Introduction
Many chemotherapy drugs in particular anthracyclines, actinomycin and high-dose methotrexate, result in both structural and functional injuries to the gastrointestinal tract (Lind, 1995; Duncan and Grant, 2003), resulting in mucositis severe enough to prevent an adequate oral intake. Although therapy is targeted at tumour cells rapidly dividing progenitor cells can also be damaged resulting in dysfunction. In particular, the epithelia of the gastrointestinal tract are adversely affected resulting in severe inflammation, lesioning, ulceration and bleeding in the oral cavity, oesophagus and lower gastrointestinal tract (Duncan and Grant, 2003). Patients experience intense pain, requiring intravenous analgesia, nausea, vomiting, diarrhoea and abdominal distension with an increased susceptibility to infection (Duncan and Grant, 2003). It is therefore frequently necessary to support the patient with parenteral nutrition, which is associated with a decreased enzyme activity and an increased intestinal permeability, potentially leading to an increased risk of bacteraemia and endotoxaemia (Guedon et al., 1986; Buchman et al., 1995). Despite various strategies, currently, treatment dose modification remains the most effective means to limit the severity of mucositis potentially resulting in a reduction in efficacy of treatment.
Glutamine is a major fuel and important nitrogen source for enterocytes and plays a key role in maintaining mucosal cell integrity and gut barrier function (Lacey and Wilmore, 1990; Gardiner et al., 1995; Buchman, 1996; Hall et al., 1996; Miller, 1999; Rhoads, 1999; van Acker et al., 1999).
Phase I studies in adults have demonstrated no toxic effects of glutamine given either parenterally or enterally (Ziegler et al., 1990) and similarly phase II parenteral studies looking at toxicity have not demonstrated any ill effects (Ziegler et al., 1992; van Zaanen et al., 1994) Studies in adult oncology patients have shown that glutamine given orally is also well tolerated (Ziegler et al., 1992; van Zaanen et al., 1994; Jebb et al., 1995; Skubitz and Anderson, 1996; Schloerb and Skikne, 1999). In one such study, a significant decrease in the severity of chemotherapy-induced stomatitis was seen using a 'swish an swallow' technique (Skubitz and Anderson, 1996) Another showed a decreased duration and severity of diarrhoea as well as a significant reduction in the need for parenteral nutrition when oral glutamine was given prior to chemotherapy (Muscaritoli et al., 1997), while a third showed a reduction in severity and duration of mucositis and a decrease in intravenous analgesia usage (Cockerham et al., 2000).
In children the maximum-tolerated oral dose of glutamine of 0.65 g kg-1 was determined by a phase I study looking at patient acceptability and plasma ammonia and glutamine levels measured at timed intervals after ingestion of a single dose of glutamine (Ward et al., 2003).
There are very few published studies looking at oral glutamine involving paediatric oncology patients (Anderson et al., 1998; Aquino et al., 2005). In a randomized double-blind cross-over study, 16 children and 8 adults, who had experienced moderate-to-severe mucositis previously with at least one previous course of chemotherapy, were given a total dose of 4 g m-2 glutamine or placebo daily for 14 days. There was a significant reduction in the duration and severity of chemotherapy-associated stomatitis (Anderson et al., 1998). In the second randomized double-blind study, 120 children undergoing haematopoietic stem cell transplantation were given a total dose of 4 g m-2 or placebo daily until 28 days post-transplant or discharge if sooner. No significant difference in mean mucositis score was observed but a significant reduction in the total number of days of intravenous analgesia and parenteral nutrition was demonstrated in the glutamine group (Aquino et al., 2005). Both studies demonstrated that smaller doses of glutamine were well tolerated in children and had some effect, although in the Aquino et al. (2005) study, no subjective reduction in oral mucositis was observed.
The dose used in the Anderson and Aquino studies was far smaller than the dose used in the phase I study (Ward et al., 2003) and if based on a 30 kg child this corresponds approximately to doses of 4 and 19.5 g day-1 respectively. Prior to this clinical study, the effect of a larger dose of glutamine in paediatric patients had not been demonstrated.
The aim of this study was to determine if 0.65 g kg-1 of glutamine given orally or enterally alongside chemotherapy is effective in reducing the incidence and severity of mucositis in certain diagnostic groups of paediatric oncology patients at risk of developing mucositis post-chemotherapy.
Materials and methods
Subjects
Children and adolescents undergoing treatment of paediatric malignancy from July 1999 until February 2005 at the Yorkshire Regional Centre for Paediatric Oncology and Haematology were recruited to the study. The protocol was designed to recruit patients until a total of 50 had successfully completed the study.
All patients were undergoing chemotherapy according to national agreed protocols, Medical Research Counsel and United Kingdom Childhood Cancer Study Group.
