrated the significant advantages of administering the probiotic *Streptococcus salivarius* ENT-K12 in delaying the onset of RIOM, substantially reducing the incidence of severe RIOM (Example 1), and completely protecting patients undergoing HSCT from developing ulcerative OM (Example 2), further studies using mice analyzed the effects of *Streptococcus salivarius* ENT-K12 on chemotherapy-induced oral mucositis, particularly on the epithelial layer. Additionally, studies were conducted to demonstrate the effect of heat-killed *Streptococcus salivarius* ENT-K12 on oral epithelial cells damaged by chemotherapy.

[0240] Preliminary studies were conducted using mouse models, which included treatment with busulfan and cyclophosphamide chemotherapy as described below.

[0241] Experimental group: (40 male C57BL / 6 mice aged 6-8 weeks) 1) Blank control group (n = 10): No intervention was performed; 2) Disease damage group (n = 10): Busulfan + cyclophosphamide combined with chemotherapy; 3) Probiotic intervention group A (n = 10): Busulfan + Cyclophosphamide + heat-killed probiotics intervention; 4) Probiotic intervention group B (n = 10): Busulfan + Cyclophosphamide + live probiotic intervention.

[0242] Material: DMSO (for dissolving busulfan and cyclophosphamide), 4% paraformaldehyde, 0.1% heparin, PBS, sodium pentobarbital, busulfan, cyclophosphamide, probiotic tablets, heat-killed probiotics, 100 µl pipette, 1 ml syringe, etc.

[0243] Reagent preparation: Dissolve 100 mg of busulfan in 25 ml of DMSO to prepare a 4 mg / ml solution for later use; dissolve 0.25 g of cyclophosphamide in 25 ml of DMSO to prepare a 10 mg / ml solution for other uses. All solutions should be wrapped in aluminum foil and stored refrigerated away from light.

[0244] Application method and dosage: i) Busulfan + Cyclophosphamide combined chemotherapy: Intraperitoneal injection: once daily at a fixed time. Weight must be measured before injection, and the dosage to be administered must be calculated (dosage: busulfan (BU) dose is 80 mg / kg, cyclophosphamide (CY) dose is 200 mg / kg).

[0245] ii) Live probiotic intervention: A probiotic solution was prepared by dissolving probiotic tablets (the study products described in Examples 1 and 2), and 100 µl of the probiotic solution was pipetted into the oral cavity of mice daily from day 1 to day 6. Mice were fasted and deprived of water for 30 minutes after drug delivery to retain the probiotics in the oral cavity as much as possible. A blank control group was administered 100 µl of physiological saline as a placebo using the same method. All mice were sacrificed on day 7.

[0246] iii) Heat-killed probiotic intervention: Oral administration as described above.

[0247] Chemotherapy administration timeframe (Busulfan for the first 4 days, and cyclophosphamide injection for the last 2 days)

[0248] tissue collection Mice were euthanized using sodium pentobarbital at a designated time point (day 7 post-treatment). Following euthanasia, the animals were perfused with 0.1 M phosphate-buffered saline (PBS) containing 0.1% heparin, followed by perfusion with 4% paraformaldehyde mixed with PBS to immediately fix the tongue tissue. After dissection, the tongue was segmented and fixed in 4% paraformaldehyde solution for 3 hours, then divided into three sections. The tissues were then cryoprotected by sequentially incubating in a series of sucrose concentrations (0.5 M, 1.0 M, and 1.5 M), then embedded in an OCT compound and stored at -80°C. The tissue sections were cut to a thickness of 12 μm and directly attached to glass slides. These slides were stored at -80°C until processing was required.

[0249] detection indicators HE staining Observation of morphological changes in taste buds.

[0250] Steps: (1) Dewaxing paraffin sections to water. Place the sections in xylene I for 10 min, xylene II for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, and 70% ethanol for 5 min, and wash with distilled water. (2) Hematoxylin staining of cell nuclei. Stain the sections with Harris hematoxylin for 3-8 min, wash with tap water, differentiate with 1% hydrochloric acid ethanol for a few seconds, rinse with tap water, then blue with 0.6% ammonia water, and rinse with running water. (3) Eosin staining of cytoplasm. Place the sections in eosin staining solution for 1-3 min. (4) Dehydration and mounting. Place the sections in 95% ethanol I for 5 min, 95% ethanol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min. (5) Microscopic examination, image acquisition and analysis.

[0251] Toluidine blue staining The tongue was stained with 1% toluidine blue in 10% acetic acid for 1 minute, followed by repeated washing with acetic acid to reveal surface erosions or ulcers (Muanza). et al., Clin Cancer Res 11 (2005), 5121). The percentage of toluidine blue-positive surface area was calculated using ImageJ software (excluding excised lesions). Tissues were then fixed in 4% (v / v) paraformaldehyde solution in PBS at 4°C for 2 hours and then embedded in paraffin or OCT. Epithelial thickness of the tongue and esophagus, and villus length of the jejunum were measured in H&E-stained tissues using ImageJ. Three mice per group, three fields of view per sample, and five measurements per field were performed blinded. Images were acquired using a Zeiss Axio Imager M1 microscope and an EC-Plan-Neofluar 920-NA 0.5 air objective, and the AXIOVISION-SE64 Rel.4.9.1 program (Bertolini). et al ., Transl Oncol 10 (2017), 612-620).

