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Jun 7, 2004 - We examined the effects of busulphan (BU) dose and patient age on toxicity and outcome in 63 children with acute leukaemia given BUCY ...
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Conditioning regimens Busulphan given as four single daily doses of 150 mg/m2 is safe and effective in children of all ages PJ Shaw1, C Nath1,2, A Berry1 and JW Earl2 1 Department of Oncology, The Children’s Hospital at Westmead, NSW, Australia; and 2Department of Biochemistry, The Children’s Hospital at Westmead, NSW, Australia

Summary: We examined the effects of busulphan (BU) dose and patient age on toxicity and outcome in 63 children with acute leukaemia given BUCY prior to allogeneic or autologous BMT. BU was administered as four single daily oral doses, based either on weight (4  4 mg/kg) or surface area (4  150 mg/m2). BU pharmacokinetic analysis was not used to dose adjust. The average daily (mg/kg) BU dose was 43% higher for the group given 150 mg/m2 compared to the 4 mg/kg dose group. This produced a median BU AUC 61% higher than with the 4 mg/kg dose. Only one child did not achieve full allogeneic donor engraftment. Regimen-related toxicity was low. Although younger children had faster BU clearance, the 4  150 mg/m2 dose ensured equivalent systemic exposure to BU, and resulted in a high frequency of engraftment without a significant increase in serious toxicity. BU, given as four single daily doses of 150 mg/m2, is appropriate and safe in all age groups of children. Given the reliable pharmacokinetics, low toxicity and high rate of allogeneic engraftment, there is no need for routine pharmacokinetic monitoring or dose modifications. This dosage regimen may be applicable for use with i.v. BU. Bone Marrow Transplantation (2004) 34, 197–205. doi:10.1038/sj.bmt.1704560 Published online 7 June 2004 Keywords: bone marrow transplant; busulphan; AML

BUCY is widely used for patients undergoing BMT for both malignant and nonmalignant diseases.1–4 In 1986 data was published from Westminster Children’s Hospital on the use of busulphan (BU) given as a single daily dose together with CY in children with a variety of genetic diseases.4,5 Early failures using a total BU dose of 8 mg/kg led to the adoption of a surface area-based dose, of 320 mg/m2 BU, with a minimum of 16 mg/kg and a suggested maximum of 20 mg/kg total dose. This was combined with

Correspondence: Prof. PJ Shaw, BMT Service, Department of Oncology, The Children’s Hospital at Westmead, Locked Bag 4001, Westmead, NSW, 2145, Australia; E-mail: [email protected] Received 2 September 2003; accepted 22 January 2004 Published online 7 June 2004

a high dose of CY (8 g/m2 over 4 days). Patients receiving bone marrow from both matched and mismatched donors showed a high rate of engraftment. Those who did not engraft received the lowest doses of BU by body weight. Subsequent pharmacokinetic studies have confirmed that younger children receiving a BU dose based on weight achieve significantly lower systemic exposure, as measured by the area-under-the-BU concentration-vs-time curve (AUC), than older children or adults.6–9 It was later suggested that an even higher surface area-based BU dose, of 600 mg/m2, may be more appropriate for children than the 16 mg/kg dose for patients with malignancy10,11 and, more recently, for patients with thalassaemia.12 The 16 mg/kg and 600 mg/m2 BU doses are generally administered over 4 days, with the total daily dose being divided into four equal doses (the q.i.d. regimen). The original reason for administering BU four times daily was to reduce the number of 2 mg tablets that had to be taken at any one time. At this centre, we follow the regimen as originally used at Westminster Children’s Hospital and give BU as four single daily doses, of either 4–5 mg/kg/day or 150 mg/m2/day. The main advantage of a single daily dose regimen is the simplicity of administration, especially in young children. We have previously demonstrated that single daily BU doses of 4 mg/kg/day and 150 mg/m2/day provide daily systemic exposure equivalent to that of the divided dose regimens.9 In the same study, we also demonstrated that the use of the surface area-based dose allowed escalation of the dose above the weight-based dose and ensured that younger children achieved a systemic exposure to BU that was equivalent to that achieved by the older children. Now, in the present study, we examine the pharmacokinetics, toxicity and efficacy of BU given as a single daily weight- or surface area-based dose in children of varying ages.