Informed consent was obtained from the parents or if the patient was aged 18 years or above from the patients themselves. The study was approved by the Local Research Ethics Committee.
Patient eligibility
Patients were eligible for recruitment to the study if they were over 1 year of age and under 22 years and had at least two identical courses of chemotherapy and were receiving chemotherapy likely to cause mucositis.
Randomization
Patients eligible for the study were used as their own controls. Patients received one course of chemotherapy with glutamine and an identical course without. Alternate patients were allocated to have the glutamine with course 1 or with course 2. In some cases patients started treatment urgently and therefore had to be allocated to have course 1 without glutamine.
Glutamine administration
The dose of glutamine was 0.65 g kg-1 and was administered orally or via an enteral feeding tube if one had been inserted.
The oral dose was administered as a once-daily dose starting on day 1 of chemotherapy and given every subsequent day for a total of 7 days. The oral dose was mixed with water at a maximum concentration of 10 g/100 ml water and a total volume of not more than 300 ml. This was flavoured with fruit cordial (not fruit juice due to its acidity). As the glutamine did not dissolve completely at high concentrations in water, the participants were advised to swirl the cup around before taking the last couple of mouthfuls. Whether taken orally or via a feeding tube the dose was given within 30 min.
Blood sampling and handling
All blood samples were taken by accessing the patients' central line. Plasma ammonia and glutamine levels were measured on the first day of each course. During the course with glutamine, the sample was taken 4 h after the oral dose had been taken. In addition, daily plasma ammonia levels were measured while the patient was in hospital having chemotherapy and glutamine to check for any cumulative effect, which had not been measured previously in the phase I study. As with the phase I study, a limit of 150 
mol l-1 was chosen as an acceptable upper ammonia level.
Due to the instability of glutamine in the blood samples, the samples were deproteinized using an equal volume of 10% sulphosalicylic acid solution that also contained the internal standard norleucine. These were centrifuged immediately. The supernatant was frozen prior to analysis by automated ion exchange chromatography (Biochrom 20). The ammonia samples were received by the lab within 30 min and analysed immediately.
Clinical measurements
The patients' weight, height, percentage weight/height and mid upper arm circumference were measured prior to each chemotherapy course and at the time of admission for febrile neutropenia.
During periods between chemotherapy courses, the following were recorded by the study investigator: (a) whether or not total parenteral nutrition (TPN) was initiated, based upon the unit's criteria for initiating TPN, and if so, the duration of feeding required, (b) whether or not the child was on enteral feeds and if so, the duration of feeding, (c) any other complications which would effect feed tolerance for example neutropenic enterocolitis, Clostridium difficile infection.
Patient-held diaries
Patients/parents were asked to grade and record symptoms of nausea, vomiting, diarrhoea, oral mucositis and abdominal pain from day 1 of chemotherapy until the next course of chemotherapy was due, approximately 21 days. Two separate diaries were kept, one for the course with glutamine and one for the course without glutamine.
Grading of symptoms was based on common toxicity criteria (CTC) scores (Appendix A).
Statistical analysis
Based on departmental data it was estimated that 21% of children treated at the Yorkshire Regional Centre for Paediatric Oncology and Haematology develop diarrhoea post-chemotherapy. The aim would be to reduce this by 50%. With a 5% significance and a power of 80%, 56 patients would be required.
Data were entered into a computerized database (SPSS Inc. & Minitab) and were analysed with courses with glutamine against courses without glutamine using Student's t-test, Mann–Whitney U-test and 
2 test. A P-value of 
0.05 was considered statistically significant.
Results
A total of 76 patients who were eligible for the study were enrolled between July 1999 and February 2005. Twenty-six patients failed to complete the study. Reasons for failing to complete are summarized in Table 1. As the study was an analysis of efficacy rather than intention to treat, the 26 patients who failed to complete the study were not included in the analysis however any results obtained from them are described in Appendix B. The characteristics of the 50 patients who completed the study are shown in Table 2.
Nineteen of the 50 patients who completed the study drank the glutamine dose. Four patients had gastrostomies in situ and 27 patients had nasogastric tubes in situ and therefore the glutamine was given via an enteral tube which was flushed well afterwards. Seventeen children (34%) had the glutamine with their first course after recruitment to the study compared to 33 (66%) who had the course without glutamine first. This was mainly due to children undergoing treatment for acute myeloid leukaemia and B-cell non-Hodgkin's lymphoma in which treatment had to be commenced without delay and therefore there was no time for a discussion regarding randomization prior to the first dose of chemotherapy. Hence only two subjects out of 10 with AML and five out of 17 with B-cell NHL were randomized to have the course with glutamine first.