[0252] Immunohistochemical staining of Ki67 To compare the effects of probiotic intervention on cell proliferation, it is a protein expressed in cells at all stages of the cell cycle except for G0 and early G1 phases.

[0253] Procedure: In this process, slides were first washed in PBS and then incubated in 10 mM sodium citrate (pH 6.0) at 95°C for 15 min to induce antigen retrieval, followed by cooling to room temperature. After washing in PBS, tissues were placed in a mixture of 5% NGS, 1% BSA, and 0.3% Triton-X 100 and blocked at room temperature for 1 hour. Rabbit anti-Ki67 primary antibody was added to the tissues, and then covered with a hybridization strip and incubated overnight at 40°C. The next day, sections were washed in PBS and then incubated in Alexa 546 goat anti-rabbit secondary antibody at room temperature for two hours. Sections were then washed in PBS and then incubated in Sytox Green for double labeling of cell nuclei. After washing again, coverslips were placed using Fluoromount G (Delay...). et al. , PLoS One 14 (2019), e0214890; Mukherjee et al ., PLoS One 12(2017), e0185473; Mukherjee et al ., PLoS One 8 (2013), e61607).

[0254] E-cadherin detection Detect the integrity of adhesion junctions in the oral mucosa.

[0255] Procedure: Paraffin-embedded tissue sections (for E-cadherin) or frozen tissue sections (for PMN) were stained with anti-E-cadherin polyclonal antibody, followed by staining with FITC-conjugated secondary antibody; or for PMN, stained with NIMP-R14, a monoclonal antibody highly specific for mouse Ly-6G and Ly-6C, followed by staining with Alexa 555-conjugated anti-rat secondary antibody. To visualize all cells, the nuclear dye Hoechst 33,258 (Bertolini) was used. et al ., TranslOncol 10 (2017), 612-620).

[0256] PLCβ2 staining Immunofluorescence labeling of PLCβ2 was used to identify type II cells in taste buds, and immunolabeling of type II cells was used to determine whether probiotic intervention affected differentiated and mature taste bud cells.

[0257] Procedure: Wash the slides in PBS and incubate with 5% NGS in blocking solution at room temperature for 1.5 hours. Then incubate the tissues overnight at 40°C with rabbit anti-PLCβ2 primary antibody diluted 1:1000. Incubate the tissues for 2 hours in the dark using Alexa 546 goat anti-rabbit secondary antibody (1:1000). Use Sytox green as a nuclear marker (Delay... et al. , PLoS One14 (2019), e0214890; Mukherjee et al ., PLoS One 12 (2017), e0185473;Mukherjee et al ., PLoS One 8 (2013), e61607; Sarkar et al ., Chem Senses (2021), 46).

[0258] SNAP-25 staining Immunofluorescence labeling with SNAP-25 was used to identify type III cells in taste buds, and the immunolabeling of type III cells was used to determine whether probiotic intervention affected differentiated and mature taste bud cells.

[0259] Procedure: The experimental protocol for SNAP-25 is the same as that for PLCβ2. Tissues were incubated overnight at 40°C with rabbit anti-SNAP-25 primary antibody. The concentration, treatment time, and Sytox labeling procedure for Alexa 546 secondary antibody were the same as described above (Delay). et al. ,PLoS One 14 (2019), e0214890; Mukherjee et al., PLoS One 12 (2017), e0185473; Sarkar et al ., Chem Senses (2021), 46).

[0260] RNA extraction and quantitative reverse transcription polymerase chain reaction (RT-qPCR) The levels of IL-6, IL-8, IL-1β, TNF-α, NF-κB, and other pro-inflammatory cytokines in the tongue were detected.

[0261] Procedure: Mouse tongues were homogenized using a POLYTRON homogenizer, and the supernatant was shaken with zirconia beads and phenol:chloroform:isoamyl alcohol. RNA was purified using the QIAgen RNeasy® Mini kit, and concentration and quality were determined using a NanoDrop device. Complementary DNA was synthesized using the SuperScript III Cells Direct® cDNA Synthesis Kit. Quantitative reverse transcription polymerase chain reaction (PCR) was performed using a Bio-Rad CFX96 PCR instrument and an iQ® SYBR Green Supermix (Bertolini). et al ., Transl Oncol 10 (2017), 612-620).

