Cyclophosphamide metabolism in children with Fanconi's ... - Nature

Bone Marrow Transplantation, (1999) 24, 123–128  1999 Stockton Press All rights reserved 0268–3369/99 $12.00 http://www.stockton-press.co.uk/bmt

Cyclophosphamide metabolism in children with Fanconi’s anaemia SM Yule1,2,3, L Price2, M Cole2,4, ADJ Pearson2 and AV Boddy5 1

Department of Haematology, Yorkhill NHS Trust, Glasgow; Departments of 2Child Health, 3Pharmacological Sciences, 4Statistics and the 5Cancer Research Unit, The Medical School, University of Newcastle upon Tyne, UK

Summary: Although patients with Fanconi’s anaemia (FA) exhibit a heightened sensitivity to DNA cross-linking agents, modified doses of CY continue to be used in their conditioning prior to BMT. We measured the pharmacokinetics and metabolism of CY in six children with FA using an established high performance thin layer chromatography technique. CY doses ranged between 5 and 20 mg/kg (median 10 mg/kg). The median CY clearance was 0.6 l/h/m2 (range 0.4–1.1 l/h/m2), t1/2 was 8.1 h (range 6.7–9.5 h) and volume of distribution was 0.19 l/kg (range 0.16–0.34 l/kg), respectively. These results contrast with those previously reported from a comparable group of non-FA children in whom the median CY clearance was 3.2 l/h/m2 (range 2–5 l/h/m2) (P = 0.035), t1/2 was 2.4 h (range 2–3.8 h) (P = 0.035) and volume of distribution 0.5 l/kg (range 0.26–0.95 l/kg) (NS). Unlike the control group in whom the presence of inactive metabolites of CY was common, metabolites could not be found in any FA patient. The enhanced sensitivity of children with FA to CY may in part result from altered drug metabolism. Keywords: Fanconi’s anaemia; cyclophosphamide; drug metabolism

Fanconi’s anaemia (FA) is an autosomal recessive disorder in which there is progressive bone marrow failure accompanied by an increased predisposition to malignancy, particularly acute leukaemia. The diagnosis is based upon increased spontaneous chromosome breakage and heightened sensitivity to DNA cross-linking agents including CY.1,2 Although patients with FA exhibit marked phenotypic variability, the majority of affected individuals develop progressive marrow failure towards the end of the first decade of life and at the present time BMT is considered the treatment of choice.3,4 During the development of BMT as a therapy for aplastic anaemia it became clear that whilst the use of CY at doses of 200 mg/kg was tolerable in the majority of individuals, patients with FA developed severe drug-related toxicity particularly mucositis, haemorrhagic cystitis and myopericarditis.5,6 These results led clinicians to reduce the conditioning dose of CY to either140 mg/kg used as a single agent, or to 20–80 Correspondence: Dr SM Yule, Department of Haematology, Yorkhill NHS Trust, Glasgow, G3 8SJ, UK Received 6 November 1998; accepted 21 February 1999