Patients and methods Toxicity and engraftment data was collected for 63 children with acute leukaemia who were administered BU (Wellcome Australia Pty. Ltd) and CY as part of conditioning for BMT from December 1987 to December 1999. The clinical data and chemotherapy regimens for these children are summarised in Table 1. Diagnosis was AML for 59 children and ALL for four children. The AML series

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198 Table 1 for BMT

Clinical data of 63 children receiving BU in preparation

Age (years) Median (interquartile range)

8.3 (2.6–11.8)

Weight (kg) Median (interquartile range)

25.0 (13.9–39.8)

Diagnosis (no. of children) AML ALL

59 4

Gender (no. of children) Male Female

30 33

Type of transplant (no. of children) Autologous Allogeneic (matched sibling donors)

40 23

Transplant conditioning (no. of children) BU (4  4 mg/kg)+CY (120 mg/kg) BU (4  150 mg/m2)+CY (120 mg/kg)

25 38

represents all consecutively diagnosed children who were referred to the hospital with a diagnosis of AML and treated on a collaborative protocol where it was planned that all patients would undergo autologous or allogeneic BMT.13,14 A total of 23 children had allogeneic BMT using HLA-matched sibling donors. The majority of these were administered standard GVHD prophylaxis with CYA and MTX (n ¼ 20), but three children had only MTX. A total of 40 children had autologous transplants. All children had pre-transplant conditioning of BU (16 mg/kg or 600 mg/m2 total dose) and CY (120 mg/kg).

Toxicity Regimen related toxicity (RRT) was evaluated as previously described.15 The medical records of all patients were reviewed and the grades of organ toxicity were determined, based on the signs and symptoms recorded during the first 28 days (100 days for pulmonary) after transplant. The presence or absence of mild (grade 1), moderate (grade 2) or severe (grade 3) toxicity in each organ system was recorded. The organ systems that were evaluated included heart, bladder, mucosa, gastro-intestinal tract, kidney, liver (hepatic), and lung. Central nervous system (CNS) toxicity was not included as part of RRT, but was considered separately as anticonvulsant therapy was not administered uniformly in this study population. Grading of organ toxicity was according to the system of Bearman et al15 with modifications in the assessments of gastro-intestinal tract toxicity and hepatic toxicity. In the assessment of gastrointestinal tract toxicity, the presence or absence of watery stools not related to infection was recorded by frequency of diarrhoea rather than by volume. Hepatic toxicity was evaluated by monitoring total bilirubin concentrations and alanine aminotransferase (ALT) activity (grade 1: maximum bilirubin of 2–6 mg/dl or a two- to five-fold increase in ALT activity from baseline, grade 2: maximum bilirubin between 6 and 20 mg/dl or a 45-fold increase in ALT activity from baseline, grade 3: bilirubin 420 mg/dl, Bone Marrow Transplantation

hepatic encephalopathy or ascites compromising respiratory function). A patient’s overall RRT was the highest score obtained for any of the organ systems, grade 4 being fatal. All nonrelapse or GVHD deaths were recorded as transplant-related mortality. The presence of veno-occlusive disease (VOD) of the liver was also determined based on MacDonald’s criteria.16

Haematological recovery Haematological recovery was assessed by standard criteria. Recovery was graded as either full (both neutrophils and platelets recovered) or partial (only neutrophils recovered fully). Rate of haematological recovery was the time interval (days) from the date of BMT to the date that the neutrophil count reached 500 and 1000  106/l (N500 and N1000, respectively) and transfusion-independent platelet counts reached 25, 50 and 100  1012/l (PL25, PL50, PL100, respectively). The number of transfusions required post transplant were also recorded. For allogeneic transplant recipients, rate of haematological recovery was determined in only those patients who achieved engraftment. One child who failed to engraft but achieved full haematological recovery was excluded from this analysis.

Engraftment For allogeneic recipients, engraftment in sex-mismatched donors was followed by metaphase or FISH analysis.17 Other markers of successful donor engraftment, such as blood group or tissue typing, were used where available. More sensitive analysis, such as RFLP PCR, was only used when clinically indicated. Graft rejection was defined as a lack of evidence of donor engraftment accompanied by the return of host lymphocytes.

Administration of BU BU doses of 4 mg/kg/day or 150 mg/m2/day were given on each of four mornings as whole or crushed tablets. Children undergoing BMT prior to November 1991 received the weight-based BU dose. A new protocol incorporating the surface area-based dose was implemented in November 1991 and thus children undergoing BMT after this date received 150 mg/m2/day BU. In younger children, including all of those younger than 3 years, the dose was administered via a naso-gastric tube. A normal diet was offered on each day of BU administration.