[0262] Statistical analysis Immunofluorescence images were acquired using a color camera mounted on a Nikon Eclipse E600 Scope and Spot acquisition software. Cell counts were performed by observers unaware of their experimental groups using the standards of Nguyen et al. Immunopositive cells were identified more clearly in Adobe Photoshop CS6 (https: / / www.Adobe.com) by adjusting brightness and RGB levels, if necessary, before quantification using NIH ImageJ (https: / / imagej.nih.gov / ij). The Ki67 percentage score was derived by dividing the number of Ki67+ immunopositive cells by the total number of basal epithelial cells or the total number of Sytox-positive taste sensory cells. Only cells located in the basal layer and those within the walls of the rimpapillary crypts containing taste buds were counted. PLCβ2 and SNAP-25 labeling data were averaged for 5–12 taste buds per mouse. The number of immunopositive cells and the total number of Sytox-green labeled nuclei within each taste bud were counted. All experimental data were collected from 3–6 mice per group.

[0263] Data from Ki67, PLCβ2, and SNAP-25 were analyzed using linear model analysis of variance (ANOVA). Independent ANOVAs were performed for each cell marker to evaluate the count of marked cells, the total number of cells in the taste buds, and the percentage of marked cells in the taste buds. All statistical tests were performed using SPSS version 26.0. Figures were generated using GraphPad Prism 8 (Delayed...). et al. , PLoS One 14 (2019), e0214890; Mukherjee et al ., PLoS One 12(2017), e0185473; Mukherjee et al ., PLoS One 8 (2013), e61607).

[0264] result Nine mice were administered busulfan at a dose of 80 mg / kg, and three died the following day. Therefore, subsequent experiments were conducted as follows: a total dose of busulfan of 120 mg / kg was administered at 30 mg / kg over four days, and a total dose of cyclophosphamide of 200 mg / kg was administered at 100 mg / kg over two days.

[0265] The chemotherapy administration timeframe (4 days of busulfan + 2 days of cyclophosphamide injection) is as follows:

[0266] Preliminary results indicate that *Streptococcus thermophilus* ENT-K12 promotes the healing of chemotherapy-induced oral mucositis in mice. Specifically, chemotherapy treatment in mice resulted in significant mucosal hypoplasia and ulceration of the tongue. Furthermore, the basal layer cells were loosely arranged, and pyknosis—the irreversible condensation of chromatin within the cell nucleus during necrosis or apoptosis—was observed. Additionally, the tongue tissue showed fewer cells in the stratum spinosum and stratum granulosum compared to healthy tissue; see also... Figure 10 A and Figure 10 B. Treatment with ENT-K12 during chemotherapy restored the integrity of the tongue mucosa and partially restored the basal, spinous, and granular layers; see also Figure 10 C. Further observation revealed that chemotherapy reduced mucosal thickness by nearly 50%, while the reduction was smaller, approximately 25%, when ENT-12 was administered during chemotherapy; see also Figure 11 A to 11C and Figure 12 .

[0267] Before conducting further experiments, the mouse model was replaced with one that included administration of 5-fluorouracil (5-Fu), as this chemotherapeutic agent is more commonly used in chemotherapy for human patients; see Example 4.

[0268] Example 4: Live and inactivated Streptococcus salivarius ENT-K12 promotes the healing of chemotherapy-induced oral mucositis in mice.

[0269] In this experiment, the effects of the chemotherapeutic agent 5-fluorouracil (5-Fu) and the administration of Streptococcus salivarius ENT-K12 on 5-Fu-damaged oral epithelial cells were analyzed.

[0270] Materials and methods: Reagent preparation: Dissolve 5 mg of 5-Fu in 100 μl of DMSO to prepare a 5 mg / 100 μl (50 μg / μl) 5-Fu stock solution. Due to the cytotoxicity of DMSO, dilute 10 μl of the 5-Fu stock solution in 10 ml of DMEM medium to prepare a 1000-fold dilution, i.e., a solution concentration of 50 μg / ml. In the intervention group, in addition to preparing a 50 μg / ml 5-Fu solution, heat-killed Streptococcus salivarius ENT-K12 solution and heat-killed Streptococcus salivarius ENT-K12 supernatant were also added.

[0271] Preparation method of heat-killed Streptococcus salivarius ENT-K12 solution: heat inactivation is performed by incubating Streptococcus salivarius at 65℃ for 60 min.

[0272] Animal model establishment: Eighteen 8-week-old mice weighing approximately 20 g were selected and randomly divided into a control group, a chemotherapy group, and a probiotic intervention group after chemotherapy (n=6). Mice in the 5-FU group and the probiotic intervention group were injected with 5-FU at a dose of 50 mg / kg for 5 consecutive days. Mice in the intervention group were orally administered 1 × 10⁻⁶ salivarius ENT-K12 probiotics collected by centrifugation via gavage twice daily. 9 CFU); while the 5-FU group mice were given sterile pure water orally twice a day.

[0273] Tissue collection and staining were performed as described in Example 3.

[0274] Analysis of the growth and proliferation activity of oral epithelial cells (HOK cells) in the presence of 5-FU In the first set of experiments, the growth activity of oral epithelial cells (HOK cells) in the presence of 5-FU was analyzed. For this purpose, 5-FU was added to 96-well plates at six different concentrations (0, 0.08, 0.4, 2, 10, and 50 μg / mL), with six wells for each concentration, and 5000 HOK cells added to each well. After loading, the HOK cells were incubated in a CO2 incubator for 24 hours. Cell proliferation was detected by live-cell analysis (LCA). The instrument was set to capture images every 2 hours for a total of 2 days; each group had 5 pairs of wells, with 4 fields of view captured per well, and images were taken at 10 × 10⁻⁶. As shown in the live-cell imaging experiments and visualization (see [link to live-cell imaging experiment]). Figure 13 After 48 hours of treatment with 5-Fu, the growth activity of HOK cells decreased.