mg/kg when combined with antithymocyte globulin and limited field irradiation.4,7–9 The increased sensitivity of FA patients to CY has been ascribed either to deficiencies in DNA repair10,11 or to disturbed oxygen metabolism with overproduction of reactive oxygen intermediates.12–14 CY is a prodrug which requires metabolic transformation to generate active alkylating species. The initial activation is thought to be mediated by a hepatic cytochrome P450 reaction. Hydroxylation at the carbon-4 position of the oxazaphosphorine ring produces 4-hydroxycyclophosphamide, which exists in equilibrium with its tautomer aldophosphamide. Spontaneous ␤-elimination of aldophosphamide releases phosphoramide mustard and acrolein. Whilst phosphoramide mustard is thought to be the active alkylating species, acrolein is an unwanted byproduct responsible for haemorrhagic cystitis.15 Alternatively, aldophosphamide may be oxidised to inactive carboxyphosphamide by aldehyde dehydrogenase.16 The other principal inactive metabolite, dechloroethylcyclophosphamide, is produced by a separate oxidative N-dealkylation reaction which is also catalysed by cytochrome P450s.15,17 In view of the enhanced toxicity of CY in children with FA we studied its pharmacokinetics and metabolism in six patients undergoing BMT. The results of this study were compared with those obtained previously from children receiving chemotherapy for malignant disease. Patients and methods The pharmacokinetics and metabolism of CY were studied in six children with FA (three females) undergoing BMT. In all cases the diagnosis of FA was made on the basis of clinical phenotype accompanied by evidence of spontaneous chromosomal fragility. Patients were aged between 7 and 12 years (median 9.5 years) and received CY as a constant rate intravenous infusion over 1 h. The administered dose varied between 5 and 20 mg/kg (140 and 560 mg/m2) (median 10 mg/kg). No patient had received CY before the study (Table 1). Prior to the infusion and at 0.5, 1, 2, 4, 6, 12, 18 and 24 h following the start of the infusion, 3–5 ml of blood was collected from an indwelling central venous catheter and anticoagulated with EDTA. Plasma was separated and frozen immediately at ⫺20°C prior to analysis. The concentration of CY and its principal metabolites were measured using an established high performance thin layer chromatography photographic-densitometry technique.17 Unfortunately this method does not reliably detect phosphoramide mustard, which requires bedside derivatization to increase its stability. Although several methods for

CY metabolism in children with FA SM Yule et al

124

Table 1

Fanconi’s anaemia patients and their conditioning regimen

Patient No.

Age (years)

CY dose

Conditioning regimen

Donor

Outcome

1

7

10 mg/kg once daily for 4 days (total dose 40 mg/kg)

CY/Campath 1G 5 mg daily for 4 days (total dose 20 mg)

MUD

Dead (graft-versus-host disease)

2

8



Matched sibling donor

Alive

3

9




Alive

4

10


CY/TBI


Dead (graft failure)

5

12

10 mg/kg once daily for 4 days (total dose 40 mg)

CY/Campath 1G 5 mg daily for 3 days (total dose 15 mg)/TBI

MUD

Dead (fungal infection)

6

12


CY/Campath 1G 5 mg daily for 4 days (total dose 20 mg)/TAI

MUD

Alive

MUD = HLA matched unrelated donor; TBI = total body irradiation; TAI = thoraco-abdominal irradiation.

measuring phosphoramide mustard have been described, none have proved reproducible.18 During the last 5 years we have studied the pharmacokinetics and metabolism of CY in a large number of children during their initial exposure to the drug. The majority of these patients were receiving CY for the treatment of malignant disease and in all cases the drug was given as a 1 h infusion. For the purpose of this investigation control patients were selected on the basis of comparable age and dose of CY to that of the FA group. Thus, seven children with known pharmacokinetic and metabolic profiles (three females), aged between 0.6 and 14 years (median 4 years), who had received a median dose of 510 mg/m2 (range 360– 560 mg/m2), were enrolled as controls. None of this group were receiving concomitant therapy with drugs known to inhibit CY metabolism, nor had they been treated with known cytochrome P450 enzyme inducers, such as dexamethasone or anticonvulsants, in the month prior to undergoing pharmacokinetic evaluation (Table 2). CY pharmacokinetics were assumed to follow a single Table 2

compartment open model with first order elimination kinetics. Estimates of pharmacokinetic parameters were obtained by maximum likelihood using ADAPT II.19 Statistical comparisons between children with FA and a control group of patients were made using the Mann–Whitney U test. Written consent from the child’s parents, and where appropriate from the subjects themselves, was obtained prior to participation in the study. The project was approved by the joint ethical committee of the Medical School of the University of Newcastle upon Tyne and the Royal Victoria Infirmary, Newcastle upon Tyne (Ref. No. 91/156). Results CY was detectable in the plasma of all six children with FA up to 24 h following its administration. The disappearance of CY from the plasma was monoexponential in all cases (Figure 1). CY clearance varied more than two-fold with a median value of 0.6 l/h/m2 (range 0.4–1.1 l/h/m2),

Concomitant medication and cyclophosphamide pharmacokinetics in children with Fanconi’s anaemia

Patient No.

