Efficacy and safety of a colostrum- and Aloe vera-based oral care protocol to prevent and treat severe oral mucositis in patients undergoing hematopoietic stem cell transplantation: a single-arm phase II study

Oral mucositis is one of the worst effects of the conditioning regimens given to patients undergoing hematopoietic stem cell transplantation. It is characterized by dry mouth, erythema, mucosal soreness, ulcers, and pain, and it may impact patient outcomes. Bovine colostrum and Aloe vera contain a wide variety of biologically active compounds that promote mucosal healing. A non-randomized phase II study was designed to assess the safety and efficacy of a combined bovine colostrum and Aloe vera oral care protocol to prevent and to treat severe oral mucositis in transplant patients. Two commercially available products were given to patients in addition to the standard protocol: Remargin Colostrum OS® mouthwash and Remargin Colostrum Gastro-Gel® taken orally. Forty-six (78.0%) patients experienced oral mucositis, 40 (67.8%) developed mild–moderate forms, and 6 (10.2%) severe ones. Comparing the study group’s outcomes with those of a homogeneous historical control group, severe oral mucositis decreased significantly (10.2% vs. 28.4%; P < 0.01), as did its duration (0.5 ± 1.9 vs. 1.5 ± 3.0 days; P < 0.01). Febrile neutropenia episodes (69.5% vs. 95.1%; P < 0.01) and duration (4.0 ± 4.7 vs. 6.2 ± 4.5 days; P < 0.01) also decreased. These findings show that the experimental protocol seems effective in preventing severe forms of oral mucositis. However, a randomized controlled trial is necessary to confirm this.


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Background Allogeneic and autologous hematopoietic stem cell transplantation (HSCT) are standards of treatment for several hematological malignancies [1][2][3]. Before stem cell infusion, the recipient is treated with a conditioning regimen that includes combinations of chemotherapy, radiotherapy, and/or immunotherapy [4,5].
The literature provides few evidence supporting strategies to prevent or treat OM [25,26]. Thus, the approaches to dealing with OM in daily practice often rely on a wide variety of products supported by scarce or anecdotal evidence [27,28]. An interest in using natural agents for OM has been increasing, since these products may be effective for symptom control and because their components can interfere with the pathobiological processes underlying OM development [29][30][31].
Bovine colostrum (BC) has a wide variety of biologically active components, including lactoferrin, lactoperoxidase, immunoglobulins, and growth factors, and its benefits on health as a dietary supplement have been widely studied [32][33][34]. The protective effects of BC on the intestinal mucosal barrier [35] and upper respiratory tract integrity [36][37][38] have been reported, as have its beneficial effects on boosting the immune system [39,40]. In addition, topical applications of BC have been effective in both wound and mucosal healing thanks to its humectant, moisturizing, re-epithelizing, antioxidant, and immunestimulant activities [41][42][43][44].
Therefore, we hypothesized that combined formulas of BC and AV, in addition to the standard oral care practice, would effectively and safely prevent and treat sOM in patients undergoing HSCT.

Study design and sample size
A single-arm, non-randomized, open label, single-center, phase II study was designed following the optimal two-stage design by Richard Simon [53]. Adult patients undergoing autologous or allogeneic HSCT were recruited; those who reported intolerance to the products' components, who were not able to use the study self-reporting tools, or patients with OM already present at admission were excluded from the study. A study group (SG) sample size of 59 recipients was calculated assuming a reduction of 50% than local benchmarking data on sOM, and considering an α error of 0.05 and a sensitivity of 0.8. The study design provided a first step of 19 participants with a cutoff for study discontinuation of more than 5 patients with sOM. After recruitment, all patients received educational intervention (interview and educational material) on study medication management and the use of the tools included in the study. The study protocol was approved by the local ethics committee (n. 2016/0030535, December 28, 2016) and it was conducted in agreement with the Helsinki Declaration of 1975 and the Guidelines for Good Clinical Practice. All patients gave written informed consent before any study-related procedure took place.

Oral care protocol
In the transplant unit where the study has been performed, a standard oral care protocol was used to prevent and treat OM. It includes oral hygiene, i.e., gentle cleansing with toothbrush and toothpaste, followed by bland saline rinses (normal saline or sodium bicarbonate), 3 times per day after each meal, with the frequency increasing after OM onset. In addition, mouthwashes with moisturizing and emollient solutions and lip balm were recommended to all patients.
Patients performed RCOS mouthwashes (1 stick pack) for 40-60 s after each oral hygiene, and RCGG (1 stick pack) was administered orally 3 times per day from the start of conditioning until OM onset (prevention phase). After OM onset (treatment phase), the frequency of intervention was increased to at least 3 to 5 times per day.