[0275] In addition, the proliferation activity of HOK cells was analyzed. 5-FU was added to 96-well plates at six concentrations (0, 0.08, 0.4, 2, 10, and 50 μg / mL), with six wells for each concentration, and 5000 HOK cells added to each well. After 48 hours of 5-FU treatment, 10 μl of CCK8 reagent was added to each well. CCK8 refers to the Cell Counting Kit-8, a highly sensitive colorimetric assay for cell viability in cell proliferation and cytotoxicity determinations, and is available, for example, from MedChemExpress (catalog number: HY-K0301).

[0276] Four hours after treatment, cells were analyzed using a standard microplate reader (e.g., Thermo Scientific Multiskan™ FC microplate spectrophotometer). Mitochondrial metabolic activity was measured to reflect cell proliferation activity, and treatment with >10 μg / mL of 5-FU for 48 hours significantly reduced HOK cell proliferation activity (see [link to study]). Figure 14 ).

[0277] Effects of heat-killed Streptococcus salivarius ENT-K12 on the proliferation of oral epithelial cells (HOK cells) As described above, a heat-killed Streptococcus salivarius ENT-K12 solution was prepared and added to HOK cells (5000 HOK cells per well) in 96-well plates at concentrations ranging from 0 mg / mL to 2 mg / mL to determine the effect of heat-killed Streptococcus salivarius ENT-K12 on their proliferation. Incubation was performed for 48 hours, after which 10 μl of CCK8 reagent was added to each well. Cells were analyzed using a microplate reader 4 hours after treatment. Figure 15In a visually perceptible manner, heat-killed Streptococcus salivarius ENT-K12 did not exhibit proliferative toxicity to HOK cells at any concentration, and heat-killed DSM 34540 treatment significantly improved HOK cell viability at concentrations >1.5 mg / mL.

[0278] Furthermore, the proliferation capacity of HOK cells was analyzed in the presence of 5-FU, and the effect of *Streptococcus salivarius* ENT-K12 on these HOK cells was evaluated. For this purpose, heat-killed *Streptococcus salivarius* ENT-K12 solution was prepared as described above and added to HOK cells (5000 HOK cells per well) in 96-well plates at concentrations ranging from 0 mg / mL to 2 mg / mL. Additionally, 10 μg / mL of 5-FU was added to each well. Incubation was performed for 48 hours, followed by the addition of 10 μl of CCK8 reagent to each well. Cells were analyzed using a microplate reader 4 hours after treatment. Figure 16 As shown, HOK cell proliferation was significantly reduced after treatment with 10 μg / mL 5-Fu. However, treatment of HOK cells with heat-killed Streptococcus salivarius ENT-K12 significantly and in a dose-dependent manner restored HOK cell proliferation.

[0279] The protective effect of live and heat-killed Streptococcus salivae ENT-K12 on the oral mucosal barrier in vivo. like Figure 17 A and 17D and Figure 18 As shown in A and 18D, 5-Fu has a significant adverse effect on the integrity and permeability of the oral mucosal barrier in mice. It is clearly visible that 5-Fu treatment reduces the oral mucosal layer area in mice, while treatment with both live and heat-killed Streptococcus salivarius ENT-K12 significantly increases it.

[0280] In addition, Ki-67 expression was analyzed. Ki-67 is a protein found in the cell nucleus and is expressed only during cell division, and is associated with cell proliferation. Figure 19 and Figure 20 As shown, Ki-67 expression in the 5-Fu group was lower than that in the probiotic group and the control group. Therefore, both live and heat-killed Streptococcus salivarius ENT-K12 significantly promoted the proliferation of oral mucosal cells.

[0281] Heat-treated Streptococcus salivarius ENT-K12 improved 5-FU chemotherapy-induced HOK cell cycle damage. Cell cycle analysis was performed using flow cytometry. For this purpose, heat-killed Streptococcus salivarius ENT-K12 solutions were prepared as described above, and CCK-8 assays and LCA live cell proliferation detection were performed.

[0282] The result is Figure 21Visualization showed that, compared to the control group, heat-killed Streptococcus salivarius ENT-K12 treatment for 48 hours did not significantly alter the HOK cell cycle; while 5-FU treatment induced S-phase and G2 / M-phase arrest—two phases closely related to rapid cell growth and protein synthesis, and which are stages in which cells prepare for mitosis and cytokinesis. Significantly, heat-killed Streptococcus salivarius ENT-K12 could recover its full cell cycle after being disrupted by 5-FU treatment.