Table 3 shows the results of the clinical data collected. There was no significant difference in the nutritional status of the patients between the two courses as indicated by percentage weight/height and mid-upper arm circumference.
Significantly higher plasma ammonia and glutamine levels were found after the courses of chemotherapy given with glutamine, compared to those without. The ammonia and glutamine levels peaked between days 3 and 4 of taking the glutamine and fell to a low level by days 5–7.
Seven out of the 50 children required TPN following the chemotherapy course given with glutamine compared to 15 out of the 50 children without glutamine. Significantly fewer children required TPN following glutamine (P=0.049) and this group required significantly fewer days of TPN (0.023).
Thirty-one patients received enteral nutrition following their chemotherapy course with glutamine compared to 25 who were enterally fed following their course without glutamine. Although a significant difference was found between the total number of days of feeding there was no significant difference in the number of children enterally fed.
Patients at risk of lower gut toxicity received an amino-acid based formula (Neocate Advance or Elemental 028 Extra, SHS International). Although this provided a source of L-glutamine it only equated to an additional 0.1 g kg-1 and would not have a significant impact on overall glutamine intake.
Table 4 shows the total number of days of grades 3 and 4 toxicity for the symptoms assessed along with the mean
s.e. and range.
No significant difference was found between the total number of days of each grade of the five symptoms or for the total number of children with each symptom (see Table 5).
Table 5 - Total number of children recording each grade of toxicity with and without glutamine and significance.
Full table No adverse effects attributed to taking the glutamine were observed or recorded in the 50 patients who completed the study.
On completion of the two courses used for the study, all the patients/parents were offered subsequent courses of chemotherapy with glutamine if they felt it had been beneficial. Twenty-nine out of the 50 (58%) took up the offer.
Discussion
The aim of this study was to determine whether or not our predetermined dose of glutamine (0.65 g kg-1) is effective in reducing the incidence and severity of mucositis in paediatric oncology patients when given alongside chemotherapy. The dose used was approximately five times greater than doses used in previous paediatric studies. However, there was no significant difference in the subjective measurements of chemotherapy toxicity as documented by the patients. All the subjective toxicity scores showed more problems if glutamine was not received (see Table 4). However this did not reach significance due to the small number of patients recruited. There was a significant reduction in the use of TPN following chemotherapy courses with glutamine both in terms of number of days and number of patients, which could be considered as an objective indicator of a decrease in the severity of lower gut mucositis. This compares to a recent randomized double-blind study by Aquino et al. (2005) in which 57 children undergoing allogeneic or autologous haematopoietic stem cell transplantation from any source were given 2 g m-2 (maximum dose, 4 g) twice daily until day 28 of transplant and 63 children received the placebo glycine. In this study, there was no significant difference in toxicity as defined by oral mucositis scores between the two groups. However, there was a significant difference between the mean number of days of TPN (P=0.01) and intravenous analgesia use (P=0.03) between the two patient groups (Aquino et al., 2005). However the study by Anderson et al. (1998), which included paediatric patients, showed a significant reduction in both duration (P=0.0005) and severity (P=0.002) of oral mucositis. The main difference between the Anderson study and that of Aquino and the current study was that Anderson selected patients who had already recorded a mucositis score of 
grade 2 and patients were therefore more susceptible to this side effect of chemotherapy. This current study did not select patients and therefore is a true reflection of the whole patient group. It may be, however, that glutamine is more beneficial in patients who are more susceptible to mucositis.
Another main difference between the current study and those of Anderson and Aquino, was that 31 out of the 50 patients took the glutamine via an enteral feeding tube rather than orally, thus avoiding the 'swish and swallow' technique which attempts to create a local effect on oral mucositis. This technique was advised specifically to participants in the Anderson et al. (1998) study making local contact in the mouth, an important variable for determining the effectiveness of glutamine against stomatitis. This significant local effect has recently been demonstrated in a study by Peterson et al. (2007). In this randomized double-blind trial of 326 breast cancer patients, a total of 7.5 g glutamine, composed in a novel drug-delivery system, or placebo was taken daily from day 1 of chemotherapy to a minimum of 14 days after chemotherapy. The study drug was orally swished for 30 s before swallowing. Patients were then crossed over to the alternate treatment during their next cycle of identical chemotherapy resulting in a significant reduction in the incidence of grade 2 oral mucositis (P=0.026) and grade 3 oral mucositis (P=0.005) in treatment cycle 1 (Peterson et al., 2007).
This novel drug-delivery system has been shown to facilitate the uptake of >100 times more glutamine by the epithelial oral mucosal cells and therefore delivers glutamine directly to mucosal cells at risk of injury from chemotherapy (Petit et al., 2000). Although the dose used in the current study was higher, the method of delivery was not effective in terms of oral mucositis. This suggests that both dose and delivery system are important in reducing mucositis.
