Introduction
Chemotherapy-induced gastrointestinal mucositis (CIGM) can not only necessitate treatment delay or cessation but also, more importantly, represent a significant burden to healthcare costs and patient quality of life [
1]. Symptoms include diarrhoea, abdominal pain and weight loss and are a direct result of damage to the epithelial layer of the gut, which increases permeability, induces inflammation and ulceration, and prevents adequate nutrient uptake. Alterations to the gut microbiome are also associated with mucositis, which can further drive the inflammatory state [
2,
3]. At present, treatment for mucositis is supportive only; new interventions intended to prevent and/or reduce mucositis are being investigated and include growth factors, anti-inflammatories, nutritional supplements and medicinal foods [
4].
Oral administration of serum-derived immunoglobulin, derived from human or animal serum or plasma, has been shown to improve symptoms of disease states with an inflammatory basis, such as viral gastroenteritis in children [
5], malnourishment in children [
6], weaning in pigs [
7], irritable bowel syndrome with diarrhoea (IBS-D) [
8,
9] and HIV-induced enteropathy [
10]. Serum-derived bovine immunoglobulin/protein isolate (SBI) is obtained through the spray-drying of bovine plasma and is available commercially as EnteraGam™ (Entera Health Inc., Cary, NC, USA), which is regulated by the FDA as a medical food in the management of IBS-D [
8,
9] and HIV-associated enteropathy [
10].
The aforementioned enteropathic conditions are associated with abdominal pain, weight loss, malabsorption and diarrhoea, and as such have a similar symptom profile to CIGM. With this in mind, the rationale for the current study was to determine whether SBI had similar positive effects on irinotecan-induced GI mucositis in a Dark Agouti rat model.
Discussion
In the current study, twice daily administration of SBI was well-tolerated and resulted in reduction in the incidence, severity and duration of irinotecan-induced clinical signs of stress and diarrhoea, as well as a significant reduction in histopathological damage in the intestines. There is evidence that the basis for these observations is modulation of the inflammatory response associated with irinotecan-induced mucositis. The results herein are consistent with results observed in similar animal intestinal studies, where SBI was utilized, and also show similar symptom reduction as seen in patient studies of enteropathies managed with SBI.
Since chemotherapy-induced GI mucositis (CIGM) has a similar etiology and symptom profile to intestinal disorders such as IBS-D and HIV-associated enteropathy, it is reasonable to suggest that patients suffering from CIGM would show similarly improved symptoms with SBI. The animal model used in this study has been well established and has many similarities to the patient setting; thus, we postulated that administration of SBI would manage CIGM.
In a pilot study of 66 patients by Wilson et al. [
8], daily administration of 10 g of SBI over a period of 6 weeks significantly reduced the severity and frequency of symptoms of IBS-D, such as abdominal pain, stool frequency, loose stools, bloating, flatulence and urgency, compared to placebo (soy protein isolate). Similarly, in a study of eight patients with HIV-associated enteropathy by Asmuth [
10], twice daily administration of 2.5 g SBI for 8 weeks resulted in reduced bowel movements and improved stool consistency in all patients. In the current study, irinotecan-treated rats gavaged with human equivalent doses of SBI also showed a significant reduction in the incidence, severity and duration of mucositis-associated symptoms, such as diarrhoea.
Since the animals in this model were administered SBI 4 days prior to irinotecan treatment, it would be interesting to see whether a longer period of pre-treatment SBI administration would further improve diarrhoea and clinical symptoms of stress. Such preparatory dosing would also be possible in patients with CIGM (if SBI was given before irinotecan administration); however, since patients with other enteropathies present with these conditions before SBI administration would be possible, benefits of preparatory dosing in these conditions will remain unknown. A future animal study to determine whether dosing with SBI at the onset of mucositis symptoms (without preparatory dosing) is able to achieve the same results as the current study would provide useful information on the value of pre-treatment and determine whether protective effects of SBI are transient or cumulative.
The lack of changes in bodyweights, organ weights, intestinal goblet cells, morphometry, apoptosis and proliferation suggest that SBI-associated attenuation of the clinical symptoms of mucositis is not dependent upon increased cell proliferation and tissue growth as is observed after treatment with growth factors designed to alleviate mucositis [
16,
17]. Similar findings were made in rotavirus-infected pigs [
18], where SBI administration did not significantly increase weight gain or alter tissue morphometry, and in weaning piglets, where SBI administration showed few changes to intestinal morphometry, and in fact resulted in lower small intestinal weight over the treatment period [
19].