Anticonvulsant prophylaxis From December 1990, patients received anticonvulsant prophylaxis during BU administration. Initially this was with clonazepam unless the patient was already on another anticonvulsant. From December 1992, all patients received clonazepam irrespective of whether they were receiving another anticonvulsant. A total of 46 children received clonazepam, two children received phenytoin, two children received carbamazepine, while 13 children did not receive

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any anticonvulsant prophylaxis. Clonazepam was given orally twice daily, from the night before the first dose of BU to the morning after the last dose.

Blood sampling and BU assay Sampling for BU analysis was offered to 48 children who had their BMT after November 1990 (when the BU assay became available). Parental consent was obtained for the measurement of plasma BU concentrations in 34 of these children (The Pharmacokinetic Group). This was done as part of a prospective study approved by the Hospital Ethics Committee between 1990 and 1994 to establish a target AUC for BU. After 1994, it was offered as part of the routine BMT service to ensure an adequate AUC was obtained, but was not done routinely. The main reasons why patients did not have pharmacokinetics performed were availability of a nurse to collect the series of blood samples and parent preference. Heparinised whole blood samples (2 ml) were collected from central venous lines for the measurement of plasma BU concentrations. The first sample was collected before the BU dose was administered and the remainder at 0.5, 1.0, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 18 and 24 h after the dose. Plasma samples were separated by centrifugation for 10 min at 41C at 3000 r.p.m., then frozen and stored at 401C until analysis. They were analysed within 2 weeks of collection. BU was measured in plasma using our previously published assay using gas chromatography with electron capture.9 The between-day coefficients of variation of the assay were 5, 5, 7, 7 and 6% for 0.5, 1, 2, 6 and 13 mM BU concentrations, respectively (n ¼ 8). The limit of detection of the assay was approximately 0.1 mM BU.

(Allogeneic Group), relapse (Autologous Group) and high AUC (45200 and 46000 mM min). The Mann–Whitney Utest was used to examine whether the two dose groups differed in single daily (mg/kg) BU dose, BU AUC, rate of haematological recovery or in the requirement for platelet or blood transfusions. The effect of younger (o3 years) or older (45 years) age on the safety and efficacy of 150 mg/m2/day BU was determined separately in allogeneic and autologous transplant recipients. Fishers exact test was used to determine whether age group significantly affected the frequencies of VOD, RRT (grade 3 or 4), engraftment (Allogeneic group), GVHD (Allogeneic Group) and relapse (Autologous Group). The Mann–Whitney test was used to examine whether age group significantly affected the rate of haematological recovery and the requirement for platelet or blood transfusions. The relationships between BU AUC and a number of transplant outcome measures were examined. BU AUCs in children who relapsed or developed GVHD were compared with those obtained for the remainder of the children in the autologous and allogeneic transplant groups, respectively, and the differences were tested for significance using the Mann–Whitney U-test. Graphical presentation and the calculation of Pearson’s correlation coefficient were used to determine whether BU AUC was significantly related to the rate of haematological recovery or to the requirement for transfusions. Due to small numbers it was not possible to test whether BU AUC was significantly higher in children with VOD and severe RRT. However, it was possible to record whether AUCs were high (46000 mM min) or normal in this subgroup.

Pharmacokinetic analysis

Results

For the 34 patients who comprised the Pharmacokinetic Group, the computer software, Kinetica 2000 (Innaphase, Philadelphia, USA) was used to determine AUC using mixed log linear rule and to generate estimates of apparent clearance (CL/F ), apparent volume of distribution at steady state (Vss/F ) and elimination half-life (t1/2). Bioavailability (F ) was unknown.

BU pharmacokinetics Figure 1 is a scatterplot showing the variability in BU concentrations in the Pharmacokinetic Group at selected time points within the 24 h dosing interval. The time to reach peak BU concentration ranged from 0.5 to 5 h (median: 2 h), leading to a large variability in concentrations at the 1 and 2 h time points. BU was almost

SPSS version 10 was employed to statistically analyse the data. The Mann–Whitney test was used to compare BU pharmacokinetic parameters in (1) children receiving 4 mg/ kg/day (n ¼ 7) and 150 mg/m2/day (n ¼ 27) BU doses and (2) children of younger (o3 years, n ¼ 10) and older (45 years, n ¼ 16) age. Fishers exact test was used to compare the frequencies of AUC determinations above 5200 mM min and above 6000 mM min for (1) the 4 mg/kg/day and 150 mg/m2/ day dose groups and (2) the younger and older age groups. The effect of dose (4  4 mg/kg or 4  150 mg/m2) on the safety and efficacy of BU was determined separately in allogeneic and autologous transplant recipients. Fishers exact test was used to determine whether BU dose significantly affected the frequencies of VOD, RRT (grade 3 or 4), engraftment (Allogeneic Group), GVHD