[0283] Streptococcus salivarius ENT-K12 reduces DNA damage levels in HOK cells induced by the chemotherapeutic agent 5-Fu. In another experiment, the level of reactive oxygen species (ROS) in HOK cells was detected using flow cytometry as described above. Figure 22 As shown, treatment with 5-Fu induced ROS levels in HOK cells, which typically reflect DNA damage during chemotherapy, while treatment with heat-killed Streptococcus salivarius ENT-K12 significantly reduced ROS levels.

[0284] In summary, it demonstrates that heat-killed Streptococcus salivarius ENT-K12 has a beneficial effect on chemotherapy-damaged oral epithelial cells, and therefore, in addition to live Streptococcus salivarius ENT-K12, heat-killed Streptococcus salivarius ENT-K12 can also be used for the prevention and treatment of oral mucositis. PCT / RO / 134 form

Claims

1. Streptococcus salivarius ( Streptococcus salivarius ( ), for the treatment or prevention of ear, nose and throat (ENT) diseases induced by or associated with cancer therapy.

2. The *Streptococcus salivarius* for the use according to claim 1, wherein, The ENT disease is an oral mucosal disorder induced by cancer therapy.

3. The *Streptococcus salivarius* for the purpose described in claim 1, wherein, The ENT disease is a respiratory infection induced by cancer therapy.

4. The *Streptococcus salivarius* for use according to any one of claims 1 to 3, wherein, The ENT disease is caused by or associated with the following: radiotherapy, chemotherapy, hematopoietic stem cell transplantation (HSCT), immune checkpoint inhibitor therapy, or any combination thereof.

5. The *Streptococcus salivarius* for use according to any one of claims 1 to 4, wherein, The ENT disease is induced by or associated with radiotherapy or concurrent chemoradiotherapy (CCRT).

6. The *Streptococcus salivarius* for use according to any one of claims 1 to 4, wherein, The ENT disease is induced by or related to chemotherapy.

7. The *Streptococcus salivarius* for use according to any one of claims 1 to 4, wherein, The ENT disease is induced by or related to hematopoietic stem cell therapy (HSCT).

8. The *Streptococcus salivarius* for use according to any one of claims 1 to 4, wherein, The ENT disease is induced by or related to HSCT following chemotherapy.

9. The *Streptococcus salivarius* for use according to any one of claims 1 to 4, wherein, The ENT disease is caused by or related to head and neck cancers treated with radiotherapy or CCRT, preferably nasopharyngeal carcinoma (NPC).

10. The *Streptococcus salivarius* for use according to any one of claims 1 to 4, wherein, The ENT disease is caused by or related to the following: treatment of hematopoietic tumors via HSCT, preferably via chemotherapy followed by HSCT.

11. Streptococcus salivarius for the treatment and prevention of ear, nose and throat (ENT) diseases, said ENT diseases being induced by or related to immunosuppression, preferably wherein said ENT diseases are oral mucosal disorders or respiratory tract infections.

12. The *Streptococcus salivarius* according to any one of claims 1 to 11, wherein, The oral mucosal disease is oral mucositis.

13. The *Streptococcus salivarius* for use according to any one of claims 1, 2, and 4 to 12, wherein, The saliva streptococci are provided in an inactivated form, preferably wherein the saliva streptococci are heat-inactivated.

14. The *Streptococcus salivarius* for the use according to claim 12 or 13, wherein, Apply the aforementioned Streptococcus salivarius: (i) Delay the onset of oral mucositis and reduce the risk of severe oral mucositis; (ii) Restoring the decreased proliferative capacity and disordered cell cycle of oral epithelial cells following chemotherapy in a dose-dependent manner; and / or (iii) To prevent the reduction of oral mucosal area after chemotherapy and to restore the integrity of damaged oral mucosa.

15. The *Streptococcus salivarius* for the use according to claim 3, wherein, Administering streptococci via salivary fluid: (i) To prevent the subject's upper respiratory tract infection from developing into a lower respiratory tract infection; (ii) The duration of respiratory infection symptoms in the subjects was shortened compared with the control group that did not receive saliva streptococcus; (iii) Compared with the control group, the mean duration of respiratory infection episodes in the subjects was shortened; and / or (iv) Reduce the need for antibiotics in the subjects compared to the control group.

16. The *Streptococcus salivarius* for use according to any one of claims 1 to 12 and 14 to 15, wherein, The oral administration of the aforementioned Streptococcus salivarius is at a daily dose of 3 × 10⁻⁶. 6 Up to 4 × 10 10 Between CFUs, 3 × 10 is preferred. 9 Up to 3 × 10 10 Between CFU or 4 × 10 9 Up to 4 × 10 10 Between CFUs.

17. The *Streptococcus salivarius* for use according to any one of claims 1 to 12 and 14 to 16, wherein, The oral administration of the saliva streptococcus is between 3 × 10 mg and 4 × 120 mg daily, preferably between 3 × 50 mg or 4 × 50 mg.

18. The *Streptococcus salivarius* for use according to any one of claims 1 to 17, wherein, During radiotherapy or chemoradiotherapy, the subjects were given the saliva streptococcus orally four times daily for a period of approximately 6 to 7 weeks.