SBI is high in glutamic acid, which is converted to glutamine. Studies have shown that free glutamine is an essential amino acid required during times of catabolic stress, such as injury or disease, and is a preferential energy source not only for enterocytes but also for rapidly proliferating immune cells [
20,
21]. It is thus linked to the maintenance of intestinal barrier function and cell differentiation and may contribute to the positive effects of SBI as observed in this study, where histopathological analyses showed that tissues from animals administered SBI were more likely to retain proper architecture and an intact epithelial layer.
One hypothesis for the positive effects of SBI in enteropathic conditions is the modulation of intestinal immune function in gut-associated lymphoid tissue (GALT) and reduced levels of pro-inflammatory cytokines [
7,
22,
23]. Oral administration of spray-dried plasma proteins was associated with a decreased level of inflammatory cytokines in organised (Peyer’s patches) and diffuse (lymphocytes in lamina propria) GALT of the ileum and colon in weaning piglets, which show similar symptoms to patients suffering from GI mucositis, including malabsorption, diarrhoea and weight loss associated with increased gut permeability [
7]. Results obtained in the current study reflect these findings, as levels of circulating and mucosal neutrophils and lymphocytes (diffuse GALT) in irinotecan-treated animals receiving SBI were lower than those in treated controls. Anecdotal evidence of less pronounced intestinal lymphatic nodules (organised GALT) and less inflamed peritoneal cavities during necropsy also supports this. Further studies to assess tissue levels of pro-inflammatory cytokines are currently underway.
Analysis of histopathological damage showed significantly lower damage scores in both colon and jejunum of irinotecan-treated animals that had received both 250 and 500 mg/kg SBI, compared to those that had not. The density of lymphoid cells present in the lamina propria was decreased in gut tissues from animals gavaged with SBI, which was reflected in comparatively lower total white cell counts and percentages of neutrophils and lymphocytes in circulating blood. Reduced damage scores were most pronounced 2 and 4 days post-treatment in tissues from animals receiving SBI compared to treated controls. This also supports the potential anti-inflammatory role of SBI, suggesting that preparatory dosing prior to irinotecan treatment is not enough to stop the initial damage but helps to attenuate the induced inflammatory state which further drives the damage.
Low-grade inflammation, either idiopathic or injury-induced, is associated with increased epithelial permeability in the gut due to degradation of occludin within tight junctions [
24]. Transepithelial bacterial translocation is then more likely to occur, thus increasing lipopolysaccharide exposure to the GALT, which then further drives the process through the release of pro-inflammatory cytokines, giving rise to clinical symptoms such as diarrhoea, malabsorption and subsequent weight loss. The process is thought to underpin chronic diseases such as IBS-D [
8] and HIV-associated enteropathy [
10], and physiologically stressful conditions such as malnutrition [
6] and weaning [
25], and is often accompanied by changes to the microbiota, which may further compound the inflammatory process. Gastrointestinal mucositis initiated by cancer therapy (chemotherapy, radiotherapy and targeted therapy) occurs through direct damage to gut epithelium and creation of reactive oxygen species; however, the subsequent mechanisms of inflammation, increased gut permeability and clinical symptoms are similar to the abovementioned conditions. It has been shown that IgG in SBI binds lipopolysaccharide from bacterial cell walls, thus protecting mucosal surfaces from bacterial antigenic exposure [
26,
27]. Similar binding effects have also been demonstrated for endotoxins [
22,
28,
23] and rotavirus [
18], further underpinning the importance of SBI in management of enteropathies [
29,
30]. Additional studies to explore the role of SBI in attenuating the intestinal inflammatory response and possible maintenance of the healthy microbiota in this animal model are currently underway.
The results from this study demonstrate that SBI shows significant promise in the management of chemotherapy-induced GI mucositis in an animal model. Positive effects of SBI in the management other enteropathies with similar symptom profiles in patients have also been demonstrated, perhaps paving the way for similar use of SBI in the supportive care of treatment-induced mucositis. Further studies to underpin exact mechanisms of action and effects on solid tumours are required, however, as well as comprehensive analyses of pro-inflammatory cytokines within gut tissue and microbiome alterations.