Busulphan concentration (µM)

Statistical analysis 40 35 30 25 20 15 10 5 0

0

5

10

15 Time (h)

20

25

30

Figure 1 Variability in BU concentrations over the 24 h dosing interval in 34 children. Bone Marrow Transplantation

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200

Table 2

10

8

Frequency

completely eliminated by 24 h, as the 24 h trough concentrations were below the 0.1 mM limit of detection for 30 (of 34) children and below 0.4 mM for the remaining four children. It was possible to accurately determine BU pharmacokinetic parameters for all children in the Pharmacokinetic Group. A histogram showing the distribution of BU AUC measurements in the Pharmacokinetic Group is shown in Figure 2. The median AUC is 6421 mM min (interquartile range: 4849–8244 mM min). The seven children who had the 4 mg/kg/day BU dose had AUCs ranging from 2465 to 5513 mM min. The 27 children receiving the 150 mg/m2/day dose had AUCs ranging from 4005 to 11278 mM min. BU pharmacokinetic parameters obtained by children receiving the 4 and 150 mg/kg/day doses are compared in Table 2. The median single daily BU dose was 5.93 mg/kg for the 150 mg/m2/day dose, which was 43% higher (Po0.001) than that of the 4 mg/kg/day dose. Median AUC for the 150 mg/m2/day dose was 61% higher than that of the 4 mg/kg/day dose (7517 vs 4675 mM min, Po0.001), while median peak BU concentration was 88% higher (23.3 vs 12.4 mM, Po0.001). Clearance normalised to body surface area was also significantly higher for the 4 mg/kg/day dose. Weight-normalised clearance, volume of distribution and elimination half-life were not significantly different for the two dose groups. The frequencies of AUC determinations above 5200 mM min and above 6000 mM min were signifi-

6

4

2

0 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 AUC (µM min)

Figure 2 Histogram showing distribution of BU AUC values in 34 children.

cantly higher for the 150 mg/m2/day dose than for the 4 mg/ kg/day dose. A total of 22 children (81%) on the 150 mg/m2/ day dose had AUCs above 5200 mM min, while 18 children (67%) had AUCs above 6000 mM min. No child on the 4 mg/ kg/day dose had an AUC above 6000 mM min and only two children (29%) had AUCs above 5200 mM min.

Comparison of the pharmacokinetics of 4 mg/kg and 150 mg/m2 BU in 34 children

Parameter

Total number of children

BU dose

Significance

4 mg/kg

150 mg/m

7

27

2

Age (years) Median (interquartile range)

7.2 (1.7–8.3)

5.3 (2.3–9.2)

BU dose (mg/kg/day) Median (interquartile range)

4.15 (4.0–4.19)

5.93 (5.38–6.36)

Po0.001a

AUC (mM min) Median (interquartile range)

4675 (3371–5242)

7517 (5591–8499)

Po0.001a

Peak BU concentration (mM) Median (interquartile range)

12.4 (10.8–16.8)

23.3 (18.3–30.8)

Po0.001a

BU concentration at 6 h after dose (mM) Median (interquartile range)

4.7 (3.2–7.2)

9.0 (5.5–10.9)

Po0.05a

CL/F (ml/min/kg) Median (interquartile range)

4.2 (3.2–5.5)

3.2 (2.8–4.4)

CL/F (ml/min/m2) Median (interquartile range)

114.9 (95.3–123.9)

83.3 (72.1–107.5)

Vss/F (l/kg) Median (interquartile range)

1.43 (0.89–1.45)

0.93 (0.81–1.18)

2.6 (1.8–3.5) 2 0

2.6 (2.3–3.1) 22 18

t1/2 (h) Median (interquartile range) No. of children with AUC45200 mM min No. of children with AUC46000 mM min a

Mann–Whitney test. Fishers exact test.

b

Bone Marrow Transplantation

NSa

NSa Po0.05a NSa NSa Po0.05b Po0.005b

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BU pharmacokinetic parameters in younger (o3 years) and older (45 years) children receiving 150 mg/m2/day BU are compared in Table 3. It was found that younger children were administered a significantly higher dose than older children (median 6.5 vs 5.5 mg/kg/day, Po0.001), but their exposure to BU, measured as AUC, was not significantly different. Weight-normalised clearance was significantly higher for younger children (median 4.4 vs 3.1 ml/min/kg). Clearance normalised to body surface area did not differ significantly for the younger and older age groups. Younger children did not have significantly greater frequencies of AUC determinations above 5200 mM min or above 6000 mM min than older children.