Endpoints and outcomes
The primary endpoint was the incidence of sOM (grade 3-4 WHO) during the study period. OM was assessed daily by the nursing staff starting from the first day of conditioning until day 21 post-transplant using the WHO scale. Secondary endpoints were the evaluation of overall OM incidence, its time of onset, and duration. OM-related pain scores were assessed daily using a 0-10 numerical rating scale (NRS). Neutropenia and febrile neutropenia (FN) duration (days) and FN events were recorded, as were some cost-related outcomes such as length of stay, antibiotic, antifungal and antiviral therapy, and days of opioid and parenteral nutrition. QoL was assessed weekly with EQ-5D-3L (not reported), and patient-reported data were collected using the Oral Mucositis Daily Questionnaire (OMDQ) (not reported). Adherence to the study protocol was monitored. Safety was assessed by collecting data on adverse events (AEs) and by monitoring blood cell count, hemoglobin, and serum levels of creatinine and bilirubin. Oral swab tests for infection were performed at admission and at day 8 post-transplant, while galactomannan serum levels were monitored weekly until day 28 post-transplant or discharge.
The study outcomes were compared with routinely collected local benchmarking data of a historical cohort of patients treated during the 22 months preceding the study start (Table 1). This cohort had undergone only the standard practice protocol to prevent and treat OM and was taken as the control group (CG). All the data were collected by the patients' electronic clinical documentation and all the nurses assessing outcomes in both groups were routinely trained to use the assessment tools.
Descriptive analysis was performed using SPSS (IBM Corp. Released 2015, IBM SPSS Statistics for Windows, Version 23.0, Armonk, NY: IBM Corp.), and the Matrix Laboratory (MATLAB) Statistical toolbox version 2008 (MathWorks, Natick, MA, USA) was used for comparative analysis. All tests with P ≤ 0.05 were considered significant.

Participants' characteristics
Seventy-one HSCTs were performed from November 2017 to September 2019 in our hematology unit: 4 patients refused to participate in this study and 8 were ineligible. Thus, 59 patients were recruited 32 (54.2%) male and 27 (45.8%) female, with mean age 52.4 years (SD ± 12.0; range . All participants in the SG were adults and had no signs of OM at admission; 6 patients (10.2%) were smokers, 10 (16.9%) experienced OM during pre-transplant therapies, and none had a history of alcohol abuse. Most participants were married (42; 71.2%) and worked (44; 74.6%). Granulocyte-stimulating factor (GCSF) was administered to all autologous HSCT patients by day 1 post-transplant in both groups; only 1 allogeneic patient in SG was treated with GCSF from day + 18 due to infection. Main clinical information on SG and CG are showed in Table 1. No significant differences between groups are reported, including some risk factors for OM development.

Oral mucositis
During the first step of the study, 15/19 participants (78.9%) experienced OM; one patient (5.3%) developed sOM (grade 3 WHO), and no serious AEs were recorded in the report form. This made it possible to continue patient recruitment.

Historical cohort comparison
Severe OM incidence decreased more significantly in SG than in CG (10.2% vs. 28.4%, respectively; P < 0.01), while overall OM incidence remained unvaried (78.0% vs 80.2%; P = 0.74) ( Table 2). No significant differences were seen in either overall or sOM time of onset with reference to HSCT and the start of conditioning. Severe OM mean duration was longer in CG (1.5 ± 3.0 vs. 0.5 ± 1.9 days; mean rank 75.9 vs. 63.1; P < 0.01), while there was no difference in overall duration of OM. Fewer patients in SG than in CG (41/59 vs. 77/81 patients, respectively; P < 0.01) developed FN, and its mean duration was shorter (4.0 ± 4.7 vs. 6.2 ± 4.5 days, respectively; P < 0.01) ( Tables 2 and 3). No differences between the groups were found regarding neutropenia duration, length of hospital stay, maximum pain score (MPS), or the duration (days) of treatment with opioids, PN, antibiotics, or antifungal medications. The mean duration of antiviral therapy was significantly shorter in SG than in the CG (2.7 ± 7.3 vs. 9.5 ± 13.0; P < 0.01).

Adherence
Overall adherence to the study protocol is described in  (Fig. 1). The development of sOM was correlated with lower adherence to RCGG during prevention (43.1% vs. 55.5%) and was associated with a decrease in adherence during the treatment phase (RCOS 52.1% vs. 70.2%; RCGG 16.7% vs. 22.5%) (Fig. 2).
No differences in adherence to the study protocol were found per type of transplant or underlying disease.