19. The *Streptococcus salivarius* for the use according to claim 18, wherein, Before the start of radiotherapy or chemoradiotherapy, administer the aforementioned Streptococcus salivarius orally four times daily for approximately two weeks.

20. The *Streptococcus salivarius* for use according to any one of claims 1 to 17, wherein, Subjects who underwent HSCT and whose hematopoietic system was reconstructed were given the aforementioned Streptococcus salivarius orally three times daily for approximately 100 days. Hematopoietic system reconstruction was defined as a platelet count > 20 × 10⁶ after HSCT. 9 Cells / L and neutrophils >0.5 × 10⁶ 9 Cells / L 21. The *Streptococcus salivarius* for use according to any one of claims 1 to 20, wherein, Streptococcus salivarius is formulated into a composition, preferably an oral composition.

22. The *Streptococcus salivarius* for use according to any one of claims 1 to 21, wherein, The composition is selected from the following oral dosage forms: tablets, capsules, gels, lozenges, chewable tablets, oil drops, and powders; preferably, wherein... The composition is an oil drop or a solid oral dosage form selected from the following: tablets, capsules, chewable tablets and lozenges, with lozenges being the most preferred.

23. The *Streptococcus salivarius* for the use according to claim 22, wherein, The oral dosage form includes at least 10 6 CFU containing Streptococcus salivarius, preferably 10 6 Up to 10 10 Salicylic streptococci between CFU, more preferably 10 9 Or 10 10 CFU (Cellular Streptococcus salivarius) 24. The *Streptococcus salivarius* for the use according to claim 22 or 23, wherein, The oral dosage form comprises 1 to 500 mg of Streptococcus salivarius, preferably 1 to 120 mg, more preferably 10 to 120 mg, and most preferably 50 mg.

25. The *Streptococcus salivarius* for the use according to any one of claims 21 to 24, wherein, The oral dosage form is a tablet.

26. The *Streptococcus salivarius* for the use according to claim 26, wherein, The tablets also include fructose, maltodextrin, magnesium stearate, and flavoring agents, preferably strawberry flavoring agents.

27. The *Streptococcus salivarius* for the use according to claim 25 or 36, wherein, The tablet is a slow-dissolving tablet, preferably wherein the disintegration time of the tablet is at least 4 to 5 minutes.

28. The *Streptococcus salivarius* for the use according to any one of claims 25 to 27, wherein, The tablets contain approximately 50 mg or 5% Streptococcus salivarius, approximately 635 mg or 63.5% fructose, approximately 290 mg or 29% maltodextrin, approximately 15 mg or 1.5% magnesium stearate, and approximately 10 mg or 1% flavoring agent.

29. A composition comprising Streptococcus salivarius for the treatment or prevention of ear, nose, and throat (ENT) diseases induced or associated with cancer therapy.

30. The composition for use according to claim 29, wherein, The ENT disease is an oral mucosal disorder induced by cancer therapy.

31. The composition for use according to claim 29, wherein, The ENT disease is a respiratory infection induced by cancer therapy.

32. The composition for use according to any one of claims 29 to 31, wherein, The ENT disease is caused by or associated with the following: radiotherapy, chemotherapy, hematopoietic stem cell transplantation (HSCT), immune checkpoint inhibitor therapy, or any combination thereof.

33. The composition for use according to any one of claims 29 to 32, wherein, The ENT disease is caused by or related to the following: (i) Treatment of head and neck cancers, preferably nasopharyngeal carcinoma (NPC), by radiotherapy or chemoradiotherapy; or (ii) Treatment of hematopoietic system tumors by HSCT, preferably by chemotherapy followed by HSCT.

34. A composition comprising Streptococcus salivarius for the treatment and prevention of ear, nose and throat (ENT) diseases, said ENT diseases being induced by or related to immunosuppression, preferably wherein said ENT diseases are oral mucosal disorders or respiratory tract infections.

35. The composition for use according to any one of claims 29 to 34, wherein, The oral mucosal disease is oral mucositis.

36. The composition for use according to any one of claims 29, 30, and 32 to 35, wherein, The composition comprises inactivated, preferably heat-inactivated, saliva streptococci.

37. The composition for use according to any one of claims 29 to 36, wherein, The composition is an oral composition.

38. The composition for use according to any one of claims 29 to 37, wherein, The composition is selected from the following oral dosage forms: tablets, capsules, gels, lozenges, chewable tablets, oil drops, and powders; preferably, wherein... The composition is an oil drop or a solid oral dosage form selected from the following: tablets, capsules, chewable tablets and lozenges, with lozenges being the most preferred.

39. The composition for use according to any one of claims 29 to 35 and 37 to 38, wherein, The composition comprises at least 10 6 CFU containing Streptococcus salivarius, preferably 10 6 Up to 10 10 Salicylic streptococci between CFU, more preferably 10 9 Or 10 10 CFU (Cellular Streptococcus salivarius) 40. The composition for use according to any one of claims 29 to 35 and 37 to 39, wherein, The composition comprises 1 to 500 mg of Streptococcus salivarius, preferably 1 to 120 mg, more preferably 10 to 120 mg of Streptococcus salivarius, and most preferably 50 mg of Streptococcus salivarius.