Table 4 shows the frequency of RRT in the total group of children. The most common side effect was mucositis, which was experienced by 92% of patients. The majority of these patients were administered a continuous intravenous narcotic drug. Hepatic toxicity, characterised by elevation in either bilirubin concentrations or in ALT activity, was experienced by 51% of patients, while renal toxicity, characterised by increases in plasma creatinine concentrations, was experienced by 13% of patients. Watery stools were the most common toxicity affecting the

Table 4 Regimen-related toxicity in 63 children receiving BUCY prior to allogeneic or autologous stem cell transplantation Organ system

Toxicity of BU given as a single daily dose Anticonvulsant prophylaxis and convulsions. As previously reported, of the first 13 children who had no anticonvulsant prophylaxis, three had convulsions.13 No child receiving oral clonazepam prophylaxis has suffered a convulsion. Neither patient who received phenytoin fitted. However, one of two children on carbamazepine fitted. As no fits have been observed with clonazepam, we have gradually reduced the dose, as it does cause acute behavioural disturbance, and our current oral dose is 0.05 mg/kg/day.

Table 3

Regimen-related toxicity

Number of children with regimen-related toxicity Grade 1

Grade 2

Grade 3

Grade 4

0 0 13 22 6 16 2

0 0 45 3 2 16 2

1 1 0 0 0 0 1

0 0 0 0 0 0 1

Heart Bladder Mucosa GI tract Kidney Hepatic Lung GI ¼ gastrointestinal.

Comparison of BU pharmacokinetics in younger (o3 years) and older (45 years) children receiving the 150 mg/m2/day BU dose

Parameter

Total number of children

Age group (years)

Significance

o3

45

10

16

Age (years) Median (interquartile range)

2.0 (1.4–2.3)

8.9 (5.8–11.5)

Po0.001a

BU dose (mg/kg/day) Median (interquartile range)

6.5 (6.3–7.4)

5.5 (4.7–5.9)

Po0.001a

AUC (mM min) Median (interquartile range)

6773 (5171–7886)

7854 (5611–8647)

NSa

Peak BU concentration (mM) Median (interquartile range)

24.2 (17.7–30.9)

22.0 (18.8–28.2)

NSa

BU concentration at 6 h after dose (mM) Median (interquartile range)

8.7 (4.3–10.4)

9.6 (5.9–11.5)

NSa

CL/F (ml/min/kg) Median (interquartile range)

4.4 (3.5–5.3)

3.1 (2.5–3.9)

Po0.01a

CL/F (ml/min/m2) Median (interquartile range)

91.5 (76.3–114.8)

89.9 (71.4–113.6)

NSa

Vss/F (l/kg) Median (interquartile range)

1.08 (0.81–1.62)

0.92 (0.82–1.07)

NSa

2.4 (2.2–3.1) 8 6

2.8 (2.5–3.1) 13 11

NSa NSb NSb

t1/2 (h) Median (interquartile range) No. of children with AUC45200 mM min No. of children with AUC46000 mM min a

Mann–Whitney test. Fishers exact test.

b

Bone Marrow Transplantation

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202 Survival of allogeneic recipients

110

Event-free survival

gastrointestinal tract and were experienced by 40% of patients. The organs which were less affected by the RRT were the bladder (2% of patients), the heart (2% of patients) and the lungs (10% of patients). A total of three children had severe (grade 3 or 4) RRT. BU AUC was determined in one of these children and was normal (5591 mM min). One child died from RRT, a girl who developed pulmonary and liver VOD after ABMT for AML in CR2. Unfortunately consent for BU pharmacokinetic analysis was not obtained for this child.

100 90 80 70

VOD of the liver 60

Only one (of 23) allogeneic transplant recipients (4%) developed VOD. This child received four daily doses of 4 mg/kg BU. Two (of 39) autologous transplant recipients (5%) developed VOD: one child had 4 days of 4 mg/kg BU while the other received 4 days of 150 mg/m2 BU. BU AUC was determined in only one of the three children who had VOD and was found to be normal (4675 mM min).