Concurrent medication

Clearance (1/h/m2)

Vd (1/kg)

t1/2 (h)

1

acy, amkiacin, chlor, cotrim, hydr, imip, itracon, mesna, ond, para, peth, ran

0.5

0.16

7.1

2

acy, granisetron, para, ran

0.7

0.16

9.2

3

acy, cotrim, flu, mesna, ond, ran

1.1

0.34

6.7

4

acy, cotrim, ond, ran

0.7

0.16

8.7

5

acy, allopurinol, chlor, cotrim, cyclizine, hydr, mesna, ond, peth, ran

0.4

0.29

9.5

6

acy, cotrim, itracon, ond, ran

0.6

0.22

7.5

Median

0.6

0.19

8.1

Range

0.4–1.1

0.16–0.34

6.7–9.5

Abbreviations as in Table 3.


Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6

10

0

CX DCCP CY

µM

µM

125

20

20

10

0 0

10

20

0

10

Time (h)

20

Time (h)

Figure 1 CY plasma concentration-time curves for children with Fanconi’s anaemia.

Figure 2 Typical CY plasma concentration-time curve of a control patient (No. 7) receiving chemotherapy for non-Hodgkin’s lymphoma. DCCP, dechloroethylcyclophosphamide; CX, carboxyphosphamide.

t1/2 was 8.1 h (range 6.7–9.5 h) and volume of distribution was 0.19 l/kg (range 0.16–0.34 l/kg) (Table 3). Inactive products of CP metabolism were not identified in the plasma of any patient with FA. The overall survival of this group is 50% at a median follow up of 5 years following BMT (range 2–8 years). The median clearance of CY in control patients was 3.2 l/h/m2 (range 2–5 l/h/m2), t1/2 was 2.4 h (range 2–3.8 h) and volume of distribution 0.5 l/kg (range 0.26–0.95 l/kg) (Table 2). One or more inactive byproducts of CY metabolism were identified in the plasma of all controls (Figure 2). CY clearance was significantly greater and half-life significantly shorter in the non-FA group of patients (P = 0.035 in each case) consistent with a more rapid rate of metabolism. No significant difference in the volume of distribution was observed between the groups.

Discussion

Table 3 Patient

This study is the first report of CY metabolism in children with FA. The results reveal wide differences in the pharmacokinetics of the parent compound and the production of inactive metabolites between patients with FA and a previously studied group of children receiving similar doses of CY for the treatment of malignant disease. Children with FA exhibit a lower clearance of CY and a longer t1/2 than non-FA patients, findings which are consistent with a slower rate of metabolism. Previous studies of CY pharmacokinetics in children with cancer have reported that, when compared with an adult population, children exhibit more rapid metabolism with greater clearance of the parent drug and a shortened t1/2.20,21 This phenomenon has also been observed for other drugs which are principally metabolised

Concomitant medication and cyclophosphamide pharmacokinetics in control children Age (years)

Dose (mg/m2)

Diagnosis

Concurrent medication

1

0.6

360

NB

2

2

490

NHL

doxo, flu, mesna, MTX, ond, pred, vcr

5

0.95

2.4

3

4

560

ALL

cotrim, cytosine, ond thioguanine

3.7

0.5

2.2

4

5

550

osteo

actinomycin D, bleomycin, cotrim, ond

2.7

0.38

2.4

5

5

510

saliv

cotrim, doxo, ond

2

0.26

2.1

6

5

440

NHL

acy, doxo, flu, mesna, MTX, ond, pred, vcr

3.2

0.56

3.2

7

14

530

NHL

doxo, flu, imip, mesna, MTX, ond, pred, vcr

2.3

0.4

3.8

cisplatin, frus, hyd, nif, ond, para, prop

Clearance (1/h/m2)