41. The composition for use according to any one of claims 29 to 35 and 37 to 40, wherein, The composition comprises 50 mg of Streptococcus salivarius, and wherein the composition is administered orally multiple times daily, preferably three or four times daily.

42. The composition for use according to any one of claims 29 to 35 and 37 to 41, wherein, The composition comprises at least 10 6 CFU of Streptococcus salivarius, wherein the composition is administered orally multiple times daily, preferably three or four times daily.

43. The composition for use according to any one of claims 29 to 42, wherein, During radiotherapy or chemoradiotherapy, the composition is administered orally to the subject four times daily for a period of approximately 6 to 7 weeks.

44. The composition for use according to claim 43, wherein, The composition is administered orally four times daily for approximately two weeks prior to the start of radiotherapy or chemoradiotherapy.

45. The composition for use according to any one of claims 29 to 42, wherein, The composition was administered orally three times daily for approximately 100 days to subjects who had undergone HSCT and whose hematopoietic system had been reconstructed. Hematopoietic system reconstruction in these subjects was defined as a platelet count > 20 × 10⁻⁶ after HSCT. 9 Cells / L and neutrophils >0.5 × 10⁶ 9 Cells / L 46. ​​The composition for use according to any one of claims 29 to 45, wherein, The composition is a tablet, preferably a tablet as defined in any one of claims 26 to 28.

47. Use of a composition comprising Streptococcus salivarius for maintaining healthy oral mucosa in subjects who are undergoing treatment with cancer or autoimmune disease therapy, during treatment with radiotherapy, chemotherapy, hematopoietic stem cell transplantation (HSCT), immune checkpoint inhibitor therapy, or any combination thereof, or who are immunosuppressed.

48. The use according to claim 47, wherein, The composition is the composition defined in any one of claims 37 to 40 and 43 to 46.

49. Use of Streptococcus salivarius or a composition comprising Streptococcus salivarius for the treatment or prevention of respiratory tract infection in a human subject, wherein, The human subjects were immunosuppressed.

50. The *Streptococcus salivarius* or composition for use according to claim 49, wherein, The immunosuppression is caused by treatment for cancer or autoimmune diseases.

51. The *Streptococcus salivarius* or composition for use according to claim 49 or 50, wherein, The cancer treatment includes HSCT and / or chemotherapy, preferably HSCT after chemotherapy.

52. The *Streptococcus salivarius* or composition for use according to any one of claims 49 to 51, wherein, Administering saliva streptococci prevented the subjects' upper respiratory tract infection from progressing to lower respiratory tract infection.

53. The *Streptococcus salivarius* or composition for use according to any one of claims 49 to 52, wherein, Compared with a control group that did not receive Streptococcus salivarius or a composition containing Streptococcus salivarius, administration of Streptococcus salivarius shortened the duration of respiratory infection symptoms in the subjects.

54. The *Streptococcus salivarius* or composition for use according to any one of claims 49 to 53, wherein, Compared with the control group, administration of saliva streptococci reduced the mean duration of respiratory infection episodes in the subjects.

55. The *Streptococcus salivarius* or composition for use according to any one of claims 49 to 54, wherein, Compared with the control group, administration of Streptococcus salivarius reduced the subjects' need for antibiotics.

56. The *Streptococcus salivarius* for use according to any one of claims 49 to 55, wherein, Streptococcus salivarius is formulated into a composition, preferably an oral composition.

57. The composition for use according to any one of claims 49 to 55, wherein, The composition is an oral composition.

58. The *Streptococcus salivarius* or composition for use according to any one of claims 49 to 57, wherein, The composition is selected from the following oral dosage forms: tablets, capsules, gels, lozenges, chewable tablets, oil drops, and powders; preferably, wherein... The composition is in the form of an ingot.

59. The *Streptococcus salivarius* or composition for use according to any one of claims 49 to 58, wherein, The composition comprises at least 10 6 CFU containing Streptococcus salivarius, preferably 10 6 Up to 10 10 Salicylic streptococci between CFU, more preferably 10 9 Or 10 10 CFU (Cellular Streptococcus salivarius) 60. The *Streptococcus salivarius* or composition for use according to any one of claims 49 to 59, wherein, The composition comprises 1 to 500 mg of Streptococcus salivarius, preferably 1 to 120 mg, more preferably 10 to 120 mg of Streptococcus salivarius, and most preferably 50 mg of Streptococcus salivarius.

61. The *Streptococcus salivarius* or composition for use according to any one of claims 49 to 60, wherein, The subjects were administered salivarious streptococci orally at a daily dose of 3 × 10⁻⁶. 6 Up to 4 × 10 10 Between CFUs, preferably 3 × 10 9 Up to 3 × 10 10 Between CFUs.

62. The *Streptococcus salivarius* or composition for use according to any one of claims 49 to 61, wherein, The subjects were given oral administration of streptococci in the form of salivarius at a daily dose between 3 × 10 mg and 4 × 120 mg, preferably 3 × 50 mg.