0

1000

Engraftment

Forty-two children are alive and well. Deaths recorded were from relapse (16), GVHD (3), bacterial sepsis complicating pre-existing cardiac failure (1) and RRT (1). Event-free survival was very satisfactory for both allogeneic and autologous BMT recipients with AML (Figures 3 and 4, respectively) as was overall survival of the 70 patients with AML referred to and treated at The Children’s Hospital from 1988 to 2000 (Figure 5). These figures are updated from our previous publication in 1994.13

Comparison of the safety and efficacy of 4  4 mg/kg vs 4  150 mg/m2 BU Tables 5 and 6 (see Supplementary information) present a comparison of the toxicity and efficacy of the 4  4 mg/kg and 4  150 mg/m2 BU doses in allogeneic and autologous Bone Marrow Transplantation

5000

6000

Survival of autologous recipients

Event-free survival

90 80 70 60 50 40 30 20 10 0 0

1000

2000

3000 4000 Time (days)

5000

6000

Figure 4 Event-free survival in 40 autologous transplant recipients.

Overall survival of 70 patients with AML

100 90 Overall survival

Outcome

3000 4000 Time (days)

Figure 3 Event-free survival in 23 allogeneic transplant recipients.

100

Of 20 children with AML undergoing allogeneic BMT, one died too early (GVHD) for an assessment of long-term engraftment. All 10 children with a sex or blood group marker showed full donor engraftment. In another nine cases, their clinical course was unremarkable, so that rigorous evidence of donor engraftment was not sought. Of three children with ALL, one showed full engraftment and one had no marker and was clinically well. The third showed slow haematological recovery with the onset of Streptococcal sepsis and encephalopathy. She only showed transient donor chimerism.17 She also had low BU concentrations (her BU AUC was 4005 mM min even though she received the 150 mg/m2/day dose) and is the only child in this series who may have benefited from an escalated dose of BU. Since this child belonged to the prospective study that was conducted between 1990 and 1994 to establish a target AUC, her BU pharmacokinetic analysis was not performed immediately and dose adjustments were not made.

2000

80 70 60 50 40 30 20 10 0 0

1000

2000

3000 4000 Time (days)

5000

6000

Figure 5 Overall survival of all 70 children referred with AML from 1988 to 2000.

transplant recipients, respectively. For both analyses, median values for BU dose (mg/kg/day) and AUC were significantly higher for the 150 mg/m2/day dose than for the 4 mg/kg/day dose. More than half of the children on the 150 mg/m2/day dose (Pharmacokinetic Group) had high

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BU AUCs (46000 mM min), while no child receiving the 4 mg/kg/day dose had an AUC above 6000 mM min. However, safety and efficacy of the 150 mg/m2/day dose was not significantly different to that of the 4 mg/kg/day dose in both allogeneic and autologous transplant recipients. In allogeneic transplant recipients, BU dose did not significantly affect the frequency of engraftment, the rate of haematological recovery, the number of blood transfusions or the frequencies of GVHD, VOD or serious RRT. In autologous recipients, BU dose did not significantly affect the rate of haematological recovery, the number of blood transfusions or the frequencies of relapse, early relapse, VOD and serious RRT. The two dose groups were matched in age and weight.

Comparison of the safety and efficacy of 4  150 mg/m2 BU in younger (o3 years) vs older (45 years) children Tables 7 and 8 (see Supplementary information) present a comparison of the safety and efficacy of 4  150 mg/m2 BU in younger (o3 years) and older (45 years) children receiving allogeneic and autologous transplants, respectively. In both of the analyses it was shown that children younger than 3 years had a significantly higher daily (mg/ kg) BU dose than children over 5 years. However, the frequency of engraftment or GVHD (Allogeneic Group), the frequency of relapse (Autologous Group) or the frequency of VOD, grade 3 or 4 RRT or the number of transfusions did not differ for the two age groups.

Relationship between BU AUC and transplant outcome measures BU AUC did not correlate significantly with either the rate of haematological recovery or the requirement for blood transfusions in allogeneic or autologous transplant recipients Of 20 children who had autologous transplants and a pharmacokinetic analysis, eight relapsed but their exposure to BU (Median AUC: 5878 mM min, Interquartile range: 5310–7906 mM min) did not differ significantly to that of the 12 children who did not relapse (Median AUC: 6773 mM min, Interquartile range: 4818–8504 mM min). Of 13 allogeneic transplant recipients with pharmacokinetic data, four developed GVHD, but their exposure to BU (median AUC: 6703 mM min, Interquartile range: 5664– 9448) was not significantly different to that obtained for the nine children who did not develop GVHD (Median AUC: 7780 mM min, Interquartile range: 3831–8493 mM min).