Vd (1/kg)

t1/2 (h)

4.1

0.65

2

Median

3.2

0.5

Range

2–5

0.26–0.95

2.4 2–3.8

Vd = volume of distribution; acy = acyclovir; chlor = chlorpheniramine; cotrim = cotrimoxazole; doxo = doxorubicin; flu = fluconazole; frus = frusemide; hyd = hydrallazine; hydr = hydrocortisone; imip = imipenem; itracon = itraconazole; MTX = methotrexate; nif = nifedipine; ond = ondansetron; para = paracetamol; peth = pethidine; pred = prednisolone; prop = propranolol; ran = ranitidine; vcr = vincristine; ALL = acute lymphoblastic leukaemia; NB = neuroblastoma; NHL = non-Hodgkin’s lymphoma; osteo = osteosarcoma; saliv = salivary gland tumour.


126

by cytochrome P450 and is thought to reflect a higher level of enzyme activity during childhood.22,23 All FA patients and several of the controls received red cell transfusions prior to the study. Routine measurements of serum transaminases were performed on all children immediately prior to the study and were less than or equal to one and a half times the upper range of normal in all cases. Ferritin levels were measured prior to the study in FA patients only and were within the normal range. Whilst we accept that these results do not exclude hepatic iron overload it is unlikely that this degree of hepatic dysfunction was sufficient to significantly inhibit CY metabolism in our patients. The effect of hepatic dysfunction, particularly in regard to circulating transaminase levels, upon CY metabolism is incompletely understood. A single adult study found no correlation between biochemical indices of liver function and CY pharmacokinetics in patients with myeloma.24 Previous studies have shown that CY pharmacokinetics may be altered by concomitant drug therapy, either with inhibitors of cytochrome P450, or following treatment with enzyme inducers.25,26 During this investigation concurrent medication was given as clinically indicated and not altered for the purpose of the study. Five children with FA and three controls received treatment with cotrimoxazole. Although cotrimoxazole potentiates the clinical effects of S-warfarin and tolbutamide, probably by reducing the activity of a single cytochrome P450 isozyme,27,28 we found no significant effect of this antibiotic on the rate of CY metabolism in a previous study.29 In addition to cotrimoxazole, several children from both groups were being treated with systemic antifungals, either fluconazole or itraconazole, whilst receiving CY. Both of these agents are inhibitors of cytochrome P450 enzyme activity.30–32 Whilst we are not aware of any investigation which has addressed the potential interaction between itraconazole and CY, a single study of CY metabolism in adults with breast cancer found no evidence of significant inhibition by concurrent fluconazole therapy.33 It is unlikely that the results of our study are systematically influenced by differences in concomitant medication as potential inhibitors of cytochrome P450 were used in both patient groups (Tables 2 and 3). Because of the relatively small doses of CY involved, the uroprotectant mesna was used in only three children in each group. Although mesna forms reversible adducts with circulating 4-hydroxycyclophosphamide it does not influence the elimination kinetics of CY itself.34 In contrast to patients in the control group the principal inactive metabolites, carboxyphosphamide and dechloroethylcyclophosphamide, were not detected in any child with FA. At the present time it is unclear whether qualitative differences in CY metabolism exist between adults and children. Direct comparisons between published studies are difficult because of different methodologies and the reliance of several adult studies on the measurement of urinary metabolites alone. Such studies do not take into account the varied stability of individual metabolites, particularly carboxyphosphamide, across a range of urinary pH.17 Although the control group was not identical in terms of age and dose administered this is unlikely to have system-