63. The *Streptococcus salivarius* or composition for use according to any one of claims 49 to 60, wherein, The composition comprises 50 mg of Streptococcus salivarius, and wherein the composition is administered orally to the subject multiple times daily, preferably three times daily.

64. The *Streptococcus salivarius* or composition for use according to any one of claims 49 to 60, wherein, The composition comprises at least 10 6 CFU of Streptococcus salivarius, wherein the composition is administered orally to the subject multiple times daily, preferably three times daily.

65. The *Streptococcus salivarius* or composition for use according to any one of claims 49 to 64, wherein, The composition was administered orally three times daily for approximately 100 days to subjects who had undergone HSCT and whose hematopoietic system had been reconstructed. Hematopoietic system reconstruction in these subjects was defined as a platelet count > 20 × 10⁻⁶ after HSCT. 9 Cells / L and neutrophils >0.5 × 10⁶ 9 Cells / L 66. The *Streptococcus salivarius* or composition for use according to any one of claims 59 to 65, wherein, The composition is an oral solid dosage form, preferably a tablet, and most preferably a tablet as defined in any one of claims 26 to 28.

67. A method for treating or preventing ear, nose, and throat (ENT) diseases induced or associated with cancer therapy in subjects with such need, wherein, The method includes administering saliva streptococci or a composition comprising saliva streptococci to the subject.

68. The method according to claim 67, wherein, The ENT disease refers to oral mucosal disorders induced by cancer therapy or respiratory infections associated with cancer therapy.

69. The method according to claim 67 or 68, wherein, The ENT disease is the ENT disease as defined in any one of claims 2 to 12.

70. The method according to any one of claims 67 to 69, wherein, Streptococcus salivarius is Streptococcus salivarius as defined in any of the preceding claims.

71. The method according to any one of claims 67 to 69, wherein, The composition is the composition defined according to any one of the preceding claims.

72. The method according to any one of claims 67 to 71, wherein, The Streptococcus salivarius and the composition are administered according to any one of the preceding claims.

73. Use of Streptococcus salivarius or a composition comprising Streptococcus salivarius in the preparation of a medicament for the treatment or prevention of ear, nose, and throat (ENT) diseases induced or associated with cancer therapy in subjects with such need.

74. The use according to claim 73, wherein, The ENT disease is an oral mucosal disorder induced by cancer therapy or a respiratory infection related to cancer therapy, preferably wherein... The ENT disease is the ENT disease as defined in any one of claims 2 to 12.

75. The use according to claim 73 or 74, wherein, Streptococcus salivarius is Streptococcus salivarius as defined in any of the preceding claims.

76. The use according to claim 73 or 74, wherein, The composition is the composition defined according to any one of the preceding claims.

77. The use according to any one of claims 73 to 76, wherein, The Streptococcus salivarius and the composition are administered according to any one of the preceding claims.

78. A method for treating or preventing a respiratory infection in a human subject, wherein, The human subject is immunosuppressed, and the method includes administering saliva streptococci or a composition comprising saliva streptococci to the subject.

79. The method according to claim 78, wherein, The subject is the subject as defined in claim 50 or 51.

80. The method according to claim 78 or 79, wherein, Streptococcus salivarius is the Streptococcus salivarius as defined in any one of claims 56 and 58 to 66.

81. The method according to claim 78 or 79, wherein, The composition is the composition defined according to any one of claims 57 to 66.

82. The method according to any one of claims 78 to 81, wherein, The Streptococcus salivarius and the composition are administered as defined in any one of claims 61 to 65.

83. A tablet comprising Streptococcus salivarius, for the treatment of ear, nose, and throat (ENT) diseases induced or associated with cancer therapy, wherein, The ENT disease is an oral mucosal disorder induced by cancer therapy or a respiratory infection related to cancer therapy, preferably wherein... The ENT disease is as defined in any one of claims 1 to 12.

84. The use of a tablet comprising *Streptococcus salivarius* for treating a respiratory tract infection in a human subject, wherein, The human subject is immunosuppressed, and preferably as defined in claim 50 or 51.

85. The tablet according to claim 83 or 84, wherein the tablet is the tablet defined according to any one of claims 26 to 28, and preferably is applied according to any one of the preceding claims.

86. The *Streptococcus salivarius* for use according to any one of claims 1 to 28, 49 to 56, and 58 to 66; the composition for use according to any one of claims 29 to 46, 49 to 55, and 57 to 66; the use according to any one of claims 47 or 48 and 73 to 77; the method according to any one of claims 67 to 72 and 78 to 82; or the tablet according to any one of claims 83 to 85, wherein... The *Streptococcus salivarius* is either *Streptococcus salivarius* K12 (American Center for Type Culture Collection (ATCC), PO P.O. Box 1549, Manassas, VA 20108, USA, accession number BAA-1024) or *Streptococcus salivarius* ENT-K12 (DSMZ-German Microbial Collection, Leibniz Institute, Braunschweig 7B, 38124, Germany, accession number DSM 34540).

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