Discussion Administration of the correct dose of BU is important for achieving the optimal transplant outcome. Overdosing can lead to severe, life-threatening, toxicity, while underdosing can lead to engraftment failure and relapse. VOD of the liver has been found to be the major dose-limiting toxicity of BMT regimens containing BUCY. This condition is also seen with single agent BU,16 but not with single agent CY.

To optimise treatment with BU a number of researchers have recommended individualisation of BU doses to achieve a target AUC.18–22 However, this study shows that, simply by administering BU as four single daily 150 mg/m2 doses, pre-transplant conditioning is optimised for children of all ages and that there is generally no need for routine pharmacokinetic monitoring or dose adjustments. We found that the single daily surface area-based dose was associated with a low frequency of serious toxicity and a high rate of allogeneic engraftment. Of 14 allogeneic transplant recipients receiving four single daily doses of 150 mg/m2 BU, only one child failed engraftment (7%), while one child had severe RRT (7%) and no child developed VOD. Of 24 autologous transplant recipients receiving the same surface area-based dose, only one child developed VOD (4%) and one child had severe RRT (4%). The toxicity and efficacy of the 4  150 mg/m2 BU dosing regimen was found to be comparable to that of the 4  4 mg/kg regimen even though there was a considerable escalation in exposure to BU. In our Pharmacokinetic Group, the median AUC obtained by 27 children receiving 150 mg/m2/day BU was 7517 mM min, while the median AUC obtained by seven children receiving the 4 mg/kg/day dose was 4675 mM min. Escalating the dose from 4 mg/kg/day to 150 mg/m2/day therefore represents a considerable escalation in exposure to BU. Grochow23 found that BU AUC above 1500 mM min was associated with an increased occurrence of VOD in patients given 1 mg/kg doses every 6 h. They observed that VOD occurred in six (of eight) patients with BU AUCs above 1500 mM min but in only one (of 27) patients with AUCs below 1500 mM min. Similiar results were recorded by Dix et al.22 When making adjustments to the 1 mg/kg BU dose to achieve an AUC within a target range, a number of researchers have used 1500 mM min22,23 as the upper limit for the target range, with the actual target AUC being 1300 mM min.24 An equivalent upper limit for the four-fold higher 4 mg/kg/day dose is an AUC of 6000 mM min, while an equivalent target AUC is 5200 mM min. In the present study, we found that the 150 mg/m2/day BU dose was frequently associated with high AUCs: 81% of children on this dose had AUCs above 5200 mM min, while 67% of children had AUCs above 6000 mM min. In contrast, no child receiving the 4 mg/kg/day dose recorded an AUC above 6000 mM min, while only 29% recorded AUCs above 5200 mM min. Thus, escalating the dose from 4  4 mg/kg to 4  150 mg/m2 results in a large proportion of children having AUCs above the 6000 mM min upper limit. In spite of this, as previously discussed, we did not observe significantly increased toxicity in children receiving the surface area-based dose. Thus, administration of BU as four single daily 150 mg/m2 doses allows an increase in exposure without a concomitant increase in toxicity. BU given as a single daily dose also allows accurate pharmacokinetic determination for all patients. We found the time to peak BU concentration ranged from 0.5 to 5 h and that BU was almost completely eliminated by 24 h. The blood sampling times for BU concentration measurement used in this study covered the whole 24 h time period and both the distribution and elimination phases were Bone Marrow Transplantation