atically influenced the results as we found no effect of these variables on CY clearance in a previous study.17 We chose not to compare CY metabolism in FA patients with children undergoing BMT for other indications because of the differences in dose administered. It is possible that the small treatment dose of CY (5 mg/kg) may have contributed to our inability to detect inactive metabolites in the plasma of FA patients 4 and 6 in that their production may have been below the level of detection of the assay used. This is unlikely to be responsible for the absence of inactive metabolites in the remaining FA patients. Our results suggest that the activity of enzymes critical for CY metabolism, particularly cytochrome P450s and possibly also aldehyde dehydrogenase, are altered in patients with FA. Unfortunately we were unable to analyse the pharmacokinetics and metabolism of CY in terms of BMT-related toxicity and outcome because of the small number of patients involved and the heterogeneity of both conditioning regimens and donors. In addition to the nature of the donor, the optimum conditioning dose of CY is also likely to be influenced by the use of limited field irradiation. A novel approach to the selection of CY dose using an in vitro assay of chromosomal breakage following exposure to CY-derived alkylating metabolites has been described.35 Following the results of this assay the authors employed a wide range of CY doses for conditioning (20–150 mg/kg) with all patients surviving 2–5 years following BMT. The success of this approach awaits confirmation in a larger series of patients. Such studies highlight the heterogeneity of FA and suggest that individualisation of CY dose, either on the basis of its metabolism or its in vitro effects, may be necessary to improve the outcome of BMT for FA in the future. Alterations in CY pharmacokinetics amongst FA patients may be linked to the presence of abnormal oxygen metabolism. Under certain conditions the cytochrome P450 enzyme complex releases potentially toxic free iron and superoxide radicals, thus abnormalities in these haemoproteins may be important in the aetiology of the FA phenotype.36,37 Using a panel of cytochrome P450 inducers and inhibitors a recent study demonstrated that cytochrome P450 enzymes play a critical role in determing free radical metabolism in FA cells. In culture, reduced enzyme activity within these cells produced abnormal intracellular oxygen metabolism with subsequent formation of multiple DNA breaks.14 These results suggest that both abnormal drug metabolism and increased free radical production in children with FA may result from cytochrome P450 abnormalities, possibly by an uncoupling of the redox cycle between cytochrome P450 oxidases and reductase. Recent experiments describing binding of the FA complementation group C protein to cytochrome P450 reductase provides further evidence of the importance of this system in this respect.38 The heightened sensitivity of FA patients to CY is well documented.5,6 Whilst it is difficult to reconcile a reduction in the clearance of the parent drug with an increase in the production of alkylating metabolites it is possible that duration of exposure is important in determining the clinical effects of CY therapy. The low rate of CY metabolism observed in children with FA may lead to sustained exposure to low levels of phosphoramide mustard, resulting


in unexpectedly severe toxicity. Alternatively, failure to generate either dechloroethylcyclophosphamide or carboxyphosphamide may contribute to the extreme sensitivity of FA patients to CY. It is also possible that altered CY metabolism occurs as an epiphenomenon and is not directly responsible for increased toxicity; rather, this results from an increase in cytochrome P450 activity following a drug ‘load’ with enhanced redox cycling. CY is known to exhibit autoinduction during fractionated dosing regimens such as those employed during BMT conditioning.39 The mechanism of this response is poorly understood but may be critical in escalating redox cycling with a subsequent increase in the production of reactive oxygen intermediates. This study demonstrates that CP metabolism is altered in children with FA. The importance of this phenomenon in determining CY toxicity in vivo is unknown. It is possible that CY metabolism is linked with an increase in the production of free oxygen radicals by a common dependence upon the cytochrome P450 system. Further pharmacological studies of drugs in children with FA are warranted.

12 13

14 15 16

17 18 19

Acknowledgements SMY was supported by the Tyneside Leukaemia Research Fund. The authors are grateful to the North of England Childrens Cancer Research Fund for their Support.

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