Safety and adverse events
Of the 76 AEs recorded in the case report form, 14 (18.4%) occurred and resolved before OM onset (prevention phase), 28 (36.4%) were observed after OM onset (treatment phase), and 34 (44.7%) manifested across both periods. Most AEs

Discussion
The safety and efficacy of BC-and AV-based oral care protocol on HSCT patients with sOM were assessed in our prospective study. Safety was monitored by analyzing data on AEs, biochemical parameters, and culture tests routinely collected during the patients' hospital stay. The compared groups were homogeneous in terms of number of participants, sex, age, type of transplant, underlying diagnosis, stem cell source, type of cell product, conditioning regimen, immunosuppressive strategy, growth factor use, and risk factors for OM development. Despite there being no difference between groups in overall OM development, a reduction of up to 60% of sOM incidence was found in SG. In addition, sOM mean duration per group appeared shorter in SG, and a correlation between the reduction in sOM incidence and the reduction in the number of FN episodes and duration appeared evident. Rathe et al. (2020) [44] explored the effects on chemotherapy-related toxicities of daily BC dietary supplementation obtaining a reduction of OM peak of severity in the treatment group more than in the control group. However, in Rathe's trial, as OM severity was a secondary endpoint, the risk of an underpowered sample was posed. Furthermore, the effects of BC supplementation on FN were not significant, while our study suggested a reduction effect. Beneficial effects of bioactive milk factors on oral mucosa exposed to chemotherapy were described in a preclinical study on hamster [54]. Significant reduction in severity and duration of OM was reported in two studies on HSCT patients undergoing chemotherapy-based conditioning regimens where whey proteins were administered as dietary supplements (systemic effect) [55] and as mouthwashes (topical effect) [56]. BC antibacterial activity conferred by lactoferrin, lactoperoxidase, and a variety of immunoglobulins [57][58][59], combined with the antimicrobial properties of AV against the Candida species [60][61][62] and type 2 herpes simplex [63], could explain some of our significant findings, such as the reduction in FN (episodes and duration) and the reduced use of antiviral medication in SG. Weak evidences of the benefits of BC on the integrity of the mucosal barrier, reducing intestinal bacteria translocation, have been reported [33,34], and AV's in vitro antiviral action has been described [63]. The immunomodulatory and antiinflammatory effects of AV were provided by the stimulation of macrophages and modulating cyclooxygenase activation pathway [47,[63][64][65], which is fundamental to OM pathobiology [11]. BC contains a range of cytokines and other non-antimicrobial substances that together modulate inflammation and maintain or improve host response under different immune system exposures [40,66].
Dietary supplementation of BC may trigger immunological events that lead to systemic effects [33]. However, the limited patient adherence to RCGG intake gave rise to several doubts on the real potency of any systemic effect in this study.
The beneficial effects of AV and of BC on wound care have been suggested by some preclinical studies [41,67,68]. Their effects on mucocutaneous issues such as pain reduction, wound healing acceleration, stomatitis healing, and QoL improvement are well known [42,43,51,52]. It has been suggested that some components of BC, such as nucleotides, epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1), promote mucocutaneous cellular growth and also help repair gene impairment [32]. The tissue regenerative properties of AV are due to its component mannose-6-phosphate (M6P), which plays a fundamental role in extracellular matrix remodelling as well as in increasing proliferation of fibroblasts and collagen and in producing some fundamental substances such as hyaluronic acid [69][70][71].
In our study, the anti-inflammatory and antimicrobial properties and the tissue healing capability of AV and BC described above were beneficial during the ulcerative phase of OM, when the oral microbial flora plays a fundamental role in amplifying gene signals, accelerating tissue damage, and increasing inflammation, pain, and the risk of systemic infections [11]. Therefore, the effects of the oral care protocol were observed primarily on sOM development and duration. Although not statistically significant, the observed reduction in the number of days of opioids use in SG might confirm this hypothesis.
The reduced compliance to the oral care protocol due to factors such as chemotherapy-related toxicities may be a limiting factor of this study. Patients affected by nausea, vomiting, and/or taste change had difficulties taking study products due to their consistence (RCGG) and/or flavor (RCOS). In particular, difficulties in RCGG swallowing were reported by these patients, especially after mucositis onset. Although our results suggest a strong effect, the oral care protocol included different strategies, such as topical and systemic interventions, which precluded any consideration on the effect of the individual products. The comparison with the historical control group may have involved the change in some undetectable variables, leading to result biases. Furthermore, the strategies for supportive therapy and care may have varied because of healthcare professionals' decisions or patients' needs.
To our knowledge, this was the first study on the combination of BC and AV for the prevention and treatment of OM in oncology setting. The oral care protocol investigated in this study showed significant results on sOM incidence without any significant AEs. Our findings may be explained by the activity of the multiple bioactive substances composing BC and AV, and secondary findings seem to confirm the antimicrobial effects of both compounds, as already suggested in the literature [33,34,47,60]. However, the study design and some limitations suggest caution when interpreting these results. A randomized controlled trial is necessary to provide evidence in favor or against the use of this approach in clinical practice. It is implemented at our institute.
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