Considering the expense of TPN, this current study demonstrates the potential for significant cost savings if oral glutamine is given alongside mucositis inducing chemotherapy. In this study, 100 more days of TPN were required following chemotherapy courses without glutamine compared to courses with glutamine. In a simple cost comparison, 7 days of TPN for a 30 kg child would cost £310 compared to £18 for 7 days of oral glutamine.
Oral glutamine did not improve the nutritional status of patients in the current study. However, the study was carried out early in the patients' treatment and over a relatively short period of time. It is therefore unlikely that significant difference in nutritional status would be apparent.
A potential concern was that hyperammonaemia might be an untoward complication of glutamine therapy, particularly in patients receiving chemotherapy, due to the possibility of hepatorenal toxicity. The previous pharmacokinetic study concluded that a single oral dose of 0.65 g kg-1 of glutamine was safe. However, this was a single dose and therefore any cumulative effect of 7 days treatment could not be excluded. For this reason, both glutamine and ammonia levels were monitored. As with the phase I study, an ammonia level of 150 mol l-1 was chosen as an upper acceptable limit because premature infants, who are more vulnerable to toxins in general, commonly tolerate plasma ammonia levels up to this level without long-term effects (Green, 1988).
The blood samples for the plasma ammonia and glutamine levels were taken 4 h after ingestion of the glutamine dose as studies have shown the levels to peak at 30–45 min post-administration of glutamine and then steadily decline to a normal range within 90–120 min (Ziegler et al., 1990; Ward et al., 2003). Although there was a significant difference between the mean plasma ammonia levels with and without the glutamine, no untoward symptoms or adverse effects were demonstrated. One subject came off the study early because the plasma ammonia level increased to 119 mol l-1 and the glutamine to 2102 mol l-1 on day 3 of taking the glutamine and was not willing to return to the regional centre for further blood samples to be taken. The pharmacokinetic profile of the courses with glutamine showed a peak plasma ammonia level on days 3–4 before the levels fell again by days 5–7. The highest ammonia level was 137 mol l-1 on day 3 before falling to 63 mol l-1 by day 6.
A significant limitation of the study is the lack of placebo due to the inability to find a suitable substance with an identical texture to the glutamine. It was therefore necessary to use the patients as their own controls. Randomization was not possible in all cases due to the necessity of starting treatment urgently in patients with acute myeloid leukaemia and B-cell non-Hodgkin's lymphoma. If patients had to start treatment before the study could be discussed, their first course had to take place without glutamine. This would have caused a bias in randomization.
A further possible cause of bias is that there may have been a positive psychological effect for the family in taking the glutamine. Including a suitable placebo would have eliminated this source of bias.
The glutamine did not dissolve completely at this high dose. The solution therefore had a gritty texture and may have aggravated nausea in some patients. Failure to take the prescribed dose of glutamine was the main reason for patients not completing the study (17%). In older patients, the required volume was as high as 300 ml and at this volume and concentration, patients struggled to take it. Patients may have found it more acceptable to take the daily dose divided into two or three smaller doses which would have increased enterocyte contact and may have proved to be more beneficial (Savarese et al., 2003).
Finally, the sample size was too small to subdivide into the main diagnostic groups to assess if the glutamine had any particular benefit after certain types of chemotherapy. As patients acted as their own controls, this study excluded patients undergoing single courses of chemotherapy such as high-dose chemotherapy and allogeneic bone marrow transplant. In these situations, mucositis can be particularly severe.
Conclusion
Although this study showed no beneficial effect of a larger dose of glutamine in reducing the incidence and severity of oral mucositis in paediatric oncology patients, when given along side chemotherapy as determined by subjective toxicity measurements, there was a significant reduction in the use of parenteral nutrition, which could reflect improvement in lower gut mucositis. The study demonstrated that a daily dose of 0.65 g kg-1 of glutamine is a safe dose to use in paediatric oncology patients with no cumulative toxicity. Due to the unpalatable nature of the suspension, it would be worth considering a divided dose in future or the use of a novel drug-delivery system, which may increase compliance and enterocyte contact. Further studies to determine the use of this dose of glutamine in children undergoing high-dose chemotherapy and bone marrow transplantation, or in other patients particularly susceptible to mucositis, are indicated.
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Appendices
Appendix A
Appendix B
Limited data were obtained from 19 of the 26 patients who failed to complete the study. Seven patients despite consenting to the study were complete non-starters and hence no data were obtained. The tables below give the results, however, as the data were not comparable, no statistics were done.
Table B3 - Toxicity outcomes grades 3 and grade 4 combined on patients who failed to complete.
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