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adequately characterised. When BU is given every 6 h, however, it may not be possible to adequately characterise the elimination phase in patients who have a longer time to peak BU. For example, Dupuis et al 24 obtained uninterpretable pharmacokinetic results in 20% of cases, usually because the concentration was still increasing at 6 h. Previous researchers have suggested that there is a relationship between BU exposure and toxicity in blood or marrow transplant recipients.21,25,26 For example, the occurrence of VOD has been shown to correlate with both high AUC estimates22,23,27 and a total BU dose exceeding 16 mg/kg.28 Average BU concentration at steady state has been found to be an important determinant of RRT18 while mean minimum BU concentrations were important determinants of transplant-related mortality21 and permanent alopecia.27 There have also been a number of reports that have failed to demonstrate a significant association between BU exposure and toxicity.19,20,28,29 A correlation between BU exposure and transplant outcome, including engraftment and relapse, has also been suggested in early studies,18,19 but not in some more recent papers, which have described larger, more uniform populations of patients.25,29 All these studies, however, are based on BU being given in multiple doses each day. Our study, in which BU was administered as four single daily doses, did not demonstrate a relationship between BU AUC and transplant outcome measures (including engraftment, relapse and toxicity). However, our numbers were small (only 34 children had a pharmacokinetic analysis) and there was a low frequency of engraftment failure and toxicity associated with this regimen. Consequently, there may be insufficient power to detect such relationships. This study, and others,6–10 have demonstrated that the pharmacokinetics of BU alters with age and that younger children tend to have higher BU clearance (normalised to weight) than older children or adults. Younger children in the study were given crushed tablets via a nasogastric tube, while older children were given whole tablets. In a previous study we have demonstrated that, compared with children taking whole tablets, children taking crushed tablets had a significantly shorter delay time, but did not differ in absorption or elimination kinetics or in AUC.9 Thus, administration of BU as crushed tablets via a nasogastric tube would not confound the higher apparent clearance in younger children. As a result of the age-related differences in BU pharmacokinetics, younger children have lower systemic exposure to BU when the dose is calculated based on body weight.6,7,9,10 This can have serious consequences on transplant outcome as insufficient dose intensity may lead to engraftment failure or relapse.18,19 Since calculating the BU dose based upon surface area rather than weight can almost double the mg/kg dose for younger children, it is important to demonstrate that the surface area-based BU dose is safe in young children. We found that, although younger children (o3 years) had a significantly higher daily (mg/kg) dose, their exposure to BU (AUC) and frequency of VOD or serious RRT was not significantly higher compared with the older children (45 years). Thus, administration of BU as four single daily 150 mg/m2 doses is safe in children of all ages. Bone Marrow Transplantation

The use of anticonvulsant prophylaxis in these patients is worthy of comment. A total of 13 patients had no anticonvulsant therapy and, of these, three had seizures.13 This is in contrast to the original regimen reported from Westminster Children’s Hospital, where none of 50 patients receiving 57 courses of single daily dose BU and CY suffered convulsions, although their daily BU dose was lower.30 All other children in our current series had anticonvulsant therapy with clonazepam, except three patients who received carbamazepine (two of whom had seizures) and two patients who received phenytoin. We therefore recommend clonazepam over other anticonvulsants because it is effective, easily administered with a simple oral regimen and it does not affect the pharmacokinetics of BU, unlike phenytoin.31 Interest in i.v. BU administered as a single daily dose is now growing.32 We have shown that the single daily, surface area-based dose of oral BU is safe and effective in children of all ages. This information may also be applicable to i.v. BU. However, it should also be noted that our study population included a relatively uniform group of children with acute leukaemia. Event-free survival was very good in this cohort of children undergoing BMT (see Figures 3–5). Patients with multiple relapses or treated with more aggressive initial therapy may be more variable with respect to the pharmacokinetics of BU and may be less tolerant of any preparative regimen. Therefore our results should not be extrapolated to patients with less favorable characteristics prior to transplant. This study has demonstrated that the two agent conditioning regimen containing four single daily doses of 150 mg/m2/day BU has comparable toxicity and efficacy with that of the regimen containing four single daily doses of 4 mg/kg/day BU in children with acute leukaemia. Use of the surface area-based BU dose also ensures that children younger than 3 years of age achieve equivalent systemic exposure to BU as do older children. Combined with 120 mg/kg CY, it is associated with a high frequency of allogeneic engraftment but is not associated with an increased frequency of serious toxicity. By providing reliable drug exposure without toxicity, we would recommend BU as a single daily dose to be the standard method of administering oral BU. Under these conditions, there is no necessity for routine pharmacokinetic monitoring and no need to add a third agent, which may only increase toxicity.

Acknowledgements CE Nath is supported by the Leukaemia Research and Support Fund. We would like to thank the Oncologists whose patients were included in the study, the nurses in the Oncology Unit for collecting blood samples and for excellent patient care.

References 1 Santos GW, Tutschka PJ, Brookmeyer R et al. Marrow transplantation for acute nonlymphocytic leukaemia after treatment with busulfan and cyclophosphamide. N Engl J Med 1983; 309: 1347–1353.

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